biotropia (2) 1988/1989: 8-11 critical period of mungbean (phaseolus radiatus l.) to weed competition i.h. utomo* tropical agricultural pest biology program, biotrop, bogor, indonesia abstract a field experiment was conducted to study the critical period of weed control on the crop of mungbean (phaseolus radiatus l.). the studies were done in the field of biotrop experimental station with the natural existing weed population. it was found that the critical period of mungbean to weed competition was from 3-6 weeks after planting. introduction competition constitutes the major noxious aspect of weeds in crop cultivation. since weeds and crops largerly use the same resources for their growth, they will compete when these resources are limited (zimdahl 1980). part of these resources is continuously available in limited amounts, for instance light, or in some extent, nutrient that becomes available by mineralization. part of these resources, however, is only available as a limited stock at a certain period during crop development. environmental factors determine competition by changing space conditions and by affecting the phenotypes of the crops and the weed concerned. when weeds emerge in a newly establishing crop, the amount of resources already utilized by the weeds or crop before competition starts is determined by their seed weight, time of emergence, relative growth rate and population density. a newly emerged crop seedling relying for food stored in the seed would experience little competition from weed seedlings at a similar growth stage, if only because at that time total demands on water and minerals are small. likewise a crop towards the end of its growth cycle, especially if it is senescing and ripening seed will often be unharmed by the presence of weeds. between these situations, there would be a period when crops are most susceptible and weed competition would be more severe. this period is known as the critical period for weed competition. this experiment was aimed at studying the critical period of an annual crop, mungbean (phaseolus radiatus l.) as basic information on when to control the weeds in such mungbean cultivation. * present address: department of agronomy, faculty of agriculture, bogor agricultural university, bogor, indonesia. 8 critical period of mungbean to weed competition-1.h. utomo materials and method the experiment was conducted at biotrop field experiment, tajur, bogor from july september 1985. the seed of mungbean (var. pr 74) was planted after land preparation in 30 plots (3 x4 m 2 ), fertilized with 50 kg urea/ha, 100 kg tsp/ha and kcl 50 kg/ha. the experiment was arranged in a randomized block design with 3 replications. the treatments of the experiment were as follows (figure 1) figure 1. the treatment of the experiment the serial of periods consisting of weed-free and weedy conditions were introduced at 1, 2, 4, 6 and 8 weeks after planting. the growth and yield performance parameters of mungbean were recorded as follows: (1) biomass at 2, 4, 6 and 8 weeks after planting (2) plant height at 2, 4, 6 and 8 weeks after planting (3) yield of mungbean, expressed as dry weight of seed (g/m 2 ). 9 biotropia no. 2, 1988/1989 results and discussion the highest yield of mungbean was obtained with treatment g (weed present only 1 wap) which was not significantly different with treatment e which was weed-free during the entire mungbean growth period. weed competition which commenced during the first week was not considered severe (table 1). table 1. yield of mungbean. treatment g/m2 *) a 63.75 de b 75.84 cde c 106.74 ab d 95.60 bc e 112.11 ab f 51.16e o 125.84 a h 88.42 bcd i 98.77 bc j 61.58e *) means followed by the same letter are not significantly different at p≤0.05. data in table 1 show that mungbean yield decreased from 112.11 to 51.16 g/m 2 when weeds were allowed to grow unchecked throughout the growing period. the competition effect of weed was evident at each stage of the growing period and the longer weed was present in the cultivation, the lower yield of mungbean. the same results were also observed in relation to the duration of weeding application, i.e. the longer the duration of weeding application, the higher the yield of crop. the highest yield (g) was not significantly different from treatments c, d, e (for respective weed-free treatments) and h, i (for respective weedy treatments). the grain yield of mungbean therefore started to decrease if weeds occurred in crop cultivation before 6 weeks after planting. thus, 4 weeks after planting is the indicated time for weeding so that the crop will produce yield comparable to plots free of weed throughout the growing period. the period of weeding time seemed not to influence the other measurements like biomass (except at 6 wap) and plant height of mungbean (table 2). 10 critical period of mungbean to weed competition-1.h. utomo table 2. plant height and biomass of mungbean. references nieto, j.h., m.a. brondo and j.t. gonzales. 1968. critical periods of the growth cycle for competition from weeds. pans (c) 14: 159-166. zimdahl, l. 1979. crop-weeds competition. international plant protection centre. 11 8.pdf 9.pdf 10.pdf 11.pdf biotropia no biotropia no. 14, 1999: 10-16 morphological comparison of three asian native honey bees (apis cerana, a. dorsata, a. florea) in northern vietnam and thailand n.v. niem and l. q. trung vietnam bee research and development center long ha, dongda, hanoi, vietnam abstract three species of asian native honey bees (apis cerana, a. florea and a. dorsata) from northern vietnam and thailand were morphologically analyzed for investigations on their geographic variations and relations. in vietnam, samples were collected from feral and managed colonies. in thailand, the collections were from feral colonies or from field bees on flowers. morphological analysis was carried out, using measurements common to honeybee taxonomy. measured characters were done under stereomicroscope with an ocular micrometer. anova program and multivariate statistical analyses were applied for treating the data. overall, a. cerana populations in northern vietnam are significantly morphologically different than from those in thailand. it may be due to their different geographic locations between the thai and vietnamese populations of a. cerana. a. florea bees from vietnam are generally bigger in size than those from thailand, but the differences are uncertain. in contrast, the body size of a. dorsata populations from thailand are bigger than those from vietnam. however, these differences are also not significant. it is necessary to take further comparative investigations of these bee species from both countries. key words: honey bee/apis cerana/apis dorsata/apisjlorea/moif>ho\ogy introduction in the genus apis, eastern honey bees (apis cerana), giant honey bees (apis dorsata), and dwarf honey bees (apis florea and apis andreniformis) are native to many countries in southeast asia. these bee species play an important role in producing large amount of valuable products for people and in pollinating agricultural crops. however, their value is not well documented. the studies of their biological and economic characteristics being useful for strains improvement are less than those of european honey bees (a. mellifera). a few publications relate to geographical variations and distribution of these honey bee species in different parts of southeast asia. ruttner (1988) reviewed the geographical distribution of a. dorsata, a. cerana, a. florea in asia. verma (1990) made an extensive investigation on the distributions of a. cerana in india. peng et al. (1989) reviewed studies on the biology and distribution of races of a. cerana in china. a morphological analysis of a. cerana and a. nigrocincta populations from southeast asia has been taken by damus and otis (1997). rinderer et al. (1995) published data on comparative morphology of the dwarf honey bees in southeast thailand and palawan, philippines. wongsiri et al. (1993) carried out a comparative investigation of some biological characteristics of a. cerana bees in china, thailand and their hybrids for the purpose of using biological measures to control varroa parasitic mites. 10 biotropia no. 14, 1999 therefore, at present, there is a big gap in information on the genetic biodiversity of asian native honey bees. it is not clear how many subspecies and ecotypes of these native bee species exist in southeast asia and how they are related to each other. the purpose of this paper is to present some preliminary results on comparative morphology of the three honey bee species: a. cerana, a. florea and a. dorsata in vietnam and thailand to contribute basic information on certain morphological parameters. materials and methods in vietnam, samples of the three honey bee species were collected from locations of four provinces in the north (haiphong, laichau, hoabinh, nghean). at each location, four to six colonies were sampled. from each single colony, a sample of 40 '-60 worker bees was collected. in thailand, a. cerana samples were collected in bangkok, partly from a feral colony, partly from field bees on flowers in the gardens. a. florea bees were also collected on flowers. a. dorsata bees were sampled in nan province by using high voltage light trap to attract the bees from 14 colonies in one tree to the light. this sample should contain bee individuals from different colonies. the sampled bees were killed with hot water (around 90°c) to obtain their stretched proboscis, then they were preserved in 70% ethanol. thirty worker bees of each location (five to ten bees per sample) were selected and analyzed morphologically on temporary slide preparations. the following morphological measurements were recorded: 1. length of proboscis 2. width of prementum 3. length of forewing 4. width of forewing 5. cubital index 6. length of femur 7. length of tibia 8. length of basitarsus 9. width of basitarsus 10. tergite 3, longitudinal 11. tergite 3, transverse 12. stemite 3, longitudinal 13. stemite 3, transverse 14. wax plate of sternite 3, longitudinal 15. wax plate of sternite 3, transverse the measurement was done according to the ruttner's methods (1988), using a stereomicroscope with an ocular micrometer. for most parts, numbers presenting lengths or widths of various structures are in millimeters. the data were analyzed using anova program, and the student's t test was used to test the null hypothesis from the two equal sources of data. a clustering analysis was also applied to analyze the relationship between two honey bee populations, and establish their tree diagrams. 11 morphological comparison of three asian native honey bees n.v. niem & l.q. tiling results the results of morphometric measurements from these honey bee species are presented in table 1. the results show that the body size of a. cerana in vietnam was bigger than that in thailand. of the 15 measured characters, the values of 11 characters were significantly bigger (1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 14) particularly the cubital index, while the width of prementum and forewing were smaller. the body size of a. florea in vietnam was also bigger than those from thailand. however, these differences were not clear. six characters (3, 4, 5, 6, 12, 14) of the bees from vietnam were significantly larger than those from thailand. the remaining characters were slightly larger (10, 13), equal (2,7,9, 15) or smaller (1, 8, 11). in contrary to a. cerana and a. florea, the body size of a. dorsata in thailand was bigger than those from vietnam. qf 15 measured characters, the value of 11 characters of a. dorsata in thailand were higher (2, 3, 4, 5, 6, 7, 8, 11, 12, 14, 15), one character was equal (10) and three characters were smaller (1, 9, 13). interestingly, while the cubital index of a. cerana and a. florea in thailand were significantly bigger than those in vietnam, this character in a. dorsata of both countries was not significantly different. discussions the differences in body parts between a. cerana populations from vietnam and thailand are reasonably significant. twelve larger measured characters of a. cerana in vietnam indicate that these two honey bee populations may be considered as separating geographical groups. according to ruttner( 1988), the characters that can be considered to distinguish bee populations into subspecies levels are lengths of basitarsus, tergite 4, sternite 3, wax plate transverse of sternite 3, and cubital index. in this study, all these characters are significantly different between the two populations. the tree linkage diagram analysis (figure 1-a) shows the bee population in vietnam is also clearly distinguished from that in thailand. all the vietnamese bee populations are ranged in a distinct group (from 1 to 15) separated from the thai group ( from 16 to 30) and there is no overlap between them. our data support the ruttner's (1988) statement that a. cerana can be distinguished into two subspecies: a. cerana cerana and a. cerana indica. the former includes the bees of vietnam, afghanistan, pakistan, north india and china. the later includes the bees of thailand, malaysia, myanmar, bangladesh, sri lanka and south india, indonesia, philippines. comparing these results to the data of peng et a/.(1989) and verma (1990), it is recognized that the a. cerana bees from vietnam are closer to the bees in southern yunnan (china) and nepal, while a. cerana bees from thailand are closer to the bees in southern india. 12 13 figure la tree linkage diagram of apis cerana bee populations from northern vietnam (1-15) and thailand (16-30). overall, the body size of a. florea from vietnam is slightly bigger than those from thailand. however, the differences between these bee populations are too small to consider them as different ecotypes. the tree diagram (figure 1-b) shows an overlap between these two populations. therefore, it is necessary to take further studies on the relationship of these bee populations between vietnam and thailand. figure ib. tree linkage diagram of apis florea bee populations from northern vietnam (1-15) and thailand (16-30). 14 biotropia no. 14,1999 contrary to a. cerana and a. florea, a. dorsata bees in thailand are bigger in body size than those in vietnam, but the differences of the measured characters and the tree diagram analysis (figure 1-c) could not support any distinction at the species levels between these two bee populations. regarding the giant honey bees (a. dorsata), maa (1953) created a genus megapis with four species: megapis breviligula, m. binghami, m. dorsata and m. laboriosa. on the other hand, ruttner (1988) stated that there is very little evidence to support the idea of distinguishing a. dorsata into different subspecies, although its distribution is in a large range of the southeast asian region. however, according to his opinion, a. dorsata in this area can be separated only into two subspecies: a. d. breviligula and a. d. binghami. in this study, the differences in morphological characteristics of a. dorsata populations from vietnam and thailand are rather low to make any conclusion at the species level of classification. figure ic. tree linkage diagram of apis dorsata bee populations from northern vietnam (1-15) and thailand (16-30). acknowledgments we would like to thank prof. dr. siriwat wongsiri, mrs. wandee wattanachaiyngcharoen (bee biology research unit, chulalongkorn university, thailand) and dr. det wattanachaiyangcharoen, mr. sawang piyapichart (faculty of agriculture, natural resources and environmental science, naresuan university, thailand) for organizing a field trip to collect samples of a. dorsata. we are grateful to prof. nguyen dinh hien (hanoi agricultural university) for helping in statistical analysis. we thank prof. t. q. hoc (vietnam national university, hanoi) for helpful suggestions on the manuscript and the correction. 15 morphological comparison of three asian native honey bees n.v. niem & l.q. trung references damus, m.s. and g.w. otis. 1997. a morphological analysis of a. cerana f. and a. nigrocincta smith populations from southeast asia. apidologie 28:309 319. maa, tsing-chao. 1953. an inquiry into the systematics of the tribe apidini or honey bees (hym.) treubia21(3)525-640. peng, y.s., m.e. nasr and s.j. loske. 1989. geographical races of a. cerana fabricius in china and their distribution. review of recent chinese publications and a preliminary statistical analysis. apidologie 20:9 -12. rinderer, i.e., b. p. oldroyd, s. wongsiri, h.a. sylvester, l.l.de guzman, j.a. stelzer and r.m. riggio. 1995. a morphological comparison of the dwarf honey bees of southern thailand and palawan, philippines. apidologie 26:387-394. ruttner, f. 1988. biogeography and taxonomy of honeybees. springer-verlag, berlin. verma, l.r. 1990. honey bee resources: biology and management: 56 69. in beekeeping in integrated mountain development. oxford & ibh publishing, new delhi. wongsiri, s,, l. chariya, p. sureerat. 1993. biological control of varroa mite: mass rearing biological control agent by crossing the chinese strain apis cerana with the thai strain a. cerana indica by instrumental insemination. asian apiculture, p. 148 -155. 16 biotropia (2) 1988/1989: 18-24 the distribution and potential problems of mimosa pigra l. in indonesia sri s. tjitrosoedirdjo tropical agricultural pest biology program, biotrop, bogor, indonesia abstract mimosa pigra l. (mimosa asperata l.) of the family mimosaceae is an introduced species from south america which is locally naturalized in indonesia. the present known distribution sites are still restricted to java, sumatra, and kalimantan. it is found in almost all provinces of java and many heavily infested areas have been noted in jakarta and west java. the introduction of the plant to other islands has not been reported yet. the separation of the islands by sea is preventing the further spread of the plant in indonesia. control is basically occasional and on an individual basis. there is no sustained effort yet to control the plant. the urgency of controlling and restricting its spread cannot be over emphasized. introduction mimosa pigra l. is an introduced species in indonesia from south america which has been locally naturalized for a long time (backer and van den brink 1963). soerjani (1979) included this species as one of the ten serious aquatic weeds in indonesia. the plant is very difficult to control because its spiny and woody character makes it very difficult to penetrate when growing very densely. recently, there is a large number of areas in jakarta and its vicinity, as well as some areas in west java infested with m. pigra. in thailand, m. pigra was introduced from indonesia in 1947. it was first planted in amphus mae taeng where it was used as a ground cover to improve soil quality and to help prevent bank erosion. within 30 years, it has infested large areas of northern thailand (lamar robert 1982). among the institutes working on m. pigra in indonesia are: the central research institute for plantation which conducted the study on its biology and ecology (sumaryono and soedarsan 1983); the biological research and development center, indonesian institute of sciences, which conducted studies on its ecology, biological control and taxonomic aspects (uji 1988, partomihardjo 1988, and kahono 1988); and biotrop which studied its potential problems at bening and saradan reservoir, east java (anonymous 1983). research on the biology, distribution, ecological role in the environment and control methods in indonesia should be intensified and the problem must be dealt with before it expands to larger areas and become more serious. 18 the distribution and potential problems of mimosa pigra l. -sri s. tjitrosoedirdjo in this paper, the present distribution of m. pigra at the known sites and the potential problems in the future are discussed and evaluated. distribution of mimosa pigra in indonesia the distribution of m. pigra in indonesia was obtained from the examination of herbarium specimens at the herbarium bogoriense, bogor, indonesia and information from several other sources (soedarsan 1980, sumaryono and soedarsan 1983). mimosa pigra was first introduced to indonesia by the bogor botanical garden from mexico (thysmann and binnendijk 1866). the earliest record of its presence was in 1844 from bogor, west java (hasskarl 1844). the oldest existing herbarium specimens were found in 1902 from ciliwung riverside in bogor identified as mimosa asperata l.; in the same year, a specimen was also found in jakarta. the present distribution of m. pigra in indonesia is still restricted to java, sumatra and kalimantan (figure 1). most of the specimens were found in java, i.e. from all the provinces, jakarta, west java, central java and east java. in sumatra it was reported from solok, west sumatra (soedarsan 1980) and sibolangit, north sumatra in 1917. the only specimen from kalimantan was found in samarinda in 1979. it seems that in kalimantan m. pigra was newly introduced. there is no report yet from other islands. according to the recent information in indonesia there are two types of the habits of m. pigra i.e. erect and prostrate stenis. field observations showed different morphological characteristics of the two types of m. pigra. the prostrate stem type is only strictly distributed around jakarta, bogor, tangerang and bekasi at an altitude of 5-260 m a.s.l. the erect stem type is widely distributed all over java (partomihardjo 1988, uji 1988). backer and van den brink (1963) indicated that in indonesia it can be found at an altitude of 1-700 m above sea level. in different places m. pigra is called by different names such as klampis air, putri main hitam (sumaryono and soedarsan 1983) andputri malu raksasa. in west java it is known as jerujut, gehgeran and cucuk buset, rondo kaget (sunda), pis kucing (java). 19 biotropia no. 2, 1988/1989 20 the distribution and potential problems of mimosapigra l.-sri s. tjitrosoedirdjo potential problems in the future there are some factors assisting the dispersal and rapid colonization of m. pigra (miller 1982). among them are the intrinsic characteristics of the plant, like the ability of whole seed pods on their segments to float on water for long periods, and the hairy nature of pods allowing segments to stick to hair or clothing, which enable them to spread easily. this is combined with dispersal both by natural uncontrollable forces and by man as well as animals. in a river, the spread of m. pigra downstream from its original location has been largely through uncontrollable water movement. its spread to isolated sites away from the river is mainly by human activity through removal of sand from the river for use in construction of roads or buildings. the ability of m. pigra to rapidly colonize areas is due to its inherent ability to flower and seed all year round under favorable conditions, the production of large quantities of seeds, their long term viability and tolerance to both flood and drought. wanichanantakul and chinawong (1979) reported that in thailand, an average plant is seeded twelve times per year. they also found that prolonged seed dormancy was due to the hard seed coat. in indonesia, (anonymous 1983) the rate of growth of m. pigra at bening and saradan reservoirs, east java, was observed. the rate of growth was 11.54 g dry weight/day -1 or 4.42% day -1 . although it is less than the rate of growth of the floating aquatic weeds, it is relatively high for a woody perennial plant. the high rate of growth is also supported by the high production of viable seeds. the number of mature seeds found in 5 cm depth of soil in a plot 25 x 25 cm 2 was 200. the laboratory experiments showed that the seed germination rate was very high, i.e. approximately 93.5% under light and 89.5% under dark conditions (anonymous 1983). sumaryono and soedarsan, 1983 reported that in bogor, the stem diameter reached up to 4.6 cm after 8 months. the first flowers appeared 4 months after seed germination. they also reported that after cutting, regeneration is very rapid. it grew better in a soil water of 43-67% and the plant could survive in flooded land although its growth was retarded. in java, m. pigra has spread faster than on other islands. colonization by m. pigra is basically through movement of seeds by water. mostly, the plants were found in wet places-such as riverside, irrigation canal or at the shore of the lake (table 1). introduction to isolated places not reached by water can also be noticed. it is also found along the road, railway or in waste lands. 21 biotropia no. 2, 1988/1989 table 1. distribution of m. pigra in indonesia based on the herbarium specimens at the herbarium bogoriense", other sources (soedarsan 19802)), anon, 19833), and own observation4). 22 the distribution and potential problems of mimosa pigra l. sri s. tjitrosoedirdjo increasing development of housing, industrial estates, highways, airports, bridges and roads in jakarta use large amount of sand. this sand comes from the rivers or places already contaminated by m. pigra seeds. heavy infestation can be found in waste lands close to housing and industrial estates, along the jagorawi highway, and along the way to soekarno-hatta airport. at ciapus bogor, there is a big sand quarry where the product is distributed mostly to jakarta and west java. m. pigra can be found easily in the areas surrounding the quarry. this condition contributes to the further spread of m. pigra elsewhere. in central java, m. pigra can be found at the roadside between yogyakarta and magelang. there are some rivers such as krasak, putih and blongkeng, between these two cities. the rivers are rich in sand from merapi mountain, which is collected and distributed to many places in central java. if the sand from these rivers is contaminated with m. pigra seeds, it can be spread further within a short time. anonymous (1983) reported that in east java, bening and saradan reservoirs are infested with m. pigra. the plants are mostly found at the lake shore. at saradan reservoir m. pigra is a dominant species. at bening reservoir where the fluctuation of the water is very high (approximately 13 m), m. pigra appears to be particularly well adapted to grow in a seasonally flooded habitat. during the dry season, m. pigra grows well and biomass production is very high. during this time at bening reservoir, the flooded area is 570 ha and there are 370 ha which can be potentially colonized by m. pigra. after flooding during the wet season, m. pigra will decompose and the nutrients released would enrich the water. its decay will cause an increase in the water's carbon dioxide content, chemical oxygen demand, ammonia and nitrate, and nitrogen, and reduce its ph and dissolved oxygen concentration. the plants recover by germination of viable seeds. anonymous (1983) suggested that a control program should be carried out during the dry season before flowering, to prevent the production of seeds. in sumatra, soedarsan (1980) reported that m. pigra was found close to the river. it reached 4 m in height. if there is no control treatment m. pigra will spread further. with the construction of the trans sumatra highway from aceh to java and crossing the sunda channel, there is a possibility that m. pigra will be spread from java to sumatra through the movement of man and vehicles. 23 biotropia no. 2, 1988/1989 summary although m. pigra has been introduced in indonesia for a long time, the existing known places are still restricted to java, sumatra and kalimantan. the introduction of the plant to other islands has not been reported yet. the separation of the islands by the sea is preventing the further spread of the plant in indonesia. in java, m. pigra is widely distributed, it is found in almost all provinces. the high rate of growth, the ability of producing large amounts of seed, the great regenerative power and also the resistance to flood and drought support the rapid colonization of m. pigra. in jakarta and west java, many places have been infested heavily by m. pigra. there is no sustained effort yet to control the plant, only occasionally by individual initiative. the urgency of controlling and restricting its spread cannot be over emphasized. references anonymous. 1983. penyelidikan tanaman air dan perikanan waduk pada proyek irigasi widas (studies on the reservoir's aquatic weeds and fishery at the widas irrigation). internal report biotrop, bogor, indonesia. backer, c.a. and r.c.b. van der brink. 1963. flora of java. vol. i. n.v.p. noordhof-groningen the netherlands. lamar robert, g. 1982. economic returns to investment in control of m. pigra in thailand. ippc document no. 42. a-82. mpc agricultural economics report no. 15. hasskarl, j.k. 1844. catalogus plantarum in horto botanico bogoriense cultarum alter. (ter lands-drukkerij : batavia). miller, i.l. 1982. the distribution and threat of m. pigra in australia proc. international symposium on m. pigra management chiang mai, thailand. partomihardjo, t. 1988. studies on the biology of mimosa pigra for supporting its control in indonesia. proceeding wssi conference vol. ii. soedarsan, a. 1980. potensi klampis air (m. pigra l.). sebagai gulma di masa depan (the potential of m. pigra as a weed in the future). menara perkebunan 48(6): 179-180. soerjani, m. 1979. recent trends in aquatic weed management in indonesia proc. 7th asian pac. weed sci. conf. sydney, australia. supp. vol. sumaryono and soedarsan, a. 1983. beberapa aspek pertumbuhan klampis air (some aspect of the growth of m. pigra). menara perkebunan 51 (6): 160-164 . thysmann, j.e. and s. binnendijk. 1866. catalogus plantarum quae in horto botanico bogoriense colunter-ter lands drukkerij: batavia. uji, t. 1988. mimosa species in java. proceeding indonesian weed science society conf. vol. i. wanichanantakul, p. and s. chinawono. 1979. some aspects on the biology on m. pigra in northern thailand. proc. 7th asian pac. weed sci. soc. conf., sydney, australia. 24 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf biotropia no. 8, 1995: 53-59 response of two sunflower (helianthus annuus l.) genotypes to va-mycorrhizal inoculation and phosphorus levels c.p. chandrashekara, v.c. patil and m.n. sreenivasa university of agricultural sciences, dharwad-580 005, karnataka, india abstract the performance of two sunflower genotypes (morden and msfh-8) with and without va-mycorrhizal fungi at three p levels (38, 56 and 75 kg p2o5 ha -1 ) in vertisol of dharwad was studied to determine the effect of mycorrhizal inoculation on plant growth, yield and p uptake. the results showed that the vam inoculation increased sunflower yield (14%), total biomass (16%), oil content (3.1%) and p uptake (30.5%) over uninoculated control. the percent root colonization and chlamydo-spore count decreased with increasing p levels. the total biomass production, seed yield and p uptake of mycorrhizal plants at 38 kg p2o5 ha -1 more than the non-mycorrhizal plants at 75 kg p2o5 ha -1 . the biomass and seed yield of mycorrhizal plants at same p level were more than the non-mycorrhizal plants. mycorrhizal plants of morden at 38 kg p2o5 ha -1 and msfh-8 at 56 kg p2o5 ha -1 produced higher seed yield, oil content and total biomass than non-mycorrhizal plants supplied with 75 kg p2o5 ha -1 . the results indicated that, va-mycorrhizal inoculation helps in saving 25 and 50 percent of recommended dose of phosphatic fertilizer (75 kg p2o5 ha -1 ) in msfh-8 (single cross hybrid) and morden (open pollinated variety), respectively. key words: mycorrhizas/plant nutrition/inoculum/glomus fasciculatum/helianthus annuus/phosphorus fertilizers/metabolism. introduction the current trend is to explore the possibility of supplementing chemical fertilizers with organic ones, more particularly biofertilizers of microbial origin. in this context, vam fungi are receiving greater attention in their beneficial effects on plant growth. vesicular-arbuscular mycorrhizae (vam) are widespread in soils, and often the growth of mycorrhizal plants will be higher in comparison to non-mycorrhizal plants. this beneficial effect on plant growth has largely been attributed to higher phosphorus (p) uptake and consequently better p nutrition of mycorrhizal plants (sanders and tinker 1973). sunflower, one of the potential oilseed crops of india has a higher p requirement. morden (open pollinated variety) and msfh-8 (single cross hybrid) are the two predominant genotypes of sunflower in karnataka. vam association have been 53 biotropia no. 8, 1995 reported in sunflower (cabello 1987), and it becomes mycorrhizal as early as at two leaf stage (iqbal and qureshi 1977). roots are heavily infected with glomus macro-carpum var. geospora under greenhouse conditions (ross and harper 1973). the sunflower roots were more colonized in bioassay tests than love grass (anderson and liberia 1987). phosphorus is usually considered to be the major problem when va-mycorrhizal infections are poor. high p levels in soil or p additions are known to reduce vam colonization of roots and sporulation (sreenivasa and bagyaraj 1989). studies have indicated that addition of an excess of readily available p eliminates beneficial mycorrhizal effects (sreenivasa et al. 1993). generally, research work on the use of mycorrhizae has been carried out under controlled conditions. pot culture trials have proved substantially that sunflower responds to inoculation with efficient strains of vam fungi at lower levels of phosphorus (jones and sreenivasa 1992). however, very few field experiments have been carried out so far. hence, the present study was undertaken to study the response of two sunflower (helianthus annuus l.) genotypes to the inoculation of vam fungus (glomus fasciculatum) and p levels, and to explore the possibility of substituting phosphatic fertilizer through vam inoculation. materials and methods a field trial was conducted at main research station, university of agricultural sciences, dharwad, on medium black soil (ph 7.6; organic carbon 0.63%; available n-0.063%; p2o5-32 kg ha-1 and k2o-295 kg ha -1) under irrigated conditions during summer season of 1993. the experiment consisted of twelve treatments comprising three p levels, two levels of vam inoculation (inoculated and un-inoculated) and two sunflower genotypes (morden and msfh-8) forming a 3 x 2 x 2 factorial experiment in a randomised complete block design with four replications each. plots of size 5.0 m x 3.6 m were prepared and spacing of 45 cm x 20 cm for morden and 60 cm x 20 cm for msfh-8 were followed with 200 and 150 plants per plot (18 m 2 ), respectively. the recommended dose of n and k were given (62.5 kg and 51.25 kg ha -1, respectively) in the form of urea and muriate of potash. the p levels consisted of 38, 56 and 75 kg p2o5 ha-1 (equivalent to 50,75 and 100% of recommended dose, respectively) as single super phosphate. glomus fasciculatum inoculum was multiplied in sterilized sand: soil mixture (1:1 by volume) using rhodes grass (sreenivasa and bagyaraj 1988). the inoculum (@ 30 g/spot) was placed 2 cm below the sunflower seeds. the inoculum consisted of colonized root fragments, hyphae and chlamydospores (126 per 50 g inoculum). 54 response of two sunflower genotypes c.p. chandrashekara, v.c. patil and m.n. sreenivasa irrigation was given at is days interval to maintain soil moisture near field capacity. five plants were uprooted 90 (morden) and 105 (msfh-8) days after planting. shoots were severed at ground level, dried at 70°c, weighed, ground to pass a 0.5 mm sieve and analysed for p by vanadomolybdate phosphoric yellow colour method (jackson 1967). roots were washed with water and representative fresh samples were stained with trypan blue and percent root colonization was estimated (phillips and hayman 1970). chlamydospore count per 50 g rhizophere soil was taken by wet-sieving and decanting technique (gerdemann and nicolson 1963) and oil content was determined by using nuclear magnetic resonance (nmr) spectrophotometer. the seed yield (kg ha-1) was computed by using net plot yield (7.2 m 2 ). the data were analysed by mstat statistical program and least significant difference was used for mean separation. results and discussion sunflower genotypes responded well to the inoculation of va-mycorrhizal fungi. vam inoculation resulted in increased total biomass (16%), seed yield (14%) and oil content (3.1 %) (table 1). vamycorrhizal fungi are associated with increased growth of many plant species, mainly through increased uptake and translocation of not only p but also other nutrients (abbott and robson 1982). in addition to p, vam also help in uptake of zn, cu, mn and fe etc., (sreenivasa et al. 1993). iqbal and qureshi (1977) reported that mycorrhizal sunflower plants were taller, stouter, had more numerous larger leaves and bore bigger capitulum producing viable seeds than non-mycorrhizal plants. va-mycorrhizal plants recorded 30 percent higher p uptake (19.91 kg ha-1) than uninoculated plants (15.25 kg ha-1), which ultimately enhanced the oil content of the seed. the percent root colonization and spore count was higher in mycorrhizal treatments than in uninoculated controls. sunflower roots were heavily infected with glomus macrocarpum var. geospora under greenhouse conditions (ross and harper 1973). this probably resulted in greater fungal host contact and greater uptake of nutrients and hence better plant growth and yield. gerdemann (1964) and daft and nicolson (1966) correlated the increased growth and yield of crops to the extent of mycorrhizal infection. the percent root colonization and spore count decreased with increasing p levels in both inoculated and uninoculated plants. bagyaraj and powell (1985) have also observed depressed vam development at recommended p level due to higher availability of phosphorus. in a pot culture study, jones and sreenivasa (1992) observed increased percent root colonization of sunflower with increase in p level up to 50% of recommended dose of p, which decreased thereafter. the total bio 55 biotropia no. 8, 1995 56 response of two sunflower genotypes c.p. chandrashekara, v.c. patil and m.n. sreenivasa mass, seed yield and p uptake of mycorrhizal plants at lower phosphorus levels (38 and 56 kg p2o5 ha -1 ) was more than non-mycorrhizal plants at 75 kg p2o5 ha -1 , which may be ascribed to higher vam-fungus root association and sporulation at lower p levels than at higher p level (table 1). the total biomass, seed yield and p uptake of mycorrhizal plants at same p level was more than the non-mycorrhizal plants. the higher efficiency of vam at lower levels of p has been ascribed to exploration of greater soil volume by mycorrhizal plants and faster movement of p ions into mycorrhizal hyphae (bolan 1991). mycorrhizal plants of morden at 38 kg p2o5 ha -1 and msfh-8 at 56 kg p2o5 ha -1 produced higher yield, oil content and total biomass than non-mycorrhizal plants supplied with 75 kg p2o5 ha -1 (table 2). this can be ascribed to the higher percent root colonization and spore count recorded with consequent increase in uptake of phosphorus. siqueria and paula (1986) found low efficiency of indigenous vam fungi. they also found maximum dry matter at 30 ppm of p2o5 in mycorrhizae inoculated soybean plants as compared to 120 to 240 ppm of p2o5 with native fungi. the maximum effect of vam fungi was found at 50 and 75% of recommended dose of p in morden and msfh-8, respectively. smith and gianinazzi-pearson (1988) found direct effect of soluble p on fungal metabolism, 57 biotropia no. 8, 1995 mainly by regulating enzymatic activities related to phosphate transfer to the host. therefore, it could be noted that further increase in p level did not enhance growth or yield of sunflower. the relative benefits of mycorrhizal fungi decreased with increasing p supply. conclusion the present study demonstrated that, the percent root colonization and spore count decreased with increasing p levels. the growth and yield of mycorrhizal plants at same p level are higher than in uninoculated plants. the differential response was obtained among the genotypes to vam inoculation. glomus fasciculatum was efficient at 38 and 56 kg p2o5 ha-1 in morden and msfh-8, respectively as compared to at 75 kg p2o5 ha-1 (100% of recommended dose); which suggests that, va-mycorrhizal inoculation helps in saving of 25 and 50 percent of recommended dose of phosphatic fertilizer (75 kg p2o5 ha-1) in msfh-8 (single cross hybrid) and morden (open pollinated variety), respectively. literature cited abbott, l.k. and a.d. robson. 1982. the role of vesicular-arbuscular mycorrhizal fungi in agriculture and the selection of fungi for inoculation. aust. j. agric. res. 33: 389-408. anderson, r.c. and a.e. liberta. 1987. variations in vam relationships of two sand praharie species. am, midland natural, 118 (1): 56-63. bolan, n.s. 1991. a critical review of the role of mycorrhizal fungi in the uptake of phosphorus by plants. plant soil. 134: 183-207. bagyaraj, d.j. and c.li. powell. 1985. effect of vesicular-arbuscular mycorrhizal inoculation and fertilizer application on the growth of marigold. new zealand. j. agric. res. 28: 169-173. cabello, m.n. 1978. vesicular-arbuscular mycorrhizae in sunflower (helianthus annuus l.) crop. resista. de la facutad de agronomia, universidad national de la plata, 63: 46-52. daft, m.j. and j.h. nicolson. 1966. the effect of endogone mycorrhizae on plant growth. new phytol. 65: 345-350. gerdemann, j.w. 1964. the effect of mycorrhizae on the growth of maize. mycologia. 56: 342-349. gerdemann, j.w. and j.h. nicolson. 1963. spores of endogone species extracted from soil by wet sieving and decanting. trans. br. mycol. soc. 46: 235-244. iqbal, j.h. and k.s. qureshi. 1977. the effect of vam association on growth of sunflower (helianthus annuus) under field conditions. biologia (pakistan). 28: 189-196. jackson, m.l. 1967. soil chemical analysis. prentice hall of india (ltd.), new delhi. jones, n.p. and m.n. sreenivasa. 1992. response of sunflower to the inoculation of va mycorrhiza and phosphate solubilizing bacteria in black clayey soil. j. oilseeds res. 10: 86-92. 58 response of two sunflower genotypes c.p. chandrashekara, v.c. patil and m.n. sreenivasa phillips, j.m. and d.s. hayman. 1970. improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. trans. br. mycol. soc. 55: 158-161. ross, j.p. and j.a. harper. 1973. hosts of vesicular arbuscular endogone sp. j. elisha mitchell sci. 89 (1/2): 1-3. sanders, f,e. and p.b. tinker. 1973. phosphate flow into mycorrhizal roots. pestic. sci. 4: 385-395. siqueria, j.o. and m.a. paula. 1986. effect of vam nutrition and phosphate use by soybean in cerado soil. revista. brasileria. de gencia do solo. 10 (2): 97-102. smith, s.e. and v. gianinazzi-pearson. 1988. physiological interactions between symbionts in vam plants. ann. rev. plant physiol. 39: 221-244. sreenivasa, m.n. and d.j. bacyaraj. 1988. chloris gayana kunth (rhodes grass) a better host for mass production of glomus fasciculatum inoculum. plant soil. 106: 289-290. sreenivasa, m.n., p.u. krishnaraj, g.a. gangadhara and h.m. manjunathaiah. 1993. response of chilli to the inoculation of an efficient vesicular-arbuscular mycorrhizal fungus. scientia hortic. 53: 45-52. sreenivasa, m.n. and d.j. bagyaraj. 1989. suitable form and level of phosphorus for mass production of va-mycorrhizal fungus, glomus fasciculatum. zentralbl. mikrobiol. 144: 33-36. 59 53.pdf 54.pdf 55.pdf 56.pdf 57.pdf 58.pdf 59.pdf biotropia no. 8, 1995: 45-52 effects of pisolithus tinctorius and laccaria fraterna on the growth and mycorrfflzal development of pinuspatula seedlings*) m. sudhakara reddy and k. natarajan centre for advanced studies in botany, university of madras, guindy campus, madras 600 025, india abstract vegetative inoculum of pisolithus tinctorius and laccariafraterna were inoculated to pinuspatula seedlings grown in both steam sterilized and unsterilized shola soil. after 4 months of seedling growth, 10 seedlings from each treatment were harvested and various growth parameters were studied. inoculation of these two fungi resulted in the production of ectomycorrhizas and increase in growth of p. patula seedlings when compared to uninoculated seedlings. laccariafraterna inoculated seedlings showed more number of mycorrhizas than p. tinctorius inoculated seedlings at the end of one year. both these fungi poorly colonized the root system in both soil treatments. there was no significant difference between these two fungi in improving the seedling growth in the nursery. key words: pisolithus tinctorius/laccaria fraterna/pinus patula/inoculum/seedlings/growth. introduction extensive work on nursery inoculation has been done with pisolithus tinctorius because of its ecological adaptation to adverse soil conditions, wide geographic distribution and broad host range, tolerance to a variety of environmental conditions and its easy propagation and manipulation in pure culture (marx et al. 1984). eventhough p. tinctorius is worldwide in distribution and has a broad tree host range it has not been found in the nilgiri hills either in pine or eucalypt plantations. the other potential ectomycorrhizal fungus laccariafraterna is wide spread throughout the world where eucalyptus and other ectomycorrhizal hosts have been introduced (tommerup et al. 1991). at certain places in nilgiri hills where eucalypt and pine plantations occur side by side, l. fraterna is mostly found to be associated with the eucalypts but rarely with pines (natarajan 1977). but under in vitro condition these two fungi were able to form mycorrhizas with p. patula seedlings. the present research study was undertaken to study the effects of these two fungi on the growth and mycorrhizal development of p. patula seedlings in the nursery. *)paper presented at the second symposium on biology and biotechnology of mycorrhizae and third asian conference on mycorrhizae (acom iii), 19-21 april 1994, yogyakarta, indonesia. 45 biotropia no. 8, 1995 materials and methods pisolithus tlnctorius culture was isolated from basidiomata collected from eucalyptus tereticornis plantations, near madras coast, tamilnadu and the culture of l. fraterna was isolated from the basidiomata collected from e. globulus plantations in nilgiri hills, tamilnadu, south india. the cultures were maintained at 25°c on potato dextrose agar medium. the mycelial inoculum of p. tinctorius and l. fraterna were prepared according to the method of marx and bryan (1975). inoculum of each fungus was grown aseptically in one litre erlenmeyer flasks containing 750 ml of vermiculite moistened with 375 ml of mmn liquid medium. the flasks were incubated at 25°c in dark. after 12 weeks of incubation, inoculum was removed from the flask and leached with cool running tap water to remove the unused nutrients. excess free water was removed by gently squeezing the inoculum wrapped in cheese cloth. fungus free vermiculite wih mmn served as control. the shola soil collected under natural vegetation of the upper region of nilgiri hills was used in the present study. the chemical constituents of the soil are as follows: ph 5.3; organic matter content -4.79%; npk levels 245, 5.2 and 35 kg/acre, respectively. the nursery experiment was conducted at the forest department nursery, ootacamund, nilgiri hills during july 1992 to june 1993. both steam sterilized and unsterilized soils were filled in polybags (about 2 kg/bag) and 10% inoculum by volume was added to the polybags separately and mixed into the upper 8-10 cm of the soil. fungus free vermiculite added to the polybags served as controls. the surface sterilized seeds of p. patula (30% h2o2 for 30 minutes) were sown in all the bags. five (5) seeds were sown in each bag. after germination, seedlings were thinned to one per bag. ten (10) replicates were kept for each treatment. after 12 months of seedling growth, 10 seedlings from each sample were harvested and the growth and mycorrhizal development were studied. the shoot height, root length, and root collar diameter were measured. after counting the mycorrhizal and non-mycorrhizal tips, the shoots and roots were dried in a hot air oven at 85°c for 48 hours. dry weights of shoot and root were determined by obtaining constant weights. the method used by zak (1973) and agerer (1986) were followed for studying the macroscopic and microscopic features of ectomycorrhizas. the colour terminology used is that of kornerup and wanscher (1978). all the data were analysed by analysis of variance and the means were compared by least significant difference (snedecor and cochran 1967) at p = 0.05 level. 46 effect of pisolithus tinctorius and laccaria fraterna m. sudhakara reddy and k. natarajan results the root examinations revealed that both the ectomycorrhizal fungi viz., p. tinctorius and l. fraterna were able to form mycorrhizas with p. patula seedlings in the nursery. the p. tinctorius type of mycorrhizas are mostly dichotomous, tetrapodials and coralloid forms, 3-6 mm long and 0.4-0.5 mm in diameter. colour of mycorrhizas are brownish yellow (5c8) to light brown (6d6). loose hyaline hyphae are associated with the surface of the mycorrhizal system. the rhizomorphs are light brown (6d6) in colour and 150-550 µm wide, composed of closely packed parallel hyphae and covered with radiating hyphae (fig. la). the transverse section showed that the mantle is 15 20µrn thick. it consists of a simple prosenchymatous tissue. cystidia, setae or sclerotia are not observed. the hartig net is composed of one or two rows of oval to globose hyphal cells which measure 5 — l0 µm in thickness and penetrate up to 4 cortical cell layers deep (fig. ib). laccaria fraterna type of mycorrhizas are mostly bipodial, rarely monopodial, 3-5 mm long and 0.3 0.4 mm in diameter. colour of the mycorrhizas are orange white (6a2) when young and brown (6d7) when old. the surface is smooth. rhizomorphs are absent (fig. ic). the transverse section showed a mantle of 1015µm thick and consists of a simple prosenchymatous tissue. the hartig net is composed of a single row of oval to globose hyphal cells which measure 3 6 µm in thickness and penetrate up to 3 cortical cell layers deep (fig. id). inoculation of p. tinctorius and l. fraterna increased the number of mycorrhizas both in sterilized and unsterilized soil. the number of mycorrhizas were more in sterilized inoculated soil than in unsterilized soil. laccaria fraterna produced more number of mycorrhizas than p. tinctorius. but there was no significant difference between these two fungi. the percentage of mycorrhizas did not differ significantly between p. tinctorius and l. fraterna inoculated seedlings in both soil treatments. the percent colonization of both inoculated fungi were lower in both soil treatments. when compared to p. tinctorius, l. fraterna inoculated seedlings showed higher percent colonization at the end of one year. no basidiomata production was seen in the case of p. tinctorius whereas l. fraterna inoculated seedlings produced basidiomata in the bags at the end of one year. the growth of p. patula seedlings was improved in both sterilized and unsterilized soil by the inoculation of the fungi. the shoot height was more in sterilized inoculated soil than in unsterilized inoculated soil. but there was no significant difference between the two fungi in improving the shoot height. the root length has increased in both soils by the inoculation of these two fungi. laccaria fraterna inoculated seedlings showed a higher root collar diameter when compared to p. tinc 47 figure 1. ectomycorrhizas of pinus patula produced by pisolithus tinctorius and laccaria fraterna. a. morphology of p. tinctorius ectomycorrhizas x 240. b. transverse section of p. tinctorius ectomycorrhizas x 320. c. morphology of l. fraterna ectomycorrhizas x 200. d. transverse section of l. fraterna ectomycorrhizas x 325. m mantle; h hartig net. 48 biotropia no. 8, 1995 effect of pisolithus tinctorius and laccaria fraterna m. sudhakara reddy and k. natarajan torius inoculated seedlings. the shoot dry weight was more in sterilized soil than in unsterilized soil in inoculated seedlings. the seedlings inoculated with l. fraterna showed maximum shoot dry weight in both soils. but there was no significant difference between the two fungi in improving the shoot dry weight. the root dry weight also did not differ significantly between the two fungi. the shoot/root ratio differed significantly in control seedlings than in inoculated seedlings in sterilized soil. in unsterilized soil there was no significant difference between the two fungi and control seedlings with respect to the shoot/root ratio (table 1). seedlings grown in sterilized soil inoculated with the two fungi showed more growth and mycorrhizal development than the seedlings grown in unsterilized soil inoculated with these fungi. laccaria fraterna and p. tinctorius did not differ significantly in improving the growth and mycorrhizal development of p. patula seedlings in the nursery. discussion since p. tinctorius and l. fraterna are known to be early stage ectomycorrhizal fungi (marx 1991; tommerup et al. 1991), these two species have been selected to evaluate their effects on the growth and mycorrhizal development of p. patula in the present study. vegetative inoculum of these two fungi improved the growth and mycorrhizal development of p. patula seedlings when compared to uninoculated seedlings in the nursery in both steam sterilized and unsterilized soil. but the difference in various growth parameters is not significant in the inoculated seedlings. the seedlings inoculated with l. fraterna seem to be marginally better than the seedlings inoculated with p. tinctorius. tommerup et al. (1991) reported that l. fraterna, an early colonizing mycorrhizal fungus of eucalyptus is a potential competitor when the seedlings were inoculated with other selected ectomycorrhizal fungi. it is interesting to note that basidiomata of l. fraterna very rarely occur in p. patula plantations in nilgiri hills and found mostly associated with eucalyptus plantations which are adjacent to the p. patula plantations. it has also been noticed that basidiomata of l. laccata, the most predominant fungus in p. patula plantations, were seldom found in the eucalyptus plantations. the results of the present experiment suggest that l. fraterna and p. tinctorius are capable of improving the growth of nursery seedlings in nilgiri conditions. the colonization of these two fungi was poor in both treatments. marx and cordell (1987) found that ph between 4.5 and 5.5 is adequate for p. tinctorius, but that a ph above 6.0 inhibits ectomycorrhizal formation. the ph of the soil used in the present study is 5.3 and hence it may not be the factor for 49 biotropia no. 8, 1995 50 effect of pisolithus tinctorius and laccaria fraterna m. sudhakara reddy and k. natarajan poor colonization of p. tinctorius in the nursery. marx et al. (1970) reported that p. tinctorius grew rapidly at 28 30°c and capable of growing at 40 42°c in pure culture and formed more mycorrhizas with p. taede at 34° c than at lower temperatures. marx and bryan (1971) reported that aseptically grown p. taede seedlings with p. tinctorius ectomycorrhizae had better survival and growth at 40°c in laboratory tests than non-mycorrhizal seedlings or those mycorrhizal seedlings with t. terrestris. temperature may be the main reason why p. tinctorius is not occurring in nilgiri hills where the temperature ranges between 5 and 20° c during different parts of the year. the isolate of p. tinctorius used in the present study was obtained from the basidiomata associated with e. tereticornis in coastal madras where the temperature in summer will raise up to 40°c. further research is needed to evaluate different environmental conditions on the growth and mycorrhizal development of p. patula by p. tinctorius. in spite of the positive growth response shown by the seedlings in the present study only further research will reveal whether these two fungi will survive when seedlings are outplanted since they are conspicuous by their absence in p. patula plantations in the nilgiri hills. references aoerer, r. 1986. studies on ectomycorrhizae. ii. introducing remarks on characterization and identification. mycotaxon, 26: 473-492. kornerup, a. and j.h. wanscher. 1978. methuen handbook of colour 3 rd ed. methuen and co. ltd. london, p. 243. marx, d.h. 1991. the practical significance of ectomycorrhizae in forest establisment. in: ecophysiology of ectomycorrhizae of forest trees. the marcuswallenberg foundation, symposia proceedings, 7: 54-90. marx, d.h. and w.c. bryan. 1971. influence of ectomycorrhizae on survival and growth of aseptic seedlings of loblolly pine at high temperature. for.sci. 17: 37-41. marx, d.h. and w.c. bryan. 1975. growth and ectomycorrhizal development of loblolly pine seedlings in fumigated soil infested with the fungal symbiont pisolithus tinctorius. for. sci. 21: 245254. marx, d.h. and c.e. cordell. 1987. ecology and management of ectomycorrhizal fungi in regenerating forests in the eastern united states. in: mycorrhizae in the next decade: practical applications and research priorities. seventh nacom, may, 3-8, 1987. gainesville, fl. ed. d.m. sylvia, l.l. hung and j.h. graham. int. of food and agric. sciences, univ. of florida, gainesville. marx, d.h., w.c. bryan and c.b. davey. 1970. influence of temperature on aseptic synthesis of ectomycorrhizae by thelephora terrestris and pisolithus tinctorius on loblolly pine. for. sci. 16: 424-431. marx, d.h., c.e. cordell, d.s. kenney, j.g. mexal, j.d. artman, j.w. riffle and r.j. molina. 1984. commercial vegetative inoculum of pisolithus tinctorius and inoculation techniques for development of ectomycorrhizae on bare-root tree seedlings. for. sci. monogr. 25: 1-101. 51 biotropia no. 8, 1995 natarajan, k. 1977. south indian agaricales iii. kavaka. 5: 35-39. snedecor, g.w. and w.g. cochran. 1967. statistical methods. the iowa state university press. iowa usa p. 593. tommerup, i.e., n.l. bougher and n. malajczuk. 1991. laccaria fraterna a common ectomycorrhizal fungus with mono-and bisporic basidia and multinucleate spores: comparison with quadristerigmate, binucleate spored l. laccata and the hypogeous relative hydnangium cameum. mycol. res. 95: 689-698. zak, b. 1973. classification of ectomycorrhizae: in: ectomycorrhizae: ecology and physiology. g.c. marks and t.t. kozlowski eds. academic press, new york and london, p. 43-78. 52 45.pdf 46.pdf 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf 5 phytochemical (elfahmi).cdr biotropia vol. 18 no. 1, 2011: 42 49 phytochemical study of cell culture jatropha curcas elfahmi , artri , komar ruslan belongs to the euphorbiaceae family which has a high economic value as a source of biofuel.. this plant has been reported containing toxic compounds such as curcin and phorbol ester and its derivatives which may become a problem if is processed to biofuel. in order to investigate the chemical constituents of this plant, a research on phytochemical and initiation of cell and organ culture has been carried out. collected from different regions in indonesia showed containing relatively the same profile of chemical contents. dominant compounds that were detected by gcms are hydrocarbon such as 2-heptenal, decadienal, hexsadecane, pentadecane, cyclooctane etc, fatty acid such as octadecanoate acid, ethyl linoleate, ethyl stearate, hexadecanoate acid and steroid such as stigmasterol, fucosterol, sitosterol. no phorbol ester and its derivatives have been detected yet by the gcms method. callus and suspension cultures of have been established using murashige and skoog medium supplemented with 4 % (w/w) sucrose, solidified with 0.9% agar and growth hormon naa 2 mg/l : bap 0,5 mg/l. , stigmasterol, fucosterol, sitosterol, phorbol ester, fatty acid 1* 1 1 1 pharmaceutical biology research group, school of pharmacy, itb, bandung, indonesia jatropha curcas j. curcas jatropha curcas j. curcas jatropha curcas abstract introduction key words: jatropha curcas j. curcas j. curcas j. curcas belongs to the family euporbiaceae which has a multipurpose use such as medicinal plant and source of biofuel. as an alternative source of biofuel, has a high economic value. the development of this plant as source of alternative energy has been supported by researches and government regulations. since this plant is very potential, it has been cultivated in many regions in indonesia. several aspects have to be considered to develop this plant not only from the view point of production capacity but also the social condition of the population. aside from a potential source of biofuel, contains secondary metabolite compounds which have pharmacological and also toxicity effects. several compounds isolated from have been reported to be toxic, they are toxalbumin, cursin and phorbol ester. toxic effects of this plant caused skin irritation, vomiting, diarhea, etc. these * corresponding author : elfahmi@fa.itb.ac.id 42 toxicities are closely related to its chemical contents. several other secondary metabolites from have been reported such as terpenoid, coumarine, flavonoid, etc. the level of these chemical contents of cause the different toxicity levels of several strains of (gadir 2003, rug m & ruppel 2000). high phorbol ester content has been proved to be responsible for the toxicity of . this toxicity effect could not be eliminated using high temperature, since phorbol ester is very stable in high temperature. chemical reactions can reduce phorbol ester content of (aregheore 2003). these toxic compounds can be a problem in processing to biofuel. attempts to detoxificate toxic compounds of have been done in order to reduce the toxic effects (haas 2000). in addition to physical and chemical detoxification, biotechnological approaches could be applied. the toxic compounds may also be eliminated or reduced by regulation of their biosynthetic pathway. several genes which are responsible for synthesis of toxic compounds have been cloned, furthermore the expression and properties of the enzyme have been studied (qin . 2005). identification of genes which are responsible for cursin production give the challenges to regulate their inhibition activities. several tools to study the biosynthesis of toxic compounds of are needed. cell, tissue and organ culture are tools that can be used to regulate the production of secondary metabolites from plants. furthermore, these tools can be used for study at genetic level. to understand the secondary metabolites in relation to its toxicity and side effects, a research concerning phytochemical study of collected from wild types and cultivated regions have been done. in addition, cell, organ and supension cultures have been initiated. the aims of this study were to investigate the phytochemical contents of and to establish the cell culture of which can be used for further study. plant materials were collected from different regions in indonesia such as padang, lampung, garut, ciamis, tasikmalaya, bantul and gunung kidul. plant organs ie. leaves, fruits and stems were separated all materials were dried at 50 c and powdered. to isolate secondary metabolites from , each plant material from different organs and collection regions was extracted using soxhlet apparatus and solvent with increasing polarity such as n-hexane, ethyl acetate and methanol. extracts were checked by thin layer chromatography (tlc). chromatogram profile was used as the first indication to choose the best system for further isolation. n-hexane extract of leaves, stems and fruits were further fractionated using vacuum liquid chromatography using silica gel 60 h as adsorbents, the eluents were the mixtures of n-hexane and dichloromethane. the compositions of the eluents consisted of n-hexane 100 %, n-hexane dichloromethane (9:1) till 1:9, and dichloromethane 100%, yielded 12 fractions (fraction 1-12). the fractions were checked by tlc. several j. curcas j. curcas j. curcas et al. j. curcas j. curcas et al. j. curcas j. curcas et al. et al j. curcas j. curcas j. curcas j. curcas j. curcas materials and method 0 extraction, isolation and identification of secondary metabolites 43 phytochemical study of cell culture elfahmi .jatropha curcas et al fractions contained the dominant compounds. identification of isolated compounds and selected fractions were conducted by gcms. spectrums were analyzed and compared with available library. the explants used in this experiment were leaves and seeds. the sterile leaves or seeds were cut into slices and callus induction was obtained using media with different compositions. these media were modifications of the murashige and skoog (murashige & skoog 1962) or the gamborg's b5 medium (gamborg 1968). medium contained the combination of macro and micro-nutrient, carbon sources and other nutritions as well as growth hormones is needed. all media are supplemented with 4 % (w/w) sucrose and solidified with 0.9% agar. the callus cultures were grown under an l/d regime (16/8 h: 3000 lux) at 26°c. the compositions of growth hormones were as follows : 1) naa 2 mg/l : bap 0.5 mg/l, 2) naa 2.22 mg/l : bap 0.5 mg/l, 3) naa 2.5 mg/l : bap 0.5 mg/l, 4) naa 2.22 mg/l:bap 0.4 mg/l. cell suspension cultures were initiated by transferring callus clumps into 100 ml sterile conical flasks with 50 ml of liquid medium of the same composition as described above but without agar. cultures were incubated on a rotary shaker (100 rpm) at 26°c under an l/d regime (16/8 h: 3,000 lux, daylight l 36w/10, osram, germany). after 1 month, 50 ml of cell suspension cultures were transferred into a 500 ml conical flask, with fresh medium yielding a total volume of 300 ml. subcultures were prepared every 3 weeks by adding 100 ml of a full-grown cell suspension culture to 200 ml of fresh medium. phytochemical screening showed that contains alkaloid, flavonoid, tanin chatecate, saponin and steroid/triterpenoid (table 1). screening results were summarized in table 1. samples from other collection regions gave the same result as shown in the table. based on the screening results, the extraction process was used as previously mentioned in materials and method section. tlc analysis resulted that chromatogram profile of n-hexane, ethyl acetate and methanol extracts from collected from different places showed relatively the same, respectively. from gcms data of fraction and isolated compounds, several spectrums showed containing hydrocarbon, fatty acid and steroid compounds. however, phorbol ester and its derivatives reported having toxic effects, could not be detected. it was probably due to low content of phorbol ester. to confirm whether these compounds did not exist in from selected regions, another gcms measurement and or using different columns and conditions should be done. from these confirmations, no phorbol esters contents were detected. based on gcms, compound 1 has molecular weight of 412 with m/z: 83, 159, 255, and 300. these fragmentation patterns of 284 with m/z 73, 129 and 241 which were similar to octadecanoic acid (fig. 2). compound 3 has molecular weight of 152 with m/z 81 which were similar to 2,4 decadienal (fig. 3). dominant callus, cell, and suspension culture of j.curcas et al. j. curcas j. curcas j. curcas results and discussion were similar to β-stigmasterol (fig. 1). compound 2 has molecular weight biotropia vol. 18 no. 1, 2011 44 crude sample alkaloid flavonoid tancin quinon saponin steroid/ terpenoidchatecate gallat leaves ciamis garut tasik + + + + + + + + + + + + + + + stem ciamis garut tasik + + + + + + + + + + + + + + + notes : + = contain secondary metabolites = did not contain secondary metabolites table. 1. phytochemical screening of collected from ciamis, garut and tasikjatropha curcas figure 1. gcms spectrum and fragmentation pattern of a) isolated compound of jatropha curcas and b). βstigmasterol compounds detected by gcms were hidrocarbon such as 2-heptenal, decadienal, hexsadecane, pentadecane, cyclooctane etc, fatty acid such as octadecanoate acid, ethyl linoleate, ethyl stearat, hexadecanoate acid etc and steroid such as stigmasterol, fucosterol, sitosterol. using leaf as the explant, callus can grow within 22 days, callus growth started with the folding of the green leaves, then callus slowly proliferated. callus was soft, compact and yellow green. the optimum callus formation was found at 82 days after inoculation. at day 90, callus started to be brownies and then died for more than 100 days. then callus were subcultured every month (fig. 4). while using seeds as the explant, callus can grow in a week after inoculation. callus was easily proliferated. formed callus was soft, compact, pale green. the optimum callus formations of seeds were found at 63 days after inoculation (fig. 5). using similar medium without agarose, cell suspension culture were grown, yielded cell suspension culture of seed 45 phytochemical study of cell culture elfahmi .jatropha curcas et al figure 2. gcms spectrum and fragmentation pattern of a) isolated compound of and b). octadecanoic acidjatropha curcas figure 3. gcms spectrum and fragmentation pattern of a) isolated compound of and b). 2,4-decadienal jatropha curcas 46 biotropia vol. 18 no. 1, 2011 a b c d e f g h figure 4. callus culture of leaf in growth phase (a) 22 days, (b) 29 days, (c) 45 days, (d) 52 days, (e) 59 days, (f) 65 days, (g) 82 days and (h) 90 days, incubation in ms medium added with naa 2 mg/l : bap 0.5 2mg/ml. j. curcas a b c d e f g h figure 5. callus culture of seeds in growth phase (a) 7 days, (b) 14 days, (c) 17 days, (d) 21 days, (e) 28 days, (f) 35 days, (g) 42 days, (h) 63 days, incubation in ms medium added with naa 2 mg/l : bap 0.5 2mg/ml. j. curcas a b c d figure 6. cell suspension culture of seeds in growth phase (a) 14 days, (b) 60 days, (c) 70 days, and (d) 80 days, , incubation in ms medium added with naa 2 mg/l : bap 0.5 2 mg/ml without agarose j. curcas 47 phytochemical study of cell culture elfahmi .jatropha curcas et al a b c d figure 7. cell suspension culture of leaf in growth phase (a) 14 days, (b) 25 days, (c) 40 days, and (d) 60 days, , incubation in ms medium added with naa 2 mg/l : bap 0.5 2 mg/ml without agarose j. curcas and leaf. but the amounts of cell suspension culture were not enough for further experiment with elicitor. scale up production of cell suspension culture is in progress (fig. 6 and 7) long time is needed to optimize callus formation (approximately 2 months for the seeds and almost 3 months for the leaves). therefore, the cell suspension cultures were not enough for the elicitor treatment. the secondary metabolites of original plant (seed, leaf) have different profiles compared to the callus and cell suspension. it could be concluded that the callus and cell suspension produced additional compounds. however, this research should be continued to determine the produced compounds of cell suspension and callus culture. some bands of tlc chromatogram of original plants were not found in the callus and cell suspension culture. analysis of n-hexane, ethyl acetate and methanol of leaves, stems and fruits of collected from different location in indonesia (sumatera barat, lampung, jawa barat dan yogyakarta) showed a similar profile (qualitatively). based on the gcms analysis of fractions and isolates of showed that the dominant compounds were hydrocarbon, fatty acid and steroid. gcms data did not show any phorbol ester compound and its derivaties which has a toxic effect. the growth of callus and suspension cultures of leaves and seeds of could be optimized in ms medium added with naa 2 mg/l : bap 0,5 mg/l. secondary metabolites content of callus and suspension culture showed different profiles. it can be concluded that the induction of callus and cell suspension can influence the production of secondary metabolites of plants. the authors would like to express their sincere thanks to the department of national education, directorate general of higher education, project of development of tropical biology, indonesia 2007, for the financial support. thanks are also due to seameo biotrop management and staff for the opportunity given to conduct this research, and to the school of pharmacy itb for providing all instruments needed for this research. conclusions acknowledgements j. curcas j. curcas j. curcas 48 biotropia vol. 18 no. 1, 2011 references aregheore em, becker k, makkar hps, 2003. detoxification of a toxic variety of jatropha curcas using heat and chemical treatments, and pr liminary nutritional evaluation with rats. s. pac. j. nat sci, 21, 50-56 gadir wsa, onsa to, ali wem, el badwi sma, adam sei, 2003. comparative toxicity of croton macrostachys, jatropha curcas and piper abyssinica seeds in nubian goats. small ruminant research 48, 61-67 gamborg ol, miller ra, oijama v. 1968. nutrient requirements of suspension cultures of soybean root cells. experimental cell research, 50, 151-158 haas w, mittelbach m. 2000. detoxification experiments with the seed oil from jatropha curcas l. industrial crops and products, 12, 111-118 murashige t, skoog f. 1962. a revised medium for rapid growth and bio assays with tobacco tissue cultures. physiologia plantarum, 15, 473-497 qin w, ming-xing h, ying x, xin-shen z, fang c. 2005. expression of a ribosome inactivating protein (curcin 2). j. biosci. 351-357. 49 phytochemical study of cell culture elfahmi .jatropha curcas et al biotropia 1 (1) 1987: 58-66 iv. storage and germination tests on shorea javanica seeds m.i.j. umboh tropical forest biology program, biotrop, bogor, indonesia keywords: germination tests, seed treatment, shorea javanica abstract seeds of shorea javanica k & v (dipterocarpaceae), were subjected to different treatments of temperature (27 ± 2°c, 20 ± 2°c and < 10°c), coating (ash and paraffin) and relative humidity (20, 66 and 86%) and their germination capability as well as moisture content assessed after 3, 7, 14 and 30 days of storage. the germination percentage of the seeds stored 30 days in a cloth bag under different conditions of temperature showed highly significant differences (a = 0.01). no significant differences were found on the effect of coating, duration of storage as well as on the interaction between these two factors. the moisture content of the seeds between 13 and 14% gave a germination percentage above 50% after 30 days. introduction like in other dipterocarpaceae, the production of seeds in shorea javanica is irregular (ashton 1960, torquebiau 1984). knowledge of seed storage technology for preserving planting stocks from one fruiting season until the next is therefore a very important prerequisite for making genetic and breeding studies of this timber species. this work is a preliminary study on the effect of storage temperature, coating, relative humidity and moisture content on the germination capacity of shorea javanica. materials and methods seeds were collected in august and september 1985 from the forest plantations near krui, lampung (sumatra). immediately after collection from the trees, seeds with complete structure (i.e. with wings) were put in a cloth bag for transport to bogor. two experiments were conducted : experiment 1 : three days after harvest, 3600 seeds were divided into 3 groups: 2 groups coated with ash or paraffin wax and uncoated control. the seeds were then stored under three different temperature conditions: room temperature (27 ± 2°c), air conditioned room (20 ± 2°c) and inside refrigerator ( < 10°c) for 3, 7, 14 and 30 days. there were 50 seeds per treatment and each treatment was in replicate. 58 iv. storage and germination test on shorea javanica seeds — umboh experiment 2 : 1800 seeds were stored in eight dessicators (200 seeds per dessicator) four of which were kept at room temperature (27 ± 2°c) and the others at air conditioned room temperature (20 ± 2°c). three of the four dessicators of each series were filled with saturated solutions of potassium acetate (kc2h3o2), sodium nitrate (nanco2) and potassium chlorite (kcl) to keep relative humidities (rh) at around 20%, 66%, and 86%, respectively (suchslands 1980). one dessicator of each series was without chemical and had approximately 67% rh at room temperature or less than 60% rh in air conditioned room. in each dessicator, the seeds were stored for 3, 7, 14 and 30 days and fifty seeds were taken at the end of each period. the germination tests were done in two sand-filled seed beds which measured 5 x 1.2 m. these seed beds were covered with plastic sheets to keep moisture at ca. 90% rh. the emergence of the shoot on the surface of sand was indicated by a small stick. the data on germination percentage of the first experiment were subjected to analysis of variance for the three factors (i.e. temperature, coating and duration of storage) with completely randomized block, whereas the data on the second experiment were presented as germination percentage for each experiment. determination of seed moisture content in relation with the germination capacity was also made. for this purpose, five seeds were added for each treatment and they were dissected and weighed before and after they were placed in an oven at 110°c for 20 hours. the formula of moisture content was on a wet weight basis : fresh weight of seed — dry weight of seed % moisture content = x 100 fresh weight of seed results experiment 1. the development of the seedling until the appearance of cotyledons and a small part of the apex needed one to two weeks after sowing. results are shown in table 1. table 1. percent germination of s. javanica seeds at different temperatures and with different duration of storage and coating (means of two replications). 59 biotropia vol. 1 no. 1, july-december 1987 effect of temperature the analysis of variance of the data showed that there were highly significant differences in the germination percentage of seeds according to the different temperature conditions (table 2). the comparison of the germination percentages of seeds stored at different temperatures was performed by tukey's identification and is shown in table 2a. there was no significant difference of germination percentage between seeds placed at 27 _+ 2°c (52.42%) and 20 ± 2°c (54.42%) but percent germination of table 2. analysis of variance on the effect of temperature, coating and duration of storage and the interaction of these factors on the percent germination of s. javanica seeds. source of variation degree of freedom sum square mean square f value blocks 1 41953.3872 41953.3872 temperature 2 35643.1078 17821.5341 39.2678 ** coating 2 172.4444 86.2222 0.1900 ns tc 4 48.2222 12.2556 0.0266 ns duration 3 876.6111 292.2037 0.6438 ns td 6 306.2222 51.0370 0.1125 ns cd 6 104.8889 17.4815 0.0385 ns tcd 12 463.7778 38.6481 0.0385 ns residual 35 15884.6178 453.8462 notes: ** = highly significant at level α = 0.01 ns = not significant at level α = 0.05 tc : interaction between temperature and coating td : interaction between temperature and duration cd : interaction between coating and duration tcd : interaction among temperature, coating and duration table 2a. tukey's identification of means of germination percentage (mean of all data of table 1 for each temperature). temperature 27±2 °c 20±2°c 10°c mean of germination (%) 52.42 54.42 6.25 ns ns: non-significant at level a = 0.05 * significant difference at level a = 0.05 60 iv. storage and germination test on shorea javanica seeds — umboh seeds placed at both temperatures compared with those at < 10°c (6.25%) was significantly different. effect of presence and absence of coating as shown in table 2, the effect of coating the seeds, and of the duration of storage as well as the interaction among factors of temperature, coating and duration of storage gave germination percentages which were not significantly different. the seeds coated with ash germinated better than those coated with paraffin which had a lower germination percentage than untreated seeds (table 2b). however, statistical test showed that the differences were not significant. table 2b. percent germination according to different coatings (means of all data of table 1, for each coating). control ash paraffin germination % means 38.25 39.25 35.58 effect of the duration of storage the means of germination percentages decreased for all storage duration of treatments 3 to 30 days (table 2c) but the differences were not statistically significant. table 2c. percent germination according to the duration of storage (means of all data of table 1, for each duration of storage). duration (days) 3 7 14 30 germination % means 42.78 38.00 35.11 33.89 experiment 2: effect of relative humidity the germination percentage of seeds stored at 27 ± 2°c with 20% rh (r20) and 66% rh (r66) went down to almost zero after 14 and 30 days of storage respectively (fig. 1). however, the seeds stored without chemicals i.e. rh aproximately 67% (rc) had a germination capacity slightly above 50% after 30 days of storage. 61 biotropia vol. 1 no. 1, july-december 1987 62 iv. storage and germination test on shorea javanica seeds umboh 63 biotropia vol. 1 no. 1, july-december 1987 this difference may be due to a negative effect on germination capability of sodium nitrite (nano2) used to maintain the rh at 66%. however, this effect still remains to be investigated. at 20 +. 2°c, the germination percentage of seeds stored at 66% rh (r66), 86% rh (r86) and 60% rh (without chemicals), were still above 80% after 30 days (fig. 2). at 20% rh (r20), the germination percentage was almost zero after 14 days. influence of temperature and relative humidity on the moisture content of the seeds : table 3 shows the values of moisture content of seeds during experiment 2. at 27 ± 2°c and 86% rh, the moisture content of the seeds was stable from 7 to 30 days, and it showed a slow decrease under the other rh conditions. at 20 ± 2°c, the moisture content of the seeds was stable up to 30 days under 60, 66 and 86% rh, while it was very low from day 7 on at 20% rh. table 3. moisture content (%) of seeds, after various durations of storage and under different temperatures and relative humidities. temperature relative humidity (%) 27 ±2°c 20 ±2°c 67 20 66 86 60 20 66 86 days 7 14.83 12.71 15.24 15.50 14.40 11.36 15.14 15.31 14 14.48 10.58 14.34 15.06 14.51 10.27 14.87 14.96 30 13.21 10.58 12.21 14.86 13.04 7.88 13.17 14.44 the value of moisture content of the seeds, when related to the germination percentages under different relative humidities, indicate that the best germination rate was achieved by those seeds with a moisture content between 13 and 14% after 30 days. results of the second experiment indicate that s. javanica seeds are preferably kept at 20 ±. 2°c (air conditioned room) under 60 to 66% rh or at 27 ± 2°c (room temperature) under 67% rh. discussion it is generally agreed that viability of seeds may be defined as the capability of the seeds to germinate under favorable conditions. the conditions which influence the germinating capacity of seeds are: temperature, moisture content, relative humidity, physiology of seed and some genetic factors. 64 iv. storage and germination test on shorea javanica seeds — umboh effect of temperature, coating and duration of storage there are interrelationships among temperature, moisture content and storage time on the life of seeds (justice & bass 1979). the germination capability of s. javanica seeds was influenced by temperature. for example, the percent germination of seeds stored at 27 ± 2°c or at 20 ± 2°c and those stored at < 10°c varied significantly (at level a = 0.01). the germination percentage of seeds at < 10°c was almost zero after 30 days of storage, or 33 days after collection from the trees. however, different coatings and storage up to 30 days had no effect on the percent germination. it is therefore considered better to store s. javanica seeds in a cloth bag at air conditioned room temperature without coating (table 1). such physiological characteristics allow one to consider shorea javanica seeds as recalcitrant seeds (chin & robert 1980). effect of temperature, relative humidity and moisture content the higher the relative humidity, the higher the moisture content of the seeds (nuhamura 1958 cited by justice & bass 1979). it is also known that, as temperature is increased when seeds are held at a constant relative humidity, the seed moisture content decreases. in the present experiment with s. javanica, temperature had a more important effect on seed germination than relative humidity. at 20°c, all seeds under 60, 66 and 86% rh still had very good germination capability (above 80%) after 30 days of storage. when the relative humidity was decreased to 20%, the moisture content decreased and it influenced negatively the germination capacity at both 27 or 20°c. this is similar with the observations of tompsett (1985) on the decrease of germinability of s. robusta seeds when their moisture content was decreased. due to the limitation of seed production at the beginning of the experiment in august and september 1985, it is recommended that further research on the effect of moisture content of the seeds on germination should be conducted. also, it is important to determine the composition of s. javanica seeds because there may be a relation between the lipid contents of a seed and its moisture content. this is based on the observation of barton (1941, cited by justice & bass 1979) i.e. seeds containing high percentage of carbohydrates, proteins, or both like rice, other grains and soybean, can have moisture contents of about 13-15% at 25°c and 75% rh whereas peanuts, which are rich in oil, would have a moisture content of approximately 9-11% under the same conditions. conclusion there were up to 30 days no significant differences in the percent germination of seeds stored at 27 ±. 2°c (52.42%) and at 20 ± 2°c (54.42%) but 65 biotropia vol. 1 no. 1, july-december 1987 the germination of seeds stored at both temperatures compared with those at 10°c (6.25%) was significantly lower. presence or absence of paraffin or ash seed coating had no effect on the percent germination of seeds. the percent germination of seeds decreased for all treatments with an increase of the duration of storage from 3 to 30 days. the seeds stored without chemicals at a relative humidity (rh) of about 67% (rc) still had a germination capacity above 50% after 30 days of storage. at 20 ±_ 2°c (room air-conditioned), the percent germination of seeds stored at 66% rh (r66), 86% rh (r86) and 60% rh (without chemicals), were still above 80% after 30 days. in all experiments, at 20% rh, the percent germination was reduced to zero after 30 days. at room temperature (27 ±. 2°q with 86% rh, the moisture content of seeds was stable from 7 to 30 days (±_ 15%), but it showed a slow decrease under other rh conditions. at room air-conditioned (20 +. 2°c), the moisture content of seeds under 60, 66 and 86% rh were about 14% up to 30 days but it was very low at 20% rh. acknowledgments i wish to thank the forest administrator of lampung utara (krui) especially mr. tajudin nur and his staff for their help in providing materials for this study. thanks are also due to mr. iwan setiawan for his technical assistance, mrs. lilian gadrinab for her kind help with english correction and dr. torquebiau for his comments. references ashton, p.s. 1980. the biological and ecological basis for the utilization of dipterocarps. bio-indonesia, 7: 43-54. chin, h.f. & e.h. roberts. 1980. recalcitrant crop seeds. tropical press son, kuala lumpur, malaysia. justice, o.c. & l.n. bass. 1979. principles and practices of seed storage. william cloves & sons limited, london, 275 pp. mahdi, a. 1982. the viability and storage characteristics of shorea stenoptera burck and citrus sinensis o. seeds. thesis presented for msc degree in birmingham university. suchsland, o. 1980. determination of sorption isotherms in minidessicator. wood science, vol. 12: n°4. tompsett, p.b. 1985. the influence of moisture content and storage temperature on the viability of shorea almon, s. robusta, and s. roxburghii seeds. can. j. for. res. vol. 15. torquebiau, e.f. 1984. man-made dipterocarp forest in sumatra. agroforestry system, 2: 103-127. 66 58.pdf 59.pdf 60.pdf 61.pdf 62.pdf 63.pdf 64.pdf 65.pdf 66.pdf biotropia no. 7, 1994: 18-29 . the possibility of controlling sclerotium rolfsii on soybean (glycine max) using trichoderma and tebuconazole*) okky s. dharmaputra department of biology, faculty of mathematics and natural sciences, bogor agricultural university; and seameo biotrop, p.o. box 116, bogor, indonesia and ina retnowati seameo biotrop, p.o. box 116, bogor, indonesia abstract the possibility of controlling s. rolfsii on soybean (glycine max) var. rinjani using t. aureoviride and tebuconazole under field conditions was studied. the experiment was conducted at the experimental plot of seameo biotrop. the pathogen was mixed with the soil (2 kg/plot) 4 days before the inoculation of the antagonist (2.25 kg/plot). the measurement of each plot was 2.5 x 6 m2. n, p and k (120 kg/ha) were applied at the same day with the inoculation of the pathogen. soybean seeds were planted 7 days after the inoculation of the antagonist. the distance between plants and between plots were 20 and 40 cm, respectively. the fungicide at concentration of 100 g/ha (in vitro concentration) and 210 g/ha (field or recommended concentration) were applied using 2 methods, i.e. 1) spraying on the planting hole at the same day as the planting of soybean seeds, and 2) spraying on the soil surrounding the plants 7 days after planting. soils that were neither inoculated with the antagonist nor the fungicide were used as controls. three replications (3 plots) were used for each treatment (including the control). the results showed that the inoculation of the antagonist, the concentrations of the fungicide, and time of application gave very significant differences in the percentages of the plants infected by the pathogen and significant differences in seed production; while the interaction between the inoculation of the antagonist and the concentrations of the fungicide, between the concentrations of the fungicide and the time of application, and between the inoculation of the antagonist, the concentrations of the fungicide and the time of application did not give significant differences either in the percentages of the plants infected by the pathogen or seed production. the percentage of plants infected by the pathogen was lower on soil inoculated with the antagonist (31.6%) than on soil not inoculated with the antagonist (52.9%). the percentage of plants infected by the pathogen was lower on soil treated with the fungicide either at in vitro concentration (37.5%) or at field concentration (37.4%) than on the soil not treated *) a part of research funded by the government of indonesia fy 1991/1992 18 18 possibility of controlling sclerotium rolfsii on soybean okky s. dharmaputra and ina retnowati with the fungicide (61.5%). nevertheless, based on statistical analysis, the fungicide at in vitro concentration was not significantly different from that at field concentration. the percentage of plants infected by the pathogen on the soil sprayed with the fungicide at the same day of seed planting was lower (30.5%) than sprayed 7 days after planting (44.4%). the seed production on the soil inoculated with the antagonist (1893.3 kg/ha) was higher than on the soil not inoculated with the antagonist (1465.7 kg/ha). the production on the soil sprayed with the fungicide either at in vitro (1758.0 kg/ha) or at field concentration (1817.1 kg/ha) was higher than on the soil not sprayed with the fungicide (1247.2 kg/ha). the production on the soil sprayed with the fungicide at the same day of seed planting (2010.9 kg/ha) was higher than sprayed 7 days after planting (1564.2 kg/ha). the combination between the inoculation of the antagonist and the fungicide application at in vitro concentration at the same day of seed planting gave higher seed production (2391.2 kg/ha) than the inoculation of the antagonist (1711.7 kg/ha) or the fungicide application either at in vitro concentration (1771.9 kg/ha) or at field concentration (1939.1 kg/ha) at the same day of seed planting. however, based on statistical analysis, the interaction among the three treatments (the antagonist, the concentrations of the fungicide, and the time of application) was not significantly different. keywords: sclerotium rolfsii, glycine max, trichoderma, tebuconazole, antagonist introduction sclerotium rolfsii is a soil-borne fungal pathogen that can cause root rot and damping-off of crops, among others, soybean (glycine max) (agrios 1988). one of the control methods of the pathogen is by using antagonistic fungi (cook and baker 1983). some soil fungi especially trichoderma have been reported to be potential biocontrol agents of soil-borne fungal pathogens. according to upadhyay and mukhopadhyay (1986) t. harzianum was able to control the disease of sugarbeet seedlings as high as 88% under greenhouse conditions. under field conditions, integration of pcnb (pentachloronitro-benzene) and t. harzianum significantly reduced the incidence of sclerotium root rot (76% disease control) and increased the root, green foliage and sucrose yield per ha. cole and zvenyika (1988) reported that t. harzianum integrated with triadimenol fungicide enhanced disease control in tobacco caused by other soil-borne fungal pathogens, i.e. rhizoctonia solani and fusarium solani. under greenhouse conditions, four strains of t. harzianum suppressed damping-off of snapbean caused by s. rolfsii (papavizas and lewis 1989). tebuconazole (folicur 250 ec) is a fungicide that can control the pathogen (bayer 1990). the objective of the study is to determine the effect of trichoderma combined with tebuconazole to control s. rolfsii on soybean. 19 biotropia no. 7, 1994 materials and methods this research was conducted at the experimental plot of biotrop, bogor, indonesia. the soil type is latosol (ph ± 5). soybean variety, isolates of s. rolfsii and trichodernna, and fungicide s. rolfsii isolate bio-1 and soybean variety rinjani which was the most susceptible variety to the pathogen, were used in this study (dharmaputra and retnowati 1992). t. aureoviride bio-5 was used as antagonist because it caused the highest percentage of inhibition to the pathogen on potato dextrose agar (pda) of ph 5 (dharmaputra and retnowati 1992). tebuconazole was used as a fungicide to control the pathogen (bayer 1990). preparation of pathogen and antagonist inocula inoculum of the pathogen was prepared based on riker and riker (1936), while the inoculum of the antagonist was based on dharmaputra and suwandi (1989). for the preparation of the inoculum of the pathogen, a mixture of sand : corn : water ( 2 : 2 : 3 ) was put in plastic bags (2.5 kg/bag), sterilized in an autoclave for 1 h, and then incubated at room temperature for one night. for the preparation of the inoculum of the antagonist, a mixture of sand : husk : water (2 : 4 : 5 ) was treated in the same manner as the preparation of the pathogen's inoculum. five pieces of the pure cultures (5 mm in diameter each) of the pathogen and the antagonist (3 days old on pda) were grown on each medium. they were then incubated at 28 °c for 7 days. inoculation of the pathogen and the antagonist, application of the fungicide, and planting of soybean the pathogen was inoculated 4 days before the inoculation of the antagonist. it was mixed homogeneously on the surface of the soil in each plot (2.5 x 6 m 2 ). the inocula of the pathogen and the antagonist were 2 and 2.25 kg/plot, respectively. n, p and k (120 kg/ha) were given at the same day as the inoculation of the pathogen. soybean seeds were planted 7 days after the inoculation of the antagonist (aia). the distance between plants and between plots were 20 and 40 cm, respectively. 20 possibility of controlling sclerotium rolfsii on soybean okky s. dharmaputra and ina retnowati tebuconazole at concentrations of 100 g/ha (in vitro concentration) and 210 g/ha (field concentration) were applied using 2 methods: a) spraying in the planting hole at the same day as planting of soybean seeds b) spraying on the soil surrounding the plants (7 days after planting). soils that were neither inoculated with the antagonist nor the fungicide were used as controls. three replications (3 plots) were used for each treatment (including the control). observation of the percentage of infected plants was carried out 14 days after planting (dap), and of the soybean production at 90 dap. factorial in randomized completely block design (rcbd) was used in this study consisting of 3 factors: a) the antagonist (not inoculated and inoculated with t. aureoviride bio-5), b) the concentration of tebuconazole (0, 100 and 210 g/ha), and c) the time of tebuconazole application. results and discussion percentage of plants infected by the pathogen analysis of variance showed that the inoculation of the antagonist and the concentrations of the fungicide gave very significant differences in the percentage of plants infected by the pathogen; the time of fungicide application gave significant difference. the interaction between the inoculation of the antagonist and the concentrations of the fungicide; the concentrations of the fungicide and the time of fungicide application; among the inoculations of the antagonist, the concentrations of the fungicide and the time of fungicide application did not give any significant difference (table 1). infected and non-infected plants at 14 dap are presented in figure 1. the percentage of plants infected by the pathogen was lower (34.1%) and significantly different on the soils inoculated with the antagonist than on the soils not inoculated with the antagonist (56.8%) (table 2). the percentage of plants infected by the pathogen was lower on the soil sprayed with the fungicide either in vitro (37.5%) or field (37.4%) concentration compared to the soil not sprayed with the fungicide (61.5%). nevertheless, the fungicide application at in vitro concentration did not differ significantly from field concentration. the percentage of plants infected by the pathogen was lower and significantly different when the fungicide was applied at the same day as the planting of soybean 21 biotropia no. 7, 1994 figure 1. non-infected (a) and infected (b) plants by sclerotium rolfsii 14 dap 22 possibility of controlling sclerotium rolfsii on soybean okky s. dharmaputra and ina retnowati table 1. analysis of variance on the effect of trichoderma aureoviride bio-5, tebuconazole and the time of fungicide application on the percentage of plants infected by sclerotium rolfsii table 2. the effect of the inoculation of trichoderma aureoviride bio-5, the concentrations of tebuconazole and the time of fungicide application on the percentage of plants infected by sclerotium rolfsii 23 biotropia no. 7, 1994 seeds (40.8%) than when the fungicide was applied 7 dap (50.1%) (table 2). it was assumed that the plants were already infected by the pathogen before spraying. it is interesting to note that the percentage of infected plants on soil sprayed with the fungicide at in vitro concentration combined with the inoculation of the antagonist (sft1a1 = 20%) was lower than on soil sprayed with the fungicide at field concentration (sf2t0a = 35.3%) (table 3), but based on statistical analysis the interaction among the inoculation of the antagonist, the concentrations of the fungicide and the time of fungicide application did not give any significant difference (table 1). figure 2 shows the plants on soil inoculated with the pathogen 14 dap; while figure 3 shows the plants inoculated with the pathogen and sprayed with the fungicide at field concentration (210 g/ha) at the same day of seed planting. the plants on soil inoculated with the pathogen, sprayed with the fungicide at concentration of 100 g/ha (in vitro concentration) at the same day of seed planting, and inoculated with the antagonist is presented in figure 4. according to upadhyay and mukhodhyay (1986), a combination of pcnb with t. harzianum was able to control basal stem rot in sugar beet caused by s. rolfsii up to 76%. seed production analysis of variance showed that the inoculation of the antagonist and the concentrations of the fungicide gave very significant differences in seed production; the time of fungicide application gave significant difference. the interaction between the inoculation of the antagonist and the concentrations of the fungicide; the concentrations of the fungicide and the time of fungicide application, and among the inoculation of the antagonist, the concentrations of the fungicide and the time of fungicide application did not give any significant differences (table 4). the seed production of the soil inoculated with the antagonist was higher and significantly different (1863.3 kg/ha) than that of the soil not inoculated with the antagonist (1351.8 kg/ha) (table 5). according to elad et al. (1980) t. harzianum was able to control the disease on bean, cotton or tomato caused by s. rolfsii and rhizoctonia solani. the antagonist was also able to increase the production of bean. the soybean production of the soil sprayed with the fungicide either at in vitro concentration (1758.0 kg/ha) or at field concentration (1817.1 kg/ha) was significantly different from that not sprayed with the fungicide (1247.2 kg/ha) (table 5). nevertheless, the soybean production of the soil sprayed with fungicide at field concentration was higher than that of the soil sprayed with the fungicide at in vitro concentration. 24 possibility of controlling sclerotium rolfsii on soybean okky s. dharmaputra and ina retnowati table 3. percentage of soybean plants infected by sclerotium rolfsii with different treatments table 4. analysis of variance on the effect of trichoderma aureoviride bio-5, tebuconazole and the time of fungicide application on the production of soybean seeds 25 biotropia no. 7, 1994 figure 2. soybean plants on soil inoculated with sclerotium rolfsii, 14 dap figure 3. soybean plants (14 days after planting) on soil inoculated with sclerotium rolfsii and sprayed with tebuconazole at concentration of 210 g/ha at the same day of seed planting 26 figure 4. soybean plants (14 dap) on soil inoculated with sclerotium rolfsii and trichoderma aure-oviride bio-5, and sprayed with tebuconazole at concentration of 100 g/ha at the same day of seed planting the soybean production of the soil sprayed with the fungicide at the same day with the planting of seeds was higher and significantly different (1756.3 kg/ha) than that of the soil sprayed with the fungicide 7 dap (1458.5 kg/ha) (table 5). the combination of inoculation of the antagonist, and fungicide application at in vitro concentration at the same day as the planting of soybean seeds (s1f1t1a = 2391.2 kg/ha) gave higher soybean production than inoculation of the antagonist (s1f0t1 = 1711.7 kg/ha) or the fungicide application either at in vitro concentration (s1f1t0a = 1771.9 kg/ha) or at field concentration (s1f2t0a = 1939.1 kg/ha) (table 6), but based on statistical analysis the interaction among the inoculation of the antagonist, the concentrations of the fungicide and the time of fungicide application did not give any significant difference (table 4). 27 possibility of controlling sclerotium rolfsii on soybean okky s. dharmaputra and ina retnowati biotropia no. 7, 1994 table 5. the effect of the inoculation of trichoderma aureoviride bio-5, the concentrations of tebuconazole and the time of fungicide application on the production of soybean seeds table 6. the production of soybean seeds with different treatments 28 possibility of controlling sclerotium rolfsii on soybean okky s. dharmaputra and ina retnowati conclusions t. aureoviride bio-5 or tebuconazole could be used to control s. rolfsii. they also could increase seed production. the fungicide was more effective when applied at the same day of seed planting than at 7 dap. the use of the fungicide at in vitro concentration combined with the use of the antagonist was more effective in decreasing the percentage of infected plants and increasing the seed production than the use of the fungicide at field concentration at the same day of seed planting. acknowledgement the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the technicians of the laboratory of plant pathology, seameo biotrop. references agrios, g.n. 1988. plant pathology. academic press, new york. bayer. 1990. folicur; systemisches fungizid technische information. cole, j.s. and z. zvenyika. 1988. integrated control of rhizoctonia solani and fusarium solani in tobacco transplants with trichoderma harzianum and triadimenol. plant pathology 37: 271-277. cook, r. j. and k.f. baker. 1983. the nature and practice of biological control of plant pathogens. the american phytopathological society, st. paul, minnesota. dharmaputra, o.s. and w.p. suwandi. 1989. substrat untuk produksi besar-besaran trichoderma. (substrate for mass production of trichoderma). annual report. research collaboration between marihat research centre and biotrop. biotrop/tagr/89/736: 44-52. dharmaputra, o.s. and i. retnowati. 1992. the possibility of controlling sclerotium rolfsii on soybean (glycine max) using trichoderma, gliocladium and tebuconazole. seameo biotrop. internal report. elad, y., i. chet and j. katan. 1980. trichoderma harzianum: a biocontrol agent effective against sclerotium rolfsii and rhizoctonia solani. phytopathology 70: 119-121. papavizas, g.c. and j.a. lewis. 1989. effect of gliocladium and trichoderma on damping-off and blight of snapbean caused by sclerotium rolfsii in the greenhouse. plant pathology 38: 277-286. riker, a.j. and r.s. riker. 1936. introduction to research and plant diseases. john swift and co., st. louis. mo. sinaga, m.s. 1986. biological control of some soil-borne fungal pathogens of soybean (glycine max (l.) merr.) with gliocladium spp. ph.d. thesis. university of the philippines at los banos. sivan, a., y. elad and i. chet. 1984. biological control effects of a new isolate of trichoderma harzianum on pythium aphanidermatum. phytopathology 74: 498-501. upadhyay, j.p. and a.n. mukhopadhyay. 1986. biological control of sclerotium rolfsii by trichoderma harzianum in sugar beet. trop. pest management 32: 215-220. 29 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf biotropia no. 3, 1989/1990: 25-40 notes on some fusarium and cylindrocarpon on dipterocarpaceae of indonesia* claire elouard laboratory of botany and forestry university of p. sabatier, 39 allees j. guesde 31000 toulouse, france abstract six species of pathogenic fungi belonging to the genera fusarium (fusarium sacchari var. sacchari, f. moniliforme, f. oxysporum, f. spl, f. sp2) and cylindrocarpon (cylindrocarpon destructans) on seeds, seedlings and saplings of dipterocarpaceae in sumatra and west java were identified. introduction the trees of the dipterocarpaceae family fill a prominent place in the southeast asian tropical forests as most of the species are used as timber trees or source of secondary products (e.g. resin "damar"). their economic value is therefore of high interest to the local population and other countries (symington 1974, ashton 1982). up to the present, the exploitation of these forests consists of extracting timber from natural stands or at a lesser extent, in traditional management of agroforestry systems (torquebiau 1984, michon 1985). the possible fungal infections of these trees have been taken into account as debilitating factor of production. during researches devoted to the dipterccarp pathology, fungal attacks on seeds, seedlings and saplings have been observed frequently. they seem to be able to induce important damages in young stands and nurseries. fungi and their damages were observed in several places in west java and sumatra (figure 1). most of them belong to the imperfect genera fusarium and cylindrocarpon (tuberculariaceae). * this study was conducted as part of a doctoral dissertation research under the supervision of prof. g. durrieu of laboratory of botany and forestry, university of p. sabatier, through the tropical forest biology programme of seameo-biotrop. 25 biotropia no. 3, 1989/1990 figure 1. location of study sites materials and methods the pathogens were isolated from diseased organs (roots, collar, stem, twigs, leaves) and cultivated on potato-dextrose-agar (pda) in petri dishes, at room temperature (26 32°c). inoculation experiments were performed on seedlings to confirm the virulence of the isolated strains. the species identification were based on recent literature (booth 1971, barnett & hunter 1972, renard et al. 1972, nirem-berg 1976) and was checked by dr. m.f. roquebert of museum d' histoire naturelle laboratoire de cryptogamie, paris. results and discussion among the pathogens isolated on dipterocarps were different species of the genera fusarium (i.e. f. sacchari var. sacchari, f. moniliforme and f. oxysporum) and cylindrocarpon (i.e. c. destructans). among the associated fungi which can 26 notes on some fusarium and cylindrocarpon — c. elouard be considered as secondary parasites were pestalotia, cylindrocladium, nigrospora and marasmius. fusarium sacchari (bull.) w, gams. var. sacchari 1. hosts vatica pauciflora (korth.) bl. this species was used to prevent fermentation of palm wine. the parasite was isolated from a sapling of about 70 cm height. shorea seminis (de vriese) sloot. the fruits are used in borneo as source of illipe butter (ashton 1982). the hosts were about 2 years old and measured 20 to 30 cm in height. shorea leprosula miq. this dipterocarp tree is one of the main sources of timber classified as light red meranti (symington 1974). hosts were saplings of about one year old and 10-25 cm'in height. both of these hosts came from the experimental plantation of cikarawang of bogor agricultural university, darmaga, bogor, west java (figure la). 2. symptoms of the disease observed in the field (figures 2 & 3) defoliation and weathering of saplings of vatica pauciflora. on shorea seminis, beige-coloured spots delimited by a brown line, at first at the apex of leaves and later on covering the whole leaf surface, leading to its fall; at the base of the twig, necrosis could be observed; the bark of both collars and roots were necrosed and became dark; some of the plants were dry from root to apex. some shorea leprosula were dry and dead, with darkish bark, cambium of collars and roots. necrosis could be observed at the base of twigs. alive plants have cracked collar's bark and darkish roots; leaves have brown spots, beginning from the extremity and later on covering the whole surface. 3. description of the pathogen (figure 4) this fusarium was isolated from the cambium of the branch of v. pauciflora and from leaves, twigs, collars and roots of s. seminis. the pigmentation of the medium was brown to vinaceous. the mycelium was cream-coloured, pink to violet, aerial, cottony to fibrous. the growth rate was 6.8 cm after 4 days (figure 5). microconidia were initially formed by simple phialides, and further more by branched conidiophores ended by polyphialides. the older the culture, the more 27 biotropia no. 3, 1989/1990 figure 2. spots on leaves of shorea seminis caused by fusarium sacchari var. sacchari figure 3. vatica pauciflora inoculated with fusarium sacchari var. sacchari 28 notes on some fusarium and cylindrocarpon c. elouard figure 4. fusarium sacchari var. sacchari figure 5. growth rate of the fungi 29 biotropia no. 3, 1989/1990 complex is the organization of conidiophores. microconidia were oval to clavate, one-celled, and measured 3 10 x 1-3 um. more often, there was no macroconidia in culture, or only few in number. macroconidia were formed by simple phialides, generally straight, 3 to 6 septate, and measured 6-55 x 1.7-2.4 um. no mycelial and conidial chlamydospores were observed. 4. associated fungi two marasmius species were associated with f. sacchari var. sacchari: m. equicrinis: it developed rhizomorphs like long black hairs, on which there were small carphophores (2-8 mm of diameter) on twigs, branches and leaves of hosts. marasmius sp. developed white himanthium on twigs, branches and leaves. fusarium monitiforme scheld. sensu wollenw. et reinking = f. verticilloides (sacc.) niremberg comb. nov. 1. hosts shorea javanica k. & v. this shorea species has been exploited in agroforestry systems for about 150 years by local population in the province of lampung, south sumatra (figure ic). trees produce a resin ("damar") which is exported to europe and america for the production of varnish, linoleum and paint. the trees were tapped and the resin was collected every month (torquebiau 1984). the parasite was collected on saplings of s. javanica of about 1 m to 1.5m height and one to two years old. 2. symptoms of the disease observed in the field defoliation and weathering of the plant; the bark of twigs and roots were black and the cambium was dry. 3. description of pathogens (figure 6) this fusarium was isolated from twigs and roots. the pigmentation of the medium was brown to violet. the mycelium was cream-coloured, pink to violet, violet-grey; at first, appearing like a small cushion and later on becoming aerial, cottony to fibrous. the growth rate after 4 days was 2.2 cm (figure 5). microconidiophores were simple, lateral to the hyphae and measured 27 — 54 x 1 5 um. microconidia were in chains and measured 5 17 x 2-4 cm; they were 30 a. microconidiophores and microconidia b. macroconidia figure 6. fusarium moniliforme one-celled, clavate to oval, sometimes with the basal cell lightly flattened. macroconidiophores were lateral branches of hyphae. macroconidia were curved, with a flat basal cell and sometimes a sharp apical cell. they were 3-8 septate when mature and measured 40-72 x 4-5 um and seldom produced in culture. no mycelial and conidial chlamydospores were observed. 4. associated fungi two parasites were found associated with fusarium moniliforme: cylindro cladium sp. from collar and pestalotia sp. from leaves with symptoms of beige spots. these two fungi were secondary parasites. fusarium oxysporum schlecht. 1. hosts shorea pinanga scheff. the fruits of s. pinanga were used as illipe-nuts. the pathogen was collected 31 notes on some fusarium and cylindrocarpon-c. elouard biotropia no. 3, 1989/1990 on saplings of about one to three years old, in population under an adult tree of s. pinanga at the arboretum of haurbentes, jasinga, west java (figure 1b). shorea javanica k. & v. this species could also be a host for f. oxysporum. it was isolated from saplings of 60 cm height at krui (figure 1c). 2. symptoms observed in the field roots, barks of collars and twigs of s. pinanga became darkish; some of the saplings were dry and dead, while others had root rot. no symptoms were observed on s. javanica. 3. description of the parasite (figure 7) this fusarium was isolated from twigs, collar and roots of saplings of s. figure 7. fusarium oxysporum 32 notes on some fusarium and cylindrocarpon — c. elouard pinanga and from twig of 5. javanica. the pigmentation of the medium was brown to violet. the mycelium was cream-coloured, violet, like a small cushion at the beginning, aerial and cottony when older. the growth rate after 4 days was 5.2 cm (figure 3). microconidiophores were simple phialides, lateral to hyphae, measuring 7-9 x 1-2 um, or branched conidiophores, until 30 um long. microconidia were variable in form, oval to ellipsoid and sometimes allantoid; they were one-celled and measured 4.7-14.4 x 1.5-4.7 um. macroconidiophores were lateral to hyphae, measuring 10—12x1 — 1.5 um. macroconidia were 3 to 5 septate, most frequently 3 septate. they were straight to lightly curved generally sharped at both ends. some macroconidia had a pedi-form basal cell and a hooked and curved apical cell. they measured 16 44( 80) x 24.5 um. chlamydospores were formed by mycelium or conidia. the mycelial chlamy-dospores were simple, of 7.7 8.9 x 6.5 8.3 um, double, of 10.6 x 6.5 um, both smooth and rough walled. fusarium sp.l 1. hosts shorea pinanga scheff. this fusarium sp. was collected on saplings of about 2 years old and 40 cm to 1 m in height in the nursery of biotrop, bogor; seeds were previously collected in the arboretum of haurbentes (figures la and b). 2. symptoms observed in the field red-brown spots developed on leaves with a grey-coloured powder on the upper surface; spots appeared at the apex of leaves and were delimited by a red line; more often, young leaves were necrosed first and then spots came into sight. development of spots on leaves led to necrosis and death of cells, and later on leaves fell. on twigs, cankers appeared and bark became darkish. on collars and roots, the pathogen caused dry rot, and collar's bark became darkish. the propagation was not aerial but by the vascular system. healthy leaves were wrapped with pieces of cellophane to avoid possible aerial contamination. spots appeared on these wrapped leaves as well as on the unwrapped ones. the proga-gation then seems to be internal, by vessels. 33 biotropia no. 3, 1989/1990 3. description of the pathogen (figure 8) this fusarium was isolated from leaves, twigs, collars and roots of s. pinanga. no pigmentation of the medium was observed. the mycelium was cream-coloured to violet, like a small cushion. the growth rate was 5 cm after 4 days (figure 5). microconidia were initially formed by long conidiophores of 45 62 x 1.2-1.8 um. microconidia were oval, cylindrical, allantoid, one-celled, and measured 1.8-17 x 1.2-3.7 um. macroconidia were formed by simple phialides or simple conidiophores, 7 -59 x 1.2-1.5 um. straight to curved, fusiform, they were 2 to 6 septate, and measured 14-41 x 1.8-5.4 um. chlamydospores were simple, 8.9x6.5 um, double, 12.4 x 7.1 um intercalary in mycelium or at the apex of hyphae. figure 8. fusarium sp.1 34 note on some fusarium and cylindrocarpon — c. elouard 4. associated fungi a secondary parasite, cylindrocladium sp., was cultured from collar with this fusarium sp. fusarium sp.2 1. host shorea javanica k. & v. this other fusarium sp. was collected on saplings of 25 cm height, at krui (figure 1c). 2. symptoms observed in the field defoliation and weathering of the plant was noted with bark darkish in appearance. 3. description of the pathogen (figure 9) it was isolated from roots and twigs. the pigmentation of the medium was pink, violet to vinaceous. the mycelium was violet, vinaceous, cottony and aerial. the growth rate after 4 days was 4.2 cm (figure 5). microconidiophores were simple phialides or short conidiophores, 21.8-22.4 x 1.6 — 3.2 um. microconidia were oval, allantoid to oblong. one-celled, they measured 2.6-16.7 x 0.9-3.7 um. macroconidia were straight to lightly curved, 2 to 7 septate, measured 10-57.6 x 2-3.5 um, and were produced by short conidiophores. chlamydospores were intercalary or terminal to hyphae, simple, of 8.3 x 7.7-9.4 um, double, of 10-14.4 x 6.9 um. 4. associated fungi a secondary parasite, cylindrocladium sp., was cultured from roots. cylindrocarpon destructans (zinssm.) scholten, anamorph of nectrica radicicola 1. hosts shorea pinanga scheff. and hopea mengerawan miq. h. mengerawan was a commercial timber tree of second class value. seeds of 5. pinanga were collected in the botanical garden (kebun raya) of bogor, west java. 35 a. microconidiophore and microconidia b. macroconidia c. chlamydospores (1) smooth wall, (2) rough wall figure 9. fusarium sp.2 saplings of h. mengerawan were 2 years old and were collected at the experimental plantation of benakat, near palembang in south sumatra (figure id). 2. symptoms observed in the field (figures 10 & 11) necrosis and weathering of the seeds of 5. pinanga were observed. the saplings of h. mengerawan had brown spots on leaves delimited by dark lines. the spots first appeared at the apex of the leaf but they covered little by little the entire surface causing the leaf to fall. thus, photosynthesis activity decreased or stopped leading to the weathering of the host. 3. description of the parasite (figure 12) c. destructans was isolated from cotyledons of s. pinanga and was responsible for damping-off. it was also cultured from roots and twigs of sapling of h. mengerawan. the pigmentation of the medium was brown to vinaceous. the mycelium was cream-coloured, violet to violet-grey, layer on medium in small cushion or powdery. the growth rate after 4 days was more than 4 cm (figure 3). 36 biotropia no. 3, 1989/1990 notes on some fusarium and cylindrocarpon -c. elouard figure 10. spots on leaves of hopea mengerawan caused by cylindrocarpon destructans. figure 11. hopea mengerawan inoculated with cylindrocarpon destructans. 37 biotropia no. 3, 1989/1990 figure 12. cylindrocarpon destructans microconidiophores were simple phialides, lateral to hyphae, 8 17 x 1.5 um, or branched conidiophores, 29 83 x 1.7 2.7 um. microconidia were oval, allantoid to oblong, with sometimes a sharp end. one-celled, they measured 5.4-21.2 x 1.8-4.8 um. macroconidiophores were phialides in groups, 13-17x2.4-3 um, or simple conidiophores lateral to hyphae, 27 x 2 um. macroconidia were thick-set straight to lightly curved, with sometimes curved ends. most of them were 3 septate and measured 18.5-33.3 x 3.5-5.2 um. chlamydospores were both conidial and mycelial; abundant, intercalary or terminal to hyphae; simple, 7-11 x 5-9 um, double 12-16 x 6.9 um, triple 21 x 7.7 um, smooth and rough walled. 4. associated fungi cylindrocladium sp. was associated with c. destructans on seeds of s. pinanga and on saplings of h. mengerawan. it was isolated from roots of h. mengerawan and can be considered as a secondary parasite. on the other hand, cylindrocladium sp. was responsible for damping-off of s. pinanga. thus, cylindrocladium sp. seems to be easily associated with c. destructans. 38 notes on some fusarium and cylindrocarpon-c. elouard conclusion these few observations showed that fusarium and cylindrocarpon were not infrequent as disease agents of young dipterocarps and their attacks very often end in death of the infected plants. if the prevalence of the fungi in natural stands is probably of less importance because of the dispersion of saplings, it may not be the same in nurseries and planted stands. the fungal attacks may become harmful on account of the heavy density of susceptible seedlings and the disease could develop at an epidemic proportion. thus, it is necessary to have better knowledge of the biology and the ecology of these pathogens, to help prevent their damages in artificial forest stands. acknowledgments this workisapartof aresearch programme supported by agrant from the french "ministere des affaires etrangeres". i want to express all my thanks to the director, prof. dr. h. sitti soetarmi tjitrosomo, the deputy director, dr. r. umaly and to the staff of biotrop, particularly to dr. okky s. dharmaputra, for the help they provided me, and to dr. m.f. roquebert, laboratoire de cryptogamie, museum d'histoire naturelle, paris, for her invaluable assistance in the identification of fusarium and cylindrocarpon specimens. references ashton, p.s. 1982. dipterocarpaceae. flora malesiana, series i, spermatophyta, flowering plants, vol. 9, part 2. p. 552. barnett, h.l. and b.b. hunter 1972. illustrated genera of imperfect fungi. burgers publishing company, third edition, 241 p. booth, c. 1971. the genus fusarium. commonwealth mycological institute, ferry lane, kew, surrey, england. 237 p. michon, g. 1985. de i'homme de la foret au paysan de 1'arbre; agroforesteries indonsiennes. these de doctorat, universit des sciences et techniques du languedoc, montpellier. 273 p. nirenberg, h. 1976. untersuchungen liber die morphologishe und biologishe differenzierung in der fusarium section liseola. mitteilungen aus der biologishen bunderanstalt fur land und forstwirt-schaft. berlin. dahlem. 117 p. 39 biotropia no. 3, 1989/1990 renard, j.l., j.p. gascon et a. bachy. dec. 1972. recherches sur la fusariose du palmier a huile-research on vascular wilt disease of the oil palm. oleagineux, 27° annee, no. 12, pp. 581 591. symington, c.f. 1974. foresters' manual of dipterocarps. malayan forest record no. 16. university malaya, kuala lumpur, malaysia. 114 text-fig., 114 planches, 244 p. torquebiau, e. 1984. man-made dipterocarp forest in sumatra agroforestry systems 2(2), pp. 103-128. 40 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf biotropia book juni revisi 14 juli 09.indd 38 rapid detection of the africanized honey bee: a tool for indonesian animal quarantine rika raffiudin(1*, anifa bintar(1, m. chandra widjaja(2, ahmad farajallah(1, bambang purwantara(3 1department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia 2perhutani-west java and banten province, bandung, indonesia 3department of reproduction and obstetrics, faculty of veterinary medicine, bogor agricultural university, bogor, indonesia abstract molecular detection methods were used to determine if africanized honey bees (ahbs) are present in populations of imported apis mellifera in indonesia. the cytochrome b (cyt b gene) was amplified from mitochondrial dna and digested with the bglii restriction enzyme (cytb/bglii). two types of animal dna extraction kits were used and found suitable for rapid preparation of dna from a. mellifera by the animal quarantine facility. results showed that all 94 colony samples from beekeepers in java produced a 485 bp pcr product from the amplification of this gene. two dna fragments of 194 and 291 bp from all samples were produced after digestion with bglii. this cytb/bglii result together with the dna sequence of cyt b showed that all collected samples of a. mellifera were the non-ahb type. hence, this study did not detect ahb in indonesia. key words : apis mellifera, molecular detection methods, dna, cytochrome b, mitochondrial genetics introduction apis mellifera currently found in australia, america, and asia (included indonesia) are imported from europe, africa and the middle east (ruttner 1988). the most favorable for beekeepers is the a. mellifera ligustica subspecies due to its tame behaviour and high honey production. besides a. m. ligustica, an aggressive african honey bee subspecies, namely apis m. scutellata was imported to brazil in the mid-1950s. since the introduction of this african subspecies into brazil, descendent of 'africanized' honey bees (ahb) have spread throughout the neotropics and into temperate north america (schiff and biotropia vol. 16 no. 1, 2009: 38 44 *corresponding author: rika_r@cbn.net.id 39 sheppard 1993). it inbred with the european honey bee (ehb = a. m. ligustica or a. m. mellifera) became a killer ahb characterized as having a deadly sting (castro et al. 1994), a high wing beat frequency (spangler 1994), sensitivity to varrhoa parasitic mites (guerra et al. 2000), and rapid colony multiplication. schiff and sheppard (1993) have surveyed 422 feral honey bee colonies from non-africanized areas in the southern united states and it revealed that over 21% of them had mitochondrial dna (mtdna) derived from a european race established in north america in the 17th century, 77% of them had mtdna common in honey bees maintained by beekeepers and about 1% exhibited african mtdna. further analysis revealed that the african mtdna was derived from a north african subspecies imported to the us in the 19th century. the ahb can be distinguished from the non-ahb bees based on the mitochondrial cytochrome b (cyt b) gene digested with bglii (cyt b/bglii). the ahb cyt b does not have a restriction site for bglii, while the non-ahb has one bglii restriction site. therefore, the ahb type produces a single band of 495 bp, whereas the non-ahb pcr product produces a double band of 194 and 291 base pairs (bp) (crozier et al. 1991). this method has been used by australian bee keepers to detect the ahb imported to australia. in the united states, cyt b has also been reported to detect the ahb (pinto et al. 2003). however, currently the indonesian animal quarantine facility does not use a molecular detection method to recognize this ahb alien species. hence, the aim of this study was to establish a rapid molecular detection method for detecting ahb status in indonesia based on the cyt b/bglii. we used two dna extraction kits designed for animal tissue for rapid dna preparation from samples. this technique is appropriate for animal quarantine officers to make a rapid and accurate decision on the presence of the ahb in imported colonies in indonesia. materials and methods apis mellifera collection imported a. mellifera were collected from apiaries located in central java (pati, kudus, jepara) and in east java (jember, malang, kediri, pasuruan), indonesia. bees were anesthetized and preserved in absolute ethanol. dna extraction genomic dna was extracted from the thorax of single bee using phenol– chloroform extraction and ethanol precipitation. in addition, we tested the genomic dna mini kit from real biotech corporation (rbc) and the genclean column genomic dna isolation kit (generay biotech). for both kits, 20 mg of bee thorax was used for dna extraction, and single thorax in a 1.5 ml microtube was immersed in liquid nitrogen for 15 min prior to being crushed using a grinder. thereafter, the protocol according to the kit manufacturer was followed. a. mellifera dna amplifi cation. part of the cyt b region was amplified using primers of cytochrome b forward: 5’-tatgtactacctttgaggacaaatatc-3’ (11400); cytochrome b reverse 5’rapid detection of the africanized honey bee – rika raffi udin et al. 40 attacacctcctaatttattaaggaat-3’ (11859) (crozier et al. 1991). numbers in brackets indicate the position of a. mellifera cyt b in the complete mtdna (accession number l06178). cycle sequencing conditions protocol was 2 min. at 94 °c for initial denaturing, 35 cycles of 30 s at 94 °c, 30 s at 55°c and 1 min. at 72 °c for dna elongation, followed by 10 min. for the dna extension. dna sequencing followed the abi bigdye automated sequencing instruction from the supplier by using the same primers of as for dna amplication. dna restriction analysis apis mellifera cyt b pcr products were digested using bglii restriction enzyme. the digestion mixture contained the cyt b pcr product, dd h2o, buffer d, bglii restriction enzyme and were incubated at 37oc overnight. dna alignment we used clustalx (thompson et al. 1997) program to align the dna sequence generated in this study with the published ahb haplotypes provided in genbank database (http://www.ncbi.nlm.nih.gov; accession number ef016646 and ef016647). results and discussions apis mellifera cyt b amplifi cation and dna restriction analysis we collected samples from 94 colonies of imported a. mellifera from big and small apiaries in central and east java (data not shown). all samples from the 94 colonies produced the same size dna band approximately 500 bp ( figure 1). pcr products from 94 samples were digested with bglii and all produced two bands of approximately 200 and 300 bp (figure 2). figure 1. cyt b pcr products from imported a. mellifera in java, indonesia; m = 100 bp dna marker, 1-10 = a. mellifera colony sample number 1-10 from java, indonesia biotropia vol. 16 no. 1, 2009 41 figure 2 cyt b/bglii restriction fragments of imported a. mellifera in java, indonesia; m = 100 bp dna marker, 1-10 = a. mellifera colony sample number 1-10 from java. apis mellifera cyt b sequence data and alignment with ahb haplotypes since all cyt b pcr product of apis mellifera gave the same result (figure 1 and 2), we only sequenced one bee, i.e. a. mellifera from colony number 9 from pati, central java (am9pt). pcr product of am9pt cyt b sequence gave a total length of 485 bp (figure 3). the bglii sites (sequence agatct) digested the pcr product into two fragments of 194 and 291 bp (figure 3) . figure 3. restriction map of a. mellifera (am9pt) cyt b/bglii. resulted for two dna fragments of 194 and 291 bp these results were in agreement with the data of crozier (1991) from a. mellifera imported to australia and data from the united states (pinto et al. 2003) which have indicated that cyt b can be used to detect the ahb. a total of 451 colonies have been screened for the ahb characters in the usa. the united states department of agriculture, animal and plant health inspection service, plant protection and quarantine used trap lines and morphological identification techniques. based on morphology, the ahb and ehb could not be distinguished by beekeepers (montesinos 1995). hence, this accurate molecular technique can help solve the problem of detecting the ahb. africanized honey bees are one of the invasive alien species that are banned from entering all nations including indonesia. therefore, this effective system for warning about invasive alien species is needed, especially due to global trade. ahb and non-ahb cyt b alignment the dna sequence alignment showed that the am9pt cyt b sequenced in this study was exactly the same as that of genbank accession l06178; crozier & crozier 1993) denoted for a. mellifera ligustica (figure 4). hence, am9pt from this rapid detection of the africanized honey bee – rika raffi udin et al. 42 research was confirmed to be non-ahb bees. the primer amplified the partial cyt b gene started at nucleotide 396 of the complete cyt b gene sequence. the bglii site was located at nuclotide 694 based on whole sequence (figure 4). cytbl06178 attcttttaatatcaatagcagctgcatttataggatatgtactaccatgaggacaaata 420 am9pt ------------------------------------tatgtactaccatgaggacaaata 24 ef016646 ------------------------------------tatgtactaccatgaggacaaata 24 ef016647 ------------------------------------tatgtactaccatgaggacaaata 24 ************************ cytbl06178 tcatattgaggtgcaacagttattactaatcttttatcagcaattccttatattggtgat 480 am9pt tcatattgaggtgcaacagttattactaatcttttatcagcaattccttatattggtgat 84 ef016646 tcatattgaggtgcaacagtcattactaatcttttatcagcaattccttatattggtgat 84 ef016647 tcatattgaggtgcaacagttattactaatcttttatcagcaattccttatattggtgat 84 ******************** *************************************** cytbl06178 acaattgtattatgaatttgaggtggattttcaattaataatgctacattaaatcgattt 540 am9pt acaattgtattatgaatttgaggtggattttcaattaataatgctacattaaatcgattt 144 ef016646 acaattgtattatgaatctgaggtgggttctcaattaataatgctaccttaaatcgattt 144 ef016647 acaattgtattatgaatctgaggtgggttctcaattaataatgctaccttaaatcgattt 144 ***************** ******** ** ***************** ************ cytbl06178 ttttctttacattttattttaccattattaattttatttatagttattcttcatttattt 600 am9pt ttttctttacattttattttaccattattaattttatttatagttattcttcatttattt 204 ef016646 ttttctttacattttattttaccattattaattttatttatagttattcttcatttattt 204 ef016647 ttttctttacattttattttaccattattaattttatttatagttattcttcatttattt 204 ************************************************************ cytbl06178 gccttacatttaactggatcatctaatcctcttggatcaaattttaataattataaaatt 660 am9pt gccttacatttaactggatcatctaatcctcttggatcaaattttaataattataaaatt 264 ef016646 gccttacatttaactggatcatctaatcctcttggatcaaattttaataattataaaatt 264 ef016647 gccttacatttaactggatcatctaatcctcttggatcaaattttaataattataaaatt 264 ************************************************************ cytbl06178 tcatttcatccatatttttcaattaaagatcttttaggattttatatcatcttatttatc 720 am9pt tcatttcatccatatttttcaattaaagatcttttaggattttatatcatcttatttatc 324 ef016646 tcatttcatccatatttttcaattaaagaccttttaggattttatattatcttatttatc 324 ef016647 tcatttcatccatatttttcaattaaagaccttttaggattttatattatcttatttatc 324 ***************************** ***************** ************ cytbl06178 tttatattcattaattttcaatttccatatcatttaggagatccagacaatttcaaaatt 780 am9pt tttatattcattaattttcaatttccatatcatttaggagatccagacaatttcaaaatt 384 ef016646 tttatattcattaattttcaatttccatatcatttaggagatccagataattttaaaatt 384 ef016647 tttatattcattaattttcaatttccatatcatttaggagatccagataattttaaaatt 384 *********************************************** ***** ****** cytbl06178 gcaaatccaataaatactccaactcatattaaacctgaatgatatttcctatttgcatat 840 am9pt gcaaatccaataaatactccaactcatattaaacctgaatgatatttcctatttgcatat 444 ef016646_1 gcaaatccaataaatactccaactcatattaaacctgaatgatattttctatttgcatat 444 ef016647_2 gcaaatccaataaatactccaactcatattaaacctgaatgatattttctatttgcatat 444 *********************************************** ************ cytbl06178 tcaattttacgagcaattcctaataaattaggaggtgtaatcggattagtaatatcaatt 900 am9pt tcaattttacgagcaattcctaataaattaggaggtgtaat------------------485 ef016646 tcaattttacgagcaattcctaataaattaggaggtgtaat------------------485 ef016647 tcaattttacgagcaattcctaataaattaggaggtgtaat------------------485 ***************************************** 1 2 3 4 5 6 7 8 9 figure 4. a. mellifera am9pt from this study aligned with genbank cyt b sequences cytbl06178 (from a. m. ligustica acc num l06178), ef016646 (from ahb haplotype 1), and ef016647 (from ahb haplotype 2). * = nucleotide homology. underlined nucleotide = bglii sites. solid boxed nucleotides = nucleotide differences between ahb-1 and ahb-2. dashed boxed nucleotides = nucleotide change causing loss of bglii site in ahb type. numbers positioned above the dna alignment were all nucleotide differences between am9 pt, and ahb haplotype 1 and haplotype 2. biotropia vol. 16 no. 1, 2009 43 dna alignment between am9pt and ahb haplotype 1 and ahb haplotype 2 from genbank (accession numbers ef016647 and ef016647, respectively) showed nine nucleotide differences (figure 4, see the numbers above the nucleotide alignment). the difference between the ahb and the non-ahb at base number 295 was shown in figure 4, labelled mutation number 5. this is the position of the restriction site for bglii; it could be observed that the nucleotide “t” in cytbl06178 and am9pt were altered to be a “c” in both ahb haplotypes 1 and 2 (ef016646 and ef016647). this mutation removed the bglii restriction site, hence shows why a single band is seen in the ahb haplotypes. based on homology analysis of the two ahbs, these two haplotypes also differ at nucleotide number 65 (figure 4). the two ahb haplotypes obtained from genbank were submitted on february 1, 2007 by szalanski, a.l. and mckern, j.a from the department of entomology, university of arkansas, usa. based on the findings of this study, we suggest to carry out cooperation between the animal sections of the agency of agriculture quarantine (balai karantina pertanian) and the apiaries association in indonesia. the cooperation could commence by performing a socialization of honey bee biology. furthermore, the molecular detection for the ahb should be implemented at the animal quarantine in every province in indonesia, particularly at soekarno hatta and ngurah rai airports, so that imported bees could be monitored. conclusions molecular analysis of imported a. mellifera based on the cytochrome b gene from mitochondrial dna digested with bglii restriction enzyme was conducted to determine whether the africanized honey bee (ahb) is present in indonesia. out of 94 colony samples from 94 beekeepers in java, all pcr products amplified from this gene were of the same size (485 bp). two dna fragments (194 and 291 bp) were produced after digested with bglii. this cyt b/bglii result showed that all collected samples of a. mellifera were of the non-ahb type. hence, no ahb could be detected in indonesia in this study. we found that two different dna extraction kits could be successfully used to extract dna for this analysis, allowing fast and effective preparation of dna for subsequent pcr analysis. references castro f., palma m., brochettobraga m., malaspina o., lazaretti j., baldo m., antila m., zuppi l., croce j. and w. cossermelli. 1994. biochemical properties and study of antigenic cross-reactivity between africanized honey bee and wasp venom. journal of investigational allergology & clinical immunology, 4:37-41 crozier r.h. 1977. evolutionary genetics of the hymenoptera. annual review of entomology, 22:263-288 crozier r.h. and y.c. crozier. 1993. th e mitochondrial genome of the honeybee apis mellifera: complete sequences and genome organization. genetics, 133:97-117 crozier y.c., koulinous s. and r.h. crozier. 1991. an improved test for africanized honey bee mitochondrial dna. experientia, 47:968-969 rapid detection of the africanized honey bee – rika raffi udin et al. 44 guerra j. and l, d. dj.goncalves. 2000. africanized honey bees (apis mellifera l.) are more effi cient at removing worker brood artifi cially infested with the parasitic mite varroa jacobsoni oudemans than are italian bees or italian/africanized hybrids. genetics & molecular biology, 23:89-92 montesinos p. 1995. managing the africanized honey bee. 2. american bee journal, 135:473-476 perez-castro e., may-itza w. and j. quezada-euan. 2002. th irty years after: a survey on the distribution and expansion of africanized honey bees (apis mellifera) in peru. journal of apicultural research, 41:69-73 pinto m.a., johnston j.s., rubink w.l., coulson r.n., patton j.c. and w.s. sheppard. 2003. identifi cation of africanized honey bee (hymenoptera: apidae) mitochondrial dna;validation of a rapid polymerase chain reaction-based assay. annual entomology society of america, 96:679-684 raffi udin r. 2002. honey bee behavioural evolution and itpr gene structure studies. phd. th esis, james cook university, australia raffi udin r., farajallah a., purwantara b., tjitrosoedirdjo s.s. and s. tjitrosemito. 2006. peranan petugas karantina terhadap pengelolaan hewan asing invasif dalam kerangka sps-wto. in: workshop pengawasan keamanan hayati hewani dan sosialisasi keamanan hayati hewani, jakarta 21-23 november 2006 ruttner f. 1988. biogeography and taxonomy of honeybees. springer-verlag, berlin ruttner f.l.t. and j. louveaux. 1978. biometrical-statistical analysis of the geographic variability of apis mellifera l. apidologie, 9:363-381 schiff n. and w. sheppard. 1993. mitochondrial dna evidence for the 19th century introduction of african honey bees into the united-states. experientia, 49:530-532 spangler h. 1994. are the wingbeat frequencies of honey bees an indicator of population or behaviour. american bee journal, 134:53-55 tegelstrom h. 1986. mitochondrial dna in natural population: an improved routine for screening of genetic variation based on sensitive silver staining. electrophoresis, 7:226-229 th ompson j.d., gibson t.j., plewniak f., jeanmougin f. and d.g. higgins. 1997. th e clustal-x windows interface: fl exible strategies for multiple sequence alignment aided by quality analysis tools. nucleic acid research, 25:4876-4882. biotropia vol. 16 no. 1, 2009 thank you for evaluating anybizsoft pdf splitter. a watermark is added at the end of each output pdf file. to remove the watermark, you need to purchase the software from http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html biotropia no. 8, 1995: 1 10 nutrient transfer in vesicular-arbuscular mycorrhizas: a new model based on the distribution of atpases on fungal and plant membranes*) f.a. smith department of botany, the university of adelaide, south australia, 5005, australia s.e. smith department of soil science, the university of adelaide, south australia, 5005, australia abstract in this paper we review the membrane transport processes that are involved in the transfer of mineral nutrients and organic carbon between the symbiotic partners in mycorrhizas. in particular, we reassess the prevailing hypothesis that transfer in vesicular-arbuscular (va) mycorrhizas occurs simultaneously and bidirectionally across the same interface and that arbuscules are the main sites of transfer. using cytochemical techniques, we and our collaborators have reexamined the distribution of atpases in the arbuscular and intercellular hyphal interfaces in va mycorrhizas formed between roots ofallium cepa (onion) and the fungus glomus intraradices. the results showed that h +-atpases have different localisation on plant and fungal membranes in arbuscular and hyphal interfaces (gianinazzi-pearson et al. 1991). while some arbuscular interfaces had h+-atpase activity on both fungal and plant membranes, in most cases the fungal membrane lacked this activity. in contrast, the plasma membranes of intercellular hyphae always had h + -atpase and the adjacent root cells did not. this suggests that the different interfaces in a va mycorrhiza may have different functions. we propose that passive loss of p from the arbuscules is associated with active uptake by the energised (atpase-bearing) plant membrane and that passive loss of carbohydrate from the root cells is followed by active uptake by the intercellular hyphae. if this model is correct, then variations in "mycorrhizal efficiency" (i.e. the extent to which mycorrhizal plants grow better than non-mycorrhizal controls) might be determined by differences in the numbers of active arbuscules as a proportion of the total fungal biomass within the root. as a first step towards investigating this possibility, we have developed methods for measuring the surface areas of arbuscular and hyphal interfaces in different fungus-host combinations, glomus spp./ allium porrum (leek). we have also measured fluxes of p from fungus to plant and have been able to partition these between the arbuscular and total (arbuscular plus hyphal) interfaces. the implications of this work, and suggestions for future investigations of the molecular mechanisms involved in nutrient transfer in mycorrhizas, are discussed. key words: mycorrhizas/glomus intraradices/atpases/allium cepa. *)paper presented at the second symposium on biology and biotechnology of mycorrhizae and third asian conference on mycorrhizae (acom iii), 19-21 april, yogyakarta, indonesia. 1 biotropia no. 8, 1995 smith, s.e., s. dickson, c. morris and f.a. smith. 1994b. transfer of phosphate from fungus to plant in va mycorrhizas: calculation of the area of symbiotic interface and fluxes of p from two different fungi to allium porrum. new phytologist. in press. smith, s.e. and f.a. smith. 1990. structure and function of the interfaces in biotrophic symbioses. new phytologist, 114, 1-38. smith, s.e. and f.a. smith. 1994. membranes in mycorrhizal interfaces: specialised functions in symbiosis. in: membranes specialised functions in plant cells. ed. by m. smallwood and d. bowles, jai press inc. (in press). tester, m. 1990. plant ion channels: whole cell and single channel studies. new phytologist, 114, 305-340. 10 biotropia no. 8, 1995 transfer of nutrients is of central importance in the functioning of the very widespread symbioses between mycorrhizal fungi and their host plants. the host gains mineral nutrients (e.g. phosphate and zinc), and in all mycorrhizas except orchid mycorrhizas the fungus gains carbohydrate. this two-way transfer occurs across interfaces which comprise the membranes of both organisms and an apoplastic region between them. the interfacial apoplast may contain considerable amounts of wall or wall-derived material, which varies according to the type of mycorrhiza and the particular interface (smith and smith 1994). in ectomycorrhizas, the interface is entirely intercellular (extracellular), whereas in some endomycorrhizas (e.g. ericoid mycorrhizas), the interface is virtually entirely intracellular. other endomycorrhizas (e.g. vesicular-arbuscular mycorrhizas) have both an intercellular interface — where the fungus penetrates between cortical cells, and an intracellular interface where the fungus penetrates the cell walls of its host and develops arbuscules. in all cases, the interface has a restricted volume. it is isolated from the external environment, and membrane transport processes in both organisms will control the physico-chemical conditions in the apoplast and hence the overall transport of solutes from one organism to the other. fig. 1 summarises the uptake and transfer of nutrients in all mycorrhizas except orchids. as well as emphasising phosphate (p) and figure 1. transfer of mineral nutrients and carbohydrates (sugars) in mycorrhizas (excluding orchids). with the exception of h + -atpases (see text), the mechanisms of membrane transport are not specified, fpm: fungal plasma membrane; rpm: root plasma membrane; pi: inorganic phosphate. 2 introduction nutrient transfer in vesicular-arbuscular mycorrhizas f.a. smith and s.e. smith zinc (zn), it shows that other mineral nutrients can be transferred from soil to the host via the fungus. in the case of nh4 + , there is conversion to organic n compounds and subsequent transfer of some of these across the interface. the transfer of sugars to the fungus is shown in fig. 1, and also the possible transfer of "growth factors" i.e. phytohormones, vitamins etc. finally, fig. 1 shows the presence of h + -transporting atpases that are present in the functional plasma membranes of all fungal and plant cells. these "energise" the membranes both electrically, via the generation of electric potential differences (pds), and chemically via the development of ph differences, and so allow selective transport of other solutes. for example, phosphate and sugars are transported into fungal and plant cells by diferrent transport proteins which catalyse co-transport of phosphate or sugar with h + (not shown in fig. 1, for simplicity). other types of transport proteins are "channels" which can open and close in response to various signals at the plasma membrane, including changes in pds and ph. for a general discussion of interfaces and transport in mycorrhizas, see smith and smith (1990, 1994). tester (1990) describes factors that control the operation of channels. membrane transport and atpases in va mycorrhizas for va mycorrhizas, the prevailing hypothesis has been that transfer of nutrients and carbohydrates occurs bidirectionally and simultaneously across the same interface and that the arbuscules, having large surface-to-volume ratio, are important as the main site of transfer. it has been suggested in the past and sometimes even now — thatp transfer can be explained by breakdown (digestion) of arbuscules, releasing p to the host. however, calculations by cox and tinker (1976) showed that this would be far too slow the account for the fluxes of p across the interface. to make these calculations, cox and tinker (1976) measured surface areas of arbuscules in the glomus mosseae/allium cepa (onion) symbiosis using electron microscopy and image analysis, the amount of infection and the total numbers of arbuscules per unit length of root. they also estimated the lifespan of the arbuscules (about 4 days), their p content and the inflow of p i.e. the uptake of p to the host per unit length of root. in this way, they showed that the p that could be released by digestion was about 100 times too small for the actual p flux; hence continuing transfer from living arbuscules is required. this is an important conclusion, and the calculated value for the p flux 13 nmol m (arbuscular surface area) -2s-1 -is also important since it is similar to values for uptake of p into cells by active transport (i.e. by h + -p co-transport), but much larger than ty 3 biotropia no. 8, 1995 pical values for efflux of p. an important question thus becomes: what is the mechanism for release of p from the fungus? this issue is discussed by smith et al. (1994a). the hypothesis of simultaneous two-way transport of p and sugar in va my corrhizas is summarised in fig. 2. details of co-transport and passive transport are shown. the hypothesis has been supported by the cytochemical demonstration of atpases on both fungal and plant plasma membranes in the arbuscular interface (marx et al. 1982), but does not take into account the potential role of the intercellular fungal hyphae in transport processes (smith and dickson 1991). likewise, the possible role of the intercellular interface was not considered by cox and tinker (1976). figure 2. diagrammatic representation of the distribution of h +-atpases and associated transport processes in va mycorrhizas that would result in bidirectional transfer of sugar and phosphate across the same arbuscular interface. based on figure 7 of gianinazzi-pearson et al. (1991). pam: periarbuscular (plasma) membrane of the root cortical cells. 4 nutrient transfer in vesicular-arbuscular mycorrhizas f.a. smith and s.e. smith spatial separation of transport? gianinazzi-pearson et al. (1991) re-examined the distribution of atpases in the arbuscular and intercellular (hyphal) interfaces in va mycorrhizas formed between roots of allium cepa (onion) and glomus intraradices. the cytochemical methods were similar to those used by others: they involved the deposition of lead phosphate at sites of atpase activity and the use of inhibitors (e.g. vanadate and molybdate) to distinguish h + -atpases likely to be involved in active membrane transport from other enzymes with atp-hydrolysing ability. the results showed that h + -atpases have different localisation on plant and fungal membranes in arbuscular and hyphal interfaces (gianinazzi-pearson et al., 1991). while some arbuscular interfaces had h + -atpase activity on both fungal and plant membranes, in most cases the fungal membrane lacked this activity. in contrast, the plasma membranes of intercellular hyphae always had h + -atpase and the adjacent root cells did not. these results suggest that the different interfaces in a va mycorrhiza may have different functions (fig. 3). the arbuscules may indeed play a major role in p transfer. passive p loss from the fungus to the inter facial apoplast would be associated with active uptake (h + -p co-transport) by the energised (atpase-bearing) plant membrane. reabsorption of p from the interfacial apoplast by the fungus may be reduced in the arbuscules, because the fungal membrane frequently appears to lack atpase activity and hence the capacity for active uptake. these figure 3. spatial separation of transfer in va mycorrhizas. a: passive loss of p from the fungus and active uptake (h + -phosphate co-transport) across the energised periarbuscular membrane. b: passive loss of sugar to the intercellular apoplast and active uptake (h +-hexose co-transport) across the energised hyphal plasma membrane. based on figure 7 of gianinazzi-pearson et al. (1991). 5 biotropia no. 8, 1995 processes would result in net transfer of p from fungus to plant. we have shown that transfer of p between the symbionts occurs at rates consistent with high passive efflux from the fungus and active uptake by the plants (smith et al. 1994a & b). gianinazzi-pearson et al. (1991) propose that the intercellular hyphae (but not the arbuscules) may be responsible for carbohydrate transfer from plant to fungus. in this case, passive loss of carbohydrate from the root cortical cells to the intercellular apoplast would be followed by active uptake by the fungal hyphae. as far as we are aware, the proposal that transfers of p and carbohydrate are spatially separated had not been made previously. arbuscules and va mycorrzhizal "efficiency" an attraction of this new model is that variations in mycorrhizal "efficiency" (i.e. the extent to which mycorrhizal plants grow better than non-mycorrhizal controls) might be explained by differences in the numbers or size of metabolically active arbuscules as a proportion of the total fungal biomass within the root. without arbuscules, the fungus would be essentially a parasite, draining carbohydrate from its host but giving no phosphate in exchange. it is well known that mycorrhizal "efficiency" does vary in different fungus/host combinations, or under different environmental conditions, but there has been little attempt to correlate these variations with numbers of arbuscules or their activity. one reason is the difficulty of measuring numbers and surface areas of arbuscules, and length and surface area of intercellular hyphae, rather than just taking the length or percent of the root that contains fungus as a measure of infection. in order to remedy this deficiency, we have developed an updated version of the methods used by cox and tinker (1976), including computer-aided image analysis techniques and the use of nitroblue tetrazolium to distinguish metabolically active fungal tissues from the total fungal biomass (smith and dickson 1991). by means of a size facility in the program, it is possible to distinguish between intercellular hyphae and arbuscules. table 1 shows data for allium porrum infected by glomus sp. 'city beach' (wum16). percentage infection was obtained conventionally and the areas of interface obtained by image-analysis. details for the experiment and methods are given by smith and dickson (1991) and smith et al. (1994b). table 1 shows that the surface area of metabolically active arbuscules per metre of root declined with time, while that of hyphae increased. after 63 days, the hyphae had the larger surface area, illustrating the danger of ignoring them as a site of nutrient transfer, irrespective of what model is adopted. table 2 shows the inflows of p (i.e. amounts of p taken up per metre of root length) into non-mycorrhizal and mycorrhizal plants, with the amount taken into 6 nutrient transfer in vesicular-arbuscular mycorrhizas f.a. smith and s.e. smith the latter via the fungus calculated by subtraction. we are aware that this calculation, which assumes that fungal infection does not affect p uptake by the host, may produce errors, but this possibility has been ignored in our calculations. table 2 shows that inflow of p decreased with time, particularly in non-mycorrhizal plants. table 3 shows the fluxes of p across the interfaces, as obtained by dividing the inflows (table 2) by the average surface areas obtained from table 1. the increasing divergence with time between the values that were obtained assuming that the flux occurred 1) only across the arbuscular interface and 2) across both arbuscules and intercellular hyphae reflects the increasing contribution of the latter to the total surface area. the range of values in table 3 (3.7-12.8 nmol m -2 s1 ) agrees quite well with the single value of nmoll m -2 s 1 that was calculated by cox and tinker (1976). 7 biotropia no. 8, 1995 although these results show that it is possible to make separate calculations for influx of p across the arbuscular interface and the total interface, they do not by themselves answer the question of whether the arbuscular interface alone is responsible for p transfer. to do this, it will be necessary to attempt to correlate p transfer with arbuscular surface area, e.g. in comparison with the total fungal biomass within the root. results that we have obtained so far suggest that where different va mycorrhizal fungi infect the same host species, there is no simple correlation. table 4 shows again the results for allium porrum infected by glomus sp. 'city beach' over 21-42 days of growth and compares them with results for allium porrum/glomus mosseae obtained at the same time and under the same conditions (smith and dickson 1991; smith et al. 1994b). g. mosseae produced a slightly larger % infection but a slightly smaller inflow of p (19.0 compared to 22.3 pmol m -1 s -1 )the areas of interfaces with g. mosseae were more than twice that with g. sp. 'city beach', and the flux of p via mosseae was accordingly much smaller than with g. sp. ' city beach', whether calculated using the area of arbuscular interface or the total interface. these results show that g. sp. 'city beach' loses p faster on a surface area basis. there are several possible explanations including not only differences in numbers of membrane transport proteins at the interface but also different rates of translocation of p through the fungi or different rates of uptake from the soil which could in turn be associated with different lengths of external hyphae. to pursue further the question of the role of the arbuscule as the sole location of p transfer in comparison with that of the intercellular hyphae (or total interface) as the location of exchange of carbohydrate, it will be necessary to do more sophisticated experiments with more fungus/host combinations. the use of va mycorrhizas in which development of arbuscules is suppressed will be particulary valuable. 8 nutrient transfer in vesicular-arbuscular mycorrhizas f.a. smith and s.e. smith conclusions in this paper we have reviewed briefly the mechanisms for transfer of nutrients in mycorrhizas mainly va mycorrhizas and have then proceeded to re-evaluate the sites of nutrient transfer, particularly the question of whether simultaneous bidirectional transfer occurs in arbuscules. we suggest that this may not be the case, and this in turn has led us to quantify the development of the arbuscular interface vis-a-vis the total interface and to attempt to calculate p fluxes across both interfaces. as well as pursuing this approach further, we are starting to attempt to identify the molecular mechanisms for transport at the interface and also to measure the physico-chemical conditions there, including the ionic conditions in the apoplast. in this way we hope that the control of nutrient transfer and its role in determining "mycorrhizal efficiency" will be better understood. acknowledgements we thank our collaborators, particularly vivienne gianinazzipearson, silvio gianinazzi, sandy dickson, christina morris, mark tester and rob reid for all their help in discussions and experiments. funding came from the australian research council, an inra international collaborative research program and the university of adelaide. references cox, g. and p.b. tinker. 1976. translocation and transfer of nutrients in vesicular-arbuscular mycorrhizas. i. the arbuscule and phosphorus transfer: a quantitative ultrastructural study. new phytologist, 77, 371-378. gianinazzi-pearson, v., s.e., smith, s. gianinazzi and f.a. smith. 1991. enzymatic studies on the metabolism of vesicular-arbuscular mycorrhizas v. is h + -atpase a component of atp hydro-lysing enzyme activities in plant/fungus interfaces? new phytologist, 117, 61-74. marx, c., j., dexheimer, v. gianinazzi-pearson and s. gianinazzi. 1982. enzymatic studies on the metabolism of vesicular-arbuscular mycorrhizas iv. ultracytoenzymological evidence (at-pase) for active transfer processes in the host-arbuscular interface. new phytologist, 90, 37-43. smith, f.a., s., dickson, c., morris, r.j., reid, m., tester and s.e., smith. 1994a. phosphate transfer in va mycorrhizas: special mechanisms or not? proceedings of the 4th international symposium on the structure and function of roots. in press. smith, s.e. and s. dickson. 1991. quantification of active vesicular-arbuscular mycorrhizal infection using image analysis and other techniques. australian journal of plant physiology, 18, 637-648. 9 1.pdf 10.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf biotropia no biotropia no. 16, 2001 : 28 38 characterization of malaysian wild bananas based on anthocyanins muhammad asif javed*, mak. chai and rofina yasmin othman division of genetics, institute of biological sciences, university of malaya 50603 kuala lumpur, malaysia abstract the male buds of 16 musa species (musaceae) populations were investigated by hplc for the occurrence of anthocyanins. the investigation was based on the presence of 6 anthocyanins. the 16 musa samples could be classified into three distinct species i.e. musa acuminata, musa violascens and musa balbisiana. musa acuminata could be divided into two subspecies : malaccensis (lowland) and tmncata (highland) according to their constituents and content of major anthocyanins. no variation was observed in the composition of the anthocyanins of kedah type ssp. siamea and selangor types ssp. malaccensis. the classification of m. acuminata into two subspecies based on anthocyanin data further supported the current taxonomic grouping of the species. key words: musa acuminata/musa violascens/musa balbisiana/musaceae /hplc /chemotaxonomy introduction the classification of the genus musa as proposed by cheesman (1947) is the only one which accords satisfactorily with the known taxonomic and cytogenetic facts. of the four named sections, eumusa is the best known partly because the plants are hardy and fairly easy to grow whereas the callimusa is the least known section. section eumusa is the most important as the progenitor of all cultivated bananas. musa acuminata (aa) and musa balbisiana (bb) belong to this section. among the cultivated bananas, different cultivars are classified according to their genomic constitution and ploidy level. malaysia is one of the centers of diversity of wild and cultivated bananas. simmonds (1955) reported four species from the malaysian peninsula. the two species musa violascens and musa gracilis are ornamental wild bananas whereas musa acuminata and musa balbisiana are the two most important species of the genus musa. musa acuminata is the most variable species of the two in malaysia being the progenitors of several local diploid and triploid banana cultivars. three different subspecies of musa acuminata suggested (simmonds 1955) were ssp. malaccensis (selangor), truncata (cameron highland) and siamea (kedah). this classification was questioned by hari (1968) and then later on shepherd (1988) based on chromosomal translocations. therefore, the nomenclature of local wild musa species has been complicated by taxonomic revisions. recent renewed interest * corresponding author: e-mail. j95asit@umcsd.um.edu.my rocketmail. asif_javed@rocketmail.com 28 biotropia no. 16, 2001 in the utilization of wild banana sources had further elucidated the need to characterize different wild musa species in malaysia. anthocyanins constitute the most important group of plant pigments. in 70 species of 33 families of angiosperms, they were found in membrane-bounded anthocyanoplasts located within the main cell vacuole. apart from contributing cyanic color to flower and fruits, the pigments also play an important role during pollination, and are of great economic and genetic importance. previous surveys of anthocyanins in the bracts of banana have been reviewed (simmonds 1954 b,c; horry and jay 1988 a,b). the distribution of various anthocyanidins among different banana cultivars gave good support to the taxonomic ordering within the genus (horry and jay 1988a,b). the present study described the relationship between different wild bananas of malaysian origin based on the distribution of anthocyanins. materials and methods sample collection wild musa species samples were collected from different parts of peninsular malaysia. the types and number of samples collected and their locations are shown in table 1. fourteen populations of wild musa acuminata were sampled. samples of musa violascens and musa balbisiana were also selected for comparative studies. ten suckers each of musa acuminata, musa violascens and musa balbisiana were collected from different mother plants and these were planted in the university farm for evaluation. suckers were planted in holes of 30-40 cm deep, spaced at 2 m x 2 m. at planting, organic compost was applied in the holes. weeding was done manually. anthocyanin extraction variation in anthocyanin pigments was studied from male buds collected from 16 samples (table 1). each banana sample consisted of three male buds as three replicates. the outermost male bracts were used for anthocyanin extraction by grinding 4-5 g of bracts to powder form in liquid nitrogen. anthocyanin pigments were then extracted by boiling the powder in 40 ml of meoh etoh (1:1) for 40 minutes. crude extracts were concentrated to 5 ml, and filtered through glass wool (horry and jay 1988a). filtrates were stored at -70°c till used for thin layer chromatography (tlc) and high pressure liquid chromatography (hplc). tlc and hplc analysis thin layer chromatography (tlc) was carried out on 0.1 mm cellulose plates (merck. 1.05716. 20x20 cm). development was accomplished with solvent mixtures prepared from analytical grades of hydrochloric acid, formic acid and deionized dis 29 characterization of malaysian wild bananas – muhammad asif javed et al table 1. samples of wild musa species ``tilled water. two solvent systems used for tlc were : a. system i. cone. hc1 formic acid water (24.9:23.7: 51.4), b. system ii. cone. hc1 formic acid water (7.1:31.4:61.5). developing tanks were lined with heavy grade filter paper and allowed to equilibrate overnight prior to use. tlc plates were kept at 80°c overnight. a base line was drawn on the loading end and a 1-2 ul of concentrated anthocyanin solutions were loaded. there were 10 samples loaded on each plate. rf values taken for each pigment according to their mobility on the tlc plate were calculated as follows; distance of migration substance rf = distance of migration of solvent front hplc analysis of anthocyanin pigments was carried out on a millipore waters™ liquid chromatograph equipped with a variable wavelength waters™ 486 detector, waters™ 600 controller and waters™ 717 plus auto-sampler was used. anthocyanin pigments separation was carried out using reverse phase c 18, inertsil ods -2 (4.6 x 1.6 mm) 5 urn column (horry and jay 1988a). hplc was run according to horry and jay (1988a), with slight modifications. two solvents used were 5% formic acid and absolute methanol (5:95, v/v). concentrated anthocyanin extracts were diluted ten times with 0.1n hc1, and filtered just before injection. flow rate was maintained at 0.80 ml/min with an injection volume of 20 ul. detection was observed at 514 nm or 240 to 600 nm with scanning detector for spectral identification of the peaks. the aglycone moiety of the 30 biotropia no. 16, 2001 anthocyanin for each hplc peak was identified according to the maxima of absorbance in the visible range (compounds 3, 6, 8: x max vis = 530, 535, 535 nm; compounds 4, 5, 7: a. max vjs = 520, 522, 522 nm) and to their retention times whereas glycosidic pattern was determined by tlc. the composition of the two solvents, flow rate and time are given in table 2. table 2. method used for anthocyanin separation by hplc system (horry and jay 1988a) the peaks showing different anthocyanin pigments (delphinidin, derivative of cyanidin, cyanidin, petunidin, peonidin and malvidin) were used for sample comparison. chemometric analysis hplc data from 16 musa samples were subjected to cluster analysis and principal component analysis (pca) using spss statistic program (spss, inc.). relative amounts of the anthocyanins expressed by percentages were used as an input matrix. results anthocyanins were extracted from the outer most bracts tested through thin layer chromatography (tlc) followed by high pressure liquid chromatography (hplc). different pigments separated by hplc were determined according to increasing retention times, and only six compounds were identified. the absence of a shoulder around 330 nm indicated the absence of acylation. the presence of a shoulder in the visible spectrum at 440 nm and the rf values obtained on tlc indicated the presence of 3rutinosides as also reported by horry and jay (1988a). thin layer chromatography on 0.1 mm cellulose layer plates clearly showed the separation of different anthocyanin pigments. rf values obtained on tlc indicated the presence of 3rutinosides namely 3-o-rutinosyldelphinidin, 3-o-rutinosylcyanidin, 3-o-rutinosylpeonidin, and 3-o-rutinosylmalvidin (table 3). high pressure liquid chromatography (hplc) of anthocyanins resolved musa samples into a maximum of nine peaks. the peaks appearing between 10 to 21 minutes were the six peaks representing the six anthocyanins according to their retention times (horry and jay 1988a,b). 31 characterization of malaysian wild bananas muhammad asif javed et al the same sample injected three times did not show any difference in the chromatograms showing the stability of the system and the anthocyanin pigments. similarly, anthocyanin components extracted from the three outermost bracts from the same male bud did not show any difference in the number of anthocyanin components. the color of the bract in the male inflorescence progressively vanishes from the outside bract (bract 1, the oldest) to the inner bracts. it was observed that the relative distribution of the anthocyanin pattern is stable from the outer bracts to the inner bracts. extracts from musa acuminata accessions, bc2 and gala were analyzed and the results are presented in table 4. six previously reported anthocyanins were observed to be piesent in different combinations and percentages among all the samples studied except flava (table 4). table 3. anthocyanin of banana bracts separated through tlc by using two different solvent systems 32 biotropia no. 16,2001 the data matrix of relative amounts of floral anthocyanins expressed by percentages for musa samples (table 4) was then subjected to cluster and principal component analysis (pca). cluster analysis was conducted by using ward's method with squared euclidean distance (fig 1). four principal components with eigenvalues more than 1.0 were obtained, which could explain 99.89% for the total variation in the data matrix. thus all samples were grouped into four based on cluster and principal component analysis (table 5). rescaled distance cluster combined figure 1. cluster analysis of musa accessions based on anthocyanins using ward's method. all musa acuminata accessions were grouped into two major clusters except for the sample flava. the highland banana bc3 was well separated from other musa acuminata accessions. similarly, gala (musa balbisiana) and bc2 (musa violascens) were also well separated. 33 characterization of malaysian wild bananas muhammad asif javed et al gala was completely separated from the musa acuminata and musa violascens by larger values in the score for third pc (table 5), and is characterized by the larger accumulation of cyanidin (84.63%) followed by cyanidin derivative (table 4). factor loadings of pigments cyanidin and cyanidin derivative for the third pc were also great (table 6). highland musa acuminata sample (bc3) was completely separated from other samples of the species gaining the highest score for the second pc (table 5), for which factor loadings of pigment petunidin were great (table 6), whereas the cyanidin derivative was also accumulated in the highest percentage compared to other accessions of m acuminata samples. however, this sample showed the lowest peonidin compared to other samples of the same species. table 5. principal component scores of musa samples table 6. factor loadings of respective pigments exiting in musa species to principal components 34 biotropia no. 16, 2001 musa violascens (bc2) showed a very different chemotype and a number of peaks produced varied from other two species samples greatly. the anthocyanins detected in the other two species were present in a very small percentage. similarly, musa acuminata sample flava was observed with a very small percentage of the six anthocyanins detected. twelve samples were classified into first group gaining the largest scores for first principal component (table 5), for which factor loadings of peonidin, cyanidin and malvidin were great (table 6), whose anthocyanin composition of the bracts seems to be the standard of the musa acuminata samples. the only variation observed among these samples was the ratio of peonidin and malvidin. different from the highland banana samples with a large accumulation of peonidin whereas, cyanidin derivative and petunidin were present in a very low percentage (table 4). as shown in fig (1) and table (5), three groups of musa acuminata were readily distinguishable. flava diverges from other two groups of samples by'the absence of anthocyanins, as it had yellow color bracts i.e. acyanic bracts. highland banana sample bc3, had a very distinct deep chocolate brown color of the male bracts, and was characterized with accumulation of the simple anthocyanins in this subspecies, whose total composition could be described as 3 rutinosides of cyanidin derivative (cyd), 3 rutinosides of petunidin (pt) (table 4). these anthocyanins were mainly present in this sample. however, a low percentage of peonidin was detected. the remaining musa acuminata accessions belonging to lowlands of malaysia showed similar anthocyanins and their bract color ranged from red and purple. similarly, all musa acuminata accessions suggested a very high ability of methylation of anthocyanidins compared to gala (m. balbisiana). discussions many cytotaxonomists have argued that a unique chemical feature should merit the same importance as an unusual leaf shape or an odd chromosome number in determining plant relationships. they have shown that chemical characters correlate in genetic surveys satisfactorily with one or more biological characters. harborne (1967) made a survey of flavonoid pigments in 45% of the genera in the family gesneriaceae against the classification proposed based on morphological grounds. the differences at the subfamily level indicated a correlation between anthocyanin chemistry and plant geography. anthocyanin components were found to be very stable and the chromatograms obtained were highly reproducible under the same conditions. although the yield of anthyocyanins in musa accessions may vary, the qualitative aspect of it remains relatively consistent, thus making them good taxonomic characters. more than nine peaks were observed in most of the samples, indicating that at least similar number of components were present. however, in the present study only six components were recognized following horry and jay (1988 a,b). from the chromatograms, all the musa species can be identified. musa acuminata accessions were characterized 35 characterization of malaysiar, wild bananas muhammad asif javed el al with partially methylated anthocyanins based on cyanidin delphinidin mixtures i.e. mixtures of cyanidin, peonidin, delphinidin, petunidin and malvidin in different proportions. flava, a yellow bracted accession, did not show significant amount of anthocyanins. simmonds (1954) reported that musajlava ridl. from malaysia was merely a yellow bracted form of musa acuminata and the yellowness was due a single recessive gene and flava was a good example of acyanic bracts (bracts without anthocyanins). musa violascens was observed with a very different chromatogram than the other two and it showed a very little amount of six anthocyanins as observed in remaining species. the anthocyanin variation observed in this species could also be related to the light pink bract color compared to the red purple color observed in other species. anthocyanin variation among these species was further suggested by their different pollinators. musa violascens were reported to be bird pollinated whereas m. acuminata and m. balbisiana flowers were pollinated by bats (simmonds 1962; argent 1976). horry and jay (1988 a, b) described a schematic sequence for anthocyanin biosynthesis in musa. they described that cyanidin is the first and simplest element in a sequence of hydroxylations whereas methylations ending at malvidin. cyanidin or peonidin always occur, suggesting that cyanidin is an obligatory intermediate. we have observed high levels of cyanidin and peonidin in all musa acuminata accessions except for the highland banana form bc3. bc3 was observed to have a very low level of peonidin whereas a new compound cyd (cyanidin derivative) was deposited in high percentage compared to all other accessions. this compound was also reported by horry and jay (1988a, b) in musa balbisiana and suggested a different biochemical pathway. it is found to be an interesting marker for highland banana forms, therefore, it merits further characterization. a similar situation was observed for petunidin, which was accumulated in a very high percentage in bc3. horry and jay (1988b) suggested that a low level of petunidin and the fact that it never accumulates without malvidin, leads to the assumption of o-methylations. these two compounds were observed in high percentage in all lowland banana samples except in bc3. in terms of evolutionary significance of anthocyanins, there is a trend for delphinidin to be replaced by cyanidin, which is considered to be the most primitive pigment. therefore, o-methylation represents an advanced feature in comparison with simple hydroxylation (harborne 1977). all lowland musa acuminata accessions were observed with low levels of petunidin compared to the high percentage of malvidin, suggesting an advanced feature of anthocyanins whereas it was different in highland banana accession showing a high percentage of petunidin. simmonds (1962); horry and jay (1988a,b) suggested that chemotype of musa balbisiana is fairly unique and possesses no "advanced" anthocyanin forms and that environmental constraints were not so extreme that specialized biochemical pathways were needed to be developed in the evolution of this species. on the other hand, musa acuminata was found to possess a broad range of chemotypes, indicating the presence of a more diversified metabolism. this suggests that musa acuminata has undergone more evolutionary changes than musa balbisiana. 36 biotropia no. 16, 2001 cluster analysis and principal component analysis (pca) produced five groupings in musa acuminata accessions based on anthocyanins. the highland banana bc3 showed completely different anthocyanin patterns and was well separated from the remaining accessions. it was also observed to be morphologically distinct. all lowland musa acuminata accessions showed variation mainly in the proportion of the peonidin and malvidin. malvidin was predominant in ri, iptj, bd1, bc1, segun, sintok, rangis, and perak whereas peonidin in kra, bd2, ppc and j-4, respectively. the discrimination found among accessions, therefore, was mainly due to the ratios of these two anthocyanins whereas overall anthocyanin distribution was found similar among these accessions and the mean chemotype proceeded from a simple readjustment of the metabolic balance between methylated anthocyanins (horry and jay 1988a). the relationship revealed by the anthocyanin fingerprints could be compared with the geographical distribution and origin of these accessions. musa acuminata accessions were collected from lowlands and highlands. a large variation in the anthocyanin composition was observed for the highland banana form (ssp. truncatd) compared to lowland samples. northern (ssp. siamea) and central malayan (ssp. malaccensis) region samples were observed with similar anthocyanin compositions. therefore, the geographical origin of lowland accessions had little effect upon the anthocyanins metabolism. whereas the highland banana was different in ecology and geography and also showed distinct anthocyanin pattern. anthocyanin data further supported the two groupings of the musa acuminata i.e. lowland (ssp. malaccensis) and highland (ssp. truncatd) as suggested by hari (1968) and shepherd (1988). acknowledgements the work was supported by a grant from the ministry of science and technology and environment, malaysia. we also would like to thank mr. gopal krishnen and mr. asokan for their kind help in running the hplc system analysis. references anderson, o. m. and g. w. francis. 1985. simultaneous analysis of anthocyanins and anthocyanidins on cellulose thin layers. j. chromatography. 318,450-454. argent, g. c. g. 1976. the wild bananas of papua new guineae. notes of royal botanical garden, edinburgh 35: 77-114. cheesman, e. e. 1947. classification of the bananas. kew bull. 3, 11-28. hari, p. c. 1968. bract imbrication as a taxonomic character in musa acuminata. trop. agric. trinidad. vol. 45, no. 2,99-108. harborne, j.b. 1967. comparative biochemistry of the flavonoids. academic press. london. harborne, j. b. 1977. flavonoids and the evolution of angiosperms. biochem. sys. ecol. 5, 5-22 37 characterization of malaysian wild bananas muhammad asif javed et al horry, j. p. and m. jay. 1988a. distribution of anthocyanins in wild and cultivated banana varieties. phytochemistry. 27, 2667-2672. horry, j. p. and m. jay. 1988b. an evolutionary background of bananas as deduced from flavonoids diversification. in: (jarret, r. l. ed.). identification of genetic diversity in the genus musa, inibap. montpellier, france, p. 158-165. shepherd, k. 1988. observations on musa taxonomy. in: (jarret, r. l. ed.). identification of genetic diversity in the genus musa, inibap. montpellier, france, p. 158-165. simmonds, n. w. 1954b. anthocyanins in bananas. nature, 173,402. simmonds, n. w. 1954c. anthocyanins in banana. ann. bot. lond. 18, 471-82. simmonds, n. w. 1955. wild bananas in malaya. malayan nature journal. 10, 1-8. simmonds, n. w. 1962. the evolution of the bananas. london. uk. longman. 38 3. imam (a species).cdr biotropia vol. 19 no. 1, 2012: 19 29 a species-specific pcr assay based on the internal transcribed spacer (its) regions for identification of , and on banana mycosphaerella eumusae m. fijiensis m. musicola iman hidayat recipient of biotrop research grant 2010/accepted 28 june 2012 a study on development of a rapid pcr-based detection method based on its region of , , and on banana was carried out. the main objecive of this study was to develop a fast and species-specific pcr-based detection method for the presence of species on banana. the methods include collection of specimens, morphological identification supported by molecular phylogenetic analysis, rflp analysis, species-specific primers development, and validation. two species of , namely, and , and one unidentified species were found in java island. three restriction enzymes used in the rflp analysis, viz, alui, haeiii, and taqi were capable to discriminate , , and . two species-specific primer pairs, viz, mfijf/mfijr and mmusf/mmusr have been successfully developed to detect the presence of and , respectively. banana, detection, fungi, leaf spot, phytopathology microbiology division, research center for biology, indonesian institute of sciences (lipi), cibinong 16911, west java, indonesia m. eumusae m. fijiensis m. musicola mycosphaerella mycosphaerella m. fijiensis m. musicola pseudocercospora m. eumusae m. fijiensis m. musicola m. fijiensis m. musicola mycosphaerella abstract introduction key words: indonesia is one of banana production zones in southeast asia. however, crop losses from global climate change and fungal pathogens pose a serious threat not only to indonesia, but also to global food security. therefore, these threats should not be underestimated. among the banana pathogens, three morphologically similar species, viz, (black leaf streak disease/black sigatoka), (yellow sigatoka disease), and (eumusae leaf spot) are well known as important plant pathogens (crous & mourichon 2002). in indonesia, these pathogens are considered as quarantine organisms (http://www.karantina.deptan.go.id/optk/ mycosphaerella fijiensis m. musicola m. eumusae * corresponding author : imanhidayat@yahoo.com 19 detail.php?id=731). therefore, it is important to prevent introduction (entry and establishment) and to limit dissemination of these pathogens in many indonesian banana-producing regions. correct and rapid identification is a fundamental step for limiting the dissemination of the plant pathogens (arzanlou 2007). failure to manage the pathogens would have far reaching effects on the industry. the 10-14 days incubation and classical isolation of the pathogens by culturing on appropriate media followed by morphological characters examination is a standard method currently used in indonesia for the imported crops inspection. however, an accurate detection and diagnosis of the and based on the conventional method are complicated due to the similarity in morphological characters (arzanlou 2007). consequently, this problem yield difficulties for indonesian quarantine in inspecting imported banana seeds or crops. many pcr-detection methods for fungi have shown to be accurate and sensitive in detection various plant pathogens (bonants 1997; mumford 2006). its sequence analysis has shown that , and are only distantly related in terms of phylogeny (crous 2002). however, the phylogeny method was still time consuming and lacked specificity to differentiate among the , and (arzanlou 2007). the lack of specificity was possibly due to the high variability among those three pathogens. therefore, it is necessary to develop a fast and specific pcr-based detection method with the aim of improving the specificity of the diagnostic procedure and increasing throughout readiness for outbreaks of the disease. fungal materials were collected from several locations in bogor and cibinong (west java), and one specimen was collected from wonosobo (central java). specimens with black leaf streak diseases symptoms of were collected during the course of field trips by using a 10×/20× magnifying lens. specimens were kept in resealable plastic bag. the bags were labelled by adding all necessary information such as location, collector/s, collection date, host name, etc. microscopic examination of materials was referred to hidayat . (2007). ascomata appearances of spp. and caespituli of anamorphic states ( spp.) on the host surface were observed by using stereo microscope (olympus szx7). detailed observations of morphological characters was carried out by means of an olympus cx31 light microscope using oil immersion (1000×). water and lactophenol were used as mounting media. measurements of all important characters and photographing/line drawings were conducted at a magnification of 1000×. single spore isolation was referred to choi . (1999). voucher specimens were deposited at the herbarium bogoriense, research center for biology, indonesian institute of sciences-lipi, cibinong, west java, indonesia. living cultures were deposited at the et al. m. fijiensis, m. musicola, m. eumusae et al. et al. et al. m. fijiensis, m. musicola m. eumusae et al. m. fijiensis, m. musicola m. eumusae et al. m. fijiensis et al mycosphaerella pseudocercospora et al materials and method fungal materials biotropia vol. 19 no. 1, 2012 20 lipimc microbial culture collection, microbiology division, research center for biology, indonesian institute of sciences-lipi, cibinong, west java, indonesia. fungal species found in this study were compared to isolate of the , , and obtained from cbs culture collection (table 1). dna from fungal cultures was extracted using cetyltrimethylammonium bromide (ctab) protocols (rogers & bendich 1994). the primers its1 (5'-gaagtaaaag tcgtaacaag-3') and its4 (5'cctccgcttattgatatgc-3') (white . 1990) were used to amplify the its area. the pcr of 10×concentrated reaction buffer containing 1.5mm mgcl taq dna polymerase (5 water . the pcr reaction was performed as follows: 1 cycle of 5 min at 94ºc followed by 40 cycles of 30s at 94ºc, 30s at 52ºc, and 30s at 72ºc. one cycle of 7 min at 72ºc was conducted. after amplification, 5 l of the reaction mixture was loaded onto a 1.0% agarose gel in 0.5×tbe buffer, separated by electrophoresis, stained with ethidium bromide, and viewed and photographed under uv light. a negative control (no dna target) was included in every experiment to test for contamination, as well as a positive control (dna from a reference strain of the pathogen). the amplicons was sequenced in both directions using the pcr primers and a dyenamic et terminator cycle sequencing kit (amersham, biosciences) according to the manufacturer's recommendations. the products were analyzed on an abi prism 3700 dna sequencer (perkin-elmer, foster city, ca). a consensus sequences were computed from the forward and reverse sequences with seqman from the lasergene package (dnastar, madison, wi). the sequences obtained from the respective primers (its5 and its4) were aligned in clustal x (thomson . 1997) and bioedit (hall 1999). phylogenetic analysis was performed in paup* (swofford 2002). ambiguously aligned sites were excluded from all analyses. unweighted parsimony (up) analysis were performed. gaps were treated as missing data. maximum parsimony analysis was performed for all data sets using the heuristic search option with 1000 random taxa additions and tree bisection and reconstruction as the branch-swapping algorithm. branches of zero length was collapsed and all multiple, equally parsimonious trees were saved. the robustness of the trees obtained was evaluated by 1,000 bootstrap replications. other measures calculated include tree length, consistency index, retention index, and rescaled consistency index (tl, ci, ri, and rc, respectively). the resulting phylogenetic tree was printed with treeview version 1.6.6 (page 1996). restriction digestion of pcr products was conducted directly without further purification with restriction endonucleases to obtain rflps; each sample was digested m. eumusae m. fijiensis m. musicola et al et al dna extraction and sequencing sequence alignment and phylogenetic analysis restriction fragment length polymorphism (rflp) analysis reaction mixture contained 5μl dna suspension; 2.5μl ; 2.5μl 600μmdntps; 0.25μl of each primer at 60μm; 0.2μl u/μl); 0.25μl internal control, and was filled up with milliq to a final volume of 25μl μ 2 pcr assay based on the its regions for identification of species on banana iman hidayatmycosphaerella 21 biotropia vol. 19 no. 1, 2012 with i, iii, i, or i in single enzyme digests. per each 20ml restriction digest, 10 ml of unpurified, amplified pcr reaction was mixed with the appropriate restriction reaction buffer and 10 u of the appropriate enzyme and then incubated for 6h at 37°c for the i, iii, or i digests or at 65°c for the i digests. restriction fragments were separated by electrophoresis in 2% (wt/vol) and 2.5% (wt/vol) sepharide gel matrix in 1× tae (40mm tris acetate, 1mm sodium edta) with etbr at 100 ng/ml in the gel and running buffer. dna bands were visualized by fluorescence under uv light and photographed. sequences obtained from its region were aligned with clustal x (thomson . 1997) dan bioedit (hall 1999). a series of species specific primers were designed using vector nti software (invitrogen, sigma-aldrich), based on sequence differences among the , , and . the robustness and specificity of various primer combinations were evaluated using dna from isolates of the , , and . dna extraction and pcr amplification of these isolates were performed as described above. alu hae taq rsa alu hae rsa taq et al m. fijiensis m. musicola m. eumusae m. fijiensis m. musicola m. eumusae development of specific pcr primers table 1. list of and obtained in this study.mycosphaerella pseudocercospora no. name origin culture collection number 1 mycosphaerella musicola (mycosphaerella sp.1) cibinong, west java, indonesia lipimc 0598 2 mycosphaerella fijiensis (mycosphaerella sp.2) cibalagung, west java, indonesia lipimc 0599 3 mycosphaerella musicola (mycosphaerella sp.3) wonosobo, central java, indonesia lipimc 0600 4 mycosphaerella eumusae unknown cbs 114825 5 mycosphaerella fijiensis cameroon cbs 120258 6 mycosphaerella musicola cuba cbs 116634 7 pseudocercospora sp. cibinong, west java, indonesia lipimc 0601 results and discussions fungal materials and phylogenetic analysis three isolates of and one isolate of were isolated from specimens collected. the cultures of species collected in this study were compared morphologically to the three species from banana obtained from cbs culture collection (netherlands). all isolates are listed in table 1. blast result from ncbi genbank database showed that sequences of sp.1 has 100% similarity to the (ay646445) (fig. 1), and sp.2 has 99% similarity to the (gq169763) (fig. 2). mycosphaerella pseudocercospora mycosphaerella mycosphaerella mycosphaerella m. musicola mycosphaerella m. fijiensis 22 the alignment data matrix of newly its sequences of three species and one speg. species from banana were aligned with sequences of , fresen., and fr. retrieved from ncbi genbank dna database. the alignment consists of 47 taxa including sequences of (fresen.) g.a. de vries and (de not.) crous & u. braun as outgroup. the data matrix yielded 510 total characters included in the analysis of which 320 characters were constant, 27 characters were variable and parsimony-uninformative and 137 characters were parsimony-informative. twentysix of the informative characters which were positioned within small insertion/deletions or ambiguous regions were excluded from the analysis. two maximum parsimonious trees were generated from the analysis. sum of minimum possible lengths is 232, and sum of maximum possible length was 1091. the best parsimonious tree selected by using kh test was generated in 360 steps (ci = 0. 644, ri = 0. 851, rc = 0.548, hi = 0. 356). the best phylogenetic trees obtained from unweighted maximum parsimony analysis is shown in figure 3 mycosphaerella pseudocercospora mycosphaerella cercospora pseudocercospora passalora cladosporium cladosporioides davidiella tassiana . figure 1. pairwise alignment showed 100% similarity between sp.1 and . (ay646445). mycosphaerella m musicola based on this analysis, three major clades were performed. these included clade with anamorph (clade i) with 61% bootstrap support. another clade was and anamorph (clade ii, 54% bootstrap support) which is also sister clade to clade with 61% bootstrap support. the last clade is and anamorph (clade iii) with 90% bootstrap support. the sp.1 from this study nested together with species in the clade with 100% bootstrap support. another species from this study, sp.3 nested together with species within clade with 92% bootstrap support. this information confirms the mycosphaerella pseudocercospora mycosphaerella cercospora mycosphaerella pseudocercospora mycosphaerella passalora mycosphaerella m. musicola mycosphaerella mycosphaerella m. fijiensis 23 pcr assay based on the its regions for identification of species on banana iman hidayatmycosphaerella biotropia vol. 19 no. 1, 2012 species name of sp.1 as , and sp.3 as . furthermore, this finding confirms that and exist in indonesian banana plantation (java). another isolate, sp.1 needs more detailed examination as this species does not form monophyletic group with any clades in the phylogenetic tree all species from banana, viz, , , and form a monophyletic clade with 52% bootstrap support. this finding has shown that species from banana is a distinct group of species among the species from various hosts. it has also indicated that the three species of from banana are host specific to the banana trees. further analysis such as pathogenicity test is necessary to carry out in order to justify the specificity of the three species from banana. mycosphaerella m. musicola mycosphaerella m. fijienssis m. fijiensis m. musicola pseudocercospora mycosphaerella m. eumusae m. fijiensis m musicola mycosphaerella mycosphaerella mycosphaerella mycosphaerella . figure 2. pairwise alignment showed two nucleotides differences between sp.2 and . (gq169763) (boxes). mycosphaerella m fijiensis restriction fragment length polymorphism ( ) analysisrflp polymorphism of fragment size of its regions was recognized as reported previously in other fungal group (gardes & bruns 1993; sreenivasadprasad 1996), and it was thought to be variable in the sequences of its region because of nucleotide deletions and insertions. in order to identify the species detected by pcr using primers its5 and its4, the rflps of the its region were generated using four restriction enzymes, namely, i, iii, i, or i. from the analysis, only i was not very useful because it did not cut the amplicon of all species et al. mycosphaerella alu hae taq rsa rsa mycosphaerella 24 from banana (fig. 4a). other restriction enzymes, namely, i, iii, and i, generated more fragment per digest, so that the its sequences of the , , and could be separated using each rflps profile. the iii had two recognition sites in the fragments of , and had three recognition sites in the fragments of and (fig. 4b). one isolate of also be separated from three other isolates of using the iii restriction enzyme because it only had two recognition sites in the fragments. a similar result was also found in the restriction fragments of generated by i and (fig. 4c-d). the majority of rflps profiles generated from iii were unique for each species. for , the given enzymes ( i and i) probably generated rflps profiles which separated isolates at the subspecies level (fig. 4c-d), but further analysis will be required to justify this result. alu hae taq mycosphaerella fijiensis m. musicola m. eumusae hae m. eumusae m. musicola m. fijiensis m. musicola m. musicola hae m. musicola alu taqi hae mycosphaerella m. musicola alu taq can figure 3. single parsimonious tree based on its nrdna sequence data representing placement of spp. and sp.1 found in this study within representatives of the family . the tree is obtained from heuristic search with 1000 random taxon addition of the sequences alignment. bootstrap values (>50%) from 1000 replicates of unweighted maximum parsimony (ump) analyses are shown above internodes. mycosphaerella pseudocercospora mycosphaerellaceae 25 pcr assay based on the its regions for identification of species on banana iman hidayatmycosphaerella biotropia vol. 19 no. 1, 2012 development of specific pcr primers understanding the banana sigatoka disease complex is a challenge for plant pathologists (arzanlou 2007). therefore, in this study we developed rapid and specific detection method with the feasibility of wide application. even though a pcr-based detection tool has been developed previously (johanson & jeger 1993), those primers could only differentiate from three speciesspecific primers were designed, namely, meuf/meur, mfijf/mfijr, and mmusf/mmusr, respectively (table 2). all primers were designed to operate at relatively high annealing temperatures (54°c-55°c), thereby preventing the coamplification of non-specific dna targets. primer sequences were compared against existing sequences in ncbi genbank data base (http://www.ncbi.nlm.nih.gov/) and ddbj dna data base of japan (http://www.ddbj.nig.ac.jp/), and a result of blast (basic local alignment search tool) showed 100% homology of the primers with sequences of strains belonging to the species of which primers were designed. single bands of correct size were obtained with species-specific primers from all strains belonging to the three species from banana. et al. m. fijiensis m. musicola. mycosphaerella table 2. primer pairs designed in this study. no. name species target notes 1 meuf (forward) (5’-catctttgcgtcggagttca-3’) mycosphaerella eumusae not species-specific (cross reactions with m. fijiensis )meur (reverse) (5’-ccgaagcgaattgaagaatcc-3’) 2 mfijf (forward) (5’-tctttgcgtcggagtttca-3’) mycosphaerella fijiensis species-specific mfijr (reverse) (5’-tccgaagcgaattgaaagatc-3’) 3 mmusf (forward) (5’-tccttaacactgcatctctacg-3’) mycosphaerella musicola species specific mmusr (reverse) (5’-tcagccgggagactttgg -3’) m. eumusae m. fijiensis m. musicola m. fijiensis m. musicola mycosphaerella m. eumusae m. fijiensis m. eumusae m. fijiensis m. fijiensis m. musicola validation assay on pure cultures of , , and showed that primer pairs of (mfijf/mfijr) (fig. 5a) and (mmusf/mmusr) (fig. 5b) are specific to the fungal pathogens as no cross-reactions with others species were observed in the amplification bands. unfortunately, primer pairs of (meuf/meur) failed to show specificity as cross-reactions were found with sequences of (fig. 5c). it is probably due to small nucleotide differences between dna sequences and from its region. the specificity of primer pairs of mfijf/mfijr to and mmusf/mmusr to is good indication for the development of molecular diagnosis technique and understanding of the sigatoka diseases complex of banana in indonesia. the molecular technique developed in this study may also significantly contribute to plant quarantine because of its reliability, specificity and simplicity. this assay could be done within 1-2 days laboratory works and analysis. 26 figure 4. restriction patterns of internal transcribed spacer (its) regions of ribosomal dna amplified from three species from banana. a. i b. iii c. i d. i (1-3: ; 4-7: ; 8-9: ). mycosphaerella rsa hae alu taq m. eumusae m. musicola m. fijiensis 27 pcr assay based on the its regions for identification of species on banana iman hidayatmycosphaerella figure 5. species-specific amplification of species from banana using specific primers. a. primer pairs mfijf/mfijr to the b. primer pairs mmusf/mmusr to the c. primer pairs meuf/meur to the and (2-4: ; 5-8: ; 9-10: ). mycosphaerella m. fijiensis m. musicola m. eumusae m. fijiensis m. eumusae m. musicola m. fijiensis biotropia vol. 19 no. 1, 2012 28 conclusions diagnosis of the banana sigatoka disease complex is a challenge for plant pathologists. rflp analysis using iii is capable in discriminating the and the rapid and specific pcr-based detection method using species-specific primers of mfijf/mfijr and mmusf/mmusr has been successfully developed to detect and , respectively, from pure cultures. further examination/validation directly on samples from infected banana leaves with diseases symptom are necessary to test the sensitiveness of this method this research was funded by seameo biotrop awarded to dr. iman hidayat. laboratory of plant molecular systematics, botanical division, research center for biology (lipi) is acknowledged for providing the authors with molecular facilities. the author also thanks sulistiani and aerma hastuty for laboratory assistance. hae restriction enzyme m. eumusae, m. fijiensis, m. musicola. m. fijiensis m. musicola mycosphaerella . acknowledgments references arzanlou m, abeln eca, kema ghj, waalwijk c, carlier j, de vries i, guzmán m, crous pw. 2007. molecular diagnostics for the sigatoka disease complex of banana. phytopathology 97: 1112-8. bonants p, hagenaar-deweerdt m, van gent-pelzer m, lacourt i, cooke d, duncan j. 1997. detection and identification of hickman by the polymerase chain reaction. eur j plant pathol 103: 345-55. choi yw, hyde kd, ho wh. 1999. single spore isolation of fungi. fungal divers 3: 29-38. crous pw, mourichon x. 2002. and its anamorph spp. nov.: causal agent of eumusae leaf spot disease of banana. sydowia 54: 35-43. crous pw, groenewald jz, aptroot a, braun u, mourichon x, carlier j. 2002. interacting morphological and molecular data sets on with specific reference to species occurring on . in: jacome l, lepoivre p, marin d, romero r, escalant jv, editors. proceedings of the 2 international workshop on leaf spot diseases. san jose, costa rica. p 43-57. gardes m, bruns td. 1993. its primers with enhanced specificity for basidiomycetes application to the identification of mycorrhizae and rusts. mol ecol 2: 113-8. hall ta. 1999. bioedit: a user-friendly biological sequence alignment editor and analyses program for windows 95/98/nt. nucleic acids symposium series 41: 95-98. hidayat i, meeboon j, to-anun c. 2007. and species (xylariaceae) from palms including sp. nov. 102: 347-54. johanson a, jeger mj. 1993. use of pcr for detection of and , the causal agents of sigatoka leaf spot in banana and plantain. mycol res 97: 670-74. mumford r, boonham n, tomlinson j, barker i. 2006. advances in molecular phytodiagnostics new solutions for old problems. eur j plant pathol 116: 1-19. page rdm. 1996. treeview, an application to display phylogenetic trees on personal computers. comput appl biosci 12: 357-58. phytophthora fragariae mycosphaerella eumusae pseudocercospora eumusae mycosphaerella musa mycosphaerella anthostomella fasciatispora f. ujungkulonensis mycotaxon mycosphaerella fijiensis m. musicola nd 29 pcr assay based on the its regions for identification of species on banana iman hidayatmycosphaerella rogers so, bendich aj. 1994. extraction of total cellular dna from plant,algal, and fungal tissues. pp. dl. 1-8. in : gelvin s, schilperoort ra, editor. plant molecular biology manual. 3 edition. kluwer academic publishers, boston. massachusetts, usa. sreenivasadprasad s, mills pr, meehan bm, brown ae. 1996. phylogeny and systematics of 18 species based on ribosomal dna spacer sequences. genome 39: 499-512. swofford dl. 2003. paup*: phylogenetic analyses using parsimony (* and other methods), version 4.0b 10. sinauer associates, sunderland, massachusetts. thomson jd, gibson tj, plewniak f, jeanmougin f, higgins dg. 1997. the clustal_x windows interface: flexible strategies for multiple sequences alignment aided by quality analyses tools. nucleic acids res 25: 4876-82. hite tj, burns t, lee s, taylor j. 1990. amplification and direct sequencing of fungal ribosomal rna genes for phylogenetics. in: innis ma, gelfand dh, sninsky jj, white tj, editors. pcr protocols: a guide to methods and application. academic press, san diego. p 315-22 rd colletotrichum w . 65 white oyster mushroom (pleurotus florida) mutant – i. djajanegara & harsoyo white oyster mushroom (pleurotus florida) mutant with altered antioxidant contents ira djajanegara1* and harsoyo2 1center of bioindustry bppt indonesia, laptiab puspitek serpong, tangerang 15314, banten, indonesia 2batan (indonesian atomic agency), patir-batan jakarta, indonesia abstract radiation using gamma ray (60co) at 0.75 kgray with dose velocity of 1.149 kgray/ hour on white oyster mushroom (pleurotus florida) mycelia yielded several mutants. based on isozyme analysis using two enzyme markers such as esterase (est) and acid phosphatase (acp) showed that 3 putative mutants (po-3, po-4 and po-5) among 5 mutants are positive. even though the isozyme patterns indicated that those 3 putative mutants are positively mutated, only po-5 showed higher productivity compared to control (po-k) which is reflected by significantly higher number of fruit bodies, higher fresh weight and dry weight yield of three successive flush periods. it was assumed that the mutation which occurred in po-3 and po-4 may affect other trait(s) of the white oyster mushroom. antioxidant analysis of those mutants indicate that mutant po-4 has significantly higher antioxidant content compared to control (po-k) and the two other mutants (po-3 & po-5). this finding leads to the possible application of white oyster mushroom as a natural antioxidant source. key words : mutant, white oyster mushroom, gamma ray, production, isozyme introduction one way to introduce genetic variability is through mutation using chemical agents or ionizing radiation. mutation may induce one or more change in characteristic of the mutated organisms. the characteristic changes may occur at the gene level which will be passed to the next generation (carlile & watkinson 1994). mutation is applied to edible mushrooms to obtain better quality and productivity. the desired mutant characteristic can be economically beneficial for example resistant to pathogen, higher yield, etc. there are two types of mutation based on the site in nucleus namely point mutation and chromosomal mutation. mutation can alter the coding region or noncoding region. mutation at coding region of a gene will cause the cell to experience silent, missense, frameshift and nonsense mutations. mutation altering the non-coding *corresponding author : idjajanegara@yahoo.com biotropia vol. 15 no. 1, 2008 : 65 73 66 biotropia vol. 15 no. 1, 2008 region will cause different protein production or have no effect on mrna maturation. other types of mutation are null and leaky mutations. these two mutations occurred at the active site of a protein (griffiths et al. 2000) gamma radiation is an ionizing radiation often applied to eukaryotic organisms. according to esser (1971), gamma radiation is an effective ionizing radiation due to its ability to penetrate cell walls of mushroom mycelia. gamma rays has higher energy which makes this type of radiation better penetrates into the target cells. several sources of gamma rays are cobalt-60, cesium-137 and technetium-99 (busby 2003). on eukaryotic cells, gamma radiation is able to break dna molecules and change the purine and pirimidine bases of the target dna (kaiser 2001). in several microbiology studies, mutation that produced higher metabolites are mostly obtained by radioisotope mutagenesis (slater 2000). ionizing radiation has been a common practice for extending storage life in mushrooms especially champignon. radiation at 1.5 – 3 kgy on champignon fruit bodies will increase storage life from 8 weeks to 12 weeks (maha & pangerteni 1989). ionizing radiation has also been used to generate mutants in several edible mushrooms. elliot (1982) used ultra violet (uv) radiation to produce an agaricus bisporus mutant strain that is resistant to fungicide. in this case, there is a possibility to produce mutant of white oyster mushroom (pleurotus florida) that has higher productivity through ionizing radiation treatment. white oyster mushroom (pleurotus florida) is an edible mushroom that gained popularity lately due to its nutritional values and ease of cultivation. this mushroom contains higher amount of protein, lipid, phosporous, iron, thiamine and riboflavin compared to other edible mushrooms. in addition, this mushroom also contains 18 essential amino acids such as isoleucine, lysine, methionine, cysteine, phenylalanine, tyrosine, tryptophan, valine, arginine, histidine, alanine, aspartic acid, glutamic acid, glycine, proline and serine (djarijah & djarijah 2001). according to bobek et al. (1989), oyster mushroom also rich in antioxidant makes this type of edible mushroom as an excellent source of natural antioxidant. the demand for natural antioxidant sources by food industries increased in the last decade due to the latest findings that indicated the harmful effect of the commonly used synthetic antioxidants such as bha (butyl hydroxy anisole) and bht (butyl hydroxy toluene). bht and bha are correlated to the damage of liver (namiki & osawa 1981). however, the antioxidant content of natural resources is very low. therefore, it will be beneficial to obtain a white oyster mushroom strain that produces higher amount of antioxidant to fulfill the food industry demand. one step to overcome this problem is to apply radiation in the form of gamma rays (60co) to create mutants with commercial characteristics such as higher antioxidant content and many others. this experiment is intended to apply gamma radiation to white oyster mushroom to obtain genetic variability which may lead to commercially superior white oyster mushroom strains. 67 white oyster mushroom (pleurotus florida) mutant – i. djajanegara & harsoyo materials and methods the mutation material used in this experiment was white oyster mushroom (pleurotus florida) local strain called tp. media used for growing the mycelia were pda (potato dextrose agar) and pdb (potato dextrose broth). all the mycelia were grown in pda and pdb media at room temperature (± 28oc). differences in mycelia diameter were observed on mycelia grown in pda media while mycelia samples for isozyme analysis were grown in pdb media. mutant and control were grown on substrates composed of saw dust (76.3%), rice husks (18.5%), chalk (2.96%) and gypsum (1.27%) as sugested by chang & miles (1989) for productivity observation. chemicals used in this experiment were chloramphenicol as an antibiotic which was added to media while ethanol 70% and 96% were used for sterilization. chemicals used for isozyme analysis were liquid n, potato starch, ascorbic acid, cysteine, tritonx-100, pvp-40, na2hpo4.2h2o, histidine monohydrate, citric acid monohydrate, tris hydoxylmethyl aminomethane, sodium phosphate, 1-naphtyl acetate, 2-naphtyl acetate, fast blue rr salt, sodium acetate, cacl2, h2o2 3%, 3-amino-9-ethylcarbasol, bromophenol blue, nad, malic acid, nbt, pms, tris-hcl, na-1-naphtyl phosphate acid, fast garnet gbg salt and aquadest. approximately 0.5 x 0.5 cm2 mycelia grown on pda media were transferred to the fresh pda media for radiation and some were reserved for stock. the same amount of mycelia (with and without radiation) were transferred to the pdb media for isozyme analysis. all the transfer and multiplication activity were done in the laminar air flow cabinet according to djarijah & djarijah (2001). radiation procedure was done according to esser (1971) with a radiation dose of 0.75 kgray using radiation velocity at 1.149 kgy/hour. radiation was applied to mycelia grown on pda media at exponential phase which is approximately 10 days after inoculation (djajanegara et al. 2004). growth curve of the white oyster mushroom (pleurotus florida ) used in this experiment was based on previous experiment by djajanegara et. al. (2004). isozyme analysis using acid esterase and acid phosphatase were done according to wendel & weeden (1989). antioxidant analysis was carried out by dpph method (yamaguchi et al. 1998) using bha (butyl hydroxy anisole) as a standard. results and discussion gamma rays radiation was done on mycelia culture occupying approximately a quarter of the agar plate (10 days after inoculation). at 12 days after inoculation, the mycelia of white oyster mushroom were in the exponential growth period (djajanegara et al. 2004). just like any other edible mushroom, the white oyster mushroom has an extensive growth pattern as long as substrate is available. mushroom cells are composed of vesicles excreting many types of ezymes and polymers to support the growth at the tip of hyphae. hyphae cells at the tip of mycelium have high metabolism rate. division on 68 biotropia vol. 15 no. 1, 2008 those cells are extensive to produce new nuclei for the newly formed cell compartments (alexopoulus et al.1996). radiation application on those cells during that stage will be expected to produce maximum effect due to maximum radio-sensitivity of the highly metabolic rate of the undifferentiated cells (prassad 1999). there are a number of publications on plant improvement using mutation. this is in contrast with strain improvement in cultivated mushroom especially the oyster mushroom which cultivation has been recently introduced. in order to verify the mutation, isozyme analysis was conducted. isozyme which is also known as multiple molecular forms of enzymes is defined as enzymes that share a common substrate but differ in electrophoretic mobility (markert & moller 1959). they are revealed when tissues extracts are subjected to electrophoresis in various types of gels and subsequently submersed in solutions containing enzyme-specific stains this type of genetic analysis may indicate that some of the variant electromorphs are encoded by alternate alleles at a single locus, in which case the allelic products are termed allozymes (prakash et al. 1969). the data in isozyme analysis show number and relative mobilities of various enzyme products when combined with appropriate genetic analysis become transformed into single or multilocus genotypes for each analyzed individual. notes : 1) control (po-k), 2) po-1, 3) po-2, 4) po-3, 5) po-4, 6) po-5 manifestations of mutation range from changes in intensity or relative intensity of bands to appearance or disappearance of bands (tyson et al.1985). in this experiment, enzyme specific stains used are acid phosphatase (acp) and esterase (est) (figures 1 & 2). according to de charisey (1985), subcellular localization of acp is various in the cell with 2 – 4 numbers of isozymes. on the other hand, subcellular localization of est is at the cytosol with 2 – 10 numbers of isozymes (wehling & schmidt-stohn 1984). isozyme analysis using those two enzyme markers will cover any mutation which occurs in the cell. + 1 2 3 4 5 6 figure 1. starch gel of pleurotus sp. assayed for acid phosphatase activity. 69 white oyster mushroom (pleurotus florida) mutant – i. djajanegara & harsoyo notes : 1) control (po-k), 2) po-1, 3) po-2, 4) po-3, 5) po-4, 6) po-5 isozyme analysis using acid phosphatase (acp) as enzyme marker in this experiment showed 2 4 isozymes (charisey 1985). in this experiment, we observed 3 bands in the control of wildtype white oyster mushroom using acid phosphatase (acp) as enzyme marker which is in agreement with the reference. those 3 bands constantly appeared in the zymogram of the wild-type white oyster mushroom sample as repeated 4 times (data not shown). there was no difference in the isozyme pattern of putative mutants po-1 and po-2 compared to control (po-k). however, there was 1 additional band present in putative mutant po-3 and po-4 compared to control , while in the putative mutant po-5 the top band that was present in control was not observed. based on this analysis the mutation occurred in po-3, po-4 and po-5. in order to be certain with the mutants, another isozyme analysis using different enzyme marker was done (fig. 2). isozyme analysis using esterase (est) specific stain was chosen to verify the gamma ray mutation. zymogram pattern of enzyme esterase (est) revealed 2 – 3 bands which is in agreement with the observation reported by wehling & scmidt-stohn (1984). there was no difference between putative mutant po-1 and control (po-k). however, there was a different isozyme pattern between control and putative mutants po-3 & po-4 in which the relative migration of the top band was shifted. in the putative mutant po-2 and po-5, additional band was observed when the pattern was compared with control (po-k). interestingly, isozyme analysis using acid phosphatase enzyme marker on the putative mutant po-2 did not show any mutation event but when the same sample was analyzed using esterase enzyme marker mutation event was observed. differences of the enzyme marker systems such as sub-cellular localization may contribute to this observation. zymogram pattern of esterase (est) revealed that mutation occurred in po-2, po-3, po-4 and po-5. figure 2. starch gel of pleurotus sp. assayed for esterase activity. 70 biotropia vol. 15 no. 1, 2008 however, based on 2 zymogram patterns of esterase (est) and acid phosphatase (acp), it could be concluded that the mutants are po-3, po-4 and po-5. the manifestation of the mutation was investigated further by growth and morphological observations. there are no morphological differences between the mutants (po-3, po-4 and po-5) and control (po-k) in term of the shape and color of the fruit bodies. data on productivity were reflected by amount of fruit bodies/bag log, diameter of fruit bodies, fresh weight (g) harvest/1 kg media (1 bag log) and fresh weight (g) of harvest/1 kg media (1 bag log). all the bag logs were treated equally and opened for aeration at the same time. harvests were conducted at 3 successive flushing periods. based on productivity observation, only mutant po-5 showed significantly higher productivity compared to control (po-k) (table 1). biological efficiency ration (ber) of mutant po-5 which is reflected by fresh weight of harvest/weight of media was higher than 10% for each harvest. the ber observed for po-5 mutant was 21% (10 weeks post-inoculation), 14% (12 weeks post-inoculation) and 10.5% (14 weeks post-inoculation), while for the wild type were 10% (10 weeks post-inoculation), 8% (12 weeks post-inoculation) and 6.5% (14 weeks post-inoculation, respectively. according to aryantha & rachmat (1999), by reaching biological ratio efficiency at least 10%, the wood mushroom cultivation will be categorized as economically feasible. the mutant po-5 also showed faster flushing period (4 days after aeration) compared to control (2 weeks after aeration). in this case, mutant po-5 is a very useful strain to increase production. table 1. growth performance of the white oyster mutants (po-3, po-4 & po-5) compared to control (po-k) types of white oyster mushroom number of fruit bodies/bag log diameter of fruit bodies (cm) fresh weight harvest (g)/ 1 kg media (1 bag log) dry weight harvest (g) / 1 kg media (1 bag log) 10 wpi 12 wpi 14 wpi 10 wpi 12 wpi 14 wpi 10 wpi 12 wpi 14 wpi 10 wpi 12 wpi 14 wpi control (po-k) 28a 26a 12a 7.5a 6.8a 7a 100a 80a 65a 13a 10.4a 8.5a mutant po-3 27a 26a 11a 8a 7a 7a 98a 78a 65a 12.7a 10.1a 8.5a mutant po-4 28a 27a 12a 7.1a 6.5a 6.8a 97a 77a 67a 12.6a 10a 8.7a mutant po-5 50b 42b 28b 9ab 9.6ab 9.5ab 210b 140b 105b 27.3b 18.2b 13.7b notes : wpi = weeks postinoculation to the bag log, a & ab = growth performance was not significantly different between genotypes based on t-test (p>0.05), b = significantly different (p<0.05) 71 white oyster mushroom (pleurotus florida) mutant – i. djajanegara & harsoyo further analysis on the mutants (po-3, po-4 and po-5) showed that mutant po-4 has significantly higher antioxidant content in the fruit body compared to control (po-k) and other mutants (po-3 and po-5). antioxidant activity of the mutant po-4 is equal to antioxidant activity of 47.627 mg/g bha (table 2). higher antioxidant activity was always correlated with higher antioxidant content (namiki & osawa 1981). as a comparison, the antioxidant content of soymilk is equivalent to 29.5mg bha/g while fermented soybean is equivalent to 72.8 mg bha/g (shahidi 1997). table 2. antioxidant content of the white oyster mutants (po-3, po-4 & po-5) compared to control (po-k) types of white oyster mushroom antioxidant content (equivalent to mg/g bha) white oyster mushroom control (po-k) 17.184a white oyster mushroom mutant po-3 16.945a white oyster mushroom mutant po-5 16.772a white oyster mushroom mutant po-4 47.627b notes: a & ab = antioxidant content was not significantly different between genotypes based on t-test (p>0.05) b = significantly different (p<0.05) several diseases are caused by strong oxidants such as cardiovascular disease, cancer and many others which can be inhibited by the use of antioxidants. most of the damaging actions of oxidants are from reactive oxygen species (ros) such as free radicals. free radicals can be produced by dust, pollution, or as a side product of metabolism. antioxidant is a chemical that is able to react with oxidant to inhibit the oxidation of bio-molecules (langseth 1995). in addition, antioxidant is also required in food industry as a substance that is able to prevent lipid oxidation which leads to decay of food. lipid oxidation is the first step for other changes in food that will have impact on nutrition value, food safety, color, flavor and texture of food (shahidi 1997). in this case, the mutant po-4 has a potential to be used commercially in the food industry as a natural antioxidant source. conclusions mutation using gamma rays (60co) on white oyster mushroom (pleurotus florida) yielded 4 putative mutants which are po-2, po-3, po-4 and po-5. isozyme analysis using acid phosphatase (acp) and esterase (est) as marker enzymes confirmed that mutation occurred at putative mutants po-3, po-4 and po-5. there were no differences in the morphology of the mycelia and fruit bodies between those mutants and control. observation on mutants po-3, po-4 and po-5 showed that the productivity of po-5 was significantly higher compared to control and 72 biotropia vol. 15 no. 1, 2008 the two other mutants (po-3 and po-4). antioxidant analysis showed that mutant po-4 has a significantly higher antioxidant content compared to control (po-k) and the two other mutants (po-3 and po-5). this leads to the possible application of this particular white oyster mushroom (pleurotus florida) mutant as a source for natural antioxidant which is beneficial for medical and food industry. references alexopoulus, c. j., c. w. mims & m. blackwell. 1996. introductory mycology. 4 th ed. john wiley & sons, inc., new york. aryantha, i. n. p. & b. rachmat. 1999. dasar-dasar usaha budidaya jamur. bio agro lestari publ., bandung. bobek et. al. 1991. effect of mushroom pleurotus ostreatus and isolated fungal polysaccharide on serum and liver lipids in syrian hamsters with yperlipidemia. nutrition 7:105-108. bueche, f. & d. l. wallach. 1994. technical physics. 4 th ed. john wley & sons, inc., new york. busby, b. 2003. radiation & radioactivity. http://www.physics.isu.edu/radinf.htm. accessed on 6 april 2003. carlile, m. j. & s. c. watkinson. 1994. the fungi. academic press, london. chang, s. t. & p. g. miles. 1989. edible jamurs and their cultivation. crc press, florida. djajanegara, i., lestari, r., harsoyo & p. wahyudi. 2004. penelitian pendahuluan mutasi dengan sinar gamma (co60) untuk meningkatkan kandungan metabolit sekunder dan analisis isozyme pada 3 varietas jaur tiram (pleurotus sp.). jurnal ilmu kefarmasian indonesia 2(1):10 17 djarijah, n. m. & a. b. djarijah. 2001. budidaya jamur tiram. penerbit kanisius, yogyakarta. de charesey, h., m. t. barreneche, m. jusuf. c. ouin and j. pernes. 1985. inheritance of some marker genes in setaria italica. theor. appl. genet. 71:57-60. elliot, t. j. 1982. genetic and breeding of cultivated jamurs in tropical jamur (ed.) s. t. chang & t. h. quimio. the chinese university press, hongkong. 35-67. elliot, t. j. & f. a. langton. 1981. strain improvement in the cultivated mushroom agaricus bisporus. euphytica 30:175-182. esser, k. 1971. application & importance of fungal genetics for industrial research in : radiation & radioisotopes for industrial microorganisms. iaea, viena : 83 – 91. griffiths, a. j. f., j. h. miller, d. t. suzuki, r. c. lewontin & w. m. gelbart. 2000. an introduction to genetic analysis. w. h. freeman, new york. hartana, a. pelatihan singkat teknik analisis dengan metode dan peralatan mutakhir di bidang hayati dan kimia. pusat studi ilmu hayati lembaga penelitian ipb, bogor. holme, d. j. & h. peck. 1994. analytical chemistry. 2 nd ed. longman scientific & technical, burnt mill. kaiser, g. e. 2001. mutation. http://student.ccbc.cc.md.us/biotutorial/protsyn/mutate.html accessed on 5 june 2003. langseth, l. 1995. oxidants, antioxidants and disease prevention. ilsi europe, belgium. larraya, l. m., g. perez, e. ritter, a. g. pisabarro & l. ramirez. 2000. genetic linkage map of the edible basidiomycete pleurotus ostreatus. appl. and environ. microbiol. 66(12): 5290 – 5300. maha, m. & d. s. pangerteni. 1989. pengawetan jamur merang (volvariella volvaceae) dengan kombinasi pemanasan dan iradiasi in risalah simposium iv aplikasi isotop dan radiasi, jakarta, 13-15 des. 1207-1215. markert, c. l., & f. moller. 1959. multiple forms of enzymes: tissue, ontogenic and species specific patterns. proc. natl. acad. sci. usa 45:753-763. moss, g. p. 1961. iubmb enzyme nomenclature: e. c. 1.1.1.37. http://www.chem.qmul.ac uk/iubmb/ biotropia book juni revisi 14 juli 09.indd 11 biotropia vol. 16 no. 1, 2009: 11 20 corresponding author: atriw@ipc.ac.id or aristri2003@yahoo.com cloning of a gene encoding protein belonging to abc transporter involved in bacterial magnetic particle synthesis in magnetospirillum magneticum amb-1 aris tri wahyudi department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia abstract magnetospirillum magneticum amb-1 synthesizes intracellular magnetic particles, magnetite (fe3o4), enveloped by membrane called magnetosome under micro-aerobic conditions. initial study of random transposon-based mutagenesis generated 62 nonmagnetic mutants of amb-1 in a mini-tn5 library. in order to identify a gene involved in bacterial magnetic particle (bmp) synthesis in the magnetic bacterium m. magneticum amb-1, a nonmagnetic mutant from the library designated as nma38-4, was analyzed. th e amino acid sequence deduced from the gene directly interrupted by transposon, orf4 (1482 bp), showed homology to atp binding cassette (abc) transporter of mesorhizobium loti with 62 % identity and 74 % similarity. it was strongly indicated by the occurrence of putative consensus sequence of atp-binding motifs (atpbinding protein). th e orf4 was subsequently cloned in pet-15b and the recombinant orf4histag fusion protein was heterologously expressed in escherichia coli bl21 (de3) plyss. a 55 kda protein corresponding to the orf4-histag fusion protein was obtained after purifi cation using ni-nta column. th is is the fi rst report describing a gene cluster containing gene encoding protein belonging to abc transporter organized in an operon which is involved in bmp synthesis. key words: magnetospirillum magneticum amb-1, bacterial magnetic particle (bmp), atp binding cassette (abc) transporter, transposon mutagenesis. introduction magnetospirillum magneticum amb-1 isolated from fresh water sediment (matsunaga 1991) synthesizes bacterial magnetic particles (bmps) of the iron mineral magnetite (fe3o4) enveloped by membrane called magnetosome, under microaerobic conditions. these intracellular single domain magnetic particles are aligned in chains with each individual crystal having a diameter of 50-100 nm enveloped by an organic membrane (gorby et al. 1988; matsunaga 1991). it is postulated that bmps confer sensitivity of the bacterium to the earth’s magnetic field lines for migration along oxygen and iron gradients. 12 biotropia vol. 16 no. 1, 2009 so far, only few magnetic bacterial strains can be cultivated under laboratory conditions in pure culture, these include m. magnetotacticum ms-1 (blakemore et al. 1979) and m. magneticum amb-1 (matsunaga et al. 1991), and m. gryphiswaldense (schleifer et al. 1991). these strains are usually used as model systems for the analysis of bmp synthesis. the capability of m. magneticum amb-1 to grow on agar plate and liquid medium both under microaerobic and aerobic conditions (matsunaga et al. 1992) makes the bacterium amenable for genetic manipulations to elucidate important metabolic mechanisms most especially for the complex process of bmp synthesis. since magnetic bacteria were fi rst discovered (blakemore 1975), only few genes were isolated namely reca (berson et al. 1989), arod (berson et al. 1991), and mam22 (okuda et al. 1996) from m. magneticum ms-1. in the previous studies, maga (nakamura et al. 1995a) and mms16 (okamura et al. 2001) genes were isolated from m. magneticum amb-1. th e maga gene functions for the iron transport across the bmp membrane. mutation of this gene rendered the cells defective in iron uptake. th e mms16 protein has a gtpase activity for the invagination of the cytoplasmic membrane for the formation of the bmp membrane. cells with inhibited gtpase activity showed that they produced disrupted bmps. previously, a non-magnetic mutant of m. magneticum amb-1, designated nma38-4, was generated by mini-tn5 transposon mutagenesis (wahyudi et al. 2001). in the present study, we have identifi ed a gene (orf4) involved in bmp synthesis in m. magneticum amb-1. th is gene encoding protein which belongs to abc transporter was cloned and heterologously expressed in e. coli as an orf4-histag fusion polypeptide. mutation within this gene rendered m. magneticum amb-1 was unable to synthesize bmps. th e aim of this study was to isolate and clone a gene (orf4) from m. magneticum amb-1 genome and express it in escherichia coli bl21 (de3) plyss. materials and methods bacterial strains and culture conditions escherichia coli dh5α was routinely cultured in luria broth (lb) (tryptone 5.0 g l-1, nacl 10 g l-1, yeast extract 5.0 g l-1) and e. coli bl21 (de3) plyss was cultured in lb supplemented with chloramphenicol 34 μg ml-1 at 37ºc. magnetospirillum magneticum amb-1 (atcc 700264) was cultured micro-aerobically in msgm at 25oc (blakemore et al. 1979). a nonmagnetic mutant, nma38-4 (wahyudi et al. 2001), was micro-aerobically cultured in msgm supplemented with kanamycin (5 μg ml-1). observation of nma38-4 a nonmagnetic mutant, nma38-4 cells (wahyudi et al. 2001) was cultured until logarithmic phase and cells of this phase were observed under light microscopy (olymphus bh2, tokyo, japan). samarium-cobalt magnet was moved in different directions near the glass slide to determine the cells magnetic response. 13 bacterial magnetic particle synthesis in magnetospirillum magneticum amb-1 – aris tri wahyudi. isolation of flanking dna and sequence analysis mutant dna fragments flanking transposon in this work was isolated by inverse pcr after ecorv digestion and circularization, using primers designed from the mini-tn5km1 sequence (primer 1: 5’-gta ccg agc tcg aat tc-3’ and primer 2: 5’-gat cct cta gag tcg ac-3’). the primers were directed outward from the transposon. a 1.3 kb inverse pcr product (fig. 1) was purified from the gel by gene clean iii kit (bio-101, carlsbad, ca.) and sub-cloned in pcr2.1 (ta cloning, invitrogen, usa), designed pcr2.1-38.4. this recombinant plasmid was transformed into e. coli dh5α. the recombinant plasmid was subsequently isolated from e. coli by qiaprep miniprep (qiagen, gmbh, germany) and used as a template for dna sequencing. the dna sequencing was performed using an automatic dna sequencer abi 377 (perkin elmer, usa). the dna sequence was subsequently aligned against the complete genome sequence of m. magneticum amb-1 (matsunaga et al. 2005). a computer software package, lasergene (dnastar, madison, wi) was used for dna and protein sequence analysis. the sequence was further analyzed by performing homology searches using program of blast (altchul et al. 1997) against the genbank and embl dna databases. gene cloning and expression in escherichia coli based on the sequence of orf4 (fig. 1), two oligonucleotide primers (primer p1: 5’-ggg gga cat atg agc gac gtc gtc gaa-3’ and primer p2: 5’-ggg gga tcc aaa tca cgt gtc gtc ccc cca-3’) were designed (underlined nucleotides indicate ndei and bamhi sites, respectively). the recombinant plasmid pet15b-orf4 was constructed by cloning of the pcr product amplified from orf4 into the ndei/bamhi site of expression vector pet15b (novagen, usa). to amplify orf4, a primer p1 with ndei site introduced at a start codon atg, and primer p2 with the bamhi site introduced downstream of the stop codon (tga) of the gene were used. the 50 μl pcr reaction mixture contained 100 ng m. magneticum amb-1 genomic dna, 2.5 u la taq (takara, tokyo, japan), 400 μm dntps, 2.5 mm mgcl2, and 0.2 μm of each of the two primers. the temperature program for pcr was one cycle of 3 min at 95 oc, 30 cycles of 1 min at 95 oc, 60 oc, and 72 oc, respectively, and one cycle for 10 min at 72 oc. the amplified fragment was subsequently isolated from the gel and purified by gene clean iii kit (bio 101). a 1.5 kb purified fragment was sub-cloned in pcr2.1 vector (invitrogen, usa), designed pcr2.1-orf4, and was subsequently transformed to e. coli dh5α. the recombinant plasmid was isolated and digested with ndei and bamhi. the fragment corresponding to the orf4 was ligated into pet15b expression vector (novagen, madison, wi) linierized with ndei and bamhi, to yield a recombinant plasmid, designed as pet15b-orf4 (fig. 1). this recombinant plasmid was introduced into e. coli dh5α and subsequently isolated and then transformed into e. coli bl21 (de3) plyss as a host strain for gene expression. transformants were plated on lb plate containing ampicillin (50 μg ml-1) and chloramphenicol (25 μg ml-1). 14 biotropia vol. 16 no. 1, 2009 bamh xho nde lac a orf4 (1482 pet15b (5.7 a or or lacpet15borf4 nde bamh ndebamh figure 1. construction of a recombinant plasmid pet15b-orf4 (~7.2 kb). purifi cation of orf4-histag fusion protein ten milliliter culture of e. coli bl21 (de3) plyss carrying pet15-orf4 was induced by 0.1 mm iptg at od600 of 0.6 for 3 h. by shaking at 37 oc. the culture was then centrifuged, and pellet was frozen at –70 oc until use. the protein was purified under denatured condition using ni-nta column (qiagen, gmbh, germany). sds-page and western blotting pellets of uninduced cells (1 ml) and induced cells (0.5 ml), solubilized lysate, or purified -orf4-histag fusion protein, were mixed with 2 x sample buffer (tris. hcl, glycerol, sds, 2-mercaptoethanol, and bromophenol blue) and denatured by boiling for 5 minutes. sds-page was performed at 12.5 % (w/v) acrylamide gel, and protein was stained with commassie brilliant blue. for western blotting, the orf4histag fusion protein polyacrylamide gel was blotted onto a pvdf membrane by electroblotting. the western blot was stained using monoclonal mouse anti-histag antibody at 1:5000 dilution. a secondary goat anti-mouse igg antibody conjugated to alkaline phosphatase was used for imaging (zymed laboratories inc.). 15 bacterial magnetic particle synthesis in magnetospirillum magneticum amb-1 – aris tri wahyudi. results observation of nma38-4 cells and colony colony of this mutant, designated as nma38-4, was grown on msgm plate. the color was white indicating the bmp was not synthesized. whereas the color of the amb-1 wild type grown on the same media was brown-black. the brown-black color indicated that bmp was synthesized. observation of nma38-4 cells under light microscopy showed that cells did not respond to the magnetic fields applied. this indicates bmp may have not been synthesized due presumably to transposon insertion into the genome, especially in the gene involved in bmp synthesis. to confirm this result, nma38-4 cells were observed by transmission electron microscopy which showed that they did not contain bmps in the cell (data not shown), indicating that bmps were not synthesized completely. dna sequence analysis of orf4 identification of the gene interrupted by mini-tn5 transposon in nma38-4 genome was accomplished by isolation of flanking dna by inverse pcr, sequencing of the flanking dna, and sequence analysis through homologous searches of major databases. figure 2 shows a gel electrophoresis of 1.3 kb inverse pcr product amplified from dna flanking the transposon of nma38-4 genome. to characterize this locus, we used sequence of dna flanking the transposon aligned against the whole genome sequence of m. magneticum amb-1 (matsunaga et al. 2005) and assembled one contig which contained mini-tn5-interrupted gene, orf4. homology search of orf4 sequence by blastx program revealed its homology with abc transporter from mesorhizobium loti (62% identity, 74% similarity). an orf directly interrupted by transposon, orf4, with location of transposon insertion site, is shown in fig. 3. putative ribosomal binding site (rbs) was found at the position of 7 bp upstream of start codon (atg). amino acid sequence analysis deduced from the genes the orf4 of m. magneticum amb-1 encodes a protein of 494 amino acids with molecular mass of 52.8 kda, as shown in fig. 4. examination of the deduced amino acid sequence revealed a high degree of homology with abc transporter. further analysis indicated that the sequence contains atp-binding sites in the orf4 and the presence of two segment walker motifs, walker a and b was identified (walker et al. 1982). the two sequences, gskkegkltcdtmlal (walker a) and kvakpghrllmvs (walker b) were localized in orf4 at position 170-185 and 309-321, respectively (fig. 2). the multi alignment of the amino acid sequences revealed a highly conserved region in segment a, whereas the consensus in segment b was less restrictive, permitting various amino acid substitutions. analysis of hydrophilicity plot of the protein deduced from orf4 using an algorithm kitedoolittle showed that orf4 protein possessed 10 putative hydrophobic transmembrane α helixes (data not shown). 16 biotropia vol. 16 no. 1, 2009 figure 2. 1.3 kb dna fragment flanking transposon mini-tn5km1 amplified by inverse pcr method (1) and dna marker 1 kb ladder (m). expression and purifi cation of histag fusion protein after recombinant plasmid pet15b-orf4 was transformed to e. coli bl21 (de3) plyss, expression of the orf4 was induced by addition of 0.1 mm iptg and under the control of promoter t7 lac. th e total protein profi le of the whole cells was analyzed by sds-page to express the histag-orf4 fusion protein. as shown in figure 4a, the protein band ~55 kda corresponding to the molecular mass of the histag-orf4 fusion protein was highly expressed in e. coli, and pure protein was obtained after denatured purifi cation. it was confi rmed to be the histag-orf4 fusion protein by western blot analysis using anti-histag antibody (fig. 4b). discussions in this study, we demonstrated that the gene encoding protein which belongs to atp binding protein is linked to bmps synthesis. mutation of this gene in nma384 generated non-magnetic cell. th e mutant did not respond to the magnetic fi elds and bmps were not completely synthesized. inverse pcr method allowed us to amplify 1.3 kb genomic fl anking dna from nma38-4 genome and align the sequence against the whole genome sequence of m. magneticum amb-1. th e contig containing the sequence of the gene directly interrupted by transposon comprised 1482 bp (fig. 3). although this gene did not contain a native promoter, it could be highly expressed in e. coli under t7 lac promoter (fig. 4). th is indicates orf4 isolated from m. magneticum amb-1 genome involved in bmps synthesis could be expressed in escherichia coli. eff ect of the transposon, not only inactivated the gene directly interrupted, but also the genes located downstream of the transposon insertion (de bruijn & lupski 1984; 17 bacterial magnetic particle synthesis in magnetospirillum magneticum amb-1 – aris tri wahyudi. kleckner et al. 1977). th erefore, orf4 may most probably have important roles during bmp synthesis. interestingly, orf encodes majority of the protein with functions which is related to atp-binding cassette (abc) transporter. involvement of abc transporter in iron transport has been reported in gram negative and gram positive bacteria such as yersinia pestis (gong et al. 2001; fetherston et al. 1999; bearden et al. 1998), streptococcus pneumoniae (brown et al. 2001a; brown et al. 2001b), neissiria meningitides (khun et al. 1998), s. pyogenes (janulczyk et al. 1999), and brachyspira hyodysenteriae (dugourd et al. 1999). taken these all together, it is therefore possible that genes with close homology to atp-binding protein identifi ed in this study may have a signifi cant role in iron transport during bmps synthesis after the iron passed across the outer cell membrane. despite the signifi cant impact of bmps to the physiological functions of the cell, information on the mechanisms and the factors aff ecting their formation is still very limited. our data suggests that a gene for atp binding cassette protein and the other genes within the same cluster may most probably be linked to bmps synthesis in m. magneticum amb-1. th is fi nding may signifi cantly contribute to the complete elucidation of the complex process of bmp synthesis, especially in m. magneticum amb-1. figure 3. dna sequence of orf4 (boxed) started with atg (start codon) and ended by tga (stop codon). 9 bases boxed show mini-tn5 insertion site. walker a (ggskkegkltcdtmlal), walker b (pkvakpghrllmvs), and ribosomal binding site (rbs) are also indicated by underline. 18 biotropia vol. 16 no. 1, 2009 figure 4. (a). sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) of protein profile of orf4 expressed in escherichia coli. lane m: marker protein. lane 1: total cell proteins from uninduced-cells of e. coli bl 21 (de3) plyss harboring pet15-orf4. lane 2: total cell proteins from induced-cells of e. coli bl 21 (de3) harboring pet15-orf4 induced by 0.1 mm iptg. lane 3: total protein cell lysate (15 μg). lane 4: purified histag-orf4 protein (6 μg). (b) lane rm: rainbow marker protein. lane 5: western blot analysis of purified histagorf4 fusion protein (6 μg) corresponding to lane 4. conclusion a gene involved in bacterial magnetic particle synthesis has been isolated from m. magneticum amb-1 genome, cloned and over-expressed in e. coli bl21 (de3) plyss. th is gene encoded protein had homology with abc transporter. a 55 kda protein corresponding to the gene (orf4)-histag fusion protein resulted from the gene expression was detected by sds-page. th e purifi ed protein was confi rmed to be orf4-histag fusion protein by western blot analysis. mutation within this gene (orf4) rendered m. magneticum amb-1 defective in bacterial magnetic particle synthesis. acknowledgments part of this work was conducted at tokyo university of agriculture and technology, japan, especially at department of biotechnology (matsunaga-takeyama laboratory). th erefore, i am grateful to prof tadashi matsunaga and prof haruko takeyama for the laboratory facilities and valuable supports. references altchul sf, madden tl, scaff er aa, zhang j, zhang z, miller w, and dj. lipman. 1997. gapped blast and psi-blast: a new generation of protein database search programs. nucleic acids research, 25:3389-3402. blakemore rp. 1975. magnetotactic bacteria. science, 190: 377-379. 19 bacterial magnetic particle synthesis in magnetospirillum magneticum amb-1 – aris tri wahyudi. blakemore rp, maratea d, and rs. wolf. 1979. isolationand pure culture of a fresh water magnetic spirillum in defi ned growth medium. journal of bacteriology, 140: 720-729. bearden sw, stagg tm, and rd. perry. 1998. an abc transporter system of yersinia pestis allows utilization of chelated iron by escherichia coli sab11. journal of bacteriology, 180: 1135-1147. berson ae, hudson dv, and ns. waleh. 1991. cloning of a sequence of aquaspirillum magnetotacticum that complements the arod gene of escherichia coli. molecular microbiology, 5: 2261-2264. berson ae, hudson dv, and ns. waleh. 1989. cloning and ccharacterization of the reca gene of aquaspirillum magnetotacticum. archives of microbiology, 152: 567-571. brown js, ogunniyi ad, woodrow mc, holden dw, and jc. paton. 2001a. immunization with components of two iron uptake abc transporters protects mice against systemic streptococcus pneumoniae infection. infection and immunity, 69: 6702-6706. brown js, gilliland sm, and dw. holden. 2001. a streptococcus pneumoniae pathogenicity island encoding an abc transporter involved in iron uptake and virulence. molecular microbiology, 40: 572-585. debruijn fj, and lupski jr. 1984. th e use of transposon tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of dna segments cloned into multi copy plasmidsa review. gene, 27: 131-149. dugourd d, martin c, rioux cr, m. jacques m, and j. harel. 1999. characterization of a periplasmic atpbinding cassette iron import system of bracyspira (serpulina) hyodysentriae. journal of bacteriology, 181: 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new method in bacterial genetic. journal of molecular biology, 116: 125-159. matsunaga t. 1991. application of bacterial magnets. trends in biotechnology, 9: 91-95. matsunaga t, sakaguchi t, and f. todokoro. 1991. magnetite formation by a magnetic bacterium capable of growing aerobically. applied microbiology and biotechnology, 35: 651-655. matsunaga t, nakamura c, burgess jg, and k. sode. 1992. gene transfer in magnetic bacteria: transposon mutagenesis and cloning of genomic dna fragments required for magnetite synthesis. journal of bacteriology, 174: 2748-2753. nakamura c, burgess jg, sode k, and t. matsunaga. 1995a. an iron-regulated gene, maga, encoding an iron transport protein of magnetospirillum sp. amb-1. journal of biological chemistry, 270: 2839228396. okamura y, takeyama h, and t. matsunaga. 2001. a magnetosome-specifi c gtpase from the magnetic bacterium magnetospirillum magneticum amb-1. journal of biological chemistry, 276: 4818348188. okuda y, denda k, and y. fukumori. 1996. cloning and sequencing of a gene encoding a new member of the tetratricopeptide protein family from magnetosome of magnetospirillum magnetotacticum. gene, 171: 99102. 20 schleifer kh, schuler d, sprins s, weinegger m, amann r, ludwig w, and m. kohler. 1991. th e genus magnetosporillum, gen. nov., description of magnetospirillum gryphiswaldense and transfer of aquaspirillum magnetotacticum to magnetospirillum magnetotacticum, comb. nov. systematic and applied microbiology, 14: 379-385. wahyudi at, takeyama h, and t. matsunaga. 2001. isolation of magnetospirillum magneticum amb-1 mutants defective in bacterial magnetic particle synthesis by transposon mutagenesis. applied biochemistry and biotechnology, 91: 147-154. walker jm, sarsate m, runswick mj, and jj. gay. 1982. distantly related sequences in the a and b-subunits of atp synthase, myosin, kinases, and other atp-requiring enzymes and common nucleotide binding fold. embo journal, 1: 945-951. biotropia vol. 16 no. 1, 2009 thank you for evaluating anybizsoft pdf splitter. a watermark is added at the end of each output pdf file. to remove the watermark, you need to purchase the software from http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html biotropia no biotropia no. 11, 1998: 22 41 nutrient stocks in four stages of a lowland rain forest at pasirmayang, jambi, central sumatra, indonesia pamuji lestari faculty of agriculture, university of bengkulu , bengkulu, sumatra, indonesia abstract studies of nutrient cycling of tropical forests should differentiate between dynamic stages of the forest. we studied the nutrient concentration (n, p, k, ca and mg) and phytomass of aboveground (living and non living parts) and belowground compartments (soil) in four dynamic stages, namely building (bl and b2), and mature (ml and m2) stages, for a lowland rain forest. nutrient concentrations in various compartments differed between the dynamic stages. bark contains higher nutrient concentration than wood parts, both in stems and branches. leaves contain higher nutrient concentration than wood parts, both in litterfall and litter. the concentration of k in throughfall is the highest, ca and mg perform similar value. throughfall exhibits lower nutrient concentration than open area rain water. the nutrient concentration of 10-20 cm is higher than in the 0-10 cm soil depth. the phytomass values are highly variable among tree parts, diameter classes and dynamic stages. the phytomass is generally the highest in m2 and significantly different from bl, ml and b2. the phytomass of leaves in litterfall and litter is higher than wood parts. more litterfall and litter are accumulated in mature than building phases. key words : nutrient / phytomass / dynamics / rain / forest introduction understanding the cycling of the original forest ecosystem mineral nutrients is essential in evaluating consequences of forest replacement to agriculture, tree plantations, or best methods by which forests can be managed for sustained timber production. reviews of overall patterns of mineral cycling in the tropics (jordan 1985; vitousek and sanford 1986; proctor 1987; bruijnzeel 1989) clearly show a wide diversity. the tropical rain forests are very rich in species with large interspecific differences in chemical composition (grubb and edwards 1982; hobie 1992), and they differ in species competition and structure over sometimes short distances (trichon 1996). moreover, the tropical forests are very dynamic systems, mosaic of regenerating and mature "phases" (halle et al. 1978; whitmore 1984). this invariably rises the question of the representativeness of field samples that are always small for obvious logistic reasons, when extrapolating the data to the whole ecosystem. the present study is part of a wider research program on nutrient patterns and variability in tropical forested lowland landscapes, both accross physiographical and 22 nutrient stock in four stages of a lowland rain forest at pasirmayang, jambi pamuji lestari successional gradients in jambi province, sumatra. it was initiated not only because there is so little information available on productivity (soerianegara 1966; schaik and mirmanto 1985) and nutrient cycling (soedjito 1988; strigel et al. 1993) of rain forest in indonesia in general, but also because the understanding of the variability of nutrient availability in such forested landscape is needed for forest land management. this study was conducted to determine whether nutrient stocks and phytomass significantly vary among dynamic stages and how the nutrient maintains the rain forest ecosystems. site description the pasirmayang permanent plot in the jambi province of sumatra (1°5's latitude and 102°10'e longitude, north muarabungo city) offers an excellent opportunity to study lowland rain forest function and dynamics since topography is very gentle, meaning that there are no or few interaction between dynamics and slopes. average altitude varies between 70 and 100 in and the plot's physiography is gentle with slopes ranging from 2 to 8% for drops of 5 to 30 m. the underlying geology is that of vast tertiary deposition basins mainly formed in sedimentary rock. these geological formations consist of layers several kilometers thick and mainly include sandstones, calcareous sandstone and marls. the soils encountered in the plot are mainly oxisols. the a horizon, located at a depth of 2 to 10 cm, contains a superficial fine root layer, and its low organic content is a characteristic feature (c/n from 8 to 15). clay content increases with depth (a-b located between 10 and 40 cm). kaolinite sensu stricto accounts for at least 80% of the clay fraction and the low loam/clay ratio is characteristic. in the brown or yellow-brown b horizon, soil acidity remains relatively constant (ph between 4 and 5). the cation exchange capacity is very low, less than 15 meq/loog of clay. these soils rank amongst the least fertile and are extremely fragile once forest has been cleared (erosion on slopes, compacting by heavy vehicles, insolation and rapid loss of surface minerals). few changes in soil characteristics are observed between the lower slopes and the top of the ridges in the plot. the litter is mostly very thin and covers a humus horizon of 2 to 3 cm thick, sometimes 15 cm deep from place to place. these thicker layers are related to dominance of certain species like dipterocarpus crinitus or d. lowii whose leaves apparently decompose very slowly. the humus horizon is extremely acid in these instances (ph 3.8 to 4). 23 biotropia no. 11, 1998 in general, the climate of the area shows an annual rainfall between 200 and 2500 mm/year. monthly mean rainfall can drop below 100 mm for one month at most, and the yearly average of rainy days varies from 120 to 150, with a mean temperature of the coldest month always above 20°c. however, as indicated by laumonier (1995) climatic variability is high and rather long dry periods (2 to 3 months) can be experienced for some years. the forest is a typical lowland dipterocarp forest as described for the eastern sumatra plains by laumonier (1995). materials and methods a. field sampling a.l. sampling unit selection in relation to forest dynamics following previous research on forest dynamics in that area by torquebiau (1986) and laumonier (1991), several criteria were taken into account for the selection of what can be seen as representative of mature and "old" building forest phases. for the particular purpose of the study a former classification of the forest dynamics made by laumonier (op.cit), was combined with whitmore (1984) tree diameter classes for mature, building and gap phases, and refined using vertical and horizontal structure criteria e.g : frequency histogram for diameters and heights, scatter and profile diagrams, crown projection, canopy cover, crown overlapping, treefall gaps and added with a concept by halle et al. (1978) that classifies trees according to their architecture: 1) trees of the "future" which are represented by the first stage of development when either conform to their original model or are regenerated by reiteration, 2) trees of the "present" are trees that survive from earlier phase of development, show no regeneration in an architectural sense as a whole and have established a new energetic balance, and 3) trees of the "past" are trees that show no particular response to the outside energy, facing an architectural chaos from disturbance and are gradually eliminated. a total of eight plots assigned in four dynamic stages with two replications in each dynamic stage were used to evaluate those vertical and structural criteria. 24 nutrient stock in four stages of a lowland rain forest at pasirmayang, jambi pamuji lestari a.2. vegetation and soil sampling a. nutrient concentration in each plot (20 m x 20 m), aboveground compartments as living parts (leaves, stems, branches and twigs), non living parts (litterfall, litter, and throughfall) and belowground (soil) compartments were sampled for the nutrients that are most likely to limit primary production and other ecosystem function (n, p, k, ca and mg). a.l. aboveground compartments living parts (trees) trees were sampled for three stem diameter classes 30 cm and up (all diameter higher than 30 cm which vary among plots), 010-29.99 cm and 0 3-9.99 cm. a composite of leaves, stems (wood, bark), branches (wood, bark), and twigs from five trees in each of the three diameter class was collected for each plot of various dynamic stages obtained from the structural analysis. an approximate of 0.5-1 kg of leaves, branches and twigs were obtained by cutting, whereas samples of stems were obtained by slashing the main stems to reduce tree damage. non living parts litterfall two semi-permanent circular litter traps (diameter 1 m) were used for litterfall collections. they were equipped with plastic mesh 1.25 m above the ground to allow proper water drainage. at emptying, the contents of each trap were put in a paper bag and sorted into leaves and woody parts. there is a total of 32 samples (8 plots; two replications in each stage with two litterfall traps in each plot and two different parts; leaves and wood). litter two litter samples (one square meter each) were taken from every plot. a total of 32 samples both for leaves and woody parts were analyzed. throughfall a total of seventeen water samples were collected for nutrient analysis. two plastic drums with a polyethylene funnel of diameter 22.5 cm in every plot were 25 biotropiano. 11, 1998 used for throughfall collection. an additional drum was put in the open for sampling bulk precipitation. a, 2 belowground (soil) a metal ring of 8.5 cm diameter and 10 cm height was used for soil collection. a total of 32 soil samples (two samples in every plot at two soil depths: 010 cm and 10-20 cm) were used for nutrient analysis. roots were not included in the present study due to logistic reason. b. phytomass living parts (trees) the phytomass of living parts in the three diameter classes has been estimated from allometric equations developed by yamakura et al. (1986) who studied plant biomass with reference to forest dynamics in east kalimantan (table 1) the phytomass calculation was based on the number of trees in each plot according to diameter classes. the results of 8 plots were then averaged and presented in each dynamic stage. table 1. regression equation for calculating biomass of tree parts developed by yamakura (1986) tree regression equation stem biomass (sb) 2.903 x 10"2(d2h)° 98'3 branch biomass (bb) i 259 leaf biomass (ib) ' -, 07266 9.146xlo"(sb+bb)° 266 sb, bb, ib, in kg d (diameter) in cm h (height) in m non living parts litterfall the equipments used are the ones used for sampling the litterfall for nutrient concentration study. the litterfall phytomass obtained is the g/m2 of samples dry weight and extrapolated into ton/ha. 26 nutrient stock in four stages of a lowland rain forest at pasirmayang, jambi pamuji lcstari litter the litter phytomass was obtained from the dry weight inside a square meter subplot (g/m ), then it was extrapolated into ton/ha. b. laboratory analysis prior to nutrient analysis, above ground and below ground materials were oven dried at 105°c for 48 hours or until constant weight. after determining oven dry weight, the samples were ground and nutrient analyzed. total nitrogen was determined using kjeldahl digestion and distillation method and phosphorus was analyzed using spectrophotometry. an atomic absorption spectrophotometer was used for potassium and calcium analysis, whereas magnesium was determined by using flame photometer. c. data analysis the nutrient concentration of different parts of trees, litterfall, litter and soil in various dynamics were compared using least significant difference (lsd) of the statistix program package version 4.0 (siegel 1992). results and discussion a. structural and dynamic classification of the plots the height of the forest canopy varies from 30 to 40 m with emergent trees rising to heights of 45 to 55 m. mature phase of the forest may have up to four distinct foliage mass layer respectively, situated at the following heights: 5 to 10 (15), 15 20(25), 25 30(35), and 35 40(45) m (laumonier 1995). a total of eight plots were selected representing mature phases and building phases (figure la b) following the former classification by laumonier (1991). eventhough it is important to study the early stages of succession i.e. treefall gaps and young aggrading phases to complete the comparison of each dynamic stage, they were excluded from the study due to logistic reasons. 27 biotropia no. 11, 1998 as shown in table 2, further analysis of the horizontal structure of these plots (number of trees, diameter, height, basal area, abundance, dominance and canopy cover), allows to group them into 4 classes corresponding to four dynamical status situation. building 1 (bl), building 2 (b2), mature 1 (ml), and mature 2 (m2). the number of trees in the four different classes are not significantly different except for b2 where a high tree density is observed. in all plots, the density is always higher for trees of the future, with the exception of ml where abundance of trees of the present is conspicuous. the trees of the past are the most abundant in m2. total, covered and overlap crown surface are the highest in bl followed by m2, ml and b2, whereas gap surface is the highest for b2 followed by ml, m2 and bl. in term of coverage, the horizontal structure is always dominated by "the trees of the present" (m1>m2>b2>b1) followed by "trees of the future" (b2>b1>m2>m1) and "past trees" (b1>m1>b2>m2). average basal area of the four different classes is similar, but more variations are observed in the vertical structure and the respective arrangement of the tree classes (table 2). b. nutrient concentration the complete results of nutrient concentration in various compartments (tree, litterfall, litter, throughfall and soil) are presented in figure 2a d. b.i. aboveground a. living parts (trees) the general pattern of nutrient concentration in trees observed in this forest is n > k > ca > mg > p. mostly n concentration in leaves is the highest compared to other parts this condition is mainly due to the physiological role of the leaves in photosynthetic activity. other nutrient concentrations are varied among parts. barks which play considerable role in transferring nutrients from trees into soil are always present in higher nutrient concentration than sap wood, both in stems and branches. b. non living parts b. 1. litterfall most nutrient concentration is varied between parts and among dynamics. the variable nutrient concentration in litterfall may be mainly the result of water penetration versus leaching from rainfall, influenced by the position of leaves in the trees, and also by the species composition. the higher nutrient concentration of 30 biotropia no. 11, 1998 leaves and wood parts in building phase may be due to the condition that in this stage plants are very active in uptaking and storing nutrients from soil. b.2. litter all nutrients in both parts are not significantly different among dynamics. the concentration of n, k and ca of leaves is significantly different from wood parts. similar to litterfall, the nutrient concentration of leaves is higher than wood parts, litter concentrates higher nutrients in building than mature stages. the fallen leaves are accumulated as forest litter. thus, the nutrient concentration of the litter may mainly come from the concentration of the forming materials. therefore, the higher nutrient concentration of the litterfall may result in the higher nutrients in the litter. moreover, in the process of decomposition more nutrients will become available, and so the concentration of nutrients in litter may be increased. by comparing aboveground living parts, it shows that the nutrient contents in leaves > litterfall > litter. the average percentage of four dynamic stages in nutrient withdrawal before litterfall is the highest for k (68%) followed by p (46%), ca (42%), n (24%) and mg (12%). the poorer quality of litter compared to litterfall may suggest that the process of decomposition is very slow. b.3. throughfall in the four phases, k exhibits the highest concentration both as input (open area) and throughfall in the forest ecosystem, whereas ca and mg show similar values. this result agrees to that of manokaran (1980) that the nutrient concentration increased as precipitation passed through the canopy as throughfall due to leaching at the canopy with conspicuous amount for k, relatively less for mg and ca. the average nutrient concentration of throughfall is higher in building than mature stage. this may be caused by the higher surface covered in building than mature stage that allows more water penetration after reaching the canopy. the values of ca and mg in the pasirmayang forest are lower than that obtained in open area. this is in contrast with strigel et al. (1993) who studied the nutrient concentration of throughfall in two forest sites in east kalimantan (table 3 ). 36 nutrient stock in four stages of a lowland rain forest at pasirmayang, jambi pamuji lestari table 3. the nutrient concentration (mg/1) of throughfall in pasirmayang, sumatra vs bukit suharto and lempake, east kalimantan throughfall open k ca mg k ca mg pasirmayang (bph) bukit suharto 1.21 1.80 0.30 0.40 0.30 0.05 1.33 0.10 0.41 0.20 0.30 0.02 lempake 1.70 0.60 0.08 0.30 0.20 0.02 in pasirmayang, the nutrient concentration of open area is higher than in throughfall, although they are not significantly different. this condition may be due to the water sample collectors'position in the open area of pasirmayang (20 m x 20 m plot size) that may still be affected by leaching of nearby canopy, i.e. increasing the nutrient concentration. in bukit suharto and lempake, the nutrient concentration of throughfall is higher than in the open area. this phenomenon may be the result of the water sampling position that is far from the forest area (100 m apart), therefore the effect of adjacent trees could be avoided. b.2. bcltm ground (soil) the concentration of p and ca is not significantly different between depths in all stages. other nutrients are varied. all nutrient concentrations are not significantly different among dynamics. at 0-10 cm soil depth, the pattern of nutrient concentration in old building (b2) and mature phase (m2) is similar with n> ca> k> p or mg. however, some differences in nutrient concentration of top soil among dynamics were observed among the plots. at 10-20 cm depth, the pattern of nutrient concentration is similar to that at 0-10 cm with n> ca> k> p or mg. the p in both soil depths are generally limited because of the small mass circulation in most forests, small input from atmosphere to compensate losses from available pools and fixation by fe and al oxides in highly weathered acid soil. the concentration of nutrients is higher in top than in the lower layer. this may be the result of higher litter decomposition process above the top soil that releases many nutrients to the soil. apparently, the concentration of nutrients in the soil of the building phase was higher than that of the mature phase. knowing, however, the extremely high variability of soil nutrient availability in the rain forest environment, it may be difficult to draw conclusions. the relationship between forest dynamics with soil 37 biotropia no. 11, 1998 variation pattern is a very complex phenomenon which is difficult to assert within the present study. c. phytomass c.l. living parts (trees) the general aboveground phytomass for the entire pasirmayang plot varies between 320 and 400 ton of dry matter per hectare (laumonier 1991) for trees with a diameter > 10 cm. undergrowth samples provide a further estimate of 15 to 20 ton/ha for individuals with a diameter ranging from 3 to 10 cm. at an average of 30 to 40 kg/m2 (extremes being 0 to 80 kg/m2), epigeal phytomass varies from 2.65 to 30.7 ton/400 m2. in the selected plots, the phytomass pattern of trees in different diameter classes (0 > 30 cm, 10 < 0 < 30 cm and 3 < 0 < 10 cm) and dynamic stages is shown in figure 3a c. among the plots, the general phytomass pattern for living parts is m2> bl> ml> b2. m2 stage exhibits the highest phytomass and differs significantly from ml, b2 and bl. for the diameter class 10 < 0 < 30 cm alone, bl > ml > b2 > m2. bl is the type of dynamic stage where fierce competition exists between a large number of "future" trees reaching the canopy. the phytomass in the 3 < 0 < 10 cm diameter class performs similar to that of pole trees with bl exhibiting the highest followed by ml, m2 and b2. c.2. non living parts a. litter/all as shown in fig. 4a, in all stages, leaves litterfall phytomass is higher than wood litterfall. the pattern of total litterfall and leaves litterfall is ml > b2 > bl > m2, whereas for wood litterfall, the pattern is slightly different with bl > b2 > ml > m2. b litter regarding utter accumulated on the ground, the pattern of leaves and total litter is the highest in m2 followed by b2, ml or bl (fig. 4b). in woody litter, the phytomass of mature is higher than in building stages. this phenomenon seems to correspond to a natural succcssional pattern of forest dynamics from building to mature phase, the "older" the phase, the more litter accumulated 38 nutrient stock in four stages of a lowland rain forest at pasirmayang, jambi pamuji lestari references bruunzeel, l.a. 1989. moist tropical forest nutrient cycling : the hydrological framework. in proctor, j. (ed). mineral nutrienu in tropical forest and savana ecosystems, p. 383-416. grubb, p.j and p.j. edwards. 1982. studies of mineral cycling in a montane rain forest in new guinea. iii. the distribution of mineral elements in the aboveground material. journal of ecology 70:623-648. halle, f.r., a.a. oldeman and p.b. tomlinson 1978. tropical trees and forests. springer-verlag, heidelberg. hobbie, s.e. 1992. effects of plant species on nutrient cycling. tree7(10):336-339. jordan, c.f. 1985. nutrient cycling in tropical forest ecosystems. wiley. london. laumonffir, y. 1991. vegetation de sumatra, indonesia . ecologie, flore, phytogeographie. th doct. etat, ups toulouse, france. 337p. laumonier, y. 1995. the vegetation and physiography of sumatra. geobotany 22. kliiwer academic, dordrecht, 234 p. manokaran, n. 1980. the nurient contents of precipitation, throughfall and stemfiow in a lowland tropical rain forest in peninsular malaysia. the malaysian forester 43(3):266-289. proctor, j. 1987. nutrient cycling in primary and old secondary forest. applied geography 7:135-152. schadc, c.p. van and e. mkmanto. 1985. spatial variation in the structure and litter production in a sumatran rain forest. biotropica 17:196-205. slegel, j. 1992. statistix version 4. analytical software. borland international inc. and fleming software. minnesota, usa. soerianegara, i. 1966. the primary productivity of selected forests in indonesia. rimba indon. 10(4):246-256. soediito, h. 1988. spatial pattern, biomass, and nutrient concentrations of root systems in primary and secondary forest trees of a tropical rainforest in kalimantan, indonesia. indonesian institute of sciences (l1pi), indonesian national mab commitee, 41-59. str1gel, g.d, ruhiyat, d. prayitno and s. sarmina. 1993. nutrient input by rainfall into secondary forests in east kalimantan, indonesia. j. trop. ecol. 10(2):285-288. torquebiau, e.f. 1986. mosaic pattern in dipterocarps forest in sumatra. agroforestry systems 2(2):103-128. trichon, v. 1996. heterogeneite spatiale des structures en foret naturelle de basse a sumatra, indonesie. doctoral de 1'universite paul sabatier. toulouse, france. vitousek, p.m and r.l. sanford, jr. 1986. nutrient cycling in moist tropical forest. ann; rev. ecol. syst. 17:137-167. whitmore, t.c. 1984. tropical rain forests of the far east. clarendon press, oxford. yamakura, t, a. hagihara, s. sukardjo, and h. ogawa 1986. aboveground biomass of tropical rain forest stands in indonesian borneo. vegetation 68:71-82 41 biotropia no. 6, 1992/1993: 45-54 review of aquaculture genetic researches in thailand uthairat na-nakorn department of aquaculture, faculty of fisheries, kasetsart university, bangkok 10903, thailand aquaculture business has been well established in thailand for more than 40 years. the most recent data indicated a total production of 260 380 tons. sixty-five percent of the total production came from coastal aquaculture, mainly tiger prawn (penaeus monodon) culture. other important species for coastal aquaculture are banana prawn (p. merguensis), cockle (anadara granosa), green mussel (perna viridis), oyster (crassostrea belcheri, saccostrea commercialis), sea bass (lates calcarifer) and grouper (epinephelus tauvina). freshwater aquaculture, although produced only 35% of the annual production, provides major protein source for people in rural areas. important freshwater species are nile tilapia (oreochromis niloticus), tawes (puntius gonionotus), sepat siam (trichogasterpectoralis), walking catfish (glorias spp.), stripped catfish (pangasius sutchi) and giant freshwater prawn (macrobrachium rosenbergii). optimum aquacultural practises, namely stocking density, nutrition requirement and water quality have been obtained in most cultured species. but genetic approach has not been considered, thus resulting in deterioration in economic traits which might be due to excessive inbreeding (reviewed by uraiwan 1989) and/or negative selection (wongsangchan 1985). the history of researches on genetics in aquaculture in thailand started in 1982 when the aquaculture genetic programme in form of a network has been established at the national inland fisheries institute, department of fisheries. this programme was supported by the international development research centre (idrc, canada) in cooperation with dalhousie university, canada (uraiwan 1989). in the same year a genetic improvement programme aiming at improving economic characters of some economic fish species has been conducted at the department of aquaculture, kasetsart university. paralelly a course in fish genetics has been offered. since then different approaches of genetics have been applied with final objectives on improving aquaculture production of the country. researches being conducted are reviewed according to the following fish species. 45 biotropia no. 6, 1992/1993 nile tilapia (oreochromis niloticus) nile tilapia was first introduced to thailand in march, 1965 as a present from the crown prince of japan to his majesty king bhumipol (duangsawasde et al. 1982 cited by uraiwan 1989). the original stock consisted of 50 pairs of fish. nowadays, it has been cultured all over the country and ranks the first in annual production in the recent years (department of fisheries 1991). in fact, most of the cultured stocks are contaminated by hybridizing with o. mossambicus introduced earlier. the only one pure strain is maintained at chitralada royal palace. main genetic problems in tilapia culture in thailand are: 1. slow growth which might be a result of inbreeding depression caused by the small number of foundation stock. unintentional negative selection might also be a cause of the slow growth (wongsaengchan 1985). 2. unacceptable appearances, namely dark colour and long shape, resemble o. mossambicus. therefore, genetic improvement programmes have been conducted to solve these problems. selection programmes using within family selection have been conducted since 1986 aiming at improving the growth of nile tilapia. sixteen families were produced from each of the chitralada strain and chitralada x nakornpathrom cross. the rotation crossing between these families was applied in each generation. after 3 generations of selection the selected lines grew an average of 17 and 13% faster than those of the control and the station seed production lines, respectively (uraiwan et al. 1989 and uraiwan et al. 1982). moreover, indirect selection for increasing growth rate has been performed by uraiwan (1989). in this selection programme offsprings of each of 5 different families were grouped into 3 categories : early maturing (21-23 weeks), medium maturing (25 27 weeks) and late maturing (> 27 weeks). selected fish from the five families were mixed together according to maturing classification. results revealed that the direct response to selection was significant. the fish selected for early maturing matured 11 to 14 days earlier than those selected for late maturing. the selection indirectly resulted in a genetic growth gain. the early maturing selected line was significantly larger and grew faster than the late maturing selected line (size 19 26% larger and growth 5 9% faster). phenotypic correlation coefficient between age and size at maturity ranged between 0.3-0.9. there were no differences in prematurity growth rates between the selected and unselected lines (uraiwan and virasith 1989). 46 review of aquaculture genetic researches in thailand u. na-nakorn thai red tilapia thai red tilapia is a hybrid of oreochromis niloticus and o. mossambicus. it has been originally observed at ubonratchathani fisheries station, in north-eastern thailand (jarimopas 1988). selection programme for better growth rate of thai red tilapia has been conducted since 1982 (jarimopas 1989). size-specific selection was used to prevent effects of asynchronous spawning and variation of mouth-brooding duration on size differences among offspring obtained from mass spawning. in each selection generation twenty-five pairs of parents were mass spawned. fry were collected for a week to obtain approximately 2000 fry. after a rearing period of about 6 weeks fry were graded. only the medium size group was kept and reared for about 8 more weeks. selection was performed when the fish were 14 weeks old. after 6 generations of selection, the selected strain of thai red tilapia was in weight and length 6.76% and 4.09% greater (p<0.05), respectively, compared to control line at 12 weeks of age (jarimopas and nukwan 1989). realized h 2 were 0.44 for weight and 0.53 for length. this selection programme is continuing. thai walking catfish (clarias spp) there are 3 species of clarias being cultured in thailand. c. macrocephalus is the most acceptable due to good meat quality, thus commands high price. culture of this species faces problems of slow growth and high disease susceptibility. c. batrachus is an alternative species for farmers who could not cope with the risk. although its growth and disease resistance is better than c. macrocephalus, it is not so acceptable due to poor meat quality, thus its price is very low. recently, african catfish, c. gariepinus has been introduced to thailand because of its high growth rate. but, again its meat is not acceptable among thai consumers. therefore, an attempt was made to combine the advantages of c. gariepinus and c. macrocephalus by hybridizing these 2 species. successful cross has been obtained between female c. macrocephalus x male c. gariepinus but not the reciprocal one (nukwan et al. 1990). hybrid has improved growth and disease resistance compared to female parent. although its meat quality and appearances could not totally replace that of c. macrocephalus, nowadays it comprises more than 90% of clarias production of the country. this hybrid has been found to be partly fertile. further studies are going on at the department of fisheries in order to study genetic properties of the hybrid for its genetic improvement in the future (lawanyavudth personal communication). 47 biotropia no. 6, 1992/1993 in fact, hybridization experiments attempting to solve the problems of c. macrocephalus were conducted before. tarnchalanukit (1986) made crosses between c. macrocephalus and pangasius sutchi which has faster growth rate and more resistance to disease than c. macrocephalus. the author reported 4 hybrid morphotypes which were described later to be actually 2 morphotypes found to be diploid and triploid hybrids (na-nakorn et al. 1992a). moreover, the authors also reported gynogenetic offsprings arisen from this cross. although these hybrids were not useful for aquaculture, they might contribute knowledge on the phylogenic relationship of these 2 species which belong to different classes (na-nakorn et al. 1992a). at present, although problems have been partly solved by culturing of hybrid clarias, more serious problems emerge which are shortage of female c. macrocephalus brooders. therefore, genetic improvement programmes of c. macrocephalus have been continued. jarimopas et al. (1989) conducted mass selection for fast growing c. macrocephalus starting in january 1986. after 3 successive selection generations the selected line was 11.8% heavier and 2.35% longer than the control line. selection response in weight and length were 26.21 g and 0.66 cm, respectively. realized heritability of weight and length was 0.47 and 0.30 respectively. selection programmes for resistance to aeromonas hydrophila which is the most serious disease were conducted at the department of aquaculture, faculty of fisheries, kasetsart university during 1987-1989 (na-nakorn et al., 1992b). c. macrocephalus was collected from 5 different localities in thailand. their performances including resistance to a. hydrophila were compared but only slight differences were observed. mass selection was performed in the f2 generation arisen from all possible crosses of the 5 strains. the 104 day-old fish were intraperitoneally injected with a. hydrophila suspension and survivors were kept as selected brooders. after one generation of selection, little improvement was observed in resistance to a. hydrophila. heritability of liability for this trait was 0.17. unfortunately the selected fish died of flexibacter columnaris which always occurred in winter and made a second generation of selection impossible. during 19871988 a cross breeding experiment was conducted hoping to benefit from heterosis. semi-diallel crosses were performed using fish collected from the southern and central parts of thailand. results showed that progenies of the cross between females from the south and males from the central grew better than the pure-breds and reciprocal cross. heterosis was shown for body weight and length at the age of 2, 3 and 3.5 months. there were no differences in disease resistance among progenies of different crosses (na-nakorn et al. undated). since this experiment was done in tanks, therefore it is being repeated in earthen ponds which is a normal practise for clarias culture. 48 review of aquaculture genetic researches in thailand u. na-nakorn chromosome manipulation has been applied to improve growth of c. macrocephalus. triploid fish were induced using cold shock (7°c, 25 minutes) applied immediately after fertilization. the shock resulted in 80% triploid fry. growth comparison showed that triploid fish grew slower than diploid ones. the survival rate of triploids was lower than that of diploids (na-nakorn and lakhaanantakun 1992). attempting to combine techniques in biotechnology and quantitative genetics na-nakorn et al. (1992d) and na-nakorn et al. (1992c) induced gynogenesis in c. macrocephalus to produce highly inbred lines for further cross breeding. meiotic gynogenesis was induced by activation of c. macrocephalus eggs using irradiated sperm of pangasius sutchi. sperm dilution (1:100 in ringer's solution) was uv-irradiated (30 w, 30 cm, 2 mins). after the activated eggs were subjected to cold shock, best results were achieved at shock temperature between 6 11 °c with shock duration of 14 minutes and starting 3.5 4.5 minutes after activation. gynogenetic fish were all females. thus, sex reversal to maleness is needed to produce male parents. recently, in my laboratory 17 alpha methyltestosterone has been orally administered to feeding fry of c. macrocephalus. results showed that administration of 30 or 60 mg mt/kg feed for either 30 or 60 days reduced growth at 60 days of mt treated fish as well as survival rate at 30 days. no changes in sex were observed, except for intersex fish found in both mt treated groups being treated for 60 days. paradoxical feminization was presumably accounted for the intersex fish. at present five different stocks of gynogenetic c. macrocephalus have been produced. sex reversal experiments are continuing dealing with lower doses of mt being administered to fry of different ages. a different kind of hormone, 11 b androstenedione is being used. thai silver barb or tawes (puntius gonionotus) being accepted among thai consumers, tawes produces an average annual production of more than 11 000 tons between 1987 1989 (department of fisheries 1991). inferior growth rate of males has been widely accepted which may be a result of their early maturing (4 months compared to 8 months in females). in normal rearing conditions females reach marketable size (500 700 g) within 8-12 months while males grow to only 300 g in the same period. thus, the production of sterile triploids, which could potentially have better growth than diploids at time of sexual maturation, may provide a practical solution to this problem. na-nakorn and legrand (1992) induced triploidy in tawes by subjecting fertilized eggs to cold shock (15°c). they found that cold shock applied immediately after fertilization 49 biotropia no. 6, 1992/1993 lasting 15 and 30 minutes resulted in 95.45 and 90.45% triploid fry, respectively, hatching percentage relative to control was 46.53 and 16.01% for shock durations if 15 and 30 minutes. triploid fish were sterile. however, growth of triploids and liploids has not yet been compared due to the small sample size. all female stock of tawes have been attempted by crossing sex-reversed male genetic female) with normal female as well as by inducing gynogenesis. sangsri 1991) reported sex-reversed tawes to maleness by oral administration of 17 alpha nethyltestosterone to 1 and 2 weeks old fry. dosages of mt used were quite high ^30 120 mg/kg feed) and numbers of sterile gonads were observed in the groups ireated for 30 days. some sex-reversed testes were histologically observed. treatment period of 60 days caused high proportion of sterile gonads. progeny testing of the presumed sex-reversed male is being performed at the department of aquaculture. stock of tawes with high percentage of females (98.7%) was produced by roongratri et al. (1992) using gynogenesis induction techniques. eggs were activated with uv-irradiated sperm of tawes 11.8 mjcm -2 ). at 1.5 minute after activation eggs were subjected to cold shock (2±0.1°c) for 10 minutes. a maximum survival rate of 61.3% of viable diploid gynogenetic offsprings (relative to control) was obtained. the gynogenetic offsprings will be sex-reversed to produce sex-reversed males which will be crossed with normal females to produce inbreeding-free all-female stock. crustaceans application of genetics to aquaculture of crustaceans is limited due to lack of techniques in broodstock rearing of most species such as penaeus monodon. however, population genetics of p. monodon in the gulf of thailand and the andaman sea was studied (sodsuk et al. 1992). this may provide useful information for genetic improvement programmes in the near future. in giant freshwater prawn (macrobrachium rosenbergii) which has been successfully cultured in central thailand, the genetic aspect of this species has recently been a matter of concern. meewan et al. (1992) estimated the heritability of carapace length of this species and reported h 2 -value of 0.04 ±0.22, 0.13 ±0.07 and 0.26 ±0.11 based on paternal, maternal and fullsib analyses, respectively. this study provided useful information for further selection programmes for fast growing stock, provided that carapace length and growth are positively correlated. 50 review of aquaculture genetic researches in thailand u. na-nakorn mollusks culture of marine mollusks in thailand is solely based on naturally produced seeds. hatchery seed production is limited at laboratory scale, thus limited genetic study of these species. however, genetic improvement of oyster has been carried out at the department of marine science, chulalongkorn university (jarayabhand and jindalikit 1992) attempting to produce a triploid small oyster, saccostrea cucullata. thermal shocks (4°c and 35°c) were applied to fertilized eggs 20 minutes after fertilization. results showed that shock duration of 6 minutes for cold shock and 6 or 15 minutes for heat shock gave an average 40% of triploids. growth and other economic important characters were not investigated. basic researches relevant to aquaculture cytogenetic researches especially karyotype studies of various fish species were conducted (tabel 1). unfortunately the application of the results was not discussed in most of the reports. biochemical genetic studies have been conducted in clarias spp. serum protein of c. batrachus and c. macrocephalus was analysed using sds-polyacrylamide gel electrophoresis. the electrophoresis patterns were found to be species-and sex-specific (jondeung and na-nakorn 1986). lawanyawut et al. (1992) conducted allozyme table 1. chromosome number of some fishes of thailand species chromosome no. (2n) references osteochilus hasselti 46 magtoon et al. (1988) puntius daruphani 50 magtoon et al. (1988) pangasius larnaudii 60 magtoon and donsakul (1988) p. sutchi 60 magtoon and donsakul (1988) p. gonionotus 50 na-nakorn and legrand (1992) clarias macrocephalus 54 donsakul and magtoon (1989) c. batrachus 100 donsakul and magtoon (1989) notopterus chitala 42 donsakul and magtoon (1990) n. blanci 42 donsakul and magtoon (1990) n. notopterus 42 donsakul and magtoon (1990) channa striata 44 donsakul and magtoon (1991) c. micropeltes 44 donsakul and magtoon (1991) c. lucius 48 donsakul and magtoon (1991) c. gachua 112 donsakul and magtoon (1991) 51 biotropia no. 6, 1992/1993 studies in c. gariepinus, c. macrocephalus and their hybrids. sixteen protein loci were resolved. three loci, pgi-2, ldh-1 and mdh-2, showed fixed differences between the parent species while the hybrids patterns were always heterozygotes. for gene transferring the growth hormone gene of the giant freshwater catfish (pangasianodon gigas), the world biggest catfish, has been sequenced, and cloned at the department of biochemistry, mahidol university (panyim personal communication). the gene has been transferred to bacteria and expression was observed. successful cloning of this gene encourages scientists to transfer the gene to economic fish species. future studies during the past 10 years aquaculture genetics was applied to only a few of completely domesticated species. genetic research and genetic improvement programmes are urgently needed to solve different problems occurring in different species, for example slow growth in sand goby (oxyeleotris marmoratus), inferior growth and less acceptability of male sepat siam, deterioration of growth of freshwater prawn, etc. intensive studies must be conducted in order to solve problems which cause a bottle neck in breeding and propagation of some economic species such as the problem of broodstock rearing in p. monodon or artificial breeding of snakehead (channa striata) etc. without these techniques genetic improvement of those species is impossible. genetic conservation of natural stocks must be emphasized. genetic stocks of wild fish are being destroyed due to intentional stocking of hatchery stock to natural water as well as unintentional escaping of hatchery stocks. genetic diversity is affected and hence, particular gene pool useful for genetic improvement programmes may be destroyed. in order to prevent undesirable genetic change, effects of introduction of new species in natural water have to be studied in the field before introduction. references department of fisheries. 1991. fisheries statistics of thailand 1989. fisheries statistics sub-division, tech. pap. no. 5/1991. dept. of fisheries, bangkok. 92 p. donsakul, t. and w. magtoon. 1989. a chromosome study of walking catfish, claries batrachus and c. macrocephalus in thailand. in: proceedings of the 27th kasetsart university conference: 426-428. kasetsart university, bangkok. (in thai). 52 review of aquaculture genetic researches in thailand u. na-nakorn donsakul, t. and w. magtoon. 1990. a chromosome study on three species of featherbacks, notop-terus chilata (hamilton), n. bland d'aubenton and n. notopterus (pallas), from thailand. in: proceedings of the 28th kasetsart university conference, 29 31 january 1990:459 466. kasetsart university, bangkok. (in thai). donsakul, t. and w. magtoon. 1991. a. chromosome study on five species channid fishes (channa, family channidae), from thailand. in: proceedings of the 29th kasetsart university conference, 4-7 february, 1991: 561-574. kasetsart university, bangkok. (in thai). jarayabhand, p. and j. jindalikit. 1992. chromosome manipulation in small oyster, saccostrea cucullata, by thermal shock : evidences from trochophore larvae (abstract). abstract of the international workshop on genetics in aquaculture and fisheries management, 31 st august 4th september 1992. asean-eec aquaculture development and coordination programme. university of stirling, scotland. jarimopas, p. 1988. thai red tilapia. thai fisheries gazette. 41 (1): 41 -43. jarimopas, p. 1989. realized response of thai red tilapia to 6 generations of size-specific selection for growth. in: final report, fish genetics project (phase ii) : 147 -164. submitted to the international development research centre. national aquaculture genetics institute, department of fisheries, bangkok. jarimopas, p., a. kumnan and j. wongchan. 1989. mass selection of clarias macrocephalus gunther for growth (3 generations). in: final report, fish genetics project (phase ii): 177 191. submitted to the international development research centre. national aquaculture genetics institute, department of fisheries, bangkok. jarimopas, p. and s. nukwan. 1989. evaluation on growth rate of selected strain thai red tilapia. in: final report, fish genetics project (phase ii) : 165176. submitted to the international development research centre. national aquaculture genetics institute, department of fisheries, bangkok. jondeung, a. and u. na-nakorn. 1986. analysis of serum proteins of clarias batrachus (linnaeus) and c. macrocephalus (gunther) by electrophoresis. the kasetsart j. 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(in thai). meewan, m., c.k. lin, s. tumwasorn and r.w. doyle. 1992. growth heritability of giant freshwater prawn (abstract). in: abstracts of the third asian fisheries forum, october 26-30, 1992, singapore: 81. na-nakorn, u. and a. lekhaanantakun. 1992. the effects of triploidy on survival, growth and feed conversion ratio of clarias macrocephalus. manuscript submitted to symposium on fish genetics and its application to aquaculture and fishery management, 8-10 december 1992, bogor, indonesia. na-nakorn, u. and e. legrand. 1992. induction of triploidy in puntius gonionotus (sleeker). fishery research bulletin no. 18. kasetsart university, bangkok. 10 p. na-nakorn, u., p. sidthikraiwong, w. tarnchalanukit and t.r. robert. 1992a. chromosome study of hybrid and gynogenetic offspring of artificial crosses between members of the catfish families clariidae and pangasiidae. manuscript submitted to environmental biology of fishes. 53 biotropia no. 6, 1992/1993 na-nakorn, u., s. chantsawang and w. tarnchalanukit. 1992b. response to mass selection for disease resistance in clarias macrocephalus. manuscript submitted to journal of applied aquaculture. na-nakorn, u., w. luangpromporn and r.a. dunham. 1992d. induction of gynogenesis in clarias macrocephalus eggs using irradiated sperm of pangasius sutchi and cold shock. paper presented in the third asian fisheries forum, october 26 30, 1992, singapore. na-nakorn, u., w. rangsin and s. witchasunkul. 1992c. suitable conditions for induction of gynogenesis in clarias macrocephalus using sperm of pangasius sutchi. paper presented in international workshop on genetics in aquaculture and fisheries management, 31st august 4th september 1992, university of stirling, scotland. na-nakorn, u., w. tarnchalanukit and c. limsuwan: undated. genetic improvement of walking catfish (clarias macrocephalus gunther) for bacterial disease resistance. report submitted to the national research council of thailand. 163 p. 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(abstract). abstracts of the international workshop on genetics in aquaculture and fisheries management, 31 august-4 september, 1992. asean-eec aquaculture development and coordination programme, university of stirling, scotland. tarnchalanukit, w. 1986. experimental hybridization between catfishes of the families clariidae and pangasiidae in thailand. env. biol. of fishes 16(4) : 317-320. uraiwan, s. 1989. aquaculture genetics improvement programs appropriate for thailand and other developing countries in asia. in: final report, fish genetics project (phase ii): 279-384. submitted to the international development research centre. national aquaculture genetics institute, department of fisheries, bangkok. uraiwan, s., m. meewan and a. kumnane. 1989. within-family selection for increasing growth rate of oreochromis niloticus (linn.). in: final report, fish genetics project (phase ii): 26-36. submitted to the international development research centre. national aquaculture genetics institute, department of fisheries, bangkok. uraiwan, s. and p. virasith. 1989. selection in thai oreochromis niloticus (linn.) by line performance growth test, (abstract) in: abstracts of the third asian fisheries forum, october 26-30, 1992, singapore : 81. uraiwan, s., r.w. doyle and m. meewan. 1992. evaluation of response to within-family selection of oreochromes niloticus (linn.) by line performance growth test, (abstract) in: abstracts of the third asian fisheries forum, october 26-30, 1992, singapore: 81. wongsangchan, a. 1985. broodstock selection and management study. technical paper in fish genetic (thailand) project. national inland fisheries institute, department of fisheries, bangkok. 36 p. 54 45.pdf 46.pdf 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf 53.pdf 54.pdf biotropia no biotropia no. 13, 1999: 18 36 spectral and textural characteristics of the lowland tropical rain forest of jambi, sumatera upik rosalina wasrin1, muchlisin arief2, and setiabudhi3 'faculty of forestry, bogor agricultural university and seamed biotrop, p.o. box 116, bogor indonesia; email: wasrinsy @ indo.net.id 2lapan, jakarta 13710, indonesia 3seameo biotrop, p.o. box 116, bogor, indonesia abstract analyses of landsat tm and spot multispectral data were performed with a very detailed description of the vegetation cover in the field to get a relevancy and consistency of digital image classification in a semi-automatic approach. three main vegetation types, i.e. primary forest, logged-over forest and secondary forest after clear cut were analyzed and the microclimatic parameters were also measured to describe the ecological condition of the vegetation. spectral and textural analysis of data obtained from field measurements and spectral reflectance values of the remote sensing data are the main topic of this report as one aspect of study on the digital method of detection and monitoring on forest ecosystem change using high resolution satellite data funded by the indonesian national research council. this study shows that spectral reflectance values alone cannot differentiate the logged-over forest from the primary forest, but it is very sharply distinguished from the secondary forest. as for the texture analysis, it is possible to distinguish the logged-over forest from the primary forest, as shown by different values of degree of entropy, although spatially, it is still doubtful. key words: indonesia/jambi/tropical rain forests/lowland areas/remote sensing/vegetation analysis/ logged-over forests/primary forests/secondary forests. introduction analysis of remotely sensed images is commonly done by calculating the multispectral reflectance values of the objects or themes, regardless of the spectral reflectance of the neighboring pixels. this has affected the mean values and variances, which is why different objects can have the same spectral reflectance or the same objects give different spectral reflectance values. digital data are principally dedicated to quickly evaluate and monitor the change and dynamics of the earth features such as land cover, land use, vegetation cover, development areas, etc. especially for the vegetation cover, the change usually occurs in a slightly dynamic phenomenon and in the form of a mosaic of gaps. the conventional photo interpretation of analog satellite imageries by using the elements of interpretation such as tone or color, size, shape, texture, pattern, shadow, site, association, becomes limited to evaluate and to monitor the change, especially if the degradation occurs in a mosaic pattern. although this conventional interpretation contains texture and pattern, it is considered only qualitatively, and is therefore sometimes inconsistent. at present, the problems of deforestation and forest degradation are becoming a concern for many people and need to be solved before forests totally disappear. in this study, we tried to detect and indicate the significant degradation from the primary forest into 18 biotropia no. 13, 1999 logged-over forest, secondary forest and finally to grassland alang-alang by using spectral and textural analyses of high resolution satellite spot and landsat tm data. in remote sensing analysis, the mosaic of degradation or perturbation of the vegetation cover can be translated into the form of textural pattern of pixels. however, not so many studies have been performed to analyze the textural phenomena of the vegetation status. most studies convert the textural phenomena of the mosaic into structural information of spectral reflectance values and consider the average/mean values and variances as a major information to be used in the classification process. to be able to understand how the texture of pixels in remote sensing data significantly correlates with ground truth, a very detailed information on the vegetation characteristics obtained from field observations and measurements are provided accordingly. study area field observations and measurements are mainly conducted inside the biotrop-barito permanent plot, in pasirmayang, muarabungo, jambi province, located between 1°1'35" 1°5'55" latitude south and 102°4'35" 102°6'45" longitude east. the observation plot for the primary forest is about 10 ha (data analyzed only from 6 ha), logged-over forest plot, 6 ha, secondary forest plot, one (1) ha and alang-alang plot varied according to the period of abandoned fallow (fig. 1). the logged-over forest plot was selectively cut in 1979/1980 and 1983/1984, whereas the secondary forest was formerly devoted to transmigration area and was figure 1. location of study site at biotrop-barito permanent plot. 19 spectral and textural characteristics upik r. wasrin et al. totally cut (clear cut) in 1978/1979. as for grassland alang-alang, its occurrence is closely related to shifting cultivation practices, therefore, the size and distribution is varied from one place to another. all these vegetation types are located inside the 2700 ha research area near the forest plantation of acacia mangium in pasirmayang. the landscape of the plot is moderately undulating with an altitude between 50 150 m above sea level. the forest is mainly considered as tropical lowland rain forest dominated by dipterocarpaceae, with a total height of the emergent tree up to about 50 m and usually composed of multilayers. laumonier (1992) described that the forest inside the biotrop observation plot consists of four dynamic phases, while torquebiau (1988) considered the mosaic of the forest is built up by four different eco-units following the dynamic phases of reorganizing, aggrading, steady state and degrading eco-units. many research activities have been done in the area, providing a detailed information on the vegetation community, structure, floristic and ecological factors such as soil and microclimate. it is,for this reason that the plots were chosen to support the study on spectral and textural behavior of the tropical lowland rain forest and its main derived vegetation types. methods vegetation measurements the characterization of vegetation based on the parameters measured in the field is done spatially to get a synopsis of mosaic or texture. the field parameters used to highlight the textural phenomena are biomass/phytomass, leaf area index (lai), tree density, structure (height) and crown projection. the biomass/phytomass and lai were obtained by using the allometric equations of ogawa et al. (1989) as follows: for primary forest for secondary forest stem biomass: ws = 0.0396 (d2h) 09326 stem biomass : ws = 0.0396 (d2h) 09326 branch biomass: wb = 0.00602 (d2h)' °27 branch biomass: wb = 0.003487 (d2h) 1027 ws ws leaves biomass: wi =————————— leaves biomass: wi = —————————— 13.75 + 0.025 ws 22.5 +0.025 ws total biomass/tree = ws + wb + wi total biomass = ws + wb + wi ws ws leaf area index lai = ——————————— leaf area index lai = —————————— 0.907 + 0.00205 ws 2.385 + 0.00465 ws the position of each tree and its crown projection are drawn inside the observation transect of 200 x 300 m2, and measurements of total height, clear bole height at the first branch, diameter at breast height are done for each single tree with total amount of 672 trees/ha or 4032 trees/6 ha in each forest type (primary forest and logged-over forest). as for the secondary forest, the observation plot size is 1 ha 20 biotropia no. 13, 1999 with a total number of 702 trees/ha. calculation was done for data gathered from the 6 ha primary forest and 6 ha logged-over forest following the size of a unit of spectral reflectance in remote sensing data, i.e. 20 x 20 m as imitating spot pixel with a total unit of 150 parcels, and 30 x 30 m according to landsat tm pixel size with a total unit of 65 parcels. the calculated vegetation parameters will further be correlated with the spectral reflectance values and will be spatially analyzed to get the textural pattern of the dynamic phenomena. spectral analysis the remote sensing data used in this study are spot cct data k/j = 272/353, july 1986; k/j = 278/354, july 1986 and k/j = 272/352, april 1990 and landsat tm data path/row = 126/061, april 1994; path/row 121/060, april 1994; and aerial photographs taken in october 1992, with scale 1 : 20 000 and its enlargement of 1:10 000 and 1:5000. a significant number of training areas (ta) were extracted from spot and landsat tm data, covering the five themes selected i.e. primary forest, logged-over forest, secondary forest, thickets and grassland alang-alang found inside the study areas. a simple statistical calculation of all ta is executed to obtain the mean value and standard deviation of each theme according to different spectral bands (spot, xs1-xs2-xs3; landsat tm, tm2-tm3-tm4). to have a good figure on the representativeness of the themes, a geographical repetition is performed by using ta of other regions of west kalimantan for landsat tm and south sumatera for spot data. a total of 179 training areas (ta) consisting of 122 536 pixels were taken from five (5) different vegetation types such as hill and lowland primary forest, loggedover forest, secondary forest, plantation/estate, mosaic of grassland and thickets, were analysed. those spectral reflectances were extracted from 3 scenes of spot and 2 scenes of landsat tm data available and tested using tukey student range procedure to know the difference between the mean spectral values of the vegetation types. textural analysis an almost representative sub-sample of training areas (ta) are taken for textural analysis. six ha field observation plots were geographically plotted in remote sensing data of landsat tm path/row = 126/061, april 1994 and spot k/j = 272/352, april 1990 (figures 2 and 3). spectral reflectance values are extracted from each band xs1-xs2-xs3 for spot and tm2-tm3-tm4 for landsat tm and presented in the form of spectral values distribution as given in the example of tm-2 landsat (table 1) and spot xs-1 (table 2). various regressions are used to get the relationships between these spectral reflectances with the field parameters especially biomass and lai which are taken as exactly as possible from the same unit/pixel. while a general feature of the vegetation mosaic is obtained from aerial photo's interpretation on scale 1 : 5769 21 spectral and textural characteristics upik r. wasrin et al. figure 2. position of primary forest plot (yellow) and logged-over forest plot (red) on landsat tm false color composite, with an enlargement of 9 times. figure 3. position of primary forest plot (yellow) and logged-over forest plot (red) on spot false color composite, with an enlargement of 7 times. 22 biotropiano. 13, 1999 table 1. spectral reflectance values of band tm2 landsat from primary forest 61 58 29 13 13 11 11 31 28 19 13 11 11 14 15 13 16 12 11 11 11 13 13 12 12 10 10 12 13 12 13 13 10 11 10 13 12 12 12 12 13 12 12 11 13 12 12 11 11 13 11 10 13 11 12 11 11 12 12 12 11 12 12 12 10 11 13 12 13 12 table 2. spectral reflectance values of band xs1 spot from primary forest 25 24 25 24 24 23 23 23 25 26 25 25 25 24 25 25 24 24 27 25 25 24 24 24 25 26 24 25 26 25 25 23 25 25 25 24 24 25 24 26 25 25 25 27 25 25 24 24 25 25 25 26 28 26 25 25 26 23 24 26 27 26 28 25 25 25 25 24 25 25 24 25 26 25 25 24 23 22 24 24 24 24 25 24 25 25 25 24 23 24 24 24 24 23 24 24 23 25 25 26 23 25 25 24 23 24 25 24 25 24 23 23 25 24 24 24 25 23 24 25 23 24 24 26 25 25 24 24 24 23 25 24 24 24 24 24 23 24 25 24 24 23 24 25 24 24 24 25 27 24 with 95% accuracy for the primary forest and 96% accuracy for the logged-over forest. in the aerial photos, the vegetation covers were divided into 4 layers i.e. the emergent frees with height from 45 55 (60) m, the canopy layer from 30 40 m, the under layer (sub-canopy) layer from 15 25 m and the ground cover/gaps from 2 10 m above the forest floor. to get the indication of textural pattern, a simple statistical approach is used to calculate the level of entropy (h') by using the equation as follows: m h' = 3.3219 (logio n 1/n ∑ ni iog10 ni) i=1 23 spectral and textural characteristics upik r. wasrin et al. where : h' = entropy level n = total number of pixels/parcels ni = number of pixels/parcels of class 1 = 1,2, 3,... m although this index is not an absolute value, it still can give a general idea on the differences between one texture of a certain object compared to the texture of the other objects. to make the value of entropy level (h') more significant, it has to be complemented with a spatial arrangement of the pixel's values. results vegetation characteristics the quantitative description of the forest according to diameter and height distributions of the trees gives an indication that the two forest types under study are considered to have a normal distribution, following j inverse for diameter distribution and cloche-like form for height distribution. therefore, there is no significant difference between these statistical and quantitative approaches. figure 4 gives a general view of the primary forest and logged-over forest from aerial photos at scale 1 : 20 000. these figures show a very different synopsis of the two forest types, where the mosaic of primary forest appears to be more rigorous than those of logged-over forest. the primary forest is still densely covered by the canopy layer and the emergent trees, while in the logged-over forest the canopy layer and the emergent trees are randomly scattered throughout the observation plot (6 ha). from aerial photographs, it is certainly possible to distinguish between the figure 4. general view of primary forest (yellow) and logged-over forest (red) on stereo-pair of aerial photos (scale ± 1 : 20 000). 24 biotropia no. 13, 1999 primary forest and the logged-over forest by looking at the ratio of percentage of the vegetation layers and other parameters such as tree density, volume and tree distribution. a similar procedure is done in the analysis of field parameters such as lai (figures 5,6, 7 & 8) and biomass (figures 9, 10, 11 & 12) of the primary forest and logged-over forest. to enhance the pattern of textural phenomena, the data are regrouped into 8 classes according to pixel sizes of landsat (30x30 m) and spot (20x20 m) for both vegetation types, primary forest and logged-over forest. 25 26 27 28 biotropia no. 13, 1999 spectral analysis the mean spectral values obtained from 3 different sites of spot images and 2 sites on landsat tm images are still varied, especially for thickets and alang-alang on spot and almost for every vegetation type on landsat tm image (table 3). although the landsat tm resolution is nearly as high as spot resolution, a relatively different mean and standard deviation or variance are recorded from the same vegetation types. it can be explained by the fact that radiometric resolution of landsat tm is not at the optimum range of radiation wavelength particularly for the vegetation response. to have a proper analysis on the mean spectral values of various vegetation types according to spectral band and images used, a procedure of tukey student range is applied to give a confidential range as given in tables 4 and 5. the tstudent range is very high in all spectral band of spot and landsat tm, indicating that statistically the mean spectral values are not significantly different from one to another. table 5 also shows no significant difference among the mean spectral values, except between primary forest and thickets and between logged-over forest and thickets, in spectral band of landsat tm-3. based on the spectral reflectance alone, it is hard to distinguish the difference between the primary forest and the logged-over forest. however, the physiognomy has a great contribution in differentiating the primary forest with the other degraded types such as secondary forest, thickets and alang-alang, and plantation or estate, both in spot and landsat tm data. the tukey student test for analyzing the mean spectral values supports this result, where primary forest and logged-over forest almost have no difference in spectral reflectance values. a quite important variability of spectral reflectance values (mean and variance) obtained in landsat tm data is probably due to inhomogeneity and different geographical location of the training areas extracted from the images (jambi and west kalimantan). figures 13 and 14 show the distribution of the spectral reflectance from different training areas (ta) taken from two different geographical location using landsat tm and spot data. no clear pattern of spectral distribution classes is shown in these graphs. therefore, this spectral analysis should eventually be complemented with textural analysis, in order to distinguish the pattern of the vegetation mosaic according to dynamic phenomena. correlation between spectral reflectance and vegetation parameters tests of correlations between spectral reflectance values and vegetation parameters such as lai and biomass are performed to know the relevancy of data gathered from vegetation measurement in the field with respect to the spectral relectance response. from tables 6 & 7, it is obvious that all parameters tested are highly correlated, especially between bands tm3 and tm4 with biomass for the primary forest as well 29 spectral and textural characteristics upik r. wasrin et al. table 3. spectral reflectance values extracted i om spot and landsat tm images spot data spot xs-1 spot xs-2 spot xs-3 no. vegetation types no. of pixels ta mean sd mean sd mean sd 1 lowland primary forest 1413 15284 1. 2. 3. 17.00 16.68 0.70 0.78 45.00 31.42 1.10 1.04 64.00 74.40 3.90 6.08 average 16.84 0.74 38.21 1.07 69.20 4.99 2 logged-over forest 6245 6726 3252 1. 2. 3. 22.50 18.20 19.00 0.80 1.54 0.70 44.10 32.80 33.00 1.24 1.28 0.70 75.40 68.50 72.00 5.40 6.01 2.80 average 19.90 1.01 36.63 1.07 71.97 4.74 3 secondary forest 1960 4801 2041 1. 2. 3. 24.00 18.00 20.00 0.90 0.67 0.80 46.00 32.60 36.00 1.00 0.85 0.90 84.00 9.60 75.00 4.20 4.17 2.60 average 20.67 0.79 38.20 0.92 79.53 3.66 4 thickets & alangalang 1004 2258 1066 1. 2. 3. 25.00 18.20 24.00 0.70 0.76 1.00 48.00 29.20 39.00 0.90 0.28 1.30 98.00 95.60 74.00 2.80 4.70 4.10 average 22.40 0.82 38.73 0.83 89.20 3.87 5 plantation/ estate 714 2730 1. 2. 3. 18.20 18.00 0.75 0.80 31.80 32.00 0.90 1.00 82.50 77.00 2.48 2.90 " average 18.10 0.78 31.90 0.95 79.75 2.69 landsat tm tm-1 tm-2 tm-3 no. vegetation types no. of pixels ta mean sd mean sd mean sd 1 low primary forest 4994 2598 1. 2. 23.30 21.60 0.91 0.93 23.06 17.50 1.08 1.06 67.20 67.60 7.09 7.01 average 22.45 0.92 20.28 1.07 67.40 7.05 2 logged-over forest 3905 6763 1. 2. 20.40 21.95 1.26 0.93 20.40 17.88 1.54 1.18 60.30 65.28 6.96 6.41 average 21.18 1.10 19.14 1.36 62.79 6.69 3 secondary fore'st 3391 1046 1. 2. 24.70 23.60 0.81 0.88 24.60 19.10 1.08 0.93 76.70 79.90 4.00 5.75 average 24.15 0.85 21.85 1.01 78.30 4.88 4 thickets & alangalang 1955 1414 -1. 2. 23.73 27.30 1.34 1.31 26.50 21.70 1.44 1.20 90.50 99.40 6.93 8.15 average 25.52 1.33 24.10 1.32 94.95 7.54 5 plantation/ estate 2128 2760 1. 2. 23.80 26.33 0.84 0.89 23.90 22.84 1.05 1.00 77.50 89.84 3.88 4.90 average 25.07 0.87 23.37 1.03 83.67 4.39 30 spectral and textural characteristics upik r. wasrin et al. table 4. tukey student range (w) values to compare mean spectral value of each spectral band spot landsat tm xs-1 18.28 tm-1 5.27 xs-2 38.92 tm-2 9.96 xs-3 75.53 tm-3 16.98 table 5. tukey student range for mean spectral values with confidential level 95% as for the logged-over forest. the other vegetation parameters like structure of tree height, tree density and crown projection are not significantly correlated. the same analysis for the correlation between the spectral reflectance of spot data with the vegetation parameters showed similar results where biomass and lai have a good correlation with spot spectral values, while no significant correlationn was found between spectral reflectance and tree height, tree density and crown projection (tables 8 and 9). however, franklin and mcdermid (1993) found that there is a significant correlation between green band (xs-2) with mean diameter at breast height, height, age and volume of the pinus forest stand, while with the infrared band (xs-3) these stand parameters showed a poor correlation. as for landsat tm data, trotter et al. (1997) examined the relationship between wood volume of the coniferous stand and landsat tm data and gave the conclusion that landsat tm only provides an acceptable information for estimating the wood volume in plantation forest of a minimum 40 ha areas. while cohen and spies (1992) obtained a correlation coefficient between 0.45 and 0.88 for the relation between hrv panchromatic texture with stand attributes such as tree bole diameter, tree height, tree density, age and a structural complexity index; while for tm wetness the coefficient correlation is between 0.51 and 0.90. he also concluded that the lowest correlation is for tree 31 biotropia no. 13, 1999 figure 13. distribution of spectral reflectance. values of the treaining areas obtained from spot scene, k/j : 272/352 and 278/354 figure 13. distribution of spectral reflectance. values of the treaining areas obtained from landsat-tm scene, path/row : 126/061 and 121/060. 32 33 33 34 34 spectral and textural characteristics upik r. wasrin et at. height. this confirms the hypothesis that structural parameter is less important to support the study on spectral reflectance behavior of the vegetation. therefore, measurement of the spectral reflectance values directly above the forest canopy is highly recommended to get a significant correlation between spectral reflectance of high resolution satellite data with the spectral reflectance obtained from field measurement and the vegetation parameters. textural analysis textural analysis was done referring to spectral reflectance values which are randomly distributed on landsat tm and spot data. likewise, the vegetation parameters, especially lai and biomass were calculated inside each parcel as imitating the pixel of landsat tm and spot spatial resolution. table 10 shows the value of entropy level as a measure of desegregation of parcels or pixels inside the 6 ha observation plot in the primary forest and logged-over forest. both in the landsat tm and spot data, the entropy level of the logged-over forest is in general greater than those of the primary forest. it means that the perturbation occurring in the logged-over forest which appears in the form of mosaic of gaps (rigorousness or smoothness of the pixels/parcels) can be expressed by the value of "entropy level". the same aspect is equally found for lai and biomass where the entropy values are greater in logged-over forest compared to those of the primary forest, indicating the heterogeneity level of the parcels analyzed. this is supported by the findings of cohen and spies (1992) where tree bole diameter, tree density, age and structural complexity index have contributed to the texture of the stand but were not significant for the tree height. table 10. level of entropy of spectral reflectance values and vegetation parameters according to the pixel size of landsat tm and spot parcel/pixel parameters primary forest logged-over forest 35 blotropia no. 13, 1999 conclusions analysis of the spectral and textural patterns on landsat tm and spot high resolution data to differentiate the logged-over forest from the primary forest mainly from the point of view of vegetation parameters and its response to radiation wavelength gave interesting results. from this study, it is obvious that the spectral reflectance alone cannot distinguish the logged-over forest from the primary forest; but in complement with the textural pattern analysis, it is possible to separate this logged-over forest from the primary forest. for the practical implementation of this findings, further study is needed to translate this formula into image classifier hi the image processing module. acknowledgement the authors would like to express their sincere gratitude to the peer-reviewers and to the indonesian national research council for providing funds which made the conduct of this research possible from 1994 until 1996. cordial appreciation is also addressed to the barito pacific timber group who has kindly provided facilities during the field work. references cohen, w.b. and t.a. spies. 1992. estimating structural attributes of douglas-fir western hemlock forest stands from landsat and spot imagery. remote sensing of environment, 41(1): 1-17. franklin, s.e. and g.j. mcdermid, 1993. empirical relation between digital spot hrv and casi spectral response and logepole pine (pinus concorta) forest stand parameters. international journal of remote sensing, 14(12): 2331-2348. laumonier, y. 1992. the vegetation of sumatera. seameo biotrop spec. pub. bogor. ogawa h., t. kira, k. yoda and k. ogino, 1989. comparative ecological studies on three main types of forest vegetation in thailand. ii. plant biomass. in: t. kira and i. iwata (eds). nature and life in sea. fauna and flora res. society, kyoto, japan. torquebiau, e.f. 1988. photosynthetically active radiation environment, patch dynamics and architecture in a tropical rainforest in sumatera. aust. j. plant physiol., 15. trotter, c.m., j.r. dymond and c.j. gotjlding. 1997. estimation of timber volume in a coniferous plantation forest using landsat tm. international journal of remote sensing, 18(10): 2209-2223. 36 microsoft word 1 biotropia no. 24, 2005 : 1 19 aspergillus fla vus infection and aflatoxin contamination in peanuts at various stages of the delivery chains in cianjur regency, west java, indonesia okky setyawati dharmaputra seameo biotrop, p.o. box 116, bogor 16001, indonesia, and department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia ina retnowati, santi ambarwati and erita maysra seameo biotrop, p.o. box 116, bogor 16001, indonesia abstract a survey to obtain information on preand postharvest handling of peanuts at farmer, collector, wholesaler and retailer levels, including aspergillus flavus infection and aflatoxin bi contamination of peanuts collected in cianjur regency, west java, was conducted during the harvest period of the wet season of february 2004. the moisture contents and physical qualities of the peanuts were also determined. thirteen and 40 dry pod samples were collected randomly from 12 farmers and 23 collectors, respectively. seven dry kernel samples were also collected from collectors. five and 45 dry kernel samples were collected randomly from 2 wholesalers and 45 retailers in traditional markets, respectively. thus, a total of 110 dry peanut pod and kernel samples were collected. the results of interviews with farmers, collectors, wholesalers and retailers, and also the moisture contents and physical qualities of the peanuts arc described in this article. the percentages of samples infected by a. flavus were highest at the wholesaler as well as at retailer levels (100%, respectively), followed by those sampled at the collectors (85.0 and 85.7%, respectively), and farmers (84.6%). the mean percentage of infected kernels in infected samples of peanuts collected from retailers was the highest (87.6%), followed by those collected from wholesalers (72.4%), collectors in the form of kernels (23.3%) and pods (17.7%), and farmers (15.2%). the range of aflatoxin bi contents in peanut samples collected from farmers (dry pods), collectors (dry pods), wholesalers (dry pods and kernels) and retailers (dry kernels) were < 3.6 -114.2, < 3.6 -2999.5 and < 3,6 34.1, < 3.6 6065.9, and < 3.6 6073.0 ppb, respectively. the highest aflatoxin b, contents at the wholesaler and retailer levels were 6065.9 ppb (in one sample) and 6073.0 ppb (in one sample), respectively. the percentage of samples contaminated with more than 15 ppb of aflatoxin bi was the highest in peanuts collected from wholesalers (80.0% of samples), followed by retailers (75.6%), farmers (38.5%) and collectors (30.0 and 14.3%). in 1999 codex alimcntarius commission determined that the maximum total aflatoxin content in peanuts intended for further processing is 15 ppb, suggesting that an alarming proportion of peanuts throughout the indonesian food chain arc in excess of this maximum limit. key words : ,4,spergillius flavus/aflatoxin/peanuts/cianjur regency introduction peanuts are next to maize and soybean as the most important secondary crop in indonesia. since indonesia has a humid tropical climate, peanuts can easily be 1 biotropia no. 24, 2005 infected by moulds (including a. flavus) under drought stress before harvest, during the drying phase in the field, or under poor storage conditions. aflatoxin has been recognized as a human and domestic animal carcinogen, and is produced following infection of peanuts by certain strains of a. flavus. pitt and hocking (1996) reported that 45% of 215 peanut samples collected from farm storage, middlemen and retailers in bogor (west java), yogyakarta (central java), and their surroundings, contained more than 50 ppb of aflatoxin, 33% more than 300 ppb, and 22 % exceeded 1000 ppb. the total annual cost of aflatoxins in peanuts in 1991 in indonesia, philippines and thailand was estimated as about $ a 158 million. indonesia incurred 84% (= $ a 132 million) of this cost (lubulwa and davis 1994). according to dharmaputra et al. (2003a), in general, aflatoxin bi contents of peanuts collected from farmer's fields/pewefoas/collectors and processed samples in the pati regency of central java were low (less than 15 ppb). the highest aflatoxin b| contents were found in raw peanut kernels collected from retailers in traditional markets, ranging from 2-124 and < 4 342 ppb during the wet and dry seasons in 2002, respectively. the percentage of raw kernel samples contaminated with aflatoxin bi (exceeding 15 ppb) collected during the wet and dry seasons was 33 and 25%, respectively. another study was also carried out by dharmaputra et al. (2005) on aflatoxin bi contents of peanuts collected from farmer's fields, collectors and retailers in the wonogiri regency and specifically the city of surakarta (central java) during the wet and dry seasons in 2003. the results also showed that the highest aflatoxin b, contents were found in raw peanut kernels collected from retailers in traditional markets, with the range of < 3.6 1859.3 and < 3.6 5511.5 ppb during the wet and dry seasons, respectively. the percentage of raw kernel samples contaminated with aflatoxin bi (exceeding 15 ppb) collected during the wet and dry seasons was 33 and 76 %, respectively. the 23rd session of the joint fao/who food standards programme held in rome, italy (28 june-3 julyl999) reported that codex alimentarius commission has adopted a maximum level of total aflatoxins in peanuts intended for further processing at 15 ppb. on 9 september 2004 the national agency for drug and food control, republic of indonesia has determined that aflatoxin bi and total aflatoxin contents in processed peanut products should not be more than 20 and 35 ppb, respectively. to minimize or to reduce aflatoxin contamination in peanuts, appropriate post-harvest handling methods in each level of peanut delivery chain (farmer, collector, wholesaler, and retailer) should be carried out. the objective of this study was to obtain information on preand postharvest handling methods, aspergillus flavus infection and aflatoxin bi contamination of peanuts collected from different points of the delivery chains in cianjur regency of west java. the moisture contents and physical quality of peanut kernels were also determined. aspergillusflavus infection and aflatoxin contamination in peanuts — okky s. dharmaputra et al. materials and methods time and location of surveys surveys were conducted during the harvest of the wet season (february 2004) at cidaun, naringgul and sindangbarang districts located in cianjur regency, and the city of cianjur, west java. based on the information obtained from the indonesian government regional office for food crops of west java province, peanut production was high in cianjur regency and it ranks second after garut regency. peanut production was high at cidaun, naringgul and sindang barang districts according to the information obtained from the indonesian government regional office for food crops of cianjur regency. the surveys comprised: • interviews, using questionnaires, with farmers, collectors, wholesalers and retailers. the questionnaires consisted of questions relating to preand postharvest handling of peanuts. • random sampling of various kinds of peanut products collected from farmers, collectors, wholesalers and retailers who were interviewed. the moisture contents, physical quality of kernels, fungal (a. flavus) infection and aflatoxin b, contents from each sample were analyzed. sampling methods based on differences of storage duration, more than one peanut sample could be obtained from each farmer, collector and wholesaler. the kind of peanuts sampled included dry pods and kernels. samples of dry pods (about 2 kg each) were divided three times manually and homogeneously to obtain working samples (about 250 g each) for analyzing moisture contents, physical quality of kernels, percentage of kernels infected by a. flavus, aflatoxin bi content, and a reserve sample. the dry peanut pods were then shelled manually. samples of dry peanut kernels (about 1 kg each) were also divided three times using a box divider to obtain working samples (about 125 g each) for analyzing moisture contents, physical quality of kernels, percentage of kernels infected by a. flavus, aflatoxin bi content, and a reserve sample. moisture content, physical quality of kernels, a. flavus and aflatoxin bi analyses moisture contents of kernels (based on a wet basis) were analyzed using a sinar tm ap 6060 moisture analyzer. the moisture contents of some samples were confirmed using the oven method (bsi 1995). two replicates were used from each sample. biotropia no. 24, 2005  physical  quality  of  kernels was  assessed  in  intact,  shriveled  and  damaged  kernels.  the  damaged  kernels included cracked, broken, discoloured, and damage caused by insects or fungi. the percentage of  each category of kernels was determined by counting them and dividing the total number of kernels used  for physical quality analysis.  the percentage of kernels infected by a. flavus was determined using a plating method (100 kernels per  sample) on aspergillus flavus and parasiticus agar (afpa) (p'metal. 1983).  aflatoxin bbt was analyzed because  it  is  the most dangerous  toxin. aflatoxin b,  contents  in  the kernels  were determined using the el1sa method (lee and kennedy 2002), with two replicates used for each sample.  results and discussion source, kind and number of samples  at the farmer level (12 farmers), 13 samples of dry peanut pods were collected, while at collector level (23  collectors),  40  samples  of  dry  pods  and  7  samples  of  dry  kernels  (1  kg/sample)  were  collected.  at  the  wholesaler and retailer levels (2 wholesalers and 45 retailers), 5 and 45 samples of dry peanut kernels were  collected,  respectively. thus,  the  total number of  samples was 110. details of  the peanut delivery  chain,  location and number of peanut samples are presented in table 1.      aspergillus flavus infection and aflatoxin contamination in peanuts — okky s. dharmaputra et at.  results   of  interviews   with   farmers,   collectors,   wholesalers   and   retailers concerning pre‐ and  postharvest handling of peanuts  interview with farmers  the results from farmer interviews (12 respondents) are presented in table 2. all farmers (100%)  planted a local variety of peanuts, with most of the seed sources from farmers (83% of respondents).  fanners harvested their peanuts at 90 to 100 days after planting. during planting, all of respondents  used fertilizer, such as urea, tsp and k.c1. weed control was conducted manually. farmers sun‐dried  peanut  pods  for  3  ‐  4  days.  most  of  the  farmers  used  woven  polypropylene  bags  (67%  of  respondents)  to  dry  peanuts.  based  on  their  experiences,  farmers  could  determine  when  their  peanuts were  fully  dried  for  safe  storage.  sixty  seven  percent  of  respondents  stored  peanuts  in  woven polypropylene bags for 1 ‐ 7 days before selling to collectors. all respondents sold peanuts to  collectors  in  the  form  of  dry  pods.  interestingly,  all  respondents were  not  aware  of  the  aflatoxin  problem in peanuts.        interview with collectors the result of interview with collectors (23 respondents) are presented in table 3.based on their experience the collectors knew whether their peanuts were fully dry. before selling to wholesalers, peanuts were stored in woven poly propylene bags (91% respondents), or by spreading them on paved floor (9% of aspergillus flaws infection and aflatoxin contamination in peanuts okky s. dharmaputra et ai. respondents) for 1-30 days. peanuts were sold to wholesalers in the form of dry pods (35% of respondents), or as dry pods and kernels to wholesalers and retailers (9% of respondents) in the city of cianjur. peanuts were also sold to wholesalers in the form of dry pods (43% of respondents), and to wholesalers and retailers in the form of kernels (4% of respondents) in the big cities of west, central and east java. all respondents shelled peanut pods using a diesel powered sheller. most collectors (87% of respondents) sorted peanuts manually before selling them to wholesalers. all respondents were not aware of aflatoxin problem in peanuts. interview with wholesalers the results of interview with wholesalers (2 respondents) are presented in table 4. at the wholesaler level peanuts were stored in woven polypropylene bags (50% of respondents), as well as in woven polypropylene and jute bags (50% of respondents). the stacks of bags containing peanuts were not placed on pallets. peanuts samples were stored for 1 month (50% of respondents) and 1-2 months (50% of respondents). fifty percents of the respondents sold only peanuts, while 50% of respondents sold peanuts and other commodities such as wheat flour and peanut oil. peanuts were sold to smaller wholesalers in the cities of bandung, bogor and jakarta. interview with retailers the results of interview with retailers (45 respondents) are presented in table 5. fifty three percent of respondents bought their peanuts from wholesalers, while 47% of respondent bought peanuts from farmers and collectors. peanuts were stored in woven polypropylene bags (71% of respondents) and jute bags (29% of respondents) for 1 7 days. containers used at the time of sampling were rectangular plastic basins (42% of respondents), winnowing trays (29% of respondents), wooden boxes (18% of respondents), round plastic basins (5% of respondents), woven polypropylene bags (4% of respondents) and jute bags (2% of respondents). aside from peanuts, retailers also sold other general commodities. peanut buyers included sellers of peanut sauce products for making gado-gado, pecel and sate, as well as house wives. retailers were also not aware of the aflatoxin problem in peanuts.   moisture contents, physical quality of kernels, the incidence of a. flavus and aflatoxin contamination  moisture contents according to diener and davis (1969) moisture content is an important factor affecting the growth of a. flavus and aflatoxin production. the range and mean moisture contents of kernels derived from various kinds of peanuts collected from farmers, collectors, wholesalers and retailers are presented in table 6 and figure 1. the moisture contents of kernels collected from farmers was relatively similar to those collected from collectors and retailers, while the moisture contents of kernels collected from wholesalers was the lowest. this was due to the storage duration of peanuts at farmer and collector levels which were relatively shorter than those at the wholesaler level (tables 2, 3 and 4). the moisture contents of kernels were always in equilibrium with the relative humidity of storage room. the range and mean moisture contents of kernels derived from various kinds of peanuts and collected from farmers, collectors, wholesalers and retailers were 5.9 9.9% and 8.4%; 7.8 -9.6% and 8.7%; 8.1 9.4% and 8.9%; 6.2 8.8% and 7.5%; 6.5 9.3% and 8.5%, respectively (table 6). in general, the mean of moisture content of peanut kernels in each delivery chain were considered safe for storage. sni (1995) determined that the safe moisture contents for storage of peanut pods and kernels were 9 and 8%, respectively. 10 aspergillus flavus infection aflatoxin contamination in peanuts – okky s. dharmaputra et al moisture content of pods and kernels are closely related with the drying process. sun-drying is the most critical postharvest handling procedure for peanuts, especially when the harvest coincides with the wet season. according to wongvirajtana et al. (1993) the duration of sun-drying can significantly affect fungal growth in grains, with the longer the period of sun-drying, the more chance for the fungi to infect the grains. physical quality of kernels range and mean of physical quality characteristics of kernels derived from various kinds of peanuts collected from farmers, collectors, wholesalers and retailers are presented in table 6, and figure 2. the percentage of intact kernels of peanuts collected from farmers was the highest, followed by those collected from collectors, wholesalers and retailers. this was probably due to the peanuts which have not being shelled using mechanical or diesel powered sheller at the farmer level, while the duration of storage of peanuts was relatively short (< 7 days). the percentages of shriveled kernels in each of the delivery chain were relatively the same (more than 22%). sni (1995) determined the maximum percentage of shriveled kernels is 4%, consequently the percentage of shriveled kernels in each delivery chain can be categorized as very high. this was probably due to the early harvest. results of interviews with farmers showed that 59% of respondents harvest peanuts 90 days after planting (table 2). peanuts harvested before full maturity will tend to produce 12 shriveled kernels after drying, consequently the kernels could be more easily infected by fungi. the percentage of damaged kernels collected from retailers was the highest, followed by that collected from wholesalers, collectors and farmers. the damaged kernels could have been caused by insects, rodents and fungal attacks, and inappropriate equipment used for the shelling of pods. the highest percentage of damaged kernels at the retailer level was probably due to the longer duration of postharvest handling from farmer up to the retailer. the range and mean percentages of intact kernels derived from various kinds of peanuts and collected from farmers, collectors, wholesalers and retailers were 59.6 83.9% and 74.8%; 42.2 83.0% and 70.0%; 56.2 77.3% and 69.0%; 37.8 -93.4% and 63.6%; 29.3 73.0% and 56.3%, respectively. the range and mean-percentages of shriveled kernels derived from various kinds of peanuts and collected from farmers, collectors, wholesalers and retailers were 14.2 36.6% and 22.9%; 13.7 56.4% and 28.0%; 19.7 41.7% and 27.9%; 3.3 50.7% and 29.0%; 15.9 -39.0% and 29.0%, respectively. the range and mean percentages of damaged kernels derived from various kinds of peanuts and collected from farmers, collectors, wholesalers and retailers were 0.6 4.9% and 2.3%; 0.6 5.6% and 2.2%; 2.1 4.5% and 3.1%; 3.2 15.6% and 7.4%; 4.1 40.3% and 14.7%, respectively. 13 biotropia no. 24, 2005 the incidence o/a. flavus the percentage of samples infected by a. flavus, range and mean percentages of infected kernels in infected samples of peanuts collected from farmers, collectors, wholesalers and retailers are presented in table 6, figures 3 and 4. 14 aspergillus flaws infection and aflatoxin contamination in peanuts okky s. dharmaputra et al. the percentage of samples infected by a. flavus was highest in peanuts collected from wholesalers and retailers (100%, respectively), followed by those collected from collectors in the form of pods (85%) and kernels (85.7%), and farmers (84.6%). the mean percentage of infected kernels in infected samples of peanuts collected from retailers was the highest (87.6%), followed by those collected from wholesalers (72.4%), collectors in the form of kernels (23.3%) and pods (17.7%), and farmers (15.2%). the highest percentage of peanut samples and mean percentages of infected kernels in infected samples collected from retailers was related to the methods of postharvest handling from farmers up to retailers, as well as the duration of storage. peanut kernels were more easily infected by fungi compared to unshelled peanuts. the damaged kernels were more easily infected by fungi compared to intact kernels. dharmaputra and retnowati (1996) observed that the range in percentage of peanut kernels infected by a. flavus in samples collected from retailers in some locations in west java during the wet season was 83 100%. aspergillus flavus was found in 98% of 256 peanut kernel samples and in 61% of all examined kernels collected from retailers in some locations in west and central java (pitt e? al. 1998). according to dharmaputra et al. (2003) 24 raw kernel samples collected from retailers in traditional markets located in bogor, pati, yogyakarta and malang were 100% infected with a. flavus during both the wet and dry seasons, respectively. dharmaputra et al. (2005) also reported that 98 and 100% of 54 raw peanut kernel samples collected from retailers during the wet and dry seasons in wonogiri regency, west java, were infected by a. flavus, respectively. aflatoxin b: contamination the range of aflatoxin b| contents in peanut kernels derived from various kinds of peanuts and collected from farmers, collectors, wholesalers and retailers is presented in table 6. the range of aflatoxin b, contents in peanut samples collected from retailers (< 3.6 6073.0 ppb) was the widest, but almost similar to those collected from wholesalers (< 3.6 6065.9 ppb). the percentage of samples contaminated with different levels of aflatoxin b| is shown in table 7. in australia the maximum allowable limit of aflatoxin in peanut and peanut products is 15 ppb (qdpi 2000). the percentage of samples contaminated with more than 15 ppb of aflatoxin bj was highest in peanuts collected from wholesalers (80% of samples), followed by retailers (75.6%), farmers (38.5%) and collectors in the form of pods and kernels (30.0 and 14.3%, respectively), although the highest percentage of samples infected by a. flavus was found in peanuts collected from retailers, followed by those collected from wholesalers, collectors and farmers. this may have been associated with differences in the existence of toxigenic strains of a. flavus. according to pitt and hocking (1997) aflatoxin production depends on the toxigenicity of strains of a. flavus. the presence of antagonistic fungi to toxigenic a. flavus could also inhibit aflatoxin production. dharmaputra et al. (2001) reported that a. niger was the most promising 15 biotropia no. 24, 2005  antagonistic  fungus  to  toxigenic  strains  of  a.  flavus,  because  it  inhibited  aflatoxin  production up to 80% under in vitro conditions. the percentages of peanut samples infected  by a. niger at farmer, collector, wholesaler and retailer levels in cianjur regency were 69.2,  80 and 85.7, 60 and 76.5%, respectively.  at  the  farmer  level, no  sample was contaminated with aflatoxin bj at more  than  1000 ppb. at  the  collector, wholesaler  and  retailer  levels, 1  sample  (2.5% of  collected  samples), 3 samples (60% of collected samples) and 7 samples (15.6% of collected samples)  contained more than 1000 ppb of aflatoxin bi, respectively. at the collector level, aflatoxin  bi content in one sample was 3000 ppb. the highest aflatoxin bi content measured at the  wholesaler and retailer level was 6066 ppb (in one sample) and 6073 ppb (in one sample),  respectively. pitt and hocking  (1996) concluded  that more  than 1000 ppb of aflatoxin  could cause acute toxic both in humans and animals.  although the percentage of samples contaminated with aflatoxin b i exceeding 15 ppb  at  the  farmer  level  (38.5%) was higher  than  that at  the collector  level  (30%), aflatoxin bi  contents more  than 500 ppb were  found  in 4 peanut samples collected  from collectors,  while the highest aflatoxin b] content at  farmer  level was 114.2 ppb. this  results  showed  that aflatoxin bi contents at the collector  level are generally higher than at the farmer  level, probably because  the duration of  storage at  the  collector  level was  longer, and  under poorer storage conditions compared to that at the farmer level (tables 2 and 3).  at the farmer level, 2 peanut samples were not infected by a. flavus, but they were  contaminated with  aflatoxin b,  (table  6).  it was  therefore  assumed  that  these peanuts  were infected by a. flavus before harvest, or during sun‐drying, and contaminated with  aflatoxin  b!  during  sun‐drying.  further  sun‐drying,  and  in  the  presence  of  antagonistic  fungi to a. flavus, could inhibit or kill a. flavus. therefore,  16    aaspergillus flavus infection and aflatoxin contamination in peanuts okky s. dharmaputra et al. no a. flavus was isolated using afpa media, but aflatoxin b, still existed. buchi and rae (1969) found that aflatoxin could only be degraded at 268 269°c. conclusions in cianjur regency farmers dried (sun-drying) peanut pods until safe moisture contents were obtained, and sold peanuts to collectors in the form of dry pods. in general, farmers stored peanuts in woven polypropylene bags for 1 7 days before selling to collectors. collectors sold the peanuts to wholesalers and retailers in the form of dry pods and kernels. collectors shelled the peanut pods using diesel powered shellers. at the wholesaler level, peanuts were stored in woven polypropylene and jute bags. in general, stacks of bags containing peanuts were not placed on pallets. retailers stored peanuts in woven polypropylene and jute bags for 1 7 days. most retailers used rectangular plastic basins as containers when the peanut sampling was being conducted, followed by winnowing trays, wooden boxes, round plastic basins, woven polypropylene bags and jute bags. the mean moisture contents of peanut kernels derived from various kinds of peanuts in each part of the delivery chain were considered to have safe moisture contents. the percentages of intact peanut kernels collected from farmers was the highest, followed by those collected from collectors, wholesalers and retailers. the percentages of shriveled kernels collected from different parts of the delivery chains were relatively similar. the percentage of damaged kernels collected from retailers was the highest, followed by that collected from wholesalers, collectors and farmers. the percentages of samples infected by a. flavus in peanuts collected from wholesalers and retailers were the highest (100%, respectively), followed by those collected from collectors and farmers. the mean percentages of infected kernels in infected samples collected from retailers were the highest, followed by those collected from wholesalers, collectors and farmers. the range of aflatoxin bi contents in peanut samples collected from retailers (< 3.6 6073.0 ppb) was the widest, but almost similar with that collected from wholesalers (< 3.6 6065.9 ppb). the percentage of samples contaminated with more than 15 ppb of aflatoxin b) collected from wholesalers (80.0% of samples) was the highest, followed by retailers (75.6%), farmers (38.5%) and collectors in the form of pods and kernels (30.0 and 14.3%, respectively). our results show that postharvest handling methods employed prior to peanuts being delivered to wholesalers and retailers will have a severe impact on the level of aflatoxin contamination in peanuts in different parts of the delivery chain. postharvest handling methods that minimize kernel infection by a. flavus and aflatoxin contamination, especially at wholesalers and retailers in traditional markets should be employed to minimize aflatoxin contamination for indonesian peanut consumers. 17 b1otropia no. 24, 2005 acknowledgements the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the indonesian government regional office for food crops of west java province, head and staff of the indonesian government regional office for food crops of cianjur regency , especially to mr. jayanudin for the information given relating to the survey; head and staff of the indonesian government subregional office for food crops at cidaun, naringgul and sindangbarang districts, cianjur regency, for their cooperation during the survey; to drs. ivan r. kennedy and nanju alice lee of the university of sydney, australia; to prof.dr. rizal syarief and dr. graeme wright who have reviewed this article; and to the technicians of the laboratory of plant pathology, seameo biotrop; mr. faizal arifin nur akbar and mr. pujo waluyo for their assistance. references bsi. 1995. oilseeds-determination of moisture and volatile matter content. british standard international. 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(eds). mycotoxin contamination in grains. aciar technical reports 37, canberra, p. 5 10. pitt, j.i. and a.d. hocking. 1997. fungi and food spoilage. blackie academic and professional, london. pitt, j.i., a.d. hocking, b.f. miscamble, o.s. dharmaputra, k.r. kuswanto, e.s. rahayu and sardjono. 1998. the mycoflora of food commodities from indonesia. journal of food mycology i (1): 41 -60. qdpi. 2000. aflatoxin in peanuts; tips to reduce the risk. crop link. queensland department of primary industries and fisheries, farming systems institute, kingaroy. (see www.qld.gov.au/ fieldcrops/3027.html). standar nasional indonesia (sni). 01-39219-1995. 1995. kacang tanah. dewan standardisasi nasional, jakarta. wongvi raj tana, p., s. soponronnarit and a. nathakaranakule. 1993. feasibility study of in-store corn drying under tropical climates. in naewbanij, j.o., a.a. manilay and a.s. frio (eds). increasing handling, processing and marketing efficiency in the grain postharvest system. proceedings of the 16'h asean seminar on grain postharvest technology. phuket, 24 26 august 1993. asean grain postharvest programme, bangkok, p. 265 283. 19 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf 10.pdf 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 4. sri sulandari (mitochondri... biotropia vol. 19 no. 2, 2012: 92 102 mitochondrial dna variation of the sumatran elephant populations in sumatera, indonesia sri sulandari and moch. syamsul arifin zein recipient of biotrop research grant 2010/accepted 11 september 2012 genetic analysis of mitochondrial dna diversity in sumatran elephant ( ) was conducted. a 630 bp segment of mitochondrial dna was amplified from 105 different sumatran elephant samples from 5 locations in sumatera (bentayan, sugihan, bukit salero lahat, seblat, way kambas) using a set of primers: mdl3 (5'-cccacaattaatgggccc-ggagcg-3') and mdl5 (5'-ttacatgaattggcagcca-accag3'). the objectives of this study were to generate mitochondrial dna d-loop sequences for all available sumatran elephant samples and to define haplotypes and nucleotide sequence diversity of the different sumatran elephant populations. the nucleotide sequence of a total of 105 pcr products were successfully determined with an average length of 616 bp. however, mitochondrial dna fragments for this analysis used the first 601 bases. six different haplotypes (bp, bt, bs, br, bx and by) were identified in sumateran elephant populations. the majority of the sampled individuals carried haplotype bt. bx and by are most likely novel derived haplotypes. all haplotypes, except for the haplotype bp belong to the sumatera clade. the haplotype bx was derived from the haplotype bt, and the haplotype by was derived from the haplotype bs by one transversion, respectively. all the other substitutions identified in this network were transitions. the haplotype bp is widely distributed from sri lanka, sumatera, peninsular malay and china. although reported to be distributed in sumatera and peninsular malay the haplotype bu was not detected among the samples analysed in this study. genetic distances within populations in bentayan, bukit salero lahat, seblat, sugihan and way kambas ranged from 0.0000 0003, and the genetic distance between the populations that is 0.0000 0.0022. the distance between haplotypes of different sumatran elephant populations was shown to be low. the diversity of haplotypes and nucleotides in sumatera island were low, the highest diversity was found in elephants sampled in the region of bukit salero lahat and the lowest was found in elephants from bentayan and sugihan. overall, the results of analysis of fu and li's f*test statistic indicates that the population of sumatran elephants in sumatra is -0.78871, which suggests that there is no inbreeding. however, the results are not significant (p> 0:10) and additional studies are required to confirm this finding. sumatran elephant, , mitochondrial dna, haplotype * research center for biology (rcb), the indonesian institute of sciences (lipi) cibinong science center, jalan raya jakarta bogor km.46, cibinong 16911, indonesia elephas maximus sumatranus elephas maximus sumatranus abstract key words: * corresponding author : ssulanda@yahoo.co.id 92 introduction the asian elephant is an endangered animal and listed on appendix i of cites (convention on international trade in endangered species) of wild fauna and flora. sumatran elephants ( ) is one of four different asian elephant subspecies in indonesia. today, the sumatran elephant is found in seven main provinces, namely nanggroe aceh darussalam, north sumatra, riau, jambi, bengkulu, south sumatra, and lampung (soehartono . 2007). the main serious threats to sumatra's elephant populations are among others forest/habitat loss, illegal hunting, poaching for ivory, and habitat degradation due to conversion of critical forest habitat to agriculture/plantation areas. also many elephant populations are trapped in small pockets which are not enough to support their life, and the condition has sparked conflict between humans and elephants. elephants raid people's crop, destroy their houses, and sometimes kill or injure people. in response, elephants are killed and the local communities persuade the government to catch and remove elephants from the wild. therefore, information on status and distribution of sumatran elephant populations, including information on genetic diversity of the remaining populations are urgently needed to determine conservation policy. molecular technology development at the moment has increased the efficiency and accuracy in the genetic characterization study among breeds of animals. mitochondrial dna (mtdna) sequences have been extensively and successfully used to determine genetic diversity. mitochondrial dna is passed to offspring by matrilineal inheritance of mitochondria via the oocyte. different regions of the mtdna evolve at different rates. the area displacement-loop (d-loop) has variation which is quite high, more polimorphic compared to other area mtdna (ishida . 1994; quinn & wilson 1993). as statement revealed by brown . (1982), quinn dan wilson (1993) and akhisinomiya (1994), d-loop area is often used for phylogenetic analysis, both inside the species and among species. therefore, much genetic variation can be expected between individuals of the same species. molecular techniques have been used to evaluate the diversity of asian elephants across or within populations. maternal inherited mitochondrial dna (mtdna) (fernando & lande 2000; fernando . 2000, 2003; fleischer . 2001; vandebona . 2002; vidya & sukumar 2005; vidya . 2005a, b, 2007; fickel . 2007) revealed several haplotypes, which belong to two haplogroups or clades. in thailand, eight haplotypes from two clades were found in 82 captive elephants (lertwatcharasarakul 2003), while mtdna control region sequences from 78 captive elephants represented 20 haplotypes (fickel . 2007). fernando . (2000, 2003) identified 27 different haplotypes within the asian elephant population by sampling over three hundred elephants from sri lanka, bhutan/north india and laos/vietnam, malaysia, thailand, bangladesh and cambodia. these haplotypes are/clustered into two well-differentiated assemblages/clades, α and β. these assemblages corresponded to the clades that were found by vidya . (2005) using mtdna sequencing. fleischer . (2001) also found these two distinct assemblages by sequencing mtdna. elephas maximus sumatranus et al et al et al et al. et al et al et al et al et al et al. et al et al et al et al 93 mitochondrial dna variation of the sumatran elephant populations – sri sulandari & moch. syamsul a.z. however, studies that analyze the sumatran elephant based on mitochondrial dna are still limited, such as the studies of fernando . (2003) and fleischer . (2001). there was also a study among 27 elephants (17 samples from alas nepal and 10 samples from way kambas) reported by okayama . (2001) on mitochondrial dna analysis of sumatran elephant, stated that sumatran haplotypes were more similar to sri lanka haplotypes than mainland asian haplotypes. haplotype frequencies were different between populations of alas nepal and way kambas. unfortunately, there were no results on genetic differentiation of sumatran population, because sampling location and size are too small to estimate genetic differentiation in sumatra. furthermore, genetic analysis of the sumatran elephant populations comprehensively sampled from aceh to lampung has not yet been reported. therefore, a research on mitochondrial dna analysis of genetic diversity within maternal lines of different populations of sumatran elephant ( ) was conducted in this study, in order (1) to generate mtdna d-loop sequences for all the sumatran elephant samples under this study and (2) to provide information on haplotypes and degree of nucleotide sequence diversity of sumatran elephant populations. to conduct the study, the highly variable d-loop region of mtdna on 105 samples of sumatran elephant from 5 locations in sumatra was analyzed in this study. all results obtained in this study should be useful for conservation strategies, particularly for sumatran elephants. it is believed that diversity is only secure when diverse conservation strategies are employed. a total of one hundred and five (105) dna material blood of sumatran elephants ( ) were collected in 5 different locations of elephant conservation center ( table 1). the samples were preserved in 96 % absolute ethanol and stored at the bank of material dna for indonesian fauna, genetic laboratory, division of zoology, research center for biology-lipi. the samples were collected in 2000 by t. okayama in collaboration between biodiversity conservation project (bcp) japan international cooperation agency (jica) and research center for biology (rcb) lipi. a list of the samples is shown in table 1. et al et al et al elephas maximus sumatranus elephas maximus sumatranus et al. materials and methods samples and study areas table 1. list of samples of sumatran elephants ( ) collected from 5 locations. elephas maximus sumatranus no. elephant conservation center province total samples 1 sugihan (su) south sumatra province 18 2 bentayan (be) south sumatra province 20 3 seblat (se) bengkulu province 21 4 bukit serelo lahat (bsl) south sumatra province 24 5 way kambas (wk) lampung province 22 total samples 105 biotropia vol. 19 no. 2, 2012 94 dna extraction, amplification and sequencing data analysis dna was extracted from whole blood using qiagen ”dneasy®blood & tissue kit. amplification of mitochondrial dna was conducted by the method of polymerase chain reaction (pcr), using thermal cycler 2700 (applied biosystems). a 630 bp segment of mtdna was amplified using a set of primers, mdl3 (5'-cccacaattaat-gggcccggagcg-3') and mdl5 (5'-ttacatgaattggcagccaaccag-3') (fernando ., 2000), and subsequent nucleotide sequencing of the amplified fragment. these primers amplify a 630-bp fragment of mtdna, including the d-loop. fernando (2000 & 2003) stated that the first 109 bp of the fragment code is for the c terminal end of cytochrome b, the next 135 bp code for threonine and proline trna's, the rest of the fragment corresponds to the non-coding control region that is the d-loop. pcr amplification was performed using a mixture of 30 l reactions containing of 50 mg sample dna, pcr buffer 1 x 200 um dntps, 2 mm mgcl and 1 unit taq dna polymerase (fermentas, native with bsa). the amplified fragment was purified, and subsequently the nucleotide sequence was determined. the nucleotide sequences obtained, were aligned, edited and analyzed including definition of genetic diversity, genetic distance (estimate of the number of nucleotide substitutions per nucleotide site between two sequences), and phylogenetic analysis by the neighbour joining method. the haplotypes determined in this study was compared to the haplotypes identified by both fernando (2000 & 2003). and vidya (2005). the accession numbers for the fernando . haplotypes as deposited in genbank ( ) are: ay245538 and ay245802 to ay245827, and for vidya haplotypes as deposited in genbank are ay365432 and ay365433. trees were rooted using wooly mammoth (genebank nc007596, krause . 2006 and dq316067, rogaev . 2006), african forest elephant (genebank jf827275, ishida ., 2011) and african savanna elephant genebank af527654, eggert . 2002 , and genebank dq316069, rogaev . 2006) as an outgroup. a fragment of the d-loop mtdna corresponding to the first 601 bp was used for analysis in this study. nucleotide sequence data was obtained after editing of the fragment d-loop sequence. sequence data was analysed with various kinds of computer software. chromas which available on http://www.technelysium.com.au/chromas.html was used for and editing the sequence result. the clustalx 1.83 was used for multiple alignment of sequences (thompson 1997; and obtained from ftp://ftpigbmc:u-strasbg.fr/pub/clustalx). macclade 4.0 was used to make the polymorphic sites (maddison and maddison, 2000 and available on http://ag.arizona.edu/ macclade/macclade.html). molecular evolutionary genetic analysis (mega) version 3.0 was used for phylogenetic and analysis of the molecular evolution (kumar . 2004, available on http://www.megasoftware.net/), while network analysis was used for illustrating haplotype diversity i.e. network 4.1.0.8 (bandelt . 1999). et al et al. et al. et al. et al et al. et al et al el al et al et al viewing et.al. et.al et.al � 2 http://www.ncbi.nlm.nih.gov mitochondrial dna variation of the sumatran elephant populations – sri sulandari & moch. syamsul a.z. 95 genetic diversity indices of sumatran elephants including haplotype diversity, haplotype frequencies, nucleotide diversity, and fu and li's f*test (1993), were carried out using dnasp version 4.0 (rozas ., 2003; available at http://www.ub.es/dnasp). a total of 105 pcr products from the samples of sumatran elephants were successfully sequenced, with an average length of about 616 base pairs. the mtdna fragments used for this analysis corresponds to the first 601 bases. table 2 and figure 1 show the six (6) haplotypes found in sumatran elephants (bp, bt, bs, br, bx and by) and identified at 23 the sites of the polymorphic (variable sites) (table 3). all haplotypes except for the haplotype bp (bsl-20 and bsl-23) are belonging to the sumatera clade. the haplotype bp which belongs to the continental clade (fig. 2) is widely distributed from sri lanka, sumatera, peninsular malay (fernando . 2000, 2003, vidya . 2009), and china (yonezawa . unpublished). although fernando . (2000 & 2003), and vidya . (2009) reported that the haplotype bu is distributed in sumatera and peninsular malay, but bu haplotype could not be detected in the samples analysed in this study. et al et al et al et al et al et al results and discussions table 2. haplotype distribution of 105 sequence of mitochondrial dna no haplotype individual with same haplotypes number haplotypes % 1 bt be8,be19,be17,be15,be5,be7,be2,be4, be10,be1,be13,be6,be16,be14,be20, be18, be3,be9,be11,be12 20 91,40% bsl17,bsl8,bsl12,bsl1,bsl7,bsl16, bsl13,bsl2,bsl4, bsl11,bsl15,bsl19,bsl14, bsl10,bsl6, bsl9, bsl3, bsl5, bsl18 19 su2, su5,su11, su6,su15,su3,su10,su17,su8,su12,su13,s u7,su9,su1,su4,su14, su16, su18 18 wk19,wk16,wk1,wk20,wk21,wk18,wk 7,wk14,wk15, wk9,wk12, wk17, wk5, wk11, wk3, wk8, wk13 19 se9,se20,se12,se4,se14,se18,se2,se13, se5,se3,se17,se15,se21,se8, se7,se6,se10,se16,se1,se11 20 2 bs wk10,wk22 2 1,90% 3 br bsl21,bsl22,bsl24 3 2,90% 4 bp bsl20,bsl23 2 1,90% 5 bx se19 1 0,95% 6 by wk4 1 0,95% total 105 100% notes: wk = way kambas, su = sugihan, se = seblat, bsl= bukit selero lahat,be = bentayan biotropia vol. 19 no. 2, 2012 96 the genetic diversity (haplotype diversity, nucleotide diversity and haplotype frequencies) were analyzed for each region (be, bsl, se,su, and k), and whole sumatera island (table 3). these analyses were carried out by using dnasp version 4.10 (rozas . 2003). based on the whole data sequence, the nucleotide diversity in sumatra is 0.00092, in way kambas (wk) 0.00056, in seblat (se) 0.00016, the bukit salero lahat (bsl) 0.00309, and zero (0) in bentayan (be ) and sugihan (su). haplotype diversity on the island of sumatra is 0.164, way kambas (wk) is 0.255, seblat (se) is 0.095, the bukit salero lahat (bsl) 0.366, and zero (0) in bentayan (be) and sugihan (su). the highest haplotype and nucleotide sequence diversity was found in elephants from the bukit salero lahat (bsl) region, and the lowest is zero (identity) which was found in elephants from the bentayan (be) and sugihan (su) regions. this means, there is a lack of mtdna diversity in elephants sampled from this region. there is only one type of haplotype, and consequently the haplotype frequency is 100%. the conditions in the region of sugihan and bentayan might indicate inbreeding. unfortunately, the analysis of fu and li's f * test cannot be performed in the regions of sugihan and bentayan, because genetic diversity = 0. overall, the results of analysis of fu and li's f * test statistic indicates that the population of sumatran elephants is -.78871, which means there is no inbreeding, but not significant at p> 0:10. the result of genetic distance calculations, showed that genetic distances within regions/ populations in bentayan, bukit salero lahat, seblat, sugihan and way kambas ranged from 0.0000 0003, is the genetic distance between the populations that is 0.0000 0.0022. thus, both genetic distance within populations and among the different populations of sumatran elephants are very low. the distance is also counted among the eight haplotypes, consisting of 6 haplotypes (bp, bt, bs, br, bx, by) which are found in the sumatran elephant, bd (borneo haplotype) and bu haplotypes reported by fernando . (2000 & 2003), and vidya (2009 in sumatra. the highest haplotype distance shows 0.17 and the lowest shows 0.0002. a limitation with this type of analysis concerns the maternal inheritance of mitochondria as well as the population structure of elephants with dominant females. asian elephants live in matriarchal societies consisting of family groups of several related females and their (juvenile) offspring. males leave these groups when they reach puberty (fernando & lande 2000; vidya & sukumar 2005). hence, although less commonly used, ychromosome characters are required in its context as male lineage (paternal lineages) just like mitochondrial dna in female lineage (maternal lineages). therefore, additional study based on y chromosome may be needed to obtain a more comprehensive results in this study. figure 2 shows the median joining network (bandelt . 1999) by using the network version 4.5.1.6 (http://www.fluxus-engineering.com). most of the sampled individuals are the haplotype bt. the haplotype bx and by are novel haplotypes. from figure 1, the haplotype bx was derived from the haplotype bt, and the haplotype by was derived from the haplotype bs by one transversion, respectively. the other substitutions in this network were transitions. et al et al et al. ) et al mitochondrial dna variation of the sumatran elephant populations – sri sulandari & moch. syamsul a.z. 97 f ig u re 1. n ei gh b o u r jo in in g t re e (n j tr ee )o f su m at ra n el ep h an ts an d o th er a si an el ep h an ts fr o m m al ay ,c o n ti n en ta la si a an d b o rn eo . s e 1 9 w k 2 1 w k 2 0 w k 1 9 w k 1 8 w k 1 7 w k 1 6 w k 1 5 w k 1 4 w k 1 3 w k 1 2 w k 1 1 w k 9 w k 8 w k 7 w k 6 w k 5 w k 3 w k 2 w k 1 s u -1 8 s u -1 7 s u -1 6 s u -1 5 s u -1 4 s u -1 3 s u -1 1 s u -1 0 s u -9 s u -8 s u -7 s u -6 s u -5 s u -4 s u -3 s u -2 s u -1 s e -2 1 s e -2 0 s e -1 8 s e -1 7 s e -1 6 s e -1 5 s e -1 4 s e -1 3 s e -1 2 s e -1 1 s e -1 0 s e -9 s e -8 s e -7 s e -6 s e -5 s e -4 s e -3 s e -2 s e -1 b s l -1 9 b s l -1 8 b s l -1 7 b s l -1 6 b s l -1 5 b s l -1 4 b s l -1 3 b s l -1 2 s u -1 2 s u m a te ra biotropia vol. 19 no. 2, 2012 98 b s l -1 1 b s l -1 0 b s l -9 b s l -8 b s l -7 b s l -6 b s l -5 b s l -4 b s l -3 b s l -2 b s l -1 b e -2 0 b e -1 9 b e -1 8 b e -1 7 b e -1 6 b e -1 5 b e -1 4 b e -1 3 b e -1 2 b e -1 1 b e -1 0 b e -9 b e -8 b e -7 b e -6 b e -5 b e -4 b e -3 b e -2 b e -1 h a p lo ty p e b t h a p lo ty p e b u h a p lo ty p e b s w k -1 0 w k -4 w k -2 2 h a p lo ty p e b r b s l -2 1 b s l -2 2 b s l -2 4 h a p lo ty p e b q h a p lo ty p e b v h a p lo ty p e b p b s l -2 0 b s l -2 3 h a p lo ty p e b k h a p lo ty p e b j h a p lo ty p e b i h a p lo ty p e b h h a p lo ty p e b m h a p lo ty p e b f h a p lo ty p e b a h a p lo ty p e b b h a p lo ty p e b l h a p lo ty p e b e h a p lo ty p e b n h a p lo ty p e b o h a p lo ty p e b d h a p lo ty p e a f h a p lo ty p e a g h a p lo ty p e a e h a p lo ty p e a h h a p lo ty p e a c h a p lo ty p e a d h a p lo ty p e a b h a p lo ty p e a a w o o ly m a m m o th fo re s t e le p h a n t s a v a n n a e le p h a n t 0 .0 0 5 9 8 9 7 9 9 6 3 5 6 6 8 7 4 6 5 5 2 6 6 8 59 6 9 6 9 7 7 4 6 0 m a la y c o n ti n e n ta l c o n ti n e n ta l b o rn e o mitochondrial dna variation of the sumatran elephant populations – sri sulandari & moch. syamsul a.z. 99 table 3. genetic diversity indices of sumatran elephants region* hd (haplotype diversity) (nucleotide diversity) number haplotipe haplotype ** way kambas/wk (22) 0.255±0.116 0.00056±0.00028 3 bs(0.09), bt(0.864), by (0.045) sugihan/su (18) 0 0 1 bt(1.00) seblat/se (21) 0.095±0.084 0.00016±0.00014 2 bt(0.952), bx (0.048) bukit salero lahat/bsl (24) 0.366±0.115 0.00309±0.00098 3 bp(0.792), br(0.125), bt(0.08 bentayan/be (20) 0 0 1 bt(1.00) sumatera (105) 0.164±0.049 0.00092±0.00032 6 bp(0.019), br (0.01), bs(0.019), bt(0.914), bx(0.01), by (0.01) note: hd: haplotype diversity (expressed as average ± 1se) nd: nucleotide diversity (expressed as average ± 1se) * the number of individuals sampled are shown within parentheses ** the frequencies of the haplotypes are shown within parentheses. the bold characters indicate the novel haplotypes. figure 2. median joining network bt bs br bp bx by continental typesumatera type wk se bsl su be transition transversion note: sampling locations: wk = way kambas, su = sugihan , se = seblat, bsl = bukit selero lahat, be = bentayan haplotypes: br, bx, bt, by biotropia vol. 19 no. 2, 2012 100 conclusions acknowledgments references based on the analysis of mitochondrial dna d-loop sequence from 105 samples of sumatran elephants, it was concluded that sumatran elephants within populations and among populations in sumatra exhibit only limited or very low genetic diversity. this study provides a first definition of mtdna diversity and is an important contribution. bp, br, bs, and bt haplotypes, and 2 new haplotypes (bx and by) are found in the sumatran elephant. the distance between haplotypes of sumatran elephant's population is also low. moreover, additional samples may be needed to obtain significant conclusions. a more comprehensive conclusion may be obtained if also y chromosome markers and autosomal markers are analyzed. many people contributed generously to this study. we are grateful to prof. m. hasegawa and t. yonesawa, ph.d for encouragement and their assistance in data analysis and a the processing of this manuscript. this study was supported by 2010 grant through seameo biotrop to sri sulandari,. we express our appreciation to t. okayama, ph.d for collecting material samples of sumatran elephants. ms. anik dhamayanthi, m.si. and ms. inda natalia, m.si. are also acknowledged for laboratory assistance. dipa akishinonomiya f, miyake t, sumi s, takada m, ohno s, kondo n. 1994. one subspecies of the red jungle fowl ( ) suffices as the matriarchic ancestor of all domestic breeds. in: proceedings national academy of sciences usa. 91: 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(2005a). population differentiation within and among asian elephant ( ) populations in southern india. journal heredity 94 (1), 71-80. vidya tnc, fernando p, melnick dj, sukumar r. 2005b. population genetic structure and conservation of asian elephants ( across india. anim conserv 8: 377-88. vidya tnc, varma s, dang nx, thanh tv, sukumar r. 2007. minimum population size, genetic diversity, and social structure of the asian elephant in cat tien national park and its adjoining areas, vietnam, based on molecular genetic analyses. conserv genet 8:1471-78. vidya tnc, sukumar r, melniek dj. 2009. range-wide mtdna phylogeography yields insights into the origins of asian elephants. proc r soc b 276: 893-902. doi:10.1098/rspb.2008.1494, published online 18 november 2008. evolution elephas maximus elephas maximus elephas maximus elephas maximus) biotropia vol. 19 no. 2, 2012 102 2. jimmy t. masagca page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 page 14 page 15 biotropia no biotropia no. 18, 2002 : 1 20 preliminary study on the palm flora of the lore lindu national park, central sulawesi, indonesia johanis p. mogea herbarium bogoriense, research center for biology, lipi, bogor. indonesia abstract the population size, structure, and composition of the palm flora in a 1350 m by 20 m rectangular plot in gunung potong and a 1500 m by 20 m rectangular plot in tongoa were measured. the total palm species from both plots numbered 33 represented by 8 genera. eight species, namely calamus omatus var. celebicus, pinanga caesia, arenga pinnata, daemonorops sp.3. calamus didymocarpus. calamus sp.4 (rapid spines), caryota mitis, andareca vestiaria have relatively high frequency values ranging from 5.46% to 10.66%. in addition, palm specimens previously collected from the park were examined at herbarium bogoriense to set up a preliminary checklist. so far, the national park is recorded as having 48 palm species represented by 11 genera which give figures of about 68% species and 58% genera of the total native sulawesi palm flora. though the number of endemic palms in sulawesi is high (72%), namely 51 out of total native 71 species, only two species are locally endemic to central sulawesi namely gronophyllum sarasinorum and pinanga sp. nov. 1 (longirachilla). so far only the latter species is endemic to the national park. key words: palm diversity/lore lindu national park/sulawesi/indonesia/endemic species introduction with emphasis on the sustainable use of biological diversity and its conservation, the indonesian-german research program had initiated a comprehensive interdisciplinary research funded by the german research council (dfg) for a duration of 15 years beginning in 2000. the program will be evaluated every three years. the research partners are two universities from germany namely, gottingen and kassel, and two from indonesia, bogor agricultural university (ipb, bogor) and university of tadulako (untad, palu). the overall goal is to identify factors which cause changes in forest margin areas. therefore, the program is known as "stability of rainforest margins" abbreviated as storma. in addition to the research program, a development of related sciences, manpower and laboratory equipment are also provided. the field of research consists of social and economic dynamics, water and nutrient turnover, biological diversity, and land use systems. the location used for the research is in and around the lore lindu national park, central sulawesi, indonesia (fig. 1). within this framework, an exploration of knowledge on palm diversity in and around the lore lindu national park is being conducted. the present paper reports a preliminary study, based mainly on the field observations made during the two weeklong field trips to the national park in november 2000 and march 2001, as well as examination of earlier herbarium collections at herbarium bogoriense. 1 biotropia no. 18, 2002 figure 1. valley study sites map in and around the lore lindu national park, central sulawesi 1. palolo, 2. sopu, 3. napu, 4. besoa, 5. bada, 6. kulawi, and 7. lindu. mountains and others: n: gunung nokilalaki, m: gunung malemo, b: boundary of the national park,f: foot path, r: road. location: km: kamarora, ll: lore lindu national park, pl: palu, pp: gunung potong plot, ra : rahmat, ro : rompo, sa : sadaunta, si : sintuwu, to : tongoa,tp : tongoa plot, tr: torire, ts: transmigration sites, ws: wuasa. 2 preliminary study on the palm flora of the lore lindu national park johanis p. mogea the lore lindu national park covers 217 000 ha of lowland and montane rainforest. approximately 70% of the park lies between elevations of 1000 to 1500 m, and less than 10% is below 1000 m. the highest place located south of tongoa is the summit of gunung (mount) nokilalaki at elevation 2355 m, the other summit is located south of the park namely gunung malemo at 2263 m (mogea & suhardjono 1981). three big rivers drain the park, namely lariang, gumbasa, and sopu. the headwaters of lariang drain at the eastern part of the park, then flow to the south and round to the west then to the north of the park. near gimpu, the river divides into two branches, one to the west ending in the west coast in south sulawesi, the other branch to the north of the park joins the gumbasa river and ends at the coast near palu. the upper gumbasa river is the outlet of lake lindu. it flows to the north around kamarora and sintuwu then unified with lariang. the lake is located at west of gunung nokilalaki. at the eastern north of the park flows sopu river, its lower reaches join at the gumbasa river. two enclaves in the park are excluded from the national park, namely lindu and besoa valleys. five other valleys are located around the park, namely palolo, sopu, napu, bada, and kulawi. there are two moderate roads available for large and public vehicles. the west is a route from palu to gimpu through kulawi valley outside the park. the east road is a route from palu to doda. the road segments near tongoa to wuasa, and torire to doda are through the eastern part of the park. the road from wuasa to rompo in november 2000 was badly damaged, therefore it can only be transversed by a four-wheel drive vehicle (fig. 1). further basic information of the park is presented in the indonesian program information sheet of the nature conservancy (tnc 2000). materials and methods the sites selected for the study were at tongoa, gunung potong, rompo, moa, gimpu, and sadaunta (fig. 1). a national park topographical map, 12xl garmin gps, and altimeter were used to locate the site and to measure the elevation. paln\ diversity was analyzed in each site using a modified mueller-dombois & ellenberg (1974) rectangular plot transect. the transects were established from the lowest elevation on each site to the highest evaluation preferably following an existing foot path. for these two trips, palm diversity was studied in two sites. the first site was in a new cruising transect of gunung potong from 850 m to 1250 m altitude, on the plot of 1350 x 20 m; and the second site was in tongoa on a foot path to the summit of gunung nokilalaki, at an average altitude of 750 m, on the plot of 1500 x 20 m. each plot consisted of many rectangular subplots of 50 x 20 m for observation of either solitary or clustered young and mature palms. for the sapling's study, each subplot consisted of one sapling square plot of 5 x 5 m and one seedling square plot of 1 x 1 m for observation of seedling coverage. through this study, species density, frequency, cover dominance, important value, structure, composition, and palm 3 biotropia no. 18, 2002 population dynamics can be calculated. during the field observation, voucher specimens were made. herbarium specimens, ripe fruits, and living seedlings were collected when necessary, particularly for proper identification and ex. situ conservation. herbarium duplicates are deposited in herbarium celebense (ceb) at the university of tadulako in palu, herbarium bogoriense (bo), netherlands national herbarium leiden branch (l), and herbarium of the royal botanic gardens kew (k). palm collecting followed standard methods; for rattans a method introduced by dransfield (1974) was followed. palm classification is in accordance with the genera palmarum which was introduced by uhl & dransfield (1987). temporary herbarium specimen preservation followed a modified schweinfurth technique (steenis 1950). identification was done in the field as well as at bo and ceb. to facilitate remembering unfamiliar or unidentified taxon in the field, a temporary nickname based on its significant morphological character was applied, hence in this paper a name like calamus sp.8 (tuberosus) or calamus sp.2 (ligule) is used. in addition, sketch drawings and digital photographs were made. field observations were supported by examination of 61 herbarium specimens at bo. new taxa will be described and published in separate papers. results and discussions during identification of palms from the study sites, it was found that 15 taxa of calamus and 4 taxa of daemonorops (table 2) need further identification particularly due to either incomplete herbarium specimens or lack of specimen types at bo, or a need for taxonomic revision. hence, naming these palms required three ways. the first is the taxon's name based on its morphological similarity to a related species, hence the epithet "aff." is used, such as calamus aff. c. zollingeri. the second is an expression of its significant character, such as when the taxon typically has long ligule, then the taxon is called calamus sp.2 (ligule). another example is where there is no spine on its knee (fig. 7-9), the taxon is called calamus sp. 1 (knee no spine). the third is when the epithet required long sentence to define the taxon, then it is called for example daemonorops sp.3 (table 2). some taxa are definitely new species, but their description is not published yet, hence the name is informal, such as calamus sp. nov.l (ahlidurii). the latter case is found as well as in caryota, gronophyllum, gulubia, and pinanga (tables 2 and 3). the number of species from both the gunung potong and tongoa plots is 33 which belong to 8 genera (table 1). calamus is represented by 20 species, among them are the well-known commercial rattans namely 'batang' (c. zollingeri), 'lambang' (c. ornatus var. celebicus), 'tohiti' (c. inops), and 'ombol' (c. symphysipus)', daemonorops represented by five species, among them is an edible young sweetest cabbage 'uwe manis' (d. macropterus); pigafetta represented by p. elata, an ornamental tree palm known as 'wanga' (fig. 6). this palm, for about three decades, was misidentified as p.filaris (dransfield 1998). 4 preliminary study on the palm flora of the lore lindu national park johanis p. mogea table 1. absolute (fa) and relative frequency (fr) of palms from 27 subplots in gunung potong (p) and 30 subplots in tongoa (t). subplot size is rectangular 50 m by 20 m 5 biotropia no. 18, 2002 6 preliminary study on the palm flora of the lore lindu national park johanis p. mogea 7 biotropia no. 18, 2002 8 preliminary study on the palm flora of the lore lindu national park johanis p. mogea while the number of species in gunung potong and tongoa is the same (23 in 7 genera), the species composition and population sizes are different (table 1). pigafetta is only found in gunung potong and korthalsia only in tongoa. thirteen species were found in both gunung potong and tongoa, among them 8 species have high absolute and relative frequency values (table 1), meaning that those species occur in most of the 57 subplots (namely 27 subplots in gunung potong and 30 subplots in tongoa). calamus ornatus var. celebicus occurred in 41 subplots (10.66%), pinanga caesia in 40 subplots (10.40%, fig. 3 5), arenga pinnata in 33 subplots (8.58%), daemonorops sp.3 in 32 subplots (8.32%), calamus didymocar-pus in 29 subplots (7.54%), calamus sp.4 (rapid spines) in 29 subplots (7.54%), caryota mitis in 28 subplots (7.28%), and areca vestiaria in 21 subplots (5.46%). of the 10 species found only in gunung potong, calamus aff. c. paspalanthus is the most significant occurring in 9 subplots (2.34%). likewise 10 species were only found in tongoa, 4 species had much higher absolute and relative frequency values namely calamus aff. c. reinwardtii occurred in 18 subplots (4.68%), arenga undu-latifolia in 14 subplots (3.64%), daemonorops sp.l (didymophylla) in 12 subplots (3.12%), and korthalsia celebica in 10 subplots (2.60%). these figures on palm diversity may change as'a result of further identification and further study as more plots are established. outside the gunung potong and tongoa plots, other species of palms were found such as calamus macrosphaerion var. macrosphaerion and c. orthostachyus in gunung malemo and moa (kramadibrata & dransfield 1992); 'ronti' (calamus leiocaulis) and 'togisi' (c. leptostachys) in moa (siebert 1997). a study on the role of rattan resources in the moa and au in the southern part of the park has been done by siebert (1997). it revealed that 'togisi' (calamus leptostachys), 'ronti' (c. leiocaulis), 'lambang '(c. ornatus var. celebicus), 'batang' (c. zollingeri), and 'noko' (daemonorops robusta) are the important rattans which produced a quite good rural cash income. other palms outside the two above mentioned plots are caryota sp. nov.l (angustifolia) in gunung malemo (zumaidar 2001), this species is solitary as c. rumphiana (fig. 2); licuala celebica and livistona rotundifolia were common and widespread throughout the park (personal observation); pinanga sp. nov.l (longirachilla), pinanga sp. nov.2 (rubiginosa), and pinanga sp. nov.3 (tenuirachis) were found in and around gimpu (sinaga 2000). the rachillae of p. caesia and pinanga sp. nov.2 (rubiginosa) are set up in many planes, the leaf sheath is dull green to bluish deep green in p. caesia (fig. 2 4), while it is reddish orange in pinanga sp. nov.2 (rubiginosa). the rachillae of pinanga sp. nov.l (longirachilla) and pinanga sp. nov.3 (tenuirachis) are set up in one plane, the leaf sheath is yellowish green in pinanga sp. nov.l (longirachilla) and it is yellow in pinanga sp. nov.3 (tenuirachis). pinanga celebica was not listed in the current sulawesi palms (table 3) due to uncertainty of its existence. the story of this taxon started in 1871, when scheffer described pinanga patula var. celebica based on the specimen of riedel s.n. the label was annotated with the plant coming from gorontalo north celebes, but in 9 biotropia no. 18, 2002 2. solitary caryota rumphiana, stem 6 m long 3. solitary pinanga caesia, stem 7 m long, 12 cm in diameter 4. solitary pinanga caesia (living collection of mogea 7467), stem 10m long, 16 cm in diameter 5. top portion of pinanga caesia (living collection of mogea 7467) shows top portion of the stem, infructescences, and leaf sheath 80 cm long, 10 preliminary study on the palm flora of the lore lindu national park johanis p. mogea 6. at the center are three tree palms of pigafetta data, the tallest is about 25 m 7. calamus sp.l (knee no spine; living collection of mogea 7466), leaf rachis 140 cm long, cirrus 100 cm long, white triangle indicates the rachillae bearing reddish brown fruit 8. top portion of infructescence of calamus sp. 1 (knee no spine, mogea 7466) bearing one ellipsoid reddish brown almost ripe fruit of 15 mm long and 7 mm in diameter 9. base portion of infructescence of calamus sp. 1 (knee no spine, mogea 7466) bearing many very young fruits, leaf sheath 15 mm in diameter 1876 he changed its status to pinanga celebica. he wrote that the plant clusters, had whitish leaves, a long inflorescence rachis with numerous rachillae, and obovoid fruits. this character diagnosis, however, is inadequate for identification to the species level as they are too general. moreover the clustered habit is not found in any of the east malesian pinanga (sinaga 2000). 11 biotropia no. 18, 2002 betel nut palm or 'pinang sirih' areca catechu and coconut palm or 'kelapa' cocos nucifera is often planted in a village garden, the former usually used for a living fence to mark boundaries. sugar palm or 'aren' or 'saguer' arenga pinnata is often grown in gardens around the park, and are often found on the forest border or in disturbed primary lowland and montane forest. hence, including these last mentioned palms, the number of species or taxa so far known to occur in and around the park is 48 represented by 11 genera (table 2) namely areca 2 species, arenga 2 species, calamus 26 species, caryota 3 species, cocos 1 species, daemonorops 5 species, korthalsia 1 species, licuala 1 species, livistona 1 species, pigafetta 1 species, and pinanga 4 species. including here are some other of unidentified genera (table 2 and appendix 1). as shown in appendix 1, many of the herbarium specimens are sterile, though it is still important to figure out the diversity but to describe or to identify up to the genus level is often inadequate. calamus are often difficult to separate from daemonorops when they are all still in the vegetative stage. on the other hand, the morphology of seedling, sapling, juvenile, and mature in the palm species is often distinct. therefore, adequate observation in the field, fertile and correct collection for the herbarium specimens is very necessary. if this field study is successfully done, it is believed that there might be some new taxa particularly of calamus and daemonorops, but also of pinanga, licuala and caryota. most calami in and around the park are clustering rattans but few of them are solitary such as in calamus inops, c. leptostachys, and c. symphysipus. stem diameter varies from 6-40 mm, with the leaf sheath diameter varying from 8 4 5 mm. the smallest cane is c. leiocaulis and the largest is c. zollingeri. petioles are mostly green, but are reddish brown in calamus sp.14 (mogea 7465, fig. 13). the leaf sheath and the petiole are covered by scarcely small spines such as in c. ornatus var. celebicus; but calamus sp.4 (rapid spines; mogea 7461) are covered by slender, rapid, greenish, large spines (fig. 10); in c. macrosphaerion var. macrosphaerion, by rapid, flat, triangular, blackish brown spines (fig. 12); and in calamus sp.14 (mogea 7465) by scarcely brown, flat, triangular comblike spines (fig. 13). the specimens of mogea 746.5 might be calamus sp. nov.l (ahlidurii) or daemonorops macropterus. when already matured, the identity of the specimen will be much easier to determine as the difference between calamus and daemonorops can only be seen after the flowering stage, namely the peduncular bract in calamus is tubular and attached to the rachis of the inflorescence while it is leafy-like and deciduous in daemonorops. calamus sp.2 (ligule; mogea 7456) has the only conspicuous ocrea or ligule (fig. 11). calamus in sulawesi are mostly cerriate, hence from this point the vegetative characters of calamus are not different from daemonorops. however, sometimes the species identity can be found based on the number of the leaflets on either side of the rachis, mostly daemonorops has regular (rarely in groups) and very narrow leaflets compared to calamus. the number of leaflets in daemonorops is mostly on the average between 40 to 80 leaflets, while in a small diameter cane of 12 preliminary study on the palm flora of the lore lindu national park johanis p. mogea calamus, the leaflets are very much less in number, usually 6 to 16 such as in calamus minahassae which has only 12 leaflets set up in 6 groups, each group consisting of one or two leaflets. when ripe, the fruit colour is pale greenish-white such as in calamus macrosphaerion (fig. 14) or glossy white (fig. 15) as in calamus sp. 1 (knee no spine) or black as in calamus zollingeri. native palms in sulawesi are represented by 71 species in 19 genera (table 3) based on observations from about 20 sites all over sulawesi (mogea 2000) updated by recent current examination of the specimens and recent field work. pending further identification, the number of genera and palm species in and around the park represents about 58% and 68% of genera and species of sulawesi's palm flora, respectively. metroxylon sagu was probably planted long ago for their edible carbohydrate and is considered here. as an introduced species. its original distribution is in moluccas and new guinea. sagu baruk (arenga microcarpa) has apparently been cultivated in district sangir talaud for 80 years already. its original distribution is the same as for the sago palm. sihombing et al. (1980) incorrectly identified it as a. obtusifolia. the sagu baruk covers about 19 890 ha or about 30% of the total agricultural area in the district (sihombing et al. 1980). 'salak pangu', salacca zalacca var. amboinense, was cultivated for some decades in pangu village, district minahasa, north sulawesi . the salak pangu now is popular as an edible fresh fruit in north sulawesi. it was said that the cultivation started in 1961. now salak pangu plantations cover about 654 ha with about 2 million individual plants (dannie 1999). other introduced palms are mostly indoor or outdoor ornamental plants. so far known, the number of the introduced palms in sulawesi is 20 (table 4), including the palm for plantation namely 'sawit' or oil palm elaeis guineensis. based on their origin, the introduced palms consist of 12 groups. these introduced palms were found in palu and on the way to the park. 13 biotropia no. 18, 2002 10. base portion of young calamus sp.4(rapid spines, living collection of mogea 7461) shows 50 cm long petioles and leaf sheaths covered by rapid slender long greenish white spines 11. top portion of young 1.5 m tall calamus sp.2 (ligule, living collection of mogea 7456) stows top of leaf sheaths bearing conspicuous young entire and old tattered ocrea 12. middle portion of calamus macrosphaerion var macrosphaerion (living collection of mogea 7470) shows leaf sheaths of 4 cm in diameter and petioles covered by rapid flat triangular blackish brown spines 13. base portion of young calamus sp. (living collection of mogea 7465) shows reddish brown petioles covered by scarcely black flat triangular combed-like spines 14 preliminary study on the palm flora of the lore lindu national park johanis p. mogea 14. nearly top portion of infructescence of calamus macrosphaerion (living collection of mogea 7470) bearing pale greenish white almost ripe ellipsoid fruits of 10 mm in diameter 15. middle portion of infructescence of calamus sp. 1 (knee no spine, living collection of mogea 7487) bearing white ellipsoid ripe fruits 8 mm in diameter table 4. checklist of introduced palms in sulawesi and their origin 15 biotropia no. 18, 2002 1) actinorhytis calapparia (blume) h. wendl. & drude ex scheti. it is listed that native palms of sulawesi consist of 71 species and can be divided into 22 groups based on local and regional plant geographical distribution, namely tropical asia, malesia, borneo; north, central, south, and southeast sulawesi; unknown locality in sulawesi, and moluccas (table 3). the number of palm species endemic to sulawesi is moderately high (70.83%) namely 51 out of total native 71 species. however, only two species namely, gronophyllum sarasi-norum and pinanga sp. nov.l (longirachilla), are endemic to central sulawesi (table 3, group 7). the latter species is so far known only from gimpu (fig. 1) the south portion of the park (table 3 and appendix 1). conclusion this preliminary study on palms in and around the lore lindu national park, including observations on 1350 m by 20 m rectangular plot sites in gunung potong, a 1500 m by 20 m rectangular plot in tongoa, herbarium specimens collected from gunung nokilalaki, sopu valley, moa, and gunung malemo revealed that there are 48 species belonging to 11 genera. calamus and daemonorops are very dominant as shrubs and climbers. the largest genus in the park-is calamus with 25 species, followed by daemonorops (6 species) and pinanga (4 species). however, more 16 preliminary study on the palm flora of the lore lindu national park johanis p. mogea fertile collections of these genera are still required. it is therefore expected that some undescribed and locally endemic palms may still be found as calamus, daemonorops, licuala, caryota and pinanga. acknowledgments the author would like to express his sincere gratitude to storma for providing funds which made the conduct of this study possible. cordial appreciation is also addressed to the officers of lore lindu national park who have kindly provided facilities during the field work and the indonesian-german research team, particularly to dr. paul kesler. thanks are also due to dr. sri s. tjitrosoedirdjo of seameo biotrop for her suggestions to improve the manuscript, and dr. arie budiman, head of the research centre for biology, lipi, bogor, for allowing the author to conduct the study. references dannie, a.t.e. 1999. salak pangu "imigran" asal bali trubus 30 9358): 69. dransfield, j. 1974. a short guide to rattans. biotrop (seameo regional centre for tropical biology). bogor. mimeographed. 69 p. dransfield, j. 1998. pigafetta. principes 42 (1): 34 40. kramadibrata, p. & j. dransfield. 1992. calamus inops (palmae: calamoideae) and its relatives. kew bulletin 47 (4): 581-593. mogea, j.p. & suhardjono. 1981. struktur dan komposisi pepohonan di gunung malemo sulawesi tengah. paper presented at the national biological seminar xv.semarang indonesia, june 26 28. mimeographed. 14 p. mogea, j.p. 2000. status konservasi palem indonesia, khususnya sulawesi. paper presented at the national biological seminar xvi, bandung, july 25 27, 2000. mimeographed. 15 p. mueller-dombois, d. & h. ellenberg. 1974. aims and methods of vegetation ecology. new york, ca 160 p. siebert, s.f. 1997. economically important rattans of central sulawesi, indonesia. principes 41 (1): 42 -46. sinaga, n.i. 2000. pinanga blume (arecaceae) in east malesia. m.sc. thesis. bogor agriculture university, darmaga, bogor indonesia, 66 p. steenis, c.g.g.j., van. 1950. the technique of plant collection and preservation in the tropics. flora malesiana i (1): xlv ixix. 17 biotropia no. 18, 2002 the nature conservancy. 2000. lore lindu national park. indonesia program information sheet sppi. jakarta. leaflet 2 p. uhl, n.w. & j. dransfield. 1987. the genera palmarum. alien press. lawrence kansas. 610 p. zumaidar. 2001. taxonomy and species relationship of indonesian caryota (palmae). m.sc. thesis. bogor agriculture university, darmaga, bogor indonesia, 44 p. 18 preliminary study on the palm flora of the lore lindu national park johanis p. mogea appendix 1. specimens examined: note: plant name: locality, elevation, phenological state (st: sterile, ffl: female flower, mfl: male flower, fl: hermaphrodite flower, fr: fruit), date, collection number, deposited hersbarium, with the exclamation point means that the author has seen the specimens) areca vestiaria: tongoa, foot path to the summit of mt. nokilalaki, alt. 750 in, fr.02-03-01, mogea 7473 (bo!, ceb!) arenga undulatifolia: tongoa, foot path to the summit of mt. nokilalaki, alt. 750 in, st.02-03-01, mogea 7474 (bo!; ceb!) calamus didymocarpus : mt. nokilalaki, toro, fr.23-04-75, meijer 9465 (bo!). calamus inops: kulawi, moa, mt. malemo, mfl.24-10-77, mogea 1474 (bo!); ditto., mogea 1475 (bo!); ditto., mogea 1476 (bo!); ditto., mogea 1477 (bo!, k). tongoa, foot path to the summit of mt. nokilalaki, alt. 750 m, ffl.05-03-01, mogea 7490 (bo!; ceb!). calamus macrosphaerion var. macrosphaerion: kulawi, mt. malemo, fr.20-10-77, mogea 1347 (bo!). calamus minahassae: sopu valley, fr.05-05-79, de vogel 5212 (bo!, l). calamus omatus var. celebicus : sopu valley, fr.26-04-79, de vogel 5054 (bo!, l); ditto., 02-05-79, de vogel 5172 (bo!); kulawi, moa, fr.22-10-77, mogea 1411 (bo!). calamus orthostachyus: kulawi, moa, mt. malemo, fr.18-10-77, mogea 1329 (bo!, k, l), ditto., mogea 1330, mfl. (bo!). calamus symphysipus: tongoa, foot path to the summit of mt. nokilalaki, alt. 750 m, st.02-03-01, mogea 7472 (bo!, ceb!). undeterminate calamus from tongoa, foot path to the summit of mt. nokilalaki, alt. 750 m: calamus aff. c omatus, st.03-03-01, mogea 7478 (bo!, ceb!), ditto, mfl.03-03-01, mogea 7479 (bo!, ceb!). calamus aff. c. reinwardtii: path to the st.02-03-01, mogea 7476 (bo!, ceb!); calamus sp.4 (rapid spines), st.18-11-00, mogea 7442 (bo!, ceb!) = mogea 7461 (see at gunung potong alt. 950 m); calamus sp.7 (tattered ligule), st.03-03-01, mogea 7477 (bo!, ceb!); calamus sp.8 (tuberosus), fr.03-0301, mogea 7486 (bo!, ceb!); calamus sp.9 mfl.05-03-01, mogea 7488 (bo!, ceb!). undeterminate calamus from gunung potong, alt. 850 m: calamus sp.8 (tuberosus), st.26-02-01, mogea 7455 (bo!, ceb!). calamus sp.2 (ligule), st.27-02-01, mogea 7456 (bo!, ceb!). calamus sp.6 (soft white spine), mfl.01-03-01, mogea 7471 (bo!, ceb!). undeterminate calamus from gunung potong, alt. 950 m: calamus sp.4 (rapid spines), st.27-02-01, mogea 7461 (bo!, ceb!) = mogea 7442 (see at tongoa ); ditto., alt. 1200 m, mfl.01-03-01, mogea 7468 (bo!, ceb!). undeterminate calamus from mt. roroka, timbu: calamus sp.10, st.20-05-79, de vogel 5482 (bo!); ditto., fr. 13-05-79, de vogel 5334 (bo!). undeterminate calamus from sopu valley: calamus sp.ll, st.05-05-79, de vogel 5209 (bo!); st.05-05-79, de vogel 5218 (bo\). undeterminate calamus from mt. nokilalaki: calamus sp.12, st.22-04-75, meijer 9438 (bo!); ditto., st. 0405-75, meijer 10021 (bo!). undeterminate calamus from kulawi, moa: calamus sp.13, st.14-10-77, mogea 1290 (bo!); ditto., st. 1810-77, mogea 1324 (bo!), ditto., mogea 1325 (bo!); ditto., mogea 1326 (bo!); ditto., mogea 1328 (bo!); ditto., st.22-10-77, mogea 1433 (bo!) 19 biotropia no. 18, 2002 caryota sp. nov.l (angustifolia); kulawi, moa, mt. malemo, alt. 1200 m, fr.23-10-77, mogea 1441 (bo!); ditto., fl.+fr.24-10-77, mogea 1473 (bo!) daemonoropx macropterus: sopu valley, fr.22-05-79, de vogel 5326 (bo!); tongoa, foot path to the summit of mt. nokilalaki, alt. 750 m, st.05-03-01, mogea 7491 (bo!; ceb!) korthalsia celebica: kulawi, moa, mt. malemo, st.,23-10-77, mogea 1462 (bo!); ditto., mogea 1463 (bo!) pinanga caesia blume: palu: sopu valley, alt. 1000 m, fr. 12-04-79, de vogel 5064 (bo!); gunung potong, alt. 950 m, fr.28-02-01, mogea 7467 (bo!, ceb!); parigi, alt. 800 m, fr.17-04-75, meijer 9378 (bo!); kulawi, gimpu, moa, alt. 1200 m, st.19-10-77, mogea 1343 (bo!); ditto., fr.l 1-10-77, mogea 1271 (bo!); ditto., fr.l 1-10-77, mogea 1274 (bo!); ditto., mogea 1275 (bo!): mt. malemo, fr.23-10-77, mogea 1443 (bo!). pinanga sp. nov.l (longirachilla): kulawi, gimpu, alt. 600 m, fr.l 1-10-77, mogea 1275 (bo!); mt. malemo, alt. 1000 m, fr.l7-04-79, mogea 1443 (bo!). pinanga sp nov.2 (rubiginosa): east of tongoa, alt. 720 m, fr.02-03-81, johannxxal, nybom & riehe 126 (bo!). pinanga sp. nov.3 (tenuirachis): kulawi, alt. 600 m, fr. 11-10-77, mogea 1271 bo!). 20 biotropia no. 6, 1992/1993: 33-44 effects of the heavy metal, zinc, on the freshwater fish tilapia nilotica l. virginia s. caring institute of biology, university of the philippines, diliman, quezon city, philippines abstract gills, gonads, and blood of tilapia nilotica exposed to different concentrations of zinc sulfate (znso4. 7h2o) exhibited histological effects. gills of posthatch larvae exposed chronically for 21 days to 2 ppm zinc sulfate and fingerlings to 10 ppm sublethal zinc concentrations exhibited hyperplasia that resulted in fusion of adjacent secondary gill lamellae. the same effects were observed in 4-hour short-term exposure to 30 ppm lethal dose. posthatch larvae subjected to 2 and 5 ppm sublethal levels of zinc for 30 days retained undifferentiated gonads with differentiation with oogonial proliferation. ovaries of control fish demonstrated healthy oocyte growth and other normal histological features after 57 days. in contrast, ovaries in treated groups exhibited excessive amounts of connective tissue, hyperemia and markedly reduced oocyte number. oocytes had wavy irregular surface outlines. deviation from normal was observed to be dose dependent. in juvenile tilapia, spermatogenesis was observed in control testes. testes of zinc-exposed fish, on the other hand, remained immature. hyperemia was markedly pronounced in both testes and ovary after 90 days exposure to zinc. blood of tilapia nilotica fingerlings exposed to sublethal concentrations of 2, 5, and 10 ppm zinc for 30, 60, and 90 days exhibited anisocytosis and poikilocytosis. there was an increase in hematocrit values in zinc-reared fish which, however, reverted to control/near control levels at day 90. hemoglobin values were inversely proportional to the level of zinc in the rearing water. the marked reduction in hemoglobin values in fish reared at the higher zinc concentrations of 5 and 10 ppm suggests the development of some degree of anemia which is also supported by the observations of anisocytosis and poikilocytosis. introduction heavy metal contamination of aquatic environments has become a current serious problem because of increased industrialization. in the philippines, data gathered by the national environmental pollution commission (nepc 1980) on eleven river systems and of researches monitoring levels of heavy metals in fishes indicate high concentrations of these substances in aquatic bodies (nepc 1980; pcarrd 1982). compared to seas and oceans, fresh water environments are more vulnerable to pollution stress inasmuch as they are smaller systems and have more limited numbers and kinds of organisms. pollution of lakes and rivers, thus, pose alarming dangers to aquatic life, as for example to fish. one fresh water fish which is presently being studied intensively insofar as its response to heavy metals contamination, is tilapia nilotica l. economically 33 biotropia no. 6, 1992/1993 important as a poor man's protein source, it is easily available and is a hardy fish species. thus, it is expected that less sturdy fishes would exhibit any effect manifested by this particular species. most of the studies that will be reported in this paper deal with the effects of zinc on posthatch larvae or fry of tilapia nilotica. this stage of development, is a good investigative material since a number of organs, e.g., the gonads have not completed full development and differentiation. the earlier developmental stages are interesting subjects, but in t. nilotica they are not too exposed to the environment inasmuch as they brood in the tilapia mother's mouth and are hence semi-protected. materials and methods preliminary 96 hr ld 50 determination was performed to obtain the approximate sublethal levels of zinc on the t. nilotica stage that would be tested. during the four days period the fish were kept in aerated water with different concentrations of znso4 and were starved to minimize the elimination of the metal together with waste products. dechlorinated tap water was used in all tests. hardness, alkalinity, and ph readings were taken twice a week on the test water in the aquarium. hardness and alkalinity were determined by titration method (apha 1971). temperature was monitored daily. zinc sulphate (znso4. 7h2o, ar merck ® ) was dissolved in deionized water and acidified to ph 2 with nitric acid (hno3). the plastic jars with 3.0 1 test water were spiked with zinc solution to bring the concentration to the desired level. posthatch fry of t. nilotica were obtained from a single source and acclimated in well aerated dechlorinated tap water for 7 days prior to experimentation. chronic as well as acute exposures to zinc were done at different time exposures. the organs to be reported on are the gills, gonads, and blood. for light microscopy, tissues were dehydrated in a series of graded ethyl alcohol, cleared in cedarwood oil and xylene, infiltrated and embedded in paraffin, and cut in ao ® rotary microtome. paraffin sections of 7 urn thickness were stained with hematoxylin-eosin and mounted in entellan ® mounting medium. resin sections were also done. tissues dissected were fixed in 2.5% glutaraldehyde in phosphate buffer, ph 7.2, followed by post-fixation in 1% osmium tetroxide. the samples were dehydrated in ethanol and propylene oxide, embedded in araldite ® , sectioned 1 2 urn thick with carl zeiss ® ultratome, stained with 1 % toluidine blue and examined under the light microscope. ultrathin sections were stained with uranyl acetate and lead citrate for electron microscopy. 34 effects of the heavy metal, zinc, on tilapia nilotica l. v.s. cariffo for blood parameters, fingerlings were reared in zinc concentrations of 2, 5, and 10 ppm for 30, 60, and 90 days (cariño and casauay 1991). blood samples were collected by gill puncture using heparinized capillary tubes and analyzed for haematocrit and haemoglobin content using capillary method (hesser 1960) and als biochemical haemoglobin procedure ® , usa, respectively. blood smears were made and stained with giemsa for erythrocyte morphology. results and discussion effects on gills in tilapia fingerlings exposed to 10 ppm and 20 ppm zinc sulfate for 21 days, the main effect was hyperplasia of the primary gill epithelia resulting in fusion of adjacent secondary lamellae (cariño & puzon 1986). hyperplasia was noted in all treatment groups, both short-term exposure (4 hours) to 30 ppm lethal concentration of zinc and long-term treatment (21 days) to 20 ppm chronic and 10 ppm sublethal levels of the metal (figs. 1 & 2). extensive cell proliferation was noted along the entire length of the gill filaments of fish exposed to the highest zinc concentration figure 1. longitudinal section of control gill filament. cc, chloride cell; sec, secondary epithelial cell; sl, secondary lamellae; pc, pillar cell; bc, blood cell; gl, gill ray. x960. 35 figure 2. longitudinal section of gill filament from 10-ppm exposed fish showing fusion of adjacent secondary gill lamellae. cc, chloride cell; or, gill ray. x480. used, 30 ppm. the overall effect of the zinc salt on the gill structure is the decrease in surface area which would otherwise make for an efficient transport of oxygen in the blood. other investigations showed that chemically induced hyperplasia of the primary gill epithelia is generally due to the increase in the number of chloride cells (ahuja 1970; crespo 1981; tuurala & soivio 1982; crespo 1984). zinc was particularly shown to induce proliferation of the chloride cells in the dogfish synliorhinus canicula (crespo 1981). increase in chloride cells have likewise been noted in the study of cariño & puzon (1986) with t. nilotica fingerlings. this indicates to invigorate osmoregulatory capacities. chloride cells are large salt secretory cells (karnaky et al. 1977) found at the base of the secondary gill filament and believed to be responsible for osmoregulation (karnaky 1980; laurent & dunel 1980). they have been shown to excrete univalent ions, though they might also be able to excrete bivalent ions (ahuja 1970). na-k-atpase is the major enzyme found in chloride cells. thickening or hypertrophy of the secondary gill epithelia was a less observable effect of zinc exposure in t. nilotica fingerlings. this was observed with 20 ppm treatment (figs. 3 & 4). 36 biotropia no. 6, 1992/1993 effects of the heavy metal, zinc, on tilapia nilotica l. v.s. carino figure 3. longitudinal section of gill filament from 20-ppm exposed fish showing fusion (f) of secondary gill lamellae and hypertropy (arrow) of secondary epithelial cell. cc, chloride cell; sec, secondary epithelial cell; gr, gill ray. x480. figure 4. longitudinal section of gill filament from 20-ppm exposed fish showing hyperplasia of chloride cell and fusion of secondary gill lamellae. cc, chloride cell; pc, pillar cell; bc, blood cell; gr, gill ray. x960. 37 biotropia no. 6, 1992/1993 hypertrophy of secondary gill epithelia has been reported as a response of fish to industrial pollutants. hughes et al. (1979) noted that there was increase in the volume of secondary epithelia in nickel exposed salmo gairdneri. chromium has similar effect on the secondary epithelia of this species (putte et al. 1981). tuurala and soivio (1982) observed that excessive hypertrophy of the secondary epithelial cells was induced by sublethal exposure of s. gairdneri to dihydroabietic acid, a major kraft pulping effluent (leach & thakore 1973). similar results have been obtained with exposure of chanos chanos fingerlings to ammonia (cruz & enriquez 1982). secondary lamellar hypertrophy increases the diffusing distance from the surrounding water to capillaries, but decreases the respiratory dead volume between the secondary lamellae and, thus, would compensate for the increase in the diffusion distance (tuurala & soivio 1982). histological observations indicate that zinc impairs gaseous exchange in fishes by increasing diffusion distance and decreasing absorption surface area. electron microscopic structure of the secondary lamella of control fish is shown in fig. 5. the lamellar blood sinus is large and circular in outline. the pillar cell and its nucleus is very distinct. fish exposed to 2 ppm zinc for 21 days were observed to have lamellar blood sinus dilation and epithelial lifting of the secondary gill lamella (fig. 6). detachment of pillar cell from the epithelium occurred at 20 ppm zinc exposure. epithelial lifting results in the enlargement of the non-tissue spaces. 38 figure 5. electron micrograph of control secondary gill lamellae. x5000. figure 6. electron micrograph of gill lamellae from 2-ppm exposed fish showing dilated blood sinus epithelial lifting. x5000. effect on the gonads gonads of 30-day posthatch control fry exhibited gonial multiplication (cariño & cruz 1990). no distinction can yet be made as to whether the gonad is testis or ovary. in contrast, all experimental fish reared for 30 days in three zinc concentrations had gonads that contained only primordial germ cells. gonial multiplication was delayed. in fish harvested after 57 days in culture, oocyte growth is observed in both control and zinc-treated fish (figs. 7a, b). a notable difference is the presence of promiscuous and dilated blood vessels densely filled with blood cells in the ovaries of zinc-treated fish. oocytes of zinc-treated fish have more nucleoli than control fish. a number of these nucleoli-studded nuclei bear on one side a quarter moon-shaped region of intensely darkly stained material which is most probably extruded rna. accumulation of maternal rna is characteristic of most growing oocytes. graded distinctions between ovaries treated with 2, 5, and 10 ppm appear inconspicuous. tests of 57-day reared control and zinc-treated fish showed spermatogonial multiplication. as noted by casauay & carifio (1988), spermatogenesis in tilapia nilotica commences later than oogenesis. testes of juvenile t. nilotica showed spermatogenic stages. in contrast, zinc-treated fish showed delay in spermatogenesis (figs. 8a, b). 39 effects of the heavy metal, zinc, on tilapia nilotica l. v.s. carino biotropia no. 6, 1992/1993 figure 7. oocyte growth from 57-day cultured fish (a) control ovary, (b) zinc treated fish ovary showing abundant blood cells. x5000. 40 effects of the heavy metal, zinc, on tilapia nilotica l. v.s. cariffo figure 8. spermatogenic stages in control juvenile t. nilotica; b. delay in spermatogenesis in zinc treated fish. x400. 41 figure 9. blood smear from a contol tilapia nilotica; b. blood smear showing "anisocyclosis", a term applied when the rbcs vary in size (arrow) after exposure to 2, 5, and 10 ppm zinc concentration, c. "poikilocytosis" exhibited by abnormal variation in shape of rbc after 2, 5, and 10 ppm exposure to zinc. xi000. 42 biotropia no. 6, 1992/1993 effects of the heavy metal, zinc, on tilapia nilotica l. v.s. carino effects on the blood anisocytosis and poikilocytosis were noted in the blood smears of zinc-treated fish (9a, b, c). in all zinc treated fish, an increase in hematocrit (%) was observed at 30 days. with the exception of fish reared at 10 ppm zinc, hematocrit values dropped to control/near control levels after 90 days. haemoglobin (g/1) content in fish reared in zinc-treated water for 90 days was inversely related to zinc concentration. exposure of tilapia nilotica fingerlings for 90 days to zinc concentrations of 5 and 10 ppm induced anemia reducing haemoglobin content of erythrocytes and not by reduced number of erythrocytes. references ahuja s.k. 1970. chloride cell and mucus-cell responses to chloride and sulphate enriched media in the gills of gambusia affinis (baird and girard) and cat/a catla (hamilton). j. exp. zool. 173: 231-250. american public health association, american water works association, and water pollution control federation 1971. standard methods for the examination of water and wastewater. 13th ed., american public health association, ny, usa. cariño, virginia s. and armando g. puzon, jr. 1986. toxic effects of zinc sulphate to tilapia nilotica l. fingerlings. natural and applied science bulletin 38(2):127134. cariño, virginia s. and arsenia a. casauay. 1991. the haemocytology of tilapia nilotica and blood studies on tilapia nilotica fingerlings after sublethal exposure to zinc. science diliman 25(1):6 22. cariño, v.s. and n.c. cruz. 1990. effects of low levels of zinc on the ovarian development of tilapia nilotica l. sci. diliman 3:34-45 casauay, a.a. and carino, v.s. 1988. gonadal sex differentiation in oerochromis niloticus. in: r.s.v. pullin, t. bhukasawan, k. tonguthai, and j.l. maclean (eds.). the second international symposium on tilapia in aquaculture. iclarm conference proceedings, department of fisheries, bangkok, thailand and international center for living aquatic resources management, manila, philippines: 121 124. crespo, s. 1982. surface morphology of dogfish (scyliorhinus canicula) gill epithelium, and surface morphological following treatment with zinc sulphate: as canning electron microscope study. mar. biol. 67:159-166. crespo, s. 1984. an in vitro study of the effects of zinc on the osmoregulatory processes. mar. pollut. bull. 15:341-342. cruz, e.r. and g.l. enriquez. 1982. gill lesions associated with acute exposure to ammonia. nat. appl. sci. bull. 34:1-13. hesser, e.f. 1960. methods for routine fish hematology. prog. fish cult. 22(4):164171. hughes, g.m., s.g. perry, and v.m. brown. 1979. a morphometric study of effects of nickel, chromium, and cadmium on the secondary lamellae of rainbow trout gills. water res. 13:665 679. 43 biotropia no. 6, 1992/1993 karnaky jr. k.j., j. karl, jr., and w.b. kinter. 1977. killifish opercular skin. a flat epithelium with a high density of chloride cells. j. exp. zool. 199:355-364. leach, j.m. and a.n. thakore. 1973. identification of the constituents of kraft pulping effluent that are toxic to juvenile coho salmon (onchorynchus kisutch). j. fish res. board can. 30:479-484. national environmental protection council. 1980. the philippine environment. manila: 52-56. philippine council for agriculture and resources research and development. 1982. state of the art environmental protection research: 1 — 11. putte, i.v.d., m.a. brinkhorst, and j.h. koeman. 1981. effecf-of ph on the acute toxicity of hexavalent chromium to rainbow trout (salmo gairneri). aquat. toxicol. 1:129— 142. tuurala, h. and a. soivio. 1982. structural and circulatory changes in the secondary lamellae of salmo gairdneri after sublethal exposures to dehydroabietie acid and zinc. aquat. toxicol. 2:21 29. 44 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf 1 dietary expo (okky).cdr biotropia vol. 18 no. 1, 2011: 1 12 1 dietary exposure assessment for aflatoxin b from processed peanut products in municipality of bogor 1 santi ambarwati , okky setyawati dharmaputra and ina retnowati a research on dietary exposure assessment for aflatoxin b (afb1) from processed peanut products in municipality of bogor was carried out. the objectives of this study were to determine the contents of afb1 in processed peanut products at retail levels, and to obtain information whether there is a risk to public health caused by the consumption of processed peanut products contaminated by afb1. survey of processed peanut product consumption was carried out by interviewing each respondent using a questionnaire of weekly processed peanut product consumption. sampling of processed peanut products was conducted at the locations where the respondents obtained processed peanut products. the number of roasted peanuts with skin pods, flour-coated peanuts and or sauces samples was 33, respectively, while the number of and sauces samples was 18 and 12, respectively. the total number of processed peanut product samples was 129. afb1content was determined using thin layer chromatography method. estimation of the dietary exposure assessment was determined using the actual survey data consisting of afb1 content, consumption data and body weight. the highest contaminated sample percentage and mean of afb1 content was found in roasted peanuts with skin pods i.e. 42% of 33 samples and 43.2 μg/kg, respectively, followed by flour-coated peanuts (30% of 33 samples and 34.3 μg/kg), and or (21% of 33 samples and 17.1 μg/kg). mean of estimated dietary exposure for afb1 found in children was 15.2 ng kg bw day and 95 percentile exposure was 38.9 ng kg bw day , while in adults 9.0 ng kg bw day and 95 percentile exposure was 27.0 ng kg bw day . the excess cancer risk of afb1 exposure in bogor from this study on children and adults was calculated as 193 and 115 cancers/year, respectively dietary exposure assessment, aflatoxin b , processed peanut products 1 1,2 1 -1 -1 th -1 -1 -1 -1 th -1 -1 * 1 2 seameo biotrop, bogor, indonesia department of biology, faculty of mathematics and natural sciences, pecel gado-gado siomay satai pecel gado-gado bogor agricultural university, darmaga campus, bogor, indonesia abstract 1 1 . key words: * corresponding author : ambarwati@biotrop.org biotropia vol. 18 no. 1, 2011 2 introduction peanuts are next to rice, maize and soybean as the most important secondary crop in indonesia. since indonesia has a humid tropical climate, peanuts can easily be infected by fungi during the drying phase in the field, or under poor storage conditions. according to sauer . (1992) fungal infection can cause a decrease in physical quality of kernels and nutritional content, rancidity, discoloration, and production of mycotoxin, among others aflatoxin. the toxin has been recognized as human and domestic animals carcinogen, and is produced following the infection of peanuts among others by certain strains of . in general, aflatoxins found in foodstuffs and their processed products are aflatoxins b , b , g and g . the most dangerous aflatoxin is b (afb1). the adverse health effects of aflatoxins can be categorized as either acute or chronic. acute aflatoxicosis occurs when moderate to high levels of the toxins are consumed and may result in hemorrhage, acute liver damage, rapid progressive jaundice, edema of the limbs, alteration in digestion, absorption and/or metabolism of nutrients, high fever, vomiting, swollen livers and possibly death (fung & clark 2004). hepatocelullar carcinoma (hcc), or liver cancer, is the third leading cause of cancer deaths worldwide, with roughly 550000-600000 new hcc cases globally each year (who 2008). it has been known for several decades that aflatoxin causes liver cancer in humans, however, the exact burden of aflatoxin-related hcc worldwide was unknown. liu and wu (2010) conducted a quantitave cancer risk assessment i.e. using global data on food-borne aflatoxin levels, consumption of aflatoxincontaminated foods, and hepatitis b virus (hbv) prevalence. aflatoxins have been classified as group 1 human carcinogen by the international agency for research on cancer (iarc) and demonstrated carcinogenic effects on many animal species, including some rodents, non human primates, and fish (international programme on chemical safety 1998). groopman . (2008) reported that specific p450 enzyme in the liver metabolize aflatoxin into a reactive oxygen species (aflatoxin-8,9-epoxide), which may then bind to proteins causing acute toxicity (aflatoxicoses) or to dna causing lesions that over time increase the risk of hepatocelullar carcinoma (hcc) or liver cancer. for cancer risk assessment, it is traditionally assumed that there is no threshold of exposure to a carcinogen below which there is no observable adverse effect. national research council (2008) stated that cancer potency factors are estimated from the slope of the dose response relationship, which is assumed to be linear, between doses of the carcinogen and cancer incidence in a population. according to ipsc/who (1998), aflatoxin risk assessment selected two different cancer potency factors for aflatoxin : 0.01 cases/ 100000/year/nanogram/kilogram body weight per day aflatoxin exposure for individuals without chronic hbv infection, and 0.30 corresponding cases for individuals with chronic hbv infection. kirk . (2005) and ok . (2007) reported that several epidemiological studies confirm that aflatoxin's cancer potency is about 30 times greater among hbv-positive than among hbv-negative individuals. et al aspergillus flavus et al et al et al 1 2 1 2 1 . . . 3 dietary exposure assessment for aflatoxin b from processed peanut products santi ambarwati .1 et al acute outbreaks of aflatoxicosis have been reported from kenya (ngindu 1982, cdc 2004), india (krishnamachari 1975) and malaysia (chao 1991, lye 1995). chronic aflatoxicosis results from ingestion of low to moderate levels of aflatoxins and the effects are impaired food convertion (shane 1993), slower rates of growth (gong 2002, 2004), and a decrease in various micronutrient levels (pimpukdee 2004). many countries have determined maximum tolerable levels of aflatoxins in peanuts and their processed products. maximum tolerable levels of aflatoxins b , b , g and g in peanuts and their processed products in australia, canada, philippines and singapore were 15, 15, 20 and 5 μg/kg, respectively (fao 2004). based on sni (2009) in indonesia, maximum tolerable limit of afb1 and total aflatoxins in peanuts and their processed products were 15 and 20 μg/kg, respectively. researches on infection and aflatoxin contamination in raw peanut kernels collected from farmers, collectors, wholesalers, retailers at traditional markets have been conducted by dharmaputra . (2005, 2007a). the results indicated that in general the highest aflatoxin contamination of raw peanut kernels was at retailers in traditional markets. dharmaputra . (2007b) reported that the high afb1 contents in raw kernels were due to among others by damaged kernels (discoloured, cracked and broken kernels). lilieanny . (2005) stated that the highest aflatoxin content was found in compared to in roasted peanuts with skin pods, roasted peanuts without skin pods, flour-coated peanuts, , and . data on aflatoxin level in processed peanut products and their consumptions are needed to prepare the dietary exposure assessment for aflatoxin. most asean countries (including indonesia) have some data on aflatoxin content in foods, however, no formal risk assessment on aflatoxin has been conducted for the region. this may be due to the lack of technical and financial resources to develop the necessary data and information needed to support or to conduct risk assessment. more data on aflatoxin contents in processed peanut products are needed. in addition, a survey of individual processed peanut product consumption should be conducted to determine aflatoxin exposure assessment (sparringa 2008). dietary assessment is a part of risk assessment, i.e. the process of estimating potential exposure of a population to food chemicals (among others aflatoxin) from the diet and comparing the potential exposure against a reference health standard for risk characterisation purpose. aflatoxin exposure assessment could be used to estimate the potential exposure/intake of the toxin, to assess the potential risk of health for a population group, and to maintain safe food supply. the objectives of this study were to determine the contents of afb1in processed peanut products at retail levels, and to obtain information whether there is a risk to public health caused by the consumption of processed peanut products contaminated by afb1. et al. et al. et al. et al. et al. et al. a. flavus et al et al et al bumbu pecel bumbu pecel enting-enting gepuk 1 2 1 2 4 materials and method pre-survey survey of processed peanut product consumption pre-survey consisted of : a. determination of data consumption survey location of processed peanut products : the location of data consumption survey was carried out at (subdistrict) bogor tengah covering 11 (lowest local government), i.e. babakan, babakan pasar, cibogor, ciwaringin, gudang, kebon kelapa, pabaton, paledang, panaragan, sempur, and tegallega. bogor tengah has the highest population density (13445 inhabitants/km ) and was selected to conduct the survey and sampling. in addition the government center including business activities are also found in this (bps kota bogor 2008). b. determination of respondents : the data of bogor tengah office showed that the population at bogor tengah until march 2009 was 116686 people. the number of respondents was determined based on the square root of population at bogor tengah, i.e. 342 respondents. in each , the number of respondents was determined proportionally based on the number of inhabitants. the respondents were grouped into two categories, i.e. children (6-15 years old, 169 respondents) and adults (16-44 years old, 173 respondents). it was assumed, that most children of 6 years and older like to eat processed peanut products, while the adults are more sensitive to hepatitic. survey of processed peanut product consumption was carried out by interviewing each respondent using a questionnaire of weekly processed peanut product consumption concerning : the kinds of processed peanut products ( or , , , , , , , or , , , and ) consumed by each respondent during the last one week the frequency of processed peanut products to be consumed by each respondent during the last week portion or product number consumed by each respondent during the last week the location where each respondent bought the processed peanut products the body weight of each respondent in addition to interviewing each respondent, observation was also conducted to obtain information about the number of processed peanut product sellers found in the surrounding of respondent domiciles. the information was used at the stage of sampling. during survey in each , the research team was accompanied by one or two staff of the who are familiar with the sites. kecamatan kelurahan kelurahan kecamatan kecamatan kecamatan kecamatan kecamatan kelurahan bumbu pecel gado-gado bumbu karedok bumbu siomay bumbu batagor bumbu satai bumbu ketoprak oncom hitam kacang garing kacang kulit kacang atom kacang telur kacang bawang kelurahan kelurahan 2 . . biotropia vol. 18 no. 1, 2011 5 sampling method of processed peanut products determination of aflatoxin b content estimation of the dietary exposure assessment for afb1 sampling of processed peanut products was conducted at the locations where the respondents obtained processed peanut products, i.e. from or , and vendors , and small shops. the number of samples of each processed peanut product was determined based on the number of processed peanut product sellers found in the surroundings of the respondent domiciles. the number of roasted peanuts with skin pods, flour-coated peanuts and or sauces samples were 33, respectively, while the number of dan sauces samples were 18 and 12, respectively. the total number of processed peanut products samples was 129. each sample consisted of 5 portions of processed peanut products in the form of sauce ( or , , and sauces) and 2 kg (= 100 small packs, weight @ 20 g) of other processed peanut products (roasted peanuts with skin pods and flour-coated peanuts). at the time of purchase, the main materials of or , and were packed separately from their peanut sauces. one portion of , and contained 75, 50 and 60 g peanut sauces, respectively. each sample was mixed manually and homogenously, and then divided into two parts to obtain working samples for afb1 content determination and a reserve sample. afb1 content was only determined in peanut sauces. afb1 content was determined using thin layer chromatography (tlc) method (aoac 2005). two replicates were used from each sample. afb1 was extracted from ground processed peanut products using methanol-h o. the filtrate was diluted using nacl solution and defatted using hexane. afb1was partitioned into chloroform, then it was removed through evaporation, and quantitated using tlc on silicagel plate by visual estimation, i.e. by comparing the spot of standard and sample. exposure assessment involves estimating the intensity, frequency, and duration of human exposures to a toxic agent. dietary exposure to aflatoxin b in bogor tengah was estimated using the afb1 concentration data, food consumption data and mean body weight. afb1 exposure was calculated on the group of average consuming (mean) and high consuming (95% percentile). consumption data were obtained from food frequency questionnaire. the estimation of dietary exposure assessment for afb1was determined based on the following formula (who 2008): warung pecel gado-gado siomay satai pecel gado-gado siomay satai pecel gado-gado siomay satai pecel gado-gado siomay satai gadogado siomay satai kecamatan 2 1 1 food consumption (kg/day) × b1 aflatoxin concentration (μg/kg) body weight (kg) ×dietary exposure = (ng/kg bw/day) 1,000 dietary exposure assessment for aflatoxin b from processed peanut products santi ambarwati .1 et al 6 results and discussion pattern of processed peanut product consumption aflatoxin b content based on five big ranks of consumers, roasted peanuts with skin pods (63% of the total respondent number) ranks first among 11 kinds of consumed processed peanut products in bogor tengah, followed by flour-coated peanuts (54.5% of the respondents ), sauce (54% of the respondents), or sauce (49% of respondent number) and sauce (34% of the respondents) (table 1). in general, either child or adult respondents, bought roasted peanuts, flour-coated peanuts, or , and from , or and vendors. in general child respondents bought from vendors, while adult respondents bought it from . the mean highest number of processed peanut product consumption of child and adult respondents was 0.0110 kg/day and 0.0149 kg/day, respectively, for or (table 2). the mean consumption number was obtained from the mean consumption frequency in one day multiplied by the mean consumption portion. afb1 contents were determined in five most consumed processed peanut products i.e. roasted peanuts, flour-coated peanuts, or sauce, and sauces. the highest contaminated sample percentage and mean of afb1 conteent was found in roasted peanuts with skin pods (42% of 33 samples and 43.2 μg/kg), followed by flour-coated peanuts (30% of 33 samples and 34.3 μg/kg), and or (21% of 33 samples and 17.1 μg/kg). (table 3). kecamatan siomay pecel gado-gado satai pecel gado-gado siomay warung warung pecel gado-gado satai warung satai pecel gado-gado pecel gado-gado siomay satai pecel gado-gado 1 respondent gender number of respondents kind of processed peanut products male female 1 2 3 4 5 6 7 8 9 10 11 childs 73 96 169 70 (21%) 12 (4%) 109 (32%) 84 (25% ) 59 (17%) 25 (7%) 11 (3%) 125 (37%) 109 (32%) 23 (7%) 9 (3% ) adults 33 140 173 97 (28%) 28 (8%) 75 (22%) 25 (7%) 58 (17%) 30 (9%) 48 (14%) 90 (26%) 77 (22.5%) 23 (7%) 19 (6% ) total 106 236 342 167 (49%) 40 (12% ) 184 (54%) 109 (32% ) 117 (34%) 55 (16%) 59 (17% ) 215 (63%) 186 (54.5 % ) 46 (13.5% ) 28 (9%) rank of the products consumed 4 3 5 1 2 note : 1 = 7 = (black oncom) 2 = 8 = (roasted peanuts with skin pods) 3 = 9 = (flour-coated peanuts) 4 = 10 = (egg-coated peanuts) 5 = 11 = (garlic peanuts) 6 = pecel/gado-gado oncom hitam karedok kacang kulit siomay kacang atom batagor kacang telur sate kacang bawang ketoprak table 1. number of respondents who consumed processed peanut products in (subdistrict) of bogor tengah kecamatan st up to 5thfrom 1 biotropia vol. 18 no. 1, 2011 7 table 2. frequency, portion and consumption number of processed peanut products in bogor tengahkecamatan respondent kind of product place of purchase*) mean of consumption frequency per day mean of portion (kg) mean of consumption number (kg/day) children roasted peanuts with skin pods warung (small shop) 0.40 0.0204 0.0082 flour-coated peanuts warung 0.31 0.0096 0.003 pecel/gado -gado sauce warung pecel/gado -gado 0.24 0.0460 0.0110 siomay sauce vendor 0.31 0.0164 0.00 satai sauce vendor 0.28 0.0224 0.00 adults roasted peanuts with skin pods warung 0.34 0.0290 0.009 flour-coated peanuts warung 0.32 0.0090 0.00 pecel/gado -gado sauce warung pecel/gado -gado 0.26 0.0572 0.014 siomay sauce vendor 0.30 0.0263 0.007 satai sauce warung satai 0.21 0.0305 0.0064 51 63 29 0 9 9 9 * small part of the respondents bought processed peanut products in traditionaland supermarkets or they prepared the products by themselves table 3. aflatoxin b content of processed peanut products at bogor tengah and maximum tolerable limit (mtl) of afb1 based on sni (2009) ) 1 kecamatan kind of processed peanut products no. of samples number (%) of samples contaminated by afb 1 number (%) of samples contaminated by afb 1 > 15 μg/kg roasted peanuts with skin pods 33 14 (42%) 14 (42%) flour-coated peanuts 33 10 (30%) 10 (30%) pecel/gado-gado sauce 33 9 (27%) 7 (21%) siomay sauce 18 2 (11%) 2 (11%) sat ai sauce 12 2 (17%) 2 (17%) mtl (sni 20 09) range and mean of afb 0 0 0 0 0 – – – – – content (μg/kg) 316.80 (43 160.00 (34.28) 197.80 (17.11) 39.90 (4.41) 198.58 (23.17) 15 .21) 1 the best way to control the presence of aflatoxins in foods and feeds is through good agricultural and manufacturing practices which could prevent fungal growth. aflatoxins are thermostable compounds and, once formed, they can persist in animal feeds and food. the usual methods of processing peanuts to make peanut butter and processing nuts for confectionery may appreciably reduce aflatoxin contamination. effective means of reducing aflatoxin contamination include removing undersized nuts, removing nuts that resist splitting and blanching, and removing discoloured nuts by hands or electric sorting (cole 1989). percentage of samples contaminated by afb1 and afb1 content of or sauce was relatively high. it was probably due to the low quality of peanuts used to prepare the sauces or containers used to store peanut kernels and to prepare the sauces were not clean. percentage of samples contaminated by afb1 and the mean of afb1 content in sauce were relatively low (11% of 18 samples and 4.4 μg/kg). the percentage of sauce samples contaminated by afb1 was also relatively low, i.e. 17% of 12 pecel gadogado siomay sate dietary exposure assessment for aflatoxin b from processed peanut products santi ambarwati .1 et al samples, but the mean of their afb1 contents was relatively high, i.e. 23.2 μg/kg (table 3). in indonesia the maximum tolerable limit (mtl) of afb1 for peanuts and their processed products is 15 μg/kg (sni 2009). percentage of roasted peanuts with skin pods, flour-coated peanuts, or sauce, sauce and sauce samples contaminated by afb1 exceeded 15 g/kg i.e. 42% of 33 samples, 30% of 33 samples, 21% of 33 samples, 11% of 18 samples, and 17% of 12 samples, respectively (table 3). based on the mean of afb1 content, among the five processed peanut products, sauce has a mean afb1 content lower than the mtl (4.41 g/kg). two out of 18 samples were contaminated by afb1 exceeding 15 g/kg. the other four processed peanut products contained mean of afb1 contents higher than mtl. consequently, risk management of the four products is suggested to be applied. lilieanny . (2005) stated that total aflatoxin contents of roasted peanuts with skin pods (47 samples), flour-coated peanuts (22 samples), (12 samples) and (4 samples) collected from several factories, supermarkets, and traditional markets in bogor, malang, pati and yogyakarta from january up to august 2002, were 1.8, 5.2, 41.6 and 20.8 μg/kg, respectively. aflatoxin was not detected in roasted peanuts without skin pods (3 samples). mean of estimated dietary exposure for afb1 in children was 15.2 ng kg bw day and 95 percentile exposure was 38.9 ng kg bw day , while in adults 9.0 ng kg bw day and 95 percentile exposure 27.0 ng kg bw day (table 4). the major contributing foods for afb1inchildren and adultswas roasted peanuts with skin pods, followed by sauce, sauce, flour-coated peanuts and sauce, while the highest contaminated level was roasted peanuts with skin pods, followed by flourcoated peanuts, sauce, sauce and sauce. the estimated highest exposures to afb1 by consuming roasted peanuts with skin pods were 44.5% (adult respondents) and 43% (child respondents) of total exposure to afb1 (table 4). further researches on the risk management of roasted peanuts with skin pods are needed. the mean dietary intake of aflatoxin for australian and swedes were 0.15 and 0.8 ng kg bw day , respectively (thuvander . 2001), for americans 0.26 ng kg bw day (jecfa 1998), for french adults (>15 years old ) was 0.1 ng kg bw day , while for children (ages 3-14 ) was 0.3 ng kg bw day. it was assumed, that bogor community consumedprocessed peanut products containinghigher content of afb1 compared to other countries. however, li (2001) reported that in guangxi, china, the probably daily intake of processed peanut products was estimated 3,680 ng kg bw day . from the five processed peanut products, the lowest dietary exposure for afb1 was sauce, either on children or adult respondents. dietary exposure for afb1 of the five products on children respondents was higher than that of adult respondents (table 4). this was due to the body weight of children which was lower than that of adults. the lower the body weight of the consumer, the higher the dietary exposure of afb1. pecel gado-gado siomay satai siomay et al bumbu pecel enting-enting gepuk pecel/gado-gado satai siomay satai gado-gado siomay et al et al. siomay estimation of the dietary exposure for aflatoxin b1 -1 -1 th -1 -1 -1 -1 th -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 biotropia vol. 18 no. 1, 2011 8 9 respondent kind of product mean of consumption number (kg/day) mean of afb1 content (μg/kg) mean of body weight (kg) dietary exposure for afb1 (n g/kg body weight /day ) percentage of total daily intake childs roasted peanuts with skin pods 0.0082 43.21 33 10.6 43.0 flour-coated peanuts 0.0030 34.28 32 3.4 13.7 pecel/gado-gado sauce 0.0110 17.11 32 5.6 22.8 siomay sauce 0.0051 4.41 32 0.7 2.8 satai sauce 0.0063 23.17 31 4.4 17.7 adults roasted peanuts with skin pods 0.0099 43.21 56 7.4 44.5 flour-coated peanuts 0.0029 34.28 57 1.8 10.7 pecel/gado -gado sauce 0.0149 17.11 56 4.4 26.3 siomay sauce 0.0079 4.41 56 0.6 3.5 satai sauce 0.0064 23.17 57 2.5 15.0 table 4. dietary exposure assessment for aflatoxin b from processed peanut products at bogor tengah 1 kecamatan risk assessment to evaluate the potential health risk of bogor community to afb1, a risk assessment of afb1 should be conducted by comparing the estimation of dietary intake and provisional maximum tolerable daily intakes (pmtdi). as afb1 is a genotoxic carcinogen, the safety factors used for non-genotoxic carcinogens cannot be applied. therefore, most agencies, including jecfa and us fda, have not determined yet a tolerable daily intake for afb1. jecfa proposed the potency value of 0.3 cancer cases per year per 100000 population per ng aflatoxin per kg body weight for hepatitis b positive individuals, while the potency value for the non-hepatitis b population was 0.01 cancer cases per year per 100000 population per ng aflatoxin per kg body weight (jecfa 1997). based on the potency value of 0.3 and 0.01 cancers per year per 100000 population per ng aflatoxin per kg body weight, prevalence of hepatitis b in bogor was 1.4% (departemen kesehatan republik indonesia 2008), while the population of bogor end 2007 was 905132 (bps kota bogor 2008). consequently, the results of this study showed that the cancer risk of afb1 exposure in bogor on children and adults was 193 and 115 cancer cases/year, respectively. therefore, cancer risk could increase due to the consumption of highly afb1 contaminated processed peanut products. afb1 is an unavoidable food contaminant. to evaluate the potential health risk of afb1 caused by food consumption, it is important to determine the natural occurrence of afb1 in food and to estimate the risk for liver cancer through dietary exposure to afb1.ok (2007) reported that the level of afb1 contamination in 28 of the 32 food products in south korea was less than 10 kg , which is the legal tolerance limit in korea. from data on daily food consumption, the exposure dose of afb1was estimated to be 6.42 x 10 mg kg body weight day .the risk of liver cancer et al. -1 -7 -1 -1 . . . . dietary exposure assessment for aflatoxin b from processed peanut products santi ambarwati .1 et al 10 for those exposed to afb1 through food intake was estimated to be 5.78 x 10 for hepatitis b-negative individuals and 1.48 x 10 for hepatitis b-positive individuals. the highest contaminated sample percentage and mean of afb1 content was found in roasted peanuts with skin pods (42% of 33 samples and 43.2 μg/kg), followed by flour-coated peanuts (30% of 33 samples and 34.3 μg/kg), and or (21% of 33 samples and 17.1 μg/kg). the percentage of sauce samples contaminated by afb1 was also relatively low, i.e. 17% of 12 samples, but the mean of their afb1 contents was relatively high, i.e. 23.2 μg/kg. among the five processed peanut products, sauce has a mean afb1 content lower than the mtl (4.41 g/kg). the other four processed peanut products contained afb1 higher than mtl. consequently, risk management of the four products is suggested to be applied. in addition, aflatoxin contamination can be minimized by using good practices, from farm up to table. mean of estimated dietary exposure for afb1 on children was 15.2 ng kg bw day and 95 percentile exposure was 38.9 ng kg bw day , while on adults 9.0 ng kg bw day and 95 percentile exposure was 27.0 ng kg bw day . the cancer risk of afb1 exposure in bogor from this study on children and adults showed 193 and 115 cancer cases /year, respectively. therefore, cancer risk could increase due to the consumption of highly afb1 contaminated processed peanut products. the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the office of for the permission in conducting the survey, to the office of and 11 offices of which belong to for the cooperation during the survey; to mr. edi suryadi, ms. amanda windyarini, mr. rahadian pratama, mr. putra hidayat nasution, ms. rena yulia vernanda, and to the technicians of the laboratory of food analysis, seameo biotrop, for their assistance. we would like also to express our appreciation to the reviewers of this manuscript. -6 -4 -1 -1 th -1 -1 -1 -1 th -1 -1 conclusions acknowledgements references pecel gado-gado sate siomay kesatuan bangsa dan perlindungan masyarakat (kesbanglinmas) kecamatan bogor tengah kelurahan kecamatan bogor tengah aoac. 2005. natural toxins. in : horwitz w, editor. official methods of analysis of aoac international. association of official analytical chemist, gaithersburg. 18 ed. ch.49. p 11 bps kota bogor. 2008. kota bogor dalam angka 2008. badan pusat statistik kota bogor, bogor. center for disease control and prevention (cdc). 2004. outbreak of aflatoxin poisoning easter and central provinces, kenya. morbid mortal weekly report 53: 790 793 th biotropia vol. 18 no. 1, 2011 chao tc, maxwell sm, wong sy. 1991. an outbreak of 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( ). sni 7385: 2009. badan standardisasi nasional, jakarta sparringa ra. 2008. total diet study and chemical risk assessment in foods. ( ). paper presented at seminar pra-2 widyakarya nasional pangan dan gizi ke-ix 2008, kelompok kerja mutu dan keamanan pangan. jakarta, 9 june 2008 thuvander a, moller t, barbieri he, janson a, salomonsson aca, olsen m. 2001. dietary intake of some important mycotoxin by swedish population. food additives and contaminant 18 : 696-706 world health organization of united nation (who), 2004. worldwide regulations for mycotoxins in food and feed in 2003; fao food and nutrition paper 81. food and agriculture organization of the united nations, rome who. 2008. the global burden of disease : 2004 update. geneva : world health organization. available at http://www.who. int/healthinfo/global_burden_disease/ 2004_reportupdate/en/index.html) who. 2008. dietary exposure assessment of chemicals in food. report of a joint fao/who consultation. annapolis, maryland, usa, 2-6 may 2005. who press, geneva yan liu, felicia wu. 2010. global burden of aflatoxin-indiced hepatocellular carcinoma : a risk assessment. environ health perspectives 118 (6): 818-24 1 batas maksimum kandungan mikotoksin dalam pangan studi diet total dan kajian paparan bahan kimia dalam pangan th . biotropia vol. 18 no. 1, 2011 12 biotropia (2) 1988/1989: 32-37 a collection of ticks (ixodidae) from sulawesi utara, indonesia l.a. durden department of entomology, nhb 165, museum support center smithsonian institution, washington d.c. 20560, u.s.a. c.h.s. watts division of natural science south australian museum, north terrace, adelaide, south australia 5000, australia abstract ixodid ticks were collected from seven species of endemic murid rodents and from vegetation in sulawesi utara, indonesia. adult ticks belonging to the species, amblyomma babirussae, a. cyprium and dermacentor (indocentor) steini were taken from the vegetation. immature ticks assignable to the genera, haemaphysalis, amblyomma and ixodes infested the murids with parauromys dominator and maxomys musschenbroekii being the two most heavily tick-infested host species. the data are compared to other tick records from sulawesi. introduction ectoparasites were collected from murid rodents live-trapped in sulawesi utara (north sulawesi), indonesia, on august 1985. we report here on the ticks (ixodi-dae) collected during this project. eighty five murids belonging to seven species, all endemic to sulawesi, were examined for ticks. the comparatively small size of these host animals made infestation by adult ticks (which are identifiable to the species level) unlikely, so adult ticks were taken from vegetation when possible to supplement the collections. collection data for the 58 ticks recovered from the murids and from vegetation are given below, listed by collection locality. specimens are currently in the rocky mountain laboratory collections (now at the smithsonian institution, washington, washington b.c., u.s.a.) and accession numbers are also given. mammal specimens are either with collections of the south australian museum or the museum zoologicum bogoriense, indonesia; their original collection numbers are presented here. 1. gunung moajat (0°45'n 124°25'e). elevation, 1800 m. from vegetation. amblyomma cyprium neumann (1q) 32 a collection of ticks (ixodidae)-l.a. durden & c.h.s. watts ex. parauromys dominator (chsw no. 114) haemaphysalis sp. (1l*) 2. dumoga-bone nat. park (0°34'n 123°54'e). elevation, 200-300 m. on grass at forest edge dermacentor (indocentor) steini schulze (1°) ex. maxomys musschenbroekii (chsw no. 19) amblyomma sp. (4l, 2n*) haemaphysalis sp. (1l) ex. m. musschenbroekii (chsw no. 21) amblyomma sp. (3l) ex. rattus xanthurus (chsw no. 28) amblyomma sp. (in) amblyomma sp. (probably)(ln) ex. maxomys hellwaldii (chsw no. 42) haemaphysalis sp. (4n) (one specimen has three acaridid mites attached to its venter). ex. rattus hoffmanni (chsw no. 47) haemaphysalis sp. (1l, in) ex. p. dominator (chsw no. 48) amblyomma sp. (12l, in) ex. m. musschenbroekii (chsw no. 54) ixodes sp. (2l) ex. m. musschenbroekii (chsw no. 55) haemaphysalis sp. (7n) ex. r. hoffmanni (chsw no. 58) ixodes sp. (1l) ex. p. dominator (chsw no. 66) haemaphysalis sp. (in) ex. r. hoffmanni (chsw no. 80) ixodes sp. (in) 3. dumoga-bone nat. park. hog's back ridge (0°35'n 123°52'e) elevation, 490 m. ex. p. dominator haemaphysalis sp. (in) ex. r. hoffmanni (chsw no. 138) amblyomma sp. (4l, 4n 2 of these nymphs belong to one species and 2 to a second species) 4. gunung mogogonipa (0°27'n 123°57'e). elevation, 1008 m. on vegetation 33 biotropia no. 2, 1988/1989 amblyomma babirussae (19) dermacentor (i.) steini schulze (19) 5. by 'lake' (0°44'n 124°27'e). elevation, 1080-1200 m. ex. p. dominator (chsw no. 13) haemaphysalis sp. (in) *l = larvae, n = nymphs. the three species of adult ticks that were collected are fairly well known. amblyomma babirussae (figure 1) represents part of the endemic tick fauna of figure 1. amblyomma babirussae (adult female). sulawesi; adults are parasitic principally on larger artiodactyls and on endemic and feral pigs (suidae) (anastos 1950, keirans and robbins 1987) while immatures are parasitic on these same hosts and also on murids (keirans and robbins 1987, van peena et al. 1974). adults of a. babirussae are also known to attach to humans (keirans and robbins 1987). amblyomma cyprium (figure 2) has a wide distribution in the indo-pacific region (anastos 1950; kohls 1957; wilson 1969; marshall 1976; kemp and wilson 1979) with adults again parasitic largerly on wild and feral pigs, but also on humans and cattle, and immatures on a variety of smaller hosts, 34 figure 2. amblyomma cyprium (adult female). particularly murids (kohls 1957; kemp and wilson 1979). dermacentor (i.) steini (figure 3) has recently been redescribed by wassef and hoogstraal (1986) who document a wide distribution for this species in indo-malaysia (including the philippines and with establishment on papua new guinea); however, they give no records for sulawesi so the present collections extend the documented range for this tick. 35 a collection of ticks (ixodidae)-l.a. durden & c.h.s. watts figure 3. dermacentor (indocentor) steini (adult female). biotropia no. 2, 1988/1989 dermacentor (i.) steini adults again appear to be parasitic mainly on wild and feral pigs (wassef and hoogstraal 1986). unfortunately, adults of an undescribed species of tick probably assignable to the genus, amblyomma, collected by durden (1986) as a single nymphal specimen (accession no. hh112,315 in the hoogstraal tick collection is now housed at the smithsonian institution, washington d.c., usa) from a murid, m. musschen-broekii, in sulawesi utara, were not collected. this immature specimen is so atypical that hoogstraal (personal communication) could not be certain of its taxonomic status and hopefully, future ectoparasite collections from sulawesi will produce adults of this fascinating tick. analysis of the prevalence of tick infestation from the murid species examined shows that no ticks were taken from the 13 bunomys fratrorum or from the 11 congeneric b. chrysocomus rats processed. the other five murid species showed various levels of infestation. rattus hoffmanni (n = 10) showed a collective tick (three genera represented) prevalence of 40% and a mean intensity of 3.0 ticks per infested animal. the other murid species had the following comparable infestation figures: r. xanthurus (n = 7)(1 tick genus), 14.3%, 2.0; p. dominator (n = 16)(2 tick genera), 31.3%, 3.4; m. musschenbroekii (n = 17)(3 tick genera), 23.5%, 4.8; m. hellwaldii (n = 11)(1 tick genus), 9.1%, 4.0. the main difference between these data and those of durden (1986) is that, in this study, no dermacentor (indocentor) sp. ticks were collected from murids. in the earlier survey, b. fratrorum and especially m. musschenbroekii were quite heavily infested by ticks of this subgenus; this difference could reflect a seasonal influence. another difference, possibly attributable to phenology, is the presence of ixodes sp. immatures from murids trapped in the lowland forests (200-300 m elevation) of dumoga-bone national park in this collection; durden (1986) did not record this genus from murids from comparable elevations in this forest. the earlier collection (durden 1986) was made from january to march, 1985, during the latter part of the monsoon season (although heavy rains were not as concentrated into this season as is typical for much of southeast asia). acknowledgments deep gratitude is extended to dr. i.e. keirans who identified the ticks described here and to dr. g.g. musser for confirming host identities. appreciation is also extended to r.g. robbins for cataloguing ticks, to dr. n.a. wilson for sup 36 a collection of ticks (ixodidae)-l.a. durden & c.h.s. watts plying separates and to the late dr. h. hoogstraal for his personal communications and earlier tick identifications. this research was undertaken as part of project wallace organized by the royal entomological society of london and the indonesian institute of sciences (results of project wallace no. 30). references anastos, g., 1950. the scutate ticks, or ixodidae, of indonesia. entomol. am., 30: 1. durden, l.a., 1986. ectoparasites and other arthropod associates of tropical rain forest mammals in sulawesi utara, indonesia. nat. geogr. res., 2: 320. kemp, d.h. and wilson, n.a., 1979. the occurrence of amblyomma cyprium (acari; ixodidae) in australia, with additional records from the southwest pacific. pac. insects, 21: 224. kohls, g.m., 1957. insects of micronesia. acarina: ixodoidea. insects micronesia, 3: 85. marshall, a.g., 1976. host specificity amongst arthropods ectoparasitic upon mammals and birds in the new hebrides. ecol. entomol., 1: 189. van peenan, p.f.d., carney, w.p., sudomo, m. and sulianti saroso, j., 1974. parasites of mammals in gumbasa valley, central sulawesi, indonesia. trop. geogr. med., 26: 352. wassef, h.y. and hoogstraal, h., 1986. dermacentor (indocentor) steini (acari: ixodidae): identity of male and female. j. med. entomol., 23: 532. wilson, n.a., 1969. ticks (metastigmata: ixodidae) collected by the noona dan expedition to the philippine and bismarck archipelagos. entomol. med., 37: 285. 37 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf microsoft word 46 biotropia no. 24, 2005 : 46 53 effect of antanan (centella asiatica) and vitamin c on the bursa of fabricius, liver malonaldehide and performance of heat-stressed broilers engkus kusnadi1, reviany wldjajakusuma2, toha sutardi3, peni.s.hardjosworo4 and arifien habibie5 'department of animal production.faculty of animal husbandry, andalas university(unand), padang, indonesia 2department of physiology and pharmacology,faculty of veterinary medicine, bogor agriculture university(ipb), bogor, indonesia 3department of animal nutrition & feed science, faculty of animal husbandry, bogor agriculture universirty(ipb), bogor, indonesia 4department of animal production, faculty of animal husbandry, bogor agriculture university (ipb), bogor, indonesia 1 deputy assistant for agroindustry-jakarta, coordinating ministry for economic affairs, jakarta, indonesia abstract high environmental temperatures may cause heat stress in poultry. this may increase water consumption, decrease feed consumption and in rum, decrease productivity level. in addition, high temperature contributes to oxidative stress, a condition where oxidant activity (free radicals) exceeds antioxidant activity. in our research, antanan (centelta asiatica) and vitamin c were utilized as anti heat-stress agents for heat-stressed broilers. we used 120 male broilers 2 -6 weeks old, kept at 31.98 ± 1.94 °c during the day and 27.36 ± 1.31 °c at night. the data collected were analyzed with a completely randomized factorial design of 2 x 3 (2 levels of vitamin c, 3 levels of antanan at 4 replications) and continued with the contrast-orthogonal test when significantly different. the results indicate that the treatments of 5 and 10% of antanan with or without 500 ppm of vitamin c and vitamin c alone significantly (p<0.05) decreased the heterophil/lymphocyte (h/l) ratio and liver malonaldchydc (mda). these treatments, however, significantly (p<0.05) increased the bursa of fabricius weight, feed consumption and body weight gain. it could be concluded that basal ration administered with 5% antanan and 500 ppm vitamin c could effectively prevent broilers from heat stress. the results support the conclusion that a basal ration supplemented with 5% antanan and 500 ppm of vitamin c or their combinations, effectivelly reduces heat stress in broilers. key words : heat stress/ centella asiatica i vitamin c introduction high environmental temperatures may result in the accumulation of body heat load so that the body suffers from heat stress. as one of the homeothermic species, poultry could maintain their body temperature relatively constant by increasing respiration rate and water consumption and/or decreasing feed consumption. as a result, their growth rate and productivity will decrease. 46 biotropia no. 24, 2005 may and lott (2001) showed that body weight gain of 3 to 7-week-old male broilers raised at a temperature of 30°c was 1869 g significantly lower than for those raised at 22°c with body weight gains of 2422 g and feed conversion decreased from 3.28 to 2.54. the lower performances of broilers raised at high temperatures may have occurred as a result of lowered secretion of thyroid hormones (geraert et al. 1996), decreased blood hemoglobin and hematocrit levels (yahave/a/. 1997), or increased excretion of some minerals (belay et al. 1992) and some amino acids (tabiri et al. 2001). in addition, heat stress may also cause oxidative stress in the body and develop abundant free radicals, promoting the occurrence of peroxidation of membrane lipids and hence attacking dna and protein membranes (rahman 2003). takahashi and akiba (1999) indicated that feeding oxidized lipids to broilers significantly decreased feed consumption, body weight gain, plasma vitamin c, and plasma a-tocopherol. in fact, the results were followed by an increase in plasma malonal-dehyde (mda) and blood heterophyl/lymphocytes (h/l) ratios as biological indices of stress in avian species. antanan/pegagan (centella asiatica (l.) urban), one of the medicinal plants containing active materials such as asiatic acid, asiaticoside, and madecasic, is readily available and evidently eliminates stress in rats (kumar and gupta 2003). shukla et al. (1999) reported that placing asiaticoside on rats wound increases curability and accelerates enzymatic and nonenzymatic antioxidant activities of new-growing tissues. in addition, vitamin c reportedly eliminated cold stress (sahin and sahin 2002) and heat stress in poultry (puthpongsiriporn et al. 2001) and showed synergism with some active materials contained in antanan (bonte et al. 1994). with this in mind, we have examined the effect of antanan (centella asiatica) and vitamin c on the bursa of fabricius, liver malonaldehide and performance in heat-stressed broilers. materials and methods this research used 2 to 6-week-old male broilers placed in several pens located in an open poultry house. each pen was flitted with a 40-watt lamp and a zinc-plate backing functioning as a heat reflector. temperature and relative humidity measurements obtained at noon and afternoon were 31.98 ± 1.28°c and 78.82 ± 5.43%, respectively. temperature and relative humidity measurements at night and early morning were 27.36 ± 0.88°c and 86.23 ± 3.93%, respectively. the levels of 500 ppm vitamin c and 10% antanan (all plant parts) of ration used were established during preliminary trials. vitamin c was dissolved in drinking water and served in the morning, two hours after the broilers received their last waterfeeding. 47 effect of antanan (centella asiatica) and vitamin c ‐ engkus kusnadi et al.  one‐hundred‐and‐twenty‐two‐week‐old male broilers were randomly allocated into 24 pens,  5 broilers  each. antanan  (5%  and  10%) was mixed with other  ingredients  to make  three  different  rations  as  follows:  1)  the  control  ration  contained  the  calory  as metabolizable  energy  (me)  3245.02  kcal/kg  and  20.84%  crude protein  ,  2) a5  =  5%  antanan  contained me  3222.95 kcal/kg and crude protein 20.91%, and 3) a10 = 10% antanan contained me 3202.87  kcal/kg  and  crude  protein  20.99%.  the  ration  formulation  and  nutrient  composition  of  treatments are presented in table 1.                  the broilers were subjected to six treatments, 20 broilers each, as follows:  1) k (control)/ration neither contained antanan nor vitamin c.  2) a5/ration was supplemented with 5% antanan  3) alo/ration was supplemented with 10% antanan  4) c, drinkwater contained 500 ppm vitamin c  5) a5c, combination of a5 and c, and  6) a10c, combination of a10 and c  48  biotropia no. 24, 2005 variable measurements: 1) relative bursa of fabricius weight taken from 4-week-old broilers, by weighing the organ and divided by body weight (puvadolpirod and thaxton 2000). 2) heterophyl/lymphocyte ratio was taken from 4-week-old broilers, by hemo-cytometer method. blood was diluted 1:101 in a red blood cell pipette with nat and herrick diluent. the total leucocyte count includes heterophils, lymphocytes, monocytes, basophils, and eosinophils is divided by the number of lymphocytes. 3) liver malonaldehyde (mda) taken from 6-week old broilers, by measuring the thiobarbituric acid (tea) value using tarladgis method (apriyantono et al. 1989). destilate from liver sample with ph: 1.5, is added to the tba reagents, covered, mixed, and incubated in boiling water bath for 35 minutes. after cooling, the absorbance of filtrate (d) was determined at 528 nm wave length. the tbars values = 7.8 d were expressed as mg/kg of malonaldehyde per kg of tissue. 4) feed consumption, body weight gain, and feed conversion are measured for 4 weeks (from 2 to 6-week-old broilers). feed consumption was determined by weighing the given ration minus the leftover. body weight gain was measured by weighing the final body weight at 6 weeks old minus the body weight at 2 weeks old. feed conversion was measured by dividing feed consumption with body weight gain. statistical analysis the collected data were analyzed using a completely randomized factorial design (crd) 2 x 3 (2 levels of vitamin c i.e. 0 and 500 ppm and 3 levels of antanan i.e. 0, 5 and 10% of rations at 4 replications), and where applicable, continued with orthogonal contrast test according to steel and torrie (1980). results and discussion the results of the effect of antanan and vitamin c administration on bursa of fabricius weight, h/l ratio and liver mda contents are presented in table 2. the data for feed consumption (fc), body weight gain (bwg), and feed conversion are presented in table 3. from table 2, the average relative of 4 week bursa of fabricius weight for controls (k) is significantly lower than those treated with a5, a10, c, a5c, or a10c. the bursa of fabricius weight is similar for all treatments from a5 to a10c. 49 effect of antanan (centella asiatica) and vitamin c ‐ engkus kusnadi et al.  these results suggest that antanan, vitamin c, or their combinations increase burs of fabricius  weight of broilers that suffered heat stress.  the ability of antanan to stimulate lymphoid gland weight was reported i stressed rats  by sharma et al.  (1996). antanan contains phenol compounds  th< potentially can prevent  peroxidation of lipid membranes including t‐ and e lymphocyte membranes. t‐lymphocytes  produce cellular immunities while e lymphocytes produce humoral immunities produced by the  bursa of fabricius in bir species. phenol compounds of tea extracts were potentially capable of  stimulatin  the production of  lymphoid cells  in  rats  (murtini et al. 2003). on  the other hanc  vitamin c acts, as a water‐soluble antioxidant capable of protecting lymphocyte from suffering  heat  stress  (puthpongsiriporn  et  al.  2001). as  a  result,  the  number  o  circulating  lymphocytes  increased so that the h/l ratio decreased.  table 2 shows a relationship between the  increase of the bursa of fabriciu weight and  decrease in h/l ratio because the bursa of fabricius is a lymphoid orgai producing lymphocytes.  thus, the smaller  the bursa of fabricius size, the  fewe  lymphocytes will be produced, and  in  turn, the higher the h/l ratio will be. increase of the relative lymphoid organ weight occurred as a  result of antanan feeding whicl was demonstrated by sharma et al. (1996), while increase of the  relative bursa o fabricius weight as a result of supplementing vitamin c to broilers was reported  b] anim et al. (2000).                                  heat stress generating oxidative stress may increase mda content as a result of lipid  peroxidation, especially for unsaturated fatty acids of membrane cells. feeding  50    biotropia no. 24, 2005 antioxidants, i.e. antanan and vitamin c, significantly decreases liver mda content. besides of being able to relieve free radicals by releasing an electron and a proton (a hydrogen ion), phenol compounds are also able to provide a chelating effect in such a way that phenols bind to transition ions. unbound metals might increase free radicals (pietta 2000). in addition, phenol compounds are characterized by flavonoids that are able to decrease fluidity of cell membranes so that it may decrease diffusion of free radicals and mda contents. this is also true for vitamin c, a water-soluble antioxidant with 2 hydroxyl groups at €2 and €3 that are readily oxidized (sediaoetama 1987).                  in addition, it is unmistakable that feeding antanan and vitamin c increases feed consumption and body weight gain of broilers over four weeks (2-6 weeks of age) but not for feed conversion (table 3). this agrees with the reports of anim et al. 2000) for broilers and sharma and sharma (2002) for rats suffering from stress. antanan contains antioxidants such as phenol compounds that are capable of eliminating oxidative stress processes (blokhina 2000), as is apparent from the decrease of h/l ratios, liver mda levels and increase of bursa of fabricius weights reported here.  conclusions feeding antanan or vitamin c or the combination of antanan and vitamin c increases bursa of fabricius weight, feed consumption, and body weight gain but decreases h/l ratio and liver mda levels. the combination of 5% antanan and 51   effect of antanan (centella asiatica) and vitamin c — engkus kusnadi et al. vitamin c tends to increase feed consumption and body weight gain and , therefore this treatment tends to be very effective in alleviating heat stress in broilers. acknowledgements the authors wish to thank the department of higher education, nations department of education, republic of indonesia and seameo searca regional center for graduate study and research in agriculture, college, lagun 4031 philippines for their financial support. references anim aj, tl. lin, py. hester, d. thiagarajan, ba. watkins and cc. wu. 2000. ascorbic aci supplementation improved antibody response to infectious bursal disease vaccination in chickens poultry sci. 79: 680-688. apryantono.a, d.fardiaz, nl.puspitasari, scdarnawati and s.budiyanto. 1989. analisis pangar departcmen pcndidikan dan kcbudayaan dircktorat jcnderal pcndidikan tinggi pusat anta universitas pangan dan gizi institut pcrtanian bogor. belay t, wicmusz cj and rg. teeter. 1992. mineral balance and urinary and fecal mineral excrctioi profile of broilers housed in thcrmoncutral and heat-distressed environments. poultry sci. 71: 104 1047. blokhina o. 2000. anoxia and oxidativc stress: lipid pcroxidation, mitochondria! functions in plant antioxidant status and mitochondria! functions in plants http://ethesis.helsinki.fi/iulkaisut/mat/bioti/vk/blokhina/anoxiaan.html. [20 dcscmbcr 2003]. bonte f, dumas m, chaudagnc c and a. mcybcck. 1994. influence of asiatic acid, madccassic acid, am asiaticoside on human collagen i synthesis. planta med. 60: 133 135. geraert pa, padilha jcf and s. guillaumin 1996. metabolic and endocrine changes by chronic hca exposure in broiler chickens: biological and endocrinological variables. br. j. nutr.75:205-216. gross wb and hs. siegel. 1983. evaluation of the heterophil/lymphocyte ratio as a measure of stres in chickens. avian dieseasc.27: 972 979. kumar vmh and yk. gupta. 2003. effect of centella asiatica on cognition and oxidative stress in ai intracerebroventricular streptozotocin model of alzheimers disease in rat. clin exp pharmaco physiol 30: 336342. may jd and bd. lott. 2001. relating weight gain and feed:gain of male and female broilers to rearini temperature. poultry sci 80: 581-58444. murtini s, murwani r, bunawan a, handharyani e and f. satrija. 2003. effects of inoculation route am dose level of tea mistletoe (scurrula oortiana) stem extract on the development of lymphoi< follicle of bursa fabricius in chick embryonated eggs. international symposium on biomedicincs 18lhand 19th september 2003. ipb. 52 biotropia no. 24, 2005 pietta pg. 2000. flavonoids as antioxidants. reviews. j nat prod 63: 1035-1042. puthpongsiriporn u, scheidelcr se, sell jl and mm. beck. 2001. effects of vitamin e and c supplementation on performance, in vitro lymphocyte proliferation, and antioxidant status of laying hens during heat stress. poultry sci 80: 1190-1200. puvadolpirod s and jp. thaxton. 2000. model of physiological stress in chickens 1. response parameters. poultry sci 79: 363-369. rahman.i. 2003. oxidative stress, chromatin remodelling and gene transciption in inflammation and chronic lung desease. j.biochcm. mol. biol. 36: 95-109. sahin k and n. sahin. 2002. efcct of chromium picolinatc and ascorbic acid dietary supplementation on nitrogen and mineral excretion of laying hens reared in low ambient temperature (7 °c). acta vet brno 71: 183-189. sediaoetama, ad. 1987. vitaminologi. jakarta: balai pustaka. sharma dnk, khosa rl, chansouria jpn and n. saha. 1996. antistrcss activity of tinospora cordifolia and centella asiatica extracts. phytotherapy-rescarch 10: 181 183. sharma j and r. sharma. 2002. radioprotcction of swiss albino mouse by centella asiatica extract. phytotherapy-research 16: 785 786. shukla a, rasik am, and bn. dhawan. 1999. asiaticoside-induced elevation of antioxidant levels in healing wounds. phytotherapy-research 13: 50-54. steel rod and jh. torrie. 1980. principles and procedures of statistic, second ed, graw-hall, book comp, new york. tabiri hy, sato k, takahashi k, toyomizu m, and y. akiba. 2000. effects of acute heat stress on plasma amino acids concentration of broiler chickens. jpn poult sci 37: 86-94. takahashi k and y. akiba. 1999. effect of oxidized fat on performance and some physiological responses in broiler chickens. j poult sci 36: 304-310. yahav s, straschnow a, plavnik i and s. hurwitz 1997. blood system response of chickens to changes in environmental temperature. poultry sci 76: 627 633. 53 46.pdf 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf 53.pdf biotropia 1 (1) 1987: 53-57 iii. mycorrhizae in agroforestry: a case-study s.t. nuhamara* tropical forest biology program, biotrop, bogor, indonesia keywords: agroforestry, mycorrhiza, resin, shorea javanica, sumatra abstract census of mycorrhizae in shorea javanica agroforests has been made periodically in the district of krui, lampung, sumatra. amanita hemibapha (amanitaceae), cantharellus cibarius (cantharella-ceae), lactarius spp., russula spp. (russulaceae) and scleroderma sp. (sclerodermataceae) were commonly encountered on the agroforest floor. these mycorrhizal fungi are naturally associated with the planted trees. the significance of mycorrhizae for the maximization of growth and sustained productivity of resin is discussed as well as the need to design well defined agroforestry systems to facilitate growth and to improve production management techniques. introduction torquebiau (1984) reported for the first time that the "kebun damar" or "resin garden" of shorea javanica k & v (dipterocarpaceae) in krui (sumatra) was a typically good example of an agroforestry system for resin production. to obtain the maximum sustained yield of resin, all the growth factors should be optimally maintained. ideally, a tree is expected to perform active and continuous chemo-ecophysiological processes for long possible periods of time. mycorrhizae play an important role at root level by dynamizing the nutrients and water uptakes. in the case of an agroforestry system, two groups of mycorrhizae can be considered: those which are directly associated with the main crop and those which are not, and/or are associated with the other component species of the system. in this paper, the author presents information related to the first group. nevertheless, new ideas proposed about the second group and the specific case of mycorrhizae in agroforestry especially the inter-relationships between the above-ground organs, e.g. leaves and the mycorrhizal roots of the system, is discussed. material and methods census of the occurrence of fruiting bodies of mycorrhizal fungi having connections with the roots of s. javanica has been made periodically at krui, * present address: faculty of forestry, bogor agricultural university, darmaga campus, bogor, indonesia. 53 biotropia vol. 1 no. 1, july-december 1987 lampung, sumatra in june and october 1985 and february of 1986. the study sites were at ngaras, gunung kemala and pahmongan, all in the surroundings of krui. soil analysis corresponding to these sites was described by sheehy skeffington in chapter 2. taxonomic identifications were made at the biotrop laboratory. this simple procedure was adopted from bakshi (1974) and based on experience with other dipterocarp species either in haurbentes, jasinga, west java or in lempake, east kalimantan. interviews with the local farmers were also conducted especially relating to seedling preparation. results and discussion based on the periodical census made so far, the following, well-known, ectomycorrhizal fungi have been recorded: amanita hemibapha amanitaceae cantharellus cibarius cantharellaceae lactarius spp. russulacea russula spp. russulaceae scleroderma sp. sclerodermataceae it was learnt from the local farmers that no artificial inoculation technique has been applied so far for mycorrhizal introduction in the agroforests. some of these mycorrhizal fungi have been artificially inoculated to shorea pinanga, s. stenoptera and hopea sangal (nuhamara et al. 1986), and developed successfully. it was also concluded that infected soil as source of inoculum performed better as compared to single mycorrhizal fungus. this suggests the need for application of infected soil to a neighboring site when a crop is to be experimented. however, on a long term basis, especially when drastic changes in agricultural practices can be foreseen, the use of pure culture will be the only advisable measure. shorea javanica is a special case because as a resin producer, the plant is expected to produce continuously. this requires optimum chemo-ecophysiological processes. to do this, both the above ground organs (e.g. leaves) and the under ground ones (roots), must function optimally. there should be a reciprocal balance of processes. it is in connection with this that the roots should be well fortified by typical mycorrhizal associations, to enable them to absorb and accumulate nitrogen, phosphorus, potassium and calcium more rapidly and for longer periods of time than non mycorrhizal ones. mycorrhizae, especially the ectomycorrhizae, appear to increase the tolerance of trees to drought, high soil temperatures, soil toxicity (organic and inorganic), and extreme low soil ph caused by high levels of sulfur or aluminium. ectomycorrhizae deter infection of feeder roots by 54 iii. mycorhizae in agroforestry: a case-study — nuhamara pathogens. hormone induced or the production of which is induced by fungal symbionts cause ectomycorrhizal roots to have a greater longevity (duration of physiological activity) than non mycorrhizal roots (marx 1973). sufficient water and other photosynthetic elements such as magnesium are essential for a proper functioning of the leaves. furthermore, according to the way carbon is fixed, plants belong to one of the c3, c4 or cam categories. figure 1. chart showing the complex ecophysiological processes in plant growth and development in relation to mycorrhiza, and possible co2 fixing ways. this implies that the different carbohydrate productions which in turn determine the different available simple carbohydrates might alter the different mycobionts. this proposed complex metabolic pattern is illustrated in fig. 1. as shown on the chart, the hormones, produced by or induced with the presence of mycorrhizae, activate the function of the rhizophere. regarding agroforestry, it is possible to manipulate the species composition and structure (michon 1983 and 55 biotropia vol. 1 no. 1, july-december 1987 torquebiau 1964) in such a way to facilitate the more stable eco-unit (oldeman 1983) which is also of good economical value for the farmers. the author suggests to construct agroforestry systems having a good combination of c3 plants like s. javanica trees and other fruit trees and c4 plants in combination with cam plants. it is believed that with such a mixed composition, a stable association can be obtained and productive compatibility achieved. another aspect of the possible manipulation of species in agroforestry concerns the relationships between host species diversity and the types of mycorrhizal association. the diagram of fig. 2 suggests that the zone where ecto and endo-mycorrhizae coexist is the best for mixed gardening or agroforestry, because the richer the mycobiont diversity, the higher the degree of productive compatibility. this practice is common in many tropical regions, e.g. java. oldeman (personal communication 1985) believed that such a model is stable and applicable to the tropical conditions. figure 2. theoretical curve showing the relationship between the host species diversity and the type of mycorrhizal association. the problem is how far it is possible to change or manipulate the symbiont composition. moving to the left of the figure would mean more endomycorrhizal plants and moving to the right a combination of ecto and endomycorrhizal plants. most of the annual herbaceous agricultural crops are endomycorrhizal, like paddy, 56 iii. mycorhizae in agroforestry: a case-study — nuhamara maize, soybean, etc, but some perennial woody agricultural crops such as coffee, cacao, oil palm are also endomycorrhizal plants. contrarily, most forest trees such as dipterocarps and pines are associated with ectomycorrhizae and there are also some important tropical forest trees such as teak and agathis which belong to endomycorrihizal plants. considering both the carbon metabolism and the mycorrhizal association of the plants, it must be possible to define the smallest agroforestry unit of good economical as well as ecological stability, and to propose it for development areas in different parts of the country. optimizing intercropping in agroforestry would mean the organizing of species composition, density and structure of both woody and herbaceous plants in order to facilitate their complementary productivity and at the same time minimizing their competitive relationships. in relation to resin production, it is suggested that further research be concentrated on tapping technique like its disturbance on the metabolic processes. the number and size of the scars affect the translocation of carbohydrates from the top down to the roots. this ultimately influences the availability of simple carbohydrates for the mycobionts. acknowledgments the author expresses his gratitude to dr. e.f. torquebiau, the french expert seconded to biotrop for his generous stimulation, inspiration as well as his invaluable criticisms on the author's ideas whithout which this paper would not be in this form. thanks are also due to dr. tri binarko suselo for his support and suggestions and to mr. koko iskandar for technical assistance. references bakshi, b.k. 1974. mycorrhiza and its role in forestry. forest research institute and colleges, dehra dun, india. 89 pp. marx, d.h. 1973. growth of ectomycorrhizal and non mycorrhizal short leaf pine seedlings in soil infested with phytopthora cinnamomi, phytopathology 63: 18-23. michon, g. 1983. village-forest-garden in west java. in: plant research and agroforestry. edited by: p.a. kuxley. international council for research in agroforestry, nairobi. 617 pp. nuhamara, s.t., s. hadi and s.s. tjitrosomo, 1986. the effect of different soil types and mycorrhizae on the growth of some dipterocarp seedlings. biotrop spec. publ. no. 26 (in press). oldeman, r.a.a. 1983. the design of ecologically sound agroforests. in: plant research and agroforestry. edited by: p.a. huxley, international council for research in agroforestry, nairobi. 617 pp. torquebiau, e.f. 1984. man-made dipterocarp forest in sumatra. agroforestry systems, 2: 103-127. 57 53.pdf 54.pdf 55.pdf 56.pdf 57.pdf biotropia no biotropia no. 12, 1999 : 1 18 the occurrence of insects and moulds in stored cocoa beans at south sulawesi okky s. dharmaputra seameo biotrop, p.o. box 116, bogor 16001, indonesia, and department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia sunjaya, muhammad amad and ina retnowati seameo biotrop, p.o. box 116, bogor 16001, indonesia teguh wahyudi the indonesian research institute for coffee and cocoa, jl. pb sudirman 90, jember 68118, indonesia abstract surveys on postharvest handling and technology processing of cocoa beans at farmer, trader and exporter levels in south sulawesi were conducted together with investigations on moisture content, pest infestation (insect and mould) and quality characteristics in terms of reducing sugar, free amino acid and free fatty acid content. surveys were conducted during dry (july 1997) and wet seasons (february 1998) in three regencies (pinrang, polewali-mamasa and luwu) and ujung pandang, south sulawesi province. interviews were carried out during surveys in the dry season. number of respondents from farmers, trailers and exporters was 38, 15 and 5, respectively. in each season, number of samples taken from farmers, traders and exporters was 9, 21 and 15, respectively. in general, farmers, traders and exporters did not carry out postharvest handling and technology processing properly. moisture content of cocoa beans collected from farmers, traders and exporters were higher than the tolerable limit recommended by sni (7.5%). moisture content of cocoa beans collected during the wet season was higher than in the dry season. insects were found on cocoa beans collected from traders and exporters. species composition and the presence of each insect species were varied among the two seasons, but the predominant species was tribolium castaneum. at trader level the percentage of insect-damaged beans during the wet season was higher than that during the dry season, while at exporter level it was lower. during the two seasons the percentage of mouldy beans at farmer level was lower than the tolerable limit recommended by sni (4%), while those from some samples at trader and exporter levels were higher than 4%, but based on the direct plating method, all of the samples at trader and exporter levels were mouldy. species composition and the percentage of beans infected by each mould species at farmer, trader and exporter levels during the two seasons were varied. the percentage of mouldy beans increased at trader and exporter levels. the predominant moulds were aspergillus flaws, eurotium amstelodami, e. chevalieri and penicillium citrinum. the predominant mould at farmer level during wet season was saccharomyces cerevisiae (yeast). reducing sugar and free amino acid content of cocoa beans collected during the dry season was higher than those collected during the wet season, either at farmer, trader or exporter levels. free fatty acid content of cocoa beans tends to be higher during the wet season than the dry season at the three levels. keywords: stored products pests/postharvest handling/technology processing/moisture content/insect/mould/reducing sugars/free amino acids/free fatty acids/cocoa/south sulawesi. 1 biotropia no. 12, 1999 introduction indonesia ranks third among the cocoa producing countries in the world after ivory coast and ghana (ico 1996). in south and southeast sulawesi cocoa production was dominated by smallholders, and generally the quality was low, i.e. high moisture content, low fat content, less flavour, and inconsistency in the quality of beans (wahyudi 1994). in relation to health and food safety to consumers due to insect infestation and mould infection, the quality of cocoa beans in indonesia is considered low (siswoputranto 1994). zaenudin and wahyudi (1996) also reported that the problem of exported cocoa beans was due to insect infestation and mould infection, so that it was subjected to automatic detention, and thus should be refumigated. the quality of cocoa beans can be increased by improving the method of postharvest handling from farmer to exporter levels. aziz (1996) assumed that the low quality of cocoa beans was attributed to farmers and traders in indonesia who did not conduct postharvest handling and technology processing properly. the objective of this study was to get information on postharvest handling and technology processing of cocoa beans at farmer, trader and exporter levels in south sulawesi province. the moisture content, pest infestation (insect and mould) and quality characteristics (reducing sugar, free amino acid and free fatty acid content) of cocoa beans were also analyzed. materials and methods time and location of surveys surveys were conducted during the dry (july 1997) and wet seasons (february 1998) in three regencies (pinrang, polmas and luwu) and ujung pandang, south sulawesi province. these districts were selected because they produce large quantities of cocoa beans, while ujung pandang was the main port for exporting of cocoa beans. range of the number of rainy days and rainfalls from those regencies during dry and wet seasons is presented in table 1. interviews using questionnaires with farmers, traders and exporters, on the spot observation at survey locations, and random sampling of cocoa beans derived from farmers, traders and exporters in selected areas were carried out during the surveys. interviews using questionnaires interviews were carried out during the dry season to get information on postharvest handling and technology processing of cocoa beans at fanner, trader and exporter levels. data were collected randomly from interviews with respondents. number of respondents from each level was different depending on the condition in 2 the occurrence of insects and moulds in stored cocoa beans okky s. dharmaputra et al. table 1. range and average number of rainy days and rainfall on location of the surveys during dry and wet seasons dry season wet season june 1997 july 1997 january 1998 february 1998 rf rd rf rd rf rd rf rd range 49-155 3-11 68-106 4-14 85-175 8-10 138-264 8-10 average 91.3 6.0 82.7 8.7 135.4 8.7 207.3 9.3 rf = rainfall (mm) rd= rainy days (day) the field during the surveys. number of respondents at farmers, traders and exporters was 38, 15 and 5, respectively. the questionnaires contain questions on postharvest handling and technology processing carried out by farmers, traders and exporters. sampling methods about 1 kg of each sample was taken randomly from farmer, trader and exporter levels. in each season, number of samples taken from farmers, traders and exporters were 9, 21 and 15, respectively. samples of cocoa (primary samples) from each stack were taken randomly from a certain number of sacks using a sampling spear. insects were separated from each primary sample using graded sieves. each primary sample was divided several times using a sample divider to obtain working samples for analyzing moisture content, insect-damage, mould and quality characteristics in terms of reducing sugars, free fatty acids and free amino acids. the presence of insects was estimated on sack surface as well as within the sacks. moisture content, insect, mould and quality characteristics analyses moisture content (wet weight) was determined based on sni 01-2323 (isc 1995). the insects were identified using the publication of haines (1991) as the main reference. insect-damaged beans were determined according to sni 01-2323 (isc 1995). mouldy beans were determined based on 2 methods, namely cut test (isc 1995) and direct plating methods on dichloran 18% glycerol agar (dg 18) (pitt and hocking 1985). the moulds were identified using the publications of pitt and hocking (1985), samson et al. (1996) as the main references. reducing sugars, free amino acids and free fatty acids were determined according to somogyi (mccready 1970), spectrometry method (lillevik 1970) and sni 01-2323 (isc 1995), respectively. 3 biotropia no. 12, 1999 results and discussion interviews with farmers, traders and exporters the results of questionnaires are shown in table 2. commonly, bulk cocoa was cultivated by smallholders in pinrang, polmas and luwu regencies (99.4% of table 2. result of questionnaires on postharvest handling and technology processing of cocoa beans at farmer, trader and exporter levels * = generally, ex fertilizer bag ** = generally, ex sugar bag 4 the occurrence of insects and moulds in stored cocoa beans okky s. dharmaputra el al respondents). immediately after harvesting, the pods were opened using a knife or a wooden billet. nevertheless, some farmers delayed opening the pods for several days (23.7%).there were only 44.7% of farmers who fermented their cocoa beans properly before drying. usually, the beans were fermented using traditional fermenting boxes. most of the traders bought wet cocoa beans from farmers (75% of respondents), consequently farmers did not dry their cocoa beans up to recommended moisture content (7.5%). method of drying used by farmers was usually sun-drying for less than 3 days (57.9%), or 3-5 days (42.1%). they spread the beans on various drying facilities, namely fish net, jute bag, polypropylene bag, wooven bamboo and pandanus mat. traders redried their cocoa beans for less than 3 days (80%), consequently the moisture content was still high and the beans could easily be infected by mould. in this condition, cocoa beans could not be stored for a long time. the common bag type used for storing cocoa beans at trader level was jute bag (33.3%) ex fertilizer and sugar bags. exporters also redried their cocoa beans by the sun-drying method, but during the wet season they used a mechanical dryer until 7.5% moisture content was attained. jute bags were usually used to store the beans. some exporters did not inspect the beans for insects and mould infestations in the warehouse. moisture content a. a. farmer level moisture content (m.c.) of cocoa beans collected from farmers during dry and wet seasons were 7.9-11.7% (mean 9.7%) and 9.3-14.0% (mean 12.3%), respectively (table 3). moisture content of the samples were higher than the tolerable limit recommended by sni (7.5%). most of the farmers (75%) sold cocoa beans in wet condition to the traders (table 2). if the cocoa beans were not dried immediately and properly by the farmers, they could readily be infected by moulds. the farmers did not pay too much attention to the m.c. of cocoa beans sold, because they want to get money quickly. their main source of living comes from cocoa beans. table3. moisture content, insect-damage, and mouldy cocoa beans collected from farmers during dry and wet seasons dry season wet season parameter mean range mean range moisture content (%) insectdamage beans (%) mouldy beans (%) cut test direct plating method 9.7 b 0 0.2 c 18.4 e 7.9-11.7 0-2 7-76 12.3 a 0 0.7 c 32.1 d 9.3-14.0 0-2 14-53 numbers followed by the same letter did not differ significantly according to duncan multiple range test at 95% confidence level 5 b1otrop1a no. 12, 1999 , based on statistical analysis, there was a significant difference between dry and wet seasons on m.c. of cocoa beans (table 3). aside from economical factors, the rainfall and the number of rainy days affected the duration of drying, so that the m.c. of cocoa beans during the wet season (12.3%) was higher than during the dry season (9.7%). differences in rainfall and number of rainy days between wet and dry seasons are shown in table 1. b. trader level moisture content of beans collected from traders during dry and wet seasons were 7.3-11.8% (mean 9.4%) and 7.3-12.9% (mean 9.6%), respectively (table 4). m.c. of cocoa beans during the wet season was higher than the dry season, but based on statistical analyses the m.c. was not significantly different for the two conditions (table 4). m.c. of almost all the samples collected (95.4% samples) during dry and wet seasons were higher than 7.5% (tolerable m.c. for storing of cocoa beans). the traders redried the cocoa beans for less than 3 days, consequently their m.c. was still high, so that they could be infected by moulds. table 4. moisture content, insect-damage, and mouldy cocoa beans collected from traders during dry and wet seasons parameter dry season wet season mean range mean range moisture content (%) insect-damage beans (%) mouldy beans (%) cut test direct plating method 9.4 a 0.2 b 1.1 d 26.7 f 7.3-11.8 0-1 0-8 5-100 9.6 a 0.5 b 2.2 c 47.6 e 7.3-12.9 ,0-2 0-7 3-100 numbers followed by the same letter did not differ significantly according to duncan multiple range test at 95% confidence level c. exporter level moisture content of cocoa beans collected from exporters during dry and wet seasons were 7.2-9.7% (mean 8.2%) and 7.5-11.5% (mean 9.4%), respectively (table 5). statistical analysis revealed that m.c of cocoa beans during the wet season was significantly higher than the dry season (table 5). m.c. of twelve samples (80%) collected during the dry season and all samples collected during the wet season were higher than 7.5%. the high m.c. of cocoa beans collected from exporters during the wet season was due to the high m.c. of cocoa beans bought from traders. however, m.c. of cocoa beans always change with the change of relative humidity in storage. m.c. of 7.5% is in equilibrium with the relative humidity of storage of 75% (wood 1985). 6 the occurrence of\jnsects and moulds in stored cocoa beans okky s. dharmaputra el al. table s. moisture content, insect-damage, and mouldy cocoa beans collected from exporters during dry and wet seasons parameter dry season wet season mean range mean range moisture content (%) insect-damage beans (%) mouldy beans (%) cut test direct plating method 8.2 b 0.7 c 3.7 d 67.4 e 7.2-9.7 0-3 0-6 8-100 9.4 a 0.5 c 1.7 d 73.4 e 7.5-11.5 0-2 0-3 41-100 numbers followed by the same letter did not differ significantly according to duncan multiple range test at 95% confidence level the presence of insects and insect-damaged beans cocoa beans could be damaged by insects during storage, mainly of the orders lepidoptera and coleoptera. kalshoven (1981) and wood (1985) reported that insect species associated with stored cocoa beans were araecerus fasciculatus (coffee bean weevil), ephestia cautella (tropical warehouse moth), and tribolium castaneum (rust red flour beetle). insect pests of cocoa beans feed directly on grain constituents. their feeding action causes loss of weight of the commodity, thus reducing its value, commercially and nutritionally. according to sni 01-2323, insect-damaged beans should not be higher than 4%. however, the quality of exported beans must be free from dead or live insects. a. farmer level there were no insect-damaged beans and live insects found on cocoa beans collected from farmers during dry and wet seasons, because most of the farmers did not store the beans for a long time. usually, soon after drying or storing for only less than 1 week, the beans were sold. b. trader level eight insect species found during the dry season were anisopteromalus calandrae, araecerus fasciculatus, callosobruchus chinensis, carpophilus sp., cryptolestes ferrugineus, liposcelis entomophila, sitophilus zeamais and tribolium castaneum. ten insect species found during the wet season were ahasverus advena, anisopteromalus calandrae, araecerus fasciculatus, c. chinensis, carpophilus sp., l. entomophila, oryzaephilus surinamensis, palorus sp., s. zeamais and t. castaneum. haines (1991) reported that these insects were major species infesting grain during storage, except a. calandrae which is a parasitic insect. tribolium castaneum was the most frequently found insect. the percentages of samples infested by t. castaneum during dry and wet seasons were 66.7 and 71.4, respectively (table 6). 7 biotropia no. 12, 1999 table 6. the number and percentage of cocoa bean samples infested by each insect species at trader level during dry and wet seasons n insect species dry season wet season number of infested samples percent of infested samples number of infested samples percent of infested samples 1 ahasverus advena 0 2 9.5 2 anisoptermalus calandrae 1 4.8 1 4.8 3 araecerus fasciculatus 5 23.8 3 14.3 4 callosobruchus chinensis 1 4.8 1 4.8 5 carpophilus sp. 1 4.8 2 9.5 6 cryptolesles fernigineus 1 4.8 0 7 liposcelis entomophila 1 4.8 1 4.8 8 oryzaephilus surinamensis 0 1 4.8 9 palorus sp. 0 2 9.5 10 sitophilus zeamais 3 14.3 4 19.0 11 tribolium castaneum 14 66.7 15 71.4 most of the traders sold various commodities in their shops which are located at traditional markets, consequently insect species of other commodities were also found on cocoa beans. sitophilus zeamais and c. chinensis are the major insects associated with maize and mung beans. their presence on stored cocoa beans was due to migration. the difference among variability of insect species between dry and wet seasons was caused by the difference in physical conditions, such as temperature, relative humidity and moisture content. haines (1991) reported that the development and behaviour of some insects could be affected by the physical condition of the environment. at high moisture content, competition between mould and other microorganisms could decrease the number of some stored insects which are replaced by fungus-feeder insects. in this study, this is shown by the presence of a. advena on 2 samples collected during the wet season. percentage of insect-damaged beans in samples collected during the wet season (0.5%) was higher than the dry season (0.2%), but they were not significantly different (table 4). c. exporter level seven insect species were found on cocoa beans collected during the dry season, they were a. fasciculatus, bracon hebetor, carpophilus sp., corcyra cephalonica, ephestia cautella, necrobia rufipes and tribolium castaneum, while during the wet season 10 insect species were found, i.e. ahasverus advena, araecerus fasciculatus, b. hebetor, carpophilus sp., corcyra cephalonica, cryptolestes ferrugineus, e. cautella, liposcelis entomophila, oryzaephilus surinamensis and t. castaneum. most of these insects were grain stored pest, except b. hebetor which is a parasitic insect of corcyra cephalonica and e. cautella. necrobia rufipes is a common insect associated with copra (haines 1997), therefore 8 the occurrence of insects and moulds in stored cocoa beans okky s. dharmaputra et al. its presence on stored cocoa beans was due to migration, because cocoa beans and copra were stored in the same warehouse. variability of insect species at exporter level during the wet season was higher than the dry season. tribolium castaneum was the most frequently found insect. the percentages of samples infested by t. castaneum during dry and wet seasons were 93.3 and 80.0, respectively (table 7). table 7. the number and percentage of cocoa bean samples infested by each insect species at exporter level during dry and wet seasons n insect species dry season wet season number of infested samples percent of infested samples number of infested samples percent of infested samples 1 ahasverus advena 0 5 33.3 2 araecerus fasciculatus 12 80.0 4 26.7 3 bracon hebetor 3 20.0 3 20.0 4 carpophilus sp. 1 6.7 2 13.3 5 corcyra cephalonica 1 6.7 2 13.3 6 cryptolestes fermgineus 0 2 13.3 7 ephestia cautella 8 53.3 1 6.7 8 liposcelis entomophila 0 1 6.7 9 oryzaephilus surinamensis 0 5 33.3 1 necrobia rufipes 1 6.7 0 1 tribolium castaneum 14 93.3 12 80.0 the variability of insects found on cocoa beans at trader level, both during dry and wet seasons was different than at the exporter level. it might be due to the difference in environmental conditions. the structures and sanitary conditions of exporter's warehouses were better than that of trader's warehouse. exporters usually stored only cocoa beans in their warehouse, while traders did not. according to sidik (1997) the presence of pests in stored grain was usually affected by the physical structure and sanitary conditions of the storage facility, and characteristic of the stored grain. the percentage of insect-damaged beans during the wet season (0.5%) was lower than the dry season (0.7%), but they were not significantly different (table 5). mouldy beans the difference between the two analyzing methods (cut test/snl and direct plating method) gave different results on mouldy beans. the percentage of mouldy beans using the cut test was lower than for the direct plating method, because the mould was invisible before it produces mycelium or spore mass inside the beans. nonmouldy beans observed using the cut test might be already infected by mould, but their intensity was very low. visual observation as well as examination of the beans using a stereoscopic microscope at low magnification also did not reveal the 9 biotropia no. 12, 1999 differences in the appearance of the beans that might be correlated with levels of mould infection. niles (1981) reported that on invisible mouldy beans which had been further redried, the mould count was 2.0 x 10s cfu/g beans. nevertheless, hanson and welty (1971) stated that the presence of microorganisms on non-mouldy beans (visual observation) has no correlation with bean quality. a. farmer level in general, farmers did not store the beans for a long period of time, therefore, the intensity of mould infection was still low. statistical analysis showed that percentage of mouldy beans by the cut test was not significantly different between dry and wet seasons, while for the direct plating method it was significantly different (table 3). the percentage of mouldy beans by the cut method during the wet season (32.1%) was higher than the dry season (18.4%). mouldy beans obtained using the cut test during dry and wet seasons were between 0-2%, respectively, while the direct plating method gave 7-76% and 14-53%, respectively (table 3). cut tests showed that 1 sample (11%) collected during the dry season and 5 samples (11%) collected during the wet season had mouldy beans. examination using the direct plating method showed that all of the samples had mouldy beans, during the two seasons. it suggests that mould infection has occurred at the farmer level, although its intensity was low. thirteen species of moulds were isolated from cocoa beans collected during the dry season, i.e. aspergillus flavus, a. niger, a. ochraceus, a. restrictus, a. tamarii, a. wentii, botryodiplodia theobromae, cladosporium cladosporioides, eurotium amstelodami, mucor piriformis, nigrospora oryzae, penicillium citrinum and saccharomyces cerevisiae (yeast). the predominant species were aspergillus flavus and a. niger. table 8 shows that the average number of beans infected by both table 8. the percentage of cocoa beans infected by each mould species at farmer level during dry and wet seasons n mould species infected beans (%) dry season wet season mean range mean range 1 aspergillus flavus 10.4 0-67 8.4 2-26 2 a. niger 10.4 0-59 6.2 0-14 3 a. ochraceus 0.1 0-1 0 0 4 a, reslriclus 0.3 0-2 0 0 5 a. tamarii 0.5 0-3 0.2 0-1 6 a. wentii 7.3 0-64 0 0 7 botryodiplodia theobromae 1.7 0-7 4.2 0-30 8 cladosporium cladosporioides 1.8 0-6 0 0 9 eurotium amstelodami 1.9 0-9 5.1 0-20 1 e. chevalier! 0 2.6 0-13 1 mucor piriformis 0.3 0-3 0 0 1 nigrospora oryzae 0.2 0-2 0 0 1 penicillium citrinum 5.6 0-19 0.2 0-2 1 saccharomyces cerevisiae 0.6 0-3 34.4 0-100 10 the occurrence of insects and moulds in stored cocoa beans okky s. dharmaputra el al. species was 10.4%. the other most frequently isolated moulds were a. \ventii (7.3%) and penicillium citrinum (5.6%). eight species of moulds were isolated from cocoa beans during the wet season, i.e. a. flavus, a. niger, a. tamarii, b. theobromae, e. amstelodami, e. chevalieri, p. citrinum and saccharomyces cerevisiae. the percentage of the beans infected by each mould species is presented in table 8. the predominant moulds were a. flavus (8.4%) and a. niger (6.2%). on some samples, yeast was most frequently isolated. however, yeast did not give negative effects, because infection was only on the shell of cocoa beans due to improper washing and drying of the beans. botryodiplodia theobromae was isolated from cocoa beans collected from farmers. the presence of this mould indicated that the pods were infected by pod rot. according to wood (1985) b. theobromae caused diplodia pod rot and infected the beans before harvest. b. trader level based on statistical analysis, the percentage of mouldy beans collected during the wet season was higher than that during the dry season and significantly different either using the cut test or the direct plating method (table 4). based on the cut test, mouldy beans obtained during the dry and wet seasons were 0-8% and 0-7%, respectively. the percentage of mouldy beans using the direct plating method was higher than the cut test. the direct plating method yielded 5-100% (mean 26.7%) mouldy beans during dry season and 3-100% (mean 47.6%) during wet season (table 4). based on the cut test, 2 samples (0.1%) collected during the dry season and 4 samples (0.2%) collected during the wet season had low quality, because the percentage of mouldy beans was more than 4%. the direct plating method showed that all samples collected either during dry or wet seasons were mouldy. the percentage of mouldy beans collected from the traders were higher than that from the farmers, because the traders did not carry out sorting properly. they also stored the beans with high moisture content for long time. nineteen species of moulds were isolated from cocoa beans collected during the dry season, i.e. aspergillus candidus, a. flavus, a. niger, a. ochraceus, a. restrictus, a. tamarii, a. •wentii, botryodiplodia theobromae, cladosporium cladosporioides, eurotium amstelodami, e. chevalieri, fusarium graminearum, f. proliferatum, libertella faginea, mucor piriformis, nigrospora oryzae, penicillium citrinum, rhizopus oryzae, and saccharomyces cerevisiae. the predominant moulds were a. niger (10.0%), followed by p. citrinum (7.6%) and a. flavus (5.6%). fourteen species of moulds were isolated from cocoa beans collected during the wet season, i.e. aspergillus candidus, a. flavus, a. niger, a. ochraceus, a. tamarii, botryodiplodia theobromae, cladosporium cladosporioides, eurotium amstelodami, e. chevalieri, libertella faginea, mucor piriformis, nigrospora oryzae, penicillium citrinum and 5. cerevisiae. the predominant moulds were e. amstelodami (15.9%) 11 biotropia no. 12, 1999 followed by a. flavus (12.6%). the percentage of cocoa beans infected by each mould species is presented in table 9. table 9. the percentage of cocoa beans infected by each mould species at trader level during dry and wet seasons c. exporter level statistical analysis showed that there was no significant difference on the percentage of mouldy beans collected during dry and wet seasons, either using the cut test or the direct plating method (table 5). the percentage of mouldy beans using the cut test and collected during dry and wet seasons were 0-16% (mean 3.7%) and 0-3% (mean 1.7%), respectively, while those using the direct plating method were 8-100% (mean 67.4%) and 41-100% (mean 73.4%), respectively (table 5). based on the cut test all samples collected during the wet season were still within the tolerable quality recommended by sni 01-2323 (less than 4%), except 1 sample collected during the dry season was higher than 4%. based on the direct plating method, all samples collected had mouldy beans in excess of 4%, either during dry or wet seasons. the percentage of mouldy beans collected from the exporters were higher than that from the traders. it was assumed that the exporters did not carry out sorting properly, or they stored the beans with high moisture contents, consequently the beans were more easily infected by mould. sixteen species of moulds were isolated 12 the occurrence of insects and moulds in stored cocoa beans okky s. dharmaputra el al. from cocoa beans during the dry season, i.e. aspergillus candidus, a. jlavus, a. niger, a. ochraceus, a. tamarii, a. wentii, botryodiplodia theobromae, cladosporium cladosporioides, eurotium amstelodami, e. chevalieri, e. repens, libertella faginea, mucor piriformis, penicillium citrinum, rhizopus oryzae and saccharomyces cerevisiae (table 10). the predominant mould was p. citrinum (37%). other moulds frequently isolated were eurotium amstelodami (31.5%) and a. niger (13.3%). the domination by p. citrinum and e. amstelodami was correlated with low moisture content of beans (about 8%). eleven species of moulds were isolated from cocoa beans during the wet season, i.e. aspergillus flavus, a. niger, a. tamarii, cladosporium cladosporioides, eurotium amstelodami, e. chevalieri, libertella faginea, mucor piriformis, m. racemosum, penicillium citrinum and rhizopus oryzae (table 10). the predominant moulds were a. flavus (20.5%) and e. chevalieri (20.9%). table 10. the percentage of cocoa beans infected by each mould species at exporter level during dry and wet seasons quality characteristics the quality requirements for both marketing and manufacturing stages can be divided into factors. firstly, the yield-determining factors are bean size, shell content, fat content, and foreign matter. secondly, the quality-determining factors are flavour, purity or wholesomeness, consistency, and cocoa butter characteristics. quality analysis of cocoa bean samples in this study were conducted in terms of reducing sugars and free amino acids, whereas cocoa butter quality was represented by free fatty acids analysis. 13 biotropia no. 12,1999 reducing sugars reducing sugars (fructose and glucose), amino acids and peptides are flavour precursors (biehl et al. 1985). reducing sugars are important flavour precursors. the role of this sugar is to perform as a degrading agent of amino acids during flavour development (rohan and stewart 1967a). hastori et al (1987) reported that reducing sugar content of cocoa beans correlated with the duration of fermentation. the results show that during the dry season they tend to be higher than the wet season, especially at farmer and trader levels (table 11). these results imply that postharvest handling of cocoa beans should be conducted more carefully at farmer and trader levels, especially during the wet season, in order to produce higher reducing sugar content. table 11. reducing sugar content of cocoa beans collected from farmers, traders, and exporters during dry and wet seasons reducing sugar content (%) dry season wet season source of cocoa beans mean range mean range farmers traders exporters 1.32 a 1.34 a 1.27 a 1.05-1.51 0.96-1.63 0.96-1.51 1.15 b 0.93 b 1. 19 a 1.04-1.34 0. 53-1.22 1.03-1.47 numbers followed by the same letter in the same row do not differ significantly according to dmrt a 95% confidence level reducing sugar content of cocoa beans collected from farmers, traders an< exporters during the dry season were 1.05-1.51% (mean 1.32%), 0.96-1.63% (mea 1.34%) and 0.96-1.51% (mean 1.27%), respectively, while during the wet seaso 1.04-1.34% (mean 1.15%), 0.53-1.22% (mean 0.93%), and 1.03-1.47% (mea 1.19%), respectively (table 11). free amino acids as mentioned above, free amino acids constitute a flavour precursor. roh and stewart (1967b) revealed that flavour development of cocoa beans correlai with amino acid synthesis during fermentation. hastori et al. (1987) reported t amino acid content could be affected by duration of fermentation. therefr fermentation must be stopped when the condition of free amino acid content reached a maximum. under-fermentation and over-fermentation cause loss of s< precursors of flavour development. the results of free amino acid analysis also show that during the dry se; they tend to be present at higher levels than the wet season. statistical analysis sh that there was a significant difference between free amino acid content of c beans collected from farmers during dry and wet seasons, but neither from tn nor exporters (table 12). 14 the occurrence of insects and moulds in stored cocoa beans okky s. dharmaputra et al. free amino acid content of cocoa beans collected from farmers, traders, and exporters during the dry season were 0.47-1.04% (mean 0.64%), 0.15-1.26% (mean 0.66%), and 0.44-1.50% (mean 0.76%), respectively, while during the wet season 0.31-0.71% (mean 0.49%), 0.41-0.84% (mean 0.59%), and 0.42-1.14% (mean 0.68%), respectively (table 12). table 12. free amino acid content of cocoa beans collected from farmers, traders, and exporters during dry and wet seasons free amino acid content (%) dry season wet season source of cocoa beans mean range mean range farmers traders exporters 0.64 a 0.66 a 0.76 a 0.47-1.04 0.151.26 0.44-1.50 0.49 b 0.59 a 0.68 a 0.31-0.71 0.41-0.84 0.42-1.14 numbers followed by the same letter in the same row do not differ significantly according to dmrt at 95% confidence level. free fatty acids the measurement of free fatty acids (ffa) provides an indication of the quality of cocoa beans to produce cocoa butter. cocoa butter with high level of free fatty acids tend to be soft, have poor crystallization properties, contain off-flavour, and have a poor shelflife (nickless 1994). the ffa level must be less than 1.0%, so that ffa in the cocoa butter produced from these beans will be less than 1.75%. this is the legal limit for cocoa butter within the eec and the limit proposed by codex (ccca 1984). the free fatty acid contents of cocoa beans collected from farmers, traders and exporters were higher during the wet season than the dry season (table 13). this might be due to incomplete or delayed drying, long period of storage under humid conditions (ccca 1984), or accidental wetting during storage, transport or shipment (wood 1985). based on statistical analysis, there were significant differences between ffa content of cocoa beans collected during the dry and wet seasons at farmer and exporter levels. these results also justified the need of a better postharvest handling technique at farmer, trader and exporter levels. free fatty acid content of cocoa beans collected from farmers, traders and exporters during the dry season were 0.25-2.19% (mean 0.90%), 0.43-2.86% (mean 0.96%), and 0.52-1.89% (mean 1.10%), respectively, while during the wet season 0.29-1.83% (mean 1.45%), 0.53-3.81% (mean 1.25%), and 0.83-2.04% (mean 1.39%), respectively (table 13). the free fatty acid contents of cocoa beans collected from farmers, traders and exporters were higher during the wet season than the dry season. this might be due to incomplete or delayed drying, long period of storage under humid condition (ccca 1984), or accidental wetting during storage, transport or shipment (wood 1985). 15 biotropla no. 12,1999 table 13. free fatty acid content of cocoa beans collected from farmers, traders, and exporters during dry and wet seasons free fatty acid content (%) dry season wet season source of cocoa beans mean range mean range farmers traders exporters 0.90 a 0.96 a 1.10 • 0.25-2.19 0.43.2.86 0.52-1.89 1.45 b 1.25 a 1.39 b 0.29-1.83 0.53-3.81 0.83-2.04 numbers followed by the same letter in the same row do not differ significantly according to dmrt at 95% confidence level. conclusions in general, farmers, traders and exporters did not carry out postharvest handling and technology processing of cocoa beans properly. moisture content of cocoa beans collected from farmers, traders and exporters were higher than the tolerable limit recommended by sni (7.5%). m.c. of cocoa beans during the wet season was higher than the dry season. insects were found on cocoa beans from traders and exporters. species composition and presence of each insect species were varied during the two seasons, but the presence of tribolium castaneum occurred at higher frequency. at trader level the percentage of insect-damage beans was higher during the wet season than the dry season, while at exporter level it was lower. based on the cut test during the two seasons the percentage of mouldy beans at farmer level was lower than the tolerable limit recommended by sni (4%), while those from some samples collected at trader and exporter levels were higher than 4%, but based on the direct plating method all of the samples collected at the two levels were mouldy. species composition and the percentage of beans infected by each mould species at farmer, trader and exporter levels during the two seasons were varied. the percentage of mouldy beans increased at trader and exporter levels. aspergillus flavus, eurotium amstelodami, e. chevalieri and penicillium citrinum were the predominant moulds. the predominant mould isolated from cocoa beans collected from farmers during the wet season was saccharomyces cerevisiae (yeast). reducing sugar and free amino acid content of cocoa beans collected during the dry season were higher than those collected during the wet season, either at farmer, trader or exporter levels. free fatty acid content of cocoa beans tends to be higher during the wet season than the dry season at the three levels. postharvest handling of cocoa beans should be conducted more carefully at farmer, trader and exporter levels, especially during the wet season, in order to produce good quality cocoa beans. 16 the occurrence of insects and moulds in stored cocoa beans okky s. dharmaputra et al. acknowledgement the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the plantation office province level, south sulawesi; plant quarantine service, ujung pandang; and dpd askindo south sulawesi for their information and cooperation during the surveys at farmer, trader and exporter levels in south sulawesi. the authors are also grateful to the indonesian research institute for coffee and cacao, for their collaboration in this study. references aziz, m.f. 1996. upaya peningkatan mutu biji kakao rakyat melalui sentralisasi pengolahan. wart a puslit kopi dan kakao 12(1): 12-17. biehl, b., e. brunner, d. passern, v.c. quesnel and d. adomako. 1985. acidification, proteolysis, and flavour potential in fermenting cocoa beans. j. sci. fd. agric. 36 : 583-598. ccca (the cocoa, chocolate and confectionery alliance). 1984. cocoa beans: chocolates manufacturer's quality requirements. 11 green street, london, 20p. haines, c.p. 1991. insects and arachnids of tropical stored products: their biology and identification (a training manual). natural resources institute, u.k. haines, c.p. 1997. insects and arachnids in indonesian food stores-biodiversity in a man-made environment. proceedings of the symposium on pest management for stored food and feed, bogor, indonesia, 5-7 september 1995. p. 95-125. hansom, a.p. and r.e. welty. 1971. microflora of raw cocoa beans. mycopath. mycol. appl. 44 : 309 316. hastori, b., djatmiko and t. wahyudi. 1987. pengaruh perlakuan pendahuluan pada fermentasi kakao (theobroma cacao l.) terhadap mutu biji keringnya. bul. teknol. industri pertanian 1 (i) : 23-40. indonesian standardization council. 1995. indonesian national standard, sni 01-2323 1995, rev. 1994: cocoa beans. international cocoa organization. 1996. production of cocoa beans by country, 1986/87 1995/96. quart, bull, of cocoa statistics 22 (3): 6. international cocoa organization, london. kalshoven, l.g.e. 1981. the pests of crops in indonesia. revised by p.a. van der laan. p.t. ichtiar baru-van hoeve, jakarta. lillevik, h.a. 1970. the analytical chemistry of the protein, peptides and amino acids. in methods in food analysis: physical, chemical and instrumental methods of analysis (ed. by joslyn, m.a.), 2nd ed. academic press, new york. p. 617-700. me cready, r.m. 1970. monosaccharides. in methods in food analysis: physical, chemical and instrumental methods of analysis (ed. by joslyn, m.a.), 2nd ed. academic press, new york. p. 475-509. nickless, h. 1994. cocoa butter quality. in proceeding of the malaysian international cocoa conference (ed. by j. selamat, b.c. lian, t.k. lai, w.r.w. ishak and m. mansor). malaysian cocoa board : 322-336. nlles.e.v. 1981. microflora of imported cocoa beans. j. stored prod. res. 17:147-150. pirr, j. i and a.d. hocking. 1985. fungi and food spoilage. academic press, sydney. roman, t.a. and t. stew art. 1967a. the precursors of chocolate aroma : production of reducing sugars during fermentation of cocoa beans. j. food sci. 32:399-402. _____. 1967b. the precursors of chocolate aroma : productions of free amino acid during fermentation of cocoa beans. j. food. sci. 32: 395-398. samson, r.a, e.s. hoekstra, j.c. frisvad and o. filtenborg. 19%. introduction to food-borne fungi. 3th ed. centraalbureau voor schimmelcultures, baam, the netherlands. 17 biotropia no. 12,1999 sidik, m. 1997. state-of-the-art of storage management. proceedings of the symposium on pest management for stored food and feed, bogor, indonesia, 5-7 september 1995. p. 11-23. siswoputranto, p.s. 1994. prospek perkakaoan dunia dan beberapa masalah yang perlu digarap. makalah disampaikan dalam gelar teknologi pascapanen kakao rakyat, samarinda, 13-14 september 1994. wahyudi, t. 1994. teknologi pengolahan untuk menghasilkan kakao bermutu baik dan usaha diversifikasi produk. makalah disampaikan dalam gelar teknologi pascapanen kakao rakyat. samarinda, 13-14 september 1994. p. 1-17. wood, g.a.r. 1985. from harvest to store. in cocoa. (ed. by wood, g.a.r and r.a. lass). longmann, london, pp.444-504. zaenudin and t. wahyudi. 1996. laporan kunjungan tim askindo ke amerika serikat dalam upaya meniadakan automatic detention terhadap kakao indonesia. warta puslit. kopi dan kakao 12(1): 44-47. 18 biotropia no. 7, 1994: 41-46 chromosomal characters of the indonesian sand goby, oxyeleotrismarmorata blkr. 1874 (eleotridae)*) jimmy t. masagca catanduanes state colleges college of agriculture and fisheries, virac, catanduanes 4800, philippines and komar sumantadinata faculty of fisheries, bogor agricultural university, darmaga campus, bogor, indonesia abstract karyomorphological investigation of sand goby or marble sleeper (oxyeleotris marmorata blkr.) from cirata reservoir, west java, indonesia was undertaken to determine the modal chromosome number and fundamental number, and to construct the karyotype from somatic metaphase cells of head kidney. a total of 30 fish samples from cirata reservoir was sacrificed for direct chromosome preparation by colchicine-citrate-aceto-methanol-giemsa staining-air drying technique. chromosome set analysis showed that the modal chromosome number of the test fish is 2n = 46, confirming previous studies. fundamental number is 50. two karyotypic formulas were found, i.e. 1) 2n = 46 (4sm + 42a); and 2) 2n = 46 (2m + 2sm + 42a). key words: freshwater fishes, chromosome analysis, oxyeleotris marmorata, indonesia introduction sand goby or marble sleeper (o. marmorata blkr.) is an important freshwater food fish in most southeast asian countries notably in thailand, indonesia and singapore (tan & lam 1973; tavarutmaneegul & lin 1988). this eleotridine gobioid is the largest in the world (smith 1965) and found naturally in streams, rivers, lakes or reservoirs and old mining pools in southeast asia (weber & de beaufort 1953; noerdin & sidik 1979; mohsin & ambak 1983). in the philippines, this fish was collected in laguna de bay lake by meyer in 1885 but no other account has been made about its range in this country (herre 1927). o. marmorata (locally known in indonesia as "ikan betutu") has been the focus of significant research because of the increasing market demand as an export *) part of a long-term research fellowship work undertaken at seameo biotrop in 1991. 41 biotropia no. 7, 1994 commodity to singapore, hongkong and taiwan. it has been cultured in thailand (supamataya 1988), vietnam (pantulu 1976) and other countries in southeast asia since the 1970's because of its suitability for aquaculture. it has wide tolerance to ph (lie 1968) and can survive at low oxygen tensions. it has a high reproductive rate and is a multiple spawner (tavarutmaneegul & lin 1988). it was introduced in saguling reservoir of west java, indonesia in 1986 (munro et al. 1990) and has spread to the adjacent reservoir of cirata. the commercial value of sand goby is increasingly recognized in many parts of southeast asia, but very little is known about its basic genetics. extensive literature search only identified the studies of arai and fujiki (1979) and manna (1989) on the chromosomal complement of sand goby and both needed further investigation. these studies utilized the gill epithelial cells as the source of chromosomes, while the present study used the kidney. this study reports on the results of karyological investigation of sand goby obtained from a population in cirata reservoir, bogor, indonesia. specifically, this study provides information on the somatic chromosome number, fundamental number or number of chromosome arms and the karyotypic formula. materials and methods live samples of sand goby were obtained from cirata reservoir in cianjur, bogor, west java and were temporarily stored at the indoor tanks of seameo biotrop laboratories. the fish specimens were identified based on the taxonomic descriptions of mohsin & ambak (1983). the methodology of chromosome preparation followed that of rivlin et al. (1985) and reddy & john (1987), with very minor modifications. six hours prior to sacrifice, the test fish was intramuscularly injected with 0.05% colchicine in 0.9% nacl at a dose of 1 ml/100 g body weight. the chromosomes were prepared directly using head kidney tissue. hypotonization was done using 0.5% sodium citrate at a duration of 30 to 40 min. and at room temperature of 25.5 to 26.5°c. prepared slides were conventionally stained with 4% giemsa at a ph of 6.5. the different chromosomes were designated as proposed by levan et al. (1964). results from 303 metaphase kidney cells of 30 pre-adult sand goby samples collected from cirata reservoir it revealed that the modal chromosome number (mcn) of 42 chromosomal characters of the indonesian jimmy t. masagca and komar sumantadinata this eleotridine goby is 46. chromosome counts varied from 38 to 50. out of the 303 metaphase cells analyzed, 117 cells (38.61%) have the characteristic count of 2n = 46; 44 cells (14.52%) have 2n = 44; 36 cells (11.88%) have 2n = 43; 24 cells (7.92%) have 2n = 42; 14 cells (4.62%) have 2n = 41; 10 cells (3.30%) have 2n = 40 and 2 cells (0.66%) have each 2n = 39 and 2n = 38 (fig. 1). frequency distribution of diploid chromosome counts of marble sleeper collected from cirata reservoir eighteen (60%) out of the 30 fish samples analyzed showed a modal chromosome number (mcn) 2n = 46; 4 samples (13.3%) have mcn 2n = 44; 3 samples (10%) have 2n-42; and only 2 samples (6.66%) have each 2n = 41 and 2n = 40. karyotypes were made based on mitotic figures in which the chromosomes were clearly recognizable. the homologous chromosome pairs were identified based on the procedures of levan et al. (1964). the chromosome set of this fish mostly consists of 2 pairs of bi-armed and 21 pairs of mono-armed chromosomes. the karyotype showed that 2 pairs of chromosomes were sub-metacentric (sm), where the first pair was larger than the second one. the test of chromosome pairs showed all acrocentrics (a). in some cells the second pair seems to have a metacentric (m) chromosome, but this was not constant. another karyotypic formula was also found in this study having 2n = 46 (2m + 2sm + 42a). 43 figure 1. biotropia no. 7, 1994 discussion previous data on the diploid chromosome number of o. marmorata analyzed from fish samples obtained in thailand (arai & fujiki 1979) and from india (manna 1989) are the same with the present investigation. a diploid number of 2n = 46 and fundamental number (nf) of 50 confirm their previous findings. the diploid number of 46 recorded in sand goby has also been recorded in' another species of the same genus, o. acanthopomus (2n = 46) from japan (arai & sawada 1974). the same diploid number was also recorded in other gobioid species, dormitator maculatus from mexico (del carmen-maldonado et al. 1985), boleophtalmuspectinorostris and periophthalmus cantonensis (nogusa 1960, cited by denton 1973). however, d. maculatus has a fundamental number of 90, which is higher than o. marmorata having an nf = 50. in the gobiidae, most of the genera have the diploid chromosome number of 2n = 44 to 2n = 48 as shown in bathygobius fuscus (2n = 48), glossogobius giurus (2n = 46) and chaetogobius annularis (2n = 44). based on cytological examination variable counts of 42, 43, 44 and 46 were also known in this study. this could be due to the technical limitations. the suggestion that possible chromosonal re-arrangement by robertsonian translocations may have occurred was discounted since the missing chromosomes in the metaphase spreads are different from each other and cannot be considered as inherent property of the test fish. some metaphase spreads have a small metacentric chromosome, but it was not constant in the cells analyzed. the earlier work of arai & fujiki (1979) reported two formulas: 1) 2n = 46 (2m + 2sm + 42st,a) and 2) 2n = 46 (1m + 3sm + 42st,a). although a metacentric chromosome was seen in few spreads, majority of the 303 cells analyzed have 2 pairs of sub-metacentric chromosomes. this study reports a new karyotypic formula of sand goby as 2n = 46 (4sm + 42a). after careful examination of the chromosome set, it seems that no heteromorphic pairs could be referred to as sex chromosomes. acknowledgement the author is grateful for the long-term fellowship granted by seameo biotrop and the permission of the catanduanes state colleges in virac, catanduanes, philippines for allowing the author to travel to indonesia while on a pasuc scholarship at de la salle university. this study is a part of the masteral thesis submitted to dlsu graduate biology department in manila. profound thanks are due to dr. ruben c. umaly (the initial supervisor of this research), dr. soekotjo 44 chromosomal characters of the indonesian jimmy t. masagca and komar sumantadinata (seameo biotrop director) and dr. sutrisno sukimin (tab programme manager). special thanks are due to mr. a.b. djunaedi, mr. widodo, ms. wati and other staff of biotrop for the assistance provided. the assistance of mr. harton of ipb is greatly appreciated. references arai, r. and a. fujiki. 1979. chromosomes of japanese gobioid fishes (iv). bull. natl. sci. mus. ser. a, 5(2): 153-159. arai, r. and y. sawada. 1974. chromosomes of japanese gobioid fishes (i). bull. natl. sci. mus. tokyo, 17: 97-102. del carmen-maldonado, m., m. uribe-abrocer, j. arreouin-espinosa and a. castro-perez. 1985. karyotypical studies on dormitator maculatus bloch and gobiomorus dormitatur lacepede (gobioidei: perciformes). cytologia, 50(4): 663-667. denton, t.e. 1973. fish chromosomes methodology. charles c. thomas publisher, springfield, illinois. herre, a. 1927. gobies of the philippines. monog. 23 bur. sci. manila. levan, a., k. fredga and a. sandberg. 1964. nomenclature for centromeric position on chromosomes. hereditas, 52: 201. lie, s.f. 1968. a study of some biological aspects of o. marmorata found in singapore. part 2. dept. of zoology, univ. of singapore. 26 p. manna, o.k. 1989. fish cytogenetics related to taxonomy, evolution and monitoring genotoxic agent. p. 21-46. in: das and jhingran (eds). fish genetics in india. today and tomorrows printers and publishers, new delhi. mohsin, m.a.k. and m.a. ambak. 1983. freshwater fishes of peninsular malaysia. penerbit universiti pertanian, malaysia, kuala lumpur. munro, j., iskandar and b.a. costa pierce. 1990. fisheries of the saguling reservoir and a preliminary appraisal of management options, p: 285-328. in: b.a. costa pierce and o. soemarwoto (eds.). reservoir fisheries and aquaculture development for resettlement in indonesia. iclarm tech. rep. 23. noerdin, n. and a. sidik. 1979. survei ikan bakut (o. marmorata blkr.) di danau jempang dan semanikarya. dinas perikanan propinsi dati i kalimantan timur. (in indonesian). pantulu, v.r. 1976. floating cage culture of fish in the lower mekong basin, p. 416-423. in: t.v.r. pillay and w.a. dill (eds.). advances in aquaculture. fao tech. conf. on aquaculture. fishing news int. redpy, p.v.g.k. and g. john. 1987. a method to increase mitotic metaphase spreads in permanent chiomosome preparations for karyotype studies of fishes, p. 199-205. in: proceedings of world symposium on selection, hybridization and genetic engineering in aquaculture, berlin, 27 30 may 1986. rivlin, k., j.w. rachlin and g. dale. 1985. a simple method for the preparation of fish chromosomes applicable to fieldwork, teaching and banding. j. fish biol., 26: 267-272. 45 biotropia no. 7, 1994 smith, h.m. 1965. the freshwater fishes of siam or thailand. tropical fish hobbyists publication, inc. usa. supamataya, m. 1988. the study of disease in sand goby (oxyeleotris marmoratus bleeker) in cage culture and some environmental factors related to infection. m.s. thesis. kasetsart university, thailand. tan, o. and t.j. lam. 1973. induced breeding and early development of the marble goby (oxyeleotris marmorata). aquaculture, 2: 411-423. tavarutmaneeoul, p. and c.k. lin. 1988. breeding and rearing of sand goby (oxyeleotris marmorata) fry. aquaculture, 69(3/4): 299 306. weber, m. and l.f. beaufort. 1953. the fishes of the indo-australian archipelago. vol. x. e. j. brill ltd., leiden. 46 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf biotropia no. 10, 1997 : 63-74 influence of media gelling agents on root biomass and in vitro va-mycorrfflzal symbiosis of carrot with gigaspora margarita alok adholeya*, anjali verma and naveen pal bhatia tata energy research institute, durban seth block, habitat place, lodi road, new delhi 110 003, india abstract an in vitro study with ri-tdna transformed roots of carrot (daucus carota) was carried out to evaluate the role of macro-elements contributed as impurities in the gelling agent (phytagel) over and above those present in the minimal (m) medium. production of root biomass was taken as a measure to quantify the influence of macro-elements added to the minimal medium. the levels of phosphorus when adjusted to 1.19 mg/1 and 1.09 mg/l, lead to dry root biomass production at par with the control. attempts made to lower the amount of impurities in phytagel by de-ionization using different alkalies, proved naoh to give the best results in terms of relatively high amount of root biomass. in an in vitro dual culture system with carrot as host and gigaspora margarita as the vesicular-arbuscular mycorrhizal fungus, phytagel impurities helped to produce maximum number of infection units and auxiliary cells when phytagel was added to the minimal medium. key words: agrobacterium rhiiogenesfdaucus caro/a/gelling agents/diaspora margarita/macro elements/vesicular-arbuscular mycorrhiza/transformed roots. introduction production of vam fungi under axenic conditions continues to be one of the most challenging goals of modem biology (becard and piche 1992). mosse and hepper (1975) were the first to report a simplified in vitro system for the study of vam development wherein they used excised roots instead of whole plants. mugnier and mosse (1987) modified the technique further by using ri-tdna transformed hairy roots as the host tissue. plants with such tumoral roots are potentialy valuable for this kind of study as they not only have exceptionally low nutritional requirements but also allow cultivation on synthetic media axenically (nuutila et al. 1995). amending an appropriate medium for co-cultivating the two components, plant roots and the fungus are the most important factor for a successful vesicular-arbuscular mycorrhizal formation in root organ culture (becard and fortin 1988). after the first *corresponding author 63 biotropia no. 63-74 report about the m medium (becard and fortin 1988), the agar component in this media was replaced by other gelling agents, such as gellen gum (gel gro, icn biochemicals, usa) or phytagel (sigma chemical co., usa). these gelling agents made the media more transparent, which facilitated observation under the light microscope. diop et al. (1992) reported that these gelling agents are polysaccharides with very few impurities. however when analyzed, they were found to contain a number of elemental ions, which significantly increased the concentration of many major and minor ions in the medium. taking these observations into account, the present study was undertaken to optimize the level of some of the major elements, which are known to help in root growth and eventually improve in vitro culture conditions towards a more stronger symbiosis hi the dual culture system. materials and methods transformation of carrot (daucus carota l.) agrobacterium rhizogenes strain (a4 wild type atcc 43057) was used to induce transformation (mugnier and mosse 1987). a loopfull of overnight grown culture was suspended in ms broth (murashige and skoog 1962) (optical density adjusted between 0.6 and 0.7). the tap root of carrot was obtained from agricultural fields and was surface sterilized for 10 min. in 30% haoi solution and rinsed three tunes hi sterile, distilled water. the cut ends of roots were then dipped in bacterial suspension for 15 min., filter soaked and put on ms medium supplemented with 500 mg/1 cefotaxime. after about 3-4 weeks, roots emerging from the cut ends were excised and put on mw medium (becard and fortin 1988). healthy white root tips with numerous laterals showing negative geotropism (unlike the non-transformed root cultures) were routinely maintained on m 0.2% medium (w/v) (becard and fortin 1988). transformation of cucumber (cucumis sativus l.) cucumber seeds were surface sterilized as described above and germinated in the dark at 27°c on moist, sterile filter paper. when radicles were approximately 2 cm long, the seedlings were transferred to ms medium slants and kept in growth room at 25°c with 16/8 h light/dark photoperiod and a relative humidity of 65±5%. once the plants became healthy, sections and slanting incisions were made on stems and leaves. cut ends were then subjected to transformation. the roots that emerged (showing negative geotropism) were subsequently maintained on m 0.2% medium (w/v). 64 influence of gelling agents on root biomass and in vitro va-mycorrhizal symbiosis alok adholeya et al. non-transformed root cultures seeds of tomato (lycopersicon esculentum mill.), were surface sterilized and the seedlings were put on ms slants. after establishment, healthy roots were excised and placed on mw medium. after 3-4 weeks, they were transferred onto m 0.2% medium (w/v) and maintained subsequently on the same medium. testing host for colonization fungal inoculum azygospores of vam fungus, gigaspora margarita becker and hall (daom 194757, deposited at the biosystematic research center, ottawa, canada), grown in pots in a greenhouse, were collected by wet sieving (gerdmann and nicolson 1963) and purified by density gradient centrifugation in 60% urografm (furlan et al. 1980). spores thus obtained were then surface sterilized with 2% chloramine t and rinsed in antibiotic solution which consisted of 1% streptomycin and 0.5% gentamycin (becard and fortin 1988). they were then stored on 1 % sterile water agar at 4°c. viability of the spores was ascertained visually by their light colour and globule filling. only viable candidates were picked up and inserted into 1 % sterile water agar and the plates were incubated in a co2 incubator (biocenter 2001, salvis, switzerland) with 2% co2 at 26°c. later on, germinated spores were transferred to m medium prepared with 0.4% (w/v) phytagel. selection of host for selection of a suitable host for subsequent studies, various root cultures of carrot, tomato, and cucumber were examined for their affinity to in vitro symbiosis with g. margarita. many of these cultures were developed in our laboratory (except for errc-i which was obtained from g. becard, errc, philadephia. usa) and classified on the basis of rate of root growth, (table 1) as a=fast (root growth > 2 cm/day), b=moderate (root growth 1-2 cm/day), and c=slow (root growth < 1 cm/day). gp-i root culture proved to be one of the fastest growing and besides being myc+ was selected for subsequent studies. table 1. host species tested for colonization study growth rate species a b c carrot errc-i gp-i gp-ii gp-iii tomato tom cucumber cu-i cu-ii cu iii 65 biotropia no. 63-74 establishment of dual culture in vitro dual culture was initiated from a single spore of g. margarita and a single healthy root tip. different root cultures of carrot, tomato and cucumber were used to establish the experimental unit. the petri dishes were kept for incubation in the dark at 26°c in order to facilitate the growth of the germ tube (becard and piche 1989). the experiment was terminated at the end of 4 weeks. the medium was dissolved and roots were recovered using 10 mm sodium citrate buffer, ph 6 (doner and becard 1991). roots were carefully picked, cleared and stained (phillips and hay man 1970). stained roots were mounted on glass slides and observed at x 200 magnification under compound microscope attached to video camera and image analyser system (leica, switzerland) loaded with quantiment 500 + software (leica, cambridge, uk) having colour option. to get the number of infectious propagule per unit length of root, the number of entry points were divided by total root length. root growth and rate limiting factors in order to identify the role of certain major nutrient elements such as nitrogen, phosphorus, calcium, magnesium, and sulphur, the basic m medium was modified in different ways (table 2). the level of each ion was altered while keeping the levels of j other ions constant (table 3). both lower and higher concentrations than what is prescribed were tested to study the role of deficiency, alternatively excess availability of the ion selected. root tip length of carrot gp-i at start of an experimental unit was 2.5 cm in various treatments. at the end of four weeks, the root system was harvested according to doner and becard (1991) and dry weight of the total root system was recorded. the experiment was laid in completely randomised block design and data! were subjected to the analysis of variance (anova) and means were separated using duncan's multiple range test (p<0.01). table 2. elemental (ionic) composition of m medium 66 influence of gelling agents on root biomass and in vitro va-mycorrhizal symbiosis alok adholeya et al. table 3. levels of selected ions attained with the basic m medium phytagel de-ionization and root growth phytagel was de-ionized by stirring it in distilled and de-ionized water in an erlenmeyer flask at 60°c (doner and douds 1995). the requisite amount of mixed bed resin, tmd-8 (sigma chemical co., usa) was then added and the mixture was incubated on a shaker at 60°c for four hours. it was then filtered through a clean cheesecloth. to the resultant turbid filtrate 200 mm koh solution was added till it became clear (ph>8.0). this clear filtrate was then poured into pre-chilled iso-propanol and the flakes were recovered by re-filtering it through a cheesecloth and were dried overnight in an oven at 50 ± 5°c. the dried de-ionized phytagel was then subjected to icp-aes (inductiviry coupled plasma-atomic emission spectroscopy) analysis to determine the concentration of ions (table 4) and to decide subsequently the extent of changes to be made in the ionic composition of elements selected for the present study. table 4. icp-aes analysis of various gelling agents 67 biotropia no. 63-74 test of de-ionized phytagel with different alkalies was done with an aim to improve rooting. in the routine procedure of phytagel de-ionization 200 mm koh is added. this addition increases the k content of the medium from 135.35 mg/1 to 188.14 mg/1. since the k level goes even higher than the impurity level of ordinary phytagel, different alternative alkalies, namely 200 mm naoh, 200 mm nahco3, 200 mm trizma and 5% nh4oh, were all tested to raise the ph of the solution. young, actively growing root tips of carrot (gp-i, 2.5 cm long) were placed in petri plates, sealed and incubated in the dark at 27°c. subsequently, at the end of four weeks, roots were recovered by the method of doner and becard (1991) and were dried in a hot air oven at 55 ± 5°c till a constant weight was achieved. test of different gelling agents and root colonization because phytagel contains significant amount of elemental impurities (table 4), various gelling agents namely extra pure (ep) agar 0.7% (w/v) (hi media, india), phytagel 0.4% (w/v), de-ionized phytagel 0.5% (w/v) and agarose 0.4% (w/v) were used to solidify the m medium. attempts were made to keep the physical state identical by varying the amount of different gelling agents in the media. in vitro dual cultures were established on these medium using a single spore of g. margarita and carrot gp-i root tip. auxiliary cells formed after 2 weeks were counted and the experiment was terminated at the end of 4 weeks. after recovering (doner and becard 1991) and staining the roots (phillips and hay man 1970), the total number of infection units formed under various treatments were counted. results and discussion amended recipes and root biomass an evaluation in terms of dry root weight production (g) for various amended recipes of nitrogen (fig. la) shows that when the basic m medium recipe was modified, there was a reduction in dry mean root weight of carrot. the percentage reduction over control was highest (50.82) in hi compared to remaining two recipies (l140.86 mg/1 and l235.86 mg/1). as none of the set concentrations performed at par with the control, it is likely that either the reduced levels of nitrogen fail to meet the biochemical and physiological demands of the roots or the disturbances in the no3 :nh4+-n ratio in the medium lowered the recovery of dry root biomass. in contrast to the basic m medium recipe, amongst all the five lower levels of phosphorus (p) tested, l1 and l2 recipes were at par with the control while l3, l4 and l5 recipes produced significantly lower root biomass (fig. 1b). of all the 68 influence of gelling agents on root biomass and in vitro va-myconhizal symbiosis alok adholeya et al. nutrients, insufficiency of p affects vam symbiosis the most (habte and aziz 1991). it is also well established that high p content is detrimental to vam establishment and decreases mycorrhizal infection (abbott and robson 1977, 1979; menge et al. 1978) and probably, reduces the uptake of some minor elements namely zn, fe, and cu. the m recipe was therefore, modified to incorporate lower levels of p as, subsequently, there is a need to optimise the p concentration in the medium for achieving better colonization. similarly, out of the four amended recipes for calcium (table 3) root growth deteriorated to a great extent with h1 and h2 recipes (fig. 1c). between the other two recipes (l1 and l2) a considerably higher dry root weight was obtained with l1 (percentage reduction over control being 39.96) and a remarkably lower recovery of root biomass was observed in case of l2. calcium is known to increase the rigidity of plant cell walls by cross-linking protein components by the formation of calcium pectate. ca ++ however, could adversely affect the biochemical and physiological processes within the root tissues as calcium at higher concentrations tends to shorten lateral branching in roots (hirrel 1981) resulting in the low recovery of root biomass and subsequently reducing the uptake of cations like mg. lower recovery at l1 and l2 levels could be because of insufficient calcium, which leads to the disorganization of the plasma and vacuolar cell membranes, resulting in increased permeability and subsequent loss of certain vital nutrient ions (burstrom 1968; simon 1978). amendments carried out in minimal (m) media recipe to achieve two of the higher levels of magnesium, namely h1 and h2 produced significantly lower values of dry root biomass (fig. 1d) when compared with the control. out of the three different recipes made by amending sulphur in the basic (m) recipe, h1 and li recipes produced significantly lower root biomass and were at par while h2 recorded 77.05% reduction over control (fig. le). therefore, the original levels of the two elements mg (87.35 mg/1) and s (97 mg/1) (in m medium and those added as impurities through phytagel) are optimal to meet the metabolic requirements of young carrot roots in vitro. the reduction in root biomass at higher levels of magnesium may be due to the ionic interactions such as ca ++ and k + , resulting in lower availability of this element. colonization of roots maximum infection units and auxiliary cells were produced on ordinary phytagel, followed by ep agar, perhaps because certain very important elements present in the phytagel added significantly towards better root growth and symbiosis. a minimum of one infection unit and three auxiliary cells were formed on the medium prepared with de-ionized phytagel, while agarose plates produced the least number of auxiliary cells and infection unit (table 5). 69 biotropia no 63-74 fig 1. effect of m medium recipes, amended for various nutrients, on in vitro root biomass. a-e, various recipes amended for nitrogen, phosphorus, calcium, magnesium and sulphur, respectively those bars followed by the same letter do not differ significantly by duncan’s multipke range test at p<0.01 70 influence of gelling agents on root biomus and in vitro va-mycorrhizal symbiosis alok adhoteya et al. table 5. test of various gelling agents for colonization of gigaspora margarita icp analysis of ep agar indicates very high sulphur and iron contents compared to phytagel. this could be detrimental to the root growth and colonization. in de-ionized phytagel the level of k rises significantly, affecting the symbiosis negatively. agarose (0.4%) is devoid of impurities and its water-holding capacity is not as high as that of other gelling agents. probably, due to this, root growth ceases after some time and affects drastically the colonization process. root biomass production in phytagel de-ionized with different alkalies the dry root biomass values obtained with phytagel de-ionized by naoh and trizma were highly significant and were at par, while the remaining alkalies tested for de-ionization produced significantly lower root biomass values (fig. 2). though sodium is one of the critical components of the medium and the extent of its uptake in plants is sometimes related to the activity of vam fungi, the level of sodium present in the medium however is quite low (1.96 mg/1), therefore, root growth was not adversely affected. moreover significantly low recovery of root biomass in case of nahco3 may be due to increase in level of na above the critical level. de-ionization of phytagel with koh definitely increases the k level in the resultant product. k + uptake by any plant is strongly influenced by the form of nitrogen available (whether no3 or nh4 + ) as well as by the cations, particularly na + . it might also be expected to be influenced by the metabolism and polymerization of phosphate (harley and smith 1983). increased k + concentration in the m medium drastically decreases percent colonization of vam fungus (becard and fortin 1988) and high k + content was also found to be detrimental to root growth (harley and smith 1983). de-ionization of phytagel with nh4oh also failed to support the root growth. there was no significant difference between nh4oh and nahco3 treatments. the significant reduction may be due to the fact that excess ammonium 71 biotropia no. 63-74 fig 2. test of different alkalies for phytagel de-ionization those bars followed by the same letter do not differ significantly by duncan's multiple range test (p<0.01) ions in m medium result in a rapid drop in the ph of the culture medium and is detrimental to root growth (becard and piche 1992). in summary, concentration of major and minor elements, ph, temperature and the biochemical or physiological demands of the tissues of the test species play a critical role in the root growth. a balance must be reached between the requirements of actively growing roots, which need a complex medium, and those of the extra-radical phase of the vam fungus, which normally grows in a rhizosphere (i.e. in a relatively nutrient-poor medium). the present study puts forth a major challenge in the task of identifying appropriate nutritional and physiological demands of root cultures and vam fungus to develop a strong symbiosis. the impurities identified hi "phytagel" makes the task of identifying an optimal defined medium much more complex. therefore, there is a need to look for an alternative while defining the requirement for vam symbiosis. 72 influence of gelling agents on root biomass and in vitro va-mycorrhizal symbiosis alok adholeya et al. acknowledgements thanks are due to mr. y.p. kalra for carrying out icp-aes analysis of various gelling agents and ms. s. krishna sundari for root transformation work. dr. david douds and dr. sadhna alstrom made critical comments on an earlier draft of the manuscript, their efforts are thankfully acknowledged. thanks are due to director teri for providing infrastructural support and the department of biotechnology govt. of india, for sponsoring the work carried out under this programme. references abbott, l.k. and a.d. robson. 1977. growth stimulation of subterranean clover with vesicular-arbuscular mycorrhizas. aust. j. agric. res. 28: 639-649. abbott, l.k. and a.d. robson. 1979. a quantitative study of the spores and anatomy of mycorrhizas formed by a species of glomus, with references to its taxonomy. aus. j. bot. 27: 363-375. bayley, j.m., j. king and o.l. gamborg. 1972a. the effect of the source of inorganic nitrogen on growth and enzymes of nitrogen assimilation in soybean and wheat cells in suspension cultures. planta 105: 15-24. bayley. j.m., j. king and o.l. gamborg. 1972b. the ability of amino compounds and conditioned medium to alleviate the reduced nitrogen. planta 105: 25-32. becard. g. and j.a. fortin. 1988. early events of vesicular-arbuscular mycorrhiza formation on ri-tdna transformed roots. new phytol. 108: 211-218. becard, g. and y. piche. 1989. new aspects on the acquisition of biotrophic status by a vesicular-arbuscular mycorrhizal fungus. gigaspora margarita. new phytol. 112: 77-83. becard. g. and y. piche. 1992. establishment of vesiculararbuscular mycorrhizae in root organ culture: review and proposed methodology. in: methods in microbiology: experiments with mycorrhizae. varma a.. j.a. norris, d.j. read (eds). academic press. new york. p. 89-108. burstrom, h. 1968. calcium and plant growth. biol. rev. 43: 287316. diop, t.a., g. becard and y. piche. 1992. long term in vitro culture of an endomycorrhizal fungus, gigaspora margarita, on ri t-dna transformed roots of carrot. symbiosis 12: 249-259. doner, l.w. and g. becard. 1991. solubilization of gellan gels by chelation of cations. biotech. tech. 5(1): 25-28. doner, l.w. and d.d. douds. 1995. purification of commercial gellan to monovalent cation salts results in acute modification of solution and gel forming properties. carbohydrate res. 273: 225-233. furlan, v., h. bartschi and j.a. fortin. 1980. media for density gradient extraction of endomycorrhizal spores. trans. br. mycol. soc. 75: 336-338. gerdemann, j.w. and t.h. nicolson. 1963. spores of mycorrhizal endogon species extracted from soil by wet sieving and decanting. trans. br. mycol. soc. 46: 235-244. habte, m. and t. aziz. 1991. relative importance of ca, n, and p in enhancing mycorrhizal activity in leucaena leucocephala grown in an oxisol subjected to stimulated erosion. j. plant nutr. 14(5): 429-442. 73 biotropia no. 63-74 harley, j.l. and s.e. smith. 1983. mycorrhizal symbiosis. academic press inc., london. hirrel, m.c. 1981. the effect of sodium and chloride salts on the germination of gigaspora margarita. mycologia 73: 610-617. menge, j.a., d. sterile, d.j. bagyaraj, e.l.v. johnson and r.t. leonard. 1978. phosphorus concentrations in plants responsible for inhibition of mycorrhizal infection. new phytol. 80: 575-578. mosse, b. and c.m. hepper. 1975. vesicular-arbuscular mycorrhizal infections in root organ cultures. physiol. pi. path. 5: 215-223. mugnier, j. and b. mosse. 1987. vesicular-arbuscular mycorrhizal infection in transformed root inducing t-dna roots grown axenically. phytopathology 77: 1045-1050. murashige, t. and f. skoog. 1962. a revised medium for rapid growth and biomass assays with tobacco tissue cultures. physiol. plant. 15: 473-497. nuutila, a.m., m. vestberg and v. kauppinen. 1995. infection of hairy roots of strawberry (fragaria x ananassa duch.) with arbuscular-mycorrhizal fungus. pi. cell rep. 14: 505-509. phillips, j.m. and d.s. hayman. 1970. improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. trans. br. my col. soc. 55: 158-161. simon, e.w. 1978. the symptoms of calcium deficiency in plants. new phytol. 80: 1-15. 74 63.pdf 64.pdf 65.pdf 66.pdf 67.pdf 68.pdf 69.pdf 70.pdf 71.pdf 72.pdf 73.pdf 74.pdf biotropia no. 9, 1996: 1-14 natural products in organic synthesis robert h. burnell departement de chimie, universite laval, quebec quebec, canada g1k 7p4 abstract a resume of four lectures presented by the author during a one month visit to biotrop in 1995. the seminars were held at the university of indonesia at depok, the technical institute in bandung, the bogor agricultural institute and at biotrop, bogor. the talks were grouped under the general title "the use of natural products in organic synthesis". key words: organic compounds/natural products. the venerable art of the chemical synthesis of naturally occurring organic compounds which has inspired and fascinated chemists for roughly one hundred and fifty years continues to be necessary today for several reasons. first, and traditionally the most important reason, is that synthesis provides the ultimate proof of structure of a substance by showing that it can be prepared in an unambiguous fashion from simple building blocks. in principle the structures of all the hundreds of new compounds isolated from nature every year should be synthesized. however, if new substances are very similar in structure they can be interrelated by conversion to one another or perhaps to products that have already been synthesized. a second reason for practicing this demanding art we call synthesis is the challenging necessity of preparing increasingly complex substances in more efficient ways and this is the most important source of innovation and invention that assures progress in the organic field. of course, synthesis can also provide an alternate to nature as the source of useful compounds. for instance, despite the effectiveness of the anti-cancer natural product taxol, it is a sobering thought that all the taxus trees in the world could not supply at present enough of the drug to treat the women actually suffering from breast and cervical cancer. and to make matters worse, the trees take almost a century to mature. furthermore it has been estimated that by the year 2000 there will be a world wide short fall of several tons of the natural anti-malarial obtained from artemesia species. the difficulty of shortages can sometimes be alleviated by synthesizing simpler and cheaper analogs. 1 biotropia no. 9, 1996 (burnell et al. 1985), maytenoquinone (burnell et al. 1988) and coleon b (burnell et al. 1983), while occasionally we were able to correct the published structures, as in the case of the nellionols (burnell et al. 1984). for structure confirmation, the simpler synthesis of the left and right handed mixture (the racemic substance) is quite adequate because as mentioned, with the exception of the optical property of rotating the plane of light, the left hand form, the right hand form or the 50:50 mixture of the two, all show the same spectroscopic behavior and these are the characteristics we use to identify them (ultra-violet, infra-red, nuclear magnetic resonance and mass spectra). strictly for proofs of structure, we have prepared molecules by total synthesis from smaller symmetrical, easy to buy or make, starting materials (burnell et al. 1993). the general strategy is shown in the upper part of scheme 6. to the rather commonplace aromatic compound 30 we attached a long chain of carbons derived from natural geraniol 29. this flexible substituent has two "double" bonds in critical positions enabling it to form two rings when attacked by a suitable "positively charged" reagent (ph-s + in our case). both the ph-s and the cn groups in the product 32 can be exchanged by oxygen and this completed a rapid synthesis of methyl nimbinone 33, a diterpenoid from the "neem" tree, azadirachta indica (ira et al. 1988 (1)). the final product from the rapid synthesis was "racemic", a 50:50 mixture of the natural substance and its mirror image and all the physical properties of our sample (except the rotation of light) were identical with those in the literature. obviously the proposed structure is correct! to compare the two approaches we also prepared methyl nimbinone from natural podocarpic acid (burnell et al. 1993). this far more arduous route is summarized in the lower part of scheme 6. the key step in this sequence was the formation and subsequent opening of the three-membered ring as a method of installing the two methyl groups on the first ring of the skeleton. in this case when the synthesized methyl nimbinone 36 was examined in the polarimeter, it did rotate the plane of polarized light in the same direction and to exactly the same extent as the product isolated from nature. this synthesis not only confirms the general structure forwarded for the diterpene, it also tells us that the substance elaborated by the neem tree is precisely and exclusively as represented in formula 36. that is, if the three rings of the skeleton are in the plane of the paper, the methyl group identified by the dark wedge between the rings, protrudes towards the reader. choosing between the rather simple straight-forward synthesis that leads to a mixture of the two image forms of methyl nimbinone or the more difficult and less rewarding preparation of the real natural diterpene from natural podocarpic acid depends on the real goal of the synthesis. but it would obviously be an enormous advantage to be able to combine the simplicity of the former with the specificity of the latter. to investigate this possiblity we selected another relatively simple compound, methyl nim 10 natural products in organic synthesis robert h. bumell bionone 44 to experiment with this "enantiomeric" style of synthesis. this diterpene is similar in name and in structure to methyl nimbinone 36 and it was also extracted from the "neem" tree (ira et al. 1988(2)). crucial to this approach is the incorporation of an asymmetric unit (technically a "chiral auxilliary") into the starting material at the outset. in theory, the "left or right-handedness" of this appendage will just be borrowed temporarily during the synthesis and it should impose its asymmetry on the reactions to induce the preferred formation of just one of the mirror image forms of the final product. at a propitious moment this helpful but perhaps costly auxilliary can be removed and recovered to be used in another synthesis. 11 biotropia no. 9,1996 we chose to attach to the necessary aromatic starting product 43, the asymmetric unit 42 (our auxilliary) invented by evans (evans el al. 1985) which we had previously prepared from the abundant natural amino acid, l-valine 41. as in the earlier synthesis, we added the ten carbon bromide 29 and immediately we knew the auxilliary was performing its function. while there are two three-dimensional structures possible for the product 45, only one of the two was formed in quantity in the reaction ! 12 natural products in organic synthesis robert h. burnell employing roughly the same steps as in the methyl nimbinone sequence (above), the intermediate 45 was cyclized to compound 46 and the two oxygen functions were introduced to afford a product which showed all the spectroscopic attributes of methyl nimbionone 47 (dumont 1993). it was encouraging that examination of the optical properties of the product in the polarimeter showed that the substance did indeed rotate the plane of light. however, the magnitude of the rotation was much lower than that recorded for the natural product which indicated that the high "left hand, right hand" selectivity we had achieved early in the synthesis (only one form of 45 was isolated), had been scrambled or lost in the later steps. we suspect that the conditions for the cyclization (45 to 46) are too severe and our efforts are now aimed at finding milder alternate methods of performing this step. the improved conditions we are searching for must provide the product in high yield, sufficient to enable the completion of the synthesis which still involves several steps. unfortunately, as yet, we have not found this more gentle procedure. acknowledgements the work described was financed partly by nsecc (the natural sciences and engineering council of canada), partly by the government of quebec (f.c.a.r.) and more recently by generous personal funding (emeritus professor charles r. engel). my sincere appreciation is extended to the following graduate students whose collaboration and talent were crucial to the study : sonia desfosses, nathalie dumont, nathalie the-berge, michel jean and david miller. the author wishes to thank the staff at biotrop for their hospitality (in particular dr. hilman affandi and dr. gloria enriquez) and the canadian international development agency (cida) for making the visit to indonesia possible. literature cited barton, d.h.r., a.g. brewster, s.v. ley, c.m. read and m.n. rosenfeld. 1981. oxidation of phenols, pyrocatechols and hydroquinones to orthoquinones using benzeneseleninic anhydride. j. chem. soc., perkin. trans. i., 1473-1476. brewster, j.h. 1959. a useful model of optical activity. j. amer. chem. soc., 81: 5475-5483. burnell, r.h., a. andersen, m. neron-desbens and s. savard. 1981. approaches to the synthesis oflyco-xanthol and similar natural products. synthesis of coleon u. can. j. chem., 59: 28202825. burnell. r.h., m. jean and s. savard. 1983. synthesis of coleon b tetramethyl ether. can. j. chem., 61: 2461-2465. 13 biotropia no. 9,1996 burnell. r.h., m. jean, d. poirier and s. savard. 1984. the structures of the nellionols. synthesis of model abieta-8,11,13 trien-7-ones. synthesis of 5-dehydronellionol trimethyl ether. can. j. chera., 62: 2822-2829. burnell, r.h., a. andersen, m. neron and s. savard. 1985. synthesis of coleon c tri-o-methyl ether. can. j. chem., 63: 2769-2776. burnell, r.h., m. jean and d. poirier. 1987. synthesis of taxodione. can. j. chem., 65: 775-781. burnell, r.h., m. jean and s. marceau. 1988. synthesis of maytenoquinone. can, j. chem., 66: 227-230. burnell, r.h., n. dumont and n. theberge. 1993. synthesis of omethyl-nimbinone. j. nat. prod., 56: 1930-1936. carriere, y., j. millar, j.n. mcneil, d.j. miller and e.w. underbill. 1988. identification of the female sex pheromone in alfalfa blotch leafminer, agromyzafrontella (rondani). j. chem. ecol., 14: 947-956. dumont, n. 1993. synthese de diterpenes naturels isoles de 1' azadirachta indica (neem). m.sc. thesis, university laval, canada. evans. d.a., d.j. mathre and w.l. scott. 1985. asymmetric synthesis of the enkephalinase inhibitor thiorphan. j. org. chem., 50: 1830-1835. furia, t.e. and n. bellanca. 1971. fenaroli's handbook of flavor ingredients. the chemical rubber co., cleveland, ohio. ira, b.s., s. siddiqui, s. faizi and b.s. siddiqui. 1988 (1). tricyclic diterpenes from the stem bark of azadirachta indica. j. nat. prod., 51: 1054-1061. ira. b.s., s. siddiqui, s. faizi and b.s. siddiqui. 1988 (2). teipenoids from the stem bark of azadirachta indica. phytochemistry, 27 : 1801-1804. king, f.e., t.j. king and j.g. topliss. 1956. synthesis of podocarpic acid. chem. and ind., 113. kupchan, s.m., a. karim and c. marcks. 1969. tumor inhibitors. xviii. taxodione and taxodone, two novel diterpenoid quinone methide tumor inhibitors from taxodium distichium. ]. org. chem., 34: 3912. miller, d., f. bilodeau and r.h. burnell. 1991. stereoselective synthesis of isomers of 3,7-dimenthyl-nonadecane, a sex pheromone of the alfalfa blotch leafminer (agromyza frontella (rondani)). can. j. chem., 69: 1100-1106. 14 biotropia no. 9, 1996 yet another motivation for synthesis stems from the fact that nature very often provides only very small quantities of the substances of the greatest physiological interest. modern analytical instruments enable the chemist to elucidate the structures of these unknown, potentially useful biological, medicinal or phytochemical compounds even if the elaborate extraction procedures afford only a few milligrams of material. however, for proper assessment of their physiological activities and their mechanisms of action, much greater amounts are probably needed and only synthesis could provide the necessary quantities. most of the interesting molecules of nature are asymmetric rather like our hands. each hand is made up of the same elements such as fingers, thumbs, palms, nails and so on and while they are very similar, they are not identical. a right-hand glove does not fit on the left hand ! in fact our hands are mirror images of one another. natural molecules made of the same atoms could be assembled in two fashions, one being the image of the other. unlike our hands, however, nature usually synthesizes only one of the two forms (chemically we call the two images "enantiomers"). since all "our human" metabolic intermediates and enzymes are also asymmetric, the physiological activity of a natural substance may be quite different from that of its mirror image. the "enantiomeric" form could just be inactive or less active than the natural product but it could have harmful effects or be highly toxic. the much quoted case of thalidomide springs to mind where one enantiomer was a benign sedative recommended for women suffering nausea during pregnancy whereas the mirror image of exactly the same molecule was teratogenic producing babies with serious physical defects. so synthesis must not only aim at preparing the target molecule with the right general structure but the methodology should also lead to the right three-dimensional structure (or stereochemistry). in simpler terms, the synthesis must give exclusively the "left" or exclusively the "right" hand molecule ! chemically producing this "left or right" hand configuration is extremely difficult. the problem is further compounded by the fact that virtually all the physical properties of the two image forms are identical which means we cannot easily tell them apart and, furthermore, we cannot separate one from the other !! however, two characteristics of the enantiomers (images) are different. first, as we have seen, they can vary greatly in their physiological activities. secondly, like all asymmetric substances, they rotate the plane of polarized light but the images turn the light in opposite directions. fortunately we can measure this in a polarimeter and under the conditions defined by the test, a fresh solution of the more prevalent form of the common sugar glucose rotates light through 112 degrees in a clockwise or right-handed fashion while its image would also turn light 112 degrees anti-clockwise or to the left. in fact we often say that a substance such as glucose is dextro-rotatory (from the latin dextrus : right) or we just abbreviate this to d-glucose where the small "d" shows that light passing through a solution of the product 2 natural products in organic synthesis robert h. burnell would be turned to the right. the image form would be levo-rotatory and called 1-glucose. (another convention describes rotation to the right as positive (+) and rotation to the left as negative or (-)). one way of assuring that the molecule targeted in a synthesis will have the right stereochemistry (left or right-handedness) is to start the synthesis from an asymmetric substance available in pure form in nature. many potentially useful, commercially available compounds exist from simple amino acids or sugars to complex steroids, alkaloids and terpenes. our work on an insect pheromone is a simple example of such an "enantio-selective" synthesis (as reaction sequences aimed exclusively at the right or left handed structures are called). the insect, the small fly agromyza frontella was accidentally introduced into canada from europe in the 1960's and its presence was rapidly detected because in its larval stage it destroyed the leaves of the popular cattle fodder, alfalfa. in their breeding ritual, the female insects attract the males by emanating a minute quantity of a chemical messenger or pheromone and although the quantity of this substance available was infinitesimal, structure 1 (or 3,7-dimethylnonadecane) was advanced for the pheromone. the structure elucidation was made possible by a skillful study of the mass spectral fragmentation after rigorous purification by preparative gas chromatography (carriere et al. 1988). potentially this natural chemical could be used to control the population of the insect by using it to divert the male flies away from the females and thus preventing reproduction ! 1 3 7 ch3-ch2-ch-ch2-ch2-ch2-ch -ch2 11 ch3 ch3 ch3 at first glance the substance is chemically uninteresting. it is just a member of the stubbornly unreactive class of organic substances known as paraffins or saturated acyclic hydrocarbons. on reflection, however, the pheromone is a synthetic challenge because the positions of the two methyl groups (chs or me groups on the third and seventh carbons of the long chain) make the molecule asymmetric so in fact there are four spatial arrangements possible, as shown in structures 2 to 5. only one will be the active pheromone but which one ? our synthetic strategy should enable us to prepare one (or better all) of them specifically. 3 biotropia no. 9, 1996 in designing a synthesis we normally work backwards from the target, hypothetically disconnecting parts we feel we can graft back onto the remaining core using chemical methods of proven specificity and geometry. our first idea for the synthesis of the agromyza pheromone 1 arose from the "retro-synthesis" shown in scheme 1. disconnections leave a central portion which contains both of the methyl branches on the long chain. many naturally occurring oils (commercially available at low price) contain compounds with this skeleton and our final choice of a "starting material" would be motivated by the need for elements of structure permitting the easy grafting of the other units. and, of most importance, the molecule should be asymmetric with at least one "left" or "right" hand centre. after considering several possibilities, pulegone 6, the main constituent of the "oil of european pennyroyal" from mentha pulegioides l. (furia and bellanca 1971) seemed ideal for the purpose. 4 natural products in organic synthesis — robert h. burnell without commenting at length on the chemical reactions involved, we constructed the pheromone as shown in scheme 2. for those interested in how the transformations were achieved, the reagents (indicated by the small letters on the arrows) are, of course, published in detail (miller et al. 1991). the steps from 6 to 11 not only add the "one carbon unit" but also induce the stereochemistry (left or right-handedness) at c.3. none of these chemical transformations could have altered the configuration of the methyl substituent of the original pulegone so the geometry at the other methyl branch, now further in the chain at c.7, survives unchanged from beginning to end. mechanistic and theoretical arguments told us that the final product from our synthesis was actually structure 3, the drawing of which implies that if the long chain zig-zags in the plane of the paper then both methyl groups represented by the dotted wedges, stick out to the rear. other considerations told us that of the four possible structures this was actually the active pheromone. when analyzed by polarimetry and using rules formulated by brewster for calculating the optical properties of hydrocarbons 5 (brewster 1959), we felt the synthetic oil also contained lesser quantities of structure 4 in which the c.3 methyl has the other configuration. 6 biotropia no. 9, 1996 natural products in organic synthesis robert h. burnell we had chosen pulegone as the starting material for the synthesis for another reason. if the disconnetion suggesting possible synthetic approaches is envisaged in another fashion as shown in scheme 3, the pheromone could be prepared by just grafting an eleven carbon fragment onto the pulegone 6. so, to check both the structure and the purity of the end product from our first synthesis, we prepared the pheromone a second time by this alternate route which as shown in scheme 4, starts from compound 8, an early intermediate in the previous synthesis (miller et al. 1991). this second sequence gave exactly the same pheromone 3 but in higher yield. remarkably in this case and as intended, the original asymmetry of pulegone (the methyl branch) is now at c.3 rather than at c.i as in the first synthesis. this "left hand : right hand" selection was only possible because the natural pulegone is asymmetric and the pennyroyal plant synthesizes only one of the two possible mirror image forms. interestingly, the image form of pulegone can be isolated in pure form from other plant species so our synthesis could also lead to the image of pheromone 3 represented by structure 2. a second example of synthesis in our laboratory starting from commercially available, naturally occurring asymmetric substances involves podocarpic acid 23 which was itself synthesized as early as 1956 (king et al. 1956). the world source of this acid is the resin obtained from podocarpus trees found here in java and new zealand. one of the molecules we hoped to synthesize was taxodione, an anti-tumoral substance shown to possess structure 28 (kupchan et al. 1969) and at that time was considered a promising drug for human therapy. when we started, other syntheses had already been reported by other groups but we felt that we could improve on existing syntheses in elegance and efficiency. podocarpic acid was a natural choice as starting material because even uninformed inspection shows that it already possesses many of the structural 7 biotropia no. 9,1996 elements of taxodione including the three angularly attached six-membered rings. but even more important, the absolute stereochemistry (the handedness) of the centres identified as c.10 and c.5 is exactly the three dimensional geometry required for "natural" taxodione ! scheme 5 is a brief outline of the crucial intermediates in our synthesis (burnell et al. 1987). the white arrows merely draw attention to the part of the molecule that will be modified in the next transformation. the key step was the use of a selenium reagent (barton et al. 1981) to oxidise the phenol 25 to the ortho-quinone 26. some of the reactions were most gratifying. for instance, while conventional wisdom would say such a change was unlikely, compound 26 rearranged spontaneously to 27 and the latter was oxidized to the final taxodione 28 by merely percolating its solution through a column of silica gel. the product was, of course, pure natural taxodione containing none of the 8 natural products in organic synthesis robert h. burnell mirror image form and this was confirmed by measuring the direction and magnitude of its ability to rotate the plane of polarized light. if we had started the synthesis from simple symmetrical chemicals, the final substance would only be half taxodione and half its mirror image and would show no rotation of the plane of light. the taxodione preparation was part of a much broader study to corroborate the structures of several naturally occurring aromatic diterpenes by synthesis. since in all cases we started from podocarpic acid (or the very similar equally affordable dehydro-abietic acid) the final products also possessed the exact stereochemistry (left or right handedness) of the natural substances. most of the structures proposed in the literature were shown to be correct as in the case of coleon u (burnell et al. 1981), coleon c 9 1.pdf 10.pdf 11.pdf 12.pdf 13.pdf 14.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf biotropia no. 8, 1995: 39-44 effects of various soil environmental stresses on the occurrence, distribution and effectiveness of va mycorrhizae a.g. khan department of biological sciences, faculty of business and technology, university of western sydney macarthur, p.o. box 555, campbelltown n.s. w. 2560, australia introduction the vesicular arbuscular (va) mycorrhizal fungi are geographically ubiquitous soil inhabitants and form universal symbiotic relationship with plants from every phylum. these fungi link host plants with host soils and their biota in the mycorrhizosphere and play an important role in plant health, productivity and soil structure. although va mycorrhizal fungi do not show any host specificity, there is increasing evidence that various climatic and edaphic environmental factors such as land use and management practices, physical, chemical and biological properties of host soils and host plant characteristics influence their occurrence, taxonomic distribution and effectiveness. the interaction of these factors with vesicular-arbuscular mycorrhizae (vam) is poorly understood except in a few cases. it is now very clear that va mycorrhizal associations are ecologically significant factors that require more attention than previously accorded. this paper discusses the occurrence, distribution and significance of vam in environmentally stressed soil conditions that limit plant growth such as drought, waterlogging and salinity. key words: mycorrhizas/environmental factors/waterlogging/soil salinity/growth development stages/ inoculum. vam and drought evidence is accumulating which supports the contention that va mycorrhizal fungi are present in arid regions in xerophytes (khan 1974) and improved resistance of plants to water stress in controlled environments (bethlanfalavay et al. 1988), as well as in the field (sylvia et al. 1993), primarily due to improved nutritional status of plants. vam are known to occur in plants growing naturally in pioneer arid habitats of sand dunes, industrial waste lands and disturbed sites such as coal tips and 39 biotrop1a no. 8, 1995 strip-mined land (khan 1978). evidence has also been accumulating, linking the presence of vam infection to survival and improved plant growth in mine soil (khan 1981). vam has great potentials in the reclamation of drastically disturbed land and arid lands for agriculture. our findings, that different vam endophytes had quantitatively different effects on growth of onions in unsterilized coal waste (khan 1981) and the extent of colonization of onion roots was dependant upon inoculum density (khan 1988), may have practical implications for the establishment of a particular endophyte in arid conditions and the extent of plant response to the infection. vam symbionts can assist in the accelerated rehabilitation of unsightly industrial waste lands. increased growth of grasses and other indigenous flora on the dump sites may be obtained by either increasing the population of ecologically adapted va endophyte strain(s) and or transplanting plants pre-inoculated with suitable strain(s) instead of applying and fertilising top soil cover. commercial quantities of vam inoculum are now available for revegetation efforts of mined or cleared land. nurseries selling plants for revegetation or transplants into fumigated soils are the immediate beneficiaries of the vam biotechnology. also, the determination of abiotic factors governing efficiency of an isolate must be made before we can realistically select superior vam fungal isolates for use in the colonization of disturbed sites. vam and waterlogging vam occur over a wide range of soil water contents (khan 1974). vam colonization has been found in plants growing in frequently waterlogged soils (khan 1993a), as well as in free floating and submerged aquatic plants (khan & belik 1994). the vam fungi are aerobic, so soil water and aeration influence their distributions and effectiveness in soils and vam spore numbers along with vam infection are positively correlated to redox potential values (khan 1993b). a relationship between characteristics of vam infection and soil moisture gradient was found in a study of a casuarina cunninghamiana-transect on a creek embankment (khan 1993a). typical vesicles and arbuscules were found in roots from drier soil. roots from relatively wet soils lacked arbuscules but contained large liquid filled intracellular vesicles. typical vesicles and arbuscules were absent in flooded creek beds where roots were associated with coenocytic intracellular hyphae with abundant lipid contents. vam roots of submerged aquatic plants also harboured vesicles and/or coenocytic hyphae only, however, arbuscules were rare or absent. it is possible that the endobiont may be existing even in the aquatic plants recorded as non-mycorrhizal in the form of mycelia, not considered by the researchers as vam infection. vam fungi become non-functional in these habitats when the soil is saturated for extended 40 effects of various soil environmental stresses a.g. khan periods of time (liberia et al. 1983). however, when sites become seasonally dry, a functional symbiosis redevelops (khan 1993c). the variations between functional and non-functional associations may be common in wet and waterlogged habitats where conditions fluctuate on a seasonal or annual basis to favour or hinder mycorrhizal formation. the nature of the periodic non-functional associations, whether benign or a mild form of parasitism, is unknown and requires further study, as suggested by anderson et al. (1994). these fluctuations may be due to a change in redox potential from reducing to oxidising by the lateral liberation of oxygen from plant roots (kludge et al. 1993). the reports of the presence of vam under waterlogged conditions may be related to the (1) internal oxygen transport from the stem to the root due to presence of aerenchyma, (2) the oxygen release from the roots of large trees to support vam fungi, (3) strains of vam fungi in the sediments that can withstand highly reduced habitats, or (4) a combination of all these factors. but these have not been demonstrated and further studies are needed. the ability of vam to tolerate submersion became evident from various studies on the commercial production of inoculum by using aerated nutrient solutions in hydroponics or aeroponics. many global studies have established that vam fungi can infect permanently submerged plants under natural condition (khan and belik 1994). however, the process of infection and various biotic and abiotic factors in the aquatic environment affecting the process are unknown and further research is needed to understand the ecological significance of vam in these habitats. an understanding of the adaptions and mechanisms of plants living in or on water is valuable in explaining the interaction between plants, vam fungi and sediment/water including its biota as a dynamic system. mycorrhizal infection of roots causes significant physiological changes in the host plant which make the mycorrhizal plant grow and respond to environmental stresses differently from a non-mycorrhizal plant. although the occurrence of mycorrhizal infection in waterlogged and submerged aquatic plants has been known for some time, very few workers have attempted to identify species involved in these associations (khan and belik 1991). various global studies suggest that environmental factors such as soil moisture, soil type and soil gases influence the taxonomic distribution of vam fungi. spores of gigaspora margaritawere reported from the mycorrhizospheres of various wet and submerged plants (khan and belik 1994), which suggests that this species may be adapted to grow in these conditions of low oxygen availability and high p,n, and phytotoxin contents of the reduced sediment. although the presence of spores in aquatic sediments suggests that they probably enter the aquatic sediments through run-off from terrestrial ecosystems, information is accumulating that support the 41 biotropia no. 8, 1995 contention that there is significant genetic and physiological diversity in population of vam fungi from dissimilar environments (bethlenfalvay 1992). further research is needed to demonstrate this 'ecological specificity'. studies are also needed to separate the effects of flooding stress on the vam fungus from those on the host root. vam and soil salinity va-mycorrhizal fungi occur naturally in saline environment (khan 1974; khan and belik 1994) and several researchers who investigated the relationship between soil salinity and occurrence of mycorrhizae on halophytes have indicated that the number of vam spores or infectivity of vam fungi based on germination and subsequent hyphal growth decreased with increasing salt (juniper and abbott 1993). saline soils pose physiological stresses such as nacl toxicity and low osmotic potentials to plants growing in them. these stresses effect the growth of plants fungus or both. nacl (750 mg nacl per kg soil) reduced the number of vesicles and arbuscules, but not the growth of hyphae in the guayule roots inoculated with glomus intraradices (pfeiffer and bloss 1998). under higher nacl concentrations, endosymbionts may be reduced to coenocytic hyphae only as under waterlogged conditions and act almost as benign parasite. there are few studies indicating that mycorrhizal fungi can increase growth of plants growing in saline habitats (ojala et al. 1983; pond et al. 1984). va-mycorrhizal fungi may have the ability to protect plants from salt stress (hirrell and gerdemann 1980; rosendahl and rosendahl 1991), but the mechanism is not fully understood. the few data available at present suggest that fungi do have a potential to enhance plant growth but further studies are needed with additional plants, fungi under variable saline conditions. va-mycorrhizal fungi most commonly observed in saline soils are glomus spp. (juniper and abbott 1993) which suggest that this may be adapted to grow in saline conditions, but ecological specificity has not been demonstrated. there is evidence that vam species distribution is markedly changed with increased salinity (stahl and williams 1986). soil salinity and waterlogging often co-occur and interact in low-lying areas but no attempts have been made to study their collective effects on vam. va-mycorrhizal infection and vamycorrhizal fungal propagules have been observed in inundated salt marshes (khan and belik 1994). a comparative study of the effects of salinity and the co-related stress of low water potential on host plant and vam fungus is desirable before plant endophyte combinations are selected for. also, it is necessary to separate na effects from other salt effects. 42 effects of various soil environmental stresses a.g. khan aforementioned studies show that flood tolerant plants and va endobiont fungi exist together. whether these fungi are adapted to such conditions play any role in alleviating plant stress brought on by flooding under aquatic environments had yet to be elucidated. some recent studies in rice-growing areas such as india, thailand and usa and our preliminary investigations in australia suggest that va mycorrhizal endobiont would benefit both wetland and upland rice. but there are many biological principles which need to be explained, ie. how the infection occurs under flooded conditions and what initiates it, how the nutrients are obtained by the vam fungi under waterlogged conditions, what vam fungi are adapted to aquatic conditions, what conditions favour the symbiosis, etc. close attention should be given to the effects of edaphic, abiotic (physical and chemical) and biotic (soil microflora and fauna, including pathogens) factors on the mycorrhizal symbiosis while researching this area. references anderson, r.c., a.c. liberta and l.a. dickman. 1984. interaction of vascular plants and vesiculararbuscular mycorrhizal fungi across a soil moisture gradient. oecologia, 64: 111-117. bethlenfalvay, g.j. 1992. mycorrhizae and crop production. in bethlenfalvay, g.j. and lindermann, r.g. (eds). mycorrhizae in substantial agriculture. asa spec. pub. 54, am. soc. agron. madison, wisconsin, p. 1-27. bethlenfalvay, g.j., r.s. thomas, s. dakessian, m.s. brown, and r.n. ames. 1988. mycorrhizae in stressed environments: effects on plant growth, endophyte development, soil stability and soil water. in whitehead, e.e. arid lands, today and tomorrow. westview press, boulder, co. p. 1015-1029. juniper, s. and l. abbott. 1993. vesicular and arbuscular mycorrhizae and soil salinity. mycorrhizae, 4: 45-57. khan, a.g. 1974. the occurrence of mycorrhizae in halophytes, hydrophytes and xerophytes, and of endogone spores in adjacent soils. j. gen. microbiol., 81: 7-14. khan, a.g. 1978. vesicular-arbuscular mycorrhizas in plants colonising black wastes from bituminous coal mining in the illawarra region of new south wales. new phytol., 81: 53-63. khan, a.g. 1981. growth responses of endomycorrhizal onions in unsterillised coal waste. new phytol., 87: 363-367. khan, a.g. 1988. inoculum density of glomus mosseae and growth of onion plants in unsterilized bituminous coal spoil. soil biol. biochem., 20 (5): 749-753. khan, a.g. 1993a. the occurrence and importance of mycorrhizae in aquatic trees of new south wales, australia. mycorrhizae. 3: 31-38. khan, a.g. 1993b. the influence of redox potential of formation of mycorrhizae in trees from wetland and water-logged areas of new south wales, australia. abst. 9th north am. conf. on mycorrhizae, aug. 812, 1993, university of guelph, guelph, ontario, p. 18. 43 biotropia no. 8, 1995 khan, a.g. 1993c. vesicular-arbuscular mycorrhizae (vam) in aquatic trees of new south wales, australia, and their importance at land-water interface. in gopal, b., hillbricht-ilkowska, a. and wetzel, r.g. (eds). wetland and ecotones: studies on land-water interaction, national institute of ecology, new delhi, p 173-180. khan, a.g. and m. belik. 1994. occurrence and ecological significance of mycorrhizal symbiosis in aquatic plants. in: varma a, hock b (eds). mycorrhiza: function, molecular biology and biotechnology. springer-verlag, heidelberg, germany, 1994 (in press). kludge, h.k., r.d. delaune and w.h. patrick jr. 1993. aerenchyma formation and methane and oxygen exchange in rice. soil sci.soc. am. j., 57: 386-391. liberta, a.e., r.c. anderson and l.a. dickman. 1983. vesicular-arbuscular mycorrhizae fragments as a means of endophyte indentification at hydrophytic sites. mycologia, 75: 169-171. ojala, j.c., w.m. jarrell, j.a. menoe and e.l.v. johnson. 1983. influence of mycorrhizal fungi on the mineral nutrition and yield of onion in saline soil. agron. j., 75: 255-259. pfeiffer, c.m. and h.e. bloss. 1988. growth and nutrition of guayale (parthenium argentatum) in a saline soil as influenced by vesicular-arbuscular mycorrhizae and phosphorous fertilisation. new phytol., 108: 315-321. pond, e.c., j.a. menoe and w.m. jarell. 1984. improved growth of tomatoes in salinized soil by vesicular-arbuscular mycorrhizal fungi collectors from saline soils. mycologia, 76: 74-84. rosendahl, c.n. and s. rosendahl. 1991. influence of vesicular-arbuscular mycorrhizal fungi (glomus spp.) on the response of cucumber (cucumis sativus l.) to salt stress. environ. exp. bot, 31: 313-318. stahl, p.o. and s.e. williams. 1986. oil shale process water affects activity of vesicular-arbuscular fungi and rhizobium four years after application to soil. soil biol. biochem. 18: 451-455. sylvia, d.m., l.c. hammond, j.m. bennett, j.h. haas and s.b. linda. 1993. field response of maize to a vam fungus and water management. agron. j. 85. 44 39.pdf 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf 1. zarina zakaria.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 biotropia no. 10,1996:1 -13 influence of moisture content and length of storage on fungal invasion of paddy rice *) danilo e. paderes college of agriculture, department of crop protection central luzon state university, nueva ecija, philippines t.w. mew department of plant pathology, international rice research institute college, laguna, philippines lina l. ilag college of agriculture, department of plant pathology university of the philippines, college, laguna, philippines abstract the relationship of moisture content and storage period to fungal population, seed germination, grain whiteness and translucency was determined. various fungal species predominated at different moisture conditions and storage periods. the fungi observed belong to the groups of aspergillus flavus-oryzae, a. glaucus, a. mdulans. a. candidus, a. versicolor, a. terreus and a. niger and an unidentified species of penicillium, trichoconiella, curvularia, fusarium, syncephalastrum and verticillium. the predominant storage fungi were a. fla\iis-oryzae and a. candidus whereas, the predominant field fungi were trichoconiella sp., cun-ularia sp. and syncepfialastrwn sp. a decrease in the number of field fungi and an increase in the number of storage fungi with storage time were observed. storage fungi were noted as early as five weeks after storage at moisture contents from 9.3 to 18.33%. the percentage germination of paddy remained high when stored at moisture contents of 9.3 to 14% but decreased with storage time at 14.5 to 18.33% moisture content. the percentage germination of paddy reached a peak at 10-15 weeks of storage. a significant negative correlation between percent germination and moisture content was observed. at 14.5-18.33% moisture content, the germination of stored paddy decreased with a marked increase of storage molds. changes in grain whiteness was not affected by moisture content. however, a decrease in percent whiteness and translucency was noted after 25 weeks of storage. keywords: stored products pests / rice / moisture content / storage / time / fungi / aspergillus sp. / penicillium sp. / trichoconiella sp. / curvularia sp. / fusarium sp./ syncephalastrum sp. / verticillium sp. *) paper presented at the symposium on pest management for stored food and feed, 5 -7 september 1995, bogor, indonesia 1 biotropia no. 10, 1997 morphological and cultural characteristics of the different groups of aspergillus a. flavus-oryzae. colonies on czapek's solution agar grew rapidly, reaching a diameter of 5.5-6.0 cm after 10 days incubation at room temperature. vegetative mycelium was largely submerged with loose texture, surface growth of long-stalked conidial structures and intermixed aerial mycelium. at first, conidial heads appeared white and later developed to a pale greenish yellow to deep yellow-green or olive-brown when mature. globose black sclerotia were produced with" age. conidial heads were predominantly large arising from a long colorless conidiophore. vesicles are typically subglobose bearing either uniseriate or biseriate sterigmata consisting of subglobose conidia. a. glaucus. colonies on czapek's solution agar reached a diameter of 1-1.5 cm after 10 days of incubation at room temperature. conidial heads were typically bluish-green. smooth-walled and brown colored conidiophore terminated to a domelike vesicle. conidia arising from a uniseriate sterigmata were typically globose to subglobose in chains. cleistothecia, yellow and subglobose were present. a, nidulans. growth in czapek's solution agar reached a diameter of 5-6 cm in 10 days at room temperature. conidial heads were usually dark green, short columnar. conidiophores were smooth and light brown in color which terminated to hemispherical vesicle bearing a biseriate sterigmata. the primary and secondary sterigmata were about equal in length. conidia were globose and echinulate. a. candidus. this organism was easily detected by having white condial heads when young and becoming yellowish cream in age. colonies in czapek's solution agar had a diameter of 2.5-3.0 cm after 10 days incubation at room temperature. conidiophores were smooth colorless bearing globose to subglobose vesicle with biseriate sterigmata. a. versicolor. the size of the colonies on czapek's solution agar reached a diameter of 1.5 cm in 10 days at room temperature. this organism has radiate to loosely columnar conidial heads. the brown conidiophore terminated to an ovate or elliptical vesicle. the echinulate conidia are attached to a strictly biseriate sterigmata. a. terreus. colonies on czapek's solution agar obtained a diameter of 4-5 cm. it can easily be identified because of its brighter color in buff, cinnamon, to orange brown compactly columnar conidial heads. the vesicles were hemispherical attached to biseriate sterigmata where globose, smooth conidia were borne. a. niger. this fungus is similar to a. flavus-oryzae by its rapid and vigorous growth on czapek's solution agar which reached a diameter of 2.5-4.0 cm after 10 days at room temperature. conidial heads were in black dark brown shades. conidiophores coming from submerged mycelia terminated to a globose vesicle. the vesicles consisted of biseriate sterigmata where subglobose, elliptical conidia were borne. 10 influence of moisture content danilo e. paderes el. al discussion moisture content is known to be the primary contributing factor in determining the kinds of fungi that invade stored seed and the degree to which they invade it (coleman and fellow 1925; and koehler 1938). in the present study, the predominant fungal species tended to vary at different moisture levels. the variation in the percent fungal incidence found either on or in the seed may probably be due to the competition among organisms. there are fast and slow invading molds (neegaard and adib saad 1962). some fungi may overgrow and obscure other species, thus preventing their identification. overgrowth of one species by another may result from an inherent growth rate difference between species (schroeder and sorenson 1961). expectedly, high percentages mold infection were observed in paddy stored at high moisture content levels. the predominant fungi found in grains with high moisture content were a. flavusoryzae, a. glaucus and a. candidus. these findings agree with those of tuite and christensen (1957), bottomley et al. (1952) and del prado and christensen (1952) on stored cereal seeds, corn and rice, respectively. the decrease in the germination of rice may be attributed to the ability of the fungi to grow on rice grains. at 14.5-18.33% moisture content, the rice grains showed a marked increase in mold infection. similar findings were reported by del prado and christensen (1952), tuite and christensen (1957) and christensen and lopez (1965) in stored rice seeds. christensen and kaufmann (1965) stated that different species of storage fungi require certain limit of moisture content for growth which determines the range over which they will predominate. they further claimed that in the starchy cereal seeds, the lower limits of moisture content that permit invasion by the common storage fungi were: aspergillus halophilicus, 13.0 to 13.2%; a. restrictus, 13.2 to 13.5%; a. amstelodami, a. chevalleri, a. repens and a. ruber, 14.0 to 14.2%; a. candidus and a. ochraceus, 15.0 to 15.2%; a. flavus, 17.5 to 18.0%. christensen and sauer (1982) also reported the different lower limits of fungi for growth in starchy cereal seeds as follows: a. halophilicus 13.0 to 14.0%; a. restrictus, a. glaucus, a. candidus and a. ochraceus, 14.5 to 16.0%; a. flavus and penicillium spp., 16.0 to 20.0%. likewise, christensen and lopez (1965) reported the invasion of storage fungi on rough rice took over at grain moisture contents of 13.4-13.8%. in the present study, storage fungi such as a. flavus-oryzae, a. candidus and a. glaucus were detected in rice grains stored at very low moisture contents (9.3-11.3%). these moisture levels are much lower than the lower limits for growth reported by other workers specially for a. candidus (christensen and kaufmann 1965; and christensen and sauer 1982). 11 biotropia no. 10, 1997 the succession of field fungi by storage fungi during storage observed in the present study agrees with the results of quitco (1982) and those at irri (1977). the percent whiteness and translucency of milled rice showed varying results. thus, the relationship of moisture content and mold count on grain whiteness and translucency needs further studies. conclusion fungi associated with stored rice were a. flavus-orywe, a. glaucus, a. nidulans, a. candidus, a. versicolor, a. terreus, a. niger, and species of penicillium, trichoconiella, curvularia, fusarium, syncephalastrum and verticillium. a high fungal population of the species of aspergillus were noted in rice paddy stored at high moisture content level. seed germination of rice remained high in rice stored at moisture contents from 9.3 to 14.0% and markedly decreased from 14.5 to 18.33% moisture content. likewise, an increase of storage fungi and decrease of field fungi was observed with increased storage period. the high mold count obtained from grain with high moisture levels favorable for growth of various species of aspergillus has contributed to decrease in seed germination. references beuchat, l.r. 1984. survival of aspergillus fla\us conidiospores and other fungi on cowpeas during long term storage under various environmental conditions. j. stored prod. res. 21: 47-52. bottomley. r.a.. c.m. christensen, and w.f. geddes. 1952. grain storage studies. x. the influence of aeration, time and moisture content on fat acidity, non-reducing sugars, and mold flora on stored yellow gram. cereal chem. 29: 53-64. christensen. c.m. 1967. germinability of seeds free of and invaded by storage fungi. proc. assoc. seed analysts?: 141-143. christensen. c.m. and h.h. kaufmann. 1965. deterioration of stored grains by fungi. ann. rev. phytopathol. 3: 69-84. christensen. c.m. and l.c. lopez. 1965. relation of moisture content and length of storage to changes in the microflora and germination percentage of rough rice. phytopathology 55: 953-956. christensen, c.m. and d.b. sauer. 1982. microflora. in storage of cereal grains and their products. c.m. christensen (ed). american association of cereal chemist inc. st. paul minnesota, p. 219240. coleman. d.a. and h.c. fellows. 1925. hygroscopic moisture of cereal grains and flaxseed exposed to atmospheres of different relative humidity. cereal chem. 2: 275-287. david. m.w., e. jay, and r.a. hill. 1985. microflora changes in peanuts (groundnuts) stored under modified atmospheres. j. stored prod. res. 21: 47-52. 12 influence of moisture content danilo e. paderes et. al del prado, f.a. and c.m. christensen. 1952. grain storage studies. xii. the fungus flora of stored rice seeds. cereal chem. 29: 456462. fandialan, i.m. and l.l. ilag. 1973. aflatoxin production ofaspergillusflavus link, isolates from rough rice, corn, sorghum, peanut and copra. phill. agric. 57: 254-263. international rice research institute. 1977. annual report. college, laguna, philippines, p. 177-178. international seed testing associations. 1976. international seed testing association rules. seed sci. and technol. 4:51-177. koehler, b. 1938. fungus growth in shelled corn as affected by moisture. j. agr. res. 56: 291-307. mallick, a.k. and b. nandl 1982. deterioration of rough rice 3. volatile compounds in short term preservation of grains. seed sci. and technol. 10: 315-320. neergaard, p. and adib-saad. 1962. some seedborne diseases and their control. indian council agr. res. 44 p. quasem. s.a. and c.m. christensen. 1958. influence of moisture content, temperature and time on the deterioration of stored corn by fungi. phytopathology 48: 544-549. qurrco, r.t. 1982. paddy deterioration from procurement to storage. naphire tech. bul. no. 2, ftt complex, mm, philippines. qurrco. r.t. and l.l. ilag. 1982. fungi as a cause of yellowing and other discolorations of rice. proc. 5th workshop grains postharvest technology, p. 118-123. raper, k.b. and d.i. fennel. 1977. the genus aspergillus. robert e. krieger pub. co., huntington, new york. 686 p. schroeder, h.w. and j.w. sorenson jr. 1961. mold development in rough rice as affected by aeration during storage. rice journal 64: 21-23. tuite. j.f. and c.m. christensen. 1957. moisture content of wheat seed in reladon to invasion of the seed by species of the aspergillus glaucus group and effect of invasion upon germination of the seed. phytopathology 47: 323-327. vaidehi, b.k. and s. waghray. 1982. storage fungi of paddy and their role in seed deterioration. national seminar on seed pathology. dept. of plant pathology. tamil nadu agricultural university. india, p. 17-19. 13 biotropia no. 10,1997 introduction rice (oryza sativa linn.) is one of the worlds major food crops. in rice producing regions where the relative humidity often ranges from 60 to 90% and the prevailing temperature is high, it has been found that these conditions are conducive for microbial growth. field and storage fungi rapidly multiply, thus affecting the quality of stored rice seeds. the dominant and often the only fungi present on stored rice paddy are species of the genus aspergillus (mallick and nandi 1982; and vaidehi and waghray 1982). like seedborne pathogens, aspergillus spp. are also responsible for the decrease in percentage of seed germination (christensen 1967) and enhance damage or discoloration of glumes (quitco and hag 1982) and aflatoxin formation (fandialan and hag 1973). studies have been conducted on the effect of physical factors on stored corn (quasem and christensen 1958x wheat (tuite and christensen 1957), peanuts (david et al. 1985) and cowpeas (beuchat 1984) but there are few studies focusing on stored rice. since most of the researches on storage fungi have been conducted under temperate and arid tropical conditions, this study identified the storage fungi affecting rice grains and determined the relation of moisture content and length of storage to the predominant mycoflora and on grain deterioration. materials and methods ir60 paddy rice seeds were used in the study. the paddy were sundried to moisture contents ranging to 9.33% and 10.56%. five 150 g paddy rice samples inside small sacks were placed in each desiccator containing super saturated solution of reagent salts, namely: potassium nitrate, potassium chloride and sodium chloride. constant temperature of 22°c was maintained by an incubator which can accommodate 12 desiccators. at every five weeks interval, the desiccators were opened and one sack in each treatment replicated three times including the control was taken and the moisture content, percent seed germination, percent whiteness, translucency and presence of fungi were determined. moisture content the high constant temperature oven method of determining seed moisture content based on the international seed testing association rules (1976) was used. twenty grams of seeds were taken in each treatment including the control and placed inside a brown paper envelope. the envelopes containing the seeds were dried inside the circulating air oven for three hours maintained at 13°c. the envelopes were cooled in a 2 influence of moisture content danilo e. paderes et al desiccator above silica gel for 30 minutes and weighed. the seed moisture content was calculated on the wet weight basis. the experimental layout consisted of a split plot design with three replications. the different salts and the control were assigned to the main plot and storage period to the subplots. percentage germination the percentage germination test was adapted from christensen and lopez (1965) with some modifications. that is, 400 instead of 100 seeds taken from each treatment including the control were used. the seeds were surface sterilized with 20% of 5.25% sodium hypochlorite for 10 min. and spaced on blotter moistened in sterile water with 100 seeds in each petri dish. the seeds were incubated at room temperature for 7 days. a seed which produced a root or coleoptile was considered to have germinated. the relationship of moisture content and percentage germination was determined. percent whiteness a kelt whiteness meter was used to determine the percent whiteness of milled rice. the whiteness meter was conditioned for 30 min. before a sample was placed inside the apparatus. approximately 35 g of milled rice from each treatment and the control were placed inside the whiteness meter. each sample was read three times in all the treatments and the control. translucency a rice meter was used to calibrate the translucency of rice grain. the rice meter was conditioned for 30 min. before a sample was placed inside the loading disk. a standard value of 83.0% was used to calibrate the sample. twenty five grams of milled rice from each treatment and the control were placed inside the loading disk. a corresponding value of the translucency of the samples was registered and recorded. each sample was read three time. a split plot design with moisture contents assigned to the main plot and storage period to the subplot was followed. storage fungi four hundred seeds from each of the treatments and the control were disinfected with 5.25% sodium hypochlorite for 1 min. and spaced in malt salt agar, usually 25 seeds in each petri dish and incubated at 25°c for 7 days. the kinds and numbers of storage 3 biotropia no. 10, 1997 fungi were observed using a stereobinocular microscope. fungi showing identical cultural characteristics were grouped together to determine the number of fungi associated on the seeds. a series of transfers were made to obtain a pure culture of the fungus. czapek's solution agar was used as the medium for identifying the isolates belonging to the genus aspergillus. three point inoculation was used as one of the basis of identification of the genus aspergillus (raper and fennel 1977). spores from the purified slants were suspended in melted agar at approximately 45°c. using a cork borer small amounts of the inoculated gelled medium were planted on desired spots on the previously plated czapek's solution agar. the plates were incubated for 10 days at 25°c. the numbers of storage molds, as affected by levels of moisture content and storage period, were analyzed using split split plot design. the levels of moisture content were assigned to the main plot, storage period to the subplot and the fungal genera to the subsubplot. results rice paddy stored in different salt solutions as well as storage period had different moisture contents (fig. 1). the equilibrium moisture content provided by the different figure 1. mean moisture contents of paddy stored in different salt solution and storage periods. bars having the same letter in each salt solution are not significantly different at 5% by dmrt. 4 moisture content (%) kcl nacl salt control influence of moisture content danilo e. paderes et. al salt treatments was highest in potassium nitrate, followed by potassium chloride, then with sodium chloride and the control after 25 weeks storage. the moisture content of the paddy stored in potassium chloride and the control varied with sampling time. however, an increasing trend of moisture content with storage period was noted in grains stored in potassium nitrate. there was no significant differences in moisture content of paddy stored in sodium chloride. effect of moisture contents and length of storage on seed germination the germination of stored paddy was affected by moisture content. at 14.5-18.3% moisture content, the percentage germination increased at 10 and 15 weeks of storage but decreased with further storage (fig. 2). rice seeds stored after 5 weeks still remained figure 2. mean percent germination of paddy stored at different time intervals. bars having the same letter at each moisture content level are not significantly different at 5% by dmrt. dormant, hence, no significant differences were observed in the percentage germination of rice seeds at all levels of moisture content. the percentage germination did not vary significantly at 15, 20 and 25 weeks of storage at moisture content level from 9.3 to 14%. however, at higher moisture levels (14.5-18.33%), the percentage germination significantly decreased at 20 and 25 weeks of storage (fig. 3). 5 biotropia no. 10, 1997 figure 3. mean percent germination of paddy stored at different moisture conditions. bars having the same letter in each storage period are not significantly different at 5% by dmrt. there was a negative correlation between moisture content and percentage germination with a correlation coefficient (r), of 0.61 (fig. 4). figure 4. relationship between percent germination and moisture contents of stored rice 6 influence of moisture content danilo e. paderes et. al effect of moisture content and length of storage on grain whiteness and translucency figure 5 illustrates the effect of moisture content and length of storage on the percent whiteness of milled rice. the percent whiteness did not vary significantly at the different moisture content levels. however, there was a decrease in percent whiteness of milled rice at 25 weeks of storage. figure 5. mean percent whiteness of milled rice stored in different moisture conditions and storage period. vertical bar (1) indicates 1 sd (0.05). the effect of moisture contents and length of storage on the translucency of grains is presented in fig. 6. there were fluctuations in the translucency of the paddy stored at moisture contents 9.3-11.3% and 12.0-14.0% with time. no significant differences were noted on the translucency of paddy at 14.5-18.33% moisture content. from 11.6 to 12.3% moisture content, the translucency of paddy taken after 5 weeks was significantly higher than the succeeding sampling periods 7 biotropia no. 10, 1997 figure 6. mean translucency of milled rice stored in different moisture conditions and storage period. bars having the same letter at each moisture content level are not significantly different at 5% by dmrt. fungi associated with rice stored in different moisture contents the majority of the fungal genera that were consistently associated with stored rice belong to the genus aspergillus. the vegetative mycelium consisted of septate branching hyphae. conidial apparatus developed as conidiophore and head from specialized, enlarged, thick walled hyphal cells. conidiophore originated from a foot cell and enlarging upward and broadening into fertile vesicles. the vesicles bore fertile cells or sterigmata producing spores or conidia borne successively from the tips of the sterigmata. further characterization of the aspergillus into group level was revealed by the different reactions of 3 point inoculation on czapek's solution agar. the cultural and morphological characteristics of the different genera were revealed by agar block technique. the isolated fungal genera were aspergillus flavus-oryzae, a. glaucus, a. nidulans, a. candidus, a. versicolor, a. terreus, a. niger, penicillium, trichoconiella, curvularia, fusarium, syncephalastrwn and verticillium. the percentage fungal incidence varied with moisture levels. there was a shift in the number of different fungal species with storage period at different moisture levels. at 5 weeks of storage, field fungi (curvularia, trichoconiella and fusarium) had higher fungal incidence and these markedly decreased with prolonged storage at all moisture levels (figs. 7 and 8). the storage fungi (a. flavus-oryzae, a. glaucus and a. candidus) slowly increased with storage period until they became the predominant fungi after 25 weeks of storage. 8 influence of moisture content danilo e. paderes et. al figure 7. fungal incidence in rice paddy stored for 25 weeks at 9.3-11.3% and 11.6-12.35% moisture contents figure 8. fungal incidence in rice paddy stored for 25 weeks at 12.014.0% and 14.4-18.3% moisture contents 9 1.pdf 10.pdf 11.pdf 12.pdf 13.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf 5. titi chandra sunarti page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 biotropia no biotropia no. 13, 1999: 1-17 integrated use of neochetina bruchi and alternaria eichhorniae in controlling water hyacinth kasno, sunjaya, asmarina s.r. putrj , okky s. dharmaputra and her! s. handayani seamed biotrop, p.o. box 116, bogor 16001, indonesia abstract the study on the integrated use of the chevroned water hyacinth weevil (neochetina bruchi mustache) and the water hyacinth blight disease (alternaria eichhorniae nag raj & ponnappa) in suppressing water hyacinth growth was carried out under field conditions at situ bagendit lake, garut, west java. the objectives of this study were (i) to investigate whether the combined use of the two control agents produced a better effect in suppressing water hyacinth growth, (ii) to evaluate whether there is a change in oviposition and feeding habit of the chevroned water hyacinth weevil if the water hyacinth is seriously infected by a. eichhorniae, and (iii) to evaluate the progress of weevil establishment in the field. the following results were obtained: (1) the concentration of tween 80 which did not interfere with spore germination was 3%. (2) there was no difference between distilled water and 1% potato dextrose broth (pdb) as a medium for fungal spores production. (3) the combined use of chevroned water hyacinth weevil and the water hyacinth blight produces a better suppressing on water hyacinth growth. (4) there was no significant effect of heavy infection by the water hyacinth blight on oviposition habit of chevroned water hyacinth weevil. heavy fungal infection only affected feeding habit of the adult chevroned water hyacinth weevil. (5) establishment of the chevroned water hyacinth weevil is in progress at situ bagendit lake, garut regency, west java. key words: indonesia/biological coraro\lneochetina bruchi/'alternaria eichhomiaeleiahhomia crassi-pes/establ ishment. introduction background the water hyacinth (eichhorniae crassipes [mart.] solm.) originated from brazil and has spread out to many subtropical and tropical countries. the plant has been considered as a noxious weed in those countries. the water hyacinth is one of the most serious aquatic weeds in indonesia (soerjani et al. 1976; tjitrosoedirdjo et al. 1993) as well as in other southeast asian countries such as malaysia (ismail 1994). recently, as a control tehnique, biological conntrol has received more attention. however, the objective is not to eradicate but to suppress the target weed population at a tolerable level. biological control can be considered as the most important component of integrated pest management (mangoendihardjo 1995). introduction 1 biotropia no. 13, 1999 of specific and potential natural enemies is the best technique for the control of exotic weeds (andreas et al. 1976; simmons 1970). according to nag raj & ponnappa (1970) and dharmaputra (1997), alternaria eichhorniae is host specific to water hyacinth and monochoria. learning from other countries, water hyacinth weevils have shown a partial control only to water hyacinth. the combined use of the two agents has produced very good results in india (gopal, personal communication 1996). for this reason, the integrated use of weevil and pathogen in indonesia was investigated. objectives the objectives of this study were (1) to investigate the possible better effect of the integrated use ofneochetina bruchi (hustache) and alternaria eichhorniae (nag raj & ponnappa) in suppressing the growth of water hyacinth; and (2) to examine the possible change of feeding and oviposition habit of the chevroned water hyacinth weevil caused by the heavy infestation of the fungus also, observations were made to monitor the establishment of the weevil in situ bagendit, garut, west java. methodology rearing of the water hyacinth weevil the chevroned water hyacinth weevil (neochetina bruchi hustache) was introduced from australia into indonesia in 1995. after a series of tests under quarantine conditions at biotrop, the release permit was issued by the government of indonesia on june, 3 1996. the weevil was then mass reared on water hyacinth grown in concrete ponds at biotrop in bogor. preparation of fungal culture and spore suspension the fungus, a. eichhorniae used in this study was an isolate collected from situ bagendit lake at garut regency, west java. to obtain fungal spores for research purposes, the fungus was grown on rice husk yeast extract agar (rhya) for at least 7 days at room temperature. spore mass was first separated from their mycelia and medium by scrubing it using a fine brush. the spore mass was then filtered using a muslin cloth of about 50 micron pore size. experiment 1. the effect of sticking agent (tween 80) on fungal spore germination the effect of tween 80 (polyoxyethylensorbitanmonooleate) in the aqua spore suspension on fungal spore germination was carried out under laboratory conditions. various concentrations of tween 80 i.e. 0, 1, 2, 3 and 4% were evaluated. the spore concentration used was 1 x 106 spores/ml. spore germination was observed under 2 integrated use otneochetina bruchi and altemaria eichhorniae kasno et al. the microsope (magnification 10 x 10) one hour after addition of tween 80. the optimum concentration with minimum side effect was used in further experiments. experiment 2. optimum spore concentration required to cause the disease (blight) various concentrations of fungal spore i.e. 2.5 x 10s, 5.0 x 105, 7.5 x 105 and 1.0 x 106 spores/ml were evaluated under greenhouse conditions. aside from spore suspension, two concentrations i.e. 2 and 3% of tween 80 as sticking agent, as well as 1% potato dextrose broth (pdb) and distilled water as carrying agents were also evaluated as to their ability to support the fungus to cause the disease in water hyacinth. inoculation of the spore suspension (1 ml/plant) was carried out on the water hyacinth surface using an atomizer at about 04.00 pm. soon after inoculation, water hyacinth was placed in the gauze cage in the greenhouse. the cages were then covered with damp jute bags for 24 hours to create a suitable condition for spore germination and penetration. the parameter used to evaluate the test was the damage severity of the water hyacinth plant due to fungal infectionn. the damage severity score was measured at 7 days after treatment. the scoring system was based on a 0 to 4 damage severity scale. the damage severity score of 0 means no infection, while score of 1, 2, 3 and 4 indicate 1-25%, 26-50%, 51-75% and more than 75% caused leaf damage or death, respectively. experiment 3. combined use of tv. bruchi weevil and spore suspension of a. eichhorniae to suppress water hyacinth growth under greenhouse conditions the target of this experiment was to obtain the optimum number of weevils in combination with spore suspension that would bring about a better effect in suppressing water hyacinth growth. the number of weevils released was 0, 1, 3 and 5 pairs/plant, while concentrations of inoculated spore were 0, 0.5, 1.0 and 1.5 times, respectively from the optimum concentration in experiment 2. water hyacinth with 6 leaves and ± 70 g wet weight was used in this experiment. the selected plants were then grown on plastic pots (1 plant/pot) containing tap water with mud at the bottom. to prevent the weevil from moving away, each pot was enclosed in transparant plastic cages with appropriate ventilation. seven days after the weevils were released, spore suspension was used to inoculate the water hyacinth (3 replications/treatment). the treated plants were then placed in cages enclosed with damp jute bags for 24 hours. parameters used to evaluate the effect of the treatments were damage severity and biomass. the scoring scales as applied in experiment 2 was also applied in this experiment. 3 biotropia no. 13, 1999 experiment 4. combined use of n. bruchi weevil and spore suspension of a. eichhorniae to suppress water hyacinth growth under field conditions a procedure similar to experiment 3 was set up under field conditions during dry and wet seasons. the number of released weevils was 0, 1, 3 and 5 pairs/plant, while the concentration of spore suspensions for inoculation was 0 and 1 x 106 spores/ml/plant. three replications were set up for each treatment (3 plants/ replication) in two seasons. selection of water hyacinth to nearly the same size was made prior to setting up the experiment. observations on the effect of the two control agents were made at 14 and 21 days after weevils were released. experiment 5. possible change in adult weevil habit by selecting feeding and oviposition sites normally, the weevil fed on the leaf surface of the water hyacinth and either the leaf tissue or petiole was used as sites for oviposition. an experiment was set up to investigate the possible effect of heavy fungal infection on the weevil in selecting sites for oviposition and feeding. three pairs of adult weevil per plant were released, while the concentrations of inoculated spore suspension were 0 and 1 x 106 spore/ml/plant. five replications were set up for each treatment. an observation was done 14 days after weevils were released. parameters used to measure the possible effect were the number of feeding scars and eggs on healthy and heavily infected water hyacinth. experiment 6. monitoring of weevil establishment in situ bagendit of garut regency, west java n. bruchii was released in situ bagendit lake on august, 28 1996 at 3 points. the number of weevils released at the first, second and third point was 75, 120 and 192 pairs respectively. release was repeated on october, 16 1977. one hundred and sixty pairs of weevil were released in the center of the lake. monitoring in the first year was done to record the dispersion of weevil from the initial point. further monitoring in the second year was done to record the presence of weevil and feeding scars. therefore, 10 samples for 0.5 m x 0.5 m of water hyacinth showing feeding scars were collected. results and discussion experiment 1. the effect of tween 80 on fungal spore germination under field conditions, rain fall as well as wind may cause fungal spores fail to penetrate into the leaf surface before germination. tween 80 may help spores to fix 4 integrated use ofneochetina bruchi and altemaria eichhomiae kasno et al. on the leaf surface for a certain period of time to avoid possible dispersal of spores due to rain fall and blowing wind. however, tween 80 may cause reduction in spore germination. such agents are often necessary to disperse fungal spores within a formulation and to support the spread and adherence of spores on the plant surface (mitchell 1988). the use of certain concentrations of such agents may influence spore germination (daigle & cotty 1991). a test to evaluate the possible negative effect of tween 80 on spore germination was made using 0, 1, 2, 3 and 4% concentrations. however, there was no significant difference in spore germination. the percentages of spore germination at concentrations of 0, 1, 2, 3 and 4% of tween 80 were 96, 95, 94, 93 and 92, respectively. considering the cost of tween 80, a 2 and 3% concentrations was selected. experiment 2. optimum spore concentration required to cause a disease (blight) based on statistical analysis, the effect of spore and tween 80 concentrations gave very significant differences to the severity score of water hyacinth leaves, while the effect of media was not significantly different (appendix 1). the severity score on leaves when measured 7 days after treatment tended to increase with the increase of spore concentration. at spore concentrations of 7.5 x 105 and 1 x 106/ml, the damage severity score was not different, i.e. 3.5 (table 1). the damage severity score on leaves increased with increase of tween 80 concentration. the scores for 0, 2 and 3% tween 80 concentration were 3.0, 3.1 and 3.7, respectively (table 2). the use of 1% pdb did not show a significant difference in the damage severity score when compared to distilled water as a medium (table 2). aqueous spore suspensions containing about 1.0 x 106 spores/ml and 3% tween 80 was selected for further experiments. table 1. the effect of a. eichhomiae spore concentration, tween 80 concentration and type of media on the severity score of water hyacinth treatment scoring spore concentration (/ml) 2.5x10' 2.8 b 5.0 x l o 5 3.3 ab 7.5x10' 3.5 a 1.0 x lo 6 3.5 a tween 80 concentration (%) 0 3.0 c 2 3.1 c 3 3.7 d type of media distilled water 3.3 e 1%pdb 3.3e numbers followed by the same letter do not differ significantly according to dmrt at 95% confidence level. 5 biotropia no. 13, 1 999 according to daigle & cotty (1991) addition of 1% pdb to each surfactant concentration increased spore germination capability compared to surfactant or 1% pdb alone. in this experiment spore germination with 1% pdb was not observed. the result showed that there was no difference in the damage severity score between distilled water and 1% pdb as the medium, and the use of 1% pdb was discontinued. experiment 3. combined use of n. bruchi weevil and spore suspension of a. eichhorniae to suppress water hyacinth growth under greenhouse conditions based on statistical analysis the variation in number of adult weevil showed a significantly different effect on the damage severity score of water hyacinth leaves caused by the presence of the pathogen, weevils and their combination when measured at 14 days and 21 days after weevil release (appendices 2 and 3). the damage severity score at 21 days after weevil release was higher than at 14 days for all treatments (tables 2 and 3). the damage severity score of water hyacinth leaves increased with the increase of weevil numbers and spore concentrations. the damage severity score due to pathogen infection was lower than that caused by weevil infestation. nevertheless, the damage severity score caused by the combination of the two agents was higher than that caused by each agent (table 3). the damage severity score of combination between spore concentrations of 1.0 x 106 and 1.5 x 106/ml; and between weevil numbers of 3 and 5 pairs were not significantly different (tables 2v and 3). these facts indicated that the optimum combination in suppressing about 70 gram size of water hyacinth growth under greenhouse conditions was 1.0 x 106 spores/ml and 3 pairs of adult weevil. there was an indication of faster infection of pathogen on undamaged leaf tissue of water hyacinth if compared to the injured one. in other case, there was an indication of feeding preference of the adult weevil on the healthy tissue rather than on infected leaf tissue of water hyacinth. table 2. the effect of adult n. bruchi and a. eichhorniae spore concentration on the damage severity score of water hyacinth leaves due to pathogen, weevil and their combination at 14 days after weevil release damage severity score spore cone. (/ml) pathogen no. of weevils (pairs) weevil combination 0 0 a 0 0 d 0 h 5.0 x 10s 2.4 b 1 1.8 e 2.5 i 1.0x10' 2.7 be 3 2.9 f 3.7j 1.5x10' 2.9 c 5 3.8 g 3-9j numbers followed by the same letter do not differ significantly according to dmrt at 95% confidence level. 6 integrated use ofneochetina bruchi and alternaria eichhomiae kasno et al. table 3. the effect of adult n. bruchi and a. eichhomiae spore concentration on the damage severity score of water hyacinth leaves due to pathogen, weevil and their combination at 21 days after weevil release damage severity score spore cone. (/ml) pathogen no. of weevils (pairs) weevil combination 0 0 a 0 0 e 0 i 5.0x10' 2.9 b 1 2.7 f 2.8j 1.0 x 106 3.3 cd 3 3.6 g 4.0k l.sxlo6 3.4 d 5 3.9 h 4.0k numbers followed by the same letter do not differ significantly according to dmrt at 95% confidence level. experiment 4. combined use of n. bruchi weevil and spore suspension of a. eichhomiae to suppress water hyacinth growth under field conditions a. dry season based on statistical analysis, the variation in number of adult weevils showed a significantly different effect on the damage severity score of water hyacinth leaves caused by the presence of the weevils and combined use of the two agents when measured at 14 days and 21 days after weevil release (appendices 4 and 5). the damage severity score at 21 days after weevil release was higher than at 14 days for all treatments (table 4). b. wet season based on statistical analysis, the number of adult weevils showed a significantly different effect on the damage severity score of water hyacinth leaves caused by the presence of the weevils and combination of the two agents when measured at 14 days and 21 days after weevil release (appendices 6 and 7). the damage severity score at 21 days after weevil release was higher than at 14 days for all treatments (table 5). the damage severity score either due to weevil, pathogen or their combination during wet season (table 5) was lower than that during the dry season (table 4). during the wet season the higher water nutrient content may provide better growth to water hyacinth plant, therefore the plant may be more resistant to the two suppressing agents. tables 4 and 5 show that the damage severity score due to combined use of the two agents, between 3 and 5 pairs of adult weevils was significantly different. this result differed from the result in experiment 3. the different result in this experiment was assumed due to the difference in the number of plants for each 7 biotropia no. 13, 1999 replication. one plant was used in experiment 3, while 3 plants were used in this experiment. charudattan et al. (1976) reported that infection of plants inoculated with acremonium zonatum was more severe in the presence ofn. eichhorniae. sanders et al. (1982) also reported that in the panama canal a positive correlation exists between a. zonatum and n. eichhorniae. table 4. the effect of adult n. bruchi and a. eichhorniae spore concentration on the damage severity score of water hyacinth leaves due to pathogen, weevil and their combination at 14 and 21 days after weevil release during dry season damage severity score pathogen weevil combination spore cone, (/ml) 14 days 21 days no. of weevil, (pairs) 14 days 21 days 14 days 21 days 0 l .oxl o6 0 a 2.7 b 0 a 3.4 c 0 1 3 5 0 d 1.9 e 2.4 f 3.4 g 0 d 2.3 f 2.9 g 3.6 h 0 i 2.9 jk 3.3 kl 3.61 0 i 3.4 kl 3.81m 4.0 n numbers followed by the same letter do not differ significantly according to dmrt at 95% confidence level. table 5. the effect of adult n. bruchi and a. eichhorniae spore concentration on the damage severity score of water hyacinth leaves due to pathogen, weevil and their combination at 14 and 21 days after weevil release during wet season damage severity score pathogen weevil combination spore cone, (/ml) 14 days 21 days no. of weevil (pairs) 14 days 21 days 14 days 21 days 0 1.0 x l o 6 0 a 2.3 b 0 a 3.1 c 0 1 3 5 0 d 1.3 e 2.3 g 3.2 i 0 d 2.8 f 2.9 h 3.6j 0 k 2.81 3.3m 3.5m 0 k 3.3m 3.8 n 4.0 o numbers followed by the same letter do not differ significantly according to dmrt at 95% confidence level. the change of water hyacinth wet weight at 21 days after weevil release in each treatment is presented in figure 1. the results show that the increase of adult weevils and concentration of spore pathogen reduced the increment of wet weight. it means that both water hyacinth control agents could suppress the growth of water hyacinth in the field. 8 integrated use ofneochetina bruchi and altemaria eichhomiae — kasno et al. figure 1 . the change of water hyacinth wet weight at 21 days after weevil release in each treatment. c=spore concentrations (/ml) s=adult weevils (pairs) (1.0 x 106 spore/ml) 0, 1, 3, 5=number of pairs based on statistical analysis, number of adult weevils and concentration of pathogen spore caused very significantly different effect on the change of water hyacinth wet weight (appendix 8). conway et al. (1978) reported that pathogen (cercospora rodmanii) could influence the growth of water hyacinth and reduce the biomass. according to galbraith (1987) acremonium zonatum caused extensive infection of individual leaves of water hyacinth, but the whole plant survived since disease development did not keep up with the production of new leaves. experiment 5. possible change of adult weevil habit in selecting feeding and oviposition sites figure 2 shows that the pathogen infection on water hyacinth did not reduce the production of weevil eggs. weevils preferred to put their eggs on the petiole than on the leaf. so the infection of pathogen did not affect the oviposition behavior of weevil. 9 biotropia no. 13,1999 experiment 6. monitoring of weevil establishment in situ bagendit, regency of garut, west java generally, the steps for biological control program of weeds are as follows: 1. to determine the conformity of weed species to be controlled biologically. 2. survey of natural enemies. 3. to study the ecology of weeds and their natural enemies. 4. to study the host specificity of natural enemies to secure that they could not become pest in the control area. 5. to introduce/release and establish natural enemies in the control area. 6. evaluation. one of the biological control program steps was to release tv. bruchi. it has been done and it is establishing now. monitoring was carried out after weevils were released to investigate the difference that may occur in the field (kasno 1989). monitoring was done at 2, 4 and 10 months after weevils were released indicating that the feeding symptom was found at distances of ± 45 m, ± 250 m and ± 2 km, respectively, from the initial point of release. however, no symptoms of attack were found on plantation crops surrounding the lake. tables 6 and 7 show the existence of adult weevils, eggs, larvae and pupae at 15 and 16 months after the weevils were released. population of adult weevils at 16 months after weevils were released was higher than at 15 months, i.e. from 0.8 to 1.4 insects per 0.5x0.5 m area. larvae and pupae were not found at 15 months after weevils were released. however, 16 months after the release larvae were also not found, but a small number of pupae were found i.e. 0.4. the number of eggs increased from 5.4 to 9.1. the average of leaf scars per second youngest leaf also increased, i.e. from 13.9 to 17.4. generally, it could be concluded that tv. bruchi weevil was still in the process of establishment, since the number of adult weevils, eggs, larvae, pupae and other parameters increased. 10 integrated use ofneochetina bruchi and altemaria eichhorniae kasno et al. conclusions 1. the integrated use of n. bruchi and a. eichhorniae as a controlling agent ofwater hyacinth caused a better suppressing of water hyacinth and its growth rather than using the agents separately. 2. the infection of a. eichhorniae on water hyacinth leaf did not change the oviposition site of tv. bruchi. 3. n. bruchi prefers to feed on intact than on the infected leaf tissue. 4. the damage severity score from all treatments increased through the time. 5. establishment of n. bruchi in situ bagendit lake, west java is still in progress. acknowledgment the authors would like to express our sincere thanks to the department of higher education, ministry of education and culture for the financial support, and to the plant quarantine department, ministry of agriculture for the release permit of n. bruchi. references andreas, l.a., c.j. da vis, p. harris & a.j. whapshere. 1976. biological control of weeds. in c.b. huffaker & p.s. messenger (eds.). theory and practice of biological control. academic press. new york. bashir, o.m. 1984. the establishment and distribution of natural enemy of water hyacinth released in sudan. tropical pest management 30(3): 320-323. charudattan, r., t.e. freeman & k.e. conway. 1976. progress in the use of plant pathogens as biological control for aquatic weeds. misc. pap. a-77-3. us army engineer waterways exp. stn, vickbury. miss. conway, k.e., t.e. freeman & r. charudattan. 1978. development of cercospora rodmanii as a biological control for eichhorniae crassipes. proc. ewrs 5* symp. on aquat. weeds. po box 14, wageningen, the netherlands, p. 225-230. daigle, d.j. & p.j. cotty. 1991. factors that influence germination and mycoherbicidal activity of alternaria cassiae. weed tech. 5: 82-86. dharmaputra, o.s. 1997. pathogenicity test of altemaria eichhorniae isolated from water hyacinth. seameo biotrop. internal report. galbraith, j.c. 1987. the pathogenicity of an australian isolate of acremonium zonatum to water hyacinth and its relationship with the biological control agent, neochetina eichhorniae. aust. j. agric. res. 38:219-229. ismail, m.a. 1994. biological control efforts towards management of some common weeds in malaysia. proc. symp. biology and management of weeds and 14* tropical weed science society conference. seameo biotrop. bogor. kasno. 1989. pengantar umum pengendalian gulma secara hayati. kumpulan materi kuliah pendidikan dan pelatihan pengelolaan gulma proyek p2tp. direktorat jenderal perkebunan, bogor, 6 nopember 2 desember 1989. seameo biotrop. mangoendihardjo, s. 1995. pengendalian hayati, komponen utama pengelolaan jazad pengganggu (biological control the main component of integrated pest management). pidato pengukuhan guru besar. universitas gadjah mada, 22 maret 1995, yogyakarta. 26 p. 13 biotropia no. 13, 1999 mitchell, j.k. 1988. gibbago trianthema, a recently described hyphomycete with bioherbicide potential for control of horse purslane (trianthema portulacastrum). plant dis. 72: 354-355. nag raj, t.r. & k.m. ponnappa. 1970. blight of water hyacinth by alternaria eichhorniae sp. nov. trans. br. mycol. soc. 55(1): 123-130. sanders, d.r., r.f. theriot & e.a. theriot. 1982. organisms impacting water hyacinth in the panama canal. misc. pap. a-82-1, us army engineer waterways exp. stn, vicksbury, miss. simmons, f.j. 1970. biological control. sompong press, bangkok. 12 p. soerjani, m. el al. 1976. proc. of the southeast asian workshop on aquatic weeds. seameo biotrop. bogor. 45 p. tjitrosoedirdjo, s., kasno & sunjaya. 1993. notes on the management of water hyacinth in rawa pening lake: 1990-1991. in proceedings of integrated pest management control component. biotrop special pub. no. 50: 147-155. 14 integrated use of neochetina bnichi and alternaria eichhorniae kasno et al. appendix 1. analyses of variance on the effect of alternaria eichhorniae spore concentration, tween 80 concentration,type of solvent and their interaction on the damage severity score of water hyacinth. source of variance f value spore concentration (a) tween 80 concentration (b) type of solvent (c) a x b a x c b x c a x b x c 5.18** 9.36** 0.09 0.64 1.06 0.64 0.15 ' = very significantly different at 99% confidence level. appendix 2. analyses of variance on the effect of neochetina bruchi numbers, concentration of alternaria eichhorniae spore and their interaction on the damage severity score of water hyacinth due to pathogen, weevil and their combination under greenhouse conditions 14 days after weevil release. source of variance pathogen f value weevil combination number of weevil pairs (a) spore concentration (b) a x b 36.50** 173.75** 4.58** 342.33** 2.33 1.56 187.30** 38.95** 4.17** = very significantly different at 99% confidence level appendix 3. analyses of variance on the effect of neochetina bruchi numbers, concentration of alternaria eichhorniae spore and their interaction on the damage severity score of water hyacinth due to pathogen, weevil and their combination under greenhouse conditions 21 days after weevil release. source of variance pathogen f value weevil combination number of weevil pairs (a) spore concentration (b) a x b 24.58** 239.95** 3.08** 491.60** 8.80** 2.43* 140.70** 50.66** 9.29** *= significantly different at 95% confidence level **= very significantly different at 99% confidence level 15 biotropia no. 13, 1999 appendix 4. analyses of variance on the effect of neochetina bruchi numbers, concentration of alternaria eichhomiae spore and their'interaction on the damage severity score of water hyacinth due to pathogen, weevil and their combination during dry season at 14 days after weevil release under field conditions. source of variance f value pathogen weevil combination number of weevil pairs (a) spore concentration (b) a x b 1..00 504.27** 1.00 90.53** 1.59 0.18 36.91** 68.57** 8.80** *= very significantly different at 99% confidence level. appendix 5. analyses of variance on the effect of neochetina bruchi numbers, concentration of alternaria eichhomiae spore and their interaction on the damage severity score of water hyacinth due to pathogen, weevil and their combination during dry season at 21 days after weevil release under field conditions. source of variance pathogen f value weevil combination number of weevil pairs (a)) spore concentration (b) a x b 2.00 874.27** 2.00 104.22** 6.00* 1.11 46.83** 112.32** 14.97** *= significantly different at 95% confidence level **= very significantly different at 99% confidence level appendix 6. analyses of variance on the effect of neochetina bruchi numbers, concentration of alternaria eichhomiae spore and their interaction on the damage severity score of water hyacinth due to pathogen, weevil and their combination during wet season at 14 days after weevil release under field conditions. source of variance f value pathogen weevil combination number of weevil pairs (a) 2.00 80.73** 75.00** spore concentration (b) 486.00** 9.80** 216.00** a x b 2.00 1.27 5.00** **= very significantly different at 99% confidence level. 16 integrated use of neochelina bruchi and alternaria eichhomiae — kasno et al. appendix 7. analyses of variance on the effect of neochetina bruchi numbers, concentration of alternaria eichhomiae spore and their interaction on the damage severity score of water hyacinth due to pathogen, weevil and their combination during wet season at 21 days after weevil release under field conditions. source of variance pathogen f value weevil combination number of weevil pairs (a) spore concentration (b) a x b 3.40 80.20** 3.40 176.00** 30.00** 4.40* 55.62** 168.23** 5.77** *= significantly different at 95% confidence level **= very significantly different at 99% confidence level appendix 8. analyses of variance on the effect of neochetina bruchi numbers, concentration of alternaria eichhomiae spore and their interaction on the change of water hyacinth wet weight. source of variance f value number of weevil pairs (a) 9.41 ** spore concentration (b) 4.31 * a x b 0.48 *= significantly different at 95% confidence level **= very significantly different at 99% confidence level 17 6. muhamad anshar amran nu anyar.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 biotropia no. 10, 1997 : 29-41 the effects of drying and shelling on aspergillus flaws infection and aflatoxin production of maize* o.s. dharmaputra seameo biotrop, p.o. box 116, bogor, indonesia; and department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia i. retnowati seameo biotrop, p.o. box 116, bogor, indonesia h.k. purwadaria department of agricultural engineering, faculty of agriculture technology, bogor agricultural university, bogor. indonesia h. susilo seameo biotrop, p.o. box 116. bogor, indonesia abstract the effects of drying and shelling on aspergillus fla\-us infection and aflatoxin production of maize stored under laboratory conditions were investigated together with the intactness of grain and change of moisture content during the storage period. fully matured maize var. arjuna and cpi-2 were harvested at 90 and 97 days after planting, respectively, after which they were unhusked and divided into 4 pans. the 1st and the 2nd parts were sun dried up to 20^ moisture content (m.c.) and then shelled and re-dried up to 17 and 14% m.c.. respectively. the 3rd part was sun dried up to 17% m.c. and then shelled but not re-dned. the 4th pan was sun dried up to 17% m.c. and then shelled and re-dried up to 14% m.c. the maize was sun dried by spreading either the cobs or the kernels on the paved floor. the nail-down wood and mechanical sheller were used for shelling the maize. after drying and shelling, maize samples were stored in the jars which were covered with muslin cloth for 3 months under laboratory conditions. a. flavus was isolated using dilution method on aspergillus flavus and parasiticus agar (afpa). the damaged kernel analysis was carried out at the beginning of storage to obtain the percentage of damaged kernel caused by shelling. the m.c. and aflatoxin were determined using oven and high performance liquid chromatography (hplc) methods, respectively. the m.c. decreased at 1 month of storage and then it was almost constant at 2 and 3 months of storage. the percentage of damaged kernels of maize var. cpi-2 was higher than those of var. arjuna. the percentage of damaged kernels of maize shelled at 20% m.c. was higher than that shelled at 17% m.c. the percentage of damaged kernels of maize shelled by mechanical sheller was higher than that shelled by nail-down wood. *paper presented at the symposium on pest management for stored food and feed, 5 -7 september 1995 bogor, indonesia. 29 the effects of drying and shelling on aspergillus flavus infection o.s. dharmaputra population of a. flavus on maize var. arjuna was higher than that of var. cpi-2. the population on maize stored at the initial m.c. of 17% was higher than that of 14%, the population on maize shelled by mechanical sheller was higher than that shelled by nail-down wood, but there was no significant difference. the population increased at 1 and 2 months of storage and then decreased at 3 months of storage. total aflatoxin bi content of maize var. cpi-2 was higher than that of var. arjuna. the content of maize dried up to 17% m.c. and then shelled but not re-dried was the lowest compared with the other methods of drying. the content of maize shelled by nail-down wood was not significantly different than shelled by mechanical sheller. the content increased with the increase of storage duration. key words: drying/shelling/x5perg///mj-/zav«j/aflatoxin introduction before and after harvest, maize (zea mays) could be infected by aspergillus flavus, a fungus that can produce aflatoxin (butler 1974; lillehoj and hesseltine 1977). the most notorious toxin is aflatoxin, as it is an extremely potent carcinogen affecting several animal species. cole et al. (1982) reported that among mycotoxins, aflatoxin was often found in maize. survey conducted by biotrop team in 1992 revealed that 35 maize samples collected from farmers and traders in lampung province contained 23-367 ppb of aflatoxin. thirty samples contained aflatoxin of more than 30 ppb (dharmaputra et al 1993). another survey conducted during 1993/94 showed that 108 maize samples collected from fanners in central lampung and kediri regencies contained 5-291 and 9-283 ppb of aflatoxin bi during dry and wet seasons, respectively. of these, 87 samples contained more than 30 ppb of aflatoxin bl. while 32 maize samples collected from traders in the two regencies contained 10-104 and 21-115 ppb of aflatoxin bl during dry and wet seasons, respectively, 26 of which contained more than 30 ppb (dharmaputra et al. 1994). the high level of aflatoxin content in most maize samples could be caused by the methods of handling that were not carried out properly. limits on the amount of aflatoxins which are permitted in foods vary between countries and products. most european countries have moved towards a limit of below 30 ppb for total aflatoxin concentration in all foods and less than 0.05 ppb of m aflatoxin hi milk (gilbert 1991). in asia, taiwan tolerates up to 50 ppb of aflatoxin bi in cereals and peanuts, while thailand has a tolerance of 20 ppb for b and g aflatoxins hi all foods and japan 10 ppb tolerance for b aflatoxin in all foods. the philippines has a 20 ppb tolerance for aflatoxin bi in coconut and peanut products for export (van egmond 1991). 30 biotropia no. 10, 1997 postharvest handling (among others, drying and shelling) could affect fungal infection. in general, the moisture content of freshly harvested maize is still high, making it a good substrate for fungal growth. shelling could cause mechanical damage, and fungal spores can infect the kernel through the damage. consequently, the kernels should be shelled using a proper tool at certain moisture content to reduce the damage of kernels. according to covanich (1991) drying is particularly a vital operation in gram handling chain, since moisture is the most important factor determining the extent grains are liable to deterioration during storage. dried grains are less susceptible to insect and mold attacks. overdrying of grains in the sun, for example, can cause breakage, loss of viability, etc. in general, the moisture content of freshly harvested maize was between 23-33%. drying of maize was conducted up to 17-20% moisture content (sfcdp 1990). most farmers in lampung and east java shelled cob of maize using mechanical sheller, while in south sulawesi fanners use a nail-down wood (sfcdp 1990). according to purwadaria (1987) the mechanized maize sheller could reduce the quantity of losses as much as 5% and lowered the shelling cost to 67-40% of the manual shelling cost. the objectives of this study were : 1) to determine the effects of some methods of drying and shelling on aspergillus flavus infection and aflatoxin production of maize stored under laboratory conditions; and 2) to determine the effect of drying and shelling on the intacmess of kernel, as well as the effect of storage period on the change of moisture content. materials and methods maize variety two maize varieties (arjuna and cpi-2) were used hi this study. they were grown at the experimental plot of the research institute for food crop biotechnology, bogor, harvested at 90 and 97 days after planting and unhusked immediately after harvest. drying and shelling cobs of maize were divided into 4 parts. the 1st and the 2nd parts were su n dried up to 20% moisture content (m.c.) and then shelled and re-dried up to 17 and 14% m.c., respectively. the 3rd part was sun dried up to 17% m.c. and then shelled but not re-dried. the 4th part was sun dried up to 17% m.c. then shelled and re-dried 31 the effects of drying and shelling on aspergillus flavus infection o.s. dharmaputra up to 14% m.c. all of the maize samples were sun dried by spreading either the cobs or the kernels on the paved floor. nail-down wood and mechanical sheller type yanmar tf 55-di with cylinder rotation of 500-700 rpm were used for shelling the maize. storing of maize and method of sampling after drying and shelling, 500 g of maize of each treatment were placed in 3.3 l jar covered with muslin cloth, and stored for 1, 2, and 3 months under laboratory conditions. two replicates were used for each treatment. the ambient temperature and relative humidity of the storage were recorded using a wilh. lambrecht thermohygrograph type 252. initial sample was derived from each replicate (jar) at the beginning of storage, and then at 1, 2, and 3 months of storage. this sample was divided twice using a sample divider to obtain working samples for moisture content. damaged kernels, population of a. flavus, and total aflatoxin bi content analyses. moisture content, damaged kernels, population of a. flavus and alfatoxin bi content analyses moisture content (based on wet basis) was determined using oven method at 130° c for 2 hours (bsi 1980). the analyses for damaged kernels were carried out at the beginning of storage to obtain the percentage of damaged kernel caused by shelling. a. flavus was isolated using dilution method on aspergillus flavus and parasi-ticus agar (afpa) (pin and hocking 1985). aflatoxin content was determined using high performance liquid chromatography (hplc) method (rodriquez and mahoney 1994). statistical analysis the data were analyzed using completely randomized factorial design with 4 factors. the 1st, 2nd, 3rd and 4th factors were maize variety, method of drying and shelling, and duration of storage, respectively. results and discussion the effect of maize variety, methods of drying and shelling, and storage period on moisture content and damaged kernel based on statistical analysis, the effects of drying, duration of storage and their interaction gave very significant differences in moisture content, while variety and shelling did not give significant differences. 32 biotropia no. 10, 1997 at the beginning of storage, moisture content on each drying method (i, ii, iii and iv) was 16.84, 14.10, 17.11 and 14.35%, respectively. at 1 month after storage they decreased (13.98, 13.51, 14.05 and 13.75%, respectively), and then they were almost constant at 2 and 3 months after storage (table 1). moisture content of maize at 2 and 3 months after storage would approach the equilibrium moisture content (emc) with relative humidity of the storage. during storage m.c. of grains would move towards the emc. according to hall (1957), henderson and perry (1976), emc was table 1. moisture content of maize treated with different methods of drying during storage numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level i = cobs of maize were sun dried up to 20% moisture content, and then shelled and re-dried up to 17% moisture content. ii = cobs of maize were sun dried up to 20% moisture content, and then shelled and re-dried up to 14% moisture content. iii = cobs of maize were sun dried up to 17% moisture content, and then shelled but not re-dried. iv = cobs of maize were sun dried up to 17% moisture content, and then shelled and re -dried up to 14% moisture content. reached when the grains did not absorb or release vapor any more. brooker et al. (1974) reported that emc of grains were affected by temperature, humidity, variety and maturity of grains. range of temperature and relative humidity of the storage were 21.75-29.25°c and 47.88-88.25%, respectively. (table 2). the effects of maize variety, drying and shelling gave very significantly differences to damaged kernels, while their interaction was not significantly different. table 2. range of temperature and relative humidity in the storage room 33 the effects of drying and shelling on aspergillus flavus infection o.s. dharmaputra the percentage of damaged kernels of maize var. cpi-2 (5.0%) was higher than that of var. arjuna (3.6%) (table 3). based on visual observation, the kernels of var. cpi-2 were bigger and less solid than var. arjuna, therefore it could be more easily broken during shelling. table 3. the effect of maize variety, methods of drying and shelling on damaged kernels at the beginning of storages * numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level *damaged kernels analysis was carried out only at the beginning of storage i = cobs of maize were sun dried up to 20% moisture content, and then shelled and re-dried up to 17% moisture content. ii = cobs of maize were sun dried up to 20% moisture content, and then shelled and re-dried up to 14% moisture content. iii = cobs of maize were sun dried up to 17% moisture content, and then shelled but not re -dried. iv = cobs of maize were sun dried up to 17% moisture content, and then shelled and re-dried up to 14% moisture content. the percentage of damaged kernels of maize shelled at 20% m.c. (drying methods i and ii) (4.7 and 5.4%, respectively) was higher than maize shelled at 17% m.c. (drying methods hi and iv) (3.7 and 3.2%, respectively). according to sfcdp (1990), in general, the percentage of damaged kernels increased if the m.c. was more than 18%. idrc (1988) reported that the maize quality deteriorated more when the shelling process took place directly after harvest at a high moisture level of grain (around 37% wb) compared to the process where maize shelling was done after the initial sun drying which brought the grain moisture content down to around 25% wb. the percentage of damaged kernels of maize shelled by mechanical shelter (5.7%) was higher than that shelled by nail-down wood (2.9%). shelling of each cob of maize using a nail-down wood did not cause friction among the cobs, while shelling using mechanical shelter created a friction. moreover, shelling done by manpower 34 biotropia no. 10, 1997 using a nail-down wood can be controlled to reduce the friction between sheller and maize. according to suprayitno (1980), the percentage of damaged kernels of maize shelled using mechanical sheller was high, because there was a friction among intact kernels, between intact kernels and the cylinder of mechanical sheller. the effect of maize variety, methods of drying and shelling, and storage duration on population of aspergillus flavus based on statistical analysis, the effects of maize variety gave significant differences to population of a. flavus; drying and duration of storage gave very significant differences; while shelling and interaction among maize variety, drying, shelling and duration of storage did not give significant differences. population of a. flavus on maize var. arjuna (697.2 colonies/g) was higher than that of var. cpi-2 (306.4 colonies/g) (table 4). wilson el al. (1983) reported that the growth of a. flavus and aflatoxin production were affected by some metabolites (groups of alcohol and aldehyde) produced by maize. population of a. flavus on maize stored at the initial m.c. of 17% (drying methods i and iii) (570.9 and 1224.4 colonies/g) was higher than that of 14% (drying methods ii and iv) (84.2 and 127.7 colonies/g) (table 4). christensen and kaufmann (1974) reported that the minimum m.c. for the growth of a. flavus on maize was 18-18.5%. according to kawashima and kawasugi (1988). grain which was sun dried immediately after shelling on concrete drying floor with m.c. less than or around 15% could be stored with low contamination of a. flavus (1.5-8%) for 56 days in middlemen's storage. if the grains (with m.c. more than 20%) were not dried after shelling, a. flavus contamination prevailed quickly. however, if m.c. was less than 17% the development of a. flavus could be inhibited. population of a. flavus on maize shelled by mechanical sheller (573.4 colonies/g) was higher than shelled by nail-down wood (430.2 colinies/g), but it did not give significant difference (table 4). also the percentage of damaged kernels shelled by mechanical sheller was higher than those shelled by a nail-down wood. the damaged kernels were more easily infected by a. flavus. dharmaputra et al. (1994) reported that there was a positive correlation between the damaged kernels and the percentage of kernels infected by a. flavus of maize obtained by farmers and village traders in central lampung and kediri regencies. population of a. flavus increased at 1 and 2 months of storage, from 48.5 colonies/g to 586.8 and 890.7 colonies/g, respectively, because the m.c. of grain, temperature, relative humidity of storage and nutrition content of grams still supported the fungal growth. garraway and evans (1984) revealed that the growth and development of fungi was very much affected by nutrition contents and substrate. according to 35 the effects of drying and shelling on aspergillus flavus infection o.s. oharmaputra chatterjee et al. (1990) a. flavus growth was highly and linearly correlated with the decrease in the percentage of healthy germ and germinability, root-shoot growth, total carbohydrate and protein of grain. pomeranz (1992) reported that during storage deterioration of grain occurred, consequently nutrients were lost because of changes among others in carbohydrates. however, at 3 months of storage, population of a. flavus (481.1 colonies/g) decreased (table 4). it was assumed that m.c., relative humidity and nutrition contents decreased and there were competitive fungi to a. flavus. according to christensen (1955), mills and abramson (1982), one kernel can be infected by more than one fungal species. in this study, the predominant fungi in addition to a. flavus were acremonium strictum, fusarium moniliforme and penicillium citrinum. zummo and scott (1992) reported that f. moniliforme inhibited a. flavus infection and aflatoxin production on maize. table 4. the effect of maize variety, methods of drying and shelling, and duration of storage on population of aspergillus flavus numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level i = cobs of maize were sun dried up to 20% moisture content, and then shelled and re-dried up to 17% moisture content. ii = cobs of maize were sun dried up to 20% moisture content, and then shelled and re-dried up to 14% moisture content. iii = cobs of maize were sun dried up to 17% moisture content, and then shelled but not re-dried. iv = cobs of maize were sun dried up to 17% moisture content, and then shelled and re-dried up to 14% moisture content. 36 biotropia no. 10, 1997 the effect of maize variety, methods of drying and shelling, and storage duration on total aflatoxin b1 content according to heathcote and hibbert (1978) production of aflatoxin was affected by strain of fungi, relative humidity, temperature, oxygen, carbohydrate and kind of commodities. in this study, four kinds of aflatoxins were determined, i.e. aflatoxin b1 , b2, g1 and g2 based on statistical analysis, the effects of maize variety, drying, interaction between drying and shelling and interaction among maize variety, shelling and duration of storage gave significant differences to total aflatoxin b1 content; duration of storage gave very significant difference, while interaction among maize variety, drying, shelling and duration of storage did not give significant difference. total aflatoxin bi content of maize var. cpi-2 (35.97 ppb) was higher than that of var. arjuna (32.36 ppb) (table 5). this could be attributed to the effect of metabolites produced by maize. total aflatoxin bi content of maize dried up to 17% m.c. then shelled but not re-dried (30.34 ppb) was the lowest compared with the other methods of drying (drying methods i. ii and iv) (36.73, 34.79 and 34.60 ppb, respectively) (table 5), although the population of a. flavus of maize was the highest. according to diener and davis (1969) aflatoxin production depended on the strain of a. flavus. total aflatoxin bi content of maize shelled by nail-down wood (32.78 ppb) was lower and not significantly different with that shelled by mechanical sheller (35.45 ppb) (table 5). it was assumed that the high damaged kernels caused by shelling using mechanical sheller were more easily infected by a. flavus, consequently, aflatoxin producing strains had more chance to infect the kernels. total aflatoxin b1 content of maize increased with longer storage. at the beginning of storage, it was 0.49 ppb, while at 1, 2 and 3 months of storage they were 8.19, 50.83 and 76.95 ppb, respectively, because aflatoxin could not be easily decomposed, thus, it accumulated. melting points of aflatoxin b1 , b2, g1 and g2 were more than 230 c, therefore, they cannot be decomposed by ordinary heating (betina 1989). as a secondary metabolite, aflatoxin production was dependent on stadia of fungal growth, aflatoxin biosynthesis pathways and on factors affecting enzymatic reaction. according to davis and diener (1983) aflatoxin production is as follows: as fungal growth and primary metabolism proceed, little or no aflatoxin was formed initially. eventually, phospha te, nitrogen, or some trace elements become limited and primary growth is retarded. as primary metabolism becomes disorganized, various primary metabolites accumulate. metabolites such as pyruvate, malonate, acetate, various amino acids, etc. trigger the development and stimulate the activity of en 37 the effects of drying and shelling on aspergillus flavus infection o.s. dharmaputra table 5. the effect of maize variety, methods of drying and shelling, and duration of storage on total aflatoxin b1 content numbers followed by the same lener do not differ significantly according to duncan's multiple range tea at 95 % confidence level aflatoxin b1. b2. g1 and g2 contents were convened into total aflatoxin b1 i = cobs of maize were sun dried up to 20% moisture content, and then shelled and re-dried up to 17% moisture content. ii = cobs of maize were sun dried up to 20% moisture content, and then shelled and re-dried up to 14% moisture content. iii = cobs of maize were sun dried up to 17% moisture content, and then shelled but not re-dried. iv = cobs of maize were sun dried up to 17% moisture content, and then shelled and re-dried up to 14% moisture content. zymes of secondary metabolism, such as those of the polyketide biosynthetic pathway that in turn promote aflatoxin biosynthesis over fatty acid biosynthesis in toxigenic isolates of a. flavus. according to betina (1984) there were acetyl co-a and malonyl co-a in aflatoxin biosynthesis. there are active ingredients of acetate and malonate which compose lipid. aflatoxin arises naturally when a toxin-producing strain of a. flavus grows on a substrate where environmental conditions are suitable for the development of the fungus. in view of the fact that a. flavus was ubiquitous and capable of development over a wide range of temperature on substrates of high carbohydrate content (heathcote and hibbert 1978). 38 biotropia no. 10, 1997 conclusions 1. moisture contents of maize decreased at 1 month of storage, and then they were almost constant at 2 and 3 months of storage. 2. maize var. cpi-2 was more resistant than var. arjuna to aspergillus flavus infection, although the percentage of damaged kernels of maize var. cpi-2 was higher than those of var. arjuna. 3. in general, the best drying method was as follows: maize was sun dried up to 17% m.c., and then shelled and re-dried up to 14% m.c. 4. a. flavus population of maize shelled by mechanical sheller was insignificantly different with nail -down wood, although the percentage of damaged kernels of maize shelled by mechanical sheller was higher than that shelled by nail-down wood. 5. population of a. flavus increased at 1 and 2 months of storage, and then decreased at 3 months of storage. 6. total aflatoxin bi content increased with the increase of storage period. acknowledgment the authors gratefully acknowledge the community service and research development project, directorate general for higher education, department of education and culture, republic of indonesia for financial support. the authors also thank dr. m. sidik, assistant to the minister for food safety, ministry of food affairs; mrs. santi ambarwati and mr. sunjaya, scientists of tropical pest biology programme, seameo biotrop, for their valuable assistance. references betina. v. 1984. mycotoxins as secondary metabolites. in v. betina (ed.). mycotoxins, productions. isolations, separations, and purifications. elsevier science publisher b.v., amsterdam, p. 3-12. betina. v. 1989. mycotoxins. chemical, biological and environmental aspects. bioactive molecules volume 9. elsevier science publisher b.v., amsterdam. brooker. d.b., f.w. bakker-arkema, and c.w. hall. 1974. drying cereal grain. the avi publishing company, inc., wesport. bsi. 1980. methods of test for cereals and pulses. part 3. determination of moisture content of cereals and cereal products (routine method). british standards institution. isbn 0 058 114333. butler. w.h. 1974. aflatoxin. in purchase, i.f.h. (ed.). mycotoxins. elsevier scientific publishing company. amsterdam, p. 1-28. 39 the effects of drying and shelling on aspergillus ftavus infection o.s. dharmaputra chatterjee. d., b.k. chattopadhyay, and s.k. mukherje. 1990. storage deterioration of maize having preharvest infection with aspergillus flavus. appl. microbiol. 11: 11-14. christensen. c.m. 1955. grain storage studies. xviii. mold invasion of wheat stored for sixteen months at moisture content below 15 percent. cereal chem. 32: 107-116. christensen. c.m. and h.h. kauffmann. 1974. microflora. in chnstensen. c.m. storage of cereal grains and their products. american association of cereal chemists, p. 158-191. cole, r.j., r.a. hill. p.o. blankenship. t.h. sandera and k.h. garren. 1982. influence of irrigation and drought stress on invasion by aspergillus flavus of corn kernels and peanut pods. reprinted from developments in industrial microbiology. vol. 23. a publication of the society for industrial microbiology. covanich. a. 1991. the grain postharvest systems. proceedings of a regional training course, grain storage warehouse control technology, indonesia. 2-23 september 1991. p. 3-9. dharmaputra. o.s.. i. retnowati. sunjaya and s. ambarwati. 1993. aspergillus flavus population and aflatoxin content on maize collected from farmers and traders in lampung province. south sumatra. indonesia. proceedings of the 12th indonesian phytopathology society congress. yogyakarta, indonesia. 6-8 september 1993. p. 560-566. dharmaputra. o.s.. i. retnowati. h.k. purwadaria. h. susilo m. sidik and sunjaya. 1994. the effect of postharvest handling on grain intactness. fungal infection and mycotoxm production of maize. i. surveys on postharvest handling, damaged kernels, fungal infection and mycotoxm contamination on maize collected from farmers and traders. grant competition research report ii/l. seameo biotrop. diener. u.l.. and n.d. davis 1969. production of aflatoxin on peanuts under controlled environments. j. stored prod. res. 5: 251-258 davis. n.d. and u.l. diener. 1983. some characteristics of toxigemc and nontoxigenic isolates of aspergillus flavus and aspergillus parasiticus. in u.l. diener, r.l. asquith. and j.w. dickens (eds.). aflatoxin and aspergillus flavus in corn. department of research information. alabama agricultural experiment station, auburn university. alabama. southern cooperative series bulletin 279, february 1983. p. 16-19. garraway. m.o. and r.c. evans. 1984. fungal nutrition and physiology. john wiley and sons inc.. new york gilbert. j. 1991. regulatory aspects of mycotoxins in the european community and usa. in "fungi and mycotoxins in stored products". aciar proceedings no. 36. p. 194-197. hall, c.w. 1957. processing equipment for agricultural products. edward brother inc., michigan. heathcote. j.g. and j.r. hibbert. 1978. aflatoxins: chemical and biological aspects. elsevier scientific publishing company, amsterdam. henderson. s.m. and r.l. perry. 1976. agricultural process engineering. john wiley and sons inc., new york. idrc. 1988. studies on the improvement of postharvest system capabilities at village unit coopera tives (kud) and private sector, national logistics agency (bulog), bogor agricultural university (ipb) and gadjah mada university (ugm), indonesia. idrc file no. 3-p-85-0338. kawashima. k. and s. kawasugi. 1988. aspergillus flavus contamination in maize during postharvest process. paper presented at kyoto international conference hall, japan, august 20-27, 1988. lillehoj, e.b. and c.w. hesseltine. 1977. aflatoxin control during plant growth and harvest of corn. in rodricks, j.v., c.w. hesseltine and m.a. mehlmen. mycotoxins in human and animal health. pathotox publishers, inc., illinois, p. 107-119. 40 biotropia no. 10, 1997 mills. j.t. and d. abramson. 1982. ochratoxigenic potential of penicillium spp. isolated from stored rapeseed and cereals in western canada. can. j. plant pathoi. 4: 37-41. pitt. j.i. and a.d. hocking 1985. fungi and food spoilage. academic press. sydney. pomeranz. y. 1992. biochemical, functional, and nutritive changes during storage. in sauer, d.b. (ed.). storage of cereal grains and their products. american associations of cereal chemist inc., minnesota, p. 55-141. purwadaria. h.k. 1987. grain post harvest technology system in indonesia: maize. soybean and groundnut in north sumatra. south sumatra and lampung. consultancy report ministry of agriculture, indonesia-fao, undp (df/fao/ins/85/004). rodriquez. s.b. and n.e. mahoney 1994. inhibition of aflatoxin production by surfactants. applied and environmental microbiology 60(1): 106-110. sfcdp report. 1990. studies on postharvest handling on corn, soybean and cassava in lampung. east java and south sulawesi, moa-aid. indonesia. suprayitno. 1980. the effect of some shelling methods on the damaged kernels of maize. faculty of mechanization and agricultural technology, bogor agricultural university. bogor. indonesia (unpublished). van egmond. h.p. 1991. regulator) aspects of mycotoxins in asia and africa. in fungi and mycotoxins in stored products. aciar proceedings no. 36: 194-197. wilson d.m.. r.c. gueldner and r.a. hill 1983. effect of plant metabolites on the aspergillus flavus group and aflatoxin production. in diener, u.l., r.l. asquith and j.w. dickens (eds.). aflatoxin and aspergillus flavus in corn. southern cooperative series bulletin 279. p. 13-15. zummo n and g.e. scott. 1992. interaction of fusarium moniliforme and aspergillus flavus on kernel infection and aflatoxin contamination in maize ears. plant dis. 76: 771-773. 41 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf 41.pdf 4. dwi andreas santosa biotropia vol. 17 no. 2, 2010: 98 104 lipid producing microalgae from several ecosystems in west and central java, indonesia 1,3*) 2)dwi andreas santosa , and sulastri , 1) department of soil science and land resources, faculty of agriculture, bogor agricultural university, bogor, indonesia 2) indonesian center for biodiversity and biotechnology (icbb), bogor, indonesia 3) seameo-biotrop, bogor, indonesia received as dipa 2008 / accepted 17 july 2010 abstract this study is aimed to get lipid producing microalgae as feedstock for biofuel production. the microalgae were isolated from 355 collected water samples which represented many distinct ecosystems such as paddy fields, rivers, agricultural dams, ponds, swampy areas and unique ecosystem of volcano and mud-volcano craters in westand central java, indonesia. a total of 267 strains of microalgae were isolated from the samples of which 221 strains of them have capability to produce lipid. there were four promising strains that produce lipid between 14.7 45.7 percent dry weight in optimal condition that were identified as chlamydomonas sp. ko-7267 and pk-7195, chlorella sp. ks-7300 and desmodesmus sp. bk-7291. keywords: microalgae, lipid, biofuel, indonesian ecosystems introduction global oil production is rapidly approaching its peak, even if natural gas liquids and expensive, destructive, and risky deepwater and polar oil are included. based on several scenarios, the peak oil will happen sometime between 2010 2030 (robert 2005). peak oil means that half of oil reserves has been exploited and used. peak production of oil in indonesia has occurred in the 1990 (full report workbook 2004). in 2004, indonesia (formerly opec member) became a net importer. the lack of stability of future energy supplies has motivated the development of alternative energy sources in order to eliminate the possibility of a future energy shortage. furthermore, one of the most environmental problems today is global warming, caused primarily due to the heavy use of fossil fuel. in the world, large amount of co 2 * corresponding author : dsantosa@indo.net.id 98 are released into the atmosphere. photosynthetic microalgae are potential candidate for substitution of the fossil energy. some strains can produce lipid which can be converted into biodiesel, while others produce petroleum like hydrocarbon. the cultivation of algae is also important to reduce the effect of climate change through utilizing excessive amount of co , since cultivated these organisms are capable of 2 fixing co to produce energy and chemical compounds upon exposure to sunlight. 2 microalgae are photosynthetic renewable resources with high lipid content, have faster growth rate than plant cells and are capable to grow in saline waters which are unsuitable for agriculture. the lipid content of microalgae on a dry cellular weight basis varies between 20 and 40%. lipid content as high as 85% has been reported for certain microalgal strains. botryococcus braunii, is a unique microalgal strain, having a long hydrocarbon chain of between 30 and 40% (dry weight basis) which produces almost similar compounds as crude oil. both physical and chemical processes are applicable in the production of liquid fuels from algal strain of high lipid content. these processes include direct lipid extraction in the production of diesel oils substitutes, transesterification in the formation of ester fuels, and hydrogenation in the production of hydrocarbons (borowitzka 1988). microalgae contain lipids and fatty acids as membrane components, storage products, metabolites and sources of energy. algal fatty acids and oils have a range of potential applications. the characteristics of algal oils are similar to those of fish and vegetable oils, and can thus be considered as potential substitutes for the products of fossil oil (as a biodiesel) (miyamoto 1997). cultivation of microalgae in a large scale can be considered as potential substitutes for the petroleum-based diesel used in transportation. the study conducted by nerl (national renewable energy laboratory, us) found that 7.5 billion gallon of biodiesel from microalgae could be produced on 200,000 ha of desert land per year. this value equals to 214 million barrels or equivalent to 65% of total annual petroleum production in indonesia. this value could never be reached by cultivating other energy producing plants. indonesian microalgae as sources of biofuel production are almost unexplored. very few related reports and publications could be found in indonesia. the exploration of indonesian microalgae as a source of sustainable energy is therefore very promising. materials and methods samples collection samples were collected from rivers, lakes, paddy fields, ponds, dams, craters and saline waters in the south area of west and central java, and also some areas in the northern coast of coastal area of central java (fig. 1). those areas represent a large variety in soils, ph, temperature and salinity. a number of 355 samples were collected from 36 different sites (table 1). 99 lipid producing microalgae from several ecosystem dwi andreas santosa et al. figure 1. the sampling sites isolation and screening of microalgae the soil and fresh water samples were cultured in 616 medium (atcc) and mj medium (modified jorgensen's media for diatoms). the samples from the saline habitats were cultured in modified noro medium (takagi et al. 2005) containing (per litre): nacl 29.2g; kno 1g; mgcl h o 1.5g; mgso .7h o 0.5g; kcl 0.2g; cacl 0.2g; 3 2. 2 4 2 2 k hpo 0.045g; tris(hydroxymethyl)aminomethane 2.45g; edta.2na 1.8g; 2 4 znso47h2o 0.087 mg;h bo 0.61 mg; cocl .6h o 0.015mg; cuso .5h o 0.06 mg; 3 3 2 2 4 2 mncl 0.23mg; (nh ) mo o .4h o 0.38mg; fe(iii)edta 3.64 mg. the ph was 2 4 6 7 24 2 adjusted to 8.0 with 1n hcl. other culture media were used in order to obtain suitable media for their growth. the algae were subjected to purification by serial dilution followed by plating. the individual colonies were isolated and inoculated into liquid omedium and incubated at 27±2 c under 1.2±0.5 klux light intensity with 12:12 hrs photoperiods. the purity of culture was ensured by repeated plating and by regular observation under microscope. the isolates were characterized with respect to lipid and/or oil production capabilities. the lipid and oil producing microalgae were determined by using nile red (nr) staining (qin 2005). a stock solution of nr was prepared by adding 2.5 mg of nr to 100ml of acetone. the solution was kept in an amber-coloured bottle and stored in the dark at room temperature. the microalgae cell was stained by placing 5 ml of culture in a petridish, then added with 200µl of nr stock solution to the dish. staining of lipids and oil was completed within 30 minutes. no destaining or rinsing was required. after staining, a drop of stained cell culture was transferred to a depression slide and cover slip added. cell culture was examined at 1000x magnification using a phase contrast microscope and epiflourescence microscope. when viewed under epiflourescent illumination, neutral lipid and oil of microalgae stained with nr will show a bright yellow/orange color (carman1991; qin 2005). 100 biotropia vol. 17 no. 2, 2010 the growth of microalgal culture was determined daily based on the appearance of green or brown color on the medium. the cell density was measured with a spectrophotometer using 680 nm wavelengths (lee et al. 1998). the terminal optical density (od) will mark at 0.2, at this point the alga biomass is equivalent to 0.37g of dry weight per liter (qin 2005). the isolates were screened based on the period of time to reach od 0.2. the total lipid production of the selected strains was measured by gravimetry methods. results and discussions sampling sample collection was conducted in west and central java from april 10 until april 14, 2008. samples were taken from diverse ecosystems i.e. sampling in mountain ecosystem in west java (gunung gede, gunung tangkuban perahu), volcanic lakes (crater of tangkuban perahu, pre-historic crater of bledug kuwu), rivers, lakes, ponds, and rice fields in west and central java, as well as swampy areas in northern part of central java. sampling has also been conducted in two agricultural dams i.e. waduk sempor, kebumen and waduk kedung ombo, purwodadi. a number of 355 water samples have been collected from those areas. the samples and sampling location are shown in table 1. table 1. samples and sampling locations code of samples sampling locations characteristic cp7041-cp7050 ciputri, pacet, cianjur, west java water from paddy fields and ponds sk7051-sk7055 sukamantri, karangtengah, west java water from paddy fields cb7056-cb7060 ciburung, padalarang, west java water from dam tp7061-tp7080 tangkuban perahu mountain, west java water from crater, hot-sulfuric water jl7081-jl7085 jaya giri, lembang, west java water from fishponds pt7086-pt7090 highway kopo, west java water from paddy fields cg7091-cg7095 limbangan street, garut, west java water from paddy fields ct7095-ct7100 ciawi, tasik, west java water from paddy fields be7101-be7110 bojongsari, cijenjing, ciamis, west java water from fishponds mh7111-mh7120 kota banjar, west java water from river pd7121-pd7130 panulisan, dayeuh harja, cilacap, central java water from paddy fields cr7131-cr7140 ciraja, kt pucung, cilacap, central java water from paddy fields jl7141-jl7145 jatilawang, banyumas, central java water from paddy fields kj7146-kj7155 kr.anyar, jatilawang, banyumas, central java water from paddy fields tb7156-tb7165 purwodadi, tambak, banyumas, central java water from paddy fields smp7166-smp7190 waduk sempor, kebumen, central java water from agricultural dam pk7191-pk7195 pekunden, kota winangun, kebumen, central java water from paddy fields pr7196-pr7200 purworejo, diy water from paddy fields tpk7201-tpk7205 taji, prambanan, diy water from paddy fields mc7210-mc7225 metese, ceper, klaten, diy water from paddy fields 101 lipid producing microalgae from several ecosystem dwi andreas santosa et al. code of samples sampling locations characteristic pw7226-pw7235 pakis, wadung getas, delanggu, klaten, diy water from paddy fields sg7236-sg7245 siwali, gondangrejo, central java water from paddy fields ls7246-ls7255 ngadul, sumbu lawang, sragen, central java water from paddy fields ko7256-ko7275 waduk kedung ombo, purwodadi, central java water from agricultural dam mt7276-mt7280 mayahan, tawangharjo, purwodadi, central java water from paddy fields bk7281-bk7295 bledug kuwu, grobogan, central java water from crater ks7296-ks7300 kuwu, grobogan, central java water from paddy fields tb7301-tb7310 tambaksari, blora, central java water from paddy fields ksr7311-ksr7315 kemadu, sulang, rembang, central java water from paddy fields bkr7316-bkr7340 banyudono, kaliori, rembang, central java water from fishponds, salt producing ponds kpr7341-kpr7350 karangpandan, rembang, central java water from fishponds gr7531-gr7360 growong, juwana, pati, central java water from paddy fields tn7361-tn7370 teban, njekulo, kudus, central java water from paddy fields tkj7371-tkj7380 tanjang, karangjati, central java water from paddy fields yw7386-yw7390 jogoloyo, wono salam, demak water from paddy fields kd7391-kd7395 pangan, kandanghaur, indramayu water from paddy fields table 1. continued isolation and screening of algae microalgae were isolated from samples collected from rivers, lakes, brackish and freshwater ponds, paddy fields, wetland soils in west and central java. the method and suitable medium for the isolation of microalgae were developed during this research. the major source of microalgae isolation (155 samples) came from paddy fields. a total of 355 samples have been used for algae isolation and 267 isolates of microalgae were obtained. some samples produced colony of algae quite fast and the medium became green after 2-3 weeks, while other samples grew slowly or did not grow. two medium have been used i.e. modified jorgensen's medium and atcc 617 medium. there were no differences between the two medium in supporting the growth of algae. the algal growth depended on the origin sample. the isolates obtained from this research were deposited at laboratory of soil biotechnology of ipb and the duplicates were deposited at icbb-culture collection of micoorganisms. distribution of microalgae based on the origin samples it was shown that origin samples play important role on the success of algae isolation. samples can be grouped into 5 : 1) paddy field, 2) river, 3) ponds, 4) agricultural dam, and 5) crater. most samples were collected from the paddy fields. the percentage of samples from paddy fields producing colony of algae is shown in table 2. 102 biotropia vol. 17 no. 2, 2010 table 2. the percentage and distribution of samples from paddy field that produced algal growth sampling site % sampling site % sampling site % west java central java cianjur 70 cilacap 85 purwodadi 80 karangtengah 60 banyumas 65 grobogan 80 bandung 80 kebumen 45 blora 90 garut 80 purworejo 70 rembang 80 tasik 83 prambanan 80 pati 63 indramayu 60 klaten 70 kudus 100 karanganyar 100 karangjati 70 sragen 80 demak 90 the geographical locations of sampling showed no significant difference in algal growth. there were also no differences between samples collected from west java and central java. samples collected from four sites produced microalgae in the range of 90 100 percent i.e. karanganyar, blora, kudus and demak. microalgae are abundant and can be found not only in paddy fields but also in all of this project studied ecosystems, both man-made ecosystems such as fish ponds and agricultural dams as well as natural ecosystems. interestingly, microalgae can also be isolated from harsh environment such as tangkuban perahu crater with very low ph (1-2) as well as bledug kuwu, a mud volcano crater that contains high concentration of salt. more than 60 percent of the samples, except samples collected from padalarang dam, produced microalgae after incubated in an appropriate media (table 3). table 3. percent growth of microalgae from different ecosystems sampling sites % sampling sites % volcano crater, tangkuban perahu 68 salt producing ponds, rembang 87 mud-volcano crater, bledug kuwu 79 river, banjar 67 dam, padalarang 20 fish pond, lembang 80 dam, sempor 64 fish pond, ciamis 60 dam, kedung ombo 78 fish pond, rembang 90 lipid producing microalgae around 267 strains of algae could be isolated from the samples. screening on lipid production of microalgae cell showed that 79 percent of the strains (212 isolates) have capability to produce lipid. those strains are then selected for further study in order to obtain the best strain which has fast growth and high lipid production to be developed further as a source of biodiesel. screening had been conducted on those strains and four promising isolates were selected i.e. pk-7195 produced 25.5 percent lipid, bk7291 produced 14.7 percent lipid, ko-7267 produced 35.7 percent lipid, and ks7300 produced 45.7 percent lipid under optimum condition. pk-7195 was isolated from a paddy field sample of pekunden, kota winangun, kebumen, central java; bk-7291 was isolated from a sample of mud-volcano crater of bledug kuwu, grobogan, central java; ko-7267 was from kedung ombo dam, purwodadi, central java and ks-7300 from sample of paddy field in kuwu, grobogan, central 103 lipid producing microalgae from several ecosystem dwi andreas santosa et al. java. based on the morphological characters, isolate ko-7267 and pk-7195 are identified as chlamydomonas sp., isolates ks-7300 identified as chlorella sp., while bk7291 is desmodesmus sp. acknowledgment authors would like to thank seameo-biotrop for funding this research. thanks are also due to the dean of faculty of agriculture, bogor agricultural university for his valuable advices and help in contractual terms. asih karyati, juleha, salmah shahab, and hartatik are acknowledged for the laboratory works. references borowitzka m.a. 1988. in” micro-algal biotechnology”. eds. borowitzka, m. a. and borowitzka l. j., p. 257287. cambridge university press, cambridge. carman k.r., thistle d., ertman s.c. and m. foy. 1991. nile red as a probe for lipid-storage products in benthic copepods. marine ecology progress series, 74: 307-311. full report workbook. 2004. statistical review of world energy. full report workbook. lee s.j., yoon b.d. and oh, h.-m. 1998. rapid methods for determination of lipid from the green botryococcus braunii. biotechnology techniques, 12:553-556. ministry of state for population and environment. 1992. indonesian country study on biological diversity. cooperation between the united nations environment programme, the republic of indonesia, and the kingdom of norway. miyamoto k. 1997. renewable biological system for alternative sustainable energy production. fao agricultural services bulletin-128. takagi m., karseno and t. yoshida. 2005. effect of salt concentration on intracellular accumulation of lipids and triacyglyceride in marine microalgae dumaliella cell. journal of bioscience and bioengineering. 101(3) : 223-226. qin j. 2005. bio-hydrocarbon from algae: impact of temperature, light and salinity on algae growth. a report for the rural industries research and development corporation. australian government. robert p. 2005. the end of oil. on the age of a perilous new world. a mariner book, boston. 104 biotropia vol. 17 no. 2, 2010 page 1 page 2 page 3 page 4 page 5 page 6 page 7 microsoft word 20 biotropia no. 24, 2005 : 20 29 phytotoxicity and field efficacy of exserohilum longirostra jc/min the control of barnyardgrass ecotypes (echinochloa crus-galli var. crus-galli(l.) beauv) abdul shukor juraimi, arifin tasrif, jugah kadir*, suhaimi napis' and soetikno slamet sastroutomo2 *faculty of agriculture, universiti putra malaysia, 43400 serdang, malaysia ; 'faculty of biotechnology and science ofbiomolecular, 43400 serdang, malaysia cab international sea regional centre, 43400 serdang, malaysia; abstract five selected ecotypes of bamyardgrass (echinochloa crus-galli var. crus-gatti) from several rice growing areas in malaysia and indonesia were tested for their susceptibility to the potential bioherbicide (exserohilum longirostratum). bamyardgrass seedlings at the 2-3-lcaf stage were treated with 2.5xl07 conidia/ml from e. longirostratum at different application frequencies (single, double and triple). in addition, aqueous extract assays were evaluated for the presence of a phytotoxic compound responsible for the virulence of the bioherbicide. results of the study showed that disease severity significantly increased 20 days after treatment and resulted in mortality of the seedlings. ecotypes from perak and lampung were most susceptible to the bioherbicide upon triple applications. percentage dry weight reductions were 86.34 and 83.14%, respectively. other ecotypes (melaka, banten and south sulawesi) were observed to have a relatively similar response. moreover, aqueous extracts of e. longirostratum increased mortality up to 92.50% of bamyardgrass seedlings. these findings suggest that regular (double and triple) applications of e. longirostratum at a concentration of 2.5xl07 conidia/ml significantly increased mortality among bamyardgrass ecotypes. mortality of the seedlings was attributed to the presence of a secondary phytotoxic metabolite. key words : field efficacy / phytotoxicity / exserohilum longirostratum i echinochloa crus-galli var. crusgalli i ecotypes. introduction bamyardgrass (echinochloa crus-galli var. crus-galli (l.) beauv) is the most important weed occurring in rice (holm et al. 1977). although various management strategies are available for bamyardgrass control in rice (e.g., cultural measures, hand-weeding, mechanical control and herbicides), each has its limitations (matsunaka 1983). following some recent successes in using plant pathogens as biological agents to control weeds (charudattan 2000), the possibility of using the bioherbicide approach to control bamyardgrass in rice-based cropping systems was investigated in the philippines and japan (zhang and watson 1997; tsukamotoe? al. 1999). as *) correspondent to : jugah kadir, department of plant protection, faculty of agriculture, universiti putra malaysia, 43400 serdang, selangor, malaysia email : kadirj2000@yahoo.com 20 biotropia no. 24, 2005 such exserohilum monoceras has since been selected to be the potential biocontrol agent for echinochloa species (zhang et al. 1997; tzukamoto et al. 1999). in addition, high virulence of e. monoceras on barnyardgrass species was due to the production of a phytotoxic compound by this fungus (zhang and watson 2000). furthermore, another species, exserohilum longirostratum was shown to be a potential biocontrol agent of itchgrass (ahmad et al. 2002). the possibility of using exserohilum longirostratum as a mycoherbicide to control barnyardgrass was investigated under glasshouse conditions. this fungus has also been recorded as a potential biocontrol agent for grassy weeds (chandramohan and charudattan 2001) and is mainly host specific to grassy weeds, while broadleaf weeds and crops are highly resistant (kadir et al. 2003). previously, we reported that e. longirostratum suppressed the growth of barnyardgrass and significantly reduced above ground biomass when applied at ixlo7 conidia/ml under glasshouse conditions (tasrif et al. 2003). based on the glasshouse studies, we decided to examine the efficacy of this pathogen in field conditions and assess the possibility that a phytotoxin may be responsible for the virulence. thus, the objective of this study was to quantify the efficacy of exserohilum longirostratum in suppressing ecotypes of barnyardgrass at different frequencies of application and its phytotoxic effect on barnyardgrass seedlings. materials and methods seed sources and inoculum production the five selected ecotypes of barnyardgrass for field experiments were collected from malaysian and indonesian rice fields. the distance between sampling locations were within 5 to 10 km (table 1). table 1. selected ecotypes of barnyardgrass for field efficacy studies ecotype location region pk-04 perak malaysia m-01 melaka malaysia l-01 lampung indonesia b-04 banten indonesia ss-01 south sulawesi indonesia small pieces of mycelium from the stock culture were aseptically transferred to potato dextrose agar (pda) in petri dishes. each pda culture was sealed with parafilm and incubated at room temperature (approximately + 30°c) for 7 days. agar plugs with mycelium (6 mm diameter) from the margins of these young 21 phytotoxicity and field efficacy of exserohilum longirostratum abdul shukor juraimi et al. colonies were used as source of inoculum. v-8 juice (v-8 juice of 200 ml, agar 18 g and distilled water 1 liter) medium were used for inoculum production following tray-mass method. the inoculum of selected potential fungal pathogen was produced on v-8 juice agar added with 3.7 mg/1 of streptomycin sulfate and 2.5 mg/1 of choloramphenicol. the antibiotic was added to prevent bacterial contamination. approximately 50 ml of mycelia suspension were sprayed on the food trays (size 20 x 30 cm) containing about 300 ml of v-8 juice agar. then the inoculated trays were incubated at room temperature (25°c to 30°c) for 2 to 3 days under 12 hour light/12 hours dark cycle. conidia were collected from a 2-day-old culture by scraping the agar surface with sterile water and rubber spatula, and filtering conidial suspension through a single sterile cheesecloth (kadir and charudattan 2000). field trials field trials were conducted in field 2 of the faculty of agriculture, universiti putra malaysia. plastic buckets (20cm diameter and 30 cm height) were filled each with 15 kg soil (jawa series) from tanjong karang paddy fields. each bucket was planted with pre-germinated seeds of barnyardgrass ecotypes. basal fertilizer (45 kg n/ha, 40 kg p/ha and 30 kg k/ha) was applied before planting (pane and masshor 1997). one week after emergence, the 2-3-leaf stage seedlings were selected and thinned to 5 seedlings/bucket. an e. longirostratum concentration of 2.5xl07 conidia/ml as counted using haemocytometer was used with 0.05% tween 20 as wetting agent diluted in water. each treatment was sprayed with 100 ml conidial solution using hand sprayer. tween 20 has been used in field trials as an additive to spore suspension to improve plant kill (klein and auld 1995). disease assessment was determined at 5-day intervals for 20 days after spraying. disease assessment was done visually based on a disease severity scale as developed by kadir and charudattan (2000). the disease ratings were: 0: 0% infection, 1: 1 to 10% leaf area are damaged, 2: 11 to 20% leaf area are damaged, 3: 21 to 30% leaf area are damaged, 4: 31 to 40% leaf area are damaged, 5: 41 to 50% leaf area are damaged, 6: 51 to 60% leaf area are damaged, 7: 61 to 70% leaf area are damaged, 8: 71 to 80% leaf area are damaged, 9: 81 to 90% leaf area are damaged and 10: 91 to 100% leaf area are damaged (plant died). environmental conditions such as relative humidity, rainfall and temperature during the study were noted. this was a factorial experiment in a randomized complete block design. barnyardgrass ecotypes (pk-04, m-01, l-01, b-04 and ss01) were selected as factor one, while the frequency of applications (single, double and triple applications) was selected as the second factor. each treatment combination was repeated four times. all control plants were sprayed with water containing 0.05% tween 20. a completely randomized design was carried out to determine the phytotoxic effect. 22 biotropia no. 24, 2005 phytotoxic effect of aqueous extracts agar plugs (6 mm diameter) of the fungus taken from the edge of 7-day-old pda cultures (inoculum) were placed into 500-ml erlenmeyer flasks containing 100 ml of modified fries liquid medium (30.0 g of sucrose, 5.0 g of kh2po4, 0.5 g of ammonium tartrate, 1.0 g of mgso4, 0.1 g of nacl, 0.1 g of cacla, 0.5 g of casein hydrolysate, and 1.0 g of yeast extract in 1000 ml of distilled water) (zhang et al. 1996). flasks were then placed on rotary shakers at 100 "rpm" and flask containing only modified fries medium served as control. after a 2-week incubation, cultures collected from each flask were separately centrifuged at 6000 "rpm" for 10 min. the supernatant was filtered through cheesecloth, followed by two layers of whatman no.2 filter paper to obtain a cell-free culture filtrate. the 3 concentrations of the e. longirostratum aqueous extract used were full-strength (v/v) (250 ml/1) half-strength (125 ml/1) and quarter-strength (62.5 ml/1). dilutions were made with sterilized distilled water. the roots of the barnyardgrass seedlings at the 2-leaf growth stage were immersed in plastic containers containing 25 ml of the aqueous extract. observations were carried out 48 hours later. disease severity was assessed based on disease incidence (percentage of diseased plant) (kadir and charudattan 2000). necrosis and wilting indicated the presence of the phytotoxic compound in the aqueous extract. statistical analysis anova was used to test for the determination of significant differences among treatment combinations, while mean separation was resolved using duncan's new multiple range test at p < 0.05 (sas 2000) for both experiments. disease severity was determined according to area under disease progress curve (audpc) (campbell and madden 1990). the audpc is the disease intensity between two periods. the higher the audpc value, the more severe the disease is. dry weight above ground biomass reduction (percent control) was taken 20 days after spraying. disease assessments were carried out based on disease index scale (0: immune, 1-2: resistant, 3-4: tolerant, 5-7: susceptible and 8-10: highly susceptible) (chang and hwang 2003). results field efficacy studies means relative humidity (%), temperature (°c) and rainfall at the experimental sites were 94%, 24 to 33°c and 8 mm, respectively. location of the trials had no significant effect on growth of barnyardgrass ecotypes. the disease severity that was observed for all ecotypes tested as expressed by audpc ranged from 590 to 1118 units 20 days after spraying (table 2). the results 23   audpc-area under disease progress curve (campbell and madden 1990); column means followed by the same small letter and row means followed by the same capital letter arc not significantly different at the 5% level by duncan's new multiple range test (dnmrt). indicate that the audpc for ecotype of pk-04 (1118) and l-01(1083) were more severely affected by treatment with e. longirostratum conidia than ecotypes m-01(775), b-04 (883), and ss-01(773). it is interesting to note that the effects of application frequencies for pk-04 and l-01 ecotypes were similar (table 2). effects of application frequency on seedling response as shown by the audpc also were observed. ecotypes pk-04 and l-01 gave similar responses for all spray frequencies and were significantly different (p < 0.05) compared to other ecotypes. ecotypes m-01 and ss01 gave similar responses at triple application frequencies. ecotype of b-04 and ss-01 had similar response at double application frequencies. the effect of application frequency on dry weight reduction of above ground biomass for all treatments is presented in table 3. the range of dry weight reduction was 37.58 to 86.34%. ecotypes of pk-04 and l-01 yielded the highest dry weight reduction (86.34 and 83.14%) at triple applications and was significantly different compared to other ecotypes. the least dry weight reduction was recorded for the ss-01 ecotype at all three levels of applications. the single and double application frequencies that reduced ecotypes biomass by more than 50% were observed for the pk-04 and l-01 ecotypes. however, double and triple applications reduced dry weight biomass below 50% for ss-01 ecotypes. in general, ecotypes pk-04 and l-01 were found to be most susceptible to. e. longirostratum injury. at triple applications, pk-04 disease severity increased up to 15 days after spraying but leveled off afterwards. at a single application, the disease severity increased up to 10 days following applications then leveled off afterwards (figure 1). however, at triple applications the level of disease severity increased beyond values obtained for those receiving double and single applications. 24     phytotoxicity and field efficacy of exserohilum longirostratum — abdul shukor juraimi et al. phytotoxic effect of aqueous extracts three aqueous extract concentrations at full, half, and quarter-strength prepared from the fungus produced necrotic, chlorosis and wilting in e. crus-galli seedlings within 48 hours after immersion. at full strength, the solution reduced living leaf area by a disease incidence of 92.5% as compared to controls and was significantly different compared to the other two treatments. half strength and quarter strength aqueous extracts caused a disease incidence of 85.0% and 57.5%, respectively (table 4). the results indicated that higher aqueous extract concentration increased seedling mortality. discussions field efficacy the virulence of the fungus e. longirostratum (upm isolate) was evaluated on 5 barnyardgrass ecotypes from different geographic locations under field conditions. among the ecotypes studied, pk-04 and l-01 were most susceptible to e. longirostratum toxicity compared to ecotypes m-01, b04 and ss-01. in this study, varied responses among barnyardgrass ecotypes in disease severity and dry weight reduction were obtained following single, double, and triple applications of e. longirostratum conidia. the conidial isolate of e. longirostratum was pathogenic to all ecotypes of barnyardgrass. the response ranged from highly susceptible (pk-04 and l-01 ecotypes) and moderately susceptible (m-01, b-04, and ss-01 ecotypes) was recorded in our field experiments. similar variability in pathogenic response among different ecotypes has also been reported for other plants. leisner and howel (1992) reported variation in symptoms from highly susceptible to resistant among 18 ecotypes of arabidopsis thaliana infected by cauliflower mosaic virus (cmv). in another study, chang and hwang (2003) reported significant differences in level of disease response among 6 adlay cultivars to fungal bipolaris coicis from different locations in south korea. 26 biotropia no. 24, 2005 e. longirostratum reduced growth and dry weight of barnyardgrass ecotypes pk-04, l-01 and b-04. however, efficacy of this fungus to control barnyardgrass depends on conidial concentration, frequency of application, and the genetic variability of barnyardgrass ecotypes. several publications on other fungal bioherbicides suggest an inoculum concentration of 106 conidia/ml to be the effective concentration in their studies (wymore and watson 1989; kadir et al. 2000). rosskopt et a/.(1999) also observed that the optimal level of control over amaranthus species by phomopsis amaranticola occurred at 6xl07 conidia/ml. accordingly, we agreed on an inoculum density of 2.5xl07 conidia/ml for e. longirostratum for our studies. regular application of certain bioherbicides has been reported to provide long-term control of the target weed. for example, kadir and charudattan (2000) reported that dactylaria higginsi provided excellent long-term control of purple nutsedge (cyperus rotundus l.) when it was applied regularly. in this study, regular application of e. longirostratum significantly controlled barnyardgrass. however, the severity of the disease caused by this fungus for barnyardgrass ecotypes in the field did not always correlate well with the aggressiveness observed in glasshouse studies. this may suggest that other factors such as increased inoculum concentration, improved formulation and barnyardgrass density influence disease severity. phytotoxic effect of aqueous extracts e. longirostratum causes leaf blight in echinochloa crus-galli ecotypes and is presently being evaluated as a potential mycoherbicide for the control of barnyardgrass. inoculation with aqueous extracts from e. longirostratum resulted in a blight-like reaction characterized by chlorosis appearing 12 hours after immersion. this was followed by rapid necrosis of the affected leaf tissue, often with an absence or weak expression of typical lesions (tasrif et al. 2003). the symptoms induced by e. longirostratum appear to be due to the presence of a phytotoxic compound. zhang and watson (2000) characterized the phytotoxic compound of e. monoceras as toxin-1, shown to be a potent and host-specific principle responsible to disease expression in echinochloa species. also, the toxin tryptophol appear to be a major metabolite in culture filtrates of drecshlera nodulosa, a pathogen of goosegrass (eleusine indica (l.) gaerth.) reported by sugawara and strobel (1987). microscopic observation following treatments with the full and half-strength aqueous extracts caused plasma-membrane breakdown in leaf tissue of the highly susceptible ecotype pk04. these results would support the presence of a phytotoxin in the aqueous extracts, that may have affected plasma membrane integrity of susceptible ecotypes in the infection process of the fungus e. longirostratum. ismail et al. (1993) have reported similar results from full-strength of aqueous extracts of legume cover crops that decreased germination and seedling growth of asystasia intrusa and paspalum conjugatum weeds attributed to an allelopathic principle. 27 phytotoxicity and field efficacy of exserohilum longirostratum abdul shukor juraimi et al. in this study, our results demonstrate that e. longirostratum at a concentration of 2.5xl07 conidia/ml effectively increased the mortality of barnyardgrass ecotypes at the 2-3-leaf stage. conclusions the fungal pathogen e. longirostratum seems to be a potential candidate as mycoherbicide on barnyardgrass. phytotoxic compound of this fungus might also enhance virulence on barnyardgrass. however, for satisfactory control on barnyardgrass, further studies need to be addressed on the increase in conidial concentration, use of different surfactant, formulation, and time of application. acknowledgement our appreciation goes to the integrated pest management for small-estate crop project (ipm-schep), ministry of agriculture in indonesia and felda foundation of malaysia who had funded this study. we also thank ms. christina stephensons for her valuable contribution in correcting the manuscript. references ahmad, a., j. kadir, m. sariah, a.s. juraimi, 2002. evaluation for drechslera sp. for potential bioherbicidc control of itchgrass (rottboellia cochinchinesis; poaccac). screening and host range test. proceedings third international conference on biopesticidcs, ed. e.m. mulla, the department of entomology, university of california, riverside, usa. p. 181 -184. campbell, c.l. and l.v. madden, 1990. introduction to plant disease epidemiology. john wilcy & son. 532 p. chandramohan, s. and r. charudattan, 2001. control of seven grasses with a mixture of three fungal pathogens with restricted host range. biological control, 22: 246-255. chang, s.w. and b.k. hwang, 2003. evaluation of virulence to adlays of korean isolates of bipolaris colds using a disease rating scale. plant disease, 726-731. charudattan, r. 2000. current status of biological control of weeds: in emerging technologies for integrated pest management. concepts, research and implementation, cds c.g. kennedy and t.b. sutton, st. paul minnesotta, aps press, p.269-288. holm, l.g., d.l. plucknett, j.v. pancho and j.p. herberger, 1977. the world's worst weeds. distribution and biology. hawaii, honolulu. ismail, b. s., a. tasrif, sastroutomo, s.s. and a. latif, 1993. allelopathic potential of legume cover crops on selected weed species. plant protection quarterly, 86: 81-87. kadir, j. b. and r. charudattan, 2000. dactylaria higginsi, a fungal bioherbicide agent for purple nutsedge (cyperus rotundas), biological control, 17: 113-124. 28 biotropia no. 24, 2005 kadir, j., r. charudattan and r.d. berger, 2000. effects of some epidemiological factors on levels of disease caused by dactylaria higginsi on cyperus rotundus. weed science, 48:61-68. kadir, j., a. ahmad, m. sariah and a.s. juraimi, 2003. potential of drechslera longirostrata as bioherbicide for itchgrass (rottboellia cochinchinensis). proceedings of the nineteenth asian pacific weed science society conference. weed science society of the philippines, manila, philippines, p 450-455. klein, t. a. and b.a. auld, 1995. evolution of twccn 20 and glycerol as additives to mycoherbicides suspensions applied to bath rust burr. plant protection quarterly, 1: 14-16. lcisner, s.m. s.h. howell, 1992. symptoms variation in different arabidopsis thaliana ecotypes produced by cauliflower mosaic virus. phytopathology, 82: 1042-1045. matsunaka, s. 1983. evolution of rice weeds control practices and research: world perspective in weed control in rice. international rice research institute. manila, philippines, p 5-17. pane, h. and m. masshor, 1997. fenoxaprop-p-ethyl is an effective herbicide in controlling red sprangletop (leptochloa chinensis l. ness). proceedings 16lb asian pacific weed science society conference, -malaysian plant protection society, ed. rajan, a. kuala lumpur, malaysia.p.272-277. rosskofpf, e.n., r. charudattan and j.b. kadir, 1999. use of plant pathogens in weed control in handbook of biological control, eds. t.s. bellows, t.w. fisher, l.e. caltagirone, d.l. dahlsten, g. gordt and c.b. huffaker, san diego, san fransisco, new york, boston, sydney, tokyo. academic press, p.891-919. (sas) statistical analysis systems, 2000. sas procedures guide version 8.0 3rd cory, nc: statistical analysis systems. sugawara, f. and g.a. strobel, 1987. tryptophol, a phytotoxin produced by drechslera nodulosum. phytochemistry 26: 1349-1351. tasrif, a., a.s. juraimi, j. kadir, s. napis and s.s. sastroutomo, 2003. mode of infection of exserohilum longirostratum on barnyardgrass (e. crus-galli vat. crus-galli). 11* national electron microscopy conference, langkawi 15-17 december 2003. p. 522-528. tsukamoto, h., m. tsuda, t. fujimori, 1999. mode of infection of echinochloa oryzicola by exserohilum monoceras. phytopathology sos. japan, 65: 553-556. wymore, l. a. and a.k. watson, 1989. interaction between velvet leaf isolate of colletotrichum cocodes and thidiazuron for velvet leaf (abutilon theophrasti) in the field. weed science 37: 478-483. zhang, w.m., k. moody and a.k. watson, 1996. responses of echinochloa species and rice (oryza saliva) to indigenous pathogenic fungi. plant disease 80: 1053-1058. zhang, w.m. and a.k watson, 1997. host range of exserohilum monoceras, a potential bioherbicide for the control of echinochloa species. canadian journal botany, 75: 685-692. zhang, w.m. and a.k . watson, 2000. isolation and partial characterization of phytotoxin produced by exserohilum monoceras, a potential bioherbicide for control of echinochloa species. proceedings of the tenth international symposium on biological control of weeds, ed. n.r. spencer, montana, usa. p.125-130. 29 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf biotropia no biotropia no. 12, 1999 : 59 77 the effectiveness of some ectomycorrhizal fungi in alginate beads in promoting the growth of several dipterocarp seedlings supriyanto seameo b1otrop, p.o box 116, bogor 16001, indonesia abstract the effectiveness of six species of ectomycorrhizal fungi (scleroderma columnare, s.dictyosporum, laccaria toccata, rhizopogon luteolus, amanita umbronata and descomyces sp.) in alginate beads in promoting the growth of four species of dipterocarp seedlings (shorea pinanga, s.leprosula, s.ovalis and hopea odorata) were studied. different sodium alginate concentrations of 5 g, 10 g, 15 g and 20 g/l were tested to find out the best bead's elasticity and spore germination. seedling height, relative field mycorrhizal dependency, percentage of mycorrhizal colonization, nutrient absorption, and the formation of hartig's net and mantle structure of dipterocarp seedlings were observed 4 months after inoculation. the best elasticity of alginate beads was found in the concentration of sodium alginate of 15 g/l. the best growth increment was found in hopea odorata inoculated with amanita umbronata (126.60 %) followed by shorea pinanga inoculated with descomyces sp. (27.10%), shorea ovalis inoculated with amanita umbronata (26.90 %) and shorea leprosula inoculated with descomyces sp. (24.20 %) over the control. the highest relative field mycorrhizal dependency was found in hopea odorata followed by shorea ovalis, s. pinanga and s. leprosula. the highest mycorrhizal colonization was obtained in shorea pinanga inoculated with descomyces sp. (75%), while inoculation with amanita umbronata on s.leprosula, s.ovalis and hopea odorata increased mycorrhizal colonization i.e. 64.5 %, 52.5%, and 46.2 %, respectively. hartig's net and mantle structures were well formed in shorea leprosula as well as s.ovalis seedlings with all mycorrhizal fungi tested, while in s.pinanga seedlings these structures were only well formed with descomyces sp. there is no clear difference in p levels in the leaves following inoculation as compared to the controls. key words: mycorrhizas/alginate beads/scleroderma columnare/scleroderma dictyosporumllaccaria laccatalrhizopogon luteoluslamanita umbronataldescomyces sp./growth/dipterocarpaceae/seedlings. introduction background industrial forest plantation (iff) is one of the government programs to improve the reduced forest productivity due to logging activities (logged-over areas) and deforestation (critical lands). during the period of the fourth to sixth five-year development plan of the government of indonesia, the area of ifp was projected to be 4.6 million hectares (departemen kehutanan 1994). high quality seedlings to replant these areas are needed. dipterocarp species make up one of the recommended species for sawn-timber production. dipterocarp is an important timber species in southeast asian countries (prosea 1994). seventy five percent of indonesian 59 biotropia no. 12, 1999 wood trading comes from dipterocarp species. currently, the rate of logging is higher than the rate of replanting. there are some problems in planting dipterocarp species. among these are the irregular flowering season, recalcitrant seeds, and the need of mycorrhizae for promoting their growth during the seedling stage. mycorrhizal research in indonesia has been promoted by the government, because of its beneficial contribution to increase the quality of seedlings via growth acceleration (marx 1973; supriyanto et al. 1992), to control root pathogenic microorganisms (marx 1973), to improve soil structure (de la cruz 1982), to improve rooting system due to hormone production (gay and debaud 1987), to increase nutrient and water uptake (boyle et al. 1987), to increase drought resistance (boyle et al. 1987; supriyanto et al. 1992), and to increase the survival rate and accelerate the xylem formation of seedlings obtained by tissue culture techniques (supriyanto 1989; and chang 1993). inoculum improvements of the potential ectomycorrhizal fungi have been achieved in some species by single spore culture, cell fusion (mating type) and protoplast culture. these techniques can be used for mass production of vegetative inoculum (fortin et al. 1988). the inoculum can be packed in several ways such as tablet, capsule, spore powder or mycelium suspension (supriyanto et al. 1992) and in alginate beads (supriyanto et al. 1994; supriyanto 1996). there are some advantages and disadvantages for each type of packaging system. alginate beads are simple, easily stored, free from climatic influences, inexpensive and are available at any time. recent technological improvements of the vegetative inoculum production promote the use of the inoculum packaging system. it is important to evaluate the effectiveness of selected tropical mycorrhizal fungi packed in alginate beads to promote the growth of dipterocarp species recommended for the industrial forest plantation program. objective the objective of the research was to compare the effectiveness of alginate inoculum of six species of ectomycorrhizal fungi on the growth of four species of dipterocarp seedlings. materials and methods seed germination dipterocarp seeds (s.pinanga, s.leprosula, s.ovalis, h.odoratd) were collected from dipterocarp experimental areas, jasinga, forest research institute and were dipped in dithane m-45 at 1% concentration for 10 minutes to eliminate the pathogenic fungi. seeds were germinated in sterilized sand medium. germination and transplanting media were autoclaved at 1.5 bar, 120°c for 30 minutes. dipterocarp seedlings, two weeks old, were transplanted into poly-bags containing sterilized sand: soil: compost (1:1:1). 60 the effectiveness of some ectomycorrhizal fungi in alginate beads supriyanto aiginate bead elasticity ectomycorrhizal fungi of scleroderma columnare, laccaria laccata, amanita umbronata, descomyces sp. were collected from dipterocarp plantation. scleroderma dictyosporum was collected from pinus merkusii plantation, while rhizopogon luteolus was collected from eucalyptus urophylla plantation. the spores of ectomycorrhizal fungi were packed in alginate beads based on le tacon et al. method (1985) modified by supriyanto et al. (1994). the elasticity tests of alginate beads were done in different concentrations of sodium alginate (5, 10, 15, 20 g/l). an amount of 2 g of ectomycorrhizal fungi spores were extracted from mature fruit bodies and diluted in each different concentration of sodium alginate (an algal extract). spore suspension was trapped in sodium alginate to form beads in 0.7m calcium chlorite solution. the alginate bead elasticity was classified into very soft, soft, elastic and hard by pressing the beads in between two fingers. to evaluate the spore viability in different concentrations of sodium alginate, 10 beads were placed in a petri dish containing modified melin-nokran medium. five replicates were used for the evaluation of each sodium alginate concentration. germination percentage of the spores in alginate beads was recorded. inoculation technique three-week old dipterocarp seedlings (one week after transplanting) were then inoculated with 5 alginate beads at a concentration of 15g/l sodium alginate. a pipette was used to aspire the alginate beads. the alginate beads were then injected into transplanting media close to the rooting system (at a depth of 2 cm). eight replicates were used for each treatment. parameters mycorrhizal seedlings were evaluated 4 months after inoculation on the basis of several parameters to determine the effectiveness of selected mycorrhizal fungi as follows: hthe high growth increment (hgi, %) = ————— x 100 %, where: he ht = height of mycorrhizal seedlings, he = height of non-mycorrhizal seedlings (control) relative field mycorrhizal dependency / rfmd (plenchette et al. 1983 in bagyaraj 1994). dwt-dwc rfmd (%) = ———————— x 100 %, where : dwc 61 blotropla no. 12,1999 dwt = dry weight of inoculated seedlings (treatment), dwc : dry weight of noninoculated seedlings (control) seedlings were dried in an oven at 70°c for 48 hours and their dry weights were determined. the leaf dry weights were separated for nutrient content analysis. number of colonized roots percentage of colonization (fortinef a/. 1982)= ——————————————xloo% total number of root samples the colonized roots were determined by the presence of hyphae enveloping the roots (external hyphae). structural analysis of mycorrhizal and non-mycorrhizal roots was done to observe the presence of mantle and hartig's net formation based on the sass microtechniques method (1958) in berlyn and miksche (1976). toluedine blue 0 of 0.1% was used for root staining. toluedine blue 0 stains specifically the mycorrhizal hyphae either mantle or hartig's net in blue pinkish color. the presence of mantle (m), hartig's net (hn) or none in the rooting system were used to determine the compatibility of inoculated mycorrhizal fungi and host plant. it was classified into semi-compatible (mantle only), compatible (hartig's net and mantle) or incompatible (no mantle and no hartig's net). good formation of mantle and hartig's net will influence the effectiveness of mycorrhizal fungi in nutrient and water absorption. the criteria of mantle formation are as follows: (negative) for no mantle formation, + (positive) for one layer or poor formation, ++ for two layers or good formation and +hfor three layers or best formation. the criteria of hartig's net formation are as follows: (negative) for no hartig's-net formation, + (positive) for one series or poor formation, ++ for two series or good formation, +++ for three series or best formation. leaf content of n, p, k, ca, fe, mg were used as a nutrient parameter. all of the seedlings were dried at 70 ° c for 48 hours for nutrient analysis. dried leaves were powdered and 250 mg leaf powder was digested with concentrated h2so4 and 30% h2o2. the leaf nutrient content was analyzed as follows: nitrogen nitrogen content was analyzed using the titration method. to 20 ml of filtered solution in the nitrogen distillate apparatus was added 15 ml of 30% naoh. nitrogen distillation was done for 10 minutes. an amount of 20 ml of 1% boric acid added to the distillate solution were combined with 3 drops of bcrm (brom cresol green and red methyl) indicator. titration of the ammonium solution was done using 0.05n h2so4. 62 the effectiveness of some ectomycorrhizal fungi in alginate beads supriyanlo phosphate phosphate content was determined spectrophotometrically at x 693 nm. phosphate solutions of 0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0 ppm were used as standards from which the phosphate content was determined. iron the wet destruction method of powdered leaf was done using hno3 + hc1o4 + hjsc^. the iron was analyzed spectrophotometrically at a. 508 nm. an amount of 0.25 ml of metal reduction solution was added to the destructed solution thoroughly to homogenize the solution. an amount of 0.5 ml of orthophenontroline and 7 ml of sodium citrate were combined. standardized iron solutions of 0, 1.0, 2.0, 4.0, 6.0 and 8.0 ppm using (nh4)2fe(so4)2 . 6 h2o were used. potassium, calcium and magnesium atomic absorption spectrophotometry was used to determine the potassium, calcium and magnesium content. standardized solutions of potassium and calcium each of 0, 2.5, 5.0, 10.0, 15.0, 20.0 and 25 ppm were prepared and used to determine potassium and calcium content. to determine magnesium content, standardized solutions of 5 ml magnesium were made from mgso4 .7 h2o, i.e. 0, 2.5, 5.0 ppm to which 5 ml lacl3 containing 4000 ppm la were added. r e s u l t s alginate bead elasticity the spore suspension was trapped using sodium alginate and formed to beads in calcium chloride solution. bead elasticity depended on the concentration of sodium alginate used. the best concentration is determined by the criteria for bead elasticity and germination percentage of trapped spores. this was 15g/l sodium alginate with the average spore germination percentage of 77% (table 1). the concentration of sodium alginate of 20 g/l produced harder beads but the germination percentage of spores trapped in alginate beads decreased. concentrations of sodium alginate lower than 15 g/l produced soft or very soft beads and can easily be dried by airflow even when its spore germination was higher. the concentration of sodium alginate at 15 g/l will be used for inoculum packaging system at seameo biotrop. this packaging system is easily handled, with high productivity and high inoculum viability. although le tacon et al. (1985) used the sodium alginate at the concentration of 10 g/l, vegetative mycelia were used instead of spores. 63 biotropia no. 12, 1999 table 1. degree of bead elasticity and germination percentage of scleroderma columnare ectomycorrhizal fungi at different concentrations of sodium alginate. no. 5g/ls.a. vs %g log/l s.a s %g 15 g/l s.a. els % g 20 g/l s.a. h %g 1 2 3 4 5 vs vs vs vs vs 70 85 80 75 8 0 s s s s s 75 75 80 90 80 els els els els els 80 75 80 75 75 h h h h h 60 65 60 50 60 average vs 78 s 80 els 77 h 59 note: vs: very soft, s: soft, els: elastic, h: harder, % c: germination percentage, s.a.: sodium alginate. high growth increment (hgi) the percentage of high growth increment (hgi) of mycorrhizal seedlings over the control (not inoculated) is presented in table 2. the best hgi was obtained at hopea odorata seedlings inoculated with amanita umbronata (126.60%). all of the mycorrhizal fungi stimulated the growth of hopea odorata seedlings, ranging from 40.6 % to 126.60 % and the growth of shorea pinanga ranging from 5.70 % to 27.10 %. the hgi of shorea pinanga and s. leprosula was promoted by inoculation with descomyces sp., whereas the hgi of shorea ovalis and hopea odorata was promoted best by amanita umbronata. scleroderma columnare, rhizopogon luteolus and laccaria laccata were not able to stimulate the hgi of shorea leprosula, and laccaria laccata was not able to stimulate the hgi of shorea ovalis. scleroderma dictyosporum consistently stimulated the hgi of four dipterocarp seedlings tested, ranging from 10.30 % to 75.00 %. table 2. percentage of high growth increment over the control of 4-month old dipterocarp seedlings inoculated with different species of ectomycorrhizal fungi. species of ectomycorrhizal fungidipterocarp species sd sc r ll am de shorea pinanga 10.30 5.70 12.90 13.60 15.50 27.10 shorea leprosula 11.10 -3.00 -13.70 -18.00 23.80 24.20 shorea ovalis 18.30 11.60 14.10 -10.80 26.90 24.10 hopea odorata 75.00 54.70 49.60 40.60 126.60 84.40 note : sd: scleroderma dictyosporum, sc: scleroderma columnare, r: rhizopogon luteolus, ll: laccaria laccata, am: amanita umbronata, de: descomyces sp. the growth response of each dipterocarp seedling inoculated with different mycorrhizal fungi can be seen in figure 1 (s. pinanga), figures 4 and 6 (s. leprosula). 64 the effectiveness of some ectomycorrhizal fungi in alginate beads supriyanto figure 1: shorea pinanga seeds (a, x 0.1), alginate beads (b, x 0.18), mycorrhizal seedlings (sc, sd, r, ll, am, de) and nonmycorrhizal seedlings (c) (c, x 0.06) and the colonization of scleroderma columnare mycorrhizal fungi at the rooting system of shorea pinanga seedling (d, x 0.2). relative field mycorrhizal dependency (rfmd) the influence of mycorrhizal inoculation can also be determined from their relative field mycorrhizal dependency (rfmd). rfmd is the dependency of a plant species on the mycorrhizal fungi at a given soil fertility. rfmd is determined on the dry weight of mycorrhizal seedlings. it is important because dry weights may reflect the efficiency of physiological processes due to the mycorrhizal inoculation and photosynthetic activity. regarding the rfmd value (table 3), hopea odorata was very dependent on the inoculated mycorrhizal fungi, ranging from 82.45% to 416.10%. shorea pinanga was dependent on scleroderma dictyosporum (17.87%), rhizopogon luteolus (8.6%), laccaria laccata (27.82%), amanita umbronata (16.82%) and descomyces sp. (34.89%). shorea leprosula was dependent on descomyces sp. (34.32%) and on amanita umbronata (27.86%), while shorea ovalis was dependent on all of 65 biotropia no. 12, 1999 inoculated mycorrhizal fungi ranging from 32.10 % to 88.20 %. since some values are negative, it is possible that in this stage of colonization, mycorrhizal fungi consumed a high amount of carbohydrate for their energy and development. generally, those carbohydrates are translocated for plant organ development. table 3. percent relative field mycorrhizal dependency over the controls (not inoculated) of 4-month old dipterocarp seedlings inoculated with different species of ectomycorrhizal fungi. ectomycorrhizal fungidipterocarp species sd sc r ll am de shorea pinanga shorea leprosula shorea ovalis hopea odorata 17.87 -25.10 43.50 394.20 -12.28 -15.30 32.10 221.10 8.60 -33.90 32.50 128.10 27.82 -225.30 7 3.20 82.45 16.82 27.86 88.20 416.10 34.89 34.32 63.20 321.40 note : sd : scleroderma dictyosporum, sc: scleroderma columnare, r: rhizopogon luteolus, ll: laccaria laccata, am: amanita umbronata, de: descomyces sp. percentage of mycorrhizal colonization percentage of mycorrhizal colonization is defined as the ability of inoculated mycorrhizal fungi to form a mantle sheet and hartig's net in the rooting system of certain host-plants. the presence of the mantle sheet can be observed by macroscopic or microscopic observation (external hyphae), while the presence of hartig's net can be observed by a microscope (internal hyphae). the presence of mantle and hartig's net in the rooting system explains the compatibility status between host plant and inoculated mycorrhizal fungi. technically, the percentage of mycorrhizal colonization is obtained from the hyphal extension that can be observed visually, while the hartig's net structure must be observed using microscopic magnification. the extent of mycorrhizal colonization of inoculated mycorrhizal fungi on four species of dipterocarp seedlings is shown in table 4. the extent of mycorrhizal colonization is classified in five categories: excellent (75 100%), good (50 74%), moderate (24 40%), poor. (1 24%) (marx and bryan 1969). based on this classification, the colonization by scleroderma dictyosporum, s. columnare, rhizopogon luteolus, laccaria laccata, amanita umbronata and descomyces sp. are table 4. percentage of mycorrhizal colonization in the root system of 4-month old seedlings of shorea pinanga, shorea leprosula, shorea ovalis and hopea odorata. mycorrhizal colonization for each mycorrhizal fungus dipterocarp seedlings sd. sc. r ll am de shorea pinanga shorea leprosula shorea ovalis hopea odorata 51.87 63.00 43.10 37.50 ,54.37 41.50 49.40 45.00 64.37 39.00 50.00 46.20 66.26 42.50 52.70 30.70 67.50 64.50 52.50 46.20 75.00 . 55.50 44.00 37.50 note : c: control, sd : scleroderma dictyopsorum, sc: scleroderma columnare, r: rhizopogon luteolus, ll: laccaria laccata, am: amanita umbronata, de: descomyces sp. 66 the effectiveness of some ectomycorrhizal fungi in alginate beads supriyanto moderate and good (30.70% to 75.00 %). excellent mycorrhizal colonization was found in shorea pinanga inoculated with descomyces sp. the ability of scleroderma dictyopsorum, s. columnare, rhizopogon luteolus, laccaha laccata, amanita umbronata and descomyces sp. to form the hartig's net and mantle structure is shown in table 5. mycorrhizal colonization in shorea pinanga and s. leprosula seedlings were generally classified in term of good and best formation, while s. ovalis and hopea odorata were classified as poor. table 5. the presence of mantle and hartig's net in the root system of mycorrhizal and non-mycorrhizal seedlings of 4-month old seedlings of shorea pinanga, s. leprosula, s. ovalis and hopea odorata. note: : no formation, + : poor formation, -h-: good formation, +++ : best formation. c: control, sd : scleroderma dictyosporum, sc: scleroderma columnare, r: rhizopogon luteolus, ll: laccaria laccata, am: amanita umbronata, de: descomyces sp. the extent of mycorrhizal colonization of the inoculated mycorrhizal fungi in dipterocarp seedlings is shown in figure 2 (s. pinanga), figures 5 and 6 (s. leprosula), figure 7a, 7b (s. ovalis). the transversal section showing the mantle and hartig's net formation in each host plant is shown in figure 3 (s. pinanga), figure 8 (s. ovalis). leaf nutrient content nutrient concentration in the leaves of s.pinanga, s. leprosula, s. ovalis and h. odorata is presented in table 6. effect of mycorrhizal inoculation on the leaf nutrient content was obtained by comparing the nutrient content of mycorrhizal and non-mycorrhizal seedlings. table 6 shows that the increment of leaf nutrient content varied depending on the host plant species and the inoculated mycorrhizal fungi. 67 biotropia no. 12, 1999 figure 2: non-mycorrhizal and mycorrhizal root system of shorea pinanga. a: non-mycorrhizal root system (x 10), b: root system inoculated with s.columnare (x4), c: root system inoculated with s.dictyosporum (x 5), d: root system inoculated with l laccata (x 5), e: root system inoculated with a. .umbronata (x 5), f: root system inoculated with descomyces sp. (x 5). 68 the effectiveness of some ectomycorrhizal fungi in alginate beadssupriyanto figure 3 : transversal section of shorea pinanga mycorrhizal roots inoculated with s.columnare (b, x 400), a. umbronata (c, x 400), rhizopogon luteolus (d, x 400), descomyces sp. (e, x 400) and non-mycorrhizal roots (a, x 400). cc = cortical cells, reec= radially elongated epidermis cells, m= mantle, hn= hartig's net. 69 biotropia no. 12, 1999 figure 4 : the growth of non-mycorrhizal seedling (a,c) and mycorrhizal seedlings (sd: s.dictyosporum, sc: s.columnare, r: r. luteolus, ll: l.laccata, am: a.umbronata, de: descomyces sp.) of 4 month old shorea leprosula (a, x 0.07) and the colonization of rhizopogon luteolus (b x 0 3 c,xl5). ' ' ' 70 the effectiveness of some ectomycorrhizal fungi in alginate beads supriyanto figure 5 : the development and colonization of mycorrhizal fungi hyphae at the root system of 4-month old shorea leprosula seedlings. a (x 0.1) and h (x 10): non-myconrhizal roots (control), b: inoculated with s. dictyosporum (x 0.1) and 5". columnare (c, x 0.1), l .laccata (d, x 0.1), descomyces sp. (e, x 0.1), rhizopogon luteolus(f,\q.l),a. umbronata(g,xq.l;l,x 15). 71 biotropia no. 12, 1999 figure 6: root system of 4-month old shorea leprosula seedlings inoculated with amanita umbronata (a, x 0.2 , b, x 0.5). figure 7a: degree of mycorrhizal colonization of s.dictyosporum (c, \ 0.4; d, x 8), s. columnare (e, x 0.4; f, x 10), rhizopogon luteolus (g, x 0.4; h, x 10), at root system of 4-month old shorea ovalis. a, b: non-mycorrhizal roots. 72 the effectiveness of some ectomycorrhizal fungi in alginate beads supriyanto figure 7b : degree of mycorrhizal colonization of i .laccata (i, x 0.4; j, x 10), a.umbronata (k, x 0.4; l, x 10), and descomyces sp. (m, x 0.4; n, x 10). figure 8 : transversal section of non-mycorrhizal and mycorrhizal roots of 4-month old shorea ovalis seedlings. a. structure of non-mycorrhizal roots (x 400), and inoculated with s.columnare (b, x 200), 5. dictyosporum (c, x 400), a.umbronata (d, x 400), descomyces sp. (e, x 400) and rhizopogon luleolus(f,x 1000). 73 biotropia no. 12, 1999 table 6. leaf nutrient content of shorea pinanga, shorea leprosula, shorea ovalis and hopea odorata mycorrhizal and non-mycorrhizal seedlings. note : values in bold means nutrient content increment and the values not in bold means no nutrient increment. c: control, sc: scleroderma columnare, sd.: scleroderma dictyosporum, r: rhizopogon luteolus, ll: laccaria laccata, am: amanita umbronata, de: descomyces sp. in s. pinanga seedlings, calcium concentration in the leaves was increased by the inoculation of laccaria laccata (1.085%) and descomyces sp. (1.111%), while the concentration of iron (fe) was increased by the inoculation of amanita umbronata ( 511.2 ppm) and rhizopogon luteolus (410.6 ppm). the concentration of n, p, k and mg was not significantly influenced by mycorrhizal inoculation. in shorea leprosula, the concentration of nitrogen in the leaves was increased by the inoculation of scleroderma columnare (1.737%), laccaria laccata (1.659%) and amanita umbronata (1.708%) and the concentration of calcium and magnesium was increased by the inoculation of scleroderma columnare (0.904%) and laccaria 74 the effectiveness of some ectomycorrhizal fungi in alginate beads supriyanto laccata (0.204%). the concentration of p, k and fe in the leaves was not influenced by the mycorrhizal inoculation. in shorea avails, the nitrogen concentration in the leaves was increased by the inoculation of scleroderma dictyasporum (1.317%), amanita umbronata (1.356%), rhizopogon luteolus (1.756%) and descomyces sp. (1.542%). the concentration of p, k, ca, fe, mg in the leaves of shorea ovalis was not influenced by the inoculation of mycorrhizal fungi. in hopea odorata, the nitrogen levels increased with inoculation of rhizopogon luteolus (2.049%) and the level of calcium in the leaves was increased by scleroderma columnare (0.903%), laccaria laccata (0.909%) and amanita umbronata (0.934%). magnesium level increased with inoculation of laccaria laccata (0.207%), amanita umbronata (0.201%) and descomyces sp. (0.200 %). iron levels in the leaves of hopea odorata was increased by the inoculation of scleroderma dictyosporum (1154.7 ppm), laccaria laccata (825.1 ppm) and rhizopogon luteolus (661.2 %). there is no clear pattern of correlation between inoculation with mycorrhizal fungi and leaf nutrient content. there is also no clear difference in p concentration in the leaves following inoculation as compared to the controls. discussion method of inoculum production of ectomycorrhizal fungi should be easily handled, cheap and effective to increase the seedling quality. alginate beads, as a carrier of inoculum of ectomycorrhizal fungi, produces an inoculum of high quality and large scale (9,000 100,000 beads/hour/machine). feldman and idczak (1994) specified two characteristics of high quality inoculum. high quality means firstly that the inoculum is able to colonize rapidly the plant root system. this is guaranteed by an inoculum of high infection. it does not necessarily mean large number of spores. secondly, the characteristic of a high quality inoculum is that it should be aseptic or contaminated with only small quantities of plant pathogenic microorganisms. low level of contamination can be ignored, for example for planting stock production in the nursery. regarding the percentage of germination of ectomycorrhizal fungi packed in alginate beads, the concentration of sodium alginate of 5 20 g/l does not prevent the spore germination, but its elasticity was different. based on our results, a concentration of sodium alginate of 15 g/l was selected because of the high spore germination percentage and good bead elasticity associated with this. s. columnare, s. dictyosporum, rhizopogon luteolus, laccaria laccata, a. umbronata, descomyces sp. that were packed in alginate beads, were able to colonize the root system of shorea pinanga, s. leprosula, s. ovalis and hopea odorata. their colonization is shown to be moderate to good. the mycorrhizal fungi formed mantle and hartig's net structures in the root system of the plant. it means that the alginate bead as a carrier of inoculum does not prevent the spore germination. their degree of 75 biotropia no. 12, 1999 compatibility can be evaluated on their capability to form the mantle and hartig's net structure. a mycorrhizal species able to form mantle and hartig's net structures in several plant species means that this mycorrhizal species has high compatibility. likely, it is reflected on its natural distribution. descomyces sp. and a. umbronata were the most possible to form those structures in the root system of dipterocarp species tested. their degree of compatibility should be followed further on their growth effect and their mycorrhizal dependency value. plants differ greatly in their need for, and response to, mycorrhizal infection. relative mycorrhizal dependency (rmd) as degree to which a plant is dependent on the mycorrhizal condition at a given level of soil fertility had been defined (gerdeman et al. 1975 in bagyaraj 1994). the rmd concept was also improved to define the mycorrhizal dependency of crops on the mycorrhizal fungi in field conditions, called relative field mycorrhizal dependency (plenchette et al. 1983 in bagyaraj 1994). based on the first and second rank of rfmd value, the dipterocarp mycorrhizal dependencies are as follows: 5. pinanga to descomyces sp. and l. lacata, s. leprosula to descomyces sp. and a. umbronata, s. ovalis to a. umbronata and l. laccata, h. odorata to a. umbronata and s. dictyosporum, respectively. those mycorrhizal fungi are important for improving the growth of dipterocarp seedlings. mycorrhizae have long been known to affect the nutrient cycling especially of p nutrition of the host plant. phosphate is the major form of p available in the soil solution and, therefore, it is not readily transported by mass flow. thus, as mycorrhizal hyphae explore the bulk soil beyond the root hairs, additionally p is taken up by hyphae and transported to the host (alien 1996). mycorrhizal fungi can also increase the uptake of mineralized p by occupying the microsites of active decomposition 'and, possibly, by being involved in the degradation of litter. mycorrhizal hyphae on a decomposing leaf and rapid transport of32 p from that leaf to a host plant via mycorrhizal hyphae had been observed (herrera et al. 1978 in alien 1996). by their role in the acquisition of nutrient resources, mycorrhizae are critical components of nutrient cycling. nutrient levels in the leaves of dipterocarp tested showed that mycorrhizal fungi could increase the nutrient concentration of some of the elements tested in the host plant. based on our results, there is no clear difference in p levels in leaves following inoculation as compared to the controls. references allen, m.f. 1996. the ecology of mycorrhizae, cambridge studies in ecology. cambridge university press. bagyaraj, d.j. 1994. vesicular-arbuscular mycorrhiza: application in agriculture. in j.r. morris, d. read and a.k.. varma (eds) : techniques for mycorrhizal research, methods in microbiology. academic press, harcourt brace & company, publishers, london-san diego-new york-bostonsydney-tokyo-toronto, p. 819-834. berlyn, g.p. and j.p. mlksche, 1976. botanical microtechniques and cytochemistry. iowa state university press, ames, iowa. 76 the effectiveness of some ectomycorrhizal fungi in alginate beads supriyanto boyle, c.d., w.j. robertson and p.o. salonius, 1987. use of mycelial slurries of mycorrhizal fungi as inoculum for commercial tree seedling nurseries. can. j. for. res. 17: 1480-1486. chang, d. 1993. the study of va mycorrhizal effects on horticultural crops. in proc. of second acom, 11-15 march 1991, chiang mai, thailand. eds. i. soerianegara and supriyanto. biotrop special publication no. 42. de la cruz, r.e., 1982. tree nutrition and fertilization . lecture note in the training course on biological aspects of silviculture, seamed biotrop, bogor, indonesia. departemen kehutanan, 1994. rencana pembangunan kehutanan pelita vi. jakarta, indonesia. feldmann, f. and e. idzcek, 1994. inoculum production of vesicular-arbuscular mycorhizal fungi use in tropical nurseries. in j.r. norris, d. read and a.k. varma (eds) : techniques for mycorrhizal research, methods in microbiology. academic press, harcourt brace & company, publishers, ondon-san diego-new york-boston-sydney-tokyo-toronto. fortln, c., j.a. fortin, g. gaulin, n. jomphe dan s. lemay, 1988. large scale ectomycorrhizal inoculation of containerized grown seedlings. in proc. of canadian workshop on mycorrhizae in forestry, 1-4 may 1988. gay j.c. and j.c. debaud 1987. genetic study on indole-3-acetic acid production by ectomycorrhizal hebeloma species : inter-and intra specific variability in homo and dikaryotic mycelia. appl. microbiol. biotechnol. 26:141-146. le tacon, f. , g. jung, g. mungier, p. michelot and c.maperin, 1985. efficiency in a forest nursery of an ectomycorrhizal fungus inoculum, produced in fermentor and entrapped in polymeric gels. can. j. bot. 63: 1644-1668. marx, d.h. and w.c. bryan, 1969. studies on ectomycorrhizae of pine in an electronical air filtered, air conditioned plant growth room. can. j. bot. 47 : 1903-1909. marx, d.h. 1973, mycorrhizae and feeder root diseases. in ectomycorrhizae : their ecology and physiology. eds. g.c. marks and t.t. kozlowski. academic press, new york. prosea, 1994. plant resources in south-east asia 5 : (1) timber trees : major commercial timbers. eds. i.soerianegara and r.h.m. lemmens, prosea foundation, bogor, indonesia pudoc-dlo, wageningen, the netherlands. supriyanto, 1989. micropropagation de pinus nigra et pinus sylvestris', application a leurs hybrides interspecifiques. phd dissertation, university of nancy i, nancy, france. supriyanto, 1996. conservation and utilization of mycorrhizal fungi genetic resources. seameo forum, vol. 3 no. 2, seameo bangkok. supriyanto, i. setiawan dan m. harahap, 1992. usaha peningkatan kualitas bibit tanaman kehutanan melalui inokulasi mikoriza (pengalaman dan program penelitian seameo biotrop). pros. seminar nasional status silvikultur di indonesia saat ini. dept. kehutanan aphi fak. kehutanan, ugm. wanagama, 27-29 april. supriyanto, i. setiawan, r.m. omon, dan e. santosa, 1994. effects of scleroderma dictyosporum obtained by protoplast culture on the growth of shorea selanica and shorea leprosula cuttings. bio-refor expert meeting. jica-frim, kangar, malaysia, 28 nov-1 77 2. dyah perwitasari page 1 page 2 page 3 page 4 page 5 page 6 microsoft word 36 biotropia no. 20, 2003: 36 48 heterologous expression of a chitinase gene from aeromonas caviaein pseudomonas fluorescens amarila mahku'+, antonius suwanto1'2'", budi tjahjono3, rob harling4 'southeast asian regional center for tropical biology (seameo biotrop), bogor, 2 department of biology, faculty of mathematics and sciences, 3 department of plant pests and diseases, faculty of agriculture, bogor agricultural university, bogor-16144, indonesia: ' 'department of crop science. scottish agricultural college, edinburgh eh9 3jg, uk. abstract a transcriptional fusion for an aeromonas caviae chitinase gene was constructed under the control of a constitutive promoter of the kanaraycin resistance gene (pkmr). the construct was inserted into a medium copy number broad host range plasmid vector to yield recombinant plasmid pam340, which harbored transcriptional fusion pkmrchi. another transcriptional fusion, ptac-chi, in a recombinant plasmid pam630, was conducted as comparison. triparental mating of e. coli carrying the recombinant plasmids with pseudomotws fluorescens 5100, a phyllosphere bacterium, was performed. pseudomonas fluorescens 5100 exconjugants were examined for constitutive expression of chitinase employing a spectrophotometric assay; they showed stronger chitin degradation activity than escherichia coli transformants. using a fungal antagonism plate assay, this chitinolytic p. fluorescens, however, could not inhibit selected phytopathogenic fungi. keywords: aeromonas caviae/ chitinase gene/transcriptional fusion/pkm'v vtac-chilpseudomonas fluorescens introduction chitinase is an enzyme that catalyzes the hydrolysis of chitin to oligomers. chitin is an insoluble linear p-1, 4-linked unbranched polymer of 7v-acetylgluco-samine, and is a major constituent of fungal cell wall. chitinase is found widely distributed amongst many organisms, but its physiological role is different among them. in chitinase-producing plants, this enzyme is used for self-defense against plant pathogens and pests, i.e. to hydrolyse chitin in the cell wall of certain fungal pathogens and cuticle of pests (boiler et al. 1983; hedrick et a\. 1988; oppenheim and chet 1992). it has been suggested that chitinase production in plants is induced by microbial infections or other injuries (hedrick et al. 1988). in chitinase-producing bacteria, extracellular chitinases are produced to digest chitin and utilize * author to whom all correspondence and reprint requests should be addressed : tel : +62 (251) 625965; fax:+62 (251) 315107 ; e-mail : asuwanto@indo.net.id + present address : department of pharmacy, faculty of mathematics and natural sciences, university oflndonesia. biotropia no. 20,2003 it primarily as a carbon and energy source (gooday 1990). the exploration and utilization of bacterial chitinase in plant protection is centred around its use to protect against pathogenic fungi as a biocontrol mechanism. this potential has been a major concern in constructing biocontrol agents against fungi. several studies regarding this have been reported (inbar and chet 1991; koby et al. 1994). we have cloned and sequenced a heterogenous chitinase gene (chi) from a soilborne aeromonas caviae isolated from a blackpepper plantation in bangka island, indonesia in our previous study ( malik et al. in press). this gene has 97% identity with the chitinase gene chia from a. caviae (uo9139) (sitrit et al. 1995). in this present study, we intended to utilize this gene under control of a constitutive promoter, i.e. the kanamycin resistance gene from tn903, in a suitable vector for expression in a phyllosphere bacterium, as a means of producing a chitinolytic biocontrol agent. we cloned this chi gene into a relatively small size and medium copy number broad host range plasmid vector, pbbrlmcs-2 (kovach et al. 1994), rather than into a high copy number plasmid as reported by koby et al. (1994). this strategy was employed to avoid accumulation of defective escherichia coll mutants during high expression of chitinase (koby et al. 1994). therefore, the aim of this work was to construct a chitinolytic p. fluorescens biocontrol strain through the introduction of a chitinase gene under the control of the kmr promoter as a transcriptional fusion (pkmr-c/j/). materials and methods bacterial strains, plasmids, and growth conditions bacterial strains and plasmids used or constructed during this study are described in table 1. e. coli strains which were used as hosts for cloning experiments throughout this study, and their derivatives also, were routinely grown in luria bertani (lb) medium (broth or solidified with 1.5% agar) at 37°c for 18 hours. ampicillin (100 ng/ml), kanamycin (25 μg/ml), trimethoprim (100 μg/ml) and gentamycin (10 μg/ml) were added to the growth media when needed. pseudomonas fluorescens strains were grown routinely in king's b (kb) 10% agar (king et al. 1954) at 25°c for 18-20 hours. spontaneous rifampicin resistant mutants (rii ) of p. fluorescens, which was done according to the method described by eisenstadt et al. (1994), were isolated by plating a 25ul aliquot of a 10-fold concentration of a stationary phase culture onto plates containing a gradient concentration from 10 to 100 ug/ml of rifampicin. dna manipulation plasmid dnas were isolated either by alkaline lysis (sambrook et al. 1989), or hv dna nurification kits (promeea wis.. or biorad, richmond, calif.). restriction heterologous expression of a chitinase gene from aeromonas caviae amarila malik el al table 1. bacterial strains and piasmids bacterial strains and plasm relevant characteristics reference or source e. coli sambrook et dh5a f", for a-complementation, general host for cloning al. 1989 top10 f", for a -complementation, general host for cloning invitrogen, inc. (carlsbad, calif.) hb101(prk2013) mod" res", general helper strain for bacterial conjugation dittae/fl/1980 top10(pam340) top10 carries fusion transcription pkmr-c/i; this study top10(pam630) top10 carries fusion transcription ptac-chi this study p.jluorescens 5100 phyllosphere biosurfactant* strain, wildtype, derived from campbell et al brassica oleracea var italica 1995 5100 rif spontaneous rifampicin resistance mutant pfsloo this study 5100(pam340) pf 5100 (rif) carries fusion transcription pkmr-c/i; this study 5100(pam630) pf 5100 (rif) carries fusion transcription ptac-chi this study aeromonas caviae ws7b chitinolytic, wildtype malik etal in press plasmids puc19 lacz. apr cloning vector sambrook et al. 1989 pas385 mcs from psl301(£cori-.eo/i) cloned into puc19 suwanto & kaplan (smal-ecorl), apr 1992 pas396 source of trimethoprim resistance gene (tpr) in pas385 suwanto & kaplan 1992 pbbrlmcs-2 broad-host range, medium copy number, kmr kovache/a/. 1994 puc4k pkmr source, kmr gene from 1n903 oknetal 1981 p34s-gm source of gentamycin resistance gene (gmr) dennis & zylstra 1998 pws506 pas385 carrying 2.9 kb xho\ + ffmdlu chitinase gene malik et al in fragment from a. caviae ws7b press pam201 c/i/-tpr dna fragment as sail cassette in pas385 this study pam330 pknf-chi in pbbrlmcs-2, tpr this study pam340 pkmr-c/i/ in pbbrlmcs-2, tp", gmr this study pam630 ptac-chi in pbbrlmcs-2, gmr this study fragments were purified from agarose gels with the gene clean kit (biolol inc., la jolla, calif.). dna filled-in reaction, utilizing t4 dna polymerase (boehringer mannheim biochemicals, indianapolis, ind.), dna fragment ligations, recombinant dna transformation, and other accessory techniques were carried out as described by sambrook et al (1989). escherichia coli transformants harboring the chitinase recombinant were selected on lb-chitin agar plates, supplemented with the appro biotropia no. 20,2003 ing pkmr, as well as ptac, were selected by a blue-white assay on lb agar plates, containing iptg (0.5mm), and x-gal (5-bromo-4-chloro-3-indolyl-b-d-thiogalacto-pyranoside) 40 mg/ml. plasmids were maintained by selection in the presence of appropriate antibiotics. bacterial conjugation recombinant plasmids pkmr-c/» and ptac-chi were introduced into p. fluorescens 5100 (campbell et al. 1995) by bacterial conjugation as described by ditta et al (1980). triparental mating was performed, employing e. coli hb101 (prk2013) (ditta et al. 1980) as helper, e. coli top10 (pam340), as well as e. coli top10 (pam630), as donor, and p. fluorescens 5100 rif as recipient. the cells of all bacteria employed in this mating were collected from mid-exponential phase cultures, harvested by centrifiiging recipient, donor, and helper cultures in a ratio of 8:1:1, washing the pellet twice in sterile saline solution, and resuspending in a small volume of lb broth (30 ul). all cell suspensions were mixed, spotted onto lb agar plates without any antibiotic supplements, and incubated at 25°c. polymerase chain reactions standard pcr reactions were performed to verify the presence of cloned chi in p. fluorescens exconjugants, followed by electrophoretic analysis. an approximately 800-bp fragment of chi (accession number aj431785) was amplified in a geneamp 2400 thermal cycler (perkin elmer). single colonies of exconjugants were resus-pended into a reaction mixture prepared with taqdna polymerase (finnzyme). the thermocycling program used in this study consisted of denaturation at 94 °c for 60 s, annealing at 54°°c for 60 s, and elongation at 72 °°c for 90 s, for 25 cycles, as a modification of the method described in chernin et al. (1997). a set of chi internal primers, which were designed based on a. caviae chitinase dna sequence (accession number aj431785), were used, i.e. forward primer 5 ' g t g a a g a a c t a c c a g g c -3', and reverse primer 5 ' g g c a g a t c a g t t g c a g c t c g -3' (genset biotech, singapore). chitinase plate assay and semimicro-quantitative assay an assay for chitinase was carried out on lb agar supplemented with colloidal chitin 1% (hsu and lockwood 1975) for e. coli derivatives, whilst for p. fluorescens derivatives this was carried out on 10% kb agar supplemented with colloidal chitin at the same amount. p. fluorescens exconjugants were selected on 10% kb agar supplemented with rifampicin (50 ug/ml) and gentamycin after introducing transcriptional fusion recombinant plasmids by bacterial conjugation. semimicro-quantitative assay was performed as described previously (malik et a/. in press). cultures of the recombinant bacteria were collected after growing for 18-20 hours both for e. coli and p. fluorescens, whilst cultures of a. caviae were heterologous expression of a chitinase gene fromaeromonas caviae amarila malik et al. collected from 15 hours. enzyme activity was measured in both extracellular and intracellular fractions, after sonicated the cell. the protein concentration was measured according to the method as described by bradford (1976). chitinase activity was assayed by a modification of methods as described using colloidal chitin azure as substrate (wirth and wolf 1990; evrall et al. 1990; hood 1990). the amount of dye released from this chromogenic substrate, remazol brilliant blue, was measured at 590 nm. a standard curve was constructed using a chitinase standard from streptomyces griseus (sigma chemical, st. louis, mo) against colloidal chitin azure as substrate, in citrate-phosphate buffer ph 6.0 as described by hood (1990), and incubation temperature 37°c for 2 hours. the enzymatic activity was expressed as units of chitinase/mg of protein. assay for antagonism to fungi an antagonism assay was performed as described by chernin et al. (1995): test bacteria were grown for 24 and 48 h for a. caviae ws7b (malik et al. in press) and p. fluorescens strains, respectively, in luria or nutrient broth at 30°c with aeration. the suspension of cells was streaked in a line at the centre of a pda plate and incubated at 30 °c for 24 and 48 h for ws7b and p. fluorescens strains, respectively. after placing 3-mm diameter agar disks of an actively growing fungal culture of either botrytis cinerea tom98ld and fusarium solani f. sp. pisi, at 3 to 4 cm away from each side of the bacterial growth area, the plates were incubated for 3 to 25 days, until mycelium growing from the two sides on a control plate came into contact. results construction of sail-cassette ofchi constitutive promoter of kanamycin resistance gene (pkmr) from tn903 used in this study possesses a unique restriction site, xhol (oka et al. 1981). this xhol site was employed to construct a transcriptional fusion of a. caviae ws7b chitinase gene chi (accession number aj431785) under the control of pkmr (fig.l). the chi gene in pws506 was cloned without its indigenous promoter (malik et al. in press). plasmid pam201-a carrying a sail cassette of chi was constructed through several intermediate-cloning steps (fig. 1). the first step was the insertion of trimethoprim resistance gene (tpr) isolated from pass 96 (suwanto and kaplan 1992), as a marker downstream ofchi fragment at hpal site in pws506. the chi-tpr fragment was then isolated subsequently employing hindhl+ecorv double digestion. the protruding end of hinalll was made blunt by means of t4 dna polymerase to obtain blunt end chi-tpr fragment, which was then ligated with blunt-end hpal of vector pas385 (suwanto and kaplan 1992), generated recombinant plasmid pam201 (fig 1). plasmid pam201-a carries c/z/-tor figure 1. schematic structure of sail cassette construction of chi. the promoter of kanamycin resistance gene (pkmr) from tn903 in puc4k is located upstream of the xhol site (oka et a/,1981) (a). a trimethoprim resistance gene (tpr) fragment, isolated from pas396 (suwanto & kaplan 1992), was inserted downstream of the chi fragment in pws506 (malik et al. in press) as marker at hpal site in polyl inker site (b). the c/i/-tpr fragment was isolated employing hindlll+ ecorv double digestion. the protruding end of ifiruh.ll was made blunt, which was then ligated with blunt-end hpal end of linearized vector pas385 (suwanto & kaplon 1992), resulting in recombinant plasmids pam201-a (chi+) and pam201-b (chi-) that carry sail cassette of c/i;-tpr (c). heterologous expression of a chitinase gene from aeromonas caviae amarila malik et al. construction of pkmr-chi transcriptional fusion in pbbrlmcs-2 a medium copy number broadhost-range plasmid vector, pbbrlmcs-2 (kovach et al. 1994), was utilized as cloning vehicle to introduce pkmr-chi transcriptional fusion into pseudomonas fluorescens 5100. the c/»/-tpr sail cassette from pam201-a was inserted into xhol site of pbbrlmcs-2 under pkmr to generate pam330, in which c/»'-tpr will be transcribed under control of kmr promoter (fig 2). bgli figure 2. restriction map of recombinant plasmid pam330 carrying pkmr-c/i; transcriptional fusion in pbbrlmcs-2. the additional gmr marker was inserted at ///«diii site downstream of tpr, which generated pam340 in addition to tpr marker, a gmr cassette isolated from p34s-gm (dennis and zylstra 1998) was used as an alternative marker by inserting the cassette downstream of tpr resulting in pam340 (fig 2), since tpr marker was not sufficient to select for pf exconjugants after mobilizing pam330 from the e. coli transformant. construction of ptac-chi transcriptional fusion in pbbrlmcs-2 a bamhl fragment of ptac isolated from pkk223-3 was inserted into bamhl site in pbbrlmcs-2, generating plasmid pam601. chitinase gene chi, which was isolated from pam202 as hindlll-ecorl c/i/-gmr fragment, was inserted subsequently into this plasmid vector, resulting in recombinant transcriptional fusion plasmid harboring ptac-chi, designated as pam630 (fig 3). plasmid pam202 was generated from pam201-b by replacing tpr with gmr. biotropia no. 20. 2003 ecori hind iii. abbreviations : km" = kanamycin resistance gene; pkmr = promotor of kanamycin resistance gene; apr = ampicillin resistance gene; tp" = trimethoprim resistance gene; gmr = gentamycin resistance gene; chi = chitinase gene figure 3. restriction map of recombinant plasmid pam630 carrying ftac—chi transcriptional fusion in pbbrlmcs-2 chitinase expression of transcriptional fusions in e. coli and p. fluorescens. screening of e. coli transformants harboring pam340, or pam630 on chitin agar plate showed only weak clearing zones observed after 7 and 11 days of incubation, respectively (table 2). on the other hand, e. coli harboring pws506 showed strong clearing zones after only 3 days of incubation. table 2. chitinolytic expression of chi on chitin agar plates. strains expression chitinolytic activity control as clearance zone* incubation time (days) e. co//top10(pws506) plac +++ 3 e. co//top10(pam340) pkmr + 7 e. co//top10(pam630) ptoc + 11 p. fluorescens 5100 (pam340) pkmr ++ 7 p. fluorescens 5100 (pam630) ptoc ++ 11 * relative chitin degradation ability was indicated as +, ++ and +++ for slightly, moderate, and very clear zone, respectively. plasmid pam340 and pam630 were mobilized into spontaneous rifampicin resistance (rif) mutants of strain pf5100. screen ino fnr pf phitmr.krf;/ heterologous expression of a chitinase gene from aeromonas caviae — amarila malik et al. selection, in conjunction with pcr techniques using chi primers as described above. approximately 800 bp bands were observed on agarose gel after electrophoresis of exconjugants (data not shown). the latter was a rapid and easy method to verify the exconjugants after isolating single colonies. constitutive chitinase expression of pkmr-chi the modified chitin azure assay used in this study could demonstrate the constitutive expression of the cloned chitinase gene by growing the e. coli recombinant and pf exconjugant without chitin. the colloidal form of chitin azure, which consists of oligomers and monomers, was fairly easy to degrade by chitinolytic activity compared to the flake form of this compound. we also constructed a transcriptional fusion of the same chitinase gene under the tac promoter (fig. 3), in order to compare the promoter activity. expression assays of both transcriptional fusions were carried out under uninduced growing conditions. the result showed that constitutive expression of chi under pkmr was slightly stronger (tables 2 and 3). by comparing chitinolytic activity of these two p. fluorescens 5100 exconjugants to the wild type bacterium a. caviae ws7b (the origin of chi gene in this study), it could be demonstrated that constitutive chitinolytic activity of these two strains of p. fluorescens were less active. this is consistent with the result of our previous study on e. coli recombinant harboring chi of ws7b in a puc vector that ws7b presumably carries more than one chitinase gene, and has a complex regulation of chitinase gene expression. table 3. chitinase activity assay chitinase assay (unit/mg protein) strains extracellular intracellular a. caviae ws7b 7.70" nd e. co//top10 0.29 0.41 e. co//top10(pws506) 2.31 4.17 eco/itop10(pam340) 0.93 1.12 e. co/;top10(pam630) 0.91 1.05 p. fluorescens 5100 rif" 0.77 0.84 p. fluorescens (pbbrlmcs-2) 0.78 0.83 p. fluorescens (pam340) 1.91 3.27 p. fluorescens (pam630) 0.97 2.53 #as positive control,collected from90 hrs of growth incubation time nd = not determined biotropia no. 20,2003 antifungal plate assay the antagonism assay of p. fluorescens strains harboring pam340, and pam630, was conducted with a. caviae ws7b as positive control for chitinolytic activity. p. fluorescens 5100 rif and p. fluorescens (pbbr!mcs2) were used as negative control strains. the inhibition zones between the pathogenic fungus botrytis cinerea tom98ld and the tested strains were observed after 3 to 24 days of incubation. the positive control, ws7b, showed an inhibition zone up to 17 mm. however, all bacteria strains tested did not show any fungal growth inhibition against fusarium solani f. sp. pisi. discussion a transcriptional fusion of a. caviae chitinase gene (chi) under kanamycin resistance gene promoter (pkmr) has been constructed. kmr gene is known to be expressed constitutively. by inserting chi under this promoter, we assume this will cause simple constitutive regulation of chi. the expression of other chitinase genes has been reported under nonindigenous promoter, ptac (koby et al. 1994; downing and thomson 2000). however, the expression of chitinase gene under the control of pkmr has previously not been reported. in this study, we demonstrated the constitutive expression of chitinase under this promoter by a simple semi micro-quantitative chromogenic assay using colloidal chitin azure as substrate. the enzyme activity was determined by measuring the amount of remazol brilliant blue dye released employing spectrophotometer. the results from the expression of chitinase transcriptional fusion assay demonstrated that both transcriptional fusions were expressed in e. coli, and were able to degrade chitin in the medium. longer incubation time for chitinolytic activity of pam340 and pam630 compared to pws506, might be due to the influence of plasmid copy number in this expression (table 1). plasmid pam340 and pam630 were constructed on a medium copy number plasmid vector pbbrlmcs-2, while pws506 was constructed on a high copy number plasmid vector pucl 9. the strategy to isolate and insert the promoters into plasmid vector pbbrlmcs-2 before fusion with chi, rather then subcloned chi under pkmr directly in puc4k, as well as under ptac in pkk223-3, was carried out to yield a broad host-range plasmid vector that carry a constitutive promoter pkmr, as well as the strong promoter ptac, which will be useful in future studies of other heterologous gene expressions. the result of the semimicro-quantitative of chitinase activity assay indicated that chi was expressed constitutively both in e. coli or p. fluorescens 5100 recombinants (table 3). however, chitinase activity of intracellular fractions were higher than the extracellular fractions which can be assumed that the chitinase product might not be well secreted in e. coli, as we have discussed in our previous study, as well as in p. fluorescens (malik et al. in press). 45 heterologous expression of a chitinase gene from aeromonas caviae amarila malik et al. the inability of transformed p. fluorescens strains to slow fungal growth is more likely caused by secretion problem, as well as low expression of chitinase product in p. fluorescens based on the results obtained from chitinolytic expression on agar plates and semiquantitative chitinase assays as shown in tables 2 and 3. the gene was isolated from a. caviae, and was cloned and expressed in e. coli and p. fluorescens, which are not indigenous hosts. nevertheless, the fact that the protein expression machinery between these three microorganisms are not the same, could explain this low chitinolytic activity. it could be also suggested that this chitinase gene (chi) in its source bacteria a. caviae ws7b, did not act alone to slow fungal growth. it might also require other antifungal mechanisms as reported previously in jones et al. (1986) and sundheim et al. (1988). conclusions transcriptional fusion of the chi gene under the control of kanamycin resistance gene promoter generated constitutive expression of chitinase in both e. coli and p. fluorescens 5100, which has previously not been reported. this chitinase gene expression might be useful to be developed further for construction of a biocontrol strain to prevent the growth of phytopathogenic fungi. acknowledgment this work was supported by graduate team research grant (urge) grant 029/htpp/ii/ urge/96 to b.t., and a higher education link award from the department for international development (dfid)/british council to r.h. and a.s. references boiler, t., gehri, a., mauch, f. and u. vogeli. 1983. chitinase in bean leaves: induction by ethylene, purification, properties, and possible function. planta 157,22-31. bradford, m.m. 1976. a rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72,248-254. campbell, l.j., marling, r., chard, j. and m. sutton. 1995. calabrese: factors controlling symptom development in bacterial spear rot. final report, project fv104b, horticultural development council, bradbourne house, east mailing, kent me 19 6dz, uk. cherain, l., z. ismailov, s.haran, and i. chet. 1995. chitinolytic enterobacter agglomerans antagonistic to fungal plant pathogens. appl. environ. microbiol. 61,1720-1726. chernin, l.s., de la fuente, l., sobolev, v., haran, s., vorgias, c.e., oppenheim, a.b. and i. chet. 1997. molecular cloning, structural analysis, and expression in escherichia coli of a chitinase gene from enterobacter agglomerans. appl. environ. microbiol. 63, 834 -839. 46 biotropia no. 20,2003 dennis, j.j. and g.j. zylstra. 1998. plasposon: modular self-cloning minitransposon derivatives for rapid genetic analysis of gram-negative bacterial genomes. appl. environ. microbiol. 64,2710-2715. ditta, g., stanfield, s., corbin, d. and d.r. helsinski . 1980. broad-host range dna cloning system for gramnegative bacteria: construction of gene bank of rhizobium melihti. proc. natl. acad. sci. 77,7347-7451. downing, k.j. and j.a. thomson. 2000. introduction of the serralia marcescensa chia gene into an endophytic pseudomonas jluorescens for the biocontrol of phytopathogenic fungi. can j microbiol 46, 363-369. eisenstadt, e., b. c. carlton and b. j. brown. 1994. gene mutation. in: gerhardt, p., r. g. e. murray, w. a. wood, n.r. krieg (eds). methods for general and molecular bacteriology. american society for microbiology, washington, d.c.: 297-315. evrall, c.c., atwell, r.w., and c.a. smith. 1990. a semi-micro quantitative assay for determination of chitinolytic activity in microorganisms. j. microbiol. methods 12,183-187. gooday, g.w. 1990. physiology of microbial degradation of chitin and chitosan. biodegradation 1, 177-190. hedrick, s.a., bell, j.n.., boiler, t. and c.j. lamb. 1988. chitinase cdna cloning and mrna induction by fungal elicitor, wounding, and infection. plant physiol. 86,182-186. hood, m.a. 1990. comparison of four methods for measuring chitinase activity and the application of the 4-muf assay in aquatic environments. j. microbiol. methods 13, 51-160. hsu, s.c. and j.l. lockwood. .1975. powdered chitin agar as a selective medium for enumeration of actinomycetes in water and soil. appl. microbiol. 29, 422-426 inbar, j and i. chet. 1991. evidence that chitinase produced by aeromonas caviae is involved in the biological control of soil-borne plant pathogens by this bacterium. soil biol. biochem. 23, 973-978. jones, j.d.g., k.l.grady, t.v.suslow, and j.r.bedbrook. 1986. isolation and characterization of genes encoding two chitinase enzymes from serratia marcescens. embo j. 5: 467-473. king, e.o., ward, m.k. and d.e. raney. 1954. two simple media for the demonstration of pyocyanin and fluoreescin. j. labor. clin. med. 44, 301-307. koby, s., schickler, h., chet, i. and a.b. oppenheim. 1994. the chitinase encoding tn7-based chia gene endows pseudomonas jluorescens with the capacity to control plant pathogens in soil. gene 147,81-83. kovach, m.e., phillips, r.w., elzer, p.h., roop ii, r.m. and k.m. peterson. 1994. pbbrlmcs: a broad-host range cloning vector. biotechniques 16, 800-802. malik, a., wenuganen, s., suwanto, a. and b. tjahjono. cloning, dna sequence and expression of aeromonas caviae chitinase gene. molec. biotech. (in press) oka, a., sugisaki, h. and m. takanami. 1981. nucleotide sequence of the kanamycin resistance transposon tn905. mol. biol 147: 217-226. oppenheim, a. b. and i. chet. 1992. cloned chitinases in fungal plant pathogen control strategies. trends biotechnol. 10,392-394. sambrook, j., fritsch, e.f. and t. maniatis. 1989. molecular cloning. cold spring harbor laboratory press. cold spring harbor. new york. usa 47 heterologous expression of a chitinase gene from aeromonas caviae amarila malik el al. sitrit, y., vorgias, c.e., chet, i. and a.b. oppenherm. 1995. cloning and primary structure of the chia gene from aeromonas caviae. j. bacteriol. 177, 4187-4189 sundheim, l., a.r. poplawsky and a.h. ellingboe. 1988. molecular cloning of two chitinase genes from serratia marcescens and their expression in pseudomonas species. physiol. mol. plant. pathol. 33: 483-491. suwanto, a. and s. kaplan. 1992. a self-transmissable narrow-host range endogenous plasmid of rhodobacter sphaeroides 2.4.1: physical structure, incompatibility determinants, origin of replication, and transfer function. j. bacteriol. 174, 1124-113 wirth, s.j. and wolf, g.a. (1990. dye-labelled substrates for the assay and detection of chitinase and lysozyme activity. j microbiol. methods 12,197-205. 48 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf 47.pdf 48.pdf 37 application method of antimicrobial substances – a. dikin et al. *corresponding author : antario_dikin@yahoo.com biotropia vol. 15 no. 1, 2008 : 37 49 application method of antimicrobial substances for the control of schizophyllum commune fr. causing brown germ and seed rot of oil palm antario dikin1*, kamaruzaman sijam2 and idris abu seman3 1agricultural quarantine agency of indonesia, jakarta, indonesia 2department of plant protection, faculty of agriculture, universiti putra malaysia, selangor d.e, malaysia 3plant pathology and weed science group, biological research division malaysian palm oil board, selangor, d.e. malaysia abstract biological seed treatment promotes to save the environment from toxic chemicals in the agricultural practices. schizophyllum commune is one of the important seedborne pathogenic fungi causing brown germ and seed rot of oil palm which required effective and efficient treatment based on environmental friendly approaches. anti-microbial substances are extracted from antagonistic bacteria of b. multivorans and m. testaceum after mass production in the liquid media. application method of anti-microbial substances for the control of schizophylllum commune was done by seed dipping for 30 minutes and vacuum infiltration at 400 mm hg. vac. for 2 min. in supernatant of anti-microbial substances diluted in sterilized distilled water with concentration ratio of 1:4. application method using anti-microbial substances from antagonistic bacteria inhibited the growth of pathogenic fungus, enhanced seed germination, and without causing any abnormal growth of oil palm seedlings. key words : anti-microbial substances, seed treatment schizophyllum commune fr. introduction seed treatment as part of integrated pest management practically minimizes the infection of any plant pathogens associated with seeds. the application of seed treatment can be used as preventive and curative method against plant pathogens. pre-treatment is normally applied to protect the seeds from soil-borne pathogens and to control seedborne pathogens. schizophyllum commune fr. causes brown germ and seed rot of oil 38 biotropia vol. 15 no. 1, 2008 palm. heavy infection of the pathogen decreased seed germination of oil palm about 60% (dikin et al. 2003). fifty two antagonistic bacterial isolates from oil palm plantation in peninsular malaysia were potential agents for suppressing the growth of s. commune. of the 52 isolates which produced anti-microbial substances burkholderia cepacia rb47, b. multivoras ru50 and microbacterium testaceum ru7 inhibited the growth of s. commune (dikin et al. 2006). formulation of active compounds in the bio-pesticide such as pyrrolnitrin from pseudomonas cepacia was developed by micro-encapsulation using gluten and casein. the micro-encapsulations protected active agents which are sensitive to direct light exposure and extreme temperature (yu and lee 1997). bacillus subtillis is an antagonistic bacteria of s. commune formulated as biopesticide in dry powder form. dry powder as the carrier of bacterial spores in the slurry is to protect seeds from the infection of rhizoctonia solani, fusarium spp., alternaria spp., and aspergillus spp. (fravel et al. 1998; desai et al. 2002). potential supernatant of anti-microbial substances from b. multivorans ru50 and m. testaceum ru7 suppressed the growth of s. commune in vitro. meanwhile, in vivo application for the control of brown germ and seed rot diseases of oil palm requires further studies. the objectives of the study were to determine the anti-microbial substances from b. multivorans ru50 and m. testaceum ru7 to control brown germ and seed rot diseases and to evaluate their potential preventive measures of oil palm seeds. materials and methods antimicrobial substance one loop-full of b. multivorans ru50 and m. testaceum ru7 isolates were inoculated to 1 liter of sterilized liquid media (10 g of neo peptone, 10 g of lactose and 10 g of peptone, 10 g of maltose, respectively, for each species of bacteria). liquid media containing the cell of each bacterial isolate were fermented in electric rotator (new brunswick scientific g-25kc) at 125 rpm for 5 days, and then added with 50 ml of absolute methanol. supernatant from fermented liquid media was separated using centrifugation and evaporated using buchi rotavopor r-200 at 45oc to reach 100 ml (dikin et al. 2006). effect of anti-microbial substances on non-germinating oil palm seeds inoculated with s. commune twenty five pre-heated oil palm seeds were artificially inoculated with s. commune by placing the seeds onto s. commune cultural plates, and then incubated in polythene bags for 7 days at room temperature for mycelial colonization. inoculated seeds were 39 application method of antimicrobial substances – a. dikin et al. treated with anti-microbial substances from mixing supernatant of b. multivorans ru50 and m. testaceum ru7. the ratios for treatment concentration of seed dipping were 1 : 2 (r2), 1:4 (r4), 1 : 6 (r6), and as control dipped into distilled water (sc-dip) for 30 minutes. other treatments of inoculated seeds were infiltrated in the anti-microbial substances of various concentrations namely 1:2 (v2), 1:4 (v4), 1:6 (v6) and as control the seeds were infiltrated into distilled water at 400 mm hg vac. for 2 min. the preheated seeds non inoculated with s. commune were used as negative control. treated non germinating seeds and the control were air dried in laminar chamber, and then incubated in polythene bags at 26±2oc. observation of seed germination was carried out at 14, 21, 28 days interval. four replicates were made for each treatment including control (dikin et al. 2003). effect of anti-microbial substances on the fungal colonization ten pre-heated oil palm seeds were treated with the supernatant of anti-microbial substances by dipping, vacuum infiltration, and seed coating. as control, the seeds were not treated with anti-microbial substances. as comparison, the seeds were dipped in a synthetic fungicide, 0.2 % of mancozeb. dipping treatment was conducted by soaking oil palm seeds in diluted anti-microbial substances 1:4 (r4) for 30 minutes. vacuum treatment was conducted by soaking and infiltration the seeds in diluted anti-microbial substances with ratio of 1:4 (v4) at 400 mm hg vac. for 2 min. seed coating was conducted by coating oil palm seeds with freeze-drying of anti-microbial substances in talc. the rate of concentration between talc and anti-microbial supernatant suspension was 1:1. seeds treated with anti-microbial supernatant were mixed with the talc powder in a polythene bag and shaken until the powder covered the whole seeds. the seeds were air dried, and then placed on the culture plate of 7day-old s. commune. plates were incubated at room temperature in polythene bags. the colonized seeds were recorded for each treatment. five replicates were made for each treatment including control. comparison of various pre-treatments of artificial inoculation of oil palm seeds on seed germination twenty five pre-heated oil palm seeds were treated with supernatant of antimicrobial substances by dipping (r4), vacuum (v4), seed coating by freeze dry solidification of anti-microbial substances in talc with concentration ratio of 1:1, dipping oil palm seeds in the suspension of peptone maltose, neo peptone lactose for 30 minutes. as negative control. as comparison oil palm seeds were dipped in the synthetic fungicide of 0.2% mancozeb. for dipping treatment seeds were shaken in diluted antimicrobial substances with concentration of 1 part of supernatant of anti-microbial substances diluted with 4 parts of distilled water (r4) for 30 minutes. for vacuum treatment the seeds were soaked and vacuumed in diluted anti-microbial substances with concentration of 1 part of supernatant of anti-microbial substances diluted with 4 parts of distilled water (v4) at 400 mm hg. vac. for 2 min. treated seeds were placed 40 biotropia vol. 15 no. 1, 2008 on cultural plates of 7–day-old s. commune and then incubated for 7 days in polythene bags at room temperature. then the seeds were collected and placed in a polythene bag for percentage of germination. seeds were incubated for 28 days at room temperature and the germination recorded. three replicates were made for each treatment. effect of pre-treated artificial inoculation of oil palm seeds on the growth of seedling a number of 10 germinated oil palm seeds from each treatment including control i.e. the protective treatment including seed dipping in diluted anti-microbial substances with ratio of 1:4 (r4) in distilled water, vacuum seed in diluted anti-microbial substances with ratio of 1:4 (v4), seed coating by freeze-dry solidification of antimicrobial substances in talc with ratio of 1:1, seed dipping in mancozeb, inoculated seeds treated with liquid solution of neopeptone, peptone, maltose, lactose as negative control and healthy seeds as positive control was sown in plastic pots (29 x 39 x 10 cm). seedling growth was maintained by watering and measuring the height of seedlings every week and the dry weight of seedlings were recorded at 12 weeks. three replicates were made for each treatment (dikin et al. 2003). results and discussion the mixed supernatant of antimicrobial substances from b. multivorans and m. testaceum were solidified in talc and kaolin as carrier by dry air heating and freeze -drying. both solidified anti-microbial substances in talc and kaolin affected the diametric clear zone of s. commune. dissolved freeze-dried anti-microbial substances in talc with distilled water 1:1 showed diametric clear zone highest formation of 21 mm, followed by freeze dried kaolin, air heating dried talc, and air heating dried kaolin with the diametric clear zone of s. commune i.e. 17.3 mm, 15.3 mm and 14.6 mm respectively. the formulation of anti-microbial substances in freeze-dried talc suppressed the growth of s. commune at dissolved ratio, 1:25, followed by freeze-dried kaolin. both freeze-dried talc and kaolin showed diametric clear zone of 14 mm and 13.6 mm, respectively. meanwhile, the air heating of dried talc and kaolin dissolved in distilled water at ratio 1:10 suppressed the growth of s. commune (table 1.). 41 application method of antimicrobial substances – a. dikin et al. table 1: clear zone diameter of s. commune against diluted anti-microbial substances in powder form formulated antimicrobial substances diluted ratio (anti-microbial substance in powder form : sterilized water)a average 1:1 1:5 1:10 1:15 1:20 1:25 1:30 freeze dry of kaolin 17.3ab 17ab 16bb 15.3cb 14.6db 13.6eb 0fa 13.4 freeze dry of talc 21aa 20ba 16.3ca 15.6da 15ea 14fa 0ga 14.5 dry air heating of kaolin 14.6ad 14.3ac 14.3ac 0bc 0bc 0bc 0ba 6.1 dry air heating of talc 15.3ac 13.3bd 12.6cd 0dc 0dc 0dc 0da 5.8 average 17 16.1 14.8 7.7 7.4 6.9 0 note: a average of three replications. means followed by different small letters within row (lsd0.05 =0.3 mm) and different capital letters within column (lsd0.05= 0.2 mm) are significantly different. among the two carriers with two drying techniques, freeze-dried talc was the best formulation of anti-microbial substances of b. multivorans and m. testaceum. dry air heating , freeze-dried talc and kaolin had different rigidity in grinding to obtain small size particles. it was easier to grind the solidified anti-microbial substances by freeze-drying technique than dry air heating. effect of supernatant of antimicrobial substances on non-germinating oil palm seeds inoculated with s. commune liquid form of supernatant of anti-microbial substances from b. multivorans and m. testaceum significantly suppressed the growth of s. commune in the oil palm seeds (p=0.05). the potential anti-microbial substances reduced the oil palm seed infection and enhanced the seed germination of oil palm. seed treatment of oil palm by dipping and vacuum showed that both treatments affected s. commune infection in the seed. seed germination of healthy oil palm seeds at 28 days after incubation was 95%. seed germination of artificial inoculated seeds dipped and vacuumed in distilled water was 17 and 56%, respectively. seed treatment with supernatant of anti-microbial substances significantly recovered seed germination. dipping (r4) and vacuuming (v4) treatment with supernatant of anti-microbial substances reached germination of 94 and 93%, respectively, and was not significantly different compared to healthy seeds i.e. 92% (table 2). seed treatment of oil palm with supernatant of anti-microbial substances suppressed fungal growth. mycelia colonized the whole seed and penetrated up to the kernel through germ pores. germ pores of seeds were blocked by compact mycelia and caused a failure in germination. the inhibition of mycelial growth of s. commune on 42 biotropia vol. 15 no. 1, 2008 the whole seeds using anti-microbial substances i.e. dipping and vacuum treatment induced seed germination. at the early stage, the germination plugs of the treated seeds release from germ pores; radicle and plumule gradually came out with soft tissue and white in colour. table 2. percentage of seed germination of oil palm seeds after incubation treatment*) days of incubation 14 21 28 control without sc 92a 92a 95a sc vac 53d 56e 56f sc dip 16f 16f 17g r2 22e 60d 78e r4 70c 82c 94ab r6 72c 82c 86d v2 72c 88b 90c v4 89ab 92a 93ab v6 86b 92a 92bc notes: average of four replications. means within column with the same small letter are not significantly different at p=0.05 according to least significant different test *) r2 = dipping treatment with one part of supernatant anti-microbial substances and added two parts of distilled water r4 = dipping treatment with one part of supernatant anti-microbial substances and added four parts of distilled water r6 = dipping treatment with one part of supernatant anti-microbial substances and added six parts of distilled water v2 = vacuum treatment with one part of supernatant anti-microbial substances and added two parts of distilled water v4 = vacuum treatment with one part of supernatant anti-microbial substances and added four parts of distilled water v6 = vacuum treatment with one part of supernatant anti-microbial substances and added 6 parts of distilled water control without sc = healthy seeds (control) sc vac = vacuum treatment of artificial inoculated seeds in distilled water scdip = dipping treatment of artificial inoculated seeds in distilled water comparison of various pre-treatments of artificial inoculation of oil palm seeds on seed germination pre-treatment of seed using anti-microbial substances was able to protect the fungal growth and was reflected by the seed germination recovery. from the different pre-treatments in the application of anti-microbial substances as preventive measures, 43 application method of antimicrobial substances – a. dikin et al. 28-day treatment after incubation showed that dipping and vacuum significantly increased the percentage of seed germination. both dipping and vacuum pre-treatments increased the germination rate, compared to seeds without artificial inoculation. both dipping and vacuum pre-treatments were also significantly different with the application of synthetic fungicide, mancozeb (p = 0.05). seed coating with freeze dry anti-microbial substances in talc was not significantly different with non-inoculated seeds and significantly different with artificial inoculated seeds treated with distilled water or liquid medium (neo peptone, lactose, peptone, maltose) as negative control (table 3.). table 3: percentage of seed germination of pre-treated seeds before artificial inoculation with s. commune*) pre-treatment days of incubation 14 21 28 vacuum 37b 72a 84a dipping 31c 72a 84a seed coating 25d 51b 80b mancozeb 26cd 51b 81b without s. commune (positive control) 73a 76a 81b inoculated s. commune + water (negative control) 11e 12c 33c inoculated s. commune + neopeptone lactose peptone maltose (negative control) 13e 13c 25d note : average of four replications. means followed by different small letters within column are significantly different (lsd p=0.05) supernatant of anti-microbial substance as seed protector against infection of s. commune supernatant of anti-microbial substances from b. multivorans and m. testaceum protected oil palm seeds from infection of s. commune (figure 1.). the anti-microbial substances in the liquid form and solidified anti-microbial substances in talc significantly inhibited colonization of s. commune on all oil palm seeds. vacuum pre-treatment of oil palm seeds significantly reduced the colonization of fungus up to 88%, followed by seed coating with solidified anti-microbial substances in talc which reduced the colonization of fungus by 66% and dipping pre-treatment reduced the colonization of fungus by 16%. the synthetic fungicide, mancozeb at 2 g/l in distilled water did not protect the seeds from the colonization of s. commune (table 4.). 44 biotropia vol. 15 no. 1, 2008 a c b d table 4: percentage of pre-treated oil palm seeds colonized by s. commune (%) pre-treatment seed colonization (%)* dipping in anti-microbial substances with concentration 1:4 84b vacuum in anti-microbial substance with dose 1:4 12d seed coating with solidified anti-microbial substances in talc 34c mancozeb 100a without anti-microbial substances (control ) 100a note : average of five replications. means followed by different small letters within column are significantly different (lsd p =0.05). d b a figure 1: a. oil palm seeds after vacuum pre-treatment with anti-microbial substances b. seed coating with freeze-dry solidified anti-microbial substances in talc c. dipping pre-treatment with anti-microbial substances d. seeds without anti-microbial substances as control 45 application method of antimicrobial substances – a. dikin et al. a b effect of pre-treated artificial inoculation of oil palm seeds on the growth of seedlings seed treatment of artificially inoculated seeds with anti-microbial substances induced seed germination. schizophyllum commune with its compact mycelia blocked the germ pores and was found to be the main cause of germination loss. when the radicle and plumule already emerged from seed, and the seed infection of s. commune took place, germinated seeds were still able to continue its growth. the anti-microbial substances showed impacts on suppressing the fungal growth during germination, and did not inhibit seedling growth. seedling height from inoculated seeds and then treated with dipping in distilled water showed growth inhibition (figure 2). seedling height at 12 weeks old showed that all treatments of vacuumed and dipped in anti-microbial substances and non inoculation of s. commune were not significantly different. mean dry weight of 12-week-old seedling treated with v2, v4, v6, r2, r4, r6, water dipping (c-wt) and water vacuum (c-cv) were significantly different. vacuum treatment (v2, v4) enhanced dry weight of seedling followed by others (table 5). figure 2. oil palm seedlings from artificially inoculated seeds with s. commune a. without treatment with anti-microbial substances b. treated artificially with anti-microbial substances 46 biotropia vol. 15 no. 1, 2008 table 5: height of oil palm seedling from the treated germinating seeds (cm) seedling age (week) treatment* c-wt no-sc v2 v4 v6 r2 r4 r6 c-vc 6 13.7d 15.7ab 15.3ab 15.4ab 15.1bc 15.4ab 16.3a 14.9bc 14.1dc 7 15.3c 17.6b 18.4ab 18.5ab 17.8ab 17.9ab 19.3a 18.5ab 17.1b 8 18.3c 19.1bc 19.1bc 21a 19.6abc 20abc 21.2a 19.9abc 20.1ab 9 19.7c 20.1bc 21.7a 21.9a 21abc 21.3ab 21.7a 20.5abc 20.9abc 10 19.7b 21.6a 22.8a 22.7a 22.6a 22.1a 22.7a 21.7a 21.4ab 11 21.4d 23.8ab 24.5a 23.6ab 23.8ab 23.7ab 23.2bc 23.3bc 22.5c 12 22.5b 24.6a 25.8a 26a 25.6a 25.1a 25.8a 25.7a 24.9a dry weight (mg) 1242b 1250ab 1425a 1431a 1265ab 1281ab 1345ab 1337ab 1368ab note : average of thirty replications. means followed by different letters within the same row are significantly different (lsd p=0.05). formulation of supernatant of anti-microbial substances from b. multivorans and m. testaceum were solidified in the carrier of talc and kaolin which affected the mic of ant-imicrobial substances against s. commune. the carrier of talc and kaolin as aggregate ingredients has the function to release and dissolve anti-microbial substances easily and faster in water without reduction of active compounds to suppress the growth of s. commune. freeze-dry solidification of supernatant of anti-microbial substances was not easy to keep it in dry powder form. the freeze-dried powder of supernatant of anti-microbial substances is hygroscopic and trapped water from the air. after 24 hours in open air, the freeze-dried powder of anti-microbial substances becomes liquid. talc and kaolin as the carriers of anti-microbial substances with concentration ratio of 1:1 formulated the anti-microbial substances to the powder form and act as bio-pesticide. the concentration ratio of anti-microbial substances in talc or kaolin was 1:1 and avoided water trapped by the formulated bio-pesticide from the air. dry technique of freeze-drying and dry air heating in the solidification of supernatant of anti-microbial substances in talc and kaolin had different values of minimum inhibitory concentration (mic). dry air heating of talc and kaolin at 60oc for 48 hours reduced the potential of anti-microbial substances to inhibit s. commune. actually, the stability of ant-microbial substances from b. multivorans and m. testaceum against the temperature in the powder form was up to 60oc within 2 hours. other limiting factors in dry air heating of solidified anti-microbial substances in talc and kaolin were hardening, difficult to grind and not easy to dissolve in water compared with freeze dry solidification of anti-microbial substances in talc and kaolin. lower 47 application method of antimicrobial substances – a. dikin et al. value of mic anti-microbial substances in talc and kaolin powder compared to the liquid form was due to reduction half of the active compounds and also the content of active compounds, such as phenazine, pyrrolnitrin and phenylpyrrol which was too small based on hplc analysis. synthetic fungicide such as thiram with a dose of 2 g/l is used for seed treatment of oil palm (turner and gillbanks 2003). although, the ratio of concentration of this synthetic fungicide was relatively high, the potential of suppressing pathogenic fungus was still effective. the pre-treatment of oil palm seeds using formulated anti-microbial substances in liquid form was effective to control s. commune. schizophyllum commune attacked oil palm seeds through direct contact between infected seeds and healthy seeds, and then the mycelia colonized the whole seed. based on the dry weight of 12-week-old seedlings, seed treatment of inoculated seeds with antimicrobial substances did not inhibit seedling growth. the liquid formulation protected the attack of s. commune by the colonizing oil palm seeds. vacuum pre-treatment was more effective than dipping pre-treatment in the application of liquid formulation as protective measure. vacuum pre-treatment quantitatively enhanced the absorption of anti-microbial substances by the oil palm seeds. mean while, dipping pre-treatment showed that the anti-microbial substances covered the whole seeds, dry faster in the open air and less absorption of anti-microbial substances. the difference in absorption quantity of anti-microbial substances on both vacuum and dipping pre-treatment in the seeds is equal to different applications of the concentration of anti-microbial substances. effective dose in the application of liquid formulation was one part supernatant of anti-microbial substances from b. multivorans and m. testaceum dissolved into four parts of distilled water. however, for the application of freeze-dry solidified antimicrobial substances in talc it was equal to one gram dissolved in two ml of distilled water. this dose of powder formulation was able to suppress the fungal growth, but the application of seed pre-treatment caused less germinating seeds, due to the formulated talc in the wet powder to cover the whole seeds which strongly blocked the germ pores for the absorption of the required oxygen in respiration activity before and after germination. coating pre-heated seeds with solidified freeze dry anti-microbial substances in talc apparently protected the oil palm seeds from contaminant fungi for long period in store. coating pre-heated seeds with synthetic fungicide is commonly used to protect oil palm seeds from contaminant fungi in the intercontinental trade. slurry pre-treated seeds were washed with water to remove the fungicide on seed surface before germination process (dikin et al. 1995). pre-treatment of seeds using supernatant anti-microbial substances from b. multivorans and m. testaceum controlled artificially inoculated seeds with s. commune. the recovery of seed germination of oil palm was due to treatment of supernatant antimicrobial substances. the effect of application of anti-microbial substances, as curative or preventive measure on seed germination and seedling growth showed no significant 48 biotropia vol. 15 no. 1, 2008 differences with healthy seeds. based on the results, the application of anti-microbial substances for the control of s. commune confirmed that the supernatant anti-microbial substances from b. multivorans and m. testaceum contain at least 3 active compounds of pyrrolnitrin, phenylpyrrol and phenazine which did not cause negative effect on seedling growth. these active compounds were reported to inhibit fungal growth (cartwright et al. 1995). conclusions supernatant of anti-microbial substances from b. multivorans and m. testaceum have been formulated in liquid form and freeze-dried talc form. both formulations had different functions for the control of s. commune as preventive and curative measures. formulated anti-microbial substances in liquid form and freeze dry solidification in talc had different values of minimum inhibitory concentration. application of the supernatant anti-microbial substances in liquid form for preventive and curative measures was conducted by seed dipping for 30 minutes and vacuum 400 mm hg vacuum for 2 minutes with ratio concentration of 1 part supernatant of anti-microbial substances diluted in 4 parts of distilled water (1:4). formulation of freeze-dry solidification of anti-microbial substances in talc was applied for seed dressing. formulation of anti-microbial substances in powder form is a preventive measure against fungal infection particularly s. commune. finally, seed pre-treatment using anti-microbial substances enhanced seed germination, and did not inhibit seedling growth of oil palm. acknowledgement part of the ph.d. thesis of the author is financially supported by irpa project, malaysian government (vote no. 54400). references cartwright, d.k., w.s. chilton and d.m. benson. 1995. pyrrolnitrin and phenazine production by pseudomonas cepacia, strain 5.5b, a biocontrol agent of rhizoctonia solani. appl. microbiol. and biotechnol., 43: 211-216. desai, s., m.s. reddy and j.w. kloepper. 2002. comprehensive testing of biocontrol agents. in biological control of crop diseases, ed. s.s. gnanamacikam. 17: 387-420. new york: marcel dekker, inc. 49 application method of antimicrobial substances – a. dikin et al. dikin, a., hermawan and z. zubir. 1995. temuan schizophyllum commune pada benih kelapa sawit asal costa rica.. prosiding kongres nasional xiii dan seminar ilmiah perhimpunan fitopatologi indonesia. mataram, indonesia. p 303-305 dikin, a., kamaruzaman sijam, zainal abidin mior ahmad and idris b abu seman. 2003. biological control of seedborne pathogen of oil palm, schizophyllum commune fr. with antagonistic bacteria. int. j. agric. and biol., 5(4): 507-512. dikin, a., kamaruzaman sijam, jugah kadir, idris b. abu seman. 2006. effect of different carbon sources and peptones on the production of antimicrobial substances from bacteria against schizophyllum commune fr. international journal of agriculture and biology. vol. 7. no. 3. 1560-8530/2005/07-3385-388. http://www.ijab.org fravel, d.r., w.j.connick jr and j. a. lewis. 1998. formulation of microorganisms to control plant disease. in formulation of microbial biopesticides, ed. h.d. burges. kluwer academic publishers, london 5:187-202. turner, p.d. and r.a. gillbanks. 2003. oil cultivation and management. the incorporated society of planters. kuala lumpur. malaysia. p.34. yu, j.j. and w.c. lee. 1997. microencapsulation of pyrrolnitrin from pseudomonas cepacia using gluten and casein. j. fermentation and bioengin., 84 (5): 444-448. biotropia book final.indd 135 taxometrics classification (hierarchical and ordination) of aquatic and semiaquatic mosses: a preliminary model to bryodiversity management m.j. loo¹*, t. a. delvalls casillas¹ and l. martin diaz¹ ² ¹unesco/unitwin/wicop..department of physical chemistry, faculty of marine and environmental sciences, universidad de cádiz – rio san pedro campus, 11510 puerto real, cádiz, spain. ¹²instituto de ciencias marinas de andalucía. csic. rio san pedro campus, 11510 puerto real, cádiz, spain. abstract bryodiversity is naturally serving the ecosystems sustainably. it serves the environments by preventing natural disaster (fl ooding), maintaining the quality of the water body and fi lter or treats the pollutants naturally. effi cient bryodiversity management is needed for environmental cost cutting and have a cost-eff ective management strategy. to achieve this, cluster and principal component analyses (pca) were manipulated to produce the linkage distance between the otus and identify the important groups of characters, respectively. in return, it becomes a guideline for bryofl ora and environmental managements. in this study, 23 otus and 156 characters were analyzed. th e output from the reliability and item analysis showed that the data set is highly reliable (cronbach’s alpha = 0.9627). from the cluster analysis, it showed that 5 clustered groups (manageable units) could be derived from the produced phenogram. th is is based on the nearest neighbour amalgation rule and euclidean distances. as for the principal component analysis, three factors were derived and explained 75.1064% of the variation with 56.0485%(pc1), 11.7346%(pc2) and 7.3233%(pc3), respectively. th e ordination showed that 5 manageable units were derived from pc1 and 3 manageable units for pc2 and pc3, respectively. in conclusion, conservation should precede any biodiversity management plans. key words: aquatic mosses, semi-aquatic mosses, cluster analysis, principal component analysis (pca), classifi cation *corresponding author: loominjet@googlemail.com biotropia vol. 15 no. 2, 2008 : 135 154 136 introduction bryodiversity management is a new discipline in management science. bryodiversity refers to the richness of bryophytes (mosses, liverworts and hornworts). management signifi es planning conservational strategy, organizing conservational plans, implementing organized conservational approach and controlling or sustaining the on-going of the implemented plan with the aim to conserve the nature (raffi eld and bingham 1994). based on stuessy (1990), biosystematics is crucial in understanding the biodiversity of a particular ecosystem. in this context the focus is on the richness of aquatic and semiaquatic mosses. without knowing the richness, no conservation plan will be implemented and thus, fl oral extinction is highly potential. in this study, aquatic and semi-aquatic mosses were studied phenetically to fi nd out the rarity and commonness among the studied populations. th is is very crucial in conservation where rare species should be urgently conserved and less threatened spesies should be sustained too. cost-eff ective is the success key in any management activities (raffi eld and bingham 1994). phenetic analysis (cluster analysis and principal component analysis) will statistically group species with the most similar characters together (scotland and carine 2000; komosinki et al. 2001; and aguilar et al. 2004) and forms few manageable units. instead of over-consuming time and costs for few related or familiar species and neglecting other populations, managing clustered group will be the solution in the successful bryodiversity management. th is new approach aims for conservation and at the same time continues serving the needs of the ecosystem. in term of costs, no artifi cial fl ood mitigator and barrier, no water quality tester and no waste water contamination might be required if aquatic and semi-aquatic mosses are present in the natural habitats (ando and matsuo 1984; frahm 1996; welch 1948; conrad 1935; whitehouse and mcallister 1954; ando 1957; grout 1912; coupal and lalancette 1976). materials and methods moss material and characterization twenty-three species or operational taxonomic units were selected and 156 characters with diff erent level of character states (table 1) were chosen for numerical classifi cation. further phenetic methodology referred to stuessy (1990), stotler and stotler (2000), frahm (2003), smith (1978), holmes (1998), tsai et al. (2002); and yamagishi et al. (2005). th e main sources for analyses were morphological and anatomical data: vegetative (gametophyte) and reproductive (sporophyte) components. both taxonomic sources were measured quantitatively and qualitatively. table 1. characters and character states for the taxometric analyses (1)plant size(0-small,1-big/large,2-others); (2)plant habitat and submergence (0-semi-aquatic,1aquatic,2-not submerged,3-sometimes partly submerged,4-occasionally submerged,5-others); (3)plant colour(0-greenish to blackish and rarely whitish,1-others); (4)ephemerality of plant(0-no,1-yes,2others); (5)plant growth form(0-acrocarpous,1-pleurocarpous,2-others); (6)plant covered by glaucous biotropia vol. 15 no. 2, 2008 137 or bluish(0-no,1-yes,2-others); (7)plant: prostate to erect(0-no,1-yes,2-others); (8)plant branching form(0-simple to pinnately branched,1-others); (9)rarity of plant(0-rare to common,1-others); (10)plant: terete or julaceous form(0-no,1-yes,2-others); (11)plant: means of asexual reproduction(0without,1-with,2-without or with,3-others); (12)plant: coarseness(0-not coarse,1-coarse,2-others); (13)plant with fl attened shoots(0-no,1-yes,2-others); (14)autoicous (autoecious)(0-without archegonia and antheridia in separate infl uorescences,1-with archegonia and antheridia in separate infl uorescences,2-others); (15)plant with innovative branches beneath infl orescences(0-no,1-rare,2often,3-others); (16)plant: more than 5mm(0-no,1-yes,2-others); (17)rhizoids(0-obvious/with rhizodal tubers,1-not obvious,2-others; (18)leaves unbordered by row of cells(0-no,1-yes,2-others); (19)leaf bordered by(0-elongate cells,1-smooth cells,2-others); (20)leaf sheathing(0-rarely,1-often,2others); (21)leaf costa ending below the apex to excurrent(0-no,1-yes,2-others); (22)leaf without hair-points(0-no,1-yes,2-others); (23)leaf hair-points(0-hyaline,1-others); (24)leaves all of one kind(0-no,1-yes,2-others); (25)leaves direction(0-homomallous,1-others); (26)leaves arrangement(0attached all around the stem,1-attached in two rows on opposite sides of the stem (distichous),2others); (27)leaf lamina(0-conspicious,1-others); (28)leaf lamina unistratose(0-no,1-yes,2-others); (29)leaf layer(0-unistratose,1-multistratose,2-unistratose to multistratose,3-others); (30)leaves inconspiciously ranked(0-no,1-yes,2-others); (31)leaves apex(0-ovate to spatulate,1-others); (32)leaves tip(0-acuminate to acute (awned),1-others); (33)leaves(0-various,1-undulate,2-straight or straight when dry,3-plicate or deeply plicate,4-plicate or not,5-concave,6-others); (34)leaf (dorsal view)(0-keeled or fl at,1-others); (35)leaves position(0-at extreme apex,1-others); (36)leaf alteration(0little altered when dry,1-others); (37)adaxial surface of the leaf costa(0-without lamellae or fi laments,1with lamellae or fi laments,2-broadly channeled or fl at,3-others); (38)leaf without cancellinae(0-no,1yes,2-others); (39)spreading leaves(0-without,1-with,2-with and wide,3-others); (40)diff erentiation of branch and stem leaves(0-strongly,1-weakly/scarcely,2-others); (41)apical cells of branch leaves(0about 1/2 length of those at midleaf,1-scarcely shorter than those at midleaf,2-others); (42)leaf bases(0-without cancellinae,1-with cancellinae,2-others); (43)leaf base with appear split(0-no,1yes,2-others); (44)sheathing base of leaves(0-without,1-rarely,2-with,3-others); (45)concave leaf bases(0-not concave,1-not concave and with a narrow insertion,2-with,3-others); (46)leaf longer than 1mm(0-no,1-yes,2-others); (47)leaf cross-section(0-recurved only on one side,1-plane to recurved,2recurved to revolute,3-revolute,4-others); (48)leaf bases never or gradually expanded(0-no,1-yes,2others); (49)propagula in leaf apices(0-without,1-with,2-others); (50)leaf apices at extreme apex(0margins entire or papillose-crenulate,1-others); (51)leaf apices(0-acuminate to bluntly acute,1cuspidate,2-cuspidate to piliferous,3-without piliferous or aristate (awn),4-with piliferous or aristate (awn),5-others); (52)channeled leaf apices(0-no,1-yes,2-others); (53)leaf cell diametry(0isodiametric,1-more or less isodiametric,2-others); (54)leaf cell surface(0-fl at, smooth and papillose,1smooth, bulging or prorulose,2-smooth,3-papillose or prorulose,4-smooth and papillose,5-fl at,6papillose (uni to pluri) or prorulose,7-rarely with minute cuticular roughenings,8-others); (55)leaf cell type(0-one type,1-others); (56)leaf cell colour(0-green,1-others); (57)size of leaf cells(0-shorts,1longs,2-others); (58)relative size of leaf cells(zero.1(-2):1,one.above about 3:1 or longer,two.4:1 or less,three.1-4(-5),four.more than 10:1,fi ve.(3-)4:1 or longer,six.others); (59)relative size of upper leaf cells(zero. more than 5:1,one. 2-6:1,two.others); (60)leaf cell papillose(0-no,1-pluripapillose,2-closely set, simple to branched papillae/simple to branched papillae,3-unipapollose to pluripapillose,4-with papillae stellate from a stipitate base to c-shaped,5-others); (61)leaf cells in obvious rows(0-no,1yes,2-others); (62)leaf cell shape(0-long-hexagonal,1-short-rectangular to linear,2-conic, clavate or branched and rarely c-shaped,3-rounded to quadrate,4-linear,5-merely rounded and not stellate,6taxometrics classifi cation of aquatic and semi-aquatic mosses – m.j. loo et al. table 1. continued 138 long, linear and hexagonal,7-rectangular,8-rectangular to long-hexagonal,9-others) ; (63)upper leaf cells(0-smooth or with low and indistinct papillae,1-smooth,2-fi rm-walled and short-oblong to rhombic,3prorulose,4-densely pluripapillose with c-shaped papillae,5-others); (64)basal leaf cells(0usually without thickened transverse walls,1-others); (65)papillose over the lumina or prorulose in leaf cells(0-no,1-yes,2-only papillose over the lumina,3-others);(66)mid leaf cells of stem leaf(0-40–120 μm long,1-others);(67)branch and stem leaves scarcely diff erentiated(0-no,1-yes,2-others);(68)leaf margin(0-single teeth or entire,1-entire,2-toothed,3-abruptly serrate at the shoulder,4-entire and denticulate,5-serrate margins whose teeth are often refl exed,6-entire or papillose-crenulate,7-with paired teeth,8-others);(69)numbers of leaf margin(0-single,1-double,2-two to multilayered,3others);(70)near midleaf or below recurved to revolute(0-no,1-yes,2-others);(71)upper leaf margins plane to revolute (with cells undiff erentiated or paler than median cells)(0-no,1-yes,2others);(72)stomates(0-absent,1-present,2-others);(73)leaf costa(0-without a costa or costa short and double, double or single with 2-3 lateral spurs,1-single,2-distinct throughout,3-others);(74)adaxial surface of leaf along costa(0-broadly channelled or fl at,1-others);(75)single leaf costa to at least midleaf(0-yes,1-others);(76)apical of leaf costa(0-subpercurrent,1-bluntly excurrent,2-excurrent to ending in the cusp,3-others);(77)transverse section of leaf costa(0-2 stereid bands,1-single and dorsal stereid band,2-diff erentiated stereid bands,3-others);(78)both dorsal and ventral stereid bands present(0-no,1-yes,2-others);(79)cells of abaxial surface of costa(0-oblong and elongate,1-quadrate to short-oblong,2-others);(80)dorsal part of leaf costa(0-smooth or toothed at back (ridged),1-not ridged,2-others);(81)size of leaf costa(0-narrow or much narrower,1-wide or broad,2others);(82)lamellae or fi laments on the adaxial surface of the costa(0-without,1-with,2others);(83)ventral costal epidermis(0-absent,1-present,2-others);(84)cells of adaxial (upper) surface of costa similar to or smaller than laminal cells in transverse section(0-no,1-yes,2-others);(85)costa more than 100μm wide at base(0-no,1-yes,2-others);(86)costa ending in the leaf apex(0-no,1-yes,2others);(87)costa ending below the apex to excurrent(0-no,1-yes,2-others);(88)costa ending in a spine(0-no,1-yes,2-others);(89)costa occupying less than 1/4 the leaf base(0-no,1-yes,2others);(90)axilliary hairs(0-hyaline,1-brown,2-others);(91)basal cells of axilliary hairs(0-slender,1others);(92)alar cells(0-scarcely diff erentiated,1-infl ated in well marked groups,2-not at all infl ated,3alar group not extending more than 20 – 40 % up leaf,4-others);(93)stem paraphyllia(0-no,1-no or lacking,2-abundant and fi lamentous,3-others);(94)stem paraphyllia foliose(0-no,1-yes,2-others); (95)foliate stem(0-sometimes complanate,1-foliate throughout and without rhizome-like connections between erect stems,2-symmetrical,3-sometimes fl attened,4-others);(96)stem form(0-erect,1occasionally branched beneath infl urouscences,2-others);(97)stem branching(0-not branched,1mostly prostrate with lateral branches,2-prostrate with erect branches bearing terminal sporophytes (cladocarpous),3-branching various (e.g. complanate-foliate, fl attened-foliate, prostrate with lateral branches and ranches curved downwards),4-others);(98)stem ranked leaves(0-without,1-with,2others);(99)stem: central strand(0-absent,1-present,2-others);(100)stem: hyalodermis(0-absent,1present,2-others);(101)stem abundance(0-sparse to abundant,1-others);(102)stem sclerodermis(0clearly diff erentiated,1-not or weakly developed,2-others); (103)stem size(0-up to 2.7mm long,1others); (104)stem epidermal cells(0-small,1-big/large,2-others); (105)stems round in transverse section(0-no,1-yes,2-others); (106)sporophytes(0-various types,1-terminal,2-not clustered,3-lateral,4others);(107)size of capsule(0-small,1-big/large,2-others);(108)capsule projection(0-long-exserted,1exserted,2-others); (109)capsule symmetry(0-symmetric,1-asymmetric,2-others); (110)capsule(0operculate or cleistocarpous,1-others); (111)capsule: valvate(0-never,1-always,2-others); (112)capsule growth form(0-erect or straight,1-inclined to pendulous,2-horizontal to pendulous,3-never furrowed biotropia vol. 15 no. 2, 2008 table 1. continued 139 or strumose,4-globose and rugulose to furrowed when dry,5-horizontal or pendulous,6others);(113)neck of capsule(0-short and inconspicious/inconspicuous,1-others);(114)surface of capsule(0-smooth,1-smooth or furrowed,2-others);(115)capsule narrower than urn(0-no,1-yes,2others);(116)position of capsule(0-distinctly terminal/terminal,1-others);(117)shape of capsule(0cylindric to oval,1-others);(118)capsule longer than 1mm(0-no,1-yes,2-others);(119)capsule apex and unlobed at base(0-no,1-yes,2-others);(120)calyptrae(0-cucullate,1-others);(121)calyptrae: plicate(0-no,1-yes,2-others);(122)covering of calyptrae(0-covering only operculum,1-covering only operculum and capsule apex,2-others);(123)calyptrae unlobed at base(0-no,1-yes,2-others);(124)size of calyptrae(0-small,1-large/big,2-others);(125)peristome(0-absent,1-present,2-present with papillose,3-others);(126)number of peristome(0-single,1-single or absent,2-single or double,3double,4-others);(127)peristome teeths(0-16,1-with teeth united in a high or rarely low,2others);(128)peristome state(0-not refl exed,1-spirally twisted above,2-with tubular basal membrane ,3-others);(129)development of peristome(0-weakly developed,1-better developed,2-strongly developed,3-others);(130)basal membrane of endostome(0-keeled,1-others);(131)segments of endostome(0-keeled and perforate,1-others);(132)endostome with cilia(0-no,1-yes,2others);(133)exostome(0-without or free of,1-with,2-others);(134)operculum(0-conic to apiculate,1others);(135)projection of seta(0-exserted,1-others);(136)size of seta(0-longer than 2mm,1-others);(1 37)perichaetial position(0-terminal,1-usually low and simple to bifi d,2-others);(138)perichaetia with papillae(0-no,1-yes,2-absent to large,3-others);(139)perichaetial surface(0-not scablike,1-scablike,2-ot hers);(140)perichaetial leaves (bract)(0-slightly or not diff erentiated,1-not diff erentiated,2-less diff erentiated,3-others);(141)shape of bract(0-never long awned,1-others);(142)propagula cup(0absent,1-present,2-others);(143)propagula(0-absent,1-present,2-with or without,3-sometimes present,4-never on leaf apices but sometimes elsewhere on leaves or in axils,5-others);(144)axillary propagula(0-without,1-with,2-others);(145)cells without nodulose-waxy walls(0-no,1-yes,2others);(146)cell characteristics(0-smooth, lax, thin-walled and hexagonal to rhombic,1others);(147)cells of old plants without colour change to bluish-green(0-no,1-yes,2-others);(148)cell walls not thickened on abaxial side(0-no,1-yes,2-others);(149)angular cells(0-not opaque,1others);(150)end walls of basal cells not thickened(0-no,1-yes,2-others);(151)hyaline basal cells (if present)(0-extending equal to costa,1-others);(152)laminal cells surface view(0-obscure,1-welldefi ned,2-others);(153)basal laminal cells(0-diff erentiated, hyaline and elongated,1-little diff erentiated, green and short-rectangular,2-others);(154)tomentum (if present)(0-restricted to extreme base of stems,1-others);(155)gemmae(0-without,1-with,2-others);(156)nerve(0-(60-) 65 – 115μm wide near base,1-others). data analysis two analyses: cluster analysis (depicting similarities among otus) (madeira et al. 1999; ferguson et al. 2000; sharma et al. 2004) and principal component analysis (pca) (non-hierarchical relationships among otus) (mcnulty 2004) were chosen and performed on the matrix data (table 2-11). for cluster analysis, single linkage amalgation rule and euclidean distances measure were manipulated for classifi cation. statistica 6.0 (by statsoft, inc. 2001) was utilized in this taxometric study (mazak and groves, 2006). further numerical taxonomic methodology followed luna et al. (2000); romero et al. (2000); and kim et al. (2003). taxometrics classifi cation of aquatic and semi-aquatic mosses – m.j. loo et al. table 1. continued 140 data validation reliability and item analysis was performed to measure the overall representation of the data analyzed and degree of bias. th is test was run with statistica 6.0 (by statsoft, inc, 2001). results and discussions th e output from the reliability and item analysis showed that the data set is highly reliable (cronbach’s alpha = 0.9627). th is means that more than 96% of the data analyzed were true score variability and refl ecting the real situation. th is value is higher than the standardized alpha (0.9612). th e character states (156 characters examined) for 23 otus (table 2–11) were analyzed and produced 22 nodes for classifi cation. table 2. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses operational taxonomic unit (otu) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 barbula bolleana (müll. hal.) broth. 1 0 0 0 0 0 1 0 0 2 3 2 2 2 3 2 1 1 bryum caespiticium hedw. 1 2 1 0 0 0 1 0 0 0 2 1 0 2 3 2 1 1 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 1 3 1 0 0 0 1 0 0 0 2 1 0 2 3 2 1 1 cratoneuron fi licinum (hedw.) spruce 2 0 1 2 2 2 1 0 1 2 3 2 2 2 3 2 2 2 didymodon tophaceus (brid.) lisa 1 0 0 0 0 0 1 0 0 2 3 2 2 2 3 2 1 1 eucladium verticillatum (brid.) bruch & schimp. 1 0 0 0 0 0 1 0 0 2 2 2 2 2 3 2 1 0 eurhynchium speciosum (brid.) jur. 1 0 0 0 1 2 0 1 0 2 3 2 2 2 3 2 2 0 eurhynchium hians (hedw.) sande lac. var. hians 1 0 0 0 1 2 0 1 0 2 3 2 2 2 3 2 2 0 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.-nann. 2 0 1 2 2 2 2 1 1 2 3 2 2 2 3 2 2 2 fissidens grandifrons brid. 2 4 1 2 2 2 2 1 1 2 3 2 2 2 3 2 2 2 fontinalis antipyretica hedw. 2 1 1 2 1 2 2 1 1 2 3 2 2 2 3 2 2 2 fontinalis duriaei schimp. 2 4 1 2 1 2 2 1 1 2 3 2 2 2 3 2 2 2 gymnostomum calcareum nees & hornsch. 1 0 0 2 0 0 1 0 0 2 2 2 2 2 3 2 1 0 hygroamblystegium tenax (hedw.) jenn. 1 1 0 0 1 2 1 0 0 2 3 1 2 2 3 2 2 1 hymenostylium recurvirostrum (hedw.) dixon 2 0 1 2 2 2 2 1 1 2 3 2 2 2 3 2 2 2 leptodictyum humile (p. beauv.) ochyra 2 0 0 0 1 2 1 0 0 2 3 2 1 1 3 2 2 0 leptodictyum riparium (hedw.) warnst. 2 0 0 0 1 2 1 0 0 2 3 2 1 1 3 2 2 0 palustriella commutata (hedw.) ochyra 2 0 0 2 2 2 1 0 1 2 3 2 2 2 3 2 2 2 philonotis fontana (hedw.) brid. 1 4 0 0 0 0 1 0 0 2 3 2 2 2 2 2 1 2 platyhypnidium riparioides (hedw.) dixon 1 1 0 0 1 2 1 0 0 2 3 2 2 2 3 2 2 0 pohlia melanodon (brid.) a.j. shaw 1 0 0 0 0 0 1 0 0 0 3 1 2 2 3 1 1 1 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 1 0 0 0 0 0 1 0 0 0 3 1 2 2 3 1 1 1 tortula marginata (bruch & schimp.) spruce 1 0 0 0 0 0 1 0 0 2 2 2 2 2 3 2 0 0 biotropia vol. 15 no. 2, 2008 141 table 3. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 barbula bolleana (müll. hal.) broth. 2 2 2 1 1 1 1 0 0 2 2 1 1 1 0 1 0 1 bryum caespiticium hedw. 0 2 1 1 1 1 2 0 0 2 3 2 0 0 0 1 1 1 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 0 2 1 1 1 1 2 0 0 2 3 2 0 0 0 1 1 1 cratoneuron fi licinum (hedw.) spruce 2 2 2 2 1 2 1 0 1 2 3 2 1 1 5 1 1 1 didymodon tophaceus (brid.) lisa 2 2 2 0 1 1 1 0 0 2 1 1 1 1 0 1 0 1 eucladium verticillatum (brid.) bruch & schimp. 2 2 2 1 1 1 1 0 0 2 0 2 1 1 0 1 1 1 eurhynchium speciosum (brid.) jur. 0 2 2 1 1 1 2 0 0 2 3 2 1 1 3 1 1 1 eurhynchium hians (hedw.) sande lac. var. hians 0 2 2 1 1 1 2 0 0 2 3 2 1 1 4 1 1 1 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.-nann. 2 2 2 2 1 2 1 1 1 2 3 2 1 1 6 1 1 1 fissidens grandifrons brid. 2 2 2 2 1 2 1 1 1 2 3 2 1 1 6 1 1 1 fontinalis antipyretica hedw. 2 2 2 2 1 2 1 0 1 1 3 2 1 1 6 0 1 1 fontinalis duriaei schimp. 2 2 2 2 1 2 1 0 1 1 3 2 1 1 6 0 1 1 gymnostomum calcareum nees & hornsch. 2 2 2 1 1 1 1 0 0 2 0 2 1 1 0 1 0 1 hygroamblystegium tenax (hedw.) jenn. 0 2 2 1 1 1 1 0 0 2 3 2 1 1 0 1 0 1 hymenostylium recurvirostrum (hedw.) dixon 2 2 2 2 1 2 1 0 1 2 3 2 1 1 6 1 0 1 leptodictyum humile (p. beauv.) ochyra 2 2 1 1 2 1 1 0 0 2 3 2 1 1 4 1 1 1 leptodictyum riparium (hedw.) warnst. 2 2 1 1 2 1 1 0 0 2 3 2 1 1 4 1 1 1 palustriella commutata (hedw.) ochyra 2 2 2 2 1 2 1 0 1 2 3 2 1 1 3 1 1 1 philonotis fontana (hedw.) brid. 2 0 2 0 1 1 1 0 0 2 0 1 1 1 2 1 1 1 platyhypnidium riparioides (hedw.) dixon 2 2 1 2 1 1 1 0 0 2 3 2 1 1 4 1 1 1 pohlia melanodon (brid.) a.j. shaw 2 2 2 1 1 1 1 0 0 2 3 1 1 1 6 1 1 0 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 2 2 2 1 1 1 1 0 0 2 3 1 1 1 6 1 1 0 tortula marginata (bruch & schimp.) spruce 1 2 2 0 0 1 1 0 0 2 0 2 1 1 6 1 1 1 table 4. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 barbula bolleana (müll. hal.) broth. 0 2 3 2 2 1 2 0 3 2 1 1 0 0 5 2 0 0 bryum caespiticium hedw. 1 2 3 2 2 2 2 0 3 2 1 1 2 1 5 2 2 0 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 1 2 3 2 2 2 2 0 3 2 1 1 2 1 5 2 2 0 cratoneuron fi licinum (hedw.) spruce 0 2 3 2 2 2 2 3 3 2 4 2 2 1 5 2 2 8 didymodon tophaceus (brid.) lisa 2 1 3 2 2 2 2 0 3 2 1 1 0 0 5 2 2 0 eucladium verticillatum (brid.) bruch & schimp. 3 1 3 2 2 2 2 0 3 2 1 2 2 1 5 2 2 6 eurhynchium speciosum (brid.) jur. 3 2 1 0 0 2 2 0 0 2 1 2 2 1 0 2 2 2 eurhynchium hians (hedw.) sande lac. var. hians 3 2 1 0 0 2 2 0 0 2 1 2 2 1 0 2 2 2 taxometrics classifi cation of aquatic and semi-aquatic mosses – m.j. loo et al. 142 operational taxonomic unit (otu) 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.-nann. 3 2 3 2 2 2 1 3 3 2 4 2 2 1 5 2 2 8 fissidens grandifrons brid. 3 2 3 2 2 2 1 3 3 2 4 2 2 1 5 2 2 8 fontinalis antipyretica hedw. 0 2 3 2 2 2 2 3 3 2 4 2 2 1 5 2 2 7 fontinalis duriaei schimp. 0 2 3 2 2 2 2 3 3 2 4 2 2 1 5 2 2 7 gymnostomum calcareum nees & hornsch. 2 1 3 2 2 2 2 0 3 1 4 0 0 1 5 2 2 6 hygroamblystegium tenax (hedw.) jenn. 0 2 2 2 2 2 2 0 3 2 1 2 2 1 0 2 2 8 hymenostylium recurvirostrum (hedw.) dixon 3 2 3 2 2 2 2 3 3 2 0 2 2 1 5 2 2 7 leptodictyum humile (p. beauv.) ochyra 1 2 2 1 1 2 2 0 1 2 1 2 2 1 0 0 2 2 leptodictyum riparium (hedw.) warnst. 1 2 2 1 1 2 2 0 1 2 1 2 2 1 0 0 2 2 palustriella commutata (hedw.) ochyra 0 2 3 2 2 2 2 3 3 2 4 2 2 1 5 2 2 7 philonotis fontana (hedw.) brid. 0 2 3 2 2 2 2 0 3 2 1 1 2 1 5 2 2 3 platyhypnidium riparioides (hedw.) dixon 1 2 3 2 2 2 2 0 3 2 1 2 2 1 5 2 2 2 pohlia melanodon (brid.) a.j. shaw 0 2 3 2 2 2 2 3 3 1 1 0 2 1 5 2 2 4 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 0 2 3 2 2 2 2 3 3 1 1 0 2 1 5 2 2 4 tortula marginata (bruch & schimp.) spruce 0 1 3 2 2 0 2 1 3 2 4 0 0 1 2 2 1 0 table 5. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 barbula bolleana (müll. hal.) broth. 0 0 0 0 1 2 3 5 1 2 1 2 0 3 2 2 2 3 bryum caespiticium hedw. 0 0 2 1 2 5 0 0 0 1 3 1 2 1 0 2 2 1 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 0 0 2 1 2 5 0 0 0 1 3 1 2 1 0 2 2 1 cratoneuron fi licinum (hedw.) spruce 0 0 2 6 2 5 2 9 5 2 3 2 2 8 3 2 2 2 didymodon tophaceus (brid.) lisa 0 0 0 3 2 2 2 2 5 0 2 1 2 0 3 2 2 2 eucladium verticillatum (brid.) bruch & schimp. 0 0 2 6 2 3 2 3 5 1 1 1 2 0 3 2 2 2 eurhynchium speciosum (brid.) jur. 0 0 2 4 0 2 4 5 1 1 3 1 2 5 3 2 2 2 eurhynchium hians (hedw.) sande lac. var. hians 0 0 2 4 0 2 4 5 1 1 3 1 2 5 3 2 2 2 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.-nann. 0 0 2 6 2 5 2 9 5 1 3 1 2 8 3 2 2 2 fissidens grandifrons brid. 0 0 2 6 2 5 2 9 5 1 3 1 2 8 3 2 2 2 fontinalis antipyretica hedw. 0 0 2 6 2 5 2 9 1 1 3 1 2 1 3 2 2 2 fontinalis duriaei schimp. 0 0 2 6 2 5 2 9 1 1 3 1 2 1 3 2 2 2 gymnostomum calcareum nees & hornsch. 0 0 2 3 2 2 2 5 5 0 1 1 2 0 3 2 2 2 hygroamblystegium tenax (hedw.) jenn. 0 0 2 6 0 5 2 9 2 1 2 1 2 5 3 2 2 2 hymenostylium recurvirostrum (hedw.) dixon 0 0 2 6 2 1 2 9 5 1 3 1 2 6 3 1 2 2 leptodictyum humile (p. beauv.) ochyra 0 0 1 4 0 5 2 6 5 1 2 0 1 4 2 2 2 2 leptodictyum riparium (hedw.) warnst. 0 0 1 4 0 5 2 6 5 1 2 0 1 4 2 2 2 2 table 4. continued biotropia vol. 15 no. 2, 2008 143 operational taxonomic unit (otu) 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 palustriella commutata (hedw.) ochyra 0 0 2 6 2 5 2 9 5 1 3 1 2 8 3 2 2 2 philonotis fontana (hedw.) brid. 0 0 2 6 2 5 2 7 3 1 3 1 2 7 3 2 2 2 platyhypnidium riparioides (hedw.) dixon 0 0 2 4 0 5 2 4 5 1 3 1 2 5 2 2 2 2 pohlia melanodon (brid.) a.j. shaw 0 0 1 5 2 5 0 8 5 1 3 1 2 0 3 2 2 2 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 0 0 1 5 2 5 0 8 5 1 3 1 2 0 3 2 2 2 tortula marginata (bruch & schimp.) spruce 0 0 2 6 2 4 2 3 4 0 1 1 2 8 0 2 1 2 table 6. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 barbula bolleana (müll. hal.) broth. 1 1 0 0 2 0 0 0 1 2 2 3 3 3 3 3 3 0 bryum caespiticium hedw. 3 1 1 3 3 2 2 0 0 1 2 2 2 2 1 2 2 2 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 3 1 1 3 3 2 2 0 0 1 2 2 2 2 1 2 2 2 cratoneuron fi licinum (hedw.) spruce 3 1 1 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 didymodon tophaceus (brid.) lisa 1 0 1 3 2 2 1 0 0 0 1 2 2 2 2 2 2 1 eucladium verticillatum (brid.) bruch & schimp. 3 1 0 3 2 1 2 0 0 2 2 2 2 2 2 2 2 2 eurhynchium speciosum (brid.) jur. 3 1 1 3 3 2 2 2 0 2 2 2 2 2 2 2 2 2 eurhynchium hians (hedw.) sande lac. var. hians 3 1 1 3 3 2 2 2 0 2 2 2 2 2 2 2 2 2 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.-nann. 3 1 1 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 fissidens grandifrons brid. 3 1 1 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 fontinalis antipyretica hedw. 0 1 1 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 fontinalis duriaei schimp. 0 1 1 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 gymnostomum calcareum nees & hornsch. 1 0 0 3 1 2 2 1 0 0 1 2 2 2 2 2 2 2 hygroamblystegium tenax (hedw.) jenn. 4 2 0 1 3 2 2 2 1 0 2 2 1 1 2 2 2 3 hymenostylium recurvirostrum (hedw.) dixon 3 1 1 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 leptodictyum humile (p. beauv.) ochyra 2 1 0 3 3 2 2 2 0 1 2 2 2 2 1 2 2 2 leptodictyum riparium (hedw.) warnst. 2 1 0 3 3 2 2 2 0 1 2 2 2 2 1 2 2 2 palustriella commutata (hedw.) ochyra 3 1 0 3 3 2 2 2 2 2 2 2 2 2 2 2 2 2 philonotis fontana (hedw.) brid. 3 1 0 3 3 2 2 0 0 2 2 2 2 2 2 2 2 2 platyhypnidium riparioides (hedw.) dixon 3 1 0 3 3 2 2 2 0 2 2 2 2 2 1 1 2 2 pohlia melanodon (brid.) a.j. shaw 3 1 0 3 3 2 2 0 0 2 2 2 2 2 2 2 1 2 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 3 1 0 3 3 2 2 0 0 2 2 2 2 2 2 2 1 2 tortula marginata (bruch & schimp.) spruce 1 1 0 2 1 2 2 0 0 2 2 1 2 2 2 2 2 2 table 5. continued taxometrics classifi cation of aquatic and semi-aquatic mosses – m.j. loo et al. 144 table 7. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 barbula bolleana (müll. hal.) broth. 1 0 1 2 0 1 0 0 1 0 1 2 1 2 2 bryum caespiticium hedw. 1 0 1 2 1 0 0 2 2 1 1 2 1 2 2 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 1 0 1 2 1 0 0 2 2 1 1 2 1 2 2 cratoneuron fi licinum (hedw.) spruce 2 1 1 1 2 2 4 2 2 2 0 2 1 2 2 didymodon tophaceus (brid.) lisa 0 0 1 2 0 0 0 0 1 2 1 0 1 2 2 eucladium verticillatum (brid.) bruch & schimp. 1 0 1 2 4 0 0 2 2 2 1 2 1 2 2 eurhynchium speciosum (brid.) jur. 1 4 1 2 4 2 1 2 2 2 1 2 1 2 2 eurhynchium hians (hedw.) sande lac. var. hians 1 4 1 2 4 2 1 2 2 2 1 2 1 2 2 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.-nann. 1 4 3 2 4 2 4 2 2 2 1 2 1 2 2 fissidens grandifrons brid. 1 4 3 2 4 2 4 2 2 2 1 2 1 2 2 fontinalis antipyretica hedw. 1 4 3 2 3 2 4 2 2 2 1 2 1 2 2 fontinalis duriaei schimp. 1 4 3 2 3 2 4 2 2 2 1 2 1 2 2 gymnostomum calcareum nees & hornsch. 1 0 1 2 3 1 0 0 1 0 1 1 0 0 2 hygroamblystegium tenax (hedw.) jenn. 1 4 1 2 3 2 1 2 2 2 1 2 1 2 2 hymenostylium recurvirostrum (hedw.) dixon 1 4 3 2 4 2 4 0 1 2 1 2 1 2 2 leptodictyum humile (p. beauv.) ochyra 1 3 1 2 4 2 2 2 2 2 1 2 1 2 2 leptodictyum riparium (hedw.) warnst. 1 3 1 2 4 2 3 2 2 2 1 2 1 2 2 palustriella commutata (hedw.) ochyra 1 1 2 2 2 2 4 2 2 2 1 2 1 2 2 philonotis fontana (hedw.) brid. 1 0 1 2 3 0 0 2 2 2 1 2 1 2 1 platyhypnidium riparioides (hedw.) dixon 1 2 3 2 3 2 1 2 2 2 1 2 1 2 2 pohlia melanodon (brid.) a.j. shaw 1 0 1 2 3 0 0 2 2 2 1 2 1 2 1 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 1 0 1 2 3 0 0 2 2 2 1 2 1 2 1 tortula marginata (bruch & schimp.) spruce 1 0 1 2 3 0 0 2 2 2 1 2 1 2 2 table 8. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 barbula bolleana (müll. hal.) broth. 0 0 0 0 0 0 0 0 0 1 1 1 2 2 0 bryum caespiticium hedw. 0 0 1 1 0 0 1 1 2 1 0 0 1 2 1 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 0 0 1 1 0 0 1 1 2 1 0 0 1 2 1 cratoneuron fi licinum (hedw.) spruce 0 2 2 2 0 0 6 1 1 2 1 1 2 2 1 didymodon tophaceus (brid.) lisa 0 0 0 0 0 0 0 0 1 1 1 2 1 0 0 eucladium verticillatum (brid.) bruch & schimp. 0 2 2 2 1 2 6 1 2 2 0 1 2 2 1 eurhynchium speciosum (brid.) jur. 3 2 2 2 0 2 6 1 2 1 1 1 2 2 1 eurhynchium hians (hedw.) sande lac. var. hians 3 2 2 2 0 2 6 1 2 1 1 1 2 2 1 biotropia vol. 15 no. 2, 2008 145 operational taxonomic unit (otu) 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.-nann. 0 2 2 2 1 2 6 1 2 2 1 1 2 2 1 fissidens grandifrons brid. 0 2 2 2 1 2 6 1 2 2 1 1 2 2 1 fontinalis antipyretica hedw. 0 2 2 2 1 2 6 1 2 2 1 1 2 2 1 fontinalis duriaei schimp. 0 2 2 2 1 2 6 1 2 2 1 1 2 2 1 gymnostomum calcareum nees & hornsch. 0 0 1 0 0 0 0 1 2 1 1 0 2 2 0 hygroamblystegium tenax (hedw.) jenn. 3 2 2 2 0 0 6 1 2 1 1 1 2 2 1 hymenostylium recurvirostrum (hedw.) dixon 3 2 2 2 1 2 6 1 2 2 1 1 2 2 1 leptodictyum humile (p. beauv.) ochyra 3 2 2 2 0 0 6 1 2 1 1 1 2 2 1 leptodictyum riparium (hedw.) warnst. 3 2 2 2 0 0 6 1 2 1 1 1 2 2 1 palustriella commutata (hedw.) ochyra 0 2 2 2 0 0 6 1 2 2 1 1 2 2 1 philonotis fontana (hedw.) brid. 1 0 1 0 0 0 4 1 2 1 1 1 2 2 1 platyhypnidium riparioides (hedw.) dixon 3 2 2 2 0 0 6 1 2 1 1 1 2 2 1 pohlia melanodon (brid.) a.j. shaw 4 0 1 0 0 0 0 0 2 1 0 1 2 2 1 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 4 0 1 0 0 0 2 0 2 1 0 1 2 2 1 tortula marginata (bruch & schimp.) spruce 1 0 1 0 0 0 6 1 1 1 1 0 2 1 0 table 9. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 barbula bolleana (müll. hal.) broth. 0 2 2 2 1 0 0 3 3 1 1 2 2 1 0 bryum caespiticium hedw. 2 2 2 2 1 4 2 3 1 0 0 1 0 2 0 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 2 2 2 2 1 4 2 3 1 0 0 1 0 2 0 cratoneuron fi licinum (hedw.) spruce 2 2 2 2 3 4 2 3 3 1 1 2 2 1 1 didymodon tophaceus (brid.) lisa 0 2 2 0 1 2 2 3 1 1 1 2 2 1 1 eucladium verticillatum (brid.) bruch & schimp. 2 2 2 2 3 4 2 3 3 1 1 2 2 2 1 eurhynchium speciosum (brid.) jur. 2 2 2 2 3 4 2 3 2 1 1 2 2 1 1 eurhynchium hians (hedw.) sande lac. var. hians 2 2 2 2 3 4 2 3 2 1 1 2 2 1 1 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.-nann. 2 2 2 2 3 4 2 3 3 1 1 2 2 1 1 fissidens grandifrons brid. 2 2 2 2 3 4 2 3 3 1 1 2 2 1 1 fontinalis antipyretica hedw. 2 2 2 2 3 4 2 3 3 1 1 2 2 1 1 fontinalis duriaei schimp. 2 2 2 2 3 4 2 3 3 1 1 2 2 1 1 gymnostomum calcareum nees & hornsch. 2 1 1 2 1 1 2 3 3 1 1 2 2 1 1 hygroamblystegium tenax (hedw.) jenn. 2 2 2 2 3 4 2 3 3 1 1 2 2 1 1 hymenostylium recurvirostrum (hedw.) dixon 2 2 2 2 0 4 2 3 3 1 1 2 2 1 1 leptodictyum humile (p. beauv.) ochyra 2 2 2 2 3 2 2 0 3 1 1 2 2 0 1 leptodictyum riparium (hedw.) warnst. 2 2 2 2 3 2 2 0 3 1 1 2 2 0 1 taxometrics classifi cation of aquatic and semi-aquatic mosses – m.j. loo et al. table 8. continued 146 operational taxonomic unit (otu) 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 palustriella commutata (hedw.) ochyra 2 2 2 2 3 4 2 3 3 1 1 2 2 1 1 philonotis fontana (hedw.) brid. 2 2 2 2 3 4 2 3 1 1 1 2 2 1 1 platyhypnidium riparioides (hedw.) dixon 2 2 2 2 3 4 2 3 3 1 1 2 2 1 1 pohlia melanodon (brid.) a.j. shaw 2 2 2 2 3 3 2 3 3 1 1 2 2 1 0 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 2 2 2 2 3 3 2 3 3 1 1 2 2 1 0 tortula marginata (bruch & schimp.) spruce 2 0 2 0 1 4 1 2 3 1 1 2 2 1 1 table 10. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 136 137 138 139 140 141 142 143 144 145 146 barbula bolleana (müll. hal.) broth. 0 0 1 0 0 0 0 5 2 1 1 bryum caespiticium hedw. 0 2 3 2 3 1 2 2 0 1 0 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 0 2 3 2 3 1 2 2 0 1 0 cratoneuron fi licinum (hedw.) spruce 1 2 3 2 3 1 2 5 2 2 1 didymodon tophaceus (brid.) lisa 1 0 2 0 0 0 2 0 2 1 1 eucladium verticillatum (brid.) bruch & schimp. 1 2 3 2 1 1 2 2 1 1 1 eurhynchium speciosum (brid.) jur. 1 2 3 2 3 1 2 5 2 1 1 eurhynchium hians (hedw.) sande lac. var. hians 1 2 3 2 3 1 2 5 2 1 1 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.nann. 1 2 3 2 3 1 2 5 2 2 1 fissidens grandifrons brid. 1 2 3 2 3 1 2 5 2 2 1 fontinalis antipyretica hedw. 1 2 3 2 3 1 2 3 2 2 1 fontinalis duriaei schimp. 1 2 3 2 3 1 2 3 2 2 1 gymnostomum calcareum nees & hornsch. 1 0 2 2 2 1 2 2 2 1 1 hygroamblystegium tenax (hedw.) jenn. 1 2 3 2 3 1 2 5 2 2 1 hymenostylium recurvirostrum (hedw.) dixon 1 2 3 2 3 1 2 5 2 2 1 leptodictyum humile (p. beauv.) ochyra 1 2 3 2 3 1 2 0 2 1 1 leptodictyum riparium (hedw.) warnst. 1 2 3 2 3 1 2 0 2 1 1 palustriella commutata (hedw.) ochyra 1 2 3 2 3 1 2 5 2 2 1 philonotis fontana (hedw.) brid. 1 2 3 2 1 1 2 2 2 1 1 platyhypnidium riparioides (hedw.) dixon 1 2 3 2 3 1 2 5 2 1 1 pohlia melanodon (brid.) a.j. shaw 0 2 3 2 3 1 2 2 1 1 1 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 0 2 3 2 3 1 2 2 1 1 1 tortula marginata (bruch & schimp.) spruce 1 2 3 2 1 1 2 2 2 1 1 biotropia vol. 15 no. 2, 2008 table 9. continued 147 table 11. matrix table showing the character states (156 characters) for the 23 otus of aquatic and semi-aquatic mosses. operational taxonomic unit (otu) 147 148 149 150 151 152 153 154 155 156 barbula bolleana (müll. hal.) broth. 2 2 1 2 0 0 0 1 2 1 bryum caespiticium hedw. 1 1 1 2 0 2 2 1 2 1 bryum pseudotriquetrum (hedw.) p. gaertn., b. mey & scherb. 1 1 1 2 0 2 2 1 2 1 cratoneuron fi licinum (hedw.) spruce 2 2 1 2 1 2 2 1 2 1 didymodon tophaceus (brid.) lisa 2 2 1 2 0 1 1 1 2 1 eucladium verticillatum (brid.) bruch & schimp. 2 2 1 2 0 2 2 1 2 1 eurhynchium speciosum (brid.) jur. 2 2 1 2 1 2 2 0 2 1 eurhynchium hians (hedw.) sande lac. var. hians 2 2 1 2 1 2 2 0 2 1 fissidens crassipes subsp. warnstorfi i (m. fleisch.) brugg.nann. 2 2 1 2 1 2 2 1 2 1 fissidens grandifrons brid. 2 2 1 2 1 2 2 1 2 1 fontinalis antipyretica hedw. 2 2 1 2 1 2 2 1 2 1 fontinalis duriaei schimp. 2 2 1 2 1 2 2 1 2 1 gymnostomum calcareum nees & hornsch. 2 2 1 2 0 2 2 1 2 1 hygroamblystegium tenax (hedw.) jenn. 1 2 2 1 2 2 2 1 2 1 hymenostylium recurvirostrum (hedw.) dixon 2 2 1 2 1 2 2 1 2 1 leptodictyum humile (p. beauv.) ochyra 2 2 0 2 1 2 2 0 0 0 leptodictyum riparium (hedw.) warnst. 2 2 0 2 1 2 2 0 0 0 palustriella commutata (hedw.) ochyra 2 2 1 2 1 2 2 1 2 1 philonotis fontana (hedw.) brid. 2 2 1 2 0 2 2 1 2 1 platyhypnidium riparioides (hedw.) dixon 2 2 1 2 1 2 2 0 2 1 pohlia melanodon (brid.) a.j. shaw 2 2 1 2 0 2 2 1 2 1 pohlia wahlenbergii (f. weber & d. mohr) a.l. andrews 2 2 1 2 0 2 2 1 2 1 tortula marginata (bruch & schimp.) spruce 2 2 1 1 0 2 2 1 2 1 th e output of the analysis is presented in phenogram (figure 1). in the fi rst, second and third node, the linkage distance value is 1.0000 for bryum caespiticium and bryum pseudotriquetrum, eurhynchium speciosum and eurhynchium hians var. hians; and, leptodictyum humile and leptodictyum riparium, respectively. th e distance linkage between pohlia melanodon and pohlia wahlenbergii is 2.0000 (node 4). as for node 5, the value is 3.0000 between fontinalis antipyretica and fontinalis duriaei.two diff erent genera of cratoneuron fi licinum and palustriella commutata showed the value of 3.7417 in node 6. fissidens crassipes subsp. warnstorfi i and fissidens grandifrons in the seventh node are distantly valued 4.0000. at the eighth node (6.4807), cratoneuron fi licinum, palustriella commutata, fissidens crassipes subsp. warnstorfi i and fissidens grandifrons were linkaged. next, cratoneuron fi licinum, palustriella commutata, fissidens crassipes subsp. warnstorfi i, fissidens grandifrons and hymenostylium recurvirostrum were clustered under node 9 with linkage distance value of 8.0000. at the tenth node, cratoneuron fi licinum, palustriella commutata, fissidens crassipes subsp. warnstorfi i, fissidens grandifrons, hymenostylium recurvirostrum, fontinalis antipyretica and fontinalis duriaei were distantly measured with value of 9.6954. taxometrics classifi cation of aquatic and semi-aquatic mosses – m.j. loo et al. 148 with distance linkage value of 10.2956, three species of leptodictyum humile, leptodictyum riparium and platyhypnidium riparioides were linkaged under node 11. next, eurhynchium speciosum, eurhynchium hians var. hians, leptodictyum humile, leptodictyum riparium and platyhypnidium riparioides were analyzed to have distance linkage value of 10.3923 (twelfth node). under node 13, there are eurhynchium speciosum, eurhynchium hians var. hians, leptodictyum humile, leptodictyum riparium, platyhypnidium riparioides and philonotis fontana (11.3137). cratoneuron fi licinum, palustriella commutata, fissidens crassipes subsp. warnstorfi i, fissidens grandifrons, hymenostylium recurvirostrum, fontinalis antipyretica, fontinalis duriaei, eurhynchium speciosum, eurhynchium hians var. hians, leptodictyum humile, leptodictyum riparium, platyhypnidium riparioides and philonotis fontana are under one cluster group (node 14 and distance linkage equals to 11.5326). under node 15, didymodon tophaceus and gymnostomum calcareum were found with 11.7898 value. for the sixteenth node, cratoneuron fi licinum, palustriella commutata, fissidens crassipes subsp. warnstorfi i, fissidens grandifrons, hymenostylium recurvirostrum, fontinalis antipyretica, fontinalis duriaei, eurhynchium speciosum, eurhynchium hians var. hians, leptodictyum humile, leptodictyum riparium, platyhypnidium riparioides, philonotis fontana and hygroamblystegium tenax were having distance value of 11.8743. with linkage distance value of 12.0831, didymodon tophaceus, gymnostomum calcareum and eucladium verticillatum were grouped under node 17. node 18 showed the combination between node 16 and 17 with 12.4097. distance linkage value of 12.4499 grouped all otus under node 18 and node 4 to become node 19. as for node 20, the combination is between node 19 and tortula marginata (12.5300). barbula bolleana and node 20 (12.8452) formed node 21. th e distance linkage value for all otus is 12.8564. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 linkage distance bryum pseudotriquetrum bryum caespiticium tortula marginata pohlia wahlenbergii pohlia melanodon eucladium verticillatum gymnostomum calcareum didymodon tophaceus hygroamblystegium tenax philonotis fontana platyhypnidium riparioides leptodictyum riparium leptodictyum humile eurhynchium hians var. hians eurhynchium speciosum fontinalis duriaei fontinalis antipyretica hymenostylium recurvirostrum fissidens grandifrons fissidens crassipes subsp. warnstorfii palustriella commutata cratoneuron filicinum barbula bolleana figure 1. dendogram showing the single linkage (nearest neighbours) clustering relationship based on euclidean distance among the 23 species of mosses biotropia vol. 15 no. 2, 2008 149 according to the principal component and classifi cation analysis, the quality of representation value is 100% or most reliable. th e fi rst three components explained 75.1064% of the variation with 56.0485%(pc1), 11.7346%(pc2) and 7.3233%(pc3) respectively (table 12). components with eigenvalues lower than 1 were eliminated and not signifi cant statistically. for pc1, the main variables are from the vegetative parts (plant and leaf ) and major morphometric characters are numbered 3, 8, 9, 26, 27, 33 (negative loading), 51 (negative loading), 54 (negative loading), 55, 56, 58 (negative loading), 60 (negative loading), 63 (negative loading), 68 (negative loading) and 143 (negative loading). less important on the reproductive part (capsule): 110, 112 (negative loading) and 126 (negative loading). pc2 showed the major variable is alar cells of leaf (92). th e focal part is on the vegetative component of the bryophyte. factor loading scores for pc3 were less correlated to the variables (characters) as compared to pc1 and pc2 . any factor score lower than 5.0000 is considered insignifi cant and eliminated from the factor loading tables. table 12. taxometric variables for the first three principal components component eigenvalue % total variance cumulative % 1 12.89116 56.04853 56.0485 2 2.69895 11.73459 67.7831 3 1.68436 7.32332 75.1064 figure 2. shows that mosses are skewed obviously to the negative side for pc1. pc1 grouped species obviously into three groups (i, j, o, d, r, n, k, l, p, q, h and g; s, v, u and f; and e, b and c) and 4 identical individual species (t, w, m and a). as for pc2 (figure 2), 2 groups were segregated into the positive (i, j, o, d, r, n, k, l, p, q, h and g) and negative (s, v, u, f, m, a, e, b and c) sides; and 2 individual species near to the intermediary part (t and w). taxometrics classifi cation of aquatic and semi-aquatic mosses – m.j. loo et al. a b c d e f g h i j k l m n o p q r s t u v w -1.00 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 1.00 factor 1 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 factor 2 figure 2. scatter diagram between pc1 and pc2 from principal component analysis of 23 species of aquatic and semi-aquatic mosses using 156 characters 150 in figure 3, pc3 grouped otus into one positive group of h, g, b, c, p, q, t, n and w; negative group of m, e, a, k, l, r, i,d, j, k, l, o, v and u; one species under intermediary line (f ); and one species near to intermediary line (s). pc1 (figure 3) segregated the individuals inconspicuously. basically, individual species grouped under intermediary area is sharing characters from positive and negative sides. figure 3. scatter diagram between pc1 and pc3 from principal component analysis of 23 species of aquatic and semi-aquatic mosses using 156 characters for cluster analysis, the algorithm chosen was hierarchical (aggromerative) (tipirdamaz et al. 2006). th is is because the main objective of this study is to group otus from smaller clusters into a larger groups (polythetic) (stuessy 1990). th e end result is to divide clustered groups for effi cient aquatic and semiaquatic bryodiversity management. th e amalgation rule for analysis was single linkage (nearest neighbour) as the purpose to study the species relationship among otus and the measurement for distance between species was based on euclidean distance. euclidean distance is the most common and easy to interpret (statistica 6.0, 2001). in this analysis, numbers of variables, cases and subcases analyzed were massive. indirectly, biases and standard deviations were minimized. as the result, the reliability of the output is more than 96%. on the other hand, clustering bryodiversity into few manageable units are very crucial and cost-eff ective. th e relationship between a cluster of mosses refl ects the generic, familial or higher taxonomic similarity. genetically, they are sharing a closer gene pool (genotypes) and morphologically, the phenotypes are signifi cant characters for identifi cation and serving the ecosystem. mosses are natural bioindicator for water quality, soil erosion controller and fi ltering the wastewater naturally (ando and matsuo 1984; biotropia vol. 15 no. 2, 2008 a b c d e f g h i j k l m n o p q r s t u v w -1.00 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 1.00 factor 1 -0.50 -0.25 0.00 0.25 0.50 factor 3 151 frahm 1996; welch 1948; conrad 1935; whitehouse and mcallister 1954; ando 1957; grout 1912; coupal and lalancette 1976). th us, this approach can help bryodiversity managers to conserve the aquatic and semi-aquatic mosses in a collective way. in a simple manner, we effi ciently manage all the clusters of mosses equally. equality helps in balancing the habitat (ecosystem) for the benefi ts of human beings. ironically, wrong management strategy can be bias to certain species, the other species will be neglected and the ecosystem will not be served naturally. from the dendogram (figure 1), there are 22 nodes. th us, management strategy can be based on the nodes in the phenogram. for node 10, one management cluster can be formed from few subclusters which consists of cratoneuron fi licinum, palustriella commutata, fissidens crassipes subsp. warnstorfi i, fissidens grandifrons, hymenostylium recurvirostrum, fontinalis antipyretica and fontinalis duriaei. subclusters of node 13 (eurhynchium speciosum, eurhynchium hians var. hians, leptodictyum humile, leptodictyum riparium, platyhypnidium riparioides and philonotis fontana), node 18 (didymodon tophaceus, gymnostomum calcareum, hygroamblystegium tenax and eucladium verticillatum), node 20 (tortula marginata, pohlia wahlenbergii and pohlia melanodon) and node 22 (bryum caespiticium, bryum pseudotriquetrum and barbula bolleana). in short, fi ve management units of bryodiversity are proposed for management. under principal component analysis (pca), management of clustered groups are strengthened (tipirdamaz et al. 2006). based on statistica 6.0 (2001), pca is reducing the numbers of variables and transform important variables into numbers of principal component. th is is benefi cial in management, where precise group of identifi able characters (in this context) are known for management and conservation. furthermore, pca is a very cost-eff ective tool for biodiversity management. in cluster analysis, bryodiversity management is based on hierachical clustered manageable unit and pca is based on group of related characters that forms one factor. in this case, we have three principal components. in the fi rst principal component (figure 2), we have three distinct groups and four independent otus (can form two minor groups). th us, 5 managable units can be formed. all otus were skewed to the negative side. statistically, it signifi es all the otus were likely characterized diff erently from the common character states. th is is very similar to the numbers of manageable units derived from cluster analysis, but with distinct species combination. as for the second component, 3 managable units were formed. it consists of a positive group that agrees with most of the common character states, a negative group that complies likely to the opposite character states and two intermediary individuals (skewed a bit to the negative side) where sharing both common and uncommon (more) character states. in figure 3, three manageable units were observed: one positive-skewed group, one negative-skewed group and one individual in the intermediary line. comparing both taxometric classifi cations, cluster analysis is useful in hierachically linked otus for relationship-based management approach. all characters have the same weight and will be used totally. as for pca, it groups otus on the scatter plot that refers to the group of important characters (principal component). th us, only critical characters are used for effi cient management. relatively, both approaches to bryodiversity management are highly appreciated. only through cluster analysis, the linkage distance will be known and important for future populational references. th is means that populations are taxometrics classifi cation of aquatic and semi-aquatic mosses – m.j. loo et al. 152 evolving and further revisions will further change the taxonomic structure. for instance, pca does not show this feature. in a nutshell, both approaches are supplementing each ones. both combinations will help in solving managerial dilemma and problems. conclusion bryodiversity in the mediterrean area of spain is relatively lower (23 species of aquatic and semi-aquatic mosses) as compared to the 46 species recorded in the tropical region of sabah, malaysia.bryofl ora conservation cum management have to be taken place. th is is critical as the bryophytes are naturally serving the ecosystems continually and sustainably. th e studied mosses were phenetically related and could be divided into 5 cluster groups through cluster analysis. th e clustered group can be managed as a manageable unit. th e rationale is that no single population will be overmanaged or neglected; and equal conservational plan to be implemented among the phenetically related units. th e manipulation of principal component analysis (pca) in this study reduced the least important characters used for management. th e output produced three components with each group contain numbers of vital characters within. th is is costeff ective for bryodiversity managers. th is analysis allowed managers to identify, manage and conserve populations based on the components. from this study, output from cluster analysis will be an alternative to the results produced from the pca. nevertheless, both outputs are highly reliable and ready to be used for management. in a nutshell, it is more meaningful to conserve natural environmental regulator rather than creating man-made mitigator. acknowledgments th e grant for this project was from european commission (ec) under the erasmus mundus programme (emp). th e author would like to express his thankfulness to mr. reymond p. echavaria iii for his comments on the statistical analyses. references aguilar d., f.x.aviles , e. querol and m.j.e. sternberg. 2004. analysis of phenetic trees based on metabolic capabilities across the th ree domain of life. journal of molecular biology 340: 491-512. ando h. 1957. notes on useful bryophytes. bulletin of the biology society of hiroshima university 7(2): 23-26. ando h. and a. matsuo. 1984. applied bryology. advances in bryology 2: 133-224. conrad h.s. 1935. mosses and soil erosion. iowa state college journal of science 9: 347-351. coupal b. and j.m. lalancette. 1976. th e treatment of waste water with peat moss. water research 10: 1071-1076. biotropia vol. 15 no. 2, 2008 153 ferguson m. e., n. maxted, m.v. slageren and l.d. robertson. 2000. a re-assessment of the taxonomy 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(1988) had studied the bioconversion of papaverine in glycyrrhiza gabra cell suspension and found the bioconversion rate into papaverinol (31.5%), papa-veraldine (4%) and demethylpapaverine was rather slow. in other case, hydro quinone was glucosylated into arbutin after addition to cell suspension cultures of datura innoxia (suzuki et al. 1987) in production rate of 2.5 gl-1 after of incubation biotropia no. 20, 2003 feeding of 3 mm l-tyrosine or phenylalanine into lithospermum flavum cultures yielded a 2-3 fold increase in 5-methoxy podophyllotoxin accumulation (van uden et al. 1990). the byconversion rates were low; 0.34 and 0.5 mg g d , respectively. it is clear that there are significant differences in bioconversion rates. several reasons include that; the rate may be dependent on the solubility of precursors and/or the intracellularly present amounts of enzyme. furthermore, the precursors can be metabolized by other reactions and even a specific transport system may be involved. funk and brodelius (1990) have used precursor feeding and addition of metabolic inhibitors techniques as a tool to evaluate the possibility that involved to the undetactable vanillin from the cultivated cells of vanilla plan/folia. they found that cinnamic acid, but not ferulic acid, is a precursor of vanillic acid in these cultivated cells of v. plamfolia. extraction of citrus grandis or pomelo peel contained almost exclusively limonene (92.6%) and myrcena (1.9%) suggests that pomelo may be utilized as source material for the production of limonene (wong 1992). the precursors for the synthesis of monoterpenoids are obtained from acetate-mevalonate pathway, which supply to mevalonic acid. mevalonic acid is the primary precursor of all the terpenoids biosynthesized by plant, including limonene. orange juice vesicles (citrus sp., rutaceae) contain an enzyme system which is capable of phospho-rylating mevalonic acid and forming the final product, a monoterpene, linalool. intact fruits and leaves convert linalooi to limonene and other cyclic monoterpenoids. linalool is considered as an immediate precursor which is expected to readily converted to limonene under favourable conditions. there are numerous reports of experiments on mevalonic acid as a precursor. moreno et al. (1993) reported that feeding mevalonic acid did not increase alkaloid accumulation in cell suspension cultures of catharanthus roseus. mevalonic acid added to cultures at concentration of 3.3 mm was reported to saturate the 030 (steroid) biosynthetic pathway in nicotiana tabaccum suspension cultures (threfall & whitehead 1988). with the aim of shortening the period for limonene production and enhancing its accumulation, the effect of feeding primary and immediate precursors was investigated. materials and methods culture conditions young c. grandis fruits about five weeks after anthesis and 4-5 cm in diameter were obtained from established plantation. sterilization technique was established by immersing the whole fruit into 20% clorox® (5.2% sodium hypochloride) for 2 hours prior to peeling and cutting. the fruits were rinsed with sterile distilled water three times and cut transversely into small segments. callus was initiated on ms (murashige & skoog 1962) basal medium supplemented with 30 mg/1 sucrose. 3.0 me/1 2.4-d 3.0 mg/1 kinetin and 0.2 mg/1 aba and incubated in the dark 24 influence of mevalonic acid and linalool on limonene accumulation nik norulaini nik ab rahman et al. hours/day at room temperature. callus were formed after 2-6 weeks with a success rate of 80-90%. callus were maintained on the media and subcultured to the same fresh media every 4 weeks interval. feeding of precursors mevalonic acid lactone ( mva) and linalool (analytical grades) were purchased from fluka ( japan). aqueous solution of mva was prepared by adding 50 ml distilled water to 1 g of mva powder. mva was filter sterilized and aliquots were added to different ms media to get a range of final mva concentrations of 0.04, 0.08, 0.38, 0.77, 1.15 and 1.54 mm in the media. various volume of linalool (~ 97% purity), 10, 50, 100, 150 and 200 μj were added to 1 liter ms media prior to autoclave. after 4 weeks, callus from maintenance media were cut into smaller pieces and transferred by spreading over the same ms medium with varying added precursors. callus growth determination callus fresh weight was taken as final weight started from the fourth week followed by fifth, sixth, seventh and eighth week of incubation period by direct measurement. to minimize the differences in growth, the initial weight of the callus prior transferring to the experimental ms media was ensured about equal according to each treatment. callus was cleaned from attached media and dried using tissue paper before weighing using analytical balance (sartorius, germany). several plates were sacrificed along the time course of the experiment, to measure the biomass accumulate. limonene determination and analysis extraction of limonene was carried out from four to eight weeks old callus. fresh callus weighing 2.0 3.0 g were extracted with approximately 70 ml methanol using soxhlet apparatus according to morris et al. (1985). identifications and quantification of limonene were made by comparing with standard purchased from fluka using gas chromatography (shimadzu gc 17a) equipped with fid detector. column used was bp-20 (0.25 (im, 30m x 0.25mm) from sge. the temperature was programmed at 60 c, 4 min, 140 c, 8 min. injector and detector temperature was 250 c respectively. the experiments were performed in triplicates and data were subjected to analysis of variance (anova). biotropia no. 20, 2003 results effect of various concentrations of mva feeding and incubation period on callus growth rate figure 1 shows the effect of various concentrations of mva feeding on callus fresh weight. comparisons of callus fresh weights, taken as an indicator of cell proliferation were determined within the eighth weeks period for all mva feeding tested. addition of exogenous mva enhanced the callus growth even at low level except for 0.04 mm compared to the control. however, as the concentration of mva feeding increased, the callus fresh weight increased concomitantly as pointed out in the figure. among the concentrations used, there was no significant difference (p>0.05) between 0.38 and 0.77 mm and between 1.15 and 1.54 mm (table 1). highest callus fresh weight was obtained with the feeding of 1.15 and 1.54 mm of mva where increase in fresh weight was about two times higher compared to control callus. influence of mevalonic acid and linalool on limonene accumulation nik norulaini nik ab rahman et al. table 1 : effects of mevalonic acid concentrations on callus growth and limonene accumulation of citrus grandis after culture for 7 weeks. mva(mm) fresh weight (g) limonene (mg/g) 0.04 3.127a 0.0015c 0.08 3.900c 0.003 le 0.38 4.868d 0.0030e 0.77 4.807d 0.0020d 1.15 5.335e o.ooisc 1.54 5.574e 0.0013c control 3.218b 0 different alphabet for each data indicates a significant difference at a = 0.05 according to tukey hsd test. the figure also reveals that mva feeding exerts positive effect on callus growth throughout the eight-week culture duration. the most active growth occurred from fourth to sixth week in incubation. callus fresh weight was measured beginning the fourth week of incubation. first measurements of fresh weight were done at week four and at this point all mva feedings showed a higher callus fresh weight than control except for 0.04 mm mva feeding. however, subsequently each feeding concentration displays similar callus growth. higher concentrations of mva feeding illustrates greater callus growth from fourth to seventh week in culture. for all corcentrations, the fresh weights achieved constant level after the seventh week. effect of various linalool feeding concentrations and incubation period on callus growth rate all feeding concentrations of linalool enhanced callus growth rate, though to a different degree. figure 2 shows that only the addition of 10 to 100 jil/1 had resulted on distinctively different (p<0.005) in callus fresh weight proportional to linalool concentrations. feeding of higher than 100 (j.1/1 showed slightly reduction in callus growth but still higher than control callus. callus showed fresh weight increment at the beginning of incubation period at the fourth week and increased in fresh weight occurred after longer incubation period until the seventh week. after this period, fresh weight remained about level indicating callus had slowed down or reached constant growth. it was about twofold increment in callus fresh weight compared to control treatment when added with 50 (il/l and above of linalool. effect of incubation period and various mva feeding concentrations on limonene accumulation the combined effect of various concentrations of mva feeding and the incubation period is shown in figure 3. compared to cultures with no exogenous mva added, other cultures with mva feeding showed limonene as early as four weeks in culture. this accumulation continues up to the seventh week, whereby limonene accumulation displays a sudden decline. this phenomenon was exhibited in all mva concentrations used. however, cultures fed with 0.08 mm mva shows a more active limonene accumulation from fourth to seventh week and this is followed by 0.38 mm mva which after the fifth week, shows a drastic increase in limonene accumulation to match that accumulated in cultures fed with 0.08 mm mva. other concentrations of mva feeding profile displayed an increase from fourth to seventh week and a decline thereafter in much lower limonene accumulation. greatest effect of mva concentrations was observed on limonene accumulation using 0.08 and 0.38 mm, whereby there is no significant difference influence of mevalonic acid and linalool on limonene accumulation nik norulaini nik ab rahman et al. between the two (table 1). outside these concentrations, the accumulation of limonene was significantly lower (p>0.05). for cultures with no exogenous mva added that is the control, no limonene was detectable. effect of various linalool feeding concentrations and incubation period on limonene accumulation results from figure 4 shows that limonene accumulation was detected from feeding of linalool at all concentrations studied. there was no particular pattern on limonene accumulation with various linalool feeding concentrations. limonene content seems to accumulate at a significant amount when 10 and 50 μl/1 of linalool was added. this amount slightly decreased with feeding of 100 μl/l of linalool and increased instantly with 150 μ.1/1 before dropped again at concentration of 200 μ.1/1 of linalool. the figure also demonstrated that limonene accumulation was highest at seventh week of incubation period. however, limonene accumulation with 200 μl/1 exogenous linalool addition gave maximum peak at week six. limonene value dropped after the seventh week of incubation regardless of linalool feeding concentrations. most rapid increase of limonene accumulation occurred between six to seven weeks of incubation and the feeding of 150 μl/1 of linalool giving the highest accumulation (table 2). influence of mevalonic acid and imalool on limonene accumulation nik norulaini nik ab rahman et al. discussion effect of precursor feeding on callus growth effect on callus growth was studied for c. grandis cultured with feeding of exogenous mva ( figure 1) and linalool ( figure 2) under various concentrations and incubation period. the growth was proportional to the concentrations of precursors used meaning that higher precursors concentrations influenced more growth on c. grandis callus culture except at very low concentration, which is 0.04 mm of mva. obvious callus growth was observed from week five to week six and began to reduce after week seven where the increase was about threefold by fresh weight. during this time, callus turned brown indicated that the accumulation of secondary metabolites in cells had caused toxicity and started to die. increase in callus growth showed that precursor supplied at the studied range did not inhibit callus growth in fact improve growth development. similar result by moreno et al. (1993) stated that feeding of the terpenoid precursors mevalonic acid, loganin, loganic acid or secologanin to suspension cultures of catharanthus roseus did not affect the culture growth, as determined by dry weight concentrations measured at 72 and 120 hrs after feeding. these results demonstrated no toxic effect for all precursors added to the cultures. decreased in callus fresh weight after the seventh week could coincide with the initiation of secondary metabolites synthesize as cell growth approached stationary phase. lindsey and yeoman (1983) concluded that there is indirect relationship between cells growth and secondary metabolites accumulation. it is expected that secondary metabolites accumulation was stimulated only when cells slow down their rate of growth i.e. at the end of the logarithmic phase and the beginning of the stationary phase. ketchum et al. (1999) showed that taxus suspension cultures accumulated maximum levels of taxoid at the end of the growth phase. this conclusion could be related to the finding of this study. interestingly, the rate of growth and highest fresh weight value attained for both precursors did not differ significantly (p>0.05). this concluded that both precursors have had same potential to serve as internal growth stimulator acting to shoot up various aspects of cell metabolism. it was suggested that rapid growth at the early stage of callus was due to synergistic effect of feeding precursors and added growth factors. this phenomenon was demonstrated when 2,4-d was reduced in immobilized cultures of catharanthus roseus cells that rised up the production of alkaloid (tom et al 1991). decrease in growth rate after week seven for fed cells was found a little greater than nonfed cells. reasonable explanation for this phenomenon is that the accumulation of limonene and linalool themselves might have caused cells toxicity which led to cell fatality (brown et al 1987; charlwood & brown 1987). biotropia no. 20,2003 effect of precursors feeding on limonene accumulation mevalonic acid lactone and linalool showed quite similar precursor feeding effects on c. grandis callus cultures. illustrations in figure 3 for mva feeding and figure 4 for linalool feeding revealed that limonene production was triggered with the introduction of mva and linalool at low concentration. limonene accumulation was detected as early as week four and continued to increase until the seventh week. the highest limonene accumulation was 0.0030 mg/g and 0.0032 mg/g obtained from cultures fed with 0.08 mm of mva for the former and 150 um of linalool for the later on the seventh week of incubation (tables 1 & 2). the results proved that precursor feeding had triggered limonene production in c. grandis cultures earlier than non fed cultures as suggested by moreno et al. (1993). they stated that exogenous supply of a biosynthetic precursor, secologanin, to the culture medium may improve metabolites, such as strictosidine and ajmalicine accumulation where the productivity is limited by lack of that particular precursor. it was showed when they added the terpenoid precursor during the growth phase, about 11-fold increase in strictosidine occurred 72 hrs after feeding and decreased to 5 fold after 120 hrs. similar pattern was observed from imbault et al. (1996) on mva feeding effects. they showed that the inhibition of endogenous mva production by pravastatin treatment blocked the alkaloid biosynthesis; supplying such cells with exogenous mva allowed accumulation of alkaloid, but did not result in incorporation of mva. however, moreno et al. (1993) did not find any significant increase in alkaloid level after mevalonic acid feeding, the basic precursor for all terpenoids, in catharanthus roseus suspension cultures. for both treatments, it is concluded that limonene production was not dependent on amount of precursors added. it showed that only a small amount of mva and linalool was needed as a stimulator for limonene production. other possibility that contributes to this finding was the facts that mva must be catalytically phosphorylated by atp and mevalonic kinase itself for the pathway to continue. therefore, the lack of those co-factor and enzyme had restricted limonene production eventhough with the presence of high amount of precursor (rohmer et al. 1996; lichtenthaler et al. 1997). the strong role of enzymes and its incorporation with feeding precursor in study of biosynthesis was briefly described by zenk (1991) in his review. besides, for accumulation of a secondary compound to occur in cultured cells, it is clearly necessary that the system retains the ability both to synthesize and to store the product. trend in limonene accumulation shows that cell maturity has direct relation to secondary metabolite accumulation with both precursor feeding. it was obvious that feeding of both precursors accumulated highest peak of same amount of limonene at the same incubation period. highest limonene accumulation at the seventh week suggested that cells had slowed down their division to prepare themselves for metabolite production. this result fulfilled the suggestion from lindsey and yeoman (1983) that rapidly growing cultures accumulate cells and not secondary products. a similar pattern of results was obtained from suspension culture of influence of mevalonic acid and linalool on limonene accumulation nik norulaini nik ab rahman et al. podophyllum hexandrum on the production of podophyllotoxin (pras 1992). both situations demonstrated that the increase in metabolite compounds accumulation occurred only at the mature-death stage of the growth curve. consideration also has to take account for limonene itself an intermediate product and would be converted to other form of monoterpenoid. this situation is definitely uncontrollable because biosynthesis is a continuous process and when and how the bioconversion occurred need further and deeper study. potential compounds synthesize from limonene are a-terpeniol, perillic acid and carveon. further experiments on identifying the accumulation of those compounds in c. grandis cultures should be carried out to prove this statement. for future work on limonene improvement, it can be suggested that study on the regulation of enzymes involved in limonene biosynthesis could impose clearer picture of the biosynthetic pathway. the use of suspension cultures for fast limonene accumulation is among the future work that can be considered. conclusion the period of limonene production in c. grandis callus culture was shortened and accumulation was enhanced by precursor feeding with mva and linalool. callus growth rate improved significantly in the presence of the precursors. this result also reconfirmed the presence of mva and linalool in limonene pathway. acknowledgement the authors would like to thank universiti sains malaysia for the research grant and the ministry of science, technology & environment (moste) for national science fellowship scheme. references brown, j.t., hegarty, p.k., & b.v. charlwood. 1987. the toxicity of monoterpenoids to plant cell cultures. plant science 48,195-201. charlwood, b.v. & j.t. brown. 1987. transport and storage of secondary metabolites in tissue cultured plant cells. biochemistry society transactions. 61-63. dorisse, p., gleye, }., loiseau, p., puig, p., edy, a.m. & m. henry. 1988. papaverine biotransformation in plant cell suspension cultures. jour. natural products 120,211-234. funk, c. & p. brodelius. 1990. influence of growth regulators and an elicitor on phenylpropanoid metabolism in 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2580. tom, r., jardin, b., chavarie, c., rho, d. & j. archambault. 1991. effect of culture process on alkaloid production by catharanthus roseus cells. jour, of biotechnology 21, 21-42. van uden, w., pras, n. & th. m. malingre. 1990. on the improvement of the podophyllotoxin production by phenylpropanoid precursor feeding to cell cultures of podophyllum hexandrum royle. plant cell tissue organ culture 23,217-224. wong, k.c. 1992. flavour compounds from local aromatic plants, in: proceedings of asean workshop on the production of natural flavours for food, p. 66 67. zenk, m.h. 1991. chasing the enzymes of secondary metabolism: plant cell cultures as pot of gold. phytochemistry 30(12), 3861-3863. 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf biotropla no biotropla no. 12,1999 . 42 52 screening for natural products of shorea spp. and anisoptera spp. of the family dipterocarpaceae from pasir mayang, jambi, sumatera hilman affandi, arif nuryadin, susilo b. prayoga seameo b1otrop, jl. raya tajur km. 6 bogor. indonesia and roger w. read department of organic chemistry the new south wales university, p.o. box / kensington, n.s.w. 2033, australia abstract screening of 12 species of shorea and 2 species of anisoptera from the forest of pasir mayang, jambi with brine shrimp (anemia salina) lethality bioassay showed that shorea gibosa and anisoptera marginata have sufficient activity for further investigation. bioassay-guided fractionation of the active extract of s. gibosa led to the isolation of stigmasterol and the shoreaphenol. bioassay-guided fractionation of the active extract of a. marginata resulted in the isolation of lupenone and 3-methoxy-4-hydroxybenzoic acid o-p-c glucopyranoside as the bioactive compound. key words: plant natural products/sftorea gibosalanisoptera marginatalactive extracts/bioassay/pasir mayang. introduction dipterocarp timbers are well known to resist biological attack from many sources. shorea robusta was shown to be highly resistant to the termites microcerotermes beesoni and heterotermes indicola (sen-sarma 1963). particle boards constructed from shorea species are also protected against cryptotermes cynocephalus (moi 1980). fresh resin of anisoptera thurifera appears to protect beenests from termites (messer 1984). dipterocarp woods cause substantial mortality to insects feeding on them. over a three-month test period, termites feeding on shorea species suffered up to 99% mortality, while termites feeding on the nondipterocarp dyera costolata showed only a 13% death rate (moi 1980). chemical factors in either resins and bark may also protect dipterocarps from microbial attack. untreated dipterocarp timbers are reported to be highly resistant to fungal invasion (bakshi et al. 1967), and volatile components of hopea papuana were shown to inhibit fungal growth (messer 1984). bacterial growth inhibitors of dipterocarp origin include essential oils of valeria indica (bhagarva and chauan 1968) and stemnoporol and alpha-copalliferol from sri lankan dipterocarps (sootheeswaran et al. 1983). 42 screening for natural products ofshorea spp. and anisoplera spp. hilman affandi el al. the research described here attempts to identify the active compounds that are responsible for their toxicities against brine shrimp (artemia salina). the crude extracts of the stem bark of twelve species of shorea and two species of anisoptera collected from pasir mayang, jambi, sumatera were screened with brine shrimp lethality bioassay. bioassay-guided fractionation of the active extracts led to the isolation of the active substances reported in this paper. methodology general 'h n.m.r spectra were recorded at 500 mhz and 13c n.m.r spectra were measured at 125.6 mhz on a bruker am 500 spectrometer. mass spectra were recorded at 70 ev with an a.e.i. ms 12 spectrometer. all measurements were performed at the school of chemistry, university of new south wales, australia. initial column chromatographic separations were made on merck 60 silica gel. merck kieselgel 60 f254 was employed for preparative tlc with a 1 mm layer of adsorbent on 20 x 20 cm glass plates and 200 mg of material applied to each plate. analytical tlc was performed on commercial aluminium-packed merck kieselgel 60 f254 art. 5554 plates, visualised under uv 254 nm light or by charring after applying a 4% h2so4/meoh spray. plant material the stem bark of twelve species ofshorea i.e. s. opalis, s. guiso, s. selanica, s. seminis, s. pinanga, s. stenoptera forma, s. stenoptera (burck)., 5. mecistopteryx, s. leprosula, s. palembanica, s. multiflora, s. gibosa and two species of anisoptera namely a. marginata, and a.costata were collected from pasir mayang, jambi, sumatera. extraction and isolation an amount of 20 g of air dried plant materials from each species ofshorea and anisoptera was extracted with methanol at room temperature. the crude extracts were evaporated under reduced pressure. sea water was poured into a small tank and shrimp eggs were added to the tank provided with an aerator. after two days the shrimp eggs hatched and mature as naupili. each crude extract was weighed (20 mg) and dissolved in dichloromethane (2 ml). from this solution 500, 50, 5 ul were transferred to small petri dishes corresponding to 1000, 100, 10 ug/ml (ppm), respectively. the solvent was evaporated by standing at ambient temperature overnight. after two days (when shrimp larvae are ready), sea water was added to each petri dish, inoculated with 10 shrimp per dish (30 shrimp per dilution), and the 43 biotropla no. 12, 1999 volume was adjusted with sea water to 5 ml/dish. the number of surviving shrimps were recorded after 24 hours. the data were analysed with a finney computer program to determine lc50 values and 95% confidence intervals (hostettmann 1991). each crude extract of the sample was tested in three replicates against a single control. the controls were treated in a similar manner without the presence of crude extracts. the results of the initial screening are compiled in table 1. table 1. relative toxicity of the crude extracts against brine shrimp at 1000, 100, and 10 ppm no species lc50 (nq/ml) (c.i. 95%)* 1 lshorea opalis 513 2 anisoptera marginata 293 3 s. guiso 1114 4 s. selanica 270 5 s. seminis 392.4 6 s. pinanga 717 7 s.stenoptera forma 691 8 s. mec/sfopterix 394.9 9 s. leprosula 259 10 s. stenopfera burck. -ve 11 anisoptera costafa -ve 12 s. palembanica 882 13 s. multiflora 346 14 s. gibosa 205 c.i. 95% = confidence intervals 95% -ve = not toxic at a concentration of more than 1000 ppm the result of the screening indicated that the crude extracts of shorea gibosa, s leprosula, and s. selanica with toxicity level (lc50) at 205, 259, and 270, respectively, showed significant activity against brine shrimp. meanwhile among anisoptera spp., a.marginata showed sufficient toxicity (lc50 = 293). in this preliminary work, however only s. gibosa and a. marginata were chosen for further investigation. therefore, large-scale extraction and fractionation of both species were carried out in the next phase of the research. extraction and fractionation of shorea gibosa. the air-dried plant material of s. gibosa was extracted with methanol. the crude extracts (100.2 g) were extracted with hexane to remove the hexane-soluble fraction (19.8 g), and the residues were chromatographed by column chromatography (silica gel 60). the initial solvent system used in the column was hexane. the polarity of the solvent was gradually increased by the addition of chloroform into the solvent system until the mixture of hexane and chloroform became 60% in hexane. the solvent system was then changed, initially by adding 5% of methanol into the chloroform until the con 44 screening for natural products ofshorea spp. and anisoptera spp. hilman affandi et al. centration of methanol became 30% in chloroform. the fractions were grouped and monitored by analytical thin layer chromatography (tlc) to obtain 14 fractions. all fractions, including the hexane fraction, were screened with the brine shrimp lethality bioassay (table 2). table 2. relative toxicity of the fractions against brine shrimp fractions lcso (ng/ml) (c.i.95%) fraction 1 fraction 2 fraction 3 fraction 4 fraction 5 fraction 6 fraction 7 fraction 8 fraction 9 fraction 10 fraction 1 1 fraction 12 fraction 13 fraction 14 -ve -ve 823 882 226 240 132 127 c.i. 95% = confidence intervals 95% -ve = not resistant at a concentration of more than the results showed fraction 9 to be the most toxic fraction, followed by fraction 14, 10, 8, and 7. however, due to time limitation and budgetary constraints only fraction 9 was subjected to further investigation. therefore, fraction 9 (1.15 g) was purified by preparative tlc, eluted initially with hexane, and later with the mixture of hexane and chloroform ( 3 : 7) to yield three fractions. fraction 1 (0.46 g) consisted of a mixture of 4 5 compounds as shown by analytical tlc. fraction 2 (0.31 g) was dissolved in a mixture of chloroform and methanol. the mixture was allowed to stand overnight and precipitation occurred. the precipitation was filtered to give solid material as a white amorphous crystal of stigmasterol (0.13 g), m.p. 169°c. meanwhile solid material present in fraction 3 (0.22 g) was filtered and recrystallised with chloroform-methanol, afforded shoreaphenol as orange crystalline compounds (13.27 mg; m.p. 240°c). extraction and fractionation of anisoptera marginata. air-dried stem bark of a. marginata (1.7 kg) were extracted with methanol. after removing the solvent under reduced pressure the crude extract (121.4 g) was extracted with hexane, to remove the hexane-soluble fraction (19.02 g). the remaining crude extract (102.38 g) was subjected to column chromatography (silica gel 60), initially eluted with 45 biotrop1a no. 12,1999 chloroform, and later with methanol by increasing the polarity at 30%, 60%, 80% and 100% levels. the fractions were pulled and monitored by analytical tlc to yield 18 fractions. fraction 1 to 10 were combined, since the components of those fractions were relatively similar judging from analytical tlc. all fractions, including the hexane fraction, were screened with the brine shrimp lethality bioassay (table 3 ). table 3. relative toxicity of the fractions against brine shrimp fractions lcjo (ng/ml) (c.i.95%) hexane fraction fraction 1 1 fraction 12 fraction 13 fraction 14 fraction 15 fraction 16 fraction 17 -ve 300 114 65 44 72 121 c.i 95% = confidence intervals 95% -ve = not resistant at a concentration of more than 1000 ppm the results indicated that fraction 14 was most toxic. fraction 14 (1.43 g) was subjected to preparative tlc, eluted twice initially with hexane followed by 30% chloroform in hexane, to yield fraction 1 (0.87 g), fraction 2 (0.13 g), fraction 3 (0.03 g); and fraction 4 (0.49 g). solid material present in fraction 1 was filtered, followed by recrystalisation in chloroform to yield the triterpenoid lupenone as a white crystalline amorf (0.21 g), m.p 160°. fraction 4 was dissolved in methanol and a crystalline solid was produced upon addition of a few drops of methanol. the solid residue was filtered and recrystallised with a mixture of chloroform and methanol to yield a colorless crystal (0.16 g), m.p. 145°. all fractions, including two compounds isolated from fraction 1 and 2, were tested with the brine shrimp lethality bioassay (table 4). table 4. relative toxicity of the fractions against brine shrimp fractions lcw (tig/ml) (c.i. 95%) lupenone fraction 2 fraction 3 glue. comp. -ve -ve -ve 7 c.i 95% = confidence intervals 95% -ve = not toxic at a concentration of more than 1000 ppm 46 screening for natural products ofshorea spp. and anisoptera spp. hilman affandi et al. results and discussion methanolic extract of the stem bark of s. gibosa, followed by fractionation by column chromatography and monitored by tlc resulted in 14 fractions. the fractions were screened with brine shrimp lethality bioassay and purification of the active fractions led to the isolation of non-biologically active compound stigmasterol. the structure of stigmasterol was established by 'h and i3c n.m.r. spectroscopy (fig. 1). the 'h n.m.r. spectrum showed two methyl singlets, 8 0.57, 1.01; three methyl doublets, 8 0.79, 0.84, 1.03; and a methyl triplet, 8 0.80. also three proton multiplets corresponding to two internally coupled, trans olefinic protons 8 5.03 (dd, j 15.1, 8.4 hz, and 5.15 (dd, j 15.1, 8.4 hz) and an isolated olefinic proton (8 5.18, m) with long range coupling were present, but no other signals occurred at higher chemical shift than 2.5 ppm. the presence of a 1,2disubstituted and a 1,1,2-trisubstituted olefin was supported by the presence of signals at 8 117.0 (ch), 129.6 (ch), 138.1 (ch) and 139.5 (c) in the "c n.m.r. figure 1. structure anisopte 1. stigmasterol 3. lupenone 4. 3-m acid of chemical constituents isolated from the ba ra marginata (3 and 4). 2. shoreaphenol ethoxy-4-hydroxybenzoic -o-p-c glucopyranoside rk of shorea gibosa (1 and 2) and 47 biotropia no. 12,1999 spectrum. the presence of a quaternary signal at 8 212.0 also confirmed the occurrence of a carbonyl group in the compound. the structure of stigmasterol was also confirmed by mass spectrometry at mlz 367 (m-c3h7), 298 (m-c8h,6), 271 (mc,0h,9), 269 (m-c10h2,), and comparative melting point analysis. stigmasterol is widely distributed in the plant kingdom. application of this compound as therapeutic agents is very limited, although extensive exploratory activities in this area have been underway during recent years (mahato'ef a/. 1992). the solid material produced from fraction 4 of the active fraction was filtered, followed by repeated crystallisation to give an orange crystalline solid of shoreaphenol. the 'h n.m.r. spectroscopy (table 5) data revealed the presence of signals at 6 8.61 (2h, br d, j 8.7hz, h-ll and h-15), 7.84 (2h, br d, j 8,7hz, h12 and h-14), 7.74 (2h, br d, j 8.8hz, h-24 and h-28), and 7.44 (2h, br d, j 8.8hz, h-25 and h-27). these signals clearly indicated the presence of two pdisubstituted benzene nuclei. the 'h n.m.r. spectrum also established signals for four m-coupled proton at 6 8.42 (1h, d, j 2.1hz, h-3), 8.11 (1h, d, j 2.1hz, h5), 7.58 (1h, d, j 2.5hz, h-19) dan 7.33 (1h, d, j 2.5hz, h-21). in addition to these signals, five table 5. 'h n.m.r. spectral data of compounds (1) (4) (ppm in cdc13 cd3od, and acetone d6) screening for natural products ofshorea spp. and anisoptera spp. hilman affandi el al. phenolic hydroxyl protons were also discernible in the 'h n.m.r. spectrum in the region 7.92 to 7.96. the i3c n.m.r. spectra (table 6) were characteristic of substituted a and (3carbons of the benzofuran nucleus. furthermore, the appearance of one proton at 5.98 (8c 54.97, d) clearly suggested the presence of-ch-coin the molecule of shoreaphenol. the mass spectrometry data are consistent with the assigned structure m/z 466. comparison of the 'h and 13c n.m.r. spectral data and its melting point of shoreaphenol with those reported (saraswathy et al. 1992) suggested close similarity. the methanolic extract of the bark of the tree of anisoptera marginata was fractionated by column chromatography using silica gel, and monitored with tlc to give 9 fractions. brine shrimp bioassay of those fractions showed the fraction eluted with chloroform-methanol (8 : 2) to be the most active fraction. the active fraction was subjected by preparative tlc, and the precipitation that occurred in fraction 1 biotropia no. 12, 1999 was filtered and crystallised with chloroform to yield the terpenoid lupenone as a white crystalline amorf (0.21 g), m.p 160°. the structure of lupenone was established from 'h and 13c n.m.r. (table 5 and 6). the 'h n.m.r. spectral data indicated the presence of five singlet resonances at 8 0.89, 0.94, 0.97, 1.07 and 1.19, arising from six methyl groups attached to quaternary carbons. there also appeared one isolated singlet at 8 1.68 due to a vinylogous methyl group, and the olefmic proton signals at 8 4.56 (dd, j 2.2, 1.4 hz) and 4.68 (d, j 2.2 hz) were consistent with a 1,1-disubstituted alkene, a propenyl group was likely. low field signals resonance at 8 3.18 (dd, j 10, 9, 5.3 hz), 2.38 (dt, j 5.7, 11.1 hz) and 1.91 (m) occurred, but there were no other olefinic resonances. the n.m.r. signal at 8 3.18 was suggestive of an axial proton adjacent to a quaternary centre and attached to a carbon bearing hydroxyl group. the 13c n.m.r. spectrum (table 5) supported the structural assignment of lupenone. in particular there appeared seven methyl signals, only two olefinic carbon signals 8 109.3 (ch2) and 151.0 (c), and a low field methine resonance ( 8 78.9) for c3. the identity of compound (1) as lupenone was confirmed by melting point and mixed melting point with an authentic sample (affandi et al. 1998). the solid material produced in fraction 4 was filtered, followed by recrystallisation in methanol to give 3-methoxy-4-hydroxybenzoic acid o-p-c glucopyranoside as a colorless crystalline amorf (0.98 g), m.p 145°. the 'h and 13c n.m.r spectral data of 3-methoxy-4-hydroxybenzoic acid-o-p-c-glucopyranoside are shown in table 5 and 6. the infrared (ir) spectrum of the compound disclosed the broad absorption at 3600 2500 cm"1 and the carbonyl stretch at 1700 cm'1. this is in agreement with the compound being an acid. the 'h n.m.r. spectrum of the compoundshowed the signals of the following protons (acetone-d6, 500 mhz): proton in 3-methoxy-4hydroxybenzoic acid, 8 3.79 (3h,s), 7.01(1h, s), 8.56 (1h, br s), 9.30 (1h, br s). proton in a glucopyranoside ring, 8 4.11 (1h, dd), 3.77 (1h, m), 3.44 (1h, dt), 3.84 (1h, m), 3.62 and 4.07 (each 1h, ddt), proton in a hydrogen bonded hydroxyl group, 8 4.87 (1h, dd), 5.43 (1h, d), 5.62 (1h, d). the 13c n.m.r, spectrum of the assigned structure indicated the presence of six quaternary carbon signals at 8 109.0, 115.7, 118.7, 139.9, 148.3, 150.9. primary carbon signals were obtained at 8 70.5, 72.3, 74.1, 79.6, 81.6, and 109.0. signals for a carbon methoxy group at 8 59.6, carbon methylene at 8 61.3 and carbon acid at 8 163.1 also were obtained. mass spectroscopy confirmed m/z 328 of the molecular formula c14h16o4. furthermore, the location of the c-glucoside ring was confirmed by the two dimensional noe spectroscopy (noesy) spectrum as described in figure 2. the long-range c-h correlation spectra with proton detection (hmbc) as described in figure 3 permits any coupling between oh protons and the carbons to which they are attached, to be detected by the appearance of cross peaks. brine shrimp lethality bioassay of this compound showed to be highly toxic against brine shrimp (lc 50 = 7), it means that the compound caused the mortality of 50 biotropia no. 12, 1999 brine shrimp more than 50% at concentration 7 ppm (jig/ml). future investigations might reveal medicinal or other uses of the compound. references affandi, h., a, nuryadin, and r.w., read 1998. studies on natural products of albizia sp., b1otropia, 11, 1 -8. bhargarvca, a.k., and c.s., chauan. 1968. antibacterial activity of some essential oils., indian j. pharm. 30:151-152. bakshi, b.k., y.n., puri, and s., singh. 1967. natural decay resistance of indian timbers. i. introduction and method. ii. resistance of sal (shorea robusta gaertn.) and teak (tectona grandifloris l.f), indian for., 93 : 305-328. hostettman, k., ed., 1991. method in plant biochemistry, vol. 6: assays for bioactivity, pergamon press, new york. mahato, s.b., a.k., nandy, and g., roy. 1992. triterpenoids, phytochemistry, 31(7), 2199-2249. messer, a.c., 1984. chalicodoma pluto : the world's largest bee rediscovered living communally in termite nests (hymenoptera: megachilidae), j. kans. entomol, soc., 57 : 165-168. moi, l.c., 1980. a new laboratory method for testing the resistance of particle boards to the drywood termite cryptotermes cynocephalus. malaysian for., 43:350-355. saraswathy, a., k.k., purushothaman, a., patra, a.k., dey, and a.b., kundu. 1992. shoreaphenol, a polyphenol from shorea robusta. phytochemistry, 31(7), 2561-2562. sen-sarma, p.k.. 1963. studies on the natural resistance of timbers to termites, i. observations on the longevity of the test termite heterotermes indicola wasm. in the saw dust from forty common indian timbers. indian for. bull. (n.s.) entomol, 220 : 1-3. sootheeswaran, s., m.v.s., sultanbawa, s., surendrakumar, and p., blandon. 1983. poly phenols from a dipterocarp species: copalliferol and stemnoporol. i. chem. soc. perkin trans. i, 699-702. 52 biotropia no biotropia no. 13, 1999: 37-48 morphometric study for identification of the bactrocera dorsalis complex (diptera : tephritidae) using wing image analysis a. adsavakulchai', v. baimai', w. prachyabrued2, paul j. grote' and s, lertlum" ' department of biology and2 department of physics, faculty of science, mahidol university, rama iv road, bangkok, 10400, thailand e-mail address: toung@ait.ac.th 3 school of biology, institute of science, suranaree university of technology, thailand 4 faculty of computer science program, chulachomklao military academy, thailand abstract the bactrocera dorsalis complex (diptera: tephritidae) used in this study included b. dorsalis, b. arecae, b. propinqua, b. pyrifoliae, b. verbascifoliae, and three new species complexes are species e, species k and species p. bactrocera tau was used as an out-group. a total of 424 adults, which emerged from pupae collected from natural populations in thailand, were prepared for wing measurements. morphometric analysis was performed on measurements of wing vein characters. wing images were captured in digital format and taken through digital image processing to calculate the euclidean distance between wing vein junctions. discriminant and cluster analyses were used for dichotomy of classification processes. all 424 wing specimens were classified to species in terms of the percentage of "grouped" cases which yielded about 89.6% accurate identification compared with the formal description of these species. after clustering, the percentage of "grouped"cases yielded 100.0%, 98.9%, 98.1%, 95.2% and 84.6% accurate identification between the b. dorsalis complex and b. tau; b. arecae and species e; b. dorsalis and b. verbascifoliae; b. propinqua and b. pyrifoliae; and species k and species p, respectively. this method of numerical taxonomy may be useful for practical identification of other groups of agricultural pests. key words: bactrocera dorsalis complex/wing image processing/morphometric/discriminant and cluster analyses. introduction the oriental fruit fly, bactrocera dorsalis (diptera: tephritidae) group of the subgenus bactrocera, has been considered one of the most important groups of agricultural pest in southeast asia because some of these species attack seed bearing organs of plants, including soft fruits and flowers (mcpheron and steck 1996). the b. dorsalis group comprises about 52 closely related species in asia, mostly southeast and south asia, with additional species in the south pacific region (drew and hancock 1994). some of these species are mophologically similar. drew (1989) postulated that the dacinae fruit flies originated in the papua new guinea area and speciated prolifically throughout the region. drew and hancock (1994) listed fourteen closely related species of the b. dorsalis complex from thailand on the basis of morphological characters. these species are b. arecae, b. carambolae, b. dorsalis, b. irvingiae, b. kanchanaburi, b. melastomatos, b. osbeckiae, b. papayae, b. propinqua, b. pyrifoliae, b. raiensis, b. thailandica, b. unimaculata and b. verbascifoliae. recently, we provided population genetic data of some members of the b. dorsalis complex (baimai et al. 1995, 1998 unpublished data; satayalai 37 biotropia no. 13, 1999 1996). nonetheless, most species of the b. dorsalis complex have limited distribution within the tropical and subtropical regions (drew 1989). the limitation of the distribution range of these species is due in part to physical, climatic and gross vegetation factors. however, it is more likely that the distribution range is correlated with the specificity on fruits of particular host plants, yet little information is available on the range of host plants of these species. the b. dorsalis complex is systematically one of the most interesting groups of insect pest (ibrahim and ibrahim 1990). because of similarity in external morphology among the members of the b. dorsalis complex and the geographic variation in morphology within each species, it has been very difficult to separate these species. consequently, such morphological variation has caused taxonomic problems (hardy 1977). thus, the most common errors are synonyms, homonyms, misidentifications and establishment of supra-specific groups based on questionable morphological characters (white and elson-harris 1992). there is still a major need for more taxonomic study in correlation with population genetic investigations of the b. dorsalis complex to address some sibling species problems. in most countries, a complete list of reference collections for identification purposes can be found, resulting from the work of trained taxonomists. it has long been obvious that fruit flies of the subfamily dacinae have major economic effects on society. therefore, economic entomologists were needed to identify the various species involved. in spite of this tireless work, however, many taxonomic problems and misidentifications accrued (mcpheron and steck 1996). some of these systematic problems can be elucidated with the aid of the recent development of taxonomic techniques such as cytotaxonomy and molecular biology as well as improved numerical taxonomy (sneath and sokal 1973). yu et al. (1992) studied morphometric analysis of linear wing measurements for identification of ichneumonid wasps using image analysis of wings. the authors outlined the procedure to digitize and to measure various wing elements with an image analyzer and the wing specimens were assigned to species by discriminant analysis and independent univariate comparisons of wing measurements. recently, weeks (1996) developed daisy (digital automated identification system) based on the idea that the pattern of veins and pigments on insect wings are distinct-like fingerprints. much of the acquisition by computer of morphological characters of insects (white and scott 1994) has involved measurement of projected images on digitizing tablets (howell et al. 1982). use of computers efficiently provides accurate measurements and saves development time. in addition, these methods can be easily repeated and made available to any user and reworked with a minimum of effort. however, these methods are costly hi terms of software development and maintenance. image analysis of morphological characters of wings may be the first step towards a completely automated insect identification technique cfuetal. 1992). in our ongoing research on the biology of fruit flies hi thailand, we attempt to employ ecological observations in the field and genetic investigations in the laboratory coupled with morphological examination of larvae, pupae and adults to help solve the problems of identifying some cryptic or isomorphic species. thus some new sibling species of the b. dorsalis complex have been found through 38 morphometric study for identification of the bactrocera dorsalis complex s. adsavakulchai et al. allozyme electrophoresis (satayalai 1996) and cytogenetic studies (baimai et al. 1995, 1998 unpublished data). in this paper we describe the methodology of image analysis to acquire and quantify morphological characters of the wing veins of eight species of the b. dorsalis complex in a computer compatible form for suitable identification of these species. materials and methods specimen collection eight species of the b. dorsalis complex used in this study include b. dorsalis (hendel), b. arecae (hardy and adachi), b. propinqua (hardy and adachi), b. pyrifoliae drew and hancock, b. verbascifoliae drew and hancock, and three new species complexes are species e, species k and species p with morphological characters different from the record of drew and hancock (1944). in addition, bactrocera tau (walker), of the subgenus zeugodacus, was used as the out-group. larval specimens of these members of the b. dorsalis complex were obtained from a wide variety of infested fruits from various parts of thailand (table 1). some larvae were processed for mitotic karyotype study which provided useful information for table 1. specimens of the eight species of the bactrocera dorsalis complex and bactrocera tau collected from different localities in thailand 39 biotropia no. 13, 1999 species identification. most of the larvae were reared either in the field or in the laboratory allowing them to pupate and finally emerge as adults. some adults were processed for electrophoretic study to confirm the genetic species as determined by mitotic chromosome markers. adults from each collection were examined morphologically for species identification in correlation with chromosomal evidence and electromorphic allozyme patterns. some adults were kept for wing specimens preparation. the framework of the research is shown in figure 1. wing preparation wings of individual adults were detached from the thorax and they were placed on a microscope slide. the wings were secured under a coverslip with canada balsam. image processing wing image processing procedure is shown in figure 2. the microscope slide with wing samples was positioned on a nikon smz-2t stereomicroscope with a low objective lens (ix). the vertical tube has a control light path switchover which allows the diversion of the right eye image to the camera. a nikon e2s digital still camera was attached with a cf projection lens (4x) that captured the whig image on the memory card. digital imaging with high-resolution of 1.3 million pixels was then transferred to application handling jpeg (joint photographic experts group) files (fig. 3). 40 morphometric study for identification of the bactrocera dorsalis complex s. adsavakulchai et al. the original color image (jpeg : raster format) was transformed into gray scale in the form of bmp (bitmap) raster file format that allows efficient localized image processing. from bmp, the image was pre-processed by low-pass filtering with average of 3x3 to create a smooth image (gonzalez and woods 1992); then the image was transformed by applying a linear function to enhance the image by calculating the ratios from pixel values of the original image divided by pixel values of the smooth image. then, the image was vectorized to create vector file format in dxf (drawing exchange file) file format and manually adjusted until suitable for measuring the 30 wing vein distances (fig. 4). the vector file was used to create 41 biotropia no. 13,1999 figure 3. showing digital image of a wing in the form of jpeg file format; 1. bactrocera arecae 2. b. dorsalis 3. b. propinqua 4. b. pyrifoliae 5. b. verbascifoliae 6. species e 7. species k 8. species p 9. bactrocera tau figure 4. diagrammatic representation of a wing of the b. dorsalis complex showing the 30 wing vein measurements. 42 bu na # 1 50 9 /2 3/ 97 4 :5 2 pm p ag e 10 morphometric study for identification of the bactrocera dorsalis complex s. adsavakulchai et al. automatically the coverage that contained 30 data sets of each sample in terms of euclidean distances. euclidean distance is the measurement between the 2 coordinates and is computed as the square root of the sum of the squared differences as shown in the following formula: euclidean distance (d) = [ (x-s)2 + (y-t)2 ] vl where euclidean distance is the distance between two points : (x,y) is co-ordinate of p; (s,t) is co-ordinate of q. statistical analysis spss for windows version 6.0 (george and paul 1995) was used for data analysis. discriminant and cluster analyses were used for dichotomy of the classification processes. all 424 wing specimens were classified to species in terms of the percentage of "grouped" cases. results and discussion our data show that the length of a vein is correlated with the size of the wing. ratios of the vein lengths provide a very effective means for recognizing trends in variation and for a quick diagnostic character. discriminant function analysis was used to derive a function as a criterion for separation of the eight species used in this study. the percentage of "grouped" cases correctly classified with the accuracy of 89.6% is shown in table 2. the linear discriminant function can completely discriminate members of the b. dorsalis complex from b. tau as follows: y = 2.76xj +9.85x2-16.74 where x! = vein2/vein22, and x2 = vein3/vein8. specimens are identified by the following rule : if y < 0 then species is bactrocera dorsalis complex if y > 0 then species is bactrocera tau the eight species of the b. dorsalis complex can also be separated by using cluster analysis (fig. 5). the results from cluster analysis show that the eight members of the b. dorsalis complex can be classified into 4 classes : (1) b. arecae and species e; (2) b. verbascifoliae and b. dorsalis', (3) b. propinqua and b. pyrifoliae; and (4) species k and species p. bactrocera tau was used as the out-group. discriminant function analysis was used to derive a function to provide maximum values for separation of these 4 classes. the "grouped" cases were correctly classified with an accuracy of 98.9%, 98.1%, 95.2% and 84.6%, respectively (table 3). the stepwise 43 biotropia no. 13,1999 table 2. classification results of the 8 species of the bactrocera dorsalis complex and bactrocera tau and percent of "grouped" cases correctly classified: 89.6%. figure 5. dendrogram using average linkage (between groups); 1. bactrocera arecae 2. b. dorsalis 3. b. propinqua 4. b. pyrifoliae 5. b. verbascifoliae 6. species e 7. species k. 8. species p 9. bactrocera tau 44 morphometric study for identification of the bactrocera dorsalis complex s. adsavakulchai et al. table 3. classification results in 4 groups of the b. dorsalis complex revealing percentage of "grouped" cases correctly classified as 98.9%, 98.1%, 95.2% and 84.6% for classes 1, 2, 3 and 4, respectively discriminant analysis procedure has certain advantages in reducing the use of a large number of variates to a small number of canonical variables. these selected variables were calculated as a ratio between variables, which were used in discriminant analysis for classification in each group afterwards. the stepwise discriminant analysis procedure was performed to take data correlation and to select variables for transformation in the next step. finally, the best ratio was selected as an index for each species as shown in table 4. the results appear to be generally satisfactory in separation of these species of the b. dorsalis complex compared with the genetic data (baimai et al. 1995, 1998 unpublished; satayalai 1996) and classical taxonomy (drew and hancock 1994). some adults were processed for electrophoretic study to confirm the genetic species as determined by mitotic chromosome markers. adults from each collection were examined morphologically for species identification in correlation "with chromosomal evidence and electromorphic allozyme patterns. however, correct 45 biotropia no. 13,1999 table 4. index for classification of the 8 species of the bactrocera dorsalis complex and bactrocera tau. (sd) = standard deviation; + = 2 or more indices must be used for classification species index value %accuraccy bactrocera tau (out-group) b. dorsalis complex vein2/vein22+ vein3/vein8 vein2/vein22+ vein3/vein8 2.82 (0.33) 0.83 (0.06) 4.59 (0.48) 0.97 (0.05) 100.0% 100.0% b. arecae species e vein!8+ vein7/vein!4+ veinl8/vein20 vein!8+ vein7/vein!4+ vein!8/vein20 25.8 (2.5) 0.39 (0.03) 1.55(0.09) 31.0(1.7) 0.4 (0.01) 1.63(0.08) 98.9% 98.9% b. dorsalis b. verbascifoliae veins /vein 17+ vein8/vein!6+ vein8/vein29+ vein!3/vein24 vein5/vein!7+ vein8/vein!6+ vein8/vein29+ veinl3/vein24 0.83 (0.05) 1.75(0.07) 1.93 (0.26) 1.15(0.04) 0.88 (0.05) 1.57(0.08) 1.85(0.26) 1.22(0.04) 98.1% 98.1% b. propinqua b. pyrifoliae vein8+ vein26+ vein7/vein!8+ vein20/vein24+ vein!3/vein20 vein8+ vein26 vein7/veinl8+ vein20/vein24+ vein!3/vein20 91.0(7.2) 10.28 (1.57) 3.09 (0.23) 0.6 (0.05) 2.02 (0.14) 85.0 (7.9) 12.92(1.27) 3.03(0.12) 0.6 (0.04) 2.03 (0.22) 95.2% 95.2% species k species p vein 23/perimeter+ veinl8/vein21+ veinl3/vein24 vein23/perimeter+ vein!8/vein21+ vein 13^6^4 0.036 (0.002) 0.77 (0.06) 1.16(0.04) 0.034 (0.002) 0.63 (0.03) 1.21 (0.04) 84.6% 84.6% identification of some species is rather low, especially species k with a correct classification of only 75%, although this species is quite distinct in external morphology. in addition, "cluster membership of cases using average linkage (between groups)" among the eight species of the b. dorsalis complex and b. tau showed some overlapping characters since there are mixed characteristics which lead to difficulty in classification (fig. 3). moreover, the dendrogram shows how the species could overlap as a result of genetic differentiation during the speciation processes (ashlock 1979). the methodology used for discrimination of members of the b. dorsalis complex proposed and employed in this study has some advantages over other tedious taxonomic techniques (e.g. cytotaxonomy and electrophoresis) for separation 46 morphometric study for identification of the bactrocera dorsalis complex s. adsavakulchai etal. of closely related species of insects. first, this method does not require fresh specimens. second, it can be operated by a person who has a minimal knowledge of taxonomy or a non-taxonomist. finally, the methodology described in this study seems to be promising for further development of on-line identification systems. it is clear that the data in the form of numerical tables can be easily stored and the computations can be rapidly made (frampton et al. 1991). the methodology of morphometric analysis described here also illustrates the rapid advance in automated methods of on-line biological classification schemes which may have implications in the field of agricultural entomology, particularly in the tropical regions. acknowledgments we wish to thank s. tigvatananont for providing most of the dried specimens of the fruit flies used in this study. we are grateful to hollywood international ltd. for providing access to use nikon e2 series (nikon digital still cameras). this work was supported by the thailand research fund (rta 3880008). references ashlock, p.o. 1979. an evolutionary systematist's view of classification. systematic zoology, 28: 441 50. baimai, v., w. trinachartvanit, s. tigvattananony, pj. grote, r. poramarcom. and i). kijchalao. 1995. metaphase karyotypes of fruit flies of thailand i. five sibling species of the bactrocera dorsalis complex. genome, 38: 1015-1022. drew, r.a.i. 1989. the taxonomy and distribution of tropical and subtropical dacinae (diptera: tephritidae). world crops pests (ed. by a.s. robinson and g. hooper). amsterdam, elsevier. p. 13-66. drew, r.a.i. and d.i. hancock, 1994. the bactrocera dorsalis complex of fruit flies (diptera: tephridae: dacinae) in asia. bulletin of entomological research supplement series: supplement, 2, 1-68. frampton, e.r., j. fry, b.p. stephenson, and j.m. cowley, 1991. a computerised system for data management during a fruit fly outbreak. the international symposium on the biology and control of fruit flies. george, d. and m. paul, 1995. spss/pc + step by step: a simple guide and reference. ca: duxbury press. belmont. gonzalez, c.r. and e.r. woods, 1992. digital image processing. addison-wesley publishing company. inc. hardy, d.e. 1977. family tephritidae. catalog of the diptera of the oriental region (ed. by delflnado m.d. and hardy d.e.). honolulu, the university press of hawaii. 854 p. howell, v.d., k. hoelmer, p. norman and t. allen, 1982. computer-assisted measurement and identification of honey bees (hymenoptera: apidae). annals of the entomological society of america, 75, 591-5944. ibrahim, r. and g.a. ibrahim, 1990. handbook on identification of fruit flies in the tropics. universiti pertanian malaysia press. mcpheron, b.a. and g.j. steck, 1996. the fourth international symposium on fruit flies of economic importance. a world assessment of their biology and management. st. lucie press. 47 biotropia no. 13, 1999 satayalai, o. 1996. electrophoretic study of natural populations of the bactrocera dorsalls complex (diptera: tephritidaej) in thailand. ph.d. thesis, bangkok. faculty of graduate studies, mahidol university. sneath, p.h.a. and r.r. sokal. 1973. numerical taxonomy: the principles and practice of numerical classification. w.h. freeman and company. weeks, p. 1996. daisy takes wing to sort out the bugs of the world. the times. july 17. white, i.m. and m.m. elson-harris, 1992. fruit flies of economic significance: their identification and bionomics. wallingford, uk, cab international. white, i.m. and p.r. scott, 1994. computerized information resources for pest identification: a review, p.129-137 in: hawksworth, d.l. (ed.). the identification and characterisation of pest organisms. wallingford, uk, cab international. yu, d.s., e.g. kokko, j.r. barron, g.b. schaalje, and b.e. gowen, 1992. identification of ichneumonid wasps using image analysis of wings. systematic entomology, 17, 389-395. 48 biotropia no. 3, 1989/1990: 1 -24 assessment of spot satellite data for tropical vegetation inventory and monitoring in sumatra y. laumonier and u..r. djailany syafii tropical forest biology programme, seameo-biotrop bogor, indonesia abstract following a previous vegetation mapping in sumatra island (indonesia), an assessment of spot satellite capability to handle specific problems related to vegetation identification and monitoring from remote sensing data has been undertaken. results of visual interpretation and multispectral analysis have shown the usefulness of spot data for the appraisal of tropical vegetation at medium scale. this was particularly striking for the swampy vegetation types including mangroves and for the secondary vegetation, for which significant improvements have been brought by multispectral classifications. a 20 m ground resolution is neither sufficient to provide information on primary forest patterns, nor to identify properly logged over areas. nevertheless, several degrees of depletion of the forest and all the serial stages have been identified, which is a considerable progress compared with previous remote sensing means. spot is a very good alternative to medium scale aerial photographs for the production of medium scale (1 : 100 000 to 1 : 250 000) vegetation and land-use maps. background and justification since 1980 biotrop, the seamed regional centre for tropical biology, has conducted not only a mapping inventory of land-use units and natural vegetation types in sumatra which involves remote sensing analysis, but also very detailed field studies on various plant formations and their dynamics, floristics, structure and ecology. results have been synthesized in the form of ecological vegetation maps (laumonier 1983, laumonier et al. 1986, 1987) and related surveys (laumonier 1981, blasco et al. 1983). the mapping was mostly done using conventional means, i.e. interpretation of a 1/100 000 scale panchromatic aerial photograph cover (1974-78) which appeared to be of rather poor quality. moreover, the landsat mss imageries have proved insufficient in discriminating many peculiar vegetation or land-use types. this necessitated an unusually detailed field work to supplement the lack of information obtained from space, but it gave researchers of the team a fairly good knowledge of the area. the second phase of the biotrop study implemented in 1988 is concerned with specific areas selected according to management problems spotted during the small-scale mapping phase. such development schemes would require an elabora 1 biotropia no. 3, 1989/1990 various shrubby secondary vegetation types identical from space, but floristically entirely different often mixed with cultivation and usually classified as "mosaics". a. visual interpretation and manual classification the visual interpretation of black and white prints allows to differentiate 19 landcover and vegetation units (table 4 and figure 3). the standard fcc gives less information compared with the black and white interpretation of the 3 bands. improved false colour composites (xs bi vgi and xs3 bi vgi) allow a better discrimination of secondary types, burnt or newly planted areas. b. digital classification for the scene of pasirmayang, a multispectral classification has been performed for two windows, one in pasirmayang area (512 x 512 pixels) corresponding mostly to natural vegetation cover, the other in sitiung area (512 x 512 pixels) consisting of cultivation and settlements. xs1, xs2 and xs3 means and standard deviations plotted on bi-dimensional diagrams provide a satisfactory discrimination of vegetation and land cover units (figure 4). in pasirmayang window, 10 classes corresponding to 6 vegetation types and 4 other land-use units were recognized (figure 4, plate 2), whereas in sitiung 5 vegetation types and 3 land-use units could be distinguished (figure 4, plate 3). table 4. classification of vegetation in muarabungo's lowland area by visual interpretation 1. lowland primary forest 2. hill forest 3. logged-over or depleted forest 4. tall secondary forest mixed with rubber trees 5. secondary growth forest mixed with remnant primary forest trees 6. secondary growth forest mixed with rubber 7. shrubby, thicket secondary vegetation 8. young secondary growth forest mixed with shrubs and herbaceous vegetation 9. newly planted rubber mixed with young secondary growth forest 10. grassland 11. riparian forest 12. fresh water swamp forest 13. swampy secondary shrubby vegetation and grassland 14. paddy field 15. newly opened, recently burnt area 16. mosaic of secondary growth and newly planted food crops (paddy, cassava, banana & fruit trees) 17. fruit trees, coconut and habitat (homestated mixed gardens) 18. rubber tree estate 19. bare soil 10 biotropia no. 3, 1989/1990 tion of larger scale vegetation and land-use maps and more precise knowledge and understanding of the land-use patterns identified on remote sensing documents. the very high ground resolution obtained with spot offers new perspectives in the mapping of those patterns. a first evaluation of such data was initiated during the peps program (programme devaluation preliminaire de spot) organized by the french space agency (laumonier et al. 1987). its aim was to assess the capabilities of spot satellite to tackle some of the most striking problems met during the first phase of the project in sumatra, specially with the identification and classification of vegetations and land-use units. the present study is a follow up of this investigation backed up by related researches (barkey 1987, djailany 1987, gastellu-etchegorry 1988) concerned with an evaluation of visual versus digital interpretations and classifications, a comparison of spot data with conventional aerial photographs and landsat mss data already analyzed in the area (deshayes 1981, ducros-gambart & gastellu-etchegorry 1984, ducros-gambart et al. 1984) and an assessment of panchromatic (p) versus multi-spectral (xs) mode. it is also supported by the ground sampling already completed by the biotrop team for the characterization of complex heterogeneous success-ional stages, primary forest patterns and land-use units in sumatra (laumonier 1981, torquebiau 1986, djailany 1987). materials and methods four scenes have been requested both in p and xs modes at the processing level 1b (table 1) corresponding to specific problem areas in central and south sumatra (figure 1). the muarabungo area (western part of jambi province) has been chosen as an example of a forested area with problems of local agricultural practices and modern plantation schemes. baturaja (south sumatra) is the test site for another type of land use where the original vegetation has almost disappeared and has been replaced by complex secondary types. lastly, the delta of musi-banyuasin was selected as a good example of management problems in swampy areas. the completion of data acquisition during the requested period (1986) occurred over a region where the cloud cover was always very high at the equator. the spot lateral viewing capability could be responsible for the relative ease in their acquisition. the materials used in the study included computer compatible tapes (cct), negative films at 1/400 000 and 1/200 000 scales for each xs band, standard false colour composites (2 scenes only) at 1/80 000 or 1/100 000 scales, and negative films at 1/200 000 in panchromatic mode. 2 biotropia no. 3, 1989/1990 figure 3. manual interpretation of vegetation types and land-use units on the black and white spot imageries, scale 1/40 000, 1986 (legend see table 4). 11 biotropia no. 3, 1989/1990 figure 4. xs3/xs2 diagram of the 15 spectral classes selected in pasir mayang scene plate 2. multispectral classification of window pasir mayang (512 x 512 pixels) 12 assessment of spot satellite data —y. laumonier & u.k. djailany-syafii legend: sitiung 1. logged-over or depleted forest 2. tall secondary forest mixed with rubber 4. rubber tree estate 11. recently burnt area 12. burnt area long time ago 13. open dry field and shrubby vegetation 14. gardens mixed with food-crops 15. habitation plate 3. multispectral classification of window sitiung (512 x 512 pixels) the multispectral analysis has brought additional information for some nonvegetated types (5,7,11,12,15; table 5) which are more or less homogeneous or difficult to separate properly on the black and white prints. the too long period of time separating data acquisition from field check and the dynamic nature of the land use unit types have hampered the proper evaluation of the results of the classification. some areas classified as "dry open fields" in july 1986 appeared to be rainfed paddy areas in october 1987. all burnt areas (shifting cultivation) face similar problems in that, the spectral response varies a lot between nearby windows (types 11 and 12 in sitiung and in pasirmayang for instance). once again, the problem of having the same spectral value for entirely different object on two different windows and viceversa hampered the classification in figure 4. the confusion between classes 8 and 14 or 9 and 13 is obvious. the haze occurring in the sitiung area may be responsible for the slightly different spectral values compared with the same object in pasirmayang. a general classification cannot be applied to these two windows. moreover, the very simple classification method used may be responsible for some difficulties in separating the objects. separation of spectral value using the box classification method faces the problem of a statistical distribution represented by the standard deviation of the centre point of the box. furthermore, 13 biotropia no. 3, 1989/1990 table 5. spectral characteristics of the 15 classes, defined on a 2 x (512 x 512 pixels) of pasirmayang scene unknown spectral values are always classified into smaller box and this results in unsolved problems in decision boundaries delineation in a bi-dimensional space. in the muarabungo scene, three windows have been chosen (sei pemunyin 1 and 2, rimbo bujang), each of 512 x 512 pixels. the first two corresponds mainly to tropical rain forest and its degradation types, the third (rimbo bujang) to agricultural landcover types found in transmigration areas. for this scene, the box classification of xs1, xs2 and xs3 means and standard deviations gathered from the three windows gives 18 classes, 13 representing vegetation types, and 5 corresponding to open areas, roads and rivers (figure 5 and table 6). results are very similar to those of pasirmayang. implementation of that classification into each window needs some adjustments to avoid misclassification. plate 4, table 7 and plate 5, table 8, are the results of such an implementation. the same problems of some pixel being not classified as in pasirmayang scene has occurred. compared with the classification by visual interpretation of the black and white prints, certain classes (11, 12, 13; table 4) are very difficult to identify properly in the digital classification process. 14 assessment of spot satellite data-y. laumonier & u.k. djailany-syafii figure 5. xs3/xs2 diagram of the 18 spectral classes selected in muarabungo scene. table 6. spectral characteristics of the 18 classes, defined on a 3 x (512 x 512 pixels) of muarabungo scene 15 biotropia no. 3, 1989/1990 plate 4. digital classification of window rimbo bujang, muarabungo table 7. digital classification of rimbo bujang, muarabungo class vegetation types pixels surface (ha) percentage (%) colour 1 unclassed 5953 238.12 2.3 black 2 logged-over or depleted forest 16283 651.32 6.2 red 3 tall secondary forest 9648 385.92 3.7 whitish mixed with rubber trees (bright) red 5 secondary growth forest 24661 986.44 9.4 pale mixed with rubber and shrubs pink 7 secondary growth forest 5940 237.6 2.3 green mixed with remnant primary forest trees 8 mosaic of food crops and 20400 816 7.8 yellowish sparse fruit trees green 9 rubber tree estate 31970 1278.8 12.2 violet 10 newly opened, recently 62956 2518.24 24 brown burnt area 11 fruit trees, coconut and 26999 1079.96 10.3 dark habitat (homestated mixed purple gardens) 15 shrubs and grassland 8315 332.6 3.2 yellow alang-alang 16 newly planted rubber 37104 1484.16 14.2 whitish mixed with young secondary grey growth forest 17 herbaceous and slightly 11915 476.6 4.5 dark open area yellow 16 plate 5. digital classification of window sei pemunyin, muarabungo table 8. digital classification of window sei pemunyin, muarabungo class vegetation types pixels surface (ha) percentage (%) colour 0 unclassed 723 28.92 0.3 black 1 lowland primary forest 21623 864.92 8.3 red 2 logged-over or depleted 62353 2494.12 23.8 pale forest pink 3 tall secondary forest 49591 1983.64 18.9 bright mixed with rubber trees red 4 young secondary forest 11824 472.96 4.5 whitish (bright) red 7 secondary growth forest 79407 3176.28 30.3 green mixed with remnant primary forest trees 10 newly opened, recently 7042 281.68 2.7 brown burnt estate 15 shrubs and grassland 11840 473.6 4.5 yellow alang-alang 16 newly planted rubber 7191 287.64 2.7 whitish mixed with young secondary grey growth forest 17 herbaceous and slightly 2885 115.4 1.1 dark open area yellow 18 river 7665 306.6 2.9 blue assessment of spot satellite data-y. laumonier & u.r. djailany-syafii 17 biotropia no. 3, 1989/1990 3. sungai sembilang — banyuasin delta sungai sembilang banyuasin delta has undergone drastic changes since it has been chosen as a pilot area for transmigration resettlement. in spite of the numerous development aid studies conducted there, it still needs an urgent and accurate monitoring. mangroves used to flourish in the area and the remaining ones are likely to suffer from high population pressure in the future if not managed properly now. besides the mangrove belt, the lowland plain is consisting of extensive peat swamps which are also very difficult to manage for agricultural purposes. vegetation types which are often difficult to identify and segregate properly are: the mangrove forest and the adjacent fresh water swamp forest just behind it (this usually leads to an over-estimation of the actual mangrove area). the fresh water swamp forest on alluvium or shallow peat and the peat swamp forest itself on deeper peat soils, altogether with various peat swamp forest types. the swampy grasslands and the paddy fields occurring within swampy areas. the various secondary shrubby swamp vegetation types and the low natural "pole" swamp forest which look identical when seen from the air. important to investigate also is the proper identification of such secondary vegetation types like the "gelam" (melaleuca cajuputi) formations. a. interpretation and manual classification the visual interpretation gives very interesting results as all the types are perfectly distinct (12 land cover types), from mangrove swamps, fresh water swamp forest and various peat swamp forest types. b. digital classification the multispectral classification performed with spot gives 9 classes among which 6 are vegetation types (plates 6a & 6b). several types or communities are visible within the mangrove itself, and the distinction between mangroves and fresh water swamp forest is easy. possible applications 1. well-drained lowlands a) primary lowland forest appraisal of the mosaic patterns was studied in panchromatic mode at 1/50 000 scale and with multispectral data. it was impossible in any case to recognize any pattern even if some degrees of natural disturbance could be pointed 18 assessment of sport satellite data – y . laumonier & u. r. djailany-syafii 19 p la te 6 a. d ig it al c la ss if ic at io n o f w in d o w s u n ga i s e m b ila n g b an yu as in d el t a ( af te r b ar ke y 1 9 8 7 ) p la te 6 b . t h em a ti c m a p o f w in d o w s u n ga i s e m b ila n g -b a n yu as in d el ta ( a ft er b ar ke y 1 9 8 7 ) biotropia no. 3, 1989/1990 out. riparian forest types, even along small rivers, have been located with accuracy. b) the logging activities have been easily spotted, but indirectly through the visualization of the logging roads. in fact, it was impossible to differentiate the undisturbed forest from the logged-over one in the surroundings. this is a major concern for an evaluation of the intensity of the depletion of forest resources in the region. however, when the human impact on the forest increases, some degrees of depletion appear. this is a real progress compared with landsat mss documents. c) for the monitoring of serial stages, spot capabilities appeared to be most striking. the complete vegetation series from newly clear-cut, burnt, newly planted to young regrowth stages mixed with rubber cultivation can be pointed out. in addition, several secondary forest types mixed with rubber trees, as well as various shrubby secondary vegetation types or low secondary forest types, usually classified as "mosaics" have been discriminated. these results constitute a very noteworthy input for agricultural development of the area. 2. swamps a) it has been sometimes difficult in the past to separate the mangrove forest from the adjacent fresh water swamp forest. this usually led to an over estimation of the actual mangrove area. the ecotone of those two formations is very clear on our spot document (especially on black and white prints). this is due to the perfect delineation of the back-mangrove belt dominated by the palm onco-sperma tigillariutn which is indirectly responsible for the perfect delineation of the mangrove area. in other places, back mangrove hills produced by mud lobsters and invaded by the fern acrostichum aureum give also a very clear white border to the inland limit of the formation. moreover, at least two if not three mangrove communities can be differentiated. b) one of the major challenges for the agricultural development of such an area is the distinction between the fresh water swamp forest on alluvium or shallow peat and the peat swamp forest itself on deeper peat soils. too deep peat areas are very difficult to manage for agricultural purposes. we were able to separate properly on the image the fresh water swamp forest from other types. it was even possible to differentiate several areas according to the thickness of the peat itself by locating easily the "fresh water swamp", "mixed peat swamp", and "peat swamp" forest types. 20 assessment of spot satellite data —y. laumonier & u.r. djailany-syafii c) natural shrubby or very low forest types, sometimes known as "padang" vegetation do occur in the area and are generally confused with secondary types. the spectral analysis has allowed to separate them perfectly. discussion and conclusion it was possible during the present study to compare the usefulness of spot xs and landsat mss data for forestry applications using research findings from previous landsat mss analysis in baturaja martapura area (ducros gambart & gastellu etchegorry 1984; gastellu etchegorry et al. 1985). some important remarks are stressed below: the number of spectral classes that can be discriminated with spot xs, i.e. 32, is much larger than the number of spectral classes that can be obtained with landsat mss, i.e. 8. this is due not only to the finer spatial resolution but also to better spectral characteristics of spot data. in order to derive a reliable and precise forest mapping (1:150 000) with landsat mss data, it was necessary to define complex algorithms (i.e. multi-textural analysis), whereas far better results are directly obtained with simple photointerpretation of 1:50 000 spot imagery. the fine resolution of spot data allows to map many earth features such as roads and rivers, which are particularly useful for an accurate spatial registration of the spot derived documents and provide opportunities for obtaining at the same time cartographic products. numerous ground control points which could not be recognized in the landsat image can easily be located in the spot image. the only advantage of landsat imagery was its larger synoptic overview. panchromatic documents were only available for two scenes and only in the form of negative films at a scale of 1/200 000 since no cct were available, the existing black and white prints were not very useful for this work as the level of information for vegetation is quite low when compared with xs data. the high resolution of spot has proved to be of high value in field checking for exact location of the image's features (e.g. small football field in a little village). however, this emphasizes the real necessity for a very fast delivery of data for proper use of remote sensing techniques in tropical environment where the character of most of the land-use and vegetation patterns is so dynamic. regarding the identification and classification of the humid tropical vegetation in sumatra for environmental management purposes, spot has brought several advantages and encouraging application prospects: 21 biotropia no. 3, 1989/1990 — forestry management several forest types, especially within mangrove and swamp forest areas and also in the uplands, have been discriminated with spot data. even if some of the needs cannot be perfectly fulfilled, e.g. proper distinction between primary and logged-over forest, the fact that degrees in the forest depletion can be pointed out will be of great interest to the forester. considerable improvement in the identification of serial stages will also facilitate the monitoring of reforestation projects. — land-use management the improvement in the identification of some land uses is also striking. such possibilities as the distinction between newly clear cut fields, burnt areas, and newly planted food crops, will allow easy monitoring of such areas where the shifting cultivation is an important feature of the landscape. — mangrove and swamps management the perfect delineation of the mangrove is one of the most beneficial results of spot. many different figures exist concerning the exact mangrove surface in the region. possibilities of studying on satellite documents what could be the mangrove communities themselves ("rhizophora type" or "sonneratia type") was another interesting aspect which should be investigated cautiously. for agricultural management of the peat area, spot provides an excellent tool for an indirect evaluation of the thickness of the peat through identification of specific vegetation types. many of the identification problems mentioned above could probably be solved by photogrammetry using aerial photographs at scale of 1:5000 to 1:10 000. however, the cost of aerial cover in such remote places prohibits in most cases their use before implementation of development project. moreover, there were often difficulties in the past for any extrapolation between the general data provided by landsat mss, and the detailed features of large scale aerial photographs. only simple digital image processing has been applied in this study. it gives nevertheless interesting results and the improvement of multispectral classifications of high resolution data is challenging for site quality assessment through vegetation appraisal. increased revisiting capabilities and lateral viewing, together with a better accuracy in the localization of samples offer additional opportunities in the data acquisition and processing. the dynamic character of most of the land-use and vegetation patterns implies that ground verification has to be carried out as soon as possible after data recording. on one hand, the use and interpretation of classes along the "brightness axis" was interesting and must be worked out in the future for the classification of spectral classes with sparse or non-vegetation cover. on the other hand, the 22 assessment of spot satellite data-y. laumonier & u.r. djailany-syafii "vegetation axis" gives information about the vegetation cover and biomass. the synthesized information that is displayed by figure 2 is undoubtedly very useful for surveying forested areas, and more especially for estimating the density of the vegetation cover of land cover classes. it can provide preliminary assessment about the vegetation cover of spectral classes that were not identified or checked in the field. finally, the validity of classes that were supposedly identified either in the field or with aerial photographs can easily be tested. for example, classes corresponding to forested areas should not be located on the "brightness axis". this study suggests that spot is certainly a very good alternative to medium altitude aerial photograph coverage (1/100 000, 1/50 000) and emphasizes its value for tropical vegetation mapping at a medium scale (1/100 000 to 1/250 000) since comparisons of the cost of spot data versus other remote sensing tools (hadjar 1987, antikidis et al. 1988) give also a striking advantage to spot. acknowledgments this study was conducted as a part of the research project supported by the peps program (programme d' evaluation preliminaire de spot) of the french space agency, in cooperation with iciv, toulouse and seameo-biotrop, bogor. the authors would like to thank dr. j.p. gastellu-etchegorry and dr. r.a. barkey for their contributions in the digital treatments of the martapura and sungai sembilang regions. references antikdis, j.p., a. hudson and j.p. gastellu-etchegorry 1988. a spot/landsat receiving station in indonesia s.c.o.t. report. 57pp. barkey, r.a. 1987. etudes des mangroves de l' indo-malaisie et application des techniques de la tele-detection a sulawesi. these doctorat de l'ups, iciv toulouse, france. biotrop-bakosurtanal 1988. pemetaan vegetasi daerah baturaja, propinsi sumatra selatan. skala 1:250 000. laporan kerjasama seameo-3iotrop dengan bakosurtanal. blasco, f., y. laumonier and purnadjaja 1983. tropical vegetation mapping: sumatra. biotrop bull, in tropical biology. no. 22. deshayes, m. 1981. traitement numerique des donnees landsat. application a la cartographic auto matique de la vegetation tropicale. these docteur-ingenieur, ups-iciv, toulouse. djailany, u.r. 1987. formations vegetales naturelles de sumatra (indonesia): structure et caractristi-ques radiometriques. these doctorat, de l'upt, iciv, toulouse, france. 23 biotropia no. 3, 1989/1990 ducros-gambart, d. and j.p. gastellu-etchegorry, 1984. automatic analysis of bi-temporal land-sat data: application to the study of the evolution of vegetation-covered areas in a tropical region. igarss symp., ref. esa sp-215: 187-192. ducros-gambart, d., t. l.e toan and m. deshayes 1984. utilisation des donnees landsat pour la cartographic des formations ve'getales tropicales dans le sud de sumatra. l'espace ge'ographique, n°3, 205-214, 290. gastellu-etchegorry, j.p. 1988. remote sensing with spot; an assessment of spot capability in indonesia. gadjah mada university press. yogyakarta. hadjar, n. 1987. forest observation by satellite. paper presented at the acrs, jakarta, a 16/1 13. laumonier, y. 1981. classification of southern sumatra forest types. biotrop unpublished report. laumonier, y. 1983. international map of the vegetation, scale 1:1 000 000; sheet southern sumatra. biotrop, bogor, iciv, toulouse. laumonier, y., purnadjaja and setiabudi 1986-87. international map of the vegetation, scale 1:1 000 000; sheet central sumatra (1986), sheet northern sumatra (1987). biotrop, bogor; iciv, toulouse. laumonier, y. u.r. djailany, j.p. gastellu-etchegorry and r. barkey 1987. assessment of spot satellite based system in tropical vegetation identification, classification and monitoring in sumatra (indonesia). spot first result in flight conference, paris nov. 87. cnes. p ? purnadjaja 1980. evolution de la vegetation 19691976 dans la region de palembang, sumatra, me'moire dea, ups-iciv, toulouse. richardson, a.j. and c.l. wiegand 1977. distinguishing vegetation from soil background information. photogram. eng. and remote sens. 43(2): 207 — 216. rouse, j.w., r.h. haas, j.a. schelland d.w. deering 1973. monitoring vegetation systems in the great plains with erts. 3rd erts symp., nasa sp-351, 1:309-317. torquebiau, e.f. 1986. mosaic patterns in dipterocarp rain forest in indonesia and their implications for practical forestry. j. of trop. ecol. 2:301 325. tucker, c.j. 1977. spectral estimation of grass canopy variables. remote sens, of environment, 6: 11-26. 24 assesment of spot satellite data – y. laumonier & u.r. djailany-syafii figure 1. location of study areas table 1. requested products and acquisition scored 3 biotropia no. 3, 1989/1990 both visual and digital analysis techniques have been used. vegetation sketch maps and land cover classifications were prepared by manual interpretation of black and white xs or standard false colour composite prints at scales varying from 1/50 000 to 1/100 000. for mangroves and swampy areas, other false colour combinations have been used such as xs2 – bi vgi or xs1 pci pc2. a supervised multispectral classification was performed simultaneously using a pericolor 1000 system in france and an ibm at/xt system in indonesia, by simple visualization of each band using gray scales or colour table and simple transformations, such as band ratios, vegetation indices and principal component analysis. the procedure is similar to the information on techniques extracting developed for soil and leaf area indices (richardson & wiegand 1977; tucker 1977; rouse et al. 1973). means, standard deviation, range of radiance values as well as coefficient of correlation for the 3 bands were computed for all samples, together with covariance matrix of all classes to assess the value of the classification. to facilitate the visualization of the field site data, xs1, xs2 and xs3 means and standard deviations were plotted on bi-dimensional diagrams. all classes have been checked in the field (one year after data acquisition by the satellite) and eventually redefined. results 1. baturaja martapura the original vegetation is much degraded with typical huge areas of grasslands dominated by imperata cylindrica, various stages of shrub savannas and pseudoclimactic forest type dominated by schima wallichii. some very degraded remnants of the original forest types and a land-use composed of irrigated or rainfed paddy fields, orchards, rubber plantations, "damar" plantations and reforestation areas are other important features of the landscape. some small depressions are common along the streams. a rather large one is found north east of the scene, around lake (lebak) datuk. the water level in these depressions fluctuates considerably and during the dry season they are mostly dry. , a. visual interpretation the classification produced is given in table 2. these results were used to produce an ecological vegetation map at a scale of 1/250 000 at the request of the indonesian national coordination agency for survey and mapping, the bakosurtanal. a total of 23 types were identified and used later as legend of the published map (biotrop bakosurtanal 1988). 4 assessment of spot satellite data y. laumonier & u.r. djailany-syafii table 2. classification of the vegetation in martapura region by visual interpretation 1. very depleted lowland forest (high secondary forest with remnant primary forest tree species) 2. high secondary forest (often mixed with rubber) 3. mosaic of high secondary forest and food crop cultivation 4. high secondary forest dominated by schima wallichii 5. shrub, secondary regrowth 7. grassland 8. swampy tall secondary vegetation dominated by melaleuca sp. 9. swampy shrubby vegetation 10. swampy grassland dominated by cyperaceae 11. rubber estate 12. coffee gardens, often overtopped by fruit trees, mixed with food crop fields 13. pepper gardens 14. orchards, fruit trees 15. newly opened field for rubber and oil palm estate 16. dry fields, food crops, mixed with secondary regrowth 17. village, habitat and fruit trees 18. rice field b. digital classification the analysis of visual displays of spot data provided the selection of thirty two spot spectral classes. computer screen displays of enhanced spot data and spot photographic products at a 1/100 000 scale of bands xs1, xs2 and xs3 allowed the identification of most of them. however, some classes (20 to 31) could be distinguished only during digital processing stages (density slicing, image enhancement, etc.). they were not detected with conventional photo-interpretation, and the question of their real existence also arises since, unfortunately, most of these classes could not be identified properly. however, qualitative values of the density of their biomass were tentatively assessed with the aid of techniques developed for extracting information on soil and leaf area indices (rouse et al. 1973; richardson & wiegand 1977; tucker 1977). they can be classified as "sparse vegetation" (29, 30, 31), "rather sparse vegetation" (26, 27, 28), "vegetation" (22, 23, 24, 25) and "green vegetation" (20, 21). the 32 classes and their characteristics are presented below (gastellu-etche-gorry 1988): they are grouped per land cover type and according to their relative position on the "vegetation axis". forest (1) primary forest: tall trees up to 45 55 m. compared to other classes, it is characterized by the smallest xs1 and xs2 radiometric values; i.e. highest absorption by vegetation. 5 biotropia no. 3, 1989/1990 (2) very depleted forest: result of former exploitation of primary forest followed by local depletion. (3) remnant primary forest and secondary growth: external features (roads, etc.) reveal some degradation. radiometries in bands xsi and xs2 are slightly larger than those of (1). (4) remnant primary forest and secondary growth. the proportion of primary forest is less important than that of (3). (5) swamp vegetation: small xs3 radiometric values: xsi and xs2 radio-metric values are larger than those of dry land forests. a "spotted" textural pattern may reveal the presence of the swampy areas. secondary forest (6) old tall secondary forest: compared to (7), the trees and texture are similar but the vegetation index (i.e. leaf area index and/or biomass density) is larger. (7) tall secondary forest: taller and older than (8); texture may be similar to that of (10). (8) low secondary forest: compared to primary forest, the xsi and xs2 radiometric values are larger and the xs3 radiometric values are similar. (9) low secondary forest: successional stage before (8), younger and smaller. rubber plantation (10) rubber plantation: homogeneous aspect on xsi and xs2 imageries. it is identified thanks to the presence of roads that usually display a geometric pattern. (11) young rubber plantation: successional stage after (12). (12) newly planted rubber trees: xsi radiometric values are between those of rubber plantation and secondary forest growth, whereas its xs3 radiometric values are smaller than those of rubber plantation, primary forest and secondary forest. settlements and mixed gardens (13) fruit tree gardens: close to settlements; should be verified in the field. (14) gardens and sparse vegetation cover (alang-alang grasses). shrubs, herbaceous vegetation and bare soil (15) herbaceous vegetation: vegetational stage after (17), it is more densely vegetated (larger vegetation index). 6 assessment of spot satellite data-y. laumonier & u.k. djailany-syafii (16) shrubs and thicket vegetation. (17) sparse vegetation cover: bare soil and more or less sparse herbaceous vegetation. (18) recently opened area: vegetational stage before (17). (19) bare soil. non-identified land cover units (assessed through their respective vegetation index; leaf area! index/biomass): (20), (21) green vegetation (22), (23), (24), (25) vegetation (26), (27), (28) rather sparse vegetation (29), (30), (31) sparse vegetation. water (32) moreover, figure 2 shows that some spectral classes which share similar spectral characteristics cannot be reliably discriminated by spectral analyses only figure 2. xs3/xs2 diagram of the 32 spectral classes selected in martapura scene. 7 biotrop1a no. 3, 1989/1990 i.e. classes (26) and (28), (21) and (11), (8) and (22), etc. in this case, knowledge of the field (if any) and the spatial and textural information which can be extracted from simple photo interpretation of spot xs1, xs2 and xs3 imageries must also be inputed (gastellu etchegorry 1988). in this study, the identification of these classes obviously required additional field checks which were conducted in june 1988. plate 1 and table 3 give an example of the reformulation of the 32 classes for one "window of the martapura scene", after field work. only 13 classes (11 vegetation types) were actually identified for that particular area, which means that several spectral values correspond actually to one vegetation types. classes 5,8,22,23,26 and 28 for instance, should be regrouped into class 7 e.g. grasslands, at least as far as identification of the types is concerned. table 3. digital classification of window batu marta, martapura vegetation types pixels surface (ha) percentage (%) colour 0 unclassed and home 14926 597.04 4.8 black stated mixed gardens (settlement) 3 secondary forest 40617 1624.68 13.2 whitish mixed with rubber bright red 4 low secondary forest 59524 2380.96 19.4 bright and schima wallichii green 6 shrub, secondary 4235 169.4 1.4 whitish regrowth (newly grey felled) 7 grassland 52676 2107.04 17.1 yellow 8 rubber tree estate 93328 3733.12 30.4 red 9 swampy shrubby 10737 429.48 3.5 orange vegetation 11 dry field, food crop, 12398 495.92 4 dark mixed with secondary green regrowth 12 longtime burnt and 6900 276 2.3 dark slightly covered by grey regrowth 13 recently burnt for 7828 313.12 2.5 brown estate area 14 swampy area 2388 95.52 0.8 violet 17 river 811 32.44 0.3 blue 18 sand sediment in the 782 31.28 0.3 bright river blue 8 plate 1. digital classification of window batu marta, martapura 2. muarabungo pasirmayang a very peculiar land-use feature is found there. local people have grown rubber trees since 1910. after about two years of rice cropping, the field is planted with rubber trees and it gradually becomes a secondary forest mixed with rubber which is rarely cleaned but still used for tapping. this leads to various stages of secondary forests mixed with rubber which are impossible to differentiate from pure serial stages on remote sensing documents. the area still harbours pieces of quite undisturbed forest and large tracks of logged over forests. the landscape is also characterized by large transmigration areas. in that area, remote sensing encountered problems in identifying and classifying the following vegetation and land cover types: the mosaic nature of the primary lowland forest: the tropical forest is highly dynamic and grows in patches, a mosaic of higher and lower forest parts which have their origin in the natural fall of trees or part of the tree crowns. the locally depleted forest (local exploitation) and the really undisturbed ones (only industrial logging is recognizable but only indirectly through the visualization of the logging roads). low secondary forest types mixed with rubber tree cultivation (managed by local farmers) and pure serial stages without human interferences. 9 assessment of spot satellite data —y. laumonier & u.r. djailany-syafii 1.pdf 10.pdf 2.pdf 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf microsoft word 30 biotropia no. 24, 2005 : 30 45 analysis of the temperature dependence of co2 assimilation rate (study case: glycine maxl. merr) tania june biotrop-jcsea, seameo biotrop, btic building, jl. raya tajur km. 6 bogor, indonesia; email: taniajune@biotrop.org; laboratory o/agrometeorology, bogor agricultural university, bogor abstract the maximum rate of carboxylation (kcmax) and maximum rate of regeneration of ribulose bisphosphate (rubp) (controlled by the rate of electron transport, jmax) arc two processes governing the photosynthetic capacity of plants. both processes are affected by temperature. this paper examines how the response of these two photosynthetic capacities to temperature determines the temperature response curve of the co2-assimilation rate for plants grown at different temperatures, by using the concept of the farquhar €3 photosynthesis model. the goal is to use photosynthetic parameters from co2 and light curves to predict the temperature dependence of the co2-assimilation rate (a) of soybean and to estimate the preferred growth temperature. analysis shows that the optimum temperature of the assimilation rate changes with the changing temperature dependence of carboxylation and regeneration of rubp. key words : temperature dependence/soybean/modeling photosynthesis/preferred growth temperature. introduction photosynthesis is strongly affected by temperature. during gas exchange measurements, the short-term temperature dependence of photosynthesis is strongly affected by other environmental factors such as light intensity and intercellular coa concentration (berry & bjorkman 1980). some of these short-term effects can be modeled by c3 photosynthesis models of farquhar et al. (1980). however, the response varies not only among species but even within an individual species subjected to changing growth temperature regimes during their developments (long-term effects) (berry & bjorkman 1980). in many species, the optimum temperature at which maximum short-term photosynthesis is obtained, shifts upwards when plants are grown at higher temperatures (lange et al. 1974; slatyer 1977; berry & bjorkman 1980; ferrar, slatyer & vranjic 1989), which is commonly known as acclimation or adaptation. the long-term temperature acclimation during growth may affect both the maximum photosynthetic rate per unit leaf area and the shape of the photosynthetic temperature response curve. how this growth temperature (long-term effect) affects the short-term temperature response of photosynthesis is still unclear. it can be attributed at the enzyme level or at the gene action-dna level. to understand the mechanism of this change in temperature dependence, it is important to know the limiting factor to photosynthesis at each measured temperature. 30 biotropia no. 24, 2005 according to the farquhar cj photosynthesis model (farquhar et al. 1980), carboxylation and regeneration of rubp are two processes governing photosynthesis. in the model, the photosynthetic rate is limited either by the capacity of rubp carboxylase (rubisco) to consume ribulose bisphosphate (rubp), denoted as fcmax, or by the capacity for rubp regeneration, denoted as jmax/4. these capacities have different temperature dependencies, in the original model and as confirmed, for example, by kirschbaum & farquhar (1984) and later by june (2002). for a fixed temperature dependence of the electron transport rate (j), farquhar & von caemmerer (1982) showed that increasing the ratio of the capacity of rubisco to consume rubp to that of rubp regeneration could change the optimum temperature (ro) by changing the relative amounts of the two components, so that the optimum temperature would be higher when the ratio is increased. june (2002) has shown that this ratio decreased with increasing temperature. however, june (2002) also showed that temperature dependence of electron transport rate is not fixed but changed, both at short-term and long-term time scales. the net effect is on the change in the ratio jmm/vcmm with temperature. in the farquhar & von caemmerer (1982) paper, the ratio of rubp consumption to rubp regeneration increased with increasing growth temperature. in june (2002), the jmm/vcmax ratio decreased as the short-term temperature measurement increased, for each of the growth condition. this paper examines how the changes in these two photosynthetic capacities with temperature affect the temperature response curve of the co2-assimilation rate for plants grown at different temperatures, using the concept of the farquhar et al. (1980) c3 photosynthesis model. the goal is to use parameters from cc>2 and light curves from june (2002) (vcmm and jmax temperature dependences) to predict the temperature dependence of the co2-assimilation rate (a) and then to test the model with independent measurements of the temperature dependence of the cc>2-assimilation rate. the plants used for testing the model were grown under the same conditions as the plants used for the parametrisation of the model in june (2002). materials and methods seeds of indeterminate soybean (glyclne max [l.] merr. cv stephen) were sown in 12 liter plastic pots containing a mixture of sand and vermiculite (1:1, v/v) and plants were thinned to one plant per pot after germination. plants were grown in a controlled environment chamber with a 14hour photoperiod of around 700 umol quanta m"2 s"', 60/70 % relative humidity day/night and three different temperature regimes: 20/15, 25/20, 32/27 day/night °c under ambient [co2], 350 umol mol'1. the lowest and highest temperature regimes were repeated with atmospheric [ccy enrichment to 700 umol mol"1. the source of light used in the growth chamber was a metalarc lamp (general electric lighting), mvr 1000/u. plants were well spaced (30 cm apart at sowing) to avoid mutual shading. rhizobial inoculation was not provided for the plants. each 31 analysis of the temperature dependence of co2 assimilation rate tania june pot was flushed every second day with full-strength herridge's solution (0.50 mm mgso4, 0.25 mm cacl2, 0.25 mm kc1, 0.125 mm kh2po4, 0.125 mm k2hpo4, 25 um ferric monosodium salt of edta, 12 um h3bo3, 3.6 um mncl2, 77 um zncl2, 76 nm cucl2, 25 nm namoo4) (herridge 1977) and watered twice daily on days when nutrients were not given. to obtain a range of nitrogen levels in the plant leaves, three different concentrations of kno3 were added to the nutrient solution (2, 5 and 16 mm). the nutrient solutions were added to each pot until they drained at the base (2.5 to 3.0 liters per pot). for gas exchange measurement, several temperature response curves of assimilation rate were measured at a light intensity of 1200 umol m~2 s"1 with co2 concentrations of 350 umol mol"1 using a photosynthetic gas exchange system developed in the environmental biology group, research school of biological sciences, australian national university. two pots of plants were used for replication. the vapour pressure difference was kept constant at around 12.5 mbar in most cases and was less than 17 mbar in all cases. for each leaf, the co2-assimilation rate was measured at six temperatures from 15°c to 40°c, holding for 15 to 20 minutes at each temperature measurement to reach a steady state condition. data obtained from measurements are then compared to simulation results where parameters are obtained from measurements by june (2002). results and discussion temperature dependence of co2 assimilation rate figure 1 shows the temperature dependence of the absolute value of the co2-assimilation rate (a) measured at 350 umol mol"1 [co2] and irradiance of 1200 umol m"2 s"1, for plants grown under 350 umol mol'1 [co2]. as expected, increasing the temperature from 15°c to 25°c increases the assimilation rate: 18 % for plants grown at 20/15°c (day/night temperature), 88 % for plants grown at 25/20°c and 98 % for plants grown at 32/27°c. increasing the measurement temperature further resulted in a reaching a maximum at the optimum temperature and then decreasing. after reaching the optimum temperature, the photosynthesis rate dropped with further increasing temperature; by 40°c the drop in a was as much as 10 % for plants grown at 20/15°c, 24 % for plants grown at 25/20°c and 10 % for plants grown at 32/27°c. it is clear that the temperature dependence of the co2-assimilation rate is different depending on the growth temperature. 32          the photosynthetic processes in soybean exhibit a capacity for temperature acclimation. plants  maintained at different day/night temperatures had different optimum photosynthetic temperatures (t0).  plants grown at 32/27°c have an optimum temperature for the co2‐assimilation rate of 32.7±0.2°c. when plants  were grown at 25/20°c, the optimum temperature was 30.1±0.8°c and when plants were grown at 20/15°c, the  optimum temperature was reduced to 28.5 ±0.9°c (table 1).  table  1. optimum  temperature  for  coz  assimilation  rate,  7"0,  and  assimilation  rates,  a,  at  optimum  temperature and at various other temperatures for plants growing at different temperatures,+  s.c. plants were grown at  the  temperatures  indicated  in  the  table, co2  concentration of 350  (imol mol"1 and nitrogen concentration of 16 mm. gas exchange measurements were conducted  at [co2]=350 (imol mol"' and light intensity, 7=1200 ̂ mol m" 2 s"'.        analysis of the temperature dependence of co2 assimilation rate tania june in a similar study with festuca arundinaceae grown at 10 and 25°c, treharne & nelson (1975) found that at measurement temperatures above 15°c, net photosynthesis rates per unit area were higher in plants grown at 25°c. the authors concluded that greater photorespiration rates were important in accounting for the low net photosynthetic rates of the 10°c plants at higher temperature measurements. however, in the photosynthesis model used in this manuscript, photorespiration is already taken into account in the form of f * (co2 compensation partial pressure in the absence of dark respiration) and c\ (co2 concentration in the intercellular air spaces). treharne & nelson (1975) further concluded that the decrease in photosynthesis above 30°c seen in both groups of plants was due primarily to a decrease in stomatal conductance, and hence c\, the decrease in c\ was not observed in my results (as seen in figure. 2) with increasing temperature. for net cc>2 uptake for plants grown at day/night temperature of 10/10°c was 12°c for agave americana and 15°c for a. deserti. when the growth temperature was raised to 30/30°c, the optimum temperature shifted upward by 7°c for a. americana and 3°c for a. deserti. shifting a. americana to the higher growth temperature caused the maximum rate of net cc>2 34 biotropia no. 24, 2005 uptake at the optimal temperature to increase, whereas the same shifting of a. deserti caused it to decrease. the results showed that a(t0), i.e. the optimum temperature for net assimilation rate or rate of photosynthesis, increase with increasing growth temperature. plants grown under 700 ^mol mol"' [co2] exhibited a similar pattern as those grown under ambient [co2], with t0 = 28.0 ± 0.4 and 32.2 + 0.7°c and a(t0) = 20.7 + 2.0 and 21.8 ± 0.7 tamol m'2 s'j for plants grown at 20/15 and 32/27°c, respectively. it is important to note that a change in c\ (the intercellular cc>2 concentration) can cause a shift in the optimum temperature of photosynthesis, with t0 increasing with ci (farquhar & von caemmerer 1982; kirschbaum & farquhar 1984). there is a linear correlation between growth temperature and the optimum temperature for co2 assimilation rate (figure 3). the linear regression from table 1 can be solved to give a "preferred" temperature for the cc>2 assimilation rate of 33°c. this concept of "preferred" temperature was initially suggested by slatyer & ferrar (1977), and is the intersection of the linear regression line and the 45° line. the "preferred temperature" was also used in june (2002) to descibe the acclimation of the electron transport rate (j) to growth temperature.   analysis of the temperature dependence of co2 assimilation rate ‐ tania june  what controls the reduction in co2assimilation rate after reaching                                     optimum  temperature?  it has been reported that high temperature  limits co2 availability, because c the physiological  responses of leaves which result in increased resistance to the ga diffusion (mukohata et al. 1971;  monson et al. 1982). high temperature also alter the substrate specificity of rubisco (jordan &  ogren  1984;  brooks  &  farquha  1985)  and  its  activity  (weis  1981;  santarius  et  al.  1991).  working  with  neriun  oleander,  badger  et  al.  (1982)  showed  that  plants  grown  at  low  temperature ha< higher activity of several photosynthetic enzymes at low temperatures but had  lowe heat stability relative to plants grown at high temperatures. temperature acclimatioi in the  electron transport system could also be the reason for the change in the co: assimilation rate at  high temperature. several studies have shown that the temperature dependence of electron  transport capacity changes with growtr temperature (armond et al. 1978; badger et al. 1982;  mitchell  &  barber  1986)  such  changes were  also  observed  in  june  (2002), where  plants  grown at highei temperature had a lower electron transport rate.  modeling the temperature dependence of the co2‐assiiniiation rate  the basic assumption underlying the modelling is that the rate of photosynthesis at any  temperature is controlled by the activity of enzyme rubp carboxylase‐oxygenase (rubisco),  denoted as vcmm, and the potential rate of regeneration of rubp, denoted as jmax/4. therefore,  at a given temperature, the net co2‐assimilation rate, a, is taken as being either the rubisco‐limited  rate, av, or the estimated rubp‐regeneration‐limited rate of photosynthesis, aj, whichever  is  smaller (units of umol m"2 s"1).  where c\ = partial pressure of co2 in the leaf, f* = co2 compensation partial pressure in the  absence of dark  respiration,  /?d  = dark  respiration by  the  leaf which  continues  in  the  light, o =  ambient  partial  pressure  of  oxygen,  and  atc  and  k0  are  the  michaelis‐menten  constants  for  carboxylation and oxygenation by rubisco, respectively. temperature dependence of c\ follows the  equations from figure 2. r^ follows this equation:  36              biotropia no. 24, 2005 rd =al+a2(t-25)+a,(t-25)2 (3) where tis leaf temperature (°c) and ,4], a2andai are the fitted parameters of the rf temperature relationship (table 2), which was obtained from june (2002). the temperature dependence of kc and k0 follows an arrhenius function as where r is the universal gas constant, 8.3144 j mol"1 k"'. ef and e0 are the apparent activation energies with the 25 subscript representing the value at 25°c. the effect of temperature on the cc>2 compensation point of photosynthesis in the absence of mitochondrial (dark) respiration follows the equation of von caemmerer et al. (1994), assuming infinite wall conductance. the temperature dependence ofj and fcmax is given by the following equations june (2002): where j(t0), t0, q, c\, c2 and c3 are the fitting parameters, specific for each set of growth conditions (table 2). the capacity of the electron transport rate can be inferred from where the light dependence of electron transport, j, follows the equation by .farquhar & wong (1984): 37 analysis of the temperature dependence of co2 assimilation rate tania june •/max is the maximum (light-saturated) rate of electron transport capacity of the leaf, 0 is the curvature factor of the light response curve of eq. (10) and a-i is the quantum yield of electron transport. / is the amount of light intensity incident on the leaf surface. /2 in eq. (9) is equal to 021. the temperature dependence of © follows the equation of june (2002): where t\= 0.93, t2 = 0.0145, and t-> = -8.13 the w4' these curvature factor parameters were obtained from june (2002), where measurement of the light curves was done with light incident on both the upper and lower surface of the leaf. biotropia no. 24,2005 simulated co2 assimilation rate the simulation results, based on table 2, at measurement temperatures of 15 to 40°c and / = 1200 umol m"2 s"1, are shown in figure 4. figure 5 shows the results for jmax, converted from j using eqs. (9) to (11). fcmax is constantly increasing with temperature, in relatively good agreement with wang et al. (1996) and other estimates (wullschleger 1993). however, ferrar et al. (1989) who investigated several species of eucalyptus grown at contrasting temperatures found that in leaves grown at high temperature, f"cmax increased with short-term temperature measurement, but in leaves grown at low temperature, kcmax did not increase as measurement temperature increased. they speculated that rubisco may be inactivated or damaged at measurement temperatures higher than the growth temperature. in my experiment, although fcmax increases with short-term temperature measurements for all growth conditions, plants grown at higher temperature have a slightly lower fcmax than plants grown at lower temperature. june (2002) showed that there is an acclimation in the electron transport rate which favours the lower growth temperature (20/15 °c). hence, if there is no stomatal effect due to different growth temperatures, then the co2-assimilation rate of plants grown at 20/15 °c will be higher than that of plants grown at 32/27 °c in the model (as seen in figure 4). the lower observed co2-assimilation rate of plants grown at 20/15°c (figure 1), and their lower assimilation rate at t0, make these plants a poor representation of this simulation (compare figure 1 and figure 4). the standard measurements of those particular leaves (standard measurement was done at light intensity of 1200 umol irfv, vpd =12.5 mbar, [ccy = 350 umol mol"', and temperature = 25°c), before starting each measurement were lower on the first day the plants were taken out from the growth chamber (the day when those measurements were done). they were increased by 19.5 % the next day. if the data (of the 20/15°c plants) were corrected with this percentage, plus taking into account the reduced ci( then the co2-assimilation rate at t0 of plants grown at 20/15°c would be higher than the other two growth temperature of plants as shown by the simulation result in figure 4. the simulation shows that differences in the temperature dependence of a are due to the differences in the processes limiting^. for example, a for 20/15 °c plants is limited by rubp regeneration below 23°c and is limited by rubisco activity at temperatures higher than 23°c, while a for 32/27°c plants is limited by rubp regeneration below 30°c and is limited by rubisco activity at temperature higher than 30°c. for plants grown at 25/20°c, a was limited by rubp regeneration below 28°c and limited by rubisco activity at temperature higher than 28°c. a co-limitation of these two capacities occurs at the optimum temperature (where av and aj lines cross in figure 4) and only one of them would limit photosynthetic rate at other temperatures, with the penalty of excess investment in the other capacity. therefore, when growth temperatures vary, changes in the organization of the photosynthetic apparatus are necessary, and this can be shown by the changed optimal ratio of jmaxs / vmaxs (farquhar & caemmerer 1982). 39 analysis of the temperature dependence of co2 assimilation rate tania june the simulated ratio of jmaxs / vcmaxs figures 4 and 5 show that between 15 and 35°c, the relative slope of the increase in jmax is higher than it is in fcmax; then the opposite happens with further increase in temperature. as the slope of increase in jmax with temperature was higher than that of fcmax within the range of 15 30°c (figures 4 and 5), the simulation predicts that the ratio of jmax/vcmax would increase with temperature within this range. figure 5 indicates that plants change the allocation of their photosynthetic resources between these two capacities as growth temperature changes. june (2002) showed that ./max/fcmax did have a lower value when plants were grown at 32/27°c compared to plants grown at lower temperatures, which is supported here by the simulation. 40 biotropia no. 24, 2005 farquhar & von caemmerer (1982) and recently hikosaka (1997) predicted that the photosynthetic rate should be co-limited by fcmax and jmax at the growth temperature for efficient nitrogen utilization of photosynthesis. figure 4 shows that the temperature where the two processes co-limit photosynthesis in the simulation (i.e. where av and aj lines cross) was close to the growth temperature. it was around 23°c for plants grown at 20/15°c, 27 °c for plants grown at 25/20°c and 30°c for plants grown at 32/27°c. the slight differences are probably due to the different light intensity used in the measurement (1200 iimol m"2 s"') compared to that in the growth chamber (600-700 umol m~2 s"'). under lower light, the electron transport rate would be lower and it might co-limit at a lower temperature than the ones from figure 4, which would improve the agreement for plants grown at the lower two growth temperatures. when j is converted to jmax using eq. (9), the value depends on which 0 value is used (as shown in figure 5). using 0 = 0.7, the estimated jmax was higher as tem 41 analysis of the temperature dependence of co2 assimilation rate tania june perature increased than the estimated jmm calculated using a θ that increased with temperature. this difference affects the ratio of jmm/ycmm, with a higher ratio obtained when θ was held constant at 0.7. now, the temperature where co-limitation occurs changes from that in figure 4. for plants grown at 20/15 and at 25/20°c, it is almost the same at 33 -35°c. plants grown at 32/27°c, were always limited by a-y figure 6 also shows that for conditions in which electron transport becomes limiting (that is, growth at high temperature), the electron transport rate will dominate the behaviour of the co2-assimilation rate, so the optimum temperature of a should shift towards the optimum temperature of jmm. conclusions the temperature response characteristics of the photosynthetic process are not fixed but depend on the prevailing conditions during growth. the present study shows how the temperature dependence of the photosynthetic rate differs between plants grown at different temperatures. the factors responsible for the difference include the change in temperature dependences of the two processes controlling photosynthesis, carboxylation and regeneration of rubp. the change in the proportion of photosynthetic resources into these two capacities can be shown by the change of the jmax/ vcmaxs ratio with growth temperature. 42 biotropia no. 24, 2005 acclimation to growth temperature occurred as shown by the changing optimum temperature with growth temperature. this acclimation is important for plants in optimising their photosynthesis rate in the environment they are exposed to in terms of the most economical way of using photosynthetic resources. for the soybean plants used in this experiment, the optimum temperature for electron transport (j) was a few degrees higher than the preferred temperature for co2-assimilation rate (33°c). hence an increase in temperature during the day to more than 33°c will still increase the electron transport rate, although not the co2-assimilation rate. for the purpose of modelling plant growth or modelling the capacity of plants in absorbing co2 in relation to the changing environmental conditions (atmospheric co2 concentration, temperature, light intensity, nitrogen, and water availability effect on supply of co2), the most useful models will be those which incorporate acclimation processes at all levels of organization within the plants. these models will be the most precise and the most responsive to changes in the physical and biological factors which control photosynthesis. however, the knowledge of the mechanisms of temperature dependence of photosynthesis is still limited and so the combination of an empirical approach with the mechanical ones in these areas where the mechanism is already established would be beneficial. references armond p. a., schreibcr u. and o. bjorkman. 1978. photosynthetic acclimation to tempe-rature in the desert shrub, larrea diraricata. ii. light harvesting efficiency and electron transport. plant physiology, 61:411-415. badger, m. r. and g.j. collate. 1977. studies on the kinetic mechanism of ribulose-l,5-bisphosphate carboxylase and oxygcnase reactions, with particular reference to the effect of temperature on kinetic parameters. carnegie institute of washington yearbook, 76:355-361. badger, m.r., bjorkman, o. and p.a. armond. 1982. an analysis of photosynthetic response and adaptation to temperature in higher plants: temperature acclimation in the desert evergreen nerium oleander l. plant, cell and environment, 5: 85-99. berry, j. and o. bjorkman. 1980. photosynthetic response and adaptation to temperature in higher plants. annual review of plant physiology, 31:491 -543. brooks, a. and g.d. farquhar 1985. effect of temperature on the coro2 specificity of ribulose-1,5-bisphosphatc carboxylasc/oxygenasc and the rate of respiration in the light. estimates from gas exchange measurements on spinach. planta, 165: 397-406. farquhar, g. d. and s. von caemmerer. 1982. modelling of photosynthetic responses to environmental conditions. physiological plant ecology. ii. encyclopedia of plant physiology, new series. o. l. lange, p.s. nobel, c.b. osmond and h. ziegler. berlin, springer-vcrlag. farquhar, g.d., von caemmerer, s. and j.a. berry. 1980. a biochemical model of photosynthetic co2 assimilation in leaves of c3 species. planta, 149: 78-90. 43 analysis of the temperature dependence of coz assimilation rate tania june ferrar, p.j., slatyer, r.o. and j.a. vranjic. 1989. photosynthetic temperature acclimation in eucalyptus species from diverse habitats and a comparison with nerium oleander. australian journal of plant physiology, 16: 199-217. herridge, d.f. 1977. carbon and nitrogen nutrition of two annual legumes. university of western australia. perth. hikosaka, k. 1997. modelling optimal temperature acclimation of the photosynthetic apparatus in c3 plants with respect to nitrogen use. annals of botany, 80: 721 -730. jordan, d. b. and w. l.ogren 1984. the co2/o2 specificity of ribulose, 1,5-bisphosphate carboxylase/oxygenase. dependence on ribulosebisphosphate concentration, ph and temperature. planta, 161:308-313. june, t. 2002. environmental effects on photosynthesis of c3 plants: scaling up from electron transport to the canopy (study case: glycine max l. merr). environmental biology, research school of biological sciences. australian national university. canberra. kirschbaum, m.u.f. and g.d. farquhar. 1984. temperature dependence of whole-leaf photosynthesis in eucalyptus pauciflora sieb. ex spreng. australian journal of plant physiology, 11: 519-538. lange, o.l., schulze, e.d., evenari, m., kappen, l., and u. buschbom. 1974. the temperature-related photosynthetic capacity of plants under desert conditions. i. seasonal 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isolated mesophyll protoplasts of vaurianella locusta (l.) betcke. photosynthetica, 25: 17-26. slatyer, r.o. 1977. altitudinal variation in the photosynthetic characteristics of snow gum, eucalyptus pauciflora sieb. ex spreng. iv. temperature response of four populations grown at different temperatures. australian journal of plant physiology, 4: 595-609. slatyer, r.o. and p.j. ferrar. 1977. altitudinal variations in the photosynthetic characteristics of snow gum, eucalyptus pauciflora sieb. ex spreng. v. rate of acclimation to an altered growth environment. australian journal of plant physiology, 4: 595-609. trehame & nelson 1975 in: plants and temperature. (eds. s.p. long and f.i. woodward). symposium of the society of experimental biologists, vol. 42. cambridge: company of biologists, p. 329-346. von caemmerer, s., evans, j.r., hudson, g.s. and t.j. andrews. 1994. the kinetics of rubisco inferred from measurements of photosynthesis in leaves of transgenic tobaco with reduced rubisco content. planta, 195: 3347. 44 biotropia no. 24, 2005 wang, k. y., kellomaki, s. and k. laitinen. 1996. acclimation of photosynthetic parameters in scots pine after three years exposure to elevated temperature and co2. agricultural and forest meteorology, 82: 195-217. weis, e. 1981. reversible heat-inactivation of the calvin cycle: a possible mechanism of the temperature regulation of photosynthesis. planta, 151: 33-39. wullschleger, s.d. 1993. biochemical limitation to carbon assimilation in €3 plants. a retrospective analysis of a/ci curves from 109 species. journal of experimental botany, 44: 907-920. 45 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf biotropia book juni revisi 14 juli 09.indd 45 biotropia vol. 16 no. 1, 2009: 45 54 corresponding author: cginting2001@yahoo.com physic nut (jatropha curcas l.) diseases in lampung province cipta ginting and tri maryono department of plant protection, university of lampung bandar lampung 34145, indonesia abstract intensifi ed cultivation of physic nut (jatropha curcas l.) could raise the importance of plant diseases. th e objectives of this research were to diagnose diseases occurring on physic nut in lampung province and to determine their intensity. field observation was conducted in four districts: south lampung, tanggamus, bandarlampung, and tulang bawang. disease intensity, whether expressed as disease incidence or severity, was recorded from plant samples determined by making diagonal lines across the fi eld on which fi ve observation spots were made. on each spot, fi ve plant samples were observed. specimens were also collected and placed individually in plastic bags for laboratory observation. th e diseases found on physic nut in lampung province were cercospora leaf spot, alternaria leaf spot, fusarium wilt, and bacterial wilt. in addition, leaf malformation fi rst thought to be viral disease was commonly found in many locations. further mechanical transmission failed to produce similar symptom on tested plants and higher population of mites were found on malformed leaves than that in normal leaves. based on the disease distribution and intensity, the most likely threatening disease in physic nut cultivation is bacterial wilt. fusarium also caused wilt, but it was only found in one subdistrict with low incidence. key words: alternaria, cercospora, fusarium wilt, bacterial wilt, jatropha curcas diseases introduction recently, eff orts to develop and utilize alternative energy sources have been increasing due to the need to meet energy demand and to reduce the dependence on fossil energy sources. utilizing plants as alternative sources is a priority because of being renewable energy resources. physic nut (jatropha curcas l.) is one of the prospective plants. it can grow on soil with medium and low fertility, its oil is inedible and of high quality for fuel (sudradjat 2006; syah 2006). physic or also known as “jarak pagar” in indonesia is used to be grown less intensive such as for fence. in the last few years, however, physic nut has been cultivated intensively in a monoculture pattern. for example, in katibang subdistrict, south lampung district, alone the acreage of physic nut was 192 ha (anon. 2007a). furthermore, the head of mining and energy department of lampung province stated that the potential area for physic nut planting in lampung province is about 5000 ha (anon. 2007b). banuwa (2007) also reported that lampung province has a good prospect to grow jatropha. 46 biotropia vol. 16 no. 1, 2009 change of planting pattern could increase the importance of some plant diseases. intensifi cation of a plant generally tends to increase the threat of pests and diseases (fry 1982; agrios 2005). plants that are grown less intensive usually do not suff er severely from pests and pathogen attack. however, when they are cultivated intensively such as in intensive monoculture pattern, plants are of high risk to be attacked severely by pests and diseases, especially when they are not properly managed. accordingly, the change in cultivation pattern of physic nut into intensive monoculture pattern should be followed by raising awareness about its pests and diseases. information about physic nut diseases in literature is very limited. lozano (2007) listed pathogens infecting the plant without mentioning their geographical distribution, including phytophthora spp., pythium spp., and fusarium spp., heminthosporium tetramera, pestalotiopsis paraguarensis, p. versicolor, and cercospora sp. it has also been reported that several pathogens infect physic nut in indonesia: ralstonia solanacearum, rhizoctonia bataticola (republic of indonesia department of agriculture 2006a), phytophthora nicotianae (republic of indonesia department of agriculture 2006b), alternaria sp. and cercospora sp., fusarium sp., botrytis and xanthomonas campestris (hambali et al., 2006; prihandana and hendroko 2006). latha et al. (2009) reported the occurrence of root and collar rot diseases of physic nut in india. to our knowledge, there is no published information on diseases of physic nut in lampung province so far. th e objectives of this research were to diagnose diseases occurring on physic nut in lampung province and to determine the intensity of the disease in the fi eld. materials and methods observation in the fi eld th e disease diagnosis started with fi eld observation of physic nut diseases. field observation was conducted in four districts, considered to be the most important areas of physic nut cultivation in lampung, and in each district two subdistricts were visited. th e districts and subdistricts were south lampung (natar and ketibung subdistricts), tanggamus (gunung alip and talang padang), bandarlampung (rajabasa and tanjung seneng), and tulang bawang (tanjung raya and simpang pematang). due to wide geographical distribution of physic nut fi elds, some fi elds were only visited once or twice. observation was focused on symptoms of diseases as they occur in the fi eld and factors that might aff ect the diseases such as plant age, plant spacing (row width), plant growth, plant canopy, and topography. to determine the intensity of diseases, plant samples were selected with the following procedure. two diagonal lines were made across the fi eld on which fi ve observation spots were made. on each of fi ve spots (x) fi ve plants were observed. for systemic diseases, the disease intensity was expressed as disease incidence determined by using the formula of di = n/n x 100%, where di = disease incidence, n = number of plants showing symptoms, and n = the total number of plants observed. 47 jatropha curcas diseases – cipta ginting et al. for local diseases, the disease intensity was expressed as diseases severity determined by the formula of where ds = disease severity, v = disease score of sample ith, n = number of plant samples with a particular score, v = the highest score, and n = total number of samples. disease score range from 0 (no symptom) to 4 (more than 50% of leaf surface showed symptoms). regarding the kinds of the diseases, in addition to the plant samples, any plants showing symptoms were observed. at fi rst the distribution of diseased plants was determined whether it was evenly distributed in the fi eld or it was in clustered (made gradient). th en, for each diseased plant it was determined whether the symptoms were local or systemic. specimens, whole or part of plant samples showing symptoms, were collected and placed individually in plastic bags. th e specimens were placed in a cooler during transportation to the laboratory for further examination. observation in the laboratory each of the specimens was observed in the laboratory soon upon arrival or the next day. if the suspected pathogen causing particular symptoms was a fungus, the specimen was incubated at relative humidity of 100% by placing sterile cotton dampened with sterile water in plastic bags or trays covered with plastic. pathogenic fungi usually form spores in 2 – 3 days at room temperature. th e spores and other structures were observed with a compound microscope. some other specimens were used to isolate infecting fungi. for this purposes, the media used was potato dextrose agar (pda) supplemented with lactic acid 1.4 ml per liter media. small pieces (1 – 2 mm) of plant tissue between the healthy and the infected tissue were taken and then placed on the media in petri dishes. fungi growing in the media was transferred into new media and identifi ed to genus. identifi cation was based on barnett and hunter (2001). diseased plants suspected to be infected by virus was diagnosed from the symptoms. to verify whether or not the causal agent was a virus, the suspected agent was transmitted mechanically. further observation was also conducted to determine whether causal agent other than virus might cause the symptoms. on the malformed leaves, mites were found. to determine whether or not the severity of leaf malformation was correlated to the population of mites, the leaves were divided into three groups, i.e.: leaves with severe symptoms, leaves with mild symptoms, and those without symptoms. from each category, the number of mites was determined. if the symptom was suspected to be caused by pathogenic bacteria, diagnosis was based on internal and external symptoms. in addition, eff ort was made to observe bacterial ooze. to do this, a few steam cuts were placed on test tubes containing tap water. about 30 minutes later, bacterial ooze was streaming out of the tissue. 48 biotropia vol. 16 no. 1, 2009 results and discussions locations and cultivation manners table 1 summarizes the locations and cultivation manners of physic nut fi elds in four districts. it shows that the cultivation system including plant age, row width, and planting pattern was similar in the subdistricts of every district. however, the cultivation system greatly varied among districts. th e area used for physic nut cultivation is generally fl at except that of ketibung, south lampung. physic nut was cultivated in a monoculture system in all fi elds except in tulang bawang district where it was grown in mixed culture with rubber and oil palm plants. physic nut generally grew well except in both fi elds of tulang bawang district and tanjung seneng where it grew poorly. in bandarlampung, only two physic nut fi elds were found and those were in rajabasa and tanjung seneng subdistricts. in addition to these two fi elds, there were a few locations where seedlings are produced for sale. in tulang bawang district, three fi elds were reported to be planted with physic nut. however, when we visited the locations it appeared that one fi eld was grown with another jathropa species locally known as “jarak kepyar”. th erefore, only two fi elds about 0.5 ha, each was observed which are located in tanjungraya and simpang pematang subdistrits. table 1. locations and cultivation manners of physic nut in the field observed in four districts in lampung province locations cultivation manners plant age (months) row width (m) planting pattern land condition south lampung district: natar subdistrict ketibung subdisrict 8 9 2 x 2 monoculture fl at 2 x 2 monoculture aslant tanggamus district: gunung alip subdistrict talang padang subdisrict 6 1 x 1 monoculture fl at 6 1 x 1 monoculture fl at bandarlampung district: rajabasa subdistrict tanjung seneng subdisrict 6 1 x 1 monoculture fl at 12 1 x 1.25 monoculture fl at tulang bawang district: tanjung raya subdistrict 2 1 x 1.25 mixed culture with rubber plants fl at simpang pematang subdisrict 2 1 x 1.25 mixed culture with oil palm plants fl at physic nut diseases th e disease and disorder of physic nut found in lampung were cercospora leaf spot, bacterial wilt, alternaria leaf spot, fusarium wilt, and leaf malformation (table 2). cercospora leaf spot was found in all fi elds except in tanjung seneng, bandarlampung. th e diagnosis of cercospora leaf spot was conducted by fi eld observation followed by 49 jatropha curcas diseases – cipta ginting et al. laboratory examination. th e form of leaf spot was irregular. th e leaf spot was whitish brown in the center with brown at the edge. th e spot was a necrotic tissue that was encircled by chlorotic tissue seen as yellowish zone (figure 1). in the laboratory, fungal reproductive structure taken from the leaf spot was identifi ed as cercospora based on the identifi cation key used (barnett and hunter 2001). cercospora is reported to cause such leaf spot on physic nut and the species is reported to be c. ricinella (hambali et al. 2006) and c. jatrophae (prihandana and hendroko 2006). based on this observation and reference, it was concluded that the disease was cercospora leaf spot caused by cercospora sp. figure 1. symptom of cercospora leaf spot. the symptom was described in the teks. alternaria leaf spot was found only in tanjung seneng. diagnosis of alternaria leaf spot was also conducted in the fi eld and confi rmed in the laboratory. th e symptoms of this disease are similar to that of cercospora leaf spot except that all spot areas are brown in color without whitish area (figure 2). in addition, after 2-day incubation at 100% relative humidity at room temperature, conidia were found in the spot area. th e conidia consisted of several dark cells. according to the identifi cation key (barnett and hunter 2001), the conidia were formed by alternaria. alternaria ricini was reported to be the causal agent of leaf spot of physic nut (hambali et al. 2006). based on the observation in the fi eld and in the laboratory by hambali et al. (2006), it was concluded that the disease was alternaria leaf spot caused by alternaria sp. figure 2. symptom of alternaria leaf spot (arrow). 50 biotropia vol. 16 no. 1, 2009 another disease of physic nut was wilt caused by a fungus, fusarium. th e disease was only found in tanjung raya subdistrict, tulang bawang district. th e symptoms of the disease were the aff ected plant became wilt and the leaves remained attached to the plants. isolation was done on pda containing lactic acid (1.4 ml/l) media and the resulted culture was whitish in color. observation under a microscope revealed that there were two kinds of conidia, i.e.: macroconidia and microconidia. according to barnett and hunter 2001, the fungus is fusarium. hambali et al. (2006) reported that f. oxysporum could cause wilt symptoms in physic nut in the seedbeds as well as in the fi eld. bacterial wilt was found at least in one subdistrict of all districts in lampung province (table 2). th e main symptoms were that the aff ected plants became wilted and the leaves remained attached to the plants. th e base stem showed necrosis. internal observation revealed that the internal tissues showed brownish color in the middle parts (figure 3). when the stem was submerged in tap water, bacterial ooze streamed out of the plant tissue. th e bacterial ooze looked like white strand but when the water was shaking the bacterial ooze soon broke and the suspension became clouded. based on the external and internal symptoms as well as the bacterial ooze, it was assumed that the disease was bacterial wilt caused by pseudomonas (ralstonia). in addition to the diseases, a disorder called leaf malformation was observed on the leaves, mostly on the young ones. leaf malformation occurred in all fi elds except the fi elds in tulang bawang district. it was thought to be caused by a plant virus because several plants showed similar symptoms when attacked by certain plant viruses (agrios 2005; semangun 2000). some farmers also believe that the problem was caused by a virus. however, mechanical transmission failed to give positive results that the inoculated plants did not show any symptom after more than 4 weeks after inoculation. since no symptoms occur in the tested plants indicating that the agent could not be transmitted mechanically, further observation was conducted in two fi elds which were natar and rajabasa subdistricts. during 5-week observation in both fi elds, some plants showed reduced symptoms and the incidence of leaf malformation was decreased (table 2). furthermore, it was observed that mites were found to be associated with the disordered plants. when the population of mite was determined on leaves with severe, mild, and no symptoms, it was consistently found that mite population on leaves with symptoms was higher than that on leaves without symptoms (table 3). table 2. locations, diseases, and disease intensity of physic nut locations (date of first observation) diseases disease intensity (%) at 1 – 5 observations1) 1 2 3 4 5 south lampung district: natar subdistrict (6 juni 2007) cercospora leaf spot2) bacterial wilt3) leaf malformation3) 22 7 98 17 4 84 13 2 58 6 4) 44 6 33 ketibung subdisrict (8 oktober 2007) cercospora leaf spot leaf malformation 10 66 9 56 tanggamus district: sinar banten subdistrict (3 september 2007) cercospora leaf spot bacterial wilt leaf malformation 20 12 80 51 table 2. continued jatropha curcas diseases – cipta ginting et al. locations (date of first observation) diseases disease intensity (%) at 1 – 5 observations1) 1 2 3 4 5 talang padang subdisrict (3 september 2007) cercospora leaf spot bacterial wilt leaf malformation 21 32 40 bandarlampung district: rajabasa subdistrict (3 juni 2007) cercospora lea1f spot bacterial wilt leaf malformation 22 12 84 22 12 84 17 12 52 25 16 40 25 20 36 tanjung seneng subdisrict (7 agustus 2007) alternaria leaf spot2) leaf malformation 4 96 5 88 tulang bawang district: tanjung raya subdistrict (19 juni 2007) cercospora leaf spot fusarium wilt3) 5 8 4 6 3 5 simpang pematang subdisrict (1 oktober 2007) cercospora leaf spot bacterial wilt 3 13 2 11 1 9 note: 1)obervation was conducted 1 – 5 times. in any fi eld observed more than once, the observation interval was 2 week. 2)cercospora leaf spot and alternaria leaf spot were expressed as disease disease severity. th e formulas used to determine disease severity is stated in materials and method. 3)bacterial wilt, fusarium wilt, and leaf malformation were expressed as disease incidence. th e formulas used to determine disease intensity are stated in materials and method. 4) means no data. figure 3. part of wilted physic nut and longitudinal section of stem revealed brownish internal tissues showed. 52 biotropia vol. 16 no. 1, 2009 table 3. population of mites on leaves with or without symptom at two subdistricts observation total number of mites per 25 leaves spots1) severe symptom mild symptom no symptom natar subdistricts 1 25 16 7 2 34 30 5 3 25 30 8 4 15 18 4 5 19 25 6 average 23.6 23.8 6.0 rajabasa subdistricts 1 24 16 5 2 33 30 7 3 25 23 7 4 19 15 6 5 18 11 4 average 23.8 19.0 5.8 1)on each observation spot, 25 leaves of each category were observed. intensity of physic nut diseases disease intensity was determined by two methods, disease severity and disease incidence, according to the kind of diseases. disease severity was used for diseases local in nature such as leaf spot and disease incidence was used for systemic in nature such as wilt diseases and leaf malformation. table 2 shows that disease severity of cercospora leaf spot on physic nut was low at tulang bawang district where the plants were 2 months old. in the other three districts, where physic nut was 6 – 12 months old, the disease severity was relatively higher than that in tulang bawang. based on the severity of cercospora leaf spot and its distribution almost all fi elds were contaminated by the disease. more attention should be paid to the diseases of physic nut cultivation. alternaria leaf spot was only found in tanjung seneng subdistrict and its severity was 4 – 5% (table 2). based on the results, it could be concluded that alternaria leaf spot is unlikely to be the major disease of physic nut. fusarium wilt was found only in tanjung raya subdistrict, tulang bawang district. its incidence was 5 – 8% (table 2). based on the incidence its occurrence was relatively low, so the fusarium wilt could be regarded as a minor disease of physic nut. since it causes wilt that is very destructive, the disease including factors aff ecting disease development and transmission should be monitored. at locations where disease intensity was measured more than once with 2 week interval, disease intensity was either remained constant or tended to decrease overtime except alternatia leaf spot. it should be noted that, the observation was made started on may 2007 when the relative humidity was relatively high, and then it was decreasing during the period of observation. based on the data, wilting diseases (fusarium wilt and bacterial wilt) also tended to decrease. th e decrease was partly due to destructive 53 jatropha curcas diseases – cipta ginting et al. observation that some wilted plants were taken and brought to the laboratory for further observation. th ese uprooted plants were not counted at subsequent fi eld observation. th e most likely to be controlled in physic nut cultivation is bacterial wilt which is suspected to be caused by pseudomonas (ralstonia) solanacearum. th e disease was found in all districts visited. its incidence was 2 – 7% in natar subdistrict, 9 – 13% in simpang pematang subdistrict, and 12% and 32% in gunung alit and talang padang subdistricts, respectively (table 2). in addition, it causes wilt so that the disease potentially devastates the plants. according to the facts gathered from this study, it is interesting to note the recovery phenomenon shown by a plant. th e plant wilted as a result of attack by pseudomonas solanacerum. however, few months later, the plant recovered. one tends to assume that the recovery phenomena found in bacterial case might be due to the dry condition. th is phenomenon needs to be studied further to determine the factor involved. information obtained from this study is relevant to formulate disease control strategy. th e observation was conducted during the dry season in lampung province so that relative humidity was low. relative humidity is an important environmental factor aff ecting disease incidence and development and low relative humidity reduce the incidence and development of plant diseases (agrios 2005; fry 1982). similar observation should be conducted during the rainy season with high relative humidity. conclusions diseases found on physic nut in lampung province were cercospora leaf spot, alternaria leaf spot, fusarium wilt, and bacteial wilt. in addition, leaf malformation was also found in many locations. th e symptom was fi rst thought to be the result of a pathogenic virus attack. however, the symptom is not mechanically transmitted and the severity of diseases seemed to be correlated with the occurrence and population of mites. th e disease severity of cercospora leaf spot and alternaria leaf spot was relatively low to moderate. disease incidences of bacterial and fusarium wilts were also considered moderate. based on the distribution and intensity of the diseases, the most likely threatening disease in physic nut cultivation is bacterial wilt. fusarium also caused wilt, but it was only found in one subdistrict with low incidence. acknowledgments th e authors would like to thank the southeast asian regional centre for tropical biology (seameo biotrop) bogor for the fi nancial support through dipa 2007 and to didik purwanto and tedy achmad rijaya for technical assistance in the fi elds and in the laboratory. 54 biotropia vol. 16 no. 1, 2009 references agrios, g.n. 2005. plant pathology. 5th ed. elsevier academic press, burlington, m.a., as. anon.. 2007a. lamsel – petani budidayakan jarak. lampung post, april 16, 2007. anon.. 2007b. bahan bakar nabati: potensi lampung 724,354 hektar. lampung post, january 24, 2007. banuwa, i.s. 2007. prospek pengembangan tanaman jarak pagar (jatropha curcas l.) di provinsi lampung. paper presented at seminar held in bandar lampung, may 29, 2009. 14 pp. barnett, h.l. and hunter, b.b. 2001. illustrated genera of imperfect fungi. 5th ed. macmillan pub. co., new york. fry, w.e. 1982. principles of plant disease management. academic press, new york. hambali, e. suryani, a., dadang, hariyadi, hanafi e, h., reksowardojo, i.k., rivai, m., ihsanur, m., suryadarma, p., tjitrosemito, s., soerawidjaja, t.h., prawitasari, t., prakoso, t., and w. purnama. 2006. jarak pagar: tanaman penghasil biodiesel. penebar swadaya, depok. latha, p., v. prakasam, a. kamalakannan, c. gopalakrishnan, t. raguchander, m. paramathma and r. samiyappan. 2009. first report of lasiodiplodia theobromae (pat.) griff on & maubl causing root and collar rot diseases of physic nut (jatropha curcas l.) in india. australasian plant disease notes 4: 19 – 20. lozano, j. a. d. 2007. jatropha. www.gvedinternational.org/fi le_117/jatropha/ pdf.eng.pdf. accesed on february 18, 2008. prihandana, r. and r. hendroko. 2006. petunjuk budi daya jarak pagar. agromedia pustaka, jakarta. republic of indonesia , department of agriculture. 2006a. penelitian dan pengembangan tanaman jarak pagar (jatropha curcas l.) sebagai bahan pembuatan enerji-bio di indonesia perlu mengikuti peta jalur yang rasional. info tek jarak pagar (jatropha curcas l.) vol. 1 no. 1. in www.deptan.go.id/berita/ update27juli06/infotek/jp/no.201/ 2006.pdf. accessed on january 17, 2006. republic of indonesia department of agriculture. 2006b. peluncuran perdana benih unggul jarak pagar (jatropha curcas l.). info tek jarak pagar (jatropha curcas l.) vol. 1 no. 7, 2006 in www.deptan. go.id/berita/update27juli06/infotek/jp/no.207/2006.pdf accessed on january 17, 2006. semangun, h. 2000. penyakit-penyakit tanaman perkebunan di indonesia. revised ed. gadjah mada university press, yogyakarta. sudradjat, h.r. 2006. memproduksi biodiesel jarak pagar, solusi hasilkan biodiesel bekualitas tinggi. penebar swadaya, depok. syah, a. n. a. 2006. biodiesel jarak pagar, bahan alternatif yang ramah lingkungan. agromedika pustaka, jakarta. thank you for evaluating anybizsoft pdf splitter. a watermark is added at the end of each output pdf file. to remove the watermark, you need to purchase the software from http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html biotropia no biotropia no. 12,1999 .31-41 vegetative compatibility groups of fusarium oxysporum, the causal organism of vascular wilt on roselle in malaysia k.h. ooi and b. salleh school of biological sciences, universiti sains malaysia, 11800 penang, malaysia abstract forty strains of fusarium oxysporvm isolated from roselle (hibiscus sabdariffa var. sabdariffa) showing vascular wilt symptoms in three states (terengganu, penang and ipoh) in the northern malaysian peninsula were used to investigate the vegetative compatibility. nitrate-nonutilizing (nil) mutants were recovered from all the strains tested and subsequently used to study vegetative compatibility groups (vcg) within the population by nit mutants pairings on minimal medium. thirteen vcgs were found and none were vegetatively compatible with those of other formae speciales (f. spp.) such as asparagi and cubense, and non-pathogenic strains from paddy and oil palm. the results indicate that there is substantial genetic diversity in f. oxysporum that causes vascular wilt disease on roselle as reflected by multiple vcgs, but the distribution of strains into the vcgs is not even as there are 26 representatives in vcg-1001m, two in vcg-1003m and vcg-1013m and only one in the other vcgs. this study may provide new insight into the establishment of a new forma specialis off. oxysporum. key words: vegetative compatibility groups/m/ mutants/fitsarium oxysporwrn/roselle/vascular wilt/ malaysia. introduction fusarium oxysporum schlechtend.: fr. is an asexual, soil-borne fungus found in agricultural soils throughout the world. this species includes many pathogenic strains, known as formae speciales (f. spp.). each forma specialis (f. sp.) is characterized by its ability to cause vascular wilt on a limited taxonomic range of host plants (booth 1971). individual strains of the fungus show a high degree of host specificity which led snyder and hansen (1940) to classify strains of the fungus into f. spp. on the basis of the host plants attacked. for example, f. oxysporum f. sp. melonis attacks muskmelon (cucumis melo l.), whereas f. oxysporum f. sp. vasinfectum is a pathogen of cotton (gossypium spp.). strains of the fungus, whether from the same or different f. spp. are usually morphologically indistinguishable (puhalla 1985). currently, pathogenicity tests are the primary means to distinguish different pathogenic fusarium strains. however, such tests do not indicate whether various strains of a given f. sp. or a physiologic race are genetically related. puhalla (1985) used nitrate-nonutilizing (nit) mutants to show that strains in different f. spp. of f. oxysporum were in distinct vegetative compatibility groups (vcg), based on the ability of complementary nit mutants to anastomose and form wild-type 31 biotropia no. 12,1999 heterokaryons. these nit mutants could be recovered without mutagen treatment from selective media containing potassium chlorate (kc1o3) (puhalla 1985). heterokaryon formation between complementary mutants was indicated by the development of dense aerial growth where mycelia of two thin nit mutant colonies touched and anastomosed when cultured side-by-side on minimal medium with nitrate as a sole nitrogen source (klittich & leslie 1988; puhalla & spieth 1985). mycelia outside the anastomosed region remained thin. diversity within pathogenic strains of f. oxysporum has been evaluated by characterizing strains in terms of vegetative compatibility. strains capable of forming a successful vegetative heterokaryon are referred to as vegetatively compatible. strains that are vegetatively compatible with one another are frequently described as members of the same vegetative compatibility group, or vcg (leslie 1993). strains from different groups would not form heterokaryons with each other. in asexually reproducing fungi such as fusarium, vegetatively compatible strains are much more likely to be genetically similar than vegetatively incompatible strains (correll et al. 1987). puhalla (1985) used nit mutants to test for vegetative compatibility among 21 strains of f. oxysporum. he found a correlation between vcg and f. sp. i.e. members of the same vcg belong to the same f. sp. vegetative compatibility tests, therefore, have been shown to be a powerful tool for studying genetic diversity in f. oxysporum (correll et al. 1987). most of the studies revealed a limited number of distinct vcgs within a f. sp. (correll et al. 1986a; jacobson & gordon 1988; and larkin et al. 1988). in contrast, 97 strains of f. o. f. sp. asparagi were found to include a minimum of 42 vcgs (elmer & stephens 1989), and 110 strains of f. oxysporum isolated from celery roots but nonpathogenic on celery included a minimum of 14 vcgs (correll et al. 1986b). vascular wilt caused by f. oxysporum is the most important soil-borne disease on roselle in malaysia (ooi et al. 1998). roselle (hibiscus sabdariffa var. sabdariffa) has recently been domesticated and is being planted in a large scale for growing juice, jam and confectionary industries in malaysia (mat isa et al. 1985; tan & said 1994). the seeds contain 17% oil similar in properties to cotton seed oil. roselle is suitable to be planted on mineral or bris soils (chin 1986). however, the cultivation of this newly domesticated crop in malaysia has been disrupted due to various diseases mainly vascular wilt caused by a soil borne fungus, f. oxysporum. the pathogenicity of f. oxysporum isolated from the roots and stems of roselle showing vascular wilt symptoms in the field was proven in the greenhouse (ooi et al. 1998). in the present study, forty strains of f. oxysporum isolated from roselle showing vascular wilt symptoms in the northern malaysian peninsula were used to investigate the vegetative compatibility among the fungal strains. 32 vegetative compatibility groups offusarium oxysporum k.h. ooi and b. salleh materials and methods strains forty strains of f. oxysporum isolated from roselle in three states of the malaysian peninsula were examined (table 1). the strains were each grown on peptone pentachloronitrobenzene (peptone-pcnb) agar (nash & snyder 1962) for 57 days. conidia from these strains were transferred to 3% water agar (wa), and individual uninucleate microconidia were isolated using a micromanipulator and transferred to potato sucrose agar (psa). colonies from these single-spore cultures were produced on psa under standard incubation conditions and the resultant colonies were stored in liquid nitrogen (salleh & sulaiman 1984; salleh & strange 1988). these parent cultures were used as the starting inoculum for all subsequent tests. table 1, complementation reactions between nitrate-nonutilizing (nit) mutants off. oxysporum1 nitl nit3 nitm nit i -o r± ±or+ nit3 ±o r + nitm + + + ' + = complementation occurs readily, = no complementation occurs, ± = weak and/or slow complementation occurs. media the basal medium (bm) was described by correll et al. (1987). minimal medium (mm) was made by adding 2.0 g of sodium nitrate (nano3) to 1 l of the basal medium. nit mutants were generated on minimal agar medium with chlorate (mmc) as described by correll et al. (1987). psa were made as described by booth (1971). peptone-pcnb medium was prepared by adding the following to 1 l of distilled water: difco peptone, 15.0 g; kh2pcv 1.0 g; mgso4, 0.5 g; pentachloronitrobenzene (pcnb 75 wp), 1.0 g; agar, 20.0 g; and streptomycin, 300 ppm (nash & snyder 1962). recovery of nitrate-nonutilizing mutants (nit mutants) nit mutants were generated on mmc at 1.5 % concentration of chlorate. mycelial transfers (approximately 2-mm3 psa blocks) of the fungus were placed on three equidistant places of three plates of mmc and incubated under standard incubation conditions (salleh & sulaiman 1984) and examined periodically (3, 5 and 33 biotropia no. 12,1999 7 days after incubation) for the appearance of fast-growing fan-shaped sectors from the initially restricted colony. all sectors were transferred to mm and those that grew as thin expansive colonies with no aerial mycelium were considered nit mutants. all nit mutants were resistant to chlorate and showed wild-type growth on psa. nit mutant phenotypes the physiological phenotypes of nit mutants recovered were distinguished by their growth on mm amended with different compounds as the sole nitrogen source (correll et al. 1987; klittich & leslie 1988). the plates were incubated as described above, and colony morphology was scored relative to the wild-type parent after four days. complementation tests vegetative compatibility was determined by observing heterokaryon formation between complementing nit mutant on mm. pairings were made by placing mycelia from each nit mutant 1-3 cm apart on mm. pairings were incubated as described above for 7-10 days and then scored for complementation. phenotypically distinct nit mutants, a nitl and a nitm, were obtained from 10 strains. these nit mutants were paired in all possible combinations to establish which strains were vegetatively compatible. nitm from one isolate in each of the vcgs thus identified was selected to serve as a tester for each group. two nitls were obtained from each of the remaining 30 strains and paired with a nitm from each of the established vcgs. from within the group of strains that did not pair with any of the testers, a second group of testers was obtained and the procedure repeated. this process was continued until all strains were assigned to a vcg. each pairing was repeated at least once (gordon & okamoto 1991). all of the nit mutants recovered from the same parent were paired with at least one nitl, one nit3, and one nitm mutant from that parent to test for self-compatibility. vegetative compatibility tests with testers of other f. spp. and non-pathogenic strains of f. oxysporum two nit mutants (one nitl and one nitm) of at least one isolate from each of the established vcgs were paired on mm with four nit testers of the following two f. spp.: asparagi (two testers) and cubense (two testers) ; and four nit testers of nonpathogenic strains of f. oxysporum from paddy (oryza saliva) (one tester) and oil palm (elaeis guineensis) (three testers). 34 vegetative compatibility groups offusarium oxysporum k.h. ooi and b. salleh results nit mutant isolation spontaneous chlorate resistant sectors were readily recovered from all 40 strains of f. oxysporum when cultured on mmc. the majority of the chlorate resistant sectors recovered were unable to utilize nitrate as a sole nitrogen source and consequently grew as thin expansive colonies with no aerial mycelium on mm. these sectors were designated nit mutants. the rate of radial expansion of these nit mutants is compatible to that of the wild types. successive subcultures of the nit mutants onto mm continue to show this thin growth habit. most of the nit mutants were stable, but a few (2-4%) of them developed small patches of heavy growth after prolonged incubation on mm. nil mutants phenotype identification recovered nit mutants could be divided into three distinct phenotypic classes by their growth and colony morphology on media containing one of five different nitrogen sources. the classes presumably reflect mutations at a nitrate reductase structural locus (nitl), a nitrate assimilation pathway-specific regulatory locus (nit3) and loci (at least five) that affect the assembly of a molybdenum-containing cofactor necessary for nitrate reductase activity (nitm). the majority of nit mutants recovered on mmc were nitl. several sectors recovered from some of the strains were resistant to chlorate but had wild-type colony morphology on mm. correll et al. (1987) have observed this type of chlorate resistant nitrate utilizing sectors in f. oxysporum. they examined in more detail such sectors to determine if these sectors resulted from a mutation in a single locus or from the heterokaryotic growth of complementary mutations in two or more different nuclei. the analysis of microconidia from these sectors indicated that the chlorate-resistant nitrate utilizing sectors could be heterokaryotic or homokaryotic. microconidia from homokaryotic sectors had a wild-type morphology on mm and presumably were mutants that were both chlorate-resistant and able to utilize nitrate (cm mutants). microconidia recovered from heterokaryotic sectors were often a mixture of nit mutants conidia, wild-type conidia, and/or crn mutant conidia. complementation tests complementation occurred between nit mutants with different phenotypes. complementation occurred more rapidly and growth of the resulting heterokaryon was more robust in pairings of nitm with nitl or nit3 mutants than in pairings of nitl with nit3 mutants (table 1). when nitl and nit3 mutants were paired, 35 biotropia no. 12, 1999 complementation reaction was weak (very little aerial mycelium). to confirm that weak reactions could not result from cross feeding without anastomosis, 10 pairs of weakly reacting strains were tested with sterilized cellophane separating the two nit mutants following a method devised by puhalla (1985). where cellophane prevented hyphal contact, there was no visible reaction. outside the cellophane barrier, where hyphae of the two nit mutants came into direct contact, a weak reaction was observed. this result was consistent with a requirement for hyphal anastomosis to produce weak reactions. in our delineation of compatibility groupings, an isolate was placed in a vcg only if it reacted strongly and developed dense aerial growth where the mycelia of the nit mutant colonies came in contact and anastomosed to form a heterokaryon, with at least one other isolate in that group. on this basis, 40 strains were assigned to a total of 13 vcgs (table 2). the largest of these included 26 strains, whereas 10 vcgs were represented by only a single strain while two vcgs were represented by 2 strains. in no case was a strain vegetatively compatible with strains from two different vcgs. table 2. strains of f. oxysporum classified by vegetative compatibility and their source strain1 source (naturally wilted roselle) l location vcg 1001m 72558% root mengabang bakung, 7erengganu t2560% root mengabang bakung, 7erengganu t2561% root kuala berang, 7erengganu t2563% root rhu 7apai, 7erengganu t2564% root rhu 7apai, 7erengganu t2566% root rhu 7apai, 7erengganu t2568% root rhu 7apai, terengganu t2577% root kuala berang, 7erengganu t2605% nonsterile seed kuala 7erengganu, terengganu 12607% rotten fruit mengabang bakung, 7erengganu 72608% rotten stem mengabang bakung, 7erengganu 72609% rotten leaf kampung pasir nering, 7erengganu 72610% rotten leaf kampung pasir nering, 7erengganu 72623% stem mengabang bakung, terengganu 72625% root kuala berang, 7erengganu 72633% rotten stem mengabang bakung, terengganu p2883% stem usm, penang p2884% stem usm, penang p2885% root usm, penang p2886% root usm, penang p2887% root usm, penang p2888% stem usm, penang p2889% rotten root usm, penang p2890% rotten root usm, penang p2893% stem usm, penang p2894% stem usm, penang 36 vegetative compatibility groups offusarium oxysporum k.h. ooi and b. salleh table 2. continued strain' source (naturally wilted roselle)2 location vcg 1002m t2567% root rhu 7apai, 7erengganu vcg 1003m 12573% root marang, 7erengganu 12618% stem kampung pasir nering, 7erengganu vcg 1004m t2575% root marang, 7erengganu vcg 1005m 72620% leaf kampung pasir nering, 7erengganu vcg 1006m t2621% leaf kampung pasir nering, 7erengganu vcg 1007m 72622% stem mengabang bakung, 7erengganu vcg 1008m 72628% root mengabang bakung, 7erengganu vcg 1009m 72629% root rhu 7apai, 7erengganu vcg 1010m 72637% healthy root rhu 7apai, 7erengganu vcg 1011m a2891% fruit ipoh, perak vcg 1012m a2882% fruit ipoh, perak vcg 1013m a2892% fruit ipoh, perak a2898% healthy root ipoh, perak 't= strains from terengganu a= strains from perak p= strains form penang '%= strains from roselle !parts of roselle where the strains were isolated heterokaryon self-incompatibility no complementation occurred between any nit mutants of eight strains tested, even after repeated attempts. furthermore, no complementation was observed when the nitl or nitm mutants were paired among themselves. the lack of complementation between phenotypically distinct nit mutants recovered from these strains lead us to designate these strains as heterokaryon self-incompatible (table 3). vegetative compatibility tests with testers of other f. spp. and non-pathogenic strains of f. oxysporum two nit mutants (one nitl and one nitm) from each of the established vcgs were paired with four nit testers from two f. spp.: asparagi and cubense', and four nit 37 biotropia no. 12, 1999 table 3. source of self-incompatible strains of f. oxysporum strain1 source2 location 12566% root rhu tapai, terengganu t2575% root marang, terengganu t2605% nonsterile seed kuala terengganu, terengganu t2608% rotten stem mengabang bakung, terengganu t2609% rotten leaf kampung pasir nering, terengganu t2637% healthy root rhu tapai, terengganu a2882% fruit ipoh, perak p2883% stem usm, penang t = strains from terengganu a= strains from perak p= strains from penang '%= strains from roselle 2parts of roselle where the strains were isolated testers from non-pathogenic strains. no heterokaryon formed (no complementation) between the testers from each vcg with any of the other f. spp. and the nonpathogenic strains. discussion fusaria are often considered to be genetically unstable because they frequently produce sectors in culture which differ from the original colony in morphology, virulence, or other characteristics (bumett 1984; puhalla 1981). a moderate degree of genetic instability could have a selective advantage for a plant-pathogenic fungus such as fusarium, allowing rapid adaptation to environmental stress such as fungicides or to the introduction of resistant genes into the hosts (sapumohotti & salleh 1992). a source of genetic variability is essential particularly for organisms such as f. oxysporum, that depend entirely on asexual reproduction. genetic instability could generate variants that are fungicide resistant, able to overcome host resistance, or tolerant to toxic wastes in the soil. a high mutation rate could be an important means of generating variability if sexual or parasexual recombination is rare (klittich & leslie 1988). heterokaryosis and other types of mycelial interactions have been recognized in the genus fusarium for at least 100 years (page 1961; stover 1959). a heterokaryon is a multinucleate cell containing genetically distinct nuclei in a common cytoplasm. such cells are often created by hyphal fusions between vegetatively compatible strains. heterokaryosis provides an opportunity for genetic recombination via the parasexual cycle in sterile, homothallic, or imperfect fungi such as fusarium (adams etal. 1987). in this experiment, spontaneous nit mutants were readily recovered without mutagenic treatment. because these mutations probably affect at least seven loci with three distinct phenotypes, the nit mutants can be used as forcing markers in the 38 vegetative compatibility groups offusarium oxysporum k.h. ooi and b. salleh formation of heterokaryon to test fungal strains for vegetative compatibility (correll et al. 1986a, 1987; gordon et al. 1986; sidhu 1986; jacobson & gordon 1988; elmer 1991). studies of fungal populations using vcgs as a means to measure diversity have become widespread in recent years. vcgs serve as a natural means to subdivide fungal populations. the vie loci and alleles that define vcgs are presumed to be selectively neutral with respect "to traits such as pathogenicity and vegetative viability. different strains are vegetatively compatible (i.e., capable of forming a heterokaryon) only if alleles at all vegetative compatibility loci are identical. vegetative compatibility groups (vcgs) are ideal markers for population studies because they occur naturally and are easy to score using spontaneous nit mutants. strains which belong to the same vcg can form heterokaryons in which cytoplasms may mix, mitotic recombination may occur, and deleterious cytoplasmic agents, such as mycoviruses, can be exchanged. vcgs, which are analogous to anastomosis groups, have been correlated with pathogenicity in many fungal species including fusarium spp. (bosland & williams 1987; correll et al. 1986a; correll et al. 1987; jacobson & gordon 1988; ploetz & correll 1988; puhalla 1985). results obtained from this study suggest that 26 (65%) of the 40 strains of f. oxysporum tested belonged to a dominant vcg designated as 1001m, but only 2 strains (5%) were grouped in vcg 1003m and 1013m, respectively. vcg 1002m and 1004m-1012m each consist of only one strain (0.025%). this result suggests that anastomosis occurs infrequently among strains in this population and implies that the wilt pathogen of roselle in malaysia is genetically diversified. but the distribution of different genotypes into vcgs among the strains is not even. there are some closely related strains and grouped into one vcg, while some others which showed no relatedness (vegetatively incompatible) with other strains were grouped into several vcgs separately as isolated groups. at least one strain from each of the 13 vcgs was tested and the results showed that they were vegetatively incompatible with the wilt pathogens, f. oxysporum f. sp. cubense and f. sp. asparagi; and also incompatible with the non-pathogenic strains from paddy and oil palm. thus, the wilt pathogen of roselle in malaysia likely appears to constitute a distinct genetic population within the f. oxysporum complex. generally, mutants deficient in the molybdenum-containing cofactor (nitm) are infrequently isolated but readily form complementing heterokaryons, making them the most suitable choices for vegetative compatibility tests. on the other hand, nit i and nit3 mutants may form weak, slow-growing heterokaryons, giving ambiguous results. we also found that the strength of a heterokaryon varied depending on the combination of the nit mutants used. the ideal combinations were my7-nitm anc nitm-nitm which form more stable heterokaryons. our study demonstrates the importance of characterizing heterokaryosis in f oxysporum. once the heteroploids are better characterized, they should be useful ir studying the effects of genetic interactions on the expression of economical!} 39 biotropia no. 12, 1999 important traits such as virulence and the production of secondary metabolites such as mycotoxins. this study may also provide new insight into the establishment of a new forma specialis of f. oxysporum, causing wilt of roselle. acknowledgments this research is supported by the government of malaysia (1rpa grant 01-0205-6003). we thank our research assistants mr. kamarudin mohd. maidin and miss wan faridah maydin for their capable assistance, the department of agriculture (state of terengganu) and the owners of the roselle sampling plots throughout the northern malaysian peninsula. references adams, o., n. johnson, j.f. leslie and l.p. hart. 1987. heterokaryons of gibberella zeae formed following hyphal anastomosis or protoplast fusion. experimental mycology 11: 339-353. booth, c. 1971. the genus fusarium. commonwealth mycological institute, london, p. 21-22. bosuand, p.w. and p.h. williams. 1987. an evaluation of fusarium oxysporum from crucifers based on pathogenicity, isozyme polymorphism, vegetative compatibility and geographical origin. canadian journal of botany 65: 2067-2073. burnett, j.h. 1984. aspects of fusarium genetics. in: the applied mycology of fusarium, edited by m.o. moss and j.e. smith, cambridge university press, cambridge, p. 39-69. chin, h.f. 1986. the hibiscus, queen of tropical flowers. tropical press, kuala lumpur. 151p. correll, j.c., c.j.r. klittich and j.f. leslie. 1987. nitrate non-utilizing mutants of fusarium oxysporum and their use in vegetative compatibility tests. phytopathology 77: 1640-1646. correll, j.c., j.e. puhalla and r.w. schneider. 1986a. identification of fusarium oxysporum f. sp. apii on the basis of colony size, virulence and vegetative compatibility. phytopathology 76: 396 400. correll, j.c., j.e. puhalla and r.w. schneider. 1986b. vegetative compatibility groups among nonpathogenic root-colonizing strains of fusarium oxysporum. canadian journal of botany 64: 2358-2361. elmer, w.h. 1991. vegetative compatibility groups of fusarium proliferatum from asparagus and comparisons of virulence, growth rates and colonization of asparagus residues among groups. phytopathology 81: 852-857. elmer, w.h. and c.t. stephens. 1989. classification of fusarium oxysporum f. sp. asparagi into vegetatively compatible groups. phytopathology 79: 88-93. gordon, t.r., j.c. correll and a.h. mccain. 1986. host specificity and vegetative compatibility in verticillium albo-atrum. phytopathology 76: 1111 (abstract). gordon, t.r. and d. okamoto. 1991. vegetative compatibility groupings in a local population of fusarium oxysporum. canadian journal of botany 69: 168-172. jacobson, d.j. and t.r. gordon. 1988. vegetative compatibility and self-incompatibility within fusarium oxysporum f. sp. melonis. phytopathology 78: 668-672. klittich, c.j.r. and j.f. leslie. 1988. nitrate reduction mutants of fusarium moniliforme (gibberella fujikuroi). genetics 118: 417-423. 40 vegetative compatibility groups of fusarium oxysporum k..h. ooi and b. salleh larkin, r.p., d.l. hopkins and f.n. martin. 1988. differentiation of strains and pathogenic races of fusarium oxysporum f. sp. niveum based on vegetative compatibility. phytopathology 78: 1542 (abstract). leslie, j.f. 1993. fungal vegetative compatibility. annual review of phytopathology 31. 127-151. mat isa, a., p.m. md. isa and a.r. aziz. 1985. chemical analysis and process of roselle (hibiscus sabdariffa l,). mardi research bulletin 13: 68-74. nash, s.m. and w.c. snyder. 1962. quantitative estimations by plate counts of propagules of the bean root rot fusarium in field soil. phytopathology 52: 567-572. ooi, k.h., b. salleh and m.h. hafiza. m.h. 1998. vascular wilt caused by fusarium oxysporum on roselle in malaysia. journal of plant protection in the tropics (submitted). page, o.t. 1961. variation in the banana-wilt pathogen fusarium oxysporum f. sp. cubense canadian journal of botany 39: 545-557. ploetz, r.c. and j.c. correll. 1988. vegetative compatibility among races of fusarium oxysporum f. sp. cubense. plant disease 72: 325-328. puhalla, j.e. 1981. genetic considerations of the genus fusarium. in: fusarium: diseases, biology and taxonomy, edited by p.e. nelson, t.a. toussoun and r.j. cook, pennsylvania state university press, university park. p. 291-305. puhalla, j.e. 1985. classification of strains of fusarium oxysporum on the basis of vegetative compatibility. canadian journal of botany 63: 179-183. puhalla, j.e. and p.t. spieth. 1985. a comparison of heterokaryosis and vegetative compatibility among varieties of gibberella fujikuroi (fusarium moniliforme). experimental mycology 9: 39 47. salleh, b. and r.n. strange. 1988. toxigenicity of some fusaria associated with plant and human diseases in malaysian peninsula. journal of general microbiology 134: 841-847. salleh, b and b. sulaiman. 1984. fusaria associated with naturally diseased plants in penang. journal of plant protection in the tropics 1: 47-53. sapumohotti, w.p. and b. salleh. 1992. nitrate reduction mutants of fusarium nygamai, the causal organism of reddish brown rot on asparagus. journal of plant protection in the tropics 9: 187-194. sidhu, g.s. 1986. genetics of gibberella fujikuroi. viii. vegetative compatibility groups. canadian journal of botany 64: 117-121. snyder, w.c. and h.n. hansen. 1940. the species concept in fusarium. american journal of botany 27: 64-67. stover, r.h. 1959. studies on fusarium wilt of banana. iv. clonal differentiation among wild type isolates of fusarium oxysporum f. sp. cubense. canadian journal of botany 37: 245-255. tan, h.h. and n.m.s. said. 1994. cultivation of roselle (hibiscus sabdariffa l.). department of agriculture, terengganu, malaysia. 18 p. 41 biotropia 1 (1) 1987: 46-52 ii. soils from cultivated stands of shorea javanica m.j. sheehy skhffington* tropical forest biology program, biotrop, bogor, indonesia keywords: soils, shorea javanica, sumatra abstract soils from shorea javanica plantations in different parts of lampung province, south sumatra were sampled to a depth of 50 cm and described. they varied from quite deep loamy alkaline soils near krui, to sticky acid clays behind ngaras. the cation content of most samples was quite high, but organic nutrients were low, suggesting volcanic origin of the soils. preliminary observations of performance in situ of 5. javanica would suggest a requirement for deep loamy, fairly alkaline soils with a moderately high exchangeable cation content for optimal yield and performance. introduction an important aspect in considering sites of s. javanica plantations is whether they can support a sufficiently productive tree crop. resin is not always abundantly provided at all existing sites and this may be a function of environmental factors, such as soil, local climate or topography. this study is proposed to deal with soil characteristics suitable for s. javanica cultivation. two approaches can be taken in the determination of the effect of soils on s. javanica performance. the first entails a survey of existing s. javanica stands, sampling the soils and gathering information on the health and resin productivity of the trees, and the second involves controlled growth experiments of seedlings on a range of soils, to estimate growth performance and thus select sites with soils yielding optimal production. this report covers the first aspect, namely analysis of soils collected at various s. javanica sites in the lampung province of sumatra. sites 1) some time was spent in the region of krui where resin production is good and villagers derive a reasonable income from its harvest. soils have already been analysed from a site ca. 5 km from krui (torquebiau 1984), but as plantations are extensive within a radius of 30 km of the town, a second sample site was chosen at palnam, 19 km from krui on the liwa road. about 3-400 m south of this road an outcrop of pink reef limestone was visible in a plantation and soil samples were taken from there. * present address: department of botany, university college, go/way, ireland. 46 ii. soils from cultivated stands of shorea javanica — skeffington 2) to the south of krui, along the coast 'road' at way biha, a plantation was found in very sandy soil about 2-300 m from the beach. the trees, most of them ca. 20-30 years old were healthy and straight, ca. 20-30 m high, but resin production is poor, the bark thick, ca. 2-3 cm, and flower and fruit production occasional. 3) further south, at ngaras, s. javanica plantations extend up the hills at the back of the village. near the village and halfway up the hill (site ngaras i) soils were brown and fairly deep. resin production is good on these lower slopes. above these, at the top of the hill (site ngaras ii), soils were redder and more acidic. resin production here is not so good. further inland on higher slopes, soils were brown and less acidic, yet resin production was no better than at ngaras ii, indicating that caution should be used when correlating resin production and soil properties. 4) two other sites were examined in lampung, one at wana, near metro where a small stand of fairly old damar trees yielded soils of good crumb structure. the other, near bakauheni was even smaller and probably disused for a longer time. in each site, soils were sampled three times and subsampled at 5 cm and 30 cm depth. methods for laboratory analyses were standard methods currently used in biotrop tropical forest biology program laboratory. results field soil descriptions these are summarized in table 1. the soils at palnam indicate a fairly rapid litter decomposition and the resulting humus grades into the clayey substratum giving a fairly uniform brown color. at ngaras, however, soils are only brown on the lower slopes (ngaras i) and these overlay a stickier yellow-brown clay. on the upper slopes they were yellowish to yellow/orange throughout and more blocky in structure, with a thin litter/humus layer above. the soil at way biha was very sandy and poorly developed below 10 cm, whereas at wana and bakauheni, soils had a good surface humic crumb structure, but beneath was reddish clay and a harsh smell of metal oxides (signs of temporary waterlogging?) was detectable. laboratory analyses the range of texture (fig. 1 and table 2) relating to particle size is quite striking but if one considers only the soils where resin production is good (i.e. at palnam and ngaras i), these are loams with a fairly even proportion of each particle size class. also among the loams are the samples from 5 cm depth at wana and bakauheni. however at 30 cm, these contain very little sand and have a high proportion of clay. the ngaras ii soils are also low in sand content, whereas the coastal soils at way biha comprise little else. the ph also shows a wide range, the most acid soils being those at ngaras, especially ngaras ii. the others are not far from neutral (ph 7). this relatively 47 biotropia vol. 1 no. 1, july-december 1987 table 1. summary of profile descriptions 48 ii. soils from cultivated stands of shorea javanica skeffington 49 biotropia vol. 1 no. 1, july-december 1987 figure 1. soil texture triangle, showing position of soils listed on table 2. high ph is borne out by a high cation exchange capacity (c.e.c) and base saturation, which are comparatively low at ngaras. analysis of individual cations (table 2) shows that calcium occupies most of the exchangeable sites in soils where the c.e.c. is high. the other three, especially k and mg are relatively high, but vary little between sites. of notable exception again are the samples from 5 cm depth at wana and bakauheni which both have a very high % base saturation and also high k and mg concentrations. the limestone bedrock must contribute to the high ph and calcium content of the palnam soils. on the other hand, the organic constituents, total carbon and nitrogen, are very low for all soils, being lowest again at the ngaras sites, which in this case differ little. the c/n ratio is also low but this is indicative that n is being mineralized, suggesting rapid decomposition, 50 s s a percent sand ii. soils from cultivated stands of shorea javanica — skeffington and thus, low concentrations of these elements are not indicative of their probable rapid turnover. inorganic phosphate is relatively low, being highest at way biha and palnam. accumulation of marine shells and detritus may be a high source of inorganic p at the coastal site of way biha. comparison with other soils because of high rainfall, tropical soils generally rapidly become acidic and poor in bases due to fast weathering and leaching (sanchez 1976). only relatively recent alluvial or volcanic soils tend to be fertile and to a lesser extent those overlying exchangeable base-rich rocks such as limestone. there is evidence that the krui soils contain volcanic ash (torquebiau 1964) and it is likely that those at ngaras may also have received ash in the past. in terms of c.e.c. and exchangeable cations all soils compare favorably with fertile andosols developed on volcanic tuffs near bogor in java (hardjosoesastro et al. 1983) where mg, k and especially ca are in fact lower than in these soils. other soils from lampung have similar chemical properties (adiningsih et al. 1983) including low carbon and nitrogen percentages, suggesting that the range of soil types presented here is relatively common in the province. in general, the soils of sumatra (and to a greater extent of java), are more fertile than those of islands with little or no recent volcanic activity or large alluvial deposits. islands such as borneo have older, more weathered soils which are more acidic with a lower cation content and exchange capacity (proctor et al. 1983). thus soils where s. javanica should grow relatively well would not be very acidic (ph > 4.5), contain a moderate to high amount of cations, but not necessarily with high carbon, nitrogen or phosphate concentrations. for a good resin yield it may be important to select fairly deep base-rich soils which also have a good mix of sand, clay and silt resulting in a loamy texture. prospects for future research the second part of the proposal outlined above, i.e. growth experiments, should follow directly from this preliminary investigation. for proper and vigorous growth of 5. javanica seedlings, they would require to be grown in fertile base-rich soils of a good loamy texture. however, some estimate of their tolerance to less fertile soils can be derived from their experimental growth in a range of soils of different ph and fertility. podzolic soils would have a ph similar to the more acid soils analyzed here and can be collected locally (e.g. at jasinga). however, these may have moderately high concentrations of exchangeable cations (adiningsih et al. 1983) and all soils thus used should be analyzed as described above to assess their comparability to the soils in the field. 51 biotropia vol. 1 no. 1, july-december 1987 performance of seedlings in poor soil conditions will probably be compounded by effects of over-shading or exposure to too much light (bravo 1960). a two-way experiment to test the combined effects of range of soils (possibly adding fertilizer to some, sundralingam 1983), and light conditions, would resolve optimal conditions for seedling growth as well as demonstrating which soil types are not conducive to healthy growth in either seedlings or adult trees. examples of more fertile soils which could be used locally are the andosols at sukamantri described in hardjosoesastroe et al. (1983). at cisarua are some brown regosols, also volcanically-derived and fertile, which may prove to be the more suitable of the fertile soils. measurement of growth performance could include size, leaf number and co2 uptake and subsequent harvest for biomass and nutrient content measurement. acknowledgments thanks are due to dr. emmanuel torquebiau for his help and advice as leader of the expedition. also thanks to mr. ahmad zainuddin for field assistance and mr. agus nasir who carried out the laboratory analysis. references adiningsih, j.s. and m. sudjadi, 1983. the effect of submergence and fertilization on some characteristics of red yellow podsolic soils from central lampung (in indonesian). pemberitaan penelitian tanah dan pupuk no. 2. pp. 1-8. bravo, p.r. 1980. germination and initial growth of shorea stenoptera and s. comoressa as affected by shading and different potting media. b1otrop report on training course in biological aspects of silviculture, sept. 1979—july 1980, bogor, indonesia. hardjosoes astro, r., h. suyanto and a.m. satari. 1983. andosol from the sukamantri area of bogor district (in indonesian). pemberitaan penelitian tanah dan pupuk no. 2. pp. 18-29. marali, m., o.k. husein and bachri, 1980. hubungan sifat tanah podsolik dengan pertumbuhan tanaman tengkawang di haurbentes, jasinga, bogor, presiding no. 1, penelitian tanah, pp. 37-44. proctor, j., j.m. anderson, s.c.l. fooden and h.w. vallack, 1983. ecological studies in four contrasting lowland rain forest in gunung mulu national park, sarawak. i forest environment and floristics. journal of ecology, 71. pp. 237-260. sanchez, p.a. 1976. properties and management of soil in the tropics. wiley, chichester. sundralingam, p. 1983. responses of seedlings of dryobalanops aromatica and d. oblongifolia to commercial fertilizers. malaysia forester 46 (1) 86-92. torquebiau, e. 1984. man-made dipterocarp forest in sumatra. agroforestry systems, 2. pp. 103-127. 52 46.pdf 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf biotropia no biotropia no. 12, 1999 : 53 58 in vitro inoculation of asparagus officinalis tissue culture shoots with fusarium prolifera tum a.k.mohd omar', n.a.r. nik norulaini2, b. salleh3 and r.a.r. iskandar' 1school of industrial technology, 2school of distance education, 3school of biological sciences, universiti sains malaysia, 11800, penang, malaysia abstract artificially inoculated asparagus tissue culture plantlets with a virulent fungus, fusarium proliferatum showed signs of infection as early as 4 days after inoculation. macroscopic observations revealed presence of early symptoms such as necrotic lesions at the affected area and light microscopic examinations clearly revealed the post-penetration events that took place including the destruction of surrounding cells. however, little is known of the hyphal activity or advancement on the host's surface at the initial stage after inoculation. scanning electron microscopic examination clearly revealed the hyphal advancement on the surface and the mode of entrance into the host tissues beneath. four days after inoculation, the fungi proceeded to spread out from the inoculation point onto the host surface which eventually developed into a sparse network of both aerial and non-aerial hyphae. non-aerial hyphae form a network of mycelium that adheres to the surface and it's movement appeared to be oriented towards the stomata. hyphal penetration occurs more often through the stomata, natural openings or wounds. in some cases, the hyphae crossed over the stomatal opening without entering the host tissues. at places where the cuticle layer is absent or not well developed the hyphae successfully grew in between the epidermal cells into the tissues beneath. key words: tissue culture/asparagus officinalis/shoots/artificial inoculstion/fusarium proliferatum. introduction in pathogen infected tissues, the preand post-penetration as well as results of infection can provide valuable knowledge regarding the virulence and epidemiology of the fungus involved. as a result of infection the physiological changes in diseased plants are usually manifested in visible symptoms which may be mild or severe, depending on the nature and aggressiveness of the pathogen and the susceptibility of the host plant. the physiological changes occurring in the diseased plant can be witnessed in the anatomical and morphological changes ('morbid anatomy') which constitute the visible symptoms. the visible symptoms such as necrosis or rot are in part related to the pathogenicity mechanisms of the pathogen, at least at the early stages of infection. histological examination very often can reveal the post-penetration development of the fungus and histological changes of the infected cells (jewell et al. 1979; nik norulaini 1992 a,b). scanning electron microscopy observations are useful in studying the development of the fungus on the epidermal surface, it's subsequent colonization on the surface and mode of penetration into the tissues beneath (ab. rahmane/a/. 1987; jacobiefa/. 1982). 53 biotropia no. 12, 1999 fusarium has been known to infect a wide range of crops in malaysia (sabariah 1987). one of the most serious diseases, namely crown rot, was described and the pathogen was identified as f. proliferatum (salleh 1990). the other casual pathogens include f. solani, f. nygamai and f. oxysporum. in the field, primary infection of f. prolifeatum is through wounding particularly those caused by insects (salleh et al. 1998). field symptoms, however, were reproduced by an artificial inoculation of asparagus in the greenhouse. the symptoms-include irregular reddish brown lesions on infected stems followed by stem girdling, crown and root rot and finally the entire plant die (salleh et al. 1998). the objective of this paper is to study the pre-penetration development of f. proliferatum, the causal agent of crown and root rot, into asparagus tissue culture shoots. materials and methods production of tissue culture shoots as hosts one year-old asparagus (asparagus officinalis l.) plants cv. mary washington 500 (mw 500) raised from seeds in the greenhouse (salleh et al. 1996) were used as source of explants. spears about 20 cm long and 0.25 cm in diameter were cut into 2.5 cm in length and surface-sterilized with 10% bleach (v/v) (5.25% sodium hypochlorite). a few drops of tween 20 emulsifier was added to enhance spread of the disinfectant. the segments were rinsed three times with sterile distilled water and blotted on sterile filter papers to remove the bleach. the sterilized segments were cut into one node segments of 1 cm each and cultured on murashige and skoog (1962) medium with the following concentrations in mgl"1 : naa 0.3, kinetin 0.1, thiamine-hcl 1.0, pyridoxine-hcl 5.0, nicotinic acid 5.0, myo-inositol 100.0, adenine sulphate dehydrate 40, sucrose 25 000 and supplemented with difco bacto malt extract 500, nah2po4.h2o 170. each culture bottle (500 ml) contained three segments and incubated at 27°c with continuous lighting at 1000 lux. multiple shoots that formed from 1 to 6 weeks were used as hosts. preparation of inoculum the fungus, f. proliferatum culture no. 851, was collected from infected asparagus plants cv mw 500 from the field. the fungus was kept in liquid nitrogen (-196°c) for several months (deposited at the fusarium culture collection unit, usm, penang) before culturing on potato dextrose agar (pda) (salleh & sulaiman 1984). four-day old cultures on pda were used as inoculum. 54 in vitro inoculation of asparagus officinalis tissue culture shoots a.k.. mohd. omar el al. inoculation of tissue culture shoots one to six weeks old shoots produced were used as hosts. three to four mm of the epidermis were carefully removed from the shoots as inoculation sites. the sites of inoculation were at the middle, basal and apex of the plantlets. mycelial plugs, about 1 mm3 taken from the periphery of the fungal colony were placed on the inoculation sites. scanning electron microscopic examination of infected shoot segments showing pathological changes were fixed in 5% glutaraldehyde for 1 hour, washed with phosphate buffer, fixed in 4% osmium tetroxide for 3 4 hours and then washed in phosphate buffer. the tissues were dehydrated through an alcohol series and critical point-dried using a critical point drier. they were then coated with gold in an ems-76m gold coater and examined with a jeol jsm35c scanning electron microscope. results and discussion twenty four hours after inoculation the hyphae began to spread progressively from the inoculum. four days later, the hyphae ramified into sparse network of mycelia that grew close to the surface of the tissue (fig. 1a). little aerial hyphae were observed. colonized stems will be overwhelmed by mycelia after six days of inoculation. removal of the mycelia from infected stems revealed early stages of necrotic lesions even at areas away from the inoculated sites (fig. ib). likewise, inoculation with conidia on mature asparagus stems with f. proliferatum showed occasional lesions (tan 1991). these reddish lesions are normally small, about 0.5 mm in length and can only be detected with a dissecting scope. the emergence of hyphae from beneath the tissues through necrotic spots were also observed, indicating existence of mycelial network within the cells. mature asparagus stems have developed a cuticle layer that inhibits the penetration of the fungus. penetration can, however, take place in cracks and crevices (nik norulaini & salleh 1990). this was observed in the basal part of the stem where the tissues were well covered with cuticle layer, or waxy deposits. the hyphae were seen entering the tissues via the stomatal opening (fig. 1c) or the natural cracks (fig. id). movement of hyphae in the groove in between cells was also apparent. no direct penetration in between cells was discovered even after exhaustive examination. mature tissues also offer resistance to any mycotoxins that might be released by the fungus. in a study by tan (1991), an incorporation of culture filtrate that contains toxins into a culture caused lesions on asparagus stems. the toxins were translocated through the base of the stem from the media. direct inoculation of the culture filtrate on the mature stem failed to induce any lesion. a significant difference in the cuticle development was observed at the apical areas of the inoculated shoots, where ramification of hyphal networks on the surface show the mycelia moving close to the surface (fig. ie). the lack of waxy deposits may influence penetration of the fungus as observed, since penetration between 55 biotropia no. 12, 1999 epidermal cells took place (fig. if). stomatal penetration of a single hypha was observed near the apical region, where the tissues were younger with little mechanical barrier, since there were only scant deposits of wax in the region. figure 1: growth and penetration of f. proliferatum on asparagus tissue culture shoots. a: ramifications of hypha into sparse network 4 days after inoculation (dai) b: necrosis (n) on inoculated stem 8 dai c: a single hypha entering the stomata d: hypha entering the tissue via natural cracks e: hypha network moving close to the surface f: penetration in between epidermal cells 56 in vitro inoculation of asparagus officinalis tissue culture shoots a.k. mohd. omar et al. although field infection is known to be via wounding, the in vitro sem examinations indicated that penetration can occur quite commonly through stomatal openings in the basal region. in younger tissues penetration can take place both between cells and through the stomata. in more mature parts of the tissue, the cuticle layer forms an effective mechanical barrier to the penetration of the fungus. conclusion although fusarium spp. have been associated with and caused many plant diseases, little has been studied on the host defence mechanisms or the fungus activities on the host. extensive review revealed the importance and potential of fusarium as disease-causing organisms, especially in the tropics (salleh & sulaiman 1984). with this preliminary finding it is hoped that this will offer a better understanding of the infection mode in nature and the pathogen's infectious capabilities on a large number of plants. further work is needed to understand the biochemical and physiological aspects of fusarium-related diseases. this approach could also lead to a screen for asparagus varieties resistant to fusarium-related diseases, such as have been used to screen non-fusarium resistant plant varieties (utkhede 1986), and to study resistance mechanisms (dolan & coffey 1985; diner et al. 1984), evaluation of susceptibility (sylvestre-guinot & delatour 1983) and surface interaction (nik norulaini & salleh 1990). the in vitro approach may offer a simplified and rapid screening method. references ab. rahman, n.n., a.m. diner, skillino, d.d. and d.f. karnosky. 1987. in vitro responses of conifer adventitious shoots and call! inoculated with gremmemella abietina. forest science 33:4: 1041-1053. diner, a.m., r.l. mott, and h.v. amerson. 1984. cultured cells of white pine show genetic resistance to axenic blister rust hyphae. science 24:407-408. dolan, t.e., and m.d. coffey. 1986. laboratory screening technique for assessing resistance of four avocade to phytophtora cinnamoni. plant disease. 70: 115-118. jacobi, w.r., h.v. amerson and r.l. mott. 1982. microscopy of cultured loblolly pine seedlings and callus inoculated with cronartiumfusiforme. phytopathology 71:138-143. jewell, f.f., p.c. jewell and c.h. walkinshaw. 1979. histopathology of the initiation of resistance-zones in juvenile slash pine to cronartium quercuum. f. sp. jusiforme. phytopathologisch meditterranica 19: 8-12. murashige, t. and t. skoog. 1962. a revised medium for rapid growth and bioassays with tobacco tissue culture. physiologia plantarum . 15:473-479. nik norulaini n.a.r. 1992a. assessment of in vitro inoculation of asparagus with conidia and culture filtrate of fusarium proliferation. proceedings of the 4* jsps-vcc seminar on integrated engineering, 13th 14th october 1992, kyoto, japan. nik norulaini n.a.r. 1992b. in vitro inoculation of asparagus with conidial suspension and culture filtrate of fusarium oxysporum schlect. f. asparag. proceedings of asia-pacific conference on agricultural biotechnology, august 20-24 1992, beijing, china. 57 blotropla no. 12, 1999 nik norulaini, n.a.r and b. salleh. 1990. in vitro inoculations of asparagus tissue culture of plantlets with vegetative hyphaof fusarium proliferatum. proceedings of the 3"* international conference on plant protection in the tropics. 20-23 march 1990, genting highlands, malaysia. sabariah, h. 1987. diseases of asparagus. b.sc. thesis, school of biological sciences, universiti sains malaysia, penang, malaysia. salleh, b. 1990. crown rot caused by fusarium proliferatum (matsushima) nirenberg; a new disease of asparagus in malaysia. proceedings of the 3rt international conference on plant protection in the topics. genting highlands, malaysia, p. 343 (abstract). salleh, b. and b. sulaiman. 1984. fusaria associated with naturally diseased plants in penang. journal of plants protection in the tropics 1: 47-53. salleh, b., a. safinat, l. julia and c.h. teo. 1996. brown spot caused by curvalaria spp., a new disease of asparagus. biotrop1a9: 26-37. sylvester-guinot, g. and c. delatour. 1983. possibility's di'appreciation de la sensibiliti'e du genre larix au lachnellula willkommii (hartig) dennis par inoculations artificielles. full annales scientia forsswiststand., 40(4) 337-354. tan, l.h. 1991. penginolculatan batang dan pucuk kultur tisu muda asparagus secara in vitro dengan ampaian konidia dan turasan kultur fusarium proliferatum (matsushima) nirenberg. b.sc. thesis, school of biological sciences, universiti sains malaysia, penang, malaysia. salleh, b., s.m. hakam, d. fachri, b.a.y. insanul, a. k.asmal, l. lahmuddin and s. nurduati. 1998. current status of asparagus diseases in southeast asia (sea). asean food journal (submitted). utkhede, r.s. 1986. in vitro screening of the world apple germplasm collection for resistance to phytophthora cactorum crown rot. scientia horticulturae, 29:205-210. 58 biotropia book final.indd 119 using streptomyces xylanase to produce xylooligosacharide from corncob anja meryandini1*, titi candra sunarti2, aprilia naomi1 and fery mutia2 1. department of biology, faculty of mathematics and natural sciences, bogor agriculture university, bogor, indonesia 2. department of agrotechno industry, faculty of agricultural technology, bogor agriculture university, bogor, indonesia abstract streptomyces 234p-16 and skk1-8 are xylanase-producing bacteria. corncob xylan were extracted using acidifi ed method. crude enzymes (produced by centrifuging the culture) were used to hydrolyze xylan from 2 varieties of corncob. crude extract activity was measured by using dns (dinitrosalisilic acid) method. xylanase from strain 234p-16 has the highest activity if cultivated in 1% hawaii xylan, whereas strain skk1-8 on 1.5% bisma xylan. skk1-8 xylanase can hydrolize corncob xylan (1% hawaii or 1.5% bisma xylan) within 4 hours and produce xylooligosacharide with polymerization degree of 4.76 and 6.37, respectively. key words: xylanase, xylooligosacharide, streptomyces. introduction besides rice, corn is the major carbohydrate sources in indonesia especially for feed and industrial raw materials. most of corn is used as raw material for food and nonfood industries. but these applications are limited to the corn kernel, and other parts of corn plant such as corncob was not utilized yet. about 30% of the corn is corncob; the rests are corn stover and kernels. data from badan pusat statistik (indonesian bureau of statistics) showed that maize production is increasing from 9.82 million ton in 2002 to 11.35 million ton in 2004. th is increase is also followed by the increase of corncob as an agricultural waste. corncob consists of protein, fat, nitrogen free extract and also hemicellulose and cellulose (johnson 1991). th e content of xylan (major part of hemicellulose) in corncob could go up to 40-g/100 g, the highest among all of the agricultural waste (yang et al. 2005). most xylan occurs as heteropolysaccharide, containing diff erent substituent groups in the backbone chain and in the side chain (beg et al. 2001). due to the corncob nutritional content, it can convert into a commercial product or be used as a medium to biotropia vol. 15 no. 2, 2008 : 119 128 *corresponding author: ameryandini@yahoo.com 120 cultivate microorganisms. in our previous research we have identifi ed xylanase-producing streptomyces (234p-16 and skk1-8). xylanase from 234p-26 has an optimum condition at ph 5 and 90 oc whereas from skk 1-8 the optimum condition is at ph 6 and 50 oc (meryandini 2005). in this research we will use this streptomyces xylanase to produce xylooligosaccharide from corncob xylan. materials and methods materials in this research we use 2 local varieties of corncobs (bisma and hawaii) and 2 acid-xylanases from indonesian streptomyces: streptomyces 234p-16 from padang and streptomyces skk1-8 from sukabumi. delignifi cation delignifi cation of corncobs methods had been investigated previously (widyani 2002). about 1000 g of corncob grits is immersed in 10 liters of 1% naocl solution for 5 hours at room temperature (28 oc). after 5 hours, the sample is decanted and rinsed by distilled water for several times and fi ltered, the solid part i.e. delignifi ed samples are then dried by oven drying at 50 oc for 48 hours. th e chemical composition of this sample will be examined as cellulose, hemicellulose, and lignin. xylan extraction & purifi cation th e method for separation of xylan using acidifi ed method from delignifi ed samples has been investigated by anggraini (2003). delignifi ed corncob grits are immersed in 15% naoh solution for 24 hours at room temperature (28 oc). th is step liberated the xylan into soluble fraction and neutralized by 6 n hcl solutions (ph 4.5-5.0). th e supernatant is centrifuged on 4000 rpm for 30 minutes to obtain residue such as xylan. purifi cation is conducted by re-dissolving the crude xylan into 4% naoh and then fi ltered. filtrate was acidifi ed using 6 n hcl (ph 4.5-5.0), and then centrifuged on 4000 rpm for 30 minutes. precipitates were dissolved in 95% ethanol and centrifuged, and then dehydrated using 50 o c drying oven. xylanase production th e streptomyces isolates are cultured on ym (yeastmalt) agar and then on oatspelt xylan agar medium (yeast extract 0.2%, sucrose 10%, k2hpo4 1.5%, mgso4 .7h2o 0.025%, nacl 0.23%, na2hpo4 .2h2o 5 %, oat spelt xylan 0.5%) for 4 days at room temperature. two cookbores of streptomyces are cultured in 100 ml liquid xylan media and incubated at room temperature using a shaking incubator with an agitation speed of 240 rpm. every day the culture should be centrifuged and the supernatans collected for enzyme assay. biotropia vol. 15 no. 2, 2008 121 xylanase production on several substrates two cookbores of streptomyces are cultured in 100 ml liquid xylan media (oatspelt and several concentration of 2 local corncob xylan) in a 500 ml erlenmeyer and incubated on a shaker for 10 to 12 days at room temperature. duration of xylan hydrolysis by crude xylanase streptomyces xylanase produced in 1% of corncob xylan was used to hydrolyze 1% of corncob xylan for 4 hours at optimum ph and temperature condition. degree of hydrolysis and degree of polymerization will be monitored every hour and the reducing sugars analyzed by dinitroasalicilycacid method, and total sugar by phenol-h2so4 method. assay of xylanase activity th e culture was centrifuged 5 minutes at 10.000 x g to obtain the xylanase crude extract. xylanase activity was measured with oat-spelt xylan as a substrate. enzyme solution (100 μl) was added to 1 ml substrate solution which contain 0.5% xylan and the mixture was incubated at optimum temperature for each enzyme (90 oc for 234p-26 and 50 oc for skk 1-8) for 30 minutes. crude extract activity was measured by using dns (dinitrosalisilic acid) method by miller (1959) with xylosa as the standard. th e reducing sugar of the references samples (substrate solution incubated without enzyme and diluted enzyme solution in buff er) were deduced from the values of the test samples. th e reducing-sugar was detected by spectrophotometer (λ = 540 nm). one unit xylanase activity was defi ned as the amount of enzyme which produces 1 μmol xylosa per minute. results and discussions xylan extraction th e chemical composition of corncob is described in table 1, and the delignifi cation eff ect of corncobs and recovery of cellulose fraction to the fi ber components are shown in table 2. table 1. chemical composition of corncobs constituent bisma variety hawaii variety ash (% db) 1.62 1.67 lipid (% db) 3.02 4.68 crude protein (%db) 2.41 4.82 crude fiber (% db) 38.07 40.65 carbohydrate (% db, by difference) 51.93 44.14 streptomyces xylanase to produce xylooligosacharide a. meryandini et al. 122 table 2. composition of fibers before and after delignification. constituent before delignifi cation (%) after delignifi cation (%) bisma variety cellulose 65.96 44.36 hemicelulose 10.82 30.38 lignin 23.74 19.21 hawaii variety cellulose 60.04 41.88 hemicelulose 18.11 30.18 lignin 16.14 15.12 elimination of lignin should be conducted since lignin will reduce the eff ectiveness of the utilization of xylan as carbon source for microbial growth and will infl uence the production of enzyme (agustine 2005). post-delignifi cation, the product composition was analyzed and small amount of lignin liberated. th e initial lignin of bisma variety (23.74%) and hawaii variety (16.14%) were reduced to 19.27% (bisma variety) and 15.2% (hawaii variety), respectively. delignifi cation cannot eliminate lignin from lignocellulosic materials completely. cellulose microfi bril was integrated in hydrophobic matrix covered by lignin, and the lignin linked with cellulose and hemicellulose in covalent linkages (agustine 2005). according to fengel and wegener (1995), lignin, cellulose and hemicellulose cannot separate completely even by special separation and purifi cation. lignin and cellulose can be detected in purifi ed cellulose and lignin. delignifi cation was conducted using 1% naocl as strong oxidant (agustine 2005). anggraini (2003) and widyani (2002) stated that hypochlorite ion from naocl can cleave the carbon linkage on lignin structure, and delignifi cation caused the opening of the linkages between lignin and other polysaccharides and infl uenced the increasing use of xylan by bacteria. xylan extraction was conducted by submerging the delignifi ed materials into 15% naoh. anggraini (2003) and widyani (2002) concluded that hemicellulose can be dissolved in alkaline solution, such as 15% naoh can produce brighter and clear powder, relatively clean from impurities, and easily dissolved in water and produced high yield. acid solution 6 n hcl was added to neutralize up to ph 4.5-5.0 to precipitate again the xylan. th e yield of xylan recovery was 9.26% from bisma variety, and 9.94% from hawaii variety. based on research results of widyani (2002) and anggraini (2003) 7.6412.94 % and 7.31-11.45 %, respectively, of xylan can be produced from corncob using the same acidifi ed methods. production of xylanase th e daily production curve of xylanase streptomyces 234p-16 and skk 1-8 assayed at ph 7.2 and 37 oc is shown in figure 1. th e highest xylanase production was reached on day-10 with the activity of 0.625 unit/ml for skk1-8 and on day-5 with the activity of 0.27 u/ml for 234p-16. th e optimum time of xylanase production was then used as the standard harvest time for the next xylanase production. biotropia vol. 15 no. 2, 2008 123 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 4 5 6 7 8 9 10 11 12 days a ct iv ity (u /m l) 234p-16 skk1-8 figure 1. production curve of streptomyces. 234p-16 and skk 1-8 xylanase assayed on 37 oc and ph 7.2. xylanase activity on several concentrations of corncob xylan 0 20 40 60 80 100 120 140 160 oat spelt hawai bisma substrate r el at iv e a ct iv ity (% ) 234p-16 skk1-8 figure 2. relative activity of streptomyces xylanases on 0.5% xylan from oat spelt, bisma dan hawaii corncob figure 2 shows diff erences in xylanase activity on several 0.5% substrates. xylanase from strain 234p-16 showed the highest activity on hawaii xylan, while xylanase from skk1-8 on oat spelt. xylanase 234p-16 showed the lowest activity on bisma xylan, whereas xylanase 45i-3 on hawaii xylan. hendarwin (2005) also reported activity diff erences in various substrates. xylanase activity in several concentrations of corn xylan and 0.5% oat spelt xylan streptomyces was inoculated in 0.5%, 1% and 1.5% bisma or hawaii xylan medium and compared with 0.5% oat spelt xylan. figure 3 shows the streptomyces 234p-16 xylanase activity on several concentrations of hawaii xylan (figure 3a) and several concentrations of bisma xylan (figure 3b ). xylanase from strain 234p-16 has the highest activity if cultivated in 1 % bisma xylan (0.57 u/ml) or in 1% hawaii xylan (0.947 u/ml) streptomyces xylanase to produce xylooligosacharide a. meryandini et al. 124 a. activity of isolate 234p-16 xylanase 0 0,2 0,4 0,6 0,8 1 4 5 6 7 8 9 10 days a ct iv iti y (u /m l) oat spelt 0,5% hawai 0,5% hawai 1% hawai 1,5% b. activity of isolate 234p-16 xylanase 0 0,2 0,4 0,6 4 5 6 7 8 9 10 days a ct iv ity (u /m l) oat spelt 0,5% bisma 0,5% bisma 1% bisma 1,5% figure 3. xylanase activities from streptomyces 234p-16 on (a) hawaii corncob ylan and (b) bisma corncob xylan compared with oat spelt xylan figure 4 shows the streptomyces skk1-8 xylanase activity on several concentrations of hawaii xylan (figure 4 a) and several concentrations of bisma xylan (figure 4b). xylanase from strain skk1-8 has the highest activity if cultivated in 1.5 % bisma xylan (49 u/ml) or in 1% hawaii xylan (5.75 u/ml). a. xylanase activity from isolate skk1-8 0 2 4 6 8 4 5 6 7 8 9 10 11 12 days a ct iv ity (u /m l) oatspelt 0,5% hawai 0,5% hawai 1% hawai 1,5% biotropia vol. 15 no. 2, 2008 125 b. xylanase activity from isolate skk1-8 0 10 20 30 40 50 60 4 5 6 7 8 9 10 11 12 days a ct iv ity (u /m l) oat spelt 0,5% bisma 0,5% bisma 1% bisma 1,5% figure 4. xylanase activities from streptomyces skk1-8 on (a) hawaii corncob xylan and (b) bisma corncob xylan compared with oat spelt xylan th e production of xylanase in 0.5% oat spelt xylan was compared with the production in several concentrations of corncob xylan. xylanase from strain 234p-16 has the highest activity if cultivated in 1% hawaii xylan, whereas strain skk1-8 on 1.5% bisma xylan (figure 3 and 4 ). th e production of xylanase is usually induced in medium containing pure xylan or xylan-rich residues. several reports showed that xylanase can be induced with lignocellulose material such as wheat bran, rice straw, corncob and sugarcane bagase (beg et al. 2001). corncob xylan is a rich carbon source with several kinds of carbon content as according to fengel and wegener (1995) lignin, cellulose and hemicellulose cannot be separated perfectly although the usage of a special separation and purifi cation methods and this content gives an eff ect on xylanase production. all carbon sources will give an eff ect on enzym production as reported by ambarawati (2005) that medium containing mannan can induce xylanase from strain 45i-3 and vice versa. th e optimum concentration to induce xylanase was 1% for hawaii xylan and for bisma xylan. th is greater result on hawaii xylan could be due to the higher concentration of hemicellulose in hawaii xylan. hydrolysis of xylan sugar composition was monitored by the increasing of reducing sugar and the decreasing degree of polymerization and the result is shown in table 3. table 3 shows the decreasing of dp during hydrolysis, while the reducing sugar continued to increase. smaller number of dp shows that polysaccharide was depolymerized into short-chains compounds. li et al. (2000) also reported that each microorganism will liberate specifi c xylanase, and showed diff erent activity on the same substrate. high diversity of xylanase may be caused by many diff erent structures of xylan in the nature. isolates skk1-8, but not 234p-16, can be used for xos production using corncob xylan within 4 hours hydrolysis time. streptomyces xylanase to produce xylooligosacharide a. meryandini et al. 126 chen et al. (1997) stated that xylo-oligosaccharide (xos) composed of 2-5 units xylose. vazque et al. (2000) reported that xos for food application should be 2-4 unit monomers, especially dp 2 xylobiose. table 3. reducing sugar, total sugar and the degree of polymerization isolate substrate time (hours) sugar production (mg/ml) total sugar (mg/ml) degree of polymerization 234p-16 1 % hawaii xylan 0 2.05 184.79 90.14 1 2.91 184.79 63.50 2 3.4 184.79 54.35 3 4.3 184.79 43.18 4 4.8 184.79 38.50 1% bisma xylan 0 1.53 151.47 99 1 2.06 151.47 73.53 2 2.39 151.47 63.38 3 3.13 151.47 48.39 4 3.39 151.47 44.68 skk1-8 1 % hawaii xylan 0 12.24 184.56 15.08 1 27.43 184.56 6.73 2 36.29 184.56 5.09 3 34.84 184.56 5.30 4 38.70 184.56 4.76 1.5% bisma xylan 0 1.36 182.61 134.27 1 2.45 182.61 74.53 2 26.84 182.61 6.80 3 24.72 182.61 7.39 4 28.65 182.61 6.37 conclusions xylanase from strain 234p-16 has the highest activity if cultivated in 1% hawaii xylan, whereas strain skk1-8 on 1.5% bisma xylan. using 1.5% of bisma xylan, xylanase from skk1-8 can produce xylooligosacharide with 6.37 degree of polymerization whereas using 1 % of hawaii xylan with 4.76 degree of polymerization within 4 hours of hydrolysis time. xylanase from strain 234p-16 is not suitable for producing xylooligosacharides. xylanase from strain 234p-16 has the highest activity if cultivated in 1% hawaii xylan, whereas strain skk1-8 on 1.5% bisma xylan. using 1.5% of bisma xylan, xylanase from skk1-8 can produce xylooligosacharide with 6.37 degree of polymerization whereas using 1 % of hawaii xylan with 4.76 degree of polymerization within 4 hours of hydrolysis time. xylanase from strain 234p-16 is not suitable for producing xylooligosacharides. acknowledgments th is research was funded by dip pusat studi regional penelitian biologi tropika (seameo biotrop) with contract agreement no 044.1/psrp/sp-pen/iv/2006 on 6th april 2006 to anja meryandini. we thank dr. ir. yulin lestari for the isolate. biotropia vol. 15 no. 2, 2008 127 references adeola o. and m.r. bedford . 2004. exogenous dietary xylanase ameliorates viscosity-induced anti-nutritional eff ects in wheat-based diets for white peckin ducks (anas platyrinchos domesticus). british journal of nutrition 92:87-94. agustine w. 2005. penentuan kondisi optimum pertumbuhan dan produksi xilanase isolat aq1. undergraduate thesis (skripsi). department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia. anggraini f. 2003. kajian ekstraksi dan hidrolisis xilan dari tongkol jagung (zea mays l.). undergraduate thesis (skripsi). faculty of agricultural technology, bogor agricultural university, bogor, indonesia ali m.k., rudolph f.b. and g.n. bennett. 2004. th ermostable xylanases 10b from clostridium acetobotylicum atcc824. journal of industrial microbiology and biotechnology 31: 229-234. ambarawati d. 2005. karakterisasi mananase streptomyces sp galur 45i-3. undergraduate thesis. 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vol. 18 no. 2, 2011: 74 80 utility of k gene to assess evolutionary relationship of genus (anacardiaceae) in indonesia and thailand mat mangifera topik hidayat , adi pancoro , and diah kusumawaty maturasek ( k) gene of chloroplast dna has served as an appropriate candidate to be a dna marker in angiosperms. using this marker, 19 species of genus , one of the ecologically important crop, collected from indonesia and thailand were analyzed. phylogenetic analysis using parsimony method revealed that the gene could clasify into three major groups, namely group i, ii, and iii. moreover, the k gene can identify species originated from thailand. although this classification system is different with the previous classification system, it can provide a new information on the current status of taxonomy. further result exhibited that dna sequences of the k of two species ( dan ) are different between indonesia and thailand specimens. : dna barcode, , k gene, parsimony, phylogenetic analysis 1,2,* 3 2 1 2 3 department of biological science, faculty of bioscience and bioengineering, universiti teknologi malaysia (utm), malaysia department of biology education, indonesia university of education (upi), bandung, indonesia school of life sciences and technology, institute technology of bandung, indonesia mat mangifera mangifera mat mangifera mangifera mat mangifera m. laurina m. macrocarpa mangifera mat received 24 november 2010/accepted 28 october 2011 abstract introduction keywords the genus l., one of the most important plant groups in deciduous forest and wet tropical rainforests including mountain forests, is one of the largest genera of the family anacardiaceae to which approximately 69 species have already been described. the genus is mostly distributed in the tropical parts of asia (india, burma, sri lanka, thailand, south tropical china, malaysia, indonesia, papua new guinea, the philippines, the solomon islands) but also in the pacific islands (kostermans & bompard 1993). in spite of their economical importance, phylogenetic relationships among species within the genus have been poorly understood due to their extremely complicated vegetative and reproductive organs. mangifera 74 e-mail: topik28@yahoo.com or topik@fbb.utm.my biotropia vol. 18 no. 2, 2011 previously, marchand (1869), pierre (1897), and kostermans and bompard (1993) have revealed classification systems for the genus based upon floral characters. however, these characters were extremely complicated in the genus and subjected to parallelism (yonemori . 2002), suggesting many taxonomic and phylogenetic problems still remain unresolved. given the shortcomings of these characters, data obtained from nucleotide substitutions of appropriate molecules are preferable for clarifying phylogenetic relationships (e.g., moritz & hillis 1996). methods for clarifying relationships in species or group organisms by using dna sequences have been proposed and initiated recent years (kress . 2005). maturasek gene of chloroplast genome served as potentially usable dna regions to flowering plants. the k gene is frequently choosen by plant systematists because the region is a single copy gene and has enough variable sites of nucleotide substitution. recently, the k gene has been widely used in phylogenetic inferences of various groups of plant (e.g. ito . 1999; ferguson sang 2001; raymond . 2002; ebihara . 2005; hidayat . 2005). phylogenetic analysis to clarify phylogenetic relationships among members of genus have been carried out using dna sequences of the k gene. a total of 19 species of was collected from indonesia and thailand, plus two species of . two members of genus (m9 and m13) were used as outgroup in phylogenetic analysis based on previous research this genus was sister group to (yonemori . 2002). detailed information of the plant is summarized in table 1. dna genome was extracted from fresh materials (young leaf) using qiagen dneasy mini plant kit with slight modification. amplification was conducted using four primers as shown in figure 1. table 2 provides detailed information on sequences of primer pairs. et al et al mat mat et al et al et al et al mangifera mat mangifera bouea bouea mangifera et al & an understanding of the evolutionary relationships in this group may contribute to the field of plant systematics or ecology. materials and methods 75 matk genetrnk trnk a b c d figure 1. strategy of amplification and sequencing of the k gene. a= k-5f, b=taa-09f, c=taa09r, dan d= k-2r. two internal primers (b and c) were designed for this study. mat trn trn utility of matk gene to assess evolutionary relationship topik hidayat .et al table 1. plant materials, their geographic origins and codes used in this study species origin code mangifera altissima blanco var bingloe indonesia m18 mangifera applanata kosterm. indonesia m14 mangifera foetida lour. indonesia m17 mangifera gedebe miq. indonesia m10 mangifera indica l. indonesia m11 mangifera laurina bl. indonesia m7 mangifera macrocarpa bl. indonesia m3 mangifera odorata griff. indonesia m16 mangifera spp indonesia m12 mangifera rufocostata kosterm. indonesia m8 mangifera similis auct. indonesia m2 mangifera caesia jack ex wall indonesia m5 mangifera casturi kosterm. indonesia m15 mangifera macrocarpa bl. thailand s1 mangifera conchinchinensis englar thailand s6 mangifera flava evrard thailand s3 mangifera gracilipes hook.f. thailand s2 mangifera caloneura auct. thailand s5 mangifera laurina bl. thailand s7 bouea oppositifolia (roxb.) meiss indonesia m13 bouea macrophylla griff. indonesia m9 76 table 2. primers used in this study name sequences trnk-5f 5’ tgggttgctaactcatgg 3’ trnk-2r 5’ aactagtcggatggagtag 3’ taa-09f 5’ggttttcccatgagtagattatcg 3’ taa-09r 5’ cgaagtagacgaagctcttgg 3’ for amplification, we used primer pairs a and d, whereas all primers used once sequencing. pcr (polymerase chain reaction) reaction included buffer pcr (1x), mgcl (2-3mm), primers (@ 0,5 mm), enzyme taq polymerase (1 u/ul), dntps mix (1,6 mm), and dna template (100-150 ng/ul). pcr was conducted according to hidayat . (2005). pcr cycles include 1 cycle at 94 c (predenaturation) for 5 minutes; 30 cycles at 94 c (denaturation) for 30 seconds, 49 c ( ) for 30 seconds, and 72 c (extension) for 2 minutes; and ended with 1 cycle at 72 c (final extension) for 8 minutes. pcr products were cloned into pgem-t easy (promega) before sending them to macrogen (korea) for sequencing. dna sequences obtained from the k gene were aligned with clustal x (thompson . 1997) and then adjusted manually. phylogenetic analyses based on the maximum parsimony criterion was performed using paup* version 4.0b10 (swofford 1998). all characters were equally weighted and unordered (fitch 1971). all the data sets were analysed by the heuristic search method with tree bisection-reconnection (tbr) branch swapping and the multrees option on, ten replications of random addition sequences with the stepwise addition option, and all most parsimonious trees (mpts) were saved. evaluation of internal support of clades was conducted by the bootstrap analysis (felsenstein 1985) utilizing 1000 replicates with tbr branch 2 et al annealing mat et al o o o o o biotropia vol. 18 no. 2, 2011 77 swapping and the multrees option off. number of steps, consistency indices (ci) and retention indices (ri) were calculated on one of the mpts in each analysis with the tree scores command in paup*. dna extraction can be done using various types of dna sources such as leaf, stem, flower, and seed. in this research, young leaf was used for dna extraction to minimize contamination that can inhibit pcr amplification. high level of concentration (600 ng/ul in average) with good ratio ( 1.750) was obtained. size and border of k gene for were determined through comparative analysis in genebank (www.ncbi.nlm.nig.gov). the results indicated that size of k gene in is about 1500 bp. multiple alignment analysis was performed by using clustalx (thompson . 1997). the aligned k comprised 1,601 characters (fig. 2). of these, 1,429 were constant and 51 were potentially informative. reconstruction of phylogenetic tree (fig. 2) using paup resulted in 23 mpts with a length of 121 steps, ci of 0.852, and ri of 0.739. the tree (fig. 3) demonstrated that the genus was monophyletic and split into three major groups. monophyletic nature of was supported by character of stoma, anomositic (hidayat, unpublished data). the three major groups found in this study is not consistent with previous classification system by mukherjee (1953), kostermans and bompard (1993) based upon morphological characters, and even yonemori . (2002) on the basis of dna sequences of internal transcribed spacer (its) region. the number of plant materials used in this study is likely to be insufficient (only 19 out of 69 recognized species). further phylogenetic analysis is, therefore, needed using more extensive sampling. however, this study has provided new information on taxonomy of . as depicted in figure 2 , (from indonesia), and were united (group i), whereas (form thailand), and were closely related (group ii). group iii was housed by the rest of species, and species which is originated from thailand was placed within group iii (fig. 2). unfortunately, no single synapomorphic character is found to support each group. moreover, this research has revealed that there are variations of k in and which come from indonesia and thailand. as seen in figure 2, (from thailand) was separated from that of indonesia (group iii; thailand specimen in group ii). similar situation has been found in : thailand in group iii and indonesia in group i. different nature between these two countries has driven the mutation in k, but this does not lead to shift the morphology. these are related with the ability of plant to adapt to the environment changes (evans 1975). as mentioned, k gene is highly conserved (e.g. ebihara . 2005; hidayat . 2005). mutation rate in this kind of gene is very slow. this is reflected by the small number of informative characters (only 51 from a total 1,601 characters) to build the results and discussion + mat mangifera mat mangifera et al mat mangifera et al mangifera m. applanata m. macrocarpa m. altissima m. laurina m. casturi, m. odorata, m. indica mangifera mat m. laurina m. macrocarpa m. laurina m. macrocarpa mat mat et al et al 78 utility of matk gene to assess evolutionary relationship topik hidayat .et al tree. as a consequence, bootstrap value in most branches of the tree are less than 50. similar condition was found in other angiosperms (e.g. raymond . 2002; ebihara . 2005; hidayat . 2005). further analysis based on the phylogenetic scheme presented here will shed more light on overlooked characters. et al et al et al figure 2. alignment process using clustalx shows the level of homology (*) biotropia vol. 18 no. 2, 2011 79 outgroup figure 3. one of the 23 mpts of based on k gene. bootstrap value of >50 are shown above each branch. * = indonesia specimen; **= thailand specimen. species inside the box are originated from thailand. mangifera mat b. oppositifolia b. macrophylla m. applanata m. macrocarpa* m. altissima m. spp m. laurina** m. kasturi m. odorata m. indica m. caesia m. foetida m. rufacostata m. gedebe m. macrocarpa** m. similis m. laurina* m. conchinensis m. flava m. gracilipes m. caloneura group i group ii group iii 100 77 55 84 61 86 conclusions this study demonstrated that the k gene classified the into three major groups. furthermore, the k identified species that is originated from thailand. the k gene in the two species, namely and , was different 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massachussets. thompson jd, gibson tj, plewniak f, jeanmougin f, higgins dg. 1997. the clusstalx windows interface: flexible strategies for multiple sequences alignment aided by quality analysis tools. nucleat acid research, 24:4876-4882. yonemori k, c honsho, s kanzaki, w eidthong, a. sugiura. 2002. phylogenetic relationships of mangifera species revealed by its sequences of nuclear ribosomal dna and a possibility of their hybrid origin. plant systematic and evolution, 231:59-75 vandenboschia radicans mat mat mangifera e . f nd biotropia no biotropia no. 16, 2001 : 10 -17 a novel integron in the genome of escherichia coli isolated from indonesian monitor lizard (varanus spp). esti puspitasari2, antonius suwanto1'2 * , amarila malik3, and walter erdelen" 'dept. of biology, faculty of science and mathematics and ivc biotechnology, bogor, agricultural university, bogor, indonesia; 2 south east asian regional center for tropical biology (seameo biotrop), bogor: indonesia 3 dept. of pharmacy, faculty of science and mathematics, university of indonesia, jakarta, indonesia; 'dept. of biology, faculty of science and mathematics, bandung institute of technology, bandung, indonesia abstract the genotype of antibiotic resistance in natural isolates of escherichia coli was determined through integron detection and characterization of the associated antibiotic resistance. e. coli sg2 isolated from varanus salvator of java demonstrated resistance to spectinomycin (50ng/ml) and streptomycin (song/ml). integron detection indicated that eight isolates out of nine e. coli isolates possessed a conserved segment of the integron. amplification of the inserted cassette of the integron in this sg2 isolate yielded a 1-kb dna fragment. sequence analyses indicated that this fragment was homologous with aad gene, which confirmed the resistance to spectinomycin/streptomycin. this is the first report on the presence of integron in the e. coli isolated from the environment. key words: integron / antibiotic resistance / escherichia coli introduction antibiotic is a substance produced by a microorganism as protection from another microorganism (madigan et al. 1997). antibiotics have a specific target site in the cell. for example, in trying to get hold of protein synthesis targeted to 30s ribosom, the presence of tetracycline, spectinomycin and streptomycin results in an invalid codon-anticodon reaction and therefore, the synthesized amino acid is also invalid ( schumm 1992). currently, there is concern on the spread of antibiotic resistance among bacteria. such dissemination is partly a consequence of the antibiotic-resistant genes mobility which reside in mobile genetic elements such as plasmids, transposons, and integrons (francia et al. 1999). integron is a new type of mobile element which has evolved by a site-specific recombination mechanism. integrons consist of two conserved segments of dna separated by a variable region containing one or more genes integrated as cassettes (levesque et al. 1995). the 5'conserved segment contained in the int gene, which encodes a polypeptide of 337 amino acids, has been shown to be homologous to other members of the integrase family and on the opposite strand, a common promoter region p1-p2 is directed toward the site of integration. the 3' conserved segment contains the qace&\, sur genes, and an open reading frame (fig.l). * corresponding author : e-mail address : asuwanto@indo.net.id 10 10 figure 1. integron structure (levesque et al. 1995) genetic changes in bacterial genome could contribute to antibiotic resistance in some bacteria. there are three mechanisms of antibiotic inactivation: (i) modification of antibiotic structure; (ii) prevention of antibiotic to get into the target site; or (iii) change in antibiotic target site (neu 1992). resistance to aminoglycoside antibiotics such as spectinomycin and streptomycin, could be due to the penetration failure, the low affinity of antibiotic to ribosom, or the inactivation of the antibiotic by enzymes such as phosphorylase, adenylase and acetylase produced by the bacteria (gian and gian 1995). desselberger (1998) reported that data on antibiotic-resistant genes in the environment were rare, whereas antibiotic-resistant genes among bacterial strains were increasing at an alarming rate. biawak monitors (varanus spp.) are reptilians lacking venomous gland, although there are some reports on biawak bites resulting in bacterial infection (auffenberg 1981). probably, this infection is caused by toxic substances produced by microbiota living in the oral of varanus spp. auffenberg (1981) identified five bacteria species that could cause infection. yogiara (1998) reported that many of e. coli isolates living in the buccal of varanus spp. are resistant to ampicillin. this study was conducted in order to understand the mechanism of the antibiotic resistance of e. coli in the digestive system of varanus spp. in this experiment, an antibiotic-resistant gene from e. coli was isolated and further characterized by dna sequence analysis. 11 a novel integron in ther genome of escherichia coli – esti puspitasari et al. material and mothods bacterial strains and plasmids the bacterial strains and p;asmids used in this research are shown in table 1. table 1. bacterial strains plasmida growth conditions antibiotic-resistant bacteria were cultured aerobic (100 rpm) at 37°c and kept overnight in luria bertani (lb) medium supplemented with one, or a combination of the following antibiotics: ampicillin (100 u.g/ml), streptomycin (50 ng/ml), spectinomycin (50 ng/ml). dna extraction bacterial isolates were grown in 10 ml lb in the presence of a selective antibiotic at 37°c overnight. alkaline lysis method (sambrook et al. 1989) was used for plasmid dna extraction, and wizard®genomic dna purification kit (promega, madison, wi) was used according to manufacturer protocol for genomic dna extraction. 12 biotropia no. 16, 2001 pcr amplification pcr were performed in gene amp® pcr system 2400 (perkin elmer, branchburg, new jersey). ready to go™ pcr beads (pharmacia biotech, uppsala, sweden) was used for each pcr reaction. each of 25 ul reaction mixtures contained pcr beads, 4 ng/ul of genomic dna, 10 ng/ul of primers (5'cs: ggcatccaagcagca ag, 3'cs: aagcagacttgacctga, qac: atcgcaatagttggcgaagt, sul: gcaaggcggaaacccgcgcc) and distilled water up to 25 ul. pcr condition for the integron was amplified in 35 thermal cycles at 94°c for 1 minute, 55°c for 1 minute, and 72°c for 3 minutes. a final extension step for 7 minutes at 72°c was also included. the primers used were 5'cs and 3'cs that amplify variable regions of integron, qac and sitll primers to amplify the conserved region. pcr product was purified using gene clean kit (bio 101, la jolla, california). cloning and transformation amplified dna was ligated into pas900 (kmr) and pgem-t easy (apr ) vector. the approximate ratio of pas900 vector to insert dna were 1:4 and that of pgem-t easy to insert dna was 1 to 16. ligation reaction consisted of vectorinsert mixture 1 ul of t4ligase, ix ligation buffer, and distilled water up to 20 ul. ligation mixture was incubated at 7°c for 16-18 h. the ligation mixture was transformed into 250 ul of chemically competent e. coli top10, followed by heat shocked at 42°c for 45 seconds. after the addition of 3 ml lb, the culture was incubated with vigorous shaking at 37°c for 90 minutes (sambrook et al. 1989). transformants were then selected on luria bertani agar (lba) supplemented with x-gal (40ug/ml) and appropriate antibiotic/s. dna sequencing ps2t double stranded templates were derived from cloning of sti in pgem-t easy. sequencing reaction consisted of 8 ul big dye-terminator, 360 ng of dna template, 8 ng/ul of primer and distilled water up to 20 ul cycle sequencing was performed using pcr machine with three-step profile for 25 cycles: a 10-second denaturation at 96°c, a 5-second annealing at 50°c, and a 4-minute extension at 60°c. cycle sequencing product was purified by ethanol-sodium acetate precipitation method. a 1.5 ml microcentrifuge tube was filled with 2 ul of sodium acetate (ph 4.6), 50 ul of ethanol 95%, and 20 ul of the cycle sequencing mixture, and then incubated at room temperature for 30 minutes. the mixture was centrifuged at maximum speed for 20 minutes. the supernatant was removed from the tube, and 250 ul of 70% ethanol was added into the tube. the mixture was centrifuged again for 5 minutes. the supernatant was removed and the dna pellet was vacuum-dried at 50 cm hg for 10 minutes. 13 a novel integron in the genome of escherichia coli esti puspitasari et al. the dna was electrophoresed according to sambrook et al. (1989). prior to electrophoresis, dna was denatured with 6 ul of loading buffer containing blue dextran and 25 mm of edta in formamide at 95°c for 2 minutes, and quickly placed on ice. approximately 1.5 ul of dna sample was loaded to each well and ran for 10 hours. the dna was compared with those in the genbank and embl databases. results and discussions integron detection by pcr amplification of dna sequence experiments using qac-sul primers indicated that there were eight out of nine e.coli isolates possessing 3'conserved segment of the integron (table 2). e. coli top 10 containing recombinant plasmid pepio did not show integron amplification, although it carries a 10-kb dna fragment originated from genomic dna of e.coli sg2 isolated from varanus spp. (table 1). we suspected that parts of the integron have been deleted when it was digested by restriction enzymes. pcr product using 5'cs-3'cs primers generated a single amplified dna band of about 1-kb. it was generated from genomic dna of e. coli sg2 isolate, which was designated as sti (table 2 and fig.2 lane 3). at the downstream end of each resistant gene cassette inserted in the variable region of integrons, there is a short imperfect inverted repeat element called the 59base element. each inserted gene has its own version of this element (levesque et al. 1995). the sti was also thought to have a 59-base element, although it was not demonstrated in this study. these 59-base elements are known to be important in the recombination events observed in the evolution of the integron. for example, in plasmid pvsi, which possesses the 5' and 3' conserved segment but no inserted gene between the conserved segments, there is no 59-base element (levesque et al. 1995). the other isolates used in this study were found to be similar to pvsi (table 2). it was reported that pvsi, a plasmid derived from pseudomonas aeruginosa, possessed integron type ino which has an unoccupied integration site and hence may be an ancestor of the more complex integrons (bissonnette and roy 1992). cloning of sti in pas900 (kmr) vector yielded recombinant plasmid called pcras. the purpose of cloning was to detect ampicillin-resistant gene in sti. e. coli top 10 harbouring the recombinant plasmid pcras was sensitive to ampicillin. this result indicated that sti was not an ampicillin-resistant gene, or it was an ampicillin-resistant gene which could not be expressed. expression of antibiotic-resistant genes in the integrated cassettes of integrons depends on cassette position. in all cases, the resistance level was the highest when the gene was present in the first cassette (collis and hall 1995). therefore, there is a need to sequence the sti for gene characterization. 14 biotroopia no. 15, 2001 figure 2. integron pcr product from total dna of pvhal isolate (lane 1), sg2 isolate (lane 3), and kb ladder as marker (lane 2). 15 a novel integron in the genome of escherichia coli — esti puspitasari et al. sequencing cloning of sti in pgem-t easy vector yielded recombinant plasmid designated as ps2t. transformant was isolated, and then sequenced. the result indicated that the sti sequence was homologous to aada gene encoding aminoglycoside adenyltransferase, which was responsible for streptomycin and spectinomycin resistance in e. coli. blast search analysis of the sti sequence indicated that the 700 nucleotides of 3'sti are integron type 3 (int 3) as in the incl/m plasmid from salmonella typhimurium (96% identity) and pnccsol plasmid from enterococcus faecalis (96% identity). the other 600 nucleotides of 5'sti were similar to the pnccsol plasmid from e, faecalis (97% identity) and the r100.1 plasmid from bacteriophage t4 (97% identity). in summary, there were eight isolates out of nine e. coli isolates possessing a conserved segment of integron and only one possessed inserted gene between the conserved segments of the integron. the sequencing analysis indicated that spectinomycinand streptomycin-resistant gene was present in the 1-kb dna fragment. acknowledgments this work was supported by dip project for biotrop 1999 / 2000 to antonius suwanto. we thank yeo chew chieng from department of microbiology, national university of singapore for conducting dna sequencing. references auffenberg,w. 1981.the behavioral ecology of the komodo monitor. university press of florida, florida bissonnette, l. and p.h. roy. 1992. characterization of ino of pseudomonas aeruginosa plasmid pvsl, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria. j. bacteriol. 174:1248-1257. collis, c.m. and r.m.hall. 1995. expression of antibiotic resistance genes in the integrated cassettes of integrons. antimicrob. agents. chemother. 39: 155-162. desselberger, u. 1998. resistance to antibiotics and other antimicrobiol agents. sgm quarterly. 25:94-95. francia, mv., j.c. zabala, f. cruz, j.m.g. lobo. 1999. the intl integron integrase preferentially binds single-stranded dna of the attc site. j. bacteriol. 181: 6844-6849. gian, g.s. dan v.h.s.gian. 1995. farmakologi dan terapi: aminoglikosid. ed. ke-4. gaya baru, jakarta. jacoby, g. 1992. exploring new strategies to fight drug-resistant microbes. science. 257:1036-1038. levesque, c., l. piche, c. larose, and p.h. roy. 1995. pcr mapping of integrons reveals several novel combinations of resistant genes. antimicrob. agents. chemother. 39:185-191. 16 biotropia no. 16, 2001 madigan, m.t., j.m. martinko, and j. parker, 1997. biology of microorganisms. 8"1 ed. prentice hall, new jersey. neu, h.c. 1992. the crisis in antibiotic resistance. science. 257:1064-1072. sambrook, j., e.k. frisch, t. maniatis. 1989. molecular cloning, a laboratory manual. ed. ke-2. cold spring harbor laboratory press. cold spring harbor, usa. schumm, d.e. 1992. intisari biokimia. terjemahan moch. sadikin. binarupa aksara, jakarta. yogiara. 1998. keragaman genetik isolat escherichia coli dari rongga mulut biawak (varanus) dan kloning gen penyandi resistensi ampisilin. skripsi. jurusan biologi fmipa ipb, bogor. 17 biotropia no biotropia no. 18, 2002 : 21 37 morphological characterization of malaysian wild banana musa acuminata muhammad asif javed*, mak chai and rofina yasmin othman division of genetics & molecular biology, institute of biological sciences, university of malaya, 50603 kuala lumpur, malaysia abstract fourteen populations of musa acuminata ranging from populations in the lowlands of northern (ssp. siamea) to central malaysian region (ssp. malaccensis) and highland banana (ssp. truncata) were characterized based on chromosome number and 46 morphological characters. a large amount of variation was observed within the populations. however, only highland bananas appeared morphologically distinct. lowland populations both from northern and central malaysia were found to be overlapping and no distinguishing pattern was observed. the morphological characters found variable within these populations were related to developmental changes and mutations. the results obtained in this study were not revolutionary. however, the survey of a large number of characters treated with multivariate techniques further sharpened the existing groupings of the musa acuminata subspecies. key words: musaceae/ malaysia / ssp. malaccensis i ssp. truncata i ssp. siamea introduction banana and plantains are one of the most important tropical fruit crops. they are staple for rural and urban consumers in the humid tropics and an important source of rural income. bananas are grown in 122 countries of the world with a cultivated area of 3.8 million ha and total production of 56.4 million metric tons both for export and local consumption (fao 1999). banana monoculture cultivation provided a powerful driving force in disease epidemics. it was observed in the case of gros michel attacked by fusarium oxysporum f. sp. cubense (foc) race 1 resulted in the collapse of the banana industry. gros michel was then replaced by cavendish bananas resistant to foc race 1. however, recent outbreak of a new foc race 4 attacking cavendish in subtropics and tropics again threatened the banana industry. there is no chemical control available and the only way is to plant cultivars resistant/tolerant to the pathogen (buddenhagen 1990). malaysia is one of the centers of diversity for both wild and cultivated bananas. musa acuminata is the most variable species and progenitor of cultivated bananas (simmonds 1962). m. acuminata was reported to be resistant to fusarium wilt race 1 (vakili 1965). a survey of local wild species has been reviewed by simmonds (1955) and kiew (1987) based on morphological characters. only a few morphological charac 21 biotropia no. 18, 2002 ters were used and characters found to be useful for subspecific classification have been further questioned (hari 1968; shepherd 1988 and 1999). therefore, the subspecific grouping of local musa acuminata has been complicated by taxonomic revisions. recent renewed emphasis on the utilization of these resources for disease resistant studies for molecular banana breeding elucidated the need to characterize different wild musa acuminata forms in malaysia. for breeding and ease of field identification, taxonomic differentiation based on morphological traits seems to be convenient and useful for evaluating germ-plasm. hence the present study aims to describe intra-and interspecies variation in different musa species, using both qualitative and quantitative morphological traits. multivariate techniques are used to describe relationship and to observe the most variable characteristics. materials and method fourteen populations of wild seeded musa acuminata species and two of musa violascens and musa balbisiana (fig. 1) were surveyed for this study (table 1). suckers collected were all planted on the university malaya farm under the same environmental conditions to reduce variation due to environmental factors. suckers were planted in holes to a depth of 30-40 cm with 2 x 2 m spacing within and between rows. weeding was done manually and fertilizer was applied occasionally. 22 marphological characterization of malaysian wild banana – muhammad asif javed et al. *geographical grouping northern peninsular malaysia = kelantan and kedah central peninsular malaysia = pahang, perak, selangor higlands = genting and cameron highlands (pahang) a mean number of five specimens were examined for each sample. the data on different morphological characters were taken during first crop cycle. all accessions planted grow well except bc3 (highland banana) and musa violascens (bc1) which were dead; therefore, morphological data on these accessions were taken in-situ. fruit bunches were also collected for the development of open pollinated seed populations. initial studies on seed germination in the greenhouse were not successful; therefore, in vitro embryo culture protocol was standardized (asif et al. 200la). ploidy was determined using flow cytometery (fcm) as previously described (asif et al. 2001). all samples used are diploid where musa acumlnata and m. balbisiana have (2n = 22) and m. violascens (2n = 20) chromosome number as suggested by simmonds (1962). the vegetative characters were scored on plants with emerged inflorescence. during this time, maximum development of vegetative parts has taken place (purseglove 1972). forty-six characters were used including those suggested by simmonds (1955) and ipgri descriptors (1996). thirty-five qualitative and 11 quantitative characters were included. simple two-state qualitative characters ordered multi-state characters were coded as series of discrete states (table 2). characters related to color were examined using a standard color chart (ipgri 1996). mean values for continuous quantitative characters for the minimum five randomly selected healthy plants per accession were calculated. phonetic analyses were carried out using spss-pc for windows version 9.0 (norusis 1985). cluster analysis and principal component analysis were carried 23 biotropiano. 18,2002 out on a data set including both metric and binary data. a distance matrix of squared euclidean distance was calculated. this distance was used as a measure of phenetic difference. clustering was carried out by single linkage method. table 2. description of morphological characteristics based on banana descriptor (ipgri 1996) and simmonds (1955). 24 marphological characterization of malaysian wild banana – muhammad asif javed et al. 25 biotropia no. 18, 2002 principal component analysis was conducted as it has the advantage of abstracting factors, one of which may represent some underlying variables like overall size, which may affect many characters. there were several significant factors but only the first four were extracted. factor scores were calculated for each otu and the first two plotted in a scatter diagram. results a single linkage cluster method grouped m. acuminata samples into four where bc2 (musa violascens) and gala (musa balbisiana) were clearly separated (fig. 2). the highland banana was clearly differentiated from lowland banana forms based on deep purple brown color of the pseudostem, petioles, and male bracts, a non-waxy petiole and lamina with both sides of lamina bases rounded and creamy colored internal bract face. the lowland populations were found to be overlapping irrespective of their geographical origin and no distinct separation was observed. flava and segun were collected from central region of peninsular malaysia while sintok and j-4 found in the northern region were grouped together and showed large morphological similarities. bd1, bd2, ri and iptj populations were from 26 morphological characterization of malaysian wild banana muhammad asif javed et al. southwest of the peninsula, while bc1, kra and ppc were from the central region (fig. 1). these accessions were morphologically similar possibly reflecting the overlapping of the geographical ranges in the central lowland regions. geographically, rangis and perak were of very diverse origin but showed high morphological similarity, differing only in bract imbrication. rangis is characterized by young bracts greatly overlapping compared to the convolute male bud observed in perak. the last group comprised gala and bc2. these were well separated from m. acuminata with little or no similarity. the position of all musa accessions on the first and second principal components as derived from the original pairwise correlations among the accessions are shown in figure 3. the first factor accounted for 36.73% variation while the second factor showed only 11.14% variation. a significant percentage decrease was observed with components three (10.36%) and four (8.79%). fig 2. phenogram of 16 musa accessions based on morphological characters and chromosome number. musa acuminata accessions were separated into four groups based on single linkage and squared euclidean distance. the highland banana bc3 was well separated at the bottom from lowland accessions. the two species bc2 and gala were also clearly separated from the musa acuminata. 27 biotropia no. 18, 2002 fig 3. principal component analysis. pca showing relative positions on the first and second principal components of 16 accessions of mum species based on 46 morphological characters and chromosome number. musa acuminata samples showed large morphological variation and grouping of the different accessions was in agreement with that of cluster analysis. component one clearly separated the samples ppc and bc3, the highland banana, from rest of the accessions of m. acuminata, whereas second component separated kra. the major groupings observed were bd1, iptj, bc1, bd2 and ri, a second group composed of segun, j-4, sintok, and flava, and finally rangis and perak (fig. 3). table 4 shows that of the 46 characters used in the analysis 13 had loadings of more than 0.5 on the first component. four of these characters i.e. chromosome number, bract apex shape, male bract lifting and fruit apex were found to be useful 28 morphological characterization of malaysian wild banana muhammad asif javed et al. in separating the three species samples m. acuminata, m. balbisiana (gala) and m. violascens (bc2), respectively. five characters could differentiate both interspecific and intraspecific accessions were bunch posture, bunch appearance, bract base shape, bract imbrication and transverse section of fruit. m. acuminata accessions could be differentiated based on these four characters e.g. rachis type, bract behaviour before falling, male flower behaviour before falling and remains of floral relicts . table 3. fruit bunch (five bunches/sample) characteristics of different wild musa acuminata bananas eight characters which accounted for much of the variation were contributed by the second component. the characters like leaf and petiole waxyness, insertion point of leaf blade clearly separated the highland form bc3 from the lowland form of m. acuminata accessions. six characters contributed largely on component three which separated the ri accession based on male bud degeneration at maturity and yellow color of compound tepal while kra and ppc were separated based on the rachis position and free tepal development. the fourth component showed four characters with loadings of more than 0.5. pseudostem color had the highest loading compared to the other three characters and clearly separated the highland banana bc3 from the other accessions. the grouping of accessions in pca corresponded well to that of cluster analysis. 29 biotropia no. 18, 2002 table 4. characters found most variable based on first four principal components. thirty characters with loadings of more than 0.5 (in bold) on components one, two three and four separating different accessions and groups of accessions of musa samples in pca. the majority of samples from the central region, bc3, bc1, bd2, ri and kra tended to be tall. perak, rangis, ppc, sintok, j4, and segun were semi-dwarf with plant heights of less than 2 m. deep purple brown color of pseudostem and petioles were found useful to differentiate highland banana form (bc3). characteristically, 30 morphological characterization of malaysian wild banana muhammad asif javed et al. highland banana showed non-waxy leaves and petioles compared to waxy types in all m. acuminata samples. generally, the leaf sheath in wild bananas is narrow on both sides forming the petiole, which is rounded beneath and channeled above, retaining the crescent shaped section of the sheath. petiole canal of perak, ppc and ri was observed to have wide and erect petiole margins compared to curve or straight erect margins in other samples. sample bc3 had both sides of the lamina pointed, whereas j-4 showed one side rounded and the other side pointed while other samples had round lamina bases. samples bci, bd1, bd2, ri, iptj and bc3 (a highland banana) were characterized with very hairy fruit peduncles. the perak, sintok, j-4 and segun samples have hairless fruit peduncles with the exception of rangis, ppc and flava having slightly hairy peduncles. ppc had a very compact fruit bunch compared to compact or lax fruit bunches in other samples. rachis position was also found to be a useful character to differentiate both kra and ppc, which had rachis falling vertically, compared to the normal horizontal position. fruits were biseriate; rachis and male bud were persistent in all accessions until the fruit maturity except the sample ri where male bud was degenerated before fruit maturity. fruit shape was straight in kra or slightly curved or curved in all other samples. observations made on fruit transverse section showed that sample ppc had a rounded transverse section, while iptj showed pronounced ridges. all samples studied showed a normal male bud (male bud and rachis persisted till fruit maturity) with the exception of ri where the male bud degenerated before fruit maturity. perak, bc3, bci, ppc and flava are characterized with medium size of bract. a strong imbrication, with young bracts overlapping at the apex of the male bud was observed in rangis, whereas flava and segun showed slight bract imbrication. other samples of m. acuminata showed convolute bract behavior. the highland banana bc3 could be differentiated from other forms with a distinct deep purple brown external and creamy color of internal bract face. similarly, flava, a yellow bracted mutant of m. acuminata, added more complexity to bract color characterization. bract curling was also found to be useful for differentiating m. acuminata accessions from m. balbisiana. all samples had basal flowers which were functionally female and contain stamens reduced to staminoids, whereas distal flowers had functionally male flowers and were female infertile. the male flowers fell off before bract dispersal in rangis, bd1 and iptj. compound tepal basic color was observed to be creamy except the sample ri (yellow). free tepal development was observed for samples kra and ppc, whereas all other samples had little or no development. number of male flowers per row ranged from 5 to 11. sample iptj had the minimum number of male flowers per row. the length of the male flowers ranged from 3 cm in the case of iptj and 7 cm for sintok. the maximum filament length was observed in the sample bci (4.4 cm) and the minimum of 1.1 cm in bc3. 31 biotropia no. 18, 2002 as agronomic characters are greatly affected by the environment, they are not useful for classification purposes but are of great importance for breeding new cultivars. the agronomic characters recorded were bunch weight, number of hands per bunch, number of fingers per hand, finger length and finger diameter. fruit bunch weight ranged from 1.96±0.02 to 9.86±0.10 kg. large fruit bunches were observed in samples bc1, bd2, bc3, kra and ppc, respectively (table 3). samples with relative large fruit hands were observed in bc3, bc1, and bd2. for fingers per hand, the maximum fruits were produced by ri (22.8+0.5) compared to a minimum of 12.8±0.5 in bd2. large finger length was observed in the samples ri (14.94±0.2 cm), bc3 (14.90+0.04 cm), and bc1 (14.0±0.32 cm). finger diameter ranged from 1.44+0.03 to 3.5±0.03 cm where large finger diameters were observed in ppc followed by bd2 and bc3, respectively. m. acuminata fruit bunches were found to contain a large number of seeds ranging 30-120 per fruit. seeds range between 5-6 mm in diameter and irregularly subglobose in shape. seeds were grayish-brown in color. germination studies in sand beds resulted into 1-2% germination. however, in vitro embryo culture resulted into more than 90% seed germination (asif et al. 2001a). iptj produced albino seedlings when cultured in vitro. discussions morphological characters used for wild banana characterization are taxonomic and not agronomic. harlan (1975) argued that it will be breeders who will make use of the classifications of crop germplasm and they will be mainly concerned with the genetic compatibility, while morphological characters will be secondary. however, engels (1986) stated that the importance of a germplasm collection to a breeding program is strongly dependent on the availability of accurate descriptions of the accessions and on the taxonomic identification of the germplasm. in this study, 10 quantitative characters were used and only four were found useful, according to pca (table 4). in general, most of the north malaysian accessions were semi-dwarf. however, this character is not useful for classification because of variation due to environmental factors. moreover, for breeding purposes, it appears that plants with semidwarf pseudostem height are preferred and capable of withstanding both stress conditions and fruit bunch weight at maturity without propping. similarly, there is less loss from wind damage, harvesting is easier and broadly, they imply high yields and easy care at a little extra expense for planting material (simmonds 1986). the number of male flowers per row was found to be varying within the same male bud similarly, flowers and style length could be modified by environmental factors. hence, these characters are not useful for characterization (ortiz & sevilla 32 morphological characterization of malaysian wild banana muhammad asif javed et at. 1997). quantitative characters might not be appropriate for such task in musa, because all were significantly affected by genotype environmental interactions. pseudostem and petiole sheath color was observed to be useful (simmonds 1955) in the identification of the highland form bc3 from the other two groups. the sheath color is the result of environment or due to genetic factors, could not be further differentiated, as the highland form did not survive under lowland conditions. lamina bases are also found to be interesting characters. skutch (1930) reported that the meristem of one lamina half frequently extends further down the petiole than the other. there is no rule as to which side is produced furthest basally. in cases of smaller size of the lamina, the inequality is much less conspicuous. the basal inequality of the lamina halves is a lasting testimony that they are of independent origins. very late appearance of the lamina halves, compared with the other portion of the leaf, also points to a phylogenetically later origin. a large amount of variability was observed in the fruit shape of different musa acuminata accessions. however, it was observed to be variable even within the same fruit bunch and thus, did not seem to be a reliable character. persistent style and staminodes were observed only in the perak. however, this character could be related to slow maturity. karamura (1999) stated that clones growing at high altitudes usually have non-persistent styles and stamens, which may take a long time to drop. rachis position could be used to differentiate kra and ppc from other accessions. the absence of the rachis and male bud observed in ri was considered a mutation (de langhe 1961). bract imbrication was regarded as an interesting character both at interspecific and intraspecific levels. bract imbrication was used by simmonds (1955) mainly to differentiate 'kedah form' (ssp. siamea) from the selangor and cameron forms. in the present study three samples, rangis, flava and segun, were observed with imbrication. rangis showed greater imbrication compared to the other two samples. in this study, three samples, sintok, j-4 and rangis, were collected from kedah and kelantan, respectively. whereas the samples flava and segun with slight imbricated bracts were collected from pahang states, the center of diversity of 'selangor form' (simmonds 1955). bract imbrication was found to vary within the accessions of 'kedah form' even within the ^ame geographical range. hari (1968) also recommended that bract imbrication should be rejected as taxonomic character in the classification of musa acuminata accessions subject to morphological changes during bud development. the data confirmed that bract imbrication could not be used as a reliable character for classification. ppc was differentiated by having a very compact fruit bunch hanging at an angle. it was observed that accessions having a pendulous rachis had compact fruit bunches. similar observations were made by de langhe (1961) where he found a pendulous bunch which commonly has fruits requiring towards the peduncle or rachis. however, karamura (1999) observed clones that showed no geotropic reaction, had pendulous bunches. a sub-horizontal bunch posture (angle of female 33 biotropia no. 18, 2002 axis) is under the control of additive gene action of dominant alleles with threshold effect (ortiz 1993). this character is of economic importance as pendulous bunch is the most symmetrical and is therefore better adapted to transportation. great variability also accounted for bract color, both at interspecific and intraspecific levels. however, the situation was more complex at intraspecific level where different samples of m. acuminata had different bract colors. highland banana sample bc3 (ssp. truncatd) was classified based on the bract color. bract color characterization based on anthocyanin analysis using high performance liquid" chromatography (hplc) showed very distinct anthocyanin pattern in highland banana compared to similar anthocyanin composition observed in lowland populations (asif et al. 200 ib). flava, a yellow bracted mutant, showed the absence of anthocyanin (simmonds 1955). during the current study, several of the observed abnormalities included the transformation of the free tepals and the different number of male flowers in alternate rows. different number of male flower rows were also varied in the same male bud of the sample bd2. in the case of sintok, two male flowers were fused together and had a total of 11 anthers instead of 10 (5 each). similarly, sample ri showed fusing of the fruits and degeneration of male bud and rachis. nair and karunakaran (1962) and jacob (1952) have reported similar abnormalities in cultivated bananas. cluster analysis grouped all m. acuminata accessions into four groups where north malaysian accessions were closely related with selangor group and highland banana sample was well separated. pca produced similar grouping of the accestsions and also showed that they were phonetically related. it thus gave a more accurate representation of how similar accessions were phenetically related to each other, although the overall picture was very similar to that of cluster analysis. the first group contained accessions flava, segun, sintok and j-4. they were collected from different geographical regions but showed large morphological similarities. most of the characters were observed to overlap with other lowland m. acuminata accessions (groups 2 & 3). this was similarly observed in the case of rangis and perak, which were collected from kelantan and perak. rangis, a typical kedah form (ssp. siamea), showed similarities with perak, a sample collected from perak (from geographical range of ssp. malaccensis). these characters were variable developmentally (hari 1968; karamura 1999). the second group comprised a large number of accessions mainly collected from the ssp. malaccensis range. they shared many morphological characters and were, as expected, closely clustered together. free tepal development and rachis position could be an interesting character for the identification of ppc and kra. the highland banana accession (bc3) growing at an altitude of 2000 m was clearly separated from the lowland accessions and showed great ecological and morphological variability from the lowland accessions. this accession was also found to be 34 morphological characterization of malaysian wild banana muhammad asif javed etal. different in growth habit, anthocyanin composition (asif et al. 200 ib) and genomic dna content (asif et al. 2001c) compared to lowland forms. it was suggested that the highland banana form ssp. truncata (simmonds 1955), could be separately classified based on morphology since the large morphological variability observed among lowland accessions made it difficult to group them into other distinctive groups. most of these accessions had overlapping morphological characteristics irrespective of their geographical relationship. the variation observed among different forms of musa acuminata could be related to their partial geographical isolation (highlands and lowlands) and changes in gene frequencies due to the small wild banana population sizes resulting from genetic drift and hybridization of different subspecific forms. mutations could also be an important factor contributing to the total variability. different subspecific forms of musa acuminata had been reported to cross frequently within the same geographical range. it had been repeatedly demonstrated that hybridization between species and subspecies belonging to the same section or between species in different sections occur readily in musa. some of these hybrids are fertile. two well-known examples include the m. balbisiana and m. textilis interspecific hybrids referred to as "canton" cultivar; and the other is m. textilis and m. balbisiana hybrid known as "pacol" (tezenas du montcel 1987). the observations based on cytological and field experiments (e.g. dodds & simmonds 1948) showed that several forms are interfertile, thus the whole assemblage formed a 'panmictic unit' (so far as geography permits). simmonds (1955) further suggested that field evidence of hybridity was most striking where populations of the ssp. malaccensis and ssp. truncata occur together. furthermore, the occurrence of mutations further increased the range of morphological variability among the populations (gonzales de leon & faure 1992). the fourteen m. acuminata populations studied consisted of a small number of individuals. small populations may not be stable because of dispersive processes which lead to the division of the populations into groups or sub-populations as observed in the case of wild bananas, where different populations observed in the same area appeared variable. secondly, it reduces the genetic variability and increases homozygosity. since in sub-populations mating between similar individuals takes place (i.e. inbreeding), genetic variability decreases within the groups, some genes are fixed and some eliminated, thus increasing homozygosity (wright 1951). this was observed in most of the wild banana populations where little variation was observed within the populations compared to among the populations and the variation observed was likely due to mutations appearing as a result of inbreeding. the development of different local populations of open pollinated plants is an example of the dispersive processes at work in natural populations. brieger (1950) found a number of local populations of maize in south america where populations were quite distinguishable among themselves but each population was highly uniform. 35 biotropia no. 18, 2002 m. acuminata produced a large amount of seeds as a result of open pollination. majority of the seeds observed had normal embryos, whereas few abnormal seeds showed the absence of embryo/endosperm or both. more than 90% embryos were germinated, thus could enable to study a large number of hybrid populations for banana breeding. bananas are cross pollinated species and it will be interesting to know the effect of inbreeding on seed bearing and germination. morphological characters studied although found useful at interspecific level was less so at intraspecific level. there were only a few morphological characters found to be useful to differentiate highland banana population from lowland, whereas there was no distinct pattern observed within lowland banana populations. therefore, it seems important that more sensitive markers be used to characterize these populations. genomic dna markers may offer the useful systems to increase our knowledge about wild banana diversity. acknowledgments the authors are grateful to the ministry of science and technology malaysia for financial support. thanks are also due to dr siti hawa jamaluddin, malaysian agriculture research development institute (mardi) for the supply of materials. references asif, m. j., mak, c. and r. y. othman 2001a. in vitro zygotic embryo culture of a wild musa acuminata ssp. malaccensis and factors affecting the germination and seedling growth. plant cell, tissue and organ culture 67 (3): 267-270. asif, m. j., c. mak, and r. y. othman. 2001b. characterization of malaysian wild bananas based on anthocyanins. biotropia 16:28-38 asif, m. j., c. mak, and r. y. othman. 2001c. characterization of indigenous musa species based on flow cytometric analysis of ploidy and nuclear dna content. caryologia 54 (2): 161 -168. brieger, f. j. 1950. the genetic basis of heterosis in maize. genetics, 35(4). buddenhagen, i.w., 1990. banana breeding and fusarium wilt. in: fusarium wilt of banana (ploetz, r.c. ed.). st. paul, mn, usa: aps press/amer. phytopathology soc., p.: 107-113. cheesman, e. e. 1947. the classification of bananas. kew bull., 2:97-117. dodds, k. s. and n. w. simmonds. 1948. sterility and parthenocarpy in diploid hybrids of musa. heredity, 2: 101-117. de langhe, e. 1961. la taxonomie du bananier plantain en afrique equatoriale. j. agric.trop. bot. appl., 8:417-449. engels, j. m. m. 1986. the identification of cacao cultivars. acta horticulturae, 182: 195-202. fao., 1999. fao 1999 yearbook, food and agriculture organization. rome. 36 morphological characterization of malaysian wild banana muhammad asif javed et al. hari, p. c. 1968. bract imbrication as a taxonomic character in musa acuminata. trop. agri. trinidad, vol. 45, no. 2:99-108. harlan, j. r. 1975. crops and man. american society of agronomy and crop science society of america, madison, wisconson. ibpgr. 1996. descriptors for banana (musa spp.). ibpgr, rome. gonzales de leon, d. and s. faure. 1992. genetic mapping of the banana diploid genome: toward an integrated approach to the study of the musa genome and the use of molecular marker technologies in musa breeding. in: proceeding of the workshop on biotechnology applications for banana and plantain improvement. san jose, costa rica. p. 29-51. jacob, k. c. 1952. madras bananas, a monograph. madras. karamura, d. a. 1999. numerical taxonomic studies of the east african highland banana (mum aaa east africa) in uganda. ph.d. thesis, department of agricultural botany, university of reading, january 1998. inibap. montpellier, france. kiew, r. 1987. notes on the natural history of the johor banana, musa gracilis holtum. malayan nature journal, 41:239-248. nair, n. r. and k. karunakaran. 1962. a note on the occurrence of staminoidy of petal in musa. indian. j. hort., 19(3&4): 176-177. norusis. m.j. 1985. spss, advanced statistics guide. new york: mcgraw hill. ortiz, r. 1993. ploidy manipulations for genetic analysis and improvement of plantain and banana (musa spp.). in: chromosome manipulations of higher plants (peloquin, s. j. and e. t. bingham. ed.). madison, usa, university of wisconsin. ortiz, r. and r. sevilla. 1997. quantitative descriptors for classification and characterization of highland peruvian maize. plant genetic resour. newslett., 110: 49-52. purseglove, j. w. 1972. tropical crops. monocotyledons. longman, london. shepherd, k. 1988. observations on musa taxonomy. in: identification of genetic diversity in the genus musa. proceedings of an international workshop held at los banos, philippines (jarret, r. l. ed.), 510 september 1988. p 158-165 shepherd, k. 1999. cytogenetics of the genus musa. international network for the improvement of banana and plantains, montpellier, france, p: 135-140. simmonds, n. w. 1955. wild bananas in malaya. malayan nature journal, 10: 1-8. simmonds, n. w. 1962. the evolution of the bananas. london, uk: longman. simmonds, n. w. 1986. classification and breeding of bananas. in: banana and plantains breeding strategies. (persley, g. j. and e. a. de langhe ed.). proceedings of an international workshop held at cairns, australia, 13-17 october, 1986. skutch, a. f. 1930. on the development and morphology of the leaf of the banana. (musa sapientum l.). amer. j. bot, 17:252-71. tezenas du montcel, h. 1987. plantain bananas. cta. paris and macmillan. london. vakili, n. g. 1965. fusarial wilt resistance in seedlings and mature plants of musa species. phytopathology, 55: 135-140. wright, s. 1951. the genetic structure of populations. ann. eugenetics, 15: 323-354. 37 blotropia no blotropia no. 11, 1998: 1 8 studies on natural products of albizia sp. hilman affandi, arifnuryadin seameo biotrop, p.o. box 116, bogor 16001, indonesia and roger w. read department of organic chemistry, the new south wales university, p.o. box 1 kensington, n.s.w. 2033, australia abstract the bark of albizia lebeckioides and albizia falcataria have been examined for their chemical constituents. a. lebeckioides has yielded the steroidal ketone, stigmasta-4,22-dien-3-one, and a triterpene alcohol as the major neutral components. a. falcataria has yielded a similar triterpene and fatty ester as its major constituents. the toxicity of the compounds was evaluated in bioassay against termites. the results showed that lupenone and stigmastadienone were toxic ioneotermes dalbergiae species. in contrast these compounds were less toxic to cryptotermes cynocephalus species. key words: insecticidal plants / albizia lebeckioides i albizia falcataria i bark / extracts / chemical constituents / toxicity / termites introduction some substances are produced by plants reasons for such as defence, growth regulation, hormonal action or as waste products. in teak (tectona grandis l.f), for example, tectoquinone is produced to protect these trees against termite attack (romanis 1887; oshima 1919; wolcott 1946). resin ofpinus possesses a number of biologically active natural products. a major constituent, a-pinene is well known for its antimicrobial and insecticidal properties (bridges 1987). among fast growing trees, albizia falcataria was reported to resist termite attack (kalshoven 1950; griffioen 1954). laboratory tests designed to study the resistance of albizia falcataria against dry wood termites (cryptotermes cynocephalus) revealed that the resistance of albizia falcataria lies somewhere between that of teak and hevea brasiliensis or even closer to teak (tarumingkeng and martawidjaja 1969). another species of albizia e.g. albizia lebeckioides was reported earlier to contain toxic alkaloids (greshoff 1914). these reports suggested that both species of albizia may contain useful chemicals that resist termite attack, therefore a search for biologically active 1 biotropia no. 11, 1998 compounds from the bark of albizia falcataria and albizia lebeckioides was undertaken. the objective of this study was to isolate and characterize the main chemical components and to determine their toxicity to termites. materials and methods general 'h nmr spectra were recorded at 500 mhz and 13c nmr spectra were measured at 125.6 mhz on a bruker am 500 spectrometer. mass spectra were recorded at 70 ev with an a.e.i. ms 12 spectrometer. all measurements were performed at the school of chemistry, the university of new south wales, australia. initial column chromatographic separations were made on merck 60 silica gel. merck kieselgel 60 f254 was employed for preparative tlc with a 1 mm layer of adsorbent on 20 x 20 cm glass plates and 200 mg of material applied to each plate. analytical tlc was performed on commercial aluminium-backed merck kieselgel 60 f254 art. 5554 plates, visualizing under uv 254 nm light or charring after applying a 4% h2so4/meoh spray. plant material samples of bark of albizia falcataria were collected from kesatuan pemangkuan hutan (kph), kediri while the bark of a. lebeckioides was taken from batutulis, bogor. extraction and isolation dried powdered bark of albizia lebeckioides (1.50 kg) was extracted with methanol to give crude extract (78.15 g). the crude extract was then re-extracted with ether to generate an ether soluble fraction (27.77 g) and an insoluble residue (50.37 g). the ether soluble fractions were carefully fractionated by column chromatography (silica gel 60) eluting with hexane containing increasing amounts of ethyl acetate to give 6 fractions. fraction 4 (1.12 g) and fraction 5 (0.98 g), as two major fractions, were subjected to purification by preparative thin-layer chromatography (ethyl acetate hexane, 1 : 9). fraction 4 gave a solid material 2 studies on natural products ofalbizia sp.hilman affandi et al. (0.85g) which crystallized from chloroform to give stigmasta-4,22-dien-3-one (2) (0.67 g) m.p. 181-184°. fraction 5 generated a crystalline solid (0.76 g). recrystallization of the latter gave lupeol (1) (0.51 g) m.p. 198-199°. the bark of a. falcataria (1.50 kg) was treated in a similar manner to the method above. re-extraction of the crude extract (65.83 g) with ether gave an ether soluble fraction (16.20 g) and a residue (49.62 g). fractionation of the ether soluble portion by column chromatography eluting with hexane generated 6 fractions. purification of the fastest moving fraction (1.28 g) by preparative thin-layer chromatography (ethyl acetate-hexane, 1:9) gave a solid material (0.93 g) which crystallized from chlorform to give fatty ester (4) (0.82 g). fraction 2 (1.04 g), upon thin layer chromatography (ethyl acetate-hexane, 1:9) gave a crystalline solid (0.78 g). recrystallization of the solid from chloroform gave lupenone (3) (0.68 g) m.p. 160-161°. termite bioassays the toxicity of the pure constituents was evaluated by bioassay using the termite neotermes dalbergiae, obtained from commercial sources in bogor, and cryptotermes cynocephalus, collected from the forest product research institute, bogor. pure compounds (25 mg) were diluted with dichloromethane (0.5 ml). the solutions were applied to 4.5 cm whatman no. 1 filter paper dish. after the solvent had been evaporated at ambient temperature, the filter papers were placed in 5 cm glass petri dishes, and 25 termites added to each container. control chambers were treated in a similar manner using pure chloroform in place of solutions of the test subtances. each pure compound was tested in four replicate experiments. at daily intervals the number of termites surviving was recorded, dead termites were removed, and the filter papers were moistened with distilled water. dishes were kept covered in a darkened room at ambient temperature. results and discussion structural analysis methanolic extracts of the bark of a. lebeckioides and a. falcataria were reextracted with ether. the ether soluble extracts were carefully fractionated by column chromatography (silica gel) and purificed by preparative thin-layer chromatography 3 biotropia no. 11, 1998 to yield lupeol (1) and stigmata-4, 22-dien-3-one (2) from die bark of a. lebeckioides, and lupenone (3) and fatty ester (4) from the bark ofa.falcataria. the structure of compound (1) was established from its microanalytical and spectroscopic data. elemental analysis and mass spectrometry indicated the molecular formula c30h50o. the 'h n.m.r. data showed six singlet resonances at 5 0.82, 0.89, 0.93, 0.98, 1.02 and 1.07, arising from six methyl groups attached to quaternary carbons. there also appeared one isolated singlet at 5 1.68 due to a vinylogous methyl group, and since the olefmic proton signals at 8 4.56 (dd, j 2.2, 1 4 hz) and 4.68 (d j 2.2 hz) were consistent with a 1,1-disubstituted alkene, a propenyl group was likely. low field signals resonated at 5 3.18 (dd, j 10,.9, 5.3 hz), 2.38 (dt, j 5.7, 11.1 hz) and 1.91 (m), but there were no other olefmic resonances. the n.m.r. signal at 8 3.18 was suggestive of an axial proton adjacent to a quarternary centre and attached to a carbon bearing an hydroxyl group. the i3c n.m.r. spectrum (table 1) -supported the structural assignment of (1). in particular there appeared seven methyl signals, only two olefmic carbon signals (8 109.3 (ch2) and 151.0 (c), and a low field methine resonance (8 78.9) for c3. the identity of compound (1) as lupeol was confirmed by melting point and mixed melting point with an authentic sample. lupeol has been found in many plant extracts, including the resin of pistacia leuticus (marner et al. 1991). compound (3) was obviously closely related to lupeol (1). its mass spectrum gave a molecular ion at m/z 424, corresponding to two fewer hydrogens than lupeol. the 'h n.m.r. spectrum (table 2) of compound (3) resembled that of (1), including the appearance of coupled olefmic signals at 8 4.57 and 4.70, and a deshielded methyl signal due to h3 in lupeol (1) was absent. instead, there appeared additional signals at 8 2.4, corresponding to protons adjacent to a multiple bond. the compound was thereby identified as lupenone (3) and the structure was confirmed by mixed melting point with an authentic sample. compound (2) differed from compounds (1) and (3). elemental analysis and mass spectrometry indicated through its molecular formula, c29h46o, that it was a nortriterpene. there appeared in its 'h n.m.r. spectrum two methyl singlets, 8 0.57, 1.01, three methyl doublets, 8 0.79, 0.84, 1.03, and a methyl triplet, 8 0.80. also there appeared a three proton multiplet corresponding to two internally coupled, trans olefmic protons (8 5 03, dd, j 15.1, 8.4 hz, and 5 15 (dd, j 15.1, 8.4 hz) and an isolated olefmic proton (8 5.18, m) with long range coupling, but no other signals at higher chemical shift than 2.5 ppm. the presence of a 1,2-disubstituted and a 1,1,2-trisubstituted olefin was supported by the presence of signals at 5 117.0 (ch), 129.6 4 studies on natural products ofalbizia sp.hitman affandi el al. (ch), 138.1 (ch) and 139.5 (c) in the 13c n.m.r. spectrum. the presence of a quaternary signal at 8 212.0 also confirmed the occurrence of a carbonyl group in the compound. structure (2) was assigned from these data and was confirmed by mass spectrometric fragments at m/z 367 (m-c3h7), 298 (m-c8hi6), 271 (m-cioh,9), 269 (mci0h2i), and comparative melting point. figure 1. structures of chemical constituents isolated fro falcataria (3) table 1. 13c n.m.r. chemical shift data of compounds (l)-( c 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 39.3 34.3 55.3 47.0 59.3 19.4 33.0 39.9 50.5 37.1 21.8 26.4 39.7 41.2 26.5 30.1 44.7 37.6 52.5 150.6 30.2 23.5 2. stigmasta 4,22-dien-3 3. lupenone m the bark of a lebeckioides (1 and 2) and a. 3) (ppm in cdclj) 2 3 37.3 31.6 212.2 71.8 42.3 121.1 33.9 31.7 50.2 36.5 20.9 38.6 42.2 56.6 24.1 28.2 56.1 11.6 19.4 40.8 21.5 138.8 39.7 34.1 218.4 47.3 54.9 19.6 33.0 40.3 50.6 36.9 21.9 26.7 39.5 41.0 26.4 30.1 44.9 37.7 52.5 150.8 30.3 23.2 5 biotropia no. 11. 1998 table 1. continued c 1 2 3 23 24 25 26 27 28 29 30 26.8 20.9 16.2 15.4 14.3 151.0 101.0 20.6 129.5 51.4 32.2 19.2 21.3 25.7 12.5 26.8 20.9 16.3 15.4 14.2 151.1 101.8 20.7 table 2. 1h n. m.r spectral data of compound 1-3 (ppm in cdc13) h 1 2 3 h-2 h-3 h-6 h-18 h-19 h-22 h-23 h-24 h-25 h-26 h-27 h-28 h-29 2.37 – 2.55 m 3.85 m 1.2 – 2.0 m 1.07 s 1.02 s 0.93 s 0.98 s 0.89 s 4.56 dd, 4.68 d 5.45 d 0.57s 1.01 1 5.15 dd 5.03 dd 0.79 d 0.84 d -1.50m 0.80 1 2.36 2.60 m 1.5 -2.0m 1.07s 1.19s 0.94s 0.97s 0.89s 4.57 dd, 4.70 d compound (4) could not be obtained pure and its structure could not be fully elucidated. from its 'h n.m.r. spectrum the substance was not terpene derived but was probably a fatty ester. there appeared in the low field region of the spectrum two triplets of equal intensity corresponding to methylene protons adjacent to oxygen (8 4.05) and a carbonyl group (8 2.28), respectively. in addition, there was a triplet resonance at 8 0.88 that could be assigned to coincident signals from the methyl groups at the ends of aliphatic chains. the remaining signals comprised two narrow multiplets at 8 1.25 and 8 1.57 corresponding to groups of similar methylene protons. the mass spectrum gave significant ions at m/z 425 and 205 that could not be assigned to any sensible structure or fragments. toxicity of compounds (l)-(3) a laboratory bioassay was conducted to assess the toxicity of pure compounds (l)(3), the tentatively assigned fatty ester (4), and the remaining extracts, to termites. the results showed that 53%, 48%, 42%, 54%, and 64%, respectively, of neotermes 6 studies on natural products ofalbizia sp.hitman aff'andi et al. dalbergiae termites that were fed these substances remained alive after the fourth day of the experiment (diagram 1). while termites in control groups moved actively when the dish was uncovered for counting, temites in treatment groups did not. this observation may suggest that termites receiving chemical treatment responded in a general rather than specific manner to pure compounds. specific responses such as death caused by direct application onto the termites were not observed. in addition the total termite mortality was not as high as expected from related studies using cryptotermes cynocephalus termites (diagram 2). this may have been due to insufficient doses of the pure compounds rather than low activity. comparison of the relative toxicity of the compounds to neotermes dalbergiae showed that lupenone and stigmastadienone were the most toxic compounds (diagram i). in contrast lupenone and stigmastadienone were less toxic against the cryptotermes cynocephalus species. this might be due to the fact that neotermes dalbergiae, a drywood termite, does not attack living trees as does the cryptotermes cynocephalus. therefore, the former are more susceptible to offensive chemicals, because they have not developed resistance mechanisms (messer 1990). % t er m in re m ai ni ng diagram 1. toxicity of the compounds against neotermes dalbergiae 7 biotropia no. 11, 1998 % t er m in re m ai ni ng diagram 2 toxicity of the compounds againts cryptotermes cynocephalus conclusion this study has shown that lupenone and stigmastadicnone, contained in the bark of albizia falcataria and albizia lebeckioides respectively, have biological activity against the drywood termite neotermes dalbergiae and that in function they may play a defensive role. toxic alkaloids, previously reported to be present, (heyne 1984) have not been found in the bark of albizia falcataria. references apsimon, j. 1973. the total synthesis of natural products, vol 2, john wiley & sons, london. bridges, j.r. 1987. effect of terpenoid compounds on growth of symbiotic fungi associated with southern pine beetle. phytopathology, p. 77. fffiser, l.f. and m. fffiser. 1959. steroids, van nostrand reinhold company, london. griffioen, k. 1964. albizia falcataria sen goede industrie houtsoort. tectona 63: 97-100. greshoff, m. 1914. indische vergiftrapporten, no. 3 in heyne, k. 1987. tumbuhan berguna indonesia, ruygrok & co., jakarta, p. 1021-1040. kalshoven, l.c.b. 1950. de plagen van de cultuurgewassen in indonesia. deel i: 145-150. marker, f.j., freyer, a., lex, j. 1991. interpenoid from gum mastic, the resin of pistacia leuticiis. phytochemistry, 30 (11). messer, a.c. 1990. chemical ecology in an indonesian context, ph.d. thesis, cornell university. oshima, m. 1919. formesan termites and method of preventing their damage. philip. sul. 15: 319-386. romanis, r. 1887. certain products from teak. chem. soc. 54: 868. tarumingkeng, r.c. and a. martawidjaja. 1969. pengujian kayu djeundjing (albizia falcataria backer) terhadap rayap kayu kering (cryptotermes cynocephalus light) secara laboratories. rimba indonesia, xiv (1-2) wolcott, g.n. 1946. what to do about pollila? univ. puerto rico, agric. exp. sta., bull. no. 68. 8 microsoft word 1 biotropia no. 22, 2004: 1-14 morphological variation of the ecotypes of echinochloa crus-galli var crus-galli (l). beauv (barnyard grass: poaceae) in malaysia and indonesia arifin tasrif' . abdul shukor juraimi*)', jugah kadir', suhaimi napis2 and soet1kno s. sastroutomo3 1 faculty of agriculture, univerxiti putra malaysia, 43400 serdang,selangor, malaysia 2faculty of food science and biotechnology, univerinti putra malaysia, 43400 serdang, selangor, malaysia 3cab international, se asia regional centre, 43400 serdang, selangor, malaysia abstract greenhouse experiments were conducted to examine the morphological traits of barnyard grass ecotypes from diverse geographic origin. seeds (caryopsis) were collected from 17 locations of rice fields throughout malaysia (11 states) and indonesia (six provinces) and were grown in pots each containing 10 kg of paddy field soil. the experiments were arranged using completely randomized design (crd) with five replicates. mean separation was calculated using duncan multiple range test at 5% probability level. unweighted pair-group method of arithmetic averages (upgma) was performed to determine the individual relationship within ecotypes of barnyard grass. twelve morphological traits such as culm, panicle, leaf, and spikelet traits were measured. the growth characters such as emergence date, heading time, and growth duration were also evaluated. the average of emergence date, heading time, and growth duration of barnyard grass collected from perils, kedah, penang, and johor were relatively earlier than other ecotypes. six groups were classified based on the cluster analysis of malaysian ecotypes of barnyard grass. principal component indicated that group six was found to be highly variable compared to others. while three groups were identified in indonesian ecotypes of barnyard grass. group one was observed to be highly variable. results demonstrated that morphological variation among ecotypes of barnyard grass showing differences between the two regions illustrate the role of geographic variation. key words : variation / ecotypes / paddy field weeds / barnyard grass. introduction the barnyard grass (echinochloa crus-galli var crus-galli l. beauv.) is an annual weed native to asia and presently can be found throughout the world (holm et at. 1977). the most widespread and economically important member of the genus is the annual e. crus-galli, which can be found within 50°n latitude and latitude 40°s. (holm at al. 1977; maun and barret 1985). barnyard grass is a noxious weed in rice field and have been shown to reduce rice yield by 40% in direct seeded rice in malaysia (azmi 1988 ) and about 21 % annually in indonesia (tjitrosemito 1994). *) correspondence to: abdul shukor juraimi, department of crop science, faculty of agriculture, universiti putra malaysia, 43400 serdang, selangor de, malaysia. e-mail a_shukor@ agric.upm.edu.my biotropia no. 22, 2004 this weed belongs to the tribe paniceae, family gramineae. the genus may include 20 to 50 species that are widely represented in tropical and warm temperate regions of the world (clayton and ronvoize 1986; martines et al. 1999; michael 1983). with an increase in direct seeding rice practices, barnyard grass is becoming a greater problem in rice fields (azmi and baki 2002; loux and barry 1991 ). in addition, continuous chemical control of barnyard grass in rice plant has caused herbicide resistance to barnyard grass (martines et al. 1999; rutledge et al. 2000). klingaman and oliver (1996) defined ecotypes as plants genetically adapted to the habitat they colonized and biotypes as plants showing a random genetic variant within an ecotype. individual plants of barnyard grass can produce thousands of seeds, but seed germination declines rapidly over time, and sometimes they have long dormancy (martinkova 1989). barnyard grass typically grows 1 to 1.5 m tall and is capable of producing a large number of seeds (azmi and baki 1995; itoh 1991). in certain conditions, it may grow up to 2 m tall (anonim 2001). in addition, the plant can grow in diverse environment (yabuno 1983; yamasue 1997), and its growth habit is strongly affected by environmental conditions. studies have shown that soil type, fertility level, and cultural regimes affect barnyard grass of some morphological traits. the production of a large number of easily dispersed seeds and the ability to flower under a wide range of photo periods contribute to the success of this weed (holm et al. 1977). phenotypic and genetic variation among barnyard grass ecotypes would be divergent resulting from the selection pressure imposed by agricultural practices, crop characteristic, geographic origin, and herbicide application. studies have shown that soil type and fertility level affect morphological characters of barnyard grass in rice field (martines et al. 1999; yamasue 1997). recent evidence has shown that substantial variation exists among seed size within plant ecotypes (marshal et al. 1986) and between geographical regions (ransom et al. 1998; sterling et al. 2000). phenotypic variability has been observed in numerous weed species, comprising half of the species reported as having biotypes or ecotypes. weeds with biotypes differing in growth and morphological characteristics include canada thistle (circium arvense l. scop) (hodgson 1964), field bindweed (convolvulus arvensis l.) (degennaro and weller 1984, quack grass (eltrygia repens l.) (westra and wyse 1981), johnson grass (sorghum helepense l.) (mcwhorter and jordan 1976), yellow nutsedge (cyperus esculentus l.) (holt 1994), leafy spurge (euphorbia esula l.) (harvey et al. 1988), and hemp dogbane (apocynum cannabinum l.) (ransom et al. 1998). differences between weed population can influence the competitive nature of weed species and may affect response to chemical or cultural control strategies. most biotype studies previously reported have been conducted under different environment. it is very important to study variation in morphological characters among ecotypes to determine how plant genotype and diverse environmental conditions could influence the plant morphology of barnyard grass under uniform conditions. thus, the objective of this morphological variation of echinochloa crus-gaili var. crus-galli l. beauv. ecotypes arifin tasrif et al. study is to determine if barnyard grass seeds collected from geographically divergent ecotypes exhibit morphological variation when grown in uniform environmental glass house conditions. materials and methods from december 2000 to march 2001, barnyard grass seeds were collected from 11 rice field locations across peninsular malaysia (100 119° e and 7°n) and 6 locations across indonesia (94 141° band 6° 8' 11° 5' s) (figure 1, table 1). at each location, seeds from a single plant were randomly collected from different sites in1 rice growing areas, so that the collected seeds would be genetically identical. seed collection biotropia no. 22, 2004 the seeds were chosen to represent the populations from which they were collected. since seeds were collected from geographically divergent locations, they can be described as ecotypes (klingaman and oliver 1996). at each location, five sampling sites were randomly selected. from each of the five sampling sites, one individual ecotype per site, but more than one ecotype were collected when morphologically different types were present at each site using the nearest-neighbor techniques (solbrig 1960) and the distance between sampling site of each location was more than 10 km (barret 1982). propagation of the ecotypes a total of 85 ecotypes of e. cruss-galli collected from the 17 locations were grown under uniform glass house conditions. before planting, seeds were soaked in tap water for 24 hours at room temperature. seeds were grown in each pot size containing 10 kg of paddy soil. basal fertilizer (0.4 g n/pot, 0.4 g p/pot, and 0.2g k/pot or equal to 40 kg n/ha, 45 kg p/ha, and 30 kg k/ha) were applied directly in each pot, while one third of nitrogen were applied at five to six weeks after seedling emergence (pane and mansor 1997). two weeks after emergence, seedlings were thinned to one plant per pot, leaving the tallest emergence individual in all replications. the pots were flooded shortly after thinning and five cm of water level of the soil surface were maintained throughout the study. environmental glasshouse morphological variation of echinochloa crus-galli var. crus-galli l. beauv. ecotypes arifin tasrif et al. conditions such as rh, temperature, and light intensity (li-cor model 185-b) were recorded and soil chemical contents were analyzed (page 1982). morphological trait assessments traits such as plant type, plant height (cm), flag leaf length (cm) and width (cm), culm diameter (cm), panicle length (cm), empty glume length (mm), spikelet length (mm), spikelet width (mm), spikelet weight (fresh weight of 100 seeds in gram), panicle awn, and number of tiller were assessed at the maturity stages. plant type (the degree of opening at the first node) and panicle awn were considered as qualitative characters visually rated on a scale from none (0), moderate (1) to severe (2) (park et al. 1995; yamasue 1997). in addition, growth characters such as emergence date, heading time and growth duration were also evaluated. specimen identification and statistical analysis specimen identification was carried out following the classification prepared by yabuno (1983), michael (1983), itoh (1991), and park et al. (1995) and compared with the specimens of the faculty of agriculture's herbarium of university putra malaysia. morphological descriptors were subjected to cluster analysis using euclidean distance coefficient of upgma analysis and associated dendrogram using ntsys-pc package version 2.1 (rohlf 2000). principal component analysis (pca) for step-wise observations among the groups of barnyard grass ecotypes was also analyzed. square root transformation was used to transform the raw data before analysis. the shan clustering program of the ntsys-pc was used to group the ecotype on the bases of those matrices. anova was used to determine significant difference in growth character of each location of barnyard grass ecotypes with five replications. means were separated using duncan's multiple range test (dmrt) at the 5% probability level using sas version 6.0 (sas 1996). results and discussion soil analysis and environmental conditions of the glasshouse a paddy field soil was taken from mardi research station, tanjong karang, selangor. the soil physical and chemical properties were as follows (sand 2%, clay 72%, dust 28%), c 3.13%, n 0.35%, ph 4.8, cec (mg/loog 29.74), c/n ratio 10 ca (bpj/loog = 8.37), and mg (bpj/loog =1.94). relative humidity of the glasshouse during experimental period ranged from 68% to 90%, and temperature ranged from 24°c to 34°c, while light intensity ranged from 660 to 930 um m^sec. these conditions were conducive for growth of barnyard grass. biotropia no. 22, 2004 variability in growth characteristics among barnyard grass ecotypes variability in growth characteristics among barnyard grass ecotypes is presented in table 2. the range of emergence was from 2.8 days to 7.2 days, that of heading time was from 42 to 56 days. while, the growth duration ranged from 89 days to 98.2 days. for emergence date, perlis and kedah ecotypes were the earliest (2.8 days to 3.0 days) and significantly different (p<0.05) from the rest of the ecotypes. the heading time (42 days and 44 days) was significantly earliest for perlis, kedah, penang, perak, kelantan and johor ecotypes. south sulawesi was the latest besides having the longest growth period of 98.2 days. while perlis, kedah, penang, perak, kelantan and johor growth were the shortest and significantly different from the rest of the ecotypes. results of the growth characteristics among barnyard grass ecotypes collected from malaysian or indonesian rice growing areas showed distinct variability. growth characteristics of the ecotypes such as heading time (42 days to 56 days) and growth duration (89 days to 98 days) were slightly similar to the growth duration of barnyard grass ( 44 dae to 45 dae and 90 dae to 95 dae) as reported by azmi et al. (1997) and itoh (1991). however, the values were slightly lower than the growth duration of barnyard grass in arkansas ( barret and wilson 1983). morphological variation of echinochloa crux-galli var. crux-galli l. beauv. ecotypes arifin tasrif el al. in general, growth characteristics variability among barnyard grass ecotypes might be affected by geographic origin. this observation was in line with the work of johnson et al. (1989) who reported that even though ecotype of some sweetvetch (hedysarum boreale nutt.) differed in their taxonomic distance values, this difference is not always closely related to specific characteristics of the collection sites. widespread species tend to have more genetic variability than close relation with narrow distribution (hamrick and godt 1990; karron 1987; maki et al. 1999). pylogenetic relationship within individual barnyard grass ecotypes within malaysian ecotypes dendrogram using upgma clustering methods from morphological traits is presented in figure 2. six groups were identified at the taxonomic distance of 0.016. the first group comprised 21 ecotypes mainly from perils, kedah, and johor. the 3rd and 4th groups comprised 14 ecotypes, respectively. groups 2, 5 and 6 consisted of 2, 3 and 1 ecotypes, respectively. two individualecotypes belonged to group 2. the only ecotype from selangor (s-02) was found in group 6. ecotypes from perlis, kedah, perak, and johor were found to be linked together and have closer relation widi each other. ecotypes from perlis and johor fell within group 1, while those of kelantan and negeri sembilan mostly belonged to group 3. penang, pahang, and selangor were dominant in group 4. principal component analysis of barnyard grass was applied to differentiate variability among the existing groups. the proportion of the first and second components were 88.76% and 7.01%, respectively (table 3). the first component was largely contributed by the size of characters such as plant height, plant type, and number of tillers. the second component was due to the shape and size characters of panicle awn, flag leaf width and spikelet length. scatter diagram between the first and second component showed that the individual plants present in different groups had characters different from the others (figure 4.a). most of them were separately distributed in the quadrants of the diagram. on the contrary, individual barnyard grass in group 6 were farther apart than the other groups, indicating that they vary largely. within indonesian ecotypes at the taxonomic distance of 0.08, three groups were classified from six locations representing three different islands. group 1 consisted of 19 ecotypes representing five locations ( two islands), except for ecotype from lampung. eight ecotypes were obtained in group 2, except for lampung ecotype. in contrast, only three south sulawesi ecotypes (ss-1, ss-2, ss-4) were found in group 3 (figure 3).   morphological variation ofechinochloa crus-galli var. crus-galli l. beauv. ecotypes arifin tasrif et al. principal component analysis among groups of barnyard grass is presented in table 3. the proportion of the first and second components were 95.22% and 61.22%, respectively. the first component was largely due to shape and size of characters such as plant height, panicle awn, and panicle length, respectively. the second component was mainly due to plant height and number of tillers. scatter diagram between the first and second components showed that plants in different groups had characters different from the others (figure 4.b). most of them were distributed in the first two components. however, ecotypes belonging to group 1 were farther distributed than other groups, indicating that group 1 had wide variation. the results of this study indicated that individual ecotype of malaysian and indonesian barnyard grass were found to be more variable (tables 2 and 3; figures 2 and 3). in general, ecotype of barnyard grass from malaysia and indonesia are separated by larger geographical distance and is expected to be different than those from the same locations. yamasue (1997) reported that variation in morphological characters were significantly different among barnyard grass collected from different habitats, even though they are from the same locality. on the contrary, weed population which are continuously associated with specific agricultural systems may evolve phonological patterns which optimize survival within the most favorable growing areas (barret 1983). the results demonstrated that genetic differentiations among ecotypes were apparently an important factor of the variation in weed management practices. the differentiation among local ecotypes was probably encouraged by self-pollinating reproduction of the barnyard grass ecotypes (honek biotropia no. 22, 2004 and martinkova 1996). on the contrary, phenotypic variations in barnyard grass ecotypes from indonesia and malaysia affected seedling emergence and growth characteristics. interestingly, south sulawesi ecotypes seemed to be specific in different habitats of different islands (figure 3). this finding is to be similar with the work of maki et al. (1999) who found that highly phenotypic and genetic differentiation of aster miyagii (asteraceae) existed among islands in japan, or high levels of genetic diversity among island populations of suzukia luchunsis (labiatae) in japan (maki et al. 2003). morphological variation of echinochola crus –galli .var.crus-galli l beauv . ecotypes – arifin tasrif et al. biotropia no. 22, 2004 morphological variations among barnyard grass ecotypes in malaysia and indonesia are not very high at present. if newer strains (biotypes) of the barnyard grass migrate from one to other regions and cross-pollinate, the genetic diversity may increase, and the control of barnyard grass in rice production may become more difficult. both genetic and environment play a role in the expression of barnyard grass phenotype. however, barnyard grass genotype maintained their general populations when grown in common site. interestingly, when barnyard grass population was grown at the uniform sites (glasshouse conditions) ecotypes from the most malaysian regions, maintained the highest plant size at short periods to reproductive maturity compared to barnyard grass from indonesian regions. the variability could be affected by local environmental heterogeneity, geographic distance and possibly agricultural practices. this finding is of importance in trying to explain the ecotype distribution among barnyard grass ecotypes from malaysian and indonesian rice fields. these results suggest that barnyard grass ecotypes have formed, and the ecotypes have adapted to specific geographic locations. the ecotype variations will affect successful management of barnyard grass using chemical or potential biocontrol agent. however, molecular traits analysis would be useful to characterize the level of genetic variability among barnyard grass ecotypes. acknowledgement acknowledgement is accorded to the integrated pest management for smallholder estate crop project (ipm-shecp) plant quarantine component and for the contribution of yayasan felda malaysia for a research studentship to arifin tasrif. we are also grateful to dr. osman osumanu, for his valuable suggestions and discussions during the preparation of this article. references anonimous .2001. echonichloa l. beauv. http:file/a:/echinochloa spp. htm azmi m. 1988. weed competitions in rice production. proc. of the national seminar and workshop on rice field management (eds. chang et al.) 141-152. azmi m. and b.b. baki. 1995. the succession of noxious weeds in tropical asian rice field with emphasis on malaysian rice ecosystem. proc. 15* apwsc.tsukuba. japan. july, 24-28, 51-67. azmi m. 1997. weed population in direct-seeded rice as affected by seeding rates. proc. 16* asian-pacific weed science society conference (rajan, eds). mpps. kuala lumpur, 251-256. azmi m. and b.b. baki. 2002. impact of continuous direct seeding rice culture on weed species diversity in the malaysian rice ecosystems. reg. symp. on env. and nat. res. 10-11 april 2002. kuala lumpur. malaysia. 11 p. barret s.c.h. 1982. genetic variation in weeds. in: biological control of weeds with plant pathogens (charudattan and walker eds). john wiley and son. 73 -98. 12 morphological variation of echinochloa crux-galli var. crus-galli l. beauv. ecotypes arifui tasrif et at. barret s.c.h. and b.f. wilson. 1983. colonizing ability in the echinochloa crusgalli complex (barnyard grass).e. seed biology. can. j. bot. 61: 556-562. barret s.c.h. 1983. crop mimicry in weeds. economic bot. 37(3): 225-282. brod g. 1968. untersuchgen zen biologic unal okologie der huknen-hirse (echinochloa crus-galli). weed res. 8: 115127. clayton w.d. and s.a. renvoize . 1986. genera grarninium. grasses of the world. p 280-281 in t.a. cope, ed. london: royal botanic garden, kew. degennaro p.p. and s.c. weller . 1984. growth and reproductive characteristics of field bindweed (convolvulus arvensis) biotype. weed sci. 32: 525528. hamrick j.l. and m.j. godt . 1990. allozyme diversity in plant species. in a.h.d. brown m.t. clegg a.l. kohler and weir b.s. 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(sas) statistical analysis sistems. 1996. sas procedures guide version 6.0, 3' rd cory, nc: statistical analysis system institute. 725 p. solbrig o.t. 1960, cytotaxonomic and evolutionary studies in die north americans species of gutierresia (compositae). contribution gray herbaceous 188: 1-61. sterling t.m., murray l.w. and y. hou 2000. morphological variation among gutierrezia sarothrae populations. weed sci. 48: 356-365. sung s.j., leather g.r. and m. hale 1987. development and germination of barnyard grass (echinochloa crus-galli) seeds. weed res. 35: 211-215. tjitrosemito s. 1994. integrated management of paddy and aquatic weeds in indonesia : current status and prospect for improvement. in: integrated management of paddy and aquatic weeds in asia. food fertilizer tcaspac.roc. taiwan. 20-31. yabuno t. 1983. biology of echinochloa species. in: weed control in rice. irri. 308-317. yamasue y. 1997. biometric analysis of echinochloa weed complex. proc. of 16lh asian-pacific weed science society conference. (ed: rajan) mpps. kuala lumpur. 233-237. westra p.h. and d.l. wyse 1981. growth and development of quack-grass (agropyron repenx) biotypes. weed sci. 29: 4452. 14 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf 10.pdf 11.pdf 12.pdf 13.pdf 14.pdf biotropia no biotropia no. 15, 2000 : 26 35 presence of hema-like and hemt-llke genes in a jsumber of anoxygenic photosynthetic bacterial isolates from indonesia and soil samples from bogor area nurul aini'and antonius suwanto' 2 -1 'laboratory of microbiology and biochemistry, inter university centre for biotechnology, bogor agricultural university, bogor-16680 indonesia. department of biology, faculty of science and mathematics, bogor agricultural university, bogor 16144, indonesia. 'south-east asian regional center for tropical biology, bogor-16001, indonesia. abstract the rhodobacter sphaeroides hema and hemt are known to encode a distinct 5-aminolevulinic acid (ala)-synthase isozyme. this enzyme catalyzes the first and rate limiting step in ala biosynthesis through the c4 pathway. this study was carried out to detect hema-\\ke and hemt-\\ke genes in twenty anoxygenic photosynthetic bacterial (apb) isolates from several wetland areas in indonesia, and four dna samples that were isolated from four soil samples obtained from bogor area. hybridization techniques of southern and dot blot were used, using hema and hemt fragment as probes. southern hybridization analyses indicated the presence of hema-\\ke gene in five of apb isolates, i.e., mb15, mb16, mb21.2, mb55 and mb6, whereas hemt-\\ke gene was detected only in mb15. dot blot hybridization analyses suggested that the soil samples from waterlogged paddy-field, dry paddy-field as well as a mud pond were predominantly occupied by prokaryotic organisms which harboured hema-]\ke gene. however, /iem7"-like sequences were also found in soil sample from dry paddy-field. key words: hema-\\ks gene / hemt-\\ke gene / southern hybridization analysis / dot blot hybridization analysis. introduction the rhodobacter sphaeroides hema and hemt genes encode a distinct 5aminolevulinic acid (ala) synthase isozyme (neidle & kaplan 1993). alasynthase catalyzes the first and rate-limiting step in ala biosynthesis through the c4 pathway. ala is the first committed precursor in the common tetrapyrrole pathway (goodwin & mercer 1986; beale & weinstein 1991; beale 1995). recently, ala has received attention as a new biodegradable herbicide (sasaki et al. 1987) and insecticide (sasaki et al. 1990). the dna sequences of hema and hemt genes and their location on r. sphaeroides physical map have been determined. the hema gene is located on the large chromosome whereas hemt gene is found on the small chromosome (neidle & kaplan 1993a). the hema and hemt genes encode peptides that are 53% similar to each other, and these peptides are also significantly similar to ala-synthase from several bacteria and eucaryotic species (neidle & kaplan 1993). the hema fragment has been cloned in r. sphaeroides (tai et al. 1988) as well as in escherichia 26 biotropia no. 15, 2000 coli (werf & zeikus 1996). the cloned hema fragment is expressed well in e. coli and able to enhance ala production (werf & zeikus 1996). this evidence shows that hema can be used as a genetic material for enhancing ala production. since hema and hemt genes have high homology to gene encoding alasynthase from other organisms, hema-v\ke and hemt-v\ke genes might be found in other bacteria that form ala through the c4 pathway. in this study, we detected the presence of hema-\\ke and hemt-\ike genes in anoxygenic photosynthetic bacterial (apb) isolates, because most member of apb use the c4 pathway to produce ala. soil, especially paddy-field soil, is known as a common habitat of apb (habte & alexander 1980: gest et al. 1985). therefore, we also carried out an experiment to detect these genes in four soil samples. the results of this study would be expected to generate some insights on the distribution and population density of apb as well as r, sphaeroides strains. in addition, the specificity of hemt would be assessed to be used as a specific molecular marker for r. sphaeroides isolates. materials and methods bacterial strains, plasmids, growth conditions and soil samples the bacterial strains and plasmids used are listed in table 1. apb isolates were grown photoheterotrophically in sistrom's minimal medium (lueking et al. 1978) in full filled screw-cap tubes, ph 7.2. e. coli were grown at 37°c in luria bertani (lb) table 1. bacterial strains and plasmids 27 presence of hema-like and hemt-like genes – nurul aini & antonius suwanto table 1. continued (sambrook et al. 1989) supplemented as needed with antibiotics. antibiotics were added at the following concentrations: 100|ag ampicillin/ml (for maintaining puc19, pui1004, pui1014, and pui612), 25|ig chloramphenicol/ml (for maintaining phfl.l). soil samples were taken from waterlogged paddy-field, dry paddy-field, lsi pond and grawida yard, bogor agricultural university, darmaga campus. all sampling areas were located at darmaga, bogor. the description of the soil samples are listed in table 2. table 2. soil samples dna isolation plasmids dna were isolated by using wizard miniprep dna purification system (promega, wise.) according to the manufacturer's instruction. genomic dna was extracted from each apb isolate using the phenol extraction method with slight modification as follows. the cell pellet was suspended in edta solution containing 15 mg lysozyme/ml, and incubated at 37°c for 1 hour. the lysis was accomplished by adding 300 ul sds buffer (0.1 m nacl; 4% sds; 0.5m tris-hcl, ph 8). the extract was freeze-thawed. the dna was phenol-extracted and ethanol 28 biotropia no. 15, 2000 precipitated as in standard protocol (sambrook et al. 1989). the e. coli genomic dna was isolated as described previously (leach et al. 1994). the dna was extracted from soil by using modified tiedje method (keller 1997, unpublished). the soil sample (10 g) was finely grinded. the 5 g grinded soil was mixed with 13.5 ml tiedje buffer (100 mm tris-hcl, ph 8; 100 mm naedta, ph 8; 100 mm na2po4, ph 8; 1.5 m nacl; 1% ctab) in 100 ml centrifuge tube, and freeze-thawed 3x. after freeze-thawing, 100 ul proteinase-k (20 mg/ml) (sigma, singapore) was added and incubated at 37°c for 30 minutes, then 10 ml 10 % sds was added and incubated at 65°c for 2 hours. the mixture was centrifuged at 6000 g for 10 minutes. the supernatant was extracted with 1 volume of chloroform (merck, jakarta) and centrifuged at 6000 g for 1 minute. the aqueous phase was transferred to a new 100 ml centrifuge tube and 0.6 volume of isopropanol (merck, jakarta) was added at room temperature. the dna was recovered by centrifugation at 16,000 g for 20 minutes at 4°c. the supernatant was discarded and the pellet was washed with 70 % ethanol. the dna was dried at room temperature and dissolved in 100 ul te buffer (omm tris-hcl, ph 8; imm edta). southern hybridization analysis the 1.2 kb bamttl (neb, singapore) fragment from pui1014 and 1.8 kb bam\\\ (neb, singapore) fragment from pui1004 were isolated for preparing hema and hemt probes. the fragments were purified from agarose gel using the gene clean kit (bio 101 inc, la jolla, calif.), and labeled with biotin-14-atp using nick translation system (gibco/brl, grand island, ny) according to the manufacturer's instruction. the unincorporated nucleotides were removed from the probes with nuctrap push columns (stratagene, la jolla, calif.). the genomic dnas extracted from apb isolates were digested with ecorl (neb, singapore), except the dna from mb 15 isolate, which was digested with bam\\\ (neb, singapore). digestions were performed in appropriate buffer at 37°c for 12 hours. the digested dna was electrophoretically separated. the dna fragments were transferred to a nylon membrane (photogene, gibco/brl, grand island, ny) by capillary action with standard method (sambrook et al. 1989). hybridization was carried out as described previously at 42°c for 12 hours (sambrook et al. 1989), followed by washing at 37°c for 2x 15 minutes each, and detection using a chemiluminescent method (photogene detection system, gibco/ brl, grand island, ny). dot blot hybridization analysis four biotinilated dna probes, i.e. hema, hemt, pucba, and i6s rrna genes were used. the probes were prepared as described above. to avoid bias in calculation, the population density of prokaryotes was based on the same amount of soil samples, and not on the same amount or standardization of dna concentration. dna isolated from soil samples were denatured as described previously (keller and 29 presence of aema-like and hemt-\ike genes nurul aini & antonius suwanto manak 1992), and applied to nylon membrane (photogene, gibco/brl, grand island, ny) by spotting directly onto the membrane. high stringency hybridization and washing conditions were used. hybridization was carried out at 42°c for 12 hours, with washing temperature of 55°c (sambrook et al. 1989). detection was done using the photogene detection system (gibco/brl, grand island, ny). results and discussion southern hybridization analyses southern hybridization analyses were performed to determine the presence of hema-like and hemt-\\ke genes in twenty apb isolates from indonesia (table 1). r. sphaeroides 2.4.1 (rsp 2.4.1) hema and hemt genes were used as probe. the analyses identified some homologous regions the homology to hema in the genomic dna of mb6, mb 15, mb 16, mb21.2 and mbs 5. the region of homology to hema probe in the genomic dna of each apb isolate is shown in figure la, lanes 3-7. the hemtprobe hybridized only to 4.8 kb bamhl fragment in mb15 genomic dna (fig. ib, lane 3). a b figure 1. a. southern hybridization analyses using hema probe. lanes (1) rhodobacter sphaeroides 2.4.1, (2) kbslell, (3) mb15, (4)mb16, (5) mb21.2, (6) mb55, and (7) mb6 b. southern hybridization analyses using hemtprobe lanes (1) rhodobacter sphaeroides 2.4.1, (2) kbslell, and (3) mb 15 30 biotropia no. 15, 2000 the results implied the presence of hetna-like gene in mb6, mb15, mb16, mb21.2 and mb55, whereas hemt-like gene was only implied in mb 15. the presence of hema-like and/or hemt-like genes in the five apb isolates indicates the ala biosynthesis in these apb isolates is employed through the c4 pathway, because the c4 pathway is mediated by ala synthase which encoded by hema and/or hemtgenes. moreover, the presence of hema-like and/or hemt-\ike genes in the five apb isolates indicates that these apb isolates could be classified under sub group cc-proteobacteria. in photosynthetic bacteria, c4 pathway is utilized by purple non-sulfur bacteria, especially sub group a-proteobacteria (avissar et al. 1989; beale 1995). the sub group a-proteobacteria contains species of genera rhodospirillum, rhodopila, rhodopseudomonas, rhodomicrobium and rhodobacter (imhoff 1995). neither hema-like gene nor hemt-like gene was identified in the other 15 apb isolates. the data suggested that the c4 pathway is not utilized by these apb isolates to produce ala. the 15 apb isolates might produce ala through c5 pathway, which does not require ala synthase. these apb isolates used are not the member of sub group a-proteobacteria, although all apb isolates studied here belong to purple non-sulfur bacteria. the hemt-like gene was only identified in mb 15. this apb isolate also carries hema-like gene. interestingly, the hema-like gene in mb 15 was detected on the same locations with rsp. 2.4.1 hema, i.e., at 1.2 kb and 2.4 kb bamhl fragments (fig. la, lane 1 and 3). the strong intensity of the hybridization signals revealed that the similarity between mb 15 hema-like gene and rsp.2.4.1 hema was very high. moreover, the color comparison of mb 15 culture with rsp. 2.4.1 culture also showed a high similarity. based on these findings, it is very likely that mb 15 was rhodobacter sphaeroides. we tentatively conclude that hemt-like gene harbors specifically in r. sphaeroides. neidle and kaplan (1993) reported that the r. sphaeroides is the only bacterial species that produces two ala synthase isozymes. however, previously ala synthase isozymes are found in some vertebrates (dierks 1990), while no previous information on bacterial ala synthase isozymes have been reported. dot blot hybridization analysis dot blot hybridization analysis was employed to detect the presence of hemalike and hemt-like genes in dna extracted from four soil samples (table 2). four dna probes, i.e., hema, hemt, pucba, and 16s rrna gene were used. this analysis also revealed the relationship of the activity of prokaryote and apb in the soil samples with the presence of hema-\ike and hemt-like genes. figure 2 shows the results of dot blot hybridization using the four probes. interpretation of these results is described in table 3. based on the hybridization using hema probe (fig. 2a), the presence of hema-like gene was detected in soils taken from waterlogged paddy field, dry paddy field and lsi pond. the presence of hema-like gene in these soil samples indicates the activity of organisms producing 31 presence of aema-like and hemt-\\ke genes nurul aini & antonius suwanto ala through the c4 pathway. the c4 pathway is utilized by animals, fungi, protozoa and sub group cc-proteobacteria (avissar et al. 1989; beale & weinstein 1991; beale 1995). figure 2. dot blot hybridization analyses using hema (a), hemt(b), 16s rrna (c), and pucba (d) as probes. dots (1) rhodobacter sphaeroides 2.4.1., (2) soil sample from, waterlogged paddy field, (2) soil sample from dry paddy field, (3) soil sample from lsi pond, and (5) soil sample from grawida yard table 3. interpretation of the result of dot blot hybridization + : detected : nodetected the number of (+) represent the degree of intensity of hybridization signal 32 biotropia no. 15, 2000 using 16s rrna probe, we identified the presence of prokaryotic microorganisms in all four soil samples (fig. 2c). the results of hybridization to pucba probe indicated that the apb could be detected in all soil samples (fig. 2d). thus, it is possible to find the ct-proteobacteria in the soil samples, because some members of oc-proteobacteria are classified as apb group. the possibility for the ct-proteobacteria to exist in the four soil samples correlates with the presence of hema-l\ke gene in these soil samples. however, in spite of the detection of the apb activities in soil from grawida yard, hema-\ike gene was not found in this soil. the intensity of hybridization signal to both 16s rrna (fig. 2c) and pucba (fig. 2d) probes shows that the population densities of both prokaryote and apb in soil from grawida yard are much lower than those found in the other three soil samples. the a-proteobacteria might not exist in the soil from grawida yard or the ccproteobacteria were actually present in this soil, but the population density is very low. therefore, presence of hema-yike gene could not be detected. soils obtained from waterlogged paddy field, dry paddy field and lsi pond were found to have high population density of prokaryotes and apb. the high population density of apb enables the a-proteobacteria to proliferate. the presence of hema-v\ke gene in these soil samples is related to the population density of apb. the population density of apb as well as other microorganisms in the soil is dependent on the availability of growth nutrient, o2 and water (brock & madigan, 1991). the fertility and the soil texture are responsible for the availability of these factors. therefore, the presence of hema-\ike gene in soil is indirectly affected by the fertility and the soil texture based on the results of hybridization to hema probe, the hema-\ike gene might be present in the soil taken from apb habitat. in nature, apb occur in moist soil, paddy field, sewage water, fresh water, brackish water, waste water, marine habitat and in extreme condition of the antartic (sasikala et al. 1985). the presence of hema-\ike gene in the soil is related to the fertility of the soil, because the availability of growth nutrient affects apb growth. the dot blot hybridization using hemt probe identified the presence of hemtlike gene only in soil taken from dry paddy field (fig. 2b, dot 3). by comparing the intensity of hybridization signal to hemt probe with the hybridization signal to hema probe, it is clear that the homologous sequence to hemt probe is present in relatively fewer amount than the homologous sequence to hema probe. the results indicate that not all organisms carrying hema-\\ke gene also carry the hemt-\ike gene. these findings confirm the data obtained from southern hybridization analyses that the presence of hemt-\ike gene is more specific than that of hema-\ike gene. the hemt or hemt-\\ke gene might be specific to r. sphaeroides. however, further studies to determine the nature of specificity of hemt or hemt-\\ke gene to r. sphaeroides should be carried out to develop in situ hybridization method to examine directly the distribution and density of r. sphaeroides in the soil or water ecosystem. 33 presence of aema-like and hemt-\\ke genes nurul aini & antonius suwanto ackowledgments this research was supported by a competitive grant (hibah bersaing v, grant 08/p21 pt/dppm/96/phbv/i/v/1996) to antonius suwanto. references avissar, y.j., j. ormerod, and s.i. beale. 1989. distribution of 6-aminolevulinic acid biosynthetic pathway among photosynthetic bacteria and related organism. arc. microbiol. 151:513-519. beale, s.i. 1995. biosynthesis and structure of porphyrine and hemes. in: r.e. blakenship, m.t. madigan, and c.e. bauer (eds). anoxygenic photosynthetic bacteria. kluwer academic publisher. netherlands, p. 847-870. beale, s.i., and j.d. weinstein. 1991. biosynthesis of of 8-aminolevulinic acid in phototropic organism. in. h. scheer (ed). chlorophylls. crc press. boca raton. p. 368-392. brock, t.d., and m.t. madigan. 1991. biology of microorganism. prentice-hall international inc. usa. p. 566-569, 703-726. dierks, p. 1990. molecular biology of eukaryot 5-aminolevulinate synthase. in h.a. dailey (ed). biosynthesis of hemes and chlorophylls. mcgraw-hill. new york. p. 210-234. gest, h., j.l. favinger, and m.t. madigan. 1985. exploitation of n2 fixation capacity for enrichment of anoxigenic photosyntetic bacteria in ecological studies. fems microbiol. ecol. 31: 317-322. goodwin, t.w., and e.i. mercer. 1986. introduction to plant biochemistry. pergamon press. oxford, p. 465-467. habte, m., and m. alexander. 1980. nitrogen fixation by photosynthetic in lowland rice culture. appl. environ. microbiol. 39: 342-347. imhoff, j.f. 1995. taxonomy and physiology of photosynthetic purple bacteria and green sulfur bacteria. in: r.e. blakenship, m.t. madigan, and c.e. bauer (eds). anoxygenic photosynthetic bacteria. kluwer academic publisher. netherland. p. 1-15. irawan, a. suwanto, p.d. tjondronegoro. 1998. isolation and screening of anoxygenic photosynthetic bacteria producing extracellular l-aminolevulinat acid. hayati. 5: 98-102. keller, g.h., and m.m. manak. 1992. dna probes. stockton press. uk. p. 1-16. kiley, j. p, and s. kaplan. 1987. cloning dna sequence and expression of the rhodobacter sphaeroides light-harvesting 8800-850-a and b800-850-p genes. j. bacteriol. 169: 3268-3275. leach, j. e., f.f. white, m.l. rhoads, and h. leung. 1994. a repetitive dna sequence differentiates x. campestris p.v. oryzae from other patovar of x. campestris. mol. plant-microb. interact. 98: 238246. leuking, d.r., r.t. faley, and s. kaplan. 1978. intracytoplasmic membrane synthesis in synchronous cell population of rhodopseudomonas sphaeroides. j. biol. chem. 253: 451-457. neidle, e.l., and s. kaplan. 1993. expression of the rhodobacter sphaeroides hema and hemt genes, encoding two 5-aminolevulinic acid synthase isozymes. j. bacteriol. 175: 2292-2303. sambrook, j.e., f. fritsch, and t. maniatis. (1989). molecular cloning. cold spring harbor laboratory press. usa. p. 9.31-9.58. 34 biotropia no. 15, 2000 sasaki, k., s. ikeda, y. nishizawa, and m. hayashi. 1987. production of 5-aminolevulinic acid by photosynthetic bacteria. j. ferment. technol. 65: 511-515. sasaki, k., n. noparatnaraporn, s. nagai. 1990. production of 5-aminolevulinic acid as a herbicide from swine waste by rhodobacter sphaeroides. ann. rep. 1c biotech. 13: 277-281. sasikala, k., ch. v. ramana, p.r. raghureer. and k.l. kovacs. 1985. anoxygenic photosynthetic bacteria: physiology and advances in hydrogen production technology. adv. app. microbiol. 28: 211-295. schleifer, k.h., w. ludwig, j. krauss, and h. festl. 1985. cloned ribosomal rna genes from pseudomonas aeruginosa as probe for conserved deoxyribonucleic acid sequences. int. j. syst. bact. 38:231-237. suwanto, a., and s. kaplan. 1989. physical and genetic mapping of the rhodobacter sphaeroides 2.4.1 genome: presence of two unique circular chromosomes. j. bacteriol. 171: 5850-5859. tai, t.n., m.d. moore, and s. kaplan. 1988. cloning and characterization of the 5-aminolevulinic synthase gene(s) from rhodobacter sphaeroides. gene. 70: 139-151. werf, m. j., and j. g. zeikus. 1996. 5-aminolevulinat production by escherichia coli containing the rhodobacter sphaeroides hema gene. appl. environ. microbiol. 62: 3560-3566. 35 2 development og gene.cdr biotropia vol. 18 no. 1, 2011: 13 23 13 comparison of three different techniques of gene transfer in humpback grouper ( ) cromileptes altivelis slamet subyakto , alimuddin , rustidja , m. sasmito jati , irvan faizal , ratu siti aliah , gemi triastutik , and komar sumantadinata humpback grouper is one of the most cultured fishes in asia, including indonesia. the main problem faced by humpback culture is its slow growth rate. one of the methods that will be more effective and efficient to solve the problem is using transgenic technique. this study was conducted to determine the effectiveness of transfection, microinjection and electroporation techniques on gene transfer in humpback grouper. transfection was performed by incubating sperm to the foreign dna (pktbp-ktgh gene construct)-transfectant complex solution, while was by injecting those complex solution into testis of mature males. microinjection was conducted in 2-4 cell stage embryos using 25 μg/ml of foreign dna solution, and duration of injection was 1, 2 and 3 seconds. electroporation by 50 v, 30 ms of pulse length, 5 of pulse number and 0.1 of pulse interval was performed to sperm using three dna concentration of 5, 10 and 20 μg/ml. the incorporation of foreign dna in sperm and embryos were analyzed using pcr method. based on pcr analysis, an optimum dna concentration for electroporation was 10 μg/ml. limited number of embryos could be microinjected during 20-30 min to reach 2-4 cell stage. microinjection for 1 second showed higher survival rate of embryos, although none or very low number of larvae was hatched. transfast was an effective dna delivery reagent for humpback grouper sperm. foreign dna could be detected in sperm from two out of ten transfected fish at least 36 hours post transfection (hpt). by transfection, foreign dna was detected in sperm at 48 hpt 25 c incubation temperature. our study revealed that transfection, microinjection as well as electroporation could be used as transgenesis methods in humpback grouper. by means of simplicity and efficacy, however, electroporation was an appropriate gene transfer method. transfection, microinjection, electroporation, transgenesis, humpback grouper 1 2 3 4 5 5 1 2 o * 1 2 3 4 5 brackishwater aquaculture development center, situbondo, ministry of marine affairs and fisheries, indonesia department of aquaculture, faculty of fisheries and marine science, bogor agricultural university, indonesia faculty of fisheries and marine science, university of brawijaya, malang, indonesia faculty of mathematics and natural sciences, university of brawijaya, malang, indonesia agency for the assessment and application of technology, jakarta, indonesia in vitro in vivo in vivo in vitro abstract key words: * corresponding author : alimuddin_alsani@yahoo.com biotropia vol. 18 no. 1, 2011 14 introduction materials and method in the last two decades, a variety of transgenic aquatic organisms have been successfully produced by researchers around the world. these transgenic organisms have been produced by introducing gene/transgene encoding protein using various transgenesis methods, that is microinjection, electroporation, transfection and particle gun bombardment (chen . 1998; sarmasik 2001). transgene can be integrated and expressed in descendants of transgenic fish. furthermore, transgenic organisms have produced the expected phenotypes. until now, most of the generated transgenic aquatic organisms are freshwater fish because these fish are relatively easy to maintain and spawn under laboratory condition. in contrast, marine fish receive very little attention despite the fact that marine fish have a high potential economic value in aquaculture. humpback grouper is one of the most cultured fishes in asia, including indonesia. the main problem faced in humpback culture is its slow growth rate. for instance, to reach consumption size of about 0.5 kg 1.0 kg, this species needs 8 up to 24 months (tucker 1991; teitelbaum 2007). its slow growth makes the operational cost of this fish aquaculture increases drastically. therefore, developing a proper method that will be more effective and efficient to solve the problem is urgently needed. application of transgenesis technique is expected to solve the problem for humpback grouper and also other marine fish, though the use of transgenic organisms in aquaculture remains controversial. several methods of transgenesis have been successfully applied to create transgenic fish. devlin (1994) utilized microinjection method to generate transgenic fish. pacific salmon, alimuddin (2005) succeeded in zebrafish. electroporation method has also been successfully used by sin . (1993) and symons . (1994) on production of transgenic salmon, while patil and khoo (1996) and rambubu (2005) succeeded in zebrafish. the transfection method was successfully applied for gene transfer in silver snapper (lu 2002) and white shrimp (sun 2005). this study was conducted to determine the effectiveness of transfection, microinjection and electroporation methods in gene transfer for humpback grouper. transfection method is one of gene transfer methods using a specific reagent that is capable of binding to and delivering foreign dna to enter the cell. foreign dna used in this study was all-humpback grouper gene construct, pktbp-ktgh, which has been constructed by rusnas (national strategic prime research) program of ministry of research and technology, republic indonesia) for genetic improvement of hump et al et al. et al. et al. et al et al et al. et al. et al. transfection back grouper research team in 2007. pktbp-ktgh contains growth hormone gene (ktgh) and β-actin promoter (ktbp) from humpback grouper. 15 gene transfer method in humpback grouper alimuddin .et al a. transfection b. transfection microinjection electroporation dna extraction preliminary experiment was conducted by two transfectanst, i.e. transfast (promega) and jetpei (polyplus) to determine their efficacy to deliver pktbp-ktgh into sperm. in this experiment, 100 μl of grouper sperm was exposed to 360 μl of physiological solution containing 10 μl pktbp-ktgh and either 30 μl transfast or jetpei, at 17ºc and 25ºc incubation temperature for 24, 48, and 72 hours transfection. plasmid pktbp-ktgh was injected into humpback grouper testes prior to spawning according to the method of lu . (2002) with slight modifications. foreign dna-transfast complex solution at the solution was injected into the right and left testes of mature broodstock, respectively. at 12, 36, 48, 60 and 72 hours post injection, dna from the transfected sperm was extracted and used as template of pcr amplification to determine the success of transfection method. microinjection was conducted as described previously (alimuddin . 2005) microinjection was carried out to fertilized grouper eggs at 2-4 cell stage. a microinjection plate that consists of ten grooves of 3% agarose gel was used to support embryos when penetrated by microinjection needle. humpback grouper embryos were gently transferred onto the groove of microinjection plate and blastodisc should face the direction of the microinjection needle. dna solution at concentration of 25 μg/ml was slowly injected into blastodisc. the volume of injected dna solution was about one-fifth of the blastodisc volume (ath-thar 2007). number of embryos that can be injected by one microinjector in each spawning time was 60-100 embryos. three durations of injection (1, 2 and 3 seconds) into each embryo were examined to obtain length of injection time resulting to higher survival number of injected embryos. electroporation is a gene transfer method that utilizes a series of electric shock to help foreign dna to enter the cell. electroporation was carried out using gene pulser xcell electroporation system (biorad) by square wave method with parameters of 50 volt, 30 ms of pulse length, 5 pulse numbers, 0.1 s of pulse interval, and using cuvettes of 0.2 and 0.4 cm gap size. these electroporation parameters were obtained from the preliminary study. concentrations of dna used in this study were 5, 10 and 20 μg/ml, respectively. each dna concentration was mixed with 25 μl grouper sperm and sodium chloride solution to final volume of 260 μl. an amount of 100 microliters of electroporated sperm was used for dna analysis using pcr method. transfected and electroporated sperm solution was subjected to centrifugation for 1 min at 3000 rpm to remove remaining dna in solvent and transfection reagent. sperm was washed two times with 200 μl physiological salt solution before performing in vitro in vivo et al et al ratio of 1:2 (1 μg of dna: 6 μl transfast) was dissolved in physiological solution to final volume of 500 μl, and then half of 16 dna extraction. sperm and the injected embryos were lyzed with 500 μl of lysis buffer for 10 minutes at 95ºc. dna was precipitated using 400 μl of ethanol 96% and then diluted with 50 μl depc water. dna solution was stored at -20ºc. pcr analysis was performed in 10 μl of 1 microgram dna extracted from sperm and embryos, 1 μl primer forward and reverse (10 pmol), 1 μl dntps mix, 1 μl ex buffer, 0.05 μl ex polymerase (takara bio) and sdw to reach the final volume of 10 μl. forward and reverse primers designed and used in this study were located at 3' terminus of ktbp promoter (fbp2) and ktgh (rgh1) sequences, respectively. the sequence of those primers was 5' ttcatccagctgatgatt gccagatgtaac-3' and 5'-agttggcttca-ggagagagtcgacattt ag-3'. a total of 35 cycles of denaturation for 30 s at 94ºc, annealing at 62ºc for 30 s, and extension at 72ºc for 1 min were conducted. two microliters of pcr product was electrophoretically separated using 1.5% agarose gel, stained with ethidium bromide, and photographed under ultraviolet light. sperms survived when transfection was conducted using transfast reagent in both incubation temperatures, while contrarily jetpei-transfected sperm died (table 1). furthermore, the results of pcr analysis showed that transfection using transfast reagent for 48 hours at 25 c incubation temperature allowed foreign dna to incorporate into sperm (fig. 1 lane 2, table 1). the forward primer located at 5' terminus of ktbp (fbp1) and ap2 reverse (5'-ctatagggcacgcgtggt-3') was used to ensure that the amplified dna product was the foreign dna. pcr product using this set primer was about 2 kb in size (fig. 1 lane 1). the result suggested that foreign dna was incorporated in sperm. pcr amplification transfection taq taq results and discussion in vitro o biotropia vol. 18 no. 1, 2011 table 1. survival and incorporation of foreign dna into transfected sperms using transfast and jetpei reagents at 17 c and 25 c incubation temperatures. in vitro o o treatments duration of transfection (hours) 24 48 72 incubation temp. 25 oc transfast sperm survived, did not carry the foreign gene sperm survived and carry the foreign gene sperm survived, did not carry the foreign gene jetpei sperm died and did not carry the foreign gene sperm died and did not carry the foreign gene sperm died and did not carry the foreign gene control sperm survived, no carry the foreign gene incubation temp. 17 oc transfast sperm survived, did not carry the foreign gene sperm survived, did not carry the foreign gene sperm survived, did not carry the foreign gene jetpei sperm died and did not carry the foreign gene sperm died and did not carry the foreign gene sperm died and did not carry the foreign gene control sperm survived,did not carry the foreign gene 17 in vivo transfection in vivo transfection was carried out using ten mature male fish by injecting dnatransfast complex to testis through a canulation tube. this technique was developed, since fish could die when directly injected by syringe into testis through urogenital pore was conducted. two out of ten broodstocks contained sperm carrying foreign gene at 36 hours and 60 hours after transfection, respectively. the size of dna band of pcr product of dna from sperm containing foreign gene was similar with that of pktba-ktgh as template (fig. 2, indicated by arrow head). kb 10,0 2,0 1,0 0,5 0,1 m 1 2 figure 1. results of pcr analysis with dna template extracted from transfected sperm using transfast. lane 1: pcr amplification product using primers fbp-1 (5'-gtgwgtgacgcyggaccaatc-3') and ap2 (5'-ctatagggcacgcgtggt-3'), lane 2: pcr amplification product using primers as described in materials and methods. arrow head indicates pcr product of foreign gh gene. transfection was carried out for 48 hours at 25ºc. m is 2-log ladder dna marker (biolabs, inc., new england). the amplified fragment is ~700 bp in size. kb 10,0 3,0 1,2 1,0 0,7 0,5 0,3 0,1 m 1 2 3 4 5 6 m figure 2. results of pcr amplification with dna that have been extracted from transfected sperm at 24 and 60 hours post transfection (hpt). lane 1 and 2: pcr product of dna from broodstock no. 2919 at 36 hpt and 60 hpt, respectively. lane 3 and 4: pcr product of dna from broodstock no. 2935 at 36 hpt and 60 hpt, respectively. lane 5: pcr product using pktba-ktgh as template, lane 6: product of pcr with no dna template. m is 2-log ladder dna marker (biolabs, inc., new england). in vivo gene transfer method in humpback grouper alimuddin .et al 18 biotropia vol. 18 no. 1, 2011 microinjection high survival rate of uninjected embryos (92%) indicated that a high quality of fertilized eggs was used for microinjection (table 2). percentage of survived microinjected embryos 14 hours after injection decreased with the increase in duration of dna injection into blastodisc (table 2). increasing the duration of injection into blastodisc increased dna volume entering the cytoplasm. thus, high dna volume in cytoplasm of blastodisc probably caused embryos to die. similar result has been obtained for catfish (at-thar 2007). in addition, embryo may also die when injection is too deep into yolksac (at-thar 2007). this might also contribute to the decrease in survival rate of injected humpback grouper embryos. as blastodisc of humpback grouper is very thin, it is highly possible that the microinjection needle enters the yolksac. furthermore, twenty microinjected embryos for each treatment at 14 hours post injection were pooled into a tube for dna analysis. pcr amplification product showed that all microinjected embryos group contained foreign dna (fig. 3). this suggested that foreign dna was transferred into blastodisc. table 2. number and percentage of developed embryos after injection of 25 μg/ml at 1, 2 and 3 seconds. treatment repetition no. developed embryos undeveloped embryos percentage developed embryos 1 second 1 42 2 95,5 2 41 14 74,5 mean 85.0±14.8 2 second 1 1 18 5,3 2 3 42 6,7 mean 6.1±1.0 3 second 1 0 31 0,0 2 0 105 0,0 mean control 508 39 0.0±0.0 92,9 electroporation in this study, application of electroporation method for transferring the gene revealed that high percentage of electroporated sperm was motile (table 3). there was no difference in sperm motility of treated and untreated dna i.e. during 5-10 minutes in water. this result suggested that electroporated sperm could fertilize the eggs as in untreated sperm. percentage of motile slight difference of water quantity in dna solution might take account to reduce percentage of motile ml dna concentration. furthermore, droop value when using 0.2 cm cuvette was slightly higher (6-12%) compared with 0.4 cm cuvette (0-6%). thus, droop seems to be affected by the size of cuvette used, although no effect on electroporated sperm motility (table 1). droop electroporated spermatozoa using dna concentration of 5 μg/ml was slightly lower (90%) compared to that of the two other treatments (100%). sperm in 5 μg/ 19 is a function of voltage reduction at the end of electric shock (v v ) from a starting voltage v (anonymous 2006). 0 t 0 table 3. actual voltage, droop and percentage of motile sperm in electroporation using different dna concentration and cuvette gap size dna concentration (μg/ml) repetition setting voltage (volt) cuvette gap (cm) actual voltage (volt) droop (%) sperm motility (%) 1 50 0.4 37 6 90 5 2 50 0.4 37 0 90 3 50 0.2 37 12 90 1 50 0.4 37 6 100 10 2 50 0.4 37 6 100 3 50 0.4 37 0 100 1 50 0.2 37 12 100 20 2 50 0.2 37 12 100 3 50 0.2 37 6 100 m 1 2 3 4 5 6 7 8 kb 10,0 3,0 1,0 0,7 0,5 0,3 0,1 figure 3. pcr amplification product of dna extracted from microinjected embryos at 14 hours post injection. lanes 1-2: 1 second injection, lane 3-4: 2 seconds injection, lane 5-6: 3 seconds injection, lane 7: pcr product with no dna template, and lane 8 is pcr product with plasmid pktbp-ktgh as template. m is 2-log ladder dna marker (biolabs, inc., new england). pcr analysis showed that electroporated sperm in all treatments contained foreign dna (fig. 4). thus, parameters of electroporation used in this study could deliver foreign dna to enter spermatozoa. in addition, semi-quantitative pcr was applied to determine whether increase of dna concentration used in electroporation could improve number of sperm carrying foreign gene. as shown in figure 4, the thickness of dna band increased by increasing concentration of foreign dna used in this electroporation method. volume of semen (25 μl, equal to about 175 million spermatozoa) utilized in electroporation and concentrations of the extracted gene transfer method in humpback grouper alimuddin .et al biotropia vol. 18 no. 1, 2011 20 dna from electroporated spermatozoa used as template in pcr amplification were similar among treatments. thus, most likely increasing of dna concentra-tion in this study increased copy number of foreign dna in spermatozoa. furthermore, higher copy number of foreign gene entering spermatozoa may raise the possibility of foreign gene to integrate to the host genome. through fertilization, those spermatozoa would then contribute to enhance the number of embryo carrying foreign gene. n 1m kb 3.0 1.0 0.5 2 3 4 5 6 7 8 9 p figure 4. pcr amplification product of dna extracted from electroporated sperm. lanes 1-3: 20 μg/ml plasmid dna concentration, lanes 4-6: 10 μg/ml plasmid dna concentration, lanes 7-9: 5 μg/ml plasmid dna concentration. m is 2-log ladder dna marker (biolabs, inc., new england). n is pcr product without dna template. p is pcr product with plasmid pktbp-ktgh as template. efficacy of sperm-mediated gene transfer (smgt) technique to produce a transgenic marine species has been proved by lu . (2002) and sun (2004). smgt method has also been applied to introduce foreign gene into humpback grouper sperm. three smgt methods (electroporation, and transfection) were examined in this study. transfection using jetpei reagent succeeds to be used in white shrimp (sun . 2005). in this study, humpback grouper sperm died when transfection was performed using jetpei reagent. any compounds of jetpei may be toxic for humpback grouper sperm. in contrast, transfection using transfast reagent allowed humpback grouper sperm to survive. transfast transfected-sperm also carried foreign gene, although this was only found when transfection was conducted for 48 hours at 25 c incubation. furthermore, transfection by injecting dna-transfast complex into testis could also deliver foreign gene to sperm. foreign gene could be detected in transfected sperm at least 36 hours post injection. transgenic sea bream could be obtained by transfection for at least 48 hous prior to spawning (lu . 2002). in the same way with sea bream, transgenic humpback grouper may also be produced. in this study, however, only two out of ten injected broodstocks carried foreign gene in their sperm. this might be due to the difference in testis maturity. in addition, by applying the spawning system for humpback grouper, screening of founder transgenic fish generated by transfection method is costly, labor-extensive and time-consuming. microinjection is generally applied to produce transgenic fish. this method has also been used to introduce foreign gene through fertilized eggs towards generation of transgenic humpback groper. as shown in figure 3, injected embryos carried the foreign gene at least until 14 hours post injection. however, in this study, none or very low number of injected embryos hatched. as in other marine finfish, eggs of et al et al. in vitro in vivo in vitro et al in vitro in vivo in vivo et al in vivo o 21 humpback grouper float in water, small in size and has unclear blastodisc. these conditions hamper microinjection precisely. in addition, the time to reach 2-cell stage of humpback grouper embryos is about 20-30 min post fertilization. so the number of embryos which could be injected by one microinjector is very limited (60100 embryos). hatching rate of uninjected embryos is high, but the survival rate of larvae is lower i.e. 5-10% in average. based on the results in zebrafish transgenic research, the number of germline transgenic f0 is 2-4% of survived fish (alimuddin . 2005; alimuddin . 2008). if we assume that similar number of germline transmitted f0 in zebrafish can be achieved in humpback grouper, the number of embryos injected will be at least 1000 embryos to obtain 2 transgenic f0. this means that microinjection should be conducted 10 times, or we need more microinjectors and technicians. thus, the use of microinjection technique to produce humpback grouper requires facilities, and is labor-extensive as well as time-consuming electroporation has been reported to be a simple and mass transgenic production method. this technique also has similar efficiency with transfection method to produce transgenic sea bream (lu . 2002). voltage (50 v), pulse length (30 μs) and number of pulse (5 pulses) were also lower compared to those of sea bream. electroporation in sea bream is conducted using 600-2000 v, 40 μs pulse length, and up to 8 pulses. this suggests that optimum level of electroporation parameters may be species specific. finally, compared to the three other methods examined for humpback grouper as discussed above, electroporation to sperm is a fast, simple, and efficient transgenic method for humpback grouper. production of transgenic humpback grouper using electroporation technique is in progress in our laboratory. electr production of transgenic grouper carrying genes regulating the important traits for aquaculture holds exciting possibilities for the future, though the cultivation of transgenic organisms remains controversial. . we thank the research team for genetic quality improvement of humpback grouper broodstock of rusnas (national strategic prime research) program of ministry of research and technology republic indonesia for providing the pktbpet al et al . in vivo et al et al . 2002). as shown in figure 4, all treatments allowed the sperm to carry the foreign gene. based on the sperm motility and results of pcr analysis, 10 μg/ml was considered as the optimum dna concentration for electroporation of humpback grouper. this dna concentration was lower than that used for sea bream, 25 μg/ml (lu oporation to sperm was considered as an appropriate approach by means of efficacy and simplicity, to generate transgenic humpback grouper. optimum dna concentration for electroporation was 10 μg/ml. conclusions acknowledgements gene transfer method in humpback grouper alimuddin .et al 22 biotropia vol. 18 no. 1, 2011 ktgh plasmid; head of the department of aquaculture, faculty of fisheries and marine sciences, bogor agricultural university for supporting laboratory facilities; our thanks are also due to the staffs of situbondo brachishwater aquaculture center, especially the grouper parent team for their cooperation and technical support in broodstock handling. references alimuddin, nugrahani w, aliah rs, sumantadinata k, faizal i, carman o and g. yoshizaki. . jurnal riset akuakultur, 2: 199-209. (in indonesian). alimuddin, yoshizaki g, carman o. and k. sumantadinata. 2003. application of gene transfer technology in aquaculture. jurnal akuakultur indonesia, 2: 41-50. (in indonesian). alimuddin, yoshizaki g, kiron v, satoh s and t. transgenic research, 14: 159 165. alimuddin, kiron v, satoh s, takeuchi t and g. yoshizaki. 2008. cloning and over-expression of a masu salmon ( ) fatty acid elongase-like gene in zebrafish. aquaculture, 282: 13-18. anonymous, 2006. instruction manual: gene pulser xcell electroporation system, biorad catalog number 74, biorad office. ath-thar mf. 2007. effectiveness of oryzias latipes using hrgfp (humanized renilla reniformis green fluorescent protein) gene as a marker in catfish clarias sp f0 generation. undergraduate thesis. department of aquacuture, faculty of fisheries and marine sciences, bogor agricultural university. (in indonesian). chen tt, kight k, lin cm, powers da, hayat m, chatakondi n, ramboux ac, duncan pl and ra dunham. 1993. expression and inheritance of rsvltr-rtgh1 complemetary dna in the transgenic common carp, . molecular biology and biotechnology, 2:88-95. devlin rh, yesaki ty, donaldson em, du sj and cl hew. 1995. production of germline transgenic pacific salmonids with dramatically increased growth performance. canadian journal of fisheries aquatic sciences, 52: 1376 1384. lu jk, fu bh, wu jl and tt chen. 2002. production of transgenic silver sea bream ( ) by different gene transfer methods. marine biotechnology, 4: 328-337. patil jg and khoo hw. 1996. nuclear internalization of foreign dna by zebrafish spermatozoa and its enhancement by electroporation. journal of experimental zoology, 274: 121-129. rambubu km, rao shn and nm rao. 2005. efficient expression of transgene in adult zebrafish by electroporation. bmc biotechnology, 5:29. rusnas of humpback grouper broodstock research team. 2007. final report on genetic improvement of humpback grouper broodstock. national strategic primer research program of the ministry of research and technology, republic indonesia. 78 p. sarmasik a, jang ik, chung cz, lu jk and tt chen. 2001. transgenic live-bearing fish and crustacean produced by transforming immature gonads with replication-defective pantropic retroviral vector , 3: 470-477. sin fyt, bartley al, walker sp, sin il, symonds je, hawke l and cl hopkins. 1993. gene transfer in chinook salmon ( ) by electroporating sperm in the presence of prsv-laz dna. aquaculture, 111:57-69. sin fyt, walker sp, symonds je and il sin. 1994. sperm-mediated gene transfer in chinook salmon. online symposium paper, international congress of fish biology. vancouver-canada, 360-365. http://www.biologybrowser.org/bb/subject/ …/index251.shtml. august 14, 2009. 2007. isolation and characterization of β-actin promoter from humpback grouper takeuchi. 2005. enhancement of epa and dha biosynthesis by over-expression of masu salmon δ6-desaturase-like gene in zebrafish. β-actin promoter from japanese medaka oncorhynchus masou cyprinus carpio sparus sarba . marine biotechnology oncorhynchus tshawytsca 23 sun ps, venzonnc, calderon fro and esaki dm. 2005. evaluation of methods for dna delivery into shrimp zygotes of ( ) . , 243:19-26. symon je, walker sp, sin fyt and l. sin. 1994. development of a mass gene transfer method in chinook salmon: optimization of gene transfer by electroporated sperm. molecular marine biology and biotechnology 3:104-111. teitelbaum a. 2007. pacific islanders gain specific knowhhow on grouper hatchery techniques.http:// www.spc.int/coastfish/news/fish_news/ 121/teitelbaum_ 121.pdf. spc fisheries newsletter #121 april/june 2007. tsai hj, lai ch and hs yang. 1997. sperm as carrier to introduce an exogenous dna fragment into the oocyte of japanesse abalone ( ). transgenic research, 6:85-95. tucker jr. and w john. 1999. species profile grouper aquaculture. srac publication no. 721. http://www.ca.uky.edu/wkrec/ grouperaquaculture.pdf. penaeus litopenaeus vannamei aquaculture , haliotis deversicolor supertexta gene transfer method in humpback grouper alimuddin .et al biotropia no biotropia no. 12, 1999 : 19-24 the establishment of procecidochares connexa in west java, indonesia : a biological control agent of chromolaena odorata soekisman thtrosemito seameo biotrop, p.o. box 116, bogor 16001, indonesia abstract kirinyu (chromolaena odorata (l.) r.m. king and h. robinson) was reported for the first time in 1934 from lubuk pakam north sumatera. it grows vegetatively during the wet season, flowers and sets a high number of fruits at the end of the wet season, and senesces during the dry season. it may be controlled manually by uprooting the weed or by slashing, and chemically by spraying with herbicides. however, this has not been successful. recently a biological control agent, procecidochares connexa was introduced at parung panjang, west java, as a biological control agent of c. odorata. this paper reports the successful establishment of procecidochares connexa. two releases of p. connexa colonies were made in parung panjang at the end of 1995. the colony was able to survive through a harsh dry season of 1997. when c. odorata was swept by fire, the emerging shoots soon were attacked by p. connexa. the population of c. odorata went down to 37.2% with 31.8% of its emerging shoots attacked by p. connexa in a 2-year period. when twigs were attacked by p. connexa, the production of cypsellas was reduced by about 50% in one season. however, the survival of emerging flies was also affected by the death of twigs upon maturation of the cypsellas. key words: biological controuchromolaena odoratalprocecidochares connexa introduction the first herbarium record in indonesia of chromolaena odorata was from lubuk pakam, north sumatera, reported in 1934 (tjitrosoedirdjo 1990). the specimen was identified as eupatorium repandum, but re-identified by van steenis as eupatorium odoratum, and is now called chromolaena odorata. it is a shrub, capable of growing up to 7 m tall when a tree is available to climb on. normally it forms a thicket of branches of 1.5-3.0 m height. when cut the stem will regrow. being a member of the compositae, flowers are arranged into clusters consisting of 30-35 uniform flower units, with 3-5 composit flowers at one terminal point. in an established community, flowers may number 3200/m2, to yield a total of 960 000/ha flowers at one time, so in one season 1 ha of c. odorata may produce more than 10 billion seeds. as a result c. odorata will spread very fast. gauitier (1993) estimated that the weight of each dried fruit is approximately 0.2 mg. with its long papus, this fruit will be able to float and reach a very high altitude as reported by white (1970) and at 500 m it will be able to spread as far as 5 km from its point of origin. c. odorata was shown to have a high relative growth rate (rgr), although it has low net assimilation rate (nar) (tjitrosemito 1996). nar is a physiologica 19 biotropia no. 12, 1999 index indicating the plant's efficiency in converting solar energy for the reduction of co2 to carbohydrate. this low nar was more than compensated for by a high value of leaf area ratio (lar). it shows that, although the nar is low, the carbohydrates produced are utilized to produce leaves. therefore, it has a very high rgr. ischaemum timorense, being a c4 photosynthetic plant has a high efficiency for its physiological activities, but a low rgr, because most of the photosynthate was used for the generation of stolon, a plant part which is not very productive. in java the common method of controlling c. odorata in plantation is by uprooting the weeds and placing the stem upside down above the soil, because if it contacts the soil, it will regrow. other methods are either chemical control or slashing conducted at prohibitively high cost (tjitrosemito 1996). considering the characteristics of c. odorata, it seems that biological control agents that attack leaves, branches, and flowers may be a good approach to control this weed. accordingly, the biocontrol agent pareuchaetes pseudoinsulata was introduced. however, despite its successful establishment in sumatera, it does not do well in java. on the other hand another biocontrol agent, procecidochares connexa established more readily, and since its release in 1995 survives in west java. rearing of p. connexa has been reported elsewhere (tjitrosemito 1996). release was also readily achieved and spread in terms of areas covered by recorded galls, occurred logarithmically. it showed an intrinsic rate of increase in the area covered as r = 1.3. this paper reports the establishment of p. connexa and its impact on the population growth of c. odorata. methodology population of procecidochares connexa the population of p. connexa was estimated by taking samples using line transects starting from the center of the field, drawn in 4 cardinal directions. along each of the line transects at intervals of 20 m, quadrants measuring 2 x 2 m2 were established. in each quadrant, the number of shoots and galls were counted. a sample of 20 shoots taken at random was obtained from each quadrant, brought to the laboratory and the presence of eggs was recorded under a binocular microscope. monitoring was carried out at 2 monthly intervals. the extent of population spread was estimated by the presence of galls. from the release sites, a line was followed to the farthest distance covered by the gall and this distance was used as the measure of spread in october 1997. 20 the establishment of procecidochares connexa in west java, indonesia soekistnan tjitrosemito impact of p. connexa on the chromolaena odorata population to evaluate the impact of p. connexa on c. odorata, the population of c. odorata in the permanent plots was counted and compared with that at the time of release, including the number of galls found on the plot. the effect of galls on seed production was measured by randomly selecting 65 c. odorata plants free of p. connexa and compared to another batch of 65 plants selected at random from those attacked by p. connexa. the number of twigs, flowerheads and cypsellas were recorded. the effect of flowerheads on the development of galls for fly emergence batches of galls with and without flowers were observed on 8 populations. the numbers of successful emergence of flies from galls were recorded and statistically evaluated. results and discussions population of p. connexa the recorded number of shoots carrying eggs of p. connexa, and the number of galls are presented in table 1. table 1. the percentage of shoots carrying eggs and noticeable galls time jan. feb. apr. jul. aug. oct. dec. eggs 30.1 27.9 27.4 31.1 52.2 39.5 34.0 galls 27.1 25.9 17.1 25.7 40.7 34.7 29.2 the precentage of shoot bearing eggs went down slightly from january (30.1%) to april (27.4%), but went up again to reach a peak in august (52.2%). it seems that during the wet season, when growth of c. odorata was rapid and shoots were abundantly available, the number of emerging flies was still too low to visit the available shoots, thus accounting for the low precentage of shoot carrying eggs. when the dry season approached c. odorata did not produce new shoots, because it began to flower. the percentage of shoot bearing eggs increased to reach a maximum value of 52.2% in august 1997, but went down again to 34.0% in december. it is interesting to note that percentage of galls was always lower than that of eggs. 21 biotropia no. 12, 1999 population spread the spread of the population was recorded in october 1997 and is presented in table 2. table 2. the population of galls in a 2 x 2 m2 quadrant at various distances measured from point or release assuming that the spread of the bioagent was exponential, when calculated from the time of release in december 1995 and an initial area of 25 m2, the rate of spread was approximately 0.69/m2/m2/month. at this rate, the spread will double the size of the area each month. it was slower than previously reported when it was observed that doubling in size of the infested area occurred each 0.5 month. since spread is also affected by the availability of c. odorata plants, this was not unreasonable. impact on c. odorata the permanent plot established in 1995 contained a very dense vegetation of c odorata. in 5 x 5 m2, 145 plants with 2333 shoots were present. the initial releases in december 1995 were made with 75 pairs of flies and repeated again with 100 pairs. in 1997 the site was swept by fire and the population of c. odorata became 54 plants/5 x 5 m2 containing 891 shoots. although the plot was burnt down, the surviving plants quickly took over, and only 4 new recruits were recorded in the permanent plot, eventhough 90 plants disappeared during a 2-year period. the new recruits were still small in december 1997, and an old surviving stump was able to regrow bearing 74 new shoots (buds) in the plot. the infestation of p. connexa remained high, as indicated by the 277 galls recorded on the permanent plot. this meant that 31.08% of all shoots were infected by flies. this was much higher than 5% reported in december 1995. the population of c. odorata was reduced leaving 22 the establishment of procecidochares connexa in west java, indonesia soekisman tjitrosemito only 37.2% of the original population, and the shoot was reduced to the same extent i.e. only 38.2% remained, with 31.08% infested by p. connexa. here, biological control was effective. the effect of galls on seed production while the number of twigs did not differ significantly among plants attacked or not by p. connexa the flower heads differed significantly in numbers. formation of galls seemed to draw nutrition from the plants, reducing flower head development from 40.95/plant to 27.7/plant (see table 3). the reduction of seed production was even more conspicuous, i.e. from 1391.5 cypsellas/plant down to 647.6 cypsellas/ plant, a reduction of about 50%. table 3. cypsellas/plant production as affected by p. connexa recorded on 65 plants characters plants with galls plants without galls statistical test number of plants 65 65 ns number of twigs 264 316 ns flowerheads/plan 27.7 40.95 p <. 0.05 cypsellas/plant 647.6 1391.5 p^o.ol the effect of flowerheads on the development of galls leading to fly emergence comparison of population of galls having flowerheads and those that did not show a very significant difference in gall development at the 1% level. galls developed on shoots without flowerheads produced 0.8 successful emergences, while galls developing on growing flowerheads produced only 0.3 successful emergences (see table 4). this is caused by the fact that twigs bearing flowerheads will soon dry up following maturation of fruits. table 4. the emergence of flies from galls (8 populations of 20 colonies of galls, with and without flowers) no. colony with flowers colony without flowers statistical test 1 0.40 0.90 2 0.30 0.70 3 0.20 0.80 4 0.35 0.90 5 0.30 0.75 6 0.35 0.90 7 0.40 0.65 8 0.40 0.95 mean 0.33 0.82 ps 0.01 23 blotropla no. 12, 1999 this finding has a practical implication as the survival of emerging adults is reduced significantly by drying out of the bearing flowerheads. when all twigs are dried up it destroys the colony of p. connexa. the condition in parung panjang seems to support the perpetuation of the colony of p. connexa, since the population has survived the harsh dry season in 1997. acknowledgement this research has been funded by aciar through aciar project no. 9110. biological control of chromolaena odorata in indonesia and the philippines. references gauitier, l. 1993. reproduction of a pantropical weed: chromolaena odorata (l.). king & robinson. condollea48: 179-193. tjitrosemito, s. 1996. the management of chromolaena odorata (l.) king & robinson in indonesia. in: prasad, muniappan, ferrar, aeschliman, de foresta (eds.). agric. ex. station. univ.guam. pub. 202: 135-142. tjitrosoedirdjo, sri. s. 1990. some notes on chromolaena odorata. proc. 10th indonesian weed sci. conf.: 40-46. white, t.c.r. 1970. dispersal of exotic weeds. austral. j. sci. 32(9): 370-378. 24 biotropia no. 3, 1989/1990: 41-49 antagonistic effect of four fungal isolates to ganoderma boninense, the causal agent of basal stem rot of oil palm okky setyawati dharmaputra and h.s. soetarmi tjitrosomo seameo-biotrop, p.o. box 17, bogor, indonesia abdul latief abadi faculty of agriculture, brawijaya university, malang, indonesia abstract four fungal isolates from soils obtained from three sites of the oil palm plantations in north sumatra were found antagonistic to ganoderma boninense, the causal agent of basal stem rot of oil palm. penicillium citrinum inhibited the growth of the pathogen and formed a zone of inhibition on the agar media. trichoderma harzianum bio 1 as well as bio 2 and t. viride not only repressed the growth of the pathogen but also caused lysis of the hyphae, and the colony was totally overgrown by the antagonists. introduction ganoderma boninense pat. is an important basal stem rot pathogen of oil palm (elaeis guineensis jacq.) in north sumatra. some control methods have been applied to overcome the disease but none seemed to be satisfactory (parnata 1974; suyoto & djamin 1981; sipayung & purba 1986). the use of antagonistic soil microorganisms to control the pathogen has not been investigated so far. taking into consideration the statement asserted by cook & baker (1983) that certain species of penicillium and trichoderma have been reported to be antagonistic to plant pathogenic fungi, we at biotrop were curious to find out whether some antagonists could be isolated from soil samples taken from the oil palm plantations in north sumatra and investigate their antagonistic properties against ganoderma boninense. this paper describes the results of an investigation on the effect of four fungal isolates on the growth of the pathogen in vitro. materials and methods isolation of fungi from soil soil samples were taken randomly from three locations (adolina, gunung bayu, and tinjowan) of the oil palm plantations in north sumatra. the dilution 41 biotropia no. 3, 1989/1990 plate method (johnson & curl 1972) was used to isolate the fungi at a concentration of 10 -2 ml. into each petri dish, 1 ml of the soil suspension was transferred aseptically and added to it was 10 – 12 ml of melted selective medium containing chloramphenicol and rose bengal. the dishes were then incubated for 5 days at room temperature. isolates of g. boninense isolates of g. boninense used in this experiment were ga, gb and gd. they were isolated from basidiocarps obtained from the basal stem of oil palm in north sumatra. the name of the isolates were based on the colour and morphology of the basidiocarp. antagonism between g. boninense isolate gb and the fungal isolates the fungal isolates obtained were tested for their antagonistic property against the pathogen using the direct opposition method as recommended by dennis & webster (1971). the promising antagonistic molds were then put aside for further studies. the direct opposition method was prepared by placing the pathogen (4 mm in diameter) on the pda medium and 2 days later at a distance of 3 cm from the pathogen the antagonist was inoculated on the same dish (figure 1). each treatment was in three replications and incubated at room temperature. observations were made on the growth of the pathogen and the presence of an inhibition zone that might develop between the two colonies (figure 2). antagonism between g. boninense isolates ga, gb and gd and the four promising isolates of fungi in this study the four isolates of fungi that were found antagonistic to the pathogen were further tested individually for their effect on the pathogen when placed together using the direct opposition method. the inoculation method of the pathogen and the antagonist is the same as previously mentioned. observations were made 3 days after the inoculation of the antagonists on the inhibition of mycelial growth of the pathogen by the antagonist using the formula of fokkema (1973): r1 r2 i = x 100% r1 where i = percentage of inhibition r1 = radius of the pathogen away from the antagonist r2 = radius of the pathogen towards the antagonist 42 antagonistic effect of four fungal isolates-o.s. dharmaputra, h.s.s. tjitrosomo & a.l. abadi figure 1. antagonistic test between g. boninense and the fungal isolate. a, inoculum of g. boninense; b, inoculum of the fungal isolate tested. figure 2. colony measurement of g. boninense (a) to calculate the percentage of inhibition by the antagonist (b). radius of the pathogen (r, and r2) and the zone of inhibition (d) were also noted. 43 biotropia no. 3, 1989/1990 for p. citrinum, the distance (d) of the zone of inhibition was also measured. hyphal interaction in the zone of confrontation between the colonies was observed by direct examination under the microscope with 10 x 200 magnification 2 days after the inoculation of the antagonist (aia). interactions observed between the pathogen and antagonist were scored according to the categories presented in table 1. table 1. score of interaction between ganoderma boninense and trichoderma. score criteria/pathogen hyphae lysed 1 ±25% 2 ±50% 3 ±90% the scores were added by 1 if pathogen hyphae were smaller than the normal ones the scores were reduced if ±10% of the antagonist by 1/2 hyphae were also lysed the scores were reduced by 1 if ± 25% of the antagonist hyphae were also lysed results and discussion the fungi isolated from the soil samples are presented in table 2: 4 isolates of penicillium, 2 isolates of aspergillus, 3 isolates of trichoderma, and 1 unidentified isolate, totaling 10 isolates obtained by the use of dilution plate method. table 2. the fungi isolated from adolina, gunung bayu and tinjowan, north sumatra. 44 antagonistic effect of four fungal isolates-o.s. dharmaputra, h.s.s. tjitrosomo & a.l. abadi cook and baker (1983) stated that penicillium and trichoderma have long been recognized as antagonists to plant pathogenic fungi. all of the ten isolates obtained were further tested on the possibility of inhibiting the growth of the pathogen (isolate gb) in vitro using the direct opposition method as recommended by dennis & webster (1971). of the 10 isolates, only four showed promising result, that is penicillium sp. 2, trichoderma sp. 1, trichoderma sp. 2, and trichoderma sp. 3 (figure 3). figure 3. antagonism between ganoderma boninense isolate gb and penicillium sp. 2, trichoderma sp. 1, trichoderma sp. 2 and trichoderma sp. 3. further identification of the four isolates showed that penicillium sp. 2, trichoderma sp. 1, trichoderma sp. 2, and trichoderma sp. 3 were respectively p. citrinum, t. harzianum bio-1, t. harzianum bio-2 and t. viride. antagonism between p. citrinum and g. boninense isolates ga, gb, and gd a zone of inhibition (d) was observed when p. citrinum was paired with the pathogen. the growth of the pathogen towards the antagonist was inhibited since the 2 nd day aia and it stopped to grow on the 6 th day aia. the distance (d) of the zone of inhibition on the 3 rd day aia were 11.25 mm, 12.88 mm and 10.88 mm for isolates ga, gb and gd, respectively. it was assumed that an antibiotic diffused into the medium. 45 biotropia no. 3, 1989/1990 statistical analysis using f indicated that i on the 3 rd day aia was not significantly different among the three isolates of the pathogen: 29.95% for isolate ga, 25.28% for isolate gb and 19.08% for isolate gd, respectively. direct examination under the microscope showed that the hyphae of the pathogen were not lysed, but they were abnormal, i.e. they had more septa and their cells were shorter than the normal ones. antagonism between trichoderma isolates and g. boninense isolates ga, gb and gd having examined all the dishes where the pathogen colonies and the antagonists were grown, it was noted that there were no inhibition zones. for all treatments, it was observed that mycelial contact between the two colonies on the same dish started on the 2 nd day aia. t. harzianum bio 2 stopped the growth of the pathogen on the 3 rd day aia, whereas t. harzianum bio1 and t. viride on the 4 th day aia. the percentage of inhibition of the pathogen mycelial growth by the antagonist is presented in table 3. in general, the three isolates of trichoderma inhibited the growth of the pathogen in vitro. the lowest percentage occurred when isolate gb was placed against t. harzianum isolate bio-2, while the highest was noted when isolate gd was inoculated against t. harzianum bio-2. it is interesting to report here that t. harzianum isolate bio 1 was able to inhibit at statistically the same degree, the growth of all isolates ga, gb, and gd, whereas the inhibition table 3. average percentage inhibition of mycelial growth of ganoderma boninense isolate ga, gb and gd by trichoderma harzianum (isolate bio 1 and bio 2) and t. viride. combination of treatment average of inhibition*) (pathogen vs. antagonist) (%) ganoderma boninense vs. trichoderma harzianum 28.96 a isolate gb isolate bio 2 g. boninense isolate gd vs. t. viride 37.71 b g. boninense isolate gb vs. t. viride 38.57 b g. boninense isolate ga vs. t. harzianum isolate bio1 39.53 be g. boninense isolate ga vs. t. harzianum isolate bio-2 39.62 be g. boninense isolate gd vs. t. harzianum isolate bio1 40.29 be g. boninense isolate gb vs. t. harzianum isolate bio1 41.57 be g. boninense isolate ga vs. t. viride 41.84 be g. boninense isolate gd vs. t. harzianum isolate bio-2 45.18 c *) means within columns followed by the same letter are not significantly different. lsd 0.05 = 6.14. 46 antagonistic effect of four fungal isolates-o.s. dharmaputra, h.s.s. tjitrosomo & a.l. abadi of t. harzianum bio 2 and t. viride was determined by the type of isolate of the pathogen. direct examination under the microscope showed that the pathogen hyphae underwent lysis; the percentage of which was determined by the type of isolate of the antagonist (table 4). in any case, the hyphae of the antagonist were also lysed in certain treatment, e.g. t. harzianum bio1 vs. g. boninense isolates ga and table 4. microscopic examination of hyphal interaction between ganoderma boninense isolates gb and gd with trichoderma harzianum (isolates bio -1 and bio 2) and t. viride. 47 biotropia no. 3, 1989/1990 gd, t. harzianum bio-2 vs. g. boninense isolates ga and gd, t. viride vs. g. boninense isolates ga, gb and gd. in general t. harzianum bio 1 had the same effectivity score against the three pathogen isolates. the ef fectivity score of t. harzianum bio 2 and t. viride was determined by the type of isolate of the pathogen. if we look at either the percentage of inhibition of mycelial growth of the pathogen by the antagonist or the capability of the antagonist to cause hyphal lyses of the pathogen, t. harzianum bio1 was the most potential antagonist. chet et al. (1979) found that t. harzianum could control damping-off disease on bean, peanuts and egg plants caused by sclerotium rolfsii and rhizoctonia solani. sivan and chet (1986) also found that t. harzianum could control fusarium spp. in cotton, wheat and muskmelon. conclusion p. citrinum, t. harzianum (isolate bio1 and bio-2), and t. viride isolated from oil palm plantation in north sumatra were antagonistic to g. boninense. t. harzianum isolate bio 1 was the most potential antagonist. p. citrinum probably produces an antibiotic substance that diffuses into the medium, while tricho-derma causes lyses of the pathogen's hyphae. acknowledgments the authors gratefully acknowledge the funds given by marihat research center, pematang siantar, north sumatra. thanks are also due to dr. r.a. samson of central bureau voor schimmelcultures (cbs), baarn, the netherlands, and dr. m.a. rifai of the research centre for development in biology, indonesian institute of science, bogor, indonesia, in having confirmed the identification of the fungal isolates. references chet, i., y. hadar, y. elad, j. katan and y. henis. 1979. biological control of soil-borne plant pathogens by trichoderma harzianum. in schippers, b. and w. gamsa (eds.). soil-borne plant pathogens. academic press, london: 585-591. cook, r.j. and k.f. baker. 1983. the nature and practice of biological control of plant pathogens. the american phytopathological society, st. paul, minnesota. 48 antagonistic effect of four fungal isolates-o.s. dharmaputra, h.s.s. tjitrosomo & a.l. abadi dennis, c. and j. webster. 1971. antagonistic properties of species groups of trichoderma. iii. hyphal interaction. trans. brit. mycol. soc. 57: 363-369. fokkema, n.j. 1973. the role of saprophytic fungi in antagonism against drechslera sorokiniana (helminthosporium sativurri) on agar plates and on rye leaves with pollen. physiological plant pathology 3: 195-205. parnata, y. 1974. control of basal stem rot of oil palm using urea in accelerating stem and stump decay. bulletin balai penelitian perkebunan medan 5(3): 89-94. sipayung, a. and r.y. purba. 1986. penelitian dan usaha penanggulangan penyakit busuk pangkal batang (ganodermd) di perkebunan kelapa sawit (research and control of basal stem rot (ganoderma sp.) in oil palm plantation). special edition. pt perkebunan vi-vii, marihat research center, marihat ulu, pematang siantar. sivan, a. and i. chet. 1986. biological control of fusarium spp. in cotton, wheat and muskmelon by trichoderma harzianum. j. phytopathology 116: 39-47. suyoto, s. and a. djamin. 1981. sistem pemberantasan penyakit busuk pangkal batang (ganodermd) di perkebunan kelapa sawit ptp vi. (system of controlling basal stem rot (ganodermd) in oil palm plantation ptp vi). paper presented at 6 th indonesian phytopathology society congress, bukittinggi. 49 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf 47.pdf 48.pdf 49.pdf biotropia book juni revisi 14 juli 09.indd 28 isolation and cloning of cdna of gene encoding for metallothionein type 2 from melastoma affine suharsono1,2, niken trisnaningrum1, lulut dwi sulistyaningsih1, utut widyastuti1,2 1research center for bioresources and biotechnology, and 2department of biology, bogor agricultural university indonesia abstract metallothionein is an important protein for detoxifying heavy metal ions. th is research was conducted to isolate and clone cdna of gene encoding for metallothionein type 2 from melastoma affi ne. total rna was isolated from young leaves. total cdna was synthesized from the total rna by reverse transcription. th e mamt2 cdna was successfully isolated by pcr technique. th e mamt2 cdna was inserted into pgem-t easy and the recombinant plasmid was successfully introduced into escherichia coli dh5α. dna sequencing analysis showed that this cdna is full length consisting of 246 pb encoding 81 amino acid residues. th is cdna is identical to mrna of atmt2 from arabidopsis thaliana. it does not contain any restriction sites found in the cloning sites of pgem-t easy. th e deduced protein of mamt2 contains 14 cysteine residues distributed in the cys-cys, cys-x-cys, and cys-x-x-cys motifs. key words: cdna, metallothionein, melastoma affine, cloning, cysteine introduction gene isolation has a very important role in the genetic improvement of crops. the genes can be isolated from genetic resources coming from microorganism, plants, or animals. melastoma malabathricum l. or m. affine d. don is an aluminum hyperaccumulator plant (watanabe et al. 2005) and used as an indicator for acid soil. since it is tolerant to acid and al stresses, it is very important to use this plant as genetic resources for tolerance genes to acid and al stresses. tolerant plants to al are very important to increase agricultural production in indonesia because indonesia has about 47.5 million hectares of pod soil yellow-red land which have low ph and high solubility of al (csar 1997). metallothionein (mt) is a protein having low molecular weight, around 4-8 kda (vallee 1991). it is constituted of 45-48 amino acid residues containing 12-17 conserved cysteine residues (kagi 1991). cobbett and goldsbrough (2002) biotropia vol. 16 no. 1, 2009: 28 37 corresponding author: sony@rcbio.org; sony-sh@ipb.ac.id, sony.suharsono@yahoo.com 29 have classified mt into four types based on distribution of cysteine residues in the c-terminal and n-terminal regions. type 1 of mt is consisted of two domains with metal binding motif cys-x-cys. type 2 is composed of two domains with metal binding motif in combination among cys-cys, cys-x-x-cys, and cys-x-cys. type 3 is also called as a phytochelatin (pc) and has a structure of (γ-glu-cys)n-gly, whereas type 4 contains rich in cysteine in repeated sequence. mt can bind heavy metal, and has an important role in the detoxifying of some heavy metal ions (clemens 2000, hall 2002), like zn (nigel et al. 1996). th e expression of bjmt2 in a. thaliana increased the seedling tolerance against co and cd (zhigang et al. 2006). th e expression of mt2 gene is induced by al stress in wheat (snowden et al. 1995), by h2o2 and metal ion in rice (zhou et al. 2005), by cu and co in a. thaliana (zhou and goldsbrough 1995), by drought stress in wild watermelon (akashi et al. 2004) but it is down regulated by small gtpase osrac i in rice (wong et al. 2004). mt2 gene had been isolated from soybean (kawashima et al. 1991), vicia faba (foley and singh 1994), rice (hsieh et al. 1995) and broccoli (yang et al. 2000). th is research has the objective to isolate and to clone the cdna of gene encoding for metallothionein type 2 from m. affi ne l. since the expression of mt2 is induced by al stress (snowden et al. 1995), we suppose that the over-expression of this gene can increase the plant tolerance to al. by this reason, this gene can be used to improve genetically the tolerance of important plant to al by over-expressing the mt2 gene. materials and methods materials young leaves of m. affine were used as plant material. pgem-t easy (promega) was used as cloning vector. escherichia coli dh5α was used as a host for recombinant plasmid. primers of actf (atggcagatgccgaggatat), and actr (cagttgtgcgaccacttgca) designed based on complete nucleotide sequence of a soybean actin gene (shah et al. 1982, genbank v00450) were used to amplify exon1-exon2 of β-actin as a control of cdna. primers of mf (tcgagaaaaatgtcttgctgtg), and m7r (cttcacttgcaggtgca agg) designed based on arabidopsis thaliana mt2a mrna (genbank nm_1117733) were used to isolate mt2 cdna of m. affine. total rna isolation young leaves (1 g) were ground with pestle in the mortar in the presence of 0.3 g sands and 10 ml warm extraction buffer (2% ctab, 2% pvp 25000, 100 mm trishcl ph8, 20 mm edta, 1.4 m nacl, 1% β-mercaptoethanol) previously heated at 65oc. this suspension was poured into 20 ml centrifuge tube, and incubated at 65oc for 10 minutes, and then added by 10 ml of chloroform:isoamylalcohol (24:1). after vortexing to mix this suspension, the tube was centrifuged at 42 000 xg (rotor sw11, sorvall ultra pro 80), 4°c for 10 mins. the upper liquid phase were collected and added by 0.25 volumes of 10 m licl. after incubation at -32oc for 2.5 hours, this suspension was centrifuged at 42 000 xg (rotor sw11, sorvall ultra pro 80), 4°c for isolation and cloning of cdna from melastoma affi ne suharsono et al. 30 10 mins. the pellet containing total rna was suspended by adding 500 ml te (10 mm tris-hcl ph 7.4, 10 mm edta). this total rna suspension was extracted by adding 1x volume of phenol ph 9, then vortexing and centrifuging at 14000 rpm (jouan br4i) at 20 oc for 10 mins. the total rna suspension was recovered from the upper phase, and then extracted by 1x volume of phenol:chloroform:isoamylalcohol (25:24:1), then centrifuged at 15 000 rpm, 20oc for 10 min. the total rna contained in upper phase was precipited by adding 0.25 volume of 10 m licl, and incubated at -32oc overnight. this total rna suspension was centrifuged at 15 000 rpm, 4oc, 10 min. total rna pellet was washed by 500 ml ethanol 70% and centrifuged at 15 000 rpm, 4oc, 10 min. after drying with vacuum dryer total rna was added by depc treated h2o to make a suspension. synthesis of total cdna total cdna was synthesized by mixing 5 μg total rna, 4 μl 5x buffer superscript iii reverse transcriptase (invitrogen), 20 pmol oligo(dt), 4 mm dntp, 10 mm dtt, 1 u enzyme superscripttmiii rtase dan depc treated h2o in the final volume of 20 μl. reverse transcription to synthesize total cdna was carried out at 52oc for 50 mins by using pcr machine (mj research tm 100). the purity of total cdna was verified by using pcr with specific primers of for cdna of exon1-exon2 of β-actin. the composition of pcr was 1 μl total cdna, 1x taq buffer, 40 mm mgcl2, 4 mm dntp mix, 20 pmol actf primer, 20 pmol actr primer, 4% dmso, 0.75 u taq dna polymerase (toyobo) and h2o in the final volume of 20 μl. the condition of pcr was pre-pcr 95°c, 5 mins, denaturizing at 94°c, 30 seconds, annealing at 57°c, 30 seconds, extension at 72°c, 1.5 min, and post-pcr at 72°c, 5 min. the pcr was conducted for 35 cycles. isolation of mamt2 cdna by pcr the composition of pcr was 1 μl total cdna, 2 μl 10x buffer taq, 4 mm dntp, 20 pmol mf primer, 20 pmol m7r primer, 4% dmso, 0.75 u taq dna polymerase (toyobo) and h2o in the final volume of 20 μl. the pcr was conducted for 35 cycles at 95°c, 5 min. for pre-pcr, 94°c, 30 seconds for denaturising, 60oc, 30 seconds for annealing, 72°c, 1.5 min. for extension and 72°c, 5 min. for postpcr. cloning mamt2 cdna into pgem-t easy mamt2 cdna of pcr product (3 ml) was mixed with 10 ng pgem-t easy (promega), 3 u t4 dna ligase (promega), 1x ligation buffer, and h2o in the final volume of 10 μl. the reaction was carried out at 4oc overnight. the ligation product was introduced into e. coli dh5α as described by suharsono (2002). selection of e. coli containing mamt2 e. coli containing mamt2 was selected based on the resistance to ampicillin and blue-white selection. the insert of mamt2 in the white ampicillin resistant colony was confirmed by pcr-colony. the colony was picked up by tooth-picker, then suspended in 5 ml h2o, and heated at 95oc, 10 mins and cooled at 15oc for 5 mins. the suspension was added by 1.5 μl 10x buffer taq, 3 mm dntp, 15 pmol biotropia vol. 16 no. 1, 2009 31 mf primer, 15 pmol m7r primer, 4% dmso, 0.75 u enzim taq dna polymerase (toyobo). pcr was conducted in the same condition as for mamt2 cdna isolation. the insert of mamt2 cdna was also confirmed by plasmid dna isolation as described by suharsono (2002). the plasmid dna was digested by ecori at 37oc for 2 hours. analysis of cdna dna was sequenced by using automated dna sequencer abi prism 3100 version 3.7. local alignment analysis of cdna was carried out by using blast2 program (http://www.ebi.ac.uk/blast2) (mount 2001). restriction sites in the cdna of mamt2 was analyzed by neb cutter program (http://www.firstmarket.com/cutter/ cut2.html). results and discussions total rna isolation total rna of m. affine had been successfully isolated from young leaves and the efficiency of isolation was 118-213 μg rna/g leaves. the absorbent ratio of λ260/λ280 was 1.4-1.6. it showed that these total rnas were probably contaminated by remaining protein and/or phenol. the purity of total rna is high if the λ260/ λ280 absorbent ratio is around 1.8-2.0 (saunders and parker 1999). electrophoresis analysis in formaldehyde denaturized agarose gel in the mops buffer showed that the total rna contained two prominent bands. these two bands were corresponded to the integrated 28s and 18s ribosomal rna (figure 1). since the total rna contained integrated 28s and 18s rrna, the integrity of total rna was also high, and could be used as template for total cdna synthesis. the integrity of mrna is very important to isolate the full length of coding sequence of dna. to use the gene for genetic improvement by over-expression, the full length of gene is indispensible. figure 1. electrophoresis of total rna of m. affine isolated in different time from young leaves. 1=10 o’clock, 2=12 o’clock, and 3=14 o’clock. total cdna synthesis by using total cdna as template and specific primer for cdna of β-actin, amplification by pcr resulted to 450 bp band corresponding to cdna of exon1-exon2 of β-actin (figure 2). the result showed that the total cdna had been successfully synthesized and this total cdna was free from the contamination by genomic dna. if genomic dna contaminated the total cdna, the amplification of β-actin would result to two bands, 450 bp and 550 bp, corresponding to amplified cdna and isolation and cloning of cdna from melastoma affi ne suharsono et al. 32 genomic dna respectively. the 550 bp dna was resulted from the amplification of exon1-intron-exon2 of β-actin, and the size of intron beetwen exon1 and exon2 is around 100 bp (shah et al. 1982). this intron is spliced during the mrna synthesis, so cdna derived from mrna does not contain this 100 bp intron. figure 2. amplification of exon1-exon2 cdna of β-actin. 1= 100 bp dna marker, 2= exon1-exon2 cdna of β-actin cdna is very important for genetic engineering of eucaryotic organisms. this dna represents the coding region of gene. the size of cdna is equal or smaller than the corresponding dna of gene. since the size is smaller, the expression of gene by using cdna is more efficient than by using dna of gene. therefore, the cdna of gene is more preferable than dna although the isolation of gene by using cdna is more difficult than by using dna in eucaryotic organisms. isolation and cloning of mamt2 cdna pcr by using total cdna as template and mf and m7r as primers of resulted around 250 bp dna (figure 3). this result was the same as predicted because the size of mt2 in several plant species is around 250 bp as found in soybean (kawashima et al. 1991), wheat (snowden and gardner 1993), a. thaliana (zhou and goldsbrough 1994), and rice (zhou et al. 2006). this cdna was then inserted in pgem-t easy, and the recombinant plasmid dna was successfully introduced into e. coli dh5α shown by the white colonies grown in the selective media containing ampicillin, x-gal and iptg. pgem-t easy plasmid contains lacz in its cloning site (cs). only e. coli containing plasmid can survive in the media containing ampicillin and only e. coli containing recombinant plasmid can grow in white colony in the presence of x-gal and iptg in the media. the insertion of cdna in the lacz found in the cloning site caused the inactivation of lacz to be expressed. on the other hand, if there are no insertions in the lacz, this gene is expressed to produce β-galactosidase and this enzyme will convert uncolored x-gal to become blue color. as a result, the color of colonies which do not contain the recombinant plasmid is blue. biotropia vol. 16 no. 1, 2009 33 figure 3. cdna of mamt2 resulted by pcr to confirm the inserted cdna of mamt2, the white colony was picked up and used as template for pcr-colony. the pcr-colony resulted 250 bp dna (figure 4). the plasmid dna was successfully isolated from this white colony. the digestion of this plasmid dna with ecori resulted two fragments of dna, one is 250 bp and the other is 3 kb corresponding to the cdna of mamt2 and pgem-t easy cloning vector, respectively (figure 4). this result showed that the white colony contained mamt2 cdna found in the pgem-t easy. figure 4. analysis of cdna insert in pgemt-easy recombinant by pcr-colony (2) and ecori digestion (3). 1= 1 kb plus dna marker. analysis of cdna of mamt2 based on sequencing from sp6 primer, the recombinant pgem-t easy contained insert dna of 257 bp consisted of 246 bp open reading frame encoding for 81 amino acid residues (figure 5). alignment analysis by blastn showed that this cdna had similarity of 100% with mrna of atmt2a of a. thaliana (accession: nm_111773.3), 90% with mrna of bjmt2 of brassica juncea (y10850.1), 89% with mrna of brmt of b. rapa (d78498.1), 88% with mrna of bcmt of b. camprestris (l31940.1), and 88% with mrna of bomt2 of b. oleracea (af200712.1). this alignment analysis showed that the cdna isolated in this research is a full length of mamt2 of m. affine containing start and stop codons. isolation and cloning of cdna from melastoma affi ne suharsono et al. 34 figure 5. nucleotide and deduced amino acid sequences of mamt2. underline sequences indicate the forward and complementary reverse primers of used to isolate mamt2, bold letters indicate start and stop codons respectively, italic letters indicate the cys amino acids. restriction site analysis showed that mamt2 contains bbvi, bsabi, btsci, tsei, foki, apeki, cspci, hpych4iii, pshai, bsrfi, sgrai, acui, btgzi, mboii, sfani, cac8i and hpy188i sites (figure 6). these sites cannot be used to clone and engineer mamt2 because they can cut mamt2 in two or more fragments. fortunately, mamt2 does not contain any restriction site found in cs of pgem-t easy. it means that, all restriction sites found in cs can be used to isolate the mamt2 cloned into pgem-t easy. figure 6. restriction site map of mamt2. to perform genetic engineering, the information of the nucleotide sequence and the restriction sites of the gene are indispensable. the nucleotide sequence information can be used to modify the sequence by in vitro directed mutagenesis. the information of restriction sites of the gene is very important to decide the restriction enzymes which will be used to cut off the gene and then to fuse with other genes or expression elements as promoter and terminator. biotropia vol. 16 no. 1, 2009 35 deduced amino acid residues demonstrate that mamt2 contains 81 amino acid residues with 14 cysteine residues. this result is in accordance with the number of amino acid residues and cysteine residues of mt type 2 from several plants (kojima 1991). analysis of cysteine sequence motif showed that mamt2 has a motif cys-cys (3rd-4th residues), cys-x-cys (8-10, 14-16, 67-69, 73-75, 78-80) and cys-x-x-cys (20-23) (figure 5). this specific cys sequence demonstrates that mamt2 has the similar motif of cys distribution with mt type 2 of several plants (robinson et al. 1993; cobbett and goldsbrough 2002). the full length of mamt2 cdna isolated in this report is the first mt family genes isolated from m. affine as the indigenous plant in tropical rain forest like indonesia. since this plant is very tolerant to al, the mamt2 can be used to improve genetically the important plant for indonesia as soybean. the genetic improvement can be done by over-expressing mamt2. we are now targeting the over-expression of mamt2 in soybean to improve the resistance to al by using strong promoter. in the near future, we also would like to over-express this mamt2 cdna in jatropha curcas. conclusions the total rna of m. affine had been isolated and converted into total cdna. the full length of cdna of mamt2 gene had been isolated from this total cdna and cloned into pgem-t easy. this mamt2 cdna contains 246 bp and encodes 81 amino acids. mamt2 contains 14 cys amino acids distributing in cys-cys, cys-xcys, and cys-x-x-cys motifs. this full length of mamt2 cdna is the first cdna of mt family gene isolated from m. affine. it can be applied to improve genetically the al tolerance of plant. acknowledgments we would like to thank biotrop-seameo and directorate of research and community services, directorate general of higher education, ministry of national education, republic of indonesia for the financial support of this research by contract agreement no. 13.1/psrp/sp-pen/iv/2005 and spk 008/hikom/ dp2m/2008, respectively. references akashi k., n. nishimura, y. ishida, and a. yokota. 2004. potent hydroxyl radical-scavenging activity of drought-induced type-2 metallothionein in wild watermelon. biochemical and biophysical research communications, 323: 72-78. clemens, s. 2001. molecular mechanisms of plant metal tolerance and homeostasis. planta 212: 475-486. cobbett c, and p. goldsbrough. 2002. phytochelatins and metallothioneins: roles in heavy metal detoxifi cation and homeostasis. annual review of plant. biology, 53: 159-182. isolation and cloning of cdna from melastoma affi ne suharsono et al. 36 csar (center for soil and agroclimate research). 1997. statistik sumberdaya lahan/tanah indonesia dan agroklimat. badan litbang departemen pertanian. jakarta. foley r.c., and k.b. singh. 1994. isolation of a vicia faba metallothionein-like gene: expression in foliar trichomes. plant molecular biology, 26: 435-444. hall j.l. 2002. cellular mechanism for heavy metal detoxifi cation and tolerance. journal of experimental botany, 53 (366): 1-11. hsieh h.m., w.k. liu, and p.c. huang. 1995. a novel stress-inducible metallothionein-gene from rice. plant molecular biology, 8: 381-389. kagi j.h.r. 1991. overview of metallothionein. methods in enzymology , 205: 613-626. kawashima i., y. inokuchi, m. chino, m. kimura, and n. shimizu. 1991. isolation of a gene for a metallothionein-like protein from soybean. plant cell physiology , 32: 913-916. kojima y., p.a. binz, and j.hr. kagi. 1999. nomenclature of metallothionein: proposal for a revision. in: metallothionein iv, c. klaassen. birkhauser-verlag. basel. mount d.w. 2001. bioinformatics: sequence and genome analysis. new york: cold spring harbor laboratory press. robinson n.j., a.m. tommey, c. kuske, and p.j. jackson. 1993. plant metallothionein. journal of biochemistry, 295: 1-10. nigel j. r., j.r. wilson, and j. s. turner. 1996. expression of the type 2 metallothionein-like gene mt2 from arabidopsis thaliana in zn2+-metallothionein-defi cient synechococcus pcc 7942: putative role for mt2 in zn2+ metabolism. plant molecular biology, 30: 1169-1179. saunders g.c., and h.p. parker. 1999. analytical molecular biology: quality and validation. teddington: lgc press. snowden k.c., and r.c. gardner. 1993. five genes induced by aluminum in wheat (triticum aestivum l) roots. plant physiology, 103: 855-861. snowden k.c., k.d. richard, and r.c. gardner. 1995. aluminium-induced genes induction by toxic metals, low cadmium, wounding and patterns of expression in root tips. plant physiology, 107: 341348. shah d.m., r.c. hightower, and r.b. meagher. 1982. complete nucleotide sequence of a soybean actin gene. proceedings national academy of science, usa, 79 (4): 1022-1026. suharsono. 2002. konstruksi pustaka genom kultivar slamet. hayati , 9(3): 67-70. vallee b.l. 1991. introduction of metallothionein. methods in enzymology, 205: 3-7. watanabe t., s. misawa, and m. osaki. 2005. aluminum accumulation in the roots of melastoma malabathricum, an aluminum-accumulating plant. canadian journal of botany, 83: 1518-1522. wong h.l., t. sakamoto, t. kawasaki, k. umemura, and k. shimamoto. 2004. down-regulation of metallothionein, a reactive oxygen scavenger, by the small gtpase osrac i in rice. plant physiology, 135: 1447-1456. zhigang a., l. cuijie, z. yuangang, d. yejie, a. wachter, r. gromes, and t. rausch. 2006 expression of bjmt2, a metallothionein 2 from brassica juncea, increases copper and cadmium tolerance in escherichia coli and arabidopsis thaliana, but inhibits root elongation in arabidopsis thaliana seedlings. journal of experimental botany, 57 (14): 3575-3582. yang c.y., y. lin, and j.f. shaw. 2000. cloning and characterization of a cdna encoding a mt2 type metallothionein from broccoli (brassica oleracea cv. green king) fl orest. plant physiology, 122: 1457. biotropia vol. 16 no. 1, 2009 37 zhou j., and p.b. goldsbrough. 1995. structure, organization and expression of the metallothionein gene family in arabidopsis. molecular and general genetics, 248: 318-328. zhou g., y.f. xu, and j.y. liu. 2005. characterization of a rice class ii metallothionein gene: tissue expression patterns and induction in response to abiotic factors. journal of plant physiology, 162: 686696. isolation and cloning of cdna from melastoma affi ne suharsono et al. thank you for evaluating anybizsoft pdf splitter. a watermark is added at the end of each output pdf file. to remove the watermark, you need to purchase the software from http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html biotropia no biotropia no. 18, 2002 : 38 51 adherence and pathogenicity assay of vibrio harveyi in tiger shrimp (penaeus monodon) larvae for screening biocontrol agent yusminah hala1, antonius suwanto23, ridwan affandi4 and muhammad zairin jr.4 'department of biology, faculty of science and mathematics, makassar university, makassar 90221, indonesia 2 department of biology, faculty of science and mathematics, bogor agricultural university, bogor, indonesia jseameo-biotrop, jl. raya tajur km 6, bogor, indonesia 4department of aquaculture, faculty of fisheries and marine science, bogor agricultural university, bogor 16680, indonesia abstract rifampicin-resistant marker was employed as a reporter to detect the adherence and colonization of v. harveyi in shrimp larvae. vibrio harveyi p1b and ya32.2 were isolated from dead shrimp larvae in besuki, northern coast of east java, while v. harveyi hb3, was isolated from pristine sea water in pacitan, southern coast of east java. vibrio metschnikovii used as biocontrol agent was isolated from healthy shrimp larvae in serang, west java. spontaneous mutation was conducted to generate v. harveyi p1b, ya32.2 and hb3 resistant to rifampicin. these mutants exhibited similar survival ability to their parental (wild type) strains. significant larval mortality was observed in shrimp larvae inoculated with ya32.2 than that of larvae inoculated with p1b. larvae inoculated with hb3 showed the lowest mortality. bacterial cell count of vibrio rf* in dead larvae were 103-104 cells/larvae. isolates of vibrio metschnikovii z and m as biocontrol candidates effectively reduced the growth and adherence ability of ya32.2 to shrimp larvae. larval mortality in rearing water inoculated simultaneously with ya32.2 and v. metschnikovii was lower than the one inoculated with ya32.2 alone. therefore, vibrio metschnikovii z or m could be developed as an effective probiotic or biocontrol agent for v. harveyi in shrimp hatcheries. key words : biological control/vibrio metschnikovii/shrimp \arvae/penaeus mwu«fon/pathogenicity assay/vibrio harveyi introduction shrimp farming is one of the most important activities in indonesia and other asian countries such as thailand, phillippines, and india (ruangpan 1998). up to 1997, indonesia was the second largest world producer of tiger shrimp (penaeus monodori) after thailand with a production of more than 99 000 metric tons of tiger shrimp (anonymous 1999). however, the exponential growth of shrimp culture in the last five years was not supported properly by a sufficient supply of shrimp larvae *corresponding author : e-mail address : asuwanto@indo.net.id: fax : 62-251-315107 38 38 biotropia no. 18, 2002 due to larval diseases and poor environmental quality of the hatcheries. among shrimp diseases, vibriosis is one of fatal bacterial diseases. this disease is caused by vibrio sp. that attacks the tiger shrimp at early larval or post-larval stage. mass mortality of shrimp larvae frequently associated with luminous vibrio (lavilla-pitogo et al. 1998; sunaryanto & mariam 1986) was identified as vibrio harveyi (karunasagar et al. 1994; lavilla-pitogo et al. 1990). although vibrio carchariae was reported as a pathogen in a brown shark (carcharinus plumbeus) that was found dead in captivity (grimes et al. 1984), it has not been reported as a pathogen of shrimp larvae. it was also found associated with a chronic skin ulcer on a shark (bertone et al. 1996). so far, it has not been determined whether luminous vibrio is a pathogen or saprophyte, even though it could frequently be isolated from dead larvae. therefore, the pathogenicity status of this group of bacteria is uncertain. pathogenicity assays based on koch's postulate (salyers & whitt 1994) is practically difficult to be conducted in tiger shrimp larvae due to its relatively small size and since so far, no germ-free larvae are available (hameed 1993). the source of luminous vibrio colonized shrimp larvae might originate from the midgut contents of the spawners (lavilla-pitogo et al 1992). bacterial pathogenicity is determined by several factors such as the ability to adhere to the host tissue to colonize and to secrete virulence factors (salmond et al. 1995). the aim of this study was to determine the adherence and colonization of vibrio isolates and their pathogenicity to shrimp larvae. bacterial isolates were molecularly tagged to distinguish it from vibrio naturally associated with the shrimp larvae. luminous vibrio isolated from hatcheries and coastal water in east java, indonesia, showed sensitivity to rifampicin (tjahyadi et al. 1994). therefore, we used rifampicin resistant (ri*) vibrio generated by spontaneus mutation as a marker for v. harveyi isolates used in this study. vibrio harveyi p1b and ya32.2 were isolated from dead larvae, while hb3 was isolated from pristine sea water (suwanto et al. 1998; teo et al. 2000). our study also indicates that adherence assay in this study could be developed to screen for potential biocontrol bacteria or probiotics against pathogenic vibrio. materials and method isolation and identification of vibrio carchariae the dead shrimp larvae were obtained in february 1997 from besuki in the northern coast of east java, indonesia. the larvae were rinsed twice with sterile sea water and placed aseptically on the thiosulphate citrate bile salt (tcbs) agar. the plate was incubated for 24 h at room temperature (28 ± 2 °c). isolated luminous colonies were restreaked'on vibrio harveyi agar (vha) (harris et al. 1996). 39 adherence and pathogenecity assay of vibrio harveyi yusminah hala et al. bacterial characterization was conducted employing cellular fatty acid analyses by microcheck, inc., microbial analyses laboratory in northfield, usa. bacteria were grown on trypticase soy broth agar (tsba) at 28 °c and then extracted for cellular fatty acid analysis (miller 1982). the fatty acid composition of each isolate was compared to a database of standard strain (v. carchariae atcc 35084) profile, using overlap coefficient which allows their identification according to their similarity index. two other v. harveyi isolates used in this study were isolated and characterized from dead larvae (p1b) and from pristine sea water (hb3) (suwanto et al. 1998). antibiotics sensitivity in order to tag vibrio with the antibiotics resistant markers, it is necessary to determine the natural antibiotic sensitivity of the isolates. all isolates used in this research were tested for antibiotic sensitivity against kanamicin (50 p.g/ml), ampicillin (50 |o.g/ml), tetracycline (10 |j.g/ml), gentamicin (20 ng/ml) and rifampicin (10 |j.g/ml) on luria bertani (lb) agar (10 g nad, 10 g tryptone, 0.5 g yeast extract, 15 g agar and 1 l distilled water). rifampicin-resistant vibrio harveyi spontaneous mutants of v. harveyi resistant to rifampicin (rf*) were selected on lb agar supplemented with rifampicin (50 (ig/ml) (eisenstad et al. 1994). the survival of rf* v. harveyi was evaluated and compared to the wild type strains in artificial larval-rearing water (3% nacl and 0.3% yeast extract in 1l distilled water= sye). growth pattern and survival assay vibrio harveyi rf" was inoculated into 100 ml sye media and incubated at 28°c. cell concentration was enumerated daily on tcbs and tcbs supplemented with 50 (j.g/ml rf (tcbs-rif). enumeration began just after v. harveyi was inoculated into sye media. the survival of v. harveyi on tcbs was compared with the survival on tcbs-rif to detect the stability of the rf* mutation. preparation of shrimp larvae two-day-old tiger shrimp postlarvae (pl2) and sea water for larval rearing were obtained from a hatchery in labuhan, west java. the larvae were maintained for acclimatization in a three-liter glass stock jar for at least two days in laboratory condition. sea water salinity was maintained at 2.5% and at a temperature of 2830°c. the larvae were fed on an artificial diet (lanzy pl; inve aquaculture, belgium). for adherence and pathogenicity assays, 50 larvae were kept in a threeliter glass jar containing 2.5 liters of autoclave-sterilized sea water. 40 biotropia no. 18, 2002 adherence assay tested bacterial strains used in the adherence assays were cultured in 50% sea water complex (swc) agar medium (ph 7.2) for 24 h at 28 °c. the culture was harvested by adding 5 ml sterile sea water onto the bacterial lawn, scraped, and collected into a tube, and then centrifuged at 5000 x g for two minutes. the bacterial cells were rinsed in sterile sea water and diluted to give a final concentration of 106 cfu/ml. the number of wild type vibrio and vibrio rf* in larval rearing water was enumerated daily on tcbs and tcbs-rif, respectively. the number of larval mortality was counted daily. the vibrio harveyi which colonized the dead larvae was enumerated daily on tcbs-rif. challenge test the isolates used for the challenge test in this study were vibrio metschnikovii isolates z and m, which were isolated from healthy zoea (z) and mysis (m), respectively (widanarni 1999). cell suspension of vibrio metschnikovii z and m at a concentration of 108 cfu/ml were inoculated into larval rearing jar 2 h before the shrimp larvae were introduced into the jar. after 6 h cocultivation of z or m isolate with shrimp larvae, pathogenic vibrio (ya32.2 rf*) were inoculated into the larval rearing jar. the treatments consisted of ya32.2 rf" which was challenged with v. metschnikovii z, m, or a combination of z and m. shrimp larvae cocultivated with ya32.2 rr* alone was employed as a control. the number of larval mortality as well as concentration of wild type vibrio and vibrio rf* in the dead larvae and in rearing water were enumerated daily after cocultivation. the total number of vibrio was enumerated on tcbs agar, while vibrio rfr was counted on tcbs-rif. each experiment was conducted in duplicates. the number of dead larvae and bacterial cell concentration were presented in average values. results isolation and identification of vibrio carchariae two vibrio isolates (ya32.2 and ya31.4) were isolated from dead larvae. colonies of these isolates were luminous, circular and appeared green with dark green in the center of the colony when grown on tcbs agar. they also grew on vha, a differential agar for vibrio harveyi (harris et al. 1996), and appeared as blue colonies due to decarboxylation of ornithin which shifted ph medium toward alkaline. when illuminated from below, yellow halos appeared, indicating cellobiose fermentation around the colonies. based on these specific characteristics, ya32.2 and ya31.4 were identified as v. harveyi. 41 adherence and pathogenecity assay of vibrio harveyi yusminah hala et al cellular fatty acid analyses of ya32.2 and ya31.4 grown on trypticase soybroth agar indicated that similarity index to v. carchariae atcc 35084 were 0.955 and 0.944, respectively. these similarity indices were much higher than those of v. parahaemolyticus or v. alginolyticus (table 1). however bergey's manual classifies v. carchariae as a junior synonym (variant) to v. harveyi (baumann et al. 1994). four vibrio harveyi isolates used in this study, i.e.: p1b, hb3, ya32.2 and ya31.2, were resistant to ampicillin (50 tig/ml) and kanamicin (50 jig/ml) but sensitive to tetracycline (10'|ig/ml), gentamicin (20 (j-g/ml) and rifampicin (10 (j.g/ml) (tabel 2). vibrio harveyi mutant rf11 an amount of 50-100 u.1 of 108 cfu/ml v. harveyi was inoculated onto lb agar supplemented with 50 (j-g/ml rifampicin. isolated colony which showed similar morphology and luminescence to the wild type was further employed for survival assay. the growth pattern and survival of three mutants p1b rf*, ya32.2 rt* and hb3 rf* were similar to their parental wild type strains (data not shown). table 1. similarity index isolates ya32.2 and ya31.4 with vibrio sp based on cellular fatty acid profiles 42 biotropia no. 18, 2002 adherence assay about 106 cfu/ml of v. harveyi rf* was inoculated into larval rearing water for adherence assay on shrimp larvae. bacterial growth profile in larval rearing water showed similar patterns to that of the wild type (fig. 1). the three strains showed bacterial growth patterns that generally increased in cell concentration at first to second or third day and decreased the following days. the highest concentration of vibrio ri* in larval-rearing water was approximately 107 cfu/ml. larval mortality in response to the three vibrio rf* strains was different as presented in figure 2. isolate ya32.2 caused the highest larval mortality on the first day and the number of vibrio rf* found in the dead larvae was 2.3xl03 cfu/larvae. on the second day after inoculation, the number of dead larvae increased to 36 animals concomitant with the highest vibrio rf* concentration at 1.2xl04 cpu/ larvae. all of the larvae were found dead on the third day with vibrio rf" found in the larvae at concentration of 1.3xl04 cfu/larvae . 43 adherence and pathogenecity assay of vibrio harveyi yusminah hala et al. figure 1. comparison of vibrio rf* and their respective parental wild type growth pattern in the larval rearing water 44 biotropia no. 18, 2002 figure 2. correlation between the cumulative number of death larvae and the number of vibrio adhered in the shrimp larvae 45 adherence and pathogenecity assay of vibrio harveyi yusminah hala et al. larval mortality in the jars inoculated with p1b occured on the second day after inoculation. the number of dead larvae found on the second day was 19 animals. larval mortality reached its highest number on the third day when the concentration of vibrio rf* in the larvae was 8.7xloj cfu/larva. the lowest larval mortality was found in the larvae inoculated with hb3: larval mortality was found only after three days of coinoculation, and until the last day of observation, larval mortality occurred in only 13 animals carrying vibrio rf* at concentration of 1.75xl02 cfu/larva (fig. 3). challenge test shrimp larvae inoculated with ya32.2 supplemented with v. metschnikovii z or m, either alone or in combination, showed less mortality than the larvae treated only with ya32.2 alone as shown in figure 4. the number of dead larvae when treated with ya32.2 in the presence of v. metschnikovii m was 11 larvae. moreover, the number of dead larvae slightly increased if ya32.2 was inoculated in the presence of z and m, although the number of vibrio rf* which adhered to the larvae was relatively similar i.e. 1.2xl02 to 3.2xl02 cfu/larva. all of the larvae treated with ya32.2 were found dead after three days of cocultivation when concentration of vibrio rf* in the larvae reached 104 cfu/larva (fig.4). figure 3. comparison between bacterial cell count of ya32.2 when challenged or when inoculated alone 46 biotropia no. 18, 2002 figure 4. correlation between the number of dead larvae and ya32.2 adherence in a challenge test experiment 47 adherence and pathogenecity assay of vibrio harveyi yusminah hala et ill. discussion fatty acid profile analyses indicated that isolates ya32.2 and ya31.4 were v. carchariae. the results indicated that vha media which was supposedly selective for v. harveyi (harris et al. 1996) actually could also support the growth of v. carchariae, although dna-dna hybridization analysis suggested that v. carchariae is a junior synonym for v. harveyi (baumann et al. 1994). in addition, ribotyping of v. harveyi and v. carchariae indicates very similar dna banding patterns (pedersen et al. 1998). physiology and genetic similarity might explain the ability v. carchariae to grow on vha media that was originally designed as a selective media for v. harveyi. one of the physiological characteristics of v. carchariae is its capability to hydrolyze urea (uh+). other reports indicated that brown shark (from where the original name of v. carchariae was derived) excreted urea for osmoregulation. it was suggested, therefore, that the adherence of v. carchariae to shark was due to urea-urease linkage (bertone et al. 1996). meanwhile, urea hydrolyzing activity was reported as an indication of potential pathogenicity of v. parahaemolyticus (kays-ner et al. 1994). in v. parahaemolitycus, uh+ is related to hemolysin production, which is a virulence factor of vibrio (osawa et al. 1996). the result of our study indicated that ya32.2, p1b and hb3 were uh+, and two of them (i.e. p1b and ya32.2) were isolated from the dead shrimp larvae. however, the shrimps were reported to excrete ammonia, instead of urea (baticados 1988). therefore, the adherence of v. harveyi to shrimp might not be mediated by urea-urease linkage. three isolates used in this research demonstrated similar sensitivity to the test antibiotics. they were resistant to ampicillin and kanamicin but sensitive to tetracycline, gentamicin and rifampicin. vibrio carchariae isolated from shark skin ulcer was sensitive to tetracycline and resistant to piperacillin and carbenicillin (bertone et al. 1996). vibrio harveyi which was the causal agent for mass mortality of shrimp larvae in india, was sensitive to tetracycline but resistant to erythromycin. on the other hand, v. harveyi generally showed similar sensitivity to gentamicin (austin et al. 1981) and rifampicin (tjahyadi et al. 1994). this study indicated that v. harveyi p1b and hb3 and v. carchariae ya32.2 were sensitive to tetracycline, gentamicin and rifampicin. this result showed that v. harveyi isolated from anywhere has similarity in sensitivity to tetracycline, gentamicin and rifampicin. bacterial cell count of v. harveyi rf* in larval-rearing water showed a different number from that of vibrio sp which occurred naturally in larval rearing water. the difference indicated the-number of vibrio sp originally colonized shrimp larvae. these vibrio sp yielded green colonies which could not be distinguished from green colony of v. harveyi rf* in rearing water. therefore, it is necessary to introduce molecular marker to distinguish the green colonies of vibrio rt* from the green colonies bf wi'd type vibrio. 48 biotropia no. 18, 2002 p1b, hb3 and ya32.2 were sensitive to rf. therefore, in this experiment, we used rf* as molecular marker to distinguish these three strains from the other v. harveyi isolates which occurred naturally in the shrimp larvae or larval rearing water. vibrio harveyi rf* mutants showed similar growth patterns to that of the wild type strain. this result indicated that the survival of v. harveyi rf* was similar to the wild type, and rf* character was stable even when it was kept in larval-rearing water. although the cell densities of each vibrio rf1* in larval-rearing water were similar at approximately 107 cfu/ml (fig. 1), larval mortality in response to the three rf* strains was different. the fastest and the highest larval mortality was due to ya32.2 inoculation which could be detected as early as one day after coin-oculation time. larval mortality due to inoculation with p1b was detected after two days of coinoculation although the number of the dead larvae which resulted from those treatments was relatively similar. probably, the two strains were potential pathogens because they were isolated from dead shrimp larvae. similarly observed mortality was found when shrimp larvae were inoculated with similar density of v. campbelli\ike bacterium (hameed 1995). our study showed that hb3 was not pathogenic. larval mortality in hb3 inoculation jar maybe due to the high density of vibrio. nonpathogenic or very weak pathogenicity of hb3 might be the reflection of its habitat where hb3 was originally obtained from pristine sea water in southern coast of java island. isolate ya32.2 was then selected for challenge test due to its potential pathogenicity. vibrio metschnikovii as a challenge strain is a vibrio species which usually adheres and colonizes shrimp larvae until the stages of post-larval stage. we assumed the strains of v. metschnikovii are not pathogen because they do not produce virulence factors (widanarni 1999). austin et al. (1995) used v. alginolyticus as a probiotic strain for a. salmonicida, v. anguillarum and v. ordalii. our results indicated that v. metschnikovii z or m could significantly reduce the growth and adherence of ya32.2 in shrimp larvae and significantly reduce larval mortality. this result demonstrated potential application of the adherence assay to screen for potential prdbiotics or biocontrol bacteria in shrimp hatcheries. acknowledgment this work was funded by grants from riset unggulan kemitraan (ruk), indonesia, and international foundation for science (ifs, a/2207-2), sweden, to antonius suwanto. 49 adherence and pathogenecity assay of vibrio harveyi yusminah hala et al. references anonymous. 1999. aquaculture production statistics 1988-1997. fao fisheries circular no 815, revision 11. fao, roma. austin b., d.a. morgan and d.j. alderman. 1981. comparison of antimicrobial agents for control of vibriosis in marine fish. aquaculture, 26:1-12. austin, b. l.f., p.a. stucken, w. robertson, 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(ed) advances in shrimp biotechnology. national center for genetic engineering and biotechnology, bangkok, p:217-224. teo, j.w.p., a. suwanto, and c.l. poh. 2000. novel p-lactamase genes from two environmental isolates of vibrio harveyi antimicrob. agents chemother. 44: 1309-1314. tjahjadi, m.r., s.l. angka and a. suwanto. 1994. isolation and evaluation of marine bacteria for biocontrol of luminous bacterial diseases in tiger shrimp larvae (penaeus numodon. fab.). as. pac. j. mol. biol. biotechnol. 2:347-352. widanarni and a. suwanto, 2000. genetic diversity of ampicilin-resistant vibrio isolated from various stages of tiger shrimp larvae development. biotropia. 15:36-47. 51 biotropia no. 5, 1991/1992: 1-9 production structure of main commercial tree species in a mangrove forest in east sumatera, indonesia cecep kusmana*) faculty of forestry, bogor agricultural university, bogor, indonesia and hiroyuki watanabe faculty of agriculture, kyoto university, kyoto 606, japan abstract production structure of main commercial tree species was studied in a mangrove forest in east sumatera, indonesia. this research was carried out in january 1991 using the estimation of standing biomass with stratified clipping method in order to know the production structure of the main commercial tree species in this mangrove forest, i.e. rhizophora apiculata, bruguieraparviflora and b. sexangula. the results obtained show that r. apiculata tended to have a sparser foliage of thicker leaves along the stem than b. parviflora or b. sexangula; therefore, r. apiculata is regarded as a shade-intolerant tree species. in contrast, either b. parviflora or b. sexangula tended to have a larger proportion of leaves and branches along the stem; consequently, those species are recognized as shade-tolerant tree species. introduction a substantial proportion of about 81 000 km coastal line in indonesia is covered by mangrove formations of various extents, from several meters to several kilometers (soegiarto 1979). in terms of tree size and extent, mangroves are more developed in the five big islands, i.e. java, sumatera, kalimantan, sulawesi, and irian jaya (darsidi 1984). in sumatera, the mangrove covers an area of 667 000 ha of which 276 000 ha are distributed in riau. recently, about 87 000 ha of mangrove forest in riau have been designated as forest concession areas belonging to the bina lestari company (42 000 ha), thai rayvithi company (40 000 ha) and silva saki company (5000 ha). for many years, the mangrove trees in riau were cut for fuelwood and used for charcoal either by forest concessionaires or local people inhabiting the region surrounding the mangroves. however, recently, most of the commercial tree species, especially bruguiera spp. and rhizophora apiculata are being exploited for the production of chipwood. *)present address: faculty of agriculture, kyoto university, kyoto 606, japan 1 biotropia no. 5, 1991/1992 it is highly necessary to establish a proper management system based upon biological knowledge to secure a sustained yield of mangrove resources. therefore, the production structure of mangrove tree species must be studied to understand the biological characteristics of mangroves. this research was undertaken in an attempt to analyze the production structure of the main commercial tree species (bruguiera parviflora, b. sexangula and r. apiculata) in the mangrove forest of talidendang besar, riau, east sumatera. study area and methods this research was carried out in january 1991 in a mangrove forest concession area belonging to the bina lestari company at talidendang besar (long. 103°28' to 103°48' e, lat. 0°21' to 1°n), which lies on the east coast of sumatera, riau province, indonesia (fig. 1). no meteorological data were available for talidendang besar. the rainfall data of mandah, the nearest area to talidendang besar, provide a good approximation of the climatic conditions (fig. 1). the average annual rainfall is about 1335 mm. according to the schmidt and ferguson system (1951), the climate of this area belongs to the type b rainfall with seven wet, two dry and three humid months. the mangrove forest in this area is characterized by the bruguiera species. the seaward fringe is strongly dominated by b. parviflora and landward zones are numerically dominated by b. sexangula. in the transition zone with a fresh-water swamp forest, the stand of b. sexangula is invaded by nypa fruticans, in which a few large individual trees of r. apiculata form an emergent layer above an even canopy of b. sexangula. in this zone, a few trees of a marginal species, ficus benjamina, occur in open areas created by fallen or dead trees and sometimes intermingled with n. fruticans along the river bank. sample trees of b. parviflora, b. sexangula and r. apiculata which have similar diameter sizes were felled. stem diameter at breast-height (dbh) and tree height of sample trees were 20.1 cm and 19.4 m for b. parviflora, and 19.2 cm and 17.6m for b. sexangula, respectively. however, the diameter at 20 cm above the highest prop-roots and the height of a felled sample tree of r. apiculata were 19.5 cm and 22.8 m, respectively. sample trees were felled at ground level and cut into horizons of 1 m length along the stem. fresh weight of stems, branches, flowers and fruits, and leaves of each horizon were separately weighed. a small sample of each organ from each horizon was taken and dried in an oven at 80°c for 48 hours to obtain a constant dry weight. the leaf area of a small sample of leaves from each horizon was measured with a leaf-area meter (type aam-7, hayashi denko). 2 production structure of main commercial tree species cecep kusmana & hiroyuki watanabe figure 1. location and climatic diagram of the research area 3 biotropia no. 5, 1991/1992 results and discussion results tables 1, 2 and 3 show the estimated aboveground biomass of anatomical organs of each tree, the leaf area and specific leaf area of a sample tree of r. apiculata, b. sexangula and b. parviflora, respectively. the aboveground biomass of sample trees of r. apiculata, b. sexangula and b. parviflora were estimated at 381 kg d.wt, 387.70 kg d.wt and 296.36 kg d.wt, respectively. compared to the sample trees of r. apiculata and b. sexangula, a sample tree of b. parviflora has a smaller aboveground biomass, although its diameter (dbh) was similar to the sample trees of the two former species. the leaves as a photosynthetic part contributed very little to biomass matters, i.e. 2.48%, 3.61% and 4.48% for r. apiculata, b. sexangula and b. parviflora, respectively. the total leaf area as well as the mean specific leaf area of r. apiculata were smaller than those of the other species. it was estimated that the mean specific leaf areas were 61.19 cmvg d.wt for r. apiculata, 77.57 cmvg d.wt for b. sexangula, and 80.80 cmvg d.wt for b. parviflora. it indicates that the leaves of r. apiculata were thickest, while the leaves of b. sexangula were thicker than those of b. parviflora. the production structure of the main commercial trfee species is shown in fig. 2. the branches and leaves of r. apiculata were shallower and sparser than those of b. sexangula and b. parviflora. the large amount of leaves and branches of r. apiculata in the stem horizon of 13 to 14 m was due to the existence of large main branches with a diameter of 13.5 cm. in addition, b. sexangula has a larger proportion of branches than the other species. discussion as indicated by the mean specific leaf area (table 1) and vertical production structure (fig. 2), r. apiculata is recognized to have a sparser foliage of thicker leaves than b. sexangula and b. parviflora. it is suggested that one characteristic of/?, apiculata is to be a shade-intolerant tree species as reported by macnae (1968). in the mangrove forest of halmahera, maluku province in indonesia, komiyama et al. (1988) reported that r. apiculata has a sparser foliage of thicker leaves than b. gymnorrhiza. similar results were also reported by ninomiya et al. (1989) for r. stylosa and b. gymnorrhiza in the mangrove forest of funaura bay, iriomote island in okinawa, and by nakasuga (1979) for r. mucronata and b. conjugata in the ryukyu islands, southern japan. 4 production structure of main commercial tree species cecep kusmana & hiroyuki watanabe table 1. estimated aboveground biomass, leaf area (la) and specific leaf area (sla) of the sample tree of rhizophora apiculata horizon aboveground biomass (kg d.wt) la sla (m) leaf flowers and branch stem prop-roots total (m2) (cm2/g d.wt) fruits 01 9.57 16.74 26.31 12 22.65 13.77 36.42 23 22.33 22.33 34 19.46 19.46 45 18.82 18.82 56 18.82 18.82 67 18.50 18.50 7g 0.07 0.01 6.68 16.59 23.35 0.36 66.09 89 16.27 16.27 9-10 0.08 0.01 0.33 15.63 16.05 0.52 62.23 10-11 14.36 14.36 11-12 14.67 14.67 12-13 12.44 12.44 13-14 5.34 0.71 63.67 10.53 80.25 21.15 50.49 14-15 0.03 0.01 0.17 5.42 5.63 0.09 67.87 15-16 5.30 5.30 16-17 0.14 0.01 0.45 4.72 5.32 0.62 50.38 17-18 0.45 0.04 1.67 4.15 6.31 2.08 50.43 18-19 0.50 0.07 2.17 3.13 5.87 2.12 68.11 19-20 1.37 0.16 4.07 2.04 7.64 6.44 63.37 20-21 0.71 0.10 1.70 1.40 3.91 3.63 62.56 21-22 0.59 0.11 1.11 0.64 2.45 2.46 63.87 22-22.8 0.17 0.03 0.18 0.14 0.52 0.68 67.68 total 9.45 1.26 82.20 257.58 30.51 381.00 40.15 673.08 average 61.19 the prop-roots constituted about 8% of the aboveground biomass of a sample tree of r. apiculata. it is supposed that in this area the stand of r. apiculata grows in estuarine conditions. lugo and snedaker (1974) also reported the low proportion of prop-roots (2%, 15%) in estuarine forest in the mangroves of florida. in addition, christensen (1978) stated that the small proportion of prop-roots of rhizophora stand growing in the estuarine conditions might be due to a better nutrient supply or reduced salinity regime. the existence of the prop-roots on the stem may be regarded as a characteristic to distinguish the production structure of r. apiculata from that of b. sexangula and b. parviflora. on the other hand, b. parviflora and b. sexangula tended to have a greater proportion of leaves and branches along the stem than r. apiculata. 5 biotropia no. 5, 1991/1992 table 2. estimated aboveground biomass, leaf area (la), and specific leaf area (sla) of the sample tree of bruguiera sexangula horizon aboveground biomass (kg d.wt) la sla (m) (m2) (cm2/g d.wt) leaf flowers and fruits branch stem total 0 1 40.52 40.52 12 23.73 23.73 23 23.79 23.79 34 21.90 21.90 45 20.44 20.44 56 0.15 0.01 0.77 18.62 19.55 1.53 97.96 67 17.16 17.16 78 0.21 0.03 2.71 18.25 21.20 2.34 79.87 89 0.46 0.05 3.84 17.16 21.51 4.36 75.73 9-10 0.27 0.04 2.07 15.70 18.08 2.55 77.36 10-11 2.86 0.37 29.68 16.79 49.70 21.22 80.49 11-12 4.09 0.61 39.65 12.05 56.40 21.48 74.75 12-13 0.14 0.02 0.89 6.75 7.80 1.51 75.29 13-14 1.69 0.27 11.21 5.48 18.65 10.86 80.41 14-15 1.62 0.24 6.93 3.87 12.66 9.59 74.99 15-16 1.56 0.34 6.84 1.61 10.35 8.83 71.12 16-17 0.11 0.12 2.04 0.91 3.88 5.54 72.84 17 17.6 0.14 0.02 0.15 0.07 0.38 0.70 70.05 total 14.00 2.12 106.78 264.80 387.70 90.51 930.86 average 77.57 conclusion while the leaves of b. parviflora were thinner than those of b. sexangula, rhizophora apiculata has a sparser foliage of thicker leaves than the two former tree species. in addition, either b. parviflora or b. sexangula has a larger proportion of leaves and branches along the stem than r. apiculata. it is suggested that one characteristic of r. apiculata is a shade-intolerant tree species whereas b. parviflora and b. sexangula are shade-tolerant tree species. 6 production structure of main commercial tree species cecep kusmana & hiroyuki watanabe table 3. estimated aboveground biomass, leaf area (la), and specific leaf area (sla) of the sample tree of bruguiera parviflora horizon aboveground biomass (kg d.wt) la sla (m) (m2) (cm2/g d.wt) leaf flowers and fruits branch stem total 0 1 28.81 28.81 12 21.11 21.11 23 18.43 18.43 34 17.76 17.76 45 17.42 17.42 56 16.08 16.08 67 15.08 15.08 78 14.41 14.41 89 0.50 0.07 4.43 13.40 18.40 5.41 109.16 9-10 4.30 0.39 23.78 12.40 40.87 31.89 73.13 10-11 0.64 0.11 2.71 9.72 13.18 5.04 81.96 11-12 8.71 8.71 12-13 0.09 0.02 0.21 7.71 8.03 0.73 99.01 13-14 1.66 0.32 9.68 8.04 19.70 14.36 85.18 14-15 1.99 0.35 8.64 4.69 15.67 12.91 75.75 15-16 1.11 0.22 4.43 3.02 8.78 6.97 71.12 16-17 2.06 0.45 6.23 1.34 10.08 13.50 85.03 17-18 0.83 0.18 1.80 0.67 3.48 5.56 73.67 18-19 0.08 0.01 0.11 0.10 0.30 0.47 68.36 19 19.4 0.03 0.01 0.01 0.01 0.06 0.15 66.47 total 13.29 2.13 62.03 218.91 296.36 96.99 888.84 average 80.80 acknowledgements we wish to thank mr. subkhan, student of the faculty of forestry, bogor agricultural university for helping in the collection of data in the field; dr. dudung darusman, dean of the faculty of forestry, bogor agricultural university for his kind assistance in providing admission letters; prof .dr. ishemat soerianegara and ir. syafii manan, msc., faculty of forestry, bogor agricultural university for their encouragement throughout this research. 7 biotropia no. 5, 1991/1992 8 f ig u re 2 . p ro d u ct io n s tr u ct u re o f a sa m p le t re e o f r . a p ic u la ta ( le ft ), b . p a rv if lo ra ( m id d le ), a n d b . se xa n g u la ( ri g h t) production structure of main commercial tree species cecep kusmana & hiroyuki watanabe references christensen, b. 1978. biomass and primary production of rhizophora apiculata bl. in a mangrove in southern thailand. aquatic botany 4: 43-52. darsidi, a. 1984. mangrove forest management in indonesia. in: soemodihardjo, s., i. soerianegara, m. sutisna, k. kartawinata, supardi, n. naamin and h. al rasyid (eds.), proceedings symposium ii on mangrove ecosystem: p. 27-37. mab, lipi, jakarta. komiyama, a., h. moriya, s. prawiroatmodjo, t. toma and k. ogino. 1988. forest as an ecosystem, its structure and function: 2. primary productivity of mangrove forest. in: k. ogino and m. chihara (eds.), biological system of mangroves: a report of east indonesian mangrove expedition 1986: 97-106. ehime university, ehime, japan. luoo, a.e. and s.c. snedaker. 1974. the ecology of mangroves. ann. rev. ecol. syst. 5: 39-65. macnae, w. 1968. a general account of the fauna and flora of mangrove swamps and forests in the indo west pacific region. adv. mar. biol. 6: 73-270. nakasuga, t. 1979. analysis of the mangrove stand. sci. bull. coll. agric. univ. ryukyus 26: 413 519 (in japanese with english summary). ninomiya, i., a. komiyama, h. moriya, y. tadaki, and k. ogino. 1989. production structure of rhizophora stylosa and bruguiera gymnorrhiza in funaura, iriomote. galaxea 8: 65-68. schmidt, f.h. and j.h.a. ferguson. 1951. rainfall type based on wet and dry period ratios of indonesia with western new guinea. verhandelingen no. 42, kementrian perhubungan, djawat an meteorologi dan geofisika, jakarta. 77 p. soegiarto, a. 1979. the indonesian marine environment: its problems and management. ber. ilmu peng. teknol. 23 (1): 9-21. 9 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf biotropia no biotropia no. 15, 2000 : 58 75 stored cocoa beans quality affected by fermentation and ephestia cautella walker (lepidoptera: phycitidae) infestation ok.ky s. dharmaputra' \ sunjaya', ina retnowati' and santi ambarwati' 'seamed biotrop, p.o. box 116, bogor, indonesia; and department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia abstract the effects of fermentation on ephestia cautella population and cocoa beans quality in terms of moisture content, fungal population, the percentage of insect-damaged and mouldy beans, lipid and free fatty acid contents during storage were investigated together with the effects of £. cautella infestation on the quality of stored cocoa beans and weight loss. fermented and unfermented cocoa beans with initial moisture contents of 7 or 9% were placed in ventilated plastic jars (ikg/jar) and stored for 6 months under room conditions. seven larvae of £. cautella instar iv (2 males and 5 females) were introduced in each jar at the beginning of storage. untreated jars contained only cocoa beans. population of £. cautella on fermented cocoa beans with either initial moisture content of 7 or 9% was lower than that on unfermented beans during storage. the population either on fermented or unfermented cocoa beans with initial moisture content of 7% was lower than that of 9%, and the population of all treatments increased during storage. moisture content of all treatments either on cocoa beans with initial moisture contents of 7 or 9% had the same pattern. the percentage of insect-damaged beans on fermented cocoa beans was lower than that on unfermented cocoa beans after 5 to 6 months of storage. the damaged beans on fermented cocoa after 6 months of storage was not different than on unfermented beans after 4 months of storage. the weight loss either on fermented or unfermented cocoa beans with initial moisture content of 9% was higher than that with initial moisture content of 7%. the weight loss on fermented cocoa beans either with moisture content of 7 or 9% was lower than that on unfermented beans during storage. the weight loss either on fermented or unfermented cocoa beans increased during storage. the percentage of mouldy beans on cocoa infested with £. cautella tended to increase during storage, while on beans not infested with the insect it fluctuated during storage. the highest percentage of mouldy beans was on unfermented and infested cocoa beans. twenty-one fungal species were isolated from all treatments of cocoa beans during storage. the total fungal population on fermented and unfermented beans had the same pattern. the population on fermented cocoa beans was lower than that on unfermented beans. total lipid content on fermented cocoa beans either infested or not with £. cautella having initial moisture content of 7 or 9%, was lower than that of unfermented beans. the content either on fermented or unfermented cocoa beans and either infested or not decreased during storage. free fatty acid content on cocoa beans infested with £. cautella was higher and significantly different than that on not infested. the content for both types increased during storage. key words : cocoa beans / fermentation / ephestia cautella i moisture content / fungal population / insect-damaged beans / mouldy beans / lipid / free fatty acid. introduction according to the international cocoa organization (1996), indonesia ranks third among the cocoa producing countries of the world after ivory coast and ghana. it has been predicted that the production of indonesian cocoa beans will 58 biotropia no. 15, 2000 increase by 4.76% per year for the period of 1995-2005. the increase is especially due to the policy which has been determined by the government of indonesia through the development and quality improvement of plant material programs (susila 1996). siswoputranto (1997) reported that in indonesia cocoa is now the fourth most important export commodity after palm oil, rubber and coffee. compared to west african cocoa beans, indonesian cocoa beans have an excessive acidic flavor, low chocolate flavor and often certain other objectionable off-flavors. consequently, they are less desired by cocoa importing countries (duncan 1990). the imperfections in the fermentation process in particular by smallholders should be improved correctly, if better markets and higher prices for well fermented beans are expected through maintaining the quality of indonesian (smallholders') cocoa (siswoputranto 1997). according to zaenudin and wahyudi (1996), insect and mould attacks were the problems of exported cocoa beans derived from smallholders, so that they were subjected to automatic detention. consequently they should be fumigated, which will need additional expenses. the problem of automatic detention could be minimized by improving the method of postharvest handling from farmer to exporter levels, and thus the quality of indonesian cocoa beans will also be improved. with regard to the low quality of cocoa beans, importing country (usa) slapped a fine of 165-180 usd per ton. south sulawesi exported 120,000-140,000 tons of cocoa beans per year with an export value of 130 million usd (kompas 1996). kalshoven (1981) and wood (1985) reported that ephestia cautella walker (lepidoptera: phycitidae) (tropical warehouse moth) is associated with stored cocoa beans. according to dharmaputra et al. (1999) e. cautella was found in some samples of cocoa beans at exporter level in south sulawesi. there is an urgent need for indonesia to improve the quality of smallholders' cocoa beans for having good markets with better prices, and for anticipating whatever changes of markets in this coming era of global economy and international free trade, where severe competitions will be faced by cocoa producing countries, despite deficits of supply which may occur. the objective of this study was to investigate the effects of fermentation on e. cautella population and cocoa beans quality in terms of moisture content, fungal population, the percentage of insect-damaged and mouldy cocoa beans, lipid and free fatty acid content during storage. the effects of e. cautella infestation on the quality of stored cocoa beans and weight loss were also analyzed. materials and methods storage of cocoa beans and insect infestation fermented and unfermented cocoa beans (bulk type) obtained from rajamandala estate crop (ptpn viii), cipatat, bandung, were used in this study. 59 stored cocoa beans quality okky s. dharmaputra et al. soon after harvest, the pods of cocoa beans were opened and removed. the first category of cocoa beans was fermented using the standard method of the state estate crops soon after removing the beans from the pods. after fermentation process, they were washed and sundried until about 7% of moisture content was reached. the second category of cocoa beans was not fermented, but they were washed directly after removing from the pods. prior to storage, cocoa beans were fumigated with phosphine at dosage rates of 2g/ton with an exposure period of five days in order to kill any stage of insect that may exist. after fumigation, the moisture content of each cocoa bean was adjusted (7 and 9%). they were then placed in ventilated plastic jars (1 kg/jar) and stored for 6 months under room conditions. seven larvae of e. cautella instar iv (2 males and 5 females) were introduced in each jar at the beginning of storage. untreated jars contained only cocoa beans. three replications were used for each treatment. methods of sampling a sample consisting of the whole content of each jar was taken before storage, and subsequently after 1, 2, 3, 4, 5 and 6 months of storage. twenty-four samples were taken at each month of storage. consequently, the number of all experiment units was 168. insects were separated from cocoa beans using graded sieves. each cocoa bean sample was then divided several times using a sample divider to obtain working samples for analyzing insect-damaged and mouldy beans, fungal, moisture, lipid and free fatty acid contents. insect and fungal population determinations insect population (larva, pupa and adult) per kg of cocoa beans derived from each jar was determined by counting the number of dead and live insects in the outer and inner parts of the beans after separation from the cocoa beans. fungal population was determined based on dilution method followed by pour plate method using dichloran 18% glycerol agar (dg18) (pitt and hocking 1997). fungal species was identified using the publications of samson et al. (1996), pitt and hocking (1997) as the main references. moisture, lipid and free fatty acid contents analyses moisture content (wet weight) of cocoa beans was determined based on sni 01-2323 (isc 1998). two replicates were used for each sample. the beans were ground and dried in the oven at 103° ± 2°c for 16 hours. the moisture content was determined using the following formula: me = (m,-m2)x 10° m| mo me = moisture content (%) 60 biotropia no. 15, 2000 m0 = the mass, in grams, of the empty dish and its lid mi = the mass, in grams, of the dish and its lid, and the test portion before drying m2 = the mass, in grams, of the dish and its lid, and the test portion after drying total lipid content was determined using soxhlet extraction method (isc 1998). the principle of this method is the extraction of free oil from the cocoa bean sample using non polar organic solvent («-hexane) which has been hydrolyzed. total lipid content was expressed as the percentage of mass and calculated on a dry weight basis, using the following formula: % total lipid content = (m1 – m2) 100 x 100 m0 100 mc me = moisture cpntent of the test sample mi = the mass, in grams, of the flask and lipid after drying m2 = the mass, in grams, of the dry flask mo = the mass, in grams, of the test sample free fatty acid content was determined using titration method (isc 1998). fat obtained from extraction is dissolved in warm ethanol and then titrated using alkali solution (naoh 0.1n). free fatty acid was calculated and expressed as the percentage of mass per mass using the following formula: % free fatty acid content = v x n x 100 m 100 mc v = the volume, in ml, of naoh n = the normality of naoh solution m = the mass, in grams, of cocoa bean lipid me = moisture content insect-damaged and mouldy beans, and weight loss determinations insect-damaged beans are those the internal parts of which contain dead insects at any stage of development, or which show damage by insects, visible to the naked eye. mouldy beans are those with fungi (mould) in the inner part. the percentages of insect-damaged and mouldy beans were determined according to sni 01-2323 (isc 1998). the percentage of dry weight loss was determined based on harris and linblad (1977) using the following formula : % weight loss = (u.nd) – (d.nu) x 100 u (nd = nu) 61 stored cocoa beans quality okky s. dharmaputra et a/. u = weight of undamaged beans nu = number of undamaged beans d = weight of damaged beans nd = number of damaged beans experimental data experimental data were analyzed using completely randomized factorial design with 4 factors. the 1st, 2nd, 3rd and 4th factors were fermentation, initial moisture content, e. cautella infestation and duration of storage, respectively. results and discussion e. cautella population the population of e. cautella on fermented cocoa beans with either initial moisture content of 7% or 9% was lower than that on unfermented beans during storage (figure 1). preliminary study on the preference test of e. cautella on fermented and unfermented cocoa beans showed that the number of insects found on fermented cocoa beans was lower than that on unfermented cocoa beans (table 1). it seems that the insects prefered unfermented cocoa beans. according to wood (1985) the unpleasant smell is caused by high content of acetic acid in fermented cocoa beans. 62 biotropia no. 15, 2000 table 1. preference test ofephestia cautella on cocoa beans treatment number of larvae found (insect/replication) fermented beans i ± 0.89 unfermented beans 10 ± 2.37 the population of e. cautella either on fermented or unfermented cocoa beans with " initial moisture content of 7% was lower than that of 9% during storage (figure 1). the change of moisture content during storage affected the growth and the development of insects. in foodstuff storage, moisture content is one of the important factors related with the growth development of insects (sinha and muir 1973;haines 1991). populations of e. cautella of all treatments increased during storage. after 6 months of storage, the lowest population was found on fermented cocoa beans with initial moisture content of 7% (24 insects/kg), while the highest was on unfermented cocoa beans with initial moisture content of 9% (122 insects/kg). moisture content moisture content of all treatments on cocoa beans with initial moisture contents of 7 or 9% had the same pattern. it decreased until 4 months of storage and then increased after 5 to 6 months of storage (figure 2). the moisture contents of fermented and unfermented beans with insects tended to decrease until 5 months of storage and increase after 6 months of storage. it was assumed that the change of the moisture content was affected by the insect activity, the presence of fungi and biochemical reactions of the commodities (sinha and muir 1973). insect-damaged beans insects are the most important cause of deterioration of stored grain ecosystems. e. cautella belongs to one of internal feeder insects, because it can move inside of the beans and cause damage to the inner parts of the beans. deterioration caused by e. cautella was determined by the presence of hole on beans, feces, silk webbing, dead bodies and frass. after 1 month of storage, the percentage of insect-damaged beans on fermented cocoa beans was not different than on unfermented ones. however, the percentage on fermented cocoa beans was lower (p < 0.05) than that on unfermented cocoa beans after 5 to 6 months of storage (figure 3). the population of e. cautella on fermented cocoa beans was lower than that on unfermented cocoa beans, consequently the insect-damaged beans on fermented cocoa beans was lower than that on unfermented cocoa beans. kresnowati (1999) reported that the percentage of damaged beans infested by araecerus fasciculatus on fermented cocoa beans was lower than that on unfermented ones. 63 stored cocoa beans quality – okky s. dharmaputra et al. storage duration (month) figure 2. moisture content of cocoa beans during storage figure 3. insect-damaged beans of cocoa during storage (note : se mean = 1.2554370) 64 biotropia no. 15, 2000 the damaged beans on fermented cocoa after 6 months of storage was not different than on unfermented beans after 4 months of storage. this showed that fermented cocoa beans could be stored for a longer period. weight loss caused by e. cautella infestation the loss agents generally fall into three classes: unavoidable (e.g. weather), human-induced (e.g., contamination, spillage, theft), and pest-induced (e.g. insects, mites, rodents) (haines 1995). the weight loss either on fermented or unfermented cocoa beans with initial moisture content of 9% was higher than that with initial moisture content of 7%. nevertheless, the weight loss of fermented beans with the two initial moisture contents was not significantly different. the highest weight loss was found on unfermented cocoa beans with initial moisture content of 9%. the weight loss on fermented cocoa beans either with moisture content of 7 or 9% was lower than that on unfermented beans during storage (figure 4). the weight loss either on fermented or unfermented cocoa beans increased during storage (figure 5). the increase of weight loss was related to the percentage of damaged beans caused by e. cautella infestation. figure 4. percentage of weight loss on fermented and unfermented cocoa beans with defferent initial moisture contents 65 stored cocoa beans quality – okky s. dharmaputra et al. figure 5. percentage of weight loss on fermented and unfermented cocoa beans during storage mouldy beans the highest percentage of mouldy beans was on unfermented and infested cocoa beans (figure 6). the percentage on fermented cocoa beans increased during storage. the percentage of mouldy unfermented cocoa beans increased after 1 month of storage and decreased until after 3 months of storage, and then increased until after 6 months of storage (figure 7). the percentage of mouldy cocoa beans infested with e. cautella tended to increase during storage, while the percentage of mouldy cocoa beans that were not infested with the insect fluctuated during storage (figure 8). species and total fungal population twenty-one fungal species were isolated from all treatments of cocoa beans during storage. fungi isolated from all treatments during storage are shown in tables 2, 3, 4 and 5. cladosporium cladosporioides. eurotium chevalieri and penicillium citrinum were always isolated from all treatments during storage. the total fungal population from all treatments during storage was relatively low (tables 2,3,4 and 5). it was due to the good quality of cocoa beans used in this study. retnowati et al. (2000) reported that the low total fungal population was also found on fermented and unfermented cocoa beans obtained from rajamandala estate crop (ptpn viii), introduced or not with a. fasciculutus. 66 biotropia no. 15, 2000 table 2. species and total fungal population of unfermented cocoa beans, infested with ephestia cautella with initial moisture content of 7 % i = infested ni = not infested table 3. species and total fungal population or fermented cocoa beans, infested and not infested with ephestia cautella with initial moisture content of 7 % i = infested ni = not infested 67 stored cocoa beans quality – okky s. dharmaputra et al. table 4. species and total fungal population of unfermented cocoa beans, infested and not infested with ephestia cautella with initial moisture content of 9 % i = inferted ni = not infested table 4. species and total fungal population of fermented cocoa beans, infested and not infested with ephestia cautella with initial moisture content of 9 % i = inferted ni = not infested 68 biotropia no. 15, 2000 figure 6. percentage of mouldy beans on fermented and unfermented cocoa infested and not infested with ephestia cautella during storage (note : se mean = 0.19107623) storage duration (month) figure 7. percentage of mouldy beans on fermented and unfermented cocoa during storage (note : se mean = 0.35747093 69 stored cocoa beans quality – okky s. dharmaputra et al. storage duration (month) figure 8. percentage of mouldy beans on cocoa infested and not infested vvith ephestia caulelta during storage (note: se mean = 0.35739599) the total fungal population on fermented and unfermented beans had a similar pattern. it decreased after 1 month of storage and increased after 2 to 5 months of storage, and then decreased again after 6 months of storage. it was assumed that the decrease of total fungal population on unfermented beans after 1 month of storage was due to the interactions among fungi infecting the beans. nevertheless, the population of fungi on fermented and unfermented beans showed no significant difference (p>0.05) during storage, except on unfermented cocoa beans after 5 months of storage (figure 9). the fungal population on fermented cocoa beans was lower than that on unfermented beans. retnowati et al. (2000) also reported that total fungal population on fermented cocoa beans was lower than unfermented ones. figure 9. total fungal population of fermented and unfermented cocoa beans during storage (note : se mean = 6.59485150) 70 biotropia no. 15, 2000 total lipid content lipids are triglycerides composed of glycerol and 3-oh group which bind 3 fatty acids (sherman and sherman 1989). according to belitz and grosch (1987) lipids are important food flavour substances and the most important component in cocoa. wood (1985) reported that the total lipid content of good cocoa beans quality is between 56 58%. total lipid content of fermented cocoa beans either infested or not with e. cautella having initial moisture contents of 7 or 9% was lower than that on unfermented beans (figure 10). it was due to the presence of fatty acids produced from fermentation process that stimulate lipid hydrolysis into fatty acids and glycerol. according to wood (1985) during fermentation process, lactic acid bacteria homofermentor transformed glucose into lactic acid. lactic acid bacteria hetero-fermentor transformed glucose into lactic acid, alcohol, acetic acid and cc>2. winarno (1991) revealed under certain conditions, such as the presence of acid, lipids are hydrolyzed into fatty acids and glycerol. this study supports the findings of retnowati et al. (2000) that the total lipid content of fermented cocoa beans was lower than that of unfermented beans. figure 10. total lipid content of fermented and unfermented cocoa beans infested and not infested with ephestia cautella having different initial moisture contents (note: se mean = 0.0648736) the lipid content of either fermented or unfermented cocoa beans with or without e. cautella decreased during storage (figure 11), due to the transformation of lipids into free fatty acids (pomeranz 1992). retnowati et al. (2000) also reported that total lipid content decreased during storage. 71 stored cocoa beans quality okky s. dhurmaputra el al free fatty acid content the presence of free fatty acids (ffa) gives an indication of the quality of cocoa beans in producing cocoa butter. cocoa butter with high levels of ffa tend to be soft, have poor crystallization properties, contain off-flavour, and have a poor shelf life (nickless 1994). figure 11. total lipid content of fermented and unfermented cocoa beans infested and not infested with ephestia cautella during storage (note: se mean = 0.1213675) ffa content of cocoa beans infested with e. cautella was significantly (p< 0.05) higher than that without insects (figure 12). the content for both types increased during storage (figure 13). the content on fermented cocoa beans with initial moisture content of 7% was significantly (p< 0.05) lower than that on the beans with initial moisture content of 9%, while the level on unfermented cocoa beans with initial moisture content of 7% was significantly (p< 0.05) higher than that on the beans with initial moisture content of 9% (figure 14). on fermented cocoa beans lipids are easier to be hydrolyzed into ffa, because there are many fatty acids produced from fermentation process. consequently, ffa on fermented cocoa beans was higher than that on unfermented beans, and it increased during storage. 72 biotropia no. 15, 2000 figure 12. free fatty aced content of cocoa beans infested and not infested with ephestia caoutella ( note : se mean = 0.00267261 storage duration (month) figure 13. free fatty acid content of cocoa beans during storage figure 14. free fatty content of fermented and unfermented cocoa beans with different initial moisture contents (note : se mean =0.00377976) 73 stored cocoa beans quality okky s. dharmaputra et al. conclusions in relation to e. cautella population and total fungal population during 6 months of storage, the quality of fermented cocoa beans and initial moisture content of ± 7% was better than that of unfermented cocoa beans and initial moisture content of ±9%. infestation of e. cautella decreased cocoa beans quality in terms of the percentage of mouldy beans, free fatty acid content, damaged beans and weight loss caused by e. cautella during 6 months of storage. fermented cocoa beans not infested with e. cautella having initial moisture content of 7% could be stored for a longer period than unfermented cocoa beans infested with the insect having initial moisture content of 9%. fermentation, insect control and initial moisture content of 7% are the important factors for maintaining the quality of cocoa beans during storage. acknowledgement the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the indonesian research institute for coffee and cocoa, jember, indonesia in providing e. cautella for mass production. the authors are also grateful to the rajamandala estate crop (ptpn viii), cipatat, bandung, for cocoa beans preparation, and to ms diana puspitasari for her assistance in conducting the experiment. references belitz, h.d. and w. grosch. 1987. food chemistry. springer verlag berlin. heidelberg. 128 p. dharmaputra, o.s., sunjaya, m. amad, i. retnowati and t. wahyudi. 1999. the occurrence of insects and moulds in stored cocoa beans at south sulawesi. biotropia (12): 1-18. duncan, r.j.e. 1990. the sime-cadbury process, background and development. paper presented at the seminar on improvements of cocoa beans processing. jakarta. 30 october 1989. haines, c.p. 1991. insects and arachnids of tropical stored product: their biology and identification (a training manual). natural resources institute. uk. haines, c.p. 1995. grain storage in the tropics. in d.s. jayas, n.d.g. white and w.e. muir(eds.). stored grain ecosystems. marcel dekker inc., new york.p. 55-100. harris, k.l. andc.j. lindblad. 1977. postharvest grain loss assesment methods. a manual of methods for the evaluation of postharvest losses. office of nutrition, agent for international development, us. international cocoa organization. 1996. production of cocoa beans by country, 1986/87-1995/96. quart. bull, of cocoa statistics 22(3): 6. international cocoa organization, london. indonesian standardization council. 1998. indonesian national standard, sn1 01-2323, rev.1995: cocoa beans. 74 biotropia no. 15 ,2000 kalshoven, l.g.e. 1981. the pest of crops in indonesia. revised by p.a. van der laan. p.t. ichtiar baru van hoeve, jakarta. kompas. 14 november 1996. kakao indonesia belum ditangani secara profesional. kompas 1996. kresnowati, k. f. 1999. kualitas biji kakao akibat fermentasi dan serangan araecerus fasciculatus de geer (coleoptera: anthribidae) selama penyimpanan. (cocoa beans quality affected by fermentation and araecerus fasciculatus de geer (coleoptera: anthribidae) infestation during storage). department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor. (in indonesian). nickless, h. 1994. cocoa butter quality. in j. selamat, b.c. lian, t.k. lai, w.r.w. ishak and m. mansor. malaysian cocoa board. proceedings of the malaysian international cocoa conference, p. 322-336. pitt, j.i. and a.d. hocking. 1997. fungi and food spoilage. black academic and professional, cambridge. pomeranz, y. 1992. biochemical, functional, and nutritive changes during storage. in d.b. sauer (ed.). storage of cereal grains and their products. fourth edition. american association of cereal chemists, inc., minnesota, p. 55 142. retnowati, i., o.s. dharmaputra, sunjaya, k.f. kresnowati. 2000. keberadaan jamur pascapanen pada biji kakao yang difermentasi dan terserang araecerus fasciculatus de geer (coleoptera: anthribidae) selama penyimpanan (the occurrence of storage fungi on fermented cocoa beans and infested by araecerus fasciculatus de geer (coleoptera: anthribidae) during storage). proc. 15* national congress and scientific seminar of the indonesian society for phytophatology. purwokerto, 16-18 september 1999. p. 519-526 samson, r.a., e.s. hoekstra, c.j. frisvad, and o. filtenborg. 1996. introduction to foodborne fungi. centraalbureau voor schimmelcultures, baarn, the netherlands. sherman, a. and sherman j.s. 1989. chemistry and our changing world. second edition. prentice hall, inc., englewood cliffs. new jersey. sinha, r.n. and w.e. muir. 1973. grain storage: part of a system. the avi publishing company, inc. connecticut. siswoputranto, p.s. 1997. indonesian cocoa. in report of the 2"d meeting of national focal point for asean cocoa club on asean cooperation and joint approaches in agriculture and forest products promotion scheme. jakarta, indonesia, 4-5 march 1997. ministry of agriculture, republic of indonesia. susila, w.r. 1996. prospek pasar kakao dunia. warta puslit kopi dan kakao 12(1):1 11. winarno, f.g. 1991. kimia pangan dan gizi. pt. gramedia pustaka utama, jakarta. 253 h wood, g.a.r. 1985. from harvest to store. in g.a.r wood and r.a. lass (eds.). cocoa. fourth edition. longman, london, p. 444 504. zaenudin and t. wahyudi. 1996. laporan kunjungan tim askindo ke amerika serikat dalam upaya meniadakan automatic detention terhadap kakao indonesia. warta puslit. kopi dan kakao 12(1): 44-47. 75 microsoft word 37 biotropiavol. 13 no. 1, 2006 : 37 -48 identification of oceanographic parameters for determining pelagic tuna fishing ground in the north papua waters using multi-sensor satellite data vlncentius slregar1'and harold joppie davido waas2) "seameo b1otrop, bogor, indonesia 2> faculty of fisheries, university ofpattimura, ambon, indonesia abstract the north papua waters as one of the important fishing grounds in the world contribute approximately 75% of world production of pelagic tunas. these fishing grounds are still determined by hunting method. this method is time consuming and costly. however, in many areas determination of fishing ground using satellited data lias been applied by detecting the important oceanographic parameter of the presence of fish schooling such as, sea surface temperature and chlorophyl. mostly these parameters are used integratedly. the aim of this study is to assess the important oceanographic parameters detected from multi-sensor satellites (noaa/avhrr, seawifs and topex poisedon) for determining fishing ground of pelagic tunas in the north papua waters at east season. the parameters include sea surface temperature (stt), chlorophyl-a and currents. the availability of data from optic sensor (seawifs: chl-a and avhrr: thermal) is limited by the presence of cloud cover. in that case, topex poseidon satellite data can be used to provide the currents data. the integration of data from multi-sensors increases the availability of the oceanographic parameters for prediction of the potential fishing zones in the study area. key words : identification, oceanographic parameter, sea surface-temperature, chlorophyl-a, multi sensors, pelagic tuna, north papua-waters introduction the north papua waters in west pacific is one of the important fishing grounds for tuna in the world, beside the east pasific waters, japan and west coast of africa. it has contributed 75 % of world production of skipjack (katsuwonus pelamis) and yellow fin (thunnus albacares) (lewis and williams 2001). the fishing activities of local fishermen and the fishing fleet of a national company in this area is characterized by hunting method, eventhough actually using echo-sounder to detect fish schools of tunas. they use natural features such as birds, floating woods, shools of dolphines and whales to locate the tuna schools. this feature having been in use since longtime, has convinced experts to consider as an indicator of the existence of pelagic tunas. however, this method is practically inefficient because it is time consuming and costly. according to previous researches, it is known that the presence of tunas in the waters is possible due to the availability of food in the area, which is indirectly trigged by the occurrence of thermal front, current (divergent and convergent) and eddys (dwivedi 2001 ; lehodey 2003). in line with the development of remote 37 integration of npp semi mechanistic-modelling t. june et al. sensing technology, these oceanography parameters, in real time, can be detected by sensors of satellite remote sensing. this study is intended to determine oceanography parameters as a key factor in the detection of potential fishing grounds of the skipjack and yellowfm tuna in north papua waters in west pasific using multi-sensors of satellite data. materials and methods the study was conducted in the north papua waters with coordinate 3° 40' s -6° 40' u and 130° e 142° e, from august to october 2003 (figure 1). satellite data used to identify oceanographic parameters for determining skipjack and yellowfm tuna fishing ground are collected from multi-sensors satellite (noaa/avrr, seawifs and topex poisedon). local area coverage (lac) of noaa/avhrr data received from lap an (indonesian agency of aerospace), biak ground station are used to map sea surface temperature (sst) distribution using the equation of me millan dan crosby (1984), as follows: sst data derived from noaa/avhrr were validated using triton mooring bouy data at the position of (02° n ; 137° e) , (05° n ; 137° e). these mooring data were received from the website 38   images of topex poisedon and seawifs received in the form of globale area coverage (gac) from website htlp://www-ccar.colorado.edu/ and http://seawifs.gsfc.nasa.gpy/. are utilized for current analysis (divergent, convergent and eddys), and chlorofill-a distribution. the analysis is performed according to pickard and emery (1990). meanwhile, fishing catch data are collected from the fishing fleet of purse seiner 1025 gt(amount :15) of pt.biak mina java. this purse seiner has dimension of 1800 m in length and 300 m in width. the fishing catch data is in the form of cpue (catch per unit of effort ton"1 per ship hour"1). 39  biotropia vol. 13 no. 1, 2006 results and discussions sst validation validation of sst derived from noaa/avhrr data using triton buoy data (in situ) showed that the sst from these two different systems has similar pattern with certain variations of value of the sst. this variation could happen depending on the oceanographic condition that controls the temperature structure at depth measurement (robinson 1985). t-test (95 %) showed that there is a strong correlation ( r=0.72) between the sst of these different systems (figure 2). therefore, we used this algorithm to map the distribution of sst for further analysis and discussion. fishing ground condition the oceanographic parameter of skipjack and yellowfm tunas fishing grounds detected from multi-satellite sensors showed that in the east season, the distribution of the fishing grounds is concentrated at warm water centroids with temperature range varying between 27.02 and 30.86°c, except during the first week of august where the temperature is lower than before, varying between 26.64 and 27.45 °c. low sst during this month is probably due to the appearance of equatorial upwelling which attains its peak in june-july (vinogradov 1981), with the lowest sst detected varying from 26.62 to 26.87 °c (waas 2004). from this range of sst, it is presumed that the appearance of two tuna species in july and august is related with the period of their spawning season, where the ideal temperature is limited to 26 °c (lehodey 2003), thus this water is a good spawning ground for skipjack and yellowfm tuna (loukos et al. 2003). chlorophyll-a (chl-a) is an indicator of the productivity of the waters which has indirect relationship with the tuna food found at high concentration in fishing grounds every month during the period of study. the concentration of chl-a detected varies between 0.21 and 0.35 ingm"3. the high concentration of chl-a 40 indenlification of oceanographic parameters v. siregar and h.j.d. waas (>0.2 mg m"3) indicates that the presence of this phytoplankton in the waters is suitable to support or to maintain the sustainability of the development of fisheries, such as skipjack and yellowfin tunas (gower 1972). lehodey (2003) and wcnno et al. (2001) found that there is a high concentration of chl-a near the coastal zone of north papua. this high concentration of chl-a is contributed not only by the upwelling (equatorial upwelling) process which occurred in that area, but also by the river flow (e.g.mamberamo river and matabori river) which bring nutrient to the north papua waters, and by halmahera eddies (lipi 1992). the surface current detected by topex poseidon satellite in the fishing grounds of skipjack and yellowfin tunas is classified as south equatorial current (sec) system, concentrated at 0° 2.5° n. the appearance of those tunas in this zone has strong relationship with the occurrence of divergent and convergent currents detected. the high concentration of phyto planktons during the equatorial upwelling at 0° 0.5° n, is predicted indirectly as a factor of the appearance of tunas at this system of currents. this condition confirms the hypothesis of uda (1973) in laevastu and hayes (1981) that the good fishing ground for tuna is found in the system of sec. oceanographic parameter derived from satellite data due to cloud cover, the number of images available from optic sensor used is limited. the number of images available are thermal: 10, chl-a distribution: 4 and currents : 12 images. those images were used for the analysis, but in this paper the number of images presented is limited to 2 for each type of data. the important parameter analysed from the available images are described as follows : thermal front analysis of thermal front using 10 images available showed that fronts could occur and develop in the north papua waters. the thermal front is detected from august until october 2003 in different forms and patterns. at the end of august the front is in the form of line streched from north of monokwari towards the northeast as long as 840 nautical miles. the position of fishing activities spread out near the thermal front in the warmest water mass. during september, the thermal front appears clearly. this front has a length varying from 550 920 nautical miles, move away off the waters of monokwari towards the northeast, forming a smaller width of meandering. intensive fishing was found concentrated at the meandering zone, which has a role as barrier, so that the phyto planktons could not move away from the front zone (dwivedi 2000). on the 2nd and 11th september (figure 3a,b), fishing grounds were clearly found near the thermal front. this phenomenon confirms narain's study (1993) that the high intensity of fishing occurs at the thermal front. the thermal front still exists during the month, move and change its form following the currents pattern. high concentrations of phyto planktons occur at the front due to two different water masses (warm and cold) and divergent currents (shixing et al. 1993). these currents can be seen from image of topex poseidon at the same date of acquisition. along the thermal front, a high 41   ^identification of oceanographic parameters v. siregar and h..i.d. waas concentration of chl-a was recorded, and at the same time, convergent current and eddys were taken place. in october, the thermal front has different pattern compared to the front of the previous month. this front forms cold water pockets with a width of ± 9.02 71.35 n.mile square and surrounded by warm water mass from the pacific ocean. the position of the fishing ground was generally concentrated outside of the thermal front zone. in the front zone, strong divergent current and eddys were taken place so that the cold water mass was spreading out following the direction of the current chlorophyll-a distribution during august and september, the images of the area were not realy clear due to the cloud cover. however, from those images and by plotting the area of fishing activities, it showed that skipjack and yellowfin tunas were found in the area of higher chl-a concentration and along the front (figure 4). in august the chl-a front is not clear. meanwhile, in october the images of chl-a is not available. in most of the cases, in order to locate accurately the potential fishing grounds, the images of chl-a distribution and sea surface temperature are used integratedly. this method could be proposed as an important technique for identifying the potential fishing ground. the method also provides indirect information on the horizontal mixing of water masses, which contributes to high productivity or high concentration of phyto plankton (chl-a) in the waters (dwivedi 2000). in this study, these images from different sensor optics (noaa/avhrr and seawifs) are also used together. currents currents (convergent, divergent and eddies) are specific oceanographic parameters in the north papua waters. these parameters play an important role in the development of thermal front, the distribution of water mass and the concentration of chlorophyll-a in the area. together, the convergent and divergent these currents are favorable for the development of marine organisms. the area with divergent currents has high productivity, and convergent current forms a mechanism of phyto plankton aggregation at the front of currents. meanwhile, eddies as the result of moving cycle (form a ring) of the water masses increase the intensity of vertical mixing of the waters followed by upwelling which occurs at the outer side of the ring, triggering the increase of water mass productivity (dwivedi 2000). according to mean and lazier (1990), currents in the study area are classified into a). sec (south equatorial current) with direction to the west b). necc (north equatorial counter current) is a narrow current with the direction to the east, and c). euc (equatorial upwelling current) is subsurface water current with the direction to the east. the dynamic of these currents is due to the coriolis force, which effects divergent and convergent currents along the borders of the currents in different directions. these currents occurred intensively in the papua waters. the illustration 43   ^identification of oceanographic parameters v. siregar and h.j.d. waas of this mechanism is shown in figure 5: (a.l) currents in the northern hemisphere have oposite direction, west-east; (a.2) shows the effect of corriolis force, the currents are divergent and the subsurface water upwell replaces the surface water as a result. this mechanism occurs in the system current of nec (northern equatorial current) and necc; (b.l) showing the convergent current as the result of necc and sec encounter; (c.l) in the equator, sec with direction towards the west will create divergent currents, inversly, with directions towards the east will c reate convergent currents (d. 1 dan d.2). the analysis of fishing ground based on available data set of topex poisedon (augustoctober) showed that the pelagic tunas are mostly catched in all types of current such as divergent, convergent and eddies . in august, most of the fishing grounds were found at the outer ring of eddys and divergent current (fig 6 a). at the end of the month, most of the fishing grounds were only found at the convergent current system. in september and october, the fishing grounds are found mostly at the boundary on divergent current system. several fishing grounds were also found at the outer ring of eddys (figure 6). from analysis of available satellite data in figure 6, it can be concluded that currents (divergent, convergent and eddies) are also an parameter important for identification of fishing ground of the pelagic tuna in the papua waters. these oceanographic parameters were derived from satellite altimeter data (microwave-radar). compared to optic data, the detection of these parameters with radar were not influenced by cloud cover. therefore, utilization of microve data which provides information on currents is proposed to be used together with optic data (visible and thermal), in order to increase the accuracy of fishing ground determination. in the case of unavailability of optic data, the use of radar data can be used as an alternative. 45   indentification of oceanographic parameters v. siregar and h.j.d. waas conclusions analysis of oceanographic parameters detected from several satellite data and fishing catch data of the north papua waters showed that the important oceanographic parameters which can be used to identify fishing ground of small pelagic tunas (skipjack and yellow fin tunas) are thermal and chlorophyl-a front, and currents (divergent, convergent and eddies). the combination of these parameters increase the accuracy of fishing ground identification. thermal (sst), chlorophyll-a and currents data are derived from optic and microwave (radar) sensors respectively. availability of data from sensor optic is limited due to cloud cover, which in this case is very frequent in papua and indonesia in general. in the case of cloud cover, where the optic data is not available, radar data can be used to provide currents information an alternative to determine fishing ground. the use of multi-sensor satellite data such as noaa-17/avhrr, seawifs and topex poseidon can be used integratedly as a promising approach in determination of potential fishing ground of the pelagic tunas in the north papua waters. references dwivedi, r.m.2000. ocean colour as a tool for potential fishing zone identification and forecast. porsec 2002. pre-conference training. national institute of oceanography. dona paula, goa -403 004, india, p. 17-22. gower, j.f.r.1972. opportunities and problems in satellite measurements of the sea. unesco tech. pap. 46, 70 p. laevastu, t., and m. l. hayes. 1981. fisheries oceanography and ecology. fishing news books ltd., 199 p. lehodey, p., 2003. sepodym application to albacore (thunnus alalunga). oceanic fisheries programme. noumea, new caledonia, p. 1-16. lewis, a.d., p.o. williams. 2001. overview of the western and central pasific ocean tuna fisheries, 2000. oceanic fisheries programme (ofp) secretariate of the pasific community noumea, new caledonia, p. 1-31. lipi, 1992. penelitian potensi pelagis dan karakteristik lingkungan perairan utara irian jaya. pusat penelitian dan pengembangan oseanologi. lernbaga hmu pengetahuan indonesia. jakarta. 113 p. loukos, h. p., l. b. monfiy., and p. lehodey. 2003. potencial change in skipjack tuna (katsuwonns pelamis) habitat from a global warming scenario : modelling approach and preliminary results. fisheries oceanography. blackwell publishing ltd., p. 474-482. mann, k.h, and j.r.n. lazier, 1991. dynamics of marines ecosystems. biological-physical interaction in the ocean. blackwell scientific publication. 446 p. mc.millin, l.m., and d.s. crosby. 1984. theory and validation of the multiple windows sea surface temperature technique. in journal of geophys. res., no. 89. p. 3655-3661. meaden, g.j. and t.d.chi, 1996. geographycal information systems, application to marine fisheries. fao fisheries technical paper. no.356 rome, fao, 335 p. 47 biotropia vol. 13 no. 1, 2006 narain, a. 1993. chlorophyll studies using remote sensing. in international workshop on applicai of satelite remote sensing for identifying and forecasting potential fishing zones in develop countries. hyderabad, india, p. 1-13. picarkard, g.l. and w. j. emery. 1990. descriptive physical oceanography: an introduction robinson, i. 1985. satelite oceanography. an introduction for oceanographers and remote sens scientists. ellis horword dim: 249 p. topex poseidon data: http://www.c.car,col(iradp,edu/ accessed on october 2003 triton bouy mooring data: ht.tjk/.;!v^vw..0iiejjioaa;goy,ia.o/j.̂ di.splay. accessed on october 2003 seawifs data: mr^zs..̂ .?lfs:.£sfc.nasa.gow accessed on october 2003 shixing, h., s. jianhua., and l. s. shuxun. 1993. remote sensing in chinese fisheries. in intematic workshop on application of satelite remote sensing for identifying and forecasting poten fishing zones in developing countries. hyderabad, india, p. 1 -4. steel, r.g.d. and j.h. torrie, 1989. principles and procedures of statistics. second edition. me gi hill int.book co., 633 p. vinogradov, m.e. 1981. ecosystems of equatorial upwelling, in: analysis of marine ecosystei a.r.longhurst (ed). london: academic press, p 69-93 waas, h.j.d. 2004. analisis daerah potensial penangkapan cakalang (katsuwonus pelamis) ( madidihang (thunnnx albacares) di perairan utara papua, pasifik barat. tesis. seko pascasarjana ips, 102 p. wenno, l.f., hadikusuma and nurhayati, 2001. hubungan antara beberapa parameter fis oseanografi terhadap distribusi kandungan klorofil-a di perairan mamberamo irian jaya, agus 2000. dalam perairan indonesia, oseanografi, biologi dan lingkungan. lp3o-li jakarta, p. 9-19. 48 37.pdf 38.pdf 39.pdf 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf 47.pdf 48.pdf biotropia no biotropia no. 16, 2001 : 18 27 induction of spawning in the tropical walking catfish (clarias batrachus) by controlling water level and temperature m. zairin jr.1*', k. furukawa2) and k. aida2) 1)department of aquaculture, faculty of fisheries and marine science, bogor agricultural university, bogor, indonesia 2)department of aquatic bioscience, faculty of agricultural and life sciences, the university of tokyo, tokyo, japan abstract this experiment was carried out to confirm the role of water level and water temperature in inducing the spawning of tropical walking catfish. mature males and females reared under 23 25 °c, were paired and induced to spawn by controlling water level and water temperature. decreasing water level and returning it to its original level resulted in a low spawning rate (less than 6.7 %) at 23 °c. decreasing water level with simultaneous increase in water temperature, followed by returning the respective levels to their originals gave high spawning rates (41.7 — 50 %); whereas the same treatment but without any water temperature decreased when the water level was returned to the initial level, gave a low spawning rate (16.7 %). increasing water level only, failed to induce spawning. a high spawning rate was obtained also when changes in water level were carried out under high temperature of 28 °c. no fish spawned in the absence of the environmental stimulation. from the results, it is confirmed that water level and temperature play important roles in inducing spawning of tropical walking catfish. changes in water temperature probably increase the sensitivity of fish to the change in water level. prolonged exposure to high water temperature could also improve the sensitivity of fish. key words: walking catfish / spawning / water level / water temperature introduction in the previous reports (zairin et al. 1992a,b), it has been suggested that under long term rearing and constant warm water temperature, female catfish maintained conditions of maturity for an extended period, and no spontaneous spawning has been seen to occur. on the other hand, from the practical point of view, one of the constraints in employing environmental factors in inducing the fish to spawn in the pond is a low spawning rate. therefore, it is necessary to investigate the ability of this fish to respond to environmental stimulation for the induction of spawning. • corresponding author: e-mail address: zairinm@indo.net.id; fax: 62-251-622541 18 18 biotropia no. 16, 2001 in nature, both environmental and physiological factors are considered as important cues in triggering final oocyte maturation, ovulation and spawning in teleosts. in tropical areas, natural water temperature is relatively high, and water level amplitude is large in association with the occurrence of the rainy and dry seasons. it has been reported that peaks of spawning activity of tropical species are often associated with rainfall and flood (lam 1983; lam and munro 1987; munro 1990). several species have been reported to spawn in relation to the change in water level, for example weakly electric fish (eigenmannia virescens) (kirschbaum 1979) and the african catfish (glorias gariepinus) (bruton 1979). moreover, mimicking environmental phenomena in nature, e.g. by regulating water level, has been commonly employed in aquaculture in southeast asian countries to induce tropical walking catfish to spawn (areerat 1987; tarnchalanukit 1987; knud-hansen et al. 1990). however, little is known on the combinatory effects of water level and water temperature in the action of inducing spawning. therefore, experiments were conducted to study the effects of water temperature and water level in the induction of spawning in walking catfish materials and methods experimental fish two month-old walking catfish were transported from bogor, indonesia to japan, and reared in an indoor concrete pond (width x length x depth = 1.5 x 3 x 0.5 m) supplied with running freshwater of deep-well origin at the fisheries laboratory, the university of tokyo, maisaka, shizuoka prefecture. during this rearing period, fish were subjected to a photoperiodic cycle of 12l(light):12d(dark), and relatively constant water temperature (23-25°c) throughout the year. fish were fed twice per day with commercially available trout dry pellets no. 5 (40% protein, chubu shiryo, co.) at a daily ration of 2-3% of body weight. under the above stocking conditions, fish began to mature at the age of 9 months and thereafter; conditions of maturity were maintained without undergoing natural spawning. two experiments (i and ii) were carried out. a week before the start of the experiment i, 15 pairs of fish were selected from the stock pond. males weighing 325-400 g and females weighing 375-500 g were used. these fish were paired and placed in each compartment. at the end of the experiment i, all fish were replaced by a new group for experiment ii. for this experiment, males weighing 250-375 g and females weighing 300-425 g were selected and paired. experimental condition five indoor concrete ponds 1 x 3 x 1 m in size were used. each pond was divided transversely into three compartments by framed plastic nets, so that every 19 induction of spawning in the tropical walking catfish m. zairin jr. et al. compartment was 1 x 1 x 1 m (fig. ib). each compartment was equipped with one spawning box, which was placed in the compartment only during the observation period of spawning (day 14-24, 31-35, 45-55 in experiment i, day 11-25, 35-45 in experiment ii). the spawning boxes 30 x 35 x 42 cm, were painted dark brown, with a hole of 12 cm in diameter (fig. la). half of the upper portion was made removable for checking for deposited eggs. an artificial plastic spawning nest made of soft plastic fibres was placed inside each box. the box was fixed in the corner of every compartment, in which 2-3 cm of its upper portion rose above that of water level. photoperiod was maintained at constant 12l:12d throughout the experiment, but water temperature and depth were varied according to the type of treatment. limitation in sample numbers did not allow statistical analysis. such experiments are very difficult to be carried out for a large number offish. treatment treatment a and a'. a sudden decrease in water level from 70 cm to 20 cm (in one hour) for 13 days (treatment a) or to 35 cm (in one hour) for 7-10 days (treatment a'), followed by an increase back to the original level of 70 cm (in one hour). water temperature was maintained at 23 ± 0.5°c during the experiments. on the day of water level increase, a spawning box was placed in each compartment. the boxes were checked everyday for deposited eggs for 10 days (treatment a) and for 4-10 days (treatment a'). for more details, refer to fig. 2. figure 1. (a) spawning box and its placement relative to water surface. (b) pond compartments and box position in each compatmentent. 20 biotropia no. 16, 2001 21 induction of spawning in the tropical walking catfish m. zairin jr. et al. treatment b. a sudden drop in water depth from 70 cm to 35 cm (in one hour), and an increase in temperature from 23 °c to 28°c (in six hours) were simultaneously carried out for 7-10 days, and after that, both water level and temperature were returned to their originals. on the day of water level increase, a spawning box was placed in each compartment. the boxes were checked everyday for deposited eggs for 4-10 days (fig. 2). treatment c. similar to treatment b, a sudden drop in water level from 70 cm to 35 cm (in one hour), and an increase in water temperature from 23°c to 28°c (in six hours) for 10 days were simultaneously carried out, but the temperature was maintained at 28°c while the water level was returned to its original (in one hour) after 10 days. on the day of water level increase, a spawning box was placed in each compartment. the boxes were checked everyday for deposited eggs for 14 days (fig. 3). treatment d and d'. only an increase in water temperature from 23°c to 28°c (in six hours) for 10 days (treatment d), and for 24 days (treatment d'), without accompanying water level manipulation. the duration of observation was 10 days. on days 11 and 35, a spawning box was placed in each compartment. the boxes were checked everyday for deposited egg for 14 days (treatment d) and for 10 days (treatment d'). for more details, refer to fig. 3. treatment e. a sudden drop in water level from 70 cm to 35 cm (in one hour) at 23°c for 10 days and then return to the original water level at 28°c. on the day of water level increase, a spawning box was placed in each compartment. the boxes were checked everyday for deposited eggs for 10 days (fig. 3). treatment f. no manipulation. this treatment is regarded as controls for all experiments. water temperature and water level were kept at 23°c and 70 cm, respectively. at days 11 and 35, a spawning box was placed in each compartment. the boxes were checked everyday for deposited eggs for 10-14 days (fig. 3). results and discussions results experiment i the results are presented in fig. 2. during experiment la, the fish were subjected to treatment a. out of 15 pairs employed, only one pair spawned (6.7%). during experiment ib, fish were subjected to treatment a' and b. from 9 pairs of fish employed in treatment a', no fish spawned. in treatment b, however, the combination of water level and temperature manipulation gave a high spawning rate (50%). out of six pairs employed, three pairs spawned or ovulated. during experiment ic, fish were subjected also to treatment a' and b. the result was still in agreement with those of experiment ib. no fish spawned in treatment a'. treatment b again gave a high spawning rate (41.7%). out of 12 pairs of fish employed, five pairs spawned. 22 biotropia no. 16, 2001 figure 3. spawning success after water level and water temperature manipulation for treatment c to f (see text for details). closed line: water level; broken line: water temperature. each of the open and closed star represents an ovulated or spawned fish, respectively. ha and lib represent the experimental period of each treatment. 23 induction of spawning in the tropical walking catfish m. zairin jr. et al. experiment ii the results are presented in fig. 3. during experiment ha, the fish were subjected to treatment c, d and f. treatment c, which was similar to treatment b, but without dropping water temperature upon increasing water level, gave a spawning rate of 16.7%. out of six pairs employed, one pair spawned. on the other hand, increasing only water temperature (treatment d) or no manipulation in water level and temperature (treatment f) could not induce spawning. during experiment lib, the fish were subjected to treatment d', e and f. manipulation of water level under 28°c (treatment e) gave a high spawning rate (50%). out of six pairs employed, three pairs spawned or ovulated. no fish spawned in treatment d' or f. in our experimental conditions, at least three days (after the placement of spawning box) were needed to reach the ovulation or spawning. the results of all treatments are summarized in table 1. table 1. spawning success after water level and water temperature manipulation 24 biotropia no. 16, 2001 discussions mimicking the environmental phenomenon to induce spawning in fish has been a common practice in aquaculture in southeast asian countries, including the use of the water level manipulation (areerat 1987; tarnchalanukit 1987; knud-hansen et al. 1990). unfortunately, no scientific effort has been made to substantiate this interesting practice. understanding this problem will add value to the development of the domestication of tropical wild edible river fish. considering the typical physical conditions in the tropic and to simplify the problem, water temperature and water level have been chosen as variables. both factors were chosen because changes in water levels of tropical river were usually accompanied by changes in water temperature. the possibility of photoperiod as an environmental cue has been omitted since our previous results showed that these fish were found mature throughout the year in japan, regardless of photoperiod (zairin et al. 1992a,b). environmental stimulation usually induces spawning by a long process through the hypothalamus-hypophysial-gonadal axis (blazquez et al. 1998). and as expected, time elapses are needed between the stimulation and the initiation of spawning. overall in this experiment, at least three days were needed for walking catfish to spawn. in the african catfish clarias gariepinus, in which fish were kept in earthen ponds, a shorter time interval was sufficient (bruton 1979). this difference may be due to the more complex physico-chemical conditions in the earthen pond. the possibility of more males needed to induce courtship behaviour has been disregarded since this fish is not a mass spawner. changes in water levels alone (treatment a) actually could induce spawning, although the spawning success was low (6.7%). this means that the fish may be responsive to the fluctuation in water level after a long time exposure to a constant temperature and photoperiod condition. a low spawning success reflects a low sensitivity of fish to water level fluctuation. this low responsiveness may be due to relatively low water temperature (23°c) employed in this experiment. this is supported by the fact that in the tropics such as indonesia, the average water temperature in places where the fish are commonly cultured, is usually higher than 23°c. from the daily observation on the performance of fish in experiment la, we considered that lowering the water level to 20 cm gave insufficient space for fish to maneuver because the depth was less than the average length of fish employed. thereafter, the minimum depth was increased to 35 cm (treatment a'). although treatment a' gave no spawned fish, we used 35 cm as the minimum depth. the results from treatment b, in which more fish spawned (41.7-50%) when the change in water level was accompanied by the change in water temperature indicate the importance of water temperature amplitude. furthermore, the results suggest that fluctuations in temperature could increase the sensitivity of fish to the water level manipulation. further supporting evidence came from treatment c and 25 induction of spawning in the tropical walking catfish m. zairin jr. etal. d. treatment c was similar to experiment b but without water temperature decrease upon the increase in water level; the spawning rate in treatment c (16.7%) was lower than treatment b (41.7-50%). no spawning occurred under treatment d and d' which indicated that increasing only the water level gave no stimulation on the fish to spawn. treatment d and d' were similar, and differed only in the duration of exposure to high water temperature. exposure to treatment d' was longer than that of treatment d. additionally, when changes in water level were carried out at a higher temperature of 28°c (treatment e), the spawning success was almost the same as those obtained when water fluctuation was accompanied by water temperature changes. these results suggest that changes in temperature could be replaced by prolonged exposure to higher temperature. furthermore, this phenomenon indicates that temperature still plays an important role in the spawning of the tropical walking catfish. from the practical point of view, the results from the best treatment of our experiments give a higher spawning success compared with those of fish farms. calculation based on figures provided by knud-hansen (1990) indicates only less than 15 % of spawning success; and in practice, 10% spawning success is regarded as an acceptable rate. therefore, for practical purposes, the role of these factors should get more attention. furthermore, we believe that combination of environmental factors with simple hormonal treatment could improve the spawning success further. research in this area should receive more attention. acknowledgment we express our thanks to all members of the fisheries laboratory of the university of tokyo, maisaka, shizuoka prefecture, for the facility for field experiments. we thank m. n. wilder for reviewing the manuscript. this study was partially funded by a grant-in-aid for scientific research from the ministry of education, science and culture, japan. references areerat, s. 1987. clarias culture in thailand. aquaculture, 63, 355-362. blazquez, m.p., bosnia, p.t., eraser, e.j. van look, k.j.w., and v.l trudeau, 1998. fish as a model for the neuroendocrine regulation of reproduction and growth. comp. biochem. physiol. partc, 199, 345364. bruton, m.n. 1979. the breeding biology and early development of clarias gariepinus (pisces: clariidae) in lake sibaya, south africa, with a review of breeding in species of the subgenus clarias. trans. zool. soc. london. 35, 1-45. 26 biotropia no. 16, 2001 kirschbaum, f. 1979. reproduction of the weakly electric fish eigenmannia virescens (rhamphichtydae, teleostei) in captivity. behav. ecol. sociobiol., 4, 331-355. knud-hansen, c.f., batterson, t.r., mcnabb, c. d., hadiroseyani, y., dana, d., and h.m eidman,. 1990. hatchery techniques for egg and fry production of clarias batrachus (linnaeus). aquaculture, 89, 919. lam, t. j. 1983. environmental influences in gonadal activity in fish. in: w.s. hoar, d.j. randall, and e.m. donaldson (eds.), fish physiology, vol. ix, part b, p. 65-116, academic press, new york. lam, t.j., and a.d. munro, 1987. environmental control of reproduction in teleost: an overview. proc. of the third int. symp. on reprod. physiol. of fish. st. john's, newfoundland, canada, p. 279-288. munro, a.d. 1990. tropical freshwater fish. in: a.d. munro, a.p. scott, and t.j. lam. reproductive seasonality in teleost: environmental influences, cec press, boca raton, florida, p. 145-239 tamchalanukit, w. 1987. induced spawning of walking catfish, clarias batrachus, by water level jegulation. proc. of the third int. symp. on reprod. physiol. of fish. st. john's, newfoundland, canada, p. 138. zairin, m., jr., furukawa, k.., and k. aida, 1992a. induction of ovulation by hcg injection in the tropical walking catfish clarias batrachus reared under 23-25°c. nippon suisan gakkaishi. 58, 1681-1685. zairin, m., jr., furukawa, k., and k. aida, 1992b. changes in ovarian maturity in the tropical walking catfish, clarias batrachus reared under 23-25°c. nippon suisan gakkaishi. 58, 2033-2037. 27 biotropia no biotropia no. 15, 2000 : 36 47 genetic diversity of ampicillin-resistant vibrio isolated from various stages of tiger shrimp larvae development widanarni a and antonius suwanto b c "department of aquaculture, faculty of fisheries and marine science bogor agricultural university, bogor 16680, indonesia b department of biology, faculty of science and mathematics, and iuc biotechnology, bogor agricultural university, bogor 16680, indonesia 'seameo-biotrop, ji. raya tajur, km 6 bogor, indonesia abstract this research was carried out to study genetic diversity of ampicillin-resistant vibrio from various stages of tiger shrimp larvae (penaeus monodon) development from,tambak inti rakyat hatchery, near labuan, west java, indonesia. a total of 25 ampicillin-resistant vibrio isolates were isolated using thiosulphate citrate bile-salt sucrose agar (tcbs-agar) and seawater complete agar (swc-agar). physiological and biochemical characterization showed that the isolates could be grouped into only two species, i.e. v. harveyi from the egg stage; and v. metschnikovii from larvae and post-larval stage (i.e nauplius, zoea, mysis, pli, pl5, pl,0, and pl,5). these isolates were also present in their respective rearing water of each stage and some natural feed. schizotyping analysis employing restriction endonuclease noll (5'-gc4ggccgc) indicated that the isolates could be grouped into at least 13 different genotypes. therefore, schizotyping was more discriminative than physiological characterization. this study showed that particular groups of vibrio colonized all stages of shrimp larvae and demonstrated closed phylogenetic relationship. these groups of vibrio might be the dominant microbiota which could suppress the development of other vibrio including the pathogenic vibrio. key words : shrimp/ampicillin-resistant k/fcno/schizotyping introduction tiger shrimp (penaeus monodori) culture has developed towards intensive levels due to high demand and significant price of this commodity. however, recently shrimp production has decreased significantly because of diseases and poor environment quality (anonymous 1994). one of the most serious problems is the disease caused by vibrio (lavilla-pitogo et al. 1990). the disease greatly influenced the sustainable supply of healthy fry (lightner et al. 1992). hameed (1993) observed that the decrease of survival rate of larvae and postlarvae was due to the concomitant increase of the bacteria population. generally, shrimps at the stadia of zoea, mysis, and the beginning of post-larvae were vulnerable to vibrio infection (rukyani et al. 1992). it was believed that these bacteria contaminated the hatchery through broodstock feces because of their presence in significant number in the midgut (lavilla-pitogo et al. 1990). therefore, * corresponding author : email address : asuwanto@indo.net.id;fax : 62-251-315107 36 biotropia no. 15, 2000 contamination of the eggs and nauplius by these bacteria was highly probable. besides the infected shrimp, these bacteria could also be isolated from seawater used for rearing water in the hatcheries (tjahjadi et al. 1994). recently, suwanto et al. (1998) demonstrated that vibrio harveyi isolated from different broodstocks were genetically very diverse and different to each other. therefore, it can be expected that vertical infection from broodstock to eggs is more likely to occur rather than horizontal transfer from larvae to larvae. in order to understand genetic diversity of vibrio at various stages of shrimp larvae development, a study on genetic diversity of vibrio isolated from egg, larvae, and post-larvae of shrimp originated from the same broodstock was carried out. a number of techniques have been developed to identify bacteria at the subspecies level. these include phage typing (stringer 1980), analyses of plasmid dna (davies et al. 1981), ribotyping (olsen et al. 1986), random amplified polymorphic dna (rapd) (welsh and mcclelland 1990), and schizotyping by pulsed-field gel electrophoresis (pfge) (suwanto and kaplan 1992). pfge analysis of large sizes of dna molecules requires a technique to isolate intact genomic dna as well as the availability of rare-cutting restriction endonucleases and the appropriate mega-base molecular size markers. under optimized condition, pfge can efficiently separate dna fragments of 100 to 10,000 kbp to distinguish strains of microorganisms which otherwise exhibit similar or identical physiological and morphological characteristics (suwanto 1994). genetic diversity analysis based upon genomic dna profile utilizing pfge has been performed for various bacteria for various aims, such as vibrio anguil-larum, a causative agent of vibriosis in fish (skov et al. 1995), genomic dna analysis of xanthomonas campestris, a causative agent of root-pustule disease of soybean (rukayadi 1995) and vibrio harveyi, a causative agent of "luminous-bacterial disease" (suwanto et al. 1998). pfge genomic dna analysis used in the experiments above have proved to be more discriminative to visualize phylogenetic diversity rather than analysis based on phenotype characterization. the purpose of this research was to study the genetic diversity of ampicillin-resistant vibrio from various stages of shrimp larvae development by pfge analyses. materials and methods isolation and identification of vibrio sp. vibrio were isolated from eggs, larvae (nauplius, zoea, and mysis), and post-larvae (pl,, pl5, pl10, and pl15) of tiger shrimp (p. monodori), natural shrimp feed (i.e. anemia and skeletonema), and rearing water obtained from tambak inti rakyat hatchery, near labuan, west java, indonesia. shrimp larvae were collected in the month of march-april, 1998. five eggs and shrimp larvae were washed gently in sterile seawater and collected into a test tube containing 1 ml sterile seawater, and homogenized by vortex agitator before it was spread on tcbs agar. appropriate 37 genetic diversity of ampicillin-resistant vibrio widanarni & antonius suwanto dilution was performed to obtain single isolated colony on tcbs agar by adding sterile seawater. the culture was incubated at room temperature, (28-31)°c, for 24 hours. isolated colonies were randomly selected for further study. biochemical and physiological characterization were conducted with microbactr analysis kit (medved science pty. ltd. australia). identification of vibrio isolates were done as described by baumann et al. (1984). seawater complete agar (5 g bactopeptone, 1 g yeast extract, 3 ml glycerol, 15 g agar, 750 ml seawater, and 250 ml distilled water) supplemented with ampicillin (50 ug/ml) (swc-ap) was employed as a selective media to screen for ampicilin-resistant vibrio isolates. the isolates were subsequently suspended in sterile seawater containing 15% glycerol (v/v) before they were stored in the freezer at -50°c. preparation of intact genomic dna and digestion purified isolates of ampicillin-resistant vibrio were grown in swc agar. one separated colony was regrown in 10 ml lb medium (10 g tryptone, 5 g yeast extract, 25 gnacl and 1 l distilled water) at 28°c overnight. one ml of bacterial suspension was centrifuged at 5,000 rpm for 30 seconds. the bacterial cell pellet was suspended in sterile piv solution (10 mm tris-cl ph 7.5, 1 m nacl). preparation of intact genomic dna was performed by embedding the bacterial cells in low melting aga-rose blocks as described previously (schwartz and cantor 1984). digestion of the intact genomic dna was done using restriction enzyme as described by suwanto et al. (1998) as follows: digestion with 10 units of not\ were performed in 150 ml of appropriate restriction buffer (1335 ul distilled water, 150 \i\ lox restriction buffer, 15 ul of 10 mg/ml bovine serum albumine). the mixture was incubated at 4°c for 15 minutes followed by incubation in 37°c overnight. dialysis of the gel plugs were performed by immersing the gel plugs in excess of ix te buffer solution (10 mm tris-cl ph 8.0, 1 mm edta ph 8.0) before placing the gel plugs into the wells of the running gel. separation of dna fragment using pfge the running gels of 1% (w/v) agarose (pharmacia) were prepared in 100 ml of 0.5x tbe (50 mm tris-borate buffer, 0.1 mm edta ph 8.0). pulsed-field gel electrophoresis was performed using chef-drii (bio rad, richmond, ca). the gels were run in 0.5 x tbe buffer, 5 volt, 14°c with ramping pulse time from 10-80 seconds for 20 hours. as molecular marker, rhodobacter sphaeroides 2.4.1 genomic digested with asel was routinely used for pfge (suwanto and kaplan 1989). dna visualization gels were stained by submerging in ethidium bromide solution (1 ug/ml) for 10 minutes. destaining were done in distilled water for 20-30 minutes. transillu-minator with uv length of 280 nm (hoefer scientific instrument, san francisco) 38 biotropia no. 15, 2000 was used to visualize the gel. photographs were taken with fast film polaroid (type 667, japan polaroid company). statistical analysis a matrix was constructed as the basis for determining the presence or absence of schizotyping bands at a given position over the size range from 30-500 kb. a cluster analysis was carried out using the unweighed pair group method with arithmetic means (upgma clustering with simple matching coefficient) of similarity coefficient for all pairs of strain and a dendrogram was generated using a computer-based taxonomy program (numerical taxonomy system, ntsys-pc version 1.60) (rohlf 1990). results and discussion physiological and biochemical characterization of vibrio isolates twenty five ampicillin-resistant isolates have been isolated and selected randomly from either tcbs or swc-ap for further study (table 1). of these isolates, 20 isolates were found to be associated with eggs, larvae, post-larvae, and the rearing water of each stage; two isolates were isolated from seawater reservoir; and the other isolates were obtained from natural feed (i.e. artemia and skeletonema). table 1. codes and sources of vibrio isolates no. code source no. code source 1. e, egg 14 st broodstock tank water 2. e2 egg 15. et spawning tank water 3. n nauplius 16. nt nauplius tank water 4. z zoea 17. zt zoea tank water 5. m, my sis 18. mt mysis tank water 6. m2 mysis 19. pl,t post-larvae 1 tank water 7. m3 mysis 20. p15t post-larvae 5 tank water 8. pl, post-larvae 1 21. pl,0t post-larvae 10 tank water 9. pl5 post-larvae 5 22. pl15t post-larvae 1 5 tank water 10. pl,o post-larvae 10 23. skt skeletonema 11. pl,, post-larvae 15 24. at, artemia 12. sw, sea water 25. at2 artemia 13. sw2 sea water 39 genetic diversity of ampicillin-resistant vibrio widanarni & antonius suwanto physiological and biochemical characterization showed that the isolates shared several similar characters. the cell was rod shape, produced lysine decarboxylase and indole, and all were able to ferment glucose (table 2). the isolates could be classified into three groups. first group, composed of e,, e2, m,, sw,, sw2, and mt shared the same characters such as: forming green colonies on tcbs, oxidase-positive, produce protease and chitinase, and able to reduce nitrate. a number of isolates were also able to utilize arabinose as a carbon source. the second group of vibrio composed of only one isolate, at2 isolated from anemia had the same characters as first group except that it was luminous. the third group (n, z, m2, pl,, pl,5, et, zt, pl5t, pl10t, skt, at,), was the dominant group of the isolates found in every larval stage except the eggs possessed completely different characters from the two groups in having nonluminous yellow colonies on tcbs, showed negative oxidase reaction, did not produce protease and chitinase, and were able to use sucrose as a carbon source. based upon baumann et al. (1984) identification, the first group was non-luminous v. harveyi, the second was luminous v. harveyi, and the third was v. metschnikovii. therefore, physiological characterization of vibrio isolated from each stage from tambak inti rakyat hatchery, west java, showed that two species of vibrio had been found. v. harveyi were associated with eggs, while v. metschnikovii were found in larvae and post-larvae (nauplius, zoea, mysis, pl,, pl5, pl,o, and pl15), these isolates were also present in their respective rearing water of each stage and some natural feed. schizotyping analysis pfge electrophoresis of the 25 vibrio isolates (obtained in this study) and of v. harveyi s14b (suwanto et al. 1998) using notl restriction enzyme (figure 1) showed discrete profiles of genomic dn a of the isolates based upon the number of fragments and migration distance (table 3). restriction with notl produced 10-16 discrete dna fragments with sizes ranging from 31-910 kb. genomic dna analysis of 25 isolates showed 13 different dna profiles. these dna profiles could be used as a unique fingerprint of each of those isolates. dendrogram of genomic dna digested with notl (figure 2) showed genetic relatedness of the isolates, which could be divided into 13 different subgroups. the sum of horizontal lines connecting the two isolates indicated genetic distance of those isolates. for example, the distance between isolate pl| and pl5 is zero indicated that these isolates are identical. of these, 13 subgroups were divided into 2 major groups. the first major group consisted of 20 isolates associated with all stages of shrimp larvae and their rearing water. the second group consisted of 5 isolates, i.e. 2 isolates from seawater reservoir, 1 isolate from broodstock rearing water, and 2 isolates from natural feed (skeletonema and anemia). some vibrio isolates originated from seawater and natural feed also could not be isolated from all larvae stages and eggs of shrimp. this result suggested that eggs were exposed to bacterial contamination not only from broodstock feces as reported by lavilla 40 biotropia no. 15, 2000 41 genetic diversity of ampiciloin-resistand vibro – widanarni & antonius suwanto 42 biotropia no. 15, 2000 43 genetic diversity of ampicillin-resistant vibrio – widanarni & antonius suwanto 44 biotropia no. 15, 2000 table 3. grouping of genomic dna profiles of 25 vibrio isolates generated by noll schizo typing no schizotype profile isolates exhibited similar schizotype 1. e, e, 2. m, m, 3. pl, pl, pl,5, pl,t, pl,,t 4. pl5t pl,t 5. z zt 6. at2 at2 7. pl,o pli(1t 8. et nt 9. m2 mt, m, 10. e2 ea 11. n n 12. sw, sw2 st 13. sk.t at, pitogo et al. (1990), but also when they were still in the ovary. our study also demonstrated that vibrio isolates from sea water or natural feed possessed distance genetic relationship to that of vibrio from eggs and larvae, indicating that vibrio from eggs and larvae may be the dominant microbiota and might play a role in suppressing the growth of vibrio from seawater or natural feed. compared to the results of suwanto et al. (1998), all of vibrio isolates in this experiment showed relatively similar genetic background. this might be due to the fact that the broodstocks in this study were obtained from the same area (aceh province), while suwanto et al. (1998) used broodstocks from different areas and provinces. this study showed that genetic analysis by pfge was more discriminative than that of physiological and biochemical analysis. from 25 isolates characterized by physiological analysis, they could be grouped into two species, while using schizotyping analysis, there were 13 different groups of genotypes. furthermore, with this analysis, v. metschnikovii, that did not produce protease and chitinase was apparently closely related to v. harveyi, a shrimp pathogen. therefore, the existence of v.metchnikovii might influence shrimp larval fitness and survival. in conclusion, physiological and biochemical characterization were able to classify 25 isolates vibrio (both luminous and non-luminous) into two species, i.e. v. harveyi and v. metchnikovii. notl schizotyping analysis showed that the isolates consisted of at least 13 different genotypes, which indicated that schizotyping was 45 genetic diversity of ampicillin-resistant vibrio widanarni & antonius suwanto more discriminative than physiological characterization. the result also demonstrated that vibrio isolates present in all stages of shrimp larvae showed close phylogenetic relationship. these groups of vibrio might be dominant and might play a role in suppressing the development of other vibrio originated from sea water, broodstock feces or natural shrimp feed. acknowledgements we express our thanks to tambak inti rakyat hatchery, labuan, west java, for supplying shrimp larvae and to allow us to use the field laboratory facility. we would like also to thank prof. dr. maggy t. suhartono and dr. dwi andreas santosa for their invaluable advice during the experiment. this research was supported by riset unggulan kemitraan (ruk) research grant to antonius suwanto and biaya pendidikan pasca sarjana (bpps) research grant to widanarni. references anonymous. 1994. problem solution alternatives of shrimp culture in java. directorate general of fisheries. jakarta. baumann p, furniss al, lee jv. 1984. facultative anaerobic gram negative rods. in: krieg nr (ed) bergey's manual of systematics bacteriology. williams and wilkins, baltimore, ma. vol. 1: 1513-1523. davies fl, underwood hm, gasson mj. 1981. the value of plasmid profiles for strain identification in lactic streptococci and the relationship between streptococcus lactis 712, ml3 and c2. j appl bacteriol 51: 325337. hameed ass. 1993. a study of the aerobic heterotrophic bacterial flora of hatchery-reared eggs, larvae and postlarvae ofpenaeus indicus. aquaculture 117:195-204. lavilla-pitogo cr, baticados cl, cruz-lacierda er, de la pena ld. 1990. occurrence of luminous bacterial diseases of penaeus monodon larvae in the philippines. aquaculture 91:1-13. lightner dv, bell ta, redman rm, mohney ll, natividad jm, rukyani a, poemomo a. 1992. a review of some major diseases of economic significance in penaeid prawn/shrimp of the americans and indopacific. in: shariff m, subasinghe rp, arthur jr (eds). diseases in asian aquaculture 1. fish health section. asean fisheries society. manila, philippines p.57-80. olsen gj, lane dj, gionvannoni sj, pace nr. 1986. microbial ecology and evolution: a ribosomal rna approach. ann rev microbiol 40: 337-365. rohlf fj. 1990. nt-sys-pc, numerical taxonomy and multivariate analysis system, version 1.60. exeter software, new york. rukayadi y. 1995. dna profile analysis of the genomes of a number of xanthomonas campestris pv. glycines isolates using pulsed-field gel electrophoresis. thesis pascasarjana program, ipb. bogor. 69p. rukyani a, taufik p, taukhid 1992. luminous vibrios in tiger shrimp hatchery and the control of the disease in shrimp larvae. j. litbang pertanian 2:1-17. 46 biotropia no. 15, 2000 schwartz dc, cantor cr. 1984. separation of yeast chromosome-sized dna by pulsed-field gradient gel electrophoresis. cell 37:67-75. skov mm, pedersen k, larsen ja. 1995. comparison of pulsed-field gel electrophoresis, ribotyping, and plasmid profiling for typing of vibrio anguillantm serovar ol. app and env microbiol 61:1540-1545. stringer j. 1980. the development of a phage typing system for group b streptococci. j med microbiol 13:133-143. suwanto a, kaplan s. 1989. physical and genetic mapping of rhodobacler sphaeroides 2.4.1 genome: genome size, fragment identification and gene localization. j bacteriol 171:1135-1145. suwanto a, kaplan s. 1992. chromosome transfer in rhodobacter sphaeroides: hfr formation and genetic evidence for two unique circular chromosomes. j bacteriol 174:1135-1145. suwanto a. 1994. pulsed-field gel electrophoresis: a revolution in microbial genetics. as pac j mol biotechnol 2:78-85. suwanto a, yuhana m, herawaty e, angka sl. 1998. genetic diversity of luminous vibrio isolated from shrimp larvae in: flegel tw (ed) advances in shrimp biotechnology. national center for genetic engineering and biotechnology, bangkok, p 2-9. tjahjadi mr, angka sl, suwanto a. 1994. isolation and evaluation of marine bacteria for biocontrol of luminous bacterial disease in tiger shrimp larvae (penaeus monodon, fab.). as pac j mol biol biotechnol 2:347-352. welsh j, me clelland m. 1990. fingerprinting genomes using pcr with arbitrary primers. nucleic acids res 18:7213-7218. 47 1. sittie aisha page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 untitled document prof. dr. mirnawati sudarmanto, faculty of veterinary medicine, bogor agricultural university, bogor (ipb), indonesia dr. ratih dewanti hariyadi, faculty of agricultural technology, bogor agricultural university (ipb), bogor, indonesia prof. dr. m.a. chozin, faculty of agriculture, bogor agricultural university (ipb), bogor, indonesia dr. soekisman s. tjitrosemito, faculty of mathematics and natural sciences, bogor agricultural university (ipb), bogor, indonesia dr. dahlia sukma, faculty of mathematics and natural sciences, the state university of jakarta (unj), jakarta 13220, indonesia dr. lisdar m. sudirman,  faculty of mathematics and natural sciences, bogor agricultural university (ipb), bogor, indonesia dr. nurita l. toruan-mathius, tissue culture laboratory, smart research institute (smartri), bogor, indonesia ir. soemaryono m.sc., biotechnology research unit for estate crops, bogor, indonesia dr. okky s. dharmaputra, seameo biotrop/faculty of mathematics and natural sciences, bogor agricultural university (ipb), bogor, indonesia dr. gayuh rahayu, faculty of mathematics and natural sciences, bogor agricultural university (ipb), bogor, indonesia dr. yadi haryadi, faculty of agricultural technology, bogor agricultural university (ipb), bogor, indonesia dr. zainal mahmud, indonesian agency for agricultural research and development (puslitbangbun), bogor, indonesia  biotropia book final.indd 95 compatibility studies of interspecific in vitro micrografting of agarwood (aquilaria malaccensis lamk.) nurita toruan-mathius*, jonner situmorang, dewi rachmawati & anida biotechnology and plant breeding, seameo biotrop, bogor, indonesia abstract aquilaria spp produced agarwood as nonwood forest production, and has high economic value. a. malaccensis is susceptible to white rot diseases and termites. on the other hand most of the community plantations are a mixed culture with rubber trees, oil palms and with high risk of contamination causing white root diseases. besides that, vegetative propagation by cuttings, stumping or air layering are still very diffi cult with low percentage of growth. th e objectives of this research were to analyze the best suitable micrograft type, changes of sds-page protein band patterns of compatible and incompatible micrografts with several combinations of gaharu planlets in in vitro condition, and histology of union area between rootstocks and scion. th e results showed that wedge or v type was the best of the micrografs. ms medium with the addition of 3 mg/l iba was the best medium for gaharu planlet growth after micrografting. acclimatization was conducted in husk chacoal and top soil (1:1) medium and grown under plastic house of 70% shading with paranet. compatible combination (ac/am) of micrografting showed that anatomy structure of union area is the same as anatomy structure of non micrograftd planlet. while incompatible (gv/am) micrografting produced necrotic layer growth from pith and parenchymateous tissues of the wood in union area along the middle of radial shoot. recovery period of union area between stocks and scion is initiated by callus formation from the pith and parenchymatous tissues of the wood. callus will diff erentiate into mature cells or tissue and become combined phloem and xylem vessels between rootstocks and scion. sds-page protein band pattern on compatible combination was the same as plants originated from seedlings. while, incompatible combination produced new protein bands with molecular weight around 21 and 30 kd. key words : agarwood, aquilaria spp, micrografting in vitro, incompatible micrografting, sdspage protein, incompatible histology. introduction a. malaccensis, is a famous species for production of good-quality agarwood. agarwood is an inducible secondary metabolite caused by interaction between defence materials like phytoalexin of gaharu trees against compatible pathogen of cells in woody biotropia vol. 15 no. 2, 2008 : 95 109 *corresponding author: nurita-toruan@smart-tbk.com 96 tissues (van der plank 1978; kunoh 1990; rahman & basak 1980; parman et al. 1996). th is secondary metabolite resin is not secreted from trees like other resins or gum, but accumulated in infected stems or branches. th is phenomenon causes the white and smooth part of wood turns to be dark and hard. th is wood becomes heavier and fragrant if it is burned (hou 1960; anonim 2002; subansenee et al. 1985). gaharu is of high economic importance in asia due to its use for the production of incense, perfumes and traditional medicines. a. malaccensis is recognized on the iucn red list as critically endangered and considered to be at risk from overexploitation. all species of aquilaria were included on the appendix ii list of the convention on international trade in endangered species of wild fauna and flora (cites) in 1994 (cites 2003) to improve control of commercial gaharu trade in all participating countries. to prevent a. malaccensis to become an endangered species, indonesia has compiled a national program for sustainable agarwood production by planting of this species in several provinces through government project and community participation (suparman 2006). th ese programs faced problems because a. malaccensis is susceptible to white root diseases and termites. on the other hand, most of the community plantations are a mixed culture with rubber trees, oil palms and with high risk of soil contamination causing white rot diseases; a. malaccensis is a relatively slow-growing plant compared with other aquilaria species; recalcitrant seed with low viability, hand–sown seeds have germination rate (67% for those planted immediately, decreasing to 47% for those sown after 1 week). to solve these problems in vitro micrografting between a. malaccensis as a scion and other species of aquilaria or gyrinops as a rootstock, or interspecifi c micrografting could be used for propagation. toruan-mathius et al. (2006 b) reported that seameo biotrop has succeeded to develop tissue culture technique for propagation of a. malaccensis, a. crassna, a. becariana and gyrinosp verstigii from selected mother plants, acclimatized and planted under fi eld conditions. th ese techniques are used as a basis for in vitro micrografting. in vitro micrografting : shoot-tip grafting in vitro (stg) consists of grafting, under aseptic conditions, a small shoot tip onto a young seedling or planlet root stocks growing in vitro. th e technique has the following steps: rootstock preparation, scion preparation, grafting procedure, growing grafted plants in vitro, and transferring to soil. possibility of micrograft less diff erentiates shoot tip tissue may also help in reducing compatibility problems between scion and stock (jonart 1986). several reports have described the application of in vitro and in vivo micrografting that may provide several advantages such as rejuvenation of mature tissues, year round plant production, enhanced compatibility studies and correlative relations between root stocks and scions, make specifi c genotypic combinations to increase productivity, and extend ecological limits of a particular plant species or cultivar to tolerate edaphic conditions (richardson et al. 1996; hartmann et al. 1997; estrada-luna et al. 2002; sobhana et al. 2001; sanjaya et al. 2006). toruan-mathius et al. (2006a) have succeeded to use in vitro and in vivo interspecifi c micrografting of cinchona ledgeriana as a scion onto c. succirbura as a rootstock to avoid white root disease. under fi eld condition, in vitro micrografting grows faster than that of in vivo grafting. da costa et al. (1991) observed that production of plants grafted on robusta coff ee was three to four times higher than that of nongrafted plants. in addition it is biotropia vol. 15 no. 2, 2008 97 also used to increase resistance to soilborne parasites or soil-borne diseases caused by pathogens such as fusarium oxysporum. grafting is also commonly used for fruit trees and in silviculture (i) to improve tree adaptation to unfavorable soil or climatic conditions, (ii) to enhance water and nutrient uptake, (iii) to increase plant vigour and extend duration of economic harvest time, and (iv) to shorten the breeding period by limiting the breeding objective for resistance to soil-borne diseases and nematodes in root stock. th ese fi ndings suggest that the existence of this technique is an eff ective means of rapid and true-to-type multiplication of desired a. malaccensis genotypes. th e objectives of this study were: (i) to determine the appropriate and reliable micrografting techniques for agarwood planlets; (ii) to determine survival, growth, rootstock suckering, and short-term compatibility of a. malaccensis scion on several species of rootstocks under in vitro conditions, and (iii) to characterize graft incompatibility based on histological and biochemical aspects of grafting. materials and methods th e study consisted of rootstock preparation, scion preparation, (a) in vitro micrografting, (b) analysis of compatibility by histology and (c) electrophoresis sds page protein of stem from scion. plant materials a. malaccensis b23 line used as scions were micropropagated following techniques established by situmorang (2000). axillary buds excised from young planlets (3-5 cm long) were initially used as explants and induced to develop shoots using murashige and skoog (1962) basal salt formulation medium adjusted at ph 5.7 and supplemented with 5.0% sucrose and 0.6% bacto agar. ba (6-benzyl aminopurine) of 1.0 mg/l was added for shoot induction and proliferation cultures. adventitious roots developed in ms medium with the addition of 3 mg/l iba (indole butyric acids). planlets of vigorous a. crassna, a. fi laria and girynops verstigii as stocks were propagated by tissue culture the same as for planlet scions. all cultures were grown in a culture room with light of 100 μmol m-2 s -1 photosynthetic photon fl ux density (ppfd) cool white fl uorescent lamps at planlet level and photoperiod of 16 h. temperature was maintained at 27 ± 2o c. micrografting under aseptic conditions, shoots of uniform length and diameter were selected from in vitro culture and used as rootstocks and scions in micrografting. stainless silver blades mounted on a handle were used for cutting the plant material. freshly picked shoot tips and in vitro regenerated shoot tips were used as microscions. to prepare the initial scions of the micrografts, the leaves from those portions were eliminated, and four types of grafting were made in each of their basal parts. for micrografting, the following method was used. when the stem axis of the plantlets reached a length of about 2 cm the upper 1.0 cm of the tips were excised and used as scion, while remaining portion was used as rootstock. in the fi rst experiment four types of grafting viz., a) wedge (v), (b) horizontal cut grafting, (c) slant-cut grafting (/) interspecifi c in vitro micrografting of agarwood – n. toruan-mathius et al. 98 and (d) cleft grafting (i) (fig.1) were made in the rootstock and scion. th e scions were placed in the incisions made in the rootstock with both the cut surfaces in good physical contact (fig. 1). th e stock and scion were held together at the point of graft with sterile (1x1 cm) light aluminum foil. for optimizing the method of micrografting in vitro plant materials and as scion only b 23 and a. crassna as a rootstock were used. on the other hand, for compatibility studies three clones of a. malaccensis as scion and three species as rootstocks were used, with the best method of micrografting, and as a control the same species of scion and rootstocks were used. th e percentage of successful and unsuccessful grafts was determined 30 days after grafting and in vitro culture. all planlets of graft treatments were cultured in ms medium + 3 mg/l iba for rooting. after three months the growth of scion as a result of graft union was observed. for incompatibilities studies, the combination of scion-stock i.e. a. malaccensis as scion and a. fi larial, a. crassna, gyrinops verstigii as stocks could be used, while as control the same species of scions and rootstocks were used. cultures were incubated in light culture room (16 hrs/days) with rh 70-80%. a b c d (i) (ii) (iii) (iv) biotropia vol. 15 no. 2, 2008 figure 1. in vitro micrografting between scion of a. malaccensis planlets with rootstocks of several species of aquilaria sp and g. verstigii, using (i) wedge, (ii) horizontal, (iii) slant, and (iv) cleft grafts. a. micropropagated shoots, b. rootstock, c. scion, and d. methods of grafts. 99 growth analysis th e in vitro micrografting success rate was determined 6 weeks after grafting by recording the number of scions still alive out of all individual treatments. number of leaves were also recorded and measured for each sample, 2 months after the date of micrografting, and recorded continuously for every month during 8 months observations. at the end of the experiment, all plants were carefully removed to study their root systems. for fi ve plants from each graft combination and the control, the stem was cut crosswise for macroscopic observation of graft union or analysis of compatibility. experimental design combination of stock-scion viz.: a.malaccensis/a.malaccensis (control 1), a. crassna/ a. crassna (control 2) , a. fi laria/ a. fi laria (control 3); a. malaccensis/ a. crassna; a. malaccensis/ a. fi laria was used. experiments used completely randomized design of 1x3 units with 15 replicates. analysis of compatibility analysis of compatibility between scion and rootstocks studies by histology and electrophoresis sds page protein of graft union, were conducted for in vitro micrografting. histology of graft a histological technique was used to observe the graft union of every combination of root stock/scion. th e graft union was cut into segment of 2 cm long and microtome was used to cut 25 μm slices from the segments. th e samples were fi xed for 24 h in solution of faa and then dehydrated in an ethanol series (50-70-80-90-95-100-100%) for 30 min. each. safranin (2%) was added in 70% ethanol and fast green (1%) in 100% ethanol for 15 seconds. th e slices were placed in a solution of 1 absolute ethanol: 1 xylene for 30 seconds and then in pure xylene (2x for 30 min. each), before being examined under the microscope (esau 1965). electrophoresis sds-page protein total protein was extracted from scion shoot (2 cm above graft line) by frozing in liquid nitrogen and stored at -40o c. plant tissues were homogenized in a mortar in the presence of liquid nitrogen and extracted using buff er containing 5 mm tris hcl, ph 8, 500 mm nacl, 2 mm ascorbic acid, 0.5 mm phenylmethylsulphonyl fl uoride and 1 mm dithiotreitol (laemmli 1970). th e homogenate was heated at 80o c for 10 min. and centrifuged at 17.000 g. supernatant were thawed and heat stable proteins were quantifi ed by the binding-dye assay (bradford 1976). protein was separated by 10-15% polyacrilamide gradient gels. heat stable proteins 5-15μg were loaded in each well with discontinuous buff er systems (laemmli 1970) using a mini protean 3-electrophoresis cell (bio-rad, hercules, ca, usa). gel lanes were loaded with equal amounts of perceptible counts; low molecular weight protein standards (bio-rad) were run as size markers. electrophoresis run with constant current of 75 ma was applied per gel. during electrophoresis the voltage increased from about 300 to 600 v. a 0.025 m tris-glycine buff er (ph 8.6) was used as the electrode buff er. when the tracking dye had migrated to the bottom of gel (after approximately 3.5 hrs) electrophoresis was stopped. gels were interspecifi c in vitro micrografting of agarwood – n. toruan-mathius et al. 100 stained with commasie brilliant blue. after staining gels were fi xed in solution containing methanol, glacial acetic acid and distilled water with the ratio of 4:1;15 (v/v/v) for 15 min. and photographed, then gels were wrapped with cellophane for documentation. th e eff ect of stock on scion was studied by the formation of new protein band or disappeared protein band compared with control (non micrografting and micrografting scion-stock with the same species). statistical analysis all data were subjected to one-way analysis of variance (anova) using sas version 6.12 (sas institute 1996). for categorical data, the normality of residuals and homoscedasticity were verifi ed to fulfi ll the requirements for anova. duncan’s multiple range test was used for mean separation. th e diff erences were considered signifi cant at p ≤ 0.05. results and discussion in vitro micrografting th e type of graft and nature of support signifi cantly infl uenced the success of micrograft. wedge or v graft gave the most successful micrografts followed by cleft graft method (tabel 1). v graft may provide a better surface for the contact of stock and scion providing good cambial joining at the graft union. it was shown that v type of micrograft also gave highly signifi cant diff erence on grafting success, except on the fi rst week of incubation because of callus formation (table 2). a good result was obtained with micrograft v type, which gave faster growth compared to cleft graft, caused by v graft which have high levels of graft union (table 2). micrografting of gaharu planlet with v graft in combination between rootstocks and scion ac/am and am/am showed the same vigour (fig. 2). (a) (b) figure 2. micrografting of gaharu planlet with v type in combination between rootstocks and scion: (a) ac/am, and (b) am/am. ac – aquilaria crassna; am – aquilaria malaccensis biotropia vol. 15 no. 2, 2008 101 table 1. effect of micrografting techniques on union and growth of scion method of grafting number of grafts grafting success (%) wedge (v graft) 25 98,5 a horizontal 20 25.6 c slant 25 28,6 c cleft grafts 25 80,7 b estrada-luna et al. (2002) reported that the best method for in vitro micrografting of prickle cactus (opuntia spp) were horizontal and wedge grafts. th e fi rm contact between rootstock and scion is extremely important at the graft junction to get fusing between them and callus formation. lukman et al. (2005) found that micrografting of two diff erent species (mundo and manggis) with v type, gave 76% succesful graft rate when the shoot tips were used directly as microscions in in vitro studies. oda (1995) said that micrografting with v type will gave a fi rm and faster union between stocks and scion. tirtawinata (2003) explained that the success rates of micrograft are highly dependent upon the cambium union of rootstocks and scion. partial contact of rootstock and scion cambium will contribute to unsuccesful joining. th ese cases are described as “translocated” graft incompatibilities (estrada–luna et al. 2002) according to hartmann et al. (1997) callus formation occurred as parenchym cells in a process of wound recovery. th ese callus will diff erentiate into cambium, xylem and phloem. th ese vessels are used for transportation of nutrition and photosyntate. kala et al. (2002) reported that in vitro micrografts rubber begin to grow weeks after micrografting. rifa’i (2003) found callus formation up to three months after micrografting. tabel 2. the effect of micrograft types on leaf number of 1-8 weekold planlets after micrografting. combination and type of micrograft on weeks .... 1 2 3 4 5 6 7 8 ac/am–v 0 0.4 a 1.56 a 2.28 a 3.00a 3.88 a 4.56a 5.08a ac/ac-l 0 0.71b 1.20b 1.58b 1.72b 2.95b 2.14b 2.23b am/am-v 0 1.27a 2.24a 3.00b 3.84 a 4.08a 4.92 a 5.48a am/gv-l 0 0.85b 1.23b 2.08b 2.84b 3.52 b 4.20b 4.84b ac/ac-v 0 1.14a 2.00 a 2.60a 3.,52a 4.,00a 4.68,a 5.32a cl/cl-l 0 0.71b 1.16b 2.00b 2.40b 3.24b 4.00b 4.48b respons tn * * * * * * * explanation ns : not significantly ; *.significantly different. numbers followed by the same letter in the same column are not significantly different according to duncan test p < 0,05 interspecifi c in vitro micrografting of agarwood – n. toruan-mathius et al. 102 acclimatization th e average of planlets survived after acclimatization in husk charcoal : sterile top soil (1 : 1) after one month incubation was 90% (tabel 3). gunawan (1992) reported that planlet from in vitro culture i.e. has wax layer on undeveloped cuticula, limited shoot lignifi cation, number of leaves palisade cells very low, undeveloped xylem, disfunction of stomata caused high transpiration it caused the scion become sensitive to evapotranspiration, attack of fungi and soil bacteria, and also to high light intensity. th at is why planlets should be acclimatized before transfering into the fi eld. table 3. survival percentage of gaharu planlet after acclimatization two months grown in plastic house. combination and graft type % of survived planlet ac/am -v 92 b ac/ac -v 90 b am/am -v 90 b ac/am -cleft 60 a ac/ac -cleft 66 a am/am -cleft 65 a explanation : numbers followed by the same letter in the same column are not significantly different according to duncan test p < 0.05 histology of micrografts th e results showed that anatomy structure of gaharu shoot is the same as anatomy structure of plantlet shoot of another dicotyledone plant which consisted of phloem, cambium and xylem. cambium was radially in between of phloem and xylem vessels. th e inner part of cambium develops into xylem vessels which function as a transportation of water and nutrition from roots to upper parts. outside the cambium is phloem which functions as a transportation of photosyntate to ground parts of plant. our observation revealed fi ve development stages during graft union formation: development of necrotic layer, proliferation of callus bridge at the graft interface, diff erentiation of new vascular cambium, restoration of new vascular tissue, and restoration of the continuity of the epidermis at the graft union. unions between parenchymatous tissues have been confi rmed 2-3 weeks after grafting. fifteen days after grafting parenchyma unions occur in all micrografts that have prospects of further development. only cells newly formed after grafting are able to unite. th e fi rst unions occur between cells originating from tissues outside the cambial region. in side-slit grafts with radial incision in the bark of the rootstocks, the fi rst union developed between callus from the rootstock cambial region in the barks fl ap, and callus from cortex, phloem rays and pith of the scion. trial incision on the rootstock unions also occurs at the incision face in a way similar to that of veneer side grafts. union of newly formed cells may develop independently of the kind of originial tissues (fig. 3a & 3b). biotropia vol. 15 no. 2, 2008 103 figure 3. histology union area of compatible and incompatible micrograft (a, b & c) radial cross section of stem at union area. (a). non micrografted stem, (b) union area of compatible micrograft, (c) union area of incompatible micrograft (d & e) longitudinal cross section of stem union area. (d) union area of compatible micrograft, (e) union area of incompatible micrograft. (a). 1cambium, 2pericycle, 3cortex (e) 1necrotic cells ; 2callus; — bars (40 μm) histological investigations concluded that callus bridging between scion and the rootstock was completed three weeks after micrografting and that both partners took part in the development of the callus. diff erentiating xylem elements within the callus bridge were observed by the fourth week but no bridging of the graft union was achieved at this stage. noticeably, each partner established new vascular system outside the callus bridge, directed towards the others. hartmann et al. (1997) explained that callus is a group of parenchym cells which has developed around wounded tissues. growth of parenchym cells is a initial phase of callus formation in union area of stocks and scion. parenchym cell has high ability to divide and develop into mature cells. cell development and anatomy of planlet will change, and its function of each tissue/cells one to another is also diff erent. undiff erentiated tissues have diff erent functions compared to diff erentiated tissues. callus will diff erentiate into mature cells or tissue and become a combined tissue between rootstocks and scion. callus growth was found from the combination of gv/am and gv/amc (fig. 3 b, c & d) but diff erent with the seedling or am/am (fig. 3). it was shown that the rootstock aff ected the growth of the scion, changes the morphology and interspecifi c in vitro micrografting of agarwood – n. toruan-mathius et al. 104 anatomical structure of the scion. in contrast, the scions have little eff ect on rootstock growth. mismatch of phloem fi ber bundles has an important eff ect on graft success (fig 3e). on the whole, the rootstocks produce a large volume of callus before union than the scions but leaf traces adjacent to cut surfaces of the scions cause locally larger formation of callus. rays that have been cut far from the phloem usually form more callus than those which have been cut close to the cambium. th e boundary between phloem and cortex is a very active zone of growth. th e cambial region plays a subordinate role as a callus producer in veneer side grafts in the rootsocks of v graft. however, the loose bark from the wood in the cambial region and the cells produce vigorous callus formation (erea et al. 2001). th is process is initiated by ray cells, but soon the major portion of the cambial cells participate if they are undamaged by grafting. callus formation from the pith and from parenchymatous tissues of the wood occurs only in union area . th e pith of the scion is particularly active when cut. since the cambium rootstocks had already entered an active stage at grafting and the rootstocks have water and nutrients supply higher than that of the scion, its cambium will be able to deposit several new rows of tracheids up at the time when union is possible. th is means that the cambium is forced to move from scion cambium which has been placed in front, but which is catching another cambium that has been placed outside (fig 3). th e fi rst unions of vascular tissues in veneer side grafts are often found between the stock fl ap and tissues situated on the short cut surface of the scion leaf traces which appear to play an important role in the establishment of vascular connections. th e cambial unions in the innermost corner of side slit grafts become complex depending on the mutual position of the tissues of the grafts components. it is not necessary for the space between the wood surfaces of the graft components to be fi lled with callus tissues, above the uppermost point of contact with the scion. th us the wound caused is callused over from all sides in the same manner as in cut branch. rifa’i (2003) found that callus formation at union area still continues up to three months after micrograft. tirtawinata (2003) reported diff erentiation process of new cambium occurred after three months of micrografting. after fourty days to 6 months of union area will make apple micrografts become fi rm (richardson 1996). estrada-luna et al. (2002) explained that compatible micrograft growth of scion planlet is very vigourous, while the incompatible one is dwarfi sh. in walnut (junglans regia) union of vascular tissues have been confi rmed after about three weeks in wellmatched grafts. when the matching has not been well done, union may require 5-6 weeks. in well-matched grafts it is common that divisions in the cambial region are able to serve in the union of vascular tissues almost at once. only a small number of short, irregular cells are then formed. when the cambial region is located further apart, they spread through intermediate parenchyma tissues toward each other by joining from one cell to the next. heteroplastic or interspecifi c micrografting is used in fruit tree breeding to produce early and abundant fl owering small trees. th e same eff ect also occurs when grafting forest trees for seed orchards. in order to avoid nematode damage to roots of coff ea arabica l. in latin america, a common practice is to apply interspecifi c grafting on c. canephora var. biotropia vol. 15 no. 2, 2008 105 robusta (pierre) rootstocks. villain et al. (1996) observed that with heavy pratylenchus sp, infestations, production of arabica plants grafted on a c. canephora var. robusta (2n=2x=44) rootstock was four times higher compared to nongrafted plants. electrophoresis sds page protein th e results showed that leaf protein of scion tested has molecular weight of 14 45 kd. generally sds-page protein band patterns of scion are the same with planlet control or non micrografting, except from am/amc and am/gv. two of the small protein molecules, 21 and 30 kd were found only in these combinations (fig. 4). th ere are several basic strategies for identifying stocks/scion interactions at the biochemical level. 2 0 0 1 1 6 9 7 2 0 0 1 1 6 9 7 k k figure 4. electrophoregram leaf protein of scion as a result of micrografting in vitro. m. marker; 1. am; 2. amc; 3. gv; 4. am/am; 5. amc/amc; 6. gv/gv; .am/amc; 8. am/gv; 9. amc/ gv th e most important step in identifying a biochemical basis for stocks/scion interaction is verifying that there is causedandeff ectrelationship between the present particular proteins. new protein formation in incompatible rootstocks/scion may be regulated by proteins because these molecules constitute the machinary of cell metabolism. when examining a development change of varietal diff erence in a metabolic process, the cause of the change or diff erence is most commonly a change in the presence, stability, or activity of particular proteins. ji-zhong et al. (2002) reported that incompatible micrografting of apple caused an increase in pod and iod enzymes activities. th ese enzymes controlled auxin translocation from root to upper parts of plant. holbrook et al. (2002) found that on compatible micrografts there was a positive relationship between rootstocks and scion. th ese conditions have been correlated with nutritions distributions, translocation of water and nutrition, and regulation of hormon transportation. raghothama (1999) explained that in interspecifi c in vitro micrografting of agarwood – n. toruan-mathius et al. m 1 2 3 4 5 6 106 compatible micrograft of arabidopsis the relationship of source and sink between stocks and scion is normal. incompatible micrografts of peach and plum planlet aff ected protein and amino acids content of the whole tissues. bertrand and etienne (2001) said that the synthesis of a conserved set of proteins, heat shock proteins, in response to water stress caused inhibition of translocation of nutrient and water. toruan-mathius et al. (1999) found that variations of protein band patterns in union area of micrograft rubber plant are caused by diff erent interactions between micrograft combinations. erea et al. (2001) explained that micrograft can be used as early detection of compatible and incompatible combination of rootstocks and scion in plants propagated by micrografts. it was clearly distinguishable that protein banding patterns signifi cantly have a relationship with the success or failure of grafting. in vitro micrografts usually fail due to incompatibility between stock and scion, oxidative browning of cut surfaces, poor contact or poor development of the root system (toruan-mathius et al. 2006a; moor 1991). sobhana et al. (2001) studied about physiological and biochemical aspects of stock-scion interaction in hevea brasiliensis and found that assimilation rate of scion is being infl uenced by the rootstock. th e considerable cv observed between the individual plants within clone in total soluble sugars, reducing sugars, phenol and amino acid contents, also indicated the existence of stock-scion interaction. toruan-mathius et al. (2006a) reported that incompatibility in interspecifi c micrografting between cinchona ledgeriana and c. succirubra showed formation of 21 and 30 kd protein and formation of stone cells in a union area. conclusions wedge or v graft was the best micrograft method for gaharu planlets, cultured in ms medium with the addition of 3 mg/l iba, acclimatized in medium of husk chacoal : top soil (1:1) and incubated in plastic house. histology structure of compatible micrografting showed that shoot union area was the same as structure anatomy of seedlings, while histology structure of incompatible micrografting has a union area and necrotic layers along radial parts of shoot. union of vascular tissues have been confi rmed after about three weeks in wellmatched grafts, and able to serve in the union of vascular tissues almost at once. only a small number of short, irregular cells are then formed. compatible combination (ac/am) of micrografting showed that histology structure of union area is the same as histology structure of nonmicrografted planlet. while incompatible (gv/am) micrografting produced necrotic layer growth from pith and parenchymateous tissues of the wood in union area along the middle of radial shoot. protein pattern in compatible combination is the same as in seedlings. while protein pattern of incompatible combination produced a new protein band with molecular weight around 21 and 30 kd. biotropia vol. 15 no. 2, 2008 107 references anonim. 2002. cultivating the world’s most expensive incense. enews 25 july 2002: 2-3. http://www.umn. edu/systemwids/enews /072502.html. 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[disertasi] bogor : program doktor institut pertanian bogor. biotropia vol. 15 no. 2, 2008 109 toruan-mathius n., sa adimihadja and i boerhendhy. 1999. rootstock-scion interaction in havea : bark protein patterns and anatomy in correlation with genetic similarities. menara perkebunan 67(l): 1-2. toruan-mathius n., lukman, agus-purwito. 2006a. in vitro micrografting technique of chincona succirubra and c. ledgeriana. menara perkebunan 74:1-10. toruan-mathius n., j situmorang, yupi-isnaini and dewi-rachmawati . 2006b. database and the diversity of aquilaria spp, gyrinops spp and fungi associated with agarwood in indonesia. laporan hasil penelitian, dipa, seameo biotrop 2006. villain l., jl. sarah, b decazy, a molina and s sierra. 1996. evaluation of grafting on coff ea canephora var. robusta, and chemical treatment for control of pratylenchus sp in c. arabica cropping systems, p.407-422. in: proc. 3rd intl. nematol. congr. gosier, guadeloupe, 7-12 july, 1996. van der plank je. 1978. genetic and molecular basis of plant pathogenesis. springer. verlag, berlin heidelberg. new york. interspecifi c in vitro micrografting of agarwood – n. toruan-mathius et al. biotropia no biotropia no. 12, 1999 : 25 30 the effects of a-chlorohydrin on the gestation of the wistar rat (rattus norvegicus) rahmaniah department of biology, university ofhaluoleo, kendari, indonesia and lien a.. sutasurya department of biology, bandung institute of technology, bandung, indonesia abstract the investigation of the effects of a-chlorohydrin on the gestation of the wistar rat has been carried out to determine whether a-chlorohydrin could interfere with the gestation of the rat and gestation period for optimal sensitivity. alpha-chlorohydrin was administered by gavage to three different groups of pregnant rats, namely on the first (pre-implantation period), sixth (early postimplantation period) or fourteenth (late post-implantation period) gestation day, at a single dose of 62.5; 75 and 90 mg/kg body weight. rats were sacrificed on the is* gestation day. number of implantations, gestation loss and post-implantation mortality were recorded. the results revealed that 75 and 90 mg/kg body weight of a-chlorohydrin significantly interfered with all the gestation periods used. for the preimplantation group, a-chlorohydrin decreased significantly the implantation number, but increased the gestation loss as well as the post-implantation mortality compared to the control groups. for the early post-implantation and late post-implantation groups, a-chlorohydrin caused a significant increase in post-implantation mortality compared to the control groups. from these results it is concluded that achlorohydrin at 75 and 90 mg/kg body weight influences the gestation of the wistar rat administered during the pre-implantation period as the most sensitive gestation period. key words : a-chlorohydrin//tattu? norvegi'ctu/rat/pre-implantation/early post-implantation/late postimplantation/implantation/gestation loss/post-implantation mortality. introduction rats comprise a major pest that often causes substantial financial loss to agriculture. there are several ways to control rat populations. for example, exposure of rats to rodenticides to kill the rats. the disadvantage of this approach is that rats will stay away from sites populated with dead rat remains. as a result, rats will move to other areas. another disadvantage is that rodenticides are likely to kill other animals. to control rat populations effectively and without detriment to other animals, it is essential to explore alternative approaches. one of the best alternatives is to use a chemical agent that would reduce fertility in rats without the need to kill them. investigations conducted during the past 35 years were carried out with steroid and non-steroid agents. among the non-steroid compounds, only a-chlorohydrin produced the ideal contraceptive effects in male rats (jones 1983). the results 25 blotropia no. 12, 1999 indicated that low dosages of a-chlorohydrin reduced fertility in males but this effect was reversible. at relatively high dosages, a-chlorohydrin effectively caused sterility in male rats. successful infertility has also been reported for a-chlorohydrin in boar (stevenson and jones 1982; 1985), rats (tsunoda and chang 1976; brown-woodman et al. 1979; paz and homonnai 1982), and macaca mullata (kirton el al. 1970). to achieve optimization of control of rat populations, successful contraception of females is essential. paz and homonnai (1982) reported that oocytes in female rats inseminated with sperm carrying low levels of a-chlorohydrin did not develop. although gao and short (1993) could not demonstrate that a-chlorohydrin produced an antifertility effect in female rats, we decided to investigate the effect of achlorohydrin on pregnancy. our results show that a-chlorohydrin significantly interfered with rat gestation. methods and materials this investigation was carried out on female rats, rattus norvegicus wistar, 90100 days old and a body weight of 180-200 gram. the antifertility drug, achlorohydrin, used in this study was supplied by sigma chemical at a concentration ofl.32g/ml. the general procedure used was as follows. female rats were mated in the afternoon, and the following day, a vaginal smear was obtained to determine the presence of sperm. when sperm was detected, the rats were designated to be in their first day of gestation. pregnant rats were divided into four groups of six rats per group. a single dose of a-chlorohydrin was administered by gavage on the first day of gestation (pre-implantation period), repeated on the sixth day of gestation (early post-implantation period) and again on the fourteenth day of gestation (late postimplantation period). one group of rats received treatments of 62.5 g/kg body weight of a-chlorohydrin, the second group received 75 mg/kg body weight and third group received 90 mg/kg body weight of the drug. the control group received distilled water. the rats were sacrified on the eighteenth day of gestation. the ovaries were removed and placed in 9% nacl. the uterus was cut opposite the implantation sites. the number of implantations, live fetuses, dead fetuses and resorption of embryos were recorded. the ovaries were microscopically examined and the corpus lutea were recorded. from these data, the following reproduction parameters were calculated for every rat: number of implantations percentage of implantation = —————————————— x 100% number of corpus lutea 26 the effects of a-chlorohydrin on the gestation of the wistar rat rahmaniah and l.a. sutasurya number of corpus lutea number of implantations percentage of gestation loss = ——————————————————————— x 100% number of implantations number of live fetuses percentage of post-implantation mortality = ——————————————— x 100% number of implantations the data were statistically analyzed for significance in difference by the lsd test. results and discussion pre-implantation period the pre-implantation period showed that at dosages of 75 and 90 mg/kg body weight gestation loss and post-implantation mortality increased significantly compared to the controls. whereas implantation showed a significant decrease (see table 1 and figure 1), the dosage of 62.5 mg/kg body weight ct-chlorohydrin produced no difference in implantation, gestation loss, and post-implantation mortality from the controls. table 1. percentage of implantation, gestation loss (gl), and post-implantation mortality (pim) for achlorohydrin treatments compared to control for all of the gestation periods gestation period dose (mg/kg b.w.) number of rats examined % implantation (x±sd) %gl (x±sd) % pim (x±sd) pre implantation (1" gd) 0 62.5 75 90 6 6 6 6 93.8 ± 0.3' 93.3 ± 0.31 79.6 ± 0.4" 77.7 ± 0.6" 6.2 ±1. 2" 6.7 ±1.3 20.4 ± 0.7" 22.3 ±1.0* 1.9 ±1.0" 6.1 ± 1.3* 16.7±1.5bc 25.3±l.lc early post implantation (e^gd) 0 62.5 75 90 6 6 6 6 94.7 ± 0.3" 94.8 ±0.3' 92.6 ± 0.8' 89.8 ±0.9* 5.3±1.5a 5.1 ± 1.4 8.0 ± 2.2' 1 0.2 ±2.3' 4.7 ±1.4* 5.4 ± 1.4' 25.0 ±1.0" 39.8±1.6c late post implantation (141" gd) 0 62.5 75 90 6 6 6 6 93.8 ± 0.3" 90.2 ± 0.71 87.6 ± 08* 89.6 ± 0.3' 6.1 ±1.4' 9.8 ± 2.1' 12.4 ±2.4* 10.4 ±1.3' 4.3 ±1.4" 5.4 ±1.4' 20.7 ±uc 24.3 ±1.0cd b.w = body weight gd = gestation day number followed by the same letter indicate that they are not significantly different (lsd test at p<0.05). 27 blotroplano. 12, 1999 figure 1. fetuses and embryo resorption for a-chlorohydrin treatments compared to control for preimplantation period (la gestation days). early post-implantation period at dosages of 75 and 90 mg/kg body weight, post-implantation mortality increased significantly, but implantation and gestation loss were not statistically different, compared to the controls (see table 1). at a dosage of 62.5 mg/kg body weight, a-chlorohydrin produced no difference in implantation, gestation loss, and post-implantation mortality from the controls. late post-implantation period at dosages of 75 and 90 mg/kg body weight, post-implantation mortality increased significantly compared to the controls. however, at these dosages implantation and gestation loss were not affected by a-chlorohydrin. at 62.5 mg/kg body weight, a-chlorohydrin had no effect on implantation, gestation loss, and postimplantation mortality. in all three implantation periods, a-chlorohydrin produced no difference in number of corpus lutea, live fetuses, and dead fetuses compared to the controls. 28 the effects of a-chlorohydrin on the gestation of the wistar rat rahmaniah and l.a. sutasurya the results obtained in this investigation clearly showed that a-chlorohydrin at 75 and 90 mg/kg body weight interferes with pregnancy when administered at all three gestation periods used, and administration during the pre-implantation period generally as the most effective stage. the agent's antifertility effect was most pronounced at 90 mg/kg body weight. at 62.5 mg/kg body weight the result was not significantly different from that of the control groups. alpha-chlorohydrin significantly decreased implantation and increased both gestation loss and postimplantation mortality. alpha-chlorohydrin decreased fertility by more than 50% and, therefore, qualifies as an antifertility drug. the mechanism of action of a-chlorohydrin in female rats is not known. in male rats, it is converted to 3-chlorolactaldehyde, which inhibits glycolysis and reduces atp synthesis in the sperm mitochondrion because less pyruvic acid is available for import into the organelle (stevenson and jones 1985). administration of a-chlorohydrin during the pre-implantation period decreased implantation and increased gestation loss. it is conceivable that the effects were caused by the drug as a result of impairment of transport of the zygote via the oviduct to the uterus, facilitated by muscle contraction and ciliar movement. this requires expenditure of a considerable amount of energy by way of atp supply (johnson and everitt 1988). this might reduce the transport rate of fertilized eggs and cause arrival of the zygote at implantation sites beyond the requisite period of 5th to 6th day of gestation. since administration of a-chlorohydrin during the pre-implantation period resulted in a decrease in implantation and increased post-implantation mortality in the course of eighteen days, this long-term effect may be attributed to storage of achlorohydrin in body fat to prolong its effect (paz and homonnai 1982). to account for the higher values of post-implantation mortality when the drug was presented in the early post-implantation period as compared to the results obtained when treatment occurred in the late post-implantation period, it is conceivable that the placental barrier, once completely formed during post-implantation treatment, may have reduced transfer of the drug and lower embryo resorption (juchau 1981). the present study supports the role of a-chlorohydrin as an effective antifertility drug in female rats. references brown-woodman, p.d.c., i.g. white and d.d. ridley, 1979. the antifertility activity and toxicity of achlorohydrin derivatives in male rats. contraception. 19: 517-527. gao, y. and r.v. short. 1993. use of estrogen, androgen or gestagen, a potential chemosterilant for control of rat and mouse population. j. reprod. pert. 97: 30-49. johnson, m. and b. everitt. 1988. essential reproduction. blackwell scientific publications. oxford. jones, a.r. 1983. antifertility actions of a-chlorohydrin in the male. aust. j. biol. sci. 36: 333-350. juchau, m.r. 1981. the biochemical basis of chemical teratogenesis. elsevier/north-holland. amsterdam. p.3 & 85. 29 biotropla no. 12,1999 kirton, k.t., r.j. ericson, j.a. ray, and a.d. forbes. 1970. male antifertility compounds, efficacy of u-5897 in primates (macaca mullata). j. reprod. pert. 21. 275-278. paz, g.f. and t.z. homonnai. 1982. a direct effect of a-chlorohydrin on rat epididymal spermatozoa. int. j. androl. 5:308-316. stevenson, d. and a.r. jones. 1982. inhibition of fnictolisis in boar spermatozoa by male antifertility agents (s>o.-chlorohydrm. aust. j. biol. sci. 35: 595-605. stevenson, d. and a.r. jones. 1985. production of 3-chlorolactaldehyde from a-chlorohydrin by boar spermatozoa and inhibition of glyceraldehyde-3-phosphate dehydrogenase in vitro. j. reprod. pert. 74: 157-165. tsunoda, y. and m.n. chang, 1976. fertilizing ability in vivo spermatozoa of rat and mice treated with achlorohydrin. j. reprod. fert. 46: 401-406. 30 biotropia no. 9, 1996: 15 25 fungi isolated from groundnuts in some locations of west java o.s. dharmaputra seameo biotrop, p.o. box 116, bogor, indonesia; and department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia i. retnowati seameo biotrop, p.o. box 116, bogor, indonesia abstract one hundred and ninety eight groundnut samples were collected from freshly harvested groundnuts (fhg), farmer storage systems (fss), middlemen warehouses (mw), wholesalers (ws) and retailer sample (rs) during the dry and wet seasons from cidolog, cianjur, sukabumi and bogor, west java, indonesia, in 1990/1991. the moisture content (m.c.), intactness of kernels, and the percentages of groundnut kernels infected by each species of fungi were analyzed. in genera), the m.c. of the samples collected during the dry season was lower than of those collected during the wet season. also, the m.c. of samples collected from fhg, fss and mw was higher than of those collected from ws and rs. the m.c. of samples collected from fhg was the highest (12.5-45.75%), but the percentages of damaged kernels were the lowest (2.5-13.8%), because the samples were shelled manually. a total of 25 species of fungi were isolated from samples collected from the 4 localities. they were acremonium strictum, aspergillus candidus, a. flavus, a. niger, a. ochraceus, a. tamarii, a. wentii, botryodiplodia theobromae, cladosporium cladosporioides, c. sphaerospermum, eumtium chevalieri, e. repens, e. rubrum, fusarium equiseti, f. longipes, f. oxysporum, f. semitectum, mucor sp., papulaspora sp., pestalotia sp., penicillium aethiopicum, p. citrinum, rhizapus sp., r. stolonifer and syncephalastrum sp. the predominant fungi in samples collected from cidolog and sukabumi during the dry season were aspergillus wentii, while those collected from cianjur and bogor were a. niger. the percentages of kernels infected by a. wentii in samples collected from cidolog and sukabumi were between 30-100% and 36-100%, respectively, while those of kernels infected by a. nigerin samples collected from cianjur and bogor were between 34-93% and 14-98%, respectively. the predominant fungi in samples collected from each location during the wet season were a. flavus. the percentage of kernels infected by the fungus in samples collected from bogor was the highest (83-100%). key words: indonesia/west java/stored product pests/groundnut/fungi/acremonium strictum/aspergillus sp./botryodiplodia theobromae/cladosporium sp./eurotium sp./fusarium sp./mucor sp./papulaspora sp./pestalotia sp./spenicillum sp/rhizopus sp./syncephalastrum sp./moisture content. 15 biotropiano. 9, 1996 introduction groundnut is a major food legume and an important secondary crop after maize and soybean in indonesia. since indonesia has a humid tropical climate, this commodity could be easily infected by fungi during storage when freshly harvested or even before harvest. there are various practices of post-harvest processing and storage that could affect the moisture content and the intactness of the kernels and thus their susceptibility to fungal infection. aspergillus and penicillium are the two common genera of fungi found on stored products. they can cause weight loss, seed discolouration, heating and mustiness, and production of mycotoxins, specially aflatoxins. the latter are toxic metabolic substances produced by a. flavus and a. parasiticus and are known to be carcinogenic agents (butler 1974). in indonesia, very few studies have been conducted on storage fungi of groundnuts and their ability to produce aflatoxin. this study shows the presence of fungi infecting goundnuts. information on the percentages of groundnut kernels infected by each species of fungi, moisture content and damaged kernels of freshly harvested, post-harvest, stored and marketed materials during dry and wet seasons are also reported. materials and methods sample collection a total of 198 groundnut samples were collected in cidolog, cianjur, sukabumi and bogor, west java, indonesia, in 1990/1991. of the total, 100 were collected during the dry season (august, september and october, 1990) from farmer storage systems (fss), middlemen warehouses (mw), wholesalers (ws) and retailer samples (rs), while 98 samples were collected during the wet season (february and march 1991) from freshly harvested groundnuts (fhg), fss, mw, ws and rs. the samples consisted of shelled and unshelled groundnuts with the latter shelled manually. about 1 kg of each sample was then divided into 4 subsamples for (1) moisture content analysis, (2) damage kernels analysis, 3) fungal analysis and 4) reserve sample. 16 fungi isolated from groundnuts os. dharmaputra & i. retnowati moisture content analysis percent moisture content of kernels (on the basis of water loss) was determined by the oven method (iso 1968). three replicates were used for each sample. the kernels were ground and dried in the oven at 130 c for 2 hours. the moisture content was determined using the formula: 100 moisture content = (mo ml) x ———— mo where : mo is the initial mass, in gram, of the test portion ml is the mass, in gram, of the dry test portion damaged kernel analysis the kernels were classified as either intact or damaged. damaged kernels are those cracked, broken, porous, shrivelled or insectdamaged. three replicates were used for each sample. the percentage of damaged kernels was determined using the formula: weight of damaged kernels damaged kernels = ———————————————————— x 100 % weight of the kernels analyzed for intact and damaged kernels fungal analysis fungi were isolated using direct plating methods on dichloran 18% glycerol agar (dg 18). according to hocking and pitt (1980), dg 18 was especially used for the isolation of xerophilic fungi. before plating, samples were individually disinfected using 1% sodium hypo-chlorite for 2 minutes. one hundred kernels were then plated (10 kernels/plate) on the medium and incubated at 25 c for 7 days. the fungi were identified using the publications of samson et al. (1984), and pitt and hocking (1985) as the main references. the percentage of kernels infected by fungal species on each medium was determined. 17 biotropia no. 9, 1996 results and discussion moisture content moisture content (m.c.) is the most important factor determining the development of storage fungi in seeds (christensen & kaufmann 1975; neergaard 1979). according to who (1979) the minimum m.c. of groundnuts for the development of aspergillus flavus was 9.0 10.0%. the m.c.'s of samples collected from different localities during the dry season are presented in table 1. the m.c.'s of samples from cidolog, cianjur, sukabumi and bogor were between 9.9 14.1%, 7.9 10.5%, 7.5 11.2% and 6.1 10.7%, respectively. it seems that the m.c.'s of samples collected from the same location were not affected by their source (fss and mw from cidolog, ws and rs from cianjur, sukabumi and bogor). the m.c.'s of samples collected from different localities during the wet season are presented in table 2. the m.c.'s of the samples from cidolog were between 12.5 -45.7% (fhg) and 10.8 18.1% (samples from fss and mw), while those from cianjur, sukabumi and bogor were between 8.7 13.9%, 7.5 10.3% and 7.7 11.1%, respectively. table 1. moisture contents and percentages of damaged kernels of groundnuts collected during the dry season 18 fungi isolated from groundnuts os. dharmaputra & i. retnowati table 2. moisture content and percentages of damaged kernels of groundnuts collected during the wet season in general, the m.c.'s of the samples collected during the dry season were lower than of those of the wet season. the moisture content of samples collected on 30 march 1991 was the highest (12.5 45.7%), because they were not yet dried. according to bulog (1981) the maximum moisture content of stored groundnuts should be 7%. in this study, the moisture contents of the samples collected from farmers storage systems and middlemen warehouses were higher than 7%. damaged kernels damaged kernels gave a chance for fungi to infect the seeds. according to christensen (1980) physical damage of seeds is one of the factors that affects fungal infestation. the percentages of damaged kernels of samples collected from different localities during the dry season are presented in table 1. the percentages of damaged kernels from cidolog, cianjur, sukabumi and bogor were between 12.0 51.8%, 10.2 38.0%, 8.7 27,9%, and 1.5 26.0%, respectively. 19 biotropia no. 9, 1996 the percentages of damaged kernels of samples collected from different localities during the wet season are presented in table 2. the damaged kernels from cidolog were between 2.5 13.8% (fhg) and 9.9 31.7% (samples from fss and mw), while those from cianjur, sukabumi and bogor were between 13.0 61.0%, 18.0 55.0%, and 6.5 -56.0%, respectively. the percentage of damaged kernels of samples collected on 30 march 1991 was the lowest because they were shelled manually, while the other samples were shelled either manually or using wooden/zinc sheller. it was assumed that handshelling prevented the occurrence of damaged kernels, but this method is time consuming. proper tools should be used for shelling groundnuts to minimize the occurrence of damaged kernels. fungal analysis twenty three species of fungi were isolated from samples collected from cianjur, sukabumi and bogor during the dry and wet seasons. the isolated fungal species belonged to the field fungi (i.e. botryodiplodia theobromae, cladosporium cladospo-rioides, c. sphaerospermum, fusarium equiseti, f. longipes, f. oxysporum, f. semitectum and papulaspora sp.), the storage fungi (i.e. aspergillus candidas, a. flavus, a. niger, a. ochraceus, a. tamarii, a. wentii, eurotium chevalieri, e. repens, e. rubrum, penicillium aethiopicum and p. citrinum), and fungi belonging to the mucorales (i.e. mucor sp., rhizopus sp., r. stolonifer and syncephalastrum sp.). aspergillus candidus, a. flavus, a. niger, a. ochraceus, a. tamarii, a, wentii, botryodiplodia theobromae, eurotium chevalieri, e. repens, papulaspora sp. and penicillium citrinum were always isolated from samples collected in each location during the dry season, while those isolated from samples collected during the wet season were always a. flavus, a. niger, a. tamarii, a. wentii, b. theobromae, e. chevalieri, papulaspora sp., penicillium citrium and r. stolonifer. according to dharmaputra and rahayu (1988) the predominant fungal species isolated from groundnut samples collected from a market in bogor, west java, indonesia belonged to the genera aspergillus, eurotium and penicillium, i.e. a. candidus, a. flavus, a. niger, a. restrictus, eurotium spp. and penicillium spp. the fungal species and the percentage of kernels infected by each species of fungi from samples collected from the three localities are presented in table 3. the percentages of kernels infected by the field fungi i.e. f. longipes (collected from cianjur during the dry season), c. cladosporioides and f. semitectum (collected from bogor during the dry season), f. equiseti and f. oxysporum (collected from, cianjur during the wet season) and c. cladosporioides, c. sphaerospermum and f. semitectum (collected from sukabumi during the wet season) were very low. they were between 0 4%, 0 8%, 0 5%, 0 1%, 0 9%, 0 3%, 0 5% and 0 2%, respectively. 20 fungi isolated from groundnuts os. dharmaputra & i. retnowati 21 biotropia no. 9,1996 among the storage fungi, a. niger was the predominant species of fungi infecting kernels collected from cianjur and bogor during the dry season. the percentage of kernels infected by the fungus in samples collected from both localities was between 3493% and 14 98%, respectively. the percentages of kernels infected by a. flavus in samples collected from both localities were between 9 98% and 8-95%, respectively. it was assumed that the moisture contents of the samples collected from the two localities were still favourable for their growth. they were between 7.9 10.5% and 6.110.7%, respectively. the predominant species of fungi infecting kernels collected from sukabumi during the dry season was a. wentii. the percentages of kernels infected by the fungus in samples collected from that location were between 36 100%, while those of a. flavus were between 10 85%. the m.c. of the samples was between 7.5 11.2%. aspergillus flavus was the predominant species of fungi infecting kernels collected from cianjur, sukabumi and bogor during the wet season. the percentages of infected kernels collected from the three localities were between 41 100%, 36 96% and 83 -100%, respectively. pitt et al. (1993) reported that the predominant fungus infecting groundnut collected from farmer storage, middlemen and retailer in thailand was a. flavus, followed by a. niger. the percentages of kernels infected by r. stolonifer and syncephalastrum sp. could not be determined, because their growth was very vigorous. twenty species of fungi were isolated from samples collected from cidolog during the dry and wet season. they were acremonium strictum, aspergillus candidus, a. flavus, a. niger, a. ochraceus, a. tamarii, a. wentii, b. theobromae, c. cladospo-rioides, e. chevalieri, e. repens, f. equiseti, f. longipes, f. semitectum, papulaspora sp., penicillium aethiopicum, p. citrinum, pestalotia sp., rhizopus stolonifer and syncephalastrum sp. (table 4). the predominant species of fungi infecting kernels collected from cidolog during the dry season was a. wentii, with an infected percentage bertween 30 100%, while the percentage of kernels infected by a. flavus was between 0 91%. the m.c. of the samples collected from that location was between 9.9 14.1%. during the wet season, the predominant species of fungi was a. flavus. the percentages of kernels infected by the fungus in samples collected from fss and mw were between 17 96%, while those in fhg were between 51 99%, and their m.c's were between 10.8 18.1% and 12.5 45.7%, respectively. according to christensen and kaufmann (1969) a. flavus can grow in seeds which have a moisture content of at least 18%. on the other hand martin and oilman (1976) reported that optimum moisture contents of natural substrate, which permitted the growth of a. flavus, were between 15 -25%. 22 fungi isolated from groundnuts os. dharmaputra & i. retnowati table 4. the fungal species and range of precentage of infected kernels of groundnut samples collected from cidolog in different storage systems the percentages of kernels infected by a. flavus in fhg were higher than in samples collected from fss and mw, because it was assumed that a. flavus infected the pods of groundnut before harves. according to kozakiewicz (1989) a. flavus is cosmopolitan in distribution. it is found soil, and decomposes vegetation in stored cereals and other seeds as well as variety of food products. dickens (1977) and pitt (1989) reported that the invasion of groundnut by a. flavus take place before groundnuts are harvested. damage of peanut kernels caused by insects and other small animals forces soil into close contact with kernels, and so facilitates the invasion of the fungus. 23 biotropia no. 9, 1996 conclusion fungi isolated from groundnuts collected during the dry and wet season from the farmer storage system (fss), middlemen warehouses (mw), wholesalers (ws), retailer samples (rs), and freshly harvested groundnut (fhg) in cidolog, cianjur, sukabumi and bogor showed the presence of a large number of species. a total of 25 fungal species were isolated from the 4 locations. the total fungal species isolated from samples collected from cianjur, sukabumi and bogor during the dry and wet season were 23, while those from cidolog were 20. there were variations in the percentage of kernels infected by each fungal species from different sources and localities. the predominant fungi in samples collected from cidolog and sukabumi during the dry season were aspergillus wentii, while those collected from cianjur and bogor were a. niger. the predominant fungus in samples collected from the 4 locations during the wet season was a. flavus. the moisture contents of groundnuts collected from fhg, fss and mw were higher than of those collected from ws and rs. there were also variations in the percentages of damaged kernels of samples collected from different sources and localities. acknowledgement the authors gratefully acknowledge the australian centre for international agricultural research for their financial support. the authors are also thankful to dr. j.i. pitt, leader of aciar project 8806; dr. ruben c. umaly and dr. gloria l. enriquez, the former deputy director and deputy director of seameo biotrop, respectively, who gave advice and suggestions; mr. sunjaya, scientist of the tropical pest biology (tpb) programme, seameo biotrop; the technicians of the laboratory of plant pathology, tpb programme, seameo biotrop; and to dr. a.d. hocking, for the confirmation of fungal identification. references bulog, 1981. bulog procurement on quality and purchasing cost of secondary crop for fy 1981/1982. letter of appoinment, head of national logistics agency, no. kep-311/ka/10/-1981. butler. w.h. 1974. aflatoxin. in purchase, i.f.h. (ed.). mycotoxins. elsevier scientific publishing company, amsterdam: 128. 24 fungi isolated from groundnuts os. dharmaputra & i. retnowati christensen, c.m. 1980. needed: research on storage molds in grains, seeds and their products. plant disease 64(12): 10671070. christensen, c.m. and h.h. kaufmann. 1969. grain storage; the role of fungi in quality loss. university of minnesota press, minneapolis. christensen, c.m. and h.h. kaufmann. 1975. control of postharvest losses caused by fungi in food, feed and grains. feedstuffs 47 (10). dharmaputra, o.s. and g. rahayu. 1988. inventory of fungi on various stored grains. biotrop special publication no. 32:55-61. hocking, a.d. and j.i. pitt. 1980. dichoran-glycerol medium for enumeration of xerophilic fungi from low moisture foods. appl. environ. microbial. 39: 488-492. iso, 1968. cereals and cereal products; determination of moisture content. iso recommendation, r712. iso/r712 1968(f). kozakiewicz, z. 1989. aspergillus species on stored products. mycol. pap. no. 161. cab international mycological institute, p. 148-158. martin, p.m.d. and g.a. oilman. 1976. a consideration of the mycotoxin hypothesis with special reference to the mycoflora of maize, sorghum, wheat and groundnuts. the ecology of mycotoxin formation. tropical product institute, london: 23-30. neergaard. p. 1979. seed pathology. vol.1. the macmillan press ltd. pitt, j.i. and a.d. hocking. 1985. fungi and food spoilage. academic press, sydney. pitt, j.i. 1989. field studies on aspergillus flavus and aflatoxins in australian groundnuts. aflatoxin contamination of groundnut: proceedings of the international workshop, 6-9 october 1987, icrisat center, india, p. 223-235. pitt, j.i., a.d. hocking, k. bhudhasamai, b.f. miscamble, k.a. wheeler and p.t. ek. 1993. the normal mycoflora of commodities from thailand. 1. nuts and oilseeds. int. j. food. microbiol. 20: 211-226. samson, r.a., e.s. hoekstra and c.a.n. van oorschot. 1984. introduction to food-borne fungi. centraal bureau voor schimmelcultures, baarn, the netherlands. who. 1979. environmental health criteria ii: mycotoxin. geneva. 25 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf 3 intraspecific (rika).cdr biotropia vol. 18 no. 1, 2011: 24 30 intraspecific variations of 16s mitochondrial gene sequences of yellow rice stem borer, (lepidoptera: crambidae) from west java scirpophaga incertulas rika raffiudin , ruth martha winnie , i made samudra yellow rice stem borer ( ) is one of the most important rice pest insects in asia, including indonesia. however, there is a lack of genetic data for this important agricultural insect. therefore, this study was conducted to explore intraspecific differentiation of partial 16s mitochondrial gene from bogor, karawang, indramayu and cirebon (west java, indonesia). here, we reported a total of 325 bp of 16s mitochondrial gene of from the obtained samples. among all dna sequences, three haplotypes of 16s mitochondrial gene were observed and submitted to genbank under accession number of gu191881, gu191882, gu191883, respectively for haplotype 1, 2, and 3. the haplotype 1 was found in all surveyed locations, except bogor. haplotype 2 and 3 were found only in from cirebon and bogor samples. these haplotype variations can be applied as dna markers for early larva detection method among other rice stem borers. hence, further explorations of the mitochondrial variations of in java and other parts of indonesia are needed moth, haplotypes, genetic differentiations, molecular identification 1* 1 2 (1 (2 department of biology, bogor agricultural university (ipb), darmaga, bogor 16680, indonesia indonesian centre for agricultural biotechnology and genetic resources research and development (icabgrd) scirpophaga incertulas s. incertulas s. incertulas s. incertulas s. incertulas s. incertulas abstract introduction . key words: yellow rice stem borer ( ) is one of the most important rice pest insects in asia, including indonesia, that highly reduces rice yield. they are dominantly spread in the north coast of java (or region, known as rice production regions), i.e. karawang, subang, indramayu and cirebon (hattori & siwi 1986). larvae of yellow rice stem borer damage the rice stem, hence disturb nutrient translocation from scirpophaga incertulas pantura * corresponding author : rika_r@cbn.net.id 24 root to leaf (pathak 1975). as a result, tillers at vegetative stage die, called dead hearth. larvae of also infest the generative stage of rice; therefore producing empty panicles, called white head. yield loss at the rice generative stage was 1-3% higher than that of vegetative stage. several methods were used to control the yellow rice stem borers such as by using pesticide, cleaning the infected area and controling their eggs. almost 57 kinds of insecticides were prohibited to be applied for rice plant because of polluting the environment and decreasing natural enemies. white rice stem borer ( ) was reported resistant to carbofuran insecticide (soejitno 1994). mitochondrial dna is a suitable tool to examine interand intraspecies genetic differences due to higher evolution rate of genes compared to those in nuclear dna (li 1997). moreover, the abundance of mitochondria in the cell provides sufficient materials for the analysis (crozier 1977). current study explores 16s mitochondrial gene of from regions. however, partial gene of 16s available in genbank are only from , the sugarcane stem borer from papua new guinea (genbank acc num ay32046) and from india acc num ay320460). those two records were published by lange (2004) to clarify the phylogenetic relationship of moth members from pyralidae and crambidae in sugarcane stem borers based on 16s and cytochrome oxidase ii (coii) mitochondrial genes (lange 2004). they also suggested that many taxonomy data based on morphology need to be re-organized. yellow rice stem borer was previously classified in the family pyralidae (pathak 1975). currently, is classified in the suborder pyraloidea; family crambidae; subfamily schoenobiinae (lange 2004). four species of rice stem borers commonly found in java are yellow rice stem borer ( ), white rice stem borer ( ), stripe rice stem borer ( ), and pink rice stem borer ( ). alteration of species domination was detected in a certain period, i.e. before 1995, in subang, west java, the dominant species was white stem borer; but later, yellow stem borer dominates the population (suharto & usyati 2005). in an attempt to build database of mitochondrial dna of yellow rice stem borers in indonesia, here we commenced the exploration of intraspecific differences of based on 16s mitochondrial gene in west java (bogor, karawang, indramayu and cirebon). the results of 16s variations of can be applied as dna markers for early larva detection methodamongother rice stemborer. yellow rice stem borer were collected from four localities in west java, indonesia. those were from several subdistricts in bogor, karawang, indramayu and cirebon (table 1). s. incertulas s. innotata et al. s. incertulas pantura s. excerptalis et al. et al. scirpophaga et al. s. incertulas s. innotata chilo suppressalis sesamia inferens s. incertulas s. incertulas materials and method s. incertulas collection intraspecific variations of 16s mitochondrial gene sequences of rika raffiudin .s. incertulas et al 25 table 1. sample locations of moth in west java, indonesia.s. incertulas biotropia vol. 18 no. 1, 2011 locations (subdistrict, district) latitude longitude sindang barang, bogor 6o34’24.61” 106o45’50.9” pedes, karawang 6o04’13.26” 107o22’32.61” arjawinangun, cirebon 6o39’08.63” 108o24’40. 47” lelea, indramayu 6o24’14.75” 108o11’12.59” dna extraction and 16s mitochondrial dna amplification dna sequencing and analysis yellow rice stem borer dna was extracted by using ctab extraction and ethanol precipitation, using thoraces as tissue source (raffiudin & crozier 2007). the 16s of mitochondrial gene of yellow rice stem borers were amplified by using forward primer 16scbf: 5'-aagattttaatgatcgaacag-3' and reverse primer 16scbr 5'tgactgtacaaaggtagcata-3' (simon . 1994). amplifications were carried out in conditions of: initial denaturation at 94 c for 2 min. then 40 cycles at : 92 c for 45s; 50 c for 60s, 72 c for 90s, and final extension at 72 c for 2 min (lange 2004). amplicon of 16s gene was sequenced by using the same primer as in the amplifications. homology of the dna sequences was analysed by using blast (http:/blast.ncbi.nlm.nih.gov/blast.cgi). dna alignments were carried out by using clustal x (thompson . 1997) and sugar stem borer 16s gene gen bank acc. num ay320460 from india and ay320461 from papua nugini (png) were used as the outgroups. all haplotypes obtained from this research were submitted to genbank. tamura-nei distance parameter implemented in mega 5.05 (tamura . 2011) was used to reveal interand intraspecific genetic distance among and 16s mitochondrial gene. this study revealed the first mitochondrial data of yellow rice stem borer 16s mitochondrial gene from west java, which was approximately 325 bp. the moth 16s rna gene sequences were at rich with 65% at. however, the value was lower than that of sugarcane stem borer 16s gene (79.01% at) (lange 2004). blast analysis of 16s gene showed 89% homology with sugarcane stem borer of 16s mitochondrial gene (e-value: 9e ). among all 16s mitochondrial gene sequences, we observed three haplotypes from bogor, karawang, cirebon and indramayu samples (table 2, fig. 1). the first 16s mitochondrial gene haplotype was the common haplotypes found from in three locations (karawang, indramayu and cirebon), i.e samples number sc14kr, sc3id, sc2cb, respectively (table 2). the second 16s mitochondrial et al et al. s. incertulas et al s. excerptalis s. excerptalis et al s. incertulas s. excerptalis s. incertulas s. excerptalis et al. s. incertulas s. excerptalis s. incertulas s. incertulas o o o o o -104 results and discussion 26 haplotypes locations s. incer tulas sample code nucleotide position (see figure 1 ) nucleotide variations genbank acc num 1 karawang sc14kr indramayu sc3id gu191881 cirebon sc2cb 2 cirebon sc15cb 154 a gcirebon sc17cb gu191882 3 bogor sc20bg 129 t ” –“ (deletion) gu191883 table 2. haplotype variations of 16s mitochondrial gene of from bogor, karawang, indramayu, and cirebon (west java) s. incertulas sc: ; notation followed “sc” = sample number and sample location, bg = bogor, kr = karawang, id= indramayu, cb= cirebon. s. incertulas 27 table 3. genetic distance based on 16s mitochondrial gene among and corrected with tamura-nei distance s. incertulas s. excerptalis gene haplotype was found in both samples from cirebon, having transition substitution at the nucleotide number 154 (fig. 1, table 2). compared to 16s mitochondrial gene haplotype 1 and 2, haplotype 3 in sc20bg sample showed deletion of one nucleotide (number 129) (fig.1). these three haplotypes were submitted to genbank under accesion number gu191881, gu191882, g 191883. intraspecific genetic distance among in west java based on 16s mitochondrial gene revealed 0.003. further analysis of intraspecific genetic distance of 16s gene between and from india and png showed 0.145 and 0.133, respectively (table 3). these values were higher than genetic distance between and , i.e. 0.063 (unpublished data). result of this study can be used for identification of yellow rice stem borer in the early instar larvae. this is due to the morphology of rice stem borers larvae which show slightly differences between yellow and white rice stem borers, hence careful examination is needed (amir 2004). phylogenetic analysis frequently establishing interand intra specific relationship between taxa and within populations as well. this was shown in several mitochondrial genes i.e. coi, nd1, and 16s that were used s. incertulas s. incertulas s. incertulas s. excerptalis s. incertulas s. innotata et al. u intraspecific variations of 16s mitochondrial gene sequences of rika raffiudin .s. incertulas et al [1] sc15cb.h2 [5] sc3id.h1 [2] sc17cb.h2 [6] sc14kr.h1 [3] sc20bg.h3 [7] se.india.ay320460 [4] sc2cb.h1 [8] se.png.ay320461 figure 1. dna alignments of 16s mitochondrial gene of from bogor (bg), karawang (kr), indramayu (id), cirebon (cb), and from india and png; “ = homology of nucleotides in the same column. sc: (yellow rice stem borer), se: (sugar cane stem borer). notation followed “sc” = sample number, sample location and haplotype number; ay320460 and ay320461 mentioned after “se” = genbank (www.ncbi.nlm.nih.gov) acc. num. for from india and png s. incertulas s. excerptalis scirpophaga incertulas scirpophaga excerptalis s. excerptalis ·” 28 biotropia vol. 18 no. 1, 2011 to construct a molecular phylogeny of butterflies belonging to the genus s.l.. s.l. has been divided into four genera in an earlier revision. the current results showed three well-supported groups within the genus, corresponding to three of the four proposed genera (zimmermann 2000). another example was on the basis of 16s ribosomal dna and coi-coii regions that revealed restriction-site variations in geographically divergent collections of tobacco budworm, (f.) tennesse, mississippi, oklahoma, and texas (roehrdanz 1994). euphydryas euphydryas et al. heliothis virescens et al. 29 s. incertulas et al. s. incertulas et al s. incertulas s. incertulas s. incertulas s. incertulas this study explored three haplotypes of in several regions in west java, however, a further exploration is needed to reveal the complete genetic intraspecific variations of this agricultural importance insect. database of mitochondrial variations from rice stem borer can be applied for further monitoring strategy to examine the dynamic population of the stem borer, to seek both the origin of this pest and the relationships among other species of rice stem borer. moreover, this result can be used as a basic data to establish enviromental safely control method such as by disrupting the mating time of the stem borer (samudra 2002) and characterise the sex pheromone of to disrupt the mating behaviour (tatsuki . 1985). this study was the first report for intraspecific differentiation of basedon 16s mitochondrial gene obtained from bogor, karawang, indramayu and cirebon (west java, indonesia) samples. three haplotypes of 16s mitochondrial gene were observed and submitted to genbank under accession number: gu191881, gu191882, gu191883, respectively for 16s mitochondrial gene haplotype 1, 2, and 3. haplotype 1 of 16s mitochondrial gene was found in almost all locations, except bogor. haplotype 2 and 3 were found only in from cirebon and bogor samples. further explorations of 16s mitochondrial gene in java and other parts of indonesia are needed to reveal complete 16s gene intraspecific differentiations of this agricultural importance insect. . funding for this research was provided by seameo-biotrop and is gratefully acknowledged. conclusions acknowledgements references amir m, kartohardjono a, siwi s, ubaidillah r. 2004. morphological species variability in the stemborer genus (lepidoptera:pyralidae) on graminous crops. treubia 33:147-163 crozier rh. 1977. evolutionary genetics of the hymenoptera. annual review of entomology 22:263-288 hattori i, siwi s 1986 rice stemborers in indonesia. 20:25-30 lange cl, scott kd, graham gc, sallam mn, allsopp pg. 2004. sugarcane moth borers (lepidoptera: noctuidae and pyraloidea): phylogenetics constructed using coii and 16s mitochondrial partial gene sequences. bulletin of entomological research 94:457-464 li wh. 1997. molecular evolution. sinauer associates, inc. publishers, massachusetts pathak mb. 1975. insects pest of rice. international rice research institute, los banos raffiudin r, crozier rh. 2007. phylogenetic analysis of honeybee behavioural evolution. molecular phylogenetics and evolution 43:543-552 scirphopaga . jarq intraspecific variations of 16s mitochondrial gene sequences of rika raffiudin .s. incertulas et al 30 biotropia vol. 18 no. 1, 2011 roehrdanz rl, lopez jd, loera j, hendricks de. 1994. limited mitochondrial dna polymorphism in north american populations of heliothis virescens (lepidoptera: noctuidae). annals of the entomological society of america 87:856-864 samudra i, emura k, hoshizaki s, ishikawa y, tatsuki s. 2002. temporal differences in mating behavior between riceand water-oats-populations of the striped stem borer, chilo suppressalis (walker) (lepidoptera: crambidae). applied entomology and zoology 37:257-262 simon c, frati f, beckenbach a, crespi b, liu h, flook p. 1994. evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers [review]. annals of the entomological society of america 87:651-701 soejitno j, samudra im, kilin d 1994. kajian ketahanan penggerek padi putih, walker terhadap insektisida karbofuran. penelitian pertanian 14:78-83 suharto h, usyati n. 2005. the stem borer infestation on rice cultivars at three planting times. indonesian journal of agricultural science 6:39-45 tamura k, peterson d, peterson n, stecher g, nei m, kumar s. 2011. mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. molecular biology and evolution 28:2731-2739 tatsuki s, sugie h, usui k, fukami j, sumartapura mh, kuswadi an. 1985. identification of possible sex pheromone of the yellow stem borer moth, scirpophaga incertulas (walker) (lepidoptera: pyralidae). . applied entomology and zoology 20:357-359 thompson jd, gibson tj, plewniak f, jeanmougin f, higgins dg. 1997. the clustal-x windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. nucleic acid research 25:4876-4882 zimmermann m, wahlberg n, descimon h. 2000. phylogeny of euphydryas checkerspot butterflies (lepidoptera: nymphalidae) based on mitochondrial dna sequence data. annals of the entomological society of america 93:347-355. . scirpophaga innotata biotropia 1 (1) 1987: 67-74 v. preliminary study on isozymes of shorea javanica u. juniarti and m. i. j. umboh tropical forest biology program, biotrop, bogor, indonesia introduction the detection of genetic variability in natural or man-made populations/ plantations is useful in both basic and applied biology. in addition to the various facets of studies on shorea javanica already initiated by torquebiau (1984) and alongside with his recommendations on focus for future research, a study on the genetic aspects of the species should be given important considerations. as the trees are tapped for resin, an important forest product, the genetic basis of the production as well as the range of variation in amount of resin production among the trees must be known. coupled with this is a thorough investigation on the differences in pest resistance/susceptability among the trees and their genetic basis. while the assumption (torquebiau 1984) that trees in natural forest areas are-rarely attacked by diseases because of mycorrhizal fungi is interesting, its confirmation is necessary. if this is true, problems would arise when plants are introduced into a new plantation site as experienced by the forest research institute (ardikoesuma 1954). thus, we need to look for pest resistant plants i.e. those that can remain healthy even in the absence of mycorrhizae. the above studies on possible genetic variation could give vital information for development of forest plantations of the species and for breeding and tree improvement strategies. by knowing the extent of genetic variation in natural population or in plantations one could be guided to maintain or increase the genetic base in these areas. biochemical characters such as isozyme banding patterns have been useful in several areas of plant biology, population genetics, evolution and breeding. isozymes are detected by starch gel electrophoresis and when their genetic control is established, they could be genetic markers in analyzing variation in morphological or physiological characters. the present study is an attempt to detect the isozymes in leaves, seeds and cotyledons of shorea javanica by gel electrophoresis. materials and methods mature leaves from six mother trees and seeds from one tree were collected from krui, lampung (sumatra) in september 1985. the leaves were kept inside a plastic ice cooling box immediately after harvest and stored in an incubator at 0°c until used for electrophoresis. fifty mg of the leaf blade, seed or cotyledons of 67 biotropia vol. 1 no. 1, july-december 1987 germinating seedlings were excised and ground in a mortar containing 20 mg of quartz sand, 20 mg of polyvinylpolyrrolidone (pvpp) and ca. 0.6 ml of an extractant containing triton-x-100, tris (0.5m), ascorbic acid (0.5m) adjusted to ph 7.0 by acetic acid. crude extract was absorbed into a 5 mm x 9 mm toyo no. 50 filter paper and the wicks were inserted into a 10% starch gel mediated with either 30 mm borate buffer (ph 8.0) or 5 mm histidine buffer (ph 6.6) at the position of 10 cm from the anodal end of the gel. the gel was prepared following smithies (1955) with a toyo starch gel moulder. electrophoresis was conducted with the use of a toyo electrophoretic apparatus containing either 0.3 m borate buffer (ph 8.0) or 0.6 m sodium citrate (ph 6.2) at a constant voltage of 300 v for borate system and at 250 v for sodium citrate system. running time was 3.5—4 hours in a toyo model is-2200 incubator at 0°c. the gel surface was covered with plastic wrap and chilled by ice throughout the run. each gel was sliced horizontally with a sharp blade and gel slices were assayed for the following enzymes: alcohol dehydrogenase (after schwartz and endo 1966), malic acid dehydrogenase (after shaw and prasad 1970), peroxidase (after endo 1978) and superoxide dismutase (after beauchamp and fridovich 1971). the zymograms were recorded by drawing. results and discussion 1. alcohol dehydrogenase (adh) a. leaves fig. 1 shows 3 anodally-moving bandmorphs. one tree showed bandmorph 1 consisting of 3 bands al, a2, and a3. another tree showed bandmorphs ii consisting of 2 bands a2 and a3. four trees showed a single band, a2. fig. 1. adh bandmorph in leaves. 68 v. preliminary study on isozymes of shorea javanica — juniart i & umboh b. seeds fig. 2 shows 5 anodally-moving bandmorphs. out of 18 seeds, 5 showed bandmorph 1 consisting of 3 bands al, a3 and a4; four showed bandmorph ii consisting of bands a3 and a4; one showed bandmorph iii consisting of band a4; four showed bandmorph iv consisting of bands a2, a4, a5 and a6 and four showed bandmorph v consisting of bands al , a2, a4 and a5. fig. 2. adh bandmorphs in seeds. c. cotyledons of germinating seeds only one adh bandmorph was found, i.e. an anodally-moving al band. 2. malate dehydrogenase (mdh) a. leaves fig. 3 shows 2 anodally-moving bandmorphs. four trees showed bandmorph i with band al, a3, a4, and a5 while two trees showed bandmorph ii consisting of bands al, a2, a3 and a4. fig. 3. mdh bandmorphs in leaves. 69 biotrop1a vol. 1 no. 1, july-december 1987 b. seeds fig. 4 shows 3 anodally-moving bandmorphs. twelve seeds showed bandmorph i consisting of bands al, a2, a4 and a5; four seeds showed bandmorph ii consisting of bands al, a2, a3, a4 and a5; two showed bandmorph iii consisting of bands al, a2 and a5. fig. 4. mdh bandmorphs in seeds. c. cotyledons of germinating seeds variation in bandmorph was also found in cotyledons. three mdh bandmorphs (fig. 5) were found; all have 3 anodally-moving bands, but migration rates differ. bandmorph i consists of bands a3, a5, a7; bandmorph ii consists of bands a2, a4, a6 and bandmorph iii consists of bands al, a3 and a5. fig. 5. mdh bandmorphs in cotyledons of germinating seeds. 3. peroxidase (pox) a. leaves three bandmorphs were detected in the leaves (fig. 6). these three have 4 similar cathodally-moving bands cl, c2, c3 and c4. bandmorph i consists of 6 anodally-moving-bands al, a2, a3, a4, a5 and a6. bandmorph ii consists of 5 anodally-moving bands al, a2, a4, a5 and a6. bandmorph iii consists of 4 anodally-moving bands al, a2, a4 and a6. 70 v. preliminary study on isozymes of shorea javanica — juniart i & umboh fig. 6. pox bandmorphs in leaves. b. seeds eight different bandmorphs were detected in the seeds (fig. 7), each one consisting of anodally and cathodally-moving bands. the range in total number of bands detected is 4-12. fig. 7. pox bandmorphs in seeds. 71 b1otropia vol. 1 no. 1, july-december 1987 c. cotyledons of germinating seeds eight pox bandmorphs were found; each one consisting both of anodally-moving and cathodally-moving bands (fig. 8). fig. 8. pox bandmorphs in cotyledons of germinating seeds. 4. super oxide dismutase (sod) a. leaves fig. 9 shows 2 sod bandmorphs i with anodally-migrating al and a2 bands and bandmorph ii with anodally-migrating al and a3 bands. four trees showed bandmoprh i and two trees showed bandmorph ii. fig. 9. sod bandmorphs in leaves. 72 v. preliminary study on isozymes of shorea javanica — juniarti & umboh b. seeds fig. 10 shows 4 bandmorphs. eight seeds showed 4 anodally-migrating bands al, a2, a3 and a4; two seeds showed 1 anodally-moving band al; four seeds showed 4 anodally-moving bands a2, a3, a4 and a5 bands and 4 seeds showed one anodally-moving a2 band. fig. 10. sod bandmorphs in seeds. following the method of starch gel electrophoresis, isozymes of 4 enzymes were detected as bands. the variation found in each of the enzymes assayed in leaves as well as among the seeds seems to be an indication of the diversity of this species in the plantations. however, considering the very limited sampling done from the plantation, the extent of this diversity could not be ascertained. knowing the number of loci involved and the genetics of the observed differences will be prerequisites in understanding the genetic diversity. the mother tree of the seeds that were analyzed were inadvertently not recorded for its isozyme bandmorphs thus the variation in bandmorphs that was exhibited by the seeds could not be analyzed in relation with the bandmorphs of the mother tree. otherwise, an insight into the breeding system of the tree could have been obtained. analysis of the isozymes in cotyledons of germinating seeds could possibly show differential gene activity at different developmental stages. suggestions for future research a sufficient number of trees must be sampled for isozyme detection from leaf extracts. to explain the genetic basis of the locus and its variation will require evidence from crosses between known bandmorphs. since this is difficult to do with this large dipterocarp tree, a progeny test could suffice. this would involve collection of seeds per mother tree as basis of known bandmorph or genotype for the 6 enzymes and raising seedlings. analysis of the enzyme pattern in the seedling leaves will have to be done. 73 biotropia vol. 1 no. 1, july-december 1987 sampling in a few plantation sites will be needed for the comparison of the genetic diversity among these sites. the analysis of the isozyme variation will provide data for quantifying the extent of the genetic variation in these sites. a possible correlation could be searched between presence/absence of certain isozyme bands or whole genotypes and soil/habitat characteristics. it would be interesting to compare the frequency of certain isozyme alleles or whole genotypes among the sites. this may give a clue to the adaptive significance of some isozyme as found in some crop plants. when linkages are found between specific isozyme alleles and genes for economically important characters, e.g. growth, yield, pest resistance, susceptibility for mycorrhizal association etc., these isozyme markers could be used for selection of planting materials to establish plantations. note the research described in this paper was originally carried out by mrs. u. juniarti and dr. m.i.j. umboh; the paper was written by mrs. l.u. gadrinab, whose assistance is gratefully acknowledged (editor). references ardikoesoemo, r.i. 1954. tanaman shorea javanica di djawa. rimba indonesia 3-4 (1954), hal. 141-151. beauchamp, c. and i. fridovich. 1971. superoxide dismutase: improved assays and assay applicable to acrylamide gels. anal. biochem. 44: 276. endo, t., b.b. shahi and c. pai. 1971. genetic convergence of the specific acid phosphatase zymograms in oryza saliva. jpn. j. genet. 46: 147. ______, 1978. a new method for peroxidase isozyme stain. ann. rep. nat. inst. genet. no. 28: 41. gadrinab, l. 1984. a biosystematic study on section pachycarpae in dipterocarpaceae: shorea macrophylla ashton and shorea stenoptera burck. biotrop internal report. gan, y. and f. robertson. 1981. isozyme variation in some rain forest trees. biotropica 13 (1): 20-28. ihara, m. 1985. a report on forest genetic studies. biotrop internal report. schwartz, d. and t. endo. 1966. the genetic control of alcohol dehydrogenase in maize-simple and compound loci. genetic 53: 709. shaw, c.r. and e. prasad. 1970. starch gel electrophoresis of enzymes. a compilation of recipes. biochem. genet. 4: 297. smithies, o. 1955. zone electrophoresis in starch gels. biochem. j. 61: 629. torquebiau, e. 1984. man-made dipterocarp forest in sumatra. agroforestry systems, 2: 103-127. 74 67.pdf 68.pdf 69.pdf 70.pdf 71.pdf 72.pdf 73.pdf 74.pdf biotropia no. 4, 1990/1991: 918 soil as a factor influencing the mangrove forest communities in talidendang besar, riau cecep kusmana forest ecology laboratory, department of forest management faculty of forestry, bogor agricultural university, bogor, indonesia abstract one transect of 10 m x 900 m was constructed perpendicular to the sea edge to know the pattern of mangrove forest communities from the sea edge through the inland area. then, one sample plot of 50 m x 50 m was established at each forest community to explore its vegetation structure and soil characteristics. the results show that in talidendang besar area, there are three mangrove forest communities stretching from the sea edge to the inland, namely bruguiera parviflora, b. sexangula and b. sexangula-nypa fruticans. the soil factors such as ph.ec (electrical conductivity), % k, % na, c-organic, n-total, nh4 (ammonia), and cec (cation exchange capacity) were regarded important in influencing the pattern of the mangrove forest communities. introduction darsidi (1987) reported that the mangrove forest in indonesia covers an area of approximately 4.25 million ha, of which about 276 000 ha are distributed in riau. most of the mangrove forests in riau are located at the east coast where the major tidal swamp land areas are under development for transmigration projects. numerous environmental factors operate in mangrove swamps, but the most important are soil type, salinity, drainage, and water currents (chapman 1975). steenis (1958) stated that the soil type is more important than the other factors in controlling the zonation of mangroves. in any area with fairly uniform climate, the chemical differences in the soil may produce marked changes in the vegetation (billing 1950). while the mangrove forest in indonesia is believed to be the largest in the world (christensen 1982), studies on the physico-chemical properties of mangrove soils are still few (soegiarto 1979). the present study was done to investigate the soil factors influencing the mangrove forest communities in talidendang besar, riau. it is hoped that the results of this research may contribute to the establishment of proper mangrove forest management in indonesia. 9 biotropia no. 4, 1990/1991 materials and methods this research was conducted in a mangrove forest concession area at tali-dendang besar belonging to the pt bina lestari which is located in the kateman district, indragiri hilir regency, riau province. geographically, this mangrove forest area is located at the east coast of sumatera with gentle topography and altitude of 03 m above sea level between long. 103° 28' to 103° 48' e and lat. 0° 21' to 1° n (figure 1). based on the systems of schmidt and ferguson (1951), the tem figure 1. location and the climatic diagram ofthe mangrove forest area of talidendang besar, riau. 10 soil as a factor influencing the mangrove forest communities c. kusmana bilahan research area has a b climate type with seven wet months, two dry months, and three humid months (badan meteorologi dan geofisika 1990). the soils of this area are organosol and glei humus (lembaga penelitian tanah 1964). to investigate the forest community from the sea edge to the inland, one transect of 10 m x 900 m was divided into 10 m x 20 m contiguous subplots and constructed perpendicular to the sea edge. within these subplots, the dbh (diameter at breast-height) of all trees (plants with dbh 10 cm up) was measured. then, one sample plot of 50 m x 50 m was established in each forest community type to explore its vegetation structure and soil characteristics. four soil samples up to a depth of 25 cm were collected randomly from the area within each adjacent sample plot of 50 m x 50 m. each soil sample was analysed for texture, ph, c-organic, ec (electrical conductivity), cec (cation exchange capacity), n-total, nh4 (ammonia), and exchangeable cations (k, na, mg, ca) at the soil laboratory of the faculty of agriculture, bogor agricultural university. the vegetation data were analysed using cox's method (1967) and the importance value index (curtis and mclntosh 1951) was used to determine the vegetational importance of a species within the forest community. results and discussion a. vegetation composition as shown in figure 2, the mangrove forest from the sea edge to the inland in talidendang besar could be divided into three different forest communities, namely, bruguiera parviflora community which occupied the area from the sea edge to about 180 m inland, b. sexangula community from about 180 m to 740 m inland, and b. sexangula-n. fruticans community from about 740 m to 900 m inland as transition area with swamp forest. table 1 shows that in the b. parviflora community, b. parviflora was considered the dominant species and b. sexangula the codominant one. in the b. sexangula community, b. sexangula was considered the dominant species and b. parviflora the codominant one, while in the b. sexangula-n. fruticans community, b. sexangula was dominant and tv. fruticans codominant. there is a marked tendency for the density and basal area of b. parviflora to decrease from the sea edge (b. parviflora community) to the inland (b. sexangula-n. fruticans community). the opposite occurred for the density and basal area of b. sexangula which tended to increase toward the inland area. in addition, the further from the sea edge, the more varied was the tree species richness. it is assumed that the less severe site conditions in the inland area give the chance to 11 figure 2. mangrove forest community from sea edge through inland in mangrove forest area of talidendang besar, riau. bp (b. parviflora), bs (b. sexangula), cd (c. decandra), hm (//. microcarpum), fb (f. benjamina), ra (r. apiculata), and nf (tv. fruticans). table 1. species density and species importance value index (ivi) of trees at three forest communities in a mangrove forest of talidendang besar, riau 12 soil as a factor influencing the mangrove forest communities-c. kusmana many species for a better growth. johnstone (1983) stated that the presence of terrestrial species in the back zone of mangal is more indicative of the salinity regime than representing an active process of colonization from the land as part of an integrated successional system. b. forest community occurrence as related to soil factors as shown in table 2, the soils which occupied each forest community have a high percentage of clay, an intermediate percentage of silt, and a low percentage of sand. this indicates that the mangrove forest area in talidendang besar receives much eroded soil containing fine soil particles through the stream flow from the upper river basin of talidendang besar. it is probably due to the extensive conversion of peat swamp forest to coconut plantation mainly by the bugis people who came from the southern part of sulawesi. the soils covered either by b. parviflora or b. sexangula communities were classified as clay, while the soils covered by b. sexangula-n. fruticans were classified as silty clay. the soils of this mangrove forest area were almost the same as those of the mangrove forest area in ujung karawang, cilacap (al rasyid 1971; soerianegara 1971), and bengkalis (dinas kehutanan propinsi dati i riau 1978). the ph of the soils which covered each of the forest community was generally neutral due to the decreasing percentage of exchangeable cation content in the soils from the sea edge of the inland area. similarly the salinity (electrical conductivity) of the soils tends to decrease toward the inland, but generally the salinity of the soils occupied by each forest community was considered low, and the adsorption site of the soils was dominated by cations in the order of ca > mg > na > k. this is suggested to be due to the fact that the mangrove forest in this area receives much fresh water through the stream flow of talidendang besar river, while the area toward inland was infrequently submerged by sea water. c-organic, nh4 (ammonia), n-total, and cec (cation exchange capacity) increased toward inland. it indicates that toward the inland area the organic matter and its decomposition process tends to increase. it is probably correlated with the maturity of the trees and the soil substrate condition in these forest communities. based on the average height and diameter of the trees in these forest communities (table 1), the inland area of the forest community appears to be occupied by more mature trees as suggested by the amount of litter fall on the forest floor. in addition, the dense aerial roots and the acrostichum aureum on the floor of the b. sexangula and b. sexangula-n. fruticans communities play an important role in trapping leaves and debris during tidal inundation, thereby contributing to the high organic matter content in these forest communities. the increasing content of n and nh4 of the soil covered by the forest community toward inland indicated a more rapid decom 13 14 soil as a factor influencing the mangrove forest communities c. kusmana position process of organic matter on sites further from the sea edge. it is probably due to the decreasing frequency of inundation of the sites toward inland so that the soils are rather stable and more or less well-drained. ponnamperuma (1972) stated that the accumulation of ammonia in anaerobic soils is due to the lack of oxygen to carry the oxidation from nitrite to nitrate, and so the mineralization of organic nitrogen in these soils stop at the ammonia stage. furthermore, broto (1984) reported that if anaerobic soil has a ph greater than 7.0 ammonia volatilization might take place through a denitrification process resulting in severe losses of nitrogen from the soils. there was a significant difference among the three forest communities for eight soil characteristics such as ph, c-organic, n-total, % k, % na, nh4, cec, and ec (table 2). these soil characteristics appeared to be important in influencing the occurrence pattern of mangrove forest communities in talidendang besar. based on a statistical test using least significant difference (table 3), the values of c-organic, cec, ec, % k, and % na were significantly different between forest communities, but the contents of n and nh4 were only significantly different between the b. parviflora and b. sexangula-n. fruticans communities. meanwhile, the ph of the soils was significantly different among the three forest communities, except between the b. parviflora and b. sexangula community. if the three mangrove forest communities were ranked according to their relative positions with regard to these important soil characteristics (table 4), the b. parviflora community tends table 3. least significant difference (lsd) test for soil characteristics showing significant difference among three forest communities no. soil characteristics community type 1 vs 2 1 vs 3 2 vs 3 1. ph ns ** * 2. c-organic ** ** ** 3. n-total ns * ns 4. nh4 ns * ns 5. % k ** ** ** 6. % na ** ** ** 7. cec ** ** ** 8. ec * ** * community type 1: bruguiera parviflora community community type 2: bruguiera sexangula community -community type 3: bruguiera sexangula-nypa fruticans community * significant difference at p < 0.05 ** significant difference at p < 0.01 ns non-significant. 15 biotropia no. 4, 1990/1991 table 4. relative position of three forest communities ranked according to mean values of eight important soil characteristics soil characteristics rank ph c-org. n-total *k %na cec nh4 ec 1 bp bs-nf bs-nf bp bp bs-nf bs-nf bp 2 bs bs bs bs bs bs bs bs 3 bs-nf bp bp bs-nf bs-nf bp bp bs-nf bp : bruguiera parviflora community bs : bruguiera sexangula community bs-nf : bruguiera sexangula-nypa fruticans community. to occupy soils which contain higher % na, % k, ec and ph, and lower c-organic, n-total, nh4, and cec compared to the others. on the contrary, the soils occupied by the b. sexangula-n. fruticans community contained lower % na, % k, ec and ph, and higher c-organic, ntotal, nh4, and cec. the b. sexangula community tends to occupy soils which contain intermediate values of these soil characteristics. it means that in talidendang besar, b. parviflora tends to grow on the rather saline and soft mud clayey soils in the area near the sea edge which is frequently submerged in sea water. b. sexangula can grow on various types of mud ranging from rather soft clayey soils in the area near the sea edge to the hard silty clayey soils in the inland area which is infrequently submerged in sea water. b. sexangula tends to grow optimally on rather hard silty clayey soil with ph 7.0 in the inland area, while the other species viz n. fruticans and ficus benjamina occur in the innermost zone of the mangrove with lower salinity. yamaha and sukardjo (1979) reported that in south sumatera, b. parviflora grows in soft mud, while b. sexangula grows mixed with b. gymnorrhiza and rhizophora apiculata in the innermost zone of mangroves on rather hard mud soil. conclusions the mangrove forest in talidendang besar could be divided into bruguiera parviflora community occupying an area from the sea edge to about 180 m inland, b. sexangula community from about 180 m to 740 m inland, and b. sexangula-n. fruticans community from about 740 m to 900 m inland. in the b. parviflora community, b. parviflora was considered the dominant species and b, sexangula the codominant one. in the b. sexangula community, b. sexangula was considered the dominant species and b. parviflora the codominant one, while in the b. sexangula 16 soil as a factor influencing the mangrove forest communities c. kusmana n. fruticans community, b. sexangula was considered the dominant species and tv. fruticans the codominant one. there were eight soil characteristics considered important in influencing the occurrence pattern of the mangrove forest community in talidendang besar, riau: ph, ec (electrical conductivity), cec (cation exchange capacity), c-organic, n-total, nh4, % k, and % na. references al rasyid, h. 1971. pemilihan jenis tanaman dalam rangka meningkatkan produksi hutan payau ujung karawang. laporan no. 134, lembaga penelitian hutan, bogor. badan meteorologi dan geofisika. 1990. keadaan curah hujan di propinsi riau. departemen perhubungan republik indonesia. badan meteorologi dan geofisika, jakarta. billings, w.d. 1950. vegetation and plant growth as affected by chemically altered rocks in the western great basin. ecology 31: 62-74. broto, k.g. 1984. waterlogged saline soils. in: snedaker, s.c. and j.g. snedaker (eds.). the mangrove ecosystem: research methods: 114-130. unesco, paris. chapman, v.j. 1975. mangrove vegetation. strauss and cramer gmbh, germany. 499 p. christensen, b. 1982. management and utilization of mangroves in asia and the pacific. fao environment paper 3. fao, rome. cox, g.w. 1967. laboratory manual of general ecology. brown company publishers, dubuque, iowa. 195 p. curtis, j.t. and r.p. mcintosh. 1951. an upland forest continuum in the prairie-forest border region of wisconsin. ecology 32 (3): 476-496. darsidi, a. 1984. pengelolaan hutan mangrove di indonesia. in: soemodihardjo, soerianegara, sutisna, kartawinata, supardi, naamin, and al rasyid (eds.). presiding seminar ii ekosistem mangrove, baturaden 3-5 agustus 1982: 19-28. mab-lipi, jakarta. dinas kehutanan propinsi dati i riau. 1979. hutan mangrove di propinsi riau. in: soemodihardjo, nontji, dan djamali (eds.). presiding seminar ekosistem hutan mangrove, jakarta 27 pebruari -1 maret 1978: 176-185. mab-lipi, jakarta. lembaga penelitian tanah. 1964. peta tanah eksplorasi sumatera bagian selatan, skala 1:1 000 000. departemen pertanian republik indonesia, lembaga penelitian tanah, bogor. johnstone, i.m. 1983. succession in zoned mangrove communities: where is the climax? in: teas, h.j. (ed.). biology and ecology of mangroves: 131-139. w. junk publishers, the hague. ponnamperuma, f.n. 1972. the chemistry of submerged soils. advances in agronomy 24: 29-96. schmidt, f.h. and j.h.a.ferguson. 1951. rainfall type based on wet and dry period ratios of indonesia with western new guinea. verhandelingen no. 42, kementerian perhubungan djawatan meteorologi dan geofisik, jakarta. soegiarto, a. 1979. the mangrove ecosystem in indonesia, its problems and management. paper prepared for the second international symposium on biology and management of mangrove, july 20-26, 1980, port moresby, papua new guinea. 17 biotropia no. 4, 1990/1991 soerianeoara, i. 1971. characteristics and classification of mangrove soils of java. rimba indonesia 16 (3,4): 141-150. steenis, c.g.g.j. van. 1958. ecology (the introductory part to the monograph of rhizophoraceae by ding hou). flora mal. 5: 431-441. yamada, i. and s. sukardjo. 1979. ecological study of mangrove and swamp forest in south sumatera, j. southeast asian studies 17 (3): 425-443. 18 9.pdf 10.pdf 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 1 growth and development of fimbristylis miliacea (l.) vahl – mahfuza begum et al.biotropia vol. 15 no. 1, 2008 : 1 11 * corresponding author: mafupaz@hotmail.com growth and development of fimbrist ylis miliacea (l.)vahl mahfuza begum1*, abdul shukor juraimi 2, rajan amartalingam 2, syed omar bin syed rastan3 and azmi bin man4 1department of agronomy, bangladesh agricultural university, bangladesh 2department of crop science, faculty of agriculture, upm, 43400 serdang, malaysaia 3department of land management, faculty of agriculture, upm, 43400 serdang, malaysia 4mardi, pulau pinang, malaysia abstract this experiment was conducted in the glasshouse of universiti putra malaysia, to determine the growth and development of fimbristylis miliacea (l.) vahl. twenty f. miliacea seeds were surface sown in ten plastic buckets of 18 cm diameter filled with 3 kg soil. after germination only one plant/bucket was retained. time of first seedling emergence, time and number of leaves appearing until first tiller formation, time of tiller formation, first inflorescence, the first 10 inflorescences appearance and their maturity were recorded for each plant. plant height and the number of inflorescence per plant was recorded weekly for up to 4 months after sowing. the first ten inflorescences for each plant were tagged after emergence, subsequently mature inflorescences were collected and the numbers of spikelets/inflorescence, seeds/inflorescence, seeds/ plant and 1000 seed weight were determined. statistical analysis was performed as complete randomized design on weekly observed plant height and inflorescence number using the sas statistical software and means were tested using tukey’s studentized range test at the 5% level of probability. fimbristylis miliacea seedlings emerged at 3 days after planting of seeds. approximate times required for the sequential production of 10 leaves, tillers, first 10 inflorescence and their maturity were 28 days after emergence (dae), 35 dae, 49 dae, 63 dae, respectively. plant height increased rapidly from 3-8 wae and maximum plant height (64.05 cm) was attained at 10 wae. this species had three important growth stages: a slow growth stage during the first 4 weeks after emergence (wae); a rapid growth stage from 4-9 wae; and finally, a maximum growth stage from 9-17 wae. within this first 4 weeks after emergence would be the most appropriate time for controlling this species with early post-emergence herbicides. each f. miliacea plant produced on average of 2.3 tillers/plant and a total of 134 inflorescences, with 84 inflorescences/plant ripening within this period. each inflorescence comprised 48 spikelets with 511 seeds and matured after 3 weeks of emergence. total seeds/plant and 1000 seed weight were 42275 and 0.035 g, respectively. time required for seed ripening was 76 days after emergence. key words: life cycle, growth, development, fimbristylis miliacea 2 biotropia vol. 15 no. 1, 2008 introduction species differ substantially in life form and timing of stages of development and reproduction. each of these processes occurs at a measurable, requisite rate. successful individuals grow rapidly or largely, develop through the various stages of their life cycle, and eventually are replaced in the environment by their progeny (radosevich et al. 1997). the components of the life cycle such as timing of germination, seedling emergence, adult survival, age of flowering, and seed numbers are pre-requisites to be determined during the life span of the plant (sabrig 1980; silvertown and doust 1993). the plant developmental phases are fundamental to the understanding of plant function, that is, the manner of interaction within the environment. fimbristylis miliacea is a serious weed in ricefields in south-east asia (moody 1989; smith 1983). in spite of the importance of this weed little is known about the species growth and development. in several annual and perennial weed species, studies on growth and developmental characteristics have increased the understanding of the weed’s morphological characteristics that aid in success, and or exploited in a control program. smith and fick (1938) studied the life history of purple nutsedge (c. rotundus) and related several growth characteristics to possible control methods. they found that effective control can be obtained if control measures are taken early in the life cycle of the plant. mcwhorter (1961) conducted similar experiments with johnsongrass [(sorghum halepense) (l.) pers.]. they concluded that eradicating johnsongrass plants must be accomplished before new rhizomes are formed (6 to 8 weeks) to be effective. chandler et al. (1977) stated that in moonflower weed the total number of seed per pods and seeds produced per plant were 2730 and 9350, respectively, with peak production occurring 5 through 7 weeks after initiation of anthesis. controlling this weed within this period is to eliminate seed production. thus, the time of flowering and seed production are thus important to know. growth and development study have been observed in several weed species such as echinochloa crus-galli (azmi 1994; itoh 1991; arifin 2004), leptochloa chinensis (pane 1997), ischaemum rugosum (bakar and nabi 2003), cyperus esculentus and c. rotundus (williams 1982). even though, biological study of f. miliacea has been reported to some extent by burkill (1935) and holm et al. (1977). but, in recent past growth and development studies on f. miliacea have not been reported. an understanding of the phenology of a weed is important for developing strategies for its control. differences in the behavior of species at each stage reveal to differences in susceptibility to control by a particular approach. information on occurrence of emergence, leaf formation, tillering, flowering, fruiting and other developmental processes would facilitate the timing of effective control measures. therefore, the study on the growth and development of f. miliacea was undertaken with the objectives of (i) to determine f. miliacea growth characteristics from seed germination to seedling establishment and subsequent seed production and (ii) to quantify the time of occurrence of all growth-developmental stages of fimbristylis miliacea. 3 growth and development of fimbristylis miliacea (l.) vahl – mahfuza begum et al. materials and methods the experiment was conducted in the glasshouse at universiti putra malaysia, from november 2003 to march 2004. seeds of f. miliacea were collected from a ricefield at the malaysian agricultural research development institute (mardi) research station, in bertam, pulau pinang on august 2003. the soil used was collected from a tanjung karang ricefield, which was a silty clay (49.71% clay, 49.56% silt and 0.73% sand) of bakau series (typic hydraquents) with ph 4.98. twenty seeds were surface sown in 18 cm diameter plastic buckets filled with 3 kg soil. ten buckets were used for this experiment. after germination, only one plant/bucket was retained. all other seedlings were removed as soon as they emerged. one third of the urea, whole triple super phosphate (tsp) and muriate of potash (mop) were applied as basal fertilizer treatments at rates equivalent to 370, 167 and 250 kg/ha, respectively. another two thirds of the urea was applied at 35 and 55 days after sowing. based on area basis each bucket received a total of 0.93 g urea, 0.42 g tsp and 0.63 g mop. the buckets were irrigated with tap water and maintained at saturation condition. malathion was applied when required to control insect pests. time of first seedling emergence, time and number of leaves appearing until first tiller formation, time of tiller formation, and first inflorescence appearance were recorded for each plant. plant height was measured weekly from the soil surface to the tip of the leaf. time of appearance of the first 10 inflorescences and their maturity was also recorded to determine the average total time to maturity of fimbristylis miliacea. the number of inflorescence per plant was recorded weekly for up to 4 months after sowing to correlate with the life span of cultivated rice. the first ten inflorescences for each plant were tagged when they emerged. these inflorescences were collected when mature and the numbers of spikelets/inflorescence were determined. these ten inflorescences were then dried for 1 week, threshed by hand and the seeds separated by winnowing. the seeds were weighed and number of seeds per inflorescence were estimated using 1000 seed weights. the number of seeds/ inflorescence was derived from the average number of seeds/10 inflorescence per plant which was as follows (charotte et al. 1990): weight of seeds from 10 inflorescence (g) no. of seeds per inflorescence = --------------------------------------------------- × 1000 seeds weight of 1000 seeds (g) × 10 thus total seeds per plant were estimated from the number of seeds per inflorescence multiplied by the number of matured inflorescence per plant. statistical analysis was performed on weekly observed plant height and inflorescence number using the sas statistical software and means were tested using tukey’s studentized range test at the 5% level of probability. 4 biotropia vol. 15 no. 1, 2008 aaab b bc d e f fg g ghh gh fgh efg def de cd bc ab a 0 10 20 30 40 50 60 70 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 week after emergence (wae) p la nt h ei gh t ( cm ) 0 20 40 60 80 100 120 140 160 in flo re sc en ce n um be r plant height (cm) inflorescence number results and discussion growth characteristics of fimbristylis miliacea growth rate as measured by increases in plant height over time was significant. initially, the plants were stunted and increase in plant height was slow for the first 2 weeks (figure 1). figure 1. weekly measured plant height and inflorescence number of f. miliacea (means within week after emergence (wae) with same alphabets are not significantly different at p ≥ 0.05 (tukeys test)) however, at the 3rd week after emergence, a significant rapid increase in plant height (10.5 cm) was observed and significantly higher weekly increasing rate was observed for up to 8 weeks. during this time the plant reached 96.57% of its maximum height. at 10 weeks after emergence the plant reached maximum height (64.05 cm) (figure 1). beyond this period, height did not increase due to onset of leaf tip senescence. a similar pattern was obtained in l. chinensis, where for the first 20 days, plants grew slowly, while during 20 – 50 days after sowing (das) the plant height increased rapidly to 100 cm and with increases of 2-3 cm/day. at 70 das the plants reached maximum height (pane 1997). pennisetum polystachyon which grows to a height of 201 cm, also showed slow initial growth for up to 8 weeks followed by a rapid increase in height at 14 weeks, resulting from the elongation of the internodes at the onset of reproduction (fernandez 1982). in contrast the fast growing weed species, purple moonflower reached 85% of its height within by 4 weeks after emergence (chandler et al. 1977) and scirpus maritimus increased it height at the rate of 2.7 cm/day during the first 40 days of growth (visperas and vergara 1978). differences in plant height are an important 5 growth and development of fimbristylis miliacea (l.) vahl – mahfuza begum et al. criteria determining species competitiveness as well as to timing of control measures. growth studies on giant burred (sparganium eurycarpum englem.), a perennial weed in annual wild rice, demonstrated that during early season growth a height difference existed between the weed and the crop which allowed for selected herbicide application ( john and ervin 1990). in some crops, efficient and safe use of herbicides, requires a height differential between the crop and the weed in order that the weed can be sprayed with minimal contact with crop foliage. tiller formation in f. miliacea was 1-4 per plant and average tiller production was 2.3 per plant (table 1). total production of inflorescences/plant was 134 within the 4-month period (table 1). positive correlation was found between tiller numbers and number of inflorescence produced (figure 2). when tiller production increased from 1 to 4, the inflorescence production showed a linear increase from 67 to 210. weekly increases in inflorescence production were significant (figure 1). the lowest inflorescences number was observed at 7-week after emergence (wae). inflorescence number then gradually increased from 10 to 16 wae. there were no significant differences between 16 and 17 wae. during the 4 months period a total of 84 inflorescences/plant matured, and percentage of ripened inflorescence was 63.22% (table 1) table 1. growth characteristics of f. miliacea plants growth parameter mean ± se plant height (cm) 64.05 ± 6.07 no. of tillers/plant 2.3 ± 0.483 no. of produced inflorescence/plant 133.8 ± 42.39 no. of ripened inflorescence/plant 84 ± 25.33 % ripened inflorescence/plant 63.22 ± 4.57 no. of spikelets/inflorescence 48.96 ± 6.82 seed no./inflorescence 511 ± 76 seed weight/plant (g) 1.374 ± 0.330 seed no./plant 42275 ± 6696 1000 seed weight (g) 0.035 ± .00215 each inflorescence was laddered with 49 spikelets and 511 seeds (table 1). the total seed and 1000 seeds weight were 1.37 g and 0.035 g, respectively. total number of seed/plant was 42 276. although in the phillipines holm et al. (1977) observed that seed production of each f. miliacea plant was 10 000. actually, seed output is a highly plastic character and is responsive to local growing conditions particularly nutrient availability, day length, and plant density. echinochloa crus-galli seed production per plant ranged from 10 776 – 47 850 (itoh 1991). whereas, azmi and itoh (1991) reported that the 6 biotropia vol. 15 no. 1, 2008 e. crus-galli complex produced less seeds 12 129 seeds/plant in the ricefield due to the impact of competition with the direct-seeded crop. most annual weeds produce a few thousand seeds per individual when growing with minimal competition, though some produce 10 000 to 25 000 seeds per plant (salisbury 1942), while others like e. crusgalli under some situations may produce over 100000 seeds per plant (young 1986; norris 1992). a few annuals (e.g. varonica hederifolia) produce fewer than 100 seeds per individual plant (salisbury 1942; boutin and harper 1991). the number of seeds produced by a plant is the product of three variables: (i) the size of the plant (ii) the proportion of photosynthate allocated to seeds, and (iii) the mean seed weight. the seed production capacity of weeds varies greatly both within and between species. williams (1982) reported that the growth of c. esculentus and c. rotundus as characterized by their total dry weight, inflorescence dry weight, root and rhizome dry weight and number of shoots per pot was similar, but they differed in the manner in which the dry weight was partitioned to reproductive structures. each species partitioned less than 2% of its dry weight into floral formation. however, c. esculentus partitioned only 38% of its dry weight to tubers, whereas c. rotundus partitioned 50% of its dry weight to fewer and larger tubers. in the case of i. rogusum, seeds/spike, seed weight/plant (g) and seed number/plant and 1000 seed weight (g) was 57, 24.5, 6000 and 4, respectively (bakar and nabi 2003), which indicates much bigger seed size and lower seed number/plant than fimbristylis miliacea. growth stages of fimbristylis miliacea emergence of f. miliacea started at 3 days after planting of seeds (table 2). approximate times required for the sequential production of leaves, tillers, first 10 inflorescence and their maturity are shown in table 2 and figure 2. at 7 days after emergence (dae) the plants were at the 3-4 leaf stage. table 2. approximate time required for seedling emergence, leaf, tiller and inflorescence formation and inflorescence ripening growth parameter mean time (days after seedling emergence) ± se 1st seedling emergence 3 ± 0 2nd leaf formation 2.82 ± 0.40 3rd leaf formation 5.27 ± 0.65 4th leaf formation 7.65 ± 0.84 5th leaf formation 10.32 ± 0.95 6th leaf formation 12.18 ± 0.75 7th leaf formation 14.5 ± 0.58 8th leaf formation 18.33 ± 0.82 7 growth and development of fimbristylis miliacea (l.) vahl – mahfuza begum et al. growth parameter mean time (days after seedling emergence) ± se 9th leaf formation 20.67 ± 0.52 10th leaf formation 25.0 ± 1.05 11th leaf formation 29.0 ± 0.82 12th leaf formation 33.43 ± 1.72 1st tiller formation 35.2 ± 3.68 2nd tiller formation 40.41 ± 3.36 3rd tiller formation 41.57 ± 3.36 4th tiller formation 42 ± 0 1st inflorescence 49 ± 2.78 5th inflorescence 57 ± 2.70 10th inflorescence 63 ± 1.94 inflorescence ripening (after formation) 17.90 ± 1.62 inflorescence ripening (after emergence) 76 ± 6.71 at 14 dae the plant had 6-7 leaves and within 28 dae the plants had reached the 10-11 leaf stage. at 4 weeks (28 days) the plant started to produce tillers. observation at 35 dae showed that the plant produced the first tiller from the 5-7 leaf node while at the 11-12 leaf stage. the first 4-6 leaves senesced before first tiller formation. obviously, leaf formation with time varies from species to species. pane (1997) observed that l. chinensis produced 4-6 leaves at 20 das and produced 2-3 tillers with 1-3 inflorescences at 30 das. whereas, a very fast growing species like e. crus-galli produced the first 2 to 3 leaves within 4 to 7 days after emergence, and seedlings started producing tillers at 14-18 days after emergence (itoh 1991; azmi et al. 1995). tiller formation stopped at the onset of the reproductive stage. all tiller formation occurred 42 days (7 weeks) after seedling emergence, followed by the emergence of 1st inflorescence within one week, whereas in i. rugosum, production of new tillers ceased 3-4 weeks after floral initiation, but resumed at 16 weeks when most of the spikes had ripened. new tillers were usually unbranched, reduced in height with less seeds/spike (bakar and nabi 2003). azmi et al. (1995) reported that e. crus-galli headed at 44-55 dae. in this study, f. miliacea produced 10 inflorescences within 63 dae (9 weeks). the inflorescence matured after 18 dae (3 weeks) of emergence (table 2 and figure 2). total time required for emergence to seed maturation was 76 days. in i. rugosum approximately 4 weeks was required for spikes to ripen (bakar and nabi 2003). in l. chinensis, panicles ripened at 50-60 days after seeding (pane,1997), while 42 – 56 days table 2. continued 8 biotropia vol. 15 no. 1, 2008 y = 43.901x + 32.827 r2 = 0.9652 0 50 100 150 200 250 0 1 2 3 4 5 tiller number in flo re sc en ce n um be r were required to heading and 89 98 days were required for maturity in e. crus-galli (itoh 1991; arifin 2004). in this study, 80% of f. miliacea leaves were dead at harvest (120 days). the time required to reach reproductive maturity in weeds varies considerably and often similar to the companion crop or sometimes considerably shorter. in the tropics, weed life cycles may be extremely short. echinochloa colona, setaria verticillata and d. aegyptium approach flowering in 30-45 days whilst rottboellia cochinchinensis produced mature seeds within 50 days of establishment (fisher et al. 1985). similar short duration life cycles may also be observed in weeds of temperate latitudes (e.g. capsella bursa-pastoris l.), but weeds of major importance tend to have an extended growing season approaching to at least 6 months. from comparative studies, m. vaginalis, c. difformis, f. miliacea and e. glabrescens have been reported to begin flowering even when plants have achieved less than 10% of their maximum biomass, with seed production continuing for more than four months (kim and moody 1989). the growth stages of f. miliacea could be classified into 3 stages (a) slow initial growth stage, from emergence to 4 weeks after emergence (wae), (b) active growth stage, from 4 to 9 wae, (c) maximum growth stage, from 9 to 17 wae (figure 2). figure 2. growth and development of fimbristylis miliacea (l.) vahl in the first 4 weeks after emergence, the plants grew slowly and attained 10-11 leaf stage. this would be the most appropriate time for controlling this species with early post emergence herbicides. during the active growing period plant height was maximum, tiller formation ceased and the plant produced up to 10 inflorescences. in annual plants the foregoing phases of the life cycle follow one another in uninterrupted sequence (larcher 1975). beyond 10 wae f. miliacea reached maximum flowering stage, and leaves, and some parts of flowering stalks and inflorescences gradually became senescent. the phase of seed production in annual plants is often associated with the rapid death of leaves and the appearance of symptoms usually associated with mineral deficiency as if the leaves have been “sucked dry” of some essential nutrient which has been trans-located to meet the greater needs of the seeds. the evolutionary development of annual plants have major activities that require resource allocation, i.e. starting from 9 growth and development of fimbristylis miliacea (l.) vahl – mahfuza begum et al. seed, and continuing with the processes of dispersal, dormancy, germination, seedling establishment, vegetative phase, resource capture, growth, flowering, seed production and lastly seed maturity (radosevich and holt, 1984). evidently, f. miliacea also has the same growth pattern. holm et al. (1977) repeatedly emphasizes the ability of weeds to compete against crop species and most of the weeds allocate a large proportion of resources to seed production. thus importance of early weed control for optimum crop production has been recognized for many years by agronomists. perhaps the basis for this view is the morphological and physiological adaptations through common evolutionary origin among weed species and crops that cause early onset of competition. species growth characteristics provide an indication of competitive ability, and understanding weed seedling growth rates is also important when considering weed management strategy that includes the use of herbicides. improper application timing of herbicide can result in poor weed control and the need for remedial treatment. conclusions fimbristylis miliacea seed emergence occurred 3 days after sowing. plant height increased rapidly from 3-8 wae and maximum plant height of 64.05 cm was attained at 10 wae. first tiller formation was at 11-12-leaf stage after 4 wae and the range of tiller production was 1-4 (average 2.3) per plant. inflorescence production was proportional to number of tillers produced. total numbers of 134 inflorescences/ plant were produced, of which the ripened inflorescences/plant and seeds/plant was 84 and 42 276, respectively. one thousand seed weight was 0.035 g. this species has three important growing stages: a slow growth stage during the first 4 wae; a rapid growth stage that generally took place from 49 wae; and finally, a maximum growth stage from 9-17 wae. the inflorescences generally developed 7-8 wae and subsequently matured 11 -12 wae. the time requirement for inflorescence maturation was 3 weeks after inflorescence emergence. agricultural weeds generally share certain properties, including small seed size, high relative growth rate, low early absolute growth rate, tolerance to stress and high reproductive capacity. these differences form the basis for a variety of weed management tactics for successful control. thus, growth and development study of a specific weed species is an organizing principle for the integration of weed management practices. acknowledgments this study was a part of ph d research work, funded as postgraduate fellowship sponsored by third world organization for women in science (twows), trieste, italy. all other research facilities were provided by the irpa project no. 01-02-040778-pr0068/05-05, universiti putra malaysia. 10 biotropia vol. 15 no. 1, 2008 references arifin, t. 2004. morphological and molecular variability amongs ecotypes of barnyardgrass (echinochloa crus-galli var. crus-galli (l.) beauv) : implication for biocontrol. ph. d. thesis. universiti putra malaysia. p. 4.14.28. azmi, m. 1994. biology and control of echinochloa crus-galli (l.) beauv. in direct seeded rice. ph. d. thesis, school of biological sciences, universiti sains malaysia. p. 333. azmi, m. and k. itoh. 1991. echinochloa crus-galli complex. in : life cycles of rice field weeds and their management in malaysia. tropical agriculture research centre, ed. itoh, k., tsukuba, japan, p. 17-27. azmi, m., m. mashhor., k. itoh and h. watanabe. 1995. life cycle and seed longevity of echinochloa crus-galli complex in direct-seeded rice in malaysia. in: proceeding of 15th asian pacific weed science conference, p. 505-511. tsukuba, japan. bakar, b.h. and l.n.a. nabi. 2003. seed germination, seedling establishment and growth patterns of wrinklegrass (ischaemum rugosum salisb.). weed biol. and manag., 3:8-14. boutin, c. and j. l. harper. 1991. a comperative study of the population dynamics of five species of veronica in natural habitates. j. ecol., 79: 199-221. chandler, j.m., r.l. munson, and c.e. vaughan. 1977. purple moonflower: emergence, growth, reproduction. weed. sci., 25(2): 163-167. charrote, v.e., l.l. edith, l.m. timothy and l.m. janis. 1990. growth and development of wild-proso millet (panicum miliaceum) biotypes. weed tech., 4: 415-419. fisher, h.h., f. lopez, l.margate, p. elliot and l. burrill. 1985. problems in control of rottboellia exaltata in maize in bukidnon province, mindanao, philippines. weed res., 25:93-102. holm, l.g., d.l. pluknett, j.v. pancho and j.p. herberger. 1977. the world’s worst weeds: distribution and biology. the university press of hawaii. honolulu. p. 273-279. itoh, k. 1991. life cycles of rice field weeds and their management in malaysia. tropical agriculture research centre, tsukuba, japan, p. 92. john, w.l. and a.o. ervin. 1990. growth and development of giant burreed (sparganium eurycarpum). weed tech., 4:849-854. kim, s.c. and k. 1989. adaptation moody, strategy and seed production of rice and weed species. korean j. weed sci., 12:183-200. larcher, w. 1975. physiological plant ecology. springer-verlag. new york. p.252. mcwhorter, c.g. 1961. morphology and development of johnsongrass plants from seeds and rhizomes. weeds, 9:558-562. moody, k. 1989. weeds reported to occur in rice in south and southeast asia. in: weeds reported in south and southeast asia. international rice research institute. los banos, laguna, p.o box 933, 1099 manila, philippines, pp. 1-80. norris, r.f. 1992. relationship between inflorescence size and seed production in barnyard grass (echinochloa crus-galli). weed sci., 40: 74-78. pane, h. 1997. studies on ecology and biology of red sprangletop [leptochloa chinensis) (l.) nees] and its management in direct-seeded rice. ph. d. thesis. universiti sains malaysia. pp. 1-235. radosevich, s.r. and j.s. holt. 1984. weed ecology: implications for vegetation management. john wiley & sons. new york. p. 265. 11 growth and development of fimbristylis miliacea (l.) vahl – mahfuza begum et al. sabrig, o.t. 1980. demography and evolution in plant populations. botanical monogr. vol. 15. oxford. blackwell sci. pub. p.222. salisbury, e.j. 1942. the reproductive capacity of plants. london: bell. silvertown, j.w. and j.l. doust. 1993. introduction to plant population biology. oxford. uk. blackwell sci. pub. p. 210. smith, e.v. and g.l. fick 1938. nut grass eradication studies: i. relation of the life history of nut grass, cyperus rotundus l., to possible methods of control. j. am. soc. agron. 10:107-113. smith, jr.r.j. 1983. weeds of major economic importance in rice and yield losses due to weed competition. in : proceedings of conference on weed control in rice. international rice research institute. los banos, laguna philippines. pp. 19-36. visperus, r.m. and b.s. vergara. 1978. autecology of scirpus maritimus l. growth characteristics and competition with rice. philip. weed sci. bull., 3: 1-13. williams, r.d. 1982. growth and reproduction of cyperus esculentus l. and cyperus rotundus l. weed res., 22: 149 -154. young, f.l. 1986. russian thistle (salsola iberica) growth and development in wheat (triticum aestivum). weed sci., 34: 901905. 2. handoko (soil water).cdr 4. agus nuryanto.cdr edited by foxit reader copyright(c) by foxit software company,2005-2008 for evaluation only. user2 callout rapd edited by foxit reader copyright(c) by foxit software company,2005-2008 for evaluation only. user2 strikeout user2 callout gen24 user2 note hilangkan spasinya edited by foxit reader copyright(c) by foxit software company,2005-2008 for evaluation only. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 biotropia book juni revisi 14 juli 09.indd 1 biotropia vol. 16 no. 1, 2009: 1 10 *corresponding author: jtmasagca@dasma.dlsu.edu.ph, jmasagca@yahoo.com. feeding ecology of tree-climbing mangrove sesarmid crabs from luzon, philippines jimmy tevar masagca de la salle university-dasmariñas cavite 4115, th e philippines abstract despite the large ecological study of tree-climbing mangrove sesarmid crabs in other countries, the philippine representatives appear to have not been investigated extensively. this paper presents the feeding ecology as to dependence on mangrove trees of sesarmids in different mangrove areas of southern luzon. this is biased on the nature of the crab habitats, arboreal climbing skills and burrowing behavior of the sesarmids: selatium elongatum and episesarma versicolor − exclusive mangrove tree climbers (emtc); sarmatium germaini − occasional mangrove tree climber (omtc); and the non-mangrove tree-climbing (nmtc) sesarmidsneosarmatium smithii, perisesarma bidens and perisesarma eumolpe. key words: mangrove crabs, sesarmid crabs, climbing skills, burrowing skills, arboreal climbing crabs, catanduanes, philippines introduction the crustaceans (e.g. brachyurans) are always a prominent and diverse element of the fauna in mangals (morton 1990). crabs are diverse in mangrove habitats and abundant in many mangals (jones 1984). several authors (e.g. jones 1984, lee 1998) have attested that brachyurans are important in the mangrove ecosystem structure and function. unfortunately, it appears that there is still a dearth of detailed published information on the occurrence of several families of this group of crustaceans in the mangrove areas of the philippines. moreover, there seems to be limited published data on the ways in which these faunal elements use the mangrove resources. the sesarmids are less documented in the philippines unlike in other countries, wherein several reports are available from singapore (sivasothi 2000; sivasothi et al. 1993; tan & ng 1994; ng & liu 1999) malaysia (tan & ng 1994; leh & sasekumar 1985), indonesia (soemodihardhjo & soerianegara 1989; rahayu & davie 2002), hong kong (poovatchiranon 1986; lee & leung 1993; lee 1998; lee 1997; kwok 1995; kwok & tang 2005; and ashton 2002). crabs have different functions and impacts on the mangrove ecosystem. firstly, they can process as much as 70% of the leaf litter, and leaf processing can turn over a litter at a rate in excess of 75 minutes (see studies of leh & sasekumar, 1985; slim 2 biotropia vol. 16 no. 1, 2009 et al. 1997; dahdouh-guebas et al. 1999; ashton 2002). secondly, most mangrove crabs feed on vascular plant materials, but they also feed on green leaves (lee 1998; skov & hartnoll 2002), including seedlings. mangrove plant seedlings grazing, particularly in the genus sesarma may also slow regeneration of mangroves. on the other hand, grapsoid crabs are the primary seed operators. emmerson & mcgwynne (1992) described the feeding and assimilation of mangrove leaves by the crab sesarma meinerti. moreover, the feeding ecology of neosarmatium smithii was studied by giddins et al. (1986) and new records from taiwan on this sesarmid crab was reported by naruse et al. (2006). the crabs of the family sesarmidae are known by various workers to have significant ecological role in mangals (lee 1998, gillikin 2000; gillikin & schubart 2004, gillikin et al. 2004). many species of these mangrove crabs do not assimilate much carbon from the mangrove leaves but rely on the sediments (bouillon et al. 2002, skov & hartnoll 2002). some species of the genus perisesarma supplement their diet with leaves (leh & sasekumar 1985, slim et al. 1997). hence, this aspect on the feeding ecology of sesarmid crabs helps to trap the energy stored in these leaves within the mangal before the tide can carry them away ( lee 1998; skov & hartnoll 2002). despite the large ecological study of mangrove sesarmids in other countries (bright & hogue 1972; hartnoll 1975; micheli, gherardi & vannini 1991; cannicci et al. 1996; slim et al. 1997; dahdouh-guebas et al. 1999; fratini, cannicci & vannini 2000, flores et al. 2003) the philippine representatives appear to have not been investigated extensively. this study on the patterns of biodiversity in aquatic systems of the philippines attempted to determine the feeding ecology of selected taxa of sesarmids as to (a) the nature of the crab habitats in the mangrove forest with reliance to the mangrove trees, trunks, roots, and leaves that offer a wide variety of ecological niches for the species under consideration; and (b) the behavior of the sesarmid crabs as revealed by their burrowing abilities and tree-climbing skills needed to explain the reliance of these crabs on the mangrove forest. materials and methods study sites the collection and observation sites of the study include: ipalsabangon mangrove area in pagbilao, quezon, philippines; ii maqueda channel in the bicol region, agojo inlet mangrove reserve area, san andres and palnab-pajo mangrove area, catanduanes, philippines. feeding ecology as to dependence on mangroves, arboreal climbing skills and burrowing behavior specimens of mangrove crabs were collected by handpicking and with the use of scoop nets and locally made traps. collections were made both in the morning and in the evening from the mangrove areas along rivers, creeks, inlets and the buffer zones or marginal strips of the coastline in one site each for the study areas from june 2005 to february 2006. 3 ecological studies on feeding behavior took place in the purposively selected study plots in palnab-pajo mangrove swamp, catanduanes during the months of october and december in 2005, while in the mangrove areas of palsabangon in pagbilao, quezon studies were done from january to february 2006. ecological studies of sesarmids were carried out in each of the mangrove areas following the methods of gillikin (2000). in the selected mangrove forests, presence or absence of crab species were determined by visual inspection in 10 m diameter plots along a transect perpendicular to the coastline, covering the full width of the forest. the study investigated at least 10 plots along a 100 to 200 m long transect in the areas under study. emergence and re-emergence of the sesarmids during fieldwork, each plot was first inspected using binoculars from a distance of approximately 6 m for 20 min. and the crab species recognized were noted and recorded. afterwards, naked eye observations were undertaken with two field workers (observers) sitting on opposite sides of the plot for at least 30 min, following hartnoll et al. (2002) and skov et al. (2002). as a modification, the researcher and field assistants were positioned in two separate paddled boats (“banca”) when the plots were observed. the researcher approached the plot and a disturbance was created (either by throwing some mud, twigs or branches of mangrove plants) so that the sesarmids, under study take refuge in the burrows, or holes, and other materials to take cover (e.g. leaves, wood, twigs etc.). the time at which the first individual of the sesarmids will emerge or re-emerge was recorded. observations for this portion of the study were undertaken during the day at low tides. diurnal and lunar cycles were not used in the distinction of the observation. the aforementioned procedures are modifications of the works of gillikin (2000), hartnoll et al. (2002) and skov et al. (2002). results and discussions reliance on the mangroves as habitats table 1 shows the summary of the observations made on the habitats of various grapsoid sesarmid crabs in the mangals of catanduanes island and pagbilao, quezon. the nature of their habitat, climbing abilities and burrowing skills could explain the feeding ecology of the mangrove crabs under investigation. table 1. summary of the feeding ecology of grapsoid sesarmid crabs from different mangals in luzon grapsoid sesarmid species behavior/nature of habitat related to feeding remarks on fi eld observations episesarma versicolor burrower and exclusive mangrove tree climbing (emtc) species migrating from burrows to the tree stems and some at the canopies; constructs burrows around soft sediments of mangrove roots and tree trunks; seen feeding on calyx and leaves; seen cropping on leaf litters and bringing fragments to the burrows. feeding ecology of tree climbing mangrove sesarmid crabs – jimmy t. masagca 4 selatium germaini occasional mangrove tree climber (omtc) species climbs on the branches of r. apiculata and r. mucronata in the mangroves of catanduanes. neosarmatium smithi burrower and non-mangrove tree climbing (nmtc) species constructing burrows on mudfl ats; not seen climbing on stems of mangrove trees in quezon and catanduanes perisesarma bidens burrower and non-mangrove tree climbing (nmtc) species seen resting on rhizophora tree (just within the water lining) during high tide apparently for avoidance and possibly for feeding purposes; also seen hiding on rocks, crevices of boulders and tree falls. perisesarma eumolpe burrower and non-mangrove tree climbing (nmtc) species on rare occasions, some crab samples were caught on the trunks possibly carried by water current during high tides. selatium elongatum arboreal climber or emtc in mangrove trunks, branches and canopies seen on main branches of rhizophora; on aerial roots; also on crevices of trunks. episesarma versicolor feed on calyx and leaves and crop on leaf litters, then bring fragments to the burrows. the grapsoid sesarmid crabs, perisesarma spp. and episesarma sp. were found to be herbivores and omnivore/deposit feeders, eating mangrove litter and water plants. moreover, the sesarmid crabs sarmatium germaini (figure 1), perisesarma eumolpe and neosarmatium smithi feed on the mangrove litter, composed of fallen mangrove leaves of rhizophora, seedlings, calyx and twigs that fall from the trees on the forest floor and into the water. this study confirmed that the leaves of the mangroves, r. marina, r. apiculata, and ceriops tagal were devoured by the generally observed herbivorous/omnivorous crabs such as perisesarma spp. the feeding habits of mangrove crabs have been divided into seven groups by jones (1984): herbivore, carnivore, omnivore, deposit feeder, omnivore/deposit feeder, specialized filterer, and filterer/omnivore. in the study of islam & uehara (2005), stomach content analysis showed that p. bidens diet consists mainly of mangrove leaves fragments, with small amounts of animal, algae and sediment matters, indicating that this sesarmid is primarily detritivorous. in chiromanthes onychophorum, malley (1998) noted that this sesarmid crab consumes fallen leaves or their fragments, incompletely digests them, and returns them to the environment as fecal matter in a more finely-divided state than when they were ingested. of recent, ya et al. (2008) reported that both perisesarma eumolpe and p. indiarum are mainly sediment grazers, but also feed on mangrove leaves and roots and occasionally animal matter. these crab species prefer avicennia alba leaves to other mangrove species, i.e., a. officinalis, a. rumphiana, r. apiculata and bruguiera gymnorhiza. dahdouhguebas (1997), ashton (2002), buck et al. (2003), thongtham & kristensen (2003) and schwamborn et al. (2006) analyzed the diets of sesarmid crabs showed that their diet mainly consisted of mangrove leaves and in addition to bustle animal matter. biotropia vol. 16 no. 1, 2009 table 1. continued 5 a. b. figure 1. sarmatium germaini from palsabangon mangrove area in pagbilao, quezon (a) and palnabpajo mangrove swamp, maqueda channel, in bicol region (catanduanes) (b). burrowing and arboreal climbing skills of sesarmids similar with other sesarmids, p. bidens are burrowers, but observations made in quezon and catanduanes indicate that some individuals tend to hide in natural refuges found in the mangals investigated. these hiding places are the crevices of rocks and boulders and in between portions of root buttresses of mangrove trees. field observation in quezon, indicates that these crabs are also having preferences in arboreal environments wherein selatium elongatum and episesarma versicolor were seen clinging on mangrove stems or rush to the mangrove stems when disturbed, while the boat is cruising in the waterways. these tree climbing crabs appear to be less antagonized during the investigation and remained to be motionless for about 1 hour. when tides are rising or lowering, these crabs appear to be unaware as observed for at least an hour. however, when the rising tide reaches their location in any of the protruding branches of the mangrove trees in the embankments, these tree-climbing crabs tend to adjust or move slowly with their heads upside down. researches indicate the possibility that these crabs cling to the mangrove stems not to find food but as avoidance from very long immersion in the water during high tides and due to the presence of predators. this could not be confirmed in the study. during field observation, it was rather easy to find p. bidens in the open sand or mudflats. behavioral differences between the male and female samples of p. bidens was observed. on the sexes, results of initial experiment suggest that there is no difference as to the aggressiveness. however, the males are more receptive to antagonistic encounters, especially when disturbed inside the aquarium prior to feeding observation. it was difficult for the researcher to catch this sesarmid due to its swiftness and alertness. one striking character of this crab is its fast action in retreating from external stimuli such as touch or other mechanical disturbance. this could be the reason why the feeding experiment did not proceed because the individuals of p. bidens placed in the glass aquaria did not feed on the pieces of r. apiculata, r. mucronata, a. marina and other associated mangrove plants. however, the report of ólafsson et al. (2002) cited that male and female crabs are likely to have different energy requirements, due to differences in the energetic costs of producing eggs or sperm (michell 1993). a study of kyomo (1992) conducted on the sesarmid crab sesarma intermedia showed that females were more specialized in their feeding habits and had higher assimilation rates than males. feeding ecology of tree climbing mangrove sesarmid crabs – jimmy t. masagca 6 n. smithii form burrows around r. mucronata and r. apiculata roots but rarely in avicennia marina found in the mangals of catanduanes. observations indicate that n. smithi feed on leaf litter but also some shrimps, specifically palaemonetes sp. this was observed during the afternoon hours when tides start to rise. observation on emergence and re-emergence of sesarmids in the burrows in general, p. bidens appears to be the most active of the three sesarmids observed during the day. as shown in table 2, the red-clawed grapsoid sesarmid crab, p. bidens emerged and re-emerged from the burrows much faster (mean of 3.48 mins) than the sesarmids n. smithi (mean of 4.15 mins) and e. versicolor (3.93 mins). all three sesarmid crab species display similar behavior of picking up the leaf fragments by first standing still in motionless for about 7 to 10 seconds. some sesarmids, e.g e. versicolor has to consume the leaf fragment where they found it, then migrate for at leas 0.5 meters and to be continued at the mouth of the burrows. some crabs move away from the areas where they got the leaf fragments and continue feeding near the burrows. some bring these fragments inside the burrows but mostly will have the tendency to leave behind when disturbance was made. there is much to be learned as to the sesarmid crab’s feeding ecology in the philippines since there are various similarities and differences in the findings of other authors. some researchers report on the unusual presence or occurrence as to their level of dependence in the different habitats, arboreal or burrows. climbing skills and burrowing abilities also vary depending on the species. while others focus on how sesarmids rush to the mangrove tree and and cling to the branches, it is still unknown whether they are apparently resting or catching some insects or invertebrates. other researchers put premium on their occurrence in the mudflats or soft sediments of mangals for feeding on some leaf litter. moreover, the sesarmids under study have been observed cropping and bustling on sand sediments and other fragments of leaf litter that some crab ecologists might have ignored and do not give emphasis. further studies can be carried out in the future in terms of the feeding ecology of these vital species of mangrove crabs in relation to morphological variation of the feeding apparatus. lastly, deeper reflections on the notions of convergent evolution (fratinni et al. 2005) and the timing of development in relation to arboreal climbing skills and burrowing abilities of sesarmids deserve further attention. table 2. data on the time of re-emergence of sesarmid crabs (p. bidens, n. smithi and e. versicolor) in burrows after disturbance sesarmid crab species time of re-emergence mean time of re-emergence (in minutes) trials (each consists of 3 repeats) (in minutes) 1 2 3 perisesarma bidens 3.35 3.65 3.45 3.4833 neosarmatium smithi 3.95 4.05 4.45 4.1500 episesarma versicolor 2.78 4.25 3.75 3.933 biotropia vol. 16 no. 1, 2009 7 conclusions based on the results presented, selatium elongatum and episesarma versicolor are exclusive mangrove tree climbers (emtc); sarmatium germaini as occasional mangrove tree climber or omtc; and the sesarmid species − neosarmatium smithii, perisesarma bidens and perisesarma eumolpe as non-mangrove tree climbers or nmtc. the sesarmid species, e. versicolor, n. smithii, p. bidens and p. eumolpe posses burrowing skills, while p. bidens appeared to be the most active of the three sesarmids investigated. acknowledgments the author acknowledges with thanks to the administration of de la salle university-dasmariñas (dlsu −d) university faculty research office (ufro) director dr. j. morta; education dean, dr. o. legaspi ; oic vvice chancellor, dr. c. cervillon; and dlsu−d br. president gus boquer for funding this research project on the mangrove crabs. sincere thanks is also given to dr. david gillikin, vrije universiteit, t brussel, belgium and dr. peter k.l. ng of the national university of singapore for sending reprints on the mangrove crabs and ms. marivene manuel, philippine national museum (pnm) for evaluating the earlier drafts of the report on this study. my special thanks to elver sison, while finalizing this paper. the assistance of my brother (rico masagca ); 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the software from http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html biotropia no. 8, 1995: 30-38 arbuscular mycorrhizal fungi from the rfflzospheres of soybean crops in lampung and west java*) k. kramadibrata research and development centre for biology indonesian institute of sciences, bogor, indonesia e.i. riyanti and r.d.m. simanungkalit central research institute for food crops, agency for agricultural research and development, bogor, indonesia abstract the occurrence of arbuscular mycorrhizal (am) fungi in the rhizospheres of field-grown soybean crops in the provinces of lampung and west java was examined. nineteen taxa of am fungi were identified as follows: acaulospora delicata, a. foveata, a. rehmii, a. scrobiculata and a. tuberculata; gigaspora cf. gigantea and gigaspora sp. 1; glomus clavisporum; glomus cf. fasciculatum, glomus micro-aggregatum, glomus sp. 1, glomus sp. 2, glomus sp. 3 and glomus sp. 4; scutellospora cf. heterogama, scutellospora cf. pellucida, scutellospora sp. 1. scutellospora sp.2. and scutellospora sp. 3. key words: mycorrhizas/soybean/rhizosphere fungi/identification. introduction there are only a few reports on arbuscular mycorrhizal (am) fungi derived from the rhizospheres of food crops in indonesia. widiastuti and kramadibrata (1992) reported on some am fungi from the acid soils of west java, among others from the rhizospheres of corn. soybean is one of the main staple foods in indonesia. it is often grown on uplands as well as on lowlands after rice in the dry season. however, the information on am species and its distribution has never been reported. schenck and smith (1982) investigated three am fungi which occurred in soybean in florida, u.s.a., i.e. glomus claroideum, g. tortuosum and gigaspora albida. the purpose of this paper is to report on species and distribution of am fungi from the rhizospheres of field-grown soybeans in the provinces of lampung and west java. *)paper presented at the second symposium on biology and biotechnology of mycorrhizae and third asian conference on mycorrhizae (acom 111), 19-21 april 1994, yogyakarta, indonesia. 30 arbuscular mycorrhizal fungi k. kramadibrata, e.i. riyanti and r.d.m. simanungkalit materials and methods the soybean-grown upland areas in lampung (central lampung) and west java (garut and bogor) have been selected for soil sampling. soil samples were collected from ten locations (four in central lampung, two in garut and four in bogor). some soil characteristics and number of am propagules of each location are described in table 1. ph of the soils measured in water suspension ranged from 4.78 to 6.94. p availability by bray-2 ranged from 2.32 to 55.59 mg/100 g soil. infective am propagules were determined by the 'most probable number' method as described by sieverding (1991). the number of am propagules ranged from 383 to 12 266 g air dried soil. a total of three to five subsamples weighing about one kg were systematically taken from each location at a depth of 0-20 cm. subsamples were then mixed 31 biotropia no. 8, 1995 thoroughly and air dried. from each sample 100 g of soil were used for spore examinations. spores were extracted by wet sieving and decanting methods in combination with 48% sucrose (walker et al. 1982), mounted on slides in polyvinyl alcohol-lactic acid-glycerol (pvlg) and then examined under a compound microscope for spore details (100looox). spores were identified by using the species description of walker (1983, 1986), walker and koske (1987) and walker and sanders (1986). melzer's reagent was used to determine spore wall reaction when necessary. spore preparations were coded krs (abbreviations of family names of the authors) and deposited partly at the central research institute for food crops and partly at the "herbarium bogoriense", research and development centre for biology-indonesian institute of sciences; both are located in bogor. results and discussion all soil samples collected from lampung and west java contained am fungal spores. however, the species were not distributed evenly as shown in table 2. thirteen were identified from the rhizospheres of soybean in central lampung, three in garut, nine in bogor. the sampling locations new sukadana and suryamataram yielded six am species (highest) each; the experimental farm cikeumeuh i and cikeumeuh ii had five species each; gantiwarno had four species; sidobinangun, experimental farm muara i and muara ii had three species each; karang pawitan and cihuni (both located in garut) had only two am species each. some spores could be identified well because they bore very specific characteristics. some identified species showed very similar characteristics with known species like gigaspora cf. gigantea, glomus cf. fasciculatum, g. cf. microaggregatum, scutellospora cf. heterogama and scutellospora cf. pellucida. the species of some spores could not be identified due to some problems such as poor condition and the limited number. species identification will be done after pot cultures of the related soil samples are established. the soil samples from sukadana contained extramatrical cells of scutellospora, while that from gantiwarno contained extra-matrical cells of gigaspora. however, the three species reported by schenck and smith (1982) on am fungi from the rhizospheres of soybean in florida were not found in soil samples collected from lampung and west java. 32 arbuscular mycorrhizal fungi k. kramadibrata, e.i. riyanti and r.d.m. simanungkalit the am fungi from the rhizospheres of soybean in lampung and west java are described as follows: 1. acaulospora delicata walker, pfeiffer & trappe the spores were globose to subglobose, yellow, 94116 x 96101 µm. the spore surface was smooth. a sporiferous saccule was not observed in any collections made during the study. collections examined: krs#41, krs#42, krs#43, krs#44 and krs#53, gantiwarno village, pekalongan subdistrict, central lampung district; krs#82, experimental farm cikeumeuh i, bogor; krs#83, experimental farm muara i, bogor; krs#94 and krs#98, experimental farm muara ii, bogor. 33 biotropia no. 8, 1995 2. acaulospora foveata trappe & janos the spore was pale yellow to greenish yellow, globose, 200 x 200 µm. the spore surface was covered with round to oblong sometimes irregular depressions with curved bottom, separated by ridges. a sporiferous saccule was not observed in any collections made during the study. collections examined: krs#60, cihuni village, wanaraja subdistrict, garut district. 3. acaulospora rehmii sieverding & toro the spores were yellow, globose to subglobose, 180 190 x 190 – 200 µm. the spore surface had labyrinthi form folds with ridges and depressions between ridges. a sporiferous saccule was not observed in any collection made during the study. collections examined: krs#66 and krs#68, experimental farm cikeumeuh i, bogor. 4. acaulospora scrobiculata trappe the spores were pale yellow to yellow, globose to subglobose, 96-125 x 96 135µm. the surface was uniformly pitted with depressions, separated by ridges with a circular, linear or y-shaped pattern. a sporiferous saccule was not observed in any collections made during the study. collections examined: krs#23, suryamataram village, sukadana subdistrict, central lampung district; krs#55, sidobinangun village, seputih banyak sub-district, central lampung district; krs#65 and krs#69, experimental farm cikeumeuh i, bogor; krs#87, experimental farm muara i, bogor. 5. acaulospora tuberculata janos & trappe the spores were light yellowish brown to light brown, globose to subglobose 220-240 x 210-240 µm. the surface was covered by tubercules. a sporiferous saccule was not observed in any collections made during the study. collections examined: krs#31, suryamataram village, sukadana subdistrict, central lampung district; krs#70, experimental farm cikeumeuh i, bogor; krs#91, krs#92, krs#93 and krs#96, experimental farm muara ii, bogor. 34 arbuscular mycorrhizal fungi k. kramadibrata, e.i. riyanti and r.d.m. simanungkalit 6. gigaspora cf. gigantea (nicol. & gerd.) gerd. & trappe the spores were pale yellow, globose to subglobose, 288 403 x 384 403 µm. the surface was smooth. a bulbous suspensor ± 40 µm diameter was found on every spore. collections examined: krs#11, krs#13 and krs# 18, new sukadana village, sukadana subdistrict, central lampung district; krs#25 and krs#27, surya-mataram village, sukadana subdistrict, central lampung district. 7. glomus clavisporum (trappe) almeida & schenck sporocarps were brown to dark brown, globose to subglobose, 300-400 x 300-380 µm. spores were brown to dark brown 100120 x 20-40 µrn, clavate to subcylindric, tapering to a cylindric subtending hypha. collections examined: krs#20, krs#21, krs#22, krs#28, krs#29 and krs#30, suryamataram village, sukadana subdistrict, central lampung district. 8. glomus cf. fasciculatum (thaxter sensu gerd.) gerd. & trappe emend. walker & koske the spores were pale yellow to pale yellow brown, globose to subglobose, 90 110 x 100110 µm. the surface was smooth. collections examined: krs#2, krs#3 and krs#19, new sukadana village, sukadana subdistrict, central lampung district; krs#63, krs#64 and krs#71, experimental farm cikeumeuh i, bogor; krs#78, krs#79 and krs#81; experimental farm cikeumeuh ii, bogor; krs#84, krs#86 and krs#88, experimental farm muara i, bogor. 9. glomus cf. microaggregatum koske, gemma & olexia the spores were found as loose aggregate. they were yellow, globose to sub-globose, 39-54 x 35-43 µm. the surface was smooth. collections examined: krs#1, new sukadana village, sukadana subdistrict, central lampung district. 10. scutellospora cf. heterogama (nicol. & gerd.) walker & sanders the spores were brown to dark brown, globose to subglobose, 200-300 x 200 — 300 µm. the surface was ornamented or covered by spines. a suspensor-like cell was found on spore, it was pale brown, ±40 µm. 35 biotropia no. 8, 1995 collections examined: krs#73 and krs#74, experimental farm cikeumeuh ii, bogor. 11. scutellospora cf. pellucida (nicol. & schenck) walker the spores were hyaline to pale yellow, pear-shaped, 160 204 x 80 – 102 µm. the surface was smooth. a suspensor-like cell was found on spore, it was hyaline, ± 40 µm collections examined: krs#76 and krs#77, experimental farm cikeumeuh ii, bogor. collections which could not be identified due to poor conditions and the small number of spores are listed below: 12. gigaspora sp.l. spores were globose to subglobose, brown to dark brown, 172.8-480 x 211.2-480 µm. the spore had one group of wall consisting of a thin unit wall, < 1 µm, hyaline and a brown laminated wall, 10-48 µm thick. the muronym is a(ul). one specimen had a germ tube. collections examined: krs#4, krs#7, and krs#17, new sukadana village, sukadana subdistrict, central lampung district; krs#45, krs#46, krs#47, and krs#52, gantiwarno village, pekalongan subdistrict, central lampung district. 13. glomus sp.l. spores were globose or sometimes subglobose, yellow, ±97µm, in groups. however, the spores of the specimens were deflated so that spore forms were not distinct, irregular; wall arrangement could not be examined. 14. glomus sp.2. spore forms could not be determined because it was deflated; it was pale yellow and in loose aggregate. collections examined: krs#49, gantiwarno village, pekalongan subdistrict, central lampung district. 15. glomus sp.3. spores were globose to subglobose, yellow; hyphae were hyaline to pale yellow. spore sizes were 100115 x 90104 µm. wall arrangements could not be analyzed. 36 arbuscular mycorrhizal fungi k. kramadibrata, e.i. riyanti and r.d.m. simanungkalit collections examined: krs#58, central seed institute karang pawitan, karang pawitan subdistrict, garut district. 16. glomus sp.4. spores were globose, pale yellow, 172 x 172 µm; wall was 2 µm thick. wall arrangements could not be analyzed. collections examined: krs#62 and krs#67, experimental farm cikeumeuh i, bogor. 17. scutellospora sp.l. spores were ellipsoid, brownish yellow, 288 x 355 µm. spore wall possibly consisted of two wall groups, a and b. wall group a consisted of two unit walls, wall 1 was thinner than wall 2, both were brownish yellow. wall group b had one membranous wall, hyaline and very thin ( < 1 µm). the muronym is temporarily a (uu) b(m). collections examined: krs#12, new sukadana village, sukadana subdistrict, central lampung district. 18. scutellospora sp.2. spores were globose or subglobose, pale brown 220.8 280 x 201.6 288µm. the wall may have been structured in two wall groups, a and b. wall group a consisted of wall 1 and 2. wall 1 was a unit wall and wall 2 was thicker than wall 1; both were hyaline. wall 2 appreared to be laminated. wall group b had one membranous wall. the muronym is a (uu) b(m). some specimens clearly had a germ shield and an initial germ tube. collections examined: krs#5, krs#6, krs#14, krs#15 and krs#16, new sukadana village, sukadana subdistrict, central lampung district; krs#57, sidobinangun village, seputih banyak subdistrict, central lampung district; krs059, karang pawitan central seed institute, garut district; krs#61, cihuni village, wanaraja subdistrict, garut district; krs#75, experimental farm cikeumeuh ii, bogor. 37 biotropia no. 8, 1995 19. scutellospora sp.3. spores were globose, brownish yellow, 326 x 326 µm. it has two wall groups, a and b. wall group a consisted of wall 1, laminated and brown. wall group b had one membranous wall. the muronym is a(l) b(m). collections examined: krs#56, sidobinangun village, seputih banyak sub-district, central lampung district. references schenck, n.c. & g.s. smith. 1982. additional new and reported species of mycorrhizal fungi (endogonaceae) from florida. mycologia 74(1): 771-92. sieverding, e. 1991. vesicular-arbuscular mycorrhiza management in tropical agrosystems. technical cooperation, federal republic of germany, eschborn. walker, c. 1983. taxonomic concepts in the endogonaceae: spore wall characteristics in species description. mycotaxon 18: 443-445. walker, c. 1986. taxomonic concepts in the endogonaceae: ii a fifth morphological wall type in endogonaceous spores. mycotaxon 25: 95-99. walker, c. & r.e. koske. 1987. taxonomic concepts in the endogonaceae: iv glomus fasciculatum redescribed. mycotaxon 30: 253-262. walker, c., c.w. maize and h.s. mcnabb jr. 1982. populations of endogonaceous fungi at two locations in central iowa. canadian journal of botany 60: 2518-2529. walker, c. & f.e. sanders. 1986. taxonomic concepts in the endogonaceae: iii the separation of scutellospora gen. nov. from gigaspora gerd. & trappe. mycotaxon 27: 169-182. widiastuti, h. & k. kramadibrata. 1992. jamur mikoriza bervesikula arbuskula di beberapa tanah masam dari jawa bar at. menara perkebunan 60: 73-77. 38 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf biotropia no. 10, 1997 : 42-62 the effects of milling degree and type of bag on fungal infection and some chemical contents of stored milled rice okky setyawati dharmaputra seameobiotrop, p.o. box 116, bogor, indonesia, and department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia abstract the effects of milling degree and type of bag on fungal infection of stored milled rice were investigated together with some chemical contents (glucose, amylose, protein and total lipid contents), and changes in moisture content. rice var. ir 64 with different milling degrees (85, 90, 95 and 100%) packed in jute and polypropylene bags (1 kg of milled rice/bag) were stored under laboratory conditions with temperature between 24.3-27.3 c and relative humidity 52.6-81.9% for 3 months. the initial moisture content (m.c.) of milled rice was ± 14%. three replications (3 bags) were used for each treatment. each bag was put individually and was arranged randomly on a wooden pallet. the results showed that in general, the increase of milling degree and duration of storage decreased the m.c. type of bag did not give significant differences on the m.c. twenty eight fungal species were isolated from rice with different milling degree and bag type during storage. the predominant species was aspergillus candidas. total fungal population decreased with the increase of milling degree and duration of storage. bag type did not give significant differences on total fungal population. in general, the increase of milling degree increased glucose content. glucose content in milled rice packed in jute bag was higher than that in polypropylene bag. glucose content tended to decrease with the increase of storage duration. the increase of milling degree increased amylose content in milled rice. amylose content of milled rice packed in jute bag was lower than that in polypropylene bag. the increase of storage duration decreased amylose content in milled rice. in general, protein content decreased with the increase of milling degree and duration of storage. 1 type did not give significant differences on protein content. total lipid content decreased with the increase of milling degree and duration of storage. total lipid content of milled rice packed in jute bag was lower than that in polypropylene bag, but based on chemical analysis the difference was not significant. based on statistical analyses, correlation between the m.c. and total lipid content with total fungal population was positive. there was no correlation between glucose, amylose and protein contents with total fungal population. rice with high milling degree can be stored safely for long period, but it has low chemical (nutritional) contents. key words: milling degree/bag type/fungal infection/chemical content/milled rice. introduction rice (oryza saliva l.) is the primary foodcrop in indonesia. the national logistics agency (bulog) buys and stores a large amount of milled rice as national stock to maintain price stability. 42 the effects of milling degree and type of bag on fungal infection okky setyawad dhannaputra during storage rice could be infested by insects, mites, microorganisms and rodents. among the microorganisms, fungi are the most important cause of deterioration of stored products (christensen and kaufmann 1974). the tropical climate of indonesia provides a favourable condition for fungal growth. according to garraway and evans (1984) the development of fungi was affected by the nutritional content of the substrate. the degree of milling is one of the physical characters of rice that may influence the fungal development. another factor that may affect the fungal development is the type of bag used to store milled rice. the objective of the study is to get information on the effects of milling degrees and bag types on moisture content, fungal population, and some chemical contents (glucose, amylose, protein and total lipid) of milled rice during 3 months of storage. materials and methods rice variety, milling degree, bag type, and storing of milled rice rice var. ir 64 was used in this study, because it is widely cultivated in indonesia. the paddy was obtained from food technology research and training centre (balai penelitian teknologi pangan) bulog, tambun, and it was stored for 2 weeks from the harvest time. the paddy was milled with different milling degrees, i.e. 85, 90, 95 and 100%. rice with 85% milling degree was enclosed by 15% of bran layer and embryo, while rice with 100% milling degree was die rice that had neither bran layer nor embryo (juliano 1972; bulog 1994). two types of bag were used, i.e. jute and polypropylene bags. rice with milling degree of 85, 90, 95 and 100% were stored in jute and polypropylene bags (1 kg/bag) under laboratory conditions for 3 months. the initial moisture content of milled rice was ± 14%. before storage rice was fumigated with phos-phine at dosage rates 2 g/tonne of rice for 6 days. three replications (3 bags) were used for each treatment. each bag was arranged randomly on a wooden pallet (figure 1). during storage the temperature and the relative humidity of the storage were recorded using a thermohygrograph. obtaining working samples initial samples were obtained from each replication (bag) of treatment at the beginning of storage, and then at 1, 2 and 3 months of storage. a sample from each bag was divided using a sample divider into 4 sub samples for (1) moisture content analysis, (2) fungal analysis, (3) some chemical content analyses, and (4) reserve sample. 43 figure 1. milled rice packed in jute (a) and polypropylene (b) bags, and arranged randomly on a wooden pallet. moisture content, fungal and chemical content analyses moisture content was determined using an oven method (bsi 1980). fungi were isolated using dilution method on dichloran 18% glycerol agar (dg 18) (pitt and hocking 1985). the glucose, amylose, protein and total lipid contents were determined using modified anthrone, williams, kjeldahl and soxhlet extraction methods, respectively (yoshida et al. 1976). identification of the fungi fungal identification was carried out according to samson et al. (1984), pitt and hocking (1985). experimental data were analysed using completely randomized factorial design with 3 factors. the 1st, 2nd, and 3rd factors were milling degree, type of bag, and duration of storage, respectively. results and discussions moisture content moisture content (m.c.) is the most important factor in determining the development of microorganism, insect and chemical reaction hi stored products (muir 1973; sinha 1973). 44 biotropia no. 10, 1997 the effects of milling degree and type of bag on fungal infection okky setyawati dharmaputra based on statistical analysis, milling degree, duration of storage, interaction between bag types and duration of storage, interaction among milling degree, bag types and duration of storage gave very significant differences on the m.c., while interaction between milling degree and bag type was significantly different. type of bag and interaction between milling degree and duration of storage were not significantly different. the increase of milling degree decreases the m.c. (table 1). the m.c of rice with 85, 90, 95, and 100% milling degree were 14.18, 14.04, 13.91 and 13.73%, respectively. table 1. the effect of milling degree, duration of storage, interaction between milling degree and bag type, interaction between bag type and duration of storage, and interaction among milling degree, bag type, and duration of storage on moisture content of rice according to dharmaputra et al. (1993) the increase in milling degree increased the m.c. of milled rice that was stored under laboratory conditions for 3 months with temperature of 25 ± 2 c and relative humidity (rh) of 80 ± 5%. in this study the result was not the same; the increase of milling degree decreased the m.c. it was assumed that the milled rice was stored at higher temperature (24.3-27.3 c) and lower rh (52.6-81.9%) (table 2). 45 biotropia no. 10, 1997 table 2. range of temperature and relative humidity at storage condition the endosperm and the embryo of milled rice were enclosed by bran layer. the layer consists of pericarp, legmen and aleuron (figure 2). the three layers contain more lipid and protein than the endosperm, while the latter contains more carbohydrate. the higher milling degree causes the higher loss of the three layers. water could be easily bound by carbohydrate compared to lipid or protein. it was assumed that the high temperature and the low rh during storage caused the evaporation of water in the grain. although the pores of polypropylene bag was smaller than jute bag, type of bag did not give a significant difference on the m.c. it was assumed that only one bag (with 1 kg of rice) was put on the wooden pallet, so that air circulation did not affect the m.c. of rice. figure 2. structure of the rice grain (cited from grist, 1969). 46 the effects of milling degree and type of bag on fungal infection okky setyawati dharmaputra during storage the m.c. of rice decreased, although the m.c. of rice at 2 months of storage was not significantly different from that at 3 months of storage (table 1). this was probably because of the higher temperature and the lower rh during storage. the lowest m.c. was found in rice with milling degree of 100% packed in polypropylene bag at 2 months of storage (13.39%); while the highest was in rice with milling degree of 85% packed in jute and polypropylene bags at the beginning of storage (14.59% respectively). species and fungal population twenty eight species of fungi were isolated from rice with different milling degrees and bag types during storage. they were aspergillus candidus, a. flavus, a. fumigatus, a. niger, a. oryiae, a. penicilloides, a. sydowii, a. terreus, a. versicolor, a. wentii, cladosporium cladosporioides, curvularia lunala, eurotium chevalieri, e. repens, endomyces fibuliger, fusarium moniliforme, nigrospora oryzae, paecilomyces varotii, penicillium sp. nr. adametzioides, p. chrysogenum, p. citrinum, p. corylo-philum, p. herquei, p. islandicum, p. raistrickii, rhizopus microsporus, synce-palastrum racemosum and wallemia sebi. the predominant fungus was a. candidus. it was assumed that the m.c. of milled rice during storage was suitable for its growth, or the fungus was able to compete with other fungal species in colonizing the substrate. among the above species, there were some species of field fungi i.e. cladosporium cladosporioides, curvularia lunata, f. moniliforme, and n. oryzae. it was assumed that the fungi infected the grain before harvest and they survived during storage. according to christensen and kaufmann (1969) field fungi need m.c. in equilibrium with the high rh (90%) or more for their growth. the presence of each fungal species on rice with different milling degree during storage is presented in table 3. a. candidus, c. cladosporioides and p.. citrinum were always isolated, while a. flavus and e. chevalieri were often isolated. most of the fungi isolated belong to genera aspergillus, eurotium, and penicillium. according to christensen and kaufmann (1974) fungi that belong to genera aspergillus and penicillium caused deterioration in grain during storage. pitt and hocking (1985) reported that eurotium is a teleomorph stage of aspergillus which is a xerophilic fungus. based on statistical analysis, milling degree, duration of storage, and interaction between milling degree and duration of storage gave very significant differences on total fungal population, while bag type, interaction between milling degree and bag type, interaction between bag type and duration of storage, and interaction among milling degree, bag type, and duration of storage were not significantly different. 47 biotropia no. 10, 1997 table 3. the presence of each fungal species on rice with different milling degree during storage total fungal population (transformed into log x) on rice with milling degree of 85, 90, 95 and 100% during 3 months of storage were 2.94, 2.50, 2.08 and 1,73 colonies/g, respectively (table 4). total fungal population decreased with the increase of milling degree. the total fungal population was related to the nutritional content of the rice. protein, lipid, some organic acids, and vitamins decreased with the increase of milling degree. they are micro and macro-elements needed for fungal growth. according to juliano (1972) the nutritional content of the rice with high milling degree was low because the outer layer of the rice was removed during milling, consequently a part of the nutrition found in this layer was lost. 48 the effects of milling degree and type of bag on fungal infection okky setyawati dharmaputra table 4. the effect of milling degree, bag type, duration of storage, and interaction between milling degree and duration of storage on total fungal population of nee type of bag did not give a significant difference on the total fungal population (table 4). the m.c. is the environmental factor in determining the fungal growth and type of bag did not give a significant difference on it, so that the bag type did not also give a significant difference on total fungal population. total fungal population decreased with the increase of storage duration (table 4). the total fungal population (transformed into log x) at the beginning of storage was significantly different from that at 1, 2 and 3 months of storage, while after 2 and 3 months of storage they were not significantly different, although their population tended to decrease. the decrease of total fungal population was presumably related to the decrease of nutritional and moisture contents in rice. the total fungal population (not transformed into log x) at the beginning of storage, and at 1, 2 and 3 months of storage were 650.48, 410.79, 390.15 and 183.81 colonies/g, respectively. the lowest total fungal population was found in rice with milling degree of 100% after 3 months of storage (32.17 colonies/g), while the highest was in rice with milling degree of 85% at the beginning of storage (1505.92 colonies/g). fungi in rice with milling degree of 85% bulog (1994) determined that rice with 85% milling degree was enclosed by 15% of bran layer and embryo. twenty four fungal species were isolated from rice with 85% milling degree at the beginning of storage, and at 1, 2, and 3 months of storage. they were aspergillus candidus, a. flavus, a. fumigatus, a. niger, a. oryzae, a. penicilloides, a. sydowii, a. versicolor, a. wentii, cladosporium dadosporioides, curvularia lunata, eurotium 49 biotropia no. 10, 1997 chevalieri, e. repens, endomyces fibuliger, fusarium moniliforme, nigrospora oryzae, penicillium sp. nr. adametzioides, p. chrysogenum, p. citrinum, p. herquei, p. raistrickii, rhizopus microsporus, syncephalastrum racemosum and wallemia sebi. population of each fungal species is presented in table 5. fungal species at the beginning of storage, and at 1, 2, and 3 months of storage were dominated by a. candidus with the population of 1474.33, 947.67, 1045.59 and 372.09 colonies/g, respectively. fungi in rice with milling degree of 90% bulog (1994) determined that rice with 90% milling degree was enclosed by 10% of bran layer and embryo. twenty one fungal species were isolated from rice with 90% milling degree at the beginning of storage, and at 1, 2, and 3 months of storage. they were aspergillus candidus, a. flavus, a. fumigatus, a. penicilloides, a. sydowii, a. terreus, a. ver-sicolor, cladosporium dadosporioides, curvularia lunata, eurotium chevalieri, e. re-pens, endomyces fibuliger, nigrospora oryzae, paecilomyces varotii, penicillium sp, nr. adametzioides, p. chrysogenum, p. citrinum, p. islandicum, p. raistrickii, rhizopus microsporus and syncephalastrum racemosum. population of each fungal species is presented in table 5. fungal species at the beginning of storage, and at 1, 2, and 3 months of storage were dominated by a. candidus with the population of 656.08, 332.41, 355.83 and 84.67 colonies/g, respectively. dharmaputra (1994) reported that milled rice with the same variety (ir 64) and milling degree (90%) stored for 3 months under laboratory conditions (in jars), was infected by eleven fungal species i.e. a. candidus, a. flavus, a. niger, a. penicilloides, a. versicolor, a. wentii, c. dadosporioides, e. chevalieri, e. repens, p. citrinum and p. herquei. the predominant species was a. flavus. the difference in fungal species that infected milled rice was presumably due to the difference of location where the paddy was cultivated. fungi in rice with milling degree of 95% bulog (1994) determined that rice with 95% milling degree was enclosed by 5% of bran layer and embryo. twenty two fungal species were isolated from rice with 95% milling degree at the beginning of storage, and at 1, 2, and 3 months of storage. they were aspergillus candidus, a. flavus, a. fumigatus, a. oryzae, a. penicilloides, a. sydowii, a. terreus, 50 the effects of milting degree and type of bag on fungal infection okky setyawati dharmapuera 51 biotropia no. 10, 1997 a. versicolor, cladosporium cladosporioides, curvularia lunata, eurotium chevalieri, endomyces fibuliger, fusarium moniliforme, nigrospora oryzae, paecilomyces varotii, penicillium sp. nr. adametzioides, p. chrysogenum, p. citrinum, p. islandicum, p. raistrickii, rhizopus microsporus and syncephalastrum racemosum. population of each fungal species is presented in table 5. fungal species at the beginning of storage, and at 1, 2, and 3 months of storage were dominated by a. candidas with the population of 211.50, 95.17, 125.00 and 50.25 colonies/g, respectively. fungi in rice with milling degree of 100% rice with 100% milling degree had neither bran layer nor embryo. it had only an endosperm (juliano 1972; bulog 1994). eighteen fungal species were isolated from rice with 100% milling degree at the beginning of storage, and at 1, 2, and 3 months of storage. they were aspergillus candidas, a. flavus, a. fumigatus, a. niger, a. oryzae, a. penicilloides, a. sydowii, a, versicolor, a. wentii, cladosporium cladosporioides, curvularia lunata, eurotium chevalieri, fusarium moniliforme, nigrospora oryzae, penicillium sp. nr. adametzioides, p. citrinum, p. corylophilum, and p. raistrickii, population of each fungal species is presented in table 5. fungal species at the beginning of storage, and at 1, 2, and 3 months of storage were dominated by a. candidas with the population of 84.17, 41.34, 35.75 and 19.34 colonies/g, respectively. glucose content most of fungi use glucose (d-glucose) for their growth (moore-landecker 1982). glucose is a form of monosaccharide with six carbon atoms (hexose). mono-saccharide is considered as carbohydrate which cannot be hydrolized into a simple form and has a very simple bond compared to polysaccharide. consequently, their chemical bond can be easily separated by enzymes compared to amylose, a polysaccharide which has a more complex chemical bond than monosaccharide (mayes 1981). based on statistical analysis, milling degree, bag type, duration of storage, interaction between milling degree and bag type, and interaction among milling degree, bag type and duration of storage gave very significant differences on the glucose content, while interaction between milling degree and duration of storage was no! significantly different. interaction between bag type and duration of storage was noi significantly different. 52 the effects of milling degree and type of bag on fungal infection okky setyawati dharmaputra glucose content increased with the increase of milling degree (table 6). the higher milling degree caused the higher loss of the bran layer and embryo. therefore, the percentage (w/w) of glucose content in rice with high milling degree was higher than that of low milling degree. glucose content on rice with milling degree of 85, 90, 95 and 100% were 2.99, 3.50, 3.27 and 3.41%, respectively. milled rice packed in jute bag had glucose content higher than packed in polypropylene bag. it was assumed that total fungal population in milled rice packed in jute bag was lower than in polypropylene bag, although based on statistical analysis it was not significantly different (table 4). glucose content tended to decrease with the increase of storage duration. it was attributed that the glucose was used for the fungal growth (moore-landecker 1982) and as energy source for grain respiration (pomeranz 1992). glucose content at the beginning and one month of storage were significantly different from that at 2 and 3 months of storage. the glucose content of milled rice at the beginning of storage, and at 1, 2, and 3 months of storage were 3.74, 3.86, 2.84 and 2.73%, respectively. table 6. the effect of milling degree, bag type, duration of storage, interaction between milling degree and bag type, interaction between nulling degree and duration of storage, and interaction among milling degree, bag type and duration of storage on glucose content of rice. milling bag degree ( % ) type glucose content ( %) duration of storage (month) 0 1 2 3 x 85 jute 3.29uvw 3.85 stu 2.57 wx 2.56 wx 3.07 ij polypropylene 3.29 uvw 3.66 stu 2.23 x 2.47 z 2.91 j 3.29 mno 3.76 kiln 2.40 r 2.52 qr 2.99 a 90 jute 3.78 stu 3.79 stu 4.06 st 3.43 tuv 3.78 g polypropylene 3.78 stu 3.68 stu 2.84 vwx 2.61 wx 3.23 ij 3.78 kl 3.74 klm 3.45 lmn 3.02 nop 3.50 b 95 jute 3.94 stu 3.43 tuv 2.47 x 2.78 vwx 3.15ij polypropylene 3.94 stu 4.30 st 2.69 wx 2.61 wx 3. 38 hi 3.94 kl 3.86kl 2.58 pqr 2.69 pqr 3.27 ab 100 jute 3.94 stu 4.89s 2.94 vwx 2.87 vwx 3.66 gh polypropylene 3.94 stu 3.24 uvw 2.93 vwx 2.50 x 2.15ij 3.94 kl 4.06k 2.93 opq 2.68 pqr 3.41 b y jute * * * * 3.41c polypropylene * * * * 3.17d 3.74 e 3.86e 2.84 f 2.73 f — numbers followed by the same letter do not differ significantly according to tukey multiple range test at 95% confidence level *not significantly different 53 biotropia no. 10, 1997 the highest glucose content was found in rice with milling degree of 100% packed in jute bag at one month of storage (4.89%), while the lowest was in rice with milling degree of 85% packed in polypropylene bag at 2 months of storage (2.23%). although a sugar other than glucose may give maximum growth of a fungus, it appears that the more closely the configuration of a sugar resembles glucose, the more fungi use it. this might be due to the ability of a fungus to use a particular sugar depending on how easy it can be converted into a phosphorylated derivative of glucose which can enter the respiratory pathways (moore-landecker 1982). amylose content most of the carbohydrate found in rice is in the form of starch (90% of dry weight) and only a very small part is in the form of hemicellulose, cellulose and some free sugars such as sucrose, raffinose, glucose and fructose (juliano 1972). starch is a polysaccharide that can be separated into two fractions depending on their ability to dissolve hi water, that is amylose (± 20%) and amylopectin (± 80%) (mayes 1981). juliano (1972) reported that based on dry weight, the amylose content of milled rice was between 7-33% depending on rice variety. based on statistical analysis, milling degree and duration of storage gave very significant differences on the amylose content, while bag type gave a significant difference. interaction between milling degree and bag type, interaction between milling degree and duration of storage, interaction between bag type and duration of storage, and interaction among milling degree, bag type and duration of storage did not give significant differences. the increase of milling degree increased amylose content (w/w) in milled rice. the amylose content of milled rice with milling degree of 85, 90, 95 and 100% were 23.84, 23.99. 24.98 and 25.50%, respectively (table 7). amylose content of milled rice packed in jute bag (24.09%) was lower than that in polypropylene bag (25.07%). it was assumed that the pores of jute bag was bigger than that of polypropylene bag, so that the air surrounding jute bag could make easier to hydrolize the amylose to form maltose or glucose. the increase of storage duration decreased amylose content in milled rice. the amylose content at the beginning of storage, and at 1, 2 and 3 months of storage were 26.72, 25.40, 24.29 and 21.90%, respectively. presumably the phenomena was due to the activities of (3-amylase and fungal enzymes which infected the molecule a-glucans and supported the hydrolization of amylose to disaccharide and monosaccharide. 54 the effects of milling degree and type of bag on fungal infection okky setyawati dharmaputra table 7. the effect of milling degree, bag type, and duration of storage on amylose content of rice amylose content (%) milling bag duration of storage (month) degree (%) type 0 1 2 3 x 85 * * * * 23.84 a 90 * * * * 23.99 ab 95 * * * * 24.98 be 100 * * * * 25.50 c y jute * * * * 24.09 d polypropylene * * * * 25.07 e 26.72 f 25.40 g 24.29 h 21.90i numbers followed by the same letter do not differ significantly according to tukey multiple range test at 95% confidence level * not significantly different protein content rice is one of the cereals which contains a higher percentage of lysine (± 4%) than other cereals. lysine is an essential amino acid which is found in very small quantities in other food stuff (juliano et al. 1973). brown rice contained ± 8% protein, 75% carbohydrate and a small amount of lipid, fibre and ash, while milled rice contained ±7% protein (juliano 1976). based on statistical analysis, milling degree and duration of storage gave very significant differences on the protein content, while interaction between milling degree and duration of storage was significantly different. bag type, interaction between milling degree and bag type, interaction between bag type and duration of storage, and interaction among milling degree, bag type and duration of storage did not give significant differences. the protein content tended to decrease with the increase of milling degree and duration of storage (table 8). the protein content in rice with milling degree of 85 and 100% during 3 months of storage were significantly different. they were 9.14 and 8.84%, respectively. according to juliano (1972) the decrease of milling degree decreased the nutritional content (including protein). dharmaputra et al. (1993) reported that total protein in milled rice var. cisadane decreased with the increase of milling degree. at the beginning of storage the protein content in milled rice was 9.44%, and then decreased to 9.18% at 3 months of storage. dharmaputra et al. (1993) reported that the increase of storage duration decreased the protein content in milled rice. 55 biotropia no. 10, 1997 table 8. the effect of milling degree, duration of storage, and interaction between milling degree and duration of storage on protein content of rice according to sinha and muir (1973), the decrease of protein content may also be caused by degradation of amino acid and non-protein nitrogen such as vitamin b and phospholipid. . the lowest protein content was found in rice with milling degree of 100% after 1 month of storage (8.28%), while the highest was in rice with milling degree of 90% at the beginning of storage (9.77%). in general, the increase of milling degree and duration of storage decreased the protein content hi milled rice. total lipid content according to juliano (1976), brown rice contained ±1.9% lipid, while milled rice contained ± 0.5% lipid. pomeranz (1974) reported that lipid was convened into free fatty acid and glycerol with the aid of lipase enzyme during storage. this con version was accelerated with the existence of storage fungi because the fungi have high ly poly tic activity. based on statistical analysis, milling degree, duration of storage, and interaction between milling degree and duration of storage gave very significant differences on the total lipid content, while bag type and interaction among milling degree, bag type and duration of storage gave significant differences. interaction between milling degree and bag type, and interaction between bag type and duration of storage did not give significant differences. total lipid content decreased with the increase of milling degree and duration of storage (table 9). total lipid content in milled rice with milling degree of 85, 90, 95 and 100% were 0.26, 0.23, 0.20 and 0.15%, respectively. juliano (1972) reported; 56 the effects of milling degree and type of bag on fungal infection okky setyawati dharmaputra table 9. the effect of milling degree, bag type, duration of storage, interaction between milling degree and duration of storage, and interaction among milling degree, bag type and duration of storage on total lipid content of nee that milling removes the embryo and the bran layer, therefore total lipid found in this pan will decrease. total lipid content of milled rice packed in jute bag (0.20%) was lower than packed in polypropylene bag (0.21 %), but chemical analysis showed no significant difference. total lipid content in milled rice at the beginning of storage, and at 1, 2 and 3 months of storage were 0.28, 0.25, 0.17 and 0.14%, respectively. dharmaputra et al. (1993) reported that the increase of milling degree and duration of storage, decreased total lipid content. according to pomeranz (1992) degradation of fat or oil in grains can be affected by several factors, such as 1) oxydative process which gives rancid odour and taste, and 2) hydrolytic process which results to free fatty acid and glycerol. the lowest total lipid content was found in rice with milling degree of 100%, packed in jute bag at 3 months of storage (0.10%), while the highest was in rice with milling degree of 85%, packed in jute bag and polypropylene bags at the beginning of storage (0.34% respectively). 57 biotropia no. 10, 1997 correlation between moisture content and total fungal population based on statistical analysis, the m.c. and total fungal population were positively related with coefficient correlation (r) = 0.52. the increase of m.c. increased total fungal population (figure 3). figure 3. correlation between moisture content and total fungal population on milled rice during storage water is required for the growth of fungi just as for other organisms. fungi usually require a film of water around their cells through which nutrients and enzyme diffuse (garraway and evans 1984). ramakrishna et al. (1993) reported that spore germination and initial growth of fungi that infected the grain were affected by water activity and temperature of storage. correlation between glucose content and total fungal population statistical analysis showed no correlation between glucose content and total fungal population. it was assumed that glucose was not only used for the fungal nutrition, but also for respiration process of grains (pomeranz 1992). 58 the effects of milling degree and type of bag on fungal infection okky setyawati dbarmaputra according to garraway and evans (1984) glucose is a good carbon source for 4-5 fungal species. most of the fungal species also used oligosaccharide and polysaccharide as carbon source. correlation between amylose content and total fungal population based on statistical analysis, there was no correlation between amylose content and total fungal population. it was assumed that amylose was hydrolized into disaccharide by fungi-infected milled rice during storage. in this study twenty eight fungal species were isolated (table 3). according to garraway and evans (1984) each fungal species may have different enzyme to hydrolize carbon sources, such as amylose. 1& correlation between protein content and total fungal population statistical analysis showed no correlation between protein content and total fungal population. it was assumed that fungi require other nitrogen source for their growth and development. according to garraway and evans (1984) most fungi are able to utilize simple inorganic nitrogen sources as well as organic sources such as amino acids. correlation between total lipid content and total fungal population based on statistical analysis, the correlation between total lipid content and total fungal population was positive with r = 0.64. the increase of total lipid content increased total fungal population (figure 4). fungi contain a specific enzyme that could hydrolize lipid from the substrate for their development and growth (patterson 1989). according to garraway and evans (1984), at first fungi hydrolized lipid with lipase enzyme, then changed them into glycerol through (3-oxidation pathway. conclusions in this study the increase of milling degree and duration of storage, decreased the m.c. of rice. the type of bag did not give a significant difference on the m.c. twenty eight fungal species were isolated from rice with different milling degree and bag type during storage. most of the fungi isolated belong to genera aspergillus, eurotium and penicillium. the predominant species was a. candidus. 59 biotropia no. 10, 1997 figure 4. correlation between total lipid content and total fungal population on milled rice during storage. total fungal population decreased with the increase of milling degree and duration of storage. type of bag did not give a significant difference on the total fungal population. glucose content of milled rice increased with the increase of milling degree. when packed in jute bag the glucose content was higher than packed in polypropylene bag, but it decreased with the increase of storage duration. amylose content of milled rice increased with the increase of milling degree. when packed in jute bag the amylose content was lower than packed in polypropylene bag, but it decreased with the increase of storage duration. protein content in milled rice tended to decrease with the increase of milling degree and duration of storage, but it was not affected by bag type. total lipid content of milled rice decreased with the increase of milling degree and duration of storage. when packed in jute bag the lipid content differed significantly than packed in polypropylene bag, but chemical analysis showed no significant difference. 60 the effects of milling degree and type of bag on fungal infection okky setyawati dharmiputn rice with low milling degree has high nutritional content, but it cannot be stored for long period. on the other hand rice with high milling degree has low nutritional content, but it can be stored for long period. to get more information on the effect of bag type on fungal infection and chemical contents of stored milled rice, a research using stack of rice is needed. acknowledgement the author gratefully acknowledges the financial support of the government of indonesia. thanks are due to dr. haryanto susilo, dr. hariyadi halid, dr. mulyo sidik, mr. amad and mrs. asmarina s.r. putri for their valuable assistance, and to dr. a.d. hocking from csiro, australia, for the confirmation of fungal identification. references bsi. 1980. methods of test for cereals and pulses. pan 3. determination of moisture content of cereals and cereal products (routine method). british standards institution. isbn 0 058 114333. bulog. 1994. tatacara teknis pemenksaan kualitas gabah, beras dan karung goni/plastik dalam rangka pengadaan pangan dalam negeri. badan urusan logistik. jakarta. (in indonesian). christensen. c.m. and h.h. kaufmann. 1969. grain storage: the role of fungi in quality loss. university of minnesota, minneapolis. ___. 1974. microflora. in c.m. chrstensen (ed.). storage of cereal grains and their product. american association of cereal chemist., inc. minnesota, pp. 158-192. dharmaputra, o.s. 1994. kapang pada beras yang berasal dari beberapa varietas padi. hayati 1(2): 37-41. ___. h. halid, h. susilo and a.s.r. putri. 1993. the effects of milling degree on fungal infection, protein and total lipid content in milled rice. proceeding 16th asean seminar on grain postharvest technology, phuket, thailand. 24-26 august 1993. oarraway. m.o. and r.c. evans. 1984. fungal nutrition and physiology. john willey & sons inc., new york. grist. d.h. 1959. rice 3 rd ed. longmans, london. juliano, b.o. 1972. rice caryopsis and its composition. in d.f. houston (ed). rice chemistry and technology. american association of cereal chemist., minnesota. ___. 1976. the chemical basis of rice grain quality. proceedings of the workshop on chemical aspect of rice grain quality. irr1. los banos, philippines. __. a.a. antonio and b.v. esmama 1973. effects of protein content on the distribution and properties or rice protein. j. sci. food agr. 24: 295-306. mayes. p.a. 1981. carbohydrate. in d.w. martin, p.a. mayes and v.w. rodwell (ed}. 18th edition. harper's review of biochemistry. lange medical publication, los altos, california. moore landecker, e. 1982. fundamentals of the fungi. 2rd edition. prentice-hall, inc., new jersey. muir. w.e. 1973. temperature and moisture in grain storage. in r.n. sinha and w.e. muir (ed). grain storage: part a system. the avi publishing company, connecticut. 61 biotropia no. 10, 1997 patterson, h.b.w. 1989. handling and storage of oilseeds, oils, fats and meal. elsevier applied science, new york. pitt, j.i. and a.d. hocking. fungi and food spoilage. academic press, sydney. pomeranz. y. 1992. biochemical, functional and nutritive change during storage. in sauer, d.b. (ed). storage of cereal grains and their products. american association of cereal chemist., minnesota, pp. 55-141. ramakrishna, n., j. lacey and j.e. smith. 1993. effects of water activity and temperature on the growth of fungi interacting on barley grain. mycol. res. 97(11): 1393-1402. samson, r.a., e.s. hoekstra and c.a.n. van oorschot. 1984. introduction to foodborne fungi. centraalbureau voor schimmelcultures, baam, the netherlands. sinha. r.n. 1973. interrelation of physical, chemical and biological variables in the deterioration of stored grains. in r.n. sinha and w.e. muir («/). grain storage: part a system. the avi publishing co. yoshida, s., d.a. forno. j.h. cook and k.a. gomez. 1976. laboratory manual for physiological studies of rice. irri, los banos, philippines. 62 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf 53.pdf 54.pdf 55.pdf 56.pdf 57.pdf 58.pdf 59.pdf 60.pdf 61.pdf 62.pdf 4 effect of physio (lazarus).cdr biotropia vol. 18 no. 1, 2011: 31 41 effect of physiological age and growth regulators on callus browning of coconut endosperm culturein vitro lazarus agus sukamto* the possibility of physiological age and growth regulators affecting callus browning of coconut endosperm was investigated. solid endosperm explants of four coconut fruits from same brunches of two coconut cultivars “samoan dwarf ” were grown on modified murashige and skoog (ms) formula with addition of 10 mg l putresine, 2.50 g l activated charcoal (ac), 1.70 g l phytagel, 0, 10 , 10 , 10 , 10 m 2,4-dichlorophenoxyacetic acid (2,4-d) or 4-amino3,5,6-trichloropicolinic acid (picloram) combined with 10 m 6-benzylaminopurine (ba). callogenesis occurred on 98.83% of explants. callus browning between different physiological ages (antipodal and micropylar tissues) of coconut endosperm at 9, 26 and 31 weeks of culture (woc) was significantly different, but not at 16 and 21 woc. auxins of 2,4-d and picloram did not affect significantly callus browning of endosperm cultures. auxin doses at 10 , 10 , and 10 m decreased significantly callus browning at 9 and 16 woc, respectively, but at 10 m browning was less significant compared to other doses at 21 woc. auxin dose at 10 m caused less significant browning compared to other doses at 31 woc. the addition of ba decreased significantly callus browning at 9 woc, but did not affect callus browning thereafter. coconut, , picloram, 2,4-d, ba research center for biology indonesian institute of sciences in vitro abstract introduction -1 -1 -1 -6 -5 -4 -3 -5 -6 -5 -4 -6 -3 key words: coconut ( l.) is a long-lived tree with a very long juvenile phase (3 5 years), generally cross-pollinated and very heterozygous. vegetative propagation through the use of various explants has been attempted but the success is mostly limited to immature unselected plants and produced very few plants. coconut endosperm is considered a mature tissue which provides large and uniform explants without damaging mother plant for culture. cocos nucifera in vitro in vitro cibinong, indonesia * corresponding author : lazarus_agus@yahoo.com 31 in vitro et al in vitro et al et al citrus grandis in vitro et al et al in vitro et al et al gasteria haworthia et al et al et al et al et al et al et al physiological age is a critical factor for the success of culture (ozyigit . 2007). the age of the endosperm at the time of culture is critical for growth (chen . 1990). explants from younger fruits responded better to culture (karunaratne . 1991). however, coenocytic (free-nuclear) endosperm from very young fruits did not survive in culture because of lack of starch (srivastava 1982). young endosperm at celliferous stage was responsive to culture in and apple (mu and liu 1978; wang and chang 1978). endosperm of 6 7 months postanthesis in coconut which solid endosperm started to form in the antipodal end of coconut fruit (tammes & whitehead 1969) was responsive to culture (kumar . 1985). whole plants are autonomous with regard to growth regulators but isolated tissues or cells require auxins or cytokinins to initiate and maintain growth until they become habituated or organized (everett . 1978). morphogenesis can be regulated by regulators (christianson & warnick 1983), skoog and miller (1957) found that the balance of auxin and cytokinin in culture medium governed morphogenesis. auxins induced callogenesis, adventitious plantlets and roots in palms (tisserat 1979; paranjothy & rohani 1982; reynolds 1982; paranjothy 1986). among auxins, 2,4-d was the most effective compared to the others in coconut culture (blake & euwens 1982; pannetier & buffard-morel 1986; karunaratne & periyapperuma 1989). auxin at high doses (10 m 10 m) was necessary for callus induction in palm, especially on medium supplemented with 1 3 g l ac (jesty & francis 1992). another auxin with properties similar to 2,4-d is picloram. picloram has been successfully applied for callogenesis in date palm (omar & novak 1990), embryogenesis in pejibaje palm (valverde . 1987), and maintained regenerative callus over long time in sugarcane (fitch . 1983). picloram was faster than 2,4-d for callogenesis, embryo induction, and final yield of embryos in and (beyl & sharma 1983). on the other hand, picloram produced more phenolics from cut surfaces and was slower for callogenesis than 2,4-d in sugarcane (fitch . 1983). the presence of cytokinins, auxins, and high doses of sucrose (0.2 m) stimulated growth of coconut and date callus (euwens 1978). combination of cytokinin at low dose with auxin at high dose was necessary for callogenesis of coconut embryos (bhaskaran 1985). srinivasan . (1985) successfully induced somatic embryos of christmas palm with 5 50 x 10 m 2,4-d and 50 x 10 m ba. ba was more effective than kinetin in stimulating callus growth in longan culture (litz 1988), increased greatly fresh weight of coconut callus and date palm callus (euwens 1978; kuruvinashetti & iyer 1980; sharma . 1984). explant browning is often associated with physiological age and failure of explants survival (krishna . 2008; guo . 2010). browning in some explants may be very severe and causes inhibition or cessation of growth (abdelwahd . 2008; misra . 2010). phenolics, tannins or oxidized polyphenols are synthesized through shikimic acid, phenylpropanoid, flavonoid, and terpenoid pathways. these substances are abundantly present in some plants and act as inhibitory agents (preece & compton 1991). phenolics, especially the most common polyphenol cause oxidative browning in explants, which lead to discoloration of the culture medium (forrest 1969; davies 1972; babbar & gupta 1986; tang & newton 2004). oxidized phenolic -5 -3 -1 -5 -5 32 biotropia vol. 18 no. 1, 2011 compounds are frequently exuded into the medium by injured woody tissues causing lethal browning or blackening of explants (alderson 1987; bhat & chandel 1991; trautmann & visser 1991). immature endosperms were more responsive in culture than mature ones (cheema & mehra 1982). this was partially due to oxidation products which were more abundant in the older explants (sugimura . 1988; preece & compton 1991; wu . 2010). activated charcoal can promote cell growth and development, it may be mainly due to its irreversible adsorption of inhibitory compounds in the culture medium and substantially decreasing toxic metabolites, phenolic exudation and browning (zhu . 1997; thomas 2008). browning can be reduced or eliminated through the use of liquid media, ac, silver nitrate, ascorbic acid, citric acid, sodium hydrosulfite, cystein, diethyldithio carbamate (dtt), potassium ethylxanthate, thiourea, benzimidazole, sodium bisulfit, polyvinylpyrrolidone (pvp), polyclar, glutathione, and bovine serum albumin, more frequent transfers, incubation with reduced illumination or in complete darkness, presoaking explants in sterile water, sealing the cut ends with paraffin wax, changing medium, avoiding high temperatures, discarding explants which show browning, reducing explants thickness, and choosing the most suitable stage (chang . 2001; wu & du toit 2004; mitsukuri . 2009). addition of auxins, particularly 2,4-d at high doses, led to browning of coconut leaf (pannetier & buffard-morel 1986). sugimura and salvana (1989) observed that 2,4-d at 2.26 x 10 m 4.52 x 10 m caused severe browning of tissues regardless of the stage and size of coconut inflorescence culture. 2,4-d at levels higher than 30 x 10 m inhibited callusing and enhanced browning of coconut embryos (karunaratne & periyapperuma 1989). callus precociously isolated from explants also caused browning and necrosis in coconut inflorescence (verdeil . 1993). addition of cytokinin, such as kinetin also caused more browning than the use of auxin in palm cultures (reynolds 1982). the objective of the study is to know the effect of physiological age (antipodal and micropylar) and growth regulators (2,4-d, picloram, and ba) on callus browning of coconut endosperm culture plant materials of four seven-month old coconut fruits were taken from two bunches of two coconut trees cultivars “samoan dwarf ” after opening inflorescences. these fruits immediately were disinfested by using alcohol 95%. the fruits were opened and their water decanted, cut horizontally with a sterile big knife. solid endosperms were aseptically cored with cork borer and scooped with a sterile spoon in laminar air flow. these endosperms were taken from either the micropylar region (upper half of fruit where embryo located) or the antipodal region (bottom half of fruit). cylindrical endosperm shapes with 8 mm diameter and 4 mm thick, used as explants, were grown on various media treatments. in vitro et al et al et al et al et al et al in vitro . -4 -4 6 materials and method explant material callus browning of coconut endosperm culture l.a. sukamtin vitro o 33 media culture experimental design callogenesis of coconut endosperm physiological age and calli browning media cultures were a modification of branton and blake formula (1986) added with 10 mg l putrescine, 2.50 g l ac, 1.70 g l phytagel, hormones 2,4-d or picloram at 0, 10 , 10 , 10 and 10 m concentrations, respectively, were combined with or without 10 m ba at 16 woc. the ph of the media were adjusted to 5.70 before they were autoclaved. the media were poured into 2.5 x 15 cm test tubes (14 ml) and autoclaved at 121 c temperature and 1 kg cm pressure for 15 minutes. media were stored for one week before use. single explant was placed into test tube with the uncut surface upright. cultures were incubated at approximately 31 c in the dark room. the experimental design was a randomized complete block design (rcbd) with each fruit as a block. treatments were factorial combinations of endosperm region (micropylar and antipodal), auxins (2,4-d and picloram) and their doses (0, 10 m, 10 m, 10 m, 10 m) and cytokinin (with or without 10 m ba) with 12 replications. callogenesis of coconut endosperm was counted as percentage on 31 woc. the tissues browning were evaluated visually at every transfer using numerical scores ranging from 0 to 3 (0 : no browning, 1 : little browning, 2 : medium browning, and 3 : high browning) at 9, 16, 21, 26 and 31 woc. data were analyzed with the general linear models (glm) and non parametric one way (kruskal-wallis test) procedure of statistical analysis system (sas). coconut endosperm explants formed callus after approximately 3 woc. callus grew predominantly on the uncut surface and on the side of endosperms. eventually, the callus grew and covered the entire explants. callogenesis occurred on almost all explants with an average of 98.83% (table 1). these results were better than those obtained by kumar . (1985) i.e. 30% of coconut endosperms, gmitter . (1990) i.e. 8 25% of endosperms, chen . (1990) i.e. 1.60 3.74% of endosperms; pannetier and buffard-morel (1982) i.e. 20% of mature coconut leaves and 50% of juvenile coconut leaves, and verdeil . (1989) i.e. 45% of coconut inflorescence. the good result could be due to genetic and sterilized factors. in addition, the plant materials used in this study were different in explants type, cultivars or plants than those used by other researchers. sterilized method was different than of kumar . (1985), this experiment only sterilized the outer skin of the fruits not the explants. therefore, the explants were more responsive in promoting callogenesis. oxidative browning (brown to black) of endosperm calli occurred during culture. browning varied within or among treatments, ranging from 0: no browning, 1: slight -1 -1 -1 -6 -5 -4 -3 -5 0 -2 0 -6 5 -4 -3 -5 results and discussion et al et al citrus maxima et al c. sinensis et al et al 34 biotropia vol. 18 no. 1, 2011 source fruit 1 fruit 2 fruit 3 fruit 4 average z y means standard error of 12 measurements means in the same group followed by the same letter in a column except for the average are not significantly different at the 5% level (based on a comparison of possible combinations between the averages of treatments) table 2. browning levels of coconut endosperm callus (0 : no browning, 1 : light brown, 2 : brown 3 : dark brown ) z y means standard error of 12 measurements means in the same group followed by the same letter in a column except for the average are not significantly different at the 5% level (based on a comparison of possible combinations between the averages of treatments) table 1. percentage of callogenesis of coconut endosperm after 31 weeks of culture 35 callus browning of coconut endosperm culture l.a. sukamtin vitro o browning 2:medium browning , and 3: severe browning (fig. 1). tissue browning by all treatments was slight (score = 1.18) at 9 woc. tissue browning increased substantially (browning level = 1.75) after 16 woc and reached maximal browning (browning level = 2.25) on 21 woc. thereafter browning decreased slightly on 26 woc (browning level = 2.23) and 31 woc (browning level = 2.18). compton and preece (1988) found that phenolic compounds exuded from excised explants were oxidized by peroxidases or polyphenolo-xidases, causing browning of both plant tissues and media. this might reduce growth or kill the tissues (preece and compton 1991). severe browning of coconut endosperm still occurred even though in young explants which were not treated by disinfectants and incubated in the dark room. whereas this condition was expected to prevent browning of ova (dias . 1994). the antipodal and micropylar tissues browning showed a steady increase at 21 woc (table 2). statistical analysis showed that browning of antipodal tissues was more significant than in micropylar tissues at 9, 26, and 31 woc. it could be due to antipodal tissues which were earlier formed (older) and thicker than micropylar tissues. these results support the findings of murashige (1974) that the influence of geonoma gami et al figure 1. browning of coconut endosperm callus at various levels i.e. 0, 1, 2, and 3 (from left to right) figure 2. a). varied colors of coconut endosperm callus, b). new yellowish white callus grown from the black callus of coconut endosperm a b 36 biotropia vol. 18 no. 1, 2011 physiological age on explants responses was not significantly different at 16 and 21 woc (table 2). explants thickness influenced browning, antipodal explants were thicker and showed more browning compared to micropylar explants. similar results were obtained by sugimura and salvana (1989) in coconut inflorescence explants of 1 mm which had 32% browning compared to 11% browning in 0.5 mm thick.coconut inflorence explants. the two types of auxins (2,4-d and picloram) did not cause any significant difference in browning of endosperm tissues. different results were reported by fitch . (1983) that picloram caused more browning than 2,4-d in cultures. the browning levels of tissues initially treated with 10 m auxin were significantly higher at 9 woc, the highest at 16 woc, and the lowest at 31 woc than those of the other auxin concentrations (table 2). the browning levels of control were higher at 9 16 woc than of those tissues treated with 10 m 10 m auxins (table 2). the tissues initially treated with 10 m auxin, showed significantly less browning than control or other auxin concentrations at 21 woc. the tissues initially treated with 10 m auxin caued significantly more browning than of the other treatments at 26 woc. this result disagreed with the findings of pannetier and buffard-morel (1986), karunaratne and periyapperuma (1989), and sugimura and salvana (1989) that 2,4-d levels higher than 3 x 10 m caused more browning than lower concentrations in coconut explants. this result agreed with the findings of phillips and henshaw (1977) in cell cultures. addition of ba significantly decreased browning at 16 woc. similar result was reported by herve . (2001) in culture. however, those of addition did not affect significantly callus browning thereafter (table 2). calli color changed progressively, from white to brown, to dark brown and to black (fig. 2a). then new yellowish white callus grew from the black callus and this sequence was repeated through many cycles (fig. 2b). severe browning did not inhibit the growth of endosperm cultures. similar result was reported by jones (1974) in oil palm and ettinger and preece (1985) in cultures. coconut endosperm probably tolerated high level of 2,4-d or picloram, even their browning were less than other treatments including control at 10 m on 31 woc (table 2), due to the presence of ac at 2.5 g l in the medium and dark incubation. it agreed with the findings of wang and huang (1976), fridborg . (1978), tisserat (1979), blake and eeuwens (1982), rao . (1987), sugimura and salvana (1989), and krikorian (1994). callogenesis occurred on 98.83% of coconut endosperm explants. calli browning increased significantly from 9 to 21 woc but not thereafter. browning of antipodal tissue-derived calli was more significant at 9, 26 and 31 woc but not at 16 and 21 woc compared to micropylar tissues. treatments of 2,4-d and picloram did not growth regulators and calli browning et al saccharum spontaneum acer pseudoplatanus et al eucalyptus gunnii hododendron et al et al -3 -6 -4 -6 -5 -5 -3 -1 r conclusions 37 callus browning of coconut endosperm culture l.a. sukamtin vitro o affect calli browning. auxins at 0 and 10 m produced significantly more callus browning than other doses at 9 and 16 woc. auxins of 10 m produced significantly less callus browning than other doses at 21 woc. calli browning was more intense at 10 m auxins at 26 woc. auxins of 10 m produced significantly less callus browning than other doses at 31 woc. addition of cytokinin ba produced significantly more callus browning at 16 woc but not thereafter. the author would like to thank dr. y. sagawa and dr. d.t. webb for their advice, while mr. r. wutzke for providing the experiment the overseas training office/ badan perencanaan pembangunan nasional (bappenas) indonesia is acknowledged for the funding support. -3 -6 -5 -3 acknowledgements references abdelwahd r, hakam n, labhilili m, udupa sm. 2008. use of an adsorbent and antioxidants to reduce the effects of leached phenolics in plantlet regeneration of faba bean. african j biotech 7(8):9971002 alderson pg. 1987. micropropagation of woody plants. in: alderson pg and dullforce wm (editors). micropropagation in horticulture, practice and commercial problems. the univ. nottingham trent print unit. p 37-52 babbar sb, gupta sc 1986. induction of androgenesis and callus formation in in vitro cultured anthers of a myrtaceous fruit tree ( l.). bot mag tokyo 99:75-83 bhaskaran s. 1985. tissue culture technology for higher vegetable oil production. in: srivastava, hc, bhaskaran s, vatsya b, menon kkg (editors). oilseed production: constraints and opportunities. oxford & ibh publishing co., new delhi. p 537-544 beyl ca, sharma gc. 1983. picloram induced somatic embryogenesis in and . plant cell tissue organ cult 2:123-32. bhat s.r, chandel kps. 1991 a novel technique to overcome browning in tissue culture. plant cell rep 10:358361. blake j, eeuwens cj. 1982. culture of coconut palm tissues with a view to vegetative propagation. in: rao a.n. 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regeneration from protoplasts of grapevine ( l.) through somatic embryogenesis by using embryogenic callus culture and activated charcoal. plant sci 123(1-2):151-57 bactris gasipaes cocos nucifera cocos nucifera in vitro citrus in vitro protea cynaroides vitis vinifera http://eng.hi138.com/?i131358 41 callus browning of coconut endosperm culture l.a. sukamtin vitro o 6 effect of plant (deden) pak... biotropia vol. 18 no. 1, 2011: 50 60 effects of plant growth regulators on shoot multiplication and root induction of cassava varieties culturein vitro deden sukmadjaja and herni widhiastuti a study on propagation of three superior cassava ( crant) varieties i.e. darul hidayah, malang-6 and adira-4 through tissue culture technique was conducted at the tissue culture lab of seameo biotrop, bogor. the objective of the experiment was to study effect of plant growth regulators on propagation, which can be used in cassava micropropagation protocol. plant materials used were auxiliary shoots of a stem node. the experiment consisted of (i) shoot multiplication, (ii) roots induction, and (iii) acclimatization. the multiple shoot regeneration was observed by using murashige & skoog (ms) basal media supplemented with 0, 0.1, 1.0 and 5.0 mg/l of benzylaminopurine (bap) combined with 0.0, 0.1 and 1.0 mg/l of thidiazuron. the root induction was observed by using 0.1 and 1.0 mg/l of iba and naa. the resulting plantlets were transplanted into plastic polybags containing soil mixed with organic fertilizer (1:1) covered with plastic sheets and transferred to a greenhouse. the result of the study showed that the highest number of shoots for darul hidayah, malang-6 and adira-4 varieties were 4.93 shoots treated with bap 1 mg/l + thidiazuron 0.1 mg/l, 4.20 shoots at bap media of 1 mg/l, and 7.20 shoots at the media of bap 1 mg/l + thidiazuron 0.1 mg/l respectively. the highest number of nodes produced was 2.9 nodes for darul hidayah at bap 5 mg/l, 5.13 nodes for malang-6 at bap 0.1 mg/l, and 6.18 nodes for adira-4 at bap 5 mg/l + thidiazuron 1 mg/l. the utilization of auxin iaa or naa could induce and accelerate the growth of roots which finally could increase the success of acclimatization process. with an average of four multiplication factors of each culture period, the potency of each cassava shoot propagated through tissue culture could produce around 37 000 plants/year. , shoot multiplication, root induction, ba, thidiazuron, iba, naa 1* 2 1 2 indonesian center for agricultural biotechnology and genetic resources research and development, jl. tentara pelajar no.3a, bogor, indonesia 16111 seameo biotrop, jl. raya tajur km.6, po box 116, bogor, indonesia manihot esculenta in vitro manihot esculenta abstract key words: * corresponding author : 50 introduction materials and method cassava ( crantz) is one of the food crops as a source of carbohydrates. in addition , cassava has a purpose used not only as a source of carbohydrates but also as raw materials for industry, cosmetics, feed and energy. as a raw material for industry, cassava could be processed into tapioca, glucose and fructose syrup, citric acid, monosodium glutamate, plywood, maltose, sorbitol and ethanol (sani 2006). meanwhile, with the increasing world prices of gasoline, cassava has now become the second source of bioethanol for premium mixture. in developing cassava production for agro-industry, the government of indonesia has issued regulations among others to optimize and to use genetic sources from the available varieties and clones of cassava. a few superior cassava varieties such as darul hidayah, adira and malang could produce 22-49 ton/ha fresh root crop. it has been reported that the productivity of darul hidayah could reach 102 ton/ha fresh root crop. the need of plant shoots exploited on a large scale will be difficult to fulfill using conventional propagation. cassava plant materials are commonly derived from vegetative propagation using stem cuttings. presently tissue culture technology is used for mass propagation of plant materials. propagation of cassava has been conducted either with shoots multiplication method or somatic embryogenesis. somatic embryogenesis was proposed as an alternative for mass cassava propagation (raemakers 1993; mathews 1993; konan 1994; ma and xu, 2002). however, it has not been really applied for large scale propagation because the frequency of plant regeneration from cassava somatic embryos is usually low. shoot multiplication method is the simplest and the safest method if observed from the point of genetic stability. this method becomes the most commonly used in the initiation step of plant propagation experiment. many authors such as kartha (1974) and konan (1997) explored the possibility to multiply cassava using microcuttings. nodal explants onto solid media provide an average of 3 to 4 microcuttings in 2 to 3 months. the objective of this study was to investigate the effects of plant growth regulators on plant regeneration of three cassava varieties in media culture. a study was conducted at the tissue culture laboratory of bioresources management center (brmc), seameo biotrop bogor. the experiment was conducted to observe the effects of several concentrations of bap combined with several concentrations of thidiazuron on multiple shoot formation and of auxin (iaa and naa) on rooting formation. plant materials applied this research were the auxiliary shoots derived from stem nodes of superior cassava clones, i.e. darul hidayah, malang and adira. isolated auxiliary shoots as explant sources were sterilized by soaking in the detergent solution for 15 min. then the explants were rinsed and soaked in 2 g/l of manihot esculenta in vitro et al et al. et al. et al. et al. in vitro explant source: 51 in vitro et almass propagation system of superior cassava clones deden sukmadjaja . both fungicide and herbicide solution for 2 hrs, respectively. the soaked explants were rinsed with distilled water three times, then soaked in 2% na-hypochlorite solution for 30 min. explants were rinsed again by distilled water which contained 50 mg/l ascorbate acid for 10 min. explants ranging in size from 2 to 3 mm were cut aseptically without damaging the apical dome (shoots growth spot). the auxiliary shoot explants were inoculated onto sterilized solid basal ms medium (murashige & skoog's 1962) supplemented with different concentrations and combinations of different plant growth regulators. aseptically explants were inoculated onto initiation shoot ms media i.e. supplemented with 30 g/l sucrose and 100 mg/l polyvinyl pirolidone (pvp). explants which have been planted in initiation media for 1-2 weeks were transferred into regeneration and shoot multiplication media. the regeneration and multiplication medium was used with ms basal media supplemented with 30 g/l sucrose, 80 mg/l adenine sulfate and different range of bap (0, 0.1, 1, 5 mg/l) combined with thidiazuron (0.0, 0.1 and 1.0 mg/l). as long as the explants were in these media, they were sub cultured into the same media every 2 weeks within 4-6 weeks. elongated micro shoots on shoot regeration media were excised and transferred to ms basal media supplemented with 30 g/l sucrose, 2 g/l activated carbon (charcoal) and two different concentrations of iaa and naa (0.1 and 1.0 mg/l) either individually. the ph of the medium was adjusted to 5.8 before gelling with agar (8 g/l) and prior to autoclaving for 15 min at120 c and at 15 lbs psi pressure. sugar was added at the concentration of 30 gr/l. medium of 25 ml was dispensed into the culture jar and plugged with autoclavable plastic cap. all the cultures were incubated in a growth room with a 16 hour photoperiod (cool, white fluorescent light -1000-1500 lux) and the temperature was maintained at 25 ± 3 c with 70-80% relative humidity in the culture room. each treatment consisted of 10 replicates and repeated three times. plantlets with well-developed roots were removed from the culture medium. the roots were washed gently under running tap water and transferred to plastic pots (polybag) for hardening which contain mixed soil and manure (1:1). the harden plantlets in the plastic polybag were covered with transparant polyethylene sheets (for about 15 days) to maintain high humidity and were kept under shade in a net house for further growth and development. the calculation of the potential number of cassava plants produced through tissue culture followed the formulation of pennell (1987): where : y = number of plantlet/plants that could be produced a = number of shootss produced at each subculture period (multiplication factor) 2 o o culture medium and condition: shoot initiation medium: shoot regeneration and multiplication medium: rooting medium: environmental condition: acclimatization and transfer of plantlets to soil: potential of cassava plant production: y = a x b x f1 x f2 x f3 n biotropia vol. 18 no. 1, 2011 52 b = number of initial explant which grow n = number of subculture at a certain period (per year) f1 = the percentage of successful culture at the stage of shoot induction f2 = the percentage of successful culture at the stage of shoot elongation f3 = the percentage of successful acclimatization experimental design: experiments were set up in a completely randomized design (crd) and each experiment usually had 10 replicates and was repeated three times. 10 explants were used per treatment in each replication. observations were recorded on the percentage of shoots, number of shoots or nodes per explant, shoot length, numberof roots per shoot and root length respectively. statistical analysis was limited to the calculation of standard errors of means. the first step of shoot initiation, after one week explants were cultured on initiation media where var. darul hidayah produced the highest number of aseptically explants compared to the other four varieties. this is assumed to be related to the source of explants. the source of darul hidayah explants was taken from the green house which has low contamination due to controlled sanitation and environmental condition compared to the two other sources of explants which were taken from the field. results and discussion shoot initiation from source of explants 53 in vitro et almass propagation system of superior cassava clones deden sukmadjaja . (a) (b) (c) (d) figure 1. (a) auxiliary shoots on ms shoot induction media, (b-d) shoots of three cassava varieties on multiplication media: var. darul hidayah, var. malang-6, and var. adira-4, respectively. 54 biotropia vol. 18 no. 1, 2011 auxillary shoot will appear after one-week old explants were cultured on ms shoot induction media supplemented with 30 g/l sucrose and 100 mg/l polyvinyl pirolidone (pvp) (fig. 1a). pvp was used to inhibit necrosis of the plant tissues which were always found in woody plants according to zdravkovic 2004. the growth rate of shoot elongation with the best visual appearance was shown by adira-4 and darul hidayah, while malang-6 showed a lower growth rate of its shoot elongation. in general, shoot induction and elongation occurred after the explants were one-week old and cultured on ms media. at the age of 4 weeks the shoots had between 3-4 nodes and sufficient enough to be sub-cultured on propagation media. among three cassava varieties, adira-1 was not used because most of the explants were contaminated after sub-cultured on regeneration and multiplication media. two steps of experiments were conducted i.e. shoot multiplication and root induction. another activity is acclimatization of plantlet in the green house. the experimental results of the role of bap and thidiazuron on the number shoots produced on regeneration and multiplication media are shown on table 1. all of bap and thidiazuron concentrations examined on darul hidayah and adira-4 showed to produce an average of higher number of shoots compared to the non plant regulators media (control). on the other hand, malang-6 explant produced higher number of shoots only on 1 mg/l bap and 0.1 thidiazuron compared to the control media. bap could encourage cell division which stimulates formation of multiply shoots. according to lu (1993) the addition of thidiazuron in media containing bap could increase the explant ability to produce shoots. in perennial woody plants among others and , thidiazuron could stimulate the formation of shoots. kerns and meyer (1986) reported that a good result was obtained at the stage of acer shooting by using combination of bap and thidiazuron. the highest shoot number of darul hidayah produced was about 4.93 shoots on media supplemented of 1 mg/l bap combined with 1 mg/l thidiazuron. the use of high concentration of 5 mg/l bap without thidiazuron resulted in high number of shoots (4.80 shoots). adira-4 has a better response and produce the average number of shoots compared to the other two varieties in all treatments examined. the average highest number of shoots of adira-4 was about 7.20 shoots on media containing 1 mg/l bap combined with 0.1 mg/l thidiazuron. the number of shoots decreased without addition of thidiazuron. the addition of thidiazuron at low concentration (0.1 mg/l) tended to increase the number of shoots produced. the average number of malang-6 shoots produced was lower than of the two varieties at all similar treatments. the highest number of shoots (4.20) was produced when treated with bap 1 mg/l. the effects of growth regulator bap and thidiazuron on the lenght of shoots are shown on table 2. the highest lenght of shoots of malang-6 were 4.33 cm obtained on media without plant growth regulator. no significant measurable effect of bap and thidiazuron were obtained on lenght of malang-6 shoots. meanwhile, on darul hidayah, the use of bap at low concentration (0.1 mg/l) with or without the addition of thidiazuron at low concentration (0.1 mg/l) resulted in higher growth of et al, in vitro prunus rhododendron the effect of media composition on shoot and root regeneration 55 in vitro et almass propagation system of superior cassava clones deden sukmadjaja . shoots compared to the other treatments. on the other hand the highest growth of shoots on adira-4 was obtained if treated with bap at 5 mg/l concentration with or without thidiazuron, i.e. between 3.13-4.19 cm (table 2). a high growth of shoots was also observed in media containing thidiazuron 1 mg/l combined with or without bap 1 mg/l, i.e. 3.44 and 3.66, respectively. nodal explants with axillary meristems derived from aseptically grown seedlings or stem cuttings were used to initiate a round compact bulb-like structure on ms medium containing 10 mg/1 bap. this latter structure, when cultured on ms medium supplemented with 0.1 mg/1 naa, 1 mg/1 bap and 0.1 mg/1 ga , produced multiple shoots. rooting of regenerated shoots exceeded 95 % in phytohormone-free ms medium. (konan . 1997). cytokinin could interact with other growth regulators to stimulate the vegetative growth of plants (maxwell & keiber 2004). in the plant physiology process, cytokinin influences cell division in order to broaden the area of the tissues and plantlet height (davies 2004). 3 et al table 1. the effect of bap combined with thidiazuron in ms media on the shoot number of darul hidayah, malang-6 and adira-4, 30 days after planting plant growth regulator (mg/l) number of shoots darul hidayah malang-6 adira-4 without plant growth regulator 2.07 ± 0.21 2.79 ± 0.30 3.00 ± 0.70 bap 0 + thi 0.1 3.93 ± 1.02 3.44 ± 1.61 6.20 ± 1.30 bap 0 + thi 1 2.87 ± 1.30 1.59 ± 0.45 5.50 ± 1.29 bap 0.1 2.20 ± 0.45 2.25 ± 0.58 4.20 ± 0.84 bap 0.1 + thi 0.1 3.06 ± 0.90 2.86 ± 1.07 3.40 ± 0.89 bap 0.1 + thi 1 3.60 ± 1.14 2.71 ± 0.90 3.60 ± 1.14 bap 1 4.75 ± 1.69 4.20 ± 0.86 6.41 ± 1.52 bap 1 + thi 0.1 4.20 ± 1.00 2.35 ± 1.44 7.20 ± 3.27 bap 1 + thi 1 4.93 ± 1.18 2.35 ± 0.91 6.00 ± 0.81 bap 5 4.80 ± 1.12 2.15 ± 0.48 5.40 ± 2.08 bap 5 + thi 0.1 3.34 ± 0.63 2.45 ± 0.58 5.20 ± 1.93 bap 5 + thi 1 3.48 ± 0.87 2.35 ± 0.59 3.67 ± 1.16 note: means are calculated by standard errors in general, the number of nodes produced in malang-6 and adira-4 varieties was higher compared to darul hidayah. in malang-6 the use of media with or without low concentration of bap combined or not with thidiazuron 0.1 mg/l produced higher number of nodes compared to other treatments (3.98-4.61 nodes). in adira-4, the use of thidiazuron l mg/l with or without addition of bap produced high number of nodes, except for bap 0.1 mg/l + thidiazuron 1 mg/l (4.83-6.18 nodes). in darul hidayah, the highest number of nodes was obtained if treated with high concentration of bap combined with or without thidiazuron, i.e. between 2.49-2.90 nodes (table 3). according to mingxia ., 2011, the cytokinin n-benzyladenine (ba) (02.0 mg l )et al −1 biotropia vol. 18 no. 1, 2011 was effective on cassava shoot regeneration. konan (1997) has conducted mass propagation system of some cassava cultivars through auxiliary shoots multiplication and stem nodes which showed that the addition of bap on basal ms media was more efficient if compared to the addition of other kinds of cytokinin. the effectiveness on organogenesis was different based on different based on different cytokinins and in combination with auxins. benzyladenine and thidiazuron stimulated more shoot organogenesis than kinetin and n-isopentenyladenine (ma 1998). et. al. table 2. the effect of growth regulator in ms media on the lenght of cassava shoot var. hidayah, malang-6 and adira-4 at 30 days after planting. plant growth regulator (mg/l) lenght of shoots (cm) darul hidayah malang-6 adira-4 without plant growth regulator 1.50 ± 0.68 4.33 ± 1.29 1.05 ± 0.37 bap 0 + thi 0.1 2.54 ± 0.71 2.18 ± 0.98 2.77 ± 0.40 bap 0 + thi 1 2.16 ± 1.52 2.31 ± 1.19 3.66 ± 0.40 bap 0.1 3.85 ± 1.49 2.17 ± 1.54 1.80 ± 0.82 bap 0.1 + thi 0.1 3.90 ± 1.50 2.36 ± 0.92 1.11 ± 0.39 bap 0.1 + thi 1 1.44 ± 0.69 2.65 ± 0.83 1.02 ± 0.33 bap 1 1.61 ± 0.76 2.69 ± 0.87 2.17 ± 0.47 bap 1 + thi 0.1 1.61 ± 0.45 1.60 ± 1.52 2.61 ± 1.16 bap 1 + thi 1 2.26 ± 0.75 2.06 ± 1.16 3.44 ± 0.58 bap 5 2.04 ± 0.28 1.24 ± 0.44 3.13 ± 1.09 bap 5 + thi 0.1 1.14 ± 0.24 1.73 ± 0.73 4.19 ± 1.85 bap 5 + thi 1 1.75 ± 0.20 1.61 ± 0.45 3.78 ± 1.25 note: means are calculated by standard errors marked differences were observed among the three varieties in the auxin requirements for root induction (table 4). darul hidayah and adira-4 showed increase of both root number and length resulted from addition of iaa to the nutrient media. no significantly effect of iaa and naa were obtained on number and lenght of root in malang-6. the length of roots of darul hidayah culture showed the highest growth compared to malang-6 and adira-4. in darul hidayah, the use of iaa and naa at the same concentrations did show a significant difference in the number of roots (table 4). the use of naa relatively produced higher number of roots compared with iaa. on the contrary, the use of iaa on malang-6 produced slightly higher number of roots compared to the use of naa. while in adira-4, the use of iaa (01 and 1 mg/l) gave a significantly different result compared to the use of naa 0.1 mg/l. ogburia (2003) has conducted a faster propagation on a number of cassava cultivars with 90 % efficiency by using ms media of 0.1 mg/l naa and 0.05 mg/l bap. the use of 0.1-1.0 mg/l iaa in rooting media of darul hidayah and adira-4 produced longer roots compared to the treatment with naa. the longest root was produced in darul hidayah treated with iaa l mg/l (5.43 cm), and for adira-4 treated 56 57 in vitro et almass propagation system of superior cassava clones deden sukmadjaja . with iaa 0.1 mg/l (3.23 cm). meanwhile, the longest root of malang-6 was produced if treated with naa 0.1 mg/l, i.e. 2.37 cm (table 5, fig 2 (a-c). mingxia ,(2011) reported the auxi ) proved to be effective on cassava root development. aladele and kuta (2008) reported that the growth rate of cassava plantlet is significantly different on various genotypes and growth environment. in acclimatization stage, rooted shoots were transplanted in the green house for hardening and their survival rate was 90% under the field condition (fig. 3). . et al n α-naphthalene acetic acid (naa) (02.0 mg l−1 table 3. the effect of growth regulator in ms media on the number of cassava nodes in darul hidayah, malang-6 and adira-4 at 30 days after planting plant growth regulator (mg/l) number of nodes darul hidayah malang-6 adira-4 without plant growth regulator 2.10 ± 0.22 4.61 ± 0.06 2.53 ± 1.52 bap 0 + thi 0.1 2.20 ± 0.65 4.78 ± 1.42 4.65 ± 0.30 bap 0 + thi 1 1.23 ± 0.28 3.44 ± 1.24 5.76 ± 0.76 bap 0.1 2.48 ± 0.84 5.13 ± 1.04 2.56 ± 1.11 bap 0.1 + thi 0.1 1.77 ± 0.84 3.98 ± 1.09 2.70 ± 1.36 bap 0.1 + thi 1 1.22 ± 0.48 3.33 ± 0.30 2.64 ± 0.43 bap 1 2.04 ± 0.58 4.08 ± 0.86 3.92 ± 0.90 bap 1 + thi 0.1 1.83 ± 0.63 3.23 ± 1.82 4.09 ± 1.91 bap 1 + thi 1 2.21 ± 0.58 3.47 ± 1.51 4.83 ± 0.96 bap 5 2.90 ± 0.50 2.86 ± 0.42 3.75 ± 0.72 bap 5 + thi 0.1 2.49 ± 0.37 3.43 ± 0.93 4.52 ± 0.62 bap 5 + thi 1 2.85 ± 0.43 3.21 ± 0.83 6.18 ± 3.48 note: means are calculated by standard errors table 4. the effect of auxin growth regulator in ms media on the number of cassava roots of darul hidayah, malang-6 and adira-4 at 15 days after planting. plant growth regulator (mg/l) number of roots darul hidayah malang-6 adira-4 iaa 0.1 4.40 ± 1.42 2.30 ± 1.77 3.60 ± 1.51 iaa 1 3.70 ± 0.89 3.30 ± 2.06 4.30 ± 1.57 naa 0.1 4.86 ± 2.19 2.00 ± 1.63 1.90 ± 1.37 naa 1 3.80 ± 1.92 2.50 ± 1.84 3.20 ± 2.10 note: means are calculated by standard errors (a) (b) (c) fig. 2. t . he occurrence of the root growth of three cassava varieties in rooting media, (a) darul hidayah; (b) malang-6; and (c) adira-4 (a) (b) fig. 3. seedlings in the green house after one week old acclimatization (a) and the performace of seedling roots (b). 58 biotropia vol. 18 no. 1, 2011 table 5. the effect of auxin growth regulator in ms media on the length of roots (cm) of darul hidayah, malang-6 and adira-4 at 15 days after planting. plant growth regulator (mg/l) length of roots (cm) darul hidayah malang-6 adira-4 iaa 0.1 4.88 ± 1.43 0.77 ± 0.62 3.23 ± 1.04 iaa 1 5.43 ± 2.49 1.88 ± 1.55 2.65 ± 0.67 naa 0.1 3.60 ± 0.76 2.37 ± 1.55 1.24 ± 1.14 naa 1 3.25 ± 2.02 1.61 ± 0.84 1.50 ± 1.54 note: means are calculated by standard errors 59 in vitro et almass propagation system of superior cassava clones deden sukmadjaja . potential of cassava plant production y = a x b x f1 x f2 x f3 one shoot explant (b) could produce an average of 4 (a) nodes or shoots for each culture period, with a frequency of 8 subcultures per year (n), and assumed that the success a the stage of shoot induction (f1) = 90%, the success at the stage of rooting (f2) = 80%, then the number of plants that could be produced per year (y) from each shoot is as follows, using the formulation of pennell (1987) : 4 x 1 x 0.9 x 0.8 x 0.8 = 37 748 plants if there are 10 initial shoot explants (b) available then the number of plants that could be produced is about 377.480. it should be noted that the number of plants produced is based on theoretical calculation; however, the implementation depends on some other related factors such as number of manpower and available facilities. each variety of clone cassava needed a different protocol to get optimum shoot initiation, shoot multiplication, root induction and elongation. the maximum number of axillary shoots and stem nodes of cassava var darul hidayah, malang-6 and adira-4 were obtained on shoot multiplication media containing bap and thidiazuron at different concentrations. the use of iaa and naa media induced and accelerated the growth of roots and increased the percentage of survival plantlet in acclimatization process. the potency of each cassava shoot could be propagated to produce about 37000plantletsper year. the authors are grateful to seameo-biotrop for providing financial support and facilities. thanks are due to the technicians of the brmc, seameo-biotrop plant tissue culture laboratory for their assistance. n 8 conclusions acknowledgements references davies pj. 2004. the plant hormones: their nature, occurrence and function in: davies pj (editor) plant hormones biosynthesis, signal transduction, action. kluwer acad press. p 1-15 kartha kk, gamborg ol, constabel f, shyluk jp. 1974. regeneration of cassava plants from apical meristems. plant sci lett 2: 107-13 konan nk, sangwan rs, sangwan bs. 1994. somatic embryogenesis from cultured mature cotyledons of cassava crantz). plant cell tiss org cult 37: 91-102 konan nk, schapke c, carcamo r, beachy rn, fauquet c. 1997. an efficient mass propagation system for cassava ( crantz) based on nodal explants and auxiliary shoot-derived meristems. plant cell rep 16(7):444-49 (manihot esculanta manihot esculenta 60 biotropia vol. 18 no. 1, 2011 kerns hr, meyer mm. 1986. tissue culture propagation of acer x freexmani using thidiazuron to stimulate shoot tip proliferation. hort sci 21(5):1209-10 lu cy. 1993. the use of thidiazuron in tissue culture. cell dev biol 29:92-96 ma gh. 1998. effects of cytokinins and auxins on cassava shoot organogenesis and somatic embryogenesis from somatic embryo explants. plant cell tissue org cult 54: 1-2 ma gh, xu qs. 2002. induction of somatic embryogenesis and adventitious shoots from immature leaves of cassava. plant cell tissue org cult 70: 281-282 mathews h, schopke c, carcamo r, chavarriaga p, fauquet lb. 1993. improvement of somatic embryogenesis and plant recovery in cassava. plant cell rep12: 328-33 maxwell bb, keiber jj. 2004. cytokinin signal transduction. in davies, pj (editor) plant hormones biosynthesis, signal transduction, action. kluwer acad press. p 321-349 pennell d. 1987. micropropagation in horticulture. growerguide no.29. grower books, london raemakers cjjm, schavemaker cm, cobsen e, visser rgf. 1993. improvement of cyclic embryogenesis of cassava crant). plant cell rep12: 226-229 sani s. 2006. kebijakan dalam strategi pengembangan ubi kayu untuk agroindustri. prospek, strategi, dan teknologi pengembangan ubikayu untuk agroindustri dan ketahanan pangan. puslitbangtan, bogor. hlm: 20-28 zdravkovic-korac s, muhovski y, druort p, calic d, rabjevic l. 2004. agrobacterium rhizogenes mediated dna transfer to l. and the regeneration of transformed plant hormones. plant cell rep 22:698-704. in vitro (manihot esculenta dalam aesculus hippocastonum fainmarinat inabuy_manuscript for biotropia 1 isolation and characterization of partial cdna of sucrose synthase putative gene in palmyra palm (borassus flabellifer) fainmarinat inabuy, adi pancoro research center for biotechnology, institute technology bandung, jl. ganesha no.10 bandung, indonesia40132. email: fimaluvbio@yahoo.com abstract intensification of biofuel resources is urgently needed considering decreased availability of world’s fossil fuel. palmyra palm (borassus flabellifer) is highly potential to be developed as bioethanol source regarding the high sucrose content in its nira. it was observed that nira produced in dry season is sweeter than that in rainy season, which presumed to be influenced by a difference in expression level of sucrose-related genes during the two seasons. study of sucrose synthase (sus) gene of palmyra are therefore required prior to study of the gene expression. palmyra sus gene sequence is currently unavailable in genbank, thereby pair of primers was designed from highly conserved region of sus proteins among monocots. a 1866 bp partial cdna fragment of sus putative gene has been succesfully isolated from rna of the young leaves of b. flabellifer. blastn and blastp aligments showed that either bfsus cdna or bfsus polypeptide has high similarity with sus cdna and proteins from diverse plant species with the highest similarity shown by tulipa gesneriana. the phylogenetic tree showed that sus protein sequences of monocot species were distinctively grouped and splitted from those of dicot species. the bfsus was clustered in monocot group, although not specifically grouped with particular monocot species. nevertheless, b. flabellifer showed nearest genetic distance with tulipa gesneriana and oncidium cv.’goldiana’. characterization of bfsus polypeptide using geneious 4.6.2 indicated the presence of sucrose synthase (sus) and glycosyl transferase (gt) domains, four putative udp-glucose binding pockets within the gt domain, and a calciumdependent ser/thr protein kinase binding site within the sus domain. these domains and motifs are highly conserved in sus proteins across plant species, confirming that the cdna fragment obtained in this study is very likely cdna encodes sucrose synthase in b. flabellifer. keywords: borassus flabellifer, nira, sucrose, sucrose synthase, sus domain, gt domain, palmyra palm. introduction palmyra (borassus flabellifer) is a palmae species with high potency to be developed as a source of bioethanol. the plant is typically grown in dry areas in strictly seasonal tropical or subtropical climate. the palmyra palm is known to be very adaptive in dry areas with only 500-900 mm annual rainfall (flach and rumawas, 1996). the main product of palmyra is the sweet liquid produced from its inflorescence, called nira, or locally known as tuak (fox, 1977). the nira of palmyra palm contains 17-20% dry matters comprises of sucrose, amino acids, proteins, vitamins, and other essential minerals. sucrose (c6h12o6) that constitutes 13-18% per liter nira is the principal material in the production of bioethanol through the process of fermentation. it was observed in east nusa tenggara, particularly in timor and rote islands, that nira tapping is more 2 preferable in dry season rather than in rainy season. beside the safety reason, it is probably because palmyra nira in dry season is sweeter than in rainy season. it becomes interesting to study whether this phenomena is solely caused by high level of water contained in the nira, by a higher rate of photosynthesis occurred in dry season, or it has something related with sucrose genes expression level. a preliminary study on sucrose related gene expressed in palmyra palm is therefore needed to answer the question. sucrose is the most important plant disaccharide; it is the principle form by which photosynthetic product is transported throughout plant tissues from the source photosynthetic tissue to the sink non-photosynthetic tissues (bush, 1999). this disaccharide consists of one molecule of glucose and one molecule of fructose that is bounded by a glycosidic bond (lodish et al., 1999). many enzymes involved in metabolism of sucrose. the closest related enzymes are sucrose-synthase (sus: ec 2.4.1.13), sucrose phosphate synthase (sps: ec 2.4.1.14), sucrose-6-phosphatephosphatase (sppase: ec 3.1.3.24), and invertase (ec 3.2.1.26). the reversible and irreversible reactions of sucrose hydrolysis are catalyzed, respectively, by sus and invertase. the biosynthesis of sucrose is catalyzed by the sequential action of sps and sppase (winter and huber, 2000). sucrose can be synthesized from hexose monophosphates by sus or sps. in the case of sus, the in vivo sucrose concentrations are always much higher than fructose or udpglucose, resulting in reaction that is essentially always towards the direction of sucrose cleavage. invertase, on the other hand, only catalyzes the cleavage of sucrose into glucose and fructose. all enzymes catalyze transfer of sugar moieties from activated donor molecules to specific acceptor molecules by forming glycosidic bonds are classified in the glucosyltranferase (gt) family of enzyme. sucrose synthase (ec 2.4.1.13) is a key enzyme involved in sucrose metabolism, included in the gt4 family regards to its role in transferring the glucose from udpglucose to fructose molecule to form sucrose or, in reverse, hydrolyze the sucrose into glucose and fructose. sus enzyme plays significant role in food storage of many plants, either in the form of starch or sugar (baud et al., 2004). sus was shown to control the mobilization of sucrose into various pathways that important for the metabolic, structural, and storage functions of the plant cell. phloem-loading, a process by which sucrose is transported from photosynthetic to nonphotosynthetic tissues, is facilitated by sus. inhibition of sus-encoding genes had shown significant reduction in sucrose import capacity of floem tissues, thereby causing less sucrose content in tomato fruit (d’aoust et al., 1999). conversion of sucrose to udp-glucose that is catalyzed by sus provides substrate for cell wall biosynthesis and starch synthesis in plant storage organs (sun et al., 1992; zrenner et al., 1995; dejardin et al., 1997; hendrik, 1990). sus activity also associated with development of nodules in legume plants and in regulation of apical meristem function (craig et al., 1999). studies on sus-encoding genes in many plant species revealed that in vitro environmental conditions, such as light, temperature, and water availability have a significant influence on the expression of sus-encoding genes. palmyra (b. flabellifer) which has higher sugar content in dry season than in wet season, probably reflects a likewise relation. sus genes have been isolated from various starch and sugar-storing plants, such as citrus (citsusa and citsus2) (komatsu et al., 2002), sugarcane (kumar et al., 2007), rice (wang et al., 1992), maize (sus1 and sh1 gene) (mccarty et al., 1986), wheat (marana et al., 1988), cotton (ruan et al., 2003), tomato (sun et al., 1992), and potato (zrenner et al., 1995).the enzyme is found in all plant tissues but is highly expressed particularly in sink tissues (baud et al., 2004). according to those studies, the full length sus gene has in average 4970 bp length, while its cdna length is only about half of it, ranging from 2400 to 2500 bp. this research was aimed to study the characteristic of sus gene in b. flabellifer. a comprehensive knowledge of the sus gene characteristics is an initial requirement to study its expression level, and further to conduct manipulation or control of those genes, which enables efforts to enhance the productivity of b. flabellifer as a potential source of sugar and bioethanol. 3 results and discussion rna isolation from borassus flabellifer total rna of b. flabellifer was isolated from the young leaves of 35-40 years old-plants. young leaves was chosen as it was reported that sus genes are commonly expressed in newly developed tissues that require supply of either sucrose or sucrose derived-compounds (kumar et al., 2007). the results of five methods tested for rna isolation from b. flabellifer is summarized in table 1. the modified method of apt et al. (1995) resulted in good quality of rna although the purity and quantity were rather low. the rna isolation was best accomplished by using trizol ® reagent (invitrogen). the reagent, a mono-phasic solution composed of phenol and guanidine isothiocyanate, is an improvement to the single-step rna isolation method developed by chomczynski and sacchi (1987). this method successfully generated two distinct bands of 18s and 28s ribosomal rna that were clearly occurred in 1% gel electrophoresis (figure 1), which indicated a good rna quality. the rna yield was relatively high, 1600 µg/ml, with low protein contamination as shown by the ratio 1.62. we noted that the success in obtaining high rna yield from borassus leaves is greatly determined by the finest powder that could be recovered from the leaves tissue. reverse transcriptionpcr a total cdna from the young leaves of b. flabellifer were obtained by cdna synthesis. they then used as template for the next pcr reaction. the annealing of sus-spesific primers to the target cdna were best achieved in temperature 52ºc. as well, 2 mm of mg2+ and 1.5 µg/50 µl of template cdna were found to be the most suitable concentration for the pcr mix. total 30 cycles of amplification using the gene specific primers, ssfw and ssrv, has successfully produced a single fragment of 1866 bp that was detected in 1% agarose gel (figure 2). according to the expected product size, this 1866 bp fragment was predicted as the target fragment of the sus gene. cloning of sus putative gene and transformation of e. coli prior to sequencing reaction, the sus putative fragment is purified from the agarose gel and then cloned into a plasmid vector, pgem®-t easy (promega), which then delivered to e. coli strain dh5α. e. coli cells that had been transformed were able to grow in the ampicilincontaining medium. transformed cells containing sus gene fragment were selected by bluewhite screening method. transformation of e. coli strain dh5α with pgem®-t containing sus fragment resulted in blue and white colonies growth on the ampicillin contained-lb media. plasmid isolation and confirmation of gene insertion plasmid isolation from the e. coli white colony and subsequent restriction cut using particular restriction enzymes are purposed to confirm the presence of the sus gene fragment within the plasmid of e. coli. when cut with ecori, the plasmid produced two unexpected fragments, ± 1300 bp and ± 500 bp, instead of 1866 bp (figure 3a). it was presumed that the sus fragment of b. flabellifer has an ecori restriction site within its sequence, thereby produced shorter fragment than expected. it was found later that the sequence of sus gene of b. flabellifer does have the recognition site for ecori. the plasmid was then cut with another endonuclease enzyme, noti. as expected, cutting with noti generated a single 1866 bp fragment (figure 3b). this result indicated that the sus gene fragment had been successfully inserted into the pgem®t easy plasmid. pcr screening with sp6 and t7 primers was further conducted to prove the presence of sus fragment within the multiple cloning sequence (mcs) region in the plasmid. those two primers 4 flank the mcs region in the pgem®-t easy plasmid. as expected, a ±1995 bp fragment was occurred (figure 3c). this length corresponds to the length of the sus fragment, ±1866 bp, plus 129 bp lengths of the sp6 and t7 mcs region. sequencing and characterization of sus putative gene and polypeptide two steps of sequencing were required since sus putative fragment of b. flabellifer, further stated as bfsus, is relatively long for a single reading by the sequencer system. the sp6 and t7 primers were used in the former sequencing, followed by specific internal primers to read the gap sequence within the fragment. the later primers were designed from the read of the former sequence. using geneious 4.6.2 program, an overlapped reading occurred from those two steps of sequencing, thus finally assembly the whole sequence of the bfsus putative gene fragment. the assembled bfsus fragment was trimmed from pgem®t vector region by using vecscreen program (www.ncbi.nlm.nih.gov), followed by determination of ssfw and ssrv primers annealing region. the final construction of bfsus partial gene, which total length is 1866 bp, was successfully obtained (figure 4). the sequence of bfsus putative gene was first analyzed by blastn program (altschul et al., 1997) to figure out the similarity level of bfsus putative gene with other sus gene sequences recorded in the genbank. the percentage of identical sites and query coverage indicated that bfsus putative gene shares quite high similarity, 70.8% – 80.6%, with sus genes from various monocots. thus, confirming that the isolated 1866 bp bfsus fragment is most likely a sucrose synthase gene. bfsus sequence shows the highest similarity with sus genes of tulipa gesneriana (80.6%), followed by x. mokara (78.8%), potamogeton distinctus (78.7%), oncidium sp. cv goldiana (78.6%), and bambusa oldhamii (78.5%). translation of bfsus cdna sequence resulted in bfsus polypeptide sequence consists of 622 amino acids (figure 5). the blastp program showed that bfsus polypeptide sequence is highly similar with sus protein sequences of many plant species, ranged from 78.3 % to 87.5%, confirming that the bfsus gene expresses sus protein. the highest similarity, 87.5%, are showed by tulipa gesneriana, a liliaceae plant widely known as tulip, and oncidium cv ‘goldiana’, a genus that contains about 330 species of orchids from the orchidaceae family. oryza sativa (86.8%), bambusa oldhamii (86.5%), zea mays (86.2%), and other monocots also showed high similarity. in order to elucidate relationship between bfsus putative protein and sus proteins from other species, and between monocot and dicot species, a phylogenetic tree is generated using neighborjoining method (figure 6). the following things can be interpreted from the phylogenetic tree. first, the similarity of sus polypeptide sequence in certain taxonomic group tends to be higher than another group. the similarity of sus among monocot species, for instance, is distinctively higher than those of dicot species. this also showed by species coming from the same genus, for instance, between solanum tuberosum and solanum lycopersicum. second, b. flabellifer is shown to be clustered with the monocot group, although not specifically grouped with particular monocot species. it is however shown that borassus flabellifer has the nearest genetic distance with tulipa gesneriana. although these two species live in distinctively different climate regions, presumably they both evolve similar mechanism to adapt to osmotic stress in their surroundings, probably by accumulating sucrose. in water stress conditions, plants are able to keep their osmotic gradient lower than their surrounding by accumulating more solutes, including sucrose, in their tissues, thereby preventing loss of water. multiple sequence alignment of sus polypeptides indicated two main domains that typically occurred in all plant species. those are a sucrose synthase domain located upstream toward the n-terminal and a glucosyltransferase domain that is located downstream towards the c-terminal 5 of the protein. these domains differs sus from other glycosyltransferase enzymes. using pfam database, it was shown that those two domains are occurred in the bfsus polypeptide albeit in partial length. the moiety of sucrose synthase and glucosyltransferase domain is found toward the n and c-terminal region, respectively, of the bfsus polypeptide. the overlapped region between these domains is spanned from asn283 to ser559. despite found to be highly conserved among plant species, the structural and functional sites and motifs within sucrose synthase domain are still unrevealed yet. instead, four putative functional motifs have been reported for the second domain, the glycosyl transferase. it is known that structure of a protein determine its function. therefore, function of a protein could be deduced by comparing protein sequences and structures with homolog proteins of known function. similar motifs between two proteins generally will have same function, especially when they are homolog (horton et al., 2006). functional motifs within the bfsus polypeptide were elucidated by finding functional conserved motifs within other enzymes of gt family which had been previously annotated. buschiazzo et al. (2004) had successfully isolated and crystallized another gt family enzyme from agrobacterium tumefaciens, the glycogen synthase (gs). gs catalyses the synthesis of the α-1,4-glucose backbone in the reaction of glycogen biosynthesis. this enzyme possesses the gt domain, a domain also found in sus enzyme, with some annotated motifs within it. according to homolog motifs in gs (buschiazzo et al., 2004), four functional motifs predicted as urasile diphosphate (udp)-binding pockets were detected in the bfsus polypeptide. the first putative udp binding pocket found in bfsus polypeptide is gly169, a residue located in a glycine rich motif, 166-dtggq-170 (figure 7) (huber and huber, 1996; buschiazzo et al., 2004). the glycine (g) residue is predicted to directly contact with phosphate group of udp molecule that binds to sus protein. buschiazzo et al. (2004) discovered that, in the close conformation of the gs protein, the glycine residues in the kxggl motif come into contact with the phosphate groups of udp. this role had been reported also in the gs of escherichia coli (furukawa et al., 1993). they reported that only the glycine residues but not the basic side chain appear to be essential for gs enzymatic activity. the dtggq motif was found to be conserved in sucrose phosphate synthase (sps), another member of gt family, which functioned as fructose-6-p binding site (huber and huber, 1996). the three subsequent udp-putative binding pockets in bfsus polypeptide are 444-mar-446 residues, n520, and t546 (figure 8 and 9). it was observed that the mar motif within the bfsus polypeptide is located at the c-terminal end of a β-strand (figure 8), a pattern that is also occurred in isr motif of gs polypeptide, albeit in different number of β-strand. this probably reflects their similar role. it was observed that the guanidinium group of r299, in gs, interacts with the phosphate group of adp via a hydrogen bonding (buschiazzo et al., 2004). the mar residues of bfsus polypeptide thereby suggested as binding site for phosphate group of udp via a hydrogen interaction. the asn520 (n520) motif of bfsus polypeptide is predicted to bind with the adenine ring of the udp molecule. according to gs, the carbonyl group of protein backbone in asn520 may be interacted with atom n6 of the adenine ring of udp molecule via a weak hydrogen interaction. the last putative udp binding pocket, the t546 was suggested to bind ribose sugar of udp molecule. the side chain of t546 may interact via hydrogen bond with o2 atom of the ribose sugar. the last conserved region of sus protein that is also found in bfsus polypeptide is the ser170 putative phosphorylation site. this essential site was found within a typical serine residue containingmotif, ‘rhlss’, which lay between arg167 and ser171. in the partial bfsus polypeptide this motif is located between arg30 and ser34 (30-rhlss-34). this motif was firstly detected in zea mays sus protein, spanned from arg159 to ser163 (hardin et al., 2003). this motif was also found in oryza sativa, saccharum officinarum (r159 to s163), bambusa 6 oldhamii, triticum aestivum (r167 to s171), and tulipa gesneriana (r161 to s165). hardin et al (2003) reported that sus protein of zea mays is phosphorylated by calcium-dependent protein kinases (cdpks) at the ser170 residue, within the rhlss motif, in addition to ser15 (asano et al., 2002). ser15 was reported as a major phosphorylation site that affects cleavage activity and membrane association, whereas ser170 is a minor phosphorylation site that may trigger enzyme degradation via the ubiquitin/26s proteasome (qiu et al., 2007). since phoshorylation of ser170 is important for enzyme degradation but not directly involved in the catalytic activity of sus, this site is suggested as one of the allosteric sites of the enzyme. those all results confirm that the isolated 1866 bp of bfsus gene fragment is most likely the sucrose synthaseencoding gene in b. flabellifer that encodes sus protein. further expression study however is required to prove that the bfsus gene actually expresses sucrose synthase protein in b. flabellifer. the sequence of partial bfsus has been submitted to the genbank with accession number gq265926. materials and methods plant material young leaves were picked from the apical shoot of 35-40 years age-plant of palmyra palm (borassus flabellifer) grown in lasiana shore area in kupang, east nusa tenggara. leaves samples were cleaned, wrapped, and immediately stored in cold condition before being taken to bandung. the whole processes of sampling were done with rnase-free standard work procedure. leaves samples were then frozen in liquid nitrogen and were stored in -800c refrigerator before rna isolation. all the laboratory works took place in the laboratory of genetics and molecular biology, in sith-itb, bandung. rna isolation total rna of b. flabellifer was extracted from the fresh young leaves that were previously stored in -80ºc. all chemicals were diluted in rnase-free water and prepared using rnase-free equipments. all equipments were formerly treated with dietylpyrocarbonate (depc) before use. grinding equipment, such as mortar, pestel, and scissors were chilled before use, either by storing in -80ºc or by soaking in liquid nitrogen. five protocols have been tested to optimize rna isolation method from young leaves of b. flabellifer. those are method of seki et al. (2002), method of khemvong and suvachittanont (2005), modified-method of apt et al. (1995), trizol-modified (i) method, and trizol modified (ii) method (invitrogen). quality of rna is determined by absorbance ratio in 260 nm and 280 nm uv light in the range of 1.80 – 2.00 which indicated low contamination of protein. appearance of two typical ribosomal rna bands, 18s and 28s, in electrophoresis gel also indicated good quality of rna. the quantity of rna was determined by rna yield (µg) per total solution volume (ml). primer design sequence of b. flabellifer sus gene is currently unavailable in the genbank, thereby pair of primer was designed from highly conserved region of sus proteins among monocot plant species. sucrose synthase protein sequences of oryza sativa, zea mays, saccharum officinarum, and other monocots, were accessed from ncbi genbank database (www.ncbi.nlm.nih.gov). multiple sequence alignment of sus proteins was conducted using clustal x software. the most conserved regions in sus proteins among those species were chosen for primer sequence 7 determination. primer sequences were deduced from those regions by choosing a particular group of species whose cdna sequence is highly conserved. this was accomplished by bioedit and codehop programs. nucleotide number 412 to 432 of sus gene of zea mays was chosen as forward primer; while reverse primer was taken from nucleotide number 2277 to 2252. primer physical characteristics were measured by sigma-genosys primer calculator. specificity of both primer pairs were tested by nucleotide blast program (altschul et al., 1997). the chosen primer sequences showed eligible characteristics, and more importantly, exhibited high specificity for sus gene (table 2). reverse transcription-pcr isolation of sucrose synthase (sus) gene from b. flabellifer was accomplished by two step reverse-transcription polymerase chain reaction (rt-pcr) method. in the first step, total cdna is synthesized from the total rna by using superscript ii cdna synthesis kit (invitrogen). in the second step, pcr amplification using sus gene-specific primers was carried out to specifically amplify fragment of sus gene from the previously isolated cdna. total cdna synthesis total cdna synthesis of b. flabellifer was conducted using superscript ii cdna synthesis kit (invitrogen). composition of each component in cdna synthesis reaction is described in table 3. all solutions are diluted in depc-treated deionized water. all steps were carried out according to rnase-free standard work procedure to avoid or minimize degradation of the rna. a master mix solution was prepared by mixing buffer rt with mgcl2, dithiothreitol (dtt), and rnase out tm recombinant rnase. separately, rna sample was mixed with oligo dt primer and dntps prior to five minutes incubation in 65ºc and then one minute in ice. the master mix was then added to the rna sample mixture and then diluted with depc-treated water to 20 µl final volume. this final mixture was incubated for two minutes in 420c for primer annealing. superscripttm iireverse transcriptase was subsequently added to the mixture, gently homogenized, and then incubated for 50 minutes in 42ºc for complete reaction of cdna synthesis. the reaction was terminated by 15 minutes incubation in 70ºc. to eliminate any remaining rna strands in the cdna solution, rnaseh were added to the solution and then incubated in 370c for 20 minutes. total cdna of b. flabellifer young leaves was obtained at the end of this step. pcr amplification for isolation of sus gene a standard pcr amplification technique was conducted following the cdna synthesis to specifically isolate the sus gene. optimization of pcr profile, especially annealing temperature, was carried out to find the best condition that favor specific amplification of the sus gene fragment. a range of annealing temperature, from 45ºc to 55ºc, was tested. concentration of cdna template, ranging from 0.5 to 2 µg /50 µl, and mg2+, ranging from 1 to 2.5 mm, were tested as well to obtain best fragment of the target gene. the pcr reaction was carried out using a long pcr enzyme mix product (fermentas). composition of the pcr mixture is described in table 4. gene cloning and transformation of e. coli the product of rt-pcr was observed by electrophoresis using 1% agarose gel. detected sus fragment was purified from the agarose gel and then cloned to the pgem-t easy® vector by employing t4 ligase enzyme. to amplify the sus fragment, the recombinant plasmids were subsequently delivered into bacterial cell of escherichia coli strain dh5α using heat-shock 8 treatment. the transformed cells of e. coli that contain recombinant plasmid were selected through screening of blue-white colony. the transformed cells were cultured for 16 hours in luria-bertany (lb) media containing 0.1 ng/µl ampicillin. the plasmids dna of the transformed cells were then extracted by alkaline lysis method (sambrook et al., 1989). insertion of sus fragment into plasmid was confirmed by cutting the plasmid with two endonuclease restriction enzymes, ecori and noti. sequencing and characterization of bfsus gene and polypeptide sus putative fragment that have been cloned to pgem-t easy plasmid were further analyzed by dideoxy chain-termination method to read the nucleotide sequence of the isolated fragment. minimum 150 ng/µl of plasmid dna is required to a single sequencing reaction. since the 1866 bp gene fragment is relatively long for a single accurate reading by the sequencer system, two steps of sequencing are needed to accomplish the total fragment reading. the first sequencing employed t7 and sp6 universal primers, which followed by second sequencing using gene internal–primers. gene-internal primers were designed from the first sequencing result. sequencing result from those four primer directions were finally assembled using geneious 4.6.2 program. all sequencing process was conducted by macrogen inc. in south korea. characterization of bfsus putative gene comprises nucleotide and polypeptide sequence analysis. the nucleotide sequence of the putative bfsus was aligned with sus cdna sequences deposited in genbank (www.ncbi.nlm.nih.gov) using blastn program in order to find their similarity. the bfsus cdna sequence was then translated into bfsus polypeptide using geneious 4.6.2, followed by searching in the genbank using blastp program. the highly conserved regions, including essential domains and motifs, were searched within bfsus putative polypeptide, either by employing geneious 4.6.2 program or by comparing the polypeptide with other related proteins that have been previously annotated. other proteins from the same family are preferable since it is expected to share similar properties with the sus. to reveal genetic relationship between borassus flabellifer and other plant species based on their sus polypeptide sequence, a neighborjoining (nj) phylogenetic tree was generated using geneious 4.6.2 tree builder. conclusion a partial cdna fragment of sucrose synthase (sus) putative gene has been successfully isolated from the young leaves of b. flabellifer, henceforth named as bfsus. the sequence has been submitted to the genbank with accession number gq265926. the sequence of bfsus cdna, and the corresponding bfsus polypeptide, is highly similar with sus cdna and sus protein from various plant species recorded in genbank, with highest similarity shown by tulipa gesneriana and oncidium sp. the bfsus putative polypeptide performs all typical characteristics of sus protein, including most conserved domains and motifs. presence of sucrose synthase (sus) and glycosyl transferase (gt) domains, four putative udp-glucose binding pockets within the gt domain, and a calcium-dependent ser/thr protein kinase binding site (ser170) within the sus domain, confirms that the bfsus cdna fragment obtained in this study is most likely encodes sucrose synthase in b. flabellifer. this also assuring that bfsus gene is expressed in the young leaves of b. flabellifer. 9 table 1. summary of five tested methods for rna isolation from young leaves of b. flabellifer. trizolmodified ii found to be the best method for rna isolation from the young leaves of b. flabellifer *) rna yield = (absorbance in 260 nm) x (42.5 µg/ml) x (dilution factor) figure 1. comparison of rna quality obtained by the five tested methods. two bands of ribosomal rna, the 18s and 28s rrna, that were occurred in electrophoresis gel indicated that total rna of b. flabellifer has been successfully obtained. trizol-modified ii method is the best method for rna isolation from the young leaves of b. flabellifer. figure 2. a single cdna fragment of 1866 bp, generated by reverse transcription-pcr, predicted as the target fragment of the sus gene of b. flabellifer. m e t h o d ratio of uv absorbance (a260/a280) rna quality in gel electrophoresis rna yield (µg/ml) *) total time (hour) trizol-modified i (invitrogen) 1.23 – 1.24 average 114.75 5 seki et al., 2002 1.44 – 1.47 poor 93.50 8 khemvong &suvachittanont (2005) 1.67 – 2.00 poor 44.625 12 apt et al. (1995)-modified 1.45 – 1.47 good 148.75 18 trizol-modified ii (invitrogen) 1.61 – 1.62 good 1600 4 28s rrna 18s rrna 1 2 3 5 4 2000 bp 1500 bp 1866 bp 10 figure 3. (a) the recombinant plasmid cut with ecori generated two unexpected ± 1300 and ± 500 bp fragments indicating gene internal cut by this enzyme; (b) plasmid cut with noti generated a single expected fragment of length ±1866 bp, indicating that sus gene of b. flabellifer had been successfully inserted in the mcs region of the plasmid; (c) pcr screening using sp6 and t7 primers resulted in ±1995 bp fragment confirmed the presence of sus fragment within the mcs region of the pgem®-t easy plasmid. table 2. pair of forward and reverse primers used to isolate sucrose synthase gene from total rna of borassus flabellifer young leaves. primer code primer sequence start primer length product size (bp) gc (%) tm (ºc) sec. struc ture blast output (σ species) susfw cttgagctggactttgagcca 412 21 52.38 62.92 weak 10 susrv cttccaggtgtacttctcctcgat ac 2277 26 1866 50.00 62.72 weak 8 table 3. chemical composition in cdna synthesis no. component final concentration 1 total rna of b. flabellifer 2 µg/20 µl 2 dntps 0.5 mm 3 oligo (dt)12-18 primer 0.025 µg/µl 4 buffer rt 1x 5 mgcl2 5 mm 6 dithiothreitol (dtt) 0.01 m 7 rnase outtm recombinant rnase 1 u/20 µl 8 superscripttm iireverse transcriptase 1u/20 µl 9 depc-treated deionized water until 20 µl 10 e. coli rnaseh 1 µl/20 µl table 4. composition of pcr mixture for sus cdna isolation no. component final concentration 1 10x long pcr buffer 1x 2 2 mm dntp mix 0.2 mm 3 25 mm mgcl2 2 mm 4 primer (forward and reverse) 0.2 µm 5 total cdna 0.03 µg/µl 6 long pcr enzyme mix 1.25 unit/ 50 µl 7 nuclease free water to 25 µl ± 3000 bp ± 1300 bp ± 500 bp a ± 1866 bp ± 3000 bp b ± 1995 bp c 11 12 13 figure 4. sequence of partial cdna of borassus flabellifer sus putative gene. the yellow arrow indicates forward primer, ssfw, and the green arrow indicates reverse primer, ssrv. the red arrow indicates internal primer p1, and the blue arrow indicates internal primer p2. the red box indicates restriction site of ecori endonuclease restriction enzyme. the brown box indicates putative phosphorylation site (30-rhlss34). the blue boxes indicate four putative udp binding pockets (166-dtggq-170; 444mar-446; n520; and t546) figure 5. the bfsus putative polypeptide sequence with total length 622 amino acids was generated by translating the sequence of bfsus cdna, using geneious 4.6.2. 14 figure 6. a phylogenetic tree based on sucrose synthase polypeptide sequence of various monocot and dicot species. similarity of sus polypeptide among monocot species is distinctively higher than those of dicot species. borassus flabellifer is clustered with the monocot group, although not specifically grouped with particular monocot species. b. flabellifer showed the nearest genetic distance with tulipa gesneriana. 15 figure 7. seven regions in bfsus polypeptide that highly conserved among monocot species: (1) sucrose synthase domain spanning from nterminal region to ser422; (2) a putative calcium-dependent ser/thr protein kinase binding site in the 30-rhlss-34 motif within the sus domain. this site may have role in triggering enzyme degradation via the ubiquitin/26s proteasome; (3)glycosyltransferase (gt) domain spanning from asn146 to the c-terminal region. four putative udp-binding pockets are detected within this domain. they are: (4) glycine residues in 166dtggq-170 motif, (5) 444mar446, (6) n520, and (7) t546. 16 figure 8. seven regions in bfsus polypeptide that highly conserved among monocot species: (1) sucrose synthase domain spanning from nterminal region to ser422; (2) a putative calcium-dependent ser/thr protein kinase binding site in the 30-rhlss-34 motif within the sus domain. this site may have role in triggering enzyme degradation via the ubiquitin/26s proteasome; (3)glycosyltransferase (gt) domain spanning from asn146 to the c-terminal region. four putative udp-binding pockets are detected within this domain. they are: (4) glycine residues in 166dtggq-170 motif, (5) 444mar446, (6) n520, and (7) t546. 17 figure 9. seven regions in bfsus polypeptide that highly conserved among monocot species: (1) sucrose synthase domain spanning from nterminal region to ser422; (2) a putative calcium-dependent ser/thr protein kinase binding site in the 30-rhlss-34 motif within the sus domain. this site may have role in triggering enzyme degradation via the ubiquitin/26s proteasome; (3)glycosyltransferase (gt) domain spanning from asn146 to the c-terminal region. four putative udp-binding pockets are detected within this domain. they are: (4) glycine residues in 166dtggq-170 motif, (5) 444mar446, (6) n520, and (7) t546. 18 acknowledgement we thank the southeast asian organization of tropical biology (seameo-biotrop) for funding this research. our work has been supported by a research grant form seameo-biotrop in the period of 2008-2009. our gratitude to the school of life sciences and technology for the facilities we were able to use in the laboratory of genetics and molecular biology. thanks to mikael tupa for his favor in obtaining the borassus flabellifer leaves in lasiana coastal area. we also thank dr. suminar achmadi and the biotropia reviewers for their careful reading, comments, and corrections of the manuscript. references altschul, stephen f., thomas l. madden, alejandro a. schäffer, jinghui zhang, zheng zhang, webb miller, and david j. lipman. 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(2002). monitoring the expression profiles of 7000 arabidopsis genes under drought, cold and high-salinity stresses using a full-length cdna microarray. plant journal 31: 279292. biotropia no biotropia no. 14, 1999 : 17 35 the occurrence of insects, fungi and organoleptic characteristics in stored coffee beans in lampung okky s. dharmaputra, sunjaya, ina retnowati and muhammad amad seamed biotrop, p.o. box 116, bogor 16001, indonesia cahya ismayadi the indonesian research institute for coffee and cocoa, jl. pb sudirman 90, jember 68118, indonesia abstract a survey on postharvest handling and technology processing of coffee beans at farmer, trader and exporter levels was conducted in west lampung and tanggamus regencies of lampung province during harvest time (july 1998). interviews and sampling of coffee beans were carried out during the survey. the number of respondents at farmer, trader and exporter levels was 22, 20 and 4, respectively, while the number of samples collected from each level was 20. all samples were analyzed for moisture content, physical quality, insect and fungal infestation, reducing sugar content, and coffee cupping. the results of the interviews indicated that postharvest handling and technology processing became better from farmers to exporters. moisture contents of coffee beans collected from farmers and traders were higher than the tolerable limit recommended by sni (13%). physical quality of coffee beans collected from exporters was higher than that collected from farmers and traders. insects were found on coffee beans collected from farmers, traders and exporters, but the number of species and the percentage of samples infested by insects from each level were relatively low. the predominant species was liposcelis entomophila. the number of fungal species on coffee beans collected from farmers was higher than that collected from traders and exporters. the predominant species at the three levels was aspergillus niger, but the lowest percentage of beans infected by this fungus was found on coffee beans collected from exporters. the lowest percentage of samples infected by all fungi was also found on coffee beans collected from exporters. reducing sugar content of coffee beans collected from exporters was lower than that from farmers and traders. aroma and flavor values tended to increase from farmers through traders to exporters, while the body decreased. some off-flavors (i.e. earthy, mouldy, fermented and woody) were encountered in a few coffee samples from farmers as well as from traders. there was no off-flavor encountered in the coffee samples from exporters. key words: stored products pests/postharvest handling/technology processing/moisture content physical quality/insect/fungi/reducing sugars/coffee cupping/coffee/ lampung. introduction people all over the world are all facing global competitiveness. with the advent of wider market access and perceived increase in demand for farm products, we face the responsibility of maintaining the sustainability of our resources through appropriate research program in postharvest. it would minimize postharvest losses which in turn should benefit our farmers in terms of higher productivity and incomes. food selfsufficiency can be attained by increasing production and minimizing food losses and quality degradation resulting from irjefficient postharvest handling. 17 biotropia no. 14, 1999 coffee is one of the important export commodities in indonesia. licht (1995) reported that indonesia is the biggest coffee producer in asia. based on data from the central bureau of statistics (1996) between january september 1996; indonesia has exported 253 027 tons of coffee beans. most jf coffee beans production is derived from smallholders. during storage, coffee beans could be infested by insects, mites, microorganisms and rodents. insects are considered the most significant cause of losses. among microorganisms, fungi are the most important cause of deterioration of stored products. the role of insects on fungal infection cannot be disregarded. aside from injuring seeds, insects also serve as carriers of fungi. furthermore, the metabolic activities of insects produce enough heat and moisture (specially during longterm storage) to allow fungal growth. no intensive study has been carried out on the occurrence of insects and fungi on stored coffee beans in indonesia. the objective of this study was to get information on postharvest handling and technology processing of coffee beans at farmer, trader and exporter levels in lampung province. the moisture content, physical quality, pest infestation (insect and fungi), reducing sugar content and coffee cupping (organoleptic test) of coffee beans were also analyzed. materials and methods time and location of surveys a survey was conducted during harvest time (july 1998) in two regencies, i.e. west lampung regency (fajar bulan and sumber jaya villages) and tanggamus regency (data-rajan, air naningan, way harong, gunung megang and tekad villages). these regencies were selected because they produce large quantities of coffee beans. interviews using questionnaires interviews were carried out during the survey to get information on postharvest handling and technology processing of coffee beans at farmer, trader and exporter levels. the number of respondents from each level was different depending on the condition in the field during the survey. the number of respondents from farmers, traders and exporters was 22, 20 and 4, respectively. the questionnaires contained questions on postharvest handling and technology processing carried out by farmers, traders and exporters, and problems encountered by them. sampling methods the number of samples taken from each level was 20. not all samples were derived from the respondents. about 1 kg of each sample was taken randomly from 18 the occurrence of insects, fungi and organoleptic characteristics okky s. dharmaputra et al. farmer, trader and exporter levels. samples of coffee (primary samples) from each stack were taken randomly from a certain number of sacks using a sampling spear. insects were separated from each primary sample using graded sieves. each primary sample was divided several times using a sample divider to obtain working samples for analyzing moisture content, physical quality, fungi, reducing sugars, organoleptic test and reserve sample. the presence of insects was estimated within the sacks. moisture content analysis moisture content of coffee beans (wet weight) was determined by the oven method (isc 1992). two replicates were used for each sample. the beans were ground and dried in the oven at 130 ± 2°c for 6 hours (first period), and then they were placed in a desiccator for,a night. they were re-dried in the oven at 130 ± 2°c for 4 hours (second period). the moisture content was determined using the formula: moisture content = p, + where : p, is the first period moisture content (in 100 g of sample) ?2 is the second period moisture content (in 100 g of sample) physical quality analysis physical quality analysis was determined based on sn1 01-2907-1992 (isc 1992). quality level of coffee bean samples was determined based on the number of defective beans from 300 g of sample. quality classification based on defective beans system and determination of the number of defective beans are shown in tables 1 and 2. table 1. quality classification based on defective beans system grade criteria of grade grade 1 maximum number of defective beans 11 grade 2 number of defective beans 12-25 grade 3 number of defective beans 26 44 grade 4-a number of defective beans 45-60 grade 4-b number of defective beans 61-80 grade 5 number of defective beans 81-150 grade 6 number of defective beans 151 225 notes : the number of defective beans was determined based on table 2. 19 biotropia no. 14, 1999 table 2. determination of the number of defective coffee beans no. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. kind of defective 1 (one) black bean 1 (one) a half of black bean 1 (one) broken black bean 1 (one) cherry bean 1 (one) brown bean 1 (one) big husk 1 (one) middle husk 1 (one) small husk 1 (one) bean with hull 1 (one) big hull 1 (one) middle hull 1 (one) small hull 1 (one) broken bean 1 (one) immature bean 1 (one) bean with one hole 1 (one) bean with more than one hole 1 (one) big branch, soil, or gravel 1 (one) middle branch,soil, or gravel 1 (one) small branch, soil, or gravel defective value 1 (one) '/2 (a half) '/2 (a half) 1 (one) 1/4 ( a quarter) 1 (one) !/2 ( a half) 1/5 (one fifth) '/2 ( a half) '/2 ( a half) 1/5 (one fifth) 1/10 (one tenth) 1/5 (one fifth) 1/5 (one fifth) 1/10 (one tenth) 1/5 (one fifth) 5 (five) 2 (two) 1 (one) notes : if one bean has more than one kind of defective, the defective value was determined based on the highest defective value. insect and fungal analyses the insects were identified using haines (1991) as the main reference. fungi were isolated using direct plating method on dichloran 18% glycerol agar (dg18) (pitt and hocking 1997). before plating, samples were individually disinfected using 1% sodium hypochlorite for 1 minute. one hundred beans were then plated (10 beans/plate) on the medium and incubated at 25°c for 7 days. the fungi were identified using samson et al. (1996), and pitt and hocking (1997) as the main references. reducing sugars analysis reducing sugars (dry weight) was determined according to somogyi nelson's method (mccready 1970). in alkalic condition, reducing sugars reduce cu 2+ to cu+. alkalies give a specific color if added with arsenicmolibdat and the absorbency of the color was measured by spectrophotometer on wave length 510 nm. reducing sugars was determined using standard curve and glucose as a reference. 20 the occurrence of insects, fungi and organoleptic characteristics okky s. dharmaputra et al. organoleptic test analysis coffee cupping was determined according to lingle's method (1986). coffee bean samples were roasted using probat roaster type pre-2 at 200°c until medium roast level (color degree of # 55 agtron/specialty coffee association of america). roasted coffee was then ground at medium grind degree. one hundred fifty ml boiling water was added to about 10 g of roasted and ground coffee. the test was carried out by 4 experienced panelists on aroma, flavor, body, and defect characteristics. evaluation for each character was determined by numerical system (scoring): 0 = none, 1 = low, 2 = low-medium, 3 = medium, 4 = medium-high, 5 = high, respectively. results and discussion interviews with farmers, traders and exporters a. farmer level the results of the questionnaires are shown in appendix 1. most of the fanners (77.3% respondents) cultivated only robusta coffee, while others (22.7%) cultivated arabica and robusta coffee, especially in west lampung. determination of harvest time was based on visual observation of the color of the berry. the ripeness of coffee berry is indicated by its red color. nevertheless, strip methods of harvesting was carried out when the number of ripe berries was about 80%. the drying process was carried out by farmers. most of the farmers (63.6%) dried the coffee directly without opening the berries, and consequently they stored the coffee in the form of dried coffee berries. the others (36.4%) opened the berries using a machine before drying. the final form obtained was called green coffee. applying this method, coffee beans would dry faster, and thus farmers could sell them faster. the drying facilities used by farmers were varied, i.e. on the ground, on a plastic (polypropylene) sheet and on paved floor. farmers revealed that in general there was no problem with insects as well as fungi in the storage. b. trader level the results of questionnaires are shown in appendix 2. traders bought coffee beans from farmers, although the moisture content was still high (20-30%). in this case, the farmers gave a discount for the undesirable moisture content of the coffee beans. most of traders (85.7%) further sun-dried their coffee beans for less than 1 day or only several hours. 21 biotropia no. 14, 1999 traders dried coffee beans on polypropylene sheet (42.9%) and paved floor (57.1%). in general, traders stored the beans for not more than 2 days by spreading them on the floor in their warehouse/store (65%) and the beans were packed using plastic bags (30%). most of traders (60%) sorted and selected the beans that will be sold to exporters, because exporters classified the price of beans based on moisture content and physical quality (i.e. percentage of extraneous matter, hulls, beans with holes and black beans). most of the traders did not have any problem with insects and fungi. nevertheless, if mouldy beans were found, no action was taken by 65% of the respondents. exporter level the results of questionnaires are shown in appendix 3. exporters in lampung obtained coffee beans from traders in lampung province, java and other provinces in sumatera. determination of price classification was based on moisture content and physical quality of the beans. the beans were packed in jute bags (91.7%) and plastic bags (8.3%) before reprocessing (further dried, sorted and sifted). the duration of storage was usually less than 1 week. further drying was carried out using a mechanical dryer up to moisture content of 12-13%. sorting was conducted using an electronic sorting machine (sortex) to discard extraneous matter and black beans. after sorting, grade quality could be determined. the beans were then packed in jute bags or containers, fumigated, and finally exported. generally, fumigation was conducted by private companies in warehouses or in the containers depending on customer demand. the fumigant used was methyl bromide (87.5% of respondents) or phosphine (12.5%) depending on customer demand and importing countries. all exporters monitored insect pest infestation. most of them (75%) did not have any problem with insect pests, while the others (25%) did. moisture content and physical quality moisture content of coffee beans collected from farmers (16.1 ± 1.3%) and traders (16.5 ± 3.3%) were higher than the tolerable limit recommended by sni (13%). all the samples (100%) collected from farmers has a moisture content of more than 13%. similar moisture content was obtained from 90% samples collected from traders (table 3). based on statistical analysis, moisture content of coffee beans from farmers was not significantly different compared to coffee beans collected from traders, but that from traders was more varied. moisture content of coffee bean samples from exporters (12.6 ± 0.5%) (table 3) was lower compared to samples from farmers and traders, but 20% of the total samples collected from exporters still exceeded the tolerable limit recommended by sni. exporters re-processed the coffee from traders to meet the standard. the 22 the occurrence of insects, fungi and organoleptic characteristics okky s. dharmaputra et at. table 3. moisture content and physical quality of coffee beans collected from farmers, traders and exporters source of coffee beans moisture content (%) grade of quality (% samples) number (%) of sam ples with moisture content > 13% farmers 16.1 ± 1.3 a 5 (10), 6 (90) 20 (100) traders 16.5 ± 3.3 a 6 (100) 18 (90) exporters 12.6 + 0.5 b 4-a(10),4-b(15), 5 (20), 6 (55) 4(20) numbers followed by the same letter did not differ significantly according to tukey's test at 95% confidence level notes : grade 4-a : samples of coffee beans with the number of defective bean 45-60 grade 4-b : samples of coffee beans with the number of defective bean 61—80 grade 5 : samples of coffee beans with the number of defective bean 81 150 grade 6 : samples of coffee beans with the number of defective bean 151 225 process included drying, mixing, grading and packaging. exporters dried coffee beans using mechanical dryers. the temperature of the dryer was set at 200°c or more; consequently, the moisture content of coffee beans from farmers and traders could be decreased from 16% up to about 13% within a short period. based on the number of defective beans, the quality of samples from traders was not better, it even tended to be of lower quality (100% of samples consisted of the lowest grade 6) compared with that from farmers (10% of samples consisted of grade 5 and 90% of samples consisted of grade 6) (table 3). traders bought coffee beans from farmers against the price which is based on moisture content, number of defective beans and extraneous matter. defective beans (black beans, brown beans, immature beans, broken beans and cherries) and the extraneous matter were counted arbitrarily by the traders. most of traders possessed an electronic moisture tester (generally cera tester) which is used to determine the moisture content for pricing. nevertheless, the tester had never been calibrated, thus the result of determination is unreliable. traders mixed the coffee from farmers. as a consequence the mixing of high as well as low quality coffee resulted in downgrading. in general, the physical quality of coffee beans from exporters was better (10% of samples consisted of grade 4-a, 15% of samples grade 4-b, 20% of samples grade 5 and 55% of samples grade 6) than that from farmers and traders (appendix 2). exporters sorted coffee beans obtained from traders in order to reduce the defects especially black beans and beans in cherry. the sorting process was conducted using electronic sorting machine. in addition, the coffee beans were also sieved with screens to get a uniform size and clean beans. 23 blotropia no. 14, 1999 the presence of insects haines (1991) reported that insect species associated with stored coffee beans were araecerus fasciculatus, hetebostrychus brwmeus and prostepanus trunchatus. the number of insect species found in coffee bean samples collected from farmers, traders and exporters, and the percentage of samples infested by the insects were relatively low (table 4). similar results were reported by the respondents. table 4. insect species found on coffee beans collected from farmers, traders and exporters 0% samples infested by insects insect species farmers traders exporters ahasverus advena 0 5 0 araecerus fasciculatus 0 0 5 liposcelis bostrycophila 10 0 30 l. enlomophila 10 10 40 sitophilus zeamais 0 5 0 in this study, insect species found on coffee beans collected from farmers were liposcelis bostrycophila (10% of samples) and l. entomophila (10%) (table 4). this result showed that insect infestation on coffee beans from farmers was relatively low, because farmers did not store the beans for a long period. the results of interviews using questionnaires showed that only 14.3% of farmers stored coffee beans for more than 3 days (appendix 1). haines (1995) reported that l. entomophila was found on 50% of samples of stored coffee beans in java and sumatera. in a tropical country like indonesia, the predominant psocid (insect of genera liposcelis) found were l. bostrycophila and l. entomophila (leong and ho 1991;syarifandhalid 1993). the role of psocids is not well understood, as to whether these insects eat or cause deterioration of stored commodities. psocids are often found in humid warehouses (syarief and halid 1993). according to sidik and cahyana (1992), high populations of liposcelis spp. made the warehouse dirty; consequently working activities were disturbed. recently it was found that liposcelis spp. were real stored product pests. their heavy infestations on stored grains occurred in indonesia, india and australia (roesli et al. 1998). the occurrence of l. bostrycophila and l. entomophila on coffee beans at the farmer level was due to the high moisture content of the beans (16.1 ± 1.3%) and the sanitary conditions of the storage facility which were not managed properly. in indonesia, populations of psocids on rice have increased drastically several months after harvest (santoso et al. 1996). three insect species were found on coffee beans at the trader level, namely ahasverus advena (5% of samples), l. entomophila (10%) and sitophilus zeamais 24 the occurrence of insects, fungi and organoleptic chaiaeteristics okky s. dharmaputra et al. (5%) (table 4). in general, s. zeamais is an insect pest found on milled rice, maize and pulses. the presence of the three insect species on coffee beans was due to migration, because the beans were stored in the stores located at traditional markets which sell milled rice and maize. ahasverus advena is a fungus feeder. its presence was due to high moisture content of coffee beans (10.5-22.7%). according to haines (1991), a. advena often found on damaged and mouldy commodities in storage is an indicator of wet storage facility. three insect species were also found on coffee beans at the exporter level. they were araecerus fasciculatus (5% of samples), liposcelis bostrycophila (30%) and l. entomophila (40%) (table 4). l. bostrycophila and l. entomophila were more often found at the exporter level than at the farmer and trader levels. this was because coffee beans stored by exporters were derived from farmers and traders and infested by those insects, resulting in their presence in greater numbers on the beans at the exporter. besides that, frequent fumigation carried out by exporters affected the population of liposcelis spp. according to roesli et al. (1998), improper fumigation, i.e. shorter exposure period or too low gas concentration, caused an increase in populations of these insects. a. fasciculatus is a major pest of stored coffee beans. its presence caused damage to the beans, thus decreasing the selling value. haines (1991) revealed that the presence of a. fasciculatus on export commodities such as coffee and cocoa beans could decrease the selling value, although the damage might not be heavy. this insect was also found on stored products in lampung and south sumatera (haines 1995). the presence of fungi based on an interview, most of the respondents said that they had no problem with fungi. their opinion was only based on visual observation. nevertheless, based on fungal isolation using dg18, all beans were infected by fungi. fifteen fungal species were isolated on coffee beans collected from farmers. they were aspergillus flavus , a. niger, a. ochraceus, a. penicilloides, a. tamarii, a. versicolor, a. wentii, cladosporium cladosporioides, eurotium amstelodami, e. chevalieri, fusarium semitectum, lasiodiplodia theobromae, mucor hiemalis, penicillium citrinum and wallemia sebi. a. niger was the dominant fungus isolated, having been found in 100% of all samples, and 83.5% of all beans examined. a. flavus was the next most common fungus, being present in 90% of the samples, but only 12.3% of all beans examined. p. citrinum was present in only 15% of the samples and infected 18.7% of all beans examined (table 5). ten fungal species were isolated on coffee beans collected from traders. they were a. flavus, a. niger, a. ochraceus, a. penicilloides, a. tamarii, a. versicolor, a. wentii, e. chevalieri, l. theobromae and p. citrinum. a. niger was also the dominant fungus isolated, being found in 100% of the samples, and 94.9% of all beans 25 biotropia no. 14, 1999 26 the occurrence of insects, fungi and organoleptic characteristics okky s. dharmaputra et al. examined. a. flcrvus, a. ochraceus, l. theobromae and p. citrinum were all present in more than 75% of the samples, but their infection on all beans examined were relatively low (14.1, 11.6, 2.3 and 30.7%, respectively) (table 5). eleven fungal species were isolated on coffee beans collected from exporters. they were a. flavus , a. niger, a. ochraceus, a. tamarii, a. wentii, c. cladosporioi-des, e. chevalieri, f. semitectum, l. theobromae, m. hiemalis and p. citrinum. the most commonly occurring species was a. niger, present in 95% of samples, and 60.1% of all beans examined. a. flavus and p. citrinum were found in 100 and 75% of samples, respectively, but infected only 11.9 and 3.7%, respectively, of all beans examined (table 5). according to scott (1994) and ono et al. (1995) a. niger and a. ochraceus could produce ochratoxin a. this toxin caused a swelling of the kidneys (nephropathy) in domestic livestock and poultry, and it has been considered as the causative agent responsible for human endemic nephropathy prevalent in areas of the balkan countries (prelusky et al. 1994) and tunisia (ccfac 1998). among the three levels, the number of fungal species isolated from coffee beans at the farmers level was the highest, because moisture content of all sample was more than 13% (table 3). the dominant fungus isolated at the three levels was the same, i.e. a. niger, but the lowest infection of all beans examined among the three levels was found at exporter level, because the exporters further dried and sorted coffee beans obtained from farmers and traders. moreover, among the three levels the lowest percentage of samples infected by all fungi was obtained from the exporter level (70.7%), while that at farmer and trader levels were not significantly different to each other (99.1 and 99.0%, respectively) (table 6). nakajima et al. (1997) reported that the dominant fungus isolated on coffee beans was a. niger. table 6. percentage of samples infected by all fungi on coffee beans collected from farmers, traders and exporters source of coffee beans samples infected by all fungal species (%) farmers' 99.1 a traders 99.0 a exporters 70.7 b numbers followed by the same letter did not differ significantly according to tukey's test at 95% confidence level reducing sugar content sugars are important components in the formation of flavor, color/pigment, caramelization and condensation products of roasted coffee. the major free sugar in coffee beans is sucrose, which reaches 6.1% in arabica coffee and 3.4% in robusta 27 biotropia no. 14, 1999 coffee (trugo 1985). besides sucrose, coffee beans also contain simple sugars including reducing ones, but in small amounts. fructose and glucose in arabica coffee constitute 0.030-0.038% and 0.023-0.030%, respectively, while in robusta coffee 0.16-0.18% and 0.19-0.21%, respectively. reducing sugars in arabica and robusta coffees are only 0.1 and 0.5%, respectively (trugo 1985). analysis of reducing sugars in the samples resulted in 2.7 ± 0.3% for coffee from farmers, 3.2 ± 0.6% for coffee from traders, and 2.3 ± 0.2% for coffee from exporters (table 7). those results were higher than the reported data. coffee beans from exporters had the lowest value compared to the coffee from farmers and traders. those lower values were probably due to the coffee that had been further dried at quite high temperature, a condition which makes the sugars caramelized or condensed through the maillard reaction, hence the amount was reduced. according to winarno (1991) caramelization of sucrose occurred when the temperature is higher than the melting point (160°c); the maillard reaction is a reaction among carbohydrates, especially of reducing sugars with primary amine groups yielding pleasant brown compounds. table 7. reducing sugar contents of coffee beans collected from farmers, traders and exporters source of coffee beans reducing sugar content (%) farmers 2.7 ± 0.3 b traders 3.2 ± 0.6 a exporters 2.3 ± 0.2 c numbers followed by the same letter did not differ significantly according to tukey's test at 95% confidence level coffee cupping (organoleptic test) aroma and flavor characters of all coffee samples were termed as low-medium to medium, while their body character ranged from medium to full (table 8). coffee from lampung is known for its unique taste with full bodied and unclean flavor. the unclean flavor is also frequently found in dry processed coffee beans. some very low off-flavors like earthy, musty, and mouldy shadowing the coffee flavor resulted in a unique character. aroma and flavor values tended to increase for the coffee at farmer (2.4 + 0.4 and 2.5 + 0.5, respectively), trader (2.8 ± 0.3 and 2.6 ± 0.3, respectively), and exporter levels (2.9 ± 0.1 and 2.9 + 0.2, respectively), while the body decreased with values of 4.3 ± 0.5, 3.6 ± 0.2, and 3.6 ± 0.2 for each coffee origin (table 8). the aromatic characters of coffee beans from exporters was the strongest, it was probably due to caramelization during the drying process which further produced specific aroma. 28 the occurrence of insects, fungi and organoleptic characteristics okky s. dharmaputra etal. tables. results of coffee cupping (organoleptic test) on coffee beans collected from farmers, traders and exporters source of coffee beans aroma flavor body off-flavor (% of samples) farmers 2.4 ±0.4 2.5 ±0.5 4.3 ±0.5 earthy : 40 mouldy : 5 fermented : 5 traders 2.8 + 0.3 2.6 + 0.3 3.6 + 0.2 earthy : 35 woody : 5 exporters 2.9 ±0.1 2.9 ±0.2 3.6 + 0.2 score: 0 = none, 1 = low, 2 = low-medium, 3 = medium, 4 = medium-high, 5 = high some off-flavors were encountered from few coffee samples from farmers as well as from traders, i.e. earthy (35% of samples), mouldy (5% of samples), fermented (5%), and woody (5%). there was no off-flavor encountered in the coffee samples from exporters. it was due to the mixing of defective beans with good beans that the off-flavor characters decreased and could not be detected by the panelists. the soil odor was absorbed by fat in the beans causing an earthy flavor. the off-flavor was also caused by fungal infection, since some fungi could produce chemical compounds with earthy flavor, like 2-methylisoborneol and geosmin. the earthy off-flavor was higher in samples collected from farmers, since most farmers dried their coffee beans on the ground with no underlayer or mat used. this made the coffee cherry become dirty and the beans were covered with soil dust after de-hulling. drying of coffee directly on the ground takes a longer time, since the moist ground hampers evaporation. it makes the coffee mouldy and tainted with the earthy and mouldy characters. the off-flavor could be avoided if the drying process was conducted on a sheet or mat, or on patio made of concrete. sanitation along with the drying process should be emphasized, since the dirty coffee would be infected by fungi. mouldy off-flavor is also referred to as musty, which is an odor taint giving the coffee beans a mouldy odor, a result of fats in the coffee beans absorbing organic material from fungi during the drying process. this off-flavor is more pronounced than the earthy off-flavor. the defect may also appear during storage, if the coffee beans had not been dried properly. if the coffee beans from farmers and traders had a high moisture content (± 16%), it could be easily infected by fungi, especially the toxigenic fungi (see fungal analysis). once the taint occurred, it would remain permanently. in order to avoid this defect, it is suggested that the coffee be dried at the very early stage of production (on the farm) properly, and the process be continued until the moisture content reaches below 13%. it is advisable not to store the coffee beans with high moisture contents. 29 biotropia no. 14, 1999 fermented off-flavor is an undesirable taste in the coffee beans that produces a strong unpleasant sour sensation or sting on the tongue. this defect is caused by the coffee cherries that had been kept or heaped for days, thus making sugars in the beans fermented and produce sour substances. fresh cherries could be easily fermented, especially the pulp and mucilage parts. the increase in temperature and microorganisms present in the cherries will hasten the fermentation. the sour substances produced would be absorbed into the beans and taint the coffee. in order to avoid this defect, the fresh cherries had to be processed immediately. the fresh cherries should be dried on the patio soon after harvest. during the drying process, the cherries were frequently turned upside-down to hasten the process and to get more uniformly dried beans. re-wetting by the rain should also be avoided. no off-flavor was encountered in the coffee samples from exporters which showed quality improvement of the coffee beans collected from farmers and traders after being re-processed. the improvement was due to fuijher drying, sorting out of bad beans, and mixing of coffee lots. the mixing of perfect and defective coffee beans could neutralize or dilute the off-flavor which makes the percentage of off-flavor beans present become very low and undetectable by panelists. conclusions most farmers cultivated robusta coffee and they carried out the drying process to obtain commercial coffee beans. postharvest handling and technology processing methods became better from farmers to exporters. physical quality, pest infestation (insect and fungi), moisture content, reducing sugar content and coffee cupping of coffee beans also became better from farmers to exoorters. recommendations extension and dissemination of good handling practices for coffee beans especially at farmer and trader levels are necessary to improve the coffee postharvest handling and technology processing. acknowledgment the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the plantation office province level, lampung and association of indonesian coffee exporters, bandar lampung, for their information and cooperation during the surveys at farmer, trader and exporter levels in lampung. the authors are also grateful to the indonesian research institute for coffee and cocoa, for their collaboration in this study. 30 the occurrence of insects, fungi and organoleptic characteristics okky s. dharmaputra etal. references central bureau of statistics. 1996. summary bulletin of cbs. jakarta. ccfac. 1998. revised position paper on ochratoxin a. paper presented at the 30"' session of the codex committee on food additives and contaminants. the hague, the netherlands, 9 1 3 march 1998. haines, c.p. 1991. insects and arachnids of tropical stored products, their biology and identification (a training manual). natural resources institute, uk. haines, c.p. 1995. insects and arachnids in indonesian food stores-biodiversity in a man-made environment. proceedings of the symposium on pest management for stored food and feed, bogor, indonesia, 5-7 september 1995. p: 95 125. indonesian standardization council. 1992. indonesian national standard. sni 01-2907-1992. coffee. leong, e.c.w. and s.h, ho. 1991. research on liposcelis bostrycophilus and liposcelis entomophilus (psocoptera: liposcelidae). in j.o. naewbanij and a.a. manilay (eds.). proceedings of the 14"' asean seminar on grain postharvest technology, 5-8 november 1991, metro manila, philippines, p: 317-327. licht, p.o. 1995. international coffee yearbook. world coffee production. lingle, t.r. 1986. the coffee cupper's handbook: a systematic guide to sensory evaluation of coffee's flavor. coffee dev. group, washington d.c. mccready, r.m. 1970. monosaccharides. in m.a. joslyn (ed.). methods in food analysis physical, chemical and instrumental methods of analysis, 2"d ed. academic press, new york. p. 475 509. nakajima, m., h. tsubouchi, m. miyabe, and y. ueno. 1997. survey of aflatoxin bi and ochratoxin a in commercial green coffee beans by high-performance liquid chromatography linked with immunoaffinity chromatography. food and agricultural immunology (9): 77 83. ono, h., a. kataoka, m. koakutsu, k. tanaka, s. kawasugi, m. wakazawa, y. ueno and m. manabe. 1995. ochratoxin a producibility by strains of aspergiltus niger group stored in ifo culture collection. mycotoxins (41): 47 — 51. pitt, j.i. and a.d. hocking. 1997. fungi and food spoilage. blackie academic and professional publ.,london. prelusky, d.b., b.a. rotter and r.g. rotter. 1994. toxicology of mycotoxins. in j.d. miller and h.l. trenholm (eds.). mycotoxins in grains; compounds other than aflatoxin. eagan press, st. paul, minnesota, p. 359403. roesli, r., r. jones and d.p. rees. 1998. factors affecting outbreaks of liposcelis (psocoptera: liposcelidae) population in grain storage. paper presented at the 7"' international working conference on stored-product protection, beijing, china, 14-19 october 1998. samson, r.a., e.s. hoekstra, j.c. frisvad and o. filtenborg. 1996. introduction to food-borne fungi. 3th ed. centraalbureau voor schimmelcultures, baarn, the netherlands. santoso, t, sunjaya, o.s. dharmaputra, h. halid and r.j. hodges. 1996. pest management of psocids in milled rice stores in humid tropics. international journal of pest management, 42 (3): 189 197. scott, p.m. 1994. penicillium and aspergillus toxins. in j.d. miller and h.l. trenholm (eds.). mycotoxins in grain: compounds other than aflatoxin. eagan press, st. paul. p. 261 -285. sidik, m. and y. cahyana» 1992. storage pest problem and its management in indonesia. biotrop special publication, (45): 75 87. syarif, r. and h. halid. 1993. teknologi penyimpanan pangan. penerbit arcan, jakarta. trugo, l.c. 1985. carbohydrates. in r.j. clarke and r. macrae (eds.). coffee volume 1: chemistry. elsevier applied science publ. london, p: 83 114. winarno, f.g. 1991. kimia pangan dan gizi. pt. gramedia pustaka utama, jakarta. 31 biotropia no. 14, 1999 appendix 1. results of questionnaires on postharvest handing and technology processing of coffee beans at farmer level no. subject % respondents 1. type of coffee cultivated: a. robusta coffee (coffea robusta) 77.3 b. arabica coffee (c. arabica) 0 c. both types of coffee 22.7 2. determination of harvest time was based on: a. color of coffee berry 100 b. size of coffee berry 0 c. others 0 3. harvesting method of coffee berries: a. selected method 9.1 b. strip method 90.9 4. duration of storing coffee berries: a. < 1 day 0.0 b. 1-3 days 35.7 c. > 3 days 14.3 5. storing method of coffee berries: a. in bag 100 b. in another container 0 c. spreading out on the floor 0 6. opening of coffee berry before drying: a. yes 36.4 b. no 63.6 7. drying facilities: a. ground 0.0 b. plastic (polypropylene) 27.3 c. paved floor 22.7 d. other 0 8. duration of drying: a. < 1 week 31.8 b. 1-2 weeks 59.1 c. > 2 weeks 9.1 9. methods for determining dried coffee beans: a. experience/organoleptic 100 b. using equipment for measuring moisture content 0 10. type of stored coffee beans: a. dried coffee berries 63.6 b. green coffee 36.4 11. duration of storing coffee beans: a. < 1 week 95.4 b. 1-2 weeks 4.6 c. > 2 weeks 0 12. type of bag used for storing coffee beans: a. jute bag 4.6 b. plastic bag (polypropylene) 86.4 c. others " 0 d. spreading out on the floor 90 32 the occurrence of insects, fungi and organoleptic characteristics okky s. dharmaputra etal. appendix 1. (continued) no. subject % respondents 13. postharvest problems: a. pest infestation (insects and/fungi) 18.2 b. processing facility 0 c. none 81.8 14. mouldy beans problems: a. yes 27.3 b. no 2.7 15. information on postharvest of coffee: a. yes 77.3 b. no _ ___22.7 number of respondents: 22 appendix 2. results of questionnaires on postharvest handing and technology processing of coffee beans at farmer level 'no. subject %respondents 1. moisture content of coffee beans bought from farmers: a. < 12% 0 b. 12-15% 0 c. 15-20% 0 d. 20-30% 100 e. >30% 0 2. methods for determining moisture content of coffee beans a. using equipment (cera tester) 75.0 b. by estimating (organoleptic) 25.0 3. price classification of coffee beans based on: a. moisture content 40.0 b. moisture content and physical quality . 60.0 c. other criteria 0 d. no classification 0 4. drying method used: a. sun-drying 93.3 b. mechanical drying 6.7 5. drying facilities used: a. ground 0 b. plastic (polypropylene) 42.9 c. paved floor 57.1 6. duration of further drying: a. < 1 day 85.6 b. 1-2 days 7.2 c. > 2 days 7.2 7. storing method of coffee beans: a. packed in jute bag 5.0 b. packed in plastic bag (polypropylene) 30.0 c. spreading the coffee beans on the floor 65.0 33 biotropia no. 14, 1999 appendix 2. (continued) no. subject % respondents 8. duration of storing coffee beans in warehouse/store: a. 1-2 days 70.0 b. 2 days -1 week 30.0 c. > 1 week 0 9. action conducted when mouldy beans were found: a. further dried 35.0 b. no action 65.0 10. pest problems encountered: a. insects infestation 10.0 b. fungal infection 0 c. none 90.0 number of respondents: 20 appendix 3. results of questionnaires on postharverst handing and technology proccesing of coffe beans at fanner level no. subject % respondents 1. price classification of coffee beans based on: a. moisture content 0 b. moisture content and physical quality 100 c. no classification 0 2. further dried coffee beans obtained from traders: a. yes 100 b. no 0 3. drying method used: a. sun-drying 0 b. mechanical dryer (oven/mason dryer) 100 4. moisture content of coffee beans after further dried: a. < 12% 0 b. 12-13% 100 c. > 13% 0 5. sorting coffee beans used: a. manpower/manually 0 b. electronic sorting machine (model sortex) 100 6. type of bag used for storing coffee beans before reprocessing: a. jute bag 91.7 b. plastic bag (polypropylene) 8.3 c. bulk system 0 7. duration of storing coffee beans in warehouse/factory: a. < 1 week 100 b. 1-4 weeks 0 c. > 4 weeks 0 8. pest monitoring: a. yes 100 b. no 0 34 the occurrence of insects, fungi and organoleptic characteristics okky s. dharmaputra etal. appendix 3. (continued) no. subject % respondents 9. fumigation of coffee beans before exported: a. yes 10 b. no 0 10. type of fumigant used: a. phosphine 125 b. methyl bromide 87.5 11. fumigation was carried out by: a. the exporters 0 b. private companies 100 12. fumigation site: a. warehousee 25.0 b. container 75.0 13. pest problems encountered: a. insects infestation * 25.0 b. fungal infection 0 c. rat infestation/others 0 d. none 75.0 14. complaint from importing countries: a. coffee beans were infested by insects 25.0 b. coffee beans were infected by fungi 0 c. physical quality was not good 0 d. coffee taste was not like desired 0 e. none 75.0 number of respondents: 4 35 biotropia book final.indd 110 effect of phytase supplementation in soybean meal based diet on nutrient digestibility and growth performance of green catfish (hemibagrus nemurus) yulisman, dedi jusadi* and ing mokoginta department of aquaculture, faculty of fisheries and marine sciences, bogor agricultural university, kampus ipb darmaga, bogor, indonesia abstract th is experiment was conducted to evaluate the eff ect of phytase supplemented to the diet on phosphorus (p) digestibility and growth performance of the green catfi sh hemibagrus nemurus. five kinds of experimental diets were used in this experiment, namely diets a, b, c, d and e. diet a, as a control, was supplemented with inorganic p, without phytase supplementation. diet b, c, d and e were supplemented with 0, 20, 40 and 60 mg phytase/100 g soybean meal (sbm), respectively, without inorganic p supplementation. fifteen fi sh with initial body weight of 6.9 + 0.2 g were stocked into each 60-l aquarium. fish were fed on the diets for 60 days. results indicated that p digestibility increased from 64.5% to 87.0% as phytase supplement increased from 0 mg in diet b to 60 mg phytase/100 g sbm in diet e. p digestibility in diet e was higher than that in diet a (77.6%). th e daily growth rate and feed conversion ratio followed similar trend. p, ca and zn concentration in the whole body and bone of fi sh fed diet e were higher than the fi sh fed diet b, c and d, but were insignifi cant compared to the fi sh fed diet a. nitrogen (n) and p loading by fi sh fed diet e were, respectively, 76% and 20% lower than those in fi sh fed the control diet. it is concluded that the inclusion of 60 mg phytase/100 g sbm in the diet of the green catfi sh could replace the utilization of inorganic p increase the digestibility of the diet thereby resulting in increased growth rate and reduced excretion of p and n into the waters. key words: hemibagrus nemurus, phytase, soybean meal, phosphorus. introduction th e expansion of global aquaculture production increases the demand for aquaculture feeds. for carnivore species as green catfi sh hemibagrus nemurus, protein can form up to 60% of the diet and fi shmeal is the main source of dietary protein. fishmeal is one of the most expensive and demanded ingredients and has become the main and most critical ingredient in aquafeed production. th e increasing cost and demand of fi shmeal has encouraged feed manufacturers to search for cheaper alternative protein source as plant protein. soybean meal (sbm) is a common plant protein source used as a substitute for *corresponding author: dedijusadi@yahoo.com biotropia vol. 15 no. 2, 2008 : 110 118 111 fi shmeal in the fi sh diet. it could replace 75% of fi shmeal as a protein source in the diet of the green catfi sh (pebriyadi 2004). a major obstacle in using sbm is their high phytic acid content. phytic acid is a substance that contains unavailable phosphorus (p) to monogastric animals, including fi sh, because these animals lack phytase, which can hydrolyze phytic acid (baruah et al. 2004). by using phytase supplement to the diet, the p digestibility can be improved. li et al. (2004) reported that 250 units of phytase per kg diet could eff ectively replace di-calcium phosphate supplement in the diet of channel catfi sh without aff ecting growth, feed effi ciency or bone phosphorus deposit. masumoto et al. (2001) showed that 0.2 g of phytase supplement to the diet containing 67% sbm increased the growth rate and feed effi ciency of the japanese fl ounder paralicthys olivaceus. th erefore, using phytase supplement in the fi sh diet could increase the bioavailability of phosphorus of sbm. hence, replacing the inorganic p of the diet, resulting in lower fecal p loss. th is experiment was conducted to evaluate the eff ect of phytase supplement to the diet on p digestibility and growth performance of the green catfi sh hemibagrus nemurus. materials and methods experimental diets five experimental diets were used in this experiment, namely diets a, b, c, d and e. diet a, as a control, was supplemented with inorganic p (nah2po4.h2o, kh2po4, ca2(po4)3) only, without phytase supplement. diet b, c, d and e were supplemented with 0, 20, 40 or 60 mg phytase (natuphos® 5000)/100 g sbm, respectively, without inorganic p supplement (table 1). th e proximate composition of experimental diets is shown in table 2. table 1. ingredients composition of experimental diet (g/kg diet) ingredients treatments a b c d e fish meal1 130 130 130 130 130 soybean meal1 640 640 640 640 640 fish oil 40 40 40 40 40 soybean oil 44 44 44 44 44 tapioca1 57 57 47,87 47,74 47,62 vitamin mix 15 15 15 15 15 choline chloride 5 5 5 5 5 l-methionine 5 5 5 5 5 taurin 6 6 6 6 6 mineral mix2 58 0 0 0 0 p-free mineral mix3 0 58 58 58 58 phytase 0 0 0.13 0.26 0.38 citric acid 0 0 9 9 9 p content (%): total p 1.46 0.55 0.55 0.56 0.58 water-soluble p 0.59 0.19 0.45 0.48 0.53 phytase for diet of green catfi sh (hemibagrus nemurus) – yulisman et al. 112 ingredients treatments a b c d e water-soluble p/total p ratio 40.29 34.87 82.79 86.06 91.13 1 crude protein (dry weight) concentration of: fish meal 70.39%, sbm 42.75%, tapioca meal 0.91%. 2 the mineral mix had the following composition (g/kg dry diet): nacl 0.5; mgso4.7h2o 7.5; nah2po4h2o 12.5; kh2po4 16; ca2(po4)3 14.49; fe-citric 1.25; filler 3.60 and trace element mix (0.5 g) had the following composition: znso4.7h2o 17.65; mnso4 8.1; cuso4.5h2o 1.55; cocl.6h2o 0.05; kio3 0.15; and filler 30.5 (takeuchi 1988). 3 p-free mineral mix had the following composition (g/kg dry diet): nacl 0.5; mgso4.7h2o 7.5; kcl 17.53; fe-citric 1.25; cacl2.2h2o 13.34; filler 30.5 and trace element mix (0.5 g) had the following composition: znso4.7h2o 17.365; mnso4 8.1; cuso4.5h2o 1.55; cocl.6h2o 0.05; kio3 0.15; and filler 30.5. table 2. proximate composition of experimental diets (% wet weight) proximate composition treatments a b c d e crude protein 31.19 29.49 29.30 30.11 29.19 crude lipid 10.58 10.62 10.18 10.30 9.92 ash 11.09 8.74 8.92 9.12 8.79 water 14.01 18.98 20.38 17.04 20.23 fiber 4.20 3.21 2.62 2.92 2.24 nfe1 28.93 28.96 28.60 30.51 30.87 total energy2 (kcal/100g) 392.73 383.71 377.03 390.53 387.38 energy/protein (kcal/g protein) 12.59 13.01 12.87 12.97 13.27 1 nitrogen free extract. 2 total energy (ge) was calculated based on equivalent values of: protein 5.6 kcal/g, lipid 9.4 kcal/g, and nfe 4.1 kcal/g (takeuchi, 1988). for the pretreatment of sbm with phytase, 640 g of sbm was mixed with 2240 ml water, and then phytase was added to the sbm-water mixed at 20, 40, 60 mg/100g, respectively, in the diets c, d and e. th e ph was adjusted to 5.5 with citric acid. th e mixtures were incubated at 37 °c for 2 h (matsumoto et al. 2001). th en, the mixtures were mixed with other ingredients and formed into granules. th e pellets were stored at -20 °c until use. feeding trial green catfi sh juveniles were obtained from the main center for freshwater aquaculture development, sukabumi, west java, indonesia. upon arrival at the laboratory of fish nutrition, of the bogor agricultural university, the fi sh were acclimated to experimental conditions for 10 days and fi sh readily adjusted to the experimental diets. th ereafter, fi fteen juveniles with an initial body weight of 6.9 + 0.2 g were stocked into each 60-l aquarium. each aquarium was part of a closed recirculation system. during table 1. continued biotropia vol. 15 no. 2, 2008 113 rearing period, each aquarium was supplied with continuous aeration and water was allowed to fl ow through at the rate of 200-300 ml/min. every day, impurities in the water of each aquarium were removed and 50% of the water was renewed to maintain water quality. from each aquarium, water was fl own to the physical and biological fi lters, then in the conditioning tank. th ereafter, the water was drained back to each aquarium by using pump. dissolved oxygen contents were 4.18-5.13 mg/l, water temperatures 28 to 30 ºc, and ph 6.30 to 6.35. each experimental diet was fed to the fi sh in three aquaria. th e diets were randomly assigned to groups of fi sh in the aquarium. th e fi sh were fed on the diets to satiation three times a day at 08.00, 13.00 and 18.00 hrs for 60 days. at the end of the feeding trial, the fi sh of each aquarium were bulk weighed from each aquarium. growth of the fi sh (as measured by the percentage of daily growth rate), feed conversion ratio (fcr), protein retention (pr) were calculated as described previously (huisman 1976; steff ens 1989; takeuchi 1988). after the fi nal weighing, fi ve fi sh were randomly sampled from each aquarium for body proximate and mineral compositions analysis, and three fi sh were randomly sampled from each aquarium for analyses of bone mineral (p, ca and zn) composition. th e proximate composition and mineral analyses were carried out according to the methods described by takeuchi (1988). digestibility trial th e eff ect of phytase supplementation to the diet on the p digestibility was determined by the indirect method with 0.5% of chromic oxide (cr2o3) as an inert reference substance. th e green catfi sh with the same size as that for the feeding trial were used in this digestibility trial. twelve fi sh were stocked into each 60-l aquarium. triplicate groups of fi sh were fed on each experimental diet to satiation three times a day for 7 days prior to collection of feces. feces were collected after 2 h of feeding by siphoning into a plastic sieve. after each collection, the samples from each aquarium were pooled, frozen at -20 ºc and stored for subsequent analyses. feces collections were conducted for 21 days. th ereafter, the samples of feces were analyzed for chromium (cr), p and protein content according to the methods described by takeuchi (1988). apparent digestibility of total nutrient, protein, and p were calculated as follows: % digestibility = 1 – (% cr in feed/ % cr in feces) x (% ingredient in feces/ % ingredient in feed) x 100 p and nitrogen (n) losses through feces were calculated as follows: p in feces (g) = undigestible p (%) x p consumption during culture period n in feces (g) = (undigestible n (%) x n consumption during culture period)/6.25 statistical methods th e data were statistically analyzed for diff erences among the means by the oneway analysis of variance. th e duncan’s test was used to compare treatment means using the statistical software spss 12 for windows. diff erences were considered signifi cant at p<0.05. phytase for diet of green catfi sh (hemibagrus nemurus) – yulisman et al. 114 results and discussion results indicated that p and protein digestibility increased from 64.5% to 87.0%, and 87.1% to 90.5%, respectively, as phytase supplement increased from 0 mg in diet b to 60 mg phytase/100 g sbm in diet e. p digestibility of diet e was higher than that of diet a (77.6%) (table 3). p digestibility was correlated with the water-soluble p. th e water-soluble p content in control diet was 0.59 %. as the phytase supplement increased, water-soluble p increases from 0.19 % in diet b to 0.53 % in diet e (table 1). it could be observed that the lowest p loss through feces was found for diet e, and the lowest nitrogen (n) loss through feces for diet d. diet e gave the second lowest n loss through feces. level of n and p loading by fi sh fed diet e were 76% and 20%, respectively, lower than those in the fi sh fed control diet (a). table 3. phosphorus (p) and protein digestibility of experimental diets parameters experiment a b c d e p digestibility (%) 77.6 64.5 70.4 73.3 87.0 p consumption (g) 10.4+0.1d 3.2+0.1a 3.2+0.1a 3..4+0.1b 4.0+0.0c p digested (g) 8.1+0.1e 2.0+0.0a 2.3+0.0b 2.5+0.1c 3.5+0.1d p output via feces (g) 2.3+0.0e 1.1+0.0d 1.0+0.0c 0.9+0.0b 0.5+0.0a protein digestibilty (%) 89.1 87.1 85.1 89.7 90.5 protein consumption (g) 257.4+2.8d 207.6+3.5a 215.6+3.5b 218.2+4.1b 251.8+1.0c nitrogen (n) digested (g) 36.7+0.4c 28.9+0.5a 29.4+0.5a 31.3+0.6b 36.5+0.2c n output via feces (g) 4.5+0.1d 4.3+0.1c 5.1+0.1e 3.6+0.1a 3.8+0.0b *) values within the same row with different superscript letters are significantly different, p<0.05. concentrations of p, ca and zn in the whole body and bone of the fi sh fed diet e were higher than those for the groups of fi sh fed on the diets supplemented with 0-40 mg phytase/100 g sbm, but were not signifi cantly diff erent from those of the fi sh fed diet a (table 4). table 4. p, ca and zn content in green catfish (% dry weight) fed experimental diets parameters experiment a b c d e whole body p 3.18 + 0.28b 2.08 + 0.27a 2.34 + 0.39a 2.97 + 0.19b 3.20 + 0.12b ca 3.26 + 0.06c 1.92 + 0.30a 2.12 + 0.07a 2.90 + 0.20b 3.30 + 0.09c zn 0.012 + 0.001b 0.009 + 0.001a 0.012 + 0.000b 0.012 + 0.001b 0.013 + 0.002b biotropia vol. 15 no. 2, 2008 115 parameters experiment a b c d e bone p 21.41+ 1.12c 13.44 + 0.26a 16.65 + 0.77b 16.99 + 1.24b 21.46 + 0.43c ca 33.33 + 0.11d 21.44 + 1.15a 24.42 + 0.76b 26.04 + 1.15c 33.60 + 1.66d zn 0.032 + 0.002b 0.021 + 0.002a 0.023 + 0.002a 0.024 + 0.003a 0.032 + 0.001b *) values within the same row with different superscript letters are significantly different, p<0.05. th e mean body weight gain of the fi sh fed diet e was higher than that of the fi sh fed with the other diets. th e daily growth rate and feed conversion ration (fcr) followed similar trend, while the fi sh fed diet b had the lowest daily growth rate and fcr (table 5). th e protein retention had the similar trend with the protein digestibility, which increased from 36.8% from 0 mg phytase/100 g sbm in the diet b to 44.4% from 60 mg phytase/100 g sbm in the diet e. table 5. initial body weight (wo), final body weight (wt), protein retention (pr), daily growth rate (dgr), feed consumption (fc), feed conversion ratio (fcr) and survival rate (sr) of green catfish parameters treatments a b c d e wo (g) 7.0+0.01 7.0+0.02 6.9+0.12 6.8+0.09 6.8+0.05 wt (g) 43.8+0.8 31.9+1.1 35.2+ 0.9 40.2+0.4 47.7+0.4 dgr (%) 3.1+0.0d 2.6+0.1a 2.8+0.1b 3.0+0.0c 3.3+0.0e pr (%) 39.5+0.2b 36.8+0.4a 40.1+1.4b 41.6+1.4b 44.4+1.9c fc (g) 709.7+7.6e 570.4+9.7a 585.9+9.5b 601.1+11.2c 688.1+2.7d fcr 1.3+0.0 c 1.5+0.1e 1.4+0.0 d 1,2+0.0 b 1.1+0.0 a sr (%) 100+0.0 100 + 0.0 100+0.0 100+0.0 100+0.0 *) values within a row with different superscript letters are significantly different, p<0.05. increasing levels of phytase level from 0 mg to 60 mg/100g of sbm resulted in increasing levels of water-soluble p, thereby increasing water-soluble p/total p ratios in the diets from 34.87% to 91.13%. diet e produced the highest p and protein digestibility, followed by diet a (control diet) (table 3). it means that phytase released p from the insositol ring of phytate to be water-soluble p (baruah et al. 2004). th e water-soluble p was digested in the intestine of fi sh, and improved the absorption and utilization of p. phytase also released some protein and amino acids from the phytic acid, hence the absorption and utilization of protein also increased. th e same results were found for other fi sh species, such as the atlantic salmon salmo salar (sajjadi and carter 2004), striped bass morone saxatilis (papatryphon and shoares 2001), rainbow trout onchorhynchus mykiss (cheng and hardy 2004), korean rockfi sh sebastes schlegeli (yoo et al. 2005), and tilapia oreochromis niloticus (liebert and portz 2005). on the other hand, the fi sh fed on the diet b had the lowest p digestibility. it appears that the concentration of phytase in diet b could not enough release all p from phytic acid in the sbm. table 4. continued phytase for diet of green catfi sh (hemibagrus nemurus) – yulisman et al. 116 table 4 shows that not only p availability in the diet could be improved by phytase supplementation, but also the other minerals as ca and zn which could be available to the fi sh. accumulation of ca, p and zn in the body and bone of fi sh also increased as the phytase levels of the diets increased from 0 to 60 mg/100 g sbm. other experiments also showed that phytase supplemented diet increased ca, p and mn contents of the african catfi sh clarias gariepinus (nwanna et al. 2005), striped bass morone saxatilis (hughes and soares 1998), and atlantic salmon salmo salar (sajjadi and carter 2004). it is known that phytic acid is the major p storage compound in plant seeds, including sbm. phytic acid is also a strong chelator of important minerals such as ca, mg, k, fe, cu, zn and forms poorly soluble complexes. apart from minerals, phytic acid also forms complexes with protein and amino acids. th e digestibility of these complexes by fi sh are very limited due to the lack of intestinal phytase (pointillart et al.1987). th e supplementation of phytase in this experiment could release those minerals and protein from phytic acid, resulted in increasing their (minerals and protein) digestibility for fi sh (table 1 & 3), hence the absorption and utilization of minerals and protein also increased (table 4 & 5). on the other hand, minerals also play a role in many processes of protein, lipid and carbohydrate metabolisms. increasing the availability of some minerals in the diet by phytase supplementation also appears to improve the protein synthesis, which can be seen from the protein retention data (table5). th e highest protein retention was found in the groups of fi sh fed on diet e, while the fi sh fed on diet b had the lowest protein retention. th e increased protein retention also improved the protein deposit in the body, hence increasing the daily growth rate (table 5). in this experiment, the fi sh fed on diet e had the highest protein retention and daily growth rate. th is indicates that phytase could increase the nutrient (protein) digestibility leading to higher protein retention, resulting in the increase of the growth rate and reduce feed conversion ratio. th e same conclusions were also indicated by other authors that the phytase supplementation to sbm improved the weight and growth rate of juvenile korean rockfi sh sebates schlegeli (yoo et al. 2005), tilapia oreochromis niloticus (liebert and ports 2005; furuya et al. 2001), rainbow trout oncorhynchus mykiss (vandenberg et al. 2003), and pangasius pangasius (debnath et al. 2005). nwanna et al. (2005) also found that supplementation of phytase to a diet containing sbm gave better feed conversion than that for diet without phytase supplementation for the african catfi sh clarias gariepinus, and the striped bass morone saxatilis (hughes and soares 1998). as earlier explained some of the diet consumed by fi sh was undigested, and will be excreted through feces. increasing phytase concentration in the diet from 0 mg to 60 mg/100g sbm reduced the levels of p and n excretion through feces. th is indicated that phytase was able to release nutrient (protein) and minerals of phytate, resulting in the improvement of fi sh growth and also reduced p and n excretion through feces. hughes and soares (1998) concluded that phytase in a diet containing high level of plant protein could reduce the p excretion of striped bass morone saxatilis, seabass dicentrarchus labrax (teles et al. 1998), japanese fl ounder paralichthys olivaceus (masumoto et al. 2001), rainbow trout oncorhynchus mykiss (vielma et al. 2002), atlantic salmon salmo salar (sajjadi and carter 2004), tilapia oreochromis niloticus (furuya et al. 2001; liebert and portz 2005), and the african catfi sh clarias gariepinus (van weerd et al. 1999; nwanna et al. 2005), thereby reducing the loading of p waste into the environment. biotropia vol. 15 no. 2, 2008 117 conclusions th e supplement of 60 mg phytase/100 g sbm in the diet improved p digestibility and the growth of the green catfi sh hemibagrus nemurus, hence reducing the loading of p and n waste to the environment. references baruah k., n.p. sahu, a.k. pal, and d. debnath . 2004. dietary phytase: an ideal approach for a cost eff ective and low-polluting aquafeed. naga, worldfi sh center quarterly 27 (3) 3 & 4 jul-dec 2004. p. 15-19. cheng zj, and r.w. hardy . 2004. eff ect of microbial phytase supplementation in corn distiller’s dried grain with solubles on nutrient digestibility and growth performance of rainbow trout, oncorhynchus mykiss. journal of applied aquaculture. 15 (3/4): 83-100. debnath d., a.k. pal, n.p. sahu, k.k. jain, s. yengkokpam and s.c. mukherjee. 2005. eff ect of dietary microbial phytase supplementation on growth and nutrient digestibility of pangasius pangasius fi ngerlings. aquaculture research (36) 2: 180 – 187. furuya w.m., g.s. gonclaves, v.r.b. furuya , c. and hayashi. 2001. phytase as feeding for nile tilapia (oreochromis niloticus). performance and digestibility. rev. bras. zootec. 30: 924 – 929. hughes k.p. and j.h. soares jr. 1998. effi cacy of phytase on phosphorus utilization in practical diets fed to striped bass morone saxatilis. aquaculture nutrition, 4: 133 – 140. huisman e.a. 1976. food conversion effi ciencies at maintenance and production levels for carp (cyprinus carpio linn) and rainbow trout (salmo gairdneri ric.). aquaculture, 9 (2): 259-273. liebert f. and l. portz . 2005. nutrient utilization of nile tilapia (oreochromis niloticus) fed plant based low phosphorus diets supplemented with graded levels of diff erent sources of microbial phytase. aquaculture, 248: 111 – 119. li m.h., b.b. manning and e.h. robinson. 2004. summary of phytase studies for channel catfi sh. mississippi agricultural and forestry experiment station (23) 23 no. 13: 1-5. masumoto t., b. tamura and s. shimeno. 2001. eff ects of phytase on bioavailabilty of phosphorus in soybean meal-based diets for japanese fl ounder (paralichthys olivaceus). fisheries science, 67:1075-1080. nwanna l.c., o.a. fagbenro and a.o. adeyo. 2005. eff ect of diff erent treatments of dietary soybean meal and phytase on the growth and mineral deposition in african catfi sh clarias gariepinus. journal of animal and veterinary advances. 4: 980 – 987. papatryphon e., and j.h. soares jr. 2001. th e eff ect of phytase on apparent digestibility of four practical plant feedstuff s fed to striped bass, morone saxatilis. journal aquaculture nutrition. 161-167. pebriyadi, b. 2004. supplementation of methionine and tryptophan in the high soybean meal diets of juvenile green catfi sh (mystus nemurus). th esis (in indonesian). graduate school, bogor agricultural university. 53 p. pointillart a., a. fourdin and n. fontaine. 1987. importance of cereal phytase activity for phytate phosphorus utilization by growing pigs fed diets containing triticale or corn. journal of nutrition 29: 907912. sajjadi m. and c.g. carter. 2004. dietary phytase supplementation and the utilization of phosphorus by atlantic salmon (salmo salar l.) fed a canola-meal-based diet. aquaculture 240: 417 – 431. takeuchi t. 1988. laboratory work-chemical evaluation of dietary nutrients. p. 179-233, in watanabe (ed) fish nutrition and mariculture. kanagawa international fisheries training. japan international cooperation agency (jica), japan. phytase for diet of green catfi sh (hemibagrus nemurus) – yulisman et al. 118 teles a.o., j.p. pereira, a. gouveia and e. gomes. 1998. utilization of diets supplemented with microbial phytase by seabass (dicentrarchus labrax) juvenils. aquaculture living resources, 11 (4): 255259. vandenberg g.w., v. dallaire, s.l. scott and j. de la noue . 2003. encapsulation of microbial phytase : eff ects on phosphorus bioavailability in rainbow trout (oncorhynchus mykiss). aquaculture nutritioncontributed papers. http://www.aquacultureassociation.ca/ac03/abstracts/nutrition.htm van weerd j.h., k.h.a. khalaf, f.j. aartsen and p.a.t. tjissen. 1999. balance trials with african catfi sh clarias gariepinus fed phytase-treated soybean meal based diets. aquaculture nutrition, 5:135142. vielma j., k. ruohonen and m. peisker . 2002. dephytinization of two soy proteins increases phosphorus and protein utilization by rainbow trout, oncorhynchus mykiss. aquaculture. 204: 145 – 156. watanabe t. 1988. fish nutrition and mariculture. jica textbook. th e general aquaculture course. department of aquatic biosciences. tokyo university of fisheries. 233 p. yoo g.y., x. wang, s. choi , k. han, j.c. kang and s.c. bai sc. 2005. dietary microbial phytase increased the phosphorus digestibility in juvenile korean rockfi sh sebastes schlegeli fed diets containing soybean meal. aquaculture 243. 315 – 322. biotropia vol. 15 no. 2, 2008 biotropia no. 6, 1992/1993: 66-70 the adsorption of imazapyr by three soil types in indonesia s. tjitrosemito seameo biotrop, p.o. box 116, bogor, indonesia s. matsunaka and m. nakata department of biotechnology, faculty of engineering, kansai university, yamate-cho, suita, osaka 564, japan and lab. pesticide science, faculty of agriculture, kobe university, rokkodaicho, nadaka, kobe 657, japan, respectively abstract the adsorption of imazapyr in three indonesian soil types was investigated with labelled 14c-imazapyr using freundlich adsorption isotherm. the availability of adsorbed imazapyr to plants as affected by washing and liming was assayed using root elongation of rice seedlings. red-yellow podsolic soil adsorbed imazapyr more than andosol and sandy soil of laladon. the adsorption was greater at lower ph. washing seemed to reduce the concentration of imazapyr as shown by the increasing length of rice roots. on the other hand liming facilitated higher concentrations of imazapyr in the solution as shown by the reduction of rice root length. the practical implication is discussed. introduction upland rice established in pot experiment by zero tillage technique on alang-alang sprayed with imazapyr at 1.5 kg a.i./ha died immediately after germination (tjitrosemito and purwanto 1991). however, imazapyr applied at 2.0 kg a.i./ha to soybean, planted using zero tillage technique in the field during the wet season, did not show any phytotoxic effect (tjitrosemito and suwinarno 1988). to understand its fate in soil, imazapyr adsorption in various soils and the effect of liming and washing were investigated. materials and methods the experiment was carried out at the laboratory of pesticide science, faculty of agriculture, kobe university, japan. three soil types, i.e. red-yellow podsolic soil (ryp), andosol and sandy soil of laladon were air dried, and sieved through a 1 mm sieve. the ph was measured in a 1:1 (w/v) soil: deionized water/slurry 66 the adsorption of imazapyr by three soil types s. tjitrosemito, s. matsunaka and m. nakata using ph meter (model com-10, dkk denki kagaku keiki co. ltd.). the ph values were 3.75; 4.83 and 5.40 for ryp, andosol and sandy soil of laladon, respectively. adsorption commercially formulated imazapyr (assault) was combined with 14 c-ring labelled imazapyr (specific activity: 43.916µci/mg) to obtain initial concentration of 5, 25, 45 and 65 µm. five grams of air dried soils (sieved through 1 mm) and 10 ml of herbicide solution were placed in 30 ml vial. the samples were equilibrated by shaking on a wrist action shaker for 24 hours at room temperature. the mixtures were transferred to centrifuge tubes and spun at 15 000 x rpm for 30 minutes. a 2-ml aliquot was removed from each tube and placed in 20 ml of scintillation cocktail and the 14 c-activity was determined by scintillation spectrophotometry. adsorption isotherms were constructed using best-fit regression equations. freundlich constant kf and 1/n were calculated from the equation: log (x/m) = log kf + (1/n) log c where, x/m = the amount of herbicide adsorbed (µmole/kg) c = herbicide concentration (µmole/i) in solution after equilibrium kf = freundlich constant, an indicator of relative adsorption of herbicide at unit concentration (i.e. c = 1 µmole/1) (1 /n) = indication relative linearity between adsorption and concentration. the phytotoxicity of imazapyr a rice bioassay was conducted by placing 5 g of air dried soil into 30-ml tubes containing commercially formulated imazapyr. the mixtures were allowed to equilibrate for 24 hr by shaking in a shaker. after equilibration, washing treatment was done by replacing the solution with deionized water and later on, 10 pre-soaked rice seeds were put in each tube. after 3 days, the rice root length was measured and analysed statistically. the treatments consisting of soil (red-yellow podsolic soil, andosol and laladon sandy soil), imazapyr (0, 0.1 and 0.2 ppm), liming (addition of caco3 at 0.1 % (-/ + )) and washing (/ +) were combined factorially and randomized completely. 67 biotropia no. 6, 1992/1993 results and discussion the relationship between the amount of adsorbed imazapyr and its equilibrium concentration in 3 types of soil is presented in figure 1. red-yellow podsolic soil, the highly weathered soil with low ph adsorbed more imazapyr than andosol or sandy soil of laladon. the addition of caco3 somehow reduced the adsorption considerably. the calculation of freundlich equation showed the characteristic of adsorption (table 1). figure 1 . the relationship between the amount of imazapyr adsorbed and its equilibrium concentration from table 1 the value of (1/n) ranged from 0.67 to 1.10 indicating a nonlinear relationship between the amount adsorbed and the equilibrium concentration. freundlich constant of ryp showed the highest value (246.0µm/kg) indicating that it has the highest affinity for imazapyr. in ryp soil alang-alang is usually found abundantly. 68 the adsorption of imazapyr by three soil types s. tjitrosemito, s. matsunaka and m. nakata table 1. relative adsorption of imazapyr (kf) and coefficient of regression (r2) on soils. soil lime kf (µ mole/kg) 1/n r2 red-yellow podsolic soil 246.0 0.84 0.99 (ph 3.75) + 98.0 0.67 0.99 andosol 120.5 0.98 0.99 (ph 4.83) + 34.7 1.05 0.98 sandy soil of laladon 17.4 1.10 0.92 (ph 5.40) + 15.9 0.95 0.77 when the values of kf and ph were examined, there was a linear relationship, i.e. y = 756.22 134.4x (r 2 = 0.98), where y = kf and x = ph value. the adsorption of imazapyr increased with the decrease in soil ph, similar results were reported by stougaard et al. (1990), wehtje et al. (1987) and arnold (1981). when the soil was limed, soil ph increased and presumably more imazapyr molecules stayed in the solution. phytotoxicity of imazapyr rice (norin no. 8) as a test plant in this bioassay showed a severe root length reduction when exposed to the soil treated with 0.1 or 0.2 ppm of imazapyr solution (table 2 a). table 2. root length (mm) of rice as affected by imazapyr and washing, liming, and soil types. treatments imazapyr (ppm) 0 0.1 0.2 a . washing no washing 32.7 18.3 14.7 lsd (5%): 5.3 with washing 35.1 28.2 20.5 b . liming no liming 35.1 27.2 18.7 lsd (5%): 6.1 with liming 31.2 19.4 15.6 c . soil redyellow podsolic 31.8 28.7 20.7 andosol 31.0 20.3 16.8 lsd (5%): 5.3 sandy soil of laladon 23.8 20.8 15.3 69 biotropia no. 6, 1992/1993 the reduction of root length was modified by washing. root length was increased from 18.3 mm to 28.2 mm and from 14.7 mm to 20.5 mm for 0.1 ppm and 0.2 ppm imazapyr treatment, respectively. it seems that washing reduced the concentration of imazapyr in the solution, so less phytotoxicity was experienced by the rice root. it indicates that imazapyr can be leached away by the flowing water. on the other hand liming (0.1% caco3) increased the phytotoxicity to the rice plant as shown in table 2 b. the soil also contributed differential response to the rice root elongation. the availability of imazapyr seems greater under the sandy soil of laladon (table 2 c). the results of this experiment may explain the different results of imazapyr on crop growth, when imazapyr is applied during or at the end of the dry season. the crop introduced in the following wet season may suffer from imazapyr phytotoxicity. however, when imazapyr is applied in the wet season, crops established one month later will escape imazapyr injury because imazapyr molecules would have been leached by the rainfall. liming will further speed up leaching if carried out long enough before crops are planted. references arnold, p.w. 1981. surface electrolyte interaction. in d.j. greenland and m.b. hayes, eds. the chemistry of soil constituents, john wiley & sons, new york: 355-404. stougaard, r.n., p.j. shea and a.r. martin. 1990. effect of soil type and ph on adsorption, mobility and efficacy of imozaquin and imazethapyr, weed sci, 38: 67-73. tjitrosemito, s. and d. suwinarno. 1988. the performance of soybean (c.v. americana) established by zero tillage technique in imperata field controlled by herbicides. biotropia (2): 1217. ______, purwanto. 1991. the performance of upland rice established by zero tillage technique on imperata field. biotropia (5): 1014. wehtje, g., r. dickens, j.w. wilcut and b.f. hajek. 1987. sorption and mobility of sulfometuron and imazapyr in five alabama soils. weed sci. 35: 858-864. 70 66.pdf 67.pdf 68.pdf 69.pdf 70.pdf biotropia (2) 1988/1989: 1-7 fruit production of a six-year old shorea stenoptera plantation at haurbentes, bogor, indonesia eizi suzuki department of biology, college of liberal arts, kagoshima university, kagoshima 890, japan lilian u. gadrinab tropical forest biology program, biotrop, bogor, indonesia abstract a six-year old plantation of shorea stenoptera at haurbentes, bogor flowered for the first time on october, 1987. in plots of 270 m2, 12 of the 32 trees had opened flowers. the average heights of flowering and non-flowering trees were 400 cm and 270 cm, respectively. the flowers and fruits were counted four times from october 28, 1987 until february 17, 1988. in october, a total of 24313 flowers existed and 1.9% of them became mature fruits on february, 1988. the fruit production was 308 kg/ha and 133 kg/ha in fresh and dry weights, respectively. introduction shorea stenoptera burck produces large and oil-rich fruits called illipe nuts. many shorea plantations are found in west kalimantan where fruits are collected for oil. there are many studies about the phenology of dipterocarpaceae (burgess 1972, cockburn 1975), mast flowering (ng 1977, yap 1987) and pollination (appanah 1981, appanah and chan 1981). there are, however, no quantitative studies on the change of the number of flowers that mature to fruits in dipterocarpaceae. there are only a few records on the age at sexual maturity of dipterocarpaceae (foxworthy 1932, ng 1966 and 1977) but none exists for s. stenoptera. thus, when a six-year old plantation of s. stenoptera in bogor began to flower, it was deemed interesting to trace the number of flowers that mature to fruits. study site and methods shorea stenoptera and other dipterocarp species have been planted in an experimental forest at haurbentes, bogor (6°32'-33's, and 108°26'e, and 200 m above sea level) which was established in 1940 by the center for forestry research and development. the total of monthly average precipitation from 1957 to 1965 was 4196 mm. 1 biotropia no. 2, 1988/1989 plots p-l to p-3 and p-4 were made in a 6-year old and 18-year old plantation of shorea, respectively. s. stenoptera seedlings had been planted every 3 m x 3 m. the plot sizes were 10 m x 10 m, 12 m x 15 m, 12 m x 15 m, and 20 m x 25 m, for p-l to p-4, respectively. p-l was a few meters distant from p-3 and about 20 m from p-2. p-2 was on a 16° slope with a north aspect while the others were on gentle slopes of less than 10°. the tree height (h) and trunk diameter at the 1.3 m height (dbh) were measured. it was noted whether the faces were in flowering or not. in p-l and p-2 all the panicles were tagged with numbered tapes and a stapler. the length of the panicles was measured and the number of flower buds, flowers, young fruits, flower scars was counted. after the flower fell, two bracteoles and the scar of the peduncle remain at the point of the panicle where the peduncle was attached. later the bracteoles also fell. a few trees were in blossom earlier than others and lost many small branches of panicles. the flower number of the former trees was therefore underestimated. on 8 december, 1987, 8 january, and 17 february, 1988 the fruits were counted again. one tree had a few new panicles in december and january. they were counted as in the above process. on 17 february, 337 fruits were collected randomly, their length, diameters and weight were measured. the calices were separated from 57 fruits and the fruits were weighed. the calices and fruits were cut into small pieces and oven dried at 105°c for 16 hours, and weighed. results and discussion the first trees of shorea stenoptera in haurbentes were grown from seeds which came from pontianak, west kalimantan about 40 years ago (masano et al. 1987). table 1 shows the amount of illipe nuts, group of shorea species, called teng-kawang in indonesia, s. stenoptera, s. pinanga scheff., and s. seminis sloot, harvested in west kalimantan from 1983 to 1987. the annual crop fluctuated greatly. tengkawang species have a characteristic of mast flowering as many other shorea species. in 1987, s. stenoptera began to flower in april in west kalimantan table 1. annual crop of illipe nuts harvested from plantations and natural forests in west kalimantan from data of the department of forestry of west kalimantan at pontianak. april 1983 march 1984 fresh weight 10 640 500 kg 1984 1985 0 1985 1986 396 900 1986 1987 1 982 980 1987 1988 7 822 480 2 fruit production of a six-year old shorea stenoptera-e\7\ suzuki & lilian u. gadrinab while at haurbentes, it was in september. the flowering season in different localities is not the same. twenty three species of dipterocarpaceae are planted at haurbentes, and most of them were flowering in september or october of 1987. in mt palung, west kalimantan, 22 species of dipterocarpaceae flowered in april of 1987 (simbolon at herbarium bogoriense, personal communication). the tree sizes and basal area (ba, summed area of stem cross sections at 1.3 m height) of 5. stenoptera in p-l to p-3 are shown in table 2. the trees were rarely shaded by neighboring trees and the areas were manually weeded. the flowering trees were significantly bigger in dbh and taller than the nonflowering ones. they began to flower after 6 years from germination. the ages at first flowering of 65 species of dipterocarpaceae in the arboretum at kepong, near kuala lumpur were from 17 to 45 years (ng 1966 and 1977), dipterocarpus baudii flowered at 6.5 years old (foxworthy 1932). 5. stenoptera is one of the earliest flowering species in dipterocarpaceae. in p-4 which is an 18 year-old-plantation all trees flowered. table 2. tree size and area of s. stenoptera in p-l, p-2 and p-3 (450 m2 in total), and p-4 (500 m2). p-l, p-2, and p-3 (6 yr old) flowering tree non-flowering all trees p-4 (18 yr old) no. 20 25 45 20 mean dbh (cm) 4.36* 2.59* 3.37 20.02 standard deviation 1.47 1.10 1.54 7.99 mean height (cm) 400* 270* 328 1568 standard deviation 105 90 116 428 basal area (m2/ha) 0.74 0.34 1.08 14.5 * the difference is significant at the level of p = 0.01. shorea stenoptera is a small tree, but is a remarkably variable species (ashton 1982). gadrinab (1984) has found two genetic types, big and small types. the big type becomes 40 m or more in height while the small type is usually shorter than 20 m. they can be distinguished even when still young and small because branches of the big type are usually thick and extended upward while those of the small type are slender and hanging down. the trees in p-l to p-3 seemed to be of the small type. a few trees in p-4 are of the big type. the changes in number from flowers to mature fruits of 12 flowering trees in p-l and p-2 are shown in table 3. the flowering season of shorea stenoptera at haurbentes started in september, and was nearing its end at the time of study which was october 28-30. s. pinanga bore fruits but no more flowers at that time. 3 biotropia no. 2, 1988/1989 eighty four percent of all flowers (24 312) had already fallen by october 30. thirteen percent of all the flowers were lost from november 1 to december 8, 1987. two trees which had only 74 flowers lost all flowers by december 8. from december 9 to february 17, 1988, 1.3% of all the flowers were lost. the mature fruits in february were 1.9% of all flowers in october, and there were only 1.7 fruits/m 2 . one tree which had 47 fruits on january 8, 1987 lost all of them by february 17. the fruits of many trees were nearly mature on january 8. hence, the real fruit set ratio (fruit no./flower no.) was from 1.9% to 2.6%. the mean fruit set ratios of 187 species of self-incompatible and 129 self-compatible hermaphroditic plants were 22.1% and 72.5%, respectively (sutherland and delph 1984). they, however did not include dipterocarpaceae. this ratio in 5. stenoptera was very low. many flowers fell soon after their opening. they might not have been pollinated. table 3. change of flower and fruit number of s. stenoptera in p-l and p-2 (270 m2) and fruit weight. no. % later flower oct. 28-30 no. of panicle 337 1987 bud 779 3.2 flower & fruits 3 181 13.1 flower scar 20 352 83.7 total 24312 100 dec. 8 young fruits 777 3.2 200 jan. 8'88 almost mature fruits 643 2.6 2 141 feb. 17 mature fruits 458 1.9 0 0 feb. 17 mean length of fruits 42 mm mean diameter of fruits 28 mm fresh fruit weight without calyx 18.16g dry fruit weight without calyx 7.84g fruit production (fresh) 308 kg/ha (dry) 133 kg/ha one tree continued to produce new panicles and flower until january 1988. the flowers in december and january were, however, 1.4% of all flowers in october. they did not contribute to fruit production in february. some trees outside the plots also continued to make a few flowers in december and january. in west kalimantan, we found a tree bearing a few flowers in november. there seemed to be some variation in the flowering period among individuals. ng (1977) has reported that s. stenoptera flowered frequently for very extended periods in kepong. 4 fruit production of a six-year old shorea stenoptera-eizi suzuki & lilian u. gadrinab the fruits without calyx were 42 mm and 28 mm in mean length and diameter respectively, and 18.16 g in fresh weight. there were significant variations among some trees in the mean length and diameters of fruits from each tree (figure 1). figure 1. relationships between mean length and diameter of fruits from each tree (solid circle) and all trees (open circle). cross lines show the confidence limits of the means (p = 0.05). the shape and length of five wings of calyx were also different among trees. the wing length varied from one third to 2 from that of fruit length. as a result, the wings have no role in wind dispersal. the productivity of fruits was 133 kg/ha in dry weight. the relationships between tree sizes and number of flowers in october are shown in figure 2. there was a rough correlation between them. the tree with the least flowers was shaded by a big tree outside the plot. the trees in p-l tended to make more fruits than trees in p-2 of the same size. only the relationships between height of the tree in p-2 and number of flowers was significantly correlated at 5% level as shown by a straight line in fig. 1 (correlation coefficient = 0.750). 5 biotropia no. 2, 1988/1989 figure 2. relationships between flower number of a tree and tree height (left figure), and dbh (right figure). the flower number is the sum of buds, flowers, young fruits, and scars in october. circle, trees in p-l. triangle, in p-2. conclusion the age at first flowering of s. stenoptera trees which are being grown at the experimental forest at haurbentes was 6 years old. two months after the start of flowering, 84% of flowers counted (24 312 flowers from 12 trees in a 270 m 2 plot) had already fallen. then after another month, 97.1% of all the flowers had fallen and by the end of another month, 98.4% of the total flowers were lost. mature fruits at this time were 1.9% of all flowers found in the first months of flowering. the productivity of fruits was 133 kg/ha in dry weight. acknowledgments the authors gratefully acknowledge the invaluable support by the japan international cooperation agency (jica) and seameo regional center for tropical biology (biotrop), and the forestry center for research and development in bogor for the permission of research in the experimental forest. thanks are also due to the field workers. 6 fruit production of a six-year old shorea stenoptera eizi suzuki & lilian u. gadrinab references appanah, s. 1981. pollination in malaysian primary forests. malayan forester, 44: 37-42. appanah, s. and h.t. chan. 1981. thrips: the pollinators of some dipterocarps. malayan forester, 44: 234-252. ashton, p.s. 1982. dipterocarpaceae. flora malaysiana, series 1, vol. 9: 237-552. martinus nijhoff pub., the hauge. burgess, p.p. 1972. studies on the regeneration of the hill forests of the malay peninsula. the phenology of dipterocarps. malayan forester, 35: 103-123. cockburn, p.p. 1975. phenology of dipterocarps in sabah. malayan forester, 38: 160-170. foxworthy, f. w. 1932. dipterocarpaceae of the malay peninsula. malayan forest records 10. gadrinab, l.u. 1984. a biosystematic study on section pachycarpae in dipterocarpaceae: shorea macrophylla ashton and shorea stenoptera burck. biotrop. masano, h. alrasyid, and z. hamzah. 1987. planting trials of dipterocarp species outside their natural distributional range in the haurbentes experimental forest, west java. proceed. third round table conference on dipterocarps (ed. a. j.g.h. kostermans), 19-37. unesco, jakarta. no. f.s.p. 1966. age at first flowering of dipterocarps. malayan forester, 29: 290-295. no. f.s.p. 1977. gregarious flowering of dipterocarps in kepong, 1976. malayan forester, 40: 126-137. sutherland, s. and l.f. delph. 1984. on the importance of male fitness in plants: patterns of fruit-set. ecol., 65: 1093-1104. yap, s.k. 1987. gregarious flowering of dipterocarps: observations based on fixed tree populations in selangor and negri sembilan, malay peninsula. proceed. third round table conference on dipterocarps (ed. a.j.g.h. kostermans), 305-317. unesco, jakarta. 7 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 4. kurniawan (melimination).cdr biotropia vol. 18 no. 2, 2011: 94 101 elimination of cvb ( ) from a range of chrysanthemum varieties by apical meristem culture following antiviral agent and heat treatments chrysanthemum virus b kurniawan budiarto*, budi marwoto, lia sanjaya, muchdar soedarjo and indijarto budi rahardjo received 06 august 2010/accepted 28 september 2011 cvb elimination for retaining healthy protocols from infected chrysanthemum plant was investigated through combined treatment of meristem culture with synthetic antiviral ribavirin or thermotherapy under conditions. the biological materials used for the experiment constituted of six commercial varieties: dewi sartika, saraswati, yellow fiji, white puma, yellow puma and white reagent. tissue culture initiation was conducted through plantlet establishment using ms supplemented with iaa. ribavirin was added in media with the concentration of 40 mg/l on cv. dewi sartika, saraswati and yellow fiji. parallel with this step, heat treatment with different durations (1, 2, and 3 weeks) was also conducted on the plantlets on white puma, yellow puma and white reagent. meristem culture was done following the chemoand thermotherapy. the experiment resumed the failure of single treatment of meristem culture in eliminating cvb from the infected chrysanthemum plantlets. under heat treatment, percentage of virus-free plantlets increased along with the duration of thermotherapy, though the survival rate of plantlets decreased in lengthened heat treatment. the best results regarding virus free plant percentage were obtained when meristem culture was applied following ribavirin or three weeks of heat treatment. (cvb), chemotherapy, heat treatment, meristem culture, virus-free indonesian ornamental crops research institute jl. raya pacet-ciheraang, po. box. 8 sdl, cianjur, west java-indonesia (43253) in vitro chrysanthemum virus-b abstract introduction key words: chrysanthemum ( kitam) is one of the major cut flowers in the world. chrysanthemum ranks the first of all cut flowers marketed dendranthema grandiflora [ramat.] * corresponding author : mbud1arto@yahoo.co 94 every year from indonesia (37.34 %) with the quantity of more than 66 million stalks in 2007 (indonesian general directorate of horticulture 2008). nowadays, however, chrysanthemum production has faced some constraints, and one of these was systemic disease attacks caused by viruses, viroids and phytoplasmic organisms. up to present, chrysanthemum virus-b (cvb) is still one of the most common viruses found in commercial growers and have caused significant economic losses. (marwoto 2004). taxonomically, cvb belongs to carlavirus wide group. genome of the virus consists of unipartite, single-stranded rna with total genome size of 7.5 kb (levay & zavriev 1991). the nucleocapsid (virions) are filamentous with no protein envelop and usually straight with a clear modal length of 685 nm (brunt . 1996). aside from chrysanthemum (compositae), cvb was also known systemically hosted in (leguminosae), nicotiana, petunia and tetragonia (megan 2001). the general symptoms of the infected plant were stunted in growth, chlorotic in leaf blades, organ structure malformation and discolored petal. degeneration in proliferation level with slower multiplication rate was also observed in the infected planlet during culture (marwoto 2004). efforts have been made to get healthy protocols by eliminating virus from the infected plants. several methods such as chemo-, thermotherapy and electrical charges have been successfully conducted for virus elimination in some crops. numerous chemicals have been tested for antiviral activity, but few were effective. the most substance is the synthetic analogue of guanosine, ribavirin (1-beta-dribofuranosyl-1-h-1,2,4-triazole-3carboxamide) added to the media in the range of 30-50 mg/l was affective against potato virus x (pvx), potato virus y (pvy), potato virus s (pvs) and potato virus m (pvm) in potato (elia . 2008), ringspot virus in citrus (sharma . 2007) and yellow leaf virus in sugarcane (parmessur & saumtally 2001). ribavirin is a member of the nucleoside anti metabolite compound that interferes with duplication of viral genetic material. though not effective against all viruses, ribavirin is remarkable as a small molecule for its wide range of activity; including important activities against both dna and rna viruses (senula 2000 . ribavirin's carboxamide group can make the native nucleoside drug resemble adenosine or guanosine, depending on its rotation. for this reason, when ribavirin is incorporated into rna, as a base analog of either adenine or guanine, it pairs equally well with either uracil or cytosine, inducing mutations in rna-dependent replication in rna viruses. such hypermutation can be lethal to rna viruses (klein & livingston 2008). heat treatment or thermotherapy was established for virus elimination related to characteristic of some viruses which showed declination in multiplication rate especially at high temperature. this method was successfully applied in the range of 35 40 c for producing virus-free potato plant (converse & tanne 1984), alstromeria (hakkaart & versluijs 1988) and apple (wang . 2006). however, both chemoand thermotherapy methods, depended on plant genotypes and viruses. varying degree of meristem cells deaths, phytotoxic causing an increase in culture time and the need for frequent transfers into fresh media coincided with the quantity of virus-free plants obtained (chen & sherwood 1991). et al. et al vicia faba et al. in vitro et al. et al et al et al. ) et al 0 95 apical meristem culture following antiviral agent and heat treatments kurniawan budiarto .et al the combination of both chemoand thermotherapy with meristem culture was dedicated to encounter the technical constraints in the single application of respective techniques. these methods were also reported to be more effective for virus destruction on sweet potato, nicotiana, lemon and cucumber (zaitlin & palukaitis 2000). the research was then conducted to find out the effect of ribavirin application in the media and heat treatment at different durations followed by meristem culture on the existence of cvb in infected chrysanthemum plants. the research was conducted at the indonesian ornamental crops research institute (iocri). six cvb infected chrysanthemum varieties i.e dewi sartika, saraswati, yellow fiji, white puma, yellow puma and white reagent were equally divided into two parallel experiments. the first three varieties served as chemotherapy using ribavirin, while the rest underwent the heat treatments. fifteen cutting samples from each variety were collected and replanted in 15 cm pot. these were then, maintained in growth chamber for 16 h. after 2 weeks, the plants were pinched and the new emerging lateral growths served as explants. shoot induction was conducted by inoculating apicals into ½ ms + 0.5 mg/l iaa. the shoots were then subcultured into ½ ms + 0.1 mg/l iaa to obtain uniform plantlets. after three weeks incubation, the plantlets were transferred into treatments media, consisting of ½ ms + 0.1 mg/l iaa and ½ ms + 0.1 mg/l iaa + 40 mg/l ribavirin. two weeks after incubation, meristematic apical of plantlets (< 0.2 mm) were dissected using binocular microscope and transferred into ms media for shoot induction. the dissections of meristematic apical of plantlets were repeated three times from newly emerging shoots. one week before heat treatments, the plantlets were preconditioned in the incubator with the daily temperature of 30 35 c. the temperature of the incubator was then increased up to 38 40 c with the duration of 1, 2 and 3 weeks. after the heat treatments, meristematic apical of plantlets was dissected and inoculated into ms media for shoot induction. after transferred into regeneration media, phenotypic performance of plantlet was recorded. randomly plantlet samples in every treatment were also selected for cvb rapid detection using direct elisa method (clark & adam 1977). an amount of 100 μl cvb igg (agdia, usa) was mixed with a ratio of 1 : 200 of the coating buffer (na co + nahco + nan ) and overnightly incubated at the temperature of materials and methods in vitr in vitro o treatment of ribavirin heat treatment elisa bioassay 0 0 2 3 3 3 biotropia vol. 18 no. 2, 2011 96 4 c. the microplates were then rinsed twice with pbs tween (nacl + kh po + na hpo + kcl + nan + tween 20 + h o) buffer of 3 minutes each. leaf samples of 0.2 g were extracted and buffered with 1 ml mixture of pbst + 0,02 % pvp (1 : 5). the 100 μl of leaf extracts were then, incubated for 2 h in 37 c and rinsed with pbs tween buffer. after labeled with alkaline phosphatase enzyme, 100 μl of igg cvb was pipetted into microplates and mixed with eci (pbst + 0,2 % bsa) with a ratio of 1 : 200. the mixture was incubated for 2 h in 37 c. after 2 h incubation, the microplates were then rinsed with pbs tween buffer. an amount of 100 μl substrate buffer containing 4-nitrophenylphospate was placed into microplates and incubated at room temperature. after the substrate color changed into yellow, the reactions was then ceased with 25 μl naoh 3m. color intensities of the substrate were measured using elisa reader (minireader ii dynatech) on 410 nm wavelength. virus free samples were obtained when the absorbent values were three times less than that of positive control. plantlet performances were affected by the application of antiviral ribavirin and meristem tip culture, however,there were no specific interaction between cultivars tested and antiviral treatments. the increase of regeneration capacity was significant in plant height, number of leaves and number of nodes of ribavirin-treated plantlets, when samples were supplemented with 40 ppm ribavirin compared to untreated plants, thus hastened multiplication rate of the plantlet of all varieties tested (table 1). 0 0 0 2 4 2 4 3 2 results and discussion chemotherapy by antiviral ribavirin table 1. plantlet height, number of leaves and nodes after 30 days of subculture, and multiplication rate of chrysanthemum varieties treated with ribavirin. treatments plantlet height*) (cm) number of leaves*) number of nodes*) multiplication rate*) ribavirin treatments on media ms + 0.1 mg/l iaa + ribavirin 9.5 a 12.6 a 12.7 a 10.75 a ms + 0.1 mg/l iaa 6.8 b 6.4 b 6.3 b 7.41 b chrysanthemum varieties dewi sartika 9.2 a 12.7 a 11.7 a 10.22 a saraswati 7.5 ab 10.2 ab 10.5 ab 8.75 ab yellow fiji 6.0 b 9.1 b 9.2 b 6.16 b * note : values followed by different letters in the same column differ significantly at lsd 5%. faster multiplication rate of ribavirin-treated plantlets indicated that cell totipotency was retained. the most putative concerns of these phenomena were related to viricidist effect of ribavirin. the antiviral might block virus replication and although existing virus might remain in the original stem sections, the new outgrowths would be virus-free or contain only very low amounts of the virus (simpkins 1981). ribavirin might be active in its triphosphate form, which inhibits the 5′ capping et al. 97 apical meristem culture following antiviral agent and heat treatments kurniawan budiarto .et al of viral rnas (dawson & lozoya saldana 1984). it inhibited virus replication at the early stage by impairing synthesis of rnadependent rna polymerase and at a later stage by impairing synthesis of the coat protein (schuster & huber 1991). with the absent of or low concentration viral particles, the interfering physiological orientation of embryonic derived cells were overruled to the maximum growth of genotype response. during the experiment, no necrotic symptom used as indication of phytotoxicity was found in all varieties. though vegetal tissues tolerance to ribavirin is genotype dependent (sidwell 2005), the chemical concentration added into the media (40 mg/l) was considered proportional for chrysanthemum. these might refer to the fact that with such concentration range, phytotoxic symptoms were observed in other crops during virus elimination thus, inhibited or decreased plant cellular metabolic activity when applied in higher concentration than 30 mg/l (verma parmessur & saumtally the survival and death rates of plantlets in every duration of thermal treatments were varied among cultivars tested, though the trend was similar. during the first week of heat treatments, 24 36 % of treated plantlets were ceased, with cv. white reagent showing the least number of death plantlets compared to the others. the number of death plantlets was slightly decreased in the following week, 12 14 % (fig. 1). a short preconditioning incubation (one week with gradual increase at 30 -35 c) before the thermotherapy was apparently not sufficient for the plantlet to make suitable adaptation on higher temperature of heat treatment. though the heat tolerance of plants was indigenously specific, the mechanical adaptation of plants was timedepended on the degree of transition in regular change. the plantlet should be provided with sufficient precondition period with small and regular increase of temperature approaching the level of heat treatment to hinder high plantlet death at the early stage (manganaris 2003). through the small gradual increase of the temperature in longer period, the plantlet could establish physiological adaptation to the temperature level of 35 to 40 c. et al. et al. 2005; 2001). et al. application of thermotherapy 0 0 heat treatment duration (weeks) 0 2 4 0 1 2 3 white reagent yellow puma white puma figure 1. plantlet death rates of three chrysanthemum cultivars after one, two and three weeks of heat treatments. 98 biotropia vol. 18 no. 2, 2011 conversely from the first and second weeks, cv. white reagent showed higher plantlet death rates in the third week compared to cv. white puma and yellow puma (fig. 1). this period was considered critical, since plantlet death rates of all cultivars tested were also highest in these week (24 38 %). wang (2006) concluded that inside the culture flask or condition, a protected environment might lead to narrower adaptation of plantlets to the extreme conditions such as high temperature. this finding suggested that at the third week the toleration limit of chrysanthemum plantlet to heat treatments was obtained. after heat treatments, apical meristematic tissue of individual plantlets was inoculated into shoot induction media. period taken for bud initiation and plantlet height after three weeks transferring into regeneration media were observed different referring to the length of thermal treatments. shortest period for bud initiation and tallest plantlets were shown by the three weeks heat treatments in all varieties tested (table 2). slower growth rate on plantlets treated by one and two weeks thermotherapy indicated that physiologically, the plantlets were still in their suboptimal potential. these growth retardations were predictably affected by virus particles remained in the tissues which systemic persistently interfered with plant metabolism (marwoto . 2004; bhatthacharyya . 1990) in vitro et al et al table 2. buds initiation and plantlet height after three weeks transferring of three chrysanthemum varieties under different period of heat treatments. cultivars period of heat treatments (weeks) 1 2 3 bud initiation (days) *) white reagent 63.8 a 64.4 a 49.8 b white puma 64.4 a 59.6 b 48.2 c yellow puma 61.6 a 59.7 a 44.4 b plantlet height alter three weeks transfering into regeneration media (cm)*) white reagent 4,32 a 5,11 a 6,34 b white puma 4,36 a 4.82 a 6.84 b yellow puma 5,02 a 6,21 b 7,13 c *) remarks : values followed by different letters in the same rows differ significantly at lsd 5 %. elisa bioassay virus detection using direct elisa method was conducted to plant samples in all treatment combinations and spectro-photometerically revealed as absorbent values (table 3). these values indicated the existence of virus particles in plant tissues. the percentage of cvb-free plantlets increased accordingly to the lengthened heat treatments and application of ribavirin within the media. three weeks thermotherapy or supplemental antiviral followed by meristem culture successfully eliminated cvb from the infected chrysanthemum plantlets. 99 apical meristem culture following antiviral agent and heat treatments kurniawan budiarto .et al 0 the success of chemoand thermotherapy in eliminating cvb from infected plants in our works inferred that cvb particles and their persistence was highly affected by heat treatments at 38 40 c and antiviral application (ribavirin 40 mg/l). the failure of single method of meristem culture for producing virus free plantlets as also presented in the absent of ribavirin treatment indicated that the isolation of meristematic sites was not sufficient to totally free the tissue from virus particles.due to its small size, virus particles could traffic cell to cell even through branched plasmodesmata and infected the meristemal cells (laimer 2003). consequently, these naturally high antiviral-content tissues were also difficult to be isolated, since they were mostly microscopic and very sensitive (brown 1988). thus, combination of thermoor chemotherapy with meristem culture for virus elimination would be more promising, especially when mixed infection occurred or for those more persistent strains. et al. table 3. cvb detection by direct elisa and percentage of virus-free plantlets under thermoand chemotherapy treatments. treatments absorbance values*) percentage of cvb-free plantlet *) (%) heat treatment 1 week 0.06 – 0.11 41.6 2 weeks 0.03 – 0.09 72.3 3 weeks 0.02 – 0.07 100 chemotherapy using ribavirin 40 mg/l without ribavirin 0.06 – 0.12 43.6 with ribavirin 0.01 – 0.06 100 positive control 0.22 0 negative control 0.01 100 *) remarks : values derived from 36 plantlet samples in each treatment combination batthacharyya p, dey s, das n, batthacharyya bc. 1990. rapid mass propagation of by callus derived from stem and leaf explans. in . biotechnology unit. department of chemical engineering iit. kharagpur. india. p. 22 25. chen wq, sherwood jl. 1991. evaluation of tip culture, thermotherapy and chemotherapy for elimination of peanut mottle virus from . journal of phytopathology, 132 (3) : 230 236. clark mf, adam an. 1977. characteristic of the microplate method of enzyme lingked immunosorbent assay for the detection of plant viruses. journal of genetic virology, 34: 475 483. conclusions references under heat treatment, percentage of 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dependent on the concentration of ribavirin, 5azadihydrouracil as well as 1,5diacetyl-5azahydouracil. biochemestry phytology pflanz, 187: 429 438. sharma s, singh b, rani g, zaidi aa, hallan v, nagpal a, virk gs. 2007. production of indian citrus ringspot virus free plants of kinnow employing chemotherapy coupled with shoot tip grafting. agriculture 8 (1) : 1 8. sidwell rw, bailey kw, wong mh, barnard dl, smee df. 2005. and influenza virus-inhibitory effects of viramidine. antiviral research, 68 (1) : 10 17. simpkins i, walkey dga, neely ha. 1981. chemical suppression of virus in cultured plant tissues. annals of applied biology, 99: 161 169. parmessur y, saumtally a. 2001. elimination of sugarcane yellow leaf virus and sugarcane bacilliform virus by tissue culture. j. of food and agricultural research, 7 (3): 121 133. verma n, ram r, zaidi aa. 2005. in vitro production of prunus necrotic ringspot virus free begonias through chemoand thermotherapy. science horticulture, 103: 239 247. wang i, wang g, tang nhr, deng x, zhang h. 2006. effect of thermotherapy on elimination of apple stem grooving virus and apple chlorotic leaf spot virus for in vitro-cultured pear shoot tips. journal of the american society for horticultural science, 41(3): 1327 1329. zaitlin m, palukaitis p. 2000. advances in understanding plant viruses and virus diseases. annual review of phytopathol, 38 : 117 143. in vitro in vitro allium sativum journal of central european , in vitro in vivo 101 apical meristem culture following antiviral agent and heat treatments kurniawan budiarto .et al biotropia no. 9, 1996: 26 37 brown spot caused by curvularia spp., a new disease of asparagus b. salleh1, a. safinat1, l. julia2 and c.h. teo3 1 school of biological sciences, universiti sains malaysia, 11800 penang, malaysia 2 laboratory of plant pathology, agricultural research centre, p.o. box 3, 89207 tuaran, sabah, malaysia 3 laboratory of plant pathology, agricultural research centre, semogok, p.o. box 977, 93720 kuching, sarawak, malaysia abstract the distribution, aetiology and symptomatology of a new disease on asparagus ferns, which we have termed brown spot, is described. descriptions of and a key to identification of the causal organisms, curvularia brachyspora, c. eragrostidis, c. lunata and c. pallescens, are also presented. pathogenicity tests showed that c. lunata was the dominant and most virulent of the four species. inoculation with conidial suspensions or mycelial transfers through wounded ferns were more effective in inducing the disease than inoculations on unwounded ferns. this is the first record of c. brachyspora in malaysia and the first report of this disease on asparagus. key words: malaysia/plant diseases/brown spotjcurvularia brachyspora/curvularia eragrostidts/curvularia lunata/curvularia pallescens/asparagus. introduction asparagus (asparagus officinalis l.) was introducted into malaysia from taiwan in the 1950s. although it is a fairly new crop, asparagus has become a preferred vegetable especially by malaysians in the higher income groups. its caloric and nutrient contents are comparable to those of the four most popular local vegetables i.e. kailan (brassica alboglabra bailey), chinese cabbage (b. chinensis l.), cauliflower (b. oler-cea l. var. botrytis) and sawi (b.juncea bailey) (anon. 1985). during a series of investigations on causes of degeneration in asparagus farms established for experimental or commercial purposes throughout malaysia in 1989-90 (salleh 1990), a new disease was observed on all varieties. in this paper we report the distribution, aetiology and symptomatology of the disease. 26 brown spot caused by curvularia spp., b. salleh et at. materials and methods disease survey and aetiology eighteen asparagus farms established for experimental or commercial purposes throughout malaysia were visited and sampled at least twice in 1989-90 (fig. 1). the last three of the farms are situated in the east malaysia (malaysian territory of the borneo island). figure 1. asparagus disease sampling sites throughout malaysia (1989-1990) plant parts showing brownish lesions were collected and the causal organisms were isolated as previously described (salleh & sulaiman 1984). single conidia of the dominant isolates (curvularia spp.) which developed from the diseased materials were placed on potato-sucrose-agar (psa) plates and incubated under standard incubation conditions described earlier (salleh & sulaiman 1984). growth of the colonies was 27 biotropia no. 9,1996 measured after four days of incubation. curvularia species were identified using the diagnostic characters proposed by ellis (1971; 1976) and sivanesan (1987), and later confirmed by the commonwealth mycological institute, uk. conidial suspensions from monoconidial stock cultures were preserved in 15% glycerol in liquid nitrogen (salleh & strange 1988), and fresh monoconidial cultures on potato-dextrose-agar (pda) slants were covered with sterile mineral oils and kept at 4°c. pathogenicity test pathogenicity tests of selected isolates of all four species of curvularia isolated from asparagus were carried out on healthy 9 month-old asparagus seedlings, varieties mary washington (mw) and uc 157. the seeds were tested for fungal contamination by plating on psa plates. apparently healthy and non-cracked seeds were sown in sterile soil in polyethylene bags (diameter 20 cm) in a greenhouse with natural lighting for nine months. the seedlings were kept healthy by spraying a mixture of a fungicide (benomyl or propineb) and an insecticide (diazinon or aldrin) monthly until the seventh month. the plants were fertilized bimonthly with a granular npk (15:15:15) fertilizer. the first method of inoculation was conducted as follows : conidial suspensions of curvularia spp. were prepared by flooding 7 day-old psa plates with 10 ml sterile water containing 0.05% tween 80. after gentle agitations, the liquid was decanted and the concentration adjusted to 1 x 10 conidia/ml. inocula were sprayed in the evening onto aseptically wounded or unwounded ferns at the base, middle, branch and tip of plants at growth stage 5 or 6 (fully developed ferns) (bansal et al. 1986). wounds were made by pricking the plants with sterile needles. parts of the ferns were surface sterilized with 70% alcohol prior to wounding. control wounded or unwounded ferns were sprayed with sterile water. a second method of inoculation was carried out by placing mycelial plugs from the plates onto similar inoculation points of the aseptically wounded ferns. the inoculation points were wrapped with sterile moistened cotton wool and tied with an adhesive plastic for 48 h. similar points on unwounded stems and branches wrapped in the same manner served as controls. for both conidial spray and mycelial plug inoculations, mixed inocula which comprised of all four species of curvularia were also used on different set of plants throughout the experiments. plants in each polyethylene bag were covered with transparent plastic bags overnight. the plastic chambers were kept moist by spraying 10 ml sterile water into each chamber after inoculation. all treatments were replicated ten times (10 plants; one in each polyethylene bag). symptom development was observed every other day. the 28 brown spot caused by curvularia spp., — b. salleh et al. length of the lesion was measured four weeks after inoculation. reisolations were made from all infected and control ferns and the resulting fungi subcultured on psa and reidentified. results and discussion disease survey and aetiology a new disease with typical greyish brown spots was observed on the ferns of all varieties of asparagus planted in all 18 sampling sites throughout malaysia (table 1). the disease was apparently more severe on asparagus planted at lower altitudes (16 sites) where mean daily temperatures were significantly higher than those of the same variety at higher altitudes (2 sites: cameron highlands and kundasang). young (<2 years) asparagus fields were generally found with only a few infected ferns while in older fields, particularly those at the lower altitudes, 10 25% of the older ferns had been attacked. it was not clear whether the increase in disease at higher temperature resulted from increased pathogen virulence or increased plant susceptibility. as is common for many necrotic diseases, both relative humidity and temperature probably play important roles in disease development. sixty percent of the fungal isolates recovered from asparagus ferns with small oval brown spots were curvularia spp., while alternaria spp. and fusarium spp. comprised the remaining 40%. the role of alternaria spp. and fusarium spp. in the asparagus brown spot complex is presently being studied and will be described elsewhere. based on cultural characteristics and conidial structures, the curvularia isolates were identified as curvularia lunata (wakker) boedijn, curvularia pallescens boedijn, curvularia eragrostidis (henn.) mayer and curvularia brachyspora boedijn. perfect stages of the fungi were not observed either in culture or on infected asparagus plants in nature. amongst the species of curvularia isolated from asparagus in malaysia, c. lunata was the most frequent (85 %), followed by c. pallescens (32.0%), c. eragrostidis (18.2%) and c. brachyspora (11.5%). the description and a key for identification of the curvularia species isolated from asparagus in malaysia are as follows : curvularia brachyspora boedijn (fig. 2) colonies blackish grey, black, cottony or hairy; growth rate 5.5 6.1 cm after four days. conidia 16.5 26.4 x 9.9 16.5 um, dark or light brown, solitary, simple, some slightly curved, clavate, ellipsoidal, broadly fusiform, 3-septate, dark bands at the septa, mostly truly median and asymmetrical. the third cell from the base is conspicuously 29 biotrop1a no. 9, 1996 larger, broader and darker than the others. conidiophores macronematous, monone-matous, straight, flexous, often geniculate, sometimes nodose, brownish and smooth. although the type species of curvularia brachyspora was originated from java, the presence of this species has never been reported in malaysia. elsewhere, it was isolated from agave, digitaria, dracaena, olea, oryza, saccharum, triticum and air (ellis 1971; sivanesan 1987). table 1. distribution of asparagus brown spot caused by curvularia spp. at 18 sampling sites in malaysia 1989-90) curvularia eragrostidis (henn.) meyer (fig. 3) colonies blackish grey, black, hairy or cottony; growth rate 5.7 6.4 cm after four days. conidia 16.5 24.75 x 9.9 16.5 um, dark brown, solitary, simple, broadly ellipsoidal, 3-septate, symmetrical, dark bands at the septa are quite thick and truly 30 brown spot caused by curvularia spp., b. salleh et al. figure 2. curvularia brachyspora boedijn a: conidia; b: conidiophores; c: cmamydospores figure 3. curvularia eragrostidis (henn.) meyer a: conidia; b: conidiophores; c: chlamydosporcs 31 31 biotropia no. 9, 1996 median. usually the second and third cells are the same size, and broader than the others. conidiophores macronematous, mononematous, mostly straight, often branched, geniculate, often nodose, brown and usually smooth. c. eragrostidis has been isolated from a wide variety of plant hosts and other substrates worldwide, including malaysia. it causes minor leaf spots on several grami-nicolous hosts (hawksworth 1990). figure 4. curvularia lunata (wakker) boedijn a: conidia; b: conidiophores; c: chlamydospores curvularia lunata (wakker) boedijn (fig. 4) colonies are dark grey or black, effuse, cottony or velvety; mycelium immersed; growth rate 6.0 7.6 cm after four days. stroma dark brown. conidia 14 28 x 7 13 µm, dark brown, solitary, often curved on the middle septum (not median); predominantly 3-septate and rounded at the apex. very few (<3%) triradiate stauroconidia. conidiophores dark gray or brownish black, macronematous and mononematous, po-lytretic, straight or flexous and smooth. c. lunata is often associated with grain moulds of sorghum and millet, and also causes a leaf spot on sorghum (singh 1982). c. lunata is common and widespread in the tropics, including malaysia, and may be recovered from many different substrates (ellis 1971). 32 figure 5. curvularia pallescens boedijn a: conidia; b: conidiophores; c: chlamydospores curvularia pallescens boedijn (fig. 5) colonies usually grey or pale grey, effuse, cottony or velvety; mycelium immersed; growth rate 7.0 7.7 cm after four days. stroma gray or pale gray. conidia 7 9 x 3 4 µm, pale or very pale brown, solitary, usually straight or only slightly curved; predominantly 3-septate. conidiophores gray or pale gray, macronematous or monone-matous, polytretic, straight or flexous, smooth and brown. c. pallescens is common on many different substrata especially in the tropics (ellis 1971; sivanesan 1987). key for the identification of curvularia spp. isolated from asparagus ferns showing brown spot in malaysia conidia remaining smooth-walled and predominantly 3-septate ............................................................................................................................ 1 1. conidia middle septum truly median ....................................................................... 2 conidia middle septum not median ........................................................................ 3 33 brown spot caused by curvularia spp., b. salleh el al. biotropia no. 9, 1996 2 conidia symmetrical, dark bands at the septa are quite thick, usually the third and the second cells are of the same size ............................................................ eragrostidis conidia asymmetrical and mostly truly median .................................... brachyspora 3. conidia straight or slightly curved, 7-9 x 3-4 u,m ....................................................................................................... pallescens conidia straight or curved, sometimes have triradiate stauroconidia, 14-28 x 7-13 (im .............................................................................................. lunata pathogenicity test pathogenicity tests showed that all isolates of the four curvularia species obtained from asparagus showing brown spot symptoms in malaysia were pathogenic to both varieties of asparagus i.e. mw and uc 157 tested. once the symptoms occurred, there was no difference in the visible symptoms caused by the four species of curvularia. the first symptom of asparagus brown spot appeared as a water-soaked lesion which later developed into a brown spot with a brownish red margin and brownish grey center. the slighty sunken spot, particularly that caused by c. lunata, gradually became dark brown and developed from 0.7 to 5.0 cm within 28 days. the spot developed upwards and sideways along the circumference and coalesced, hence girdling the stem. at this stage, the entire stem appeared brown with irregular dark-brown patches (fig. 6), quite similar to the symptoms observed on rice stem infected by blast (pyricularia oryzae). under favourable conditions, heavily infected ferns were easily broken and eventually killed. no symptoms were observed on the spears and crowns. inoculations of wounded or unwounded plants with either conidial suspensions or mycelial plugs on fully developed asparagus ferns resulted in symptoms identical to those seen in the field. c. eragrostidis, c. pallescens and c. brachyspora only caused milder symptoms on ferns inoculated through wounds and the first symptoms appeared later ( ± 23 weeks) than those induced by c. lunata. isolates of these three species did not produce visible symptoms on unwounded plants even up to 4 weeks after inoculation. reisolation from artificially infected ferns showing brown spot symptoms yielded only the pathogens that were used for inoculation. reisolation from plants infected with a mixture of all four species always yielded c. lunata. therefore we concluded that c. lunata was the most dominant and virulent species of curvularia in this context. these greenhouse results are similar to the disease incidence and severity seen under field conditions. wound inoculation using fungal mass was the best (p<0.01) of the four methods tested since the first typical symptom of brown spot i.e. watersoaked spot, caused by c. lunata was observed within seven days of inoculation. the ino 34 figure 6. an advanced stage of brown spot on asparagus stem caused by curvularia lunata. note the irregular dark-brown patches. culation was more effective on the upper parts and branches than on the base and middle parts of the ferns. a wound was not an absolute requirement for disease development since the pathogens also may enter through stomata, and/or natural and accidental wounds. in other diseases of asparagus, e.g. dead stem caused by fusarium culmorum, inoculation succeeded only when the stems were wounded (van bakel & krom-kerstens 1974). infection by the purple,spot pathogen, stemphylium vesicarium on asparagus cv. mw was more numerous arfd occurred at shorter wetting durations on wounded than on 35 brown spot caused by curvularia spp., b. salleh et al. biotropia no. 9,1996 unwounded plants (johnson & lunden 1986). they also showed that wounds produced by blowing sand could serve as entry points for the infection of the pathogen. in scanning electron micrographs of fusarium proliferation, the causal agent of reddish-brown rot of asparagus, nik norulaini & salleh (1990) clearly showed that this pathogen successfully penetrated its host only through wounds and stomata. in all asparagus fields visited during the present survey, we observed numerous bark-eating caterpillars and mollusks that could easily have created wounds that curvularia spp. and other pathogens could use to enter the host. therefore, effective pest control probably will lead to significant reduction of brown spot and other diseases of asparagus in the field. yield losses occurring as a result of infections by curvularia spp. on asparagus in malaysia have not been ascertained, but based on our present survey we would estimate between 15 35%. based on these symptoms and aetiology, we proposed that this disease complex on asparagus ferns denoted as brown spot. this report is the first on brown spot of asparagus in malaysia, and to our knowledge, anywhere in the world. acknowledgements this research was supported by the government of malaysia (irpa 123/31037 2401). we thank the owners and persons in charge of the eighteen asparagus sampling sites. references anon. 1985. tanaman asparagus tanah rendah. risalah pertanian bil. 55. jabatan pertanian semenanjung malaysia, kuala lumpur. 27 p. bansal, r.k., s.a. menzies and p.o. broadhurst. 1986. screening of asparagus species for resistance to stemphylium leaf spot. new zealand journal of agricultural research 29: 539-545. ellis, m.b. 1971. dematiaceous hyphomycetes. commonwealth mycological institute, london. 452-459 pp. ellis, m.b. 1976. more dematiaceous hyphomycetes. commonwealth mycological institute, london. 404-411pp. hawksworth, d.l. 1990. cmi description of fungi and bacteria. mycopathologia 111: 109-130. johnson. d.a. and j.d. lunden. 1986. effects of wounding and wetting duration on infection of asparagus by stemphylium vesicarium. plant disease 70: 419-420. 36 brown spot caused by curvularia spp., b. salleh et al. nik norulaini, n.a.r. and b. salleh. 1990. in vitro inoculation of asparagus tissue culture plantlets with vegetative hyphae of fusarium proliferatum. in: proceedings of the 3 rd international conference on plant protection in the tropics, genting highlands, malaysia, pp. 127-129. salleh, b. 1990. crown rot caused by fusarium proliferatum, a new disease of asparagus in malaysia. in: proceedings of the 3 rd international conference on plant protection in the tropics, genting highlands, malaysia, p. 343 (abstract). salleh. b. and b. sulaiman 1984. fusaria associated with naturally diseased plants in penang. journal of plant protection in the tropics 1: 47-53. salleh, b. and r.n. strange. 1988. toxigenicity of some fusaria associated with plant and human diseases in the malaysian peninsula. journal of general microbiology 134: 841-847. singh, r.s. 1982. plant pathogens-the fungi. oxford and ibh publishing, london. 342pp. sivanesan, a. 1987. graminicolous species of bipolaris, curvularia, drechslera, exserohilum and their teleomorphs. mycological papers 158. pp. 261. van bakel, j.m.m. and j.j. krom-kerstens. 1974. dead stem disease of asparagus caused by fusarium culmorum. netherlands journal of plant pathology 80:104-109. 37 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf microsoft word 54 biotropia no. 24, 2005 : 54 61 effect of 3, 5, 3'-triiodothyronine (t3) hormone on nucleic acid and protein content of the muscle and the growth of giant gouramy, osphronemus gouramy lac. sri handayani1, m. zairin jr.2, ing mok.oginta2 and maria bintang3 ' department ofaquaculture, faculty of fisheries and marine science, mulawarman university, samarinda, indonesia 2 department ofaquaculture, faculty of fisheries and marine science, bogor agricultural university, bogor, indonesia 3 department of biochemistry, faculty of science and mathematics, bogor agricultural university, bogor, indonesia abstract this experiment was conducted to study the effect of 3, 5, 3'-triiodothyroninc (tj) hormone on nucleic acid and protein content of the muscle and the growth of giant gouramy, osphronemus gouramy lac. five experimental diets, which contain isocaloric diets, but different in t3 hormone level were used in this experiment (0.0, 2.5, 5.0, 7.5, and 10.0 mg t3 hormone/kg diet). the experimental diets were tested to three different groups offish for sixty days feeding trial. fish body weight in groups i, ii, and iii were 0.39-0.42 g/fish; 19.11 -21.99 g/fish, and 37.52-40.79 g/fish, respectively. the results showed that the highest rna, dna concentration and rna/dna ratio of the muscle were produced by 10.0 mg t3 hormone/kg diet for group i and ii; and 2.5 mg t3 hormone/kg diet for group iii. similar results also were found for the protein content of the whole body, protein retention, and the daily growth rate of the fish. key words : osphronemus gouramy/ti hormone/nucleic acid/protcin/growth introduction giant gouramy is well known as a slow growing fish that has been traditionally cultured for a long time in west java, indonesia. intensification of the culture of this fish has been started recently. however, our studies indicated that the growth of the fish could be enhanced by fulfilling their nutrient requirements (mokoginta et al. 1994, 1995a & b, 1996). in addition to fulfilling their nutrient requirements, growth could be enhanced by hormone treatment as suggested by matty (1985). some studies on thyroid hormone treatment revealed that the hormone plays an important role on somatic growth of fish (donaldson et al. 1979; fagerlund et al. 1984; matty et al. 1982). it was assumed that thyroid hormone could activate protein synthesis in liver and muscle cells (jackim and la roche 1973; narayansingh and bales 1975; medda and ray 1979; matty et al. 1982) by increasing rna and dna concentration of liver and muscle cells. woo et al. (1991) showed that administration of 3, 5, 3'triiodothyronine (t3) hormone in diet 54 biotropia no. 24, 2005 of red sea bream increased the growth rate, feed efficiency, digestive enzymes activity in the intestine and activities of other key enzymes in various pathways of carbohydrate metabolism. t3 affects protein synthesis by biphasic mode i.e., anabolic at lower dose, but catabolic at higher dose (matty and lone 1985; medda and ray 1979). medda and ray (1979) showed that the administration of thyroxine (t4) hormone at the dose of 2 or 4 mg/g body decreased protein accumulation and rna content in the liver and brain, whereas dosage of 1 mg/g body weight increased protein accumulation and rna content in liver and brain. therefore, in this experiment, we study the effect of the administration of tj hormone in diet on nucleic acid and protein content of the muscle and the growth of giant gouramy. materials and methods fish experimental fish were obtained from a fish farmer in parung. the fish were divided into three groups based on body weight. group i was of 0.39 0.42 g body weight, group ii was of 19.11 21.99 g body weight and group iii was of 37.52 -40.73 g body weight. fish were reared in aquaria (50 x 30 x 30 cm, 20 1 volume) with ten fish in each aquarium (group i), while for fish of group ii and iii with one fish in each aquarium. fish were fed three times daily to satiation for 60 days. to maintain water quality, 70 80 % of total water volume was replaced and feces were siphoned out every day. aquaria were cleaned and water was totally replaced once a week. this experiment was conducted at water temperature of 27 30°c; ph 7.5 8.0; do 6.28 7.2 ppm; and ammonia 0.08 0.13 ppm. diets semi-purified diets were used in this study. the fish were fed with a diet of 43 % protein level (group i), while the other fish (groups ii and iii) were fed with 32 % protein level. all diets have protein-energy ratio of 8 kcal de/g protein. the different protein levels between groups were based on the protein requirement of giant gouramy (mokoginta et al. 1994,1995 a & b, 1996). the diet was supplemented with 0, 2.5, 5.0, 7.5, and 10 mg of t3/kg. before mixing, t3 was dissolved in 70 % alkaline alcohol (33 ml 90% ethanol 12 ml 0.1 n naoh) and the solution was sprayed on the diet. the control diet was sprayed with alkaline ethanol without t3. following supplementation of t3, the diets were air dried and then stored in a freezer (-20°c) until feeding. the composition of diets for fish in group i, ii and iii are shown in tables 1 and 2, respectively. 55 data collection and chemical analysis each group of fish was weighed and taken for proximate analysis at the beginning and the end of the experiment. rna and dna concentrations were determined at the end of the experiment. proximate analyses were determined using methods as described by takeuchi (1988). analysis of rna concentration was determined by the orcinol method, while for dna concentration by diphenilamin method (plummer 1979). calculation and statistical analysis daily growth rate (dgr) was calculated according to equation of huisman (1976) : wt = (wo + 0,01 a)1 where wo and wt are initial and final mean of body weight, respectively, t is the number of days, and a is daily growth rate (%/day). protein retention was calculated according to the equation of takeuchi (1988): protein retention (%) =( total protein content of body (final) total protein content of body (initial)) (g)/ total protein consumed (g) x 100. the study was designed using complete randomized design with five treatments and three replications. the effect of t3 hormone on protein retention, rna and dna concentration, and rna/dna ratio and growth rate were evaluated by using analysis of variance followed by polynomial orthogonal test (steel and torrie 1993). 56 biotropia no. 24, 2005 results and discussion rna and dna concentrations and rna/dna ratio in muscle are shown in table 3. the table shows that rna/dna ratio increased following t3 levels supplemented into the diet. increasing t3 level from 2.5 mg t3/kg diet to 10 mg t3/kg diet increased rna/dna ratio in groups i and ii. the highest level of t3 hormone (10 mg t3/kg diet) caused an increase in rna/dna ratio to 10.8 % and 4.4 % over control in groups i and ii, respectively. in group iii, increasing rna/dna ratio (2.3% over control) was obtained at the level of 2.5 mg t3/kg diet, but the effect of 3,5,3'-triidothyronine (t3) hormone sri handayani et al. several studies showed that rna concentration and rna/dna ratio were closely related to growth and nutritional status of fish. therefore, rna concentration and rna/dna ratio could be used as an indicator of fish growth and nutritional status. it is commonly accepted that rna concentration in liver or muscle of fish may reflect protein synthesis, where increase in rna concentration was followed by a rise in protein synthesis (yang and dick 1993). rna/dna ratio is index of metabolic activity and change in rna/dna ratio reflects change in protein synthesis (brafield 1985; widler and stanley 1983). correlation between administration of t3 and protein synthesis might be explained by the effects of t3 hormone on the increase of rna concentration through interactions between t3 hormone and receptor in nucleus which activated enzyme for rna synthesis (matty etal. 1982; djojosoebagio 1990). this study showed that administration of 10 mg t3/kg diet in groups i and ii increased rna and dna concentration, rna/dna ratio to 26.6 %, 14.4% and 10.8% over control, respectively, for group i; and 9.6%, 4.95%, and 4.4% over control, respectively, for group ii. while in group iii, the highest value of rna/dna concentrations and its ratio were reached at 2.5 mg t3/kg diet i.e. 5%, 2.5% and 2.3% over control, respectively (table 3). as mentioned above, increasing rna concentration in muscle was followed by a rise in protein synthesis, rna/dna ratio and growth rate of fish. alien et al. (1979) defined growth as increasing mass by two fundamental biological processes; protein accretion and proliferation cell. this study, clearly showed that administration of 10 mg t3/kg diet in groups i and ii increased rna concentration (26.6 % and 9.6% over control, respectively) and protein retention (30 % and 16.5 % over control, respectively). the ability to retain the body protein was followed by an increase in growth rate offish (9.3% and 14.4 % over control, respectively) and also reflected by an increase in rna/dna ratio (10.8 % and 4.4% over control, respectively) (tables 3 and 4). in group iii, higher rna concentration was attained at 2.5 mg t3/kg diet and followed by increasing body protein level (5 % over control). furthermore, protein retention and growth attained a higher value, an increase of 10 % and 17.5 % over control, respectively (tables 3 and 4). the effect of t3 hormone on protein synthesis is biphasic, i.e. anabolic at low doses and catabolic at higher dose ( medda and ray 1979). as rna and protein concentrations in a cell depend upon the synthetic and degradation rate, and as the thyroid hormones increase the turnover rate of this cellular constituents, it is possible that at higher doses the turnover rate becomes much higher and the degradation rate surpasses the synthetic rate (goldberg et al. 1980 in matty et al. 1982). in this study, biphasic effect of t3 was only shown in group iii, but not in other groups. in conclusion, all groups in this study showed that the effect of t3 hormone administration on rna and dna concentration, rna/dna ratio, body protein level, protein retention, and daily growth rate depend on fish size, in which the difference of potential growth rate was produced by fish size. 58 * percentage of increase/decrease compared to control (treatment a). 59 effect of 3,5,3'-triidothyronine (t3) hormone sri handayani et al. references alien, r.e., r.a. mcrkel and r.b. young. 1979. cellular aspect of muscle growth : myogenic cell proliferation. j. anim. sci. 49 : 115 127. brafield a. e. 1985. laboratory studies of energetics budgets. in tytler, p. and p. calow (eds), fish energetics, new perspectives. croom helm, london, p. 257-281. djojosocbagio s. 1990. fisiologi kclenjar endokrin i. pusat antar universitas ilmu hayat. institut pertanian bogor. 247 p. donaldson e.m., u. h. m. fagcrlund, d.a. higgs, j. r. mcbridge. 1979. hormonal enhancement of growth. in fish physiology. i w s woar, d. j. randall, and j.r. brett (eds.), vol. 8. academic press, new york. p. 453-597. fagerlund u. h. m., i. mccallum, d. a. higgs and j. r. mcbride. 1984. diet composition as a factor in the anabolic efficacy of 3, 5, 3'triiodo-l-thyronine administrated orally to steelhead trout (salmo gairdneri). aquaculturc, 36 : 49-59. huisman, e.a. 1976. food convcrtion cfficicnces at maintenance and production levels for carp, cyprinus carpio l and rainbow trout, salmo gairdneri. aquaculturc, 9 : 259 273. jackim and la roche. 1973. protein synthesis in fundulus leteroctilus muscle. comp. biochem. physiol. a., 44:851-866. matty a. j. 1985. fish endocrinology. croom helm, london. 267 p. matty a. j. and k. p. lone. 1985. the hormonal control of metabolism and feeding. in: fish energetics. tytler p. and p calow (eds.). new perspectives, croom helm, london, p. 185-209. matty a. j., m. a. chaudhry, and k. p. lone. 1982. the effect of thyroid hormone and temperature on protein and nucleic acid content of liver and muscle of sarotherodon mossambica. gen. comp. endocrinol., 47 : 497 507. medda, a. k. and a. k.. ray. 1979. thyroxine and muscle of lata fish, gen. comp. endocrinol., 9 : 473-480. mokoginta i., m. a. suprayudi, and m. setiawati. 1994. kcbutuhan nutrisi ikan gurame (oshpronemus gouramy lac.) untuk pertumbuhan dan rcproduksi. lap. pen. hb. ii/2, dirbinlitabnas, dikti, dcpdikbud. ipb. mokoginta i., m.a. suprayudi and m. setiawati. 1995a. kebutuhan optimum protein dan energi makanan bcnih ikan gurame (oshpronemus gouramy lac.). jur. perikanan indonesia, 1 (3) : 82 94. mokoginta i., m. a. suprayudi, and m. setiawati. 1995b. kebutuhan nutrisi ikan gurame (oshpronemus gouramy lac.) untuk pertumbuhan dan reproduksi. lap. pen. hb. 11/3, dirbinlitabnas, dikti, depdikbud. ipb. mokoginta i., m. a. suprayudi, and m. setiawati. 1996. kebutuhan nutrisi ikan gurame (oshpronemus gouramy lac.) untuk pertumbuhan dan reproduksi. lap. pen. hb. ii/4, dirbinlitabnas, dikti, dcpdikbud. ipb. narayansingh t. and j. g. eales. 1975. effect of thyroid hormones on in vivo [1 14c] l icucine incorporation in plasma and tissue protein of brook trout (salvelinus fontinalis) and rainbow trout (salmo gairdneri). comp. biochem. physiol. b., 52: 399 405. 60 biotropia no. 24, 2005 plummer, d.t., 1979. an introduction to practical biochemistry. mcgraw-hill. new delhi. 362 p. steel r.; g. d., and j. h. torrie. 1993. prinsip dan proscdur statistika. pt gramcdia pustaka utama, jakarta. 748 p. takcuchi, t. 1988. laboratory work chemical evaluation of dietary nutrients. in: fish nutrition and mariculturc. watanabc (ed.). jica text book. the general aquaculture course, japan, p. 179-190. widlcr i. b. and j.g. stanley. 1983. rna dna ratio as an index to growth in salmonid fishes in the laboratory and in stream contaminated by carbaryl. j. fish biol., 22 : 165-172. woo, n. y. s., a. s. b. chung, and t. b. ng. 1991. influence of oral administration of 3, 5, 3'-triiodothyroninc on growth, digestion, food conversion and metabolism in the undcrycarling red sea bream (crysophrys major). j. fish biol., 39 : 459-468. yang x. and t. a. dick. 1993. effect of dietary fatty acids on growth, feed efficiency and liver rna and dna content of arctic charr, salve/inns alpinus (l). aquaculture, 116 : 57 70. 61 54.pdf 55.pdf 56.pdf 57.pdf 58.pdf 59.pdf 60.pdf 61.pdf biotropia no. 7, 1994: 30-40 notes on some growth characteristics of mikania cord at a (burm. f.) b.l. robinson*) b.t. mercado institute of biological sciences, university of the philippines at los banos, college, laguna, philippines abstract mikania follows a sympodial dichotomy pattern of branching. both stem (branches) and leaves give rise to new plants with relative ease. internodes also root easily but do not give rise to new plants. flower formation and seed germination are strongly influenced by light. numerous seeds are produced but only few are filled; still fewer are the seeds that germinate. the period from early emergence to about the 3-leafed stage is most critical for survival of the new plant. keywords: mikania cordata, growth, weed physiology introduction mikania cordata (burm. f.) b.l. robinson, a creeping woody perennial is popularly known by its local names of climbing hempvine and mile-a-minute plant. it is described systematically in the compendium of the world's worst weeds (holm et al. 1977). the stem (and its branches) and the mature leaves easily form roots when these come in contact with the soil (figs, la & b). the leaves on the creeping stem or branch are strongly negatively geotrophic. if a portion of the stem or branch is twisted to bring the leaves to face the soil, overnight the leaves would be upright, held up by the petioles that had curved upward (fig. 2). pattern of branching branching follows a sympodial dichotomy (fig. 3). the stem produces a terminal shoot which dries up after some time. at its closest node emerges the first pair of branches which elongates. on these branches are borne several nodes. the *) the project was supported by a grant from the national research council of the philippines (nrcp). 30 notes on some growth characteristics of mikania cordata b.t. mercado la ib fig. 1. new plants arising from stem (a) and leaf (b) cuttings fig. 2. the upright bended petioles and nodal roots pointing upward indicate the strong negative geotrophic characteristic of leaves and stem 31 biotropia no. 7, 1994 fig. 3. the sympodial dichotomous branching in mikania. the numbers indicate approximate length of the branches axillary buds on these nodes emerge to form new shoots. in most cases however, only one of the pair develops into a new branch, on whose nodes again emerge pairs of shoots, again with only one of the pairs elongating into new branches. this pattern is repeated thereafter, ultimately resulting in the entangled mass of branches which may be circular to the right or circular to the left (holm et al. 1977). the terminals of the main branches, on the other hand, die off after some time, permitting the side branches to elongate rapidly. thus this pattern of branching makes possible the continuous growth and rejuvenation of the plant. with time the stem progressively becomes woody. considering the numerous branches formed, and the bulk of leaves borne by these branches, this growth pattern allows the plant to produce a dense mat of foliage in quite a short time. 32 144.2 notes on some growth characteristics of mikania cordata b.t. mercado rate of stem elongation of plants from leaf and stem cuttings the data in figure 4a represent the observations from 10 plants grown from leaf cuttings, while those in figure 4b, from 13 plants grown from stem cuttings. in both cases, the first branch that came out from the rooted cutting was tagged for the daily or weekly measurements. the branches of the leaf cuttings elongated much more rapidly than the similar branches of the stem cuttings. these were at its longest on the 26th day after the first measurement. these ceased to elongate after this date. three (3) tagged branches of the stem cuttings died off 39 days after the initial measurement, while the rest, after the 68 th day. fig. 4 rate of elongation of the first branch from leaf (a) and stem cutting (b) 33 biotropia no. 7, 1994 effect of sunlight on rate of stem elongation three plants raised from seeds were grown under direct sunlight and another three plants, under partial shade. (a makeshift enclosure roofed by 2 layers of fishnets served this purpose). the plants were observed daily for 21 days after the 5-leafed stage. as before, the first branch that emerged was tagged and measured daily until the elongation of the branch decreased or stopped in any of the light treatments. under partial shade (fig. 5a) the branches elongated much more rapidly than those under direct sunlight (fig. 5b). its elongation continued even after the 20th day, while the elongation of branches under direct sunlight completely stopped on the 18th day after the initial measurement. the number of nodes borne on the tagged branches of both sets of plants did not differ very much from each other, although node count was slightly higher in the branches of the plants under partial shade. fig 5. elongation of the first branch from a seed-grown plant exposed to (a) partial shade and (b) direct sunlight 34 notes on some growth characteristics of mikania cordata b.t. mercado the biomass productions after a six-month growth period under partial shade or direct sunlight of plants grown from cuttings are given in table la (stem cutting) and in table 1b (leaf cutting). in both light treatments, the shoot dry weight of the plants under partial shade was 2-5 times higher than those of the plants grown under direct sunlight. light apparently did not influence the dry matter production of the roots. surprisingly, however, the root system of the plant was not profuse and long as might be expected of a plant capable of producing such dense vegetative growth (fig. 6a). this may be due in part to the fact that the creeping stem or branch readily produces two kinds of roots: the usual nodal roots, and the internodal roots (fig. 6b). undoubtedly, anchorage as well as absorption of nutrients and water is a function well carried out by these stem-borne roots. there are not very many creeping plant species, succulent or woody, that develop internodal roots. on the whole, the plants grown under partial shade are much more vegetative, luxuriant and green than the plants grown under direct sunlight. the plants under partial shade remain vegetative. on the other hand, those which are exposed to sunlight start to flower in mid november until late january. field observation also showed that even in the same plant, the shaded portion remains vegetative while the upper portion exposed directly to sunlight produces flowers profusely. the seed the seeds are elongated, 1.5-1.8 mm in length and about 0.3-0.4 mm at its widest. these are black, with a longitudinal ridge in the middle, tufted at one end with pappus (about 40 50 hairs). the pappus adds about 3.0 mm to the length of the seed. the seed is filled with white endosperm. red ants are common foragers of ungerminated seeds. the seed dries up easily if kept in the open air. it has a very short period of viability. table la. dry weight (g/p) of mikania grown from stem cuttings under partial shade or direct sunlight (6 feb.-sept. 1991) plant partial shade direct sunlight no. shoot root shoot root 1 135.7 3.2 38.5 2.2 2 136.0 2.3 17.2 1.4 3 176.6 3.1 27.0 2.1 4 95.6 2.2 30.0 3.0 5 176.6 1.4 25.6 1.4 average 144.1 2.44 27.66 2.02 35 biotropia no. 7, 1994 table 1b. dry weight (g/p) of mikania grown from leaf cuttings under partial shade or direct sunlight (11 march-9 sept. 1991) plant partial shade direct sunlight no. shoot root shoot root 1 170.5 2.5 46.0 2.0 2 81.5 2.3 40.0 1.3 3 63.5 1.4 43.0 2.3 4 65.6 2.2 35.0 1.6 5 85.3 3.8 43.5 2.9 average 93.28 2.44 41.5 2.02 36 fig. 6a. the main root system of mikania plant fig. 6b. the nodal and internodal roots of a creeping stem of mikania seed count and seed germination a panicle has as many as 134-428 seeds (table 2), but the number of filled seeds is very low (6.52 27.62%). similar observation was reported by wirjahardja (1976) in the three mikania species common in indonesia. fifteen (15) filled seeds were sown in petri dish lined with moist filter paper. four (4) dishes were completely wrapped with two layers of carbon paper, while another four (4) uncovered dishes were placed on a lighted laboratory table. germination took place within three days in the light-exposed dishes. the carbon paper-wrapped dishes, opened seven days after sowing the seeds, showed complete failure of germination. the germination of the light-exposed seeds (table 3) was very low. repeated trials failed to exceed 50% germination even when the germination period was extended for another week. soil germination trials showed that seeds slightly buried into the soil (sticking the whole 1.5 — 1.8 mm length of the seed into the moist soil) failed to germinate, while seeds, laid flat on its side on the surface of moist soil germinated within a few days. . , • 37 notes on some growth characteristics of mikania cordata b.t. mercado biotropia no. 7, 1994 table 2. number of seeds (cypsela) per panicle panicle no. filled seeds unfilled seeds total % filled 1 21 301 322 6.52 2 41 227 268 15.29 3 25 183 208 12.02 4 25 317 342' 7.31 5 37 97 134 27.61 7 43 385 428 10.04 8 39 239 278 14.03 table 3. germination of mikania seed (15 seeds per petri dish) replication no. of seeds germinated germination % 1 4 2 4 3 6 4 6 26.6 26.6 40.0 40.0 the above observations suggest that although a mikania plant (one exposed to sunlight) has the propensity to produce numerous seeds which could be dispersed at great distance, only a handful of these seeds are filled, and only a few of them are viable. for the viable seeds to germinate light is indispensable. the seedling the seedling emerges from the pole directly opposite the pappus-bearing pole of the seed. the thin, long primary root comes out first. the root tip is distinctly covered with a black root cap. conspicuous is the tuft of short, fine and sticky roots borne at the base of the hypocotyl. these roots would later on constitute the root system of the plant, replacing the long primary root that dies off after some time. the hypocotyl is much bigger in diameter than the spindly primary root but a little shorter. the hypocotyl of the newly emerged seedlings had an average length of 0.97 cm. in another 12 days, however, it had already an average length of 21.83 38 notes on some growth characteristics of mikania cordata b.t. mercado cm, about twice longer than the primary root. at one end of the hypocotyl the pair of round, green, succulent cotyledons is borne. the cotyledons remain intact on the seedling until the young plant has attained the 3-leafed stage. the seedling, from germination to the early early 3-leafed stage is most vulnerable to wilting. however, once it has survived this critical period it develops rapidly into a mature and highly vegetative plant. summary mikania has a well developed and efficient means of vegetative reproduction (fig. 7). a portion of the mature stem or branch (iv-1), as long as it bears a node, easily gives rise to a new plant. with similar ease, the mature leaves can also give rise to new plants (iv-3). an internodal stem or branch (one which does not bear a node), easily forms roots but does not give rise to a new plant (iv-2). fig. 7. the life cycle of mikania cordata 39 biotropia no. 7, 1994 the development of the plant, whether derived from cuttings (i) or from seeds (iii) is strongly influenced by light. the plant grown under partial shade elongates very rapidly, is dark green and luxuriant. it remains however, vegetative (i-b). in contrast, the plant grown under direct sunlight, develops less densely but comes to profuse bloom in mid-november until late january (i-a). numerous seeds are produced but only a few are filled, and fewer still are viable. the pappus-bearing, minute seeds (ii) are dispersed by wind. for the seed to germinate, light is indispensable. for the seedling to survive it must be located in a moist and shaded area. references . holm, l.g., d.l. plucknett, j.v. pancho, and j.p. herberger, 1977. the world's worst weeds. university press. hawaii, honolulu. wirjahardja, j. 1976. autecological study of mikania spp. proceedings of the fifteenth asian-pacific weed science society conference. tokyo, japan. asian-pacific weed science society. 40 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf 74 biotropia vol. 15 no. 1, 2008 recovery of more than 10 years-drying monascus cultures and its purification methods from fungal and bacterial contamination nandang suharna microbiology division, research center for biology, indonesian institute of sciences (lipi), cibinong, jakarta, indonesia abstract this study was carried out to understand the recovery capability of more than 10 years drying monascus cultures. a new simple purification technique from fungal contamination using ethanol-soaking treatment was also reported as a part of this study. the result showed that all drying cultures were recovered well and retained their characters such as good growth, pigmentation and production of fruit bodies (ascomata), sexual spores (ascospores) and asexual spores. several cultures showed its good growth in 20% ethanol medium. this study also reported successful purification of cultures from fungal contamination using ethanol-soaking treatment. this self-drying method, therefore, could be suggested as a good long-term preservation method for monascus cultures. moreover, purification method from fungal contamination soaked in ethanol 70% or 95% was successfully effective. key words: recovery, preservation, monascus, drying, purification introduction monascus becomes recently popular as this fungus is used in food, specially chinese red rice (angkak), red wine, rice wine, kaoliang beer, soya cheese and pigment, food colorant in asia, mainly china, philipines, japan, thailand and indonesia (steinkraus 1983). the major mold that plays this important role is m. purpureus. this fungus is mainly used in the production of angkak. this product mostly is known by its value as food or drink colorant since it can be used as an alternative for synthetic chemical based on its health concern. monascus purpureus is also known as a producer of monacolin, a statin substance which can inhibit 3-hydroxyi-3-methylglutaryl coenzyme a (hmg-coa) reductase in cholesterol biosynthesis (endo 1979). hence, monacolin production by this fungus is being extensively studied recently. corresponding author: nsuharna@yahoo.com biotropia vol. 15 no. 1, 2008 : 74 85 75 recovery of more than 10 years-drying monascus culture – nandang suharna. accordingly, on account of its highly benefits, maintenance and preservation of living monascus cultures become extremely important so as to ensure its continuous use without risk of loss of its capability. currently, three primary methods of culture preservation are already known such as continuous growth, drying, and freezing. continuous growth methods, in which cultures are grown on agar medium, generally are used for short-term storage. such cultures are stored at temperatures of 5 °c-20 °c, or they may be frozen to increase the interval between subcultures. the methods are simple and inexpensive because specialized equipment is not required (smith 1993; smith and onion 1994; nakasone et al. 2004). in short-term storage, routine maintenance is usually not preferable, since it is laborious and has higher risk of loss of capability due to frequent transfer. whilst, long term storage much reduces transfer frequency and risk loss of capability. therefore, the long-term storage will supply stock culture more safely with good quality. drying is the most useful method of preservation for cultures that produce spores or other resting structures. drying methods are technically simple and also do not require expensive equipment (smith 1993; smith and onion 1994; nakasone 2004). freezing methods, including cryopreservation, are versatile and widely applicable. with or without cryoprotectants, most fungi can be preserved in liquid nitrogen or in standard home freezers. with freeze-drying, or lyophilization, the fungal cultures are frozen and subsequently dried under vacuum condition. the method is highly successful with cultures that produce mitospores. freeze-drying and freezing below -135°c are excellent methods for permanent preservation, and we highly recommend them. however, both methods require specialized and expensive equipment, as described in the next section. the choice of preservation method depends on the species concerned, the resources available and the goal of the project (nakasone et al. 2004). we experienced with yearly storage of monascus cultures preserved by grown on agar slant but then let dry naturally at room temperature. these cultures were tested for its recovery from years of incubation period. preservation of monascus culture using liquid drying started last year. however, our findings facilitate many things including handling of the microorganism. the objective of this study is to know the recovery of monascus cultures after drying for more than 10 years. this study also covered purification of drying monascus cultures from microbial contamination. in this study, we also report our experience with purification of monascus cultures from fungal contamination besides bacterial contamination. this report also covers a new simple method to purify monascus cultures from fungal contamination. 76 biotropia vol. 15 no. 1, 2008 materials and methods monascus strains seventeen strains of monascus were used in this study (table 1). these strains were preserved on slope agar and underwent drying during storage at room temperature for 10 years. monascus cultures are maintained on taoge (germinates of phaseolus radiatus l.) extract agar 6% (ta) slope at room temperature. ta medium preparation referred to saono et al. (1969). this medium contained extract of germinating beans (phaseolus radiatus l.), 6% sucrose and 15% agar bacto. cultivation medium media used for cultivation was malt extract agar (mea) 2% (difco ltd.) which composed of malt extract (2%), peptone (10%), glucose (10%) and agar bacto (15%). water agar (wa) media used for cultivation of bacterial-contaminated culture of monascus contained agar bacto (15%) and tap water; and ethanol liquid medium which was composed of ethanol 20%, peptone 10%, glucose 20% and yeast extract 20% (pgy ethanol 20%) were used for cultivation in high concentration of alcohol. water soaking treatment prior to re-cultivation of drying monascus cultures water soaking treatment is to soak dry cultures in ta medium by adding sterile distilled water three hours before re-cultivated on a new fresh ta medium. recultivation of drying monascus cultures re-cultivation was carried out by transferring a small part of culture after treated by water soaking treatment on to mea 2% plate. this plate is then incubated at room temperature. purification from bacterial contamination cultivation on water agar (wa) medium is very effective method to separate fungal colony from bacteria (watanabe 2004). therefore, this method was applied to purify monascus culture from bacterial contamination. before cultivation the contaminated culture was pre-treated by soaking in distilled water for three hours in order to soften and to re-hydrate the agar. subsequently, a small part of the culture was removed and transferred into wa medium. after few days of incubation, the separation of fungi and bacteria can be observed. isolation of monascus colony free from bacterial colony was carried out and monascus culture was re-cultivated onto a new fresh mea 2% medium. 77 recovery of more than 10 years-drying monascus culture – nandang suharna. purification from fungal contamination by ethanol soaking treatment to eliminate fungal contamination, a small part of the contaminated dry culture was soaked into ethanol 70% or 95% for one minute prior to cultivation on mea 2%. incubation was done at room temperature. after three days incubation period, monascus culture which was free from the fungal contaminant was transferred under aseptic condition to a new fresh medium. the effect of one minute-ethanol soaking treatment on monascus growth the effect of one minute-ethanol-soaking treatment on monascus growth was studied. each sample of 14 drying monascus cultures was soaked into ethanol before cultivation in agar medium. prior to this treatment, the sample was soaked in water for three hours for re-hydration and to soften the agar so as to facilitate the fungus to grow. the cultures were then ready to be soaked in ethanol for one minute before cultivation on new fresh agar medium without rinsing using water. observation on retained important characters of monascus a. observation on colony growth was determined by its growth intensity as + (good growth) or – (poor growth). b. observation on pigmentation is carried out by observing the occurrence of pigmentation visually. c. production of cleistothecium (ascomata/fruit bodies), ascospores (sexual/generative spore) and aleurispores (asexual/vegetative spore). after making slide preparation, microscopic observation was carried out and each production determined as + (good production) or (no or poor production). d. growth in 20% ethanol medium. this is a test to know its growth capability of monascus strains in high concentration of ethanol medium. table 1. list of monascus collection maintained on slope agar that underwent drying during ten years storage. strain code fungus name year cultured & stored source of strains a70.1.2 monascus sp. 1994 shrimp specimen preserved in ethanol coel monascus sp. 1994 coelenterata specimen preserved in ethanol ka15.1(i) monascus sp. 1994 shrimp specimen preserved in ethanol ka15.2 monascus sp. 1994 shrimp specimen preserved in ethanol ka15.3 monascus sp. 1994 shrimp specimen preserved in ethanol ka30.1 monascus sp. 1994 shrimp specimen preserved in ethanol ka30.2 monascus sp. 1994 shrimp specimen preserved in ethanol ka30.3 monascus sp. 1994 shrimp specimen preserved in ethanol ka70.1 monascus sp. 1994 shrimp specimen preserved in ethanol 78 biotropia vol. 15 no. 1, 2008 strain code fungus name year cultured & stored source of strains ka70.3 monascus sp. 1994 shrimp specimen preserved in ethanol ka70.4 monascus sp. 1994 shrimp specimen preserved in ethanol ka70.4 nr4 monascus sp. 1995 shrimp specimen preserved in ethanol ka70.5 monascus sp. 1994 shrimp specimen preserved in ethanol ktb monascus sp. 1993 coelenterata specimen preserved in ethanol myom monascus sp. 1994 maibua fasciata specimen preserved in ethanol myot monascus sp. 1994 myotis ultisima specimen preserved in ethanol ngk m. purpureus 1993 chinese red rice results and discussion after pre-treated by soaking in sterile water for three hours and subsequently recultivated on new fresh agar medium, 17 drying monascus cultures successfully recovered. one culture was contaminated by bacteria, three were contaminated by molds (aspergillus and penicillium), and 13 cultures were in pure condition (table 2). recovery capability (table 2) indicated that 15 cultures were in good and the other two in bad condition. however, poor growth of these two cultures might be affected by the growth of the contaminant. after purification (table 4) these cultures could grow well. all monascus growth could be observed after three days incubation at room temperature (25-31°c). the purpose of purification work was to get rid of bacterial contamination from culture with code strain myot, by re-cultivation the monascus colony on water agar medium and the other four monascus such as m. purpureus srb6.1, m. purpureus slc, m. purpureus mlgb, and m. purpureus bdg 2.1 as well (table 3). those four cultures were also known to suffer from bacterial contamination. this additional work was intended to enrich the data. the result indicated that this purification was much possible since the bacteria grew very restricted, but monascus grew rapidly leaving bacterial colony. after isolation and re-cultivation on the new medium this monascus colony grew well (table 3). as the three monascus cultures were contaminated by other molds, purification attempt was carried out using ethanol-soaking treatment before its cultivation on agar medium. this technique could eliminate mold contamination from the three cultures (table 4). however, this technique did not work for bacterial contamination. the purification of culture with code strain myot contaminated by bacteria could not be carried out, as the bacteria were not effectively inhibited by ethanol. therefore, this technique can be used for purification from fungal contamination. table 1. continued 79 recovery of more than 10 years-drying monascus culture – nandang suharna. after pre-treated by one minute-ethanol (70% or 95%) soaking on monascus drying cultures before cultivation on mea 2% showed that all cultures could grow well (table 5). visually, there was obviously no effect on the monascus growth. figure 1 shows the growth of monascus sp. a70.4 on mea 2%. without pre-treated by soaking in ethanol, the fungal contaminant grew well and no growth of the monascus was observed when this contaminated culture was cultivated in mea 2%. when this contaminated culture was pre-treated by soaking in ethanol at 70% or 95%, the monascus culture grew well and there was no growth of fungal contaminant observed. therefore, this treatment enables to purify monascus cultures from fungal contamination. hence, it is recommended to use this simple technique to deal with fungal contamination of monascus cultures especially m. purpureus, m. ruber or maybe other osmophilic monascus. observation under light microscope of all monascus strains indicated that all strains still produced ascomata abundantly, ascospores and aleuriospores (table 6). the color of monascus colony was whitish shade, except ngk strain was blood red. based on these results, at least drying more than 10 years did not change the above characters observed. cultivation in 20% ethanol medium showed that 11 monascus strains were able to grow but not the other nine strains (figure 2). the growth of these 11 strains was observed at various days (7-12 days) of incubation on pgy ethanol 20% (table 6). this treatment aimed at the capability of growing monascus strains in ethanol at extreme concentration showed that the 11 strains which originated from degraded ethanol still retained their viability. table 2. recovery of monascus drying culture after 10 years of storage. strain code viability purity recovery capability (colony growth) a70.1.2 viable pure good coel viable pure good ka15.1 (i) viable pure good ka15.2 viable pure good ka15.3 viable pure good ka30.1 viable pure good ka30.2 viable contaminated by aspergillus (overgrowth) good ka30.3 viable pure good ka70.1 viable pure good ka70.3 viable pure good ka70.4 viable contaminated by penicillium (overgrowth) good 80 biotropia vol. 15 no. 1, 2008 strain code viability purity recovery capability (colony growth) ka70.4 nr4 viable pure good ka70.5 viable contaminated by aspergillus (overgrowth) poor ktb viable pure good myom viable pure good myot viable contaminated by bacteria (overgrowth) poor ngk viable pure good table 3. recultivation of monascus colony contaminated by bacteria. strain code growth (3 days old) mea 2% wa monascus sp. myot m r m. purpureus srb6.1 m r m. purpureus slc m r m. purpureus mlgb m r m. purpureus bdg 2.1 m r notes : m: bacterial and monascus growth mixed; direct monascus isolation was not possible. r: bacterial growth was very restricted, direct monascus isolation was possible. table 4. purification from fungal contamination using ethanol-soaking treatment strain code ethanol treatment 0% 70% 95% growth condition, purity ka30.2 poor, aspergillus good, pure good, pure ka70.4 poor, penicillium good, pure good, pure ka70.5 poor, aspergillus good, pure good, pure myot poor, bacteria poor, bacterial contamination poor, bacterial contamination table 2. continued 81 recovery of more than 10 years-drying monascus culture – nandang suharna. table 5. the effect of 1 minute-ethanol soaking treatment on monascus grown on malt extract agar 2% after three days incubation at room temperature. strain code ethanol concentration 0% 70% 95% monascus sp. a70.1.2 + + + monascus sp. coel + + + monascus sp. ka15.1 (i) + + + monascus sp. ka15.2 + + + monascus sp. ka15.3 + + + monascus sp. ka30.1 + + + monascus sp. ka30.2 + + + monascus sp. ka30.3 + + + monascus sp. ka70.1 + + + monascus sp. ka70.3 + + + monascus sp. ka70.4 + + + monascus sp. ka70.4 nr4 + + + monascus sp. ka70.5 + + + monascus sp. ktb + + + monascus sp. myom + + + monascus sp. myot + + + m. purpureus ngk + + + note : + = good growth figure 1. monascus sp. a70.4 pre-treatment with and without soaking in ethanol for one minute before cultivation on mea 2% and incubated for 7 days at room temperature. the green colony is penicillium growing from inoculation point of monascus without pre-treatment; no monascus colony observed (a). two whitish colonies are monascus, no fungal contaminant colony observed after the pre-treatment (the upper right was pre-treated by 70% ethanol) (b); below one was pre-treated by 95% ethanol (c). 82 biotropia vol. 15 no. 1, 2008 table 6. the retained cultural and morphological properties of monascus drying cultures and their resistance to 20% ethanol after stored more than 10 years. strain code growth on mea 2% (room temperature) grown on pgy ethanol 20% (room temperature) recovery (day) production colony pigmentatiom recovery (day)ascmt ascpr aleu a70.1.1 3 + + + white yellowish 7 a70.1.2 3 + + + white yellowish no growth coel 3 + + + white yellowish 10 ka15.1 3 + + + white yellowish no growth ka15.2 3 + + + white yellowish no growth ka15.3 3 + + + white yellowish 10 ka30.1 3 + + + white yellowish 10 ka30.2 3 + + + white yellowish 10 ka30.3 3 + + + white yellowish no growth ka70.1 3 + + + white yellowish no growth ka70.3 3 + + + white yellowish 7 ka70.4 3 + + + white yellowish 7 ka70.4 nr4 3 + + + white yellowish no growth ka70.5 3 + + + white yellowish no growth kns15.1 3 + + + white yellowish 10 ktb 3 + + + white reddish orange 12 mm 3 + + + white reddish orange no growth myom 3 + + + white yellowish 10 myot 3 + + + white yellowish 12 ngk 3 + + + red blood no growth note : ascmt= ascomata, ascpr= ascospores, aleu= aleurispores, + = good production the above test obviously showed good stability of the drying monascus cultures after ten years storage at room temperature, although from the growth tests in 20% ethanol only 55% were able to grow well (table 6). figure 2 shows 5 monascus strains growing well in 20% ethanol medium. this study clearly showed that all drying monascus cultures over 10 years could recover well and still retained its production of ascomata, ascospores and aleurispores, but not all monascus tested including one m. purpureus strain showed its capability to grow in liquid medium containing ethanol at high concentration. 83 recovery of more than 10 years-drying monascus culture – nandang suharna. good recovery of monascus after 10 years storage in dry condition is in fact caused by the presence of ascospores produced by monascus. these ascospores can germinate rapidly after one-day incubation period (suharna 1999). furthermore, these typical spores are abundantly produced (hawksworth and pitt. 1983; suharna 1999). without these spores, particularly m. purpureus, the fungus cannot survive in dry condition for years. we ever came across with failure of one drying collection of m. purpureus to grow after incubation for 5 years because of lacking of ascospores. this fact showed that the presence of ascospores is very vital for monascus survival in dry condition for long time. the result showed that all drying monascus cultures for 10 years could recover well with 100% viability. however, without prior treatment by soaking in water for three hours before cultivation these recoveries would not succeed. it is known that monascus is commonly found in substrate with low water activity. the monascus collection tested here originated from dry substrate. twenty monascus isolates were isolated from deteriorated ethanol. this indicated that the 20 isolates were typical fungi with their survival capability in dry condition. this result indicated important information at least an alternative for the maintenance of monascus not only cheap and simple but also long-term storage (10 years) in particular. therefore, it is recommended to use this method for the maintenance of m. purpureus or m. rubber in fermentation industry. it is well known that one of the very simple methods to preserve mold is to use agar medium which comprises serial sub-culturing from poor medium to rich medium. figure 2. five monascus strains show its growth in liquid medium containing ethanol 20% after incubated for two weeks at room temperature. the growth indicated that the five monascus strains still have the capability to grow in medium containing alcohol at very high concentration. 84 biotropia vol. 15 no. 1, 2008 the use of medium depended on the fungal strain (smith 1993; smith and onion 1994). these methods need routine maintenance because the fungal cultures could only be stored for a short-term period before subculture. this storage period ought to be taken into account in maintaining fungal cultures to avoid late handling causing the death of the cultures. several molds are known having its storage period between 2 and 4 weeks before sub-culturing. whereas, the majority of fungi have the storage period between 2 and 4 months before re-cultured and others can be maintained until 12 months (smith 1993; smith and onion 1994). therefore, preservation method using agar causes routine maintenance and take much time. during storage cultures kept on agar slope usually underwent drying because of evaporation. the nutrient content of the medium is already low and the fungal growth of cultures is in late phase (linear phase). in this condition most microbes including bacteria and fungi usually produce resistant spores. these spores are engineered for survival of its life. it is suggested that this kind of spores can survive for tens or hundreds or may be thousands years under dry condition. drying cultures can also be obtained by letting cultures kept on agar medium to dry in room temperatures. monascus purpureus cultures kept on slope agar will dry quickly several weeks at room temperature. during its growth this fungus produces ascospores masses abundantly. this typical spore has a great function for survival of its life. this spore can survive from agony condition such as very low nutrient or drought of water. however, it is of interest using more monascus species in this study to know recovery capability of the monascus originated from wet habitat such as m. sanguneus and m. pallens from dry condition compared to the other monascus species which originated from dry habitat such as m. purpureus and m. rubber. monascus sanguneus and m. pallens were firstly described by cannon et al. (1995). these 2 species were isolated from the surface sediment of a river in iraq and showed a non-osmophilic nature (cannon et al. (1995). most monascus species are showing osmophilic affinity (pitt & hocking 1997). conclusions ten-year drying cultures of 20 monascus strains were well recovered (the viability is 100%) with good retained characters such as growth, pigmentation and production of ascomata, ascospores and aleurispores. the capability to grow in alcohol at high concentration was shown by 60% of monascus strains. the purification method from fungal contamination by soaking in ethanol 70% or 95% was successfully effective. the study on monascus species showed long survival in dried agar medium, while there was no change in their cultural and morphological characteristics. 85 recovery of more than 10 years-drying monascus culture – nandang suharna. references cannon, p.f., s.k. abdullah, and b.a. abbas. 1995. two new species of monascus from iraq, with a key to known species of the genus. mycol. res., 99(6): 659 662 endo, a. 1979. monacolin k; a new hypocholererolemic agent produced by a monascus species. j. antibiotic, 32(8), 852-854) hawksworth, d.l. and j.i. pitt. 1983. a new taxonomy for monascus species based on cultural and microscopical characters. aust. j. bot., 31:51 61 nakasone kk, sw. peterson, and s jong, 2004 preservation and distribution of fungal cultures in biodiversity of fungi: inventory and monitoring methods.. elsevier academic press, amsterdam. p: 37-47 pitt ji, hocking ad, 1997. fungi and food spoilage. 2nd ed. balnckie academic & professional, london, united kingdom saono s., i. gandjar, t. basuki, and h. karsono. 1969. mycoflora of ragi and some other traditional fermented food of indonesia annales bogorienses 5, part iv : 187 204 smith d. 1993. notes on the preservation of fungi for small culture collections. international mycological institute. egham, surrey, united kingdom. 36 p. smith d. and a.h.s. onions. 1994. the preservation and maintenance of living fungi. imi technical handbooks no.2. international mycological institute. egham, surrey, united kingdom. 120 p. steinkraus, k.h. 1983. handbook of indigenous fermented foods. marcel dekker, inc. new york : 547 553 stchigel am, jf canon, sk abdullah and j guarro. 2004. new and interesting species of monascus from soil, with a key to the known species. studies in mycology 50: 299-306 suharna n. 1999. pengaruh perendaman di dalam air sebelum pemindahan terhadap pemulihan biak-biak monascus spp. yang mengering. jurnal mikrobiologi tropika, 74 80 watanabe, t. 2004. personal communication. biotropia no. 8, 1995: 23-29 sources of mycorrhizal infection of shorea acuminata seedlings under laboratory conditions*) lee su see forest research institute of malaysia kepong 52109 kuala lumpur malaysia abstract uninoculated dipterocarp seedlings raised in normal field soil in nurseries were always found to have mycorrhizas after a few months. this study set out to determine whether dipterocarp seedlings could continue to grow and develop in the absence of mycorrhizas and also to determine possible sources of mycorrhizal infection of dipterocarp seedlings raised under laboratory conditions using shorea acuminata as a typical example. seedlings were planted in capped or uncapped perspex boxes containing sterile or non-sterile field soil and watered daily with sterile water or tap water. seedling growth and development of mycorrhizas were monitored at monthly intervals for up to seven months. seedlings grown in sterile soil remained uninfected after seven months while infection was found in some of the seedlings grown in normal soil regardless of whether they had been watered with tap water or sterile water. this showed that field soil (i.e. under grass) far from the forest contained suitable inoculum for forest tree seedlings. tap water and the air were not important sources of infection. however, mycorrhizal infection was very uneven indicating that the inoculum was probably very unevenly distributed in the soil or that the inoculum density was rather low. seedlings grown in sterile soil showed better growth than those grown in normal soil and infection of roots by parasitic fungi in the latter was also observed. key words: mycorrhizas/plant pathology/lnfections/shorea acuminata/seedlings. introduction enrichment planting is often conducted in logged forests in malaysia to ensure adequate stocking of dipterocarps for future rotations. for this purpose, dipterocarp seedlings mostly of shorea species are raised in nurseries whenever seed is available. shorea species, especially of the red meranti group are important for their valuable hardwood timber. under normal practice, freshly collected seeds with their wings removed are sown into boxes or beds containing washed sand or a soil/sand mixture. the seeds germinate very quickly, usually after five to seven days, and at the age of one month are transplanted into polybags containing an unsterilised mixture of *)paper presented at the second asian conference on mycorrhizae, acom '91, march 12-17, 1991. chiang mai, thailand. 23 biotropia no. 8, 1995 sand and top soil. the seedlings can be maintained in the nurseries without any fertilizer application for quite some time before outplanting without any visible symptoms of poor health. plants of over one year-old have been observed to do well in the nursery. no mycorrhizal inoculum is added but the roots of the seedlings are usually found to be ectomycorrhizal by about three months (unpubl. data; lee 1988). an account of the mycorrhizal association of the dipterocarpaceae is given by lee (1990). smits et al. (1988) reported that dipterocarp seedlings could not be successfully raised in the nursery in east kalimantan and that seedlings transplanted into secondary forest usually died. they stated that dipterocarps are obligately ectomycorrhizal and are thus unable to survive unless associated with their specific fungal partner(s) (smits 1983, 1985; noor and smits et al. 1987). moreover, smits et al. (1988) advocated various methods of inoculation of dipterocarp seedlings to ensure survival and successful establishment in the nursery and forest. this study was conducted to determine whether dipterocarp seedlings could continue to survive and develop in the absence of mycorrhizas and also to determine possible sources of mycorrhizal infection of seedlings raised under laboratory conditions similar to those found in the nursery. materials and methods experimental design the experimental design was a randomised 2.2.2 factorial: ± steamed soil, ± cap, ± watering with sterile tap water. each treatment consisted of five replicates. plant material shorea acuminata dyer seeds were obtained from trees growing in field 3 in the compound of the forest research institute of malaysia (frim) and germinated in boxes containing washed sand. s. acuminata is common in mixed dipterocarp forest and yields timber classed in the light red meranti group. soil mixture and planting top soil was collected from under grass at the farm in universiti pertanian malaysia and sieved to remove stones, roots and other organic debris before mixing with vermiculite in the ratio of 7:3. one portion of the soil mixture was stored for later use while the other portion was steamed in a soil steamer for one hour 24 sources of mycorrhizal infection of shorea acuminata seedlings lee su see on two consecutive days. the steamed and cooled soil mixture was then incubated in a tightly closed black plastic bag in the greenhouse for two weeks before further use. perspex boxes consisting of two sheets of clear perspex measuring 30 cm x 20 cm separated by a 1.5 cm polystyrene spacer and bound together with "cling film" were used as pots for the seedlings. a one month-old seedling was transplanted into each box with its roots in contact with one sheet of perspex. the boxes were then wrapped in black plastic and placed in plastic basins in the laboratory under a bank of fluorescent tubes emitting cool white light at an intensity of about 2000 lux. the daily temperature ranged from a maximum of 32°c to a minimum of 23°c. caps for the boxes were made from aluminium foil with a perforation to allow the seedling to emerge. caps were also fitted with a glass tube to facilitate watering. the plants were watered daily with either sterile or normal tap water. measurement and analysis at fortnightly intervals seedling height was measured and the roots inspected for formation of ectomycorrhizas. when present mycorrhizas were clearly visible through the clear perspex under the stereomicroscope. the experiment was terminated after seven months. relative growth rates were used to compare the performance of the seedlings under the various treatments. results and discussion all s. acuminata seedlings grown in steamed soil remained non-mycorrhizal but healthy after seven months. leaving the boxes uncapped did not produce any infection in the seedlings; neither did watering with normal tap water. it appeared that tap water and the air were not important sources of infection as had earlier been postulated and that steaming the soil mixture was effective in preventing infection. the results indicated that dipterocarp seedlings could easily survive and develop normally for up to seven months in the absence of ectomycorrhizas and fertilizer application. ectomycorrhizal infection of seedlings grown in normal soil appeared to be a random process. at least one seedling in each treatment (± cap, ± sterile tap water) was infected after seven months (table 1). since the soil mixture was the only common factor in all the treatments, and steaming the soil eliminated ecto-mycorrhizal infection, it appeared that the soil was the main source of infection. 25 biotropia no. 8, 1995 the random pattern of infection and the low infection rate was probably an indication of the random natural distribution and low density of the mycorrhizal inoculum in the field soil. it also appeared that soil under grass located several kilometers from the forest contained suitable dipterocarp mycorrhizal inoculum. it had previously been assumed, and in fact smits and co-workers (1988) reported, that such areas would not contain any suitable dipterocarp inoculum in the absence of dipterocarp host trees. earliest infection occurred at week 10 while infection was last observed to occur at week 17 (table 1). two main types of ectomycorrhizas were observed. these mycorrhizas have also been commonly observed on roots of nursery seedlings (un-publ. data). uninfected seedlings continued to grow normally but were generally smaller than the infected seedlings as reflected in the relative growth rates shown in figure 1. it appeared that ectomycorrhizas enhanced the growth of infected seedlings, the effect being more dramatic the earlier the infection. table 1. ectomycorrhizal infection of s. acuminata seedlings grown in normal soil under various treatments treatment number of seedlings infected infected at week tap water, uncapped 1 10 1 (2/5) 17 tap water, capped 1 d/5) 11 sterile tap water, capped 1 (1/5) 17 sterile tap water, uncapped 1 13 1 (2/5) 17 * figures in parentheses indicate the total number of infected seedlings in each treatment at the end of 7 months. roots of a number of seedlings grown in normal soil were attacked by root pathogenic fungi such as cylindrocladium sp. resulting in death of roots, the cessation of growth and yellowing of three seedlings after 10 weeks and the death of one seedling after 17 weeks. cylindrocladium spp. are relatively common in local soils and have been observed to be associated with death of dipterocarp seedlings in the natural forest (unpubl. data). attack by such root pathogenic fungi could have caused the death of the dipterocarp seedlings reported by smits and co-workers (1988) in indonesia. in comparison, all seedlings grown in steamed soil were healthy with normal green foliage until the end of the experiment and had much more extensive but 26 sources of mycorrhizal infection of shorea acuminata seedlings lee su see figure 1. relative growth rates of shorea acuminata under different treatments in normal unsterilised soil: -i tap water, capped; * tap water, uncapped; x sterile water, capped; . sterile water, uncapped; mean (± s.d.) for uninfected seedlings. normal uninfected root systems than seedlings grown in normal soil. steaming the soil before planting probably killed all potentially pathogenic organisms resulting in better seedling survival. the mean relative growth rates of seedlings grown in unsterilised or steamed soil were not significantly different (p ≤ 0.05) after 29 weeks (fig. 2). although the use of steamed soil for planting may results in higher survival rates and generally produce healthier plants, the economics of the practice must be given serious consideration before it can be adopted on a routine basis. based on the results of this study it would appear that the potting medium used for planting dipterocarp seedlings in the nursery is the main source of ecto-mycorrhizal inoculum and that earliest formation of ectomycorrhizas in the field normally takes a period of about 10 to 12 weeks. the inoculum found in field soil was also compatible with the seedlings and appeared to promote better growth. therefore, there appears to be no absolute necessity to inoculate dipterocarp seedlings in the nursery unless local conditions are such that absolutely no suitable inoculum 27 biotropia no. 8, 1995 figure 2. mean relative growth rates of shorea acuminata in steamed soil (•) and in normal unsterilised soil ( + ). is present. this would be highly unlikely since the results of this study show that soil located several kilometers from forest areas without dipterocarp host trees could still initiate ectomycorrhizal infection producing enhanced growth in infected plants. it should further be pointed out that nursery-raised dipterocarp seedlings offfopea and shorea spp. have been successfully planted, growing into normal trees in parks and urban areas. this further suggests that survival and normal development of dipterocarp seedlings can be achieved without inoculation with specific fungi. however, this does not mean that mycorrhizal inoculation of dipterocarps should be ignored as it has been shown that mycorrhizal infection definitely enhances the growth of seedlings. efforts should be made to find the most suitable mycorrhizal fungi and methods of inoculation for the economic production of dipterocarp seedlings suitable for planting in logged-over forests and other cleared areas to ensure the best chances for their survival and growth. 28 sources of mycorrhizal infection of shorea acuminata seedlings lee su see acknowledgements i would like to thank seameo biotrop for sponsoring my attendance at this conference, the international foundation for science for a grant that made this study possible, the dean of the faculty of forestry, universiti pertanian malaysia for use of facilities during my tenure there, and the director-general of the forest research institute of malaysia (frim) for permission to present this paper. references lee, s.s. 1988. do mycorrhizas play a role in the growth and development of parashorea densiflora sloot & sym.? in: m. mohinder singh (ed.). agricultural and biological research priorities in asia. proceedings of the ips symposium of science asia '87. 14-17 october, kuala lumpur, p. 167-171. lee, s.s. 1990. the mycorrhizal association of the dipterocarpaceae in the tropical rain forest of malaysia. ambio 19(8): 383-385. noor, m. and w.t.m. smits. 1987. pengaruh intensitas cahaya dan suhu tanah terhadap ektomikoriza dan pertumbuhan anakan shorea polyandra. in: komar soemarna, harun alrasyid, ishemat surianegara dan achdiat (eds.). presiding simposium hasil penelitian silvikultur dipterocarpaceae, 24 november 1987. jakarta, indonesia, p. 11-30. smits, w.t.m. 1983. dipterocarps and mycorrhiza: an ecological adaptation and a factor in forest regeneration. flora malesiana bull. 36: 3926-3937. smits, w.t.m. 1985. specificity of dipterocarp mycorrhiza. in: r. molina (ed.). proc. 6th nacom. 25-29 june, 1984. bend, usa. smits, w.t.m., d. leppe and m. noor. 1988. metode inokulasi untuk persemaian dipterocarpaceae. edisi khusus no. 5, balai penelitian kehutanan samarinda. 29 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf biotropia book final.indd 129 pollination effectiveness of apis cerana fabricus and apis mellifera linnaeus (hymenoptera: apidae) in jatropha curcas l. (euphorbiaceae) tri atmowidi*, puji riyanti, andeng sutrisna department of biology, faculty of mathematics and natural sciences, bogor agricultural university, darmaga campus, bogor 16680, indonesia abstract pollinators are well known to provide key ecosystem. animal pollinators are thought to contribute between 15 and 30% of global food production and bees are recognized to be the most important taxon. th e pollination eff ectiveness of two species of bees, apis cerana and a. mellifera (hymenoptera: apidae) in jatropha curcas (euphorbiaceae) was studied. th ree cages, made of insect screen were set up. each cage contains three individual plants. one colony of a. mellifera and a. cerana were placed in the fi rst and second cage, while the third cage was used for control plants. bee colonies were placed during one month in the fi rst and second cages. fruit set of plants pollinated by a. mellifera, a. cerana, control plants, and open plants were counted and compared. pollination by honeybees increased the number of fruits per plant and fruits per raceme of j. curcas. based on measuring of fruit produced by plants, pollination eff ectiveness of a. mellifera was higher than a. carana. key words: pollination, eff ectiveness, apis cerana, a. mellifera, seed set, jatropha curcas. introduction th e honeybees are the principal species used for crop pollination worldwide (free 1993). th e true honeybees (tribe apini, genus apis) consist of nine species i.e. a. mellifera linnaeus, a. cerana fabricus, a. dorsata fabricus, a. laboriosa smith, a. fl orea fabricus, a. andreniformis smith, a. koschevnikovi buttel-reepen, a. nigrocincta, dan a. nuluensis. genus apis primarily tropical was restricted to the old world until a. mellifera was introduced worldwide. both species, a. cerana and a. mellifera consist of medium -sized (10-11 mm) species with multiple combs in cavities, dances on vertical surfaces of combs in the dark (michener 2000). th e combs are built under the ceiling of the cavity and attached to the cavity’s walls. th e arrangement and distribution on the combs are similar in all races of each species (koeniger 1995). colonies of a. cerana are relatively small (6000-7000 workers), but a. mellifera colonies consist of 100, 000 or more individuals (winston 1987). * corresponding author: atmowidi@ipb.ac.id biotropia vol. 15 no. 2, 2008 : 129 134 130 comparative foraging behaviour of a. cerana and a. mellifera on apple orchard had been reported by verma (1995). worker bees of a. cerana started their foraging activity signifi cantly earlier in the morning (06.03 h) than a. mellifera (06.27 h), but duration of foraging trip of a. mellifera (17.92 minutes) was signifi cantly longer than that of a. cerana (11.85 minutes). th e peak of foraging activities of a. cerana was between 09.00 h and 13.00 h with a temperature range of 15.5-21oc, and that of a. mellifera was between 11.00 h-13.20 h with a temperature range from 21 to 25oc. worker bees of a. mellifera carried signifi cantly heavier pollen loads than a. cerana throughout the day. a. cerana contacted the stigma on average 3.09 stigmas per visit and spent 5.90 seconds on each fl ower, whereas these values for a. mellifera were 3.33 stigmas per visit and 6.63 seconds on each fl ower, respectively (verma 1995). selection for eff ective pollen transfer and receipt has been considered to be the principal force in the evolution of the angiosperm fl ower. most fl owers, however, allow access to a variety of visitors (faegri & van der pijl 1979). diff erences among visitors in morphology, physiology, and foraging behavior may result in diff erences in their eff ectiveness as pollinators (young 1988). wallace et al. (2002) also reported that the natural pollinators were extremely effi cient in producing fertilization and fruit set of jeff ersonian virgata. in male-sterile oilseed rape (brassica napus), steff an-dewenter (2003) reported that insect pollination can positively aff ect several yield components. however, these eff ects are dependent on cultivar and growing conditions. total yields are often not increased due to the considerable compensatory capacity of oilseed rape (westcott & nelson 2001). jatropha curcas (euphorbiaceae) is a perennial, deciduous shrub or treelet. th e plant produces fl owers in racemose infl orescences, with dichasial cyme pattern. th e fl owers are unisexual, and male and female fl owers are produced in the same infl orescence. normally, the infl orescences produce a central female fl ower surrounded by a group of male fl owers. in some cases the female fl owers are substituted by male fl owers. numerically, 1–5 female fl owers and 25–93 male fl owers are produced per infl orescence. th e average male to female fl ower ratio is 29 : 1. each infl orescence, once it begins fl owering, fl owers daily, and the fl owering lasts for 11 days. th e fl owering pattern showed that the male fl owers are produced earlier (compared to the female fl owers) and will produce fl owers daily until the male buds are exhausted. th e female fl owers bloom between the second and the sixth day. th e fl oral base contains nectar in trace amount, which is 0.3 ml per fl ower. th e fl owers open daily during 0530–0630 h. th e unpollinated fl owers fall off on the fourth day, while the pollinated ones remain in place. th e sepals and petals gradually enlarge and protect the growing fruit until the latter reaches its full size (raju & ezradanam 2002). here, we studied the pollination eff ectiveness of two species of honeybees, apis cerana and a. mellifera in j. curcas. th e eff ectiveness of the pollinators was measured by the number of seeds set, and seeds weight. materials and methods study site th e study was conducted in jatropha plantation located at indramayu district, west java from may to september 2007. biotropia vol. 15 no. 2, 2008 131 measuring of the pollination eff ectiveness of honeybees th e plants selected for measuring pollination eff ectiveness were about three years old. we set up three cages made by of insect screen. th e size of each cage was 9x3x2.5 m3 and each cage contains three plants of j.curcas. one colony of a. cerana and a. mellifera were placed in the fi rst and second cages for approximately 1 month (june 22 to july 25, 2007) to pollinate the fl owers. th ird cage was used as a control (no bee application). at this time, bees were fed with honey that placed in a cup. each infl orescence observed was tagged by color ribbon. after the end of the fruiting period, the number of fruits per plant, fruits per raceme, seeds per fruit, and seed weight were counted. observation of fl ower visitors species composition of fl oral visitors was observed visually at jatropha plantation using scan sampling method (martin & bateson 1993) for a few days. after observation, insect visitors were caught by sweep netting for species identifi cation in the laboratory. data analysis fruits set of j. curcas pollinated by a. cerana and a. mellifera were compared with control plants and open plants (plants pollinated by natural pollinators). diff erences in fruit numbers of j. curcas pollinated by bees, control plants, and open plants were analysed by analysis of variance (anova) and scheff e test at the 95% level by using systat 10 for windows. results and discussion pollination eff ectiveness of honeybees in j. curcas pollination eff ectiveness of two species of honeybees in j. curcas varied. pollination eff ectiveness was shown by the number of fruits produced by plants. result showed that the mean number of fruits produced per plant as a result of pollination by a. cerana (17 fruits), a. mellifera (19 fruits), and open plants (16 fruits) were higher than that of control plants (5 fruits) (table 1). although, based on statistical tests, they were not signifi cantly diff erent. table 1. reproductive success of j. curcas pollinated by a. cerana, a. mellifera, open plants, and control plants plant reproductive success numbers (+st.dev) control plants a. cerana a. mellifera open plants number of fruits per plant 17a (+7.94) 19a (+11.93) 16a (+12.15) 5a (+5.13) number of fruits per raceme 2.04a(+1.65) 3.05a (+1.75) 2.45a (+1.47) 2.29a (+1.25) number of seeds per fruit 2.76a (+0.51) 2.61a (+0.50) 2.47a (+0.63) 2.63a (+0.52) seed weight (g) 0.47ac (+0.17) 0.39b (+0.14) 0.51a (+0.16) 0.41cb(+0.11) note: different lower case letters in the same row indicate different values based on analysis of variance (anova) and scheffe test at 95% level (p<0.05). eff ectiveness of bees to pollination of jatropha – t. atmowidi et al. 132 th e number of fruits per raceme of plants pollinated by a. cerana was lowest (2.04 fruits) compared to plants pollinated by a. mellifera (3.05 fruits), open plants (2.45 fruits), and control plants (2.29 fruits). pollination by bees increased the number of fruits per plant and fruits per raceme of j. curcas. mean number of seeds per fruit of plants pollinated by a. cerana, a. mellifera, open plants, and control plants were 2.76, 2.61, 2.47, and 2.63 seeds, respectively. th e number of seeds per fruit ranged from 1 to 3 seeds (table 1). results showed that a. mellifera was the superior pollinator compared to a. cerana. in addition, the number of fruits per plant and fruits per raceme of plants pollinated by a. mellifera were higher than that pollinated by a. cerana. verma (1995) stated that some characteristics of a. mellifera were better than a. cerana, i.e. duration of foraging trip was signifi cantly longer and worker bees carried signifi cantly heavier pollen loads throughout the day. th e increased yields of j. curcas pollinated by bees was probably caused by cross pollination. as stated by westcott & nelson (2001), insect pollination leads to earlier cessation of fl owering and more synchronous pod and seed ripening, thereby possibly increasing the weight of seed harvest. th e natural pollinators were extremely effi cient in producing fertilization and fruit set of p. virgata (wallace et al. 2002). in male-sterile oilseed rape (b. napus), steff an-dewenter (2003) reported insect pollination could positively aff ect several yield components. however, these eff ects are dependent on cultivar and growing conditions and total yields are often not increased due to the considerable compensatory capacity of oilseed rape (westcott & nelson, 2001). similarly, atmowidi et al. (2007) reported that mustard (brassica rapa) pollinated by insects (mostly by bees) increased the number of pods, seeds per pod, and seeds per plant. lower fruit set of j. curcas plants caged with insect screen could be caused by other factors, such as the number of plants observed, size of plants, and screen eff ect. th e number of plants observed (three plants for each application) very limited. size of plants observed was quite uniform, but individual variation in size occurred. th e screen may be aff ected to plant photosynthetic rate, but most probably this eff ect is small, because the screen transmitted the light intensity easily. th e natural fruit set rate indicates that j. curcas does not suff er seriously if there are no other pollination agents. flowers of the species are unisexual, and male and female fl owers are produced in the same infl orescence. th e production of female fl owers in small number, surrounded by a large number of male fl owers seems to be a strategy to ensure pollination to the maximum extent. th e stigma receptivity lasting three days also additionally provides opportunities for pollination, if not pollinated on the fi rst and second day. however, the plant with predominant xenogamy requires mostly xenogamous pollen for more fruit set, after selective elimination of growing fruit. th erefore, pollen transfer between conspecifi c has a great infl uence on the net percentage of natural fruit set (raju & ezradanam 2002). diversity of fl ower visitors based on fi eld observations, the fl ower-visitors of j. curcas belong to bees and ants (hymenoptera), butterfl ies and moths (lepidoptera), beetles (coleoptera), thrips (th ysanoptera), and fl ies (diptera). bee species belonging to a. cerana, ceratina sp, trigona sp., and hylaeus sp. were found visiting the fl owers. at least, three species of biotropia vol. 15 no. 2, 2008 133 butterfl ies (nyctemera sp., eurema hecabe, and neptis hylas) also visited the fl owers. snout beetle and some species of diptera (syrphus balteatus, sarcophaga sp., and musca domestica), and thrips were also found visiting the fl owers. in general, insect pollinators visit the fl owers in the morning. on brassica rapa (brassicaceae), the peak abundance of insect pollinators occurred between 08.30-09.30 h (atmowidi et al. 2007). similar visitation was shown by two species of pollinators (trigona carbonari: apidae and leiopcoctus speculiferus: colletidae) visited persoonia virgata (proteaceae) occurred before 12.00 h (wallace et al. 2002). foraging time of insect pollinators related to plant resources, especially pollen and nectar. th e increase of plant resources such as during mass fl owering showed an increase in available resources so that the foragers could collect more rewards per unit time. if resources are limited, the foragers require longer searches and travel time (wesphal et al. 2006). generally, both pollen and nectar content of most plants are higher in the morning. conclusions pollination by honeybees, a. cerana and a. mellifera increased the number of fruits per plant and fruits per raceme of j. curcas. in j. curcas, pollination eff ectiveness of a. mellifera was higher than that a. cerana. naturally, fl owers of j. curcas are visited by bees and ants (hymenoptera), butterfl ies and moth (lepidoptera), beetles (coleoptera), thrips (th ysanoptera), and fl ies (diptera). acknowledgments th is project was funded by biotrop dipa 2007. we greatly acknowledge dr. sih kahono, dr. rosichon ubaidillah, dr. yayuk rahayuningsih, woro nurjito,m.sc. and pudji aswari, m.sc. of zoological museum, indonesian institute of science, cibinong for their help in identifi cation of insect pollinators. we are also grateful to dian sari and sunaryo for the assistance in collecting the data. finally, the authors are grateful to the two anonymous referees for their comments of this manuscript. references atmowidi t, buchori d, manuwoto s, suryobroto b, hidayat p. 2007. diversity of insect pollinators and seed set of mustard (brassica rapa: brassicaceae). hayati 14:155-161. faegry k, van der pijl l. 1971. th e principles of pollination ecology. ed ke-2. braunschweig: pergamon press. free jb. 1993. insect pollination of crops. san diego: academic press. koeniger n. 1995. biology of the eastern honeybee apis cerana (fabricus 1773). in: kevan pg, editor. th e asiatic hive bee: apiculture, biology, and role in sustainable development in tropical and subtropical asia. ontario: enviroquest ltd. pages 29-39. eff ectiveness of bees to pollination of jatropha – t. atmowidi et al. 134 martin p, bateson p. 1993. measuring behaviour: an introductory guide. ed ke2. cambrige: cambrige univ. press. michener dm. 2000. th e bees of the world. baltimore: johns hopkins univ. press. raju ajs, ezradanam v. 2002. pollination ecology and fruiting behaviour in a monoecious species, jatropha curcas l. (euphorbiaceae). current science 83:1395-1398. steff an-dewenter i. 2003. seed set of male-sterile and male-fertile oilseed rape (brassica napus) in relation to pollinator density. apidologie 34:227–235. verma lr. 1995. apis cerana: biometric, genetic, and behavioural aspects. in: kevan pg, editor. th e asiatic hive bee: apiculture, biology, and role in sustainable development in tropical and subtropical asia. ontario: enviroquest ltd. hlm 41-53. wallace hm, maynard gv, trueman sj. 2002. insect fl ower visitors, foraging behaviour and their eff ectiveness as pollinators of persoonia virgata r. br. (proteaceae). australian journal of entomology 41: 55– 59. wesphal c, steff an-dewenter i, tscharntke t. 2006. foraging trip duration of bumblebees in relation to landscape-wide resource availability. ecological entomology 31:389–394. westcott l, nelson d. 2001. canola pollination: an update, bee world 82:115–129. winston ml. 1987. th e biology of the honey bee. cambridge: harvard univ. press. young hj. 1988. diff erential importance of beetle species pollinating dieff enbachia longispatha (araceae). ecology 69:832-844. biotropia vol. 15 no. 2, 2008 12 biotropia vol. 15 no. 1, 2008biotropia vol. 15 no. 1, 2008 : 12 24 somatic embryogenesis from meristem explants of ginger otih rostiana* and sitti fatimah syahid *division of plant breeding, indonesian medicinal and aromatic crops research institute jalan tentara pelajar no. 3 bogor 16111, indonesia abstract the use of planting materials from in vitro culture, especially derived from somatic embryos has some advantages such as genetically stable and pathogen-free. meristem culture of ginger through somatic embryogenesis could be a potential method for producing pathogen-free planting materials. somatic embryogenesis on ginger was performed to obtain vigorous plantlets having the same rhizome size as the mother plant. callus was induced from meristem tissue of inner bud of indonesian ginger rhizome var. cimanggu-1 and consecutively subcultured into certain media at each steps of experiments. the vigorous embryogenic calli were observed on ms medium containing 100 mgl-1 glutamine and 2% sucrose with addition of 1.0 mgl-1 2,4-d + 3.0 mgl-1 ba. the highest number of somatic embryos (about 82.0.g-1 friable calli) was achieved on that medium, 4 weeks after culturing. furthermore, the optimum growth of embryogenic calli containing somatic embryo was obtained on ms medium enriched with 6% sucrose. the highest number of mature somatic embryos (57.2 embryos) was achieved on ms medium, 18 days after incubation. the regeneration potency of somatic embryos obtained from ginger meristem was 51.20%.g-1 friable callus. the valuable result of this study was the achievement of normal rhizome size of regenerated plantlets, instead of micro rhizome. key words: zingiber officinale rosc., meristem culture, somatic embryogenesis. introduction ginger (zingiber officinale rosc.) is one of the most important export commodities of indonesia. this crop is also one of the important components of indonesian herbal medicine, locally called jamu, phytopharmaca and is contributing to the indonesian foreign exchange and work opportunity for labors. the lack of market stock and serious damages due to pest and disease, have caused high fluctuations to indonesian ginger exports and prices within the last decades. to overcome the limiting factors, the use of healthy planting material is necessary to be developed. plant tissue culture has been adopted for the purpose of in vitro mass propagation on various crop species. in case of the big-white ginger cultivar of indonesian variety cimanggu-1, the use of shoot bud-derived plantlets, either by direct organogenesis or through callusing, has been developed to obtain disease-free planting materials (mariska * corresponding author : otihrostiana@yahoo.com 13 somatic embryo development of ginger meristem – o. rostiana & s.f. syahid and syahid 1994). in further field experiments, the healthy plantlets and vigorous plants were not able to produce normal rhizomes (the same-big rhizome as their mother plants), eventhough they were repeatedly planted in three consecutive generations (syahid and hobir 1996). it is suggested that genetic alteration or epigenetic change during the in vitro culture and regeneration have been performed. in order to eliminate the genetic change during the in vitro culture, other regeneration pathway should be considered. somatic embryogenesis has been accomplished in some species for producing millions of seeds. the form of somatic seeds through somatic embryogenesis, is more efficient than organogenesis. besides, somatic embryogenesis is more preferable in plant genetic improvement through in vitro culture and genetic transformation as well, because single cell derived-plant is eassier to be controlled as somatic embryo derived-plant. in general, somatic embryogenesis and meristem derived plants are true-to-type and genetically identical to the mother plants (evans and sharp 1986; jimenez 2001), however, certain differences might appear according to the plant species characteristics. the true-to-type of somatic embryo derived plantlet has been found in in vitro culture of picea abies (heinze and schmidt 1995). on the other hand, among recalcitrant species such as serealia crops and conifers, good results have been performed by the induction of somatic embryogenesis. achievements in inducing somatic embryogenesis in monocots are mostly derived from generative explants. meanwhile, in dicotyl vegetative explant, such as leaves, are commonly applied for inducing somatic embryo derived-plantlet. leaf explant of orchard grass completely induces somatic embryo through the formation of embryogenic calli with addition of a strong auxin like dicamba (bhojwani and razdan 1996). on the other hand, kackar et al. (1993) performed the somatic embryo culture of indian ginger var. eruttupetta by using an aseptic leaf-explant with the addition of 2,4-d and dicamba. the same explant source was also performed in inducing somatic embryogenesis of fingerroot (boesenbergia rotunda l.) and galangal (kaempferia galanga l.) (tan et al. 2005; rahman et al. 2004). this study was aimed at obtaining the normal-size of rhizome (the same size as their mother plant) through induction of somatic embryos from meristem explant of indonesian var. cimanggu-1. most meristematic cells of meristem tissue derivedsomatic embryo are genetically stable and not easy to be mutated (bach and pawlowska 2003). therefore, to eliminate somaclonal variation and other genetic alteration during the in vitro culture, meristem tissue will be applied for the induction of somatic embryogenesis on ginger. 14 biotropia vol. 15 no. 1, 2008 materials and methods explant preparation the inner shoot bud (meristem) of indonesian var. cimanggu-1, was excised and simultaneously sterilised by using sterilizing agents such as 70% etoh, 5% sodium hypochlorite and 0.2% mercury chloride, for about 5-10 minutes, followed by rinsing with sterile-aquadest. callus induction sterilized meristems were placed on ms basal medium (murashige and skoog 1962) consisting of 8% agar, 2% sucrose, 100 mg l-1 l-glutamine, 1.0 to 3.0 mg l-1 2,4-dichlorophenoxy acetic acid (2,4-d) and 0 to 5.0 mg l-1 n6-benzyl adenin (ba). a single factor experiments were arranged in completely randomized design, replicated three times. subculturing. embryogenic calli were formed 8 weeks after culturing on callus induction medium, and then transferred on to hormone-free ms or n6 basal media with the addition of 3% mannitol for calli proliferation. a single factor experiments were arranged in completely randomized design, replicated four times for each treatments. pro-embryos were then subcultured into the best basal medium, either ms or n6 basal media, according to the best result of the previous stage of experiment, with the addition of 6% sucrose (6s) for obtaining mature embryo. single-factor experiments were arranged in completely randomized design, replicated four times. regeneration mature embryos (torpedo-like structure-embryos) were subcultured into regeneration medium for further development. ms basal medium with addition of 3% sucrose and 200 mgl-1 l-proline were applied in combination with pgrs such as ba at the concentration of 0, 0.1, 0.5 and 1.0 mgl-1, ga3 at the concentration of 0, 0.1, 0.3, 0.5 and 1.0 mgl-1. single-factor experiments were arranged in completely randomized design, replicate three times. histology the callus at each stage of development was subjected to histological analysis according to sass (1951) by using paraffin-embedded callus dissection following formaldehyde-glacial acetic acid-alcohol (faa) fixation series. statistical analysis all collected data were analyzed by using anova (p0.05) followed by duncan’s multiple range test (p. 0.05) (windows computers, 2000). observation results on embryo proliferation were analyzed by using t-test according to furlong et al. (2000). 15 somatic embryo development of ginger meristem – o. rostiana & s.f. syahid results and discussion callus induction callus initiation was on tract when the basal-edge of the meristems enlarged, followed by the change of shoot-dome colour from white to yellowish, at two weeks after culturing. eight weeks after culturing, friable callus (embryogenic callus) formation of about 93.33%/explant was formed on ms basal medium enriched with 1 mg l-1 2,4-d in combination with 3 mg l-1 ba. though, that combination was statistically the same as the treatment without ba (table 1). table 1. percentage of embryogenic calli from ginger meristem cultured on ms medium enriched with various concentration of 2,4-d and ba, 8 weeks after culturing treatment percentage of embryogenic calli (%) 2,4-d (mg.l-1) ba (mg.l-1) 1 0 86.67 a*) 1.0 8.33 e 3.0 93.33 a 5.0 70.00 b 2 0 46.67 c 1.0 25.00 d 3.0 63.33 b 5.0 61.67 b 3 0 28.33 d 1.0 36.67 cd 3.0 63.33 b 5.0 61.67 b note: numbers followed by the same letters are not significantly different according to dmrt (5%). the application of 2,4-d on ginger meristem culture showed that the higher the concentration applied, the more compact calli were observed. furthermore, when 2,4-d was applied at 2-3 mg.l-1 browning and necrotic calli were observed. an addition of 1.0 mg.l-1 ba into medium containing 2,4-d neither vigorous embryogenic calli were obtained, low quantity and compact calli were observed (table 1). embryogenesis from the subcultured callus for obtaining globular somatic embryos structure the formed embryogenic calli were cultured on either ms or n6 basal media with the addition of 3% mannitol. transparent globular embryos were developed on both media in one week after subculturing. the shape of ginger embryos at globular phase was generally round or oval (fig. 1a). 16 biotropia vol. 15 no. 1, 2008 a c e f g b d mm mm cm cm cm mm mm figure 1. the development of ginger meristem into newly regenerated plant with normal rhizome size through somatic embryogenesis. a. the structure of globular somatic embryo of ginger, 4 weeks after subculturing into proliferation medium (30 times enlargement). b. globular embryo, 2 weeks after proliferation (40 times enlargement). protoderm layer started to differentiate (arrowed). c. the structure of torpedo somatic embryo of ginger, 18 days after subculturing into maturation medium (10 times enlargement). d. torpedo embryo, 18 days after subculturing into maturation medium (40 times enlargement). arrowed: differentiated procambium. e. embryo somatic-derived seedlings on ms medium supplemented with 1 mgl-1 ba (left) and embryo somatic-forming adventitious roots on hormone free-ms medium (right), 30 days after subcultured (1 : 1.4 scaled). f. embryo somatic-derived normal plantlet, 8 weeks after subcultured on to hormone-free ms medium (1: 1.3 scaled). g. normal rhizome size of ginger-regenerated plants obtained from meristem culture through somatic embryogenesis. 17 somatic embryo development of ginger meristem – o. rostiana & s.f. syahid numbers of globular phase of somatic embryos increased due to the age of cultures. the highest number of somatic embryo was obtained at 4 weeks after subculturing, 82.00 ± 12.25 embryo.g-1 embryogenic callus on ms medium and 70.00 ± 19.26 embryo.g-1 embryogenic callus on n6 medium. statistical analysis showed that ms basal medium was more capable in inducing higher numbers of somatic embryo than that of n6 basal medium (p0.05). the number of somatic embryo decreased by the increase of age of cultures (table 2). histological analysis conformed that the somatic embryo was induced from cortex tissue and that the globular phase developed at 2 weeks after culturing on proliferation medium (fig. 1b). table 2. number of somatic embryos derived from ginger meristem culture on ms and n6 basal media enriched with 3% mannitol period (weeks) average number of somatic embryos/g of embryogenic callus ms n6 1 32.75 ± 11.97 39.75 ± 5.31 2 54.50 ± 20.16 51.50 ± 9.66 3 70.50 ± 31.97 48.75 ± 15.16 4 82.00 ± 12.251) 70.00 ± 19.261) 5 53.75 ± 16.32 36.25 ± 9.28 6 51.50 ± 10.92 22.75 ± 15.28 7 43.50 ± 19.55 11.00 ± 7.65 note: based on t-test at 5% level. mature embryo formation was performed on ms basal medium, enriched with 6% sucrose. cylindrical form of mature embryo started to develop into 2 different domes, i.e. cotyledons and apical shoot-forming dome as well as root-forming dome. two days after subculturing into ms medium enriched with 6% sucrose, about 10.60 ± 4.76 torpedo-shape somatic embryos were initiated from 1 g of embryogenic calli (table 3). 18 biotropia vol. 15 no. 1, 2008 table 3. number of mature somatic embryos on ms basal medium supplemented with 6% sucrose age(day) average number of mature embryo/g of embriogenic calli1) 2 10.60 ± 4.76 6 33.40 ± 6.74 10 55.80 ± 12.73 14 38.60 ± 6.53 18 57.20 ± 15.99 22 45.80 ± 10.50 26 28.00 ± 8.65 30 16.20 ± 8.91 the highest number of torpedo-shape somatic embryo (57.20 ± 15.99 embryos) was obtained at 18 days after subculturing. mature somatic embryo was capable to form two different domes, i.e. shoot and root (fig. 1c-d). however, in some cases apical meristem structure was more dominant than shoot meristem. histological section showed that during the formation of torpedo-shape somatic embryo, root-apical, procambium and scutellum differentiations were performed (fig. 1c-d). regeneration germination of somatic embryo was characterized by the formation of the triangle-shape at meristem apical dome with yellow greenish in color, at 6 days after subcultured. at the second week of culturing, germinating seeds started to appear and developed to become normal seedlings/plantlets, especially when ba was added into ms basal medium. three weeks after culturing, various shapes of somatic embryos either a single normal or fused embryos, had been differentiated into plantlets. meanwhile, the abnormal embryos underwent necrosis and then aborted. embryo germination was indicated by the presence of shoot and root buds. four weeks after culturing, the morphogenesis of somatic embryos were clearly visible, nevertheless the low ability of somatic embryos to germinate was still a limiting factor in meristem culture of ginger. ms basal medium supplemented with 1 mgl-1 ba was found to be the best culture medium for obtaining normal plantlets at the age of 30 days, to which the highest number of seedlings was obtained (22.1 seedlings). on the other hand, instead of growing plantlet, vigorous adventitious roots were obtained on hormone-free ms medium (fig. 1e). furthermore, it was found that both ba and ga3 significantly affected the somatic embryo regeneration (p 0.05). however, the higher the concentration of ga3, the lower the number of seedlings was obtained (table 4). 19 somatic embryo development of ginger meristem – o. rostiana & s.f. syahid table 4. number of seedlings derived from somatic embryo of ginger meristem cultured on ms medium supplemented with ba and ga3 treatment average number of seedlings ba (mgl-1) ga3 (mgl-1) 0 0 7.3 cd* 0.1 16.4 ab 0.3 16.4 ab 0.5 9.3 bcd 1.0 9.3 bcd 0.1 0 7.3 cd 0.1 10.0 bcd 0.3 3.7 de 0.5 16.1 ab 1.0 11.5 bc 0.5 0 0.5 f 0.1 0.5 f 0.3 0.5 f 0.5 12.8 abc 1.0 17.4 ab 1 0 22.1 a 0.1 12.6 abc 0.3 12.5 abc 0.5 0.5 f 1.0 1.2 ef notes : numbers followed by the same letter are not significantly different according to dmrt (5%). for statistical analysis data were transformed into √(y + 0.5). based on this study, it was found that regeneration potency of meristem culture of ginger through somatic embryogenesis was 51.20%.g-1 of embryogenic calli when cultured on ms medium supplemented with 1 mg l-1 ba. the addition of ga3 into ms basal medium produced only 34.13% of regeneration potency, and 1.13 to 31.84% of regeneration potency when ba was combined with ga3. therefore, to gain an optimal germination of ginger somatic embryo, higher concentration of ba (> 1 mg.l-1) without addition of ga3, was necessary. germinating somatic embryos from ms medium supplemented with either 1 mg l-1 ba, 0.5 mg l-1 ba, 0.1 mg l-1 ba + 0.3 mg l-1 ga3 or 0.1 mg l-1 ga3 were then transferred onto hormone-free ms medium with the addition of 3% sucrose. among them, the most vigorous plantlets were obtained from ms + 0.1 mg l-1 ba + 0.3 mg l-1 ga3 culture medium-derived seedlings (fig. 1f ), at 8 weeks after subculturing. however, the number of embryo somatic-generated plantlets remained low, of which only about 2-6 plantlets were observed. 20 biotropia vol. 15 no. 1, 2008 abundant embryogenic calli (93.33%/explant) from meristem culture of ginger were achieved on ms basal medium supplemented with 1 mg l-12,4-d in combination with 3 mg l-1 ba, and 100 mg l-1 l-glutamine, at 8 weeks after incubation. this result showed that 2,4-d was necessary for initiation of embryogenic callus of ginger, with the addition of ba at proper concentration. the same result was also found in embryogenic callus initiation of kaempferia galanga l. (vincent et al. 1992). callus proliferations were obtained on hormone-free ms medium consisting of 3% mannitol. a globular-shape pro-embryo started to develop at the first week after subculturing into proliferation medium. quantities of globular embryo increased due to the age of culturing up to seven weeks, then gradually decreased. ammonium-rich basal media such as ms, which consists of high concentration of nh4no3 and myo-inositol, supported well the differentiation of ginger somatic embryo, though an adverse effect was observed on other grammineae species (talwar and rashid 1989; adkins et al. 2002). salt nutrients in ms medium with addition of 3 to 12% sucrose are usually sufficient to support embryo differentiation (raghavan 2003; anbazhagan and ganapathi 1999). an addition of 3% mannitol into ms basal medium, assumed that it acted as an osmotic regulator which in turn affected the rate of cell differentiation and stimulated embryogenic cells morphogenesis in meristem culture of ginger. torres et al. (2001) reported that the development of synchronized embryo and the increase of its quantity were observed when mannitol was added into ms medium. the same results were also found on embryogenic culture of papaya, celery, alfalfa and maize (ziv 1999; bronsema et al. 1997). somatic embryo maturation in meristem culture of ginger was indicated by the change of embryo color and formation of the brown spots. these brown spots suggest the presence of an active substance i.e. amylum, protein and lipid, secreted by somatic embryo from the osmotic cell pressure. the increase of secretion of active substances suggests its correlation with the desiccation process when somatic embryo enters the germination phase. bach and pawlowska (2003) suggested that the presence of amylum, protein and lipid around the vascular cells was an indicator in somatic embryo development. similarly, oropeza et al. (2001) reported that the development of embryogenic calli was associated with the number and type of intracellular protein and callus cells. hence, somatic embryo maturation from proliferated calli of ginger meristem is interrelated with the type and number of active substance among the embryogenic cells. the growth and development of ginger mature somatic embryo into complete plantlets (seedlings) sometimes go along with the abnormal germinated seeds, such unexpected growth of hairy roots, hereafter we named as an early germinating seeds. according to bronsema et al. (1997), somatic embryo maturation is associated with the presence of scutellum, a coleoptile and root-bud like structures. somatic embryo maturation was also affected by the composition of applied culture medium. the presence of ammonium ion and nitrate at a high concentration is a prerequisite in ginger somatic embryo development. high concentration of ammonium ion supported the process of 21 somatic embryo development of ginger meristem – o. rostiana & s.f. syahid somatic embryo differentiation into normal plantlets (george 1993). these results were in agreement with the findings of krikorian (1995), adkins et al. (2002) and ramage and williams (2002). however, the needs of ammonium ion for the growth of somatic embryo and its morphogenesis depend on the explant sources and the initial plant growth regulators applied. according to percy et al. (2000), high osmotic pressure in culture medium would increase the somatic embryo formation. sucrose, usually applied as a carbon source, is also a potent osmotic agent (van creij et al. 1999). another potential osmotic agent is abscisic acid (aba), that at a proper concentration it would enable to control the medium osmolarity (raghavan 2003; mohan and krishnamurthy 2002). to overcome the early seeds germination (the formation of unexpected hairy roots) in somatic embryogenesis of ginger, an experiment on addition of aba, aside from sucrose, into culture medium of somatic embryo maturation is now in progress. somatic embryo germination was indicated by the simultaneous formation of shoots and roots. goh et al. (1999) stated that embryo germinated when the plumullae started to emerge. in this research, adventitious roots formation was observed at first stage of regeneration and increased by the increase of age of culture up to the fourth week. addition of plant growth regulators into regeneration medium would accelerate the germination process and increase the quantity and quality of seedlings. in this research, a high percentage of somatic embryo germination was observed on the medium containing 1 mg.l-1 ba, at 30 days after culturing. regeneration potency of ginger meristem somatic embryo reached 51.20%.g-1 of embryogenic calli cultured on ms basal medium enriched with 1 mgl-1 ba, of which the number of seedlings was 8.98 times higher than that of the control medium. meanwhile, ga3 alone or in combination with ba, remained ineffective in supporting somatic embryo germination of ginger. however, to obtain the optimum growth of plantlet, the presence of ba is a prerequisite in meristem culture derived somatic embryo of ginger. similar result was also found in the development of ginger leaf derived embryogenic callus (kackar et al. 1993), though simultaneous development of somatic embryo derived either root or shoot meristems remained unclear. the development of somatic embryo into normal plantlet commonly occurs through four stages i.e. callus induction, callus proliferation, embryo maturation and germination. in case of somatic embryo derived from ginger meristem culture, its development into normal plantlet needs a transfer process of germinated seeds from the regeneration medium into new medium. such new medium is called growth medium for plantlet development. in this experiment the medium used for the growth of plantlet was hormone-free ms medium with the addition of 3% sucrose. although the development of protocol in ginger somatic embryogenesis required more researches, the results of this experiment, somehow, have a definite advantage in in vitro studies of ginger, especially in generating healthy planting material with normal rhizome size, a bigger size than the usual micro rhizomes resulted from shoot 22 biotropia vol. 15 no. 1, 2008 tip-derived in vitro plantlets (fig. 1 g). therefore, the encouraging results of this study gave great possibility for development of ginger resistant variety either through in vitro selection, somatic hybridization or genetic transformation. conclusions the addition of 1 mg.l-1 2,4-d and 3 mg.1-1 ba into ms basal medium enriched with 100 mg.l-1 of glutamine and 2% sucrose, was effective in inducing embryogenic calli from meristem explants of indonesian ginger. in further embryo development, ms medium was found to be more capable to increase mature somatic embryo than n6 medium. the regeneration potency of somatic embryos obtained from indonesian ginger meristem was 51.20%/g friable callus. though, further researches are needed to be conducted, especially to eliminate early germinating seeds. the protocol for generating somatic embryo derived plantlet from meristem culture of ginger was developed. the most valuable result of this study was the achievement of normal rhizome size of regenerated plantlet, instead of micro rhizome. acknowledgement the authors would like to thank dr. ika mariska for her assistance and encouragement during the research work and the manuscript preparation and also to dr. d. sitepu for his critical reading of the manuscript, and ms. r.r. sitinjak for her contribution on the research work. references adkins, s.w., a.l. adkins, c.m. ramage and r.r. williams. 2002. in vitro ecology: modification of headspace and medium conditions can optimize tissue and plant development. in: taji a, r. williams (eds) the importance of plant tissue culture and biotechnology in plant sciences. university of new england unit, australia, pp. 55-77. anbazhagan v.r. and a. ganapathi. 1999. somatic embryogenesis in cell suspension cultures of pigeon pea (cajanus cajan). plant cell, tissue organ cult. 56: 179-184. bach, a and b. pawlowska. 2003. somatic embryogenesis in gentiana pneumonanthe l. acta bio. crac. series bot. 45 (2): 79-86. bhojwani, s.s. and m. razdan. 1996. plant tissue culture: theory and practice. elsevier amsterdam, oxford, new york, tokyo. 23 somatic embryo development of ginger meristem – o. rostiana & s.f. syahid bronsema, f.b.f., w.j.e. van oostveen and a.a.m. van lammeren. 1997. comparative analysis of callus formation and regeneration on cultured immature maize embryos of the inbred lines a188 and a632. plant cell, tissue organ cult. 50: 57-65. evans, d.e and w.r. sharp. 1986. somaclonal and gametoclonal variation. in: evans et al. (eds) hand book of plant cell culture, vol. 4, technique and application. macmillan pub. co., new york, pp. 97-132. furlong, n.e., e.a, lovelace and k.l. lovelace. 2000. research methods and statistics an integrated approach. harcourt college, tokyo. george, e.f. 1993. plant propagation by tissue culture. 2nd ed. exegetics ltd, england. goh, d.k.s., n. michaux-ferriere, o. monteuuis and m.c. bon. 1999. evidence of somatic embryogenesis from root tip explants of the rattan calamus manan. in vitro cell. dev. biol. plant. 35: 424-427. heinze, b and j. schmidt. 1995. monitoring genetic fidelity vs somaclonal variation in norway spruce (picea abies) somatic embryogenesis by rapd analysis. euphytica 85: 341345. jimenez, v.m. 2001. regulation of in vitro somatic embryogenesis with emphasis on the role of endogenous hormones. r. bras. fisiol. veg. 13 (2): 196-223. kackar, a., s.r. baht, k.p.s. chandel and s.k. malik. 1993. plant regeneration via somatic embryogenesis in ginger. plant cell. tissue organ cult. 32: 289-292. krikorian, a.d. 1995. hormones in tissue culture and micropropagation. in: p.j. davies (ed.) plant hormones: physiology, biochemistry and molecular biology. kluwer academic, dordrecht, boston, london, pp. 774-793. mariska, i and s.f. syahid. 1994. propagation of ginger through meristem culture. j. indust. crops. res. 7 (1): 1-6. murashige, t and f. skoog. 1962. a revised medium for rapid growth and bio assays with tobacco tissue cultures. physiol. plant. 15: 473-497. mohan, m.l and k.v. krishnamurthy. 2002. somatic embryogenesis and plant regeneration in pigeon pea. biol. plant. 45 (1): 19-25. oropeza, m., a.k. marcano and e. de garcia. 2001. proteins related with embryogenic potential in callus and cell suspensions of sugarcane (saccharum sp.). in vitro cell dev. biol. plant. 37: 211-216. percy, r.e., k. klimaszewska and d.r. cyr. 2000. evaluation of somatic embryogenesis for clonal propagation of western white pine. can. j. for. res. 30: 1867-1876. raghavan, v. 2003. one hundred years of zygotic embryo culture investigations. in vitro cell. dev. biol. plant. 89: 437-442. rahman, n.n., m.n. amin, t. ahamed, m.r. ali and a. habib. 2004. efficient plant regeneration through somatic embryogenesis from leaf base-derived callus of kaempferia galanga l. asian j. plant sci. 3 (6): 675-678. ramage, c.m and r.r. williams. 2002. mineral nutrition and plant morphogenesis. in vitro cell. dev. biol. plant. 38: 116-124. sass, j.e. 1951. botanical microtechnique. iowa state university press, ames. 24 biotropia vol. 15 no. 1, 2008 syahid, s.f and hobir. 1996. the growth and rhizome yield of ginger derived from in vitro culture. j. indust. crops. res. 2 (2): 95-100. talwar, m and a. rashid. 1989. somatic embryo formation from unmerged inflorescences and immature embryos of a graminaceous crop echinochloa. ann. bot. 64: 195-199. tan, s.k., r. pippen, r. yusof, h. ibrahim, n. rahman and n. khalid. 2005. simple one-medium formulation regeneration of fingerroot [boesenbergia rotunda (l.) mansf. kulturfl.] via somatic embryogenesis. in vitro cell dev. biol. plant 41: 757-761. torres, a.c., n. mfëe-ze and d.j. cantliffe. 2001. abscisic acid and osmotic induction of synchronous somatic embryo development of sweet potato. in vitro cell. dev. biol. plant. 37: 262-267. van creij, m.g.m., d.m.f. kerckhoffs, s.m. de bruijn, d. vreugdenhil and j.m. van tuyl. 1999. the effect of medium composition on ovary-slice culture and ovule culture in intraspecific tulipa gesneriana l. crosses. available at <http://www.liliumbreeding.ne/crey-med.htm>[12/06/04]. vincent, k.a., m. hariharan and k.m. mathew. 1992. embryogenesis and plantlet formation in tissue culture of kaempferia galanga l. a medicinal plant. phytomorphology. 42 (3 & 4): 253-256. ziv, m. 1999, developmental and structural patterns of in vitro plants. in: soh w, bhojwani ss (ed.) morphogenesis in plant tissue culture. kluwer academic, dordrecht, boston, london, pp. 235-253. biotropia (2) 1988/1989: 25-31 removal and leaching of nutrients by salvin1a molesta mitchel and eichhornia crassipes (mart.) solms y.c. wee and h.h. yeoh department of botany, national university of singapore, singapore 0511, republic of singapore abstract profuse growth of eichhornia crassipes and salvinia molesta in singapore reservoirs required their regular manual removal as their prolonged presence can lead to deterioration in the quality of the potable water. clearing of the reservoir catchments, together with regular removal of the weeds and dumping them away from the catchments, should, in the long term, reduce their presence in the reservoirs. laboratory experiments showing the removal of chloride, sulphate, phosphorus and nitrate from the growing medium and the release of chloride, phosphorus and nitrate by rotting plants should convince the administrators of the benefit of proper management of the problem. introduction eichhornia crassipes (water hyacinth) and salvinia molesta (water spangle) are two of the most troublesome water weeds of southeast asia. the former is bulky and grows profusely in eutrophic waters, doubling its number every eight to ten days (wolverton and mcdonald 1976). the latter, a water fern of oligotrophic waters, is similarly profuse in growth, with a doubling time under favorable growth conditions of four to ten days (mitchell and tur 1975). both plants were introduced into the country: water hyacinth as an ornamental in 1893 and water spangle as a teaching specimen around the 1950s (wee 1986, wee and corlett 1986). water hyacinth existed in rural fish ponds where it was grown as a feed for pigs. it became a problem when the kranji reservoir was constructed in 1970 by the barraging of the kranji river. as the catchment of this reservoir was agricultural areas, pollutants from pig farms and run-off fertilizers from fruit and vegetable farms provided enough nutrients to trigger a population explosion five years later. problems of water spangle was seen in the seletar reservoir. its catchment of secondary forests ensured less pollution but, agricultural pollutants still found their way into the water, resulting in proliferation of the plants around 1978. removal of these plants has traditionally been by mechanical means but use of herbicides like 2,4-d and paraquat has proven to be more efficient (penfound and earle 1948, widyanto 1976). as both bodies of water were reservoirs, use of herbicides was not considered. the use of beetles to control water spangle, as reported in austra lia (room 1984) has yet to be tried in this region. thus, at the 25 biotropia no. 2, 1988/1989 two reservoirs, mechanical removal of the weeds was regularly undertaken, as the presence of large quantities of the weeds besides being aesthetically objectionable could cause deterioration of the quality of the potable water. this work investigated the removal and leaching of nutrients by these plants under laboratory conditions, as under proper supervision, these plants could be used to our advantage, to remove the pollutants from the water. materials and methods water spangle and water hyacinth were obtained from the garden of the department of botany, national university of singapore. they were grown in rain water contained in concrete tanks. for experiments, 35 x 29 x 12 cm plastic trays were used to contain the plants. uniform sized plants were first washed in tap water a few times, then rinsed twice with distilled water. five to ten water spangle plants (total weight 250 g) and five water hyacinth plants (total weight 350 g) were placed in each basin containing 5 1 hoagland's complete nutrient solution (hewitt 1952). nine replicates per plant material were set up. the basins were left in the greenhouse partially exposed to sunlight. loss of water through evaporation was made up by the addition of distilled water up to the original level. samples of water were removed at regular intervals and analyzed for sulphate, chloride, nitrate and phosphorus using standard chemical methods (anonymous 1973). the nutrient uptake by the plants was then calculated, based on the amount of nutrient left in the medium. a parallel series of experiments were conducted using tap water (43.6 ppm chloride, 656 ppm sulphate, 3 ppm phosphorus) instead of the complete solution. the fresh weight of water hyacinth used here was 612 g while that of water spangle was 555 g. leaching experiments were done by leaving 100 g fresh weight each of water hyacinth and water spangle in basins to rot. at regular intervals the rotting plants were washed with 300 ml distilled water and the water analyzed for sulphate, chloride, nitrate and phosphorus. results and discussion figure 1 shows the removal of chloride, sulphate, phosphorus and nitrate from the complete media by water spangle and water hyacinth on a per gram fresh weight basis with time. chloride removal was rapid, 0.54 mg and 0.29 mg were removed 26 removal and leaching of nutrients – y.c. wee & h.h. yeoh figure 1. removal of nutrients (mg g fr wt-1) by water hyacinth (•) and water spangle (o) from hoagland's complete solution with time. 27 biotropia no. 2, 1988/1989 by water spangle and water hyacinth, respectively, by the third day. the former removed 0.57 mg of the chloride after 16 days, after which there was a net decrease in chloride removal, perhaps due to the leaching of the anion into the growing medium from decaying leaves. water hyacinth however, showed a constant and gradual increase in removal of the anion with time, resulting in 0.57 mg removal after 40 days. sulphate was also efficiently removed; within three days water spangle removed 7.88 mg and water hyacinth 4.43 mg of the sulphate present. phosphorus was slowly removed from the nutrient solution by both plants. water spangle took 33 days to remove some 0.53 mg of the phosphorus while water hyacinth removed about 0.43 mg. nitrate removal showed a rather erratic course. it was rapidly removed during the first 12 days by both plants, after which there was a net decrease in nitrate removal, attributed to leaching of the nutrient from the plants before a further increase. the data showed that the anions, chloride, sulphate and nitrate were rapidly removed while phosphorus uptake by these plants was generally slower. water spangle and water hyacinth grown in tap water also showed uptake of chloride and sulphate (figure 2), but the profiles were different from those of plants grown in figure 2. removal of nutrients (mg g fr wt-1) by water hyacinth (•) and water spangle (o) from tap water with time. 28 removal and leaching of nutrients-y.c. wee & h.h. yeoh the complete medium. it took water spangle and water hyacinth 22 days to remove 0.19 mg and 0.23 mg of the chloride respectively after which removal was very gradual. sulphate removal, however, was slightly more efficient. by 16 days, 5.22 mg and 5.07 mg were removed by water hyacinth and water spangle respectively. there were no further removals of sulphate after this period. no data were available for phosphorus and nitrate as there was only a trace of the former and no trace of the latter at all in the tap water. rotting plants were also observed to release chloride, phosphorus and nitrate into the water (figure 3). these were found to increase with time over a period of figure 3. nutrients leached into distilled water (mg g fr wt-1) during decay of water hyacinth (•) and water spangle (o). 29 biotropia no. 2, 1988/1989 18 days, after which the plants were totally disintegrated. sulphate released by both plants was negligible. the general profiles for both water spangle and water hyacinth were much the same. the use of aquatic plants to bring eutrophic waters into proper nutrient balance is well known (steward 1970). so far, most have been with water hyacinth. their abilities to remove phosphorus from sewage effluent (ornes and sutton 1975) and nitrogen and phosphorus from eutrophic waters (duningan, phelan and shamsuddin 1975) have also been reported. wolverton and mcdonald (1976) showed that in an experimental lagoon enriched with sewage effluent, these plants can reduce the pollutant level by 75-80%. the plants can also absorb toxic heavy metals like gold, silver, cobalt, strontium, cadmium, nickel, lead and mercury (wolverton and mcdonald 1976). the excessive presence of nitrogen and phosphorus is the main cause of eutrophication and the consequent prolific growth of aquatic weeds (sheffield 1967). thus removal of these primary nutrients can significantly reduce weed growth. a single water hyacinth plant has been shown capable of absorbing over 3 mg of phosphorus per day (rogers and davis 1972). however, haller and sutton (1973) showed that the maximum accumulation of 9.07 mg phosphorus per g dry plant weight was seen after four weeks when the phosphorus content of the growing medium was around 40 ppm. results from the experiments show that the presence of water spangle and water hyacinth in the two reservoirs can be put to advantage if they are allowed to proliferate, regularly harvested and the harvested plants dumped. the ability of these plants to remove nutrients as well as pollutants rapidly from the water means that with every harvesting, the water gets a little cleaner. in time, the purity of the water would be such that it would not be able to support the excessive growth of these weeds. however, low nutrient status does not necessarily mean that growth will be eradicated as it has been shown that water spangle can survive for long periods under conditions of severe limiting nutrient status (gaudet 1973). the above practice will need to go hand in hand with the cleaning of the reservoir's catchments, which, since 1977, has been in progress as a result of resettlement of pig farms elsewhere (wee and corlett 1986). however, the past practice of dumping the harvested plants along the edge of the reservoirs just to save cost of transportation was counter-productive in that, nutrient leached from the rotting plants gets back into the water to support growth of a new crop of plants. data collected in the above experiments should go a long way in convincing administrators the advantages of investing in transportation costs in the long term. 30 removal and leaching of nutrients-y.c. wee & h.h. yeoh acknowledgments this project was funded by a grant from the singapore institute of biology. references anonymous, 1973. chemical methods of water testing. bdh chemicals ltd., poole, england. 47 pp. 2nd ed. dunioan, e.p., r.a. phelan, and z.h. shamsuddin, 1975. use of waterhyacinths to remove nitrogen and phosphorus from eutrophic waters. hyacinth control journal 13: 59-61. gaudet, jj. 1973. growth of a floating aquatic weed, salvinia, under standard conditions. hydro-biologia, 41: 77-106. haller, w.t. and d.l. sutton. 1973. effect of ph and high phosphorus concentrations on growth of water hyacinth. hyacinth control journal 11: 50-61. hewitt, e.j. 1952. sand and water culture methods used in the study of plant nutrition. commonwealth bureau of horticultural plantation crops, tech. comm. no. 22. mitchell, d.s. and n.m. tur. 1975. the rate of growth of salvinia molesta (s. auriculata auct.) in laboratory and natural conditions. journal of applied ecology 12: 213-225. ornes, w.h. and d.l. sutton. 1975. removal of phosphorus from static sewage effluent by water-hyacinth. hyacinth control journal 13: 56-58. penfound, w.m. t. and t.t. earle. 1948. the biology of the water hyacinth. ecological monograph 18: 448-472. rogers, h.h. and d.e. davis. 1972. nutrient removal by waterhyacinth. weed science 20: 423-427. room, p.m. 1984. biological control of floating weeds in australia. paper presented at sympos. in weed science, april 1984, bogor, indonesia. 2 pp. sheffield, c.w. 1967. water hyacinth for nutrient removal. hyacinth control journal 6: 27-30. steward, k.k. 1970. nutrient removal potentials of various aquatic plants. hyacinth control journal 8: 34-35. wee, y.c. 1986. aquatic weed problem in singapore's freshwater reservoir. in: biotrop special publication no. 24, symposium weed science (eds. j.v. pancho, s.s. sastroutomo and s. tjitrosemito) 25-29. wee, y.c. and r. corlett. 1986. the city and the forest: plant life in urban singapore. singapore university press. widyanto, l.s. 1976. studies on the growth and control of waterhyacinth (eichhomia crassipes (mart.) solms). proceedings of the 5th asian-pacific weed science society conference, tokyo. 429-434. wolverton, b. and r.c. mcdonald. 1976. don't waste waterweeds. new scientist 71: 318-320. 31 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf biotropia no. 5, 1991/1992: 22-25 antioxidant metabolism in water stressed peanut treated with diniconazole g.c. rivero institute of biology, university of the philippines, diliman, philippines and d.m. orcutt department of plant physiology, virginia polytechnique institute and state university, blacksburg, va 24061-0331, usa abstract the response of alpha-tocopherol, glutathione and ascorbate was studied in peanut (arachis hypogea l., cv. nc-17) subjected to water stress and treated with a triazole fungicide, diniconazole (dini). there was no significant difference in alpha-tocopherol levels between control and dini treated plants. however, a 14% increase was observed in alpha-tocopherol level in dini treated, water stressed plants compared to water stressed plants. total glutathione in dini treated stressed plants increased by 13 and 31% over control and water stressed plants, respectively. ascorbate levels decreased significantly in all treatments compared to the control. these results indicate that dini alleviates the oxidative damage caused by water stress by increasing total glutathione levels. however, dini does not seem to affect alpha-tocopherol and ascorbate levels in peanuts under water stress. introduction protection against harmful oxidation of biomembranes is provided by several antioxidative systems, including a large array of enzymes and a variety of small molecules such as glutathione, beta-carotene, and the vitamins c and e (finckh and kunert 1985). in plants, vitamin e (alpha-tocopherol) is located mainly in the chloroplast (bucke 1976) and a high concentration of the lipid-soluble vitamin is found in the chloroplast envelope (lichtenthaler et al. 1981). vitamin c (ascorbate), a reducing agent, is present in substantial amount in higher plants (jones and hughes 1983), and the water-soluble vitamin is generally regarded as a normal cellular constituent (finckh and kunert 1985). the tripeptide glutathione is widely distributed in plant cells (rennenberg 1982). it is implicated in the adaption of plants to environmental stress such as drought and extremes of temperature (alscher 1989). glutathione, ascorbate and alpha-tocopherol are of special interest because of their demonstrated association with stress resistance. the present study evaluated the response of alpha-tocopherol, glutathione and ascorbate in water stressed peanut plants treated with diniconazole. 22 antioxidant metabolism in water stressed peanut g.c. rivero & d.m. orcutt materials and methods peanut (arachis hypogea l., cv. nc-17) seedlings were grown in controlled conditions, i.e. continuous illumination (392 uem-2sec-l) and controlled temperature (28 ± 2°c). treatments consisted of control, water stressed, dini treated and dini treated stressed plants arranged in a completely randomized block design with 10 replications. dini was applied foliary at a rate of 4 mg/plant (recommended field rate) every 2 weeks for 7 weeks commencing when the plants were 6 weeks old. drought stress treatment began 4 days following initial dini treatment and consisted of applying 125 ml of distilled water to plants subjected to drought every 3 days versus 250 ml to non-stressed plants. harvested plants (117 days old) were frozen in liquid nitrogen, freeze dried and stored at -23°c until analyzed for alpha-tocopherol, glutathione and ascorbate. alpha-tocopherol was extracted in 80% ethanol partitioned with hexane and the hexane layer analyzed using a hewlett packard model 1090 hplc, equipped with a fluorescence detector (294 nm excitation, 325 nm emission) following modified procedures of cort et al. (1983) and grumbach (1983). the hexane ran through a silica column (hypersil 5 um, 200 x 4.6 mm, hewlett packard). glutathione and ascorbate extraction was accomplished by homogenizing leaf tissue in 2% metaphosphoric acid. the homegenate was centrifuged at 17 000 g and the supernatant analyzed for glutathione (griffith 1980) and ascorbate (foyer and halliwell 1977) using a beckman du-65 spectrophotometer at wavelengths 412 nm and 523 nm, respectively. results and discussion the following table reflects the responses of alpha-tocopherol, glutathione and ascorbate in water stressed peanut plants treated with dini. alpha-tocopherol, total glutathione and ascorbate levels in water stressed peanut (arachis hypogea l., cv. nc-17) plants treated with diniconazole trt vitamin e glutathione vitamin c (% dw) (nmoles/gdw) (umoles/gdw) c 0.57a 835.01b 7.28a t 0.43b 880.40ab 3.91b cs 0.49ab 719.86c 4.65b ts 0.50ab 946. 14a 4.74b c = control; t = treated w/ dini; cs = water stressed; ts = treated w/ dini and water stressed. value with different letters are significantly different at α = 0.05 (lsd). 23 biotropia no. 5, 1991/1992 there was no significant difference in alpha-tocopherol levels between control and dini treated plants. however, a 14% increase was observed in alpha-tocopherol level in dini treated stressed plants compared to water stressed plants. earlier reports (senaratna et al. 1985, mackay et al. 1987) on antioxidant levels in stressed plants indicated an increase on the levels of total antioxidants. mackay et al. (1987) evaluated the antioxidant potential of microsomal membranes and reported an increase in antioxidant potential of triazole (s-3307) treated wheat plants exposed to ozone. however, their analysis was made on total antioxidant potential of the lipid fraction, as the ability of the lipid extract to inhibit the in vitro oxidation of exogenous linoleic acid. the oxidation reaction was similar to that seen for alpha-tocopherol and thus, the antioxidant capacity of their samples were expressed as alpha-tocopherol equivalents (mackay et al. 1987). in our study, alpha-tocopherol was measured directly by hplc. ascorbate levels decreased significantly in all treatments compared to the control. there is evidence that ascorbate represents a reservoir of antioxidant potential to regenerate directly, under conditions of stress, the lipid-soluble primary antioxidant alpha-tocopherol (packer et al. 1979; leung et al. 1981). this could explain the significant decrease in ascorbate levels in water stressed and dini treated water stressed plants compared to the control. total glutathione in dini treated stressed plants increased by 13 and 31% over control and water stressed plants, respectively. in pea, a chilling-resistant species, total glutathione decreased only slightly, but was still higher than that in cucumber, a chilling-sensitive species (wise and naylor 1987). the results presented here indicate that dini alleviates the oxidative damage caused by water stress by increasing the total glutathione levels. summary total glutathione levels increased in stressed peanut plants treated with diniconazole and could be a factor in drought resistance. diniconazole does not seem to affect alpha-tocopherol and ascorbate levels in peanuts under water stress. 24 antioxidant metabolism in water stressed peanut g.c. rivero & d.m. orcutt acknowledgement this study was part of a research conducted during the senior author's us-aid post-doctoral fellowship at the department of plant pathology, physiology and weed science, virginia polytechnique institute and state university, blackburg, va 24061, usa from october, 1989 to august, 1990. special thanks are due to dr. r. madamachi and ms. n.r. hopkins from the same department for their invaluable assistance. references alscher, r.g. 1989. physiol. plant. 77: 457-464. bucke, c. 1976. phytochemistry 7: 693-700. cort, w.m., t.s. vicente, e.h. waysek and e.d. williams. 1983. j. agric. food chem. 31: 1330 1333. finckh, b.f. and k.j. kunert. 1985. j. agric. food chem. 33: 574-577. foyer, c.h. and d.o. hall. 1976. planta 133: 21-25. _____ and b. halliwell. 1977. phytochemistry 16: 1347-1350. griffith, o.w. 1980. anal. biochem. 106: 207-212. grumback, k.h. 1983. z. naturforsch. 38c: 996-1002. jones, e., and r.e. hughes. 1983. phytochemistry 22: 2493-2499. leung, h.w., m.j. vong and r.d. mavis. 1981. biochem. biophys. acta. 664: 266-272. lichtenthaler, h.k., u. presenzel, r. douce and j. joyard. 1981.. j. biochim. biophys. acta 641: 99-105. mackay, c.e., t. senaratna, b.d. mckersie and r.a. fletcher. 1987. plant cell physiol. 28 (7): 1271-1278. packer, j.e., t.f. slater and r.l. willson. 1979. nature (london) 278: 737-738. rennenberg, h. 1982. phytochemistry 21: 2771-2781. seneratna, t., b.d. mckersie and r.h. stenson. 1985. plant physiol. 78: 168-171. 25 22.pdf 23.pdf 24.pdf 25.pdf biotropia no biotropia no. 17,2001 : 9 17 characterization of three benzoate degrading anoxygenic photosynthetic bacteria isolated from the environment dwi suryanto1, antonius suwanto2'3*, and anja meryandini3 ; dept. of biology, faculty of science and mathematics, north sumatra university, medan, indonesia 2 south east asian regional center for tropical biology (seameo-biotrop), bogor, indonesia 3 dept. of biology, faculty of science and mathematics, bogor agricultural university, bogor, indonesia abstract three anoxygenic photosynthetic bacteria, ds-1, ds-4 and cas-13, have been examinated for their morphological and physiological properties. all strains were rod-shape cells with a swollen terminal end, gram negative, motile, non-halophilic, non-alkalophilic and non-acidophilic, and capable of utilizing benzoate aerobically and photo-anaerobically. sequence analysis of part of 16s rrna genes showed that ds1 and cas-13 were closely related to rhodopseudomonas palustris strain 7 with a similarity of 97%, whereas ds-4 may not be closely related to the former two strains with a similarity of 78% based on the constructed phylogenic tree. spectral analysis indicated that the three bacteria had bacteriochlorophyl a and normal spirilloxanthin series. growth in medium enriched with vitamin and supplemented with benzoate as their sole c-sources was better than in medium without vitamin. benzoate degradation in medium with vitamin was accelerated. the ability to grow on benzoate without added vitamins indicated that the bacteria were able to synthesize their own vitamins. key words: anoxygenic photosynthetic bacteria/ benzoate degradation/ 16s rrna gene. introduction some toxic compounds are slowly degraded in polluted aerobic zones and, therefore, may leach into anaerobic subsurface environments (kohring et al. 1989). anaerobic degradation of such compounds may play an important role in eliminating toxic substances. anaerobic bioremediation has been proposed as an inexpensive method for in situ removal of organic contaminants in the environment (kuo and genthner 1996). recent concern about the environmental fate of industrially produced organic compounds has prompted a resurgence of interest in the anaerobic degradation of aromatic compounds (harwood and gibson 1988). anoxygenic photosynthetic bacteria (apb) are nutritionally versatile in their ability to utilize diverse sources of carbon ranging from simple aliphatic organic acids to complex polysaccharides (hiraishi et al. 1995). the occurrence of these bacteria as common inhabitants in aquatic and some terrestrial habitats in nature ' corresponding author: e-mail address : asuwanto@indo.net.id 9 biotropia no. 17, 2001 (hiraishi et al. 1995) might be explained partly by the fact that these bacteria survive on various modes of energy-generating systems (hiraishi et al. 1995). the ability of phototrophic purple non-sulfur bacteria to grow aerobically and anaerobically in the presence of diverse aromatic compounds makes these good candidates for potential biodegradation of harmful compounds (hanvood and gibson 1988; wright and madigan 1991; shoreit and shaheb 1994). commercialization of these bacteria for purification treatment plants was initiated about 15 years ago (kobayashi and kobayashi 1995). not many phototrophic purple non-sulfur bacteria species are known to degrade aromatic compounds. rhodopseudomonas palustris (harwood and gibson 1988; gibson and gibson 1992; shoreit and shaheb 1994), rhodomicrobium vannielii (wright and madigan 1991), rhodobacter capsulatus (blasco and castillo 1992; shoreit and shaheb 1994), rs. blastica, and rhodospirilium rubrum (shoreit and shaheb 1994) are able to degrade a variety of monocyclic aromatic compounds with or without other c-sources. benzoate and its derivatives are among the common aromatic compounds that can be completely mineralized (harwood and gibson 1988; wright and madigan 1991; shoreit and shaheb 1994). however, blasco and castillo (1992) noted that rhodobacter capsulatus e1f1 is able to degrade mononitrophenol and dinitrophenol with acetate as its carbon source. rs. palustris utilized several phenolic compounds, hydroxylated and methoxylated aromatic acids, aromatic aldehydes, and hydroaromatic acids (harwood and gibson 1988). other diverse aromatic compounds have also been reported to be utilized by this group (harwood and gibson 1988; wright and madigan 1991; shoreit and shaheb 1994). degradation of aromatic compound by phototrophic purple non-sulfur bacteria in general was emphasized in this investigation based on their ability to utilize diverse aromatic compounds. to date, this group of bacteria has not been subjected to intensive examination (harwood and gibson 1988). this study focused on the ability of three new isolates of apb to utilize benzoate. identification of the bacterial isolates based on their 16s-rrna genes as well as their morphological and physiological properties was also carried out. materials and methods bacterial cultures and cultivation three isolates of apb, designated as ds-1, ds-4, and cas-13 were studied. the two former strains were isolated from java, and the last was isolated from moluccas. all isolates were maintained on modified sistrom medium with benzoate as the sole carbon source. to determine the ability of the bacteria to utilize and degrade aromatic compounds, the isolates were grown in modified sistrom by omitting all carbon sources, including nitrilo-triacetic acid, supplemented with 5 mm benzoate as the c 10 characterization of three benzoate degrading anoxygenic photosynthetic bacteria -dwi suryanto et al. source with or without vitamins in 100 ml completely filled screw-capped tubes. isolates also were grown in modified sistrom supplemented with vitamins and 5 mm succinate, or 5 mm acetate as their carbon sources to serve as a means of comparison. for cultures grown on benzoate, the inocula were obtained from 3-day old cultures of bacteria grown in modified sistrom with benzoate with an initial cell number of 5xl06 cells/ml. for cultures to test other c-sources, inocula were taken from 2-day old cultures grown in sistrom media with succinate as c-source. unless mentioned otherwise, all media were adjusted to ph 7.2. growth condition, measurement of growth, and quantitation of benzoate cultures were illuminated with a 40 w tungsten bulb at a distance of 30 cm. growth rates were determined by measuring turbidity at 660 nm every 24 hours. for cultures grown in 5 mm succinate and 5 mm acetate, growth rates were measured every 12 hours. for cultures grown in benzoate, residual benzoate levels were measured at 120 hours of incubation. benzoate concentration was measured at 276 nm using a hitachi model u-2010 uv/vis spectrophotometer (hitachi instrument, inc. japan) according to shoreit and shabeb (1994). cell density of the isolates grown in other c-sources was measured after 72 hours of inoculation. spectral analysis cells were harvested from 7-day old anaerob-phototrophic cultures grown on modified sistrom supplemented with vitamins and 5 mm succinate as sole carbon source, and suspended in icm buffer (10 mm phosphate buffer ph 7.0 and 1 mm naedta ph 7.0). sonication was carried out using soniprep 150 (msb, uk) at an amplitude of 2 u. for 2 minutes, three times with a time interval of 1 minute. cell extracts were centrifuged at 3000 rpm for 30 minutes at 4°c. protein concentration was determined according to the pierce bca* protein assay kit (rockford, iii, usa). spectral analysis was performed using hitachi u-2010 in protein concentration of ± 100 (ig/ml. amplification and sequencing of part of 16s rrna genes to sequence part of the 16s-rrna genes, the 16s-rrna genes were amplified by pcr using specific primers of 63f and 1387r from genomic dna (200 ng) on readyto-go pcr beads (pharmacia-biotech, uppsala, sweden). phenol-chloroformisoamylalcohol (25:24:1) treatment, ethanol precipitation, and agarose gel electrophoresis were used to purify the genomic dna. total volume of the pcr reaction (25 ul) consisted of 1.5 u tag dna polymerase, lomm tris-hcl (ph 9 at room temperature), 50 mm kc1, 1.5 mm mgcl2, 200 um of each dntps and stabilizer including bsa. the reaction was incubated in a gene amp pcr system 2400 thermocycler (perkin-elmer cetus, norwalk, conn.). 11 biotropia no. 17, 2001 part of 16s-rrna gene was sequenced to infer the closest related organism from the ribosomal database project (rdp) maintained at the university of illinois, urbana-champaign. the sequencing reactions were done by using the big dye ready reaction dye deoxy terminator kit, purified by ethanol-sodium acetate precipitation. the reactions were run on an abi prism 377 dna sequencer (pe applied biosystems, foster city, ca.). construction of phylogenetic trees for the construction of phylogenetic trees, cluster analysis of 16s-rrna gene was done by a computer program from the european bioinformatics institute (http://www.ebi.ac.uk). the treecon computer program (yves van de peer of the department of biochemistry, university of antwerp) was used to determine their phylogenetic relatedness based on their nucleotide sequences and mflp profiles obtained from pulsed-field gel electrophoresis. results and discussion the three new isolates described in this study were similar in their morphological properties. all were gram negative, non-halophilic, non-alkali or acidophilic, aerobic and anaerob-phototrophs that have motile rod-shaped cells with terminal swellings. under anaerobic photothrophic growth conditions, all new isolates produced brick-red and pink cultures in succinate and benzoate, respectively. spectral analysis of cell free extracts (figure 1) of photosynthetic pigments showed absorption maxima for ds-1 at 374, 493, 587, 803 and 852 nm, ds-4 at 371, 501, 585, 803 and 848 nm, and cas-13 at 373, 502, 585, 802 and 871 nm indicating the presence of bacteriochlorophyl a and normal spirilloxanthin series including lycopene and rhodopin (imhoff 1995). in this respect, the three isolates were nearly identical to each other and might be considered as one group. however, these isolates clearly differed from rhodobacter sphaeroides 2.4.1., which has different absorption maxima (376, 451, 453, 477, 507, 587, 746, 799, and 850 nm), with yellow-brown colonies grown anaerobically in succinate. the results from the analysis of partial sequencing of 16s-rrna (c.a. 500 bp) of ds-4, ds-1, and cas-13 demonstrated that ds-4 and cas-13 were closely related (figure 2). partial sequencing of the first 500 bp of 16s rrna gene could lead to a main line of descent (stackebrandt and rainey 1995). comparison with the rdp database of the university of illinois indicated that both ds-1 and cas-13 demonstrated 97% similarity with rs. palustris strain 7, and of 83% and 84% with rb. sphaeroides il106, respectively (table 1). similarity of those of ds-4 to rs. palustris strain 7 was 78%. these results suggested that ds-1 and cas13 are likely to be one species, rs. palustris. ds-4, however, is significantly different from ds-1 and cas-13 by 22%, and from rb. sphaeroides il106 by 30%. complete sequence analysis is needed to give more definitive information about the taxonomic position of these three isolates. 12 characterization of three benzoate degrading anoxygenic photosynthetic bacteria -dwi suryanto et al. figure 1. absorption spectrum of cell extracts ds-1, ds-4, cas-13, and rb. sphaeroides 2.4.1. figure 2. phylogenetic tree 16s rrna gene sequences. the number at the tree lines represented bootstrap values. 13 biotropia no. 17, 2001 table 1. percent similarity of the ds-1, ds-4, and cas-13 with other relative members of anoxygenic photosynthetic bacteria. unlike rb. sphaeroides 2.4.1, all of the three isolates were able to metabolize benzoate (figure 3). among the members of apb, rs. palustris is the most common species capable of utilizing benzoate (harwood and gibson 1988; gibson and gibson 1992; shoreit and shaheb 1994). the ability to grow in the presence of aromatic compounds such as benzoate without vitamins (figure 3) may suggest that the organisms were able to synthesize their own vitamins. however, supplemented vitamins could increase their potential in metabolizing benzoate. the rate of benzoate utilization for ds-1, ds-4, and cas-13 were 0.024 mm/hour, 0.02 mm/hour, and 0.03 mm/hour in media with vitamin supplements compared to 0.017 mm/hour, 0.012 mm/hour, and 0.018 mm/hour in media without vitamins. a relatively similar pattern of extended lag phase was observed in growth of the cultures. time was needed in preparation of producing a number of enzymes. carbon availability might affect cell growth. cell density was observed to be higher in media with benzoate (c7) (figure 3), followed by succinate (c4) and acetate (c2) (figure 4). relatively low cell density of all isolates was shown in 5 mm benzoate with no vitamins. vitamins were certainly necessary for their metabolic activity. 14 characterization of three benzoate degrading anoxygenic photosynthetic bacteria -dwi suryanto et al. figure 3. growht of ds-01, ds-4 and cas-13 in 5 mm benzoate with vitamins (above) and without vitamins (below). rb. sphaeroides 2.4.1. was used as negative control. 15 biotropia no. 17, 2001 figure4. growth of ds-1, ds-4, cas-4, cas-13, and r6. sphaeroides 2.4.1. in 5 mm succinate (above) and 5 mm acetate (below) with vitamins. 16 characterization of three benzoate degrading anoxygenic photosynthetic bacteria -dwi suryanto et al. acnowledgement this research was funded by the center for microbial diversity, faculty of science and mathematics, bogor agricultural university, bogor indonesia. references blasco, r. and f. castillo. 1992. light-dependent degradation of nitrophenols by the phototrophic bacterium rhodobacter capsulatus e1f1. appl. environ. microbiol. 58: 690-695. gibson, k. j and j. gibson. 1992. potential early intermediates in anaerobic benzoate degradation by rhodopseudomonaspalustris. appl. environ. microbiol. 58: 696-698. harwood, c. s. and j. gibson. 1988. anaerobic and aerobic metabolism of diverse aromatic compounds by the photosynthetic bacterium rhodopseudomonas palustris. appl. environ. microbiol. 54: 712-717. hiraishi, a., k. muramatsu, and k. urata. 1995. characterization of new denitrifying rhodobacter strains isolated from photosynthetic sludge for wastewater treatment. j. fermen. bioengine. 79: 39-44. imhoff, j. f. 1995. taxonomy, phylogeny and general ecology of anoxygenic phototrophic bacteria. in anoxygenic photosynthetic bacteria. ed. blakenship, r.e., m.t. madigan, and c.e. bauer. kluwer academic publishers. the netherlands, p. 1-15. kobayashi, m. and m. kobayashi. 1995. waste remediation and treatment using anoxygenic photosynthetic bacteria. in anoxygenic photosynthetic bacteria. ed. blakenship, r.e., m.t. madigan, and c.e. bauer. kluwer academic publishers. the netherlands, p. 1269-1282. kohring, g., x. zang, and j. wiegel. 1989. anaerobic dechlorination of 2,4-dichlorophenol in freshwater sediments in the presence of sulfate. appl. environ. microbiol. 55: 2735-2737. kuo, c. and b.r.s. genthner. 1996. effect of added heavy metal ions on biotransformation and biodegradation of 2chlorophenol and 3-chlorobenzoate in anaerobic bacterial consortia. appl. environ. microbiol. 62: 23172323. shoreit, a. a. m. and m. s. a. shaheb. 1994. utilization of aromatic compounds by phototrophic purple nonsulfur bacteria. biodegrad. 5: 71-76. stackebrandt, e. and f.a. rainey. 1995. partial and complete 16s rdna sequences, their use in generation of 16s rdna phylogenetic trees and their implications in molecular ecological studies. in molecular microbial ecology manual. ed. akkermans, a.d.l., j.d. van elsas, and f.j. de bruijn. pp. mmem -3.1.1/1-17. wright, g. e. and m. t. madigan. 1991. photocatabolism of aromatic compounds by the phototrophic purple bacterium rhodomicrobium vannielli. appl. environ. microbiol. 57: 2069-2073. 17 biotropia no. 10, 1997 : 14-28 influence of insect and seed sample size and heat treatment on the infestation of callosobruchvs chinens1s (l.) on mungbean, vigna radiata (l) wilczek *) elisa m. bucruanon 1 and belen morallo-rejesus 2 1 crops research division, philippine council for agriculture, forestry and natural resources research and development, 4030 los banos, laguna, philippines 2department of entomology, college of agriculture, university of the philippines at los banos, 4031 college, laguna, philippines abstract the influence of different insect and seed sample size and heat treatment on the infestation of bean weevil, callosobruchus chinensis on mungbean,vjg/m radiata (l.) wilczek, was studied. insect and seed sample size as well as varieties/genotype had significant influence in obtaining large responses in the number of eggs and progenies of the bean weevil. use of at least 10 adult weevils to infest test samples containing at least 40 seeds for a 5-day oviposition period should produce reliable results when infesting mungbean seeds with unsexed weevils. dry heat treatment was very effective in disinfesting mungbean seeds from the bean weevil in different developmental stages. it improved germination depending upon the condition of the seed before treatment and certain temperature limits. a suggested treatment for mungbean dismfestation using dry heat would be 60°c and 70°c for two-and one-hour treatments, respectively at 12% moisture content. for seeds in bulk, 60°c is much preferred. key words: callosobruchus chinensis/ insect and seed sample size/mungbean infestation/heat treatment introduction bruchids are world-wide pests of mungbean (vigna radiata (l.) wilczek) in storage. the bean weevil, c. chinensis is one of the three bruchid species that infests mungbean in the field and during storage. of the two given periods of attack, it is during storage that infestation results in greatest loss. measures to control the pest include among others the use of bruchid resistant cultivars and selective insecticides. much effort has been directed towards the screening or evaluation of chemical insecticides, vegetable oils and plant products effective hi controlling bruchids, as well as in the development of resistant varieties (duguet and wu 1986; davis et al 1984; varma and pandey 1978; pandey et al. 1981; epino and morallo-rejesus 1982). * paper presented at the symposium on pest management for stored food and feed, 5 -7 september 1995, bogor, indonesia. 14 biotropia no. 10, 1997 during the process of screening, however, certain ecological and biological factors may have their individual and interactive effects on the test insect. current practice in routine screening studies particularly against bean weevils differs largely in the number of insects and seeds used and the length of time the weevils are allowed to oviposit on the seeds. the optimum number of insects and seeds to use and the minimum oviposition period that should produce reliable results when infesting mung-bean seeds with bean weevils need to be determined. this has been established for maize and rice weevils (widstrom el al. 1972) but not for bean weevils in mungbean. inasmuch as seeds should be clean and free from insects before use in all screening and other related studies, disinfestation is of prime importance and it must therefore be very effective to ensure that the seeds used for screening are clean. it becomes even more important with the bean weevil which has a particular way of development that prevents easy detection. heat treatment at 70°c for 1 hour has been reported to be very effective in disinfesting maize seeds in china (dr. gonzalo de leon, personal communication, 1988, cimmyt entomologist). nevertheless, it needs to be further tested for its possible effect on the general condition of seed especially with legumes. this study therefore, was undertaken to determine the influence of insect and seed sample size and heat treatment on the infestation of bean weevil hi stored mungbean. materials and methods influence of insect and seed sample sizes on infestation of bean weevil on stored mungbean two mungbean genotypes, v2802 and vc 1973a, which had shown resistance and susceptibility to bean weevil attack, respectively were used in the experiment (talekar 1988; avrdc 1986). four sample sizes of mungbean seeds (10, 20, 40 and 60 seeds per sample) were tested. likewise, four weevil sample sizes (10, 20, 30 and 40 per sample) were tested. weevils were introduced into cups containing seeds of different sample sizes and held at 25°c, 75% rh. a designated male/female ratio of adult weevils was not used hi this experiment because earlier studies reported a sex ratio (males:females) of 1.06:1.00 (bato and sanchez 1972) and 6:5 (raina 1970) and therefore, it was assumed that there was a high probability of equal number of males and females in the weevil samples. moreover, emergence of adult weevils took place at night and mating occurred within an hour after emergence from the seed. one mating is enough to ensure egg fertilization and laying starts a day after emergence. the weevils were allowed to oviposit for three periods: 3, 5 and 10 days. after each oviposition period, 15 influence of insect and seed sample size and heat treatment e. m. buctuanon and b. morallo-rejesus the weevils were removed and the eggs were counted. the cups were checked daily for progeny emergence. the two mungbean genotypes, four bean weevil sample sizes, tiiree oviposition periods, and four mungbean seed sample sizes were combined in a factorial arrangement requiring % treatment combinations per replication. three replications provided three blocks for analysis under a completely randomized design. seed damage and number of eggs and progeny per adult weevil in all treatments were determined. results from the analysis of variance were used to determine which combination(s) of the main effects influenced the measurement of bean weevil biology and mungbean quality. comparisons between the responses within each factor were performed using the least significant difference (lsd). effect of dry heat treatments on different stages of bean weevil mungbean varieties, v2709 and mg-9, a resistant and a susceptible line, respectively were used. each sample consisted of 2000 seeds which were exposed to 1000 adult bean weevils for oviposition. for the egg stage disinfestation experiment, mungbean seeds were exposed to oviposition by the weevil for five days. after oviposition, seeds were mixed thoroughly and divided equally, and packed in small cotton bags before heat treatment. for the larval stage disinfestation test, the eggs laid on the seeds were allowed to hatch. the seeds were then mixed thoroughly, and divided equally into a sample size consisting of 100 seeds before dry heat treatment. for the adult stage disinfestation study, twenty-five bean weevil adults were placed in small bottles containing 100 seeds per replication. the bottles were then placed in the oven for heat treatment. heat treatments for disinfestation of all the developmental stages of the bean weevil were at 70°c and 80°c for one hour, and 60°c and 50°c for two hours. the treatments including the untreated control were replicated 4 times. mungbean seeds had 12% moisture content before heating. each of the samples was transferred into small bottles with a screen cover and arranged hi a completely randomized manner under room conditions at 25°c and 75% rh. effect of dry heat treatments on mungbean seed vigor germination tests according to the aosa (1981) "rules for testing seeds" was conducted to evaluate the damage on the seeds because of the exposure to heat. seedling growth rate (sgr) vigor test was carried out. 16 biotropia no. 10, 1997 results and discussion influence of insect and seed sample sizes on the infestation of bean weevil on stored mungbean main effects and first-order interactions that significantly influence the number of eggs laid by the bean weevil, progeny produced per adult parent weevil, and percent damaged seeds are shown in table 1. of the four principal factors tested in this study, mungbean variety or genotype (v), bean weevil sample size (b), and seed sample size (s) were the most significant factors influencing weevil infestation. table 1. summary of f values for die main effects and interaction in the analysis of variance of factors mat influenced the number of bean weevil eggs and progeny per adult and percent damaged seeds1. source of variation df no. of eggs peradulr no. of progeny peradulr2 percent damaged seeds3 variety (v) bean weevil samples (b) seed samples (s) oviposition period (o) v x b 1 3 3 2 3 462.05** 36.94** 41.04** 2.63ns 15.3** 2720.19** 225.18** 181.54** <1 ns 83.87** 4217.7** 4.1** 2.69* <1 ns 4.1** v x s 3 2.43ns 61.64** 2.69* v x o 2 <1 ns <1 ns <1 ns bxs 9 <1 ns 3.57** 1.87ns b x o 6 <1 ns <1 ns 1.23ns s x o 6 2.61* 1.05ns 1.87ns v x b x s 9 <1 ns 1.53ns <1 ns v xb x o 6 <1 ns <1 ns 1.23ns v x s x o 6 <1 ns <1 ns <1 ns b x s x o 18 <1 ns <1 ns <1 ns v x b x sx o 18 <1 ns <1 ns <1 ns error 192 * = significant at 5% level ** = significant at 1 % level ns = not significant 1 = average of the three replicates 2 = based on data analyzed using the log (x + 1) transformation 3 = based on data analyzed using the arc sine transformation four significant interactions involved seed sample size. the number of eggs per adult parent weevil was significantly influenced by the interaction of seed sample size and duration of oviposition (fig. 1). eggs per adult markedly increased with larger 17 influence of insect and seed sample size and heat treatment e. m. buctuanon and b. morallo-rejesus seed sample size. this is probably due to the greater number of seeds available for oviposition with more seeds thus lessening competition among the weevils. comparison among the three oviposition periods (3, 5 and 10 days) revealed that there was no significant differences between 3-day and 10-day oviposition period. the highest number of eggs laid by the weevils occurred at the 40-seed and 60-seed samples and at the 5-day oviposition period. although the highest egg count was obtained at the 60-seed sample this was comparable with that of the 40-seed sample at 5-day oviposition period and the latter was preferred. it was the minimum seed sample size and shortest duration of oviposition that yielded a high number of eggs per adult parent weevil. fig. 1. number of bean weevil eggs laid per adult with increasing mungbean seed sample size for three oviposition periods the interaction of the varying level of mungbean seed and parent bean weevils strongly influenced the number of progeny obtained per adult (fig. 2). the highest number of progeny (14/parent weevil) was obtained from 10 weevils/60 seeds. nevertheless, at 10 weevils/40 seeds, the same trend was observed, i.e. number of 18 biotropia no. 10. 1997 progenies was still high (8/parent weevil). the highest number of progenies per adult weevil obtained at 10-weevil sample size was suspected to be due to competition between larvae that existed at higher weevil sample size (20, 30 and 40). this resulted in only a few surviving adults. fig. 2. number of bean weevil progenies per adult with increasing seed sample size for four bean weevil sample sizes a highly significant effect was obtained in the number of progenies per adult as a result of the interaction between seed sample size and genotype (fig. 3). many bean weevil progenies emerged from the susceptible seeds. however, it was quite interesting to note that both genotypes received, more or less the same number of eggs as shown by their nonsignificant f-value (table 1). the marked difference in the number of progenies suggests failure of eggs to hatch or failure of larvae to develop into adult weevils in the resistant mungbean genotype. the effect of seed genotypes and seed sample size was also significant in terms of percent damaged seeds (fig. 4). complete damage in susceptible seeds indicated that all seeds had eggs deposited on them. in the resistant genotype, however, percent 19 influence of insect and seed sample size and heat treatment e. m. buctuanon and b. morallo-rejesus fig. 3. number of bean weevil progenies per adult with increasing seed sample size for two mungbean varieties damaged seeds significantly decreased at higher seed sample size. this shows that some seeds of the resistant genotype were missed by the females or eggs were laid but failed to develop into adult weevils. the proportion of these seeds without eggs increased as the number of seeds increased. the absence of eggs or progeny on some seeds of the resistant genotype, however, indicates that they are unsuitable for oviposition or larval growth and development. the reason why some seeds of the resistant genotype are unsuitable for oviposition is not yet clear. four highly significant interactions involved bean weevil sample size (table 1). the seed genotype x bean weevil sample size interaction (fig. 5) strongly influenced the number of eggs per adult. as the number of adult parent weevils increased, the number of eggs laid decreased in both susceptible and resistant genotypes. interference among weevils or eggs laid early by the weevils could make the bean testa less attractive (rougher) for oviposition. probably, it could be due to the effect of crowding of weevils or competition for oviposition sites. ten weevils/40 or 60 seeds seem to be the ideal insect and seed sample sizes to use in the interaction/screening studies for 20 biotropia no. 10, 1997 fig. 4. mean percent of damaged seeds as influenced by increasing seed sample size for two mungbean varieties bean weevils in mungbean since the highest number of eggs and progenies per adult occurred in this level. the degree of egg aggregation at the 10, 20 and 30-weevil samples on the resistant genotype was less than the susceptible counterpart but rose significantly between 30and 40-weevil samples. the reason for this significant increase is not yet clear. the number of progeny per adult weevil was strongly affected by bean weevil sample size and genotype interaction (table 1). the number of adult progeny emerging decreased as bean weevil number increased in both susceptible and resistant genotypes (fig. 5b). the result is consequent with the trend of result on the number of eggs (fig. 5a). adult weevil production depends on the hatchability of eggs which in turn is influenced by collisions between adults and laid eggs (nwanze el al. 1975). egg hatchability and larval growth and development may have been affected also by some resistance factors present in the seed. the susceptible and resistant genotypes varied in their reaction to bean weevil numbers as indicated in the percentage of damaged seeds (fig. 5c). the seeds of the susceptible genotype were completely 21 influence of insect and seed sample size and heat treatment – e. m. buctuanon and b. morallo-rejesus 22 biotropia no. 10, 1997 damaged. in the resistant genotype, the percent damaged seeds in the 10-, 20and 30-weevil samples were invariably less than the 40-weevil samples. it was noted, though, that there were significantly more eggs laid at 40-weevil sample in the resistant genotype (fig 5a), yet the resulting number of progenies did not give the same trend of result (fig. 5b). not many weevil progenies were produced at 40-weevil sample. this result is consistent with the previous discussion that progeny production on resistant seeds was suppressed due to some resistance factors present in the seeds. effect of dry heat treatments on the different stages of bean weevil in the egg stage disinfestation experiment, results show that heat treatment significantly influenced the number of adult bean weevils that emerged from the disinfested mungbean seeds. no adult weevils emerged from seeds treated at 60°c, 70°c and 80°c, and exposed for 2-hour and 1-hour, respectively (fig. 6a). weevils survived from dry heat treatment at 50°c for two hours. likewise, temperatures at 70°c and 80°c applied for one hour and 60° c for wo hours killed all the larvae (fig. 6b). higher number of larvae survived from 50°c for 2-hour exposure which was comparable to the control. moreover, no adult weevils survived after exposure to 70°c and 80°c for one hour and at 60°c for 2 hours (fig. 6c). only 64% and 79% of adult weevils were killed at 50°c for 2-hour exposure in susceptible and resistant lines, respectively. these results indicated that effective control was achieved using dry heat treatments at 60°c, 70°c and 80°c for twoand onehour exposure, respectively. insect eggs, larvae and adults were completely killed at these temperature levels and duration of exposure, thus indicating that this temperature range could be used in disinfesting mungbean seeds depending upon its effect on the viability and vigor of seeds which is discussed in the succeeding topic. effect of dry heat treatments on mungbean seed vigor the effect of dry heat temperatures on the vigor of seeds of two mungbean genotypes based on the percentage of normal seedlings, plumule length and dry matter weight is shown in table 2. results showed that dry heat treatments increased the percentage of normal seedlings in both susceptible and resistant genotypes (fig. 7a). highest and lowest percentages of normal seedlings from the resistant genotype were obtained at 70°c and 80°c, respectively for one hour exposure. lower temperatures (50°c and 60°c) but of longer duration of exposure (two hours) gave results comparable to that of the control and those exposed at 70°c. the most pronounced increase in the percentage of normal seedling (50.5%) with the susceptible genotype was 23 influence of insect and seed sample size and heat treatment e. m. buctuanon and b. morallo-rejesus 24 biotropia no. 10, 1997 table 2. summary of f values of the analysis of variance on the effect of the different treatment levels and varieties on the percentage of normal seedlings, mean plumule length and weight 1 obtained from seeds exposed to 80°c. no germination occurred from among those seeds in the control. there were hard seeds recovered from the susceptible genotype indicating the seeds to be probably dormant were broken by heat treatment specifically at higher temperature. results clearly indicated that dry heat treatment tremendously improved germination of seeds. this may probably be due to the increased water permeability at the strophiole of the mungbean seeds. mott and mckeon (1979) observed this phenomenon with dry heat treatments of stylo seeds, s. humilis. the effect of different dry heat temperatures and genotype interactions on plumule length was highly significant (table 2). differences in plumule length were markedly evident between genotypes (fig. 7b). in the resistant genotype, the plumule length tended to be longer, comparable with the control treatment, from seeds exposed to higher temperatures (70°c and 80°c) for one hour. seeds from the control treatment failed to germinate. there was no significant difference in the dry matter weight obtained between genotypes, and in all levels of dry heat temperatures in the resistant genotypes (fig. 7c). highest dry matter weight was obtained in 60°c treatment for two hours in the susceptible genotype as compared to the 80°c-1 hr treatment. heating mungbean seeds with dry air generally improved germination. however, certain important factors and/or seed conditions should be considered before treatment. in this particular study, resistant variety seemed to respond well to heat treatments especially at higher temperatures, but only to a maximum of 70°c. dry heat temperatures of 80°c significantly damaged the seeds as indicated by the lowest percentage of germination and normal seedlings obtained from this treatment level. variety can be a factor in heat damage resulting perhaps from differences in hardness, protein content or the variety as such (ghaly and taylor 1982). 25 influence of insect and seed sample size and heat treatment e. m. buctuanon and b. morallo-rejesus 26 biotropia no. 10, 1997 mungbean seed vigor as evaluated, based on the four parameters, seemingly is not affected detrimentally by dry heat treatments up to certain limits. dry heat treatment of seed at 60°c is safer to use. the 60°c temperature level was found to be equally effective when compared with 70°c and 80°c in disinfesting mungbean seeds from the bean weevil, c. chinensis. summary and conclusion the influence of insect and different seed sample sizes and heat treatment on the infestation of bean weevil, callosobruchus chinensis (l.) on mungbean was determined. insect and seed sample sizes as well as variety/genotype had significant influence in obtaining high production of eggs and progenies of the bean weevils. these different parameters are tp be considered in screening studies. bean weevil eggs laid per adult increased with high seed numbers and the number of progenies decreased with increasing numbers of parent weevil. the weevils per 60 or 40 seeds in 5-day oviposition periods provided the greatest frequency of significant responses in the number of eggs and progeny per adult weevil and percentage of damaged seeds. these are the minimum number of weevils necessary in a parent population and the practical seed sample size that would support the minimum number of days of oviposition to obtain maximum oviposition and/or infestation for screening studies. the number of weevil progenies differed in the resistant and susceptible genotypes, but no variation was observed in the number of eggs laid. the number of eggs laid and emerging progenies decreased as the number of parent weevils introduced was increased in both susceptible and resistant genotypes. seeds of susceptible genotypes were completely damaged in all seed density levels. however, percent damaged seeds decreased at higher seed densities in resistant genotypes. dry heat treatment was found to be very effective in disinfesting mungbean seeds from the different development stages of bean weevil. dry heat treatment improved germination and is not detrimental to the seeds regardless of genotype, depending however, on the condition of the seed before treatment which affects the dry heat temperature requirement and/or limits of the seeds. dry heat treatment of seeds at 60°c for 2 hours and 70°c for 1 hour exposure at 12% moisture content give complete insect control and is safer to use. heat treatment at 60°c however, is much preferred in disinfesting big bulk of seeds. 27 influence of insect and seed sample size and heat treatment e. m. buctuanon and b. morallo-rejesus literature cited aosa. 1981. rules for testing seeds. 6(2): 30, 113. avrdc, 1986. avrdc progress report. 1984. asian vegetable research and development center (avrdc), shanhua. taiwan, 480 p. bato, s.m. and f.f. sanchez. 1972. the biology and chemical control of callosobruchus chinensis (linn.) (coleoptera: bruchidae). philipp. ent. 2(3): 167-182. davis, r., j. boczek, d. pankiewicz-nowicka and m. kruk. 1984. efficacy of tricalcium phosphate as a legume grain protectant. proc. third int. working conf. on stored-product entomology. kansas state university, manhattan, kansas, p. 256-261. duguet, j.s. and g.x. wu. 1986. assessment of activity of deltamethrin against callosobruchus chinensis l. and callosobruchus maculatus fab. (bruchidae). int. pest control. 28: 36-41. epino. p.b. and b. morallo-rejesus. 1982. mechanisms resistance of mungbean (vigna radiata (l.) wilczek) to callosobruchus chinensis (l.). philipp. ent. 5: 447-462. ghaly. t.f. and p. a. taylor. 1982. quality effects of heat treatment of two wheat varieties. jour. agric, and eng'g. res. (27): 227-234. mott. j.j. and g.m. mckeon. 1979. effect of heat treatments in breaking hardseededness in four species of stylosanthes. seed sci. & tech. 7: 15-25. nwanze. f.f., e. horber and c.w. pms. 1975. evidence for oviposition preference of c. maculatus for cowpea varieties. environ. ent. 4: 409-412. pandey. g.p.. r.b. doharey and b.k. varma. 1981. efficacy of some edible oils for protecting greengram against the attack of callosobruchus maculatus i.fabrj. indian jour. agric. sci. 51: 910-912. raina, a.k. 1970. callosobruchus spp. infesting stored pulses (grain legumes) in india and a comparative study of the biology. indian jour. entomol. 32: 303-310. talekar. n.s. 1988. biology, damage and control by bruchid pests of mungbean. proc. of the second int. symp. on mungbean. p. 329-342. varma, b.k. and g.p. pandey. 1978. treatment of stored greengram seed with edible oils for protection from callosobruchus maculatus (fab.). indian jour. agric. sci. 48: 72-75. widstrom. n.w., l.m. redlinger. and w.j. wiser. 1972. appraisal of methods for measuring com kernel resistance to sitophilus zeamais. jour. econ. ent. 65: 790-2. 28 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf biotropia no biotropia no. 14, 1999: 1-9 genetic diversity analysis of thermophilic bacteria from candradimuka crater in central java employing pcr-rflp of 16s-rrna gene temmy desiliyarni1+, antonius suwantoaaao2, 3 4*, maggy t. suhartono3, and tresnawati purwadaria5 'graduate school, bogor agricultural university, bogor, indonesia 2 department of biology, faculty of science and mathematics, bogor agricultural university, jl. raya pajajaran, bogor, indonesia 3 inter university center for biotechnology, jl. puspa tromol pos 1 darmaga, bo gor agricultural university, bogor, indonesia 4seameo-biotrop, jl. raya tajur, km. 6 bogor, indonesia ''research institute for animal production, po box 221 bogor 16002, indonesia abstract the specific primers for bacteria (63f and 1387r) were used to amplify the 16s-rrna genes from total community genomic dna of thermophilic bacteria. the total community genomic dna was obtained from muds and water samples of candradimuka crater, dieng plateau, central java. pcr products were cloned into vector pcr*2.1-topo (3.9 kb) and transformed into escherichia coli topic. two tetrameric restriction endonucleases rsal and hhal were employed to generate restriction fragment length polymorphisms (rflp) paterns. these enzymes yielded 10 and 9 groups of 16s-rrna profiles or otu (operational taxonomic units) from 27 16s-rrna gene clones. rsal was found to be more discriminative in differentiating the clones than hhal. rsal-rflp indicated that otu 7 and otu 3 represented the most abundant clones, i.e. 6 and 5 clones respectively. the distribution of 16s-rrna gene clones could indicate relative distribution of specific groups of thermophilic bacteria in their natural habitat. analysis of diversity at the dna level could represent both culturable and unculturable bacteria in the environment. similarity analysis showed that at level 0.600 there were 8 different groups from 10 rflp profiles generated by rsal digestion. this study indicated that there were at least 8 groups of different thermophilic bacteria occupying candradimuka crater. key words: thermophiles, 16s-rrna, candradimuka crater. introduction sustainability of the biosphere on our planet depends on microbial activities. however, we know very little about the microbial world. the reason for this poor understanding lies in the fact that microbes are tiny and individually invisible to the eye. until recently, microbial identification required isolation of pure cultures followed by a series of physiological tests and biochemical characterization. the pure culture approach has limited our view of microbial diversity in the world, since ' corresponding author; e-mail address : asuwanto@indo.net.id * present address : mercu buana university, jl. meruya selatan, jakarta. 1 biotropia no. 14, 1999 it has been known that approximately only 1% of microorganisms in the environment could be cultured (amann et al. 1995; pace 1997; borneman et al. 1996). microorganisms are involved in many important activities such as soil formation, toxin removal, biogeochemical cycles of carbon, nitrogen, phosphor and others, conservation of more complex species, and to serve as valuable materials to many industries. environmental stresses can alter microbial population and therefore endanger biosphere health. in the absence of systematic approaches to studying and ensuring survival of diverse microorganisms, perhaps the best approach is to preserve as many habitats as possible, especially those from extreme environment which may have unique microorganisms (fox 1994; borneman et al. 1996). one of the extreme environments is thermal environment which could be found in natural and artificial systems such as volcanic region, coal refuse piles, hydrothermal vents and geothermal power plants (stetter 1995). species diversity of the thermophilic organisms is found in both the domain of bacteria and archaea. thermophilic bacteria exhibit a wide range of nutritional capability such as phototrophy and chemotrophy; autotrophy and heterotrophy; chemolitotrophy and chemo-organotrophy; aerobiosis and anaerobiosis (brock 1986). the application of molecular approaches to assess bacterial diversity is now entering the exponential growth. these approaches have overcome the requirement for prior cultivation. the most widely used technique to amplify the gene coding for 16srrna from environmental samples relies on the application of pcr (service 1997; marchesi et al. 1998). for identification purposes, hypervariable regions of rkna molecule are particularly useful due to relatively high differences between species but relatively low variability within species. currently, there are more than 4000 16srrna entries in the database, covering about 1800 species which continue to grow (bottger 1996; amann et al. 1994). hypervariable regions are also the regions with the most concentrated polymorphism for restriction fragment length polymorphisms (rflp) purposes (green 1998). the highly conserved regions of the rrna molecule can serve as primer binding sites for in vitro amplification by pcr (amann 1994). pcr primers designed and evaluated by marchesi et al. (1998) i.e. . 63f (5'-cag gcc taa cac atg caa gtc) and 1387r (5'-ggg cgg wot gta caa ggc) could amplify 1300 bp of a consensus 16s-rrna genes from bacteria. in our study, we attempt to explore genetic diversity of thermophilic bacteria with pcr-rflp of 16s-rrna genes from environmental dna. rflp analysis was conducted using tetrameric endonucleases rsal (5'-gtlac) and hhal (5'-gcgic). these enzymes along with bstul were described by moyer et al. (1996) as the most efficacious at detecting and differentiating bacterial small sub unit rrna genes on the basis of their ability to correctly classify operational taxonomic units (otu). in this study, we have also analyzed partial 16s-rrna sequences of two dominant otu in candradimuka crater. 2 genetic diversity analysis of thermophilic bacteria temmy desiliyarni et al. materials and methods sample collection candradimuka crater is located in dieng plateau (above 2000 m from sea level), central java, indonesia. sample was collected from a small pool (cl site) characterized by almost boiling hot spring with temperature of 88°c and ph7 (fig. 1). water and sandy-mud were collected in a 250 ml sterile bottle, tightly sealed, and transported within 24 hours for immediate processing. figure 1. profile of cl site in candradimuka crater, dieng plateau, central java. extraction and purification of genomic dna approximately 5 g of sample was extracted as described by zhou et al. (1996) and modified by tiedje (1997, unpublished). the crude dna obtained was purified with the prep-a-gene kit (biorad, richmond, ca). amplification and cloning of 16s-rrna genes the 16s-rrna genes were pcr-amplified by specific primers for bacteria (63f and 1387r) from purified genomic dna (200 ng) using ready-to-go pcr 3 biotropia no. 14, 1999 beads (pharmacia-biotech). total volume of pcr reaction contained 1.5 u tag dna polymerase, lomm tris-hcl (ph 9 at room temperature), 50 mm kc1, 1.5 mm mgcl2, 200 um of each dntps and stabilizer including bsa. the reaction was incubated in gene amp pcr system 2,400 thermocycler (perkin-elmer cetus, norwalk, conn.)the pcr protocol was: pre-pcr at 94°c for 2 min, denaturation at 92°c for 30 s, annealing at 55°c for 30 s, elongation at 75°c for 1 min and post-pcr at 75°c for 5 min with a total of 30 cycles (marchesi et al. 1998). the pcr product was cloned into pcr®2.1-topo (3.9 kb), and transformed into competent e. coli top 10 using topo ta cloning kit (invitrogen corp.). the positive clones were screened for a-complementation and selected as white colonies. rflp analysis of 16s-rrna genes recombinant plasmids were isolated using the wizard plus sv miniprep system (promega, madison, wi). £cori and rsal were employed to digest the plasmids in order to verify the cloned dna and to classify according to their restriction patterns. the reason for selecting these two enzymes was based on the fact that the insert dna was cloned between two sites of ecori and there are 3 rsal sites in the vector plasmid. clones that contained approximately 1,300 bp of 16s-rrna genes were chosen for rflp analysis. these clones were subsequently pcr-amplified, restricted by tetrameric endonuclease rsal and hhal to detect the different restriction profiles. restriction enzyme digestion was performed at 37°c for 6 hours before they were separated by gel electrophoresis in 2% agarose gel, stained with 0.5 ug of ethidium bromide per ml and visualized by uv transilluminator (sambrook et al. 1989). rflp profiles obtained from rsal digestion were used to generate dendrogram reflecting their genetic relationship employing upgma clustering method from ntsys program (rohlf 1990). dna sequencing of 16s-rrna genes genes for 16s-rrna from two dominant otus were partially sequenced to infer the closest related organism from rdp database. the sequencing reactions were done using the big dye ready reaction dye deoxy terminator kit and purification using ethanol-sodium acetate precipitation. the reactions were run on an abi prism 377 dna sequencer (perkin-elmer cetus, norwalk, conn.). results and discussion screening for a-complementation yielded 36 white colonies (transformants). recombinant plasmids from all of these transformants were extracted. verification of the clones containing an insert dna (16s-rrna gene) using rsal generated 19 groups of restriction profiles. thus, we used these 19 groups for subsequent rflp 4 genetic diversity analysis of thermophilic bacteriatemmy desiliyarni et al. analysis. a total of 16 groups (representing 27 clones) contained the entire 1.3 kb to 1.35 kb 16s-rrna insert after pcr-amplification using amplimers 63f and 1387r. two tetrameric restriction endonucleases were employed to differentiate rflp profile from 16s-rrna genes of 16 groups mentioned above in order to identify every object to be classified as otu or a strain in most bacteriological works (logan 1994). the rflp profiles resulted from rsal digestion generate 10 otus (fig. 2) which were: otu 1 (clone number 1, 2, 5, 31), otu 2 (number 3), otu 3 (number 4, 10, 14, 21, 32), otu 4 (number 6), otu 5 (number 9), otu 6 (number 7, 8), otu 7 (16, 18, 19 25, 26, 34), otu 8 (number 17, 20), otu 9 (number 12, 15, 27, 36) and otu 10 (number 30). two dominant otus are otu 7 (6 clones) and otu 3 (5 clones). in evaluating these profiles, we identified discrete patterns that are specific for a clone and could be distinguished one from another as an otu. results from these data infer that bacteria belonging to otu 7 and otu 3 were distributed relatively wide and dominant at the habitat of candradimuka crater. figure2. rflp profile of 16s-rrna gene digested with rsal. molecular standard is 100 bp dna ladder (m). the second digestion of 16s-rrna gene using hhal produced 9 otu (fig. 3). the grouping of restriction profiles was the same as rsal except that otu 7 and otu 8 (from foal-rflp) were in the same group in ///zal-rflp. the result of rflp using these two enzymes indicated that rsal was more discriminative than hhal to differentiate the clones, in this case for bacterial strains from candradimuka crater belonging to otu 7 and otu 8. this result was in agreement with the one conducted by moyer et al. (1996) that hhal, rsal and bstul were the most 5 m 17 18 20 21 27 30 34 36 biotropia no. 14, 1999 figure 3. rflp profile of 16s-rrna gene digested with hhal. molecular standard is 100 bp dna ladder (m). efficacious enzymes at detecting and differentiating 16s-rrna genes originated from environmental dna. our result indicated that at least 10 otus were present in candradimuka site cl which reflected the relative abundance of bacteria in this extreme habitat, in contrast to only two culturable clones obtained on thermophilic maintenance medium incubated at ph 1 and temperature of 70°c under aerobic condition. research conducted by huber et al. (1991) with samples taken from three sites of candradimuka crater obtained two hyperthermophilic archaea, i.e. thermoproteus and desulfurococcus and novel anaerobic obligate bacteria having chemolitotrophy h2/no3". the use of amplimers 63f and 1387r in this study would limit the amplification of 16s-rrna genes only for bacteria. consequently, one should keep in mind that assessment of prokaryote diversity in this study did not include members from archaea. the reduction of 16 groups of restriction profiles to 10 otus from /faal-rflp profiles could be explained with the possibility that the gene insert may have two different orientations when ligated to the vector. the difference could be determined from recombinant plasmid restriction profiles, but not for intact 16s-rrna genes obtained from pcr. the 10 otus of thermophilic bacteria were analysed further using cluster analysis (fig. 4). this dendrogram showed that at level 0.600 there were 8 groups from 10 otus, in that otu 4 and otu 5 were in one cluster as well as otu 7 and otu 8. from this dendrogram, it could also be shown that at level 0.205, there were two groups of genetic relationship. one group consisting of otu 7, 8, 9 and 10, were in the same group with it-08 isolate obtained from gunung pancar, west java 6 m 2 3 < 6 9 1 0 16 m 17 ib 20 2t 27 30 31 34 36 8 bp (tan 1999) and cr1.2 (isolate from 16s-rrna gene library of candradimuka crater at site 4). isolate of it-08 has the dna sequence similar to bacillus thermoleovorans (98%) whereas cr1.2 has the dna sequence similar to brevibacterium thermoruber (99%). the other group shows that otu 1 was closely related to e. coli (16s-rrna genes in pkk3535 constructed by brosius et al., 1981 in herawati, 1996). one possible explanation is the construction for otu 1 and e. coli involved 3 restriction fragments which have two similar band sizes. another explanation 'is that otu 1 is a new bacterial isolate which yielded dna profile similar to e. coli when restriction enzyme rsal was employed in this study. in a habitat like candradimuka crater, there may be some dominant bacteria occupying that extreme habitat. they are physiologically active among the slow grower and dormant bacteria. therefore, estimating genetic diversity through isolation of dna from environmental sample is able to represent all of the organisms including unculturable ones. although there are some biases such as different cell lysis methods and amplimer sets resulting in limited comparability to other studies, the data of 16s-rrna genes from environmental libraries demonstrated the presence of hitherto unidentified bacteria. only a minority of sequences retrieved from directly isolated soil dna were reported to be closely related to cultured organisms so that bacterial communities in the environment were composed mainly of uncultured species (felske et al. 1998). partial sequencing of otu 3 (520 bp) and comparison to ribosomal database project (rdp) database from the university of illinois indicate that otu 3 demonstrated 96% similarity to ocrobactrum anthropi (iam 14119) whereas otu 7 (450 bp) had 86% similarity to pseudomonas flavescens (b62(t)). dna sequencing provides more accurate data because nucleotides are the basic unit of information, straightforward approaches for inferring phylogenetic history and very reliable to compare specific dna fragment. besides the advantages, this technique carries inherent drawbacks, such as relatively high cost operation and impractical for 7 biotropia no. 14, 1999 routine application long dna molecule. on the contrary, pcr-rflp technique is simple, rapid, low cost, for a 50 3000 bp dna fragment range could be assayed, and especially more appropriate when many individuals need to be sampled (hillis et al. 1996). to assess the diversity of an environmental sample using pcr-rflp study, we do not have to use the most accurate technique like dna sequencing. therefore, the simpler and faster method could be valuable in taxonomic study for bacterial in situ detection and routine investigation. acknowledgments this research was financially supported by hibah tim project (urge) no. 005/htpp/iv/urge 1999/2000, and dip seameo-biotrop bogor 1998/1999 to antonius suwanto. references amann, r.i., w. ludwig, and k.h. schleifer. 1994. identification of uncultured bacteria: a challenging task for molecular taxonomists. asm news. 60:360-365. amann, r.i., w. ludwig, and k.h. schleifer. 1995, phylogenetic identification and in situ detection of individual microbial cells without cultivation. microbiol. rev. 59:143-169. anonim. 1997. topo ta-cloning® manual. invitrogen corp. san diego, california. bomeman, j., p.w. skroch, k.m.o'sullivan, j.a. palus, n.g. rumjanek, j.l. jansen, j. nienhuis and e.w. triplett. 1996. molecular microbial diversity of an agricultural soil in wisconsin. appl. environ. microbiol. 62:1935-1943. bottger, e.c. 1996. approaches for identification of microorganisms. asm news. 62:247-250. brock, t.d. 1986. an overview of the thermophiles. in brock, t.d. thermophiles: general, molecular and applied microbiology. john wiley & sons. new york. herawati, e. 1996. physiology and genetic characterization of luminous vibrio causing disease on tiger prawn (penaeus monodon fab.). thesis, aquaculture program, faculty of fisheries, bogor agricultural university, bogor. 'felske, a., a. wolterink, r. van lis and a.d.l. akkermans. 1998. phylogeny of the main bacterial 16srrna sequences in drentse a grassland soils (the netherlands).appl. environ. microbiol. 64:871-879. fox, j.l. 1994. microbial diversity: low profile, immense breadth. asm news. 60:533-536. green, e. k. 1998. restriction fragment length polymorphisms. in rapley, r. and j.m. walker. molecular biology methods handbook. humana press. totowa, new jersey. hillis, d. m., b. k. mable, a. larson, s. k. davis and e. a. zimmer. 1996. nucleic acids iv: sequencing and cloning. in d. m. hillis, c. moritz and b. k. mable (ed.). molecular systematics. sinauer associates, inc. massachusetts. huber, g., r. huber, b.e. jones, g. lauerer, a. neuner, a. segerer, k.o. stetter and e.t. degens. 1991. hyperthermophilic archaea and bacteria occurring within indonesian hydrothermal areas. system. appl. microbiol. 14:397-404. 8 genetic diversity analysis of thermophilic bacteria temmy desiliyami et al. logan, n. a. 1994. bacterial systematics. blackwell scientific publ. london. marchesi, j.r., t. sato, a.j. weightman. t.a. martin, j.c. fry, s.j. hiom and w.g. wade. 1998. design and evaluation of useful bacterium-specific pcr primers that amplify genes coding for bacterial 16srrna. appl. environ. microbiol. 64:795-799. moyer, c.l.. j.m tiedje, f.c. dobbs, d.m. karl. 1996. a computer-simulated restriction fragment length polymorphism analysis of bacterial small-subunit rrna genes: efficacy of selected tetrameric restriction enzymes for studies of microbial diversity in nature. appl. environ. microbiol. 62:2501-2507. pace, n. r. 1997. a molecular view of microbial diversity and the biosphere. science. 276:734-739. rohlf, c. l., c.c. yeo and l. tay. 1990. ntsys-pc, numerical taxonomy and multivariate analysis system, version 1.60. exeter software. new york. sambrook, j., e.f. fritsch and t. maniatis. 1989. molecular cloning. a laboratory manual. second edition. cold spring harbor laboratory press. service, r. f. 1997. microbiologists explore life's rich, hidden kingdoms. science. 275:1740-1742. stetter, k. o. 1995. microbial life in hyperthermal environments. asm news. 61:285-290. tan, i. 1999. characterization of a thermophilic xylanolytic bacterium isolated from gunung pancar hotspring. bogor. master thesis, graduate study, bogor agricultural university, bogor zhou, j., m.a. brunns, and j. m. tiedje. 1996. dna recovery from soils of diverse composition. appl. environ. microbiol. 62:316-322. 9 biotropia (2) 1988/1989: 12-17 the performance of soybean (c.v. americana) established by zero tillage technique in imperata field controlled by herbicides s. tjitrosemito tropical agricultural pest biology program, biotrop, bogor, indonesia d. suwinarno department of agronomy, faculty of agriculture bogor agricultural university, bogor, indonesia abstract a field experiment was conducted to investigate the performance of soybean (c.v. americana) when established with zero tillage technique on imperata dominated area. four different techniques of alang-alang control i.e. imazapyr (20 kg ai/ha), glyphosate (25 kg ai/ha), glufosinate (3.0 kg ai/ha) and manual cultivation were arranged factorially with time of plantings i.e. 1,2 and 3 months after treatments. the alang-alang damages varied with herbicides and times, imazapyr (20 kg ai/ha) showed slow appearance of damage at 3 months after application it was only 69%, while that of glufosinate was already down to 48% due to regrowth. no phytotoxicity was recorded, but the yield was low. introduction soybean, glycine max l. merrill, is promising and a proven source of plant protein and edible oil, containing 39-45% (dry matter basis) high quality protein with excellent amino acid pattern and 20-23% (dry matter basis) edible oil with considerable unsaturated fatty acids (hittle 1974). in indonesia, the soybean production in 1973 was 541 000 tons with the domestic consumption of 505 000 tons. indonesia was exporting 36 000 tons, however, since 1975 indonesia has been importing an increasing amount of soybean reaching more than 500 000 tons in 1983, spending a considerable amount of precious devisa. actually, the indonesian production of soybean had been from 492 000 tons annually during pelita i (first five year development plan), to 568 000 tons annually during pelita ii (second five year development plan) and 622 000 tons during pelita iii (third five year development plan); however, this production could still not meet the domestic demand and a considerable amount of soybeans has to be imported (sihombing 1985). 12 the performance of soybean (c.v. americana)-s. tjitrosemito & d. suwinarno various efforts have been tried to increase the production of soybean in indonesia; these are through intensification as well as extension program. areas available for further agricultural expansion are, unfortunately, those classified as marginal land (driessen & soepraptohardjo 1974); some of those areas are dominated by alang-alang (imperata cylindricd). this type of areas has been given a high priority for agricultural development (sastrosuwarno 1981); therefore, it is imperative to develop techniques applicable to areas dominated by this weed to utilize the area into an agricultural production system. manual and mechanical cultivation were reported to be successful, when done during the dry season (sembiring and supardjo 1981; mangunsong 1976), but the hazard of erosion can be of considerable magnitude (suwardjo 1986, coaster 1936), so manual and mechanical cultivation are considered less favorable and zero tillage may be a good alternative to those techniques. tjitrosemito et al. (1983), reported that glufosinate at 3 kg ai/ha was able to control alang-alang well; and the subsequent work in pots indicated that even glufosinate 1-3 kg ai/ha was enough to control alang-alang followed by establishment of soybean (tjitrosemito and wiroatmodjo 1983). however, the regrowth of alang-alang under treatments of glufosinate was usually quite high after 3 months. as pointed out by tjitrosemito (1985) there are several factors affecting the success of this technique among others, the appropriate planting time after herbicide application. ideally, crops should be planted immediately after spraying to take advantage of dying imperata with no germination of other weeds yet; but it may have to consider the residual activity of herbicide and avoid the possible phytotoxi-city of crops by that herbicide. this work reports on the effects of different planting time after spraying from 1-3 months on the growth performance of soybean (c.v. americana). materials and method the work was carried out from august 1985 to march 1986, at the biotrop experimental field, which had been grown with alang-alang for the last three consecutive years. the field was divided into 36 plots measuring 4 x 5 m 2 with pathways of 1 m wide in between plots. the pathway was cleared from alang-alang during the whole experimentation by slashing. the experimental design was split-plot. the main plot consisted of four different methods of alang-alang control, i.e. foliar application of herbicides imazapyr (20 kg ai/ha), glyphosate (2.5 kg ae/ha), glufosinate (3.0 kg ai/ha) and manual cultivation, while the subplot consisted of three different time of planting, i.e. 1, 2 and 3 months after spraying/treatment and replicated 3 times. 13 biotropia no. 2, 1988/1989 before planting, the standing dead alang-alang was flattened by rolling a drum over the plots. the soybean (c.v. americana) was planted at a distance of 40 x 20 cm by dibbling three grains in each hole. plastic strings were extended across the plot 40 cm apart and a very shallow furrow was made along the extended string to facilitate fertilizer application before dibbling. the fertilizer was applied as basal treatment as tsp, urea and k2so4 at 60 kg p2o5, 45 kg n, and 50 kg k2o/ha. to protect the soybeans from agromysa phaseoli, furadan 36 was applied and azodrin at 2 cc/1 was sprayed weekly for 4 weeks. the spraying was repeated whenever necessary. manual weeding was done 2 weeks after planting at all treatments by cutting weed at ground level. the percentage of damage to alang-alang at planting time was recorded and analyzed statistically. the growth performance of soybean was evaluated in terms of plant height, leaf number, leaf area, yield and weight of 100 grains of soybean. results and discussion 1. percentage of damage to alang-alang at planting time the percentage of damage to alang-alang is presented in table 1. table 1. the average percentage of damage to alang-alang at planting time. time after spraying (months) method of control 1 2 3 1. imazapyr (2.0 kg ai/ha) 18.3 30.0 69.3 2. glyphosate (2.5 ae/ha) 30.0 86.0 88.3 3. glufosinate (3.0 kg ai/ha) 99.3 84.3 48.3 4. manual 81.7 60.0 31.7 lsd (cxt) : 17 the average percentage of damage reflected the character of the herbicide used. imazapyr (2.0 kg ai/ha) produced a slow appearance of visual damage. one month after spraying, alang-alang exhibited only 18.3% damage increasing to 69.3%, two months later. glyphosate (2.5 ae/ha) caused a faster appearance of visual damage i.e. 30.0% at one month and 88.3%, 2 months later. these two herbicides are both systemic, affecting the synthesis of amino acids (shaner et al. 1985). glufosinate 14 the performance of soybean (c.v. americana)-s. tjitrosemito & d. suwinarno showed a very rapid action in terms of visual symptoms which were almost complete one month after spraying. however, the affected alang-alang recovered soon and regrowth was quite extensive 3 months after spraying. it was not different from manual control. glufosinate is a strong inhibitor of glutamine synthesis in plants, inducing a rapid built-up of ammonia which reaches toxic level (kochler and lotzsch 1985). no phytotoxicity was observed in all treatments. this was contrary to the previous work on imazapyr where at 1.5 kg ai/ha it affected the growth of soybean. this may be due to possible leaching under field condition which did not occur in pots since they were not perforated and soybean exploited soil in pots extensively (less soil in pots). it is also possible that the variety americana is more tolerant to imazapyr than orba, the variety used in the previous work. further studies in this line are needed. time of planting after treatment did not affect the height of soybean plant as indicated by the absence of phytotoxicity. one month after spraying was sufficient to avoid the phytotoxic effect of glyphosate, glufosinate or imazapyr in terms of vegetative growth. contrary to the time of planting after treatment, alangalang control affected the growth of soybean considerably. at two weeks old, soybean under manual cultivation grew shorter (10.1 cm) compared to those with herbicide treatment (table 2). at 8 weeks, the growth of so.ybean under glufosinate (3.0 kg ai/ha) slowed down, but it did not differ from those under manual cultivation, while those under glyphosate (2.5 kg ai/ha) grew tallest (53.4 cm). table 2. the average plant height (cm) at 2, 4 and 8 weeks after planting. time after planting (weeks) method of control 2 4 8 1 . imazapyr (2.0 kg ai/ha) 11.9 b 19.6 b 51. 3 bc 2 . glyphosate (2.5 kg ae/ha) 12.0 b 20.2 b 53.4 c 3 . glufosinate (3.0 kg ai/ha) 12.3 b 19.2 b 48.1 ab 4 . manual 10.1 a 17.2 a 44.7 a nb: numbers in a column followed by the same letter do not differ significantly at p≤0.05. 2. the development of leaves the soybean performance in terms of leaf number and area is presented in table 3. 15 biotropia no. 2, 1988/1989 table 3. the average leaf number/plant and leaf area recorded 8 weeks after planting. leaf method of control number leaf area index 1 . imazapyr (2.0 kg ai/ha) 10.9 b 1.9b 2 . glyphosate (2.5 kg ae/ha) 9.8 ab 1.7 ab 3 . glufosinate (3.0 ai/ha) 8.6 a 1.5 a 4 . manual cultivation 8.9 a 1.4 a nb: numbers in a column followed by the same letter do not differ significantly at p≤0.05 the vegetative growth in terms of plant height, leaf number and leaf area indicated an excellent growth exhibited by soybean when alang-alang was controlled by imazapyr (2.00 kg ai/ha). the growth of soybean under glufosinate was reasonable and was as good as those under manual cultivation. 3. yield of soybean the yield of soybean is presented in table 4. table 4. the yield of soybean (kg/ha) under various techniques. method of control kg/ha 1. imazapyr (2.0 kg ai/ha) 750 2. glyphosate (2.5 kg ae/ha) 750 3. glufosinate (3.0 kg ai/ha) 690 4. manual cultivation 680 the yield of soybean gave a very different picture from its vegetative growth. the yield of manual cultivation was as high as those under glyphosate treatment. soybean grown with zero tillage when alang-alang was controlled with imazapyr was lower than manual control of glyphosate. it was rather surprising that the vigorous growth of leaves did not produce a high yield as was expected. plant population and calculated leaf area index did not support the contention that this low yield was due to overcrowding. it may be worth to mention that this experiment was carried out during the very wet season, it probably affected the availability of par, thereby reducing the yield. 16 the performance of soybean (c.v. americana)-s. tjitrosemito & d. suwinarno references coster, c. 1938. bovengrondse afstroming en erosie op java. tectona 31: 613-729. driessen, p.m. and soepraptohardjo. 1974. soil for agricultural expansion in indonesia. bull. no. 1. soil research institute. hittle, c.n. 1974. soybeans around the world. soybean production protection and utilization. intsoy 6: 6-17. hymowitz. 1970. on the domestication of soybeans. econ. bot. 24(4): 408-421. ismail, inu g., w.s. ardjasa and s. effendi. 1981. prospek penggunaan herbisida roundup dalam pengelolaan lahan alang-alang untuk tanaman pangan. pres. higi ke 6: 9-14. koecher, h. and k. loetzsch. 1985. uptake, translocation and mode of action of the herbicide glufosinate-ammonium in warm climate weed species. proc. xth apwss conf.: 193-198. mangunsong, m., d. siregar and h. harahap. 1975. pemberantasan alang-alang dalam skala besar pada tanah podsolik merah kuning. symp. pencegahan dan pemuliaan tanah kritis dalam rangka pengembangan wilayah. 20 hal. morse, j. 1950. history of soybean production. in soybeans and soybean products, vol. 1, k.s. markly, ed. interscience publishers, inc. ny. 3-59. sastrosuwarno, s. 1980. padang alang-alang sebagai prioritas pembukaan daerah transmigrasi. pros. p.p. padang alang-alang, biotrop, bogor, 7 hal. sembiring, e.n. and s. supardjo. 1981. studi penyiapan lahan alang-alang secara khemis dan mekanis untuk pemukiman transmigrasi. proc. vlth indonesian weed science society conf. l-88a. shaner, d.l., p.l. anderson, m.a. stidham, m. muhitch, m.l. reider, p.a. robson and t.r. peoples. 1985. the mode of action of ac 252, 925. proc. xth apwss: 185-192. sihombing, d.a. 1985. prospek dan kendala pengembangan kedelai di indonesian, dalam kedelai, puslitbangtan, bogor. suwardjo, h. and n. sinukaban. 1986. masalah erosi dan kesuburan tanah di lahan kering podsolik merah kuning di indonesia. pros. lokakarya usaha tani konservasi. 17 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf biotropiano. 7, 1994: 12-17 bioecology of dioryctria abietella denis and schiff. a pest of conifers in the north-western himalaya t.d. verma and r.k. gaur department of entomology dr. y.s. parmar university of horticulture and.forestry nauni-solan-173 230, h.p., india abstract cones and seeds of conifers, such as pinus roxburghii, p. wallichiana, p. gerardiana, cedrus deodara, abies pindrow and picea smithiana are seriously damaged by dioryctria abietella denis and schiff. (lepidoptera : pyralidae) in the north-western himalayan region of india. bioecological studies carried out during 1991 '92 revealed that the females laid whitish, elliptical eggs singly on the depressed surface of the young cones. the average egg size was 1.00 ± 0.11 x 0.60 ±0.08 mm and this stage lasted for 3 5 days. the larval stage passed through five instars. all the instars differed in appearance, size and duration and larvae became full-fed in an average of 24.8 ± 1.9 days. the full-fed larva spun a cocoon around itself, sealed it with white papery membrane and pupated inside the cone or any other outside protected place. the prepupal and pupal periods lasted for 7-8 and 10-14 days, respectively. the pupa was dark brown, 13.8 ± 0.07 mm in length. total period from egg to adult varied from 46 to 59 days (52.7 ±4.8 days). adults were dirty brown in appearance and were 13.59±0.115 mm long with an average wing expanse of 29.0 ± 1.00 mm. they lived for 4 to 5 days. under laboratory conditions, the pest completed two generations per year and full-fed larvae of 3rd generation overwintered during september october. two larval parasites belonging to order hymenoptera and diptera, and a fungal pathogen (fusarium sp.) were found associated with this pest. the detailed biology, nature and extent of damage, along with its distribution have been discussed and methods of control suggested. key words: insect biology, insect ecology, dioryctria abietella, coniferae, north-western himalaya, forest pests introduction production of healthy seed is the backbone of afforestation programmes. unfortunately this important aspect has so far been ignored in india. a high percentage of cone and seed crop is destroyed by insects every year resulting in great losses and adverse effect on the natural regeneration. at times the insect attack is so severe that the whole seed crop is destroyed. cone and seed insects of indian conifers have not been studied in detail. some of the important insects are the cone worms, dioryctria abietella denis and shiff. (lepidoptera : pyralidae) and cateremna cedrella hampson (lepidoptera : pyralidae). beeson (1941) and browne (1968) gave brief accounts of the insects 12 bioecology of dioryctria abietella t.d. verma and r.k. gaur damaging cone and seed of conifers. recently, bhandari (1988) discussed the insect pests of cone and seeds of conifers and their control. in this paper an account of the life processes of d. abietella is given. materials and methods insect culture was raised from field collected overwintering larvae. the adults emerging from these larvae were held on 2nd year green cones in wooden cages for mating and oviposition. observations on the pattern, time and number of eggs laid and their incubation period were recorded. freshly emerged larvae were carefully transferred with camel hair brush to small plastic jars, provided with fresh green cones. to avoid excess moisture inside the jars, a circular hole was cut in the centre of the lid and a piece of muslin cloth was fixed. the morphological characters and behaviour of the larval instars and the duration of each instar was recorded. the size of head capsule was also measured with the help of calibrated ocular micrometer which was taken as a criterion for differentiation of different instars. full-fed larvae were transferred to glass jars (15 x 20 cm) containing soil and cone scales and needles, for pupation. parasites and pathogens associated with the insect were recovered from the culture in the laboratory and the percent parasitization was recorded. in addition, field surveys were also carried out at monthly intervals in coniferous forests to record the incidence, distribution and level of infestation of the pest. to determine the level of infestation fifteen cones were randomly observed from twenty trees selected at random in each locality. results and discussions d. abietella is a borer of cones and seeds of pinus roxburghii, p. wallichiana, p. gerardiana, picea smithiana, cedrus deodara and abies pindrow in himachal pradesh. the biology of this pest on p. gerardiana, p. roxburghii, p. wallichiana, p. smithiana and c. deodara studied under laboratory condition revealed that the eggs were singly glued to the surface of cones generally in the depressions on the scales. mathur et al. (1958) also reported that d. abietella eggs were whitish, elliptical and flattened, turning reddish brown after two days. the length and breadth of the eggs ranged from 0.86 1.10 mm (av. 1.00 ± 0.11 mm) and 0.54 0.73 mm (av. 0.60+0.08 mm), respectively. the egg stage lasted for 3 5 days (av. 4.4 ± 0.22 days). the larvae came out of the eggs by making a hole in the chorion in 30 40 minutes and entered the cone in about four hours. the larva is reddish brown with black head capsule. the width of the head capsule ranged from 0.31 -0.34 mm 13 biotropia no. 7, 1994 (av. 0.33 ±0.00 mm) (table 1). the first instar larva measured 1.2-1.5 mm (av. 1.34±0.11 mm) in length and the average duration was 3.6 days (table 2). the second instar larva was light brown, 3.5-4.0 mm (av. 3.72 + 0.18 mm) in length. the head capsule measured 0.51-0.56 mm (av. 0.53±0.01 mm) in width. the second instar lasted for 4-5 days (av. 4.5±0.16 days). the third and fourth instar larvae were dark brown with reddish brown head capsule. the width of head capsule of 3rd and 4th instar ranged from 0.80-0.95 mm (av. 0.87 ±0.06 mm), respectively. the body length varied from 9.5012.00 mm (av. 10.59±0.85 mm) and 14.00-16.00 mm (av. 15.30±0.67 mm) in two instars, respectively. the third instar lasted for 45 days (av. 4.70±0.15 days) and fourth instar for 5 -6 days (av. 5.40±0.16 days). the fifth instar larva was dirty brown along with dark brown head capsule. the width of head capsule ranged from 2.37-2.45 mm (av. 2.42±0.01 mm) and measured 22.00-25.00 mm (av. 23.55± 1.21 mm) in length. this stage lasted for 6-7 days (av. 6.7±0.52 days). table 1. the width of head capsule of larval instar of d. abietella (data based on 10 observations) width of head capsule (mm) ratio of head instar range mean capsule width i 0.31-0.34 0.33 ±0.00 11 0.51-0.56 0.53 ±0.01 1.60 iii 0.80-0.95 0.87 ±0,01 1.63 iv 1.39-1.47 1.45 ±0.06 1.65 v 2.37-2.45 2.42 ±0.07 1.67 table 2. duration of different developmental stages of d. abietella (mean of 10 observations) mean duration (days) stage range mean ± s.e. egg 3-5 4.4 ±0.22 larva (instar) i 3-4 3.6±0.16 ii 4-5 4.5±0.16 iii 4-5 4.7±0.15 iv 5-6 5.4±0.16 v 6-7 6.7±0.15 prepupa 7-8 7.6±0.16 pupa 10-14 12.5±0.40 adult 4-5 4.3±0.15 14 bioecology of dioryctria abietella t.d. verma and r.k. oaur the larvae became full-fed in 22 27 days (av. 24.8 + 1.93 days). mathur et al. (1958) reported that mature larvae were about 20 mm long, light reddish with blackish yellow head. the full-fed larva constructed a cocoon before entering the pre-pupal stage. the cocoon was later sealed from all sides by means of white papery membrane. pupation took place inside or on the surface of infested cones or on soil surface. the cocoon measured 16.00-20.00 mm (av. 18.80±2.20 mm) in length and 8.0010.00 mm (av. 9.00± 0.81 mm) in width. the pre-pupal period lasted for 7 8 days (av. 7.60 ± 0.16 days) and pupal period for 10-14 days (av. 12.50 ± 0.40 days). the pupa was dark brown, 13.50+14.10 mm (av. 13.80±0.07 mm) in length. however, mathur et al. (1958) reported it to be light brown and about 10 mm long. adults were dirty brown in appearance and were 13.00 14.00 mm (av. 13.59 ± 0.11 mm) long with wing expanse of 28.00-30.00 mm (av. 29.00 ± 1.00 mm). beeson (1941) reported the wing expanse of the moth as 22 34 mm. adults lived for 4 5 days (av. 4.30 ±0.15 days). the egg laying started 1 -2 days of their emergence. a female laid 30-50 eggs (av. 42.40 ± 6.78 eggs) during her life time. total period from egg to adult varied from 46-59 days (av. 52.7 ±4.80 days). the insect completed two generations annually and full-fed larvae of third generation overwintered during the last week of september. the sex ratio in the laboratory reared population was 1 male to 1.5 female. the natural enemies of the pest included two larval parasites (one each belonging to hymenoptera and diptera). out of 160 larvae of the host, 39 hymenopteran and 15 dipteran parasites were collected which accounted to 24.37 and 9.38 percent infestation. a fungal pathogen fusarium sp. was also isolated from the full-fed larvae. the pest was found to be distributed throughout the inner and outer himalayas of himachal pradesh. it infested c. deodara, p. wallichiana, p. smithiana, p. gerardiana, p. roxburghii in the region (table 3). the infestation was maximum (89.06%) in cedrus deodara cones around shimla and by 70% at chail in solan table 3. incidence of d. abietella on cone and seed of different conifers in himachal pradesh district locality/ percent infestation forest division c. deodara p. wallichiana p. smithiana p. gerardiana p. roxburghii shimla kasumpati 89.06 thanedhar 52.00 11.00 mashobra 30.00 25.00 35.00 solan chail 70.00 nauni 14.72 kinnaur sharbo 30.43 15 biotropia no. 7, 1994 district. in p. wallichiana, the maximum infestation (52%) was recorded at thanedhar. however, in p. smithiana the infestation level was only 11% in this locality. whereas, at mashobra in the shimla district the infestation was 35% in p. smithiana, 30% in c. deodara and 25% in p. wallichiana. the infestation in p. gerardiana was 30.43% in the kinnaur district of inner himalayas. the incidence of this insect was comparatively low in p. roxburghii cones throughout the outer himalayas, being maximum (14. 72%) at nauni in the solan district (fig. 1) figure 1. incidence of dioryctria abietella in coniferous forests 16 bioecology of dioryctria abietella t.d. verma and r.k. gaur it is practically difficult to control cone and seed insects in the natural forests. however, the pest population can be managed by collecting the infested cones and disposing them off together with the use of bio-control agents under the northwestern himalayan conditions. conclusion a high percentage of cone and seed crop is destroyed by insects every year resulting in great losses and adversely affecting the natural regeneration of conifers. numerous insects have been observed feeding casually on cone and seeds. but exceptionally d. abietella was found to be dominant. under laboratory conditions, the insect had a total life span of 46 59 days and completed two generations in a year. during the course of investigation the larval parasites of hymenoptera and diptera suppressed the population to a great extent. survey studies of this pest species revealed that d. abietella was widely distributed throughout the inner and outer himalayan ranges of himachal pradesh especially on cones of cedrus deodara and pinus gerardiana. continuous surveillance of this pest and its natural enemies is important in order to manage this pest. acknowledgements the authors are thankful to the indian council of forestry research and education, dehradun for funding the present studies and dr. o.p. bhalla, head, department of entomology for providing the necessary facilities. references beeson, c.f.c. 1941. ecology and control of forest insects of india and neighbouring countries. vasant press, dehradun: 1007 p. bhandari, r.s. 1988. insect pests of cone and seed of conifers and their control. tree protection: 342-347. browne, f.g. 1968. pests and diseases of forest plantation trees. clarendon press oxford: 1330 p. mathur, r.n., b. sinoh, and k. lal. 1958. insects pests of flowers, seeds and fruits of forest trees. indian for. bull. no. 223 (new series) entomology: 103 p. 17 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf biotropia no. 6, 1992/1993: 55-65 shading effects on growth and partitioning of plant biomass in paspalum conjugatum berg. i.b. ipor universiti pertanian malaysia, kampus bintulu, p.o. box 396, 97008, bintulu, sarawak, malaysia and c.e. price imperial college, silwood park, ascot, berkshire sls 7py, england abstract glasshouse studies were carried out to determine the effect of shading on the growth and partitioning of plant biomass in paspalum conjugatum berg. the invidual leaf rate expansion, final leaf are, specific leaf area, and the whole plant vegetative growth pattern, dry-matter production, leaf area as well as biomass partitioning were significantly influenced by shading. at the 75% level of shading, p. conjugatum produced the highest values of leaf weight ratio, specific leaf area and leaf area ratio. individual leaf assessment revealed that shading significantly increased the final leaf area, duration of leaf expansion and specific leaf area. introduction weeds are often considered as very competitive plants. weeds competing for light, water and nutrient have adverse effects on crop growth and yield. the availability of these resources influences the physiological, morphological and competitive responses of both weeds and crops. the effects of light on weed growth vary between species. generally, these effects can be distinguished on the basis of their physiological characteristics. among the common response of plants upon shading in general, are the alteration of leaf size (bjorkman and holmgren 1966), internode extension, number of branching (vince-pruce 1977) and stem elongation rate (morgan and smith 1981). paspalum conjugatum berg, is an important, stoloniferous creeping perennial weed in malaysia and in many regions of southeast asia. it grows well in the moist, fertile soils of agricultural fields, roadsides and wastelands. p. conjugatum grows profusely in most estates such as rubber, oil palm, cocoa and coconut (holm et al. 1977). it can tolerate, survive and grow under shade condition (beetle 1974; ward and woolhouse 1986). p. conjugatum possesses several important characteristics 55 biotropia no. 6, 1992/1993 that enable it to interfere with crop growth: rapid growth with extensive stolon formation, early and profuse tillering, heavy seed production and readily rooting at most nodes whenever in contact with the soil surface. however, little information has been reported on the morphological and physiological response of p. conjugatum when exposed to different environmental conditions. the present study was conducted to determine the effects of shade on the growth of p. conjugatum in different shade levels. materials and methods plant materials and growing conditions uniformed size seedlings of two leaf stage were transplanted into 10-cm diameter pots filled with john innes no. 1 compost mixture. watering was carried out daily and 15 ml of nutrient solution ( 7 : 7 : 7 npk foliar fertilizer) was given twice a month. the temperature of the glasshouse was maintained at 30±40°c during the day and 12±3°c at night. the plants were exposed to sunlight and supplemented with 12 hours of fluorescent white light each day. these plants were grown under three shade levels: 0% (control), 50% and 75%. single and double layers of green nettings (netlon, uk) were used to cover the cages to obtain 50% and 75% shade, respectively. the light intensities of the shading regimes were regularly checked with skye light meter (skye instrument limited, uk). harvesting and growth analysis ten plants from each light regimes were used to determine weekly increments in plant height or length, leaf number, internode length, stolon and node numbers. the other group of plants were harvested at 35 and 42 days after transplanting to determine the leaf area and total dry weight (65°c, oven-dried). in addition, dry weights of plant parts were also determined on the 42nd-day of harvest. dry-matter production and biomass allocation patterns of the three shade levels were compared using mathematical growth analysis techniques (kvet et al. 1971; patterson et al. 1979). data from 42nd-day harvest were used to calculate leaf weight ratio, shoot weight ratio, specific leaf area and leaf area ratio. calculation of dry-matter production, net assimilation rate and leaf area duration were based on 35th-day and 42nd-day harvests. all experiments were repeated twice and data were subjected to analysis of variance. duncan's multiple range or least significant difference (lsd) test was used to determine significant differences among the mean values for each treatment. 56 shading effects on growth and partitioning of plant biomass i.b. ipor & c.e. price growth of individual leaf the leaf shape was traced on a white paper and cut out with scissors. the area traced was taken as the leaf area and determined with an optomax tracer. leaf measurements were made on alternate leaves, starting from the second leaf till the sixteenth leaf. measurements were made at the time of leaf appearance, i.e. when the leaf was sufficiently distinct from the apical bud so that it could be measured without damaging the bud. the measurement was continued until the area remained constant after three successive readings. an example of the measurements on one plant is given in figure 1. the individual leaf was finally harvested and oven dried at 65°c for determination of dry weight. two sets of experiments were arranged in completely randomized blocks with ten replicates. final leaf size, duration of leaf expansion, weighed mean growth rate and specific leaf area of each individual leaf were calculated and subjected to variance analysis. means were compared using the lsd test. figure 1. example of measurement of leaf area of individual leaves of p. conjugatum. each number indicates leaf position. 57 12 16 20 24 28 32 36 40 44 days after transplanting 48 52 56 60 biotropia no. 6, 1992/1993 results and discussion the number of leaves recorded for p. conjugatum was significantly influenced by light intensity (figure 2). thus, plants at 0% shade had significantly more leaves than those provided with 50% and 75% shade. however, height of plants at 75% shade was significantly greater than those at 0% and 50% shade, 35 days after transplanting (figure 3). the shortest plant height were all recorded at 0% shade. reducing light intensity to 75% had significantly decreased the number of stolons (figure 4). plants at 0% shade produced more stolons as compared with those at 50% shade, 35 days after transplanting. bjorkman (1968) and ishimine etal. (1985) noted that reduced light levels to a certain degree would result in increased stem extension of solidago virgaurea l. and height of vaseygrass (paspalum urvillei steud.), respectively. however, further reduction of light suppressed the plant growth. this pattern of growth was also reported by soerjani (1970) and moosavi and dore (1979) in their work on imperata cylindrica. decreasing the light intensity tends to increase the total dry weight of p. conjugatum (table 1). plants grown under 0% shade produced the highest total dry weight, followed by 50% and 75% figure 2. effect of shading on leaf number of p. conjugatum. 58 shading effects on growth and partitioning of plant biomass i.b. ipor & c.e. price figure 3. effect of shading on the height of p. conjugatum. figure 4. effect of shading on stolon number of p. conjugatum. 59 biotropia no. 6, 1992/1993 table 1. effect of shading on vegetative growth, leaf area production and biomass allocation in p. conjugatum (42nd day harvest). shade levels. total dry matter of three populations of cogon grass [imperata cylindrica (l.) beauv.] decreased three fold at 56% of full sunlight and 20 fold at 11% of full sunlight (patterson 1980b). imperata cylindrica that grew with 11% of full sunlight had a leaf area ratio 2.5 times greater than those grown in full sunlight. the leaf area of the p. conjugatum increased correspondingly with increase in shade levels (table 1). the highest leaf area was 433 cm 2 from 50% shade, followed by 344 cm 2 from 75% and 274 cm 2 from 0% shade. p. conjugatum is a c4 grass native to shaded habitats or shade tolerant species (ward and woolhouse 1986) which normally produced linear thin leaves. the highest value of leaf areas of plants from 50% shade was possibly due to a sufficient number of large leaves as compared with those from 75% shade. leaves at 0% shade were reduced in size although the number exceeded from those in the other light regimes. the reduction in total leaf area with 75% shade may have resulted from a reduction in the leaves. partitioning of plant biomass into roots and leaves differed significantly among the plants under the three shade levels. at 0% and 50% shade levels, the plants partitioned more biomass into roots and stems than the other two light regimes (table 1). rwr (root weight ratio) and swr (shoot weight ratio) were highest at 0% and 50% shade. plants at 75% shade, partitioned most biomass as leaf area was also greatest in 75% shade, as indicated by their high specific leaf area and leaf area ratio values. the values differed significantly among the three shade levels. plants at 75% shade had the highest leaf area ratio followed by those at 50% shade. leaves produced under shade conditions were generally thinner than those produced in unshaded treatment (blackman and black 1959). the concomitant increases in specific leaf area and leaf weight ratio resulted in substantial increases in the leaf area ratio which means that the amount of leaf area per unit of plant 60 shading effects on growth and partitioning of plant biomass .i.e. ipor & c.e. price weight was increased by shading. morgan and smith (1978) reported that the lar increased by 38%, after 32 days in the shade treatment (15% sunlight) of cyperus rotundus grown for 30 days compared with full sunlight conditions. leaf area ratio and specific leaf area of veronica chamaedrys, v. montana and v. offlcinalis were also increased at low radiance (dale and causton 1992). increase in leaf area ratio with shading was an adaptation to low light at the whole-plant level because it reflected a greater allocation of plant biomass to photosynthetic tissue and a greater distribution of this tissue as light-intercepting structure in the form of the leaf area (patterson 1980b). analysis of the components of dry-matter production, net assimilation rate and leaf area duration at the three shade levels indicated that dry-matter production and net assimilation ratio showed the highest value at 0% and 50% shade for leaf area duration (table 2). leaf area duration significantly differed among the three shade levels. table 2. effect of shading on dry-matter production, net assimilation rates, leaf area duration of p. conjugatum during the 35th-to-42nd-day interval. shade dmp nar lad level (g) (mg.cm2.day) (cm2. day) 0% 3.74a 0.70a 5443c 50% 2.88ab 0.32b 8546a 75% 1.42b 0.21b 6701 b within each column, values sharing the same letter are not significantly different at 5% level, according to duncan's multiple range test. in general, decreasing the light intensity tends to increase the final leaf areas (figure 5). the largest size of individual leaves was always found at 75% shade. newton (1963) observed a similar trend as reduction in light significantly reduced the final areas of individual leaves of cucumber. the final areas of individual leaves in all treatments showed a characteristic variation with leaf position with an increase in size up to about leaf six. final leaf sizes of different leaves across shading treatment were significantly different except for leaf 12 and 14 under 0% and 50% shade. leaf areas were maximum at leaf 6 of plants at 75% and 50% shade while this was at least 4 of plants at 0% shade. dennet et al. (1979) observed that the final leaf area increased with leaf position (numbered from the base) up to the 8th leaf of faba beans. a similar general pattern of variation in final leaf area with leaf position had been reported for sunflower (yegappan et al. 1982), maize (thiagarajah and hunt 1982) and sugar beet (milford et al. 1985). 61 biotropia no. 6, 1992/1993 figure 5. effect of shading on final leaf area of p. conjugatum. the duration of expansion increased significantly with increasing shade and decreased with increasing leaf number (figure 6). leaves at both 50% and 75% shade took a significantly longer period to reach their maximum size. initially, the duration of expansion was increased with increasing leaf number but decreased after leaf 6 for 50% and 75% shade. the results revealed that leaves from higher shading particularly at the earlier growth stage needed longer time to reach their maturity. much of the variations in final leaf area with leaf position or shade level were associated with changes in the weighed mean growth rate rather than with changes in the duration of expansion. weighed mean growth tended to increase with decrease in light intensity (figure 7). at 75% shade, the weighed mean growth rate was significantly higher than at 0% and 50% shade. weighed mean growth rate reached a maximum of 1.3 cmvday for leaf 6 at 75% shade. the specific leaf area was significantly increased by increase in shade (figure 8). for example, specific leaf area of leaves under 75% shade differed greatly compared with other shade treatments. maximum specific leaf areas were 299, 318 and 334 for 0%, 50% and 75% shade, respectively. it was also observed that the 62 shading effects on growth and panitioning of plant biomass i.e. ipor & c.e. price figure 6. effect of shading on the duration of expansion of p. conjugatum leaves figure 7. effect of shading on weighed mean growth rate of p. conjugatum leaves 63 biotropia no. 6, 1992/1993 figure 8. effect of shading on specific leaf area p. conjugatum increase in a specific area depended on the leaf position that tended to decrease as the leaf number increased. plants grown in shade typically have lower maximum photosynthetic rates than plants grown in full light condition. patterson (1980a) showed that shaded plants normally have lower rates of dark respiration which result in conservation of the photosynthates produced. as a result of the lower respiration rates, shade-grown plants typically have a lower light compensation point, the light level at which respiratory carbon dioxide loss equals photosynthetic carbon dioxide uptake and no net carbon dioxide exchange occurs. references bjorkman, o. 1968. further studies on differentiation of photosynthetic properties in sun and shade ecotypes of solidago virgaures l. physiol. plant, 21: 84-89. bjorkman, o., and p. holmgren. 1966. adaption to light intensity in plants native to shaded and exposed habitats. physiol. plant, 19: 854-914. 64 shading effects on growth and partitioning of plant biomass i.b. ipor & c.e. price blackman, g.e. and j.n. black. 1959. physiological and ecological studies in the analysis of plant environment. xi. a further assessment of the influence of shading on the growth of different species in the vegetative phase. ann. bot. 23: 51-63. dale, m.p., and d.r. causton, 1992. the ecophysiology of veronica chamaedrys, v. montana and v. officinalis. ii. the interaction of irridiance and water regime. journal of ecology 80(3): 493 504. dennet, m.d., j., elston and j.r. milford. 1979. the effects of temperature on the growth of individual leaves of vicia faba l. in the field. annals of botany, 43: 197-208. holm, l.g., d.l. plucknett, j.v. pancho, and j.p. herberoer, 1977. the world's worst weed. distribution and biology., university press of hawaii, p. 609. ishimine, y.k., k. miyazato, and s. matsumoto. 1985. physiological and ecological characteristics of weeds of sugarcane fields in the ryukus islands. 3. effects of shading on growth and seed production of paspalum urvellei steud. weed res. (japan) 30: 148-150. kvet, j., j.p. ondok, j. necas and p.o. jarvis. 1971. method of growth analysis in plant photo-synthetic production : manual of methods, eds. z. sestak, j. catsky and p.o. jarvis, w. junk. n/v publ. the hague: 343-391. milford, g.f.j., t.o. pocock, j. riley and a.b. messem, 1985. an analysis of leaf growth in sugarbeet. leaf expansion in field crops. annals of applied biology 106: 187-203. moosavi-nia, h. and j. dore. 1979. factors affecting glyphosate activity in imperata cylindrica l. beauv. and cyperus rotundus l. ii. effect of shade. weed res. 19: 321-327. morgan, d.c. and h. smith. 1978. linear relationship between phytochrome photoequilibrium and development in light grown chenopodium album l. planta 142: 187-193. morgan, d.c. and h. smith. 1981. control of light. the effect of light quantity (total influence rate) and light quality (red : far-red ratio) new phytologist 88: 239-248. newton, p. 1963. studies on the expansion of the leaf surface. ii. the influence of light intensity and photoperiod. j. exp. bot. 14: 458-482. patterson, d.t. 1980a. light and temperature adaption p. 205-235. in predicting photosynthesis for ecosystem model eds. j.d. hesketh and j.w. jones, vol. 1 crc press, inc. boca raton, f.l. patterson, d.t. 1980b. shading effects on growth and partitioning of plant biomass on cogon grass (imperata cylindrica) from shaded and exposed habitat. weed sci. 28: 735-740. patterson, d.t., c.r. meyer, e.p. flint and p.c. quimby, 1979. temperature response and potential distribution of itchgrass (rottboellia exaltata) in the united states. weed sci. 27: 77 82. soerjani, m. 1970. alang-alang, imperata cylindrica (l.) beauv. (1812). patterns of growth as related to its problem of control. biotrop bull. no. 1. thiagarajah, m.r. and l.a. hunt, 1982. effect of temperature on leaf growth in corn (zea mays). canadian j. of botany 60: 1647-1652. vince-pruce, d. 1977. photo control of stem elongation in light growth plants of fuchsia hybrids. planta 133: 149-56. ward, d.a. and h.w. woolhouse, 1986. comparative effects of light during growth on the photosyntetic properties of nadp-me type c4 grasses from open and shaded habitats. gas exchange, leaf, anatomy and ultra structure. plant, cell and environment 9: 261 -270. yegappan, t.m., d.m., patton, c.t. gates and w.j. muller, 1982. water stress in sunflower (helianthus annuus l.) ii. effect of leaf cells and leaf area. annals of botany 49: 63-68. 65 55.pdf 56.pdf 57.pdf 58.pdf 59.pdf 60.pdf 61.pdf 62.pdf 63.pdf 64.pdf 65.pdf 1. rudolf v kuhn.cdr biotropia vol. 20 no. 1, 2013: 1 9 diversity of plasmodial myxomycetes from anda island, pangasinan, philippines rudolf v. kuhn , anton oliver m. javier , coleen p. rodillas , christian m. parra , liwayway hiyas m. corpuz , anthony t. buaya , and thomas edison e. dela cruz * received 20 december 2011/accepted 10 january 2013 the unique life cycle and fascinating fruiting bodies of myxomycetes make them ideal model organisms for the study of cellular differentiation and ecological patterns. our research study then focuses on the diversity and abundance of myxomycetes found in anda island, pangasinan in northern philippines. a total of 180 moist chambers were prepared from ground leaf litter and twigs collected from a 15 m quadrat within the study site. twenty four species of myxomycetes belonging to 11 genera were collected and identified from the moist chambers: (2), (1) (1) (2), (1), (2) (1) (1), (1) (11), and (1) of all moist chambers, 55% yielded myxomycetes. ground leaf litter (29%) yielded more myxomycetes than twigs (26%). assessment of species diversity (h =1.15), richness (h =5.33) and evenness (e=0.56) showed rich assemblage of myxomycetes. among the collected species, one for each of the genera , , and were recorded to be abundant. interestingly, three species of myxomycetes are new records for the philippines: and . this is the first report of myxomycetes in anda island, pangasinan, philippines. myxomycetes, species abundance, diversity, biogeography, philippines 1 1 1 1 1 2 1,2 2 department of biological sciences, college of science and fungal biodiversity and systematics group, research center for the natural and applied sciences university of santo tomas españa 1015 manila, philippines arcyria collaria , comatricha , craterium diachea diderma , didymium , elaeomyxa perichaena , physarum stemonitis . arcyria craterium diderma, physarum, craterium microcarpum, physarum decipiens elaeomyxa miyazakiensis 1 2 abstract introduction s g key words: the philippines is a tropical archipelago of 7107 islands. the geographic isolation of many of these islands resulted in various unique flora and fauna. it has been estimated that more than 6000 species of animals and plants are endemic to the country (myers . 2000). therefore, it is not surprising that new species of plants and animals are still being discovered recently in the country, e.g. forest mice ( et al apomys * corresponding author : tedelacruz@mnl.ust.edu.ph 1 aurorae a. banahao a. brownorum a. magnus a minganensis a. sierrae a. zambalensis) et al. (varanus bitatawa) et al. (nepenthes attenboroughii) et al. (drosera ultramafica) et al. craterium retisporum et al. et al et al. hemitrichia serpula, perichaena depressa, physarella oblonga, stemonitis fusca arcyria cribraria, lycogala stemonitis et al. et al. arcyria globosa comatricha robusta, craterium atrolucens, lamproderma cacographicum, oligonema schweinitzii perichaena microspora , , , , , , and (heaney 2011), monitor lizard (welton 2010), giant pitcher plant (robinson 2009), sundew plant (fleischmann 2011) including those belonging to the myxomycetes, e.g. (moreno 2009). however, studies on island myxomycetes are relatively few in spite of their role in wood decay and nutrient cycling in temperate forests and in bio-accumulation of heavy metals (zhulidov . 2002; takahashi 2004). rojas and stephenson (2008) surveyed different habitat elevations and substrates types in cocos island, costa rica and reported 41 species belonging to 19 genera. beltran (2010) also evaluated the species diversity of myxomycetes between arid and semi-arid zones in the canary islands in spain and reported 63 species belonging to 21 genera. the study also highlighted the similarities between slime molds collected from the arid and semi-arid zones in the canary islands to the desserts of central america. in the philippines, and species of , and were reported from the islands of palawan and palaui, cagayan (reynolds 1981; quimio 2002). dela cruz (2011) also reported 30 species of myxomycetes from hundred islands national park, pangasinan. macabago (2012) identified 45 species and 13 genera from lubang island, occidental mindoro. interestingly, this geographically isolated island yielded six new records for the philippines: , and . in this research study, we reported 24 species including three new records of myxomycetes from specimens collected in anda island, pangasinan, philippines. this is the first report of myxomycetes in anda island, pangasinan in northern philippines. anda island (16°17'00.16”n, 119°58'00.08”e; land area= 83.80 sq.km) is located near the town of bolinao in the province of pangasinan, northern philippines. climate data indicate an average monthly rainfall range of 6.1 mm in february to 608.6 mm in august. the island has an average minimum and maximum temperature of 23.3 °c and 32.4 °c, respectively, and average monthly humidity between 71 85%. it has a type i climate season, i.e. wet from june to november and dry from december to may. the study site is characterized by a tropical secondary forest, with patches of rice fields and established residential houses. a small patch of this secondary forest along the local highway and near a rice field was used as the collection site. the island is also inhabited by man and may be useful in looking at the effects of human civilization on myxomycete assemblages. materials and methods study site 2 biotropia vol. 20 no. 1, 2013 collection of substrates and preparation of moist chamber set-ups and herbaria characterization and identification of plasmodial myxomycetes moist chamber productivity and ecological analysis ground leaf litter (30) and twigs (30) were collected in brown paper bags within a 15 m quadrat. prior to the preparation of moist chambers, the collected substrates were allowed to air-dry for few days. a total of 180 moist chambers were then set-up following the protocol of stephenson and stempen (1994). the moist chambers were placed inside wooden cabinets, not directly exposed to sunlight, and incubated at room temperature for up to 8 weeks. all moist chambers were observed regularly (at least twice a week) for the presence of plasmodia and/or fruiting bodies. the number of moist chambers with plasmodia and/or fruiting bodies was recorded and used in ecological analysis. fruiting bodies with their respective substrate were removed from the moist chamber set-up, allowed to air dry and glued on previously prepared herbarium boxes. herbarium boxes were then labeled with the collection number, substrate, collection sites and dates, the species name and the names of the collector. all herbaria were deposited at the pure and applied microbiology laboratory, research center for the natural and applied sciences, university of santo tomas. to identify the collected myxomycetes, the specimens for each species were described based on their fruiting body descriptions and spore morphology under a dissecting and a compound light microscope. identification was done following comparison of these morphological characters with published literature (stephenson & stempen 1994; keller & braun 1999), web-based identification keys (http://slimemold.uark.edu/) and an electronic, computer-based identification key, (mitchell 2008). to check for the current valid names of the identified myxomycetes, an online nomenclatural information system (http://nomen. eumycetozoa.com) was used. the productivity of the moist chambers (mc) was computed by determining the percent yield for the collection site and each of the substrate types. plasmodial/or fruiting body growth on a moist chamber was counted here as positive for myxomycetes and was recorded as one positive collection. then, the total positive collection was divided by the total number of moist chamber prepared. thus, the percentage yield (py) was computed as follows: percent yield (py) = (1) total number of mc prepared then, the relative abundance (ra) was also determined for each of the collected species of myxomycetes. initially, the number of moist chambers positive for a species of myxomycetes was counted and divided by the total number of collections. 2 synkey number of mc positive for myxomycetes 3 diversity of plasmodial myxomycetes from anda island rudolf v. kuhn– et al. the relative abundance was computed as follows: relative abundance (ra) = (2) total number of myxomycete collection abundance indices (ai) were then assigned to all of the species represented among the collections. initially, the percentage of a particular species among the total number of collections, i.e. the relative abundance, was determined. then, a “breaking point” was assigned based on these percentage values (s. l. stephenson, pers. comm., 04 january 2009). each species was then categorized as: (1) abundant (a) if their relative abundance (ra) is ≥10% of the total collections, (2) common (c) if ra is ≥5% but <10% of the total collections, (3) occasionally occurring (o) if ra is ≥3 but <5 of the total collections, and (4) rare (r) if the myxomycetes had an ra of <3% of the total collections. finally, the myxomycete diversity of the study site was also calculated using different diversity indices as described by dagamac . (2012). these indices were computed based on ra and as follows: shannon index diversity (3) where p = is the proportional abundance of the th species gleason index of species richness (4) where n = the total number of species n = the total number of individuals in th species pielou's index of species evenness / (5) where h = shannon index of diversity h = the maximum value of hs of 180 moist chambers set-up from ground leaf litter and decaying woody twigs collected from anda island, 55% yielded myxomycetes (fig. 1a). fruiting bodies were recorded more in moist chambers than plasmodia. between the two collected substrates, ground leaf litter (29%) yielded more myxomycetes than twigs (26%) (fig. 1b). schnittler (2002) also found a higher occurrence of myxomycetes on the ground leaf litter, though macabago (2010) recorded a higher yield in aerial than ground leaf litter. it was previously reported that myxomycetes associated with woody twigs are not necessarily same as those associated with ground litter in terms of their occurrence and abundance (stephenson 2008). stephenson (1989) implicated that the myxomycetes assemblage in ground leaf litter was mediated by factors that is favorable for plasmodial or amoeba development. these factors included acidic ph of the decaying leaf litter, soil nutrients, moist and the available food microorganisms, bacteria, yeast and algae. as shown in this study, the high myxomycetes yield recorded in ground leaf litter as compared to twigs indicated that ground leaf litter is a favorable substrate for myxomycetes. number of collections for a particular species of myxomycetes et al et al. et al. et al. e.g. (h ) = -∑ (p ln p ) (h ) = n -1/ln n e = h h percent yield s i i i g p i s max i p i s max i i results and discussion 4 biotropia vol. 20 no. 1, 2013 5 b a figure 1. percent yield of myxomycetes in moist chambers (a) and between the two collected substrates (b) d b ca e f figure 2. representative species of myxomycetes recorded in anda island, pangasinan: (a) (b) , (c) and (f) craterium retisporum, diachea leucopodia perichaena pedata, (d) elaeomyxa miyazakiensis, (e) perichaena depressa physarum bivalve diversity of plasmodial myxomycetes from anda island rudolf v. kuhn– et al. 6 species occurrence, abundance and diversity arcyria collaria , comatricha , craterium diachea diderma , didymium , elaeomyxa perichaena , physarum stemonitis . physarum phy. bivalve, phy. bogoriense, phy. cinereum, phy. compressum, phy. decipiens phy. echinosporum arcyria cinerea, collaria arcyrionema diachea leucopodia, diderma hemisphaericum, didymium nigripes, elaeomyxa miyazakiensis, per. depressa, comatricha stemonitis fusca craterium cra. retisporum cra. microcarpum, craterium microcarpum, physarum decipiens elaeomyxa miyazakiensis a. cinerea, cr. microcarpum, diderma hemisphaericum, phy. echinosporum arcyria physarum stemonitis et al. et al et al et al. collaria arcyrionema diachea leucopodia, physarum decipiens stemonitis fusca comatricha craterium retisporum, diderma ., didymium nigripes, elaeomyxa miyazakiensis, physarum bivalve, phy. bogoriense, phy. cinereum, phys. compressum, physarum physarum physarum physarum diderma comatricha et al. arcyria, physarum, diderma stemonitis et al. craterium craterium leucocephalum craterium et al , a total of twenty four species of myxomycetes were collected from anda island, pangasinan (table 1). the identified species belong to the following genera: (2), (1) (1) (2), (1), (2) (1) (1), (1) (11), and (1) of the collected species, six were identified as and . , sp. and were also reported in anda island. two species of , i.e. and were also noted among collected substrates. interestingly, three species of myxomycetes are new records for the philippines: and . these species have been previously reported in other countries but not in the philippines until now. of all collected myxomycetes, and were the most abundant (table 1). species of , and were also reported to be widely abundant in temperate and tropical forests, and in island habitats occurring in ground leaf litter or parts of living trees such as twigs and old bark (schnittler 2002; stephenson . 2004; everhart & keller 2008; stephenson . 2008; tran 2008; adamonyte & kastanje 2011). , and were recorded as common in this study, while most of the species were recorded as rarely occurring (table 1). sp.1, sp sp.1, sp.2, sp.8, sp.9, and two unidentified myxomycetes were all recorded as rare. similarly, species of and has been found to be abundant in temperate than in tropical forests (stephenson 1989; novozhilov 2000). the genera and are also abundantly diverse and distributed in island habitats (beltran 2004; rojas & stephenson 2008). rojas and stephenson in 2008 described the genus as rare in cocos island, costa rica; however, adamonyte and kastanje in 2011 abundantly recorded in the island of saaremaa, estonia. differences in climatic conditions in these two islands play a crucial part in the abundance and distribution of species. this may also be a factor for the differences in myxomycete distribution in anda island. though anda island is inhabited by man, the influence of anthropogenic activities to myxomycete distribution was not correlated, and therefore, is recommended for future studies. in this study, the computed diversity indices of myxomycetes in anda island, pangasinan showed high value for species diversity (h =1.15), species richness (h =5.33) and species evenness (e=0.56). similarly, high species diversity was also recorded in lubang island, occidental mindoro (macabago . 2012). however it was observed that geographic isolation does not greatly influence species composition, and endemism seems not to occur in for myxomycetes since species recorded in an island habitat also occur in mainland (eliasson 1991; rojas & s g biotropia vol. 20 no. 1, 2013 7 stephenson 2008). but the wide range of diverse habitats in island ecosystems provides diverse microhabitats for myxomycetes (rojas & stephenson 2008). thus, the diverse assemblage of myxomycetes recorded in this study further supports other studies conducted in island ecosystems. a total of 24 species of myxomycetes belonging to 11 genera were collected and identified in anda island, pangasinan philippines: ( var. ) , , and conclusions arcyria cinerea a. cinerea digitata , collaria arcyrionema, craterium microcarpum, cra. retisporum diachea leucopodia, diderma hemisphaericum didymium nigripes, elaeomyxa miyazakiensis, perichaena depressa, physarum bivalve, phy. bogoriense, phy. cinereum, phy. compressum, phy. decipiens, phy. echinosporum, table 1. species abundance of myxomycetes collected from anda island, pangasinan taxa % abundance arcyria cinerea (a. cinerea var. digitata) a arcyria sp. 1 o collaria arcyrionema c comatricha sp. 1 r craterium microcarpum a craterium retisporum r diachea leucopodia c diderma hemisphaericum a diderma sp. r didymium nigripes r elaeomyxa miyazakiensis r perichaena depressa o physarum bivalve r physarum bogoriense r physarum cinereum r physarum compressum r physarum decipiens c physarum echinosporum a physarum sp. 1 r physarum sp. 2 r physarum sp. 3 o physarum sp. 8 r physarum sp. 9 r stemonitis fusca c unidentified myxo 6 r unidentified myxo 7 r note : abundant indices, where abundant (a) = ≥10% of the total collections, common (c) = ≥5% but <10% of the total collections, occasional (o) = ≥3 but <5 of the total collections, and rare (r) = <3% of the total collections a diversity of plasmodial myxomycetes from anda island rudolf v. kuhn– et al. 8 biotropia vol. 20 no. 1, 2013 stemonitis fusca. arcyria comatricha diderma physarum craterium microcarpum, physarum decipiens, elaeomyxa miyazakiensis, physarum arcyria craterium diderma one species each of , , and five species of were identified up to the genus level only. three species, i.e. and are new records for the philippines. higher productivity was also recorded in the study as well as in ground leaf litter than twigs. eleven species of and one species for each genus , , and were recorded abundantly. the authors would like to thank the research center for the natural and applied sciences, university of santo tomas for the research grant, prof. dr. steven l. stephenson, university of arkansas, usa for his assistance in the identification of our species, and nikki heherson a. dagamac and edward e. dela cruz for their assistance in collecting our substrates. acknowledgments references adamonyte g, kastanje v. 2011. myxomycetes of the island of saaremaa, estonia. folia cryptog estonica fasc 48:1-4. beltran e, lado c, barrera j, gonzález e. 2004. myxomycetes diversity in the laurel forest of garajonay national park (canary islands, spain). syst geogr pl 74: 159-73. beltran e, mosquera j, lado c. 2010. myxomycete diversity from arid and semiarid zones of the canary islands (spain). mycotaxon 113:439-42. dela cruztee, kuhnrv, javieraom, rodillascp, parracm, corpuzlhm, mchugh rd. 2011. occurrence and distribution of plasmodial myxomycetes in hundred islands national park, pangasinan, philippines. acta manilana 59:65-74. eliasson uh. 1991. the myxomycete biota of the hawaiian islands. mycol res 95: 257-67. everhart se, keller hw. 2008. life history strategies of corticolous myxomycetes: the life cycle, plasmodial types, fruiting bodies, and taxonomic orders. fungal div 29: 1-16. fleischmann a, robinson as, mcpherson s, heinrich v, gironella e, madulid da. 2011. 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(nepenthaceae) pitcher plant from central palawan, philippines. bot j linn soc 159: 195-202. rojas c, stephenson sl. 2008. myxomycete ecology along an elevation gradient on cocos island, costa rica. fungal div 29: 117-27. schnittler m, lado c, stephenson sl. 2002. rapid biodiversity assessment of a tropical myxomycete assemblage maquipucuna cloud forest reserve, ecuador. fungal div 9: 135-67. stephenson sl. 1989. distribution of myxomycetes in temperate forests ii. patterns of occurrence of bark surface of living tress, leaf litter, and dung. 81: 608-21. stephenson sl, schnittler m, lado c. 2004. ecological characterization of a tropical myxomycete assemblage maquipucuna cloud forest reserve, ecuador. 96: 488-97. stephenson sl, stempen h. 1994. myxomycetes: a handbook of slime molds. usa: timber press inc. stephenson sl, urban la, rojas c, mcdonald ms. 2008. myxomycetes associated with woody twigs. sociedad mexicana de micología 27: 21-28. takahashi k. 2004. distribution of myxomycetes on different decay states of deciduous broadleaf and coniferous wood in a natural temperate forest in the southwest of japan. syst geogr pl 74:133-42. tran htm, stephenson sl, hyde kd, mongkolporn o. 2008. distribution and occurrence of myxomycetes on agricultural ground litter and forest floor litter in thailand. mycologia 100:181-90. welton lj, siler cd, bennett d, diesmos a, duya rm, dugay r, rico elb, van weerd m, brown rm. 2010. a spectacular new philippine monitor lizard reveals a hidden biogeographic boundary and a novel flagship species for conservation. biology letter 6: 654-8. zhulidov da, robarts rd, zhulidov av, zhulidova ov, markelov da, rusanov va, headley jv. 2002. zinc accumulation by the slime mold (l.) wiggers in the former soviet union and north korea. j environ q 31:1038-42. craterium asean regional center for biodiversity conservation, laguna nepenthes mvcologia mycologia fuligo septica myers n, mittermeier ra, mittermeier cg, da fonseca gab, kent j. 2000. biodiversity hotspots for conservation priorities. nature 403: 853-858. diversity of plasmodial myxomycetes from anda island rudolf v. kuhn– et al. biotropia vol. 1 no. 1, july-december 1987 multidisciplinary research on shorea javanica foreword it was april 1985, under the leadership of dr. ir. z. goto, then tropical forest biology program manager, that it was decided to launch in biotrop a multidisciplinary research on shorea javanica. this followed the publication in 1984 by e.f. torquebiau of a paper describing the traditional planting of this tree for resin production by farmers near the small town of krui, in lampung province, southern sumatra (man-made dipterocarp forest in sumatra. agroforestry systems, 2: 103-127). a proposal was subsequently made to develop in biotrop different research topics around this species in order to promote it for plantation forestry. the choice of this species was justified by the important knowledge from its traditional uses and planting, while in the long term, it is hoped that the development of plantations of this species will promote the use of other dipterocarps and native trees for plantation forestry. 41 biotropia 1 (1) 1987: 42-45 i. introduction e.f. torquebiau* tropical forest biology program, biotrop, bogor, indonesia the plantations of shorea javanica k.&v. (dipterocarpaceae) in the district of krui (lampung province, sumatra; see fig. 1 for situation map and main climatic data) are remarkable examples of successful land development after deforestation and shifting cultivation which was mentioned in the indonesian forestry literature as far back as 1937 (rappard 1937). this tree is a white meranti which is locally found in the natural forest and tapped for its beautiful, crystalline resin, or "damar". the local name of the tree is "damar mata kucing", which means "cat's eye resin". one of the traditional cultivation systems in the area is shifting cultivation ("ladang"): rain-fed rice is grown during one or two years and then coffee, other crops, and damar trees are planted to convert the ladang into a permanent agricultural field. the damar trees close their canopies above the other crops after some years and can be tapped for resin after about 15 years and during a rotation of approximately 50 years. they constitute dense stands of 40—50 m high trees called "kebun damar" (damar gardens) which look like a natural rain forest. seeds for planting stock were formerly obtained from the surrounding natural forest but nowadays they come from the pre-existing plantations which cover an area of approximately 1000 ha (scholz 1983). fruiting seasons are occasional and irregular, often several years spaces, so that the farmers manage large nurseries of seedlings which can be maintained for several years and transplanted to the plantations when needed. transplantation of bare-rooted seedlings is easy. other useful trees (e.g. clove trees), are simultaneously planted in the ladang at the time of planting the damar trees, so that, although the latter largely dominate, the resulting stand is a multi-layered, mixed one, comprising of different useful plants (fruits, vegetables, medicinal plants, etc.). the whole cultivation system, from the shifting cultivation stage to the establishment of a permanent tree plantation, constitutes an efficient agroforestry system which is extensively described by michon 1984, 1985; michon et al 1984; and torquebiau 1984. the resin of shorea javanica is traditionally used for torches, caulking boats, batik coloring, etc., and is now exported to industrial countries where there is market for uses such as food additives, cosmetics, paints, varnishes and * present address: icraf, p.o. box 30677, nairobi, kenya. 42 i. introduction e.f. torquebiau figure 1. situation map and main climatic data (torquebiau 1984). 43 43 biotropia vol. 1 no. 1, july-december 1987 medications. however, the resin is exported as raw material and there is no industrial unit yet for resin processing in indonesia. although the only significantly large area of damar mata kucing plantation in sumatra is in the surroundings of krui, some other localities were found in southern sumatra, indicating that at one time, the damar business was probably a very attractive one. these localities are: — bakauheni, at the very southern tip of sumatra, 10 km from bakauheni, to the north of the trans-sumatra road, is a small stand of fairly old, disused damar trees. other damar trees can be recognized here and there in the landscape, and there are probably other plantations in this area. — sukadana, near way kambas nature reserve. this locality was reported by meijer (1975), but only one [huge!] tree is now left in the village and is disused. people said the other damar trees have been felled for timber! — wana (sribowhono), some 25 km east from the above. approximately 1 ha stand of fairly old damar trees is present. other stands are said to exist in the surroundings. — hujan mas, near muaraenim. some damar trees in home gardens not far from the trans-sumatra road were noted. — batu raja, some km to the north of the town. damar trees in home gardens can be seen from the trans-sumatra road. — localities near semangka bay and kota agung, mentioned in meijer (1975), could not be visited. some localities are probably also to be found in southern bengkulu province (rappard 1937). it is important to collect seeds or seedlings when a survey is made of these different localities, so that the gene pool of damar trees can be diversified. resin quality may differ between localities (and between trees!). tapping techniques are also different according to the zone. the best tool to scrape the resin from the trees as said by rappard (1937) is the "damar axe" of krui, while further north in lais, a different tool causing more damage to the trees is used. the aim of biotrop's multidiciplinary research project on shurea javanica is to gather sufficient information on the silviculture of this tree to serve as basis for proposing it as a species for plantation forestry and agroforestry. up to now, only some of these topics could be dealt with and the first results of the studies are presented in the following chapters. a sub-project on tissue culture and water culture is also going on, but has not produced any significant result yet. aseptic germination of embryo has been successfully achieved and will be used as basis for new vegetative propagation experiments. data and material gathering in the field were conducted in the surroundings of krui during field trips attended by different staff members from 44 i. introduction — e.f. torquebiau biotrop in june, september and october 1985 (ripe seeds could be collected then!), and february 1986. although much research work is still to be done, it is thought that there is sufficient knowledge on the silvicultural management of this species to warrant the establishment of experimental plantations now. executive proposals have been submitted to the directorate of reforestation and land rehabilitation (rrl) and to p.t. kutai timber indonesia with the hope that such trial plantations could be started as soon as possible, with the scientific collaboration of biotrop. however, these proposals have not received any answer so far. references meuer, w. 1975. indonesian forests and land-use planning. thomas hunt morgan school of biological sciences, univ. of kentucky, lexington, kentucky 40506/missouri botanical garden. mimeogr. 113 pp. michon, g. 1984. prospects for the use of agroforestry systems in regional forest management: examples from indonesia. in: biotrop symposium on forest regeneration in south east asia. may 1984, biotrop, bogor, indonesia. biotrop special publication no. 25. michon, g. 1985. de phomme de la foret au paysan de i'arbre: agroforesteries indonesiennes. thesis, university of montpellier, france, 273 pp + figs. michon, g., f. mary, j.m. bompard & p. lombion, 1984. traditional agroforestry in indonesia. lipi, jakarta, mimeogr. rappard, f.w. 1937. de damar van bengkoelen. tectona, dl. xxx, pp. 697-915. scholz, u. 1983. the natural regions of sumatra and their agricultural production pattern. vol. 1, text: crifc, bogor, indonesia, vol. 2, maps: sarif, padang, indonesia. torquebiau, e.f. 1984. man-made dipterocarp forest in sumatra. agroforestry systems, 2: 103-127. 45 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf biotropia book juni revisi 14 juli 09.indd 21 viability and plasma membrane integrity of the spotted buffalo epididymal spermatozoa after thawing with the addition of dextrose into the extender yulnawati 1, h. maheshwari 2, herdis 3, m. rizal4 1* rc. biotechnology, lipi, jl. raya bogor km. 46, cibinong, 16911, 2 dept of anatomy, physiology and pharmacology, faculty of veterinary medicine ipb, jl. agatis ipb campus, darmaga, bogor, 16680 3 bppt , jl. mh. th amrin kav. 8, jakarta 4dept. of animal husbandary, faculty of agriculture, pattimura university, jl. ir. m. putuhena, kampus pokka, ambon abstract th e objective of this study was to obtain the viability and plasma membrane integrity of the spotted buff alo epididymal sperm after addition of dextrose into andromed® extender. spermatozoa that have been collected from cauda epididymis were diluted with andromed® extender as control (k) and andromed® + 0.2% dextrose (p1) and andromed® + 0.4% dextrose (p2) as treatments. th e results showed that the quality of epididymal spermatozoa decreased during cryopreservation process. th e percentage of motility after thawing in p1 (46%) and p2 (46.67%) were signifi cantly higher (p<0.05) compared to k (41%) as well as the percentage of live sperm in p1 (58.8%) and p2 (60%) compared to k (52.2%). th e percentage of membrane integrity in p1, p2 and k were 67.4; 66.8 and 68 %, respectively. in conclusion, the addition of 0.2 and 0.4% of dextrose into andromed® acted as an extra cellular cryoprotectant and could maintain the viability and membrane integrity of the spotted buff alo epididymal spermatozoa after thawing. key words: epididymal sperm, cryopreservation, dextrose, spotted buffalo introduction indonesia is worldwide known to have high biodiversity that need to be considered for the conservation of the flora and fauna specific to this country. spotted buffalo is one of the indonesian biodiversity that need a particular attention as its population tends to decrease every year. this animal is thought to bring fortune and is usually used for the veneration in traditional ceremony in tana toraja, south sulawesi. according to the local people believe, spotted buffalo can only survive in its natural habitat, which is in tana toraja. the farmer put a special treatment for the spotted buffalo; particularly the male is maintained separated from the female biotropia vol. 16 no. 1, 2009: 21 27 corresponding author : yulnawati@yahoo.com 22 to prevent it from reproduction activities. this condition is, therefore, thought to make the conservation effort of this animal become more difficult. one of the alternative attempts to prevent this animal from extinction is to utilize the gonad, which are the testis and the epididymis of the male that is sacrificed in the traditional ceremony. the epididymis can be used as a source of potential spermatozoa that can conserve the genetic material. the fertility of the spermatozoa taken from the cauda epididymis is more or less the same as the spermatozoa from the ejaculate (hafez and hafez 2000). collection of the epididymal spermatozoa has been done in various species such as primate (feradis et al. 2001), sheep (rizal 2006), cattle (graham 1994), cat (tsutsui et al. 2003, yulnawati and setiadi 2005), pig (kikuchi et al. 1998), rhinocerous (lubbe 1999), equine (squires et al. 2000; papa et. al. 2008), deer (soler et al. 2003), dog (setiadi et al. 2007, hori et al. 2004) and african buffalo (herold et al. 2004, herold et al. 2006). spermatozoa collected from cauda epididymis is then stored in the form of liquid and/or frozen sperm. furthermore, epididymal sperm could be used for assisted reproductive technology such as artificial insemination (ai), in vitro embryo production (ivep) or intra cytoplasmic sperm injection (icsi). in order to maintain the quality of the spermatozoa during storage and after thawing for further process, the extender with a certain composition is needed. during the freezing process, the important material in the extender is the cryoprotectant that can protect sperm from coldshock. cryoprotectant can directly prevent the formation of ice crystal in the cell, as well as working extracellularly as a membrane protector. both the intracellular and extracellular cryoprotectants can be added at the same time in a certain ratio into the extender. sugar is used in the research as one of the extracellular cryoprotectant that should be added to the glycerol-as intracellular cryoprotectant-contained andromed® extender. it is hoped that the addition of dextrose sugar can maintain the viability dan integrity of plasma membrane of the spotted buffalo’s epididymal spermatozoa after thawing. materials and methods cauda epididymis of the spotted buffalo was collected during the rambu solo’ funeral ceremony in the pangli village, sesean, north toraja. the cauda epididymis was flushed and stored in 0.9% of nacl before the collection of spermatozoa. epididymal spermatozoa was collected using combination technique, slicing/ flushing and pressing all parts of the cauda’s tissues (rizal et al. 2004) using andromed® solution as an extender. evaluation of the quality of fresh spermatozoa observed was percentage of progresive motility, percentage of live spermatozoa, concentration, percentage of abnormality and percentage of plasma membrane integrity (mpu). collected spermatozoa was centrifuged at 500 g for 20 min. at room temperature. the supernatant was discharged and the pellet that contained spermatozoa was re-diluted with andromed® (minitub, germany) extender. the volume of the extender used is based on the concentration previously calculated. basic extender used in this research was andromed®. andromed® is a commercial extender that contains glycerol as an intracellular cryoprotectant. andromed® without biotropia vol. 16 no. 1, 2009 23 any addition of substance was grouped as control, whereas the addition of 0.2 and 0.4% w/v of dextrose (merck, germany) into andromed® were grouped as treatments. the diluted epididymal spermatozoa were placed in 0.25 ml of mini straw with 60 million of motile spermatozoa per straw and equilibrated in the refrigerator at 5°c for 3 hours. freezing of the epididymal spermatozoa was started by arranging the straw 10 cm above the surface of the liquid nitrogen with the temperature of -130°c for 15 min. afterwards, the straw was plugged into the liquid nitrogen with the temperature of -196°c and stored in the container. later, each frozen epididymal spermatozoa sample was thawed for quality evaluation. thawing process was carried out by placing the straw into the water bath at 37oc for 30 seconds. the percentage of sperm motility, live sperm, and plasma membrane integrity of post diluted, equilibrated and thawed epididymal spermatozoa were recorded. the percentage of progresive motility (move forward) of the sperm was counted subjectively at eight different visual fields, using light microscope with the magnitude of 400x. the percentage of live sperm was counted using eosin staining. live spermatozoa were characterized by white colour of the head, whereas the dead sperm had red head (toelihere 1993). plasma membrane integrity (mpu) was characterized by circular tail or inflated, whereas the broken one was characterized by by straight tail when the sperm was exposed to the hypoosmotic solution and incubated at 37oc for 60 min (revell and mrode 1994). for each parameter observed, a sum of minimum 200 sperma was evaluated using light microscope with the magnitude of 400x. data obtained were analyzed using random analysis of variance (anova) with three treatments and five replications. difference between treatments was tested using duncan multiple range test (steel and torrie 1993). results and discussions the quality of fresh epididymal spermatozoa of the spotted buffalo obtained from this research is shown in table 1. the concentration of spermatozoa collected from the cauda epididymis using flushing technique and pressure, was 10445x106 spermatozoa/ml. theoretically, this was reliable because cauda epididymis functions as a spermatozoa storage before ejaculation and in the cauda and the spermatozoa has not have seminal plasma addition (garner and hafez 2000). the sperm concentration in bull cauda epididymal was 3593 4406,7 x 106 sperm/ml (suhendra 2002) and 6,26 x 109 sperm/ml in buffalo cauda epididymal tissue (toelihere 1993). table 1. the avarage quality of fresh spotted buffalo epididymal spermatozoa parameters mean ± st.dev concentration (x 106 sperma/ml) 10445 ± 43.62 volume (ml) 0.45 progressive motility (%) 65.0 ± 0.00 live (%) 79.3 ± 1.30 abnormality (%) 15.0 ± 2.24 plasma membrane integrity (%) 80.8 ± 0.43 epididymal spermatozoa of the spotted buff alo – yulnawati et al. 24 the percentage of the epididymal spermatozoa motility obtained from the research was 65 % and this value is still in the range of the requirements for using it in the artificial insemination (ai) application. the percentage of the abnormality of this epididymal spermatozoa was also high (15 %), however this value is still in the normal range and reliable to be used for fertilization process, since the normal fertile semen contains not more than 20 % abnormal spermatozoa (ax et al. 2000). the percentage of abnormal morphology of 8-10 % did not give any significant effect to the fertility, however, if the abnormality form one ejaculate which is more than 25 %, the decrease of the fertility could not be anticipated (bearden and fuquay 1997). generally, after undergoing three steps of freezing process, the research indicated that the percentage of the motility, viability and plasma membrane integrity of the treatment groups were better compared to the control group. the percentage of motility after thawing in the control group, dextrose 0.2 and 0.4 %, were 41, 46 and 46.67 %, respectively. there was a significant difference (p<0.05) between treatment groups which had dextrose addition and the control group. on the other hand, there was no significant difference (p>0.05) between treatment groups and the control group of the percentage of plasma membrane integrity (table 2). it could be concluded that the value of post thawing epididymal sperm still met the requirements for ai, ivf or icsi application. table 2. the mean (±stdev) percentage of motility and viability of epididymal spermatozoa of spotted buffalo at different stages of freezing processes frozen stage parameters control dextrose 0,2% dextrose 0,4% dilution % m 65,00 ± 0,00a 65,00 ± 0,00a 65,00 ± 0,00a % live 76,00 ± 2,83a 82,00 ± 1,41b 82,67 ± 1,25b % mpu 78,67 ± 0,47a 77,33 ± 2,87a 80,00 ± 0,82a equilibration % m 50,00 ± 0,00a 56,67 ± 4,71a 56,67 ± 4,71a % live 70,33 ± 0,47a 73,00 ± 1,89a 72,67 ± 1,25a % mpu 72,00 ± 0,82a 71,33 ± 0,94a 73,00 ± 0,82a th awing % m 41,00 ± 2,00a 46,00 ± 2,00b 46,67 ± 2,36b % live 52,20 ± 2,48a 58,80 ± 1,83b 60,00 ± 0,82b % mpu 68,00 ± 1,10a 67,40 ± 1,36a 66,80 ± 1,47a note: m: motility, mpu: plasma membrane integrity, a,b different superscript in the different column showed significantly different (p<0,05). dextrose is produced by the conversion enzyme found in cornstarch and undergoes demineralization ion exchange process. dextrose is the form of d-glucose that can be metabolized by the cell through biochemical process called glycolysis to be an energy source. in general, the addition of the sugar into the spermatozoa extender can affect the motility, viability and plasma membrane integrity of the spermatozoa. the addition of sugar into the extender can diminish the damage of the spermatozoa acrosome (yildiz et al. 2000). sugar is known as an extracellular cryoprotectant. the mechanism of how dextrose works in this research is assumed as a protector for the plasma membrane rather than to prevent the formation of the ice crystal in the cell. biotropia vol. 16 no. 1, 2009 25 whereas, glycerol already contained in the andromed®, play a role as intracellular cryoprotectant,to prevent the formation of ice crystal during freezing process. there are no reports of experiments on the epididymal sperm of spotted buffalo freezing and thawing process. therefore, none of the publications reported on the use of sugar that was added into the basic extenders in spotted buffalo sperm cryopreservation process. for the references, the use of sugar as cryoprotectant in different animal species were reported, such as sheep (aisen et al. 2002; molinia et al. 1994), dog (yildiz et al. 2000; rigau et al. 2001), pig (de los reyes 2000) and bull (woelders et al. 1997). generally, sugars play a role as the capacity agent of cryoprotectant and works as the spermatozoa plasma membrane protector during freezing process. it was also known that the sugars have dehydration activity and be able to interact with the cell membrane (aisen et al. 2000). conclusions in conclusion, recovered spermatozoa from cauda epididymis of spotted buffalos that undergo freezing processes using andromed with the addition of 0.2 or 0.4% dextrose are suitable for ai. acknowledgments this research was finacially supported by dipa biotrop 2008, no: 047.1/ psrp-sp/iii/2008. special thank goes to the head of animal division in north toraja, dr. yulius and mr. slamet sumitro for supplying the cauda epididymis samples and using the laboratory facilities. references aisen e.g., alvarez h.l., venturino a. and j.j. garde. 2000. effect of trehalose and edta oncryoprotective action of ram semen diluents. theriogenology, 53(5): 1053-1061. aisen e.g., medina v.h. and a. venturino. 2002. cryopreservation and post-thawed fertility of ram frozen semen in different trehalose concentrations. theriogenology, 57:1801-1808. ax r.l., dally m., didion b.a., lenz r.w., love c.c., varner d.d., hafez b. and m.e. bellin. 2000. semen evaluation. in: hafez b 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1993. principles and procedures of statistic. london: mc graw-hill book co. inc. pub. ltd. squires e.l., gomez-cuetara c. and j.k. graham. 2000. effect of seminal plasma on cryopreserving epididymal and ejaculated stallion spermatozoa. proceedings 14th international congress on animal reproduction. stockholm, 2-6 july 2000; 2, p.166. suhendra. 2002. the study about some parameters of bull sperm quality in different part of epididymis. bachelor thesis. bogor: faculty of veterinary medicine, bogor agricultural university. toelihere m.r. 1993. artificial insemination in animals. bandung: angkasa. tsutsui t., wada m., anzai m. and t. hori. 2003. artificial insemination with frozen epididymal sperm in cats. j. vet. med. sci. , 65 (3): 397-399. woelders h., matthij a. and b. engel. 1997. effects of trehalose and sucrose, osmolality of the freezing medium, and cooling rate on viability and intactness of bull sperm after freezing and thawing. cryobiology, 35:93-105. yildiz c., kaya a., aksoy m. and t. tekeli. 2000. influence of sugar supplementation of the extender on motility, viability and acrosomal integrity of dog spermatozoa during freezing. theriogenology, 54(4): 579-585. yulnawati and m.a. setiadi . 2005. motility and membrane integrity of cat epididymal sperm during storage at 4°c. med. vet j., 21(3): 100-104. epididymal spermatozoa of the spotted buff alo – yulnawati et al. thank you for evaluating anybizsoft pdf splitter. a watermark is added at the end of each output pdf file. to remove the watermark, you need to purchase the software from http://www.anypdftools.com/buy/buy-pdf-splitter.html http://www.anypdftools.com/buy/buy-pdf-splitter.html biotropia no. 8, 1995: 11-22 effects of soil sterilization on the formation and function of two strains of pisolithus tinctorius on eucalyptus urophylla*) nelly s. aggangan, bernie dell biological and environmental sciences, murdoch university, perth, western australia 6150 nick malajczuk csiro division of forestry, private bag p.o. wembley, western australia 6014 reynaldo e. de la cruz biotech, university of the philippines at los ratios, college, laguna, philippines 4031 abstract to examine the effects of soil microbial population on mycorrhizal development and function, eucalyptus urophylla seedlings were inoculated with two pisolithus tinctorius isolates and grown in sterile, partly sterile and non-sterile soil. the two isolates of pisolithus were an effective isolate (h445) collected from under eucalypts in australia and an isolate (h615) collected from under eucalypts in the philippines. soils used were infertile acid soils collected from field sites in pangasinan, luzon and surigao, mindanao. in both soils, the australian pisolithus h445 improved the growth of e. urophylla seedlings more than philippine isolate h615. the uninoculated seedlings exhibited stunted growth typical of p deficiency. height at 8 weeks was significantly taller in sterile than in non-sterile soil. a significant interaction effect of inoculation and soil sterilization on height at harvest was observed only in surigao soil. soil sterilization had a varied effect on mycorrhizal formation. in pangasinan soil, root colonization by h445 was significantly greater in non-sterile soil than in sterile soil. whereas in surigao soil, root colonization was significantly reduced by 54% from partly sterile to non-sterile soil. on the other hand, h615 showed significant mycorrhizal colonization in non-sterile soil compared from those in partly sterile and sterile soils. the degree of infection did not necessarily correspond to growth promotion in e. urophylla seedlings. these results indicate that the performance of the h445 was markedly affected by the microbial flora of the two soils. thus, its potential use in the philippines needs to be thoroughly tested in the field before its widespread use in any inoculation program. key words: mycorrhizas/soil sterilization/pisoto/ws tinctorius/eucalyptus urophylla. *)paper presented at the second symposium on biology and biotechnology of mycorrhizae and third asian conference in mycorrhizae (acom hi), 19-21 april 1994, yogyakarta, indonesia. 11 biotropia no. 8, 1995 introduction eucalypts are important reforestation species grown in the philippines to provide raw materials for the pulp and lumber industries. their fast growth and adaptability to low nutrient soils might be dependent on the presence of symbiotic mycorrhizal associations. for example, previous inoculation work carried out in the philippines on pisolithus and eucalypts indicate growth enhancement with a pisolithus isolate collected from under pinus in the philippines (de la cruz et al. 1990, 1991). pisolithus species have been shown to increase survival and growth of a wide range of tree species such as oaks, pines and eucalypts. in the tropics, lee su see et al. (1994) found that a pisolithus isolate colonized the roots and subsequently improved the growth of two dipterocarp seedlings grown in the nursery. recent work conducted by burgess et al. (1994a) found that growth stimulation of e. grandis seedlings varied greatly among 20 isolates of pisolithus. an isolate (h445) collected from under eucalypts was one of the best growth promoters. this same isolate has also been reported to dramatically increase the growth of e. globulus and e. diversi-color in other studies (burgess et al. 1993). however, not all isolates are beneficial to plant growth. for example, tonkin et al. (1988) observed severe root reduction and retarded shoot growth of e. marginata when inoculated with certain pisolithus isolates in aseptic condition. one factor influencing the response of eucalypts to inoculation with pisolithus is the host from which the isolate was isolated. pine isolates of pisolithus, for example, have been shown to form poor associations with eucalypts (malajczuk et al. 1982, 1990; burgess et al. 1994a). in the development of an inoculation program for nurseries in the philippines, it is essential to introduce compatible ectomycorrhizal isolates that can improve the growth of eucalypts. isolate h445 presents a possible candidate for such introduction. introduction of pisolithus isolates into the philippines from australia requires a thorough testing of the behaviour of these fungi on roots of eucalypt seedlings growing in soil collected from field sites in the philippines. in addition, it is necessary to compare the performance of introduced australian isolates with local philippine pisolithus isolates. the glasshouse screening experiments conducted, so far, for pisolithus and eucalypts have used pasteurized soil (burgess et al. 1993, 1994a). it is important to understand factors that affect inoculum survival and persistence of fungi on eucalypt roots in the field. predicting the effectiveness and competitiveness of introduced ectomycorrhizal fungi in the field, and comparison with previous glasshouse experiments, requires preliminary testing of the fungi in the presence or in the absence of native soil microflora. in a sterile condition, all the native microorganisms 12 effects of soil sterilization nelly s. aggangan et al. are eliminated by sterilization, thus leaving the introduced mycorrhizal fungi to function freely without interference from other microbes. in non-sterile soil, on the other hand, the introduced fungi must compete with other microorganisms in order to survive and function. this would represent conditions similar to those in the field. it is also possible that partial sterilization such as incorporation of a small amount of non-sterile soil to a fumigated one, can stimulate an isolate to develop and absorb more nutrients. in this paper, the effectiveness of an australian (h445) and a philippine pisolithus isolate (h615) on the formation of ectomycorrhizas and growth stimulation of e. urophylla seedlings was compared. plants were grown in soils collected in the philippines and subjected to three sterilization treatments (non-sterile, partly sterile and sterile soil). materials and methods seed germination and mycorrhizal synthesis seeds of e. urophylla (seedlot # 18094 from mt. egon, flores island, indonesia), obtained from csiro australian tree seed centre (canberra, australia), were aseptically germinated on agar plates. seeds were washed with 70% ethanol containing tween 20 for one minute, surface sterilized with 10% sodium hypochlorite for 5 minutes and washed three times with sterile water. two isolates of pisolithus were selected: a) h445 an isolate provided by csiro division of forestry, collected under e. marginata in western australia and which has been shown to stimulate the growth of eucalypts in field trials, and b) h615 collected under e. camaldulensis in the philippines but probably associated with pinus (burgess et al. 1994b). ectomycorrhizas were synthesized aseptically in petri dishes by transferring 7 day-old germinants onto the surface of 10-day old hyphal mats growing on modified melin norkrans medium (marx 1969) with low glucose concentration (1.75 g/l) for two weeks (malajczuk et al. 1990). plates were incubated in a growth room set at 12 hrs photoperiod and temperature of 25°c. at 10 days they were transplanted into undrained pots containing 2 kg soil. the surface of each pot was covered with aluminium foil with four holes where the seedlings were inserted. the foil reduced water loss from the soil and minimized spore contamination. after four weeks, the seedlings were thinned to two plants per pot. 13 biotropia no. 8, 1995 soil treatments soils (015cm depth) were collected at sites in: a) labrador, pangasinan, luzon and b) in bislig, surigao sur, mindanao, referred to in figures as pangasinan and surigao soil, respectively. both sites are grasslands which previously were monsoon forests dominated with dipterocarps. the pangasinan soil has a ph of 4.1 (1:1 soil, 0.005m cacl,), 2.02% organic matter (walkey-black method), 0.11% total n (modified kjeldahl method), and 0.84 ppm available p (bray no. 2) while the surigao soil has a ph of 5.9, 2.48% organic matter, 0.06% total n and 0.21 ppm available p. textural class of the two soils is silt loam. the soils were air dried, pulverized and passed through a 2 mm screen. two kg dry soil were dispensed into plastic pots with polyethylene bag liners. soil treatments were: sterile, partly sterile and non-sterile. pots designated as sterile and partly sterile were fumigated with methyl bromide (one canister per m 3 soil) in a fumigation chamber for 3 days. the non-sterile designated pots were left unfumigated. the partly sterile soil treatment was prepared by mixing 1% unfumigated soil with 99% methyl bromide, fumigated soil. addition of fertilizer one week after fumigation, all pots received the following basal nutrients (per kg soil): 16 mg ca(h2po4)2.h2o, 270 mg nh4no3, 233 mg k2so4, 71.3 mg cacl2, 21.4 mg mg so4.7h2o, 10 mg znso4.7h2o, 5 mg cuso4.5h2o, 0.36 mg coso4.7h2o, 0.7 mg h3bo3 and 1.62 mg na2moo4.2h2o. manganese and iron were not included because the soils had high concentration of these elements (dell, unpublished). the nutrient solutions were added evenly on the soil surface after sterilization. the soil was mixed thoroughly and watered to 80% field capacity and incubated in benches inside a screenhouse for five days prior to planting. seedling maintenance and growth monitoring two weeks after transplanting, 2.5 ml nitrogen solution (54g nh4no3/l h2o) pot -1 week -1 was added. the same rate was added for the next two weeks after which the rate of nitrogen was increased to 5 ml pot -1 week -1 for a period of 7 weeks. all pots were watered to field capacity by weight when necessary. height was monitored twice a month. 14 effects of soil sterilization nelly s. aggangan et al. harvest and mycorrhizal assessment after 12 weeks, the seedlings were harvested. shoots were cut 1 cm above the soil surface and the root systems were gently washed under running water. fine roots (diameter less than 0.5 mm) were separated from the coarse roots, blotted dry in between paper towels and cut into 1 cm lengths. fine root samples of 0.2 g fresh weight were further chopped into 1-2 mm lengths and were fixed in 70% ethanol for counting mycorrhizal root infection. fine roots were cleared and stained as described by phillips and hayman (1970). stained roots were spread evenly over a petri dish and were examined under a stereomicroscope. ten randomly selected fields of view were chosen and all roots crossing a hair-line were examined for the presence or absence of infection. fully colonized root tips were scored as mycorrhizal. experimental design and statistical analysis the experiment was conducted in a screenhouse at biotech, up los banos, philippines following a two factor (fungi and soil treatment) factorial in randomized complete block design (rcbd) with three replicates. pots were rearranged in benches once a week. within each soil type, all data collected were analyzed statistically using two way anova. treatment means were compared using duncan's multiple range test at p< 0.05. results plant growth in pangasinan soil, inoculation (as factor a) significantly (p< 0.01) affected heights at 6, 8,10 and 12 weeks (at harvest) after planting, and dry matter yields (shoot, root, fine root, coarse root and total biomass). generally, the australian pisolithus h445 was more effective in increasing height growth of e. urophylla seedlings from 6 weeks to 12 weeks after planting, dry matter yields and phosphorus uptake (data not shown) than the philippine isolate h615. inoculation with the australian isolate increased (p< 0.05) height at 6 weeks until 12 weeks (harvest) after planting. h615 significantly increased seedling heights from 6 weeks to 10 weeks after planting compared with the uninoculated plants but significantly shorter than those inoculated with h445. at harvest (12 weeks) however, height of seedlings inoculated with the philippine isolate was not significant compared with the uninoculated counterpart. likewise, soil sterilization (as factor b) significantly affected 15 biotropia no. 8, 1995 height at 6 weeks (p< 0.01) and at 8 weeks (p< 0.05) and fine root dry matter yield (p < 0.05). height at 6 and 8 weeks after planting and fine root dry weight obtained in sterile soil were significantly higher than in non-sterile soil. heights and fine root dry weight in partly sterile soil were intermediate and these were not significant compared with those in sterile soil as well as those in non-sterile soil. there was no significant interaction effects of inoculation and soil sterilization on any of the periodic height measurements (fig. 1a). height at harvest due to inoculation with h445 was increased by 4x, 5x and 8x in sterile, partly sterile and in non-sterile soil, respectively, relative to the uninoculated seedlings. on the other hand, h615 increased seedling heights by 1.2x, 2x and 3x in the same soil treatment order. although the mean height of h445 plants were two times taller than those of h615, there was no significant differences among treatments. the uninoculated seedlings had the shortest height in all the three soil treatments. height of uninoculated plants was reduced by 2 folds while total dry weight (data not presented) by 10 folds from sterile to non-sterile soils. whereas, the inoculated seedlings did not differ much irrespective of soil treatment. shoot dry weight (data not presented) showed similar trends to plant height. a significant interaction effect was observed only on fine root dry weight. fine root dry weight was dramatically increased (p< 0.05) by h445 in sterile (fig. 2a) compared with h615 and uninoculated treatments. fine root dry weight was greater (p< 0.05) in sterile than in non-sterile soil. in surigao soil, inoculation (as factor a) significantly affected height at 6 to 12 weeks after planting, coarse root dry weight, root p concentration and uptake and shoot p uptake. h445 and h615 inoculated seedlings were of similar height which was significant compared with height of the uninoculated ones from 6 to 10 weeks after planting. at harvest, h445 inoculated plants were significantly taller than those inoculated with h615 and the uninoculated ones. although, height at harvest, coarse root dry weight and root p concentration obtained by h615 inoculated plants were twice those of uninoculated counterpart, the differences were not statistically significant. soil sterilization (factor b) significantly affected height at 8 weeks and shoot p concentration. height in sterile soil was significantly taller than in partly sterile and non-sterile. the latter soil treatments (partly sterile and non-sterile soil) yielded similar height growth response. shoot p concentration in partly sterile soil was significantly higher than in non-sterile soil. unlike in pangasinan soil, there was a significant interaction effect of inoculation and soil treatment. in sterile, h445 was consistent in promoting a significant increase in height (p<0.01) from 6 weeks until harvest (fig. 1b) compared with the uninoculated seedlings. two way analysis revealed that height growth obtained by seedlings inoculated with h445 in sterile soil was significant compared with those inoculated with h615 and the uninoculated ones. in partly sterile soil, height obtained by h445 16 effects of soil sterilization nelly s. aggangan et al. figure 1. effects of mycorrhizal inoculation on the accumulated height growth of e. urophylla seedlings grown in pangasinan (a) and surigao (b) soils subjected to three sterilization treatments. letters represent results of duncan's multiple range test after two way anova on height at 12 weeks on each soil treatment. lsd bar = 56. 17 biotropia no. 8, 1995 inoculated seedlings was significantly taller than the uninoculated seedlings but not significant as compared with those inoculated with h615. in non-sterile soil, height differences between the inoculated and uninoculated seedlings was not significant. h615 inoculation gave intermediate height and shoot dry weight in sterile and in partly sterile soil which were not statistically different from the uninoculated seedlings. unlike seedlings grown in pangasinan soil, there was no significant interaction effect of inoculation and soil sterilization on fine root dry weight (fig. 2b). however, plants had much larger shoot and root systems in the surigao than in the pangasinan soil. mycorrhizal development in pangasinan soil, there was a significant difference between the three inoculation treatments and soil sterilization significantly affected mycorrhizal development. mycorrhizal infection by h445 was significantly higher (41%) than h615 (21%). the uninoculated seedlings had 7% infection. mycorrhizal infection in non-sterile soil was significantly higher (30%) than in sterile soil (18%). root colonization in partly sterile soil was intermediate which was not significant compared with those obtained in sterile or in non-sterile soils. two way analysis indicates that in sterile and non-sterile soils, h445 and h615 had similar infection levels (fig. 2c). however, in partly sterile soil, h445 had greater (p<0.01) root colonization than h615. root colonization by h445 was significantly higher in non-sterile than in sterile soil but not significant compared with those in sterile and in non-sterile soils. uninoculated seedlings had low levels (not more than 11 %) of infection in the two soils (fig. 2c). in surigao soil, mycorrhizal development formed by h445 and h615 (as factor a) was not significantly different from each other but significant compared with the uninoculated treatment. root colonization was significantly higher in non-sterile and in partly sterile soils than in sterile soil. there was a significant interaction effect of inoculation and soil sterilization treatments. in non-sterile soil, h615 formed higher (p<0.05) infection compared with h445 (fig. 2d). greatest mycorrhizal formation by h445 was observed in partly sterile soil which was reduced (p<0.05) in sterile and in non-sterile soils. percent mycorrhizal infection by h445 in non-sterile soil was similar to that of uninoculated plants grown in either of the three soil treatments. root colonization by h615 was higher (p<0.01) in non-sterile (65%) than in partly sterile soil (34%). infection in sterile soil was intermediate and not significantly different to percent mycorrhizal infections observed in non-sterile and in partly sterile soils. correlation analyses revealed a low relationship (r 2 = 0.6) between percent mycorrhizal infection and height or shoot and fine root dry weight. 18 effects of soil sterilization nelly s. aggangan et al. figure 2. fine root dry matter weight (a and b) and mycorrhizal infection (c and d) of e. urophylla seedlings inoculated with two pisolithus isolates grown on sterile, partly sterile and non-sterile acid soils. letters represent results of duncan's multiple range test after two way anova on each soil. lsd bar = 0.116. 19 biotropia no. 8, 1995 discussion growth of e. urophylla seedlings in the two philippine soils was stimulated by inoculation with two isolates of pisolithus. growth of plants inoculated with the australian eucalypt isolate, h445, was enhanced more than those inoculated with the philippine isolate, h615. previously, h615 was classified as a pine isolate (burgess et al. 1994b) on the basis of polypeptide patterns identified by id sds-page. the effectiveness of a pine pisolithus isolate in promoting growth of eucalypts has been demonstrated in field trials (de la cruz et al. 1990, 1991). potentially, the introduction of australian isolates in any nursery inoculation programs in the philippines would provide an advantage for growth promotion of eucalypts. it remains to be determined whether these specific eucalypt isolates would persist and outperform the indigenous pine pisolithus isolates in the field. it is interesting to note that garbaye et al. (1988) observed the replacement of an introduced pine isolate (marx strain 270), by an indigenous scleroderma species within 2 years after outplanting. in this experiment, mycorrhizal root development was markedly affected by sterilization treatment in one philippine soil (surigao). mycorrhizal infection by h445 was reduced by 54% in non-sterile soil. in contrast, h615, was unaffected by soil sterilization treatments. this would suggest a possible role of soil microflora in affecting the infection process. several studies have suggested that soil microflora play an important role in influencing the inoculation process of seedling roots by mycorrhizal fungi (garbaye and bowen 1987; marx et al. 1982, 1984). such effect may be inhibitory or stimulatory to mycorrhizal formation (bowen and theodorou 1979; garbaye and bowen 1989; fitter and garbaye 1994). it would appear that the introduction of new strains of ectomycorrhizal fungi from australia and screening of endemic strains requires a thorough testing of the performance in a range of soils. it is anticipated that no single species would be appropriate for all soil types in the philippines and the introduction should include a wide range of physiologically distinct species. introduced ectomycorrhizal fungi should be able to survive and tolerate ecological variation within a site and progressively colonize the new roots after outplanting (garbaye 1982). most importantly, the introduced fungi must enhance host performance to a greater degree than the native fungi (danielson 1988). competitiveness or persistence of the association would depend on three factors: the host, mycorrhizal fungus and soil microbes wherein each factor is subject to environmental influences. further work is required to examine the influence of the composition of the microbial population on ectomycorrhizal formation on a range of philippines soil. 20 effects of soil sterilization nelly s. aggangan et at. acknowledgements this work was supported by the australian international development bureau and the australian center for agricultural research while dr. nelly s. aggangan was doing her postgraduate degree. references burgess, t., n. malajczuk and b. dell. 1994a. variation in mycorrhizal development and growth stimulation of 20 pisolithus isolates inoculated onto eucalyptus grandis w. hill ex maiden. new phytol (in press). burgess, t., n. malajczuk and b. dell. 1994b. variation in pisolithus based on basidiocarp and basidiospore morphology, culture characteristic and polypeptide analysis using id-sds page (in press). burgess, t., n. malajczuk and t.s. grove. 1993. the ability of 16 ectomycorrhizal fungi to increase the growth and phosphorus uptake of eucalyptus globutus labill and e. diversicolor f. muell. plant and soil 153: 155-164. bowen, g.d. and c. theodorou. 1979. interactions between bacteria and ectomycorrhizal fungi. soil. biol. and biochem. 11: 119-126. danielson, r.m. 1988. mycorrhizae in forestry: the state-of-the-art in land reclamation. in canadian workshop on mycorrhizae in forestry, 1 -4 may 1988, universite laval, ste-foy, que. edited by m. lalonde and y. piche. universite laval, ste-foy, que. p. 39-41. de la cruz, r.e., n.s. aggangan, j.t. zarate and r.p. yecyec. 1991. improved reforestation technologies in the philippines. pcarrd book series 121: 65-78. de la cruz, r.e., e.b. lorilla and n.s. aggangan. 1990. ectomycorrhizal tablets for eucalypthus species. in fast growing tress and nitrogen fixing tress. eds. d werner and p muller, gustav fisher verlag, stuttgart, new york. p. 371. fitter, a.h. and garbaye, j. 1994. interactions between mycorrhizal fungi and other soil arganisms. plant and soil 159: 123-132. garbaye, j. 1982. quelques aspects de la competitive des souches ectomycorhiziennes. in les mycorhizes, panic integrante de la plante: biologic et perspectives d'utilisation. eds. s gianinazzi, v gianinazzi-pearson and a trouvelot. pp. 303-312. proceedings of a seminar, 5-6may 1982 atm dijon, france, les colloques de finra, no.13. garbaye, j. and g.d. bowen. 1987. effect of different microflora on the success of ectomycorrhizal inoculation of pinus radiata. can.j.for. res. 17: 941-943. garbaye, j. and g.d. bowen. 1989. stimulation of ectomycorrhizal infection of pinus radiata by some microorganisms associated with the mantle of ectomycorrhizas. new phytol. 112: 383-386. garbaye, j., j.c. delwaulle and d. diangana. 1988. growth response of eucalypts in the congo to ectomycorrhizal inoculation. forest ecology and management 24: 151-157. lee su see, f. lapeyrie and mohd. yazid zanip. 1994. techniques for controlled ectomycorrhizal inoculation of dipterocarp seedlings and cuttings. in programs and abstract third asian conference on mycorrhizae, 19-21 april 1994, yogyakarta, indonesia, p. 4-6. 21 biotropia no. 8, 1995 malajczuk, n., f. lapeyrie and j. garbaye. 1990. infectivity of pine and eucalypt isolates of pisolithus tinctorius on roots of eucalyptus urophylla in vitro. new phytologist 114: 627-631. malajczuk, n, r. molina and j. trappe. 1982. ectomycorrhizal formation in eucalyptus. i. pure culture synthesis, host specificty and mycorrhizal compatility with pinus radiata, new phytol. 91: 467-482. marx, d.x. 1969. the influence of ectotropic mycorrhizal fungi on the resistance of pine roots to pathogenic infection. i. antagonism of mycorrhizal fungi to root pathogenic fungi and soil bacteria. phytopathology 59: 153-163. marx, d.h., j.l ruehle, d.s. kenny, c.e. cordell, j.w. riffle, r.j. molina, w.h. pawuk, s. navratil, r.w. tinus and o.c. goodwin. 1982. commercial vegetative inoculum of pisolithus tinctorius and inoculation techniques for development of ectomycorrhizae on container-grown tree seedlings for. sci. 28(2): 373-403. marx, d.h., c.e. cordell, d.s. kenny, j.g. mexal, j.d. arthman, j.w. riffle and j.w. molina. 1984. commercial vegetative inoculum of pisolithus tinctorius and inoculation techniques for development of ectomycorrhizae on bare-root tree seedlings. for. sci. monogr. no. 25. phillips, j.m. and d.s. hayman. 1970. improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. trans. brit. mycol. soc. 55: 158-160. tonkin, n.c., n. malajczuk and j.a. mccomb. 1988. ectomycorrhizal formation by micro-propagated clones of eucalyptus marginata inoculated with isolates of pisolithus tinctorius. new phytologist 111: 209-214. 22 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 6. annisa satyanti page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 page 14 page 15 microsoft word 62 biotropia no. 24, 2005 : 62 68 molecular phylogenetic analysis of monascus fungi based on internal transcribed spacer region n. suharna1, y. kikuchi2'3 and t. fukatsu3 'research center for biology, indonesian institute of sciences, bogor, indonesia 2natural history laboratory, faculty of science, ibaraki university, mito 310-8512, japan 3national institute of biological functions and resources, advanced industrial science and technology (aist), tsukuba 305-8566, japan abstract a molecular analysis of internal transcribed spacer region has been carried out to reveal the relationship among 16 strains of monascus spp. a primer set comprised primer its1 and its4 was used to amplify this region in which they were cloned and scqucnccd. we also compared the sequence result with m. purpureus af458473, m.ruber af458470, m. kaoliang af451859, m. araneous af458471 and m. pilosus af451856 and one outgroup species thermoascus crustaceus u18353. the result showed that 16 monascus spp. were divided into two large clades while m. ruber af458470 was basically separated from all those monascus. one of the two large clades included the seven m. purpureus strains, m. purpureus af458473, m. araneosus af458471 and m. kaoliang af451859. another large cladc included the six monascus sp. strains which typically have whitish colonies, the three m. ruber strains and m.pilosus af451856. however, even outstanding morphological differences possessed by several white monascus and one whitish m. purpureus strain, all monascus strains were suggested to be very closely related with similarity >99% almost 100%. although this its analysis could not discriminate cultural and morphological differentiation of monascus strains studied, yet there is still little genetic variation within these strains. key words : molecular genetics/monascus spp./fungi introduction hawksworth and pitt (1983) cited that monascus species (monascaceae) are important for producing asian fermented foods particularly red rice (ang-kak), rice wine and kaoliang brandy, soy bean cheese and food colorants; for their antibacterial properties; production of mycotoxin; and a major component of silage mycofloras. lakrodi et al. (200) cited that one of these world wide distributed fungi, m. purpureus is known as the red rice fungus that has been used for over a thousand years by the chinese as a traditional herbal medicine. however, this fungus was firstly isolated from chinese red rice (ang-kak) in java. in indonesia, we isolated and collected m. purpureus from ang-kak in java and sumatra and from other several monascus species isolated from deteriorated invertebrate specimens. based on morphological observation on growth in agar media, we found some interesting not ordinary properties in m. purpureus such as two m. purpureus isolates one of which has unique character such as bigger ascomata and the other one has white colony. while the other four monascus 62 biotropia no. 24, 2005 isolates have very remarkable properties such as they resist ethanol at very extreme concentration and their morphological characters are also unique. so, it is of interest to know the genetic relationship rather than morphological relationship among those isolates within monascus species. actually, dna sequence analysis based on the d1/d2 regions of lsu rrna genes of monascus species have been conducted by park and jong (2003). they suggested that m. lunispora, m. floridanus, and m pallens were separated in different clades. however, five monascus species such as m pilosus, m. purpureus, m. ruber, m. eremophilus and m. sanguineus were reflected in monophyletic relationship (park and jong 2003). these five species were definitely different species based on morphological characters on agar media by hawksworth and pitt (1983) (m. pilosus, m. purpureus, m. ruber), hocking and pitt (1988) (m eremophilus) and cannon et al. (1995) (m sanguineus). as park and jong (2003) have suggested no separation among the five species of monascus, we intended to do analysis on internal transcribed spacer region of monascus. this region is known for its various nucleotides sequences so it might be more perspective rather than on lsu rrna genes. this similar work was not only to reveal genetic diversity of monascus in indonesia, but also to understand the relationship among monascus species and direction of mutation by analysis materials and methods monascus strains a number of 16 monascus strains used in this study were isolated and identified by suharna in 2002 and 2003 (table 1). molecular phylogenctic analysis of monascus fungi — n. suharna et al. cultivation and purification of monascus strains all fungi were cultivated on ym agar plate and for purification of cultures water agar 2% was used. incubation was carried out at room temperature (25°c) for three days. a little amount of mycelial mass of each fungus was picked up using toothpick then transferred into centrifuge tube containing 10 ml of ym broth medium and incubated at 30°c for three days. the mycelial mass of each fungus was then harvested for dna extraction. dna extraction the harvested mycelial mass of each fungus was put onto paper to remove excess of medium then subsequently put onto mortar before freezing by pouring liquid nitrogen and grounded by mortar and pestle. the following step of dna extraction was carried out using qiaamp tissue kit (qiagen). the yield of dna samples was quantified and qualified by both spectrophotometer and gel electrophoresis. pcr, cloning and sequencing of its region its region was amplified by pcr with specific primer set, its1 (5'tccgtaggtgaacctgcgg-3') and its4 (5'-tcctccgcttattgatatgc-3') (white et al. 1990). pcr was conducted using amplitaq dna polymerase (roche, basel, switzerland) and supplemented with buffer system under a temperature profile of 94 °c for 4 min. followed by 30 cycles of 94 °c for 30 sec, 55 °c for 1 min. and 72 °c for 1 min. the pcr products were directly cloned with ta-cloning vector pt7blue (takara) and escherichia coli dh5a competent cells (takara) using ampicillin and x-gal blue-white selection system. to check the length of the inserted dna fragment, white colonies expected to contain inserted plasmid were directly subjected to pcr using the primers univl9 (5'-gttttcccagtcacgacgt-3') and rev20(5'agctatgaccatgattacgc -3'). when a pcr product of expected size (600 bp) was obtained, two clones of which were cultured overnight in 3 ml of lb medium containing ampicillin, and subjected to plasmid extraction using qiaprep-spin miniprep kit (qiagen). the purified plasmids were eluted with 50 ul distilled-water and used for sequencing. dye terminator-labelled cycle sequencing reaction was conducted with dna sequencing kit fs (perkin elmer) and four sequencing primers univl9 and rev20 under a temperature profile of 94 °c for 4 min. followed by 30 cycles of 94 °c for 30 sec, 50 °c for 1 min. and 60 °c for 4 min. the products were analyzed by abi prism 377 dna sequencer (perkin elmer). 64 biotropia no. 24, 2005 molecular phylogenetic analysis the its region sequences determined were subjected to molecular phylogenetic analysis together with those retrieved from the ddbj nucleotide sequence database. a multiple alignment of the its region sequences was generated by the program package clustal w (thompson et al. 1994). phylogenetic trees were constructed by the neighbor-joining method using clustal w (thompson et al. 1994). bootstrap tests (felsenstein 1981) were performed with 1000 replications. results figure 1 shows that amplification by primer set comprised its1 and its4 were successful for all monascus strain tested. the size of amplified dna products of all monascus isolates by the primer set was 700 base pair each as shown in figure 1. molecular phytogeny figure 2 shows that thermoascus crustaceus u18353 used as outgroup was significantly separated from all monascus species that integrated in one main clade. this monascus clade consists of very similar two large clades, while m. ruber af458470 was basically separated from all those monascus. one of the two large 65 molecular phylogenetic analysis of monascus fungi n. suhama el al. clades includes the seven m. purpureus strains, m. purpureus af458473, a araneosus af458471 and m. kaoliang af451859. another large clade includes th six monascus sp. strains which typically have whitish colonies, the three m. rube strains and m.pilosus af451856. the similarities within the seven m. purpurei> strains and m. purpureus af458473, m. araneosus af458471 and m. kaolian af451859 are very high more than 99% or almost 100%. however, m. purpure^ pkb5 shows a little more different. the six monascus sp. strains, the three m. rube and m. pilosus af451856 also showed very high similarity. but lesser difference i shown by monascus sp. ka30.1. discussions with regard to the level of very high similarity more than 99% almost nearl; 100% among all monascus fungi we analyzed, indicated that there is little mutatioi on its region of monascus species. concerning genetic diversity it seemed tha there is a diverse mutation tendency as several monascus strains such a 66 biotropia no. 24, 2005 m.purpureus pkb1, m.purpureus prba, m.purpureus jms, m.purpureus af458473, m. rubber cka1 and monascus sp. ka30.1 appeared more different though these differences were so slight. therefore, this phylogenetic analysis revealed very close relationship among monascus strains so we suggested that all m.purpureus were identical to each other as shown by m.purpureus ngk-j, m.purpureus jmba, m.purpureus pkb1, m.purpureus pkb5, m.purpureus srba, m.purpureus af458473 and m. araneosus af458471 in the same clade. in another clade, all indonesian m. ruber strains and m. pilosus were identical to each other. however, m. ruber af458470 was separated in another clade. this work also confirmed that m. araneosus af458471 and m. kaoliang af451859 are the same species with m. purpureus. previously, hawkswoth and pitt (1983) have included m. araneosus and m. kaoliang as m. purpureus synonym based on morphological characteristics. it is also of interest to note that m. purpureus srba has a uniqueness of its morphological features such as having bigger ascomata (generally 90 um in diameter) than "normal" m. purpureus. the normal m. purpureus has ascomata up to 70 um in diameter. the other white isolates such as monascus sp. mm, monascus sp. coel, monascus sp. ktb, monascus sp. myom and monascus sp. myot have different morphological characters. these white isolates at least have two different morphological characters compared to m. ruber such as shorter ascospores, cleisthothecium wall transparent. moreover, monascus sp. myom and monascus sp. myot have fusiform aleurispores, while m. ruber has no this spore form(suharna 1999). besides d1/d2 regions of lsu rrna genes, phylogenetic analysis on its region was also made by park and jong (2003) to know the relationship among monascus strains accessed from genebank. the phylogenetic tree constructed showed similar result with our analysis on its sequence of monascus where m. ruber and m. pilosus were very closely related and all m. purpureus strains were included together in the same clade. though it is still suggested that the two clades were very closely related, interestingly, all indonesian m. ruber strains were in the same clade with m. pilosus af451856. while m. ruber with accession number af458470 retrieved from ddbj was separated in another clade. this indicated that three m. ruber isolates and six white monascus isolates showed little difference from m. ruber af458470, but identical to m. pilosus af451856 except for monascus sp. ka30.1 and m. ruber cka3 with genetically little difference. however, despite of this finding, it is still much surprising in regard with the outstanding morphological differences of several strains as mainly possessed by whitish strains such as monascus sp. ka30.1, monascus sp. myom, monascus sp. ktb, monascus sp. myot, monascus sp. mm, monascus sp. coel. park and jong (2003) also reported similar result as their comparison of two very distinct species based on morphological observation such as m. ruber and m. pilosus. therefore, we are in concordance with park and jo (2003) that it is needed to reveal in more details about molecular phytogeny to elucidate molecular taxonomy included in the identification of monascus species. 67 molecular phylogenctic analysis of monascus fungi — n. suharna et al. acknowledgments this research was funded by the general exchange program, japan societ the promotion of science and conducted in early 2004 at the national institu biological functions and resources, advanced industrial science and techno (aist), tsukuba 305-8566, japan. references cannon, p.p. , s.k. abdullah and b.a. abbas. 1995. two new species of monascus from iraq, \ key to known species of the genus. mycol. res. 99: 659-662 fclsenstcin, j. 1981. evolutionary trees from dna sequences : a maximum likchood approach. j. evol 17: 368-376 hawskworth, d.l. & j.i. pitt. 1983. a new taxonomy for monascus based on cultural and micros characters. aust. j. bot. 31: 51-61 lakrodi, k., c. chaisrisook, b. yongsmith and d.z. skinner. 2000. rapd analysis of genetic vari within a collection of monascus spp. isolated from red rice (ang-kak) and sofu. mycol. res (4): 403-408 park, hg., jong, s-c. 2003. molecular characterization of monascus strains based on the d1/d2 re of lsu rrna genes. mycoscicnce 44: 25-32 thompson, j.d., d.g. higgins, and j.j. gibson. 1994. clustal w: improving the sensitivity of progrc multiple alignment through sequence weighting, position-specific gap penalties and weight it choice. nucleic acids res. 22:4673-4680 white, t. j., t. bruns, s. lee, and j. w. taylor. 1990. amplification and direct sequencing of fi ribosomal rna genes for phylogcnctics. pp. 315-322 in: pcr protocols: a guide to method: applications, eds. innis, m. a., d. h. gelfand, j. j. sninsky, and t. j. white. academic press, new york. suharna, n. 1999. a study on the fungal occurrences in several animal specimens preserved in alcohc gakuryoku 5 (3): 139-144 68 62.pdf 63.pdf 64.pdf 65.pdf 66.pdf 67.pdf 68.pdf biotropia no. 7, 1994: 1-11 biological control of damping-off fungi of agoho (casuarina equisetifolia l.) using antagonistic bacteria f.a. dela peña mariano marcos state university, batac, ilocos norte philippines 2906 a.k. raymundo, e.p. militante, m.u. garcia and l.u. dela cruz university of the philippines at los banos, college, laguna philippines 4031 abstract a series of laboratory and nursery experiments were conducted specifically to determine the efficacy of 85 strains of bacillus species and 15 actinomycetes against six fungal pathogens isolated from damped-off agoho. these damping-off fungi were: fusarium oxysporum schet., rhizoctonia solani kuhn., phytophthora parasitica dastur, pythium debaryanum hesse, and two unidentified pathogens temporarily designated as unk 1 and unk 2. preliminary test using the agar-plug technique revealed that 18 of the bacterial isolates could suppress two or more of the six damping-off fungi. fusarium oxysporum was inhibited by 17 bacterial isolates, r. solani by 8 isolates, p. parasitica by 14 isolates and p. debaryanum by 15 isolates. the unidentified damping-off fungi unk 1 and unk 2 were inhibited by 13 and 9 isolates, respectively. further screening using the agar-diffusion method disclosed that 10 isolates were effective antagonists with bacillus subtilis (code no. r060), bacillus sp. (code no. r071), and streptomyces sp. (code no. r086) as the consistent and most effective inhibitors. application of the three most promising antagonistic bacteria as seed treatment s howed that they effectively inhibited the growth of the damping-off fungi in the laboratory as exhibited by an increase in percent germination. bacillus subtilis however, was not able to antagonize the effect of p. debaryanum in this particular experiment. seed germination and seedling survival were likewise improved with the application of the three most promising antagonistic bacteria as seed treatment. this was shown after three months under nursery conditions. there were possible mechanisms of control by the antagonistic bacteria against the damping-off fungi. the mycelium and spores of the pathogenic fungus may have been attacked and parasitized by the antagonist when they were simultaneously grown in culture media. there must have been a competitive interaction between the two microorganisms. any essential requirement of microorganisms can serve as potentially possible basis for competition. another was antibiosis which is an inhibitory effect exerted by an organism upon another organism through the production of antibiotic compounds. moreover, several strains of bacteria are effective in lysing cell walls of pathogenic fungi under laboratory conditions. lysis is often attributed to production of cell wall degrading enzymes like chitinase and gluconase that may hydrolyze major constituents of fungal cell walls. 1 biotropia no. 7, 1994 furthermore, several strains of bacteria are effective in lysing cell walls of pathogenic fungi under laboratory conditions. lysis is often attributed to production of cell wall degrading enzymes like chitinase and glucanase that may hydrolyze major constituents of fungal cell walls. as demonstrated by kapoor and kar (1979), hyphae showed lysis in addition to thickening of hyphae as well as formation of chlamydospores in abundance as an effect of bacillus sp. on f. oxysporum f. sp. lycopersici. inhibitory substance diffused into the media may initiate fungal cell wall lysis. most bacteria are effective fast-growing colonizers which make such microorganisms efficient biological control agents of plant diseases. seed treatment gave the antagonist the opportunity to be the first colonizer of the plant roots of the seedlings (elad and chet 1987) thus having an advantage for any competition for nutrients supplied by roots. seed exudates probably occurred in the interaction between the antagonistic bacteria and damping-of f fungi in the root and is responsible at least to a degree for the observed biological control by introduced bacteria. large populations of bacteria established on planting material and roots become a partial sink for nutrients in the rhizophere, thus reducing the amount of carbon and nitrogen available to stimulate sporulation of fungal pathogen or for subsequent colonization of the root (weller 1988). in addition, cell wall degrading enzymes are activated by nutrient deprivation imposed by microbial activity. oospore germination by some damping-off fungi has already been shown to be affected by exogenous nutrients (howell and stipanovic 1980). nutrients supplied by root exudates stimulate oospore germination in the rhizosphere. it therefore, appeared that bacteria may compete with germinating oospore for available carbon or nitrogen sources, and by eliminating these resources, the bacteria reduce the percentage of oospore germination. in addition, elad and chet (1987) stated that there was a significant positive correlation observed between inhibition of oospore germination and disease reduction by bacteria. conclusion the promising antagonistic bacteria therefore, could reduce the incidence of damping-off disease of agoho. its application as biological control agent could be a good substitute for soil fungicides. although soil fumigation is very effective for the control of damping-off pathogens, it is very expensive, impractical and may create problems like environmental pollution and pathogen resistance. 10 biological control of damping-off fungi of agoho f.a. dela pena et al. references dayan, m.p. and v.o. sinohin. 1989. diagnosing forest tree diseases (fungal). ecosystem research and development bureau, college, laguna. 29 p. dela cruz, a. 1986. microbial antagonists of the potato soft-rot pathogen, erwinia carotovora pv. carotovora (jones) bergey, harrison, breed, hammer, and hutton. (unpublished ms thesis, university of the philippines at los banos, college, laguna). dickson, j.g. 1956. diseases of field crops. me. graw hill book, co., new york, 517 p. elad, y. and i. chet. 1987. possible role of competition for nutrients in biocontrol of pythium damping-off by bacteria. phytopathol. 1: 375-396. garret, s.d. 1965. toward biological control of soil-borne plant pathogens: 4-16. in: k.f. barker and w.c. snyder (eds.), ecology of soil-borne plant pathogens-, prelude to biological control. university of california press, california. henis, y. and i. chet. 1975. microbiological control of plant pathogens. in: d. perlman (ed.) advances in applied microbiology. vol. 19. academic press, new york. 332 p. henis, y. and m. imbar. 1968. effect of bacillus subtilis on growth and sclerotium formation by rhizoctonia solani. phytopathol. new york. howell, c.r. and r.d. stipanovic. 1980. suppression of pythium ultimum induced damping-off of cotton seedlings by pseudomonasfluorescens and antibiotic pyoluteorin. phytopathol. 70: 712 715. jones, r.k. 1985. fungicides for bedding plants. bedding plants inc. news 16(2): 3-4. kapoor, i.j. and b. kar. 1989. antagonism of azotobacter and bacillus to fusarium oxysporum f. sp. lycopersici. indian phytopathol. 42(3): 400-404. larson, r. 1987. growing concerns. pest control: how much is enough. marketletter 2: 5. national research council. 1984. casuarinas: nitrogen fixing trees for adverse sites. national academy press, washington d.c. 118 p. olsen, c.m. and k.f. baker. 1968. selective heat treatment of soil and its effects on the inhibition of rhizoctonia solani by bacillus subtilis. phytopathol. 58: 79-87. raymundo, a.k., e.t. serrano, g.d. reyes and t.o. zulaybar. 1985. isolation and identification of antibiotic-producing bacillus from the soil. phil. agr. 68: 393-402. weller, d.m. 1988. biological control of soilborne plant pathogens in the rhizosphere with bacteria. ann. rev. phythopathol. 26:379-407. 11 biotropia no. 7, 1994 pre-emptive colonization was also a possible mechanism of control. seed treatment gave the antagonist the opportunity to be the first colonizers of the plant roots and moved along the roots of the seedlings thus having an advantage for any competition of nutrients. most bacteria are effective fast-growing colonizers which make such microorganisms efficient biological control agents of plant diseases. key words: biological control. fungal diseases, casuarina equisetifolia, antagonistic bacteria introduction agoho (casuarina equisetifolia l.) is a good fuel wood species in scores of tropical countries like the philippines. it has particularly been valuable for stabilizing sand dunes as it withstands salt sprays, thrives/grows in infertile soil and is drought-tolerant (national research council 1984). these indispensable characteristics make it highly recommended for reforestation and afforestation purposes. damping-off disease however, is the major threat in the production of sufficient and quality planting stocks. damping-off is a term applied to any disease that results in the decay of seeds in the soil before seedling emergence, or rapid rotting usually at the soil line of recently emerged seedlings (dayan and sinohin 1989). it is caused by a number of soil-inhabiting fungi that are facultative parasites and not specialized as to host. the ubiguity of damping-off fungi necessitates effective control measures because there are times when seedlings in the entire seedbed are wiped out resulting to shortage of supply for field planting. chemical control is usually not feasible due to the expensive price of fungicides. moreover, they can cause environmental pollution and may even induce pathogen resistance (jones 1985; larson 1987). biological control perhaps offers one of the best alternatives. as defined by garret (1965), biological control is any condition or practice under which survival or activity of a pathogen is reduced through the agency of any organism (except man himself) with the result that there is a reduction in the incidence of the disease caused by the pathogen. one of the ways that biological control may operate to suppress pathogen is introduction of biocontrol agents or antagonists. it is for this purpose that a study on the biological control of damping-off fungi of agoho using antagonistic bacteria was conducted. specifically, the study aimed to determine the efficacy of promising antagonistic bacteria in the laboratory and under nursery conditions. 2 biological control of damping-off fungi of agoho f.a. dela pena et al. materials and methods the antagonistic activity of 100 bacterial isolates consisting of 85 strains of bacillus species and 15 actinomycetes was determined against six fungal pathogens isolated from damped-off agoho. the damping-off fungi were: fusarium oxysporum schet., rhizoctonia solani kuhn., phytophthora parasitica dastur, pythium debaryanutn hesse, and two unidentified pathogens temporarily designated as unk 1 and unk 2. preliminary screening using the agar-plug technique involved simultaneous inoculation of the pathogen (damping-off fungus) and the antagonist (bacterial isolate). bacterial isolates showing good antagonistic activity were then subjected to the agar-diffusion method and further tested in the laboratory and under nursery conditions as seed treatment. agar-plug technique the cell suspension of bacillus species was prepared by growing the organism in tryptone glucose yeast extract agar (tgya) in a flat bottle for 24-48 hr under room temperature. after incubation, the growth was flooded with sterile distilled water and then aseptically dislodged from the agar. the resulting suspension was adjusted to 0.30 optical density (od) at 540 nm using a spectronic 20 to have the desired population of 109 cells/ml (dela cruz 1986). the cell suspension of actinomycete was prepared by growing the organism in yeast-malt extract broth (ymb) for 48 72 hr on a shaker at room temperature. the broth culture was then pooled to make the necessary amount needed and homogenized using an osterizer for 5 min. a 10% suspension was made by diluting 100 ml of the prepared actinomycete suspension into 900 ml sterile distilled water (dela cruz 1986). agar plugs were prepared by initially growing the damping-off fungus on potato dextrose agar (pda) under room temperature for seven days or until sufficient growth was obtained. a sterile 1-cm-diameter cork borer was used to obtain the agar plugs from these culture plates. the agar plugs inoculated on agar plates were initially prepared by plating 1 ml of bacterial cell suspension on tgya for bacillus species and yeast-malt extract agar (yma) for actinomycetes. inoculation of the agar plugs was done in an upside-down position to provide a direct contact between the pathogen and the antagonist. the assay plates were incubated at room temperature for 3 to 5 days, after which growth of the pathogen was observed. this experiment utilized a completely randomized design (crd) with three replications. 3 biotropia no. 7, 1994 agar-diffusion method cultures (24 to 48-hr-old) of the potential antagonistic bacteria were used to inoculate tryptone glucose yeast extract broth (tgyb) for bacillus species and ymb for actinomycetes. these were then incubated at room temperature on a shaker. after 48 hr (for bacillus spp.) and 72 hr (for actinomycetes) incubation, the broth cultures were centrifuged at 14 000 rpm, 0°c, for 15 min. the supernatant was used for the microbial assay. one loopful of growth from 48-hr-old culture of the damping-off fungus was suspended in 2 ml sterile distilled water, mixed and the suspension inoculated into 100 ml pda top agar. this was used to seed the pda plates and then allowed to solidify (raymundo et al. 1985). following the solidification of the top agar, four cup cylinders were positioned on the plate and these were then filled with 0.1 ml of appropriate supernatant from each potential antagonist. after incubation for 48 hr or until sufficient growth was obtained, the assay plates were examined and the diameter of zones of inhibition was measured. this experiment utilized a crd with three replications and three subreplications. significant values were further subjected to duncan's multiple range test (dmrt). antagonistic bacteria showing potential to control the damping-off fungi in the agar-diffusion method were further screened as seed treatment in the laboratory and under nursery conditions. laboratory experiment newly prepared cell suspensions of the antagonistic bacteria were used to coat the agoho seeds by soaking for at least 15 min. before sowing onto sterile germination plates lined with tissue paper. the germination plates were initially inoculated with 5 ml fungal cell suspension prepared by suspending a loopful of growth of a 48-hr-old culture onto 100 ml sterile distilled water. twenty agoho seeds treated with the antagonistic bacteria were sown in each inoculated germination plate. untreated seeds sown in an uninoculated germination plate served as the control. all seeds sown were disease-free and previously surface-sterilized. the plates were incubated at room temperature for 15 days. watering was regulated using sterile distilled water. seed germination and the occurrence of damping-off disease were regularly assessed. the experiment utilized a crd with three replications. 4 biological control of damping-off fungi of agoho f.a. dda pena et al. nursery experiment the promising antagonistic bacteria were further screened as seed treatment under nursery conditions. a newly prepared bacterial cell suspension corresponding to each treatment was used to coat the agoho seeds. this was done by soaking for at least 15 min. before sowing into a pot with naturally infested soil. untreated seeds sown in naturally infested soil served as the control. another treatment made use of untreated seeds sown in a soil medium subjected to sterilization in an autoclave for 1 hr at 15 psi for three consecutive days. there were 20 disease-free and previously surface-sterilized seeds sown in every pot. seed germination and the occurrence of damping-off disease were assessed. the percentage of survival of the seedlings was determined after three months when the study was terminated. the experiment utilized a crd with five replications. significant values were further subjected to dmrt. results and discussion in vitro assay of the antagonistic activity of the 100 bacterial isolates using the agar-plug technique revealed that only 18 inhibited two or more of the damping-off fungi of agoho (table 1). fusarium oxysporum was inhibited by 17 bacterial isolates, r. solani by 8 isolates, p. parasitica by 14 isolates, and p. debaryanum by 15 isolates. the unidentified damping-off fungi unk 1 and unk 2 were inhibited by 13 and 9 isolates, respectively. of those that showed inhibitory effects, 15 belonged to the genus bacillus while three were actinomycetes. further screening using the agar-diffusion method disclosed that 10 were promising antagonists with b. subiilis (code no. r060), bacillus sp. (code no. r071), and streptomyces sp. (code no. r086) as the consistent and most effective inhibitors (table 2). some of the strains listed for inhibitory effects gave values below that of the reference antibiotic mycostatin (200 ug/ml) while others did not produce any inhibition zone, thus there was no antimicrobial activity observed. effect of the three promising antagonistic bacteria on percent germination of agoho seeds inoculated with the damping-off fungi in the laboratory is shown in table 3. the control which was not inoculated with any of the six pathogens gave the highest seed germination of 81.67%. it was however, surpassed though not significantly different by seed treatment with bacillus sp. (code no. r071) against p. parasitica resulting to 83.33%. inoculation with any of the damping-off fungi and with no seed treatment with any antagonist gave the lowest percent germination. bacillussubtilis (code no. r060) however, was not able to antagonize 5 biotropia no. 7, 1994 the effect of p. debaryanum in this particular experiment. application or treatment with b. subtilis may not have been of optimum concentration to affect an increase in percent germination of seeds inoculated with p. debaryanum. this could also be due to the fact that this pathogenic fungus is considered very virulent (dickson 1956). under nursery conditions, the antagonists suppressed the growth of the damping-off fungi (table 4 and fig. 1). seed treatments gave percent germination values of 74, 69 and 78 for b. subtilis, bacillus sp., and streptomyces sp., respectively. seed coating with streptomyces sp. gave a comparable value with untreated seeds sown in sterilized soil (83 %). table 1. inhibitory effects of the bacterial isolates on growth of the damping-off fungi of agoho based on agar-plug techniquea codeb classificationc inhibitory effectd __________ number f. oxysporum r. solani p. parasitica p. debaryanum unk 1 unk 2 r013 b + + + + rom b + + + r059 b + + + + r060 b + + + + + + r063 b + + + + + r070 b + + + + r071 b + + + + + + r072 b + + + + r073 b + + + r074 b + + + + r075 b + + + r076 b + + + + r078 b + + r079 b + + r080 b + + + + r086 a + + + + + + r095 a + + + + + + r099 a + + + + total number of active isolates = 18 17 8 14 15 13 9 a results based on three replications b treatments with no antimicrobial activity against any of the six damping-off fungi are not listed c either belonging to the genus bacillus (b) or an actinomycete (a) d positive sign ( + ) means with inhibitory effect: negative sign (-) means no inhibitory effect 6 biological control of damping-off fungi of agoho f.a. dela pena et al. table 2. in vitro assay of the antagonistic activity of the different bacterial isolates against the damping-off fungi of agoho using agar-diffusion method inhibition zoneb (mean diameter in mm) f. oxysporum r. solani p. parasitica p. debaryanum unk 1 unk 2 control 0 f 0 d 0 d 0 e 0 d 0 e r013 0 f 0 d 0 d 0 e 11.97c 0 e r060 21.65c 29.63a 27.05a 9.31b 19.21a 17.85c r071 29.22b 19.09b 10.06a 11.16a 14.30b 34.67a r072 0 f 0 d 9.89c 0 e 0 d 0 e r074 0 f 10.71c 0 d 0 e 0 d 11.37d r07s 9.73e 11.80c 0 d 0 e 11.20c 10.21d r078 9.62e 0 d 0 d 0 e 0 d 0 a r079 0 f 12.58c 0 d 0 e 0 d 0 e r086 31.42a 27.69a 18.43b 0 e 14.56b 25.26b r095 9.75e 11.96c 9.24c 9.00c 0 d 11.04d m200 d 14.05d 12.64c 9.43c 8.67d 11.84c 11.96d a treatments with no antimicrobial activity against any of the six damping-off fungi are not listed b results based on three replications with three subreplications c means with the same letter within the column are not significantly different at 1 % level using dmrt d reference antibiotic mycostatin (200 ug/ml) table 3. effects of the promising antagonistic bacteria on percent germination of agoho inoculated with damping-off fungi under laboratory conditions treatments percent germination (means) f. oxysporum r. solani p. parasitica p. debaryanum unk 1 unk 2 control 91.67a 87.67a 81.67a 81.67a 81.67a 81.67a pathogen alone 18.33d 1.67b 38.33c 0 d 0 e 26.67d pathogen + r060 65.00b 68.33a 66.67b 0 d 71.67b 58.33b pathogen + r071 50.00c 66.67a 83.33a 63.33b 60.00c 41.67c pathogen + r086 75.00ab 76.67a 61.67b 30.33c 51.67d 71.67ab means with the same letter within the column are not significantly different at 1 % level using dmrt furthermore, table 4 and fig. 1 show that percent survival of seedlings was increased by seed treatment with values of 94, 85, and 95 for b. subtilis, bacillus sp. and streptomyces sp., respectively. these were significantly different with the control (62%). seed coating with b. subtilis and streptomyces sp. gave comparable values with untreated seeds sown in sterilized soil (99%). 7 treatmentsa biotropia no. 7, 1994 figure 1. effects of the three promising antagonistic bacteria on percent germination and survival (after three months) of agoho under nursery conditions. bars with the same fill marked with different letter(s) are not significantly different at the 5% level using dmrt 8 biological control of damping-off fungi of agoho f.a. dda pena et al. table 4. effects of the three promising antagonistic bacteria on percent germination and survival (after three months) of agoho under nursery conditions treatments germination survival a 57d 62c b 83a 99a c 74bc 94a d 69c 85b e 78ab 95a means followed by a common letter are not significantly different at 5% level by dmrt. legend: a untreated seeds sown in naturally infested soil b untreated seeds sown in sterilized soil c b. subtilis treated seeds sown in naturally infested soil d bacillus sp. treated seeds sown in naturally infested soil e streptomyces sp. treated seeds sown in naturally infested soil results of the assay methods revealed that a considerable number of bacillus sp. and actinomycetes could inhibit the growth of damping-off fungi in culture. the mycelium and spores of the pathogenic fungi may have been attacked and parasitized by the antagonists when they were simultaneously grown in culture media utilizing the agar-plug technique. there must have been a competitive interaction between the two microorganisms as supported by studies of henis and chet (1975). any essential requirement of microorganisms can serve as potentially possible basis for competition. competition for nutrients between the antagonist and germinating spores of the pathogenic fungus could have taken place. olsen and baker (1968) added that b. subtilis which developed along the mycelial wall of r. solani apparently obtained nutrients either from wall components or contents leaking from senescent fungal cells. in the agar-diffusion method, antibiosis which is an inhibitory effect exerted by an organism upon another organism through the production of antibiotic compounds may have likely occurred. the supernatant used in the assay demonstrated the possible role of antibiotics in biological control. moreover, most strains of b. subtilis or any other species of the genus bacillus and actinomycetes produce a wide variety of antibiotics which inhibit filamentous fungi. bacillus subtilis in particular, produces a subtilinlike antibiotic (henis and chet 1975) in addition to bulbiformin, mycosubtilin and bacillomycin (henis and inbar 1968) which have antifungal activity. some of the antibiotics produced by such strains may be extracellular, diffusable in solid agar, and could inhibit the growth of fungal pathogens. 9 1.pdf 10.pdf 11.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf 25 the quality of physic nut (jatropha curcas l.) r.l. worang et al.biotropia vol. 15 no. 1, 2008 : 25 36 the quality of physic nut (jatropha curcas l.) seeds packed in plastic material during storage rantje lilly worang1 okky setyawati dharmaputra2,3 rizal syarief4 and miftahudin2 1 faculty of mathematics and natural sciences, manado state university, tondano, north sulawesi, indonesia 2 department of biology, faculty of mathematics and natural sciences, bogor agricultural university, darmaga campus, bogor, indonesia 3 seameo biotrop, jl. raya tajur km. 6, po box 116, bogor, indonesia 4 department of food science and technology, faculty of agricultural technology, bogor agricultural university, darmaga campus, bogor, indonesia abstract the effect of storage duration on fungal population, moisture content, lipid and free fatty acid contents, lipase activity, viability and vigor of physic nut seeds was investigated. physic nut seeds with initial moisture content of 7.9% were stored in plastic bags under warehouse conditions. samples of physic nut were collected before storage, and subsequently after one to six months of storage. the results showed that the moisture contents increased after one month of storage, and became relatively constant up to six months of storage. the range of moisture contents (7.9 – 8.4%) was safe for storage of physic nut seeds. sixteen fungal species were isolated from physic nut seeds during six months of storage. fungal population decreased with the increase of storage duration. at the beginning of storage, most of the fungi that infected the seeds were classified as field fungi, such as colletotrichum sp., cladosporium spp., and fusarium spp.. their populations decreased with the increase of storage duration. after three months of storage, the existence of field fungi was generally replaced by storage fungi, such as aspergillus spp., and penicillium spp. dominate the population. lipid contents, viabilities and vigors decreased with the increase of storage duration, while free fatty acids and lipase activities increased. under uncontrolled conditions, physic nut seeds packed in plastic material can be stored up to one month for seeds to be planted, while it can be stored up to five months for producing oil. key words : storage duration, fungi, lipid, free fatty acid, lipase, viability, vigor, physic nut, jatropha curcas l. corresponding author : okky@biotrop.org 26 biotropia vol. 15 no. 1, 2008 introduction with the increasing demand of fuel products, and the decreasing of fossil fuel sources, the world prices of petrol and diesel are increasing, including in indonesia. it is assumed, that the fossil fuel reserve will be exhausted within 10 15 years in indonesia. hence alternative sources of energy for running our generators, automobiles, etc are being considered (hambali 2006). the possibility of obtaining oil from plant resources to be a supplement or replacement of fossil fuel has aroused a great interest. in several countries including in indonesia, non-edible vegetable oil can be used as biodiesel, which is a nontoxic and biodegradable oil. physic nut (jatropha curcas l.) seeds is known to be a good source for biodiesel. the oil contents in the seeds and in the kernels are 25 – 30% and 50 – 60%, respectively. the oil contains 21% saturated fatty acids and 79% unsaturated fatty acids (gubitz et al. 1997). other uses of physic nut extracted oil are for making soap, biopesticide, and medicinal substance for skin disease treatment. the physic nut oil has the requisite potential of providing a promising and commercially viable alternative to diesel oil, since it has desirable physicochemical and performance characteristics comparable to diesel. cars could be run with physic nut without requiring much change in design (hambali et al. 2006; warsiki et al. 2007) physic nut seeds can be used as seeds and oil producer. according to adikadarsih and hartono (2006) the use of physic nut seeds for seedlings should derive from the fruits which skin is yellow up to blackish yellow in colour, because they have high percentages of viability and vigor, i.e. 89 and 81 %, respectively. wanita and hartono (2006) reported that seeds originated from physic nut fruits which skin is yellow up to blackish yellow in colour produce seeds with highest oil content, i.e. 2830 %. to obtain good quality of physic nut seeds, some factors should be taken into consideration, i.e. the degree of fruit maturity, safe seed moisture content, appropriate container and storage condition, duration of storage, good viability and vigor of seeds. consequently, appropriate postharvest handling is needed. christensen and kaufmann (1969) reported that during storage seeds or grains could be infected by fungi which cause a decrease in viability, discolouration, various biochemical changes, heating and mustiness, loss in weight, and production of toxins when it is consumed may be injurious to human and domestic animals. in many cases, fungi infecting seeds are seed-borne pathogens. they play an important role in the transmission of numerous pathogenic species to seedlings as well as to the soil (chelkowski 1991). the objective of this study was to determine the effects of storage duration on fungal population, moisture content, lipid and free fatty acid contents, lipase activity, viability and vigor of physic nut seeds. 27 the quality of physic nut (jatropha curcas l.) r.l. worang et al. materials and methods origin of physic nut seeds physic nut seeds were obtained from fresh harvested fruits which fruit skin was yellow up to blackish yellow in colour. the fruits were obtained from plants (lampung accession) cultivated in loyang village, cikedung subdistrict, indramayu regency, west java in may 2007. fruit peeling and seed drying the fruit skins were peeled using a knife, then the seeds were air-dried on the selves in a shaded place up to moisture contents of about 8 %. packaging and storing of physic nut prior to packaging, damaged seeds were hand picked from the batch. sound seeds were packed in plastic packaging materials (2 kg/bag) under normal oxygen concentration. they were then placed randomly on wooden shelves and stored for one, two, three, four, five and six months ( june – november 2007) under warehouse conditions. the composition of the plastic packaging material was ny15/pe15/ lldpe70. according to dharmaputra et al. (2007) total fungal population of peanut kernels packed in three different types of plastic packaging materials (opp30/pe15/ lldpe80, ny15/pe15/lldpe80, and ny15/pe15/lldpe70 ) under low oxygen concentration were not significantly different. the characteristic of plastic packaging material is presented in table 1. the plastic packaging bag was produced by pt interkemas flexipack in tangerang, west java. table 1. the characteristics of plastic packaging material name of product code compositition thickness (mm) wvtr (water vapour transmission rate) (g/ m2/24 hrs) o2tr (oxygen transmission rate) (cc/m2/24 hrs) trl.vacuum bag 03 ny70 ny 15/pe 15/lldpe 70 0.0974 0.6340 1.8734 note : analyzed by test and calibration laboratory, institute for chemical and packaging, jakarta, indonesia opp : oriented polypropilene lldpe : linear low density polyethylene the experiment was arranged in completely randomized design.three replications (= bags) were used for each storage duration (including at the beginning of storage). each bag was used to pack physic nut seeds with different storage duration and 28 biotropia vol. 15 no. 1, 2008 replications. the ambient temperature and relative humidity of the storage room were recorded using a tinytag data logger. sampling method samples of physic nut seeds were collected from each bag before storage (at the beginning of storage) and subsequently every month thereafter until 6 months of storage. each sample was divided three times using a box divider to obtain working samples for moisture content, fungal population, lipid and free fatty acid contens, lipase activity, viability and vigor of seeds, and reserved sample. determination of moisture content, fungal population, lipid and free fatty acid contens, lipase activity, viability and vigor moisture contents of seeds (based on wet basis) were determined using oven method (ista 1999, with a slight modification). two replicates were used for each sample. fungi from each sample was isolated and enumerated using serial dilution method followed by pour plate method on dichloran 18% glycerol agar (dg18) (hocking and pitt 1980; pitt et al. 1992). fungal identification was conducted based on samson et al. (1996), pitt and hocking (1997) using czapek yeast extract agar (cya) and cya containing 20% sucrose (cy20s). lipid and free fatty acid contents were determined based on soxhlet and titration methods, respectively (aoac 1999). lipase activity was determined based on moore (1973, with a slight modification). viability (percentage of germination) of seeds from each sample was determined by growing 25 seeds in rectangular plastic containers containing sand (5 kg/container) 7 and 10 days after planting (dap) under green house conditions (prihandana and hendroko 2006). normal germination was observed 7 dap, while normal and abnormal germination were observed 10 dap. determination of seed vigor from each sample was conducted using rice straw paper method (sadjad 1994). vigor, less vigor, non-vigor, and death were observed 7 dap. statistical analysis standard anovas were used to analyse the results followed by duncan’s multiple range test at the 5% probability level. results and discussion moisture contents moisture content is the most important environmental factor that influence fungal growth in stored grains (christensen et al. 1992). storage duration gave very significant differences on the moisture contents of physic nut seeds. the moisture 29 the quality of physic nut (jatropha curcas l.) r.l. worang et al. contents of physic nut after one month of storage (8.41%) were higher and significantly different from those at the beginning of storage (7.89%). the moisture contents decreased after two, three, four, five, and six months of storage, i.e. 8.14, 8.12, 8.14, 8.06 and 8.16%, respectively (table 2). according to the directorate general of plantation crop (2006) the safe moisture content for storage of physic nut seeds was 7 – 9 %. the moisture content of grains is in equilibrium with the relative humidity of the storage. range of temperature and relative humidity are presented in table 3. the moisture content of the seeds was correlated with the relative humidity of the storage. the type of plastic packaging material also affects the moisture contents of physic nut seeds. plastic packaging bags used in this study are made from good material for storing peanut kernels (dharmaputra et al. 2007). according to warsiki et al. (2007) the moisture contents of physic nut seeds packed in bags were higher than those packed in polypropylene bags. table 2. moisture content, total fungal population, lipid and free fatty acid contents, lipase activity, viability and vigor of physic nut seeds during storage duration of storage (month) moisture content (% wet basis) total fungal population (cfu/g dry basis) lipid content (% dry basis) free fatty acid content (% dry basis) lipase activity (u/ml) viability (%) vigor (%) 0 7.89 a ± 0.07 8589.0 a ± 1359.3 41.2 a ± 0.34 0.10 a ± 0.01 0.23 a ± 0.08 89.33 a ± 8.33 83.33 a ± 5.77 1 8.41 d ± 0.02 6519.6 a ± 3224.5 37.9 b ± 0.69 0.60 b ± 0.01 0.35 ab ± 0.03 74.67 b ± 4.62 70.00 b ± 0.00 2 8.14 c ± 0.04 5917.7 a ± 1026.8 37.2 bc ± 0.66 0.62 b ± 0.01 0.42 bc ± 0.04 71.33 bc ± 1.15 63.33 bc ± 5.77 3 8.12 bc ± 0.02 1405.4 b ± 1196.7 35.9 cd ± 0.56 0.67 bc ± 0.03 0.51 cd ± 0.05 68.00 bc ± 2.00 56.67 c ± 5.77 4 8.14 c ± 0.03 1297.3 b ± 377.2 35.1 de ± 0.54 0.79 c ± 0.06 0.58 d ± 0.01 64.67 cd ± 2.31 36.67 d ± 5.77 5 8.06 b ± 0.02 1145.6 b ± 761.9 33.5 ef ± 0.86 1.00 d ± 0.09 0.71 e ± 0.07 60.00 de ± 3.46 33.33 de ± 5.77 6 8.16 c ± 0.03 225.4 b ± 28.4 31.7 f ± 2.06 1.10 d ± 0.14 0.93 f ± 0.15 53.33 e ± 2.31 26.67 e ± 5.77 note : means followed by the same letter in the same column are not significantly different according to duncan’s multiple range test at the 5% level (p > 0.05). 30 biotropia vol. 15 no. 1, 2008 table 3. range of temperature and relative humidity during storage duration of storage (month) temperature (oc) relative humidity (%) range mean range mean 1 24.4 28.3 26.3 55.2 – 82.8 74.1 2 25.1 -28.7 26.5 48.2 -70.2 70.2 3 25.1 – 29.1 26.5 55.2 – 78.5 69.4 4 25.1 – 28.3 26.7 46.0 – 80.7 68.1 5 25.1 – 28.7 26.7 55.6 – 82.4 73.1 6 24.4 – 28.3 26.1 65.6 – 85.4 76.5 according to desai (2004) modern packaging materials and methods maintain seeds at their original quality from the time of their packaging to the time they are used for planting. the best way to maintain the viability and vigor of many kinds of seeds is to store them in a dry and cold place. packages designed to protect most physical qualities of seeds, such as weight, size, colour, moisture content, and purity (freedom from weeds, inert matter, disease organisms and damage), as well as their physiological aspects, like viability, vigor and dormancy, are made up of materials that have sufficient tensile strength, bursting strength, and tearing resistance to withstand normal pressures and handling procedures. polyethylene is the most extensively used thermoplastic film. conventional low-density films are generally used in seed packages. this packaging material is ideal for providing seale-storage conditions in a humid climate, giving protection against high humidity in a seed store. fungal population sixteen fungal spesies were isolated during six months of storage (table 4). they were aspergillus flavus, a. niger, a. ochraceus, a. penicillioides, a. restrictus, a. tamarii, a.wentii, cladosporium sp., c. cladosporioides, colletotricum sp., eurotium chevalieri, e .rubrum, fusarium moniliforme, f. semitectum, penicillium citrinum, and p. oxalicum. aspergillus flavus, a. niger, cladosporium sp., f. moniliforme, f. semitectum, and p. citrinum were always isolated during storage. table 4. fungal population of physic nut seeds during storage no. fungi fungal population (cfu/g dry basis) duration of storage (month) 0 1 2 3 4 5 6 1 aspergillus flavus 6.5 1.1 6.5 88.2 8.7 17.4 9.8 2 a. niger 3.3 2.2 4.4 3.3 1.1 2.2 3.3 3 a. ochraceus 0.0 0.0 1.1 0.0 0.0 0.0 0.0 4 a. penicillioides 0.0 0.0 0.0 0.0 0.0 730.9 92.6 5 a. restrictus 0.0 50.2 61.0 0.0 695.6 0.0 0.0 31 the quality of physic nut (jatropha curcas l.) r.l. worang et al. no. fungi fungal population (cfu/g dry basis) duration of storage (month) 0 1 2 3 4 5 6 6 a. tamarii 2.2 1.1 2.2 0.0 1.1 1.1 1.1 7 a. wentii 1.1 1.1 2.2 4.4 2.2 0.0 0.0 8 cladosporium sp. 427.7 462.9 90.4 37.0 20.7 44.6 17.4 9 c. cladosporioides 169.4 41.5 47.9 21.8 0.0 0.0 15.2 10 colletotrichum sp. 5609.6 1880.1 1415.2 68.6 61.0 0.0 0.0 11 eurotium chevalieri 0.0 0.0 0.0 1.1 0.0 0.0 2.2 12 e. rubrum 0.0 0.0 0.0 2.2 0.0 63.1 3.3 13 fusarium moniliforme 1652.4 3703.5 580.2 1007.8 409.3 156.6 15.2 14 f. semitectum 247.5 10.9 209.0 14.1 3.3 6.5 3.3 15 penicillium citrinum 276.8 291.5 3496.6 156.7 94.7 124.0 63.2 16 p. oxalicum 193.2 73.2 0.0 0.0 0.0 0.0 0.0 total 8589.7 6519.3 5916.6 1405.1 1297.6 1146.4 226.5 duration of storage gave very significant differences on total fungal population. total fungal population after one, two, three, four, five and six months of storage did not coincide with moisture contents at the same months (table 2 ). total fungal population decreased with the increase of storage duration. total fungal population at the beginning of storage (8589.0 cfu/g.db) was higher and significantly different from that after six months of storage (225.4 cfu/g.db). the highest total fungal population was found at the beginning of storage, because the high population of field fungi, such as cladosporium spp., colletotricum sp., f. moniliforme, and f. semitectum . cladosporium spp. produces abundant conidia. storage fungi found in physic nut seeds were species of aspergillus and penicillium. based on their occurrence in the seeds, christensen and kaufmann (1969) divided two groups of fungi that cause deterioration of seeds, i.e. field fungi and storage fungi. it was assumed that the decrease of fungal population during storage was due to the low of moisture contents in the seeds after one month up to six months of storage. in general the dominant fungal species during storage was field fungi. according to sauer et al. (1992) fields fungi have high water requirements for their growth, compared to storage fungi. it was also assumed that the decrease of total fungal population was due to the decrease of oxygen during storage. according to moore-landecker (1996) most fungi are obligate aerobes and require at least some free molecular oxygen in the atmosphere. physic nut seeds also need o2 found in plastic packaging bags for their life. garraway and evans (1984) concluded that the type of response by fungi to oxygen concentration depends on the species. table 4. continued 32 biotropia vol. 15 no. 1, 2008 lipid contents storage duration gave very significant differences on lipid contents of physic nut seeds. the lipid contents decreased with the increase of storage duration (table 2). after one month of storage, lipid contents (37.9% db) were lower and significantly different from those at the beginning of storage (41.2% db). after six months of storage, lipid contents (31.7% db) were lower and significantly different from those at the beginning of storage (41.2% db), after one (37.9% db), two (37.2%db), three (35.9% db), and four (35.1% db) months of storage. physic nut seeds contain high lipid content. the lipid contents in the seeds and in the kernels are 25 – 30% and 50 – 60%, respectively. the lipid contains 21% saturated fatty acids and 79% unsaturated fatty acids (gubitz et al. 1997). lipid contents of physic nut used in this study were high (37.95%), because the seeds were derived from fruits which fruit skin is yellow up to blackish yellow. although after five months of storage lipid contents (33.5% db) were significantly different from those at the beginning of storage (41.2% db), they were still able to be used for producing oil. lipid contents are affected by the degree of fruit maturities at the time of harvesting. wanita and hartono (2006) reported that the lowest (10.93 %) and the highest (29.38 %) lipid contents were found in physic nut seeds derived from fruits which fruit skins are green and yellow, respectively. free fatty acid contents and lipase activity due to hydrolysis process, fat and lipid in seeds are broken down into free fatty acids (ffa) and glycerol by lipases. rapid increases in concentrations of ffa frequently are observed in deteriorating seeds, particularly when the temperature and moisture content are high. many field and storage fungi produce lipases. seeds also produce lipases, but these commonly are produced during germination, and are not present in stored seeds (pomeranz 1992). storage duration gave very significant differences on ffa contents of physic nut seeds. ffa contents increased with the increase of storage duration (table 2). after one month of storage, ffa contents (0.60% db) was higher and significantly different from those at the beginning of storage (0.10 db %). after five (1.00 % db) and six months of storage (1.10 % db) ffa contents were higher and significantly differents from those at the beginning of storage (0.10% db), after one (0.60% db), two (0.62 % db), three (0.67% db) , and four months of storage (0.79% db). according to st. angelo and ory (1983) lipase and lipoksigenase are important enzymes to degrade lipid in seeds. storage duration gave very significant differences on lipase activity of physic nut seeds. lipase activity increased with the increase of storage duration (table 2). after two months of storage, lipase activity (0.42 u/ml) was higher and significantly different from those at beginning of storage (0.23 u/ml). after five and six months of storage lipase activity (0.71 and 0.93 u/ml, respectively) were higher and significantly different 33 the quality of physic nut (jatropha curcas l.) r.l. worang et al. from those at the beginning of storage. after six months of storage, lipase activity was higher and significantly different from those after five months of storage. as the moisture contents were considered low (table 2) and the total fungal population decreased (table 4) during storage, the lipase activity could be due to seed enzymes rather than fungal enzymes. seed viability and vigor according to pomeranz (1992) fungal infection can decrease seed viability and vigor of seeds during storage. priestley (1986) reported that the most obvious, though not the most informative, indication of the quality of a seed lot is its germinability. the speed of germination, which has long been recognized as an indicator of seed vigor, is usually a more sensitive measure of seed deterioration than loss of viability. storage duration gave very significant differences on the percentages of germination and vigor. the percentages of germination and vigor decreased with the increase of storage duration (table 2). the percentages of germination after one month of storage (74.67%) were lower and significantly different from those at the beginning of storage (89.33%). directorate general of estate crops (2006) determined the germination of physic nut seeds to be planted should be more than 80%. after one month of storage, the percentage of germination (74.67%) was significantly different from those at the beginning of storage (89.33%). nevertheless, based on statistical analyses, the percentage of germination after one month of storage was 74.67 ± 4.62%. consequently, under uncontrolled conditions, physic nut seeds packed in plastic material can be stored up to one month for seeds to be planted. after six months of storage, the percentages of germination (53.33 %) were lower and significantly different from those at the beginning of storage (89.33). according to copeland and mc donald (2001) seeds containing high lipid content decrease their viability faster compared to those containing high carbohydrate content. the seed germination percentage in this study was more than 80 %, because the seeds were derived from fruits which fruit skin is yellow up to blackish yellow in colour as required for good quality of physic nut seeds. the percentages of vigor after one month of storage (70.00%) were lower and significantly different from those at the beginning of storage (83.33%). after six months of storage, the percentages of vigor (26.67%) were lower and significantly different from those at the beginning of storage (83.33 %), after one (70.00%), two (63.33 %), three (56.67%) and four months (36.67%) of storage. according to adikadarsih and hartono (2006) the lowest percentages of germination (7 %) and vigor (4.33 %) were found in physic nut seeds derived from fruits which fruit skin is green in color ; while the highest percentage of germination (91.67 %) and vigor (84.67 %) were found in seeds derived from fruits with fruit skin is yellow in color. 34 biotropia vol. 15 no. 1, 2008 under conditions that favour them to grow, storage fungi invade the germs or embryos of seeds prefentially, and sometimes exclusively. very often, and especially if the moisture content of the seeds is at or just slightly above the lower limit that allow a given species of storage fungi to grow, the germs may be invaded to the point of near decay, with no evidence of molding being evident outwardly, even with microscopic examination, and little or no invasion of the endosperm immediately adjacent to the germ. the first effect of this invasion is weakening of the germ, followed by death (sauer et al. 1992). conclusions duration of storage affected the quality of physic nut seeds. after one month of storage, the moisture content of physic nut seeds increased, and became relatively constant up to six months of storage. during storage the moisture contents were still safe for storing physic nut seeds (7.89 – 8.41%). sixteen mould species were isolated from physic nut seeds during six months of storage. fungal population decreased with the increase of storage duration. at the beginning of storage most of fungi infected the seeds were field fungi (colletotrichum sp., cladosporium spp., and fusarium spp.). their populations decreased with the increase of storage duration and replaced by storage fungi (penicillium spp. and aspergillus spp.). lipid contents, viabilities and vigors decreased with the increase of storage duration, while free fatty acids and lipase activities increased. under uncontrolled conditions, physic nut seeds packed in plastic material can be stored up to one month for seeds to be planted, while for producing oil, they can be stored up to five months. acknowledgements the authors gratefully acknowledge dr. theresia prawitasari† for her suggestions and encouregement. we also thank to the staff member of plant pathology and service laboratory, seameo biotrop, who have in one way or another contributed to this research. references adikadarsih, s. & j. hartono. 2006. pengaruh kemasan buah terhadap mutu benih jarak pagar (jatropha curcas l). paper presented at lokakarya ii: status teknologi tanaman jarak pagar. pusat penelitian dan pengembangan perkebunan, departemen pertanian. bogor, 29 november 2006. 35 the quality of physic nut (jatropha curcas l.) r.l. worang et al. aoac. 1999. in horwits w (ed). vol. ii. official methods of analysis of food composition; additives; natural contaminants. 16th ed. association of official analytical chemist, gaithersburg. chelkowski, j. 1991. fungal pathogens influencing cereal seed quality at harvest. in chelkowski, j. (ed.). pp. 53 – 66. cereal grain; mycotoxins, fungi and quality in drying and storage. elsevier, amsterdam. christensen, c.m. and h.h. kaufmann. 1969. grain storage: the role of fungi in quality loss. university of minnesota press, minneapolis. christensen, c.m., b.s. miller and j.a. johnston. 1992. moisture and its measurement. in sauer, d.b. (ed.). pp. 39 – 54. storage of cereal grains and their products. american association of cereal chemists. inc., st. paul. copeland, l.o. and m.b. mc donald. 2001. principles of seed science and technology. 4th ed. kluwer academic publishers, boston. desai, b.b. 2004. seeds handbook; biology, production, processing, and storage. 2nd ed. marcel dekker, inc., new york. dirjenbun. 2006. pedoman mutu benih jarak pagar sistem dan prosedur pembangunan sumber benih dan peredaran benih jarak pagar. direktorat jenderal perkebunan, jakarta. dharmaputra, o.s., i. retnowati and s. ambarwati. 2007. three different types of plastic packaging materials: their effects on mould infection and aflatoxin contamination. biotropia 14(1): 9 – 23. garraway, m.o. and r.c. evans . 1984. fungal nutrition and physiology. john willey & sons, new york. gubitz, g.m, m. mittelbach and m. trabi. 1999. exploitation of the tropical oil seed plant jatropha curcas l. bioresource technology 67 : 73-82. hambali, e. 2006. proses pengembangan tanaman jarak pagar untuk biodisel dan produk turunan lainnya. paper presented at workshop pendirian kebun bibit sumber, demplot dan feasibility study untuk perkebunan jarak pagar. bogor, 16-17 may 2006. sbrc, lppm-ipb. hambali, e. et al. 2006. jarak pagar tanaman penghasil biodisel. penebar swadaya, jakarta. hocking, a.d. and j.i. pitt. 1980. dichloran-glycerol medium for enumeration of xerophilic fungi from low-moisture foods. applied environmental microbiology 39 : 488-492. ista. 1999. international rules for seed testing. proceedings international seed testing association 31 (1), wageningen. moore, t.c. 1973. research experiences in plant physiology. a laboratory manual. sprigerverlag, new york. moore – landecker, e. 1996. fundamental of the fungi. 4th ed. prentice – hall, inc, new jersey. pitt, j.i., a.d. hocking, r.a. samson and a.d. king.1992. recommended methods for mycological examination of foods. in samson, r.a., a.d. hocking, j.i. pitt and a.d. king, eds. pp. 365-368. modern methods in food mycology. elsevier, amsterdam. 36 biotropia vol. 15 no. 1, 2008 pitt, j.i. and a.d. hocking. 1997. fungi and food spoilage. blackie academic & professional, london. pomeranz, y. 1992. biochemical, functional and nutritive changes during storage. in sauer, d.b. (ed.). pp. 55 – 141. storage of cereal grains and their products. 4th ed. american association of cereal chemists, inc., st paul. priestley, d.a. 1986. seed aging; implications for seed storage and persistence in the soil. comstock publishing associates, cornell university press, ithaca. prihandana, r. and r. hendroko. 2006. petunjuk budidaya jarak pagar. pt agromedia pustaka, jakarta. sadjad, s. 1994. kuantifikasi metabolisme benih. pt gramedia widiasarana indonesia, jakarta. samson, r.a., e.s. hoeksttra, j.c. frisvad and o. filtenborg. 1996. introduction to food-borne fungi. 3th ed. centraalbureau voor schimmelcultures, baarn. sauer, d.b., r.a. meronuck and c.m. christensen. 1992. microflora. in sauer, d.b. (ed.). pp. 313 – 340. storage of cereal grains and their products. 4th ed. american association of cereal chemists, minnesota. st. angelo a.j. and r.l. ory. 1983. lipid degradation during seed deterioration. phytopathology 73: 315. wanita, y.p. and j. hartono. 2006. pengaruh tingkat kemasakan buah terhadap kadar mi nyak jarak pagar (jatropha curcas l.) (the effect of degree of fruit maturity on oil content of jatropha curcas l). paper presented at lokakarya ii: status teknologi tanaman jarak pagar. pusat penelitian dan pengembangan perkebunan, departemen pertanian. bogor, 29 november 2006. warsiki, e., d. sumangat and w rismawati. 2007. pengaruh bahan dan kondisi pengemasan terhadap mutu biji jarak pagar. paper presented at konferensi jarak pagar menuju bisnis jarak pagar yang feasible. sbrc, lppm, institut pertanian bogor, bogor, 19 june 2007. result and discussion production and utilization of cellulase from biotropia no. 25, 2005 : 50 – 59 trichoderma viride r. hidayat1, s. wulandari2, k.g. wiryawan3 and suryahadi3 1department of animal nutrition and feed science, padjadjaran university, bandung, indonesia. 2faculty of animal science, jember agricultural polytechnique, jember, indonesia. 3department of animal nutrition and feed science, bogor agricultural university, bogor, indonesia. abstract an appropriate preservation technology for forage such as silage needs to be developed in order to overcome the shortage of tropical forage during the dry season. a good quality of silage is obtained by decreasing the fibre contents (neutral detergent fibre, acid detergent fibre and lignin). the research was conducted in two stages: 1). production and activity test of crude enzymes from trichoderma viride and 2). comparative test using crude enzymes from trichoderma viride strain qm 9414 (cetv) and commercial cellulase (cellulase “onozuka r-10”, heidelberg) on rice straw silage. the treatments were arranged as follows : p0=untreated rice straw silage, p1=treated with commercial cellulase at 10 iu kg-1, p2= treated with cetv at 3.8 iu kg-1, p3=treated with cetv at 7.6 iu kg-1 and p4= treated with cetv at 11.4 iu kg-1 of fresh rice straw. all treatments were enriched with 5% molasses. the result of the research indicated that: 1). the activity of crude enzymes from trichoderma viride strain qm 9414 (cetv) was 1.52 iuml-1. 2). the addition of both enzymes did not decrease dry matter of rice straw silage; however, organic matter significantly decreased (p<0.05) by addition of commercial cellulase. 3). the addition of both enzymes decreased fibre contents (ndf), furthermore increased (p<0.05) in sacco digestibility of dry matter and organic matter of rice straw silage. keywords : cellulase/trichoderma viride/rice straw/silage/fibre introduction the main problem of improving ruminant production in indonesia is the shortage of high quality forage during dry season causing the animal production become inefficient, because of an increase in feed cost. on the other hand, the forage is abundant during the wet season. application of forage preservation technology in the farm may solve the problem, as the forage becomes more available all the year. there are two forage preservation techniques namely dry preservation (hay) and wet preservation (silage). for some matters, ensiling has many advantages, as forage quality may be maintained for a few months. however, the silage production process still has weakness, as the dry matter decreases and the fibre content of the forage is not influenced by the process. rice straw is one of the agricultural by-products which is a very potential source of energy for ruminants. availability of rice straw increases as the rice straw silage production increases. rice straw is usually abundant at the same time as the 50 period of high forage production occurring in the wet season. farmers choose forage as feed rather than rice straw. farmers store rice straw to be used during dry season when forage production is low. rice straw as feed has been used generally in the tropic and sub-tropical areas (jackson 1977), especially in the hot season. in indonesia, rice straw has been used for feed at least 31-39%, but most of it was burnt or used as fertilizer (36-62%) and the rest is used for industries (komar 1984). biotropia no. 25, 2005 rice straw has low nutritive values. rice straw contains 80% organic material which can be potentially digested; but in reality, it can be digested by ruminants only 45-50% (lubis 1963; jackson 1977). according to doyle et al. (1986), rice straw nutrient content is generally low consisting of crude protein 2.2-9.5%, hemicelluloses 21-29%, cellulose 35-49%, with coefficient digestibility value of organic materials 31-59%, and lignin 4-8%. dry storage condition (hay) makes the quality of rice straw becomes worse, therefore, a better method is needed to store the rice straw. the present techniques in silage production are focused mainly on feed stuff preservation rather than improvement of its quality. it needs an effort to improve the process, and ensiling may be improved by both preservation and increase of the quality of forage. addition of enzymes as an active substance may disperse cell wall (ndf and adf compounds) (stokes and chen 1994), increase digestibility and release the soluble carbohydrate portion of the forage. enzymatic pre-hydrolysis of forage component may promote bacterial growth, enhance lactic acid production and decrease ph of silage (bolsen and sapienza 1993). the present experiment is focused on application of commercial cellulase enzyme and crude enzyme produced by trichoderma viride to improve nutritive value of rice straw. materials and methods materials the materials used in this experiment were: rice straw (cultivar ir 64), a commercial cellulase (onozuka r-10 from trichoderma viride with activity 1.0 umg-1 produced by serva electrophoresis gmbh d-69115 heidelberg carl-benzstr.7) and trichoderma viride strain qm 9414. growth media for trichoderma viride growth media composition that were used as substrates for trichoderma viride to produce raw enzymes were : media i, one litre media contains : 5 g nh4no3, 0.5g kcl, 0.01g feso4. 7h2o, 0.5g mgso4.7h2o, 0.001g cuso4.2h2o, 1g yeast extract and 6g pepton ,the ph of media was 5.45. media ii, one litre media contains : 5g nh4no3, 0.5g kcl, 0.01g feso4 .7h2o, 0.5g mgso4.7h2o, 0.001g cuso4.2h2o, 1g yeast extract and 15g rice straw. (montesqrit 1998). 51 experimental design production and utilization of cellulose from trichoderma viride – r. hidayat et al. the research was conducted in two stages as follows: stage i: production and activity test of crude enzymes from trichoderma viride. one loop of trichoderma viride was cultured in a reaction tube containing 3-5 ml media i for four days and then it was transferred to an erlenmeyer glass containing 250 ml media ii and incubated for 14 days with shaking. the crude enzymes were obtained by centrifugation at 4000 rpm, at 50 c for 30 minutes and the supernatant was used as an additive in the production of rice straw silage. stage ii: a comparative test using crude enzymes from trichoderma viride strain qm 9414 (cetv) and a commercial cellulase (cellulase “onozuka r-10”, heidel-berg) on rice straw silage. the experiment used completely randomized design with 5 replicates. a total of 25 silos with 1 kg per silo of fresh material were prepared. the treatments were based on enzyme activity and arranged as follows : p0=untreated rice straw silage, p1=treated with commercial cellulase at 10 iu kg-1, p2= treated with cetv at 3.8 iu kg-1, p3=treated with cetv at 7.6 iu kg-1 and p4= treated with cetv at 11.4 iu kg-1 of fresh rice straw. all treatments were enriched with 5% molasses to provide ready available carbohydrate (rac) as a stimulant fermentation. the ph before ensiling was normal. silage preparation the rice straw silage was made as many as 1 kg for each sample. the ensiling procedure is as follows: the rice straw was chopped into pieces of ± 5 cm length. the fresh and chopped rice straw was mixed with either commercial enzymes or crude enzymes from trichoderma viride (spraying). the mixture was pressed and compacted by hand, and placed into a silo (glass jar). then the silo was closed tightly so that the condition became free from o2, air and waterproof. chemical analysis silage samples were collected for chemical analysis on the 30th day of ensiling. the physical characteristics; colour, presence of fungi and smell of the silage were studied. hence, the following parameters were measured : dry matter (dm), organic matter (om), crude protein (cp) according to proximate analysis, ph (ph-beckman model φ 40). ndf, adf and lignin content were analysed according to van soest and wine (1967). lactic acid was determined with an hplc technique (shimazu, japan 3081-09202-20atd-e). dm and om digestibility were determined by nylon bag technique (in sacco) in a fistulated buffalo. the size of nylon bag is 140 x 90 mm. one gram sample of silage was placed into nylon bag and incubated in the rumen for 24 hours. one week before and during the experiment, the buffalo was fed on 40 kg elephant grass and 5 kg rice bran per day. 52 statistical analysis biotropia no. 25, 2005 the data were subjected to analysis of variance (anova). when the ftest was significant (p<0.05), the contrast orthogonal test for paired comparisons was used. results and discussions production and activity test of crude enzymes from trichoderma viride on medium i, fungal growth was visible at the second day of cultivation. on medium ii, fungal growth was detected at the second day which was shown by the mycelial growth in the media after transferred into erlenmeyer glass. on the fourth day, the growth of mycelium was maximum and it was harvested. from 25 glasses of erlenmeyer, 21 of them were harvested and others were thrown away because they were contaminated by unexpected micro-organisms or had imperfect growth. the cellulase activity of crude enzymes from trichoderma viride (cetv) was 1.52 iuml-1. this result was higher than previous results (montesqrit 1998) by the same procedure, which showed that the cmc-ase activity from t. viride fermented for 14 days was 0.673 iuml-1. this was caused by a different strain of t. viride used, while the present experiment used t. viride strain qm 9414. comparative test using crude enzymes from trichoderma viride (cetv) and commercial cellulase on rice straw silage. the influence of treatments on the physical quality of rice straw silage generally, rice straw silage had the same physical characters, either in colour, smell, texture or the presence of fungi. rice straw silage had the same colour that was yellow brownish and moulds were seen on the top of the silage. the colour of silage was not different from the fresh rice straw. the silage had good smell, fresh and crumb texture for all treatments. it showed that the fermentation had taken place by producing lactic acid. the utilization of dry rice straw as silage material also had positive impact on silage quality i.e. the silage liquid was little and there was not much soluble nutrient wasted into silage liquid. other reports indicated that making silage in wet condition produced much liquid (mcdonald et al.1991), so dryer condition was needed to make easy handling and produced higher and drier silage. 53 the influence of treatments on the chemical quality of rice straw production and utilization of cellulose from trichoderma viride – r. hidayat et al. 1. nutrients content of rice straw silage table 1 presents the nutrients content of rice straw silage. the dry matter content of rice straw silage treated with enzymes (p1, p2, p3 and p4) was not significantly different from dry matter of untreated rice straw silage. this result was different from rice straw directly treated by t. viride in which it caused degradation of rice straw dry matter 8.4% (soetjiharto 1997). however, dry matter of rice straw silage treated by commercial enzymes was significantly (p<0.05) reduced compared to that of silage treated by cetv. table 1. nutrient content of rice straw silage treatments nutrients p0 p1 p2 p3 p4 dry matter (%) 24.3±0.4 23.9±0.6 24.6±0.1 24.6±0.2 24.9±0.6 organic matter (%) 79.1±0.2 79.2±0.2 79.2±0.3 79.6±0.1 79.5±0.4 crude protein (%) 5.9±0.5 5.8±0.3 5.4±0.2 5.3±0.3 5.6±0.3 p0=untreated rice straw silage, p1=treated with commercial cellulase at 10 iu kg-1, p2= treated with cetv at 3.8 iu kg-1, p3=treated with cetv at 7.6 iu kg-1 and p4= treated with cetv at 11.4 iu kg-1 of fresh rice straw. organic matter content of rice straw silage treated with enzymes (p1, p2, p3 and p4) was 79.4 %, and higher (p<0.05) compared with control (p0=79.1 %). this result was different from direct microbiological treatment as stated by djunaidi (1988) that organic matter content of rice straw silage put on the ground was descending. organic matter content of rice straw silage treated with commercial enzymes (79.2 %) was not significantly different from organic matter of rice straw silage treated with cetv (p2, p3, and p4 = 79.4%). it was different from stokes and chen (1994) observation that the increase of commercial cellulase addition to corn silage decreased the silage organic matter content. crude protein of rice straw silage treated with enzymes (p1, p2, p3 and p4) was 5.5 % and significantly lower (p<0.05) than crude protein of untreated rice straw silage (p0=5.9%). this was caused by cetv which still contained protease, so that some crude protein of rice straw was degraded to become ammonia. a different result was reported by stokes and chen (1994) in which crude protein content of corn silage treated with commercial cellulase was increased. this was caused by microorganism growth as source of protein. 2. fibre content of rice straw silage the effects of addition of commercial cellulose and cetv to fibre content of rice straw silage are presented in table 2. the neutral detergent fibre (ndf) 54 content of rice straw silage treated with enzymes (p1, p2 ,p3 and p4) was 72.3 %, and significantly lower (p<0.05) compared to untreated silage (p0=79.0%). reduction of ndf content of rice straw silage treated with cetv followed the equation of y= 77.75-1.37x. corn silage treated with cellulase had lower ndf content (46.7%) compared to untreated corn silage (53.1 %) (stokes and chen 1994). biotropia no. 25, 2005 there is no significant difference (p<0.05) between acid detergent fibre (adf) content of rice straw silage treated with enzymes (p1, p2 ,p3 and p4=56.2 %) and control (p0=59.5%), but the adf content of rice straw silage treated with enzyme tended to be lower (p<0.05) than adf content of untreated rice straw. enzyme treatment decreased adf content of rice straw silage about 5.6% from control. table 2. fibre content of rice straw silage treatments p0 p1 p2 p3 p4 significance ndf (%) 79.0±3.4 76.4±1.2 74.2±2.7 71.2±3.1 67.4±2.9 p < 0,05 adf (%) 59.5±4.4 59.2±2.9 57.6±7.6 53.6±2.7 54.2±2.1 ns lignin (%) 7.7±.0.8 8.4±.1.7 6.6±.1.7 6.2±.1.5 5.7±.0.5 ns ndf= neutral detergent fibre, adf=acid detergent fibre, p0=untreated rice straw silage, p1=treated with commercial cellulase at 10 iu kg-1, p2= treated with cetv at 3.8 iu kg-1, p3=treated with cetv at 7.6 iu kg-1 and p4= treated with cetv at 11.4 iu kg-1 of fresh rice straw. the acid detergent fibre (adf) content of rice straw silage treated with cetv (p2, p3, and p4) was 55.1 %, and it was not significantly different from adf content of rice straw silage treated with commercial enzyme (p1=59.2%). this result was different from that reported by stokes and chen (1994), that adf content of corn silage treated by commercial enzyme decreased about 11 – 13 %. it appears that adf content of rice straw silage treated with cetv was lower compared with commercial enzyme treatment. this means that cetv had higher potential than commercial cellulase in degrading adf of rice straw silage. rice straw is difficult to be degraded because rice crop is harvested at mature stage with high cell wall content and perfect lignification level. the lignin content of rice straw silage treated with enzymes (p1,p2,p3 and p4= 6.6%) was not different from control (7.7%). however, the content of rice straw silage lignin treated by commercial enzyme (p1=8.4%) was significantly higher compared with that treated by cetv (6.2 %). the decrease in lignin content by cetv treatment might be caused by: crude enzymes cetv which possess many enzymatic activities including lignase, and production of organic acid during ensilage. 55 lignin can be depolymerised by microbial enzymes produced by trichoderma sp. (paterson 1986). on the other hand, commercial enzyme is purely cellulase enzyme that has no lignase activities, so increasing the level of commercial enzyme did not affect the lignin content of rice straw silage. production and utilization of cellulose from trichoderma viride – r. hidayat et al. 3. ph and lactic acid production of rice straw silage there were no differences between ph of rice straw silage treated with enzyme (p1, p2, p3 and p4 = 3.72) than that of control (p0 = 3.72). this result is in line with stokes and chen (1994) who reported that enzyme application has no effect to corn silage ph. nevertheless, rice straw silage ph treated with commercial enzyme (p1 = 3.68) had significantly lower (p<0.05) ph than rice straw silage treated by cetv (3.73). the effect on the addition of commercial cellulase and cetv to ph and lactic acid production of rice straw silage is presented in table 3. table 3. ph and lactic acid production of rice straw silage treatments p0 p1 p2 p3 p4 significance lactic acid (%) 0.23±0.05 0.35±0.04 0.27±0.01 0.26±0.03 0.25±0.04 p < 0.05 ph 3.72±0.03 3.68±0.02 3.71±0.01 3.75±0.01 3.74±0.02 ns p0=untreated rice straw silage, p1=treated with commercial cellulase at 10 iu kg-1, p2= treated with cetv at 3.8 iu kg-1, p3=treated with cetv at 7.6 iu kg-1 and p4= treated with cetv at 11.4 iu kg-1 of fresh rice straw. ph of rice straw silage treated with commercial enzyme had the lowest ph. this was related to silage lactic acid production, where silage treated with commercial enzyme had the highest concentration of lactic acid (0.35 %). rice straw silage ph treated with cetv tended to increase as the enzyme levels were increased. this might be due to a carry-over of some buffers from the media of trichoderma viride during centrifugation for enzyme preparation. in addition, protease activity of cetv could degrade protein into ammonia. the increase in ph of rice straw silage treated with cetv follows the linear equation of y=3.696+ 0.0072x. in general, rice straw silage ph was very good, i.e. under 4. this means that rice straw silage can be stored in silos for a long time. good silage had ph between 3.5 and 4.0 (aak 1983). lactic acid production of rice straw silage treated with enzyme (p1, p2, p3 and p4) was 0.29 % higher than the control (p0=0.25 %). addition of enzyme could improve lactic acid production of rice straw silage. low moisture content probably influenced the relative growth of homofermentative and heterofermentative lactic acid bacteria during ensiling, which in turn lowered the ph of silage, minimizing 56 deterioration (mcdonald et al. 1991). a different result was reported by man and wiktorsson (2002) in which lactic acid production of cassava and gliricidia tops silage was 0.95-0.99 %, respectively. biotropia no. 25, 2005 lactic acid concentration of rice straw silage treated with commercial enzyme (p1=0.35 %) was significantly higher than lactic acid concentration of rice straw silage treated with cetv (p2,p3 and p4=0.26 %). the lactic acid concentration was parallel to rice straw silage ph. the higher production of lactic acid, the lower became the silage ph. ph of rice straw silage treated with commercial enzyme was the lowest. while the ph of rice straw silage treated with cetv increased parallelly to cetv dosages. digestibility of dry matter and organic matter of rice straw silage the digestibility of dry matter (ddm) of rice straw silage treated with enzyme (p1, p2, p3 and p4=37.5%) was significantly higher than the digestibility of dry matter of untreated rice straw silage (p0=28.0%). a similar result was obtained for digestibility of organic matter (dom) (table 4.). the increased degradation might have to do with the increased damage of feed surface caused by exogenous enzyme treatment of feed before feeding (nsereko et al. 2000). hong et al. (2003) stated that nutrient degradation rate and effective degradability of dm increased by addition of enzyme mixture consisting of mainly cellulase and xylanase. the enzyme applied to forages immediately before in vitro incubation also improved digestion of dm and ndf, suggesting that fibrolitic enzymes applied for feeding (direct fed) may enhance digestion of forage by cattle (feng et al. 1996). beauchemin et al. (2001) reported that applying fibrolytic enzymes prior to feeding enhances ruminal fibre digestion by altering the structure of the feed, thereby making it more susceptible to degradation. exogenous enzymes treatment, prior to ingestion, increase bacterial colonization and thereby improves dm disappearance of forage (yang et al. 1999) because exogenous enzymes increased microbial attachment of ruminal microbes to feed and increased activity of enzymes associated with feed particles (wang et al. 2001). the digestibility of dry matter of rice straw silage treated by cetv (p2, p3 and p4=37.1%) was not significantly different from the digestibility of dry matter of rice straw silage treated with commercial enzyme (p1=38.8%). the improvement in the digestibility of silage treated with enzymes was caused by decreasing ndf, adf and lignin content in the silage. decreasing fibre content (adf and ndf) and lignin of rice straw silage made the silage easier to be degraded by rumen microbes. the increase of ddm of rice straw silage treated with cetv followed the linear equation of y=21.31+3.14x. meanwhile, dom followed the equation of y=17.43+3.46x. it showed that cetv dosage had not reached the maximum level as the digestibility was still increasing. 57 table 4. digestibility of dry matter and organic matter of rice straw silage production and utilization of cellulose from trichoderma viride – r. hidayat et al. treatments digestibility of dry matter (%) digestibility of organic matter (%) p0 28.6±3.3 25.6±3.6 p1 38.8±2.5 36.8±2.6 p2 28.3±2.0 25.0±2.5 p3 38.9±1.3 37.0±1.7 p4 44.0±3.5 42.3±4.0 p0=untreated rice straw silage, p1=treated with commercial cellulase at 10 iu kg-1, p2= treated with cetv at 3.8 iu kg-1, p3=treated with cetv at 7.6 iu kg-1 and p4= treated with cetv at 11.4 iu kg-1 of fresh rice straw. digestibility of dry matter (ddm) and organic matter (dom) of rice straw silage treated with enzyme (p1, p2, p3) was significantly higher than digestibility of dry and organic matter of untreated rice straw silage (p0). the present study clearly indicates that the most promising method to improve the nutritive value of agricultural by-products in terms of increasing the digestibility is to treat rice straw with cellulase enzyme addition during ensiling. conclusions fungi of trichoderma viride strain qm9414 produced crude enzymes with high cellulolytic activity i.e. 1.52 iu/ml. cetv was able to improve the biological value of rice straw silage and it was better than commercial cellulase. addition of commercial cellulase (onozuka r10 from trichoderma viride ) and crude enzymes from trichoderma viride (cetv) have positive influence on physical quality of rice straw silage and did not cause negative effect on dry matter and organic matter content of rice straw silage. acknowledgement the researcher would like to thank seameo searca for financing this research. references aak. 1983. the forage. yayasan kanisius. yogyakarta. beauchemin, k.a., d.p. morgavi, t.a. mcallister, w.z. yang and l.m. rode. 2001. the use of enzymes in ruminant diets. in: recent advances in animal nutrition (ed. p.c. garnsworthy and j. wiseman). nottingham university press. nottingham. p. 298-322. bolsen, k. k., sapienza. 1993. silage technology. planting, making and feeding to the livestock. pioneer seeds. usa. 58 djunaidi, i.h. 1988. improvement of rice straw through degradation of fungi. bogor agricultural university, bogor (desertation). biotropia no. 25, 2005 doyle, p.t., c. devendra, g.r. pearce. 1986. rice straw as a feed for ruminant. idp, australian universities and college, canbera. feng, p., c.w. hunt, g.t. pritchard and w.e. julien. 1996. effect of enzyme preparations on in situ and in vivo degradation and in vivo digestive characteristics of mature cool-season grass forage in beef steers. j. anim. sci. 74:1349-1357. hong, s.h., b.k. lee, n.j. choi, s.s. lee, s.g. yun and j.k. ha. 2003. effect of enzyme application method and levels and pre-treatment times on rumen fermentation, nutrient degradation and digestion in goats and steers. asian-aust.j.anim.sci. vol 16, no. 3:389-393. jackson, m.g. 1977. the alkali treatment of straw, anim. feed sci. and tech. 2:105-130. komar, a. 1984. technology of rice straw preservation as feed. yayasan dian grahita. indonesia, bandung. lubis, d.a. 1963. feed science. yayasan pembangunan jakarta. man, n.v. and h. wiktorsson. 2002. effect of molasses on nutritional quality of cassava and gliricidia tops silage. asian-aust. j. anim. sci. 5(9):1294-1299. mcdonald, p., a.r. henderson., s.j.e. heron. 1991. the biochemistry of silage. chalcombe publication, 13 highwoods drive, mariow bottom, marlow, bucks sl73pu. montesqrit, 1998. cellulase extraction from the mold and its application to improve fibrous waste product for ruminant. m.sc. thesis. post graduate programe. bogor agricultural university. bogor. nsereko, v.l., d.p. morgavi, k.a. beauchemin and l.m. rode. 2000. inhibition of ruminant feed enzyme polysaccharidase activities by extracts from silage. can. j. anim. sci. 80:523-526. paterson, a. 1986. biodegradation of lignin and cellulosic material. expert consultation on biotechnology for livestock production and health. rome. aga:biot/86/24. soetjiharto, m. 1997. isolation of mold-degrading lignocellulase and its application to improve nutritive value of fibrous feed. bogor agricultural university. bogor. stokes, m.r. and j. chen. 1994. effect of enzyme-inoculant mixture on the course of fermentation of corn silage. j. dairy sci. 77:3401-3409. van soest, p.j., r.h. wine. 1967. use of detergents in the analysis of fibrous feeds. iv. determination of plant cell wall constituents. j. assoc. of anal. chem., 50:50. wang, y., t.a. mcallister, l.m. rode, k.a. beauchemin, d.p. morgavi, v.l. nsereko and a.d. iwaasa. 2001. effect of an exogenous enzyme preparation on microbial protein syntesis, enzyme activity and attachment to feed in the rumen simulation technique (rusitec). br. j. nutr. 85:325-332. yang, w.z., k.a. beauchemin and l.m. rode. 1999. effect of enzyme feed additives on extent of digestion and milk production of lactating dairy cow. j. dairy sci. 82:391-403. 59 r. hidayat1, s. wulandari2, k.g. wiryawan3 and suryahadi3 abstract introduction the main problem of improving ruminant production in indonesia is the shortage of high quality forage during dry season causing the animal production become inefficient, because of an increase in feed cost. on the other hand, the forage is abundant during the wet season. silage preparation chemical analysis results and discussions production and activity test of crude enzymes from trichoderma viride the influence of treatments on the physical quality of rice straw silage 1. nutrients content of rice straw silage 2. fibre content of rice straw silage hong, s.h., b.k. lee, n.j. choi, s.s. lee, s.g. yun and j.k. ha. 2003. effect of enzyme application method and levels and pre-treatment times on rumen fermentation, nutrient degradation and digestion in goats and steers. asian-aust.j.anim.sci. vol 16, no. 3:389-393. jackson, m.g. 1977. the alkali treatment of straw, anim. feed sci. and tech. 2:105-130. komar, a. 1984. technology of rice straw preservation as feed. yayasan dian grahita. indonesia, bandung. lubis, d.a. 1963. feed science. yayasan pembangunan jakarta. man, n.v. and h. wiktorsson. 2002. effect of molasses on nutritional quality of cassava and gliricidia tops silage. asian-aust. j. anim. sci. 5(9):1294-1299. mcdonald, p., a.r. henderson., s.j.e. heron. 1991. the biochemistry of silage. chalcombe publication, 13 highwoods drive, mariow bottom, marlow, bucks sl73pu. montesqrit, 1998. cellulase extraction from the mold and its application to improve fibrous waste product for ruminant. m.sc. thesis. post graduate programe. bogor agricultural university. bogor. nsereko, v.l., d.p. morgavi, k.a. beauchemin and l.m. rode. 2000. inhibition of ruminant feed enzyme polysaccharidase activities by extracts from silage. can. j. anim. sci. 80:523-526. biotropia book final.indd 87 occurrence and distributional range of mangrove vascular flora of catanduanes island, luzon, philippines jimmy tevar masagca* professional education department, college of education de la salle university-dasmariñas cavite 4115, the philippines abstract mangroves play very significant roles not only on the economic aspects but also on the ecological aspects as biobelting for tidal surges and tsunamis. the loss of human lives due to the deadly tsunamis in east asia and the unabated destruction of coastal vegetation have resulted in a renewed focus on the mangrove resources. the purpose of this paper is to report the existing mangrove database of the typhoon-prone island province of catanduanes in luzon, philippines which will be used as bases in determining the appropriate educational management initiatives of various sectors for mangrove rehabilitation and regeneration. a total of 37 species of mangrove vascular flora (13 species of major mangrove elements, 10 species of minor mangrove elements and 14 associated mangrove species) were identified in the island under study. two species of the genus avicennia (a. marina and a. officinalis) were noted in the island. the other genera (bruguiera, ceriops, sonneratia and rhizophora) are well distributed in the designated eco-zones. nypa fruticans is the most important mangrove plant species and a member of the screwpine family (pandanaceae), pandanus tectorius which is an associated mangrove species is well-distributed all throughout the island. key words: mangroves, vascular flora, catanduanes, philippines, occurrence and distribution. introduction in recent years, there had been an increased interest on the status of mangrove environment and the degradation of mangrove ecosystems in the south east asian (sea) region. the rapid depletion of mangrove cover in the philippines was due to the over-all exploitation by coastal dwellers and conversion to agriculture and aquaculture industries (zamora 1989a, 1991b; bennagen & cabahug 1991; primavera 2001). the coastal-dwelling communities of many countries of the region have been utilizing the mangrove resources in many different ways. they depend on wood and non-wood products as well as the economically important species of mangrovebiotropia vol. 15 no. 2, 2008: 87 94 *corresponding author: jtmasagca@dasma.dlsu.edu.ph 88 associated fauna. on the utilization and conservation of mangroves, masagca (2007) observed the general agreement of science teachers and coastal dwellers on their perceptions as to the ecological values such as protection from floods, erosion and other climatic factors. japar (1994) compiled the information on plant species associated with mangals in the country based on the papers of gomez (1980), zamora (1987), and calumpong (1994). there are 31 to 34 exclusive mangroves species and 40 to 65 non-exclusive categories. these numbers vary slightly from country to country. studies on mangroves in the philippines are numerous, in bukidnon for instance, quimpang et al. (1987) described the mangrove forest structure in gingoog city. the team of vega et al. (1991) assessed the mangroves of san miguel bay in camarines sur using the transect plot method in their fieldwork as outlined by dartnall & jones (1986). de leon (1992) in negros oriental noted that avicennia dominates talabong mangrove forest with 251-stems/10 sq m on the average. in guimaras, babaran & ingles (1997) assessed the mangrove forests and nipa swamps. a total of 26 mangrove plant species were identified in the island. this paper reports the mangrove-associated flora of catanduanes island, luzon, philippines. the said typhoon-prone island is located in the bicol peninsula wherein mangroves play very significant roles not only on the economic aspects but also on the ecological aspects as biobelting for tidal surges and tsunamis. materials and methods location of the study catanduanes is an island province (total land area of 1,483 sq km) of the eastern part of the philippine archipelago (13.5˚ to 14.1˚n lat. and 124˚ to 125˚ e long.). highest elevation of the island is 803 m above sea level. the monsoonal climate of the province consists of two distinct dry season and wet season. typical diurnal range of temperatures is 25˚ to 32˚c with minimal variations throughout the year. relative humidity varies from 75 to 89%. the most likely current that affects the island is the north equatorial current moving westward across the pacific. research methods, sources of data and sampling procedures heywood (1995) propounded that characterizing biodiversity involves the observation and characterization of the main units of variation (e.g. genus and species), as well as the quantification of variation within and between them (e.g. taxonomic relatedness). ocular surveys in the present study cover mangrove areas in river mouths, creeks and buffer zones or marginal strips of the coastline found in the different study zones (see table 1). the transect surveys were employed in gathering data and collection of voucher specimens for the mangrove floristic components of the mangroves in the island. primary data were from direct systematic surveys or observation of the mangrove areas in the island. laboratory observations were done to identify floral specimens not identified in situ. biotropia vol. 15 no. 2, 2008 89 the study sites cover the whole island, except for a landlocked town and a coastal town without a distinct mangrove area suited for scientific observation. there were 6 study zones established in the province for the purpose of showing the whole province as an ecological unit for analysis as shown in table 1. table 1. arbitrary ecological zones on catanduanes island, luzon zone zonal location municipality* code i northern pandan pan-06 ii northwestern caramoran car-04 iii northeastern panganiban viga bagamanoc pag-07 vig-09 bag-01 southern virac vrc-10 v southeastern bato baras gigmoto bat-03 bar-o2 gig-05 *also termed as town study stations for the transect surveys were selected based on the availability of mangrove forests stands suitable for the sampling method, i.e. pcqm (pointcentered quarter method). two to three transect lines were established from the forest margin at right angles to the edges of the mangrove forest. these were made landward to seaward direction with the use of magnetic compass. data gathering procedures the predominant plants in the mangrove ecosystem are the trees or shrubs and these species were generally observed in the mangrove sites. some plant specimens not identified in the site were collected from the mangrove sites and were plant pressed. some samples of leaves, flowers and fruits were also obtained and photographed in the laboratory. taxonomic diagnoses were noted following the reports of ding hou (1960), tomlinson (1986) and santisuk (1985). the works of morton (1990) and calumpong & meñez (1997) were used as guides. tomlinson (1986) who arbitrarily categorized mangrove flora into major, minor, associated and specialized mangal elements used fairly rigid criteria to distinguish the mangrove floral species. since there is no standard way of categorizing the mangroves, tomlinson’s categories were employed in the present work. taxonomic identification and counting of the different species of flora were accomplished using the aforementioned guides. some specimens were dispatched to the philippine national museum (pnm) in manila for confirmation of the identity. herbal collections of pnm were also used as reference materials to identify the taxa of plant specimens. results and discussion tables 2, 3 and 4 present the checklist of the major, minor and associated mangrove flora, respectively found in catanduanes island. occurrence and distributional range of mangrove – jimmy tevar masagca 90 a total of 13 species of major mangal elements (table 2), 10 species of minor mangal elements (table 3), and 14 associated mangrove species (table 4). three species of specialized mangrove plants were also identified as caesalpinia crista, derris trifoliate and asplenium sp. table 2. list of major mangal elements recorded in catanduanes island, luzon, philippines family genus species code used avicenniaceae avicennia avicennia marina am avicennia offi cinalis ao combretaceae lumnitzera lumnitzera racemosa lr lumnitzera littorea ll arecaceae nypa nypa fruticans nf rhizophoraceae bruguiera bruguiera gymnorhiza bg bruguiera sexangula bs ceriops ceriops decandra cd ceriops tagal ct rhizophora rhizophora apiculata ra rhizophora mucronata rm sonneratiaceae sonneratia sonneratia alba sa total: 5 families 7 genera 13 species table 3. list of minor mangal elements recorded in catanduanes island, luzon, philippines family genus species code bombacaceae camptostemon camptostemon philippinensis cp euphorbiaceae excoecaria excoecaria agallocha er lythraceae pemphis pemphis acidula pa meliaceae xylocarpus xylocarpus moluccensis xm xylocarpus granatum xg myrsinaceae aegiceras aegiceras corniculatum ac aegiceras fl oridum af pteridaceae acrostichum acrostichum aureum aa acrostichum speciosum as sterculiaceae heritiera heritiera littoralis hl total: 7 families 7 genera 10 species table 4. list of associated mangal elements recorded in catanduanes island, luzon, philippines family genus species code acanthaceae acanthus acanthus ebracteatus acanthus illicifolius ae ai apogonaceae cerbera cerbera manghas cm bignoniaceae dolichandrone dolichandrone spathacea ds combretaceae terminalia terminalia catappa tc cyperaceae cyperus cyperus malaccensis cm lecythidaceae barringtonia barringtonia racemosa br biotropia vol. 15 no. 2, 2008 91 family genus species code leguminoceae aganope ipomoea pongania aganope heptaphylla ipomea pes-caprae pongania pinnata ah ip pp malvaceae hibiscus hibiscus tiliaceus ht th espesia th espesia populnea tp palmae corypha corypha elata ce pandanaceae pandanus pandanus tectorius pt total: 10 families 13 genera 14 species 90 there are 94 species of vascular flora in mangals and associated coastal communities of the philippines (zamora 1995). tomlinson (1986) noted that there are 18 species of major mangal elements, 12 species of minor elements, and 24 specialized mangal elements. the number of species obtained in catanduanes is comparatively lower than the reports from other areas of the country, but slightly higher than the number of species obtained from guimaras island. the categorization of mangrove flora by tomlinson (1986) indicates that there are 34 species of major mangrove species worldwide; 20 species of minor element; and 46 mangal associates. considering this available reference data, catanduanes island shares 38% of the major elements (13 out of 34 species) and 72% (13 out of 18 from zamora’s list). for the minor elements, the province shares 50% (10 out of 20 species of tomlinson’s list) and 83 % (10 out of the 12 species from zamora’s list). table 5 shows the comparison of the various listings. table 5. category and number of mangrove floral species recorded in catanduanes island, philippines compared to other reports/listings. group of mangroves tomlinson (1986) zamora (1995) this study elements world-wide philippines catanduanes island major 34 17 13 minor 20 12 10 associated 46 40 14 total 100 69 37 distributional range of mangroves in catanduanes island globally, mangrove ecosystems are thought to contain about 60 species of trees and shrubs and more than 20 additional species frequently associated with the mangrove flora but not necessarily restricted to it (barth 1982; hamilton & snedaker 1984). some 70 species of the mangrove plants are recognized from various regions of the world, with the highest concentrations of species being found in sea and australia (spalding et al. 1997). according to fortes (1989), the most diverse single stand of mangrove forest is found in pagbilao, quezon (luzon, philippines) with a total of 29 species. the report of cadiz & de leon (1994, cited by calumpong & meñez 1997) revealed that the mangrove forest of misom in baliangao, misamis occidental consisted of 21 species, while in bais bay in negros island (visayas, philippines) a total of 14 species were noted by calumpong (1992). babaran & ingles (1997) table 4. continued occurrence and distributional range of mangrove – jimmy tevar masagca 92 indicated that not all species of major as well as minor elements are simultaneously present in all municipalities of guimaras, philippines. in the genus avicennia, 2 species (a. marina and a. officinalis) are found in catanduanes. the species, a. eucalyptifolia as reported by zamora (1991) is not found in this province. the other genera, e.g. bruguiera, ceriops, sonneratia and rhizophora are also well distributed in this island. the most important major mangal species in the province is the palm, nypa fruticans. a member of the screwpine family (pandanaceae), pandanus tectorius (an associated mangrove species) is distributed all throughout the province. however, this is not true in other places of the philippines. in guimaras, babaran & ingles (1997) made no report about its presence there. likewise, calumpong & menes (1997) did not also mention pandanus in their report. among the members of family pandanaceae, p. tectorius is the most well known species. this is the commonest and the most widespread (gruezo & zamora 2000; stone 1976). moreover, terminalia catappa (locally known as “tarisoy”) was described by calumpong and meñez (1997), and earlier included by dr. p. zamora in his previous papers. this species of the combretaceae family is well distributed in back mangals of the southern and western portions of island under study. among the minor mangrove elements, the genus acanthus, represented by the species, acanthus ebracteatus and a. illicifolius are well distributed in the different zones of the island from the northern to southern zones and eastern to western zones. of the major elements, sonneratia and avicennia are well distributed all throughout the island. among the minor elements, excoecaria agallocha and aegicera corniculatum are distributed in almost all zones of the island. conclusions a total of 37 species of mangrove vascular flora (13 species of major mangal elements, 10 species of minor mangal elements and 14 associated mangrove species) were identified in the island under study. two species of the genus avicennia (a. marina and a. officinalis) are present in the study sites. the other genera (bruguiera, ceriops, sonneratia and rhizophora) are well distributed in the designated eco-zones. the palm, nypa fruticans is the most important mangrove plant species in the island under study and a member of the screwpine family pandanaceae , pandanus tectorius which is an associated mangrove species is well-distributed all throughout the island. acknowledgment the author expresses his sincere thanks to the administration of de la salle university-dasmariñas for giving the impetus in carrying out the study on the mangroves while investigating the mangrove brachyurans of catanduanes and quezon. earlier studies on the mangroves of the island were carried out by the author while teaching in the premier state college. this work was completed with the assistance of various individuals. likewise, profound thanks is also given to raul evasco, ma joie estopin, biotropia vol. 15 no. 2, 2008 93 elver sison, macoy leyba, igo de castro and others from molino, cavite for the assistance provided during the writing of the final drafts of the report. references babaran, r.p. and j.a. ingles. 1997. philippine marine coastal habitats at risk: a case study of guimaras island. institute of marine fisheries and oceanology, university of the philippines in the visayas institute of integrative and development studies-marine affairs program, up press. barth, h. 1982. the biogeography of mangroves. in: d.n. sen and k.s. rajpurohit (eds.). tasks for vegetation science: contributions to the ecology of halophytes. the hague: dr. w. junk. bennagen, m.e.c. and d. cabahug jr. 1991. natural resources accounting: mangroves. technical report no. 2. natural resources accounting project final workshop. national institute of geological sciences, 15 oct. 1991. university of the philippines, diliman, quezon city. calumpong, h.p. 1992. systematic studies on the red algal genus laurencia (ceremiales: rhodomelaceae) in the philippines. phd dissertation, university of california, berkeley, california, usa. calumpong, h.p. 1994. status of mangrove resources in the philippines. in: c.r. wilins, s. sudara and c.l. ming (eds.) proceedings of the 3rd asean-australian symposium on living coastal resources, chulalongkorn university, thailand held on 16-20 may, 1994. calumpong, h.p. and e. g. meñez. 1997. field guide to the common mangroves, seagrasses and algae of the philippines. bookmark, inc.: makati city, philippines. dartnall, a.j. and m. jones (eds.). 1986. a manual of survey methods: living methods in coastal areas. asean-australia cooperation program on marine science handbook. townsville, australia: aims. de leon,r.o.d., j.a.u. nuque and r.j. raymundo.1992.leaf litter production and tidal export of rhizophora apiculata, r.mucronata, avicennia marina and sonneratia alba from talabong mangrove forest in bais, negros oriental. philippine council for agriculture, forestry and development. los banos, laguna. pccard series no. 127, pp. 84-90. ding hou, 1958. rhizophoraceae. flora malesiana. series 1, 5:429-493. fortes, m. 1989. mangrove and seagrass study. living resources in coastal areas: phase i terminal report. university of the philippines, quezon city, philippines. gruezo, w.sm. and p.m. zamora. 2000. conservation status of philippine biota. 2. sararanga philippinensis merr., family pandanaceae. asia life sciences, the asian international journal of life sciences, 9(2):189=204. hamilton, l.s. and s.c. snedaker (eds.). 1984. handbook for mangrove area management. unesco and east west center, environment and policy institute, hawaii. japar, s.b. 1994. mangrove plant resources in the asean region. in: c.r. wilins, s. sudara and c.l. ming (eds.) proceedings of the 3rd asean-australian symposium on living coastal resources, chulalongkorn university, thailand held on 16-20 may, 1994. masagca, j.t.2007. perceptiones de los maestros de ciencia y habitants costeros sobre conservacion de la biodiversidad de manglar (science teachers and coastal dwellers perceptions on mangrove biodiversity conservation). revista de educacion en ciencias, journal of science education, 7(1):24-29. morton, j. 1990. the shore ecology of the tropical pacific. jakarta: unesco rotsea. primavera, j. 2001. mangroves. haring ibon, 3(2):16-21. occurrence and distributional range of mangrove – jimmy tevar masagca 94 quimpang, v.t., h.c. porquiz, i. gurea, l.a. samaniego. 1987. the community structure of the mangrove forest in pangasihan, gingoog city. cmu journal of agriculture, food and nutrition, 9(1):84-102. tomlinson, p.b. 1986. the botany of mangroves. cambridge university press, cambridge. santisuk , t.1985. explanatory keys to terrestrial trees and shrubs recorded from the mangrove formations in thailand. proceedings of a training course in life history of selected species of flora and fauna in mangrove ecosystems. thailand, vol. 1. spalding, m.f., m.f blasco and c. field (eds.). 1997. world mangrove atlas. international society for mangrove ecosystems. the world monitoring center and international tropical timber organization. stone, b.c. 1976. studies in malesian pandanaceae, xiv. notes on philippine taxa. kalikasan, the philippine journal of biology, 5(1):1936. vega, m.j.m., l.r. garces, q.p. sia iii and r.g.g. ledesma. 1994. assessment of mangrove resources in san miguel bay. in: g. silvestre, c. luna and j. padilla (eds.) multidisciplinary assessment of the fisheries in san miguel bay, philippines (1992-1993). iclarm technical report. zamora, p.m.1984. philippine mangroves: assessment of status, environmental problems, conservation and management strategies. in: e. soepadmo, a.n. rao and d.j. macintosh (eds.), pages 606-707. asian symposium on mangrove environment research and management. unversity of malaya, kuala lumpur, malaysia. zamora, p.m. 1989. mangroves of the philippines. biotrop special publication (bogor, indonesia), 37:43-65. zamora, p.m. 1991. managing the philippine mangal for long-term human survival. transactions of the national academy of science and technology (nast), 12:89-125. biotropia vol. 15 no. 2, 2008 biotropia no. 9, 1996: 53 61 growth inhibition of pathogenic root fungi by extracts of ectomycorrhizal fungi or picea glehnii inoculated with ectomycorrhizal fungi* maria catarina megumi kasuya dep. de microbiologia, universidade federal de vicosa, vicosa, mg, 36570-000, brazil satoshi tahara dept. of applied bioscience, faculty of agriculture, hokkaido university, kita-ku, sapporo 060, japan and tsuneo igarashi dept. of forest science, faculty of agriculture, hokkaido university, kita-ku, sapporo 060, japan abstract this work sought to verify the presence of compounds with antimicrobial properties in extracts of ectomycorrhizal fungi or in picea glehnii inoculated with ectomycorrhizal fungi. extracts from pisolithus tinctorius, scleroderma flavidum, amanita pantherina and paxillus sp., grown in liquid culture media, and from p. glehnii seedlings inoculated or not with the above ectomycorrhizal fungi and cultivated in in vitro condition, were processed to obtain two fractions, water and ethyl acetate solubles. these fractions were tested for the presence of inhibitory constituents against fusarium roseum, pythium sp. and rhizoctonia solani. direct bioautography technique on tlc or paper disc technique was used, depending on the extract and pathogenic fungi tested. the results showed the production on inhibitory components, not only by ectomycorrhizal fungi, but also by p. glehnii inoculated or not with ectomycorrhizal fungi. the sensitivity varied considerably according to the type of fungus or extract. key words: japan/mycorrhizas/inh|bition/extracts/antimicrobial compounds/amanita pantherina/pisolithus tinctorius/paxillvs spjscleroderma flavidum/fusarium roseum/pythium spjrhizoctonia solani. introduction damping-off has been responsible for considerable losses of tree species in nurseries. research has shown that these losses can be reduced by inoculation of effective * paper presented at the second symposium on biology and biotechnology of mycorrhizae and third asian conference on mycorrhizae (acom iii), 19 21 april 1994, yogyakarta, indonesia 53 biotropia no. 9, 1996 ectomycorrhizal fungi (sinclair et al 1982, chakravarty and unestan 1987; sampangi et al. 1985; duchesne et al. 1989). it has been postulated that root protection by ectomycorrhizal fungi results from antibiotic synthesis which could either be by the myco-symbiont or by the host plant; a barrier effect caused by the presence of a fungal mantle around roots; or a nutrient competition in the rhizosphere (zak 1964; and marx 1973). none of these can be viewed as universal mechanism (harley and smith 1983), but several mechanisms may act simultaneously and/or synergistically to suppress disease (schisler and linderman 1987). the synthesis of antimicrobial substances by plants in response to ectomycorrhizal fungi has been reviewed and the use of ectomycorrhizal fungi in biological control has been reported (duchesne et al. 1987: 1989b). since the protection provided by these fungi is also influenced by environmental conditions including tree species and pathogens involved (marx 1969; stack and sinclair 1975; sylvia and sinclair 1983a,b; perrin and garbaye 1983; sampangi and perrin 1986; chakravarty and unestan 1987a,b; duchesne et al. 1989), a screening of ectomycorrhizal fungi that is able to produce antimicrobial substances is useful for a better understanding of the mechanism of disease suppression by ectomycorrhizal association. a better understanding of involved mechanisms may lead not only to field manipulations of environmental conditions to enhance disease protection, but also help the design of optimal screening programs. it may also favour the discovery of new pesticides which lead to novel methods of disease control (duchesne et al. 1989b). this study sought to verify the presence of compounds possessing antimicrobial properties in the extracts of ectomycorrhizal fungi or in p. glehnii inoculated with ectomycorrhizal fungi. materials and methods fungal cultures the ectomycorrhizal fungi, pisolithus tinctorius (pers.) coker & couch, scleroderma flavidum e.&e., amanita pantherina (dc.:fr.) krombh. and paxillus sp., were isolated from basidiocarps collected under different forest species in hokkaido. all of them were proven to be mycorrhizal fungi by in vitro condition with p. glehnii (unpublished data). the phytopathogenic fungi, fusarium roseum, rhizoctonia solani and pythium sp. were isolated from p. glehnii seedlings, collected from nursery beds, and kindly provided by y.y. cha, of lab. of sylviculture, fac. of agriculture, hokkaido university. the ectomycorrhizal fungi were maintained on modified melin norkran 54 growth inhibition of pathogenic root fungi m.c.m. kasuya et al. (mmn) solution a (kottke et al. 1987) and the pathogenic fungi on pda (potato dextrose agar), both on petri dishes at 26°c in the dark. extracts from ectomycorrhizal fungi the ectomycorrhizal fungi were grown in mmn at 16°c, in the dark. after 34 days, a volume of 90% methanol (meoh) equal to that of growing cultures was added to each culture. this mixture was filtered after 24 hours, and the meoh in the filtrate material was evaporated under vacuum condition at 40°c. the constituents in the concentrate were partitioned between water and ethyl acetate (etoac). these extracts were stored in the dark at-10°c. in vitro seedlings cultivation the growth system for the in vitro seedlings cultivation consisted of a modified method of sylvia and sinclair (1983a) and duchesne et al (1988a). the test tubes (200 x 30 mm) were lined with filter paper (100 x 100 mm), immersed in mmn solution a (kottke et al. 1987). seeds of p. glehnii were superficially disinfested for 30 min. in h2o230%, rinsed with autoclaved distilled water, and germinated for 7 days in petri dishes before transferring to the test tubes containing the nutrient solution. three seedlings per tube were transferred to each tube and the roots were placed between the filter paper and test tube wall. a week after the seedlings had been transferred, they were inoculated by using one disc (7 mm diameter) containing ectomycorrhizal fungi's myce-lia. the discs were placed between filter paper and test tube wall close to the roots. twenty-five replications per treatment were taken. extracts from seedling the extraction was done on six-month-old seedling. the in vitro synthesis material was divided into two parts: (a) the liquid part, containing nutrient solution and exudates from root and fungi, and (b) the solid part, containing root systems, mycelia and filter paper. an equal volume of etoac was added to the liquid part, and the mixtures were shaken to yield water and etoac soluble fractions. to the solid part, initially about 20 ml of 90% meoh was added per synthesis in vitro. twenty-four hours later, the mixture was filtered. the meoh of the filtrate was evaporated under vacuum condition, at 40°c, and the constituents in the concentrate were partitioned between water and etoac soluble fractions. 55 biotropia no. 9, 1996 direct bioautography technique on tlc this technique was applied to etoac soluble fractions and pathogenic fungi, fusarium roseum and pythium sp. that produce large quantities of spores. the spores of cladosporium herbarum were used as the control to verify the presence of fungitoxic substance due to its high sensibility. samples of etoac extracts equivalent to 10 and 20 mg of mycelia or seedling (dry weight basis) were subjected to tlc using silica gel 60 f254 plates with a thickness of 0.25 mm and a mixture of chloroform-meoh (20:1). the constituents were viewed under uv 245nm (quenching) and 356nm (fluorescent) light, and then the chromatograms were sprayed with a spore suspension of c. herbarum, f. roseum and pythium sp. (romans and fuchs 1970). the plates were incubated in a moisture chamber at 28°c for 5 days to detect growth inhibition spots. the relative distance (rrf) of each inhibition spot was calculated as the relation between the distance of inhibition spot from the starting line and the distance of remarkable fluorescent spot presented by the extract from p. glehnii (b) from the starting line, which was detected close to the front line. paper disc technique the effects of etoac soluble fractions on r. solani, and on all water soluble fractions on phythium, f. roseum and r. solani were examined using paper disc technique. extracts equivalent to 10 and 20 mg (dry weight basis) of seedling material and a. pantherina, and 10 and 13.8; 10 and 16.6; and 10 and 11.8 mg for p. tinctorius, s. flavidum and paxillus sp., respectively, were aseptically imbibed in 8 mm paper discs. the smaller quantities (<20 mg) were used since the densities of extracted fraction for those fungi exceeded the absorption capacity of the paper discs. to verify the inhibition zone produced by the extracts, two doses of each extract were placed on the same petri dish but opposite each other, and the pathogenic fungus mycelia disc (7mm) was located in the center of the dish (fig. 1). the incubation time defined according to the growth rate of pathogenic fungi in previous assays was 3, 8 and 20 days for pythium, f. roseum and r. solani, respectively. the relative inhibition radius (rlr) was calculated as the relation between the radius of inhibition and the radius of paper disc (fig. 1). there were 2 replications per treatment. results and discussion some constituents of etoac extract inhibited the growth of pathogenic fungi. however, these substances acted differently on the individual pathogens, as verified 56 growth inhibition of pathogenic root fungi m.c.m. kasuya et al. figure 1. scheme of the paper disc technique. a and b are 8 mm disc imbibed, respectively, with equivalent to 10 and 20 mg of ectomycorrhizal fungi or p. glehnii extract. c is 7 mm containing pathogenic fungi inoculum; a and b are the radius of inhibition zone. shaded area represents colony growth of pathogenic fungi. from the different rrf on inhibition spots (table 1), except for the extract of a. pantherina x p. glehnii. for this host-fungus combination the same spot inhibited both c. herbarum and f. roseum. some spots visible under uv 254nm and 365nm were fungitoxic, but not all fungitoxic spots were detected under uv light. the fungitoxic substances were more frequently detected in the liquid part of extracts obtained from the seedlings, suggesting that many antibiotic components are present in the exudates. duchesne et al. (1988a) observed that fungitoxic materials were present in the rhizosphere but not in plant tissue of pinus resinosa inoculated with p. involutus. in the water fractions (table 2), fungitoxic compounds were detected in the solid materials (root system + filter paper + mycelia). all ectomycorrhizal fungi tested were capable of producing some kind of fungitoxic substance (table 1). inhibition of phytopathogenic fungi by cell free culture media of ectomycorrhizal fungi was tested by kope and fortin (1989). the authors verified that 7 ectomycorrhizal fungi inhibited 20 out of 24 pathogenic fungi tested. it is possible to produce fungitoxin againts pythium when p. glehnii inoculated with p. tinctorius was synthesized by the latter, since the same spot was present in the extract of this fungus (table 1). the presence of the host plant could have stimulated the synthesis of the fungitoxin, considering the smaller quantity of mycelia present in the 57 biotropia no. 9, 1996 table 1. inhibition spots (rrf*) against cladosporium herbarum, fusarium roseum and pythium sp. when extracts equivalent to 10 and 20 mg of mycelia or plant material (dry weight basis) was applied to direct bioautography on the tlc plates. extract of the inoculated seedling. duchesne et al. (1988b) observed that sporulation of f. oxysporwn was depressed by p. involutus and this suppression was more pronounced when the exudate of pinus resinosa was added, suggesting that the presence of root exudate can stimulate synthesis of antimicrobial substances produced by ectomy-corrhizal fungi. in in vitro studies, perrin and garbaye (1983) verified a strong suppression of pythium ultimum, possibly as an effect of diffusible or volatile inhibitory substances produced by ectomycorrhizal fungus hebeloma crustuliniforme. although we are unable to determine whether ectomycorrhizal fungi or host plant or both were responsible for the synthesis of antimicrobial compounds, the results from this study suggest that mycorrhizal association stimulated production of new fungitoxic compounds (table 1). laccaria laccata was able to inhibit fusarium spp. in vitro, but production of phenolics, induced by inoculation of these ectomycorrhizal fungi might be the basis of root protection of pseudotsuga menziesii (sylvia and sinclair 1993b; and chakravarty and hwang 1991). according to buscot et al. (1992) not only production of 58 growth inhibition of pathogenic root fungi m.c.m. kasuya et al. fungistatic phenolics compounds, but also the greater vigor of mycorrhizal plants is perhaps the cause of resistance to phytopathogenic fungi. earlier work by stack and sinclair (1975) showed that inoculation of laccaria laccata spores protected p. menziesii against f. oxysporum, even without mycorrhizae. complementary results obtained by the paper disc technique (table 2) suggest that some fungitoxins were also present in the aqueous extracts, and the degree of inhibition varied according to the origin of extract. table 2. inhibition radius (rrf)* of fusarium roseum, pythium sp., and rhizoctonia solani, when extracts equivalent to 10 and 20 mg** of mycelia or plant material (p.m.) (dry weight basis) were applied using paper disc technique. p. glehnii could produce antimicrobial compounds in the absence of ectomycorrhizal fungi (table 2). since these compounds were not detected when the plant was associated with ectomycorrhizal fungi, it is speculated that the mycosymbionts degraded these compounds or transformed them into other metabolite(s). 59 biotropia no. 9, 1996 the growth of r. solani was not affected by any etoac fraction tested. however, the water fraction presented some compounds that delayed pathogenic mycelial growth. no antagonistic effect on the growth of r. solani was observed when a. pantherina or p. tinctorius were confronted on the pda media (zhao et al. 1989). kope and fortin (1989) verified that p. tinctorius cell free media showed high inhibition against r. pra-ticola, but such inhibition was very low against r. solani (kope and fortin 1989). although the methodologies used to test inhibition by zhao et al. (1989) and kope and fortin (1989) differed from those used in this study, sensitivity of r. solani to compounds produced by ectomycorrhizal fungi seems very low especially in comparison with pythium (table 2). the results of this study although not field tested suggest that inoculation of ectomycorrhizal fungi even when the association with the host plant is not established, may protect plants from pathogenic fungi by production of antibiotic substances. it was also verified that new antimicrobial compounds are produced directly by the ectomycorrhizal fungi or by the host plant in response to mycosymbiont association as reported by zak (1964) and marx (1973) for other mycorrhizal systems. further research is needed : to detect whether these fungistatic compounds are produced in the field; and to monitor whether these ectomycorrhizal fungi are still able to protect plants against pathogenic fungi tested when they are interacting with other microorganisms (krupa et al 1973; schisler and linderman 1989; and chen et al. 1988) or with different environmental conditions (harley and smith 1983). acknowledgements the authors sincerely thank r.m.c. muchovej for her invaluable suggestions as well as for improving the english, and to cnpq (conselho nacional de desenvolvimen-to cientifico e tecnologico brazil) for the scholarship granted to the first author. references buscor, f, g. weber and f. oberwinkler. 1992. interactions between cylindrocarpon destructans and ectomycorrhizas ofpicea abies with laccaria laccata and paxillus involutes. trees, 6: 8390. chakravarty, p. and s.f. hwano. 1991. effect of an ectpmycorrhizal fungus, laccaria laccata, on fusarium damping-off in pinus banksiana seedlings. eur. j. for. path., 21: 97-106. chakravarty, p. and t. unestan. 1978a. mycorrhizal fungi prevent disease in stressed pine seedlings. j. phytopathology, 188: 355-340. chakravarty, p. and t. unestan. 1987b. differential influence of ectomycorrhizae on plant growth and disease resistance in pinus sylvestris seedlings. j. phytophatology, 120: 104-120. 60 growth inhibition of pathogenic root fungi m.c.m. kasuya et al. duchesne, l.c., r.l. peterson and b.e. ellis. 1987. the accumulation of plant-produced antimicrobial compounds in response to ectomycorrhizal fungi: a review. phytoprotection, 68: 17-27. duchesne, l.c., r.l. peterson and b.e. ellis. 1988a. interaction between the ectomycorrhizal fungus paxillus involutes and pinus resinosa induces resistance to fusarium oxysporum. can. j. bot., 66: 558-562. duchesne, l.c., r.l. peterson and b.e. ellis. 1988b. pine root exudate stimulate the synthesis of antifungal compounds by the ectomycorrhizal fungus paxillus involutus. new phytol., 108: 471476. duchesne, l.c., r.l. peterson and b.e. ellis. 1989a. the time course of disease suppression and antibiosis by the ectomycorrhizal fungus paxillus involutus. new phytol., i l l : 693698. duchesne, l.c., r.l. peterson and b.e. ellis. 1989b. the future of ectomycorrhizal fungi as biological control agents. phytoprotection, 70: 51-57. harley, j.l. and s.e. smith. 1983. mycorrhizal symbiosis. new york, academic press inc., 483 p. homans, a.l. and a. fuchs. 1970. direct bioautography on thin layer chromatograms as a method for detecting fungitoxic substances. j. chromatog., 51: 327-329. kope, h.h. and j.a., fortin. 1989. inhibition of phytopathogenic fungi in vitro by cell free culture media of ectomycorrhizal fungi. new phytol., 113: 57-63. kottke, i., m. guttenberger, r. hamp and f. oberwinkler. 1987. an in vitro method for establishing mycorrhizae on coniferous tree seedlings. trees, 1: 191-194. marx, d.h. 1969. the influence of ectrotophic mycorrhizal fungi on the resistance of pine roots to pathogenic infection. i. antagonism of fungi to root pathogenic fungi and soil bacteria. phytopathology, 59: 153-163. marx, d.h. 1973. in: marks, g.c. and kozlowski (eds.). ectomycorrhizae: their ecology and physiology. academic press, new york. pages 351-382. perrin, r. and j. garbaye. 1983. influence of ectomycorrhizae on infectivity of fyf/z/wm-infested soils and substrates. plant and soil, 71: 345-351. sampangi, r. and r. perrin. 1986. attempts to elucidate the mechanisms involved in the protective effect of laccaria laccata against fusarium oxysporum. in : gianinazzi-pearson and s. gianinazzi (eds), proceedings of the 1st european symposium on mycorrhizae. dijon, france, july 1-5, 1985. p. 799-806. schisler, d.a. and r.g. linderman. 1989, response of nursery soil microbial populations to volatilize purge from soil around douglas-fir ectomycorrhizae. soil biol. biochem., 21: 397-401. sinclair, w.a., d.m. sylvia and a.o. larsen. 1982. disease suppression and growth promotion in douglas-fir seedlings by the ectomycorrhizal fungus laccaria laccata. for, sci., 28: 191-201. stack, r.w. and w.a. sinclair. 1975. protection of douglas-fir seedlings against fusarium root rot by a mycorrhizal fungus in the absence of mycorrhizal formation. phytopathology, 65: 468472. sylvia, d.m. and w.a. sinclair. 1983a. suppressive influence of laccaria laccata on fusarium oxysporum and douglas-fir seedlings. phytopathology, 73: 384-389. sylvia, d.m. and w.a. sinclair. 1983b. phenolics compounds and resistance to fungal pathogens induced in primary roots of douglas-fir seedlings by the ectomycorrhizal fungus laccaria laccata. phytopathology, 73: 390-397. zhao, z.p., kuo s.c. and bi. k.c. 1989. selection of ectomycorrhizal fungi with resistance to rhizoctonia solani. agriculture, ecosystems and environments, 28: 575-579. zak, b. 1964. role of mycorrhizae in root disease. ann. rev. phytopath., 2: 377-392. 61 53.pdf 54.pdf 55.pdf 56.pdf 57.pdf 58.pdf 59.pdf 60.pdf 61.pdf biotropia no biotropia no. 14, 1999 : 36 51 litter fall in a primary and two logged-over lowland tropical rainforests in pasirmayang, jambi. upik rosalina wasrin* and agus eka putera ** 'senior scientist and "research assistant, remote sensing and ecology laboratory, seamed biotrop, p.o. box 116, bogor 16001, indonesia abstract litter accumulation in a primary and a logged-over lowland dipterocarp forest at pasirmayang, jambi was measured using the litter trap method. in the primary forest, traps were placed in four distinct areas, reflecting the succession stages of the forest from building to maturation. in the logged-over forest, litter production was measured at two different sites, one cut in 1979/1980 and the second in 1983/1984. in the primary forest, average litter production during the observation period was 925 g m"2yr'. in the logged-over forest, average litter production was 721 g m'2 yr1 for the site cut in 1979/1980 and 706 g m'2 yr1 for the site cut in 1983/1984. leaves comprised the major contributor of litter with 67% of total litter produced in the primary forest, 67% of total litter in the 1979/1980 cut logged-over forest, and 65% of total litter in the 1983/1984 cut logged-over forest. the purpose of the study was to use litter fall as a measure of forest productivity to assess the recovery of logged-over forests and, to provide a basis for comparison of forest-derived land practices for appropriate forest management strategies. key words: litter production/primary forest/logged-over forest/forest productivity/pasirmayang/jambi. introduction knowledge of forest productivity is a basic requirement for determining appropriate management strategies for forests, to maintain or even increase productivity. forest productivity is based on the capture of solar energy and soil-derived nutrients to form chemical compounds used to synthesize various compounds for plant growth and reproduction (binkley 1986). therefore, litter fall can be used as an indicator of productivity (medwecka-kornas 1970). forest productivity is influenced by a range of factors such as nutrient status of the ecosystem, organic matter decomposition, nutrient cycle in the plant (biochemical), nutrient cycle in the ecosystem (biogeochemical), and nutrient cycle between ecosystems (geochemical), which are very complex, since it is also influenced by climate and the availability of nutrients from parent rocks. present address : * lecturer, faculty of forestry, bogor agricultural university, campus darmaga, bogor, indonesia email : wasrinsy @ indo.net.id " ** staff, bali barat national park, bali, indonesia 36 biotropia no. 14, 1999 twigs, bark, flowers, and fruit or seed (small to fine litter), and branches and fallen stems (big litter) to a lesser extent (medwecka-kornas 1970). litter fall can be measured by using litter traps in the form of a basket, box or funnel (medwecka-kornas 1967; newbould 1967; korcagin 1960). for tropical forests, kira & schidei (1967) suggested to use baskets with a circular diameter of 50 cm (medwecka-kornas 1970). the frequency of litter collection from the trap depends on the phenology and the method used. nevertheless, measurement of litter production should represent a minimum period of one year of the total growth season, and should be monitored continuously over several years for at least 3 years (medwecka-kornas 1970). although there is hardly any primary lowland forest left in sumatera, we aimed at collecting litter fall data in one of the last remnants, to provide a basis of comparison for forest-derived land use practices. selective cutting to harvest forest timber practiced up to recent days has resulted in a mostly degraded vegetation structure such as pioneer trees with low canopy (dynamic /), a group of young trees with low canopy (dynamic 2), trees with low branches and small crowns (homeostatic 1 ), and if left undisturbed for sometime (10 years ), this yiejds tall trees with high branches and large crowns similar to the primary forest (homeostatic 2). based on the different vegetation structures following various logging practices and its impacts, we hypothesize that litter fall will differ in quantity and quality (plant components) among various gaps of successional stages of lowland forest, while the litter fall of old logged-over forests (10 years old) equals that of the primary forest. materials and methods to measure litter production, 4 traps were made of nylon with a 1 mm x 1 mm mesh. the circular trap mouth has a diameter of 1 meter and is fixed by 3 iron pipes about 100 cm from the ground, and the lowest part of the trap located about 20 cm from the ground (fig. 1). the circular form avoids the margin effect of the plot. trap mouth nylon wire iron pipe figure 1. illustration of the li 37 litterfall in a primary and two logged-over forests upik r. wasrin & agus eka putra in the primary forest, measurement was conducted inside a 3 ha area (100 m x 300 m) which is divided into 20 m x 20 m plots, arranged systematically at 25 traps per hectare (fig. 2) fig. 3. vegetation dynamics phases inside the litter production observation plot at a primary forest site after laumonier (1997) fig. 2. location of litter traps inside the 3 ha plot of a primary forest litter trrap arrangement of traps was based on the four vegetation dynamics phases used b laumonier (1997) and defined as follows : dynamic 1 : dominated by pioneer trees with very low canopy. dynamic 2 : dominated by trees of the future with low canopy. homeostatic 1 : dominated by trees of the present with low branches and small crowns. homeostatic 2 : dominated by tall trees of the present with high branches and large crowns. 38 biotropia no. 14, 1999 in the logged-over forest, litter production was assessed in two different forest areas, one cut in 1979/1980 and the second in 1983/1984. the traps were systematically placed inside a square plot with distance between traps of about 50 m. the total number of traps was 25 representing about a 200 m x 200 m area (fig. 4). it was assumed that the logged-over forest only had one vegetation dynamic phase. litter collection from traps was .carried out monthly. collection from the primary forest began from august 1991 until november 1993 and was resumed during the period of june 1994 until july 1996. collections from the 1979/1980 cut logged-over forest were made from july 1994 july 1996, and from the 1983/1984 cut logged-over forest during march 1994 july 1996. figure 4. location of litter traps inside a 200 m x 200 m plot of a logged-over forest site. litter was classified as leaves, flowers, fruits, stipules, twigs, bark and others. litter was oven-dried at a temperature of 105°c for 48 hours and dry weight was measured according to wasrin et al. 1995. results and discussion a. litter production at the primary forest measurement of litter production at the primary forest was conducted from august 1991 november 1993 interrupted from october 1993 to may 1994, due to a technical problem. a total of 54 measurements were obtained that fluctuated during the observation period, as shown in fig. 5. litter production in gm'2 was obtained upon conversion of litter production in g trap"1, by dividing the amount by 39 litter trap litterfall in a primary and two logged-over forests upik r. wasrin & agus eka putra trap size (0.785 m2). litter production shown is the monthly average value collected from 75 traps. the highest amount of litter production recorded was 169 gm~2 in october 1991. the smallest amount was 39.22 gm"2 in february 1996. average litter production during the observation period was 77 gm~2month~' or 925 gm~2year~'. this value is lower compared to the number given by wanner in jordan (1983) in the rain forest of north borneo (1070 gnv2year') and those of kira in jordan (1983) in a tropical rainforest of malaysia (1055 gnv2year~'), but higher compared to the rainforest in java (810 gm"2year~'). in general, leaves contributed the largest portion of monthly litter production with an average of 48 gm^month"1 or 62 % of total litter production, followed by twigs (22 %), others (litter components that cannot be identified or classified further due to their small size) (6 %), bark (4 %), fruit (3 %), stipules (2 %), and flowers (1%). month figure 5. total litter production per month (average of 75 traps) in the primary forest during 54 months of observation (august 1991 -ju ly 1996) fig. 6, 7, and 8 show fluctuations in litter production attributed to specific plant parts over the observation period. leaves significantly influence the results for total litter production as leaves comprise the major constituent. thus, the high amounts attributed to leaves (fig. 6) correspond to those recorded for total litter (fig. 5) with highest values in august 1991, october 1991, september 1993, august through october 1994, and september 1995. compared to rainfall levels at the study site (fig. 9) there is a good correlation between high amounts of leaf litter and relatively low levels of rainfall. on the other hand, leaf litter decreased during months of high levels of rainfall. 40 biotropia no. 14, 1999 figure 6 . leaf and twig litter production during 54 months of obserbation in the primary forest site. figure. fruit and flower litter production during of months of observation in the primary forest site. 41 litterfal in a primary and two logged-over forest – upik r. wasrim & agus eka putra figure 8. stipule, bark and other litter component production during 54 months of obserbation in the primary forest site figure 9. average variability rainfall per month derived from monthly rainfall data between 1985 -1994. total litter production is also high, albeit less than half of the highest total value for january 1992, march 1993 and december 1994. this could be attributed to the increase in fruit litter in january 1992 and twigs litter in march 1993 and december 1994. fig. 7 compares litter attributed to flowers to that of fruit. the pattern of accumulation for each over time is relatively similar where periods of increased 42 biotropia no. 14, 1999 flower litter are followed by periods of increased fruit litter three months later as would be expected. for example, flower litter was high in october 1991 and october 1994 and fruit litter was optimal in january 1992 and february 1995. similarly, a relatively high level of fruit litter occurred from january 1995 till april 1996, preceded by a moderate accumulation of flower litter in november and december 1995. litter from other plant parts like twigs, stipules, and bark did not follow clear patterns of accumulation. fig. 10 compares litter production to the vegetation dynamics phases of a primary forest. the average monthly data of litter produced was obtained from 11 traps for the dynamic 1 phase, 26 traps for the dynamic 2 phase, 11 traps for the homeostatic 1 phase, and 27 traps for the homeostatic 2 phase (see fig. £ and fig. 3). in general, litter produced in the homeostatic 2 phase was higher than that produced in the other phases, with a total amount of 68 gm'2 month"1 compared to 62 gm"2month'' for homeostatic 1, 58 gm"2 month"1 for dynamic 2, and 53 gm"2 month"' for dynam ic 1. figure 10. litter fall in a primary forest based on vegetation dynamics phase. to assess the relationship between litter production, the rate of litter decomposition, and the vegetation dynamics phases, the data were statistically analyzed using anova (setyawan 1999). the results indicated that the data for the four vegetation dynamics phases, the logged-over forest cut in 1979/1980, and the logged-over forest cut in 1983/1984 were significantly influenced by litter production. when the data for the vegetation dynamics phases of the primary forest are 43 litterfall in a primary and two logged-over forests upik r. wasrin & agus eka putra compared to data for the logged-over forests, the highest amount of litter was produced in the homeostatic 2 phase, followed by the homeostatic 1, dynamic 2, and dynamic 1 phases, the logged-over forest cut in 1979/1980, and the loggedover forest cut in 1983/1984 with the lowest amount (setyawan 1999). the results suggest that litter production increases with stability or maturity of the forest ecosystem, considering that the vegetation dynamics phases of the primary forest from dynamic 1 to homeostatic 2 reflect a succession process toward stability or maturity characterized by the increase in tree structure. increased stability or maturity of a forest ecosystem results from the increase in forest structure, both horizontally (species distribution) and vertically (stratification). according to ewel (1983) forest structure increases with maturity. this increase in forest structures characterized by an increase in tree crown density, species diversity, tree age, and biomass that influences increased litter production (setyawan 1999). odum in jordan (1983) suggested that net production of litter increases as vegetation becomes larger and leaf area index goes up. litter production is optimal during the climax vegetation stage, and changes in the course of the succession process. litter production varies as it can be influenced by season, tree species, crown density, light, temperature variation during day and night, nutrient availability, disease, age of trees, and forest habitat size (alrasjid 1986; heald 1971). the lower value obtained for litter production in a logged-over forest compared to the primary forest, suggests that the logged-over forest structure is still in the building phase. this would mean that the logged-over forest structure is still unstable, and that its vegetation dynamics phase is more juvenile compared to the primary forest. the lower tree biomass resulting from the logging activity may also account for the lower value of litter production. soerianegara in setyawan (1999) suggested that logging activity might change the forest environment from climax to the unstable condition. in turn, this could affect tree growth. ecologically, logging activity could reduce biodiversity and soil fertility, increase soil erosion and surface run-off, thus decreasing the environmental quality in general. nutrient supplies decrease and forest stability is disturbed. b. litter fall in logged-over forest. the observation period for litter fall in a logged-over forest was shorter than in the primary forest, i.e. 29 months for the logged-over forest cut in 1983/1984 and 24 months for the logged-over forest cut in 1979/1980. litter fall in the logged-over forest cut in 1979/1980 and 1983/1984 are 60 gnrtnonuy1 or 721 gm^year1 and 59 gm"2month"' or 706 gm~2year~', respectively. if we compare litter production of the primary forest to that of the logged-over forest over the same observation period (fig. 11), the logged-over forests show relatively small differences in litter fall compared to the primary forest. litter fall amounts for the logged-over forest cut in 1979/1980 approach those for the primary forest, whereas litter fall levels for the logged-over forest cut in 1983/1984 are lower. this would suggest that the longer 44 biotropia 14, 1999 figure 11. litter fall of a primary forest and logged-over forest between july 1994-july 1996. time period following logging activity allowed the logged-over forest to recover its ondition toward climax, and begins to resemble the primary forest. the proportional distribution of litter components from the total in the loggediver forest cut in 1979/1980 and cut in 1983/1984 are presented in figures 12 and figure 12. percentage of litter component (average monthly dry weight) in a logged-over forest out in 1979/1980 45 litterfall in a primary and two logged-over forests upik r. wasrin & agus eka putra 13. like in the primary forest, leaves and twigs still comprise the major form of litter in the logged-over forest. however, the proportion of leaf litter to the other components in the logged-over forest is slightly higher than in the primary forest. the dynamics of leaf litter production in the logged-over forest cut in 1979/1980 and cut in 1983/1984 are shown in figures 14 and 15. in general, the highest production of leaf litter occurred in september october, while the lowest amount was in february april. this pattern was reflected in the total litter production,dynamics of both forest types (figures 16 and 17) as leaf ijtter yields the largest contribution to total litter produced. if we compare this with the average monthly rainfall data recorded between 1985 1994 (fig. 9), highest leaf litter production coincides with months of relatively low rainfall or toward the end of the dry season, while the lowest levels occurred in months with relatively high rainfall. this pattern is similar with that for the primary forest. this relates to the increase in availability of sun light in the dry season that increases photosynthetic activity. photosynthesis produces carbohydrate as a source of biomass increase and maturation of leaves and other parts of the plant, thus contributing to the increase in litter weight. the relation between flower and fruit litter production in the logged-over forest cut in 1979/1980 is not obvious from the data obtained. fig. 18 shows that figure 13. percentage of litter component (average monthly dry weight) in a logged-over forest out in 1983/1984 46 biotropia no. 14, 1999 figure 14. leaf and twig litter production between july 1994 – july 1996 in the logged-over forest cut in 1979/1980 figure 14. leaf and twig litter production between march 1994 – july 1996 in the loggedover forest cut in 1983/1984 47 litterfall in a primary and two logged-over – upik r. wasrin % agus eka putra figure 16. total litter production between july 1994 – july 1996 in the logged-over forest out in 1979/1980 figure 17. total litter production between march 1994 – july 1996 in the logged-over forest 1983/1984 48 biotropia no. 14, 1999 figure 18. fruit and flower litter production between july 1994 july 1996 in the logged-over forest cut in 1979/1980. relatively high production of fruit litter occurred in march 1995 and march 1996, whereas peak production of flower litter was recorded in august 1995 that might be responsible for the increase of fruit litter production in march 1996. referring to the rainfall data in fig. 9, highest flower litter production occurred in the months with the lowest monthly rainfall between october december, while highest fruit litter production occurred in the months with the highest rainfall between january march or during the wettest months. there is a similarity with the primary forest. however, to observe the dynamics of fruit and flower litter production in more detail, longer period of observation of the logged-over forest is needed for at least the same period of observation as that for the primary forest. in the logged-over forest cut in 19&3/1984 the highest amounts of leaf litter were produced between august october 1994 and in september 1995, while the lowest amounts were produced in june 1994, april 1995 and march 1996. the highest amounts of fruit litter were produced in march 1994, march 1995, and march 1996,.whereas the highest amounts of flower litter were produced in october 1994, july 1995 and november 1995. fig. 19 also shows that the increased production of flower litter influences fruit litter production proportionally. 49 litterfall in a primary and two logged-over forests upik r. wasrin & agus eka putra figure 19. fruit and flower litter production between july 1994 july 1996 in the logged-over forest cut in 1983/1984. acknowledgments this research is part of a joint research program between biotrop and barito pacific timber group (bptg) at the pasirmayang permanent plot, jambi. the authors gratefully acknowledge mr. husin and mr. harmen and their staff at pt. ifa-bptg for their assistance and support during the field work. we also wish to thank mr. musa for his assistance in the field and mr. waluyo for his assistance in the laboratory. our sincere thanks are also due to prof. dr. ralph ockerse, academic advisor and team leader hep-2 yogyakarta, for his generous assistance in rewriting the manuscript. references alrasyid, h. 1986. pelepasan unsur c organik dan unsur hara mineral lainnya selama pelapukan serasah di areal tegakan sisa hutan alam mangrove, sungai sepada, kalimantan barat. buletin penelitian hutan 503 : 29-44. binkley, d. 1986. forest nutrition management. a wiley-lnterscience publication. new york. 290 p. ewel, j. 1983. succession. in. golley, f.b. (eds.). 1983. ecosystem of the world 14a. tropical rain forest ecosystems : structure and function. elsevier scientific publishing company. amsterdam. 381 p. jordan, c.f. 1983. productivity of tropical rain forest ecosystems and the implications for their use as future wood and energy sources. in : ecosystems of the world 14a.tropical rain forest ecosystems : structure and function. elsevier scientific publishing company. amsterdam, p. 117-132 kira, t. & t. schidei. 1967. primary production and turn over of organic matter in different forestecosystem. ecology. vol. 17, no. 3 : 70 -87. 50 biotropia no. 14, 1999 korcagin, a.a. 1960. methods of determination of the seed productivity of forest trees and forest communities. field geobotany ii. acad. of science of the ussr press. laumonier, y. 1997. the vegetation and physiography of sumatra. geobotany 22. kluwer academic publisher. medwecka-kornas. 1967. ecosystem studies in a beech forest and meadow in the ojcao national park. in: studia nature. ser. a medwecka-kornas. 1970. plant litter production. unesco. newbould, p.j. 1967. methods for estimating the primary production of forests. ibp handbook no. 2. blackwell scientific publications. oxford. 62 p. setyawan, t.p. 1999. hubungan produktivitas dan laju dekomposisi serasah pada hutan alam primer dan hutan bekas tebangan di hutan alam produksi pasirmayang, jambi. skripsi. jurusan manajemen hutan, fakultas kehutanan, institut pertanian bogor. bogor. 67 p. wasrin, u.rosalina, a. eka putra and i. setiawan. 1995. optimalisasi produktivitas lahan hutan alam produksi di areal pengusahaan hutan pt. ifa-barito pacific timber group, di pasirmayang, jambi. laporan penelitian. kerjasama antara seameo-biotrop dengan barito pacific timber bogor. 52 p. 51 biotropia no. 6, 1992/1993: 1-32 notes on the family ampullariidae (gastropoda: prosobranchia) in the philippines: i. digestive, circulatory, and excretory systems roberto c. paoulayan and elpidio a. remigio institute of biology, college of science, university of the philippines, diliman, quezon city, philippines abstract a total of 232 ampullariid snails collected from 23 sites covering 7 islands in the philippines were compared conchologically and 200 alcohol-preserved specimens were dissected for anatomical characteristics. conchological comparison of the shells of the collected snails with that of identified lots from the senckenberg natur-museum, frankfurt, germany, the british museum for natural history, london, england, the koninklijk belgisch instituut voor natuurwetenschappen, brussels, belgium, and the rijksmuseum voor natuurwetenschappen, leiden, netherlands, revealed the presence of 5 species in the collected samples. these are: p. conica, p. ampullacea, p. mainitensis, p. quadrasi, and p. vittala. the latter 3 species were previously reported as being indigenous to the philippines. aside from characteristics of the shell, the morphology of the stomach may be useful for species discrimination. p. quadrasi and p. vittata, however, do not seem to differ anatomically from p. conica. introduction information on the taxonomy of the philippine snails belonging to the family ampullariidae (pilidae) is very deficient. there is a dearth of critical scientific data available aside from the initial reports on pilids from the philippines written more than a century ago by philippi (1851) and reeve (1856). although a number of species has been named and described, an accurate account of the number of existing species and a more reliable system of identification have yet to be clearly established. species differentiation has been based primarily on a very limited set of shell samples with rather polymorphic conchological features. examination of type and collected specimens in the following museums: the senckenberg natur-museum (smn), frankfurt a.m., germany; the british museum of natural history (bmnh), london, england; the koninklijk belgisch instituut voor natuurwetenschappen (kbin), brussels, belgium; and the rijksmuseum voor natuurwetenschappen (rvn), leiden, netherlands, as well as extensive literature search revealed that 9 nominal species of pila have been recorded from the 1 biotropia no. 6, 1992/1993 figure 6. a. the alimentary tract of p. conica. bar = 3 mm. b. jaws of p. conica. bar = 1 mm. abbreviations to figures see appendix 2. also roughly the boundary of the buccal mass with the esophagus. at this region, the roof of the buccal cavity is slightly thickened and is furrowed longitudinally. from this region the cavity leads to the esophagus dorsally and into the radular sac ventrally. the junction between the two entrances being an extended, furrowed flap (ff), corresponding to the elliptical pad of p. globosa (prashad 1925). the chitinous jaws (fig. 6b) are connected to each other on their dorsal edges by a very thin membrane (jm). each jaw is roughly oval in outline, with a thickened slightly serrated cutting edge (jce) which is also the most anterior edge of the jaw. a ridge (jr) is present that serves as the attachment of the jaws to the sphincter muscle. the cutting edge itself is slightly curved inwards. the more posterior portion of each jaw is thinner and bears a slight projection at its ventral edge. except for differences in size, which is dependent on the size of the snail, the jaws of the other species examined are similar to that of p. conica. there are two pairs of cartilages, the superior and lateral cartilages. the superior cartilages (fig. 8d: sc) are small somewhat triangular structures on the lateral sides of the anterior portions of the radula. medial and posterior to them 10 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio figure 7. representative radula of the tentatively identified species. a. p. conica: lake mainit, surigao del norte. b. p. ampullacea: lake manguiao, palawan. c. p. ampullacea: magallanes, sorsogon. d. p. mainitensis: oacao, palo, leyte. e. p. vittata( = p. conica): sta. cruz, marinduque. f. p. quadrasi ( = p. conica): lamluad valley, lake sebu, so. cotabato. bar = 100µm. 11 biotropia no. 6, 1992/1993 figure 8. buccal mass musculature of p. conica. a. side view b. dorsal view c. ventral view d. longitudinal section abbreviations in figures see appendix 2. see text for numbered muscles. bar = 1 mm. are the bigger lateral cartilages (lc). these are somewhat s-shaped and obliquely situated structures, having a thickened ventral edge. the relatively thinner upper portion curves inward and partly overlaps that of the opposite side. externally the lateral cartilages can be seen as ridges on the ventro-lateral sides of the buccal mass between the muscles, the bulk of the cartilages being embedded in the buccal mass. in all the specimens examined, the cartilages are of similar shape and number. the radula (fig. 8d: r) is a ribbon shaped structure, located on the floor of the buccal cavity, bearing the teeth that are arranged in a definite pattern in 33 41 similar transverse rows. the radula is taenioglossate (fig. 7a) consisting of seven teeth in the transverse row, one central (rhachidian) flanked on each side by one lateral and two marginals. the general radular formula is 2-1-1-1-2. the marginals, laterals, and to a certain extent the rhachidians overlap and obscure from view their structures. during preparation, individual teeth were deflected in order to reveal and clearly observe their fine features. the rhachidian teeth have horizontally elongated body from which 7 cusps project. the anterior margin of the body is grooved. the central cusp is the largest, triangular, spade-like, or dagger-like in form. there are two lateral teeth per row. each has a base that is elongated obliquely, with prominent ridges on the dorsal surface. the body is basically shouldered and typically bears five cusps: a large mesocone, a small endocone and ectocone, a basal 12 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio cusp at the antero-medial part of the base and hidden from view by the body, and another cusp lateral to the ectocone. the mesocone is the largest cusp, dagger-like in form, and has smoothly curved or straight lateral edges terminating in a pointed structure. there are four marginal teeth to a row. they are smaller than the laterals. immediately outer to the laterals are inner marginal teeth with narrow bases and longitudinal ridges. an inner marginal tooth has two cusps, a small inner and a large outer cusp. a basal lobe is present at the base projecting posteriorly and firmly attached to the redular membrane. the outer marginal is essentially identical to the inner one but lacks a basal lobe and is smaller. the radular sac (fig. 8a: rs) is a short, rather tubular, and somewhat rigid structure projecting ventrally out of the posterior end of the buccal mass from the midventral line. internally, the more posterior portion of the radula lies in this sac as a broad ribbon whose posterior end reaches the bottom of the sac. the inner wall of the radular sac facing the teeth appears somewhat fleshy and is in close contact with it. this part of the wall also extends to the buccal cavity terminating as the elliptical pad that forms the junction between the entrances to the esophagus and radular sac. the muscular system of the buccal mass (fig. 8) is highly organized in accordance with its complex movements. the action of some of the muscles is however sometimes difficult to interpret. as with the buccal cartilages, the musculature of the buccal mass in all specimens examined is similar. they are listed in table 1 according to the terminology of prashad (1925) and demian (1964). there are two salivary glands (fig. 8: sgl), one on each side, which lie on the postero-dorsal limit of the buccal mass. a short duct (sold) starts near the ventral, anterior edge of each gland and enters the buccal mass muscles. the ducts open into the buccal cavity near the flap forming the junction of the esophagus and the radular sac. the cream colored esophageal pouches (esp), one on each side, lie ventrolateral to the salivary glands. by careful dissection, they can be separated from the salivary glands. the pouches on each side has an opening into the buccal cavity near the junction of the buccal cavity and the esophagus. the internal walls of the pouches are simple with few, if any, longitudinal ridges. the esophagus (fig. 6a: eso) is a thin-walled tube arising from the postero-dorsal edge of the buccal mass. it continues posteriorly below the floor of the pallial cavity, arching to the left in a wide bend as it enters the visceral mass, slightly narrowing as it continues to the stomach at the level just below the pericardium. no difference in the form of the salivary glands, esophageal pouches, and esophagus was noted for the other species examined. the stomach (figs. 6a: st & 9a) is a somewhat complicated pouch located behind the posterior kidney chamber. it is seen externally as a maroon patch partly 13 biotropia no. 6, 1992/1993 table 1. terminology of the muscular system of the buccal mass according to prashad (1925) and demian (1964). 14 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio table 1. continued 15 biotropia no. 6, 1992/1993 figure 9. stomach of representative species (opened along the exposed margin). a. p. conica: u.p. lagoon, quezon city b. p. ampullacea: lake manguiao, palawan c. p. mainitensis: gacao, palo, leyte d. p. vittata( = p. conica): marinduque e. p. quadrasi ( = /". conica): lamluad valley, lake sebu, so. cotabato abbreviations in figures see appendix 2. 16 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio embedded in the digestive gland. upon exposure by slitting through the patch, the stomach shows a broad j-shaped cavity with a very muscular wall. the innerside is lined by longitudinal ridges. the cavity is divided into two areas, the gizzard (gi) at the anterior portion where the esophagus enters and the narrower, tubular, and relatively straight posterior style sac (ss). the esophagus enters the stomach near the midsection of the smaller (inner) curvature. posterior to the entrance of the esophagus, at the bend of the curvature, is the semilunar pit (ve), whose wall is elaborated by ridges.. in this pit are openings for the two ducts of the digestive gland (dgo). the margin of the semilunar pit arises as a prominent gastric shield (gs). the gastric shield, together with the wall of the stomach on the other side, prevents food coming from the esophagus, from entering the semilunar pit. the gastric shield at its posterior end continues to the style sac as the major typhlosole (mat). the wall of the style sac is marked by transverse ridges. running parallel to the major typhlosole is another ridge, the minor typhlosole (mit). the major and minor typhlosole border the intestinal groove (ig) between them. dorsal to the minor typhlosole, at the level of the beginning of the pyloric portion of the stomach, lie two stomach pouches (sp), one smaller than the other. the stomach pouches are somewhat flattened towards the external wall of the stomach and their lumina are separated by a relatively thin vertical septum. no crystalline style in the style sac was found in any of the specimens examined. the digestive gland (fig. 4: dg) is a conspicuously greenish mass occupying the upper portions of the spire and partly covering the stomach. externally, the digestive gland seems to be made up of two distinct lobes, an anterior and posterior lobe, the demarkation made by the position of the stomach as well as that of the two main ducts. upon dissection, however, the apparently "distinct" lobes are actually fused, having no internal demarkation between them. the border between the stomach and the intestine is situated just below the pericardium. this junction is a sharp u-shaped loop with a pleated caecum (fig. 6a & 9a: 1c). the intestine then runs between the stomach and the posterior chamber of the kidney, following the latter's margin to its posterior end. at this point, the intestine turns back and upwards ventral to the posterior kidney chamber. the intestine then makes several loops and seems to lie inside the cavity of the posterior kidney chamber (fig. 11b: in). the intestine then unloops, turning anteriad, running along the opposite margin of the posterior kidney chamber and along the gonoduct on the dorsal side as the rectum. before the rectum terminates into the rectal papilla, the duct of the rectal gland (fig. 6a: rcg) enters it on its dorsal side. the gland is branched and is situated at the dorsal side of the rectum on the level of the anterior portion of the gill. the anal papilla (anp) is a cylindrical 17 biotropia no. 6, 1992/1993 tube with a fringe opening projecting into the mantle cavity dorsal to the genital opening, as previously described above. interspecific variation in the alimentary tract: in p. ampullacea, each spadeshaped rhachidian tooth has a central cusp flanked by two pairs of smaller lateral cusps (figs. 7b & 7c). aberrant rhachidian teeth without lateral cusps were also observed in some snails. the lateral teeth have relatively smaller mesocones with smooth lateral edges. there are 35 37 rows of teeth in the radula. the rhachidian teeth of p. mainitensis (fig. 7d) is similar to that of p. ampullacea but the central cusps are more triangular, resembling an arrow head. the lateral teeth have larger dagger-like mesocone with straight lateral edges. there are 35-36 rows of teeth in the radula. the radular teeth of p. vittata and p. quadrasi (fig. 7e & f) are similar to those of p. conica. with respect to the morphology of the stomach, in p. ampullacea (fig. 9b), the more anterior of the stomach pouches (sp) is not markedly demarkated, giving the stomach an appearance of having only one pouch. it also has a relatively shorter style sac. the stomach of p. mainitensis (fig. 9c) is similar to that of p. ampullacea, although, the two stomach pouches are distinct. the stomach of p. vittata and p. quadrasi (fig. 9d & 9e) is similar to that of p. conica. no difference in the morphology of the intestine, rectum, and anal papilla was noted between the species examined. 3. the circulatory system of p. conica the pericardial cavity (fig. 10a: p) is situated on the left side of the body whorl lying between the posterior limit of the pulmonary sac and the kidney on the anterior and the stomach and the digestive gland on the posterior. its cavity communicates with that of the posterior kidney chamber through the renopericardial duct (fig. 1 ie: rpo) which runs in the septum that separates the pericardial cavity and posterior kidney chamber. the opening of this duct into the pericardial cavity can best be seen by deflecting the heart to the more anterior side of the pericardium. the opening of the duct is at the anteroventro-lateral wall of the pericardium. as the other monotocardians, pila conica has a single auricle and a single ventricle (figs. 10a & 10b). the auricle (a) is a thin walled sac capable of great distensions. internally, fine muscle fibers (am) in the form of thin bundles runs in various directions. however, most of the muscle fibers run longitudinally from the wall of the entrance to the auricle wall. the ventricle (v) is ovoid in form with a thick muscular wall. the auricle opens into the ventricle through the auriculoventribular opening. this opening is guarded by two valves (aw) attached by thin muscular fibers to the wall of the ventricle. as seen from the inner side, the wall of the ventricle is crisscrossed by large muscle bundles forming a coarse meshwork. 18 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio figure 10. a. general scheme of the circulatory system of p. conica. vessels drawn in solid lines are the more superficial vessels. vessels drawn in broken lines are the deeper vessels passing through the head foot region and the visceral mass. bar = 5 mm. b. heart (opened to show the internal structures). bar = 1 mm. abbreviations in figures see appendix 2. the cavity of the ventricle appears to be reduced, because of these muscular bundles. the ventricle in turn leads to the very short aortic trunk. the entrance of the ventricle to the aortic trunk is also guarded by two smaller valves (vav). the morphology of the pericardial cavity and the heart in the other species do not differ from that of p. conica. the ventricle leads to a short aortic trunk at its posterior end. this trunk then divides into the anterior (aa) and posterior aorta (ap). the former turns anteriad and expands into a pouch-like ampulla (am) occupying the most posterior end of the pericardium. the anterior aorta, shortly after leaving the ampulla, continues in its course anteriad, embedded in the muscular tissue and forming the leftventrolateral wall of the pallium. as the aorta reaches the level of the anterior portion of the esophagus, it splits into two big branches, one branch (aa2) turning right, making an exit from the wall, and emerging into the head sinus (the space surrounding the buccal mass and the anterior portions of the esophagus), the other big branch (aa1) continues its course anteriad eventually breaking into branches that supply blood to the left mantle wall, the osphradium, the left siphon, as well 19 biotropia no. 6, 1992/1993 philippines by several workers (philippi 1851; reeve 1856; kobelt 1911; bequaert and clench 1939). these species are: 1. pila conica (gray 1828) 2. pila luzonica (reeve 1856) 3. pila lubrica (reeve 1856) 4. pila vittata (reeve 1856) 5. pila ampullacea (linnaeus 1758) 6. pila moellendorffi (kobelt 1911) 1. pila mainitensis (kobelt 1911) 8. pila quadrasi (kobelt 1911) 9. forbesopomus atalanta (bequaert and clench 1939) up to the present, there are few local studies which have dealt with the other aspects of the snail's biology. palomino and jueco (1983) published data on the radula of some freshwater snails including pila luzonica. they showed the conservative structure of the ampullariid radula but the description lacks important structural detail. garibay et al. (1987) studied the life history as well as early embryogeny of laboratory-reared pilid snails which were identified as p. luzonica. this study hopes to give additional anatomical data which may be of use towards a better understanding of the ampullariid snails in the philippines. materials and methods the study was based on specimens collected in 1986, 1988, 1989 and 1990 from different geographical areas in the philippines as shown in figure 1. brief descriptions of these sites are listed in appendix 1. the shells of the snails collected in this study were compared with those of the identified museum lots in order to come up with a tentative identification of the former. such identification was based on ocular comparison of the shells. snail relaxation prior to dissection snails were first menthol-relaxed before killed and dissected or preserved in 70% ethyl alcohol. this was done by placing the snails in a small basin with two inches of water. a plastic cover was then placed on top. this plastic cover had a gauze sachet filled with menthol crystals taped on the underside. caution had been applied to prevent the menthol crystals from falling on the water as the snails are very sensitive and upon contact with menthol will immediately withdraw inside their shells. relaxation of the snails through "breathing" menthol vapor may be slow but produces the desired result. normally overnight relaxation was enough. bigger snails required more time. relaxed snails were then killed by immersion in 80°c water for 30 seconds prior to dissection or preservation. 2 biotropia no. 6, 1992/1993 as to the surrounding tissue of the left side of the mantle. the branch of the anterior aorta that emerges into the head sinus turns obliquely anteriad and after a short distance crosses the esophagus dorsally as it also gives branches to it. shortly after crossing to the right side of the esophagus, it subdivides into two branches. the right branch penetrates the right side of the sinus and within this wall, it turns anteriad to supply branches to the right siphon (egestion siphon), the tissue of the right side of the mantle, as well as the copulatory organs. the other branch turns ventrad and supplies blood to the buccal mass, the snout, and the foot region. the posterior aorta subdivides immediately after leaving the aortic trunk. one branch (fig. 10a: api) leads to the stomach and, through smaller branches, supplies blood to the various parts of the stomach. the other branch (ap2) can be seen just underneath the mantle epithelium, crossing the junction between the stomach and the intestine, running a course posteriorly between the intestine and the posterior kidney chamber. this artery gives off branches to the intestine, digestive gland, and the gonoduct in this region. the arterial system of the other species examined is similar to that of p. conica. the venous system made up of sinuses consists of the head-foot sinus, (fig. 10a: hf). the main trunk of the foot sinus can best be seen by removing the pedal-pleural ganglia complex. ventrally lies the opening of the foot sinus to the large head sinus. the main trunk of the foot sinus is rather large and situated medially collecting tributaries of smaller sinuses from all parts of the foot. the head sinus is relatively the largest of all sinuses in the snail. it lies above the foot and below the floor of the mantle cavity surrounding the anterior part of the alimentary tract. some of the blood from this large sinus goes to the tributaries of the pallial vein, through small openings along the left and right walls. these tributaries then unite, on each side, to form the main pallial veins (pv). the right pallial vein joins the afferent gill vein (agv) and the left pallial vein opens into the afferent lung vein (alv). the rest of the blood exits from openings on the more posterior portion of the right wall of the head sinus. these openings lead to the sinus through which the sub-intestinal-visceral connective nerve also passes. this sinus (fig. 1 ib: akv) continues as the main branch of the afferent kidney vein as it enters the viceral mass. afferent kidney vein: (fig. 11b: akv). the main branch of this vein turns obliquely inward, becoming larger in diameter. it follows a course along the posterior wall of the columellar muscle, just anterior to the digestive gland where it receives branches from the digestive gland, stomach, and the anterior portions of the intestine, to a level between the junction of the posterior and anterior renal chambers. here it then divides, one branch to become the afferent vessel of the posterior kidney chamber (akpv) and the other to become the afferent vessel of the anterior kidney chamber (akav). 20 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio figure 11. the excretory system of p. conica. a. dorsal view of the anterior and posterior chambers. bar = 5 mm. b. posterior chamber (opened along its margin). bar = 1 mm. c. anterior chamber (opened to show the internal structure). arrow points to the extension of its base. bar = 1 mm. d. lamella of the anterior chamber showing position of blood vessels. bar = 1 mm. e. opening between the anterior and posterior chambers cut to show the opening of the reno-pericardial canal. bar = 1 mm. abbreviations in figures see appendix 2. 21 biotropia no. 6, 1992/1993 branchio-renal complex: this is a system of vessels leading to the gill or in part to the pulmonary sac (fig. 10a: agv & alv). there are two efferent branches from the anterior kidney chamber (fig. 10a, 11c & 1 id: ekav). one branch can be seen prominently along its median axis and exiting to the afferent gill vessel. the other branch is located on the posterior edge of the chamber and receives blood from this chamber as well as some blood from the posterior kidney chamber and the intestines. at the anterior tip of the anterior kidney chamber, this branch receives blood coming from the rectum through a short vessel (rcv), then exits to the afferent gill vessel. the afferent gill vessel (agv) is a prominent vessel with a relatively large diameter that runs alongside the gill, giving branches to the gill leaflets. at its main trunk it receives two to three short vessels from the rectum on the right side (rcv). at the most anterior end of the afferent gill vessel, it receives blood from the genital and rectal papilla as well as the right pallial and visceral vein. the visceral vein (vv) collects blood from the intestines, and the digestive and genital glands then runs alongside the gonoduct ventrally, collecting blood from it along the way. at its anterior end, it seems to join with the pallial vein shortly before entering the afferent gill vein. the afferent gill vein has also an anterior connection with the afferent pulmonary vessel, running along the margin of the mantle. at the left and right side, the afferent pulmonary vessel (apv) receives branches coming from the osphradium (osv), the copulatory organ (cov), and branches from the mantle edge (mv). the vessels leading to the auricle of the heart are as follows: a), the efferent vessel of the posterior kidney chamber (ekpv), whose main trunk and tributaries can be best seen at the inner side of the roof of the chamber. it runs a course through the pericardium entering the extreme right edge of the auricle, its entrance (fig. 10a & b: ekpv) being partly obscured by the fine longitudinal muscles of the auricle; b). the efferent lung-gill vessel (fig. 10a: elgv) this is a relatively big vessel located beneath the skin on the dorsal side running between the edges of the gill and the pulmonary sac. it collects aerated blood from the pulmonary sac and the gill leaflets through a series of small vessels opening on its left and right walls, respectively. it enters the auricle prominently more or less at its tip. some differences in the venous system were noted in the other species examined. in p. mainitensis and p. ampullacea, one of the branches of the efferent branch from the anterior kidney chamber (ekav1) runs along the margin of the anterior kidney, somewhat alongside the afferent gill vessel. as to the vessels entering the auricle, in p. mainitensis, there seems to be a short efferent lung vessel that collects blood from the more posterior portion of the sac, aside from the efferent 22 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio lung-gill vessel. this vessel may, however, be simply a part of the efferent lung-gill vessel as it joins the latter before the latter enters the auricle. the venous system of p. vittata and p. quadrasi do not differ from that of p. conica. 4. the excretory system of p. conica the renal organ or kidney (fig. 11) consists of two chambers an anterior and a posterior chamber. the anterior chamber (fig. 11 a: ak) could be seen on the upper side of the animal as a reddish brown elongated structure lying to the right of the pericardium after removal of the shell. its floor projects somewhat into the mantle cavity, and opens into it to the right of the epitaenia. it can be differentiated from the posterior chamber by the lamellated appearance of its inner wall which is seen through the thin mantle epithelium at its dorsal surface. on opening the chamber by making a slit along the roof margin and then deflecting the resulting flap, two longitudinal, whitish, and medially placed structures with triangular lamella arising from their sides could be observed (fig. 1 ib). these were the main branches of the afferent vessel of the anterior kidney chamber (akav1 & akav2) coming from the main trunk of the afferent vessel of the kidneys described above. one branch (akav1) is placed on the floor of the chamber while the other (akav2) is located at the inner dorsal roof. these vessels divide into smaller branches as they enter the lamellae (fig. 1 id). at the periphery of the lamellae, the tiny branches enter the efferent kidney veins of the chamber (ekav1 & ekav2). one of these (ekav1) is found along the posterior margin of the chamber forming a part of the branchio-renal complex. the other (ekav2) courses along the dorsal surface of the anterior kidney chamber. the anterior kidney chamber also forms a branched appendage, projecting from its posterior edge near the junction with the posterior kidney chamber (fig. 11 a: arrow). it is also lamellated and forms the dorsal roof of the opening of the anterior kidney chamber into the mantle cavity (fig. 11b: ok). at this level, but more medially placed on the floor of the chamber, is a longitudinal slit (o). this is the opening through which the posterior kidney chamber communicates with the anterior chamber. the posterior kidney chamber: (fig. 11 a: pk). the roof of the posterior kidney chamber prominently lies to left of the rectum as a broad brownish structure with a hooked posterior portion. on making a cut along the margin of the roof and deflecting the flap anteriorly, the spacious cavity of the chamber can be seen (fig. 1 ib). the soils of the intestine, parts of the genital duct, and portions of the digestive gland can be seen projecting into the cavity. the floor of the chamber consists of a very thin membrane separating the cavity from the organs projecting into it. in 23 biotropia no. 6, 1992/1993 contrast, the roof of the cavity appears to be thicker and spongy due to the thick plexus formed by the relatively large and branched posterior kidney vessels. there are two main vessels the afferent (akav) and efferent (ekav) vessels of the posterior chamber, and a minor vessel system (fig. 10a: arrow) that is found at the right margin of the organ and exits into the branch of the efferent vein of the anterior kidney chamber on that side. the main trunk of the afferent and efferent vein of the posterior chamber is seen prominently placed at the median portion of the chamber's roof. the posterior kidney chamber is separated from the pericardial cavity by a thin vertical septum. the renopericardial canal connecting the pericardial cavity to the posterior kidney chamber is found in this septum. the canal opens at the tip of a very short papilla very near the longitudinal slit communicating the posterior with the anterior kidney chamber described above (fig. 1 ie: rpo). except for differences in size, which is dependent upon the size of the snail, the morphology of the kidney of the other species examined does not differ from that of p. conica. discussion distinct difference in mantle or body pigmentation was not observed. although some snails have shells that have lighter color, yellow or orange without bands, have been observed, such seem to be independent of mantle pigmentation. snails with lighter colored shells still possess normal black pigmentation of the mantle and the other parts. all members of the family ampullariidae are amphibious. such an adaptation is due to their possession of a ctenidium and a lung sac which are both functioning. this has enabled some members, especially pila, lanistes, and pomacea, to exist in a wide range of habitats (mozley 1939; mandahl-barth 1954; andrews 1965a). they can, in addition, withstand seasonal drought by aestivating (meenakshi 1956). their distribution, however, seems to have a latitudinal limit, being widely distributed only along the tropical and subtropical belt. in turbinicola, asolene, lanistes, pomacea, and marisa (scott 1943; kretschmann 1955; demian 1958; andrews 1965a), the downgrowth of the mantle which forms the lung sac is relatively extensive. the same observation has been noted in the present study on pila. in afropomus balanoides, the lung-sac is formed by a simple circular fold of the roof of the mantle cavity (berthold 1988). as previously observed in other pila species , the lung sac of the snails in this study has a wide slitlike aperture, unlike the small aperture reported for pomacea and lanistes. the 24 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio development of a small lung sac aperture thus seems to represent convergence towards a small and more efficiently manageable aperture (andrews 1965a). due to its peculiar position, the gill seems to correspond to the osphradium on the left with which, however, it is not homologous (prashad 1925). as with other monotocardian prosobranchs, the gill of ampullariid snails is actually the left gill which is shoved to the right side by the development of the extensive pulmonary sac (fretter & graham 1962). one structure that probably plays an important role in the direction of water currents in the mantle cavity, as well as in separating the pulmonary sac from the gill and in providing protection by effectively closing of the ctenidium when the water condition turns foul, is the epitaenia (andrews 1965a). the general morphology of the alimentary tract as described is similar in all the species of pila examined. the characteristics of the radular teeth, especially the differences seen in the number and form of the cusps, have a limited use for species differentiation. this is due to (1) the high probability of the mechanical destruction or modification in the form of the cusps and (2) the observed fusion of cusps in some individuals of the same species. the basal lobe at the inner marginal tooth was described by moretto and nahabedian (1983) for pomacea canaliculata and by berthold (1988) for afropomus balanoides. it was observed in all the snails here examined. such observation supports berthold's statement that a basal lobe is present in the inner marginal teeth of all the other pilid genera. the general morphology of the rest of the buccal mass as well as the salivary glands, esophageal pouches, and the anterior portion of the esophagus is also similar to that described for p. globosa, m. cornuarietis, p. canaliculata, and a balanoides (prashad 1925; scott 1957; demian 1964; berthold 1988). there are two stomach pouches in all the snails examined. the presence of two stomach pouches has also been observed in marisa cornuarietis (demian 1964) and afropomus balanoides (berthold 1988) and in the thai ampullariidae (keawjam 1987). the stomach pouches in pila, in this study, were not as big as keawjam's illustration for the thai pilidae. andrews (1965b) reported only one pouch ("glandular pouch") in pomacea canaliculata. it is possible that the anterior pouch, as from my observations in p. ampullacea, is not so extensive. rod-like fecal material in the dissected style sac of some of the snails examined were noted. compaction of fecal and excretory material seems to occur at the style sac (andrews 1965b). as previously observed in other genera, there is no crystalline style in the style sac of pila. a caecum which seems to be divided into transversely positioned chambers was found in all the snails dissected in this study. it was also described in the other ampullariid genera such as in afropomus (berthold 1988) and in pila, turbanicola, 25 biotropia no. 6, 1992/1993 and pomacea (andrews 1965b). it may be that the presence of this anterior caecum is a synapomorphy shared by the members of the family ampullariidae. short, semi-compact, pellet like material were noted in the portion of the intestine just after the caecum, giving credence to the suggestion that pellet formation is brought about in the caecum in pila (andrews 1965b). the coiling of the intestine, seen only when the edges of the posterior kidney chamber is cut open, probably needs more attention. in pila, here examined, and in afropomus (see berthold 1988, p. 154,f.bc), the pattern of coiling is similar, while that of pomacea (see andrews 1965b, p.21,f.l) is simpler. the rest of the posterior portion of the alimentary tract shows no difference from that described by andrews (1965b) for pomacea. an anal gland has not been described for afropomus by berthold (1988). it may have been overlooked, as was the case for p. globosa by prashad (1925) and for the thai ampullariidae by keawjam (1987). a similar gland appears in some members of the prosobranchia, including archeogastropods and neogastropods (hyman 1967). the description of the anal glands of some stenoglossans (fretter 1946) resembled that of pila. the anal glands probably play a role in the elimination of excess calcium and iron salts and purines, of which its development in ampullariids may be correlated with their freshwater habits (andrews 1965b). as a final note on the digestive gland of pila, ranjah (1942) proposed that originally there were two lobes of the digestive gland but the anterior (post-torsional left) lobe is supressed at an early stage in development. two distinct ducts were observed in all the specimens dissected. their separated location from each other precludes the presumption that they represent two main branches from a single lobe of the digestive gland. andrews (1965b) observed that in young pomacea, there were two distinct lobes, although a single lobe was observed in mature snails. apparently, the single lobe seen is the result of fusion of the two lobes, such that any border which formerly existed became indistinguishable due to the very close contact between the two lobes. such a close contact is expected when one considers a very limited space available in the visceral hump for the development of a large structure such as a multifunctional digestive gland. in all the species examined, the outline of the venous system appeared to be similar, except for that minor difference in the efferent lung-gill vein of p. mainitensis where a short "pulmonary sinus" is located very near the entrance to the auricle. such a peculiar position gives it an appearance of an independent pulmonary vein. this peculiar sinus, however, opens into the efferent lung-gill vein and not directly into the auricle; therefore it may still be a part of the latter. prashad (1925) described a distinct pulmonary vein for p. globosa, but this was later refuted by andrews (1965a). observations on the venous system of the species of pila examined here are similar to that described by andrews (1965a). 26 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio blood from the posterior kidney chamber exits from two points: (1) the larger efferent vessel of the chamber that opens directly into the auricle and (2) two to four small sinuses from the more anterior right margin of the chamber and joining the efferent vein of the anterior chamber at that area. andrews (1965a) postulates that the larger efferent vein of the posterior chamber is actually the vein from the degenerate nephridial gland. this vein has been enlarged to drain a larger area of the chamber and the actual efferent vein of the chamber is that vessel that joins the second branch of the efferent vessel that drains the anterior chamber. the basic plan of the arterial system of pila here examined is similar to that described for other pilids like p. globosa (prashad 1925), p. canaliculata, t. saxea, l. ovum bangweolicus (andrews 1965a) and a. balanoides (berthold 1988). berthold, however, described a slightly thickened ampulla for afropomus and added that such a form seems to be plesiomorphic compared to the bigger globular ampulla of the other species mentioned above. the members of the family ampullariidae seem to be peculiar in having a kidney made up of two chambers which are morphologically distinct from each other. the general shape as well as position with each other of the anterior and posterior kidney chambers in all the species of pila here examined is similar to that described by prashad (1925) and illustrated by michelson (1961) for p. globosa. summary 1. species identification of the ampullariid snails in the philippines was previously based only on characteristics of the shell, hence, an approach was initiated in this study towards basing identification on as many characters as possible. 2. tentative identification of the collected snails based on conchological comparison with museum specimens were done and five species from the collection were identified. these are: p. conica, p. vittata, p. quadrasi, p. mainitensis, and p. ampullacea. 3. dissection of the soft anatomical parts revealed few character states in the organ systems here studied which may be useful for species identification. these are: (a) the number of osphradial and gill leaflets, (b) the morphology of the cusps of the rachidian teeth (although caution must be exercised in using this character as the radula is prone to mechanical modification), (c) the morphology of the stomach, and (d) the position of the efferent branch of the anterior kidney chamber (ekav1). no difference was observed in the other organs here studied. 4. p. vittata and p. quadrasi have been observed to be anatomically indistinct from p. conica based on comparison of the digestive, circulatory, respiratory, and excretory systems. 27 biotropia no. 6, 1992/1993 acknowledgements during these studies the authors had the support of several private persons and institutions. technical assistance or advise was received from mr. manny sapuay of nsri (photography) and the staff of u.p. n.e.c. (scanning microscope). prof. dr. edmund gittenberger, dr. jackie van goethem, dr. ronald janssen, and dr. peter mordan for allowing me to examine their collection of philippine ampullariid snails. dr. rita triebskorn for sending me much needed literature and dr. tomas berthold for sharing his insights on the group of snails studied. tus and macy mamaril and our assistant, richard kho for the company and lively discussions during the collection trips. dr. gloria l. enriquez, dr. claus meier-brook, and dr. ruben umaly for their critical advices and encouragements. the german academic exchange service (daad) for supporting the senior author's research scholarship in germany. the office of research coordination for the research grant that allowed us to collect snails. we also wish to express our gratitude to our countless colleagues in the institute of biology and outside the university who have in one way or another contributed to this work. literature cited andrews, e.b. 1965a. the functional anatomy of the mantle cavity, kidney, & blood system of some pilid gastropods (prosobranchia). j. zool. 146: 70-94. ______. 1965b. the functional anatomy of the gut of the prosobranch gastropod pomacea canalicuta and of some other pilids. proc. zool. soc. london 145: 19-36. bequaert, j.c. & w.j. clench. 1937. forbesopomus, a new genus in the family pilidae (ampullariidae), from the philippine islands. proc. new engl. zool. club 16: 53-56. berthold, t. 1988. anatomy of afropomus balanoides (mollusca: gastropoda: ampullariidae) and its implication on phylogeny and ecology. zoomorphology 108: 149159. demian, e.s. 1958. on the respiratory system and the mechanisms of respiration in lanistes bolteni chemnitz. ain shams sci. bull. cairo, 3: 301-316. ______. 1964. the anatomy of the alimentary system ofmarisa comuarietis (l.) goetoborgs vetensk. vitterhets-samh. handh. 6 ser. b 9(2): 1-75. fernando, w. 1931. the development of the kidney in ampullaria (pila) gigas. proc. zool. soc. london 62: 745-750. fretter, v. 1946. the pedal sucker and anal gland of some british stenoglossa. proc. malac. soc. london 27: 126-130. fretter, v. and a. graham, 1962. british prosobranch mollusk, their functional anatomy and ecology. ray society, london. garibay, j.l., r.c. pagulayan and b. cruz. 1987. studies on pila luzonica reeve: hatchability and growth under laboratory conditions. nat. appl. sci. bull. 39(1): 81-90. 28 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio hyman, l.h. 1967. the invertebrates. volume vi. mollusca i. mcgraw hill, new york. keawjam, r. 1987. the apple snails of thailand: aspects of comparative anatomy. malacol. rev. 20: 69-90. kobelt, w. 1911. die gattung ampullaria. in: vol. i, pt. 29 ii (neue folge) of systematisches conchylien-cabinet von martini und chemnitz. ed. w. kobelt, von bauer, und raspe. nurnberg: 76-99. kretschmann, i. 1955. untersuchungen ueber bau und function der atmungsorgane bei ampullarien. wiss. z. humboldt-univ. berlin 4: 109-119. mandahl-barth, g. 1954. the freshwater mollusks of uganda and djacent territories. ann. mus. roy. congo bel. tervuren, sciences zool. 32: 1-206. meenakshi, v.r. 1956. physiology of hibernation of the apple snail pila virens (lam.). curr. science 25: 21-322. michelson, e.h. 1961. on the generic limits in the family pilidae (prosobranchia: molluska). breviora 133: 1-10. moretto, h.j.a. and d.e. nahabedian, 1983. the radula of ampullaria canaliculata (prosobranchia: mollusca). comun. mus. arg. cienc. nat. bernardino rivadaria inst. nac. invest. cienc. nat (arg.) (hydrobiol.) 2: 107-117. mozley, h. 1939. the freshwater molluska of the tanganyika territory and zanzibar protectorate and their relation to human schistosomiasis. trans. roy. soc. edinb. 59: 687-744. palomino, m.l.p. and n.l. jueco. 1983. radular pattern in three edible freshwater snails. kalikasan, phil. j. biol. 12: 174-176. philippi, r.a. 1851. die gattung ampullaria. in: vol. 1, pt. 20 of systematisches conchylien-cabinet von martini und chemnitz. h. kuster, von bauer, und raspe (eds.) nurnberg. prashad, b. 1925. anatomy of the common indian apple snail, pila globosa. mem. indian mus. 8: 91-152. ranjah, a.r. 1942. the embryology of the indian apple snail pila globosa (swainson), mollusca, gastropoda. rec. indian mus. calcutta 44: 217-322. reeve, l. 1856. monograph of ampullaria. conchologica iconica 10. london. scott, m.i.h. 1943. sobre la organizacion de ampullaria (asolene) megastoma sowerby. not. m\is. la plata 8, zool. no. 70: 269-280. _______. 1957. estudio morfologica y taxonomica de los ampullaridos de la republica argentina. rev. mus. argent. cienc. nat. (zool.) 3: 233-333. 29 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio both menthol-relaxed, alcohol preserved snails and menthol-relaxed, freshly killed snails were used for anatomical studies. the effect of alcohol fixation, particularly on the size of the organs and tensus were noted accordingly. dissection of the soft parts approximately 200 snails were dissected and anatomically examined. the following steps were done in the removal of the soft parts from the shell without crushing the shell. to remove the operculum, a blunt edged, spatula-like probe was inserted in the space between the operculum and its attachment on the dorsal side of the foot. while holding the snail's body steady, the operculum was loosened by pressing at the attachment area with the probe. for the rest of the soft body, the probe had been inserted in the space between the columellar muscle and the shell. by exerting a little pressure, without puncturing the tissue, the muscle was pried loose from its attachment. a pair of forceps was used to grasp the snail's foot and to pull the body out while rotating it, following the coiling of the shell. dissection was done under a wild zoom stereomicroscope m7. exposure of the mantle cavity was accomplished by a cut made beginning at the right mantle edge, dorsal to the exhalant siphon, between the gill and the rectum up to the level of the anterior chamber of the kidney. in exposing the anterior portions of the digestive tract, a longitudinal slit was made starting from the dorsal tip of the snout and continued posteriorly to the area of exit of the esophagus to the visceral mass. after these two major incisions, most of the organs could be seen through the thin mantle and could be reached without much difficulty. they could, therefore, be exposed in situ. camera lucida drawings were done with a wild drawing tube. radulae were obtained by extracting buccal masses from the snails and immersing them in 10% sodium hydroxide for 24 hours. the radulae were further cleaned by sonication while immersed in 70% ethanol and subsequently processed for microscopic examination. some of the radulae were immersed in 10°7o sodium hypochlorite to dissolve the radular membrane and facilitate the observation of the lateral lobes of the inner marginal teeth. the rest were eventually air-dried, mounted in specimen blocks, sputter coated with gold using a jeol fine coat ion sputter jfc-1100, and examined under a jeol jsm-35c s.e.m at the national engineering center, in u.p. diliman. voucher specimens are presently lodged at the invertebrate museum, institute of biology, u.p. diliman, q.c. and at the mollusc division, national museum, manila. 3 biotropia no. 6, 1992/1993 appendix 1 30 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio appendix 1. (continued) 31 biotropia no. 6, 1992/1993 appendix 2. list of abbreviations a = auricle of the heart j = jaws aa = anterior aorta jce = cutting edge of jaw aabm = branch of the anterior aorta to the buccal jm = membrane connecting the two mass jaws agv = afferent gill/ctenidial vessel jr = ridge of jaw ak = anterior kidney chamber lp = labial pulps akav = afferent kidney vein to the anterior chamber ls = lung/pulmonary sac akl = lamella of the anterior kidney chamber lso = opening of the lung sac into akpv = afferent kidney vein to the posterior the mantle cavity chamber mat = major typhlosole akv = main vessel of the afferent kidney vein mit = minor typhlosole alv = afferent lung vein me = mantle edge am = auricular muscles mo = mouth amp = ampulla of the anterior aorta mv = mantle veins anp = anal/rectal papilla o = slitlike opening between the ap = posterior aorta anterior and posterior kidney aw = auriculo-ventricular valves chambers bm = buccal mass ok = opening of the kidney bmc = cavity of the buccal mass chamber into the mantle c = columellar muscle cavity co = copulatory organ opl = operculiferous lobe on dorsal cov = vessels from the copulatory organ side of the foot do = digestive gland os = osphradium dgd = duct of the digestive gland osv = osphradial vein e = eyes ov = ovary ekav = efferent kidney vein from the anterior p = pericardium chamber pk = posterior kidney chamber ekpv = efferent kidney vein from the posterior pv = pallial vein chamber r = radulla elgv = efferent lung-gill vein rcg = rectal/anal gland ep = epitaenia/mantle fold rcv = rectal vein es = egestion/exhalant siphon rpo = renopericardial opening eso = esophagus rs = radular sac esp = esophageal pouches sc = superior cartilage of the buccal mass sgl = salivary gland f = foot sgld = salivary gland duct g = gill/ctenidium so = subradular organ gi = gizzard sp = stomach pouches gp = genital papilla ss = style sac of the stomach gs = gastric shield st = stomach hf = head-foot sinus t = testis hm = muscles of the internal wall of the heart te = tentacle 1c = intestinal caecum v = ventricle ig = intestinal groove va = ventriculo-aortic valve in = intestine ve = vestibule is = ingestion/inhalant siphon vm = ventricular muscles vv = visceral vein 32 biotropia no. 6, 1992/1993 results species found in the collection sites are map-pinpointed in figure 1. representative photographs of the shells of the snails examined are presented in figures 2 & 3. figure 1. species noted in the collection sites. a-paoay, ilocos norte; b-tuguegarao, cagayan; c-san isidro, isabela; d-caraen, pangasinan; e-u.p. lagoon, quezon city; f-tanay & montalban, rizal; g-magallanes, sorsogon; h-pinamalayan, oriental mindoro; i-boac & sta. cruz, marinduque; j-alimodian, iloilo; k-palo, leyte; l-mainit, surigao del norte; m-lake sebu, marbel, & koronadal, south cotabato; n-lake manguiao, palawan. 4 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio figure 2. representative shells of the snails identified as pita conica from the following sites: a-tuguegarao, cagayan; b-paoay lake, ilocos norte; c-san isidro, isabela; d-carmen, pangasinan; e-u.p. lagoon, quezon city; f-tanay, rizal; g-pinamalayan, oriental mindoro; h-gacao, palo, leyte, 1-hubang, palo, leyte; j-alimodian, iloilo; k-lake mainit, surigao del norte; l-lake sebu, so.cotabato; m-lamluad valley, so. cotabato; n-marbel, so. cotabato; o-koronadal, so. cotabato. bar = 5 mm. 5 figure 3. representative shells of the other species collected. a.-p. ampullacea: lake manguiao, palawan; 3-p. ampullacea: magallanes, sorsogon; c-p. mainitensis: gacao, palo, leyte; d-p. vittata ( = p. conica): sta. cruz, marinduque; e-p. quadrasi ( = p. conica): lamluad valley, so. cotabato. bar = 5 mm. anatomical studies 1. external morphology and description of the mantle cavity of p. conica the body of the animal is divisible into three regions (fig.4) head, foot, and visceral mass. the head is prolonged into a contractile snout. the elongated snout terminates anteriorly on its two sides into a pair of short labial pulps (lp) or tentacles. posteriorly the head bears a pair of long contractile tentacles (te). when fully extended the tentacles measure about 50 mm in length, thicker at its base but gradually tapering to a filamentous structure at the tip. when contracted, as when the animal is disturbed or as in the case of preserved specimens, the tentacles are reduced to about one-fourth of their length. the eyes (e) are situated on small stalks situated on the outer sides of the bases of the tentacles. projecting anteriorly on the two sides of the head are two lobes. the left lobe forms the inhalant siphon (is) and the right lobe forms the exhalant siphon (es). 6 biotropia no. 6, 1992/1993 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio figure 4. external features of the body of p. conica. a. left view; b. right view female; c. right view male; d. view of the mantle cavity. bar = 3 mm. abbreviations in figures see appendix 2. the foot (f) is triangular in outline with the apex directed backwards. the sides are more or less arched in broadly rounded angles. the operculum is attached to the dorsal surface of the posterior half of the foot (fig. 5c:opl). the columellar muscle (fig. 5c) is seen as a broad band arising from its attachment to the columella and running longitudinally through the upper part of the foot to the operculiferous lobe (opl) on the postero-dorsal surface. 7 r . eigv agv ak co iso os dg biotropia no. 6, 1992/1993 figure 5. a. osphradium of p. conica. bar = 1 mm. b. gill leaflet of p. conica. bar = 1 mm. c. collumellar muscle of p. conica. arrows point to area of attachment to the shell. bar = 6 mm. abbreviations in figures see appendix 2. dorsally, the mantle encloses the mantle cavity in which the structures of the pallial complex are situated (fig. 4d). the mantle is thick along its free edges (me). as seen dorsally, pigmented streaks run longitudinally from this free edge eventually coalescing to form a more or less united pigmented patch covering the mantle roof. the pedunculate osphradium (fig. 4d: os & 5a) hangs from the roof of the left side of the mantle close to the edge. it is elongate-oval in shape, somewhat pointed along its left margin and rounded on the right inner end. it is bipectinate and consists of an average of 35 fleshy leaflets arranged along its broad axis. the osphradium hangs transversely to the course of the respiratory water current as it flows into the mantle cavity. posterior to the osphradium and occupying the greater part of the mantle cavity is the pulmonary or lung sac (fig. 4d: ls), arising from the ventral mantle wall as a more or less circular unpigmented folding with a ventral opening into the mantle cavity (lso). the tissue of the pulmonary sac is moderately thickened and is richly supplied with blood sinuses. 8 notes on the family ampullariidae in the philippines r.c. pagulayan & e.a. remigio the gill or ctenidium (fig. 4d: g & 5b) is composed of approximately 170 triangular leaflets running alongside and to the right of the pulmonary sac. each triangular gill leaflet is attached along the ctenidial axis and has two free sides of unequal sizes, consequently, the slightly rounded apex is skewed to the right. the epitaenia (fig. 4d: ep) arises from the floor of the mantle cavity. it runs from the left side to the right, ending at the base of the left margin of the exhalant siphon, anteriorly. inside the right wall of the mantle, the rectum runs parallel to the gill on the right side opening to the anal papilla (fig. 4d: anp), and extended projection of the mantle roof with a fringed opening. to the right of the rectum running parallel to it, lies the pallial portion of the reproductive duct terminating into the genital papilla (gp). the genital papilla in males is a somewhat conical structure crossing the anal papilla ventrally. its tip, in which the genital opening is located, is in close contact, but not attached, to the base of the copulatory organ. in females, the pallial oviduct opens into a broad and short genital papilla located just beside the anal papilla. in egg-laying females, the genital opening is enlarged and the papilla is almost obliterated. in all snails examined, a copulatory structure (co) consisting of a penis enclosed in a penis sheath hangs from the right mantle roof. although present, the copulatory organ is degenerated in females. interspecific variation in the structures of the pallial complex: in p. ampullacea, an average of 260 gill leaflets were counted, and in p. mainitensis, an average of 245 leaflets. as to the number of osphradium leaflets, p. ampullacea has about 58 68 leaflets, and p. mainitensis has about 52 54 leaflets. no apparent difference in pigmentation of the head-foot region was observed between the species, although, in p. ampullacea and p. mainitensis, pigmentation of the penis sheath was observed. such pigmentation was not observed in the other species. the pallial complex of p. vittata and p. quadrase do not differ from that of p. conica. 2. the alimentary tract of p. conica the buccal mass which forms the first part of the alimentary tract (fig. 6a) is found inside the head sinus. the buccal cavity (fig. 8d: bmc) is subdivided into different regions. the anterior region is rather small, bounded anteriorly by the mouth opening (mo) and posteriorly by the jaws (j). the skin forming its walls are thickened, partly pigmented, and with longitudinal folds. from the opening of the jaws, the cavity continues as a very short tube, then flattens with its margins arched ventrad and posteriad following the countour of the subradular organ (so) and the more anterior portions of the radula. the cavity then widens a little on the level of the junction of the opening of the esophageal pouches. this region is 9 1.pdf 10.pdf 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 2.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf 3.pdf 30.pdf 31.pdf 32.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf microsoft word 11 biotropia no. 20, 2003: 11 23 potency of vibrio isolates for biocontrol of vibriosis in tiger shrimp (penaeus monodon) larvae widanarni1'2. a. suwantou',sukenda2,and b. w. lay4 'department of biology, faculty of science and mathematics, bogor agricultural university, bogor 16144, indonesia 2 department of aquaculture, faculty of fisheries and marine science, bogor agricultural university, bogor 16680, indonesia 3seameo-biotrop, jl. raya tajur km 6, bogor 16720. indonesia ^faculty of veterinary science, bogor agricultural university, bogor 16680, indonesia abstract this study was carried out to obtain vibrio isolates able to function as biocontrol of vibriosis in shrimp hatchery. thirty one vibrio isolates were isolated from tiger shrimp larvae and hatchery environments, i.e. labuan, pangandaran, and lampung, indonesia. pathogenic v. harveyi mr5339 was obtained from maros, south-sulawesi and was made as a rifampicin resistant mutant (rfr) to screen for those 31 vibrio isolates in in vitro assays and to allow us to monitor their presence in shrimp larvae and larval rearing water. almost all vibrio isolates could inhibit the growth of pathogenic v. harveyi mr5339 rfr. skt-b isolate from skeletonema was the most effective to inhibit the growth of v. harveyi mr5339 rf* and significantly reduced larval mortality in pathogen challenge assays. these prospective biocontrol bacteria, at concentration of 10" cfu/ml, did not show pathogenicity to shrimp larvae. skt-b was gram negative, short rod-shape, exhibited yellow colonies on tcbs and swarming on swc-agar media, motile, utilized glucose and sucrose but not lactose: produced extracellular protease and amylase, but did not produce chitmase. partial sequencing of 16s-rrna gene skt-b showed skt-b similarity to vibrio alginofyticus. keywords: shrimp larvae / biocontrol bacteria / vibriosis. introduction tiger shrimp (penaeus monodori) culture in indonesia has become more intensive and extensive because of high demand and economic value of this export commodity. however, the shrimp culture industry is associated with multiple problems such as diseases and poor environmental quality, which became the main constraint to reach the target of production. bacterial disease that attacked at hatchery stage is the most serious threat and often caused mass mortality in shrimp larvae which greatly influenced the sustainable supply of healthy fry. this disease is often caused by a luminous bacterium identified as vibrio harveyi (lavilla — pitogo et al. 1990; pedersen et al. 1998). to prevent such mass mortalities, shrimp hatcheries routinely use antibiotics. however, widespread antibiotics applications could result in antibiotic resistant pathogens (karunasagar et al. 1994; tjahjadi et al. 1994; tendencia and de la pena * corresponding author: asuwanto@indo.net. id biotropia no. 20,2003 2001). not only can resistant bacteria proliferate after an antibiotic has killed off the other bacteria, but also they can transfer their resistant genes to other bacteria which have never been exposed to the antibiotic (verschuere et al. 2000). furthermore, this approach is not predictable since v. harveyi strains that attack shrimp larvae are genetically diverse (suwanto et al. 1998). the development of vaccine was hampered due to the lack of immune response-memory or the degradation of the absorbed vaccines (alabi et al. 1999). an alternative method of controlling pathogenic bacterial strains in shrimp cultures could be supplementation of pure cultures of natural bacterial isolates (biocontrol) which might produce chemical substances inhibiting the growth of pathogens. the approach basically employs the activity of microorganism that could suppress or inhibit the growth of v. harveyi without causing bad impact on the equilibrium system in a particular microbial community. this has been an established practice in husbandry of terrestrial animals (fuller 1992; ohhira et al. 1996), and has only recently been applied to aquatic systems such as fish culture (gildberg et al. 1995) '-nd crustacean culture (riquelme et al. 1997; rengpipat et al. 1998). widanarni and suwanto (2000) reported the presence of several vibrio isolates associated with the shrimp from the egg stadia to post larva, as well as their rearing environment. based on their physiological and genetical characters, the isolates were distinguishable from v. harveyi that has been proved to be pathogenic on shrimp larva. therefore, further studies should be conducted to evaluate its potency as biocontrol in shrimp hatchery. similar finding was also reported by riquelme et al. (1997) in which vibrio isolates associated with scallop larvae were proved to be a potential probiotic in scallop culture in chile. the purpose of this research was to obtain new bacterial isolates as improved biocontrol agents which can be applied to solve the problem of bacterial disease in shrimp culture, and to study their ability to inhibit the growth of v. harveyi. materials and methods isolation of vibrio candidates for biocontrol vibrio isolates for biocontrol were isolated from tiger shrimp larvae and hatchery environments, i.e. labuan, pangandaran, and lampung, indonesia. samples were taken from eggs, larvae (nauplius, zoea, and mysis) and post-larvae of tiger shrimp, natural shrimp feed (i.e. artemia and skeletonemd), seawater, and rearing water of each stage of shrimp larvae. all samples were spread on thiosulphate citrate bile salt agar (tcbs, oxoid). the culture was incubated at room temperature (28-31)°c, for 24 hours. subsequently, different morphological types of colonies were randomly selected for further study. pathogenic v. harveyi mr5339 was obtained from balitdita (research institute for coastal aquaculture) maros, south-sulawesi. 12 potency of vibrio isolates for biocontrol of vibriosiswidanarni et al. sensitivity test of vibrio to rifampicin purified isolates of vibrio candidates for biocontrol and pathogenic v. harveyi mr5339 were grown in seawater complete agar (swc-agar) (5 g bactopeptone, 1 g yeast extract, 3 ml glycerol, 15 g agar, 750 ml seawater, and 250 ml distilled water) supplemented with rifampicin (rf) 50 u.g/ml. after overnight incubation at room temperature (28-31)°c, the bacteria were scored for their antibiotic sensitivity by streaking on the appropriate antibiotic-containing media. rifampicin-resistant mutant of v. harveyi one milliliter of a 24 hour culture of v. harveyi mr5339 was centrifuged at 5000 rpm for 1 minute. following the removal of the supernatant, the bacterial pellet was resuspended in 100 (il of sterile seawater. the suspension was then spread onto swc-agar containing rf (50 u.g/ml). colonies of luminescent bacteria grown on swc-agar + rf were restreaked onto new media to obtain isolated v. harveyi mr5339 resistant to rifampicin. determination by in vitro test of biocontrol vibrio the inhibitory effects of each isolate candidate for biocontrol were tested against v. harveyi mr5339 rf*, known to be a pathogen of shrimp larvae. tested isolate candidates for biocontrol was added to 10 ml of swc-broth in test tubes at 106 cells/ml. onto the same test tubes were also added overnight liquid culture of v. harveyi mr5339 rf* at 102cell/ml. all test tubes were incubated overnight at room temperature. appropriate dilutions will be chosen for seeding the mixed culture on swc-agar supplemented with rf (50 |ag/ml). if the number of colony forming unit (cfu) of v. harveyi mr5339 rf* from the control tubes (i.e. tubes inoculated only with v. harveyi mr5339 rf1*) is larger than the number of v. harveyi of the mixed cultures (i.e. v. harveyi mr5339 rf* which is mixed with the candidate biocontrol isolate), this could identify potential candidates of biocontrol isolates that inhibit the growth of the v. harveyi mr5339 rf* pathogen. pathogenicity assay of biocontrol bacteria to shrimp larvae shrimp larvae were incubated with the isolates which showed positive growth inhibitory activity against v. harveyi mr5339 rf*. bacterial isolates as candidate of biocontrol agent were resuspended in sterile seawater. shrimp larvae were cultured in 2-liter shrimp rearing tank (20 individuals per tank). bacterial suspension was added into the tank at the final concentration 106 cfu/ml. after incubation at room temperature, survival rate of larvae at 5 days was recorded. biotropia no. 20, 2003 pathogen challenge test this test was carried out to study the efficacy of vibrio candidate biocontrol isolates towards pathogenic v. harveyi in shrimp larvae. the best three biocontrol isolates, based on in vitro test, were selected for pathogen-challenged assay. suspension of each isolate was placed in the shrimp rearing tank for 6 hours before shrimp larvae were introduced into the tank. after cocultivation of isolates with shrimp larvae for 6 hours, v. harveyi mr5339 rf* was introduced into the culture tank. this experiment was performed in three replications. tanks treated with only v, harveyi mr5339 rf* or without bacteria were employed as controls. total vibrio population (counted on tcbs media), v. harveyi mr5339 rf* (counted on tcbs media + rf) in the rearing media, larval mortality monitored daily for 5 days and the survival rate of shrimp larvae were determined as described previously (hala 2002). microbiological and physiological properties cell shape, motility, and gram staining were evaluated using an olympus bh2-rfc microscope. a number of physiological characteristics were analyzed using microbact kit test (medved science pry. ltd. australia) and production of extra-cellular protease, chitinase and amylase monitored on swc-agar supplemented with skim milk, colloidal chitin and starch were tested, respectively. the presence of extra-cellular hydrolytic enzymes could be used to provide further information on the pathogenicity or virulence of the isolates. amplification and sequencing of 16s-rrna gene since many strains of marine bacteria share similar physiological and morphological characteristics, identification of bacteria strictly based on such parameters is not reliable. therefore, it is essential to perform dna sequencing of the 16s-rrna gene in order to provide reliable additional information for bacterial identification. modified phenol-chloroform-isoamylalcohol treatment and ethanol precipitation were used to extract the genomic dna (sambrook et ul. 1989). the 16s-rrna genes were amplified by pcr using specific primers of 63f and 1387r from genomic dna provided by ready-to-go pcr beads (pharmacia-biotech, uppsala, sweden). these primers were successful to work with a broad range of environmental samples (marches! et al. 1998). a part of 16s-rrna gene was sequenced to infer the closest related organism from ribosomal database project (rdp) maintained in the university of illinois, urbana-champaign. the sequencing reactions were done by using the big dye ready reaction dye deoxy terminator kit, purify with ethanol-sodium acetate precipitation. the reactions were run on an abi prism 377 dna sequencer (perkin-elmer cetus, norwalk, conn). potency of vibrio isolates for biocontrol of vibriosis — widanami el al. results and discussion isolation of vibrio candidates for biocontrol thirty one vibrio isolates were isolated from tiger shrimp larvae and hatchery environments, i. e. labuan, pangandaran, and lampung (table 1). of these isolates, 25 isolates were found to be associated with eggs, larvae, post-larvae, and the rearing water of each stage; 1 isolate was obtained from seawater reservoir: 4 isolates were obtained from natural feed (i.e. 2 isolates from artemia and 2 isolates from skeletonema). all of the isolates were non-luminous yellow colonies on tcbs and swarming on swc-agar media. table 1. codes and sources of vibrio isolates no. code source location 1. sw seawater labuan 2. n-a nauplius labuan 3. nt nauplmstank water labuan 4. z-a zoea labuan 5. pl,,-a post-larvae 6 labuan 6. pl,,-b post-larvae 6 labuan 7. pu-c post-larvae 6 labuan 8. pl,,-d post-larvae 6 labuan 9. pln-a post-larvae 1 1 labuan 10. pl,,-b post-larvae 1 1 labuan 11. plnt-a post-larvae 1 1 tank water labuan 12. plnt-b post-larvae 1 1 tank water labuan 13. skt-a skeletonema labuan 14. skt-b skeletonema labuan 15. pl2 post-larvae 2 pangandaran 16, pl2t-a post-larvae 2 tank water pangandaran 17, pl2t-b post-larvae 2 tank water pangandaran 18. pl, post-larvae 3 pangandaran 19. pl,t post-larvae 3 tank water pangandaran 20. pl,, post-larvae 6 pangandaran 21. pl,,t-a post-larvae 6 tank water pangandaran 22. pl,,t-b post-larvae 6 tank water pangandaran 23. pl7 post-larvae 7 pangandaran 24. pl7t-a post-larvae 7 tank water pangandaran 25. pl7t-b post-larvae 7 tank water pangandaran 26. at-a artemia pangandaran 27. at-b anemia pangandaran 28. e egg lampung 29. n-b nauplius lampung 30. n-c nauplius lampung 31. z-b zoea lampung 32. mr5w) (v.harveyi) infected shrimp larvae maros-sulsel biotropia no. 20,2003 pathogenic v. harveyi mr5339 was obtained from balitdita (research institute for coastal aquaculture) maros, south-sulawesi and was isolated from infected shrimp larvae. this isolate formed green colonies on tcbs and became luminous on tcbs or swc-agar media. v. harveyi mr5339 was further employed for both in vitro and pathogen challenge assay to screen for potential biocontrol bacteria. sensitivity test of vibrio to rifampicin sensitivity testing of vibrio isolates including v. harveyi mr5339 showed that all of the isolates were sensitive to rifampicin. therefore, a rifampicin resistant mutant of v. harveyi mr5339 was employed in the assay for biocontrol in vitro and pathogen challenge to screen for potential biocontrol bacteria. tjahjadi et al. (1994) reported that almost all of the bacteria that were isolated from seawater and hatchery-rearing water at kalianget, east java, including v. harveyi, were resistant to various antibiotics except rifampicin. rifampicin, a bactericidal antibiotic, is active against gram-positive and some gram-negative bacteria. this drug interferes with transcription processes in bacteria and thus far was never used in shrimp hatchery. in vitro assay for biocontrol activity the inhibitory effects of each vibrio isolate candidate for biocontrol were tested against v. harveyi mr5339 rf (figure 1). three out of 31 isolates tested i.e. skt-b, pl2t-a and n-c were isolated from skeletonema, post-larvae 2 rearing water and nauplii showed the best result. after 24 hours of incubations, only very few colonies (102-103 cells/ml) of v. harveyi mr5339 rf* were grown on culture media inoculated with v. harveyi mr5339 rf* at concentration of 102 cells/ml and vibrio candidates for biocontrol at concentration of 106 cells/ml. the number of colonies in the control experiments (inoculated only with v. harveyi mr5339 rf*) could reach 5x108 cells/ml. the other tested isolates could not significantly inhibit the growth of mr5339 rf, therefore, only sktb, pl2t-a, and n-c were further studied in the pathogenicity challenge experiments. before performing challenge test with v. harveyi mr5339 rfr in shrimp larvae, some potential isolates candidate for biocontrol were analysed for their pathogenicity to shrimp larva. after 5 days of exposure with biocontrol isolates at 1q6 cfu/ml, it had confirmed that skt-b, pl2t-a and n-c isolates were not pathogenic to shrimp larvae. these facts were indicated by relatively similar values of survival rate to those of control group (table 2). pathogen challenge test three of the most potential vibrio isolates i.e. skt-b, pl2t-a and n-c for biocontrol based on in vitro test, were assayed for their activities in inhibiting colonization of v. harveyi mr5339 rf* in shrimp larvae. observations were carried out on larval survival rates and total population of vibrio and v. harveyi mr5339 rr* both in the rearing water and in the dead larvae. results showed that each of the three biocontrol candidates significantly (p<0.05) could increase survival rates of shrimp larvae reared in seawater inoculated with 103 cells/ml of mr5339 rf". larval survival rates in each of biocontrol treatment, i.e. skt-b, pl2t-a and n-c were 93%, 90% and 82%, respectively. on the other hand, treatment with mr5339 rf* inoculation without biocontrol isolate showed only 68% survival rate of shrimp larvae (figure 2). the isolates presumably increased survival rates of shrimp larvae as survival rates of control group (without addition of biocontrol or v. harveyi mr5339 rfr isolates) were lower than treatments with biocontrol isolates. however, observation on shrimp larvae fitness after inoculation is required to determine the physiological condition of larvae. rengpipat et al. (1998) reported the use of bacillus strain s l l as a probiotic administered in enriched artemia to larvae of the black tiger shrimp (p. monodon). at two weeks, p. monodon survival was significantly different between the control groups (85%) and the treated groups (89%). when challenged with a pathogenic v. harveyi strain d331, the shrimp treated with probiotics showed a higher survival (13%) when compared to the control group (4%). haryanti et el. (2000) also reported that inoculation with by-9 strain at a dose of 106 cfu/ml to shrimp larvae, resulted to a survival rate of 59.3%, while for the control only 14.7%. the increased survival rate might be due to the growth inhibition of v. harveyi mr5339 rf* on shrimp larvae by biocontrol bacteria. although no significant difference was observed in the number of v. harveyi mr5339 rf* either in culture media (figure 3) or dead larvae (figure 4), high mortality in shrimp larvae for treatment without biocontrol isolates indicated the effect of the inhibition. however, the way how the mechanisms occurred need to be studied further. according to verschuere et al. (2000), action mechanism of probiotic bacteria or biocontrol could be divided into several ways as follows: (1) production of inhibitory compounds, (2) competition for chemicals or available energy, (3) competition for adhesion sites, (4) enhancement of the immune response, (5) improvement of water quality, (6) interaction with phytoplankton. to get an insight about action mechanism of probiotic bacteria, especially on their competition for adhesion sites, biocontrol bacteria should be tagged with molecular marker so that the existence of bacteria in shrimp larvae could be figure 3. number of colonies of vibrio sp. and v. harveyt mr5339 rľ in larva rearing thanks   potency of vibrio isolates for biocontrol of vibriosiswidanarni et al. detected. one of the molecular markers extensively used for studying bacterial activity in the e nvironment is gf'p (green fluorescent protein) gene isolated from a jellyfish (aequorea victoria) (manning 1997). if expressed, the gene will produce green fluorescence gfp protein under uv light, so that the bacteria could be easily observed. moreover, as molecular gene marker, gfp could provide several advantages such as no requirement for exogenous substrate or energy source for their visualization. gfp assays were reported to be sensitive, stable, non toxic, and did not disturb cell function and growth (josenhans et al. 1998; ling et al. 2000). characterization and identification of biocontrol bacteria skt-b was gram negative, short rod-shape, produced yellow colonies on tcbs, and exhibited swarming activity on swc-agar. this isolate was motile, could utilize glucose and sucrose but not lactose; produced protease and amylase, but did not produce chitinase. partial sequencing (500 bp of the 5'-end) of 16s-rrna gene of skt-b showed that the isolate showed similarity to vibrio alginolyticus (83% of similarity). complete sequencing of the 16srrna gene, however, should give more definitive information about the taxonomic position of this isolate. austin et al. (1995) reported that v. alginolyticus was effective in reducing diseases caused by v. anguillarum and v. ordalii, however, other strains of this bacterium has been associated with vibriosis in shrimp. therefore, characterization and identification of biocontrol strain were crucial steps to be carried out to assess the pathogenicity of biocontrol bacteria to the shrimp larvae. moreover, characterization and identification of biocontrol strain were also important for mass production, quality control and patenting to protect commercial interest (gomezgil et al. 2000). according to riquelme et al. (1997), vibrio might be more promising to be developed as probiotics when compared to other probiotic species for hatchery applications, since vibrios are commonly associated with larvae in culture, and auto-inhibition could limit the growth of pathogenic vibrio. in conclusion, vibrio isolates from tiger shrimp larvae and hatchery environments have the ability in reducing shrimp larvae mortality in pathogen challenge assays. the isolates are potential to be developed as a biocontrol agent and therefore as an alternative to chemical treatment in preventing luminous bacterial diseases in shrimp hatcheries. acknowledgments this research was supported by dip-funded research biotrop 2001/2002 to as. biotropia no. 20, 2003 references alabi, a.o., d.a. jones and j.w. latchford. 1999. the efficacy of immersion as opposed to oral vaccination of penaeus indicus larvae against vibrio harveyi. aquaculture, 179:1-11. austin. ii l. f., p. a. stucken, w. robertson, i. effendi and d. r. w. griffith. 1995. a probiotic strain of vibrio alginofyticus effective in reducing diseases caused by aeromonas salmonicida, v. anguillarum and k ordain. j. fish diseases, 18:93-96. fuller, r. 1992. history and development of probiotics. in r. fuller (ed) probiotics the scientific basis, p.p: 1-8. chapman and hall. london. gildberg, a., a. johansen, and j. bogwald. 1995. growth and survival of atlantic salmon (salmo salar) fry given diets supplemented with, fish protein hydrolysate and lactic acid bacteria during a challenge trial with aeromonas salmonicida. aquaculture, 138:23-34. gomez^jil, b., a. roque and j.f. turnbull. 2000. the use and selection of probiotic bacteria for use in the culture of larval aquatic organisms. aquaculture, 191:259-270. hala, y., a. suwanto, r. afemdi, and m. z. zairin. 2002. adherence and pathogcnicity assay of vibrio harveyi in tiger shrimp (penaeus monoclori) larvae for screening biocontrol agent biotropia, 18:8-51. josenhans, c., s. friedrich and s. suerbaum. 1998. green fluorescent protein as a novel marker and reporter system in helicobacler sp. fems microbiol. lett., 161:263-273. karunasagar, i., r. pai, g. r. malathi, and i. karunasagar. 1994. mass mortality of penaeus monodon larvae due to antibiotic-resistant vibrio harveyi infection. aquaculture, 128:203-209. lavilla-pitogo, c.r., l.l. baticados, e.r. cruz lacierda and l.d. de la pena. 1990. occurrence of luminous bacterial diseases of penaeus monodon larvae in the philippines. aquaculture, 91:1-13. ling, s.h.m., xh. wang, l. xie, t.m. lim, and k.y. leung. 2000. use of green fluorescent protein (gfp) to study the invasion pathways of edwardsiella tarda in in vivo and in vitro fish models. microbiol., 146:7-19.. manning, e. 1997. glow fish: an unusual glowing molecule from jelly fish is helping to illuminate cellular events. bioscience, 47:135-138. marchesi, j.r., t. sato, a.j. weightman, t.a. martin, j.c. fry, s.j. hiom, and w.g. wade. 1998. design and evaluation of useful bacterial-specific pcr primers that amply genes coding for bacterial 16s rrna. appl. environ. microbiol. 64:795-799. ohhira, i., t. tamura, n. fujii, k. inagaki, and h. tanaka. 1996. antimicrobial activity against methicillin-resistant sk/phyhcoccus aureus in the culture broth of enterococcus faecalis th10, an isolate from malaysian fermentation food. temph. japanese j. dairy and food sci., 45:93-96. pedersen, k.l., l. verdonck, b. austin, d.a. austin, a.r. blanch, p.a.d. grimont, j. jofre, s. koblavi, j.l. larsen, t. tiainen, m. vigneulle, and j. swings. 1998. taxonomic evidence that vibrio carchariae grimes et a!.. 1985 is a junior synonym of vibrio harveyi (johnson and shunk, 1936), baumann et al, 1981, int. j. sys. bacteriol., 48:749758. rengpipat, s., s. rukpratanporn, s. piyatiratitivorakul, and p. menasveta. 1998. probiotics in aquaculture: a case study of probiotics for larvae of the black tiger shrimp (penaeus monodon). in flegel, t.w.(ed.) advances in shrimp biotechnology. the national center for genetic engineering and biotechnology. thailand. potency of vibrio isolates for biocontrol of vibriosis — widanarni et al. riquelme. c., r. araya, n. vergara, a. rojas, m. guaita, and m. candia. 1997. potential probiotic strains in the culture of the chilean scallop argopecten purpwatus (lamarck, 1819). aquaculture, 154:17-26. sambrook. j., e.f. fritsch, and t. maniatis. 1989. molecular cloning. cold spring harbor laboratory press, cold spring harbor, new york. suwanto. a., m. yuhana, e. herawaty, and s.l. angka. 1998. genetic diversity of luminous vibrio isolated from shrimp larvae. in flegel t.w. (ed) advances in shrimp biotechnology. national center for genetic engineering and biotechnology. bangkok. tendencia, e.a. and l. dela pena. 2001. antibiotic resistance of bacteria from shrimp ponds. aquaculture, 195:193-204. (abstract). tjahjadi, m.r., s.l. angka and a. suwanto. 1994. isolation and evaluation of marine bacteria for biocontrol of luminous bacterial diseases in tiger shrimp larvae (penaeus monodon fab.) aspac. j. mol. biol. biotechnol. 2:234-352. verschuere. l., g. rombaut, p. sorgeloos, and w. verstraete. 2000. probiotic bacteria as biological control agents in aquaculture. microbiol. mol. biol. rev., 64:655-671. widanarni and a. suwanto. 2000. genetic diversity of ampicillin resistant vibrio isolated from various stages of shrimp larvae development. biotropia, 15: 36^47. 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf biotropia no biotropia no. 15, 2000 : 1 – 7 control of bagras (eucalyptus deglupta) damping-off by fungicides emilio o. anino northern mindanao state institute of science and technology 8600 ampayon, butuan city, philippines abstract selected fungicides were tested to control damping-off affecting bagras seedlings in the central nursery of the paper industries corporation of the philippines (picop), surigao del sur, philippines. the fungicides, at three concentrations each, were applied once before seed sowing to control pre-emergence damping-off and applied again after germination to control post-emergence damping-off. ajax detergent (2g/l h2o), benlate (0.5 g/1 h2o), brassicol (1.5 gv'l h2o), and fungitox (1.0 g/1 h2o) provided the best level of control against the disease. ajax detergent is the most practical among the best chemicals because it is cheap, locally available, not a health hazard, and less polluting. key words : eucalyptus deglupta/ seeds/nursery/flwzoctoma so/am'/fungicides/application introduction anino (1983) reported that the fungus causing damping-off in bagras seedlings in the central nursery of the paper industries corporation of the philippines (picop), surigao del sur, philippines is rhizoctonia solani. r. solani is a soil-inhabiting fungus belonging to class deuteromycetes (fungi imperfecti) under order mycelia sterilia and genus rhizoctonia (bernett and hunter 1972). the pathogen also caused web blight in falcata (paraserianthes falcataria), mangium (acacia mangium) and bagras (eucalyptus deglupta). the fungus started inciting post-emergence damping-off when bagras germinants were about 3 days old. for about 20 days, thereafter, the pathogen fatally infected 60% of all seedlings in a lightly irrigated sowing bed (anino 1983). the objective of this study is to screen for chemicals that can control the damping-off disease. 1 biotropia no. 15, 2000 materials and methods treatments the chemicals and concentrations tested were as follows : the soil was sterilized by fire-heating for 24 hours. experimental design the treatments, in three replicates, were arranged in crd at the shedhouse in the picop experimental nursery. evaluation parameters were as follows: a. number of emerging seedlings for determining the effects of the treatments on pre-emergence damping-off. b. total number of healthy seedlings for determining the effects of the treatments on pre-post and post-emergence damping-off. data in percent were transformed first into arc-sine values before analysis in crd. all treatment means were compared using duncan's multiple range test. 2 control of bagras (eucalyptus deglupta) emilio o. anino seed box preparation seventy-eight seed boxes for 26 treatments in 3 replicates were prepared. seventy-five boxes were filled with unsterilized soil, while three boxes for control b were filled with fire-sterilized soil. the boxes were arranged in crd, 30 cm apart atop the sowing table in the shedhouse. the table was rid of ants. the soils in all boxes (except control b) were sprayed with mycelial suspension of r. solani and watered daily for 15 days to successfully contaminate the soil with live pathogen. chemical application fifteen days after inoculation, the soils in test boxes (except controls) were drenched with one liter of their prescribed treatments. the treated boxes were not watered for 5 days. thereafter, about 1100 bagras seeds taken from seedlots stored at 0°c with germination capacity of 90% were sown into each box. the approximate viable number of seeds was 1056. the boxes were then watered once daily. the amount of water was just enough to wet the seeds and the soil medium at about 2-cm deep to avoid leaching of the chemicals. determining treatment effects on pre-emergence damping-off five days after sowing, the number of emerging seedlings on each box was determined by: a) establishing three 5-cm wide sampling strips; b) making 100% tally of seedlings in each strip; c) taking the area (cm2) of the strip and converting the average number of seedlings to per cm2, and d) taking the inner area of the box and multiplying it with seedlings/cm2 to get the total number of seedlings on the box. the percentage of healthy seedlings on each box was used as indicator of the effects of the chemicals on the pathogen and was based on the 1056 viable seeds, which were equivalent to 90% germination capacity. determining treatment effects on post-emergence damping-off the same seedlings on the boxes were used as samples for testing the effects of the treatments on post-emergence damping-off. when most of the treated boxes showed one or more infected seedlings, the boxes were rid of the diseased seedlings and re-sprayed with 130 ml of their assigned chemicals. re-spraying was conducted 13 days after the first spraying. for 20 days, the infected seedlings per box per treatment were counted and subtracted from the number of emerging seedlings to get the total number of plants protected by the treatments after two spraying schedules. 3 biotropia no. 15, 2000 result and discussion protection on pre-emerging seedlings table 1 shows the treatments and their effects on damping-off table 1. effects of test chemicals on damping-off cv = 30% cv = 32 % note : 1. control a-soil not sterilized and not chemically treated but inoculated with r. solani. 2. control b-soil was sterilized but not chemically treated and inoculated with r. solani. 3. means followed had by the same latter(s) are not significantly different at the 5 % confidence level. the seedlings, which had successfully amerged from below or had grown at the soil surface, were considered to have been protected from the pre-emergence damping-off pathogen (r. solani) by the test chemicals. the numbr of healthy 4 control of bagras (eucalyptus deglupta) emilio o. anino seedlings per box per treatment concentration was, therefore, indicative of the fungicidal effectiveness of the test chemicals. table 1 shows that among 8 chemicals, 4 were effective as shown below: the number of seedlings protected by the above treatments was about equal because they were not significantly different at 5% confidence level. the above results suggest that the four chemical treatments were highly effective in sterilizing the r. solani infested soils, thus allowing a high percentage of seeds to germinate without infection. protection on 5-to 20-day-old seedlings table 1 also shows the effects of the chemicals on post-emergence damping-off after re-spraying 130 ml of each of the said chemicals onto the 5-day-old seedlings. as expected, the treatments effective in controlling pre-emergence damping-off had consistently protected significant number of growing seedlings. for 20 days, the infection incidence in treated seedlings ranged from 0 to 8.20%, while the incidence in the untreated ones (control-a) reached as high as 9.24%. based on the number of seedlings protected after two spraying schedules, the best fungicides were as follows: note : values followed by the same letters are not significantly different at 5% confidence level. 5 biotropia no. 15, 2000 in the fire-sterilized but not chemically-treated soil (control b), the number of emerging and healthy seedlings was 74.16% only or 11.36% lower than the 85.52% in the non-sterilized but pathogen-inoculated soil and subsequently treated with 2 g/l/h2o of ajax detergent. the sterilized soil was apparently contaminated later with r. solani because of the relatively lower number of emerging seedlings and subsequent infection of 0.56% of the plants (table 1). moreover, the current practice of sterilizing soil with fire will not always protect the seedlings from severe infection if greater amount of pathogen propagules were inadvertently introduced into the soil during routine watering. in fact, the incidence of damping-off, which occurred at a seed box in the greenhouse, reached as high as 95%. conclusion beniate (0.5 g/i h2o), ajax detergent (2 g/1 h2o),'brassicol (1.5 g/1 h2o), and fungitox (1.0 g/1 h2o), in that order, are effective fungicides against damping-off pathogen (r. solani) infecting bagras seedlings. spraying 130 ml of ajax detergent and beniate solutions onto a sowing box with slight infection incidence can protect 85.52% and 88.51% of the seedlings, respectively, for 20 or more days. being very cheap, less polluting, and locally available, ajax detergent is the practical fungicide for nursery use. it is recommended that ajax detergent (2 g/1 h2o) can be used for sterilizing soil medium for seed sowing. methods of application are as follows: spray evenly 1 liter of water mixed with completely melted 2 g ajax detergent onto the soil inside a sowing box up to dripping point. let the treated soil stand for 3 days before seed sowing. after sowing, water the seed boxes once a day and just enough to wet about 2 cm deep of the soil. irrigate only when absolutely necessary and allow soils to partially dry before re-irrigating. inspect daily all sowing boxes for any symptom of damping-off. if 1 or 2 seedlings were infected, spray 130 ml of ajax solution (about 4 passes of a plastic mist sprayer) onto all seedlings in the box. do not wait for the disease to spread because the seedlings could not be saved any more once infected. seedlings affected by damping-off fall into the soil. their stems and leaves are rotten, watery and "melting". do not water the seedlings on the day treatment is applied. water on the next day when absolutely necessary. if infection recurred, spray again the detergent solution. the same techniques, as enumerated, shall be followed if beniate, bassicol, and fungitox at their prescribed concentrations were applied instead of ajax detergent. acknowledgements the author is grateful to the paper industries corporation of the philippines for giving him the chance to conduct this experiment. 6 control of bagras (eucalyptus deglupta) emilio o. anino references anino, e. o. 1983. control of bagras damping-off by fungicides. progress report of frd-picop study number 303-04-04 (unpublished). bernett, h. l. and b. b. hunter, 1972. illustrated genera of imperfect fungi. 3rd ed.burgess publishing company, minneapolis, minnesota. 241 p. 7 5. aurellia tatipata.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 blotropla no blotropla no. 11, 1998: 9-21 integrated management of chromolaena odorata emphasizing the classical biological control soekisman tjitrosemito seameo biotrop, p.o. box 116, bogor 16001, indonesia abstract chromolaena odorata, siam weed, a very important weed of java island (indonesia) is native to central and south america. in the laboratory it showed rapid growth (1.15 g/g/week) in the first 8 weeks of its growth. the biomass was mainly as leaves (lar : 317.50 cm'/g total weight). it slowed down in the following month as the biomass was utilized for stem and branch formation. this behavior supported the growth of c. odorata into a very dense stand. it flowered, fruited during the dry season, and senesced following maturation of seeds from inflorescence branches. these branches dried out, but soon the stem resumed aggressive growth following the wet season. leaf biomass was affected by the size of the stem in its early phase of regrowth, but later on it was more affected by the number of branches. the introduction of pareuchaetes pseudoinsulata to indonesia, was successful only in north sumatera. in java it has not been reported to establish succesfully. the introduction of another biological control agent, procecidochares conneca to indonesia was shown to be specific and upon release in west java it established immediately. it spread exponentially in the first 6 months of its release. field monitoring continues to evaluate the impact of the agents. other biocontrol agents (actmole anteas and conotrachelus) wilt be introduced to indonesia in 1997 through aciar project on the biological control of chromolaena odorata in indonesia and papua new guinea. keywords: indonesia / north sumatra / west java / biological control / chromolaena odorata i pareuchaetes pseudoinsulata i procecidochares conneca. introduction the nature of c. odorata (siam weed) as a weed in agricultural production systems is well recognized (tjitrosemito 1996; tjitrosoedirdjo et al. 1991) not only for perennial plantation crops (syamsudin et al. 1993), for forest teak plantations in java island (setiadi 1989), but recently also for forest plantations outside of java island. sagala (1994), for example, pointed out the role of c. odorata in providing inflammable fuel, leading to 2-4 m high flames in forest fires. moses (1996) also emphasized the important infestation role of c. odorata in reducing pasture productivity in east nusa tenggara in a recent regional symposium in kupang, east nusa tenggara. in baluran national park, east java, the presence of the weed caused a severe reduction in herbage yield in the park, leading to the reduction of population of protected wild animals such as banteng (bosjavanicus) and deers (cervus timorensis, 9 biotropia no. ii , 1998 muntiacus muncak). the situation here is quite severe, since the infestation of acacia nilotica, reaching a population of 1337 trees/ha (alikodra 1987) covered a major part of the grassing land. the park manager, following extensive mechanical control of a. nilotica using bulldozers, anticipated the return of native grasses, but c. odorata instead dominated the open space. another line of approach has recently been explored by the agroforestry research institute. following the report by slaats (1995) in africa, c. odorata may be useful as a fallow crop . the report by kasniari 1996, indicated a high yield of shoot biomass when the soil was fertile (12.44 mg ha"1), but not as high when the soil was less fertile (9.04 mg ha"'). when burnt, most of these biomass was reduced (0.77 mg ha'1). it further stressed the role of c. odorata as a weed in agricultural production systems with a very strong competitive power. the role of c. odorata as a weed is particularly severe in the newly planted or established plantations (oil palm, rubber, coconut) particularly in sumatera and kalimantan (sipayung and de chenon 1995) with quite substantial loss of investment. various control techniques have been pursued such as physical (manual, mechanical) as well as chemical approaches, and both are costly. in an attempt to find a better method for c. odorata management, biological control of c. odorata in indonesia has been done since 1989 (sipayung and de chenon 1985), supported further through the aciar project no. 9110: biological control of chromolaena odorata in indonesia and the philippines from january 1, 1993 to december 31, 1995. pareuchaetes pseudoinsulata rego barros (lepidoptera: arctiidae) was imported from guam, and after sufficient testing, this biocontrol agent was allowed to be released in the field in 1992. p. pseudoinsulata did establish in north sumatera, but not in java. although it was established, its effect was not as expected and another agent, i.e. procecidochares connexa (diptera: tephritidae) was introduced in indonesia in 1993. following extensive testing it was allowed to be released in 1995 by the minister of agriculture. this paper reports the results of procecidochares connexa (diptera: tephritidae) introduction to java island and its spread in the field. materials and methods a. the growth of c. odora ta the experiments were carried out in: parungpanjang, west java, in a forest area developed for bee farming. the total area was about 1000 ha planted mainly with 10 integrated management ofchromolaena odorata soekisman tjitrosemito acacia mangium and about 10 ha was planted with kapok trees (ceiba petandra) infested with shrubs of various kinds including c. odorata, mimosa invisa, hyptis sp., melastoma affine, ficus sp., with coppice schima walichii, and lianas such as meremia sp., mikania micrantha, passiflora sp., etc. experiment i: plant structure since october 1995 plots measuring 4 m2 were slashed at monthly intervals, and the stumps were mapped. the diameter was measured in mm, and recorded. the biomass was separated into leaves and stems, dried at 80°c for 48 hours or until the weight was constant and the weight recorded. the data were analyzed to characterize the plant structure of the established mature c. odorata population. experiment ii: the growth of c. odorata coppice the growth of coppice was observed at monthly intervals on plots following slashing treatment described in experiment i without destroying the plants. particular attention was given to emergence of coppice from small diameter stumps. the data were recorded in terms of coppice number and height of each stem, and number and position of branches. the data were analyzed statistically. b. rearing and release of procecidochares connexa b.l. rearing a. gall collection the stem cuttings bearing gall were collected from marihat research station, north sumatera, indonesia, on my 18, 1995. at biotrop, bogor, the galls were dissected. there were 17 galls altogether producing 48 pupae. the pupae were yellowish white when young and turned dark brown when mature. the pupae were subsequently reared on petridishes lined with moist filter paper and put in a rearing cage made of a wooden frame with fine plastic mash measuring 0.4 x 0.4 x 0.4 m3 kept in an air conditioned room. from 48 pupae, 38 imago emerged consisting of 19 females and 19 males. female iinagos were easily differentiated from the males by its conspicuous ovipositor. b. oviposition the emerged imago was soon paired up, in an 8 cm test tube to mate, and released into caged potted chromolaena odorata plants the following day in the 11 biotropia no. 11, 1998 green house. the cages of medium size, 0.5 x 0.5 x 1 m, were made of wooden frames with fine plastic mash, housing plastic pots of 51 capacity containing soil mixed with worm casting at 1:1 ratio as medium to support the growth of c. odorata. the potted c. odorata plant should have at least 20 shooting buds, preferably more, upon which the female fly will oviposit. the cages potted c. odorata with the pair of flies were kept approximately one week or until the flies died to ensure that the female oviposits its eggs on the c. odorata plant. the potted plants were then immediately removed and put under the sun with plenty of water and fertilizer. c. life cycle it takes 50 days for the gall to develop and mature. the maturity of the gall was indicated by the appearance of a window opening on the gall, which was still closed by a very thin cover of transparent lining gall epiderm used to allow emerging fly to escape. when most of the galls showed the window opening, the galls were harvested and dissected. from 19 pairs of flies, 53 galls were produced and after dissection yielded 142 pupae. from 142 pupae, 118 emerged (83%) in a period of 11 days (sept. 26 oct. 6, 1995), with a female to male ratio of 64 : 54. d sex ratio from 64 females of the first generation in the laboratory, 407 galls were produced yielding more than 1200 flies comprising 640 males and 629 females. in the second generation, the mature galls were not dissected, but the harvested gallbearing stem cuttings were collected and reared in the rearing cages by dipping the stem cutting in jars of water to keep the cuttings fresh. e. mass production the emergence took place from december 12, 1995 up to january 12, 1996. some emerged later. the 3rd generation of p. connexa in biotrop produced 3232 flies consisting of a female to male ratio of 1646 to 1586. the rearing procedure in the laboratory at biotrop was slightly modified and standardized as follows: the emerging flies were collected by sucking them with a small modified vacuum cleaner and reared for at most 2 days in a rearing cage. the population of images in 2 days can reach 100 pairs of flies. these paired imagos must be released immediately to new potted c. odorata plants having at least 20 shooting buds each. the potted c. odorata plants were housed in a big cage made of iron frame with a 12 integrated management of chromolaena odorata ~ soekisman tjitrosemito fine plastic screen measuring 2 x 3 x 3 m3. the plastic screen was lifted one week later, or until all flies died. the potted c. odorata plants were provided with adequate water and fertilizer. approximately 50 days later the emerging flies were sucked with the modified vacuum cleaner as mentioned above. about 250 pairs of flies obtained that way were once more released in parungpanjang and 32 galls were sent to yogyakarta. since emergence took more than 1 month, between february 26, 1996 and april 7, 1996 at biotrop, everyday there were new emerging imagos of p. connexa. the 4th generation emerged from may 10, 19% to june 28, 1996. there were more than 3835 galls, producing more than 4000 flies. b.2. release experiment hi. release a. permanent plot permanent 5 x 5 m plots were made in the area densely populated by c. odorata, marked with wooden poles of 4 m at the corner of the plot, delineated with steel wire. inside the plot individual plants of c. odorata were tagged with numbered alluminium tags attached to the base of the plants. the precise location of c. odorata plants was plotted on grid paper. the number of plants and their growing shoots were recorded. the permanent plots were made in parungpanjang and sukabumi. b. release release of p. connexa was done in the permanent plots at parungpanjang and sukabumi. b.l parungpanjang the release was conducted on december 19, 1995 with 75 pairs of mated imagos, and repeated again on december 30, 1995, with 100 pairs of additional mated imagos. b.2. sukabumi the release was carried out directly from the cage containing 100 pairs of mated imagos in the permanent plots on may 28, 1996, and when no gall was 13 b10tropia no. 11,1998 observed in the field on june 17, 1996, the following day, an additional 65 pairs of mated imagos were released. c. evaluation the evaluation was carried out by observing the presence of galls in the permanent plots and in the surroundings. c. 1. permanent plots in parung panjang every single plant was inspected on december 30, 1995, while in sukabumi this was done on september 14, 1996. the galls were counted and recorded. c. 2. surrounding permanent plots outside the permanent plots the observation were out using the line transect method. four lines at four cardinal directions were followed using measuring tape. along the way any c. odorata plants directly under the line was inspected for the occurrence of any galls. the number of galls and the distances were recorded and used to estimate the area infected by p. connexa these data indicated the population growth and dispersal of the fly. c. 3. data a nalyses data on weed growth were analyzed using multiple regression, while the area infected by the fly was analyzed using exponential growth to estimate the rate of spread. results and discussion a. the growth of c. odora a.l. the plant structure the data of the mature, well established c. odorata community slashed in october were analyzed to see how the leave biomass was affected by other variables (i.e. plant height, shootbase diameter, number of branches, and stem dry weight). using linear multiple regression analysis with data from 52 plants, the following regression equation was obtained : 14 integrated management of chromolaena odorata — soekisman tjitrosemito y= i.2258 2.8075 x 10'3 x, + 4.561 x 10'2 x2 3.2034 x 10'3 x3 + 0.18373 x< (t2 = 0.7349) where: y = leaf biomass(g) (= 3.252 g) xi = plant height (cm) (=159.100 cm) x2 = shootbase diameter (mm) (= 4.981 nun) x3 = number of branches (=10.520) x< = stem dry weight (g) (= 14.000 g) further analysis of the regression coefficient indicated that only the x4 variable was significant in affecting the total leaf biomass, because this population of c. odorata just sprouted to produce coppice along the denuded stem after undergrowing leaf shading during the dry season following flowering and fruiting. it is interesting to compare the growth behavior of c. odorata in the laboratory, where in the first two months of growth most of the photosynthate is utilized to produce leaves consequently it has a relatively high relative growth rate (rgr) =1.15 g/g/weeks (tjitrosemito 1996). it seems that this character is consistent whether it grows from seeds or when recovering from drought. at this time of the year the growth of c. odorata was still recovering from drought. the tops of stem and some branches that were carrying inflorescences were dried off following the dispersal of seeds as indicated by the negative sign of the coefficients. the greater the number of branches the lesser the biomass of leaf because those branches did not carry leaves just yet since the top part of those branches were dead. the situation was similar for plant height, as the higher the plant the lesser the leaves, as the top part was still dried off. the plant size was relatively small having an average height of 159.10 cm with leaf biomass of only 3.253 g/plant and total stem dry weight of 14.00 g/plant. the number of branches was relatively high i.e. 10.520 g/plant, but still small and some were only carrying 2 leaves. when slashing was done in may, the following regression line was obtained: y = -12.567 + 3.6413 x 10'2 x, + 6.8146 x 10'1 x2 + 0.9645 x3 + 2.9480 x 10'2 x4 where: y = leaf biomass (g) (= 5.583 g) x, = plant height (cm) (=199.1cm) x2 = number of branches (= 7.243) x3 = stem diameter (mm) (= 5.608 mm) x, = stem dry weight (g) (= 28.53 g) further analysis of regression coefficients indicated that the x2 variable, i.e. number of branches, was highly significant in affecting the biomass of the leaf. x3 15 biotropia no. 11, 1998 and x4 were also significant in affecting the biomass of the leaf. the number of branches represent new growth, where leaves are located. therefore, the greater the number of branches the higher the biomass of the leaves they carry and the sign is positive. stem dry weight and stem diameter were indicating the size of the plant. the taller the plant the higher the biomass of the leaves, and the signs are positive. however, the constant carried a negative sign, indicating that the growth of this population was very thick with many leaves at the top of the population so that some leaves at the lower level were shaded and some of them already senesced. this plant structure was different from those in october when the population was still recovering from the drought. the size of plant already reached a mature stage. stem dry weight reached 28.53 g/plant with the leaf biomass of 5.839 g/plant, and the average plant height was 199.1 cm. the number of branches stabilized at an average of 7.243 g/plant and these branches were effective viable branches which will bear inflorescences when mature. a.2. the growth of c. odorata coppice the growth of coppice was recorded on all stumps from as small as 2 mm to 10 mm when slashing was done during the wet season, i.e. from october june, while from july to september regrowth was recorded only on plants with a diameter of 5 mm or greater. at this period, most of seedlings (if seeds happen to germinate) will also die due to the drought. it seems that the regrowth of coppice of c. odorata is mostly affected by soil moisture. it was unfortunate that we did not collect data on soil moisture. this information will be collected in future observations. the above data revealed the plant structure in terms of biomass distribution. to understand the plant structure in physical terms the data on coppice growth was quite revealing. when observations were carried out at 2 and 3 months after slashing (during the wet season) the following data were obtained (average of 78 plants) (table 1). at two months after slashing as the space was stil available, the plant utilized its energy for producing branches, reaching a total of 26.07 branches/plant. the plant height varied reaching a maximum of 240 cm but the average was 162.5 cm, while the branches were distributed slightly skewing to the left, i.e. indicating that the plants were still investing their energy for growth. the peak of branch number was observed at a height of 80-100 cm above ground. at three months after slashing, however, there was a stabilization in number of branches. leaves of lower branches were shaded out and the branches were sacrificed 16 integrated management of chromolaena odorata — soekisman tjitrosemito by the plant, in favor of the new branches at the top of the plants. the plants grew taller reaching a maximum of 300 cm but the average was 220 cm. the branches were distributed slightly skewing to the right since at this time the lower branches and leaves were dying off. the plants formed a population architecture in such a way that most of the leaves were grown on the top branches to harvest sunlight as much as possible at die expense of the lower leaves and branches. this is the final structure when the population will produce flowers. when the time permits and moisture is available the plant will grow to form a very dense thicket of c. odorata. table 1. the distribution of branches in the c. odorata community at different stages of growth harvested 2 months after slashing harvested 3 months after slashing plant height (cm) 162.50 220.600 total living branches 26.07 18.95 branches from : 0-20 cm height 0.5714 0.1818 20-40 cm height 1.9290 0.3939 40-60 cm height 8.6790 1.0150 60-80 cm height 17.0000 2.3030 80100 cm height 22.4000 3.3940 100-120 cm height 20.7100 6.4550 120-140 cm height 18.2500 9.1360 140-160 cm height 11.9300 12.0000 160-180 cm height 6.8570 12.5200 180-200 cm height 3.0360 13.7000 200-220 cm height 0.9286 10.9500 220-240 cm height 0.4286 7.0450 240-260 cm height 2.9090 260-280 cm height 0.6364 280-300 cm height 0.1212 b. laboratory rearing of p. connexa from table 2, it appears that the time of emergence varies considerably from 9-25 days (from pupation/dissection to emergence). the emergence was about 79% with a sex ratio of 1 : 1. so this initial culture of p. connexa at biotrop was 19 pairs of imago. 17 biotropia no. 11, 1998 table 2. the emergence of imago of p. connexa from pupae no. date of emergence total female male 1. 07-27-1995 3 2 1 2. -282 1 1 3. -294 2 2 4. -303 1 2 5. -31 6. 08-01-1995 7 4 3 7. -02 8. -033 1 2 9. -041 1 . 10. -05. . 11. -06. . 12. -073 2 13. -082 1 14. -093 : 1 15. -103 2 16. -112 1 17. -122 1 total : 38 19 19 assuming that pupae represented the egg laid by a female, each female in generation i, on average, laid 7.5 eggs, this was lower than reported by sipayung and de chenon (1995), being 16, and much lower than the total number of eggs that may be laid by a female of p. connexa (69 eggs). however, for the following generation the estimated eggs laid was higher i.e. 19 per female. for the initial colony each gall contained about 3 pupae per gall. this was maintained up to generation n at biotrop. it dropped at generation iii and iv to about 1 pupae per gall. this condition may be attributed to the stage of the plant occurring mostly in the flowering stage at this time of the year (table 3). table 3. the outcome of the rearing in the laboratory emergence imago remarks gall pupa period total female male initial colony 17 48 17 38 19 19 dissected generation i 53 142 11 -118 64 54 stem cutting in jars modified vacuum cleaner generation ii 407 (nr) 30 1269 640 629 generation hi 3177 (nr) (nr) 3232 1586 1646 generation iv 3835 (nr) (nr) 4027 2023 2004 nr: not recorded 18 integrated management of chromolaena odorata soekisman tjitrosemito c. field experiment c.i. parungpanjang the population of c. odorata in parungpanjang, west java, a permanent plot of 5 x 5 m2, was 145 c. odorata plants with 2333 + 8.7 shoots. the c. odorata vegetation had a very dense thicket of shoots. in the permanent plot of 5 x 5 m2, on december 30, 1995, 128 galls were found. it is about 5% from the available shoots. this infestation is still low, in terms of the efficiency of controlling c. odorata. the spread of p. connexa into the surrounding area may be judged from data in table 4. table 4. the distribution of galls in parungpanjang the northern side of the permanent plot was bordered by a forest of young eucalyptus, mixed with calliandra callothyrsus and ceiba petandra. this forest seems to form a barrier for p. connexa to spread. the rate of spread may be estimated from the distance where galls were recorded. assuming that the forest on the northern side of the plot constitutes a barrier, the available area of spread was half circular as in figure 1. so the area is approximately '/i m2 (where r = distance of galls found). in the first month, the area covered by the infestation of p. connexa estimated from the distance of recorded galls, was about 150 m2. the following 2 months the spread covered an area of approximately 2203 m2 and at 6 months after release the area covered was 120 580 m2. the mode of spread seems to follow an exponential pattern, with relative spread rate of 1.1 in a month. 19 figure 1. diagram of the position of the permanent plot in parungpanjang c.2. sukabumi the population of c. odorata in the permanent plot of 5 x 5 m2, consisted of 82 c. odorata plants with 2140 + 18.5 shoots. this c. odorata vegetation was under a coconut plantation. it seems that this weed has been neglected for some reason. although the population was less than that of parungpanjang, the number of shoots was almost the same. in fact, the average for sukabumi was 26.1 shoots/plant, higher than that of parungpanjang (16.1 shoots/plant). two weeks after the release in sukabumi no gall was observed, since c. odorata plants were in the process of fruit maturation, where shoots were dried out. so c. odorata plants were slashed and p. connexa was released again on june 18, 1996 with 65 pairs of flies. on august 8, 19%, 120 galls were recorded. the rate of infestation is about 5%, similar to the infestation rate in parungpanjang, while the observation outside the permanent plot is still being evaluated. in contrast to pareuchaetes pseudoinsulata, p. connexa survived and reproduced well at java island. this result is very encouraging. although the rate of infestation is still low (5%), it is hoped that with the coming wet season the infestation will be higher. the impact of p. connexa on the performance of c. odorata such as its growth, seed production and seed viability are still under investigation and it is expected that one or two more biological control agents could be introduced in indonesia. references aldcodra, h.s. 1987. the exotic plantation of acacia nilolica and its problem on the ecosystem of savana baluran national park. duta rimba 79/80/xiii/1987: 30-34. kasniari, d.n. 1996. the role of chromolaena odorata in increasing soil fertility in alang-alang field. msc. thesis. university of brawijaya, malang. 20 integrated management of chromolaena odorata soekisman tjitrosemito moses, b.c. 1996. a. concept of integrated agriculture, with reference to the subsector of animal husbandry. a, paper presented to the regional symposium. university of nusa cendana, march 26, 1996. saoala, ap.s. 1994. land fire control, experience from riam kiwa plantation area established on alang-alang grassland. in: tampubolon el al. (eds.) from grassland to forest: profitable and sustainable reforestation of alang-alang grassland in indonesia. 97-108. setiadi, d. 1989. notes on weeds under teak forest. proc. 9*. indonesian weed sci. conf. ill : 74-78. sffayung, a. and r. desmier de chenon. 1995. procecidocrtares connexa to control chromolaena odorata. a paper presented to the meeting on release of procecidochares connexa, july 17, 1995. research centre of oil palm, medan. slaats, jj.p. 1995. chromolaena odorata fallow in food cropping system: an agronomic assessment in south-west ivory coast. phd. thesis. agricultural university, wageningen. the netherlands. 117 p. syamsuddin, e., t.l. toeing and r.a. lubis. 1993. integrated pest management in oilpalm plantation in indonesia. biotrop spec. publ. 50. 137-145. tjitrosemrro, s. 1996. the management of chromolaena odorata (l.) r.m. king & h. robinson in indonesia in: prasad et al. (eds.). 1996. proc. 3rd. international workshop on biological control and management of chromolaena odorata: distribution, ecology and management of chromolaena odorata. agricultural experiment station, univ. guam. mangilao, guam, usa.: 135-142. tjmosoedirdjo, s., s.s. tjitrosoedirdjo and r.c. umaly. 1991. the status of chromolaena odorata (l.) r.m. king & h. robinson in indonesia. biotrop spec. publ. 44: 57-66. 21 microsoft word 1 biotropia vol. 13 no. 1, 2006 : 1 10 transposition and expression of gfp gene in the genome of vibrio harveyi to monitor its adherence in shrimp larvae wldanarni0, antonius suwanto^sukenda1', and blbiana wldiati lay3) '' faculty of fisheries and marine science, bogor agricultural university, bogor 16680, indonesia '' faculty of science and mathematics, bogor agricultural university, bogor 16680. indonesia ''faculty of veterinary science, bogor agricultural university, bogor 16680, indonesia abstract expression of green fluorescent protein encoded by gfp gene in vibrio harveyi was investigated to understand the ability of the gene as a molecular marker for adherence of this pathogenic vibrio in shrimp larvae. the gfp gene was inserted into pl/clsnot and putmini-tn.') to generate a recombinant plasmid pwg02 and p\vg03, respectively; which was transferred into the three isolates of v. han'eyi employing diparental mating. recombinant e. coli carrying p\vg02 and pwg03 resulted in green-fluorescent colonies and cells due to the production of gfp. however, all of mini-tn.j, including mini-tn.5-gfp were not successfully transferred to v. harveyi. therefore, we used mini-tn/fl (plofkm-gfp) for inserting of gfp gene into v. han'eyi genome. although we could obtain relatively high (10's) transconjugans employing tn/rt, only one of tnjo derived isolate of v. harveyi g3 (g3-tn/flgfp) showed gfp expression and was further employed for adherence assay. fluorescent g3-tn70gfp cells could be observed inside the digestive tract of shrimp larvae and could be distinguished from vibrio that naturally exist in shrimp larvae. key words: gfp gene, vibrio han'eyi, gene expression, shrimp larvae, molecular marker introduction vibrio harveyi was identified as a causative agent of mass mortalities of shrimp larvae and were frequently associated with luminous vibrio (lavillapitogo et al. \ 990; karunasagar et al. 1994; ruangpan 1998, suwanto et al. 1998). luminescent vibriosis in shrimp larvae is characterized by lethargy, anorexia, muscle opacity, bacterial masses in the hemocoel, and luminosity of the larvae (lavilla-pitogo et al. 1990). electron microscopy observation has revealed that bacteria colonize the feeding apparatus, forming bacterial plaques in heavily infected larvae, therefore, it is highly probable that the mouth is the main entrance for colonization of inner tissues (lavilla-pitogo et al. 1990). pathogenicity assays based on koch's postulates (madigan et al. 2003) were practically difficult to be conducted in shrimp larvae due to their relatively small size and lack of availability of vibrio-free larvae (widanarni and suwanto 2000). investigations into adherence and pathogenicity processes of this disease might ' corresponding author: e-mail: asuvvanto@indo.net.id biotropia vol. 13 no. 1, 2006 greatly facilitate if a visible marker could be introduced into the bacterial cells. pathogenic v. harveyi have observed their attachment to crustacean larvae by epifluorescence microscopy using 5-(4,6-dichorotriazin-2-yl) aminofluorescein (5-dtaf, d-16) as a marker (soto-rodriguez et al. 2003). one visible molecular marker which has extensively been used for studying bacterial activity in the environment is gfp, i.e. a gene encoding green fluorescent protein (gfp) from a jellyfish (aequorea victoria) (manning 1997). as a molecular gene marker, gfp has some advantages, such as no requirement for exogenous substrate or energy source for their visualization, sensitivity of detection, high stability, lack of toxicity, and no disturbance in cell function and growth (josenhans et al. 1998; ling et al. 2000). gfp as a molecular marker has been used to demonstrate the mechanism of edwardsiella tarda infection on epithelial cells of giant gouramy (ling et al. 2000); and pseudomonas plecoglosicida infection in ayu (plecoglossus altivelis) (sukenda and wakabayashi 2001). gfp was also successfully used as a marker in lactic acid bacteria (lactobacillus plantarum and l. lactis) to study the possibility of using the bacteria as live vaccine carriers (geoffroy et al. 2000). a broad host-range plasmid expressing gfp gene (pwgol) was constructed and has been successful to tag k harveyi (widanarni et al. 2005). however, under non selective longterm experiments without antibiotic pressure, pwgol was highly unstable. therefore, transposon insertion may provide an alternative method to insert gfp gene directly into the genomic dna of v. harveyi in order to yield stable recombinants. in this report, we describe the construction in tn vector and expression of v. harveyi carrying gfp gene in the genomic dnas to monitor its adherence in shrimp larvae. materials and methods bacterial strains and plasmids bacterial strains and plasmids used in this study and their relevant characteristics are described in table 1. escherichia coli and v. harveyi were grown in luria bertani (lb) medium at 37°c and seawater complete (swc) medium at 28°c, respectively. lb medium was made as previously described (sambrook et al. 1989) and swc medium contained 5 g bactopeptone, 1 g yeast extract, 3 ml glycerol, 15 g agar, 750 ml seawater, and 250 ml distilled water. plasmid construction and molecular techniques recombinant plasmid pwg02 which has the lac promoter was constructed and used to drive the expression of gfp. the promoter and gfp gene were isolated from psklol using £cori sites and ligated into puclsnot linearized with £cori. the recombinant plasmid vector (pwg02) (figure 1) was digested with notl and then promoter and gfp gene were ligated into putmini-tnj linearized with noil resulting transposition and expression of gfp gene widanami et al. in recombinant plasmid (pwgos) (figure 2). the recombinant plasmid vector was transformed into e. colt dh5a using a standard heat shock transformation (sambrook et al. 1989) and the colonies carrying pwg02 and pwg03 were examined for green fluorescence under uv-transilluminator at 260 nm (biometra ti 1, gottingen). plasmid extraction, restriction enzyme digestions, agarose gel electrophoresis, gel isolated dna fragment purification, and ligation were carried out using standard methods (sambrook et al. 1989), and following the manufacturer's instructions. restriction endonucleases and other enzymes were obtained from new england biolabs inc (beverly, ma, usa). bacterial mating to transfer recombinant plasmid pwg03 harboring mini-tn5gfp into v. harveyi we used diparental mating (suwanto and kaplan 1992). escherichia coli dh5a (pwgos) donors were grown overnight in lb medium supplemented with spectinomycin and streptomycin (sp/sm) 50 ugmt1 at 37°c; whereas v. harveyi recipients were grown in swc medium at 28°c. each 1.5 ml of the donor and recipient were pelleted in a micro-centrifuge at maximum speed for 1 min, and then the cells were washed with 1.0 ml 0.85% nacl, re-centrifuged, and suspended in 40 ul of lb medium before being spotted onto a filter (1 cm diameter; pore size 0.45 urn; millipore) on lb medium agar. the bacteria were allowed to conjugate at 28°c for 16 to 18 hours. at the end of the mating period, the filter containing the bacterial transposition and expression of gfp gene — widanarni et al. mixture was transferred into 1.5 ml microfuge tube containing 0.8 ml of 0.85% nacl. the bacterial cells were suspended thoroughly by agitation on a vortex mixer. the transconjugants were selected on thiosulphate citrate bile salt (tcbs, oxoid) medium supplemented with sp/sm (50 jugm!"1). the selective medium tcbs was used to inhibit the growth of e. coli, while allowing v. harveyi transconjugants harboring tnjgfp (resistant to spectinomycin and streptomycin) to grow. the same method was conducted to transfer recombinant plasmid plofkm-gfp (resistant to kanamycin) into v. harveyi and the transconjugants were selected on tcbs medium supplemented with kanamycin (100 (agml"1). some of v. harveyi transconjugants were analyzed by pulsed-field gel electrophoresis (pfge) with notl restriction enzyme (suwanto and kaplan 1992; widanarni and suwanto 2000) to show the place of tnlogfp inserted in the genomic of v. harveyi. gfp stability and pathogenicity assay vibrio harveyi strains harboring g/p both in the plasmid pwgol (widanarni et al. 2005) and in the genomic's dna were grown overnight in swc broth supplemented with kanamycin. sequential propagation under non selective conditions were performed by inoculating with 1:100 (v/v) to assess gfp existence by comparing duplicate colony counts on selective and non selective plates. for pathogenicity assay, two groups with three duplicates of shrimp post-larvae (pl4) were immersed for 30 min in 106 cfuml"1 of gfp recombinants and wild type of v. harveyi (final concentration), respectively, and then placed in a 2 l shrimp rearing tank. a control group was immersed in sterile scawater. daily survival rate of shrimp larvae for 5 days were recorded and compared with the control group. adherence assay samples of shrimp larvae from control and treatment groups were directly observed under a fluorescence microscope. samples from dead shrimp larvae were also inoculated onto swc plates containing kanamycin (100 figrnl"1) to show that the dead shrimp larvae were infected by recombinant v. harveyi. results and discussion gfp-containing plasmid construction the gfp-plasmid vector was constructed for molecular marker in v. harveyi. two gfp vectors i.e. pwg02 and pwgos, were constructed with lac promoter to drive the expression of gfp. recombinant e. coli carrying pwg02 or pwg03 (mini-tnjgfp) resulted in green-fluorescent colonies and cells due to the production of gfp (figure 3). however, all of mini-tn5, including mini-tnj-gfp was not successfully transferred to v. harveyi. the same results were observed by stretton e? biotropia vol. 13 no. i, 2006 a b figure 3. fluorescence micrograph of (a) e. coli dh5a (pwg02) and (b) e. coli dhsct (pwg03) al. (1998). therefore, we used mini-tn70 (plofkm-gfp) that has been constructed by stretton et al. (1998) for inserting ofgfp gene into v. harveyi genome. construction of v. harveyi gfp+ employing mini-tn/0 (plofkm-gfp) we could obtain relatively high transconjugants employing tnlo (table 2). pulsed-field gel electrophoresis (pfge) analysis of some v. harveyi transconjugants demonstrated that tn70gfp was randomly inserted in the gcnomic v. harveyi g3 (figure 4). however, only one of tnlo derived isolate of v. harveyi g3 (g3-tn70gfp) resulted in green-fluorescent colonies and cells due to the expression of gfp and that fluorescence levels qualitatively was almost the same with g3 (pwgol) (figure 5). this result occurred due to the fact that gfp in plofkmg/p was constructed promoterless, so its expression depended on promoter strength in the insertion site within v. harveyi genome. gfp stability and pathogenicity assay colonies of v, harveyi g3 (g3-tn70gfp) that were grown on media with or without antibiotic exhibited uniform fluorescence appearance. this was not the case transposition and expression of gfp gene widanarni et al. m figure 4. pfge profiles of genomic dna of v. harveyi g3 (lane m: axel-digested genotnic dna of r. sphaeroides 2.4.1 as a molecular size marker (suwanto and kaplan 19 89). lane 1 and 8: g3 wild type, lane 2: g3 tn/flgfp (kmr and showed gfp expression), lane 3 -7: mutants 03 (kmk) figure 5. fluorescence micrograph of (a) v. harveyi g3 (pwgoi) and (b) g3-tn/ogfp for v. harveyi g3 (pwgol). their colonies grown on antibiotic-containing media exhibited uniform fluorescence appearance, whereas those grown on media without antibiotic showed mixture of fluorescent and non fluorescent colonies which might indicate plasmid loss. the stability of the gfp on v. harveyi g3 both in plasmid and in the genomic's dna were investigated during sequential propagation in the absence of antibiotic selection for five successive days. under nonselective long-term experiments without antibiotic pressure, pwgol was highly unstable but tn/0gfp was stably maintained in v. harveyi g3 strain (figure 6). insertion of tn70gfp also did not show alteration in g3 pathogenicity to shrimp larvae (figure 7), so that it was further employed for adherence assay. adherence assay sample from dead shrimp larvae showed that the dead larvae were infected by v. harveyi. vibrio harveyi g3-tn/0gfp could be isolated from dead shrimp larvae placed on tcbs+km media and fluorescent g3-tn70gfp could also be observed directly in the carcasses of dead larvae. fluorescent g3-tnjogfp cells were observed in the oral region at 15-30 min after inoculation and could be observed inside the digestive tract at 2-3 h (figure 8v the concentration of v. harveyi g3-tnlogfp used in this study was 10 cfuml". soto-rodriquez et al. (2003) reported that 105 cfuml"1 v. harveyi labeled with 5-(4,6-dichlorotriazin-2-yl) aminofluorescein (5-dtaf, d-16) could be observed in the oral region of litopenaeus vannamei mysis at 0 and 2 h after ingestion. after 4 h inoculation, individual cells could already be seen inside the middle intestine, and at 18 or 24h, the fluorescent v. harveyi were observed throughout the intestinal tract. when presented at higher densities of bacterial cells (exclusively), fluorescent v. harveyi could be easily observed along all regions of zoea digestive tracts after 30 min (soto-rodriquez et al. 2003). 8 conclusions recombinant v. harveyi harboring gfp both in plasmid pwgol and in the genomic dnas produced green-fluorescent cells. however, tn7(?gfp was stably maintained in the genomic v. harveyi so that it could be used to monitor adherence and pathogenicity of v. harveyi in shrimp larvae. acknowledgments this work was supported by dip-funded research of seameo-b1otrop (2000-2002) and international foundation for science grant (ips, a/2207-2) to as. references geoffrey m.c., guyard c., quatannens b., pavan s., lange m. and a. mercenier. 2000. use of green fluorescent protein to tag lactic acid bacterium strains under development as live vaccine vectors. appl. environ. microbiol., 66: 383-391. herrero m., de lorenzo v. and k.n. timmis. 1990. transposon vectors containing non-antibiotic resistance selection marker for cloning and stable chromosomal insertion of foreign genes in gram negative bacteria. j. bacteriol., 172:6557-6567. josenhans c., friedrich s. and s. suerbaum. 1998. green fluorescent protein as a novel marker and reporter system in helicobacter sp. fems microbiol. lett., 161:263-273. karunasagar i., pai r, malathi g.r. and i. karunasagar. 1994. mass mortality of penaeus monodon larvae due to antibioticresistant vibrio harveyi infectioa aquaculture, 128:203-209. lavilla-pitogo c.r., baticados m.c.l., cruz-lacierda ejr. and l.d. de la pena. 1990. occurrence of luminous bacterial diseases ofpenaeus monodon larvae in the philippines. aquaculture, 91:1-13. ling s.h.m., wang x.h., xie l, lim t.m. and k.y. leung. 2000. use of fluorescent protein (gfp) to study the invasion pathways ofedwardsiella tarda in in vivo and in vitro fish models. microbiol., 146:7-19. madigan m.t., martinko j.m. and j. parker. 2003. brock biology of microorganisms. prentice-hall pearson education inc. usa., p. 12-13. biotropia vol. 13 no. 1, 2006 manning e. 1997. glow fish: an unusual glowing molecule from jelly fish is helping to illuminate cellular events. bioscience, 47:135-138. ruangpan l. 1998. luminous bacteria associated with shrimp mortality. in flegel tw (ed) advances in shrimp biotechnology. national center for genetic engineering and biotechnology, bangkok. sambrook j.. fritsch e.f. and t. maniatis. 1989. molecular cloning. cold spring harbor laboratory press. new york. usa. soto-rodriguez s.a., simoes n., jones d.a., roque a. and b. gomez-gil . 2003. assessment of fluorescent-labeled bacteria for evaluation of in vivo uptake of bacteria (vibrio spp.) by crustacean larvae. j. microbiol. methods, 52:101114. stretton s., techkamjanaruk s., mclennan a.m. and a.e. goodman. 1998. use of green fluorescent protein to tag and investigate gene expression in marine bacteria. appl. environ. microbiol., 64:2554-2559. sukenda and h. wakabayashi. 2001. adherence and infectivity of green fluorescent protein-labeled pseudomonasplecog/ossicida toayu (plecoglossus altivelis). fishpathol., 36:161-167. suwanto a. and s. kaplan. 1989. physical and genetic mapping of the rhodobacter sphaeroides 2.4.1 genome: genome size, fragment identification, and gene localization. j. bacteriol., 171:5840-5849. suwanto a. and s. kaplan. 1992. a self-transmissible, narrow-host-range endogenous plasmid of rhodobacter sphaeroides 2.4.1: physical structure, incompatibility determinants, origin of replication, and transfer functions. j. bacteriol., 174:1124-1134. suwanto a., yuhana m. , herawaty e. and s.l. angka. 1998. genetic diversity of luminous vibrio isolated from shrimp larvae. in flegel tw (ed) advances in shrimp biotechnology. national center for genetic engineering and biotechnology, bangkok. widanami and a. suwanto. 2000. genetic diversity of ampicillin resistant vibrio isolated from various stages of shrimp larvae development. biotropia, 15:36-47. widanami, suwanto a., sukenda and b.w. lay. 2005. construction of recombinant vibrio hai-veyi to study its adherence in shrimp larvae. in p walker r, j lester, mg bondad-reantaso (eds). diseases in asian aquaculture v, p. 465-474. fish health section, asian fisheries society, manila. 10 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf 10.pdf 5. agus nuryanto (molecular).cdr 5. sri wilarso.cdr biotropia vol. 20 no. 1, 2013: 38 49 bacteria from arbuscular mycorrhizal fungi spores sp. and sp. : their antagonistic effects towards soilborne fungal pathogens and growth stimulation of sp. gigaspora glomus gigaspora in vitro sri wilarso budi * and nunang lamaek may recipient of biotrop research grant 2010/accepted 21 may 2012 this research was aimed at obtaining bacterial isolate from arbuscular mycorrhizal fungi (amf) spores showing antagonistic effects against fungal pathogens and yet compatible with amf. seven isolates of bacteria were isolated from surface-sterilized amf spores of sp. (gg) and five isolates of bacteria isolated from sp. (gl). all bacterial isolates were identified based on its morphological methods and biochemical reaction and revealed that 9 isolates belong to genus , while the other 3 isolates belong to genus , and respectively. the tests to the antagonists against fungal pathogens and stimulation of amf hyphal development of sp. showed that there were 3 isolates of bacteria ( gg1, gg5 and gl3) had the ability to inhibit the growth of pathogens and to enhance the development of amf hyphae sp . these three bacteria isolates potentially can be used to enhance the quality of amf inoculants as biocontrol and biofertilizers. enzymatic activity test showed that there were 7 isolates of bacteria that produced cellulase and protease activities, i.e. gg1, gg3, gg6, gg7, gl2, gl4 and gl5. bacteria, arbuscular mycorrhizal fungi spores, antagonistic effects, stimulation effects, fungal pathogens 1 2 1 2 department of silviculture, faculty of forestry, bogor agricultural university, bogor 16680, indonesia faculty of forestry, the state university of papua, west papua, indonesia gigaspora glomus bacillus pseudomonas proteous enterobacter gigaspora in vitro bacillus subtilis pseudomonas diminuta enterobacter hormaechei gigaspora in vitro bacillus subtilis bacillus cereus bacillus laterosporus bacillus pasteurii proteus penneri bacillus firmus bacillus cereus abstract key words: * corresponding author : wilarso62@yahoo.com 38 introduction many kind of microorganisms have been observed to be associated with mycorrhizosphere of different host plants. the functional significance of the microbial associate in the ecosystem has been well documented (budi 2012) including associative n -fixing bacteria (cruz & ishii 2011), plant growth-promoting rhizobacteria (siddikee . 2010), phosphate solubilizing bacteria (khan . 2009), and antagonists of plant pathogens (artursson . 2006, bakhtiar 2010). in addition, the bacteria associated with spores of amf and (roesti . 2005), (cruz . 2008, cruz & ishii 2011, horii & ishii 2006, budi 2012), and (bharadwaj . 2008) have been reported. these amf spores associated bacteria has been reported significantly increased amf hyphal growth and suppress fungal pathogens (cruz & ishii 2011). associated microorganisms may complement mycorrhizal activities, particularly in biological control and biofertilizer in agricultural systems. nowadays, the awareness of international community to food safety has increased due to negative impacts of the use of chemical substances in crop production. the movement of “back to nature” of international community increases and organic food has become a new trend for their life style. intensive agriculture in many countries including indonesia have led to excessive inputs of agrochemicals (fertilizers, pesticides) but agricultural policies are now tending towards more organic for sustainable agricultural systems. sustainability in agriculture has been defined as the successful management of resources for agriculture to satisfy enhancing the quality of the environment and conserving resources (ladha 1992). in this respect, the government of indonesia c.q ministry of agriculture has declared “go organic in 2010”. it implies better soil management to avoid modifications that have adverse effects on chemical and biological processes supporting plant growth. one approach is the decrease in chemical inputs to rational (economic but non-polluting) levels through the development of alternative strategies to ensure acceptable yield. an increasing demand for low-input of chemical substance in agriculture has resulted in greater interest in soil microorganisms that increase soil fertility or improve plant nutrition and health including the use of amf as biofertilizer and biocontrol agents. the use of amf for sustainable agriculture has been widely used and reported significantly increased plant productivity (wu & zou 2009; siddikee . 2011), but the avaibility of high quality inoculants is very limited due to the production of this fungi as biofertilizer. up to now, biocontrol is carried out in open pot culture which is sensitive to contamination by other organism or soil borne plant pathogen. therefore, the development of amf inoculum production is needed in order to obtain high quality and purity of amf inoculum. one approach to produce a high quality and purity of amf inoculum is by using amf spore associated with bacteria. the objective of this study was to acquire bacteria from surface-sterilized spores of amf that showed antagonistic effects against fungal pathogens and yet compatible with amf which potential to improve inoculum quality. et al. et al et al et al et al. glomus constrictum glomus geosporum et al gigaspora margarita et al et al. glomus intraradices glomus mosseae et al et al 2 39 bacteria from arbuscular mycorrhizal fungi spores sp. and sp. sri wilarso budigigaspora glomus – et al. materials and methods isolation and identification of bacteria from the amf spores antagonistic effects of isolated bacteria towards soil borne plant pathogen stimulation effect of bacteria on hyphal growth of amf spores the spores of amf sp. and sp. originally from the collection of silviculture laboratory, faculty of forestry ipb were sieved by wet sieving according to method of gardeman and nicholson (1963). hundred spores of each species were surface sterilized according to the method described by budi . (1999a). the surface sterilized spores were crushed using sterile needles and transferred to petri dishes containing sterile agar base (sigma-aldrich) and nutrient agar (oxoid) and then incubated in the dark at 30 c for 24 h. the bacteria that grew were transferred to another petri dishes containing the same media until single colonies were obtained. a single colony was then identified based on morphological methods using biochemical reaction developed by biomerieux and carried out at laboratory of identification and determination, school of life science and technology, bandung institute of technology, bandung, indonesia. isolated bacteria were tested toward soil borne plant pathogen . the common soil borne plant pathogens like sp., sp. and sp. originally from the laboratory of pest and diseases, faculty of forestry ipb, were used in this study. the test was carried out according to the method of varese . (1996). the pathogens were grown on petri dishes containing potato dextrose agar (difco) media. colony of bacteria were placed about 1.5 cm from the pathogen. the petri dishes were then sealed with parafilm, incubated in the dark at 25 c for 7 days. the radial growth of mycelium were measured and compared to the control treatment. the experiment was carried out in a completely randomized design composed of 12 bacterial treatments and one control in triplicates for each pathogenic fungi. data were analysed by one-way anova. the amf sp. spores were collected and surface sterilized according to the method of budi . (1999a). the spores were then placed in petri dishes containing sterile zeolit medium supplemented with nutrient. ten spores were put on sterile filter paper saturated with 50 l suspension of bacteria and were then placed on each petri dish. the control sterile filter paper was saturated with sterile water. petri dishes were then sealed with parafilm, incubated in the dark at 25 c for 21 days. the hyphal growth were recorded starting from germ tube and only the main hyphae was measured (fig. 1) and compared to the control treatment. the experiment was performed in a completely randomized design and composed of 12 bacterial treatments and one control in triplicates. data were analysed by one-way anova. gigaspora glomus et al pseudomonas in vitro slerotium rhizoctonia ganoderma et al gigaspora et al o o o in vitro in vitro biotropia vol. 20 no. 1, 2013 40 plate assay for enzymatic activities test isolated bacteria from amf spores bacterial isolates were grown in erlenmeyer flasks for 24 h on a culture shaker at 30 c in nutrient broth buffered medium. ten micro liters of each bacterial suspension ±10 cfu/m/l was spotted onto plates containing the substrate of the enzyme to be tested and grown at 25 c for 48 h. cellulolytic activity was assessed as described by teather and wood (1982) using a solid medium, containing mgso 7h o (0.1 g/l), cacl 2h o (0.2 g/l), feso 7h o (0.04 g/l), nacl (0.2 g/l), kh po4 (0.3 g/l), k hpo4 (0.5 g/l), cmc (carboxymethylcellulose) (sigma) (5 g/l), yeast extract (0.1 g/l) and bacto agar (15 g/l). for visualization of -d-glucan hydrolysis, the agar medium containing cmc was flooded with an aqueous solution of congo red (1 mg/ml) for 15 min (budi . 2000). the clear zones was measured after 48 h incubation. the proteolytic activity test was performed according to the method described by dunne . (1997). the experiment was performed in a completely randomized design and composed of 12 bacterial treatments and one control with triplicates. data were analysed by one-way anova. a total of 12 bacteria isolates were isolated and identified from surface sterilized spores of am fungi. there were 7 isolates of bacteria isolated from sp. and 5 isolates isolated from sp. the morphological characteristics of each isolate of bacterial colony and its identification are shown in table 1. 0 8 0 4 2 2 2 4 2 2 2 β et al et al gigaspora glomus results and discussion figure 1. hyphal growth measurement 41 bacteria from arbuscular mycorrhizal fungi spores sp. and sp. sri wilarso budigigaspora glomus – et al. source of isolate isolates code morphological characteristics of bacterial colony (colour, colony surface and type) gram species name gigaspora sp. gg1 positive bacillus subtilis gg2 positive bacillus licheniformis gg3 positive bacillus cereus gg4 positive bacillus brevis gg5 negative pseudomonas diminuta gg6 positive bacillus laterosporus gg7 circular, entire, umbonate, cream, opaque circular, undulated, umbonate, cream, opaque circular, serrate, raised, opaque circular, undulated, raised, surface dried, opaque circular, entire, convex, transparent, yellowish circular, filamentous, raised, opaque circular, undulate, filamentous, raised, opaque positive bacillus pasteurii glomus sp. gl1 negative bacillus circulans gl2 negative proteus penneri gl3 negative enterobacter hormaechei gl4 positive bacillus firmus gl5 circular, entire, umbonate, translucent circular, entire, convex, translucent, yellow circular, entire, convex, transparent irregular, undulate, raised, transluent circular, serrate, raised, opaque, positive bacillus cereus table 1. species identification and morphology characteristics of isolates of bacteria isolated from amf spores of sp. and sp.gigaspora glomus antagonistic effects of isolated bacteria against fungal pathogen. the results indicated that there are 2 bacterial isolates originated from sp. spores ( gg1 and gg5) and 2 bacterial isolates from sp. spores ( gl2 and gl3) have the ability to inhibit the growth of three pathogens tested. the percentages of inhibition gigaspora bacillus subtilis pseudomonas diminuta glomus proteus penneri enterobacter hormaechei biotropia vol. 20 no. 1, 2013 42 varied among pathogens tested. the bacterial isolates of gg1 and gg5 inhibited the growth of mycelium of sp. by 7980%. while four isolates of bacteria ( gg1, gg5, gl2, and gl3) showed inhibitory effects to by 46-54%. testing against sp. showed that bacterial isolates gg1, gg5, gl2 and gl3 gave inhibitory effects of 59-90 %. (table 2). among the 12 isolates of bacteria tested, one isolate ( gg5) had a significantly higher effect than the control, while 6 other isolates ( gg1, gg2, gg3, gg4, gg6 and gl3) increased amf hyphal development higher than the control treatment but were statistically not different. of the remaining 5 isolates, 2 isolates significantly inhibited the amf hyphal growth, i.e. gg7 and gl5 while the other 3 isolates inhibited the amf hyphal growth but were not significally different from the control treatment (table 3). ten out of the 12 isolates of bacteria produced cellulase activities, i.e. gg1, gg2, gg3, gg5, gg6, gg7, gl2, bacillus subtilis pseudomonas diminuta sclerotium bacillus subtilis pseudomonas diminuta proteus penneri enterobacter hormaechei rhizoctonia ganoderma bacillus subtilis pseudomonas diminuta proteus penneri enterobacter hormaechei pseudomonas diminuta bacillus subtilis bacillus licheniformis bacillus cereus bacillus brevis bacillus laterosporus enterobacter hormaechei bacillus pasteurii bacillus cereus bacillus subtilis bacillus licheniformis bacillus cereus pseudomonas diminuta bacillus laterosporus bacillus pasteurii proteus penneri enterobacter stimulation effect of isolated bacteria on hyphal growth of amf spores sp. enzymatic activity characterization of bacteria gigaspora in vitro table 2. yradial growth of pathogenic fungal m celium in vitro no isolates radial growth of mycelium (cm)* and % inhibition sclerotium sp. % rhizoctonia sp. % ganoderma sp. % 1 control 9.00 a 0 9.00 a 0 9.00 a 0 2 gg1 1.70 d 81.11 4.17 d 53.66 1.04 c 88.44 3 gg2 9.00 a 0 9.00 a 0 9.00 a 0 4 gg3 9.00 a 0 9.00 a 0 9.00 a 0 5 gg4 9.00 a 0 9.00 a 0 9.00 a 0 6 gg5 1.88 d 79.11 4.35 d 51.66 1.12 c 87.55 7 gg6 9.00 a 0 9.00 a 0 9.00 a 0 8 gg7 9.00 a 0 9.00 a 0 9.00 a 0 9 gl1 9.00 a 0 4.65 b 48.33 9.00 a 0 10 gl2 2.99 c 66.77 4.16 d 53.77 0.92 d 89.77 11 gl3 3.86 b 57.11 4.86 c 46 3.68 b 59.11 12 gl4 9.00 a 0 9.00 a 0 9.00 a 0 13 gl5 9.00 a 0 9.00 a 0 9.00 a 0 *values in a column followed by the same letter do not differ significantly from each other at value of 0.05.p 43 bacteria from arbuscular mycorrhizal fungi spores sp. and sp. sri wilarso budigigaspora glomus – et al. table 3. stimulation of amf hyphae of sp. growth after 21 days incubation by bacteria gigaspora no isolates hyphal length (μm)* % increased/ decreased (-) 1 control 202.44 bc 2 bacillus subtilis gg1 286.31 ba 41.43 3 bacillus licheniformis gg2 369.84 ba 82.69 4 bacillus cereus gg3 231.79 bc 14.50 5 bacillus brevis gg4 287.37 ba 41.95 6 pseudomonas diminuta gg5 499.19 a 146.59 7 bacillus laterosporus gg6 363.89 ba 79.75 8 bacillus pasteurii gg7 19.62 e -90.31 9 bacillus circulans gl1 139.46 bcd -130 10 proteus penneri gl2 160.68 bcd -20.62 11 enterobacter hormaechei gl3 324.47 ba 60.28 12 bacillus firmus gl4 181.46 bc -11.56 13 bacillus cereus gl5 156.38 bcd -22.75 note : * values in a column followed by the same letter do not differ significantly from each other at value ofp hormaechei bacillus firmus bacillus cereus bacillus subtilis bacillus cereus bacillus brevis bacillus laterosporus bacillus pasteurii proteus penneri bacillus firmus bacillus cereus bacillus subtilis bacillus cereus bacillus laterosporus bacillus pasteurii proteus penneri bacillus firmus bacillus cereus et al. et al. et al. et al. gl3, gl4, and gl5, while 8 isolates produced protease activities, i.e. gg1, gg3, gg4, gg6, gg7, gl2, gl4, and gl5. there were 7 isolates that produced both cellulase and protease activities, i.e. gg1, gg3, gg6, gg7, gl2, gl4 and gl5, as indicated by clear zone (halo) around the colony (table 3, fig. 2). bacteria have been observed to live in close association with amf (gopal 2012), and have been isolated from different amf spores (xavier & germida 2003; roesti 2005; horii & ishii 2006; cruz 2008; bharadwaj 2008;, figure 2. celullase activity (a) and protease activity (b) as indicated by clear zones (halo) biotropia vol. 20 no. 1, 2013 44 cruz & ishii 2011). cruz and ishii (2011) found 2 bacteria sp. and isolated from liquid present inside amf spore of , while budi (2012) found four bacteria sp. and ksc_sf9c isolated from surface sterilized amf spores . xavier and germida (2003) found 5 bacteria, , sp. , and sp. from surface sterilized amf spores of . this study demonstrated different bacteria species isolated from surface sterilized amf spores of sp. and sp. we found 7 bacteria from sp. and 5 bacteria from sp. as shown in table 1. as reported by roesti . (2005), the bacterial community associated with the amf spores was more influenced by the amf identity (amf species) than by the host plant. the difference in composition of the spore walls or exudates of amf species may have played a major role in the selection of bacterial populations living on the spore (roesti . 2005). bacteria associated with amf spores have been reported to play an important role in antagonistic effect of soil borne plant pathogens, amf spore germination and hyphal growth (horii & ishii 2006; cruz & ishii 2011). the interesting thing from this study was that among the 12 isolate bacteria tested, 3 bacterial isolates ( gg1, gg5 and gl3) have the ability to inhibit the growth of three pathogens and stimulate hyphal growth of sp. (table 2 and 3). this finding was in line with cruz and ishii (2011), who found that sp., and isolated from surface sterilized bacillus b. thuringiensis gigaspora margarita et al. bacillus bacillus megaterium, bacillus subtilis bacillus flexus strain g. margarita alcaligenes bacillus burkholderia flavobacterium pseudomonas glomus clarum gigaspora glomus gigaspora glomus et al et al bacillus subtilis pseudomonas diminuta enterobacter hormaechei gigaspora bacillus b. thuringiensis paenibacillus rhizosphaerae table 4. enzymatic activities of bacteria isolated from sp. and sp. as indicated by clear zones diameter after 48 h incubation gigaspora glomus no bacteria isolates enzymatic activities ( ø clear zones, cm) celluase protease 1 bacillus subtilis gg1 4.80 cd 4.80 ba 2 bacillus licheniformis gg2 2.83 e 0.00 c 3 bacillus cereus gg3 5.70 b 6.50 a 4 bacillus brevis gg4 0.00 f 5.77 a 5 pseudomonas diminuta gg5 5.10 cb 0.00 c 6 bacillus laterosporus gg6 5.87 b 6.20 a 7 bacillus pasteurii gg7 7.17 a 5.83 a 8 bacillus circulans gl1 0.00 f 0.00 c 9 proteus penneri gl2 5.07 cb 4.73 ba 10 enterobacter hormaechei gl3 2.97 e 0.00 c 11 bacillus firmus gl4 3.23 e 3.73 b 12 bacillus cereus gl5 4.10 d 4.73 ba * values in a column followed by the same letter do not differ significantly from each other at value of ,0.05p 45 bacteria from arbuscular mycorrhizal fungi spores sp. and sp. sri wilarso budigigaspora glomus – et al. amf spores had antagonistic effect to the pathogenic fungi and and stimulate hyphal growth of . many studies have reported that has the potential to be used as biocontrol agent against soil borne plant pathogens (nalisha . 2006; velmuragan . 2009). phae . (1990) found that produced , a bioactive compound that are active as antifungal agents against several plants pathogenic fungi as well as antibacterial. the production of hydrolytic enzyme protease by sp. isolated from mycorrhizosphere that caused cell wall degradation of pathogenic fungi have also been reported (budi 2000). in this experiment, also produced hydrolitic protease enzyme. we assume that combination of protease enzyme with the other active compounds produced by could inhibit pathogenic fungal growth. other bacteria such as and have been reported effectively control diseases caused by soil borne plant pathogens (thomashow . 1990; chermin . 1995, barea . 1998). these bacteria produced bioactive compound phenazine-1-carboxylic acid responsible for suppression of soil borne plant pathogenic fungi (thomashow . 1990). in this study only gg5 produced hydrolytic protease enzyme but not gl3, indicating that the role of these enzymes in controlling the growth of soil borne plant fungal pathogens is not clear and it seems that the inhibitory effect of these bacteria to soil borne plant fungal pathogen development is probably derived from more than one mechanism (budi . 2000). further investigation is necessary to isolate bioactive compound and its mode of action from these 3 bacteria isolates. the stimulation of amf hyphal growth by amf spores associated with bacteria have been reported by several researchers (budi 1999b; cruz & ishii, 2011). in this study gg1, gg5 and gl3 have the ability to stimulate hyphal growth of sp. and supressed several soil borne fungal pathogens . four others isolates only have the ability to stimulate hyphal growth of sp. (table 3). the exact mechanism of hyphal growth stimulation by spores associoated with bacteria is not known, but cruz and ishii (2011) reported that bacteria released volatile compounds and exudates involved in stimulating hyphal growth. futher study is needed to investigate the possible isolate volatile compounds and exudate released by these 3 bacteria isolates. certain arbuscular mycorrhizal fungi hyphae produce hydrolytic enzymes which hydrolyses the biopolymers such as protein, chitin and cellulose that will help the amf to degrade and infect the plant cell walls (gopal . 2012). furthermore, the hyphae and spores of amf also harbour associated bacteria that produce hydrolytic enzyme (budi . 1999b). in this experiment, 7 isolates produced both cellulase and protease activities, i.e. gg1, gg3, gg6, gg7, gl2, gl4 and gl5, as indicated by clear zone (halo) around the colony (table 3, fig. 2). as reported by budi (2012) isolated from surface sterilized amf spores have shown to increase mycorrhizal root colonization of neem seedling in the nursery and also produced cellulase and protease activities. gigaspora margarita rosellimia necatrix, phytium ultimum, fusarium oxysporum rhizoctonia solani g. margarita b. subtilis et al et al et al b subtilis iturin paenibacillus et al. b. subtilis b. subtilis pseudomonas enterobacter et al et al et al et al pseudomonas diminuta enterobacter hormaechei et al et al. bacillus subtilis pseudomonas diminuta enterobacter hormaechei gigaspora in vitro gigaspora in vitro et al et al bacillus subtilis bacillus cereus bacillus laterosporus bacillus pasteurii proteus penneri bacillus firmus bacillus cereus et al. bacillus subtilis g. margarita biotropia vol. 20 no. 1, 2013 46 production of volatile compounds that can positively influence germination of amf spores, provision of nitrogen through nitrogen fixation, solubilization of soil phosphate sources, detoxification of the fungal microhabitat, change of ph level of siderophores are also some of the proposed mechanisms underlying stimulation of mycorrhization (bharadwaj 2012). further study is needed to test the consistency effects in the field, and to investigate the mechanisms involved in the antagonism towards fungal pathogens as well as stimulation effect to the hyphal growth of am fungi. there is a possibility that 3 isolate bacteria ( gg1, gg5 and gl3) isolated from surface sterilized spores of am fungi sp. and sp. have the ability to inhibit the growth of 3 pathogens and stimulate hyphal growth of sp. . these bacteria have a great opportunity to be used as biocontrol agents for soil borne plant pathogens as well as for improving the amf inoculum quality, since up to now the production of these fungi for biofertilizers and biocontrol is carried out in open pot culture sensitive to contamination by other organisms or pathogens. this research was supported by biotrop through the competitive research grant no 050.7/psrp/spk-pnlt/iii/10. the authors would 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growth and root nutrient status of citrus subjected to salt stress. 35: 388-91. xavier ljc, germida jj. 2003. bacteria associated with spores influence mycorrhizal activity. soil biol biochem 35: 471-8. bacillus subtilis bacillus licheniformis in vitro in vivo science asia glomus clarum 49 bacteria from arbuscular mycorrhizal fungi spores sp. and sp. sri wilarso budigigaspora glomus – et al. 4. rini (dengue).cdr biotropia vol. 19 no. 1, 2012: 30 41 dengue early warning model using development stages of mosquito and climate information aedes aegypti rini hidayati , rizaldi boer , yonny koesmaryono , upik kesumawati , and sjafrida manuwoto received 10 january 2012/accepted 15 may 2012 this study aims at developing a dengue early warning model using climate information. the model was developed through three steps of analyses. the first step was to determine the length of periods used in prediction and optimal time for eradicating mosquito's breeding sites. the second step was to identify the best prediction model of dengue incidence rate (ir). the third step was to develop an early warning model using stochastic spreadsheet. it was found that the best predictor for predicting dengue incidence rate at week-n (ir ) was (1) rainfall index with two weeks lead time (ich ). the rainfall index of week-n is a function of three week moving averages rainfall (ch3), . (ch3 -1.155*ch3 +0.702*chn-2), and (2) ir with one week lead time (ir ). the ir model prediction was ir = 0.795*ir +0.067*ich with r =76.6%. these models (models resulted from the first and third steps) can be used to provide early warning on optimum time for controlling the mosquito's breeding sites and the need for fogging action in order to prevent the dengue incidence rate beyond the critical limit as defined by the ministry of health. , early warning model, breeding site, time control, climate variability, climate change 1* 2 1 3 4 th 2 1. 2. 3. 4. departement of geophysics and meteorology, bogor agricultural university (ipb), darmaga, bogor, 16680, indonesia centre for climate risk and opportunity management in southeast asia and pacific (ccrom-seap), bogor, 16143, indonesia veterinary health entomology laboratory, veterinary faculty, ipb, bogor, 16680, indonesia entomology laboratory, dept. of plant protection, ipb, bogor, 16680, indonesia. aedes aegypti i.e aedes aegypti abstract introduction n n-2 n n-1 n-1 n n-1 n-2 key words: since mid 20 century (christopher 1960), until the beginning of the 21 century, the disease of dengue haemorragic fever (dhf) has been given attention worldwide. data of 2007 indicated that the number of cases and death rate of the th st * corresponding author : rinihidayatigfm@gmail.com 30 patients, as well as the number of cities infected by this disease keeps on increasing. according to ipcc (2007), the attack will increase due to climate changes at high confidence level. the use of early warning system and epidemic prediction is one potential of adaptation options to reduce the impact of weather and climate in the field of health (mc michael . in kovats . 2000). the life of vector and the transmission process of dhf disease are closely related with climate conditions before disease incidence; thus, studies related to climate have been increasing. the current study, however, has been directed to the relation between this disease and climate information related to climate change issues (hales . 2002; reiter 2001) and climate variability in order to anticipate both seasonal and annual incidence of such a disease (focks . 1995; schreiber 2001; peterson . 2005; sasmito 2006; sintorini 2006). the models have been developed consisting of a mechanistic models (fock . 1993, focks . 1995, reiter 2001, and sintorini 2006), as well as empirical models (hales . 2002, schreiber 2001; peterson . 2005; sasmito . 2006). however, there has not been any prediction model on the number of dhf incidence used by the department of health in indonesia. this is due to the consideration that the model is inaccurate, complicated in application, relatively costly in obtaining input parameter, or related to a specific location only. model to be developed is a simple model that will be easy to apply. the model will be a combination of mechanistic and empirical models. input variables are easily obtained. the prediction model of dhf based on climate information is expected to be used as materials for an early warning system that is beneficial to develop strategies for anticipation and control for such disease; particularly the one in indonesia. a number of reasons for this are, among others: (1) there is a climate observation station, particularly for rainfall, in every city/regency (2) climate observation has been carried out routinely and institutionally, and (3) climate determines the breeding places, activities of the vector ( ) and pathogen ( virus) so that the incidence of dhf needs climate pre-condition. in general, this study was aimed at developing dengue early warning model as the basis for determining the anticipation and control step upon dengue incidence which is related to climate occurrence. this research was carried out through three stages. the first stage was determining the length of mosquito's life cycle period. the second was developing a prediction model on dengue incidence rate by using climate variables. while, the third stage was an early warning model by using stochastic spreadsheet. et al et al et al et al et al et al. et al et al et al et al et al aedes aegypti dengue materials and method 31 dengue early warning model using development stages of rini hidayatiaedes aegypti et al. determining the length of development stages period for mosquito and dengue virus developing a prediction model for dengue incidence rate (ir) developing early warning model using stochastic spreadsheet psn ir aedes aegypti the prediction on the length of periods for mosquito's development stages at one particular location was determined based on heat unit. the period length of one stage in mosquito's development (n days) was counted from heat unit value (hu c days), divided by the difference between the daily mean air temperature (t c) in the study area and basic temperature t by using the following formula: such length of period was used as: (1) basis for determining the period for controlling mosquito's breeding sites (psn) which consisted of the eradication of immature mosquito breeding sites (tpn, days) and mature mosquito's resting places (snd, days), and (2) basis for determinating of predictor's length period based on climate variables to predict ir. incidence rate of dengue (ir) data availability was weekly. therefore, the analysis of climate variables to predict ir were also used in weekly period. predictors of climate variables were grouped into wetness and thermal data. the length of wetness period was determined from immature stage (pd) mosquito phase length in weekly (n /7), whereas thermal length period was determined by the length of virus extrinsic incubation period (eip), also in weekly period (n /7). weekly wetness data (in cm/week) was obtained from the sum of daily rainfall data. weekly thermal factors that consist of mean, maximum and minimum temperature (tr, tn and tx, in c), were obtained by averaging daily data. further analysis was conducted to calculate the data in the form of moving averages. determining the period for moving averages analysis were based on pd for wetness data and eip for thermal data. further analysis was also conducted to construct predictive models in the form of principal component regression of the best predictors obtained from the previous analysis. the data used were the ones from indramayu regency (2002 2006). meanwhile, the data used in validation process were taken from indramayu (2007), bogor (2003 2006), north jakarta (1998 2002), and padang (2003 -2005). the early warning model discussed here was the prediction of the optimum time model for implementation, dengue incidence rate ( ), and the need for fogging. model was developed in the form of stochastic spreadsheet by using crystall ball program package. the prediction of the optimum time for psn implementation refered to optimum time for the implementation of cleaning water tanks where immature mosquito (tpn) lives, and optimum time for the implementation of cleaning adult mosquito's breeding sites (snd). each of tpn and snd was predicted based on the number of days in the period of pd mosquito and eip virus using the daily mean air temperature as an input. in the case of pd and eip prediction, the average daily temperature in a week was 0 0 o a b pd eip n = hu/(t -t ).a b 32 biotropia vol. 19 no. 1, 2012 considered to represent the average daily temperature during the period of pd or eip, so the prediction for the next few days could be done. prediction of dengue incidence rate was conducted by using the best prediction model. the need for fogging was determined based on the critical boundary of ir figure determined by the minister of health namely, 20 patients per 100 000 inhabitants per year for an endemic regency. focks . (1993) have shown that the higher the air temperature, the faster the gonotropic cycle and virus extrinsic incubation. in accordance to heat unit concept, hidayati . (2007) revealed that the higher the air temperature, the faster the egg hatches and grows into adult mosquito, or, the faster the mosquito complete all of their life cycle stages, including their age. furthermore, the heat unit of each development phase was relatively constant, thus, the higher the air temperature, the faster the heat unit that needs to be reached. pd and eip were determined based on the following formulation: results and discussions the length of development stages period for mosquito and dengue virus aedes aegypti et al et al � �ba tt hu n � � hu nd tb were heat unit and basic temperature which are suitable for immature stage and extrinsic incubation period. t was the daily mean air temperature in the study region. basic temperature and heat unit value in mosquito's various development phases and their relation to dengue virus transmission are presented in table 1 a . a table 1. basic temperature and heat unit at various stages of mosquito's life phase tb (0c) heat unit (hu in c0days) average cv(%) immature 15.0 224 27.4 eip virus 17.0 128 5.0 gonothropic 17.5 36 6.4 age 10.0 544 24.6 cv : variation coefficient (source: hidayati 2007).et al. d . hf endemic regions are generally located at low altitude or city with dense population and relatively high temperature. data on temperature in 4 research locations; namely, indramayu, north jakarta, bogor and padang were considered to represent data on air temperature of several big cities /regencies with endemic areas of dhf in indonesia. in the weekly period, the average length of pd and eip in four locations were nearly the same (table 2); that is, 3 weeks for the phase of mosquito immature stage (pd) and 2 weeks for virus extrinsic incubation period (eip) 33 dengue early warning model using development stages of rini hidayatiaedes aegypti et al. table 2. t s he average length of the phase of mosquito immature stage and virus extrinsic incubation period in four research location t . t . a he most influential climate variable in pd period of mosquito was the existence of wetness (rain), while the dominant influence in eip was temperature. accordingly, to develop a relation model between dengue incidence rate and climate variables, there is a need to have 3 weeks moving average analysis, like the length of pd mosquito period for wetness variable, and 2 weeks moving average analysis, like the length of eip for thermal variable he interview with the head of disease eradication and prevention department, health office of indramayu regency (august 2006) revealed that the shortest period for about 2 weeks was required to be able to utilize the result of early warning in determining operational steps for controlling the diseases. therefore, in selecting the best climate predictor variable, an interval of two weeks between climate occurence and diseases incidence was used results of the analysis of all available climate variables, showd that the best subset of predictive models predictors in dhf incidence rate of two weeks after the climate incidence were rainfall (ch) and temperature (t) in the form of , , , , , and . number 2 or 3 attached to variable ch (rainfall) and temperature including tr (average temperature), tx (maximum temperature), and tn (minimum temperature) were weekly periods for moving averages analysis. index indicates the time of occurrence during week before the ir occurrence that has been predicted. actually, the rainfall and air temperature data were the easiest climate variable to be obtained. ll climate information consisting of those rainfall and temperature were calculated in terms of the relation between climate variable with dengue incidence rate of dhf by using the principle component regression analysis. by using this method, multikolinieriti among the climate data could be ignored. the result of the principle component regression analysis was: (equation 1) (r adjusted = 52.1%; dws = 0.634). p s only p )rediction model for dengue incidence rate (ir rediction based on climate variable i ch3 ch3 ch3 tr2 tx2 tn2 ir = 0.920 + 0.157*ch3 0.052*ch3 + 0.066*ch3 + 0.826*tr2 0.387*tx2 0.492* tn2 n-2 n-4 n-5 n-2 n-2 n-2 n n-2 n-4 n-5 n-2 n-2 n-2 n-i 2 district/city mean air temperature (oc) immature stage(pd) extrinsic incubation period (eip) days weeks days weeks indramayu 27.3 21 3 12 2 jakarta utara 28.0 20 3 12 2 bogor 27.0 21 3 13 2 padang 27.1 21 3 13 2 34 biotropia vol. 19 no. 1, 2012 these results indicate that rainfall from 7 until 2 weeks before the week of disease incidence and air temperature at 3 and 2 weeks before the period of the disease incidents would determine the amount of ir. ir will be high if the rainfall in week 7, 3, and 2 is high, while in contrary low at week 6, 5 and 4. redictors comprised the combination of climate variables of two weeks before the dengue occurrance period, and ir one week before the disease incidence. by including ir aside from the climate variables consisting of rainfall, average temperature, maximum temperature, and minimum temperature, the equation of relation model obtained was reasonably good with the following form: r the next analysis result obtained by ignoring air temperature data did not reduce the model quality. if only rainfall was used as predictor, the equation produced would also be good. the form of its equation model is: , where (equation 3) if the model is developed based on predictor that consists of the combination of climate and ir at two weeks before the incidence, temperature has to be calculated together with rainfall in order to obtain a good equation model: (r : ; . (equation 4) (r adjusted = 78.0% ; dws = 2.202 ; ~ n(0; 0.33; p:0.064) et al the equation model obtained was not good enough with a determination coefficient only 52%, the dws value stayed far below 2.0, that means that residual model still contained an obvious autocorrelation value at level 1 so that the model was not adequately stable when used for prediction (selvanathan . 2004). the obstacle on the low accuracy of prediction model which only includes climate variables was also found by not only schreiber (2001) for the weekly-scale model, but also sasmito (2006) and sintorini (2006) for the monthly-scale model. a better prediction model can be obtained by including non climate factor; that is, ir before prediction period. ir was chosen as a predictor variable by considering that ir was relatively easy to be obtained by the health office in regency/city, and able to describe the level of inhibitant's susceptibility. p . p d rediction based on the combination between climate variables and ir before predictionperio ir = 0.795*ir + 0.067*ich3 ir = 0.612*ir + 0.111*ich3 + 0.315*it2 ir = 0.744*ir + 0.070*ch3 0.073*ch3 + 0.042*ch3 + 0.199*tr2 0.115*tx2 0.079*tn2 . ( ich3 = ch3 1.155*ch3 + 0.702*ch3 ich3 = ch3 0.715*ch3 + 0.244*ch3 , and it2 = tr2 0.577*tx2 0.397*tn2 n n-1 n-2 n-4 n-5 n-2 n-2 n-2 n-2 n-2 n-4 n-5 n-2 n-2 n-4 n-5 n-2 n-2 n-2 n-2 2 2 2 adjusted = 78.4%; dws = 2.151; ε ~ n(0; 0.33; p:0.094). corrected = 70.3%; dws : 1.182; ε ~ n(0; 0.38; p:0.040). the form of the equation model is ε n n-1 n-2 n n-2 n-2 n-2 wnwnwnwnwnwnwn-wnch3n ch3n ch3n t2nirn-1 ir note: w: week, ch: rainfall period, t:temperature period, ir: incidence rate period figure 1. time frame period of ir prediction, climate variables and ir as predictors 35 dengue early warning model using development stages of rini hidayatiaedes aegypti et al. t . t . he rainfall in indonesia is, in fact, the most fluctuative climate variable which influences other climate variables including temperature, relative humidity, and incoming radiation. in developing ir model with a climate variable, the most obvious predictor was the rain. hidayati . (2008) found that the pattern of monthly dengue proneness index in major parts of indonesia was the same as the pattern of their monthly rainfall, except in some big cities where their maximum index experienced an interval two or three months after the maximum monthly rainfall. the temperature, indeed has an obvious impact, however, the data on temperature was relatively difficult to obtain. based on the equations of prediction models on dengue incidence rate, equation 3 was the most potential to be used as a model for early warning since the information on rain together with ir could be used to predict the incidence of diseases for the next period he result of model validation showed that the model from equation 3 resulted in good ir prediction, not only in indramayu, but also in bogor, north jakarta and padang. rmse relative value was in average less than 1, and the correlation value between prediction result and actual data was significantly high (table 3). furthermore, actual ir plot and ir resulted from the prediction did not show significant difference. however, this model has been underestimated in predicting the high ir et al n-1 table 3. result of prediction model validation based on equation 3 in cities where the altitude was higher (> 500 m asl), or where mean air temperature was significantly lower (< 24 c), the length of period for moving average calculation of climate variable needs to be adjusted to the heat unit concept. furthermore, wetness variable also has to be adjusted to the length of pdn period, and thermal variable has to be tailored to the length of eip. as an example, a place with an air temperature average of 21 c, or at the height of about 1000 m asl, the moving average for wetness variable was approximately 5 weeks, and its thermal period was 4 weeks. thus, the rainfall variables that can be used in developing predictive models of ir are five weekly rainfalls ( ). if we include the temperature in the model, then the form of temperature variables are 4 weekly temperature or (modification of fig. 1). however, the prediction model equations cannot be presented here in detail, because it is not sufficient supporting data are available. in general, a city located at an elevation > 500 m asl is not an endemic area. 0 0 ch5 t4 n n regency/ city year rmse rmse/average correlation coefficient indramayu 2007 1.272 0.601 0.75 bogor 03 – 06 0.930 0.496 0.83 north jakarta 98-02 1.693 0.603 0.90 padang 03 – 05 0.724 0.601 0.75 36 biotropia vol. 19 no. 1, 2012 d teveloping early warning model using stochastic spreadshee early warning dengue model . i stochastic spreadsheet model was developed using weekly time frame, consisting of input cell, assumption cell, formula cell, and prediction cell. model inputs comprised the number of dengue cases one week before prediction period, the number of population, average weekly air temperature (tr ) one week before prediction period (mm). the assumptions used in ir model were error model which was distributed normally with mean value of 0.00 and standard deviation of 0.33. the heat unit of immature mosquito stage was distributed normally with an average of 224 and with a standard deviation of 61. likewise, the eip heat unit was normally distributed with an average of 128.4 and standard deviation of 6.6. this assumption is built from the analysis of the mosquito life cycle heat units (table 1) and the distribution of the ir. thus, this assumption can be applied to most of the dengue endemic areas. the formula cell was used to define ir at the present week, rainfall index, ir the week after, and the averages of pd mosquito and eip virus periods n addition, input, assumption and formula previously defined were then used to conduct a simulation in the dengue early warning model. simulations which were carried out for several times would produce optimum prediction period for tpn and snd, ir value for the following week, and decision whether or not fogging at various n-1 confidence levels should be conducted. each of the optimum period for psn implementation was determined based on the length of both pd mosquito and eip with confidence level (exceeded probability) of 90% or 75% in accordance with level of interest. such value was obtained by filling out the certainty column with the display of simulation result. fogging was suggested if the ir in this week was > 0.5 and predicted to raise in the following week; or if ir prediction for the following week was > 1 table 4. an example of the number of input for early warning dengue model . input parameter value average daily air temperature in the last one week 27.5 number of patients in the last one week 15 number of population 1.500.000 weekly rainfall (mm) in … week before (wb) 7 wb 6 wb 5 wb 4 wb 3 wb 2 wb 0 mm 20 mm 75 mm 100 mm 80 mm 150 mm a . s an illustration, the number of input as shown in table 4, produced prediction values as presented in figure 2. with confidence level of 90%, optimum period of tpn and snd cleaning were conducted in 7 days and 4 days after simulation time, or 14 and 11 days after previous cleaning, and ir prediction value in the following week would exceed the number of patients i.e. 1.6 persons per 100 000 population. consequentrly, fogging is highly recommended 37 dengue early warning model using development stages of rini hidayatiaedes aegypti et al. figure 2. output of dengue early warning model from 1000 times simulations anticipative steps and control of dhf disease incidence i . b . t . n order to control dhf disease, the decree of minister of health (kepmenkes) no: 581 year 1992 suggested to prioritize activities on prevention and society empowerment by performing what is called: psn 3 m plus (eradicating mosquito's breeding site drying, covering, burying, plus adding larvaside, raising fish, using mosquito net, and spraying). in wet tropical regions where the vegetation and fauna are varied, it was relatively difficult to eradicate mosquito. in order to suppress the number of dhf patients, therefore, psn has to be carried out regularly and entirely. so that, mosquito's life cycle can be totally cut. as an effort to control mosquito, the optimum time for psn implementation as resulted from the simulation can be used as guidance ased on the decree of the minister of health (menkes) 581/92 fogging was carried out when there was an outbreak, that is, when the number of cases doubled from the previous period, or when a case occurred in the region where previously none of such case was found. in order to suppress the number of patients not exceeding the critical limit as determined by the ministry of health, fogging is suggested to be carried out when the ir value early in the week is > 0.5. based on rainfall data, it is predicted that ir will increase; and/or ir prediction in the following week is > 1 he model operationalization should be conducted every week, particularly during rainy season, so that the ir prediction value and information whether or not fogging was performed will be available in each of the previous week. dhf disease season generally occurs from the beginning to the end of rainy season. unlike the ir prediction and fogging, psn simulation has to be performed one week after psn was carried out aedes aedes 38 biotropia vol. 19 no. 1, 2012 t . i . he step for anticipation and control of the disease could make use of the result of ir value simulation one week after. such a superficial model can be utilized by personnels who work in hospitals and city/regency health offices as the basic for calculating both structure and infrastructure that need to be provided for nursing and treating the patients in the following week n the beginning of rainy season, where the number of dhf patients starts to rise, it is suggested to carry out psn at time period in accordance with exceeded probability of 90%. in the season where the ir is relatively low, psn period at probability level of 75% can be chosen (fig. 3) 2928272625 14 12 10 8 6 4 2 temperature (cdeg) d ay af te r a w ee k te m p m ea su re m en t tpn75 tpn90 snd75 snd90 variable scatterplot of tpn75; tpn90; snd75; snd90 vs t ( c)o figure .3 period (days) of controlling immature mosquito breeding sites (tpn) and mature mosquito breeding sites (snd) recommendation for the dry season (75) and rainy season (90) t . he result of ir value prediction simulation is needed as the base of calculation upon structure and infrastructure for controlling dhf diseases. since treating dhf patients has to be immediately carried out, both supporting structure and infrastructure should be available in an adequate number, or even, higher number. to accommodate such needs, ir prediction is suggested to be below the exceeding probability levels; namely 10%, or 25% at the highest the implementation of fogging is highly suggested if the probability level to gain value of 1 in the simulation is higher . each of the value, either 1 or 0 described the need to carry out fogging. from such description, it can be identified that the higher the ich, the higher confidence value upon the suggestion to carry out fogging. conversely, fogging will not be suggested if, naturally, climate supports the decrease of ir cases. accordingly, although ir prediction is high , but naturally there will be a decrease untill less than 1, fogging is not really suggested. the fogging of superficial model result (fig. 4) has not been able to describe the characteristics of fogging: focused (on the selected/limited area), or mass (on an extensive area). fogging can be carried out by the community or funds provided by 39 dengue early warning model using development stages of rini hidayatiaedes aegypti et al. figure 4. level of fogging requirement the local government. such a built model can only predict the incidence of one week ago (for the next week). as a result, fogging activities are suggested to be prioritized by the community independently under supervision or as a program of city/regency's local health office. conclusions early warning models can be developed based on (1) the weekly ir model prediction and the optimum time for controlling mosquito's breeding sites (psn implementation) models using heat unit concept, (2) for controlling immature mosquito's breeding site, and (3) for controlling moquito's resting places. the rainfall index of week-n , is a function of three weeks moving averages rainfall (ch3), . (ch3 -1.155*ch3 + 0.702*chn-2), and ir is ir one week lead time. model was developed in the form of the stochastic spreadsheet by using crystall ball program package, so that the confidence level of the model outputs can be demonstrated. rom the beginning of rainy season to the beginning of dry season, optimum period for psn implementation of model superficial result is used with 90% exceeded probability, but from the middle to the end of dry season, simulation result with 75% exceeded probability can be used. the provision of both structure and infrastructure is recommended to be in line with ir prediction value at high estimated value with 10% exceeded probability, minimal at low estimated value in accordance with ir prediction value with 25% exceeded probability. fogging activity is suggested if the simulation result of exceeded probability is more than or the same as 90%. a routine simulation model in the endemic region in the low altitude should be conducted every week, particularly in endemic regions. in mid and high altitude, models need to be evaluated ir = 0.795*ir +0.067*ich , n (days) = 256 cdays/((ta-15) c) n (days) = 128 cdays/(ta-17) c ich n n-1 n-2 n i.e. i.e 0 0 0 0 th n n-1 n-1 f . 40 contour plot of fogging vs irn-1, ich3n-2 biotropia vol. 19 no. 1, 2012 references christophers sr. 1960. (l) the yellow fever mosquito. its life history, bionomics and structure. cambridge univ. press. cambridge. focks da, haile dg, daniels e, mount ga. 1993. dynamic life table model for (l.) (diptera: culicidae). analysis of the literature and model development. j med entomol 30: 1003-17. focks da, daniels e, haile dh, keesling je. 1995. a simulation model of the epidemiology of urban dengue fever: literature analysis, model development, preliminary validation, and samples of simulation results. am j trop med hyg 53(5):489-506. ales s, de wet n, maindonald j, woodward a. 2002. potential effect of population and climate changes on global distribution of dengue fever: an empirical model. lancet. 2002. hidayati r, kesumawati u, manuwoto s, boer r, koesmaryono y. 2007. kebutuhan satuan panas untuk fase perkembangan pada nyamuk (diptera: culicidae) dan periode inkubasi ekstrinsik virus dengue (heat unit requirement for development stages of aedes aegypti and extrinsic incubation period of dengue virus). j ekol kes 6(3):648-58. hidayati r, boer r, koesmayono y, kesumawati u, manuwoto s. 2008. sebaran daerah rentan penyakit dbd menurut keadaan iklim maupun non iklim (distribution of vulnerable region of dengue fever disease based on climate and non-climate condition). j agromet xxii (1) : 61-9 ipcc 2007: climate change 2007: impacts, adaptation and vulnerability. contribution of working group ii to the 4 assessment report of the intergovernmental panel on climate change, m.l. parry, o.f. canziani, j.p. palutikof, p.j. van der linden and c.e. hanson, eds., cambridge university press, cambridge, uk, 976pp. kovats, r.s., menne, b., mcmichael, a.j., corvalan, c., bertollini, r. 2000. climate change and human health: impact and adaptation. pp:28 kusriastuti r. 2006. kebijaksanaan penanggulangan demam berdarah dengue di indonesia. makalah disampaikan pada pelatihan klinis, lampung, 19 april 2006 peterson at, martinez-campos c, nakazawa y, martinez-meyer e. 2005. time-specific ecological niche modeling predicts spatial dynamics of vector insects and human dengue cases. transactions of the royal society of tropical medicine and hygiene (2005) 99, 647 – 55. reiter p. 2001. climate change and mosquito-born disease. environ health perspect, 109(1): 141-61 sasmito a, tim bmg. 2006. protipe model kewaspadaan dini bahaya demam bedarah dengue (dbd) di wilayah dki jakarta. makalah disampaikan pada seminar hasil penelitian pengembangan meteorologi dan geofisika tahun 2005. badan meteorologi dan geofisika. jakarta schreiber kv. 2001. an investigation of relationship between climate and dengue using a water budgeting technique. int j biometeorol (2001) 45: 81-9 selvanathan a, selvanathan s, keller g, warrack b. 2004. australian business statistic-abridged 3rd edition. thomson, australia. sintoroni mm. 2006. model dinamika sistim penularan demam berdarah dengue dalam kaitan dengan pola variabilitas iklim di jakarta. disertasi ilmu kesehatan masyarakat, ui. jakarta (unpublished) wmo. 1981. guide to agricultural meteorology practices (wmo-no:134). secret of wmo. geneva. switzerland aedes aegypti aedes aegypti aedes aegypti h . th 41 dengue early warning model using development stages of rini hidayatiaedes aegypti et al. biotropia no. 4, 1990/1991: 1-8 growth and rooting system of acacia mangium obtained by tissue culture iwan setiawan, m.i. umboh and supriyanto seameo biotrop, p.o. box 17, bogor, indonesia introduction since 1980/1981, the government of indonesia through the ministry of forestry has started to reforest logged-over, alang-alang, unproductive areas and to convert them to forest industry plantation. the target is 300 000 ha per year. it means, 750 million seedlings should be provided per year (planting distance 2 m x 2 m). the tree species to be planted in forest industry plantation should have shorter life cycle (8 10 years), good stem-form, good rooting system, and should be fast growing. acacia mangium has been selected as one of the important tree species for forest industry plantation due to its growth, quality of fiber wood (pulp and paper industry) and rooting system (produce a lot of secondary root and nitrogen fixater) (soebardjo 1986). the reforestation of logged-over dipterocarp forests in malaysia with a. mangium has also been considered (appanah and weinland 1989). generally, reforestation with a. mangium is done with seedlings obtained by seed germination. a. mangium produce a lot of seeds but its production is still limited by the season, while the conventional method of vegetative propagation through cuttings gave very low percentage of rooted-cuttings (1%) (umboh and syamsul yani 1989). the micropropagation of a. mangium through tissue culture is a promising method. the production of a. mangium plantlets through that method has been done at the forest genetic laboratory, tropical forest biology, seameo biotrop (situmorang 1988, umboh 1988, umboh et al. 1989, 1990). these rooted-plantlets (plantlings) were first put in the green house (acclimatization) before planting in the field. field tests of some agricultural plants have been done but information on forest trees species is still lacking because the production of plantlings through tissue culture is still limited as there are still problems of their rooting. in fact, the progress of reproducing woody plants by tissue culture has been much slower than with herbaceous plants. the major reason for this limited success with forest trees appears to be due to lack of efforts and because it takes longer time than herbaceous plants (durzan and campbell 1974). generally, rooted-cuttings or rooted-plantlets do not produce tap root, but secondary roots only. the most frequent origin of adventitious roots are the cambium, phloem and pericycle. it is less frequent for 1 biotropia no. 4, 1990/1991 the cortex, pith and xylem (haissig 1974). the time and place of initiation of induced root primordia vary greatly among species (kramer and kozlowski 1979). the rooting system will influence the growth pattern of the rooted-cuttings or plantlings. the objective of this study is to observe the growth and rooting system of a. mangium obtained by tissue culture in the field. the information gathered will be used for sylviculture and genetic study of a. mangium plantlings. materials and method the plantlings of a. mangium were obtained by tissue culture method described previously by umboh and syamsul yani (1990). during the acclimatization period, the plantlings were planted in mixed soil-sand medium (1:1) for one month and green house conditions. after 4 months in the green house, the plantlings were transferred to the field (figure 1). forty plantlings (50-60 cm high) were planted in the field at 3 m x 3 m planting distance. to avoid strong direct sunlight, simple shading with alang-alang leaves was implemented. figure 1. four-month old a. mangium plantlings before planting in the field. the experimental site is located at the forest research and development experimental garden, cikarawang, darmaga, bogor. the area was dominated by imperata cylindrica (alang-alang), open area, 244 m a.s.l. and 3552 mm/year rainfall. the soil type is red brown latosolic and is very fertile up to 1 m soil depth. 2 growth and rooting system of acacia mangium-i. setiawan, m.i. umboh & supriyanto the height and diameter growth rate were measured once a month. the clear-bole height was also recorded. to confirm its rooting system, two trees were dug and compared with other a. mangium trees obtained from seed germination, of the same age, at 2 m x 2 m planting distance, situated 150 meters from the field test and planted by the forest research and development center, gunung batu, bogor. this plantation is designated for provenance trials of a. mangium. results and discussion the height and diameter growth rate of a. mangium plantlings are shown in figures 2 and 3. during the 2.5 years of field test, some important developments were observed: during the first six months, the plantlings were trying to adapt to field conditions. the mean height growth was slow (0.5 m whithin 6 months) and diameter increment was very low. during the second six months the stems were more vigorous and some branches were formed. the mean increase in height was 1.5 m within 6 months. it was figure 2. height growth of a. mangium tree obtained by tissue culture. a : growth during adaptation period b : growth after adaptation period c : growth during maturation period 3 biotropia no. 4, 1990/1991 figure 3. diameter growth of a. mangium tree obtained by tissue culture. faster than during the first six months and the mean diameter increment was more evident (2.5 cm within 6 month). after one year of planting, the mean height increased remarkably (3.5 m within 6 months) and 4.5 cm within 6 months for mean diameter growth rate. many branches at the lowest level broke off naturally (natural pruning). the natural pruning of branches can reach up to 3/4 of the tree height among seed germinated trees because the planting distance is very close (figures 4a and 4b). since the planting distance among the a. mangium plantlings was greater, lower natural pruning (1/2 of tree height) was observed together with more branching (figure 5a). the mean height, diameter and height clear-bole of a. mangium plantlings after 2.5 years of field test were 10.3 m, 15 cm and 5 m, respectively. in the philippines, the height of 3-year old a. mangium (from seed germination) reached up to 8.3 m and 9.3 cm in diameter, without mentioning its soil type. while in bangladesh, a. mangium (from seed germination) planted in good soil condition grew up to 8 m in height and 15 cm in diameter (soebardjo 1986). the height and diameter growth of a. mangium obtained by tissue culture were better than those obtained from seed germination. this might be caused by its rooting system and soil fertility. a tree has a good rooting system if the secondary roots develop well and has some vertical roots. the volume of soil occupied by the roots is an important factor in determining the amount of minerals and water available to trees. roots of forest trees tend to 4 growth and rooting system of acacia mangiumi . setiawan, m.i. umboh & supriyanto figure 4a. plantation of a. mangium, 2.5 years old, 2 x 2 m planting distance, obtained from seed germination. provenance trial at the research institute of forestry cikarawang, bogor. figure 4b. the rooting system of a. mangium obtained from seed germination, 2.5 years old, planting distance 2 x 2 m. 5 figure 5a. the plantation of a. mangium produced by tissue culture, 2.5 years old, 3 x 3 m planting distance. form a dense mat on the surface of soil intercepting minerals released by decaying litter (russel 1973). based on the observations of the root formation and development, a. mangium trees obtained by tissue culture produce a lot of lateral roots (secondary roots) but no tap root. three to four adventitious roots initiated in vitro developed as main roots and some vertical roots were also formed (figure 5b). the number of secondary roots (bigger than 0.5 cm diameter) was 20-30. in the nursery, the formation of secondary roots can be stimulated by cutting the tap root (abod 1984). a. mangium trees obtained from seed germination formed tap roots and less lateral roots. since the tap root is still being formed, the formation of lateral roots was inhibited. consequently, the mineral absorbtion in this system is less efficient than in trees obtained by tissue culture. sylviculturally, this field test gives better understanding of the growth pattern, rooting system and ecological adaptation of a. mangium produced by tissue culture. since its growth (height and diameter) and rooting system are very good, seedlings production through tissue culture should be used in reforestation programme specially for forest industry plantation. 6 biotropia no. 4, 1990/1991 figure 5b. the rooting system of a. mangium obtained by tissue culture, 2.5 years old, planting distance 3 x 3 m. these results should be supplemented either by cytological or electrophoretic studies before proceeding to the clone trees in bigger quantity because most seedlings obtained by tissue culture (plantlings), specially callus culture, produce high genetic variability (d'amato 1975; berljak 1984). from the wood technology point of view, the wood qualities of a. mangium that need to be examined include hardness and the fiber length. since tissue culture of forest trees is relatively new, there is little information on wood quality of trees produced by tissue culture. the main constraint is the fact that forest trees take longer time to grow than agricultural plants (fruit trees). fortunately, the life cycle of a. mangium is very short compared to other forest trees (6 — 8 years for albizia falcataria, 35 year for dipterocarps, 6-100 years for tectona grandis). since a. mangium is used also for pulp and paper industry, the examination of fiber quality is also needed, i.e. possible improvement of fiber length. conclusions 1. regarding the height and diameter growth after 2.5 year of field test, a. mangium trees produced by tissue culture grow better than those obtained by seed germination. 2. the rooting system of a. mangium produced by tissue culture is very compact, massive, and with a lot of secondary roots. although there is no tap root, 3-4 adventitious roots developed vertically and assumed the same function as tap root. 7 growth and rooting system of acacia mangium-l. setiawan, m.i. umboh & supriyanto biotropia no. 4, 1990/1991 references abod, s.a. 1983. the effect of restricted watering, root pruning and their combination on root regenerating potential and the growth of pinus merkusii seedlings. research report, seameo biotrop, bogor, indonesia. berljak, j.c. 1984. micropropagation of black pine (pinus nigra arn). abst. msc. thesis, univ. zagreb. djamato, f. 1975. the problem of genetic stability in plant tissue and cell cultures in: crop genetic resources for today and tomorrow (eds. frankel, o.h. and j.g. hawkes). cambridge univ. durzan, d.j. and r.a. campbell. 1974. prospects for the mass production of improved stock of forest trees by cell and tissue culture. ca. j. for. sci. 4: 151-174. haissio, b.e. 1974. origins of adventition roots. n.z. j. for. sci. 4: 229-310. kramer, p.j. and t.t. kozlowski. 1979. physiology of woody plants. academic press inc., new york, san francisco, london. russel, e.w. 1973. soil conditions and plant growth. 10th ed. longmans, green, new york. soebardjo. 1986. penanaman eucalyptus sp. dan acacia mangium di areal hph pt. arara abadi kumpulan makalah seminar dan reuni iv fakultas kehutanan, universitas gadjah mada, yogyakarta 11-12 september 1986. umboh, m.i.j. 1988. production of acacia mangium willd. plantlets by in vitro vegetative propagation. in: the application of tissue culture techniques in economically important tropical trees. biotrop special publication no. 35. seameo biotrop, bogor: 87-96. umboh, m.i.j. and syamsul a. yani. 1989. micropropagation of some selected acacia mangium trees from subanjeriji forest plantation. in: seminar on forest tree tissue culture, organized by winrock int. and plantek int. ltd., 17-18 august 1989, singapore. umboh, m.i.j., iwan setiawan, hanif kamil, syamsul a. yani and j. situmorano. 1989. l'application de techniques de culture in vitro a la multiplication d'especes forestieres tropicales en indonesie. bull. soc. bot. francais, 136, actuall bot. (3/4): 179-184. umboh, m.i.j., syamsul a. yani and j. situmorang. 1990. pengadaan bahan tanam acacia mangium dan eucalyptus urophylla dari beberapa pohon pilihan di hutan tanaman subanjeriji. in: seminar on biotechnology for forestry, held by pau, university of gadjah mada, 12-13 february 1990, wanagama, yogyakarta. 8 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf doi: 10.11598/btb.2015.22.2.450 diversity of epiphytic orchids and host trees (phorophytes) in secondary forest of coban trisula, malang regency, east java, indonesia siti nurfadilah purwodadi botanic garden – indonesian institute of sciences, j surabaya-malang km. 65 purwodadi pasuruan east java 67163alan , , , indonesia received 19 december 2014/accepted 18 november 2015 abstract epiphytic orchids are an integral component of forest ecosystems that contribute to a high proportion of plant diversity. the aim of this study was to investigate the diversity of orchids and their host trees (phorophytes) in a secondary forest of coban trisula (the trisula waterfall) of bromo tengger semeru national park javain east province . e fifteen, indonesia. two line transects were established ach transect was 150 m long and composed of 10 x 10 m plots, resulting in the total number of 30 sampling plots at the study site. the following data were recorded in each plot: species name and individual numbers of epiphytic orchids, species name and individual numbers of the phorophytes and vertical distribution of the orchids on the phorophyte. 15 epiphytic orchid species there were found from 13 genera in the secondary forest of coban trisula. was the most abundant appendicula angustifolia epiphytic orchid species (relative abundance = 52 4%), followed by (29 9%). all recorded orchids . .trichotosia annulata grew on 21 individuals from nine phorophyte species. and castanopsis javanica (mean = 589.5 individuals/tree) engelhardia spicata (mean = . )425 67 orchid individuals/tree were phorophytes hosting the largest number of individual orchids, respectively. the greatest abundance of epiphytic orchids was on the basal and the middle part of phorophyte branches (zone 3 and zone 4). this study indicated that orchid conservation management is required in the coban trisula to protect the survival of orchids in this area from potential human disturbances, as coban trisula is one of tourist destination. keywords: coban trisula diversity, epiphytic orchid, phorophyte, secondary forest, introduction epiphytes are known as one of important components in forest ecosystems, contributing to a high proportion to floral diversity (wolf 2005). the role of epiphytes is vital as the habitat for canopy invertebrates and as nutrient sources in the forest canopies (nadkarni 2004; cardelus et al. & mack 2010). contribution of epiphytes is also important to the total biomass and nutrient pools in the forest ecosystems (nadkarni 2004). et al. the family orchidaceae is among the most dominant groups of vascular epiphytes (johansson 1974; gentry dodson 1987; & annaselvam parthasarathy 2001; kromer & et al. 2005; zotz schultz 2008)& . orchidaceae is one of the biggest families containing around 25 000-35 000 species , , (dressler 1981; 1993), representing 1/10 of the total vascular plant species. however, orchidaceae is also one of the threatened plant families (iucn/ssc orchid specialist group 1996; mondragon elliott 2013), due to & overexploitation, overcollection, deforestation and fire (koopowitz dixon 2003). orchids & are h ly eigh sensitiv to environmental changes (newman 2007) and high dependen on et al. ly t other organisms (mycorrhizal fungi and insects as pollinators) for their survival (swarts dixon & 2009). orchid conservation efforts need to be done by considering , and the biology ecology the nature of threats . towards the orchids * corresponding author : siti.nurfadilah@lipi.go.id; biotropia vol. 22 no. 2, 2015: 120 128 120 mailto:siti.nurfadilah@lipi.go.id; coban trisula is a waterfall in bromo tengger semeru national park (btsnp), which is administratively located in ngadas village, malang regency, east java . this province waterfall is a frequent tourist destination in east java, usually visited as part of regular tours to mount bromo and mount semeru. coban trisula can be reached a pathway a secondary through in forest lined by epiphytic orchids growing on trees (host trees or phorophytes) the orchids in , making this area vulnerable to human disturbances due to illegal orchid collection by visitors/tourists. some studies showed the negative impact of tourism and recreation on vascular plants, various especially on orchids family (orchidaceae) (pickering hill 2007; ballantyne pickering & & 2013; rankin 2015). rankin (2015) et al et al. . reported that more than 45 plant families have species listed as orchids were threatened, in which the most common species listed as at risk from threats. the most common threat plant is collection by visitors in protected areas. the aim of this study was to investigate the diversity of epiphytic orchid and the phorophytes along the s pathway to coban trisula to develop conservation management of orchids in that area. materials and methods study site this study was conducted along the pathway to coban trisula located in secondary forestthe , village ngadas, malang regency, east java province, indonesia ( s and 08 00'213"o 112 8 e) with elevation of , m above o 7'82" , 1 475 sea level (asl). the site under the management is of bromo tengger semeru national park (btsnp). the dominant trees in this forest are macropanax dispermus lithocarpus (pampung), sundaicus engelhardia spicata(pasang) and (danglu). data ollectionc records of diversity of epiphytic orchids and the phorophytes were in two line-conducted transects. each transect was 150 m long and composed of 15 plots (each plot 10 x 10 's size is m), resulting in the total number of 30 sampling plots at the study site (annaselvam & parthasarathy 2001; focho 2010). species et al. name and individual number of the epiphytic orchids and the phorophytes were recorded. further, the vertical distribution of epiphytic orchids on the phorophytes within the five zones determined by johansson (1974) was recorded (fig 1). . biotropia vol. 22 no. 2, 2015 vertical distribution of epiphytic orchids on the host tree in five zones: zone 1 : the bottom part (1/3) of the main stem zone 2: the upper part (2/3) of the main stem zone 3: the bottom part of the branches zone 4: the middle part of the branches zone 5: the outer part of the branches. (johansson 1974) figure 1 division of the phorophyte into five ones johansson (1974) z determined by 121 diversity of epiphytic orchids and host trees (phorophytes) nurfadilah – data nalysisa parameters z weremeasured and analy ed relative frequency of phorophyte (% ft), relative abundance of orchids (% fo), the average number of individuals of orchids of each phorophyte species (ji/jt), the average number of epip ytic h orchid species on a phorophyte species (js/jt), and the vertical distribution of the orchids on the phorophytes (yulia budiharta 2012a; 2012b).& a. relative requency of phorophyte (% ft)f nt % ft = x 100% total number of all phorophytes where: nt = the number of trees in the plot hosting a particular orchid species b a. relative bundance of orchid (% fo) no % fo = x 100% total number of all orchid species where: sno = the number of individual of a particular orchid species within the plot c . the average number of orchid individuals on a phorophyte species = ji jt where: orchid ji = the number of individuals sjt = the number of individual of each phorophyte species d orchid . the average number of species on a phorophyte = species js jt where: speciesjs = the number of orchid jt = the number of individual of each phorophyte species e. vertical distribution of epiphytic orchids on the phorophyte s specie were determined by mapping vertical distribution of each epiphytic orchid species on the phorophyte, from the trunk to outer branches in five zones (zone 1, zone 2, zone 3, zone 4 and zone 5) and by calculating the average number of individuals of epiphytic orchids in each zone. results and discussion the ccur ence of piphytic rchids on the o r e o phorophytes there were 15 epiphytic orchid species and 9 phorophyte species (table 1). the recorded results of the present study showed that the number of phorophyte species each hosting epiphytic orchid varied 1 5 phorophyte from to species (table 1). some epiphytic orchid species occu red on a single phorophyte species; such as r appendicula elegans (100 individuals) found were exclusively on phorophyte ; lithocarpus sundaicus bryobium hyacinthoides (10 individuals) were recorded only on phorophyte ; engelhardia spicata and (50 individuals) dendrobium luxurians were only observed on phorophyte castanopsis javanica (table 1). other epiphytic orchid species occurred on multiple phorophyte species; such as appendicula angustifolia gr on four phorophyte ew species ( , macropanax dispermus lithocar pus sundaicus drypetes sumatrana castanopsis , and javanica parapteroceras odoratissimum). was hosted by 5 in the study site i.e. phorophyte species engelhardia spicata ficus syzygium , sp , sp ,. . lithocarpus sundaicus drypetes sumatrana (table and 1). the results of the present study were similar to other studies showed that the number of which phorophyte species epiphytic orchids hosting varied from a single to multiple phorophyte species. adhikari (2012) reported that the et al . orchid occur ed on one rdendrobium nobile phorophyte species, while the orchid rhynchostylis retusa was found on many different phorophyte species. rosa-manzano (2014) also reported et al . that most epiphytic orchids in tropical dry forests of yucatan, mexico occu red on a single tree r species. tremblay (1998) also showed a et al . puerto rican orchid, lepanthes caritensis hosted by one phorophyte species ther epiphytic , while o orchids were reported to occur in many phorophyte spec ies. annaselvam and parthasarathy (2001) reported that epiphytic orchids in tropical evergreen forest at varagalaiar, western ghats, india g w on many phorophyte re species. trapnell and hamrick (2006) also showed 33 hosting phorophyte species epiphytic orchid laelia rubescens at one site. 122 epiphytic rchidso the present study showed the secondary that forest of coban trisula contained 15 epiphytic orchid species lower , in which the diversity was compared to the diversity of epiphytic orchids in other areas within the bromo tengger semeru national park; such as in resort senduro that had 42 epiphytic orchid species (utama 2005). the lower diversity of epiphytic orchids in coban trisula compared to resort senduro may have been caused by the in difference ecosystem types in which coban trisula is secondary forest, while resort senduro is a primary forest. the difference of primary forest and secondary forest in terms of their plant diversity has been widely studied. primary forest ha higher plant diversity than s that of secondary forest, including epiphytic orchid diversity (barthlott 2001 kubota 2005). et al et al . .; studies in the venezuelan andes and japan comparing the diversity of vascular epiphytes in biotropia vol. 22 no. 2, 2015 table 1 the epiphytic orchid species and the phorophytes in the secondary forest of coban trisula no epiphytic orchids number of phorophyte species phorophyte species 1 appendicula angustifolia blume 4 macropanax dispermus (blume) kuntze lithocarpus sundaicus (blume) rehder drypetes sumatrana (miq.) pax & k.hoffm castanopsis javanica (blume) a.dc. 2 appendicula elegans rchb.f 1 lithocarpus sundaicus (blume) rehder 3 bryobium hyacinthoides (blume) y.p.ng & p.j.cribb 1 engelhardia spicata var. colebrookeana (lindl. ex wall. ) koord. & valeton 4 bulbophyllum odoratissimum (sm.) lindl. ex wall. 1 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton 5 ceratostylis brevibrachiata j.j. sm . 2 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton actinodaphne procera 6 cymbidium sp. 1 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton 7 dendrobium luxurians j.j.sm. 1 castanopsis javanica (blume) a.dc. 8 dendrobium spathilingue j.j.sm. 2 lithocarpus sundaicus (blume) rehder castanopsis javanica (blume) a.dc. 9 dendrochilum abbreviatum blume 1 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton 10 mycaranthes oblitterata blume 1 castanopsis javanica (blume) a.dc. 11 parapteroceras odoratissimum (j.j.sm.) j.j. wood 5 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton ficus sp. syzygium sp. lithocarpus sundaicus (blume) rehder drypetes sumatrana (miq.) pax & k.hoffm 12 schoenorchis juncifolia reinw. ex blume 3 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton lithocarpus sundaicus (blume) rehder ficus grossularioides burm.f. 13 thrixspermum subulatum (blume) rchb.f 1 castanopsis javanica (blume) a.dc. 14 trichotosia annulata blume 4 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton lithocarpus sundaicus, (blume) rehder drypetes sumatrana (miq.) pax & k.hoffm castanopsis javanica (blume) a.dc. 15 vanda tricolor lindl. 2 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton castanopsis javanica (blume) a.dc. 123 primary forests and secondary forests showed that primary forests had higher diversity of epiphytic orchids compared to secondary forests (barthlott et al et al . . ; 2001 kubota 2005). the present study also showed that the most abundant epiphytic orchid was appendicula angustifolia with r (% fo)elative abundance of 52 41%, followed by % . with trichotosia annulata fo 29 9% (table 2). the most abundant of . orchid in this study site, is a appendicula angustifolia sympodial orchid, that ha continuous s lateral growth of the the stems through rhizome, which is an effective vegetative reproduction to grow a large number of individuals. appendicula angustifolia also ha many flowers along the stems, s with each rachis bear 10–15 flowers (comber s 1990; 2001). the large number of a. angustifolia flowers and seedincrease the chance for the fruit set to produce large population. all these s characters of support the a. angustifolia high abundance of at the study appendicula angustifolia site. comber (1990) also reported a tree covered by high density in indonesia,of a. angustifolia. appendicula angustifoli a is i and widely distr buted found all over java and sumat ra, from 800e to 1 700 m asl (comber 1990), elevation . host ree (phorophyte) peciest s the results of the present study also showed that there were 9 phorophyte species in coban trisula, which the diversity is lower than that in resort senduro located within the same national park ( ) bromo tengger semeru national park . utama (2005) reported 16 phorophyte species in resort senduro. the lower diversity of phorophyte in coban trisula compared to resort senduro caused by different may have been ecosystem types (secondary forest in coban trisula and primary forest in senduro). resort other studies also showed similar results. t able 2 the epiphytic orchid species in coban trisula and the parameter values no epiphytic orchid species nt no % ft % fo 1 appendicula angustifolia blume 6 2,056 15 52 .4 2 appendicula elegans rchb.f 1 100 2.5 2 .55 3 bryobium hyacinthoides (blume) y.p.ng & p.j.cribb 1 10 2.5 0 .25 4 bulbophyllum odoratissimum (sm.) lindl. ex wall. 1 120 2.5 3 .06 5 ceratostylis brevibrachiata j.j. sm. 2 50 5 1 .27 6 cymbidium sp. 1 2 2.5 0 .05 7 dendrobium luxurians j.j.sm. 1 50 2.5 1 .27 8 dendrobium spathilingue j.j.sm. 2 16 5 0 .41 9 dendrochilum abbreviatum blume 1 75 2.5 1 .91 10 mycaranthes oblitterata blume 1 100 2.5 2 .55 11 parapteroceras odoratissimum (j.j.sm.) j.j. wood 9 74 22 .5 1 .89 12 schoenorchis juncifolia reinw. ex blume 4 41 10 1 .05 13 thrixspermum subulatum (blume) rchb.f 1 50 2.5 1 .27 14 trichotosia annulata blume 5 1,172 12 .5 29 .9 15 vanda tricolor lindl. 4 7 10 0 .18 notes: nt = the number of trees in the plot hosting a particular orchid species no = the number of individuals of a particular orchid species within the plot s% ft = relative frequency of phorophyte % fo = relative abundance of orchids 124 diversity of epiphytic orchids and host trees (phorophytes) nurfadilah – biotropia vol. 22 no. 2, 2015 barthlott (2001) reported that the diversity et al . of phorophytes in the secondary forest was lower than that of the primary forest in the venezuelan andes. the individual number of orchids growing on a phorophyte ranged from to 589.5 6.5 individuals/tree (ji/jt) (table 3). the largest individual numbers of epiphytic orchids were found on (ji/jt = 589.5 castanopsis javanica individuals of orchids/tree), followed by engelhardia spicata (ji/jt = 425.6 individuals of orchids/tree). and castanopsis javanica engelhardia spicata not only had a large individual number of epiphytic orchids, also had the highest species but richness of epiphytic orchids. eight orchid species ha been recorded growing on the phorophyte d engelhardia spicata and seven species on the phorophyte . other castanops i s javani ca phorophytes such as and actinodaphne procera macropanax dispermus only hosted one orchid species. were all recorded phorophytes specialized on sub-montane and montane areas (hardyanti & hakim 2014). most of them such as castanopsis javanica, engelhardia spicata, macropanax dispermus and ha rough or fissured lithocarpus sundaicus d bark. this bark structure might support litter accumulation and therefore build a nutrient and , humidity reservoir providing a comfort habitat for epiphytic orchids (annaselvam pathasarathy & 2001). the thickness of organic substrates layer on the tree bark varied within the vertical distribution of a single tree species from bare bark (thin substrates < 1 cm) to 5 cm thick substrates. the variety of substrat thickness covering the e phorophyte was also observed by johansson (1974) in west african rainforests and barthlott et al. (2001) in the venezuelan andes. in our study the highest abundance of orchids occur ed on thick substrates. this is consistent r with the results of the study conducted by annaselvam and parthasarathy (2001) showing that most thick branches densely covered were with vascular epiphytes, accumulating substantial amounts of humus, nutrients and moisture. rosamanzano (2014) also reported that bark et al . roughness and substrate were the most area important phorophyte characteristics affecting the epiphytic orchids abundance. vertical istribution of piphytic rchids on d e o p specieshorophyte the present study showed a range of vertical distribution of epi hytic orchid species from p zone 2 to zone 5 (table 4). the most abundant orchid, was found to have appendicula angustifolia, the widest vertical distribution ranging from zone 2 to zone 5. the ability of to occupy a. angustifolia a large area and different zones supported population growth resulting in the abundance of table 3 the phorophyte species in coban trisula and the parameter values no phorophyte species jt js ji js/jt ji/jt 1 actinodaphne procera nees 1 15 2 castanopsis javanica (blume) a.dc. 3 drypetes sumatrana (miq.) pax & k.hoffm. 4 engelhardia spicata var. colebrookeana (lindl. ex wall.) koord. & valeton 5 ficus grossularioides burm.f. 6 ficus sp. 7 lithocarpus sundaicus (blume) rehder 8 macropanax dispermus (blume) kuntze 9 syzygium sp. 1 4 2 3 2 1 5 2 1 7 3 8 1 1 6 1 1 2,358 13 1,277 30 10 175 35 10 1 1.75 1.5 2.67 0.5 1 1.2 0.5 1 15 589.5 6.5 425.67 15 10 35 17.5 10 notes: jt = the individual number of each phorophyte species js = number of orchid species orchidji = number of individuals js/jt = the average number of epiphytic orchid species on a phorophyte species epiphytic orchid phorophyte ji/jt = the average number of individuals on a species 125 the orchids. sp. and cymbidium dendrobium luxurians were observed to occur in zone 5 only (table 4). most epiphytic orchid species grew on zone 3 and zone 4. a small number of orchid species occu red on the trunk (only two orchid r species on zone 2 and no orchid species grew grew on zone 1. the pattern of vertical distribution of epiphytic orchids in the forest of coban trisula was similar to that of other regions africa, i.e. in south america and mexico where vascular epiphyte abundan were higher in zone 3-5 tree ce ( crown than in zone 1-2 along the trunk ) ( ) due to better light the crown intensity nearby tree (johansson 1974 kromer 2005 rosa-; ; et al . manzano 2014)et al. . implications for onservationc coban trisula is one of tourist destination within area of bromo tengger semeru the national park. the tourist number bromo to tengger semeru national park very high was (table 5). the number of visitors increased sharply from 2011 to 2014, with the peak of tourist number reached 551 644 visitors in 2013 , (table 5). table 4 zones of the occur ence of epiphytic orchids on their phorophytes and the number of rchid individuals in each r o zone no epiphytic orchid species zone 1 zone 2 zone 3 zone 4 zone 5 1 appendicula angustifolia blume 2 appendicula elegans rchb.f 3 bryobium hyacinthoides (blume) y.p.ng & p.j. cribb 4 bulbophyllum odoratissimum (sm.) lindl. ex. wall. 5 ceratostylis brevibrachiata j.j. sm. 6 cymbidium sp. 7 dendrobium luxurians j. j. sm. 8 dendrobium spathilingue j. j. sm. 9 dendrochilum abbreviatum blume 10 mycaranthes oblitterata blume 11 parapteroceras odoratissimum (j. j. sm.) j.j. wood 20 5 12 schoenorchis juncifolia reinw. ex blume 13 thrixspermum subulatum (blume) rchb.f 14 trichotosia annulata blume 15 vanda tricolor lindl. 796 10 20 45 11 33 34 25 152 3 800 100 10 100 5 5 30 33 35 6 50 1,020 77 total number of orchid individuals 0 25 1,129 2,271 200 2 50 33 10 2 297 table 5 the number of visitors bromo tengger semeru national park to visitors year 2011 2012 2013 2014 domestic foreign total visitors 103,091 22,380 125,471 249,577 26,297 275,874 518,746 32,898 551,644 512,887 23,451 536,338 source: balai besar taman nasional bromo tengger semeru 126 diversity of epiphytic orchids and host trees (phorophytes) nurfadilah – biotropia vol. 22 no. 2, 2015 management of orchid conservation is required to protect the orchids in coban trisula and other places within bromo tengger semeru national park rom population and loss f decrease of diversity. this is related to the impact of tourism and recreation the survival of activities on plants, especially orchids (pickering hill 2007; & rankin 2015). he most common type of et al. t threat in area isthe tourism and recreation plant collection by visitors which can decrease plant species diversity . (rankin 2015; calderon-et al aguilera 2012). a recommendation to et al . protect the epiphytic orchids in this area from human disturbances is required. conclusions fifteen epiphytic orchid species from 13 genera were found in the secondary forest of coban trisula, bromo tengger semeru national park. nine phorophyte species were found to be the host the epiphytic orchid species in coban of trisula. the most abundant orchid species was appendicula angustifolia and the phorophyte species hosting the largest number of orchids was castanopsis javanica. management of orchid conservation is required to protect the survival of orchids in coban trisula. acknowledgements the present study was funded by dipa. my sincere thanks to pak tarmudji and pak went tatang (purwodadi botanic garden) and pak sukiyono (bromo tengger semeru national park) for the assistance in the field. references adhikari yp, fischer hs, fischer a. 2012. host tree utilization by epiphytic orchids in different land-use 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16911. indonesia 2research center for biotechnology, indonesian institute of sciences abstract the preservation of milk with additional antibacterial and aromatic supplements, produced in indonesia, was investigated. organoleptic performances of milk with the addition of 10% supplements, made as juices, were tested by panellists, and the total bacteria, protease activities, lipase activities and acidities, were detected by total plate counts, azocasein method, modified dole extraction and base-acid titration, respectively. out of the 27 supplemented skim and whole milk samples, 15 whole milk samples and 10 skim milk samples were selected as acceptable, based on their better organoleptic performances, their lower bacterial counts, protease and lipase activities, and their acidities percentages which were not significantly different, compared to that of control, at 5 days after the expiry date (p<0.05). these 15 whole milk samples contained honey, cinnamon, citronella, ginger, turmeric, galingale, wild ginger, nutmeg, pepper, clove, galangale, green tea, bamboo leaf, garlic leaf and aloe vera; and the 10 skim milk samples contained honey, cinnamon, citronella, ginger, galingale, pepper, galangale, green tea, bamboo leaf and aloe vera. key words: milk, preservation, antibacterial and aromatic supplements, protease, lipase introduction pasteurised milk spoils at refrigerated temperatures due to the activities of psychotropic bacteria, pseudomonas spp., and the main species is pseudomonas fluorescens (deeth et al. 2002; chandler et al. 1990; craven and macauley 1992). to ensure the quality of the milk during storage, the milk is stored at a temperature of 4-7oc (chandler et al. 1990), and the average shelf life of stored milk is approximately 7 days (khusniati et al. 2006). however, heo (1989) examined commercial milk samples stored at 7.20c and found that after 10 days of storage 91% of the milk was acceptable, whereas after 14 days, 82% of the whole milk was still acceptable. the qualities of pasteurised milk in refrigerated storage were affected by the growth of psychrotrophic bacteria, especially pseudomonas spp., and to keep the quality biotropia vol. 15 no. 1, 2008 : 50 64 51 the preservation of milk t. khusniati & y. widyastuti of the milk at storage, the milk were stored at temperature of 4-7oc (chandler et al. 1990). the psychrotrophic bacteria began to grow at the expiry date, and times after the expiry date. the bacteria, especially pseudomonas spp grew rapidly in the stored milk. furthermore, the different qualities of milk may have resulted from the different types of milk in storage. the quality of whole milk was higher than that of skim in the refrigerated storage, due to the higher lipid content in whole milk than that in skim ( janzen et al. 1982). the main problem with milk, after the pasteurisation process, was recontamination. during the pasteurisation process in factories, psychotrophic bacteria are killed by the process. however, after pasteurisation, particularly if hygiene within the processing factory is poor, these psychotrophic bacteria can re-contaminate milk (craven and macauley 1992). during storage after the expiry date, the psychotrophic bacteria in the milk grew rapidly, and the longer the time of storage, the higher the growth and enzyme activities of psychotrophic bacteria within the milk (chandler et al. 1990; deeth et al. 2002) the spoilage of pasteurised milk may result in microbial and chemical changes in the milk (reinheimer et al. 1993). the biochemical changes of the milk at spoilage may have resulted from the activities of extracellular enzymes, especially protease, which degrades protein (deeth et al. 2002; janzen et al. 1982); and lipase, which degrades lipid (deeth et al. 2002; bucky et al. 1986). the addition of antibacterial and aromatic supplements, to reduce the spoilage, can be added to the milk. it is widely known that there are a lot of commercial antibacterial and aromatic plant materials produced in indonesia, such as: honey, cinnamon, citronella (sweet), ginger, radish, turmeric, galingale, zingiber (roots), wild ginger, nutmeg, cardamom, cumin, pepper (seeds), garlic, clove, javanoni, galangale (bubs); green tea, laurellike (dried leaves); bamboo leaf, banana leaf, guava leaf, avocado leaf, betel vine, celery, garlic leaf, aloe vera (fresh leaves). these twenty-seven plant materials contain antibacterial and aromatic compounds, which may inhibit the growth and enzymatic activities of the psychotrophic bacteria within the stored milk. honey contains ”inhibine”, hydrogen peroxide and flavonoid (lusby et al. 2005); cinnamon contains cinamic acid, cinnamon oil, ethanol, methylene chloride, eugenol and benzyl benzoate (tabak et al. 1999). the root materials of ginger contains “gingerin”, gingerols, α-zingiberene, shorgaols and ginger oil (kikuzaki. 2000); radish has some glucosinolates and hexenyl acetate (tirranen et al. 2001); turmeric has turmeric oil (lantz et al. 2005); galingale contains aromatic galingale (anon 1999). nutmeg contains diphenylpropanoids and ethyl acetate (sherry et al. 1982); cardamon contains aromatic cardamom (mummenhoff and hurka 1991); and pepper, adipic acid, piperine and oleorecine (chitwood et al. 2003). garlic contains ‘allicin’, free phenolics, p-coumaric, ferulic, p-hydroxybenzoic, garlic oil and vanillic acid, and specific garlic aromatic (tirranen et al. 2001); and javanoni contains anthraquinon and aromatic javanoni (anon 2001). 52 biotropia vol. 15 no. 1, 2008 citronella contains citronella oil and kingisidic acid; zingiber contains zingiber oil, acyclic oxygenated and monoterpenes; wild ginger roots contain wild ginger oil; clove contains clove oil, isobiflorin,biflorin, eugenol, phenolic compound and ferulic acid; cumin contains glycosides of 2-c-methyl-d-erythritol and cumin oil ;and galangale contains galangale oil (cineol, α-pinene, eugenol, camphor, methyl cinnamate and sesquiterpenes) (deans and ritchie 1987). the dried leaves of green tea contains polyphenol, phenolic acid, catechins, caffeine, flavonoid, epicatechin and ascorbic acid (an et al. 2004); and laurellike that has aromatic laurellike (anon 2005). the fresh leaves of bamboo contains cinnamic derivatives (tachibana et al. 1992); banana has starch phosphorylase, polycyclic aromatic hydrocarbon and non-volatile organic acid (palmer and wyman 1965); guava contains aromatic guava leaf (settheeworrarit et al. 2005); and avocado contains (r)-2-hydroxy4-oxohenicosan-1-yl acetat, aldehydes, ketones, alcohols, terpenoides, estragole and 2-hexenal (carman and duffield 1995). the fresh leaves of celery contain apiole, 3-butylphthalide, sedanenolide, monoterpene hydrocarbon, phthalates and limonene (macleod and ames1989); garlic containing garlic leaf lectin (tirranen et al. 2001); aloe vera has glucomannans, uronic acid, glycoproteins, anthraquinon, saccharrides and phenolic compounds (ni et al. 2004); pandan contains pandan oil, monoterpene hydrocarbon and sesquiterpene hydrocarbon, while betel vine contains betel vine oil, eugenol, 1, 3 benzodioxol (5)-2-propenyl and anethole (deans and ritchie 1987). the differences in the structure of the antibacterial compounds may have caused variation in the effectiveness of antibacterial substances and the aromatic materials in inhibiting spoilage bacteria and/or pathogenic bacteria. in the indonesian dairy market, the supplements of strawberry and chocolate were added into the commercially pasteurised milk, and stored at refrigerated temperatures in cartons or plastic packages. the addition of these supplements was mainly to create more tastes and flavours for the consumers. these milk are produced in factories, located mainly in java, and distributed not only to local areas but also outside java. the shelf life of these milk is known to be around 7 days as is the shelf life of fresh milk. this may be due to the lack of antibacterial compounds within the supplements. to make longer the shelf life of the commercial milk distributed both in the local area and outside the factories locations, antibacterial and aromatic materials may be used. it is expected that these materials, when added to milk in storage can suppress the growth and enzymatic activities of psychotrophic bacteria, thus increasing the quality and shelf life (the time started after completion of the pasteurisation process up to the time of expiry date), of pasteurised milk. this paper reports on the preservation of milk with the addition of antibacterial and aromatic supplements produced in indonesia. 53 the preservation of milk t. khusniati & y. widyastuti materials and methods milk samples the same types, of commercially batched pasteurised milk, at a temperature of 85oc for 30 minutes, in 1 and 2 litre cartons were used. the milk samples, stored at 4oc until use, were transported on ice to the laboratory, and a 100-ml aliquot of each sample was then transferred aseptically into 200-ml sterile bottles. preparation of antibacterial and aromatic materials the collection of the 27 supplements from supermarkets and traditional markets was carried out in the bogor area, indonesia. all the materials selected were clean and in good condition . juice supplements extracted from antibacterial and aromatic materials juice supplements were made by the extraction of juices from the antibacterial and aromatic materials. fifty (50)-grams of the materials were then made into juice by the addition of 500 ml of boiled water. the materials were then homogenized by using a blender. the juices produced were subsequently filtered through a sterilized stainless steel filter with ø (diameter) 0.5 ml, and the cleared juices were kept at a refrigerated temperature until ready for use as supplements. pasteurised milk with the addition of supplements one and two litre cartons of commercially pasteurised milk were used. the addition of extract after pasteurisation instead of before was because of: (1) the total bacterial counts after pasteurisation were lower than that of before. (2) the pathogenic bacteria were killed after pasteurisation, but the bacteria weren’t killed before pasteurisation (3) materials extracted were used for inhibiting psychotrophic bacteria, which caused the spoilage of the milk at storage. the milk was then transferred aseptically from the big bottles into 200 ml sterile bottles. one representative of each of the twenty-seven different liquid supplements was segregated into separate 200-ml aliquots of the batch of pasteurised milk. the samples, with the addition of the supplements, together with milk without supplements (controls), were incubated at 4oc for up to 5 days after the expiry date. at 5 days before the expiry date, samples of controls and the supplemented twenty-seven milk samples were organoleptically assessed. at 5 days after the expiry date, all the milk samples were investigated for total aerobic counts, acidities, and used for production of supernatants to be assayed for protease and lipase activities. the results presented for bacterial counts, protease activities, lipase activities and acidities are mean values for the three replicates. 54 biotropia vol. 15 no. 1, 2008 sensory assessments the sensory evaluation was conducted by 18 panellists, and assessed by a modified ranking test. the ranking test was as follows; the tastes, colours, flavours and homogeneity, of the milk’ samples, were organoleptically evaluated with scores of: 1.00<2.00 (unacceptable), 2.00-<2.50 (less acceptable), 2.50-<2.65 (acceptable), and >2.65 (more acceptable). the characteristics of organoleptic assessments were tastes (acidic, sweet, plain, milky or others), colours (white, green, red or others), flavours (plain, acidic, sweet, milky or others), and homogeneity (homogeneous, non-homogeneous or others). the equation between the scores and the acceptability ranking was based on the results of the scores and the comments of the sensory evaluation of the milk samples. total aerobic bacterial counts ten-fold serial dilutions of the milk samples were made and spread plate counts performed according to australian standard as 1766.1.4 using nutrient agar. the plates were incubated for 2-3 days at 30oc. acidity measurement the acidities of the milk’ samples were measured by base-acid titration. 10 ml of each milk sample was poured into erlenmeyer 100 ml and several drops of phenolphthalein were added. the solutions were titrated by naoh 0.1n un-till the colours changed to pink. the formula of acidity (based on lactic acid) was a.b. 90.1000/c [a: volume of na oh used (ml); b: concentration of naoh standardized (n); c: volume of milk titrated (ml); d: be lactic acid (g/equivalent)]. preparation of the supernatants (crude enzymes) bacterial cells were removed from the incubated milk samples (with and without the additional supplements) by centrifugation at 24 000 g for 10 min. at 40c. the resulting supernatants (crude enzymes) were collected and stored at -200c in sterile bottles until assayed for enzyme activities protease assay proteolytic activity was then assayed by the use of the azocasein method, using sulphanilamide-azocasein (sigma chemical co., usa) as the substrate, according to the method of christen & marshall (1984), with some modifications. the reaction mixture contained 2 ml of azocasein (10g/l, dissolved by heating 0.1 m-tris-hcl buffer ph 7.4 (sterile) containing 2mm-cacl2 at 630c for 30 minutes) and 0.5 ml crude enzyme solution (supernatant) was incubated for 1 h at 370c. one unit of proteolytic activity was defined as the volume of enzyme solution (ml) required for producing an absorbance increase at 345 nm of 0.01 a.u. in an hour under the assay conditions. 55 the preservation of milk t. khusniati & y. widyastuti lipase assay the lipase activity assay was carried out based on the assay procedure of a modified dole extraction procedure (deeth et al. 1975). the supernatants (crude enzyme; 0.5 ml) , 0.25 ml buffer (2 m-diethanolamine-hcl, ph 8.5), 3 ml uht cream, and 1 ml sterile water (in a stoppered test tube) were incubated at 40oc for 2 hours in a shaking (100-rpm) water-bath, and lipase activities were then measured by titration. the reaction mixture was added with 10 ml of extraction mixture (isopropanol: petroleum ether: 4n h2so4)(40:10:1), 6 ml of petroleum ether, and 4 ml of water. the mixture was then shaken for 15 sec., and an-aliquot (8 ml) of the upper layer was transferred to a 50 ml conical flask and 0.5 ml of 0.02% bromothymol blue indicator was added. the free fatty acid (f.f.a) was titrated with 0.02n methanolic koh. the activities of the supernatants are expressed as µequiv.ml-1h-1 using the formulae: n (ttest/ptest – tcontrol/pcontrol) x103 ___________________________ v x h where n is the normality of the methanolic koh, ttest and tcontrol are the titration volumes for the test and the control respectively, ptest and pcontrol are the proportions of the upper layers titrated, v is the volume of enzyme solution and h is the incubation time in hours. statistical analysis all treatments of all milk samples, with and without the addition of supplements, were statistically analysed by anova with factorial complete randomised design (snedecor and cochran 1989) using general linear model with three replications. results and discussion the organoleptic performances of all supplemented milk were generally better than that of the controls (without supplements), although the panellists classed some of them as organoleptically un-acceptable. of the 27 supplemented whole milk samples 20, 4 and 3 samples at 5 days before the expiry date were classed as acceptable, less acceptable and unacceptable by panellists, while of the 27 supplemented skim milk samples 14, 8 and 5 samples were classed as acceptable, less acceptable and unacceptable by panellists at the same date, respectively (table 1). the 20 supplemented whole milk samples contained honey, cinnamon, citronella, ginger, turmeric, galingale, zingiber, wild ginger, nutmeg, pepper, clove, galangale, green tea, laurellike, bamboo leaf, banana leaf, guava leaf, avocado leaf, betel vine and aloe vera. 56 biotropia vol. 15 no. 1, 2008 the 4 supplemented whole milk samples contained cumin, garlic, celery and garlic leaf, while the rest i.e. 3 supplemented whole milk samples contained radish, cardamom and javanoni. the 14 supplemented skim milk samples contained honey, cinnamon, citronella, ginger, galingale, pepper, galangale, green tea, bamboo leaf, banana leaf, guava leaf, avocado leaf, betel vine and aloe vera. the 8 supplemented skim milk samples contained turmeric, zingiber, wild ginger, nutmeg, garlic, clove, laurellike and celery, while the 5 supplemented skim milk samples contained radish, cardamom, cumin, javanoni and garlic leaf. therefore, the 20 supplemented whole milk samples and the 14 supplemented skim milk samples were classed as acceptable, based on organoleptic assessments. to further classify their quality and shelf life, these acceptable milk, were then counted for total bacteria and measured for enzymatic activities and acidities the preservation of the whole milk (table 2) and skim (table 3) can be explained by the lower total bacterial counts, proteolysis and lipolysis, and the acidities percentages which were not significantly different, than those of controls. this is caused by the lower production of psychotrophic bacteria within these milk. the total bacterial counts, protease and lipase activities, of the 20 acceptable whole milk, and the 14 acceptable skim, were significantly lower than that of the controls (without supplements), at the time of storage 5 days after the expiry date (p<0.05). table 1. the organoleptic performances of skim and whole milk with the addition of antibacterial and aromatic supplements, at 5 days before the expiry date no. antibacterial and aromatic supplements whole milk skim milk 1 honey > acceptable > acceptable 2 cinnamon > acceptable acceptable 3 citronella > acceptable > acceptable 4 ginger > acceptable > acceptable 5 radish unacceptable unacceptable 6 turmeric > acceptable < acceptable 7 galingale > acceptable acceptable 8 zingiber acceptable < acceptable 9 wild ginger acceptable < acceptable 10 nutmeg acceptable < acceptable 11 cardamon unacceptable unacceptable 12 cumin < acceptable unacceptable 13 pepper > acceptable acceptable 14 garlic < acceptable < acceptable 15 clove acceptable < acceptable 57 the preservation of milk t. khusniati & y. widyastuti no. antibacterial and aromatic supplements whole milk skim milk 16 javanoni unacceptable unacceptable 17 galangale > acceptable acceptable 18 green tea acceptable acceptable 19 laurellike > acceptable < acceptable 20 bamboo leaf > acceptable acceptable 21 banana leaf > acceptable acceptable 22 guava leaf > acceptable acceptable 23 avocado leaf > acceptable acceptable 24 betel vine acceptable acceptable 25 celery < acceptable < acceptable 26 garlic leaf < acceptable unacceptable 27 aloe vera > acceptable > acceptable 28 milk (control) > acceptable > acceptable notes: > acceptable (>2.65); acceptable (≥ 2.50); < acceptable (≥2.00); unacceptable (<2.00) the total bacterial counts of the 27 supplemented milk at storage 5 days after the expiry date, were in the range of 6.0x102-8.4x104 cfu/ml (whole milk) and 8x102-9.4x105 cfu/ml (skim milk), while the total bacterial counts of milk without supplements (controls) were 8.5x105 cfu/ml (whole milk) and 9.7x106 cfu/ml (skim milk) at the same times, respectively (table 2-3). the total bacterial counts of the twenty (20) acceptable whole milk, and the fourteen (14) acceptable skim, were in the range of 6.0x102-8.5x103 cfu/ml and 8.0x102-9.3x104 cfu/ml, respectively. therefore, the 20 whole milk and the 14 skim milk categorized as acceptable were based not only on organoleptic performances, but also on total bacterial counts. the protease activities of the 27 supplemented milk were in the range of 0.200.40 u/ml (whole milk) and 0.30-0.50 u/ml (skim milk), at storage 5 days after the expiry date, while that of without supplements (controls) were 0.50 u/ml (whole milk) and 0.60 u/ml (skim milk), at the same times, respectively (table 2-3). the protease activities of the 20 acceptable whole milk, and the 14 acceptable skim milk, were in the range of 0.20-0.35 u/ml and 0.30 0.45 u/ml, respectively. furthermore, the protease activities of the 15 out of the 20 acceptable whole milk were lower than that of the other 5, while the protease activities of the 10 out of the 14 acceptable skim milk were lower than that of the other 4 (p<0.05). the protease activities of these 15 acceptable whole milk (added with honey, cinnamon, citronella, ginger, turmeric, galingale, zingiber, wild ginger, nutmeg, pepper, clove, galangale, green tea, bamboo leaf and aloe vera) were in the range of 0.20-0.30 u/ ml, at 5 days after the expiry date, while the protease activities of these 10 acceptable table 1. continued 58 biotropia vol. 15 no. 1, 2008 skim milk (added with honey, cinnamon, citronella, ginger, galingale, pepper, galangale, green tea, bamboo leaf and aloe vera) were 0.30-0.40 u/ml. thus, the 15 whole milk and the 10-skim milk categorized as acceptable were based not only on organoleptic performances and total bacterial counts, but also on protease activities. the lipase activities of the 27 supplemented skim and whole milk were significantly lower than those without supplements (controls), and the lipase activities in supplemented whole milk were significantly higher than that in skim, at the storage 5 days after the expiry date (p<0.05)(tables 2-3). the lipase activities of the supplemented milk were in the range of 0.17-0.27 µequiv. ml-1 h-1 (whole milk) and 0.11-0.22 µequiv. ml-1 h-1 (skim milk), at storage 5 days after the expiry date, while the lipase activities of the milk without supplements (controls) was 0.29 µequiv. ml-1 h-1 (whole milk) and 0.24µequiv. ml-1 h-1 (skim milk), at the same times, respectively. the lipase activities of the 20 acceptable whole milk and the 14 acceptable skim were in the range of 0.170.24 µequiv. ml-1 h-1 and 0.12-0.20 µequiv. ml-1 h-1, respectively. furthermore, the lipase activities of the 15 out of the 20 acceptable whole milk were lower than that of the other 5, and that of the 10 out of the 14 acceptable skim were lower than that of the other four (p<0.05). the lipase activities of the 15 acceptable whole milk (added with honey, cinnamon, citronella, ginger, turmeric, galingale, zingiber, wild ginger, nutmeg, pepper, clove, galangale, green tea, bamboo leaf and aloe vera) were 0.16-0.22 µequiv. ml-1 h-1, at 5 days after the expiry date, while the 10 acceptable skim (additional honey, cinnamon, citronella, ginger, galingale, pepper, galangale, green tea, bamboo leaf and aloe vera) were 0.11-0.17 µequiv. ml-1 h-1. thus, the 15 whole milk and the 10 skim classified as acceptable were based not only on organoleptic assessments, total bacterial counts and protease activities, but also on lipase activities. the acidities of the 27 supplemented milk, at storage 5 days after the expiry date, were in the range of 0.27-0.36% (whole milk) and 0.29-0.39% (skim milk), while the acidities of milk without supplements (controls) were 0.26% (whole milk) and 0.28% (skim milk), at the same times, respectively (tables 2-3). the acidities of the 20 acceptable whole milk and the 14 acceptable skim at 5 days after the expiry date were in the range of 0.27-0.34% and 0.29-0.37%, respectively. furthermore, the acidities of the 15 out of the 20 acceptable whole milk, and the 10 out of the 14 acceptable skim, at the same date, were in the range of 0.27-0.38% and 0.29-0.36%, respectively. the acidities of the 15 acceptable whole milk, and the 10 acceptable skim milk, at 5 days after the expiry date, were not significantly different to that of the controls (p<0.05). 59 the preservation of milk t. khusniati & y. widyastuti table 2. preservation of whole milk with the addition of antibacterial and aromatic supplements at 5 days after the expiry date no. antibacterial and aromatic supplements total bacterial counts (cfu/ml) protease activities (u/ml) lipase activities (µequiv. ml-1h-1) acidities (%) 1 honey 6x102 (k) 0.20i 0.17 klj 0.27k 2 cinnamon 7x103 (fg) 0.25 h 0.18 ijk 0.28jk 3 citronella 6.3x103 (gh) 0.20i 0.17 klj 0.30hij 4 ginger 5x103 (i) 0.20 i 0.17 klj 0.27k 5 radish 8.3x104 (c) 0.40 d 0.26 bc 0.35cde 6 turmeric 7.2x10 3 (ef ) 0.20i 0.19 hij 0.29ij 7 galingale 7.2x103 (ef ) 0.20i 0.19 hij 0.29ij 8 zingiber 8.4x103 (d) 0.30g 0.21 fgh 0.32fgh 9 wild ginger 8.3x103(d) 0.30g 0.20 ghi 0.32fgh 10 nutmeg 8.3x103 (d) 0.30g 0.20 ghi 0.32fgh 11 cardamon 8.4x104 (c) 0.40 d 0.27 ab 0.36bcd 12 cumin 8.3x104 (c) 0.40 d 0.26 bc 0.27bcd 13 pepper 7x103 (f ) 0.25h 0.19 hij 0.28jk 14 garlic 6x103 (h) 0.20 i 0.19 hij 0.27k 15 clove 8.3x103 (d) 0.30g 0.20 ghi 0.32fgh 16 javanoni 8.4x104(c) 0.40 d 0.26 bc 0.33efg 17 galangale 8.3x103 (d) 0.30 g 0.22 efg 0.28jk 18 green tea 8.3x103 (d) 0.30 g 0.21 fgh 0.31ghi 19 laurellike 8.3x103 (d) 0.35 e 0.24 cde 0.33efg 20 bamboo leaf 8.3x103 (d) 0.30 g 0.21 fgh 0.31ghi 21 banana leaf 8.4x103 (d) 0.35 e 0.24 cde 0.34def 22 guava leaf 8.5x103 (d) 0.35 e 0.24 cde 0.34def 23 avocado leaf 8.4x103 (d) 0.35 e 0.24 cde 0.33efg 24 betel vine 8.4x103(d) 0.35 e 0.24 cde 0.33efg 25 celery 8.4x104 (c) 0.40 d 0.25 bcd 0.35cde 26 garlic leaf 8.3x103 (d) 0.30 g 0.22 efg 0.32fgh 27 aloe vera 6x103 (h) 0.20i 0.16 klm 0.27k 28 whole milk (control) 8.5x105 (b) 0.50 b 0.29a 0.26k note: numbers followed by different letters were significantly different (p<0.05) 60 biotropia vol. 15 no. 1, 2008 table 3. preservation of skim milk with the addition of antibacterial and aromatic supplements at 5 days after the expiry date no. antibacterial and aromatic supplements total bacterial counts (cfu/ ml) protease activities (u/ml) lipase activities (µequiv. ml-1h-1) acidities (%) 1 honey 8x102 (j) 0.30 g 0.12 op 0.30hij 2 cinnamon 8x103 (de) 0.35e 0.13 nop 0.31ghi 3 citronella 7.2x103 (ef ) 0.30 g 0.12 op 0.33efg 4 ginger 6x103 (h) 0.30g 0.12 op 0.29jk 5 radish 9.4x105 (ab) 0.50 b 0.21 fgh 0.38ab 6 turmeric 8.1x103 (c) 0.40 d 0.15 lmn 0.34def 7 galingale 8x 103 (d) 0.30g 0.14 mno 0.32fgh 8 zingiber 8.6x104 (d) 0.40 d 0.16 klm 0.36bcd 9 wild ginger 8.4x104(c) 0.40 d 0.15 lmn 0.36bcd 10 nutmeg 8.5x104 (c) 0.40 d 0.16 klm 0.36bcd 11 cardamon 9.4x105(ab) 0.50 b 0.22 efg 0.39a 12 cumin 9.3x105 (ab) 0.50 b 0.21 fgh 0.39a 13 pepper 8x103 (d) 0.35 e 0.14 mno 0.31ghi 14 garlic 7.1x103 (f ) 0.30g 0.14 mno 0.29jk 15 clove 8.5x104 (c) 0.40 d 0.15 lmn 0.36bcd 16 javanoni 8.6x105 (b) 0.45 c 0.19 hij 0.37abc 17 galangale 8.1x103 (c) 0.40d 0.17 klj 0.31ghi 18 green tea 8.4x104 (c) 0.40 d 0.16 klm 0.36bcd 19 laurellike 8.7x104 (c) 0.45 c 0.19 hij 0.37abc 20 bamboo leaf 8.4x104 (c) 0.40 d 0.17 klj 0.36bcd 21 banana leaf 9.0x104 (c) 0.45 c 0.18 ijk 0.37abc 22 guava leaf 9.3x104 (c) 0.45 c 0.19 hij 0.37abc 23 avocado leaf 8.9x104 (c) 0.45 c 0.19 hij 0.36bcd 24 betel vine 9.2x104(c) 0.45 c 0.20 ghi 0.36bcd 25 celery 9.4x105 (ab) 0.50 b 0.20 ghi 0.38ab 26 garlic leaf 8.4x104(c) 0.40 d 0.17 klj 0.36bcd 27 aloe vera 7x103 (fg) 0.30g 0.11p 0.29jk 28 skim milk (control) 9.7x106 (a) 0.60 a 0.24 cde 0.28k note: numbers followed by different letters were significantly different (p<0.05) 61 the preservation of milk t. khusniati & y. widyastuti the differences in the acceptability of the 15 acceptable whole milk and the 10 acceptable skim, may be due to the antibacterial and aromatic compounds of these supplements which were more effective in inhibiting bacterial growth and enzymatic activities of psychrotrophic bacteria in whole milk than that in skim. the better organoleptic performances of the 15 acceptable whole milk than that of skim, at 5 days before the expiry date (table 1), may be due to the higher lipid contents of the acceptable whole milk than that of the skim which may result in the better organoleptic performances of acceptable whole milk than that of skim. it has been reported that at 5 days before the expiry date (the designated expiry date), the tastes, flavours, colours and homogeneity of pasteurised milk were fresh due to, at that time, psychotrophic bacteria of pseudomonas spp. hadn’t grown yet (bishop and white 1986; chandler et al. 1990; craven and macauley 1992). the lipid contents of the whole milk were higher than that of the skim and the shelf life of the whole milk was higher than that of the skim, as supported by chandler et al. (1990) and deeth et al. (2002). at storage 5 days after the expiry date, the antibacterial and aromatic compounds in the 15 acceptable whole milk, and the 10 acceptable skim, can suppress the bacterial counts, protease and lipase activities of psychrotrophic bacteria in skim and whole milk (table 2-3). the differences in the total bacterial counts, protease and lipase activities between these 15 acceptable whole milk and 10 acceptable skim may be due to the differences in the type of the antibacterial and aromatic compounds, and the nutritional and organic acid compounds, of these acceptable skim and whole milk. it is known that the various antibacterial and aromatic compounds, nutritional compounds and acidities, of aromatic supplements, may have resulted in the different bacterial counts, protease and lipase activities between the supplemented skim and whole milk, at 5 days after the expiry date. after the expiry date, the longer the time of storage may result in the higher total bacterial counts in refrigerated, pasteurised milk (bishop and white. 1986; chandler et al. 1990; craven and macauley 1992), and the longer the time of storage, the higher the growth of psychotrophic bacteria, especially pseudomonas spp. in refrigerated milk (bishop and white 1986; chandler et al. 1990; craven and macauley 1992). furthermore, the protease activities of refrigerated pasteurized milk increased at times of storage, and the longer the time of storage the higher the protease activities of refrigerated milk ( janzen et al. 1982), and the lipase activities in refrigerated milk increased at the time of storage, also the higher the times of the storage the higher the lipase activities of refrigerated milk (bucky et al. 1986). for the aromatic supplements, there are various examples, with different ph, which may affect the acidities of the skim and whole milk. the lower bacterial growth of the 15 acceptable whole milk, at storage 5 days after expiry date, than that of the skim may be due to the increased effectiveness of the 62 biotropia vol. 15 no. 1, 2008 antibacterial compounds, in the acceptable whole milk, in inhibiting psychrotrophic bacteria, than that of in the skim. the higher lipid contents of the supplemented whole milk than that of the skim may have resulted in the stronger protection of bacterial attack, in the whole milk than that in skim. bacterial growth of whole milk, in refrigerated storage, was lower than that in the skim (chandler et al., 1990). the lower protease activities of the 15 acceptable whole milk at storage, than that of the skim (tables 2-3), may be due to the effectiveness of the antibacterial compounds, in the acceptable whole milk, in inhibiting protease activities of the psychrotrophic bacteria, than that in the skim. the higher lipid contents of the supplemented whole milk, compared to the skim, may have resulted in the stronger inhibition of the protease activities of psychrotrophic bacteria, on the supplemented whole milk than that on the skim. the protease activities of psychrotrophic bacteria in the whole milk at refrigerated storage were lower than that of the skim (deeth et al. 2002; janzen et al. 1982). on the contrary, the higher lipase activities of the 15 acceptable whole milk in storage than that of the skim may be due to the higher lipid contents of the supplemented whole milk than that of the skim. the lipase activities in the whole milk, at refrigerated storage, were higher than that in the skim (deeth et al. 2002; bucky et al. 1986). the differences in the acidities between the 15 acceptable whole milk and the 10 acceptable skim in the storage, may be due to the differences in the organic acidic compounds between these acceptable skim and whole milk. conclusions the study on the preservation of milk with an additional 10% of antibacterial and aromatic supplements, produced in indonesia showed that 15 out of the 27 supplemented whole milk, and 10 out of the 27 skim (preserved milk) were tested and classified as acceptable. this classification was based on their better organoleptic performances, their lower bacterial counts, protease and lipase activities, and their acidities percentages which were not significantly different, compared to that of milk without supplements (unpreserved milk), at 5 days after the expiry date (p<0.05). these 15 acceptable whole milk contained honey, cinnamon, citronella, ginger, turmeric, galingale, zingiber, wild ginger, nutmeg, pepper, clove, galangale, green tea, bamboo leaf and aloe vera, and the 10 acceptable skim milk contained honey, cinnamon, citronella, ginger, galingale, pepper, galangale, green tea, bamboo leaf and aloe vera. acknowledgement the authors would like to acknowledge (1) jsps-ronpaku for providing the scholarship and (2) biodiversity projects of research centre for biology, indonesian institute for sciences for partly providing the funds for this research. 63 the preservation of milk t. khusniati & y. widyastuti references an, bj., kwak, jh., son, jh., park, jm., lee, jy., jo, c. & m.w. byun. 2004. biological and antimicrobial activity of irradiated green tea polyphenols. food chem., 88, 549-555 anonymous. 1999. greater galangale (alpinia galangal [l.] willd). spice pages: greater galangale (galangal, khaa, laos). anonymous. 2001. javanoni, bad flavours which contain medicinal substances. kompas (newspaper). june. 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jose a. ordonez2 'center for multidisciplinary research and linkages, graduate school of education, arts and sciences de la salle university-dasmarinas, philippines 2 biological sciences department, college of science de la salle university-dasmarinas, philippines abstract the karyomorphology of glossogobius giuris (gobiidae) obtained from lake taal and some rivers of cavite in luzon island, philippines was described. metaphase chromosome analysis (colchicine-sodium citrate-carnoy's fixationgiemsa staining procedures) of the hematopoitetic cells in the anterior kidneys revealed that the diploid chromosome number was 2n=46 (46a). fundamental number (fn) is also 46, since all chromosomal morphology were acrocentrics without any distinguishable heteromorphic pair of chromosomes in the metaphase spreads from both dry and wet preparations. this study confirms previous reports on the chromosomal sets of g. giuris from india. keywords : philippines / lake taal / genetics / karyomorphology / glossogobius giuris introduction gobies such as glossogobious giuris and g. celebius have been the subjects of rapidly increasing number of pollution and genetic investigations because of their readily adaptable characteristics to laboratory studies. the present karyomorpho-logical study of this popular food fish (locally named as "biya" in the philippines) was conducted to fill in the wide gaps on fish cytogenetics in the country. several species of gobies and other gobioid fishes (e.g. eleotrids) have been described in terms of their karyomorphological characters such as boleophthalmus pectinirostris, gobius abei and periophthalmus cantonensis (kirpichnikov 1981; nogusa 1960). this study sought to provide relevant information on the basic cytogenetics of glossogobius giuris (gobiidae), specifically, 1) to ascertain the chromosome number and fundamental number of g. giurus; 2) to construct the tentative karyo-type or ideograms of representative samples of the test fish under consideration; and 3) to describe the karyomorphological characters of the test fish. glossogobius giuris was the test fish selected for our chromosomal investigation since no study so far has been reported on its karyomorphology in the philippines. selecting g. giuris from the teleosts of lake taal and rivers of cavite can be justified since cytogenetical investigations of these fishes would explain possible changes in the genetic constitution brought about by incipient speciation in our ongoing chromosome evolution studies. moreover, empirical data gathered in this study will add to the growing body of literature on fish chromosomes that are 11 biotropia no. 21,2003 needed in future studies related to cytotaxonomy, aquaculture breeding and genetoxic testing with the use of chromosomes as biomarkers. methodology fish samples of g. giuris were obtained in the waters off the volcano island or "pulo" in lake taal and some rivers of cavite (dasmarinas-indang area and maragondon riverine area). the methods used in the study basically followed the rapid flame drying techniques (with colchicinesodium citrate-carnoy's fixation-giemsa staining procedures) used in the previous works of masagca (2000) and masagca & sumantadinata (1994). sample preparation fish specimens were pre-treated by intra-muscular injection with colchicine (0.05% in 0.8% nacl) at 1 ml/loog body weight and allowed to swim in well-aerated glass aquaria (40 liters) for 5-6 hours (h). after treatment, the specimens were sacrificed by decapitation or hypothermia (in cracked ice) and kidneys were dissected out, carefully cleared of blood vessels and placed in a petri dish with 0.5-0.6% sodium citrate for hypotonization. anterior kidney tissues were minced into smaller pieces for 16 to 20 minutes in the dish with the hypotonic solution. cell suspensions are transferred to a 10-ml polypropylene tubes and centrifuged for 4-5 minutes at 2500-3500 rpm. the supernatant was removed using a pasteur pipette without disturbing the cell pellet or cell button. about 4 ml of the cold freshly prepared carnoy's fixative (3 absolute methanol: 1 glacial acetic acid) was poured into the tube. after 15 minutes of periodic agitation, the cells were centrifuged again, the supernatant was removed and replaced with fresh fixative. the cell pellet was disturbed gently with a fine point needle of a disposable syringe or dissecting needle, the tube was labeled and stored in a refrigerator for 24 to 30 h before slide plating. slide plating, staining and chromosome analysis pre-cleaned microscope glass slides previously soaked in a 50% ethanol and chilled in the refrigerator overnight were used. after final centrifugation, cells were re-suspended in a small volume of the fixative (about 0.5 to 0.95 ml, depending on the size of the cell button). three to four drops of the suspension were plated on the chilled slide with a pipette and air-dried. wet and dried slides were stained by dipping them into staining jars containing 4% giemsa stock solution (at ph 6.8) for 30-40 minutes. stained glass slides were rinsed with de-ionized water and dried for 12 karyomorphology of the philippine rock goby jimmy t. masagca & jose a. ordonez 30 minutes in an improvised slide dryer. the slides are then placed in a xylene (or xylol) for 10 minutes. slides were air dried for 15 minutes. some slides were mounted using entellan b. stained slides were examined under lpo (10 x) and hpo (loox) to locate well-spread metaphase chromosomes. suitable or well-spread chromosomes were screened to count the diploid chromosomes. results and discussion table 1 shows the frequency distribution of chromosome counts of g. giurus fish samples obtained from lake taal and selected rivers of cavite. using three (3) samples of g. giurus obtained from the dasmarinas-indang riverine areas, a total of 71 well-suited metaphase cells were screened to determine the chromosomal number (cn) as shown by the modal chromosome count. out of this number, 52 cells or 73.24% showed a characteristic count of 2n=46; 12 cells or 16.9% with 2n=44; 5 cells with the characteristic count of 45; and 1 cell each for counts 43 and 47. of the six (6) fish samples of g. giurus obtained from maragondon area, there were 126 metaphase plates with 104 or 82.54% showing a cn of 2n=46, 8 or 6.35% with 2n=44, 10 or 7.94% with 2n=45; and 4 cells with 47. from the 12 samples of g. giurus from lake taal, a total of 314 well-spread metaphase cells were obtained for chromosomal analysis. out of this number, 246 cells (78.1%) have the characteristic count of 2n=46; 39 cells or 12.4% with 2n=45; 12 or 3.8% with 2n=47; 11 or 3.5% with 2n=44 and 6 cells or 1.9% with a chromosome count of 43. in sum, of the 511 metaphase cells (71 metaphase cells from fish samples obtained in dasmarinas-indang areas, 126 metaphase cells from maragondon areas and 314 metaphase cells from lake taal) screened, there were 402 cells or 78.7% have the diploid number of 46; 54 cells (10.5%) have 45; 31 cells (6.1%) have 44; 17 cells (3.3%) have 47 and 7 cells (1.4%) have 43 chromosomes. modal chromosome number based on the data presented, the chromosomal number of the philippine rock goby, g. giurus obtained from 3 areas showed that the diploid modal chromosome number is 2n=46. nf is also 46 (fn=46), since all chromosomes are mono-armed. the predominant chromosome number from the 3 locations was consistently observed at 2n=46 (figure 1). this finding confirms the work of manna (1989) from indian samples and earlier reports of kaur & srivastava (1965, as cited by denton 1972). although characteristic counts of 43, 44, 45 and 47 were noted in wet preparations of slides, the majority of the metaphase cells showed the chromosome count of 46. the chromosomal count of 45 was known in 39 cells or 12.42% of the total number of metaphase spreads. in counting chromosomes, there are instances when 13 biotropia no. 21,2003 overlapping cannot be avoided. there are also possible technical reasons like a missing chromosome during slide plating. denton (1972) reported that within family and genus there seems to be a tendency of reduction in chromosome number to parallel speciations. furthermore, gold (1979) surmised that chromosome numbers and variations in chromosome number do distinguish certain taxonomic groupings, as in the case of salmoniformes (e.g. salmo truttd). generalizations have already been made on chromosome numbers among the members of orders cyprinidontiformes, cypriniformes, siluri-formes and perciformes (rishi 1989). however, it seems that gobies and eleotrids tended to show the common chromosome number of 44 to 46. karyomorphology of the philippine rock goby jimmy t. masagca & jose a. ordonez figure 1. chormosome counts of g. giuris from different locations in cavite and lake taal. the karyotype of g. giuris consists of 46 acrocentric chromosomes. since an acrocentric chromosome is counted as only one arm, the total number of arms will also be 46. the centromere of acrocentric type of chromosome is terminal on which produces a chromosome with one long arm. the karyotypes of the gobies boleoph-thalmus pectinirostris, gobius abei and periophthalmus cantonensis were also found to have 2n=46 (all acrocentrics) as reported in the studies of kirpichnikov (1981) and nogusa (1960). karyomorphological characters of g. giuris as shown in tables 1 and 2, the diploid chromosome number, 2n=46 obtained for g. giuris was consistent in 3 locations (dasmarinas-indang areas, maragondon areas and pulo (i and ii) in lake taal. in the gobiidae family, most of the genera have chromosome number of 2n=44 to 2n=48, such as in bathygobius fuscus (2n=48) and chaetogobius annularis (2n=44). the chromosomes of gobies and eleotrids show variability from 2n=43 to 62 (masagca 2000), with most of the chromosome numbers are 2n=44, 46 and 48. in another study, the karyotype of g. microdon has a diploid number of 2n=56 and nf of 66 with a chromosome formula of 4m+6sm+46st, a. 15 biotropia no. 21, 2003 variability in chromosomal counts would lead to certain generalization of the possibility of changes in chromosomal number due to fusions, translocations and other mechanisms. however, this is not conclusive in the present study since there is a need for further chromosomal banding studies and constancy of variation in the counts. in some studies, like the paedomorphic goby, aphia minuta (gobiidae) wherein the diploid complement ranged from 44 to 41 due to robertsonian fusions (nf=44). data on spermatogenesis suggest that structural heterozygotes are fertile and that these chromosomal changes are not involved in speciation process (caputo et al. 1999). table 2. summary of chromosome counts for g. giuris from 3 locations. location diploid (2n) chromosome number total no. of metaphase cells 43 44 45 46 47 dasmarinnas indang area 1 12 5 52 1 71 maragondon areas 0 8 10 104 4 126 pulo i & 11, lake taal 6 11 39 246 12 314 total 7 31 54 402 17 511 percent (%) (1.4) (6.1) (10.5) (78.7) (3.3) (100) in this study, characteristic counts of 41, 42, 43, 44 and counts higher than 46 in the test animals (g. giurus) were observed. variations in chromosome number maybe attributed to several factors: (1) handling techniques; (2) chemically induced; and (3) inherent genetic characteristic of the test fishes. handling techniques would explain the variability in chromosome counts. the karyotypes of two other teleosts, g. giurus previously described in japan, india and elsewhere could permit the researchers to have further comparison using the conventionally stained chromosomes and in the future the banded chromosomes from fully elongated chromosomes. diploid chromosome number of the philippine common goby, g. giuris is 2n=46 and nf of 46. all of the chromosomes are acrocentric (mono-armed). the chromosome number of 46 is common to the order perciformes. diploid chromosome number of oxyeleotris aporos is 2n=46, which is also similar to the chromosomes of selene setapinnis (family carangidae) as described by netto & pauls (2000). in fishes, 48 rod-like chromosomes have been considered to be the modal number as shown in the works of nogusa (1960), roberts (1967) and ohno & atkin 16 karyomorphology of the philippine rock goby jimmy t. masagca & jose a. ordonez (1968). recently manna (1989) advocated that 48 chromosomes mixed morphology and only rods were the modal ones from which the evolution of different karyotypes can be envisaged. conclusion this study concluded that the philippine rock goby, g. giuris has a diploid chromosome formula of 2n=46 (a) and having the fundamental number of 46. karyomorphological characters of the goby under study reveal that majority of the chromosomal spreads consist of all acrocentric chromosomes, which are common among the gobiids and eleotrids. no heteromorphic pair of chromosomes was observed in the ideograms prepared. characteristic chromosome counts of g. giuris range from 41 to 48. acknowledgment the authors express their thanks to the dlsu-d administrators (dr. h.d. torres, dr. mf. ramos, dr. v.l. de leon, dr. n.m. medina, ms. c. cervillon and dr. j. samonte for the ufro grant. references arai, r. and y. sawada. 1974. chromosomes of japanese gobioid fishes (i). bulletin of natural science tokyo 17:97-102. arai, r. and a. fujiki. 1979. chromosomes of japanese gobioid fishes (iv). bulletin of the natural science museum. series a 5 (2): 153-159. caputo, v., m. l. caniglia and n. machella. 1999. the chromosomal complement of aphia minuta, a paedomorphic goby. the fisheries society of the british isles, p. 455-458. denton, t.e. 1972. fish chromosome methodology. charles c. thomas publisher, springfield, illinois. 166 p. kaur, d. and m.d.l. shrivastava. 1965. the structure and behavior of chromosomes in freshwater teleosts. cited by denton, t.e.1973. fish chromosome methodology. berlin: charles c. thomas publishing. springer verlag. kirpichnikov, v.s. 1981. genetic bases for fish selection. springer verlag: berlin. kligerman, a.d. and s.e. bloom. 1977. rapid chromosome preparations from solid tissues of fishes. journal of the fisheries research board canada, 23:266-269. levan, a., k. fredga and a. sanberg. 1964. nomenclature for centromeric position on chromosmes. hereditas, 52:201. manna, g.k.i 989.fish cytogenetics related to taxonomy, evolution and monitoring genotoxic agent. pages 2146, in: das and jhingran, eds. fish genetics in india. today and tomorrow printer & publishers: new delhi, india. 17 biotropia no. 21,2003 masagca, j.t. 2000.karyology of the south-east asian marble sleeper, oxyeleotris marmorata bleeker 1852. asian international journal of life sciences journal, 9(2):181-188. masagca, j.t. and k. sumantadinata. 1994. chromosomal characters of the indonesian sand goby, oxyeleotris marmorata blkr. 1874 (eleotridae), biotropia-southeast asian journal of tropical biology (7): 41-46. masagca, j.t. 1993. karyotypic differentiation among natural populations of sand goby. o. marmorata blkr. (eleotridae) in the indo-malayan region. proceedings of the symposium on fish genetics and its application to fishery management held on dec. 8-11, 1992, seameo-biotrop bogor, indonesia. medina, f.i.s. iii. 1984. a simple method for studying the mitotic metaphase of tilapia nilotica. proc. symp. research trends phils. nrcp bull. no. 98. netto, m.r.c.b. and e. pauls. 2000. family carangidae: cytogenetical and evolutionary aspects (international workshop on marine genetics, 1998 (brazil). nogusa, s. 1960. a comparative study of the chromosomes in fishes with particular considerations on the taxonomy and evolution. memoir of hyogo university of agriculture 3(1):1.cited by denton, t.e.i973.fish chromosome methodology. charles c. thomas publishing. springer verlag, berlin. 166 p. ohno and atkin. 1968. evolution from fish to mammals by gene duplication. hereditas, 59:169-187. pagulayan, r.c., n.c. lopez and f.s. magbanua. 1997. littoral fishes of lake taal. sylvatrop tech. j. of philipp. ecosystems and mat res. 7(l&2):84-93. reddy, p.v.g.k. and g. john. 1987. a method to increase mitotic metaphase spreads in permanent chromosome preparations for karyotype studies of fishes. pages 199-205. in: proceedings of the world symposium on selection, hybridization and genetic engineering in aquaculture held in bordeaux. 27-30 may 1986. vol. ii. rishi, k..k. 1989. current status of fish cytogenetics. pages 1-20, in: das and jhingran (eds.), fish genetics in india. today and tommorow's printers and publishers, new delhi, india. rivlin, k., j.w. rachlin and g. dale. 1985. a simple method for the preparation of fish chromosomes applicable to field work, teaching and banding. journal of fish biology, 26:267-272. roberts, e.l. 1967. chromosome cytology of the osteichthyes. progressive fish culturist, 29:75-83. sumner a.t., h.j. evans and r.a. buckland. 1971. a new technique for distinguishing between human chromosomes. nature new biol. 232:31-32. 18 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf microsoft word 122 biotropia vol. 13 no. 2,2006 : 122 -131 assessment of coastal land use changes in banten bay, indonesia using different change detection methods puvadol doydee¹ and vlncentius slregar² 'department of fishery management, faculty of fisheries, kasetsart university, bangkok, 10900, thailand 2seameo biotrop, jl. raya tajur km 6, p.o box 116, bogor indonesia e-mail:vincents@biotrop.org abstract many types of the coastal land use in banten bay have been assessed in order to know the change as evidently detected by landsat imagery in 1994 and 2001. image processing such as, supervised classification and various change detection techniques are performed to the satellite images. red green method showed the best result for detecting the coastal land use change. this method is suitable for detecting the increasing areas of the paddy fields and settlement. image differencing method is capable to detect the increasing areas in agriculture, decreasing in fishponds and natural areas. image ratioing method can be considered suitable for detecting the increasing area of fishponds, decreasing of paddy fields and agriculture areas. each coastal land use type has increased, except for the natural area/brushwood. most of agriculture and paddy fields areas have been converted to fish ponds. key words: coastal land use/land cover, change detection method, remote sensing introduction as a transition area between land and sea, coast is characterized essentially by geologic nature of the land. sometimes, coastal zone is defined depending on the project purposes, for example loicz project (holligan and de boois 1993). this zone in many cases is relatively unstable and fragile (carter 1989), but became an important place for the human activities, such as culture, economic, fisheries, industry, tourism and social (rais and de boois 1997). more than 60 % of the human population lives in coastal area and it will increase in the future, hi many cases, coastal area is the best place or the only available space for city development, agriculture, fisheries, industry and tourisms. this condition will cause the increasing demand for space for such activities. however, in coastal zone a land use change is obviously a phenomenon (loicz 1994; williams et al 1997; noaa 1998) this change is an essential matter that should be monitored for planning or avoiding any further changes that can damage or harm the environment. remote sensing technique is one of the effective tools in monitoring phenomena that change continuously overtime and cover large areas (aronoff 1991). one of the major applications of remote sensing technique is change detection method. many readymade algorithms are available to perform that method. despite of their differences in identification, an accurate spatial registration of the various dates of imagery is a requirement for all methods used. corresponding author: vincents@biotrop.org 122 asessment of coastal land use changes p. doydee and v. siregar this study is intended to assess the capability of four methods of change detection in detecting the change in coastal land use, and to compare the result of those methods with each other. location of this study is the coastal zone of banten bay. banten bay is an area most frequently used for various researches, since in this region many activities are taken place, such as agriculture, fishing and mariculture, harbor, and industry. there is no doubt that these activities will influence the coastal habitats and resources. materials and methods the study area is located in banten bay at the northern coast of banten province (figure 1). banten bay has an area of about 120 km2 with water depth of less than 13 m. geographically, this bay lies on 05°55' s-06°05' s and 106°05'e -106°15'e and the distance from jakarta is about 175 km. this bay is also characterized as a lowland area (saptarini 2000). data used comprised satellite data of landsat-5 tm (with the path/row 123/064 and acquisition date of april 6, 1994), and landsat-7 etivt (with path/row 123/064 and acquisition date of august 7, 2001, nasa, 2002), topographic and geomorphological maps. different software have been used such as image processing er mapper (er mapper 1997), banten bay information system 3.0 and geographic information system. supervised classification method used has the objective to identify and classify the features in an image quantitatively (richards 1995; lillesand and kiefer 1994; congalton and green 1999; yusuf 2001). this involves the analysis of multi-spectral image and the application of statistically based decision rules for determining the coastal land use type of each pixel on an image (middlekoop and lif 1991). many different techniques have been developed for change detection. these methods involve change extraction and change classification. (coppin et al. 2002; jensen 1986). the use of change detection techniques in this study comprised four methods, including red green, image differencing, image ratioing and principal component analysis methods. image analysis was performed to assess the coastal land use change between two different dates by overlaying the images of landsat-5 tm in 1994 and landsat-7 etm+ in 2001, and applying the change detection techniques as stated above. prior to the application of change detection techniques , the images utilized have to be normalized using atmospheric correction technique of histogram adjustment in order to eliminate error due to different atmospheric conditions of the images of different periods (wallace and campbell 1996). image of 1994 was rectified to image of 2001 which has been already corrected (geodetic datum wgs 84 and map projection sutm 48). 123 coastal land use in 1994 and 2001 the type of coastal land use in 1994 and 2001 are grouped into 5 classes such as, paddy fields, fishponds, settlement, agriculture and natural areas (brushwood). the maps of coastal land use were prepared from the result of supervised classification using the band combination of 542/rgb (figure 2.) change detection techniques red green method this method involves displaying simultaneously one dataset in green and one dataset in red, namely image in 1994 was displayed as red and image in 2001 was displayed as green. the red color means that these areas only exist in image 1994 and the green color in image 2001. for example in lontar district (figure 3) as zoomed-in, paddy fields in 1994 have been converted to fishponds in 2001. the red color is represented as paddy fields before and those that have been converted into fishponds in 2001. this could be confirmed by using geomorphological map and thematic coastal land use map resulted from supervised classification. 124 asessment of coastal land use changes p. doydee and v. siregar image differencing method the digital number value of image in 2001 was subtracted from digital number value of image in 1994. the basic concept of this method is that, in the raster data, each band has its digital value (dn). therefore, if it is subtracted from the same dn value of each pixel in both images, the output is equal to 0. the output might be minus or plus in the case that those pixels do not have the same dn values, this means that there is a change of value between the two images. this change might be due to the change of characteristic of the object. the output of this method was displayed as pseudo color area (figure 4-left). prior to data overlay, the raster data should be converted from raster cells into vector polygon and then overlaid that vector (change portion) with the original image so that the raster and vector polygon could be distinguished (original image), the result is presented in figure 4 (right). figure 4. the result of image differencing method. 125 biotropia vol. 13 no. 2, 2006 image ratioing method actually, this is a simple method as image differencing method where in this method the formula is changed from subtraction to division. the data from two registered images are divided pixel by pixel. therefore, the areas in different dates that have the same value of dn is equal to 1, it means that those areas do not change. bands of tm1, tm2 and tm3 are highly correlated. the lower the correlation between two bands the greater the information content. ratio of tm2 and tm5 provide some subtle wetland information (jensen 1986). the result of this method is presented in figure 5. figure 5. the result of image ratioing method principal component analysis method the pc#2 has maximum variance of the original data set, therefore it is used for analysis. pc#2 data consist of two bands inputs, where input 1 and input 2 were images of 1994 and 2001, respectively. the result showed that the bright blue color appears covering the areas (figure 6-left). the output should be converted from raster cells into vector polygon in order to facilitate the interpretation (figure 6right). figure 6. the result of principal component analysis method 126 asessment of coastal land use changes p. doydee and v. siregar change analysis the entire study area was 46 785.69 ha consisting of 23 437.26 ha terrestrial zone and 23348.43 ha water. the classification of coastal land use (type) between two dates was processed by post-supervised classification method (cho 2000) and the result is presented in table 1 . table 1 shows that the area of each land use type has increased, except for the natural area/brushwood which shows the contrary. the main factor caused by this change is the increasing numbers of the population and human activities in this area. from the field check, it was observed that some parts of agriculture and paddy fields are converted into fishponds. it is known that in 1990's fishponds were very promising, and for that reason most of villagers and farmers converted their agriculture area and paddy fields into fishponds in the coastal zone. according to table 2, there are two types of changes, that is the increasing and the decreasing area. obviously, the total areas of coastal land use converted was 7706.79 ha .these changes not only happen from one type of land use to other types, but also in the area of the same land use type. for example of decreasing area, it can be noted as follows: paddy fields as in 1994 have been converted into different land use types, such as fishponds (812.46 ha), settlements (70.16 ha), and agriculture areas (712.66 ha), respectively in 2001. fishponds as in 1994 have been converted only into settlement (8.15 ha) in 2001, while for settlement there is no change in terms of land use type and the area between the two periods. agriculture as in 1994, has been converted into paddy fields (1015.06 ha), fishponds (761.39 ha) and settlement (317.18 ha), respectively in 2001. natural areas (brushwood) in 1994 have been converted into paddy fields (998.51 ha), fishponds (727.27 ha), settlement (320.19 ha) and agriculture (1963.76 ha) in 2001. meanwhile, the increasing areas of the land use types from 1994 to 2001 are as follows: paddy fields are increased as 127 biotropia vol. 13 no. 2,2006 much as 2013.57 ha from agricultural (1015.06 ha) and natural areas (998.51 ha); the area of fishponds are increased to 2301.12 ha from paddy fields (812.46 ha), agricultural (761.39 ha) and natural areas (727.27 ha); settlement areas are increased 715.68 ha which comprised paddy fields (70.16 ha), fish ponds (8.15 ha), agriculture (317.18 ha) and natural areas (320.19 ha) and; agriculture areas are increased to 2,676.42 ha that is from paddy fields 712.66 ha and natural areas about 1903.76 ha. the total of land use type (7706.79 ha) then is considered as a reference for selecting the best method of change detection to be applied. using the four converted detection methods, the converted areas of each method were as follows: the red green method (7094.97 ha), image differencing method (6185.25 ha), image ratioing method (8490.96) ha and principal component analysis (pc#2) (5920.47 ha), respectively. from the result presented in table 3, it could be concluded that red green method is better for detecting the coastal land use change compared to other methods. saptarini (2000) has also concluded that red green method is simple and easy method to detect the change of areas in coastal zone of banten bay. this method shows that the total number of converted areas closely agrees with the number of converted areas of the reference. as stated above, the total of each land use type has increased from 1994 to 2001, except for natural areas/brushwood which show the contrary. 128 asessment of coastal land use changes p. doydee and v. siregar the increasing and decreasing areas of each type of land use were derived from four change detection methods. for increasing area in general, red green method is the best compared to the other methods. but, if the increasing area of each type of land use is observed, we found that only pc#2 has no land use types which has a wide close to the reference (table 4). it means that each change detection method is suitable to detect certain land use type. table 4. the increasing area of each land use type using the four change detection methods compared to the reference. type red green image differencing image ratioing pc#2 references paddy fields 1,969.86 1,680.39 2,088.15 1,802.88 2,013.57 fishponds 2,161.82 1,806.74 2,394.97 1,988.55 2,301.12 settlement 804.84 439.41 827.13 493.20 715.68 agriculture 2,158.44 2,258.72 3,180.70 1,635.85 2,676.42 total 7,094.97 6,185.25 8,490.96 5,920.47 7,706.79 therefore, it could be concluded that red green method is suitable for detecting the increase of paddy fields and settlements. differencing method is better in detecting the increasing agriculture areas. meanwhile, the ratioing method could be considered as the best method to detect the increasing fishpond areas (table 5). for decreasing areas, all the change detection methods used showed that the area of each type of land use is far less under the area of reference (table 6). meanwhile, if the decreasing area of each type of land use provided by the change detection methods used is compared with the reference, it could be 129 biotropia vol. 13 no. 2,2006 concluded that image differencing method is better for detecting the decreasing area of fishponds and natural areas compared with other methods and image ratioing method could be considered as the better method for monitoring the decrease of paddy fields and agriculture areas (table 7). this study showed that no specific change detection method could be used to detect all land use types. each method could detect better a specific land use type compared to the other methods. conclusions supervised classification of remotely sensed data (landsat data in!994 and 2001) of the coastal zone of banten bay provides 5 land use types such as paddy fields, fishponds, agricultural area, settlement and brushwood (natural area). these land use types served as the reference for land use change during those two periods. to detect the change of land use types in those areas, several change detection methods are performed to the landsat satellite data. change analysis showed that among various change detection methods used, the red green method has provided the best result for detecting the coastal land use change. this method is suitable for detecting the increasing areas of the paddy fields and settlements. meanwhile, image differencing method is capable to detect increasing areas in agriculture, decreasing in fishponds and natural areas. image ratioing method could be considered suitable for detecting the increasing area of fishponds, decreasing of paddy fields and agriculture. each coastal land use type in the study areas had increased, except for the natural areas, and most of agriculture and paddy fields areas have been converted to fishponds. certain change detection methods used has shown a good performance for detecting the change of certain coastal land use types in banten bay, however, several tests in different areas are needed to be conducted in order to know the level of accuracy of each method used. acknowledgments the authors would like to acknowledge the funding support of the seameo regional center for graduate study and research in agriculture (seameo searca), and technical support and assistance of mr. nyoman sukmantalya in providing all the data needed. 130 asessment of coastal land use changes p. doydee and v. siregar references aronoff, s. 1991. geographic information systems. a management perspective, wdl publications, ottawa, 294 p. carter, r.w.g. 1988. coastal environment. an introduction to the physical, ecological and cultural systems of coastlines, academic press, toronto, 617 p. cho, s.h.2000. digital change detection by postclassification comparison of multi-temporal remotely sensed data. university of idaho, departement of forest resources. journal of the korean society of remote sensing col 16, no 4, p. 367-373. www.ksrs.or.kr/iournal full/1 6 4%20ftp/8josung.pdf congalton, r. g. and k. green. 1999. assessing the accuracy of remotely sensed data: principles and practices, lewis publishers, boca raton, 137 p coppin, p., lambin, e., jonckheere, l., and b. muys 2002. digital change detection methods in natural ecosystem monitoring. in analysis of multi-temporal remote sensing images. bruzzone,l.and smiths,p.eds. world scientific, new jersey. 440 p. er mapper 5.5 .1997. er mapper workbook level one, training workbook, 25 august 1997, california, 382 p. holligan, p.m. and h. de boois. 1993. land-ocean interactions in the coastal zone (loicz). global change report (25). igbp. stockholm, 50 p, url: http:// ww\v, cs.utk. edu/~l ucas/nubl icat ions/publ ications , html jensen, j.r. 1986 introductory digital image processing. a remote sensing perspective. prentice-hall, englewood cliffs, 3 18 p. lillesand t. m. and r. w. kiefer .1994. remote sensing and image interpretation (3 rd edition), john wiley and sons, 750 p. loicz .1994. land-ocean interactions in the coastal zone, core project of the international geosphere biosphere programme of the international council of scientific unions, p. 6. middelkoop, h. and j. lif. 1991. implemention on temporal relationship in: knowledge base system of satellite image, photogrammetric engineering and remote sensing. vol.57, july 1991, p. 937 945. nasa .2002. landsat 7 science data users handbook, url: http://ltpwww.gsfc. nasa.gov/ias/handbook/handbook_htmls/chapter8/chapter8.html noaa .1998. shoreline, coastal zone glossary, url: http://www.csc.noaa.gqv/ sho reline/glossarv.html rais, j., dutton, m., pantimena, l., plouffe, j. and r. dahuri, 1997. integrated coastal and marine resource management, proceedings of international symposium, 25-27 november 1997, batu, itn, bakosurtanal and proyek pesisir, malang. p. 273-276. richards, j.a. 1995. remote sensing digital image analysis, springerverlag, berlin, p. 54-57. saptarini, d. 2000. coastline changes detection using remote sensing technique: banten bay study case, master in information technology for natural resources management, bogor agriculture university (1pb), bogor, 53 p. wallace, j. and n. campbell. 1996 evaluation of the feasibility of remote sensing for monitoring, national state of the environment indicators, technical paper series, department of the environment, canberra, p. 30. williams, s.j., k. dodd k, and k.k. gohn .1997. coast in crisis, usgs homepage. url: http://www.usgs.gov/http://www.usgs.gov/ yusuf, d.n. 2001. remote sensing and gis for landuse change analysis, case study in kab. bandung, master in information technology for natural resources management, bogor agriculture university (ipb), bogor, 68 p. 131 122.pdf 123.pdf 124.pdf 125.pdf 126.pdf 127.pdf 128.pdf 129.pdf 130.pdf 131.pdf biotropia no biotropia no. 16, 2001 : 1 9 in vitro propagation of angiopteris evecta using spores damien cupitt, poonam bhatia* and nanjappa ashwath primary industries research centre, school of biological and environmental sciences, central queensland university, rockhampton, qld 4702, australia abstract techniques of establishing angiopleris evecta plants in vitro were studied. soaking of a. evecta spores in water for 24 hours markedly reduced spore contamination. soaking of the spores in 1 -2 % of sodium hypochlorite for less than 5 minutes allowed satisfactory disinfestation without affecting spore viability. lower concentration of minerals (1/4 ms), presence of charcoal in the medium and exposure of the spores to light were crucial for spore germination and gainetophytc development of a. evecta. keywords: angiopleris evecta i king fern / spore / ms medium / bleach / light / tissue culture / sporophyte / gamctophytc abbrevations ba benzylaminopurine; bb bold's basal medium; pcpa para-chlorophenoxyacetic acid; 2,4-d 2,4-dichlorophenoxyacetic acid; iaa indoleacetic acid; iba indolebutyric acid; kinetin -6-furfurylaminopurine; mm moss's mineral medium; ms murashige & skoog media; naa naphthaleneacetic acid; noa naphthoxyacetic acid. introduction the fern and fern allies are the surviving members of the earliest lineages of vascular plants. none of these primitive plants produce either seeds or flowers; instead they reproduce via single-celled spores. ferns are usually found in moist terrestrial or aquatic environments and their size may vary from a few centimeters to many meters. angiopteris evecta is known as king fern or giant fern and it belongs to the family marattiaceae. it is a magnificent fern, the fronds are reputedly largest in the world, and are about 5 m long, arching, semi-weeping, bi-pinnate, and glossy (figure la). lower pinnules have earlike lobes at the base. sporangia always occur `* corresponding author : e-mail address : p.bhatia@cqu.edu.au fax: 61-7-4930 9255 1 biotropia no. 16 , 2001 figure 1. a. mature sporophyte with well developed fronds; b. pinnule showing sori; c. sporangia in dense clusters; d&e. developing gametophytes in ms vi c medium; f. germinating sporophyte from gametophyte. 2 in vitro propagation of angiopteris evecta using spores damien cupitt et al. in dense clusters of five to eight opposite pairs (figure ib & ic). the king fern is found in australia (queensland and new south wales), malaysia, polynesia and southern and northeastern parts of india. it also occurs in new guinea and indonesia (s. marsterson; department of e.p.a., rockhampton, pers. com.). in northeastern parts of india, the massive stem is cooked and eaten by the tribes. an intoxicating drink called 'ruchshi' is also made out of it. the plant yields aromatic oil that is used for perfuming coconut oil in south sea islands (manickam and irudayaraj 1992). conventionally, king ferns are almost impossible to propagate from spores but may be reproduced vegetatively from the fleshy earlike projections which are commonly known as auricles. plant development is very slow and it may take 12 months or more before a small frond and sufficient roots develop to support the plant (blomberry and maloney 1994). tissue culture can prove to be the fastest and the most effective method of multiplication of the king fern. studies on in vitro spore propagation and culture media requirements of different ferns have been conducted by bernabe et al. (1999); amoroso and amoroso (1998); fernandez et al. (1997); goller and rybczynski (1995) and dong and su (1993). since spores remain the only source of fern propagation, considerable importance has been placed on spore disinfestation and culture media selection for different ferns. fernandez et al. (1997) observed optimal growth of blechnum spicant gametophytes in ms (murashige and skoog 1962) liquid medium spread over a 2% agar and exposed to 16 hours of light. borelli et al. (1990) used both calcium and sodium hypochlorite solution for disinfesting spores and they obtained the best results with 2% sodium hypochlorite solution. borelli et al. (1990) also observed better germination of the ferns cyathea schanschin and dicksonia sellowiana on john and knop's media after 4-8 weeks, with the prothalli developing 30-40 days later. in a similar study, goller and rybczynski (1995) disinfested spores of cyathea australis using 3% chloramine with tween. they reported better germination of spores on anderson's medium supplemented with 80 mg t1 of adenine sulfate solidified by 0.8% agar. no studies are currently available for a. evecta either for spore disinfestation or for selecting suitable culture medium. the present study was carried out to optimize spore disinfestation procedures and to determine suitable culture medium and growth conditions for establishment of a. evecta in tissue culture. materials and methods collection and storage of spores recently, matured pinnules containing sori were collected fresh from the rockhampton city council botanical garden, rockhampton, australia. after a close microscopic examination, closed sori containing spores were isolated from the 3 biotropia no. 16, 2001 fronds for disinfestation. another set of pinnules was stored in a brown paper bag to allow drying without fungal infection. effect of soaking spores on contamination the sori were separated from the pinnulus by scraping with a scalpel. the released sporangia were then collected onto a piece of paper. these sporangia were exposed to three treatments. in treatment 1, sporangia were filtered through a 106 um sieve using demineralised water and the filtrate was collected in a glass jar. the sporangia were allowed to soak in water for approximately 24 hours. once soaked, the spore solution was microfuged (x 10,000 rpm) for five minutes to concentrate the spores prior to sterilisation (sieved and soaked). the spore solution (1 ml) was inoculated onto agar plates to test for contamination and germination. in treatment 2, the unsieved sporangia were used, and they were soaked in sterile water for 24 hours before microfuging as in treatment 1 (not sieved but soaked). in treatment 3, plant debris were removed by sieving and the spores were not soaked in water (sieved but not soaked). the rest of the operations were the same as for treatment 1. in treatment 4, spores were neither sieved nor soaked (neither sieved nor soaked). spore disinfestation two concentrations (1% and 2%) of sodium hypochlorite were used for disinfestation, with the exposure times of 3, 5, 7 and 9 minutes. a known volume (1 ml) of solution containing sporangia was placed in an eppendorf tube and 1.5 ml of 1% or 2% naocl was added. after 30 seconds of soaking, the eppendorf tube was centrifuged for 3 minutes. one milliliter solution was removed from the top of the eppendorf tube and a milliliter of sterile water was added. the contents were centrifuged again for 2 minutes. the washing with sterile water was repeated 4 times, to remove all traces of naocl prior to inoculation. inoculation using aseptic bench top tunnel technique, culture tubes (50 ml capacity, plastic) were inoculated with two drops of spore solution and the tubes sealed before being placed in a controlled environment room. media media with low nutrient content were selected, because the spores, like seeds, contain all required nutrients for early growth. wide ranges of media were used. these include ms, msc (ms media + 1 g/l charcoal), ms%c (ms medium with v* nutrients +'/« sugar + 1 g/l charcoal), ms low (ms medium + low hormones*), ms'/z low (ms medium with vi nutrients + low hormones*), ms% low (ms medium 4 in vitro propagation of angiopteris evecta using spores damien cupitt et al. with 1/4 nutrients + low hormones*), fn (fern normal), bold's basal (bischoff and bold 1963) and moss's mineral media. *here de fossard's (1981) broad spectrum low hormones were used. low hormones consisted of auxins (iaa+iba+naa+noa+2,4-d+pcpa; 0.1 um each) and cytokinins (bap+kinetin; 0.1 um each). incubation one set of inoculated tubes was randomly stacked in a clear plastic bag and incubated in a controlled environment room (cer) at 30 °c with a light intensity of 100 uem~2s~'. the cer was maintained at a photoperiod of 12 hours light and 12 hours dark. the second set of tubes was sealed within a black plastic bag to simulate dark environment that is often found in dense rainforest floor where mature ferns are usually found. both sets of tubes were placed side by side in the cer to ensure that they were exposed to similar climatic conditions except for light. results effect of soaking on contamination and spore germination pre-soaking of spores for 24 hours before disinfestation reduced contamination. the contamination rate could be reduced to as low as 3% with pre-soaking treatment alone. most of this contamination was caused by fungi (penicillium sp. and aspergillus sp.) and rarely by bacteria. contamination was as high as 75% when the spores were neither sieved nor soaked. the sieving and soaking reduced the contamination rate to 13% (table 1). sieving had very little effect on contamination, but the soaking markedly reduced the contamination rate. table 1. effect of pre-soaking treatments on contamination and spore germination of a. evecta 5 biotropia no. 16, 2001 response of a. evecta spores to naocl the degree of contamination varied from 0% to 38%, with 1% naocl and from 0% to 9% at 2% naocl (table 2). exposure of king fern spores to 1 or 2% naocl for either 7 or 9 minutes resulted in complete disinfestation. however, none of these spores germinated when the exposure time exceeded 5 minutes. thus, the treatment of king fern spores with 2% naocl solution for less than 5 minutes appear to provide the best results. the tubes that were inoculated with untreated spores were fully contaminated suggesting that disinfection is a must for king fern spores. table 2. contamination status of a. evecta spores in response to naoci concentration and time of exposure (number of tubes used per treatment varies from 8 to 38) effect of culture media on spore germination the highest germination (69%) was observed in msvic medium followed by moss's mineral medium (25%), hold's basal medium (17%), ms 'alow (4%) and ms'/tlow (0%) (table 3). spores failed to germinate on full strength ms medium, either with or without hormones and charcoal. reduced concentration of ms medium and the presence of charcoal maximized king fern spore germination (figure ld& le). table 3. effect of culture media on spore germination of a. evecta (please see methods for description of acronyms) 6 in vitro propagation of angiopteris evecta using spores damien cupitt et al. role of light in germination of a. evecta spores light played a major role in 'the germination of a. evecta spores. irrespective of soaking treatment, the length of exposure to naocl, or the type of medium used, almost all the spores failed to germinate in the absence of light (table 4). gametophytes that grew well in tissue culture conditions produced sporophytes within 6 months (figure if). the sporophytes were then successfully transplanted and raised in a potting mix. table 4 the role of light on spore germination of a. evecta light dark number of tubes inoculated 170 49 number of tubes showing spore germination 15 2 percentage of tubes showing spore germination 9 2* * tubes germinated only after the cultures were exposed to light discussion pre-disinfestation treatments are usually beneficial in reducing contamination, to a certain level, without harming the explant. this experiment gave a very useful clue for the use of pre-disinfestation treatment for decontaminating spores of angiopteris evecta. contamination percentage was reduced to as low as 3% when sporangia were soaked in water for 24 hours. it is likely that the bacteria and fungi that were adsorbed onto the fern spores become more susceptible to naocl treatment if they are soaked in water prior to disinfestation. prior to soaking of sporangia, the whole material was passed through 106 urn sieve (treatment 1) with the aim of reducing the quantity of material to be disinfested, as it included a considerable amount of sori cases and dried leaf pieces. sieving did not seem to make any difference to contamination rates both in soaked and unsoaked treatments. our results revealed that 2% naocl is highly effective in disinfesting a. evecta spores at both 3 and 5 minutes. these results are in agreement with those of borelli et al. (1990) who obtained best results with 2% sodium hypochlorite for cyathea schanschin and dicksonia sellowiana spores. in contrast, goller and rybczynski (1995) obtained better results when they disinfested intact leaves containing the sori of cyathea australis with 3% chloramine and tween for 30 minutes. the length of exposure of a. evecta spores to sodium hypochlorite also had a considerable bearing on the success of germination. our results showed that three minutes exposure to sodium hypochlorite reduced contamination to a considerable 7 biotropia no. 16, 2001 extent whereas, 7 and 9 minutes exposure proved to be lethal as the spores failed to germinate. based on these results, we recommend the exposure of a. evecta spores to high concentration (1-2%) of naoclfor short duration (<5 minutes). the pattern of spore germination in various media compositions appears to reflect the need for low concentrations of mineral nutrients and the presence of charcoal for a. evecta spore germination. this observation is in consistence with the findings of khoo and thomas (1980), who found that high mineral salt concentrations tended to retard spore germination and sporophyte formation of adiantum raddianum cv tassel. however, the current findings contrast with those of fernandez et al. (1997) who reported that ms full strength liquid media being optimal for growth of blechnum spicant gametophyte. apart from ms media, andersen's media for cyathea australis (goller and rybczynski 1995), jone's media and knop's solution, respectively, for cyathea schanschin and dicksbnia solviana (borelli et al. 1990) and knudson's media for cyathea spinulosa (agrawal et al. 1993) have also been advocated. similar to our findings, wardle et al. (1998) recommended the use of charcoal for the spore germination. spores are analogous to seeds and they contain all the required nutrients for early growth, therefore it can be justified to use low nutrient media during initial stages of germination. light is an important factor and it plays a significant role in the germination of a. evecta spores. the results of this study concur with the field observations (s. marsterson; department of e.p.a., rockhampton, pers. com.) where the king fern seedlings usually occur in patches of rainforests that have been exposed to light possibly due to fire or other sources of damage to canopy. light has been found to govern the development stage of whole gametophyte (turnwald et al. 1999). kiss and kiss (1998) reported that light frequency in the red region is found to promote spore germination more than in far-red region. light not only regulates the growth mechanism in haploid gametophyte, but it also affects development of sporophyte due to involvement of a photoreceptor phytochrome (christensen et al. 1998). presence of light has been shown to increase nuclear dna and this increase is known to have an influence on fern spore germination (raghavan 1993). this study has demonstrated the importance of soaking of a. evecta spores to minimize contamination, sensitivity of king fern spores to high mineral concentration of the media and the need for light and charcoal for its spore germination. these data highlight the desirable conditions for disinfestation and germination of king fern spores. references agrawal dc, ss pawar, af mascarenhas. 1993. cryopreservation of spores of cyathea spinulosa wall, ex. hook. f. an endangered tree fern. j plant physiol 142: 124-126. amoroso cb, vb amoroso. 1998. spore culture studies on some economic ferns of mindanao, philippines. acta horticulturae 461: 231-235. 8 in vitro propagation of angiopteris evecta using spores — damien cupitt et al. bernabe n, g williams-linera, m palacios-rios. 1999. tree ferns in the interior and at the edge of a mexican cloud forest remnant: spore germination and sporophyte survival and establishment. biotropica31: 83-88. bischoff h, hc bold .1963. some soil algae from enchanted rock and related algal species. phycological studies iv. university of texas publication no. 6318, austin p. 9-13. blomberry am, b maloney .1994. propagating australian plants. kangaroo press, nsw. 112 p. borelli fp, cef castro, lap de matthes, afc tombolato, v nagai .1990. propagacao de pteridofitas in vitro e in vivo atraves de esporos. bragantia 49: 205-219. christensen s, y tokuoka, j silverthorne, m wada. 1998. phytochrome regulation of expression of mrna encoding the major light-harvesting chlorophyll a/b-binding proteins of photosystem ii in the haploid phase ofadiantum capillus-veneris. plant and cell physiology 39: 647-654. de fossard ra. 1981. tissue culture for plant propagators. the university of new england. armidale, nsw. p. 10-11. dong l, xh su. 1993 .study on the spore propagation of the ostrich fern, matteuccia struthiopteris todaro. acta horticul.turae sinica 20: 274-278. fernandez h, am bertrand, i feito, r sancheztames. 1997. gametophyte culture in vitro and antheridiogen activity in blechnum spicant. plant cell tissue and organ culture 50: 71-74. goller k, jj rybczynski. 1995. in vitro culture used for woody fern cyathea australis (r.br.) domin vegetative propagation. acta societatis botanicorum poloniae 64: 13-17. khoo si, mb thomas. 1980. studies on the germination pf fern spores. the plant propagator 26: 11-15. kiss hg, jz kiss. 1998. spore germination in populations of schizaea pusilla from new jersey and nova scotia. international journal of plant sciences 159: 848-852. manickam vs, v irudayaraj. 1992. pteridophyte flora of the western ghats south india b. i. publications, new delhi p. 56-57. murashige t, f skoog. 1962. a revised medium for rapid growth and bioassays with tobacco tissue cultures. physiologia plantarum 15: 473-497 raghavan v .1993. chloroplast activities of dark-imbibed and photo induced spores of the fern onoclea-sensibilis. protoplasma 175: 75-84. turnwald s, r scheuerlein, m furuya .1999. phytochrome-dependent modulation and re-induction of growth of the first rhizoid in dryopteris paleacea sw. planta. 208: 98-106. wardle da, o zackrisson, mc nilsson. 1998. the charcoal effect in boreal forests: mechanisms and ecological consequences. oecologia 115: 419-426. 9 biotropia no. 5, 1991/1992: 41-52 histopathology of the telencephalon and diencephalon of t1lapia nilotica exposed to sublethal dose of malathion s-[1,2-di-(ethoxycarbonyl ethyl) dimethyl phosphorothiolothionate] edna a. amparado institute of biology, university of the philippines, diliman, quezon city, philippines abstract a 35-day exposure of tilapia nilotica embryos to sublethal doses of 3.0 ppm and 0.3 ppm malathion, s-[l,2-di-(ethoxycarbonyl ethyl) dimethyl phosphorothiolothionate], commercial grade, ec 57, produces cellular and ultrastructure changes in the brain. a number of nuclear centers of the treated animals are markedly larger than those of the control. aberrant features observed in day-45 embryos are the neoplastic masses and increased vascularization. ultrastructure defects include the presence of nuclear blebs, cytoplasmic vacuolations and increased lysosomal bodies. introduction the reliance of agriculture on pesticides to increase crop yield has resulted in the extensive and intensive use of these chemicals. since the ban on the use of the organochlorine, organophosphates have been used increasingly to control pest infestation. though neither as deadly nor as persistent as the organochlorines, organophosphates are potent inhibitors of acetylcholinesterase (rao et al. 1984; anthony 1986; antwi 1987). a number of studies have shown that these chemicals cause, too, morphopathologic effects. among these are vertebral malformations, parrot beak syndrome in fowl (van leewen et al. 1986), curved body, axis, blister formation and delayed melanogenesis in frog (pawar et al. 1983), proliferation of type ii pneumocytes, proliferation of interstitial cells and delayed septal and capillary development in lungs (koizumi et al. 1988), atresia of follicles (ansari et al. 1986), and demyelination and degeneration of axon (hoffman et al. 1984), disruption of oogenesis in pontius conchonus (kumar and pant 1988) and vacuolation of tubules and degeneration of glomeruli in glorias batrachus (mandal 1987). this study focuses on the histopathology of the telencephalon and diencephalon of tilapia nilotica, exposed to commercial grade malathion. 41 biotropia no. 5, 1991/1992 materials and methods a. chemical malathion s-[l,2-di-(ethoxycarbonyl ethyl) dimethyl phosphorothiolothionate], commercial grade, 57 ec, used in the study for this concentration is utilized in agriculture, poultry and livestock spray. it is a deep brow~n to yellow liquid, slightly soluble in water (145-147 ppm), mixable with many organic solvents including alcohol, esters ketones, ethers, aromatic and alkylated hydrocarbons and vegetable oils. it has a limited solubility in certain paraffin hydrocarbons. it has a boiling point of 156 157°c; melting point of 2.9°c; density of 1.23 g/cc. it is hydrolyzed at ph > 7.0 or < 5.0 but is stable in aqueous solution buffered to ph 5.26 (merck index 1983). b. collection and rearing of fry tilapia nilotica eggs, approximately 1000, were obtained from the bureau of fisheries and aquatic resources in tanay, rizal and allowed to hatch in finger bowls. the hatchlings were divided into control and experimental groups, each consisting of 300-350 larvae and reared in 1.0 x 1.0 x 1.5 m concrete ponds in dechlorinated tap water until day 45, post-fertilization. malathion, commercial grade, ec 57, diluted to sublethal doses of 0.3 ppm and 3.0 ppm served as pond water for the experimental group. pond water was changed every week, taking care to maintain the dose concentration in the experimental pond. histological studies a. paraffin series five fry from each group were harvested every three days until day 45, fixed in zenker's solution, dehydrated in alcohol series, sectioned serially at the brain region and stained in delafied's hematoxylin-eosin for light microscopic examination. the sizes of nuclear centers in each brain region at a particular developmental stage were measured using an ocular micrometer (fig. 1a and 1b). statistical analyses of the data were done by the use of two-way anova. 42 figure 1a. paraffin transverse section of themedial olfactory area of untreated, day-19, tilapia nilotica. the olfacto-somatic area (os) consisted of small neurons. x 400 figure 1b. paraffin transverse section of the thalamus of untreated, day-45. tilapia nilotica. the nucleus rotondus (nr) is a large nuclear center in the lateral region of the thalamus. x 400 b. resin sections specimens were decapitated and heads were fixed overnight at 20°c in 2.5% glutaraldehyde in sodium phosphate buffer, ph 7.2. brains were dissected and washed in millonig's buffer for 45 minutes and fixed for one hour at 20°c in 1% osmium tetroxide. after a 45-minute buffer wash, they were dehydrated through an acetone series, embedded in araldit (luft's araldit formula) at 40-50°c for 24 h each and at 60°c for 3 days. sections for light microscopy were cut at 0.5 um using the lkb ultramicrotome and stained with 1% toluidine blue. for electron microscopy, ultrathin sections, 100 um, were made with the same ultramicrotome, stained with 1% uranyl acetate, counterstained with lead citrate, and examined with joel, jem 100u electron microscope. 43 histopathology of the telencephalon and diencephalon edna a. amparado biotropia no. 5, 1991/1992 results light microscopy a. telencephalon the early histologic aberration which occurred among the free-swimming larvae, days 16-19, was the early differentiation of the olfacto-somatic area. unlike in the control group, this nuclear center of embryos exposed to 0.3 ppm malathion was composed of large polyploid neurons (fig. 2). at this stage, too, the olfactory bulb and a number of nuclear centers had larger sizes than those of the control group which size differences persisted until day 35 (table 1). thereafter, the nuclei of treated embryos showed two types of changes: i) increased areas of olfactory bulb and olfacto-somatic areas and ii) decreased areas of nucleus olfactorius pars bulbaris, nucleus pars commissuralis, preoptic nuclei and somatic area (table 1). day 45 specimens showed a number of histopathologic effects.the medial olfactory area of 3.0 ppm-dosed embryos showed hypertrophy and increased vascularization (fig. 3). table 1. comparative sizes (um) of the nuclear centers in the telencephalon of tilapia nilotica at specific developmental stage, post-fertilization 44 biotropia no. 5, 1991/1992 table 1. continued nuclear center 36-45 days a b c nucleus olfactorius 3505.30+138.80 2202.58+370.88 3341.79+462.10 pars bulbaris nucleus olfactorius 1428.92+29.10 1560.00+39.30 1992.25+ 58.40 pars commisuralis nucleus preopticus 375.63+26.40 333.29+38.40 258.56+27.50 olfacto-somatic area 190.85+17.50 121.59+11.10 181.25+269.00 somatic area 20927.40+591.40 19836.80+820.90 19396.40+835.80 nucleus taenia 843.60+77.80 618.87+40.60 819.26+67.30 table 2. comparative sizes (um2) of selected nuclear centers in the diencephalon of tilapia nilotica at specific developmental stage, post-fertilization 45 histopathology of the telencephalon and diencephalon edna a. amparado table 2. continued b. diencephalon larger nuclear centers than those of the control group were observed as early as day 16 and the disparity in sizes persisted until day 45, post-fertilization. table 2 shows the sizes (um) of selected diencephalic nuclei at specified developmental stage. during the late free-swimming stage, day 16-19, the epithalamic centers were larger than those of the control while other nuclear centers were smaller. on days 21-28, nuclear masses were much larger than those of the control. on days 36-45, a number of nuclear centers exhibited regression in sizes with the notable exception of hypophysis and nucleus geniculatus lateralis which showed steady increase in sizes. in the dorsal thalamus, the region lateral to the nucleus ventrolateralis thalami showed hypertrophy (fig. 4). 46 figure 2. paraffin transverse section of the medial olfactory area of day-19 tilapia nilotica exposed to 0.3 ppm malathion since day 10, post fertilization. the olfacto-somatic area (os) consisted of large polyploid neurons. x 400 figure 3. paraffin transverse section of the medial olfactory area of day-45 tilapia nilotica exposed to 3.0 ppm malathion since day 10, post fertilization. the brain showed hypertrophy (arrow) and increased vascularization (arrowhead). x 400 figure 4. paraffin transverse section of the thalamus of day-45 tilapia nilotica exposed to 3.0 ppm malathion since day 10, post fertilization. the lateral region showed hypertrophy (arrow). x400 47 histopathology of the telencephalon and diencephalon edna a. amparado biotropia no. 5, 1991/1992 electron microscopy ultrastructure defects were observed in the telencephalic and diencephalic neurons of pesticide-exposed fishes. the electromicrograph of normal neurons of t. nilotica in day-45 is shown in fig. 5. in both 0.3 and 3.0 ppm malathion exposed fish, the nucleus showed blebbing, ruffling and nucleolar disintegration (fig. 6). extensive vacuolations and lysosomal bodies were observed in the cytoplasm (fig. 7 and 8). discussion the present study describes the impact of chronic exposure to commercial grade malathion on the neurons of tilapia nilotica embryos. membrane deformations are similar to the necrosis in the brain of pacific herring larvae, clupea harenguipallasi, exposed to crude oil (cameron and smith 1980), larvae hatched from eggs that have been incubated in varying concentrations of zinc (somasundaram et al. 1984) and in paraclithys lethosigma exposed to mercury (trump et al. 1975) and mouse brain exposed to the neurotoxicant trimethyltin (chang et al. 1982). necrosis is attributed to the binding of the toxicant to the sh group in the neuronal membrane (trump et al. 1978), failure of the neuronal plasma membrane atpase system (chang et al. 1982) and interaction of the lipophilic pesticide with the cell membrane (triebskorn and kunast 1990), and increase in acetylcholine release that induced increased membrane permeability to sodium, potassium, and calcium ions (kabayashi et al. 1988). these authors contend that changes in fluid and electrolyte balance resulted in membrane damage. while these researchers categorized nuclear damage as effects caused by lethal concentrations, in this study, nuclear blebs are induced by sublethal concentrations. interestingly, these cytopathologic conditions are observed only on older embryos, day 45 to day 60 specimens. chronic exposure may have exhausted the defense mechanisms such as the depletion of energy resources needed to initiate protective processes (recio et al. 1988). other histopathologic effects include hypertrophy of a number of nuclear centers and increased vascularization. such aberrations were observed in the gill epithelium of teleosts exposed to acute doses of heavy metals such as zinc (khangarat 1982), cadmium (norgren et al. 1988), copper, silver and lead (simula 1988). similarly, thickening of the basement membrane of the digestive tract of the garden slug resulted from exposure to lethal dose of carbamate molluscide chloethocarb (triebskorn and kunast 1990). the observations that hypertrophy and increased vascularization were visible in day 45 embryos may indicate that prolonged exposure to sublethal concentrations eventually produce histologic aberrations induced by lethal exposure. these pathomorphologic changes may be due to prolonged 48 histopathology of the telencephalon and diencephalon edna a. amparado figure 5. electromicrograph of normal neurons in day-45 tilapia nilotica. the nuclear membrane is intact and the cytoplasm does not exhibit vacuolization. x 6000 49 biotropia no. 5, 1991/1992 figure 6. electromicrograph of neurons in day-45 tilapia nilotica exposed to 3.0 ppm malathion since day 10, post fertilization. neurons exhibited nuclear blebs (arrow), x 6000 figure 7. electromicrograph of neurons in day-45 tilapia nilotica exposed to 3.0 ppm malathion since day 10, post fertilization. cytoplasmic vacuolations were extensive in both the telencephalon and diencephalon. x 6000 figure 8. electromicrograph of neurons in day-45 tilapia nilotica exposed to 3.0 ppm malathion since day 10, post fertilization. lysosomal bodies were rampant in the cytoplasm, x 6000 50 histopathology of the telencephalon and diencephalon edna a. amparado deprivation of acethylcholinesterase which is required for cellular interactions in the maintenance of the nervous system structure (chase and kankel 1988). the findings that pesticides cause numerous neuronal pathology have repercussions on the reproductive functions and life span of tilapia nilotica. that a very low dose is capable of producing various neuron aberrations, evaluation on the intensive and extensive use of pesticides is an urgent necessity. references anthony, j., e. banister and p.c. oloffi. 1986. effects of sublethal levels of diazinon: histopathology of liver. bull. environ. contain. toxicol. 37: 501-507. antwi, l.a.k. 1987. fish head acetylcholinesterase after aerial application of tenephos in two rivers in burkina faso, west africa. bull. environ. contain. toxicol. 37: 501-507. chase, b.a. and d.r. kankel. 1988. on the role of normal acetylcholine metabolism in the formation and maintenance of the drosophila nervous system. dev. biol. 125: 361-380. chang, l.w., t.m. triemeyer, g.r. wenger, p.e. mcmillan, k.r. reuhl. 1982. neuropathology of trimethyltin intoxication. ii. electron microscopy study on the hippocampus. env. res. 29: 445-458. chang, l.w. 1983. neuropathology of trimethyltin intoxication iii. changes in the brain stem neurons. env. res. 30: 399-411. delpire, e., c. duchene, g. goessens and r. gilles. 1985. effects of osmotic shocks on the ultra structure of different tissues and cell types. ex. cell. res. 160: 106-116. khangarot, b.s. 1982. histopathological changes in the branchial apparatus of pontius sophore (hamilton) subjected to toxic doses of zinc. arch. hydrobiol. 93: 352-358. kumar, s. and s.c. pant. 1988. comparative sublethal ovarian pathology of some pesticides in the teleost. pontius conchonius hamilton. bull. environ. contain. toxicol. 41: 227-231. mandal, p.k. and a.k. kulshrestha. 1980. histopathological changes induced by the sublethal smithion in glorias batrachus l. indian j. exp. biol. 18: 547-552. norgren, l.k., p. runn, c. haux and l. forlin. 1985. cadmium induced changes in the gill morphology of zebrafish, brachydamio nerio (hamilton-buchanan) and rainbow trout, salmo gairdneri (richardson). j. fish biol. 27: 81-95. pant, j.c. and t. singh. 1983. inducement of metabolic dysfunction by carbamate and organophos phorous compounds in a fish, pontius conchonius. pestic. biochem. physiol. 20: 294-298. rao, d.m. and a.s. murthy. 1980. toxicity, biotransfortmation and elimination of endosulfan to the indian major carp, catla catla with special reference to some biochemical changes induced by the pesticide. pestic. biochem. physiol. 33: 220-229. recio, a., a. marjgomez, e. angulo and j. moya. 1988. zinc treatment of the digestive gland of the slug arion ater l. 2. sublethal effects at the histological level. bull. environ. contain. toxicol. 41: 865-871. sunila, i. 1988. acute histological responses of the gill of mussel, mytilus edulis to exposure by environmental pollutants. j. inverteb. pathol. 52: 137-141. 51 biotropia no. 5, 1991/1992 triebskorn, r. and c. kunast. 1990. ultrastructural changes in the digestive system of deroceras reticulatum (mollusca: gastropoda) induced by lethal and sublethal concentrations of the carbamate molluscicude cloethocarb. malacologia 32: 87-104. van leeuwen. c. j., t. helder and w. seinen. 1986. aquatic lexicological aspects of dithiocarbamates and related compounds. iv. teratogenicity and histopathology in rainbow trout (salmo gairdnerf). aq. toxicol. 9: 247-259. wester, p.w. and j.h. canton. 1986. histopathological study of oryzias latipes after long-term b-hexachlorocyclohexane exposure. aq. toxicol. 9: 21-45. 52 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf spatial pattern in beta diversity of echinoidea and asteroidea communities from the coastal area of tomia island, wakatobi marine national park, indonesia kangkuso analuddin , nasaruddin , andi septiana , wa ode sarliyana ,1 2 1 1 1, * agus nurlyati , wa masa nd saban rahim1 1 2a 1department of biology, faculty of mathematics and natural sciences, halu oleo university, kendari 93232 indonesia, 2the museum and research center of wallacea, halu oleo university, kendari 93232 indonesi, a received 15 december 2013/accepted 16 january 2015 abstract t e purpose of this was elucidate the spatial pattern in the beta diversity of marine benthic echinoidea h study to and asteroid that inhabit the coastal area of island wakatobi marine national park, indonesia wo ea tomia , . t transect placed lines of 460 and 260 m in length with small quadrats of 1 m were at the open and protected beaches 2 perpendicular to the coastlines. similarity ind ( )ly the importance value index and ex si of organisms on these taxa were calculated along each transect. was the most dominant echinoid at open and protected echinometra mathaei beaches, while was the most dominant asteroid both areas. most si values of echinoidea the protoreaster nodusus in at open beach were estimated less than 50%, which was lower than si values of organisms protected beach. to be at the on the other hand, most of si values of asteroidea at both areas were estimated more than 70% reflecting high degree of similarity of its species composition among sites. the dis imilarity index of organisms taxa of s in the echinoidea and asteroid increased significantly with increasing distance between sites, which suggested the ea that pattern in beta diversity of these taxa was associated with spatial heterogen ity e . keywords: asteroidea eta diversity, echinoidea, similarity ndex, wakatobi marine national park, b i introduction now days, biodiversity of marine organisms a is declin global scale (gatson 2000; roberts ing on et al. 2002), and therefore, attention more should be directed to conservation of marine biodiversity. many scientists use the term beta diversity as a key component of biodiversity survey (gray 2000). beta diversity defined as the changes in composition of organi ms or species diversity s between habitats (whittaker 1960 . beta diversity ) is known to provide useful information on marine area relationship or connectivity, which reflects the processes operating in those areas, s and ha been considered to be essential in environmental and conservation-based censuses and establishment of nature reserves (purvis & hector 2000; cleary 2003; tuomitso 2003; et al. koleff 2003). marine organisms play very et al. important roles of coastal for stabilizing ecology ecosystem (menge 1999), stabili inshore et al. zing environments (jie 2001), regulat et al. ing atmospheric processes (murphy duffus 1996), & providing forfood and pharmaceuticals human (hunt vincent 2006), as well as & functioning as recreational and aesthetic aspects (ponder et al. 2002). however, human activities such as over fishing (jackson 2008), bottom trawling and dredging (pauly 2005), as well pollution of et al. as coastal waters (halpern 2008) are known to et al. have negative impact on bent ic ecosystems h sustainability. wakatobi marine national park often called an underwater paradise is among the orld's most w popular marine parks. this marine park has very high resource potential, in terms of biodiversity* corresponding author : zanzarafli@gmail.com biotropia vol. 22 no. 1, 2015: 33 43 doi: 10.11598/btb.2015.22.1.355 33 mailto:zanzarafli@gmail.com biotropia vol. 22 no. 1, 2015 34 both species and uniqueness, and has become a leading t urist destination and focus of research o activities. however, most studies in this region have been done for ecological asse sment on coral s reef and fishes only, while few data are available s regarding ecological organization for marine be thic echinoidea and asteroidea, which are n fundamental for sustainable conservation of this marine park becau e of their significant role in the s coastal food chain. these taxa also have high aesthetic value for tourism. therefore, analyses of the spatial pattern in the beta diversity of these taxa may be useful for the potential ecotourism and conservation of biodiversity and the coastal environment of wakatobi marine national park, indonesia. echinoids are important food source for human aily exploitation of these organisms . d may threaten some species by over exploitation as well as habitat destruc ion. most people living at t the coastal area at the wakatobi marine national park capture fish and also some species of marine benthic organisms. therefore, understanding the spatial trends in beta diversity for echinoidea and aster idea taxa may help to preserve their habitat o and to ensure their future sustainability. many ecologists realize that good understanding of the marine organisms in relation to enviro mental n condition essential to generate effective is conservation schemes and guidelines for the sustainable exploitation of natural resources. in this effort determination of beta diversity of marine organisms plays an essential role (gaston 2000; lubchenco 2003; tuomitso 2003). et al. et al. understanding the distribution and complexity of benthic habitats will provide important information for management goals (kendall et al. 2005) and conservation strategy (fortin et al. 2005), while identifying habitat characteristics for particular species are being increasingly taken up by marine ecologists to describe patterns of benthic diversity (barrett 2001).et al. in the present study the spatial pattern in the , beta diversity of marine benthic organisms of echinoidea and asteroidea inhabiting the open and protected beaches of tomia island, wakatobi marine national park . he spatial was elucidated t trend in abundance, similarity and di similarity s index and ordination pattern of each taxon es according to their distribution across the seashore gradient . the spatial patterns of was described beta diversity on these taxa were elucidated in relation to environmental condition open and s at protected beaches and distance from the beach. to know whether distance between or sites patches affects the similarity index in each taxon, the relationship between distance and disimilarity index among sites . th analyses were analyzed ese were estimating performed by interval ordination from randomly selected paired , and then sites generating regression model between interval ordination and dissimilarity index. materials and methods study ites the present study was carried out along the coast of tomia island, wakatobi marine national park located at the 5 46' s and 123 55' e of 0 0 s province . .outheast sulawesi , indonesia (fig 1) the study was december 2010. conducted in meteorological data taken from kendari station showed that annual temperature range was c24-33 , while the minimum and maximum 0 temperature during study period were 24 c 0 c s ,and 32 , re pectively. annual rainfall was 2 000 0 mm at mountain, while it was 200 mm at the the coastal area. in addition, annual relative moisture ranged 75% to 84%, whereas from relative moisture was 76% at the study period with six days. the beach condition of tomia rainy island provides an excellent site for ecological study of marine invertebrates. the beach provides a unique panorama as it is surrounded by some small islands, which protect the beach from heavy wave action of banda ea, while other the s areas are exposed to direct heavy wave action. the substratum is composed of rock and sand. however, some species of seagrasses occur at the study site, such as cymodocea rotundata, cymodocea serulata, halophila minorenhalus acoroides and . spatial attern n eta iversity f ommunitiesp i b d o echinoidea and asteroidea c – et al.kangkuso sampling method the two transect lines were placed perpendicularly to the costal area of tomia island at the wakatobi marine national park. transect i with a length of 460 m was placed open beach at and divided into 13 sites or patches (400 m wide 2 each), while transect ii with length of 260 m was placed protected beach and divided into 7 sites at (400 m wide each). furthe more, five small r2 quadrats (1 m each) were placed purposively wide 2 in each site both open and protected beaches, at respectively. the name of species and their individual number for echinoidea and s asteroidea in each small quadrat were directly observed in the field using shirai (1997), while unknown species was brought to the aboratory l for identification. for determining the organic content, the sediment samples were taken from both transects and brought to the laboratory of ecology and taxonomy, halu oleo university, kendari. the sediment samples were air dried for seven days hen 10 g . t of the air-dried sediment samples were taken cand oven dried at 105 for 0 24 hours and weighed. the samples then dryashed c at 700 for 2 hours and weighed. the 0 percentage of organic matter content of sediment samples was estimated according to brower 1997et al. ( ). data analysis the similarity and dissimilarity indexes of organisms among s s for echinoidea and ite asteroidea both transects open and in at protected beaches were calculated using formula of bray and curtis (1957). in addition, ordination analyses were applied for elucidation the of multidimensional pattern. the spatial pattern in the beta diversity of each taxon was elucidated in figure 1. study sites of open and protected beaches ( ) in the coastal areas of tomia island, wakatobi marine national circled park, indonesia 35 protected beach open beach relation to the condition of open and protected beaches, distance from shore and substrate types of the beach. to know whether interval among sites affects the similarity for each taxon, the index relationship between distance among sites and di similarity each taxon . s index of were analyzed th analys s performed by estimating the ese e were interval ordination from randomly selected paired sites, and then regression model generated between ordination and dissimilarity for the index both taxa. the coefficient correlation of regression line was estimated, and a statistical test was done for determining whether there s any wa or no correlation between distance among sites and dis imilarity index for each taxon.s results and discussion the present study showed a little variation of organic matter content of sediment among habitats: 1 sandy areas with the seagrasses ) estimated at 14.62%; 2 rocky habitat at 14.43%; ) and 3 mixed rocky and sand habitat at 13.78%. ) however, diversity of the marine benthic for echinoid and asteroid communities inhabited the coastal area of tomia island, wakatobi marine national park varied at open and protected beaches. species of echinoids occurred on nine open beach, while eight species were found only at protected beach in the coastal area of tomia island (table 1) . was the dominant echinoid echinometra mathaei both open and protected beaches of tomia at island, indicating its high important role for susta nability of coastal ecosystem. on the other i hand, was the rarest echinoid in diadema setosum this coastal area, which might be less adaptable with environmental condition (fig 2) . . meanwhile, few aster ds species were found oi in this coastal area with only four species on both open and protected beaches (table 2). protoreaster nodusus was the dominant steroid a in both areas, while was the rarest archaster typicus a .steroid (fig 3). differences in species compostion of marine benthic at present study might be determined by habitat characteristics and organic matter content. beaman (2005) et al. found that habitat characteristic affect marine ed benthic composition. rocky reef habitats influence the behavior of benthic organisms, d and edin turn influenc higher level processes of population dynamics and community structure (knight morris 1996). sandy habitats & are important for burrowing organisms and their predators and provide foraging areas for species which may also use nearby firm substrate areas for sheltering purposes (ross 2007). et al. therefore, spatial pattern in the community composition of echinoidea and asteroidea might be associated with substrate types at small patches in particular the coastal area of tomia island. biotropia vol. 22 no. 1, 2015 table 1 species composition of open and protected beaches in the coastal area of tomia island, wakatobi . echinoidea at marine national park, indonesia no. family species open beach (transect i) protected beach (transect ii) 1 echinometridae echinostrephus aciculatus echinometra mathaei echinometra sp. a echinometra sp. b 2 temnopleuridae salmacis sphaeroides temnopleurus alexandrii 3 diadematidae astropyga radiate diadema setosum echinotrix calamaris 4 toxopneustidae toxopneustes pilieolus tripneustes gratilla 5 brissidae brissus latecarinatus note (present), (absent)s : √ 36 the similarity indexes of echinoid community both open and protected beaches are described at in tables 3 and 4, respectively. as shown in table 3 the similarity indexes of echinoids varied among sites, while three values of imilarity ndexes (si) were estimated more s i than 85%, i.e. the si values of paired sites i-ii, vvii and of paired sites i-xii. however, twentythree si values paired sites of echinoids were of estimated more than 50%, while more than half of the si values of echinoids were less than 50%, even five si values paired sites were zero. these of results suggested that there was a lower degree of similarity species of echinoids among sites at open beaches of tomia island. on the other hand, table 4 shows that more than half the si values of of echino ds protected beaches were estimated i at to be more than 50%, indicating a high degree of similarity among sites for echinoids protected at beaches, although two si values were estimated as zero or completely different. these trends demonstrate the change in the echino ds species i composition across open beaches, which might be due to heavy wave action. there was only small change in species composition of echinoids across protected beach from heavy wave action. 0 50 100 150 200 0 50 100 150 200 250 300 protected beach echinometra mathaei echinometra sp a echinotrix calamaris echinostrephus aciculatus temnopleurus alexandrii echinometra sp b brissus latecarinatus diadema setosum distance from the land (m) figure 2. spatial trends in the mportance alue ndex ivi of echin idea community at open beach (upper) and protected i v i ( ) o beach (below) in the coastal area of tomia island, wakatobi marine national park, indonesia table 2 species composition of astero open and protected beaches in the coastal area of tomia island, wakatobi . idea at marine national park, indonesia no. family species open beach (transect i) protected beach (transect ii) 1 ophidlasteridae protoreaster nodusus linckia laevigata 2 oreasteridae culcita novaeguineae 3 archasteridae archaster typicus 4 astropectenidae astropecten scoparius note (present), (absent)s : √ 37 spatial attern n eta iversity f ommunitiesp i b d o echinoidea and asteroidea c – et al.kangkuso biotropia vol. 22 no. 1, 2015 0 50 100 150 200 0 50 100 150 200 250 300 350 400 open beach linckia laevigata protoreaster nodusus archaster typicus culcita novaeguineae distance from the land (m) 0 50 100 150 200 0 50 100 150 200 250 300 protected beach linckia laevigata protoreaster nodusus archaster typicus astropecten scoparius distance from the land (m) figure 3. spatial trends in the mportance alue ndex ivi of asteroidea community at open beach (upper) and protected i v i ( ) beach (below) in the coastal area of tomia island, wakatobi marine national park, indonesia table 3. similarity and i similarity of chinoids open beaches in the coastal area of tomia island, wakatobi d s indexes e at marine national park, indonesia si\di i ii iii iv v vi vii viii ix x xi xii xiii 1 12.5 61.46 28.27 62.57 56.25 53.33 46.96 100 54.58 70.83 62.5 14.89 2 87.5 61.46 21.19 67.98 61.67 58.75 52.38 100 60 70.83 50 26.36 3 38.55 38.54 66.47 61.46 58,33 61.46 53.91 67.71 38.55 67.71 61.46 52.78 4 71.73 78.81 33.53 46.79 44.44 37.55 38.10 100 63.41 84.52 50 41.8 5 37.43 32.05 38.54 53.20 36.23 13.44 42.58 63.19 57.78 89.01 47.83 62.23 6 43.75 38.33 41.67 55.56 63.77 47.22 25.24 63.89 51.11 61.11 58.33 55.91 7 46.67 41.25 38.55 62.45 86.56 52.78 53.57 76.62 57.78 100 49.58 57.27 8 53.04 47.62 46.09 61.91 57.42 74.77 46.43 75.71 49.60 58.57 70.71 46.62 9 0 0 32.29 0 36.81 36.11 23.38 24.29 63.33 45.83 55 100 10 45.42 40 61.46 36.59 42.22 48.89 42.22 50.40 36.67 57.78 78.89 39.7 11 29.17 29.17 32.29 15.48 10.99 38.89 0 41.43 54.17 42.22 79.17 70.83 12 37.5 50 38.54 50 52.17 41.67 50,42 29.29 45 21.11 20.83 76.36 13 85.11 73.65 47.22 58.20 37.77 44.09 42.73 53.38 0 60.30 29.17 23.65 38 s indexes ( )table 5 hows the imilarity si for a atsteroids open beaches. the si values of asteroids were less variable with only a few si values of steroids estimated less than 50%, a to be while many si values were estimated more to be than 80%, and even some si values estimated to be indicatednearly 100%. these results that there was high similarity of species composition of a atsteroids open beaches of tomia island. a similar trend in the si values of steroids was a found protected beaches.at as shown in table 6 there only three si is values of steroids protected beaches were a at estimated less than 50%, while more than to be half of si values of steroidea were estimated a to be above 70%, indicating the high degree of similarity in species of steroids protected a at beaches of tomia island. multidimensional patterns of the benthic community of chinoids and steroids at the e a study site were elucidated by ordination analyses. the ordination pattern of chinoids at open e beach (fig 4, upper) showed four groups: 1) . group a consisted of four sites or patches including sites i, ii, iv and sites xiii; 2) group b consisted of five sites (sites iii, vi, viii, x and sites xii); 3) group c consisted of sites v and vii; and 4) group d consisted of sites ix and xi. the sites located at the same groups indicated that chinoids might have e species similar or matching resource requirement and enviro mental preferences. the ordination n pattern of chinoids along transect ii or e protected beaches (fig , below) could be also . 4 divided into 4 groups, though the sites in each group differed as compared to the ordination pattern of chinoids open beaches. these e at wefour groups re as follows: 1) group a consisted of sites iii and v; 2) group b consisted of sites i, ii, and iv; 3) group c consisted of site viii only; and 4) group d included sites vi and vii. table 4. similarity and is imilarity of chinoids protected beaches in the coastal area of tomia island, d s indexes e at wakatobi marine national park, indonesia si \ di i ii iii iv v vi vii viii 1 24.78 47.69 36.52 59,83 47.81 35.31 76.97 2 75.22 46.43 11.74 58,57 35.42 35.42 64.58 3 52.31 53.57 46.43 24,29 58.57 58.57 70.71 4 63.48 88.26 53.57 58,57 43.18 43.18 58.33 5 40.17 41.43 75.71 41.43 58.57 58.57 82.86 6 52.19 64.58 41.43 56.82 41.43 20.83 100 7 64.69 64.58 41.43 56.82 41.43 79.17 100 8 23.03 35.42 29.29 41.67 17.14 0 0 table 5. similarity and is imilarity of steroids open beaches in the coastal area of tomia island, wakatobi d s indexes a at marine national park, indonesia si \ di i ii iii iv v vi vii viii ix x xi xii 1 22.5 24.28 34.16 14.16 14.16 20.83 18.33 22.12 18.75 36.66 36.67 2 77.5 19.82 25.41 22.5 22.5 25.41 38.75 24.20 27.5 38.75 38.75 3 75.71 80.17 7.38 36.07 36.07 37.73 24.28 44.02 33.57 33.57 38.75 4 65.83 74.58 92.61 19.16 19.16 20.83 18.33 27.12 18.75 33.57 38.75 5 85.83 77.5 63.92 80.83 19.16 20.83 18.33 27.12 18.75 41.66 41.67 6 85.83 77.5 63.92 80.83 100 20.83 32.5 85.45 21.25 31.11 22.5 7 79.16 74.58 62.26 79.16 79.16 79.16 32.5 19.09 21.25 31.11 22.5 8 81.66 61.25 75.71 81.66 67.5 67.5 84.16 8.03 4.166 20.83 100 9 77.87 75.79 55.97 72.87 14.54 80.90 91.96 77.87 11.66 14.54 38.75 10 81.25 72.5 66.42 81.25 78.75 78.75 95.83 88.33 89.54 14.54 17.36 11 63.33 61.25 66.42 58.33 68.88 68.88 79.16 63.33 85.45 75 27.5 12 63.33 61.25 61.25 58.33 77.5 77.5 74.58 61.25 82.61 72.5 85.13 39 spatial attern n eta iversity f ommunitiesp i b d o echinoidea and asteroidea c – et al.kangkuso biotropia vol. 22 no. 1, 2015 table 6. similarity and i similarity of steroids protected beaches in the coastal area of tomia island, wakatobi d s indexes a at marine national park, indonesia si \ di 1 2 3 4 5 6 7 1 57.38 45 40 31.66 55.53 58.33 2 42.61 20.79 24.28 26.07 17.14 6.19 3 55 79.20 44.72 36.08586 36.08 15.55 4 60 75.71 55.27 50 40 29.16 5 68.33 73.92 86.66 50 41.36 26.67 6 44.46 82.85 63.91 60 58.63 23.33 7 41.66 93.80 84.44 70.83 73.33 76.66 figure 4. ordination pattern of community at open beach ( ) and protected beach ( ) in the coastal area of echinoidea left right tomia katobi island, wa marine national park, indonesia statistical analysis regression model using d s(fig. 5) showed that the i similarity indexes (di) of echinoids among sites both open and at protected beaches increased significantly with increasing interval ordination or separation of sites or patches ( < 0.001). this means that the p degree of similarity of echinoids both open at and protected beaches decreased with increasing distance sites.among the ordination pattern of asteroids on open beach (fig. 6) could also divided into 4 group: (1) group a consisted of site xii only; (2) group b consisted of sites iii, iv, v, vi, vii, ix and site xi; (3) group c included sites i and ii; and (4) group d consisted of site viii only. the same ordination pattern of asteroids was found at the transect ii (figure 6), although the sites in esch froup were different as compared to the grouping in the transect i. these groups at transect ii are: (1) group a contained sites, ii, iv and vi; (2) group b consisted of site iv only; (3) group c contained site i only; and (4) group d included sites iii and v. statistical analysis a regression model using d i(fig. 7) showed that issimilarity ndexes (di) atof asteroids among sites both open and protected beaches also increased significantly with increasing interval ordination or separation of sites ( < 0.01). this mean that the degree of p s similarity of asteroids among sites open and at protected beaches decreased with increasing distance sites both open and protected among at beaches. these spatial heterogeneities might be correlated to the environmental condition of the beach, such as substrate types and distance from the offshore. this because the happened echinoids and asteroids communites showed high similarity among sites in general both open at and protected beaches. the degree of similarity in some sites for both taxa were completely different although they responded to the same sets of substrate types, which might influence the community structure of marine benthic community in the tomia island. the community composition of echinoids and asteroids in each site might be significantly associated with substrate types in small patches. for species that have limited dispersal abilities, distance or spatial component expected to be is 40 0 20 40 60 80 100 0 20 40 60 80 100 open beach interval ordination 0 20 40 60 80 100 0 20 40 60 80 100 protected beach interval ordination figure 5 relationship of i o tion to the i similarity ndex of echinoidea community at the oastal area of . nterval rdina d s i c tomia island, indonesia figure 6 ordination pattern of asteroidea community at open beach ( ) and protected beach ( ) in the coastal area of . left right tomia island, wakatobi marine national park, indonesia 41 spatial attern n eta iversity f ommunitiesp i b d o echinoidea and asteroidea c – et al.kangkuso important structuring factor in community similarity, which would result in assemblages of sites close together being more similar which are than assemblages further of those which are apart. many previous studies revealed that distance between sites can be a function of differences in spatially explicit environmental variables (borcard . 1992; harrison . 1992; et al et al ohmann spies 1998). the present result clearly & found a significant association between distance of sites and community similarity for echinoids and asteroids both at open and protected beaches, because they showed a highly significant relationship between interval ordination and di similarity indexes for those two taxa. many s previous studies that pattern in beta indicated diversity can differ among taxa due to communitywide differences in dispersal ability and other taxon-specific factor (gatson 2000; reyers . et al 2000). beta diversity of organisms in many taxa ha been affected by not only distance between s sample stands but also enviro mental variables n (ellingsen gray 2002; clearly . 2004). in & et al addition, barros . (2004) found that et al sedimentary bedforms such as ripples, sand and waves are known to affect the abundance and distribution of benthic organisms. abiotic factors such as human disturb nce might be factor the individual number, a reducing and even the species number of these benthic communities. it is known that sea urchins are frequently collected by people for consumption, which might have strong effects on their abundance and distribution. on the other hand, open beach protected beach 42 biotropia vol. 22 no. 1, 2015 interaction wit in benthic community itself s h become biological control for the community stability. meanwhile, steroids act as predator for a s sea urchin in general, and might reduce the number of sea urchin in the coastal of tomia island. the association with distance itself may also be due to distance-dependent environmental variable. several studies showed that the distribution of chinoderms french polynesia e at beach was primarily influenced by the concentration of carbonates (adjeroud 1997). however, ellingsen and gray (2002) also found in a norwegian coastal area that sea urchins were , more restricted in their distribution than other macrobenthos, and the beta-diversity of echinoderms had the weakest association with environmental variables of all the observed taxa. conclusions spatial patterns of the benthic community of echinoidea and asteroidea that inhabit the coastal area of tomia island, wakatobi marine national park varied habitat. ultidimensional pattern by m of each taxon created four groups in general , although the sites or patches in each group were quite different. the trend in beta diversity of echinoidea and asteroidea seem toed be associated with spatial heterogen ity because e there was a significant correlation between separation of sites and community similarity of benthic community. this information is fundamental for conservation wit in the h wakatobi marine national park. acknowledgements edwe thank halu oleo university for financial support this research. the for we also thanked staff of wakatobi marine national park for coopera-tion and support our . we of field work would also like to thank the student and general s volunteers who had provided assistance during the field work. references adjeroud m 1997 factors influencing spatial patterns on . . coral reefs around moorea, french polynesia. mar ecol progr ser 159: 105 19. beaman rj, daniell jj, harris pt 2005 geology-benthos . . relationships on a temperate rocky bank, eastern bass strait, australia. mar fresh res 56 (7) 943-58. : barros f, underwood aj, archambault p 2004 the . . influence of troughs and crests of ripple marks on the structure of subtidal benthic assemblages around rocky reefs. estuar coast shelf sci 60 (4) : 781-90. barrett n, sanderson jc, lawler m, halley v, jordan a. 2001 mapping of inshore marine habitats in south . eastern tasmania for marine protected area planning and marine management. technical report. 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was conducted at situ bagendit lake, garut, west java. a release and recapture method was employed to study the mode and rate of dispersal of the weevils under field conditions. regular sample collection at two-month interval was done to evaluate the pattern of distribution and to assess the age structure under field condition. another two months regular observation was done to assess damage severity due to adult weevils and the fungus, a. eichhorniae, on water hyacinth. results showed, that the weevils seem to disperse actively to all directions following the presence of water hyacinth. the data also showed that the dispersal rate of the weevils was about a few meters a week. under field conditions at situ bagendit lake, the weevils were about evenly distributed throughout water hyacinth mass. the density of the weevils fluctuated from time to time, but the trend slightly increased. the survival rate of the weevils at situ bagendit lake was estimated not more than 5%. it was suspected that various limiting factors such as various predators have caused the low population increase under field conditions. the population increase through time confirmed that the weevils have established at situ bagendit lake. field data showed that there were no interaction between the damage severity of the weed caused by both weevils and the fungus. introduction water hyacinth, eichhornia crassipes (mart.) solms laubach (pontederiaceae), is one of the most important aquatic weed and causes various serious problems in indonesian water bodies as well as in some other tropical and subtropical regions of southeast asia. various efforts to control water hyacinth have been attempted including the use of biotic agents. biotrop initiated researches on biological control of water hyacinth by conducting a seminar on aquatic weed management in 1974 followed by introducing water hyacinth weevil, neochetina eichhorniae warner (coleoptera : curculionidae) from florida in 1975. aside from n. eichhorniae, biotrop in cooperation with aciar and csiro has also imported in 1994 another weevil species, i.e., neochetina bruchi hustache (coleoptera : curculionidae) for the same purpose. both water hyacinth weevils have also been tested and released in the fields of many countries including indonesia where this weed has been introduced to those countries (julien and griffith 1998; barley 1990). 18 biotropia no. 17, 2001 research activities on biological control of water hyacinth have been conducted in many countries, generally using insects, fungi and fish as biological control agents (harley 1990). the significant role of neochetina spp. as biocontrol agent of water hyacinth has been reported from different countries such as queensland (wright 1979; room 1986), sudan (bashir 1984), bangalore (jayanth 1988) and louisiania (goyer and stark 1984). other studies showed that better control of water hyacinth may be achieved by the integrated use of water hyacinth weevils with other biocontrol agents such as fungi (saraswati 1980; charudattan 1986; galbraith 1987; gaunter and mohamed 1992). the release of n. eichhorniae has been conducted at some release sites in indonesia but after some time only partial control of water hyacinth has been achieved. for experimental purposes n. bruchi has been released at situ bagendit lake in august 1996 and october 1996 consisting of 100 and 125 pairs, respectively. further releases in the same site were done in september 1998, december 1998, january 1999, august 1999, december 1999 consisting of 265, 600, 262, 307, 700 adults ofn. bruchi and n. eichhorniae. another biological control agent is a fungus, alternaria eichhorniae nag raj & ponappa (hyphomycetes), found in the lake, but its role has never been recorded. the role of the fungi to control water hyacinth at experimental scale combined with n. bruchi resulted in a more serious damage. the aims of the study were : to study the distribution pattern of neochetina spp. at the release site and the composition amongst the developmental stages of the weevils; to evaluate the establishment of neochetina spp. at the release site; and to study the relationship between water hyacinth damage severity due to neochetina spp. and a. eichhorniae at the release site. materials and methods mode and rate of dispersal and distribution of the water hyacinth weevils under open field the release of the weevils was done at situ bagendit lake, garut regency. the water body of the lake was originally 124 ha, but when the research was carried out the estimated water body varied from 30 ha (in dry season) to 50 ha (in rainy season). there were few blocks of water hyacinth mats. the biggest water hyacinth mat was about 0.5 ha on which the experiment was carried out. the aim of this experiment was to evaluate the mode, dispersal rate and distribution pattern of the weevils at the release site. a total of 307 adults of neochetina spp. marked with spotted white color on their elytra consisting of 174 males and 133 females were released in the center of situ bagendit lake as the first release. three months later, the second release consisted of 350 marked adult males and 350 marked adult females. fortnight observation was conducted using transect method. from the release point, transects 19 establishment of neochetina spp. kasno et ai were drawn to the four cardinal directions, and hence the presence of adult weevils marked with spotted white color was observed. the parameters observed were distance from the release point and the number of adults at 1 m2 area. age structure of water hyacinth weevils in the field the structure of developmental stages or the age structure of neochetina spp. was determined at situ bagendit lake. the aim of this experiment was to assess the age structure of those weevils at the release site. ten units of water hyacinth samples measuring 50 x 50 cm each were collected for further examination on the number of eggs, larvae, pupae and adults. each sample was taken from the systematic points of diagonal imaginary lines crossing the compact floating water hyacinth mat. regular observations at monthly interval were conducted starting from june 1999 until the end of january 2000. the experiment was also conducted to prove the hypotheses that there were predaceous fish affecting the survival trend of the weevils under field conditions of the lake. floating screen cages, measuring 125 x 175 x 75 cm each, were used as unit plot of the experiment. each cage contained water hyacinth plants covering about 0.25 % of the water surface. each cage was provided with 2 pairs of adult weevils, 23 eggs and 25 larvae. there were 10 cages split into two groups. group 1 was equipped with screen net on the root areas under water surface in such a way that the net did not affect the root development, while group ii was not. the plots were arranged in a randomized layout into two rows. the space between the rows was about 1 m and space between cages in each row was about 0.5 m. observations were done on the number of adult weevils and pupae in each cage on the 60* and 120th day after setting. relationship between the attacks of water hyacinth weevils and leaf blight using the same collected samples given above, the damage severity of each sample unit was assessed by estimating the area of the leaves attacked by the weevils and the fungus. the collected data were then analyzed on its correlation. results and discussion mode and rate of dispersal and distribution of the water hyacinth weevils under open field condition when an exotic biotic control agent of a weed is released in an open field, they are free to express their true behavior. the release using adult weevils, both n. eichhorniae and m bruchi, instead of other developmental stages is due to the fact that adult weevils are the most suitable as well as practical in handling. 20 biotropia no. 17, 2001 if the phytophagous insects are collected from the field, and cultured under artificial condition in the laboratory, they are generally to some extent, suffering from stress. culturing insects under such condition for a longer period may reduce their vitality, the longer the worse. how many generations of insects suffering a significant reduction in its vitality vary from species to species (donnelly 1997). adult weevils of either n. eichhorniae or tv. bruchi to be released in an open field were originated from outdoor screen cage cultures. in the outdoor screen cage, the weevils were expected to start adapting to the new environment. such treatments were intended to provide opportunity to recover the loss of vitality, because good quality is more important than quantity of a weed biotic control agent (julien et al. 1999) . as soon as the weevils were released in the open field, they might express whatever they like such as feeding on the preferred available host plants, breeding and spreading naturally. it was expected that soon after the release the adult weevils would not move far away from the release point and settle far from each other. if they disperse far from each other it may reduce the opportunity to meet each other for a mating process. data on the dispersal of released weevils collected from the experimental release site and recapture showed that the adult weevils spread through all cardinal directions (figure 1). figure 1. distribution of adult n. eichhorniae and n. bntchi after 2 (*) and 4 (o) weeks from the release time at situ bagendit lake 21 establishment of neochetina spp. kasnoe/a/. data from the first release of 307 marked weevils showed that two weeks after release the weevils dispersed to all cardinal directions with various distances. data collected on the 4th week after release showed a similar pattern but with longer distance. the longest distance on most cardinal direction almost reached the border of the water hyacinth mat within four weeks after release. outside of the water hyacinth mat, one of the border areas was swampy but with no water hyacinth, the two other borders were grassland and agricultural land and the rest of the borders (west and north) was the open water surface of the lake. with the assumption that the rate of dispersal from the release point was about the same, if the area of water hyacinth mat was larger, it is estimated that the adult weevils would have dispersed about 15 m within 4 weeks. besides, the limited size of the water hyacinth mat prevented spotting of the marked adult weevils. therefore, the plan to conduct observation on the 6th week was not carried out. the release of 700 marked adult weevils showed similar results. however, some parts of the water hyacinth mat on the western and northern parts were moved away by the wind before the first and second observation, respectively. therefore, there was no data representing weevil's distribution on western and northern parts from the release point. within a fortnight they were observed to move several meters away from the release point farther from the original site. it seemed that the adult weevils moved by crawling from plant to plant amongst the connected plants instead of flying. probably they could fly to move to disconnected water hyacinth plants. the data indicated that within four weeks after release, the adult weevils spread unevenly. the presence of young leaves which started to open from folded stage probably attracted the weevils. the young leaf is softer than the older , because the nitrogen content of young leaf is much higher than the older leaf (center and wright 1991). the difference in nutrient content was suspected to be one of the reasons of feeding preference. young leaf stage was most preferred by the adults during the day. after about two and a half years from the release of the first batch in the situ bagendit lake, adult feeding scars on the leaves could be found on most of water hyacinth plants growing on almost all parts of the lake. generally, adult weevils prefer to feed on young laminas which have just started to open from closed stage. assuming that water hyacinth suffered from adult feeding scars, the weevils have spread almost all over the water hyacinth area. it seems that within a few weeks after the release most of the adult weevils were still confined in one area where the distance between their standing position is relatively close. with such position, it is believed that the possibility of mating is still high. therefore, releasing water hyacinth weevils in a new environment by liberating few hundred adults of mixed sex at one site is safe. age structure of the water hyacinth weevils in the field after releasing in an open field the weevils have to adapt to the local conditions as well as to encounter various limiting factors. the ability to adapt to the 22 biotropia no. 17, 2001 new environment may be shown by some indicators such as the presence of damage symptom as the result of feeding activity, the presence of various developmental stages, the sustainable generations, and the most important indicator is the increased population density through time. monthly interval sample collection was carried out to evaluate the age structure of the weevils at the rdease site. it was indicated that the weevils have spread to most water hyacinth plants in the lake. figure 2 shows the population trend of each developmental stage of the weevils under field conditions. a trend of increase through time was shown by adult and a slight increase was also shown later by the pupal stages. figure 2. population trend of each developmental stage of neochetina spp. from june 1998-february 1999 in situ bagendit lake the number of adults was mostly higher than the pupae because there was an accumulation of the survived adult of the overlapped generations as the average longevity of the adult was much longer than the average developmental period of both pupa and larva. it was questioned why the increase in the number of adults in september was not followed by the increase in the number of both eggs and larvae. the trend of the number of larvae from month to month was more or less stable which might be an indicator of the limit of carrying capacity of water hyacinth plant in the lake. the decreasing trend from the number of eggs to larvae and pupae is shown in figure 3. such curve may provide information on the age structure and the survival rate of the weevils at the release site. about 5 % of eggs could survive up to the 23 establishment of neochetina spp. kasno et al. pupal stage and less than 5 % may survive up to the adult stage. this estimated survival rate was much lower than the reproductive potential determined under laboratory condition. tjitrosoedirdjo et al. (1999) reported that the potency of weevil reproduction could reach about 50%. figure 3. structure of developmental stages ofneochetina spp. at situ bagendit lake. the difference between potential survival rate of the weevils in the laboratory and the fact in the field may be attributed to various limiting factors resulting in low survival rate. it was suspected that some predaceous fish could cause the low survival rate of the weevils in the situ bagendit lake. an experiment was then set up to proof the possible attack from predaceous fish. the results are presented in table 1. table 1. average number of pupal and adults stages of water hyacinth weevils and damage severity under simulation to test the role of predators in inhibiting the population increase 24 biotropia no. 17, 2001 table 1 indicates that the number of both pupae and adult weevils under screen cages equipped with screen net on its base after two and three and a half months were higher than the cages without screen net on the base. the cages which were equipped with screen net prevented from possible predation of the weevils. unpublished data collected from a simulation experiment showed that various fish may consume root-bearing pupae under laboratory conditions. there were various fish such as common carp (cyprinus carpio), tilapia, oreochromis mosambicus, cat fish, found in situ bagendit lake. spiders were reported to predate and prey on tv. bruchi larvae (tjitrosoedirdjo 1998). at situ bagendit lake, spiders were often found, so they might be one of the natural enemies of water hyacinth weevils. the slow population increase of the weevils at the release site may lead to a conclusion that neochetina spp. are not effective control agents. it was reported that effectiveness of an biotic control agent may be affected by various factors such as by climate (jayanth and visalakshy 1990; grodowitz et.al. 1991). although the population increase in situ bagendit lake was very low, the increasing trend during the observation period confirmed that neochetina weevils have established in the lake. therefore, additional releases of same biological control agents are not necessary. relationship between the attacks of water hyacinth weevils and fungus a plant pathogenic fungus may disperse from an initial point of infection to other host plants through a direct contact with the infected parts. aside of that, spores and mycelia may be transferred to other host plants by carrying agents. an adult weevil of neochetina may act as a carrying agent of the spores and mycelia of the fungus, a. eichhorniae. a monthly interval observation carried out to assess the damage severity of water hyacinth plants by each biological control agent in situ bagendit lake is presented in table 2. it was obvious that there was no interaction between the attack of adult weevils and the fungus. data obtained from laboratory experiment showed that a higher damage severity of water hyacinth due to both biotic control agents caused a more serious damage (kasno et al. 1998). probably the level of both weevil populations and the fungus colony were not high enough to cause such speculated effect. according to kasno et al. (1998) various fish such as tilapia and common carp at the feeding test showed that those fish predated on pupae which were in the roots of water hyacinth. aside from fish, frogs and eel fish might also attack the weevils. under the simulated experiment, the weevil's elytra were found in the crop of the frog. at situ bagendit lake, spiders were often found, so they might be the natural enemies of the weevils. according to tjitrosoedirdjo (1998) spiders were able to catch and prey on n. bruchi larvae. 25 establishment of neochetina spp. kasnoetal. correlation between the damage severity of water hyacinth due to the weevils and fungus the percentage of weevil attack and the number of feeding scars on the second youngest leaf of water hyacinth is shown in table 2 and appendix 1. the attack level of the pathogenic fungi a. eichhorniae and the weevil neochetina spp. at situ bagendit lake did not show significant correlation in causing damage to water hyacinth, because the attack was relatively low. table 2. correlation between damage severity of water hyacinth plants due to the attacks of weevils and fungus at situ bagendit lake by the year 1999 2000 note: figures followed by the same letter in the same column are not significantly different according to duncan multiple range test at 95% confidence level. conclusions in the spatial distribution, adult of water hyacinth weevils actively dispersed in all cardinal directions following the presence of water hyacinth. the distribution pattern of the adults of water hyacinth weevils was initially contagious or clumped, but later on they tended to spread uniformly. neochetina. bruchi and n. eichhorniae released at situ bagendit lake since 1996 have become widely established. the survival rate of water hyacinth weevils at situ bagendit lake being estimated by the age structure showed a much lower rate than its reproduction potential found in the laboratory. the damage severity of water hyacinth plants due to a. eichhorniae and neochetina spp. at situ bagendit lake was not significantly correlated. 26 biotropia no. 17, 2001 references bashir, m.o. 1984. the establishment and distribution of natural enemies of waterhyacinth released in sudan, short communication. tropical pest management 30(3):320-323. caunter, i.g. and s. mohamed. 1992. effect ofneochetina eichhorniae on waterhyacinth in malaysia and its interaction with myrothecium roridum. in: proc. of the 3rd international conference on plant protection in the tropics (eds. p.a.c. ooi. g.s. lim and p.s. teng) 6:261-264. mapps, kuala lumpur, malaysia. center, t.d., steward, k.k., and m.c. bruner. 1982. control of waterhyacinth (eichhorniae crassipes) with neochetina eichhorniae (coleoptera:curculionidae) and a growth retardant. weed science 30(5):453-457. center, t.d. and a.d. wright. 1991. age and phytochemical composition of water hyacinth (pontederiaceae) leaves determine their acceptability to neochetina eichhorniae (coleoptera: curculionidae). j. environ. entomol. 20(l):323-334. charudattan, r. 1986. integrated control of waterhyacinth (eichhorniae crassipes) with a pathogen, insects, and herbicides. weed science 34 (suppl.) 1):26-30. donnelly, g. 1997. mass rearing insects for biological control of weeds. in biological control of weeds: theory and practical application. m. julien and g. white (eds). aciar monograph no 49. canberra: 89-96 galbraith, j.c. 1987. the pathogenicity of an australian isolate of acremonium zonatum to waterhyacinth, and its relationship with the biological control agent, neochetina eichhorniae. australian journal of agricultural research 38:219-229. goyer, r.a. and j.d. stark. 1984. the impact of neochetina eichhorniae on waterhyacinth in southern louisiana. journal of aquatic plant management 22:57-61. grodowitz, m.j., r.m. steward and a.f. confrancesco. 1991. population dynamics of waterhyacinth and the biological control agent neochetina eichhorniae (coleoptera: curculionidae) at a southern texas location. environmental entomology 20(2):652-660. harley, k.l.s. 1990. the role of biological control in the management of water hyacinth, eichhornia crassipes. biocontrol news and information 11(1): 11-22 jayanth, k.p. 1988. successful biological control of waterhyacinth (eichhorniae crassipes) by neochetina eichhorniae (coleoptera: curculionidae) in bangalore, india. tropical pest management. 34(3):262266. jayanth, k.p. and p.n.g. visalakshy. 1990. studies on drought tolerance in water hyacinth weevils neochetina eichhorniae and n. bruchi (coleoptera: curculionidae). j. biological control 4(2): 116119. julien, m.h and w.w. griffith. 1998. biological control of weeds: a world catalogue of agents and their target weeds. 4th ed. cabi, uk. 223p. julien, m., w.w. griffith and a.d. wright. 1999. biological control of water hyacinth. aciar monograph no.60. canberra. 87p. kasno, okky s. dharmaputra, sunjaya, a.s.r. putri and h.s. handayani. 1998. establishment of water hyacinth weevil, neochetina bruchi. seamed biotrop. 26p. 27 establishment of neochetina spp. kasno et al. saraswati, r. 1980. the possible use of the combination neochetina eichhorniae warner — myrothecium roridum tode ex fr. to control waterhyacinth (kemungkinan penggunaan kombinasi kumbang moncong neochetina eichhorniae wamer dan jamur penyakit myrothecium roridum tode ex fr. untuk pengendalian eceng gondok eichhornia crassipes (mart.) solms. faculty of mathematic and natural science, padjajaran university, bandung. 44 p. tjitrosoedirdjo, s.s. 1998. biological control of water hyacinth, water fern and giant mimosa using insect natural enemies (pengendalian hayati eceng gondok, kiambang dan klampis air dengan menggunakan serangga musuh alami.) research report of competitive grant of higher education, ii/5. directorate general of higher education. department of education and culture. wright, a.d. 1979. preliminary report on damage to eichhornia crassipes by an introduced weevil at a central queensland liberation site. proceeding of the seventh conference of the asian pasific weed science society, p: 227-229. 28 biotropia no. 17, 2001 appendix 1. analysis of variance on the percentage of damage severity due to weevils and fungi, and the number of spots at the second youngest leaves of water hyacinth at situ bagendit, garut regency 29 microsoft word 49 biotropia vol. 13 no. 1, 2006 : 49 55 localization of gfdd4-1 expressed protein in physcomitrella patens cells diah ratnadewi faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia abstract the expression of a new dehydration-related gene of physcomitrella patens, gfdd4-i, was traced for its localization in the plant cells. this revelation is useful to predict the possible roles of the protein in plant tolerance to environmental stress. this gene was fused to gfp marker gene and transfected into the plant protoplasts. under a confocal laser microscope, it was detected that the gfdd4-1 protein associated with the off started to generate at the cell periphery and developed more intensively inwards to cytoplasm, forming vesicles and cystemal structures or network. the protein might be membrane protein which may involve directly in membrane maintenance or cellular protection against stress conditions. key words : protoplast transformation, protein localization, dehydration-related gene, gfp, physcomitrella patens introduction abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity. diverse environmental stresses often activate a number of cell signalling pathways and cellular responses, such as production of stress proteins, up-regulation of antioxidants and accumulation of compatible solutes (vierling and kimpel 1992). in plant species which is tolerant to a stressing condition, the metabolic responses activated in response to the stress may contribute to the mechanism of tolerance. the production of heat-shock proteins (hsps) and chaperons (ingram and bartels 1996; bray et al. 2000), osmoprotectants and free-radical scavengers (bohnert and sheveleva 1998) are among the examples of substances that may involve in the protection of cell membranes and proteins. physcomitrella patens is a bryophyte which was reported to be highly tolerant to various abiotic stresses, in particular to salt, osmotic, and dehydration stresses (frank et al. 2005). genes ppshpl and ppshp2 in p. patens which demonstrate homology to rci2a and rci2b , respectively, of arabidopsis thaliana, encoding highly conserved small hydrophobic proteins, have been confirmed to be up-regulated by desiccation, salt, sorbitol, cold, and abscisic acid (kroemer et al. 2004), while an original dehydration related gene of p. patens, gfdd4j(genefishmg differential display clone 4-1), demonstrated to be activated additionally by cold and aba (ratnadewi and frank 2005). this indicated that p. patens has a number 49 corresponding author: dratnadewi@yahoo.com biotrop1a vol. 13 no. 1, 2006 of genes regulated by those diverse stress conditions conferring its high level ot tolerance, and whose pathways are overlapping and complementary one to the other. under light stress condition, sepl and sep2 encoded proteins (stress-enhanced proteins, seps) in a. thaliana have been detected to localize in thylakoid membrane of chloroplasts; they might function in one or another way as plant protector to high-intensity light and are not likely light harvesting (heddad and adamska 2000). by attaching the open reading frame of the gene gfdd4-1 to gfp marker gene, we attempted to investigate the localization of the encoded protein in p patens cells, the protein site may indicate its possible function in plant tolerance tc environmental stress. materials and methods isolation of cdnas this research employed copy dna of the gfdd4-1 gene. searching of cdism clones was carried out in the ppest database (basf-albert ludwigs university o: freiburg) (rensing el al 2002) using gfdd4-1 gene sequence as query. the cdnj* cloned in bacterial cultures have been retrieved as glycerol stocks from the clom depository. the clones were subject to pcr reactions, sequencing and selection foi the full length one. plasmid reconstruction the coding region of the selected cdna clone was amplified througl pcr reactions. the primers used were supplemented with bamhi at the 5 prime and with kpnl at the 3' prime: the forward primer wa 5'-ggatccatgaattccgagggtctt-3' and the reverse primer wa 5'ggtaccatgaccaccacgactattc-3' to obtain the expected dw fragment size of about 600 bp. the pcr product was subsequently loaded on ai agarose gel (1.0% wv"1) and the appropriate band was isolated using qiaexi purification kit for gel extraction (qiagen). the cloning of the dna fragment wa performed in pcr*4-topo* vector (invitrogen), and the dnas were then harvestei and purified through mini-preparation procedure (qiagen). when the concerne< fragment would be used as insert, digestion by bamhi and kpnl followed by dm; isolation and purification using qiaexii kit were executed again. plasmid pmav4 contains gfp gene and was selected as vector to fuse the dn/ fragment to the reporter gene. it was linearized at the bamhl and kpnl sites whei the dna fragment would be inserted (figure l).the ligation was executed with t-dna ligase (mbi fermentas). 35s promoter drove the expression of these tandei genes. 50 bacterial transformation escherichia coli competent cells clone xl1 blue mrf" (100 ul) were proceeded for transformation by inoculating the new pmav4 plasmid construct (2 to 8 ul). the bacterial mixture was incubated on ice (30 seconds), in water bath at 42°c (90 seconds) and on ice again (2 min). one millilitre of lb medium was added to the mixture prior to incubation in 37°c shaking water-bath for one hour. an amount of 100 ul of the transformed bacteria was plated on lb+amphycilin agar medium (sambrook and russel 2001). the rest was spinned down at 5000 rpm for 3 min; most of the supernatant was removed and the more concentrated solution was spread over a fresh medium of the same composition. bacterial colonies were expected to grow after an overnight incubation at 37°c. the appropriate ligated plasmid was isolated through dna maxi-preparation (qiagen) from a single colony derived cell suspension. protoplasts isolation and transformation moss plants at protonema stage were harvested from 200 ml bioreactor liquid culture, which corresponds to about 10 mg dry weight. aseptically the moss was proceeded for protoplasts isolation according to the protocol described by rother et al. (1994) with slight modification. in a glass tube, 100 ul of dna solution (0.5 ug ul~' in ca(no3)2) was added to 250 ul of protoplast solution (1.2 x 10* protoplasts ml"1); 350 ul of peg 4000 solution (40%) was then incorporated into the mixture. it was mixed gently by rolling the tube between fingers. the subsequent procedure followed exactly the method cited in hohe et al. (2003). the transformed protoplasts were re-suspended in 3 ml of regeneration medium. it comprised 0.25 gl"1 kh2po4, 0.25 el'1 kc1, 0.25 gr1 mgs04-7h20, 1 gr1 ca(n03)2, 12.5 mgl'1 feso4-7h20, 50 gp and 30 gl'1 mannitol. the ph of the medium was adjusted to 5.8 with koh and its osmolarity to 51 biotropia vol. 13 no. 1, 2006 approximately 540 mos using mannitol. the protoplast solution (1.5 ml) was transferred into 3 cm-well of culture plate. the protoplast cultures were incubated under dim-light at 25°c. observations were carried out at 1,2, 4, and 8 days after transfection. transgenic cells were detected visually under a uv light microscope. when the expected green fluorescent intact cells were visible, the images were taken in more detail under a confocal laser scanning microscope. results and discussion cdna isolation and plasmid reconstruction from searching in the ppest database with gfdd4-1 gene sequence as query, three hits came out from there, e.g. clones no. ppoo1088038, pp004071329 and pp004083128. the three cdna clones were retrieved from the depository to proceed to pcr for amplification using standard primers ml3-20 and m13-rev. dna sequencing revealed that the clone pp004071329 is the full-length cdna and was used further in this work. by using bamhl and kpnl, insert of 600 bp dna fragment and linearized pmav4 were obtained prior to their ligation to a new construct. figure 2 demonstrates the pcr products resulted from these digestions. gfdd4-l/gfproteins in transformed cells one day after the transfection, few green protoplasts were already seen under uv-light microscope, and they looked multiplying in number and intensity over the days of observation. the green fluorescence was assumed to be the protein expressed by the gf'p gene that was closely associated to the gfdd4-1. it has been proven that the intrinsic fluorescence of gfp allows for non-invasive and monitoring to track the expression and location of proteins and other structures within cells or organism without killing or destroying the biological samples. this makes gfp efficient as a transformation marker. at the first till the fourth day, the green structures scattered unevenly along the periphery of the cells (figure 3). chloroplasts are represented by the red auto fluorescence; it was obvious that the protein was not generated in the plastid membrane. when a barrier filter was used to block the red fluorescence of chlorophyll, typical green fluorescence appeared more clearly. at the fourth and eighth day, the protein was more abundant, extending into the cytoplasm, forming vesicles and cysternal structures in some cases, and in some other cells it formed a network. 52 abiotic stresses such as drought, salinity, cold, or heat are often interconnected and cause disruption of osmotic and ionic homeostasis as well as damage of functional and structural proteins and membranes (wang et al. 2003). plant responses to abiotic stress are quite complex due to the fact that, in one hand, it involves many genes and biochemical-molecular mechanisms, on the other hand, different stressing stimuli can induce only a single gene. in their response to environmental stresses, plants activate a large set of genes leading to the accumulation of specific stress-associated proteins. the product of stress-related genes can be classified into two major categories: 1). those that directly involve in the cellular protection against environmental stresses, such as hsp and lea proteins and chaperons, various osmo-protectants and detoxification enzymes, 2). those that play roles in signalling cascades and in transcriptional control, such as transcription factors and protein kinase (seki el al. 2003; wang ef a/. 2003). upon water, salinity, and/or extreme temperature stress, plants produce predominantly hsps and lea proteins. hsp70s are found in several cellular compartments such as in cytoplasm, in the lumen of endoplasmic reticulum (er), in the matrix of mitochondria, as well as in chloroplasts. they have been shown to act as molecular chaperons that function in the stabilization of proteins and membranes, and in assisting protein refolding under stress conditions (vierling 1991). accordingly, in this preliminary work, the gfdd4-1 protein associated to the gfp was observed spreading intensively over the cell. the protein in the peripheral area and the structures of vesicles and cysternae exhibited that the protein might be membrane protein which may involve in membrane maintenance or cellular protection against stress conditions. conclusions the gfdd4-1, a dehydration-related protein in physcomitrella patens, locates at cell membranous system. it may function directly or indirectly in cellular protection against environmental stresses. 54 localization of gfdd4-1 expressed protein d. ratnadewi acknowledgement the author would like to gratefully acknowledge the german academic exchange service (daad) for the great opportunity given to do a part of her research in germany. high appreciation also goes to prof. dr. ralf reski and dr. wolfgang frank who permitted me to join their research group at the institute of plant biotechnology, albert-ludwigs university of freiburg. references bohnert, h.j. and e. sheveleva. 1998. plant stress adaptations making metabolism move. curr. opin. plant biol., 1:267274. bray, e.a., j. bailey-serres and e. weretilnyk. 2000. responses to abiotic stresses. in: gruissem w., buchanan b., jones r. (ed). biochemistry and molecular biology of plants. rockville md: amer. soc. plant physiologists, p. 11581249. frank, w., d. ratnadewi and r. reski. 2005. physcomilrella patens is highly tolerant against drought, salt and osmotic stress. planta, 220:384-394. heddad, m. and i. adamska. 2000. light stress-regulated two-helix proteins in arahidopsis thaliana related to the chlorophyll a/2>-binding gene family. pnas 97(7):3741-3746. hohe, a., t. egener, j.m. lucht, h. holtorf, c. reinhard, g. schween and r. reski. 2003. an improved and highly standardised transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, physcomitrella patens. curr. gen., 44:339-347. ingram, j. and d. bartels. 1996. the molecular basis of dehydration tolerance in plants. annu. rev. plant biol., 47:377-403. kroemer, k., r. reski and w. frank. 2004. abiotic stress response in the moss physcomitrella patens: evidence for an evolutionary alteration in signaling pathways inland plants. plant cell rep., 22:864-870. ratnadewi, d. and w. frank. 2005. ekspresi gen gfdd4-1 pada physcomitrella patens dan gen homolog pada arahidopsis thaliana dalam responsnya terhadap cekaman abiotik. hayati, 12(4):127-130. rensing, s.a., s. rombauts, y. van de peer and r. reski. 2002. moss transcriptome and beyond. trends plant sci., 7:535538. rother, s., b. hadeler, j.m. orsini, w.o. abel and r. reski. 1994. fate of a mutant macrochloroplasts in somatic hybrids. j. plant physiol., 143:72-77. sambrook, j. and d.w. russel. 2001. molecular cloning: a laboratory manual 3"' ed. cold spring harbor: cold spring harbor laboratory press. scki, m., a. kamei, k. yamaguchi-shinozaki and k. shinozaki. 2003. molecular responses to drought, salinity and frost: common and different paths for plant protection. curr. opin. biotech., 14:194-199. vierling,e. 1991. the roles of heat-shock proteins in plants. annu. rev. plant biol., 42:579-620. vierling, e and j.a. kimpel. 1992. plant responses to environmental stress. curr. opin. biotech., 3:164-170. wang, w., b. vinocur and a. altaian. 2003. plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. planta, 218:1-14. 55 49.pdf 50.pdf 51.pdf 52.pdf 53.pdf 54.pdf 55.pdf 6. arida (genetic) revisi daf... biotropia vol. 20 no. 2, 2013: 122 133 genetic variation, heritability and correlation between resin production character of high resin yielder (hry) pinus merkusii arida susilowati *, iskandar z. siregar , supriyanto , imam wahyudi and corryanti (kopertis wilayah i sumut-nad) recipient of biotrop research grant 2010/accepted 18 april 2013 tree improvement programs for high resin yielder were started in 2006 through a series of survey and morphological identification of candidate trees with high resin production. specific information about genetic parameter of resin yielder candidates in cijambu seedling seed orchard is still not determined yet, although based on resin distribution trend this sso has the highest mean of resin production. in this research, individual and family heritability, coefficient genetic variation and genetic-phenotypic correlation were estimated for resin production and growth data from 15 open polinated families of planted in 1982 (set 1) 1983 (set 2) in cijambu sso. the results showed high value of coefficient genetic variation (cvg: 14.5-28.43%), individual narrow sense heritability values for resin production character (0.58-0.77) which resemble with previous researches. this indicates that genetic factor was dominant for resin production and selection activities were effective to get high yielder superior candidate. phenotypic and genotypic correlation found that bark thickness, crown length and stem diameter character was positively significant correlated to resin production, whereas severity attack level of pests-diseases and number of branches were negatively significant correlated to resin production. selection, high resin yielder, , heritability, phenotypic 1 2 2 3 4 1 2 3 4 coordinatior of private higher education region i north sumatera-aceh, indonesia silviculture department, faculty of forestry, bogor agricultural university, bogor, indonesia forest product department, faculty of forestry bogor agricultural university, bogor, indonesia research and development center of perum perhutani, bogor, indonesia p. merkusii pinus merkusii p. merkusii, variation abstract key words: * corresponding author : arida_iswanto@yahoo.co.id doi: 10.11598/btb.2013.20.2.1 122 introduction pinus merkusii et al p. merkusii p. merkusii pinus merkusii is known as an important industrial species for pulp and paper and sawn wood, gum rosin production, as well as considerable species for reforestation and land rehabilitation in indonesia (suhardi . 1994). one valuable product of resin and very demanded by international market is gondorukem (gum rosin). gondorukem is a potential product, grouped under pine chemical products and it plays an important role as non timber forest product in indonesia because it provides high national income about us$ 50 million/year as well as more job opportunities (fachrodji 2010). problems faced in gondorukem export was lower productivity so that indonesia still ranks as third position after china and brazil as gum rosin producer (cunningham 2006). in order to sustain indonesian gondorukem export, several activities have been undertaken to increase resin production through tree breeding activities, application of improved silvicultural techniques, improvement of tapping techniques and management (fahrodji 2010). breeding activities focusing on resin production is the most prospective way to be developed since early studies conducted in 2006, have resulted to several plus trees producing high yield of resin (high resin yielder). high resin yielder is the term given for pine genotype producing over 50 g/tree/3 days (fakultas kehutanan ugm 2006) higher than the current production (21 g/tree/3 days). breeding activities in indonesia were started since 1976 through a series of progenies test and descendants of plus tree selection which was focused on stem straightness character (soeseno 1988), while resin production is only a side product. in considering high value of resin products, pine breeding activities were recently conducted to obtain plus trees for resin production (soeseno 2001). for breeding activities concentrating on resin yielder, it is important to collect information on genetic parameters such as coefficient of genetic variation, heritability and association of characters related to resin production for directing efficient selection. several researches on conducted in early establishment of seedling seed orchard (sso) in java and other pine species showed that resin production character has high heritability value, but specific information of cijambu sso is not determined yet. therefore, research on "genetic variation, heritability and correlation between resin production characters of high resin yielder (hry) in cijambu seedling seed orchard (sso)" is needed. the result will be used to provide best possible trees for further genetic improvement as well as to establish plantations using improved genetic materials. experiment was carried out in cijambu sso, sumedang. data for this research were obtained from progenies test plantation established in 1982 (set 1) and 1983 (set 2). at early plantation the research focused on stem character, where 200 families materials and methods 123 genetic variation, heritability and correlation between resin production character arida susilowati– et al. were planted/year designed in completely randomized block design, 5-tree line plot with ten blocks as replication at a spacing of 3 x 3 m. together with selective thinning leaving only 2-3 trees per plot. second selection focused on resin production conducted in 2006 found 96 families with 110 plus trees in progenies trial planted in 1978-1983. planting year 1982 involved 20 open pollinated families and in 1983 involved 25 open pollinated families of resin yielder candidates spread in 2 different blocks. for cvg and heritability estimation, 15 selected open polinated families were used. in this research two assumption and 2 types of estimation were used, those were: 1) resin production was not different at the age 29 and 30 years, for these reason we combined 2 set of families (1982 and 1983) into one estimation and 2). resin production was different at the age of 29 and 30 years so we used separate estimation using set 1 and set 2. characters studied in this research were resin production and other quantitative characters which assume to be related to resin production those were: total height (th), clear bole height (cbh), diameter, crown length (cl), crown width (cw) number of branches (nb), first branch angle (fba), bark thickness (bt), severity attack level from pest and disease (sl), clear bole volume (vbc) and total volume (vtot). all characters were measured based on previous methods for forest trees developed by bacilieri (1996); cantini (1999); kremer (2002); ginwal (2004); weber and montes (2005); baliuckas (2005) and devagiri (2007). resin production was measured by calculating resin tapping weight for 3 days. analyses of variance (anova) were carried out on single tree plot design according to isik (2008) using individual tree basis for each progeny test with following linear model: y : μ+ b+ f+e where: y : individual tree observation; μ: the overall mean; b : the effect of i block; f :theeffectof j family; e :withinploterror.only2replicationswere includedin the analyses. variance and covariance component were calculated by equating the means square or mean cross product to their expectation. as progeny trial assumed to be half-sib, heritabilities and standar error (s.e) were estimated by using single site analyses formula: heritability of family means: h f/ + /nb+ ), with s.e h f: (s.e f)/ + /nb+ ), individual tree heritability: h : 4 + ), with s.e. h : (4s.e f)/ ( + ) where: h f: family mean heritability; h :individual tree heritability f: the component due to family means : the component of block : the component of variance within plot. the standard error of family component variance (s.e f) was calculated by anderson & bancroft (1952) hardiyanto (1996): s.e f: [2/k (ms) / (df +2)] , et al. et al. et al. et al. et al. et al. ( ( in data analysis ijk i j ijk ijk i j ijk f b e f b e f b+ e f b+ e b e i i i th th 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0.5 f : σ σ σ σ σ σ σ σ f/ (σ σ σ σ σ σ ; σ ; σ ; σ σ σ δ δ biotropia vol. 20 no. 2, 2013 124 i: i. where: k is the coefficient of family means square; ms the i means square used to estimate n component; df:the number of degree of freedom for ms criteria for heritability f) was calculated according to cotteril and dean (1990). estimates of genetic variation coefficient performed using cornelius (1994) formula as follows: genetic and phenotypic correlation between resin production character and growth character estimated using statistical formula as follows: estimates of variances and allied statistics for contributing sources of variability are presented for each trait in table 1. based on cornelius (1994) classification, genetic variation in cijambu sso showed variation value with total character contribution ranging from 4.05% (total height character) to 28.70% (severity attack level from pest and disease). this result indicated that not all of the characters observed in cijambu sso were influenced by genetic factors. resin production and severity attack level from pest-disease character have high value of coefficient genetic variations. diameter, crown, bark thickness, number of branches, clear bole volume, total volume, have moderate value of coefficient genetic variations, while total height, clear bole height and branching angle in set 2 have lower value of coefficient genetic variations. high cvg value represents resin production character (14.5-28.43%) and severity attack level from pest-disease (24.028.70%). it indicates that genetic factors will have a big impact on the appearance of characters. high value for resin production character was obtained compared to previous research conducted by roberds (2003) in sso (cvg:13.7%). high value for severity attack level from pestdisease was also compared to bastein & alia (2000) in (cvg:26.8%). in order to tree improvement focused on resin production in cijambu sso, these characters are important to be studied in detail because of the influence of genetic factors. cvg value for total height, clear bole height, crown width, crown length and number of branches character has lower value. it indicated that these characters belong to vegetative characters which is influenced by environmental factors. th th 2 (h et al. p.taeda p. sylvestris coefficient genetic variation (cvg) results and discussion genetic variation, heritability and correlation between resin production character arida susilowati– et al. 125 heritability based on cotteril and dean (1990) classification, heritability value for resin production character at cijambu sso was high (h2f:0.700.09-0.820.08 and h2:0.580.08-0.770.08), severity attack level from pest-disease also has high heritability value (h2f: 0.640.02-0.800.16 and h2:0.640.13-0.690.04). stem diameter, bark thickness, crown length, number of branches and volume have lower to moderate heritability value (table 1). characters σ2f σ2b σ2e cvg (%) h2f se h2 se 29 and 30 years old resin production days (g/3) 581.8 338.1 86.1 21.6 0.70 0.09 0.58 0.08 total height (m) 1.94 2.58 11.07 6.0 0.14 0.05 0.12 0.05 clear bole height (m) 3.40 7.0 3.39 6.6 0.33 0.23 0.25 0.17 diameter (m) 0.002 0.007 0.002 10.1 0.29 0.72 0.20 0.52 bark thickness (cm) 0.36 0.89 0.26 8.8 0.34 0..83 0.24 0.58 number of branches 55.92 69.15 33.17 12.8 0.45 0.10 0.35 0.08 crown length (m) 1.507 3.122 1.74 10.6 0.31 0.30 0.24 0.23 crown width (m) 1.272 2.96 1.55 12.5 0.30 0.30 0.22 0.21 first branch angle 105.97 34.32 58.12 11.2 0.58 0.10 0.53 0.09 clear bole volume (m3) 0.002 0.009 0.04 13.9 0.35 0.22 0.33 0.20 total volume (m3) 0.007 0.34 0.14 9.4 0.18 0.60 0.12 0.42 severity attack level from pest and disease 139.91 15.75 49.86 28.2 0.71 0.13 0.68 0.12 set 1 (29 years old) resin production days (g/3) 200.70 53.94 46.82 14.5 0.72 0.13 0.67 0.19 total height (m) 0.67 3.5 21.83 3.3 0.04 0.01 0.03 0.03 clear bole height (m) 1.69 3.5 6.08 4.89 0.21 0.11 0.15 0.13 diameter (m) 0.001 0.0005 0.002 7.9 0.47 0.15 0.36 0.04 bark thickness (cm) 0.27 0.36 0.12 6.5 0.39 0.15 0.36 0.54 number of branches 15.73 77.16 40.13 13.2 0.14 0.03 0.12 0.09 crown length (m) 2.42 3.02 1.13 13.0 0.40 0.48 0.37 0.18 crown width (m) 1.11 1.56 3.26 10.3 0.26 0.18 0.19 0.05 first branch angle 6.4 36.15 106.2 2.8 0.07 0.01 0.04 0.02 clear bole volume (m3) 0.02 0.002 0.07 13.1 0.31 0.16 0.19 0.06 total volume (m3) 0.06 0.07 0.23 13.8 0.25 0.15 0.17 0.37 severity attack level from pest and disease 95.59 5.29 37.83 24.0 0.80 0.16 0.69 0.04 table 1. family f), block variance b), within plot error e), coefficient of genetic variation (cvg), family heritability (h f) and individual tree heritability for resin production and growth characters variance (σ (σ variance (σ 2 2 2 2 biotropia vol. 20 no. 2, 2013 126 p. merkusii et al. p. eliotii et al. p. pinaster et al. p. taeda et al. et al. et al. et al. et al. et al. et al. heritability for resin production shows high value (0.580.08-0.770.08). it seems to be slightly different than previous research results (0.69) conducted by leksono (1990) at cijambu and sempolan on 12 years old progenies test plantation. this result was higher than reported by zhang (2010) on (0.37), tadesse (2001) on (0.5) and roberds (2003) on (0.44 to 0.59), and similar value was also found at stem diameter and branching quality. higher value in this research compared to previous researches at early sso establishment suggested that the materials used for this research originated from second selection focused for resin production. for high resin production heritability values, wenger (1984); burczyk (1998); kassuth (1984); mergen 1955, gill (1998) explained that resin production character is controlled by gene. this indicates that improvement program for resin production character through genetic selection would provide higher genetic gain not affected by other interaction factors. heritability value for height and stem diameter were slightly different from previous researches conducted by hardiyanto (1996) who focused on stem straightness, and leksono (1996) for resin yield at 12 years old progenies test (h2f: 0.40 dan h2f: 0.43). heritability value for branching trait character is lowe in set 1 (0.12 and 0.07) but in set 2 the heritability value is high (0.44 dan 0.71). the changes of heritability value in long rotation crops such as a tree is not surprising since genes involved in growth may change with age (namkoong 1980; monteuis 2011), and these changes also may be related to different growth phases (franklin 1979). change of heritability value for diameter, tree height and bark thickness character at different age probably is influenced by silvicultural practices such as thinning and other management practices (gwaze 1997 & lopez-upton 1999). result from cvg and heritability estimation in cijambu sso showed high value for resin production, it indicated that more dominant genetic factors determine this character. based on this value, tree improvement program for high resin yielder can be initially conducted by mass selection of individual trees with high resin production. characters σ2f σ2b σ2e cvg (%) h2f se h2 se set 2 (30 years old) resin production (g/3 days) 1012.5 151.2 5 151.3 28.43 0.82 0.084 0.77 0.08 total height (m) 1.15 1.02 0.86 4.64 0.46 0.06 0.38 0.52 clear bole height (m) 3.08 6.75 0.33 4.91 0.45 0.09 0.30 0.06 diameter (m) 0.002 0.01 0.001 8.49 0.19 0.07 0.12 0.411 bark thickness (cm) 0.09 0.19 0.38 10.93 0.16 0.01 0.14 0.29 number of branches 66.50 14.09 5.61 8.12 0.84 0.05 0.70 0.41 crown length (m) 0.82 0.05 1.007 7.81 0.44 0.06 0.44 0.55 crown width (m) 0.50 0 0.48 7.84 0.51 0.09 0.51 0.01 first branch angle 201.95 18.75 75 5.26 0.71 0.10 0.68 0.10 clear bole volume (m3) 0.0090.04 0.03 12.04 0.62 0.09 0.50 0.06 total volume (m3) 0.14 0.33 0.009 13.06 0.38 0.08 0.26 0.07 table 1. continued genetic variation, heritability and correlation between resin production character arida susilowati– et al. 127 biotropia vol. 20 no. 2, 2013 this condition agrees with tadesse . (2001) who stated that when a population has high heritability value for a character, mass selection method would be more efficient for improving the character. furthermore, white . (2007) stated that mass selection was appropriate to be implemented in early selection and for high heritability value characters because the phenotypes of individual trees describe its genetic ability. phenotypic and genotypic correlation (table 2) showed low to moderate coefficient value, indicated that not overall phenotypic appearance describing genetic expression because interaction between environment and genetic factor also influencing phenotypic expression of trees. phenotypic correlation showed resin production character was positively significant correlated with stem diameter, bark thickness and crown length. furthermore, number of branches and severity attack level from pest -disease character was negatively significant correlated with resin production. positive correlation between resin productions with some characters, indicated that resin production will increase equally with the increase of character value. on the other hand, a negative correlation between resin production with some characters indicates that resin production will decrease with high value of character components. genetic correlation between resin production and stem diameter, bark thickness, and crown length indicated that resin production will increasedequally with the increase of these characters. on the contrary resin production will decrease equally with the increase of branching number and severity attack level of pest-disease. different results were obtained in set 2 (30 years old progenies trial), in set 2 we found negative correlation between resin production and tree height, it indicates that resin production will decrase with the increase of tree height. correlation between resin production with stem diameter and crown length are in accordance with previous research results conducted by pswaray (1996); coppen (1984) on westbork (2011) on and tadesse (2001) on . panshin & de zeeuw (1984) also explained that a good tree is characterized by large diameter and large crown size because trees need more wider light absorbance for photosynthesis process. correlation between resin production and stem diameter have been reported also by coppen 1984 who stated that wider diameter trees have wider annual ring and giving great chance to have more resin ducts and produce higher resin yield than smaller diameter trees. number of branches and severity attack level from pest and disease have negatively significant effect to resin production meaning that resin production decreased with the increasing number of branches and severity attack level from pest. papajiannopoulos (2002) in also found that tree with canopy openes (lower branch number) have higher resin yield compared with higher branch number which assume to be related to photosyntetic process, resin viscocity and accumulation. correlation between resin production and severity attack level from pest-disease have been deeply studied in other pine species and conifers. previous researches conducted et al et al et al. et al. p.elliottii; p.taeda et al. p.pinaster naval store et al. p.halepensis phenotypic and genetic correlation between growth character and resin production 128 c ar ac te r p ro d t h c b h d b t c l c w n b f b a sl v b c v to t se t 1 d an se t 2 p ro d 1 0. 01 4 -0 .1 12 0. 58 4* * 0. 29 9 0. 17 6 0. 09 6 -0 .4 96 * -0 .0 23 -0 .5 17 * -0 .2 84 -0 .2 54 t h 0. 12 1 1 0. 20 4 -0 .0 43 -0 .2 83 0. 31 6 0. 71 6* 0. 15 5 0. 25 2 0. 04 7 0. 16 3 0. 41 1 c b l 0. 10 6 0. 44 5 1 -0 .3 44 -0 .4 35 -0 .1 87 0. 03 3 0. 24 0 -0 .2 00 0. 30 6 0. 49 0* -0 .0 .8 1 d 0. 65 0* 0. 27 2 0. 23 6 1 0. 60 2* * 0. 12 8 -0 .1 05 -0 .0 96 -0 .1 43 -0 .2 41 0. 18 3 0. 38 7 b r 0. 18 6 0. 77 4 0. 67 2 0. 41 5 1 -0 .0 63 -0 .1 44 -0 .1 45 -0 .0 11 -0 .1 19 0. 06 5 0. 24 5 c l 0. 12 9 0. 54 0 0. 64 9 0. 29 0 0. 24 8 1 0. 32 3 -0 .1 50 0. 05 3 -0 .2 04 -0 .1 59 0. 06 9 c w 0. 13 5 0. 17 4 0. 49 0 0. 30 2 0. 25 4 0. 50 0 1 0. 14 1 0. 28 1 0. 19 8 0. 03 0 0. 28 6 n b -0 .0 52 0. 21 9 0. 19 0 0. 11 7 0. 33 4 0. 23 3 0. 24 3 1 0. 04 7 0. 35 7 0. 32 1 0. 25 6 f b a 0. 04 4 0. 18 6 0. 16 2 0. 10 0 0. 28 4 0. 19 8 0. 20 7 0. 08 1 0. 11 4 0. 03 8 0. 23 3 sl 0. 04 1 0. 17 4 0. 15 1 0. 23 6 0. 26 5 0. 16 2 0. 19 3 0. 07 0. 06 1 0. 31 8 0. 25 4 v b c 0. 36 6 0. 15 2 0. 13 2 0. 47 2 0. 23 2 0. 16 2 0. 16 9 0. 25 4 0. 30 5 0. 32 2 1 0. 75 1* * v to t 0. 28 0 0. 11 6 0. 10 1 0. 62 3 0. 17 8 0. 12 4 0. 12 4 0. 20 4 0. 40 9 0. 40 0 0. 35 6 1 se t 1 (3 0 ye ar s o ld ) p ro d t t b t b c d t k b p t l t c p t sc p h p v b c v to t p ro d 1 0. 32 9 0. 29 7 0. 42 9* 0. 40 3* 0. 42 6* 0. 12 2 -0 .3 55 * 0. 23 8 -0 .3 21 * 0. 56 4 0. 46 9 t h 0. 20 8 1 0. 19 8 0. 57 5 0. 07 0 0. 19 5 0. 15 6 0. 28 5 0. 64 2 -0 .1 79 0. 49 8 0. 83 8* c b l 0. 16 4 0. 68 1 1 0. 05 9 -0 .3 39 0. 36 9 0. 44 4 -0 .1 88 -0 .1 24 -0 .0 86 0. 72 0* 0. 08 5 d 0. 56 1* 0. 42 4 0. 33 6 1 0. 46 9 0. 08 2 0. 16 0 -0 .2 68 -0 .4 28 -0 .1 92 0. 73 1* 0. 93 3* * b r 0. 34 5 0. 10 9 0. 66 4 0. 53 3 1 -0 .5 06 -0 .0 97 0. 19 0 0. 56 0 0. 34 9 0. 09 7 0. 33 8 c l 0. 15 0 0. 62 7 0. 31 1 0. 30 7 0. 68 9 1 0. 28 2 -0 .2 90 -0 .0 71 -0 .4 51 0. 05 8 0. 28 1 c w 0. 18 3 0. 76 2 0. 49 7 0. 37 3 0. 85 1 0. 55 1 1 0. 20 4 0. 14 5 -0 .0 47 0. 15 7 0. 54 9 n b -0 .0 94 0. 31 1 0. 31 1 0. 19 2 0. 61 9 0. 28 4 0. 34 5 1 0. 17 4 -0 .1 90 0. 14 1 0. 00 4 f b a 0. 11 8 0. 39 3 0. 39 0 0. 24 0 0. 49 4 0. 35 6 0. 55 1 0. 22 3 1 0. 04 5 0. 18 0 -0 .0 59 sl -0 .0 60 0. 25 4 0. 19 8 0. 31 5 0. 31 5 0. 18 1 0. 22 0 0. 11 3 0. 14 2 1 -0 .1 79 0. 17 4 v b c 0. 25 2 0. 21 9 0. 17 4 0. 50 5 0. 27 6 0. 15 9 0. 19 3 0. 66 7 0. 12 4 0. 63 1 0. 68 3 v to t 0. 37 8 0. 15 7 0. 12 5 0. 55 2 0. 19 8 0. 11 4 0. 13 8 0. 57 6 0. 68 9 0. 45 5 0. 40 0 1 t ab le 2. p h en o ty p ic (a b o ve d ia go n al ) a n d ge n et ic co re la ti o n (b el o w d ia go n al ) b et w ee n re si n p ro d u ct io n ch ar ac te r an d o th er co m p o n en t genetic variation, heritability and correlation between resin production character arida susilowati– et al. 129 biotropia vol. 20 no. 2, 2013 c ar ac te r p ro d t h c b h d b t c l c w n b f b a sl v b c v to t t ab le 2. c o n ti n u ed se t 2 (2 9 ye ar s o ld ) p ro d t t b t b c d t k b p t l t c p t sc p h p v b c v to t p ro d 1 0. 04 5 -0 .1 26 0. 55 5* 0. 09 4 0. 39 7* 0. 06 0 -0 .7 14 * -0 .1 62 -0 .6 66 * 0. 47 4 0. 45 6 t h -0 .0 56 1 0. 17 8 -0 .4 91 0. 35 6* 0. 34 9 0. 30 0* -0 .0 29 0. 55 0 -0 .1 15 0. 40 0 0. 44 2* c b l -0 .0 76 0. 23 5 1 -0 .4 23 -0 .0 97 -0 .5 16 -0 .4 28 0. 01 4 -0 .6 41 -0 .0 94 0. 03 1 0. 04 2 d 0. 49 9* 0. 10 7 0. 29 6 1 0. 65 3 -0 .1 87 -0 .2 18 -0 .1 87 -0 .2 59 -0 .0 84 0. 22 7 0. 33 3 b r 0. 35 5 0. 05 1 0. 51 9 0. 11 6 1 -0 .2 83 -0 .6 47 0. 29 8 -0 .1 81 0. 42 8 0. 66 4 0. 58 0 c l 0. 26 5 0. 26 7 0. 19 1 0. 34 5 0. 10 5 1 0. 40 5 0. 08 0 0. 15 2 -0 .1 25 -0 .5 79 -0 .2 54 c w 0. 06 5 0. 29 1 0. 29 1 0. 40 6 0. 13 5 0. 32 0 1 -0 .2 05 0. 63 0 0. 11 5 -0 .1 43 0. 23 9 n b -0 .4 89 0. 09 1 0. 47 3 0. 43 5 0. 04 3 0. 13 3 0. 33 3 1 0. 09 6 0. 53 2* 0. 30 1 0. 32 6 f b a 0. 07 5 0. 08 7 0. 08 7 0. 33 5 0. 13 2 0. 09 4 0. 20 1 0. 01 92 1 0. 11 5 -0 .1 43 0. 23 9 sl -0 .4 96 0. 06 5 0. 13 5 0. 08 5 0. 23 5 0. 19 2 0. 43 4 0. 45 3 0. 19 8 1 0. 19 3 0. 16 9 v b c 0. 12 6 0. 03 5 0. 07 9 0. 42 1 0. 25 6 0. 13 2 0. 17 7 0. 15 6 0. 25 3 0. 06 7 1 0. 68 7 v to t 0. 27 8 0. 23 0 0. 21 7 0. 43 5 0. 29 9 0. 34 3 0. 19 2 0. 25 7 0. 19 8 0. 09 9 0. 18 8 1 n o te : * *: si gn if ic an t d if fe re n t at 99 % * : s ig n if ic an t d if fe re n t at 95 % 130 in temperate regions by kleinhentz . (1998); blada (2000); kim . (2003); rafael . (2005) concluded that pest and disease caused significant decrease of resin production quantities. furthermore, raffa & berryman (1982) in also found severity attack level which caused loss of resin yield quantities. however, specific studies about correlation between resin production and severity attack level in cijambu sso have not been conducted yet. from this research we found that both resin production and severity attack level in cijambu sso have high cvg value, it indicated that selection activities focused for resin production also can escorted together with resistance for pest and disease. although some characters have a correlation to resin production, further research still need to be conducted because phenotypic observation is influenced by growth phase and environment, so it could not differentiate recessive genotype with resemble morphological appearance such as secondary metabolites (finkeldey 2005). to overcome this problem molecular marker such as rflp, rapd, aflp and microsatellite can be used. results from genetic variation and heritability estimation of resin yielder candidates character in cijambu sso showed high coefficient of genetic variation value (cvg: 14.5-28.43%) and heritability value (h2:0.580.08-0.770.08) for resin production character. it indicated that genetic factor strongly affected resin production character. genetic and phenotypic correlation found that stem diameter, bark thickness and crown length character were positively significant correlated to the resin production, whereas severity attack level from pest-disease and number of branches was negatively significant correlated to resin production. it indicated t genetically improvement on stem diameter, bark thickness and crown length increasing resin production. whereas, higher severity attack level from pest-disease and number of branches decreased resin production. this research was part of arida susilowati's studies toward doctorate degree from bogor agricultural university (ipb). i would like to express my sincerest thank to seameo-biotrop for supporting my research with and development center of the perum perhutani for the access to their experimental plot in cijambu sso and in providing samples as well as for technical assistance during the fieldwork. et al et al et al p.taeda phd research grant. my sincerest appreciation also goes to research conclusions acknowledgments genetic variation, heritability and correlation between resin production character arida susilowati– et al. 131 biotropia vol. 20 no. 2, 2013 references baliuckas vt, lagerstrom i, norell and g eriksson 2005. genetic variation among and within populations in swedish species of l. and l. assessed in a nursery trial. , 54: 1-8. bastein c, alia r 2000. what might be useful measures of genetic variability for adaptive characters within populations of scots pine . burczykj, lewandowskai, chojnackib 1998. resin production of scots pine trees may be associated with multilocus allozyme 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tectona grandis commonw. for rev pinus elliottii elliottii silvae genetica pinus pinaster hylobius abietis ann. for. sci . environ. entomol can. j. for. res pinus merkusii et in pinus l. in plant resources of south-east asia 5(1) pinus pinaster forest genetic silvae genet forest genetic genetic variation, heritability and correlation between resin production character arida susilowati– et al. 133 microsoft word 1 biotropia no. 20, 2003: 1-10 synergistic activity of enzymes produced by eupenicillium javanicum and aspergillus niger nrrl 337 on palm oil factory wastes tresnawati purwadaria', noni nirwana 2, pius p. ketaren', dyah iswantini pradono2, and yantyati widyastuti3 ' research institute for animal production, p. o. box 221, bogor 16002, indonesia, 2 department of chemistry, faculty of science and mathematics, bogor agricultural university, jl raya pajajaran bogor, indonesia, 3 research centre for biology-lipi. jl. juanda 18, bogor 16002, indonesia. abstract the use of palm kernel cake (pkc) and palm oil mill effluent (pome), substances from palm oil factory wastes, for monogastric is limited by their high cellulose and mannan contents. hydrolytic enzymes have been supplemented to increase the nutrient digestibility. the maximal digestibility was obtained in the synergistic action of all enzyme components including b-d-endoglucanase (cmcase), b-d-glucosidase, b-d-mannanase, p-d-mannosidase, and oc-dgalactosidase. two kinds of enzymes produced by eupenicillium javanicum and aspergillus niger nrrl 337 on the submerged culture containing 3% coconut meal were selected to hydrolyze pkc or dry pome. enzyme from e. javanicum contained higher cmcase, b-d-mannanase, and a-d-galactosidase activities, while that from a. niger nrrl 337 contained more p-d-glucosidase and p-d-mannosidase activities. saccharification (hydrolytic) activities of enzyme mixtures on pkc and pome were determined at ph 5.0, the optimal ph for p-d-mannanase from e. javanicum, and at 5.4 the optimal ph for a-d-galactosidase from e. javanicum and p-d-glucosidase from a. niger nrrl 337. the enzyme proportions of e. javanicum and a. niger nrrl 337 were 100 : 0, 80 : 20, 60 : 40, 40 : 60, and 0 : 100%. the highest saccharification activity on both substrates was observed on the mixture of 80% a. niger nrrl 337. the ph levels did not significantly affect saccharification activity. fiber components in pkc were more digestable than in pome. further analysis on the reducing sugar components using thin layer chromatography showed that more monomers were produced in the 60 or 80% of a. niger nrrl 337. the glycosidases of a. niger nrrl 337 played more important role in the saccharification activity. keywords: synergistic activity/ palm kernel cake/palm oil mill effluent/ eupenicillium javanicum/ aspergillus niger nrrl 337 introduction feeding costs account for 65-70% of the total cost of animal production. therefore, it is important that most part of the feed can be utilized. feeding of commercial chickens in indonesia usually follows the formula applied in their original country (united states). normally, the feed contains corn, soybean meal, and fishmeal. these feedstuffs are not abundant in indonesia and have to be imported. on the other hand, rice bran, coconut meal, and palm oil factory wastes: palm kernel cake (pkc) and palm oil mill effluent (pome) are available. pkc is a residue of the oil extraction of palm fruit and constitutes up to 45% of palm oil bunches, while pome is the sludge of crude palm oil process and amounted to 2% biotropia no. 20, 2003 of palm oil bunches. the fiber content of pkc exceeding 20% could occur if shells and fruit fiber are not removed in the extraction process and over its half part is neutral detergent fiber (ndf) containing galactomannan and mannan (swick and tan 1995). the crude fiber (cellulose and lignin) of steamed pkc was 21.7% (supriyati et al. 1998). the cellulose, hemicellulose (galactomannan and mannan), and lignin of steamed pome were 17.3, 22.0, and 15.8% (purwadaria et al. 1998). supplementation of mannanase (galactomannanase) enzyme complex to improve the nutritional value is potential for improving feed quality. the components of the enzymes are pd-endoglucanase (cmcase), p-d-glucosidase, p-d-mannanase, p-d-mannosidase, and a-dgalactosidase. eupenicillium javanicum isolated from palm seed (purwadaria et al. 1994) produced less p-d-glucosidase and more p-d-mannanase, p-d-mannosidase, and a-dgalactosidase than aspergillus niger nrrl 337 known as mannanolytic fungus (araujo and ward 1990; haryati et al. 1997). the different activities of the enzyme component open up the possibilities of using the enzyme mixtures to improve the saccharification (hydrolysis) activity in pkc and pome. the synergistic effect using enzyme mixture of trichoderma viride and a. ustus on alkali treated bagasse had been reported (manonmani and sreekantiah 1987). the saccharification on the substrate was 63% when using enzyme mixture of 1:1, while when individual enzymes were used 13.5 and 22.9%, respectively. the information on optimal mixture of such enzyme composition will be useful in enzyme application for animal feeding. the objective of the present study is to determine the synergistic activity of enzymes produced by e. javanicum and a. niger nrrl 337 to hydrolyze pkc and pome. materials and methods palm kernel cake (pkc) and palm oil mill effluent (pome) pkc was obtained from palm oil factory by solvent extraction, while pome was obtained from the centrifugation of palm oil mill effluent. after centrifugation pome was dried under the sun. both dry materials were ground to a fine powder (0.5 mm) using wiley mill. enzyme production enzymes were produced by e. javanicum (riap collection) or a. niger nrrl 337 in the medium containing yeast extract 3g/l, coconut meal 30g/l and minerals in g/1 (nh4)2so4 1.4, kh2po4 2.0, mgso4 0.3, urea 0.3, and cacl2 0.3 and in ppm feso4 5, mnso4 16, znso4 14, and cocl2 20. molds were cultivated in 50 ml medium in a 250 ml flask at 29°c using reciprocal shaker (150 rpm), after inoculation with 2 ml of spore suspension (5 x 1013 and 30 x 1013/ml for e. javanicum and a. niger, respectively) from five-day pda culture slant. the synergistic activity of enzymes on oil palm factory wastes — tresnawati purwadaria et al incubation time for e. javanicum was five days, while that for a. niger was six days (haryati et al. 1997). sodium azide was added at 0.2% final concentration. the culture was then centrifuged (12000 rpm, 20 min, 4°c) and supernatant was collected for enzyme assays and saccharification. enzyme activities the activity of carboxymethylcellulase (cmcase) and β-d-mannanase were assayed by determining the reducing sugars produced from cmc and gum locust bean (mannan) as glucose or mannose, respectively (haggett et al. 1979; araujo and ward 1990). one unit was defined as enzyme which liberates one p.mol glucose or mannose per minute. the glycosidase activities (β-dmannosidase, β-d-glucosidase, and α-d-galactosidase) were assayed using nitrophenyl glycosides as substrates and one unit was defined as enzyme which liberates one umol nitrophenol per minute (ide et al. 1983). specific activity of all enzymes was calculated in unit/mg extracellular protein. determination of protein and fiber component concentration protein concentration was determined by bradford method (1976) and bovine serum albumin was used as a standard. the concentrations of fiber components (cellulose, hemicellulose, lignin and silica) were calculated as neutral and acid dietary fiber according van soest and robertson (1968). determination of optimum ph and temperature the activities of both enzymes were determined at 50°c at different ph (4.6, 5.0, 5.4, 5.8, and 6.2) to obtain the optimum ph, while for determination of the optimum temperature, the enzyme assays were carried out at maximum ph and different temperatures (35, 40, 45, 50, 55, and 60°c). saccharification activity towards pkc and dry pome saccharification activities were determined following determination of avicelase (haggett et al. 1979) using pkc and dry pome as substrates. the incubation time of the reaction was two hours and reducing sugars produced was determined with dns method (miller 1959). the activity value was expressed in unol glucose/ml liberated in one minute. this reaction was also used to determine the synergistic activity of the enzyme mixtures. several proportions of enzymes from e. javanicum and a. niger nrrl 337 (0:100, 20:80, 40:60, 80:20, and 100:0%) were used. aside from the concentration of reducing sugar produced in the reaction, the sugar components of the product were also determined using thin layer chromatography (lestari et al. 2001). the samples and standards (glucose, cellobiose, mannose, mannobiose, and biotropia no. 20, 2003 galactose) were spotted on plates of silica gel 60 (20 x 20 cm). the reducing sugar concentrations of spots from samples were 3-8 чg, while the concentration of each standard was 8 чg. elution was carried out using the mixture of ethanol, n-propanol and water (30:150:20). the elution was stopped when the solvent reached 2 cm from the top. the gel was then dried, and the elution was repeated to get better resolution. spots were detected by spraying the plates by a mixture of aniline (1 ml), diphenylamine (1 g), 80% h3po4 (7.5 ml) in 50 ml acetone and heated at 100°c for 1 hour. results and discussion the activities of enzyme components (cmcase, (β-d-mannanase, (β-d-manno-sidase, β-dglucosidase, and α-d-galactosidase) involved in the fiber hydrolysis were determined (table 1). the enzymes produced from e. javanicwn contained higher activities of cmcase, β-dmannanase, and a-d-galactosidase, while those produced by a. niger contained higher β-dmannosidase and p-d-glucosidase activities. it is already known that aspergillus spp. produce more glycosidases (ghose et al. 1985; manonmani and sreekantiah 1987; haryati et al. 1997). the different major enzyme components of both enzymes suggested the possibility of combination between enzymes. table 1. specific activities of cmcase, p-d-mannanase, p-d-mannosidase, p-d-glucosidase, and a-d-galactosidase of enzymes pr oduced by e. javanicum and a. niger nrrl 337 grown on coconut meal. specific activities (u/mg) molds [protein] cmcase (ug/ml) p-d-man nanase p-dgluco sidase a-dgalacto sidase p-d manno sidase e. javanicum 435 18.6 1172.2 1.5 16.8 1.1 a. niger nrrl 337 498 9.9 14.1 3.1 2.3 3.3 the optimal synergistic activity was obtained when the optimal ph and temperature conditions were applied. the optimal ph and temperature of e. javanicum β-d-mannanase and αd-galactosidase were at 5.4 and 50°c, and 5.0 and 55°c respectively, while those of a. niger βd-glucosidase were at 5.0 and 55°c (figures 1 and 2). the temperature difference gave less effect than the ph difference. a temperature increase from 50 to 55°c reduced β-d-mannanase activity at 22.7%, while the temperature reduction from 55 to 50°c reduced activities of α-d synergistic activity of enzymes on oil palm factory wastes tresnawati purwadaria et al. galactosidase and (β-d-glucosidase at 11.3 and 10.9%, respectively. an increase of ph from 5.0 to 5.4 increased 39.5% of the β-d-mannanase activity, and reduced 24.9 and 13.5% α-dgalactosidase and β-d-glucosidase, respectively. further analyses at these conditions were used to determine saccharification activity towards pkc and pome (table 2). biotropia no. 20,2003 enzymes produced by e. javanicum showed higher saccharification activity towards pkc or pome than those from a. niger due to high activities of e. javanicum cmcase, β-dmannanase and α-d-galactosidase in hydrolyzing cellulose and hemicellulose compounds. the hydrolysis activity upon pkc was also higher than pome which contains higher fiber and minerals. the cellulose and lignin contents of pkc were 14.2 and 20.5%, respectively, while those of pome were 20.8 and 25.6%. the hemicellulose (mannan and galactomannan) contents of pkc and pome were almost similar, being 5.3 and 5.6%, respectively. the microenvironment of ph influenced more significantly saccharification activity than the temperature, therefore the synergism activities towards pkc and pome were carried out at ph 5.0 and 5.4 which were the optimum ph for glycosidases and β-d-mannanase, respectively. both ph conditions were applied at 50°c. all mixtures of enzymes at ph 5.0 or 5.4 showed the synergistic saccharification either towards pkc or pome (figures 3 and 4). the highest synergistic action of the mixture was obtained when higher volumes of a. niger (60 or 80%) were used. for example, in the mixture of 80% a. niger the hydrolysis upon pome at ph 5.0 produced the reducing sugar at 162 uj/ml (figure 4a). without synergistic activity the reducing sugars produced from the 80% of a. niger (86.4 uj/ml) and 20% of e. javanicum (17.8 uj/ml) would be 104.2 uj/ml. the synergistic activity increased the reducing sugars up to 55%. the better activity in the higher a. niger rations indicated that its β-d-glucosidase and β-d-mannosidase play more important role in the hydrolysis system. it was already known that the digestion of disaccharides (cellobiose and mannobiose) by glycosidases reduced the feed back inhibition effects on endoglucanase and endomannanase. it was reported that the highest synergistic action in the culture mixture of t. reesei d-16 and a. went// pt 2804 to produce cellulase and hemicellulase was also observed at composition of 1 (t. reesei): 4 (a. wentii) (ghoseetal. 1985). the optimum ph of e. javanicum β-d-mannanase and α-d-galactosidase were at 5.4 and 5.0, respectively, while that of a. niger β-d-glucosidase was at 5.0 (figure 1). which of the enzyme component had more important role in the synergistic activity was not clearly indicated by varying ph conditions. all ph conditions and substrates produced similar pattern and showed the highest synergistic activity at 60 or 80% a. niger application. moreover, considering the reducing synergistic activity of enzymes on oil palm factory wastes tresnawati purwadaria et a 0:100 20:80 40:60 60:40 80.20 100:0 a. niger : e. javanicum (%) 0:100 20:80 40:60 60:40 80:20 1 a. niger : e. javanicum (%) figure3. synergistic saccharification towards pkc by enzyme mixtures of a. niger and e. javanicum at ph 5.0 (a) and 5.4 (b). the dotted lines represent the theoretical reducing sugar values expected for a non-synergistic saccharification. 0:100 20:80 40:60 60:40 80:20 100:0 a. niger : e. javanicum (%) 0:100 20:80 40:60 60:40 80:20 100:0 a. niger : e. javanicum (%) fiaure4. syncrmstic saccharification towards pome by enzyme mixtures of a. niger and e. javanicum at ph~5.0 (a) and 5.4 (b). the dotted lines represent the theoretical reducing sugar values expected for a non-synergistic saccharification. sugar produced by pkc and pome at ph 5.4 by 100% a. niger (143 jg/ml and 130 uj/ml, respectively) was higher than that of 100% e. javanicum (78 uj/ml and 69 fg/ml, respectively), then the proportion of a. niger should be higher than 80% on pkc and 60% on pome (figures 3 and 4). although the optimum ph of a. niger β-d-glucosidase was at 5.0, only saccharification upon pome showed a better synergy at ph 5.0, while that upon pkc was better at ph 5.4. the obtained result might be due to the high mineral concentration in pome that influenced the activity of each enzyme component differently. the ash and silica contents of pkc were 8.5 and 1.7%, respectively, while those of pome were 17.6 and 9.5%. the high mineral or cation contents in pome might have influenced the buffering capacity of the substrate and affected the ph in the reaction. biotropia no. 20,2003 the role of every enzyme component in the synergistic action ws difficult to be detected since all enzyme components took part in the reaction. detailed successive reaction in the synergistic action had been clearly observed in the pure enzyme components (purwadaria 1995). however, results from tlc plates indicated that different concentrations of sugar components were produced on pkc and pome by the hydrolysis action of the mixtures of e. javanictan and a, niger (figure 5). k5 85 ke s« figure 5. thin layer chromatogram of reducing sugars produced from synergistic sachanfication of e. javanicum and a. niger at ph 5.0. saccharification activities were determined towards pkc (a) and pome (b). glucose (gi), cellobiose (gi), mannose (mi), mannobiose (m2), and galactose (ga) were used as reference. the concentration of each standard was 8 \%. ki, kj, k3, kj, k5, and k,, were controls without incubation, while si, s2, s3, s4, s5, and s6 were samples from the composition of a. niger : e. javanicum at 0:100, 20:80,40:60, 60:40, 80:20, and 100:0%. the chromatogram of pkc digestion was clearer than that of pome due to its higher mineral content which had disturbed the separation (figure 5). the reducing sugars of controls produced from the mixture of enzymes and substrate without incubation were compared with samples from the mixture of enzymes and substrate with incubation. smaller amounts of reducing sugars were detected from the controls (substrates) compared to the samples. oligosaccharides (trimers and more) resulted from endoglucanase or endomannanase activities especially by higher composition of e. javanicum in the incubated samples (figure 5a-s], 82, and 83) were much more than those of controls. the oligosaccharides produced were further digested by glycosidases of e. javanicum and the addition of glycosidases from a. niger (figure 5a and b). addition of 60 or 80% a. niger to the enzyme mixtures produced more monomers and dimers including mannose, glucose, galactose, and cellobiose. the possibility of higher hydrolysis activity resulted from synergistic action will contribute a beneficial effect in the enzyme application on monogastric. the synergistic activity of enzymes on oil palm factory wastes ~ trtsnawati purwadaria tl at, incorporation of carbohydratases known as inducer enzymes is inhibited by the feed soluble sugar content prepared for energy source. the addition of glycosidases produced by a. niger (β-dglucosidase and β-d-mannosidase) on top of α-d-galactosidase from e.javanicum might have increased the digestion of the dimmers such as cellobiose and mannobiose that reduced the inhibition effect of the dimmers (short olygosaccharides) on endoglucanase and endomannanase. therefore, the application of enzyme cocktails in animal di»ts is considered to be more appropriate than single enzyme. references araujo, a. & , o. p. ward. 1990. extracellular mannanases and ;.alactanases from selected fungi. j. ind. microbiol. 6:171178. bradford, mm. 1976. a rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. anal. biochem. 72: 248-254. ghose, t.k., t panda, and v. s, bissau. 1985. effect if culture phasing and mannanase on production of cellulose and hemicellulase by mixed culture ot trichoderma reesei d 1-6 and aspergillus wendtii pt 2804. biotechnol. bioeng. 25: 1353-1361. haggett, k.d., p.p. gray and n.w dunn. 1979. crystalline cellulose degradation by a strain of cellulomonas and its mutants derivatives. eur. j. appl. microb. biotechnol. 8: 183-190. haryati, t, t. purwadaria, j. darma, and b. tangendjaja, 1997. production of extracellular glycosidase by eupenicillium javanicum and aspergillus niger nrrl 337 on the coconut meal substrate. proc. second conf. on agricultural biotechnology, jakarta, indonesia, 13-15 juni 1995.aard, indonesia, p. 517-522 ide, j.a., j. m. daly, and p.a.d. rickard. 1983. production of glycosidase activity by cellulomonas during growth on various carbohydrate substrate. eur. j. appl. microb. biotechnol. 18: 100-102. lestari, p., a. a. danvis, k. syamsu, n. richana, and d.s. damardjati. 2001. analisis gula reduksi hasil hidrolisis enzimatik ubi kayu oleh a-amylase termostabil dari bacillus stearothermophilus til 12 (reducing sugar analyses on the enzymatic hydrolytic product of cassava by thermostabil a-amylase from bacillus stearothermophilus til 12). j. mikrobiol. indon. 6: 23-26. manonmani, h.k. and k.r. sreekantiah. 1987. saccharification of sugar cane bagasse with enzymes from aspergillus ustus and trichoderma vinde. enzyme microb. technol. 9: 484-488. miller, g.l. 1959. use of dinitrosalicylic acid reagent for determination of reducing sugar. anal. chem. 31:426-428. purwadaria, t. 1995. synergism in the hydrolysis of cellulose by endoglucanase i and ii (endo 1 and ii) and cellobiohydrolase (cbh i) purified from cellulomonas cs1-17. annalcs boaoricnses. 3: 12-24. purwadaria, t., a.p. sinurat, t. haryati, i. sutikno, supriyati and j. darma. 1998. korelasi antara aktivitas enzim mananase dan selulase terhadap kadar serat lumpur sawit hasil fermentasi dengan aspergillus niger (the correlation between mannanase 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vol. 21 no. 2, 2014: 71 81 high turbidity affects filtration rate and pseudofaeces production of the mud clam (solander 1786) (bivalvia: corbiculidae) polymesoda erosa francis albert t. argente *, senona a. cesar and danilo t. dy received 18 october 2012/accepted 1 october 2014 is an economically and ecologically important bivalve which thrives in brackish water mangroves or zones. unpredictable weather conditions and unregulated anthropogenic activities in mangrove area can lead to high turbidity conditions and possibly affect the filtering capacity of . this study will aid in the development of culture technique of this species in the region. results could also be used to facilitate adequate stock management for potential commercial exploitation in central philippines. a laboratory experiment was conducted to determine the effects of turbidity concentration and body size on the filtration rate and pseudofaeces production of . filtration rates significantly increased with higher turbidity concentration up to 750 mg/l ( < 0.05). pseudofaeces production also increased with increasing turbidity concentration ( < 0.05). body size did not affect the filtration and pseudofaeces production of . results suggested that was resilient to highly disturbed turbid environments and therefore, could be a potential candidate species for aquaculture. biodeposit, bohol, , pseudofaces production, silt, suspension filter-feeders ,1,3 2,3 3 1 2 3 pangasinan state university-binmaley campus, binmaley, pangasinan, philippines visayas state university, baybay city, leyte, philippines university of san carlos, cebu city, philippines abstract introduction polymesoda erosa nypa p. erosa p. erosa p p p. erosa p. erosa nypa polymesoda erosa,keywords: development in a country is coupled with increasing demand for food that could be sourced out from aquatic ecosystems. among many aquatic species, bivalves are now heavily cultured to meet such demand, yet there are still some fisheries that heavily rely on the wild stock, but with high potential for mariculture (nuryanto & susanto 2010). understanding the physiological and ecological attributes of a species is crucial in deciding its mariculture feasibility. such species include the mangrove or * corresponding author : faargente@gmail.com doi: 10.11598/btb.2014.21.2.1 71 mud clam, (solander 1786), synonymously known as (morton 1984). is widely distributed in the indo-pacific region (morton 1984, nuryanto & susanto 2010). it is also found in northern australia as component of earth mounds (gimin 2004, brookwell 2006). possesses some characteristics that make it both an economically and ecologically important species. this clam is a potential source of bioactive compounds exhibiting antiviral (chatterji 2002) and antimicrobial (sharma 2009) properties. its congeneric species, is found in 16 estuaries in mexico and is part of mexico's commercial fishery (wakida-kusunoki & mackenzie 2004) while is also heavily exploited in the artisanal fishery in india (clemente & ingole 2009), malaysia (hamli 2012), indonesia (hardinsyah 2006), northern australia (gimin 2005) and philippines (germano 2003, laureta 2008). is noted to be harvested and traded in bohol, central philippines (argente 2012). biological studies showed that is dioecious and hermaphrodite (morton 1988), with no external dimorphism (clemente & ingole 2009) but with high polymorphism (nuryanto & susanto 2010). it can attain a size of 110 mm shell length (morton 1988). in goa, india where it is an exploited fishery product, adult shell density is 9 ind./m , described to be of non-random, patchy distribution (clemente & ingole 2011). clam harvest is still done by hand gleaning between thickets. thrives in extreme adverse environmental conditions like low ph (morton 1976); in waters with high heavy metal concentration (modassir 2000), and high turbidity (yap & mohd azri 2009). it is an oligohaline species (morton & chan 1986). field studies have reported that thrives in brackish water environments with salinity ranges of 7-31 ppt (clemente & ingole 2009, yap & mohd azri 2009) but probably prefer higher salinity range since the low tide mark does not support adult population (clemente & ingole 2011). it can be found in areas only inundated during very high spring tides (clemente & ingole 2011), with ability to rapidly resume filter feeding activity when inundated (yap & mohd azri 2009). thrives well in true mangrove dominated area or in dominated estuarine area. is a filter feeder, referred as suspensivore by wakida-kusunoki and mackenzie (2004), with ability to remove particles from water current by bio-filter mechanism accomplished by the gills (ruppert & barnes 1994). this form of nutrient acquisition significantly contributes to bentho-pelagic coupling (barnes 2006, jones 2011). the removal of suspended particles in the water column by filter feeding results in the production of biodeposits (clavier & chauvaud 2010); particles coated with mucus which are processed in the mantle cavity and ejected through the inhalant siphon or along the ventral mantle margin (berg 1996). in loay-loboc river, bohol, central philippines, natural population of may experience high turbidity condition due to unregulated anthropogenic activities and erratic weather disturbances. currently, it is not yet known up to what extent can tolerate the turbidity concentrations that are normally encountered in natural as well as in disturbed mangrove or environments. there are evidences that variations in body size and concentration of suspended particles influence their physiological responses (rajesh 2001, rueda & smaal 2002, hatton 2005). thus, a laboratory experiment was conducted to determine the effect of body size and polymesoda erosa geloina coaxans p. erosa et al. p. erosa et al. et al. p. caroliniana p. erosa et al. et al. et al. et al. polymesoda p. erosa nypa p. erosa p. erosa p. erosa nypa fruticans p. erosa et al. et al. p. erosa p. erosa nypa et al. et al. 2 72 biotropia vol. 21 no. 2, 2014 turbidity concentrations on the filtration rate and pseudofaeces production of collected in loay-loboc river, bohol, central philippines. this study will aid in the development of culture techniques of this species in the region. results could also be used to facilitate adequate stock management for potential future commercial exploitation in central philippines. at the moment, the populations in central philippines are harvested and considered as low level artisanal fishery for sustenance consumption (laureta 2008, argente 2012). the clams were collected from a zone in masayon, calvario, loay-loboc river, bohol (9.60853° n, 124.01265° e) (fig. 1). the estuarine river is known for different anthropogenic activities such as fishing, gastropod and bivalve gleaning, recreational boat cruise and sand quarry. water samples were taken from the collection site to determine the prevailing salinity and total suspended solids (tss) condition. substrate samples were also collected for use as suspended particles during the experiment. the clams were brought to the laboratory (university of san carlos marine station, mactan island) within 24 hours after collection and acclimated in a basin filled with filtered brackish water (7 ppt) for 12 hours prior to the experiment. the clams were not fed during the acclimation period. two size classes of , smaller-sized clams (47.4±4.3 mm) and larger-sized clams (58.0±2.4 mm) were used during the experiment. substrate samples mostly composed of sand and mud were wet sieved to separate the silt (< 63 µm diameter). the silt were air-dried, pounded and oven-dried (75 °c) to constant dry weight. experimental concentrations of suspended silt were based on the prevailing tss condition at the collection site. a range finding test was conducted to determine the turbidity concentrations used in the experiment. consequently, four higher turbidity concentrations (250, 500, 750 and 1,000 mg/l) and a control (40 mg/l) were used. the experiment employed a randomized complete block design (rcbd) with the experimental replications as the blocking variable. an experimental unit (fig. 2) included an individual clam glued on a bamboo stick using aquatic epoxy and placed in a plastic container filled with 1 l filtered brackish water (7 ppt) and silt particles of the established experimental turbidity concentrations. the container was aerated from the bottom to ensure suspension of silt particles throughout the experiment. a piece of plastic was placed above the air source to avoid clam distress which may affect filtration activity. six replications were conducted for each experimental turbidity concentration per size class. each clam was randomly allocated to an experimental unit and a wait-period of approximately five minutes was observed before the actual timing of the incubation experiment which lasted for 120 minutes (2 hours). after the experiment, the clams were removed from the experimental units. pseudofaeces clinging from the bamboo p. erosa p. erosa nypa p. erosa materials and methods 73 filtration rate and pseudofaeces production of francis albert t. argentepolymesoda erosa – et al. sticks, clam shells and walls of the containers were collected and placed in pre-labeled petri dishes and oven dried (75 °c) to constant dry weight. the brackish water in the experimental units were filtered using pre-weighed (constant dry weight) whatmann™ gfc 47 mm filters. the filters with residues were air-dried for 24 hours and oven-dried at 75 c to constant dry weight o . 74 biotropia vol. 21 no. 2, 2014 figure 1. map of loay-loboc river, bohol, central philippines. collection site is shown with a marker (star). inset map a is the philippines, b is bohol figure 2. experimental unit of the study bamboo stick clam plastic container piece of plastic air stone air hose the filtration rate (fr; mg/minute/ind) and pseudofaeces production (pp; mg/minute/ind) of the were determined using these equations: fr = [tss (filter filter )] / time elapsed (1) pp = wt of pseudofaeces / time elapsed (2) where: tss = initial quantity (mg) of total silt in the experimental unit filter = initial weight (mg) of the filter filter = weight(mg) offilter with residue time elapsed = 120 minutes a two-way anova (analysis of variance) with replications was used to determine the effects of body size and turbidity concentration on the filtration rate and pseudofaeces production of the experimental clam. raw data were logtransformed (log+1) to satisfy parametric statistical assumptions. in cases of significant differences, the tukey hsd test was used as post hoc test. the significance level was set at 95% ( = 0.05). in this study, the prevailing salinity in the sampling area was 7 ppt, while the prevailing total suspended solids (tss) was 40 mg/l. total organic matter content of substrate was 30%. table 1 shows the results of two-way anova on the effects of turbidity concentration (tc) and body size (bs) to the log-transformed fr and pp of . tc significantly influenced fr and pp ( < 0.05). however, bs showed no significant effect on fr and pp. likewise, no interaction effect between tc and bs was observed. the log-transformed data showed that the fr of significantly increased with higher tc up to 750 mg/l, beyond which no significant increase was observe (fig. 3). similarly, the log-transformed pp of also showed significant increased with higher tc (fig. 4). however, the variations in pp within each tc were wider compared to fr. bivalves tend to escalate their filter feeding behavior with higher turbidity conditions (bayne 1993, rajesh 2001). in highly turbid waters, these filter feeders consume much energy to filter suspended particles (hibbert 1977). consequently, bivalves have different threshold limits in their filter feeding activities (berg 1996, rajesh 2001). exceeding beyond the threshold may have negative effects in the physiological responses of these species within their environment. in the work of morillo-manalo and del norte-campos (2010) on a burrower bivalve, , the initial increase in filtration rate followed by a decrease at higher p. erosa p p. erosa p p. erosa p. erosa et al. et al. et al. et al. paphia undulata i r i i i r results and discussion water quality measurement results filtration rate (fr) and pseudofaeces production (pp) of p. erosa 75 filtration rate and pseudofaeces production of francis albert t. argentepolymesoda erosa – et al. 76 biotropia vol. 21 no. 2, 2014 effect ss df ms f p-value log-transformed fr tc 14.114 4 3.528 501.100 0.000* bs 0.004 1 0.004 0.580 0.448 bs*tc 0.031 4 0.008 1.100 0.369 error 0.352 50 0.007 log-transformed pp tc 17.064 4 4.266 130.495 0.000* bs 0.021 1 0.021 0.640 0.427 bs*tc 0.079 4 0.020 0.604 0.661 error 1.635 50 0.033 table 1. anova results on the effects of turbidity concentration (tc) and body size (bs) to the log-transformedfiltration rates (fr) andpseudofaecesproduction (pp)of p. erosa figure 3. log-transformed filtration rates of larger-sized (a) and smaller-sized (b) at different turbidity concentrations p. erosa (bars indicate standard deviation) l o g -t ra n sf o rm ed f il tr a ti o n r a te s (m g /m in u te /i n d .) turbidity concentration (mg/l) 1.8 40 2.0 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 a 250 500 750 1,000 1.8 2.0 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 40 250 500 750 1,000 b concentration, was attributed to the inherent nature of the clam to stimulate valve opening and filtration activity when exposed to lower concentration. then, at higher concentration, this may result to overloading of the ctenedia, the filter apparatus that led to closing of the valve, thus reduction in fr. however, in this experiment, the plateau of fr was not yet reached. ecologically, it could mean that can still withstand higher turbidity level, as in the case during heavy rains of which the loayloboc river receives water from tributaries as far as carmen (the interior part of bohol). the unpredictable weather disturbances and the increasing human perturbations can result in turbid aquatic environments. bivalves with higher threshold of filtration would be more resilient in disturbed waters. in this study, showed potential in adapting to such situation. the fr of the clam significantly increased in turbidity conditions of up to 750 mg/l. this was a remarkable feat as compared to , and which could filter in turbidity concentrations ranging from 10 to 570 mg/l (bayne 1993, navarro & widdows 1997, rueda & smaal 2002). p. erosa p. erosa mytilus edulis cerastoderma edule spisula subtruncata et al. 77 figure 4. log-transformed pseudofaeces production of larger-sized (a) and smaller-sized (b) at different turbidity concentrationsp. erosa (bars indicate standard deviation) filtration rate and pseudofaeces production of francis albert t. argentepolymesoda erosa – et al. l o g -t ra n sf o rm ed p se u d o fa cc es p ro d u ct io n (m g /m in u te /i n d .) turbidity concentration (mg/l) a1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 -0.2 2.0 40 250 500 750 1,000 40 250 500 750 1,000 b 78 biotropia vol. 21 no. 2, 2014 p. erosa p. erosa et al dreissena polymorpha spisula subtruncata paphia undulata et al. p. erosa p. erosa perna canaliculus perna viridis crassostrea madrasensis et al. paphia undulata paphia undulata p. erosa et al. et al. et al. et al. p. erosa et al p. erosa p. erosa the pp of showed increasing rate at higher tc. morillo-manalo and del norte-campos (2010) stated that the production of pseudofaeces implies maximum energy gain for the bivalves during feeding. produced more pseudofaeces in higher tc to optimize energy gain instead of utilizing more to metabolize excess particles. this is also a mechanism to regulate ingestion rate as well as preventing saturation of the gills. iglesias . (1998) equated fr to ingestion rate, when all filtered material is ingested with no pseudofaeces production. similar condition was encountered with other bivalves such as , and (bayne 1993, rueda & smaal 2002, morillo-manalo & del norte-campos 2010). the bs of did not affect the filtration and pseudofaeces production. during the experiment, the valves of opened all throughout the incubation period indicating that they were actively filtering regardless of size. in other bivalves such as , and , these physiological responses tend to increase with larger body size (rajesh 2001, hatton 2005). smaller individuals of these species may have little chance of survival in highly turbid waters. on the other hand, the short-necked clam showed a reduction of fr with increasing bs (morillo-manalo & del norte-campos 2010). the higher fr by smaller is supposedly needed to support its fast growth and development (del norte-campos & villarta, 2010), compared to which is considered to be a long lived species (morton 1988). another important consideration here was the range of shell length between the two treatment levels used in the study. a wider range (i.e. juvenile size vs. mature size) in future experiment might reveal important information on the effect of clam size. in this study, we experienced difficulty in obtaining small clams in the collection site. ecologically, the filtration of suspended particles and pseudofaeces production are physiological functions of bivalves (berg 1996, higano 2004). the processing of suspended materials in the water column carried out by suspension filter feeders was significant in assessing trophic relationships in freshwater, estuarine and marine ecosystems (prins 1996, espinosa 2008, manganaro 2009, sarikhani & javanshir 2010, mamun & khan 2011). this study suggested that natural population of in loay-loboc river was resilient in their environment, which agreed to experiments conducted by yap and mohd azri (2009) and by bayen . (2005). in this time of climate change and rising human population, the search for new and better adapted species for aquaculture is deemed necessary (de silva & soto 2009). the resiliency of to adverse environmental conditions makes it a potential candidate species for aquaculture population in loay-loboc river was resilient with the ability to respond to highly turbid waters. the filtering activity and pseudofaeces production in the population tended to escalate at higher turbidity concentrations up to 750 mg/l. size is a non-factor in its physiological responses suggesting high survivability in disturbed, conclusions 79 turbid waters compared to other bivalves. as a result of its resiliency to adverse environmental conditions, would be considered as potential candidate species for aquaculture in central philippines. we thanked the university of san carlos, marine biology section for logistic support. we also thanked mr. henry palaca for collecting the clams used in this study. the scholarship grants were given by the department of science and technologyphilippine council for agriculture, aquatic and natural resources research and development (dost-pcaarrd) to fata and sac were hereby acknowledged. this was a marine science contribution of psu, vsu and usc. p. erosa acknowledgements references argente fat. 2012. commercially important mangrove bivalves of the visayas, philippines: diversity and fishery. in: 21 annual fr. heinrich schoenig biology: symposium: 2012 march 3. philippines. university of san carlos talamban campus, cebu city. barnes p. 2006. . vancouver(ca): centre for shellfish research, malaspina university-college, british columbia. bayen s, wurl o, karuppiah s, sivasothi n, lee hk, obbard jp. 2005. persistent organic pollutants in mangrove food webs in singapore. chemosphere 61: 303-13. bayne bl, iglesias jip, hawkins ajs, navarro e, heral m, deslous-paoli jm. 1993. feeding behaviour of the mussel, : responses to variations in quantity and organic 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b ril 60; c conventional logging; and d) primary forest ) ) as control total plot permanent area was 48 ha and was measured periodically every 2 years within 17 years after . about logging. for data analysis purpose, trees were divided into dipterocarps and non-dipterocarps. two major groups, i.e. range of mortality rates for all species in logged-over forest were 2.5-29.3% per ha per 2 years which was close very to primary forest at year-5 after logging. while range of ingrowth rate for all species in logged-over forest were 1.3-21.3% per ha per 2 years which were higher than those for the primary forest within 17 years. the mortality and ingrowth rates fluctuation of dipterocarps species group were different from those of non-dipterocarps. : keywords dipterocarps, ingrowth, logged-over forest, mortality introduction lowland tropical rain forest is a natural forest with trees having typical characteristics, harboring the greatest species diversity in the world (whitmore 1990; richards 1996) and having numerous variations of tree's dimensions (prodan 1968). lowland tropical rain forests in southeast asia are dominated by dipterocarpaceae (ashton 1982), therefore, it is often referred to as the dipterocarp forests. dipterocarp forest is a tropical rainforest inhabiting type a and type b climate types area, covering sumatera, kalimantan, sulawesi, north maluku and papua with the highest layer of forest canopy filled with family dipterocarpaceae, especially genus shorea, dipterocarpus, dryobalanops hopea and (ashton 1982). dipterocarp forest in west malesia region is the most productive tropical forest types based on timber value (fao 2001). in indonesia, dipterocarpaceae is the largest contributor (over 25%) to commercial timber forests in decades, with volume of 50-100 m per ha, especially in 3 kalimantan (sist . 2003)et al . in primary and logged-over natural forest, the stand stand conditions having differences in structure, species composition, tree density, canopy structure, mortality and ingrowth, will have varied growth rates depending on tree the age after logging (silva . 1995; lewis . 2004; et al et al ishida . 2005). recovery of a logged-over et al forest happens in a long period after logging (smith & nichols 2005), which varies depending on deforestation rate and environmental carrying capacity (muhdin . 2008). natural production et al forests in indonesia have more than 50% of logged-over forests (ditjen planologi kehutanan 2011). therefore, it is very important to know * corresponding author : fhsusanty@gmail.com biotropia vol. 22 no. 1, 2015: 11 23 doi: 10.11598/btb.2015.22.1.297 11 mailto:fhsusanty@gmail.com biotropia vol. 22 no. 1, 2015 12 about the variation of tree characteristics in a logged-over forest. biology and ecology information of dipterocarps is needed as scientific basis for developing effective forest management policies (naito . 2008).et al forest biometric characteristics is among quantitative approaches to study the properties or characteristics of forest trees in size (metric) for a specific biological dimension as the user identification by ratio and interval scale (prodan 1968). input variables to determine quantitative tools are mostly provided by classical forest inventories on plots (vanclay 2003; gourletfleury . 2005). most of the early biometric et al research are found in studies on plantations and temperate forests that do not have such complexity as tropical forests. the heterogeneity and complexity of obstacles occur in the forms of diversity and variation of conditions as well as limitations or lack of long term data observation. average rate of mortality and its correlations to several reliable and measurable variables in size or site characteristics as input factors mostly determine the mortality model (keister 1972; hamilton & edwards 1976; monserud 1976; hamilton 1994; monserud & sterba 1999; cited flewelling & monserud 2002). according to chertov . (2005), a new paradigm in achieving et al sustainable forest management requires prediction of effective growth forest tree dynamics involving aspects of ecological characteristics. to achieve sustainable forest management, preparation of quantitative management tools such as yield regulation models becomes very important. important variables needed to build the models are mortality and ingrowth rates of forest trees. this aimed to determine study mortality and ingrowth rates of dipterocarps and non-dipterocarps spesies groups for 17 years after being logged, which rates will be used to formulat biometric characteristics variability e of dipterocarp forest in logged-over forests based on time series data. materials and methods study site this study was carried out at labanan research forest station (1˚49'-2˚10' n and 116˚7'-117˚27' e) located in berau region, east kalimantan province. according to schmidt and ferguson climate classification (1951), the study site was within type b climate (q = 14.3–33.3%). based on koppen system classification the study site was within type afa climate with many rainy days over in a year with mean annual precipitation about 1,800-3,000 mm/year. the highest monthly mean precipitation happened in january (242.5 mm) and the lowest is in august (90.9 mm). maximum temperature rate happens in september and november (35 ºc) and the lowest was in february and august (21 ºc), with average temperature of 26 ºc. the study site was located at 500 m above sea level (asl) and it was a relatively hilly forest. soil type in labanan research forest station consisted of ultisol (87.3%), entisol (10.7%) and incenptisol (2.0%). labanan research forest station as a low land tropical forest is dominated by family dipterocarpaceae, consisted of 7 genera i.e. anisoptera, cotylelobium, dipterocarpus, dryobalanops, parashorea, shorea vatica and . besides family dipterocarpaceae, other dominant genera are also present in labanan research forest station such as sapotaceae, meliaceae, moraceae, ebenaceae, sapindaceae leguminaceae and . among landscapes at the labanan research forest station was a swamp forest dominated by and lophopetalum shorea balangeran (saridan and susanty 2005. figure 1. study site-labanan research forest station, berau, east kalimantan province mortality and ingrowth pattern of dipterocarps in forest recovery in east kalimantan farida h. susanty – et al. data collecting permanent plots were set up in the logged-over forest as well as in the primary forest. the size of each plot was 200x200 m (4 ha) which was divided into 4 square subplots with size of 100x100 m (1 ha). the permanent plots were built in 4 condition variations with total area of 48 ha. measurements were carried out by census method for all species with limit diameter of 10 cm including number of trees, tree species, stem diameter (diameter at breast height or 20 cm above buttresses) and number of dead trees. repeated measurements were performed every two years. reduced impact logging can be defined as logging technique to minimize environment impact on forest trees and soils (dykstra 2008). this technique is needed to preserve ecological aspect of forest trees and to ensure sustainable yield of production forest in the future. data used in this study were data collected from 1990 to 2008. treatments applied on research plots were as follows: a) ril 50: reduced impact logging techniques with limit diameter of 50 cm, skid trail planning was based on contour maps and tree position as well as supervision of tree felling and skidding (3 plots). b) ril 60: reduced impact logging techniques with limit diameter of 60 cm, skid trail planning was based on contour maps and tree position as well as supervision of tree felling and skidding (3 plots). c) cnv: conventional logging techniques with limit diameter of 60 cm, no skid trail planning, conducted without considering contour line maps or tree position, felling was done by loggers experiences (3 plots). d) pf: primary forest as controls (3 plots). data analysis data organization was carried out using database software microsoft visual foxpro 9.0, while data analysis was performed by using spreadsheet and spss 15.0. data were analyzed based on tree density and tree structure (stems per ha) as well as basal area (m per ha) with two major 2 species groupings e.g. dipterocarps and nondipterocarps species. calculations of mortality and ingrowth rates were performed every 2 years. residual tree characteristics assessment was done by comparing variations in forest conditions by using different test mean values (t-test), analysis of variance (anova) and regression analysis. regression equation tested was linear equations, polynomials, exponential and logarithmic. criteria used for selecting the best equation were based on the regression coefficient (r), determination coefficient (r ) and the highest 2 value of the smallest standard error (se) (steel & torrie 1995) results and discussion tree density the dynamics of logged-over forest within 17 years were represented by number of trees per hectare and basal area per hectare against tree fluctuations after-logging using different logging techniques (fig. 2). mean values of tree density at the initial conditions (pre-harvest) were compared using t-test and the results showed no significant differences in all study plots (t <t ). fluctuations of tree density calc tab(0:05; 6) and basal area of logged-over forest would increase up to the initial conditions before logging (primary forest). logged-over dipterocarp forest recovery reached the conditions close to the primary forest at 9 years after-logging based on tree density and at 11 years after-logging based on basal area. according to gourlet-fleury . et al (2005) and kao and iida (2006), tree density extremely increases at 5 to 7 years after-logging, which was explained by the increasing growth of open canopy in after-logging periods. range of tree density in the studied loggedover forest was 461-647 stems per ha with average value of 531 stems per ha. sianturi and kanninen (2005) stated that at the period of 2 years afterlogging in jambi forest, the tree density reached 90% compared with tree density at the same forest before logging. tree density in a primary forest in sangai, central kalimantan (478-738 stems per ; ha average of 583 stems per ) was similar to tree ha density in labanan forest (susanty 2006). this study results were also similar to the condition in eastern amazon forest showing average tree density of 480±96.6 stems per ha (sist & ferreira 2007). while gourlet-fleury . (2005) et al presented higher value of tree density for french guiana forest (625 stems per ha). 13 range of basal area value in the studied logged-over forest was 19.35-31.84 m per ha. as 2 comparisons, basal area value in a several years after-logging forest in central kalimantan ranged from 16.4 to 26.7 m per (krisnawati 2001). a 2 study conducted by setiawan (2013) showed that basal area value in a logged-over forest in muara wahau, east kalimantan ranged from 12.63 to 32.57 m per ha, while basal area value in a 2 primary forest in muara wahau, east kalimantan ranged from 27.80 to 32.57 m per ha. basal area 2 value in amazon forest had average of 28±4 m 2 per ha (sist & ferreira 2007). in logged-over forests, tree density was about 93-102% and basal area were about 81.0-88.8% compared with initial condition or before logging condition. based on this result, the recovery pattern of forest seemed to have positive trend with similar variations. the tree density afterlogging variable is important to know ecological sustainability (sist . 2003; smith & nichols et al 2005; muhdin 2012). this variable may indicate the level of all logged-over forest which have well recovered, with assumption no interference or disturbance, thereafter. different recovery patterns of logged-over forest influenced ecological factor and forest tree condition. growth monitoring on a 24 ha permanent sample plots in silviculture experiments in amazônia (1980-1989) indicated that even in the same block of concession area, forest recovery has different characteristics and patterns shown by fluctuations of tree dynamic (silva 1995). initial et al. succession process in logged-over forests started from the end of logging operational, followed by growing process in a time series function. mortality rates the mortality level in different logging techniques is influenced by logging intensity based on number of trees and volume per hectare which were felled by logging. logging intensity in ril 50 had felled trees of 10.7 4.9 stems per ha + with volume of 96.8 66.5 m ha per; ril 60 had + 3 felled trees of 7.0 3.0 stems per ha with volume + of 56.5 23.3 m per ha and cnv had felled + 3 trees of 10.1 4.2 stems per ha with volume of + 107.2 59.6 m per ha (sist & bertault 1998). + 3 these results indicated that logging intensity had a tendency to increase from ril 60, ril 50 and cnv logging techniques, respectively. in the logged-over forest, the mortality rates for all species ranged from 2.5 to 29.3% per ha per 2 years (table 1). the highest mortality rates occurred in year-1 and year-3 after logging then declined or was similar to mortality rates at the primary forest in year-5 after logging. the mortality rates in this study were higher than those in some studies; for example mortality rate in year-2 after logging in east kalimantan forest was 2.5% per year (primack . 1985; nguyen-et al the . 1998); mortality rate in a logged-over et al forest in papua new guinea was 2.5% per ha per year (mex 2005); mortality rate in a mixed dipterocarp forests in asia was 1.5% per year (nguyen-the . 1998). compared to other et al forest types, dipterocarp forest has lower mortality rate than that of peat swamp forest which was 6.13% per year and of heath forests which was 4.26% per year (nishimua . 2006).et al mortality rates in forest with different logging techniques indicated higher logging intensity for both dipterocarps and non-dipterocarps (fig. 3). biotropia vol. 22 no. 1, 2015 14 figure . forest trees for all species with different logging techniques based on (a) average tree density (stems per ha) and (b) 2 average basal area (m per ha)2 non-dipterocarps species have a tendency to have higher mortality rate than dipterocarps. mortality rate for both dipterocarps and nondipterocarps in primary forest ranged from 2.0 to 6.0% per ha per 2 years with average of 3.29% per ha per 2 years. the increasing logging intensity was negatively correlated with stem densities, species abundance and biodiversity, where prior changes in the logged-over forest for the first 5–10 years after logging were gradually occurred in successive samples (kariuki . 2006). the et al mortality rates of trees in a forest is relatively high within 7 years, indicating that damaging effects of logging would permanently influence trees, either directly or indirectly. in other words, within 7 years after logging, the remaining trees will begin to recover. fluctuations of mortality rate within 17 years after logging indicated that the mortality rates decreased over years for both dipterocarps and non-dipterocarps. a theoretical frame for ingrowth limitations, climatic variation or many factors became the constraints to predict treelevel change (harcombe . 2002). several et al variables causing differences of mortality rates were logging intensity, initial structure and dominant species composition. the mortality degree is different for every tree's stage in the forests. ingrowth rates ingrowth rates in logged-over forest have the same tendency with mortality rates that correlate with level of logging intensity (table 2). in this case, conventional logging technique can still control the total felled trees to only cut down trees having limit diameter of 60 cm. the ingrowth rates in logged-over forest (1.3-21.3% per ha per 2 years) were higher than those in primary forest (0.6-4.7% per ha per 2 years). the highest 15 table . tree mortality rates (% per ha per 2 years) for all species1 forest condition pal1 pal3 pal5 pal7 pal9 pal11 pal13 pal15 pal17 (% per ha per 2 years) ril50 mean 23.7 10.9 4.3 5.8 3.0 2.9 2.7 3.4 3.3 sd 10.6 6.2 1.4 1.2 0.7 1.1 1.1 1.7 1.5 ril60 mean 22.3 8.3 4.4 5.5 3.3 2.5 2.5 2.5 3.5 sd 8.1 3.5 1.1 1.3 0.9 0.8 1.1 1.3 2.0 cnv mean 29.3 12.8 2.8 6.8 3.3 3.6 2.8 2.9 4.3 sd 9.2 8.0 1.3 2.3 0.9 2.1 1.0 1.9 0.4 p1 p3 p5 p7 p9 p11 p13 p15 p17 pf mean 2.9 3.2 4.7 6.0 3.2 3.4 2.7 2.0 3.2 sd 1.0 1.3 5.1 1.8 1.2 1.5 1.5 1.4 2.0 notes : pal = period after logging (years) sd = standard deviation p = monitoring period (years) figure . mortality rates (stems per ha per 2 years) of trees with different logging techniques for species group (a) 3 dipterocarps and (b) non-dipterocarps mortality and ingrowth pattern of dipterocarps in forest recovery in east kalimantan farida h. susanty – et al. ingrowth rate for all species occurred at year-5 to year-7 (table 2). after logging, the ingrowth rates increased substantially during the first 5 years. during the next five years, there was a sharp decrease. hardiansyah . (2005) stated that at et al year-1 after logging in jambi, ingrowth rate was 0.19-2.89% per year with average 2.1% per year. meanwhile, in papua new guinea mex (2005) stated that at year-13 after logging the trees had smaller ingrowth rate with mean of 41 stems per ha. in the primary forest, ingrowth rate fluctuation were relatively low i.e. 2.0-4.7% per ha per 2 years with average of 2.5% per ha per 2 years. fluctuations of ingrowth rates for dipterocarps and non-dipterocarps species in logged-over forest with different logging techniques had different patterns (fig. 4). in this study 68 tree families had been identified (appendix 1), in which there were 8 genera of dipterocarps (appendix 2) namely anisoptera, cotylelobium, dipterocarpus, dryobalanops, hopea, parashorea, shorea, vatica and consisted of 92 species. non-dipterocarpaceae trees dominating the logged-over forest were especially pioneer species such as sp. this result was macaranga similar to the research results in brazilian amazon forest which showed that the ingrowth rate increased in the first 8 years after logging provided there were more light in the under storey which stimulated ingrowth (silva . et al 1995). ingrowth can soar through the canopy opening after logging provided there were more growing space and ingrowth would decrease in line with the competition among trees (gourlet-fleury . 2005). logged-over forests et al would have good recovery with high growth rate at year-3 after logging (kao & iida 2006). the first highest ingrowth rate occurred in year-7 indicating high growth regeneration which in line with the beginning of mortality rate decline in year-7. the second highest ingrowth rate occurred in year-15 which might be caused by recovery process of forest trees after being logged-over. biotropia vol. 22 no. 1, 2015 16 table . tree ingrowth rates (% per ha per 2 years) for all species2 forest condition pal1 pal3 pal5 pal7 pal9 pal11 pal13 pal15 pal17 (% per ha per 2 years1) ril50 mean 1.8 9.0 21.3 14.5 8.8 2.8 3.4 6.5 0.6 sd 0.6 4.9 25.3 8.9 4.4 2.4 1.7 2.7 0.2 ril60 mean 2.1 6.3 9.0 10.5 7.9 1.6 3.5 9.8 0.7 sd 1.0 2.3 2.3 3.3 3.8 1.0 1.4 5.7 0.1 cnv mean 1.3 12.7 19.1 19.6 7.8 3.5 5.2 5.4 0.7 sd 0.6 8.5 13.4 8.2 4.8 1.2 2.6 1.9 0.2 p1 p3 p5 p7 p9 p11 p13 p15 p17 pf mean 2.1 3.8 3.7 4.7 2.0 1.1 1.6 4.5 0.7 sd 1.0 1.9 1.9 2.2 1.1 1.1 0.9 2.7 0.6 figure . ingrowth rates (stems per ha per 2 years) of trees with different logging techniques for species group (a) 4 dipterocarps and (b) non-dipterocarps notes : pal = period after logging (years) sd = standard deviation p = monitoring period (years) species grouping regression equation r r2 se d lof y(m) = -0.039x3 + 1.295x2 13.177x + 43.501 0.8955 0.8020 4.855 y(i) = 0.011x3 0.3676x2 + 3.2295x + 0.2946 0.5326 0.2837 3.552 pf y(m) = 0.0073x3 0.2272x2 + 1.8366x + 0.8951 0.6448 0.4158 1.577 y(i) = 5.107e-0.133x 0.4106 0.1686 0.371 nd lof y(m) = -0.0779x3 + 2.6842x2 28.4x + 101.3 0.9071 0.8229 9.985 y(i) = 0.0667x3 2.1667x2 + 18.792x 11.094 0.6317 0.3990 14.765 pf y(m) = 0.0196x3 0.5982x2 + 4.8012x + 4.3734 0.6118 0.3743 4.142 y(i) = 13.873e-0.082x 0.4900 0.2401 0.339 all species lof y(m) = -0.1174x3 + 3.9793x2 41.577x + 144.81 0.9160 0.8391 13.686 y(i) = 0.0778x3 2.5343x2 + 22.022x 10.799 0.6363 0.4049 17.288 pf y(m) = 0.0269x3 0.8254x2 + 6.6378x + 5.2685 0.6375 0.4064 5.474 y(i) = 17.636e-0.078x 0.4775 0.2280 0.3359 canopy gap occurred in a logged-over forest can function as a catalyst for tree ingrowth until certain condition. this phenomenon would then decrease due to competition in population (gourlet-fleury . 2005; kao & iida 2006), et al which would explain why natural ingrowth formed in the period of 11-17 years in the forest recovery. different species group will be affected variably to competition for light based on respective tolerable range (harcombe . 2002). et al research on forest tree in peninsular malaysia showed variations in regenerated tree by class diameter of trees in the logged-over forest area (seng . 2004).et al period after logging to mortality and ingrowth pattern results of analysis of variance (anova) showed that different harvesting techniques had no significant effect on mortality rate and ingrowth rate for all species. however, descriptively, the mortality and ingrowth rates were higher at year-1 and year-3 after logging (tables 1 and 2). from this study it was indicated that ril 50 seemed to be the best technique to increase ingrowth rate, but ril 60 was the selected technique to decrease the mortality rate after logging. regression equations were developed to show relationship between after-logging period to mortality and ingrowth rates for each species group (table 3). these equations showed that after-logging period correlated with mortality rate in logged-over forest for both dipterocarps and non-dipterocarps. recovery period after logging will affect the level of mortality and ingrowth rates for dipterocarps, non-dipterocarps and all species groups (fig. 4). all species group in logged-over forest had polynomial regression pattern for mortality and ingrowth rates, but ingrowth rates on primary forest had negative exponential pattern. high mortality rate was shown to happen in dipterocarps in early years of logging and seemed to be caused by dipterocarps being a major commercial species. meanwhile, high mortality rate for non-dipterocarps seemed to be due to forest area clearing related to skidding trails and logging yard as well as to logging operation. mortality rates for dipterocarps and nondipterocarps as either direct or indirect effect of logging was continued until the third year. ingrowth rates of non-dipterocarps species group was higher than that of dipterocarps. this was correlated with general growth or increment characteristics of total tree basal area increment of non-dipterocarps which was higher than that of dipterocarps species groups (silva . 2002; susanty & suhendang et al 2013). 17 table . regression equations between after-logging period (x) to mortality rates (m) and ingrowth rates (i) for dipterocarps 3 (d), non-dipterocarps (nd) and all species notes: lof : logged-over forest; pf : primary forest; x : after-logging period (years); y(m) : mortality rates (stems per ha per 2 years); y (i) : ingrowth rates (stems per ha per 2 years) mortality and ingrowth pattern of dipterocarps in forest recovery in east kalimantan farida h. susanty – et al. results of regression analysis (table 3; fig. 5) showed that after-logging period was significantly correlated to mortality rates for both dipterocarps and non-dipterocarps. the function of recovery time after logging would reduce mortality rates from logging effect into natural mortality rates. ingrowth rates after logging for both dipterocarps and nondipterocarps could not be explained only from recovery time after logging. opportunity for seedlings and saplings to grow is influenced by biocharacteristics of each species and by distribution pattern (husch 2003; adam & et al. kolbs 2005; gersonde & o'hara 2005). natural characteristics of mortality and ingrowth rates shown by primary forest conditions indicated that mortality rate tended to be higher than ingrowth rate within the 17 years of measurements for both dipterocarps and non-dipterocarps. in natural forest, growth will increase the dimension of individual trees while reducing the number of trees due to competition of growing space. results from this research provided the first quantitative information for management planning in labanan forest. to achieve higher ingrowth rate and minimize mortality rate until year-3 after-logging, the reduced impact logging technique (ril 50 and ril 60) was better than conventional logging technique. these data serve as basic information needed to start forest management planning and provide useful guide for choosing suitable planning system. this study highlighted the need to consider silviculture treatment to stimulate biotropia vol. 22 no. 1, 2015 18 figure . patterns of mortality and ingrowth rates (stems per ha per 2 years) on logged-over forest (lof) and primary forest 5 (pf) for species groups (a) dipterocarps; (b) non-dipterocarps; (c) all species growth rates in forest areas, especially to increase wood production. the methods described in this study can be enhanced to involve a more detail species grouping for increasing model accuracy and to accommodate high species diversity (phillips . 2002; valle . 2006). in et al et al dipterocarp forest, valuation of tree recovery could be assessed by tree structure model, mortality rate and ingrowth rate, involving species characteristic by species grouping. conclusions residual trees in dipterocarp forest in year-17 after-logging based on had been well recovered density and basal area value that reached more than 85% close to the initial condition. mortality rates in logged-over forest would be similar to that in the primary forest at year-7 after-logging, while ingrowth rate in logged-over forest would be similar to that in the primary forest at year-17 afterlogging. fluctuations in mortality and ingrowth rates in logged-over forest reached equilibrium conditions at year-7 after-logging. in the subsequent years, ingrowth rate was higher than mortality rate. fluctuations of mortality and ingrowth rates in the primary forests were relatively low. recovery period for trees in logged-over forest had an important effect to decrease mortality rates for both dipterocarps and non-dipterocarps. different types of logging techniques (ril 50, ril 60 and conventional logging) with variations in logging intensity 7-14 stems per hectare did not have significant influence on recovery of density and basal area value, as well as on mortality and ingrowth rates. the reduced impact logging technique was better than conventional logging technique to achieve higher ingrowth rate and minimize mortality rate until year-3 after-logging. 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project. jakarta(id): cirad-forêt-forda-pt inhutani i. p 139-61. sist p, fimbel r, sheil d, nasi r, chevallier mh. 2003. towards sustainable management of mixed dipterocarp orests of south-east asia: oving f m beyond minimum diameter cutting limits. environ conserv . 30(4): 364 74 sist p, ferreira fn. 2007. sustainability of reduced-impact logging in the eastern amazon. forest ecol manag 243: 199-209. smith rgb, nichols jd. 2005. patterns of basal area increment, mortality and recruitment were related to logging intensity in subtropical rainforest in australia over 35 years. forest ecol manag 218: 31928. steel rgd, torrie jh. 1995. prinsip dan prosedur statistika: suatu pendekatan biometrik. sumantri b, penerjemah. jakarta (id): pt gramedia pustaka utama. terjemahan dari: principles and procedures of statistics. edisi ke-2. susanty fh, suhendang e. 2013. riap individu dan tegakan periodik hutan dipterocarpaceae setelah penebangan. in: ngatiman, wahyudi a, editor. restorasi ekosistem dipterokarpa dalam rangka peningkatan produktivitas hutan. prosiding: 2013 oktober 22; samarinda (id): kementerian kehutanan. balai besar penelitian dipterokarpa. p156-67. vanclay jk. 2003. growth modelling and yield prediction for sustainable forest management. the malaysian forester 66(1):58-69. valle d, mark s, edson v, james g, marcio s. 2006. identifying bias in stand-level growth and yield estimations : a case study in eastern brazilian amazonia. forest ecol manag 236: 127-35. whitmore tc. 1990. tropical rain forest dynamics and its implications for management. in: gomez-pompa a, whitmore tc , hadley m, editor. rain forest regeneration and management: man and the biosphere series volume 6. paris(fr): parthenon publishing group. p 67-89. 21 mortality and ingrowth pattern of dipterocarps in forest recovery in east kalimantan farida h. susanty – et al. appendix 1. list of species by family in labanan forest family famil y famil y famil y actinidiaceae dilleniaceae moraceae saxifragaceae alangiaceae dipterocarpaceae myristicaceae simaroubaceae amonaceae ebenaceae myrsinaceae sonneratiaceae anacardiaceae elaeocarpaceae myrtaceae sterculiaceae annonaceae euphorbiaceae ochnaceae symplocaceae apocynaceae fagaceae olacaceae theaceae aquifoliaceae flacourtiaceae oleaceae thymelaeaceae araucariaceae clusiaceae oxalidaceae tiliaceae bignoniaceae hypericaceae polygalaceae ulmaceae bombacaceae icacinaceae proteaceae urticaceae burseraceae juglandaceae rhamnaceae verbenaceae caesalpiniaceae lauraceae rhizophoraceae celastraceae lecythidaceae rosaceae chrysobalanaceae fabaceae rubiaceae combretaceae loganiaceae rutaceae connaraceae lythraceae sapindaceae convolvulaceae magnoliaceae sapotaceae crypteroniceae melastomataceae sapuidaceae datiscaceae meliaceae sarcotheca biotropia vol. 22 no. 1, 2015 22 47 shorea bentongenensis 48 shorea confusa 49 shorea exelliptica 50 shorea faguetiana 51 shorea falciferoides 52 shorea fallax 53 shorea guiso 54 shorea hopeifolia 55 shorea inappendiculata 56 shorea johorensis 57 shorea laevis 58 shorea lamellata 59 shorea leprosula 60 shorea leptoderma 61 shorea longisperma 62 shorea macrophylla 63 shorea macroptera 64 shorea maxwelliana 65 shorea mecistopteryx 66 shorea multiflora 67 shorea ochracea 68 shorea ovalis ssp ovalis 69 shorea parvifolia 70 shorea parvistipulata appendix 2. list of dipterocarps species no species no species 1 anisoptera costata 2 anisoptera laevis 3 anisoptera sp 4 cotylelobium melanoxylon 5 cotylelobium sp 6 dipterocarpus acutangulus 7 dipterocarpus caudiferus 8 dipterocarpus confertus 9 dipterocarpus conformis 10 dipterocarpus costulatus 11 dipterocarpus elongatus 12 dipterocarpus fusiformis 13 dipterocarpus glabrigemmatus 14 dipterocarpus gracilis 15 dipterocarpus grandiflorus 16 dipterocarpus hasseltii 17 dipterocarpus humeratus 18 dipterocarpus mundus 19 dipterocarpus pachyphyllus 20 dipterocarpus palembanica 21 dipterocarpus stellatus 22 dipterocarpus tempehes 23 dipterocarpus verrucosus 24 dipterocarpus sp 25 dryobalanops beccarii 26 dryobalanops lanceolata 27 dryobalanops sp 28 hopea bracteata 29 hopea cernua 30 hopea dryobalanoides 31 hopea ferruginea 32 hopea mengarawan 33 hopea nervosa 34 hopea pachycarpa 35 hopea rudiformis 82 shorea sp 83 vatica albiramis 84 vatica micrantha 85 vatica nitens 86 vatica oblongifolia 87 vatica odorata 88 vatic a rassak 89 vatica sarawakensis 90 vatica umbonata 91 vatica vinosa 92 vatica sp 36 hopea sangal 37 hopea semicuneata 38 hopea sp 39 parashorea malaanonan 40 parashorea smythiesii 41 parashorea sp 42 shorea agamii ssp agamii 43 shorea almon 44 shorea angustifolia 45 shorea atrinervosa 46 shorea beccariana 71 shorea patoiensis 72 shorea pauciflora 73 shorea pinanga 74 shorea scrobiculata 75 shorea semicuneata 76 shorea seminis 77 shorea smithiana 78 shorea superba 79 shorea symingtonii 80 shorea virescens 81 shorea xanthophylla 23 mortality and ingrowth pattern of dipterocarps in forest recovery in east kalimantan farida h. susanty – et al. microsoft word 85 biotropia vol. 13 no. 2,2006 : 85 98 bacterial community shifts of a high mountain lake in response to variable simulated conditions: availability of nutrients, light and oxygen munti yuhana1, thomas horath2 and kurt hanselmann2 'department ofaguamlture, faculty of fisheries and marine science, bogor agricultural university, kampus ipb darmaga 16680 bogor, indonesia. 1 department of microbiology, institute of plant biology, university of zurich, zollikerstrasse 107, 8008 ziirich, switzerland. abstract we studied bacterial population composition shifts by exposing natural water samples to variable simulated environmental conditions. the samples were taken from lake jori xiii (2640 m a.s.l), an oligo-to mesotrophic cold freshwater lake, located in the eastern swiss alps. the jori lakes are characterized as remote, unpolluted high mountain lakes with a long period of ice cover and typically low nutrient concentrations. culture independent techniques (pcr-based analyses) were used for detection and molecular characterization of a large number of bacteria most of which are still uncultivable. bacterial community shifts over three ecological conditions (nutrients, light and oxygen availability) were detected by using temporal temperature gradient gel electrophoresis (ttge) of a pcr-amplified part of the 16s rrna gene. the bacterial populations responded differently to the variable conditions, as revealed by ttge pattern shifts during the experiment. key words: temporal temperature gradient gel electrophoresis (ttge), arb, small subunit ribosomal rna gene (ssu rrna gene), alpine freshwater lake jori xiii, pcr introduction lake jori xiii is located in canton graubiinden, switzerland. this lake has a maximum depth of 10.9 m and its surface area is approximately 1 5400 m2. lake xiii presently is one among 22 medium and small lakes in the jori catchment. some of them are several thousand years old, and others were formed when the glacier retreated during the last 150 years. the habitats are still evolving. they are situated in the eastern part of swiss alps, at 46°46'n latitude and 9°58'e longitude, at altitudes between 2489 m to 2750 m above sea level. the iron-rich area is almost free of vegetation. several lakes (for example lakes i, ii and iii) are still influenced by streamlets from the melting glacier, whereas many others, which are mostly situated on higher places, are no longer under glacier influence, including lake jori xiii (figure 1). remote, unpolluted high mountain lakes are ecosystems in which their hydrochemical conditions are characterized by extremely variable fluctuations and often sudden changes. these intrannual and interannual environmental conditions corresponding author: myuhana@botinst.unizh.ch or hanselma@botinst.unizh.ch 85 biotropia vol. 13 no. 2,2006 photo by kurt hanselmann figure 1. view of lakes jori i, ii, iii, xii and xiii during summer, ice-free period. can be related to the atmospheric or climate variability. the alpine lakes can be considered as relatively simple aquatic ecosystems with short food chains, restricted environmental variables and low species diversity. organisms living in these habitats are challenged by harsh temperatures (below freezing to +15°c maximally), by seasonal and diurnal radiation differences between darkness and high levels of uv, by nutrient deprivation and bursts during snow melt phases, and by anoxia and darkness in the hypolimnion below a long lasting ice cover. during the short summer period, the epilimnion experiences high diurnal temperature fluctuations, strong light penetration and high uv radiation (bothwell et ol. 1994). maximal water temperature during this period can reach 15°c but it could suddenly drop to 0°c in case of snow fall. bacteria are known to thrive under extremely broad-range conditions including such that would kill other organisms. they are further able to perform various metabolic processes, and their capabilities are more diverse compared to higher organisms. therefore, we focused on the adaptation of the bacterial community in this extreme habitat. we were interested in how the bacterial population composition shifts under fluctuating environmental conditions. normally, in the environments which get exposed to diurnal changes and annual variation in physicochemical and trophic conditions, the communities are forced to adapt quickly. the bacterioplankton community structure changes were studied in vitro by simulating three environmental determinants i.e. light, oxygen, and nutrients. temporal temperature gradient gel electrophoresis (ttge) has been used to follow the bacterial community composition changes. ttge is a method which does not require a cloning step for profiling microbial populations, therefore, it is a fast monitoring method. the small subunit (ssu) ribosomal rna gene is widely used as a comparative phylogenetic marker (ward et al. 1990; muyzer et al. 1993). the ribosomal rna gene has several features that make it an ideal phylogenetic marker. first, as the backbone inside the ribosome it is ubiquitous in the living world. 86 bacterial community shifts m. yuhana et al. further it has the same function in all organisms, it is functionally homologous. also it is more sensitive to mutations than most of the other genes since its gene product is used directly without any further translation into a protein where some mutations could keep silent. the gene sequence itself has regions of broad homology but also of heterogeneity compared with different organisms (it is moderately well-conserved) which makes it easy to align it in almost all levels of phytogeny. concerning the nucleic acid length of the rrna gene, the sequence of the ssu rdna has the advantage that it is long enough for a reasonable alignment but quite shorter than the sequence of the large subunit rdna (lsu rrna gene) which makes it cheaper and faster to work with. the community shifts were monitored by comparing the changes of dna fingerprints of pcr-generated 16s rdna fragments in a ttge gel. the electrophoretic separation of the community dna molecules in ttge is based on the temperature gradient. the separated bands can then be excised, pcr-amplified, and sequenced to identify the members of the community. this technique has been applied already for assessment and monitoring of bacterioplankton community dynamics in diverse aquatic environments (bosshard et al. 2000; pearce 2000; casamayor et al. 2002). the three environmental determinants chosen nutrient supply, light, and redox condition strongly affect biological processes. combinations of these factors were used for in vitro simulations. the results illustrate the population flexibility and further support the validity of the selective adaptation hypothesis for ecosystems under harsh and highly variable environmental conditions (elena and sanjuan 2003). materials and methods sample collection sampling was carried out in late autumn in october 2002 in lake jb'ri xiii. samples were taken from the lake water column, at 5 different depths of 1, 3, 5, 7, and 9 meters, respectively. water samples were collected with a 500 ml niskin sampler. the sampler was pre-rinsed with the lake water from the desired depths. the actual water samples were filled in sterilized 500 ml glass bottles and were kept at low temperature in a cooling box for transportation. sub samples for the experiments under the different conditions were taken in the laboratory (table 1). volumes of 100 ml for each experiment were placed aseptically in sterile 200 ml glass bottles. all samples were incubated at 4°c under a combination of the following conditions: with or without additional nutrients, in the light and in the dark, with oxygen (stirring) and anoxically (figure 2). the final concentration after adding nutrients was 10 fold diluted luria bertani (lb) medium consisting of: tryptone 0.5 g i"1, yeast extract 1.0 g i"1, and nacl 0.5 g i"1. the ph was adjusted to 7.2. a sample set was incubated in the dark (the bottles were wrapped in aluminum foil). samples that needed light were incubated under two 15 watt tube lamps (osram) i n a 3 0 x 3 0 x 7 0 cm3 refrigerator, with continuous illumination. aerobic incubation was done on stirrer plates. each bottle of this 87 biotropia vol. 13 no. 2,2006 figure 2. incubation of samples treated under a series of different simulated conditions at 4°c. treatment was stirred continuously using a magnetic bar and the bottles were covered by sterile cotton plugs. for anoxic treatment, the bottles were purged with o2-free n2 gas, completely filled and tightly sealed. resazurin was used as redox indicator in the anaerobic cultures. nucleic acid extraction, pcr, and ttge analysis for community profiling preparation for dna extraction of initial samples (without treatment) was processed directly in the field laboratory. water samples of 75 ml were filtered 88 bacterial community shifts m. yuhana et al. aseptically through 0.22 fim pore size filters (millipore gvwp, 25 mm in diameter) using a sterile syringe and a "swinnex®" disc filter holder (millipore, sxoo 025 00). the filters were placed in sterile 1.5 ml tubes and kept at -20°c until further processing, i.e. dna extraction, pcr amplification and ttge analyses. after 2 weeks of incubation, the cultures were harvested for total genomic dna extraction, pcr amplification, and further ttge analysis. cells were collected by filtration using 0.22 urn pore size filters (millipore, gvwp, 25 mm in diameter). the sample quantity varied, depending on the turbidity of the culture. normally, 40 to 50 ml from the treatments with nutrients and 75 ml from cultures without added nutrients were filtered. samples were treated applying protocol b of the qiagen dneasy® tissue kit for gram positive bacteria with an additional bead beating lysis step. the microorganisms on the filters were rinsed with lysis buffer and further processed according to the qiagen protocol. the cells were lysed with lysozyme and disrupted physically by bead beating. bead beating was done by adding 100 mg of glass beads (0.1 mm in diameter) to each tube and shaking at 80% of maximum speed in a retsch mm2000 bead beater (f. kurt retsch gmbh & co. kg, 42781 haan, germany) for 1 min. the extracted dna was diluted in 100 ^1 sterilized h2o. tubes containing extracted genomic dna were stored at -20°c until further processing. to amplify the 16s rrna gene fragments from the total community dna, we used the general bacterial primers 27f (= s-d-bact-0008-b-s-20; 5'-aga gtt tga tcm tgg ctc ag) and 1524r (= slightly modified 1525r, s-d-bact-1524-a-a-18; 5'-aag gag gtg atc car ccg) (lane 1991). pcr reactions were prepared in a volume of 25 ul, containing (final concentration): dh2o, taq buffer (ix) (sigma), 0.1 mg ml"1 dnase-free bovine serum albumin (amersham, pharmacia biotech inc.), 0.2 mm dntps, 200 nm of each primer, 40 u ml'1 taq polymerase (sigma), and approximately 50-100 ng template dna. pcr was performed with a techne thermocycler (techne ltd, duxford cambridge, u.k.). pcr was run under the following conditions: initial denaturation at 94°c for 130 sec. the next steps were 10 cycles of 94°c for 15 sec, 63°c for 30 sec and lowering the temperature by 0.5°c in every cycle (touch down), then 72°c for 80 sec with increasing the duration by 1 sec every cycle. these steps were followed by 20 cycles of 94°c for 15 sec, 56°c for 30 sec, 72°c for 90 sec with increasing the period by 1 sec every cycle followed by a final extension step at 72°c for 10 min. pcr products were analyzed by electrophoresis in a 1% agarose gel and 0.5x tab running buffer [20 mm trizma base, 10 mm glacial acetic acid, and 1 mm na2edta]. first pcr amplifications yielded approximately 1'500 bp length of the 16s rrna gene (data not shown). the primary pcr products were used as templates for the nested pcr. this method was used to increase the sensitivity and signal strength of the pcr amplification. universal primers 3 57f (slightly modified) (= s-d-bact-341-b-s-17; 5'-cct acg gga ggc agc ag) and gc-907r (= gc-univ-907-a-a-20; 5'-cgc ccg ccg cgc gcg gcg ggc ggg gcg ggg gca cgg ggg g ccg tca att cmt ttr agt tt) (lane 1991; muyzer et al. 1998) were used to amplify 89 biotropia vol. 13 no. 2, 2006 the 16s rrna gene fragments. pcr was performed with initial denaturation at 94' for 5 min, followed by 75°c for 15 sec. the next steps were 20 cycles of 94°c f 20 sec, annealing at 65°c lowered by 0.5°c every cyclefor 30 sec (touch dowr and elongation temperature 72°c for 1 min. these steps were followed by 15 cycl< of 94°c for 20 sec, 52°c for 30 sec, 72°c for 70 sec, and a final extension step ; 72°c for 10 min. nested pcr amplification yielded 16s rrna gene fragments c approximately 560 bp length (figure 3). figures. products after nested pcr with 357f and gc-907r of the 16s rrna gene fragments. only a part of the pcr products from several samples in this study is shown. m: marker (a dna digested with ecorl/himllu). (1-5: samples from depth 1 m; 1: initial, 2: hdo, 3: hlo, 4: ndo, 5: nlo; 6-10: samples from depth 3 m, 6. initial, 7: hda, 8: hla, 9: nda, 10: nla, 11-15: samples from depth 5 m; 11: initial. 12: hdo, 13: hlo. 14: ndo, 15: nlo; 16-20: samples from depth 7 m, 16: initial, 17: hda, 18: hla, 19: nda, 20: nla). initial: initial population, before treatments were applied. h: 10-fold diluted lb medium added, n: no nutrients added, d: incubation in the dark, l: incubation in the light, a: anoxic condition, o: oxic condition. 90 bacterial community shifts — m. yuhana et al. ttge was carried out in a dcode™ mutation detection system (bio-rad iboratories). 10 ul of the pcr samples and 10 ul of 2x loading buffer (70% [v/v] ycerol, 0.05% [w/v] bromophenol blue) were loaded onto 6% polyacrylamide gels :rylamide: n,n'-methylene bis-acrylamide 37.5:1 [w/w]; 7 m urea, ix tab). the is were run at temperatures starting at 54°c and raising to 64°c, temperature ramp te was +1.1°c h"1, the voltage was 90 v (4.0 v/cm), and running time was about 9 the gels were stained in 1 ng ml"1 ethidium bromide solution for 15 min, stained in water for 45 min, visualized, and photographed under uv insillumination (x = 312 run). •quencing of the 16s rrna genes from pcr-ttge bands and phylogenetic ee construction the representative bands of the dominant bacteria which appeared distinctly in fge gels were excised and dna was recovered by putting the gel fragments gether with 20 ul of h2o into a 1.5 ml tube, freezing twice, and taking the .pernatant. pcr amplification was performed by using 341f and the non-gc-907r imers. pcr products were bidirectionally sequenced using abi prism® big dye™ !.0 (applied biosystems). the pcr products were purified by centrifugation in icrocon filter devices (microcon ym 100, millipore, bedford, mass., usa), and ere used for a 10 ul-single pcr reaction: 5 to 20 ng dna template, 3 (jl big dye applied biosystems) and 3 |al of 1.5 um primer were used. after the sequencing 3r, the products were purified with sephadex g-50 (amersham, pharmacia iotech ab) and loaded onto the sequencing machine (abi prism 377 dna squencer). the blast search tool available from ncbi (http://www.ncbi.nlm.nih.gov/ ast) (altschul et al, 1998) was used to list the closest neighbors of the sequences, he new 16s rrna gene sequences were imported to the ssu rrna database :trieved from the technical university of munich (release january 2004) using the rb phylogeny program (http://www.arb-home.de/) (ludwig et al. 2004) and ligned automatically employing the fast aligner vi.03 of the are software nvironment. subsequently the alignment was corrected manually according to the jcondary structure. the sequences were then added to the general tree of the arb su rrna database by calculating distances with a maximum-parsimony algorithm nd using the appropriate sai (sequence associated information) filters. results and discussion during the short summer period and autumn, the lake was homothermic i.e. the ike water masses were completely mixed. therefore, the physical and chemical larameters of the lake water column did not differ much among various depths table 2). the initial ttge profile from depth of 1 m is the same as those from lepths of 3 m, 5 m, 7 m, and 9 m. the community patterns of lake jqri xiii differ 91 biotropia vol. 13 no. 2,2006 generally, it can be seen that incubations under high nutrient conditions showed fewer bands than the initial community and also fewer when compared to those without nutrients addition. low numbers of ttge bands indicate a limited number of species, i.e. only those microorganisms from the community which possess the highest competitive ability, are growing well. environments with low nutrients lead to a more diverse species composition, and community shifts between the initial and the final state are less pronounced. cultures kept in the light did not yield distinctly different patterns from those ones kept in the dark regardless whether they were grown with or without nutrient addition, except for the aerobic condition with no additional nutrients. certain bacterial communities develop different life strategies as an adaptation to various environmental conditions. their population would be present predominantly even under markedly different environmental pressures which results in similar ttge banding patterns. however, it cannot be ensured that those co-migrating bands have the same sequence identity. therefore, it is helpful to identify the communities appearing in the ttge bands. in this experiment, the representative ttge bands of dominant communities were recovered and sequenced. the bands are phylogenetically affiliated to alpha-, and beta-proteobacteria, actinobacteria, chloroplasts, and candidates of division op 10 (figure 5). the communities are phylogenetically diverse and respond quickly to various environmental fluctuations. some sequences represented by bands a, b, c, 6 and 8, showed very low similarity values (86 to 94 % sequence similarities) to known 16s rdna sequences of cultured bacteria present in the databank (table 3). cultivation efforts for those microorganisms could become valuable concerning their physiological properties. the results suggest that community competition and adaptation processes are common in ecosystems exposed to strong physicochemical fluctuations. physiological abilities of the microorganisms present in their gene pool allow the community to respond rapidly to environmental changes and thus ensure the functioning of microbially-mediated processes in the habitat. microorganisms possessing a high physiological flexibility, so-called niche generalists, will dominate the population in their communities under various conditions (elena and sanjuan 2003), while niche specialists will get selected under more specific conditions. environmental changes express themselves as physical and chemical instabilities, which exert selection pressures for different microorganisms. these changes promote the dynamic microbial community transitions in fluctuating environments (rainey and travisano 1998). 94 bacterial community shifts m. yuhana el al. added) rather than to nlo. under low nutrient conditions, the majority of ttge bands still appeared. some became less intense after the two-week incubation period. a few bands are missing at the end of the experiment (e. g. bands a and b). initial: initial population, before treatments were applied. h: 10-fold diluted lb medium added, n: no nutrients added, d: incubation in the dark, l: incubation in the light, a: anoxic condition, o: oxic condition. s: start of gels, e: end of gels. the bacterial diversity decreases when the community is exposed to high nutrient concentrations and anoxic conditions. this can be detected from experiments designated hda and hla at depths of 3 m and 7 m, respectively. in these treatments, light apparently is an essential determinant. in the light, bands 8 (3m depth) and 10 (7m depth) became prominent, in the dark, band 7 (3m depth). based on ncbi-blast of 16s rrna gene sequences, the closest known relative of band 7 (community exposed to hda treatment) was an anaerobic, sulfate reducing bacterium, desulfotomaculum sp. dem-kme99-2, aj276565 (with 88% sequence similarity). in the hla culture, band 8 dominated the community, which is phylogenetically related to the alphaproteobacterium sphingomonadaceae bacterium n, dq497241 (99% sequence similarity). the sequence of band 10 was closest related to the betaproteobacterium li2-55 from lake loosdrecht water column, netherlands, aj964892 (97% sequence similarity). 93 biotropia vol. 13 no. 2, 2006 generally, it can be seen that incubations under high nutrient conditions showed fewer bands than the initial community and also fewer when compared to those without nutrients addition. low numbers of ttge bands indicate a limited number of species, i.e. only those microorganisms from the community which possess the highest competitive ability, are growing well. environments with low nutrients lead to a more diverse species composition, and community shifts between the initial and the final state are less pronounced. cultures kept in the light did not yield distinctly different patterns from those ones kept in the dark regardless whether they were grown with or without nutrient addition, except for the aerobic condition with no additional nutrients. certain bacterial communities develop different life strategies as an adaptation to various environmental conditions. their population would be present predominantly even under markedly different environmental pressures which results in similar ttge banding patterns. however, it cannot be ensured that those co-migrating bands have the same sequence identity. therefore, it is helpful to identify the communities appearing in the ttge bands. in this experiment, the representative ttge bands of dominant communities were recovered and sequenced. the bands are phylogenetically affiliated to alpha-, and beta-proteobacteria, actinobacteria, chloroplasts, and candidates of division op 10 (figure 5). the communities are phylogenetically diverse and respond quickly to various environmental fluctuations. some sequences represented by bands a, b, c, 6 and 8, showed very low similarity values (86 to 94 % sequence similarities) to known 16s rdna sequences of cultured bacteria present in the databank (table 3). cultivation efforts for those microorganisms could become valuable concerning their physiological properties. the results suggest that community competition and adaptation processes are common in ecosystems exposed to strong physicochemical fluctuations. physiological abilities of the microorganisms present in their gene pool allow the community to respond rapidly to environmental changes and thus ensure the functioning of microbially-mediated processes in the habitat. microorganisms possessing a high physiological flexibility, so-called niche generalists, will dominate the population in their communities under various conditions (elena and sanjuan 2003), while niche specialists will get selected under more specific conditions. environmental changes express themselves as physical and chemical instabilities, which exert selection pressures for different microorganisms. these changes promote the dynamic microbial community transitions in fluctuating environments (rainey and travisano 1998). 94 bacterial community shifts m. yuhana et figure 5. phylogenetic affiliation of representative dominant ttge bands of water samples from lake jori xi11 incubated at different environmental conditions. ne\v sequences are written in bold (band_xx, accession no., length ibpj). closest known relatives are written in italic, followed by their accession nos. and sequence lengths (bp). a yeast 18s rrna gene of saccharomyces cerevisiae is used as outgroup. the scale bar indicates 10% sequence divergence 95 biotropia vol. 13 no. 2,2006 conclusions it is possible to follow the bacterial community shifts by applying the pcr-based molecular technique of temporal temperature gradient gel electrophoresis (ttge). the physicochemical determinants applied in the experiments triggered the changes in the bacterial community composition originating from a high mountai bacterial community shifts m. yuhana et al. lake jo'ri xiii. 16s rrna gene sequencing and phylogenetic analysis revealed that the predominant populations are affiliated to the alphaand betasubgroups of the proteobacteria, to actinobacteria (high gc gram + bacteria), chloroplasts and candidates of division op 10. acknowledgments we are grateful to hanne grob, ignachio fernandez and gabriela iqbal-nava for excellent technical assistance. we thank the reviewers for helpful comments on the article. references altschul, s.f, t.l. madden, a.a. schaffer, j. zhang, z. zhang, w. miller and dj. lipman. 1997. gapped blast and psi-blast: a new generation of protein database search programs. nucleic acids res, 25: 3389-3402. bosshard, p., r. stettler and r. bachofen. 2000. seasonal and spatial community dynamics in the meromictic lake cadagno. arch. microbiol., 174: 168-174. bothwell, m.l, d.m.j. sherbot and c.m. pollock. 1994. ecosystem response to solar ultraviolet-b radiation: influence of trophic level interactions. science, 265: 97-100. casamayor, e.o, c. pedrds-alio, g. muyzer and r. amann. 2002. microheterogeneity in 16s ribosomal dna-defmed bacterial populations from a stratified planktonic environment is related to temporal changes and to ecological adaptations. appl. environ. microbiol., 68: 1706-1714. elena, s.f. and r. sanjuan. 2003. evolution: climb every mountain? science., 302: 2074-2075. hollibaugh, j.t, p.s. wong, n. bano, s.k. pak, e.m. prager and c. orrego. 2001. stratification of microbial assemblages in mono lake, california, and response to a mixing event. hydrobiologia., 466: 45-60. iqbal-nava, g. 2003. microbial ecosystem evolution: biogeochemical nutrient scavenging is a prerequisite. diploma thesis, university of basel, switzerland. lane, d.j. 1991. 16s/23s rrna sequencing, p. 115-175. in e. stackebrandt and m. goodfellow (ed.), nucleic acid techniques in bacterial systematics. john wiley & sons ltd., chichester, united kingdom. ludwig, w., o. strunk, r. westram, l. richter, h. meier, y. kumar, a. buchner, t. lai, s. steppi, g. jobb, w. forster, i. brettske, s. gerber, a.w. ginhart, o. gross, s. grumann, s. hermann, r. jost, a. konig, th. liss, r. lubmann, m. may, b. nonhoff, b. reichel, r. strehlow, a. stamatakis, n. stuckmann, a.vilbig, m. lenke, th. ludwig, a. bode, and k.-h. schleifer. 2004. arb: a software environment for sequence data. nucleic acids res, 32:1363-1371. muyzer, g, b.c. de waal and a.g. uiterlinden. 1993. profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16s rrna. appl. environ. microbiol., 59: 695-700. muyzer, g. and k. smalla. 1998. application of denaturing gradient gel electrophoresis (dgge) and temporal temperature gradient gel electrophoresis (ttge) in microbial ecology. antonie leeuwenhoek, 73: 127-141. muyzer, g, t. brinkhoff, u. niibel, c. santegoeds, h. schafer and c. wawer. 1998. denaturing gradient gel electrophoresis (dgge) in microbial ecology, p. 1-27 in a. d. l. akkermans, j. d. van elsas, and f. j. de bruijn (ed.), molecular microbial ecology manual. kluwer academic publishers, dordrecht, the netherlands. muyzer, g. 1999. dgge/tgge, a method for identifying genes from natural ecosystems. curr. opin. microbiol, 2: 317-322. ogier, j.-c, o. son, a. gruss, p. tailliez and a. delacroix-buchet. 2002. identification of the bacterial microflora in dairy products by temporal temperature gradient gel electrophoresis. appl. environ. microbiol, 68: 3691-3701. 97 biotropia vol. 13 no. 2,2006 pearce, d.a. 2000. a rapid, sensitive method for monitoring bacterioplankton community dynamics, applied to atlantic freshwater lakes. polar biol. 23:352-356. rainey, p.b. and m. travisano. 1998. adaptive radiation in a heterogeneous environment. nature., 394: 69-72. ward, d.m., r. weller and m.m. bateson. 1990. 16s rrna sequences reveal numerous uncultured microorganisms in natural community. nature, 345: 63-65. 98 85.pdf 86.pdf 87.pdf 88.pdf 89.pdf 90.pdf 91.pdf 92.pdf 93.pdf 94.pdf 95.pdf 96.pdf 97.pdf 98.pdf biotropia no biotropia no. 17,2001 : 9 17 characterization of three benzoate degrading anoxygenic photosynthetic bacteria isolated from the environment dwi suryanto1, antonius suwanto2'3*, and anja meryandini3 ; dept. of biology, faculty of science and mathematics, north sumatra university, medan, indonesia 2 south east asian regional center for tropical biology (seameo-biotrop), bogor, indonesia 3 dept. of biology, faculty of science and mathematics, bogor agricultural university, bogor, indonesia abstract three anoxygenic photosynthetic bacteria, ds-1, ds-4 and cas-13, have been examinated for their morphological and physiological properties. all strains were rod-shape cells with a swollen terminal end, gram negative, motile, non-halophilic, non-alkalophilic and non-acidophilic, and capable of utilizing benzoate aerobically and photo-anaerobically. sequence analysis of part of 16s rrna genes showed that ds1 and cas-13 were closely related to rhodopseudomonas palustris strain 7 with a similarity of 97%, whereas ds-4 may not be closely related to the former two strains with a similarity of 78% based on the constructed phylogenic tree. spectral analysis indicated that the three bacteria had bacteriochlorophyl a and normal spirilloxanthin series. growth in medium enriched with vitamin and supplemented with benzoate as their sole c-sources was better than in medium without vitamin. benzoate degradation in medium with vitamin was accelerated. the ability to grow on benzoate without added vitamins indicated that the bacteria were able to synthesize their own vitamins. key words: anoxygenic photosynthetic bacteria/ benzoate degradation/ 16s rrna gene. introduction some toxic compounds are slowly degraded in polluted aerobic zones and, therefore, may leach into anaerobic subsurface environments (kohring et al. 1989). anaerobic degradation of such compounds may play an important role in eliminating toxic substances. anaerobic bioremediation has been proposed as an inexpensive method for in situ removal of organic contaminants in the environment (kuo and genthner 1996). recent concern about the environmental fate of industrially produced organic compounds has prompted a resurgence of interest in the anaerobic degradation of aromatic compounds (harwood and gibson 1988). anoxygenic photosynthetic bacteria (apb) are nutritionally versatile in their ability to utilize diverse sources of carbon ranging from simple aliphatic organic acids to complex polysaccharides (hiraishi et al. 1995). the occurrence of these bacteria as common inhabitants in aquatic and some terrestrial habitats in nature ' corresponding author: e-mail address : asuwanto@indo.net.id 9 biotropia no. 17, 2001 (hiraishi et al. 1995) might be explained partly by the fact that these bacteria survive on various modes of energy-generating systems (hiraishi et al. 1995). the ability of phototrophic purple non-sulfur bacteria to grow aerobically and anaerobically in the presence of diverse aromatic compounds makes these good candidates for potential biodegradation of harmful compounds (hanvood and gibson 1988; wright and madigan 1991; shoreit and shaheb 1994). commercialization of these bacteria for purification treatment plants was initiated about 15 years ago (kobayashi and kobayashi 1995). not many phototrophic purple non-sulfur bacteria species are known to degrade aromatic compounds. rhodopseudomonas palustris (harwood and gibson 1988; gibson and gibson 1992; shoreit and shaheb 1994), rhodomicrobium vannielii (wright and madigan 1991), rhodobacter capsulatus (blasco and castillo 1992; shoreit and shaheb 1994), rs. blastica, and rhodospirilium rubrum (shoreit and shaheb 1994) are able to degrade a variety of monocyclic aromatic compounds with or without other c-sources. benzoate and its derivatives are among the common aromatic compounds that can be completely mineralized (harwood and gibson 1988; wright and madigan 1991; shoreit and shaheb 1994). however, blasco and castillo (1992) noted that rhodobacter capsulatus e1f1 is able to degrade mononitrophenol and dinitrophenol with acetate as its carbon source. rs. palustris utilized several phenolic compounds, hydroxylated and methoxylated aromatic acids, aromatic aldehydes, and hydroaromatic acids (harwood and gibson 1988). other diverse aromatic compounds have also been reported to be utilized by this group (harwood and gibson 1988; wright and madigan 1991; shoreit and shaheb 1994). degradation of aromatic compound by phototrophic purple non-sulfur bacteria in general was emphasized in this investigation based on their ability to utilize diverse aromatic compounds. to date, this group of bacteria has not been subjected to intensive examination (harwood and gibson 1988). this study focused on the ability of three new isolates of apb to utilize benzoate. identification of the bacterial isolates based on their 16s-rrna genes as well as their morphological and physiological properties was also carried out. materials and methods bacterial cultures and cultivation three isolates of apb, designated as ds-1, ds-4, and cas-13 were studied. the two former strains were isolated from java, and the last was isolated from moluccas. all isolates were maintained on modified sistrom medium with benzoate as the sole carbon source. to determine the ability of the bacteria to utilize and degrade aromatic compounds, the isolates were grown in modified sistrom by omitting all carbon sources, including nitrilo-triacetic acid, supplemented with 5 mm benzoate as the c 10 characterization of three benzoate degrading anoxygenic photosynthetic bacteria -dwi suryanto et al. source with or without vitamins in 100 ml completely filled screw-capped tubes. isolates also were grown in modified sistrom supplemented with vitamins and 5 mm succinate, or 5 mm acetate as their carbon sources to serve as a means of comparison. for cultures grown on benzoate, the inocula were obtained from 3-day old cultures of bacteria grown in modified sistrom with benzoate with an initial cell number of 5xl06 cells/ml. for cultures to test other c-sources, inocula were taken from 2-day old cultures grown in sistrom media with succinate as c-source. unless mentioned otherwise, all media were adjusted to ph 7.2. growth condition, measurement of growth, and quantitation of benzoate cultures were illuminated with a 40 w tungsten bulb at a distance of 30 cm. growth rates were determined by measuring turbidity at 660 nm every 24 hours. for cultures grown in 5 mm succinate and 5 mm acetate, growth rates were measured every 12 hours. for cultures grown in benzoate, residual benzoate levels were measured at 120 hours of incubation. benzoate concentration was measured at 276 nm using a hitachi model u-2010 uv/vis spectrophotometer (hitachi instrument, inc. japan) according to shoreit and shabeb (1994). cell density of the isolates grown in other c-sources was measured after 72 hours of inoculation. spectral analysis cells were harvested from 7-day old anaerob-phototrophic cultures grown on modified sistrom supplemented with vitamins and 5 mm succinate as sole carbon source, and suspended in icm buffer (10 mm phosphate buffer ph 7.0 and 1 mm naedta ph 7.0). sonication was carried out using soniprep 150 (msb, uk) at an amplitude of 2 u. for 2 minutes, three times with a time interval of 1 minute. cell extracts were centrifuged at 3000 rpm for 30 minutes at 4°c. protein concentration was determined according to the pierce bca* protein assay kit (rockford, iii, usa). spectral analysis was performed using hitachi u-2010 in protein concentration of ± 100 (ig/ml. amplification and sequencing of part of 16s rrna genes to sequence part of the 16s-rrna genes, the 16s-rrna genes were amplified by pcr using specific primers of 63f and 1387r from genomic dna (200 ng) on readyto-go pcr beads (pharmacia-biotech, uppsala, sweden). phenol-chloroformisoamylalcohol (25:24:1) treatment, ethanol precipitation, and agarose gel electrophoresis were used to purify the genomic dna. total volume of the pcr reaction (25 ul) consisted of 1.5 u tag dna polymerase, lomm tris-hcl (ph 9 at room temperature), 50 mm kc1, 1.5 mm mgcl2, 200 um of each dntps and stabilizer including bsa. the reaction was incubated in a gene amp pcr system 2400 thermocycler (perkin-elmer cetus, norwalk, conn.). 11 biotropia no. 17, 2001 part of 16s-rrna gene was sequenced to infer the closest related organism from the ribosomal database project (rdp) maintained at the university of illinois, urbana-champaign. the sequencing reactions were done by using the big dye ready reaction dye deoxy terminator kit, purified by ethanol-sodium acetate precipitation. the reactions were run on an abi prism 377 dna sequencer (pe applied biosystems, foster city, ca.). construction of phylogenetic trees for the construction of phylogenetic trees, cluster analysis of 16s-rrna gene was done by a computer program from the european bioinformatics institute (http://www.ebi.ac.uk). the treecon computer program (yves van de peer of the department of biochemistry, university of antwerp) was used to determine their phylogenetic relatedness based on their nucleotide sequences and mflp profiles obtained from pulsed-field gel electrophoresis. results and discussion the three new isolates described in this study were similar in their morphological properties. all were gram negative, non-halophilic, non-alkali or acidophilic, aerobic and anaerob-phototrophs that have motile rod-shaped cells with terminal swellings. under anaerobic photothrophic growth conditions, all new isolates produced brick-red and pink cultures in succinate and benzoate, respectively. spectral analysis of cell free extracts (figure 1) of photosynthetic pigments showed absorption maxima for ds-1 at 374, 493, 587, 803 and 852 nm, ds-4 at 371, 501, 585, 803 and 848 nm, and cas-13 at 373, 502, 585, 802 and 871 nm indicating the presence of bacteriochlorophyl a and normal spirilloxanthin series including lycopene and rhodopin (imhoff 1995). in this respect, the three isolates were nearly identical to each other and might be considered as one group. however, these isolates clearly differed from rhodobacter sphaeroides 2.4.1., which has different absorption maxima (376, 451, 453, 477, 507, 587, 746, 799, and 850 nm), with yellow-brown colonies grown anaerobically in succinate. the results from the analysis of partial sequencing of 16s-rrna (c.a. 500 bp) of ds-4, ds-1, and cas-13 demonstrated that ds-4 and cas-13 were closely related (figure 2). partial sequencing of the first 500 bp of 16s rrna gene could lead to a main line of descent (stackebrandt and rainey 1995). comparison with the rdp database of the university of illinois indicated that both ds-1 and cas-13 demonstrated 97% similarity with rs. palustris strain 7, and of 83% and 84% with rb. sphaeroides il106, respectively (table 1). similarity of those of ds-4 to rs. palustris strain 7 was 78%. these results suggested that ds-1 and cas13 are likely to be one species, rs. palustris. ds-4, however, is significantly different from ds-1 and cas-13 by 22%, and from rb. sphaeroides il106 by 30%. complete sequence analysis is needed to give more definitive information about the taxonomic position of these three isolates. 12 characterization of three benzoate degrading anoxygenic photosynthetic bacteria -dwi suryanto et al. figure 1. absorption spectrum of cell extracts ds-1, ds-4, cas-13, and rb. sphaeroides 2.4.1. figure 2. phylogenetic tree 16s rrna gene sequences. the number at the tree lines represented bootstrap values. 13 biotropia no. 17, 2001 table 1. percent similarity of the ds-1, ds-4, and cas-13 with other relative members of anoxygenic photosynthetic bacteria. unlike rb. sphaeroides 2.4.1, all of the three isolates were able to metabolize benzoate (figure 3). among the members of apb, rs. palustris is the most common species capable of utilizing benzoate (harwood and gibson 1988; gibson and gibson 1992; shoreit and shaheb 1994). the ability to grow in the presence of aromatic compounds such as benzoate without vitamins (figure 3) may suggest that the organisms were able to synthesize their own vitamins. however, supplemented vitamins could increase their potential in metabolizing benzoate. the rate of benzoate utilization for ds-1, ds-4, and cas-13 were 0.024 mm/hour, 0.02 mm/hour, and 0.03 mm/hour in media with vitamin supplements compared to 0.017 mm/hour, 0.012 mm/hour, and 0.018 mm/hour in media without vitamins. a relatively similar pattern of extended lag phase was observed in growth of the cultures. time was needed in preparation of producing a number of enzymes. carbon availability might affect cell growth. cell density was observed to be higher in media with benzoate (c7) (figure 3), followed by succinate (c4) and acetate (c2) (figure 4). relatively low cell density of all isolates was shown in 5 mm benzoate with no vitamins. vitamins were certainly necessary for their metabolic activity. 14 characterization of three benzoate degrading anoxygenic photosynthetic bacteria -dwi suryanto et al. figure 3. growht of ds-01, ds-4 and cas-13 in 5 mm benzoate with vitamins (above) and without vitamins (below). rb. sphaeroides 2.4.1. was used as negative control. 15 biotropia no. 17, 2001 figure4. growth of ds-1, ds-4, cas-4, cas-13, and r6. sphaeroides 2.4.1. in 5 mm succinate (above) and 5 mm acetate (below) with vitamins. 16 characterization of three benzoate degrading anoxygenic photosynthetic bacteria -dwi suryanto et al. acnowledgement this research was funded by the center for microbial diversity, faculty of science and mathematics, bogor agricultural university, bogor indonesia. references blasco, r. and f. castillo. 1992. light-dependent degradation of nitrophenols by the phototrophic bacterium rhodobacter capsulatus e1f1. appl. environ. microbiol. 58: 690-695. gibson, k. j and j. gibson. 1992. potential early intermediates in anaerobic benzoate degradation by rhodopseudomonaspalustris. appl. environ. microbiol. 58: 696-698. harwood, c. s. and j. gibson. 1988. anaerobic and aerobic metabolism of diverse aromatic compounds by the photosynthetic bacterium rhodopseudomonas palustris. appl. environ. microbiol. 54: 712-717. hiraishi, a., k. muramatsu, and k. urata. 1995. characterization of new denitrifying rhodobacter strains isolated from photosynthetic sludge for wastewater treatment. j. fermen. bioengine. 79: 39-44. imhoff, j. f. 1995. taxonomy, phylogeny and general ecology of anoxygenic phototrophic bacteria. in anoxygenic photosynthetic bacteria. ed. blakenship, r.e., m.t. madigan, and c.e. bauer. kluwer academic publishers. the netherlands, p. 1-15. kobayashi, m. and m. kobayashi. 1995. waste remediation and treatment using anoxygenic photosynthetic bacteria. in anoxygenic photosynthetic bacteria. ed. blakenship, r.e., m.t. madigan, and c.e. bauer. kluwer academic publishers. the netherlands, p. 1269-1282. kohring, g., x. zang, and j. wiegel. 1989. anaerobic dechlorination of 2,4-dichlorophenol in freshwater sediments in the presence of sulfate. appl. environ. microbiol. 55: 2735-2737. kuo, c. and b.r.s. genthner. 1996. effect of added heavy metal ions on biotransformation and biodegradation of 2chlorophenol and 3-chlorobenzoate in anaerobic bacterial consortia. appl. environ. microbiol. 62: 23172323. shoreit, a. a. m. and m. s. a. shaheb. 1994. utilization of aromatic compounds by phototrophic purple nonsulfur bacteria. biodegrad. 5: 71-76. stackebrandt, e. and f.a. rainey. 1995. partial and complete 16s rdna sequences, their use in generation of 16s rdna phylogenetic trees and their implications in molecular ecological studies. in molecular microbial ecology manual. ed. akkermans, a.d.l., j.d. van elsas, and f.j. de bruijn. pp. mmem -3.1.1/1-17. wright, g. e. and m. t. madigan. 1991. photocatabolism of aromatic compounds by the phototrophic purple bacterium rhodomicrobium vannielli. appl. environ. microbiol. 57: 2069-2073. 17 6. iwan saskiawan page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 biotropia no. 9,1996: 38-52 the genus mimosa with special reference tom. quadrivalvis l. var. leptocarpa (d.c.) earnedy, a new species record for the weed flora in malaysia *) baki, b. bakar, h. noorma wati and m.a. haji mohamed botany dept., university of malaya, 59100 kuala lumpur, malaysia abstract an exploratory floristic survey of the genus mimosa was conducted in 1993 to ascertain species diversity and their spatial patterns of distribution in peninsular malaysia. a new species record of uncertain indigene, m.. quadrivalvis was recorded for the first time in restricted localities along the roadsides in pekan darat and bertam, seberang perai, gurun and bedong in kedah in addition to widely distributed and seemingly ubiquitous presence of m. invisa mart. ex. colla and m. pudica l. the latter two species were mostly found in open, disturbed and derelict habitats, agricultural areas and ex-mining lands. both species exhibited largely contagious and overdispersed distribution patterns with positive peaks in pattern intensity values although regularity or underdispersed distribution patterns do manifest in certain localities. the giant mimosa, m. pigra inhabited in clustered thickets, large pockets of lands in the urban and sub-urban localities in the states of penang, perak, kelantan, kuala lumpur and negeri sembilan. in other states, m. pigra was confined to a few localities in smaller patches. except for m. quadrivalvis, the other species of mimosa are serious weeds in the agricultural, recreational and residential and derelict areas. a key to the mimosa species is constructed along with brief descriptions on their morphology and ecology. key words: malaysia/weed ecology/mimosa invisa/mimosa pigra/mimosa pudica/mimosa quadrivalvis/ weed distribution/weed anatomy and morphology. introduction the genus mimosa, in the family mimosaceae (syn. leguminosae or fabaceae) is large and cosmopolitan with 300-500 species worldwide (holm et al. 1977; nielsen 1981, 1983, 1992). the intricacy of this genus has made it a subject of continued research interest and taxonomic revisions (e.g. ridley 1925; corner 1952; henderson 1959; burkill 1966; metcalfe & chalk 1972; nielsen 1992). according to index kewensis (1991), there are well over 1800 binomials of mimosa worldwide with heavy concentrations in tropical america. in malesia, there are three introduced and naturalised species. recent studies by nielsen (1992) gave extensive accounts of the genus *) paper presented at the symposium on biology and management of weeds and fourth tropical weed science conference, 22 24 november 1994, bogor, indonesia 38 38 the genus mimosa baki, b. bakar et al. on the world scale. in malaysia, the genus mimosa is known for its weedy elements viz. m. invisa, m. pigra and m. pudica infesting large areas of agricultural, residential, recreational, roadsides and derelict areas and ex-mining lands. ridley (1925) gave the first account of the family in the malay peninsula while burkill (1966) described a brief history of the various species that has been introduced into the malay peninsula. waterhouse (1993) enlisted the three weedy species of mimosa with the 15 to 19 ratings among the 140 most important weed species as appropriate targets for biological control in southeast asia in general and in malaysia in particular. this paper reports briefly on the morphology and distribution patterns of mimosa spp. with emphasis on m. quadrivalvis l. var. leptocarpa (dc.) barneby. materials and methods an exploratory floristic survey was conducted in 1993 in peninsular malaysia to ascertain species diversity and spatial distribution patterns of the genus mimosa. for such purpose, samplings were made at random in selected vegetable gardens, derelict areas, immature rubber and oil palm estates, ex-mining lands and roadsides. in addition, the list count quadrat (1m x 1m) method of kim & woody (1983) was also used. in the case of m. pigra, the line transect method was used with quadrat size of 2m x 2m. only adjacent and accessible areas close to the roads were sampled. a total of 72 quadrat samples for each species were obtained (table 1). the spatial distribution date for each species were subjected to pattern analysis (allinson 1981) with pattern intensity values used as indices to measure the departure from randomness. detailed morphological comparisons were made between species. table 1. plot size,number of transects and quadrats transects employed in pattern analysis of plant population of mimosa species 39 biotropia no. 9, 1996 results and discussion pattern of distribution figure 1 illustrates the extent of infestation of the four species of mimosa in peninsular malaysia. generally, heavy concentrations of m. pigra are found in ex-mining lands, ex-padi farms and derelict areas both in rural and urban localities in the states of penang, perak, kelantan, kuala lumpur and negeri sembilan, often in clustered thickets and large pockets. mimosa invisa and m. pudica are widely distributed and seemingly ubiquitous mostly in open, disturbed and derelict habitats, agricultural areas and ex-mining lands. the soils range from rather wet of the inseptisol, vertisol and entisol types or the relatively dry soils of the oxisol or ultisol types. soil samples taken in areas where rampant infestations of mimosa spp. occur registered ph values ranging from 4.0 5.0. mimosa invisa and m. pudica are also found on the bris soil habitat in terengganu, pahang, kelantan and pantai remis areas of perak. clump sizes of adult populations vary considerably according to the species of mimosa. in the case of m. pigra, it ranges from 1.0 m to 6 m in diameter while for m. pudica and m. invisa these were about 0.25 m 2.0 m. mimosa quadrivalvis has a relatively smaller clump size of about 0.2 1.0 m (table 2). genet populations range from 0.2 1.0 plant/m2 for m. pigra; 0.5 4.0 plants/m2 (m. invisa and m. pudica) to 1.0 5.02 plants/m2 for m. quadrivalvis, thereby emphasizing different morphology and growth habits of the different species of mimosa (table 2). table 2. clump size and genet population density of mimosa spp. in peninsular malaysia species clump size (m) genet population density (per m 2 ) m. invisa 0.25 2.0 0.5 4.0 m. quadrivalvis 0.20-1.0 1.0-5.0 m. pigra 1.0 -6.0 0.2-1.0 m. pudica 0.25 2.0 0.5 4.0 the spatial distribution of the four species of mimosa differed accordingly, showing pattern at different block sizes in different regions of survey. in the case of m. pigra population in the northern states of penang, perak and kedah (region a), pattern was observed with peak pattern intensity values at block size 12. similar clusterings were observed among m. pigra populations in malacca, negeri sembilan, johore (region c) and kelantan (region d). mimosa pigra populations in the federal territory, 40 the genus mimosa baki, b. bakar el al figure 1. the distribution of (a) mimosa invisa, (b) m. quadrivalvis, (c) m. pigra, and (d) m. pudica in peninsular malaysia. each symbol represents at least one record within 5 m radius. 41 biotropia no. 9, 1996 selangor (region b) and southern perak showed pattern at block sizes 8 and 16. a tendency towards regular undispersed spatial distribution was observed among m. pigra populations in region b at block size 1 (figure 2a). individual genets among m. invisa population for all regions showed regularity for block sizes 1, 2 and 3. contagious overdispersed individuals were observed at block size 6 for region c and block size 8 for regions a, b and d (figure 2b). regularity in spatial distribution of individuals of m. pudica was recorded at block sizes 1, 2 and 3 for all regions saved region b. positive peaks of pattern intensity values were recorded at block size 6 for regions a, b and d and block sizes 6 and 16 for region c (figure 2c). the spatial distribution of individual genets of m. quadrivalvis showed pattern at block size 6 but exhibited regularity at block sizes 1, 2 and 3 (figure 2d). no concrete arguments can be forwarded to explain the spatial heterogeneities exhibited by different populations of mimosa pigra, m. invisa, m. pudica and m. quadrivalvis. differential growth rates and differences in morphology among the species studied are too simple a notion to help illustrate such differences. further analyses on the possible roles of environmental variables such as soil, ph, nutrient status, soil types, etc. in explaining the spatial heterogeneities and distribution patterns of the different mimosa species are needed. morphology and species description morphological illustrations and habit of m. quadrivalvis are shown in figures 3 -6. comparative morphological traits and characteristics of the species are shown in tables 3 and 4. 1. mimosa invisa auct. non mart, ex colla: backer & bakh. f., fl. java 1 (1963) 561; burkill, diet. econ. prod. mal. penins. 2 (1966) 1498; nielsen, fl. camb., laos, vietnam 19 (1983) 34, pi. 5: 1-7; nielsen, fl. malesiana ser. 1, 2, 1 (1992) 184-185. a procumbent perennial shrub, growing up to 3 m high. stem conspicuously 4-angled, up to 4 m long, densely distributed with prickles. leaves bipinnate, primary leaflets 4 to 9 pairs, leaflets opposite sessile 12 to 30 pairs, lanceolate, 6 to 12 mm long, 1.5 mm wide. inflorescence a globular head, axillary, flowers pinkish violet, corolla 4-lobed with 8 pinkish violet exserted stamens. fruit softly spiny, 3to 4-seeded borne as clustered axillary, flattened pods, 1.0 to 3.5 cm long, 6 to 10 mm wide, splitting along the valves into 1-seeded segments when mature. seeds flattened, ovate light brown, rather glossy, 2 to 3 mm long. 42 the genus mimosa baki, b. bakar et al figure 2. pattern intensity values of spatial distribution of (a) m. pigra, (b) m. invisa, (c) m. quadrivalvis, and (d) m. pudica plants at different block sizes. lower case letters on the curve denote regions of occurrence (see text for details). 43 biotropia no. 9, 1996 figure 3. mimosa quadrivalvis. 44 the genus mimosa baki, b. bakar et al. figure 4. mimosa quadrivdvis. (a) stamens, (b) axillary inflorescence, (c) opened flowers. 45 biotr0pia no. 9, 1996 figure 5. mimosa quadrivalvis. (a) mature pod densely covered with long, fine prickles, (b) opened pod, (c) young pod, (d) seeds. 46 the genus mimosa baki, b. bakar et al. figure 6. mimosa quadrivalvis. (a) seeds, (b & c) emergence of hypocotyl, (d & e) hypocotyl with cotyledons, (f) emergence of once-pinnate, first true-leaf, (g) young plant. 47 biotropia no. 9, 1996 48 the genus mimosa baki, b. bakar et al. 49 biotropia no. 9, 1996 2 mimosa quadrivalvis l. var. leptocarpa (dc.) barneby. a straggling shrub. mature stem angular, prickles sparsely distributed; younger stem slightly rounded. leaves evenly bipinnate, primary leaflets 1 2 pairs, 0.5 1 cm apart from each other, leaflets opposite, 9-16 pairs, almost sessile, 2 8 mm long, 0.5 -2.0 mm wide, margin entire, pubescent, oblanceolate-oblong, both abaxial and adaxial surfaces sparsely distributed with glands and sparsely hairy but abaxial surface of outermost leaflet usually more hairy compared to abaxial surface of the inner leaflets. secondary veins obscure, apex mucronate-truncate, base truncate. secondary rachilla covered with short fine hairs, sparsely distributed. inflorescence a globular head, axillary, 1 8 2 0 flowers per inflorescence. stamen numerous, pinkish, 1.0 1.5 mm long, arranged in several rows forming a globular head. bracts present, hairy. fruits in axillary flattened pods, 1 3 in a group, usually the third one abortive, 4.0 9.5 cm long with prominent raised ridge along the edges, apex sharply pointed, tapered to a point 1.0 1.5 cm long, almost linear, moderately to densely covered with long, fine prickles, pedicel 0.8 1.1 cm long, hairy; mature fruits dehisce along sutures. seed ovate to rhomboid in shape when dry, brownish-black, about 3 mm long. 3. mimosa pigra l., backer & bakh. f., fl. java 1 (1963) 561; nielsen, fl. camb. laos, vietnam 19 (1981) 34, pi. 5: 8-14; nielsen, fl. malesiana ser. 1, 2,1 (1992) 185. mimosa sepiaria auct. non benth.: ridley, fl. mal. pen. 1 (1922) 656, p.p.; burkill, diet. econ. prod. mal. pen. 2 (1966) 1499. an erect, perennial woody shrub, growing up to 5 m high. stem woody, up to 3 m long, sparsely distributed with prickles. leaves bipinnate, primary leaflets about 15 pairs, leaflets sessile, lanceolate. inflorescence a globular head, axillary, flowers pink or mauve, corolla 4-lobed with 8 pink stamens. fruits densely hairy, 20to 25-seeded axillary, borne as clustered flattened pods, 6.5 to 7.5 cm long, 7 to 10 mm wide, breaking into 1-seeded segments when mature. seed brown or olive green, oblong, flattened, 4 to 6 mm long, 2 m wide. 4. mimosa pudica l., backer & bakh. f., fl. java 1 (1963) 561; nielsen, fl. camb. laos vietnam 19 (1981) 35, pi.; nielsen, fl. malesiana ser. 1, 2, 1 (1992) 185-186. a procumbent, perennial, creeping shrub growing up to 50 cm high. stem rounded, stiff, up to 150 cm long, prickles scattered along the internodes. leaves bipinnate, primary leaflets 1 to 2 pairs, leaflets opposite, 12 to 25 pairs, oblong or linear, 9 to 12 mm long, 1.5 mm wide, margins hairy. inflorescence an ovoid or a globular head, borne on prickly axillary stalks, flowers purplish pink, corolla 4-lobed, stamens 4, purplish pink. fruits oblong, flattened 1to 5-seeded pod, 1 to 2 cm long, 3 to 6 mm wide, edge with prickles, borne in cluster on leaf axils, breaking into 1-seeded segments 50 the genus mimosa baki, b. bakar et al. when mature. seeds light brown, flattened with a finely granular surface, 2.5 to 3 mm long. key to mimosa species in peninsular malaysia 1. primary leaflets 1-2 pairs 2. leaflets almost sessile, pubescent, 2 8 mm long, 0.5 2.0 mm wide. stem angular. flowers pink, in globular heads, stamens pink. pods 4.0 9.5 cm long, apex sharply pointed tapered to a point 1.0 1.5 cm long, covered with long, fine prickles ..................................................................................................... m. quadrivalvis 2. leaflets sessile, 9 1 2 mm long, 0.5 mm wide, margin hairy. stem rounded. flowers purplish pink, in ovoid or globular head, stamens purplish pink. pods 1 2 cm long, edge with prickles ............................................................................ m. pudica 1. primary leaflets more than 2 pairs 3. primary leaflets 4 9 pairs, leaflets lanceolate. stem conspicuously 4-angled with numerous prickles. flowers pinkish violet. pods 1.0 3.5 cm long, 3 to 4-seeded. seed light brown, glossy, ovate, 2 3 mm long ................................................ m. invisa 3. primary leaflets about 15 pairs, leaflets lanceolate. stem woody, prickles sparsely distributed. flowers pink or mauve. pods 6.5 7.5 cm long, 20to 25-seeded. seeds brown or olive green, oblong, 4 6 mm long .......................... m. pigra acknowledgements the authors are extremely thankful to dr. g.p. lewis of kew botanical gardens, u.k. for identifying the species and mr. anuar pandak and mr. zainal mustafa for their technical assistance. thanks are also due to mr. tan lip hong for typing the manuscript. references allinson, j.m. 1981. numerical and experimental analysis of species relationships in grassland. phd thesis, university of wales. backer, c.a. and r.c. bakhueen van den brink. 1963. flora of java. vol, 1. rijksherbarium. leyden. 547-565. 51 biotropia no. 9, 1996 burkill, i. h. 1966. a dictionary of the economic products of the malay peninsula. 2nd ed. the crown agent for the colonies, london. corner, e.j.h. 1952. wayside trees of malaya. 2nd ed. govt. print. off. singapore, p 413. henderson, m.r. 1959. malayan wild flowers. dicotyledons. malayan nature society, p 100. holm, l.g., d.l., plucknett, j.v. pancho and j.p. herberger. 1977. the world's worst weeds. distribution and biology. univ. press of hawaii, honolulu. index kewensis 1991. oxford university press. kim, s.c and k. woody. 1983. comparison of some methodologies for vegetation analysis in transplanted rice. korean journal of crop science 28(3): 310-318. metcalfe, c.r. and l. chalk. 1972. anatomy of the dicotyledons. vol. 1. oxford, clarendon press, p 487. nielsen, i. 1981-83. flora of cambodia, laos and vietnam, vol. 19. nielsen, i.c. 1992. mimosaceae (leguminosae mimosoideae). flora malesiana ser. 1, vol. 2, prt. 1. ridley, h.n. 1922. flora of malay peninsula vol. 1. reeve and co. london . waterhouse, d.f. 1993. the major arthropod pests and weeds of agriculture in southeast asia: distribution, importance and origin. aciar, canberra. 141 p. 52 38.pdf 39.pdf 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf 564 (analyn tiger).cdr tiger beetles (coleoptera: carabidae: cicindelinae) of compostela valley province, mindanao island, philippines 1* 2* analyn anzano cabras , milton norman dejadena medina and 3jürgen wiesner 1college of arts and sciences, university of mindanao, davao city, philippines 2 research and publication center, university of mindanao, davao city, philippines 3dresdener ring 11, d-38444 wolfsburg, germany received: 25 november 2015/accepted: 16 september 2016 abstract the first faunistic record of tiger beetles (coleoptera: carabidae: cicindelinae) in compostela valley province, mindanao with notes on their habitat is presented. opportunistic sampling, standard light trapping and photo documentation were conducted in selected areas from january to april 2013 and in september 2015. one hundred four tiger beetles belonging to twelve species and seven genera were recorded. an astonishing (100%) endemicity was observed identified to subspecies level. thopeutica and calomera species, specifically thopeutica anichtchenkoi wiesner, 2015 and calomera mindaoensis cassola, 2000 were observed to be abundant in the samples mostly found in the mountainous and forested open riparian ecosystem of cagan, new bataan. cylindera (ifasina) discreta elaphroides (dokhtouroff 1882) was the most common species and was found in different habitat types, but the majority of the species showed a narrow and specialized habitat preference. protection and conservation of remaining forest areas in cagan is especially important in preserving endemic species. keywords: compostela, conservation, endemics, fauna, tiger beetle introduction the philippine tiger beetle fauna is one of the most unique in the world that despite its small land area, philippines ranks fifth in terms of tiger beetle richness and third on the level of endemism after madagascar and australia (cassola & pearson 2000; cassola & ward 2004; cassola 2011). based on endemism per unit area, philippines ranks number one. with very limited entomological expeditions, the tiger beetle fauna of the philippines is poorly known. early collections date back to 1859 1865 by c.g. semper and 1913-1919 by g. boettcher. these tiger beetles were made known by european entomologist, schaum (1860, 1862, 1863) and horn (1907, 1908, 1909, 1923, 1924, 1937). recent publications on the tiger beetle fauna of the philippines were made by bogenberger (1988), cassola (2000, 2011), cassola and ward (2004), cassola and zettel (2006), deuve (2015), dheurle (2015), naviaux (1992, 2002) and wiesner (1980, 1988a, 1988b, 1989, 1992a, 1992b, 2015). since the early 1990's, new tiger beetle species have been described from the different, but poorly explored islands such as palawan, leyte, mindoro and mindanao. to date, there are 139 tiger beetle species known from the philippines, of which 111 (86.3%) are endemic to this country with a limited geographic range (santos 2014). in 1998, an international workshop on the natural history of the philippines held at berlin, germany was conducted where insect studies were greatly emphasized hurdling the concern on increasing loss of philippines forest through deforestation (cassola 2000). since the majority of the species are forest dependent, loss of the forest has caused a tremendous threat to different species and others may have gone extinct without being known to science. biotropia 3 2 6 133 139 vol. 2 no. , 201 : doi: 10.11598/btb.2016.2 . .3 2 564 * corresponding author: mnd_medina@umindanao.edu.ph; ann.cabras24@umindanao.edu.ph 133 tiger beetles are often used as good bioindicators of the microhabitat changes of an ecosystem. since many faunal elements including several tiger beetles are associated with forest ecosystems and have a limited habitat range, they are most vulnerable to deforestation (cassola & ward 2004). the philippines had already lost 75% of its forest in the past century, so many tiger beetles may have disappeared without being described. compostela valley province belongs to the eastern biodiversity corridor which is known as one of the remaining biodiversity hotspot of the philippines. however, there is a great concern over its biodiversity, as the province is experiencing major anthropogenic disturbances including illegal logging and mining. this paper presents the baseline data of tiger beetle fauna of compostela valley province with notes on the habitat preference of tiger beetles. this information is important in drafting conservation efforts for this insect group. materials and methods opportunistic sampling, light trapping and photo documentation were conducted for diurnal and nocturnal tiger beetles in selected areas in compostela valley province from january to april 2013 and in september 2015. seven areas were sampled: (1) cagan, new bataan (07°41'n; o o126°08'e), (2) sampao (07 42' n, 125 59' e), (3) mayaon, montevista (07°48'n; 126°02'e), (4) o obarangay andili, mawab (07 30' n, 125 56' e), (5) pasian, monkayo (07°56'n; 125°58'e), (6) san o oisidro (07 36' n, 125 58' e) and (7) cabalinan, nabunturan (07°36' n; 125°57' e). specimens were obtained using insect net during the day and light trapping during the night. the specimens were killed using ethyl acetate, dried and stored in the first authors' collection. results and discussion a total of 104 individuals belonging to 12 species and seven genera of tiger beetles were recorded. three species were recorded for each of the genera therates and thopeutica, while two species were recorded for the genera calomera and cylindera. prothyma, tricondyla and neocollyris were represented only by one species. seven species (58%) widely distributed from luzon to mindanao are philippine endemics (table 1, while five species have no known record except in mindanao and at the moment they are recognized as mindanao endemics (table 1, fig. 2) unless new records say otherwise. two species out of three calomera present in mindanao were recorded in compostela valley province namely calomera lacrymosa (dejean 1825) and calomera mindaoensis (cassola 2000). majority of the species were collected close to the riverbanks using light trapping method. species of the genera thopeutica, prothyma, 134 biotropia vol. 23 no. 2, 2016 figure 1 map of sampling sites in compostela valley province, philippines 135 – tiger beetles of compostela valley province, mindanao island, philippines cabras et al. t ab le 1 sp ec ie s di st ri b ut io n , h ab it at a n d ab un da n ce o f t ig er b ee tl es in t h e c o m p o st el a v al le y p ro vi n ce sp ec ie s h ab it at t yp e g eo gr ap h ic d is tr ib ut io n c g c b si sm m a a n p a n p er ce n ta ge c al om er a m in da na oe ns is (c as so la 20 00 ) o p en , r iv er in e sa n d y ar ea s in m o un ta in o us s ec o n da ry f o re st m in da n ao e n de m ic 13 1 7 21 20 .1 9 c al om er a la cr ym os a (d ej ea n 1 82 5) o p en , r iv er in e, lo w la n d sa n dy a re as m ix ed a gr ic ul tu ra l e co sy st em p h ili p p in e en de m ic 3 3 2. 88 c yl in de ra ( if as in a) m ou th ie zi (d h eu rl e 20 15 ) b o th f o un d in t h e o p en , r iv er in e sa n dy ar ea s o f a se co n da ry f o re st in m o un ta in o us a re a an d lo w la n d m ix ed ag ri cu lt ur al e co sy st em m in da n ao e n de m ic 1 4 1 6 5. 77 c yl in de ra ( if as in a) d is cr et a el ap hr oi de s (d o kh to ur o ff 1 88 2) b o th f o un d in t h e o p en , r iv er in e sa n dy ar ea s o f a lo w la n d s ec o n da ry f o re st a n d m ix ed a gr ic ul tu ra l e co sy st em p h ili p p in e en de m ic 4 1 16 6 27 25 .9 6 n eo co lly ri s (h et er oc ol ly ri s) si m ili or (h o rn 1 89 3) sh ad ed a re as a n d b ra n ch es o f sh ru b s in th e m ix ed a gr ic ul tu ra l e co sy st em p h ili p p in e en de m ic 3 3 2. 88 p ro th ym a (s ym pl ec th ym a) he te ro m al lic ol lis h et er om al lic ol lis (h o rn 1 90 9) o p en , r iv er in e sa n dy a re as in a m ix ed ag ri cu lt ur al e co sy st em m in da n ao e n de m ic 1 1 0. 96 t he ra te s co ra cin us c or ac in us (e ri ch so n 1 83 4 ) sh ad ed r iv er in e sa n dy a re as a n d sh ru b s in m ix ed a gr ic ul tu ra l e co sy st em p h ili p p in e en de m ic 1 1 2 1. 92 t he ra te s fa sc ia tu s fa sc ia tu s (f ab ri ci us 1 80 1) sh ad ed r iv er in e sa n dy a re as a n d sh ru b s in m ix ed a gr ic ul tu ra l e co sy st em p h ili p p in e en de m ic 1 2 3 6 5. 76 t he ra te s fu lv ip en ni s ev er et ti (b at es 1 87 8) sh ad ed r iv er in e sa n dy a re as a n d sh ru b s in m ix ed a gr ic ul tu ra l e co sy st em p h ili p p in e en de m ic 3 1 4 3. 85 t ho pe ut ica ( t ho pe ut ica ) an ich tc he nk oi (w ie sn er 2 01 5) o p en , r iv er in e sa n d y ar ea s in s ec o n da ry fo re st in m o un ta in o us a re a m in da n ao e n de m ic 24 24 23 .0 7 t ho pe ut ica ( t ho pe ut ica ) ro la nd m ue lle ri (c as so la 2 00 0) o p en , r iv er in e sa n d y ar ea s in s ec o n da ry fo re st in m o un ta in o us a re a an d lo w la n d m ix ed a gr ic ul tu ra l e co sy st em m in da n ao e n de m ic 3 1 4 3. 84 t ri co nd yl a (t ri co nd yl a) e lo ng at a (h o rn 1 90 6) sh ad ed a re as a n d b ra n ch es o f sh ru b s in th e m ix ed a gr ic ul tu ra l e co sy st em p h ili p p in e en de m ic 3 3 2. 88 t o ta l 10 4 n o te s: c g = c ag an ; c b = c ab al in an ; s i = s an i si dr o ; sm = s am p ao ; m a = m ay ao n ; a n = a n di li; p a = p as ia n 136 biotropia vol. 23 no. 2, 2016 cylindera and calomera were all found in the open and sandy riverbanks and caught using both light trapping and hand netting methods. these four genera were found to inhabit open and sandy riverine ecosystems, agreeing with dangalle et al. (2012). species of the genera therates, neocollyris and tricondyla were often found perching or running in the shrubs and trees near rivers preferring shade rather than exposed open areas. these species were easily caught using a hand net or by hand. species from the genus therates were especially found in close proximity to river banks while the species from the genera neocollyris and tricondyla were found along the roads and some were near creeks and rivers. the most common species found to be widespread in compostela valley were cylindera (ifasina) discreta elaphroides, therates fasciatus fasciatus and calomera mindanaoensis (table 1). cylindera (ifasina) discreta elaphroides) was the most dispersed species found in four municipalities, while therates fasciatus fasciatus and calomera mindanaoensis were found in three municipalities indicating the wide distribution of these species in the province. thopeutica anichtchenkoi was abundant but exclusively found in a single locality in cagan which is characterized by mountainous secondary forest. other species from the genera therates, tricondyla, neocollyris and prothyma were exclusively distributed in the lowland riparian ecosystems with mixed agricultural vegetation, while species from the genera calomera, cylindera and theopeutica were found either in riverine ecosystems with secondary forest in mountainous areas as well as in lowland open riverine ecosystem with mixed agricultural vegetation. among the areas sampled, mayaon had the highest species richness. the lowland riparian ecosystem with mixed agricultural vegetation with patches of remaining secondary forests was the most preferred habitats by eight species of tiger beetles including calomera mindanaoensis, calomera lacyrmosa, cylindera (ifasina) mouthiezi, cylindera (ifasina) discreta elaphroides, therates coracinus coracinus erichson, 1834, therates fasciatus fasciatus, therates fulvipennis everetti and thopeutica rollandmulleri. cagan which is a mountainous ecosystem was represented by four species of tiger beetles namely calomera mindanaoensis, thopeutica anichtchenkoi, thopeutica rollandmuelleri and cylindera (ifasina) mouthiezi. among the species mentioned only thopeutica anichtchenkoi was exclusively found in cagan which inferred the high habitat preference of this species to forested riverine ecosystems in mountainous areas. the species of the genera calomera, cylindera and thopeutica showed a high preference to mountainous and forested riverine ecosystems as indicated by their high abundance in cagan, whereas the species of the genera therates, neocollyris, tricondyla and prothyma showed high preference to lowland riparian ecosystem with a b figure 2 endemic species (a) calomera mindanaoensis; (b) prothyma (symplecthyma) heteromallicollis heteromallicollis 137 mixed agricultural vegetation. the arboreal nature of the species of the genera therates, neocollyris and tricondyla agreed with cassola (2000) and dangalle et al. (2014) about the habit of these beetles. the preference of the tiger beetles to live in close proximity to the water enables them to be near water and food and provides a good habitat for their reproduction (bhargav & uniyal 2008). the high abundance of cylindera (ifasina) discreta elaphroides being found in five sampling sites as compared to other species could indicate the high adaptability of the species to habitat modification since it was found inhabiting the mountainous forested riverine ecosystem of cagan as well as the low land river banks with mixed agricultural ecosystems in mayaon, san isidro, cabalinan and pasian. on the contrary, thopeutica anichtchenkoi was abundant but exclusively found in a single locality in cagan which was characterized by mountainous secondary forest. the exclusive distribution and high abundance of this species could indicate its high preference to mountainous forested riparian ecosystem. many species of tiger beetles are known for their association with forest habitats and limited distributional range as well as narrow habitat preferences as related to temperature, exposure to sunlight, ph and soil moisture and salinity (dangalle et al. 2014). the other species recorded in cagan such as calomera mindanaoensis, thopeutica rollandmulleri and cylindera (ifasina) mouthiezi were also found in the lowland riverine ecosystems with mixed secondary forest and agricultural vegetation indicating the species association with forested habitats. this also implied the species ability to adapt and thrive in habitats with human encroachment brought by conversion of land to farming. santos (2014) mentioned that further studies should be conducted in tiger beetles' ability to adapt to the changing environmental conditions brought by human disturbances and non-forested habitats. cagan which is a mountainous forested ecosystem had the highest abundance of tiger beetles with 41 (39.2%) individuals belonging to four species and three genera followed by mayaon with 29 (27.88%) individuals recorded belonging to eight species and four genera. pasian had the third highest abundance of tiger beetles with seventeen individuals recorded belonging to five species and three genera. the high abundance of tiger beetles in cagan may indicate the preference of the two most abundant species i.e. calomera mindaoenses and thopeutica anichtchenkoi for riparian ecosystems in mountainous and forested ecosystem due to its vegetation, soil moisture and ph which are important factors that determine tiger beetles' habitat preference (dangalle et al. 2014). the high abundance could be due to the ample availability of food resources which is a key factor in most insect taxon abundance. although mayaon had the highest species richness, species abundance was quite low indicating the high competition of food among the different species of tiger beetles. species coexisting in a locality had been observed. calomera lacrymosa and calomera mindaoensis as mentioned by cassola (2000) can occur sympatrically. both were found occurring in mayaon. calomera lacrymosa was most common in mayaon, while calomera mindaoensis was most abundant in cagan and pasian. these two species can be easily mistaken as one species. however, upon closer inspection it could be found that calomera lacrymosa was smaller at 10 11 mm with paler and dull elytra with the two discal dots connected to each other by a narrow lineole, while calomera mindaoensis was bigger with a size that ranged about 13 14 mm and had velvety and darker elytra with elytral punctuation poorly or not visible at all and the two discal dots not connected. in cagan calomera mindanaoensis and thopeutica anichtchenkoi were the most dominant. however, thopeutica rolandmulleri and cylindera (ifasina) mouthiezi were also found, but in very low number. mayaon which was inhabited by seven species of tiger beetles was dominated by cylindera (ifasina) discreta elaphroides with 16 recorded individuals, while the other species were only represented by low abundance of around 1 4 individuals. the high species richness in mayaon could be attributed to the optimum sunlight and brightness, which were necessary in the predatory activities as well as thermoregulation of the different species of tiger beetles (knisley 1984). areas sampled with the lowest species richness and abundance were cabalinan, barangay. andili and sampao with only one to two species recorded. these riparian areas have a mixed agricultural ecosystem with few standing forest and high anthropogenic disturbances which can inhibit the diversity by reducing the habitat. – tiger beetles of compostela valley province, mindanao island, philippines cabras et al. 138 biotropia vol. 23 no. 2, 2016 conclusions most philippine species of tiger beetles showed a consistent habitat preference. species of the genera tricondyla, neocollyris and therates were arboreal species found in shrubs and trees near the rivers and roads, while other species of the tiger beetles were found in open riparian ecosystems. a narrow distribution of thopeutica anichtchenkoi was observed and found exclusively in cagan. cylindera (ifasina) discreta elaphroides was the most dispersed species found in almost all areas sampled indicating its high adaptability. immediate conservation efforts should be conducted for the tiger beetles thopeutica anichtchenkoi, cylindera (ifasina) mouthiezi and thopeutica rollandmulleri which were highly associated with forested riparian ecosystems and had narrow distribution. references bhargav vk, uniyal vp. 2008. communal roosting of tiger beetles (cicindelidae: coleoptera) in the shivalik hills, himachal pradesh, india. cicindela 40(1-2):112. bogenberger jm. 1988. two new species of tiger beetles from palawan (coleoptera, cicindelidae). mitteilungen der münchner entomologischen gesellschaft 78:109-14. cassola f. 2000. studies on tiger beetles. cii. the cicindelidae collected by roland a. müller in the philippine 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contribution towards the knowledge of cicindelinae. mitteilungen des internationalen entomologischen vereins 40(1/2):1-8. – tiger beetles of compostela valley province, mindanao island, philippines cabras et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 diversity of actinomycetes from eka karya botanical garden bali**, shanti ratnakomala , puspita lisdiyanti , ,1 1 1 2* nita r. prayitno , evi triana yulin lestari , ratih d. hastuti , , misa otoguro3 4 1 5yantyati widyastuti , katsuhiko ando5 4 and endang sukara1, 1research center for biotechnology cibinong 16911, indonesia, indonesian institute of sciences, 2research center for biology cibinong 16911, indonesia, indonesian institute of sciences, 3 aculty of mathematics and natural sciences, i p b ,680department of biology, f nstitut ertanian ogor, bogor 16 indonesia 4soil research institute, bogor 16002, indonesia 5 inite biolog cal resource center (nbrc), 2-5-8 kasuzakamatari, kisarazu, chiba, japan received 1 july 2015/accepted 6 may 2016 abstract a total of 2 actinomycetes were isolated 29 strains of and identified by full sequence of 16s rrna gene analysis. samples ed from , bali island,consist of 18 soil and 20 leaf litter were collected eka karya botanical garden indonesia. two isolation methods, sdseast xtract (sy) and rehydration-centrifugation (rc) used . i.e. y e were in this study based on 16s rrna gene analysis, isolated actinomycetes may be grouped into 2 genera. b 8 ased on molecular analysis of 16s rrna gene similarities showed that eka karya botanical garden . isolated actinomycetes of origin diverse is analysis on from soil samples, resulted in is most 144 isolates 24 genera and more than 87 species. the streptomyces dominant gen where or from isolated actinomycetes belong to this genus. it was followed by us 65 isolates 45% actinoplanes (25 isolates = 17%). from leaf-litter 85 isolates 9 genera samples, the total number of may be group into ed and more than 41 species. the most dominated genus is (42 isolates = 49%) (16 actinoplanes catenuloplanesfollowed by isolates = 19%). eka karyakeywords: 16s rrna gene analysis, actinomycetes, biodiversity, botanical garden introduction actinomycetes are microorganisms belong to gram positive bacteria which are often saprophytic while some of them produce spores and mycelium. they play t roles n degrading importan i and decomposing organic compounds in the soil. they also produc secondary metabolites may e such as antibiotics, enzymes and other bioactive compounds for human welfare. actinomycetes constitute a significant component of the microbial population in most soils and counted of over 1 million per gram of soil. oil is, cells s therefore this , the most prolific source of particular group. s oil represents the most intensively studied habitat of actinomycetes. actinomycetes are thought to be most also the significant in the degradation of relatively group complex, recalcitrant polymers naturally in found plant litter and soil (hopwood 2007; baskaran et al. 2011). the preliminary characterization of the actinomycetes isolates was colony appearance, observing of zoospore bearing isolates, and dap analysis. dap analysis is a biochemical itself analysis of cell walls to observe the dap isomers of g the cell wall of actinomycetes. most of ram positive bacteria have lysine instead of dap in their cell wall , . through this method actinomycetes could be into 3 groupsgrouped . according miyadoh (2004), actinomycetes that to have ll-dap in their cell wall generally belong to genus and ; while those streptomyces streptacidiphilus with -dap type in their cell wall generally meso belong to the non or so called rare streptomyces actinomycetes. actinomycetes that have both biotropia vol. 23 no. 1, 2016: 42 51 doi: 10.11598/btb.2016.2 . .5043 1 * c orresponding author: yahoo.comshanti_ratna@ ** this paper was presented at symposium on recent advances on microbiological researches and its application (8-9 november 2011). serpong, indonesia. 42 ll-dap and meso-dap usually belong to genus kitasatospora. therefore, the dap analysis could differentiate those three types of actinomycetes. some actinomycetes share the same ability to release flagellated zoospores at a certain stage in their life cycle (cross 1986). current classification of motile-spored actinomycetes can identify at least six suborders containing zoosporic genera, including micromonosporinae, micrococcinae, frankinae, pseudonocardinae, kineosporiineae and streptosporanginae et al (stackebrand . 1997). these t zoospores bearing actinomycetes have been associated with river, lake and fresh water, river sediments, desert soil (garrity . 1996 bredholt et al ; et al. et al 2008 sibanda . 2010), decaying plant ; materials submerged in streams and cast up on lake shores (kudo 1998 tamura . 2010), et al. et al; blades of grass inhabiting streams and soils (hasegawa 1991). it has become increasingly apparent that motile actinomycetes can produce a variety of antibiotics and other bioactive metabolites or be used for biochemical conversion of complex compounds (hasegawa 1991 garrity . 1996 khamna . 2010 ; ; ;et al et al khanna . 2011).et al according to hayakawa (2000), et al. actinomycetes could be divided into two types based on spores produced by the non-motile and motile. actinomycetes which bear non-motile spores that are not generally form flagella, for example, are , , , streptomyces nocardia micromonospora and so on. and are actinoplanes catenuloplanes motile zoospores bearing actinomycetes and the zoospores can move. several actinomycetes genera such as actinoplanes, amycolatopsis, catenuloplanes, dactylosporangium, kineospor a, i microbispora, micromonospora nonomuraeaand are often very difficult to isolate and cultivate due to their slow growth and those rare belong to the actinomycetes (hayakawa 2008). eka karya botanical garden, bedugul bali in , island is a unique plant , indonesia ex situ conservation site for plant species of high elevated eastern tropical rain forest of indonesia, adjoining with the tropical forest of batukahu nature reserve. this garden is located at 1,2501,450 m above sea level, with area of 157.5 hectares (389 acres). temperature is about 17 25 c in daytime and is dropped to 10-15 c at o o night with 70-90% humidity (mukaromah & suparta 2007). based on the uniq ness of the ue above location, we stud about the diversity of ied actinomycetes this location. this study wasin intended to be done as pioneer research in eka karya botanical garden. several stud on ies diversity of actinomycetes were to be done in indonesia, such as from lombok island (lisdiyanti et al. 2012) and cibinong science center (widyastuti et al. 2013). to obtain new strains that poten ially produce new can t metabolites, it is still necessary to conduct exploration and examination of obtained samples from diverse habitats and environments. few parts of the research had been orally presented in 2011 during symposium on recent advances on microbiological researches and its application, conducted by indonesian society for microbiology (permi) in serpong. materials and methods sampling ethodsm soil samples were obtained from eka karya botanical garden located at 1,250-1,450 m above sea level, 115 9'0-58” e and 8 15-17'0-59” n, o o with 5-10 cm depth from soil surfacesoil ph of . the soil samples was between 6.0-6.5. the samples were put in plastic bag ecaying immediately to . d leaf-litter samples were collected from soil surface were put in. the samples immediately to paper bag. all samples were air dried at room temperature for 1-2 weeks, ground using blender and filtered with 200 m mesh filter paperµ . sds-yeast extract (sy) solation ethodi m sy isolation method described by was widyastuti (201 ) a combination of 0.05% et al. 3 . sds (sodium dodecyl sulphate) as a germicide to eliminate soil bacteria, 6% yeast extract as spore activating agents and heating at 40 c for 20 o minutes increase the recovery of could also s actinomycetes from various soil samples. this method was used for isolating general actinomycetes. rehydration and centrifugation (rc) i msolation ethod the rc isolation method was used for isolati . the sample is ng motile actinomycetes rehydrated by air-dried container in 10 mm phosphate buffer containing 10% soil extract, at 43 diversity f ctinomycetes rom eka karya botanical garden balio a f , – et al.shanti ratnakomala 30 c for 90 minutes, followed by centrifugation at o 1,500 x g for 20 minutes (hayakawa . 2000 et al ; otoguro 2001 widyastuti . 201 )et al. et al; 3 . humic acid with vitamins (hv) ediumm hv medium contained (in liter) 1 g humic was acid, 0.02 g caco , 0.01 g feso .7h o, 1.71 g 3 4 2 kcl, 0.05 g mgso .7h o, 0.5 g nahpo , 5 ml of 4 2 4 vitamins solution, 50 mg cycloheximide, 18 g agar, ph 7.2. the composition of vitamins solution was 0.5 mg thiamine hcl, 0.5 mg riboflavin, 0.5 mg niacin, 0.5 mg pyridoxine hcl, 0.5 mg inositol, 0.5 mg ca-panthotenate, 0.5 mg p-aminobenzoic acid and 0.25 mg biotin in 5 ml water and sterilized by 0.22 m filtration (hayakawa & nonomura 1987). this vitamin solution was added after autoclave sterilization. analysis of diaminopimelic acid (dap) preliminary biochemical test performed is the dap determination using a method of thin layer chromatography (tlc) on cellulose to separate isomers of dap (hasegawa . 1983). three et al loops of the cells was put in screw cap plastic tube, added with three drops of 6n hcl, autoclaved at temperature of 121 c, 1 atm for 15 minutes, and o then applied to cellulose chromatography plate. tlc eluent solution used was mixture of methanol: water: 6n hcl: pyridine (80: 26: 4: 10 v/v), and eluted for 12 hours. after that, the spots were sprayed with ninhydrin solution (0.3 g ninhydrin in 100 ml of butanol + 3 ml of acetic acid), and heated at 100 c for 3 minutes.o preparation of emplate dna and pcr t a gmplification of 16s rrna ene chromosomal dna was extracted as described by saito & miura (1963) from 14-dayold cell cultures grown on yg agar medium by using dneasy plant maxi kit (qiagen). 16s rrna gene replication reaction was performed using primer pair, 9f (forward: 5'-gagtttgatcctggctcag-3' positions 9-27) and 1541r (reverse: 5'-aaggaggtgatccagcc-3' position 1541-1525) of numbering escherichia coli system (brosius 1978). pcr amplification et al. was performed used taka a ex taq with total r volume of 50 l, consisting of 0.4 mm of each primer, 1 ng of dna template, 2.5 mm of dntp, 1 of ta a a pcr buffer, and 5u of taq k r polymerase in final volume. pcr conditions was 95 c for 3 minutes to denaturate the target dna, o then by 30 cycles at 95 c for 3 seconds for o denaturation again, 55 c for 15 minutes for o primer annealing, and 72 c for 1 minute for o primer extension, and subsequently, 1 cycle at 72 oc for 5 minutes to complete the process of amplification. pcr reaction was conducted using a geneamp pcr system 9700 (applied biosystem). pcr products were examined by electrophoresis on agarose 2%, to assure that the target dna had been amplified. pcr products were then purified using the gfx-96 pcr purification kit (amersham pharmacia biotech), with reference to the protocol. 16s rrna ene equencingg s pcr products that had been purified were cycle sequenced using the bigdye terminator sequence with version 3.1 cycle sequencing kit. this reaction used 6 primers to amplify 1,500 bp of 16s rrna gene, which is 9f, 515f (5'gtgccaagcagccgcggt-3' position 515531), 1099f (5'-gcaacgagcgcaaccc-3' position 1099-1114), 536r (5'-gtattaccgcggctgcttg-3' positions 536-519), 1115r (5'agggttgcgtcgttg-3' position 1115 1100), and 1541r of numbering escherichia coli system (brosius . 1978). in total 10 l of et al reaction sequence containing 2.0 l of big dye terminator premix, 1.0 l of 5 big dye sequencing buffer, 0.8 l of each primer (1 pmol/l), and 0.5 l of template dna were synthesized of the chain by using a geneamp pcr system 9700 (applied biosystem) with the following conditions predenaturation at 96 c for 1 minute, 45 cycles at a o temperature of 96 c for 10 seconds for o denaturation, 50 c for 5 seconds for primer o annealing, and 60 c for 90 seconds for primer o extension, and subsequent to storage at 16 c. the o product was purified using dyeex 96 kit (qiagen) and sequenced using abi prism 3700 (applied biosystem) dna sequencer. sequence ata nalysis and lignment d a a search 16s rdna sequence was translated from the 16s rrna gene by using atgc sequencing analysis software version 7.3 (abi prism) and corrected manually. nucleotide sequence data of the isolates was searched the closest homology 44 biotropia vol. 23 no. 1, 2016 with other strains in the 16s rrna gene data base using blast (http://www.ncbi.nlm.nih. gov) . results and discussion from t 38 he total number of samples consist of ed 18 soil samples and 20 leaf-litter samples 409 actinomycetes were isolated, . a total of 229 isolates based on the colony appearance, were selected and used study. in this from 229 isolates, 144 were isolated from soil samples and 85 were isolated from leaf-litter samples. from soil samples, 60 and 84 actinomycetes were isolated by sy and rc isolation method, respectively; and 85 from leaf-litter samples by using rc were isolated isolation method ( ). the dap analysis table 1 showed that within the actinomycetes isolated from soil source by sy isolation method, 18 isolates had ll-dap, 31 isolates had /ll-meso meso/oh dap, 11 isolates not but the rest d id have dap polymers containing . actinomycetes isolated by rc isolation method showed that 24 isolates had ll-dap, 48 isolates had -meso dap/ll/oh in their cell wall, and the rest meso 12 isolates d not have dap id containing polymers using. from leaf litter source rc isolation method, 3 isolates had ll-dap, 66 isolates had dap/lloh on their cell meso meso/ wall, and 16 isolates not dap containing d have id polymers. no suborder no family no genus blast result >99 % 98 % 97 % <96 % total 1 corynebacterineae 1 nocardiaceae 1 nocardia 2 5 2 9 2 rhodococcus 1 1 2 frankineae 2 cryptosporangiaceae 3 cryptosporangium 1 1 3 kineosporiaceae 4 kineosporia* 7 3 10 3 micrococcineae 4 intrasporangiaceae 5 lapilicoccus 1 1 5 promicromonosporaceae 6 promicromonospora 1 1 4 micromonosporineae 6 micromonosporaceae 7 actinoplanes* 8 33 22 4 67 8 catellatospora 1 1 9 catenuloplanes* 12 4 16 10 dactylosporangium* 3 3 11 krasilnikovia* 3 3 12 micromonospora 3 4 1 8 13 verrucosispora 1 1 5 propionibacterineae 7 nocardioidaceae 14 kribbella 2 2 15 nocardioides 1 1 2 6 pseudonocarnineae 8 actinosynnemataceae 16 actinokineospora* 1 1 17 saccharothrix 1 1 9 pseudonocardiaceae 18 amycolatopsis 1 2 3 19 pseudonocardia 3 1 4 20 saccharomonospora 1 1 7 streptomycineae 10 streptomycetaceae 21 kitasatospora 5 2 7 22 streptomyces 42 24 4 3 73 8 streptosporangineae 11 nocardiopsaceae 23 nocardiopsis 4 4 12 streptosporangiaceae 24 acrocarpospora 1 1 25 nonomuraea 1 2 2 5 26 streptosporangium 1 1 13 thermomonosporaceae 27 actinocoraliia 1 1 28 actinomadura 1 1 73 98 41 17 229 45 diversity f ctinomycetes rom eka karya botanical garden balio a f , – et al.shanti ratnakomala table 1 number of isolated and selected actinomycetes from eka karya botanical garden, indonesia sampling site source no. of samples isolation method selected isolates dap isomer ll m/ll-m/oh nd eka karya botanical garden soil 18 sy 60 18 31 11 18 rc 84 24 48 12 leaflitter 20 rc 85 3 66 16 38 229 45 145 39 table 2 actinomycetes isolated from eka karya botanical garden, bali, 2003 indonesia, note: * = zoospore-bearing actinomycetes http://www.ncbi.nlm.nih. identification of 229 isolates based on 16s rrna gene sequencing showed that the isolates belong to 8 suborders, 13 families and 28 genera of the lass actinomycetales (table 2). the largest c group of actinomycetes found belong .to ( ) genus 73 isolates the second streptomyces largest group genus 67 belong to (actinoplanes isolates he third largest group ) belong to . t genus 16 isolates . about 58 catenuloplanes ( ) isolates (25%) may be new species or new genus, because it has <98% of 16s rrna gene similarity compared to the known strains in the database. and cultures sy isolation method incubation on hv agar plates containing nalidixic acid introduced by hayakawa and nonomura (1989) improve the possibilities of isolating d actinomycetes while decreasing the number of bacterial . this method proved to be an colonies effective tool for isolating act nomycete . i s rc isolation method described hayakawa . by et al ( ) and ( found to et al2000 otoguro . 2001) was also be an effect ve tool for the ion ofi isolat zoospore from the genera of , , actinoplanes actinokineospora actinosynnema catenuloplanes, dactylosporangium, , geodermatophylus kineospor a.iand the phosphate buffer-soil extract solution significantly promoted liberation of motile zoospores from the source material, and the centrifugation eliminated streptomyces and other non-motile actinomycetes. in general actinomycetes isolated , using sy were by many non-method dominated motile actinomycetes, while those isolated using the rc method we motile re dominated by actinomycetes. rc is an isolation method developed isolat motile zoospore for ing (hayakawa . 2000).et al weall 229 selected isolates re identified using m procedure olecular identification based on full sequence of 16s rrna gene (±1,500 bp). the isolates were identified into genus and further species level by blast and phylogenetic tree construction. currently, actinomycetes consisted of 24 families, 80 genera and 500 species (liu . et al 2009). in study, identify 8 suborders, our we could 13 families and 28 genera ( )table 3 . we predicted that there more than were 109 species. this is the first comprehensive study of actinomycetes conducted in eka karya botanical garden, bali island, indonesia. table 3 diversity of actinomycetes in soil samples note: * = zoospore bearing actinomycetes 46 biotropia vol. 23 no. 1, 2016 no suborder no family no genus no of species rc sy total 1 corynebacterineae 1 nocardiaceae 1 nocardia 4 1 8 9 2 rhodococcus 1 1 1 2 frankineae 2 kineosporiaceae 3 kineosporia* 1 1 1 3 micrococcineae 3 promicromonosporaceae 4 promicromonospora 1 1 1 4 micromonosporineae 4 micromonosporaceae 5 actinoplanes* 12 25 25 6 catellatospora 1 1 1 7 dactylosporangium* 1 3 3 8 krasilnikovia* 1 3 3 9 micromonospora 1 2 2 10 verrucosispora 1 1 1 5 propionibacterineae 5 nocardioidaceae 11 kribbella 2 2 2 12 nocardioides 2 2 2 6 pseudonocarnineae 6 actinosynnemataceae 13 actinokineospora 1 1 1 14 saccharothrix 1 1 1 7 pseudonocardiaceae 15 amycolatopsis 2 1 2 3 16 pseudonocardia 3 2 1 3 7 streptomycineae 8 streptomycetaceae 17 kitasatospora 5 6 1 7 18 streptomyces 36 33 32 65 8 streptosporangineae 9 nocardiopsaceae 19 nocardiopsis 4 2 2 4 10 streptosporangiaceae 20 acrocarpospora 1 1 1 21 nonomuraea 3 2 3 5 22 streptosporangium 1 1 1 11 thermomonosporaceae 23 actinocoraliia 1 1 1 24 actinomadura 1 1 1 87 84 60 144 diversity of ctinomycetes on oil amplesa s s from soil samples, we obtained 144 isolates of actinomycetes that had been identified by 16s rrna gene analysis and preserved well in liophilized form. the isolates contained 24 genera and more than 87 species. the most dominated genera in the soil samples was (65 streptomyces isolates = 45%) and the next was (25 actinoplanes isolates = 17%). based on the isolation methods, 15 genera (60 isolates) were by sy isolation successfully isolated method and 16 genera (84 isolates) were isolated by rc isolation method. , genera of nocardia rhodococcus catelatospora micromonospora kribbella, , , , nocardioides streptosporangium actinocoralia, , and actinomadura were easily isolated using sy isolation method; while rc method was useful for isolating genera , , actinoplanes krasilnikovia dactylosporangium verrucosispora actinokineospora, , and . most of soil actinomycetes saccharothrix isolated by rc isolation method belong to zoospore bearing actinomycetes. by using different isolation method, the dominant species of actinomycetes were also differe . in isolated nt this study, we proved that actinomycetes isolated using the rc method were dominated by groups of zoospore bearing actinomycetes. this result is similar to that described by et al.hayakawa (2000) and otoguro (2001).et al. several ecological factors that played a role in the distribution of genera actinomycetes included humus content and ph of the soil (nonomura & hayakawa 1988), climate may influence the specific type of soil-inhabiting actinomycetes (hayakawa . 2010). soil of eka karya et al botanical garden a humus-rich soil with ph is range from 6 to 6.5. this soil type is suitable for the growth of actinomycetes. some of actinomycetes are distributed in iverse plant species plant rhizosphere soils. d found in the garden should also support the growth of actinomycetes . actinomycetes have been found to play an important role in rhizosphere soil (suzuki . 2000 el-tarabily & et al ; sivasithamparam 2006). there is a possibility that these microorganisms can protect plant roots from plant pathogen and promote plant growth. figure 1 phylogenetic position based on 16s rrna sequences of several isolates under the genera from eka karya nocardia botanical garden. bar, 1 substitutions per 200 nucleotides 47 diversity f ctinomycetes rom eka karya botanical garden balio a f , – et al.shanti ratnakomala for plant root protection, the modes of action of actinomycetes include antibiosis, parasitism, the production of extracellular hydrolytic enzymes and competition for iron (getha . 2005 ;et al errakhi . 2007). sy isolation method was et al particularly successful for isolating common actinomycetes such as spp. in natural streptomyces habitats, streptomycetes are common and are usually a major component of the total actinomycetes population. kim (1984) reported that within population in the soil, actinomycetes are dominated by (95 43%).streptomyces . d indicate identification by molecular approach that actinomycetes obtained from eka karya botanical garden have potential should value as a source find newto new species or genus. based on the analysis of 16s rrna gene, <97% sequence were in homology with the closest species on blast searching compared to the current database. n ew species and new genus among the strains studied re obviouswe . the16s rrna gene sequence of strain id03-0848 and id03-0856 were aligned with those of the type species of the major and other actinomycete lineages. nocardia the resulting phylogenetic tree is shown in figure 1. strain id03-0848 and id03-0856 formed a coherent clade within the lineage, clearly nocardia distinguished from other described strains with highly bootstrap value. this was suspected to be new genus or new species in the lineage.nocardia diversity of ctinomycetes on eaf-litter a l samples meanwhile, from the leaf-litter as a source material, we obtained 85 isolates of actinomycetes that had been identified by 16s rrna gene analysis and preserved well in liophilized form. the isolates contained 9 genera ( ) and table 4 more than 41 species. the most dominated actinoplanesgenus was (42 isolates = 49%) and catenuloplanesthe next was (16 isolates = 19%) and (9 isolates = 10%). the same as in kineosporia soil samples, most of the leaf-litter actinomycetes isolated by rc method belong to the zoospore bearing actinomycetes. this finding is in agreement with other reports which mentioned that actinomycetes belonging to genera actinoplanes, catenuloplanes kineosporia and were frequently isolated from leaf-litter samples (pagani 1978 kudo . 1998 & parenti ; ;et al hayakawa . 2000 ratnakomala 2011).et al et al.; they showed very similar characteristics such as possession of motility, absence or rarity of hydrophobic aerial hyphae and formation of orange colonies, similar to the color of fallen leaves (van hop 2011).et al. et al. et alxu (1996) and meliani . (2012) reported that there was a positive correlation between diversity of actinomycetes with vegetation. land of primary forest has higher diversity of actinomycetes compared with land of secondary forest and agricultural land. on dry, barren and cold land, there are less actinomycetes found (xu 1996 garrity . 1996). search et al. et al; of new active compounds, especially from actinomycetes requires a large number of isolates. it would be more promising if sampling and isolation techniques more specific (lo . are et al 2002). therefore, it is essential to look for unique types of vegetation where the soil sample will be taken new taxonomical for finding ly important actinomycetes. it is also to find important table 4 diversity of actinomycetes from leaf-litter samples note: * = zoospore bearing actinomycetes 48 biotropia vol. 23 no. 1, 2016 no suborder no family no genus no of species rc 1 frankineae 1 cryptosporangiaceae 1 cryptosporangium 1 1 2 kineosporiaceae 2 kineosporia* 1 9 2 micrococcineae 3 intrasporangiaceae 3 lapilicoccus 1 1 3 micromonosporineae 4 micromonosporaceae 4 actinoplanes* 21 42 5 catenuloplanes* 2 16 6 micromonospora 6 6 4 pseudonocarnineae 5 pseudonocardiaceae 7 pseudonocardia 1 1 8 saccharomonospora 1 1 5 streptomycineae 6 streptomycetaceae 9 streptomyces 7 8 41 85 a metaboli . cti omycete with new c propertiesn s t find a new actimomycete here is a possibility to species for the tion of newproduc antibiotics or other secondary metabolites. these microbes will specifically generate new secondary metabolites which allow them to degrade toxic compounds from these plants (park 1999 ho et al. ; et al . 2000). eka karya botanical garden is one place for conservation of tropical ex situ plant forests in indonesia understood that high . it is diversity of actinomycetes will likely to be found in place.such selection of proper method of isolation is crucial to obtain new actinomycetes species. wasit obvious from our study that the use of rc method was useful to isolate new significantly species from leaf-litter samples, especially from genus kineosporia . the 16s rrna gene sequences of 10 strains (id03-0578, id03-0677, id03-0678, id03-0683, id03-0684, id03-0714, id03-0716, id03-0722, id03-0739 and id03-0760) were aligned with those of type species of the major kineosporia and other actinomycete lineages. as shown in , strain id03-0739 and id03-figure 2 0714 were moderately related to the type strain k. succinea ab003932. strain id03-0683 and id03-0760 were related to type strain k. rhizophila ab003933. strain id03-0684 was closely related to type strain d86937. strain id03-k. aurantiaca 0677 and id03-0678 shared the same branching position and formed a single clade with id030716 and id03-0722. these four strains were clearly distinguished from other described strains with highly bootstrap value. this was suspected to be new genus or new species in the kineosporia lineage. this study is significantly important to describe the diversity of actinomycete in s indonesia. there are ample space to use isolated s actinomycete for the benefit of society. further s research on several important taxa including proposing new species or genus is mandatory. more data on phenotype, biochemical characterization, dna-dna hybridization and chemotaxonomic are required. conclusions selection of proper isolation method is crucial to obtain a new actinomycetes species. using sy isolation method, this research was successfully isolated 2 new species of actinomycetes from eka karya botanical garden. this study is significantly important to describe the diversity of actinomycete in indonesia. there are ample s space to use isolated actinomycete for the benefit s of society. f on important urther research some taxa including for proposing new species or genus is mandatory more. data on phenotypic, biochemical characterization, dna hybridization and chemotaxonomic data are required to describe the other actinomycetes candidates as new species. figure 2 phylogenetic position based on 16s rrna sequences of several isolates under the genera from eka kineosporia karya botanical garden. bar, 1 substitution per 100 nucleotides 49 diversity f ctinomycetes rom eka karya botanical garden balio a f , – et al.shanti ratnakomala 0.01 kax 1000 1000 1000 1000 1000 665 641 938 995 644 532 438 540 567 536 1000 861 709 999 629 id03-0739 id03-0714 id03-0760 id03-0683 id03-0684 id03-0678 id03-0722 id03-0716 id03-0677 id03-0578 kineosporia succinea (ab003932) kineosporia rhizophila (ab003933) kineosporia aurantiaca (d86937) kineosporia aurantiaca (x87110) kineosporia aurantiaca (ab003931) kineosporia aurantiaca (af095336) kineosporia rhamnosa (ab003935) kineosporia rhamnosa (ab003934) kineospria radiotolerans (af247813) kineosporia aurantiacus (ab007420) kineococcus mikuniensis (x92618) cryptosporangium japonicum (d85466) cryptosporangium sp. (ab006168) cryptosporangium arvum (d85465) cryptosporangium aura (ab047490) cryptosporangium minu (ab037007) streptomyces lavendulae (d85116) acknowledgements this study was conducted under the joint research project between department of biotechnology, national institute of technology and evaluation, japan and the indonesian institute of sciences (lipi) representing indonesian government research institutes. the authors thanked eka karya botanical garden, lipi and technician in nite and research s center for biotechnology lipi for their assistance. references baskaran r, vijayakumar r, mohan pm. 2011. enrichment method for the isolation of bioactive actinomycetes from mangrove sediments of andaman islands, india. malay j microbiol 7(1):26-32. bredholt h, fjærvik e, johnsen g zotchev sb. 2008. , actinomycetes from ediments in the trondheim s fjord, norway: iversity and iological ctivity. d b a mar drugs 6(1):12 24. brosius j, palmer ml, kennedy pj, noller hf. 1978. complete nucleotide sequence of a 16s ribosomal rna gene from . proc natl acad sci escherichia coli 75(10):4801-5. cross t. 1986. the occurrence and role of actinoplanetes and motile actinomycetes in natural ecosystems. in: megusar f, gantar m, . perspectives in (eds) microbial ecology. proceedings of the iv international symposium on microbial ecology. p 265 70. el-tarabily ka, sivasithamparam k. 2006. nonstreptomycete actinomycetes as biocontrol agents of soil-borne fungal plant pathogens and as plant growth promoters. soil biol biochem 38: 1505-20. errakhi r, bouteau f, lebrihi a, barakate ml. 2007. evidences of biological control capacities of streptomyces sclerotium rolfsi i spp. against responsible for damping disease in sugar beet ( l.). beta vulgaris world j microbiol biotechnol 23:1503-9. garrity gm, heimbuch bk gagliardi m. 1996. isolation of , zoosporogenous actinomycetes from desert soils. j ind microbiol 17:260-7. getha k, vikineswary s, wong wh, seki t, ward a, goodfellow m. 2005. evaluation of sp. streptomyces strain g10 for suppression of wilt and fusarium rhizosphere colonization in pot-grown banana plantlets. j indian microbiol biotechnol 32:24-32. hasegawa t, takizawa m, tanida s. 1983. a rapid analysis for chemical grouping of aerobic actinomycetes. j gen appl microbiol 29:319 22.hasegawa t. 1991. studies on motile arthrospore-bearing rare actinomycetes. actinomycetol 5(2):64-71. hayakawa m, nonomura h. 1987. efficacy of artificial h a s n aumic cid as a elective utrient in hv gar used for the solation of oil ctinomycetes. j ferment i s a technol 65(6):609-16. hayakawa m, nonomura h. 1989. a new method for the intensive isolation of actinomycetes from soil. actinomycetol 3(2):95-104. hayakawa m, otoguro m, takeuchi t, yamazaki t, iimura y. 2000. application of a method incorporating differential centrifugation for selective isolation of motile actinomycetes in soil and plant litter. antonie van leeuwenhoek 78:171-85. hayakawa m. 2008. studies on the isolation and distribution of rare actinomycetes in soil. actinomycetolo 22:12 9.hayakawa m, yamamura h, sakuraki y, ishida y, hamada m, otoguro m, tamura t. 2010. diversity analysis of actinomycetes assemblages isolated from soils in cool-temperate and subtropical areas of japan. actinomycetol 24:1-11. ho cc, tan gya, seow i, ajam n, tan ei, goodfellow m, ward ac, brown r, wong nk, lo cw, cheah hy, lai ns, suzuki ki. 2000. isolation, characterization and biological activities of actinomycetes isolated from dipterocarp rain forest soils in malaysia. in: nnga bh, tan hm, suzuki k-i, editor. microbiology diversity in asia. singapore: world scientific. hopwood da. 2007. , streptomyces in nature and medicine the antibiotic makers. uk: oxford university press, inc. khamna s, yokota a, peberdy jf, lumyong s. 2010. indole3-acetic acid production by sp. isolated streptomyces from some thai medicinal plant rhizosphere soils. eurasia j biosci 4:23-32. khanna m, solanki r, lal r. 2011. selective isolation of rare actinomycetes producing novel antimicrobial compounds. int j adv biotechnol res 2(3):357-75. kim cj. 1984. isolation and screening of actinomycetes from natural environments. swed n: genetic e engineering research institute, kist. kudo t, matsushima k, itoh t, sasaki j, suzuki k. 1998. description of four new species of the genus kineosporia kineosporia succinea kineosporia : sp. nov., rhizophila kineosporia mikuniensis sp. nov., sp. nov. and sp. nov., isolated from kineosporia rhamnosa plant samples, and amended description of the genus . int j syst bacteriol 48:1245 55.kineosporia liu n, wang h, liu m, gu q, zheng w, huang y. 2009. streptomyces alni sp. nov., a daidzein-producing endophyte isolated from a root of alnus nepalensis d.don. int j syst evol microbiol 59:254-58. lo cw, lai ns, cheah hy, wong nki, ho cc. 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received 24 june 2014/accepted 9 november 2016 abstract ralstonia solanacearum is an important disease of tomato. an alternative method to control the disease is the application of biocontrol agents. plant growth-promoting rhizobacteria (pgpr) could be used as potential biocontrol agents. pgpr with siderophores is among compounds having important role in disease suppression. this experiment was conducted to select and characterize the siderophore-producing rhizobacteria from tomato and to determine their potential as antagonistic agents for r. solanacearum. candidates of the pgpr were isolated from tomato grown in west java province, indonesia. the isolates were detected as siderophore-producing bacteria using cas medium. among 29 isolates producing siderophore and having negative result on hypersensitivity reaction, two isolates provided the widest diameter of inhibition zone toward r. solanacearum. both isolates were cp1c and cp2d with diameter of inhibition zone up to 3.6 and 7.0 mm, respectively. based on the sequence of 16s rdna, isolate cp1c was identified as brevundimonas sp., while isolate cp2d was identified as enterobacter sp. both bacteria did not cause negative effect on the increasing plant height and dry weight of the plants, compared with control. keywords: bacterial wilt, biocontrol, brevundimonas, enterobacter, pgpr biotropia 4 2 7 85 93 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 2 406 * corresponding author: asnawangsih@yahoo.com 85 introduction bacterial wilt of tomato caused by ralstonia solanacearum is one of important diseases of tomato in tropics and subtropics area (jeung et al. 2007). the bacteria survive for a long time in the soil (wang & lin 2005). the bacteria also multiply in the xylem as well as attacking the xylem and affect the water and nutrient translocation, causing wilting and death of the plant (agrios 2005). among the control methods to control the disease, is the application of biological control (biocontrol) agents (yuliar et al. 2015). according to jenifer et al. (2013), one of the most important mechanisms responsible for suppressing pseudomonas sp. (a plant pathogen) is siderophoremediated competitions for iron. siderophores (from the greek “iron carriers”) are small ferric-ion-specific chelating agents produced by bacteria and fungi which grow in low iron condition causing them to scavenge iron from the environment and to make iron available to the microbial cell (neilands 1995; pal & gokarn 2010). siderophores are also known to bind molybdenum and lead (pal & gokarn 2010). sayyed et al. (2005) reported that siderophoreproducing pseudomonas sp. plays vital role in stimulating plant growth and in controlling several plant diseases. in pseudomonas fluorescens the pigment produced is a siderophore. pigment production is increased in the presence of sodium, potassium, lead, molybdenum, cadmium and ammonium sulphate ( (nh ) so ) 4 2 4 (bhattacharya 2010; sayyed et al. 2005). adding exogenous amino acid did not significantly increase the production of siderophore (hu & xu 86 biotropia vol. 24 no. 2, 2017 java province, indonesia. from each village, 1,500 g of rhizosphere soil samples (with tomato roots) were collected from five different plots (300 g per plot as subsample) and thoroughly mixed using trowel, until becoming a composite sample. from each composite sample, 10 g of rhizosphere soil was immersed in 90 ml 0.85% nacl. after a serial dilution, 0.1 ml of each dilution was inoculated on chrome azurol sulphate (cas) medium (gross 1990; louden et al. 2011). inoculation was repeated three times. plates containing the inoculation were incubated at room temperature (±28 c). numbers of o colony of siderophore-producing rhizobacteria were calculated at 24 48 hours after incubation, based on the production of orange zone around colony of the bacteria. successfully isolated bacteria were transferred to king's b agar medium and separated from each other to obtain pure culture. each isolate was preserved in nutrient broth (nb) with 20% glycerol and kept in -20 c environment. for daily maintenance, o isolates were preserved in nb and kept at room temperature (±28 c)o . hypersensitive reaction test hypersensitive reaction (hr) test was conducted to select the nonpathogenic bacteria as candidates of biocontrol agents. the test was 8 9conducted by inoculating 10 – 10 cfu/ml of rhizobacteria on tobacco leaves. one milliliter of suspension was injected into tobacco leaf using 5 ml sterile syringe. successful injection was indicated by water soaked zone around the point of injection. inoculation of each isolate was conducted in duplo. inoculated leaves were oincubated at room temperature (±28 c) for 24 hours. leaves showing necrotic symptoms before 24 hours were counted as having positive reaction to the hypersensitive reaction and thus, be eliminated. antagonistic activities of siderophoreproducing rhizobacteria against in vitro ralstonia solanacearum bacteria isolates having negative reaction on the previous hypersensitive test were tested on their antagonistic activity toward r. solanacearum. both bacteria, i.e. r. solanacearum and rhizobacteria, were grown on a king's b agar 2011). sayyed et al. (2005) also reported that mn, hg and co showed inhibitory effect on siderophores growth and production. presence of potassium, magnesium and calcium had little inhibitory effect on siderophores production compared to controls (battacharya 2010). sayyed (2005) reported that production of et al. siderophores by bacteria was affected by growing media. in growing media such as nutrient broth (nb) and mackonkey's broth (mb), siderophores were not produced because both media are luxurious media having high content of fe. fefree succinic acid medium (sm) was found to provide maximum (92.25%) siderophores production in comparison to 88.00% in barbhayya rao broth (br), 79.00% in cassamino acid broth (caa) and 48.00% in enrichment medium (em) (sayyed 2010). a study on ph et al. effect to the production of siderophores by rhizobium sp. showed that the growth and production of siderophores started at ph 4.5, reaching maximum at neutral ph; at ph 10.0, there was no siderophores production (sridevi et al. 2008). study on genetic diversity of tobacco rhizosphere which produces siderophore demonstrated that 85% of the total 354 isolates produced siderophores in iron limited liquid medium; some of them are , pseudomonas enterobacter serratia pantoea erwinia , , , and stenotrophomonas γ-proteobacteria which belong to (tian 2009).et al. this experiment was conducted to select and characterize the siderophore-producing rhizobacteria from tomato and to determine their potential as antagonistic agents for r. solanacearum. materials and methods isolation and quantification of siderophoreproducing rhizobacteria siderophore-producing rhizobacteria were isolated from rhizosphere samples taken from healthy tomato plants, collected from tomato field in cipanas sub-district (cianjur district) and lembang sub-district (west-bandung district). in cipanas sub-district, samples were collected from three villages, while in lembang sub-district samples were collected from two villages. both sub-districts are the center of tomato field in west 87 siderophore-producing rhizobacteria and potential antagonistic activity toward ralstonia solanacearum – nawangsih et al. plate. five hundred micro liter suspension of r. solanacearum (10 10 cfu/ml) was spread on the 8 9 surface of the king's b agar plate. after being air dried, three sterilized filter papers having 0.5 cm diameter were placed besides the agar plate with 2 cm distance from each other. filter paper in the center was inoculated with 50 µl of sterilized distilled water and designated as control. the two other filter papers were each inoculated with 50 µl suspension of one isolate of rhizobacteria. each treatment was repeated three times. inoculated plates were incubated at room temperature (±28 c). the antagonistic activity o was observed at 24 hours after incubation. antagonistic activity was indicated by the production of inhibition zone around the filter paper inoculated with siderophore-producing rhizobacteria. e f f e c t o f s i d e r o p h o r e p r o d u c i n g rhizobacteria on the viability of tomato seeds tomato seeds (arthaloka and ratna varieties) were dipped in 10 10 cfu/ml suspension of 7 8 siderophore-producing rhizobacteria for 16 hours before being planted on sterilized mixture of soil and compost (1 : 1 ratio). the soil mixture was put in 30 50 cm polyethylene pot tray having 128 holes. the experiment was arranged as completely randomized factorial design with 7 isolates of biocontrol agents and one control (without bacteria) as the first factor and two tomato varieties (arthaloka and ratna) as the second factor. thus, 16 treatments were applied in this experiment. each treatment was replicated three times. there were 48 units in total; each unit contained 15 seeds. one tomato seed was grown in one hole of polyethylene pot tray. the isolates of biocontrol agents used in this experiment were cp1c (code of one isolate of bacteria), cp2b, cp2d, cp3e, lb1a, lb1c and lb1l. seeds dipped in sterilized distilled water were used as control. layout of the treatments was shown in table 1. total of the emerging seedlings were calculated every day. seed viability (sv) was calculated using the following formula: total normal seedlings sv (%) = x 100% total seeds sown e f f e c t o f s i d e r o p h o r e p r o d u c i n g rhizobacteria on the height, fresh and dry weight of tomato plants an experiment to test the effect of siderophore-producing rhizobacteria toward the height, fresh and dry weight of tomato plants was conducted in a green house. tomato seeds of arthaloka and ratna varieties were dipped in suspension of siderophore-producing rhizobacteria (10 10 cfu/ml) for 16 hours 7 8 before being planted in 10 x 15 cm polybag filled with 2.5 – 3 kg of sterilized mixture of soil and compost (1 : 1 ratio). the experimental design applied was completely randomized factorial design having similar layout with the one presented in table 1. the only difference was that each unit contained 5 seeds. plant height was measured every 5 days starting from the day when the first leaf was fully opened. total area under height of plant growth curve (auhpgc) was calculated using table 1 layout of experiment to test the effect of siderophore-producing rhizobacteria toward the viability of tomato seeds tomato varieties isolate’s code cp1c cp2b cp2d cp3e lb1a lb1c lb1l control arthaloka repl 1 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds repl 2 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds repl 3 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds ratna repl 1 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds repl 2 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds repl 3 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 15 seeds 88 biotropia vol. 24 no. 2, 2017 modification of formula reported by van der plank (1963): where: y = increasing of plant height at the next i+1 observation y = increasing of plant height at the time of i observation t = the next observation (ii, iii, …, vi) i+1 t = time of observation (i, ii, …, v)i two months after planting, all plants (five plants) from each replication of each treatment were rooted and weighted using digital balance. the average of the five plants represented data of fresh weight for each replication. dry weight of plant was determined by drying the fresh plants in the oven at 100 c. the weight of the sample was o checked periodically until the weight of the sample was constant. characterization and identification of the siderophore-producing rhizobacteria seven isolates of the siderophore-producing rhizobacteria were characterized based on microscopic and colony appearances, as well as on physiological and biochemical properties, following the methods of klement . (1990) et al and schaad . (2001). two isolates of the et al siderophore-producing bacteria having potential as biocontrol agents were genetically identified by sequencing the 16s rdna gene. dna was extracted from log phase culture using phenolchloroform extraction procedure (sambrook & russel 2001). the 16s rdna gene was amplified using universal primer for prokaryotes which were the forward primer 27f (5'-agagtttgatcctggctcag-3') and reverse primer 1492r (5'ggttaccttacgactt-3'). total volume reaction for polymerase chain reaction (pcr) was 25 µl consisted of 1 µl of dna template; 12.5 µl of 1x ready mix pcr, 1.5 mm mgcl , 0.2 mm dntps and taq 2 polymerase 5 units/reaction; 9.5 µl of ddh o; 1 2 µl of 120 pmol primer 27f; and 1 µl of 120 pmol primer 1492r. pcr was performed under the following conditions: one cycle of preo denaturation at 95 c for 5 minutes, followed by 35 o cycles of denaturation at 95 c for 1 minute, o annealing at 55 c for 1 minute and extension at o72 c for 2 minutes. the reaction was terminated owith a final extension at 72 c for 10 minutes. the pcr products were sent to the first base laboratory, malaysia for sequencing. blast searches were performed for sequences obtained to find the similarity with sequence data in genebank. data analysis data of the bacterial population was analyzed using t test of minitab program version 13.3. effects of the bacteria to the plant growth were statistically analyzed using anova for completely randomized factorial design with isolates of biocontrol agents as the first factor and tomato varieties as the second factor. treatment means were compared using the dmrt test at 5% level of significance. sas program version 9.1 was used for performing statistical analyses for completely randomized factorial design and for the dmrt test. results and discussion abundance and antagonistic activities of siderophore-producing bacteria based on the t test, the average of siderophoreproducing bacteria isolated from cipanas district 7 was 1.98 10 cfu/g, which was not significantly different with those from lembang district 7 having average of 5.3 x 10 cfu/g. colonies of the siderophore-producing bacteria on cas medium were shown in figure 1. siderophore production was indicated by the orange color around the colony of the bacteria. figure 1 shows that each colony produced different amount of siderophores, indicated by the diameter of orange area around each colony of bacteria. among the 60 isolates of siderophoreproducing rhizobacteria, 31 isolates positively showed hypersensitive reaction on tobacco, while 29 others showed negative reaction. bacteria were also tested further for their antagonistic activities against . based on the ralstonia solanacearum antagonistic test, 16 isolates positively produced inhibition zone (fig. 2) having diameter between 0.5 to 7.0 mm. among those 16 isolates, 9 isolates were hr positive which had to be eliminated from being candidate of biocontrol agents. the auhpgc n – 1 y y i i+ +1 2 t i +1 – tis( (( ( 89 other 7 isolates were hr negative, having respective diameter of the inhibition zone of cp1c (3.6 mm), cp2b (2.3 mm), cp2d (7.0 mm), cp3e (5.0 mm), lb1a (1.6 mm), lb1c (1.6 mm) and lb1l (0.5 mm). the widest diameter of inhibition zone was produced by isolate cp2d which was isolated from cipanas (table 2). microorganisms growing under aerobic conditions need iron for a variety of functions, including reduction of oxygen for atp synthesis, reduction of ribotide precursors of dna, for formation of heme and for other essential purposes. a level of at least one micromolar iron is needed for optimum growth (neilands 1995). figure 1 production of siderophores by tomato rhizobacteria on cas agar was indicated by yellow-orange color around colony of bacteria figure 2 production of inhibition zone (arrow sign) by the isolate of siderophore-producing rhizobacteria (note: the inset shows magnification of the inhibition zone) table 2 isolates of siderophore-producing rhizobacteria which produced inhibition zone against r. solanacearum on kings’s b agar isolate code1) diameter of inhibition zone (mm) isolate code diameter of inhibition zone (mm) isolate code diameter of inhibition zone (mm) cp1b2) cp1c cp2b cp2c cp2d cp2h 6.8 3.6 2.3 0.6 7.0 4.5 cp2l cp2s cp3e cp3m cp3t lb1a 1.6 2.6 5.0 2.2 4.0 1.6 lb1c lb1d lb1e lb1l 1.6 0.5 0.5 0.5 note: 1) cp = isolates from cipanas; lb = isolates from lembang 2) isolates written in bold were positively causing hypersensitive reaction (hr) siderophore-producing rhizobacteria and potential antagonistic activity toward ralstonia solanacearum – nawangsih et al. table 3 effect of siderophore-producing rhizobacteria on tomato seed viability of arthaloka and ratna varieties treatment seed viability (%)*) var. arthaloka control cp1c cp2b cp2d cp3e lb1a lb1c lb1l var. ratna control cp1c cp2b cp2d cp3e lb1a lb1c lb1l 93.33 ab 86.63 abc 88.83 abc 84.40 abc 88.87 abc 75.50 abc 100.00 a 95.53 a 66.60 c 75.53 abc 79.97 abc 84.43 abc 75.53 abc 68.83 bc 66.63 c 84.30 abc note: *) means in the same column followed by the same letter are not significantly different according to duncan multiple range test (p < 0.05) 90 biotropia vol. 24 no. 2, 2017 table 4 effect of siderophore-producing rhizobacteria on the increase of tomato plant height of arthaloka and ratna varieties treatment plant height increase (cm)1) auhpgc3) (cm days)i 5 dap2) ii 10 dap iii 15 dap iv 20 dap v 25 dap vi 30 dap var. arthaloka control cp1c cp2b cp2d cp3e lb1a lb1c lb1l var. ratna 1.04 abcde 0.99 bcde 1.51 abc 1.69 ab 1.23 abcd 1.66 ab 1.71 ab 1.87 a 3.57 a 1.42 a 2.30 a 2.32 a 2.78 a 2.48 a 2.43 a 2.69 a 2.03 b 3.83 a 2.04 b 2.24 b 2.00 b 2.25 b 1.81 b 1.81 b 2.86 a 2.49 a 2.65 a 2.61 a 2.58 a 3.05 a 2.70 a 2.61 a 4.34 a 4.53 a 4.74 a 4.66 a 3.93 a 4.61 a 4.44 a 4.19 a 6.85 a 7.50 a 7.20 a 7.81 a 6.43 a 6.98 a 6.63 a 6.65 a 83.75 a 82.56 a 80.40 a 82.90 a 75.53 a 83.53 a 77.78 a 77.78 a control cp1c cp2b cp2d cp3e lb1a lb1c lb1l 0.79 cde 1.09 abcde 0.29 e 0.48 de 0.87 bcde 0.91 bcde 0.53 de 0.40 de 1.99 a 0.99 a 1.51 a 1.91 a 1.23 a 1.78 a 1.45 a 2.00 a 1.89 b 3.73 a 1.89 b 2.03 b 1.87 b 1.46 b 2.0 b 1.81 b 1.97 a 2.11 a 1.31 a 1.85 a 1.86 a 1.57 a 1.44 a 1.35 a 3.19 a 3.98 a 2.85 a 2.97 a 2.89 a 2.29 a 2.57 a 2.27 a 5.09 a 6.47 a 3.81 a 4.37 a 4.23 a 3.67 a 3.77 a 3.55 a 59.90 a 72.92 a 48.05 a 56.00 a 52.00 a 46.97 a 48.17 a 47.09 a note: 1) means in the same column followed by the same letter are not significantly different according to duncan multiple range test (p < 0.05) 2) dap = days after planting 3) auhpgc = area under height of plant growth curve seven isolates of the bacteria were tested for their effects on seed viability, plant height and the fresh and dry weight of two varieties of tomato plants, i.e. arthaloka and ratna, as presented in table 3, 4, and 5, respectively. data in table 3, 4, and 5 show not only that the isolates of bacteria did not significantly increase the seed viability, plant height, fresh and dry weight of tomato compared to control, but also they did not have harmful effects to the tomato plants. the characteristics of colony morphology, physiology and biochemistry aspects of the seven isolates were presented in table 6 and 7, respectively. 91 table 6 morphological characteristic of colony of the seven isolates of siderophore-producing rhizobacteria on king's b agar isolate code1) colony characteristic diameter color elevation edge form cp1c cp2b cp2d cp3e lb1a lb1c lb1l ± 1 mm ± 1 mm ± 1 mm ± 1 mm ± 1 mm ± 3 mm ± 1 mm white broken white broken white dark yellow greenish white pale white broken white convex domed convex convex convex umbonate convex wavy entire entire entire entire curly wavy circular circular circular circular circular irregular circular note: 1) cp = isolates from cipanas; lb = isolates from lembang table 7 physiological and biochemistry characteristics of siderophore-producing rhizobacteria having potential as antagonist for r. solanacearum isolate code1) fluorescence gram reaction phosphate solubilization resistance to 80 oc cp1c + + cp2b +++ + cp2d + + cp3e lb1a + + lb1c + + + lb1l + + note: 1) cp = isolates from cipanas; lb = isolates from lembang table 5 effect of siderophore-producing rhizobacteria on fresh and dry weight of tomato plants treatment1) fresh weight (g/plant)2) dry weight (g/plant) var. arthaloka control cp1c1) cp2b cp2d cp3e lb1a lb1c lb1l var. ratna control cp1c cp2b cp2d cp3e lb1a lb1c lb1l 15.439 a 14.266 a 15.070 a 15.265 a 9.515 a 12.557 a 12.572 a 11.335 a 10.109 a 8.748 a 6.887 a 10.744 a 9.566 a 7.880 a 6.887 a 5.319 a 3.381 a 2.587 a 2.900 a 2.986 a 2.381 a 2.721 a 2.579 a 2.742 a 2.360 a 1.954 a 1.801 a 2.281 a 2.006 a 1.547 a 1.436 a 1.535 a note: 1) cp = isolates from cipanas; lb = isolates from lembang 2) means in the same column followed by the same letter are not significantly different according to duncan multiple range test (p < 0.05) based on the inhibition zone production, effect on seed viability, effect on plant height increase, and effect on fresh and dry weight of tomato product, two isolates of siderophoreproducing bacteria having the best effects were selected. the two isolates were cp1c and cp2d. the sequence of 16s rdna of those isolates were referred to the gene bank. using the blast program the isolate of cp1c was identified as brevundimonas sp., while the isolate cp2d was siderophore-producing rhizobacteria and potential antagonistic activity toward ralstonia solanacearum – nawangsih et al. 92 biotropia vol. 24 no. 2, 2017 · = enterobacter sp. cp2d = brevundimonas sp. cp1c figure 3 phylogenetic tree of isolates enterobacter sp. cp2d and brevundimonas sp. cp1c enterobacter sp. mth17 mth17 gi323218729 enterobacter sp. b49 b49 gi389827949 enterobacter ludwigii b-5 carrot gi444438257 enterobacter sp. acc2 acc2 gi399936203 enterobacter sp. m.d.e.na4-3 m.d.e.na4-3 gi326635001 enterobacter sp. e6-pcai-t2p21 e6-pcai-t2p21 gi358365187 enterobacter sp. enrichment culture clone gi355343574 cp2d brevundimonas sp. 13630g 13630g gi206581409 brevundimonas nasdae 13636e gi206581436 brevundimonas sp. mc8-1 mc8-1 gi300253164 cp1d brevundimonas vesicularis l17 gi375004753 blackwater bioreactor bacterium bw23 bw23 gi16589030 uncultured bacterium gi253770558 uncultured bacterium gi253770269 100 84 100 100 80 100 73 100 69 100 41 46 75 0.05 table 8 maximum score, e value and percentage of similarities of siderophore-producing bacteria isolate species homolog identity max score query cover e value accession number cp1c brevundimonas sp. 13630 g 16s ribosomal rna gene, partial sequence 95% 2021 92% 0.0 eu741063.1 cp2d enterobacter sp. enrichment culture clone dwsr 106 16s ribosomal rna gene, partial sequence 88% 998 69% 0.0 jn944751.1 identified as enterobacter sp. with percentage of similarity of 92% and 93%, respectively. maximum score, e value and the percentage of similarities of siderophore-producing bacteria were presented in table 8. phylogenetic tree of the related bacteria is presented in figure 3. tian et al. (2009) reported that enterobacter and pseudomonas were dominant in the rhizosphere of tobacco, with 44.5% and 24.7% total frequency, respectively. siderophores are produced by various bacteria and fungi, usually classified by the ligands used to chelate the ferric iron. the major groups of s iderophores include the catecholates (phenolates), hydroxamates and carboxylates (e.g. derivatives of citric acid) (saharan & nehra 2011). rachid & ahmed (2005) reported that streptomycin and penicillin added to succinate medium acts differently on the siderophores production. streptomycin reduced siderophores production below 10 µm in different iron concentrations, while penicillin increased the production of siderophores in the presence of excess iron (above 100 µg/ml). the growth of p. fluorescens and siderophore production were inhibited by the occurrence of heavy metals (lead, mercury and cadmium), especially in iron-limited condition . 93 conclusions the average of siderophore-producing bacteria isolated from cipanas district (1.98 x 10 7 cfu/g) was not significantly different from those isolated from lembang district (5.3 x 10 cfu/g). 7 the highest diameter of inhibition zone to ralstonia solanacearum was 7.0 mm, produced by isolate cp2d. the selected bacteria producing the inhibition zone did not significantly affect seed viability, plant growth, fresh weight and dry weight of tomato compared to control. based on the characteristics of colony morphology, physiology, biochemistry and partial sequence of 16s rdna, two selected isolates, i.e. cp1c and cp2d were identified as sp. and sp., brevundimonas enterobacter respectively. acknowledgements this research was funded by dipa ipb with scheme of decentralized research program (program penelitian desentralisasi: hibah bersaing), contract no. 27/i3.24.4/spk/ pd/2010, on 5 march 2010. the authors also thank dr kikin hamzah mutaqin for the guidance in several molecular tests activities. references agrios gn. 2005. plant pathology. fifth edition. new york (us): academic press. 992 p. bhattacharya a. 2010. siderophore mediated metal uptake by pseudomonas fluorescens and its comparison to iron (iii) chelation. cey j sci (bio. sci.) 39(2):147-55. gross m. 1990. siderophores and fluorescent pigments. in: klement z, rudolph k, sands dc, editors. methods in phytobacteriology. budapest (hu): akadémiai kiadó. 568 p. hu qp, xu jg. 2011. a simple double-layered chrome azurol s agar (sd-casa) plate assay to optimize the production of siderophores by a potential biocontrol agent . afr j microbiol res bacillus 5(25):4321-7. jenifer mra, reena a, aysha os, valli s, nirmala p, vinothkumar p. 2013. isolation of siderophore producing bacteria from rhizosphere soil and their antagonistic activity against selected fungal plant pathogens. int j curr microbiol app sci 2(1):59-65. jeung y, kim j, kang y. 2007. genetic diversity and distribution of korean isolates of ralstonia solanacearum. plant dis 91(10):1277-87. klement z, rudolph k, sands dc. 1990. methods in phytobacteriology. budapest (hu): akadémiai kiadó. 568 p. louden bc, haarmann d, lynne am. 2011. use of blue agar cas assay for siderophore detection. j microbiol biol educ 12(1):51-3. neilands jb. 1995. siderophore: structure and function of microbial iron transport compounds. j biol chem 270(45):26723-6. pal rp, gokarn k. 2010. siderophores and pathogenicity of microorganisms. j biosci tech 1:127-34. rachid d, ahmed b. 2005. effect of iron and growth inhibitors on siderophores production by pseudomonas fluorescens. afr j biotechnol 4(7):697702 . ava i l ab l e on l ine a t h t tp ://www. academicjournals. org/ajb. saharan bs, nehra v. 2011. plant growth-promoting rhizobacteria: a critical review. life sciences and medicine research, volume 2011:lsmr-21. http://astonjournals. com/lsmr [retrieved on 24 october 2011]. sambrook j, russell dw. 2001. molecular cloning. a laboratory manual. third edition. new york (us): cold spring harbor lab pr. p.6-62. sayyed rz, badgujar md, sonawane hm, mhaske mm, chincholkar sb. 2005. production of microbial iron chelators (siderophores) by fluorescent pseudomonads. indian j biotechnol 4:484-90. schaad nw, jones jb, chun w. 2001. laboratory guide for identification of plant pathogenic bacteria. third ed i t ion . s t . pau l (us) : the amer ican phytopathological society. 373 p. sridevi m, kumar kg, mallaiah kv. 2008. production of catechol-type of siderophores by rhizobium sp. isolated from stem nodules of sesbania procumbens (roxb.) w and a. res j microbiol 3(4):282-7. tian f, ding y, zhu h, yao l, du b. 2009. genetic diversity of siderophore-producing bacteria of tobacco rhizosphere. braz j microbiol 40:276-84. wang jf, lin ch. 2005. integrated management of tomato bacterial wilt. the world vegetable center. h t t p : / / w w w. a v r d c . o r g / p d f / p r o d 5 management_bacterial_wilt.pdf. [retrieved on 26 september 2011]. yuliar, nion ya, toyota k. 2015. recent trends in control methods for bacterial wilt diseases caused by ralstonia solanacearum .. microbes environ. 30(1):1-11 siderophore-producing rhizobacteria and potential antagonistic activity toward ralstonia solanacearum – nawangsih et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 511 (sri wida potency).cdr potency of rhizosphere bacteria to promote rice growth under saline condition * sri widawati and i made sudiana research center for biology, indonesian institute of sciences csc-lipi, bogor 16911, indonesia received: 8 july 2015/accepted: 25 july 2016 abstract saline soil is a common problem in coastal paddy field, especially in indonesia. salinity affects rice growth and the activities of soil functional microbes, including functional bacteria, which play roles in plant growth. some of these microbes are associated with rice plants and are able to survive under saline condition. the presence of functional microbes is also important to improve soil quality. nitrogen and phosphate are essential soil nutrients and is available in soil due to the activities of nitrogen-fixing bacteria and free-living plant-associated bacteria. the objective of the present study was to obtain nitrogen-fixing, phosphate solubilizing and indole acetic acid (iaa)-producing bacteria that are able to survive and promote the growth of rice under saline conditions. from rice and peanut rhizosphere, caphosphate (ca-p) solubilizing and nitrogen-fixing bacteria were isolated separately using specific media. then, the cap solubilizing ability, phosphomonoesterase activity and iaa-producing ability were quantitatively examined. based on the abilities, 20 strains were selected and identified as burkholderia cepacia-complex, burkholderia anthina, burkholderia cenocepacia, bacillus cereus-complex (three strains), achromobacter spanius, azospirillum sp. (four strains), azotobacter sp. (three strains), rhizobium leguminosarum, rhizobium sp. (two strains), and pseudomonas sp. (three strains). the inoculation of several single strains or the mixture of the selected strains promoted the growth of rice under saline conditions. these inoculants could be potential as biofertilizer in saline paddy fields. keywords: indole acetic acid production, phosphate solubilization, plant growth promoting bacteria, nitrogen fixation, rhizosphere, rice introduction most of the fertile paddy fields in indonesia are located in coastal area and experiences soil salinization due to seawater intrusion (djufry et al. 2011). salinity affects not only the growth of rice (oryza sativa linn.), but also the activities of functional soil microbes, including bacteria, that play roles in mineralization of macro and microelements for plant growth (balser et al. 2006). some of these bacteria are associated with rice plants and are able to survive under saline condition. the activity of soil microbes is an important aspect of biogeochemical cycles of carbon, nitrogen, sulfur, phosphorus, etc. (banig et al. 2008). the presence of functional microbes is also important to improve the quality of soil (wijebandara et al. 2009). nitrogen and phosphate are essential nutrients and are available in soil due to the activities of nitrogen-fixing bacteria and free-living plant-associated bacteria (steenhoudt & vanderleyden 2000). several bacteria belonging to the genera rhizobium, azotobacter and azospirillum are able to fix nitrogen and solubilize phosphate (nosrati et al. 2014). some members of these genera also produce plant growth promoting hormone such as indole acetic acid (iaa), gibberellins and cytokinins (bhattacharyya & jha 2012). therefore, these genera are regarded as important components of biofertilizer (rao 1994; bhattacharjee & dey 2014). introduction of growth promoting bacteria can increase nitrogen availability for plants and enhance crop productivity. however, very little information is available for the effect of salinity on bacteria that have beneficial functions, such as nitrogen fixation, phosphate solubilization and the production of plant growth hormone (pliego et al. 2011; lugtenberg et al. 2013; nakbanpote et al. 2014). the purpose of this study is to obtain nitrogen-fixing, phosphate solubilizing and iaa producing bacteria that are able to survive and biotropia 3 2 6 116 123 vol. 2 no. , 201 : doi: 10.11598/btb.2016.2 . .3 2 511 * corresponding author: widadomon@yahoo.com 116 promote the growth of rice under saline conditions. materials and methods bacterial sources bacterial sources were obtained from collected rhizosphere of rice (oryza sativa) and peanut (arachis hypogaea) cultivated in the research field of cibinong science center, west java province, indonesia. the physical and chemical properties of this research field indicated that the soil is infertile soil (table 1). isolation of bacteria phosphate solubilizing bacteria were screened following the method of park et al. (2011). halo zone formation around colonies after 7 daycultivation at 30 °c on pikovskaya medium was used as an indicator of ca-phosphate (ca-p) solubilization (nguyen et al. 1992). nitrogenfixing bacteria were isolated targeting the genera rhizobium, azopirillum and azotobacter according to mubarik et al. (2011) and salamone et al. (2012) as well as aquilanti and clementi (2004), respectively. determination of ca-p solubilization ca-p solubilizing ability of the isolated strains was quantitatively determined according to chen et al. (2006) by measuring orthophosphate in the culture fluid after 7 days of cultivation. orthosphosphate determination was conducted according to vassileva et al. (2000). determination of phosphatase activity extracellular phosphomonoesterase (pmease) activity of the strains was determined following the method of tabatabai and bremner (1969) using p-nitrophenyl phosphate. the unit of the pmease activity was defined as µmol/hof pnitrophenol released in 1 ml of extracellular enzyme solution that was fractioned from 1.0 ml of the culture fluid after 7 days of cultivation. determination of iaa production the iaa production of the strains was investigated after 7 days of cultivation, following the methods of crozier et al. (1988) and gravel et al. (2007). selection and identification of bacteria based on the ca-p solubilization, pmease activity and iaa production abilities, a total of 20 strains were selected from the above strains for rice growth assays. identification of the 20 strains was performed following the method of otsuka et al. (2008) based on the 16s rrna g e n e s e q u e n c e w i t h 1 6 s 9 f ( 5 gagtttgatcctggctcag-3) and 16s1510r (5-ggctaccttgttacga-3) primers. rice growth assay at the stage of germination under saline condition one strain out of 10 taxonomic groups was selected and subjected to a root and shoot growth assay of rice at the stage of germination based on zaller (2007) and cerabolini et al. (2004). briefly, ten seeds of three rice cultivars, inpara-3, inpari-13 and inpara-6 were soaked in sterile water for 5 hours in their respective containers. these 10 rice seeds were then arranged based on their respective cultivars on top of filter paper which was put inside a petridish (20 cm in diameter). fifteen milliliter of 4.0 g/l nacl solution was poured onto the filter paper inside table 1 physical and chemical properties of soils in the research field parameter p (%) k (%) c (%) n (%) c/n ratio ca (%) exchangable mg (%) exchangable na (%) exchangable al dd (%) soil ph amount of bacteria population characteristic 0.173 0.045 1.303 0.36 3.61 11.41 0.57 0.30 0.04 5.8 104-105 determined accoding to rowell (1994) very low very low low moderate very low high low low low acid infertile 117 promoting rice growth using rhizosphere – widawati and sudiana each petridish, on which 10 rice seeds were lined up, followed by 1.0 ml of bacterial inoculant 9 suspension containing 10 cells/ml. root and shoot lengths were measured at 7 days after germination. this experiment was set up using complete randomized design with three replications. rice growth assay at 45 days after planting under saline and non-saline conditions ten strains out of 20 isolates tested on germination test were then subjected to rice growth assay for 45 days under saline condition with 0.4% nacl. the number of cells for each 7 treatment was adjusted to about 3.2 x 10 . this value was selected based on the number of bacteria commonly found in paddy field soil. in a preliminary test (rice growth assay at the stage of germination), inpari-13 and inpara-6 could not grow well under the same saline condition. therefore, only inpara-3 was used in this assay. four seeds of inpara-3 were planted to experimental pots (0.5 gallon pots) containing sterile sands (1.5 kg) flooded with water(field capacity of sands = 24% or 360 ml ). treatments applied were: 1. saline condition (adding 360 ml of 0.4% nacl (6 g nacl) to the 0.5 gallon pots) and 2. non-saline condition (without 0.4% nacl). into each pot, 5 ml of bacterial inoculant suspension was added. the result of experiment is shown in table 5. after 7 days, the second inoculation with the same amount of bacterial suspension was conducted. the water level in pot was regulated by adding sterile water to compensate water decrease due to evaporation. the electrical conductivity (ec) value of the assay media under saline condition was kept at 7.5 ms/cm. at 45 days after planting the growth of rice was evaluated. this experiment was set as complete randomized design performed with three replications. results and discussion composition of the strains the selected 20 strains, originated from the rhizosphere of rice and peanut, belonged to the genera burkholderia, bacillus, achromobacter, pseudomonas, azospirillum, rhizobium and azotobacter (table 2). the selected strains were originated from non-saline soil. the reason for the selection was to compare the physiological characteristics of microbes isolated from saline and non-saline soil. the result of this study showed that the functional microbes for table 2 list of bacteria isolated from rice and peanut rhizosphere isolate code* phylum/class** taxon source (rhizosphere) csc p1 csc p2 csc p3 csc p4 csc p5 csc p6 csc p7 csc p8 csc p9 csc p10 csc p11 csc p12 csc n1 csc n2 csc n3 csc n7 csc n8 csc n9 csc n10 csc n11 proteobacteria/beta proteobacteria/beta firmicutes/bacilli proteobacteria/beta firmicutes/bacilli proteobacteria/gamma proteobacteria/alpha proteobacteria/alpha proteobacteria/alpha proteobacteria/gamma proteobacteria/gamma proteobacteria/beta proteobacteria/alpha proteobacteria/gamma proteobacteria/alpha proteobacteria/alpha firmicutes/bacilli proteobacteria/gamma proteobacteria/gammaproteobacteria/alpha rice rice rice rice rice rice rice rice rice rice rice rice peanut peanut peanut peanut peanut peanut peanut peanut notes: * = a strain with p in its code were isolated as ca-p solubilizing bacteria, and that with n were isolated as nitrogen fixing bacteria ** = alpha-, beta and gammadenote the classes alphaproteobacteria, betaproteobacteria and gammaproteobacteria, respectively burkholderia cepacia-complex burkholderia cenocepacia bacillus cereus-complex achromobacter spanius bacillus cereus-complex pseudomonas sp. azospirillum sp. azospirillum sp. rhizobium sp. azotobacter sp. azotobacter sp. burkholderia anthina rhizobium sp. pseudomonas sp. rhizobium leguminosarum azospirillum sp. bacillus cereus-complex azotobacter sp. pseudomonas sp. azospirillum sp. 118 biotropia vol. 23 no. 2, 2016 promoting rice growth were not different from that reported by susilowati et al. (2015). phosphate solubilizing ability of the strains ca-p solubilizing ability of the strains is shown in table 3. the difference in the strength of ca-p solubilizing ability was not related to the taxonomic property. all strains formed halo zone around colonies, and the area ratio of the halo zone to a colony was variable (data not shown) indicating the ability to solubilize ca-p differed among the strains. this was reflected in the ca-p solubilizing ability which was quantitatively determined (table 3). the highest ca-p solubilization ability was shown by pseudomonas sp. csc n2 and the lowest was shown by achromobacter spanius csc p4. the activity of pmease is shown in table 3. pseudomonas sp. cscn2 again showed the highest pmease activity, and achromobacter spanius csc p4 seemed to have no extracellular pmease activity. nitrogen-fixing ability of the strains all strains belonging to rhizobium, azotobacter and azospirillum genera were able to grow on nitrogen-limited media implying that these strains were able to fix nitrogen (chien et al. 1992). iaa production of the strains iaa production of the strains is shown in table 3. the amount of iaa produced varied depending on strains. the highest production was achieved by azospirillum sp. csc p8 and azospirillum sp. csc p7. the lowest iaa production was detected in achromobacter spanius csc p4. effect of bacterial inoculation on the rice during the 7-day germination assay with 0.4% nacl, the effect of bacterial inoculation varied depending on the strains (table 4). the best growth was obtained by the mixture of strains on inpara-3, with 7.46 cm and 6.5 cm in shoot and root length, respectively. medium level effect was observed in burkholderia cepacia-complex, bacillus cereus-complex, pseudomonas sp., azospirillum sp. and azotobacter sp. however, inoculation of burkholderia cenocepacia, achromobacter spanius, rhizobium sp., burkholderia anthina and rhizobium leguminosarum had no effect on shoot and root length. cultivars inpara-6 and inpari-13 could not grow without any inoculant (control) or with five single-strain-inoculants. in the 45-day growth assay (table 5), the growth of rice cultivar inpara-3 under saline burkholderia cepacia-complex burkholderia cenocepacia bacillus cereus-complex achromobacter spanius bacillus cereus-complex pseudomonas sp. azospirillum sp. azospirillum sp. rhizobium sp. azotobacter sp. azotobacter sp. burkholderia anthina rhizobium sp. pseudomonas sp. rhizobium leguminosarum azospirillum sp. bacillus cereus-complex azotobacter sp. pseudomonas sp. azospirillum sp. table 3 ca (po ) solubilization ability, pmease activity and iaa production of the strains3 4 2 isolate code taxon phosphate solubilization (mg/l)* pmease (unit)* iaa production (mg/l)* csc p1 csc p2 csc p3 csc p4 csc p5 csc p6 csc p7 csc p8 csc p9 csc p10 csc p11 csc p12 csc n1 csc n2 csc n3 csc n7 csc n8 csc n9 cscn10 cscn11 8.72 ± 0.89 1.06 ± 0.16 10.54 ± 0.16 0.30 ± 0.68 1.51 ± 0.11 11.26 ± 0.58 7.39 ± 0.42 6.68 ± 0.37 2.28 ± 0.63 5.71 ± 0.53 1.57 ± 0.95 0.47 ± 0.47 1.18 ± 0.05 11.39 ± 0.53 4.94 ± 0.32 2.00 ± 0.32 0.89 ± 0.95 0.83 ± 0.89 10.08 ± 0.26 1.86 ± 0.47 0.63 ± 0.71 0.13 ± 0.52 0.82 ± 0.85 0.01 ± 0.04 0.10 ± 0.86 0.75 ± 0.26 0.60 ± 0.86 0.68 ± 0.10 0.51 ± 0.70 1.27 ± 0.68 0.49 ± 0.67 0.10 ± 0.12 2.01 ± 0.34 2.22 ± 0.93 0.31 ± 0.27 0.47 ± 0.03 0.12 ± 0.88 0.45 ± 0.09 0.85 ± 0.89 0.14 ± 0.59 8.67 ± 0.92 2.63 ± 0.16 8.16 ± 0.90 1.94 ± 0.21 5.46 ± 0.58 8.27 ± 0.67 9.45 ± 0.06 9.56 ± 0.16 6.08 ± 0.42 8.75 ± 0.98 6.21 ± 0.32 2.13 ± 0.16 3.82 ± 0.89 8.16 ± 0.90 8.61 ± 0.10 7.61 ± 0.39 2.73 ± 0.68 8.39 ± 0.06 8.33 ± 0.84 8.09 ± 0.22 note: values represent mean±standard deviation (n = 3) 119 promoting rice growth using rhizosphere – widawati and sudiana table 4 the effect of bacterial inoculants on root and shoot length of rice (three cultivars) at the stage of seed germination isolate code taxon rice cultivar shoot length (cm)* root length (cm)* control csc p1 csc p2 csc p3 csc p4 csc p6 csc p8 csc n1 csc n9 csc n12 csc n3 mix (control: no inoculation) burkholderia cepacia-complex burkholderia cenocepacia bacillus cereus-complex achromobacter spanius pseudomonas sp. azospirillum sp. rhizobium sp. azotobacter sp. burkholderia anthina rhizobium leguminosarum mixture of strain inpara-3 inpari-13 inpara-6 inpara-3 inpari-13 inpara-6 inpara-3 inpari-13 inpara-6 inpara-3 inpari-13 inpara-6 inpara-3 inpari-13 inpara-6 inpara-3 inpari-13 inpara-6 inpara-3 inpari-13 inpara-6 inpara-3 inpari-13 inpara-6 inpara-3 inpari-13 inpara-6 inpara-3 inpari-13 inpara-6 inpara-3 inpari-13 inpara-6 inpara-3 inpari-13 inpara-6 4.03 a dead dead 5.55 de 4.23 ab 4.37 ab 4.16 ab dead dead 5.54 de 4.51 abcd 4.59 abcd 4.09 a dead dead 5.58 de 4.55 abcd 4.79 abcd 6.10 e 4.80 abcd 4.94 abcd 4.30 ab dead dead 5.56 de 4.82 abcd 4.31 ab 4.03 a dead dead 4.34 dead dead 7.46 f 4.98 abcd 4.96 abcd 0.51 a dead dead 4.43 ghi 2,00 bcde 1.39 abc 1.47 abcd dead dead 4.24 ghi 2.99 defg 2.04 bcde 2.90 cdefg dead dead 4.70 hi 3.41 efgh 3.22 efgh 5.00 i 3.65 fghi 3.11 efgh 3.95 fghi dead dead 4.44 ghi 2.83 cdefg 2.39 bcdef 1.25 ab dead dead 3.62 dead dead 6.50 j 4.01 ghi 4.13 ghi note: values followed by the same letter in the same column are not significantly different based on duncan's multiple range test at 5% level condition was less than that under no saline condition. under saline condition, rice cultivar inpara-3 inoculated with the mixture of strains showed the best growth with 29 cm in plant height and 5.5 cm in root length. as a single isolate inoculation, pseudomonas sp. csc n6 showed the best effect. twenty strains with ca-p solubilizing, extracellular pmease producing and iaa producing abilities were successfully obtained, with an exception of a. spanius csc p4 that did not show clear pmease activity. these strains did not lean to a specific taxonomic lineage and composed of members of the phyla proteobacteria (the classes alphaproteobacteria, betaproteobacteria and gammaproteobacteria) and fermicutes. the fact that the strains were isolated as nitrogen-fixing bacteria including ca-p solubilizing members indicated that ca-p solubilizing ability was common among bacteria, at least among those living in the rhizosphere. it was also possible that pmease activity was common among 120 biotropia vol. 23 no. 2, 2016 table 5 the effect of bacterial inoculants on the growth of rice cultivar inpara-3, in sterile sand media under saline and non-saline conditions 45 days after planting isolate code inoculant salinity condition total dry biomass (g) plant height (cm) root length (cm) – csc p1 csc p2 csc p3 cscp4 csc p6 csc p8 csc n1 csc n9 cscn12 csc n3 – no bacteria burkholderia cepacia-complex burkholderia cenocepacia bacillus cereus-complex achromobacter spanius pseudomonas sp. azospirillum sp. rhizobium sp. azotobacter sp. burkholderia anthina rhizobium leguminosarum mixture of all the isolates non-saline saline non-saline saline non-saline saline non-saline saline non-saline saline non-saline saline non-saline saline non-saline saline non-saline saline non-saline saline non-saline saline non-saline saline 0.02 a 0.01 a 0.09 cde 0.07 abcd 0.06 abcd 0.04 abc 0.09 cde 0.08 bcd 0.04 abc 0.02 a 0.13 e 0.09 cde 0.09 cde 0.07 abcd 0.07 abcd 0.04 abc 0.08 bcd 0.07 abcd 0.06 abc 0.02 a 0.05 abc 0.02 abc 0.14 e 0.12 de 14.25 ab 13.00 a 27.50 ghi 26.25 ghi 26.00 fgh 22.75 cdefg 27.30 ghi 26.00 fgh 25.50 efgh 17.00 abc 29.50 hi 26.88 ghi 27.50 ghi 26.25 ghi 24.50 defgh 20.75 cde 26.50 ghi 26.25 ghi 21.00 cdef 17.15 abc 23.00 defg 19.00 bcd 31.00 i 29.00 hi 1.50 ab 1.00 a 5.00 jk 4.00 hij 3.50 fgh 2.65 de 4.50 ij 3.75 ghi 3.50 fgh 3.00 ef 6.15 l 5.00 jk 4.25 ij 4.00 hij 3.25 fg 2.25 cd 4.50 ij 3.75 ghi 3.50 fgh 2.25 cd 3.25 fg 2.00 bc 6.50 l 5.50 k rhizosphere bacteria. interestingly, the strains with higher ca-p solubilizing ability generally showed higher pmease activity. iaa production was also reported as common among soil bacteria (hasan 2002; xin et al. 2009), which was supported by the present study. saline environment inhibits rice growth. this is because rice is a saline sensitive plant (ashraf & + +harris 2004); also because the uptake of ca , k 2 and inorganic n and p are disrupted under high na concentration (ashraf & harris 2004). in addition, the salinity also affected soil enzyme activities (siddikee et al. 2011), which could indirectly affect rice growth. the inoculation of the selected strains affected germination of rice under saline condition (table 4). the inoculation of mixture of the strains resulted in the best rice growth. as single strain, azospirillum sp. csc p8 and pseudomonas sp. csc p6 provided the best and the second best rice growth support, respectively. it was possible that the inoculants supported the growth of rice by supplying phosphate and iaa. azospirillum sp. is a potential nitrogen fixer and the mixture of the strains also includes nitrogen fixers. therefore, it was possible that nitrogen fixed by the inoculants might also promote rice growth. rice cultivars inpari-13 and inpara6 did not grow without the existence of inoculants. the present study showed that the inoculation of five strains and the mixture of strains enabled these cultivars to grow. this indicated that the inoculation not only promoted rice growth by supplying nutrient and iaa, but also enhanced rice tolerance towards salinity. it was interesting that some strains isolated from peanut rhizosphere could promote and support rice growth. among the rice cultivars tested in the present study, only inpara-3 grew in saline condition without inoculation of the strains. therefore, inpara-3 was then subjected to rice growth assay with 0.4% nacl. in this assay, the inoculation of the mixture of strains, pseudomonas sp. csc p6 and azospirillum sp. csc p8 provided notes: values followed by the same letter in the same column are not significantly different by duncan's multiple range test at 5% level. non-saline condition = 360 ml freshwater in 0.5 gallon pots. saline condition = 360 ml freshwater in 0.5 gallon pots was added with 0.4% nacl (6 g nacl). 121 promoting rice growth using rhizosphere – widawati and sudiana the best, the second best, and the third best rice growth support, respectively. these inoculants may be promising as biofertilizer to support rice growth in saline paddy fields. conclusions twenty strains of rhizosphere bacteria with ca-p solubilizing ability and iaa production were successfully obtained in this study. those bacteria mainly belonged to burkholderia cepacia-complex, burkholderia anthina, burkholderia cenocepacia, bacillus cereus-complex, achromobacter spanius, azospirillum sp., azotobacter sp., rhizobium leguminosarum, rhizobium sp. and pseudomonas sp. potential nitrogen fixing bacteria are azospirillum sp., azotobacter sp., rhizobium leguminosarum and rhizobium sp. most strains had pmease activity. some strains showed growthpromoting effect on rice under saline conditions and produced plant growth hormone. these strains could be candidates for biofertilizer for rice in saline paddy field. it is also important to consider using combination of inoculants and rice cultivars to obtain maximum result. acknowledgements this work was funded by jica-jst satrep 2011-2016. we thank dr shigeto otsuka from university of tokyo for research guidance. we are grateful to senlie oktavitana, anis mutirani and rinatu siwi for their assistance in laboratory works. references aquilanti lff, clementi f. 2004. comparison of different strategies for isolation and preliminary identification of azotobacter from soil samples. soil biol biochem 36(9):1475–83. ashraf m, harris pjc. 2004. potential biochemical indicators of salinity tolerance in plants. plant sci 166 (1):3–16. balser tc, mcmahon kd, bart d, bronson d, coyle dr, craig n, flores-mangual ml, forshay k, jones se, kent ae, shade al. 2006. bridging the gap between micro and macro-scale perspectives 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sudiana page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 microsoft word 111 biotropia vol. 13 no. 2, 2006 : 111 121 antagonistic bacteria against schizophyllum commune fr. in peninsular malaysia antarjo dikin', kamaruzaman sijam', jugah kadir' and idris abu semanz 'department of plant protection, faculty of agriculture, universiti putra malaysia, 43400 upm serdang, selangor d.e, malaysia 2plant pathology and weed science group, biological research division malaysian palm oil board, 43000 kajang, selangor, d.e. malaysia abstract schizophyllum commune fr., is one of the important fungi, causes brown germ and seed rot of oil palm. biodiversity of antagonistic bacteria from oil palm plantations in peninsular malaysia is expected to support in development of biopesticide. isolation with liquid assay and screening antagonistic bacteria using dual culture assay were carried out in the bioexploration. a total of 265 bacterial isolates from plant parts of oil palm screened 52 antagonistic bacterial isolates against 5. commune. bacterial isolates were identified by using biolog* identification system i.e. bacillus macroccanus, b. thermoglucosidasius, burkholderia cepacia, b. gladioli, b. multivorans, b pyrrocinia, b. spinosa, corynebacterium agropyri, c. misitidis, enterobacter aerogenes, microbacterium testaceum, pseudomonas aeruginosa, p. citronellolis, rhodococcus rhodochrous, serratia ficaria, serratia sp., s. marcescens, staphylococcus sciuri, sternotrophomonas maltophilia. key words : schizophyllum commune, biodiversity, antagonistic bacteria introduction schizophyllum commune fr. causes brown germ and seed rot. heavy infection decreased seed germination of oil palm about 60 percent (dikin et al. 2003). proper seed treatments are required for the control of s. commune in the oil palm seeds. some synthetic fungicides were applied to reduce the loss of germination due to this pathogen, but the negative impact from toxic chemicals to the environment was difficult to avoid. the utilization of bacteria as biological control agents successfully controlled plant pathogen (sharga and lyon 1998; bapat and shah 2000). many studies in exploration of beneficial organisms have been carried out such as pseudomonas fluorescent for the control of fusarium wilt of tomato (dekkers et al. 1998). streptomyces halstedii (k122) and s. coelicolor (k139) to inhibit the fungi belonging to oomycetes, zygomycetes, deuteromycetes, ascomycetes and basidiomycetes (frandberg and schnurer 1998). bacillus subtilis suppressed phytopathogenic microorganism (phae et al. 1990). the isolation of antagonistic bacteria was early stage for development of biopesticide such as pseudomonas fluorescent from rhizospheres (dekkers et al. 1998), bacillus licheniformis from leaves of citrus orchard at letaba estates, corresponding address : antario_dikin@yahoo.com 111 biotropia vol. 13 no. 2,2006 tzaneen (jager and kosten 1998), streptomyces halstedii (k122) and s. coelicolor (k139) from cereal grains (frandberg and schnurer 1998), bacillus subtilis from the composts (phae et al. 1990), and burkholderia cepacia from infected oil palm seeds (dikin et al. 2003). the liquid assay technique was a simple method for isolation of bacteria. fluorescent pseudomonas from pythiumdiseased tulip roots was isolated by extraction of infected root in sterilized water (weststeijn 1990). malaysia is well known as a mega-biodiversity country with complex microbial association. the exploration of beneficial bacteria from oil palm plantations is expected to utilize the antagonistic bacteria from the same ecology of the oil palm pathogen itself. the purposes of the study were to isolate and to screen the antagonistic bacteria from different plant parts of oil palm for the control of schizophyllum commune. materials and methods cultural schizophyllum commune fr. and plant part of oil palm the culture of s. commune was isolated from heavy infection of oil palm seeds. the fungus was confirmed based on their morphological characteristics and the pathogenic fungus of oil palm (alexopoulus et al. 1996). the fungal isolate was sub-cultured onto pda medium for further study. randomized samples of plant parts such as fruits, seeds, rhizosphere, and plant debris were collected from oil palm fields in selangor (upm, bangi, kajang and seri kembangan), guthrie, layang-layang, johor in peninsular region, malaysia. plant parts were used for isolation of potential bacteria. isolation of antagonistic bacteria plant parts of oil palm such as fruits, seeds, rhizospheres and plant debris were rinsed with tap water to remove the adhered soil on surface. mesocarp of fruits, endosperm of seeds, and rhizosphere were sliced into 0.5-1.0 cm2 and then 50 g sliced plant parts were placed into 250 ml erlemeyer flask added with 100 ml distilled water. plant debris with 5. commune was collected under oil palm tree. ten g of plant debris were cut off in size 1 cm and then transferred into a 250 ml erlemeyer flask added with 100 ml sterilized water. sample materials in flasks were placed on electric rotator at 100 rpm overnight at 26 ± 2°c. fold serials (lo^-lo"4) dilutions of suspension were made, 0.5 ml suspension from each diluted suspension was streaked on king's b (kb) and nutrient agar (na) agar media plates. plates were incubated at 26-28°c for 48 hours. bacterial colonies on plate were purified by streaking single bacterial colony onto na medium plates. each pure culture of bacteria was screened for the antagonistic bacteria based on dual culture (dikin et al. 2002;montealegree/a/. 2003). 112 antagonistic bacteria a. dikin et al. screening the antagonistic bacteria screening of bacterial antagonist was carried out using dual culture assay. one 6-mm diameter of s. commune agar plug was placed at the centre of pda medium in a petri dish with 9 cm diameter. bacterial isolate was streaked on pda medium with a distance of 2.5 cm between s. commune agar plug and bacterial isolate. plates were incubated for 7 days at 26 ± 2°c. the percentage of radial inhibition growth was measured with the formula: pirg (%) = (1 (fungal growth near to bacterial isolate /fungal growth other side at the same plate as control)) x 100%. each treatment was replicated 3 times. receded data were analyzed using sas® software. treatment effect was tested by anova and the means compared using least significant different test at 5% probability level (okamoto et al. 1998; anonymous 1999; montealegreefa/. 2003). identification of antagonistic bacteria potential antagonistic bacterial isolates were identified by biolog® identification system which followed the biolog's procedures. bacterial suspension was inoculated into gn or gp micro plates depending on gram reaction cluster, 145 ul per well using the 8-channei repeating pipette. microplate was covered with its lid and incubated at 28-30°c for 24 hours to allow the utilization of carbon sources. reading result was directly done after inserting the incubated microplate into the biolog's reader apparatus and its installed micro soft ware of biolog® identification system for identifying bacteria up to the species level (anonymous 2001). results and discussion isolation of antagonistic bacteria the number of bacterial isolates was extracted from samples of seeds, fruits, rhizospheres and plant debris of oil palm which grew on kb and na media. a total of 265 bacterial isolates from plant parts of oil palm were found from different locations, peninsular region, malaysia. separation of bacterial isolates was based on the morphological colony performance such as colony colour, elevation, the margin of colony and colony surface (hayward 1983). isolation of potential bacteria from plant parts of oil palm using liquid assay was effective and simple technique. the liquid assay and the agar plate media are commonly used for isolation of pathogenic bacteria from infected plant parts. bacterial isolates from different plant parts of oil palm on na and kb media grew well on the cultural plates. dual culture assay screened 52 out of 265 bacterial isolates against s. commune. the number of antagonistic bacteria from each location isolated from different plant parts is presented in table 1. 113   antagonistic bacteria a. dikin et al. based on table 1, 96 bacterial isolates as the highest number were obtained from the rhizosphere followed by plant debris, 89 isolates. the average number of bacterial isolates from rhizosphere was 9.6 followed by 6.8 from plant debris, 6.1 from fruits, and 5.2 from seeds. the bacterial isolates obtained from plant debris were more diverse with the number of bacterial isolates higher than other plant parts such as rhizophere, fruit, and seed. eighteen out of 89 antagonistic bacterial isolates were obtained from plant debris, followed by 15 out of 96 isolates from rhizosphere, 10 out of 43 isolates from fruit, and 9 out of 37 isolates from seed. the probability for isolation of antagonistic bacteria from each plant part was 20.2 percent, 15.6 percent, 23.2 percent, and 24.3 percent, respectively. more dominant bacteria in the rhizosphere and plant debris than seeds and fruits were due to the different available nutrition and the requirement for bacterial growth. dominance of bacteria in the rhizospheres and plant debris was due to complex interaction between microorganisms and plant parts. plant debris such as decayed empty bunch, fronds, and rachis were good media for the fungal growth. blotching symptom with water soak and brown colour in the fruiting bodies of s. commune was the indication of interaction between fungus and bacteria. dual culture assay of s. commune against antagonistic bacteria is presented in figure 1. figure 1. a. burkhoderia multivorans (bacterial code-50) inhibits the growth of s. commune on dual culture of pda medium at 7-day after incubation at 26 ± 2°c b. burkholderia cepacia inhibits the growth of s. commune on dual culture of pda medium at 7-day after incubation at 26 ± 2°c among 52 isolates from different plant parts and different sampling locations inhibited the mycelial growth of s. commune with various percentages of radial inhibition. each bacterial isolate with radial growth inhibition of s. commune is presented in table 2. the range of radial growth inhibition of antagonistic bacteria was 3.3 percent up to 95.2 percent from the bacterial code 29 and 10, respectively. in table 2, there are 7 bacterial isolates with highest mean percentage of radial growth inhibition with the bacterial code 10, 8, 9, 14, 50, 7, and 2 with the percentage of inhibition of 95.2, 115 biotropia vol. 13 no. 2,2006 90.6, 83.2, 83.1, 83, 81.8, and 81.5, respectively. a number of antagonistic bacteria were isolated from plant parts with varied mean percentages of radial growth inhibition against s. commune. the bacterial isolates had high radial growth inhibition which were obtained from plant debris, rhizospheres, and fruit. many authors have reported that certain antagonistic bacteria suppressed the growth of pathogenic fungus. in vitro study showed that burkholderia cepacia from tomato's rhizospheres suppressed the growth of fusarium oxysporum f.sp. lycopersicae stronger than s. commune. in contrast, b. cepacia from rhizhosperes of oil palm suppressed s. commune stronger than f. oxysporum f.sp. lycopersicae (kamaruzaman and dikin 2005). in this case , targeted potential antagonistic bacteria against s. commune should be isolated from the area of oil palm plantation. identification of antagonistic bacteria identification of antagonistic bacteria against s. commune based on biolog® identification system is presented in table 3. 116 antagonistic bacteria a. dikin et al. table 3. antagonistic bacteria based on biolog* identification system plant part bacterial code bacteria rhizosphere 1 burkholderia cepacia 6 corynebacterium agropyri 7 microbacterium testaceum 17 pseudomonas citronellolis 20 bacillus macroccanus 23 b. cepacia 24 burkholderia spinosa 47 b. cepacia 49 staphylococcus sciuri 50 burkholderia multivorans 51 burkholderia pyrrocinia 52 b. cepacia fruit 3 b. cepacia 5 serratia marcescens 9 c. agropyri 25 pseudomonas aeruginosa 34 p. aeruginosa 38 p. aeruginosa 40 p. aeruginosa seed 4 serratia sp. 12 bacillus thermoglucosidasius 18 b. pyrrocinia 22 b. cepacia 26 p. aeruginosa 37 sternotrophomonas maltophilia plant debris 2 s. mallophilia 8 burkholderia gladioli 10 b. gladioli 11 b. cepacia 14 c. agropyri 16 serratia ficaria 19 enterobacter aerogenes 28 b. gladioli 30 p. aeruginosa 33 corynebacterium masitidis 42 m. testaceum 43 rhodococcus rhodochrous table 3 presents the identified bacteria and non-identified bacteria by using biolog identification system. in the rhizosphere 12 identified species and 3 non-identified species were found. the identified species from rhizosphere were b. cepacia, c. agropyri, m. testaceum, p. citronellolis, b. macroccanus, b. spinosa, s. sciuri, b. multivorans, and b. pyrrocinia. among 9 species, the dominant identified species in the rhizosphere was b. cepacia. 117 biotropia vol. 13 no. 2,2006 seven identified species and 3 non-identified species of antagonistic bacteria were found in fruits. the identified species from fruits were b. cepacia, s. marcescens, c. agropyri, and p. aeruginosa. among the 4 species, the dominant identified species in the fruit was p. aeruginosa. p. aeruginosa was isolated from the rhizospheres and plant debris. this bacteria was recognized as the supplier of mineral which was required for metabolism process of plant from the access of bacterial metabolites (hofte et al. 1993; abdullah et al. 2003). there were complex microorganisms around rhizospheres to compete with each other for survival which showed the synergism and antagonism interaction. infected plant debris with s. commune around the rhizosphere was to bait the potential antagonistic bacteria. six identified species and 3 non-identified species of antagonistic bacteria in seeds were found. the identified species from fruits were serratia sp., b. thermoglucosidasius, b. pyrrocinia, b. cepacia, p. aeruginosa, and s. maltophilia. in the plant debris 12 identified species and 6 non-identified were found. the identified species were s. maltophilia, b. gladioli, b. cepacia, c. agropyri, s. ficaria, e. aeogenes, p. aeruginosa, c. masitidis, m. testaceum, and r. rhodochrous. the dominant species from plant debris was b. gladioli. b. cepacia and b. gladioli were dominantly found in the rhizospheres and plant debris, respectively. the presence of b. cepacia in the rhizospheres of oil palm was the same evident with the presence of b. cepacia in the rhizospheres of banana to protect plant infection caused by fusarium oxysporum f. sp cubense. b. cepacia colonizes the surface of hyphae and fungal macrospores (pan et al. 1997). b. gladioli was found in the rhizospheres and potential antagonistic bacteria against s. commune, however the implication for biological control was less recognized. the identified species of antagonistic bacteria from different plant parts were bacillus macmccanus, b. thermoglucosidasius, burkholderia cepacia, b. gladioli, b. multivorans, b pyrrocinia, b. spinosa, corynebacterium agropyri, c. misitidis, enterobacter aerogenes, microbacterium testaceum, pseudomonas aeruginosa, p. citronellolis, rhodococcus rhodochrous, serratia ficaria, serratia sp., s. marcescens, staphylococcus sciuri, and sternotrophomonas maltophilia. out of these species were new recorded species of antagonistic bacteria i.e. b. thermoglucosidasius, b. multivorans, b. spinosa, c. agropyri, c. misitidis, enterobacter aerogenes, p. citronellolis, rhodococcus rhodochrous, serratia ficaria, and staphylococcus sciuri. however, b. cepacia, b pyrrocinia p. aeruginosa, s. marcescens and s. maltophilia were reported as biocontrol agents (burkhead et al. 1994; kobayashi et al. 1995; suparman et al. 2002; szczech and shoda 2004). avirulent isolate of b. gladioli strain 1064a is used for suppressing the incidence of bacterial seedling blight of rice caused by b. plantarii (miyagawa 2000). p. aeruginosa is grouped as fluorescent pseudomonads based on the production of a fluorescens pigment on kb medium (sand et al. 1980). the bacterium produces siderophores as plant growth promoter (hofte et al. 1993) and broad spectrum antagonistic bacteria against pathogenic fungi (haas et al. 1991). 118 antagonistic bacteria a. dikin et at. several species of fluorescent pseudomonads were known to be antagonistic bacteria and used as biological control agents. p. aeruginosa 7nssk2 was able to suppress pythium splendens, the causal pre and post-emergence damping-off and root rot of many crops such as tomato (tambong et al. 1998). p. aeruginosa and s. marcescens were isolated from plant part of oil palm, these isolates were confirmed as biocontrol agent for suppressing sclerotium rolfsii and rhizoctonia solani (ordentliche/a/. 1987). conclusions a number of bacterial isolates from plant part such as seeds, fruits, rhizospheres, and plant debris under oil palm trees were potential antagonistic bacteria against s. commune. a total of 52 out of 265 bacterial isolates were identified as the antagonistic bacteria against s. commune. the identified antagonistic bacteria using biolog® identification system were as follows : agrobacterium agropyri, bacillus macroccanus, b. thermoglucosidasius, burkholderia cepacia, b. gladioli, b. multivorans, b pyrrocinia, b. spinosa, corynebacterium agropyri, c. misitidis, enterobacter aerogenes, microbacterium testaceum, pseudomonas aeruginosa, p. citronellolis, rhodococcus rhodochrous, serratia ficaria, serratia sp., s. marcescens, staphylococcus sciuri, and sternotrophomonas maltophilia. some of bacterial isolates were recognized as biocontrol agents of plant pathogenic fungi and the rest of isolates have yet to be studied for their status. all of these species are required for further studies on the production of their secondary metabolites which might be potential substances to inhibit the growth of s. commune. acknowlegdment the study is partially supported by irpa project, malaysian government (vote no. 54400) as part of the ph.d. thesis of the first author. references abdullah, h., h.m. saud and c.e. fong. 2003. evaluation of the combined effect of pseudomonas aeruginosa and penicillmm sp. on the development of bacterial wilt caused by ralstonia solanacearum on tomato. abstract. third federal of asia pacific microbiology societies conference. kuala lumpur, october 15-18,2003 alexopoulos, c.j., c.m. mims and m. blackwell. 1996. introductory mycology. phyllum: basidiomycota. order: aphyllophorales. polypores, chantharelles, tooth fungi, coral fungi, and corticioids. 19 : 563-597. anonymous. 1999. sas* software. system release version 8 (ts ml), sas institute trial site, gary, north california, usa. anonymous. 2001. a powerful 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no. 2, 2006 : 99 110 alimentary canal anatomy and histology of the worker termite neotermes bosei leksono ekopuranto hariprabowo, rika raffiudin and taruni sri prawasti department of biology, faculty of mathematics and sciences, bogor agricultural university, jalan ray a pajajaran, bogor, 16144 indonesia abstract as social insects, termites live in a colony that consist of reproductive (drone and queen), and non-reproductive (soldiers and workers) castes. workers obtain their food directly from wood, humus, and other substances that contain cellulose. the objective of this study was to examine the alimentary canal of the neotermes bosei workers. observations of gut transverse section were carried out through the length, perimeter, and area of each alimentary canal region. the results showed that total length of n. bosei alimentary canal was 13.71+1.28 mm. the canal was divided into fore-, mid, and hindgut which were 24, 28, and 48%, respectively of the gut total length. two types of alimentary canal epithelial cells were found, i.e. the squamous and transitional cells. areas covered with thick muscular tissues were crop, proventriculus, and rectum. proventriculus was characterized with six large dentitions. there was no gastric caeca in n. bosei midgut, which commonly occurred in chewing insect. secretory cells .were observed at proventriculus and ventriculus regions. cardiac valve was found at the anterior end of ventriculus. area with the largest outer perimeter was the rectum pouch. enteric valve had three internal folds. key words: drywood termites, alimentary canal, histology, kalotermitidae introduction termites were classified into the order of isoptera, which consist of six families i.e. mastotermitidae, kalotermitidae, hodotermitidae, rhinotermitidae, serritermitidae, and termitidae (krishna 1969). as social insects, termites live in a colony. each colony comprises reproductive (drone and queen) and non-reproductive (worker and soldier) castes. worker termites obtain their food directly from wood, humus and other materials that contain cellulose. soldier castes obtain the food from the workers through trophalaxis (stuart 1969). the ability of termites, particularly of the members of kalotermitidae to digest the cellulose is due to the mutualistic symbiosis with the flagellate protist (claveland 1925). hence, the food digestion process in termites is different with that of most other insects (tokuda et al. 2000; inoue etal. 2000). the alimentary canal is divisible into stomodeum (foregut), mesenteron (midgut) and proctodeum (hindgut). digestion process occurred in the midgut (dow 1986; inoue et al. 2000), whereas hindgut is the most important part in food absorptions (inoue et al. 2000). neotermes bosei (figure 1) is classified into the family of kalotermitidae (ahmad 1965). n. bosei is known as damp wood termites, because it lives in the dry and living wood (scheffrahn & su 1994). this species was selected for this current corresponding address : rika rafiudin.net.id 99 biotropia vol. 13 no. 2, 2006 termite histology study due to lack of information on kalotermitidae alimentary canal . the objective of this study is to examine the anatomy and histology of the termite n. bosei worker alimentary canal. materials and method neotermes bosei collection the n. bosei soldiers and workers imagoes were collected from the fishing shop in several traditional markets in bogor, west java (i.e. pasar anyar, pasar bogor, pasar ciawi, and pasar empang). the soldiers of n. bosei were needed for identification purpose up to species level. observation of n. bosei alimentary canal length of alimentary canal ten n. bosei alimentary canal were taken by dissectioning the lateral part of termite thorax and abdomen. termite alimentary canal was observed under stereo microscope (nikon fdx-35) and photographs were taken by using nikon smz1000 camera. the photographs were digitized for analysing canal total length (crop up to anus), the length of fore-, mid-, and hindgut. the measurements were carried out with imagej sofware (http://rsb.info. nih.gov/ii). diameter and the cell structure ofn. bosei alimentary canal six series of serial transversal section of the canals were made through paraffin embedding method (thickness of 6 um). the sections were stained by using double staining of haematoxylineosin (he). photographs were taken with compound microscope (olympus ch20) and olympus dp 12 camera. inner and outer peripheri 100 alimentary canal anatomy and histology l. e. hariprabowo et al. (ip and op), lumen area (la), and gut area (ga) were measured by imagej sofware (http://rsb.info. nih.gov/ij). embedding block with paraffin method (gray 1952) neotermes bosei alimentary canals were fixed with bouin fixative for two hours. then, they were washed and dehydrated in series of ethanol i.e. 70, 80, 90 %, and absolute ethanol, 15 min for each step. for slide clearing it was immersed in xylol for 1.5 hours. infiltration paraffin step was performed for three times, each step in 45 min and subsequently stained with he. results and discussion foregut the tv. bosei foregut was a slender canal. the mean of length from the crop up to proventriculus is 3.32 + 0.57 mm (figure 2). figure 2. the alimentary canal of n. bosei worker termite oesophagus oesophagus was the canal that connected termite's pharynx and its crop. this region consisted of several layers, i.e. intima, squamous cells (at the lumen wall), columnar cell (at vili), and thin muscle layer (figure 3). the ip, op, la, and ga values were listed in table 1. figure 3. oesophagus transversal section of n. bosei worker termite 101 biotropia vol. 13 no. 2,2006 crop neotermes bosei worker crop was divisible into three parts, i.e. the anterior, median, and posterior regions (figure 4). the anterior crop had six large vili which covered almost the entire lumen. this region had intima layer, columnar epithelial cells (at the vili) and squamous epithelial cells (at the lumen wall), circular and longitudinal muscle cells. mean of the ip, op, la and ga were listed in table 1. figure 4. crop transversal section of n. bosei worker termite (a) anterior, (b) median, (c) posterior region, (d) cell layers of the posterior crop note: oeso= oesofagus; crl= anterior crop; cr2= median crop; cr3= posterior crop; pvl= anterior proventriculus; pv2= posterior proventriculus; vl= anterior ventriculus; v2= posterior ventriculus; pl= short channel; p2= enteric valve; p3= rectum pouch; p4= colon; p5= rectum; = data not available 102 alimentary canal anatomy and histology l. e. hariprabowo et al. large number of vili covered the median region of the crops compared to that in the anterior region. squamous epithelial cells in the median crop were shorter than those in the anterior (figure 4b). the ip, op, la and ga values of median crop regions were higher than those in the anterior (table 1). the posterior crop region contained the thickest muscle, intima layer, squamous epithelial cells, circular and longitudinal muscle layers (figure 4c and 4d). proventriculus proventriculus was the last part of the foregut (figure 2) consisted of anterior and the posterior regions (figure 5a,b). four dentitions were observed at the lumen wall at the anterior proventriculus region (figure 5a), whereas six dentitions were found in the posterior region (figure 5b). figure 5. proventriculus transversal section of .m bosei worker termite (a) anterior region, (b) posterior region, (c) cell layers composed the anterior region, (d) layers composition of the posterior region; 1,2,3,4 = dentition type in the anterior proventriculus proventriculus ofn. bosei worker was covered by a thick intima, transitional epithelial cells, and a thick muscle layer (figure 5c,d). several secretory cells (figure 5d) were also found in this region. they were characterized by a cluster of larger cells compared to the other epithelial cells surrounding them. anterior and posterior proventriculus measurements were shown in table 1. as a chewing insect n. bosei worker has proventriculus canal (miller 1965). the proventriculus occurred in the chewing insect such as moth larva hofmannophila pseudospretella (lepidoptera: oecophoridae) (gerard 2002) and beetle dendroctonus (coleoptera: scolytidae) (diaz et al. 2003). the intima layer in the proventriculus acts as the food grinder (wigglesworth 1972). no proventriculus 103 biotropia vol. 13 no. 2,2006 occurs in the haustellate insect such as in the fly bactrocera dorsalis (diptera: tephritidae) (lee et al. 1998). midgut ventriculus the length ofn. bosei worker midgut (figure 2) was 3.86+0.61 mm, composed of only a region, the ventriculus. it was characterized by the occurrence of transitional epithelial cells and thin muscle cells. this was congruent with the result of noirot and noirot-timothee (1969) that ventriculus has one cell type, the transitional epithelial cell, as the absorption and secretion cells. however, nasutitermes takasagoensis worker ventriculus consisted of columnar cells (tokuda et al. 2001). a variety of cell occurs in the dendroctomts (coleoptera: scolytidae) ventriculus as well (diaz et al. 2003). the anterior ventriculus had a cardiac valve (figure 6a), which was an imagination structure at the foregut towards the midgut. this structure was not found in the posterior ventriculus (figure 6b). the cardiac valve covered the constricted region, hence it had low ip, op, la and ga values (table 1). antiperistaltic movement was prevented by the cardiac valve, a constriction structure in the anterior ventriculus as shown in n. bosei. it prevented the food at the midgut to flow back to the foregut (snodgrass 1935). this character was also reported in n. takasagoensis (termitidae) (tokuda et al. 2001). hence, in termites and most other insects, the digested food always moves f rom anterior towards the posterior region of the alimentary canal. there is no gastric caeca in the midgut of n. bosei worker; this structure is the same in n. takasagoensis worker (tokuda et al. 2001). in most chewing insects, gastric caeca lays at the anterior region of the midgut (romoser 1973) to enlarge the food absorption (wigglesworth 1972). the absence of gastric caeca in worker termites showed an adaptation of termite digestion system as well. termites do not 104 alimentary canal anatomy and histologyl. e. hariprabowo et al need an additional space in the midgut for food absorption because the cellulose will be degraded subsequently in the hindgut, particularly in the rectum pouch. hindgut the length ofn. bosei worker hindgut was 6.56+0.91 mm, composed of intima layer, columnar, transitional epithelial cells, and muscle cells. this alimentary canal region consisted of five parts, i.e. short channel that connected the midand the hindgut, enteric valve, rectum pouch, colon and the rectum. short channel this region could not be determined in the fresh alimentary canal, unless a histology preparation was made (figure 7a, b). this is due to the similar diameter and the same color with that of ventriculus region. short channel was covered with columnar epithelial cells and muscle layers (figure 7b). intima layer was not found in this region. the mean value of ip, op, la, and ga of these regions were shown in table 1. enteric valve enteric valve was the second region in the hindgut constructed of three longitudinal folds facing towards the lumen, performed a clep structure (figure 8a). it composed of intima layer, columnar epithelial cells, and muscle cells (figure 8b). mean of enteric valve op was 0.530+0,039 mm (table 1). rectum pouch the third part of the hindgut was the rectum pouch (figure 9a), consisted of intima layer, transitional epithelial cells, and thin layer of muscle cells (figure 9b,c). rectum pouch had the largest op value compared to others, that was 3.859+1.807 mm. 105 biotropia vol. 13 no. 2,2006 colon colon was the fourth part in the hindgut region (figure 10a,b), that composed of the same layers as shown in the rectum pouch. the op value of n. bosei worker colon was 1.508+0.370 mm. rectum rectum was the last part in the hindgut region (figure 11) with a thick muscle layer. other layers were the same as shown in the rectum pouch. there was no distinct region which separated the circular and longitudinal muscle layer, hence both were linked (figure lib). the op value of n. bosei worker rectum was 2.036+0.569 mm, it was the largest op diameter (see also figure 2). this big pouch in termite hindgut is an important region as mentioned by inoue et al. (2000), the hindgut harbours symbiotic flagellates which assist in cellulose digestion. cellulose is a polimer of 5000 -10 000 anhidrid glucosa connected with p(l,4) glycoside (feighl and hill 1983). most organism could not use p(l,4) glycoside as the energy source. those flagellates produce cellulase enzyme that degrade the cellulose into acetic acid, co2, and h2. hence, the enlargement of the termite hindgut is an adaptation mechanism of its behaviour and physiology. another difference of n. bosei worker to other termites is that it did not have a mixed segment. mixed segment has a variety of histologic structures, located between the midand the hindgut. it is reported to exist in the termitidae as an important character for identification (bignell 1994). in addition, the anatomy of n. bosei worker alimentary canal showed several differences compared to other insect alimentary canals. the hindgut of n. bosei worker was the longest region, which comprised 48% of the total length, whereas 24 and 28% for foregut and midgut, respectively. in most insects, the midgut is the longest part (wigglesworth 1972) as shown in fruit fly bactrocera dorsalis 106   biotropia vol. 13 no. 2,2006 (diptera: tephritidae) (lee et al. 1998; hung et al. 2000) and moth larva, hofmannophila pseudopretella (lepidoptera: oecophoridae) (gerard 2002). in dendroctonus (coleoptera: scolytidae) no difference in length among the fore-, mid-, and the hindgut were found (diaz et al. 2003). high variety value was observed in the outer and inner perimeter ratio (op/ip). in most measurements, ip was higher than op, because large vili were facing to the lumen. however, at the crop, ip was lower than op; this was due to the small vili size at the crop. ip was lower than op which also occurred at the anterior ventriculus as well. this is due to the existence of cardiac valve. muscle thickness in n. bosei worker alimentary canal was varied as well. thin muscle was found in oesophagus, ventriculus, and rectum pouch. this character showed that mechanic digestion was not a dominant process in those regions. thin muscle in oesofagus helps the peristaltic movement to bring the food towards the crop (snodgrass 1935; elzinga 1978). in ventriculus there was no thick muscle which means that the cells did not digest the food mechanically. on the other hand, food had been mechanically digested in the proventriculus. 108 alimentary canal anatomy and histology l. e. hariprabowo et al. the thick muscle layer occurred in the crop, proventriculus, and rectum. food were mixed with saliva and stored in the crop (wigglesworth 1972). thus, the thick muscle layers in the crop were needed to enhance the muscle contraction to bring the food forward. in the proventriculus, the thick muscle layer together with six dentitions in the proventriculus ground the food. moreover, the function of the thick layer in the rectum was to draw the feces out of the colon. in future studies, we need to collect more information about the anatomy and histology of other termite caste ofn. bosei alimentary canal, such as the soldier and reproductive castes. then, one can make a comparative study among those termite castes and determine the conserve canal structure. one can also examines the plasticity of the structure(s), if any, due to the different behaviour and physiology of each termite caste. for example, soldiers termites obtain their food from the workers through trophalaxis. based on the different types of food, do the crop of the soldier termites have the same histology structure as those of the workers? this basic histology study will be fruitful as well for a further researches such as to examine the influence of several pesticide at the digestive canal of worker termites. one can see whether the pesticide is able to change the cells structure and intima layer covered the worker termites gut. conclusions the total length of n. bosei alimentary canal was 13.71+1.28 mm. the canal was divided into fore-, mid-, and hindgut which were 24, 28, and 48%, respectively of gut total length. two types of alimentary canal epithelial cells were found, i.e. the squamous and transitional cells. areas covered with thick muscular tissue were crop, proventriculus, and rectum. proventriculus was characterized with six large dentitions. there was no gastric caeca in n. bosei midgut which commonly occurred in chewing insect. secretory cells were observed at proventriculus and ventriculus regions. cardiac valve was found at the anterior end of ventriculus. area with the largest outer perimeter was the rectum pouch. enteric valve had three internal folds. references ahmadm. 1965. termites (isoptera) of thailand. bull. amer. nat his. suppl., 131: 33-195. bignell, d.e. 1994. soil-feeding and gut morphology in higher termites. in: hunt jh, nalepa ca, editor. nourishment and evolution in insect societes. san fransisco: westview pr. p 131-158. borror d.j., delong d.m. and c.a. triplehorn . 1981. an introduction to the study of insects. 5th ed. new york: cbs college publ. chapman r.f. 1982. the insects structure and function. 3rd ed. cambridge: harvard univ pr. claveland l.r. 1925. the feeding habit of termite castes and its relation to their intestinal flagellates. biol. bull., 48:295-308. diaz e., arciniega o., sanchez l., cisneros r, and g. zuniga . 2003. anatomical and histological comparison of the alimentary canal ofdendroctonus. ann.entomol. soc. am., 96:144-152. dow j.a.t. 1986. insect midgut function. in: tokuda g, nakamura t. murakami r, yamoka i. 2001. morphology of the digestive system in the wood-feeding termite nasutitermes takasagoensis. zool. sci., 18:869-877. elzinga r..j. 1978. fundamentals of entomology. new delhi: prentice hall of india private limited. 109 biotropia vol. 13 no. 2,2006 emerson a.e. 1929. communication among termites. in: wilson eo. 1974. the insect societies. cambridge: the belknap univ pr. feighl d.m. and j.w. hill. 1983. general, organics, and biological chemistry. 2nd ed. edina: burgess publ. gerard p.j. 2002. the digestive system of the keratin-feeding larvae of hofinannophila pseudospretella (lepidoptera: oecophori-dae). new zealand j. zool., 29:15-22. gray p. 1952. handbook of basic microtechnique. philadelphia: the blakiston company. hung c.n., lin t.l. and w.y. lee . 2000. morphology and ultrastructure of the alimentary canal of the oriental fruit fly bacterocera dorsalis (hendel) (diptera: tephritidae)(2): the structure of the midgut. zool. studies, 39: 387-394. hungate r.e. 1939. experiment on the nutrition of zootermopsis (iii) the anaerobic carbohydrate dissimilation by the intestinal protozoa. in: mauldin jk, smythe rv, baxter cc. 1972. cellulose catabolism and lipid synthesis by the subterranean termite, coplotermes formosanus. insect biochem., 2: 209-217. inoue t., kitade o., yoshimura t. and t. yamoka . 2000. symbiotic association with protist. in: abe t, bignell de, higashi m, editor. termites: evolution, sociality, symbioses, ecology. dordrecht: academic publ., p. 275-288. krishna k. 1969. introduction. in: krishna k, weesner fm, editor. biology of termites. vol 1. new york: acad pr., p. 1-17. lange a..b. and i. orchard . 1998. the effects of schistoflrfamide on contractions of locust midgut. peptides 19:459-467. lee w.y., chen m.e. and t.l.lin. 1998. morphology and ultrastructure of the alimentary canal of the oriental fruit fly bacterocera dorsalis (hendel) (diptera: tephritidae)(l): the structure of the foregut and cardia. zool. studies, 37:95-101. mcfarlane. nutrition and digestive organs. in: blum ms, editor. fundamentals of insect physiology. new york: john wiley and sons. mcmahan e.a. 1969. feeding relationship and radioisotope techniques. in: krishna k, weesner fm, editor. biology of termites. volume ke-1. new york: academic pr., p. 1-17. miller a. 1965. the internal anatomy and histology of the imago of drosophila melanogaster. in: demerec m, editor biology of drosophila. new york: hafner publ co., p. 425-439. noirot c.h. and c. noirottimothee . 1969. the digestive system. in: krishna k, weesner fm, editor. biology of termites. volume ke-1. new york: academic pr., p .49-88. ribbands c.r. 1953. the behaviour and social life of honeybees. in: wilson eo. 1974. the insect societies. cambridge: the belknap univ pr. romoser w.s. 1973. the science of entomology. new york: macmillan publ co, inc. sheffrahn r.h. and n.y. su . 1994. keys to soldier and winged adult termites (isoptera) of florida. florida entomologist, 77:460-470. snodgrass, r.e. 1935. principles of insect morphology. new york: mcgraw-hill book company, inc. stuart a.m. 1969. social behaviour and communication. in: krishna k, weesner fm, editor. biology of termites. vol 1. new york: academic pr., p. 193-232. tokuda g., nakamura t., murakami r. and i. yamoka i. 2001. morphology of the digestive system in the wood-feeding termite nasutitermes takasagoensis. zool. sci., 18:869-877. wigglesworth v.b. 1972. the principles of insect physiology. london: chapman and hall. wilson e.o. 1965. chemical communication in the social insects. in: wilson eo. 1974. the insect societies. cambridge: the belknap univ pr. wilson e.o. 1974. the insect societies. cambridge: the belknap univ pr. 110 99.pdf 100.pdf 101.pdf 102.pdf 103.pdf 104.pdf 105.pdf 106.pdf 107.pdf 108.pdf 109.pdf 110.pdf sri-13 mei 2017-387 (lazarus histological).cdr histological analysis of cultured in vitro coconut endosperm lazarus agus sukamto* research center for biology, indonesian institute of sciences, bogor 16911, indonesia received 2 april 2014/accepted 4 november 2016 abstract coconut is a very important plant for the livelihood of people in tropical countries. it is also used as an icon of tropical region. coconut fruit is very heavy and can cause injuries if the fruit falls down and hits somebody who happens to be underneath a coconut tree. in order to avoid the accident, the coconut fruits have to be regularly cut off. coconut tree originated from in vitro cultured endosperm is a triploid plant that produces seedless fruit (without endocarp). coconut fruit without endocarp is not heavy. the objective of this study was to investigate plant regeneration of fresh and in vitro cultured coconut endosperms. the fresh and developed in vitro cultured coconut endosperms were observed using histological analysis. solid endosperm of seven month-old postanthesis coconut from “samoan dwarf ” cultivar was freshly picked up and cultured in vitro on modified branton & blake formula. histological study of fresh coconut endosperm showed that the endosperm consisted of parenchyma cells, which were relatively uniform in shape and size, with some nuclei consisted of 1 – 5 nucleoli. three month-old calli of in vitro grown coconut endosperm in semi solid media showed that its cells varied in shape and size, characterized by high nucleus to cytoplasm ratio, high starch, protein and lipid contents which underwent many divisions. seven month-old calli of in vitro grown coconut endosperm in liquid media showed embryogenic cells which resembled proembryos. fourteen month-old bud-like structure of coconut endosperm in semi solid media showed a meristematic layer, tunica-corpus structure, cortex-like region and tracheids of xylem. these results indicated that the bud-like structure was an early stage of shoot bud formation in coconut endosperm. this is the first report of early stage of shoot bud formation occurring on coconut endosperm cultured in vitro. keywords: bud-like structure, calli, coconut endosperm, cultured in vitro, histological analysis introduction coconut is one of the most important palm crops in the world because all parts of coconut plant are very useful for humankind. coconut is planted by 10 million small-holder farmers on 12 million ha of land in the world (nguyen . et al 2015). the decrease of coconut production is mainly caused by palm senescence and diseases; therefore, there is a great demand of coconut plant having high productivity and resistance to diseases (sandoval-cancino . 2016). coconut et al tree is a tall tree which fruit weighs about 1.44 kg. coconut fruit consists of exocarp, mesocarp and endocarp parts. the exocarp and mesocarp parts make up the husk of coconut fruit which is very light in weight. the endocarp part (seed) of coconut is heavy and contains shell, nut, embryo and water. endosperm is a triploid tissue. coconut plant originated from endosperm is a triploid plant and produces seedless fruits. seedless fruit of coconut does not have endocarp and thus, it is not heavy. coconut tree should be maintained by regularly cutting off the fruits, which is quite costly. therefore, triploid coconut tree will be more economical to maintain. coconut (cocos nucifera l ) is a long-lived plant . with 3 5 years juvenile phase. coconut is generally cross-pollinated and very heterozygous (sukamto 2011a). studies on the development of coconut cultured in vitro was reported for the past sixty years, mostly using embryos (immature unselected plants). coconut remains a recalcitrant species that reflected mainly in the slowness of in vitro morphogenesis. there is a difficulty to biotropia 4 1 7 1 8 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 1 387 * corresponding author: lazarus.agus@gmail.com 1 monitor the ability of the meristematic cells of coconut to actively divide; coconut also has heterogeneous response of various explants (sandoval . 2003; fernando 2010). before their et al differentiations are visually recognizable, many changes occur at the cellular and tissue levels. cells could undergo a series of orderly divisions, to form callus, proembryo, embryo and bud-like structure. cell aggregation consists of small, isodiametric, thin-walled, micro-vacuolated cells, highly basophilic, densely staining nuclei and cytoplasm (thorpe & murashige 1970; reinert et al. 1977). they may also contain numerous starch grains or lipid deposits (thorpe & murashige 1970; ross . 1973; villalobos . et al et al 1985; arnold & hakman 1988; kanchanapoom & tinnongjig 2001; liu 2013), and produce proembryos that may develop to become embryos, organs or vascular tissues (reinert . et al 1977; thorpe 1978). few studies were conducted on coconut endosperm culture, which only produced low callus and failed to develop further morphogenesis (fisher & tsai 1978; bhallasarin & bagga 1983; kumar . 1985). recent et al improvements have been achieved using endosperm explants and they are succeeded to obtain quicker and higher callus formation, also embryo-like structures (sukamto 2011b). the calli formed an embryo structure after 21 weeks of culture and grew to become bud-like structure after 1 month-old of culture, showing three 4 protuberances on the top. this study was aimed at understanding morphogenesis at cellular and tissue levels using histology of fresh coconut endosperm and cultured coconut in vitro endosperm. materials and methods plant material solid endosperm of seven month-old postanthesis coconut from “samoan dwarf ” cultivar was picked up freshly from coconut palm trees in oahu island, hawaii. the study was conducted at tissue culture laboratory, horticulture department of hawaii university at manoa, usa. the fresh cuts of solid endosperm were used as control of plant materials for histological analysis. the solid endosperms were aseptically cored with cork borer and scooped with a sterile spoon inside a laminar air flow cabinet. cylindrical endosperm having 8 mm diameter and 4 mm thickness, used as explants, were grown on various media treatments. the endosperm formed callus after seven weeks of culture. three month-old calli cultured in semi solid medium, seven month-old calli cultured in liquid medium and 14 month-old bud-like structure (10 x 8 mm in size) cultured in semi solid medium, were used for histological analysis. culture media the culture media were a modification of branton and blake formula (branton & blake 1986) with addition of 10 mg/l putresine, 2.50 g/l activated charcoal (ac), 1.70 g/l phytagel, 0, -6 -5 -4 -3 10 , 10 , 10 , 10 m 2,4-dichlorophenoxyacetic acid (2,4-d) or 4-amino-3,5,6-trichloropicolinic -5 acid (picloram) combined with 10 m 6benzylaminopurine (ba) at 16 woc. the ph of the media was adjusted to 5.70 before being autoclaved. the media were poured into 2.5 x 15 cm test tubes (having volume of 14 ml) and were o 2 autoclaved at 121 c and 1 kg/cm for 15 minutes. media were stored for one week before being used. single explant was placed in a test tube with the uncut surface upright. cultures were o incubated at approximately 31 c in a dark room. data interpretation fresh solid endosperm were thinly cut with a sharp and clean razor blade, stained with toluidine blue, then covered with a cover glass. the specimens were examined and photographed using zeiss photomicroscope. plant materials of calli and bud-like structure derived from in vitro cultured coconut endosperm were obtained using protocol developed previously by sukamto (2011b). several three month-old calli cultured in semi solid medium were cut with a sharp and clean razor blade and put inside a petri dish containing a small volume of glutaraldehyde fixative (2.75% w/v). thin tissue sections (1 – 2 mm) were vacuumed two times; 1 hour for each vacuum session. tissues were washed three times with sorensen phosphate buffer for 10 minutes each time and stored in refrigerator overnight. the tissues were fixed with 2% osmium tetroxide and 2 biotropia vol. 24 no. 1, 2017 a combination of toluidine blue and acid fuchsine, feulgen-fast green stained dna and counterstained against light green, or periodic acid schiff (pas) stained polysaccharides. acid fuchsine stained most cells components, especially mitochondria; toluidine blue was used as a counter stain; pas was used to stain carbohydrates (gurr 1965). results and discussion fresh coconut endosperm and calli cultured in semi solid medium fresh coconut endosperm from young coconut fruit consisted of parenchyma cells, which were relatively uniform in shape and size (fig. 1a). its nuclei had one to five nucleoli (fig. 1a, 1b). the similar result was reported by popielarskakonieczna . (2013) in cereal endosperm. et al after being cultured , the cells grew into in vitro various shape and size. the results of this study showed that calli of the three month-old culture had darker at the right side compared cytoplasm to those at the left side (fig. 1c). similar result was reported by fernando . (2003) in coconut et al plumule cultured and by popielarska-in vitro konieczna . (2013) in cereal endosperm et al cultured . the dark areas of cytoplasm in vitro contained accumulation of lipid bodies and dense cytoplasm (fig. 1d). this result agreed with ceniza . (1992) who studiedet al fatty acids in callus of in vitro culture coconut endosperm d . the majority shape of lipid bodies were rod-like shape and the others were droplet/globular shape, which were similar to study results obtained by liu (2013) on ripe chili pepper fruits. this study also showed that intercellular spaces of callus tissues were connected with fibrous and reticular structures. similar result was reported by popielarska . (2006) in endosperm-derived et al callus of kiwifruit. in regards to the light area of cytoplasm, the study showed that the cytoplasm had fewer lipid bodies and more frequent cell divisions in many planes; some nucleoli appeared long (fig. 1e). formation of four-cell proembryo structures developed from coconut endosperm culture (fig. 1f) was similar to proembryo formed by segmenting division of coconut inflorescence culture (verdeil . 1994)et al . washed three times with 0.2 m sorensen phosphate buffer at ph 7 – 7.4 for 20 minutes each time. specimens were dehydrated in a graded series of ethanol concentrations (10, 20, 30, 40, 50, 60 and 70%) for 15 minutes in each ethanol concentration. specimens were immersed overnight in 70% ethanol inside capped vials which were stored in refrigerator. the specimens were furthermore dehydrated with graded series of ethanol concentrations (70, 80, 90, 95 and 100%) for 30 minutes in each ethanol concentration. the dehydrated specimens were transferred into capped vials containing 5 ml of 100% ethanol and 1 ml historesin (hr), without using any transition solutions and were mixed by swirling. these specimens were then stored overnight at room temperature. hr was added 3 ml in day 1, 3 ml in day 2 and 4 ml in day 3. upon completing those steps, the specimens underwent two times changes of 100% hr at 5 ml each time. the specimens were then sectioned with a rotary microtome at 5 – 10 µm. several seven month-old calli cultured in liquid medium were directly stained as samples. cell suspensions were transferred using pipette onto a glass slide. the sample was stained using a few drops of 0.25 – 0.5% potassium iodide-iodine (iki) and was then covered with a cover glass (berlyn & miksche 1976). the specimens were examined and photographed with a zeiss photomicroscope. an embryo structure of the 21 week-old culture grew to become bud-like structure after 14 month-old culture. the bud-like structure was cut longitudinally into three parts which were immediately fixed in a 50 ml mixture of 1% acrolene, 2% glutaraldehyde, 2% para formaldehyde and 0.05 m sodium cacodylate buffer at ph 7.6. this specimen was vacuumed twice for 2 days each time. after this process, the fixative agent was decanted; the tissue was washed three times with 0.05 m cacodylate buffer for 30 minutes each time and then stored in the refrigerator for three days. the specimens were transferred to peel-a-way molds containing embedding medium. the containers were then closed to exclude oxygen and vacuumed overnight. polymerized blocks were glued on wood blocks. the embedded samples were then sectioned with a rotary microtome at 5 – 10 µm. the sections were stained with 1% toluidine blue, 3 histological analysis of cultured coconut endosperm – sukamtoin vitro calli cultured in liquid medium seven month-old calli cultured in liquid medium showed development of proembryo structure from coconut endosperm (fig. 2a). the proembryo resembled the embryo-like structure (els) in soybean anther culture (rodrigues et al. 2005). proembryo composed of embryogenic cells characterized by high nucleus to cytoplasm ratio, high starch and protein reserves (fig. 2b, 2c, 2d). this result resembled to embryo and callus formation of pollen grain culture of coconut (perera et al. 2008). formation of proembryo structures could happen without suspensor (fig. 2e) or with suspensor (fig. 2f). suspensor of proembryo was important to increase embryo survival on the development of early globular embryos (paolicchi et al. 2003). bud-like structure cultured in semi solid medium an embryo occurred on callus treated with -6 picloram10 m after 21 weeks of culture (fig. 3a). the embryo grew to become bud-like structure after 14 months of culture (fig. 3b). histology of bud-like structure showed an apical meristem tip between cotyledonary notch, vascular strand and dermal layer (fig. 3c). this structure was primary meristematic state of apical meristem, tended to organize shoot primordial of coconut embryo development (guzman et al. 4 figure 1 fresh endosperm cells shows: a). relatively uniform shape and size (bar = 4 µm) and b). nuclei consists of 4 – 5 nucleoli (bar = 10 µm). fragmentation of the three month-old calli consists of c). dark and light areas (bar = 10 µm); d). dark area composed of cells with dense cytoplasm and a lot of lipid components (bar = 2 µm); e). light area composed of highly vacuolated cells composed of many divisions of cells with few lipid components (l) (bar = 2 µm); f). formation of proembryos structures with several number of cells (bar = 20 µm) biotropia vol. 24 no. 1, 2017 a b c d e f 1978). it resembled shoot bud proliferating on surface of pinus eldarica (wagley et al. 1987) and revealed a well-structured shoot meristem towards whole coconut plant (verdeil et al. 1994). there were tracheids of xylem, inside of vascular strand and lipid droplets in the bud-like structure (fig. 3d). similar lipid droplets were reported by kanchanapoom & tinnongjig (2001) in embryoid cells of oil palm and liu (2013) in plastoglobular shape of chili pepper fruits. in this study, somatic embryogenesis occurred on the cultured coconut endosperm. many protuberances were visible in the mantle, one of which resembled early stage of proembryo (fig. 3e). similar structure was reported by dawayati et al. (2012) in embryonic callus of date palm. the peripheral cells, especially the protuberances, differed from cells in the adjacent internal region. the mantle cells were smaller, had denser cytoplasm and had prominent dark nuclei. this result was similar to cells of leaf and hypocotyl cultures of torenia fournieri and anagallis arvensis, which destined to become embryos, buds and shoot apices (reinert et al. 1977). this study revealed that the protuberance had one layered tunica covering a large group of irregularly arranged cells which could be the corpus (fig. 3f). this was found in regenerated shoot bud, which was similar to organogenesis in leaf-derived callus of black pepper (sujatha et al. 2003) and endospermderived callus of kiwifruit (popielarska et al. 2006). this tunica-corpus resembled bud meristem in foeniculum vulgare callus (anzidei et al. 1996). 5 a a b c e f d figure 2 a) formation of proembryo composed of seven cells (bar = 4 µm); b). formation of cells containing starch grains (bar = 4 µm); c). formation of proembryo composed of seven month-old calli cultured in vitro in liquid medium (bar = 4 µm); d). formation of proembryonal cell complex composed of several proembryo units, showing segmented division (bar = 2 µm); e). formation of three-celled structures of proembryos cultured in liquid medium (bar = 4 µm); f). formation of five-celled structures of proembryo with suspensor in liquid medium (bar = 2 µm) histological analysis of cultured coconut endosperm – sukamtoin vitro histological analysis of bud-like structure of coconut endosperm showed clearly that there were a meristematic layer with a dermal layer, cortex-like region and central vascular tissue. it was likely that shoot organogenesis occurred in the endosperm calli of coconut. this is potentially important because caulogenesis has not been reported yet in coconut endosperm culture. these results are encouraging and warrant further studies to get plant regeneration. the plant is traditionally propagated by seed and is general ly cross pol l inated. therefore, micropropagation is needed in order to get the best genotype with abundant offspring. the micropropagation can also support the easy exchange of coconut germplasm. although coconut micropropagation from various tissues had been attempted, it was mostly hindered by embryo tissue that was not true to type (fernando & gamage 2000; sukendah & cedo 2005; ake . 2007; saenz . 2010; koffi . et al et al et al 2013; muhammed . 2013). it is more difficult et al to have successful tissue culture of adult coconut explants, especially on inflorescence (blake & euwens 1980; sandoval-cancino . 2016), stem et al apice (blake & eeuwens 1982) and leaf (raju . et al 1984; buffard-morel . 1992; verdeil . et al et al 1994). 6 a b c d e f t l figure 3 a). embryo structure occurred after 21 weeks of cultured (bar = 1 mm); b). embryo grew to became bud-like structure (bar = 1 mm) after 14 months of culture; c). cross section of the bud-like structure showing cotyledonary notch (s) and vascular strand (v) toward apical meristem (t) (bar = 30 µm); d). enlargement of vascular strand showing parenchymatous cells, tracheids (t) of xylem and lipid droplet (l) (bar = 10 µm); e). somatic embryogenesis on meristematic area showed globular proembryo (e) (bar = 10 µm); f). protoderm layer of tunica (t) and corpus (c) in meristematic area (bar = 50 µm) biotropia vol. 24 no. 1, 2017 conclusions solid endosperm of seven month-old postanthesis coconut could produce calli, proembryos and bud-like structure. histological analysis showed that the calli composed of highly vacuolated cells and small cells with dense cytoplasm, high nucleus to cytoplasm ratio, high starch, protein and lipid. calli cells underwent many divisions and formed proembryo structures. the proembryo structure grew to become budlike structure, which showed cotyledonary notch, tunica-corpus structure and central vascular strand. these results indicated that 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3 page 4 page 5 page 6 page 7 page 8 microsoft word 75 biotropia vol. 13 no. 2,2006 : 75 84 preovulatory changes and ovulation in cattle undergoing spontaneous or cloprostenol-induced luteolysis bambang purwantara1'3, rene h0ier2, mette schmidt2, and torben greve2 1 department of veterinary clinic, reproduction and pathology, faculty of veterinary medicine, bogor agricultural university, darmaga 16680 bogor, indonesia ²department of clinical studies, section reproduction royal veterinary and agricultural university, bulowsvej 13, 1870 frederiksberg c. denmark ³seameo biotrop, jl. raya tajur km 6, p.o. box 116bogor, indonesia abstract the follicular population, diameter of the ovulatory and subordinate follicles, corpus luteum (cl) size, concentration of progesterone and estradiol-17p were studied following spontaneous or cloprostenol-induced luteolysis. a total of 14 heifers received cloprostenol treatment on day 9-11 of the cycle to synchronize their estrus. subsequently, they were divided into two groups, one group which was allowed to undergo spontaneous luteolysis and the other group in which estrus was induced from days 9 to 12. in the induced-group, transrectal ultrasonography were performed daily started two days prior to injection until the onset of estrus. in the spontaneous-group, ultrasonography was done daily from day 15 until the onset of estrus. in both groups scanning were performed every 4 h from the onset of estrus until ovulation was ascertained. small (sf, 2-4 mm), medium (mf.5-9 mm ) and large (lf,>9mm) size follicles were recorded. the diameter of largest and subordinate follicles were measured and blood were drawn from jugular vein at approximately around scanning and the plasma were used for measurement of progesterone (p4) and estradiol-17p (e2) concentration. there was no different in term of number of sf, mf and lf (p>0.05) between the two groups. similarly, no effect of side (left vs. right ovary) and cl position (ipsivs contralateral to the ovary) was found (p>0.05). however, it was demonstrated that mean number of ovulatory follicles was higher (po.01) in the spontaneously ovulating group while the regressing-cl size was larger in the cloprostenol induced animals (p<0.05). occurrence of time of ovulation in relation to initial signs of estrus was observed in both groups of animals. this variation could be attributed to the existence of a large preovulatory follicle which enhanced the time ovulation. conversely, when subordinate follicles showed grow up and replace the large follicle the interval from heat to ovulation was prolonged. progesterone experienced a more steep decrease than the spontaneous group of animals and a positive correlation was observed between the diameter of cl and the p4 concentration. for e2 there was a positive correlation between the e2 and follicular size. it is concluded that the variation in follicular among animals contributed to the variability in timing of ovulation, particularly prostaglandin-induced animals. the diameter of ovulatory follicle in spontaneous group was larger as compared to inducedgroup. key words : follicular development, ovulation, cloprostenol,cattle reproduction introduction preovulatory follicular development and its regulation in cattle has so far remained unclear (assey et al. 1993; lindsay et al. 1996; ireland et al. 2000; evan 2001; burn et at. 2005). detailed studies of the pattern of follicular development during the estrous cycle has been based on slaughter house material (rajakoski 1960; dufour et al. 1972) and ultrasonography (pierson and ginther 1989a; pierson corresponding author: b.purwantara@biotrop.org biotropia vol. 13 no.2,2006 and ginther 1989b; fortune et al. 1988; rivera et al. 2001; ginther et al. 2001; webb et al. 2003) however, the processes by which the ovulatory follicles is selected is still not clear (staigmiller and england 1982; pierson and ginther 1988 and quirk et al. 1986; mihm et al. 2000; evan 2003; kobayashi et al. 2006), although india ink marking of preovulatory follicles combined with cauterization of certain larger follicles (matton et al. 1981) has improved the understanding of the underlying mechanisms. the development of clinical ultrasonogaphy (quirk et al. 1986; pierson and ginther 1989a; pierson and ginther 1989b; savio et al. 1988; sirois and fortune 1988; purwantara et al. 1993; purwantara et al. 1994; ginther et al. 2001; frike et al. 2002) which allows visualization of ovarian follicles over time, has given even a better insight. it is, however, still an open question when the follicle destined to ovulate can be recognized and how this affect the subordinate follicular population. the timing of ovulation in cattle has in most studies been determined in spontaneous non superovulated cows/heifers either by rectal palpation (schams et al. 1977) or by ultrasonography (larsson 1987), and in superovulated cattle (savio et al. 1990; fortune et al. 1991; purwantara et al. 1994). however, no detailed studies are available concerning a possible relationship between the occurrence of ovulation and preovulatory hormonal parameters (progesterone and estradiol 17p in neither spontaneous nor prostaglandin induced luteolysis and ovulation. the aim of this study was, therefore during the preovulatory periods in spontaneous vs. induced luteolysis (1) to monitor the development of ovulatory and non-ovulatory follicles at various times prior to ovulation (2) to characterize the regression of the corpus luteum, and (3) to determine progesterone (p4) and estradiol (e2) profiles and relate those to the time of ovulation. materials and methods animals fourteen heifers of mixed beef cattle breed between 2-3 years of age and weighing between 300-500 kg were used in the experiment. the animals had normal estrous cycles at least once prior to the study. subsequently, the animals were divided equally into two groups, the spontaneous-group and the induced-group. spontaneous-group: following induced estrus using a single injection of cloprostenol (estrumat vet., cooper animal health ltd., denmark) and ovulation, the animals were left alone for approximately 19-20 days at which time close observation for a spontaneous heat was performed three times daily. induced-group: on day 9-12 of estrous cycle (day estrus = day 0) the heifers received a single injection of cloprostenol (estrumat vet., cooper animal health ltd., denmark) to induce estrus and ovulation. 76 preovulatory changes and ovulation in cattle b. purwantara et al. ultrasonography a real-time b-mode diagnostic ultrasound equipments (concept ultrasound scanner, dynamic imaging ltd., uk) equipped with a linear array 7.5 mhz transrectal transducer was used. detailed procedures of the ultrasonographic examination of the ovaries were performed as described earlier by purwantara et al. (1994) and the schedule was as follows: in the spontaneous-group, scanning was performed daily from day 15 of the estrous cycle until onset of heat and then every 4 h. in the induced-group daily ultrasonographic scanning were done two days prior to cloprostenol injection and until the onset of heat. from the onset of heat and until ovulation was confirmed the animals were scanned every 4 h. the number of small (sf, 2-4 mm), medium (mf, 5-9 mm), and large (lf, >10 mm) size ovarian follicles were counted and the diameter of the largest, subordinates and the diameter of corpus luteum were measured by using built-in integral caliper. corpus luteum integrity were scored as 3 (highly intact), 2 (moderate) and 1 (less intact, difficult to discriminate) depending on the gray shade and the demarcation line between the corpus luteum and the stroma. each ovarian scan was recorded by means of video recorder and diagram of each ovary were drawn. individual preovulatory and its subordinate follicles were closely examined. ovulation was defined as the disappearance of the largest non-echogenic area (dominant follicles) between two consecutive examinations. blood sampling and hormone measurement daily blood sampling were drawn from jugular or coccygeal vein using heparinized vacuum tube. blood plasma was separated by centrifugation at 3000 rpm for 15 min and stored at -20°c until analysis was performed. progesterone (p4), estradiol-17p (e2) and lh concentration were analyzed in all samples by ria according to h0ier (1989), respectively. the interand intra-assay coefficient of variance (cv) of p4 measurement was 3-10% and 715% respectively, and <10% for e2 depending on the position of displacement of curve. the sensitivity (least detectable concentration) was 0.25 ng/ml and 4 pg/ml for p4 and e2 measurement, respectively. statistical analysis data were analyzed using general linear model for repeated measurement analysis of variance of statistical analyses system (sas 1986). the effect of group (spontaneous vs. induced) and day on mean number of sf, mf, lf and total follicle (tf) were determined. the similar effect were examined on the diameter of ovulatory follicle and its subordinate, corpus luteum diameter, progesterone (p4) and estradiol-17p (e2) concentration. regression analysis and pearson correlation coefficient were determined to relate cl diameter and p4. correlation analysis were also performed to examine relationships between the occurrence of ovulation and the rate of cl regression and the changes on diameter of ovulatory follicle. 77 biotropia vol. 13 no.2, 2006 results and discussion occurrence of estrus was subject to a great deal of individual variation but it was in general observed on between day 18 and 23 of the estrous cycle in heifers undergoing spontaneous luteolysis and on the third day after cloprostenol injection. in the spontaneous-group, the interovulatory interval ranged from 19 d 2 h to 23 d 10 h. in the induced-group, ovulations occurred from pg-injection, ranging from 64 (62-66) h to 126 (124-128) h. in 4 animals where the lh surge was clearly identified, the interval between lh surge to ovulation ranged from 24 to 26 h. a pronounced individual variation occurred in terms of the time which lapsed between prostaglandin injection (induced-group) or cl regression (spontaneous-group) and subsequent ovulation. it is anticipated at least in the induced-group that the stage of follicular development plays an important role on these differences. savio et al. (1990) and ginther et al. (2001) observed that the majority of ovulation originated from the follicles which was dominant on day 7 at the time when prostaglandin was injected. moreover, fortune el al. (1991) and evan (2003) confirmed that the majority of the dominant follicle undergo ovulation when luteolysis was induced during the growing or early plateau phase. in contrast, when prostaglandin was injected during the late plateau phase or atresia phase, the largest follicles failed to ovulate and its subordinate became the ovulatory follicles. in two heifers ovulating within 62-66 h (<3 days) after cloprostenol injection, the ovulatory follicle was generally not difficult to determine as a growing larger follicle where the subordinate had diameters less than 5 mm during the last 3 days prior to ovulation. no other large and medium size follicle were detected in these animals, the follicle which became the largest follicle during the 3 days prior to ovulation. this follicle underwent ovulation while the other became atretic. the other two of these late-ovulating groups developed two large follicles >10 mm which both increased in size until ovulation. ovulation occurred from the follicles which remained the largest during the last 5 days prior to ovulation. in the animals where ovulation occurred within 3 and 4 days after cloprostenol injection shift in dominance was observed only in one animal, while in the remaining, the dominant ovulatory follicles suppressed their subordinates which underwent atresia. evidence from this study showed that a shorter interval between injection of cloprostenol and ovulation occurred when a single large follicle with no medium size subordinate follicles were present during the period of luteolysis. in contrast, the interval was longer when the dominant follicle had to be or was replaced by one of the subordinate or when more than two large follicles developed during the phase of luteolysis. this is in agreement to larsson (1987) who found an increase in the length of proestrus period when 2 follicles greater than 10 mm in diameter were present in that period. some developmental changes during the transitional phase from early to late plateau phase of growth, characterized by a loss in the ability of the dominant follicle to ovulate may contribute to this variation (sirois and fortune 1990; ginther et al. 2001; evan 2003; burn et al. 2005). ovulation was not dependant upon the side. ovulations occurred on the right ovary in 55 % of the induced-group (n=ll) and in 56 % of the spontaneous-group 78 preovulatory changes and ovulation in cattle b. purwantara et al (n=14). the trend was not statistically significant (p>0.05). ovulations occurred ipsi-lateral or contra-lateral to the corpus luteum in 55% or 45%, respectively of the spontaneous ovulations and 64% or 36%, respectively of the induced ovulations. one heifer developed two large size growing follicles on the same ovary and they both ovulated. these ovulations occurred within a 4 h interval and no extended preovulatory period was observed. no significant difference were demonstrated on the mean number of sf, mf and lf between the groups (p>0.05). it was noticeable as seen on fig. 1 (a), that the ovulatory follicle in the spontaneous-group attained a larger mean diameter than the ovulatory follicles in the induced-group. mean diameter of ovulatory follicle was different between group (p<0.01) and day (p<0.01). in the spontaneous-group as seen on fig. 1 (b), the mean diameter of subordinate follicles remained fairly stable until 2 days prior to ovulation at which time they decreased, a pattern which was contrary to the induced group the interovulatory interval was subject to a wider variation in the group undergoing spontaneous ovulation. perhaps, the number of waves has influenced this variation. it was reported by ginther et al. (1989a) that the length of the interovulatory interval was in average of 2.4 days longer in 3-wave than in 2-wave cycles. other studies on the other hand (savio et al. 1988; sirois and fortune 1988) were unable to find differences in the interovulatory interval between the 2-wave and 3-wave pattern animals. since this study was limited to the preovulatory period, these contradictory findings could not be confirmed. in this particular period, however, it was reported that the length of interval from luteal regression to ovulation (ginther et al. 1989a; ginther et al. 1989b) and growth of follicle during luteolysis (savio et al. 1988; sirois and fortune 1988) did not differ between 2-wave and 3-wave pattern animals. the number of small, medium and large size follicle was not different between the two groups and days. this indicated a similar pattern of folliculogenesis in spontaneous and induced ovulations. in addition, during luteolysis no major changes 79 biotropia vol. 13 n0 .2, 2006 in follicular population was observed except the disappearance of ovulatory follicles at the time of ovulation. no ovulation occurred when the diameter of follicle was less than 10 mm. the larger mean diameter of ovulatory follicles which was observed in spontaneous-group is not in agreement to quirk et al. (1986) who found no significant difference of ovulatory follicle development in spontaneous vs. cloprostenol-induced ovulation. this emphasizes that the size alone is not a good indicator of readiness to undergo ovulation. an interesting pattern was observed concerning the mean diameter of subordinate follicles in the spontaneous and induced-group. in the spontaneous group, the mean diameter remained stable until proestrus and then decreased dramatically, while in the induced -group the subordinate follicles tended to increase in size until estrus and then decreased slightly. it is speculated, therefore, that the ovulatory follicles in the spontaneous-group had exhibited a clear dominancy several days before estrus, while those in the induced-group was not selected until the latest stage of the cycle. in this study ovulations occurred with an equal frequency on left and right ovary. this is different from previous studies (rajakoski 1960; pierson and ginther 1989b; purwantara et al. 1992) which reported that ovulations occurred more frequently on the right than the left ovary. in addition, we found no ipsior contralateral effect of cl on ovulation in the spontaneous-group and only a slightly greater number of ovulation from ipsilateral cls in the induced-group. this is conflicting to rajakoski (1960) who found a significant greater number of ovulation occurred from cl bearing ovary. as depicted in fig. 2 (a), the mean diameter of corpus luteum was affected significantly by the group (p< 0.01) and the induced-group maintained a higher diameter. a similar trend was indicated on the effect of day (p<0.01). 80 the cl was scored as 3 at the day of cioprostenol injection, then it changed to 2 sometime between the second and third days and reached score 3 at the day or 1 day before -ovulation. in the spontaneous group score 3 existed 5-4 days before ovulation, then attained score 2 during the following 2-3 days and reached score 1 at 1-2 days prior to and until the day of ovulation. the mean concentration of p4 decreased following cioprostenol injection as seen on fig. 2 (b) and more dramatically in the induced-group. however, in some heifers, p4 concentration did not decline to reach a level under 1 ng, which is defined as the level compatible with luteal regression. on the other hand, the concentration of e2 increased and reached a maximum level 48 h prior to ovulation, and then decreased (fig. 3). a positive correlation was found to exist between p4 concentration and cl diameter during the regression period (r=0.51, po.01). similarly was plasma p4 and cl integrity (r=0.49, p<0.01), and cl diameter and cl integrity (r=0.86, po.01). the mean diameter of the regressing cl was larger in the induced-group compared to spontaneous-group and although subject to great variation, the concentration of p4 was also greater in inducedgroup until 3 days prior to ovulation. it is interesting to note that the dramatic decrease of p4 in the induced-group between day 5 and day 3 prior to ovulation was not accompanied with a similar trend in the decrease of cl diameter, but rather related to the cl integrity. we speculate that the decrease of p4 concentration during luteolysis in the induced-group occurred ahead of decrease in cl diameter, and was different from the spontaneous luteolysis. concentration of e2 reached the peak 2 days prior to ovulation or about 1 day before the lh surge which it is in agreement to other studies (harrison et al. 1985; dutchen et al. 1994; rhodes et al. 1995; singh et al. 1997; valdez et al. 2005). conclusions it was concluded that the timing of ovulation following luteolysis varied between animals both in spontaneous and inducedgroup. this variation was predominant in the induced-group and it was shorter when the single large 81 biotropia vol. 13 no.2,2006 preovulatory follicle existed, and tended to occur later when a subordinate follicle had to replace a large follicle and became the preovulatory follicle. no effect of side (left or right ovary) and ipsior contralateral position of cl to the ovulatory follicle was found. population of small, medium and large size follicles was not different between induced and spontaneous-group. however, mean diameter of ovulatory follicle was larger in spontaneous compared to induced-group. moreover, the diameter of subordinate follicles and cl regression varied between the two treatment groups. acknowledgments this study was supported by animal biotechnology research center, royal veterinary and agricultural university copenhagen denmark and danida. the authors also would like to thank mr. ben s01oy for providing assistance for the hormonal analysis. references assey, r.j., b. purwantara, t. greve, p. hyttel and m. schmidt. 1993. corpus luteum size and plasma progesterone levels in cattle after induction of luteolysis. theriogenology, 39 (6): 1321-1330. burns, d.s., f. jimenez-krassel, j.l.h. ireland, p.o. knight and j.j. ireland. 2005. numbers of antral 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functional correlates. j. reprod. fertil., 109:35^4. sirois, j. and j.e fortune. 1988. ovarian foilicular dynamics during the estrous cycle in heifers monitored by real-time ultrasonography. biol. reprod., 22. 308-317. staigmiller, r.b. and b.c. england. 1982. folliculogcnesis in the bovine. theriogenology, 17: 43-52. 83 biotropia vol. 13 no.2, 2006 valdez k.e., s.p. cuneo, p.j. garden and a.m. turzillo. 2005. the role of thecal androgen production in the regulation of estradiol biosynthesis by dominant bovine follicles during the first folicular wave. j anim. sci., 83: 597-603 webb, r., b. nicholas, j.g. gong, b.k. campbell, c.g. gutierrez, h.a. garverick and d.g. armstrong. 2003. mechanism regulating follicular development and selection of dominant follicle. reprod. suppl., 61:71-90. 84 75.pdf 76.pdf 77.pdf 78.pdf 79.pdf 80.pdf 81.pdf 82.pdf 83.pdf 84.pdf 5. dwi retno (arbuscular).cdr biotropia vol. 20 no. 2, 2013: 112 121 arbuscular mycorrhizal fungi associated with ( )bisbul diospyros blancoi dwi retno ningsih , kartini kramadibrata , and agustin wydia gunawan received 15 september 2013/accepted 10 december 2013 ( ) is a kind of edible fruit and could be beneficial as inhibitor for skin ageing process. majority of root plants have symbiotic associations with arbuscular mycorrhizal fungi (amf); however, amf in has never been reported. the objective of this study was to observe amf colonies and to identify amf spores in bisbul tree rhizospheres and in potcultured . roots and soil samples from rhizospheres were collected from three locations in bogor area. roots were stained using trypan blue 0.05%. soil samples were air dried, then parts of them were used for spore isolation and the rest were used for pot cultures. spores were isolated by wet sieving and decanting method and then continued with centrifugation. the results showed that the structures of amf colonies in roots were arbuscules, vesicles, coiled hyphae, and internal hyphae. isolated amf spores were identified as , . and . this is the first report of mycorrhizal infection in the root of and amf association with root. am fungi, symbiosis, 1 2 1,3* 1 2 3 department of biology, faculty of mathemathics and natural sciences, institut pertanian bogor, darmaga campus, bogor 16680, indonesia herbarium bogoriense, botany division, research center for biology lipi, jalan raya jakarta-bogor km 46, cibinong 16199, indonesia faculty of biotechnology, atma jaya catholic university of indonesia, jalan jendral sudirman 51, jakarta 12930, indonesia bisbul diospyros blancoi bisbul pueraria javanica bisbul bisbul acaulospora longula, a. scrobiculata, a. tuberculata, claroideoglomus geosporum, funneliformis etunicatum, gigaspora candida, g. ramisporophora, glomus albidum g glomerulatum, scutellospora calospora bisbul bisbul diospyros blancoi abstract introduction key words: at present, researches on the diversity of arbuscular mycorrhizal fungi (amf), which have mutualistic symbiotic associations with fruit trees, are starting to gain attention. one of the fruit plant considered to have economic value is . ( ) is a kind of red fruit indigenous to the philippines and was bisbul bisbul diospyros blancoi * corresponding author : agustinwydiagunawan@yahoo.com doi: 10.11598/btb.2013.20.2.2 112 introduced to bogor botanical garden in 1881. the wood from trees can be used as material for the roof and handicraft. the fruit contains vitamint beneficial to make the skin smooth, maintain healthy eyes, and prevent constipation (coronel 1992). symbioses of amf with fruit plants in indonesia have been reported in salak ( ) (retnaningsih 1998); persimmon ( ), mango ( ), papaya ( ), and soursop ( ) (septyarini 1999); durian ( ) (chairani 2002); rambutan ( ) (muliawan 2002); mangosteen ( ) (lucia 2005); banana ( ) and tomato ( ) (duaja & jasminarni 2008); and jambu-jambuan ( sp.) (setiadi & setiawan 2011). however, there has been no report on amf in tree yet. therefore, the first step to examine the symbiosis of amf in roots is to observe their amf colonies and to identify amf spores in rhizospheres as well as in pot cultures with as the host. the samples of roots and soil were collected from three locations in bogor, i.e. ipb darmaga campus, cibinong, and ciampea. three trees were selected from every location, and sampling was replicated two times for every tree. for each replication ± 500 g sample was taken from each position, which was 100 cm from the tree and at a depth of 15-20 cm. the samples were then mixed to make one composite sample. root samples were immediately cleaned and immersed in 70% alcohol solution, while soil samples were air-dried for spore isolation and pot cultures. amf colonies were observed using trypan blue stain (phillips & hayman 1970) with a modification in cell clearing with 10% koh for 120 min. the structures of amf colonies were observed in 20 randomly picked root cuts. the roots that showed the structures of arbuscules, vesicles, coiled hyphae, and internal hyphae indicated the occurrence of symbiosis between the roots and amf. these mycorrhizal roots were examined to calculate the percentage of am in roots using the following formula : pot cultures with as the host were prepared with planting media as follow: 50 g sterile zeolite, 100 g soil sample from rhizosphere, and then 50 g sterile zeolite as the cover layer. three pot cultures were prepared from each rhizosphere soil sample of every tree, so there were 18 pots. then, the plants bisbul salacca edulis diospyros kaki mangifera indica carica papaya annona muricata durio zibethinus et al. nephelium lappaceum et al. garcinia mangostana musa paradisiaca solanum lycopersicum syzygium bisbul bisbul bisbul pueraria javanica bisbul bisbul p. javanica bisbul bisbul material and methods sampling of roots and soil amf colonies in roots pot cultures bisbul number of fields-of-view containing mycorrhizae total observed fields-of-view × 100% 113 arbuscular mycorrhizal fungi associated with ( ) dwi retno ningsihbisbul diospyros blancoi – et al. were maintained for three months in order to produce amf spores. each pot was watered daily with sterile water. fertilizer containing 5% p was used; the amount of water for each plant was 100 ml. fertilizing was done when the plants were two weeks old, and then repeated every week until they were three months old. after three months, the plants were left to dry for three weeks. . amf spores were isolated from 100 g rhizosphere soil sample for every tree, so the total amount of soil sample for every location was 300 g. likewise for the pot cultures, 100 g was isolated from every pot culture so there was 300 g soil sample for each tree, or 900 g for each location. amf spores were isolated employing wetsieving and decanting method and centrifugation (walker 1982). collected spores were preserved by mounting them on slides in as media. identification was conducted based on morphology, while the nomenclature followed schü & walker (2010). roots are pigmented, so it took up to 120 min to clear the cells using 10% koh in order to observe the internal structures of arbuscular mycorrhizae clearly. the structures of the observed roots were found to be arbuscules, vesicles, coiled hyphae, and internal hyphae (fig. 1) which indicated that the roots had symbiotic associations with amf. the average of colonization was 41% in roots collected from ipb darmaga campus, 49% from cibinong, and 64% from ciampea. bisbul bisbul et al. polyvinyl alcohol lacto glyserol bisbul bisbul bisbul bisbul spore isolation and identification amf colonies in roots βler results and discussion bisbul vesicles coiled hyphae coiled hyphae arbuscules figure 1. arbuscular mycorrhizal structures (arrow) in rootsbisbul . biotropia vol. 20 no. 2, 2013 114 the occurrence of the structures proved that roots had symbiotic association with amf, and that amf spores could be found in the soil of rhizospheres. its close relative, (septyarini 1999), (hawley and dames 2004), and (delvian 2010) were also reported to have symbiotic association with amf. the arbuscular mycorrhizae observed in roots were classified as arum-paris type. different number of spores were isolated from the soil of rhizospheres collected from three locations in bogor (table 1). amf spores collected from rhizosphere soil in ipb darmaga campus had the highest quantity as well as diversity, 17 isolated spores consisted of 7 amf spesies: and . spores in rhizospheres from cibinong were and 2 other species different from those found in darmaga, they were and amf spores found in rhizospheres in ciampea were and bisbul bisbul diospyros kaki d. nigrocartex (zhao et al. 2001), d. scabrida d. pendula bisbul bisbul bisbul acaulospora longula, a. scrobiculata, a. tuberculata, gigaspora ramisporophora, funneliformis etunicatum, claroideoglomus geosporum, glomus glomerulatum bisbul c. geosporum, g. glomerulatum, glomus albidum scutellospora calospora. bisbul acaulospora longula, a. scrobiculata, c. geosporum. amf spores table 1. total spores of arbuscular mycorrhizal fungi (amf) obtained from rhizospheres and pot cultures with as the host bisbul pueraria javanica amf total amf spores darmagaa cibinonga ciampeaa brb pcc brb pcc brb pcc acaulospora longula 1 13 0 16 4 16 acaulospora scrobiculata 4 8 0 16 1 7 acaulospora tuberculata 3 2 0 3 0 4 claroideoglomus geosporum 3 85 2 34 1 48 funneliformis etunicatum 2 3 0 2 0 9 glomus albidum 0 7 3 17 0 24 glomus glomerulatum 2 4 1 6 0 9 scutellospora calospora 0 2 1 6 0 2 total 15 124 7 105 6 123 when the soil from bisbul rhizospheres was used in pot cultures with as the host, the amf species observed in rhizospheres were also found in their pot cultures. this study produced a high quantity of amf spores, so it was possible to conduct the identification in order to confirm the identity of the amf obtained directly from bisbul rhizospheres, and also to trap amf propagules which spores could not be isolated directly from the soil of rhizospheres. p. javanica bisbul gigaspora candida bisbul notes: br: bisbul rhizospheres; pc: pot culture soil ph: darmaga (5.8-6.0), cibinong (5.8-6.0), and ciampea (6.2-6.6) amf spores collected per 300 g soil of br, amf spores collected from 300 g pc media, which was an average of 3 pot culture replications of each bisbul rhizosphere soil in 1 location. a b c 115 arbuscular mycorrhizal fungi associated with ( ) dwi retno ningsihbisbul diospyros blancoi – et al. 1. spain & n.c. shenck the spores were globose to subglobose, yellow to brown in color, measured 90132 × 93-150 μm. spore wall was yellow to brown and without ornamentation. total wall thickness was < 6-9 μm. was successfully obtained from darmaga (ph 5.7-5.9) and ciampea (ph 6.2-6.6). chairani (2002) reported that this species was found in durian in bogor with ph 4.2-6.3 and fahriny (2013) in rhizospheres at ph 5.9. it seemed that this species could survive in a wide range of acidic ph. had similarities in color and form compared with those first described by schenck . (1984). the spore size in this study was bigger compared to that reported by schenck . (1984), (60-)70fahriny (2013), 126-14 × 96-117 μm. in this study, the sporiferous saccule of was not found. schenck . (1984) reported that sporiferous saccule was found at the terminal hyphae and after the spores matured, the sporiferous saccule would be left empty. specimens examined: drn 6, drn 18, drn 19, drn 20, drn 25, drn 26, drn 36, drn 38, drn 39, drn 47, drn 50, drn 51, drn 52, drn 68, drn 70, drn 76, drn 77, drn 80, drn 82, drn 97, drn 98, drn 103, drn 105, drn 109, drn 111, drn 112, drn 113, drn 114, drn 118, drn 119, drn 120, drn 121, drn 122, drn 123, drn 127, drn 132, drn 136, drn 139, drn 144, drn 146, drn 160, drn 171, drn 173, drn 175, drn 176, drn 178, drn 179, drn 182, drn 184, drn 187, drn 191, drn 195, drn 193, drn 194, drn 196 and drn 199. 2. trappe the spores were globose to subglobose, yellow in color, and measured 90-115 × 93-112 μm. spore surface had ornamentation of evenly spaced, linear elliptical crescent-like forms. spore wall was hyaline to yellow. total wall thickness was 6-9 μm. was obtained in darmaga soil at ph 5.7-5.9 and ciampea (ph 6.2-6.6). this species had been reported by chairani (2002) from durian rhizospheres in bogor at ph 4.2-6.5 and fahriny (2013) from rhizospheres at ph 6.7. had the same color, form, and ornamentation as those described by trappe (1977). this spore size was smaller compared with that reported by trappe (1977), 100-240 × 100-220 μm; widiastuti and kramadibrata (1992) 100-200 × 100200 μm; septyarini (1999) 105.6-211.2 × 96-220.8 μm; as well as haerida and kramadibrata (2002) 108.9-144 × 108.9-144 μm; kramadibrata (2009) 90-(130)-250 × 100-(120)-250 μm. however, it was bigger compared to that reported by lucia (2005), i.e. 82-125 × . specimens examined: drn 3, drn 10, drn 20, drn 29, drn 30, drn 33, drn 34, drn 35, drn 37, drn 41, drn 43, drn 44, drn 45, drn 56, drn 65, drn 72, drn 85, drn 99, drn 111, drn 118, drn 119, drn 124, drn 125, drn 127, drn 130, drn 131, drn 132, drn 136, drn 137, drn 140, drn 142, drn 143, drn 144, drn 146, drn 148, drn 149, drn 150, drn 152, drn 156, drn 157, drn 158, drn 159, drn 167, drn 176, drn 178, drn 181and drn 182. acaulospora longula acaulospora longula et al. areca a. longula et al et al a. longula et al acaulospora scrobiculata acaulospora scrobiculata et al. areca a. scrobiculata 90(-110) μm, and 67-120 μm 116 biotropia vol. 20 no. 2, 2013 3. janos & trappe. the spores were globose to subglobose, yellow to brown in color, measured 114272 × 111-272 μm. spore surface had ornamentation of smooth beads, dense, and uniform. spore wall was yellow to brown. total wall thickness was 9-12 μm. was successfully obtained from darmaga (ph 5.7-5.9). chairani (2002) reported the presence of this species in durian in bogor with ph range of 4.2-6.5 and it had the same form, color, and ornamentation as those described by janos and trappe (1982). the spore size was relatively smaller compared to that reported by janos and trappe (1982), i.e. 255-327 × 255-340 μm. likewise, spores reported by septyarini (1999), chairani (2002), lucia (2005) and kramadibrata (2009) were smaller than that of janos and trappe (1982). specimens examined: drn 2, drn 44, drn 55, drn 70, drn 80, drn 101, drn 103, drn 122, drn 124, drn 126, drn 145, drn 150, drn 155, drn 177, drn 180, drn 186, drn 190, drn 192, drn 193 and drn 199. 4. (w.n. becker & gerd.) c. walker & a. schüßler the spores were globose to subglobose, yellow to brown in color, and measured 81-108 × 81-111 μm. spore wall was yellow to brown. total wall thickness was 3-9 μm. (syn./formerly: ) was observed in darmaga at ph range of 5.7-5.9. chairani (2002) was successful in obtaining this species at ph range of 4.2-5.0. this species had the same color and form as those described by becker dan gerdemann (1977). the spore size in this study was relatively the same or bigger compared to that reported by becker dan gerdemann (1977) which was 68144( × kramadibrata . (2007) 48-77(-156) × 48-77(-156) μm and kramadibrata (2009) 150 × 150 . specimens examined: drn 8, drn 10, drn 15, drn 46, drn 56, drn 77, drn 86, drn 87, drn 97, drn 103, drn 107, drn 121, drn 123, drn 153, drn 159, drn 169, drn 170, drn 175, drn 176, drn 178, drn 179, drn 181, drn 184, drn 187, drn 190, drn 192, drn 193, drn 194 and drn 197. 5. (t.h. nicolson & gerd.) c. walker & a. schüßler the spores were globose to ellipsoid, brown to red in color, and measured 75-158 × 72-132 μm. spore wall was brown. total wall thickness was 6-15 μm. was found in all study area, at ph range of 5.7-6.6. these spores had the same color and form as those described by walker (1982). the spore size in this study was smaller than what was reported by walker (1982), which measured 110-190 μm (syn./formerly ). however, the spore size was relatively the same or bigger than lucia (2005) which measured 99-124 × and smaller than kramadibrata (2009) which measured 100 × 280 . specimens examined: drn 4, drn 9, drn 11, drn 12, drn 17, drn 22, drn 23, drn 24, drn 25, drn 26, drn 27, drn 28, drn 29, drn 30, drn 31, drn 32, drn 33, drn 34, drn 35, drn 36, drn 37, drn 38, drn 39, drn 40, drn 42, drn 43, drn 47, drn 48, drn 49, drn 52, drn 53, drn 54, drn 55, drn 56, drn 57, drn 59, drn 60, drn 61, drn 62, drn 63, drn 64, drn 66, drn 67, drn 69, drn 71, drn 72, drn 73, drn 74, drn 75, drn 76, drn 79, drn 81, drn 82, drn 83, drn 86, drn 87, drn 88, acaulospora tuberculata acaulospora tuberculata et al. a. tuberculata et al. claroideoglomus etunicatum claroideoglomus etunicatum glomus etunicatum et al. et al funneliformis geosporum funneliformis geosporum glomus geosporum -162) μm, lucia (2005) 47-107 43-99 μm, μm 92-107 μm μm 117 arbuscular mycorrhizal fungi associated with ( ) dwi retno ningsihbisbul diospyros blancoi – et al. drn 89, drn 90, drn 91, drn 92, drn 93, drn 94, drn 95, drn 96, drn 97, drn 100, drn 101, drn 103, drn 104, drn 106, drn 107, drn 108, drn 109, drn 110, drn 113, drn 114, drn 115, drn 116, drn 122, drn 123, drn 125, drn 128, drn 130, drn 131, drn 132, drn 133, drn 136, drn 137, drn 138, drn 139, drn 140, drn 141, drn 149, drn 150, drn 151, drn 152, drn 153, drn 155, drn 157, drn 159, drn 161, drn 162, drn 165, drn 166, drn 168, drn 169, drn 172, drn 174, drn 175, drn 174 , drn 178, drn 179, drn 180, drn 183, drn 184, drn 187, drn 188, drn 190, drn 191, drn 192, drn 193, drn 194, drn 195, drn 196, drn 197, drn 198, drn 199 and drn 200. 6. bhattacharjee, murkeji, j.p.tewari & skoropad the spores were globose to subglobose, greenish white in color, and 90-225 × 90228 μm in size. spore wall was white. total wall thickness 3-9 μm. bulbous suspensor was white to yellow and 27-42 × 21-39 μm in size. had the same color and form as those described by bhattacharjee and mukerji (1982). the obtained spores were 90-225 × 90-228 μm in size, this is smaller compared to that described by bhattacharjee and mukerji (1982), i.e. 200-300 μm. the suspensor was also smaller in size, compared to that reported by bhattacharjee and mukerji (1982), i.e. 30-50 μm. specimens examined: drn 150, drn 155, drn 170 and drn 171. 7. spain, sieverd. & n.c. schenck the spores were globose to subglobose, brown to reddish brown in color, and 150372 × 165-372 μm in size. spore wall was brown. total wall thickness 6-9 μm. bulbous suspensor was yellow to brown and 27-36 × 21-39 μm in size. only 2 spores of were found in darmaga at soil these spores had the same color and form as those described spain (1989). the spore size was 150-372 × 165-372 μm, still in the same range as that described by spain (1989), i.e. (143-)150-400 × 200-450(-501) × 288-595 μm, and lucia (2005) 182-317 × (1989) sized (32-)40-60(72) × . specimens examined: drn 1, drn 2, drn 126, drn 128, drn 132, drn 133, drn 136, drn 138, drn 139, drn 144, drn 148, drn 149, drn 170, drn 181and drn 185. 8. c. walker & l.h. rhodes the spores were globose to subglobose, hyaline to yellowish white in color, and 69120 × 66-135 μm in size. spore wall was hyaline to yellow. total wall thickness was < 39 μm. was found in cibinong soil with ph range of 5.9-6.0. fahriny (2013) reported that this species associated with at ph 6.9. had the same color and form as those described by walker and rhodes (1981). in this study, the spore size was smaller compared to that reported by walker and rhodes (1981) which measured (85-) 95-168(-198) × (85-)95-168(-177) μm and fahriny (2013) which measured 48-144 × 66-171 μm and kramadibrata (2009) which measured 85-160 × 85-160 μm. gigaspora candida gigaspora candida gigaspora ramisporophora gigaspora ramisporophora ph 5.7-5.9. by et al. et al. et al. glomus albidum glomus albidum areca g. albidum μm, septyarini (1999) 230-576 172-317 μm. the obtained suspensor relatively smaller than that reported by spain (50-) 60-83 μm 118 biotropia vol. 20 no. 2, 2013 glomus glomerulatum glomus glomerulatum scutellospora calospora s. calospora et al bisbul a. scrobiculata a. tuberculata, g. ramisporophora specimens examined: drn 13, drn 14, drn 29, drn 41, drn 43, drn 44, drn 45, drn 58, drn 61, drn 62, drn 84, drn 91, drn 99, drn 101, drn 111, drn 117, drn 118, drn 119, drn 121, drn 123, drn 125, drn 126, drn 127, drn 128, drn 129, drn 135, drn 136, drn 137, drn 141, drn 143, drn 144, drn 145, drn 146, drn 148, drn 150, drn 152, drn 153, drn 154, drn 155, drn 156, drn 158, drn 159, drn 160, drn 161, drn 163, drn 164, drn 166, drn 168, drn 169, drn 171, drn 175, drn 176, drn 177, drn 179, drn 190, drn 192, drn 193, drn 196, drn 197 and drn 199. 9. sieverd the spores were globose to subglobose, yellow to brown in color, and 70-117 × 70123 μm in size. spore wall was yellow to brown. total wall thickness was 6-9 μm. was successfully obtained from darmaga (ph 5.7-5.9) and cibinong (ph 5.9-6.0). the specimens had the same color and form as those described by sieverding (1987). the size of these spores was bigger compared to that reported by sieverding (1987) which measured 40-70 μm, while in this study the spores were 70-117 × 70-123 μm in size. specimens examined: drn 7, drn 9, drn 46, 52, drn 61, drn 73, drn 77, drn 97, drn 98, drn 99, drn 103, drn 105, drn 107, drn 114, drn 117, drn 118, drn 121, drn 122, drn 123, drn 136, drn 139, drn 140, drn 163, drn 164, drn 170, drn 175, drn 176, drn 177, drn 179, drn 180, drn 181, drn 186, drn 190, drn 194 and drn 196. 10. (t.h. nicolson & gerd.) c. walker & f.e. sanders the spore was globose to subglobose, greenish yellow to brown in color, and 135234 × 141-210 μm in size. spore wall was hyaline to yellow. total wall thickness was 618 μm. bulbous suspensor was hyaline to brown and 27-45 × 24-39 μm in size. the quantity of amf spores was the lowest, only 1 spore was found from cibinong at ph (5.9-6.0). it had the same color and form as those described by koske and walker (1986). the spore size was same range as that described by koske and walker (1986), which was 114-285(-511) × 110-412(-511) μm, but this spore was bigger than that reported by kramadibrata . (2007), which measured 86-134 × , kramadibrata (2009) 150 × 150 um (globose) and 100-160 × 165-250 μm (oblong). the suspensor was 27-45 × 24-39 μm specimens examined: drn 16, drn 83, drn 84, drn 85, drn 119, drn 126, drn 133, drn 134, drn 138 , drn 139, drn 144, drn 146, drn 159, drn 164, drn 185 and drn 192. ten species of amf spores were found in the rhizosphere of in bogor, also eleven species of amf were found in persimmon rhizosphere in cibinong (septyarini 1999), but only 3 same species of amf were recorded from both plants i.e. , and . the quantity and diversity of amf species obtained from every location were different from each other. it could be influenced by different locations and soil types. ipb darmaga campus and cibinong soil belong to ultisol (dominated by clay), while in 86134 μm in size, relatively smaller than what was reported by koske and walker (1986), which measured 33-48 μm. 119 arbuscular mycorrhizal fungi associated with ( ) dwi retno ningsihbisbul diospyros blancoi – et al. ciampea is alfisol (dominated by fine clay) (soil survey staff 2010). widiastuti and kramadibarata (1992) suspected that the soil dominated by clay fraction was suitable for the development and growth of spp. spores, while koske (1987) reported that the spores from genera and were found in large quantity in sandy soil. amf species diversity can also be influenced by the soil ph which was different in each location (moreira-souza 2003). was the dominant genus in rhizospheres and pot cultures. carvalho (2001) reported that this species (formerly . ) was the dominant fungi in saline soil. furthermore, gai (2006) also reported that the spread of (including syn. and ) was wider in tropical and subtropical regions compared to other genera. this study is the first report on mycorrhiza and it showed that amf was associated with trees in the form of arum-paris type. the spores found in tree rhizospheres were identified as , . and . glomus gigaspora scutellospora et al. funneliformis geosporum bisbul et al. g geosporum et al. glomus claroideoglomus funneliformis bisbul bisbul bisbul acaulospora longula, a. scrobiculata, a. tuberculata, claroideoglomus geosporum, funneliformis etunicatum, gigaspora candida, g. ramisporophora, glomus albidum g glomerulatum, scutellospora calospora conclusions references becker wn, gedermann jw. 1997. sp. nov. . 6:29-32. bhattacharjee m, mukerji kg. 1982. structure and hyperparasitism of a new species of . . 78(1):184-188. carvalho lm, cacador i, loucao mam. 2001. temporal and spatial variation of arbuscular mycorrhizas in salt marsh plants of the tagus estuary (portugal). 11:303-309. doi:10.1007/s00572-001-0137-6. chairani, gunawan aw, kramadibrata k. 2002. mikoriza durian di bogor dan sekitarnya. 7(2):44-46. coronel re. 1992. edible fruits and nuts. in plant resources of south-east asia 2. edited by verheij ewm, coronel re bogor (id): prosea foundation. p 151-152. delvian. 2010. keberadaan cendawan mikoriza arbuskula di hutan pantai berdasarkan gradien salinitas. j ilmu dasar. 11(2):133-142. duaja md, jasminarni. 2008. isolasi dan karakterisasi cendawan mikoriza arbuskular di rhizosfer beberapa jenis tanaman di kebun percobaan fakultas pertanian, universitas jambi. . 12(2):34-38. fahriny ra. 2013. ragam cendawan mikoriza arbuskula pada di kebun raya bogor. [ ]. bogor (id): institut pertanian bogor. gai jp, christie p, feng g, li xl. 2006. twenty years of research on community composition and species distribution of arbuscular mycorrhizal fungi in china: a review. . 16:229-239. doi:10.1007/s00572-005-0023-8. haerida i, kramadibrata k. 2002. identifikasi jamur mikoriza arbuskula pada rizosfer tanaman jagung manis di jawa. . 2(2):33-37. hawley gl, dames jf. 2004. mycorrhizal status of indigenous tree species in forest biome of the eastern cape, south africa. . 100(11):633-637. glomus etunicatus mycotaxon gigaspora trans br mycol soc mycorrhiza. j mikrobiol indones. . j agron areca skripsi mycorrhiza floribunda south africa j sci 120 biotropia vol. 20 no. 2, 2013 janos dp, trappe jm. 1982. two new species of tropical america. . 15:515-522. koske re, walker c. 1986. species of ( ) with smooth-walled spores from maritime sand dunes: two new species and redescription of the spores of and . 27:219-235. koske re. 1987. distribution of va mycorrhizal fungi along a latitudinal temperature gradient. . 79(1):55-68. kramadibrata k. 2009. recovered from cacao soil. reinwardtia 12(5): 357-371. kramadibrata k, prastyo h, gunawan aw. 2007. jamur arbuskula pada bambu di jawa. . 8(6):531-536. lucia y. 2005. cendawan mikoriza arbuskula di bawah tegakan tanaman manggis dan peranannya dalam pertumbuhan bibit manggis ( l.). [thesis]. bogor (id): institut pertanian bogor. moreir-souza m, trufem sfb, gomes-da-costa sm, cardoso ejbn. 2003. arbuscular mycorrhizal fungi associated with (bert.) o. ktze. . 13:211-215. doi:10.1007/s00572-0030221-1. muliawan j, gunawan aw, kramadibrata k. 2002. mikoriza rambutan di bogor dan sekitarnya. 7(1):24-25. phillips jm, hayman ds. 1970. improved procedures for clearing roots and staining parasitic and vesiculararbuscular mycorrhizal fungi for rapid assessment of infection. . 55(1):158-161. retnaningsih e. 1998. biodiversitas cendawan mikoriza arbuskula pada rizosfer salak. [ ]. bogor (id): institut pertanian bogor. schenck nc, spain jl, sieverding e, howeler rh. 1984. several new and unreported vesicular-arbuscular mycorrhizal fungi ( ) from colombia. . 76(4):685-699. schü 2010. . edinburgh (uk): royal botanic garden edinburgh. septyarini. 1999. cendawan mikoriza arbuskula di kebun plasma nutfah puslitbang bioteknologi-lipi, cibinong. [ ]. bogor (id): institut pertanian bogor. setiadi y, setiawan a. 2011. studi status fungi mikoriza arbuskula di areal rehabilitasi pasca penambangan nikel (studi kasus pt inco tbk, sorowako, sulawesi selatan). . 3(1):88-95. sieverding e. 1987. a va-mycorrhizal fungus, sp. nov., with two hyphal attachments and spores formed only in sporocarps. 29:73-79. soil survey staff. 2010. . 11th ed. washington (us): us department of agriculture natural resource conservation service :85-573. spain jl, sieverding e, schenck nc. 1989. : a new species with novel sporophores from brazil. . 34(2):667-677. trappe jm. 1977. three new endogonaceae: and . . 6(2):359-366. walker c, mize cw, mcnabb jr hs. 1982. population of endogonaceous fungi at two locations in central iowa. 60(12):2518-2529. doi:10.1139/b82-305. walker c, rhodes lh. 1981. : a new species in the . . 12(2):509-514. walker c. 1982. species in the : a new species ( ) and a new combination ( ). . 15:49-61. widiastuti h, kramadibrata k. 1992. jamur mikoriza bervesikula arbuskula di beberapa tanah masam di jawa barat. 60(1):9-19. zhao zw, xia ym, qin xz, li xw, cheng lz, sha t, wang gh. 2001. arbuscular mycorrhizal status of plants and the spore density of arbuscular mycorrhizal fungi in the tropical rain forest of xishuangbanna, southwest china. . 11:159-162 . acaulospora mycotaxon scutellospora endogonaceae scutellospora pellucida scutellospora calospora. mycotaxon mycologia glomeromycota berita biol garcinia mangostana araucaria angustifolia mycorrhiza j mikrobiol indones. trans br mycol soc skripsi endogonaceae mycologia the glomeromycota: a species list with new families skripsi j silvikul trop glomus glomerulatum mycotaxon. keys to soil taxonomy gigaspora ramisporophora mycotaxon glomus constrictus, sclerocystis clavispora, acaulopsora scrobiculata mycotaxon can j bot. glomus albidus endogonaceae mycotaxon endogonaceae glomus occultum glomus geosporum mycotaxon menara perkebunan. mycorrhiza βler a, walker c. 121 arbuscular mycorrhizal fungi associated with ( ) dwi retno ningsihbisbul diospyros blancoi – et al. the growth of cenococcum geophilum effect of mancozeb 80% concentrations on the growth of cenococcum geophilum fr. under in vitro condition biotropia no. 25, 2005 : 22 – 28 supriyanto seameo biotrop, p.o. box 116, bogor 16001, indonesia, and department of silviculture, faculty of forestry, bogor agricultural university, bogor, indonesia ujang susep irawan laboratory of silviculture, seameo biotrop, p.o. box 116, bogor 16001, indonesia abstract fungicides, such as mancozeb 80% are used in nurseries to prevent the plant root against pathogenic fungi. these fungicides may have negative impacts on beneficial organisms such as ectomycorrhizal fungi. cenococcum geophilum is an important ectomycorrhizal fungus associated with some forest trees species. an in vitro experiment was conducted in laboratory condition. cenococcum geophilum was cultured on solid modified melin nokrans’ (mmn) medium containing mancozeb 80 % at different concentrations (0, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μm). a completely randomized design was used with 8 replicates petri dishes. mancozeb 80 % decreased the growth of mycelia of c. geophilum. the radial growth of mycelia was not inhibited by mancozeb 80 % at 0 to 400 μm concentrations. fungi-static effect of mancozeb 80 % was found at 500 to 600 μm concentrations, meanwhile fungi-toxic effect of mancozeb 80 % was obtained at concentration more than 700 μm. a lethal level of mancozeb 80% to the growth of c. geophilum was not found. key word : fungicide/mancozeb 80 % /cenococcum geophilum/fungi-static, fungi-toxic introduction background seedlings production is one of the important processes for the success of forestry programs. good quality seedlings must be provided in sufficient number and in appropriate time prior to the planting activities. seedling quality is affected by genetic and environmental factors. the presence of mycorrhizal fungi on the root system of seedlings is one environmental factor that has been proven to increase plant growth. mycorrhizal research in indonesia has been promoted by the government, because of its potential to increase the quality of seedlings via growth acceleration (marx 1973), to control some root pathogenic microorganisms (marx 1973), to improve rooting due to hormone production (gay and debaud 1987), to increase nutrient and water uptake (boyle et al. 1987), to increase drought resistance (boyle 22 et al. 1987), and to increase survival and accelerate xylem formation of plants obtained by tissue culture techniques (supriyanto 1989, chang 1993). biotropia no. 25, 2005 the incidence of disease in tropical nurseries can lead to loss of nursery stock. the damping off diseases, caused by fusarium sp., rhizoctonia sp., or pythium sp., are of most concern. these fungi destroy the root system and curtail water and nutrition absorption. therefore, fungicides are commonly used in nurseries to protect the plant root against these pathogenic fungi. concentrations of mancozeb 80% normally used in forest nurseries, such as in pinus merkusii nurseries, are between 1.8 – 2.0 gram/liter. fungicides may inhibit the growth of the pathogenic fungi or kill them. however, non-target microorganisms, especially the beneficial ones such as mycorrhizal fungi, may also be killed. cenococcum geophilum is an important mycorrhizal fungus in the nursery. this fungus belongs to imperfect fungi, and is commonly found in the nursery as mycorrhizal fungi. cenococcum geophilum doesn’t produce a fruiting body. its isolation started from isolated mycorrhizal root tips. in nursery techniques for seedlings production, the nursery man used commonly pesticide and fertilizer intensively. therefore, it is possible that c. geophilum could survive under high concentration of pesticide and fertilizer, high water retention, and less oxygen. in such condition, c. geophilum became a promising mycorrhizal fungi to be used for seedlings production of forestry species. the use of fungicides such as mancozeb 80% (dithane m-45) could have a negative effect on the growth of c. geophilum. several studies have pointed out the negative effect of pesticides on the growth and development of mycorrhizal fungi (marx and rowan 1981). in pure culture, mancozeb 80 % has a strong toxic effect on the growth of mycorrhizal fungi including pisolithus tinctorius, laccaria laccata, rhizopogon luteolous, and corticium bicolor (thapar 1987). however, so far no information is available for c. geophilum. aside from the effect of fungicides on the growth of mycorrhizal fungi, the work on herbicides indicates opposite growth responses in scleroderma aurantius and pisolithus tinctorius (lake et al. 1981). hence, the objectives of this research were : to study the effect of different concentrations of mancozeb 80% on the growth of c. geophilum and to determine the fungi-toxic level of mancozeb 80% to c. geophilum under in vitro conditions. materials and methods source of culture come from the laboratory of silviculture seameobiotrop, that was isolated from pinus merkusii seedlings. cenococcum geophilum was propagated on solid modified melin norkrans’ medium (marx 1969). small pieces of dense mycelia (1 cm in diameter) were cultured on mmn media containing mancozeb 80 % at 0, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 and 23 1000 μm concentrations. mancozeb 80% was added in the culture media and autoclaved at 120o c, 1.5 bar, 30 minutes. the experiment was carried out in an incubator. the temperature in the incubator was 28o c to 30o c and in dark condition. radial growth of mycelia was observed every 5 days for two months. measurement of radial growth of c. geophilum mycelia was conducted at 8 different directions. the colour change of mycelia was also observed. morphological change of mycelia from different fungicide treatments was observed under a light microscope. the experiment was arranged in completely randomized design with 8 replicates petri dishes. analysis of variance was done followed by duncan’s multiple range test. effect of mancozeb 80% concentrations – supriyanto and ujang susep irawan results and discussions radial growth of mycelia of cenococcum geophilum the effects of different concentrations of mancozeb 80 % on the growth of mycelia of c. geophilum is shown in figure 1. at 0, 50, 100, 200, 300 and 400 μm concentrations, the growth of mycelia was always better than the other concentrations ( > 500 μm ). inhibition of mycelial growth was found clearly at 30 days after treatment. the mycelia of c. geophilum grew very well at 5 days after treatment, especially at 0, 50, and 100 μm concentrations. however, in general the increase of fungicide concentration significantly inhibited the growth of mycelia. finally, the effects of mancozeb 80 % on the growth of mycelia of c.geophilum can be classified into two groups as follows : fungi-static and fungi-toxic effects (figure 1). unit in μm 0 5 10 15 20 25 30 5 10 15 20 25 30 35 40 45 50 55 60 time (day) r ad ia l g ro w th (m m ) 0 50 100 200 300 400 500 600 700 800 900 1000 24 figure 1. effect of mancozeb 80 % on the growth of cenococcum geophilum over 60 days on mmn agar figure 2 shows the radial growth of two-month-old c. geophilum mycelia in different concentrations of mancozeb 80 %. biotropia no. 25, 2005 0 5 10 15 20 25 30 0 50 100 200 300 400 500 600 700 800 900 1000 r ad ia l g ro w th (m m ) concentration of fungicide (μm) 26.3 a 27.3 a 24.8ab 22.8ab 22.4ab 21.8abc 19.9 c 16.3 c 4.1 d 3.8 d 2.2 d 1.1 d figure 2. radial growth of two-month old cenococcum geophilum mycelia in different concentrations of mancozeb 80 % the radial growth of c. geophilum mycelia in different concentrations of mancozeb 80 % was divided into three groups as follows: (1) the radial growth of mycelia at concentration of 0-400 μm (21.8 – 27.3 mm), (2) the radial growth of mycelia at concentration of 500-600 μm (16.3 -19.9 mm), (3) the radial growth of mycelia at concentration of 700 – 1000 μm (1.1 4.1 mm). inhibition effect of mancozeb 80% to radial growth of c. geophilum mycelia in the solid medium of mmn was not significant at 50 to 400 μm concentrations. the fungi-static effect (the concentration in which fungicide starts to inhibit the growth of mycelia) of mancozeb 80 % was started at 500 μm up to 600 μm concentrations. while, the fungi-toxic effect (the concentration in which fungicide starts to kill the fungi) of mancozeb 80 % was found at 700 μm concentration. figure 3 shows that c. geophilum mycelia could not grow in solid medium of mmn containing mancozeb 80% of 700 – 1000 μm. in this case, mancozeb 80 % disturbed cellular organ of c. geophilum. moreover, the normal process of fungal intracellular digestion did not run well. it will cause the dead of the fungi or disturb the fungal growth (finholt 1952). fungicides will cause the decrease of membrane permeability surrounding fungal hyphae. three systemic fungicides, benlate, aliette, and ridomil, showed 25 different effects on mycorrhizal infection. benlate reduces or eliminates mycorrhizal infection, aliette increases, while ridomil has been reported to have no consistent effects on the percentage of mycorrhizal infection (jabaji -hare and kendrick 1987). benlate, affected all aspects of fungal development, not only the fraction of the root length infected but also the total root length (carey et al. 1992). benlate severely reduced the number of intercellular hyphae and arbuscules per section and there was a small but non-significant effect with respect to vesicles (sukarno and smith 1993). this condition will give a negative effect on the intracellular digestion process. the decrease in membrane permeability caused inhibition of liquid material absorption through the fungal membrane. moreover, the fungicide could dis-activate extracellular enzymes (finholt 1952). enzyme availability is very important for fungal activity in decomposing its nutrient source to the simplest form to be absorbed. the inactivity of this kind of enzyme will inhibit the fungal nutrient absorption that may cause the disturbance on the fungal growth. finally, this condition will cause the death of the fungi. the effects of mancozeb 80 % on the performance and structure of mycelia of cenococcum geophilum is shown in figure 4. effect of mancozeb 80% concentrations – supriyanto and ujang susep irawan figure 3. radial growth of two-month old cenococcum geophilum mycelia in different concentrations of mancozeb 80 % in solid medium of mmn in solid medium of mmn containing mancozeb 80% most of the hyphae became fragmented dark black color thick and branching while in the control 26 treatment their hyphae were light black slurry healthy and no thickening of cell walls. biotropia no. 25, 2005 thickening of cell wall figure 4. two-month-old cenococcum geophilum mycelia in solid medium of mmn (40 x) without fungicide (a) and with fungicide addition (b) conclusions an in vitro experiment showed that mancozeb 80 % reduced the growth of cenococcum geophilum mycelia at more than 700 μm concentration as fungi-toxic level. mancozeb 80 % affected also the mycelial morphology (fragmented, dark black) of cenococcum geophilum. references boyle, c.d., w.j. robertson and p.o. solanius. 1987. use of mycelial slurries of mycorrhizal fungi as inoculum for commercial tree nurseries. can. j. for. res. 17 : 1480 –1486. carey pd, fitter ah, watkinson ar. 1992. a field study using the fungicide benlate to investigate the effect of mycorrhizal fungi on plant fitness. oecologia 90 : 550-555. chang, d. 1993. the study of va mycorrhizal effects on horticultural crops. in proc. of second acom, 11-15 march 1991, chiang mai, thailand. eds. i soerianegera and supriyanto. biotrop special publication no. 42. finholt, r.w., weeks, m. and hathaway, c. 1952. new theory on wood preservation. ind. eng. gay j.c. and j.c. debaud. 1987. genetic study on indole-3-acetic acid production by ectomycorrhizal hebeloma species : inter-and intra specific variability in homo and dikaryotic mycelia. appl. microbiol. biotechnol. 16 : 141-146. 27 jabaji-hare sh. kendrick wb. 1987. response of an endomycorrhizal fungus in allium porrun l to different concentrations of the systemic fngicides metalaxyl (rindomil) and fosetyl-al (alliete). soil biology and biocemistry. 19 : 95-99. lake d.b. ippoliti d.g., and brandow c.c. 1981. effect of herbicides on the growth of pisolithus tinctorius and scleroderma aurantrium in pure culture. fifth north american conference on mycorrhiza, quebec, canada. august 16-22, 1981. pp 66. effect of mancozeb 80% concentrations – supriyanto and ujang susep irawan marx, d.h. 1969. phytopathology 59, p 152-163. marx, d.h. 1973. mycorrhizae and feeder root diseases. in ectomycorrhizae their ecology and physiology. eds. g.c. marks and t.t. kozlowski. academic press, new eds. g.c. marks and t.t. kozlowski. academic press, new york. marx, d.h. and rowan s.j. 1981. fungicides influence growth and development of specific ectomycorrhizae on loblolly pine seedlings. for. sci. 27 : 167 – 176. sukarno, n. and smith, s.e. 1993. effect of fungicides on vesicular –arbuscular mycorrhizal symbiosis. the effect of vesicular-arbuscular fungi and plant growth. the new phytologist v. 123 (1) p 139147. supriyanto. 1989. micropropagation de pinus nigra et pinus sylvestris. application a leurs hybrides interspecifiques. phd dissertation, university of nancy i, nancy, france. thapar, h.s. 1987. effect of fungicides on ectomycorrhizal fungi in vitro. proceedings of the workshop on mycorrhiza round table, held in new dehli, 13-15 march 1987. p. 155-159. 28 supriyanto abstract background results and discussions conclusions references fungal infection and aflatoxin contamination in stored nutmeg ( ) kernelsmyristica fragrans at various stages of delivery chain in north sulawesi province okky setyawati dharmaputra , santi ambarwati , ina retnowati1,2* 1 1 and nijma nurfadila1 1seameo biotrop, jalan raya tajur km 6, bogor 16134, indonesia 2 16680, adepartment of biology, faculty of mathematics and natural sciences, institut pertanian bogor, bogor indonesi received 22 january 2015/accepted 21 may 2015 abstract fragrant nutmeg ( ) is an important commodity that has been used in food and pharmaceutical myristica fragrans industries, hence its quality should be monitored. the objectives of the research were obtain information on the (a) to postharvest handling of nutmeg investigate the occurrence of fungi (includ ) and aflatoxin kernels; (b) to ing a. flavus contamination in stored nutmeg oisture content and percentage of damaged kernels and (c) mto measure nutmeg kernels. methods used in this study included survey, interviews and sample collection in the delivery chain. this study was ions conducted in april and may 2013, in three reg (north minahasa, siau tagulandang biaro (sitaro) and sangihe talaud) and two cities (bitung and manado). the total number of nutmeg samples collected from kernels different point of the delivery chain was 76. it consisted of samples collected from farmers (25 samples), collectors s (22) and exporters (29). the results showed that , the moisture content of nutmeg kernels collected from north sulawesi province was not higher than the maximum limit of moisture content determined by indonesian national standard or sni (10%). nutmeg kernels collected from farmers and collectors had a high percentage of damaged kernels. and were the dominant fungi infecting nutmeg kernels collected from aspergillus niger endomyces fibuliger farmers and collectors, while was the dominant fungus found infecting nutmeg kernels stored by eurotium repens the exporters. aflatoxin b and total aflatoxin contents nutmeg kernels samples collected from farmer and exporter1 s s of were based on statistical analysis using non-parametric analysis, the effect of delivery chain did not give relatively high. any significant difference on moisture content, percentage of damaged kernels, total fungal pulation and s nutmeg po total aflatoxin content. our study suggested that methods the postharvest handling of nutmeg kernels conducted by farmers, collectors and exporters in north sulawesi province (north minahasa, sitaro and sangihe talaud regions), bitung and manado cities should be improved to minimize aflatoxin b contamination1 and total aflatoxin . keywords: aflatoxin, delivery chain fungi, , north sulawesi rovince, p , nutmeg kernelsmyristica fragrans introduction nutmeg is native to the moluccas islands of indonesia, but nutmeg is also grown in nowadays penang island in malaysia, in the caribbean (particularly grenada), in the southern state of kerala in india and in zanzibar islands. fragrant nutmeg ( ) is an important myristica fragrans commodity widely used in food and pharmaceutical industries, therefore , it is important monitor its quality (direktorat to jenderal perkebunan 2012). based on statistical data from the directorate general of estate crops in indonesia, area s planted with nutmeg were 75,062 ha . the in 2008 distribution areas covered 19 provinces. the largest plant area was in north ation of nutmeg moluccas (33%), followed by n aanggroe ceh d (23%), north sulawesi arussalam or nad (18%), moluccas (12%), west java (5%) and the rest (9%) in other provinces. indonesia contributes 75% (8,943 tonnes) of nutmeg production in the world (revitalisasi perkebunan pala siau, sulawesi utara 2010). north sulawesi province is one of the most important nutmeg producing in . nutmeg from provinces indonesia* corresponding author : okky@biotrop.org biotropia vol. 22 no. 2, 2015: 129 139 129 doi: 10.11598/btb.2015.22.458 mailto:okky@biotrop.org biotropia vol. 22 no. 2, 2015 130 this is exported to t netherlands, italy, province he japan and vietnam. during and storage s, postharvest period nutmeg kernels can be infested by insects and colonized by microorganisms. among microorganisms, fungi important are the most cause of stored foodstuff deterioration. fungal infection in foodstuff can cause discolouration and mycotoxin contamination, as well as decrease in physical quality and nutritional content (sauer et al. 1992). aflatoxins are toxins produced by certain fungi, such as aspergillus flavus a. and parasiticus are considered dangerous . aflatoxins due to associat with various diseases their ion in human and animals, such as aflatoxicoses and liver cancer. there are four naturally occurring aflatoxins in many stored commodities, i.e. aflatoxins b , b , g and g . the most common 1 2 1 2 and toxic aflatoxin is aflatoxin b (1 (afb ) basappa 1 2009). the european union has determined maximum tolerable limits (mtl) of b and af 1 total aflatoxin in nutmeg kernels to be 5 and 10 ppb, respectively (fao 2004). since nutmeg is among expor t impor tant ag r i cu ltu ra l commodit for indonesia, it is important to ies conform to nutmeg kernels importation rules the from countries. the first step in the importing postharvest handling is to monitor and improve the current procedure of nutmeg storage. kernels in relation to storage monitoring, several objectives in this study were set as follow : to (a) s obtain information on postharvest nutmeg handling methods to (b) ; investigate fungal population, fungal diversity (including aspergillus flavus of stored ) and aflatoxin contamination nutmeg kernels collected from different points of the delivery chain in north sulawesi province and (c) to determine and percentage moisture content of damaged nutmeg kernels in the postharvest chain. materials and methods time and ocation of urveyl s s of nutmeg kernels weresurvey and sampling conducted in april and may different 2013 at point of delivery chain s , i.e. at farmer, collector and exporter levels in five locations in north sulawesi province. the five locations chosen for the survey based on recommendation from the agricultural service of north sulawesi province were north minahasa, siau tagulandang biaro (sitaro) and sangihe talaud regions; bitung and manado cities (table 1). interview using uestionnairesq interviews during the survey were conducted to collect information on postharvest nutmeg handling at different point the delivery chain s in (farmer collector ). the , and exporter levels questionnaires on contained questions postharvest handling carried out by procedures farmers, collectors , and exporters as well as problems encountered by them umber of . the n respondents from each level of delivery point was different depending on the conditions in the field during the survey. sampling ethodm s the samples were collected from the places where the respondents obtained the nutmeg kernels. the number of samples nutmeg kernels at each level determined of delivery point was proportionally, based on the number of farmers, collectors and . exporters as much as shelled kernels 500 g of nutmeg and , in-shell kernels were1 000 g of nutmeg collected randomly from each respondent ach . e sample packed in a was clean plastic (polyethylene) and then was double packed in bag hermetic bags to minimize any changes to the nutmeg kernels samples due to long distance transportation between the location of sampling and laboratory in bogor were, where the samples analyzed. method for btaining orking ampleso w s the nutmeg shelled manually to in-shell s were obtain the skernel . each sample was mixed homogeneously, then was into four parts. divided one part was used to determine the percentage of damaged nutmeg kernels and as a reserve sample, while the other three parts were ground using a mill powder tech model rt 04 the ground . nutmeg samples were then divided into eight parts to determine moisture content, fungal population, dominant fungal species infecting kernels aflatoxin ontent.and c fungal infection and aflatoxin contamination in stored nutmeg ( ) dharmaputra – et al. myristica fragrans determination of moisture content, percentage of damaged kernels, fungal p a n copulation and flatoxi ontent moisture content of (based on wet nutmeg basis) w determinedas using the distillation method (sni 1993). weretwo replicates used for each sample. the percentage of damaged kernels was calculated from the weight of damaged the kernels ed by the and divid the weight of working sample damaged kernelfrom which the s were taken damaged kernels included cracked, . broken, shriveled and mouldy kernels. werefungi isolated using serial dilution the method followed by pour plate method with the dichloran 18% glycerol agar (dg18) (hocking & & pitt 1980 pitt hocking 2009). each fungal ; species identified according to pitt and were hocking (2009) as the main reference. aflatoxin b (afb ) and total aflatoxin1 1 contents were determined using hplc with post-column derivatization (vicam 2007). data collection and analysis data collection on nutmeg postharvest handling methods at farmer, collector and exporter levels were carried out by conducting interviews with farmers, collectors and exporters. data of moisture content (mc), percentage of damaged kernels, fungal total population and total aflatoxin content were collected from farmers (25 samples), collectors (22 samples) and exporters (29 samples) at north sulawesi province. total samples at farmer and collector levels were collected from north minahasa, sitaro and sangihe talaud regions. meanwhile, total samples at exporter level were collected from bitung and manado cities. data of mc, percentage of damaged nutmeg kernels, total fungal population and total aflatoxin content were analyzed with nonparametric one way kruskal-wallis test. 131 tabl 1 , samplese level of delivery chain sub-district origin of the sample and the number of nutmeg kernels at various stages of delivery chain in north sulawesi province region/ city level of delivery chain sub-district origin of the sample number of samples north minahasa farmer kauditan 16 collector kauditan 17 exporter siau tagulandang biaro (sitaro) farmer east siau 1 west siau 1 collector east siau 3 exporter sangihe talaud farmer kendahe 2 central tabukan 1 tamako 1 east tahuna 1 west tahuna 1 mangawito 1 collector kendahe 2 exporter bitung farmer collector exporter halmahera island 1 lembeh island 1 kauditan 1 some locations 1 bitung 2 manado farmer collector exporter siau 12 tahuna 1 manado 10 total number of samples 76 results and discussion results of with armers, interviews f c eollectors and xporters the total number of respondents was 26 (14 farmers, 8 collectors and 4 exporters). interviews related to nutmeg postharvest handling at farmer and collector levels were conducted only in north minahasa, sitaro and sangihe talaud reg , ions while interviews at exporter level were conducted in bitung and manado cities. method of collecting nutmeg at farmer level, farmers picked generally nutmeg fruits from the trees . they also (±50%) collected nutmeg fruits which had fallen on the ground . armers collected nutmeg (±50%) f also fruits that had fallen on the ground, because this method was easier to be conducted compared to harvesting nutmeg fruits directly from the trees. price of nutmegs picked directly from the trees was similar to price of nutmegs that had fallen on the ground. ,therefore farmers did not pay attention to the method of proper nutmeg postharvest handling. at collector level, a total of 57% of collectors bought nutmegs from farmers in three conditions, i.e. wet, semi-dry and dry conditions; for both shelled and in-shell .nutmeg s if a collector bough nutme st gin-shell , then a wooden stick was used for conducting the shelling process. as much as 12.5 and 87.5% of farmers sold nutmeg s in semi-dry and dry conditions respectively. in , general farmers sold nutmeg in dry condition, , s because price higher than sold in the was those semi-dry or wet conditions. at exporter level, up to 75% of exporters bought shelled nutmegs from collectors, while 25% of exporters bought a mixture of shelled and in-shell . nutmegs most of exporters bought shelled s the weight of shelled nutmeg , because nutmegs was that of lighter than in-shell nutmegs, which could the costdecrease transportation . method of drying and storing n s utmeg at the farmer level, 87.5% of farmers dried shelled and in-shell nutmegs using sun-drying method by spreading the nutmegs on tarpaulin placed on the ground. the dried nutmegs were subsequently stored in plastic bags. after 1 kg of nutmegs were collected and shelled, they were sold to collectors. in general, farmers did not do any attempts to sort dried nutmegs based on physical quality, because they wanted to sell dry nutmeg as soon as possibles . at the collector level, drying of nutmegs was conducted using two methods, i.e. sun-drying and smoke drying methods. in general, collectors used smoke drying method, especially during rainy season. the smoke drying method used coconut shells as fuel. up to 57% of collectors dried both shelled and in-shell nutmegs using sundrying method on tarpaulin placed on the ground. a total of 43% of collectors smoke-dried nutmegs on wire and wooden racks, followed by sun-drying method. for better result, collectors dried the nutmeg kernels using wire and wooden racks at elevated position ±1 m from the floor, to avoid contamination by animal faeces or dirt. in the night or when it rained, the semi-dried and wet nutmeg kernels were packed in the same plastic bags. as much as 71% of collectors sold nutmeg kernels to exporters, while 29% of nutmeg kernels were sold to large traders, without being sorted. before being sold, the nutmeg kernels were stored for about a month. during storage, 71% of collectors did not monitor nutmeg kernels for aflatoxin contamination, due to lack of aflatoxin detection equipment, although they knew the impact of aflatoxin on human and animal health. at the exporter level, 75% of exporters grouped the nutmeg kernels manually based on qualities. the exporters usually stored the sorted nutmeg kernels in gunny and plastic (polypropylene) bags. those were exporters aware of potential aflatoxin contamination and the impact on human and animal health. they monitored the nutmeg kernels for aflatoxin contamination using a long wave ultraviolet lamp, in which aflatoxin contaminated nutmeg kernels would produce blue green yellow fluorescence and be removed prior to export. source and umber of amplesn s the total number of samples nutmeg kernels collected from different points in the delivery chain was 76 consisted of nutmeg kernels . this samples from farmers (25 samples), collectors (22 samples) and exporters (29 samples) (table 1). 132 biotropia vol. 22 no. 2, 2015 moisture content the moisture content (mc) of foodstuff at the beginning of storage is one of the important factors influencing the quality of foodstuff during storage. a high initial mc value at the beginning of storage provides an opportunity for growth and development of spoilage and mycotoxigenic fungi. according to indonesian national standard or sni, maximum moisture content value for nutmeg kernels and their processed products should be 10% (sni 01-0006-1993). the range and mean mc values of nutmeg kernels collected from farmers, collectors and exporters in north minahasa, siau tagulandang biaro (sitaro) and sangihe talaud regions; bitung and manado cities are presented in table 2. the highest and the lowest mc values of nutmeg kernels collected from farmers in north minahasa region were 18.00 and 8.00%, respectively. for collectors, those mc values were 15.50 and 7.50%, respectively. the mean mc value of nutmeg kernels collected from farmers was similar to that collected from collectors, i.e. 10.88 and 11.07%, respectively. the mean mc value of nutmeg kernels collected from farmers and collectors were higher than the maximum limit determined by . the high mc value of sni nutmeg kernels collected from farmers was probably due to the short period of drying after the harvesting process, low intensity of sun light during the sun-drying process, limited storage space and short duration of storage. the highest and lowest mc values of nutmeg kernels from farmers in sitaro region were 10.50 and 10.00%, respectively, while the highest and lowest mc values of nutmeg kernels from collectors were 11.50 and 6.50%, respectively. the mc values of nutmeg kernels from farmers in sitaro region exceeded the maximum limit determined by . sni in sangihe talaud region the range and mean of mc values obtained from the farmers' and collectors' samples were lower than the maximum limit determined by . the highest and sni lowest mc values of nutmeg kernels from farmers were 9.00 and 6.98%, respectively; while the highest and lowest mc values of nutmeg kernels from collectors were 8.00 and 7.99%, respectively. the range of mc values of nutmeg kernels collected from exporters in manado (7.00 11.50%) was wider than that collected from bitung (7.50 10.00). the mean mc value of nutmeg kernels from exporters in bitung (8.75%) and manado (9.48%) cities were lower than the maximum limit determined by . sni 133 tab 2 , undamaged and damaged kernelsle range and mean of moisture content of nutmeg collected from farmers, collectors and exporters in north sulawesi province region / city level of delivery chain range (mean) of moisture content (% wet basis) range (mean) of undamaged kernels (%) range (mean) of damaged kernels (%) north minahasa farmer 8.00 – 18.00 (10.88) 0 – 88 (22.78) 12.00 –100 (77.22) collector 7.50 – 15.50 (11.07) 0 – 81.42 (21.20) 18.58 – 100 (76.12) exporter siau tagulandang biaro (sitaro) farmer 10.00 – 10.50 (10.25) 52.91 – 61.88 (57.40) 38.13 – 47.09 (42.61) collector 6.50 – 11.50 (8.83) 0 – 47.85 (15.95) 52.15 – 100 (84.05) exporter sangihe talaud farmer 6.98 – 9.00 (7.85) 0 – 61.54 (36.71) 38.46 – 100 (63.29) collector 7.99 – 8.00 (8.00) 0 – 58.78 (29.39) 41.22 – 100 (70.61) exporter bitung farmer collector exporter 7.50 – 10.00 (8.75) 0 – 75.31 (35.25) 24.69 – 100 (64.75) manado farmer collector exporter 7.00 – 11.50 (9.48) 0 – 71.33 (26.42) 28.67 – 100 (73.58) fungal infection and aflatoxin contamination in stored nutmeg ( ) dharmaputra – et al. myristica fragrans the mean mc value of nutmeg kernels collected from farmers and collectors in north minahasa region were higher than that collected from other delivery chains in north sulawesi province. overall, the highest mean mc value was recorded from nutmeg kernels collected from collectors in north minahasa region, followed by those collected from farmers in sitaro region, from exporters in manado city, from collectors in sitaro region, from exporters in bitung city and from farmers and collectors in sangihe talaud region. nutmeg kernels are very hygroscopic, therefore, storage at semi-dried condition caused the kernels to absorb moisture which led to initiation of mould growth, spoilage and mycotoxin contamination. based on non-parametric statistical analysis, d significantly influenceelivery chain did not moisture content (mc) value kernelss of nutmeg ( ).table 3 percentage of u damaged ndamaged and kernels damage to nutmeg kernels may contribute to infection by mycotoxigenic fungi such as a. flavus and increase the chances for aflatoxin contamination during the postharvest storage. the r nge of damaged nutmeg kernels percentage a in north minahasa region from farmers and collectors were 12.00 – 100 and 18.6 – 100%, respectively. in sitaro region, the range of damaged nutmeg kernels from farmers and collectors were 38.1 – 47.1% and 52.2 – 100%, respectively. means of damaged nutmeg kernels percentage from farmers and collectors were 42.6 and 84.1%, respectively. in sangihe talaud region, the mean of damaged nutmeg kernels percentage collected from farmers (63.29%) was lower than that collected from collectors (70.6%). in bitung city the percentage of damaged nutmeg kerne s collected from exporters was l 64.8%. in manado city the percentage was 73.6% (table ). means of damaged nutmeg kernels 2 percentage collected from farmers and collectors in north sulawesi p were higher than rovince those collected from exporters. this might be due to improper sorting regimes used by farmers and collectors. in addition, many farmers and collectors still manually shelled the nutmegs using wooden stick, which can increase the percentage of damaged nutmeg kernels. based on statistical analysis using nonparametric analysis, elivery chain did not d significantly influence the percentage of damaged nutmeg table 3kernels ( ). fungal population diversity and dominance the highest diversity of fungal species was found in nutmegs collected from farmers and collectors in north minahasa region, 13 and i.e. 12 species, while the lowest number respectively, of fungal species were observed in samples obtained from farmers in sitaro region (7 species) and those obtained from collectors in sangihe talaud region (2 species; tables 4, 5, 6). aspergillus flavus was found in 56% samples from farmers and in 53% samples from collectors in north minahasa region. fungal population isolated from samples from farmers was more contaminated than those from collectors (table 5). the dominant fungi found in nutmeg samples from farmers was (81%) penicillium citrinum followed by (69%) and a. niger eurotium repens (63%). three dominant fungi found in nutmegs collected from collectors were endomyces fibuliger (76%), (76%) and (76%) which a. niger p. citrinum may have been caused by high moisture content. 134 table 3 the effect of nutmeg delivery chain on moisture content (mc), percentage of damaged nutmeg kernels, fungal total population and total aflatoxin level of delivery chain mc (%) percentage of damaged kernels (%) fungal total population (cfu/g wet basis) total aflatoxin content (ppb) farmer 9.98 ± 2.62 a 70.55±29.66 a 3.9x105±1.7x106 a 141.10±392.11 a collector 10.49 ± 2.52 a 76.70±32.39 a 1.3x106±3.2x106 a 2.15±4.54 a exporter 9.33 ± 1.13 a 71.75±30.27 a 9.9x103±1.1x104 a 50.63±213.43 a note: means in the same group followed by the same letter in a column are not significantly different at 5% level biotropia vol. 22 no. 2, 2015 no was isolated from nutmeg samples a. flavus obtained from collector in the sitaro region. at farmer level, a. niger, a. penicillioides, eurotium repens p. citrinum and were isolated from all samples, dominated by only a a. penicillioides. small number of nutmeg samples were colonized by in the sangihe talaud region. no a. flavus nutmeg samples from collectors appeared to contain . all samples of nutmegs a. flavus obtained from farmers and collectors in all sampling sites contained a. niger. the total fungal population and diversity of samples from exporters were lower in bitung city than in manado city (table 7). in bitung city, a. flavus was only isolated from 2% of samples. the dominant fungi were xerophilic spoilage fungi such as (all samples) followed by e. repens e. chevalieri (83%). in manado city, much higher number of samples contained (39%). a. flavus again, the dominant fungal population isolated were (87%), followed by (70%) e. repens a. niger and (65%).a. penicillioides aspergillus flavus was found in 56% samples from farmers and in 53% samples from collectors in north minahasa region. fungal population isolated from samples from farmers was more contaminated than those from collectors (table 5). the dominant fungi found in nutmeg samples from farmers was (81%) penicillium citrinum followed by (69%) and a. niger eurotium repens (63%). three dominant fungi found in nutmegs collected from collectors were endomyces fibuliger (76%), (76%) and (76%) which a. niger p. citrinum may have been caused by high moisture content. no was isolated from nutmeg samples a. flavus obtained from collector in the sitaro region. at farmer level, a. niger, a. penicillioides, eurotium repens p. citrinum and were isolated from all 135 table 4 fungal populations and diversity in nutmeg samples obtained from farmers collectors in north minahasa and region no fungi number (%) samples infected by fungi range (mean) of fungal population in nutmeg (cfu/g wet basis) farmer collector farmer collector 1. aspergillus flavus 9 (56) 9 (53) 0.5 x 10 – 8.8 x 103 (1.5 x 103) 0.7 x 10 – 5.2 x 103 (7.8 x 102) 2. a. niger 11 (69) 13 (76) 0.2 x 10 – 2.2 x 104 (4.3 x 103) 0.1 x 102 – 8.8 x 103 (2 x 103) 3. a. ochraceus 1 (6) 0.7 x 10 (0.7 x 10) 4. a. penicillioides 2 (13) 3 (18) 2.2 x 102 – 1.3 x 103 (7.8 x 102) 4.5 x 10 – 2.7 x 102 (1.7 x 102) 5. a. tamarii 8 (50) 4 (24) 0.8 x 10 – 1.2 x 103 (3.3 x 102) 1.5 x 102 – 7.3 x 102 (3.7 x 102) 6. a. sydowii 2 (13) 1 (6) 1.2 x 10 – 1.8 x 103 (9.2 x 102) 8.2 x 102 (8.2 x 102) 7. a. wentii 1 (6) 1.2 x 102 (1.2 x 102) 8. endomyces fibuliger 7 (44) 13 (76) 5.2 x 10 – 8.4 x 106 (1.3 x 106) 1.5 x 10 – 1.1 x 107 (2.2 x 106) 9. eurotium chevalieri 5 (31) 3 (18) 1.5 x 10 – 0.4 x 104 (1.2 x 103) 3.3 x 10 – 7.3 x 103 (2.6 x 103) 10. e. repens 10 (63) 9 (53) 0.5 x 10 – 6.8 x 103 (1.7 x 103) 1.3 x 10 – 3.2 x 103 (9.0 x 102) 11. e. rubrum 7 (41) 0.2 x 10 – 3.5 x 103 (9.4 x 102) 12. penicillium citrinum 13 (81) 13 (76) 0.3 x 10 – 0.6 x 105 (6.3 x 103) 1.3 x 10 – 0.4 x 104 (7.8 x 102) 13. rhizopus sp. 2 (13) 2.5 x 102 – 7.2 x 102 (4.8 x 102) 14. syncephalastrum racemosum 1 (6) 0.2 x 103 (0.2 x 103) 15. trichoderma sp. 2 (13) 0.1 x 102 – 1 x 103 (5.1 x 102) notes: number of samples collected from farmers: 16 number of samples collected from collectors: 17 fungal infection and aflatoxin contamination in stored nutmeg ( ) dharmaputra – et al. myristica fragrans samples, dominated by only a a. penicillioides. small number of nutmeg samples were colonized by in the sangihe talaud region. no a. flavus nutmeg samples from collectors appeared to contain . all samples of nutmegs a. flavus obtained from farmers and collectors in all sampling sites contained a. niger. the total fungal population and diversity of samples from exporters were lower in bitung city than in manado city (table 7). in bitung city, a. flavus was only isolated from 2% of samples. the dominant fungi were xerophilic spoilage fungi such as (all samples) followed by e. repens e. chevalieri (83%). in manado city, much higher number of samples contained (39%). a. flavus again, the dominant fungal population isolated were (87%), followed by (70%) e. repens a. niger and (65%).a. penicillioides nutmegs imported from india, sri lanka, indonesia and brazil were infected by , a. niger a. flavus rhizopus stolonifer and . the dominant fungi in these samples were (mandel 2005). a. flavus the water availability of semi-dried and damaged nutmeg kernels provided environmental conditions which are conducive to xerophilic and xerotolerant fungi, including mycotoxigenic species. the boundary conditions for growth and mycotoxin production sug gested that aflatoxigenic and ochratoxigenic fungi may be able to th ve under storage conditions (lacey & ri magan 1991; sanchis & magan 2004; magan & aldred 2007). 136 tabl and siau tagulandang e 5 fungal population and diversity in nutmeg samples obtained from farmers collectors in biaro (sitaro) region no fungi number (%) samples infected by fungi range (mean) of fungal population in nutmeg (cfu/g wet basis) farmer collector farmer collector 1. aspergillus niger 2 (100) 2 (67) 0.2 x 10 – 2.5 x 10 (1.3 x 10) 5.5 x 102 – 4.2 x 104 (2.1 x 104) 2. a. penicillioides 2 (100) 1 (33) 1.8 x 10 – 2.0 x 103 (0.1 x 104) 3.7 x 102 (3.7 x 102) 3. endomyces fibuliger 1 (50) 0.5 x 10 (0.5 x 10) 4. eurotium chevalieri 1 (50) 1 (33) 6.7 x 10 (6.7 x 10) 2.8 x 10 (2.8 x 10) 5. e. repens 2 (100) 1 (33) 0.2 x 10 – 3.3 x 102 (1.7 x 102) 1.8 x 10 (1.8 x 10) 6. e. rubrum 1 (50) 1 (33) 1.7 x 10 (1.7 x 10) 0.3 x 10 (0.3 x 10) 7. penicillium citrinum 2 (100) 0.2 x 10 – 0.8 x 10 (0.5 x 10) notes: number of samples collected from farmers: 2 number of samples collected from collectors: 3 tab and sangihe talaud ionle 6 fungal population and diversity in nutmeg samples obtained from farmers collectors in reg no fungi number (%) samples infected by fungi range (mean) of fungal population in nutmeg (cfu/g wet basis) farmer collector farmer collector 1. aspergillus flavus 1 (14) 0.2 x 10 (0.2 x 10) 2. a. niger 7 (100) 2 (100) 0.7 x 10 – 2.4 x 105 (5.1 x 104) 4.8 x 10 – 0.1 x 105 (5.4 x 103) 3. a. penicillioides 3 (43) 0.1 x 102 – 3.7 x 102 (1.3 x 102) 4. endomyces fibuliger 1 (14) 1.5 x 105 (1.5 x 105) 5. eurotium repens 4 (57) 0.5 x 10 – 8.3 x 102 (2.2 x 102) 6. e. rubrum 3 (43) 0.3 x 10 – 0.5 x 103 (1.7 x 102) 7. fusarium solani 1 (14) 5.2 x 104 (5.2 x 104) 8. penicillium citrinum 3 (43) 1 (50) 0.3 x 102 – 1.8 x 104 (6.8 x 103) 4.3 x 102 (4.3 x 102) notes: number of samples collected from farmers: 7 number of samples collected from collectors: 2 biotropia vol. 22 no. 2, 2015 based on statistical analysis using nonparametric analysis, elivery chain did not d significantly influence the total fungal population ( ).table 3 aflatoxin t a contentb and otal flatoxin s1 the range of afb and total aflatoxin content 1 in nutmeg samples from farmers in north sulawesi province were 0.40 – 1,632.19 ppb and 0.58 – 1,831.48 ppb, respectively. survey conducted at farmer level provided information a that postharvest handling was not conducted properly, i.e. mixed nutmeg picked the farmers s from the tree th fell on the ground s with ose ; consequently afb and total t the aflatoxin conten1 in these samples (table 8)were high . according to horn (2003) soil serves as a reservoir for and that a. flavus a. parasiticus produce aflatoxins in agricultural commodities. aflatoxigenic fungi reside in soil as conidia, sclerotia and hyphae, while act as primary inocula for directly infect peanuts (and possibly nutmeg ing fruit whic fell on the ground). rh ange of afb 1 and total aflatoxin in nutmeg samples content obtained from collectors in north sulawesi province were 0.11 – 14.59 ppb and 0.11 – 16.65 ppb, respectively. sun-drying method was faster and more effective than smoke-drying method. however, if the weather is extreme, sun-drying method is not recommended, because it could reduce the quality of atsiri oil in nutmeg kernels. therefore, many collectors used smoke-drying method, because the temperature can be controlled. smoke-drying the weakness using method the of long drying duration, includes need which may allow colonization of spoilage and mycotoxigenic fungi. based on statistical analysis using nonparametric analysis elivery chain did not , d 137 table 7 fungal population and diversity in nutmeg samples collected from exporters in citbitung and manado ies no. fungi bitung city manado city number (%) samples infected by fungi range (mean) of fungal population in nutmeg (cfu/g wet basis) number (%) samples infected by fungi range (mean) of fungal population in nutmeg 1. aspergillus flavus 2 (33) 0.5 x 10 – 7.7 x 102 (3.9 x 102) 9 (39) 0.1 x 102 – 5.3 x 102 (1.3 x 102) 2. a. niger 3 (50) 0.3 x 10 – 1.7 x 102 (7.1 x 10) 16 (70) 0.7 x 10 – 0.4 x 105 (3.1 x 103) 3. a. penicillioides 2 (33) 0.5 x 102 – 3.5 x 103 (1.8 x 103) 15 (65) 1.3 x 102 – 2.9 x 104 (6.6 x 103) 4. a. sydowii 1 (17) 0.5 x 10 (0.5 x 10) 5. a. tamarii 1 (17) 0.2 x 103 (0.2 x 103) 1 (4) 2.2 x 102 (2.2 x 102) 6. a. versicolor 2 (9) 4.5 x 102 – 1.2 x 103 (8.1 x 102) 7. endomyces fibuliger 2 (33) 1.7 x 102 – 6.7 x 102 (4.2 x 102) 4 (17) 2.8 x 102 – 5.3 x 103 (1.7 x 103) 8. eurotium chevalieri 5 (83) 0.2 x 10 – 2.5 x 103 (5.4 x 102) 5 (22) 0.5 x 102 – 3.7 x 103 (1.1 x 103) 9. eurotium repens 6 (100) 0.1 x 102 – 8.8 x 102 (3.6 x 102) 20 (87) 2.5 x 102 – 1.8 x 104 (0.5 x 104) 10. e. rubrum 1 (17) 6.8 x 102 (6.8 x 102) 15 (65) 2.8 x 10 – 0.2 x 104 (5.2 x 102) 11. paecilomyces variotii 1 (4) 4.5 x 102 (4.5 x 102) 12. penicillium citrinum 4 (67) 0.5 x 10 – 9.3 x 102 (2.7 x 102) 11 (48) 1.8 x 10 – 1.3 x 103 (2.4 x 102) note : in bitung city 6s number of samples collected from exporters : in manado city 23 number of samples collected from exporters : fungal infection and aflatoxin contamination in stored nutmeg ( ) dharmaputra – et al. myristica fragrans (cfu/g wet basis) significantly influence total aflatoxin content of nutmeg ( ).table 3 several exporters in north sulawesi province possess facilities for drying, shelling, sorting and storage facilities. thus, aflatoxin contamination could be minimized. in this research, we also collected a sample of sorted nutmeg kernels visually assessed using the long wave ultraviolet lamp. this potentially contaminated sample from an exporter contained a total aflatoxin content of 0.18 – 1,112.58 ppb. tabata (1993) reported et al. that aflatoxin was found in 3,054 foodstuff and their product samples, especially nutmeg samples. the highest aflatoxin contamination was found in nutmeg (80%), while afb was also found in 1 pistachio nuts (1,382 ppb). takahashi (1993) reported that in 1986 until 1991, as much as 29 (43%) of 67 nutmeg samples collected from japan, were contaminated by aflatoxin. according to okano (2012) the distribution of et al. aflatoxigenic fungi in 25 imported indonesian nutmeg samples were contaminated with aflatoxins b or b and g. the incidence of aflatoxigenic fungi in the samples contaminated with high levels of aflatoxin was significantly higher than that in the samples with low levels of the toxins (r = 0.752). the toxin production of isolates from the samples in cultures of yeast extract sucrose broth was examined by means of tlc and hplc analyses. the ability of isolates to produce aflatoxins did not correlate with the contamination levels of aflatoxin in the samples. overall, the postharvest handling procedures need to be standardized to minimize aflatoxin contamination. conclusions generally, moisture content (mc) of nutmeg samples collected from north sulawesi province was below the maximum recommended limit of indonesian national standard or sni. nutmeg samples collected from farmers and collectors generally had a higher percentage of damaged kernels. and were the a niger e. fibuligerspergillus 138 tabl b e 8 aflatoxin and total aflatoxin contents in nutmeg collected from farmers, collectors and exporters in north 1 sulawesi province region/city level of delivery chain number of samples number (%) samples contaminated by aflatoxin range (mean) of afb1 content in contaminated samples (ppb) range (mean) of total aflatoxin content in contaminated samples (ppb) north minahasa farmer 16 6 (37.50) 0.40 – 762.24 (128.52) 0.58 – 910.48 (153.28) collector 17 5 (29.41) 0.19 – 14.59 (4.70) 0.19 – 16.65 (5.67) exporter siau tagulandang biaro (sitaro) farmer 2 2 (100) 1.03 – 1.44 (1.23) 1.44 – 1.55 (1.49) collector 3 3 (100) 0.11 – 0.69 (0.40) 0.11 – 1.34 (0.58) exporter sangihe talaud farmer 7 7 (100) 1.65 – 1 632.19 (335.92) 1.65 – 1831.48 (371.99) collector 2 2 (100) 3.28 – 13.94 (8.61) 3.28 – 13.94 (8.61) exporter bitung farmer collector exporter 6 3 (50) 1.40 – 799.25 (267.74) 1.40 – 1112.58 (372.18) manado farmer collector exporter 23 15 (65.22) 0.10 – 266.72 (18.37) 0.18 – 334.49 (20.69) biotropia vol. 22 no. 2, 2015 dominant fungi in nutmeg kernels from farmers and collectors, while was the dominant e. repens species in samples obtained from nutmeg exporters in north sulawesi province. aflatoxin b and total aflatoxin contents in nutmeg samples 1 collected from farmers and exporters were relatively high. although based on statistical analysis using non-parametric analysis, elivery d chain did not significantly influence moisture content ( ) nutmeg mc , percentage of damaged kernels, total fungal population and total aflatoxin content of nutmeg, the method of nutmeg postharvest handling especially at farmer and , co l lector levels should be conducted ap p ro p r i a t e l y t o mi n i m iz e a f l a tox i n contamination. acknowledgements the authors would like to acknowledge seameo biotrop for providing financial support through dipa 2013. thanks due to were the indonesian government's office of plantation crop of north sulawesi province in manado and to cv multi rempah sulawesi for their information and cooperation during the survey; to mrs ratnaningsih, mr edi suryadi and mr. iswadi for their technical support. references basappa sc. 2009. . aflatoxins; formation, analysis and control new delhi (in): narosa publishing house. direktorat jenderal perkebunan. 2012. peningkatan produksi, produktivitas dan mutu tanaman rempah dan penyegar. pedoman teknis perluasan tanaman pala tahun 2012. jakarta. 20 hal. fao. 2004. worldwide regulations for mycotoxins in food and feed in 2003. fao food and nutrition paper 81. rome (it): food and agriculture organization of the united nations. 165 p. hocking ad, pitt ji. 1980. dichloran-glycerol medium for enumeration of xerophilic fungi from low moisture foods. appl env microbial 39: 488-92.. horn bw. 2003. ecology and population biology of aflatoxigenic fungi in soil. toxicol rev 22 j toxin (2 & 3): 351-79. lacey j, magan n. 1991. fungi in cereal grains: their occurrence and water and temperature relationship. in: chelkowski j, editor. cereal grain, mycotoxins, fungi and quality in drying and storage. amsterdam (nl): elsevier. p 77-118. magan aldred n, d. 2007. post-harvest control strategies: minimizing mycotoxins in the food chain. jint of food microbiology. 119 (1-2): 131-9. mandel qa. 2005. fungal contamination of some imported spices. mycopathologia 59 : 291-8. okano k, tomita t, ohzu y, takai m, ose a, kotsuka a, ikeda n, sakata j, kumeda y, nakamura n, ichinoe m. 2012. aflatoxins b and g contamination and aflatoxigenic fungi in nutmeg. shokuhin eiseigaku zasshi 53 (5): 211-6. pitt ji, hocking ad. 2009. new yorkfungi and food spoilage. (us): springer. revitalisasi perkebunan pala siau, sulawesi utara. 2010. warta penelitian dan pengembangan pertanian 32 (1): 4-6. sauer db, meronuck ra, christensen cm. 1992. microflora. in: sauer db, editor . storage of cereal grains and their product 4 editionth (us). . minnesota : american association of cereal chemist. p 313 – 40. sanchis v, magan n. 2004. environmental conditions affecting mycotoxins. in: magan, n, olsen m, editors. mycotoxins in food: detection and control. florida (us): crc press. p 496. standar nasional indonesia. biji pala1993, . sni 01-00061993. jakarta : badan standardisasi nasional. (id) tabata s, kamimura h, ibe a, hashimoto h, iida m, tamura y, nishima t. 1993. af latoxin contamination in foods and foodstuffs in tokyo: 1986-1990. j aoac int 76 (1): 32-5.ern. takahashi t. 1993. aflatoxin contamination in nutmeg: analysis of interfering tlc spots. j food sci 58: . 197-8. vicam. 2007. aflatest instruction manual for hplc. watertown : vicam (us) . 139 fungal infection and aflatoxin contamination in stored nutmeg ( ) dharmaputra – et al. myristica fragrans microsoft word 57 biotropia no. 19, 2002 : 57 64 molecular identification of egg parasitoid, telenomus spp. (hymenoptera: scelionidae) from several locations in java using rapd -pcr netti yuliarti1, purnama hidayat 2, and damayanti buchori3 'agricultural polytechnic, andalas university, payakumbuh, west sumatera, indonesia mj department of plant pests and diseasestfaculty of agriculture, bogor agricultural university, bogor, indonesia abstract random amplified polymorphic dna (rapd) amplified by the polymerase chain reaction (pcr) was used to determine the differences of four telenomus species and five populations of t. rowani from several locations in java. amplification of genomic dna by using primer p2 (amersham pharmacia biotech) indicated that each telenomus species had a unique set of rapd bands. two bands which characterized the genus are estimated to be 300 and 430 bp. each species had three specific bright bands except t. dignoides which only had two specific bright bands. however, no bands are unique to any of the five populations of t. rowani and all of the bands are less than 500 base-pair. cluster analysis using upgma (unweighed pair group method with arithmatic mean) showed that the four telenomus species consist of two groups, t. rowani and t. remus in one cluster and t. dignus with t. dignoides belonging to another cluster. key words : pcr-rapd / clustering / telenomus spp. introduction telenomus (hymenoptera: scelionidae) are minute wasps, often black, about 1 mm in length, and feed exclusively on the eggs of other insects. the genus particularly attacks lepidoptera and hemiptera but they are also known to attack diptera and neuroptera (johnson 1984). the host range is relatively broad but the degree of host-specificity varies between species (polaszek & kimani 1990). telenomus often plays an important role in the natural control of insect pest populations. some biological control programs are presently being carried out on pyralid populations using egg parasitoids of the genus telenomus with good results. in the ivory coast, for instance, these parasitoids are used to control eldana saccharina wlk., maliarpha separatella rag., and scirpophaga melanoclysta mey., on sugar-cane, rice and maize; in india against chilo sacchariphagus (boyer) on sugarcane and in bolivia against diatraea rufescens box and d. saccharalis (f.) on sugarcane. the biology of these egg parasitoids is even more interesting and has a potential for preventing an outbreak of the pest population. some of them are phoretic upon the adults of their host (bin & johnson 1982). more than 500 species of telenomus are currently known. however, biological studies about the genus are few in number when compared to trichogramma (polaszek & kimani 1990; honda & trjapitzin 1995). there are a number of 57 biotropia no. 19,2002 unresolved taxonomic problems at species-level in this genus because of its small size and general morphological uniformity (nixon 1937 and polaszek 2001). the taxonomy of the genus has been based on morphological traits. however, these characters may display a great level .of plasticity and do not always reflect the phylogenetic relationships among species. molecular techniques provide efficient tools for the study of natural population genetics (hoy 1994). random amplified polymorphic dna polymerase chain reaction (rapdpcr) has been used to survey mosquito species and population (kambhampati et al. 1992) and population genetics of parasitoid wasp diaeretiella rapae (hymenoptera: braconidae) (vaughn & antolin 1998). previously, landry et al. (1993); masutti (1994) and meilin (1999) used rapd technique for tricho-gramma species identification. rapd-pcr is particularly valuable for genomic mapping in species for which little genetic information is available (hoy 1994) such as the genus telenomus. so far, no study has been done on the differentiation between and within telenomus species in indonesia. we employed molecular technique to see species differences and to see whether there are genetical variations within the same species from different locations. our objective was to differentiate four species of telenomus and five populations of t. rowani collected from several locations in java using the rapd technique. materials and methods sample collection and identification egg parasitoids of telenomus spp. were collected from various crops, e.g soybean, sugarcane and paddy in west java, central java and east java. parasitized lepidoptera and hemiptera eggs were collected and brought to the laboratory for subsequent parasitoid development. identification of parasitoid was based on nixon (1937), nishida and torii (1970) dna extraction dna was extracted using a technique described by goodwin et al. (1994) with the following modifications. one to three wasps of each telenomus species and five populations of t. rowani were crushed with a glass rod in 125 ul ctab (2 %). dna preparations were stored at 4 °c in 30 ul distilled water. pcr amplification pcr was performed using gene amp r pcr system 9700 (pe applied biosystem). ready to go ™ rapd bead (pharmacia biotech) was used for each pcr reaction. each of 25 ul reaction mixture contained a rapd bead, 5 ul of genomic dna, 5 ul of 25 pmol primer p2 (5' -d[gtttcgctcc]-3' ) and 15 ul 58 molecular identification of egg parasitoid, telenomus spp netti yuliarti et al. distilled water. pcr condition for the integron was amplified in 45 thermal cycles at 95 °c for 1 minute, 36 °c for 1 minute and 72 °c for 3 minutes. the pcr products were then held at 4 °c until recovered. dna visualization the dna fragment was separated using an agarose gel. agarose gel of 1.5 g was prepared in 100 ml of 1 x tbe (90 mm tris-borate buffer, 2 mm edta ph 8.0) and then heated until boiling point using microwave. electrophoresis eight ul of pcr product was mixed with 2.0 ul of gel-loading buffer (30% glycerol, 0.25% bromophenol blue and 0.25% xylene cyanol) and electrophoresed on 1.5% agarose gel (tbe) at 100 volt for about 2 hours. dna bands were stained with ethidium bromide and photographed with fast polaroid film using transluminator uv light. dna band size was determined by comparing with a 100 base-pair dna ladder run on gel. data analysis the data matrix was constructed with each line in the gel containing the identified species and rapd-pcr bands were scored 1 if it is present and 0 if absent. a cluster analysis was carried out using the unweighed pair group method with arithmetic means (upgma clustering) of similarity coefficient for all pairs of species and population. the phenogram was generated using a computer-based taxonomy program numerical taxonomy system, (ntsys-pc version 2.1) (rohlf 2000). results and discussion based on morphological characters, four telenomus species have been identified, t. rowani, t. remits, t. dignus and t. dignoides (yuliarti 2002). we have screened three primers (pharmacia biotech) as follows: pi (5'-d[ggtgcgggaa]-3'), p2 (5'-d[gtttcgctcc]-3') and p3 (5'-d[gtaga.cccgt]-3-). the results showed that primer p2 gave the best polymorphic bands. the results of genomic dna amplification using primer p2 (5' d[gtttcgctcc]-3') indicated that the four telenomus species can be genetically differentiated (fig. 1). each species of telenomus has a unique set of rapd bands. several clear and well-amplified bands were used for analysis. the result of the amplification showed that the size range of the bands are between 200 base-pair (bp) and 1500 bp. two bands approximately 300 and 430 bp seem to indicate the specificity of the genus telenomus. these two bands are monomorphic bands (fig. 1). in contrast, the polymorphic bands which indicate the specificity of each species are shown by different band size as seen in fig.l. 59 biotropia no. 19,2002 fig. 1 shows that each telenomus species has three specific bands except t. dignoides which has only two specific bands. specific bands of t. rowani are estimated to be 650, 850 and 1100 bp, whereas t. remus are 600, 850 and 1150 bp. unique bands of t. dignoides are estimated to be 700 and 1450 bp, whereas t. dignus are 550, 800 and 1050 bp. although only slight differences occur between each telenomus species, our results showed that rapd procedures can reveal species-specific banding pattern that can be used for a rapid and easy identification of telenomus species. this technique can potentially be very important for identification purposes, since polaszek and kimani (1990) stated that due to its smallness, telenomus spp. are most often difficult to identify correctly which then resulted in confusion and errors in identification. to overcome these difficulties, molecular technologies can be applied to allow the detection of diagnostic markers which are necessary to resolve ambiguities in taxonomical identification and systematics in the genus telenomus (polaszek 2001). 60 molecular identification of egg parasitoid, telenomus spp — netti yuliarti et al. rapd-pcr has some limitations i.e uncertain reproducibility, fragments of the same size are not necessarily of the same sequence and reveal dominant marker only. previously, masutti (1994) and meilin (1999) used rapd markers for trichogramma species identification. even though rapd-pcr has its own drawbacks, i.e bands of the same size are not necessarily of the same sequence, these bands are normally inherited as dominant traits. therefore, rapd is still useful for generating species-specific markers. hoy (1994) stated that rapd-pcr may be able to detect small differences in the genomes of individual insects or mites, different insect populations or species. in this study we could not find differentiation among the five t. rowani populations by using primer p2 (5 -d[gtttcgctcc]-3) (amersham pharmacia biotech), despite the fact that those populations originated from different locations in java (fig.2). we found no variations in dna banding patterns for all populations. in other words, all of the populations revealed the same bands with the same molecular weight and only three bright bands were estimated to be in the range of 300-480 bp. there are two interpretations that could be derived from these results. first, the null difference can be due to the limitations of rapd technique; second, the result reflected real condition of t. rowani populations. in this case rapd-pcr did not allow differentiation of t. rowani populations because there are no specific bands that can be detected using this particular primer. however, screening was conducted with only one primer, and it is likely that unique rapd markers could be found that discriminate among the populations if additional primers are tested. however, these studies can be used as a framework on which to base further studies which are required to fully elucidate species and population of telenomus. this result was contrary to previous studies on some populations of trichogramma evanescens (masutti 1994); t oidea armigera and t oidea cojuangcoi (meilin 1999) by using rapd-pcr in which differentiation among the populations were found. in another study, vaughn & antolin (1998) reported that diaretiella rapae (hymenoptera: braconidae) populations are genetically subdivided into a small spatial scale that corresponds to host-use patterns. cluster analysis cluster analysis based on upgma revealed that four telenomus species formed two groups (fig. 3). the dendrogram showed that t. remits and t. rowani belong to one cluster, whereas t. dignus and t. dignoides belong to another group. however, the two groups in this dendrogram were different in coefficient level (table 1). the dendrogram showed that t. rowani was closely related to t. remus, whereas t. dignus was closely related to t. dignoides. however, t. remus and t. rowani were rather far to t. dignus and t. dignoides. masutti (1994) stated that rapd banding patterns might be informative for the phylogenetic relatedness. in addition, nixon (1937) reported that t. dignoides was certainly very closely related to t. dignus. 61   references bin f, johnson nf. 1982. some new species of telenomus egg parasitoids of tropical pyralid pests. redia 65: 229-252 (+ 5 plates). goodwin dh, xue bg, kuske cr, sears mk. 1994. amplification of plasmid dna to detect plant pathogenic mycopiasma like organism. ann appl biol 36. 124-127. johnson nf. 1984. systematics of nearctic telenomus : classification and revisions of the podisi and phymatae group. bull of the ohio biological survey 6 (3): 1-113. honda jy, trjapitzin sv. 1995. a species description and biological comparison between a new species of telenomus haliday (hymenoptera: scelionidae) and trichogramma platneri nagarkatti (hymenoptera: trichogrammatidae): two egg parasitoids of sabulodes aegrotata (guenee) (lepidoptera: geometridae). pan pacific entomol 71 (4): 227-236. hoy ma. 1994. insect molecular genetics. an introduction to principle and applications. san diego. academic press. kambhampati s, black iv wc, rai ks. 1992. random amplified polymorphic dna of mosquito species and population (diptera: culicidae). techniques statistical analysis and applications. j med entomol 29 (6): 939-945. landry bs, dextraze l, boivin g. 1993. random amplified polymorphic dna markers for dna fingerprinting and genetic variability assessment of minute parasitic wasp species (hymenoptera: mymaridae and trichogrammatidae) used in biological control programs of phytophagous insects. genome 36: 580-587. masutti fv. 1994. molecular identification and phylogeny of parasitic wasp species (hymenoptera: trichogrammatidae) by mitochondrial dna rflp and rapd markers. insect mol biol 3 (4): 229-237. meilin a. 1999. keragaman karakter morfologi dan genetika populasi parasitoid telur, trichogramma spp. and trichogrammatoidea spp. (hymenoptera: trichogrammatidae) dari daerah geografis yang berbeda di pulau jawa. (tesis). bogor: program pascasarjana ipb. nishida t, torii. 1970. a handbook of field methods for research on rice stem-borers and their natural enemies. london. international biological programme. 63 biotropia no. 19,2002 nixon gej. 1937. some asiatic telenominae (hymenoptera: proctotrupoidea). ann & mag nat hist 10 (20): 444-475. polaszek a, kimani sw. 1990. telenomus species (hymenoptera: scelionidae) attacking eggs of pyralid pest (lepidoptera) in africa: a review and guide top identification. bull of entomol res 80: 57-71. polaszek a. 2001. an overview of the parasitoids ofspodoptera exigua. workshop on the management ofspodoptera spp. in vegetable crops. kuala lumpur. 14-16 march 2001. rohlf fj. 2000. ntsys pc, numerical taxonomy and multivariate analysis system, version 2.1. exeter software. new york. vaughn tyt, antolin mf. 1998. population genetics of an opportunistic parasitoid in an agricultural landscape, j heredity 80: 152-162. yuliarti n. 2002. karakter morfologi dan molekuler parasitoid telur, telenomus spp. (hymenoptera: scelionidae) dari beberapa daerah di jawa. (tesis). bogor. program pascasarjana ipb. 64 57.pdf 58.pdf 59.pdf 60.pdf 61.pdf 62.pdf 63.pdf 64.pdf 583 stalis norma (genotypic).cdr genotypic and phenotypic characterization of alcaligenes javaensis g j 3 potential as an effective biodegrader stalis norma ethica , oedjijono , endang semiarti , jaka widada 1,2* 3,4 2,3 2,5 and tri joko raharjo 2,6,7 1 , , indonesiafaculty of nursing and health sciences, universitas muhammadiyah semarang semarang 50273 2biotechnology study program, graduate school, universitas gadjah mada, yogyakarta 55281, indonesia 3 faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia 4faculty of biology, universitas jenderal soedirman, purwokerto 53122, indonesia 5 faculty of agriculture, universitas gadjah mada, yogyakarta 55281, indonesia 6faculty of mathematics and natural science, universitas gadjah mada, yogyakarta 55281, indonesia 7 alppt unit iii laboratory, universitas gadjah mada, yogyakarta 55281, indonesi received 18 december 2015/accepted 09 november 2017 abstract utilization of glycerol by lipase producing bacteria offers great benefits for fat and oil waste degradation and waterwaste treatment. nevertheless, there have been lack of reports about the availability of non-pathogenic, lipase producing bacteria, which could naturally degrade glycerol produced from the lipolysis process by lipase. this study reported a newly identified species of rhizobacteria, alcaligenes javaensis jg3, which is not only able to produce high level of lipase, but also able to degrade glycerol molecules. identification of strain jg3 was carried out using sem (scanning electron microscope), bd phoenix 100 automated microbiology system and 16s rrna gene analysis to determine its taxonomy status. the ability of the strain to metabolize glycerol was investigated both genotypically and phenotypically using degenerate pcr and a glycerol minimal medium. identification test results showed that strain jg3 belongs to genus alcaligenes, with the closest relationship with a. faecalis and a. aquatilis (96% nucleotide similarity maximum). degenerate pcr resulted in a 248-bp sequence showing 93% similarity with glpk of candidatus sodalis pierantonius sope, a key gene involved in glycerol metabolism. in vitro glycerol utilization test result showed that alcaligenes sp. jg3 was able to grow on glycerol aerobically, but not anaerobically. it is concluded that alcaligenes sp. jg3 possesses genes coding for glycerol metabolism and this trait is phenotypically expressed, thus making the strain potential to be used as an effective fat and oil biodegrader. keywords: alcaligenes, biodegradation, degenerate pcr, glpk, glycerol metabolism introduction strain jg3 is a soil rhizobacterium originated from root of zea mays cultivated in an agricultural land in purwokerto, central java province, indonesia. previously regarded as azospirillum species, the strain could grow in a mixture of bran and cassava medium containing fats for 8 weeks (oedjijono 2003; ethica et al. 2013a, 2013b).et al. strain jg3 is an attractive research object because strain jg3 is capable of producing quite high level of lipase (lestari et al. 2009, 2016). enzyme activity of crude lipase extract produced by strain jg3 was 25 u/ml, which was considered higher than those previously reported about bacillus subtilis 168 bcl1002 (15 u/ml) and other wildtype rhizobacterial strains isolated from soil, such as pseudomonas spp., bacillus sp., staphylococcus aureus and micrococcus lutens (10 to 17 u/ml) (lestari et al. 2009; lesuisse et al. 1993; charulatha et al. 2012). nevertheless, the taxonomy status of strain jg3 is unclear because a polyphasic study has not been conducted. biological utilization of glycerol has been observed in lipase producing bacteria, such as anaerovibrio glycerini sp. nov., serratia marcescens and * corresponding author: norma@unimus.ac.id biotropia 5 1 8 1 10 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.1.583 1 pseudomonas aeruginosa (schauder & schink 1989; prasad & manjunath 2012). however, not all lipase producing bacteria can utilize glycerol released from the lipolysis process allowing effective degradation of fat and oil waste. for example, bacterial strain burkholderia arboris sl1b1, which secretes lipase and assimilates fatty acids cannot utilize glycerol as a carbon source (matsuoka et al. 2009). on the other hand, applications involving glycerol metabolism have been reported species of genera , from klebsiella citrobacter enterobacter clostridium lactobacillus, , , , bacillus propionibacterium anaerobiospirillum , and (yazdani & gonzales 2007). , the unfortunately potential uses of these organisms limited due are to issues including pathogenicity (murarka et al. 2008). key enzymes in glycerol metabolism of bacteria are g3pdh (glycerol-3-phosphate dehydrogenase) and gk (glycerol kinase) coded by structural glpd and glpk genes (pettigrew et al. 1988; holmberg et al. 1990). expression of two g enes encod ing g l yce ro l -3 -phospha te dehydrogenase and glycerol kinase, the glpd and glpk, was necessary and sufficient to enable growth of corynebacterium glutamicum on glycerol as the sole carbon and energy source (litsanov et al. 2012). williams . (1994) reported that a gram-et al negative strain, nm48, has pseudomonas aeruginosa ability to hydrolyse diacylglycerol using its extracellular lipases to form glycerol and fatty acids and also has ability to perform glycerol uptake. results of the study by williams . et al (1994) demonstrated that washed cells of strain p. aeraginosa nm48 prepared from cells grown on batch culture with glycerol as carbon source exhibited high glycerol uptake, glycerol kinase and glycerol-3-phosphate dehydrogenase activities. t of strain jg3 he ability to produce lipase enzyme the strain is an indication that could potentially be microbial degradation of used in fats and oils, which is important for the treatment of wastewater from restaurants and food industries (matsuoka 2009). also, et al. investigation of new strain which is nonpathogenic, facultative anaerobic and easy-togrow with adequate genetic information enabling genetic manipulation is important to lead toward effective microbial degradation. therefore, this study was aimed identify key genes involved in to glycerol metabolism and to investigate the ability of strain jg3 in performing glycerol metabolism as an effective biodegrader. materials and methods bacterial strain rhizobacterial strain jg3 used in this study was kindly provided by mr oedjijono (as generous gift) from microbiology laboratory, faculty of biology, universitas jenderal soedirman, indonesia. subculture and cultivation of glycerol degrading bacterial strain strain jg3 previously received as culture on nutrient agar medium was immediately stored at 15 °c. several plates and tubes containing instant 20 g/l na (nutrient agar), 15 g/l tsa (tryptic soy agar) with 5% sheep blood, 20 g/l macconkey and 8 g/l nb (nutrient broth), were prepared. strain jg3 was then purified using single-cell colony technique. colonies having circular, regular and flat shapes with whiteyellowish color were selected and grown on both na and nb media at 30 °c for 24 hours. single colonies from the pure culture were also inoculated on tsa having 5% sheep blood and macconkey plates at similar temperature condition to identify the possibility of strain jg3 in exhibiting pathogenic characteristics, one of which is causing blood haemolysis. pure cultures on na were used for direct colony pcr (pcr using bacterial cells as template without prior dna extraction). phylogenetic analysis a cladogram was created using neighbor-joining algorithm developed by saitou and nei (1987) based on the obtained sequence of 16s rrna gene of bacterium jg3. its homologs in other rhizobacteria retrieved from genbank database were created using mega 6.0 software. the estimates calculations on evolutionary divergence among sequences were determined using maximum composite likelihood model (tamura et al. 2004, 2013). all required alignments were carried out using clustalw (thompson et al. 2002). 2 biotropia vol. 25 no. 1, 2018 mncl ·4h o, 0.02 g/l cacl ·2h o, 0.018 g/l 2 2 2 2 feso ·7h o and 1 g/l yeast extract. into 100 ml 4 2 of the minimum medium, 1 l starter was μ introduced in two larger tubes under aseptic condition, incubated at 37 °c for 48 hours. one tube was put in an anaerobic jar (anaerocult, merck) which cap was loosely placed on the tube to allow gas exchange, but avoiding evaporation; the jar was placed on a shaker. the other tube was also put on a shaker with tightly placed cap. this experiment was carried out in triplicates. the fermentation was non-ph controlled and nonstirred, due to the low reaction volume. after 48 hours of fermentation, the 10-ml fermentation sample was acidified to ph = 2.0 with 17% h po . the ph was checked using ph indicator 3 4 strips (ph 2.0 to 9.0, merck). the acidified fermentation sample was centrifuged at 5,000 rpm (3,836 x g) for 20 minutes in a beckman avanti j-251 centrifuge at room temperature. after centrifugation, supernatants were collected and filtered through a 0.45-m-pore-size filter (milipore, denmark). as much as 1 ml supernatant of fermentation sample was subjected to gas chromatography – mass spectroscopy (gc-ms) assay. gc-ms assay of glycerol utilization gas chromatography-mass spectroscopy (gc-ms) assays were performed to detect possible growth strain jg3 in minimal glycerol of broth under aerobic condition and to identify glycerol fermentation products which might be expressed by its glycerol genes involved in metabolism composition of two samples . (aerobic and anaerobic isolates) was analyzed using gc spectrophotometer gd 2010 shimadzu equipped with ms detector, a rastek rxi-5ms column, and he polysiloxilane (helium) as the carrier gas for the presence of any possible fermentation products. each sample was prepared by adding 10 l of 6n hcl to a 900 l μ μ of cell-free sample (kim 1991). helium gas as the carrier gas was used. column the temperatures of oven, 40 , injector and detector were °c 310 °c and 250 °c, respectively. the temperature gradient was as follows: 40 °c for 5 minutes, ramped at 10 °c to 280 °c per minute, with 31 minutes hold time mode was split. the injection , column flow was 0.56 ml/minute totaland the flow was 40 ml/minute. degenerate primer d esign sequences of glpks required to design degenerate primers were obtained from genbank. global alignments were performed using clustalw (thompson et al. 2002) and were used as input for primaclade (gadberry et al. 2005). a pair of primers having the least possibility of hairpin formation, selfcomplementarity and dimerization was selected. gene isolation amplification by colony pcr using the designed primers aiming to amplify the glpk partial region (gkf and gkr) was performed in a 25 μl reaction volume at annealing temperature of 50 °c with other pcr parameters set as previously described (ethica et al. 2013b, 2017). the isolated dna from this process was purified, followed by sequencing. the sequencing of all pcr products was conducted using bigdye® terminator v3.1 sequencer system (applied biosystem, usa). the obtained sequences were ready for analysis. blast and sequence analysis the sequence of partial glpk and 16 rrna genes were deposited in genbank to obtain accession number. homology analyses were performed using blastn and blastx (altschul et al. 1997). the alignments based on deduced amino acids were conducted using clustalw (thompson et al. 2002). the phylogenetic relationship of the obtained sequence with other sequences referred by blast from other organisms already deposited in genbank was determined using mega 6.0 (tamura et al. 2004, 2013). in vitro glycerol utilization test in vitro a small-scale glycerol utilization test was performed to identify the ability of jg3 isolate in utilizing glycerol both in aerobic and anaerobic conditions. for starter, cultured bacterial cells (1 μl 24-hours) from nb medium was added into a reaction tubes containing10 ml of autoclaved minimal medium loaded with 90 g/l glycerol, 0.26 g/l mgcl , 0.01 g/l namoo ·2h o, 10.9 2 4 2 g/l kh po , 2.84 g/l k hpo , 0.08 g/l 2 4 2 4 (nh ) so , 0.66 g/l nh cl, 0.016 g/l 4 2 4 4 3 genetic characterization of alcaligenes javaensis jg3 potential as an effective biodegrader ethica et al. phenotypic characterization phenotypic characterization was conducted based on analysis of bacterial cell morphology, biochemical tests, tests of the ability of the strain to degrade glycerol substrate. morphological tests were conducted to examine the colony morphology of strain jg3 on nutrient, macconkey and tsa agar media including the shape, color, end, elevation and structure of the colonies. observation on strain's cell morphology was conducted using scanning electron microscope (sem) with 7,500x magnification. for sem observation, fresh bacterial cells were suspended in a phosphate-buffered salt solution. the cells were then fixed with 0.5% glutaraldehyde, washed several times and dehydrated in a series of ethanol concentrations. after the cells were sputter coated with goldpalladium, they were observed with an sem (model jsm 6300 f; jeol, japan) at 3 kv. gram staining was performed based on previously reported method (hucker 1921). for biochemical test automated system bd , an phoenix 100 with protocols suggested was utilized by the manual of the instrument. culture from solid medium was first suspended in phoenix id tm broth to adjust in 0.5 – 0.6 mcfarland turbidity using a crystal spec nephelometer. a drop of phoenix ast indicator solution was added to each phoenix ast broth tube prior to inoculation tm with 25 µl of the suspension in a final concentration of 5 x 105 cfu/ml). nmic/id-5 phoenix panels were inoculated within 30 tm minutes of initial preparation. panels were scanned and placed into the phoenix instrument tm for incubation at 35 °c , followed by reading. phylogenetic characterization of glycerol degrading bacterial strains g e n o m i c d n a f o r p h y l o g e n e t i c characterization was extracted and purified using purelink® quick gel extraction kit following instructions of its manufacturer (promega 2010). the 16s rrna genes were amplified using pcr w i t h p r i m e r s o f 2 7 f ( 5 ' agagtttgatcctggcctcag-3') and 1492r (5'-ggttaccttgttacgactt-3'). the 16s rrna gene amplification was carried out using genomic dna of the strain as template (turner 1999). pcr (thermal cycler applied et al. biosystem) was performed to amplify the 16s rrna genes in a final volume of 25 l, which μ consisted of genomic dna (50 ng/ l) 0.5 l, μ μ 8f1492r primers (10 m) 1 l each, 12.5 μ μ polymerase kit and 10 l dh o. amplification μ 2 was carried out at 95 °c for 4 minutes; 30 cycles at 95 °c for 30 seconds, 30 cycles at 55 °c for 30 seconds, 30 cycles at 72 °c for 2 minutes and the final extension at 72 °c for 7 minutes. the sequences of the 16s rrna genes from each isolate were used as query to determine the genes and species of its closest proteobacterial relative using blastn (altschul 1990). et al. subsequently, sequences were aligned using clustal x program developed by thompson et al. (1994). phylogenetic trees were inferred by the neighbor-joining method (saitou & nei, 1987) with the phylogenetic analysis package mega 6.0, which included the use of tools to plot the tree topologies. to provide confidence estimates for branch support, a bootstrap analysis was performed in 1,000 replications (felsenstein 1985). reference sequences of 16s rrna genes were obtained from genbank, which were included in the phylogenetic analysis (chaerun et al et al. 2012; sya'di . 2017). results and discussion taxonomy status of strain jg3 taxonomy status of strain jg3 was determined using polyphasic approach involving morphological, biochemical and molecular identifications. growth of single colonies of strain jg3 was first obtained from colony purification using na medium showing different results on three plates containing na, tsa with 5% sheep blood and macconkey agar. morphology of colonies grown on the three media is displayed in figure 1. strain jg3 could grow on na, a common medium for a wide variety of bacteria, displaying round, irregular, smooth, flat shapes with yellowish color (fig. 1). strain jg3 could also grow on tsa media containing blood without causing haemolysis typically exposed by pathogenic bacteria attacking blood. on tsa medium, strain jg3 showed punctiform, dry, irregular shapes with greyish color. after the gram-staining process, strain jg3 showed characteristics as a gram-negative bacterium. the 4 biotropia vol. 25 no. 1, 2018 ability of strain jg3 to grow on various media without the need of strict temperature condition or specific treatment showed that the rhizobacterium was easy to grow. however, it did not show growth on macconkey agar plate .) macconkey medium is commonly used to cultivate gram-negative, enteric, pathogenic bacteria (allen 2005). inability of the bacteria to grow on macconkey agar medium is also a typical sign that the bacteria could not ferment lactose (allen 2005) . results of this study showed that strain jg3 was able to grow both on complex and blood containing media without causing blood haemolyses, indicating that the strain could be categorized as an opportunistic organism, but not significantly pathogenic. cellular morphology of strain jg3 was further examined using scanning electron microscopy (sem) showing the appearance of strain jg3 cells as rods or coccobacilli having width of 0.7 1.0 µm (fig. 2). according to sem result, typical cells of strain jg3 were slightly varied in size, but basically were rod-shaped occurring as single, in pairs or short chains. in terms of morphological characteristics, the punctiform-shaped colonies of strain jg3 on tsa medium matched the characteristics of gram-negative rhizobacteria, , alcaligenes belonging to sub-phylum . cells betaproteobacteria of strain jg3 did not show any curved or vibrioid shapes, yet only straight rods or coccobacilli having sizes of 0.7 – 1.0 x 0.5 – 2.6 µm, which were smaller than most members. the bacillus bergey's manual states that are alcaligenes members of the class .betaproteobacteria biochemical assay on strain jg3 was carried out using bd (beckton dickinson) phoenix 100 automated microbiology system. 5 genetic characterization of alcaligenes javaensis jg3 potential as an effective biodegrader ethica et al. a b c figure 1 observed growth of bacterial strain jg3 on various agar media after 24-hour incubation at 30 °c: a. growth on nutrient agar; b. growth on tsa agar plate containing 5% blood; c. no growth was observed on macconkey agar plate figure 2 cells of bacterial strain jg3 observed under scanning electron microscope (sem; jeol, 5310-lv; 20kv) 6 biotropia vol. 25 no. 1, 2018 figure 3 bd phoenix 100 screening report on strain jg3 forty-four biochemical characteristics of strain jg3 were shown as the output of bd phoenix 100 automated microbiology system (fig. 3). based on the bd phoenix screening readout, strain jg3 was detected as species alcaligenes faecalis rhizobacterium with 99% confidence. genotypic identification of strain jg3 relied on the analysis of 16s rrna gene using universal or species-strain specific primers. amplification of partial 16s rrna gene from strain jg3 using genomic dna as template (seen as a band at ~4,000 bp) resulted in single dna band on tae-electrophoresis gel with a size of ~1,500 bp (fig. 4). the dna band was excised from gel, purified and sequenced resulting in 1,511-bp nucleotide sequence, which was then deposited in genbank under accession number of ab914514. figure 4 gel electrophoresis analysis of pcr products using gkf and gkr primers: m = marker, lane 1 = amplified dna fragment using gkf1 and gkr primers 7 genetic characterization of alcaligenes javaensis jg3 potential as an effective biodegrader ethica et al. results of identification tests revealed that strain jg3 belongs to genus, namely alcaligenes alcaligenes sp. jg3. although the results of morphological and biochemical tests showed that the strain resembled having alcaligenes faecalis coccobacillus cell shapes and having bd phoenix confidence of 99%, the phylogenetical features of the strain based on 16s rrna exposed only had 96% similarity to the closest species in phylogenetic tree (fig. 5). the similarity level was below the suitable similarity cut-off for the identification of new taxa at genus and species levels based on 16s rrna gene, which are 97% and 99%, respectively (drancourt 2000).et al. based on phylogenetic analysis and results of morphological and biochemical tests, in accordance wi th bergey 's manual of determinative bacteriology (garrity 2005), a et al. novel species of the genus isolated from alcaligenes root of cultivated in a land of central java zea mays province, indonesia, strain jg3 alcaligenes javaensis is proposed. detection of glpk degenerate pcr colony using gkf (5'atcggcatcaccaaccagc-3') and gkr (5'ggycacrtcctcgccatc -3') primers designed using primaclade resulted in a single band on gel electrophoresis corresponding to dna size of around 271 bp (fig. 5). after sequencing, it was obtained as 248-bp nucleotide sequence deposited in genbank database under accession number of ab894421. result from blastn search indicated that the sequence shared 93% similarity with of glpk candidatus sodalis pierantonius sope atcc 15264, suggesting that it is likely part of gene encoding glycerol kinase. this result was in line with the previous study revealing the presence of in glpd strain jg3, another key gene responsible for glycerol utilization (ethica 2013a) revealing et al. that genotypically strain jg3 has ability to metabolize glycerol. glycerol degradation by strain jg3 results of the test showed that in vitro alcaligenes sp. jg3 could grow on minimal medium containing glycerol as carbon source aerobically, but not anaerobically. after 48 hours, turbidity of medium in aerobic sample reached od = 1 as sign of bacterial growth, which was 600 later confirmed by results of gc assays. the obtained gc chromatogram (fig. 6) of both aerobic and anaerobic samples were aligned showing shifted retention time (tr) by 3.3 minutes between two single peaks of both samples, which were identified as glycerol by mass spectra showing significance identity (si) level of 95 and 96%, respectively, with glycerol compound based on mass spectral matching with reference library. there was no significant substance other than glycerol detected by gc on both aerobic in anaerobic samples. the size of figure 5 a cladogram showing position of strain jg3 strain based on 16s rrna gene sequence relative to other bacteria (note: this cladogram was created using mega 6.0, a program developed by tamura et al. 2013) 8 biotropia vol. 25 no. 1, 2018 glycerol peak area of anaerobic sample was larger than that of aerobic sample indicating that the concentration of glycerol in anaerobic sample was higher than that in aerobic sample. since there was no growth in anaerobic sample, it was assumed that glycerol quantity in anaerobic samples represented the initial glycerol quantity of the samples. thus, the lower glycerol concentration in aerobic sample after 48 hours incubation was a sign that glycerol in the medium was aerobically utilized by strain jg3. identification on strain jg3 using polyphasic approach consisting of phenotypic and genotypic tests has been conducted in this study. by evaluating morphological, biochemical and phylogenetical features of strain jg3 based on information from bergey's manual, the rhizobacterium was classified as genus , alcaligenes namely sp. jg3. as stated by drancourt alcaligenes et al. (2005), 99% similarity is a suitable cutoff for identification at species level and 97% similarity is suitable cutoff for identification at genus level to determine new species based on 16s rrna gene. this means that strain jg3 has high level of novelty, both in genus and species levels. results of glycerol utilization tests for strain jg3 showed that the strain, which was previously known as lipase producer, also has an active aerobic glycerol metabolism. as stated in bergey's manual, and its subspecies ( a. faecalis a. faecalis subsp. and subsp. ) faecalis a. faecalis parafaecalis usually have no ability to grow on glycerol. a. latus was found to have the ability to utilize glycerol, but no associated data was found for the rest members of genus (garrity 2005). alcaligenes et al. therefore, strain jg3 has uniqueness, distinguishing the strain from other members of genus in terms of glycerol utilization.alcaligenes glycerol utilization property of strain jg3 also shows the ability of the strain to effectively degrade fat. it is possible for strain jg3 to degrade fat using lipase produced by itself and subsequently metabolize glycerol resulted from this degradation process. the ability of strain jg3 to produce lipase enzyme along with the ability to utilize glycerol aerobically is a strong indication that the strain could potentially be used as an effective degrader of oils and fats. such ability is essential for application in wastewater treatment including in grease-traps installed for the figure 6 chromatogram of liquid containing glycerol minimal medium and bacterial strain under aerobic (top) and anaerobic (below) conditions for 48 hours 9 genetic characterization of alcaligenes javaensis jg3 potential as an effective biodegrader ethica et al. treatment of wastewater from restaurants and food industries as initially developed by matsuoka et al. (2009). conclusions a new taxa of genus alcaligenes identified in this study, alcaligenes sp. jg3, possesses a gene involved in glycerol metabolism and has active glycerol metabolism shown by its ability to grow on a minimal glycerol medium. strain jg3 also has ability to produce lipase enzyme indicating that strain jg3 could potentially be used as effective microbial degraders of fats and oils for application in wastewater treatment. acknowledgements the manuscript was dramatically improved after receiving technical support from clinical program on articles writing in international journal, directorate of intellectual property management, ministry of research, technology and higher education (kemenristek dikti) of the republic of indonesia. the authors also acknowledge and thank overseas seminar assistance program, directorate general of research and development reinforcement of kemenristek dikti, for the support in publishing this paper. deep gratitude is due to mr tri joko raharjo from laboratorium penelitian pusat terpadu (lppt), universitas gadjah mada, yogyakarta, indonesia, for financial support and supervision to carry out this study. great gratitude is also extended to mr oedjijono from microbiology laboratory, faculty of biology, universitas jenderal soedirman, indonesia, for providing living materials used in this study. references allen me. 2005. macconkey agar plates protocol. washington (us): american society for microbiology. p. 1-4. altschul sf, madden tl, schäffer aa, zhang j, zhang z, miller w, lipman dj. 1997. gapped blast and psi-blast: a new generation of protein database search programs. nucleic acids res. 25:3389–402. chaerun sk, hasni s, sanwani e, moeis mr. 2012. mercury (hg)-resistant bacteria in hg-polluted gold mine sites of bandung west java province. indonesia microbiol 6:57-68. charulatha r, prabhakar s, sivamani p, kandeepan c, tiagarajan v. 2012. characterization of clinical app l i c a t ion o f ex t r ace l l u l a r l i pa se by 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gonzalez r 2008. . fermentative utilization of glycerol by escherichia coli and its implications for the production of fuels and chemicals. app environ microbiol 74 1124-35: . oedjijono, ryandini d, permiarti. 2003. formulasi biofertilizer dari kultur campuran bakteri pemfiksasi nitrogen dan pelarut fosfat pada medium onggok dan dedak [research report, unpublished]. retrieved from fakultas biologi universitas jenderal soedirman. pettigrew dw, ma dp, conrad ca, johnson jr 988 . 1 . escherichia coli glycerol kinase, cloning and sequencing of the glpk gene and the primary structure of the enzyme. j biol chem 263 135-9: . prasad mp, manjunath k. 2012. effect of media and process parameters in the enhancement of extracellular lipase production by bacterial isolates from industrial effluents. int j microbiol res 4:308-11. promega 2010 , . . wizard® genomic dna purification kit technical manual (tm050), section, 3.g. isolating genomic dna from gram-positive and gramnegative bacteria . madison 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and plastids by small subunit rrna sequence analysis. j euk microbiol 46:32738. william sg, greenwood ja, jones cw. 1994. the effect of nutrient limitation on glycerol uptake and metabolism in continuous cultures of pseudomonas aeruginosa. microbiology 140(11):2961-9. yazdani ss gonzalez r 2007 anaerobic fermentation , . . of glycerol: a path to economic viability for the biofuels industry. curr opin biotechnol 18ion : 213 9. 10 biotropia vol. 25 no. 1, 2018 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 3. suryo wiyono.cdr biotropia vol. 20 no. 1, 2013: 19 28 powder formulation of antagonistic yeasts, and as biofungicides cryptococcus albidus cryptococcus terreus suryo wiyono* and widodo recipient of biotrop research grant 2009/accepted 14 may 2012 this study aimed 1) to investigate the compatibility of yeast antagonists i.e. and 2) to obtain suitable carrier in powder formulation for those two yeasts, 3) to get appropriate formulation additives for those yeasts, and 4) to obtain optimum powder formulation consisting of yeasts, carrier materials and additives. compatibility of and were tested through bio-assay against on detached banana fruit and on detached tomato leaves. compatibility was indicated by no reduction of antagonistic activity. benomyl-resistant mutant of and cycloheximide-resistant mutant of were mixed with sterilized tapioca, talc and kaolin to get initial yeasts density of 8.5 log cfu/g and water content of 15%, then packed in plastic bag and stored under room temperature. survival of formulated yeasts was assessed monthly by planting on pda medium containing 150 ppm cycloheximide for , and 150 ppm benomyl for . yeasts population was expressed in log cfu/g materials. to determine the effect of carrier materials on antagonistic activity, io-assay of formulated yeasts against pathogens was conducted after 3 months of storage. tested additives cacl , pure chitin and crab shell powder were added into suspension of and to get concentration of 1.25%, 0.5 % and 0.1% (w/v). sterilized distilled water and yeasts without additives was used as control. then, the treatments were examined for the antagonistic activity through bio-asaay on detached banana fruits and tomato leaves. appropriate additive(s) was determined by its ability to increase antagonistic activity of yeasts. storability and antagonistic activity of and in the mixture of best carrier and additive were examined. was compatible to . talc was the best carrier material supporting highest survival of , without contamination of other microorganisms. talc was the best carrier material for formulation by maintaining its survival for six months of storage. additives able to increase antagonistic activity of were cacl 0.1 %, pure chitin 0.1 % and crab shell powder 0.5%. all of tested additives materials did not affect antagonistic activity of supplementation of chitin and crab shell, both at the rate of 1.25% into talc-base powder formulation increased survival of and . formulation, yeast antagonists carrier agent, talc, tapioca, kaolin, cacl , chitin, crab shell powder 1 department of plant protection, faculty of agriculture, bogor agricultural university darmaga, bogor-indonesia c. albidus c. terreus, c. albidus c. terreus lasiodiplodia theobromae alternaria solani c. albidus c. terreus c. terreus c. albidus b i.e. c. albidus c. terreus c. albidus c. terreus cryptococcus terreus c. albidus c. terreus c. albidus c. terreus c. albidus. c. terreus c. albidus , cryptococcus terreus, cryptococcus albidus, abstract 2 2 2 key words: * corresponding author : suryow@hotmail.com 19 introduction a group of novel promising biocontrol agents of plant disease are antagonistic yeasts. the advantage of yeast as biocontrol agent is dry and heat tolerance, therefore, adaptable on leaves and other aerial plant parts. it is fast growing, easy to be mass cultured and socially more acceptable (spadaro 2003). research in foreign countries showed the effectiveness of yeast in controlling plant disease. a yeast antagonist was reported effective to control apple rot disease caused by (spadaro 2003). moreover, near harvest application of effectively control post harvest disease of grapefruit (karabulut . 2003). on tomato plants was reported to be effectively controlled by the use of epiphytic yeasts strains 101 and us 7 and (saligkarias . 2002). and collected by the authors have been screened for their antagonistic activity , bio-assay , field test and partially characterized. and were effective yeast antagonists against stem end rot of mango caused by and petal blight of orchid caused by , respectively (wiyono 2008; sugiprihatini 2009). recently, the second yeast is also effective tested against leaf blight of tomato caused by and chrysanthemum white rust caused by . in addition, fan and tian (2001) reported that is also effective in controlling apple post harvest diseases i.e. grey mold and blue mold. after obtaining superior isolates of antagonists, technology for mass production and formulation is required to develop an antagonist as biocontrol agent on commercial scale. since those two yeasts are relatively easy to be mass cultured in a cheap medium (potato dextrose broth pdb), formulation technology is a critical step. suitable formulation technology is the main limiting factors in developing microbial pesticides in indonesia, even high numbers of effective antagonists had been isolated (santoso . 2005). appropriate formulation technology will facilitate storage, transportation, application technique and also bio-performance of antagonists. solid formulation is chosen to be developed for easier transportation and handling. formulation of beneficial microbes contains microbes, carrier and additives. combination of microbes will broaden the target of bio-fungicide. optimization of whole components ensures the quality of formulation and further commercial application. some minerals such as kaolin and talc can be used as carrier in formulation of and (kinay & yildiz 2008). additives play also an important role in biocontrol performance. calcium chloride and chitin were used as additive enhancing antagonistic activity of yeasts antagonists and . compatibility of the two yeast antagonists, appropriate as carrier materials and additives for and has not been investigated yet. the objectives of the research were: 1) to assess the compatibility of two yeast antagonists and , 2)to obtain cheap material as carrier agent which ensure long time survival and bio-performance of and 3) to get formulation additives supporting storability and bioperformance of yeast antagonist metschnikowia pulcherrima penicillium metschnikowia fructicola et al botrytis cinerea candida guilliermondii candida oleophila et al cryptococcus albidus cryptococcus terreus in vitro in vivo cryptococcus albidus cryptococcus terreus lasiodiplodia theobromae curvularia pallescens alternaria solani puccinia horiana crypococcus albidus et al metschnikowia pulcherrima pichia guilliermondii candida guilliermondii, pichia membranefaciens, cyptococcus laurentii c.albidus c. terreus c. albidus c. terreus c. albidus c. terreus, 20 biotropia vol. 20 no. 1, 2013 c. albidus c. terreus cryptococcus albidus c. terreus c. albidus c. terreus lasiodiplodia theobromae alternaria solani lasiodiplodia theobromae alternaria. solani c. albidus c. terreus l. theobromae a. solani c albidus, c. terreus c albidus c. terreus c. albidus c. terreus and ., and 4) to obtain optimum solid formulation in combination with yeasts, carrier material and additives antagonistic yeasts used were and obtained from the author's collection. for formulation purpose, benomyl-resistant mutant of which is similar to antagonistic activity was generated. in addition, resistant to cycloheximide was used in formulation experiment. and collections of the author were used for bio assay of yeasts. both of the two yeast antagonists were cultured in potato dextrose broth (pdb difco) and harvested at early stationary phase of growth, centrifuged at 5000 g (jouan centrifuge br4i), washed with sterilized distilled water, mixed with saline solution adjusted to appropriate density. was cultured on pda (difco) ph 5.5 for seven days and the conidia harvested by soaking water on the surface and filtered with cheese cloth. was cultured on s-medium and incubated for seven days under nuv exposure to induce sporulation (abadi 1987), prior to harvesting of conidia io-assay of yeast antagonists was conducted based on previous technique developed by the authors using detached banana fruits and detached leaves of tomato. the detached organs were placed on moistened plastic pans (30 cm x 25 cm x 5 cm), one plastic pan contained five banana fruits or tomato leaves. detached banana fruits were dipped in cell suspension of at 7 log cfu /ml, while the detached leaves were dipped in cell suspension of 7 log cfu/ml, both added with wetting agent tween 20, 0.005 %. then the treated banana fruits and tomato leaves were air dried. sterilized distilled water was used as control. conidia suspension at 50 µl of and , both at the density of 10 conidia /ml was placed on the surface of fruit and leaves, respectively. inoculated fruits and leaves were stored under dark condition for 24 hours and then incubated under room temperature (27 c) and photoperiods of 12: 12 (d:l). disease severity was assessed by estimating of necrosis part at five days after inoculation and expressed in percent. effective treatment was indicated by low disease severity he treatment consists of , a mixture of and , and untreated (water). each treatment was replicated five times. yeast concentration used for compatibility test was 5 x 10 cfu/ml for and 5 x 10 cfu/ml , therefore obtaining final concentration of 7 log cfu/ml, with ratio of 1:1, and added with wetting agent tween 20, 0.005 %. bio-assay was conducted on detached fruit of banana and detached leaves of tomato (see bio-assay of antagonistic activity). . . b . t materials and methods yeast and pathogen preparation bio-assay of antagonistic activity of yeasts compatibility of and 4 o 6 6 c. albidus c. terreus 21 powder formulation of antagonistic yeasts as biofungicides suryo wiyono– et al. if the two yeasts are compatible, it will be further tested in a formulation experiment aterials tested for carrier were kaolin powder, talc powder and tapioca. each material was regarded as treatment and replicated five times. the materials were sterilized by standard autoclaving. suspension of two yeast antagonists and its mixture ( preparation mentioned above) was mixed by spraying (hand sprayer yoto 1-l, made in indonesia) yeast suspension on the tested materials in running blender, then air dried to get final yeast density of 8.5 log cfu/g with 15% of water content of formulation. materials containing yeast were then packed in plastic bags and stored under room temperature for 6 months. each material containing yeast was assessed for yeast survival and antagonistic activity every 30 days for six months. survival of yeast in each carrier materials was determined by plating on pda (difco) ph 5.5 containing 150 ppm benomyl for , and 150 ppm cycloheximide for colony isolated from each carrier material of three month storage was then tested for its antagonistic activity using the technique as described in bioassay of antagonistic activity aterials screened for additives are pure chitin (sigma), natural material containing chitin i.e. crab shell powder, and calcium chloride. methods for testing the formulation additives are based on previously developed technique (wiyono . 2008). each material was tested with water-based suspension/solution at the rate of 1.25 %; 0.5%; 0.1% (w/v). suspension of and at density of 7 log cfu/ml mixed with the tested additives and final concentration of additives was adjusted to the tested concentration. yeast suspension without addition of tested materials was used as control. yeasts with various additives treatment was furthermore tested for their antagonistic activity using the technique as described in bioassay of antagonistic activity est results obtained from previous experiment with yeasts compatibility, carrier materials and additives were then continued to test for suitable powder formulation . combination of yeasts, selected carrier materials, selected additives in powder formulation was conducted. the survival and antagonistic activity were also assessed every 30 days for four months he research showed that was compatible to . effectiveness . m . m . b . t screening for carrier materials screening for additives combination of suitable yeast composition, carrier materials and additives compatibility of dan c. albidus c. terreus. et al c. albidus c. terreus c. terrreus c. albidus results and discussion c. terreus c. albidus 22 biotropia vol. 20 no. 1, 2013 23 of the two yeast antagonists did not decrease in mixed application (tables 1 and 2). the compatibility of the two yeasts make broader spectrum of the mixture. one important advantage of antagonists combination is the possibility to obtain a broader spectrum (burges & jones 1998). survival of dan in various carrier materialsc. terreus c. albidus c . e . t arrier materials are important component of formulation for maintaining microbes survival and antagonistic activity. among materials tested for , tapioca provided the highest survival. overall, could survive in all tested carriers for four months. during five months of storage could not be detected anymore in all of the tested materials (table3). talc powder provided highest survival for (table 4). up to six months of storage, talc powder still resulted in relatively high survival (4.60 log cfu/g) ven though tapioca is the best material for storage, it has high contamination level of other fungi. tapioca is an organic flour, hence it can act as nutrition for some other fungi and bacteria. this was not the case with mineral powder such as kaolin and talc. kinay and yildiz (2007) stated that talc in granular formulation is able to provide storability of antagonistic yeasts and for more than 6 months. storability of talc was better than kaolin. storability of talc in this experiment was lower than that reported by kinay and yildiz (2007), because this experiment used powder formulation instead of granular form he use of talc as carrier material in powder formulation is also able to maintain bio-performance of the two yeasts in storage (tables 5 and 6). antagonistic activity of c. terreus c. terreus c. terreus c. albidus c. albidus c. terreus metschnikowia pulcherrima pichia guilliermondii table 1. compatibility of and in controlling fruit rot of banana caused by cryptococcus albidus c. terreus l. theobromae treatment disease severity (%) untreated 34.53 b cryptococcus albidus 26.00ab cryptococcus terreus c. terreus+ c. albidus 7.53 a note: numbers followed by same symbol are not significantly different at p<0.05 with drmt test table 2. compatibility of and in controlling alternaria leaf blight of tomato caused by cryptococcus albidus c. terreus alternaria solani treatment disease severity (%) untreated 10.58 b cryptococcus terreus 5.55 a c. terreus+ c. albidus 5.48 a note: numbers followed by same symbol are not significantly different at p<0.05 with drmt test powder formulation of antagonistic yeasts as biofungicides suryo wiyono– et al. 24 biotropia vol. 20 no. 1, 2013 table 3. survival of on various carrier materials in powder formulationc. terreus 5 carrier materials yeast density (log cfu/g) in i-th month 0 1 2 3 4 tapioca 8.70 a 7.06 b 7.23 b 7.36 b 4.18 b talc 8.65 a 6.00 a 6.54 a 5.69 a 4.50 b kaolin 8.65 a 6.67 a 6.24 a 6.26 a 3.82 a note: = undetected numbers followed by same symbol are not significantly different at p<0.05 with drmt test table 4. survival of on various materials in powder formulationc. albidus carrier materials yeast density (log cfu/g) in i-th month 0 1 2 3 4 5 6 tapioca 8.24 a 5.95 a 6.98 a 4.21 a talc 8.72 a 5.70 a 6.65 a 5.98 b 6.17 4.20 4.65 kaolin 8.60 a 5.25 a note: -= undetected numbers followed by same symbol are not significantly different at p<0.05 with drmt test table 5. antagonistic activity of formulated with different carrier materials against alternaria leaf blight of tomato (after 3 months storage) c. terreus carrier materials alternaria leaf blightseverity (%) control (water) 7.64 b unformulated fresh yeast 2.13 a tapioca 0 a talc 2.12 a kaolin 1.13a note: numbers followed by same symbol are not significantly different at p<0.05 with drmt test table 6. antagonistic activity of formulated with different carrier materials against fruit rot of banana (after 3 month storage) c. albidus carrier materials fruit rot severity (%) control (water) 16.23 b unformulated fresh yeast 5.24 a tapioca 9.14 a talc 5.22 a kaolin note: numbers followed by same symbol are not significantly different at p<0.05 with drmt test 25 c. albidus c. terreus pseudomonas fluorescens et al c. terreus c. albidus, and in all tested materials (except kaolin) did not decrease after 3 months of storage indicated by no significant difference to unformulated fresh yeast ormulation additives such as additional nutrients are often important in formulation of beneficial microbes. some formulation additives are able to increase antagonistic activity of antagonistic microbes, for example zinc and manganese can improve antagonistic activity of b5 (wiyono . 2008). among tested additives, ca cl 0.1%, pure chitin 0.1% and crab shell powder 0.5% significantly increase antagonistic activity of (table 7). even though there was no significant difference among the three treatments, crab shell powder provided highest increase of antagonistic activity dditives treatment did not increase significantly antagonistic activity of however cacl 0.5%, crab shell powder 1.25% and pure chitin 1.25% tend to increase . f . a additives-mediated enhancement of antagonistic activity of and . c. terreus c. albidus 2 2 table 7. effect of additives on the antagonistic activity of c. terreus treatment conc. (% w/v) alternaria leaf blightseverity (%) water 25.45 e yeast without additives 9.52 cd ca cl2 1.25 7.12 abcd ca cl2 0.5 5.35 abc ca cl2 0.1 4.52 ab chitin 1.25 10.00 d chitin 0.5 8.35 bcd chitin 0.1 4.25 ab crab shell powder 1.25 4.80 ab crab shell powder 0.5 3.14 a crab shell powder 0.1 3.94 ab note: numbers followed by same symbol are not significantly different at p<0.05 with drmt test table 8. effect of additives on the antagonistic activity of c. albidus treatment conc. (% w/v) fruit rot severity (%) water 55.00 c yeast without additives 35.00 ab ca cl2 1.25 41.25 ab ca cl2 0.5 21.25 a ca cl2 0.1 43.75 ab chitin 1.25 15.75 a chitin 0.5 53.75 b chitin 0.1 43.75 ab crab shell powder 1.25 21.25 a crab shell powder 0.5 41.25 ab crab shell powder 0.1 43.75 ab note: numbers followed by same symbol are not significantly different at p<0.05 with drmt test powder formulation of antagonistic yeasts as biofungicides suryo wiyono– et al. 26 biotropia vol. 20 no. 1, 2013 antagonistic activity (table 8). furthermore, crab shell powder 1.25% was used in powder formulation containing mixture of and , because its effect is not significant to chitin and its price is far cheaper than pure chitin the research result was in line with previous researches in other yeasts. antagonistic activity of yeast (various species or isolates) can be enhanced by addition of calcium chloride (tian . 2002; abadias . 2003), pure chitin (vivekananthan . 2004; yu . 2007). calcium chloride could increase the biocontrol efficacy of tested yeast due to its ability to enhance plant resistance against plant diseases (biggs . 1997; droby . 2003). the same mechanism is similar for chitin (yu . 2007). this research resulted in a new finding that crab shell powder, a chitin-containing material, provides the same level of enhancement compared to pure chitin. the mechanism how crabshell increase antagonistic activity of is not exactly known, probably it involves induction of plant resistance. harti (2010) reported induced resistance of banana against fusarial wilt diseases after treated with crab shell powder. aside from chitin, crab shell contains protein, calcium, phosphate, and other elements such as iron, manganese and zinc (multazam 2002), therefore effect of composing elements of crab shell powder on antagonistic activity could not be ignored. this is an advantage since using crab shell powder is cheaper than pure chitin. the use of crab shell powder does not need chemicals for processing, so it is costeffeective. ne strategy to improve storability and bio-performance of formulated microbes is by providing additives (burges & jones 1998; fravel 2005; wiyono 2008). some additives of antagonistic yeasts are metal ion (calcium), sugar (trehalose), biopolymer (chitin) and calcium (abadias . 2003). addition of crab shell 1.25% into talc-based c. terreus c. albidus et al et al et al et al et al et al et al c. terreus et al. et al . o effect of crab shell powder as additives in talc-based powder formulation of and c. terreus c. albidus table 9. survival of in talc formulation and supplemented with additivesc. terreus yeast density (log cfu/g) in i-th month 0 1 2 3 talc 8.7 a 6.15 a 6.54 a 5.02 a talc + chitin 8.19 a 7.89 b 6.75 a 5.02 a talc +crab shell powder 8.73 a 7.00 ab 6.07 a 6.07 b numbers followed by same symbol are not significantly different at significant level of 0.05 with dmrt test table 10. survival of in talc formulation and supplemented with additivesc. albidus yeast density (log cfu/g) in i-th month 0 1 2 3 talc 8.87 a 6.06 a 6.50 a 5.26 a talc + chitin 9.31 a 7.56 b 6.48 a 5.14 a talc + crab shell powder 9.56 a 7.30 b 6.22 a 6.06 b numbers followed by same symbol are not significantly different at p<0.05 with drmt test 27 formulation increased survival of and (tables 9 and 10). further studies on the exploration and optimization of various additives are needed to prolong survival rate of formulated yeasts is compatible to therefore it can be used in a mixture formulation. best carrier materials in powder formulation of was talc, able to maintain yeast survival for four months of storage without contamination of other microorganisms. talc powder provided best survival of , with survival more than 5 months of storage. crab shell at concentration of 1.25% can be used as additive in powder formulation containing a mixture of and based on its enhancement of antagonistic activity on and survival of both antagonistic yeasts he authors would like to acknowledge seameo biotrop for providing financial support through dipa 2009. moreover, the authors express their gratitude to people involved in this research, ms ratih munawaroh and mr. dadang surahman at the laboratory of plant mycology dept of plant protection, faculty of agriculture, bogor agricultural university c. terreus c. albidus . terreus c. albidus, c. terreus c. albidus c. terreus c. albidus, c, terreus . . t . conclusions acknowledgments references c abadi al. 1983. antagonism between leaf surface microorganism and causal agent of early blight of tomato ( ). master thesis. graduate school bogor agricultural university, bogor (in bahasa indonesia). abadias m, usall j, teixidó n, viñas i. 2003. liquid formulation of the postharvest biocontrol agent cpa-1 in isotonic solutions. phytopathology 93:436-42. biggs ar, el-kholi mm, el-neshawy s, nickerson r. 1997. effects of calcium salts on growth, polygalacturonase activity, and infection of peach fruit by plant dis 81:399-403. burges hd, jones ka. 1998. trends in formulation of microorganisms and future research requirements. in: burges, hd (ed.), formulation of microbial biopesticides, beneficial microorganisms, nematodes and seed treatment. kluwer academic publication, dordrecht, pp. 311-332. droby s, wisniewski m, el ghaouth a, wilson c. 2003. influence of food additives on the control of postharvest rots apple and peach and efficacy of the yeast-based biocontrol product aspire. post harvest biol technol 27: 127-35. fan q, tian sp. 2001. postharvest biological control of grey mold and blue mold on apple by (saito) skinner. postharvest biol technol 21: 341-50. fravel dr. 2005. commercialization and implementation of biocontrol. ann rev phytopathol 43: 337-59. harti h. 2010. control of fusarial wilt ( f.sp. (e f smith)) of banana by using crabshell powder and chitosan. master thesis. bogor agricultural university, indonesia. alternaria solani, lycopersicon esculentum candida sake monilinia fructicola. cryptococcus albidus fusarium oxysporum cubense powder formulation of antagonistic yeasts as biofungicides suryo wiyono– et al. karabulut oa, smilanick jl, mlikota gabler f, mansour m, droby s. 2003. nearharvest applications of , ethanol, and sodium bicarbonate to control postharvest diseases of grape in central california. plant dis 87:1384-9. kinay p, yildiz m. 2008. the shelf life and effectiveness of granular formulations of and yeast isolates that control postharvest decay of citrus fruit. 433-40. multazam. 2002. prospect for utilization of shell of crab ( sp.) as supplement of fish feed. department of fishery product technology. bogor agricultural university. bogor (in bahasa indonesia). saligkarias id, gravanis ft, epton has. 2002. biological control of on tomato plants by the use of 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pelleting process. biol control 46 : 348-57. wiyono. s. 2008. biological control of petal blight of dendrobium caused by research report (in bahasa indonesia) departemen of plant protection faculty of agriculture ipb. bogor. vivekananthan r, ravia m, saravanakumara d, kumarb n, prakasama v, samiyappan r. 2004. microbially induced defense related proteins against postharvest anthracnose infection in mango. crop prot 23: 1061-7. yu tl, yin wy, wang y, zheng x. 2007. effect of chitin on the antagonistic activity of against in pear fruit. 122: 44-8. metschnikowia fructicola metschnikowia pulcherrima pichia guilliermondii portunus botrytis cinerea candida guilliermondii candida oleophila botryodiplodia theobromae rhizopus stolonifer pseudomonas fluorescens curvularia pallescens. cryptococcus laurentii penicillium expansum biol control 45: biol control plant patho int j food microbiol biotropia vol. 20 no. 1, 2013 28 response lonkida ( l.) of towards nauclea rientaliso mycorrhiz al inoculum in waterlogged condition faisal danu tuheteru , cecep kusmana , irdika mansur1, 2* 3 3, and iskandar4 1tropical silviculture major, post-graduate chool of s institut pertanian bogor, bogor 16680, indonesia 2department of forestry, faculty of forestry and environmental science, halu oleo university , indonesia 3department silvi ultur institut pertanian bogor, bogor 16680, indonesiaof c e, faculty of forestry, 4department of soil sciences and land resources, faculty of agriculture, institut pertanian bogor, bogor 16680, indonesia received 1 august 2014/accepted 6 october 2014 abstract w . aterlogged condition is one of environmental conditions not favorable for plants and mycorrhizal fungi the tolerant are more adaptive to ed condition arbuscular mycorrhizal species waterlogg . the documented effects of fungi ( ) esides increasing the tolerance of plants, amf inoculation on wetland plant performance are inconsistent. b amf waterlogged . was carried out also causes plants depression in condition this study to examine the morphoanatomical adaptation, survival, growth, biomass and nutrient uptake of onkida ( l.) from different l nauclea orientalis habitats inoculated with arbuscular mycorrhizal fungi (amf) in waterlogged condition for 90 days. experiment procedures include : seed germination, preparation of inoculums and inoculation of and waterlogg d amf ed treatment. ling adventitious rootsthe results showed that lonkida seed s formed lenticels (100%), (41%) and a under waterlogg seedling dry landerenchym ed condition. s from habitats, both mycorrhizal and non-mycorrhizal, had lower height growth rate than those from other habitat types. the same results happened relative growth rates for for shoot (rgrs), root (rgrr) and total (rgrt) and the ratio of root shoots. treatments of temporal dry weight swamp habitat without ed condition and with ed condition and treatment in savannah with waterlogg waterlogg waterlogg plant dry weight root shoot and ed condition had rgrt greater than those in other treatments. in general, ( , total between swamp with ) and total n in roots and shoots were greater in the interaction temporal habitat nonmycorrhizal in ed condition. waterlogg however, lonkida seedling from dry land habitat required amf under waterlogged to improve biomass and n a cumulation in roots. , waterloggcondition c independently ed condition increased the pool of average height gain (18%), diameter (46%), leaf area (40%), leaf length (17%) and leaf width (21%). nonetheless, ed condition treatment decreased the number of leaves (9%) colonization waterlogg and amf (71%) the results of this study indicate that seed s from temporal had good growth and high biomass in . d ling swamp condition with or without ed condition waterlogg . : keywords arbuscular mycorrhizal fungi (amf), morpho-anatomical adaptations, ., lonkida, lnauclea orientalis swamp ed condition, waterlogg introduction waterlogged is one of environmental conditions not favorable for plants and mycorrhizal fungi (helgason filters 2009). & waterlogged condition causes anaerobic condition indicated by hypoxia and anoxia (elzenga van veen 2010). these conditions can & affect growth, development and adaptability of plant (kreuzwieser gessler 2010; species & parolin wittman 2010) as well as species & composition of forest stands (kozlowski 1984, 1997). effects of waterlogged condition is highly varied depending on genetic and age of the plants, time and duration of inundation (kozlowski 1984) as well as depth of inundation (iwanag a & yamamoto 2008). plant can tolerate waterlogged condition through morpho-anatomical and physio logica l adaptat ion mechani sms. physiological adaptation can be done by accumulating reserves in the root in the form of carbohydrates and alcohol fermentation as an * corresponding author : faisaldanu_28@yahoo.com biotropia vol. 22 no. 1, 2015: 61 71 doi: 10.11598/btb.2015.22.1.416 61 mailto:faisaldanu_28@yahoo.com biotropia vol. 22 no. 1, 2015 62 alternative (kreuzwieser gessler 2010; parolin & & wittman 2010), while morpho-anatomical adaptation is done by forming lenticels, adventi ous root and erenchyma (folzer . ti s a et al 2006; parolin wittman 2010)& . plant tolerance to ed condition varies waterlogg and is determined by the (kozlowski species 1984). the tolerant are more adaptive to species waterlogg ed condition (kogawara 2006). et al. however, differences in habitat of the species also largely determine the success of the plant to survive in inundation condition (ferreira . et al 2009; nielsen . 2010). several studies have et al reported that growing naturally in the species inundated area are more tolerant to waterlogged condition, such as (keeley 1980), nyssa sylvatica female (nielsen 2010) and populus angostifolia et al. himatanthus sucuba et al. (ferreira 2009). information on tropical tolerance and species adaptation to inundation still needs to be studied (parolin 2009). in indonesia, research on tolerant species toward different types of inundated habitat is still very limited. lonkida ( l.) is a tropical nauclea orientalis multipurpose tree species that grows naturally in a variety of habitat types including wetlands (petty & douglas 2010; kartikasari 2012) and has et al. natural distribution in indonesia (whitmore et al. 1997; keβler 2002). this species has potential et al. uses for agroforestry, phytoremediation and rehabilitation of degraded land, including wetlands (marghescu 2001; amihan-vega & mendoza 2005; mawaddah 2012) and medicinal plant (lim 2013). a preliminary study of lonkida response to ed condition greenhouse waterlogg in scale had been conducted and it was confirmed that this species was tolerant to waterlogged condition (kurniawati 2011). however, studies related to growth response, adaptation, and plant nutrient uptake ability of lonkida from various habitats under waterlogged condition had not been reported. this was also reported to be colonized species by arbuscular mycorrhizal fungi ( amf) glomus mossea in india (sudha ammani 2010) and & showed good growth response to the application of amf in waterlogged condition (kurniawati 2011). tolerance to waterlogged condition could be enhanced through inoculation. amf association of inundated plants with is part amf of strategy to improve nutrient uptake and oxygen circulation (elzenga & van veen 2010). in waterlogged condition, could amf association improve growth (osundina 1998; fougnies et al. 2007), biomass and nutrient uptake, especially phosphorus (muok ishii 2006; fougnies & et al. 2007), the growth of root systems (qiang-sheng et al. 2013) and the structure of plant communities (bauer 2003). however, colonization, et al. abundance and benefits of in edamf waterlogg condition can vary among plant species (bauer et al. 2003) due to of used (secilia species amf & bagyaraj, 1994; sah 2006), availability of et al. phosphorus (stevens 2002; garcia 2008) et al. et al. and distance from the rhizospher (keeley amf 1980). biomass of mycorrhizal nyssa sylvatica seedlings from inundated seed sources were higher than that from dry areas (keeley 1980). the purpose of this study was to examine the morpho-anatomical adaptation, survival, growth, biomass production and nutrient uptake of amfinoculated lonkida originated from different habitat types in waterlogged condition. materials and methods experiment procedures seed germination lonkida seeds were collected from mother trees grown in different habitat types (swamps, temporary , temporary swamps savannah swamps and dry land areas) in c andkendari ity, konawe south konawe district (fig. 1), southeast sulawesi rovincep . lonkida seeds were germinated in plastic boxes having dimension of 20x20x5 cm containing sterile soil media (±1 kg) and were grown until reaching seedling stage at age of 150 days before being used in this experiment. there were 144 seedlings ready to be used in this experiment. inoculum preparation and amf inoculation amf inoculum used was mycofer ipb containing 4 (four) species of amf, namely glomus etunicatum, glomus manihotis, acaulospora tuberculata gigaspora margaritaand . the number of spores per 5 g of inoculum was 57-125 spores. before inoculation, polybags having size of amf 15x20 cm were filled with sterile soil media (1.5 kg soil media/bag). there were 72 lonkida seedlings having age of 150 days inoculated with amf inoculum and subsequently were called mycorrhizal seedlings. the other 72 lonkida response o lonkida ( l.) mycorrhiz i condition faisal – et al. f owards al noculum n aterloggednauclea orientalis t i w seedlings were not inoculated with amf inoculum and served as control treatment; subsequently were called non-mycorrhizal seedlings. chemical properties of the sterile soil media were ph 5.2, organic c (walkey black) & 0.27%, total n (kjeldahl) 0.06%, c/n ratio 5, available p (bray i/i ) 2.5 ppm, exchangeable ca, i mg, k, na, and cec were 10.32 cmol/kg, 1.49 cmol/kg, 0.66 cmol/kg, 1.76 cmol/kg and 19.79 cmol/kg (extract buffer 1.0 n nh oac ph 7.0), 4 respectively. waterlogged treatment mycorrhizal and non-mycorrhizal seedlings at age of 150 days after planting were then treated with ed condition. each seed was waterlogg ling placed in a polybag having size of 26.5x23 cm containing sterile soil media. a total of 72 seedlings inside polybags were immersed in water, while the other 72 seedlings inside polybags were not immersed, but watered every day as control (garcia . 2008). water level was monitored et al every 3 days. experimental design this study used factorial in a completely randomized design (crd) consisting of 3 factors: 1. treatment a= habitat origin of lonkida consisted of: a =dry land;0 a = ; 1 swamp a =temporal ;2 swamp a = temporal savannah.3 swamp 2. treatment b = amf inoculum treatment consisted of: b = uninoculated non-mycorrhizal);0 ( b = inoculated with amf inoculum 1 (mycorrhizal). 3. treatment c = ed treatment waterlogg consisted of: c = (non-waterlogged);0 control c =waterlogged.1 each treatment was replicated 3 times and each replication consisted of test plants. the 3 experiment was conducted for 90 days. at the end of experiment, the seedlings' age was 240 days old. data collection growth measurements were conducted at the end of the experiment (at the end the 90-day experiment) for seedling height, seedling diameter, number of leaves per seedling and plant . survival measurement of leaf area was conducted using green leaf area meter ga-5 model. relative growth rates (rgr) for total (rgtt), shoots (rgts) and roots (rgtr) for control dry mass and waterlogged condition were then calculated using the formula of (200 ):mendoza 5et al. 63 figure 1. distribution map of lonkida tree sampling rgri = (ln wt ln w )/(t -t )f f oto where: rgr= relative growth of plants; rate i= total dry weight, shoots and roots; wt = dry weight shoots and roots f plant (total, ) at the end of the (seedlings' experiment age was 240 days old); wt = dry weight o plant at the beginning of experiment period (seedlings' age was 150 days old); t -t = the difference between the and the f o initial total period growth (experiment of period was 90 days). dry weight and in plant tissuenutrients at the end of 90 days, the shoots and roots were harvested. fresh shoots and roots were ovendried at 70 c for 48 hours (garcia . 2008). o et al determination of p was carried out using hno -3 hclo method, and n by kjeldahl (balai 4 penelitian tanah 2009). for n and p, two expressions of nutrient uptake efficiency was calculated con entration per unit of dry weight i.e. c and total (a cumulation) uptake per unit of dry c weight. morpho-anatomical adaptation observation and counting of the number of lenticels and were done every adventitious roots week . using non destructive samples the data of lenticel and adventitious roots obtained at s were the end of . the proportion data of experiment lenticels number and adventitious roots the were ratio between the number of sample forming s lenticel and adv ootss entitious r . aerenchyma observations for the lenticles and adventitious roots were made by following the method of freezing microtome . roots of 3 (longstreth 1879) cm long (diameter <3 mm) were immersed in 70% alcohol solution ( ours the roots were then 24 h ). cut using scissors into root pieces of 0.5 cm long. the root pieces were then put in yamato mc802a electro freezer for 2-3 minutes at -15 c. o the roots were then cut using yamato rv-240 microtome with thickness of 20 µm. before the roots were placed on object glasses, several drops of 20% glycerol solution were applied on the object glasses, while the roots were stained with safranin. aerenchyma observation was made using axio imager microscope a1m/axiocam mrc5 x magnification . (200 ) susceptibility (si) index for all measured plants variables, and soil s index (si) was calculated using the usceptibility method of hiler (1972):et al. si = 1 (waterlogged plant/ )non-waterlogged plant si value will be positive if the waterlogged condition decreases the variables measured. on the other hand, si value will be negative if waterlogged condition increases the variables measured. amf spore density and colonization thirty grams of soil samples were collected from soil medium in polybags used as mychorrizal lonkida medium. spores were extracted from soil using wet sieving and decanting method (gerdemann & nicolson 1963) followed by centrifugation; supernatant acquired was added with 50% sugar solution (walker 1982). amf et al. spores extracted were observed and counted under a dissecting microscope with 35x magnification. root colonization was observed by using root staining technique of brundrett et al. (1996). fresh roots cleaned in 10% koh for were 2 days, then were immersed in h o for 10-20 2 2 minutes and were rinsed thoroughly with water. the roots were then soaked in hcl 0.2% for 20 minutes and then were stained with trypan blue solution . . ten root samples (1 cm long) (0 05%) from each plant were observed under a microscope with 200x magnification. oot r colonization was calculated using the formula of brundrett (1996):et al. % = root colonization number of fields-of-view containing mycorrhizae total observed fields-of-view data analysis data were analyzed using analysis of variance (anova). abnormal data were normalized using logarithmic transformation. the data were further analyzed using duncan's multiple range test (dmrt) for means comparison. data analyses were computed using sas 9.1.3 portable program. biotropia vol. 22 no. 1, 2015 64 x 100% 65 table . measurement of the observed variables for lonkida seedlings grown in ed condition and in non-1 waterlogg waterlogged condition variables measurement for lonkida seedlings grown in waterlogged condition (mean±se) measurement for lonkida seedlings grown in non-waterlogged condition (mean±se) si effect of waterlogged condition plant growth variables survival 100±0.00 a 100±0.00 a ns ns height (cm) 8.13±2.45 a 6.18±1.177 b -0.32 + diameter (mm) 1.81±0.31 a 1.24±0.25 b -0.46 + total of leaves (blade) 13.1±0.838 b 14.3±1.183 a 0.09 leaf area (cm2) 9.8±3.045 a 7.0±0.659 b -0.40 + leaf length (cm) 8.2±0.878 a 7.0±0.363 b -0.17 + leaf width (cm) 2.9±0.431 a 2.4±0.132 b -0.21 + rgrs (per day) 0.0084±0.0032 a 0.0060±0.0015 b -0.42 + rgrr ( per day) 0.0075±0.0035 a 0.0069±0.0020 a ns ns shoot/root ratio 1.89±0.25 a 1.77±0.32 a ns ns adaptation to waterlogged condition total adventious root 1.16±0.552 a 0.0±0.00 b + adventious root (%) 48.6±17.005 a 0.0±0.00 b + total lenticels 30.9±2.91 a 1.3±0.35 b -22.7 + lenticels (%) 100±0.00 a 81±16.95 b -0.23 + amf symbiosis amf colonization (%) 11.5±2.64 b 29.1±6.25 b 0.61 nutrient uptake n in root (%) 1.091±0.13 a 1.185±0.10 a ns ns n in shoot (%) 0.73±0.059 b 0.83±0.061 a 0.12 p in root (%) 0.208±0.14 a 0.203±0.016 a ns ns total p in root (mg) 0.255±0.05 a 0.199±0.033 b -0.28 + p in shoot (%) 0.124±0.02 a 0.108±0.013 a ns ns total p in shoot (mg) 0.276±0.11 a 0.178±0.029 b -0.55 + le ad a b c notes: 1) figures followed by the same letters in different columns are not significantly different according to dmrt at 95 % confidence level 2) differentns=not significantly 3) susceptibility index=1-(waterlogged plant/non-waterlogged plant) si/ figure . lonkida magnification aerenchyma2 cross-section of root tissue (x200 ) showing the presence of in (a) nonwaterlogged (b waterlogged (c) formation adventitious roots condition; ) condition; of lenticels (le) and (ad) response o lonkida ( l.) mycorrhiz i condition faisal – et al. f owards al noculum n aterloggednauclea orientalis t i w results and discussion mor o anatom adaptationph ical seed of lonkida have morpho-anatomical lings adaptation mechanisms to survive in ed waterlogg condition. the results showed that lonkida seedlings d , lenticels produce adventitious roots and erenchyma (fig. and table ). lenticels a 2 1 began to form within 3 to 5 days after being in the waterlogged condition. all seed under lings waterlogged condition formed 100% lenticels (table 1). lonkida seedlings grown in results and discussion mor o anatom adaptationph ical seed of lonkida have morpho-anatomical lings adaptation mechanisms to survive in ed waterlogg condition. the results showed that lonkida seedlings d , lenticels produce adventitious roots and erenchyma (fig. and table ). lenticels a 2 1 began to form within 3 to 5 days after being in the waterlogged condition. all seed under lings waterlogged condition formed 100% lenticels (table 1). lonkida seedlings grown in w ed aterlogg condition had an increase in lenticels and adventitious roots compared with lonkida seedlings grown in noned conditionwaterlogg (table 1). aerenchyma was formed in the root cortex of lonkida in both waterlogged and nonwaterlogged conditions. aerenchyma proportion in root of lonkida grown in waterlogged tissue condition was greater than that of lonkida grown in noned condition (fig. )waterlogg 2 . seed s of lonkida adapt to waterlogged ling ed condition by modifying the morphology and anatomy of the body through lenticels, adventitious root and aerenchyma formations. in the treatment where lonkida seedlings were grown in non-waterlogged condition and swamp habitat, the lenticels percentage was below 100 %. the same finding was found in aerenchyma in root tissue. a formation were absent in dventitious root treatment where lonkida seedlings were grown in non-waterlogged condition. the adaptation mechanism improves the oxygen uptake ability and oxygen transfer to plant tissues in waterlogged condition (yin 2012). thus, et al. morphological and anatomical adaptations could reduce hypoxic condition and contribute to the restoration and maintenance of aerobic respiration of seedlings in waterlogged condition (ashraf 2012). lenticels were crucial in the diffusion of oxygen and anaerobic metabolism resulting products (ethanol, co and ch ) and 2 4 played a role in plant water homeostasis (ashraf 2012). the of plants tolerant to adventitious root waterlogged condition was formed to play the role as the main root replacement to maintain water and mineral supplies when the main roots were not functioning normally, to maintain aerobic respiration of seedlings and to initiate stomata opening (kozlowski 1997; ashraf 2012). network of aerenchyma contributed to the life and growth of plants in a long term waterlogged condition (ashraf 2012). adaptation mechanisms mentioned above were found both in plants tolerant or intolerant to water log ged condit ion. lent ice ls and adventitious root woody plant, were found in such as calophyllum brasilienses camb (de oliveira & joly 2010), (medina . 2009), erythrina speciosa et al larix laricina populus (islam & macdonald 2004), deltoids et al salix gracilistylabéjaoui ( . 2012), (nakai et al salix martiana. 2010) and (parolin 2009). aerenchyma formation in root also occurs in erythrina speciosa et al. melaleuca (medina 2009), cajuputi et al. quercus petraea (tanaka 2011) and (folzer 2006). in addition to the above et al. mechanisms, the roots of lonkida seedlings also appeared on the surface of the media and out through the holed polybags. these facts indicated that the roots could grow well in waterlogged condition and could absorb oxygen and nutrients for the plants. the formation of leaves and roots in ed condition was thought to support waterlogg growth through the activity of oxygen uptake, increased water and nutrient contents and increased photosynthesis activity (kozlowski 1997). increased seedling growth in ed waterlogg condition indicated that lonkida seed s had ling high efficiency in the improvement of biomass per each nutrient absorbed and maintained aerobic respiration (kozlowsk 1997; tanaka .i et al 2011 ). plant growth he t of s ling igh lonkida eed s in temporal swamp and savannah habitats without mycorrhiza showed significant difference from other treatments, except for treatment in swamp habitats either with or without mycorrhiz ( . a fig 3a waterlogged). lonkida seedlings grew better in condition those in non-water-compared with logged condition, with h igh growth differences e t of 32%, diameter differences of 46%, leaf area differences of 40%, leaf length differences of 17% and leaf width differences of 21% between the two treatments (table ). nonetheless, 1 waterlogged condition reduced the number of leaves 9%. temporal habitat by swamp on waterlogged condition had the highest rgrt value (0.0125 g/day) and the lowest rgrt value occurred in the dry land ed under waterlogg condition (0.0047 g/day) . . treatment (fig 3b) of biotropia vol. 22 no. 1, 2015 66 waterlogg ed condition did not significantly affect rgrr, but rgrs increased by 42% . (table 1) non-mycorrhizal lonkida seedlings had higher rgrs, rgrr and rgrt compared with mycorrhizal lonkida seedlings the (table 3). results of this study indicated that edwaterlogg condition did not inhibit vertical growth (root and shoot) and horizontal growth (diameter) of plant. lonkida seed s originated from ling dry land habitats have lower growth rate, rgrs, height rgrr , rgrt and root shoot ratio compared with lonkida seedlings originated from other habitat types, for both mycorrhiz and non-mycorrhiza al l treatments. the results of this study indicated that lonkida originated habitat seedlings from dry land were not suitable to be planted in ged waterlog condition. several studies have reported the same results, such as study on the female p uop lus angustifolia that grew naturally in inundated area was more tolerant to waterlogged condition within 15 weeks compared to male p. angustifolia male which originated from the dry land (nielsen et al. et al 2010). ferreira . (2009) reported that himatanthus sucuba originated from non-flooded areas (terra-firme) was intolerant to waterlogged condition compared with that originated from inundated area (varzea) in amazonian flood plain. however, shoot root ratio of seedlings originated from dry habitats were smaller than those from other habitats. the low was shoot root ratio assumed as being a protective mechanism from dehydration through transpiration (van splunder et al. 1996). another strategy that might occur was that seed s from dry habitats had more c ling land translocation to the roots than to the shoots (martinez 2012).et al. in contrast to the dry habitats, lonkida seedlings originated from temporal swamp habitat in waterlogged and non-waterlogged conditions and lonkida seedlings originated from savannah habitat waterlogg in ed condition had greater rgrt compared with rgrt in other treatments. the average height growth of lonkida seed s ling from temporal swamp and savannah habitats were quite high, indicating that lonkida seedlings from both habitats were tolerant to waterlogged condition; which also indicated that the physiological adaptation mechanism of the seedlings was not disrupted and morphoanatomic change was formed well in the al waterlogged condition. in addition, lonkida seed s in the temporal grew well in dry ling swamp condition and in ed condition. parolin waterlogg (2009) explained that the differences in the type of tolerance to waterlogged condition were strongly associated with ecotype differences among habitats due to natural selection. the results of this experiment indicated that lonkida is a species having relatively broad ecological distribution, compared to most species from inundated area, which generally had relatively small ecological amplitude (parolin 2009). plant biomass table 2 shows significant difference on shoot dry weight was shown for mychorrizal lonkida seedlings originated from dry land in waterlogged condition, non-mychorrizal lonkida seedlings originated from swamp in waterlogged condition, non-mycorrhizal lonkida seedlings originated from savannah in waterlogged condition and mycorrhizal lonkida seedlings originated from savannah in non-waterlogged condition. the same table also shows that root dry weight of mycorrhizal lonkida seedlings originated from dry land in waterlogged condition was significantly different from all other treatments. significant difference on total dry weight was shown for mycorrhizal lonkida seedlings originated from dry land in waterlogged condition, non-mycorrhizal lonkida seedlings originated from savannah in waterlogged condition and mycorrhizal lonkida seedlings originated from savannah in nonwaterlogged condition (table 2). hootratio s root of non-mycorrhizal lonkida seedlings was higher (1.95) than that of mycorrhizal lonkida seedlings (1.65) . (table 3) the results of this study showed that lonkida seed s had various adaptation strategies to ling overcome environmental stress. seedlings from dry habitats required mycorrhizae fungi in waterlogged condition. some studies also indicated that amf improved plant tolerance in waterlogged condition (osundina 1998; neto et al. et al. the role of amf 2006; fougnies 2007). in waterlogg condition ed can be seen from increase of stevens 2002; nutrient uptake especially p ( et al. muok and ishii 2006; ; et al.fougnies 2007 garcia et al. ) enhanc growth ( 2008 in ing secilia & 67 response o lonkida ( l.) mycorrhiz i condition faisal – et al. f owards al noculum n aterloggednauclea orientalis t i w biotropia vol. 22 no. 1, 2015 68 bagyaraj 1994 osundina 1998 fougnies ; ; et al. 2007 secilia & ) and plant biomass (keeley 1980; bagyaraj 1994). nutrient uptake the highest total n in root occurred in mycorrhizal lonkida seedlings originated from dry land habitat in ed condition. the highest waterlogg total n in shoot occurred in non-mycorrhizal lonkida seedlings originated from temporal swamp in waterlogged condition the (table 2). results of this study revealed that was amf needed by seedlings of habitat in dry land waterlogged condition. n uptake by in amf waterlogg ed condition had also been reported in aster tripolium et al. (neto 2006). in contrast to the dry habitats, seed s of habitat ling temporal swamp did not require mycorrhizae i waterlogged n condition. non-mycorrhizal lonkinda seed lings from temporal swamp in waterlogged condition had higher n accumulation in leaves and roots. total p in root of lonkida seedlings grown in waterlogged condition was higher than that of lonkida seedlings grown in non-waterlogged condition (table 1) which was in line with research on plants of mill. (mendoza . lotus glaber et al 2005; garcia . 2008).et al arbuscular mycorrhizal fungi (amf) colonization and spore density a fungi (amf)rbuscular mycorrhizal colonization in waterlogged condition was 61% lower than that in non-waterlogged condition (table ). amf structures found in the roots of 1 lonkida were internal hyphae, external hyphae, hyphal coils, vesicles and arbuscular. the internal structure of the hyphae commonly amf was good without waterlogg . the ed condition numbers of vesicles per 1 cm roots were more in lonkida seedlings grown in condition waterlogged (table ). inhibit 2 waterlogged condition ed colonization and spore density of amf. amf colonization believed to decrease the density was of spores in waterlogged condition due to anaerobic environment (escudero & mendoza 2005; mendoza 2005; garcia 2008; et al. et al. stevens 2011; 2013). the low et al. et al.wu number of spores in this study attributed to the fact that the species of amf used were not originated from areas with waterlogged condition (qiang-sheng 2013), or oxygen -deficient et al. condition (kozlowski 1997), limited or had supply of c from the host (neto 2006). thus, et al. the amf was not able to sporulate and had limited external hyphae resulting from waterlogg condition (secilia & bagyaraj 1994; ed sah 2006). nevertheless, other studies et al. showed higher colonization of amf in waterlogg condition (sah 2006; neto ed et al. et al. 2006; faougnies 2007). in addition to the et al. species of amf, plant species (bauer 2003), et al. availability of p (stevens 2002; garcia et al. et al. 2008) distance from the amf rhizospher (keeley , 1980) as well as depth and duration of waterlogg also contribute to the ed condition d amf symbiosis (graham . 2013).et al arbuscular mycorrhizal structure found were the internal and external hyphae, vesicles, arbuscular, coil and spores. arbuscules found and the numbers of vesicles per cm root were more in waterlogged condition in non waterlogged-than condition. arbuscule is a structure of amf in the root that acts as a terminal for the transfer of nutrients from the host to the amf and the transfer of carbon to fungi (smith read 2008) & . vesicles are storage structures and can support the growth of the internal hyphae (smith read & 2008). n , vesicle i waterlogged condition structures found in large quantities. the were results of this study indicate that the amf save d d a lot of energy in the vesicles needed to adapt to the (garcia 2008). waterlogged condition et al. this fact ha also been reported in the roots of d lotus tenuis et al. citrus junos (mendoza 2005) and (qiang-sheng 2013). ow et al. l number of arbuscules the root of lonkida also show that in ed the transfer of c from plants to fungi was low and inhibit sporulation processed conclusions lings hadlonkida seed high survival rate and adapt to waterlogg by modifying ed ed condition the morpho-anatom through formation of ical lenticels, and aerenchym . adventitious roots a lonkida seed s inundated habitat tend ling from ed to have higher growth and biomass. in waterlogged condition, amf treatment increased biomass and n accumulation in roots of lonkida seedlings from dry land habitat. originated a fungi (amf)rbuscu la r mycor rh iza l colonization in waterlogged condition was lower 69 response o lonkida ( l.) mycorrhiz i condition faisal – et al. f owards al noculum n aterloggednauclea orientalis t i w than that in non-waterlogged condition. based on these results, lonkida has the potential to be used for rehabilitating degraded mining lands. references amihan-vega b, mendoza jd. 2005. benefits rom ree f t g t d u e rrowing in he egraded plands: mpirical ealities f t :rom tabango, leyte, he philippines. in harrison s, herbohn j, suh j, mangaoang e vanclay j editors., , ac iar sm a l l ho l der fores t r y pro j ec t redevelopment of a timber industry following extensive land clearing : from tproceedings he e p w . nd-ofroject orkshop ormoc city, the philipines , 19-21 august 2004. p 93-106. ashraf ma. 2012. waterlogging stress in plants : a review. afr j agric res. 7(13): 1976-81. balai penelitian tanah. 2009. petunjuk teknis analisis kimia tanah, tanaman, air dan pupuk . bogor (id) : balai besar litbang sumber daya lahan pertanian, balai penelit ian dan pengembangan pertanian departemen pertanian. bauer cr, kellogg ch, bridgham sd, lamberti ga. 2003. mycorrhizal olonization across hydrologic c gradients in restored and reference freshwater wetlands. wetlands 23(4): 961-68. béjaoui z, albouchi a, lamhamedi ms, abassi m, el aouni mh. 2012. adaptation and morpho-physiology of three marsh. x l. clones after populus deltoides p. nigra preconditioning to prolonged waterlogging. agroforest syst 86:433-42. brundrett m, n bougher, b deu, t grove, majalaczuk . 1996. working with mycorrhizas in forestry and agriculture ( ) :. canberra au australian centre for international agriculture research. de oliveira vc, joly ca. 2010. flooding tolerance of calophyllum brasili ense camb. 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yamanoshita t, tange t. 2011. morphological and anatomical changes of melaleuca cajuputi under submergence. trees 25: 695-704. van splunder i, voesenek lacj, coups h, de vries xja, blom cwpm. 1996. morphological responses of seedlings of four species of salicaceae to drought. can. j. bot 74: 1988-95. walker c, mize cw menabb hs jr. 1982. population of , endogonaceous fungi at two location in central iowa. 60:2518-29.can. j. bot. 71 response o lonkida ( l.) mycorrhiz i condition faisal – et al. f owards al noculum n aterloggednauclea orientalis t i w 3. loukrakpam (two) revisi.cdr biotropia vol. 21 no. 2, 2014: 91 98 two new fischer, 1894 (nematoda: tylenchina) from manipur, northeast india aphelenchoides loukrakpam bina chanu and naorem mohilal received 25 february 2014/accepted 1 october 2014 two new nematode species of discovered from different localities of manipur, north east india had been described in the present study. sp. nov. has a long stylet and four lateral lines which are gradually merged into two at the tail region. sp. nov. has a small body and short tail with a stylet length of 8.6 μm. sp. nov., sp. nov., manipur, northeast india * parasitology section, department of life sciences, manipur university canchipur 795003, manipur aphelenchoides aphelenchoides longistylus aphelenchoides neominoris aphelenchoides longistylus aphelenchoides neominoris abstract introduction keywords: aphelenchoides aphelenchoides aphelenchoides spp. are soil dwelling nematodes and often found in decaying plant materials. their characteristic features include slender stylet with narrow lumen and usually with small basal knobs or swellings with presence of post uterine sac. female tails are of medium length, conoid, with a tip pointed or rounded and often mucronate terminus. most important characteristics of males include paired spicule, separate, rose thornshaped or derived from them with absence of gubernaculums. a number of spp. have been reported from india and from throughout the world. but description of new species of the genus is increasing due to intensive survey from different parts of the world including the biodiversity hot spot areas of north east india. the present manuscript deals with descriptions of two new species of spp. from manipur, north east india. the discovery of the specimens is an added asset to the knowledge of the rich biodiversity of the region. * corresponding author : bina.chanu@gmail.com doi: 10.11598/btb.2014.21.2.3 93 materials and methods results and discussion nematodes were extracted from collected soil samples by baermann funnel technique. collected nematodes were fixed in taf (triethanolamine, formaldehyde and water) and processed by glycerol-ethanol method of seinhorst (1959). specimens were mounted in dehydrated glycerine. after slide preparation, measurements were taken using an ocular micrometer fixed on nikon, trinocular research microscope, model eclipse e200 and diagrams were drawn under a drawing tube attached to the same microscope. sp. nov.aphelenchoides longistylus 94 biotropia vol. 21 no. 2, 2014 table 1 morphometric data of species of sp. nov.. aphelenchoides longistylus characters holotype ♀ paratypes ♀ s paratype ♂s n 1 12 4 stylet 24.22 24.22 24.22 length 0.59 0.59 – 0.66 (0.625±27.79) 0.562 – 0.620 (0.59 ±0.02) a 34.4 34.4 35.85 (35.02±0.61) 38.2 – 42.02 (40.11±1.91) b 8.6 8.33 – 9.81(9.08±0.52) 7.24 –8.33 (7.78±0.54) b´ 6.03 6.03 – 6.49 (6.27±0.18) 3.25 – 5.603 (4.42±1.17) c 14.33 13.67 – 14.58 (14.19±0.38) 13.54 – 17.45 (15.22±1.43) c´ 6.00 5.6 – 6.14 (5.91±0.22) 4 – 6.2 (4.92±0.86) g1 40.69 39.39 – 43.08(41.05±1.52) v 69.76 67.78 – 69.76 (69.08±0.92) pvs/ v-a% 11.25 11.25 – 12.25(11.71±0.41) t 92.61 – 102.84 (97.56±3.66) max. body width 17.3 17.3 – 19.03(17.87±0.815) 13.38 lip diameter 5.19 5.19 5.13 lip height 1.73 1.73 1.73 oesophagus 98.61 98.61 – 102.07 (99.76±1.63) median bulb length 13.84 13.84 13.84 –15.57 (14.55±0.73) median bulb diam. 10.38 8.65 10.38 (9.51±0.86) 8.65 –10.38 (9.53±0.70) ovary length 242.2 242.2285.45 (257.35±19.88) testis 335.62 – 342.45 (339.14±1.91) sperm theca 48.44 48.44 pus 15.57 8.65 – 18.65 (16.74±1.35) pus/vbd 0.9 0.9 – 1.07 (0.96±0.07) rectum 6.92 6.92 spicule 24.22 tail 41.52 41.52 – 48.44 (44.16±3.05) 41.52 – 46.23 (43.89±1.91) abd 6.92 6.92 – 8.65 (7.49±0.81) 10.32 note: all measurements are in μm except length is in mm description female: male: type habitat and locality: type material: differential diagnosis: body is straight to curved ventrally upon fixation, 0.59 0.66 (0.62±27.79) mm in length and about 34.4 35.85 (35.02±0.61) times greatest body width long. cuticle is finely annulated which is about 0.7 μm wide. lateral fields with 4 longitudinal lines which merge into 2 lines at around tail regions. cephalic region is indistinctly set off from body, appearing smooth and with 6 equal lips. stylet is 24.22 μm long with indistinct basal knobs. cylindrical portion of the stylet is slightly longer than anterior conical section. procorpus is wider anteriorly, gradually narrowing at posterior part, 48.44 59.1 (52.48±4.15) μm long. median bulb is spherical to pyriform in shape and occupying four-fifth of body width, 13.84 μm in length and 8.65 10.38 (9.51±0.86) μm in diameter. oesophageal glands forms a non overlapping lobe with intestine, about 34.6 38.06 (36.60±1.28) μm in length. nerve ring is about one-half of body width behind median bulb, encircling the anterior end of oesophageal glands. excretory pore is at base of median bulb, 74.39 83.04 (78.33±3.22) μm long from anterior body region forming a lobe at dorsal side of nerve ring. hemizonid, deirids and phasmids were not seen. vulva is a transverse slit, two-fifth of body width long and is located approximately at one-half body length from anterior end. genital tract is monoprodelphic, outstretched, usually extending up to the oesophageal gland lobe. oocytes are arranged in a single row, post uterine sac about 2.15 2.31 (2.21±0.07) μm times anal body diameter long. tail is 5.6 6.2 (5.9±0.22) μm times anal body diameter long, tapering gradually into a cylindrical tube terminating in a ventral prong tip. found in abundance as is female, is slightly smaller than female and is more straight upon fixation. cephalic region, stylet and oesophagus are as described for females. tail is slender with a single terminal mucro. spicules are about 24.22 μm long. testis is single, 335.62 342.45 (339.14±2.51) μm long. collected in december 2012 from soil around rhizospheric regions of coconut plant, linn. at ninghsing khul, jiri, imphal west district, manipur, india. holotype female on the slide fsb a -1/♀ sp. nov., paratype females on the slides fsb a 212/♀ sp. nov., paratype males on the slides fsb a 14/ ♂ sp. nov. and deposited in the nematode collection center of parasitology section, department of life sciences, manipur university, canchipur 795003, manipur, india. sp. nov. differs from all other species of fischer, 1894 in possession of the longest stylet and 4 lateral lines which gradually merged into 2 at the tail regions. cocos nucifera aphelenchoides longistylus aphelenchopides longistylus aphelenchoides longistylus aphelenchoides longistylus aphelenchoides 3 3 3 95 two new fischer, 1894 (nematoda: tylenchina) loukrakpam bina chanuaphelenchoides – et al. however, it comes close to (hussain & khan 1967); (chawla & khan 1979); (tandon & singh 1974) and (hooper 1958). sp. nov. differs from (hussain & khan 1967) in having 4 lateral lines throughout body length, longer body length, larger values of a, b, c´, stylet and longer spicule; smaller values of c, v and indistinct stylet knobs (lateral lines =3, body length = 0.39 0.45, a= 30 33, b= 4 4.5, c´=3, stylet = 11 13, spicule = 15 19, c= 16 20, v = 69 77 and presence of stylet knobs in (hussain & khan 1967). sp. nov. comes close to (chawla & khan 1979) in having similar range of a, b, c and v values. but, it differs from (chawla & khan 1979) in having 4 lateral lines gradually merging into 2, larger values of body length, c value, stylet length and absence of stylet knobs (lateral lines= 3, body length = 0.4 0.5 mm, stylet = 10 -11 μm and presence of stylet knobs in (chawla & khan 1979). sp. nov. also comes close to (tandon & singh 1974) in having similar range of body length, a, b, c values and spicule length. but, it differs from tandon & singh, 1974 in having larger value of c´, smaller value of v, longer stylet and 4 lateral lines which merged into 2 and indistinct stylet knobs (c´= 2.9, v = 71 73, stylet = 11 14 μm, 4 lateral lines and presence of stylet knobs in (tandon & singh 1974). similarly, sp. nov. comes close to (hooper 1958) in having similar range of body length, a, b, c and v values. but it differs from (hooper 1958) in having 4 lateral lines, smaller values of b and longer stylet with indistinct stylet knobs and larger values of spicules (lateral lines = 3, b= 10 14, stylet = 11 μm, spicules = 15 20 μm and presence of stylet knobs in (hooper 1958). sp. nov. body is finely annulated, tapering towards both extremities, straight and slightly curved ventrally on the tail region upon relaxation. lateral fields have 4 incisures. cephalic framework is smooth, set off from the body, about 5.19 μm wide and 2.53 μm high. stylet is prominent with distinct stylet guards, 8.65 μm in length with distinct stylet knobs. oesophagus has zig-zagly coiled procorpus, has strongly developed and rounded corpus with median sclerotised plates and elongated gland lobe overlying the intestine dorsally at a distance behind the bulb which is equal to about 3 4 times body width long. neither deirids nor phasmids were seen. excretory pore is close behind the nerve ring. vulva is prominent, protruding both lips with vagina inclined. large and elongated spermatheca, about 65.74 70.24 (67.99±2.25) μm long, oocytes are arranged in single row reaching up to the oesophageal bulb. post-vulval uterine sac is about one-half vulval body width and is empty. anterior lip of anus is protruding, tail is bluntly rounded, 12.11 25.95 (19.03±6.92) μm in length with a small hair like mucro. aphelenchoides absari aphelenchoides chalonus aphelenchoides lanceolatus aphelenchoides sacchari aphelenchoides longistylus a. absari a. absari aphelenchoides longistylus a. chalonus a. chalonus a. chalonus aphelenchoides longistylus a. lanceolatus a. lanceolatus a. lanceolatus aphelenchoides longistylus a. sacchari a. sacchari a. sacchari aphelenchoides neominoris description female: 96 biotropia vol. 21 no. 2, 2014 97 male: type habitat and locality: type material: differential diagnosis: not found. collected in september 2013 from soil around the rhizospheric regions of orange plant, from sibilong, chandel district, manipur, india. holotype female on the slide fsb-a 2/ ♀ sp. nov., paratype females on the slides fsb a 1, 3 10/♀ sp. nov. and deposited at the nematode collection center of parasitology section, department of life sciences, manipur university, canchipur 795003, manipur, india. sp. nov. differs from all other species of fischer, 1894 in having the shortest body length. but, it comes close to (ebsary 1991); (hussain & khan 1967) and (masleen 1979) in several other characters. 4 4 aphelenchoides neominoris aphelenchoides neominoris aphelenchoides neominoris aphelenchoides aphelenchoides minoris aphelenchoides absari aphelenchoides vaughani two new fischer, 1894 (nematoda: tylenchina) loukrakpam bina chanuaphelenchoides – et al. characters holotype paratypes n 1 10 stylet 8.65 8.65 length 0.439 0.351 – 0.439 (383.48 ± 39.71) a 36.29 36.29 – 40.6 (38.44± 2.16) b 6.51 4.23 – 6.51 (5.37± 1. 14) b´ 6.03 6.03 – 6.49 (6.27±0.18) c 36.29 13.53 – 36.29 (24.90± 11.37) c´ 1.75 1.75 – 3.75 (2.75±1) g1 50 41.87 – 50.0 (45.93±4.06) v 71.65 69.95 – 71.65 (70.8±0.85) lip width 5.19 5.19 lip height 2.53 2.53 oesophagus 67.47 67.47 – 83.04 (75.25±7.78) median bulb width 8.65 6.92 – 8.63 (7.49±0.81) ovary 219.71 147.05 – 219.71 (183.38±36.33) spermatheca 65.74 65.74 – 70.24 (67.99±2.25) pus 15.57 12.11 – 15.57 (13.84±1.73) pus/vbd 1.28 1.28 – 1.4 (1.34±0.06) rectum 5.19 5.19 tail 12.11 12.11 – 25.95 (19.03±6.92) abd 6.92 6.92 table 2. morphometric data of female species of sp. nov.aphelenchoides neominoris note: all measurements are in μm except length is in mm 98 biotropia vol. 21 no. 2, 2014 legends to figures a female anterior body b male anterior body c female entire body d female reproductive system e female lateral lines f female tail region g male entire body h male tail region i l. sp. nov. i female anterior body j female reproductive system k female lateral lines and l female tail region. aphelenchoides neominoris figure 1: a h. sp.nov.aphelenchoides longistylus 99 aphelenchoides neominoris aphelenchoides minoris aphelenchoides minoris a. minoris aphelenchoides neominoris a. absari a. absari a. absari aphelenchoides neominoris aphelenchoides vaughani a. vaughani a. vaughani no. sb/ft/ls-113/2013 sp. nov. comes close to (ebsary 1991) in having smaller body length and a ventral mucro. but, it differs from (ebsary 1991) in having 4 lateral lines, larger values of a, c; smaller values of b, pus, stylet and indistinct stylet knobs (lateral lines = 3, a= 26 29, b= 89, c= 15, pus= 5 vbd, stylet= 10 μm and presence of stylet knobs in (ebsary 1991). sp. nov. comes close to (hussain & khan 1967) in having almost similar body length, 4 lateral lines and presence of ventral mucro. but, it differs from hussain & khan, 1967 in having larger values of a, b and c but smaller values of c´, stylet length, presence of post uterine sac and indistinct stylet knobs (a= 30 33, b= 4.0 4.5, c= 16 20, c´=3, stylet = 1113 μm, absence of post uterine sac and presence of stylet knobs in (hussain & khan 1967). sp. nov. is similar to (masleen 1979) in having almost similar body length, presence of 4 lateral lines and ventral mucros. but, the species differs from (masleen 1979) in having larger values of a, c and g and smaller values of b, stylet length, tail length and in having a simple ventral mucro (a= 2431, b= 6.3 9.2, c= 12.1 16.9, g = 32 44, stylet = 9.5 11 μm, tail = 23.5 38.5 μm with a ventral mucro minutely multi-papillate at its tip in (masleen 1979). the authors thanked the head, department of life sciences, manipur university, canchipur for providing necessary laboratory facilities. the corresponding author also acknowledged the science and engineering research board, department of science & technology, govt. of india for financial support in the form of fast track young scientist fellowship (serb ). 1; 1 acknowledgements references aebsary ba. 1991. . canada: agriculture canada. iv. chawla ml, khan e.1979. two nomenclatorial corrections. indian j nematol 7: 100. fisher m. 1894. über eine clematis -krankheit. bericht aus dem physiolischen laboratorium des landwirthschaftlichen, instituts der universitat halle (11)3: 1 11. hooper dj. 1958. n. sp. and n. sp. (nematoda: aphelenchoidea). nematologica 3: 228 35. hussain si, khan am.1967. on the status of the genera of the superfamily aphelenchoidea (fuchs, 1937). proc helm soc wash 34: 167 74. maslen nr. 1979. six new nematode species from maritime antarctic. nematologica 25: 288 308. tandon rs, singh sp. 1974. nematode parasites of the common sponge gourd, from lucknow geobios 1: 24 7. seinhorst jw.1959. a rapid method for the transfer of nematodes from fixative to anhydrous glycerine. nematologica 4: 67 9. catalog of the order tylenchida (nematoda) aphelenchoides dactylocercus a. sacchari luffa cylindfrica . two new fischer, 1894 (nematoda: tylenchina) loukrakpam bina chanuaphelenchoides – et al. 2. tri (binary).cdr biotropia vol. 21 no. 2, 2014: 80 90 binary vector construction and -mediated transformation of lysozyme gene in seaweed agrobacterium tumefaciens kappaphycus alvarezii tri handayani , alimuddin , utut widyastuti , emma suryati and andi parenrengi received 19 november 2013/accepted 6 september 2014 ice-ice disease is the biggest problem in the cultivation of seaweed . the disease is caused by bacterial infection and induced by drastic changes of water quality. lysozyme has the ability to break down bacterial cell wall. the purpose of this research was to construct of a binary vector pmsh1-lys carrying chicken lysozyme (lys) gene and to introduce pmsh1-lys on . the binary vector expression was transformed into lba4404 by triparental mating. thallus was inoculated with carrying pmsh1-lys and then the transformed thallus was selected by adding 20 mg/l hygromycin to the culture medium. polymerase chain reaction (pcr) analysis showed that the construction of the binary plasmid pmsh1-lys was established. percentage of pmsh1-lys transformation on was 23.56%, while the efficiency of regeneration was 11.32%. pcr analysis showed that three of the regenerated thallus contained lysozyme gene. thus, transgenic was successfully produced. these findings can be useful for studying the mechanisms of seaweed defense against bacterial infection. , genetic transformation, , lysozyme 1,2* 1 3 4 4 1 department of aquaculture, faculty of fisheries and marine sciences, bogor agricultural university, bogor 16680, indonesia research center for oceanography,indonesian institute of science, jakarta 14430, indonesia research center for bioresources and biotechnology, bogor agricultural university and department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor 16680, indonesia research institute for coastal aquaculture,maros,south sulawesi, indonesia kappaphycus alvarezii k. alvarezii agrobacterium tumefaciens a. tumefaciens k. alvarezii k. alvarezii agrobacterium tumefaciens kappaphycus alvarezii 2 3 4 abstract introduction keywords: kappaphycus alvarezii (doty) is economically important red tropical seaweed highly demanded for its cell wall polysaccharides, being the most important source of kappa* corresponding author : umuamel@gmail.com doi: 10.11598/btb.2014.21.2.2 82 carrageenan in the world (bixler 1996). carrageenan is used in industries including food, textile, cosmetic, pharmaceutical and photography (yu . 2002). kappacarrageenan is used as stabilizers, thickeners and emulsifiers (bixler 1996). seaweed production can be increased by extensification and improvement of cultivation method. however, ice-ice disease is the biggest problem in seaweed cultivation. ice-ice infection can spread in broad area of cultivation for a week, and cause thallus damage about 60-80% within 1-2 months (sulistijo, personal communication, 2002). carrageenan content in the ice-ice infected seaweed decreases (amiluddin 2007), and this leads to loss for farmers (yulianto & mira 2009). bacteria are suspected as causative agents of ice-ice disease, including , and . shows the highest pathogenicity (nasution 2005). largo (2002) also found sp. on ice-ice infected thalli. furthermore, aris (2011) reported five bacteria on ice-ice infected thalli, namely and . genetic engineering techniques can be used to make seaweed resistant to bacterial infections. as the first step, takahashi . (2010) had transformed glucuronidase gene into thallus by particle bombardment. huddy . (2012) had also successfully introduced the lacz gene into the thallus by particle bombardment. in plant transgenic production, a foreign gene is generally transferred using . the method has advantages such as having relatively low cost, small copy number of gene and reproducibility (hiei . 1997). contains binary expression vector. the first vector is part of the virulent but without helper t-dna, while the second vector is smaller and contains a t-dna gene to be inserted (loeidin 1994). lysozyme has been used to generate transgenic fishes that are resistant to bacterial infections, such as zebrafish (yazawa . 2006), shrimp ( ) (burge . 2007) and salmon ( l.) (fletcher . 2011). lysozyme is small ubiquitous antibacterial enzyme that hydrolyzes gram-positive bacteria (li . 2008). in addition, lysozyme is also able to kill gram-negative bacteria infecting bivalves and shrimp (burge . 2007). yazawa (2006) established a transgenic zebrafish strain expressing chicken lysozyme gene. in the challenge experiment, 65% of the f2 transgenic zebrafish survived when infected by and 60% fish survived when infected by , whereas all non-transgenic fish were died. the lysozyme lytic activity of f2 in transgenic salmon was 40% greater than that in non-transgenic siblings (fletcher . 2011). chicken lysozyme possessed lytic activities against and (yazawa . 2006). thus, chicken lysozyme is an important component of immune defense against diverse bacterial infections that can be exploited to increase seaweed resistance against pathogens. the aims of this study were to construct a binary plasmid carrying chicken lysozyme gene and to transfer it into thallus by using . et al pseudomonas nigricaciens p. fluorescens, vibrio granii, bacillus cereus v. agarliquefaciens vibrio agarliquefaciens vibrio k. alvarezii flavobacterium meningosepticum, v. alginoliticus, pseudomonas cepacia, p. diminuta plesiomonas shigelloides et al porphyra yezoensis et al gracilaria gracilis agrobacterium tumefaciens et al a. tumefaciens a. tumefaciens, et al litopenaeus vannamei et al salmo salar et al et al et al et al. flavobacterium columnare edwardsiella tarda et al micrococcus lysodeikticus, flavobacterium columnare, aeromonas hydrophilla vibrio anguillarum et al k. alvarezii a. tumefaciens β-1, 4-linked glycoside bonds of peptidoglycan, a major cell wall component of 83 binary vector construction and -mediated gene transformation tri handayaniagrobacterium tumefaciens – et al. materials and methods kappaphycus alvarezii and sterilization construction of lysozyme-expressing plasmid kappaphycus alvarezii kappaphycus alvarezii et al. not spe not spe escherichia coli et al e.coli not spe thalli with green color were obtained from lampung, sumatera island, indonesia. thalli were maintained in fiber tanks with a flow-through water system, aerated and under a 12/12 hours (light/dark) photoperiod at room temperature. thalli were cut about 3 cm in length and then sterilized by 1% iodine solution and detergent. the thalli were maintained in prevasoli (pes) medium liquid until they were ready to be transformed. binary vector pmsh1 (nara institute of science and technology, japan; fig. 1) was used for the construction of lysozyme (lys) expression vector. lys gene was amplified from pjfker-lys (yazawa 2005; fig. 2) using specific pcr primers lysf: 5'-gca cta gtg gca aca tga ggt ctt tgc-3' and lys-r: 5'-ttg cgg ccg ctc ctc aca gcc ggc agc-3'. pcr was performed with 2 minutes initial denaturation at 94 c and then 35 cycles were run at: 30 seconds denaturation at 94 c, 30 seconds annealing at 64 c (at 59 c for nos terminator) and 1 minute of extension at 72 c. the amplified products were electrophoresed on a 2% agarose gel with 1x tae buffer. the dna of pmsh1 and pcr product of lys gen were cut by restriction enzymes i and i. lys gene was ligated with i and i-digested pmsh1. this recombinant binary vector was designated as pmsh1-lys. plasmid pmsh1-lys was then transformed into . 2002). transformant lys specific primer, and restricted using i and i enzymes. o o o o o dh5α by heat shock (suharsono dh5α was identified by pcr method using 84 biotropia vol. 21 no. 2, 2014 figure 1. map of the t-dna plasmid pmsh1 (naist, japan). npt ii: neomycin phosphotransferase ii, a selection marker gene. hpt: hygromycin phosphotransferase, a selection marker gene. mcs: multi cloning site, regions of target genes that is controlled by the cauliflower mosaic virus 35s promoter (camv 35s) and terminator (t) nopaline synthase (nos), containing i, i, i, i, i, i, i, i xba xho sac sma kpn spe not bamh transformation of pmsh1-lys into agrobacterium tumefaciens plasmid pmsh1-lys was transformed into by tri-parental mating (liberty . 2008). tri-parental mating was carried out using , hyg ) as the donor strain, dh1 carrying prk2013 (kan ) as a. tumefaciens et al e. coli e. coli dh5α carrying pmsh1-lys (kan r r r 85 figure 2. map of pjfker-lys (yazawa . 2005). chicken lysozyme gene is controlled by the promoter of keratin (keratin) japanese flounder ( ). sv40: simian virus 40 terminator. npt ii: neomycin phosphotransferase, gfp: green flourescense protein et al paralichthys olivaceus binary vector construction and -mediated gene transformation tri handayaniagrobacterium tumefaciens – et al. the helper strain, and lba4404 (strep ) as the recipient. was grown at room temperature on lb medium with 50 μg/l streptomycin. c on lb medium with 50 mg/l kanamycin and 50 mg/l hygromycin. dh1 bearing helper plasmid prk2013 was grown at 37 c on lb medium with 50 μg/l kanamycin. about 20 μl bacteria consisted of dh1 and were cultured together (conjugation=”konjugasi”) for 36 hours at room temperature. conjugated product was identified by antibiotic selection (kanamycin 50 mg/l, hygromycin 50 mg/l and streptomycin 50 mg/l), and pcr method using specific primers: for lys gene was lys-f: 5'-gca cta gtg gca aca tga ggt ctt tgc-3' and lys-r: 5'-ttg cgg ccg ctc ctc aca gcc ggc agc-3'; for 35s camv promoter was 35scamv-f: 35s-f: 5'-atg gct gga gta tta gct ggg-3' and lys-r: 5'-ttg cgg ccg ctc ctc aca gcc ggc agc -3; for nos terminator was lys-f: 5'-gca cta gtg gca aca tga ggt ctt tgc-3' and nos-r: 5'-ctc ata aat aac gtc atg cat tac a-3'. pcr was performed with 2 minutes initial denaturation at 94 c and then 35 cycles were run at: 30 seconds denaturation at 94 c, 30 seconds annealing at 64 c (at 59 c for nos terminator) and 1 minute extension at 72 c. the amplified pcr products were electrophoresed on 2% agarose gel with 1x tae buffer. a single colony of containing pmsh1-lys was incubated in 5 ml of lb medium and grown for 36 hours on a 200 rpm shaker at room temperature. the bacterial culture was refreshed in 10 ml of lb medium and grown for 18 hours on a 200 rpm shaker at room temperature to an od600 of 0.5-1.0. the bacterial culture was centrifuged at 5,000 rpm and the pellet was resuspended in 25 ml of liquid suspension medium containing pes and 100 μm acetosyringone. the suspension was used for thalli transfection. a. tumefaciens a. tumefaciens e. coli e. coli e. coli e. coli a. tumefaciens a. tumefaciens a. tumefaciens r o o o o o o o dh5α containing pmsh1-lys was grown at 37 dh5α, preparation of suspension for co-cultivationagrobacterium 86 biotropia vol. 21 no. 2, 2014 production of transgenic detection of lysozyme gene in transgenic construction of lysozyme expressing plasmid kappaphycus alvarezii k. alvarezii et al a. tumefaciens k. alvarezii e. coli not spe e. coli e. coli e. coli a. tumefaciens transgenic were produced as previously reported (cheney . 2001). seaweed thalli (1-2 cm in length) were wounded by sterile syringe and infected by carrying pmsh1-lys for 30 minutes in suspension medium containing pes and 100 μm acetosyringone. the infected thalli were transferred to co-cultivation medium (pes medium containing 100 μm acetosyringone) for 3 days and then transferred to recovery medium (pes medium without acetosyringone) for 7 days. finally, transgenic thalli were selected in pes medium containing hygromycin 20 mg/l for 14 days. genomic dna was extracted from putative bud of transgenic using ctab reagent (doyle & doyle 1987). dna sample (1 μl) was used in a 10 μl pcr mixture. the pcr primers for lys detection were lys-f and lys-r; 35s camv-f and lys-r; and lys-f and nost-r. pcr was performed with 2 minutes initial denaturation at 94 c and then 35 cycles were run at: 30 seconds denaturation at 94 c, 30 seconds annealing at 64 c (at 59 c for nos terminator)and 1 minute extension at 72 c. the amplified pcr products were electrophoresed on a 2% agarose gel with 1x tae buffer. construction of binary plasmid was done by ligating chicken lys gene (460 bp) and pmsh1 (12,986 bp). result of the ligation was 13,449 bp (fig.3a lane 1). verification of containing pmsh-lys was performed by digesting pmsh1-lys with i and i restriction enzymes. the restriction products were two fragments in size of 12,986 bp and 460 bp (fig.3a lane 2). the 12,986 bp fragment was the size of pmsh1, and 460 bp was lys gene fragment. , respectively (fig. 3b). based on the results, it was concluded that the binary vector pmsh1-lys was established. pcr verification and enzyme restriction performed above also confirmed that transformation of pmsh1-lys into d been successful. plasmid pmsh1-lys contained genes encoding protein resistant to antibiotics. thus, bacterial colony survived and grew in selective medium containing kanamycin and hygromycin was a pmsh1-lys transformed ould . k. alvarezii o o o o o results and discussion dh5α dh5α carrying pmsh1-lys was also identified using pcr with primers lys-f and lys-r; 35s-f and lys-r, and lys-f and nos-r. the results of pcr analysis using those primers were 460 bp, 670 bp and 580 bp dh5α ha dh5α. the pmsh1-lys c then be used for transformation into 87 figure 3. a. pattern restriction of pmsh1-lys using i and i enzymes. m: 1 kb dna ladder marker (fermentas). lane 1: plasmid of pmsh1-lys, lane 2: i and i digested pmsh1-lys and lane 3: pcr product of lysozyme gene (lys). b. identification of lysozyme gene using pcr with primers lys-f and lys-r (lanes 1, 2 and 3), 35s-f and lys-r (lanes 4 and 5) and lysf and nos-r (lanes 6 and 7). m: 100 bp dna ladder marker (fermentas), lanes s. lane 2: positive control, pjfker-lys. lanes 3, 5 and 7 are the negative c not spe not spe escherichia coli dh5α containing 1, 4 and 6 are dh5α containing pmsh1-ly ontrol (non-transformant dh5α) figure 4. triparental mating. a. results of tri-parental mating grown on la medium without antibiotics. b. lba 4404 transformants on selective medium containing 50 mg/ hygromycin, 50 mg/ kanamycin and 50 mg/ streptomycin. c. lba 4404 non-transformants did not grow on selective medium agrobacterium tumefaciens a. tumefaciens l l l binary vector construction and -mediated gene transformation tri handayaniagrobacterium tumefaciens – et al. transformation of pmsh1-lys into agrobacterium tumefaciens transformation of pmsh1-lys into was performed by tri-parental mating (fig.4). plasmid pmsh1-lys in (as a recipient) through the conjugation process by prk2013 in dh1 as a helper (fig. 4a labelled as “konjugasi”=conjugation). wa was resistant to streptomycin, but susceptible to kanamycin and hygromycin. thus, bacteria grew on the selective medium was containing pmsh1-lys derived from tri-parental mating (fig.4b). non-transformant was not grown on the selective medium (fig. 4c) a. tumefaciens e. coli a. tumefaciens e. coli e. coli a. tumefaciens a. tumefaciens a. tumefaciens dh5α (as a donor) was transferred into dh5α containing pmsh1-lys s resistant to kanamycin and hygromycin, but susceptible to streptomycin. . figure 5. identification of containing pmsh1-lys by pcr method. lanes 1, 2 and 3: pcr product using primers lys-f and lys-r. lanes 4, 5 and 6: pcr product using primers 35s-f and lys-r. lanes 7, 8 and 9: pcr product using primers lys-f and nos-r. m: 100 bp dna ladder marker (fermentas). lanes 1, 4 and 7 are ontaining pmsh1-lys). lanes 3, 6 and 9 are a negative control (nontransformant lba4404) agrobacterium tumefaciens a. tumefaciens a. tumefaciens lba4404 from tri-parental mating. lanes 2, 5 and 8 are a positive control (dh5α c identification of transformants was performed by pcr method. as shown in figure 5, pcr amplification products using lys-f/lys-r primer was 460 bp (lanes 1 and 2), using primers 35s-f/lys-r was 670 bp (lanes 4 and 5), and using lysf/nos-r primer was 580 bp (lanes 7 and 8). this result showed that contained pmsh1-lys, and it could then be used for transformation into . a. tumefaciens a. tumefaciens k. alvarezii 88 biotropia vol. 21 no. 2, 2014 transformation of pmsh1-lys into kappaphycus alvarezii k. alvarezii a. tumefaciens a. tumefaciens gracilaria changii k. alvarezii agrobacterium et al thalli were adapted on the pes culture medium (liquid and solid), and then were transformed with pmsh1-lys by (fig.6). the result showed that after 30 minutes infection with at od600 of 0.5 to 0.8, the transformed thalli could grow in pes medium containing 100 acetosyringone (fig.6). the transformed thalli could grow on selective medium containing 20 mg/l hygromycin (fig. 6a-c). non-transformant thalli was gradually dead on selective medium containing 20 mg/l hygromycin (fig. 6d-f). wild type thalli could grow on pes medium (fig. 6g-i). in total, 53 out of 225 transformed thalli (23.56%) grew on hygromycin selective medium (table 1). this number was lower compared to percentage of gene lacz transformation on using particle bombardment methods (80-94%). the result found in this study was thought to be affected by the difference of transformation method. in the next study, genetic transformation protocols of need to be improved for increasing the transformation percentage. there were 3 (11.32%) thalli succeeded to germinate. efficiency of putative sprouting ratio was determined by the number of putative sprouting thalli grew on hygromycin selective medium. the result showed that the efficiency of putative germination transformed thalli was lower than those of non-transformed ones (22%). it might be caused by infection and antibiotic selective medium. this finding was in accordance with suma . (2008) that the addition of antibiotics in selective medium μm figure 6. development of transformed thalli at 4 (a), 8 (b) and 12 (c) weeks culture in selective medium containing 20 mg/l hygromycin. non-transformant thalli at 4 (d), 8 (e) and 12 (f) weeks culture in selective medium containing 20 mg/l hygromycin. non-transformant thalli in the medium without hygromycin at 4 (g), 8 (h) and 12 (i) weeks culture. in d-f, green color indicates survived thalli, while the white color indicates dead thalli agrobacterium tumefaciens kappaphycus alvarezii table 1. transformation percentage and putative bud of containing lysozyme gene kappaphycus alvarezii treatment number of thalli number of hygromycin resistant thalli percentage of transformationa) number of putative bud number of positive by pcr efficiency of putative budb) transformation 225 53 23.56% 6 3 11.32% control -1) 50 0 0 0 0 0 control +2) 50 0 11 0 22% notes: the percentage of the number of hygromycin-resistant thalli versus the total number of thalli transformed the percentage of the number of thalli that sprouts putative versus number of hygromycinresistant thalli non-transgenic thalli in hygromycin selective medium non-transgenic thalli in non-hygromycin medium a) b) 1) 2) 89 binary vector construction and -mediated gene transformation tri handayaniagrobacterium tumefaciens – et al. ba c d e f g h i can cause thallus growth and development decrease. optimum infection duration and different treatment methods on selective medium in future research is expected to result in higher germination efficiency. transformation percentage in this study was 23.56% (table 1). figure 7. lysozyme gene detection in transformed thalli by pcr method. lanes 1, 2 and 3: pcr products using lys-f and lys-r primers; lanes 4, 5 and 6 using 35s-f and lys-r primers; lanes 7, 8 and 9 using primers lys-f and nos-r primers. m: 100 bp dna ladder marker (fermentas). lanes 1, 4 and 7 are transgenic thalli; lanes 2, 5 and 8 are positive control (plasmid of pmsh1-lys); lanes 3, 6 and 9 are negative control (wild type of seaweed). agrobacterium tumefaciens kappaphycus alvarezii presence of lys gene in was confirmed by pcr method using three primers set. as shown in figure 7, transgenic thalli (lanes 1, 4 and 7) possessed pcr amplicon in the same size with the positive control of pmsh1-lys plasmid (lanes 2, 5 and 8), while the non-transgenic thalli showed no amplicon. this indicated that the putative bud derived from transformation was transgenic carrying lys gene. k. alvarerzii a. tumefaciens 90 biotropia vol. 21 no. 2, 2014 this study is the first transgenic production using infection method and lys gene as the transgene. analysis of lys gene activity in transgenic againts ice-ice bacterial agent is in progress. this transgenic can also be useful for studying the mechanisms of seaweed defense against bacterial infection. in addition, transgenic seaweed resistance to ice-ice disease can be useful to ensure seaweed production in the season when ice-ice disease frequently infects. transformation method developed in this study is beneficial to produce other transgenic seaweed expressing protein that regulates important traits in aquaculture. examples of interesting genes that can be used to generate transgenic seaweed are cooper/zinc superoxide dismutase (cuzn-sod) and omega-3 highly unsaturated fatty acids (n-3 hufa) metabolic enzymes. those enzymes had been used in plant transgenic production. cuzn-sod had successfully been transformed in to increase its resistant to environmental stress (hannum 2012). transgenic expressing n-3 hufa metabolic enzymes had also been generated (robert . 2005). construction of the binary plasmid pmsh1-lys was established with size of 13,449 bp. mediated transformation of lysozyme gene could produce transgenic . the efficiency of putative bud was 11.32 %, while percentages of positive transformants were approximately 23.56%. k. alvarezii a. tumefaciens k. alvarezii k. alvarezii nicotiana tabacum arabidopsis et al agrobacterium tumefaciens kappaphycus alvarezii conclusions 91 binary vector construction and -mediated gene transformation tri handayaniagrobacterium tumefaciens – et al. acknowledgements references this research was supported by the collaboration between research institute for coastal aquaculture, maros and research center for bioresources and biotechnology, bogor agricultural university. our thanks to department of aquaculture, faculty of fisheries and marine science, bogor agricultural university for technical support. amiluddin nm. 2007. a study of growth and carrageenan content of seaweed that are affected by ice-ice disease in pari island, thousand islands. . bogor (id): institut pertanian bogor. aris m. 2011. identification, pathogenicity of bacteria and the use of gene 16s rrna for ice-ice detection onseaweed aquaculture ( ). . bogor (id): institut pertanian bogor. bixler h.1996. recent developments in manufacturing and marketing carrageenan. hydrobiologia 326/327: 3557. burge ej, madigan dj, burnett le, burnett kg. 2007. lysozyme gene expression by hemocytes of pacific white shrimp , after injection with vibrio. fish shelfish immun 22: 327-39. cheney d, metz b, stiller j. 2001. -mediated genetic transformation in the macroscopic marine red algae . j.phycol 37: 11-2. doyle jj, doyle jl. 1987. a rapid dna isolation procedure for small quantities of fresh leaf tissue. phytochem. bull. 19: 11-5. fletcher gl, hobbs rs, evans rp, shears ma, hahn al, hew cl. 2011. lysozyme transgenic atlantic salmon ( l aquaculture res 42: 427-40. hannum s. 2012. isolation, cloning, and expression analysis of gene coding copper/zinc superoxide dismutase (cuzn-sod) from l. . bogor (id): institut pertanian bogor. hiei y, komari t, kobu t. 1997. transformation of rice mediated by . plant mol. biol 35: 205-18. huddy sm, meyers ae, coyne ve. 2012. transformation of lacz using different promoters in the commercially important red alga, . afr. j. biotechnol 11(8): 1879-85. largo db. 2002. recent developments in seaweed diseases. in: the national seaweed planning workshop. proceeding: 2001 aug 2-3; 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''storma (stability of rainforest margin) scientists hltp:/avww. storma.de abstract net primary production, npp, is one of the most important variables characterizing the performance of an ecosystem. it is the difference between the total carbon uptake from the air through photosynthesis and the carbon loss due to respiration by living plants. however, field measurements of npp are time-consuming and expensive. current techniques are therefore not useful for obtaining npp estimates over large areas. by combining the remote sensing and gis technology and modelling, we can estimate npp of a large ecosystem with a little ease. this paper discusses the use of a process based physiological sunshade canopy models in estimating npp of lore lindu national park (llnp). the discussion includes on how to parameterize the models and how to scale up from leaf to the canopy. the version documented in this manuscript is called netpro model, which is a potential npp model where water effect is not included yet. the model integrates cis and the use of remote sensing, and written in visual basic 6.0 programming language and map objects 2.1. netpro has the capability of estimating npp of cs vegetation under present environmental condition and under future scenarios (increasing [co2], increasing temperature and increasing or decreasing leaf nitrogen level). based on site-measured parameterisation of vam* (photosynthetic capacity), /jj (respiration) and leaf nitrogen oni), the model was run under increasing co2 level and temperature and varied leaf nitrogen. the output of the semi-mechanistic modelling is radiation use efficiency (?). analysis of remote sensing data give normalized difference vegetation index (ndvi) and related leaf area index (lai) and traction of absorbed photosynthetically active radiation (/m>ak). climate data are obtained from 12 meteorological stations around die parks, which includes global radiations, minimum and maximum temperature. co2 absorbed by vegetation (gross primary production, gpp) is then calculated using the above variables and parameters with the following equation: estimating npp, while ecosystem respiration is set as a function of temperature for estimating nee. under present condition, the net absorption of co> by the vegetation of lore lindu national park (npp) is 1330.31 gcm"2year"' and at double co2 and temperature increased of 3.5 "c, it increased by 23 %, reaching 1638.80 gcm'2 year'1. key words : npp semi-mechanistic model, photosynthesis, carbon sequestration, net primary-production, tropical forest 22 biotropia vol. 13 no. 1,2006 introduction the global carbon balance has become an issue of great concern during the last decade due to its impact on climate. ipcc (2001) has implicated that the increase in co2 concentration from 285 ppm in year 1780 to 360 ppm in year 2000 has resulted in 0.6 °c global average temperature increase and it is projected that in the year 2100, the increase will be up to 5.8 °c. the concern over this problem has resulted in international agreements (e.g., rio 1992; unfccc 1994; and kyoto 1997) to reduce co2 concentration in the atmosphere. net primary production (npp) is an important quantitative characteristic of an ecosystem (churkina et al. 1999). it refers to the net production of organic carbon by plants in an ecosystem usually measured over a period of a year or more. it is the difference between carbon gain through photosynthesis and carbon loss through respirations. it constitutes the total annual growth increment (both above and below ground) plus the amounts grown and shed in senescence, reproduction or death of short-lived individuals in a stand plus the amounts consumed by herbivores. seasonal changes in npp will influence seasonal changes in net ecosystem exchange (nee; npp-soil respiration), and it is a principal cause of seasonal changes in atmospheric co2 (keeling et al. 1996). tropical forests are important sink of co2, representing 59 % of the global carbon pool in forests (dixon et al. 1994). although the area of tropical forests is only 22 % of the global forest area, they account for 32 43 % of the world's potential terrestrial npp (melillo et al. 1993; field et al. 1998). because conventional measurements (periodical destructive harvesting) of npp of all ecosystems at all times are impractical, models are needed to estimate npp. the model discussed in this manuscript is based on farquhar and caemmerer (1982), june (2002) and de pury and farquhar (1997). most part of the model equations are mechanistic and some are semi mechanistic. when using such models in estimating npp, prediction of environmental effects to npp become more reliable. this manuscript contained theoretical background in developing the models, at leaf and canopy level; parameterisation and integrating remote sensing (rs) technology for data input and geographical information system (gis) for display. parameterisation of model was conducted using photosynthesis system adclc4am. the model is run to answer the following question: (i). how much co2 is absorbed by vegetation in a protected forest like lore lindu national park ; (ii). how would changes in temperature, light, and co2 concentration in the atmosphere affect the absorption? model parameters and estimated npp of lore lindu national park are presented. integration of the model with remote sensing data and gis are done using map object 2.1 and visual basic 6.0 in an application software netpro. 23 integration of npp semi mechanistic-modelling t. june et al. modelling net primary production (npp) plant photosynthesis and net primary production plants fix carbon dioxide from the atmosphere during the process of photosynthesis. all the carbon fixed during photosynthesis is called gross primary production (or gross primary productivity in terms of rate, for example, tonnes of carbon per hectare per year, usually called gpp). some of the fixed carbon is used by the plants themselves for metabolic processes (largely respiration) and in this process, carbon dioxide is returned to the atmosphere. the carbon that is not used in respiration remains on the plant and adds to the biomass of the plant. this is net primary production (npp). npp represents the net new carbon stored as biomass in stems, leaves or roots of plants. it is the difference between the carbon assimilated during photosynthesis by plant leaves and carbon consumption through respiration by leaves, stems and roots. it is a quantitative measure of plant growth and carbon uptake. the knowledge of npp distribution provides information on the productivity of croplands, forest and grasslands and thus helps improve management strategies for sustainable development of natural resources. at the national scale, npp allows the estimation of the contribution of landmass to the global carbon budget which is important in global change studies. in agricultural system and forestry, npp is usually defined as the increase in the standing biomass plus losses through litterfall and through consumption by herbivore. there are three main ways of estimating net primary productivity first involves the estimation of biomass production and second through the measurement of gas exchange and modelling or third through co2 flux measurement using surface tower over forest canopy or agroforestry system or using mast installed with instrument directly measuring co2 fluxes over short type of vegetation like grassland or crop. npp modeling and data requirements. an attractive approach for estimating npp was firstly proposed by mont'ith (1972; 1977), in which he determined dc/dt (carbon accumulation over time;wnen time = 1 year dc/dt = npp) as a product of the efficiency of the canopy (e, mol co2 mol"1 par or in unit gc mjt1), fraction of photosynthetically active radiation, par (/apak) absorbed by the canopy and the daily par reaching the top of the canopy (par) as: the original approach of monteith considered the value of e as a constant and based on net co2 absorption (determined through increased in biomass). on reality, 24 biotropia vol. 13 no. 1,2006  e changes  through  time due  to  the changing climatic and plant variables  like leaf area  index, nitrogen level, and water status. in order to make the model to be responsive to  the changing environmental condition, or  to be used  for a climate change prediction  effect, e has to be mechanistically or semi mechanistically modelled using the approach  introduced in june (2002).  the outline of the semi‐mechanistic model to produce the e value introduced in this  manuscript is shown in figure 1.  01 i\rr scim mcciiaiiimii-inuueiuug — i. june tfi ui. i light intensity incident on leaf surface (nmolm"2 s"1) j rate of actual electron transport (̂ imol m"2 s"1) •/ma* maximum electron transport rate (̂ imol m'2 s"1) ke michaelis-menten constant for carboxylation by rubisco (|.ibar) ka michaelis-menten constant for oxygenation by rubisco (mbar) o ambient partial pressure of oxygen (mbar) r universal gas constant, 8.3144 j mol4 k"1 rt dark respiration of leaf which continues in the light (nmol m"2 s"1) t leaf temperature (°c) k™« maximum rate of rubisco activity in the leaf (umol m"2 s"') &„ nitrogen extinction coefficient k light extinction coefficient af0 leaf nitrogen concentration on top of canopy (mmolm"2) ^c.kai total par absorbed by the canopy and leaf (umol m"2 s"') npp net primary production gpp gross primary production nee net ecosystem exchange rveg respiration by vegetation (0.45 gpp) reco respiration of ecosystem (as a function of temperature) the model used is a c^ photosynthesis model. it is chosen due to the fact that €3 plants dominate 95 % of earth vegetation. it is shown in the model that the responses of c3 leaf photosynthesis to light, temperature and co2 concentration can be described by the biochemical properties of just two steps in the process, the carboxylation reaction (shown by kcmax) and the regeneration of the acceptor for carboxylation (shown by j,mu). this mechanistic model, has been widely validated as an accurate predictor of photosynthetic carbon uptake by leaves with variation in environmental conditions. the scaling up to canopy to estimate npp is done using sun-shade model (de pury and farquhar 1997; june 2002). in the simulation, supply of cck (fj) into the leaf is modelled as 0.7 of ambient cck (ca). this is a condition where water is not a limiting factor and vapour pressure deficit is around 12.5 mbar. 7max is taken as 2.1 fcmax. to run the model the following groups of data are needed: (1) fixed parameters of leaf and canopy photosynthesis for €3 plants (table 1); (2) photosynthetic parameters based on measurements ( table 2) ; (3) hourly climate data of maximum and minimum air temperature and global radiation. these hourly data are generated from daily data. ra was determined by extrapolation of a linear regression at the lower end of the par response curve (at par = 0-100 ^mol m"2 s"1) (figure 2) and fcmm was estimated from the lower end of the q response curve at q around 100 jibar (table 2). 26 b1otropia vol. 13 no. 1,2006 to scale up the model result for the whole national park, input data (lai and /apar) derived from ndvi (normalized difference vegetation index) observed from satellite images (landsat tm) are used as follows: where nir and red is the amount of reflected light of visible near infrared, and red wavelengths, respectively; /apar is fraction of absorbed photosynthetically active radiation. both equations (3) and (4) are developed for tropical forest. model development with study case lore lindu national park netpro model is a prototype of potential net primary production model where water deficit effect is not included yet. the model integrates the use of remote sensing in obtaining leaf area index (lai) through a relationship with ndvi. the mathematical equations showing the linear regression between lai with ndvi and lai with /apar were obtained from tropical forest area. the lai is used as input to the photosynthesis model, while the fapm is used as input for eq. (1) to estimate 27 integration of npp semi mechanistic-modelling t. june et al. table 1. fixed photosyiithetic parameters for c3 plants and canopy parameters parameters value sources r* co: compensation partial pressure in the absence of dark respiration (|*bar) (at 25 42.75 bemacchi et al. (2001) e activation energy for carboxylation, oxygenation, respiration, rubisco activity and coi compensation point (j mol"') 79430,36380, 46390, 65330,37830 bemacchi et al. (2001) k, michaelis-menten constant for carboxylation by rubisco (pa) 40.49 bemacchi et al. (2001) ka michaelis-menten constant for oxygenation by rubisco (pa) 27840 bemacchi etal. (2001) r universal gas constant (j mol"' k"1) 8.3144 goudriaan (1977) p« canopy reflection coefficient for diffuse par 0.036 goudriaan (1977) k, diffuse and scattered diffuse par extinction coefficient 0.715 goudriaan (1977) ph reflection coefficient of a canopy with horizontal leaves 0.041 goudriaan (1977) *' beam and scattered beam par extinction coefficient (for random orientation of leaves) 0.69/sinp june (2002) n,, base level of nitrogen not associated with photosynthesis (rnmol n m"') 29 antene/a/. (1995) x. ratio of rubisco capacity to leaf nitrogen content 1.63 june (2002) pi the leaf reflection coefficient for par 0.10 de pury and farquhar (1997) t, the leaf transmissivity to par 0.05 de pury and farquhar (1997) 0, curvature factor of the light response curve 0.7 june (2002) note: 4-i/ip is sun elevation. 28 biotropia vol. 13 no. 1,2006 study site: lore lindu national park a. site information the netpro v. 1.0 is run for lore lindu national park, central sulawesi the whole national park is located in lat. 1° 107-1 °50s and long. 119°50?120°20?e (figure 5). vegetation that occurs in tnll includes species dominating the lower area (200-1000 m) such as mussaendopsis beccariana, ficus sp., myristica sp., pterospermum sp., canangium odoratum, arrenga pinata and species dominating the higher area (1000-2500 m, 90 % of tnll) such as castanopsis argentea and lithocarpus sp. other species includes podocarpus sp., elaeorpus sp., adinandra sp., litsea sp., callohylhim sp., eucalyptus deglupta and palmae (kartawinata 1985; mogea 2002). b. software used and data input for netpro sofware used for data preparation and to run netpro includes visual basic 6.0, ermapper 6.4, arcview 3.3 and mapobject 2.1. data input into netpro v 1.0 includes (1) shapefile of polygon with different characteristics of ndvi, minimum 29   biotrop1a vol. 13 no. 1, 2006 figure 5. lore lindu national park in central sulawesi, with red dots showing 12 meteorological stations around the park and red square shows the location the meteorological tower where direct measurement of co: fluxes are conducted. and maximum temperature and par classes of llnp; (2) daily averaged climate data (global radiation, maximum and minimum temperature) from year 2001-2005. ndvi is derived from landsat tm 7 dated 21 august 2001. the climate data, global radiation and daily temperature are zoned into several polygons ( figure 6). ndvi, leaf area index (lai), and /apar resulting from image analysis for year 2001 are shown in figures 7, 8 and 9. ndvi values are divided into 4 classes and based on the equation of ibrahim (2001), lai ranges from 0 to 10.04. lai values are used as input to photosynthesis model to obtain e value. /apar distribution (ranges from 0 to 65 %) is shown in figure 9, where these values are used as input to the model to obtain npp and nee. classes of average ndvi, temperature and global radiation are overlayed to form polygons with different characteristic of climate and ndvi. c. net primary production (npp) and net ecosystem exchange (nee) of llnp the value of simulated e changes with changing environmental conditions such as changes in global radiation, temperature, atmospheric co2 concentration, and nitrogen level of the leaf, and therefore result in varied npp and nee values (table 3). 31     integration of npp semi mechanistic-modelling t. june et al. conclusions this framework of model in estimating npp using remotely sensed ndvi combined with semi-mechanistic modeling is first introduced by the author at the scientific meeting on climate and weather prediction, center for climate and atmospheric science applications, national institute of aeronautics and space (lapan) in bandung, indonesia on 31th july 2002 where the proceedings was published in early 2003. the idea was then used by several undergraduate and post graduate students for thesis researches, using a constant value of radiation use efficiency (e) under the author's supervision for study sites in sumatera and sulawesi. this research idea is further extended to include a mechanistic estimation of the radiation use efficiency, and in 2004 it was approved by the integrated research award secretariat of the indonesian institute of sciences to be funded for the period of 2004-2006. however with limited availability of budget during that period, the funding was then cancelled after being approved by the reviewer panel. through seamed biotrop dipa2004, the development of the framework was realized and the prototype of the application software netpro is produced. model parameterization was conducted in lore lindu national park early 2005 supported by storm a sfb 552 (stability of rainforest margin) project. this work still needs improvement in display and modeling. it also needs inclusion of water effect, although application for lore lindu national park the assumption that water is not a limiting factor can be used. to be applicable to different site and type of vegetation, site specific parameterization (vcmax, e) and site variable measurement and estimation like leaf nitrogen, lai, /apar. climate data (diurnal global radiation, minimum and maximum temperature, diffuse radiation, vapour pressure deficit) are required. it is also important to develop relationship between ndvi and lai and /apar from different type of vegetation. direct measurement of co2 flux and e from eddy correlation and micrometeorology techniques for validation of model are needed. application of 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res., vol. 99, (3): 5263-5283. von, caemmerer, s.j.r. evans, g.s. hudson and t.j. andrews (1994). the kinetics of rubisco inferred from measurements of photosynthesis in leaves of transgenic tobaco with reduced rubisco content. planta, 195: 33-47. 36 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 702 sujan balami (effect of invasive) revisi tambah gambar.cdr effect of invasive on species ageratina adenophora richness and composition of saprotrophic and pathogenic soil fungi sujan balami, lal b. thapa and sanjay kumar jha * central department of botany, tribhuvan university, kirtipur 44618, nepal received 29 september 2016/accepted 3 september 2017 abstract belowground modification of soil microbial community by invasive plants is well evident. similar instances of ageratina adenophora invasion have been reported. this study was aimed to determine the effect of a. adenophora invasion on species richness, species or community composition and occurrence frequency of soil fungi. these parameters were analyzed using culture method on invaded and uninvaded soils. species richness of soil fungi was lower in the a. adenophora invaded soil compared to the uninvaded soil. the occurrence frequency of particular fungi was different for those two soil conditions. a. adenophora also altered soil fungi species composition in the invaded soil by replacing saprophytic fungi and accumulating pathogenic fungi. thus, a. adenophora is associated to lower species richness of saprophytic soil fungi and high occurrence frequency of pathogenic soil fungi. this study concluded that the invasive a. adenophora modifies belowground soil fungi communities as one of the mechanisms involved in the successful invasion of a. adenophora. keywords: belowground modification, soil fungi, species composition, species richness introduction uncoupling of ecosystem dynamics and consequent threat to biodiversity loss are attributed to invasion of alien plants (iaps) (webster et al. 2006; boy & witt 2013). the invasive alien species have adopted multiple strategies to proliferate (holzmueller & jose 2009) in an introduced range and able to compete with native plants for resources and space (mack & d' antonio 2003). various experimental evidences showed that modification in soil biota is one of strategies involved in the invasion of iaps (xiao et al. 2014a). the iaps release myriads of chemicals from their above and belowground parts into soils by leaching process and exudation (dayakar et al. 2009). these chemicals are capable of altering soil biophysical properties and ultimately, plant diversity (garbeva 2008; doornbos 2012; thapa et al. 2016a). in response to this, aboveground biota sharing the same niche with the iaps invariably regulates the changes (darrah 1993) by applying a feedback system (van der putten et al. 1993). soil microbial community has either negative or positive feedback to the plants (bonanomi et al. 2005). for example, negative feedback may involve pathogenic effect, production of bioactive compounds and production of allelochemicals, while positive feedback involves root fungus mutualism and secretion of plant growth promoting substances (bias 2006). et al. on the other hand, regulation in nutrient cycle is a result from plant-soil feedback system (johnson et al. 1997). such a plant-soil feedback system determines diversity and relative abundance of the above and belowground organisms (van der putten 1993).et al. plant soil microbe studies in iaps shows that they can modify soil microbial communities (kourtev et al. 2002; van der putten et al. 2007) for successful invasion (reinhart & callaway 2004; li et al. 2006; boudiaf et al. 2013). negative feedback mechanisms due to iaps may result to the decrease in competitive abilities of native species (ehrenfeld et al. 2005). the negative feedbacks are also recognized in biomass and growth of native * corresponding author: lal_thapa25@yahoo.com biotropia 4 3 7 212 219 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 3 702 212 plants (stinson et al. 2006), which might be hostspecific, leading to a decline of native plants population (didham et al. 2007). ageratina adenophora (spreng.) r. m. king and h. robinson, an invasive alien plant of asteraceae family, is a perennial, erect or decumbent subshrub (tiwari 2005), natively found in et al. mexico and costa rica and spread worldwide (niu 2007; xue 2010; inderjit 2011). et al. et al. et al. this species contains an array of bioactive constituents such as terpenoids, flavonoids, phenyl propanoids and their derivatives (kundu et al. 2013). several mechanisms have been suggested as the reasons of successful invasion of iaps including in their novel range. most a. adenophora studies on iaps focused on aboveground vegetation changes (levine 2003). a wide et al. range of belowground biotic interactions can play important role in determining plant interactions and ecosystem function (callaway 2001). et al. therefore, it is important to understand the interactions between iaps and soil biotic community and their interactions (wolfe & klironomos 2005). moreover, an integrated understanding of how aboveground and belowground biota interact with iaps is necessary to manage and restore alien invaded native communities (wolfe & klironomos 2005). there are some studies on modifications in the belowground biotic community associated with a. adenophora invasion, for example mangla and callaway (2008) and niu (2007). in nepal, et al. severe invasion of occurred from a. adenophora tropical to subtropical regions affecting native schima-alnus and other types of vegetation (tiwari 2005; thapa 2015; 2016b; 2017). et al. et al. hence, this study hypothesized that a. adenophora is responsible for changing species richness, species or community composition and occurrence frequency of the native soil fungi as invasion mechanisms of in a. adenophora nepalese forests. materials and methods the experiment was designed for comparing species richness of soil fungi, species composition and occurrence frequency between a. adenophora invaded and uninvaded soil. the invaded soil consisted of invaded non-rhizosphere soil and rhizosphere-contained invaded soil. the experiments were conducted at the central department of botany, tribhuvan university, kathmandu, nepal in march and august 2014. sampling site and method of soil sampling soil samples were collected from champadevi community forest located at southwest of kathmandu valley, nepal. altitude of the location varied from 1,400 – 2,300 m asl with annual mean otemperature of 18 c and annual precipitation of 1,343 mm. schima wallichii, alnus nepalensis, myrsine capitelata, maesa chisia, castanopsis indica and quercus sp. were common native trees in the forest. the forest was invaded by a. adenophora for 40 50 years and the invasion has become a serious challenge for the native diversity of the area due to its severe colonization (thapa et al. 2016b). uninvaded soil was collected from the forest site located at 27˚42ʹ06″ n and 85˚19ʹ14″ e with altitude of 1,600 m. rhizosphere-contained invaded soil was collected by uprooting a. adenophora. a total of 30 plants were uprooted from three sampling sites of the invaded patches nearby the uninvaded site, 10 plants from each sampling site (distance between each sampling site was at least 50 m). the soil around root surfaces was collected in sterile plastic bags and a composite sample was prepared. invaded non-rhizosphere and uninvaded soil samples were collected from 10 random points at the invaded and uninvaded patches of the forest. soil samples were collected from soil depth of 10 cm below soil surface. composite sample of each invaded non-rhizosphere and uninvaded soil was prepared. freshly collected soils were sieved separately through sterile mesh (2 mm osize), and stored in a refrigerator at 4 c for 5 days, until use. fungi culture, isolation and identification serial dilution method (benson 2002) followed by pour plate technique were adopted for culturing and isolating fungi from all types of -3 -5composite soil samples. dilutions of 10 and 10 were used for plating (aneja 2003). czapek dox agar [with 30 mg/l (amoxicillin)] and potato dextrose agar were used for culture, isolation and pure culture. the culture plates were oincubated at 25±5 c (gallenkamp economy incubator size 1) for 15 days. there were 90 replication plates for each soil sample, where the presence or absence (1/0) of particular fungi was 213 effect of invasive ageratina adenophora on saprotrophic and pathogenic soil fungi balami et al. followed by three species from class zygomycota, one species from basidiomycota and one actinomycetes. species richness of soil fungi in the uninvaded soil was greater (28 species) than that in the invaded soil ( < 0.05). twenty soil fungi species p were recorded in the rhizosphere-contained invaded soil. twenty two soil fungi species were recorded in the invaded non-rhizosphere soil (table 1). these results indicated that a. adenophora reduced species richness of soil fungi. these findings also clarified the interactions between soil fungi and invasion of , a. adenophora which was still contradictory (mangla callaway & 2008). czapek dox agar medium was used to culture and isolate soil fungi targeting saprophytic or pathogenic soil fungi, as this media was proven to be appropriate for culturing and isolating common saprophytic and pathogenic fungi (abildgren et al. 1987). this study proved that soil fungi enumeration favored czapek dox agar medium. other studies showed that a. adenophora increased the abundance of mycorrhizal soil fungi which suggested that mycorrhizal recorded in each replication plates. fungi grown in the plates were observed at day 3, 7 and 15 and identified based on standard literature (barnett & hunter 1960; gilman 1975; watanabe 2010). a data matrix of species was prepared statistical analysis species richness of soil fungi in different soil samples was compared using one-way analysis of variance (anova). frequency rank curve was used to compare the occurrence frequency of fungi found in different soil samples. species composition of soil fungi was analyzed using non-metric multidimensional (nmds) technique. the analyses were carried out using r software (version 2.15.3) (r core team 2015). the acceptable significance level was p < 0.05. results and discussion species richness a total of 34 soil fungi species were found in soil samples (table 1). four species twenty-nine were reported from division ascomycota biotropia vol. 24 no. 3, 2017 214 table 1 total numbers of soil fungi species found in different soil type soil type total species uninvaded soil 28a a. adenophora invaded non-rhizosphere soil 22b a. adenophora invaded rhizosphere soil 20b figure 1 species composition of soil fungi in the invaded soil, rhizosphere-contained invaded soil and uninvaded soil based on nmds analysis (final stress = 0.0002163944, k = 2, distance measure = “jaccard” distance, trymax = 1,000) colonization induced positive feedback to enhance the invasiveness of a. adenophora (niu et al. 2007; yu et al. 2011; and xiao et al. 2014b). comparing the results of those studies with the results of present study, it is suggested that a. adenophora can replace certain groups of soil fungi commonly found in the uninvaded soil. analysis of soil fungi species composition (fig. 1; table 2) and frequency ranking curve (fig. 3) showed the response of soil fungi species toward rhizosphere-contained soil, invaded soil and uninvaded soil. species composition and occurrence frequency nmds analysis showed that species composition of soil fungi varied in accordance with soil types. species compositions recorded in the invaded non-rhizosphere soil and the rhizosphere-contained invaded soil were different from that in the uninvaded soil. soil fungi species such as curvularia sp., alternaria alternata, colletotrichum sp., acremonium sp., aspergillus fumigates, verticillium sp. and fusarium moniliforme occurred more frequently in the rhizosphere-contained invaded and the invaded non-rhizosphere soils. other soil fungi species such as absidia sp. and chaetomium funicola were exclusively occurred in the uninvaded soil (fig. 1 & 2). species belonging to genera alternaria, colletotrichum, fusarium, verticillium, acremonium 215 effect of invasive ageratina adenophora on saprotrophic and pathogenic soil fungi balami et al. table 2 soil fungi species and respective species codes depicted on nmds space in figure 1 sn fungi name abbreviation 1 absidia sp. abs 2 acremonium sp. acrs 3 alternaria alternata alta 4 alternaria sp. alts 5 aspergillus a aspa 6 aspergillus b aspb 7 aspergillus fumigates aspf 8 aspergillus niger aspn 9 aspergillus fumigates aspf 10 bipolaris sp. bips 11 curvularia sp. curs 12 cladosporium sp. clas 13 melanospora sp. mels 14 cunninghamella sp. cuns 15 fusarium oxysporum fuso 16 fusarium moniliforme fusm 17 fusarium ciliatum fusc 18 gliocladium sp. glis 19 mucor sp. mucs 20 penicillum a pena 21 penicillium b penb 22 penicillium c penc 23 penicillium d pend 24 penicillium e pene 25 rhizophus sp. rhis 26 rhizoctonia sp. rhizs 27 staphylotrichum sp. stap 28 streptomyces sp. stre 29 trichoderma harzianum trih 30 trichoderma koningii trik 31 verticillium sp. verts 32 gonytrichum sp. gons 33 chaetomium funicola chaf 34 colletotrichum sp. cols note: a, b, c, d and e are arbitrarily given specific names for unidentified fungi (consis ting more than one species) to species level and curvularia were contributors in species composition of soil fungi in the invaded soil, whereas the species belonging to genera aspergillus, penicillium, absidia, chaetomium were major contributors in forming species composition of soil fungi in the uninvaded soil (fig. 1 & 3). these results supported hypothesis of soil biota alteration as proposed by various researchers (wolfe & klironomos 2005; si et al. 2013). goodness of fit for environmental factors (represented by soil types) in figure 1 was 2 significant in ordination space (r = 0.29, p < 0.01). permutational anova (permanova) showed that species composition of soil fungi significantly varied in accordance with soil types (p < 0.05). nmds analysis showed that species composition of soil fungi was changed by the invasion of a. adenophora (fig. 1). alteration of species composition could be caused by differences in species richness in the invaded and uninvaded soils. species composition of soil fungi in the rhizosphere-contained invaded soil and invaded non-rhizosphere soil was not different (fig. 1). species richness of soil fungi in those two soil types was similar (table 1). frequency rank analysis showed that a. alternata, penicillium sp., curvularia and fusarium oxysporum were the frequently occurred soil fungi in the rhizosphere-contained invaded soil (fig. 3). similarly, f. oxysporum frequently occurred in the invaded non-rhizosphere soil followed by curvualria sp., penicillium sp. and a. alternata (fig. 3). rhizophus sp. and cunninghamella sp. were the least frequent in rhizospherecontained soil. rhizoctonia sp., rhizophus sp., verticillium sp. were the least occurred soil fungi species in the invaded non-rhizosphere soil. trichoderma harzianum was the most frequently found soil fungi in the uninvaded soil followed by penicillium t. knoningii 3 sp. and (fig. ). the least 216 biotropia vol. 24 no. 3, 2017 figure 3 frequency rank curve of (a) rhizosphere-contained invaded soil; (b) invaded non-rhizosphere soil; and (c) uninvaded soil figure 2 fungal plate of chaetomium funicola frequently found soil fungi species in the uninvaded soil were sp. sp.curvularia , alternaria , staphylotrichum aspergillus .sp. and sp occurrence frequency in this study showed a similar tendency of accumulation of pathogenic soil fungi in the invaded soil. nmds and frequency analysis showed that fusarium, colletotrichum alternariaand species were very frequently found in the invaded soil (fig. 1, 3 & ) 4 and these genera are common pathogens (chalermpongse 1987). previous studies in warm tropical humid monsoonal climate of india also reported that and a. adenophora chromolaena odorata accumulated pathogenic fungi (mangla & callaway 2008; mei 2014).et al. on the other hand, soil fungi belonging to genera and were penicillium, aspergillus chaetomium common decomposers (fu-qiang 2004) et al. found in the uninvaded soil (fig. 1 & ). this 3 indicated that saprophytic soil fungi in the invaded soil might be reduced by where a. adenophora pathogenic soil fungi increased. the reduction of saprophytic soil fungi might be important mechanism behind the invasion and affliction of native species by .a. adenophora plant species could determine rhizosphere microbes (both bacteria and fungi) via root exudates, phytoanticipins, phytoalexins or allelochemicals secreted by the plants (bever et al. 2010). presence of allelochemicals in root exudates or leachates from aerial parts such as leaf and litter of (wan 2011; zhang a. adenophora et al. et al. 2013) could be responsible for the decrease of species richness, the alteration of soil fungi species composition and the alteration of occurrence frequency of saprophytic fungi. inderjit (2011) found that is et al. a. adenophora responsible for higher mortality of native species in china and india. similarly, thapa (2017) et al. reported that reduced the growth a. adenophora and development of native seedlings nepal. this study suggested that changes in species richness and species composition of soil fungi might also be responsible in seedling mortality, growth and development of aboveground native plant. in the field, is found in thick a. adenophora stands in the invaded areas of nepal. a. adenophora deposits litter in the soil and leaches substances from aerial parts, including green leaves, during rainy days (thapa 2017). litter et al. accumulation, decomposition in soil and leached substances from aerial parts may have antifungal properties against certain fungi (broeckling et al. 2007). various allelochemicals in aerial parts of a. adenophora such as phenolics, sesquiterpens (zheng 2012) might affect soil microflora or et al. alter soil quality (katherine 2006). et al. additionally, might alter the cycle of a. adenophora soil nutrient which could have affected the abundance or distribution of particular soil fungi species or community in the soil. plant-soil feedback mechanism is important to explain vegetation dynamics and ecosystem function including plant invasiveness. positive feedback is evident through colonization of mycorrhizal fungi by (niu 2007; a. adenophora et al. yu 2011; xiao 2014 ). there might be et al. et al. b negative feedback to the native species through accumulation of pathogenic soil fungi and decrease of saprophytic soil fungi in the invaded soil by a. adenophora. conclusions a. adenophora invasion led to the decrease of soil fungi species richness and altered soil fungi species composition. the invasion facilitated the 217 effect of invasive ageratina adenophora on saprotrophic and pathogenic soil fungi balami et al. figure 4 fungal plates of fusarium (a) and alternaria (b) (a) (b) 218 biotropia vol. 24 no. 3, 2017 occurrence of saprophytic fungi as well as accumulated several pathogenic soil fungi. the accumulation of pathogenic fungi could have detrimental effect on the growth and development of native species of higher plant. changes in soil fungi species and communities could be one of the mechanisms involved in the successful invasion of a. adenophora. acknowledgements the authors express sincere gratitude to prof dr pramod kumar jha, former head of central department of botany, tribhuvan university (cdb-tu), kathmandu, nepal, and prof dr mohan s iwakot i , head cdb-tu, for encouragement and providing 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of the serine green fluorescent protein (sgfp) pyricularia grisea gene nto ra dc4 i ce isolated from using digitaria ciliaris agrobacterium tumefaciens-mediated genetic transformation stephanie , utut widyastuti and suryo wiyono1 2,3* 4 1program study of graduate school, institut pertanian bogor; bogor 16680, indonesiabiotechnology, 2research center for bioresources and biotechnology, institut pertanian bogor; bogor 16680, indonesia 3department , faculty of mathematics and natural science , of biology s institut pertanian bogor; bogor 16680, indonesia 4department of , faculty of agriculture, institut pertanian bogor, bogor 16680, indonesiaplant protection r 4eceived 24 july 2013/accepted 12 september 201 abstract blast disease (caused by ) has long been known as a serious problem for up rice. now, it also pyricularia grisea land attacks lowland rice. however, the mechanism facilitating this range expansion is still unknown. one option is to insert a marker into so that it can be used to monitor the spread of infection. (p. grisea p. grisea s sgfp erin green fluorescent protein ) gene of the study of has been used to monitor gene expression specific tagged proteins for fungal cell. genome of dc 4 from in this study, the gene has been integrated into the the sgfp igitaria c . p. grisea d iliaris p using thelasmid was introduced into triparental mating method (tpm). genetic transformation sgfp a. tumefaciens was performed by co-cultivating spore dc4 with lba4404–pcamb-s . dc4 s of the p. grisea a. tumefaciens gfp p grisea. transformant was selected by using selection medium 300 µg/m hygromycin. the integration of a containing l of the the the c thesgfp sgfp sgfp gene into genome was confirmed by pcr using 's spe ific primer pair, -nos terminator primer pair and -tubulin primer pair as internal control. expression of from the an the gene the β sgfp p. grisea dc4 transformant w detected with blue light fluorescent microscopas y. keywords: agrobacterium tumefaciens, digitaria ciliaris, pyricularia grisea , erine green mediated transformation dc4 s fluorescent protein introduction pyricularia grisea magnaporthe grisea (teleomorph ), synonymous with cav, is a rice pyricularia oryzae plant pathogen in many countries and is known as the agent causing last disease (rho 2001). et al.b the disease is estimated to be responsible for 30% of annual yield loss, the equivalent in food volume to meet the needs of 60 million people (dagdas 2012). in indonesia, blast infestation et al. reached 36,727 ha of the total 13,153,080 ha area of rice cultivation in 2012 (ministry of agriculture 2013). blast disease has been known as a serious problem for up rice, however, land recent results showed that the last pathogen also b attacks rice planted in low s or irrigated land land (sobir 2003). to date, the mechanism of blast et al. disease transmission from upland rice to lowland land has not been examined. p griseayricularia is a pathogen on more than 50 species of wild grass in the vicinity of rice field (couch & khon 2002 ). has a wide variety p. grisea of hosts besides rice plant; among them are triticum aestivum zea mays eleusine coracana, , (finger millet), (cultivated grass), setaria italica brachiaria mutica et al (couch & khon 2002; listiyowati . 2011). because of this wide host range, cereal grasses and wild grasses around the area of cultivation are also prone to infection and p. grisea therefore, could become alternative hosts as well as alternative inocula sources for spreading disease.* corresponding author : ututsuharsono2002@yahoo.com biotropia vol. 22 no. 1, 2015: 73 79 doi: 10.11598/btb.2015.22.1.329 73 mailto:ututsuharsono2002@yahoo.com biotropia vol. 22 no. 1, 2015 74 research showed that grasses growing around rice field could become temporary hosts for fungi that cause last disease (listiyowati 2011). et alb . those fungi were capable of infecting rice plants and showed some genetic structure alterations from the original grass isolate. isolated p. grisea from grass (dc4) is able to infect rice d. ciliaris plants that are moderately resistant and susceptible plants. this isolate of dc4 that switches host undergoes genetic alterations, which are marked by changes in scar marker for cut1, pwl2, erg2 as well as physiological race based on the ability to infect differential rice varieties in indonesia (listiyowati 2011). this et al. is presumed as a genomic adaptation response of the fungi isolate to the new host. however, the mechanism that causes such change is still unknown. therefore, as an initial step it is necessary to insert a marker into to p. grisea monitor infection.p. grisea according to lorang (2001), the et al. gfp gene could be used as a tool in studying interactions between fungi and plants. has gfp also been used to monitor the virulence of blastcausing fungi. gene has several advantages as gfp a marker. gene detection does not require any gfp substrate addition to obtain the visualization; it does not require special treatment of the tissue and its presence in the cell is not harmful to the cell itself. these advantages make a great gfp marker in gene transformation and expression (lorang 2001). today, the gene has been et al gfp. shown to be expressed in 16 species of 12 genera of fungi including (lorang 2001). m. grisea et al . the gene has been successfully used as gfp important marker for several fungi that cause diseases and is among them (balhadere & p. grisea talbot 2001). sgfp gene transfer into fungi genome requires a biological vector. has agrobacterium tumefaciens been long used as a biological vector for gene transfer in plants. besides transferring genes to its host plant, is also able to transfer a. tumefaciens dna to yeasts and filamentous fungi (combier 2003). the objective of this research was to introduce gene into dc4 sgfp p. grisea isolated from using d. ciliaris a. tumefaciens-mediated genetic transformation. subsequently, there would be a gene introduction into the fungi sgfp genome. the success of this gene introduction could be developed to facilitate molecular studies of pathogen-host interactions. materials and methods triparental mating the introduction of binary plasmids that contain gene into was donesgfp a. tumefaciens with the tpm assay described by hanum (2011). three bacteria: dh donor ing e. coli 10b contain pcamb plasmids, dh1 ing sgfp e. coli contain helper prk2013 plasmids and a. tumefaciens lba4404 as the recipient strain were used in tpm assay. the three bacteria were grown in solid lb to prevent conjugation. a. tumefaciens sgfp transformants were then identified using colony pcr with specific primers -f and -r.sgfp sgfp fungal strains and culture conditions p. grisea d. ciliaris race dc4 isolated from was provide by d mycology laboratory, department of biology, faculty of mathematics and natural s c i e n ce s , i n s t i t u t pe r t a n i a n bo g o r. fungal cultures were grown on oatmeal agar medium (oma: 30 g of oatmeal for 1 l). fungi were grown in oa medium and incubated for 710 days at 28 ºc. for production of conidia, fungal cultures were grown under light of a nearuv lamp for 5 6 days to using sterilized water ( ).munandhar 1998et al. transformation one colony of containing a. tumefaciens pcamb plasmids was grown on 2 ml of sgfp minimal medium (hooykaas 1979) et al. sup lemented withp 100 µg/ml streptomycin, 60 µg/ml kanamycin, and 50 µg/ml hygromycin at 28 c for 48 hours with 250 rpm rotation ino no light condition. the bacterial culture was diluted with 5 ml induction medium (im) containing 200 µm acetos ringone toy an optical density of 0.15 at a600. the bacterial culture of a. tumefacien -sgfps was grown for 4-6 an additional hours at 28 c with 250 rpm rotation o to reach an a600 . (betts 2007) of 0 5 . . approximately et al 100 µl of dc4 spores (10 spores/ml) was p. grisea 6 mixed with 100 µl of cells a. tumefaciens-sgfp (a600 = 0.5) then added with a etosyringon 200c e µm, and incubated for 30 minutes with no light. the co-cultivated cultures were then into plated im medium and incubated at 28 c for 48 hours. o the co-cultivation result was transferred into complete medium containing 300 µl/ml introduction of the ( ) gene nto serine green fluorescent protein sgfp pyricularia grisea i stephanie – et al. hygromycin and 200 µg/ml cefotaxime and incubated at 28 c for 5-7 days. pore o single s selection was done by spreading 100 l µ conidia suspension on oa medium that contained 300 µg/ml h gromycin and incubated for 5-7 days y until dc4 appeared (rho p. grisea et al single spores . 2001 modified by betts 2007).et al. analysis of transformant genomic dna of mycelium from p. grisea transformant and non-transformant were p. grisea isolated for verification using pcr. dna isolation was done with the method described by listiyowati (2011) using 2% et al etyl rimethyl . c t a b mmonium romide (ctab). the isolated dna sgfp gene as then w amplified through pcr using -f and -r primers as well as sgfp sgfp combined primers of f and nos-r. the sgfppcr mixture used consisted of 1 µl (100 ng) genomic dna, 0.5 mm forward primers, 0.5 mm reverse primers, 5 µl pcr mix (fermentas) and added with ddh o up to 10 l of total volume. 2 µ the pcr program to amplify fragments sgfp consisted of: pre-denaturation at 94 ºc for 1 minute, denaturation at 94 c for 1 minute, o annealing at 53 c for 30 seconds, elongation at o 72 c for 1 minute and final elongation at 72 c o o for 5 minutes; this process was run in 35 cycles. the pcr results were through visualized electrophoresis 1% (b/v) agarose gel at 100 on volt for 30 minutes in tae 1 and continued with gel immersion in 0.5 mg/l etbr for 20 minutes and were visualized under uv transluminator (shanti 2008). m yicroscop to visualize , p. grisea transformants they were subcultured in oa medium containing 300 µg/ml hygromycin. the culture was incubated at room temperature for 6 days. the transformants were grown under light of the a near-uv lamp for 5 6 days. the acquired spores were then to observed using an olympus bh2-rfch microscope on the fluorescent setting with a 515 bandpass emission fillter (blue light) . results and discussion triparental mating (tpm) the plasmid camb was introduced p sgfp into using the tpm method (fig. 1). a. tumefaciens plasmids in dh10b were contained e. coli moved into lba4404 through a. tumefaciens conjugation with the help of prk2013. e. coli triparental mating generated several colonies growing in a medium containing 50 µg/ml kanamycin, 50 µg/ml streptomycin and 50 µg/ml hygromycin. the only bacterium able to grow in that selective medium was a. tumefaciens lba4404 containing pcamb plasmids. sgfp colonies from tpm that were able to obtained grow in the selective medium were then analyzed with pcr. the results of the pcr analysis showed that colonies of with -f and -a. tumefaciens sgfp sgfp r primers produced amplicon with the size of 643 bp (fig. ). the results of this amplification 1 had the same size as the positive control (fig. ). 1 this showed that pcamb plasmid w sgfp as successfully introduced into a. tumefaciens through the tpm method. this method was previously used by hanum (2011) to move the binary plasmid pmsh into mmcuzn-sod a. tumefaciens et al.. according to wise (2006) tpm is a quite efficient method for transferring gene in a non-conjugated plasmid into a. tumefaciens. 1000 bp 750 bp 500 bp 250 bp 643bp figure . pcr identification of the gene introduced into lba4404 colon using tpm1 ies . m: marker 1 kb; sgfp a. tumefaciens k+: plasmid pcamb (positive control); k-: ddh o (negative control); 1-5: transformant sgfp a. tumefaciens2 sgfp 75 transformation p. griseagenetic transformation of dc4 using a. tumefaciens w s achieved using the method a described in rho (2001) and betts (2007). et al et al. . one hundred microliter (100 µl) of spores (10 6 per ml) were co-cultivated with 100 µl of bacterial cells in an induction medium that had been added to 200 m acetosyringone for 48 µ hours. an acetosyringon concentration of e 200 µm is important in fungal transformation to induce vira genes of so that t-a. tumefaciens dna transfer may take place (knight . 2009). et al an acetosyringon concentration of 200 µm is e commonly used in fungal transformation with a. tumefaciens as a biological vector and it has been shown to have consistent results (covert . et al 2001; dos reis . 2004; knight . 2009; et al et al xiaoran . 2012)et al . p. grisea dc4 grown in hyg omycin and r cefotaxime selective medium was then cultured to produce spores. the spores were then spread in oa medium containing 300 µg/ml hygromycin and incubated for 5-7 days until some single spores that were transformed were obtained (fig. 2). selection of spore was done to single s eliminate false transformant fungi (betts et al. 2007). mycelium growth on hygromycin medium indicated the success of transformation (tucker & orbach 2007). some s that single spore were not able to grow on hygromycin medium because the genes having important roles in growth might undergo some damage or their activities might be interrupted by the presence of foreign genes. p. griseasingle s s pore of dc4 transformant and p. grisea dc4 non-transformant were cultured in a medium containing 300 µg/ml hygromycin then the growth diameter was observed at day 7, 14, and 21. the growth of transformant p. grisea increased rapidly which was 14.8 mm at day 7 became 28.7 mm at day 14 and became 37.3 mm at day 21. non-transformant growth was p. grisea slower which was 6.5 mm at day 7, 12.55 mm at day 14 and 17.3 mm at day 21 (fig ). . 3 hygromycin added into the oa medium was able to inhibit the growth of non-p. grisea transformants compared to dc4 p. grisea transformant (fig 4). this was caused by sgfp . the inhibiting activity of hygromycin on the p. grisea dc4 non-transformant. the use of an appropriate selection marker is essential for the success of transformation. selection using an antibiotic is performed to eliminate nontransformant cells as well as to ensure the resistance level carried by the transformant (frandsen 2011). in this research dc4 p. grisea transformants were resistant to hyg o-sgfp r mycin because of the addition of the hygromycin-resistance gene contained in the pcamb plasmid. betts (2007) used sgfp et al. the concentration of 300 µg/ml for the selection of both transformant and non-m. grisea transformant. in contrast, shanti (2008) used a concentration of 225 µg/ml hygromycin to inhibit 173 (originated from rice plant) p. grisea non-transformant and transformant generated through speroplas. the difference in growth rate between the transformant on hygromycin medium and non-hygromycin medium (fig. 4) was likely due to the influence of the number of gene copies integrated, however, further evidence for this is required. f 2 single spore race igure . of dc4 at day 5 grown in oa medium containing 300 µg/ml hygromycinp. grisea biotropia vol. 22 no. 1, 2015 76 figure . average growth rate of dc4 non-transformant and transformant in oa selective medium 3 p. grisea sgfp containing 300 µg/ml hygromycin figure 4. growth of dc4 non-transformant (nt) and transformant (t) in oa medium containing 300 µg/ml p. grisea hygromycin (hgr+) and without hygromycin (hgr-) analysis of ransformantt putative transgenic dc4 from grass that p. grisea had been selected in hygromycin medium was then analyzed using pcr with specific primers sgfpsgfpsgfp-f and r as well as f and nos-r primers to detect the presence of gene. sgfp pcr molecular analysis generated amplicons of 643 bp and 899 bp which were in alignment with the positive control amplicon (fig. ). pcr 5 of non-transformant dna did not generate sgfp gene amplicons. this result showed that the sgfp gene was inserted into the fungal genome. the gene was used as an internal b-tubulin control to ensure the amplified dna was in good condition. the pcr using bt1af and b-tubulin bt1ar primers generated amplicons of 550 bp (fig ). 5 . p. grisea sgfp dc4 with gene insertion was the bright green fluorescence observed using a fluorescence microscope (fig. 6). this showed that the gene was well integrated in the sgfp fungal genome and constitutively expressed in p. grisea sgfpdc4. the use of requires a promoter which is needed for expression. the sgfp pcamb plasmid receiving gene sgfp sgfp insertion from the pct74 plasmid was able to express the gene under the control of the sgfp toxa promoter (lorang 2001).et al. 77 introduction of the ( ) gene nto serine green fluorescent protein sgfp pyricularia grisea i stephanie – et al. conclusions pathogenic dc4 from had been p. grisea d. ciliaris successfully transformed with the gene sgfp using -a. tumefaciens-mediated genetic trans formation. the gene had been integrated sgfp into the genome. mycelia had p. grisea fluorescence been observed under a fluorescence microscope. acknowledgements thanks to dr. osbourn from john innes institute, england who provided plasmid pcamb. thanks to margaret cargill and sgfp pattric o'connor for early reading this manuscript. this research was funded by imhere b2c titled “biological role of rice blast disease to develop rice plant tolerance to rice blast” under the name of dr utut widyastuti, agreement letter no : 12it3.24.4/spp-i-mhere/2012. references balhadere pv, talbot nj. 2001. pde1 encodes a p-type atpase involved in appressorium-mediated plant biotropia vol. 22 no. 1, 2015 78 figure . pcr results of dc from transformation5 4 . m: marker 1 kb; k+ dan : plasmid pcamb-p.grisea sgfp sgfp-nos sgfp -tubulin p. grisea p. grisea , k+ : ras 173 (positive control); k-: ddh2o (negative control); 1: dna dc4 β nontransformant; 2-3: dna dc4 transformant p. grisea sgfp figure . dc4 transformant mycelium 7 hours after spore harvest observed under microscope without and with 6 p. grisea sgfp fluorescent infection by the rice blast fungus . magnaporthe oryzae science direc 44:1035-49t . betts mf, tucker sl, galadima n, meng y, patel g, li l, donofrio n, floyd a, nolin s, brown d .et al 2007. development f igh hroughput ransformation o a h t t s f i m iystem or nsertional utagenesis n magnaporthe oryzae. science direct 44:1035 49. . combier jp, melayah d, raffier c, gay g, marmeisse r. 2003 . -mediated agroba ct er ium tumefac i ens transformation as a tool for insertional mutagenesis in the symbiotic ectomycorhizal fungus hebeloma cylindrosporum. microbiol lett 220:141-48. couch bc, khon lm. 2002. a multilocus gene genealogy concordant with host preference indicates segregation of a new species, , magnaporthe oryzae from mycologia 94(4):683-93.m. grisea. couch bc, fudal i, lebrun mh, tharreau d, valent b, van kim p, notteghem jl, kohn lm. 2005. origins of 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2007. -mediated agrobacterium transformation to create an insertion library in magnaporthe grisea. ronald pc, lorence a, editor . plant-pathogens interaction methods and protocols; methods and protocols volume 354 methods in olecular iology . : m b totowa (us): human pr . 57-67.ess p wise aa, liu zz, binns aw. 2006. three methods for introduction of foreign dna into . agrobacterium methods in mol iol 43-54b . 79 introduction of the ( ) gene nto serine green fluorescent protein sgfp pyricularia grisea i stephanie – et al. introduction in vitro conservation of coconut (cocos nucifera l.) embryos biotropia no. 25, 2005 : 11 – 21 in culture media sukendah1 and ma l. o. cedo2 1dept. of agronomy, faculty of agriculture, pembangunan nasional “veteran” university, surabaya, indonesia 2 assistant professor, dept. of horticulture, uplb, laguna 4031, los baños, philippines the possibility of delaying the germination of mature coconut embryos in vitro for medium-term conservation of coconut was investigated. embryos of coconut was stored in sugar free, full-strength and half-strength ms media for 3 months with minimal loss in viability. germinability of embryos decreased with prolonged storage. at the end of 9-month storage period, embryos kept in full-strength media were all non-germinable, while those stored in half-strength media still gave 15% germination. addition of mannitol up to 0.3m did show any significant effect on germination of embryos stored for 3 months. however, longer period of storage helps to a certain extent in the preservation of embryo viability. the presence of mannitol in the storage medium also induced morphological abnormalities in seedlings derived subsequently from the stored embryos. mannitol at 0.4m was totally deleterious to the embryo. significance of these findings on coconut genetic resources conservation are discussed. keywords: in vitro conservation/coconut embryo. introduction the safe conservation of genetic resources of coconut is faced with various problems. coconut is a species with a truly recalcitrant seed which has no dormancy (engelmann et al. 1995). because of this characteristic, there is no possibility of storing it ex situ even for short periods of time. genetic resource collections (germplasm) are conserved as special field genebanks (living plants) in national or regional centers. field genebanking, however, run the risk of being damaged by natural disasters, pests and diseases. it also requires more space. moreover, many sources of plant genetic materials (indonesia, the pacific, and philippines) are still almost unexplored, and the small proportion of coconut germplasm currently available in existing field collections and certain accessions are now in danger of dying. the in vitro technique addresses all these concerns. it offers the possibility of providing duplicate, healthy collections for storage and use with less space. since vegetative propagation of coconut is as yet not feasible, embryo culture technique is a promising alternative. coconut embryo culture protocols are now available in the literature (batugal and engelmann 1998). in all these protocols, embryo germination commences soon after the embryos are placed in the culture medium. subsequently, fully developed seedlings ready for acclimatization may be obtained within 5-7 months. for in vitro conservation, such quick growth response to the medium is not desired. thus, efforts 11 are now focussed on modifying the medium and other culture conditions in order to delay the germination while at the same time preserving the germination potential of the embryos during storage. biotropia no. 25, 2005 techniques which can be used to maintain collection in delaying the embryo germination are: the use of reduced temperature, minimized light, and modifications in media. ashmore (1997) was able to store sugarcane shoots at 18ºc on reduced mineral salts, and banana shoots at 16ºc in the presence of plant growth subtances. chatty and tissouni (1999) showed that the suitable temperature for major palm species is about 25ºc, whereas lack of germination was observed at the extreme temperature of 5ºc. the american palm germinated better at temperatures ranging from 25ºc to 35ºc, but did not germinate at 15ºc. meanwhile, ng and ng (1991) reported to slow down the growth rate, cultures are basically incubated under reduced light intensity or total darkness in conjunction with low incubation temperature. however, the modification of temperature and/or light to preserve cultures required a high cost because of electricity and equipment. ng and ng (1991) suggested that an alternative approach to retard growth was to use a reduced-growth medium. recent publications on slow-growth media indicate that a variety of techniques are still being utilized, such as the reducing concentration of mineral salt, increasing, decreasing or eliminating sucrose, adding mannitol or sorbitol and growth retardant to the culture medium (ng and ng 1991). one of the advantages of this method is that the modification of the culture medium requires no special equipment. preliminary studies on storage media conducted by assy-bah and engelmann (1993) have indicated that embryos of coconut can be stored for 6 months on a medium devoid of sucrose and containing 2 g/l activated charcoal with eventual germination of stored embryos at 100%. related study done by damasco (2000) on tagnanan tall showed that aba and mannitol at low concentrations (0.05 to 0.1m) might not be effective to inhibit growth in culture. however, high concentration of mannitol (0.2 and 0.3m) significantly reduced percent germination and subsequent growth of germinated embryos. in this report, results on the use of reduced-nutrient media and mannitol for coconut embryo storage are presented. materials and methods collection, sterilization, and excision of embryos mature (11-12 months) coconut palm variety ‘tagnanan tall’ were collected from the philippine coconut authority zamboanga research center. a cylinder of endosperm embedding the embryo was extracted from each nut using a 2-cmdiameter cork borer. the endosperm cylinders were washed in 95% ethanol (1-2 minutes) then sterilized in 100 % commercial bleach (5-6 % sodium hypochlorite) for 20 minutes. 12 under aseptic condition, the embryos were excised from the solid endosperm cylinders, then sterilized in two changes of 10% commercial bleach for 5 minutes at each time and finally rinsed three times with sterile distilled water. sterilized embryos were transferred to a sterile flask containing sterile distilled water for size selection. the same size embryos (± 1 cm) were then inoculated singly onto liquid storage medium according to the treatments. control of culture media for in vitro conservation of coconut – sukendah and ma l.o. cedo culture media and arrangement of treatments the composition of the storage medium used in the present experiment was that of assy-bah (1986), at full-strength or half-strength. the medium contained activated charcoal and had no sucrose. after the required period of storage (3, 6, and 9 months), embryos were germinated following the embryo culture protocol of the university of the philippines at los baňos or uplb (batugal and engelmann 1998). the experiment was conducted by culturing the embryos in test tubes containing 10 ml of full-strength or half-strength ms media without or with mannitol at 0.10 m, 0.2 m, 0.30 m, or 0.4 m. each treatment was replicated three times, each consisting of 35 embryos. the cultures were randomized and incubated in a dark room with temperature maintained at 25°c. observation of embryos viability of embryos was evaluated based on the following parameters: length of embryos, measured every 2 months since embryos have been incubated; percent germination, it was done after the embryos have been preserved for 3, 6, and 9 months of storage. the stored embryos from the experiment were transferred to the germination medium described in the uplb protocol; normal and abnormal seedlings, observed 3 months after embryos have been transferred to the germination medium. seedlings which showed well-developed roots & shoots were considered normal, and seedlings with no shoot, no root or stunted seedlings as abnormal seedling. data were collected based on: a) percent normal seedlings and abnormal seedlings, and b) morphological features, observed by taken their photograph. observations of shoot length, number of leaves, leaf width, leaf length, length of primary root, and number of adventitious roots for normal seedlings were done 5 months after the embryos have been transferred to the germination medium. results and discussions coconut embryos that preserved in full-strength or half-strength ms basal medium significantly increased in length even up to 6th month of storage. in overall, embryos maintained in half-strength media were significantly longer than embryos in the full-strength media (table 1). previous studies on reducing the mineral salt concentration showed that the cultures in general grow slowly or not at all (ashmore 13 1997). apparently the coconut embryos did not have the same response. no further study has reported on the response of coconut embryos treated in low-salt medium. interestingly, at the end of each storage period, it was noted that the final volumes of the spent half-strength media were lower than those of the full-strength media (data not presented). this supports the view that greater growth (in length) of embryos in the half-strength media could have resulted from greater nutrient and water uptake by embryos in those media. biotropia no. 25, 2005 by contrast, addition of mannitol to the storage medium tended to inhibit elongation of embryos during storage. increasing amounts of mannitol (0.1m to 0.4 m) increasingly reduced the final lengths of the embryos, particularly during the first two months of storage (table 1). beyond 6 months of storage, no significant difference on final length among embryos was noted. table 1. effects of level of nutrients and concentration of mannitol on elongation growth of coconut embryo during storage period of storage (month) treatments 2 4 6 final length of embryo (mm) level of nutrients: full-strength half-strength 9.76b* 10.37a 11.01ns 11.56 11.33ns 11.71 concentration of mannitol (m): 0.0 0.1 0.2 0.3 0.4 10.55 a 10.65 a 10.17 a 9.58 ab 9.38 b 11.85ns 11.83 11.28 11.37 10.12 12.27 ns 12.23 11.50 11.45 10.15 * means followed by the same letter in a column for each factor are not significantly different from each other using lsd at 5% level of significance when the stored coconut embryos are placed in a germination medium such as y3 (eeuwens medium), they would start germinating or may exhibit a slight increase in size but still remain ungerminated or the embryos may take a deteriorative path and eventually dies. table 2 shows that storing coconut embryos for 3 months in the modified ms medium, either at full-strength or half-strength, resulted in significant 10-30% reduction in percent germination. prolonging the storage period to 6 months reduced even further the germination to 10-30% in five of the treatments and to zero (no germination) in the other five treatments. with 9 months of storage all embryos kept in full-strength media become non-germinable, while those kept in half-strength media containing low amounts of mannitol showed 13-20% germination. mannitol when added to half-strength (but not to full strength) 14 table 2. percent germination of stored embryos observed 2 months after transfer to the modified y3 germination medium control of culture media for in vitro conservation of coconut – sukendah and ma l.o. cedo period of storage (month) level of nutrients mannitol concentration (m) 3 6 9 full-strength 0.0 0.1 0.2 0.3 0.4 80.00 bc* 70.00 c 80.00 bc 90.00 ab 20.00 e 30.00 b 00.00 d 20.00 cb 00.00 d 00.00 d 00.00 c 00.00 c 00.00 c 00.06 c 00.00 c half-strength: 0.0 0.1 0.2 0.3 0.4 100.00 a 80.00 bc 100.00 a 90.00 ab 40.00 d 00.00 d 20.00 cb 30.00 b 10.00 cd 00.00 d 00.00 c 20.00 b 20.00 b 13.33 b 00.00 c unstored embryos 100.00 a 100.00 a 100.00 a * means followed by the same letter in a column are not significantly different from each other using dmrt at 5% level of significance. media up to 0.3m appeared to help preserve the viability of the stored embryos. it is possible that the hypertonic condition created by the addition of mannitol at this level could have helped maintain membrane integrity, thus prevent rapid cell death which otherwise occurs in untreated coconut embryos during prolonged storage. gomez et al. (1995) reported that mannitol significantly prevented ep-catalyzed hydrolysis of 40 and 50 kda polypeptides. ep (endopeptidase) is a monomeric enzyme (gomez et al. 1994) that has an important role in the digestion of cell wall protein. however, addition of high mannitol concentration (0.4m) significantly inhibited the germination of embryos. after the required storage period, embryos were transferred to the y3 germination medium. although high percentage of the stored embryos exhibited the early signs of germination and they were considered germinated (table 2), many of these embryos did not develop into normal seedlings. in fact, some 10-70% of them showed abnormal growth such as produced well-developed roots but without shoots or well-developed shoot but little elongation of roots or stunted seedling even up to the third month after transfer to the y3 medium (table 3 & fig. 1). of the remaining embryos, normal seedlings were obtained from those stored in media without mannitol or with mannitol at 0.1m (fig. 2). the presence of higher levels of mannitol (0.2 and 0.3m) in the storage medium seemed to have inhibited seedling development. seedlings tended to produce shorter shoots whether stored in full-strength or half-strength media. with 0.4m mannitol, development of shoot and root totally failed (table 4). several authors have reported similar inhibitory effect of mannitol on growth of several crops (ashmore 1997; babu et al. 1999; and damasco 2000). 15 table 3. percentage of normal and abnormal seedlings developed from embryos stored for 3 months in full and half-strength media with or without mannitol biotropia no. 25, 2005 abnormal seedlings mannitol concentration (m) total number of embryos cultured un germinated embryos normal seedling no shoot (a) no root (b) stunted (c) total abnormal seedlings (a +b+ c) fullstrength medium: 0.0 0.1 0.2 0.3 0.4 10 10 10 10 10 20 30 20 10 80 80 70 50 30 0.0 0.0 0.0 20 30 20 0.0 0.0 0.0 10 0.0 0.0 0.0 10 20 0.0 0.0 0.0 30 60 20 halfstrength medium: 0.0 0.1 0.2 0.3 0.4 10 10 10 10 10 0 20 0 10 60 80 70 60 20 0.0 0.0 0.0 20 50 30 10 0.0 0.0 0.0 0.0 10 10 20 20 10 20 10 40 70 40 cba figure 1. a. abnormal seedling with stunted growth; b. abnormal seedling with extensive root system but without shoot; c. abnormal seedling with two sword leaves and an enlarged base but without fully formed root 16 table 4. shoot and root development of seedling grown from embryos stored for 3 months in halfand full-strength ms medium with various concentration of mannitol control of culture media for in vitro conservation of coconut – sukendah and ma l.o. cedo concentrations mannitol (m) shoot length (cm) total no. of leaves/plant average leaf width (cm) average leaf length (cm) length of primary root (cm) no. of adventitious root full-strength medium: 0.0 0.1 0.2 0.3 0.4 half-strength medium: 0.0 0.1 0.2 0.3 0.4 unstored embryos 11.23 ns 10.58 8.05 8.15 0.00# 11.48ns 10.02 10.90 6.80 0.00# 17.66* 2.50ns 2.17 2.00 3.00 0.00# 2.50ns 2.33 2.50 1.00 0.00# 3.00 1.47ns 1.43 1.13 1.20 0.00# 1.40ns 1.50 2.08 0.40 0.00# 3.03** 7.20ns 8.08 6.18 6.92 0.00# 8.33ns 6.93 6.68 2.40 0.00# 10.06 8.93ns 8.10 5.88 6.60 0.00# 6.08ns 5.13 6.25 2.40 0.00# 10.57 2.00ns 1.17 1.25 2.00 0.00# 1.00ns 1.17 1.50 1.00 0.00# 1.20 ns = not significantly different among the treatments in the same column * = significantly different from stored embryos in the same column ** = highly significantly different from stored embryos in the same column # = embryos remained germinated figure 2. normal five-month-old seedlings in full-strength medium with various concentrations of mannitol 17 inhibitory effect of osmotically active agents such as mannitol at high level has appeared during the storing period of embryos (table 1). these embryos, then were not able to germinate even though supported by enough nutrient. mannitol is a metabolically inactive sugar alcohol (dodd et al. 1991). it is one of compatible solutes that can decrease plant water potential by osmotic adjustment (the accumulation of compatible solutes that promotes acclimation to dry) (buchanan et al. 2005). the large amount of mannitol in the cell could result in the increase of the osmotic pressure of the medium drastically. a high osmotic potential in the external medium can induce the protoplast to lose water, causing the cells to become plasmolyzed. if kept in this medium for extended period, it can cause cells to become permanently damaged. study on leaf disks of maize under hyperosmotic condition by del longo (1993) revealed the cellular damage in both the sensitive and the resistant maize line. damage as indicated by, for example, the extension of lipid peroxidation, the destruction of chlorophyll and carotenoids, the decrease in levels of protein sulfhydryl groups and the leakage of electrolytes from cells was apparent in leaf disks as a consequence of the applied stresses (0.5 m mannitol). biotropia no. 25, 2005 it should be mentioned that the stored embryos were not washed off before they were transferred to the germination media. so that the mannitol in the storage media was carried over to the new media. this could have caused the abnormal growth of the seedling. the washing step was not done, however to avoid the removal of important metabolites from possibly already leaky cells as suggested by withers (1986; cited by benson 1990). microscopic observations of the paraffin sections of the embryos showed that high mannitol concentration caused plasmolysis of cells in the region of the apical meristem, which could have led to damage of shoot or root development (fig. 3). evidence from some researches showed that the damage occurred on the hypocotyls figure 3. plasmolyzed cells in 0.3 m mannitol medium 18 or in a critical number of cells of the axis would produce abnormal seedling or the germination would be retarded. thus, the symptoms of slow growth, abnormal growth, or no growth could result from fundamental changes in membranes and macromolecules. control of culture media for in vitro conservation of coconut – sukendah and ma l.o. cedo the results showed that delaying the germination of coconut embryos of the variety ‘tagnanan” was effective for up to 3-month-storage period. after 3 months, the deterioration of embryos occurred very fast. by the end of 9 months, most of the embryos showed significant loss in viability. however, some embryos still showed viability potential after 9 months of storage (indicated by percent germination of about 20-30%). clearly, there is still a need for further studies on the culture media of coconut embryos to increase the viability and germinability of embryos longer than 3 months period of storage. addition of mannitol at a low concentration, lower than 0.2 m, gave a high germination rate (70-100%) and relatively high percentage of normal seedlings (5060%), both in the full-strength and half-strength media free of sucrose. damasco (2000) reported that addition of mannitol at 0.05 to 0.1 m to the medium containing high level of sucrose (60 g/l) induced germination and growth of coconut embryos of the variety ‘tagnanan’ with the same germination rate as the control treatment. it appeared that mannitol was not effective in delaying germination. since the present study was able to delay the germination up to 3 months and maintain high viability, further studies using other substances which could delay germination should be done. sucrose analysis showed that coconut embryo contains very low amount of sucrose, only 42.49 mg per 100 g dry weight. apparently, addition of some amount of sugar in the storage medium may still be important. the study indicated that it is possible to prolong the inactive metabolic phase of coconut embryos before the active germination phase for at least 3 months by controlling the culture medium.technique of delaying the germination may be used as a complementary conservation strategy of coconut germplasm. this technique may have a role in one or more activities in the conservation-use cycle such as in in vitro collecting, in vitro exchange and distribution of coconut germplasm, and in vitro conservation. conclusions the overall results indicate that it is possible to delay the germination of coconut embryo in vitro for up to 3 months without loss in viability. ms medium at half-strength was found to be a better medium for embryo storage than the full-strength medium. addition of mannitol at low concentration (0.2m or lower) did not have any significant effect on storage. however, with 0.3 m or higher concentration of mannitol, the germination fell drastically to 30%. furthermore, high concentration of mannitol induced abnormality in the resulting seedlings (formation of shoot without root, root without shoot, and stunting). 19 acknowledgments biotropia no. 25, 2005 the author would like to express her sincere gratitude to searca and ipgri for the financial support to endure this study. she is also deeply grateful to the director and staff of department of horticulture, uplb, philippines for the assistance and facilities and also to mr. carlos b. carpio, deputy administrator, ardb, pca for the supply of the coconut embryos. references ashmore, s.e. 1997. status report on the development and application of in vitro techniques for the conservation use of plant genetic resources. ipgri. rome. 67p. assy-bah, b. 1986. culture in vitro d’embryos zygotiques de cocotiers. oleagineux 41:321-328. assy-bah, b. and f. engelmann. 1993. medium-term conservation of mature embryos of coconut (cocos nucifera l.). plant, cell, tissue and organ cult. 33:19-24. babu, k.n., s.p. geetha, d. minoo, p.n. ravindran and k.v. peter. 1999. in vitro conservation of cardamom (elettaria cardamomum maton) germplasm. plant genetic resources newsletter. 119:41-45. batugal, p.a and f. engelmann. 1998. coconut embryo in vitro culture. proceedings of the first workshop on embryo culture 27 –31 october 1997 banao, guinobatan, albay, philippines. benson, e.e. 1990. free radical damage in stored plant germplasm. international board for plant genetic resources. rome. buchanan, b.b., w. grussen, and r.l. jones. 2005. biochemistry and moleculer biology of plants. chapter 22: responses to abiotic stresses. aspb (american society of plant biologists). new york. chatty, y. and t. tissouni. 1999. efect of temperature on germination of ornamental palm tees in tunisia. in: ruano,m. c. (ed.). proc. of the 2nd int. symp. on ornamental palms and other monocots from the tropics. acta hortic. 486, ishs. damasco, o.p. 2000. utilization of embryo culture technology for gemplasm conservation: development of medium term conservation for coconut zygotic embryos. paper report of the 2nd inter. coconut embryo culture workshop, merida, 20-23 march, 2000. mexico. del longo, o.t., c.a. gonzalez, g.m. pastori, and v.s. trippi. 1993. antioxidant defences under hyperoxygenic and hyperosmotic conditions in leaves of two lines of maize with differential sensitivity to drought. plant cell physiol. 34 (7): 1023-1028. dodds, j.h., z. huaman and r. lizarraga. 1991. potato germplasm conservation. in: dodds, j.h. (ed.). in vitro methods for conservation of plant genetic resources, chapman and hall. london. p. 93-109 engelmann, f., b. assay-bah, s. bagniol, d. dumet, and n. michaux-ferriere. 1995. cryopreservation of date palm, oil palm, and coconut. in: bajaj, y.p.s. (ed.).biotecnology in agriculture and forestry. springer-verlag, berlin. p. 148-167. 20 gomez, l.d., l.m. casano, m.rouby, m.s. buckeridge, and v.s. trippi. 1994. proteolytic activity associated with cell wall. isolation and partial characterization of a protease from the extracellular fluid of bean hypocotyls. agriscientia. 11:3-11. control of culture media for in vitro conservation of coconut – sukendah and ma l.o. cedo gomez, l.d., l.m. casano, and v.s. trippi. 1995. effect of hydrogen peroxide on degradation of cell wall associated proteins in growing bean hypocotyls. plant cell physiol. 36 (7):1259-1264. ng, n.q. and s.y.c. ng. 1991. reduced-growth storage of germplasm. in: dodds, j.h. (ed.). in vitro methods for conservation of plant genetic resourcess. chapman and hall. london. p. 11-40. 21 introduction observation of embryos biotropia no. 4, 1990/1991: 19-30 strain differences in two species of callosobruchus (coleoptera: bruchidae) developing on seeds of cowpea {vigna unguiculata (l.)} and green gram {v. radiata (l.)} rennie roesli department of agronomy, faculty of agriculture bogor agricultural university, bogor, indonesia phil dobie storage department, odnri, slough, uk brian m. gerard school of agriculture, faculty of science university of edinburgh, edinburgh, uk abstract ovipositional behaviour, development period, and density effect on adult survival of c. maculatus strains from indonesia, nigeria, and yemen, and c. chinensis strains from indonesia and kenya on cowpea and green gram were studied at 20°c and 70% relative humidity. variations on ovipositional behaviour were found among c. maculatus as well as among c. chinensis strains. variations on developmental period were found only among c. maculatus strains. the developmental period of callosobruchus spp. was shorter on green gram than that on cowpea. density effect was remarkably found only on adult survival of c. maculatus yemen strain. these results make useful contribution to the species biology, and have important implication if strains of these species are accidentally imported to countries, or when new legume crops are introduced. introduction beetles belonging to the family bruchidae are the most important insect pests of stored legumes. infestation by bruchids causes losses of weight, nutritional value and germination potential, and therefore the commercial value of the commodity may be reduced (southgate 1978; dick and credland 1986). the most economically important and widespread bruchids species are the cowpea seed beetle, callosobruchus maculatus (fabricius), and the adzuki bean beetle, c. chinensis (linnaeus) (southgate 1978; tdri 1984). the use of resistant varieties of cultivated legumes is one of the recommended control methods of bruchid infestations. varietal resistance against callosobruchus has been reported in cowpeas and chickpea (dobie 1981; raina 1971; singh 1978). 19 biotropia no. 4, 1990/1991 however, there were variations reported in the response of geographically different strains of c. maculatus to a resistant variety of cowpea (dick 1984; dick and credland 1986). further studies on variation on geographically different strains of c. maculatus revealed the occurrence of differences in their biology and behaviour (credland et al. 1986; credland and dick 1987; credland 1986). this study was made to seek more information on the occurrence of geographical variations among c. maculatus and c. chinensis strains, especially to compare strains from indonesia (asia) with those from other tropical countries. materials and method three strains of c. maculatus viz. strain from the iita cultures, nigeria (labeled as iita), yemen, and indonesia; and two strains of c. chinensis viz. strains from indonesia and kenya were used. they were obtained from the culture of odnri, slough, uk. two types of seeds were used as hosts i.e. californian black eyed cowpea (vigna unguiculata (l.)) and australian green gram (vigna radiata (l.)). daily egg production the surface area of cowpea seeds is larger than that of green gram. as oviposition of the species is assumed to be influenced by the surface area, the cowpea seed number used should therefore be different from that of green gram. after measuring the surface areas of both seeds, it was decided that the surface area of one cowpea seed was approximately equal to the surface area of three green gram seeds. one kernel of cowpea or three kernels of green gram were introduced into a 2.5 cm diameter and 5 cm high glass tube. a pair of adults (age < 1 day) of each strain was added into the tube, and the tubes were covered with foam bungs. twenty five replicates were made on both types of seed and all insect strains. the whole set of experiment was kept in the laboratory at 27°c and the r.h. at 70%. the following day, the number of eggs laid by each female was counted and recorded, and egg-laden seeds were replaced by fresh seeds. the observation and seed replacement were done daily until the 10th day. effect of seed availability on egg production different numbers of seeds i.e. 1 (low), 3 (medium), and 10 (high) kernels of cowpea; and 3 (low), 9 (medium), and 30 (high) kernels of green gram were introduced into the tubes. one pair of adults (age < 1 day) of each strain was placed 20 strain differences in two species of callosobruchus-th. roesli, p. dobie & b.m. gerard in each tube and the tube was covered with foam bungs. twenty replicates were made of all treatments and all insect strains, and kept in the experimental room. observation on the number of eggs laid by each female was done on the 7th day after treatment. developmental period eight to 10 adults of each insect strain were introduced into each petri dish containing cowpea or green gram seeds. the dishes were covered with the lids and kept in the experimental room for one night the adults were removed the following day. under a binocular microscope, seeds bearing a single egg were taken out and individually put into tubes. the tubes were covered with foam bungs and kept in the experimental room. twenty five replicates were made of all insect strains. observation on adult emergence were started on the 20th day after treatment. emergence of fresh adults was recorded daily until no more adults emerged. adult survival from seeds bearing different number of eggs to obtain cowpea seeds bearing 1, 2, 3, 4, and 5 eggs, the same procedure as in the developmental period experiment was applied. however, selection was done not only of seeds bearing 1 egg, but of those bearing 2, 3, 4, and 5 eggs as well. the replicate number of each treatment was designed to be inversely proportional to the egg density (giga 1982). thus, the number of eggs used were approximately the same in all treatments (table 1). table 1. the design of the experiment on adult survival from seed bearing different number of eggs no. of eggs/seed 1 2 3 4 5 no. of replicates 20 10 7 5 4 no. of eggs 20 20 21 20 20 emergence of adults was observed and recorded daily starting from the 20th day after treatment. result daily egg production some females were found to lay eggs on the seeds and on the tube walls and so both were recorded and analyzed separately. 21 biotropia no. 4, 1990/1991 the general pattern of daily egg production of all strains on seed was the same. high number of eggs were laid during the early period of the female's life. the maximum number was reached either on the second or third day, and then it gradually decreased (fig. 1). the sum of 10-day egg production per female of all strains was analyzed by two factors analysis of variance. oviposition on seed, on tube wall, or the total, over the period of 10 days was influenced by insect strain (table 2). c. maculatus iita strain was found to have the highest fecundity followed by c. chinensis indonesian, c. maculatus yemen, c. chinensis kenyan, and c. maculatus indonesian strain (table 2, column "total"). differences in the fecundity were found between each strain of c. maculatus, as well as between each strain of c. chinensis (table 2 and fig. 2). c. chinensis indonesian strain laid the highest number of eggs on seed, followed by c. maculatus iita strain. lower numbers were laid by c. maculatus yemen and c. chinensis kenyan strains with the lowest number laid by c. maculatus indonesian strain (table 2). large number of eggs on the tube wall were especially laid by c. maculatus iita and yemen strains. the rest of the strains laid very small number of eggs on the tube. the mean egg number was not significantly different from one another, but they differ significantly from those laid by c. maculatus iita and yemen strains. seed species did not affect oviposition of all strains over 10 days, either on seed, on tube wall or the total (table 3). table 2. means and standard errors of egg product ion of five strains of callosobruchus maculatus over 10 days on seed, on tube wall, and the total insect strains on seed on tube wall total cm* iita 63 .90 a ±4.57 17.02 a ±2.55 80.92 a ±4.98 cm yemen 45.90 b ±4.11 6.06 b ±1.35 51.96b ±4.67 cm indonesia 32.08 c ±2.08 0.50 c ± 0.30 32.55 c ±2.15 cc* indonesia 68.24 a ±1.86 0.47 c ±0.25 68.74 a ±1.91 cc kenya 38.33 bc ±3.20 0.02 c ± 0.02 38. 35 be ±3.20 a, b and c indicate level of significance at 5%; relevant only down the column. * cm for callosobruchus maculatus cc for callosobruchus chinensis. table 3. means and standard errors of insect oviposition over 10 days on seeds, on tube wall, and the total seed species on seed on tube wall total cowpea 48.91 ns ± 2.56 4.89 ns ± 0.96 53.80 ns ± 2.86 green gram 51.08 ns ± 2.38 4.98 ns ± 1.09 56.06 ns ± 2.77 ns : non significant. 22 strain differences in two speciess of callosobruchus – r. roesli, p. dobie & b.m. gerard figure 1. oviposition over 10 days of c. maculatus iita (1), yemen (2), indonesia (3) and c. chinensis on cowpea (cp) and green gram (gg) 23 biotropia no. 4, 1990/1991 figure 2. effect of seed availability on the fecundities of c. maculatus iita (1), yemen (2), indonesia (3) and c. chinensis on cowpea (cp) and green gram (gg). 24 strain differences in two species of callosobruchus-r. roesli, p. dobie & b.m. gerard effect of seed availability on egg production some females were also found to lay eggs on the tube wall. eggs laid on seeds and on tube walls were analyzed separately. tables 4, 5, and 6 show that the oviposition of callosobruchus over 7 days was influenced by seed availability and insect strain, but not by the seed species. the total number of eggs increased significantly with the increase of seed availability. c. maculatus iita produced the highest total number of eggs, followed by c. chinensis indonesia. lower number was produced by c. chinensis kenya, and the lowest number was produced by c. maculatus yemen and indonesian strains. the number of eggs laid on the seed increased significantly with the increase of seed availability (table 4). in contrast, the number of eggs laid on the tube wall decreased with the increase of seed availability (table 4), however the number of eggs laid on tubes with low seed number did not significantly differ from that laid on tube with medium number of seeds. table 4. mean and standard errors of callosobruchus spp. oviposition on seed, on tube wall, and the total at different seed availabilities seed number on seed on tube wall total low medium high 35.42 c±1.50 50.22 b±1.56 59.88 a ±1.46 8.55 b±1.14 .31 b±1.03 3. 63 a ±0.62 43.96 c± 1.78 57.51 b±1.81 63.51 a±1.54 a, b, and c indicate level of significance at 5%; relevant only down the column. table 5. mean and the standard errors of five strains of callosobruchus spp. ovipositions on seed, on tube wall and the total insect strains on seed on tube wall total cm* iita 58.98 a ±2.52 18.68 a ±1.85 77.64 a ±2.39 cm yemen 36.54 c ± 1.85 11.50 b ± 1.36 48.04 c ± 2.21 cm indonesia 34.10 c ± 1.48 1.21 c ±0.24 35. 29 d ± 1.48 cc* indonesia 63 .05 a ±1.92 0.84 c ± 0.32 63. 89 b ± 1.90 cc kenya 49.85 b ±1.31 0.24 c ± 0.07 50.09 c ± 1.33 a, b, c, and d indicate level of significance at 5%; relevant only down the column. * cm : c. maculatus cc : c. chinensis table 6. mean and standard errors of callosobruchus spp. oviposition on seed, on tube wall, and the total of two kinds of seed. seed kind on seed on tube wall total cowpea 49.29 ns ± 1.43 4 . 39 b ± 0.56 53 . .67 ns ± 1.51 green gram 47.72 ns ± 1.30 8 . .60 a ± 0.95 56 . .32 ns ± 1.45 a and b indicate the level of significance at 5%; relevant only down the column. 25 biotropia no. 4, 1990/1991 c. maculatus iita laid the highest number of eggs on tube wall followed by c. maculatus yemen. the rest of the three strains laid only a small number of eggs on tube wall and they did not differ significantly (table 5). the number of eggs laid on the tube containing green gram was higher than that on tube containing cowpea (table 6). the effect of seed availability on egg production of individual strains on cowpea and green gram is shown in figure 2. the effect of seed availability is remarkably seen in c. maculatus yemen and indonesian strains. development period the results indicated that there were differences in the development rate among the insect strains (table 7). the development period of c. maculatus iita strain was the longest, and was significantly longer than the others. development periods of c. maculatus yemen and indonesian strains did not differ significantly. the difference in the development period of the two strains of c. chinensis was not significant, however they were significantly shorter than the development periods of all c. maculatus strains (table 7). seed species significantly influenced c. maculatus and c. chinensis development periods. most of the insect strains developed faster on green gram than on cowpea, except the c. maculatus strain from yemen (table 8). table 7. mean and the standard errors of development periods of 5 callosobruchus strains table 8. mean and standard errors of development period of callosobruchus on cowpea and green gram a and b indicate the level of significance at 5%. 26 strain differences in two species of callosobruchus-r. roesli, p. dobie & b.m. gerard adult survival from seeds bearing different number of eggs the results indicated that egg density, from 1 to 5 eggs per seed, did not influence significantly the number of adult survival of most strains, except on c. maculatus yemen strain (fig. 3). remarkable decreases were noted on adult survival means of seeds bearing 4 and 5 eggs. figure 3. adult survival from seeds bearing different number of eggs of 5 different callosobruchus strains. discussion some females, especially those of c. maculatus females, were found to lay eggs on tube surface. however, there seemed to be less preference for oviposition on tube wall because the number of eggs laid on the tube wall was usually large only when there was a shortage of seeds for oviposition, such as during the peak days of the ovipositional period (fig. 1), or when there were few seeds for oviposition (fig. 2). females of callosobruchus spp. have been reported to control the successful development of their progenies by choosing an appropriate site for oviposition and distributing their eggs more or less equally over the available seeds. they avoid laying eggs on seeds with a rough surface and on seeds already bearing bruchid eggs if noninfested seeds are still available. if noninfested seeds are unavailable, the 27 biotropia no. 4, 1990/1991 females will lay eggs on seeds already bearing eggs, but will choose the bigger ones first. the females can also detect small differences in egg density, and prefer to oviposit on seeds bearing smaller number of eggs (yoshida 1961; avidov et al. 1965; nwanze et al. 1975; messina and renwick 1985a, b). the discriminating ability of female callosobruchus in uniformly distributing their eggs is highly developed, however, in some species the discrimination in choice of seeds suitable for larval growth is not developed. in other words, the ovipositional behaviour was not related to the suitability of seeds for the development of larvae (avidov et al. 1965; bhattacharya et al. 1977). therefore, the apparent preference for oviposition on the tube wall under these conditions could possibly be more accurately described as a strong repellence to ovipositing on seeds that are already bearing numerous eggs. it is difficult to say if the differences in the number of eggs laid on the tube wall by different strains of c. maculatus were due to differences in the ability of females of different strains to discriminate between suitable sites for larval development or simply because they have different fecundities. for example, the fecundity of c. maculatus iita female was the highest, and that strain female also laid the highest number of eggs on the tube wall. probably the female of that strain had relatively greater seed shortage problem than the other two strains. in contrast, c. chinensis appears to have more developed discriminating ability to choose suitable sites for larval development. it was found that although females of the c. chinensis indonesia had higher fecundity than those of the c. maculatus yemen and indonesian strains, fewer eggs were laid on the tube by c. chinensis indonesia. the number of eggs oviposited by females c. maculatus indonesian and yemen strain was suppressed when only a small number of seed was available. however, those oviposited by the other strains were not remarkably suppressed with the reduction of seed availability. credland (1986) stated that the conditions that determine the maximum fecundity differ within and between strains. the reduction in female fecundity as a response to low seed availability is perhaps due to deterrence effects, chemically or physically, of eggs already laid on the seed (messina and renwick 1985a). the development period of callosobruchus, in this experiment was slightly shorter on green gram than that on cowpea. this suggests that green gram is a slightly better host for callosobruchus. giga and smith (1978) reported that of the several pulses tested, including green gram and cowpea, green gram was the most favourable food species for oviposition, speed of development and survival of c. maculatus. when suitable host seeds are infested at numbers above the population's optimum density, there is a reduction in the number emerging of adults due to mortality which primarily took place during the larval stage (utida 1941; mitchel 28 strain differences in two species of callosobruchus-r. roesli, p. dobie & b.m. gerard 1975; giga 1982; dick 1984). the reduction in the number of adults produced due to high density was more pronounced in the c. maculatus yemen strain than those of other strains used in this experiment. variations in the effect of density on the number of adults produced have also been reported to occur among strains of c. maculatus. strains from brazil and nigeria (iita) can produce more than ten adults from a seed with numerous eggs, whereas a strain from yemen rarely produced more than three (dick 1984; dick and credland 1984; credland et al. 1986). the density effect on the number of adults observed in c. maculatus indonesian strain, c. chinensis indonesian and kenyan strains seems to be similar to that in c. maculatus iita. however, higher densities than the maximum density recorded in this experiment should have been used to be able to see the effect more clearly. the geographical variations on the biology and behaviour of c. maculatus and c. chinensis found in this experiment are possibly the result of either genetic evolution of a population which occupies a particular environment and is therefore subjected to that environment selection pressures; or genetic divergence among populations which is caused by chance fluctuations in its allele frequency; or the change in the gene pool (dick 1984; credland 1986). the occurrence of geographical variations among populations of an insect species should be noted when studying or referring to the species biology or behavioral characteristics. attention should also be given to the possibility that a less important species population might become a serious pest if a better plant host species or variety were introduced to the area. acknowledgement the authors would like to thank dr. c.p. haines for his assistance and advice, mr. d.j.b. calverley, head of storage department, odnri, slough, uk. for providing laboratory space and facilities, and the british council for the research funds. reference avidov, z., s.w. applebaum, and m.j. berlinger, 1965. physiological aspects of host specificity in the bruchidae ii: ovipositional preference and behavior of callosobruchus chinensis (l.). ent. exp. & appl. 8: 96-106. bhatacharya, a.k., p.k. pathak, and s.p. shah, 1977. oviposition and development of callosobruchus chinensis (linn) (coleoptera : bruchidae) on several host species. bull. grain technol. 15: 38-41. 29 biotropia no. 4, 1990/1991 credland, p.p. 1986. effect of host availability on reproductive performance in callosobruchus maculatus (f) (coleoptera : bruchidae). j. stored prod. res. 22: 49-54. credland, p.p. and k.m. dick. 1987. food consumption by larvae of three strains of callosobruchus maculatus coleoptera : bruchidae). j. stored prod. res. 23: 31-40. dick, k.m. 1984. bionomic variation among populations of southern cowpea weevil, callosobruchus maculatus and their responses to different varieties of the primary host. ph.d. thesis, bedford college. 305p. dick, k.m. and p.f. credland. 1984. egg production and development of three strains of callosobruchus maculatus (f.) coleoptera : bruchidae). j. stored prod. res. 20: 221-227. dick, k.m. and p.f. credland. 1986. variation in the response of callosobruchus maculatus (f.) to a resistant variety of cowpea. j. stored prod. res. 22: 43-48. dobie, p. 1981. the use of resistant varieties of cowpea (vigna unguiculata) to reduce losses due to post harvest attack by callosobruchus maculatus. in: the ecology of bruchids attacking legumes (pulses): 185-192. (ed. v. labeyrie). dr. w. junk publisher. london. giga, d.p. 1982. the comparative biology of four callosobruchus species with particular reference to competition in c. rhodesianus and c. maculatus. ph.d. thesis, university of reading. giga, d.p. and r.h. smith, 1987. egg production and development of callosobruchus rhodesianus pic and callosobruchus maculatus (f.) (coleoptera : bruchidae) on several commodities at two different temperatures. j. stored prod. res. 23: 9-15. messina, f.j. and j.a.a. renwick, 1985a. ability of oviposition seed beetles to discriminate seeds with differing egg loads. ecological entomology 10: 225-230. messina, f.j. and j.a.a. renwick, 1985b. mechanism of egg recognition by cowpea weevil callosobruchus maculatus. entomol. exp. appl. 37: 241-245. mitchel, r. 1975. the evolution of oviposition tactics in the bean weevil. callosobruchus maculatus (f.). ecology, 56: 696-702. raina, a.k. 1971. comparative resistance to three species of callosobruchus in a strain of chickpea (cicer arietinum l.). j. stored prod. res. 7: 213-216. singh, s.r. 1978. resistance to pests of cowpea in nigeria. in: pests of grain legumes: ecology and control: 267-279. (ed. singh et al.). academic press. london. southgate, b. j. 1978. the importance of bruchidae as pests of grain legumes, their distribution and control. in: pests of grain legumes: ecology and control: 219-229. (ed. singh et al.). academic press. london. tdri. 1984. insect and arachnid of tropical stored products : their biology and identification (atraining manual). tropical development and research institute. slough. utida, s. 1941. studies on experimental population of the azuki bean weevil callosobruchus chinensis (l.) i: the effect of population density on the progeny population. mem. coll. agr. kyoto imp. univ. 48: 1-31. yoshida, t. 1961. oviposition behaviour of two species of bean weevils and interspecific competition between them. mem. fac. lib. arts and educ., miyazaki univ. 11: 41-65. 30 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 349 (hery seed germi).cdr seed germination of avicennia marina (forsk.) vierh. by pericarp removal treatment *hery purnobasuki and edy setiti wida utami department of biology, faculty of sciences and technology universitas airlangga, surabaya 60115, indonesia received 12 november 2013/accepted 28 september 2016 abstract avicennia marina is one species used for building coastal ecosystem stabilization in surabaya shores because of its tolerance to high salinity level and resistance to wave energy. their existence is threatened by various factors i.e. land conversion, pollution and interisland bridge existence. growth of a. marina is currently limited by seed dormancy. in an effort to increase propagation efficiency, seed viability and vigor were compared based on collection dates and pericarp removal methods. the results showed no relationship between salinity levels of soaking water or seed buoyancy and seed viability or seed vigor. collection dates may influence seed viability and vigor. seeds collected in october and november germinated more quickly than those collected in december, although there were no differences in total germination. hand-peeled seeds germinated more slowly than soaked seeds for all collections, but with no differences in total germination. seeds collected in october and november grew more vigorously than those collected in december. keywords: avicennia marina, propagation eficiency, seed viability introduction avicennia marina (the gray mangrove) produces recalcitrant seeds, dispersed by tidal currents. the dispersal unit or propagule of a. marina is the fruit, which usually consists of a single embryo surrounded by a thin pericarp (tomlinson 1986). the fruit is considered to be cryptoviviparous, a condition where the hypocotyl does not enlarge sufficiently to rupture the pericarp while attach to the parent (hutchings & saenger 1987). seeds of a. marina are cryptoviviparous and do not have a dormant stage resulting in germination possibility while still attach to the parent plant (tomlinson 1986; farnsworth 2000). a. marina seeds possess a hydrophilic pericarp, which seed is abscised, shed upon contact with water (tomlinson 1986). the seeds are dispersed tidally and may remain viable while afloat in agitated seawater for up to one year, although viability decreases over time (usda 2009). typical a. marina seed germination initiates when a propagule comes to rest on a suitable substrate such as tidal mud flat, beach or within a spartina spp. marsh (lewis 2000). this epigeal germination is first observed with the extension of a geotropic root radical into the soil where the seed comes to rest. the hypocotyl extends to become a vertical stalk supporting the cotyledon (tomlinson 1986). the propagule then extends its epicotyl, from which the first true leaves emerge, allowing the plant to independently photosynthesize the seed. the cotyledons are then desiccated and abscised (tomlinson 1986). seed germination is controlled by a number of mechanisms and is necessary for growth and development of the embryo, resulting in the eventual production of a new plant. under unfavorable conditions, seeds may become dormant (secondary dormancy) to maintain their germination ability. however, when the conditions are favorable, seeds can germinate (miransari & smith 2014). among factors controlling seed germination and dormancy is seed pericarp removal (sari et al. 2006). seed pericarp is responsible for seedcoating dormancy, affecting seed germination and seedling establishment by preventing water biotropia 3 2 6 74 83 vol. 2 no. , 201 : doi: 10.11598/btb.2016.2 . .3 2 349 * corresponding author: hery-p@fst.unair.ac.id 74 absorption, producing chemical inhibitors, preventing leaking of inhibitors from the seed and inhibiting the radical protrusion (simpson 1990; baskin & baskin 1998; ogawa & iwabuchi 2001; takos 2001; sari et al. 2006). the effect of the pericarp on germination depends on seed size and water availability (chac¢on & bustamante 2001). reproductive success in plants is highly dependent on the survival of their seeds during dispersal. the conditions in which seeds reside after leaving the parent tree towards establishment, and the adaptations of seeds for surviving these conditions are crucial. seedling establishment is considered difficult for mangroves because of the unstable and varied substrates as well as tidal influence (tomlinson 1986). hydrological regimes are particularly important in controlling the survival and subsequent growth of the mangrove seedlings (murray et al. 2003). mangrove habitats in surabaya shores are currently vulnerable to subsidence and sea level rise. as the integrity of coastal salt marsh in surabaya degrades, a. marina habitat is threatened by many activities such as land conversion, pollution and interisland bridge existence. as a result, mangrove stands may be permanently inundated or drained; neither of which are conducive to survival and establishment. the objectives of this research were to determine the optimal time to collect seeds and the pericarp removal methods to support the highest germination rate and to obtain the most vigorous seedling growth of avicennia marina. materials and methods seed source avicennia marina seeds were collected from mature trees near suramadu bridge, surabaya (fig. 1) on 15 october 2011, 15 november 2011 and 15 december 2011. five hundred and fifty (550) seeds were transported in dry buckets to the biology department, universitas airlangga and were maintained indoors at room temperature for 24 hours. the day of seed collection was assigned as day zero for all timedelineated treatments. seed preparation two treatments were applied to the seeds, one day after being collected. the two treatments were: 1. seeds pre-soaked in seawater having various levels of salinity until the pericarps were self-removed, and 2. hand-peeled dry seeds. in the first treatment, there were 5 levels of seawater salinity i.e. 0, 5, 10, 15 and 20 ppt. instant sea salt was used to make these levels of salinity. ten seeds were immersed in each of salinity level on the first day after seeds collection (day 1). the intention of seeds immersion was to self-remove the pericarps and to establish seeds figure 1 sampling locations for a. marina seed harvests (a, b, c, d and e) near suramadu bridge 75 seed germination of avicennia marina by pericarp removal treatment – purnobasuki and utami buoyancy. s seed buoyancy was determined after 48 hours . based on the buoyancy, seeds were categorized as floaters or sinkers. in the second treatment, pericarps of 5 seeds were hand-peeled on day 1. the peeled seeds were categorized as peeled. after the pericarps were removed in the first and second treatments, the seeds were weighed and measured along the longest axis. seed planting prior to planting, individual polybags (7.5 x 22 cm) were filled with planting media consisted of sphagnum moss and vermiculite mix. polybags were placed in raceway tables in a greenhouse at the biology department of universitas airlangga. the raceway tables were kept submerged in seawater allowing planting media to be saturated with seawater prior to planting, thus simulating natural germination conditions (alleman & hester 2010). thermometers were placed in between the submerged raceway tables and at the ends of the tables. water temperature was recorded within 30 minutes interval for the duration of the study. air temperature was maintained between 35 and 37 ºc using thermostat-controlled heaters and exhaust fans. the pericarp-peeled seeds were placed on top of the planting media. one seed was placed in each polybag located on raceway tables. each seed was placed similarly oriented, standing vertically to facilitate equal growth of the radicle into the planting medium. seed germination to determine the effect of seed collection dates on seed germination, the percentage of seeds germination was compared based on seed collection dates and the seed treatments i.e. handpeeled, floater (0 ppt) and sinker (0 ppt). five replications were applied to compare the percentage of seeds germination. the comparison was carried out on day 48 i.e. on 15 march 2012 to allow the earliest planting of a. marina in surabaya coastal area. germination rate avicennia marina produces cryptoviviparous propagules which means that embryonic axis/hypocotyl of the developing embryo does not penetrate the seed coat. therefore, seed germination was recorded when the epicotyl's true leaves were visible beyond the cotyledon (fig. 2). propagule viability was recorded daily for the first 28 days for each treatment by observing the vigor and growth. germination rate is determined by calculating the germination percentage of different time interval after planting and then plotting these data. the cumulative germination over time was compared among treatments. the germination rate of each treatment was calculated using formula as follows (ranal & de santana 2006): germination percentage germination percentage is the number of germinated seeds from a seed population. germination percentage of seeds was compared among treatments after 28 days of study. percentage of seed germination was calculated for each replication, from which the average was calculated for each treatment. figure 2 basic post-germination anatomy of an avicennia marina propagule (crumbie 1987) seedling vigor seedling vigor was quantified by measuring seedling height (mm) and stem length (mm) from the soil surface to the terminal bud. mean height of plants for each seed collection date was measured three times during the growing period. seedlings from seeds collected in october were 76 biotropia vol. 23 no. 2, 2016 measured at 62, 75 and 165 days since collected (dsc). seedlings from seeds collected in november were measured at 43, 70, 92 and 133 dsc and seedlings from seeds collected in december were measured at 79, 104 and 126 dsc. seeds that had not germinated were not included in the seedling vigor analysis. data analysis analysis of variance (anova) and duncan's multiple range test (dmrt) at p<0.05 were carried out for data analysis. data were analyzed using ms excel and spss version 10 to explore possible treatment variations. anova was carried out to determine the treatments effects on seed germination percentage as well as on height and diameter of the seedlings. dmrt was used to compare the mean of germination percentage, height and diameter in each pre-sowing treatment. results and discussion seed germination is a response characterized by three parameters i.e. percentage, rate, and uniformity (hartmann et al. 2002). germination percentage is the number of germinated seeds from a seed population. germination rate is the "speed or velocity" of germination and can be expressed as the time needed for a defined percentage of seed to germinate. germination uniformity is a measurement of the time needed for all seeds to germinate. the mean weight and length of seeds among the treatments varied slightly (table 1). however, the largest and the smallest seeds were sinkers and there was no evidence of seed size effect among treatments. germination percentage was not linear over time and became asymptotic after 40 days of seeds collection (fig. 3). after 28 days, the hand-peeled seeds treatment had the lowest (21%) germination percentage (fig. 3 and 4). germination rates for the floaters and sinkers soaked in 0 ppt treatments were not different from those soaked in other salinity levels. therefore, the subsequent germination trials only used seeds soaked in 0 ppt salinity and handpeeled seeds for pericarp removal (fig. 3). seed size is commonly used as an indicator of seed viability and vigor for many plant species (murali 1997), but the relationship is highly varied and therefore, typically abandoned as reliable indicator outside agricultural applications. seed size and buoyancy have been examined in mangrove species as indicators of seedling performance, but primarily as factors influencing seed dispersal (rabinowitz 1978a, 1978b, 1978c). the studies of rabinowitz (1978a) suggested that the buoyancy and seed size of avicennia and other mangrove species may determine the zonation of seed deposition, but make no indication of viability and vigor. in this study, although seed size varied slightly, there were no differences in germination rate among seed sizes. therefore, selection of a. marina seeds were not differentiated by size. table 1 mean (±se) of seed weight (g) and length (mm) for seeds collected in october treatment weight (g) floaters sinkers peeled 0 ppt 4.1±0.08ab 3.9±0.37ab 5 ppt 4.4±0.23ab 3.8±0.15ab 10 ppt 4.1±0.10ab 4.5±0.72a 15 ppt 3.9±0.09ab 3.2±0.22b 20 ppt 3.8±0.11ab 3.6±0.24ab peeled 4.2±0.07ab length (mm) 0 ppt 28.9±0.28a 29.1±1.33a 5 ppt 29.9±0.28a 29.4±0.55a 10 ppt 28.4±0.37a 28.7±0.49a 15 ppt 27.6±0.38a 24.7±0.89b 20 ppt 28.7±0.29a 27.3±1.02ab peeled 29.4±0.22a notes: numbers followed by the same letters in the same column are not significantly different at p<0.05 77 seed germination of avicennia marina by pericarp removal treatment – purnobasuki and utami germination rates for the floaters and sinkers soaked in 0 ppt treatments were not different from those soaked in other salinity levels. therefore, the subsequent germination trials only used seeds soaked in 0 ppt salinity level and hand-peeled seeds for pericarp removal (fig. 3). germination percentages of seeds soaked in various levels of salinity were not significantly different. therefore, treatment comparison for all collected seeds only used seeds soaked in 0 ppt salinity level (fig. 4). mean of seed sizes varied among treatments and collection dates, but there were no obvious trends among pericarp removal treatments, salinity levels or collection dates (table 2). germination rate was relatively different for seeds collected in october, november or december (fig. 5). seeds collected in october germinated faster (78%) than seeds collected in december (11.3%) (fig. 6). however, on 15 march 2012 (48 dsc), the averages of germination percentage were different among treatments (fig. 7). figure 3 cumulative germination percentage at 48 dsc (days since collected) for floaters, sinkers and peeled treatments for a. marina seeds collected on 1 october 2011. si = sinkers exposed to 0 ppt; fl = floaters exposed to 0 ppt; pe = peeled seeds; sob = combinations of seeds soaked in various salinity levels and buoyancy treatments; vertical line indicates 28 dsc for seeds collected on 1 october 2011 figure 4 mean (±se) of germination percentage per treatment in 28 dsc for seeds collected on 1 october 2011. f = floaters; s = sinkers; p = peeled seeds; the numbers following either f or s represents salinity levels. bar charts having the same letters on top are not significantly different at p<0.05 78 biotropia vol. 23 no. 2, 2016 seeds peeling to expose embryos have been carried out by other researchers in different experiments (arrillaga et al. 1992). seeds collected in october and november germinated sooner than those collected in december, however, the total germination was not different in 48 dsc. seeds collected in october and november grew to similar sizes in 48 dsc and both grew more vigorously than those collected in december. duration and seawater temperature most likely influenced the seed germination and growth rates of seeds collected in december. further study is warranted to determine the relationship between duration, seawater temperature and propagule performance. hand-peeled seeds germinated later compared to seeds soaked in various levels of salinity, but with no differences in total germination. the stem length average of handpeeled seed was nominally greater for all seeds collected by 15 march 2012. agitation of soaking seawater at various salinity levels was observed to delay germination in avicennia germinans (mcmillan 1971). the delay of germination displayed in peeled seeds might indicate that there were other stimulus besides immobilization as an initiator of germination in this species, such as seed moisture, pericarp abscission and seed abscission. the potential for delayed germination of cryptoviviparous seed is justification for further investigation. this table 2 mean (±se) of seed length (mm) and weight (g) for floaters (0 ppt), sinkers (0 ppt) and peeled seeds for seeds collected in october, november and december treatment collected in october november december length (mm) floaters 29.6±0.28ab 31.2±0.29a 28.4±0.42b sinkers 29.4±1.33ab 29.9±0.34ab 29.9±0.50ab peeled 31.2±0.29a 29.6±0.29ab 30.7±0.31ab weight (g) floaters 3.0±0.08ab 3.3±0.09a 2.4±0.13b sinkers 2.9±0.37ab 3.0±0.09ab 2.9±0.12ab peeled 3.2±0.07a 3.0±0.07ab 3.2±0.11a figure 5 cumulative germination percentage for seeds collected in october, november and december in 48 dsc, in 0 ppt salinity level. october collection is represented by black lines; november collection is represented by light gray lines; december collection is represented by dark gray lines. hand-peeled seeds are represented by short dashes, floaters are represented by solid lines and sinkers are represented by long dashes 79 seed germination of avicennia marina by pericarp removal treatment – purnobasuki and utami condition was not consistent with research results of hu et al. (2009) who reported that removal of pericarp improved the germination percentage up to 90% from 44% in seeds of hedysarum scoparium and 60 100% in seeds of oryza sativa (miyoshi & sato 1997). pericarp removal did not provide significant effects on seedling performance in terms of dry masses of epicotyls and roots of seedlings. this result was slightly different from the study results of liu et al. (2012). this might be caused by mechanical constraints rather than chemical inhibitors. it is generally accepted that epicotyl dormancy of acorns in the field is mainly caused by inhibitors in cotyledons, embryos or pericarps (liu et al. 2012). this was consistent with most research (robertson et al. 2006, finney 2011) who reported pericarp-imposed dormancy on seeds. our study suggested that pericarp was responsible for seed dormancy because seeds with intact pericarp were very dormant; therefore, removal of the pericarp significantly increased germination percentage and resulted in complete germination of viable seeds. figure 6 mean (±se) of germination percentage of a. marina seeds in 48 dsc for seeds collected on 1 october 2011, 1 november 2011 and 1 december 2011. floaters are represented by black bars; sinkers are represented by gray bars; hand-peeled seeds are represented by white bars. bar charts having the same letters on top are not significantly different at p<0.05 figure 7 mean (±se) of germination percentage in 48 dsc for seeds collected on 1 october 2011, 1 november 2011 and 1 december 2011. floaters are represented by black bars; sinkers are represented by gray bars; hand-peeled seeds are represented by white bars. bar charts having the same letters on top are not significantly different at p<0.05 80 biotropia vol. 23 no. 2, 2016 the means of seedlings stem lengths for seeds collected in october and november were not significantly different in 48 dsc among treatments. however, seeds collected in december had significantly shorter seedlings stem length in 48 dsc (fig. 8). range of seawater salinity in the mangrove habitats of surabaya coast is 17 30 ppt all year round. seawater salinity can widely vary due to pollution, hurricane and flood which introduce large volumes of seawater or freshwater to the coastal estuaries. the studies of rabinowitz (1978 a, b, c) and mckee (1995) indicated that the salinity level of the soaking seawater used to immerse seeds might affect viability duration during dispersal. however, results of this study indicated that there were no correlations between buoyancy or salinity level of soaking seawater used to immerse seeds and seed viability or vigor. this study, however, suggested that seeds collection date might influence seed viability and vigor, such as shown in the germination of avicennia germinans (finney 2011). seeds collection date is commonly used in horticulture and agriculture to predict seed viability and crop yield as well as to observe the relationship between seed maturity and viability (basra 1995). however, correlations between early and late seed collections are not consistent among species. seeds collected in later dates and their maturity had been shown to positively influence seed quality and vigor in soybeans glycine max (tekrony et al. 1984) as well as other cultivated crops and trees (basra 1995). maximum seed viability and vigor occurred earlier in seed maturation for tomatoes solanum lycopersicum (demir & ellis 1992). relationships between propagule and seed maturity as well as viability among viviparous mangrove genera are poorly defined (farnsworth 2000). this research indicated that salt is not needed in the soaking process to remove pericarp. seeds collected in earlier date experienced quicker germination and subsequently produced larger plants at the earliest time of field planting. however, longer maintenance was required to grow the seedlings. seed collected in later date produced smaller plants, but required less greenhouse time with no difference in germination viability. germination can be delayed for a short time by hand-peeling the pericarps from dry seeds with equal or greater germination and growth rates, however, it requires more labor (finney 2011). conclusions removal of seed pericarp improved germination. there were no correlations between buoyancy or salinity level of soaking seawater used to immerse seeds and seed viability or vigor. seeds collection date might influence seed viability and vigor. seeds collected in october and november germinated more quickly than those collected in december, although there was no figure 8 mean (±se) of seedlings stem length for seeds collected in october, november and december in 48 dsc. floaters are represented by white bars; sinkers are represented by gray bars; hand-peeled seeds are represented by black bars. bar charts having the same letters on top are not significantly different at p<0.05 81 seed germination of avicennia marina by pericarp removal treatment – purnobasuki and utami significant difference in total germination. seeds collected in october and november had similar growth and both grew more vigorously than those collected in december. simple alterations in seed treatment can enhance seed germination. acknowledgements we thank djoko suwondo for his great support in our field research and prof mitsuo suzuki phd. for his helpful comments and criticis on the manuscript. we also thank ade hermawan and syaiful yahya for their field-sampling assistance. references arrillaga i, marzo t, segura j. 1992. embryo culture of fraxinus ornus and sorbus domestica removes seed dormancy. hort science 27:371. alleman lk, hester mw. 2010. refinement of the fundamental niche of black mangrove (avicennia germinans) seedling in louisiana: applications for restoration. wetl ecol manag 19:47-60. baskin cc, baskin jm. 1998. seeds: ecology, biogeography, and evolution of dormancy and germination. san diego, ca (us): academic press. 666 p. basra as. 1995. seed quality: the basic mechanisms and agricultural implications. binghamton, ny (us): haworth press. 376 p. chac¢on p, bustamante ro. 2001. effect of seed size and pericarp on seedling recruitment and biomass in cryptocarya alba (lauraceae) under two contrasting moisture regimes. plant ecol 152:137-44. crumbie mc. 1987. avicennia marina: the grey mangrove: general notes and observations. bull emirates nat hist gr (abu dhabi) 32:2-13. demir i, ellis rh. 1992. changes in seed quality during seed development and maturation in tomato. seed sci res 2:81-7. farnsworth e. 2000. the ecology and physiology of viviparous and recalcitrant seeds. ann revue ecol systematics 31:107-38. finney w. 2011. comparative growth and propagule viability of louisiana-harvested black mangrove, avicennia germinans. master thesis. faculty of sciences in marine and environmental biology, nicholls state university, thibodaux, la (us). 34 p. hartmann ht, kester de, davies jr ft, geneve rl. th2002. plant propagation: principles and practices. 7 ed. upper saddle river, nj (us): prentice hall. 869 p. hu xw, wang yr, wu yp. 2009. effects of the pericarp on imbibition, seed germination, and seedling establishment in seeds of hedysarum scoparium fisch. et mey. ecol res 24(3): 559-64. hutchings p, saenger p. 1987. ecology of mangroves. st. lucia (au): university of queensland press. 388 p. lewis rr iii. 2000. ecologically based goal setting in mangrove forest and tidal marsh restoration. ecol engin 15:191-8. liu y, liu gq, li qm, liu y, hou ly. 2012. influence of pericarp, cotyledon and inhibitory substances on sharp tooth oak (quercus aliena var. acuteserrata) germination. plos one 7(10): e47682. mckee k. 1995. seedling recruitment patterns in a belizean mangrove forest: effects of establishment ability and physico-chemical factors. oecologia 101:44860. mcmillan c. 1971. environmental factors affecting seedling establishment of the black mangrove on the central texas coast. ecology 52:927-30. miransari m, smith dl. 2014. plant hormones and seed germination. environ exper bot 99:110-21. miyoshi k, sato t. 1997. removal of the pericarp and testa of seeds of japonica and indica rice (oryza sativa) at various oxygen concentrations has opposite effects on germination. physiol plant 99(1):1-6. murali ks. 1997. patterns of seed size, germination and seed viability of tropical tree species in southern india. biotropica 29:271-9. murray mr, zisman sa, furley pa, munro dm, gibson j, ratter j, bridgewater s, minty cd, place cj. 2003. the mangroves of belize part 1. distribution, composition and classification. for ecol manage 74:265-79. ogawa k, iwabuchi m. 2001. a mechanism for promoting the germination of zinnia elegans seeds by hydrogen peroxide. plant cell physiol 42(3):286–91. rabinowitz d. 1978a. dispersal properties of mangrove propagules. biotropica 10:47-57. rabinowitz d. 1978b. mortality and initial propagule size in mangrove seedlings in panama. j ecol 66:45-51. rabinowitz d. 1978c. early growth of mangrove seedlings in panama and an hypothesis concerning the relationship of dispersal and zonation. j biogeogr 5:113-33. ranal ma, de santana dg. 2006. how and why to measure the germination process. revista brasil bot 29(1):1-11. robertson aw, trass a, lasley jj, kelly d. 2006. assessing the benefits of frugivory for seed germination: the importance of the deinhibition effect. funct ecol 20:58–66. sari a, ogu b, bilgic a. 2006. breaking seed dormancy of laurel (laurus nobilis l.). new for 31:403–8. 82 biotropia vol. 23 no. 2, 2016 simpson gm. 1990. seed dormancy in grasses. new york (us): cambridge university press. 297 p. takos ia, 2001. seed dormancy in bay laurel (laurus nobilis l.). new for 21:105–14. tekrony dm, egli db, balles j, tomes l. 1984. effect of date of collected maturity on soybean seed quality and phomopsis sp. seed infection. crop science 24:189-93. tomlinson pb. 1986. the botany of mangroves. new york (us): press syndicate of the university of cambridge. 419 p. united states department of agriculture (usda). 2009. natural resource conservation service. plant materials center. plant guide for black mangrove. 1-3. 83 seed germination of avicennia marina by pericarp removal treatment – purnobasuki and utami page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 microsoft word 19 biotropia no. 21, 2003 : 19 31 antagonistic effect of three fungal isolates to aflatoxin-producing^spergiy/hs/javhs okky setyawati dharmaputra seamed biotrop, p.o. box 116, bogor, indonesia and faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia asmarina s.r. putri, ina retnowati and santiambarwati seameo biotrop, p.o. box 116, bogor, indonesia abstract aflatoxin contamination in preharvest peanuts can be controlled among others by using antagonistic fimgi to aflatoxin-producing fungi. aspergillus flavus is one of the fungal species where certain strains can produce aflatoxin. informations regarding the type of interactions between antagonistic fungi and toxigenic a. flavus, and the effects of culture filtrates of the test fungi on the growth and aflatoxin production of toxigenic a. flavus are necessary, before antagonistic fungi could be used as biocontrol agent. three fungal isolates (nontoxigenic a. flavus bio 2127, a. niger bio 2129 and trichoderma harzianum bio 19130) were tested for their antagonistic properties against toxigenic a. flavus bio 2132 using direct and indirect confrontation methods. on direct confrontation method, four kinds of agar media were used, i.e pda (potato dextrose agar), mea 1% (malt extract agar 1%), smkya (sucrose 200 g, mgso47h2o 0.5 g, kno3 3 g, yeast extract 7 g, and block agar aa 20 g), and the mixture of mea 1 % + smkya (1:1). the results indicated that the type of interactions between toxigenic a. flavus either with nontoxigenic a. flavus or with t. harzianum was b type. in this type of interaction, the growth of both toxigenic a. flavus and the test fungi inhibited each other (mutual inhibition) with the zone of inhibition < 2 mm. type of interaction between toxigenic a. flavus and a. niger depended on the kind of media. on smkya and mea 1% + smkya media, the interaction was b type, while on pda and mea 1% media it was d type. in this d type of interaction, toxigenic a. flavus and a. niger inhibited each other (mutual inhibition) at a distance > 2 mm. culture filtrates derived from nontoxigenic a. flavus and a. niger grown on me 1%, smky and me 1% + smky inhibited the growth (based on dry weight) of toxigenic a. flavus, except culture filtrates derived from t. harzianum grown on smky and me 1% + smky media stimulated the growth of toxigenic a. flavus. culture filtrates of nontoxigenic a. flavus, a. niger and t. harzianum inhibited aflatoxin b\ production of toxigenic a. flavus. culture filtrates of a. niger and t. harzianum with conidial concentrations of ixlo6, 2xl06 and 3xl06 per ml inhibited aflatoxin b, production up to 100%. the percentage of inhibition of aflatoxin bi production increased with the increase of conidial concentrations of nontoxigenic a. flavus. the highest percentage of inhibition of aflatoxin bi production (62.5%) was obtained from conidial concentration of 3xl06 per ml. aspergillus niger was the most potential fungus in inhibiting the growth of toxigenic a. flavus, either on agar media or on culture filtrates of test fungi. culture filtrate of a. niger was also the most potential filtrate in inhibiting aflatoxin bi production of toxigenic a. flavus. keywords: antagonistic effect / aspergillus flavus i aspergillus niger i trichoderma harzianum i aflatoxin 19 biotropia no. 21,2003 introduction aflatoxin contamination in peanuts occurs when kernels become infected by aspergillus flavus, a. parasiticus and a. nomius, under drought stress before harvest, during the drying phase in the field, or under unsuitable storage conditions. pitt et al. (1991) reported that a. flavus (and a. parasiticus) are able to grow as commensals in developing peanut plants, and from there the fungi can invade developing peanuts. levels of 1000 ppb of aflatoxin could cause acute liver damage in man and animals. lower levels of aflatoxin in peanut products if consumed could cause liver cancer and premature death in humans, as well as reducing productivity of livestock (pitt and hocking 1996). according to pitt and hocking (1996), 45% of 215 peanut samples collected from retailers in bogor and yogyakarta contained more than 50 ppb of aflatoxin, 33% more than 300 ppb, and 22% exceeding 1000 ppb. in foodstuff, the limit of aflatoxin content determined by u.s. food and drug administration was 20 ppb (park 1993). codex alimentarius commision adopted the maximum level of total aflatoxin content in peanuts intended for further processing at 50 ppb (fao and who 1999). during the past 20 years, a number of approaches have been advocated and tested for reduction of aflatoxins in peanuts: resistant cultivars, thickened shells, waxy testa, improved farm management techniques and postharvest procedures involving drying and storage. some of these approaches have merit, but despite the expenditure of large sums of research funding aflatoxin in peanuts remains a serious commercial problem. other approaches such as using antagonistic fungi to aflatoxin-producing fungi are still needed (pitt 1999). one of the more promising method is the concept of biocontrol by competitive exclusion (pitt 1999). this involves the use of competitive fungi to reduce the possibility of toxigenic fungi present in the soil entering developing peanuts and then producing aflatoxins in them. according to dorner et al. (1992), nontoxigenic a. parasiticus can be used as biocompetitive agent to control aflatoxin contamination in peanuts before harvest. dharmaputra et al. (2001) reported that among fungi isolated from the soil of peanut farms at wonogiri regency (central java), nontoxigenic a. flavus bio 2127, a. niger bio 2129 and trichoderma harzianum bio 19130 were antagonistic to toxigenic a. flavus bio 2128. the objectives of this study were to get information on the type of interactions between each of the three fungal isolates (nontoxigenic a. flavus, a. niger and t. harzianum) and toxigenic a. flavus; and to investigate the effects of culture filtrates of the test fungi on the growth and aflatoxin production of toxigenic a. flavus. materials and methods fungal isolates fungal isolates used were toxigenic a. flavus bio 2132, isolated from peanut farms in pati regency, central java, in january 2002; nontoxigenic a. flavus bio 20 antagonistic effect of three fungal isolates okky s. dharmaputra et al. 2127, a. niger bio 2129 and t. harzianum bio 19130, isolated from soils of peanut farms at wonogiri regency, central java, in may 2000 (dharmaputra et al 2001). the four fungal isolates belong to the plant pathology laboratory culture collection, seameo biotrop. each fungal isolate was subcultured on potato dextrose agar (pda) medium and incubated at room temperature (28 ± 2°c) for 7 days. test of antagonism between three fungal isolates and toxigenic a. flavus 1. direct confrontation between fungal colonies nontoxigenic a. flavus, a. niger and t. harzianum isolates were tested for their antagonistic property against toxigenic a. flavus using direct opposition method (dennis and webster 1971) on four agar media, i.e. pda, me a 1% (malt extract agar 1%), smkya (sucrose 200 g, mgso4.7h o 0.5 g, kno3 3 g, yeast extract 7 g, block agar aa 20g) and the mixture of mea 1% + smkya (1:1). toxigenic a. flavus (4 mm in diam) was placed on the four media in a petri dish (diam 9 cm). at the same time at a distance of 3 cm from toxigenic a. flavus inoculum, each test fungus was inoculated on the same dish. the plates were incubated at room temperature. three replications were used for each treatment. observations were carried out seven days after incubation on the inhibition of mycelial growth of toxigenic a. flavus by the test fungi using the formula of fokkema (1973): r i r 2 1= x l o o % r1 where i = percentage of inhibition f] = radius of toxigenic a. flavus away from the test fungi r2 = radius of toxigenic a. flavus towards the test fungi the type of interactions between toxigenic a. flavus and each of the test fungus was determined based on wheeler and hocking (1993), adapted from magan and lacey (1984), as presented in table 1. 2. indirect confrontation 2.1. preparation of cultural filtrates of three test fungal isolates isolates of nontoxigenic a. flavus, a. niger and 7". harzianum were grown respectively on pda medium in petri dishes (9 cm in diam) and incubated at room temperature for 7 days. fungal conidia were suspended in sterile distilled water. two ml of each test fungal isolate suspension with three conidial concentrations (ixlo6, 2xl06 and 3xl06 conidia/ml) were'grown on 100 ml me 1%, 100 ml smky, and 100 ml of the mixture of me 1% + smky (1:1) liquid media in 250 ml erlenmeyer flask, respectively, and then incubated at room 21 biotropia no .21,2003 antagonistic effect of three fungal isolates okky s. dharmaputra et al. temperature for 14 days. the concentrations of conidia were determined by microscopically counting the number of conidia using a haemacytometer. after incubation, the culture filtrates of the fungi were separated from their colonies using sterile filter paper no. 1, followed by sterilization of the culture filtrates using millipore (0.45 jam pore size). 2.2. effect of culture filtrates of test fungi on the growth and aflatoxin production of toxigenic a. flavus one ml of conidial suspension of toxigenic a. flavus (ixlo6 conidia/ml) was inoculated into 50 ml of sterilized culture filtrates derived from each treatment combination in a glass bottle volume 100 ml. they were then incubated at room temperature for 10 days. as control, toxigenic a. flavus was grown on me 1%, smky and in the mixture of me 1% + smky liquid media. three replications were used for each treatment (including the control). observations were made on the dry weight of mycelia of toxigenic a. flavus and aflatoxin production. the dry weight of mycelia was determined by drying the fungal colonies in an oven at 95°c until a constant weight was attained (gourama and bullerman 1995). aflatoxins were analyzed using a thin layer chromatography method (bainton et al. 1980). in this case, aflatoxin in culture filtrates was extracted using chloroform and identified by two dimensional tlc using standard comparison. aflatoxin content produced by the most potential test fungal isolate was confirmed using a high performance liquid chromatography method (rodriguez and mahoney 1994). statistical analysis the data were analyzed using a completely randomized factorial design with 2 factors. the first and the second factors were the isolates of test fungi (nontoxigenic a. flavus, a. niger, and t. harzianum) and conidial concentration of test fungi (1x106, 2x106 and 3xl06 conidia/ml), respectively. results and discussion type of interaction and percentage of growth inhibition between toxigenic a. flavus and test fungi on various agar media two types of interactions (b and d types) were found between each of the three test fungi and toxigenic a. flavus after 7 days of incubation (table 2). on the type of interaction b, the test fungi and the toxigenic a. flavus inhibited each other's growth with a zone of inhibition < 2mm. this type of interaction was found on the interaction between the toxigenic and the nontoxigenic a.flavus on pda, mea 1%, smky a, and mea 1% + smkya media; between the toxigenic a. flavus and 23 biotropia no. 21,2003 a.niger on smkya and mea 1% + smkya media; between the toxigenic a. flavus and t. harzianum on pda, mea 1%, smkya, and mea 1% + smkya media. on the d type of interaction, the test fungi and the toxigenic a. flavus inhibited each other's growth with a zone of inhibition > 2 mm. this type of interaction was found on the interaction between the toxigenic a. flavus and a. niger on pda and mea 1% media. on the two media a zone of inhibition was found between the colony of a. niger and toxigenic a. flavus. it was assumed that a. niger produced antibiotic. according to jeffries and young (1994), production of extracellular metabolites (such as antibiotics and lytic enzymes) was one of the mechanisms of antagonism between two fungal isolates. dharmaputra et al. (2001) reported that the type of interaction between toxigenic a. flavus isolate 557 and t. harzianum, and between a. flavus isolate 102 and t. harzianum were a and b types, respectively; between toxigenic a. flavus isolate 23j and nontoxigenic a. flavus isolate 18t was b type; between toxigenic a. flavus isolate 102 and a. niger was d type. the kind of media did not give significant differences on the percentage of growth inhibition (based on mycelial growth) of toxigenic a. flavus by the three test fungal isolates. nevertheless, the highest percentage of growth inhibition was found on smkya medium (43.77%) (table 3). the test fungal isolates gave very significant differences on the percentage of growth inhibition of toxigenic a. flavus on smkya medium. trichoderma harzianum caused the highest growth inhibition (49.09%) compared to toxigenic a. flavus on smkya medium (table 4). 24 antagonistic effect of three fungal isolates okky s. dharmaputra et al the effect of culture filtrate of test fungi on the growth and aflatoxin production of toxigenic a. flavus the effect of culture filtrates of nontoxigenic a. flavus and a. niger on the percentage of growth inhibition of toxigenic a. flavus (based on the dry weight of mycelia) was not significantly different. nevertheless, the highest percentage of growth inhibition (69.44%) of toxigenic a. flavus caused by the culture filtrate of nontoxigenic a. flavus was on me 1%, while that of the culture filtrate of a. niger (72.51%) was on smky (table 5). it indicated that nontoxigenic a. flavus was more competitive to toxigenic a. flavus on me 1% medium compared with those of smky and me 1% + smky media, while a. niger was more competitive on smky medium compared with those on me 1% and me 1% + smky media. culture filtrate derived from t. harzianum grown only on me 1% medium inhibited the growth of toxigenic a. flavus (63.64%), while on smky and me 1% + smky media the filtrate stimulated the growth of toxigenic a. flavus (38.0 and 31.58%, respectively) (table 6). it indicated that culture filtrate of t. harzianum was only effective in inhibiting the growth of toxigenic a. flavus on me 1% medium. using tlc method, only aflatoxin b, was detected by toxigenic a. flavus. as aflatoxin was not produced on me 1 % medium, consequently only smky and me 25 biotropia no. 21, 2003 1% + smky media were used to study their effects on the growth (based on dry weight of mycelia) and aflatoxin production of toxigenic a. flavus. the kind of culture filtrates of test fungi on the growth of toxigenic a. flavus on smky media was significantly different. conidial concentration of test fungi and the interaction between the kind of the culture filtrates and conidial concentrations were not significantly different. the percentage of growth inhibition of toxigenic a. flavus caused by culture filtrate of a. niger (72.51%) was higher than that of nontoxigenic a. flavus (45.62%). on me 1% + smky medium, culture filtrates and conidial concentration of test fungi, and their interaction were not significantly different. nevertheless, the percentage of growth inhibition of toxigenic a. flavus caused by culture filtrate of a. niger (68.68%) was higher than that of nontoxigenic a. flavus (64.0%). antagonistic effect of three fungal isolates okky s. dharmaputra et al. the inhibition percentage of aflatoxin b, production caused by culture filtrate derived from nontoxigenic a, flavus grown on smky medium (41.67%) was not significantly different with that of me 1% + smky medium (58.33%). culture filtrate derived from a. niger either grown on smky or me 1% + smky media completely inhibited aflatoxin production effectively (100%) (table 7). culture filtrate of t. harzianum grown on the two media also completely inhibited aflatoxin bi production up to 100% (table 7), but also stimulated the growth of toxigenic a. flavus (table 6). the kind of culture filtrates derived from test fungi and conidial concentration of test fungi and their interaction gave significantly different effects on the percent inhibition of aflatoxin production using smky medium. on me 1% + smky medium, the kind of culture filtrates of the test fungi gave very significantly different effect on aflatoxin b) production, while conidial concentrations and the interaction between kind of culture filtrates and conidial concentrations were not significantly different. culture filtrates derived from a. niger with the concentrations of 1x106, 2xl06 and 3xl06 conidia/ml grown on smky medium inibited aflatoxin bt production up to 100% (table 8). the percent inhibition of aflatoxin b, production increased with the increase of conidial concentrations of nontoxigenic a. flavus. the culture filtrate of nontoxigenic a. flavus with the concentration of 1x106 conidia/ml was not significantly different with that of 2x106 conidia/ml against the inhibition percentage of aflatoxin bj production, while that of 3xl06 conidia/ml was significantly different. the culture filtrate derived from a niger grown on me 1% + smky medium inhibited aflatoxin b] production higher (100.00%) than that of nontoxigenic a. flavus (56.25%). aflatoxins produced by the most potential test fungal isolate were confirmed by using high performance liquid chromatography (hplc). two kinds of aflatoxins (bi and g|) were found using this method. 27 biotropia no. 21,2003 the results of test of antagonism using direct confrontation showed that smkya medium was the best medium for antagonism study between toxigenic a. flavus either with nontoxigenic a. flavus, a. niger or t. harzianum. this is because the highest percentage of growth inhibition of toxigenic 'a. flavus caused by the three test fungal isolates occurred on smkya medium (table 3). on smkya medium the highest percentage of growth inhibition of toxigenic a. flavus was caused by t. harzianum followed by a. niger, while the lowest was caused by nontoxigenic a. flavus (table 4). however, the results of test of antagonism study using indirect confrontation showed that the culture filtrate derived from t. harzianum grown on smky medium stimulated the growth of toxigenic a. flavus (table 6). therefore, a. niger was the most potential test fungus in inhibiting the growth and aflatoxin b, production of toxigenic a. flavus, either on agar media or on culture filtrates of the test fungi. confirmation of aflatoxin contents produced by toxigenic a. flavus grown on culture filtrate of a. niger cultivated on smky and me 1% + smky is presented in table 9. the results showed that there were differences in aflatoxin bi contents analyzed using tlc and hplc methods, because different methods could have different results. nevertheless, based on the two methods used, culture filtrates derived from a. niger cultivated either on smky or me 1% + smky liquid media with different conidial concentrations completely inhibited aflatoxin b] production of toxigenic a. flavus. 28 antagonistic effect of three fungal isolates okky s. dharmaputra et al. table 9. aflatoxin bj contents produced by toxigenic a. flavus grown on culture filtrate derived from a. niger cultivated on smky and me 1% + smky liquid media with various conidial concentrations. aflatoxin bl content (ppb) conidial concen tration of a. niger (conidia/ml) media tlc method hplc method 1 x 106 culture filtrate (using smky) 0 0 control (smky) 160 97.57 culture filtrate (using me 1% + smky) 0 0 control (me 1% + smky) 120 60.79 2x10" culture filtrate (using smky) 0 0 control (smky) 160 98.89 culture filtrate (using me 1% + smky) 0 0 control (me 1% + smky) 120 69.37 3x 10' culture filtrate (using smky) 0 0 control (smky) 160 103.58 culture filtrate (using me 1 % + smky) 0 0 control (me 1% + smky) 120 73.68 tlc = thin layer chromatography; hplc = high performance liquid chromatography conclusions the type of interactions between toxigenic a. flavus either with nontoxigenic a. flavus or with t. harzianum was b type, respectively. in this type of interaction, mutual inhibition on contact or space between the test fungi and toxigenic a. flavus was small (< 2 mm). the type of interactions between toxigenic a. flavus and a. niger depended on the kind of media. on smkya and mea 1% + smkya media, the interactions were b type, while on pda and mea 1% media, it was d type. in this d type of interaction, toxigenic a. flavus and a. niger inhibited each other (mutual inhibition) at a distance > 2mm. culture filtrates derived from nontoxigenic a. flavus and a. niger grown on me 1%, smky and me 1% + smky inhibited mycelial growth (based on dry weight) of toxigenic a. flavus, except those derived from t. harzianum grown on smky and me 1% + smky media which stimulated the growth of toxigenic a. flavus. 29 biotropia no. 21,2003 the culture filtrates of nontoxigenic a. flavus, a. niger and t. harzianum inhibited aflatoxin bi production of toxigenic a. flavus. the culture filtrates of a. niger and t. harzianum with the conidial concentrations of 1x106, 2xl06 and 3xl06 per ml could inhibit aflatoxin b] production up to 100%. the percent inhibition of aflatoxin b, production increased with an increase of conidial concentrations of nontoxigenic a. flavus. the highest percent inhibition of aflatoxin b\ production (62.5%) was obtained from the conidial concentration of 3x106 per ml. aspergillus niger was the most potential fungus in inhibiting the growth of toxigenic a. flavus, either on agar media or on culture filtrates of the test fungi. the culture filtrate of a. niger was also the most potential filtrate in inhibiting aflatoxin bi production of toxigenic a. flavus. the results of this study gave important informations on antagonistic effect of three fungal isolates (nontoxigenic a. flavus, a. niger and t. harzianum) before they could be used to control aflatoxin-producing a. flavus in peanuts grown under green-house and field conditions. acknowledgements the authors gratefully acknowledge the financial support of the government of indonesia. we also thank the technicians of the plant pathology laboratory, seameo biotrop, who have in one way or another contributed to this research. references bainton, s.j., r.d. coker, b.d. jones, e.m. morley, m.j. nagler, r.l. turner. 1980. mycotoxin training manual. tropical products institut (tpi), london. dennis, c and j. webster. 1971. antagonistic properties of species groups of trichoderma. iii. hyphal interaction. trans. brit. mucol. soc. 57: 363 369. dharmaputra, o.s., a.s.r. putri, i. retnowati and s. ambarwati. 2001. soil mycobiota of peanut fields at wonogiri regency, central java: their effect on the growth and aflatoxin production of aspergillus flavus in vitro. biotropia no. 17: 30 59. dorner, j.w., r.j. cole and p.d. blankenship. 1992. use of a biocompetitive agent to control preharvest aflatoxin in drought stressed peanuts. journal of food protection 55 (11): 888 892. fao and who. 1999. codex aliraentarius commision. food standard programme. report of the 23'd session, rome, 28 june 3 july 1999. food agriculture organization and world health organization, rome, italy. fokkema, n.j. 1973. the role of saprophytic fungi in antagonism against dreschslera sorokiniana (helminthosporium sativum) on agar plates and on rye leaves with pollen. physiological plant pathology 3: 195-205. gourama, h. and l.b. bullerman. 1995. inhibiton of growth and aflatoxin production of aspergillus flavus by lactobacillus sp. j. food protection 58 (11) 1249 1256. 30 antagonistic effect of three fungal isolates okky s. dharmaputra et al. jeffries, p. and t.w.k. young. 1994. interfungal parasitic relationship. cab international, wallingford. magan, n. and j. lacey. 1984. the effect of water activity, temperature and substrate on interaction between field and storage fungi. trans. br. mycol. soc. 92: 83 93. park, d.l. 1993. controlling aflatoxin in food and feed. food technology 47 (10): 92 96. pitt, j.i. 1999. controlling aflatoxins in peanuts by competitive exclusion of toxigenic fungi. in: dietzgen, r.g. (ed). elimination of aflatoxin contamination in peanut, pp. 21 -22. aciar proceedings no. 89, canberra. pitt, j.i. and a.d. hocking. 1996. current knowledge of fungi and mycotoxins associated with food commodities in southeast asia. in: highley, e. and g.i. johnson (eds). mycotoxin contamination in grains, pp. 5 -10. aciar technical reports 37, canberra. pitt, j.i., s.k. dyer and s. mccammon. 1991. systemic invasion of developing peanut plants by aspergillusflavus. letters in applied microbiology 13: 16 20. rodriguez, s.b and n.e. mahoney. 1994. inhibition of aflatoxin production by surfactants. applied and environmental microbiology 60 (1): 106 110. wheeler, k.a. and a.d. hocking. 1993. interactions among xerophilic fungi associated with dried salted fish. j. appl. bacteriology 74: 164 169. 31 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 4. rima hs (identification).cdr biotropia vol. 20 no. 2, 2013: 104 111 identification of anatomical characteristics of in its interaction with aquilaria microcarpa fusarium solani rima hs siburian , ulfah j siregar , iskandar z siregar , erdy santoso ,and imam wahyudi phd reseach grantee 2011/accepted 12 march 2013 is one of agarwood-producing plants. interaction of pathogenic fungi may cause physiological changes that have an impact to cells, tissues or organs of plants. this study aimed to determine the differences in anatomical characteristics of wood between inoculated and uninoculated with . result showed that traits of inoculated and uninoculated wood in general were partly different. the differences were found in wood color, odor, deposit inside the lumen vessel and the frequency of included phloem. chemical compounds in fusarium-inoculated wood were elemol, baimuxinal, 3-phenyl-2-butanone, and chromen-4-one. anatomical characteristics pathogenic fungi . 1* 2 2 3 2 1 2 3 post graduate student faculty of agriculture, bogor agricultural university, bogor, indonesia faculty of forestry, bogor agricultural university, bogor, indonesia center for research and development conservation and rehabilitation ministry of forestry, bogor, indonesia aquilaria microcarpa aquilaria microcarpa fusarium solani aquilaria microcarpa aquilaria microcarpa, , , fusarium solani abstract introduction key words: a. microcarpa fusarium et al et al is one of gaharu-producing plants, contains a distinctive color (brown-black) and levels of mastic (dewan standard nasional 1999). agarwood is formed as a reaction of plant to the disruption due to biotic or abiotic factors. the most widely biotic disturbances reported are the role of fungus in the formation of agarwood impaired by sp. (gong & shun 2008; siregar 2009; isnaini . 2009; mohamed . 2010). in the interaction process between the fungus with its host, the fungus pathogenicity greatly affect the response given by the plant (mendgen & deising 1993). the response is a plant defense mechanism that serves as a physical biochemistry of plant barriers in cells and tissues that would kill or inhibit the growth of pathogens (groenewald 2005). * corresponding author : rhsiburian@yahoo.com doi: 10.11598/btb.2013.20.2.4 104 biochemical resilience is a series of biochemical reactions that occur in cells and tissues of plants in order to produce toxic substances to pathogens or create conditions that inhibit the growth of pathogens in the plant (agrios 1996). biochemical changes that may occur including the synthesis and accumulation of salicylic acid (wobbe & klessig 1996) or fitoaleksin (beynon 1997), the compound result of secondary metabolites toxic to viruses, bacteria, and fungus-like fatty acid (lowton . 1992), and issuance of oligosaccharide elicitor by the plant (nothnagel . 1983). these compounds may not only protect plants against pathogens comprehensively but can also suppress the development of pathogens without reducing production. in addition, the plant can defend itself without producing metabolites that are required by the pathogen in order to inhibit the developmental process of pathogen. research on the interaction of plant with sp. has been conducted by examining young plants/seedlings (rahayu 2009; putri 2009) but the extent of the difference between the anatomical characteristics and its content at inoculated and uninoculated plants have not been studied and reported. therefore, this study aimed to identify differences in the anatomical characteristics and content of chemical compounds between fusarium-inoculated and uninoculated of plants. plant material used was that had been inoculated three years prior to the research initiation and formed tree aloes compound at tree id# 5. samples were also selected from un-inoculated plants at the same approximate age and grown in location close to inoculated plants with id# 22. sampling was carried out by drilling trunk of wood at four cardinal directions (east, west, north and south). chemicals used were technical alcohol, technical glycerine, faa solution (37% formaldehyde: glacial acetic acid: 70% alcohol = 5:5:90), a solution of n-butanol, gifford solution (glacial acetic acid: 60% ethanol: technical glycerin = 20:80:5), 4% formaldehyde, 100 mm k hpo , 2% safranin, 1% i ki, and a solution of copper acetate [cu (ch coo) 2 h o] 50%. all of these chemicals were used to make preparative slides. the tools used were foto-microscope (nikon obtiphot 2), microscope (nikon afx-dx labophot -2), rotary microtome (yamato rv-240), sliding microtome (hm 400 microm r) and frozen microtome (yamato rv-240). observations were conducted through the microtome specimens after being stained by safranin to identify the differences in anatomical characteristics between inoculated and un-inoculated trees. wood samples were then evaluated by gas chromatography using gas chromatography mass spectrometry (gcms). wood samples from inoculated and uninoculated plants were used for anatomical observation. the samples were soaked into 70% ethanol immediately, infiltrated with 20% polyethylene glycol 2000 in ethanol, dried for 3-4 days in the oven of 60ºc, and et al et al a. microcarpa fusarium a. microcarpa a. microcarpa materials and method 2 4 2 3 2 microtome specimen preparation 105 identification of anatomical characteristics of rima hs siburianaquilaria microcarpa – et al. then sliced to a thickness of 12-20 in transverse, radial and tangential directions. the thin specimen obtained was stained using 2% safranin for 1-2 hours, washed and then dehydrated with ethanol 30%, 50%, 70%, 90%, and absolute consecutively for 10 minutes each. furthermore, the specimens were immersed in xylol for about 1 minute and then mounted on the object glass. after mounting, the specimen was put on a slide warmer at 40-50ºc for 1 day and ready to be observed. the procedure was based on richter (1990) after modification. characteristics of both inoculated and un-inoculated wood were observed by using photomicroscope (nikon obtiphot 2) and microscope (nikon labophot afxdx -2). the observed characteristics consist of wood color and odor, growth ring, vessel elements (shape, distribution, diameter, length, arrangement and type of perforation plate), ray parenchyma (type, width, height and frequency), axial parenchyma, intercellular canals, sediment or tyloses and mineral inclusions as well as the frequency of included phloem. results obtained in each sample were described qualitatively. encountered exudates were further tested by using gcms to determine the chemical compounds. analysis was only performed on wood sample containing sediment, using pyrolysis gcms of shimadzu gcms-qp2010. helium (0.8 ml/min.) was used as a carrier, equipped with capillary column db-5 ms (60 mm x 0.25 mm, film thickness 0.25 ), operated by electron impact (ei) mode at 70 ev and ion source temperature of 2000ºc. identification of chemical compounds was conducted on retention time and ms analysis. it was observed that anatomical characters of wood from the inoculated and uninoculated of were partly different. the difference was found in wood color, odor, deposits in vessel-element and the frequency of included phloem. in case of inoculated plants, the color of wood especially around the injection area is slightly darker than that of inoculated plants. wood odor was also somewhat different: the inoculated wood has a little bit distinct odor compared to that of un-inoculated wood. it was also observed that inoculated wood has a gold-plated resin inside their lumen. such kind of resin was not observed in the uninoculated wood. some similarities and differences in anatomical features of inoculated and un-inoculated of wood were presented in table 1. resin produced by tree was not exudated out of wood, but deposited and infiltrated inside the tissues. it then contributes to the changes in tissue color from white to dark brown in general. according to rao . (1992), a large amount of resin in case of was accumulated in included phloem, but in small amount in other parts such as trachea xylem elements, fiber cells and ray parenchyma. included phloem is a secondary phloem located in the secondary xylem (mauseth 1988). network in which the resin accumulated was well known, but the networking μm μm observation results and discussion a. microcarpa a. microcarpa a. microcarpa et al a. agalocha aquilaria biotropia vol. 20 no. 2, 2013 106 for its secretion was still unknown (mandang & wiyono 2002). it was assumed that secreting tissue of parenchyma phloem was composed by the living cells capable to store starch, fat, organic compounds, and some secondary metabolite materials such as tannins and resins (fahn 1991). table 1. observation of anatomical characters of wooda. microcarpa characters observed a. microcarpa inoculated uninoculated wood color wood odor growth ring vessel elements distribution perforation plate deposit in pores frequency of included phloem wood scent light to dark brown distinct odor unclear mostly in radial multiple and partly solitaire simple present higher scented white (light) regular unclear mostly in radial multiple and partly solitaire simple none lower unscented identification of anatomical characteristics of rima hs siburianaquilaria microcarpa – et al. 107 aquilaria aquilaria aquilaria pinus et al et al et al fusarium included phloem was produced during secondary growth. according to blanchette (2005), included phloem is a dedicated networking capable to secrete resin. in addition to the , the similar structure was also found in nyctaginaceae and amaranthaceae. included phloem on has the same function as the resin glands on sp. it was referred as a place to resin being accumulated (nagy . 2000). included phloem that located in the secondary xylem (mauseth 1988) is a complex structure formed by the cambium to the inside, made up of filter elements, companion cells, parenchyma tissues, and fibers (rao . 1992). cell wall of included phloem components is very thin, less of lignin content so it was not stained by the safranin, and has a plate-type sieve foraminate (rao . 1992; nobuchi & siripatanadilok 1991). if close to or surrounded by the included phloem, parenchyma cells will die and function as the resin storage (fahn 1991). response of inoculated plant to the infection of sp. occurred at cellular level, tissues or organs. it was observed that in case of inoculated plant there was a large number of included phloem in which the terpenoid compound and others were accumulated as the deposit but has no plate-gold resin inside the lumen of vessel elements (figs. 1 & 2). biotropia vol. 20 no. 2, 2013 108 figure 1 cross section of (a) uninoculated and (b) inoculated. a. microcarpa figure 2 included phloem islands in (a) uninoculated (b) inoculated. a. microcarpa chemical components of a. microcarpa . the chemical compounds detected from by using gcms analysis are listed on table 2. table 3 shows similarity and also variation in the chemical composition of several agarwood oil samples. generally, agarwood oils are mixtures of sesquiterpenes, sesquiterpene alcohols, oxgyenated compounds, chromone derivatives and resins. some important compounds are agarospirol, jinkohol-eremol, jinkohol and kesenol that may contribute to aroma of agarwood (nakanishi 1984; ishihara 1993). yuan (1995) found differences in chemical components between high and low quality of gaharu. agarol was the first sesquiterpene compound isolated from aloe (yuan 1995), and oxoagarospirol, an aloe fragrance components resulting from infection of fungus on the wood. the oxoagarolspirol content in increased two months after inoculation. according to ishihara . (1991), kusunol, dihidrokanon, karanon, and oxo-agarospirol were a sesquiterpene compound isolated from low-quality gaharu. benzilaseton is a constituent of aloes identified by yang and cheng in (burfield 2005). these compounds and the compounds of kromone (also known as a constituent of aloes (yagura . 2005; konishi . 2002) start to be detected since the third day of inoculation. agarwood is a type of tree defense fitoaleksin compounds triggered its formation after the attack, then the formation of kromone benzilaseton compounds. this prompts the induction of defense responses against tree. a. microcarpa a. sinensis a. sinensis et al a. sinensis et al et al f bulbigenum table 2 agarwood components from inoculated a. microcarpa identification of anatomical characteristics of rima hs siburianaquilaria microcarpa – et al. 109 table 3 similarity and variation in chemical compounds of several spp.aquilaria compounds authors elemol baimuxinal 3-phenyl-2-butanone chromen-4-one chen h 2011 (. et al. aquilaria sinensis lour.) chen h 2011 (. et al. aquilaria sinensis lour.) faridah s. 2009 (aquilaria maleccencis) faridah s. 2009 (aquilaria maleccencis), dai h 2009 (. et al. aquilaria sinensis lour.) biotropia vol. 20 no. 2, 2013 110 conclusions references the difference in anatomical features of plants that interacted with sp., was distinctively found in wood color, deposited material deposition in pore, the smell/aroma of wood and sedimentation in included phloem. further, examination of deposited material in pores by using gcms in inoculated plants interacting with sp., its aloe contains several compounds that forming the basic structure of seskuiterpenoid such as baimuxinal, elemol, 6-methoxy-2-(2-phenylethyl)-4hcromen-4-one and 3-phenyl-2-butanone. fusarium fusarium burfield t. 2005. agarwood chemistry. www.cropwatch.org. [2 february 2010]. chen h, yang y, xue jian, wei j, zhang z and chen h 2011.comparison of compositions and antimicrobial activities of essential oils from chemically stimulated agarwood, wild agarwood and healthy (lour.) gilg trees. 2011, , 4884-4896; doi:10.3390/molecules16064884. dai hao f, liu j, zeng y, han z, wang h, mei w. 2009. a new 2-(2-phenylethyl) chromone from chinese eaglewood. , , 5165-5168; doi:10.3390/molecules14125165 fahn a. 1991. 4th ed.. oxford: butterworth-heinemann. faridah s. 2009. analysis of agarwood oil composition via preparative thin layer chromatography. [thesis] universiti malaysia pahang. groenewald s. 2005. biology, pathogenecity and diversity of fusarium oxysporum f.sp. cubense [thesis]. universitas of pretoria; pretoria. hou d, 1960. thymelaeaceae. in: van steenis, c.g.g.j. (ed), flora malesiana series 1, volume 6. wolternoordhoff publishing, groningen, the netherlands, p 1-15. ishihara m, tomoyuki t, tsuneya, kenji u. 1991. fragrant sesquiterpenes from agarwood. phytochem 33: 11471155 konishi t, konoshima t, shimada y, and kiyosawa s. 2002. six new 2-(2-phenylethyl) chromones from agarwood. 50(3) 419-422 kunoh h 1995. cytological approaches in understanding host-parasite interaction signal transduction mandang yi, wiyono b. 2002. anatomi kayu gaharu ( lamk.) dan beberapa jenis sekerabat. 20: 107-126. aquilaria sinensis molecules 16 molecules 14 . plant anatomy. . chem. pharm. bull. japan. . aquilaria malaccensis buletin penelitian hasil hutan identification of anatomical characteristics of rima hs siburianaquilaria microcarpa – et al. 111 mauseth j d. 1988. plant anatomy. pearson education. glenview il. mendgen k, h deising. 1993. infection structures of fungal plant pathogena cytological and physiological evaluation. 124: 193-213. mohamed r, jong pl, s zali. 2010. fungal diversity in wounded stems of fungal divers 43:67-74 nagy ne, franceschi vr, solheim h, krekling t, christiansen e. 2000. wound-induced traumatic resin duct development in stems of norway spruce (pinaceae): anatomy and cytochemical traits. american journal of botany 87: 302-313. nakanishi, t., yamagata, e., yoneda, k., nagashima, t., kawasaki, i. yoshida, t., mori, h. and muira, i. 1984. three fragrant sesquiterpenes of agarwood. 23(9):2066-2067 nobuchi t, siripatanadilok s. 1991. preliminary observation of wood associated with the formation of aloewood 63:226 -235. nofryanti e. santoso e, sitepu i, turjaman m. 2010. kajian kimia gaharu hasil inokulasi fusarium sp pada hutan vol. vii no. 2 : 175-188. pojanagaroon s, kaewrak. 2002. mechanical methods to stimulate aloewood formation in (kritsana) trees [abstract]. acta horticul 676:161-166 qi shu-yuan. 1995. spesies: culture and production of eaglewood (agarwood). in: bajaj yps, editor. , : springer. p 36-46. rahayu g, santoso e, wulandari e. 2009. efektifitas dan interaksi antara sp dan sp dalam pembentukan gubal gaharu pada . workshop pengembangan teknologi produksi gaharu berbasis pada pemberdayaan masyarakat di sekitar hutan. bogor. rao, k.r., dayal, r. and ramesh-rao, k 1992. the secondary xylem of (thymeleaceae) and the formation of agar. iawa bull. 13(2): 163-172 richter hg. 1990. wood and bark anatomy of lauraceae iii. aspidostemon rohwer & richter. iawa bull. (n.s.). 11. 47-56. wong, t.m. 1977. wood structure of the lesser known timbers of peninsular malaysia. malayan forest record no. 28. forest research institute. kepong. yuan qs 1995. species: culture and the production of eaglewood (agarwood) dalam bajaj yps editor. biotecnology in agriculture and forestry 33: medicinal and aromatic plants viii. berlin. springer. p. 36-46 yagura t, shibayama n, ito m, kiuchi f, honda g. 2005. three novel diepoxy tetrahydrochromones from agarwood artificially produced by intentional wounding. 46 : 4395-4. new phytol aquilaria malaccensis. . phytochemistry . aquilaria crassna . bulletin of the kyoto university forest aquilaria microcarpa. info aquilaria crassna . aquilaria in vitro biotechnology in agriculture and forestry 33 medicinal and aromaticplant viii acremonium fusarium aquilaria microcarpa aquilaria agalocha . aquilaria in vitro . tetrahedron letters biological remediation of cyanide: a review karamba kabiru ibrahim, mohd arif syed, mohd yunus shukor and siti aqlima ahmad* faculty of biotechnology and biomolecular sciences, universiti putra malaysia, 43400 upm serdang, selangor, malaysia received 8 april 2014/accepted 9 november 2015 abstract cyanide and its complexes are produced by industries all over the world as waste or effluents. biodegradation is considered to be the cheapest and the most effective method to clean-up cyanide from the environment. several studies on different types of microorganisms that can degrade cyanide in the environment have been carried out. hydrolytic, oxidative, reductive and substitutive/transfer reactions are some of the common pathways used by microorganisms in cyanide degradation. biodegradation of cyanide can occur aerobically or anaerobically depending on the environmental conditions. immobilized enzymes or microorganisms prove to be very effective method of degradation. microorganisms such as , , , klebsiella oxytoca corynebacterium nitrophilous brevibacterium nitrophilous bacillus spp., spp. and ukmp-5m have been reported to be very effective in biodegradation of pseudomonas rhodococcus cyanide. : keywords biodegradation, cyanide, environment, microorganisms introduction carbon and nitrogen elements form cyanide. they are ubiquitously found in the environment. amygdalin, which is found in vegetables, fruits, seeds, cashew nuts, cherries, apricots and bean sprouts is a natural source of hydrogen cyanide (hcn) (mark . 1999). chemical treatment is et al the most widely used method in the degradation of industrial effluents containing cyanide. but, this treatment is very expensive and sometimes inefficient in the process and thus requires an alternative means of treatment (patil & paknikar 2000). researches on microorganisms have been carried out to see if they can be an alternative means of cyanide degradation. bioremoval has been reported to be less expensive than physical and chemical methods of cyanide degradation and faster than natural oxidation (ozel . 2010; et al dash . 2009). destruction of cyanide in et al wastewaters and tailing solutions by capable microorganisms has been proved to be an alternative to the long-practiced chemical methods for the removal of cyanide. biological methods of treating industrial effluents have been reported to have high capital cost but low operating cost. therefore, these methods are more profitable than the traditional process. biological process that could satiate the need for extraction and environmental control is now practiced in some countries that understood the process (dash . 2009).et al cyanide degrading microorganisms biological treatment process facilitates growth of microorganisms that are essential for treatment (akcil 2003). it has been discovered that cyanide naturally occurs in the environment via the degradation of plant cyanogenic glycosides. a lot of microorganisms are able to detoxify simple forms of cyanide (gadd 2001). pseudomonas fluorescens ncimb 11764 has been reported to be able to utilize potassium cyanide (kcn) in fed-batch culture (kunz . 1998). et al pseudomonas fluorescence has also been reported to degrade ferrocyanide (arzu & zumriye 2000). the growth of ncimb pseudomonas fluorescens 11764 on medium containing potassium cyanide (kcn) has also been reported (kunz . 1998). et al cyanide degradation by , alcaligenes, escherichia coli * corresponding author : aqlima@upm.edu.my biotropia vol. 22 no. 2, 2015: 151 163 151 doi:10.11598/btb.2015.22.2.393 mailto:aqlima@upm.edu.my 152 acinetobacter bacillus and species have also been reported in a number of studies. it has been reported that strains of bacteria from genus are very effective in the klebsiella bioremediation of cyanide and thiocyanate. k. oxytoca isolated from an industrial waste containing high level of cyanide proliferates well with the cyanide as the only nitrogen source (kao et al k. oxytoca. 2003). it has been reported that is effective in biodegradation even at concentrations higher than 1 mm of cyanide (chena . 1999). et al the microorganism biodegrades cyanide to products that are nontoxic using cyanide as the only nitrogen source aerobically or anaerobically (kao . 2003; stephen 2004). seven bacterial et al strains isolated from gold mine environment in korea have been found to be very effective in the biodegradation of thiocyanate with the initial concentration of 150 mg/l within sixteen days of incubation (lee . 2003). four strains from the et al genus , one strain of bacillus corynebacterium nitrophilus brevibacterium and two from have been found to effectively degrade cyanide. pseudomonas pseudoalcaligenes cect5344 is an alkaliphilic strain of bacteria that is reported to be capable of biodegrading cyanide. the organism was isolated from sludge of guadalquivir (cordoba, spain). it proliferates at an optimum ph of 9.5 and utilizes 2 mm of cyanide as the only hydrogen source (luque-almagro . 200 ; et al 5 huertas . 2010). the organism can also utilize et al complex of cyano-metal, cyanate and residue from jewellery industry as the sole nitrogen source (luque-almagro . 200 ). this strain has been et al 5 described to possess a cyanide-insensitive respiration system, which includes cytochrome bdtype alternate oxidase (aox) that replaces the cytochrome c oxidase (quesada . 2007)et al . general pathway reaction for biodegradation of free cyanide there are four common pathways involved in the bioremoval of cyanide i.e. hydrolytic reaction, oxidative reaction, reductive reaction and substitution/transfer reaction. more than one pathway can be used for cyanide degradation by certain microorganisms (raybuck 1992; ezzimufaddal & lynch 2005). the pathway to be used depends on various factors such as oxygen availability, ph level of the environment, concentrations of the cyanide and cyanide bioavailability and solubility in the soil water system (aronstein . 1994).et al hydrolytic reaction the following equations show the reactions that occur during hydrolytic pathway for cyanide degradation (david . 2006):et al hydrolytic reactions contain nitriles with r denotes either analiphatic or aromatic group. hydrolytic reactions are catalyzed by cyanide hydratase, founding a formamide or cyanidase and yields formate and ammonia. cyanide hydratase is principally a fungal enzyme and is extremely preserved between species (barclay et al. 2002). cyanide dihydratase (cyanidase) is produced chiefly by bacteria. cyanide hydratase and cyanidase have, in recent times, been presented to have certain resemblances at both the amino acid and structural stages to nitrilase and nitrile hydratase enzymes (reilly & turner 2003). enzymes that utilize nitrile have been established in a wide variety of fungal, plant and bacterial species. nitrilases and nitrile hydratases modify both aliphatic and aromatic nitriles to the equivalent acid or amide, respectively, but indicate less substrate specificity than cyanide hydratase and cyanide dihydratase. for instance, conversion of with the cyanide hydratase gene from e. coli fusarium lateritium permits the proliferation of nitriles as the only source of nitrogen. sitedirected mutagenesis of this gene stops the activity of both cyanide hydratase and nitrilase, signifying that cyanide hydratase possesses nitrilase activity, as well (nolan . 2003). the et al variety of enzymes in this fantastic family and their diverse catalytic act and substrate specificities grant significant chance for biotechnological improvement, encompassing the bioremediation of industrial nitrile waste (rezende . 2000; dias . 2001).et al et al oxidative reaction in the oxidative reaction, the cyanate formed by the enzyme cyanide monoxygenase is changed to ammonia and carbon dioxide by the same pathway as cyanate and thiocyanate (david . et al 2006). biotropia vol. 22 no. 2, 2015 substitution/transfer reaction this is referred to as assimilatory pathway. several genera of bacteria are said to assimilate cyanide. this reaction is more extensively researched in . other chromobacterium violaceum bacteria that utilize this reaction include escherichia coli, bacillus megaterium, citrobacter freundii and (david . 2006). the enterobacter aerogenes et al substitution/transfer reaction uses cyanoalanine synthase enzyme as the catalyst using oacetylserine (oas) as substrate. the cyana te produced by cyanide monoxygenase is changed to nh and co by 4 2 + very similar pathway as the cyanate from thiocyanate (stephen 2004). thiocyanate biodegradation reactions of cyanide with pyretic materials in effluents lead to the production of thiocyanate. the enzyme that is responsible for the production of thiocyanate is sulfurtransferase via the in vivo action of thiosulfate-cyanide. its biodegradation can be achieved by at least two pathways namely, cyanate and carbonyl pathways (david . 2006; et al kwon . 2002; plessis . 2001; sorokin . et al et al et al 2001; yamasaki . 2002).et al three species of thiocyanate-degrading bacteria obtained from very high alkaline soda lagoon soils and sediments produce high amounts of cyanate when grown at ph 10 with thiocyanate as the only source of nitrogen. the activity of cyanase that converts cyanate to carbon dioxide and ammonia is also high (sorokin . 2001). et al the fungus produces sulphate acremonium strictum and ammonia from thiocyanate devoid of the production of cyanate (kwon . 2002). in the et al cyanide is converted to cyanate by cyanide monoxygenase with cyanate standing as the catalyst for conversion of cyanate to ammonia and carbon dioxide dependent on bicarbonate. cyanases have been identified in several bacteria, fungi, plants and animals (guilloton . 2002).et al the assumed task of cyanase has, for a long time, been as a defense against poisoning by cyanate (raybuck 1992). as cyanate is not a familiar metabolite, new essential roles in favor of cyanases in nitrogen and bicarbonate/carbon dioxide metabolism have been suggested. more proposed roles for plant cyanases comprise ammonia absorption as a result of cyanate bioremediation and a task in the concentration and deliverance of carbon dioxide for photosynthesis (guilloton . et al 2002). another oxidative pathway makes use of cyanide dioxygenase to produce ammonia and carbon dioxide directly (stephen 2004). moreover, in strain bcn6 and ncimb e. coli p. fluorescens 11764, the production of cyanohydrin complexes is reportedly essential for oxygenase-mediated cyanide biodegradation (kunz . 1998; figueira et al et al. 1996). reductive reaction the reductive pathway results from the act of nitrogenase enzyme and the products ensuing from transfer of pair of electrons (stephen 2004; david . 2006).et al studies have been carried out in two bioreactors in cassava wastewaters and synthetic wastewater. this indicates a clear reductive process on biodegradation (paixao . 2000; annachhatre & et al amornkaew 2000). the studies indicate that the anaerobic bioreactors prop up the proliferation of methanogenes which can result in the production of biogas. the increase in the cyanide concentration inhibits methanogenesis from anaerobic biogranules. this effect could be a result of high cyanide concentration in the feed stock (annachhatre & amornkaew 2000). biological remediation of cyanide: a review ibrahim – et al. 153 carbonyl pathway, the thiocyanate is transformed to ammonia and carbonyl sulphide. the confirmation for this pathway is as a result of the identification of thiocyanate hydrolase that is responsible for the transformation in the chemolithotroph and the thiobacillus thioparus genes encoding this enzyme have been acknowledged in other thiocyanate-degrading bacterial cultures (yamasaki . 2002)et al . aerobic biodegradation of cyanide the process of biodegradation that requires the use of oxygen is termed as aerobic system of biodegradation. under aerobic conditions, cyanide is broken down by cyanide-oxidizing bacteria into harmless compound. the process degrades hydrogen cyanide and produces hydrogen cyanate, which then undergoes hydrolysis to form ammonia and carbon dioxide as shown in the equation below: reports have indicated that aerobic process of biodegradation is many times faster and better than anaerobic degradation (stephen 2004). algae such as ,arthrospira maxima scenedesmus obliquus, and spp. have also been reported in the chlorella detoxification of cyanide (dwivedi . 2011; et al gurbuz . 2009).et al anaerobic biodegradation of cyanide anaerobic system of biodegradation is the process that occurs in the absence of oxygen (dwivedi . 2011) fedorak and hurudey in et al . 1989 was the first to report biotreatment of cyanide anaerobically using semi continuous batch cultures and hydraulic retention time (hrt) of 25 days for one liquid volume replacement (fallon . 1991). anaerobic biodegrada-et al tion can only occur in the presence of hs or h s 2 2 and is restricted to reduce portion of the heap environment. the sulphur species that is present depends on the ph. at ph ≥ 7, hs is the species 2 that is dominant and at ph ≤ 7 although h s is the 2 more dominant species of sulphur in other phs (dwivedi . 2011).et al the hcns will undergo hydrolyses to produce nh , h s and co .2 2 2 anaerobic cyanide biodegradation has been proven to be a concomitant method of biogas generation that is of economic benefit (stephen 2004). under anaerobic condition, biodegradation results in the formation of nitrogen as the end product. furthermore, anaerobic treatment is not fast and it is more vulnerable to toxic upsets ensuing from exposure to other elements present in the solution undergoing the treatment. in addition, cyanide toxicity threshold for anaerobic bacteria is only 2 mg/l, while aerobic bacteria have 200 mg/l threshold. therefore, anaerobic bioremoval is considered to be less effective method of bioremoval mechanism (stephen 2004). effect of immobilization on cyanide biodegradation immobilization has been reported to be a very effective and efficient tool of biodegradation. this tool offers many economic and technical advantages over the free cell type as it offers the possibility of maintaining the cells in a stable and viable condition with high specific surface area for microbial proliferation (dursun & aksu 2002). immobilized cells are less vulnerable to compounds that are toxic and exhibit high tolerance towards distress in the reaction environment (chen . 2008) moreover, et al . immobilized cells are more advantageous when compared with the use of free cells because of the ability to support higher concentration of cell density and eliminate the difficult, timeconsuming and expensive process of cell recovery and recycling (zhou . 2007). et al bioremediation by cct candida guilliermondii 7207 was very effective on immobilized cells (dias . 2001). this is a remark that reiterates et al previous researches indicating that immobilized microorganisms or enzymes make available an active stage for bioremediation (stephen 2004). effective biodegradation of cyanide compounds by immobilized on zeolite has been p. fluorescens reported (suh . 1994). m. graca campos in et al 2005 reported effective, feasible and efficient detoxification of cyanide by immobilized f u s a r i u m o x y s p o r u m c c m i 8 7 6 a n d methylobacterium sp. rxm ccmi 908 using a packed bed reactor for integrated biodegradation biotropia vol. 22 no. 2, 2015 154 155 of cyanide (campos . 2006) maegala et al et al.. (2012) reported effective biodegradation of cyanide by immobilized cells of rhodococcus ukmp. oxygenation in immobilized cell culture oxygenation is a major challenge in immobilization. researchers have reported critical challenges in supplying sufficient and adequate concentration of oxygen in order to keep high amount of viable and productive cells throughout a culture period (meuwly . 2005). in packed et al bed reactors it was observed that if there is low supply of oxygen, there will be decrease in viability of culture, metabolic activity and productivity until optimal supply of oxygen is achieved. this is because oxygen has a very poor solubility in cell culture medium (fassnacht & portner 1999). factors affecting cyanide biodegradation in environment the presence of microorganisms that have the physical and metabolic abilities to degrade the contaminants in the polluted environment ensures the success of biodegradation. cyanide compounds are found broadly in natural surroundings and the metabolic degradation of these compounds by microorganisms is thus possible. however, the following factors affect the process (kao . 2006; baxter & cummings et al 2006; dash . 2009):et al  cyanide concentration in the environment can have significant effect in the treatment. for instance, high concentration of acetonitrile has been proved toxic to by causing klebsiella oxytoca damage to nitrile hydratase, which is the nitriledegrading enzyme and by preventing bioremoval of the compound by the microorganism.  biodegradation of cyanide compounds can be affected by the availability of nutrients. carbon has been recognized as the restrictive factor in the biodegradation of cyanide compounds, which may make the biodegradation of industrially polluted soils not feasible.  aerat ion is ver y important in the biodegradation of cyanide as oxygen is required during the degradation pathways.  cyanide toxicity can be paramount to anaerobic bacteria principally methanogens. other contaminants present at the polluted areas may also affect bioremoval.  existence of high concentration of other pollutants can have negative effect on degradation of cyanide by swaying the native population and possibly hindering the proliferation of specific organism. biodegradation of free/complex cyanide and thiocyanate in the biotreatment of cyanide, bacteria have the capacity to change free and metal cyanides to bicarbonate and ammonia, whereas the free metals are adsorbed within the biofilm or unconfined as precipitates in solution. alkali metal cyanides such as potassium cyanide (kcn) and sodium cyanide (nacn ) are easily being 2 degraded by different forms of bacteria across the globe. in 1969 in usa, free cells of bacillus megaterium was reported to degrade potassium cyanide (castric & strobel 1969). in the same year in canada, free cells was reported bacillus pumilus to degrade 2.5 mg/l of potassium cyanide at the ph of 8.5 9.0 and temperature of 40 – °c (skowronski & strobel 1969). in 1972, free cells of which is a pathogenic fungus stemphylium loti, of the cyanogenic plant 'bird's foot-trefoil' ( l.) (fry & mills 1972) was lotus corniculatus reported to degrade 0.97 m of potassium cyanide at ph of 6.5 7.5 and temperature of 25 with – °c the removal efficiency of 77 nm (fry & mills 1972). several reports have been reviewed across the world up to the year 2012. in malaysia rhodococcus ukmp-5m in both free and immobilized forms was reported to degrade potassium cyanide at 30 temperature °c condition with the removal efficiency of 64 and 96%, respectively (maegala . 2011; maegala et al et al. 2012). this removal capacity is not only limited to bacteria but other fungal organisms such as fusarium solani, which in 1997 its free cells were reported to degrade potassium cyanide concentration of 0.5 0.8 mm at the ph of – 9.2 10.7 and temperature condition of 30 in – °c france (dumestre . 1997). in 1975, free cells et al of nca 1503 was reported to b. stearothermophilus degrade nacn and nahso with initial 2 3 concentration of 5 mm and 50 mm at a ph of 7.8 and temperature of 27±2 with the removal °c efficiency of 5 8 g/l/hour (atkinson 1975). the – simplicity within which the complexes of metal biological remediation of cyanide: a review ibrahim – et al. 156 biotropia vol. 22 no. 2, 2015 cyanide are being degraded commonly follows the sort of general stability, with free cyanides being the readily degradable while cyanide of iron being the least degradable. zinc (zn), nickel (ni) and copper (cu) are moderate in terms of degradability of their metal cyanides (young & jordan 1995; botz 2001). a study conducted in korea in 1994 reveals that p. fluorescens immobilized on zeolite was able to degrade tetracyano nickelate ii at 30 (suh . 1994). °c et al studies in turkey in 1999 showed that immobilized was able to degrade p. fluorescens ferrous (ii) cyanide complex (dursun . 1999) et al while free cells of was able to degrade p. fluorescens 100 mg/l ferrous (ii) cyanide complex at ph 5 and temperature of 25 at degradation rate °c of 30 mg/g/hour (dursun . 1999). free cells et al of spp. and spp. were citrobacter pseudomonas reported in india to degrade 52 mg/l metal cyanide complexes such as copper cyanide and zinc cyanide with the removal efficiency of > 99.9% (patil & panikar 2000). in the united kingdom in 2005, free cells of sp.trichoderma and sp. were reported to degrade 2,000 fusarium mg/l metallo cyanide at ph 6.5 and temperature of 25 , with the removal efficiency of 2,000 °c ppm (ezzi-mufaddal & lynch 2002). degradation of complex cyanide is not limited to only bacteria because in the united kingdom in 1998, mix cultures of ,fusarium solani, t. polysporum f. oxysporum scytalidium thermophilum , and penicillium miczynski were reported to degrade k ni(cn) and k fe(cn) at a concentration 2 4 4 6 of 0.75 mm and 0.25 1 mm concentrations and – ph 4.5 7.0 and temperature conditions of 25 – °c with the removal efficiency of 50 56%, – respectively (barclay . 1998). in 2001 in south et al africa, some free cells of microorganisms were reported to degrade thiocyanate at the concentration of 500 mg/l with the removal efficiency of 0.5 mg/l/h (sorokin . 2001). in et al the same year in russia, free cells of alkaliphilic bacteria were reported to degrade 40 mm initial concentration of thiocyanate at a ph 10 and temperature of 28 with removal efficiency 4 °c mm (plessis . 2001). in 2002 in korea, et al acremonium strictum was reported to degrade 7.4 g/l concentration of thiocyanate at a ph 6 with 100% removal efficiency in 85 hours (kwon . et al 2002). in 2002 in tokyo, japan, free cells of thiobacillus thioparus thi115 was reported to degrade thiocyanate with initial concentration of 0.1 g/l at a temperature of 30 (yamasaki . °c et al 2002). there is no doubt from this review that pseudomonas sp. has an upper hand in the degradation of both free and complex cyanides in quite a number of countries across the globe. table 1 summarizes the degradation potentials of some microorganisms on free and complex cyanides and thiocyanate. 157 biological remediation of cyanide: a review ibrahim – et al. biotropia vol. 22 no. 2, 2015 158 159 biological remediation of cyanide: a review ibrahim – et al. biotropia vol. 22 no. 2, 2015 160 conclusions certain economic and physical factors chiefly limit the use of biotechnologies in the degradation of cyanide. biodegradation is potentially the cheapest means to get rid of cyanide but factors such as ph, temperature and nutrients concentration of the cyanide affect the process. bioremoval can be achieved under aerobic and anaerobic conditions. free and immobilized cells have proven to be very effective and efficient methods of biodegradation. the microbes potentially have certain enzymes that can change cyanide into naturally occurring compounds. four types of pathways are used by the microorganisms to biodegrade and one or combination of two pathways can be employed in the process by microorganisms (raybuck 1992; ezzi-mufaddal & lynch 2002). spp. shows great pseudomonas capability 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pseudomonas a c i d o v a r a n s influence of ph and phenol. indian j environ health 27: 1–8. sirianuntapiboon s, chuamkaew c. 2007. packed cage rotating biological contactor system for treatment of cyanide waste water. bioresource technol 98: 266–72. skowronski b, strobel ga. 1969. cyanide resistance and cyanide utilisation by a strain of . bacillus pumilus can j microbiol 15: 93–8. sorokin dy, tourova tp, lysenko am, kuenen jg. 2001. microbial thiocyanate utilisation under highly alkaline conditions. appl biochem microbiol 67: 528–38. stephen e. 2004. biological degradation of cyanide compounds. curr opin biotechnol 15: 231–6. suh y, park jm, yang j. 1994. biodegradation of cyanide compounds by immobilised pseudomonas fluorescens on zeolite. enzyme microb tech 16: 529–33. white dm, schnabel w. 1998. treatment of cyanide waste in a sequencing batch biofilm reactor. water res 32: 254–7. yamasaki m, matsushita y, namura m, nyunoya h, katayama y. 2002. genetic and immunochemical characterization of thiocyanate-degrading bacteria in lake water, appl biochem microbiol 68: 942–6. young ca, jordan ts. 1995. cyanide remediation: current and past technologies, in proceedings of the 10 th annual conference on hazardous waste resources. p. 104– 29. zhou x, liu l, chen y, xu s, chen j. 2007. efficient biodegradation of cyanide and ferrocyanide by na-alginate beads immobilised with fungal cells of . can j microbiol 53: 1033–7trichoderma koninji . 163 biological remediation of cyanide: a review ibrahim – et al. 4. decky (exotic).cdr biotropia vol. 21 no. 1, 2014: 38 52 exotic plants in halimun salak corridor:micro-environment, detection and risk analysis of invasive plants decky indrawan junaedi , dodo received 18 june 2013/accepted 2 june 2014 research of exotic plant species detection and risk analysis of invasive plant was conducted in halimun-salak corridor area. this study aimed to conduct inventory of exotic plant species in this area and to perform risk analysis of invasive to the exotic plants found. the invasion risk assessment of detected exotic plants was analyzed using weed risk assessment procedure (wrap) method. moreover, analysis of multi-dimensional scaling (mds) based on inequality was performed on relative humidity, light intensity, and soil ph. there are eleven exotic plant species consisted of three tree species and eight species of herbs/shrubs. and are the exotic species with the highest and the lowest wrap score, respectively. mds analysis shows that exotic tree species have similar environmental variables. moreover, environmental variables of are relatively different from other exotic species found in the halimun-salak corridor. recommendations for the management of invasive exotic plant species in the area are: immediate management implementation, priority of eradication to exotic plants that have not been abundant but have high risk score, two management options (gradual eradication or containment) should be considered for exotic plants with very high wrap score, such as and . plant ecology, halimun-salak corridor, multi-dimensional scaling, weed risk assessment, invasive plant, conservation 1 2 1 2 cibodas botanical garden, indonesian institute of sciences (lipi), po. box 19 sdl sindanglaya, cipanas, cianjur 43253, centre for plant conservation, bogor botanical garden, indonesian institute of sciences (lipi) austroeupatorium inulifolium camellia sinensis clidemia hirta ageratina riparia, austroeupatorium inulifolium chromolaena odorata abstract key words: * corresponding author : deqee82@gmail.com doi: 10.11598/btb.2014.21.1.4 38 introduction halimun-salak corridor ecosystem is important because it is not only a mountain forest ecosystem, but also acts as a connecting corridor between mount salak and mount halimun. moreover, halimun-salak corridor has several risk factors associated with the threat to ecosystem integrity. along with habitat degradation, fragmentation and disturbance from human activities, invasive alien species (ias) (including invasive alien plants) being one of the factors that poses a threat to the integrity of the halimun-salak corridor ecosystem (jica 2005). within an 11 years period (19902001), the width of the halimun-salak corridor has decreased from 1.4 km (1990) to 0.7 km (2001) (cahyadi 2003). on the other hand, there are lack of studies on invasive species in south east asia, where the biodiversity hotspot lies in (peh 2010). considering invasive species issues, detection and risk analysis of invasive plants are needed in the management of invasive plants. upon the detection, alien species which potentially become invasive could be detected and managed earlier so that management cost becomes cheaper. by doing risk assessment, existed exotic plants, either invasive or not invasive can be estimated and ranked due to its invasiveness rates or invasiveness possibilities. therefore, management prioritization of exotic invasive plants will be accomplished easier with risk assessment. risk assessment reflects the chances of an exotic species to become invasive in the future or reflects the rate of invasiveness itself. weed risk assessment procedure (wrap) (virtue 2008) is a risk assessment system used to assess the risk of invasive plant species from an exotic species that are already existed in a new host area. the objective of this study was to conduct inventory of exotic plants (either invasive or not invasive (yet)) that are already existed in the halimun-salak corridor. this study also aimed to look at the risk of exotic plants to become invasive in the halimun-salak corridor. in addition, this study also aimed to analyze the diversity of habitat environment (micro-environment) variables (ph, light intensity and air humidity) of areas where exotic species were detected. halimun-salak corridor is the area that connects mount salak and mount halimun. halimun-salak corridor included as a part of halimun-salak national park based on minister of forestry decree no. 175/kpts-ii/2003 dated june 10, 2003. northern part of the corridor is part of administrative region of bogor covering area as large as 1662.72 hectares. southern part of the corridor is included in the district of sukabumi which covered 2533 hectares. the altitude range of halimun-salak corridor is from 1000 m above sea level to 1400 m above sea level. annual rainfall in the halimun-salak corridor ranges from 4000 mm to 5000 mm per year (jica 2005). et al. materials and methods study site 39 exotic plants in halimun salak corridor decky indrawan junaedi– et al. exotic plants detection exotic species inventory was conducted at 27 observation points in 7 different locations in the halimun-salak corridor. sampling areas covered by the detection sampling are: bivak, legok buluh, cigorowek-3, palahlar, pasir panjang, pasir bedil and kubang (table 1). tabel 1. detection sampling location in halimun-salak corridor location coordinates altitude (m asl) sampling number ar/fi s e bivak 06044'59.8" 106036'47.9" 1100 6 ar and fi legok buluh 06044'53.0" 106037'07.0" 1068 6 ar and fi cigorowek-3 06044'42.1" 106038'01.3" 1015 4 ar and fi palahlar 06044'53.1" 106037'22.6" 1021 2 ar pasir panjang 06045'11.2" 106037'12.6" 1061 3 ar and fi pasir bedil 06045'06.9" 106036'54.6" 1069 2 ar kubang 06045'17.6" 106037'52.2" 1042 4 ar and fi notes: ar= adjacent to footpath, fi= forest interior detection samplings were conducted in two types of locations. the first location type is the area adjacent to forest access road into the woods with the assumption that the exotic species introduction pathway is via humans (carried away to the forest by humans). the second location type is the area inside the forest interior with the assumption that in addition to human pathways, exotic species can also be dispersed by the help of animals, wind and other vectors. at each sampling point, exploratory observation of the exotic plant species was conducted within a radius of 3 meters from the point of observation. exotic plant species were observed and recorded. invasiveness risk assessment exotic plants that have been detected were then assessed by using australia weed risk assessment procedure (wrap) (virtue 2008) to assess the risk of invasiveness. wrap is a framework used to analyze the risk of an invasive exotic plant that are already existed in an invaded area. wrap consists of questions that should be answered and then scoring was performed based on the answers. the scoring decision was conducted fully based on available scientific information provided from online global invasive plant database such as pacific island ecosystem at risk (pier) and invasive species specialist group (issg), iucn database. total accumulated value of all wrap scoring answer was expressed as a risk score. the risk scores are quantitative and can be compared between one species and another in the analysis. wrap used in this study were adapted and modified to match the conditions and circumstances in et al. biotropia vol. 21 no. 1, 2014 40 the study locations as provided in annex 1. accuracy of the risk assessment results is measured using logistic regression analysis to generate the roc curve (receiver operating characteristic, using mysystat 13) that describes how accurate risk analysis model used (quinn & keough 2002). logistic regression follows the equation: e / (1 + e )...........................................................................................................1) whe p score) ants obtained (real data) was defined as the relative frequency (fr) more than 10%. on every detection sampling plot, ecological (micro-environment) data were recorded for analysis of ecological habitats. habitat ecological data recorded include: air relative humidity, light intensity, and soil ph. analysis of multi-dimensional scaling (mds) was based on inequality using xlstat software (microsoft excel add-in) and was conducted based n micro-environment data. mds analysis was used to examine the degree of differences among detected exotic species based on its microenvironment/ micro-habitat variables measured. based on the sampling detection of exotic plants in 27 locations of the study site, species area curve is presented in figure 1. the curve indicates the relation between the numbers of sampling plots which was taken with the addition of new exotic species detected. π (x) = re: g (x) = β0 + β1 (wra and π (x) is the opportunity of exotic species (i) will become invasive. definition of invasive exotic pl g (x) g (x) i micro-environment results and discussion detected exotic plants in the halimun-salak corridor based on modified wrap scoring results are presented in table 2. figure 1. species-sampling relation curve that describes the relation between the number of sampling (x axis) and new exotic species counted (y axis) 41 exotic plants in halimun salak corridor decky indrawan junaedi– et al. t ab le 2. d et ec te d ex o ti c p la n t s p ec ie s in h al im u n -s al ak co rr id o r, o cc u rr en ce fr eq u en cy in al l t o ta l s am p lin gs an d ri sk as se ss m en t s co re b as ed o n m o d if ie d w ee d r is k n o sp ec ie s n am e a u th o r o cc u re n ce f re q u en cy f k f r r el at iv e o cc u r re n ce f re q u en cy (% ) in va si ve (i ) / n o t (n ) m o d if ie d w r a p sc o re 1 a ge ra ti na ri pa ri a (r eg el ) 1 0 .0 4 0 .0 1 1 .2 8 n 23 2 a us tr oe up at or iu m in ul ifo liu m (k u n th ) r .m .k in g & h .r o b. 19 0 .7 0 .2 1 26 .9 2 i 24 3 b el lu ci a pe nt am er a n au d in 11 0 .4 1 0 .1 2 15 .3 8 i 18 4 c al lia nd ra ca lo th yr su s m ei sn . 9 0 .3 3 0 .1 12 .8 2 i 16 5 c am el lia si ne ns is (l .) k u n tz e 1 0 .0 4 0 .0 1 1 .2 8 n 9 6 c hr om ol ae na od or at a (l .) r .m .k in g & h .r o b. 9 0 .3 3 0 .1 12 .8 2 i 19 7 c in ch on a pu be sc en s v ah l 1 0 .0 4 0 .0 1 1 .2 8 n 16 8 c lid em ia hi r t a (l .) d .d o n 8 0 .3 0 .0 9 11 .5 4 i 17 9 l an ta na ca m ar a l . 2 0 .0 7 0 .0 2 2 .5 6 n 16 10 m ae so ps is em in ii e n gl . 4 0 .1 5 0 .0 4 5 .1 3 n 14 11 s pe rm ac oc e al at a a u b l. 6 0 .2 2 0 .0 7 8 .9 7 n 16 42 biotropia vol. 21 no. 1, 2014 roc curves that illustrate the accuracy of the risk analysis model are presented in figure 2. the value of area under the roc curve is 0.817.this value showed that the accuracy of the logistic regression model is approximately 81.7% figure 2. roc ( ) curve that determines the accuracy of the logistic regression conducted in the risk assessment receiver operating characteristic figure 3. mds configuration of three environment variables of detected exotic plants: soil ph, light intensity and air humidity. dimension 2 was used in the configuration. detected exotic species are, ai: , bp: , cc: , co: , ch: , lc: , me: and sa: austroeupatorium inulifolium bellucia pentamera calliandra calothyrsus chromolaena odorata clidemia hirta lantana camara maesopsis eminii spermacoce alata 43 exotic plants in halimun salak corridor decky indrawan junaedi– et al. mds analysis results based on three environmental variables (ph, light intensity and air humidity) are presented in figure 3. kruskal stress value of 0.002 indicates that the model configuration presented in figure 3 is valid. the valid kruskal stress value is smaller than 0.1 (quinn & keough 2002). shepard diagram of mds analysis is presented in figure 4. shepard diagram indicates the reliability of the data used in the mds analysis. based on the detection results, from totally 11 exotic species detected, four of them are tree species: , , and . the rest of other seven species are herbaceous/shrubs. based on detection sampling data, two exotic tree species having fr value above 10% are and . is native to south america (backer & backhuizen 1963). this plant is likely to spread rapidly in the halimunsalak corridor because of rapid spread through the animal's assistance that eats the fruit. exotic species can become invasive when it got past the “barrier” that exists in the invaded locations (theoharides & dukes 2007). in general, exotic plants have become invasive when it is said to have had offspring and form new colonies that are relatively numerous and have an adverse impact to invaded ecosystems (richardson 2000). however, not all exotic species detected are able to reach interior part of the forest where the forest canopy cover is still relatively intact. most of the exotic plants of the asteraceae found in the exterior forest areas or in the forest gaps (forest openings). mds analysis showed that two asteraceae species ( and ) have different positions relative to the other exotic species due to different types that formed on the configuration. the different position in the mds maesopsis eminii cinchona pubescens calliandra calothyrsus bellucia pentamera bellucia pentamera calliandra calothyrsus bellucia pentamera et al. c. odorata a. inulifolium figure 4. shepard diagram of mds analysis of three environment variables: soil ph, light intensity and air humidity 44 biotropia vol. 21 no. 1, 2014 configuration refers to different micro-environment preferences. therefore, and have relatively different micro-environment preferences compared to other exotic plants that are clustered in different quadrants (fig. 3). even though has the highest wrap value and refers to high risk to become invasive, but the environmental constraints (the intensity of light/canopy shade availability) becomes the limiting factor for these two species to become very invasive. however, if there is a gap occurred in the forest area, this species will be able to establish and invade. another interesting point is the grouping of exotic tree species in the same quadrant of the mds analysis result ( and ) (fig. 3). habitat preference (relative humidity, light intensity, and soil ph) were similar among these three exotic tree species in halimun-salak corridor. these three exotic tree species were mostly found in the interior forest, not in the forest edges. indeed, these three exotic tree species have not reached invasive stage. however, after a period of adaptation time (lag time), these species will quickly become invasive and will have an impact on the halimun-salak corridor ecosystem. time lag determines exotic plant species to become invasive (hobbs & humpries 1995). ias management in halimun-salak corridor should be focused on species that are already existed in the forest interior. effective and efficient management can minimize the cost of subsequent management (radosevich 2007). 's micro-environment preference (three variables: soil ph, light intensity and relative humidity) is very different from almost all exotic species recorded. backer and backhuizen (1963) mentioned that naturalized in west java, especially around bogor. in its native range in south america and the caribbean, dominates open areas, forest edges and forest openings/ disturbed forest and is rarely found under the shade-covered forests floor (dewalt ., 2004). but dewalt . (2004) also have indicated that is more shade tolerant in the invasion region than in the native range. included as one of the 100 worst invasive species in the world (issg 2006). and is an interesting case in this study. wrap values of and are relatively high, while the results of the detection sampling populations is relatively low (table 2). there are several factors that may explain the low number of sampling populations of and . first, the number of sampling may be relatively small compared to the pattern of spread of this species in the halimun-salak corridor. with the equal number of sampling units, the representation of the types of populations obtained with the clumped distribution pattern is more adequate than diffuse or random dispersal patterns. secondly, is not (or not yet?) penetrating the ecological constraints that exist in the halimun-salak corridor forest to reach the invasive stage. is still in the process of establishment/early naturalization stage. naturally occurs in open areas and the seed germination is inhibited by low light intensity (gentle & duin 1997, 1998). c. odorata a. inulifolium a. inulifolium maesopsis eminii, calliandra calothyrsus bellucia pentamera clidemia hirta c. hirta c. hirta et al et al c. hirta c. hirta ageratina riparia lantana camara a. riparia l. camara a. riparia l. camara l. camara l. camara l. camara 45 exotic plants in halimun salak corridor decky indrawan junaedi– et al. conclusions acknowledgements references there are 11 species of exotic plants in the halimun-salak corridor area consisted of three woody tree species and eight herbs/shrubs species. is an exotic species that has the largest wrap value (24) and is an exotic species with the lowest wrap value (-4). exotic tree species have similar environmental preferences. is an exotic species that has relatively different environment variables compared to other exotic species found in the halimun-salak corridor. based on the risk assessment of invasiveness and field observation conducted, some recommendations for the management of invasive exotic plant species in the halimun-salak corridor are: (1) management of invasive species can be done with several options, but the more immediate the implementation, the cheaper the management cost and the simpler the management methods will be, (2) eradication can be implemented to exotic plant species that have not been abundant in population but have relatively high wrap value such as and . whenever possible, eradication should start from the individual which exist in the forest interior and continue toward the outside of the forest, (3) gradual eradication or control (containment) are two possible management options for exotic plant species that have a very high wrap score such as , and . future study should be focused on how to conduct ias management implementation that gives the smallest impact to the ecosystem of halimun-salak corridor. it is important to conduct ecological study of exotic species micro-habitat as well as investigating the impact of these exotic species to halimun-salak corridor ecosystem. authors thank soekisman tjitrosoedirdjo for significant recommendations and review on the methodology section of this work. authors also thank sri astutik for valuable discussion during field data collection, megawati for dedicated plant identification and to pak uci and pak atma for their field assistance and guidance. this study funded by grant “program prioritas nasional 9 program penelitian, penguasaan, dan pemanfaatan iptek: pembangunan kebun raya daerah” (national priority program, 9 programs for research, control, and utilization of science and technology on the development of district botanical garden). lantana camara camellia sinensis clidemia hirta bellucia pentamera cinchona pubescens ageratina riparia chromolaena odorata lantana camara backer ca, backhuizen rcvdb. 1963. flora of java (spermatophytes only) volume i. groningen: n.v.p. nordhoff. 46 biotropia vol. 21 no. 1, 2014 cahyadi i. 2003. analisis spasial struktur dan fungsi koridor hutan antara taman nasional gunung halimun dengan hutan lindung gunung salak. bogor: program pascasarjana ipb, tesis. dewalt sj, denslow js, ickes k. 2004. natural-enemy release facilitates habitat expansion of the tropical shrub . ecology 85(2): 471-483. gentle cb, duggin ja. 1997. l. invasions in dry rainforest open forest ecotones: the role of disturbances associated with fire and cattle grazing. australian journal of ecology 22: 298-306. gentle cb, duggin ja. 1998. interference of by with nutrient enrichment in mesic forests on the central coast of nsw. plant ecology 136: 205-211. heikkila j. 2011. a review of risk prioritisation schemes of pathogens, pests and weeds: principles and practices. agricultural and food science 20: 15-28. hobbs rj, humphries se. 1995. an integrated approach to the ecology and management of plant invasions. conservation biology 9: 761-770. invasive species specialist group (issg). 2006. global species invasive database: clidemia hirta. accessed 10 january 2013 from jica team, endangered species. 2005 ecological study halimun-salak corridor mount halimun-salak national park. accessed 10 september 2011 from . peh ksh. 2010. invasive species in southeast asia: the knowledge so far. biodiversity and conservation 19: 10831099. quinn gp, keough, mj. 2002. experimental design and data analysis for biologist. cambridge: cambridge university press. richardson dm, pysek p, rejmanek m, barbour mg, panetta fd, west, cj. 2000. naturalization and invasion of alien plants: concepts and definitions. diversity and distributions 6: 93-107. radosevich, sr 2007, plant invasions and their management, chapter 3 in cipm (ed.), invasive plant management: cipm online textbook, bozeman, mt: center for invasive plant management. accessed 2 may 2011 from . theoharides ka, dukes js. 2007. plant invasion across space and time: factors affecting nonindigenous species success during four stages of invasion. new phytologist 176: 256-273. virtue s, spencer, rd, weiss, je, reichard, se. 2008. australia's botanic gardens weed risk assessment procedure. plant protection quarterly 23: 166-178. clidemia hirta lantana camara choricarpia leptopetala lantana camara . http://www.issg.org/database/species/ecology.asp?si=53&fr=1&sts=sss& lang =en. http://sci.kagoshima-u.ac.jp http://www.weedcenter.org/textbook/index.html 47 exotic plants in halimun salak corridor decky indrawan junaedi– et al. annex 1. modified weed risk assessment procedure used in the study (virtue 2008)et al impacts score 1. weed history – what is the weed history of the plant? it is a weed in your region 4 it is a weed elsewhere in indonesia 3 it is a weed overseas 2 it is not known to be weedy, but other forms of the plant and/or plants in the same genus are weeds in indonesia and/or overseas 1 the genus is not known to be weedy 0 2. competition – how well does the plant out-compete other types of plants? if not controlled it can grow to dominate the following three size classes of plants: 4 trees (or emergent aquatics) shrubs (or surface aquatics) ground covers (or submerged aquatics) if not controlled, it can grow to dominate one of the above size classes of plants. or 2 at certain times of the year it can dominate two size classes if not controlled, it can dominate one of the above size classes at certain times of the year 1 it is not competitive, and is readily dominated by most other plants if they are not controlled 0 3. health – is the plant a health risk to people and/or animals? is it highly toxic and has caused deaths 3 it can cause significant physical injuries or illness 2 it can cause slight physical injury or mild illness with no long-lasting effects 1 it is not a health risk to animals or humans 0 4. movement – if the plant escapes does it have the potential to block the movement of people, animals, vehicles or water? a group of plants is very tall, thorny, tangled and/or dense & impenetrable year-long 3 a group of plants is rarely impenetrable, but does significantly undergo slow physical movement year-long 2 a group of plants is never impenetrable but can significantly undergo slow movement for part of the year 1 plant has no significant effect on movement 0 48 biotropia vol. 21 no. 1, 2014 impacts score 5. environmental effects– does the plant have attributes that, at high density, can cause detrimental changes to the environment? the plant has two or more of the following attributes: 2 highly flammable leaves fixes nitrogen salty leaves habitat or food source for pest animal invasion high water use removes habitat or food source for native animals it has one of the above attributes 1 it has none of the above attributes 0 6. ease of control how easy is the plant to kill? hard. it readily tolerates or reshoots after herbicide application, cutting, cultivation, grazing or fire 3 medium. one herbicide application or cultivation kills the plant, but not cutting, grazing or fire 2 simple. plants are killed by hand-pulling, cutting, grazing or fire 1 potential distribution 7. hardiness – how well is the plant adapted to the local climate? in the garden it needs no maintenance to establish, grow and flower, if planted at the right time of year. the localclimate is similar to other places where it grows in the world. or, it is a weed of drier, exposed land in the region or in other parts of indonesia or overseas with similar climate types 3 it needs some maintenance to establish when planted in the garden, but will then grow and flower well without further maintenance. the local climate is harsher in some aspects to what the plant is originally adapted to. or, it is a weed of wetter, sheltered land in the region or in other parts of indonesia or overseas with similar climate types 2 it needs some maintenance to establish when planted in the garden, but can then survive unassisted. however, it grows poorly in the local climate and needs occasional maintenance during stressful periods (e.g. watering in dry months). or, it has naturalised occasionally in the region in specialist habitats (i.e. it has a narrow ecological amplitude) but hasn’t formed high-density infestations. it grows in distinctly different climates elsewhere in indonesia or overseas 1 it needs frequent maintenance and/or special conditions to grow and flower in the garden. or, the local climate isvery different to where the plant grows elsewhere in the world and it has not been recorded as naturalised in the region or in other parts of indonesia or overseas with similar climate types 0 annex 1. continued 49 exotic plants in halimun salak corridor decky indrawan junaedi– et al. annex 1. continued invasiveness 8. reproduction – how well does the plant reproduce? the plant has all three of the following attributes: 4 short time to seeding (within 3 years from planting for herbaceous plants, 5 years for woody plants) ‘volunteer’ seedlings commonly come up in the garden, or as a weed, seedlings are commonly seen nearby mature plants. mature plants produce at least 5 new plants by vegetative means per year it has two of the above attributes 3 it has one of the above attributes 2 it has none of the above attributes, but is able to reproduce by itself 1 it sets no seed in any natural circumstances and has no vegetative spread. the plant can only be propagated with human assistance (eg. shoot cuttings). it will not naturalise. (low weed risk) 9. natural spread – are the plant’s propagules (seed or vegetative) likely to spread long distances to susceptible habitats by natural means? propagules are likely to be dispersed by two or more of the following: 4 flying animals ground animals water wind and susceptible habitats (e.g., farmland, native vegetation, road and rail corridors, waterways) are within 1 km of the gardens propagules are likely to be dispersed by two or more of the above means and susceptible habitats are within 5 km of the gardens, or, propagules are likely to be dispersed by one of the above means and susceptible habitats are within 1 km of the gardens 3 propagules are likely to be dispersed by one of the above means and susceptible habitats are within 5 km of the gardens 2 propagules are not normally spread long distances from parent plants but the botanical garden abuts susceptible habitats 1 propagules are not normally spread long distances from parent plants and the botanical garden is from susceptible habitats by urban developmentbuffered 1 10. human spread – are the plant’s propagules (seed or vegetative) likely to spread long distances due to human activities? the plant is appealing and easily to propagate. visitors to the gardens may intentionally take propagules (e.g., seeds, cuttings, fruit etc.) to grow elsewhere 3 propagules are likely (or known) to be dispersed by two or more of the following means: 2 people (accidentally) vehicles/machinery contaminated produce in soil propagules are likely (or known) to be dispersed by one of the above means 1 propagules are not normally spread long distances from the parent plant 0 score lwr 50 biotropia vol. 21 no. 1, 2014 annex 2. detection sampling data from 27 plots. date plot number species name altitude (m asl) soil ph air relative humidity (%) light intensity (x 2000 lux) 20/06/2012 1 austroeupatorium inulifolium 1110 6 50 322 2 austroeupatorium inulifolium 1103 6.2 55 219 3 austroeupatorium inulifolium 1100 6.8 56 1270 4 austroeupatorium inulifolium 1100 6.8 56 1270 calliandra callothyrsus 5 calliandra callothyrsus austroeupatorium inulifolium 6 bellucia pentamera 21/06/2012 7 austroeupatorium inulifolium 1050 6 43 62 bellucia pentamera 8 spermacoce alata 1068 6.8 52 594 austroeupatorium inulifolium bellucia pentamera 9 austroeupatorium inulifolium 1019 6.5 47 364 calliandra callothyrsus 10 maesopsis eminii 1056 6 51 433 maesopsis eminii 12 austroeupatorium inulifolium 1099 6.8 37 1010 spermacoce alata maesopsis eminii bellucia pentamera austroeupatorium inulifolium 23/06/2012 13 chromolaena odorata 1046 6 49 209 bellucia pentamera 14 bellucia pentamera 1014 6.2 49 219 calliandra callothyrsus 15 calliandra callothyrsus 1041 6 51 697 bellucia pentamera 16 none 1003 6.5 55 267 24/06/2012 17 austroeupatorium inulifolium 1005 6.5 50 3390 chromolaena odorata bellucia pentamera 18 austroeupatorium inulifolium 1049 6 51 1627 lantana camara clidemia hirta 51 exotic plants in halimun salak corridor decky indrawan junaedi– et al. date plot number species name altitude (m asl) soil ph air relative humidity (%) light intensity (x 2000 lux) chromolaena odorata spermacoce alata 25/06/2012 19 lantana camara 1036 6.2 57 90 clidemia hirta spermacoce alata austroeupatorium inulifolium chromolaena odorata 20 cinchona succirubra 1028 6.5 57 8.6 bellucia pentamera 21 austroeupatorium inulifolium 1010 6 58 6.1 chromolaena odorata bellucia pentamera 26/06/2012 22 austroeupatorium inulifolium 1061 6 47 29 calliandra callothyrsus clidemia hirta chromolaena odorata spermacoce alata 23 austroeupatorium inulifolium 1020 6.4 50 1311 clidemia hirta bellucia pentamera 27/06/2012 24 calliandra callothyrsus 1028 6.8 45 1590 austroeupatorium inulifolium camellia sinensis clidemia hirta spermacoce alata chromolaena odorata 25 maesopsis eminii austroeupatorium inulifolium clidemia hirta 26 calliandra callothyrsus 1028 6.8 45 1590 clidemia hirta 27 austroeupatorium inulifolium 1028 6.8 45 1590 calliandra callothyrsus clidemia hirta chromolaena odorata ageratina riparia annex 2. continued 52 biotropia vol. 21 no. 1, 2014 602 phakpoom (optimization and efficien).cdr optimization and efficiency improvement of in house immunoaffinity column ku-af02 for aflatoxin detection phakpoom kooprasertying , thanapoom maneeboon , panrapee 1,5 2 iamtaweejaroen , ratchanee hongprayoon1,5 3 and warapa mahakarnchanakul 4* 1the graduate school, kasetsart university, bangkok 10900, thailand 2 scientific equipment and research division, kasetsart university research and development institute, kasetsart university, bangkok 10900, thailand 3 at kamphaeng saendepartment of plant pathology, faculty of agriculture , kasetsart university, nakhon pathom 73140, thailand 4 department of food science and technology, faculty of agro-industry, kasetsart university, bangkok 10900,thailand 5 center for advanced studies for agriculture and food (casaf), kasetsart university bangkok 10900, thailand, received 4 january 2016/accepted 26 april 2017 abstract in-house immunoaffinity column for aflatoxins (afs) detection is firstly developed by kasetsart university since 2011 i.e. the first iac prototype, ku-af01. detection of aflatoxin b (afb ) up to 200 ng is considered significantly 1 1 efficient, but the production cost is considered high. the high cost was due to the cost of monoclonal antibody specific to aflatoxin and its supporting materials i.e. cnbr-activated sepharose 4b. therefore, this study was aimed to improve the efficiency of in-house immunoaffinity colums by replacing cnbr-activated sepharose 4b with other supporting materials and to lower the ratio of antibody to supporting materials. response surface methodology (rsm) was applied to determine the optimum alternative supporting materials and the ratio. results revealed that when compared with cnbr-activated sepharose 4b, all materials tested could recover higher than 80% afb , except the 1 activated ch sepharose 4b. results also indicated that the optimum ratio was 0.4 mg anti-aflatoxin monoclonal antibody to 204 µl cnbr-activated sepharose 4b. cnbr-activated sepharose 4b was considered to provide the best precision in recovering afb . ku-af02 increased the ability to detect afb to 500 ppb. the recovery of afs in the 1 1 reference materials using ku-af02 was more than 96% successful, with horrat value range of 0.34 – 0.76. the reference materials used were 2 levels of afs in peanuts, i.e. 47.68 and 72.14 ng/g. in terms of quality control in iac production, there were no significant differences among the 5 batches produced (p > 0.05). ku-af02 demonstrated ostable and constant percent recovery at 4 c for up to 12 months (tested with standard 200 ppb afb ). this study 1 indicated that ku-af02 could lower the ratio of supporting materials to the antibody and that ku-af02 has high stability and has good ability for cleaning up afs. therefore, ku-af02 is recommended to be used as an in-house immunoaffinity column for aflatoxin detection in thailand. keywords: aflatoxins, immunoaffinity column, in-house method, mycotoxin detection * corresponding author: fagiwpm@ku.ac.th 114 biotropia 4 2 7 114 126 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 2 602 introduction aflatoxins (afs) are secondary metabolites called difuranocoumarins, produced by aspergillus flavus a. parasiticus and , commonly found in food and feed (calvo 2002). afs are toxic and may et al. have been the most investigated mycotoxin. afs have been associated with aflatoxicosis in livestock, domestic animals and humans worldwide. at present, the maximum limit of afs in food is set at 20 ppb by the united states food and drug administration (us fda) (anukul . et al 2013), whereas only 4 ppb is allowed in food by the european union (european union 2006). countries worldwide have established their respective limit of afs. usually, the contamination of afs has high prevalence, but low concentration at the level of part per billion (ppb). in the last couple of years, several thai exported products were rejected due to afs contamination; for example 40 ppb of afs in rice (chinaphuti & aukkasarakul 2009) and 31 ppb of afs in chili pouch packed together with instant flavoured noodles (rapid alert system for food and feed 2012). a s analysis in food is normally carried out by f extracting food samples with organic solvents such as methanol, followed by afs detection. the food extract solvent, however, may contain afs together with other impurities which consequently will interfere with the results of analyses. to increase the reliability of analysis results, the impurities have to be removed from the food extract solvent prior to analysis, using immunoaffinity column (iac). at present, iac is the common requirement in mycotoxin detection and quantification. unfortunately, in thailand most of iac are imported which resulted to high analysis cost. in 2007, the first prototype of iac developed from polyclonal antibody was conducted at kasetsart university and was succeeded in recovering 20 ppb afb (wongsuttichot 2007). 1 this prototype was initially meant to be used as an in-house iac to reduce analysis cost. later, the polyclonal antibody of this prototype iac was replaced with monoclonal antibody to improve the efficiency, but the production cost was still considered costly (mahakarnchanakul 2011). et al. since antibodies and supporting material contribute as a major part of the production cost, these components have to be optimized to reduce the production cost of iac for cleaning up afs. lower cost production may be encouraged using ku-af02 as in-house iac. it is expected that the increasing success in detecting aflatoxins will result to safer food in thailand. materials and methods materials and chemicals cyanogen bromide-activated sepharose 4b (cnbr-activated sepharose 4b) as supporting material used for antibody immobilization was purchased from ge healthcare (sweden). the apparatus for iac production such as solid phase extraction (spe) empty tube 1 ml, 20 m loose μ frits, spe outlet cap and spe inlet cap (vertical chromatography, thailand) were used for packing the coupled medium. chrome grade acetonitrile and methanol were purchased from mallinckrodt (usa). the monoclonal antibody (mab) specific to afs was kindly provided by department of plant pathology, faculty of agriculture at kamphaeng saen, kasetsart university, thailand. acetic acid, sodium chloride and sodium bicarbonate were purchased from merck (germany). standard afb and sodium 1 acetate were purchased from sigma (spain). tris hydrochloride was purchased from vivatis (usa). ground peanuts containing aflatoxins (as the reference material) were prepared by mycotoxin laboratory, at scientific equipment and research division, kasetsart university research and development institute (kurdi), kasetsart university, thailand. preparation of solution according to cnbr-activated sepharose 4b manual (ge healthcare 2009), coupling buffer solution (0.1m nahco containing 0.5m nacl, 3 ph 8.3) was prepared by dissolving 8.40 g nahco and 29.20 g nacl in 1 l filtered 3 deionized (di) water. then, the blocking buffer solution (0.1m tris-hcl, ph 8.0) was freshly prepared by dissolving 15.76 g tris hydrochloride in di water. before adjusting volume, ph was adjusted to 8.0. this step was followed by preparing the washing buffer solution (0.1 m acetic acid/sodium acetate, ph 4.0 containing 0.5 m nacl and 0.1 m tris-hcl, ph 8.0 containing 0.5 m nacl) which was prepared by mixing 90 ml 0.2m naac with 410 ml of 0.2m acetic acid, added with 29.20 g nacl followed by adjusting the total volume to 1,000 ml. phosphate buffer saline 10x (paa, austria) was diluted with di water to achieve 1x pbs (ph 7.4) to re-suspend the medium. pbs with 0.02% nan 3 was prepared by dissolving 0.02g nan in 100 ml 3 pbs. all solutions were filtered through 0.45 µm nylon filter before usage. karnovsky solution, as fixative for electron microscopy, was prepared by mixing 2 g of paraformaldehyde with 25 ml of distilled water. the solution was heated in 60 °c with stirrer plate. then the solution was added with 1m optimization and efficiency improvement of immunoaffinity column for aflatoxin detection – kooprasertying et al. 115 the medium was then collected using sintered filter flask, washed from the excess antibody using ±50 ml coupling buffer and transferred into ±50 ml blocking buffer (0.1m tris-hcl, ph 8.0). the medium in blocking buffer was shaken using orbital shaker for another 2 hours, washed with washing buffer (0.1 m acetic acid/sodium acetate, ph 4.0 containing 0.5 m nacl and 0.1 m tris-hcl, ph 8.0 containing 0.5 m nacl). this was conducted for 3 cycles. each cycle used ±50 ml washing buffer. the medium was then resuspended in 50 ml pbs. finally, 0.2 ml medium was packed in each spe empty tube with polyethylene frits and the column was filled up with pbs containing ±0.6 ml 0.02% nan in 3 each column. (modified from ge healthcare 2009) and stored at 4 °c until usage. optimization of antibody and cnbr– activated sepharose 4b to develop iac, the major cost of iac production depended on the amount of antibody a n d c n b r – a c t i v a t e d s e p h a r o s e 4 b (mahakarnchanakul et al. 2011). thus, the amount of these 2 components has to be optimized. response surface methodology was applied to determine the proper ratio. the experiment was designed as central composite design and the data obtained were analyzed using minitab (version 14). the alpha value was face centered. the experimental treatments are summarized and shown in table 1. the recovery percentage of afb , standard 1 deviation (sd) and relative standard deviation (rsd) were the parameters for response surface determination to optimize volume of antibody and cnbr–activated sepharose 4b. the seven columns from each treatment (table 1) were tested with 15 ml 30% methanol (containing 200 ng of afb , which was prepared 1 from stock of 10 ppm afb ). the solution was 1 passed through iac at a flow rate of 1 2 drops per second (dps). after the whole solution had been passed through the iac, the column was washed twice with 10 ml deionized water (di water) at the same flow rate. the captured afs were eluted with 1 ml methanol at 1 dps, and then filtered (nylon syringe filter, whatman) and subjected to high performance l iquid chromatography (hplc) evaluation. naoh until the solution was clear. finally, 5 ml 50% glutaraldehyde and 20 ml 0.2m cacodylate buffer (ph 7.2) were added into the solution. to prepare 0.2m cacodylate buffer (phv7.2), 8.56 g sodium cacodylte, 25 g calcium chloride and 2.5 ml 0.2n hcl were dissolved with 200 ml of di water. amplification of monoclonal antibody specific to aflatoxins monoclonal antibody (mab) specific to aflatoxins from hybridoma clone c10 was produced using integra celline flasks (cl1000) based on the manual from the manufacturer (integra biosciences ag ch-7205 zizers, switzerland). briefly, the hybridoma was maintained between the space of membrane 6(initial cell amount was 2 x 10 cells/ml which had > 80% of viable cell) in the hybridoma serum free medium (hsfm). the flask was filled with hsfm up to 250 ml. at certain times, cells were 7collected, leaving 3 x 10 cells/ml to be maintained in the flask. the flask was then refilled with fresh hsfm up to 250 ml. the collected medium was then centrifuged (2,000 rpm for 5 minutes). the supernatant was purified by aktaprime™ system (ge healthcare, sweden) using hitrap protein g column (ge healthcare, usa). the purified antibody concentration was determined by measuring od 280 nm and calculated as the following equation. ○then, mab was lyophilized and stored at 4 c until usage. iac preparation to prepare 50 columns of iac, purified 50 mg mab was dissolved in coupling buffer solution (0.1m nahco containing 0.5 m nacl, ph 8.3) 3 (1 mg/ml) then coupled with 10.2 ml cnbractivated sepharose 4b. the mixture was then shaken using orbital shaker (boekel scientific, ○usa) at 150 rpm, 4 c, for 18 hours. at the end of the incubation period, the mixture was left at 26 °c to separate the medium (cnbr-activated sepharose 4b coupling with mab). the supernatant was then measured at od 280 nm, in order to determine the excess of mab using the following formula: 116 biotropia vol. 24 no. 2, 2017 concentration of antibody (mg.ml) = od 280 nm value x dilution factor 1.4 the results were transformed to percent recovery and the optimum ratio of antibody and cnbr-activated sepharose 4b volumes was determined. percent recovery was calculated using the following formula: after calculating the recovery percentage, the standard deviation (sd) and relative standard deviation (rsd) were determined. rsd was calculated to describe the precision and the repeatability of each treatment. rsd was calculated using formula: the recovery percentage, sd and rsd were then used as suitable variables in the response surface analysis, performed by minitab version 14. high performance liquid chromatography condition to determine the amount of afb , afb , 1 2 afg and afg , the hplc with fluorescent 1 2 detector (separation module e2695 with fluorescence detector 2475, waters, usa) was used. a symmetry c18 column (5 m, 4.6 x 150 μ mm) with c18 guard column (water corporation, usa) was used to separate the aflatoxins. photochemical derivatization (phred, aura industries, usa) was used to enhance the afb 1 and afg detection by fluorescence detector. 1 the column was equilibrated with a mixture of water:acetonitrile:methanol [60:20:20 (v/v)] which was also the mobile phase. flow rate at 1 ml/minute was used and the column oven temperature was 35 c. the excitation and ○ emission wavelength of the fluorescence detector were set at 365 and 445 nm, respectively. improvement of the efficiency of in-house iac the cnbr-activated sepharose 4b was replaced by ch-activated sepharose 4b, hitrap nhs-activated sepharose 4b or nhs-activated sepharose 4b having spacer arm length of 8, 10 and 14 atoms, respectively. the iac efficiency were compared by passing 200 ng afb in 15 ml 1 30% methanol through the iac and then evaluated by hplc. beside the recovery percentage, the distributions of antibody on the supporting material were observed by transmission electron microscopy. the coupled supporting material was then attached with gold labeled anti-mouse igg (h+l) system (kpl, usa). the wet gel was dehydrated with rising concentrations of ethanol (30, 50, 70, 90, and l00%, v/v), absolute acetone and embedded in activated micropal. after sedimentation of the gel particles occurred, the 117 table 1 coupling percentage, actual amount of antibody and recovery percentage of 200 ng afb from iac containing 1 different volumes of antibody and supporting material (cnbr-activated sepharose 4b) column experimental design results volume of antibody to supporting material ratio (mg/ml) coupling percentage (%) actual antibody (mg) afb1 recovery*, a (%) expected antibody (mg) cnbr-activated sepharose 4b (µl) 1 0.3 153 1.96 97.26 0.31 79.66 ± 2.03f 2 0.4 153 2.61 90.02 0.37 82.70 ± 2.63e 3 0.5 153 3.26 90.95 0.47 85.82 ± 0.83d 4 0.3 204 1.47 93.29 0.29 86.64 ± 2.51d 5 0.4 204 1.96 97.26 0.42 103.36 ± 2.31b 6 0.5 204 2.45 90.95 0.47 88.62 ± 3.70d 7 0.3 255 1.17 92.51 0.29 87.83 ± 2.13d 8 0.4 255 1.46 93.14 0.38 94.80 ± 4.23c 9 0.5 255 1.96 97.26 0.52 107.13 ± 1.25a note: * = values in the same column followed by different letters are significantly different at p < 0.05 a = mean ± sd from 7 replicates percent recovery = concentration of afb1 after passing through the iac column concentration of afb1 before passing through the iac coloum x 100 relative standard deviation (%) = standard deviation mean x 100 optimization and efficiency improvement of immunoaffinity column for aflatoxin detection – kooprasertying et al. 118 biotropia vol. 24 no. 2, 2017 mixture was cooled down to 4 °c. the lower part of the stiffened gelatin containing the sepharose beads was fixed at 4 °c in karnovsky solution for 30 minutes, washed in cacodylate buffer (ph 7.2, 0.2 m), dehydrated and embedded in activated micropal. ultrathin sections were then made with the lkb ultratome i11 (lkb-produkter, stockholm). the sections were viewed with electron microscope (ht 7700, hitachi, japan) at an accelerating voltage of 80 kv with 10,000x magnification. iac capacity testing the capacity of the optimized iac was tested by passing various amounts of standard afb (1, 1 10, 100, 200, 300, 500 and 1,000 ng) through the iac column. total afs recovery was also conducted at 50 ng for each of afb , afb , afg 1 2 1 and afg (total afs amount was 200 ng). the 2 results were then transformed into recovery percentage. rsd and horwitz ratio (horrat) value were then determined. the horrat value was calculated using formula: the predicted rsd was calculated from horwitz ratio (horrat) using formula: (-0.15)predicted rsd = 2c , where: c = concentration found or added, expressed as dimension less mass fraction (thompson 2007). ground peanuts as reference material containing afs at 47.68 ng/g (afb 38.25 and 1 afb 9.75 ng/g) and 72.14 ng/g (afb 56.28 and 2 1 afb 15.86 ng/g) were extracted and passed 2 through the optimized and aflatest iac columns based on the aflatest hplc instruction manual (vicam 2009). briefly, 25 g reference material with 5 g methanol were blended at high speed (for 2 minutes at 26 °c) with 125 ml methanol:water (70:30), then filtered through filter paper no.1 (whatman). fifteen milliliters of the filtered extract was diluted with 30 ml di water, filtered through glass microfiber filter (whatman) and 15 ml was passed through the iac column. efficiency comparison between in-house iac and commercial iac the afb and afs concentration resulted 1 from reference material clean-up by the in-house iac and commercial iac were compared with statistical analysis including correlation (spss version 17), test (spss version 17) and bland-taltman plot (sigma plot version 12). the stability of iac production and shelflife testing five batches of optimized iac were produced in order to check the quality of producing different batches. seven columns from each batch were tested with 200 ng afb . the results were 1 analyzed to obtain data on the quality difference among batches productions using spss software version 17. for shelf-life testing, iac was produced and stored at 4 °c. the iac was tested within batches with 200 ng afb after 1, 3, 6, 9 1 and 12 months of storage duration. the results were analyzed to obtain data on the difference among storage duration and acceptable range of recovery percentage using spss software version 17. results and discussion optimization of materials using iac production after mab was coupled with cnbr-activated sepharose 4b, the excess amount of the antibody was measured to determine the coupling percentage. all treatments gave excellent coupling percentage (> 90%), ranging from 90.02 to 97.26% (table 1). the amount of bound antibodies did not different from the expected amount of antibodies in each column. the highest coupling percentage (97%) was obtained when the volume ratio of the antibody to the supporting material was 300 µl:153 µl, 400 µl:204 µl, 500 µl:255 µl. under these conditions, ratio of antibody and supporting material were much lower than the ratio recommended by the operating procedure (ge healthcare 2009) i.e. at least 2.5 5 times. the operating procedure recommended 5 10 mg of horrat = rsd from the results rsd predicted from horwitz equation 119 protein per ml of cnbr-activated sepharose 4b, which is quite large amount of antibody to be used. when the amount of antibody was higher or lower than 2 mg of antibody per 1 ml of cnbractivated sepharose 4b, the coupling percentage decreased as shown in table 1. the supplier's recommended ratio of mab and supporting material might not be suitable for specific purpose or antibody. for instance, in developing iac for zearalenone and ochratoxin clean-up, some research found that 1 mg antibody coupled with 1 ml of cnbr-activated sepharose 4b, as supporting material, provide 40 – 50% stability toward high concentration of acetonitrile and methanol (uchigashima et al. 2009; uchigashima et al. 2012). the ratio used was obviously lower than the recommended ratio, but still the recovery percentage were satisfactorily ranging from 80 to 100%. in some cases, however, the supplier's recommended ratio is useful. for example, in developing iac for bisphenol a in serum, some research found that the ratio of the polyclonal antibody and supporting material (14 mg to 2 ml) provided coupling percentage of 86±3% (zhao et al . 2003). in the case of iac for quinolone and sulfonamide clean-up, it was found that the ratio of 8 mg antibody to 1 ml supporting material provided coupling percentage of 91.1% (li et al. 2008), which was lower compared to coupling percentage provided by our study (97%). high coupling percentage, however, is not the guarantee for the iac efficiency. therefore, the efficiency should be evaluated as recovery percentage. according to the codex alimentarius commission (1995), the acceptable recovery percentage of mycotoxin analysis is 80 110%. 1all conditions could recover afb in the acceptable range, except the ratio of 300 µl antibody to 153 µl cnbr-activated sepharose 4b (table 1). although our study provided high coupling percentage (97%), the efficiency to recover afb was not acceptable (79%).1 in terms of extraction step, basically 70 – 80% methanol or acetonitrile is sufficient to be used as the solvent for afs extraction. to allow antibody completely capture the toxin, the organic solvent in extraction process should be diluted to 23 – 26%. the highest organic solvent concentration to be passed through iac column was 30% (scott & trucksess 1997). in our study, the specificity test was conducted by passing through 200 ng afb1 diluted in 15 ml 30% methanol in the iac column. the concentration of organic solvent before being passed through the column was slightly higher than the recommendation provided by vicam manual (23 26%). when the amount of supporting materials asw varied toward a constant 0.3 mg of antibody, the results showed significantly different recovery percentage ( < 0.05). recovery percentage p increased from 79 to 87% as a result of increasing the amount of cnbr-activated sepharose 4b from 153 to 255 µl (table 1), which might have been caused by the steric hindrance between the tightly packed of antibody on the surface of each bead in the column. other possible cause was that higher amount of supporting material (at a constant amount of antibody) would provide higher surface and extended reaction time between the antibody and the toxin. similar results were obtained when the amount of antibody was increased to 0.4 and 0.5 mg. on the contrary, equal amount of supporting materials toward varied amount of antibody would increase recovery percentage. increasing the amount of antibody did not increase the coupling percentage, which indicated that the excess of antibody would lose during the coupling process. also, excess amount of antibody might cause the occurrence of steric hindrance which resulted to the less specificity to toxin. therefore, ratio of antibody to the supporting material is the critical factor to develop the iac. the ratio should be balanced to achieve the best efficiency in toxin detection. anova results in our study showed that the amount of antibody and cnbr-activated sepharose 4b as supporting material had significant effect on the recovery percentage ( < p 0.05), in which our study used 200 ng afb for 1 testing the efficiency of iac column. therefore, our study chose the 400 µl:204 µl ratio of the antibody to the supporting material or 2 mg antibody per 1 ml supporting material as the expected optimum condition to provide high coupling percentage. to confirm the ratio of these two coupling substances, the response surface methodology was used to determine the optimum coupling condition. before the optimization process was optimization and efficiency improvement of immunoaffinity column for aflatoxin detection – kooprasertying et al. 120 biotropia vol. 24 no. 2, 2017 conducted, the recovery percentage, standard deviation and relative standard deviation variables were analyzed to select suitable variables for plotting the response surface. suitable variables were chosen based on the r value > 85% and 2 p value < 0.05. the selected suitable variables had r 2 of 88.2% and values of 0.033 for antibody and p 0.049 for supporting materials. the value of recovery percentage was determined to be the optimum condition of coupling step based on contour plot of the response surface methodology (fig. 1). based on setting criterion of 100%, recovery percentage was selected between the areas of 400 µl of antibody and 204 µl of supporting material. improving the efficiency of the in-house iac this experiment was aimed to improve the efficiency of iac by varying the spacer arm length between the cnbr and sepharose 4b. within the affinity of chromatography system, normally the binding site of the target toxin may be located at the surface. some binding site, however, may be located deep within the molecule hindering the antibody to access the target toxin. increasing spacer arm length may overcome the hindrance by interposing antibody and supporting material. length of the spacer arm may provide better affinity, but the length increase must be cautiously done. previously, the experiment was designed to vary the spacer arm length between the sepharose 4b and cnbr, due to the limited availability of cyanogen bromide in local market, under the permission from the thailand ministry of defence (royal thai government gazette 2008). this situation made it impossible to vary the length of spacer arm within the same supporting material. therefore, we used four supporting materials available in local market i.e. cnbractivated sepharose 4b, ch-activated sepharose 4b, hitrap nhs-activated sepharose 4b and nhs-activated sepharose 4b. recovery percentage of the coupling step with different supporting materials are shown in table 2. coupling monoclonal antibody with three supporting materials (cnbr-activated sepharose 4b, hitrap nhs-activated sepharose 4b and nhs-activated sepharose 4b), resulted in successful recovery of afb (96.6 101.1%, 88.9 1 98.0% and 95.9 103.9%, respectively), except the ch-activated sepharose 4b which provided much lower recovery (59.70 84.47%). low amount of afb was possible to be recovered using the ch-1 activated sepharose 4b as supporting material, which might be due to the limited length of spacer arm. based on the specification of each supporting material, the recovery percentage may decrease depending on the number of spacer arms. the ch-activated sepharose 4b used in our figure 1 contour plot of recovery percentage determined by antibody and supporting material (gel) 121 study contained 10 atoms spacer arm and provided the lowest recovery percentage. among the other three supporting materials, cnbractivated sepharose 4b exhibited the lowest sd value (table 2). therefore, cnbr-activated sepharose 4b was selected as material for iac production. table 2 shows similar values of coupling percentage and quite different values of recovery percentage. cnbr-activated sepharose 4b, hitrap nhs-activated sepharose 4b and nhs-activated as supporting materials had moderate active role on the surface to capture the antibody and the target toxin, resulting to high recovery percentage (93 – 101%). the developed iac was named as ku-af02 and was further tested for capacity, reproducibility and stability. the distribution of antibody on each supporting materials by transmission electron microscope (tem) was conducted to investigate the relationship between the affinity of antibody on supporting material to the coupling percentage. unfortunately, the illustrations presented in this manuscript did not exhibit the relevance of the affinity of antibody and supporting material to the coupling percentage table 2 recovery percentage of afb recovered by iac containing different supporting material1 supporting material spacer arm (n of atom) coupling percentage (%) recovery percentagea (%) cnbr-actiavated 4b (0) 98.21 98.67 ± 1.80 ch-activated sepharose 4b 6-aminohexanoic acid (8) 97.56 71.83 ± 9.78 hitrap nhs-activated 4b 6-aminocaproic acid (10) 98.40 93.85 ± 3.63 nhs-activated sepharose 4b 6-aminocaproic acid (14) 96.08 100.59 ± 3.06 anote: = mean and sd from 5 replicates (a) (b) (c) (d) (e) figure 2 the transmission microscopy illustration of (a) cnbr-activated sepharose 4b and monoclonal antibody distribution on (b) cnbr-activated sepharose 4b © ch-activated sepharose 4b (d) hitrap nhs-activated 4b and (e) nhs activated sepharose 4b at 10,000x magnification optimization and efficiency improvement of immunoaffinity column for aflatoxin detection – kooprasertying et al. 122 biotropia vol. 24 no. 2, 2017 (fig. 2). the dark color indicates the uniform distribution of antibody (fig. 2b), which was the coupling of antibody with cnbr-activated sepharose 4b. the condensed color was distributed from the outer surface of supporting material to the inner surface. the antibody which should have been shown as dark color was not found at the inner or the outer layer of sepharose (fig. 2a), although the sepharose was stained with gold label. the dark color shown in figure 2c was the coupling of antibody with ch-activated sepharose 4b, which was located at the inner or outer surface; the color was slightly toned down from the outer to the inner surface of the supporting material. the dark color shown on figure 2c, which was the coupling of antibody with activated ch sepharose 4b, was located at the surface or outer and the color slightly dropped from the outer to the inner of supporting material. in case of hitrap nhs-activated sepharose 4b and nhs-activated sepharose 4b (fig. 2d and 2e), the antibody seemed to be equally distributed to the inner surface, but the color intensity was less than that in figure 2a. the coupling percentage of antibody on four supporting materials was shown to be similar (> 90%) (table 2), which might be presented in figure 2c. the density of antibody on the surface of ch-activated sepharose 4b seemed to be higher than that on other supporting materials. it was expected that ch-activated sepharose 4b had potential to enhance the affinity of antibody to the supporting material. however, the steric hindrance of the condensed antibody on chactivated sepharose 4b, caused the lower capture of the tested toxin. the steric hindrance might interfere the affinity of antibody resulting to the lowest recovery percentage for ch-activated sepharose 4b. the capacity of iac recovery percentages of various amount of afb (1 to 1,000 ng) in 15 ml 30% methanol 1 obtained using the developed iac ku-af02 (coupling condition: 0.4 mg of antibody with 204 µl of cnbr-activated sepharose 4b) are shown in table 3. the capacity of ku-af02 achieved the standard of analysis when 1 to 500 ng was tested. the recovery percentages were all above 80%. particularly at 500 ng afb , 1 the recovery percentage was 87% with horrat value less than 2 (table 3). as expected, the recovery value slightly decreased when the amount of afb increased. recovery percentage 1 was lower than 80% when 1,000 ng of afb was 1 loaded. at this amount of afb , although the 1 horrat value was less than 2, the recovery percentage was considered not satisfying for the capacity of iac. thus, the developed iac ku-af02 was successful in increasing the recovery of afb up to 500 ng compared to the 1 previous iac, kuaf-01 (mahakarnchanakul et al. 2011). ku-af02, was also successful in recovering other derivatives of afs i.e. afb , afg and 2 1 afg . the experiment was carried out by mixing 2 50 ng of each afb , afb , afg and afg in 1 2 1 2 15 ml 30% methanol and passing them through the ku-af02. the iac column of ku-af02 was capable to recover 45.37 ± 1.87 ng of afb1, 40.64 ± 2.63 ng of afb 43.41 ± 1.93 ng of afg2, 1 and 33.75 ± 2.20 ng of afg , with total aflatoxin 2 recovery percentage of 81.6%. table 3 recovery percentage at different amount of afb recovered by optimized iac1 amount of afb1 (ng) recovery percentagea (%) calculated rsd predicted rsd horrat value 1 102.86 ± 6.42 6.25 22.39 0.28 10 103.81 ± 1.93 1.86 15.85 0.12 100 95.41 ± 2.27 2.38 11.22 0.21 200 95.51 ± 1.71 1.79 10.11 0.18 300 90.07 ± 2.19 2.43 9.52 0.25 500 87.06 ± 3.57 4.10 8.81 0.47 1000 75.35 ± 6.04 8.02 7.94 1.01 anote: = mean and sd from 7 replicates 123 the results showed that iac ku-af02 recovered afb , afb and afg higher than 1 2 1 80%, except for afg the recovery percentage 2 was only 67%. although preliminary test showed that our monoclonal antibody had specificity with single toxin test when loading afb , afb and 1 2 afg and afg separately in iac, the recovery 1 2 percentage of each test obtained was > 98% and was not significantly different (p > 0.05) among those recovery percentages. after mixed afs were loaded, iac ku-af02 could recover the parent form of b and g aflatoxins (b , 90.74% and g , 1 1 86.81%) better than the others (b , 81.28% and g2 2 67.50%). similar to the results when tested with commercial iac, higher recovery percentage of afb , afb and afg (79.18%, 72.68%, 78.39%) 1 2 1 over afg (27.25%) were observed.2 the remaining afg in test samples 2 apparently reduced performance of total afs detection by iac, either in the developed iac or the commercial iac. therefore, samples contaminated with high amount of afg should 2 be carefully handled because the afg2 may decrease the recovery of other forms aflatoxins. afg is the highest contaminant in unpolished 2 rice (5.5 ppb) (jankhaikhot 2005). further tests were done using reference materials i.e. two-level contaminant contained in defatted ground peanuts. the reference materials were extracted and passed through optimized iac. the results showed that the recovery percentage of afb , afb and afs obtained 1 2 from reference material no. 1, representing medium contamination (47.68 ng/g) and reference material no. 2, representing high contamination (72.14 ng/g), were between 80 110% with horrat value of 0.8 and ± 0.4, respectively. these results also showed the capacity of ku-af02 in detecting contaminated food using peanuts as the representative. similar to the test in pure toxin solution, developed iac ku-af02 recovered parent forms (b and g) of aflatoxins from the reference material better than those from the derivative forms, particularly in mixed forms. efficiency comparison of in-house and commercial iac seven replicates of medium and high contaminants aflatoxins in peanut (reference materials no.1 and no.2) were quantified using hplc to determine the amount of afb and afs. 1 the correlation between recovery percentage of afb and afs using ku-af02 iac column and 1 recovery percentage using commercial iac column were determined (fig. 3). close correlation (r) between the obtained amount of afb and the afs using the developed 1 and commercial iac columns were 0.961 and 0.962, respectively. the r values representing 2 the confidence values of x and y variables were figure 3 correlation (linear-regression) between clean-up samples with commercial iac column (x-axis) and optimized iac column (y-axis) for afb ( ) and afs ( ) detection in peanut reference materials1 80 75 70 65 60 55 50 45 40 35 30 30 40 50 60 70 80 concentration from commercial iac (ppb) y = 0.8854x + 4.6804 2r = 0.9237 y = 0.9451x + 4.1188 2r = 0.926 afb1 afs afs afb1 concentration from optimized iac (ppb) optimization and efficiency improvement of immunoaffinity column for aflatoxin detection – kooprasertying et al. 0.923 and 0.926, respectively. close correlations of the linear regression equations indicated similar trend of analysis, but might not be directly compared the efficiency of these tested two iac columns. tthe paired samples -test and bland-altman plot were used to compare the results. the -value t of afb data (1.283) was lower than critical twot 1 tailed value (2.160), which meant no difference between two data sets resulting from the two tested column ( > 0.05). a similar result was p found in case of afs. apart from -test, the bland–altman plot was t used to determine the agreement between two tested methods, for example between modified and standard methods (bunce 2009; eken 2009; hanneman 2008). the use of this method has been reported in the application of medical instrument (zaki . 2013), computed et al tomography and ultrasound prostate volume measurements (gloi . 2008). in 2013, tansakul et al et al. (2013) compared between elisa and lcms/ms for fumonisin detection in corn samples using bland-altman analysis and found no difference between these two methods at a range of 0 20 mg/kg. 1in our study, the amount of afb and afs obtained from the developed iac ku-af02 and commercial iac columns were compared using bland–altman plot (fig. 4a and 4b). none of these data were located outside the limit line (±1.96 sd). thus, the clean-up of contaminated peanut samples using the developed iac kuaf02 or commercial iac column provided similar results, ranging from 35 to 60 ppb for afb or ranging from 40 to 80 ppb for afs (fig. 1 4). the stability of batch production and shelflife of developed iac the stability of iac production was determined in 5 batches. the results showed that all batches could recover afb higher than 95% 1 (98.32, 99.68, 101.04, 101.27 and 102.38) and there were no significant difference among the batches produced ( >0.05 . thus, the quality of p ) each batch had no effect on the iac efficiency. stability of recovery percentage of ku-af02 stored for 1 12 months, are shown in table 4. along the storage duration, recovery percentage decreased from 95 to 85%, but still greater than 80%. there were significant differences of recovery percentage among storage durations (table 4). the horrat values for all storage duration were less than 2 (table 4). based on the result, the developed iac ku-af02 had shelf-life up to 12 months when stored at 4 °c. the long stability will encourage the use of the developed iac ku (a) (b) figure 4 bland–altman plots of afb (a) and afs (b) concentration after being cleaned-up using the developed iac ku-1 af02 and commercial iac columns 124 biotropia vol. 24 no. 2, 2017 average concentration of afb 1 from developed and commercial iac (ppb) d if fe re n ce c o m m er ci al a n d d ev el o p ed i a c mean + 1.96sd mean mean 1.96sd 6 4 2 0 -2 -4 -6 -8 30 35 40 45 50 55 60 65 8 6 4 2 -2 -4 -6 -8 0 40 45 50 55 60 65 70 75 80 average concentration of afs from developed and commercial iac (ppb) mean mean + 1.96s d mean 1.96s d d if fe re n ce c o m m er ci al a n d d ev el o p ed i a c af02, as in-house method. this developed iac ku-af02 may also be distributed to other laboratories. conclusions the production cost of the developed iac ku-af02 was successfully reduced compared to the previous iac (ku-af01) by reducing the ratio of antibody to cnbr-activated sepharose 4b. the optimized coupling condition was determined by response surface methodology (rsm) and the range of recovery percentage was acceptable. cnbr-activated sepharose 4b was determined to be the suitable supporting material in this study. the capacity of ku-af02 was higher than that of the commercial and previous iac (ku-af01). ku-af02 was successful to recover afs in two reference materials. no significant difference was observed when five batches of ku-af02 were produced. optimized iac had a shelf-life of 12 months when produced and stored at 4 °c. there were no significant difference in using the developed iac ku-af02 and commercial iac to clean-up afb and afs from ground peanuts reference 1 materials. acknowledgements the authors acknowledge the financial support received from the center for advanced studies for agriculture and food (casaf), kasetsart university. we are greatfully thankful to the scientific equipment and research division, kasetsart university research and development table 4 recovery percentage, rsd and horrat value of 200 ng afb obtained from recovery process using the developed 1 iac ku-af02 stored at different storage duration storage duration (months) recovery percentage*, a (%) rsd horrat value 1 95.01 ± 2.62a 2.75 0.27 3 94.57 ± 3.73a 3.95 0.39 6 93.18 ± 1.19a 1.27 0.13 9 90.52 ± 0.87ab 0.96 0.10 12 85.61 ± 5.18c 6.06 0.60 note: * = values in the same column followed by different letters are significantly different at p < 0.05 a = mean and sd from 7 replicates institute, kasetsart university, thailand for their kind support of the facilities. thanks are also extended to the department of plant pathology, faculty of agriculture at kamphaeng saen kasetsart university for providing monoclonal antibody specific to aflatoxins. references anukul n, vangnai k, mahakarnchanakul w. 2013. significance of regulation limits in mycotoxin contamination in asia and risk management programs at the national level. j food drug anal 21(3):227-41. bunce c. 2009. correlation, agreement, and bland-altman analysis: statistical analysis of method comparison studies. am j ophthalmol 148(1):4-6. calvo am, wilson ra, bok jw, keller np. 2002. relationship between secondary metabolism and fungal development. microbiol mol biol rev 66(3):447-59. chinaphuti a, aukkasarakul s. 2009. contamination of aflatoxin b1 and ochratoxin a in brown rice and white rice. [abstract]. thai rice research journal 3:57-65. eken c. 2009. bland-altman analysis for determining agreement between two methods. j emerg med 36(3):307. european union. 2006. commission regulation (ec) no 1881/2006 of 19 december 2006 setting maximum levels for certain contaminants in foodstuffs. http://eur-lex.europa.eu/lexuriser v/lexuriserv. do?uri=oj:l:2006:364:0005:0024:en:pdf . [retrieved on 20 june 2015]. ge healthcare. 2009. cnbr-activated sepharose 4b. instruction affinity media 1-12. gloi a, mccourt s, zuge c, goettler a, schlise s, cooley g. 2008. a bland-altman analysis of the bias between 125 optimization and efficiency improvement of immunoaffinity column for aflatoxin detection – kooprasertying et al. computed tomography and ultrasound prostate volume measurements. med dosim 33(3):234-8. hanneman sk. 2008. design, analysis, and interpretation of method-comparison studies. aacn adv crit care 19(2):223-34. jankhaikhot n. 2005. aflatoxin in unpolished rice and exposure and estimation to aflatoxin in unpolished rice consumption in bangkok. ms thesis. salaya (th): mahidol university. li c, wang z, cao x, beier rc, zhang s, ding s, li x, shen j. 2008. development of an immunoaffinity column method using broad-specificity monoclonal antibodies for simultaneous extraction and cleanup of quinolone and sulfonamide antibiotics in animal muscle tissues. j chromatogr a 1209(1-2):1-9. mahakarnchanakul w, hongprayoon r, kladpun s, puthpongsiriporn t, malichan s, maneeboon t, yangyune p, sinprasopchai l, saijit p. 2011. development of immunoaffinity column to reduce costing of aflatoxin determination. research report. bangkok (th). rapid alert system for food and feed [internet]. 2012. rasff portal reference 2012.bza, 2012. h t t p s : / / w e b g a t e . e c . e u r o p a . e u / r a s f f window/portal. 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[retrieved on 20 june 2015]. thompson m. 2007. limitations of the application of the horwitz equation: a rebuttal. trends analyt chem 26(7):659-61. uchigashima m, saigusa m, yamashita h, miyake s, fujita k, nakajima m, nishijima m. 2009. development of a novel immunoaffinity column for aflatoxin analysis using an organic solvent-tolerant monoclonal antibody. j agric food chem 57(19):8728-34. uchigashima m, yamaguchi murakami y, narita h, nakajima m, miyake s. 2012. development of an immuno-affinity column for ochratoxin analysis using an organic solvent-tolerant monoclonal antibody. methods 56(2):180-5. vicam. 2009. aflatest: instruction manual for hplc use. watertown (us): vicam. wongsuttichot p. 2007. development of an immunoaffinity column from polyclonal antibodies specific to aflatoxin b1 in peanut determination. ms thesis. bangkok (th): kasetsart university. zaki r, bulgiba a, ismail r, ismail na. 2013. a systematic review of statistical methods used to test for reliability of medical instruments measuring continuous variables. iran j basic med sci 16(6): 803–7. zhao m, liu y, li y, zhang x, chang w. 2003. development and characterization of an immunoaffinity column for the selective extraction of bisphenol a from serum samples. j chromatogr b analyt technol biomed life sci. 783(2):401-10. 126 biotropia vol. 24 no. 2, 2017 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 microsoft word 13 biotropia no. 23, 2004 : 13 39 fisheries associated with mangrove ecosystem in indonesia: a view from a mangrove ecologist sukristijono sukardjo the centre for oceanological research and development, indonesian institute of sciences, jl.pasir putih 1 ancol timur, p.o.box 4801 jktf jakarta 11048, indonesia. e-mail: s_sukardjo@telkom. net and s_sukardjo@yahoo. com abstract blessed with mangrove area of some 9.6 million ha in extent, indonesia represents an important country with fishery resources being a source of food and nutrients. the fishery resources utilized by man, such as fishes, crustaceans and mollusks that are found in the mangrove ecosystem/swamp area arc enormous. there is a range of species caught in the mangrove and surrounding areas with over 70 species. however, commercially valued species are limited to a few such as rabbit fish, snapper, grouper, marline catfish, fringe-scale sardine, and anchovy. leaf detritus from mangroves contribute a major energy input into fisheries. but information about the study on the relationship between fishery species and mangroves, ecologically and biologically, arc scanty. the mangrove is a physiographic unit, the principal components of which arc organisms. therefore, the problems are predominantly of a biological nature (e.g., mangroves fishery relationship). positive correlation between the mangrove area and penaeid shrimp catch found in indonesia, the philippines, australia and mexico. finally, the most important part of the variance of the msy (maximum sustainable yield) of penaieds (53% of the variance) could be explained by a combination of area of mangrove habitats and latitude. keywords : indonesia/mangrove/ecosystem/fisheries/ecology/coastal areas/fishes/molluscans/ crustaceans. introduction indonesia (6° n 10° s and 95° e 142° e) is an archipelagic state in the tropical area with 18,110 islands and almost more than 108,000 km of coastlines (sukardjo 1997; anonim 2003). these areas form an important and valuable natural resource with potential economic value, and a potentially important production area for food. people have long relied on coastal waters as a source of food. the coastal zone of the indonesian waters includes a number of bays and gulfs into which large and small rivers empty, creating estuarine conditions in the inshore areas. the extent of the estuarine zone in the sea varies according to the size of the river and the volume of its freshwater discharge, the steepness of the gradient, and the tidal range. also, these coastal zones contain diverse productive ecosystems that include mangroves, sand beaches, several riverbasins and freshwater bodies. each of these ecosystems has components that are exploited in a wide variety and used with different intensities because of socio-economic and socio-cultural demands. therefore, indonesia, is considered as one of the largest maritime country in the world. 13 biotropia no. 23, 2004 with almost more than 75% of indonesian territories being seas, marine fishery is one of the important renewable resources for the nation. for instance, 1,450,000 tons of pelagic fish had been caught for whole indonesia (eps 1995). ironically, depletion of the marine resources is starting to become evident in indonesia. the fishery resources utilized by man, such as fishes, crustaceans and mollusks (oyster, mussel, cockle), that are found in the mangrove swamp area are enormous. indigenous people in indonesia have exploited the biota of mangrove waters for centuries, and fish and shrimp are still one of the major products harvested from this mangrove swamp (schuster 1952). thus, aquatic resources play a vital role in the social, economic and ecological well-being of coastal communities in indonesia. furthermore, the value of the mangrove ecosystems is not just of academic interest, it also relates to their cultural and commercial value. the full economic value should be determined in order to make intelligent decisions about their future use. thus the understanding of both the roles and the functions of mangrove ecosystem in terms of fishery in indonesia is also of considerable importance. the following paper is, therefore, an effort to review and methodize existing information on the mangrove-fishery association subject in indonesia. the indonesian mangroves: a fishery feature situated at the two continents, asia and australia, and two oceans, pacific and indian, the indonesia archipelago, has an ecologically and economically strategic position with distinct culture that took shape on these 18,110 islands. the climate of indonesia is tropical and generally governed by the monsoonal situation, resulting in fairly regular climate variations. during the winter, the northern part of the earth has air pressure around asia higher than that of australia. north of the equator, the wind blows from north and east the ne monsoon, at and near the equator, it becomes more north and north nw monsoon. south of the equator the winds turns from nw via w to sw the west monsoon. consequently, indonesia is blessed with high annual rainfall (>1500 mm/year) and comfortable tropical temperatures. moreover, indonesia's waters are homogeneous to an unusual degree. the country lies entirely within the tropics, straddling the equator from roughly 5°n to 10°s latitude. oceanographic conditions are uniform in this equatorial zone, characterized by lack of seasonal changes and consistently warm waters. the indonesian waters are ideal monsoon region. these are very favorable for mangroves development. consequently, large areas of the mangrove ecosystem in indonesia are to be found and estimated at about the range of 4.25 million ha (1982) to 9.6 million ha (2002). therefore, mangrove forests are a major feature of the coastline in many islands of indonesia. however, this figure and its distributional extent is still in debates for further clarifications. this type of forest vegetation is common in the malesia (comprising the political states of indonesia, malaysia, singapore, brunei darussalam, the philippines and png) and has stimulated the interest of various systematics and ecologists as can be learned from comprehensive 14 fisheries associated with mangrove ecosystem in indonesia — sukristijono sukardjo studies by watson (1928) in malay peninsula, and by brown and fisher (1918) in the philippines, and by becking et al. (1922) in indonesia. a substantial body of information exists on the biology, ecology and other aspects of mangroves or mangrove ecosystem or mangrove forests or mangals (macnae 1968). the bibliography lists of mangrove forests in indonesia can be found in e.g., rollets (1997). however, recent records of mangrove literatures can be learned through a series of the proceedings of the national seminar on mangrove ecosystem (i-vi) and others. but our knowledge of the mangrove fish fauna in contrast, is limited to a handful of papers that deal with inventory. a similar state of knowledge exists on the association between mangroves and fisheries (see: macnae 1974 for general information); and the economic value of the fisheries function of mangroves in the bintuni bay, irian jaya, has been discussed by ruitenbeek (1991). commercial fisheries in indonesia or indonesia's artisanal commercial fisheries are largely dependent upon coastal and estuarine fishes. in indonesia, mangrove forests reach their greater structural and floristic diversity in all biggest islands e.g., sumatra, kalimantan, and irian jaya, and tropical forests generally have higher rates of litter production than temperate forests (bray and gorham 1964). mangrove forests in indonesia are floristically rich with at least 94 species represented (sukardjo 1994), and produce large quantities of litterfall e.g., in east kalimantan estimated to be about 21.10 to 29.35 dry weight t/ha/year (sukardjo 1995). they produce a large amount of fixed carbon which is used by man for timber, firewood and charcoal. it is hypothesized that this carbon may also support detritus-based food webs both in the swamp and in surrounding coastal waters. in a global context, mangrove swamps have been recognized as extremely productive ecosystems, which not only have a high rate of primary productivity, but also export organic matter and support a variety of aquatic organisms (odum and heald 1972); and most of the aquatic organisms are valuable economically for man. it is generally supposed that mangroves play an important role in supporting a wide range of marine life in near-shore waters and in sustaining coastal fisheries. these highly productive mangrove forests provide critical habitat and food for many organisms, including important fisheries species. however, human population concentrates in the coastal zones, imposing enormous pressure on these mangrove forests. in indonesia moreover, mangrove swamp communities or mangals (after macnae 1968) occupy warm coastal areas where land is becoming increasingly valuable for farming, mariculture, and recreation. thus men's use of mangrove areas in indonesia for the establishment of ponds for the culture offish and prawns, and other fishery activities, and for timber etc., is practically common. in indonesia, mangrove ecosystems or mangroves are found in tidal wetlands under varying conditions of salinity, water-logging, anaerobic substrate, often hyper-saline (>33 %) and acidic. many of the largest mangrove areas of indonesia occupy a significant portion of the deltas of rivers, large and small. here, mangrove ecosystems are delicate and complex, intensely dynamic and fragile; and their principal components are the physical and chemical environments, the biotic elements (flora and fauna), and human interferences. each component of the 15 biotropia no. 23,2004 environment namely, climate, salinity, freshwater supply, siltation, erosion, substrate and nutrients, act on flora and fauna, and, in-turn, these influence the environment. for instance, reduced freshwater flow through a mangrove forest probably not only reduces the nutrient input from surrounding terrestrial areas, but also results in higher soil salinities. thus, it becomes a stress for mangroves itself and their associated meiofauna. mangrove ecosystems are open systems and the cycles of material transport in mangrove forests are driven by physical and biological factors that control the rate of input and output of inorganic and organic components. changes in the quality of water entering and leaving the mangroves are particularly important in determining natural cycles. in addition, they support a characteristic fauna which includes crabs, shrimps and molluscs. the waterways support a varied fauna of fish, shrimp and plankters. the general acceptance is prawn fisheries are important commercial fisheries in many developing countries and developed countries. an ecological importance of the mangrove ecosystem fishery aspects 1. ecological features of mangroves as fish habitat mangrove forests are a common type of pan-tropical coastal vegetation. in indonesia, characteristics of mangrove sites varied provincially and locally, but it does have the same serve as fish habitats and refuges for a large number of species of conservation concern. for instance, in the estuary of the rokan river in sumatra, hardenberg (1950) has reported the occurrence of some 175 species, including occasional migrants. the number of individuals is, however, high. in indonesia, there are large marine mangal areas in an environment characterized by tidal ranges of 2-5 m, fluctuating salinities of 28-33%o, high atmospheric humidity and constant subequatorial temperatures; and mangrove forests of embankments, lagoons, and harbors are less dominated by hydrological factors. for instance, the coast of java experiences strong semi-diurnal tides, with a range of 5 m and above during extreme spring tide. the coast may be classified as a high mesotidal environment. the waters are well mixed vertically, and salinities range from 20 to 32 %o. major dilution occurs from the discharge of freshwater by 11 rivers. also, wind blowing over the sea surface can cause the disturbance of hydrodynamic sea stability. this condition, furthermore, will result in a rising of sea water towards the surface from the below regions and oceanographically known as upwelling; and it is very beneficial for fishery. in sumatra and java, the color of estuarine water may be tea-brown or blackish, due to outflow from enormous peat forests (hardenberg 1950). the ph of this water is reported to be as low as 5 or less. there is a marked increase in the number of planktonic organisms as a result of increase in nutrient salts. copepods diminish to be replaced by an increased amount of diatoms and noctiluca. finally, mangrove ecosystems by their nature include an aquatic element; and 16 fisheries associated with mangrove ecosystem in indonesia — sukristijono sukardjo marine species are replaced by coastal forms. for instance, the position of burrowing animals within the substrate is most closely linked to the state of the tide. this may be merely the temporary intrusion of seawater along open coasts at high tide, or it may be the waters of tidal creeks or estuaries surrounded by mangroves. thus, the unique association of animals and plants, for instance, that dwell upon the roots of mangroves in indonesian seas are interesting and has long been attracting the attention of naturalists and ecologists. the structural-functional diversity of the true-mangrove species complex is probably best expressed in the remarkably high diversity of animals for which the ecosystem provides a variety of physical habitats and food sources. for example, the rhizophora mangroves that predominate the fringe habitat have a well-developed prop-root system that is flooded semi-diurnally by tides and may provide habitat to fishes. also, some structurally heterogeneous habitats in tidal waters have been shown to provide fish with protection from predators and enhanced feeding opportunities (rozas and odum 1988) as can be learned that a large proportion of fish found in mangroves are considered juveniles and species of a small size at maturity (bell et al. 1984). thus, the mangroves-fisheries connection may, therefore, lie in the nursery function through provision of food and shelter from predation (hatcher et al. 1989); and protection from predation is offered by physical structure (e.g., mangrove roots, substratum, and turbidity), in combination with for example, shrimp behavior such as hiding and burying (dall et al. 1990). moreover, the estuaries of the indonesian coastal zone support a varied fish fauna, but it is rather poor in species. the fish fauna consists of species that spend all or a major part of their life in the estuaries, and marine or freshwater species which migrate seasonally into or through the estuaries (table 1). consequently, the fish or ichthyoids fauna occurring in and/or within mangroves and/or mangrove environment, have been placed in various ecological groups: as permanent residents, temporary residents or rare species. table 1. types of fish species recorded from apar mangrove bay areas, east kalimantan, and from banyuasin mangrove estuary areas, south sumatra . type estuarine species marine species riverine species total *) adults 73 69 25 145 juveniles**) 45 31 15 68 total 73 75 26 151 notes : * as some species can be classified under several headings, totals given arc not the sums of columns. ** as the specific identification of juvenile fish is frequently difficult the numbers of species given mere approximations. 17 biotropia no. 23, 2004 the mangrove forest and aquatic ecosystems are independent, with biological production processes different from those of purely terrestrial forest ecosystems. due to their characteristic location, the mangroves play an important role in the ecology of the coastal zone area and in support of the marine species that utilizes the mangrove environment during part or all of their life cycles. for instance, some prawn species may breed and complete their life cycle in shallow coastal mangrove waters. many species require the more saline deep offshore waters to spawn. the larvae then migrate back to the food rich and protective coastal mangrove waters to mature. adults of the commercially important penaeid prawns usually spawn at sea and after a short larval life (2 to 3 weeks), the post-larval stage settles in the near inshore areas and estuaries (dall et al. 1990). roles of the mangrove forests in interactions with neighboring coastal and marine ecosystems in indonesia are summarized in table 2. there are features in common, including physical, chemical characters and effects of seasonality, of the mangrove areas as habitats for fish and other aquatic animals. for certain species of fishes, areas of higher salinities may provide suitable habitat for developing juvenile fishes. also, the fish of muddy estuaries show affinities to deep-sea fauna in some of their features. some genera of clupeids and polynemids have prolonged fin-rays, and others have small-diminutive eyes. hardenberg (1950) has recorded even a species of blind sole from the rokan estuary. the organic nitrogen compounds of the falling leaves of mangroves are either incorporated into the sediment or converted into peat for detritus feeding shellfish, or broken up to become food for bacteria, fungi and finfish. the importance of bacteria both as a food source in marine and estuarine sediments and as mineralizing agents has been widely commented upon. thus exported detritus also provides an important habitat for juvenile prawns in near-shore sub-tidal mudflats (daniel and robertson 1990). johannes (1978) pointed out that many fish in tropical marine environments move inshore to spawn. in this context, he mentioned the gerridae, sparidae, engraulidae, pomadasydae and sciaenidae, all of which are demersal coastal forms and spawn in estuaries. for example, in the palau (8° n, 135° e) spawning migrations, only herklotsichthys sp. migrated from its usual seagrass and/or lagoon habitat to spawn in mangrove creeks (johannes 1978). one common mangrove-deniser, lutjanus argentimaculatus, migrated to the lagoon and reef slope to spawn. lethrinus sp and siganus lineatus were the only other fish which occasionally inhabit mangroves that migrated to spawn on reef slopes. estuaries play a role in energy transfer between a river and the sea, which is especially important for many commercial coastal fishes whose fish larvae and juveniles are dependent on the estuary as a nursery and feeding grounds. as discussed by hambrey (1996a, b), the nursery function of mangrove ecosystem is likely to be highly variable, with some areas being of far greater value, and others of far lower value than the global estimates would suggest. for example, mangrove swamps of reef flats are generally important as nurseries for mullet (mugilidae) and baitfish (clupeidae and others), and, together with inland mangroves, are a source of crab (for instance scylla serrata and sesarma spp.). the dominant mullet in all 18 fisheries associated with mangrove ecosystem in indonesia sukristijono sukardjo mangrove creeks is liza subviridis. large mullet, valamugil sp and l. argentea appear to be more abundant in the open main channels of indonesian mangrove system. ambassis gymnocephalus is the dominant zoo-planktivore in mangrove creeks. thus, mangroves are often considered to be important to coastal fisheries both in terms of their role as breeding and nursery grounds for various fish and prawn species, and as a source of food. table 2. roles of the mangrove forest in indonesia: an experience with the mangrove forests in south china sea, east kalimantan, south sumatra and segara anakan cilacap. physical interactions nutrient interactions biotic interactions 1.1. filtering terrestrially derived sediment, and 1 .2. reduction in sediment load reaching the sea. 1. export of dissolved and paniculate organic matters (dom, pom). 1 . export of mero-planktonic larvae to marine food chains. 2.1. buffering salinity changes. 2.2. reduction in volume of freshwater reaching the sea. 2. sinks for paniculate organic matters (pom). 2.1. provision of feeding habitat (pfh) -marine invertebrates and fin fish (diurnal and seasonal migration). 2.2. pfh seabirds, shorebirds, marine mammals. 3.1. provision of juvenile (nursery) habitat (pjh) crustaceans (shrimp, lobster) and mollusks. 3.2. pjh fin fish (reef fish, sea-grass bed fish, pelagic). 3.3. pjh — seabirds and shorebirds nesting (also roosting). 3.4. pjh marine mammals (dolphins). 2. aquatic ecology the ecological importance of mangrove forests (e.g., thayer et al. 1987; robertson and duke 1987; 1990) in estuarine and coastal ecosystems throughout the world is well established. estuaries in indonesia contain diverse habitats, including mangrove forests, sea-grass beds, mudflats and open water channels, all of which may differ in depth, structural heterogeneity, substratum type and tidal exposure. it is widely accepted that mangrove communities are important components of estuarine ecosystems (odum 1961; odum and de la cruz 1967). elsewhere in indonesia, mangroves are associated with estuaries; and most estuaries in indonesia are mangrove-lined for much of their lengths. thus, mangrove forests contribute a great amount of structural heterogeneity to these estuaries. mangroves in the estuaries are able to absorb inorganic compounds from fresh water runoff for photosynthesis and thus play an important role as primary producers. mangroves are 19 biotropia no. 23, 2004 a source of primary productivity for food webs that serve as a base for the production of marine and estuarine organisms such as lobsters, finfish and shellfish. mangrove estuaries and creeks usually have significant salinity fluctuations. the great changes in salinity occurring during rainy seasons may cause periodic mass mortality of marine organisms. on the other hand, changes in salinity will also result in soil salinity of root zones in the mangrove forests. the mangrove forests and/or association in indonesia may be considered of great importance too, for example, the south china sea, including the estuaries, adjacent coastal waters and the offshore waters, as its high productivity probably makes it the major source of organic matter. these communities act as nurseries for many organisms and produce large quantities of organic matter (e.g., litter-fall) which forms the base of estuarine food chains. moreover, their detritus and nutrients are exported out of the ecosystem through tidal flushing and these form a food base for marine micro-organisms which in turn support the valuable estuarine and near-shore fisheries. the vast mangrove system in indonesia may be considered as very important in terms of high productivity and as a major source of nutrients to the ecosystem of the estuaries, the adjacent waters and the offshore waters. in apar bay east kalimantan, sukardjo (1995) found that the total flux of organic matters (woody detritus and other mangrove litter components) estimated to be about 21.11 to 29.34 dry weight t/ha/year. this is exported to adjacent waters in the form of fine suspended materials. in florida, ray (1974) found that 90% of water-borne debris within the mangrove zone is derived from mangrove vegetation. about half of this is exported to adjacent waters in the form of fine suspended material so that about 35-60% of all suspended matter on the off-shore olithic banks is of mangrove origin. the waters in the deltas, as well as those inshore, carry a large amount of suspended, as well as large plant materials, such as leaves, branches and trunks. teredinid mollusks play an important role in many mangrove swamps by rapidly breaking down wood and releasing nutrients into the food chain. since teredinids use wood as a substratum for burrowing and, in many species, as food (turner 1966, rayner 1977), the numbers of teredinids may be directly related to the amount of wood available. on the other hand, the major food of sesarmid crabs in mangrove forests is leaf litter (e.g., malley 1978). these crabs carry leaves down their burrows as well as consuming them on the sediment surface, and macnae (1968) suggested that feeding by sesarmid crabs may account for the scarcity of leaves on the floors of mangrove forests. it is interesting than that in inland mangroves most of the detritus must be broken down in situ. the high productivity of associated estuaries and banks is due to such suspended matter. in indonesia, my field observations have shown that fish in the mangrove environment utilize virtually all sources of food available, as follows : 1. insects and fruits from mangroves and from terrestrial sources (e.g., toxotes and some catfish). 20 fisheries associated with mangrove ecosystem in indonesia sukristijono sukardjo 2. detrital mud (e.g., scatophagus, mullets, some catfish). 3. small invertebrates (e.g., gudgeons, gobies, ambassids, anchovies, etc.). 4. prawns and crabs (e.g., polydactylus, johnius, pristia, lates calcarifer, kurtus, scutengraulis, some catfish). 5. mollusc (e.g., cinetodus,acanthopagrus, tokifugu). 6. fish (e.g., carcharhinus, lates, johnius, polydactylus, some catfish). some fish are primarily estuarine, with the major portions of their population residing in the mangrove areas. others appear to enter the estuaries from either the sea or rivers mainly to forage, while others again pass through the area on breeding migrations or breed within the area or in adjacent coastal waters. those forms which breed within the mangrove belt or in the adjacent sea frequently utilize the area as nursery grounds for juveniles during various stages of development. marine and riverine forms penetrate the mangrove zones to varying degrees either as small peripheral populations or occasional individual stragglers. table 2 summarizes the data available in the reports. it gives some idea of the inter-linking of the mangrove community with the adjacent marine and riverine communities and the presence of juveniles indicates which species utilize the mangrove area for breeding or nursery grounds. thus, the productivity of the mangrove ecosystem is not measured simply by the productivity of plant material by mangroves, but must include the production of fisheries that are dependent on the mangrove detritus. 3. ecological productivity of mangroves as habitat and fisheries in indonesia, mangroves represent the dominant soft bottom plant communities of the marineterrestrial transition in the coastal zone; and considered to be areas of high primary productivity which support economically important detrital-based marine food webs. as a consequence of the range of spatial and temporal variation in physical and chemical factors found in such environments, the biological communities display remarkable adaptations which permit them to survive under such harsh environmental condition. for instance, the extreme ranges of ph, do, temperature and salinity are caused by freshwater influx or conversely, by the shallowness of mangrove waters and high incident radiations are common features. the plant species are members of terrestrial families which have adaptations to survive under conditions of high salinity, low oxygen and nutrient availability in the soil, wind and wave action, and substrate instability. the animals are, like the plants, representatives of largely terrestrial groups. in contrast the aquatic animal community is dominated by members of essentially marine families which are adapted to viable salinity, turbid conditions and to feeding directly or indirectly on materials from the dominant primary producers. in a global context, mangroves are one of the more productive ecosystems in the world in terms of both primary productivity and the productivity of marine animals. the primary productivity is manifested in terms of the accumulation of 21 biotropia no. 23, 2004 biomass (wood, root etc.) and the production of leaves. in indonesia, sukardjo < yamada (1992) reported that the biomass of r. mucronata amounted to be 93.73 dry weight/ha and mangroves produce a large quantity of organic debris in the fo of leaf litter, viz 7.56-10.82 dry ton/ha/year (sukardjo 1995). generally, the tc biomass, height, litterfall, decomposition, and fresh water turnover increase fn dwarf to riverine mangrove forests (pool et al. 1975, 1977; brown and lugo 198 the significance of this detritus in the food web has been recently focused upon some workers (leh and sasekumar 1980; thong and sasekumar 1980). mullet a some juvenile fishes, and penaeid shrimps utilize this material in mangrove estuar almost exclusively. leaves which are continually being shed by mangroves under decomposition and result in pom (particulate organic matters) and do (dissolved organic matters) that enter into the near-shore estuarine or mari environment. the pom, which consists of small particles, is consumed by larval a; juvenile marine organisms that utilize the mangrove habitat as a nursery and feedii ground. the dom, which consists of a wide range of soluble organic compounds, consumed by filter feeders (e.g., clams, oysters, mussels, etc.) that are common the near-shore environment, and who retain the phyto-nano-and micro-plankton th is vastly composed of primary producers who need dom to synthesize organ matter. the production of bacterial biomass from dom is an important step carbon and energy flow through marine environments and potentially serves as tl mechanism by which dilute dissolved organics become available to metazoc trophic levels (azam et al. 1983). in addition, any organic material that transported seaward is flocculated by the increasing salinity. the flocculated organ matter (fom) becomes a substrate for benthic feeders and scavengers. therefor mangroves are known to be very productive in terms of primary organic mated! and recycling of mineral nutrient (lugo and snedaker 1974). also, the richness c mangroves in organic materials provides a wide variety of food at different levels c the food web for organisms that will , in turn, serve as fish food (beumer 1978 mugil cephalus and cyprinodon variegatus are mentioned as such. also, mud crat scylla serrata occupies a niche between the primary producers and tertiar producers and is thus, an important organism in the energy-flow of the mangrov ecosystem. sukardjo (1995) reported that the canopy closure by mangrove trees range 87.70-99.00%. macnae and kalk (1962) considered that shading by mangrove tree protects animals, particularly juvenile and larval forms, in shallow water (e.g. mangrove creeks) from direct sun. in addition, the complex and entangled mangrovi roots and stems offer refuge and protection from predators. thus, the importance o mangroves as habitat for fish and prawns continues to be a topic of debate amonj marine ecologists (macnae 1974; robertson and duke 1987). the detritus is eithei refractory and sinks to the bottom, presumably never enters the food chain, or is dispersed over a large area without any local impact (e.g., magnification of benthic secondary production). however, it should note that marine ecologists generall} agree that inshore areas constitute a crucial part of the life support systems oi offshore populations by providing nursery and feeding areas (jansson et al. 1988). 22 fisheries associated with mangrove ecosystem in indonesia sukristijono sukardjo therefore, biologists have recently paid much attention to the mangrove areas as nursery areas for shrimp and fish, such as certain species of penaeids that are dependent on mangrove forests during their juvenile stages. these species include penaeus monodon, p. indicus, p. merguiensis and most species of metapenaeus, p. semisulcatus and p. latisulcatus spend larval and juvenile stages. the life cycle of these prawns and environmental factors influencing it, are summarized in figure 1. it has been shown that the mangroves and the environment they create is vital to the continuation of many commercial penaeid prawn fisheries. also, many commercially important fishes such as chanos sp., mtigil sp., hilsa sp., and pomadasys sp. use mangrove water ways as nursery grounds. moreover, mangrove communities function as a solar-powered, tidal subsidized and pulse stabilized ecosystem (odum 1974). there is a large energy surplus in mangroves in the form of detritus. the detritus forms the basic component in the marine primary consumers diet, and thus, a basic support for many commercial fisheries. therefore, the role of mangroves as a habitat for fish and prawn is interesting, even today. p. merguiensis is an important component of the commercial catches of penaeid prawns throughout indonesia. the population dynamics of juvenile p. merguiensis in estuarine systems in indonesia have been studied intensively by toro and sukardjo (1990), and mangrove-lined mud banks have been shown to be the main nursery area for this species. however, until recently, little research had been carried out on the behavior of juvenile p. merguiensis or the degree to which they utilize the mangroves. from the inventory of various sources, hundreds species of fish and prawn belonging to residents and non-residents will be found in the mangrove environment in indonesia (table 2). but not all non-resident fish found in mangroves inhabit them as fry or juveniles. in indonesia, six habitat groups can be identified in the mangrove environment. not all of the species will be found in any one of the habitat group. different resident species occupy different habitat in the mangrove ecosystem (table 3). for instance, in indonesia generally, the mudflat fish community consisted of mainly ambassids, ariids, clupeids, cynoglossids, engraulids, mugilids and sciaenids. five residents species found only on the mudflats were the catshark hemiscyllium indicum, the grey mullet liza argentea, the silver pennah croaker pennalia argentata, the spotted croaker protonibea diacanthus and the anchovy stolephorus macroleptus. also, the mangrove inlets and creeks were dominated by schooling fish species belonging to the families ambassidae, eleotridae, engraulidae, clupeidae, leiognathidae and mugilidae. among those, the dominant species in terms of weight were arius sagor, ambassis gymnocephalus, liza subviridis, toxotesjaculator, sphyraena barracuda and lates carcarifer. by using gillnet, 70% of the total catches consisted of sardinella melanura, thryssa kammalesis, t. hamiltonii and stolephorus indicus. also, several demersal fish species reportedly inhabit the creeks. thus, only 29 species are recorded to be commonly found in all of the habitat groups (table 4). 23 figure 1. summary of the factors afecting penaeid prawn in the mangrove ecosystem       biotropia no. 23, 2004 table 4. (continued) species family mangrove creeks & inlets mudflat near inshore waters far inshore waters 14. saurida tumbil synodontiidae + + + + 15. sccutor insidiator leiognathidac + + + + 16. sctipinna taty clupcidae + + + + 17. sillago sihama sillaginidac + + + + 18. stigmatogobius sadanundio gobiidac + + + + 19. stolcphorus tri engraulidae + + + + 20. tcnulosa sinensis clupeidac + + + + 21. thcrapon jarbua theraponidae + + + + 22. thryssa hamiltonii engraulidae + + + + 23. t. kammalcnsis engraulidae + + + + 24. trichiurus savala trichiuridac + + + + 25. trypauchcn vagina gobiidae + + + + 26. mctapenacus affinis penaeidac + + + + 27. m. brevicornis pcnacidae + + + + 28. parapcnacopsis sculptilis penaeidac + + + + 29. pcnacus merguiensis penaeidac + + + + discussions and concluding remarks: an indonesian perspective a positive correlation between near-shore catches of shrimp or fish and mangrove area has been documented for indonesia (martosubroto and naamin 1977), malaysia (gedney et al. 1982), australia (staples et al. 1985), the philippines (camacho and bagarinoa 1987) and elsewhere (cf. table 5). also, there exists an obvious link between the fishery in the immature mangrove system and the one in the adjacent marine system. fish populations in both estuaries and mangrove ecosystems can be abundant with a wide diversity of species. it is well known to fishermen that many species of fishes occur in mangrove estuaries and creeks, and it has been observed many times that these systems act as nursery areas for larval and juvenile fishes. a comprehensive listing of fish species that actually spend their juvenile years in mangrove environments has yet to be made. nor is it known to what degree the relationship is obligate or facultative for particular species. much of the evidence is circumstantial. moreover, the structural heterogeneity of habitats in tidal waters has been shown to provide fish with protection from predators and 28 fisheries associated with mangrove ecosystem in indonesia sukristijono sukardjo table 5. quantified relationships between mangroves and coastal resources. no. study site formula references remarks 1. indonesia y = 0.1 128 x + 5.473 (r2 = 0.79, n = na) martosubroto and naamin 1977 y = shrimp production (x 1000 tons) x = mangrove area (x 10,000 ha) 2, philippines y = 0,8648x + 0.0991 (1^ = 0.66^= 17) paw and chua 1989 y = log 10 of penacid shrimp catch (tons) x = log 1 0 of mangrove area 3. 38 regions of the world log 10msy = 0.4875 logam-0.0212l + 2.41 pauly and ingles 1986 msy = maximum sustainablc yield of pcnacids am = area of mangroves l = degrees of latitude 4. australia y= 1.074x + 218.3 (r2 = 0.58, n = 6) staples et al. 1985 y = banana prawn catch (tons) x = mangrove shoreline (km) 5. northeastgulf of mexico and in lousiana y= 1.96x-4.39 (r2 = 0.92, n = 7) turner 1 977 y = percentage of brown shrimps x = % of saline vegetation in an hydrological unit 6. 27 locations in asia, america and africa no equation given (r2 = 0.69, n = 5; 8) in: turner 1977 y = annual shrimp yield x = hectare of vegetated estuary 7. gulf of mexico ln y = 0.496lnx + 6.070 (r2 = 0.48,n= 10) yancz-arancibia et al. 1985 y = fish capture (tons) x = coastal marshes in km2 enhanced feeding opportunities (rozas and odum 1988). indeed, the supposed connection between mangroves and juvenile nekton is often advanced as one of the key arguments for the conservation of mangrove forests in indonesia. unfortunately, the use of fringing mangrove habitats by commercial and recreational fishery in indonesia has not been well documented. also, no reliable catch statistics are available for both commercial and recreational fish production from mangroves. moreover, in indonesia, very little and/or no listing is made of various teleost families, such as the apogonidae, bothidae, chandidae, chanidae, dorosomidae, elopidae, engraulidae, gerridae, latidae, leiognathidae, megalopidae, platy-cephalidae, pseudomugilidae, soleidae, syngnathidae and toxotidae, all of which 29 biotropia no. 23, 2004 contain species occurring in mangrove systems. while the ichthyo fauna occurring within mangrove environment may be broadly categorized into those fishes that exist there permanently, those that intermittently enter them as adults, and those that seasonally occur there as eggs, larvae, or juveniles. thus, one of the principal reasons for rehabilitating mangrove ecosystem in indonesia is to increase biodiversity and conserve the ecosystem. mangroves are often considered as an ecosystem per se, due to their strong specificities (twilley et al. 1996) but they belong to intermittently brackish tidal zone and can be seen as a part of estuarine systems. young fish utilize estuaries, near shores marine areas and mangrove ecosystem in order to benefit from the availability of food and perhaps also to gain protection from predators. in indonesia, estuaries can be considered ecosystems because they are composed of numerous subsystems or habitats. the term habitat refers to the place occupied by an entire community of organisms. thus estuaries are known to be important as feeding, nursery, or habitat areas, in the life cycles of many fish species, while the role of estuaries, particularly mangrove, in producing commercially important fishes is being recognized, their value for fishes of little or indirect commercial or angling importance has not been accepted. it seems that estuaries, all over the world are mainly nursery for many marine animals. as a matter of fact, the problem of exact relationship existing between ichthyology and mangroves is far from being solved. even in temperate estuaries there are groups of important fishes which spend a part of their life either in the estuary itself or in the ocean during their life cycles. in recent years, the importance of the mangroves is well established (e.g., critical feeding, rearing and nursery habitat for economically important marine species, providing a buffer against storms, filtering pollutants from upstream sources, and preventing coastal erosion). also, quantitative relationships between fish yield and area of mangrove have been well established. for instance, according to turner (1977) who has analyzed the data for 27 locations in america, asia and africa, the abundance of penaeid shrimp is directly related to the absolute area and type of estuarine vegetation. however, in indonesia, the degree of importance to commercial ecological functions of the mangroves are often under pressure from economic uses of this coastal area. for instance, conversion of mangrove forest which destroys the natural nursery grounds of fish and shellfish aggravated by the conversion of inland freshwater swamp forests which diminished the area of freshwater habitat and affects the supply of water and nutrient to the spawning and nursery areas. studies in mangrove communities (e.g., austin 1971; odum and heald 1972; lasserre and taffort 1977; janez-aranbicia et al. 1980; bell et al. 1984) have generally concluded that the fish fauna has a low species diversity and high proportion of temporary residents occurring mainly as juvenile. there is however, little evidence of fish spawning or breeding inside mangrove areas. recorded evidence refers only to gobiid fishes (penridge 1971) including mudskippers (boleophthalmus, periophthalmus, periophthalmodon, scartelaos). the abundance 30 fisheries associated with mangrove ecosystem in indonesia — sukristijono sukardjo and species composition of fish and crustaceans in mangroves and their adjacent near-shore habitats (sea-grass, sand-flats, mudflats etc.) at several sites in particular islands of indonesia are necessary to answer the questions regarding mangroves-fishery relationship. for instance, the correlation between offshore prawn catches and area extent of mangroves (macnae 1974; turner 1977; martosubroto and naamin 1977) has been suggested to be indication of the dependence of juvenile prawns on mangroves rather than of a food chain link (hatcher et al. 1989). though prawns offshore did not carry the mangrove carbon signal, it was shown that several species of juveniles prawns collected from a mangrove inlet consumed mangrove -derived carbon (rodelli et al. 1984). another example is, the environmental conditions in the estuaries are not conducive to the survival of the first-stage zoea of scylla serrata and berried females, therefore, migrate to sea to spawn. thus, program aimed to test the belief that mangroves as major nursery grounds for juvenile's fish and crustaceans are needed. in indonesia, since mangrove forests sustain marine life in estuaries and ponds and act as shelter belts against tropical cyclones, the fresh water needs of mangrove forests should be evaluated in water management practices along the coasts. moreover, the estuarine is one of the common features in the coastal mangrove belts, and represent an important zone for many marine organisms to feeding, breeding, spawning, and other behavior. in addition, it is essential to determine, for a given local fishery, the real dependency of fish resources on estuarine environment by answering the following questions in regard to the relation with fishery, as follows: 1. is the estuarine zone essential for a given species? 2. are there alternative areas for its development? 3. which parameters are critical in its life-history? 4. what are its trophic relationships with other species? in indonesia, major fishing grounds are located in the coastal areas in east sumatra, south and east kalimantan, and south irian jaya where there are extensive virgin mangrove forests (table 6) (chong et al. 1990). fortunately, ecological information for fisheries fauna associated with mangroves in indonesia is scant. fisheries resources in the mangrove environment have usually been examined in terms of their economic potential, and thus only the commercial species such as p. monodon has received only detailed bioecological study (e.g., toro and sukardjo 1990, 1995). the prawn species of creeks were represented by juvenile penaeus penicillatus, p. merguiensis, p. indicus, p. monodon, metapenaeus brevicornis and m. ensis (affinist). also, sediment type and organic carbon have been known to influence the distribution of prawns (e.g., william 1958; branford 1981; toro and sukardjo 1997), as do the effects of salinity (e.g., gunter et al. 1964; mair 1980 ; dall 1981) and the presence of coastal vegetation (e.g. young 1978; de freitas 1986). i he fluctuations and success of prawn fisheries appears more dependent on the juvenile stage, and the conditions in the mangroves which influence it (e.g., salinity, water discharge, sediment type etc.), than any other part of its life history. biotropia no. 23, 2004 thus, the small number of prawn species as observed in mangrove habitats in indonesia could be due to the following three factors: 1. niche occupation being limited to the epibenthic surface of the substrate. 2. the substrate being comparatively homogenous (in this case, a largely silt-clay one), and 3. a lack of euryhaline species. traditionally mangrove ecosystems have provided some of the richest fishing grounds for much of the islands in the country. also, fishery activity within the mangroves is mainly at the subsistence level (cf. table 6). for instance, crabs belonging to different genera and species are abundant along the estuarine shores and mangrove swamps, but those of economic importance affording minor fisheries are the swimming crabs belonging to the genera scylla and neptumis. scylla serrata, neptunus pelagicus and n. sanguinolentus are the most common forms. therefore, methods and sampling time, and type of nets will catch different species composition by weight and diversity. fishing gear utilized includes fixed traps (sero and kelong), hook and line, cast nets, gillnets, beach seine, trammel net, stationary lift net and stationary tidal seine (logo), e.g. in muna (table 7). the gill nets for instance, were not effective in catching the demersal fish and prawns. main demersal fish caught are ariidae, carangidae (excluding megalaspis spp. and decapterus spp.), leiognathidae, lutjanidae, polynemidae, sciaenidae, serranidae. main pelagic fish caught are rastrellinger spp., megalaspis cordyla, decapterus spp., and sardines (clupeidae). multiple tidal nets are usually set up in the main river streams for catching shrimp.trammel nets are used in river mouths for catching coastal shrimp such as p. merguiensis, p. monodon and m. brevicornis. traps (sero) are placed at river mouths or at the edge of the mangrove forests. crab nets are used at the river edge or in small canals and streams for catching mangrove crabs. for instance crab nets are very valuable and efficient economically for fishermen in muna (tables 7, 8). thus, the people who live within the mangrove ecosystems and exploit them have adapted their life-style and landuse patterns in a variety of ways along the indonesian coasts, e.g., in south sumatra for general illustration (table 9). the results of investigations of fisheries fauna associated with mangroves in indonesia enable some generalizations to be drawn regarding the structure and composition of mangrove associated fish communities. such communities appear to be characteristically composed of 13 to 14 families with only 10 families recorded being dominant (tables 2-4). some fish families, whose taxonomy is still under review, such as mugilidae (thomson, in lal et al. 1984), leiognathidae and lutjanidae (alien, in lal et al. 1984) and sphyrocnidae (rose, in lal et al. 1984). fao (1994) reported that 64 fish species, 74 crustacean species and 71 mollusk species were harvested from the mangrove area in malay peninsula. the main facts that emerged from the discussions on the fisheries associated with mangroves were as follows : 32 fisheries associated with mangrove ecosystem in indonesia sukristijono sukardjo 1. the life cycles, reproduction cycle, food habits and growth rates of mangrove species are poorly known and have only recently been the object of attention in relation to the mangrove ecosystem. the species being investigated are those of economic importance, such as sesarmids crabs, penaeid prawn, fin fish and certain molluscs. 2. research on food webs indicates complex pathways, which qualitative measurements of conversion efficiency are available. 3. migrations, behavior, eco-physiology and habitat requirements of species are partly known but generally poorly understood. 4. studies on the relationships with adjacent ecosystems such as sea-grass beds and coral reefs are being studied, but only a few are described in any detail. 5. the question was addressed as to what extent forest structure has an effect on fauna populations and trophic relationships, and it was re-emphasized that more studies are urgently needed on this area. 6. the often quoted factors which influence fisheries potential and production in mangrove systems are only described, seldom experimented upon and not quantified. 7. the level of assimilation efficiency has been measured for a few species of animals but measurements are still crude. at this stage we are still looking at quantitative rather than qualitative changes. 33 biotropia no. 23, 2004 table 6. fishery regulations (applied to all of indonesia): gear and vessel restriction. zone (distance from shoreline) vessel size and power maximum gear not allowed in zone 1 : 0-3 miles 5 gross tons, 10 hp purse seines, nets > 120 m length 2: 3-7 miles 25 gross tons, 50 hp nets > 300 m length 3: 7-12 miles 1 00 gross tons, 200 hp nets > 600 m length 4:> 12 miles no limit no limit table 7. fishery production in the mangrove forest in muna project sites in 1996. fishing gear (100% used in the mangrove area) number of fishing gear (unit) number of fishermen (h.hold) production (t/year) economic value (rp/year) 1. togo (multiple tidal net) 60 40 87.70 500,976,000 2. jala (cast net) 8 8 0.52 2,254,000 3. rokkang (crab net) 1,300 260 135.20 540,800,000 table 8. fishery product from mangrove forest by species in muna fish collector in 1996. species production (ton/year) prize (rp/kg) economic value (rp/year) 1. shrimps 7.30 5,500 40,150,000 2. scylla scrrata 60.00 4,500 270,000,000 3. portunus pclagicus 57.36 1,000 57,360,000 4. sea cucumber (teripang) 8.50 25,000 212,500,000 fisheries associated with mangrove ecosystem in indonesia sukristijono sukardjo table 9. coastal zone resource use compatibility matrix: compatibility of resource use options in south sumatra (numbers correspond to resource use). c = complementary, h = harmful, e = exclusive sector item 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 fisheries 1 . coastal fishery * 2. inland fishery * 3. aquaculturc h e * forestry 4. large scale forestry h c * 5. husbandry of nipa h e * 6. husbandry of nibung c h e c * 7. husbandry of jelutung c e c c * 8. crocodile hunting c c e e c c c * 9. monitor lizard trapping h * agriculture 10. transmigration scheme h e h h h * 1 1. irrigation scheme e # transport 12. port construction h h * tourism 13. small scale tourism c c c * conservation 14. habitat & endangered species c c e e c c h h h h c * 15. migratory shore birds c c e c c h h h c c * 1 6. breeding water birds c c e c c h h c c c * acknowledgments the author is very grateful to anonymous reviewers who read critically the manuscript. biotropia no. 23, 2004 references anonim. 2003. kebijakan dan stratcgi pengelolaan pesisir dan laut (ocean policy). makalah disajikan pada lokakarya 10 tahun pengelolaan lingkungan pesisir dan laut di indonesia: 1993-2003. jakarta 20-21 oktober2003. austin, m.h. 1971. a survey of the ichthyofauna of the mangroves of western puerto rico during december 1967august 1968. caribbean j. ofsci. 11:427-455. azam, f., t. fenchel, j.g. field, j.s. gray, l.a. meyer-reil and f. thingstad. 1983. the ecological role of microbes in the sea. mar. ecol. 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(cd.), estuarinc perspectives, 465-485. ny and london: acad. press. yanez-arancibia, a., g. soberon-chavcz and p. sanchcz -gill. 1985. ecology of control mechanisms of natural fish production in the coastal zone. in: yancz-arancibia, a (cd.), fish community ecology in estuaries and coastal lagoon towards an ecosystem integration, 571-595. unam press, mexico. young, p.c. 1986. morcton bay, queensland: a nursery area for juvenile penaeid prawns. aust. j. mar. freshwater res. 29:55-75. 39 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf abiotic factors influencing mantangan ( ) invasion in bukit barisan selatan merremia peltata national park jani master , soekisman tjitrosoedirdjo and ibnul qayim1* 2 3 1department of biology, faculty of mathematics and natural sciences, universitas lampung, bandar lampung 35145, indonesia 2weed science society of indonesia 3department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, bogor 16680, indonesia received 20 january 2015/accepted 27 january 2016 abstract bukit barisan selatan national park merremia peltatasome areas in have serious problems related to (family convolvulaceae) invasion. the abiotic factors influencing invasion were investigated in this study. this m. peltata research compared abiotic factors in three forest locations with different invasion levels i.e. severe, moderate and mild invasions. abiotic factors measured were percentage of tree canopy coverage, micro climate factors (temperature and humidity), elevation above sea level and physical and chemical properties of the soil surface. in this study, canopy coverage is classified as abiotic factors because it influences the intensity of light that can reach the forest floor. a posthoc duncan's multiple range test (dmrt) was employed to determine significant differences ( <0.05) among p abiotic factors. in addition, a test of correlation and multiple linear regression were conducted to determine the relationships between abiotic factors and invasion. regression testing showed that canopy coverage multiplem. peltata significantly ( <0.05) influenced invasion. based on the generated model, a 1% addition of canopy coverage p m. peltata would decrease invasion by 2.8% multiple linear regression analysis was used to predict the relationship m. peltata . between soil nutrients and invasion. the c/n ratio and p o , ca, mg and na concentration significantly ( <0.05) 2 5 p influenced invasion level. again, based on the generated model, the increase in the c/n ratio as well as in p o and ca 2 5 concentrations were correlated with the of invasion; while the increase of mg and na reduction m. peltata concentrations were correlated with the increase of invasion. invasion in bukit barisan selatan m. peltata m. peltata national park was influenced by opened forest canopy and was correlated with high concentration of na and mg in the soil of the invaded forest areas. : bukit barisan selatan national park, invasive species, mantangan, keywords merremia peltata introduction indonesia is a country with abundant flora and fauna, some of which are conserved and protected in conservation areas such as national parks. existing national park areas in indonesia are threatened by alien plant species that invade and threaten flora and fauna biodiversity. alien species that have the ability to grow quickly so as to impact biodiversity in invaded areas can be referred to as invasive alien species (ias) (pyšek & richardson 2010). broadly, an ias is a species that has the potential to harm the economy, environment and/or cause damage to human, animal or plant health (national invasive species council 2006). merremia peltata mantangan or in the local language, is an invasive species of conservation areas. this species causes severe problems in bukit barisan selatan national park (bbsnp) with more than 7,000 ha covered by m. peltata (master 2013). infestation of the weed is et al. suspected to be the cause of animal migrations, such as tiger, elephant and sumateran rhinoceros, to rural areas up north (irianto & tjitrosoedirdjo 2010). analysis of landsat and advanced land observing satellite (alos) images indicated that m. peltata invasion is found from south part of biotropia vol. 23 no. 1, 2016: 21 27 21 doi: 10.11598/btb.2016.2 . .3 1 457 * c orresponding author: gmail.comj.janter@ 22 bbsnp running 33 km to the north of the national park. it is also found in small outbreaks as far as 50 km north of bbsnp (master 2013).et al. the bbsnp is the third biggest conservation area in sumatera island covering an area of 356,800 ha. administratively, this area belongs to lampung barat, pesisir barat and tanggamus districts of lampung province and the bengkulu district of bengkulu province, (4 31' – 5 57' s; 0 0 103 34' – 104 43' e) (gaveaua 2007).0 0 et al. m. peltata is a liana (climbing plant) of the convolvulaceae et al. family (paynter 2006), and it is distributed throughout madagascar, the mescarenes, seychelles, malay peninsula, malaya islands, philippines, new guinea, northern au s t r a l i a a n d t h r o u gh o u t po l yn e s i a (ooststroom & hoogland 1953). it is similar to sweet potato in morphology, with a wide heart shaped leaf connected by peltate petiole, in contrast to the petiole connection of sweet potato. the hairless plant stem grows to 20 m in length and excretes white liquid when injured. it may grow over other plants (stone 1970). the corolla is white or yellow, 5–6 cm in length and forms a bell-shape corolla (fosberg & sachet 1977). spreading of this plant occurs generatively, both by seeds or vegetatively from roots along its stems which touch the ground. egetative growth v rates reach 16 cm/weekcan (pengembara 2014). environmental factors, both biotic and abiotic, determine the presence of a species in a particular location (huang 2003). et al. accordingly, not all alien species introduced to an area will become invasive; this can be influenced by factors including environmental fitness and the presence or absence of predators or competitors (theoharides & dukes 2007). the rapid growth of in bbsnp is probably caused by a m. peltata number of abiotic factors. the objective of this research was to determine abiotic factors influencing invasion in bbsnpm. peltata . materials and methods erremiam peltata is found mostly in southern regions of bbsnp, but not all of in this m. peltata region grows densely. to determine abiotic factors influencing plant invasion, environmental factors were recorded in three forest locations with different invasion levels, i.e. severely invaded, moderately invaded and mildly invaded forest (fig. 1). first location (severely invaded forest) is close to the village enclave way haru, bandar dalam and tampang. based on land cover maps, this site has a decreasing forest cover for an area of 2,565.54 ha from 2000 to 2009. presumably these changes were the result of forest clearing biotropia vol. 23 no. 1, 2016 figure 1 map of research location of invasion in bukit barisan selatan national park, lampung, sumatera m. peltata (master 2013)et al. abiotic factors influencing mantangan ( ) invasion jani mastermerremia peltata – et al. 23 for plantations (prasetyo 2011). the second et al. location (moderately invaded forest) is a post-fire forest in 1997; fires that occurred mostly on the forest floor affecting the lining of plant and open up some forest canopy (wcs-ip 2001). the third location (mildly invaded forest) is a primary forest. selection and classification of these invasion levels were based on coverage of from m. peltata landsat and alos image analyses in 2002 and 2008 (master 2013). the final selection was et al. conducted by recording the percentage of m. peltata canopy coverage by using plot of 20 x 20 m area. three line transects of 1,000 m each, with minimum of 200 m interval distance between each line transect, were made in each forest location. plots of 20 x 20 m were sampled along each line transect, at 100 m intervals, thus totaling 30 plots in each location. invasion was m. peltata estimated based on the percentage of plot that was covered by . environmental factors m. peltata recorded for each plot were: 1. the percentage of tree canopy coverage (tree canopies that were more than 4 m in height) which was estimated using a concave densiometer; 2. microclimate (temperature and humidity) were measured at noon, 1 m above the ground using thermometer and hygrometer on each plot; 3. elevation above sea level; and 4. physical and chemical properties of top soil using composite method (crozier . et al 1998). soil physical and chemical properties observed were soil texture, ph (h o), kcl, c-2 organic, n-kjedahl, cation exchange capacity (cec) and exchangeable cations (k, na, ca, mg). soil texture fraction comparisons were separated as follows: 1. sand fraction having particles with diameter of 2 mm 50µ; 2. loam fraction having particles with diameter of 50µ 2µ; and 3. clay fraction having particles with diameter of <2µ) (hardjowigeno 2010). data for each environmental factor from each of the three locations were analyzed using anova and the mean differences were tested using the duncan's multiple range test (dmrt) at <0.05). tests of correlation and linear p regression were carried out using spss 12 program to determine any relationships existed between abiotic factors and invasion.m. peltata results and discussion percentage cover of invading m. peltata forests m. peltata the mean coverage percentage of in the mildly invaded forest ranged from 1 to 15%. in comparison, the mean coverage percentage in the moderate invaded forest ranged from 27 to 55%. in the severe invaded forest, the mean coverage percentage was 44%, while in some location the percentage was up to 100%. abiotic factors (non-soil) and m. peltata invasion levels of the six abiotic factors observed i.e. air temperature, air humidity, ground temperature, soil humidity, soil ph and percentage of tree canopy coverage (canopy above 4 m), only the percentage of tree canopy coverage was significantly varied ( <0.05) among the three p locations (table 1). forest canopy coverage had significant negative correlation to m. peltata invasion. based on a generated model, every 1% addition of canopy coverage would decrease m. peltata invasion by 2.8% (table 2). the severely invaded forest locations had more open tree canopy coverage. the open canopies facilitate access of to solar m. peltata radiation such that the weed dominated these table 1 summary of abiotic factors at the three research locations location air temperature (oc) air humidity (%) ground temperature (oc) soil humidity (%) soil ph canopy coverage (%) mildly invaded forest 26.41 a 90.72 a 25.65 a 47.93 a 6.75 a 92.31 a moderately invaded forest 27.93 b 83.79 b 26.10 b 47.65 a 6.65 a 81.51 b severely invaded forest 27.15 ab 88.84 a 26.00 ab 54.05 a 6.51 a 69.89 c note: numbers followed by the same letters in same columns are not significantly different ( <0.05)p 24 biotropia vol. 23 no. 1, 2016 areas. in the mildly invaded forest, mean canopy coverage was denser (92.31%). denser canopy coverage prevented extensive growth m. peltata and development such that it only grew through gaps of canopy where there were collapsed trees. this result is consistent with other findings which reported that grew on the edges of m. peltata forest, especially in degraded land in fiji (kirkham 2005), and that this plant grew densely in logged forest and open forest in other parts of sumatera (irianto & tjitrosoedirdjo 2010). air temperature, air humidity and ground temperatures were all significantly ( <0.05) higher p in moderately invaded forest than in mildly invaded forest (table 1). the increase in these factors was most likely related to tree canopy coverage, such that more open canopies would increase solar radiation, air and ground temperatures, as well as rainfall reaching the forest floor (sasaki & mori 1981). severely invaded forest had more open tree canopy coverage, but air temperature, air humidity and ground temperatures in that location were not different to mildly invaded forest. this is probably because the ground coverage of m. peltata in both levels of invaded forest produced similar microclimate. there were no relationship between elevation above sea level and the infestation size of m. peltata. m. peltata this is despite the fact that is known to occupy lowland habitats to 300-400 m above sea level in samoa and fiji (meyer 2000, kirkham 2005). soil abiotic factors and invasion m. peltata levels beside the aforementioned environmental factors, the physical and chemical properties of the soil were also analyzed in the three research locations. the mildly invaded forest was dominated with a sandy-clay-loam soil texture, while the moderately and severely invaded forest had clay and clay-loam soil texture (table 3). soil in the secondary and invaded forests had higher clay content than soil in the primary forest. the clay fraction is the most important table 2 results of multiple linear regression between percentages of coverage and abiotic factorsm. peltata coefficientsa model unstandardized coefficients standardized coefficients t sig. collinearity statistics b std. error beta tolerance vif 1 (constant) 89.020 53.253 1.672 0.099 air temperature -2.841 3.189 0.148 -0.891 0.376 0.356 2.812 air humidity -2.782 1.668 0.281 -1.668 0.100 0.345 2.897 ground temperature 6.209 7.892 0.119 0.787 0.434 0.432 2.317 soil humidity -0.603 0.386 0.236 -1.562 0.123 0.431 2.320 soil ph -23.790 8.175 0.448 -2.910 0.005 0.414 2.418 canopy coverage -1.218 0.301 0.427 -4.047 0.000 0.882 1.134 note: a = dependent variable: percentage of m. peltata coverage table 3 soil texture samples in the three research locations location texture percentage (%) mildly invaded forest sandy clay loam 33.33 clay 55.56 clay loam 11.11 moderately invaded forest clay 44.44 clay loam 55.56 severely invaded forest clay 55.56 clay loam 44.44 25 component because its particles have more surface area than sand particles. soil with higher clay content has higher cation exchange capacity (cec) than sandy soil due to greater overall surface area of clay particles (munawar 2011). the cec value was higher in the severely invaded forest (25.67 cmol /kg), followed by moderately c invaded forest (19.07 cmol /kg), with the lowest in c the mildly invaded forest (13.51 cmol /kg) (table c 4). the cec is a chemical property closely related to soil fertility. soil with higher organic and clay contents had higher cec values than soil with low or g a n ic co nt ent an d/or s a ndy so i l s (hardjowigeno 2010). further, soil with higher cec values is able to better retain and provides nutrients (cations and anions) than soil with low cec values. the lack of cation exchange capacity in low cec soil results in the nutrients being easily washed through the soil with underground water movement (infiltration, percolation) such that these nutrients are no longer available for plant growth. the severely invaded forest had a higher alkaline density (base saturation) value (71.11%) than the other two forest locations. contributing to this higher alkaline density were the alkaline cations ca, mg, k and na. these alkaline cations are important soil nutrients required by plants. the presence of organic substances in the soil can improve soil fertility chemically, physically and biologically. the amount of organic carbon in the severely invaded forest was higher (3.46%) than that in the mildly invaded forest. based on soil fertility criteria, c-organic in the severely invaded forest belonged to high category. nitrogen (n) is a crucial soil nutrient, often found with low availability. total nitrogen in the severely invaded forest was significantly ( <0.05) p higher (0.35%) than that in the other two locations. there was no significant difference ( >0.05) of total nitrogen between the mildly p invaded forest (0.17%) and the moderately invaded forest (0.21%). higher nitrogen values contribute to low c/n ratios such that more nitrogen is available to microorganisms to decompose carbon containing material, recycling the nitrogen quickly (munawar 2011). higher nitrogen contents in plants will result in faster vegetative growth. was rarely found to m. peltata flower in severely invaded forest, and this may have been caused by high nitrogen contents in the soil. regression analyses between soil factors and invasionm. peltata double linear regression analyses were conducted to predict whether soil nutrient status was correlated with invasion. c/n ratio and concentration of p o , ca, mg and na 2 5 significantly ( <0.05) contributed to p m. peltata invasion (table 5). based on a generated model, the increase of c/n ratio and concentration of p o and ca may reduce invasion; while 2 5 m. peltata the increase of mg and na concentration may increase invasion. invasion in m. peltata m. peltata the severely invaded forest was correlated with table 4 average soil chemical properties in three research locations soil chemical property mildly invaded forest moderately invaded forest severely invaded forest value level value level value level c-organic (%) 2.07 a middle 2.62 ab middle 3.46 b high n (%) 0.17 a low 0.21 a middle 0.35 b middle c/n ratio 11.66 a middle 11.88 a middle 9.88 b low p2o5 (mg/100 g) 24.22 a middle 18.44 b low 24.88 a middle k2o (mg/100 g) 28.33 a middle 31.11 a middle 39.77 a middle p-bray (ppm) 4.00 a very low 2.76 a very low 6.53 b very low ca (cmolc/kg) 4.59 a low 5.50 a low 11.11 b middle mg (cmolc/kg) 3.04 a high 3.96 a high 6.28 b high k (cmolc/kg) 0.57 a middle 0.50 a middle 0.67 a high na (cmolc/kg) 0.41 a middle 0.46 b middle 0.51 c middle cec* (cmolc/kg) 13.51 a low 19.07 a middle 25.67 b high base saturation (%) 62.55 a high 55.11 ab high 71.11 b high notes: cec = cation exchange capacity numbers followed by the same letters in the same row are not significantly different ( <0.05)p abiotic factors influencing mantangan ( ) invasion jani mastermerremia peltata – et al. 26 biotropia vol. 23 no. 1, 2016 low c/n ratios and high concentration of mg and na in the soil. sodium has an important role in determining soil characteristics and plant growth, especially in areas close to a beach. the severely invaded forest in this study is located close to a beach. this is a suspected cause of the high na content in the soil in this location. excessive na content can also be toxic for plants (hanafiah 2007), influencing cell membrane and organelle functions, as well as disrupting plant metabolism sequences (hamim 2008). soil sodium concentrations significantly ( <0.05) increased as invasion levels of p m. peltata increased in this study. this weed is suspected of being a halophyte, being able to grow in a land with high salt content. another soil nutrient correlated with m. peltata invasion was mg. soil samples from the severely invaded forest had significant ( <0.05) higher p content of mg (6.28 cmol /kg) than either mildly c invaded forest (3.04 cmol /kg) or moderately c invaded forest (3.96 cmol /kg) (table 4). c magnesium is a crucial element used in chlorophyll formation. like other soil nutrients, magnesium deficiency results in a typical color change to leaf material. premature leaf fall is also caused by magnesium deficiency (hanafiah 2007). magnesium is a macro element in soil when found in its organic form (sutcliffe & baker 1975) and has important role in phosphate availability (agustina 2004). conclusions abiotic factors influencing invasion m. peltata in bukit barisan selatan national park are opened forest canopy coverage and high content of na and mg in the soil. acknowledgements this study was part of master thesis of the first author, financially supported by seameobiotrop dipa 2011. the author thanked artha graha peduli and wildlife conservation society indonesia program for their assistance in conducting the field work. references agustina l. 2004. . jakarta dasar-dasar nutrisi tanaman (id): pt rineka cipta. crozier cr, heiniger rw. 1998. soil facts: soil sampling for precision farming systems. north carolina (usa): north carolina state university. fosberg fr, sachet mh. 1977. flora of micronesia, part 3, convolvulaceae. contributii botanice 36:1-34. gaveaua dla, wandono h, setiabudi f. 2007. three decades of deforestation in southwest sumatera: have protected areas halted forest loss and logging, and promoted re-growth biol conserv 134:495-. 504. table 5 result of multiple linear regression between the percentage of coverage and soil nutrient factorsm. peltata coefficientsa model unstandardized coefficients standardized coefficients t sig. collinearity statistics b std. error beta tolerance vif 1 (constant) 50.768 144.015 0.353 0.729 loam 0.797 1.181 0.117 0.675 0.509 0.315 3.173 clay -0.569 0.905 0.140 -0.629 0.538 0.190 5.277 h2o -11.183 23.641 0.128 -0.473 0.642 0.128 7.818 cn ratio -8.535 2.739 0.370 -3.116 0.006 0.668 1.498 p2o5 -2.721 1.197 0.377 -2.273 0.036 0.342 2.924 ca -5.902 1.694 0.888 -3.485 0.003 0.145 6.899 mg 18.726 4.721 0.970 3.966 0.001 0.157 6.351 na 210.264 74.615 0.390 2.818 0.012 0.493 2.029 base saturation (%) 0.735 0.459 0.292 1.601 0.128 0.284 3.522 note: a = dependent variable: coverage of merremia peltata 27 hamim. 2008. jakarta (id): universitas fisiologi tumbuhan. terbuka. hanafiah ka. 2007. . jakarta (id): dasar-dasar ilmu tanah pt. raja grafindo persada. hardjowigeno s. 2010. jakarta (id): akademika ilmu tanah. pressindo. huang w, pohjonen v, johansson s, nashanda m, katigula mil, luukkanen o. 2003. species diversity, forest structure and species composition in tanzania tropical forest. for ecol manage 173:11-24. irianto r, tjitrosoedirdjo s. 2010. invasi (l.) merremia peltata merr., convolvulaceae di taman nasional bukit barisan selatan, indonesia. journal gulma dan tumbuhan invasi tropika 1:65-70. kirkham ws. 2005. valuing invasives: understanding the merremia peltata invasion in post-colonial samoa. dissertation. austin (usa): the university of texas at austin. master j, tjitrosoedirdjo ss, qayim i, tjitrosoedirdjo s. 2013. ecological impact of (l.) merremia peltata merrill invasion on plant diversity at ukit barisan b selatan national park. biotropia 20(1):29-37. meyer jy. 2000. preliminary review of the invasive plants in the pacific islands. in: sherley g, editor. invasive species in the pacific: a technical review and draft regional strategy. samoa (ws): south pacific regional environment programme. p 85-114. munawar a. 2011. . kesuburan tanah dan nutrisi tanaman bogor (id): ipb press. national invasive species council. 2006. invasive species definition clarification and guidance white paper. https://www.invasivespeciesinfo.gov/docs/council / isacdef.pdf (accessed october 20 , 2014).th ooststroom van sj, hoogland rd. 1953. convolvulaceae. flora malesiana i 4:452-3. paynter q, harman h, waipara n. 2006. prospects for biological control of . . new merremia peltata report zealand: conservation international. pyšek p, richardson dm. 2010. invasive species, environmental change and management and health. ann rev environ resour 35: 25-55. pengembara t, master j, yulianty, rustiati el, subiakto a. 2014. laju ertumbuhan antangan (merremia p m peltata l. merr.) ang umbuh elalui egenerasi y t m r vegetatif. proceedings of seminar nasional pengembangan teknologi pertanian. lampung (id): politeknik negeri unila. prasetyo a, hikmat a, prasetyo lb. 2011. pendugaan perubahan cadangan karbon di tambling wildlife conservation taman nasional bukit arisan b selatan. media konservasi 16: 87 – 91. sasaki s, mori t. 1981. growth responses of dipterocarp seedlings to light. malaysian forester 44: 319-45. stone bc. 1970. the flora of guam. micronesica 6:1-659. sutcliffe jf, baker da. 1975. . london plant and mineral salts (uk): edward arnold publishing. theoharides ka, dukes jf. 2007. plant invasion across space and time: factors affecting non indigenous species success during four stages of invasion. j new phytol 176:256-73. wildlife conservation society – indonesia program (wcsip). 2001. taman nasional bukit barisan selatan dalam ruang dan waktu. . bogor (id): research report phka/wcs-ip. abiotic factors influencing mantangan ( ) invasion jani mastermerremia peltata – et al. https://www.invasivespeciesinfo.gov/docs/council 1. jimmy (occurance).cdr biotropia vol. 18 no. 2, 2011: 61 73 occurrence of arboreal-climbing grapsids and other brachyurans in two mangrove areas of southern luzon, philippines jimmy t. masagca received 06 august 2010/accepted 02 may 2011 despite the obvious importance to ecosystem functioning, the most prominent groups belonging to the grapsidae are generally regarded as less studied in the philippines. in this study, the occurrence of arboreal-climbing grapsids and other brachyurans associated with the mangals of quezon and catanduanes was considered including some aspects on climbing, burrowing and feeding behaviour of selected grapsids represented by and . the non-grapsoid taxa are represented by varunidae ( ), portunidae ( , , , ); and eriphiidae ( ). (white 1847) [ ] is an exclusive mangrove tree climber (emtc), while (a. milne edwards 1873) is described here as occasional mangrove tree climber (omtc). (de haan 1835) is a non-mangrove arborealclimbing species (ntc) only seen on crevices of the mangrove areas. creates burrows most often than . likewise, the study provides information on the presence of the portunid orange mud crab ( ); the green mud crab ( ); the varunid ( ); and extremely abundant xanthiid crab, in the mangroves of catanduanes but not in pagbilao, quezon. grapsid crabs, brachyurans, mangroves, philippines catanduanes state colleges, virac 4800, catanduanes, the philippines; pacific island institute for pedagogy, technology, arts & sciences, inc. (pacifictech) 82 a constantino street, virac 4800, catanduanes, philippines; de la salle university-d, cavite, philippines) hemigrapsus, pseudograpsus metopograpsus ptychognathus charybdis portunus scylla thalamita epixanthus metopograpsus latifrons grapsus pseudograpsus elongatus hemigrapsus (hemigrapsus) penicillatus [grapsus (eriocheir)] p. elongatus m. latifrons scylla olivacea s. paramamosain ptychognathus altimana epixanthus dentatus abstract introduction key words: the brachyurans are interesting to study in terms of their association with mangrove flora, behavior, feeding and ecology (khan . 2005). this group makes up as much as 80% of the macro−faunal biomass in mangroves and densities can even reach to 80 to 90 sq. m. reports attest that the mangrove forest constitutes the habitat et al * corresponding author : pacifictechjtm@yahoo.com 61 with the richest diversity of land dwelling crabs (hartnoll 1988, fratini . 2005). it is also indicated that the most important functional role of mangrove crabs which received greater attention is their ability to process as much as 70% of the leaf litter (leh & sasekumar 1985, slim . 1997, dahdouh-guebas . 1999, ashton 2002). it was reported by jones (1984) and lee (1998) that brachyurans are important in the mangrove ecosystem structure and function. members of family grapsidae are possibly one of the most important components of the fauna of mangrove forests globally, in part because of their influence in nutrient cycling by feeding on litterfall (salgado-kent & mcguinness 2010). unfortunately, it appears that there is still a dearth of detailed published information on the structural components as to the occurrence of these grapsoidal families like grapsidae and other brachyurans in the mangroves of the philippines. there are few reports in this country that deal with the ways in which these crabs use the mangrove resources compared to other southeast asian countries wherein numerous crab literature are available (sivasothi 2000, sivasothi . 1993, tan & ng 1994, ng & liu 1999, leh & sasekumar 1985, soemodihardjo & soerianegara 1989, rahayu & davie 2002, poovachiranon 1986, lee & leung 1993, lee 1998, kwok & tang 2005). except for some previous reports (mcnae 1968, banaag 1972, zamora 1989a, zamora 1989b, dolar 1991, dólar 1991), there are scanty reports on occurrence, ecology and physiology of the mangrove-dwelling arboreal-climbing grapsid crabs in the philippines. this paper presents the occurrence of arboreal-climbing grapsid crabs and other brachyurans associated with the mangrove areas in quezon and catanduanes island, luzon (philippines). some insights on the dependence to mangrove trees as habitats, climbing skills and burrowing behavior of these grapsids are also noted. crab specimens were handpicked and scooped using nets and locally made traps during daytime and at night time in two mangrove areas of southern luzon, philippines: (1) palsabangon mangrove area, pagbilao, quezon; and (2) and the palnab-pajo mangrove area in catanduanes island. the collection sites include areas along rivers, creeks, inlets and the buffer zones or marginal strips of the coastline from june 2005 to february 2006. in addition nearby rivers, backshores and inside mangrove forests were also surveyed in may 2007 for these arboreal-climbing grapsid crabs. one female and several male specimens were obtained in each study area. measurements of the crab specimens were represented by maximum carapace width ( ); carapace length ( ); body height ( ); and chelar palm height (cph). ratios of cal/mcw, boh/mcw and cph/mcw were computed. all measurements are made with vernier calipers and ratios are in two decimal places following ng and liu (1999) as used by masagca (2009). observations on the feeding ecology of the crabs under study were carried out each of the mangrove study areas modifying the methods of gillikin (2000). in the mangrove areas covered, presence or absence of crab species were determined by visual inspection in 10 m diameter plots along a transect et al et al et al et al et al. agojo inlet mangrove reserve project mcw cal boh materials and methods 62 biotropia vol. 18 no. 2, 2011 perpendicular to the coastline, covering the full width of the forest. the study investigated at least 10 plots along a 100 to 200 m long transect in the study areas. table 1 presents arboreal-climbing mangrove grapsid crabs described in the present study, while table 2 shows the other brachyurans (or non-grapsids) occurring in the areas under investigation. based on the field surveys conducted in banks of the streams or rivers, backshores and inside mangrove forests, the families of brachyurans included in this report are the (1) grapsidae, (2) portunidae, (3) varunidae and (4) eriphiidae. as shown in the said tables these brachyurans include 3 genera ( , and ) for family grapsidae; a single genus ( ) for varunidae; 4 genera ( , , and ) for portunidae and a single genus ( ) for eriphiidae. results and discussion occurrence and some taxonomic diagnosis descriptions on the morphometry metopograpsus pseudograpsus hemigrapsus ptychognathus scylla thalamita portunus charybdis epixanthus table 1. summary of the different taxa of grapsoid sesarmid crabs identified in selected mangrove areas in quezon and catanduanes. brachyurans in two mangrove areas of southern luzon, philippines jimmy t. masagca 63 family genus species occurrence/location grapsidae hemigrapsus metopograpsus pseudograpsus hemigrapsus penicillatus metopograpsus latifrons pseudograpsus elongates quezon catanduanes quezon table 2. summary of taxa of non-grapsoid sesarmid crabs obtained from different locations. family genus species occurrence/location varunidae ptychognathus ptychognathus altimana catanduanes, quezon portunidae charybdis portunus scylla thalamita charybdis affinis portunus pelagicus scylla serrata scylla olivacea thalamita crenata quezon, catanduanes quezon quezon, catanduanes quezon quezon, catanduanes eriphiidae epixanthus epixanthus dentatus catanduanes based on field surveys, the different arboreal-climbing grapsid crabs associated with the mangrove areas under study include the three genera: (1) 1851 (2) h. milne edwards, 1853 ) and (3) h. milne edwards, 1837 these grapsid crabs were known to occur both in the lowland portions of streams, estuaries, and backshores of the. the grapsid (white 1847) [ ] in quezon and catanduanes island was observed to be associated with the sesarmid crabs, (de haan 1835) and h. milne edwards, 1853 [ ]. this means that these mangrove crabs occupy the same spots in the mangrove habitats that include feeding as shown in their climbing and burrowing behavior. hemigrapsus dana, (hemigrapsus penicillatus), metopograpsus (metopograpsus latifrons pseudograpsus (pseudograpsus elongatus). m. latifrons grapsus perisesarma bidens neosarmatium smithii sesarma biotropia vol. 18 no. 2, 2011 64 the same observation that greater number of grapsid crabs occur in the banks of the stream and at the backshore of the mangrove rather than inside or within the forests of the 2 mangrove areas confirming the previous made by tam and wong (2000), showing a significant difference in occurrence or diversity of grapsoid sesarmids and other brachyurans. the succeeding paragraphs present some taxonomic descriptions and morphometry of the arboreal climbing grapsids and other brachyurans. = h. milne edwards, 1853 (type species forskal, 1775, subsequent designation by davie, 2002; gender masculine) white, 1847 [nomen nudum] h. milne edwards, 1853 de haan in herklots, 1861 (nomen nudum) de man, 1879 a. milne-edwards, 1867 metopograpsus metopograpsus latifrons grapsus h. milne-edwards, 1853 (grapsidae) (white, 1847) [ ] metopograpsus cancer messor = grapsus latifrons = metopograpsus maculatus = grapsus (grapsus) dilatatus = grapsus (grapsus) dilatatus = metopograpsus pictus figure 1. from a maqueda channel mangrove area (palnab-pajo mangrove) in catanduanes island metopograpsus latifrons this arboreal-climbing grapsid has squarish carapace, slightly converging backwards; with 3 maxilliped not meeting in the middle line; and one tooth behind the antero-lateral one. carapace of appears to be converging backwards. this grapsid attacks the collector during several field works in quezon and catanduanes. this crab is conspicuously found in sluice gates of fish ponds in the mangrove area, prop roots of and tree trunks. as an arboreal climbing grapsid crab, this opportunistic animal was observed to assume an inverted or downward position (facing the water) when found on trunks of mangrove trees. some samples were also collected in crevices of trees during low tides. this grapsoid crab feeds on leaves, algae mollusks (vannini . 1997) and crustaceans (jones 1984). it is also stressed that in another species of the genus is less dependent on the leaves of mangrove plants (dahdouh-guebas 1999). in singapore, 3 species ( and havebeen the subjectof several reports. h. milne edwards, 1837 (type species latreille, 1817, subsequent designation by holthuis, 1977; gender masculine) rd m. latifrons rhizophora et al metopograpsus, m. oceanicus et al. m. gracilipes,m. frontalis m. latifrons) = pseudograpsus grapsus penicilliger genus pseudograpsus h. milne edwards, 1837 65 = pachystomum nauck, pachystomum philippinense = pseudograpsus erythraeus pseudograpsus elongates mcw boh 1880 (type species nauck, 1880, by monotypy; gender neuter) kossmann, 1877 table 3 shows the mean values of morphometric data of grapsid crab from the mangroves under study. males tend to be larger in terms of body size as to maximum carapace width ( ) and body height ( ). pseudograpsus elongatus heterograpsus(a. milne-edwards, 1873) [ ] table 3. mean values ( ) of the morphometry of from catanduanesin mm p. elongatus sex of crabs mcw cal cal/mcw boh boh/mcw cph cph/mcw male 32.95 37.23 1.13 18.62 0.56 14.82 0.45 female 31.19 37.82 1.21 10.67 0.34 11.62 0.37 legend: mcw=maximum carapace width; cal=carapace length; boh= body height; cph= chelar palm height (all values are in mm). table 4. mean values ( ) of some of the morphometrics of female crab samples of , showed a mean mcw = 31.19 mm, while males = 32.95 mm. in terms boh, females mean showed mean boh of 10.67 mm and males gave a mean boh of 18.62 mm. body form for females (boh/mcw = 10.67mm/31.19 mm = 0.34± 0.01, n=2), while for males (boh/mcw = 18.62 mm/32.95 mm = 0.56± 0.01, n=8) the body form is relatively vaulted. chelipeds equal and sexually dimorphic. male chelae larger (cph/mcw = 14.82mm/ 32.95mm = 0.45) and more strong than females (cph/mcw = 11.65mm/31.19mm = 0.37). identifying characters of are two distinct teeth behind the anterolateral one, carapace converging backwards. carapace squarish, slightly converging backwards; 3 maxilliped not meeting in the middle line; legs and carapace not hairy, carapace slightly convex; two distinct teeth behind the antero-lateral one. = dana, 1851 (type species dana, 1851, subsequent designation by rathbun, 1918; gender masculine) = a. milne-edwards, 1869 (type species h. milne edwards, 1837, subsequent designation by rathbun, 1918; gender masculine) = yokoya, 1928 p. elongatus p. elongatus hemigrapsus hemigrapsus crassimanus lobograpsus cyclograpsus crenulatus brachynotus brevidigitatus rd genus hemigrapsus hemigrapsus penicillatus grapsus eriocheir dana, 1851 (de haan, 1835) [ ( )] in mm h. penicillatus sex of crabs mcw boh boh/mcw cph cph/mcw male 28.72 17.02 0.59 9.39 0.33 female 27.29 12.07 0.34 9.02 0.33 legend: mcw=maximum carapace width; boh= body height; cph= chelar palm height (all values are in mm). table 4 shows the identity of the grapsid, de haan, 1858 from catanduanes which was confirmed by ms. marivene manuel from the pnm in manila. h. penicillatus brachyurans in two mangrove areas of southern luzon, philippines jimmy t. masagca biotropia vol. 18 no. 2, 2011 66 varunidae h. milne edwards, 1853 genus stimpson, 1858 rathbun, 1914 [ ] ptychognathus ptychognathus altimanus varuna = stimpson, 1858 (type species stimpson, 1858, by monotypy; gender masculine) [opinion 85, direction 37] = nauck, 1880, (type species coelochirus crinipes nauck, 1880, by monotypy; gender masculine) table 5 shows the summary of the mean values of selected morphometrics of males are bigger than females. carapace pitted but glabrous, a little broader than long ( =39.54/36.08); lateral margin with two sharp teeth; legs fringed on the last 3 joints. male chelipeds are larger (cph/mcw=0.38) than the females (cph/mcw=0.22). ptychognathus ptychognathus glaber coelochirus p. altimanus. mcw/cal table 5. mean values ( ) of the some morphometrics ofin mm p. altimanus sex of crabs mcw cal cal/mc w boh boh/mcw cph cph/mcw male 39.54 36.06 0.91 15.56 0.56 15.02 0.38 female 33.09 32.56 0.98 15.36 0.34 7.37 0.22 legend: mcw=maximum carapace width; cal=carapace length; boh= body height; cph= chelar palm height (all values are in mm). samples were collected near the canals connected to a small stream inundated during the high tides. this varunid crab is abundant in the backshore portions of the mangroves. found in the back mangroves of quezon and catanduanes (near the rice paddies) and at the edges near the areas where freshwater streams are flowing. ng . (2008) notes that is being revised by n.k. ng p.k.l. ng. several groups of species are now recognizable and new genera will be established for them. = forskal, 1775 = herbst, 1794 = marion de proce, 1822 = var. shen, 1932 as shown in table 6, males of are bigger than females. buccal frame rectangular; carapace much wider than long (mcw/cal 116.57/48.44= 2.41), bow fronted, and much serrate, drawn out into lateral spikes. chelae strong but slender. buccal frame rectangular, last pair of walking legs paddle-like; 9 antero-lateral spines. samples of this portunid crab were obtained at the outer margins of the mangrove forest areas in quezon and catanduanes. the use of baited lift nets (local name= “bintol”) allowed for the collection of this crabs. et al ptychognathus cancer pelagicus cancer cedonulli portunus denticulatus portunus pelagicus sinensis p. pelagicus portunidae rafinesque, 1815 subfamily portuninae rafinesque, 1815 genus weber, 1795 linnaeus, 1758) [cancer] portunus portunus (pelagicus) pelagicus 67 table 6. mean values ( ) of the some morphometrics ofin mm p. pelagicus sex of crabs mcw cal cal/mcw boh boh/mcw cph cph/mcw male 116.57 48.44 0.42 25.73 0.22 17.78 0.152 female 112.65 48.65 0.43 20.38 0.18 17.35 0.154 legend: mcw=maximum carapace width; cal=carapace length; boh= body height; cph= chelar palm height (all values are in mm). genus forskal, 1775) [ ] scylla de haan, 1833 scylla serrata ( cancer = scylla cancer serratus achelous crassimanus scylla tranquebarica oceánica lupa lobifrons de haan, 1833 (type species forskal, 1775, subsequent designation by rathbun, 1922; gender feminine) = macleay, 1838 = var. dana, 1852 = h. milne edwards, 1834 a b figure 2. (a, carapace) pagbilao quezon and maqueda channel catanduanes.scylla serrata as presented in table 7, male samples of obtained from the study areas are smaller than the female samples. samples obtained were heavy with moderately convex carapace ( 63.54/40.46 = 1.57), with 4 teeth; antero-lateral margin with 7 teeth, periopods/pleopods smooth, no hairs. table 7. mean values ( ) of the some morphometrics of s. serrata mcw/cal in mm s. serrata sex of crabs mcw boh boh/mcw cph cph/mcw male 63.54 21.56 0.34 8.07 0.13 female 67.85 23.45 0.35 9.23 0.14 legend: mcw=maximum carapace width; boh= body height; cph= chelar palm height (all values are in mm). scylla olivacea cancer(herbst, 1796) [ ] summary data on selected morphometrics of are presented in table 8. ratios obtained for boh/mcw and cph/mcw show almost the same values, which may indicate that sexual dimorphism is not that intense. s. olivacea brachyurans in two mangrove areas of southern luzon, philippines jimmy t. masagca biotropia vol. 18 no. 2, 2011 68 table 8 mean values of the some morphometrics of. (in mm) s. olivacea sex of crabs mcw boh boh/mcw cph cph/mcw male 62.02 20.34 0.33 7.09 0.11 female 63.52 21.57 0.34 7.26 0.12 legend: mcw=maximum carapace width; boh= body height; cph= chelar palm height (all values are in mm). genus charybdis harybdis (charybdis) affinis de haan, 1833 c dana, 1852 =? gordon, 1931charybdis barneyi figure 3. general view of from quezon. table 9. mean values ( ) of the some morphometrics of charybdis affinis in mm c. affinis carapace of more or less hexagonal (mcw/cal 40.09/29.63= 1.353), antero-lateral margins diverging backwards, fronto-lateral much less than maximum width, bow-shaped front cut into 6 teeth. table 9 shows that the ratio of cph/mcw for both male and female samples are almost the same. ward (1941, cited by ng 2008) described from davao as: carapace is broader than long, bare and glossy, granulated under lens. c. affinis et al. c. philippinensis sex of crabs mcw boh boh/mcw cph cph/mcw male 43.34 16.8 0.39 17.78 0.41 female 40.09 15.64 0.39 16.52 0.41 legend: mcw=maximum carapace width; boh= body height; cph= chelar palm height (all values are in mm). genus latreille, 1829 ruppell, 1830 [ , sic] thalamita thalamita crenata talamita = thalamita cancer adnete = thalamonyx goniosoma danae t. crenata mcw cal latreille, 1829 (type species herbst, 1803, by monotypy; gender feminine) a. milne edwards, 1873 (type species a. milne edwards, 1869, subsequent designation by rathbun, 1922; gender masculine) as shown in table 10, carapace of (figure 4) is much more or less hexagonal ( = 50.63 mm, = 32.02 mm), but antero-lateral margins sub-parallel; fronto-orbital not much less than maximum carapace width; chelipeds strong ( =11.35 mm, male; 12.06 mm, female); transverse ridges usually distinct. carapace is rounded with five antero-lateral teeth. cph figure 4. from pagbilao, quezon. table 10 mean values of the some morphometrics of thalamita crenata . (in mm) t. crenata sex of crabs mcw cal cal/mcw boh boh/mcw cph cph/mcw male 50.63 47.01 0.93 17.93 0.35 11.34 0.22 female 31.04 32.02 1.03 17.43 0.56 12.06 0.39 legend: mcw=maximum carapace width cal=carapace length; boh= body height; cph= chelar palm height (all values are in mm) superfamily eriphioidea macleay, 1838 family eriphiidae macleay, 1838 genus heller, 1861 (white, 1848) [ ] epixanthus epixanthus dentatus panopeus = heller, 1861 (type species heller, 1861, by monotypy; gender masculine) = de man, 1879 = haswell, 1881 the mangrove crab, (figure 5) displays two visible spines on the upper internal face of claw carpus; 5 big teeth on the antero-lateral carapace margins, carapace widely mottled. celipeds are unequal (right larger than left). table 11 shows that chelipeds of male samples (0.40) are larger compared to the female samples (0.26) epixanthus epixanthus kotschii epixanthus dilatatus panopeus acutidens e. dentatus a b c figure 5. (carapace) from palnab-pajo mangrove area in virac, catanduanes (a), frontal view (b) and forcept-like claws (c). epixanthus dentatus 69 brachyurans in two mangrove areas of southern luzon, philippines jimmy t. masagca table 11. mean values ( ) of the some morphometrics ofin mm e. dentatus sex of crabs mcw cal cal/mcw boh boh/mcw cph cph/mcw male 45.02 32.41 0.72 19.32 0.43 18.21 0.40 female 50.01 29.45 0.59 17.05 0.34 13.01 0.26 legend: mcw=maximum carapace width; cal= carapace length; boh= body height; cph= chelar palm height (all values are in mm). samples of were obtained under drift woods, buried on the mud. the right claw of this crab is stout and consists of a special tooth which it uses to open gastropods. plate 17b (fig. 5) shows the forcept-like claw of the crab. this crab is omnivorous (dahdouh-guebas . 1999), butpreysmostlyon crabs (cannicci . 2008). on the arboreal-climbing behavior, several individuals of the mangrove crabs, (white, 1847) were observed as exclusive tree-climber (emtc) in mangrove canopies. this grapsid, invariably stays longer on the branches of mangrove trees with mostly upside down position. during the study, climbing height range of 50 grapsid crabs (in each study area) observed from 0.065 m to 2.35 m above the water lining. majority of these grapsid crabs climb at the main trunks of and sometimes on the branch of when the tide is rising and when insects (e.g. spiders) are also found, since they are omnivorous feeders (jones 1984). although some are seen on the lateral branches, these are only happening when these grapsids were antagonized. it was observed that when are being caught by hand picking at the bottom of the trunk of the mangrove tree submerged in water, some of these crabs rushed to the upper portion of the trunk evading from the capturist. in catanduanes, a greater number of occur in the mangrove areas studied supporting the high biomass and density report in segera anakan, indonesia (geist 2011). this climbing behavior wasnotobserved in thegrapsid crabsof quezon. another observation refers to the tendency of the grapsid, to climb in the fronds of when chased or antagonized while they are in the water. individuals of this grapsid, (and also the sesarmid crab, ) tend to escape or evade the researchers by climbing fast to the trees. the other grapsid, is known to be an occasional mangrove tree-climber (omtc), while is non-arboreal species (nas) that was seen only in crevices of the mangrove areas. on burrowing behavior, the grapsid also creates burrows and so with , but the former is more active compared to the latter. burrowing activities have a pronounced effect on sediment properties, contributing immensely in rendering changes in the properties of mangrove sediments. as noted by nagelkerken (2008) changes in biochemical processes can be observed by enhancing the porosity and water flow through the sediment, assisting in flushing toxic substances. crab burrows provide an efficient mechanism for exchanging water between the anoxic substrate and the overlying tidal water. jones (1984) described the feeding habits of mangrove crabs and divided into seven groups: herbivore, carnivore, omnivore, deposit feeder, omnivore/deposit e. dentatus et al et al m. latifrons rhizophora sonneratia m. latifrons m. latifrons m. latifrons et al. m. latifrons nypa fruticans m. latifrons selatium elongatum p. elongatus, h. penicillatus p. elongatus m. latifrons et al. arboreal-climbing, burrowing and feeding behavior of the grapsid crabs 70 biotropia vol. 18 no. 2, 2011 feeder, specialized filterer, and filterer/omnivore. the grapsids are herbivores and omnivore/deposit feeders, eating mangrove litter and water plants. nordhaus (2011) described extensively the food preferences, diet and food consumption of grapsoid crabs in indonesia. this will become an important reference for studying further the food and feeding habits of grapsids in the philippines. a total of 3 genera belonging to the family grapsidae ( and ) are reported here possessing tree-climbing abilities. (white 1847) [ ] is an exclusive mangrove tree climber (emtc), while (a. milne edwards 1873) is described here as occasional mangrove tree climber (omtc). (de haan, 1835) on-mangrove arboreal-climbing species (ntc) only seen on crevices of the mangrove areas. the non-grapsoidal brachyurans are represented by 3 families [(varunidae ( ), portunidae ( , , , ); and eriphiidae ( )]. to what extent this tree-climbing abilities of the said grapsid crabs in quezon and catanduanes relate to the feeding behaviour of the grapsid crabs reported in the present study awaits further studies. likewise, food preference, diet and consumption of these grapsid crabs from the philippines indicate future needs. the author acknowledges with thanks to the de la salle university-dasmariñas, university faculty research office (ufro) for funding a research on the mangrove crabs. profound thanks to asst. prof. rico masagca of dlsu-manila and president & ceo of pacifictech, mrs. rose m. gianan, tersy m. flores, mrs. margie m. sabino, mrs. elsie m. almonte, yule, mark, kate and marby jean for their unending support in this work on mangrove brachyurans. the head of the catanduanes state colleges, dr. minerva morales (suc president iii) is also greatly appreciated. m. latifrons, p. elongates and n. penicillatus et al. hemigrapsus, pseudograpsus metopograpsus metopograpsus latifrons grapsus pseudograpsus elongatus hemigrapsus (hemigrapsus) penicillatus [grapsus (eriocheir)] is a n ptychognathus charybdis portunus scylla thalamita epixanthus conclusions acknowledgements references a . b . c shton ec. 2002. mangrove sesarmid crabfeeding experiments in peninsular malaysia. journal of experimental marine biology and ecology, 273: 97119 anaag jf. 1972. vegetational composition and association in a mangrove forest ecosystem in puerto galera, oriental mindoro. natural and applied science bulletin, 29(1-2): 1-40 annicci s, burrows d, fratini s, smith tj, offenberg j, dahdouh-guebas f. 2008. faunal impact on vegetation structure and ecosystem function in mangrove forests: a review. aquatic botany, 89 (2): 186-200. 71 brachyurans in two mangrove areas of southern luzon, philippines jimmy t. masagca chen gc, ye y. 2010. changes in properties of mangrove sediment due to foraging on leaves by crabs (grapsidae: sesarminae). dahdouh-guebas f, giuggioli m, oluoch a, vannini a, cannicci s. 1999. feeding habits of non-ocypodid crabs from two mangrove forests in kenya. 64: 291297. dolar mll. 1991. a survey on the fish and crustacean fauna of the seagrass beds in north bais bay, negros oriental, philippines, p. 367-377. : proceedings of the regional symposium on living resources in coastal areas. quezon city: university of the philippines marine science institute. dolar mld, alcala ac, nuique j. 1991. a survey on the fish and crustaceans of the mangroves of the north bais bay, philippines, p. 513-519. : alcala, a.c. 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(eds.), proceedings of a symposium on mangrove management: its ecological and economic considerations held in bogor, indonesia, august 9-11, 1988. biotrop special publication no. 37. tam fy, wong ys. 2000. hong kong mangroves. city university of hong kong press. tan cgs, ng pkl. 1994. an annotated checklist of mangrove brachyuran crabs from malaysia and singapore. 285: 75-84. vannini m, oluoch a, ruwa rk. 1997. the tree-climbing crabs of kenyan mangroves. : (b. kjerfve, b.l. de lacerda and e.s. diop, eds.), p. 325338. unesco technical papers in marine sciences. new york: unesco. zamora pm. 1989a. management policies and political awareness, p. 189-191. : cd field and m. vanucci (eds.) proceedings of the symposium on new perspectives in research and management of mangrove ecosystems, 11-14, nov. 1986, sri lanka. chiromanthes bidens c. maipoensis in proceedings of the second international marine biological workshop: the marine flora and fauna of hong kong and southern china, hong kong perisesarma crustaceana, in mangrove fisheries and connection terebralia palustris journal of experimental marine biology and ecology, in et al hydrobiologia, in mangrove ecosystems studies in latin america and africa in zamora pm. 1989b. mangroves of the philippines. biotrop special publication (bogor, indonesia), 37: 43-65. 73 brachyurans in two mangrove areas of southern luzon, philippines jimmy t. masagca sri-13 mei 2017-453 (nabila isolation).cdr isolation of endophytic fungi from the coasta (solanum sp.) l plant terong pungo and its against oral antibacterial activity pathogenic bacteria** nabila ukhty , kustiariyah tarman , and iriani setyaningsih 1,2 1,3* 1 1department of aquatic products technology, faculty of fisheries and marine sciences, institut pertanian bogor, bogor 16680, indonesia 2department of fisheries, faculty of fisheries and marine science, universitas teuku umar, meulaboh, aceh 23681, indonesia 3marine biotechnology division, center for coastal and marine resources studies, institut pertanian bogor, bogor 16680, indonesia received 8 january 2015/accepted 8 august 2016 abstract coastal plant locally known as terong pungo (solanum sp.) was used by aceh community as traditional medicine for toothache. the objectives of this study were to isolate endophytic fungi from the leaves of terong pungo and determine the antibacterial activity of the fungi against bacteria causing dental caries and infection. eight endophytic fungi were isolated. antagonism test among the isolates was performed to select the most dominant fungus. tp6 was the selected fungus based on the antagonism assay. crude extracts of the fungus were macerated from the culture broth using ethyl acetate. the strongest antibacterial activity of the extracts was obtained when the fungus was at 12 days of cultivation. the fungal crude extract was strongly active against staphylococcus aureus, staphylococcus epidermidis and pseudomonas aeruginosa with inhibition zone diameter of 20, 21 and 23 mm, respectively. keywords: endophytes, medicinal plant, toothache, traditional medicine introduction in the oral cavity, bacteria are often associated with the oral and dental diseases. caries and periodontal diseases seem to appear following an imbalance in the oral resident microbiota, leading to the emergence of potentially pathogenic bacteria (marcote & lavoie 1998). streptococcus mutans pseudomonas aeruginosa, , staphylococcus aureus staphylococcus epidermidis and often times can be isolated from the mouth of a patient. these bacteria are known as normal bacteria in the mouth, however, they might become pathogenic because of predisposing factors such as changes in the quantity of bacteria and decreasing immune system, which cause dental caries and infection (al-bayati & sulaiman 2007). there are several health products available commercially as anti-microbiota for oral cavity and dental, but the products mostly contain alcohol as antibacterial, which may cause side effect such as vomit, diarrhea and stained teeth (more . 2008). the carcinogenic properties of et al alcohol may happen when there is formation of acetaldehyde in the oral cavity (bean . 2007; et al mccullough & farah 2008; lachenmeier . et al 2009). these side effects of alcohol trigger efforts in finding safer and more effective active compounds in oral health products. terong pungo ( sp.) (fig. 1) is a coastal solanum plant, usually used by aceh community to treat toothache. biotropia 4 1 7 9 15 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 1 453 * corresponding author: kustya@gmail.com ** this paper was presented at international conference on integrated coastal management and marine biotechnology, emphasizing on icm practices and applications. 29 30 november 2016. bogor, indonesia. 9 the ethyl acetate crude extract of terong pungo leaf showed antibacterial activity against escherichia coli staphylococcus aureus and (hardjito 2008). the ethanol extracts of solanum surattense showed the antibacterial activity against staphylococcus aureus bacillus subtilis escherichia coli, , , pseudomonas aeruginosa salmonella typhi shigella , , dysenteriae vibrio cholerae and (sheeba 2010). endophytic fungi associated with terong pungo plant produce active compounds which are similar to the compounds produced by its host. strobel . (2004) stated that the endophytic et al fungi can be isolated from seed, leaf, flower, branch, stem and root of the host plant. jena and tayung (2013) reported that several endophytic fungi isolated from could produce solanum ubrum secondary metabolites as source of antimicrobial and antifungal compounds. this study was aimed at isolating endophytic fungi from terong pungo leaf and observing the potential of the fungal bioactive compounds as antibacterial for oral pathogenic bacteria. materials and methods collection of sample the leaves of terong pungo ( sp.) was solanum collected from the coastal area of pidie district, aceh province, indonesia. mature healthy plant leaves were collected from different parts of the trees. leaf samples from each plant were randomly cut off and placed separately in sterile plastic bags and stored in low temperature (4.5 ºc) for two days until being used for endophytic fungi isolation. isolation of endophytic fungi the solanum leaves were sterilized using 70% alcohol for 2 minutes and then rinsed with sterile water for 1 minute. the sterilized leaves were cut into 1 x 1 cm pieces and then put the leaf pieces in petri dish containing potato dextrose agar (pda), four pieces for each petri dish. petri dishes containing pieces of solanum leaves were then oincubated at room temperature (27 29 c) for 12 14 days. these pieces of leaves were checked once a day to observe the growth of hyphal tips of the endophytic fungi. the growing hyphal tips were immediately transferred into other petri dishes containing fresh pda and kept incubated at 27 – 29 °c to get pure isolate (arnold et al. 2003). isolation process was conducted in a clean bench (thermo scientific 1300 series a2). antagonism test of endophytic fungi the antagonism assay was performed by inoculating 2 3 isolates of potential endophytic fungi on pda medium. the inoculum was a 5 mm piece of isolate on pda medium. the 10 biotropia vol. 24 no. 1, 2017 figure 1 terong pungo (solanum sp.) showing leaves and fruit culture collection of the laboratory of microbiology, universitas indonesia. this assay was performed using agar well diffusion technique, which was modified from holo et al. (1991). the antibacterial assay used two bacteria ( and ) s. mutans p. aeruginosa to test the periodic for obtaining the optimum time of fungal culture. the fungus was then cultured based on this result. was grown on blood agar streptococcus mutans (ba), while , and p. aeruginosa s. aureus s. epidermidis were grown on nutrient agar (na). those bacteria were then incubated at 37 c for 24 hours. o furthermore, was suspended in brain s. mutans heart infusion (bhi), while , p. aeruginosa s. aureus and were suspended in nutrient s. epidermidis broth (nb). these bacteria were then incubated at 37 c for 24 hours. o optical density (od) was measured using a uv vis spectrophotometer (uv-2500). the optical density (od) used was approximately 0.5. an amount of 20 µl bacteria was inoculated on mha+sterile sheep blood for and on s. mutans mha for , and . p. aeruginosa s. aureus s. epidermidis mha medium containing test bacteria were then prepared with 8 wells for positive and negative controls and 3 extracts with different concentrations (0.5, 1 and 2 mg/well). all concentrations and controls were performed in two replications. the positive controls used were amoxicillin and chloramphenicol with concentration of 300 µg/well. the negative control was ethyl acetate as the solvent for extraction process. the incubation was performed at 37 c for 24 hours. inhibition zone o appeared around the well was measured. the data were analyzed descriptively into the bar chart. results and discussion endophytic fungi isolated from terong pungo ( sp.)solanum eight isolates obtained from the endophytic fungi were tp1, tp2, tp3, tp4, tp5, tp6, tp7 and tp8. guo et al. (2000) stated that the grouping of fungal isolates can be based on morphospecies i.e. based on the similarity of color and surface texture of the colony, pigmented hyphae, exudates, edge shape, growth rate and sporulation inoculum was incubated at 27 29 c (room o temperature) (sudantha & abadi 2007). the purpose of this test was to determine which isolates grow dominantly. characterization of endophytic fungi the 7 days old endophytic fungi were characterized based on macroscopic and microscopic morphology. the macroscopic observation included the fungal shape, surface, color and edge form. the microscopic properties were observed using microscope (cole parmer) with 400x magnification. fungal isolates were identified based on their morphological characters, such as conidiospore structures using standard identification manuals (barnett & hunter 1972; gandjar . 2000).et al cultivation of endophytic fungi the selected endophytic fungi were mass cultivated on potato dextrose broth (pdb) by placing agar blocks of actively growing pure culture (3 mm in diameter) in 250 ml erlenmeyer flask at 28 °c, which was then mixed using orbital shaker at 150 rpm (srikandace . 2007). et al harvesting was conducted by filtering the mycelia using sterile filter paper to separate the mycelia from the culture medium. mycelia were then dried and measured as the biomass of endophytes. the culture medium was used for extraction of bioactive compounds. fungal growth and ph of the culture were observed every 3 days for 21 days. extraction of bioactive compounds culture medium of the harvested fungi was macerated using ethyl acetate for 3 x 24 hours by shaking the medium at 150 rpm (tarman 2011). the results of the extraction were separated using a separating funnel, then concentrated using a rotary vacuum evaporator (heidolph vv 2000) at 45 °c to produce the crude extracts. the crude extracts were used for antibacterial bioassay. antibacterial activity against oral bacterial pathogens antibacterial assay was performed in vitro against streptococcus mutans, pseudomonas aeruginosa, staphylococcus aureus and staphylococcus epidermidis. all bacterial cultures were obtained from the 11 isolation of endophytic fungi from terong pungo and its antibacterial activity – ukhty et al. structures. based on the morphospecies, the endophytic fungi obtained from the leaf of terong pungo were identified as aspergillus spp., trichoderma spp. and fusarium spp. selection result of endophytic fungi antagonism assay was conducted to determine which isolates grow dominantly (fig. 2). in this research, the antagonism assay was performed by direct opposition method, i.e. three fungal isolates co-cultured in the same plate. based on the antagonism test, tp6 isolate was the most dominant among other isolates. it is generally assumed that the dominancy of an organism related to the production of secondary metabolites which are important as chemical defense. therefore, tp6 isolate was selected for further research step. characteristics of isolate tp6 macroscopic and microscopic characteristics of the selected fungal tp6 isolate are presented in figure 3. figure 4 presents the fungal growth for 21 days. 12 biotropia vol. 24 no. 1, 2017 5 6 1 3 5 6 2 5 4 figure 2 antagonism test among endophytic fungi of solanum sp. (notes: isolate numbers: 1 = tp1; 2 = tp2; 3 = tp3; 4 = tp5; 5 = tp6; 6 = tp7) a b conidiophore figure 3 tp6 isolate on pda medium (a) and 400x magnification (b) figure 4 growth curve of tp6 isolate during 21 days (notes: = dry biomass, = ph ) d ry b io m as s (g /m l ) cultivation period (days) the macroscopic characteristics of tp6 isolate were grey mycelia, uneven surface with abundant hypha growing at the colony edge and greyish green as the reverse color. the microscopic observation at 400x magnification showed that the fungus has long conidiophores. however, the tp6 isolate has not been identified into the genus or species. the tp6 isolate was then cultured on potato dextrose broth (pdb) for 21 days at room temperature with orbital shaking at 120 rpm. the fungus growth was determined using dry weight of the mycelia which were harvested every 3 days for 21 days cultivation. according to meletiadis et al. (2001) fungal growth has 4 phases consisting of lag, log, stationary and death phases. tp6 fungus was in logarithmic phase until day 12; the stationary phase was reached on day 12 until day 18; and finally on day 18 tp6 started to reach death phase. growth phase is closely related to the fungal nutrient sufficiency in their media (srikandace et al. 2007). according to kusumaningtyas et al. (2010) the pdb medium contains potato dextrose as carbon source which is the crucial component for microbial growth. the primary substances of the microbe cells are carbon and nitrogen. therefore, if the carbon is unavailable, the growth will be disrupted. lack of carbon also causes cell death. antibacterial activity against oral bacteria the test organisms in this study were streptococcus mutans, pseudomonas aeruginosa, staphylococcus epidermidis and staphylococcus aureus. streptococcus mutans attaches to the tooth surface by producing glucuronyl transferase enzyme which causes plaque and colonies on the surface of the tooth ( . pseudomonas aeruginosa shulman et al. 1992) can be found in the oral cavity (komiyama et al. 1985) and is an opportunistic bacterium that can cause severe infection in susceptible patients (iversen et al. 2007). staphylococcus epidermidis and staphylococcus aureus are normal flora bacteria which can produce toxin when their normal habitats are disturbed (torabinejad & walton 2009). these bacteria cause toothache. antibacterial activity of the crude extract of tp6 fungus was observed every 3 days for 21 days cultivation (day 3, 6, 9, 12, 15, 18 and 21) to determine the optimum fungal growth which produced the highest antibacterial activity. the antibacterial activity of the fungal crude extracts against and is shown in p. aeruginosa s. mutans figure 5. crude extract of endophytic tp6 fungus showed antibacterial activity against p. aeruginosa. however, it was not active against s. mutans because the inhibition zone was not detected. fungal crude extract obtained from culture broth harvested at day 12 showed the highest antibacterial activity against with p. aeruginosa inhibition zone diameter of 22.5 mm for 2 mg extract/well; 20 mm diameter for 1 mg extract/well; and 15.5 mm diameter for 0.5 mg extract/well. based on this result, the tp6 fungus was cultured for 12 days. figure 5 antibacterial activity against pseudomonas aeruginosa using ethyl acetate crude extract of tp6 isolate ( = 0.5 mg concentration, = 1 mg concentration, = 2 mg concentration and = positive control) (note: the data presented are mean±sd of 2 replications) 13 isolation of endophytic fungi from terong pungo and its antibacterial activity – ukhty et al. secondary metabolites are commonly produced in stationary phase. secondary metabolites are not essential for the cultures growth, but serve survival functions in nature and are usually produced during the late growth phase (demain & fang 2000). simanjuntak . (2002) et al stated that secondary metabolites are produced by fungus with the highest intensity at the end of exponential phase or at the beginning of stationary phase. therefore, in this study, the antibacterial activity of the extracts harvested more than day 12 was decreasing. the crude extract was not active against s. mutans which may have been caused by the developing resistance of the bacterium. according to millar . (2001) bacterial et al resistance against antibacterial compound is formed by three mechanisms, such as insufficient drugs, inactive drugs and physiological change of bacteria. further antibacterial assay against , s. mutans s. aureus s. epidermidis p. aeruginosa , and was performed for the fungal crude extracts which were cultured for 12 days. the antibacterial activity of the crude extracts against all test bacteria is presented in figure 6. the crude extracts showed antibacterial activity against , staphylococcus aureus staphylococcus epidermidis pseudomonas aeruginosa and with inhibition zone diameter of 20, 21 and 23 mm for 2 mg extract/well, respectively. this antibacterial activity was as strong as the inhibitory ability of the positive control used i.e. chloramphenicol, with inhibition zone diameter of 22 mm for 300 µg per well. whereas the amoxicillin as the positive control for antibacterial activity against s. mutans was stronger as the inhibitory ability than the crude extracts with inhibition zone diameter 25 mm for 300 µg per well. antibacterial assay showed that crude extracts possessed a broad spectrum of antibacterial activity since the extracts inhibited the growth of gram-positive and gram-negative bacteria. in this study, and represented s. aureus s. epidermidis gram-positive bacteria, while p. aeruginosa represented gram-negative bacteria. however, the extracts were not active against . this s. mutans may have been caused by the resistance properties developed in s. mutans. conclusions eight isolates of endophytic fungi having different morphology were collected i.e. tp1, tp2, tp3. tp4, tp5, tp6, tp7 and tp8. fungal isolate tp6 showed antibacterial activity against oral pathogenic bacteria such as staphylococcus aureus, staphylococcus epidermidis and pseudomonas aeruginosa. the optimum activity was exhibited when the fungus was cultivated for 12 days. references al-bayati fa, sulaiman kd. 2007. in vitro antimicrobial activity of salvadora 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plants of similipal biosphere reserve, india and their antimicrobial prospective. japs 3(4):s7-s12. komiyama k, tynan jj, habbick bf, duncan de, liepert dj. 1985. in the oral cavity and pseudomonas aeruginosa sputum of patients with cystic fibrosis. oral surg oral med oral pathol 59(6):590-4. kusumaningtyas e, natasia m, darmono. 2010. potensi metabolit kapang endofit rimpang lengkuas merah dalam menghambat pertumbuhan escherichia coli dan staphylococcus aureus dengan media fermentasi pdb dan pdy. prosiding seminar nasional teknologi peternakan dan veteriner. 20 mei 2010. bogor, indonesia. p. 819-24. lachenmeier dw, gumbel-mako s, sohnius em, keckwilhelm a, kratz e, mildau g. 2009. salivary acetaldehyde increase due to alcohol containing mouthwash use: a risk factor for oral cancer. int. j can 125:730-5. marcote h, lavoie mc. 1998. oral microbial ecology and the role of salivary immunoglobulin a. microbiol mol biol rev 62(1):71-109. mccullough mj, farah cs. 2008. the role of alcohol in oral carsinogenesis with particular reference to alcohol-containing mouthwashes. aust dent j 53:302-5. meletiadis j, meis fgm, mouton jw, vermeij pe. 2001. analysis of growth characteristics of filamentous fungi in different nutrient media. j clin microbiol, 39(2): 478-84. millar mr, walsh tr, linton cj. 2001. carriage of antibiotic-resistant bacteria by healthy children. j antimicrob chemother 47:605-10. more g, tshikalange te, lall n, botha f, meyer jjm. 2008. antimicrobial activity of medicinal plants against oral microorganism. j ethnopharmacol 119(3):4737. sheeba e. 2010. antibacterial activity of solanum surattense burm. f. j sci.eng. tech kathmandu university 6(1):1-4. shulman st, phair jp, somers hm. 1992. the biological and clinical basis of infectious disease fifth . edition. philadelphia (us): wb.saunders company. p. 110-6. simanjuntak p, melliawati r, soekmanto a, parwati t, bustanussalam. 2002. pengembangan bahan baku zat bioaktif anti malaria dari kapang endofit tumbuhan 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no. 4, 1990/1991: 31-40 potential role of wild crucifers in the preservation of diadegma eucerophaga horstman (hymenoptera: ichneumonidae), a parasitoid of the diamondback moth, plutella xylostella linnaeus (lepidoptera: plutellidae) utomo kartosuwondo faculty of agriculture bogor agricultural university bogor, indonesia sunjaya tropical agricultural pest biology programme, seameo biotrop bogor, indonesia abstract laboratory and field experiments were conducted to evaluate the potential role of two species of wild crucifers in the preservation of diadegma eucerophaga horstman, a parasitoid of the diamondback moth, plutella xylostella linnaeus. in the laboratory, d. eucerophaga developed quite well on p. xylostella larvae fed on leaves of two species of wild cruciferous plants, nasturtium heterophyllum bl. and cardamine hirsuta l. these wild crucifers may serve as food and oviposition sites for p. xylostella. in the field, n. heterophyllum and c. hirsuta planted adjacent to insecticide-treated cabbage plots provided refuge for d. eucerophaga parasitoids. introduction the diamondback moth, plutella xylostella linnaeus, is a major pest of cole crops in indonesia (and other parts of the world). in indonesia, an introduced parasitoid, diadegma eucerophaga horstman, plays an important role as a natural control agent of diamondback moths. the parasitoid was established in the cabbage-growing regions in west java where the rate of parasitism was reported to be in the range of 5-86% (sudarwohadi 1984). in the absence of insecticide application, d. eucerophaga parasitoids can often give an effective natural control of the diamondback moth. since insecticides are regularly applied to cabbage fields, the role of d. eucerophaga in suppressing diamondback moth population may be severely affected by intensive insecticide applications. under such unfavourable condition, a sound ecological measure should be undertaken in order to preserve d. eucerophaga parasitoids. preservation of natural enemies can be achieved, among other means, through the provision of refuge for natural enemies by managing wild host plants growing 31 biotropia no. 4, 1990/1991 in the surrounding of the main-crop field. preservation of natural enemies through appropriate management of wild host plants is a feasible means which can be incorporated in any crop pest management (van emden 1976). wild alternative host plants growing in the vicinity of the main-crop field can enhance the survival of a limited number of pest individuals and its natural enemies in the absence of the main host plant (ullyett 1947 in van den bosch and telford 1973). it has been known that a number of wild cruciferous plant species can serve as alternative hosts for p. xylostella (harcourt 1957). these wild crucifers may serve as refugium for the d. eucerophaga parasitoids when the main crop is being treated with insecticides or during a fallow period, provided that the parasitoid can develop on diamondback moth larvae feeding on wild crucifers. preservation of natural enemies is an important component of pest management programme (luckmann and metcalf 1975). natural enemies can be affected by insecticide application in two ways: they die of insecticide intoxication or due to lack of food (hosts or preys) which are eliminated by insecticides. in order to assure the availability of food for natural enemies, it is necessary to adopt a control approach that will leave a pest residue, below its economic threshold. in the case of the diamondback moth, the pest residue may find refuge in the wild cruciferous plants growing in the surrounding of the main-crop field and in turn, the diamondback moth residue can support the development and survival of d. eucerophaga parasitoids. in java, it was reported that p. xylostella can live on the following cruciferous plants: capsellabursa-pastoris, cardamine hirsuta (everaarts 1981) and nasturtium heterophyllum (backer and van slooten 1924). the potential role of the last two wild crucifers, which can be found in bogor and pacet highland, west java, in the preservation of d. eucerophaga parasitoids was evaluated in this study. laboratory and field experiments were carried out to study: 1) the oviposition preference of the diamondback moth to n. heterophyllum, c. hirsuta and cabbage, 2) the development of the diamondback moth on n. heterophyllum and c. hirsuta, 3) the development and survival of d. eucerophaga parasitoids on diamondback moth larvae fed on leaves of n. heterophyllum and c. hirsuta, and 4) the occurrence of d. eucerophaga on n. heterophyllum and c. hirsuta planted adjacent to cabbage plots. materials and methods laboratory experiments were carried out in the tropical pest laboratory, seameo biotrop, bogor, from november 1988 to february 1989, while field 32 potential role of wild crucifers in the preservation of d. eucerophaga horst. u. kartosuwondo & sunjaya experiments were conducted in the horticultural experimental farm, segunung, facet, from january to april 1989. insects and food plants larvae, pupae and adults of the diamondback moth were collected from cabbage fields around facet (1100 m in altitude), cianjur, west java. insects were maintained in the laboratory on brassica juncea. d. eucerophaga parasitoids were collected from the same area as diamondback moth and reared in the laboratory on diamondback moth larvae. both adults, p. xylostella and d. eucerophaga were fed on 10% honey solution soaked in cotton wool. experimental host plants, cabbage (brassica oleracea var. capitata), nasturtium heterophyllum and cardamine hirsuta, were planted in an insecticide-free area in a green house at seameo biotrop, bogor. when used, the age of plants was about 40-60 days old. experiments the study consisted of three laboratory experiments (1-3) and two field experiments (4-5). the first three experiments served as a preliminary study for the last two. experiment 1 oviposition preference of the diamondback moth to n. heterophyllum, c. hirsute and brassica oleracea var. capitata n. heterophyllum, c. hirsuta and cabbage plants kept separately on small vials containing water were placed in a wooden-framed muslin cloth cage (30x30x30 cm). a pair of mated diamondback moth adults was then introduced into the cage. the number of eggs deposited by the female on the three plant species was recorded daily until the female died. food plants were replaced daily. each treatment (plant species) was replicated eight times and the experiment was arranged in a completely randomized design. experiment 2 development of the diamondback moth on n. heterophyllum, c. hirsuta and brassica oleracea var. capitata eggs deposited by diamondback moth females maintained in cages containing larval food plants were transferred separately into a plastic cage (15 cm in diameter and 20 cm high) containing the corresponding food plants. food plants were replaced daily. insect development was followed until adult emergence, and the duration of 33 biotropia no. 4, 1990/1991 the egg, larval and pupal development stages were recorded. the experimental design was the same as in experiment 1. experiment 3 survival of d. eucerophaga on diamondback moth larvae fed on leaves of n. hetero phyllum and c. hirsuta ten third-instar diamondback moth larvae were confined in a plastic cage (25 cm in diameter and 50 cm high) containing n. heterophyllum or c. hirsuta plants as food. a pair of mated adult of d. eucerophaga parasitoids was then released into the cage. the female parasitoid was allowed to oviposit on diamondback moth larvae for five hours and then the parasitoid pair were removed from the cage. the number of parasitoid adults emerged from parasitized diamondback moth larvae was recorded. the percentage of emergence was calculated based on the number of diamondback moth larvae used. the experiment was arranged in a completely randomized design with two treatments (two species of wild crucifers) and eight replications. experiment 4 occurrence of d. eucerophaga on n. heterophyllum, c. hirsuta and b. oleracea var. capitata in the field n. heterophyllum, c. hirsuta, and cabbage plants were planted separately on thirty field small plots (1x1 m) which were divided into ten blocks. thus, each block consisted of three small plots containing the three plant species (one species/plot). plants were not treated with insecticides. the experimental plots were examined regularly for evidence of diamondback moth infestation. infested plant leaves (containing diamondback moth larvae) were removed and transferred to plastic cages (25 cm in diameter and 50 cm high) in the laboratory. collected diamondback moth larvae were fed on the same food plants as their origin and food plants were replaced daily. diamondback moth larvae were examined for evidence of parasitization and the number of adult d. eucerophaga parasitoids which emerged was recorded. a randomized block design was used to arrange this experiment. experiment 5 occurrence of d. eucerophaga on n. heterophyllum, c. hirsuta and insecticide treated b. oleracea var. capitata in the field this experiment was similar to experiment 4, but in this experiment cabbage plots were treated with baythroid 50 ec at a rate of 0.4 kg a.i./ha for each application. at the time of sampling of infested plant leaves, cabbage plots had been treated with the insecticide four times. the experimental design and the parameter observed were the same as in experiment 4. 34 potential role of wild crucifers in the preservation of d. eucerophaga horst. u. kartosuwondo & sunjaya results and discussion oviposition preference of the diamondback moth to n. heterophyllum, c. hirsuta and brassica oleracea var. capitata the average number of eggs deposited by the female diamondback moth per day over a seven-day oviposition period on two wild crucifers and cabbage is presented in table 1. table 1. average number of eggs deposited by the female diamondback moth on three cruciferous plant species over a seven-day oviposition period day plants 1 2 3 4 5 6 7 n. heterophyllum 11.21) 2.50 2.75 0.75 0.25 0.12 0 c. hirsuta 16.7 9.12 3.62 6.87 2.25 1.75 1.87 b. oleracea 25.6 10.60 11.20 4.25 2.00 0.12 0 1) each value in the table is an average of eight replications. the number of eggs deposited on day-1 was the highest among the seven-day oviposition period. this result is in agreement with that obtained by vos (1953). the oviposition preference of diamondback moth females to three cruciferous plant species is shown in table 2. table 2. average number of eggs deposited by the diamondback female on three cruciferous plant species 1) means in the same column followed by a common letter were not significantly different (p = 0.05), using least significant difference test; data were transformed to √ x+ 1 before analysis; each value in the table is an average of eight replications. in the laboratory, the three cruciferous plant species could serve as oviposition sites for diamondback moth females, with the order of preference: cabbage > c. hirsuta >n. heterophyllum. apparently, the three plant species contain secondary compound(s) that act as oviposition stimulant for diamondback moths, although 35 biotropia no. 4, 1990/1991 it may not be in the same amount. in crucifers, a compound that stimulates ovi-position in diamondback moths is known as mustard oil glucoside (gupta and thor-steinson 1960; harborne 1988). development of the diamondback moth on tv. heterophyllum, c. hirsuta and brassica oleracea var. capitata it was known from experiment 1 that diamondback moth females could lay eggs on the two wild crucifers tested, especially on c. hirsuta. results of experiment 2 indicated that the diamondback moth developed quite well on those wild crucifers. egg plus larval and pupal periods of the diamondback moth reared on three cruciferous plant species are indicated in table 3. table 3. average egg plus larval and pupal periods (days) of the diamondback moth reared on three cruciferous plant species food plants egg plus pupal period larval period1) n. heterophyllum 11.0 a 4.75 b c. hirsuta 10.38 a 3.0 c b. oleracea 11.0 a 4.0 b 1)means in the same column followed by a common letter were not significantly different (p = 0.05), using least significant difference test; each value in the table is an average of eight replications. the three host plants did not affect the combined duration of egg and larval stages of the diamondback moth significantly. the effect of host plants on the diamondback moth development was manifested in the duration of the pupal stage. the pupal period of the diamondback moth maintained on n. heterophyllum was comparable with that on cabbage, whereas on c. hirsuta the pupal period was the shortest. the obtained data suggest that the nutritional contents of the three cruciferous plant species can adequately support the growth and development of the diamondback moth. like in oviposition, a secondary compound mustard oil glucoside can also act as feeding stimulant for diamondback moth larvae (gupta and thorsteinson 1960; harborne 1988). survival of d. eucerophaga on diamondback moth larvae fed on leaves of tv. heterophyllum and c. hirsuta results of experiment 2 showed that diamondback moth larvae developed quite well on two wild crucifers, n. heterophyllum and c. hirsuta. from this experiment it can be inferred that diamondback moth larvae feeding on those wild 36 potential role of wild crucifers in the preservation of d. eucerophaga horst. u. kartosuwondo & sunjaya crucifers could support the development of its parasitoid, d. eucerophaga. the percentage of emergence of adult parasitoids resulting from a five-hour exposure often third-instar diamondback moth larvae to a mated parasitoid pair is presented in table 4. the data indicated that d. eucerophaga could survive on diamondback moth larvae feeding on wild cruciferous plants. the yield of food plants of diamondback moth larvae did not show a significant effect on the percentage of emergence of adult parasitoids. the percentage of emergence could be higher if the female parasitoid was allowed to oviposit on its host for more than five hours. table 4. average percentage of emergence of d. eucerophaga adults from diamondback larvae reared on two species of wild crucifers food plants of percentage of emergence diamondback larvae of parasitoid adults1) n. heterophyllum 31.63 a c. hirsuta 40.0 a 1) percentage of emergence was calculated based on the number of diamondback moth larvae used. means in the same column followed by a common letter were not significantly different (p = 0.05), using least significant difference test; data were transformed to arcsin x before analysis; each value in the table is an average of eight replications. occurrence of d. eucerophaga on n. heterophyllum, c. hirsuta and b. oleracea var. capitata in the field the average number of d. eucerophaga adults emerging from its host collected from three host plant habitats is shown in table 5. the number of d. eucerophaga adults which emerged from its host feeding on three cruciferous plants did not differ significantly from each other, although table 5. average numbers of d. eucerophaga adults emerged from its host collected from three cru ciferous plant habitats 1) means in the same column followed by a common letter were not significantly different (p = 0.05), using least significant difference test; data were transformed to √x + 1 before analysis; each value in the table is an average of ten replications. 37 biotropia no. 4, 1990/1991 cabbage and c. hirsuta seemed to provide better habitats than n. heterophyllum. the data suggested that in the field, diamondback moths can lay eggs on the wild crucifers, n. heterophyllum and c. hirsuta. the larvae of diamondback moths can feed on the wild crucifers aside from cabbage. occurrence of d. eucerophaga on tv. heterophyllum, c. hirsuta and insecticide-treated b. oleracea var capitata in the field in experiment 5, cabbage plots were sprayed with insecticide, while wild crucifers plots were not. the results of experiment 5 are shown in table 6. the average number of d. eucerophaga in insecticide-treated cabbage plots was the lowest among the three plant habitats, although it did not differ significantly from that in n. heterophyllum habitat. the average number of d. eucerophaga in c. hirsuta habitat was the highest and significantly different from the other two. data from experiment 4 and 5 suggested that there might be a movement of diamondback moths and d. eucerophaga parasitoids from insecticide-treated cabbage plots to adjacent wild crucifer plots. thus, wild crucifers growing in the surrounding of the cabbage field may provide refuge for d. eucerophaga parasitoids when unfavourable conditions prevail. table 6. average numbers of d. eucerophaga adults emerged from its host collected from two wild cruciferous and insecticide-treated cabbage plant habitats 1) means in the same column followed by a common letter were not significantly different (p =0,05), using least significant difference test; data were transormed to √x+1 before analysis; each vaue in the table is an average of ten replications the experimental results discussed above provide information on the potential role of wild cruciferous plants in the preservation of d. eucerophaga, a parasitoid of the diamondback moth, p. xylostella. two species of wild crucifers, tv. heterophyllum and c. hirsuta, can provide oviposition sites for diamondback moths and can serve as larval food. d. eucerophaga parasitoid can survive on its host feeding on the aforementioned wild crucifers. 38 potential role of wild crucifers in the preservation of d. eucerophaga horst. u. kartosuwondo & sunjaya p. xylostella is an oligophagous insect feeding on a number of plant species of cruciferae (thorsteinson 1952) which contain a secondary compound known as mustard oil glucoside (thorsteinson 1952; gupta and thorsteinson 1960; harborne 1988). this compound serves both as oviposition and feeding stimulant for the diamond-back moth. p. xylostella larva is a specific host for d. eucerophaga parasitoids (vos 1953). therefore, in order to enhance the survival of the parasitoid under unfavourable conditions, such as during insecticide application or in the absence of cabbage plants in the field, it is necessary to ensure the availability of wild cruciferous plants refuge for the pest residue. this necessity was supported by data from the last experiment. when cabbage plants were treated with insecticide, the diamondback moth in wild crucifers could support the survival of d. eucerophaga parasitoids. the wild crucifer c. hirsuta seems to be more suitable than n. heterophyllum as refuge for the diamondback moth and its parasitoid. acknowledgement the research work was supported by seameo biotrop. the authors wish to thank prof. dr.ir. amris makmur for making possible to carry out the experiment and to prof.dr.ir. h. sitti soetarmi tjitrosomo for her guidance and criticism. references backer, c.a. and d.f. van slooten. 1924. javaansche thee onkruiden. batavia drukkerijen ruygrok & co. 287 p. everaarst, a.p. 1981. weeds of vegetables in the highlands of java. lembaga penelitian hortikultura, pasar minggu, jakarta. 121 p. gupta, p.d. and a.j. thorsteinson. 1960. food plant relationship of diamondback moth (plutella maculipennis curt.). ii. sensory regulation of oviposition of the adult female. ent. exp. appl. 3: 305-314. harborne, j.b. 1988. introduction to ecological biochemistry. academic press, london. 356 p. harcourt, d.g. 1957. biology of the diamondback moth, plutella maculipennis curt. (lepidoptera: plutellidae), in eastern ontario. ii. life history, behaviour, and host relation-ships. can. entomol. 89: 554-564. luckmann, w.h. and r.l. metcalf. 1975. the pest-management concept. in introduction to insect pest management (r.l. metcalf and w.h. luckmann, eds.), p. 3-35. john wiley & sons, new york. 587 p. 39 biotropia no. 4, 1990/1991 sudarwohadi, s. 1984. status pengendalian hayati hama plutella xylostella oleh parasitoid diadegma eucerophaga di jawa barat. seminar hama dan penyakit sayuran di cipanas, 29-30 mei 1984. 17 p. thorsteinson, a.j. 1952. the chemoctactic responses that determine host specificity in an oligophagous insect (plutella maculipennis curt., lepidoptera). can. j. zool. 31: 52-72. van den bosch, r. and a.d. telford. 1973. environmental modification and biological control. in biological control of insect pests and weeds (p. debach, ed.): 459-488. chapman and hall, ltd., london. 844 p. van emden, h.f. 1976. pest control and its ecology. edward arnold. 59 p. vos, h.c.a.a. 1953. introduction in indonesia of angitia cerophaga grav., a parasite of plutella maculipennis curt. cont. gen agric. res. stat., bogor. 32 p. 40 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf microsoft word 1 biotropia no. 21, 2003 : 1 10 isolation and characterization of a novel benzoate utilizing serratia marcescens dwi suryanto1 and antonius suwanto2'3* 'department of biology, faculty of mathematics and natural sciences, north sumatra university, medan, indonesia . department of biology, faculty of science and mathematics, bogor agricultural university, bogor, indonesia . 3seameo-biotrop, p.o. box. 116, bogor, indonesia abstract a new benzoate-utilizing strain, serratia marcescens ds-8, isolated from the environment was characterized. the strain was enterobacilli, gram negative, mesophilic, non halophilic, and aerobic bacterium that showed motile ovalerod shaped cells. the isolate produced extracellular chitinase, protease, and prodigiosin (a red pigment produced by several serratia strains yielding bright red or pink colonies). a physiological assay using microbact* test showed that the strain was closely related to klebsiella ozaenae (49.85%) and serratia liquefaciens (24.42%), respectively. however, 16s rrna sequence analysis indicated that the strain was closely related to s. marcescens dsm 30121 with similarity level of 98%. ds-8 strain was able to synthesize its own vitamins. optimum growth in benzoate was obtained at ph between 7-8.5 and nacl concentration of 1-1.5% (w/v). the isolate could grow in benzoate-containing medium up to 10 mm. other carbon sources that could support the growth of ds-8 were casamino acid, glutamate, glucose, acetate, potato starch, and ethanol. keywords: serratia marcescens/aromatic degradation/168 rrna sequence introduction a large amount of monocyclic hydrocarbon aromatic and its derivatives, as well as polycyclic hydrocarbon aromatic, have been deliberately introduced to the environment. many of these compounds, particularly the chlorinated derivatives, are toxic and carcinogenic even at low concentrations. the metabolism of these hydrocarbon aromatic compounds in nature depends on the ability of catabolic reaction of particular microorganisms (semple and cain 1996). of these microorganisms, bacteria of the genera such as alcaligenes, bacillus, pseudotnonas, bulkholderia, and rhodococcus (guerin and boyd 1995; lenke et al. 1992; mars et al. 1996; shen and wang 1995) are the main degrading microbes. several other microorganisms such as fungus (gurujeyalakshmi and oreil 1989), and algae (semple and cain 1996) have also been reported to aerobically catabolize aromatic hydrocarbons. one of the important monocyclic aromatics introduced to the environment is benzoate. it has been introduced through some herbicides application or other industrial 'corresponding author, e-mail: asuwanto@indo.net.id biotropia no. 21, 2003 practices (werwath et al. 1998). hence, it is also found as one of the important intermediate in metabolic pathway of many aromatic compounds (powlowski and shingler 1994). the increasing need of this aromatic compound for industrial purposes and its application should make us aware of its environmental potential hazard. so far, a study on aerobic benzoatc and its derivative degradation has been done in acinetobacter sp. strain 4cb1 (adriaens et al. 1989), amycolatopsis and streptomyces spp. (grund et al. 1990), and pseudomonas (altenschmidt et al. 1993). in the group of enterobacteriaceae, klebsiella pneumonia was reported to degrade 3and 4-hydroxy-benzoate (suarez et al. 1991), and salmonella typhimurium was able to metabolize m-hydroxybenzoate and gentisate (goetz et al. 1992). diaz et al. (2001) noted the involvement of escherichia coli in the metabolism of phenylacetic acid, 3and 4-hydroxyphenylacetic acid, phenylpropionic acid, 3hydroxyphcnylpropionic acid, and 3-hydroxycinnamic acid and amines (phenylethylamine, tyramine, and dopamine). in this study, another member of enterobacteriaceae, serratia marcescens strain ds-8 was found capable of using benzoate as its sole c-source. we would like to examine further the ability of s. marcescens ds-8 to utilize benzoate and to characterize its physiological properties as well as its taxonomic identity. materials and methods bacterial isolate and culture condition the isolate was previously isolated from household sewage water in bogor, west java, indonesia. screening was carried out by growing the isolate in modified salt medium (dong et al. 1992) supplemented with 5 mm na-benzoate as sole carbon source. the isolate was maintained in 12.5% glycerol at -70° c. benzoate and other c source utilization test benzoate utilization was determined by growing the isolate in a modified salt medium supplemented with or without vitamins with 5 mm na-benzoatc as a carbon source. escherichia coli top 10 was used as a control. to determine the degrading ability in different conditions, the isolate was grown in modified salt medium with different initial ph, nacl concentration, and benzoate concentration. growth in other c sources was performed in modified salt medium supplemented with vitamins with either 5 mm succinate, 5 mm glucose, 1% casamino, 5 mm citrate, 5 mm glutamate, 5 mm acetate, 3% ethanol, or 1% potato starch as carbon sources. cell, density in other c source utilization test and benzoate utilization test were measured turbidimetrically at 660 nm after 72 hours and 120 hours of incubation time, respectively. benzoate-utilizing serratia marcescens — dwi suryanto & antonius suwanto growth conditions, measurement of growth, and quantification of benzoate utilization all liquid cultures were cultivated aerobically in 250 ml erlenmeyer. cultures were grown in 200 rpm at 30°c. growth was measured turbidimetrically at 660 nm. benzoate concentration was measured at its absorption maximum of 276 nm using hitachi model u-2010 uv/vis spectrophotometer (hitachi instrument, inc. japan) following the establishment of standard curve relating benzoate concentration to uv absorbance (shoreit and shabeb 1994). for all inoculations, the seed cultures were taken from 2-day-old culture of modified salt medium with 5 mm na-benzoate as carbon source. the cultures were grown with the initial cell concentration of 5xl06 cell/ml. unless otherwise indicated, all media were adjusted to ph 7.2. benzoate solution was filter-sterilized. examination of cell morphology and physiological properties cell shape, motility, and gram staining were evaluated using a nikon ys2-t microscope. physiological characteristics were analyzed using microbact kit test (medvet science pty. ltd., adelaide, australia). test of production of extracellular protease and chitinase was monitored on salt medium agar supplemented with colloidal chitin and skim milk. emb agar was used for preliminary screening for enterobacteriaceae isolates. the appearance of the colonies was observed in luria bertani (lb) agar supplemented with either 50 (-ig/ml ampicillin, 50 ng/ml spectinomycin/streptomycin, 10 ng/ml trimethoprim, 5 mm benzoate, 5 mm salycilate, 5 mm gentisate, or 5 mm phenol. a test of growth in different temperatures was monitored on lb agar. amplification and sequencing of part of 16s rrna gene the 16s-rrna genes were pcr-amplified using specific primers of 63f and 1387r from genomic dna (200 ng) using ready-to-go pcr beads (pharmacia-biotech). modified phenolchloroform-isoamylalcohol treatment, ethanol precipitation, and agarose gel electrophoresis were used to purify the genomic dna. the total volume of pcr reactions (25 \i\) consisted of 1.5 u taq dna polymerase, lomm tris-hcl (ph 9 at room temperature), 50 mm kc1, 1.5 mm mgcl2, 200 \m of each dntps, and stabilizer including bovine serum albumin. the reaction was incubated in a gene amp pcr system 2,400 thermocycler (perkin-elmer cetus, norwalk, conn). part of the genes for 16s-rrna were sequenced to infer the closest related organism from ribosomal database project (rdp) maintained in the university of illinois, urbana-champaign. the sequencing reactions were done by using the big dye ready reaction dye deoxy terminator kit and purification with ethanol biotropia no. 21,2003 sodium acetate precipitation. the reactions were run on an abi prism 377 dna sequencer (perkin-elmer cetus, norwalk, conn.). construction of phylogenic tree cluster analysis of 16s-rrna gene was done using the computer program from european bioinformatics institute (http://www.ebi.ac.uk). the treecon computer program (yves van de peer of department of biochemistry, university of antwerp) was used to determine the relatedness in phylogenic tree based on the nucleotide sequences. results and discussion the new benzoate-utilizing bacteria, ds-8, described in this study was a motile, ovale-rod shaped, gram negative, mesophilic, non halophilic, and aerobic bacterium. instead of microscope observation, the motility could be seen by its swarming activity. on lb solidified with 1.2% agar, the strain swarm on the agar surface, colonized the entire plate with expansion rate of c.a. 19 mm/hours. eberl et al. (1999) also observed a similar behavior in s. liquefaciens mg1. an auto-induction phenomenon might be involved in this swarming activity (eberl et al. 1999;lindumefa/. 1998). based upon microbact test (table 1), ds-8 was closely related to k. ozaenae (49.85%) and s. liquefaciens (24.42%), respectively. however, the test might have no significant match to the available characteristics in microbact data of bacterial species. this might occur since the test is designed for hospital purposes. any other specific characters for isolates might not be incorporated in this detection kit. growth on emb media indicated that the isolate was a member of enterobacteriaceae. partial sequencing (c.a. 500 bp of the 5'-end) of 16s rrna gene of ds-8 showed that the isolate might likely be one species of s. marcescens dsm 30121 (98% of similarity) (figure 1). the isolate showed lower similarity (94%) to strain k. ozaenae. complete sequencing of the 16s rrna gene, however, should give more definitive information about the taxonomic position of the isolate. the isolate produced a red or pink pigmentation on lb agar, or on lb agar supplemented with ampicillin, and streptomycin and spectinomycin, trimethoprim, tetracyclin, skim milk, or chitin (table 2). on the former antibiotic-containing media, the pink color appeared later in the growth stage. no red or pink color was produced when grown in media containing 1 mm phenol, 5 mm salycilate, 5 mm gentisate, or in lb agar supplemented with gentamicin. the red pigment might be prodigiosin, a red pigment produced by the genus of serratia. we did not examine whether the media affected the prodigiosin production, or a quorum sensing might take an effect. other interesting physiological traits were that the isolate produced extracellular chitinase and protease. a study on genetic and cloning of gene encoding chitinase of s. marcescens has been done by watanabe et al. (1997). benzoate-utilizing serratia marcescens — dwi suryanto & antonius suwanto the ability of the isolate to grow on structurally similar aromatic compounds like benzoate, gentisate, salycilate, and phenol, can be explained as it might have similar cell membrane transport system or similar catabolic pathway after ring fission (shimp and pfaender 1987). the different ability to grow in benzoate, gentisate, salycilate, and phenol (table 2) might be due to different uptake efficiency (altenschmidt et al. 1993). ds-8 was able to grow aerobically in 5 mm benzoate with or without vitamin supplement (figure 2). however, different responses of growth in these media were significant. bacterial growth in media supplemented with vitamins was faster than they were without vitamins. ds-8 clearly required vitamins for optimal growth in benzoate. the generation time in media with vitamins and media without vitamins were 11.4 hours and 16.8 hours, respectively. consequently, benzoate degradation also occurred much faster in media supplemented with vitamins. in media with benzoate-utilizingserrarta marcescens dwi suryanto & antonius suwanto vitamin supplementation, benzoate utilization rate was 0.16 mm/hour compared to 0.04 mm/hour in media without vitamin supplement. the ability to grow in the aromatic compound without vitamins indicated that the isolate was capable of synthesizing its own vitamins. relatively low cell density (absorbance at 660 nm) in this study might be a result of using poor media ( figure 3a). minimal media for this purpose are usually supplemented with 0.01% yeast extract or other common c sources (adriaens et al. 1989; gurujeyalakshmi and oriel 1989; shimao et al. 1989). as shown in figure 3, optimum ph and nacl concentration for bacterial growth was between 7-8.5 and 1-1.5%, respectively. the optimum range of ph and nacl concentrations showed that the isolate was not acidophilic or halophilic. optimum growth temperature was in the range of 20-35°c. this isolate could not grow at 40°c. at 37°c, no red pigment appeared. andreeva and ogorodnikova (1999) observed that prodigiosin was not accumulated by growing s. marcescens at this temperature. ds-8 grew very poorly in media with benzoate concentration exceeding 10 mm. a common concentration for benzoate degradation test was up to 5 mm (altenschmidl et al. 1993; bundy et al. 1998). an increase in cell tolerance against toxic substrates was crucial to improve the degradation capabilities. alteration of cm to trans-fatty acid of cell membrane might improve cell tolerance to toxic substrates (heipieper et al. 1992). relatively poor growth of the isolate was also observed in 5 mm benzoate without vitamin supplement. hence, vitamins were needed for optimum growth. benzoate utilization might indirectly depend on ph, nacl concentration, as well as benzoate concentration, since the cell growth was affected by initial ph, nacl concentration, and benzoate concentration (figure 3). the experiment with other organic compounds showed that the isolate grew well in media with casamino acid and glutamate (figure 4). the isolate grew poorly in glucose, acetate, potato starch, and ethanol and failed to grow in succinate or citrate. it seemed that the availability of organic nitrogen in casamino acid and glutamate supported the growth of ds-8. eberl et al. (1999) showed that the doubling time of s. liquefaciens mg1 was significantly increased even in only 0.01% casamino acid. the inability of ds-8 to utilize succinate and citrate might be due to the lack of special transport system. for example, an inducible transport system for malate, succinate, and fumarate was present in rhodobacter (rhodop-seudomonas) sphaeroides (gibson 1975). acknowledgements this research was funded by the center for microbial diversity, faculty of science and mathematics, bogor agricultural university, bogor, indonesia.   benzoate-utilizing serratia marcescensdwi suryanto & antonius suwanto references adriaens, p., kohler, h-pe., kohler-staub, d., and d.d. focht. 1989. bacterial dehalogenation of chlorobenzoate and coculture biodegradation of 4,4'-dichlorobiphenyl. appl. environ. microbiol., 55, 887-892. andreeva, i.n. and t.i. ogorodnikova. 1999. the effect of the cultivation conditions on the growth and pigmentation on serratia marcescens. zh. mikrobiol. epidemiol. immunobiol., 3,16-20. (abstract). altenschmidt, u., oswald, b., steiner, e., herrmann, h., and g. fuchs. 1993. new aerobic benzoate oxidation pathway via benzoyl-coenzyme a and 3-hydroxybenzoyl-coenzyme a in a denitrifying pseudomonas sp. j. bacteriol. 175,48514858. bundy, b.m., campbell, a.l., and e.l. neidle. 1998. similarities between the ont4sc-encoded anthranilate dioxygenase and the ie«^5c-encoded benzoate dioxygenase of acinetobacter sp. strain adp1. j. bacteriol. 180, 4466-4474. eduardo diaz, e., ferrandez, a., prieto, m.a., and j.l. garcia. 2001. biodegradation of aromatic compounds by escherichia coli. microbiol. mol. biol. rev. 65, 523-569. dong, f., wang, l., wang, c., cheng, j., he, z.,.sheng, z., and r. shen. 1992. molecular cloning and mapping of phenol degradation genes from bacillus stearothermophilus fdtp-3 and their expression in escherichia coli. appl. environ. microbiol. 58,2531-2535. eberl, l., molin, s., and m. givkov. 1999. surface motility of serratia liquefaciens mg1. j. bacteriol. 181,1703-1712. gibson, j. 1975. uptake of c4 dicarboxylates and pyruvate by rhodopseudomonas sphaeroides. j. bacteriol. 123,471480. grund. e., knorr, c., and r. eichenlaub. 1990. catabolism of benzoate and monohydroxylated benzoates by amycolatopsis and streptomyces spp. appl. environ. microbiol. 56,1459-1464. biotropia no. 21,2003 guerin, w.f. and s.a. boyd. 1995. maintenance and induction of naphthalene degradation activity in pseudomonas putida and an alcaligenes sp. under diiferent culture conditions. appl. environ. microbiol. 6, 4061-4068. gurujeyalakshmi, g. and p. oriel. 1989. isolation of phenol-degrading bacillus stearothermophi/us and partial characterization of the phenol hydroxylase. appl. environ. microbiol. 55,500-502. hiepieper, h.j., diefenbach, r., and h. kuweloh. 1992. conversion of cis-unsaturated fatty acids to trans, a possible mechanism for the protection of phenol-degrading pseudomonas putida p8 from substrate toxicity. appl. environ. microbiol. 58,18471852. lenke, h., pieper, d.h., bruhn, c., and h. knackmuss. 1992. degradation of 2,4-dinitrophenol by two rhodococcus erythropolis strains, hl 24-1 andhl 24-2. appl. environ. microbiol. 58,2928-2932. lindum, p.w., anthoni, u., christofferson, c., eberl, l., molin, s., and m. givskov. 1998. af-acyl-l-homoserine lactone autoinducers control production of an extracellular surface-active lipopeptide required for swarming motility ofsetratia liquefaciens mg1. j. bacteriol. 180,6384-6388. mars, a., houwing, j., dolfmg, j., and d.b. janssen. 1996. degradation of toluene and trichloroethylene by bulkholderia cepacia g4 in growth-limited fed-batch culture. appl. environ. microbiol. 62, 886-891. powlowski, j. and v. shingler. 1994. genetics and biochemistry of phenol degradation by pseudomonas sp. cf600. biodegrad. 5,219-236 semple, k.t. and r.b. cain. 1996. biodegradation of phenols by the alga ochromonas danica. appl. environ. microbiol. 62,1264-1273. shen, h and y. wang. 1995. simultaneous chromium reduction and phenol degradation in a coculture of escherichia coli atcc 33456 and pseudomonas putida dmp-1. appl. environ. microbiol. 61, 2754-2758. shimao, m., onishi, s.; mizumori, s., kato, n., and c. sakazawa. 1989. degradation of 4-chlorobenzoate by facultatively alkalophilic arthrobacter sp. strain sb8. appl. environ. microbiol. 55,478^182. shoreit, a.a.m. and m.s.a. shaheb. 1994. utilization of aromatic compounds by phototrophic purple nonsulfur bacteria. biodegrad. 5,71-76. suarez, m., gibello, a., allende, j.l., martin, m., ferrer, e., and a. girrido-pertierre. 1991. degradation of 3and 4hydroxybenzoate by k/ebsiellapneumoniae. appl. microbiol. biotechnol. 34,677-682. shimp, r.j. and f.k. pfaender. 1987. effect of adaptation to phenol on biodegradation of monosubstituted phenols by aquatic microbial communities. appl. environ. microbiol. 53,1496-1499. watanabe, t., kimura, k., sumiya, t., nikaidou, n., suzuki, k., suzuki, m., taiyoji, m., ferrer, s., and m. regue. 1997. genetic analysis of the chitinase system of serratia marcescens 2170. j. bacteriol. 179, 7111-7117. werwath, j., arfrnann, h., pieper, d.h., timrnis, k.n., and r. wittich. 1998. biochemical and genetic characterization of a gentisate 1,2-dioxygenase from sphingomonas sp. strain rw5. j. bacteriol. 180,4171-4176. 10 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf 10.pdf biotropia no. 5, 1991/1992: 15-21 effect of light qualities and storage periods on the germination of pennisetum polystachion seeds*) verapongs kiatsoonthorn and soekisman tjitrosemito department of biology, faculty of science, srinakhrinwirot university (prasanmitr), bangkok 10110, thailand and seameo biotrop, jl. raya tajur km 6, p.o. box 17, bogor, indonesia. abstract seeds of the yellowish inflorescence strain of pennisetum polystachion, collected from the field in indonesia, were kept in the dark for 30 days, then germinated in 12-h light and 24-h light under various light qualities, namely, white, black, blue, red and far-red. there was no effect of photo-period to seed germination. percent of seed germination under white, red, far-red, blue and dark were 49, 43, 22, 11 and 2%, respectively. white and red light did not cause any difference to seed germination. seeds kept in 12-h light alternating with 12-h dark and 24-h dark for 15 and 30 days were tested for germination. results showed that light condition during seed storage did not effect seed germination. long storage period resulted in more seed germination. during seed germination test, effect of light played a great role on increasing seed germination. introduction pennisetum polystachion, or mission grass in english (wssa 1984), in thailand had two distinctive forms of immature inflorescence colour, namely purplish and yellowish. studies concerned with seed germination of the yellowish inflorescence strain had been reported (van rooden et al. 1970; noda et al. 1985; suppaphon et al. 1987; arunpu et al. 1991). fernandez (1980) without specifying the immature inflorescence colour, stated that seed of this species collected in indonesia was photosensitive for germination, especially in the first 70 days of storage after harvest; whereas, noda et al. (1985) reported that the response of this seed species to light was very low. arunpu et al. (1991) reported that this species was non-sensitive to light. in order to clarify these discrepencies, the following experiments were conducted. *)this article is part of a six-month research fellowship report submitted to seameo biotrop in december 1991. 15 biotropia no. 5, 1991/1992 materials and methods 1. light qualities and durations p. polystachion seeds harvested on 8 july 1991 from biotrop experimental field were removed from bristles, screened through a 1-mm-wire mesh to obtain uniform seed sizes and kept in the dark at room temperature (25 29°c) for 30 days until germination test. one hundred seeds were placed on a filter paper moistened with 4ml distilled water, in a 9-cm petridish covered with lid. treatments were designed in factorial of 2 factors: first factor was light period i.e. 12-h light and 24-h light, and another factor was the light qualities, namely white, red, far-red, blue and dark. for light quality treatments each petridish was wrapped with a blue, a red, a blue plus a red transparent polyethylene to obtain blue, red and far-red treatments, respectively (fernandez 1980). the dishes of dark treatment were wrapped with aluminium foil whereas the white lighted treatment was unwrapped. light transmission through those transparent polyethylene, measured by specto-photometer is shown in fig. 1. these dishes were placed in a 25-29°c room illuminated with 24-h light by cool fluorescent tubes philips tld 36 w. light fig. 1. percentage transmission of light through red, blue, and red plus blue transparent polyethylenes from tungstar light source 16 effect of light qualities and storage periods verapongs kiatsoonthorn & soekisman tjitrosemito intensity reaching the dishes through red, blue, and red plus blue transparent polyethylene was 0.066, 0.073, 0,004 uem -2 s -1 , respectively, whereas the controlled light was 0.775 uem -2 s -1 . for the 12-h lighted treatment, those dishes were covered with an opaque container for 12-h dark alternating with 12-h light. during the experimental period the dishes were randomly rotated in order to reduce the effect of different light intensities. germination was tested 6 days after treatments and was transformed to arcsine square root before statistical analysis. 2. storage period, light conditions during seed storage and germination test 2.1. effect of lighted conditions during storage and storage period lighted the experiment was replicated 4 times in factorial design with 2 factors: the first factor was the storage period of 15 and 30 days; the second factor was light conditions during storage consisting of the dark and light/dark conditions. p. polystachion seeds collected from the same location as in experiment 1, on 20 august 1991, were kept in a 24-h dark (dark) or 12-h light alternating with 12-h dark (light/dark). for the dark treatment, seeds were placed in polyethylene bag and wrapped with aluminium foil; while for the light condition, the polyethylene bag was not wrapped with aluminium foil. both treated seeds were separately kept in 9-cm petridishes, placed on a table near the window for 15 and 30 days until germination. germination test was done in a 30°c controlled incubator illuminated with 12-h light alternating with 12-h dark. light intensity during the day was 1.32*10 -4 uem -2 s -1 . the detailed methods were the same as mentioned in experiment 1. accumulated number of seed germination at 8th day were transformed to arcsine square root before statistical analysis. 2.2. effect of lighted conditions during storage and germination methods seeds stored in light/dark and dark conditions for 30 days, as in experiment 2.1, were used. the experiment was replicated 4 times in factorial design with 2 factors: the first factor was light condition, consisting of the dark, and the light/dark condition for 30 days; the second factor was germinating condition consisting of alternating 12-h lighted with 12-h dark with data collected every 2 days up to 8 days (light/2), dark condition with data collected every 2 days up to 8 days (dark/2), and dark condition with data collected 8 days later (dark/8). for seeds germinated in the dark/2 and dark conditions, petridishes were wrapped with aluminium foil. germinated seed counting was done in dark room under green light (40 watt incadescent bulb wrapped with a green transparent polyethylene). in both experiments, accumulated data of seed germinated at 8 days after treatments were transformed to arcsine and statistically analyzed. 17 biotropia no. 5, 1991/1992 results and discussion 1. effect of light qualities and durations results of this experiment showed that exposure of 24-h light and 12-h light did not cause any difference on seed germination. percent germination of seeds under white, red, far-red, blue light and dark conditions were 49, 43.1, 21.9, 11.1 and 1.6%, respectively, whereas red and white light treatments similarly effected seed germination (figure 2). percent (%) far + red l light qualities fig. 2. germination of p. polystachion seeds under various light qualities (30 days after seeds collection) light qualities and photo-period did not interact to effect seed germination (figure 3). the result of seed germination under various light qualities in this experiment was similar to fernandez's result (1980) except the effects of blue and far-red which were significantly different in this result but not different in fernandez's. one form of phytochrome in seed, phytochrome far-red (pfr), which is an active form for seed germination, can be transformed from phytochrome red (pr) when irradiated with red light; and pfr can be transformed back to pr when irradiated with far-red light (taylorson 1987). pfr can also degenerate to pr in the 18 0 blue dark red white effect of light qualities and storage periods verapongs kiatsoonthorn & soekisman tjitrosemito fig. 3. seed germination of p. polystachion effected by light qualities and photo-periods (nonsignificant between the interaction factors) dark. this explains why red and white lights showed higher germination than the dark condition. blue and red transparancies apparently permitted transmission of red light better than blue transparancy, therefore the former stimulated more germination than the later. although blue transparancy transmitted wavelength between 430-550 nm (fig. 1), this light has not been reported to affect germination. in fernandez's method, the light intensities were different from the other because some petridishes were covered with various transparancies, while the distance between the light source and petridishes were similar. 2. storage period, light conditions during seed storage and germination test 2.1. effect of lighted conditions during storage, and storage period p. polystachion seeds kept in room temperature for 15 days had lower germination percentage than those kept for 30 days (table 1). however, different light conditions during seed storage did not show any different effect on seed germination. light condition and period during seed storage did not interact to affect seed germination. 19 biotropia no. 5, 1991/1992 table 1. percent germination of seeds kept in two different lighted conditions for 15 and 30 days (8 days after treatments) 2.2. effect of lighted conditions during storage and germination methods the result of this experiment supports that of experiment 2.1., i.e. the light condition during seed storage did not affect seed germination. however, different germinating methods effected seed germination differently. more seeds germinated in light/2 than in dark/2 or dark/8. in dark condition, those seeds observed for germination every 2 days had higher germination than those observed only once at 8 days after germination (table 2). the green transparancy may be able to transmit some red light, which stimulates germination. however, both factors of light conditions during seed storage and of germinating methods did not have any interaction on the number of germinated seeds. the difference in seed germination under light and dark condition found in this experiment was similar to fernandez's result (1980) and noda et al. (1985); but was contrary to arunpu et al. (1991). it was reasoned that seeds used in the experiment of arunpu et al. were kept for too long (6 months at 5°c dark condition) so photo-sensitivity of seed was lost. table 2. percent germination of seeds effected by lighted conditions during storage and germinatimg methods (8 days after treatments) 20 effect of light qualities and storage periods verapongs kiatsoonthorn & soekisman tjitrosemito references arunpu, s., v. kiatsoonthorn and y. yingwiwatanapong. 1991. effect of some environmental factors on seed germination of p. setosum (swartz) l.c. rich. kasetsart journal (abstract in english). in press. fernandez, d.b. 1980. some aspects on the biology of pennisetum poylstachyon (l.) schult. philippine journal of weed science 7: 1-10. noda, k., l. chaiwiratnukul, s. kanjanajirawong and m. teerawatsakul. 1985. some biological characteristics of pennisetum spp. in thailand. proceeding of 10tn asian-pacific weed science society conference: 75-80. suppaphon, j., p. roengapapong and chitpong. 1987. certain characteristics of flower and seed germination of pennisetum setosum. in: report of workshop on pennisetum setosum, held by prince of songkla university and ministry of agriculture and cooperatives in cooperation with agricultural science association of thailand under the royal patronage, pesticide business association of thailand, and weed science society of thailand: 19-32. (abstract in english). taylorson, r.b. 1987. environmental and chemical manipulation of weed seed dormancy. in: review of weed science. weed science society of america vol. 3: 135-154 van rooden, j., l.m.a. akkermans and r. van der veen. 1970. a study on photoblastism in seeds of some tropical weeds. acta. bot. neerl. 19(2): 257-264. wssa. 1984. composite list of weeds. weed science vol. 32 (supplement 2). 21 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf biotropial(l) 1987: 1-25 studies on the breeding structure of tree species in the tropical rain forest. i: family clumps and intrapopulation differentiation kan-ichi sakai *, toru endo *, shinya iyama* national institute of genetics, mishima, japan yasusada miyazaki*, shigesuke hayashi*, yoshiya shimamoto * kyushu university (tukuoka), kagoshima university (kagoshima) and hokkaido university (sapporo), japan respectively lilian u. gadrinab & ulfah juniarti tropical forest biology program, biotrop, bogor, indonesia abstract breeding structures of two tropical rain forest tree species, altingia excelsa in java and agathis borneensis in kalimantan were investigated. assuming that similarity in the assortment pattern of the isoperoxidase bands tells genetic relationship between trees, on the one hand, and that inbreeding increases smaller values of the disagreement counts, on the other, it has been concluded that inbreeding occurs considerably in altingia excelsa and to some extent in agathis borneensis. finding that trees showing very low disagreement counts are located close to each other, they were grouped as an assumptive family. it was found that different families were quite dissimilar with respect to isoperoxide constitution and in several leaf characters as well. the distance between two trees at which they can mate is estimated to be 16 to 18 meters or 16.5 meters and the area one family occupies is 200 to 250 m ,̂ assuming that a family clump can be a breeding unit in altingia excelsa, within which trees mate at random. some families were distributed mixed with each other within the mating distance, but they were found still genetically differentiated from each other. this reproductive isolation among families is interpreted to be due to genetic differences between families in flowering time. in agathis borneensis, there was no indication of family clump formation. related trees may have been widely scattered in the forest, and the inbreeding of the species may be due to self-fertilization of individual trees and not to outcrossing between relatives. introduction one of the characterisitic features of a tropical rain forest is its high species diversity. it stimulates our interest to inquire into two relevant problems. one is how trees could propagate in such a forest with an extremely low population density for each species, or what the breeding system of tropical tree species is. * temporarily seconded to biotrop. 1 biotropia vol. 1 no. 1, july-december 1987 another problem is how speciation takes place in such a tropical rain forest of high diversity of species. in the meantime, we recollect two hypotheses already advanced concerning the speciation problem: one is the so-called genetic drift hypothesis by fedorov (1966), and another is the selection hypothesis by ashton (1969). fedorov assumes that a very small number of conspecific trees with asynchronous flowering among them should naturally induce each individual tree to propagate by self-fertilization which after many generations results in speciation by genetic drift. ashton, on the contrary, assumes that notwithstanding more or less difficulty in outbreeding, it would still occur frequently enough to allow gene exchange throughout populations in a continuous habitat causing speciation as a result of allopatric differentiation between populations in response to differential selection pressures. this paper describes results of an investigation into the breeding structure of two species, altingia excelsa noronha (hamamelidaceae) in java and agathis borneensis warb. (araucariaceae) in kalimantan,with an aim to find a way to pierce an opening to the problem of speciation. materials and methods two natural forests were investigated. the stand in which altingia excelsa was investigated was located on the outskirts of the village of ciwidey near bandung in western java. all trees with diameter at breast height (dbh) exceeding 6 cm, growing in quadrat of 50 x 50 m in the stand were measured for their growth, and their position in the quadrat was mapped on a section-paper. in all, 148 trees of various species were counted, among which 38 were trees of aliingia excelsa. twigs with mature leaves were collected from each of these 38 trees for laboratory research. the mature leaves from each tree were divided into two parts, one for measurement of several leaf characters while the remaining part for the electrophoretic analysis of the isoperoxidases. preliminary studies showed that the zymogram patterns for acid phosphatase and esterase were rather simple and thus only peroxidase was used in this study. agathis borneensis was sampled from a quadrat of the same size plotted in a natural forest of international timber corporation indonesia in kalimantan. there were about 230 trees with dbh exceeding 6 cm of various species in the quadrat, of which 21 were agathis borneensis. growth measurement and mapping of individual trees were made and their leaves were collected for the study of leaf characters and eletrophoretic analysis. leaf characters measured in both species were leaf length, leaf width, leaf size (length x width), number of veins, and length and thickness of petioles. 2 breeding structure of tree species in the tropical rain forest — sakai et al. methods applied for the electrophoretic analysis were as follows: enzyme extractant m4 containing 10% triton x-100, 0.5 m nacl tris and 0.2 m ascorbic acid, was adjusted with acetic acid to ph 7.5. one hundred mg of finely cut leaf pieces were crushed in a mixture of 0.5 ml of the extractant, 100 mg of quartz sand and 50 mg of polyvinyl-polypyrrollidone (endo 1981). this system proved to be generally suitable for the extraction of peroxidase in altingia as well as in agathis. starch gel electrophoresis was subjected to the system modified by brewer (1970). the gel buffer contains 0.42 g of histidine hc1 and 0.072 g of naoh per 0.5 1 mixed with 60 to 66 g of hydrolyzed potato starch, with the final ph of the solution around 6.1. the tray buffer was with a ph of about 6.0 and it contained 117.6g(or 0.6 m) trisodium citrate and 9.4 g of citric acid per liter. the gel mold of toyo model ha-1 devised by endo (1968) was used. electrophoretic run was done under constant 250 voltage per 20 cm for 4.5 hours in a refrigerator with an ice cooling box. reaction mixture of peroxidase stain contains 4 mm eugenol, 4 mm 3-amino-9-ethylcarbazole in 10% acetone solution at final concentration, and 0.03% hydrogen peroxide, and 0.02m tris-acetic acid buffer, ph 4.0 (endo 1978). the figure 1. schematic explanation for measuring the disagreement count between x and y. 3 biotropia vol. 1 no. 1, july-december 1987 isoperoxidase zymograms of 38 altingia excelsa and 21 agathis borneensis trees are schematically drawn in appendices 1 and 2. on the assumption that the pattern of the isoperoxidase bands of a tree would be nothing but the reflection of its genetic make-up, genetic similarity was measured among trees with the aid of the disagreement count which gives the total number of missing mate to each band between a given pair or trees (sakai & miyazaki 1972). in practice, there may occur variation in activity of bands, but it was neglected for the present study. figure 1 explains the method of calculation of the disagreement counts between two zymograms. detection of family structure of a tree species in a natural stand with the aid of the disagreement counts was made on the following premises: (1) it was assumed that the plural isoperoxidase bands appearing in the population would be distributed at random among individual trees if mating occurs at random, while inbreeding, either self-fertilization or mating between relatives, would tend to produce the isoperoxidase combination of the parental type. (2) the disagreement counts obtained between trees of the same family were thus lower in value than those obtained between trees not related with each other. it should be borne in mind, however, that converses are not always true; that is, trees with low disagreement count cannot immediately be regarded as sib-members of a family. (3) propagated sib-trees would be apt to form a clump around their mother-tree. (4) trees growjng in a clump would have a higher chance of mating with each other than with trees of a separate group, particularly in the tropical rain forest with low population density. (5) trees belonging to a single family would be more alike in their biochemical as well as vegetative characters, and it may naturally enlarge character variation among families in contrast to its curtailment within families. results and discussion i. altingia excelsa (a) formation of family clumps individual variation of dbh of 38 trees is presented in table 1. the table shows that trees exceeding 61 cm in dbh were five among 38, or 13%. in order to determine if the actual distribution of disagreement counts occurs according to the random assortment of the isoperoxidase bands in individual trees, we calculate the theoretical expectation of the latter (appendix 3). 4 breeding structure of tree species in the tropical rain forest — sakai et al. the comparison between theoretical expectations and observed disagreement of bands by the method described is given in table 2. data presented in table 2 show an apparently excess occurrence of observations in the lower classes of 0, 1 and 2, suggesting that the fertilization in altingia excelsa is not panmictic, but a fair amount of inbreeding could be occurring. 5 biotropia vol. 1 no. 1, july-december 1987 in figure 2 we find that actual observations exceeded the theoretical expectations in such lower classes as 0, 1 and 2, and also in those of higher classes exceeding 8. that disagreement counts of several smallest and largest values appeared more frequently than theoretical expectation suggests that inbreeding on the stand tended to construct two contrary groups of trees, one with very similar isoperoxidase patterns and another with highly dissimilar ones. inbreeding in a natural forest can occur in two ways: self-fertilization of individual trees and crossing among relatives. for the latter to occur, trees belonging to the same family growing together in a clump would be more advantageous. are consanguineous trees of the same family really growing in a clump? in order to answer this question, the relationship between inter-tree distance and disagreement counts was investigated (table 3). table 3. relationship between disagreement counts and inter-tree distances in altingia excelsa. inter-tree distance (m) disagreement count total x 2 p 0.1 2.3 4.5 6< 0-10 expectation 6.7 21.6 30.5 33.3 observation 15 25 25 27 92 13.17 <0.01 11-20 expectation 123 39.8 56.3 61.6 observation 11 29 70 60 170 6.43 >0.05 21-30 expectation 14.3 46.4 65.5 71.7 observation 15 42 58 83 198 3.05 >0.20 31< expectation 17.7 57.2 80.8 88.4 observation 10 69 80 85 244 5.90 >0.10 table 3 shows that the difference between expected and observed value was statistically significant only for the smallest inter-tree distance of 0-10 m. it was found that the difference was in the conspicuous excess of observations in the 0 and 1 classes of the disagreement counts, that is, trees growing nearby or so close to each other at a distance of less than 10 m were likely to bear a striking resemblance in the pattern of the isoperoxidase zymograms, giving the impression that genetically, closely related trees grow together in small groups. in other words, tree pairs showing low disagreement count as 0 to 2 and growing nearby in natural forest may be considered as members of a family. thus five assumptive families: a, b, c, d and e were depicted in figure 3. broken lines connecting individual trees with arabic numerals 0, 1 or 2 in the figure are presumed ties between parent and child trees, the numerals indicating the corresponding disagreement counts. until trees in question are proven to be related members of genetically separate 6 breeding structure of tree species in the tropical rain forest — sakai et al. figure 3. family clumps inaltingia excelsa. arabic numerals represent disagreement counts, while those in parentheses disagreement counts between trees of presumed relation. 7 biotropia vol. 1 no. 1, july-december 1987 families, the "assumptive families" designated above will be called merely as tree "groups" for the time being. of the five groups, two, i.e. b and e are worthy of note because both have the largest groups, each with 7 trees. the largest trees of both groups were connected with small disagreement count 1 or 2, indicating that the two groups are more or less related or maybe descendants of a common progenitor. of more interest is that both groups appeared to show a within-group segregation in width of leaves (table 5). although we are not yet in a position to speak definitely, it is supposed that b and e groups might be both heterozygous for a gene probably with major effect on leaf width. (b) differentiation in the constitution of isoperoxidase components the occurrence of various isoperoxidase bands in trees of five groups was examined (table 4). table 4. interpopulation differentation in distribution of isoperoxide bands *) the maximum number of isoperoxidase bands per tree in altingia excelsa was 16. it was found that trees of each group were characterized by specific isoperoxidase bands. for instance, c group is very peculiar in not having such bands as a and / which are rather ubiquitous in altingia excelsa. instead, it possessed e, g, and k bands which are more or less uncommon to the other groups. table 4 shows that those five groups were very variable in their biochemical characteristics demonstrating an intrapopulation genetic differentiation. (c) intrapopulation differentiation in vegetative characters now we should further inquire into differentiation among groups in some leaf characters. six leaf characters were investigated and their variation is given in a form of frequency distribution in table 5. it was noted that in almost all characters, variation among these groups was apparent. b group had the biggest size in many leaf characters, e the next, while c and d groups were the smallest, leaving a intermediate. the analysis of variance of the leaf characters is shown in table 6. 8 breeding structure of tree species in the tropical rain forest — sakai et al. table 5. variation of six leaf characters in five family groups of altingia excelsa 9 biotropia vol. 1 no. 1, july-december 1987 significant at the 5%* and 1%** levels, respectively. ns) non-significant. from table 6 we find that vein number was not significantly variable among groups, but measurements in leaves and petioles were all significantly variable. from the facts described above, one can conclude that groups a to e defined up to now are clumps of trees of single families or family clumps, each of which is being genetically differentiated from others. for genetic differentiation among clusters within a population to occur, it is necessary that propagation is by mating within the same, but not between different families. if one assumes that trees of a single family form a breeding group within which mating occurs at random, then what would be the area occupied by a group in a natural stand? of the five families depicted in figure 3, families a and c are not included in the following discussion because of too small number of trees in them. one notices in figure 3 that in each of the three remaining families, one tree always stood relatively far from the others of the same family. thus, the inter-tree * inter-tree distance. 10 breeding structure of tree species in the tropical rain forest — sakai et al. distance was measured in two ways: one for all trees, and another for all trees but one standing apart. the results are presented in table 7 and figure 4. figure 4. distribution of inter-tree distances in three families of altingia excelsa. black circles are those between the farthest tree and the remaining ones in each family. the distribution of inter-tree distances in three families is shown in figure 4, wherein distances involving the farthest tree are depicted in black circles. excluding the black circles, one may state that the mean distance of the mean plus one standard deviation as the family distance for e = 10.79 + 5.71 = 16.5 m (table 7). this distance is expected to include 84% of cases in a theoretical normal distribution and could be taken as the mating distance in altingia excelsa, if we assume 16 to 18 meters as the mating distance, then the area as a circle of a family clump in the species is roughly estimated to be π r 2 = 200 m 2 to 250 m2 with r = 8 to 9 meters. if 16 to 18 meters is the mating distance, then why do b and c families look genetically isolated from each other in spite of growing mixed together in the forest (figure 3)? we have already concluded that the two families b and c are very different biochemically as well as morphologically. table 8 shows the distribution of the disagreement counts obtained between trees within and between b and c families, based on which we have distinguished b from c. it indicates that both families are very dissimilar in the assortment pattern of the isoperoxidase bands. 11 biotropia vol. 1 no. 1, july-december 1987 in order to find whether trees of both families could be regarded as growing mixed together in an area of the mating distance, the inter-tree distances have been measured among six trees excluding the farthest one of the b family and three of the c. the data are presented in table 9. table 9. inter-tree distances of six trees* in family b and distances between three trees of family c and six of family b. inter-tree distance (m) 0.1 2.6 5.1 7.6 10.1 12.6 15.1 17.6 total mean sd 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 within b 2 3 3 4 1 2 0 0 15 7.09 3.71 between 0 0 3 3 3 4 4 1 18 12.25 3.98 b and c * one farthest tree was discarded. the inter-tree distance between the two families are from 5 to 20 meters, but 13 of 18 or 72% of the inter-tree distances was less than 15 meters, and 17 of 18 or 94% was less than 17.5 meters, which are for the present understood as within the range of the mating distance for altingia excelsa. why are trees of both families isolated sexually in spite of growing within the range of 16 to 18 meters? at present we guess that the two families may not flower at the same time inducing sexual isolation between them. details of the argument is given in the discussion. ii. agathis borneensis a comparison was made between theoretical distribution of disagreement counts and the actual observations (table 10). from table 10 one notes that the number of actual observations appeared to exceed the expectations of the disagreement counts in the respective classes of 0 and 1, and that of 6 or more although the excess is not enough to reach the level of statistical significance. in figure 5, we find that the general situation of the curve is 12 breeding structure of tree species in the tropical rain forest — sakai et al. figure 5. comparison between observed and expected frequencies of disagreement counts in agathi.s borneensis. table 10. comparison between theoretical expectations and actual observations of disagreement counts in agathis borneensis similar with that of altingia, and it is considered that inbreeding occurs to some extent in agathis borneensis, too. the next step is to examine if the occurrence of low disagreement counts was related to small inter-tree distances (table 11). disagreement counts in agathis borneensis looked to be randomly distributed and quite independent of inter-tree distances. thus, one can conclude that in agathis borneensis parental and child trees do not form the clusters of a single family group in a natural stand. occurrence of inbreeding in the species may have been due to self-fertilization of individual trees. 13 biotropia vol. 1 no. 1, july-december 1987 table 11. distribution of disagreement counts in relation to inter-tree distances in agathis borneensis. discussion plants growing in a population naturally involve related and nonrelated individuals more or less intermingled with each other. in fact, there is an indication that a plant population can be divided into these two groups. for instance, levin (1977) gave a brief description in his paper dealing with the distribution of genetic variation in the annual herb phlox drummondii hook that crossing between plants growing within 2 meter distance gave about three times more degenerative embryos than those from plants growing farther than 15 meters. price and waser (1979) in their crossing experiment with plants of delphinium nelsonii in wild populations collected pollen from plants growing at different distances of 0 (self), 1, 10, 100 and 1000 meters. it was found that the cross between plants growing at an intermediate outcrossing distance of 10 meters gave the best results in seed set and survival rate of seedlings, while cross with plants growing nearby seemed to show inbreeding depression. coles and fowler (1976) found in picea glauca that seeds obtained from crossing between trees growing within a radius of 100 meters showed some inbreeding effect in seed set, germination and seedling growth. how do parental and child trees distribute in a forest? hubbell (1979) found in a tropical dry forest that there were three groups of tree species with respect to the distribution pattern of the adult and juvenile densities around given adult trees. of the 30 species examined, 15 belonging to the first group showed the highest mean juvenile density in the 0 to 5 meters annulus, closest to the adult. the ten 14 breeding structure of tree species in the tropical rain forest — sakai el al. species of the second group showed horizontal density curves, while the remaining 5 species constituting the third group showed the maximal juvenile density in the area between 5 and 15 meters from the adult. there are few papers describing that seedlings are not likely to grow in the neighborhood of their mother trees. for instance, janzen (1970, 1971) described the effect of herbivores in preventing growth of saplings around their mother trees in tropical forests. webb, tracy and haydock (1968) found an autoallelopathic effect of subtropical rain forest trees that produced a substance toxic to seedlings of the same species. watkinson (1978) observed that in an annual grass species vulpia fasciculata (gramineae) the seed drops first around the mother plant (first phase), but later they migrate or are buried in the soils (second phase). the present study aims at detection of family clumps, if any, in two tree species, altingia excelsa noronha and agathis borneensis \varb. both are the tropical rain forest tree species having not a few conspecific trees in the same stand. the number of trees of altingia in the 50 x 50 m quadrat in the ciwidey natural forest that was investigated was 38 among a total 140 trees. this seems to be not exceptional for the species, because federov (1966) wrote in his paper that altingia excelsa was one of those rare species which could attain a relatively large population density in the tropical rain forests in east java. another species, agathis borneensis also had a relatively large population density, i.e. 21 among 230 trees in the 50 x 50 m quadrat in kalimantan. for detection of familial structure in these two species, the results of isoperoxidase analysis have been used. two of the present authors, sakai and miyazaki (1972) published a paper reporting the analysis of family groups in the natural forests of thujopsis delabrata sileb. et zucc. by means of the so called disagreement counts which measures the degree of dissimilarity in the assortment pattern of isoenzyme bands between individual trees. based on the accepted views that the behavior of isoenzymes is monopolitically controlled by genes, and that isoenzymes have nothing to do with adaptability and are thus free from natural selection, it is interpreted that the degree of dissimilarity in the assortment pattern of isoenzymes could tell the genetic dissimilarity. as a matter of fact, schwartz and armitage (1983) have hypothesized in the study of wild marmots that related individuals should have a higher average electrophoretic genetic similarity than unrelated individuals, although their case is a little different from ours. this disagreement counts of isoenzymes described above have thenceforth been employed for detecting familial structure in fagus crenata blume by hashizume and sugimoto (1980), cryptomeria japonica d. don by hashizume and sugimoto (1982), chamaecyparis obtusa endl. by hashizume, watanabe and ookita (1983) and abies sachalinensis mast, by matsuura (1983). 15 biotropia vol. 1 no. 1, july-december 1987 all these studies depend on a few premises, among which is that random mating between trees of a population would give rise to random assortment of individual bands of the isoenzymes in individual trees, while inbreeding would induce resemblance in the combination pattern of their bands. it was found in the present study that in altingia, the disagreement counts of such a low value as 0, 1 or 2, i.e. trees with very high similarity, were more significant than the theoretical expectation calculated on the basis of random assortment of isoenzyme bands. in other words, trees similar in the assortment pattern of the isoperoxidases were more than randomly expected, indicating that propagation by inbreeding has most probably taken place in the stand. further analysis of the distribution of the disagreement counts in relation to the inter-tree distances has shown that the lowest counts were found mostly in the distances of 0 to 10 meters between trees. this means that trees with similar assortment patterns of isoperoxidase bands are located very close to each other, or that related trees form a small subgroup or clump in the stand. in this connection, it is interesting to remember the work of hashizume and sugimoto (1980) on fagus crenata blume. they found that the disagreement counts measured between mother-trees and their respective offspring were mostly so small as 0 and 1. thus presumptive families have been depicted for altingia excelsa in figure 3, where five groups, a, b, c, d and e were considered. in some papers concerning formation of small groups of trees in a forest, roe (1967) reported that in picea engelmannii, the seed dispersal occurs in the vicinity around the mother trees. fedorov (1966) wrote that in the tropical rain forest, "not only rare species, but dominant species too, usually form small populations". ashton (1969) wrote that "contagious distribution of individuals is in fact general among rain-forest trees". according to ashton, the clumping is related to the means of dispersal on the one hand, and to the chance of saplings of a single species to take their place in a gap, on the other hand. what role would these small groups of trees in a forest play with respect to the reproduction and propagation of trees? sarvas (1967) divided pollen dispersal of tree species into 4 levels: (1) within individual trees, (2) between trees within a subpopulation, (3) between sub-populations within a population and (4) between populations. he investigated the problem in wind-pollinating pinus silvestris by the pollen catch technique and found that 50% of pollen affecting fertilization came from outside the population. contrary to sarvas, however, langner (1953) made an experimental study on the fertilization problem in picea abies (l.) karst. he made use of the "aurea" mutant and found that pollination occurred mostly between immediately neighboring trees in the stand. 16 breeding structure of tree species in the tropical rain forest — sakai et al. ashton (1969) described that in the tropical forest, group or clump is likely to be the principal breeding group in most tree species although outcrossing with other groups also occurs. chan (1980) observed that in shorea leprosula, trees in a cluster produced more fruits than isolated ones. appanah (1980) described that in the tropical rain forest, inter-tree movements of pollinators occurs mainly between neighboring trees in a clump, suggesting short distance pollen transfer. if reproduction occurs within a clump, or the effective breeding group is very small and with little exchange of genes between clumps, genetic differentiation should be great among them (wright 1946). in a perennial herb liatris cylindracea, schaal (1975) and schaal and levin (1978) found striking variation in gene frequencies of isoenzymes and in plant growth characters among subpopulations. levin (1977) investigated distribution of genetic variation in wild populations of phlox drummondii hook., a complex annual species consisting of six subspecies. the isoenzyme studies showed that most genetic variation was found within populations, less between populations within subspecies and least among subspecies. on the contrary, schaal and smith (1980) investigated isoenzymes in populations of a leguminous allogamous species desmodium nudicaule and found that no genetic differentiation within the population or no genetic substructuring was found in the species. in tree species, curies and ledig (1982) found in 11 populations of pinus rigida that 97% of genetic diversity was among individual trees within populations and only 3% was between them. a similar result was obtained by hiebert and hamrick (1983) in pinus longaeva bailey. linhart, mitton et al. (1981) investigated genetic variation at seven isoenzyme loci in six clusters of trees in a population of pinus ponderosa laws. they found that the tree clusters differed significantly from each other in the isoenzyme loci as well as in several gross morphology. in pinus monticola dougl., rehfeldt (1979) grew 8 full-sib families from each of 12 populations to investigate variation in several growth characters. he found significant variation among families within populations, but litt le or no differentiation among populations. in pseudotsuga menziesii (mirb.) franco, yeh and o'malley (1980) investigated 21 isoenzyme loci in 11 populations. they found that almost all genetic variation was found within populations and very little among them. in the tropical rain forest, gan, robertson et al. (1977) have found spatial heterogeneity in several isoenzymes as well as in some leaf characters in a population of shorea leprosula and xerospermum intermedium. they attributed this heterogeneity to the short-range pollen flow and fruit dispersal causing spatial isolation between subpopulations. in aitingia excelsa, it was found that each of the five presumptive families or groups was apparently different from others in several respects. as seen in table 4, every group possesses its specific bands which other 17 b1otropia vol. 1 no. 1, july-december 1987 groups do not. the groups were also variable in several leaf characters. these facts tell us that the five groups were genetically differentiated, suggesting that each group had been genetically separated from others. in other words, reproduction may have occurred mainly within each group without exchanging genes with others. thus, the five groups are admitted to be separate as breeding groups, and because of very low values of disagreement counts within each group, each of them is certainly considered to be a single family. assuming that trees within a single family clump could mate freely, we are able to estimate the mating distance in the species as 16 to 18 meters or 16.5 meters. for estimating the area occupied by the family clump as a breeding group, it would be a circle of 200 or 250 m 2 with a radius of 8 or 9 meters. a panmictic unit of breeding or a group of plants in which their gametes may come together has been defined by wright (1946) as a neighborhood in a plant population. schaal and levin (1978) estimated the area of the neighborhood of a perennial herb, liatris cylindracea to be 33 m 2 on the basis of flying distance of insects and the distance of seed dispersal. the family clump in the present paper may correspond to the neighborhood in some respects, because the two concepts equally emphasize an occurrence of random mating among members of a group. a remarkable difference between these two is breeding in the family clump, mating occurs among related individuals, while in the neighborhood it occurs among all individuals. attention should be paid to the two families b and c (figure 3). granting that 16 to 18 meters are the mating distance of aitingia excelsa, a question arises as to why trees of both families could be so much differentiated from each other in electrophoretic as well as in morphological characters, in spite of their mixed distribution in the forest. in nature, there may be various kinds of reproductive barriers. an extreme case may be found among provenances from a wide region. for instance, dogra (1981) found in pinus wallichiana a.b. jacks native to himalayas that flowering time was different from provenance to provenance, particularly between low and high elevation. the mountain ridge could also be a very effective barrier. in the present case of altingia, however, this geographical barrier was not present in the population. flowering time of a plant is of course dependent on environmental conditions, on the one hand, and genetic constitution of the plant, on the other. for the genetic control on flowering of plants, stern and roche (1974) have given forcible discussions in their book, "genetics of forest ecosystems". stam (1983) took a view of natural selection of environments on within-population differentiation of flowering time in plants. 18 breeding structure of tree species in the tropical rain forest — sakai et al. it is often stated that the lack of seasonality in the tropical forest leads to irregularity and lack of coincidence in flowering among not only related species, but also among individual trees of the same species (fedorov 1966, ashton 1969, frankie 1975, chan 1980). this maybe due to shortage in the tropics of environmental effect like day/night length or vernalizing temperature which serves as an incentive to flower induction. thus, in the tropical rain forest where climatic control is absent, genetic make-up or genotype should be responsible for the initiation of flowering in each tree. here we can recollect that trees of the same family are likely to have many of the same genes in common. so, it is to be expected that they will show synchronized flowering so far as they belong to the same family, flowering time of which, however, may be different from trees of the other families. the two families b and c would have been sexually isolated from each other though their trees grow mixedly distributed within the same area of the mating distance. thus different families in altingia excelsa would be subjected to the effect of genetic drift. (additional investigations are needed). as described previously, two opposite views have been proposed for reproductive system of trees in the tropical rain forest. one is that of fedorov (1966) who thinks that in the tropical rain forest, tree species reproduces predominantly by inbreeding, probably by self-fertilization due to scarcity of conspecific individuals combined with asynchronous flowering among them, leading to genetic drift and finally to speciation. another view is that of asthon (1969) who looks that the mode of speciation in the tropical rain forest is essentially not different from other terrestrial plant ecosystems, the speciation being expected to occur as a result of outcrossing and gene recombination combined with natural selection. data obtained from the present study on altingia excelsa seem to favor fedorov although we take a view of intra-family propagation instead of fedorov's self-fertilization hypothesis. in agathis borneensis, inbreeding occurs to some extent but there is no sign of clustering of a group of trees or formation of family clumps as detected in altingia excelsa. it is assumed that related trees are separated widely in the forest without forming a small group per family. effect of the inbreeding observed may be attributed to self-fertilization of individual trees. conclusion from the present study it has been found that a population of altingia excelsa involved several breeding groups, each of which consisted of related trees of a single family. families were very variable biochemically as well as 19 biotropia vol. 1 no. 1, july-december 1987 morphologically, suggesting that they were genetically differentiated subgroups within a population. the mating distance, that is, the distance within which trees can mate at random has been estimated to be 16 to 18 meters in altingia excelsa. if trees of different families were mixedly growing within the mating distance, they still maintained their peculiarities suggesting the presence of inter-familial sexual isolation due perhaps to genetic difference in flowering time among families. in agathis borneensis, formation of no family clump was noticed, though there was an indication of occurrence of inbreeding to some extent. inbreeding in this species may probably be due to self-fertilization in individual trees. literature cited appanah, s. 1980. pollination in malaysian primary forests. tropical ecology and development. p. 177-182. ashton, p.s. 1969. speciation among tropical forest trees: some deductions in the light of recentevidence. biol. j. linn. soc. 1. p. 155-196. brewer, g.j. 1970. "introduction to isozyme techniques". academic press (new york). 186 p. chan, h.t. 1980. reproduction biology of some malaysian dipterocarps. tropical ecology and development, p. 169-175. coles, j.f. &d.p. fowler. 1976. inbreeding in neighboring trees in two white spruce population. silv. gen. 21 (1): 29-34. dogra, p.d. 1981. variability in biology of flowering in blue pine provenances of northwestern himalayas in relation to reproductive barriers and gene flow. proc. symp. on flowering physiology. xvii iufro world cong. p. 8-15. endo, t. 1968. zymography (in japanese). sci. rep. toyo kagaku sangyo 26: 1-5. endo, t. 1978. a new method for peroxidase isozyme stain. ann. rep. nat. inst. gen. 28: 41. endo, t. 1981. an extraction system for leaf zymographic analysis in woody plants. ibid. 31: 41. fedorov, an.a. 1966. the structure of the tropical rain forest and speciation in the humid tropics. j. ecol. 54: 1-11. frankie, g.w. 1975. tropical forest phenology and pollinator plant coevolution. "coevolution of animals and plants", ed. by l.e. gilbert and p.h. raven. univ. texas press, p. 192-209. gan, y.y., f.w. robertson, p.s. ashton, e. soepadmo & d.w. lee. 1977. genetic variation in wild population of rain forest trees. nature 269 (5626): 323-325. curies, r.p. & f.t. ledig. 1982. genetic diversity and population structure in pitch pine (pinus rigida) mill.). evolution 36 (2): 387-402. hashizume, h. & s. sugimoto. 1980. a study of the breeding system in natural forests of buna (fagus crenata blume) using the peroxidase isozyme technique. hardwood research 1: 59-71. hashizume, h. & s. sugimoto. 1982. genetic and thremmatologic studies on the natural forest of sugi (cryptomeria japonica d. don) in the chugoku mountain district. (i) the reproductive system of naturally grown sugi. bull. fac. agr. tottori univ. 34: 73-81. hashizume, h., y. watanabe & m. ookita. 1983. genetic studies on natural forests of hinoki (chamaecyparis obtusa endl.) in the chugoku mountain district. (i) studies of genetic 20 breeding structure of tree species in the tropical rain forest — sakai et al. variability and reproductive system in naturally grown hinoki using the peroxidase isozyme technique. ibid. 35: 24-33. hiebert, r.d. & j.l. hamrick. 1983. patterns and levels of genetic variation in great basin bristle cone pine, pinus longaeva. evolution 37 (2): 302-310. hubbell, s.p. 1979. tree dispersion, abundance, and diversity in a tropical dry forest. science 203 (4387): 299-1307. janzen, d.h. 1970. herbivores and the number of tree species in tropical forests. amer. nat. 104 (940): 501-528. janzen, d.h. 1971. escape of juvenile dioclea megacarpa (leguminosae) vines from predators in a deciduous tropical forest. amer. nat. 105 (942): 97-112. langner, w. 1933. eine mendelspatung bei aurea-formen von picea abies (l.) karst. als mittel zur klarung der befruchtungs-verhaltnisse im walde. z. f. forstgenetic 2 (3): 49-51. levin, d.a. 1977. the organization of genetic variability in phlox drummondii. evolution 31 (3): 477-494. linhart, y.b., j.b. mitton, l.b. sturgeon &m.l. davis. 1981. genetic variation in space and time in a population of ponderosa pine. heredity 46 (3): 407-426. matsuura, t. 1983. family analysis in natural forest of abies sachalinensis. "ecological-genetic studies in natural forests and their practical applications" hoppo ringyo-kai (sapporo). p. 265-281. price, m.v. & n.m. waser. 1979. pollen dispersal and optimal outcrossing in delphinium nelsonii. nature 277: 94-297. rehfeldt, g.e. 1979. ecotypic differentiation in population of pinus monticola in north idaho – myth or reality? amer. nat. 114 (5): 627-636. roe, a.l. 1967. seed dispersal in a bumper spruce seed year. u.s. for. serv. res. paper 1nt-39. p. 1-10. sakai, k.i. & y. miyazaki. 1972. genetic studies in natural populations of forest trees. ii. family analysis. silv. gen. 21 (3): 149-154. sarvas, r. 1967. pollen dispersal within and between subpopulations: role of isolation and migration in microevolution of forest tree species. proc. xiv cong. 1ufro, vol. 3: 332-345. schaal, b.a. 1975. population structure and local differentiation in liatris cylindracea. amer. nat. 109(969): 511-528. schaal, b.a. & d.a. levin. 1978. morphological differentiation and neighborhood size in liatris cylindracea. amer. j. bot. 65 (9): 923-938. schaal, b.a. & w.g. smith. 1980. the apportionment of genetic variation within and among populations of esmodium nudiflorum. evolution 34 (2): 214-221. schwartz, o.a. & k.b. armitage. 1983. problems in the use of genetic similarity to show relatedness! evolution 37 (2): 417-420. stam, p. 1983. the evolution of reproductive isolation in closely adjacent plant populations through differential flowering time. heredity 50 (2): 105-118. stern, k. & l. roche. 1974. "genetics of forest ecosystems". springer-verlag (berlin). 330 p. watkinson, a.r. 1978. the demography of a sand dune annual: vulpiafasciculata. iii. the dispersal of seeds. j. ecol. 66 (2): 483-498. 21 biotropia vol. 1 no. 1, july-december 1987 webb, l.j., j.g. tracy & k.p. haydock. 1968. a factor toxic to seedlings of the same species associated with living root of the nongregarious subtropical rain forest tree grevillea robusta. j. appl. ecol. 5 (1): 13-25. wright, s. 1946. isolation by distance under diverse systems of mating. genetics 31: 39-59. yeh, f.cn.-h. & d. o'malley. 1980. enzyme variations in natural populations of douglas f ir , pseudotsuga menziesii (mirb.) franco, from british columbia. i. genetic variation patterns in coastal populations. silv. gen. 29 (3-4): 83-92. 22 breeding structure of tree species in the tropical rain forest — sakai et al. appendix 1 23 b1otropia vol. 1 no. 1, july-december 1987 appendix 2. 24 breeding structure of tree species in the tropical rain forest — sakai et al. appendix 3 calculations of the theoretical expectations of disagreement counts according to the random assortment of isoperoxidase bands in individual trees let the frequency of occurrence of i-th band in the population be designated as xj. the probability that the disagreement count will be either zero or one for the band is given in the following: probabiliy that the ppulation will give zero or one disagreement count for the i-th isoperoxidase band the probability of getting zero disagreement count for the i-th band is given as (xj + (1 — xj) 2 ), and that of getting one is given as 2xi(l — xi), since plus meeting with plus or minus meeting with minus ( + /+ or — / — ) gives zero, while minus meeting with plus or the inverse ( + / — or — / + ) gives disagreement count of value one. let the total number of isoperoxidase bands which are assumed to be genetically independent with each other be p, then the probability of getting zero disagreement count in the population will be (x1 2 + (1— x 1 2 ) x . . . . . . . . . . x (x 2 i + (1— xi) 2 ) x . . . . . . . . . . x (x 2 p + (1-x 2 p) = p π i = 1 (x2 i + (1-x j )2) the probability that the disagreement count gets the value of one in the population with p bands will be given by: p p σ π i = 1 [2xi (-xj) j = 1 (x2j + (1-xj)2)] the probability that the disagreement count may take the value larger than 2 can be calculated in the similar way. 25 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf 10.pdf 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf 6. ristianti (notes) rev.cdr biotropia vol. 18 no. 2, 2011: 123 128 notes on the distribution of invasive freshwater snail (lamarck, 1822) and ( d'orbigny, 1835) in indonesia pomacea canaliculata p. insularum ristiyanti m. marwoto & nur r. isnaningsih received 24 november 2011/accepted 10 december 2011 the freshwater snails and have been reported as important invasive species causing damage to crops and predominantly wetland rice in asia. these snails are known as “golden apple snail” (gas), an introduced species from argentina. or known as “keong mas, keong murbei” was introduced in indonesia around 1983, and after more than 20 years, it now can be found very abundant at various habitats such as marshes, ponds, irrigations, lakes and rice fields in almost all places in indonesia. based on the collections of these snails deposited in the mzb (museum zoologicum bogoriense, research center for biology) and secondary data (references), the distribution of these two snails was studied. is widely distributed, while is only found at lake semayang and lake balikpapan in kalimantan. the distribution map is presented and will be useful as a basic information to manage these invasive snails. distribution, snail, invasive, indonesia research center for biology, gedung widyasatwaloka, jalan raya jakarta bogor km 46, cibinong, bogor 16911 pomacea canaliculata p. insularum pomacea canaliculata pomacea canaliculata p. insularum pomacea canaliculata, p. insularum, abstract introduction key words: the freshwater snails and have been reported as important invasive species causing damage to crops and predominantly wetland rice in asia. reports on the serious damage to rice in malaysia, philippines, japan, vietnam and indonesia have been published (hyunh 2006; cuong 2006; adalla & magsino 2006; wada 2006; suharto . 2006; yahaya . 2006). these snails are known as “golden apple snail” (gas), an introduced species from argentina. or named as “keong mas, keong murbei” was introduced in indonesia around 1983, and after more than 20 years, the snail has spread and became pomacea canaliculata p. insularum et al et al pomacea canaliculata * corresponding author : rist001@lipi.go.id 123 very abundant in various habitats such as marshes, ponds, irrigations, lakes and rice fields in almost all places in indonesia. in 2004, the first author visited lake semayang, lake loa kang and lake balikpapan in east kalimantan and collected shells of and unexpectedly shells that have similar characters with (d'orbigny 1835) were found which have not been recorded before. this finding added the number of invasive snails in indonesia to two species. hayes (2008) did not list from indonesia but he reported that this snail is present in singapore and malaysia. rawling (2007) already stated that this snail is distributed widely in southeast asia and assumed that it was introduced to indonesia from malaysia. the occurrence of in indonesia also have been reported by suharto 2006; isnaningsih & marwoto (2011), but based on our examination, the shells are similar to as mentioned by cowie 2006 who also noted that has not been detected in asia. detailed study on the occurrence of in indonesia is still needed. damage to ricefields in indonesia caused by have been reported in medias such as newsletters, radios, and televisions (see appendix). presently, even the data on their distribution in indonesia is largely a speculation. the public awareness about the danger of the gas (golden apple snails) in general is very limited. identifying the freshwater snails found in rice fields or irrigations is also difficult for the farmers and the local people. usually they only recognised the occurrence of based on the color of the egg capsules which are pink or bright reddish. preliminary study on the distribution of and is needed to evaluate the distribution of these two species. the aim of this study is to present a basic data on the distribution of and in indonesia. the data would be necessary for future studies and also to manage the invasive snail becoming pest in rice and threatens some native snails. the author used about 500 specimens deposited in the mzb (museum zoologicum bogoriense, research center for biology) and secondary data from references dated from 2005 up to 2011. all information of the localities where the snails have been collected from 1990 to 2011 were recorded and marked on the distribution map. yellow dots represent the localities based on references and red data for the localities based on mzb's specimens of , while the occurrence of was indicated by star. of mzb collection came from 90 localities in indonesia, distributed from the northern sumatra (aceh, bukit kese, bengkulu sibaganding,manggung pariaman, danau kerinci, jambi, danau ranau, lampung, sigarung-garung, tanggamus, lampung, krakatau), java (bogor, tasikmalaya, pomacea, p. insularum et al. p. insularum p. paludosa et al. p. canaliculata et al. p. paludosa p. paludosa p. canaliculata p. canaliculata p. canaliculata p. insularum p. canaliculata p. insularum p. canaliculata p. insularum pomacea canaliculata materials and methods results and discussion 124 biotropia vol. 18 no. 2, 2011 figure 1: map of the distribution of and in indonesiap. canaliculata p. insularum the distribution map (based on mzb collections & secondary datas) shows that is more widely distributed compared to that of found only in lake loa kang and semayang in balikpapan, kalimantan. according to reports from newspapers and magazines the invasive snail has damaged about 10 ha of ricefields in some places such as in java, sumatra, sulawesi, kalimantan, sumbawa, lombok or totally about more than 100 ha mainly in aceh north sumatra and some areas in java. it seems that possesses less physiological adaptability compared to and need a specific habitat such as big lakes with muddy substrates and water hyacinth or water plantations like in lake loa kang, semayang and balikpapan. cazzaniga (2006:39) explained about the pattern of distribution and habitat of in argentina basically tropical and subtropical and fail to thrive in salty, very alkaline, poorly vegetated environments, with high risk of desiccation. furthermore, he concluded that the fast expansion of this snail in southeast asia might be caused by some biological reasons but the main factor is human actions, since the snail has potential economic value. can be usually found in irrigated rice fields areas, or marshes and ponds. the populations will increase in rainy season and the juveniles or young snails will spread widely. the expansions of in indonesia are usually caused by human activities, especially in jawa, sumatra and sulawesi where people have cultured the snail to be consumed (as edible snail) without realising the risk of the invasive snail (isnaningsih & marwoto 2011). on the contrary, the local people in balikpapan, east kalimantan, generally prefer to consume freshwater fishes rather than snails. p. canaliculata p. insularum p. canaliculata p. insularum p. canaliculata p. canaliculata pomacea canaliculata p. canaliculata cianjur, sukabumi, krawang, depok, bekasi, rawapening, tuban, jogya), bali (lake tamblingan, lake bratan), sulawesi (maros, manrepo, buton, palu, bone, pangkep), kalimantan (tau lumbis, lake semayang, lake balikpapan, malinau), and papua (wamena, biak). while was only recorded from three locations i.e lake semayang, lake loa kang and lake balikpapan in east kalimantan. the distribution of and is presented on figure 1. p. insularum p. canaliculata p. insularum notes on the distribution of invasive freshwater snail ..... ristiyanti m. marwoto .et al 125 conchological et al. p. canaliculata p. insularum p. canaliculata p. insularum et al. p. canaliculata et al. pomacea canaliculata . the shell morphology description is based on general description to compare the two species, for detailed morphology and anatomy study see cowie 2006. the invasive snail and have similar shells. the shell differ mainly in having relatively higher spire and less broadly in compared to that of (rawling 2007), and the shell size of is relatively smaller (cowie 2006). the shell characters of both species are described as follows :. (lamarck, 1822) shell (fig.2) globose, somewhat thin or transparent with smooth surface. dextral coiling. yellowish or dark brown, around the suture shell's color become pale. sometimes there are dark spiral bands that become brighter at the body whorl. spire high and pointed. whorls 5.25 5.50, and rapidly increasing in size, the body whorl more globose. umbilicus perforate. suture curved to form deep channel. apertural shape elongate cylindrical and the columellar lip not thickened. measurements: height of shell 12.58 69.66 mm; width of shell 4.94 64.90 mm; height of body whorl 11.20-61.20 mm; length of aperture 8.58 49.7 mm; width of aperture 6.50 34.31 mm fig. 2. ariation of from different localities.pomacea canaliculatashell v 126 biotropia vol. 18 no. 2, 2011 pomacea insularum (d'orbiny, 1839) m . shell (figs.3) globose, somewhat thick compared to . dextral coiling. brown and become darker near the umbilicus.spire low and commonly erroded. whorls 5, and rapidly increasing in size, the body whorl more globose. umbilicus perforate. suture with shallow channel. apertural shape elongate cylindrical and the columellar lip not thickened. easurements: height of shell 66.16-85.00 mm; width of shell 63.32-79.6 mm; height of body whorl 64.60 -77.55 mm; length of aperture 46.05-59.25 mm; width of aperture 33.24-44.45 mm p. canaliculata figure 3. shell variation of from lake loa kang. adalla cb, magsino e. 2006. understanding the golden apple snail ( ): biology and early initiatives to control the pest in the philippines. : global advances in ecology and management of golden apple snails. rc joshi & ls sebastian (eds), 199-213. philrice. philippines. cazzaniga nj. 2006. harmless and useless in its natural realm (argentina). global advances in ecology and management of golden apple snails. rc joshi and ls sebastian (eds), 3760. philrice. philippines. cuong dn. 2006. the golden apple snail in vietnam. global advances in ecology and management of golden apple snails. rc joshi and ls sebastian (eds), 243-254. cowie rh, hayes ka, thiengo sc. 2006. what are apple snails? confused taxonomy and some preliminary resolution. : global advances in ecology and management of golden apple snails. rc joshi and ls sebastian (eds), 3-23. philrice. philippines. hayes ka, joshi rc, thiengo sc, cowie h. 2008. out of south america: multiple origins of non-native apple snails in asia diversity and distributions, 14 (4), 701-712. pomacea insularum pomacea canaliculata in pomacea canaliculata : in: in in . acknowledgements we would like to express our thanks to the anonymous reviewers who gave the comments and corrections on the first draft of this manuscript. this work was supported by dipa of research center for biology lipi (2009). references 127 notes on the distribution of invasive freshwater snail ..... ristiyanti m. marwoto .et al huynh nk. 2006. golden apple snails in vietnam. : global advances in ecology and management of golden apple snails. rc joshi and ls sebastian 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mn, sivapragasam a. 2006. golden apple snails in malaysia. global advances in ecology and management of golden apple snails. rc joshi and ls sebastian (eds), 215230. philrice. philippines. in pomacea in pomacea canaliculata in: in: 128 biotropia vol. 18 no. 2, 2011 5. shahabuddin (effectiveness... biotropia vol. 21 no. 1, 20114: 48 58 effectiveness of dung beetles as bioindicators of environmental changes in land-use gradient in sulawesi, indonesia shahabuddin , uswah hasanah ,and elijonnahdi received 13 june 2013/accepted 09 november 2013 bioindicators have been widely accepted as useful tools for monitoring and detecting changes in the environment or habitat condition. by using bioindicators, it is possible to assess the impact of human activities on the biota, instead of examining the entire biota. in this paper we analyzed diversity of dung beetles (coleoptera: scarabaeidae) across land use gradient in central sulawesi and tested the suitability of dung beetles as bioindicators for environmental changes. ninety baited pitfall traps were placed and several habitat parameters were measured at five land-use types ranging from natural forest to cacao agroforestry systems to open areas in 2009 and 2012. the effectiveness of dung beetles as bioindicators of environmental changes was evaluated by the method, a method combining the specificity and fidelity of certain species with particular types of habitat or environmental conditions. surprisingly, the results showed that the diversity of dung beetles in two types of cacao plantations were similar to the forest sites and were significantly higher than the open cultivated area. of the 16 dung beetles species analyzed only four species could be suggested as indicator (characteristic) species while the majority of collected species were categorized as detector species. two of them ( and ) were associated with natural forest and cacao agroforestry system, thus were suggested as the indicator of shaded and cooler habitats whereas and can be suggested as indicator of unshaded and warmer habitats (bare land area). bioindicators, diversity, scarabaeidae, habitat preferences, . 1* 1 2 1 2 department of agrotechnology faculty of agriculture university of tadulako, palu 94118, central sulawesi, indonesia department of biology faculty of mathematics and science university of tadulako, palu 94118, central sulawesi, indonesia indval copris saundersi onthophagus forsteni o. limbatus o. trituber indval abstract keywords: * corresponding author : shahabuddin_slh@yahoo.com doi: 10.11598/btb.2014.21.1.5 48 introduction the conversion of natural habitat to other land uses and its consequences on environmental changes has been acknowledged as the main driver of biodiversity loss at the southeast asia and global scales (sodhi . 2004) and by 2100 the impact of land use changes on biodiversity is likely to be more significant than that of climate change, nitrogen deposition, species introductions and changing atmospheric concentrations (sala 2000; young 2009). therefore, detecting environmental changes due to land-use change is needed in order to avoid the continuous loss of biodiversity. bioindicators have been proven to be useful tools for monitoring and detecting changes in the environment. species have different ecological requirements and their reactions to environmental variation are different from one another. therefore, some species are better indicators than others (dufrêne & legendre 1997). some species are generalists occurring in a wide range of habitats (ubiquitous), while others are more specialized, requiring certain habitat characteristics ( ). a bioindicator can be defined as a species or a species group that reflects the abiotic or biotic state of the environment ( ), represents the impact of environmental change on a habitat, community or ecosystems ( ), or indicates the diversity of other species ( ) (mcgeoch 1998). one of the reasons for using bioindicators is their cost-effectiveness. by using bioindicators it is possible to assess the impact of human activities on the biota, instead of examining the entire biota. especially useful are species that provide early warning of change (spellerberg 1993) environmental changes can cause different kinds of effects in the indicator, including physiological changes or changes in species number or abundance. the response of the species can be seen within the organism (e.g. heavy-metal concentrations), at the species level (species number and abundance) or at the community level (relations between species, e.g. pestpredator). increase or decrease or abundance of species number might be directly caused by change in abiotic and/or biotic factors or indirectly by change of species assemblage of other species (davis . 2001; rainio & niemela 2003; hambler . 2011; gerlach . 2013). a good bioindicator must fulfill several criteria. it has to be well-known taxonomically and ecologically, be distributed over a broad geographic area, have specialization to certain habitat requirements, provide early warning of change, be easy and cost-effective to survey, be relatively independent of sample size. its response should reflect the response of other species, one should be able to differentiate between natural cycles or trends and those induced by anthropogenic stress, and it should be of potential economic importance (e.g. noss 1990; pearson & cassola 1992). however, it is difficult to find species or species groups which would have all of these criteria (noss 1990; pearson & cassola 1992). requirements needed depend on the goal of the survey and the sensitivity to the anthropogenic disturbance is the most important criteria for monitoring environmental changes (kremen . 1993). et al et al stenotopic environmental indicator ecological indicator biodiversity indicator . et al et al et al et al . 49 effectiveness of dung beetles as bioindicators of environmental changes in land shahabuddin– et al. in this study dung beetles (coleoptera: scarabaeidae) was selected as a bioindicator group because it has been widely known as one of the best bioindicator groups (e.g. halffter & favila 1993; mcgeoch 2002). dung beetles have been proven to be very suitable to assess effects of disturbances on tropical ecosystems (nichols . 2007; shahabuddin 2010) and human habitat modification (shahabuddin 2010; harvey 2006) due to their abundance, highly varied with respect to species traits, and rapid responses to environmental change (slade . 2011; shahabuddin . 2005 & 2010; spector 2006; slade . 2011). another advantage is their relatively complete species inventories and their data on the abundance of individual species can be achieved rapidly with standardized methods (larsen & forsyth, 2005). recently, dung beetles have been identified as one of the most cost-effective group for biodiversity survey in tropical forests (kessler . 2011) and contributors in improving the level of soil carbon stocks (kessler . 2012). one method used to quantify the 'bioindicator value' of a range of taxa is the indicator value ( ) method developed by dufrene and legendre (1997). this method combines measurements of the degree of specificity of a species to an ecological state, for example a habitat type, and its fidelity within that state (dufrene & legendre 1997). species with a high specificity and high fidelity within a habitat will have a high indicator value. high fidelity (frequency of occurrence) of a species across sample sites is generally associated with large abundance of individuals. both characteristics facilitate sampling and monitoring, which are important requirements for a useful bioindicator (kremen . 1994; mcgeoch . 2002). the indicator value method is important to conservation biology because it is conceptually straightforward and allows researchers to identify bioindicators for any combination of habitat types or areas of interest, e.g. existing conservation areas, or groups of sites based on the outcome of a classification procedure (mcgeoch & chown 1998). accordingly, method has become the most robust and popular method used to measure indicator species analysis (mc.geoch . 2002; aydin & kazak 2010; negro . 2011). in central sulawesi, forest habitats especially in the interior of lore lindu national park (llnp) are still relatively undisturbed while the margins of the park are characterized by a mosaic land-use type such as near-primary forests, secondary forests, forest gardens and plantations of cacao, maize and paddy rice fields (gerold . 2004). this study aimed to evaluate the effectiveness of dung beetles as bioindicator of environmental changes across land-use gradient in the margins of llnp. this study was carried out at the northern margin of the llnp in central sulawesi, indonesia. the park, a local biodiversity hot spot covers an area of 229,000 ha and is located southeast of palu, the capital of central sulawesi province. all study sites were selected at the surrounding of the palolo valley in the vicinity of the villages of bobo et al. et al et al. et al. et al et al et al et al et al indval et al et al indval et al et al et al materials and methods study area 50 biotropia vol. 21 no. 1, 2014 (01 07'0.46" s 119 59'702" e) and were situated at an altitude between 790 and 985 m asl. dung beetle communities were studied in five land-use types: natural forest (nf); secondary forest (sf); cacao agroforestry systems (cacao cultivated under natural shade tree at the forest margin (ac); cacao plantation under monospecific shade tree dominated by (cp); and open area (oa), cultivated either by or . three replications sites for each land-use type were selected with distance at least 50 m from each other dung beetles were sampled in 2500 m plots at 15 sites all using baited pitfall traps as described in shahabuddin (2010). six traps were set up at the centre of each plot and placed with an interval of 10 m. the traps were baited with ca. 30 g of fresh cattle ( ) dung and exposed six times from april to july in 2009 and march to june in 2012. cattle dung has been widely used as bait for dung beetles, aside from human faeces (e.g. erroizi 2004; andresen 2005; mendoza 2005). our previous study also showed that at the same weight of bait (ca. 30 g) cattle dung attracts the dung beetles with similar species composition found in the dung of anoa ( ), an endemic herbivore of sulawesi (shahabuddin . 2010). the trapped specimens were removed after two days and preserved in scheerpelz solution (krell 2007). later on, the samples were identified in the laboratory using available identification keys (e.g. balthasar 1963) and by comparing to the reference collection of the center for biodiversity research tadulako university. species which could not be identified, were sorted to morphospecies. several habitat parameters (i.e. vegetation structure and microclimate) affecting the dung beetles diversity (see davis . 2001; shahabuddin 2010) were measured to characterize the land-use types including air temperature, relative humidity, canopy cover, and herb layer coverage. the relative humidity at the start and end of the exposure period were measured using a digital thermo-hygrometer (corona model: gl 99) 1 m above ground while the canopy cover was visually estimated at four locations per site for a corridor of ca. 10 m inside the plot. the herb coverage was estimated at four plots of 2x2 m randomly placed at ca. 5 m inside the plot. based on the environmental variables measured all land-use types were then grouped using a two-dimensional scaling (clarke 1993; statsoft 2001). the three most widely used measures of species diversity were species richness, shannon-wiener index and simpson index (si = ∑ pi ) (lande 1996). species richness of dung beetles was estimated using the second-order jackknife extrapolation method (colwell, 2004), one of the best species richness predictor with respect to accuracy (e.g. brose . 2003). as units for estimating the total species richness of land-use type, samples from all traps and replicates were pooled for individual sample o o 2 r 2 2 gliricidia sepium zea mays morus alba et al. bos taurus et al. et al. bubalus deppresicornis et al et al et al specimens collection environmental variables measured data analysis . 51 effectiveness of dung beetles as bioindicators of environmental changes in land shahabuddin– et al. times ( = 6) due to the close proximity between each trap and site. effects of habitat type on diversity were tested using one-way anova. abundance data were transformed by log (n+1) before analysis (zar 1999). statsoft 6.0 software (2001) was used to perform all statistical analyses. all diversities measured were computed with estimates version 7.00 program (colwell 2004) by randomizing the ranking of samples 50 times. only species sampled in both sample periods (2009 and 2012) were analyzed. the effectiveness of each dung beetles species as bioindicators were identified for each habitat type using the indicator value ( ) method (dufrene & legendre 1997).this method combines measures of specificity and fidelity and provides an for each species, as a percentage (dufrene & legendre 1997). specificity measure: = / where is the mean number of species across sites of group , and is the sum of the mean numbers of individuals of species over all groups. fidelity measure: = / where is the number of sites in cluster (habitat) j where species is present, and is the total number of sites in that cluster. the percentage indicator value for species in cluster (habitat) is then: = × × 100. the indicator values are the highest (100) when all individuals of a species are found in a single habitat (high specificity) and when the species occurs in all samples of that habitat (high fidelity). species with between 50% to less than 70% is categorized as the detector or generalist species while those species with significant of greater than 70% were regarded as characteristic indicator species for the particular habitat type (mc.geoch . 2002). a total of 1996 dung beetles specimens were collected during the study period. they belongs to four genera (dominated by ) and 28 species (for complete species list see shahabuddin 2013). however, only 16 species were recorded in both sample periods (2009 and 2012). the diversity of dung beetles changed from natural forest, to agroforestry cacao and to open area. interestingly, the diversity of dung beetles in both forest types and the two types of cacao plantations tend to be similar but significantly higher than that in open cultivated area (table 1). hence, this study showed that secondary forest and agroforestry system may support a high portion of tropical dung beetles species than in the bare land and thereby in line with the findings by nichols . (2007) and our previous study (shahabuddin . 2010). the fact that agroforestry system has high potency for conserving high biodiversity supported by previous study (e.g. mcnely & scroth 2006; schulze . 2010). however, the results may also be related with the spatial distribution of our study sites. the agroforestry cacao sites were closer to the natural and secondary forest than to the open area and has a high opportunity to be colonized by dung beetles coming from the forest sites. therefore, the high diversity of dung beetles at the cacao agroforestry system is also related to their close proximity to the forest sites. it has n indval indicator value a nindividuals nindividuals nindividuals i j nindividuals i bij nsites nsites nsites i nsites i j indval a b indval indval et al onthophagus et al et al et al ij ij i ij i ij j ij j ij ijij results and discussions diversity of dung beetles 52 biotropia vol. 21 no. 1, 2014 been reported that neighboring forest or isolation from forest may determine insect communities in tropical land-use systems (tscharntke . 2005; klein . 2006). agroforestry systems can be part of the habitat for many forest species using it for foraging, but they may also harbour largely independent populations. nonetheless, this study suggests that the preservation of environmental heterogeneity should be encouraged for conserving dung beetles in the llnp, central sulawesi. this is important because high diversity of dung beetles in tropical land-use will enhance its ecosystem function and this ecological services will be diminished by increasing human dominated land-use (shahabuddin 2011; slade . 2011; kudavidanage 2012). in more natural and heterogeneous habitats, such as natural forest and agroforestry system, dung removal, biological control and seed dispersal activities of dung beetles were higher than in homogenous or disturbed habitats (slade . 2007; nichols . 2008; shahabuddin 2011; slade . 2011). because the dung beetles diversity at the forest sites (natural forest and secondary forest) and the cacao plantation sites (agroforestry cacao and cacao plantation) were similar to each other but significantly higher than in the open area (table 1), the indicator species analysis land-use type is only classified into three groups, : forest, agroforestry cacao and open area. the high similarity of environmental parameter measured among these three groups of land-use types was supported by anova showing that temperature and herb coverage decreased from the natural and secondary forest to the open area while canopy and humidity showed a reverse pattern. however, a highly significant difference of environmental parameter measured was only recorded in the open area (fig. 1). this land-use type grouping was also supported by the ordination technique using multidimensional scaling (fig. 2). based on analysis of 16 dung beetles species collected in two sampling years (2009 and 2012), this study recorded four species having less than 70% and therefore, can be used as indicator (characteristic) species that are: at forest sites, at cacao plantation, as well as and at open area (table 2). and are suggested to be used as indicators of cooler and shaded habitats such as forest sites and cacao agroforestry et al et al et al et al. et al et al et al i.e indval indval copris saundersi onthophagus forsteni o. trituber o. limbatus copris saundersi onthophagus forsteni indicator species table 1. diversity of dung beetles at five habitat type. nf = natural forest; sf = secondary forest; ac = cacao agroforestry system ; cp = cacao plantation; oa = open area land-use type estimated species richness (jack-2) (f4,10=6.27, p <0.05) number of species recorded f4,10 =3·60, p < 0·005 h' simpsom nf 14.3a 9ab 1.6 4.2 sf 14.4a 10.7ab 1.7 4.2 ac 15a 11a 1.7 4.3 cp 13.6a 10ab 1.7 4.5 oa 8.7b 6.3b 1.3 3.1 53 effectiveness of dung beetles as bioindicators of environmental changes in land shahabuddin– et al. system, while and most likely indicate warmer and unshaded habitats (e.g. open cultivated area). these characteristic species ( of > 70%) are unlikely to move from their requisite to other habitat types, even under changing conditions within this habitat. accordingly, populations of these species need only to be monitored within the specific habitat. beside indicator species, this study has recorded several generalist or moderate species with between 50% to less than 70%. these species were therefore, not characteristic species, as they do not have high of more than 70% for any particular habitat. species meeting these criteria are unlikely to respond very rapidly to changing habitat conditions. they can invade either close canopy or moist habitat e.g. natural forest, cacao agroforestry or cacao plantation but also open or warmer environment such us open area. furthermore, these species are less likely to become more vulnerable than indicator species, because a variety of habitats or ecological states, rather than only a single one, provide suitable resources for them and accordingly this group of species will be useful for longer-term monitoring. o. trituber o. limbatus indval indval indval figure 1. the effects of land use change on a) temperature (anova: f(4, 10)=25.15, p<0.01), b) relative humidity (anova:f(4,10)=62.88, p<0.01), c) canopy cover (anova:f(4,10)=139.81, p<0.01), and d) herb layer coverage (anova: f(4, 14)=52.15,p<0.01). all variables were averaged per land-use type. nf= natural forest; sf= secondary forest; ac= agroforestry cacao; cp= cacao plantation; oa= open area. significant differences between habitat types were indicated by different letters over the standard error (based on tukey's hsd post-hoc test) 54 biotropia vol. 21 no. 1, 2014 nf1 nf2 nf3 sf1sf2sf3 ac1 ac2 ac3 cp1cp2cp3 oa1oa2oa3 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 dimension 2 -1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 d im e n s io n 1 stress=0.007 open area forest sites agroforestry cacao figure 2. multidimensional scaling (mds) placing the five land-use type studied in three groups based on several habitat parameters measured. nf= natural forest; sf= secondary forest;, ac= agroforestry cacao; oa= open area table 2. indicator values ( percentage) for dung beetle species recorded in three group of habitats. species with > 70 % is categorized as a characteristic species (c) while species with 50 ≤ 70 % is a detector species (d) of certain habitat type indval indval indval species* forest sites agroforestry cacao open area species category 2009 2012 2009 2012 2009 2012 copris macacus 67.4 13.3 22.6 20.0 0.0 0.0 copris punctulatus 12.1 22.2 30.3 44.4 24.2 0.0 copris saundersi 95.8 91.7 0.0 0.0 0.0 0.0 c onthophagus cf.wallacei 22.4 41.5 10.5 41.5 67.0 1.5 onthophagus forsteni 0.0 3.7 33.3 74.1 0.0 0.0 c onthophagus fulvus 0.0 16.7 53.6 61.8 7.1 21.5 d onthophagus rectecornutus 0.0 0.0 0.0 33.3 66.7 0.0 onthophagus ribbei 60.5 32.4 8.4 37.1 0.4 1.0 d onthophagus rudis 5.3 39.5 29.3 51.2 13.3 9.3 d onthophagus scrutator 1.3 37.5 67.8 57.1 5.8 0.0 d onthophagus sp.1 63.6 62.8 19.4 30.2 6.9 4.7 d onthophagus sp.2 33.3 0.0 0.0 33.3 0.0 0.0 onthophagus sp.3 0.0 46.9 66.7 38.6 0.0 14.5 d onthophagus trituber 0.0 4.2 7.1 4.2 92.9 75.0 c phaechrous emarginatus 58.3 35.7 0.0 41.9 0.0 22.4 onthophagus limbatus 0.7 0.3 0.2 19.6 97.3 77.3 c * only species recorded in both sample years (2009 and 2012) were included in the analysesindval 55 effectiveness of dung beetles as bioindicators of environmental changes in land shahabuddin– et al. conclusions acknowledgements references the fact that diversity of dung beetles at secondary forest and agroforestry system has no significant differences with natural forest has important implications on landscape management aiming at maintaining a high biodiversity. besides natural forest sites, certain agroecosystems like cacao agroforestry also have potency to maintain a high local diversity. dung beetles 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s178-s186. zar jh. 1999. biostatistical analysis. 4th edition. prentice-hall inc. new jersey.usa et al biodiversitas biological conservation . 58 biotropia vol. 21 no. 1, 2014 biotropia 1 (1) 1987: 26-40 studies on interference among trees in a plantation of altingia excelsa kan-ichi sakai*, andreas rumbino** national institute of genetics, mishina, japan and cenderawasih university, manokwari, irian jaya, indonesia respectively shinya iyama* and lilian u. gadrinab national institute of genetics, mishima, japan and tropical forest biology program, biotrop, bogor, indonesia respectively abstract a study was made with the use of a 50-year old altingia excelsa noronha plantation with 260 standing trees for separating components of density and intraspecific competition. a component of density effect causes overall decrease in growth while that of competition results in a contrasting effect in growth between any pair of neighboring trees. to detect this density effect, it is most appropriate to use an area of ca. 100 m2 irrespective of the experimental area, e.g. circular or quadratic. competition effect cannot be detected when two individuals are apart more than two meters. an application of the density and competitive ability to tree breeding is briefly mentioned. introduction growth of trees standing in a monospecific population is under considerable influences of associate trees growing in the proximity. the first is the general effect of thickly planted trees and the second is the specific effect of competition between a given pair of neighboring trees. in some cases the so-called allelopathic effect goes on with them though in the present species no sign of its occurrence has been noticed. the density response and the inter-tree competition are very ubiquitous and consequently silviculturally very important, although the two have often been treated by plant ecologists and agronomists as phenomena of the same category. the senior writer of the present paper, however, is of the belief that the density effect is biologically different from the inter-tree competition. therefore, in the present study, the writers intend to investigate the density response and the intraspecific competition in a plantation planted more than fifty years ago with seedlings of a tropical tree species altingia excelsa noronha of the * temporarily seconded to biotrop. ** biotrop ten-month research training scholar on forest plant genetic resources, 1 september 1980—30 june 1981. 26 interference among trees in a plantation of a/tingia excelsa — sakai et al. family hamamelidaceae. the species is locally called rasamala and is one of the valuable timber species native to indonesia. it grows in natural stands in western java and is also planted for afforestation. its wood is heavy, hard and fine-grained and the color is red to blackish brown. it produces a yellow scented rasamala resin which is used in perfumery. it grows in an area of elevation of 500 to 1500 meters above sea level. the rasamala plantation is on a slow increase in the mountainous area of western java. materials and methods materials for the study have been obtained from an afforested plantation of altingia excelsa located in the mountainous region of bandung in the western part of java, indonesia. according to a brief document of the regional forestry office, saplings were initially planted at a distance of 3 x 1 meters in 1927. the first thinning was applied in 1932 (5 years after planting), while the second thinning in 1938 or 11 years after planting. detailed record, however, is not available at present. after 1939, the stand has been left without artificial thinning, though some trees died from the attack of insects or mammals such as forest pig, or due to some other reasons. data were taken in february 1981 when the number of trees still growing in the 60 x 90 meters rectangular plot was 268. measurement was taken on tree height, clear bole height and diameter at breast height (dbh) and exact location of trees. estimation of the effect of tree density on the growth of trees has been made in two ways: (1) effect of number of trees growing within a given radius on the growth of a tree standing in the center of the circle (circular plot) and (2) effect of number of trees within a quadrat or a circle on their average growth (quadrate plot or circular plot). the estimation is based on the assumption that the effect of density would be general for every tree standing in a neighborhood. for estimating the effect of tree density by measuring the central tree, various sizes of the circle from a radius of 2.5 to 7.5 meters have been taken, while the effect of tree number on their average growth has been measured in a quadrat of 10 x 10 meters or in circle of a radius of 5 to 6 meters. for an investigation of intraspecific competition, correlation of dbh between two nearest neighboring trees, on the one hand, and difference in dbh between them, on the other, have been measured for various inter-tree distances. this is on the assumption that the effect of competition on the growth of a pair of adjoining trees would be antagonistic, i.e. favoring one at the expense of another to yield negative correlation or to enlarge the difference between their dbh's. 27 biotropia vol. 1 no. 1, july-december 1987 results of the study trees of altingia excelsa in the present plantation have grown after 55 years of planting to an average height of 32.6 meters, though some trees exceeded 40 meters. the dbh has grown to 33.3 centimeters on the average, with the biggest tree with a value of more than 60 centimeters. the variations in the tree height and dbh are presented in tables 1 and 2, and graphically but with an increased number of class intervals in figures 1 and 2. figure 1. frequency histogram of tree height of an even-aged stand of altingia excelsa. the frequency histogram of the tree height is negatively skewed and leptokurtic (fig. 1), while that of dbh is positively skewed with an intuitive impression of some multimodality (fig. 2), though both of the tree height and the dbh were positively correlated with r = 0.6769** for 266 degrees of freedom. 28 i. growth of trees interference among trees in a plantation of altingia excelsa — sakai et al. figure 2. frequency histogram of diameter at breast height (dbh) of an even-aged stand of altingia excelsa. 29 biotropia vol. 1 no. 1, july-december 1987 ii. density effect at first, the density effect was measured by the dbh of a central tree being surrounded by various number of trees growing within a given area of a circular plot. the circular plots were constructed with various radii from 2.5 to 7.5 meters. if the tree density does exert some hindering effect on the growth of trees, the number of trees within a given circle should be negatively correlated with dbh of the central tree because the higher the density, the poorer is the growth of the central tree would be. the correlation coefficients between them are shown for various sizes of the circle (table 3). it is found that a smaller circle with a radius less than 4 meters is too small to include sufficient number of trees to yield apparent density effect. it looks that the most reliable size of the circle for detection of the density effect is a radius of 5 to 6.5 meters with the average of 4.5 to 7 trees growing within the plot. the distribution of dbh of a central tree, averaged for each class, against various number of trees within the radius of 5.5 meters, as an example, is shown in figure 3. figure 3. effect of number of trees coexisting within a radius of 5.5 meters on the dbh growth of a central tree. x: mean value. 30 interference mong trees in plantation of altingia excelsa sakai et al. 31 biotropia vol. 1 no. 1, july-december 1987 the second approach to the estimation of the density effect was made by measuring the average growth of all trees growing within a given circle. the size of the circular plot was confined to the radii ranging from 5 to 6.5 meters on the basis of the foregoing analysis. the correlation coefficients between the number of trees within a plot of a given radius and their average growth are shown in table 4. it shows that the most reliable size of a circle for estimating density response is with a radius of 5.5 to 6.0 meters. the 10 x 10 meter quadrat just corresponds to the area of a circle of a radius of 5.64 meters, giving an equivalent correlation of —0.351. the distribution of dbh in relation to the number of trees within a limited area with a corresponding regression line is depicted for a circle of 5.5 meter radius in figure 4 and 10 x 10 meter quadrat in figure 5. it is concluded from these studies that the response to density in the plantation of altingia excelsa can be properly detected by measuring mean dbh figure 4. effect of number of trees coexisting within a radius of 5.5 meters on their dbh growth. x: mean value. growth of trees in relation to their number growing within a radius of 5.5 to 6.0 meters or in quadrats of 10 x 10 meters, area of which being approximately between 95 and 110 m 2 or just 100 m 2 in the case of the quadrat. measurement of dbh of a single tree for detection of density effect is more or less inaccurate because of high fluctuation in measurements as found in figure 3 (compare with 32 interference among trees in a plantation of altingia excelsa — sakai et al. ** significant at the 0.01 level figure 4). granting that the coefficient of regression described in figure 4 is correct, we can mention that the response to tree density in altingia excelsa is -0.75 centimeters in dbh against an increase of one tree in the stand. figure 5. effect of number of trees coexisting in a 10 x 10 meter quadrat on their dbh growth. x: mean value. 33 biotropia vol. 1 no. 1, july-december 1987 iii. intraspecific competition as shown in figures 3 to 5, growth of dbh of trees decreases linearly as the number of trees in a limited area increases. competition between adjoining trees may interrupt the linear decrease in dbh due to the density effect. if competition occurs between two adjoining trees, the growth of the one is favored while that of another is handicapped on the basis of their inherent competitive ability. thus, a negative correlation results between them. table 5 shows that the correlation of dbh between two adjoining trees is negative if the inter-tree distance is less than 2 meters. the positive correlation found at the inter-tree distance larger than 2.01 meters indicates that inter-tree competition in altingia excelsa appears to come into play within a distance of two meters. in the right column of table 5 and also in table 6, the differences in dbh between two adjoining trees in relation to inter-tree distances are described. it is expected naturally that if competition occurs between two adjoining trees, the difference in dbh between them should be greater by the effect of competition than otherwise. it is found from table 5 that at a distance less than two meters, the difference in dbh is greater than others; while from table 6, one notes that at a distance of two meters or less, the distribution of various values of difference in dbh is not at random, but larger values than 11 are more often observed than theoretically expected. table 5. inter-tree competition in relation to inter-tree distance in altingia excelsa. inter-tree distance in meters number of pairs correlation of dbh between two adjoining trees difference in dbh between two adjoining trees in centimeters <1.75 11 — 0.4139† 14.71 ±7.96 1.75—2.00 16 —0.2276j† 12.63 ±7.62 2.01—2.25 14 + 0.6035* 9.65 ±7.93 2.26—2.50 28 + 0.1275† 12.25 ±8.08 2.51—3.00 30 + 0.4349* 9.94 ±7. 65 3.01—3.50 27 + 0.5858** 7.21 ±5.10 3.51—4.00 32 + 0.3703* 8. 35 ±7.65 4.01—4.50 30 + 0.0259† 11.12±7.38 4.51—5.00 25 + 0.6222** 6.84 ±5.27 5.01—5.50 15 + 0.1285† 7. 85 ±6.92 5.51—7.50 21 + 0.6155** 7. 56 ±5.78 † falling short of the significance level. *, ** significant at the 5 and 1% levels, respectively. 34 interference among trees in a plantation of altingia excelsa — sakai et al. figure 6 shows the distribution of mean differences in dbh between two adjoining trees at different inter-tree distances from 1.5 to 7.5 meters. the theoretical curve to fit the data can be obtained from the following formula : y = 20.574/x°6 0 figure 6. mean differences in dbh against distances between two adjoining trees. the curve represents theoretical one calculated by y = 20.574/xo-60. 35 biotropia vol. 1 no. 1, july-december 1987 where y and x are dbh difference and inter-tree distance, respectively. the curve suggests that the competitive effect is apparent only when the inter-tree distance is small or less than 2 meters. discussion as early as some 30 years ago, koyama and kira (1956) investigated the frequency distribution of plant weight in experimental populations of several kinds of plants and found that the distribution of girth of trees or weight of annual plants generally showed a positive skewness or the l-shaped distribution though in the initial stage of their growth it took the form of a normal distribution. they concluded from a mathematical study that, assuming normal distribution in the initial plant weight and also in the growth rate, the distribution of plant weight in later stages had to become positively skewed or to show the l-type distribution without additional assumption of interaction among plants. they further found that the frequency distribution of plant height, on the contrary, showed a normal curve or in some cases negative skewness. ford (1975) investigated the frequency histograms of girth of trees or weight of annual plants in even-aged plant monocultures and concluded that positive skewness and platykurtosis showing bimodality were characteristic of their distribution. he also observed an even-spatial distribution of large plants in the population. he attributed such an occurrence of positive skewness with bimodality and the even-spatial distribution to the effect of intraspecific competition. in contrast to these observations, tanaka (1983) recently published a paper describing an apparently negative, instead of positive, skewness in the distribution histogram of dbh in 42-year-old cryptomeria japonica plantation. in altingia excelsa of the present investigation, the tree height showed highly significant negative skewness together with an apparent peakedness, whereas the dbh showed a significant positive skewness as depicted in figure 2. in addition, the histogram of dbh seems to be indicative of multimodality rather than bimodality. as described above, ford is of the opinion that bimodal distribution is an evidence of intraspecific competition, but we hold another view from ford in that multimodal distribution would be more general than bimodal one under the influence of competition. this is because the competitive ability of a plant is the joint product of environmental or non-heritable effect and the inherent genetic effect of the plant. the environmental effect involves, in addition to those generally regarded factors, such non-heritable effect as an acquired advantage due to earlier germination than others. that competitive ability of a plant is a genetic character and is mostly controlled by the so-called polygenes having so low a heritability as 0.3 or less has been repeatedly demonstrated (sakai 36 interference among trees in a plantation of altingia excelsa — sakai el al. 1955, oka 1960, akihama 1967, 1968). as a matter of fact, it is considered that the competitive ability of plants may involve a complex of capacities concerning exploitation of various kinds of resources with or without interference with their associates. in other words, plants in a seed-propagated population are expected to segregate for various morphological and physiological characters which could be positively or negatively responsible for competitive ability. taking such genetic complexity for granted and assuming random combination of genotypes for competitive ability in a pair of adjoining trees, multimodal distribution of tree girths will be more acceptable than bimodality. in the present study, density response has been measured by growth of dbh of a single tree growing in the center of a circular plot being surrounded by its associates, on the one hand, and by the mean dbh of all trees coexisting inside a circle, on the other. inter-tree competition, on the contrary, has been measured by either the correlation or the difference between dbh's of two neighboring trees. as briefly described in the opening paragraph of this paper, there has been a conflict of opinion among ecologists, agronomists and geneticists on the relation between density effect and inter-plant competition. agronomists and ecologists generally appear to take a view that competition is the response of plants to density induced shortages of common resources on the assumption that plants under such circumstances inevitably scramble for the short resources. for instance, kira, ogawa and sakazaki (1953) defined the effect of plant associates growing in a limited area on the growth of a plant as the "competition-density effect" without making a distinction between the two. not a few papers dealing with problems of inter-plant competition from a similar viewpoint have been published (see for example harper 1961). recently jacquard (1973) set forth his view that the density effect is one aspect of inter-plant competition and should be treated as of the same category. in this connection we can introduce a certain evidence from our experiment conducted before. sakai, mukaide and tomita (1963) investigated inter-tree competition in 13 plantations of cryptomeria japonica d. don., three of which were being planted with vegetatively propagated clonal strains and the remaining ten with seed-propagated ones. in this investigation, correlation of stem diameter between two adjoining trees was measured with an expectation that the correlation coefficient would theoretically be minus if competition occurred between them, because it should promote growth of one tree with higher competitive ability, and hinder that of another with lower ability. in practice, the effect of competition to make the inter-tree correlation minus may often counterpoise the plus effect of environments which is due to similarity in environmental conditions in neighborhood. the correlation coefficients between two adjoining trees in the clonal stands were found to be between 0.57 and 0.31 with a mean of 0.437 ± 0.130, while in seed-propagated stands, they were between 37 biotropia vol. 1 no. 1, july-december 1987 0.33 and -0.28 with a mean of 0.024 ± 0.195. thus, it has been concluded that in a clonal population, growth of individual trees may be suppressed by the effect of density, but little sign of inter-tree competition is observed. of interest in this connection is that kira, ogawa and sakazaki (1953) did find in their soya-bean experiment that in a population of a single cultivar, "the correlation coefficients between the weight of an individual and the mean weight of the nearest six plants were for the most part positive" going against their expectations. the values in the population were as high as 0.645 or 0.735. this result increased if inter-plant competition did not occur in the population of a soya-bean cultivar. two years later, some of them (hozumi, koyama & kira 1955) conducted a similar experiment with maize and found that the correlation between neighboring plants was negative. to the present writers, this appears quite reasonable because soya-bean is a highly self-fertilized plant and a cultivar is most likely a genetically homogeneous population, while maize is an allogamous plant and accordingly a single cultivar may without doubt be genetically heterogeneous. those who emphasize inter-plant scramble for common resources as the main cause of competition reason that the most widespread and intensive competition would occur in a group of plants with high genetic similarity, because the genetic resemblance among individuals will bring about the similarity in their requirements (sammeta & levin 1970). needless to say, the genetic resemblance among groups of plants is highest in a population of vegetatively propagated plants from a single parent (a clone) and a cultivar of self-fertilizing plants continuously propagated from a single plant in each generation. results of the experiments just mentioned above show that the reasonings are not correct, suggesting that the gene-to-gene bound competition must be an actual occurrence in plant populations. fasoulas and tsaftaris (1975) take the view that "density brings about competition" which suppresses the yield of plants either evenly (in a monogenotypic population) or unevenly, and defined the latter as "allo-competition" and the former as "isocompetition" instead of saying "no-competition". we have to add here the conclusion drawn from a spacing and mix-planting experiment of 12 barley cultivars conducted by sakai and lyama (1966). in the spacing experiment it was demonstrated that all cultivars equally showed a very poor growth at a very high density, but as the density decreased, they showed various patterns of growth promotion showing cultivar-specific density response. in the mix-planting experiment with the same cultivars, the twelve barley cultivars were found to be variable for competitive ability among them. by comparing both experiments, it was found that the density response and competitive ability were only partly correlated but not reaching the 0.05 level of statistical significance. 38 interference among trees in a plantation of altingia excelsa — sakai et al. thus, it was concluded that the response to plant density and competitive ability were different characters controlled by mostly different groups of genes. we finally state that the effect of plant density upon growth of plants is the general effect of decreasing growth for all plants concerned, though the amount of decrease may be more or less different due to genotypes. the effect of intraspecific competition is the specific effect of the interaction of genotypes for competitive ability in a given pair of plants to favor growth of one plant at the expense of another. so far not much attention seems to have been paid to the silvicultural significance of genetic variability of density response and intraspecific competition in forest trees, but the problem of density response and competition will be worthy of notice in future tree breeding, particularly in cross-fertilizing tree species with a rapid growth rate as found in the tropical forest trees. conclusion from the analytic study of density response, on the one hand, and competitive ability, on the other, in a plantation of more than 50-year-old altingia excelsa noronha trees, the following results have been obtained: (1) for estimation of density response in the species, measurement of average dbh of all trees growing in circular plots with radii of 5.5 or 6.0 meters or in 10 x 10 meter quadrate plots is effectual. in the present plantation it is expected that an increase of one tree would decrease growth of dbh of neighboring trees by 0.75 centimeters. (2) it has been found that inter-tree competition which favors, two adjoining trees, one tree at the expense of another has occurred in the plantation when two trees grow within a distance of two meters. (3) the writers hold the view that density response and competitive ability of trees are different characters each probably controlled by different groups of genes though the problem is seen differently among ecologists, agronomists and geneticists. (4) it is considered that density response as well as competitive ability will be important in future silviculture, particularly in cross-fertilized tree species with high growth rate. acknowledgments this study has been conducted as a part of the research activities of the tropical forest biology program of biotrop (seameo regional center for tropical biology), bogor, indonesia. the writers have to express their gratitude to 39 biotropia vol. 1 no. 1, july-december 1987 prof. dr. ishemat soerianegara, the former director of biotrop, and also to prof. dr. zoefri hamzah, the former tropical forest biology program manager of biotrop for their valuable advice and help in the study. literatures cited akihama, t. 1967. estimation of competitive ability and its relation to fitness in rice hybrid population. jap. j. breed. 17: 262-265. akihama, t. 1968. inheritance of the competitive ability and effects of its selection on agronomic characters. ibd. 18: 12-14. fasoulas, a. and a. tsaftaris. 1975. an integrated approach to plant breeding and field experimentation. dept. genet. &p1. br., aristotelian univ. of thessaloniki, greek, pub. no. 5: 5-37. ford, e.d. 1975. competition and stand structure in some even-aged plant monocultures. j. ecol. 63(1): 311-334. harper, j.l. 1961. approaches to the study of plant competition. symp. soc. exp. biol. 15: 1-39. hozumi, k., h. koyama and t. kira. 1955. intraspecific competition among higher plants. iv. a preliminary account on the interaction between adjacent individuals. j. inst. polytech., osaka city univ., d 6. p. 121-130. jacquard, p. 1973. glossary of terms and definition: fodder crops section, "competition and fodder crop breeding" (6th issue, april 1973, eucarpia). kira, t., h. ooawa and n. sakazaki. 1953. intraspecific competition among higher plants. i. ompetiton-yield-density interrelationship in regularly dispersed population. j. inst. polytech., osaka city univ., d 4. p. 1-16. koyama, h. and t. kira. 1956. intraspecific competition among higher plants. viii. frequency distribution of individual plant weight as affected by the interaction between plants. ibd., d. 7. p. 73-94. oka, h.i. 1960. variation in competitive abilities among rice varieties. jap. j. breed. 10: 61-68. sakai, k.i. 1955. competition in plants and its relation to selection. cold spring harbor symp. quant. biol. 20: 137-157. sakai, k.i. and s. iyama. 1966. studies on competition in plants and animals. xi. competitive ability and density response in barley. jap. j. breed. 16 (1): 1-9. sakai, k.i., h. mukaide and k. tomita. 1968. intraspecific competition in forest trees. silvae genetica 17 (1): 1-5. tanaka, k. 1983. changes in diameter and height distribution in a forest stand. j. jap. for. soc. 65: 473-476. 40 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf 4. jani.cdr biotropia vol. 20 no. 1, 2013: 29 37 ecological impact of (l.) merrill invasion on plant diversity at bukit barisan selatan national park merremia peltata jani master *, sri s. tjitrosoedirdjo , ibnul qayim , and soekisman tjitrosoedirdjo received 25 march 2013/accepted 13 june 2013 bukit barisan selatan national park (bbsnp) is the third largest protected area in sumatra. unfortunately, the area is now invaded by or mantangan occupying about 7000 ha of the area. the aim of this study was to determine the ecological impacts of mantangan on plant species composition at bbsnp. three sites with different disturbance regimes were selected for vegetation analysis and assessment on plant species composition : primary forest representing undisturbed area, secondary forest representing burned area, and invaded forest representing forest invaded by mantangan. three line transects were constructed at each locations along 1 km, and the nested sampling plots were set up every 100 m with the following quadrant's size: 20 m x 20 m for trees, 10 m x 10 m for poles, 5 m x 5 m for sapling, and 2 m x 2 m for seedling. data collection covered the degree of mantangan invasion, trees species and diameter. results showed that invaded forest has lower plant diversity index (1.90) than the other two forests. this was caused by the invasion of mantangan as the percentage of coverage in the invaded forest reached 44% compared to secondary and the primary forest, which were 27.11% and 1.00%, respectively. percentage of mantangan coverage and diversity showed a strong negative correlation (-0.988). invasive species, impact, , mantangan, bukit barisan selatan national park 1 2 3 2 1 2 3 department of biology, faculty of mathematics and natural science,lampung university south east asian regional center for tropical biology (seameo biotrop), bogor, indonesia department of biology, faculty of mathematics and natural science, bogor agriculture university, bogor, indonesia. merremia peltata merremia peltata abstract introduction key words: bukit barisan selatan national park (bbsnp) is considered as the third largest protected area in sumatra, located at the southern tip of sumatera island covering an area of 356800 ha, extending from the southern of lampung province up north to bengkulu province (btnbbs 1999). the park also has the distinction of being . * corresponding author : j.janter@gmail.com 29 one of the few remaining tropical rain forests that harbors rhinos, tigers, and elephants. it is also a home for over 300 bird species, sun bears, bearded pigs, tapirs, gibbons, leaf monkey, great argus pheasants, rare orchids, and . presently, it was reported that bbsnp has lost more than 20% of its original forest area and it has been facing with serious threats of deforestation (suyadi 2011). this valuable remaining forest will become highly fragmented into isolated patches if current deforestation and encroachment rates continue. beside threatened with rapid deforestation and encroachments, this area has now been invaded by or called as mantangan occupying about 7000 ha. mantangan is a local plant species native to asia. it has wide distribution, extending from indian ocean islands of pemba, madagascar, mauritius, reunion, and the seychelles, throughout malesia, northern australia and eastwards into polynesia to the society islands (smith 1991). the mantangan invasion in bbsnp has been reportedly disturbing the natural habitat of a number of wild animals. irianto and tjitrosoedirdjo (2010) reported that the wild animals migrated up to the north approaching human settlements due to declining of food resources. it was argued that habitat loss and poaching impacted badly on the population of wild animals. poaching reduced the number of tigers and rhinos in the park. tiger and human conflicts have been increasing around the park, leading to losses on both sides (irianto & tjitrosoedirdjo 2010). irianto and titrosoedirdjo (2010) reported a heavy invasion of mantangan in tambling village, an area inside the bbsnp close to the seashore; mantangan grew prolifically, the young shoot of mantangan is brown purplish in color with a leaf diameter up to 40 cm and yellow flower. trees found in the area were among others sp., pule ( ), jabon ( ) and others as well as shrubs covered heavily by the climbing , giving a view of mantangan invasion in the area. this study assessed the ecological impacts of mantangan invasion on plant diversity at bbsnp. the observation was conducted from february 2011 to january 2012. herbarium studies were conducted in the herbarium bogoriense (bo), and seameo biotrop (biot), bogor, indonesia. sampling site for vegetation analysis was divided into three categories purposively based on invasion regimes. the first location has very little invasion of mantangan, an average of only 1%. the second location has a moderate invasion (5% 50% with an average value of 27%). the third location is the location with the highest invasion, up to 100% with an average of 44%. invasion of mantangan was calculated by estimating the percentage of the plot covered by mantangan. the second location was a post-fire forest in 1997, fires that occur mostly on the forest floor which affect the lining of plant down and open up some of the forest canopy (wcs-ip 2001). the third location lies close to the village enclave way haru, bandar dalam and tampang. based on land cover maps, this site has been decreasing forest cover an area of 2565.54 ha from 2000 to 2009. presumably these changes are rafflessia amorphophallus titanum merremia peltata ficus alstonia scholaris anthocepalus cadamba m. peltata materials and methods 30 biotropia vol. 20 no. 1, 2013 the result of forest clearing intended for plantations (prasetyo . 2011), while the first location is never disturbed. accordingly, the first location is referred as primary forest, the second as secondary forest and the third location as invaded forest (fig.1). line transects were constructed and replicated three times at each location. each line transect was drawn from the edge of the forest up to the forest along 2 km in distance, and square plots were made every 100 m. the nested sampling plots were constructed with the following measurement: 2 x 2 m for seedling, 5 x 5 m for sapling, 10 x 10 m for poles, and 20 x 20 m for trees (fig. 2). the data collected were diameter of the trees, trees species (include poles, sapling and seedling). computation on density, dominance, frequency and importance value indices were made from trees, pole, sapling and seedlings. diversity index of shannon-wiener diversity, evenness index and similarity index were calculated. et al figure 1. map of study site figure 2. plot design of the vegetation analysis 31 2 x 2 m : plot size for seedling (height < 1,5 m) 5 x 5 m : plot size for seedling (height > 1,5 m and dbh< 10 cm) 10 x 10 m : plot size for poles (dbh 10 – 20 cm) 20 x 20 m : plot size for trees (dbh > 20 cm) 100 m ecological impact of invasion jani mastermerremia peltata – et al. results and discussion based on vegetation analysis that was conducted on three sampling sites of 1 ha sample area, the primary forest had 153 species of trees with diameter above 10 cm. in the secondary forest, there were 103 species of trees with diameter above 10 cm, while the invaded forest had 116 species of trees with diameter above 10 cm. based on the observation, species composition within the three sample sites was relatively high compared to a number of research findings taken from other lowland tropical rainforests. by comparison, trees with diameter above 10 cm within 1 ha sample plot in kayan mentarang national park, east kalimantan had 106 species of plants (purwaningsih 2009); in pasoh malaysia, there were 683 species of plants recorded from a sample area of 50 ha; in baro colorado island (bci) panama, there were 229 plant species in a sample area of 50 ha (condit 1996); in costa rica, there were 561 plant species in a sample area of 23.4 ha (lieberman . 1996); and in uppangala, ghats, india, there were 103 plant species in a sample area of 28 ha (pascal 1996). the tree density in primary forest indicated that the forest was still in good condition than the secondary forest and invaded forest. tree density with diameter > 10 cm in primary forest was 403 trees per hectare, while in secondary and invaded forests; there were 365 and 305 trees per hectare, respectively. according to campbell (1992), tree density with diameter >10 cm in tropical areas was about 245 859 trees per hectare. meanwhile, according to sheil . (2002), tree density with diameter over 10 cm in tropical rainforests was about 400 600 trees per hectare. invaded forests had fewer species than primary and secondary forests due to mantangan invasion. the highest percentage of mantangan coverage was in the invaded forest with an average of 44.00%, followed by the secondary forest with an average of 27.11% and the primary forest as the lowest coverage with an average of 1.00%. in line with the number of species' data and mantangan coverage, it can be obviously seen that the more the forest was invaded, the less the plants lived there (fig. 3). not only the number of species, but also the number of individuals in the invaded forest was less than in primary forest. et al. et al et al. et al 32 biotropia vol. 20 no. 1, 2013 figure 3. the number of individuals on three research sites 33 tropical rainforests had vegetation composition that illustrated the dynamics of regeneration which occurred naturally, in which the vegetation at seedling stage had the highest number of species. furthermore, the number of species started to decrease at sapling and timber stages. however, it does not occur in the invaded forest. figure 3 shows that the number of seedlings was significantly less than the number of saplings. besides, the number of seedlings on this invaded forest was less than those of two other sites. each research site was dominated by different species, either seedling, sapling, pole, or tree. trees of the primary forest was dominated by and . meanwhile, the secondary forest was dominated by , while the invaded forest was dominated by that was a plant with the second-highest ivi on the secondary forest (table 1). the dominant trees on the secondary and invaded forests were fast growing trees, such as and it indicated that the forests were ever in a disturbed condition. moreover, another indication was vegetation of the family datiscaceae such as that could survive on burned land (goldammer 1996). in the invaded forest, there were still some vegetation of the family dipterocapaceae. this was demonstrated by the presence of as the second-highest ivi in that site. this tree showed high capability to withstand againts invasion of mantangan. in addition, there were other five members of dipterocapaceae, for instance , and . pole stage at the primary forest was dominated by . the species composition at this stage was similar to the tree stage. meanwhile, secondary forest was dominated by , different species with tree stage in this location. in the invaded forest, dominate this stage. the highest ivi in the secondary forest was and in the invaded forest. furthermore, at sapling stage, the primary forest was dominated by , while the secondary and invaded forests were dominated by the domination of at sapling stage in the primary forest also occurred at seedling stage. at seedling stage dominated the secondary forest, while dominated the invaded forest (table 1). research findings showed that plant community in the primary forest was more steady than secondary and invaded forests. species domination at tree stage at secondary and invaded forest was not followed by other stages. this condition enables structure and species composition revolution in the future. if the dominant trees on both forests were fallen, so it would be substituted by other dominant species from the stages underneath. in terms of shannon-wiener's diversity index, tree, pole and sapling in three forest types did not differ one from the other (table 2). however, when all stages were considered, diversity in invaded forests was less than primary and secondary forests. the low diversity is negatively correlated with invasion of mantangan (-0.988, p value = 0.097), high invasion of mantangan led to a low diversity of plant. shanon-wiener diversity index is calculated from numbers of species, without considering if the species which make up the number is different , when the species making the number is considered and compared each other using index of similarity, strombosia javanica dipterocarpus palembanicus tetrameles nudifora cananga odorata c. odorata, t. nudiflora glochidion arborescens. t. nudiflora d. palembanicus dipterocarpus retusus, shorea javanica, anisoptera costata vatica obovata strombosia javanica t. nudiflora dillinea exelsa bridelia monoica croton argyratus popowia bancana dillenia excelsa. p. bancana cleisthantus myrianthus aglaia macrophyla ecological impact of invasion jani mastermerremia peltata – et al. 34 biotropia vol. 20 no. 1, 2013 table 1. the three highest ivi at each research site family spesies ivi primary forest secondary forest invaded forest tree sterculiaceae strombosia javanica 31.70 dipterocarpaceae dipterocarpus palembanicus 25.11 22’,90 anacardiaceae dracontomelon dao 23.80 datiscaceae tetrameles nudiflora 43.75 annonaceae cananga odorata 35.16 36.14 euphorbiaceae glochidion arborescens 14.31 17.14 pole sterculiaceae strombosia javanica 33.64 euphorbiaceae croton argyratus 17.25 37.82 meliaceae aphanamixis humile 15.77 euphorbiaceae bridelia monoica 34.55 27.59 dilleniaceae dillenia excelsa 32.60 annonaceae cananga odorata 30.50 dipterocarpaceae dipterocarpus palembanicus 22.48 sapling annonaceae popowia bancana 41.07 13.12 euphorbiaceae glycosmis pentaphylla 24.90 annonaceae popowia pisocarpa 21.26 dilleniaceae dillenia excelsa 28.41 39.25 lauraceae endiandra rubescen 15.47 euphorbiaceae croton argyratus 25.81 euphorbiaceae cleisthantus myrianthus 19.84 seedling annonaceae popowia bancana 24.61 euphorbiaceae glycosmis pentaphylla 22.58 sterculiaceae pterospermum javanicum 9.49 euphorbiaceae bridelia monoica 10.76 euphorbiaceae cleisthantus myrianthus 35.20 sapindaceae xerospermum noronhianum 10.72 meliaceae aglaia macrophyla 19.39 euphorbiaceae bridelia monoica 15.59 dilleniaceae dillenia excelsa 14.42 they are different one from the other (dyke 2003). according to those arguments, similarity index (is) was calculated by using steinhaus coefficient. its result was low index value (close to 0) that means those three sites at all stages had different species composition (table 3). mantangan devastated the habitat by covering other plants so that it reduced the availability of light for other plants. 35 table 2. shannon-wiener diversity index (h') and the average percentage of covermantangan location shannon-wiener’s diversity index (h’) a) % average of coverage mantangan tree pole sapling (g) seedling (g) all stage primary forest 3.84a 3.61a 3.51a 3.51a 2.49a 1.00 secondary forest 3.31a 3.12a 3.74a 2.75b 2.21b 27.11 invaded forest 3.46a 3.23a 3.46a 2.66b 1.90c 44.00 note : a) numbers followed by same letter on the same column are not obviously different based on t-test at 5% significance level table 3. steinhaus similarity index on three research sites phase of growth location secondary forest invaded forest tree primary forest 0.35 0.29 secondary forest 0.43 pole primary forest 0.24 0.19 secondary forest 0.41 sapling primary forest 0.36 0.36 secondary forest 0.35 seedling primary forest 0.24 0.13 secondary forest 0.10 erythrina variegata coffea arabica mantangan climbed up the canopy for sunlight, then rapidly grew and covered the entire canopy. the covered trees did not receive enough sunlight and hold the mantangan's weight as well. this condition caused those covered trees gradually died. another case was that mantangan would twist the trunk so the growing process of the twisted tree would be devastated. however, this case was rarely found. mantangan invasion was calculated by percentage estimation of its cover. in the primary forests, the coverage of mantangan was very little with an average of only 1% and the highest value of coverage was 15%. in the secondary forests, the average coveriage was 27% and the highest value of coverage was 55%. in addition, the invaded forests were the forests with the highest percentage of mantangan coverage. it reached an average of 44% coverage. in some locations, they were fully covered by mantangan (100%) (figure 2). mantangan invasion made possible because of the forest devastation caused by forest fragmentation. forest fragmentation might occur due to several activities such as illegal logging, farming, and land clearing for road construction. in several invaded forests, there were remnants types of crops that was abandoned for a long time such as dadap ( ) and coffee ( ). moreover, some logged trees were found. it was assumed that this location was a farming area. ecological impact of invasion jani mastermerremia peltata – et al. 36 biotropia vol. 20 no. 1, 2013 forest devastation in bbsnp already happened since 1960s. according to satellite image analysis from 1972 to 2006, bbsnp had lost 22% of its forest cover and left about 67.225 ha of total area 356.800 ha of national parks (suyadi 2011). forest devastation in bbsnp was mostly done by the farmers who live in and around the forest (suyadi 2011). on an invaded forest, there was an enclave that was a district in the administrative area of way haru village, west lampung regency. furthermore, on this location there was a four-meter-dirt road for nature tourism activities and the conservation of sumatran tigers. the existence of enclave and dirt road in this national park became the causes of forest fragmentation. forest fragmentation might cause some impacts, one of which was the edge effect (watson 2004). edge effects on forest ecosystem occurred in a form of microclimate changes, species composition changes, the decline of plants diversity, the abundance and density of species. this might cause local extinction of species because they were unable to adapt to the changing of condition there (meffe 1994). these edge effects became worse with the presence of invasive species, such as mantangan. it was alleged that at the edges of forest fragments, mantangan was able to live well and start invading into the forest. at the edges of open forests, soil moisture would decrease (sizer 1999). in this condition, a liana like mantangan was able to live better than other trees (londre 2006). invasion of mantangan was feared to have an effect on forest regeneration in the future, because in the invaded forest, the number of seedlings was significantly less than the number of saplings, pole and tree. furthermore, the diversity and number of seedlings was lower than in the two other forests. hence, it is necessary to do efforts to control the invasion in bbsnp. this study was part of the master thesis of the first author financially supported by seameo-biotrop dipa 2011 through dr. sri s. tjitrosoedirdjo. the author wishes to thank artha graha peduli and wildlife conservation society indonesia program for their facilities in conducting the field works. et al. m. peltata conclusions acknowledgments references [btnbbs] balai taman nasional bukit barisan selatan. 1999. rencana pengelolaan taman nasional bukit barisan selatan, buku ii. kota agung, lampung : btnbbs. campbell dg, stone jl, rosas a jr. 1992.a comparison of the phytosociology and dynamics of three floodplain (varzea) forest of know ages, rio jurua, western brazilian amazon.bol j of linnaian soc 180: 213-37. 37 condit r, hubbell sp, lafrankie jv, sukumar r, manokaran n, foster rb, ashton ps. 1996. species area and species individual relationship for tropical tree: a comparison of three 50-ha plots. j ecology 84 : 549-62. dyke fred van. 2003. conservation biology: foundations, concepts, applications. international edition. new york. usa: mcgraw-hill companies. goldammer jg, b siebertand, w schindele. 1996. fire in dipterocarp forest. in: a schulteand d schöne, editor. dipterocarp forest ecosystem: toward sustainable management. singapore: world scientific. irianto r, tjitrosoedirdjo s. 2010.invasi (l.) merr., convolvulaceae di taman nasional bukit barisan selatan, indonesia. j gulma dan tumbuhan invasi tropika 1:65-70. lieberman d, lieberman m, peralta r, hartshorn gs. 1996. tropical forest structure and composition on a large scale altitudinal gradient in costa rica. j ecology 84: 137-52. londre ra, schnitzer sa. 2006. the distribution of lianas and their change in abundance in temperate forests over the past 45 years. j ecology 87:2973-8. meffe gk, carrol cr. 1994. principles of conservation biology. sunderland: sinnauer assoc. pascal jp, pelissier r. 1996. structure and floristic composition of a tropical evergreen forest in southwest india. j trop ecol 12 : 191-214. prasetyo a, hikmat a, prasetyo lb. 2011. pendugaan perubahan cadangan karbon di tambling wildlife conservation taman nasional bukit barisan selatan. media konservasi 16 : 87 91. purwaningsih. 2009. analisis vegetasi hutan riparian dataran rendah di tepi sungai nggeng, taman nasional kayan mentarang, kalimantan timur. berita biologi 9 : 254-9. sheil d, ducey mj, sidiyasa k, samsuddin i. 2002. a new type of sample unit for the efficient assessment of diverse tree communities in complex forest landscapes. bogor: cifor. sizer n, tanner evj. 1999. response of woody plant seedlings to edge formation in lowland tropical rainforest, amazonia. biol conserv 91: 135-42. smith ac. 1991. flora vitiensis nova. volume 5, lawai, kauai, hawai'i, national tropical botanical garden. 4950. suyadi. 2011. deforestation in bukit barisan selatan national park, sumatre, indonesia. j biol indonesia 7: 195206. watson jem, whittaker rj, dawson tp. 2004. effects of deforestation and forest modification on understorey bird in central sulawesi, indonesia. bird conserv int 15:257-73. [wcs-ip] wildlife conservation society indonesia program. 2001. taman nasional bukit barisan selatan dalam ruang dan waktu. laporan hasil penelitian 2000 2001. bogor : phka/wcs-ip. merremia peltata ecological impact of invasion jani mastermerremia peltata – et al. microsoft word 69 biotropia vol. 13 no. 2,2006 : 69 74 embryo reconstruction by transplantation of the donor inner cell mass to the recipient bovine blastocyst arief boediono laboratory of embryology, department of anatomy, physiology and pharmacology, faculty of veterinary medicine, bogor agricultural university, darmaga 16680bogor, indonesia abstract in an attempt to produce the interspecies embryo transfer, this study was conducted to evaluate the efficacy of the production of reconstructed blastocyst by transferring the donor icm into the recipient trophoblast. icm cells were isolated from the donor blastocyst by immunosurgery method. zona-free blastocysts were incubated in the medium (tcm-199) containing 20% of the heat-inactivated rabbit anti-bovine-serum. the embryo reconstruction was produced by three different methods. recipient blastocyst was maintained on the holding pipette by gentle suction, with the icm in a 9 o'clock position to have the possibility of developing incorporate icms (method i), the icm was in a 3 o'clock position to break the original icm during injection (method 2); cutting the original recipient icm followed by insertion of the donor icm (method 3). reconstructed blastocysts were then cultured overnight and examined morphologically according to the re-expansion of the reconstructed blastocyst with or without developed donor icm. according to morphological observation in this study, 37.9% of the reconstructed blastocyst developed with the incorporation of two icm originally from recipient and donor (method i), 66.7% of the reconstructed blastocysts developed with a single icm (method 2), and 80.0% of the reconstructed blastocyst developed from the icm originally from donor icm (method 3). these results showed that the reconstructed blastocyst is better produced by cutting the original recipient icm followed by the insertion of the donor icm (method 3). key words: embryo reconstruction, immunosurgery, icm transfer, bovine. introduction embryo reconstruction for chimera production has been used in experiments oriented towards animal science such as the production of interspecies pregnancies in domestic animals (fehilly et al. 1984a; polzin et al. 1987). so far, chimeras have been obtained by the aggregation of the blastomeres (brem et al. 1984; boediono et al. 1993; boediono et al. 1999) or by inner cell mass transplantation (butler et al. 1987; polzin et al. 1987; picard et al. 1990). the aggregation of cell from embryos results in embryos with a more randomly distribution contribution of cells from each donor to the trophoblast and icm. fehilly et al. (1984b) in their production of interspecific sheep-goat chimeras produced one kid by the injection of a goat icm into a sheep blastocyst and one lamb from the reciprocal injection. rorie et al. (1994) produced two normal lambs after transferring nine reconstructed embryos into three recipient goats. the reconstructed embryos comprised trophoblast cells from the embryo recipient species (goat) and icm cells from the embryo donor species (sheep). the present study was conducted to evaluate alternative procedures corresponding author: abl@cbn.net.id 69 biotropia vol. 13 no. 2, 2006 for production of the reconstructed embryo by transplantation of the donor inner cell mass to the recipient bovine blastocyst. materials and method in vitro embryo production embryos were produced by standard in vitro maturation, fertilization and culture procedures (boediono et al. 2003). bovine ovaries were collected from a nearby abattoir and transported (32-35°c) to the laboratory in 0.9% physiological saline solution immediately following collection. follicular oocytes were allowed to mature for 22 hr at 38.5°c under 5% co2 in air. frozen-thawed sperm was washed twice with 2.5 mm caffeine in brackett-oliphant medium (b-o; brackett and oliphant 1975) without bovine serum albumin. sperm concentration was adjusted to 5 x 106 spermatozoa per ml in b-o supplemented with 0.3% bovine serum albumin (bsa, sigma) and 20 ̂ g/ml heparin (shimizu, japan). a100 ul aliquot of the sperm suspension was pre-incubated for 1 hr. in v//ra-matured oocytes were transferred into fertilization droplets for insemination (20-25 oocytes/droplet). after 18 hr of sperm exposure, oocytes were washed and transferred to a polystyrene dish (4-well multidish; nunclon, roskilde, denmark) containing tcm199 supplemented with 5% superovulated cow serum (boediono et al. 1994), 5 ng/ml insulin (wako, osaka, japan) and 50 ng/ml gentamicin sulfate for further development. adherent cumulus cells surrounding the embryos were removed by repeated pipetting 48 h post-fertilization. the cumulus cells adhering to the surface of the culture dish were not disrupted, and embryos were cultured on this somatic cell monolayer. the culture medium was replaced with fresh medium 96 h post-fertilization. icm isolation blastocysts produced in vitro were used in this experiment. an immunosurgical method for icm isolation was carried out by the similar method described by softer and knowles (1975) with modification. the zona pellucida was removed by a short incubation (<5 min) in tcm-199 containing 0.5% pronase. for immunosurgery, the embryos were incubated for 1 h in the medium (tcm-199) containing 20% of the heat-inactivated rabbit anti-bovine-serum. they were then washed in three successive baths of tcm-199 + 20% pcs and were transferred into tcm-199 containing 10% of reconstituted guinea pig complement for 10 min followed by two successive washes in tcm-199 + 20% pcs. embryos were aspirated into a pipette of a diameter slightly larger than the icm to remove any degenerating trophoblastic cell. embryo reconstruction the manipulator was arranged with a beveled injection pipette with an outer diameter of approximately 40-50 \\m on one side and an embryo holding pipette 70 embryo reconstruction a. boediono with an outer diameter of 150 urn on the other side (method 1 and 2). the donor icm was drawn into the injection pipette and held directly opposite the recipient blastocyst on the holding pipette. the embryo reconstruction was produced by three different methods. recipient blastocyst was maintained on the holding pipette by gentle suction, with the icm in a 9 o'clock position to have the possibility of developing an incorporate icms (method 1, figure 1a-b), the icm was in a 3 o'clock position to break the original icm during injection (method 2, figure 1c-d); cutting the original recipient icm followed by insertion of the donor icm (method 3, figure 1e-f). with a swift precise forward motion the tip of the injection pipette was introduced into the blastocoele and the icm was injected. reconstructed embryos were cultured overnight and then examined morphologically according to the re-expansion of blastocyst with the presence of original and donor icms (method 1 and 2) and the re-expansion of the blastocyst with or without donor icm (method 3). results and discussion embryo reconstruction was performed by injection of a total of 78 icm isolated from donor blastocysts into blastocoel recipient blastocysts. in an attempt to produce interspecies pregnancy, the development of the donor icm with the original trophoblast is most important to produce a baby originating from donor icm. according to morphological observation , 37.9% of the reconstructed blastocyst produced by method 1 developed from the incorporation of two icm originally from recipient and donor, while 34.5% developed with the corporation of two icm (table 1). using the same method, fehilly et al. (1984b) in their production of inter 71 biotropia vol. 13 no. 2,2006 species sheep-goat chimeras, produced one kid that developed from the donor icm, two chimeras and 10 lambs after transfer of 22 reconstructed blastocysts into 14 recipients. none of the reconstructed blastocyst produced by method 2 developed with two incorporated icm. a total of 66.7 percent of the reconstructed blastocysts developed with a single icm. the purpose of this method was to break the original recipient icm and insert the doonor icm. however, we are not sure that the broken original icm would be destroyed and the developed icm come only from donor icm. there was a possibility that the original icm could recover and develop 72 embryo reconstruction a. boediono incorporating both icms from donor and recipient. the transfer of these embryos may result in chimeric animals. to avoid the development of the original recipient icm into the reconstructed blastocyst, the removal of icm recipient blastocyst by cutting followed by insertion of the donor icm (method 3) is the most definite way to produce interspecies pregnancy. in this study, 80.0% of the reconstructed blastocyst developed with the icm originally from donor icm. the reconstructed blastocysts have been transferred to the three recipients resulting in one pregnancy. this method would be useful for production of the interspecies embryo transfer to rescue the endangered animals or for nuclear transfer produced embryo (sansinena et al. 2005). conclusions attempts to produce interspecies embryos for transfer showed that the reconstructed blastocyst is better produced by cutting the original recipient icm followed by the insertion of the donor icm (method 3 in this study) and transferring into recipient with the same species as recipient blastocyst. acknowledgments the author would like to thank dr. robert a. godke, professor of the department of animal science, louisiana state university, usa for his suggestions and helpful evaluation of the manuscript. references butler, j.e., anderson, g.b., bondurant, r.h., pashen, r.l, and m.c. penedo. 1987. production of ovine chimeras by inner cell mass transplantation. j. anim. sci., 65:317-324. boediono, a., oe, m, yamamoto, m, takagi, m., saha, s. and t. suzuki. 1993. production of chimeric calves by aggregation of in vitro bovine embryos without zonae pellucidae. theriogenology, 40:1221-1230. boediono, a., takagi, m., saha, s. and s. suzuki. 1994. the influence of day 0 and day 7 superovulated cow serum during development of bovine oocytes in vitro. reprod. fertil. dev., 6:261-264. boediono, a., suzuki, t., li, l.y. and r.a. godke. 1999. offspring born from chimeras reconstructed from parthenogenetic and in vitro fertilized bovine embryos. mol. reprod. dev., 53:159-170. boediono, a., suzuki, t. and r.a. godke. 2003. comparison of hybrid and purebred in v/fro-derived cattle embryos during in vitro culture. anim. reprod. sci.,78:l-l 1. brackett, b.c. and g. oliphant. 1975. capacitation of rabbit spermatozoa in vitro. biol. reprod., 12. 260-274. brem, g., tenhumberg, h., and karausslich. 1984. chimerism in cattle through microsurgical aggregation of morula. theriogenology, 22:609-613. fehilly, c.b., willadsen, s.m. and e.m. tucker. 1984a. experimantal chimaerism in sheep. j. reprod. fertil., 70:347-351. fehilly, c.b., willadsen, s.m. and e.m. tucker. 1984b. interspecific chimerism between sheep and goat. nature, 307:634-636. picard, l, chartrain, i., king, w.a. and k.j. betteridge. 1990. production of chimaeric bovine embryos and calves by aggregation of inner cell masses with morulae. mol. reprod. dev., 27:295-304. polzin, v.j., anderson, d.l., anderson, g.b., bondurant, r.h., butler, j.e., pashen, r.l., penedo, m.c.t. and j.d. rowe. 1987. production of sheep-goat chimeras by inner cell mass transplantation. j. anim. sci.. 65:325-330. 73 biotropia vol. 13 no. 2,2006 rorie, r.w., pool, s.h., prichard, j.f., betteridge, k.j. and r.a. godke. 1994. a simplified procedure for making reconstructed blastocysts for interspecific and intergeneric transfer. vet. rec., 135:186-187. sansinena, m.j., hylan, d., hebert, k., denniston, r.s. and r.a. godke. 2005. banteng (bos javanicus) embryos and pregnancies produced by interspecies nuclear transfer. theriogenology, 63:1081 1091. solter, d. and b.b. knowles. 1975. immunosurgery of mouse blastocyst. proc. natl. acad. sci. usa., 72:5099-5102. 74 69.pdf 70.pdf 71.pdf 72.pdf 73.pdf 74.pdf biotropia no. 5, 1991/1992: 26-40 effects of carbofuran on the reproductive capacity of a freshwater snail, radix quadrasi, under laboratory conditions imelda f. pagulayan, gloria l. enriquez and virginia s. caring college of science, university of the philippines diliman, 1001 quezon city, philippines abstract the effects of 4 sublethal concentrations of carbofuran (250, 500, 1000 and 2000 ppm) on the reproductive capacity of r. quadrasi was determined. results showed that incubation period is delayed and inhibited by 1000 and 2000 ppm carbofuran but not by lower concentrations. the hatching period is longer in treated snails and not all eggs hatch in the 1000 and 2000 ppm treatment. the percentage of hatching is inversely proportional to the carbofuran concentration. oviposition was delayed in all the treated stages and at all dosages. the higher the carbofuran concentration, the later the onset of oviposition. the reproductive period is shortened. fecundity was decreased in snails treated at emb and sm. however, only the 2000 ppm carbofuran concentration showed an adverse effect on the snails exposure at psm. introduction pesticides are continuously finding widespread use and increasing amounts are being introduced into the aquatic environment. researchers worry that this increasing use of pesticides and the farming industry's dependence on it may eventually lead to health problems for both consumers of farm products and farm workers (allmon 1985). moreover, this will pose a potential hazard not only to human beings but also to aquatic organisms of economic importance (mane et al. 1979). invertebrates constitute a very large portion of the fauna in both the aquatic and terrestrial system. from an ecological viewpoint, these animals play an important role in the energy flow in these ecosystems. the feeding habit of invertebrate animals varies from detritus feeding to herbivorous and carnivorous food consumption. disruptions caused by pesticide stress in one or more organisms may directly interfere with other inter-dependent components of the food chain (gunther et al. 1968). carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl-n-methylcarbamate) developed by the niagara chemical division of fmc corporation is commercially known as furadan. it is a systemic insecticide, nematicide with broad spectrum activity (metcalf et al. 1968; yu et al. 1974; cremlyn 1980; handa 1980) although hydro 26 antioxidant metabolism in water stressed peanut g.c. rivero & d.m. orcutt lyzed rapidly in water. it is an effective contact toxicant and is non-corrosive and compatible with other pesticides (rangaswany et al. 1976). carbamate insecticide is a popular choice since it is less toxic, less persistent and has lower residual effects in animals than other classes of pesticides. nevertheless, it is accumulated to a certain extent. this work aims to determine the levels of carbofuran concentrations that are toxic or deleterious to the embryonic (emb), presexually mature (psm), and sexually mature (sm) r. quadrasi; to determine the effects of sublethal concentrations of carbofuran on the fecundity and reproductive period of the snail; and to determine the particular age group that is most sensitive to carbofuran. materials and methods organism radix quadrasi, (fig. 1) the snail used in this study, were obtained from stock cultures started in 1981 and have been maintained and reared continuously in the snail room of the natural sciences research institute, university of the philippines, diliman, quezon city. 27 figure 1. radix quadrasi biotropia no. 5, 1991/1992 laboratory maintenance and culture of snails, maintenance of eggs and young snails followed that of de lara and enriquez (1981) and pagulayan and darvin (1986). preparation of working solutions carbofuran in the form of 3g was prepared by dissolving the granules first in acetone before adding distilled water. the solution was aerated overnight before use. concentration parts in per million (ppm) were used. all glasswares and plastic containers were acid washed and rinsed with de-ionized water. petri dishes were oven-dried before use. experimental set-up thirty egg masses were used as the source of snails that were observed for fecundity and reproductive period. ten of these egg masses were allowed to hatch under normal conditions (control). the other experimental groups consisting of 10 egg masses each were treated with 250 ppm and 500 ppm carbofuran for a period of one week in separate petri dishes. two-week treatment durations and 1000 and 2000 ppm concentrations were not used because preliminary work showed that survival of experimental snails would be less than 50%. a. treatment at the larval stage two weeks after the eggs hatched into young snails, 48 snails were taken at random from the untreated egg masses and were placed in groups of four in 12 separate basins. the same was done for egg masses treated with 250 and 500 ppm. the average size and age of the snails at first and last oviposition were recorded. the total number of egg masses laid per group were recorded weekly in the entire length of the reproductive period. the length of the reproductive or egg laying period was recorded. b. treatment at week four (psm) forty five newly hatched snails from egg masses under normal laboratory conditions were separated at the age of two weeks. they were placed in 9 separate basins in groups of five. the above procedure was followed. however, the snails were exposed to the four carbofuran concentrations applied at the end of the 4th week to the end of the 6th week after hatching. 28 effects of carbofuran on the reproductive capacity imelda f. pagulayan et al. c. treatment at week seven (sm) the same procedure was followed except that the snails were treated at the end of the 7th week to the end of the 9th week after hatching. in the two-week duration of treatment, the medium was changed after the first week. possible toxic material in the medium like the feces were removed regularly. results effects of fecundity fecundity is the total number of the eggs produced by the individual during its reproductive period. reproductive period, on the other hand, is defined as the number of days from the first to the last day of egg-laying. a. treatment at embryonic stage (emb) table 1 shows the size range and average at the onset and end of oviposition, the reproductive capacity and the reproductive period of r. quadrasi that were hatched from untreated embryos and embryos exposed to 250 and 500 ppm concentrations of carbofuran. the average size length: 10.62, 10.56 and 10.45 mm for the control, 250 ppm and 500 ppm treatment respectively, are not significantly different from each other. however, the age range and average in days of the treated snails differ greatly from the untreated. the earliest oviposition took place in the control at day 29, although the average age was 39 days. the earliest oviposition for both treatments was 43 days with an average age of 49.5 days. the treated snails oviposited at a much older age but the size of the snails were almost the same as that of the untreated ones. the average size length at last oviposition differ between the control and the treated snails; for the control it took place at an older age range with an average of 224 days. oviposition stopped at an average age of 196.5 days for the 250 ppm treatments; and 193 days as average age for the 500 ppm treatment. there is no significant difference in the size and average for the two treatments. the number of egg masses, number of eggs (table 1 and fig. 2) and average number of eggs per egg mass are highest in the control followed by the 250 ppm and lowest in the 500 ppm treatment. the reproductive period was shorter in the treated snails. 29 biotropia no. 5, 1991/1992 30 t ab le 1 . s iz e an d a g e at t h e o n se t an d e n d o f o v ip o si ti o n , re p ro d u ct iv e p er io d o f r . q u a d ra si t h at w er e h at ch ed f ro m e m b ry o s ex p o se d t o tw o c o n ce n tr at io n s o f ca rb o fu ra n efects of carbofuran on the reproductive capacity – imelda f. pagulayan et al. 31 f ig u re 2 . t o ta l n u m b er o f eg g m as se s an d e g g s p ro d u ce d i n r . q u a d ra si t re at ed a t e m b b y 2 c ar b o fu ra n c o n ce n tr at io n biotropia no. 5, 1991/1992 b. treatment at week 4, pre-sexual maturity (psm) table 2 shows the effect on the size and age of the snails treated at the end of week 4 to the end of week 6 at the onset and end of oviposition. the total number of egg masses, eggs (fig. 3) and the average eggs per egg mass observed during the entire reproductive period is also shown in the table. with the exception of the 2000 ppm treatment wherein the average snail size at initial oviposition is smaller, the size average of the rest of the treatments and control are not significantly different from each other. the average age for initial oviposition in the control is 40.44 days, while the treated snails at 4 carbofuran concentrations are 53.36, 55.0, 56.5 and 55.84 days. this is an indication that treated snails oviposited at an older age. the average size in the last oviposition of the treated snails is smaller than in the control except in the 2000 ppm treatment. the average age at last oviposition is higher in the control and significantly lower in the treated. the 500, 1000 and 2000 ppm treatments were not significantly different from each other but significantly different from the 250 ppm group. the number of egg masses and eggs in the snails exposed to 2000 ppm is significantly low. there is no significant difference though in the total number of eggs and egg masses among the other experimental set-ups including the control (fig. 3). the reproductive period is longest in the control, and decreasing with higher concentrations. those exposed to 500, 1000 and 2000 ppm are lower but not significantly different from each other. c. treatment at week 7, sexual maturity (sm) table 3 shows the effect of the 4 carbofuran concentration on the size of the snails treated at week 7 at the onset and end of oviposition. the total number of egg masses laid and the total number of eggs for the entire reproductive period is also shown (fig. 4). the average number of eggs per egg mass at the end of the observation period is likewise indicated. it is shown in table 3 that untreated snails start to oviposit at an average length of 9.6 mm. the average size of the snails at the onset of oviposition in all four treatment groups as well as in the control does not significantly differ from each other. the average age, however, was older in the treated snails, indicating that while sizes are more or less the same, oviposition occurred at an older age. there are snails in the untreated that oviposited in as early as 35 days of age, while the youngest snail that oviposited in the experimental group was 45 days old, or a difference of 10 days. some snails did not oviposit: 1 snail in the untreated, 10 in the 250 ppm treatment, 8 in the 500 ppm treatment, 6 in the 1000 ppm treatment and 32 . effects of carbofuran on the reproductive capacity – imelda f. pagulayan et al. 33 t ab le 2 . s iz e an d a g e at t h e o n se t an d e n d o f o v ip o si ti o n , eg g -l ay in g c ap ac it y a n d r ep ro d u ct iv e p er io d o f r . q u a d ra si e x p o se d to fo u r co n ce n tr at io n s o f ca rb o fu ra n a t w ee k 4 ( p re -s ex u al m ar tu ri ty ) fo r a d u ra ti o n o f tw o w ee k s t ab le 3 . s iz e an d a g e at t h e o n se t an d e n d o f o v ip o si ti o n , eg g l ay in g a n d r ep ro d u ct iv e p er io d o f r . q u a d ra si e xp o se d t o f o u r co n ce n tr a ti o n s o f ca rb o fu ra n a t w ee k 7 ( s ex u a l m a tu ri ty ) fo r a d u ra ti o n o f 2 w ee ks ) biotropia no. 5, 1991/1992 34 fi gu re 3 . t o ta l n u m b er o f eg gm as se s an d e gg s p ro d u ce d b y r . q u a dr a si t re at ed a t th e p sm b y 4 c ar b o fu ra n c o n ce n tr at io n s effects of carbofuran on the reproductive capacity imelda f. pagulayan et al. 35 f ig u re 4 . t o ta l n u m b er o f eg g m as se s an d e g g s p ro d u ce d i n r . q u a d ra si t re at ed a t s n b y 4 c ar b o fu ra n co n ce n tr at io n s biotropia no. 5, 1991/1992 7 in the 2000 ppm treatment. average age at first oviposition in the untreated is 42 days, while in the treated the range is from 58.4 to 60.73 days (table 3). the average size of the snails at last oviposition was smaller in the treated but not significantly different from control and from each other. the average age at last oviposition, however, is highest in the control (169.42 days) and lowest in the 2000 ppm treatment (120.36 days) in a decreasing order with increasing concentration. the total number of egg masses and eggs was highest in the untreated snails, 1284 and 76559, respectively. the number of egg masses and eggs produced in the treated was lower and significantly different from the control but not significantly different from each other. the average number of eggs per mass is highest in the control and lower in the treated. the numbers of snails that reached the stage of oviposition in the treated set-ups were reduced. the reproductive capacity of the treated snails increases weeks after treatment. the observation is more evident in the treated than in the untreated snails. however, the above increase is still not enough to equal the number of egg masses and eggs produced by the control group. the reproductive period in the treated snails is significantly lower than in the control. d. comparative fecundity results in the three designated stages (emb, psm, sm) table 4 is the summary of the effects of all the treatments on the size and age at the onset and end of oviposition, on the reproductive capacity and length of the reproductive period in the three stages of treatment. the size range in days at the start of oviposition was 29 50 days for the emb control; 35 52 days for the psm control; and 35 50 days in the sm control. the total range using 138 snails was from 29-52 days. the smallest in size average at the same time oldest in average for the initial oviposition in all the treatments was on the sm treated snails, followed by the psm and the largest but the youngest to oviposit was in the emb. while table 4 shows that the psm group is the earliest to stop ovipositing, it cannot be concluded that it is the most sensitive to the treatment since the control group follows the same trend. the reproductive capacity is highest in the emb treated snails. while sm treated snails had a longer reproductive period than the psm, their reproductive capacity is very much less. 36 effects of carbofuran on the reproduction capacity – imelda f. pagulayan et al 37 t ab le 4 . c o m p ar at iv e ef fe ct o f ca rb o fu ra n a p p li ed o n t h e th re e d es ig n at ed s ta g es i n t h e li fe o f r . q u a d ra si o n i ts f ec u n d it y biotropia no. 5, 1991/1992 discussion the results of previous work (de lara and enriquez 1981) gave a range of 35 -42 days under controlled condition as the start of oviposition. in the current work, however, the total range in these 138 snails was 29-52 days. the current results did not exactly sustain the earlier findings, perhaps because of the effect of cold temperature when these experiments were done, since the expected oviposition periods coincided with the cold months of december and january. boray (1964) observed in his work that no copulation occurred in l. tomentosa in temperatures below 16°c in the laboratory cultures or in the field. the variations apparently reported in the reproductive biology of lymnaeids in general may be due to the different culture methods used. however, remigio and pagulayan (1984) contradict this finding. start of oviposition was delayed in all the treated snails while size range and average do not differ much in both the untreated and treated snails in the initial oviposition. the treated snails are older which means that it took a greater number days to reach the ovipositing size. it seems that it is more the size rather than age that determines the onset of oviposition. this is true in all the 3 age treatments. since growth is delayed by the treatment, delay in the oviposition could be attributed to it. it can be stated that the higher the concentration, the later is the start of oviposition. this could also be due to a delay in the rate of gonadal maturation. the higher the concentration, the earlier the cessation of oviposition. the number of egg masses and eggs produced at initial oviposition is highest at the control and lowest at the 2000 ppm treatment. the treatment not only delayed but also inhibited the reproductive capacity of most of the snails. inhibition increased with increasing carbofuran concentration. however, the actual number of egg massesat first oviposition could not be correctly determined. the fact that the number of egg masses at its first appearance is less than the number of snails clearly indicates that not all oviposited. there is no way of determining which of the snails in the basin did or did not oviposit since they are in groups of 4's and 5's. it is possible that the first oviposition in some may have been included in later weeks and counted as later ovipositions. while it is true that there is no accurate method of counting the number of egg masses for first oviposition, there is no other way, unless observations will be done on individual snails cultured in separate basins; such condition, however, is not conducive to fecundity. the results showed that the carbofuran maybe more effective in a particular age group, and the results could have altered the individual's competitive ability, i.e. food getting, mating. this could be the reason for the decreasing fecundity with increasing concentration in the emb; there was a drastic effect only 38 effects of carbofuran on the reproductive capacity imelda f. pagulayan et al. of the 2000 ppm on the egg laying capacity of the psm treated snails, and a drastic effect of all the treatments on the egg laying capacity of the sm treated snails. it may also be possible that the psm treated snails could have recovered after the effect of the 250, 500 and 1000 ppm carbofuran concentrations had diminished. recovery brought them back on equal footing with the untreated ones, thus the egg production was higher, counteracting the low egg production at the start. the initial effect, therefore, seems to be delaying in this particular stage instead of totally inhibiting with the exception of the 2000 ppm which partially inhibited the egg laying capacity. godan (1983) stated that the periods of greatest sensitivity occur when the gastropods are very young and again when they are adults, at the peak of their reproductive activity. resistance is greatest among juveniles before the onset of sexual maturity. this reinforces the results of this work, as shown by the effect on fecundity of the sm treated snails. sexual maturity is the most critical stage to carbofuran treatment when it comes to reproductive capacity. the reproductive period is shorter in the treated snails and is again inversely proportional to the carbofuran concentration. the snails had to attain a bigger size and older age before oviposition took place. the snails used as control in the emb, psm and sm treatments had a wide range of variation for the first oviposition. the said control set-ups were not done at the same time. that could explain the wide variation in 29 50 for the emb control; 35 52 for the psm control; 35 50 for the sm control. the final average, though, for all the control is still 40.48 days which still supports the upper range of the results of earlier works, 35-42 days (de lara and enriquez 1981). the pesticide decreased the reproductive capacity of the snails and shortened the reproductive period. unlike its effect on growth, the sm treated snails were the ones adversely affected. there were snails that did not oviposit at all and a great number did not reach the size/age of oviposition. the carbofuran, depending on the concentration and/or the stage of application must have direct and subtle impacts on behaviour that influences the snail's competitive success i.e. small changes in courtship or mating rituals induced by the carbofuran may lead to mating failure, thus affecting the reproductive capacity and period. references allmon, f. 1985. pesticides and unhealthy dependence. science 6(8): 14. cremlyn, r. 1980. pesticides: preparation and mode of action. john wiley and sons, new york. de lara, a.v. and g.l. enriquez. 1981. life history of laboratory reared radix quadrasi (gastropoda: pulmonata). kalikasan 10(2-3): 242-254. 39 biotropia no. 5, 1991/1992 godan, d. 1983. pest slugs and snails, biology and control. springer-verlag, berlin. gunther, f.a., w.e. westtake and p.s. jaolan. 1968. reported. handa, s.k. 1980. spectrophotometric determination of carbofuran residues in water. assoc. chem. 64(2): 200-201. mane, u.h., m.s. kachole, and s.s. pawar. 1979. effect of pesticides and narcotants on bivalve mollusks. malac. 18: 347-360. metcalf, r.l., t.r. fukoto, c. collins, k. berck, s. abd el-aziz, r. munoz and c.c. cessrl. 1968. metabolism of 2,2-dimethyl-2,3-dihydrobenzofuranyl-7-n-methyl carbamate (furadan) in plants, insects, and mammals. j. agr. food. chem. 16(2): 300-311. pagulayan, i.f. and d. darvin. 1986. the systematic relationship of radix quadrasi and mixas cumingiana (pulmonata: lymnaeidae). part iii genetics and part iv ecology. final report on a research project submitted to the natural sciences research institute, diliman, quezon city. rangaswany, j.r., y.d. vijayashanakar, and s.r. prakish. 1975. a simple spectrophotometric method for the determination of carbofuran residues. j. aoac 59(6): 1276-1278. yu, c., g.m. booth, d.j. hansen, and l.r. larden. 1974. fate of carbofuran in a model ecosystem. j. food chem. 22(3): 431-434. 40 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf microsoft word 56 biotropia vol. 13 no. 1,2006 : 56 67 current microbiological aspects in high mountain lake research munti yuhana0* and kurt hanselmann2)* "faculty of fisheries and marine science, bogor agricultural university, bogor, indonesia. 21 institute of plant biology, university of zurich, 8008 zurich, switzerland abstract remote and normally unpolluted high mountain lakes provide habitats with no or very limited anthropogenic influences and, therefore, their hydrodynamics are mostly regulated by the natural conditions. researches in high mountain lakes deal with measuring and modeling the response of the habitats to environmental changes especially correlated to acid deposition, pollutants influx and climatic variability. the microbial world has also become a focus in many studies of these extreme ecosystems. despite the pressure of harsh and extreme conditions, microbial communities living in these habitats developed flexible strategies and show quick adaptation to climate oscillation. new aspects in microbiological studies in recent high mountain lake research are presented in this paper. key words : high mountain lake, extreme environment, microbial communities, microbial diversity, psychrotolerant microbe, molecular microbe introduction in recent years, interests in the presence and survival of microorganisms which inhabit extreme environments have been increasing. attempts to discover novel microorganisms possessing special metabolic characters have encouraged microbiologists to intensive researches on microorganisms which are able to survive and sustain in extreme habitats. extremophiles dwelling in extreme environments have attracted the interest of microbial ecologists because of the uniqueness of peculiar physiological properties. life strategies of microbes evolved in low temperatures and nutrient limited environments have also attracted the attention of those who are studying conditions for life on other planets, such as mars (monastersky 1997). naturally cold ecosystems, such as antarctic and arctic regions, cryoconite holes, ice-covered lakes, the deep sea, high altitude mountain environments and glaciers (morgan-kiss et al. 2006) may represent analogs on earth to the cold extra terrestrial habitats. high mountain ecosystems are also of ecological interest because of their richness in natural resources, their biodiversity, the clean water, healthy climate, original soil, and diverse geology which are relevant to issues of environmental protection. on the other hand, the utilization of the mountain areas e.g. for the tourist industry is an issue of socio-economic importance. * corresponding authors: myuhana@botinst.unizh.ch or hanselma@botinst.unizh.ch 56 biotropia vol. 13 no. 1, 2006 microbiologists are mainly interested in the following questions: • what microbial diversity is present in high mountain lake habitats? are there unknown microorganisms which are characteristic for the extreme conditions? • are psychrotolerant organisms phylogenetically closely related to each other? • do microbes present in oligotrophic cryo-habitats possess special life strategies? • do the communities possess physiological and ecological flexibilities with which they can respond to the fluctuating physico-chemical conditions? • what are the ecological parameters determining the community structures and how rapidly do they respond to environmental changes? in tropical regions, the extreme cold environment can be found for instance in areas at high elevations of ice-covered mountains, i.e. mount jayawijaya in papua island, indonesia, mount kilimanjaro in tanzania, and the tropical andes in south america. despite of the cold temperature and oligotrophic characteristics, these ecosystems are different to those found in temperate and polar regions. they exist only at altitudes of 4500 m a.s.l. and higher, whereas in temperate and polar regions they normally begin at lower altitudes (e.g. > 1 400 m a.s.l) depending on the season. strong uv radiation, because of the continuous sun ray exposure throughout the year and thinner ozone layer at higher altitudes, might influence the physico-chemical characteristics of tropical mountain habitats. in addition, atmospheric deposition of pollutants (acidity and toxic air pollutants) that strongly influences the ecology of temperate high mountain lakes (mosello et al. 2002), may occur less in tropical mountain habitats, because of the higher elevation. so far, very limited information is available on (sub) tropical high mountain lake habitats. therefore, this article presenting various studies on temperate high mountain areas may encourage for new research and exploration of our tropical high mountain lake habitats. high mountain lake habitats as indicators of environmental changes global warming indicators for decades mountain areas have been used as sites to evaluate possible effects of global warming and its consequences. they are sensitive indicators for the early detection of the impacts of rapid climatic changes on the hydrological cycle, water chemistry and ice melting. even a small temperature change can strongly influence their hydrological cycle (schindler et al. 1990). the climatic warming observed in the last 25-30 years seems to be most pronounced in the mountain regions all over the world (beniston et al. 1997; rogora et al. 2003). the temperatures measured in these areas have increased by 1.5 2.0°c on the average since 1980, whereas an increase of approximately 0.5°c was observed on a global scale (beniston et al. 1997). the climatic changes and environmental fluctuations particularly affect open waters of high mountain lakes (psenner and schmidt 1992; sommaruga-wograth et al. 1997; koinig et al. 1998). in addition, other factors such as the atmospheric dust 57 current microbiological aspectsm. yuhana et al. deposition (psenner 1999) and long periods of anthropogenic impacts (kamenik et al. 2000) also shape the ecology of these areas. the duration and extent of the snow cover in a lake catchment area are dominant factors governing the release of weathering products from rocks and soils into the water (wright and schindler 1995). a recent study shows that the effect of climate warming has influenced the weathering rate in the mountains (rogora et al. 2003). through the chemical and physical degradation of rocks and soil minerals, solute contents increased. warmer temperatures are also enhancing biological processes by increasing the primary productivity. biochemical characteristics and dynamics of high mountain lake habitats trophic condition of high mountain lake habitats high altitude lakes are normally situated above the timberline and have catchment areas with little or no vegetation. consequently, they are often oligotrophic as defined by the concentration of nitrogen and phosphorus. examples include the lakes which became subject of the european project molar (mountain lake research) such as in the high tatra mountains (slovakia, 2 655 m a.s.l.) (kopacek et al. 1995), and the sumava mountains (czech, 1456 m. a.s.l.); schwarzsee (austria, 2799 m a.s.l.), lake paione superiore (italy, 2269 m a.s.l.) (mosello et al. 2002), yellow belly lake (u.s.a, 2157 m a.s.l.) (pilati and wurtsbaugh 2003), gossenkollesee (austria, 2417 m a.s.l.) (kamenik et al. 2000), jori lakes iii and vii (switzerland, 2557 m a.s.l.) (hinder et al. 1999a). lake jori xiii does not meet the oligotrophy criteria especially in summer season. low n:p ratios were measured at the end of the ice cover period, whereas the highest ratios were measured during the ice free period (iqbal-nava 2003). the physico-chemical conditions strongly depend on the duration of the ice and snow cover. the nutrient conditions in lake jori xiii initiate high biomass and remarkable microbial activities and population dynamics. atmospheric deposition influences mountain lakes' hydrochemistry atmospheric depositions have been demonstrated to be one of the key factors to influence longterm changes in the chemical characteristics of high mountain lakes (kamenik et al. 2000). airborne desert dust depositions contain major basic cations, carbonates, sulphates and other anions of nutrient value for residents of remote lakes (psenner 1999). the high calcium contents of the saharan aerosols, which are also found in aerosols from other deserts, might significantly contribute to the biogeochemical cycles involving acid neutralization (de angelis and gaudichet 1991). airborne dusts in snow and rain have also contributed to the high buffering capacity of some mountain lakes and to the elevated seasonal nutrient levels (de angelis and gaudichet 1991; psenner 1999). 58 biotropia vol. 13 no. 1, 2006 environmental changes demonstrated by microbial community succession physicochemical conditions in high mountain ecosystems are exposed to strong environmental fluctuations, seasonally as well as diurnally. these habitats (figure 1) are good models for studies of microbial dynamics of cold environments with a simple and short food web. during the long ice-cover period, they are exposed to cold temperatures, nutrient limitation, darkness below the thick ice cover, and anoxic conditions at larger water depths. whereas in the short summer season, they are exposed to high sun radiation, oxic conditions and wind-driven water masses upwelling. despite low nutrient input and commonly limited concentrations, some lakes also showed self-trophication capability, which is particularly observable during the summer season (iqbal-nava 2003). these environmental pressures provide heterogeneous ecological niches in the habitats. therefore, the microbial community adapts itself by continuously selecting for those members which are best adapted to the particular conditions in the ecosystem. a recent study observes dynamic changes in planktonic bacterial community composition, seasonally as well as spatially (yuhana 2005). these changes are a consequence of strongly fluctuating environmental conditions in which the communities show a quick response to fulfill the requirements for various ;**. photo by munii yuhami ecological niches. their dynamics were not only demonstrated by the seasonal changes in the genera diversities, but also their abundances. the quick adaptation was also observed in the planktonic microalgal populations. the autotrophic and heterotrophic flagellate as well as ciliate communities showed dynamics and high activities as indicated by high [3h]thymidine and [3h]leucine or [14c]leucine uptake rates during the short ice-free period (felip et al. 1995; hinder et al. 1999a). their population was supported with the availability of organic matters as nutrients which originate from allochthonous and autochthonous sources. two different external 59 current microbiological aspects m. yuhana et al sources can contribute to nutrient enrichment inside a lake: atmospheric input or input from the catchment. the input can consist of dissolved organic matter, organisms, particulate matter, vegetation debris including pollen grains, and insects (fclipetal. 1995). the role and the physiological adaptation of microorganisms in high altitude habitats the role of microorganisms in high altitude habitats in aquatic ecosystems, the bacterioplankton community is an integral part of the food web, which is essential for protists and mctazoans (cho and azam 1988). temporal and spatial alterations of the microbial community in pelagic food webs of high mountain lake habitats have been studied (felip et al. 1995). recent studies focus on more specific subjects such as the effect of the uv radiation on the bacteriovory (sommaruga et al. 1996), microbial diversity and activity (alfreider et al. 1996) and palaeolimnology (koinig et al. 1998). compared to eutrophic ecosystems, the pelagic food webs in these oligotrophic ecosystems appeared to be less complex and the microbial loops might play an essential role in recycling nutrients for the higher trophic levels (hinder et al. 1999b). the significance of the pelagic microbial assemblage increases with the level of oligotrophy of the lake water (hahn et al. 1999). this is true for pelagic food webs in circumcontrol as well as in acidified high mountain lakes (wille et al. 1999). microorganisms in high mountain habitats play a key role in biogeochemical cycles such as subglacial rock weathering and nutrient cycling. microorganisms living in phosphate-limited mountain lakes are able to mobilize particulate iron phosphates from sedimentary deposits and biofilm-associated microbes can develop on iron-phosphate-oxyhydroxide coated surfaces (amberg-brunner 2002). microbially mediated pyrite oxidation occurs at low temperature and glacier bed microbial communities contribute to sulfate release to the environment (sharp et al. 1999). from these biochemical processes, furthermore the microbial population derives energy from the oxidation of reduced mineral or organic carbon within the sediments. physiological adaptation to low temperature organisms that live and actively grow at near freezing temperatures and limited-nutrient conditions face a number of growth constraints. under the cold conditions the enzyme reaction rates are generally lower, uptake and transport systems function more slowly, membranes become less fluid and nucleic acid structures become more stable (feller et al. 1996; graumann and marahiel 1996). however, microorganisms have evolved various strategies to adapt to these hindrances. they range from molecular level to cell and ecosystem levels (gerday et al. 1997). the evolution of cold shock and antifreeze proteins, the modulation of the kinetic key enzymes, and the development of more fluid biological membranes 60 biotropia vol. 13 no. 1,2006 through the accumulation of polyunsaturated fatty acyl chains are among the means of adaptation to low temperature (morgan-kiss et al. 2006). adaptation to high-level uv exposure attenuation coefficients (kd) of the photosynthetically active radiation (par) and uv were used for indirect determination of the chlorophyll concentrations. kd values, which are based on the upper and lower uv radiation intensities, are used to illustrate the strength of uv intensity at different wave lengths i.e. 305, 320, 340, and 380 nm. results from iqbal-nava's study (2003) showed that the diffuse attenuation coefficients (kd) measured in swiss alps lake jori xiii (ca. 2640 m a.s.l.) were ranging from 0.68 to 1.54. whereas kj values of the austrian lake gossenkolle at 2417 m a.s.l were ranging from 0.14 to 0.32 (sommaruga and psenner 1997), and the mean kd values of 13 oligotrophic lakes in the bariloche region in argentina (ca. 2000 m a.s.l.) were between 0.3 and 0.8 (morris etal. 1995). microorganisms living at high altitudes are challenged by intense uv radiation. uv b radiation (280-320 nm) is potentially the most damaging for living cells (morris et al. 1995). the uv b level in the winter cover of high mountain lakes can reach up to 50% higher than at sea level (psenner and sattler 1998). the strong uv exposure can lead to growth inhibition of benthic diatoms (bothwell et al. 1994); causes damage in heterotrophic flagellates (sommaruga et al. 1996), and inhibits the rate of nutrient uptake by bacterioplankton in the water column (sommaruga et al. 1997). some adaptive strategies against strong uv radiation, that were found in permanently low temperature high mountain habitats are, for instance, the capability to screen uv and the photosynthetically active radiation (par) by chlamydomonas sp. in addition, chloromonas sp. posseses uv-screening pigments called mycosporine-like amino acids (maa), whereas some cyanobacteria like nostoc, phormidium, and anabaena spp. segregate a mucopolysaccharide matrix (morgan-kiss et al. 2006). molecular approaches applied to study the microbial succession in high mountain aquatic ecosystems pcr-based methods provide information on the nucleic acid composition of microorganisms isolated from different habitats which mostly are still not (yet) cultivable. when carl woese introduced the use of 16s rrna sequences for molecular phylogeny (woese 1987), only 12 microbial phyla could be phylogenetically compared. currently, 26 phyla of approximately 52 identifiable major phyla within the bacterial domain have cultivated representatives (rappe and giovannoni 2003). the number of environmentally retrieved bacterial 16s rrna genes has been increasing rapidly and now is exceeding 30 000 (rappe and giovannoni 2003; wagner 2004). carl woese's approach has also uncovered a new domain of life, the archaea. formerly, archaea were thought to exclusively consist 61 current microbiological aspects m. yuhana et al. of thermophiles, halophiles, and strictly anaerobic methanogenic microorganism inhabiting extreme environments. today, archaea are recognized as ubiquitou microorganisms, also present in high mountain lake habitats (yuhana 2005). in recent years, advanced techniques have been developed to study thi composition of microbial communities (figure 2). pcr-based community fingerprinting techniques such as denaturing gradient gel electrophoresis (dgge or temporal temperature gradient gel electrophoresis (ttge) allow us to monitoi the microbial structure based on community banding patterns. these techniques an based on the separation of the pcr products of genes isolated from mixec populations possessing different nucleotide sequences (muyzer et al. 1993). nucleic acid is extracted from the natural samples and dna amplification is performed by pcr with primers targeting for instance the small subunit ribosomal rna genes. the pcr products are subsequently analyzed by loading them onto a polyacrylamide gel containing a linearly increasing gradient of denaturants (in dgge) or a linearly increasing temperature gradient (in ttge). by using dgge or ttge, the diversity and microbial community composition can be described without enriching them or performing the cloning techniques (muyzer and smalla 1998; muyzer 1999). the number of bands on a gel may not accurately reflect the number of different species in their habitat, but the dna of the most abundant representatives of the communities normally gets amplified and should be represented in the band pattern (muyzer et al. 1993). these techniques can also be applied for the screening of clone libraries (e.g. bosshard et al 2000). both techniques provide an alternative way for microbial community fingerprinting without application of cloning strategies which is more time consuming. dgge and ttge combine a direct visualization of community diversity and the opportunity for subsequent identification of microbial population members by sequence analysis or hybridization experiments using taxon specific probes (muyzer 1999). this community fingerprinting technique has been successfully applied in high mountain lake ecosystems. the population dynamics and the community succession can be followed seasonally as well as spatially which has been done for example in the alpine shallow lake jori xiii (10.4 m, maximum depth). while this lake was temperature-stratified, it showed distinct ttge banding patterns, whereas the patterns were identical at all depths during summer upwelling events (yuhana 2005). a different study was carried out in the meromictic lake cadagno (bosshard et al. 2000). this lake shows a permanent stratification, chemically as well as physically, represented by an oxic mixolimnion, a chemocline water column and an anoxic, monimolimnion of high salinity. the spatial community structure, as revealed by ttge banding patterns, of the chemocline and the monimolimnion were highly similar whereas the mixolimnion showed a distinctly different pattern. for the temporally community shift, the authors reported that community structure of these three zones varied with time. in the mixolimnion and the chemocline, the community composition changed greatly during the sampling period, whereas in the monimolimnion, the microbial community shift was less distinctive. the distribution of microbial populations was mainly correlated to the spatial or temporal fluctuation 62 biotropia vol. 13 no. 1,2006 in their micro-environmental conditions, such as organic matter concentration (crump et al 2003) or nutrient availability (yuhana 2005). it has also been demonstrated that the spatial distribution of the phototrophic community present in a dense layer changed diurnally by active vertical movement in response to the light conditions and the chemical gradient (egli et al. 2004). a direct and rapid detection technique to taxonomically identify the members of microbial communities is fluorescent in situ hybridization (fish). the use of specific rrna-targeted oligonucleotide probes allows one to visualize the morphotype and size of hybridized cells; as well as to determine quantitatively the species, sub groups, or domains among the dapi-stained cells (amann et al. 1990; amann et al. 1995). the in situ assessment for the abundances and the microbial composition has been widely applied to various habitats. different studies were carried out to investigate the community compositions of habitats from high mountain lakes (alfreider et al. 1996; pernthaler et al. 1998). alfreider et al. (1996) 63 current microbiological aspects m. yuhana et al. studied the microbial composition in the different snow, slush, and pelagic layer c lake gossenkolle in the tyrolean alps (2417 m a.s.l.). they reported that th application of probes specific for the alpha, beta, and gamma subclasses c proteobacteria and the cytophagaflavobacterium group showed a very distinc bacterial community composition within different habitats (snow, slush, and lak water). the community, in most cases, was dominated by members of the bet subclass (6.5 to 11.6% of the bacteria detectable with the probe hub). the pcr-based (culture-independent) analyses, however also have thei limitations. without having appropriate culture representatives, it is quite difficult ti predict phenotypic properties of the detected but uncultured microorganisms. it i still a challenge for microbial ecologists to assign functions and activities t< populations within those communities in complex ecosystems (wagner 2004; paei and steppe 2003). the microbial communities, which consist of mixed groups o microbial species having different functions and metabolic activities, are responsibli for maintaining the ecosystem fitness (paerl and steppe 2003), i.e. microbiall; mediated chemical transformations and habitat alterations (boetius et al. 2000). ii spite of extreme conditions, highly active and diverse microbial communitiei occupying the ice and snow cover of high mountain lakes have been demonstratec (felipefaz. 1995). conclusions although mountain environments have been exploited for a number 01 purposes, the giant microbial gene pool present in these extreme habitats has beer neglected so far. their aquatic areas are environments often characterized by simple food chains, low in species diversity and species richness. appreciation of microbiai diversity, their genomic richness and metabolic capabilities is very much supported by advances in molecular techniques. these techniques allow us to phenotypically and genotypically characterize microbes, as well as to assign their ecological roles, without the need for cultivation processes. hence, the remote mountain habitats which typically contain low nutrient concentrations can no longer be underestimated as genetic pools. this untouched reservoir of genomes might be exploited for biotechnological purposes, one day, e.g. for cold active enzymes. acknowledgments we would like to thank thomi horath for his valuable suggestions on the article. the fruitful comments from the reviewers were also extremely appreciated. references alfreider, a., j. pernthaler, r. amann, b. saltier, p.o. glockner, a. wille and r. psenner. 1996. community analysis of the bacterial assemblages in the winter cover and pelagic layers of a high mountain lake using in situ hybridization. appl. environ. microbiol., 62: 2138-2144. 64 btotropia vol. 13 no. 1, 2006 amann, r.l, l. krumholz and d.a. stahl. 1990. fluorescent-oligonucleotide probing of whole cells for determinative, 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65.pdf 66.pdf 67.pdf biotropia no. 4, 1990/1991: 41-48 the role of haltica sp. (coleoptera: halticidae) as biological control agent of polygonum chinense kasno faculty of forestry bogor agricultural university, bogor, indonesia s. tjitrosemito and sun jay a tropical agricultural pest biology programme seameo biotrop, bogor, indonesia abstract the role of haltica sp. (coleoptera: halticidae) with emphasis on host specificity and damage potential in controlling polygonum chinense was evaluated under laboratory condition. starvation test of the weevil on 33 weeds and 14 crop plant species indicated that only 6 weed species were attacked: polygonum chinense, p. nepalense, p. barbatum, p. longisetum, ludwigia octovalvis and l. parennis with p. chinense as the most preferred host plant. preliminary damage potential test indicated that a population of 0, 1,2 and 3 pairs of adult weevil reduced the percentage of fresh weight increment of p. chinense by 0; 46.2; 74.7 and 75.5% respectively. field observations indicated that the larvae as well as adult weevils are potential biological control agents of p. chinense. further studies are, however, on the host-range of this weevil. introduction polygonum chinense l. is one of the weeds growing amongst tea plantations in west java but it has never been reported as a serious problem in the area. locally it is known as "titiwuan" (backer & slooten 1924) and in thailand it is called "phayaadong" (harada et al. 1987). it was reported to be distributed also from india eastward to japan and it grows mostly on highland or open forest, coffee and tea plantations (harada et al. 1987). although west java is classified as a populated province, the utilization of labour to control weeds manually in large tea plantations is considered costly. chemical control with herbicides is considered cheaper and more practical. on the other hand there is a strong demand to save the environment from any pollutant for better living. therefore, it is necessary to develop other control methods in line with minimizing the side effects of herbicides and lowering the cost. in the tea producing areas of west java, there are many kinds of insects associated with p. chinense. haltica sp. (coleoptera: halticidae) seems to be one of the most common and it causes considerable damage to the weed. a study on the potential role of the insect as biological control agent is needed. 41 biotropia no. 4, 1990/1991 materials and methods haltica sp. was collected from a tea plantation of ptp xii, gunung mas located about 30 km on the way from bogor to bandung. they were brought to biotrop laboratory at bogor for rearing and further observations on its life cycle, host specificity and damage potential. some behavioural aspects of haltica sp. were casually observed in the field. the insects were reared in petri dishes and fed with fresh cut leaves of polygonum chinense. the room temperature and relative humidity of the laboratory varied from 24-31°c and 48-82% respectively. starvation test of newly hatched larvae and newly emerged weevil of haltica sp. was done against 14 species of crop plants and 33 species of weeds (appendix 1). five heads of larvae and adult weevils in separate petri dishes were reared with a single fresh cut leaf of test plants. replacement of fresh cut leaves of the test plants was done every day during the test. p. chinense was used as the control. the response parameter of the insect on the test plants was mainly the presence of feeding scars. observation was done every 24 hours till most of the test insects died. when the test insects produced feeding scars, the test was continued till the sixth day to make sure that they can survive by consuming the cut leaves of the test plants. the positive test plants were tested further for food preference. preferential test using larvae of the insect was carried out against six species of weeds namely polygonum chinense, p. longisetum, p. barbatum, p. nepalense, ludwigia octovalvis and l. parennis. six heads of haltica larvae were released in petri dish containing six arranged pieces of the weed species. the inner space of each petri dish was equally divided into six radial sectors (figure 1) and each piece of fresh cut leaves of test plants was put into each sector. six heads of test weevil were released at the center of the petri dish to allow them to select freely the test leaves. the response parameter of the preferential test was the level of feeding scars on each test leaf. preliminary damage potential test of the weevils against p. chinense was carried out by releasing various numbers of adult weevils on 20 day-old plants. the average fresh weight of newly cut p. chinense before being transplanted into plastic pots was 14.30±2.27 gram and the average leaf number was 11.85 ± 1.59 pieces. the pots were 15 cm in height, 10 cm and 15 cm in bottom and upper diameters, respectively. the pots were fully filled with light soil. the potted plants were allowed to grow for 20 days. watering was done daily with tap water. to avoid attack of any insect, the potted plants were covered singly with screen cage. various numbers of weevils e.g. 0, 1, 2 and 3 pairs were released on a single pot plant and kept for ten days. the test was run following complete randomized design with 42 figure 1. six radial sectors of the petri dish used for preferential test. 5 (five) replications. the response parameter of the test was mainly based on the percentage of reduction of fresh weight compared with the control at the end of the test. results and discussion under laboratory conditions of 24 31 °c temperature and 48 82% relative humidity, respectively, the life cycle of the insect varied from 26 to 28 days. the incubation, larval and pupal periods were 5-6, 13-15 and 7 days, respectively. during the larval period, it molted three times, i.e. it had four larval instars. other biological data such as adult longevity and egg production of an adult female have not been precisely observed. however, it seemed that the adult longevity was more than a month and egg production of a female was more than a hundred. the adult females laid their eggs in groups of ten on the abaxial surface of the leaves of p. chinense. usually the early larvae feed on the outer tissues of the leaf where the adult female oviposited but when they have developed bigger, they feed on most of the leaf tissues. 43 the role of haltica sp. as biological control agent-kasno, s. tjitrosemito & sunjaya biotropia no. 4, 1990/1991 the adult weevils feed mostly on leaves and soft stem tissues of p. chinense. the adults feed randomly but the larvae produced local spot symptom. both larvae and adults caused defoliation of p. chinense. starvation test of both larvae and weevils of haltica sp. against 14 crop species and 33 weed species showed that the insects feed on six of the test weeds: p. chinense, p. barbatum, p. nepalense, p. longisetum, ludwigia octovalvis and l. parennis. p. chinense was the most preferred by the larvae. the rank of preference of the larvae is presented in appendix 2. it seemed that the insect is polygophagous but feeds especially on polygonum spp. it is strongly suspected that p. chinense, p. barbatum, p. nepalense and p. longisetum contain the key feeding stimulant for haltica sp. biological control using insects has a risk of changing from control agent to pest of crop plants in the future. luckily in indonesia, there is no known crop plant belonging to polygonum sp. and ludwigia sp. there are some closely related insects to haltica sp. namely h. caerulea oliv, h. cyanea (weber) and h. caerulea which were reported as promising biological control agents of water primrose (ludwigia spp.). a total of 123 plant species have been tested by cibc (sankaran et al. 1967 cited by mangoendihardjo et al. 1977) which reported that these insects feed on nicotiana tabacum under laboratory condition (rao et al. 1977). h. cyanea has also been reported to feed on leaves of some forest trees such as ammonia baccifera, a. rotundifolia, terminalia myriocarpa in india (beeson 1941), and some food crops in indonesia (kalshoven 1981). haltica sp. that was observed in this study was formerly suspected as h. caerulea but due to the supporting data on its host range, the authors of this paper name it haltica sp. for the moment. the correct name of the insect will be confirmed later. preliminary damage potential test using weevils on p. chinense grown in plastic pots indicated that the insect produced serious damage on the weed. populations of 1, 2 and 3 pairs of adult weevil caused a reduction on the fresh weight increment of 46.19, 74.33 and 75.53%, respectively within the last 10 days of the 30-day experiment. conclusion and recommendations conclusion haltica sp. (coleoptera: halticidae) is a promising candidate as biological control agent of polygonum chinense. 44 the role of haltica sp. as biological control agent-kasno, s. tjitrosemito & sunjaya recommendations 1. further studies are needed especially on its host range before deciding to recommend it as a biological control agent of p. chinense. 2. confirmation of the correct identification of the insect is necessary. references backer, c.a. and d.f.v. slooten. geillustreerd handboek der javaansche theeonkruiden en hunne beteekenis voor de cultuur. drukkerijen ruygrok & co. jakarta. beeson, c.f.c. 1941. the ecology and control of the forest insects of india and the neighbouring countries. dept. of agriculture. dehra dun. harada, j., y. paisooksantivatana and s. zungsontiporn. 1987. weeds in the highlands of northern thailand. project manual no. 3 nat. weed science res. inst. project. bangkok. kalshoven, l.g. 1981. the pests of crops in indonesia. revised edition by p.a. van der laan. pt ichtiar baru van hoeve. jakarta. mangoendihardjo, s., o. setyawati, r.a. syed and s. sosromarsono. 1977. insects and fungi associated with some aquatic weeds in indonesia. proc. sixth asian pacific weed science society conference. jakarta. rao, v.p., m. a. gani and t. sankaran. 1971. a review of the biological control of insects and other pests in south east asia and the pacific regions. techn. communication no. 6. cibc, trinidad. 45 biotropia no. 4, 1990/1991 appendix 1. response of the larvae and adults of ha/tica sp. on several test plants during starvation test no. plant species response amaranthaceae 1. amaranthus spinosus l. asteraceae 2. ageratum conyzoides l. 3. bidens pilosa l. 4. crassocephalum crepidioides (benth.) s. moore 5. chromolaena odorata (l.) r.m. king & h. robinson 6. eupatorium riparium reg. 7. eleutheranthera ruderalis poit. 8. mikania micrantha h.b.k. 9. tridax procumbens l. balsaminaceae 10. impatiens platypetala lindl. carparaceae 11. cleome rutidosperma dc cruciferaceae 12. nasturtium heterophyllum bl. cyperaceae 13. cyperus rotundus uphorbiaceae 14. phyllanthus niruri l. gramineae 15. imperata cylindrica (l.) raeuschel 16. paspalum conjugatum berg. 17. sporobulus berteroanus trin. melastomataceae 18. oxalis barrelieri l. 19. oxalis corniculata l. onagraceae 20. ludwigia octovalvis (jacq.) raven + 21. ludwigia perennis l. + 22. ludwigia peruviana (l.) polygonaceae 23. polygonum barbatum l. + 24. polygonum chinense l. + + + + 25. polygonum longisetum de br. + + 26. polygonum nepalense meissn. + + + 46 the role of haltica sp. as biological control agent-kasno, s. tjitrosemito & sunjaya appendix 1. (continued). no. plant species response rubiaceae 27. borreria data (aubl) dc 28. borreria laevis griseb. 29. diodia sarmentosa swartz. verbenaceae 30. lantana camara l. 31. stachytarpheta indica (l.) crop species amaryllidaceae 1. a ilium cepa l. 2. allium fistulosum l. araceae 3. colocasia esculenta (l.) brassicaceae 4. brassica oleraceae l. convolvulaceae 5. ipomoea batatas poir. gramineae 6. oryza sativa l. 7. zea mays l. leguminosae 8. arachys hypogea l. 9. glycine max (l.) 10. phaseolus vulgaris l. musaceae 11. musa paradisiaca l. solanaceae 12. capsicum annuum l. 13. solarium lycopersicum l. theaceae 14. cammelia sinensis (l.) o.k. note: — no feeding scars + feeding scars present. 47 biotropia no. 4, 1990/1991 appendix 2. response of larvae of haltica sp. on several weed species during preferential test no. weed species response 1. polygonum chinense + + + + 2. p. nepalense l. + + + 3. p. longisetum de br. + + 4. p. barbatum l. + 5. ludwigia octovalvis (jacq.) raven + 6. l. perennis l. + 48 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf 47.pdf 48.pdf 615 triono bagus (phnotypic).cdr phenotypic and molecular characterization of m hoots d in transgenic ultis evelopment phalaenopsis amabilis arboring 35s::knat1 (l.) blume h (knotted-like arabidopsis thaliana 1) *triono bagus saputro , endang semiarti and1 2 3 aziz purwantoro 1 departement of biology, institut teknologi sepuluh nopember, surabaya 60111, indonesia 2faculty , of biology universitas gadjah mada, yogyakarta 55281, indonesia 3 faculty of agriculture, universitas gadjah mada, yogyakarta 55281, indonesia received 28 january 2017/accepted 07 june 2017 abstract phalaenopsis amabilis (l.) blume is one of indonesian natural orchid which has an aesthetic flower and possesses high economic value. the low multiplication rate and long periods of life cycle are the main obstacles to conventionally propagate this orchid. the aims of this research were to analyze the stability of transgenic plant p. amabilis harboring 35s::knat1 based on morpho-genomic characterization. knat1 gene is reported as a gene that involved in the shoot formation, and it had been successfully introduced into phalaenopsis amabilis (l.) blume genome. after seven times regeneration, the confirmation of the transgene existence in the genom is needed to ensure whether the plant could consistently maintain the transgene in its genome and to characterize the shoot development. the experiment was carried out in 3 steps: 1) co-integration analysis of 35s::knat1 into p. amabilis genom; 2) phenotypic analysis on the multiplication rate, morphological variation and venation pattern; and 3) protein profile analysis of transgenic plants. the results showed that the survival rate of putative transgenic was 58.7% on np0 medium and 62.5% on np sim medium. pcr analysis confirmed that 82.5% transgenic growth on np0 and 93.33% on np sim contained dna fragment of knat1 gene, nptii gene and trnl-f intergenic spacer, indicating that those plants are positive transgenic. the 35s::knat1 transgenes and phytohormone were independently involved in multishoots formation of p. amabilis transgenic plants. the phenotypic of plantlets were classified into six main criteria, i.e. normal shape, lobed leaves, rosette, elongated stem, cup shoot and widened leaves. the normal type was the most abundant type of variation (± 29%) in both medium. protein profile showed that all transgenic plants produced 45,8 kda protein and that was equivalent with molecular weight of knat1 protein. taken together, all those data indicated that 35s::knat1 transgene were consistently integrated into the transgenic plant genome. keywords: 35s::knat1 (knotted-like arabidopsis thaliana 1), multishoots, phalaenopsis amabilis, transgene introduction orchid is an ornamental plant that has high economic value and high market sharing (da silva 2013). phalaenopsis amabilis (l.) blume or known as moth orchid is an indonesian native plants (semiarti et al. 2007). p. amabilis commonly used as parental line for breeding purpose in terms to produce superior hybrid varieties (semiarti et al. 2011a). p. amabilis is a monopodial orchid that having single growth node that emerging from the central part of a plant (weissenhofer et al. 2013). for large scale propagation, monopodial orchid has many difficulties to meet the market demands since the orchid only has one single stem (dresler 1981). the long periods of life cycle, 2-3 years, are also the emerging challenge in the cultivation of p. amabilis (mercuriani et al. 2012). the damages of natural habitat and collection activities for commercial purposes are also significantly decline the amount of this orchid in nature (weissenhofer et al. 2013). genetic engineering using modified dna is needed to obtain transgenic plants having better characters compared to its parental line. the knat1 gene (knooted-like arabidopsis thaliana 1) * corresponding author: endsemi@ugm.ac.id; esemiart@gmail.com biotropia 5 1 8 1 21 vol. 2 no. , 201 : 1 doi: 10.11598/btb.2018.25.1.615 11 from arabidopsis thaliana plant was successfully inserted into .several plants i.e in rice (sakamoto et al 2006), p. (semiarti et al. 2007), . amabilis celosia plumosus l the bp/knat1 gene (sun et al. 2011). can be expressed in several spot plants but s in , is primarily expressed in the region around the shoot apical meristem (sam) and regulates positively sam development in a. thaliana (kerstetter & poethig 1998; byrne et al. 2000). the expression of inserted transgene can be decreased by its growth condition repetitive and the series of subculture the s. based on that fact, the stability of transgene function in the transformant sneed to be observed re. the aims of this study we : 1) to study integration existence in transgenic plants; 2) to observe the effect of auxin-cytokinin hormone in sim medium; 3) to conduct protein profiling of transgenic p. amabilis harboring 35s::knat1 transgene. this research is important to maintain the continuous function of transgene in transformant plant. materials and methods the study was conducted in the laboratory of plant tissue culture, faculty of biology and gene t i c eng inee r i ng labo r a to r y o f biotechnology pau ugm. this study used phalaenopsis amabilis (l.) blume (java forms) which was inserted with 35s::knat1 by semiarti et al. in 2007. the plant was maintained in in vitro conditions for seven generations before being used as material. the code of transgenic plants was # 7.121. media used for the in vitro maintenance was new phalaenopsis (np), which was modified by islam et al. (1998). selection of putative transgenic in medium containing kanamycin resistance rate of the materials were observed in np0 medium and shoot induction medium (np sim) added with kanamycin as the selection agent. the concentration of kanamycin added to the media was 200 ppm. observations were made three times at the subculture to determine the increment of resistance to kanamycin at each subculture. the subculture in those two media was observed for 12 weeks. duration for three observations at the two subcultures was 36 weeks or 9 months. the usage of 200 ppm kanamycin concentration was based on research conducted by yu et al. 2001 who was successful in making ( ) selection to gain putative transgenic in orchidaceous plants, madame thong-dendrobium in. confirmation of knat1 gene co-integration into p. amabilis genome using pcr techniques the surviving plants in media containing 200 ppm kanamycin selection agent was used in subsequent observations. only plants that have survived in the last subculture were subjected into the confirmation of transgene integration, 35s::knat1. plant dna genome were isolated using ctab 3% methods (saputro et al. 2016; hasan et al. 2012) and then analyzed using pcr. pcr techniques were conducted using three primer pairs i.e. knat1 primers, nptii primers and trnl-f primers. knat1 gene primer pairs, knat1f1 (5'-ccgagaattgcttccg atctg-'3) and knat1r1 (5'-cttgagtt cccgatcttcggc-'3), nptii gene primer pairs, nptii f (5'-cacgacgggcgttcctt gc-'3), nptiir (5'-gtggtcgaatgggca ggtagc-'3), and universal primers for intergenic spacer t rnl-f, t rnl.e (5 ' ggttcaagtccctctatccc-'3) and trnl.f (5'-atttgaactggtgacacgag-'3). pcr were conducted using pcr thermocycler boeco thermal cycler tc-sq. pcr performed with this following system: pre-denaturation at 94 °c for 5 minutes, followed by 30 cycles of denaturation: annealing: extension (94 °c for 1 minute, 55 °c for 1 minute and 72 °c for 2 minutes). the next stage was the post extension at 72 °c for 5 minutes and the final stage was cooling (hold) at 4 °c for 15 minutes. pcr products were then separated with 1% agarose gel electrophoresis, colored using ethidium bromide and visualized under uvtransluminator. multiplication rate of transgenic plant harboring 35s::knat1 onto medium np0 and np sim the subculture plantlet in previous studies was divided based on its performance. plants that were able to show the formation of multiple shoots were used as starting materials. 12 biotropia vol. 25 no. 1, 2018 mixture of 14% glacial acetic acid (ch cooh) : 3 84% ethanol (c h oh) solution. the leaves 2 5 were then incubated overnight at room temperature. the specimens were then dehydrated twice in 70% ethanol (1 hour for each dehydration) and twice in 99.5% ethanol (1 hour for each dehydration). furthermore, the specimen was immersed overnight in a mixture of chloral hydrate clearing solution (c h cl o ). 2 3 3 2 leaf specimens were finally observed under a n eschenbach microscope with 20x magnification & . (berleth jurgen 1993) protein profile analysis of 35s::knat1 transgenic plants using sds-page phalaenopsis amabilis leaves were cut. leaves sample was determined to be 150 mg. the samples were added with 200 ml pbs and finely ground until homogen. all homogenates were collected and then put into a sterile tube. homogenates were centrifuged at 6000g for 5 minutes. the supernatant was transferred into a new sterile tube and stored at 4 °c. protein profile of plants were analyzed using sds-page (holme & peck 1998). results and discussion selection of putative transgenic in medium containing kanamycin the selection process to ensure the candidate of transgenic plants was conducted by growing all candidates in np0 and np sim media containing 200 ppm kanamycin. detail information of the resistance is shown in table 1. the data indicated that the percentage of surviving plants were improved along the subculture process. furthermore, the obtained materials were then planted onto np0 and npsim media containing kanamycin as the selection agent. the concentration of kanamycin added into the media was 200 ppm. subculture process was performed 3 times, 12 weeks each with total duration of 36 weeks or 9 months. growth patterns of transgenic plants harboring 35s ::knat1 the observation of growth patterns were conducted by observing the formation of shoots in transgenic and non-transgenic plants. nontransgenic plants were grown on np0 and np sim media without kanamycin; while transgenic plants were grown on np0 and np sim media containing kanamycin. shoot formation was observed every two weeks to determine the average of shoot formation. observations were made on the last subculture, performed on five non-transgenic and transgenic plants. phenotypic alteration in transgenic plants the population of putative transgenic plants obtained in the last subculture was classified based on their morphological variations. the classification of morphological variation was based on the shape change of plant organs compared with non-transgenic plants. the observation of variation was separated based on grown medium. the effect of knat1 genes into the alteration of venation pattern of transgenic plants the first leaves of non-transgenic and transgenic were fixed in a phalaenopsis amabilis 13 phenotypic and molecular characterization in transgenic phalaenopsis amabilis (l.) blume saputro et al. table 1 resistance level of transgenic p. amabilis and non-transgenic p. amabilis toward kanamycin after 12 weeks observation phase resistance toward kanamycin (%) np0 np sim non transgenic transgenic non transgenic transgenic 8th subculture 0 17.95 0 15.38 9th subculture 0 48.15 0 46.88 10th subculture 0 58.70 0 62.50 the resistance of putative transgenic in media containing kanamycin were caused by expression of selectable marker gene i.e. nptii gene. the increment of plant resistance to kanamycin is shown in table 1. confirmatio of transgene 35s::knat1 co-n integration on the p. amabilis genome using pcr technique trnl-f amplicon was produced in all lines (fig. 1). line a and b were the example of control (non-transgenic plants). line c and d were transgenic plants having only nptii gene attached on its genome. line e and f had three dna fragment i.e. 200 bp (nptii gene), 500 bp s (trnl-f intergenic spacer) and 616 bp of knat1 gene. t hese plants considered as putative transgenic ran. the putative t sgenic having those three bands was determined as positive transformant. d percentage of positive etail transformant is show in table 2.n wasthe co-integration percentage shown in table 2. the non-transgenic plants were used as a control an show zero percentage of co-d ed integration. the highest percentage 93 33% of . were obtained by putative transgenic grown in np sim. utative co-integration percentage of p transgenic planted onto np0 medium only was 85 50%. . 14 biotropia vol. 25 no. 1, 2018 fedcbam mfedcbam m figure 1 electrophoregrams of pcr result: m = gene ruler of dna ladder mix; a-b = non-transgenic of p. amabilis; c-d = fragment of nptii gene; e-f = positive transgenic table 2 co-integration percentage of 35::knat1 transgene into p. amabilis chromosomes plants medium genotype character co-integration percentage (%) trnl-f trnl-f, nptii trnl-f, nptii, knat1 non transgenic np0 10 0 0 0/10 (0.00%) np sim 10 0 0 0/10 (0.00%) putative transgenic np0 40 7 33 33/40 (82.50%) np sim 30 2 28 28/30 (93.33%) multiplication rate of transgenic plant 35s::knat1 onto np0 and npsim media shoot apical meristem (sam) requires a high concentration of cytokinin and low concentration of gibberellin to maintain its growth and meristematic function to continuously produce indeterminate organs. this condition can be achieved by introducing knat1 gene. results of this study showed that transgenic plants remained thto have high stability in the 10 subculture. the stabilizing function of transgen in transformant can be observed by its ability to produce multi shoots. the multiplication rate of p. amabilis (transgenic or non-transgenic) was relatively higher when planted onto sim medium compared to np0 medium. the amount of shoots formation is shown in table 3. np sim medium provided higher shoot formation compared to np0 medium. transgenic plants also provided higher shoots formation compared to non-transgenic plants. transformant planted in np0 medium showed 398% in multiplication rate, while transformant planted in np sim showed higher rate (567%). those results indicated that cytokinin-added medium was more suitable to provide large amount of positive transformant. statistical analysis showed that np sim medium was significantly provided higher shoot formation compared to np0 medium. transgenic plants also provided higher shoots formation compared to non-transgenic plants. furthermore, the combination of those two factors did not show any interactions in increasing the shoots formation. growth pattern in transgenic plant harboring 35s::knat1 plants inserted with 35s::knat1 produced earlier shoots compared to the non-transgenic plants (table 4). in transgenic plant, the shoots were already formed in four weeks after planting, while the non-transgenic plants started to produce shoot in six weeks after planting. in np sim medium, the addition of new shoots was higher than in np0 medium. 15 phenotypic and molecular characterization in transgenic phalaenopsis amabilis (l.) blume saputro et al. table 3 shoot formation of p. amabilis within three subcultures plants medium number of planted explants after number of dead plants number of surviving plants number of shoots multiplication rate (%) 8th subculture non-transgenic np0 30 0 30 30 100.00 np sim 30 0 30 49 163.33 putative transgenic np0+ kanamycin 39 32 7 27 385.71 np sim+ kanamycin 39 33 6 32 533.33 9th subculture non-transgenic np0 30 0 30 30 100.00 np sim 30 0 30 62 206.67 putative transgenic np0+ kanamycin 27 14 13 54 415.38 np sim+ kanamycin 32 17 15 83 553.33 10th subculture non-transgenic np0 30 0 30 33 110.00 np sim 30 0 30 53 176.67 putative transgenic np0+ kanamycin 46 19 27 106 392.59 np sim+ kanamycin 24 9 15 74 493.33 16 biotropia vol. 25 no. 1, 2018 knat1 gene was isolated from arabidopsis thaliana plant. knat1 gene is expressed in shoot apical meristem (jackson et al. 1994) which is the indeterminate organ. knat1 genes are down regulated when leaf initiation process occurrs. these expression pattern describes the important role of knat1 gene to maintain an indeterminate form of meristem and repressing the differentiation process (byrne et al. 2000). knat1 gene was inserted in p. amabilis under the control of 35s strong promoter from cauliflower mozaic virus (camv) causing the occurrence of over-expression of knat1 gene. over-expression of knat1 gene in arabidopsis was known to cause the formation of new shoots on upper and lower surfaces of arabidopsis leaves, an alternation of the normal leaf shapes into lobed shape (lincoln 1994; et al. chuck 1996) as well as in tobacco plants et al. (nishimura 2000). insertion of knat1 et al. gene into was expected for further p. amabilis observation about the function of knat1 gene and its influence on the shoot growth. the multiplication level of shoots is shown in figure 2. there was only one shoot formed in nontransgenic plants, while the transgenic plants were able to produce multishoots. phenotypic alteration in transgenic plants over-expression of knat1 gene leads to many changes in plant . over-phenotype expression of knat1 gene causes leaf alteration, ectopic meristems, inflorescence-like structures form , , ation dwarfishness apical dominance loss and generation of meristematic tissues in leaf ; (williams-carrier et al. 1997 sun et al. 2011). the usage of knat1 gene as a selectable marker was successfully conducted by luo (200 ).et al. 6 in this study, an interesting phenomenon was also found i.e. six alterations of plant morphology occurred in plants inserted with knat1 gene. those six alternations were table 4 shoot formation of p. amabilis plants medium numbe r of shoots number of new shoots in weeks after planting 0 2 4 6 8 10 non-transgenic np0 5 0 0 0 0 0 0 np sim 5 0 0 0 1 1 1 transgenic np0+kanamycin 5 0 0 1 2 2 3 np sim+kanamycin 5 0 0 2 3 4 4 2 1084 6 12 16 a b 2 1084 6 12 162 1084 6 12 16 a b figure 2 growth pattern in shoot formation of phalaenopsis amabilis 16 weeks after planting: a. non-transgenic plants; b. transgenic plants using 35s::knat1 (scale for 2 6 weeks = 1 mm; scale for 8 16 weeks = 0.5 cm) 17 normal leaves, lobed leaves, rosette, elongated stem, cup-shoot and widened leaves. in this study, the normal leaves was the most abundant variants found. it is important to obtain information whether the alterations are inherited in p. amabilis progenies. in lettuce plants (frugis et al. 2001 ) and celosia plumosus (sun et al. 2011), the harboring knat1 gene were reported to inherit morphological alteration of the first generation of transformant plants (t0). the selfing results showed that 75% of filial (t1) denoting the alteration in the shape of its first leaf, but not changing the percentage of seed germination, the emergence time of the first leaf and its phylotaxis (frugis et al. 2001). all the variations in this research were caused by the over-expression of 35s::knat1 (tabel 5). venation patterns of non-transgenic and transgenic plants were observed to ensure the anatomic performance of leaves. the results showed that the alteration also occurred in the venation of leaf (fig. 3). the alterations were detected from the very early stage of leaf development. the over-expression of knat1 gene are able to disturb hormon concentration in sam leading to imbalance status. the details of alteration occurred every two weeks are shown in figure.3. in lobed leaves variant, the venation pattern was also shown. the results showed that there was a fundamental change concerning the phenotypic and molecular characterization in transgenic phalaenopsis amabilis (l.) blume saputro et al. table 5 percentage of morphological variation in p. amabilis transgenic 35s::knat1 plant phenotype variations number of plants normal (%) lobed leaves (%) rosette (%) elongated stem (%) cup shoot (%) widened leaves (%) np0 106 29.25 11.32 14.15 9.43 19.81 16.04 np sim 74 29.73 13.51 12.16 9.46 29.73 5.41 figure 3 the differentiation of leaf venation patterns observed in 2, 4, 6, 8, 10, and 12 weeks: a. regular (normal) venation of leaf; b. irregular venation (scale = 0.5 cm) 18 biotropia vol. 25 no. 1, 2018 development of leaf vascularization compared to non-transgenic plants. the appearance of lobus in leaf's lamina causes the irregular venation. furthermore, in severe level, the alterations produce the emergence of two mid-vein in one leaf's lamina. protein profile analysis of 35s::knat1 transgenic plants using sds-page materials used in this profiling is the proven materials that carry knat1 gene and represents various types of morphological variants. knat1 protein contains 398 amino acids with 45,835.3 daltons. one hundred percent of tested plants all had bands of 45 kda in size indicating knat1 protein (fig. 4). different intensity was caused by p. amabilis growth phase. co-integration analysis of 35s::knat1 transgen into phalaenopsis amabilis genome the material used in this study is phalaenopsis amabilis orchid which had been transformed with plasmid containing the construction of knat1 cdna gene (knooted-like arabidopsis thaliana 1). transformation of 35s::knat1 on moth orchid mediated by agrobacterium tumafaciens was established semiarti et al (2011b). by . the results of regeneration showed that not all of plants are positive transgenic harboring 35s::knat1 plants in their genome. this can be caused phenomenon by many factors mong others, chimera is the . a main factor causing diversity in plant genome. chimera may occur in the transformation because not the whole plant cells are being infected by agrobacterium tumafaciens during co-cultivation. chimera can be reduced by conducting repetitive subcultures on selection medium. selection marker gene which is often used in the plasmid construction is an antibiotic resistance gene or herbicide resistance gene. the selection marker gene that were used in the plasmid construction in this purpose is an antibiotic resistance gene, nptii (neomycin phosphotransferase) gene. npt gene showed high resistance over kanamycin antibiotic. kanamycin gene expression lead a transgenic p. amabilis to have a survival performance in the medium containing kanamycin, while non transgenic plants could not survive. the surviving plants in antibiotic selection medium were isolated using its genome dna and subjected to pcr. the results obtained from pcr showed that not all surviving plants on selection medium was transgenic plants harboring 35s::knat1, sometimes only harboring nptii gene. plants carry only the antibiotic resistance genes because the plants have defense mechanisms that prevent the introduction of foreign objects. when infected to the plant cells, t-dna was protected by the complex of vire2 and vip1 (vire2 protein interacting). the figure 4 electrophoregrams of p. amabilis protein profile: m = protein marker, broad range, biolabs; nt = non transgenic; a = normal variant of transgenic plants; b = lobed leaves; c = rossete; d = elongated stem; e = widened leaves; f = cup shoot 19 integration process requires t-dna in free condition which means that t-dna does not form a complex with vire2 and vip1. the vire2 and vip1 need to be eliminated first. vire2 protein is required in the t-dna transfer process (rossi et al. 1996). plants activates the f-box proteins associated with the defense i.e. vbf (vip1-f-box binding protein). vbf protein makes the complex vire2 and vip1 becomes unstable through the mechanism of proteosom degradation mediator by scf mediator (skp, cullin, f-box containing complex). the ideal mechanism to eliminate the vire2 and vip protein occurs when t-complex is already approach the target chromatins. on the other hand, when the elimination process occurs just before t-complex approaches the chromatin, the transient expression and t-dna degradation by nuclease and several part of those construction gene are integrated into plants genomes (zaltsman et al 2010).. protein profile of transgenic plants indicates that protein with molecular weight of 45.8 kda exists. this protein is predicted as knat1 protein. the results showed that all transformant plants produce knat1 protein. were able to non-transformant plants u to produce were nable knat1 protein. knat1 protein is a transcription factor for plant growth, so the high amount of knat1 protein changes the hormonal balance in sam region. sam plays a dynamic and pivotal role in guiding plant growth. the disproportion of hormonal concentration may lead to morphological alteration in transformant plant. hormone concentration was regulated in the synthesis process, transport and conversion into a more or less active form (veit 2009). auxin, gibberellin and cytokinin together are known as the hormones having key role in determining plant morphology. auxin and gibberellin are the mandatory hormones mediating the formation of lateral organs or determinate organs. cytokinin has contrary activities and associated with indeterminate growth programs of cells, including maintaining the number of indeterminate cell population in sam. the center region of sam has relatively low concentration of ga caused by high concentration of knat1 protein which represses gene transcription encoding ga20 oxidase (hay et al. 2002). bolduc and hake (2009) concluded that the regulator elements in the near 3' end of first introns of ga2ox1 gene binded by kn1 in maize. knox proteins on tobacco plants, nth15 protein can directly suppress the transcription of the ntc12 gene, which is the gene that encodes ga20-oxidase required in ga biosynthesis (sakamoto et al. 2001). knat1 gene activity in the central zone of sam is able to increase the concentration of cytokinin. the increment of cytokinin due to knox protein is capable to activate isopentenyl transferase genes. isopentenyl transferase involves in catalyzing a step in the cytokinin biosynthesis. higuchi et al. (2004) reported that the -over expression of gene that encoding c cytokinins ytokinins oxidase can decrease concentrations in cells. this condition leads the to reduction of meristem size and is able to initiate the formation of leaf r . and othe tissues on the other hand, in several cases, the decrement of endogenic s vel causcytokinin le es meristem abortion (werner et al. 2003). knox protein is the main regulators controlling hormonal balance, either at dicotyls or monocotyls plants (sakamoto et al. 2006; hay et al. 2002). conclusions 35s::knat1 transgene was consistently integrated with phalaenopsis amabilis genome until ththe 10 subculture. there were many alterations in plant morphology classified into six main groups: normal, lobed leaves, rossete, elongated stem, cup shoot and widened leaves. the plant morphology was dominated by normal variant. all transgenic plants harboring 35s::knat1 produced protein with molecular weight of 45.8 kda, equivalent with knat1 protein size. it is important to have confirmation on amino acids sequence to ensure whether it is the knat1 protein. it is suggested that further confirmation is required for transgene expression at the mrna to determine the level of transcription in every transgenic plant lines harboring 35s:: knat1. references berleth t, jürgens g. 1993. the role of the monopteros gene in organising the basal body region of the arabidopsis embryo. development 118:575-87. phenotypic and molecular characterization in transgenic phalaenopsis amabilis (l.) blume saputro et al. bolduc n, hake s. 2009. the maize transcription factor knotted1 directly regulates the gibberellin catabolism gene ga2ox1. the plant cell preview: 1-12. byrne me, barley r, curtis m, arroyo jm, dunham m, hudson a, martienssen ra. 2000 asymmetric . leaves1 mediates leaf patterning and stem cell function in . nature 408 967-71.arabidopsis : chuck g, lincoln c, hake s. 1996. knat1 induced lobed leaves with ectopic meristem when overexpressed in 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kojima m, yamamato y, nagasaki h, inukai y, ... matsuoka m. 2006. ectopic expression of knotted1-like homeobox protein induces expression of cytokinin biosynthesis genes in rice. plant physiol 142:54-62. saputro tb, finariyah f, dianawati s, sholihah nf, ermavitalini d. 2016. in vitro election of ocal s l m s gaize (zea mays) on nacl tress and its enetic characterization using rapd. biosaintifika 8(3) 344-51.: semiarti e, indrianto a, purwantoro a, isminingsih s, suseno n, ishikawa t, … machida c. 2007. agrobacterium-mediated transformation of the wild orchid species plant biotechnol phalaenopsis amabilis. 24:265-72. semiarti e, indrianto a, purwantoro a, machida y, machida c. 2011a. agrobacteriumediated m t oransformation of indonesian rchids for m g t . in: icropropagation, enetic ransformation a l v a r e z av a i l a b l e f r o m : m , e d i t o r . http://www.intechopen.com/books/genetictransfor mat ion/ag robacter 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gamba. werner t, motyka v, laucou v, smets r, van onckelen h, schmulling t. 2003. cytokinin-deficient transgenic arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. plant cell 15:2532–50. williams-carrier re, lie ys, hake s, lemaux pg. 1997. ectopic expression of the maize kn1 gene phenocopies the ooded mutant of barley. h development 124 3737-45: . yu h, yang sh, goh cj. 2001. mediated agrobacteriumtransformation of a with class1 dendrobium orchid knox gene doh1. plant cell rep 20:301-5. zaltsman a, krichevsky a, kozlovsky sv, yasmin f, citovsky v. 2010. plant defense pathways subverted by for genetic transformation. plant agrobacterium signal behav 5:1245-8. 21 phenotypic and molecular characterization in transgenic phalaenopsis amabilis (l.) blume saputro et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 6. dyah (testing) revisi.cdr biotropia vol. 21 no. 2, 2014: 125 130 testing reliability of serum samples as a dna source on captive breeding longtailed macaques ( )macaca fascicularis dyah perwitasari-farajallah and achmad farajallah received 24 may 2013/accepted 14 september 2014 the serum reliability was tested in captive breeding conditions on the long-tailed macaques ( ). serum and buffy coat were applied to standard protocol of dna extraction following the amplification of microsatellite dna locus of d2s1777 by polymerase chain reaction (pcr) and visualized pcr products by means of polyacrylamide gel electrophoresis (page) with silver staining. the amount of genomic dna extracted from serum was sufficient for genotyping individuals at dna microsatellite locus of d2s1777 with allele size of approximately 160 bp. the study showed that serum could provide reliable alternative for obtaining dna when taking blood using anticoagulant was impracticable. serum, dna source, captive breeding, monkeys 1,2* 1 1 department of biology, faculty of mathematics and natural sciences, bogor agricultural university, bogor 16680, indonesia primate research center, bogor agricultural university, bogor 16151, indonesia macaca fascicularis 2 abstract introduction keywords: primates play an essential role in biomedical research. the study of primates has also contributed to our understanding of basic biological phenomena such as reproduction and several diseases, to the development of drugs, and vaccines (nathanson & maithieson 2000; sibal & samson 2001). as in other model organisms, genetic background in primates such as long-tailed ( ) and rhesus macaques ( ) is an experimental variable that affects the response of other study variables (stevison & kohn 2008; hernandez . 2007). a standardized system for genetic testing would be beneficial for the management of breeding colonies and also facilitate the characterization of other important traits, including mhc type, resistant to simian immunodeficiency virus (siv) and the susceptibility to many human pathogens (satkoski . 2008). moreover, data on reference of hematologic and serum parameters are essential for macaca fascicularis m. mulatta et al et al * corresponding author : witafar@yahoo.com doi: 10.11598/btb.2014.21.2.6 125 evaluating results obtained from laboratory animals and this baseline biological data are needed before they are used for scientific purposes (bonfanti . 2009). in fact, to fulfill the entire criteria mentioned, the analyses relied on the availability of blood samples. upon the purposes of studies, ordinarily blood samples are taken from the animals without anticoagulant or with anticoagulant, such as heparine and sodium citrate (especially for genetic characterization). however, when obtaining blood samples with anticoagulant is impracticable for some reasons, available samples particularly serum or clot samples will be an alternative samples to be utilized. the lack of published description for detailed protocol on serum application, however, facilitated the remarkably approach for genetic analyses of captive breeding monkeys. as a result, our study aimed to examine the applicability and reliability of serum samples as a dna source. blood samples were collected in conjunction with routine health physical examinations of individuals in captive breeding population of long-tailed macaques ( ) housed at primate research center, bogor agricultural university. two kinds of blood samples were available for analyses: one using heparin as anticoagulant and the other without heparin. blood with anticoagulant were centrifuged at 3,000 rpm for 15 minutes. erythrocyte and plasma were stored at -20 c for other purposes, and buffy coat was directly applied to dna extraction protocol. along with processing blood with anticoagulant, serum was obtained after samples were allowed to clot. in total 38 sera and buffy coat were used as dna sources. dna extraction was carried out following the method of kan . (1977) with minor modification for serum and buffy coat separately to avoid potential contamination. serum or buffy coat was washed with 0.9% nacl1mm edta. centrifugation was performed at 2,000 rpm for 10 minutes. supernatant was discarded and pellet was suspended in 2.5 ml ste buffer (salted tris-edta), 200 μl 10% sds and digested with 40 microl proteinase k (5 mg/ml) for a minimum of 2 hours at 37 c. then genomic dna was extracted with standard phenol-chloroform method. as dna microsatellites are the most common neutral nuclear marker applied in genetic diversity studies, we tested microsatellite amplification to validate amplification success from the tested sources. the test was based on amplification of d2s1777 microsatellite marker, a monomorphic dna microsatellite locus on genetic study of long-tailed and pig-tailed macaques (perwitasari-farajallah . 2007; perwitasari-farajallah 2010). allele size in contemporary long-tailed macaques was 160 bp. pcr was conducted in a ptc 100 mj research inc. in 12.5 μl reaction volumes using pcr buffer (containing 25 mm mgcl l 2.5 mm dntp. each reaction contained 25 pm of each primer flanking the microsatellite region (f:5'-tccccaagtaaagcattgag-3';r:5'et al macaca fascicularis et al et al et al. materials and methods o o + 2),1.0u/μ taq polimerase (promega), 10xpcr buffer, 126 biotropia vol. 21 no. 2, 2014 gtatgtaggtagggaggcagg-3') and approximately 10-100 ng of dna. after 3 minutes initial denaturation at 94 c, pcr was performed in a total of 30 cycles using the following conditions: 30 seconds denaturation at 94 c, 60 seconds annealing at 48 c, and 60 seconds elongation at 70 c (perwitasari-farajallah . 2004). with a final elongation of 5 minutes at 72 c, pcr was terminated. strict precautions against pcr contamination were performed. separate area were utilized for the preparation of amplification reactions, the addition of dna template, and the carrying out of amplification reactions. negative water pcr blanks were included in every analysis. two microliters of each pcr products were electrophoresed on 6% polyacrylamide gels and visualized by silver-staining following the technique described by tegelström (1986). allele sizes were verified using dna size standard of 100 bp ladder (biorad). dna was successfully extracted from almost all the samples tested. seventy four percent of the serum samples ( = 15) and 100% of the buffy coat ( = 19) were successfully amplified and gave strong amplification products (fig.1 & 2). it revealed that dna derived from buffy coat samples have sufficient dna for repeats genotyping attempts. in contrast, insufficient dna yielded in serum samples may in part be attributed to inadequate amount of nucleated cell inside the samples. o o o o o et al n n results and discussion figure 1. amplification of the d2s1777 microsatellite locus from ten long-tailed macaques serum and buffy coat. m: size standard, 100-bp dna ladder; lanes 1-5, dna extracted from serum; lanes 6-10, dna extracted from buffy coat. 127 testing reliability of serum samples as a dna source on captive breeding dyah perwitasari– et al. dna size standard of 100 bp (biorad) and pcr products on 6% page stained with silver demonstrated that the concentration of the pcr products of isolated dna from serum samples and from buffy coat were lower and higher compared to the dna size standard, respectively. although the dna extracted from serum samples yielded low concentration of pcr product, we conceived that it revealed reliable results for further analyses. present study demonstrated that d2s1777 locus revealed to be monomorphic (fig.1&2) as detected in our previous research with the allele size of approximately 160 bp. although false alleles or amplification artefact are typically not only of low frequency, but also sporadic in occurrence, it cannot be disregarded as a potential source of error in microsatellite genotyping (taberlet 1999). despite report by fernandes . (2007) that the use of species-specific primers reduces the risk of false species assignment; the results obtained in this study consisted of apparent allele repeatedly amplified from several samples. moreover, the d2s1777 is a humanderived microsatellite locus used in our laboratory for the routine genotyping of longtailed macaques ( ) using buffy coat as dna source (perwitasari-farajallah . 2010; perwitasari-farajallah 2007). many genotyping studies depend on cross-species amplification, that is, the application of primers derived in one species for characterization of individuals in another, usually closely related species (bradley . 2008). therefore, a variety of strategies to determine dna quality and detect or reduce genotyping errors should be determined (paetkau 2003). utilization of serum samples was described previously by jiminez and tarantal (2003). they showed that fetal gender can be reliably determined in the early first trimester from maternal serum samples of rhesus macaques ( ) by real-time pcr. they adapted and applied the idea of some researchers (lo . 1997, 1998; costa . 2001; honda . 2002) regarding fetal gender determination in human by et al. et al m. fascicularis et al et al. et al m. mulatta et al et al et al figure 2. amplification of the d2s1777 microsatellite locus from ten long-tailed macaques serum and buffy coat. size standard, 100-bp dna ladder; lanes 1-4, dna extracted from serum; lanes 5-12, dna extracted from buffy coat. s = serum; bc = buffy coat 128 biotropia vol. 21 no. 2, 2014 real-time pcr analysis of maternal serum samples. even though they used serum for different purposes if compared to what we performed in our study, it appeared to be all of them obtained satisfactory results. our study provided, probably (to our knowledge), the first description of serum applicability as a source of dna for future genetic studies of long-tailed macaques captive breeding population when obtaining blood samples using anticoagulant is impracticable. we were grateful to primate research center, bogor agricultural university for making available samples and to division of animal systematic and ecology, department of biology, bogor agricultural university for laboratory assistance. drs randall c. kyes (university of washington, seattle, usa) and betsy ferguson (oregon health and science university, usa) provided important comments and suggestions. we thanked anonymous reviewers for helpful suggestions to improve this manuscript. conclusions acknowledgements references bradley b, doran-sheehy d, vigilant l. 2008. genetic identification of elusive animals: re-evaluating tracking and nesting data for wild western gorillas. j zool 275:333-40. bonfanti u, lamparelli d, colombo p, bernardi c. 2009. hematology and serum chemistry parameters in juvenile cynomolgus monkeys ( ) of mauritius origin: comparison between purpose-bred and captured animals. j med primatol 38:228-35. costa jm, benachi a, gautier e, jouannic jm, ernault p, dumez y. 2001. first-trimester fetal sex determination in maternal serum using real-time pcr. prenatal diagnostic 21:1070-4. fernandes ca, ginja c, pereira i, tenreiro r, bruford mw, santos-reis m. 2007. species-specific mitochondrial dna markers for identification of non-invasive samples from sympatric carnivores in the iberian peninsula. 9:681-90. hernandez rd, hubisz mj, wheeler da, smith dg, ferguson b, rogers j. 2007. demographic histories and patterns of linkage disequilibrium in chinese and indian rhesus macaques. science 316:240-3. honda h, miharu n, ohashi y, samura o, kinutani m, hara t, ohama k. 2002. fetal gender determination in early pregnancy through qualitative and quantitative analysis of fetal dna in maternal serum. human genetic 110:75-9. jimenez df, tarantal af. 2003. fetal gender determination in early first trimester pregnancies of rhesus monkeys ( ) by fluorescent pcr analysis of maternal serum. j med primatol 32:315-9. kan yw, dozy am, trecartin r, todd d. 1977. identification of a nondeletion defect in thalassemia. n engl j med 297:1081-4. macaca fascicularis conservation genetics macaca mulatta 129 testing reliability of serum samples as a dna source on captive breeding dyah perwitasari– et al. lo ym, corbetta n, chamberlain pf, rai v, sargent il, redman cw, wainscoat js. 1997. presence of fetal dna in maternal plasma and serum. lancet 350:485-7. lo ym, tein ms, lau tk, haines cj, leung tn, poon pm, wainscoat js, johnson pj, chang am, hjelm hm. 1998. quantitative analysis of fetal dna in maternal plasma and serum: implications for noninvasive prenatal diagnosis. am j human genetic 62:768-75. nathanson n, maithieson bj. 2000. biological consideration in the development of a human immunodeficiency virus vaccine. j infec dis 182:579-89. paetkau d. 2003. an empirical exploration of data quality in dna-based population inventories. mol ecol 12:1375-87. perwitasari-farajallah d, kyes rc, iskandar e. 2010. microsatellite dna polymorphisms for colony management of long-tailed macaques ( ) population on the tinjil island. biodiversitas 11:55-8. perwitasari-farajallah d. 2007. human short tandem repeat (str) markers for paternity testing in pig-tailed macaques. hayati 14:39-43. perwitasari-farajallah d, farajallah a, kyes rc, sajuthi d, iskandriati d, iskandar e. 2004. genetic variability in a population of long-tailed macaques ( ) introduced onto tinjil island, indonesia: microsatellite loci variations. hayati 11: 214. satkoski j, george d, smith dg, kanthaswamy s. 2008. genetic characterization of wild and captive rhesus macaques in china. j med primatol 37:67-80. sibal lr, samson kj. 2001. nonhuman primates: a critical role in current disease research. ilar journal 42:7484. stevison ls, kohn m.h. 2008. determining genetic background in captive stocks of cynomolgus macaques ( ). j med primatol 37:311-7. taberlet, p, waits lp, luikart g. 1999. noninvasive genetic sampling: look before you leap. trends in ecol evol 14 323-7. tegelström, h. 1986. mitochondrial dna in natural populations: an improved routine for the screening of genetic variation based on sensitive silver staining. electrophoresis 7:226-9. macaca fascicularis macaca fascicularis macaca fascicularis : 130 biotropia vol. 21 no. 2, 2014 1054-fiks.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 doi: 10.11598/btb.2015.22.2.367 effect of substrate moisture and invasive grass competition on native fig ( ) seedling ficus fistulosa recruitment in limestone quarries annisa satyanti* center for plant conservation, bogor botanical garden, indonesian institute of sciences, bogor 16003, indonesia received 10 february 2014 /accepted 4 february 2015 abstract this study was conducted to determine the possibility of native fig ( ) for rehabilitating degraded ficus fistulosa habitats in limestone quarries. a greenhouse experiment was carried out to test the effect of different substrate moisture levels and competition between native fig ( ) and invasive grass on the ficus fistulosa pennisetum polystachyon native fig's growth and survival. was chosen as the studied species because it has high importance value ficus fistulosa index (ivi) compared to other fig species tested in ciampea limestone hill. the results showed that the substrate moisture levels did not affect the native fig biomass and the invasive grass biomass was not reduced by drought. the interaction of substrate moisture levels and competition from invasive grass reduced overall fig biomass, but not the leaf numbers and individual plant size. this study suggested that for quarry rehabilitation, invasive species management should be advocated along with soil treatment. keywords:ficus fistulosa competition , quarried limestone, rehabilitation, substrate moisture introduction limestone regions over the world along with their biodiversity are threatened mainly by limestone quarrying causing land degradation problems due to soil depletion and alteration of land topography. vegetation removal and lack of available soil on steep slopes induce a very high risk of erosion in limestone ecosystems (clemente . 2004). the common method of et al limestone quarrying increases drainage, physical and chemical erosion of the substrate which hinder natural germination and establishment of seedlings, thus delaying re-colonization (clemente et al. 2004). during the rainy season, water run-off is high, while the open sites are easy to dry up. plant recruitment in newly disturbed areas, like in an over-quarried limestone, is determined by water availability and soil moisture (soliveres 2012). the large number of abandoned quarries in many countries presents challenges for restoration of these degraded habitats (yuan . 2006). et al revegetation is among common strategies to restore the abandoned quarries (zhang chu & 2011). limestone quarries revegetation is challenging because not all plants are able to cope with relatively barren rock cliff and absence of topsoil or humus. substrate moisture is the most limiting environment variable influencing seedling emergence and survival, particularly in arid ecosystems (soliveres 2012), such as of these abandoned limestone quarries. in addition to water availability, competition against fast growing invader such as exotic grass can be critical during the seedling stage of the desired native plants. understanding factors that restrict the establishment and growth of native plant species can aid efforts for species conservation in their habitats and can expand options for species recovery, including restoration, translocation and ex-situ et al. conservation (maschinski 2004). native plant species is favoured for rehabilitating disturbed ecosystems (khater et al. 2009). is potential for limestone quarries ficus rehabilitation. in the abandoned limestone quarries in ciampea, bogor, west java, indonesia, * corresponding author : annisa.satyanti@lipi.go.id biotropia 2 95 101 vol. 22 no. , 2015: 95 mailto:annisa.satyanti@lipi.go.id 96 ficus is abundant in the remaining limestone forests (satyanti & kusuma 2010). previous study conducted by satyanti and kusuma (2010) ranked fourteen species ficus from ciampea limestone hill, i.e. , ficus annulata f. fistulosa f. grossularoides f. hirta f. montana f. , , , , pinnata f. sagittata f. septica , , and six other unidentified species based on their ficus importance value index (ivi). was ficus fistulosa found to have the highest ivi value in the limestone habitat in ciampea (satyanti & kusuma 2010) and hence, it is selected as the studied species in the current experiment. this study was conducted to determine the possibility of native fig ( ) for ficus fistulosa rehabilitating degraded habitats in limestone quarries. a greenhouse experiment was carried out to test the effect of different substrate moisture levels and competition between native fig ( ) and invasive grass ficus fistulosa pennisetum polystachyon .on the native fig's growth and survival materials and methods study site the limestone site in ciampea is a long coral reef raised to above 350 m above sea level, located at 106°41'00.0" e and 06°33'00.0" s. the formation of limestone is of the bojongmanik formation which is equal to a middle mioscene age (effendi . 1998). the forest used to have et al n u m e r o u s d i p t e r o c a r p u s h a s s e l t i i (dipterocarpaceae), stelechocar pus burahol (annonaceae) and a number of diospyros (ebenaceae), but no single species was dominant (van steenis 1931 in whitten & soeriaatmadja 1996). study species native fig reinw. ex. blume ( )ficus fistulosa common yellow stem fig (moraceae) is an evergreen tree and grows to about 10–15 cm in trunk diameter. it is native to malesia floristic region, including indonesia. it has a straight trunk with smooth bark ranging from light grey to yellowish in colour. the young twigs are hollow and easily breaks. leaves of this tree are alternately arranged, the young leaves are pale pink in colour. figs are pear shaped ±2.5 cm wide, borne on 2.5–5 cm long stalks and in cluster on woody knobs on the trunk and branches. figs ripened from yellow to green yellow. the flowers are found within the figs; with male and female flowers located at different trees. individual trees bears 4–7 crops a year. in java and borneo, fig is pollinated by fig wasps, and ceratosolen constrictus seed dispersal is mainly carried out by bats (genus dyacopterus ficus fistulosa). does not have aggressive roots like other strangler . hence, it is an ficus excellent tree to attract wildlife back to both native habitat and urban environment. it is also ethno-botanically important; in some parts of indonesia the young leaves are eaten as salads. a decoction of the leaves is given to women after childbirth and the latex has been used to treat headache (corner 1988). invasive grass schult )( ( )l.pennisetum polystachyon pennisetum polystachyon or widely known as mission grass is native to tropical africa. it is an annual or perennial plant; the culm is simple or branched; the branches are often flowering. spikelet length is 3–5 mm; false spike is 8–10 mm, excluding the bristle; the longest bristle is 15–55 cm long. when mature the spikelets break off at the control axis together with the bristles. it produces few tillers per plant. the distribution of this grass is throughout the tropics up to 1,500 m asl (above sea level). this grass requires high rainfall, but it is also grown in semi-arid regions. it tolerates both acid and alkaline soils. pennisetum polystachyon spreads readily by seeds which survive annual burning. seedling vigour is good, even in poor soil conditions (clayton . 2006).et al in indonesia it is considered invasive species (tjitrosoedirdjo 2005). seed germination of the native fig and the invasive grass fruits of the native fig and panicles of the invasive grass were collected from the limestone quarry in ciampea, bogor-west java. the limestone hill in this site is a long coral reef raised to above 350 m asl and has been quarried for more than thirty years. (dipterodipterocarpus hasseltii carpaceae), (annonaceae) stelechocarpus burahol and a number of (ebenaceae) species diospyros were used to be abundant in this limestone forest, but no single species was dominant (van steenis 1931 in whitten & soeriaatmadja 1996). the biotropia vol. 22 no. 2, 2015 e o s m a i g c o n fffect f ubstrate oisture nd nvasive rass ompetition n ative ig satyanti – above mentioned species are now infrequently present in this habitat. ficus fistulosathe fruits of and panicles of the grass and limestone substrate were subsequently brought to the greenhouse of the bogor botanical garden, where further experiment was conducted. the fruits of the native fig were then cut open and seeds were collected. seeds were then air dried for 1–2 hours prior to sowing. seeds were sown on sand in a tray. after 10–14 days seeds of the native fig were germinated and were kept to grow in the sowing tray before being transplanted to limestone substrate for competition experiment. in parallel, panicles of the invasive grass were sown on sand in different sowing tray. after 3–4 weeks, seeds were germinated and grass seedlings were kept until leaf sheaths reached 5–10 cm length or about a week after sowing. fig and grass seedlings were subsequently transplanted to the treatment pots and watered until substrates were fully saturated and substrate moisture treatment was applied from the next watering top-up. competition experiment factors involved were substrate moisture and competition. substrate moisture treatments were 34 ml/week and 68 ml/week, inducing water stress (drought) for the low moisture treatment. this water volume was chosen based on personal observation that 68 ml/week was able to keep the substrate well saturated and the surface was adequately moist. each pot contained limestone substrate weighing 250 g. limestone substrates were collected from the quarried in ciampea. the substrate from these abandoned quarries was in the form of a mixture of coarse and fine gravel of limestone. two levels of competition were employed, i.e. with and without grass competition. in a competition pot, one seedling of native fig was planted with two seedlings of invasive grass. one-month-old native fig seedlings with 2–3 true leaves, ±2–3 cm tall were transplanted to limestone substrates in 9 cm (diameter) and 12 cm (depth) free draining pots for competition experiment. invasive grass seedlings were transplanted to competition pots at the same time. the experiment set–up was a factorial block design assigning each treatment and its combination within each block. for each treatment combination, six replicates were used and all were arranged in five blocks. thus, each block consisted of 24 pots, in which six pots of low soil moisture and no competition, six pots of low soil moisture and with competition, six pots of high soil moisture and no competition, and six pots of high soil moisture and with competition, leading to a total number of 120 pots. the competition experiment were maintained and observed for eight months. at the end of the experiment, measurements were made for native fig seedlings' height, leaf number and biomass. grass above and belowground biomass were also measured. data analysis in order to disentangle the effect of substrate moisture and competition on native fig seedling growth, a two-way anova was carried out. in addition, the interactions between substrate moisture and competition against invasive grass were also tested to determine whether or not those two factors affect the growth of native fig. for competition pot, invasive grass aboveand below-ground biomass were also tested against substrate moisture. subsequently, correlation between the native fig and invasive grass biomass was analysed. all calculations were performed using software spss ver. 15.0. results and discussion substrate moisture effect on native fig and invasive grass growth of native fig during the eight month observation was relatively slow period considering their nature as a woody tree growing in tropical climate. watering at 68 ml/pot/week significantly reduced native fig biomass compared to the 34 ml/pot/week but the effect of substrate moisture on native fig plant size and leaf number was on the other hand benign (fig. 1). grass biomass, however, was independent from the substrate moisture suggesting that invasive grass might be quite resistant to drought or water availability fluctuation (fig 2). . 97 biotropia vol. 22 no. 2, 2015 98 34 ml/ week 68 ml/ week substrate moisture 0 1 2 3 34 ml/ week 68 ml/ week substrate moisture 0 1 2 3 34 ml/ week 68 ml/ week substrate moisture 0 1 2 3 34 ml/ week 68 ml/ week substrate moisture 2 4 6 8 fig ure 1 p 38.537 173.251 native fig preferred lower water regime between competition treatments (f = 462.02, = 0.16 and f = 250.605, for aboveand below-ground, respectively). there is no effect of substrate moisture on fig seedling length and leaf number between competition treatments 34 ml/ week 68 ml/ week substrate moisture 0,0 0,2 0,4 0,6 34 ml/ week 68 ml/ week substrate moisture 0,0 0,2 0,4 0,6 fig ure aboveand below-ground grass biomass were not affected by the substrate moisture2 99 even though the pots were freely drained, the bulky and clayish nature of the limestone substrate may lead to biomass reduction of the native fig. , in general, tends to grow better in ficus aerated substrate and intolerant to submersion, even temporarily (parolin & wittmann 2010). there was no significant effect of substrate moisture to aboveand below-ground ficus biomass when grass was present. however, there was a significant reduction in biomass under higher moisture treatment in the absence of grass (fig. 3). effect of presence of invasive grass on native fig native fig aboveand below-ground biomass, leaf number and seedling length were reduced because of the presence of invasive grass (fig. 3 and 4). from the competition point of view, invasive grass contribution in suppressing native fig growth can either be from water and nutrients competition or light competition. moreover, at the end of the experiment seedlings height ficus were mostly exceeded by grass leaf sheaths. however, there was no correlation between native fig and invasive grass growth (table 1). it might be that other variables that were not measured had more influence. for instance, allelopathy from exotic grass had more influence on native fig biomass than merely moisture and neighbour growth. grass roots are known to have pennisetum allelopathic chemicals (zain 2013) and et al. adversely affects (tan . 2012), cyperus indica et al leptochloa chinensis hedyotis verticillata and (norhafizah . 2012). is, on the other et al ficus hand, known to have phytotoxic effect on grasses (siddiqui . 2009).et al figure 3 invasive g rass significantly reduced native fig aboveand below-ground biomass (f = 592.572, = 0.000 and 49.353 p f = 322.514, = 0.000, respectively). both aboveand below-ground biomass were very small at the presence 222,965 p of grass. there was a significant interaction effect between competition and substrate moisture to fig aboveground (f = 469.997; = 0.000) and below-ground biomass (f = 252.918; = 0.000). for both aboveand 39.144 174.851 p p below-ground biomass, there was no significant effect of substrate moisture when grass was present, while there was a significant reduction in biomass under higher moisture treatment in the absence of grass table 1 significance value for relationship between biomass biomass r2 p significance grass below-ground – fig below-ground biomass 0.010 0.436 ns grass below-ground – fig above-ground biomass 0.002 0.769 ns grass above-ground – fig above-ground biomass 0.001 0.770 ns grass above-ground – fig below-ground biomass 0.009 0.477 ns e o s m a i g c o n fffect f ubstrate oisture nd nvasive rass ompetition n ative ig satyanti – 34 ml/ week 68 ml/ week substrate moisture 34 ml/ week 68 ml/ week substrate moisture 100 biotropia vol. 22 no. 2, 2015 interaction effect of substrate moisture and competition to fig growth and survival it was apparent that the interaction of substrate moisture and presence of competition from grass reduced biomass of fig above-ground and below-ground (fig. 3). there was no significant interaction effect on fig seedling size and leaf number even though it was evident that fig seedling leaf was reduced under the presence of grass (fig. 4). in general, native fig seedling length and number of leaf were not adversely affected by substrate moisture (fig.1), but rather by competition (f = 60.054; = 0.000 and 64.827 p f = 76.056; = 0.000, respectively; fig. 4). our 182.533 p current study showed that the effect of grass competition was larger than the differences in substrate moisture (fig. 1, 3 and 4). under greenhouse conditions, the native fig seeds germinated well on sand. the seedlings were able to grow on limestone substrates without addition of humus, but it was unknown whether seeds could germinate in novel soils under field conditions. until the end of the experiment, all fig seedlings survived regardless their small growth increment. it is not clear whether or not the seedlings will be able to cope further when the experiment is prolonged or when they are transplanted in the field. field trial showed that seedling establishment in limestone quarries varies dramatically with abiotic and biotic factors and it is often difficult to successfully reintroduce the seedling in the field (maschinski 2004).et al. as the native fig species showed very limited growth in this study, one perhaps would argue to use other fast growing species regardless the fact that they are non-indigenous. the use of native rhizobia-symbiosis forming plants can be used to enhance the success rate of limestone revegetation (jha . 1995). nevertheless, the et al use of native plants has many benefits in restoration schemes, such as their high adaptability to environmental stresses and low ecological risk (ballesteros 2012; kirmer et al. et al. 2012). in the field, it is quite challenging to find recognizable number of native fig recruitment in the quarried areas. besides inhospitable soil moisture and surface characteristics (soliveres et a l . 2012) na tura l e st ab l i shment an d reintroductions on unoccupied limestone field has probably been limited by poor natural dispersal. conclusions this study showed that the aboveand belowground native fig biomass was reduced mainly due to competition against invasive grass. the effect of grass was also pronounced to other fig traits, leaf number and seedling size. grass biomass, however, was not affected by substrate moisture. the effect of grass competition was larger than the differences in substrate moisture. f 4 igure competition against grass also significantly reduced the size of seedling and leaf numbers (f = 60.054; = 0.000 64.827 p and f = 76.056; = 0.000, respectively). there was no interaction effect between competition and substrate 182.533 p moisture to fig seedling size 34 ml/ week 68 ml/ week substrate moisture 34 ml/ week 68 ml/ week substrate moisture 101 study on the effect of allelopathy from invasive grass and neighbouring plants in a field experimental set-up as well as other potential native plants for limestone revegetation need to be carried out to develop better management of quarried limestone hill. acknowledgements the study was supported by grant no. 46.06.08 “an impact assessment of limestone quarries on flora diversity in ciampea, bogor” from the rufford foundation, uk. references ballesteros m, cañadas em, foronda a, fernándezondoño e, peñas j, lorite j. 2012. vegetation recovery of gypsum quarries: short-term sowing response to different soil treatments. app veg sci 15: 187-97. clayton wd, vorontsova ms, harman kt, williamson h. [internet]. 2006 onwards. grassbase – the online world grass flora. [cited 2013 april 15]; available from: http:// www.kew.org/data/grasses-db.html clemente as, werner c, máguas c, cabral ms, martinsloução ma, correia o. 2004. restoration of a limestone quarry: effect of soil amendments on the est ab l ishment of nat ive medi te r ranean sclerophyllous shrubs. restoration ecol 12: 20–8. corner ejh. 1988. wayside trees of malaya third edition vol 1 – 2. kuala lumpur (my): malayan nature society. effendi ac, kusnama k, hermanto b. 1998. geological map of bogor quadrangle, java. bandung (id): geological research and development centre. jha pk, nair s, gopinathan mc, babu cr. 1995. suitability of rhizobia-inoculated wild legumes argyrolobium flaccidum astragalus graveolens indigofera gangetica , , and lespedeza stenocarpa in providing a vegetational cover in an unreclaimed limestone quarry. plant soil 177: 139–49. khater c, martin a, maillet j. 2009. spontaneous vegetation dynamics and restoration prospects for limestone quarries in lebanon. appl veg sci 6: 199-204. kirmer a, baasch a, sabine t. 2012. sowing of low and high diversity seed mixtures in ecological restoration of surface mined-land. appl veg sci 15: 198-207. maschinski j, baggs je, sacchi cf. 2004. seedling recruitment and survival of an endangered limestone endemic in its natural habitat and experimental reintroduction sites. am j bot 91 (5): 689–98. norhafizah mz, ismail bs, chuah ts. 2012. herbicidal activity of (napier grass). afr j pennisetum purpureum biotechnol 11(23): 6269–73. parolin p, wittmann f. 2010. struggle in the flood: tree responses to flooding stress in four tropical floodplain systems. aob plants 1-54. satyanti a, kusuma ywc. 2010. ecological study in two quarried limestone karsts hills in bogor west java: vegetation structure and floristic composition. biotropia 17 (2): 115–29. siddiqui s, yadav r, yadav k, wani fa, meghvansi mk, sharma s, jabeen f. 2009. allelopathies potentialities of different concentration of aqueous leaf extracts of some arable tree on germination and radicle growth of var. c-235. glob j cicer arietinum mol sci 4 (2): 91-5. soliveres s, monerris j, cortina j. 2012. irrigation, organic fertilization and species successional stage modulate the response of woody seedlings to herbaceous competition in a semi_arid quarry restoration. appl veg sci 15(2) : 175-86. tan pw, chuah ts, ismail bs. 2012. allelopathic potential effect of on . in: pennisetum purpureum cyperus iria the 2 international conference on environmental nd and agriculture engineering. proceedings: 2012 june 29-june 30; jurong west (sg): iacsit press. p 109–13. tjitrosoedirdjo ss. 2005. inventory of the invasive alien plant species in indonesia. biotropia 25: 60–73. whitten aj , soeriaatmadja rs. 1996. the ecology of java and bali. singapore: periplus editions. yuan jg, fang w, fan l, chen y, wang d-q, yang z-y. 2006. soil formation and vegetation establishment on the cliff face of abandoned quarries in the early stages of natural colonization. restor ecol 14: 349–56. zain nm, yew oh, sahid i, seng ct. 2013. potential of napier grass ( ) extracts as a pennisetum purpureum natural herbicide. pak j bot 45: 2095–100. e o s m a i g c o n fffect f ubstrate oisture nd nvasive rass ompetition n ative ig satyanti – http:// http://www.kew.org/data/grasses-db.html 1. sutomoo (plnat comunity).cdr 6. c hanny wijaya.cdr biotropia vol. 20 no. 1, 2013: 50 71 flavour of papaya ( l.) fruitcarica papaya c. hanny wijaya * and feng chen received 28 august 2012/accepted 18 december 2012 papaya is included in five major tropical fruits of the world after banana, mango, and pineapple. indonesia is one of the leading countries of papaya production after india and brazil. the centre of tropical fruits study (pkbt) at bogor agricultural university (ipb), indonesia, has started a longterm breeding program since 2003 in order to improve the quality of local papayas. fruit exports are being targeted at more specialized consumers, who are either seeking products either raised in more environmentally and health-conscious ways or those with a significant outstanding flavour. flavour continues to be the predominant quality characteristic important for a successful international marketing. to understand the biosynthesis pathways are becoming more important for the flavour industry in recent years, as this could aid in the production of the flavour volatiles in the same manner as the natural biosynthesis. it is also necessary to understand the aroma active compounds and their changes during processing because unexpected changes in aroma may cause a product unmarketable even if other quality factors are acceptable. understanding the changes of aroma active compounds in the papaya fruit, such as loss of desirable flavour and development of offflavour during processing will be very helpful for determining proper processing condition. the flavour composition of papaya fruit is reviewed in this paper. this paper presents an overview of important publications regarding the characteristic features of the biology of the fruits, consumption worldwide, commercial application in food processing, though the review of biogenesis of volatiles is still the main focus. papaya ( l.), flavour, review, volatiles, tropical fruit, biogenesis 1 2 1 2 department of food science and technology , faculty of agricultural technology bogor agricultural university (ipb), bogor 16680, indonesia department of food, nutrition, and packaging sciences, clemson university, clemson, sc 29634, usa carica papaya abstract introduction key words: excellent in vitamin c, pro-vitamin a, minerals (wall 2006) as well as rich in dietary fibre, papaya ( l.) is emerging as a popular fresh fruit which offers health benefiting properties. by the mid-19 century, papaya had spread over the tropical region, from florida, hawaii, india, sri lanka, malaysia, indonesia, to africa (morton & macleod 1990). carica papaya th * corresponding author : hazemi@indo.net.id 50 et al et al et al et al et al et al et al et al wide variability is shown by papaya grown in various countries. colour, texture, size, and particularly, flavour continue to be the predominant quality characteristics important for a successful international marketing of horticultural crops (picha 2006). fruit exports are being targeted at more specialized consumers, who are either those seeking products either raised in more environmentally and health-conscious ways or those who are prepared to pay a significant price for outstanding flavour (zoe 2006). there are a number of numerous literatures which discussed the volatiles present in the papaya fruits (flath & forrey 1977; idstein . 1985 ; mohammed . 2001; pino . 2003; almora . 2004). papaya possesses a characteristic aroma, which is due to several volatile components, such as alcohols, esters, aldehydes, and sulphur compounds (marostica & pastore 2007). like many other climacteric fruits, papaya undergoes a variety of physical and chemical changes after harvest (shiota 1991). the biogenesis of flavours in fruits has been found to take place mostly during the ripening stage. the understanding of the biosynthesis pathways have been of increasing importance in the flavour industry in recent years, as this could aid in the production of the flavour volatiles in the same manner as the natural biosynthesis. this is crucial in allowing the flavours to be labelled as natural. pino . (2003) reported that based on more than 40 years of intensive research toward the volatile profiles of various papaya cultivars, almost 400 volatiles have been identified. the identified volatile profiles were various depending on the various methods used to extract and isolate the compounds and the species of the papaya used in the analysis (pino . 2003; devitt . 2006). furthermore, as mentioned before, the flavour of papaya fruit is a result of a complex interactions among sugars, organic acids, minerals, and aroma volatile compounds, which may vary with cultivar and production location. when the papaya fruits are subject to processing, there would be qualitative and quantitative alterations in the overall composition of volatile compounds (mohammed . 2001). the processing of the papaya fruits involves the major structural changes in the fruit (e.g. slicing, pulping, heating and freezing) that can result in a significant change in the sensory characteristics. it is also necessary and crucial to understand the aroma active compounds and their changes during processing because unexpected/unpleasant changes in aroma may make a product unmarketable even if the other quality factors are acceptable. therefore, understanding the changes of aroma active compounds in the papaya fruit, such as loss of desirable flavour and development of off-flavour during processing will be very helpful for determining proper processing condition. in this paper, the flavour composition of papaya fruit is reviewed. however, the information is not limited to a compilation of reported volatile compounds. this review will also present an overview of important publications and a description of the characteristic features of the biology of the fruits, consumption worldwide, commercial application in food processing, though review of volatile compounds will still be the main focus which that includes two aspects, i.e., the analytical methodology used for the determination of the volatile compounds and the contribution of individual components to the characteristic flavour. future researches will be also highlighted. b 51 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. biology and worldwide consumption the papaya, l., is a member of the small family caricaceae with genus and species l. according to its taxonomy classification, papaya is under the superkingdom , kingdom , phylum , sub phylum , division , sub division , super class class , order , and the family (trace 2012; paull & duarte 2011). however, a recent taxonomic revision proposed that some species formerly assigned to were more appropriately classified in the genus (badillo 2002). papaya is a fast growing tree like herb that can grow at the rate of 6 to 10 ft (1.8-3 m) in the first year and reaching 20 or even 30 ft (6-9m) in height, with a hollow green or deep-purple stem becoming 12 to 16 inch (30-40 cm) or more thick at the base and roughened by leave scars. the leaves emerge directly from the upper parts of the stem in a spiral on nearly horizontal petioles 1 to 3 ½ ft (30-105 cm) long, hollow, succulent, green or more less dark purple. the blade, deeply divided into 5 to 9 main segments, each irregularly subdivided, varies from 1 to 2 ft (30-60 cm) in width and has prominent yellowish ribs and vein. the life of a leaf is 4 to 6 months. both the stem and leaves contain copious white milky latex (morton 1987). the five-petal led flowers are fleshy, waxy and slightly fragrant. papaya flowers are born on inflorescences which appear in the axils of the leaves. female flowers are held close against the stem as single flowers or in clusters of 2-3 (chay-prove . 2000). male flowers are smaller and more numerous and are born on 60-90 cm long pendulous inflorescences (nakasone & paull 1998). bisexual flowers (hermaphrodite) are intermediate between the two unisexual forms (nakasone & paull 1998). the functional gender of flowers can be altered or reversed, depending on environmental conditions, particularly temperature. some trees bear only short-stalked female flowers or bisexual (perfect) flowers also on short stalks, while other may bear only male flowers, clustered on panicles 5 or 6 ft long. some trees have both male and female flowers. at certain seasons the trees produce short-stalked male flowers, while at other times perfect flowers. certain varieties have a propensity for producing certain types of flowers. male or bisexual trees may change completely to female trees after being beheaded. only hermaphrodite (bisexual flowered) papaya tree can bear fruit in a single tree by self-pollinating. the female papaya tree can also produce fruit from cross pollination by either bisexual or male trees. although male tree may sometimes bear fruit, it is not edible (fao 1992). the fruit is melon-like with an oblong or elliptic shape. fruits from female trees are spherical, whereas the shape of fruit from bisexual trees is affected by environmental factors, particularly temperature that modifies floral morphology during early development of the inflorescence (nakasone & paull 1998). fruits are ready to be harvested five to six months after flowering, which occurs five to eight months after seed germination (chay-prove . 2000). ripe papaya fruits have smoothed, thin green-yellow-orange coloured skin. depending on the cultivar, flesh thickness varies from 1.5 to 4 cm (nakasone & paull 1998) and flesh colour may be pale yellowish-orange to red (villegas 1991; nakasone carica papaya carica carica papaya eukaryota virdiplantae streptophyta embryophyta tracheophyta spermatophyta magnoliophyta, rosidae brassicales caricaceae carica vasconcella et al et al 52 biotropia vol. 20 no. 1, 2013 & paull 1998). the central cavity of mature fruits is containing numerous grey-black spherical seeds of 5 mm in diameter (villegas 1991). the fruits range in size from 7-30 cm long and vary in mass from about 250 to 3000g (oecd 2003). according to rivera (2005), papaya varieties generally can be divided into two groups, the small fruits weighing 500 grams below and big fruits weighing from 500 g up to 10 k. whereas, cosidine and cosidine (1982) divided papaya into four categories according to its size, i.e., small size weighing 0.3-.4 kg, medium size weighing 0.4-0.45 kg, big size weighing 0.45-0.9 kg, and very big size weighing more than 0.9 kg. in order to improve the quality of local papayas, the centre of tropical fruits study (pkbt) at bogor agricultural university (ipb), indonesia, has started a longterm breeding program since 2003. this program targets at developing new cultivars which will have outstanding specifications and characteristics that will be desirable for international market demand, resistant to biotic and abiotic stress and have high yield (anonymous 2003). until 2005, the program has screened 75 genotypes of papaya collected from several areas in indonesia and those introduced from abroad. this papaya genetic bank collection has been divided into two categories based on size: small papaya and medium-big papaya, or on its specific utilization purpose as vegetable/fruit papaya or papain produced papaya (anonymous 2005). among the 24 genotypes that have been morphologically characterized, 'eksotika', 'sunrise solo', 'bangkok', 'red king' and 'california' were recommended by pkbt as outstanding varieties of vegetable/fruit papaya. there are also other three outstanding genotypes obtained from open pollinated breeding released by pkbt named 'papaya arum bogor' (small-type), 'papaya prima bogor (medium-type) and 'papaya wulung bogor (papain producer). some other superior genotype candidates such as ipb 3, ipb 8, ipb 6, ipb 9 are still under evaluation (anonymous 2005; sriani 2008-personal communication). healthy, low in calories and a rich source of vitamins, calcium and phosphorous, the papaya is one of the most easily digested fruits. the ripe papaya fruit is consumed as fresh table fruit. sometimes it is cut in wedges and served with lime or lemon juice, also in few cases, a few seeds (just a few) are left attached to a peppery flavour. the firm-ripe flesh is often cubed or shaped into balls and served in fruit salad or fruit cup as well as seasoned and baked for consumption (morton 1987). however, in many asian countries, especially japan, malaysia, vietnam, indonesia and thailand, the fruit is consumed as grated vegetable while still in the green stage. papaya cuisine is now becoming popular in european society. there is also an array of processed papaya products available prepared by minimal processing, drying, canning, pickling and freezing. ripe flesh is commonly made into sauce for shortcake or ice-cream sundaes, or is added to ice cream just before freezing. it can also be cooked as pie, pickled, or preserved as marmalade or jam, papaya cubes with other fruits, covered with sugar syrup, may be quick-frozen for later serving as dessert. papaya juice and nectar may be prepared from the fruit and are sold fresh in bottled or canned. half-ripe fruits are sliced and crystallized as a sweetmeat (morton 1987). green papaya puree can also be utilized as chilly-sauce thickener. young leaves of papaya can be cooked and eaten as vegetables in several countries in asia. papaya leaves contain the bitter alkaloids, carpaine and pseudeocarpaine, 53 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. which can be destroyed by heat. sprays of male flowers are sold in asian including indonesian and new guinea markets after being boiled with several changes of water to remove the bitterness and eaten as a vegetable. in africa, the young stems are cooked and served. papaya seeds are sometimes found as an adulterant of whole black pepper (morton 1987). according to philippine council for agricultural, forestry and natural resources research and development (pcarrd-dost 2004), papaya is grouped in five major tropical fruits after banana, mango, pineapple which shared 6 percent of on estimated world production of tropical fruits in 2004. evans and ballen (2012) reported that based on fao statistical division data 2012, the global papaya production in 2010 was estimated 11.22 metric tons or 15.36 percent of the total tropical fruit production, ranked third. however, other sources said that it is impossible to obtain reliable estimates of total world papaya production, since the home-grown crops are unregistered. this phenomenon clearly occurs in indonesia, although it is believed that the annual production must be several million metric tons. the leading global papaya producing countries for period 2002-2010 were india (36.6%), followed by brazil (17.5%) and indonesia (6.9%). while sidhu (2006) reported the leading country of papaya production is brazil, followed by nigeria, india, mexico and indonesia. according to the national agricultural statistics service (nass 2007), only hawaii in the united states produces 45.9 million pounds of papaya fruit on about 2,320 acres in 2002, however, the production is declining every year. the production was only 28.7 million pounds on about 2,095 acres in year 2006 (nass 2007). most of the papaya grown in hawaii is consumed as fresh fruit (93%), only small amounts were processed into juices or other processed foods. the determination of aroma substances by instrumental technique generally consists of two stages. the first phase is the isolation of analytes from the complicated food matrix based on two primary principles-volatility and/or solubility. the second phase is the identification of the analytes. it is essential to isolate the desired volatile compounds in order to eliminate interfering signals coming from the complex food matrix. hence, it is important to select an appropriate sample preparation method so that the isolated product can be possibly representative. currently, there are many isolation techniques that can be used depending on the properties of the food product. generally, there are two common extraction techniques used in extracting volatile compounds from papaya, namely the simultaneous distillation/solvent extraction (sde) (macleod & pieris 1983; morales & duque 1987; almora . 2004) and the dynamic headspace analysis (mohammed . 2001; flath . 1990). these two flavor in papaya ( )carica papaya methods of extraction, isolation and identification of papaya aroma compounds et al et al et al 54 biotropia vol. 20 no. 1, 2013 methods are also commonly used for the study of volatiles compounds of other fruits such as apples (argenta . 2004; shashirekha . 2008), mangoes (sakho . 1998; torres . 2007), mangosteens (macleod & pieris 1982), peaches and nectarines (lavilla . 2002). the principal of sde technique is based on the differences in volatility and polarities among the analytes and other non-volatile components present in the food matrix (teixeira . 2007). an appropriate solvent also should be chosen to facilitate the extraction of the important volatile components, while excluding/limiting the components that could interfere with the analysis. in sde method, the sample is simultaneously distilled and extracted from steam condensate by organic solvents reflux, such as diethyl ether (almora . 2004) and ch cl (morales &duque1987) in a likens and nickerson apparatus. the concentrated extracts will then be analyzed by gco and/or gc-ms. the advantages of sde analysis of volatile compounds are obvious that only two single main operations (extraction and concentration) are required and yet give a relatively wide spectrum of chemical compounds detected (peng . 2004), and due to the continuous recycling, a relatively small quantity of organic solvent is used. in addition, it minimizes the possibility of artefact introduction from this source (schultz . 1977). dynamic headspace technique uses a ”purge and trap” method involving the passing of carrier gas through a liquid sample, followed by trapping of the volatile analytes on an absorbent material, and the analytes are flushed onto the column for analysis by gc or gc-ms (snow 2002). mohammed . (2001) had collected the volatiles by using tenax as the absorber, and used purified nitrogen gas to flush sample onto the tenax trap. the aliquots collected were then injected into gc-ms. recent study on the papaya volatiles has been reported by ulrich and wijaya (2010). two different sample preparation methods which are liquid-liquid extraction and stir bar sorptive extraction (sbse) have been utilized in the analysis. there were clear different results obtained using these two methods. it has been known that the utilization of sbse as well as solid phase microextraction (spme), although they are more rapid isolation compared to liquid-liquid or sde, only strong substances will be discriminated. the sample preparation using sbse is effective and usable in the aroma comparison research topic. however, it is not a proper approach for aroma identification since it will not cover all character impact compounds responsible for the wholesome aroma of the sample. generally, selecting different extraction techniques might lead to the identification of different volatile compounds. this could be due to inadequate sensitivity (i.e., only the most abundant volatiles can be detected and/or unable to detect trace compounds) and different in selectivity of the trap or solvent used (i.e., non-polar compounds can hardly be extracted by polar solvents/trap absorbent). for instance, larrayoz (2001) reported that the dynamic headspace technique combined with a purge & trap device could extract more highly volatile compounds than the sde method. in contrast, the sde is more efficient for extracting low-volatile components such as phenols, free fatty acids, lactones and longer-chain aldehydes, ketones, alcohols and esters. et al et al et al et al et al et al et al et al et al et al et al. 2 2 55 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. aroma active compound synonym (s) odor descriptors molecular mr (g/mol) b.p. (oc) cas registry no. butanoic acid butyric acid, octyl butyrate pungent unpleasant odor, acrid-taste c4h8o2 88.106 163.5 107-92-6 4-methyloctane 4-methyloctane somewhat pungent, citrus-like c9h20 128.255 142.4 2216-34-4 hexanoic acid caproic acid; pentane 1carboxylic acid; hexylic acid; hexoic acid strong fruity odor, pineapplelike c6h12o2 116.16 205 142-62-1 benzenemethanol phenyl carbinol; alphahyroxytoluene; benzoly alcohol; phenyl methanol mild-sweet aroma like c7h8o/ c6h5ch2o h 108.1 205 100-51-6 translinalool oxide (furanoid) (2r,5r)-5 isopropyl 2-methyl-2vinyltetrahydrofuran earthy-leafy cislinalool oxide (furanoid) (2r,5r)-5 isopropyl 2-methyl-2vinyltetrahydrofuran sweet, floral,creamy, fruity benzylacetate acetic acid benzyl ester; acetic acid phenylmethyl ester; alphaacetoxytoluene; benzyl ethanoate; phenylmethyl acetate fruity, apple and pear-like ch3cooc h2c6h5 150.18 215 140-11-4 linalool cispyranic oxide na citrus-like cyclohexane hexamethylene; hexanaphthene; hexahydro-benzene; benzenehexahydride; sweet-fruity c6h12 84.16 80.7 695-06-7;57129-701 (±)form; 6335795-9 (r) -form; 41035-07-8 (s) form: (£)-form gammahexalactone 4-hexanolactone; 4-caprolactone; 4-ethyl butrolactone;4hydroxyhexanoic acid lactone;4hexanolide sweet, creamy, lactonoic, fruity, coumarin-like with green coconut nuances c6h10o2 114.44 216 gammaoctalctone 5-butyldihydroxy -2 (3 h)furanone;4octanolide;4buthyy butyrolactone coconut-like odor, sweet, lactonoic, fruity creamy, coumarin c8h14o2 142.197 117 108943-45-9 (r) form deltaoctalactone 5-octanolide;5hydroxyoctanoic acid lactone sweet, creamy, fatty with tropical and dairy nuances, fruity c8h14o3 142.197 140 108943-46-0 (s) form 80 104426-32-6 (± ) form 126 table 1: aroma active compounds as reported by different researchers found in papaya 56 biotropia vol. 20 no. 1, 2013 c10h18o2 170.2487 222.6°c at 34995-77-2 c10h18o2 170.2487 na 60047-17-8 c15h24o3 252.3493 356.1°c at 14009-71-3 760 mmhg 760mmhg et al et al et al carica papaya et al approximately 150-200 volatile components of ripe papaya pulp have been reported in various papers (flath & forrey 1977; macleod & pieris 1983; idstein & schreier 1985; schreier . 1985). these volatiles are released by harvested intact fruit in small amount per unit time, and some of the reported volatile compounds are only generated in quantity from non-volatile precursors due to the disruption of fruit tissue (flath . 1990). volatile constituents from papaya extracted by both the simultaneous distillation/solvent extraction method and dynamic headspace analysis are listed in table 1. volatiles identified in these studies included heterocyclic compounds, terpenoids, aromatic hydrocarbon, alcohol, acids, esters and ketones. a fairly wide range of different types of compounds have been identified in different studies (as shown in table 1), partly due to papaya species aside from the type of extraction and analysis technique used. for instance, the volatiles of sri lanka papaya were dominated by esters (macleod & pieris 1983), whereas terpenoids (mainly linalool and linalool oxides) provided the most abundant group of volatiles for hawaiian papaya (flath & forrrey 1977). on the other hand, different method approaches might be able to show the same tendencies. for instance, the volatile components of papaya (solo variety) which were recovered by four different methods: e.g., vacuum trapping train (distillation under low temperatures with liquid nitrogen traps), co-distillation-extraction (vacuum), distillation, and co-distillation-extraction (1 atm.), showed that, in spite of great variations due to the recover method, linalool was always the major compound detected in all the methods (flath & forrey 1977). mohammed . (2001) had identified a total of 26 aroma volatiles emanating from fresh cut papaya ( l. cv. solo) using the dynamic headspace technique followed by gc-ms, where some had not been reported by schreier and winterhalter (1986). however, some of these newly identified volatiles such as 4methyl-1-octane, tetrahydro-3-furfuryl-furan and 2,4-dimethylhexane may have arisen due to the mechanical damage sustained by the tissue during preparation (mohammed . 2001). in addition, 11 out of these 26 aroma volatiles were identified as aroma active compounds, namely, butanoic acid, 4-methyloctane, hexanoic acid, benzenemethanol, trans-linalool oxide, linalool, benzyl acetate, linalool cis-pyranic oxide, cyclohexane and benzaldehyde. it was also shown in the study that the volatile extract obtained from papaya slices changed from a distinct fresh sweet and flowery odour to slightly less evident, then to an unpleasantly dry, pungent and fruity odour which could be explained by the changes in the distribution of these aroma active compounds during storage. this in turn illustrated the aroma of papaya due to a complex and dynamic integration of compounds which changed with time as well. papaya is a climacteric fruit that undergoes the ripening process upon harvesting, until the stage where a fully ripened papaya gives fully developed flavours and odours which then perceived and recognized by human as an indication that the papaya is ripened. hence, the papaya fruit gives its characteristic fruity, balsamic, sweet odours aroma-active compounds of papaya 57 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. after undergoing a variety of physical and chemical changes during the ripening process. herein, aroma components that give the aroma characteristic of a food are called aroma active compounds or character impact compounds. mohammed (2001) reported that the dominating aroma-active compounds in papaya were linalool and benzaldehyde. this claim is also supported by chan (1973) who reported that the major odorous compounds from fresh, ripened papaya ( ) were linalool, with smaller amount of ethyl acetate, 1-butanol, two configurational isomers of the linalool oxides (2-methy-2-vinyl-2-(hydroxyl-2-propyl) tetrahydrofuran), and benzyl isothiocyanate. also, gamma-hexalactone, gamma octalactones and delta octalactones were reported in other literatures (sidhu, 2006) as aroma-active compounds contributing to the overall perceived characteristic aroma of the papaya. other prominent volatiles like butanol, 3-methylbutnol, benzyl alcohol an terpineol in papaya ( l., maradol roja) at its mature stage were also reported by almora . (2004), who also mentioned a decrease in benzyl isothiocyante, and increase in linalool, terpinen-4-ol, esters (methyl butonoate, ethyl hexanoate and ethyl dodecanoate). the combination of the different esters is associated with the detected fruit concentrations during ripening (almora . 2004). the major aroma-active compounds present in cut papaya was identified to be linalool, benzaldehyde and benzyl isothiocyanite (bitc). linalool is the most dominant aroma-active compound present in the papaya ( l.). the chemical name is 3,7-dimethyl-1,6-octdine-3-ol. (±)-linalool (22564-99-4), like the individual enantiomers, a colourless liquid. it is an acylic terpene with a flowery and fresh odour reminiscent of lily of the valley (bauer . 1997). however, the enantiomers differ slightly in odour (klein & ohloff 1962). the aroma threshold for linalool was found to be within 4 to 10 ppb. at 30 ppm, it has a taste characteristic of floral, woody, and sweet, with a green spicy tropical nuance. hence, the presence of linalool at different concentrations in a wide range of foods might give a slightly different characteristic sweet-flowery aroma in the foods. in the , whilst hydrogenation of linalool yields tetrahydrolinalool, which is often used as a fragrance compound. also, linalool can be converted into linalyl acetate by reacting with ketone or excess of boiling acetic anhydride. benzaldehyde is also the dominating aroma-active compound in papaya, having a characteristic of bitter almond oil and imparting a bitter-nutty and almond odour (mohammed . 2001). it is also named benzene carboxaldehyde and almond artificial essential oil. benzaldehyde undergoes auto oxidation in the absence of inhibitors to perbenzoic acid, which then react with a second molecule of benzaldehyde to benzoic acid. upon hydrogenation of benzaldehyde, benzyl alcohol will be formed. benzyl isothiocyanate (bitc) is found in enzymic hydrolysates of extracts from various plant families: namely, , , , , and (ettlinger & hodgkins 1956; ettlinger & et al. et al. carica papaya carica papaya et al et al carica papaya et al et al cruciferae moringaceae capparidaceae tropaeolacea, caricaceae gyrostemonaceae salvadoraceae d α y note (arctander 1969), whilst balsamic note could be attributed to benzyl alcohol and α-terpineol presence of acids, linalool isomerizes readily to geraniol, nerol and αterpineol. it is oxidized to citral by chromic acid. oxidation with peracetic acid gives linalool oxides 58 biotropia vol. 20 no. 1, 2013 kjaer 1968), or can be synthesized in bruised or injured papaya. it possesses pungent, off-flavour aroma. as revealed by tang (1971), benzyl isothiocyanate is a natural metabolite emanating from intact green papaya. also, he mentioned that the concentration of benzyl isothiocyanate (or its glucosinolate precursor) decreased in the flesh of papaya whilst it increased in the seeds with maturity. despite the decreasing concentration of bitc along with maturation, it can make significant sensory impression since it has a very obnoxious nature of low threshold. on top of the elaborated aroma-active compounds, other aroma active compounds that are present in smaller amounts (shown in table 1) also contribute to the overall perceived aroma of ripe papaya. hence, each individual aroma active compounds has its own vital role in giving our sensory a full picture on the papaya aroma. in another investigation, linalool was detected in relatively low concentration in the solvent-extracted volatiles of fresh papaya pulp from sri lanka, while esters represented the majority. the authors attributed the characteristic sweaty note of this papaya fruit mainly to methyl butanoate. phenylacetonitrile was also found in high amounts (17.7%) combined with lower concentration of benzyl isothiocyanate (1.5%), which played a critical role in the aroma of papaya (macleod & pieris 1983). oxygenated terpenoids derived from linalool might play an important role in brazilian papaya aroma. several oxygenated derivatives of linalool were identified in the solvent-extracted samples, such as the two diasteroisomers of 6,7epoxylinalool:2,6-dimethyl-octa-1,7-diene-3,6-diol and 2,6-dimethyl-octa-3,7-diene2,6,-diol (winterhalter 1986). fifty-one volatile components from intact hawaiian papayas of different ripeness were recovered by tenax using the “trap and purge” method. as expected, the largest number of components was found in the fully ripe fruits. linalool, followed by linalool oxide a, linalool oxide b, and ethyl acetate were the major components in the fully ripe fruits. it is worth noting that several compounds, e.g., linalool and all aldehydes, exist in all four ripeness stages (flath . 1990). another investigation reported the esters as the predominant volatile components of the maradol variety (about 41% w/w of the total volatiles) (pino . 2003). the major representative compounds in the simultaneous steam distillationsolvent extraction were methyl butanoate and ethyl butanoate. previous work described the esters as the predominant compounds among the volatiles; papaya, for example from sri lanka and colombia had 52 and 63% of esters in the total volatiles, respectively (macleod & pieris 1983; morales & duque 1987). studies on the diversity of papaya volatiles in different cultivars and new breeding lines have been conducted by ulrich and wijaya (2010). character impact compounds such as hexanal, cis-2-pentenol, nonanal, cis-linalool oxide, linalool, butanoic acid, verbenone, phenyl methyl of butanoic acid and others have been detected. the volatiles patterns differ significantly among the genotypes resulting in very different sensory quality. the gained obtained knowledge will contribute to the breeders' ability in developing new papaya varieties with acceptable flavour properties (ulrich & wijaya 2010). et al et al 59 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. despite the significant diversity in aroma compounds of papaya fruit, the volatiles in papaya are mainly derived from three major metabolism pathways: the catabolism of fatty acid, amino acid, and carbohydrates. the biosynthesis pathways are usually interlinked, with products from one pathway serving as the precursors for another pathway. these degradation reactions account for many aroma compounds, as they result in the formation of a host of low molecular weight products which has significant sensory properties. this catabolism is associated with fruit ripening, during the climacteric rise in respiration, and the rate of flavour formation reaches a maximum during the post-climacteric ripening phase (reineccius 2006). the metabolism pathways will be elaborated in the following sections. aroma compounds may be formed from lipids via several different pathways. the primary pathways involved in the aroma of papaya include the enzymatic conversion of acyl lipids through n/lipoxygenase pathways (reineccius, 2006). these pathways generate short branched-chain aldehydes, alcohol, ester and ketones. fatty acid derived volatiles play important regulatory roles in defence d tissues can form volatiles via the oxylipin biosynthesis pathways (devitt . 2006). in papaya, methyl and ethyl ester derivates of lipid catabolism have been identified as strong contributors to aroma (macleod & pieris 1983; morales & duque 1987), examples include ethyl butyrate, ethyl acetate, methyl butyl acetate, ethy butanoate and methyl butanoate. significant amount of butanol has also been reported (morales & duque 1987). cules, one atp, one fad, one water molecule, and one nad to form an acetyl coa molecule and an acyl coa molecule. using acetyl coa as precursor, several other compounds such as esters, sterols, and most terpenes can be synthesized (reineccius 2006). the acetyl coa molecule can also undergo reaction to reform into fatty acid such as butyric acid which contributes to the papaya aroma. as mentioned above, the biogenesis of certain volatiles in papaya can be further explained by a process known as the oxylipin pathway, which in brief, is the metabolism of polyunsaturated fatty acids (pufas) by lipoxygenase (linoleate oxygen oxidoreductase; lox) and the subsequent reactions after it. stumpe (2005) and chehab (2007) reported that: “ this pathway is initiated by the action of lipases on complex membrane lipids in the fruit, causing the release of unesterified fatty acids. the free polyunsaturated fatty acids which contain a ( )-1,4-pentadiene structure then act as substrates for the enzyme lox introducing molecular oxygen to them to form their corresponding hydroperoxy derivatives. these derivatives then undergo further enzymatic reactions to give rise to fatty acid hydroperoxides, hydroxyl fatty acids, epoxy fatty acids, keto fatty acids, volatile aldehydes and cyclic compounds, which are collectively known as oxylipins. some of these products are responsible for the unique aroma of the papaya fruit papaya flavour biogenesis βoxidation and oxylipi and plant development. fatty acid volatiles are formed in intact fruit via the βoxidation pathway while cut or damage in the papaya plant, β-oxidation occurs in the peroxisomes, organelles which contain enormous amounts of enzymes. the substrate of the reaction is an acyl. the acyl lipid reacts with two coa mole et al et al. et al. cis, cis . 60 biotropia vol. 20 no. 1, 2013 et al et al et al cis et al unstable allene oxides are formed from the activity of allene oxide cyclase (aos). they can either undergo nonenzymatic hydrolysis leading to alpha-and gamma-ketols, or be metabolized to 12-oxophytodienoic acid (opda) by aoc. subsequently, opda is transported to the peroxisomes where its cyclopentenone ring is reduced by opda reductase to form opc:8. finally, the opc:8 is subjected to three rounds of beta-oxidation to yield jasmonic acid (ja) (chehab . 2007). ja and its precursor opda are signalling compounds that play a role in plant development and response to injury (stumpe . 2005). ja is also converted to a variety of derivatives like its ester, methyl jasmonate, which contributes to the aroma of the papaya fruit. the other major metabolic route that is dominant for the direct production of important compounds of the characteristic aromas of many fruits including papaya is hydroperoxide lyase (hpl) pathway (yilmaz 2001). hpl catalyzes the oxidative cleavage of fatty acid hydroperoxides, producing volatile aldehydes and oxoacids. the hpl enzyme can yield c-6 aldehydes and c-12 omega-oxoacids from the 13hydroperoxide derivatives (kalua . 2007). the c-6 aldehydes include saturated aldehyde, hexanal from linoleic acid and the unsaturated aldehyde, -3-hexenal from linolenic acid. this unsaturated aldehyde is unstable and undergoes rapid isomerisation to a stable compound, trans-2-hexenal. the c-6 aldehydes formed through the hpl activity can also be further reduced by alcohol dehydrogenase to form the corresponding calcohol, like cis-3-hexen-1-01 (matsui 2006), which is one of the constituents of papaya's volatiles. furthermore, alcohol acetate transferase catalyses the formation of acetate esters through acetyl coa derivatives together with alcohol. acetate ester, esters of alcohols with other fatty acids, as well as the aldehydes and alcohols produced in this pathway are important constituents of papaya fruit which give it a distinct aroma by virtue of the volatiles. many of the aliphatic esters, alcohols, acids, and carbonyls found in the fruit are derived from the oxidative degradation of linoleic and linolenic acids. amino acid metabolism generates aromatic, aliphatic, and branched chain alcohol, acids, carbonyls, and esters that are important to the flavour of papaya (reineccius 2006). variations of the free amino acid content in fruits have been known to occur during ripening, corresponding to the period of maximum production of aroma compounds. this is evidence that amino acids are precursors of aroma compounds generated during fruit ripening, and is an important source of volatile compounds contributing to their aroma (tomas-barberan . 1997). the amino acids present in plants in general are classified under two categories, namely the non-aromatic (absence of benzene ring) amino acids (cysteine, methionine, leucine, isoleucine and valine) and the aromatic amino acids (phenylalanine, tryptophan, tyrosine). phenylalanine and tyrosine are believed to be the chief precursors of aromatic volatile compounds in plants, synthesized by the shikimic acid pathway. following the shikimic acid pathway, phenylalnine can enter other two pathways, one of which produces benzyl glucosinolate, the precursor of benzyl isohiocyanate (one of the major compounds in papaya); the other is the cinnamic acid metabolism pathway which produces aromatic alcohols, acids, esters and carbonyls. aroma compounds can also be synthesized from non-aromatic amino acids precursors (eg. cysteine, methionine, leucine, isoleucineand valine). 61 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. the aromatic amino acids are not naturally present in plants and have to be synthesized by a metabolic pathway, namely the shikimic acid pathway. the shikimic acid pathway converts simple carbohydrate precursors derived from glycolysis and the pentose phosphate pathway to the aromatic amino acids. in the initial steps, d-glucose is phosphorylated. after a series of enzymatic transformations, it is subsequently converted into phenolic compounds, ultimately yielding the active precursor amino acids. following the shikimic acid pathway, the other main pathway which phenylalanine enters is the cinnamic acid metabolism (phenyl propanoid pathway). in this pathway, the amino acids are further transformed into aromatic alcohols, acids, esters and carbonyls. benzaldehyde, 4-methylbenzaldehyde, acetophenone, benzophenone, bdamascone, b-ionone (trace), and benzyl alcohol and phenylacetonitrile (10-50 ppb), which are aromatic compounds found in papaya (idstein . 1985a) are hypothesized to be generated in this manner. out of the three aromatic amino acids generated through shikimic pathway, phenylalanine has been stipulated as the chief precursor of aromatic aroma compounds derived from amino acid metabolism (lamikanra 2002). there are two main pathways which phenylalanine can react to form other compounds. the first of these two main pathways is responsible for forming benzyl glucosinates in the papaya plant. the synthesis of volatile aroma compounds with non-aromatic amino acids as precursors takes place through a completely different pathway. the pathway follows the citric cycle instead of the shikimic acid pathway. the first step of transamination occurs when glutamic acid is produced from 2-oxoglutarate. the next step is the decarboxylation of 2oxoacid formed after amino acid transamination. this produces only aliphatic and its branched aroma compounds particularly methyl-branched (aliphatic) alcohols, acids, esters and carbonyls. as mentioned above, esters are very important contributors to the aroma of papaya (macleod & pieris 1983; morales & duque 1987). they are formed by the reaction between alcohols and acyl coa's derived from fatty acid and amino acid metabolism. this reaction is catalyzed by acyl alcohol transferase (aat) (sanz . 1997; perez . 1996; ueda . 1992). according to a study done by morales and duque (1987), the papaya aroma extract's main constituents are carboxylic esters, dominated by the ethyl and butyl esters of the c2-c8 saturated-chain carbocylic acids, with the exception of c5. saturated normal-chain c4, c6, c7, and c8 methyl esters were also found, as well as other saturated aliphatic and aromatic esters. some unsaturated esters were also detected: ethyl but-2-enoate, n-butyl but-2-enoate, ethyl oct-2-enoate, and n-butyl oct2s, i.e., ethyl-3-hydroxybutyrate and n-butyl 3-hydroxybutyrate. alcohols, including ethanol, butanol, hexanol, and octanol, were the another group of important components observed in the papaya fruit. these alcohols corresponded to the alcohol part of the identified ester. there are few flavour constituents that come directly from carbohydrate metabolism. however, terpenes for example, which arise both from carbohydrate and lipid metabolism, also play very important roles in plant's aroma. et al et al et al et al enoate together with two βhydroxy ester 62 biotropia vol. 20 no. 1, 2013 terpenes are classified by the number of isoprene units (c5). in one particular study by flath and forrey (1977), terpenoids constituted 81% of the total volatiles detected, with the monoterpene alcohol, heidlas . 1984; flath . 1990). of particular interest are linalool and its pyranoid and furanoid oxides due to their prominence as flavour components of papaya (schreier & winterhalter 1986; flath . 1990). linalool and its oxides are biosynthesized through geranyl diphospate which produce linalyl cation and linalyl pyrophosphate. linalyl pyrophosphate are the amount of free linalool is relatively small in papaya, instead, it often occurs in ripe papaya fruits in a glycosidic bound form, from which it is released through cell disruption (heidlas 1984). linalool glycoside is synthesized by the enzyme glycosyltransferse, and its concentration versus time increased with the maturity of the plant organ. this accumulation is generally coupled with the production of the corresponding free forms. the degradation of carotenoids, particulary the oxygenated xanthophylls, contributes . 2003; flath & forrey 1997). the co-oxidative cleavage of carotenoid polyene chains to form ionones and damascenones occurs enzymatically through the action of oxidases including lipoxygenases, peroxidases, and dioxygenases, released during fruit ripening. glucosinolates are thioglucosides mainly found in the botanical family cruciferae, although rare occurrence outside this family is also documented. papaya is one of the best examples of a nonplant which contains benzyl glucosinolates, and hence produces benzyl isothiocyanates among its volatiles, by enzyme actions (macleod & pieris 1983). flath and forrey (1977) confirmed the presence of benzylglucosinolate in papaya fruit based on their discovery of relatively large amounts of both benzylthiocyanate and phenylacetonitrile (another product of benzyl glucosinolate degradation) among the volatile components. benzyl glucosinate is a compound that is continually formed during the growth of plant, from seed to fruit. it is present in a high concentration in the young papaya plant, especially in the leaves and roots. as the papaya plant grows, the compound is gradually transported to the fruit (bennet . 1997). from phenylalanine, benzyl glucosinolate is synthesized via three stages: (1) amino acid chain elongation, (2) synthesis of the glucosinolate from the amino acid, and (3) chain modifications. although it does not contribute much to the flavour of the papaya fruit, it is crucial to the formation of benzyl isothiocyanate (phytochemical containing sulphur and linalool, representing 68% of the total emissions. other terpene hydrocarbons like myrcene, ocimene, limonene, sabinene and neoalloocimeme, and terpene alcohols like αterpineol, nerol, and geraniol also contribute to the major volatiles in papaya ( also formed other monoterpenes such as α-terpineol, limonene, and pinene. e.g. endogeneous βglucosidase activity during to the aroma profiles of many fruits and vegetables as well. trace amounts of the ketone β-ionone have been identified previously as papaya volatile component (pino et al et al et al et al. et al cruciferae et al synthesis of benzyl glucosinolate 63 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. nitrogen) of which is a major flavour volatile of the papaya fruit with specific sensory) (flath & forrey 1977; flath . 1990). benzyl isothiocyanate has a protective function in the papaya fruit as well. it is produced by the enzymatic hydrolysis of benzyl glucosinolate during fruit injury (patil . 1973). the formation of benzyl isothiocyanate, which are formed from the aglycone by a lossen re-arrangement involving the migration of the side chain from the oxime carbon to the adjacent nitrogen, is catalysed by myrosinases (halkier &and gershenzon 2006). in fact, even intact papaya fruit releases traces of benzyl isothiocyanate vapour (patil & tang 1974). like the other soft pulp fruits, papaya is highly perishable & susceptible to fungal attack during storage. most of the volatiles formed after storage are mainly due to the mechanical damage sustained by the tissue during preparation such as cutting or fungal attack. the cellular disruption allows enzymes and substrates that are previously sequestered separately within the cells to be able to be interacted, mediated qualitatively and quantitatively and altered, which results in the synthesizing of volatile compounds including 4-methyloctane, tetrahydro-3-furfuryl-furan, 2,4dimethylhexane, benzene-methanol, isopelletierins, 5-6-dimethydecane, cyclohexylazide, undecane, n-tridecane, butyl ketone, 7-tridecanone, 5-9-undecadien2-one & benzyl tiglate (mohammed . 2001). schreier & winterhalter (1986) has also reported the presence of 6,7-epoxy-linalool, 2,6-dimethyl-1,7-octadiene-3,6-diol, 2,6-dimethyl-3,7-octadiene-2,6-diol, 2,6-dimethyl-3,7-octadiene-2,6-diol, cis and trans-2,6-dimethyl-2,7-octadiene-1,6-diol and 2,6-dimethyl-7-octene-2,3,6-triol as well as four diastereoisomeric epoxy-linalool oxides in their furanoid and pyranoid forms as well. although low temperature is used to maintain the freshness of the ripen papaya, continuous changes of the volatile compounds composition were observed during this storage. on the third day of storage, there were major changes in the odour perceived in the ripen papaya (mohammed . 2001). the freshly cut papaya initially has a mixture of flowery, fresh, bergamot-like, fruity, bitter, nutty, almond odour, mainly due to the presence of the dominant odouractive compounds, linalool, which gives flowery and fresh odour reminiscent of lily and benzaldehyde which contributes to bitter nutty and almond odour (bauer . 1997). there are also other trace volatiles such as and -linalool oxides, cyclohexane, hexanoic acid and benzenemethanol which contribute to the fruity flavour (bauer . 1997). furthermore, the presence of cyclic ester, -and linalool also contribute to the earthy and slightly bergamot-like odour in papaya (bauer . 1997). these flavours perceived from the freshly cut ripen papaya would last for the first two days of storage and change to the different volatiles. the sweet flowery odour perceived initially was lost on the third day of storage due to a significant decrease of about 50% of linalool, while benzaldehhyde, linalool cispyranic oxide and cyclohexanebeing vanished (mohammed . 2001). on the other et al et al et al et al et al trans cis et al cis trans et al et al flavor changes during storage and processing 64 biotropia vol. 20 no. 1, 2013 hand, benzyl acetate was the dominant aroma volatile at the storage interval and together with un-pleasant earthy flowery odour of trans-linalool and un-pleasant coffee like aroma taking place. the significant increase of butanoic acid present on day 3, also causes the impartment of dry-flowery lily-like odour, thus intensifies the non pleasant odour of the papaya after 3 days storage. upon realizing the undesirable changes of the aroma active compounds during storage of the sliced ripen papaya, it would be advisable that ripe papaya should be consumed immediately after the cutting of the papaya. otherwise, the papaya should be left without processing which would prevent the formation of non-pleasant volatile compounds due to the cellular disruption. papaya puree is the major semi-processed product that is used in juices, nectars, fruits cocktails, jams, jellies and fruit leather (salunkhe & kadam 1995; somogyi . 1996). commercially, papaya puree is produced by pushing the whole fruit into a pulpier fitted with a 0.033 inch screen. the puree is not heated to inactive the enzymes and even contain pulverized peel and & seeds. the next step is subjected to subsequent thawing and freezing to dispatch and preserved the puree. as mentioned above, squeezing of the whole fruit into the pulpier will cause the cellular disruption that allows enzymes and substrates to be able to interact, resulting in a similar sweet flowery and fruity odour due to the release of a large amount of linalool, benzaldehyde and other trace compounds. however, using the commercial method on producing papaya puree cannot maintain the sweet flowery and fruity odour, and in turn resulted in the off-flavour perceived in the papaya puree. the formation of off flavour was mainly due to the reaction induced by naturally occurring enzymes in the papaya during processing and frozen storage. it is also due to the microbial action in the centre of the puree mass prior to the freezing process and at the periphery of the mass during the thawing process (chan . 1973). both enzymatic and microbial action causes the formation of butyric, hexanoic, and octanoic acid and their methyl esters. the formation of these free acids which mainly has pungent-rancid odour might contribute to the off-flavour. furthermore, during the puree process, the content of benzyl isothiocyanate increases significantly higher than the linalool content due to the hydrolysis of glucosinolate by enzyme. this proportion will suppress the sweet flowery odour and intensify the pungent cabbagelike odour as well. to minimize the off-flavour formation, an improved method for processing puree, by acidification to ph 3.55 and thermal in-activation of enzyme, has been developed (chan . 1973). aside from being made into puree, as has been mentioned above, papaya can be processed into other products to meet consumer's needs. in processing of all these products, thermal treatment or thermal processing is ubiquitously applied as an essential part, for the purposes of in-activating enzymes, softening of texture, concentration or drying of product, and sterilization. however, the thermal treatment also brings detrimental effects to the product quality, especially with regard to the aroma profile of papaya. the volatile aroma active compounds present in papaya will be inevitably lost to different extent during the thermal treatment. in a previous summary of the processed papaya products and their processing conditions (salunkhe & kadam 1995; somogyi . 1996), it seems that the et al et al et al et al 65 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. temperatures involved in the thermal treatments of various processing's are below the boiling points of most aroma active compounds (table 2). it indicates that during the thermal treatments, cycloxehane will probably be the one which loses most due to its low boiling point, while other compounds will be partly lost due to evaporation. the loss of cyclohexane and other aroma active compounds will lead to the deterioration of olfactory quality of papaya. therefore, the processed products are often perceived as less sweet or fruity in aroma than the fresh-cut, non-processed fruits. based on the boiling point and likelihood of evaporation, the aroma active compounds listed in table 2 can also be classified roughly into three categories. cyclohexane which has the lowest boiling point and thus most easily evaporated; and the second group are benzaldehyde, butonoic acid and 4-methyloctane, with the medium boiling point, evaporate at a moderate rate; while other aroma compounds, including linalool, bezyle isothicynate, hexanoic acid, benzenemethonal, cis-linalool oxide, benzyl acetate and gamma-hexalactone, with relatively high boiling points, evaporate at a slow rate. during the heat treatment, besides loss due to evaporation, some of the aroma active compounds may be oxidized with the presence of oxygen.among the 15 aroma active compounds identified in papaya, linalool and benzaldehyde, the two major aroma compounds in fresh papaya, are the most susceptible to oxidation. linalool undergoes autoxidation with o to form hydroperoxides, of which 7-hydroperoxy3,7-dimethyl-octa-1,5-diene-3-ol is the most important one (skold 2006; backtrop . 2006) this would result in reduction of linalool and in turn the sweet smell of the papaya aroma in the processed product. benzadehyde also undergoes autoxidation to perbenzoic acid and converts to benzyl alcohol upon hydrogenation (bauer . 1997). as a result, the bitter almond aroma contributed by benzaldehyde will decrease in the processed papaya product. to reduce loss of aroma in processed papaya products, irradiation is proposed as an alternative to heat treatment when the purpose is to kill fruit flies, prevent fungal growth or inactivate enzymes. irradiation is able to achieve the same or better et al et al 2 table 2. summary of boiling points of several aroma active compounds in papaya aroma compounds boiling point (oc) aroma compounds boiling point (oc) linalool 198 cis-linalool oxide 188 benzaldehyde 178 benzyl acetate 214 benzyle isothicynate 243 linalool cis -pyranic oxide not available butonoic acid 164 cyclohexane 81 4-methyloctane 142 gamma hexalactone 220 hexanoic acid 223 gamma octalactones not available benzenementhonal 205 delta octalactones not available trans-linalool oxide not available 66 biotropia vol. 20 no. 1, 2013 outcomes as heat treatment, with much less damage to the sensory qualities of papaya (moy 1993). however, irradiation cannot replace heat treatment if the product requires be softening or dehydrating. in addition, the resistance to irradiated foods of consumers is still strong in the current context, which poses big challenge to the promotion of irradiation in food products. papaya is a climacteric fruit that undergoes the ripening process upon harvesting, until the stage where a fully ripened papaya gives fully developed flavours which then is perceived and recognized by human as ripening indication. the flavour of papaya fruit is a result from a complex interactions between sugars, organic acids, minerals, and aroma volatile compounds, which may vary with cultivar and production location. when the papaya fruits are subject to processing, there would be qualitative and quantitative alterations in the overall composition of volatile compounds due to the processing conditions. volatiles identified in these studies included heterocyclic compounds, terpenoids, aromatic hydrocarbon, alcohol, acids, esters and ketones. a fairly wide range of different types of compounds was identified in different studies. these volatiles are released by harvested intact fruit in small amount per unit time, and some of the reported volatiles are only generated in quantity from non-volatile precursors due to the disruption of fruit tissue. despite the significant diversity in aroma compounds of papaya fruit, the volatiles in papaya are mainly derived from three major metabolism pathways: the catabolism of fatty acid, amino acid, and carbohydrates. the biosynthesis pathways are usually interlinked, with products from one pathway serving as the precursors for another pathway. these degradation reactions account for many aroma compounds, as they result in the formation of a host of low molecular weight products which has significant sensory properties. moreover, papaya is one of the best examples of a nonplant which contains benzyl glucosinolates. although this compound does not contribute much to the flavour of the papaya fruit, it is crucial to the formation of benzyl isothiocyanate (phytochemical containing sulphur and nitrogen with specific sensory), which is a major flavour volatile of the papaya fruit. benzyl isothiocyanate has a protective function in the papaya fruit. this manuscript has been accomplished during the same program 2012 funded by dghe-ministry of education and culture, ri. thanks should be also addressed to mr. i kadek putra yudha prawira for his technical support. conclusions acknowledgments cruciferae 67 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. references almora k, pino ja, hernandez m, duarte c, gonzalez j, roncal e. 2004. evaluation of volatiles from ripening papaya ( l., var maradol roja). food chem 86(1): 127-30. anonymous. 2003. riset unggulan buah tropis indonesia. 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125-56. torres jd, talens p, carot jm, chiralt a, escriche i. 2007. volatile profile of mango ( l.), as affected by osmotic dehydration. food chem 101: 219-28. trace-molecular biology database [ ] (accessed october 23 , 2012). ueda t, waverczak w, ward k, sher n, ketudat m. 1992. ulrich d, wijaya ch. 2010. volatile patterns of different papaya ( l.) varieties. j appl bot food qual 83:128-32. villegas vn. 1991. l. in: plant resources of south-east asia no. 2: edible fruits and nuts. edited by verheij ewm, coronel re (eds). wageningen: pudoc 108-112. carica papaya carica papaya carica papaya j agric food chem annona squamosa asimina triloba medicago truncatula cistus ladanifer mangifera indica mutations of the 22and 27-kd zein promoters affect transactivation by the opaque-2 protein. plant cell 4: 701-09. carica papaya carica papaya the fragrance chemical β-caryophyllene http://www.bats.ch/trace/?file5=coreforms/ taxonomy.php&record_ number=19tt th 70 biotropia vol. 20 no. 1, 2013 wall mm. 2006. ascorrbic acid, vitamin a, and mineral composition of banana ( .) and papaya ( ) cultivars grown in hawaii. j food composit anal 19: 434-45. winterhalter p, katzenberger d, schreier p. 1986. 6,7-epoxy-linalool and related oxygenated terpenoids from fruit. phytochemistry 25: 1347-50. yilmaz e. 2001. the chemistry of fresh tomato flavor. turk j agric 25: 149-55. zoe c. 2006. the fruits of foreign trade. taiwan review 56 (11): 50-5. musa sp carica papaya carica papaya 71 flavour of papaya ( l.) fruit c. hanny wijayacarica papaya – et al. ichtyofauna at cijalu river, cilacap regency central java province, indonesia agus nuryanto, dian bhagawati, m. nadjmi abulias and indarmawan faculty of biology, jenderal soedirman university, purwokerto 53122, indonesia received 22 january 2014/accepted 21 june 2016 abstract cijalu river is located in western cilacap regency central java . the river runs through forestry, of province housing and farming areas. this condition lead to a prediction that the river has altered its physico-chemical s been on characteristics inhabited by variety of fish species. this study aim to collect data about fish causing the river to be a ed species inhabit cijalu river and its distribution. a survey method has been done with ing clustered random sampling technique the river was divided into three different areas, i.e. upper-, middle and downstreams. species diversity was . measured as the number of species, while distribution was measured as the presentation of fish species in each site . nineteen fish species of 10 families were identified from cijalu river. the 10 families were anabantidae bagridae, , balitoridae, , osphronemidae, and sisoridae cyprinidae was channidae, cichlidae, cyprinidae loricariidae, poecilidae . the family spec , followed by ( species), channidae having the highest number of species (6 ies) bagridae 3 (2 species) and osphronemidae a complex (2 species). the remaining respective families were represented by one species. pattern of fish distribution was observed during study. and were only found at the glyptothorax platypogon channa gacua the upper-streams, while was obtained in downstreams species could be anabas testudineus . the other remaining observed either middleto downstreams, or even from upperto downstreams. d pattern of species from the ifferent distribution could be caused by physico-chemical character s from upper to lower parts of the river, istic variation especially substratewater velocity and types. ci lu , , diversi , keywords: ja river distribution ty fish introduction diversity indicate the presence of species at s certain ecosystem. in macrotaxonomy, diversity measurement is a measure of number of species in an area, whereas in ecology, diversity is represented by species number and its abu ances. igh species diversity prove that an nd h s ecosystem is in an equilibrium state and play s important role n keeping ecosystem in an i equil br um condition. according to odum i i (1971) high diversity show by no dominant is n species in an ecosystem. biodiversity is an integral part of nation development after brundtland report was first published in 1987 (unep 1987). igh support of h international community on that report and followed by global consultation has led to the establishment of the convention on biological diversity in 1994, which provides guidance related to global issue on biodiversity and biodiversity initiative (nguyen de silva 2006) & . in general, biodiversity initiative focused on terre trial ecosystem. however, at present days, s aquatic ecosystem become among the most s discussed s topic , especially coral reef and about wetlands ecosystems. later on, resea cher are r s also interested to study biodiversity aspect of freshwater ecosystem including freshwater fish diversity (nguyen de silva 2006). previous & report showed that a total of 13,000 freshwater fish species ha been described and belong to ve 2,513 genera (du geon . 2006; leveque . d et al et al 2008). the majority of freshwater fish species inhabit tropical areas, mainly in asia (nguyen de & silva 2006). asia harbor approximately 121 s freshwater fish families, higher than those in freshwater ecosystems in africa and latin america which have only 50 and 55 fish families, respectively. on species level, 28 up to 32% of the 13,000 described freshwater fish species were biotropia vol. 23 no. 1, 2016: 1 9 1 doi: 10.11598/btb.2016.2 . .3623 1 * c orresponding author: yahoo.comanuryanto2003@ 2 recorded from asia with approximately 462 spesies are threatened. the number is equivalent to 17.5% of total number of threatened species in the world (l vequ . 2008). a sum of 66 e e et al species were critically end gered or end gered an an and 32 species of them belong to yprinidc . indonesia is among 12 sian ountries with the a c highest number of threatened fish species (nguyen de silva 2006).& high diversity of fish species in asia is especially found in big rivers and their floodplains (welcomme 2000; cbd 2003; coates . 2003). et al in is detail, asia's freshwater fish dominated by c ,yprinid group (±1 000 spesies) and followed by balitoridae and cobitidae (±400 species, recently, balitoridae divided intois balitoridae, gas t r omyzon t idae , el l opos to mat idae , vaillantellidae, barbuccidae, serpenticobitidae and nemacheilidae s; while cobitidae become cobitidae and botiidae kottelat 2012 gobiidae ( ;) (300 species), catfishes bagridae (±100 species) and osphronemidae (85 species) (nguyen de & silva 2006). specifically, kar . (2006) also et al reported that cyprinidae is a domina t freshwater n fish in india. family previous studies reported freshwater fish diversity in africa (albaret . 2004; leveque et al 1997; harrison whitfield 2006) europe & and (collares-pereira . 2002). several studies ha et al d also been done in indonesia, however, those studies were mostly performed outside of java; such as in kalimantan (haryono 2004 sulistyarto ; et al. 2007) and sumatera (duya 2008). studies in smaller rivers in java ha also been done in ve cileumeuh and cikawung rivers. a study in cileumeuh 2 spe ies river found a total of 2 fish c within families in iver, 10 . cileumeuh r cyp inidae was the speciose family with 0r 1 species bagridae and followed by with four spe ies (nuryanto . 201 ). et al et al.c 2 nuryanto (2015) also recorded a total of 19 species and nine families from cikawung river. cijalu river is located in western cilacap regency, central java . administratively, province this river belong to district with s majenang its headwater padontelu . located in mountains cijalu river empties into cijalu cileume h river. u r siver belong to citanduy watershed together with cileumeuh and cikawung rivers. there have been no data o fish diversity d sn an it distribution at ja cijalu ci lu river. in addition to the fact that river is subjected to several impacts from forestry, farming and house waste which alter the physico-chemical characteristics of the river, cijalu river is overfished. , it is therefore important to study fish diversity at river to cijalu develop database on freshwater fish species in banyumas region which is important for further study, such as conservation strategy. this study aim collect on ed to data fish diversity and distribution cijalu river, cilacap egencyat r . materials and methods c random sampling technique was lustered performed by dividing cijalu river into three clusters i.e. upper , middle and downstream (fig. 1) . amples , based on substrate types fish s were collected at sampling sites:eleven five at upper-, three at m ddleand three at downstreams i using and nets12 volt with 0.5, electr shockeric biotropia vol. 23 no. 1, 2016 figure 1 sampling sites across ci riverjalu (108 04' and 109 30', 7 03' and 7 52') o o o o notes : 1= m11 sa pling site numbers = border among partsriver's 3 0.75 and 1 inch in mesh sizes. sampling was performed from april to july 2012. electricshocker and 0.5 inch nets were used during the sampling in the upper-stream, whereas 0.75 and 1 inch nets were used at the middleand downstreams. sampling efforts performed for one were hour netting and electroshocking each siteat . biological variables and physico-chemical characteristics were examined during the study. b were number of iological variables measured fish distribution in species (s) and fish cijalu r . physic -chemical measured iver o parameters were water depth, light intensity, water temperature, water acidity (ph), c rbon dioxide, a water velocity and substrate river . methods used in measuring physic -chemical areo parameters summarized n table 1i . fresh fish directly preserved samples were in labelled plastics bag filled with 70% ethanol. direct fixation eth nol was due using a carried out to technical difficulties obtain formalinin ing . upon arrival in the laboratory, the samples were washed running water and the used soaking under ethanol was replaced the fresh 70% ethanol. by for permanent preservation, the samples were fixed inside bottles containing fresh 70% ethanol (diluted from 100% pro-analysis ethanol). samples were identified according to identification key from kottelat . (1993) and et al f b (froese pauly 2012)& .ish ase results and discussion species diversity a total of 19 fish c were spe ies of 10 families found during sampling at cijalu river (table 2) which is considered as high diversity based on guideline from ncdenr (2006). this study found lower number of species (19) compared to previous study in cimanuk river which found 40 species ( . sjafei . 2001)et al cimanuk river was chosen as compar the ison because cimanuk river is located in the same biogeographic region in the same island and is with cijalu river ifferen result between . d t s this study and study cimanuk at cijalu river the at river could be caused by different ecological characteristics river river such as length, size and annual water volumes. based on survey at cijalu river in 2012 and at cimanuk river in 2011 (nuryanto sugiharto 2011), cijalu river is & shorter and smaller than cimanuk river. cijalu river is only about 40 km in length, spanning only from orthern to outhern part of majenang n s district in cilacap regency, whereas cimanuk river has more than 100 km in length spanning from garut regency up to offshore of java the sea in indramayu regency, west java . province moreover, cijalu river is appr ximately 40 m in o width, while cimanuk river reaches 100 m in width at the lower part. in addition, cimanuk river has constant annual water volumes more than cijalu river. according to kottelat . et al (1993) longer and wider ecosystems usually are assumed to have higher microhabitat variation than smaller and shorter areas igh habitat . h variation supports high variety of inhabitants. therefore, it is reasonable that cimanuk river which is longer and wider than cijalu river has high number of species. these conditions agree with woo ton (1991) t who noted that larger streams are inhabited by higher number of species due to higher microhabitat variety than the smaller one. similar phenomenon was also reported by clavero . (2004) in 27 iberian et al mediterranean river basins, where wider rivers (tajo, guadiana and jucar rivers) support ed higher number of fish species than the r maining e twenty four smaller rivers. in addition mazeika , et al reported . (2006) that stream geomorphic, including stream size, has significant effect on fish community diversity. table 1 p o parameters mea methods hysic -chemical surement physico-chemical parameter measurement methods water depth (m) light intensity (m) water temperature (°c) water ph carbon dioxide (co2) water velocity (m/sec) substrate sechi disk sechi disk thermometer universal ph papers apha 1985 linear measurement visual ichtyofauna t cijalu river central javaa , province agus nuryanto, indonesia – et al. 4 biotropia vol. 23 no. 1, 2016 species diversity comparison to the studies outside indotropic or oriental region showed complex pattern of similarit and differences. ies for example, our study obtained similar number of species with a study clavero . carried out by et al (2004) in guadalquivir rivers. however, different phenomena were observed when comparing our study to other studies. in one hand, our species record in cijalu river was lower compared to the reported by et alstudy clavero . (2004) conducted in three iberian mediterranean river basins. other hand, a slightly higher species on the number was recorded in our study compared to a study r r t epo ted by cassa ti (2005) from the morno do diabo state park, southeastern brazil. our result also recorded higher species number than the collection by . reported t al clavero e (2004). s erethe above comparison w not equal since our study and those studies from cl v ro . a e et al (2004) and cassa ti (2005) re located in t we different biogeographic regions. these differences might cause different inhabitants. therefore, it is reasonable to find that results on fish species composition from our study conducted in indotropic or oriental region showed results from the studies different conducted in brazil (neotropic region) and europe (palearctic region) according to gaston . and williams (1997), each biogeographic region has their own organisms and most of the organisms are different among regions. this study at cijalu river found 10 famil ies i.e. anabantidae, bagridae, channidae, balitoridae, c i ch l i d ae , c y p r i n i d ae , l o r i c a r i i d a e , osphronemidae, d sisoridaepoeciliidae an . cyprinidae was the most speciose family with 6 species fol owed by with species, and , l bagridae 3 then channidae and osphronemidae with two species, respectively. the remaining six families only had one species, respectively (table ) 2 . comparison on family level also showed similarit and difference among studies. ies s our study at cijalu river recorded lower number of families compared to a study conducted at cimanuk river which recorded 20 families (sjafei et al. 2001). this difference could be caused by different ecological characteristics as previously discussed. however, both studies agreed on cyprinidae being the most speciose family high . number of cyprinids species was also repo ted r from musi river kejalo curup bengkulu with , , seven species (duya 2008). it seems that high number of cyprinids species is a common phenomenon in the river systems. this phenomenon was also reported in previous studies at different river systems either in indonesia or outside indonesia n . i rawa table fish species at ci l river2 and distribution ja u no species distribution upper middle lower 1 anabantidae a. anabas testudineus + 2 bagridae a. mystus gulio b. mystus micracanthus c. hemibagrus nemurus + + + + + + 3 balitoridae a. nemacheilus fasciatus + + 4 channidae a. channa striata b. channa gachua + + + 5 cichlidae a. oreochromis niloticus + 6 cyprinidae a. labiobarbus kuhlii b. mystacoleucus obtusirostris c. osteochilus vittatus d. barbodes binotatus e. barbodes microps f. rasbora argyrotaenia + + + + + + + + + + + + + + 7 loricariidae a. pterygoplichthys pardalis + + 8 osphronemidae a. osphronemus gouramy b. trichopodus trichopterus + + + 9 poeciliidae a. poecilia reticulata + + + 10 sisoridae a. glyptothorax platypogon + notes: = ; = absent + present family 5 lebak there wererungan river 19 cyprinidae species number recorded (sulistyarto . 2007). aet al of 26 cyp inids species was in bukit recordedr batikap central kalimantan (haryono 2002), , while a study in kayan mentarang national park, east kalimantan 19 c of recorded spe ies c prinidae java island, several y (haryono 2004). in stud also found number of cyp inids ies various s r species. ll stud reported that cyp inidae the a ies isr dominant family (cileumeuh river (nuryanto . et al 2012); cikawung river (nuryanto . 2015)). et al our present study and those previous studies agree with nguyen and de silva (2006) who reported that cyprinid group dominated freshwater fish species in asia. ahmad 2014 et al. ( ) also reported that cyprinid dominated fish species in sungkai wildlife reserve. the dominance of cyprinidae was also reported in the river basins throughout europe (reyjol . 2007). however, et al the trend of cyprinidae dominance was not observed in southeastern brazil where the speciose familiy were characidae followed by cichlidae and loricariidae no cyprinid ; there were species recorded (langeani in southeastern brazil et al. 2005). it was not possible to compare this study at cijalu river wi conductedth the study in the iberian mediterranean rivers at family level, because there was no family information availa le b in study (clavero the iberian mediterranean rivers et al. 2004). as it was discussed previously, the different results were caused by these studies being conducted in different biogeographic regions. however, the comparison itself is important to enrich our knowledge of other taxa (species and family) occurred in other biogeo raphic regions.g resultedanother important information from this study was that 3 19 species were of found non-native or introduced or exotic species i.e. pterygoplichthys ardalisp (page robins 2006; levin & et al poecilia reticulata o. niloticus et . 2008), and (paller al. 2011). therefore, high species diversity cijalu at river could not be used as an indicator of river health it is inhabited by three exotic because species which might become problem for native species, though maitland (2004) that if the al stated number of exotic species less than 25%, the is ecosystem is still .in healthy condition ofnevertheless, we have to be aware the presen of and in cijalu ce p. pardalis o. niloticus river it become a major problem in because may the future. it has been reported by maitland (2004) and pimentel . (2005) that exotic species might et al threaten native species through ecological alteration. moreover, introduced species has caused native species declining due to in numbers competition, disease transmi sion etc. (gozlan s et al. 2010). previous studies negative had shown impact of introduce species hermoso . d . et al (2011) reported that the declining numbers of native species in iberian streams was due to high abun ance of exotic species. a study from albins d and hixon (2008) had also a negative shown impact of exotic species on native with species 79% a aver ge value of reduction. it has been reported that nile tilapia in becomes competitor an ecosystem (cag uan 2007) or predator for a native species (morgan 2004). nile tilapia et al. win the competition because this species is mores a gre sive and voracious (morgan . 2004). a g s et al study from hoover . (2004) has also reported et al a negative impact of to native species p. pardalis in usa waters. in indonesia, yuniartiningisih (2011) has reported that nile tilapia has caused . .r argyrotaenia r lateristriata and declining to be in numbers in pelus river, purwokerto, central java province decrease. this was due to niche overlap among those species. in case of cijalu river, high awareness should be paid because introduced species have higher biological advantages than those native of species, so they can better adapt to aquatic habitat with poor water quality than that native of species. for instance, mouth sucker catfish ( ) live well in aquatic ecosystem with p. pardalis s low oxygen content because this species is equipped with arborescent organ for effective respiration in such ecological condition this . species is also well adapted to aquatic ecosystems with high organic matter content because it fe d e s on detritus and algae (page robin 2006). & whereas, nile tilapia ( ) well adapted to o. ni o icusl t is poor water quality ecosystems (figueredo & giani 2005). therefore, it is necessary to control the developmenet of and p. pardalis o. niloticus population in cijalu river to minimize the threat to native species. fish istributiond s f species was collectedive at the upperstreams se. ,the species were barbodes binotatus rasbora argyrotaenia, poecilia reticulata, channa gac ua glyptothorax platypogon. h and sampling conducted the middle part of ci river at jalu ichtyofauna t cijalu river central javaa , province agus nuryanto, indonesia – et al. 6 biotropia vol. 23 no. 1, 2016 obtained a 6 fish total of 1 species the obtained . species were pterygoplichthys pardalis mystus gulio, , mystus micracanthus hemibagrus nemurus hemibagrus , , nemurus, trichopodus trichopterus, , channa striata oreoch omis niloticus, labiobarbus kuhlii, mystacoleucus r obtusirostris, barbodes binotatus barbodes microps, , osteochilus vittatus rasbora argyrotaenia poecilia , , reticulata osphronemus gouramyand . at the downstream area, total of 1 fish species a 5 was found during the study, i.e. pterygoplichthys pardalis, mystus gulio mystus micracanthus hemibagrus , , nemurus nemacheilus fasciatus, trichopodus trichopterus, , channa striata, labiobarbus kuhlii, mystacoleucus obtusirostris, barbodes binotatus barbodes microps, , osteochilus vittatus rasbora argyrotaenia poecilia , , reticulata anabas testudineus and . it seems that different species compositions were observed in each part. there was also a river tendency that some species only at occurred certain part of the river, such as in g. platypogon upper-stream and in down-streamsa. testudineus areas; while the other species were widely distributed at most part , such as river s l. kuhlii and or even along the river i.e.m. obtusirostris b. binotatus r. argyrotaenia and (table 2). this complex distr bution pattern could be i caused by some species hav specific preference to certain ing ecological condition, while the other species can adapt to wide range of ecological factors. for a example, prefer ed upstream area of g. platypogon r the river with stong current and stony substrate. g. platypogon is equip ed with ventral disc to attach p itself on the substrate in strong current upperstream area. anabas testudineus typical downstream species, were found. the finding was already assumed before sampling since s ecological everal characteristics observed at the lower part of cijalu river such as average water temperature range , d from 28 to 30 ºc, ph 5.7 to 6.8, ranged from carbon di xide range between 0.1 and 0.5 ppm o d , as well as s ,muddy and sandy sub trates fit well with the need of that species. this species adapted to poor aquatic habitat since it is equipped with arborescent organ for effective respiration in such ecological condition. this finding was congruent with tay . (2006)the finding from who et al reported sa. testudineus that inhabit swamp areas, stagnant water bodies, estuaries and ponds. the finding of along part of the b. binotatus river was not surprising since this species normally inhabits wide range of habitats from strong to weak current. a similar finding was also reported in cileumeuh river (nuryanto 2012) and et al. cikawung river (nuryanto . 2015). et al b binotatus . commonly inhabit mountain stream, river and s lake. this mean that can adapt to b. binotatuss a wide range of habitat conditions. the remaining cyprinidae species was found at middleand downstream of cijalu river, ranging from stone to sandy bottom and from high to low water velocity. this means that cyp inids species were distributed in all part of r s the river. the finding was normal since it is agreed with nikolsky (1963) who has the finding from noted that cyprinidae commonly inhabits river with either high or low velocity. g platypogon . a typical upstreams species were found during the sampling. this result was different to nuryanto . (2012; 2015) who et al find g. platypogondid not in cileumeuh and cikawung rivers. the difference could be caused by different fishing gear used to collect samples at the upstream areas in the present and previous studies. the in present study, we used electricshocker during sampling at the upstream, while nuryanto . (2012; 2015) only used et al nets. both sampling gears had different effectiveness to used atbe the areaupstream . electricshocker more effective than nets is because electricshocker can induce fish in any water velocity conditions and in any fish-hiding places to . the electricity induction caused fish become weak and easily to be caught. in contrast, strong current at upstream area washed away the nets before fishes. according to even trapping lapointe . (2006), sampling ef ectivity at each et al f part of river depend upon the fishing geara s , while the gear fish electricshocker is best for sampling upstream . at area mystus nigriceps h. nemurus bagridaeand ( ) were only found at the middle and lower part of s cijalu r conducted at serayu iiver. n another study river mystus nigriceps, it has been reported that and h. nemurus along the r were distributed iver (setijanto sulistyo 2008). the differen could & ce be due to different microhabitat of cijalu and serayu rivers. fcijalu river has many plain or lat and shallow areas with sandy substrates, while serayu river has many sites with swamp and y deep areas muddy susbtrates (nuryanto with & sugiharto 2011). these types of habitats in serayu river most likely the reason that both bagrids are species were distributed along iver.serayu r 7 physico-chemical characteristics average values of physico-chemical parameters in cijalu rivers are presented in table 3. water velocity and substrates were the two parameters showing quite differences among parts of cijalu river; while the remaining parameters were almost similar among part of s the river. therefore, the discus ion focused on s species distribution comparison based on was those two parameters. fthe di ferences on water velocity and substrates were suggested to e responsib b le for different ish distribution along the river. f pattern it has been well known that water velocity or current is a key factor caus differences among ing river parts (odum 1971) affect distribution, ing movement and adaptive behaviour of riparian organisms (brown 1975). in this study, g platypogon c gac ua. . h were found and only at the u per part of the river with high water velocity p and stony substrate. this may be due to the adaptation capability of these species to river part which has strong current and stony substrate. according to ng and rachmatika (2005) glyptothorax is able to survive in strong water current because it is equipped with thoracic adhesive organ attach on stony , so that it can substrate. whereas, is adapted to strong c. gac uah current and stony substrate by hiding inside crevices or between stones. t he finding of ,p pardalis t trichopterus a . . . and testudineus parts of cijalu in the middle and lower river agreed with our expectation since those three species adapt to water ecosystem with can low oxygen content, high c rbon diox de, low a i acidity and muddy substrate. this finding was in agreement that of stating with pethiyagoda (1991) that well adapted to poor habitata. testudineus is quality. inhabit swamps areas and t. trichopterus s ponds with high density of freshwater vegetations low lim 2012). in addition, ( & pethiyagoda (1991) that reported p. pardalis, t. trichopterus a. testudineus are able and to live in poor habitat because they tohave the ability directly breathe oxygen from the air using additional respiratory organ. the remaining species were not specific site, so they were distributed almost along the river. the ir distribution along part the rivers is well s of supported ecological char cteristic which by a s are relatively d ra . mo e te, especially water velocity conclusions nineteen species offish 10 families were collected during the at cijalu riverstudy , which indicated that the river has high fish diversity. cyp inidae speciose family with six species. r is the some species were distributed along the river, while other species were limited at certain parts of the rivers. the d ce in species ifferen distribution could be due to various physicochemical characteristics parts of the river, along especially water velocity and substrate. acknowledgements we would like to thank jenderal soedirman university for the funding. we greatly appreciated students who involved during the sampling were and laboratory works. we also greatly appreciated fishermen and driver for their help during the study. tabl e 3 average value of physico-chemical characteristics of cijalu river physico-chemical characteristic upper middle lower depth (m) 0.66 0.48 0.60 light intensity (m) 0.47 0.35 0.39 temperature (°c) 28.0 27.0 30.0 velocity (m/s) 1.47 0.67 0.33 acidity (ph) 6.80 6.3 5.7 carbon dioxide (co2) (ppm) 0.1 0.5 0.3 substrate big to medium stones small stones to gravel sand to mud ichtyofauna t cijalu river central javaa , province agus nuryanto, indonesia – et al. references ahmad ab, fahmi-ahmad m, rizal sy. 2014. fish diversity in sm ll streams of sungkai wildlife reserve, perak, a malaysia. j wildlife and park 29:13-21 . albaret jj, simier m, darboe fs, jean-marc e, raffray j, de morais lt. 2004. fish diversity and distribution in the gambia estuary, west africa, in relation to environmental variables. aqua liv resour 17(1): t ing 35-46. albins ma, hixon ma. 2008. invasive indo-pacific lionfish pterois molitans reduce recruitment of atlantic coralreef fishes. mar ecol prog series 367:233-8. american public health association (apha). 1985. 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( our common future. geneva (ch the world ): commission on environment and development commission for the future. welcomme rl. 2000. fish biodiversity in floodplains and their associated rivers. in: gopal b, junk wj davis , ja, editor. biodiversity in wetlands: assessment, function and conservation, volume 1 leiden (nl): . backhuys publishers p 61–87.. wootton rj. 1991. ecology of teleost ishes, fish and fisheries 1.f london (uk): chapman & hall. yuniartiningsih s. 2011. analysis of ec logical impact of o introduced species on cyprinidae diversity. master thesis. purwokerto (id): postgraduate program of jenderal soedi man universityr . 9 ichtyofauna t cijalu river central javaa , province agus nuryanto, indonesia – et al. 4. yadi page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 2. okky ds (aspergillus).cdr biotropia vol. 20 no. 2, 2013: 81 88 aspergillus flavus population and aflatoxin b content in processed peanut products in municipality of bogor, west java, indonesia 1 okky setyawati dharmaputra *, santi ambarwati , ina retnowati and amanda windyarani received 10 february 2013/accepted 22 october 2013 the objective of this study was to document the population of and aflatoxin b content of five processed peanut products collected from different retailers in (subdistrict of central bogor), municipality of bogor. a total of 129 samples of processed peanut products were collected. the products consisted of roasted nut-in shell peanuts (33 samples), flour-coated peanut kernels (33), sauce (18), sauce (33) and sauce (12). sample size varied from 2000 g for roasted nut-in shell peanuts and flour-coated peanut kernels, to 1500 g for sauce, sauce as well as sauce samples. the samples were mixed homogeneously. it was then divided into two parts manually, and then each part was also divided into two parts to obtain working samples to determine population, afb1 content and a reserve sample. kernels of roasted nut-in shell peanuts and flour-coated peanut kernels were obtained by shelling their skin pods and removing the seed coat and the batter coat of tapioca flour manually, respectively. in peanut processed products was isolated using a serial dilution method, followed by pour plate method on aspergillus flavus and parasiticus agar (afpa). afb content was determined using thin layer chromatography method. two replicates were used for each sample.the results showed that the population of in roasted nutin shell peanuts, flour-coated peanut kernels, sauce, sauce and sauce were 0.3, 0.1, 0.3, 13.2 and 0.4 cfu/g (wet basis), respectively. the highest afb content of 43.2 ppb was found in roasted peanut nut-in shell, followed by flour-coated peanut kernels (34.3 ppb), sauce (23.2 ppb), sauce (17.1 ppb) and sauce (4.4 ppb). aflatoxin b , processed peanut products, municipality of bogor 1,2 1 1 2 1 2 seameo biotrop, jalan raya tajur km. 6, bogor 16134, indonesia department of biology, facultas of mathematics and natural sciences, bogor agricultural university, darmaga campus, bogor16680, indonesia a. flavus kecamatan bogor tengah siomay pecel/gado-gado satai siomay pecel/gado-gado satai a. flavus aspergillus flavus a. flavus siomay pecel/gado-gado satai satai pecel/gado-gado siomay aspergillus flavus, abstract 1 1 1 1key words: * corresponding author : okky@biotrop.org; okky_sd@yahoo.com doi: 10.11598/btb.2013.20.2.5 81 introduction in indonesia peanuts ( ) is the third most important secondary crop after maize and soybean (bps 2013). they are consumed as human dietary supplements and processed into various snack foods such as, roasted nut-in shell peanuts, flour-coated kernels, siomay, and sauces. according to bps (2010) in indonesia in 2009 the production of peanuts was 763 507 tonnes. in tropical and humid countries such as indonesia, fungal infection can occur before as well as after harvest. according to sauer . (1992) fungal infection after harvest could decrease the physical quality and nutritional content, discolouration of seeds (grains) and mycotoxin production, among others aflatoxin. aflatoxin can cause liver cancer in human and domestic animals, produced by certain strains of . there are four kinds of aflatoxins which are generally found in foodstuff and their processed products, i.e. aflatoxins b , b , g and g . the most dangerous of the toxins is afb1. lilieanny . (2005) reported on the percentage of infection and total aflatoxin content in roasted nut-in shell peanuts (47 samples), flour-coated peanut kernels (22), sauce (12), and (4) obtained from several factories, supermarkets, and traditional markets in bogor, malang, pati and yogyakarta. the percentages of samples infected by in roasted nut-in shell peanuts, flourcoated peanut kernels, sauce and were 38.3, 27.3, 50.0 and 100%, respectively. total aflatoxin content in the products was 1.8, 5.2, 41.6 and 20.8 ppb, respectively. in indonesia the maximum tolerable limit of afb1 content in peanuts and their processed products is 15 ppb (sni 2009). the objective of this study was to investigate the population of and afb content of five processed peanut products collected from retailers in (subdistrict of central bogor), municipality of bogor. based on rankings given by 342 respondents in 11 subdistricts of central bogor who consumed 11 processed peanut products, roasted nut-in shell peanuts was ranked as first choice, followed by flour-coated peanut kernels; , and sauces (dharmaputra . 2010). the five processed peanut products were obtained from 11 shops in (subdistrict) of central bogor from 23 july until 21 august 2009. subdistrict of central bogor was selected for conducting survey and sampling, because it is the most populated subdistrict in the municipality of bogor. roasted nut-in shell peanuts, flour-coated peanut kernels and sauce were obtained from (small shops). was obtained from traveling salesman, was obtained from both arachis hypogaea gado-gado satai et al aspergillus flavus et al a. flavus pecel enting-enting gepuk a. flavus pecel enting-enting gepuk a. flavus kecamatan bogor tengah kelurahan siomay pecel/gado-gado satai et al kelurahan pecel/gado-gado warung siomay satai 1 2 1 2 1 materials and method processed peanut products location of sampling 82 biotropia vol. 20 no. 2, 2013 warung pecel gado-gado satai siomay pecel gado-gado satai siomay siomay pecel/gado-gado satai siomay pecel/ gado-gado satai pec l gado-gado siomay satai a. flavus and traveling salesman. and are salad made from cooked vegetables. is small pieces of meat roasted on skewer. is steamed ravioli filled with meat and open on the top. , , and are served with peanut sauces. a total of 129 processed peanut products were collected. they consisted of roasted nut-in shell peanuts (33 samples), flour-coated peanut kernels (33), sauce (18), sauce (33), and sauce (12). roasted nut-in shell peanuts and flour-coated peanut kernel samples were a labeled products, while pasta sauces samplings were collected randomly without knowing their processing and their sanitations during processing. at the time of purchasing, the sauces of , and were packed separately from the main materials. locations of sampling, thekinds andnumbersofprocessedpeanutproducts arepresented in table1. each sample consisted of five portions of processed peanut products in the form of pasta ( e / , and sauces) and 2 kg (100 @ 20 g packets) of roasted nut-in shell peanuts and flour-coated peanut kernels. one portion of gadogado, siomay and satai sauces contained 75, 50 and 60 g of peanut sauces, respectively. each sample was mixed manually and homogeneously, and it was then divided into two parts manually. each part was also divided into two parts to obtain working samples for the determination of population and afb1 content, and a reserve sample. peanut kernels of roasted nut-in-shell peanuts and flour-coated peanut kernels were obtained by shelling their skin pods and peeling the batter coat of tapioca flour manually, respectively. sampling and to obtain working samples 83 table 1. location of sampling, kinds and number of processed peanut product samples location of sampling number of samples total roasted nut-in shell peanuts flourcoated peanut kernels siomay sauce pecel/ gado-gado sauce satai sauce kelurahan tegallega 3 3 2 3 1 12 kelurahan babakan 3 3 2 3 11 kelurahan sempur 3 3 3 3 2 14 kelurahan panaragan 3 3 1 3 3 13 kelurahan gudang 3 3 1 3 3 13 kelurahan kebon kelapa 3 3 2 3 1 12 kelurahan ciwaringin 3 3 1 3 10 kelurahan cibogor 3 3 3 9 kelurahan babakan pasar 3 3 1 3 10 kelurahan pabaton 3 3 2 3 2 13 kelurahan paledang 3 3 3 3 12 total 33 33 18 33 12 129 note : = no sample aspergillus flavus – et alpopulation and aflatoxin b content in processed peanut products okky s. dharmaputra .1 determination of population determination of afb1 content aspergillus flavus aspergillus flavus et al a. flavus siomay pecel/gado-gado satai aspergillus flavus a. flavus a. flavus was isolated using serial dilution method followed by plating method on aspergillus flavus and parasiticus agar (afpa) (pitt . 1983, 1992). the kernels derived from roasted nut-in-shell peanuts and flour-coated peanuts were ground. a 25 g of ground sample was placed in an erlenmeyer flask (volume 500 ml), and then sterile distilled water was added to make up the volume to 250 ml. this process resulted in dilution of 1 : 10. the erlenmeyer flask with the suspension was shaked vigorously using a shaker kottermann 4020 for two minutes as much as 250 times to obtain a homogeneous suspension. a 10 ml of the suspension was taken using a volumetric pipet, then it was placed in a separate 250 ml erlenmeyer flask containing 90 ml distilled water, to make a 1 : 100 dilution. the same step was repeated to obtain a serial dilution of 1 : 1 000.two replicates were used for each sample. a 1 ml of each dilution of each sample was transferred in a petri dish (9 cm in diameter) using a volumetric pipet, then 15 ml afpa media (45 c) was poured into the dish. three petri dishes were used for each dilution . the petri dishes were shaked manually to obtain a homogeneous dispersion of the suspension in the media, they were then incubated at room temperature (28 c) for 4 days. population of per gram kernels of roasted nut in-shell peanuts or that of flour-coated peanut kernels; , or sauces (based on wet basis, w.b.) from each replicate was determined using the following formula: x x y where: afp= population per gram each processed peanut product per replicate x = volume of each processed peanut product suspension placed in each petri dish y = dilution which gives colony separately z = mean of colony number of from three petri dishes afb1 content was determined using thin layer chromatography (aoac 2005). this method is applicable to determine 5-25 ng/g afb1 in processed peanut products. dilution is needed for higher concentration of afb1. limit of detection for this method is 0.5 ng/g, while that of recovery and precision are 87-101% and 12.6%, respectively (as rsd intra laboratory study). afb1 stock standard solution 1000 ppm was made from 1 mg crystalline afb1 using 1 ml methanol. lower concentrations of afb1 (100 and 20 ppm) was prepared from 1000 ppm stock solution and were verified using a spectrophotometer (aoac official method 970.44). extraction: as much as a mixture of 1100 g of kernels of roasted nut-in shell peanuts or flour-coated peanut kernels of each sample, 1500 ml distilled water and 22 g nacl were ground using a blender for three minutes. a 200 g of this mixture was then packed in a polyethylene bag and stored in a freezer as retain samples. about 130 g of each kernel sample which has been in the form of pasta and each sample of processed peanut o o 84 biotropia vol. 20 no. 2, 2013 afp = cfu/g (w.b.) 1 85 products in the form of sauces was placed in a 250 ml erlenmeyer flask. fifty ml nacl 2.2%, 150 ml methanol p.a. and 100 ml nhexane were added and the mixture was stirred using a magnetic stirrer for 30 minutes. it was then left for 30 minutes to obtain a good separation. as much as 25 ml methanol phase was taken using a volumetric pipette, it was then filtered and placed in a separated funnel (volume 250 ml). this part was extracted using 25 ml chloroform p.a. after separation, chloroform fraction in bottom layer was placed in a 100 ml vial. the liquid as the result from extraction was evaporated until it was almost dried. the obtained residue was dissolved again using chloroform p.a., it was then transferred into a vial and re-evaporated. before processing for tlc, the residue left after evaporation was dissolved again using 500 ul chlorofom solution p.a. identification was carried out using a chromatography tank containing eluent, i.e. chloroform p.a. : acetone p.a. (9 : 1). a 5 and 10 ul samples of aliquots were spotted on chromatography plate using a 10 ul microsyringe. on the same plate afb1 standard solutions 1-10 ul were also spotted. the known concentrations of aflatoxin b standards used were also spotted between 1-4 ul. chromatography plate was placed in chromatography tank containing eluent, it was then eluted from the bottom up to the top until the eluent attained the limit of the top. the result of elution was then dried using a hair dryer and was observed under long wave (365 nm) ultra violet length. qualitative test was conducted by comparing the retention factor (rf) of sample and standard spots, while quantitative test was carried out by comparing the fluorescence intensity of sample spot and standard spot. if afb1 was not detected, the fluorescence intensity of sample spot was compared with that of standard spot. if the fluorescence intensity of sample spot was too intense to match the standards, the sample extracts should be diluted and re-chromatographied.afb1content was determined using the following formula: aflatoxin b content (ppb) = w x z where: s = volume of afb1 standard (µl) which gives fluorescence equivalent with z ul of sample y = concentration of standard afb1 (µg/ml) z = volume of sample extract (µl) required to give fluorescence equivalent with s µl afb1 standard w = weight (g) of extracted sample v = volume of the solvent (µl) required to diluted final extract fp = dilution factor 150/25. . the percentage of roasted nut-in-shell peanuts and flour-coated peanut kernels; , and sauce samples infected by were 15.2, 6.1, 5.6, 1 1 s x y x v x fp results and discussion aspergillus flavuspopulation and afb1contentof processed peanutproducts siomay pecel/gado-gado satai a. flavus aspergillus flavus – et alpopulation and aflatoxin b content in processed peanut products okky s. dharmaputra .1 86 biotropia vol. 20 no. 2, 2013 57.6 and 8.3%, respectively, while those contaminated by afb were 42.4, 30.3,11.1, 27.3 and 16.7%, respectively. the percentage of processed peanut product samples infected by and those of non-detected afb contents are presented in table 2. all product types were infected by , but the highest population of was found in sauce, followed by and sauces, roasted nut-in shell peanuts and flour-coated peanut kernels. the range and the mean of population, afb1 content and the percentage of processed peanut product samples contaminated by afb1 exceeded 15 ppb are presented in table 3. afb1 contents in processed peanut products were varied. the mean of highest afb1 content was found in roasted nut-in shell peanuts (43.2 ppb) (table 3). the percentage of roasted nut-in shell peanuts samples containing afb1> 15 ppb was also 42.4%. the percentage of processed peanut product samples contaminated by afb1exceeded 15 ppb were as follows: roasted nut-in shell peanuts 42.4%, flourcoated peanut kernels 30.3%, sauce 11.1%, sauce 21.2%, and sauce 16.7%. 1 1a. flavus a.flavus a. flavus pecel/gado-gado satai siomay a. flavus siomay pecel/gado-gado satai table 2. percentage of processed peanut product samples infected by , contaminated by aflatoxin b , and non-detected aflatoxin b content a. flavus 1 1 * = afb1 content < detection limit for afb1 (0.5 ppb) using thin layer chromatography method table 3. range and mean of population, aflatoxin b content, and percentage of processed peanut product samples containing aflatoxin b exceeded 15 ppb a. flavus 1 1 processed peanut products number of sample of samples infected by a. flavus number (%) of sample contaminated by afb1 number (%) of non-detected afb1 content samples (0.0 ppb)* roasted nut-in shell peanuts 33 5 (15.2) 14 (42.4) 19 (57.6) flour-coated peanut kernels 33 2 (6.1) 10 (30.3) 23 (69.7) siomay sauce 18 1 (5.6) 2 (11.1) 16 (88.9) pecel/gado-gado sauce 33 19 (57.6) 9 (27.3) 24 (72.7) satai sauce 12 1 (8.3) 2 (16.7) 10 (83.3) processed peanut products range (mean) of a. flavus population (cfu/g (w.b)) range (mean) of afb1 content (ppb) samples containing afb1 exceeded 15 ppb (%) roasted peanuts with skin pods 0.0 – 3.3 (0.3) 0.0 – 316.8 (43.2) 42.4 flour-coated peanuts 0.0 – 1.7 (0.1) 0.0 – 160.0 (34.3) 30.3 siomay sauce 0.0 – 5.0 (0.3) 0.0 – 39.9 (4.4) 11.1 pecel/gadogado sauce 0.0 – 255.0 (13.2) 0.0 – 197.8 (17.1) 21.2 satai sauce 0.0 – 5.0 (0.4) 0.0 – 198.6 (23.2) 16.7 87 fungi can be killed by heating, while aflatoxins could not be easily degraded by heating, because they have high melting point. the melting point of afb1 is 267 c (buchi & rae 1969). therefore, afb1 was able to be detected on the processed peanut products used in this study, although they were heated during processing (table 2). although the percentage of samples infected by was low, the processed peanut products could be contaminated by afb1 due to the toxin produced during post-harvest handling (drying and shelling). afb1 can only be produced by toxigenic strains of . afb1 contents were lower than 0.5 ppb in some samples. the mean of highest population was found in sauce, followed by and sauces, roasted nut-in shell peanuts and flour-coated peanut kernels (table 3). dharmaputra (2010) reported that population in raw peanut kernels collected from two traditional markets in bogor was as high as 4865.8 cfu/g wet basis. in this study population in the processed peanut products were lower than those in raw peanut kernels, because the kernels were heated during processing. population in sauce was much higher compared to that in the other processed peanut products (table 3). at the time of purchasing, sauce was not ready to be consumed, but it was prepared by the seller when people come to buy . it was assumed, that was not only derived from the peanut kernels, but it also from the ingredients and tools that could have been contaminated, for example, a mortar to grind the peanut kernels or a glass jar to store the kernels could be the source of contamination.the level of population in the products could also be due to the sanitation during processing, which was not tested in this study. the mean of population in roasted nut-in shell peanuts and flour-coated peanut kernels, and sauces were very low, i.e. < 1 cfu/g (w.b.), because raw peanut kernels were already heated. he roasted nut-in shell peanuts had high aflatoxin levels as a consequence aflatoxin production could have occurred in the post-harvest storage was still found in processed peanut products, although they were heated during processing. certain fungal species have dormant structures. fungi can also contaminate processed peanut products during production, packaging and transportation. the mean of highest afb1 content was found in roasted nut-in shell peanuts, i.e. 43.2 ppb (table 3). afb1content was affected among others by the quality of the peanuts and the method of processing. bankole and eseigbe (2004) reported that 43.4% of 106 fried peanuts without oil samples collected in nigeria were infected by . the range of afb1 content of 64.2% of all samples positive contaminated by the toxin was 5 106 ppb. the peanuts were in the form of kernels and they were processed traditionally by roasting using hot sands on fire place made from clay. o a. flavus a. flavus a. flavus pecel/gado-gado satai siomay et al. a. flavus a.flavus aspergillus flavus pecel/gado-gado pecel/gado-gado pecel/gado-gado a. flavus a. flavus a. flavus siomay satai t aspergillus flavus a. flavus aspergillus flavus – et alpopulation and aflatoxin b content in processed peanut products okky s. dharmaputra .1 88 biotropia vol. 20 no. 2, 2013 conclusions acknowledgements references the population in roasted nut-in shell peanuts, flour-coated peanut kernels / and sauces were relatively low (0.1-13.2 cfu/g wet basis). the highest afb1 content was found in roasted nut-in shell peanuts (43.2 ppb), followed by flour-coated peanut kernels (34.3 ppb), sauce (23.2 ppb), sauce (17.1 ppb) and sauce (4.4ppb). to minimize aflatoxin contamination in processed peanut products, it is important to conduct a good handling practice from farmer up to table. the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the office of ( ) for the permission in conducting processed peanut products sampling, to the office of and 11 offices of belongs to for the information and cooperation during the sampling, and to mrs. elly sunarsih and mrs. ratnaningsih for their assistance in conducting afb analyses. of a. flavus , siomay, pecel gado-gado satai satai pecel/gadogado siomay kesatuan bangsa dan perlindungan masyarakat kesbanglinmas kecamatan bogor tengah kelurahan kecamatan bogor tengah 1 [aoac] association of official analytical chemist. 2005. natural toxins. di dalam: horwitz w, editor. . ed ke-18. gaithersburg: aoac. p 11. [bps] badan pusat statistik. 2013. . jakarta: bps. bankole sa, eseigbe da. 2004. aflatoxins in nigerian dry-roasted groundnuts. 34(6):268-271. büchi g, rae id. 1969. the structure and chemistry of the aflatoxins. in: goldblatt la, editor. . new york: academic pr. p 55-75. dharmaputra os, retnowati i, ambarwati s, windyarani a. 2010a. population and aflatoxin b content of raw peanut kernels collected from traditional markets in bogor, west java, indonesia. paper presented at international mycotoxin conference, penang, malaysia, 1 4 december 2010. dharmaputra os, ambarwati s, retnowatii. 2010b. dietary exposure assessment for aflatoxin b from processed peanut products in municipality of bogor, west java, indonesia. 18(1):1-8. lilieanny, dharmaputra os, retnowati i, putri asr. 2005. . (population of storage mold and aflatoxin content of processed peanut products). 10(1):17-20. pitt ji, hocking ad, glenn dr. 1983. an improved medium for the detection of and . 54: 109-114. pitt ji, hocking ad, samson ra, king ad. 1992. recommended methods for mycological examination of foods. in: samson ra, hocking ad, pitt ji, king ad, editor. . amsterdam: elsevier. p 365-368 [sni] standarnasionalindonesia. 7385:2009. 2009. . jakarta: badan standardisasi nasional. sauer db, meronuck ra, christensen cm. 1992. microflora. in: sauer db, editor. . ed ke-4. minnesota: american association of cereal chemist. p 313-340. official methods of analysis of aoac international produktivitas padi dan palawija di indonesia nutr food sci aflatoxins; scientific background, control, and implications aspergillus flavus biotropia populasi kapang pascapanen dan kandungan aflatoksin pada produk olahan kacang tanah j mikrobiol indones aspergillusflavus a. parasiticus j appl bacteriol modern methods in food mycology batas maksimum kandungan mikotoksin dalam pangan storage of cereal grains and their product 1 1 biotropiano. 5, 1991/1992: 10-14 the performance of upland rice established on alang-alang dominated area after various techniques of alang-alang control s. tjitrosemito seameo biotrop, p.o. box 17, bogor, indonesia and a. purwanto pt. monagro kimia, wisma kosgoro jl. thamrin, llth floor, jakarta, indonesia abstract pot experiment to investigate the performance of upland rice in a previously alang-alang dominated area was conducted under greenhouse condition at biotrop, bogor, indonesia from november 1986 to may 1989. the treatments were factorially combined, replicated 5 times and randomized completely. the first factor was alang-alang control consisting of 5 different techniques, i.e. (1) glyphosate applied at 2.2 kg a.e./ha; (2) imazapyr applied at 1.5 kg a.e./ha; (3) dalapon applied twice at 7.4 + 7.4 kg a.i./ha; (4) slashing followed by soil cultivation; (5) slashing of alang-alang only; while the second factor was nitrogen fertilizer at 4 different levels, i.e. (1) 0 kg n/ha, (2) 60 kg n/ha, (3) 120 kg n/ha given twice, 60 kg n/ha at planting time and 60 kg n/ha at 38 dap, (4) 180 kg n/ha given twice, 90 kg n/ha at planting and 90 kg n/ha at 38 dap. plant height (cm), tiller number/pot, productive tiller (%), panicle length (cm), spikelets/panicle, empty spikelet (%), weight 1000 grains (g) and grain yield (ton/ha) were observed. upland rice grown with zero tillage technique using glyphosate (2.2 kg a.i./ha) or dalapon (14.8 kg a.i./ha) performed as good as or even better than manual cultivation. imazapyr at 1.5 kg a.e./ha was phytotoxic to rice planted 1 month after spraying. the application of n fertilizer lower than 60 kg n/ha was not sufficient, but more than 60 kg n/ha was too high; it stimulated the production of too many tillers, with high percentage of unproductive tillers and empty grains. introduction areas under alang-alang (imperata cylindrica (l.) beauv.) have recently been utilized for transmigration schemes in indonesia. the transmigrants from java and bali used to cultivate their land before planting upland rice. this practice, however, is very tedious, especially on areas dominated by alang-alang. they are usually able to cultivate only 0.4-0.7 ha of land manually in this alang-alang dominated area. 10 the performance of upland rice established s. tjitrosemito & a. purwanto the area under alang-alang is known to be infertile, producing a very low rice yield, insufficient to support the transmigrant's family. it forces them to find additional income, and when the area is close to the forest, they join the group of illegal loggers. it is imperative to find ways to raise the transmigrant's income to improve their condition. in this context pot experiments were carried out under greenhouse condition to investigate the growth and yield of upland rice in alang-alang dominated areas. material and methods pot experiments were conducted under greenhouse condition at biotrop, bogor, indonesia from november 1986 to may 1989 to investigate the performance of upland rice grown in a previously alang-alang dominated area established by various techniques at different levels of nitrogen fertilizer. the alang-alang plants were first established in plastic pots of 20 cm diameter and 21 cm height by 5 single-node cuttings/pot for approximately 9 months. for the experiment, 100 pots were utilized. the treatments were factorially combined, replicated 5x and randomized completely. the first factor was alang-alang control consisting of 5 different techniques, i.e. (1) glyphosate applied at 2.2 kg a.e./ha; (2) imazapyr applied at 1.5 kg a.e./ha; (3) dalapon applied twice at 7.4 + 7.4 kg a.i./ha; (4) slashing followed by soil cultivation; (5) slashing of alang-alang only; while the second factor was nitrogen fertilizer at 4 different levels, i.e., (1) 0 kg n/ha, (2) 60 kg n/ha, (3) 120 kg n/ha given twice, 60 kg n/ha at planting time and 60 kg n/ha at 38 days after planting, (4) 180 kg n/ha given twice, 90 kg n/ha at planting and 90 kg n/ha at 38 days after planting. dalapon was sprayed twice at 7.4 kg a.i./ha each time at intervals of 3 weeks; the other herbicide treatments were done on the same days as the second spraying of dalapon, while slashing and cultivation treatments were carried out on the same day of seed planting. the sprayings were carried out using cp20 at high pressure with yellow nozzle calibrated to deliver an equivalent amount of 600 1/ha. upland rice (oryza sativa c.v. sentanu) seeds were selected for uniformity and planted one month after the last herbicide spraying, by dibbling 5 seeds/pot at about 2 cm deep. the plants were later thinned to 3 plants/pot. the basal fertilizer consisting of kc1 and triplesuper-phosphate (tsp) at 60 kg k2o/ha and 60 kg p2o5/ha, respectively, were given to all pots by dibbling about 5 cm deep at a distance of 5 cm from the rice hill. the nitrogen fertilizer in the form of urea was also given in the same manner. 11 biotropia no. 5, 1991/1992 during the experiment, the soil was kept moist and plants were prevented from pests by spraying with sevin 855 and from pathogens by spraying with dithane m-45. the data collected were plant height at harvest time, number of tillers, number of panicles, length of panicle, number of spikelets/panicle, percentage of empty spikelets, the yield of grain/pot at 14% moisture content, weight of 1000 grains and yield/ha equivalent. the data were analysed statistically using f-test and the means were compared with dmrt. results and discussion upland rice plants grown on pots previously treated with imazapyr at 1.5 kg a.e./ha were killed. it seems that 1.5 kg a.e./ha of imazapyr even 1 month after application was still too toxic for upland rice in this pot condition. similar results were also reported by tjitrosemito & tentamia (1986) with soybean; but it was contrary to the results of field experiments on soybean reported by tjitrosemito & suwinarno (1988), where application of imazapyr at 2.0 kg a.e./ha to alang-alang fields when planted with soybean 1 month later showed no symptoms of phyto-toxicity. it is important, therefore, to study further the behaviour of imazapyr molecules in the soil and to specify what conditions may increase or decrease its availability to plants to become toxic. the performance of upland rice is summarized in tables 1 and 2. table 1. the performance of upland rice established in pots previously occupied by alang-alang after various control techniques variables rice establishment after alang-alang control glyphosate + zero tillage dalapon + zero tillage imazapyr + zero tillage soil cultivation slashed + zero tillage plant height (cm) 85.2 c 76.9 b 0.0 a 78.1 b 69.7 b tiller number/pot 5.5 c 4.9 b 0.0 a 5.6 c 4.9 b productive tiller (%) 76.4 c 69.4 be 0.0 a 67.8 be 63.3 b panicle length (cm) 19.1 c 18.8 c 0.0 a 17.0 be 16.0 b spikelets/panicle 76.5 d 65.3 c 0.0 a 61. 8 c 55.4 b empty spikelet (%) 18.7 b 18.6 b 0.0 a 16.6 b 16.6 b weight 1000 grain (g) 23.6 b 22.9 b 0.0 a 23.0 b 23.2 b grain yield (ton/ha) 0.81 d 0.58 c 0.0 a 0.67 cd 0.39 b nb: numbers in a column followed by the same letter do not differ significantly at 5% level 12 the performance of upland rice established s. tjitrosemlto & a. purwanto table 2. the effect of n-fertilizer on the performance of upland rice established on land previously dominated by alang-alang variables rate of nitrogen fertilizer (kg n/ha) 0 60 120 180 plant height (cm) 38.5 a 53.0 b 57.2 b 58.0 b tiller number/pot 3.5 a 4.3 b 6.5 c 6.6 c productive tiller (%) 51.4 a 55.8 b 50.8 a 48.5 a panicle length (cm) 9.7 a 12.3 b 13.0 bc 13.5 c spikelets/panicle 33.1 a 64.6 b 79.5 c 81.7 c empty spikelet (%) 13.3 b 9.6 a 10.9 a 10.7 a weight 1000 grain (g) 14.3 a 15.3 c 14.4 ab 14.7 bc grain yield (ton/ha) 0.16 a 0.44 b 0.57 bc 0.65 c this local upland rice variety produces only a small number of tillers. its performance was quite well when established by zero tillage after alang-alang had been cleared using glyphosate at (2.2 kg a.e./ha). it produced 0.81 ton/ha of grain which was as good as that produced when the rice planted after alang-alang was slashed followed by manual soil cultivation. under dalapon treatment it also showed quite a good yield i.e. 0.58 ton/ha which was not different from that under manual cultivation. when alang-alang was only slashed before dibbling the seed, the rice performed badly. it produced only 0.39 ton/ha equivalent. this was expected because it had to compete against regrowth of alangalang from rhizomes. the tillering was somehow very limited under dalapon treatment. there were only 4.9 tillers/pot which was the same as that under slashing, while under glyphosate it was 5.5 tillers/pot. under glyphosate treatment, this upland rice also showed the highest percentage of productive tillering (76%) and the highest number of spikelets/ panicle. it was not surprising to see that the performance of this upland rice with grain yield of 0.81 ton/ha equivalent was the best under glyphosate treatment in this experimental condition. the effect of nitrogen fertilizer is shown in table 2. the application of nitrogen fertilizer (60-180 kg n/ha) increased the performance of upland rice. when unfertilized, rice grew short (38.5 cm) with a small number of tillers, a small part of which became productive. the panicle was also short with a few spikelets and the panicle had a high percentage of empty spikelets. morever, the grain was light, therefore, the total grain yield was the lowest among the treatments. it is clear that n-fertilizer is badly needed for this rice production. the application of 60-180 kg n/ha increased the height and the tiller number' considerably (p < 0.01); however not all of these increased tillers developed into productive tillers. when this upland rice was fertilized with 120 and 180 kg n/ha, 13 biotropia no. 5, 1991/1992 apparently too many tillers were produced to develop further into productive tillers. in fact, under 180 kg n/ha, less than 50% of the tillers produced developed into productive ones, which is worse than rice not treated with fertilizers. nitrogen fertilizer application at 60 kg n/ha increased the tiller number and the proportion of tillers developing into productive ones. n-fertilizer rate at 60 kg n/ha is probably the appropriate rate. nitrogen fertilizer application increased the number of panicles. treatments with 120 and 180 kg n/ha increased the spikelet number/panicles more than double, i.e. 79.5 and 81.7 respectively. but, again, this increase was too much to fill up the spikelet which gave lighter grain as assessed by the weight of 1000 grains. nitrogen fertilizer application at 60 kg n/ha showed the heaviest grain among the treatments, which supports further the conclusion that the appropriate n-fertilizer application is 60 kg n/ha in this condition. with the available results, it is obvious that food crops can actually be established in alang-alang dominated area using zero tillage technique, provided that alang-alang is controlled with appropriate herbicides and an appropriate rate of fertilizer is applied. references tjitrosemito, s. and tentamia. 1986. the growth performance of soybean under the influence of herbicide residue. biotrop internal report. _____, and d. suwinarno. 1988. the performance of soybean (c.v. americana) established by zero tillage technique in imperata field controlled by herbicides. biotropia 2: 12-17. 14 10.pdf 11.pdf 12.pdf 13.pdf 14.pdf 2. lulut (phylogenetical).cdr 7 iiventory on the alien invasif plants species of indonesia inventory of the invasive alien plant species in indonesia biotropia no. 25, 2005 : 60 – 73 sri sudarmiyati tjitrosoedirdjo dept. of biology, faculty of science and mathematics, bogor agricultural university, jl. raya pajajaran, bogor, and south east asian regional center for tropical biology (seameo biotrop) p.o. box 116, bogor, indonesia sudarmiyati@biotrop.org abstract an inventory of the alien plant species in indonesia based on the existing references and herbarium specimens concluded that 1936 alien plant species are found in indonesia which belong to 187 families. field studies should be done to get the complete figures of alien plant species in indonesia. based on the existing figures of the plant species, the invasive alien plant species can be identified, followed by studies on the assessment of losses, biology, management and their possible utilizations. alien plant species are imported to indonesia for cultivation, collection of the botanical garden, as experimental plants or other curiosities. aside from plants purposely imported, there are also introduced plant propagules contaminating imported agricultural products. these alien plant species can be beneficial or have a potential of being invasive. the alien cultivated species consisted of 67% of the total number. more than half of the cultivated plants are ornamental plants. some of the species are naturalized or escaped from cultivation and become wild and invasive. some other naturalized species, adapted well without any problems of invasion. there are 339 species or 17% of the species recorded as weeds. the highest record of weeds is found in the family of poaceae (57 species), followed by asteraceae (53 species) and cyperaceae (35 species). there are 6 families having more than 10 species of weeds: amaranthaceae, asteraceae, cyperaceae, euphorbiaceae, poaceae, and rubiaceae. three families have more than 100 species: asteraceae 162 species, poaceae 120 species, and papillionaceae 103 species. five species of aquatic and 20 species of terrestrial plants considered as important alien plant species in indonesia were identified and some of their distributions noted. keywords : alien plant species/invasive alien plants/weeds/environmental weeds/aquatic and terrestrial alien plants introduction indonesia, with its many large and small islands, has a great diversity of plants. however, natural disasters, logging and other activities destroy the habitat and reduce biodiversity. as people move around the world, they bring together plants with them (weber 2003). expanding global trade in agriculture, forestry and other industries that depend on raw materials has allowed the transport of species to various parts of the world including indonesia. not all of the alien species are harmful. most of them are crops or ornamental plants. some species are naturalized and adapted well to the local environment, while some become invasive. biological invasion by alien plant species constitutes one of the leading threats to natural ecosystems and biodiversity, and they also impose an enormous cost on 60 agriculture, forestry, fisheries, and other human enterprises, as well as human health. the effects of alien species on native species and ecosystems are numerous and usually irreversible. the impact is sometimes massive but often subtle. natural barriers such as oceans, mountains, rivers, and deserts that allowed the intricate coevolution of species and the development of unique ecosystems have been breached over the past five centuries, especially during the twentieth century, by rapidly accelerating human trade and travel. plane, ships, and other forms of modern transportations have allowed both intentional and unintentional movement of species between different parts of the globe, often resulting to unexpected and sometimes disastrous consequences. biotropia no. 25, 2005 the invasive plant species; some of them have been well recognized as weeds in agricultural production systems, while weeds in natural habitat have been recognized as environmental weeds. agricultural weed problems have received more attention than in other areas. weeds in agro-ecosystems differ in their ecology from invasive plants because the invaded ecosystems are different. agro-ecosystems are highly artificial and represent simple, species poor habitats with environmental homogeneity and predictable disturbance regimes. in contrast, natural habitats are mostly species-rich, environmentally heterogeneous and often unpredictable. plants invading agroecosystems represent mostly herbaceous species, often adapted to the crop system, whereas plants invading natural habitats comprise the full range of life forms (weber 2003). environmental weeds have received increasing attention, because their impact on native biodiversity has been increasingly recognized and the direct costs of controlling them have increased (groves 1999). invasive alien species are taxonomically diverse. many local species are extinct or at the risk of being out-competed by invasive alien plants species, and many native ecosystems have been irreversibly lost to invasion. alien plant species introductions might become invasive, and degrade the ecosystems as well as economic income. degraded environment due to alien plant invasions is not easy to rehabilitate. the extinction of local organisms is not easily renewable. the united nations convention on biodiversity or convention on biological diversity (cbd) which was declared in 1992 was ratified by the indonesian government in 1994. protecting our biodiversity will be very valuable to development in the future. seameo biotrop has been working on weeds for quite sometime. at present, not only weeds cause considerable agricultural losses, degrading catchments areas and fresh water ecosystem, invasive alien plant species which constitute one of the leading threats to natural ecosystem and biodiversity are studied in cooperation with the indonesian ministry of environment since 2003 (tjitrosoedirdjo & tjitrosemito 2005). 61 the records on the invasive alien plant species in indonesia have been dispersed in many publications. however, there are very few comprehensive information on invasive alien plant species. inventory of the invasive alien plant species in indonesia sri s. tjitrosoedirdjo there are some problems in studying the invasive alien plant species in indonesia i.e.: 1). people are not aware of the harmful effects of invasive alien species and the government is not prepared to handle the problems yet. 2). the limited information scattered among various institutes; fail to impress the government on the urgency of the matters although the government ratified cbd in 1994. 3). it takes a long time among the departments to lead coordination. it is now recognized that the focal point for invasive alien species is the ministry of environment which currently coordinates the activities. 4). the ministry of environment is currently studying the principles of handling invasive alien species. the objective of this work is to identify the invasive alien plant species and their distribution in indonesia materials and methods inventory and distribution a large amount of literature has been searched for inventory. the main sources of the list include the following: flora of java, flora malesiana, weeds of rice in indonesia, weed leaflets papers of the weed science society conference proceedings, various other publications in edited books and journals. other additional informations were searched from common floras and from the internet. the list included the number of families, species and the origin of each species. some of the important invasive alien plant species were analyzed for their distribution. the distributions were noted from the references and examination of the herbarium specimens conducted at the herbarium bogoriense (bo) and seameo biotrop herbarium (biot). important indonesian invasive plant species the decision on which species to consider as important is based on the availability, importance and distribution. the species included here is only a fraction of all invasive species in indonesia. only the species considered as seriously problematic and regarded to deserve attention in terms of monitoring and control are included. 62 results and discussions inventory based on the inventory of alien plant species as of january 2005, there are 1936 species belonging to 187 families extracted from the compilation of tjitrosoedirdjo & tjitrosemito 2005, as shown in table 1. the inventory was conducted from the existing references and information from the herbarium specimens. field studies were not conducted yet. if the field studies were conducted, the number of the species recorded will increase with additional records from the fields and new records will be available. new species and varieties of plants have been brought into indonesia since the colonization era. the demand to import new plants continues in a global environment of free trade and this demand is likely to increase. some of these alien species are beneficial and contribute to the quality of the indonesian life, a similar proportion have naturalized and become weeds in agricultural ecosystems or the natural environment. as indicated in table 2, the cultivated one and the ornamental plants are the highest containing 63% of the total numbers, followed by weed species 17.5%. more than half of the cultivated plants are ornamental plants. some of the alien species are naturalized or escaped from cultivation and become wild. there are 81 species whose benefits are unknown. table 1. introduced plant families in indonesia no. family alphabet name of the family no. of families no. of species 1. a acanthaceae, acoraceae, agavaceae, aizoaceae, alismataceae, alliaceae, aloaceae, amaranthaceae, amaryllidaceae, anacardiaceae, annonaceae, anthericaceae, apiaceae, apocynaceae, aponogetonaceae, araceae, araliaceae, araucariaceae, arecaceae, aristolochiaceae, aslepiadaceae, asparagaceae, asphodelaceae, asteraceae, aucubaceae, azollaceae 26 426 2. b balsaminaceae, basellaceae, begoniaceae, berberidaceae, betulaceae, bignoniaceae, bixaceae, bombacaceae, boraginaceae, brassicaceae, bromeliaceae, buddlejaceae, burseraceae, butomaceae 14 111 3. c cactaceae, caesalpiniaceae, campanulaceae, cannaceae, capparaceae, caprifoliaceae, caricaceae, caryophyllaceae, casuarinaceae, chenopodiaceae, chloranthaceae, clusiaceae, cochlospermaceae, colchiaceae, combretaceae, commelinaceae, convallariaceae, convolvulaceae, costaceae, crassulaceae, cucurbitaceae, cupressaceae, cyanastraceae, cycadaceae, cyclanthaceae, cyperaceae 26 237 biotropia no. 25, 2005 63 table 1. continued no. family alphabet name of the family no. of families no. of species 4. d dioscoreaceae, dipsaceae, dracaenaceae 3 8 5. e ebenaceae, elatinaceae, eriocaulaceae, ericaceae, erythroxylaceae, escalloniaceae, euphorbiaceae 7 56 6. f fagaceae, flacortiaceae 2 6 7. g geraniaceae, gesneriaceae, gleicheniaceae, goodeniaceae 4 15 8. h haemodoraceae, haloragaceae, hamamelidaceae, heliconiaceae, hemerocallidaceae, hyacinthaceae, hydrangeaceae, hydrocharitaceae, hydrophyllaceae, hypericaceae 10 35 9. i iridaceae 1 28 10. j juncaceae 1 1 11. l lamiaceae, lauraceae, lecythidaceae, lentibulariaceae, linaceae, loasaceae, loganiaceae, lythraceae 8 76 12. m magnoliaceae, malphigiaceae, malvaceae,marantaceae, marsileaceae, melastomataceae, meliaceae, menispermaceae, menyanthaceae, mimosaceae, molluginaceae, moraceae, moringaceae, musaceae, myoporaceae, myristicaceae, myrsinaceae, myrtaceae, 18 179 13. n nelumbonaceae, nyctaginaceae, nymphaeaceae 3 8 14. o oleaceae, onagraceae, orchidaceae, oxalidaceae 4 156 15. p pandanaceae, papaveraceae, papilionaceae, passifloraceae, pedaliaceae, philesiaceae, philydraceae, phormiaceae, phytolaccaceae, pinaceae, piperaceae, pittosporaceae, plantaginaceae, plumbaginaceae, poaceae, polemoniaceae, polygalaceae, polygonaceae, pontederiaceae, portulacaceae, primulaceae, proteaceae 22 315 16. r ranunculaceae, resedaceae, rhamnaceae, rhizophoraceae, rosaceae, rubiaceae, rutaceae 7 80 17. s salicaceae, salviniaceae, sapindaceae, sapotaceae, saururaceae, saxifragaceae, scropulariaceae, simarubaceae, simmondsiaceae, solanaceae, sterculiaceae 11 95 18. t taccaceae, tamaricaceae, taxodiaceae, theaceae, theophrastaceae, thymelaeaceae, tiliaceae, trapaceae, tropaeolaceae, turneraceae 10 18 19. u urticaceae 1 4 20. v velloziaceae, verbenaceae, violaceae, vitaceae 4 38 21. x xanthorrhoeaceae, xyridaceae 2 2 22. z zamiaceae, zingiberaceae, zygophyllaceae 3 12 total 187 1936 inventory of the invasive alien plant species in indonesia sri s. tjitrosoedirdjo 64 based on table 1, the introduced families which have 10 or more weedy species are listed in table 3. there are 17 families having more than 5 species of weeds, namely amaranthaceae, asteraceae, caecalpiniaceae, carryophyllaceae, cyperaceae, euphorbiaceae, lamiaceae, lytraceae, onagraceae, papillionaceae, polygonaceae, poaceae, rubiaceae, solanaceae and verbenaceae. three families have more than 100 species, asteraceae, poaceae and papillionaceae. the highest number was recorded from asteraceae 162 species, followed by poaceae 120 species and papillionaceae 103 species. biotropia no. 25, 2005 the highest number of cultivated plants is found in asteraceae. some 77% of the cultivated plants of asteraceae are ornamental plants. papillionaceae has 61 species of cultivated plants, followed by poaceae 42 species. the highest record of weeds is found in the family of poaceae 57 species, followed by asteraceae 53 species, cyperaceae 35 species, euphorbiaceae 16 species, rubiaceae 11 species, amaranthaceae 10 species, while the other families have only less than ten species (table 3). table 2. alien plant species in indonesia, their cultivation and weedyness no. cultivated/weedy ness number 1. cultivated 551 2. cultivated as ornamental 671 3. naturalized 252 4. escaped 60 5. weeds 339 6. curiosity 6 7. unknown 63 total 1936 origin of the alien species the alien plant species came from almost all parts of the world and continents, europe, africa, asia, australia, pacific and america. most of the species came from tropical america or other parts of america. the highest number came from america 40%, mostly from tropical america (figure 1), followed by asia 26%, and africa 12.8%. the lowest number of alien plant species came from australia and new zealand only 4%. 65 table 3. the families of introduced species with 10 or more weed species inventory of the invasive alien plant species in indonesia sri s. tjitrosoedirdjo no. family cultivated cultivated as ornamental plants naturalized escaped weeds not known total no. 1. amaranthaceae 0 3 3 0 10 0 16 2. asteraceae 19 63 20 4 53 3 162 3. caecalpiniaceae 15 22 5 0 5 6 53 4. carryophyllaceae 0 12 2 1 7 0 22 5. cyperaceae 0 2 0 0 35 2 39 6. euphorbiaceae 12 5 10 0 16 0 43 7. lamiaceae 12 10 12 0 8 0 42 8. lytraceae 5 3 2 0 7 1 18 9. onagraceae 7 0 3 0 5 0 15 10. papillionaceae 49 12 24 5 9 4 103 11. polygonaceae 6 1 2 1 5 0 15 12. poaceae 31 11 19 2 57 0 120 13. rubiaceae 15 19 2 0 11 0 48 14. solanaceae 14 5 17 2 5 1 44 15. verbenaceae 3 19 3 1 6 0 32 africa america tropic america (others parts) asia australia & new zealand europe not known 25 % 15 % 26 % 12.80 8.20 % 9 % 4 % fig 1. origin of the alien plant species in indonesia 66 important invasive alien plant species in indonesia biotropia no. 25, 2005 important invasive alien plant species in indonesia are classified into two different habitats: aquatic and terrestrial, as shown in tables 4 & 5. tabel 4. important aquatic alien invasive plants species no. species family origin 1. eichhornia crassipes (mart.) solms pontederiaceae south america 2. hydrilla verticillata (l.f.) royle hydrocharitaceae asia 3. mimosa pigra l. mimosaceae tropical america 4. pistia stratiotes l. araceae not known 5. salvinia molesta d.s. mitchell salviniaceae south america for a long time, we have been aware of the existence of infestation problems of aquatic alien plant species in open waters, such as natural lakes, man-made lakes, irrigation channels, fish ponds, and others (tjitrosoedirdjo & widjaja 1991). the first record of aquatic plants in java, bali and sumatra was made during the year 1928 to 1939 by the “deutschen limnologischen sunda expedition” which recorded over 80 species present in some open waters in indonesia (van steenis & ruttner 1933). most of the records came from the open waters in java with only few records from outside java. pancho and soerjani (1978), soerjani (1979) and tjitrosoedirdjo & widjaja (1991) reviewed that eichhornia crassipes, hydrilla verticillata, mimosa pigra, pistia stratiotes and salvinia molesta were the most important species of invasive aquatic plants in indonesia. there were some species specific to certain open waters such as polygonum barbatum which invaded ir. p.m. noor reservoir in south kalimantan (hisbi 1990); hanguana malayana invaded lake kerinci in sumatra and lake semayang in east kalimantan (staff university of indonesia 1970; sastroutomo & utomo 1985); phragmites karka invaded lake rawa danau, banten and lake curug, west java (sastroutomo & utomo 1985). the only native species is phragmites karka, while other species belong to the alien species. these alien species easily established themselves in their new environment, and spread so rapidly that native species were sometimes suppressed. this indicates that these plants possess a high power of adaptation when accompanied by a lack of natural predators. another characteristic of these plants is their rapid reproduction both vegetatively and generatively. 67 table 5. important invasive terrestrial alien plant species inventory of the invasive alien plant species in indonesia sri s. tjitrosoedirdjo no. species family origin 1. acasia nilotica (l.) willd. ex del. mimosaceae africa & asia 2. austroeupatorium inulaefolium (kunth) r.m. king & h. rob. asteraceae tropical america 3. chromolaena odorata (l.) king & h. rob. asteraceae central & south america 4. cryptostegia grandiflora r. br. asclepiadaceae india & madagascar 5. dicranopteris linearis (burm. f.) gleicheniaceae tropical america & subtropic 6. eupatorium sordidum less asteraceae mexico 7. jatropha gossypifolia l. euphorbiaceae central & south america 8. lantana camara l. verbenaceae tropical america 9. mikania micrantha kunth asteraceae central & south america 10. melastoma affine d. don melastomataceae asia 11. mimosa diplotrica c. wright ex sauvalle mimosaceae brazil 12. panicum maximum jacq. poaceae tropical africa 13. passiflora ligularis a. juss passifloraceae south america 14. pennisetum polystachion (l.) schult.) poaceae africa tropic 15. piper aduncum l. piperaceae south america 16. sida rhombifolia l. malvaceae asia 17. stachitarpeta indica (l.) vahl verbenaceae tropical america 18. stachytarpeta jamaicensis (l.) vahl verbenaceae tropical america 19. themeda arguens (l.) hack. poaceae not known 20. tribulus terrestris l. zygophyllaceae warm temperate most of the species listed in table 5 have been recorded as important weeds in agricultural crops. however, some additional species which have not been reported before are the important environmental weeds such as piper aduncum, jatropa gossiphifolia. two species, cryptostegia grandiflora, and tribulus terrestris are also included in table 5. rubber vine or c. grandiflora is included here, since it has become an important invasive species in australia after their introduction. new records in indonesia have to be monitored, although up to now, it is only reported in java. the other species t. terrestris also have to be monitored. t. terrestris is cultivated as medicinal plants in central java. the spread or escape from cultivation has to be prevented. passiflora ligularis is included due to its invasiveness in mount gede pangrango, where its climbing habit covers the crown of the trees and suppresses their growth. this species will be more problematic in the future than they are at present, if there is no effort to control it. 68 distribution of some important invasive alien plant species biotropia no. 25, 2005 the distribution of some important invasive alien plant species in indonesia are discussed in this paper. distribution of invasive alien plant species is necessary to be studied to prevent the spread to other areas and for the plant quarantine to decide which species is considered as optk a2 (organisme penganggu tumbuhan karantina a2)/ the invasive alien plant species which are found in indonesia but not widely distributed and being officially controlled and prevented from entering other parts of the islands. eichhornia crassipes it was introduced to beautify the ponds of bogor botanical garden in 1886. soon afterwards this plant had already spread all over the country (tjitrosoedirdjo & widjaja 1991). it is recorded in sumatra: south sumatra, lampung, west sumatra, jambi and north sumatra; kalimantan; south kalimantan; nusa tenggara: bali, lombok and flores; papua: jayapura and merauke. almost all open waters are infested by water hyacinth. the most well known and striking example is the water hyacinth problem at rawa pening lake, central java where the problem up to now is still unsolved. hydrilla verticillata it is recorded in three islands: java, sumatra and sulawesi. it is found in sumatra: lampung, south sumatra, west sumatra and jambi; sulawesi: south sulawesi; and no record yet from kalimantan and papua (figure 2). mimosa pigra it was first introduced in indonesia from mexico for botanical curiosity by the bogor botanical garden (thysman & binnendijk 1866). the earliest record of its presence was in 1844 from bogor, west java, (hasskal 1844) and it was spread and naturalized in java (tjitrosoedirdjo 1988). in sumatra, it was reported from solok, west sumatra and sibolangit in 1917. now it has spread widely from aceh to south sumatra (partomihardjo 1987). in kalimantan, it was reported since 1979 in samarinda at the lakes semayang, jempang and melintang as well as at the river sides of mahakam at east kalimantan (tjitrosemito 1997). in papua, it was firstly noticed in 1995 at the riverside of maro river between desa (village) poo and toray. apparently, the seeds contaminated materials and equipment transported by boat for constructing the people’s settlements at the places nearby maro river (barano 1999). m. pigra infested the riverside of maro river at the area of wasur national park up to wango river near the border of papua new guinea. in 1999 it was estimated that m. pigra covered approximately 15.6 ha, forming a discontinuous belt of 10 m wide (purba 1999). 69 as shown in figure 2 in indonesia, it has been recorded in sumatra: lampung, south, west and north sumatra, aceh; kalimantan: central, south and east kalimantan; papua: merauke: there is no record yet from sulawesi. inventory of the invasive alien plant species in indonesia sri s. tjitrosoedirdjo salvinia molesta it is recorded in java, sulawesi, sumatra, kalimantan, and papua. it is distributed in sumatra: lampung; kalimantan: south and east kalimantan; sulawesi: lake tempe; papua: danau sentani. acasia nilotica a native of africa and continental asia, it was introduced to java in 1850 and since long time out of cultivation and spread also outside java. java: west and east java; nusa tenggara: timor, and papua: wasur national park (figure 2). introduced in 1969 in baluran, east java, where a. nilotica was planted as fence to protect the teak forests near baluran national park. shortly after that, rapid expansion was begun, more than 5000 ha was occupied by a. nilotica. baluran is the only conserved savanna area left in java providing herbivore feed for banteng (bos javanicus). the invasion of a. nilotica reduces the herbage yield as well as the population of banteng. there is no record yet from sumatra, kalimantan and sulawesi. austroeupatorium inulaefolium a native of tropical america, it is commonly found in tea plantations in west java and naturalized at mount gede-pangrango. in jambi, bengkulu and west sumatra it is found in highlands. chromolaena odorata a native of central and south america, it is an aggressive invader. in central and south america, the genus chromolaena has about 165 species, but c. odorata is the only species which is now pan-tropically distributed. it is found for the first time in indonesia in 1934 in lubuk pakam, north sumatra, in tobacco plantations. after the war of independence, it had become quite common and very distinctive by its white violet flowers. it spreads very quickly all over the islands in indonesia from aceh, sumatra to papua. eupatorium sordidum a native of mexico, it was introduced in west java as ornamental plant. running wild it becomes a problem at gede-pangrango national park in west java at 1400-17 000 m altitude at the forest trails, forest-borders and waste places 70 biotropia no. 25, 2005 legend: mimosa pigra acacia nilotica hydrilla verticillata figure 2. distribution of acacia nilotica, hydrilla verticillata and mimosa pigra in indonesia mikania micrantha the genus mikania has about 400 species mainly of the warmer parts of the new world. the only indigenous species in asia is mikania cordata while mikania micrantha is the only new world species to have been introduced there. the latter species readily takes to disturbed areas and tends to be weedy (parker 1972). in 1949 it was imported from paraguay and planted in the bogor botanical garden. in 1956, this species was introduced as a non-legume ground cover in rubber plantations due to the scarcity of legume seeds. wirjahardja (1976) reported that by 1976, it occupied the greater part of rubber plantations and abandoned agricultural areas in west and east java and south sumatra. now it is widely spread in almost all the islands of indonesia. although m. cordata is a native species of southeast asia, m. micrantha is a more aggressive species and causes a lot of problems, especially the maintenance and harvest of the crop become difficult. it is also climbing the trees at the edge of the forest in open areas. in indonesia, it is now difficult to find m. cordata. their growth is suppressed by m. micrantha. the specimens can only be found at the herbarium bogoriense (bo) and biotrop herbarium (biot) (tjitrosoedirdjo 2002). 71 conclusions inventory of the invasive alien plant species in indonesia sri s. tjitrosoedirdjo based on the inventories of the existing references and herbarium specimens there are a total of 1936 alien plant species belonging to 187 families in indonesia. field studies should be done to get the complete figures of the alien plant species in indonesia. based on the existing figures of the alien plant species, the invasive alien plant species can be identified, followed by studies on assessment of losses, biology, management and their possible utilizations. most of the alien plant species are cultivated as ornamental plants. there are 67% of the total species number. the weed species consists 17.5% of the total species number. the alien plant species came from all continents of the world, the highest number of their origin was america mostly the tropical part, followed by asia, africa, europe and the smallest number from australia and new zealand. there are three families with more than 100 species, asteraceae 162 species, poaceae 120 species and papillionaceae 103 species. the highest number of the weedy species is found in the family of asteraceae followed by poaceae and cyperaceae. twenty-five important aquatic and terrestrial alien plant species in indonesia were identified and some of their distributions were noted. field studies should be conducted for completing the records. acknowledgements this work was supported by tropical biology development sub project (d.i.p. project funded by goi) through seameo biotrop 2004 and the ministry of environment, indonesia. thanks are due to ms. nenah suminah for her assistance in compiling the data and to mr. setiabudi for preparing the distribution map. references barano, s.s. 1999. persoalan dan penanganan eceng gondok, putri malu raksasa, kirinyu dan rusa di taman nasional wasur. proceeding lokakarya introduksi species asing dan permasalahannya, jayapura 29-30 july, 1999(in indonesian) . groves, r.h. 1999. environmental weeds-past, present and future. plant protection quarterly vol. 14 (3): 92-99 hasskarl, j.k.1844. catalogus plantarum in horto botanico bogoriense cultarum alter. ter lands drukkerij, batavia. hisbi, d. 1990. problems and control of polygonum barbatum in ir. p.m. noor reservoir, south kalimantan, indonesia. biotrop special publication no. 40: 63-68 mangunsoekarjo & kadnan. 1973. mikania eradication in immature rubber. proceedings 2nd indonesian weed science society conference. 72 partomihardjo, t. 1988. observation of some biological aspects of mimosa pigra to support its control in indonesia. proceeding ixth indonesian weed society conference, bogor, indonesia biotropia no. 25, 2005 purba, m. 1999. masalah penangan flora fauna eksotik di taman nasional wasur dan sekitarnya. proceeding lokakarya introduksi species asing dan permasalahannya, jayapura 29-30 july, 1999 (in indonesian). sastroutomo, s.s. and i.h. utomo. 1985. aquatic weeds problems in lake rawa danau, west java. biotrop internal report. soerjani, m. 1979. recent trend in aquatic weed management in indonesia. proceedings 7th apwss conference. supplement volume. steenis, c.g.g.j. van and f. ruttner. 1933. die pteridophyten und phanerogamen der deutschen limnologischen sunda expedition. archiv. fur hydrobiologie, suppl. band xi. tropische binnengewasser band iii. e. schweizerbartsche verlachhandlung, stuttgart. thysmann, j.e. & s. binnendijk. 1866. catalogus plantarum guae in horto botanico bogoriense colunter. ter lands drukkerij, batavia. tjitrosemito, s. 1997. infestation of mimosa pigra in mahakam river systems, east kalimantan, indonesia. weedwatcher 27:1-2. tjitrosoedirdjo, s.s. & f. widjaja. 1991. aquatic weeds management in indonesia. biotrop special publication no. 40: 25-36. tjitrosoedirdjo, s.s. 2002. notes on the asteraceae of sumatera. biotropia. no. 19: 65-84. tjitrosoedirdjo, s.s. & s. tjitrosemito. 2005. compilation of alien plant species in indonesia. research report cooperation between ministry of environment, republic of indonesia and seameo biotrop. doc. no.: biotrop/capsi/2005/1019. universitas indonesia staff. 1979. survei ekologi danau kerinci. laporan akhir tahun 1978-1979. lab. biologi wilayah, fmipa, universitas indonesia. doc. no. 05/lbw/79 (in indonesian). weber, e. 2003. invasive plant species of the world. a reference guide to environmental weeds. cabi publishing. cab international. wallingford. oxon ox 10 8de, uk. wirjahardja, s. 1976. autoecological study of mikania spp. biotrop internal report. 73 abstract introduction materials and methods inventory and distribution important indonesian invasive plant species results and discussions inventory origin of the alien species distribution of some important invasive alien plant species eichhornia crassipes hydrilla verticillata mimosa pigra salvinia molesta acasia nilotica austroeupatorium inulaefolium mikania micrantha acknowledgements references 678 irang wahyu (an snp).cdr an snp marker potentially linked to somatic embryogenesis of oil palm ( )elaeis guineensis irang wahyunanto , diana e waturangi , nurita toruan-mathius and1,2* 1 2 adi yulandi 1 1 faculty of biotechnology, atma jaya catholic university of indonesia, jakarta 12930, indonesia 2plant production and biotechnology division, pt smart tbk., jakarta 10350, indonesia received 28 december 2016/accepted 14 april 2017 abstract oil palm (elaeis guineensis) is one of the most important oil-bearing crop in the world. this crop can be vegetatively propagated only using tissue culture technique. oil palm tissue culture technique has low efficiency, with callogenesis and embryogenesis stages as the limiting factors. genetic factor has a major role in determining the success rate of these two stages. the use of molecular markers which represent the rate of embryogenesis or callogenesis has the potential to improve the efficiency of oil palm tissue culture process. in this study, snp mining was conducted on embryogenesis transcriptome data, oil palm cdna database, oil palm genome database, and oil palm snps marker database in ncbi. the objective of this study was to obtain snp marker which represents the embryogenesis potential, to be further used in marker assisted selection of oil palm ortets. one snp (emb6) showed significant association with embryogenesis rate. this snp was found in one of auxin response factor (arf) family gene. thnucleotide replacement from adenine to guanine changed the 307 amino acid from isoleucine to methionine. oil palms with adenine homozygote (a/a) pattern on the emb6 showed 8-fold higher chance to produce significantly higher embryogenesis rate than adenine-guanine heterozygote (a/g). keywords: callogenesis, elaeis guineensis, snp, somatic embryogenesis introduction oil palm (elaeis guineensis) is among the most important crop which produces vegetable oil. vegetative propagation for oil palm can only be conducted using tissue culture methods to obtain highly productive oil palm seeds (wong et al. 1997). tissue culture technique is beneficial for individual propagation of oil palm having high productivity and/or certain trait of interest. the tissue culture technique can increase oil palm production by 30% compared to the commercial seed dura x pisifera (dxp) on a large scale field trials (cochard et al. 1999; wahid et al. 2005). tissue explants of oil palm resulted from in vitro culture are developed through callus formation or normally termed as indirect embryogenesis (te-chato & hilae 2007). however, not all of the cultured explants have the potential to be developed into embryogenic callus. those explants which are not successfully developed into embryogenic callus usually form a nonembryogenic callus, that will remain in the form of callus without any possibility to develop into ramet (rohani et al. 2000). corley and tinker (2003) reported that the level of callogenesis in oil palm is still low (around 19%), while the ability to form somatic embryoid is only 3 6% (wooi 1995). one of the main factors that determine the ability of oil palm tissue culture is the genetic factor, which is indicated by the fact that some genotypes are more productive than the others (wooi 1995). identif ication of single nucleotide polymorphism (snp) in oil palm genome had been attempted in several studies. identification of quantitative trait loci (qtls) associated with callogenesis and embryogenesis in oil palm with broad range of markers from afrlps, rflps, and ssrs has been done (ting et al. 2013), but studies to determine the process of de-differentiation and embryogenesis with * corresponding author: irangwahyunanto@hotmail.com biotropia 4 2 7 153 160 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 2 678 153 specific snp markers in plant genomes are limited. in this study, the identification of candidate snp associated with somatic embryogenesis in oil palm were done in silico, which was continued to the validation process based on the phenotype data of oil palm tissue culture. this study was aimed to obtain snp marker candidates which represents the embryogenesis potential, to be further used in marker assisted selection of oil palm ortets, and thus increase the tissue culture process efficiency. materials and methods in silico selection of snps marker target genes were derived from expressed sequence tags (ests) from low et al. (2008), and lin et al. (2009) which were deposited in genbank database ey396120-ey413718 and gh635901gh637767. ests were subsequently assembled using cap3 program (huang & madan 1999). contigs and singletons were annotated to oil palm cdna database from mpob's research (genomsawit.mpob.gov.my) (singh et al. 2013), using the blast tool (altschul et al. 1990) to obtain a list of genes that was expressed during the process of embryogenesis. the candidate genes were further aligned to the oil palm genome sequence (genomsawit. mpob.gov.my) (singh et al. 2013) to get the fulllength gene sequence. the alignment process was done using sim4db program (walenz & florea 2011). the full-length gene sequence was generated using getfastabed tool in galaxy platform (quinlan & hall 2010). then, the fulllength gene sequences were used as the database for blast alignment of oil palm snp sequences obtained from mpob (genomsawit.mpob.gov. my) (ting et al. 2014), and ncbi (teh et al. 2016) as the queries (fig. 1). ten candidate snp markers were selected based on the function, expression and sensitivity of the mutation position. dna fragment sequences were aligned using unipro ugene (okonechnikov 2012). primers of each snp et al. marker were designed using primer-blast tool from ncbi, which combined the algorithm of primer3 (untergrasser 2012) and ncbi et al. blast. primer quality was analyzed and selected using netprimer tools from premier biosoft. snps marker validation thirty ortets were kindly provided by the tissue culture laboratory of pt smart tbk. these ortets were selected based on the tissue culture productivity data, including callogenesis and embryogenesis rate. genomic dna were isolated and purified from leaf tissue using nucleospin plant ii kit (macherey-nagel gmbh & co kg, duren, germany). the quality of extracted dna was measured using 1% agarose gel electrophoresis and nanodrop spectrophotometer (ther mosc ient i f ic, massachusetts, usa). the dna was amplified by figure 1 oil palm embryogenesis snp mining bioinformatics workflow 154 biotropia vol. 24 no. 2, 2017 contigs and singletons from assembly process were aligned to two sets of mpob's cdna database (genomsawit.mpob.gov.my), namely v1 and v2 (table 2). the genes were selected based on its correlation to embryogenesis function via text mining methods. selection of the embryogenesis related gene candidates' selection was according to elhiti et al. (2010), resulting in 423 embryogenesis related genes. these genes only work as reference for this study. there might still be other genes involved in embryogenesis due to the complexity of embryogenesis process. the sequences were aligned to mpob's genome database using sim4db program to locate the index position of the intact gene (intron+exon). the index position in gff3 format was then used as input to generate the intact gene's fasta sequence. the whole genomic sequence of candidate genes was used as the database to align the oil palm snp markers. from mpob's 1,766 snp positions, there were 12 snps with positive hit to target genes, 10 snps were in introns, while the other two were synonymous codon variant in exons. in ncbi, which has 112,360 snp positions, there were 1,575 positive hits to target designed primers using pcr. the pcr products were confirmed using agarose gel electrophoresis. pcr products were purified using qiaquick pcr purification kit (catalog no.28104, qiagen, hilden, germany). purified pcr products were sequenced, then statistically analyzed and compared with the callogenesis and embryogenesis productivities data using spss 20.0 (spss inc., chicago, usa). for odds ratio analysis, tissue culture productivity data (callogenesis and embryogenesis rate) were converted into categorical data (low callogenic, high callogenic, low embryogenic and high embryogenic), then compared with nucleotide variation using n cochran's and mantel-haenszel cross tabulation statistics in spss. the tissue culture productivity data variation was analyzed with one-way anova. results and discussion a total of 19,471 embryogenesis related ests were obtained from low et al. (2008) and lin et al. (2009). the ests libraries were assembled using cap3 program resulting to 13,020 sequences of contigs and singletons (table 1). table 1 ests assembly results source subjects ests contigs + singletons low et al. (2008) nec 6,498 3,760 ec 2,717 2,130 lin et al. (2009) emb 8,389 5,456 initiation 949 854 proliferation 918 820 note: source of ests: non-embryonic callus (nec), embryogenic callus (ec), embryoid (emb), embryoid initiation and embryoid proliferation table 2 embryogenesis related candidate genes number subjects contigs + singletons v1 (genes) v2 (genes) nec 3,760 2,269 1,841 ec 2,130 1,085 821 emb 5,456 3,640 3,074 initiation 854 530 437 proliferation 820 561 506 note: source of ests: non-embryonic callus (nec), embryogenic callus (ec), embryoid (emb), embryoid initiation and embryoid proliferation 155 an snp marker potentially linked to somatic embryogenesis of oil palm – wahyunanto et al. table 3 snps with positive hits to target genes snps database functional consequence amount (hits) mpob intron variant 10 mpob synonymous codon 2 ncbi intron variant 19 ncbi downstream variant 61 ncbi upstream variant 99 ncbi 3’utr variant 36 ncbi 5’utr variant 11 ncbi splice-donor variant 1 ncbi synonymous codon 34 ncbi missense variant 53 ncbi undefined 1,261 table 4 snps marker for oil palm embryogenesis snps gene amino acid variation used for further sequencing emb1 elaeis guineensis e3 ubiquitin-protein ligase ring1-like, transcript variant x2, mrna leu → ser yes emb2 elaeis guineensis auxin-responsive protein iaa10-like, mrna arg → ser yes emb3 elaeis guineensis ethylene-responsive transcription factor 1like, transcript variant x2, misc_rna ile → val yes emb4 elaeis guineensis protein phosphatase 2c and cyclic nucleotide-binding/kinase domain-containing protein, transcript variant x5, misc_rna thr → ala yes emb5 elaeis guineensis probable wrky transcription factor 70, mrna glu → gly yes emb6 elaeis guineensis auxin response factor-like, mrna ile → met yes emb7 elaeis guineensis cytochrome p450 85a1-like, transcript variant x3, mrna pro → ser yes emb8 elaeis guineensis coatomer subunit beta-1-like, mrna ile → leu → val no emb9 elaeis guineensis coatomer subunit beta-1-like, mrna pro → ala → ser no emb10 elaeis guineensis dna-binding protein bin4, transcript variant x4, mrna thr → pro → ala yes emb11 elaeis guineensis auxin response factor-like, transcript variant x1, mrna met → val → leu yes emb12 elaeis guineensis probable cellulose synthase a catalytic subunit 1 [udp-forming], mrna ile → val no emb13 elaeis guineensis glutathione s-transferase zeta class-like, mrna asp → his yes 156 biotropia vol. 24 no. 2, 2017 genes, which includes 1,261 undefined variants, 61 downstream variants, 19 intron variants, 1 splicedonor variants, 99 upstream variants, 36 3'utr variants, 11 5'utr variants, 34 synonymous codon variants and 53 missense variants (table 3). in this study, we focused on missense snps. however, the selection of snps marker had functional consequence only as a priority scale adjusting to the scope and research resources. snp variations in other functional positions remain a potential determinant of embryogenesis rate (ting . 2013). from 53 missense variant et al snps, 40 snps were eliminated because the cdna annotation of the ests did not match with the gene locus defined in the snp information. thirteen snps position used for further process were described in table 4. from all thirteen selected snps, only 10 were selected for further dna sequencing process. from dna sequencing process, we obtained data of snp variations, which were further statistically analyzed using cross tab chi square, odds ratio analysis and one-way anova (spss 20.0) (table 5). cross tab chi square was used to analyze the degree of dependency between snp variations with embryogenesis rate. one-way anova was used to observe the difference in embryogenesis rate between snp variations. odds ratio analysis was used to compare the relative odds of the snp variations to the occurrence of high embryogenesis. from 10 snp positions, only one snp, in auxin response factor family (emb6), showed significant result in cross tab chi-square and in one-way anova. odds ratio analysis showed 8 times chance of higher embryogenesis when the snp at emb6 is adenine homozygote (a/a) compared to adenine-guanine heterozygote (a/g). sample size of population in this study is still relatively small. further observation with larger population is needed for marker revalidation. there is also a possibility of other genes influencing embryogenesis of oil palm, which needs further investigation. table 5 statistical result of snp variation and embryogenesis rate gene code snp*) indels**) cross tab chi square one way anova odds ratio e3 ubiquitin protein ligase ring 1 emb1 0 no independent not significant n/a auxin responsive protein iaa 10 emb2 8 no independent not significant n/a ethylene responsive transcription factor emb3 1 no independent not significant n/a pp2c emb4 1 yes independent not significant n/a wrky transcription factor emb5 4 yes independent not significant n/a auxin response factor a emb6 3 yes dependent (p = 0.01) significant (p = 0.015) aa = 8x ag (p = 0.014) cytochrome p450 85 a 1 like emb7 0 no independent not significant n/a dna binding protein bin 4 emb10 1 no dependent (p = 0.04) not significant n/a auxin response factor b emb11 1 no independent not significant n/a glutathione s transferase emb13 1 yes independent not significant n/a 157 an snp marker potentially linked to somatic embryogenesis of oil palm – wahyunanto et al. auxin response factors (arf) family has been suggested to play a key role in regulating the expression of auxin response genes (liscum & reed 2002). gliwicka et al. (2013) found that the expression of over half of aux/iaa and arf g enes were chang ed dur ing somat i c embryogenesis in arabidopsis. auxins play critical roles in most of the major growth responses throughout different developmental stages of plants; such as organogenesis, vascular tissue differentiation, apical dominance, root initiation, and tropism; as well as cellular level processes including extension, division, and differentiation (guilfoyle & hagen, 2007; mockaitis & estelle, 2008; su et al. 2014). a large number of potentially auxin regulated candidate genes, which function in growth and developmental processes, have been identified in arabidopsis and other plant species (rosado et al. 2012; liu et al. 2014; di et al. 2015; guilfoyle 2015). arf regulation is well-studied (salehin et al. 2015). at low auxin levels, aux/iaa proteins form dimers with arfs to inhibit arf activity resulting in the repression of auxin-responsive genes. at high auxin levels, aux/iaas bind to the tir1/afb scf complex and subsequently become ubiquitinated and degraded by the 26s proteasome. the arf is then released and regulate the transcription of its target auxin response genes (wang & estelle 2014) (fig. 2). most of the arf proteins consist of an nterminal b3-type dna binding domain (dbd), a variable middle region that functions as an activation domain (ad) or repression domain auxin response geneauxin response element auxin response factor aux/iaa auxin (low auxin) auxin response geneauxin response element auxin response factor aux/iaa auxin (high auxin) off on auxin auxin auxin auxin tir1/afb scf complex ub ub ub ub proteasome ub ub auxin response geneauxin response element auxin response factor aux/iaa auxin (low auxin) auxin response geneauxin response element auxin response factor aux/iaa auxin (high auxin) off on auxin auxin auxin auxin tir1/afb scf complex ub ub ub ub proteasome ub ub figure 2 auxin signalling pathway (adopted from da costa . 2013; salehin 2015; wang & estelle 2014)et al et al. figure 3 emb6 amino acid variation position 158 biotropia vol. 24 no. 2, 2017 (rd) and a carboxy-terminal dimerization domain (ctd:domain iii/iv), which is involved in protein– protein interactions by dimerizing with auxin/indole-3-acetic acid (aux/iaa) family genes or between arfs (kim et al. 1997; guilfoyle & hagen 2007; piya et al. 2014). emb6 thwas located at the 307 amino acid, inside the conserved domain of arf family (fig. 3). the position was in the middle region which is critical in determining arf function. the variations found in this study were a/a and a/g, which change the amino acid from isoleucine to methionine. although both amino acids are hydrophobic and have similar size, methionine has a unique structure as a sulphur-containing amino acid, which might change the protein folding due to its sulphuric bond. conclusions in this study, bioinformatic analysis of snp markers was performed by comparing the snp database with the genes related to embryogenesis. one snp located in an auxin respone factor family gene showed a significant association with the embryogenesis rate of oil palm tissue culture. this snp may be used as a marker to select sample source to increase the efficiency of oil palm tissue culture process in the near future. further observation with a larger population is needed for marker revalidation. acknowledgements this research was conducted at biotechnology department laboratory; the samples and tissue culture productivity data were obtained from the tissue culture department. both places are part of the plant production and biotechnology division of pt smart tbk. references altschul sf, gish w, miller w, myers ew, lipman dj. 1990. basic local alignment search tool. j mol biol 215:40310. cochard b, durand-gasselin t, amblard p, konan ek, gogor s. 1999. performance of adult oil palm clones. in ariffin d, chan kw. sharifah, editors. emerging technologies and opportunities in the next millennium. agriculture 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8:28897. walenz b, florea l. 2011. sim4db and leaff: utilities for batch spliced alignment and sequence indexing. bioinformatics 27(13):1869-70. wang r, estelle m. 2014. diversity and specificity: auxin perception and signaling through the tir1/afb pathway. curr opin plant biol 21:51–8. wong g, tan cc, soh ac. 1997. large scale propagation of oil palm clones – experiences to date. acta hort (ishs) 447:649-58. wooi kc. 1995. oil palm tissue culture-current practice and constraints. in rao v, henson ie, rajanaidu n, editors. proceedings of the 1993 isopb international symposium on recent developments in oil palm tissue culture and biotechnology: 24–25 september 1993. kuala lumpur (my). p. 21-32 . 160 biotropia vol. 24 no. 2, 2017 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 3. nunik s (contrasting).cdr biotropia vol. 18 no. 2, 2011: 81 93 81 contrasting arboreal and terrestrial bryophytes communities of the mount halimun salak national park, west java nunik s. ariyanti and sulistijorini bryophytes are frequently neglected due to its small size and their economical value is not much known. however, a recent study (harris 2008) listed about 150 ethnobotanical species of bryophytes; about 27 percent of those species are used in traditional chinese medicine including which is used for treating nervous disorder and cardiovascular disease. this species is also found at mount gede pangrango national park (hasan & ariyanti 2004). bryophytes occupy a wide range of habitats, colonizing various terrestrial substrates, tree trunks and tree canopies. bryophytes represent important components of forest floor and epiphyte communities in many ecosystems, contributing to forest diversity, structure and ecosystem-level processes. even bryophytes especially liverworts are abundant and dominant in “cloud” or “mossy” forest. department of biology, faculty of mathematics and natural sciences, bogor agricultural university recipient of biotrop research grant 2009 / accepted 30 april 2011 bryophyte communities were compared between arboreal (trunk bases) and terrestrial habitats in primary forest mount halimun salak national park, west java. the communities were analyzed based on species diversity, abundance, and biomass. a total of 150 bryophytes species were identified, including 67 species of mosses (bryopsida) and 83 of liverworts (hepaticopsida). both bryophyte groups varied in diversity and abundance between arboreal and terrestrial communities as well as among different elevations. species diversity of arboreal habitats (116 species) was higher than that of terrestrial habitats (64 species). moss species were more abundant in terms of coverage in terrestrial habitats whereas liverworts species were more abundant in arboreal habitats. species richness in both terrestrial and arboreal habitats decreased towards higher elevation, whereas the abundance increased. : bryophytes, mosses, liverworts, terrestrial habitat, arboreal habitat abstract introduction key words rhododendron giganteum corresponding author : nuniksa@gmail.com biotropia vol. 18 no. 2, 2011 82 despite of their traditional medicinal use, bryophytes have a range of important roles to play in the environment. bryophytes may serve as substrate for other plants and offer shelter to small animals. changes in epiphytic bryophyte assemblages affect other canopy dwelling biota, such as vascular epiphytes, invertebrates, and especially foraging birds (sillet 1994; andrew . 2003). the moist environment created by the bryophytes is also quite favorable to the establishment and growth of important groups of microorganisms, such as the nitrogen-fixing blue green algae (gradstein 2001). the abundance of liverworts in “cloud” or “mossy” forest is considered an important factor in eliminating the deteriorating effect of heavy rains, including helping to prevent soil erosion and adding to hill stability (pócs 1980). bryophytes may serve as potential indicators of climate changes since they are closely assosiated with climatically sensitive habitat and ecosystem (gignac 2001). in addition, bryophytes have important contributions to forest nutrient cycling, particularly to n-cycling (longton 1984; turetsky 2003). structure and floristic composition in tropical rain forests vary considerable. the bryophyte flora of tropical rain forest changes significantly with elevation, different taxa often occur in the different forest belts. bryophytes are very useful indicators of life zones and forest types in tropical mountain regions, since they have relatively modest number of species and genera, very wide geographical ranges, and great variation in biomass (frahm & gradstein 1991). studies dealing with diversity and abundance of bryophytes are more frequently referred to temperate region and tropical america region (e.g. mcgee & kimmerer 2002; sillet . 1995; acebey . 2003; nöske . 2008) than to tropical asia. among a few ecological studies of bryophytes in tropical asia (frahm 1990; sporn . 2009; gradstein & culmsee 2010) is the diversity and abundance of epiphytic bryophytes assessed in primary and secondary submontane rain forest and cacao agroforestry in sulawesi (ariyanti . 2008). however, the study did not simultaneously compare the community structures and contributions of both arboreal (epiphytic) and terrestrial bryophyte species in the same ecosystem. although java was considered well inventoried, recent publications dealing with the bryophytes of java are very few and a modern checklist for mosses is lacking. in contrast, checklists of both mosses and liverworts of others major islands in malesian region (borneo, sulawesi, philippines) have been provided (ariyanti & gradstein 2007; ariyanti . 2009; gradstein . 2005; menzel 1988; tan & engel 1986; tan & iwatsuki 1991; suleiman . 2006). söderström . (2010) published a first modern checklist of the liverworts and hornworts of java and reported more than 600 species for the island. of these, 479 are accepted species as currently understood based on revision and monographs; the remaining species are little known or of doubtful status, or invalid names. this research examines bryophyte communities in understory forest of mount halimun, west java, indonesia. mosses of mount halimun have recently been inventoried (tan ., reinwardtia 12(3): 205-214. 2006) but liverworts and hornworts of the mountain have not yet been reported. there are two primary aims: to assess and compare bryophyte communities in two different substrate type (arboreal and terrestrial) and different altitudes of study sites. et al et al. et al et al et al et al et al et al et al et al et al et al 83 arboreal and terrestrial bryophytes communities nunik s. ariyanti .et al materials and methods results study area terrestrial sampling arboreal sampling data analysis species diversity the study took place at mount halimun which is located in mount halimun salak national park, west java province, indonesia 106º12’ 106º45’ east, 06º32 06º55 south. the four study sites were established at different altitudes: at 1730 m (site 1), 1250 m (site 2), 1100.m (site 3) and 1000. m (site 4). at each of the four study sites, a plot of 30 x 30 m was established. at each plot, terrestrial and arboreal bryophyte diversity were estimated based on species richness and abundance (cover cm and frequency %); the total biomass of the several most dominant species were surveyed; the forest stand and vascular plant characteristics were inventoried. field samplings were done between july and august 2009. methods for terrestrial diversity sampling referred to (botting . 2008) with few modifications. at each plot of 30 x 30 m, two parallel 30 m transects were established 10 m apart, along which the bryophytes were surveyed in 1 x 1 m subplot placed at five equi-distant points on the transect. each species present was recorded and collected for future identification; the coverage (cm of cover) and frequency (%) of each species occurred in the subplots were recorded based on the quadrat 20 x 30 cm. arboreal bryophytes were sampled on five selected trees per plot of 30 x 30 cm following ariyanti . (2008) and botting . (2008) with modifications. selected arboreal sampling trees supporting bryophytes are the trees of more than 20 cm of diameter breast high. five quadrats of 20 x 30 cm were placed at different directions of 0-200 m high of tree trunk. each species present on the quadrat was recorded or collected for future identification. the coverage (cm cover) and frequency (%) of the species in a quadrat of 20 x 30 cm were also recorded. species identification was done using the following literature: eddy (1988; 1990; 1996) and bartram (1039) for the mosses, draft version of gradstein (2011) for the liverworts. an analysis of variance (anova) with the main fixed effects of altitude (plots at different altitude) and substrates type (arboreal and terrestrial) were used to evaluate bryophyte communities in the understorey forest with respect to species richness, coverage. in total, 150 bryophytes species were identified across the terrestrial and arboreal (trunk bases) habitats in the primary forest of the mount halimun salak national park. they consist of 67 species of mosses (bryopsida) and 83 species of ’ ’ 2 2 2 2 2 et al et al et al 84 liverwort (hepaticopsida). in this research we did not found any hornwort species (antocerotopsida) in the plots. hornwort commonly occurs in humid, non-forest terrestrial habitats such as river bunk or the open slope of forest margins. arboreal bryophytes with 116 species found on the base of tree trunks seems more diverse than those of terrestrial bryophytes with 64 species, identified from various substrates such as rotten log, root of trees, rock, and humus. thirty three species were identified from both terrestrial and arboreal substrates. the lists of species found in the study sites is presented in appendix 1. the total number of species found in the plots of 30 x 30 m ranged from 46 species in plot iii to 79 species in plot iv. species richness of arboreal bryophytes in the plots was higher than that of terrestrial bryophytes and varied from 38 to 64 (fig. 1). among the arboreal bryophytes, the number of liverworts species in each plot mostly was higher than those of mosses species. on the other hand, the terrestrial bryophytes composed of more mosses than liverworts species. species richness and abundance biotropia vol. 18 no. 2, 2011 26 9 32 21 9 27 22 7 25 41 7 47 12 15 22 18 9 24 16 9 21 23 16 32 0 10 20 30 40 50 60 70 80 a t a+t a t a+t a t a+t a t a+t plot i plot ii plot iii plot iv figure 1. the species number of liverworts ( ) and mosses ( ) on arboreal (a) and terrestrial (t) substrates in plot i, ii, iii, and iv (30 x 30 m) that located at different altitudes � � sp ec ie s ri ch n es in th e p lo ts o f 30 x 30 m 0 3 6 9 12 15 18 21 plot i plot ii plot iii plot iv figure 2. the average of species number of liverworts ( ) and mosses ( ) of arboreal bryophytes in each tree trunk base in plot (30 x 30 m) i, ii, iii, and iv � � t h e av er ag e o f sp ec ie s n u m b er 85 terrestrial bryophytes arboreal bryophytes plot / species coverage cm 2 plot / species coverage cm 2 plot i plot i hypnodendron sp. 1 60.1 ± 17.8 syrrhopodon tristichus 119.7 ± 61.2 trismegistia regida 48 ± 33.2 plagiochila frondescens 95.5 ± 52.4 bazzania sp. 1 24.8 ± 15.5 schistochila sciurea 95.3 ± 50.2 pogonatum macrophyllum 24 ± 24.0 bazzania tridens 94.4 ± 52.6 trichosteleum elegantissimum 19 ± 14 .0 plagiochila dendroides 85.3 ± 49.5 bazzania vittata 18.0 ± 12.4 bazzania vittata 81.2 ± 43.7 plot ii plot ii callyscostella papillata 58.6 ± 29.3 spruceanthus polymorphus 21.4 ± 10.7 heteroscyphus argutus 26.9 ±15.66 plagiochila sciophyla 18.8 ± 12.4 lejeunea anisophylla 18.6 ± 18.6 heteroscyphus argutus 17.7 ± 9.6 telanarea neesii 15.8 ± 10.9 exostratum blumei 14.2 ± 13.5 vesicularia reticulata 15.5 ± 9.2 leucophanes massartii 14.2 ± 10.9 distichophyllum schmidtii 11.8 ± 8.3 plagiochila javanica 12.2 ± 8.5 plot iii plot iii callyscostella papillata 65.8 ± 57.4 lejeunea anisophylla 36.0 ± 18.2 heteroscyphus argutus 30.8 ± 26.4 mitth yridium junquilianum 28.9 ± 15.9 achanthorrinchium papillatum 30.5 ± 18.4 radula javanica 20.0 ± 9.6 vesicularia reticulata 24.3 ± 16.3 mitthyridium flavum 19.4 ± 10.1 lejeunea anisophylla 23.3 ± 18.4 acanthorrhyncium papillatum 10.4 ± 7.0 telanarea neesii 18.6 ± 15.5 lepidozia wallichiana 10.2 ± 10.2 plot iv plot iv trichosteleum boschii 33.1 ± 19.0 syrrhopodon muelleri 19.6 ± 13.3 achanthorrynchium papillatum 31.7 ± 31.7 plagiochila propingua 14.5 ± 10.8 isopterygium albesce ns 17.6 ± 12.5 thysananthus retusus 13.9 ± 8.8 heteroscyphus argutus 14.0 ± 7.1 radula javanica 13.1 ± 8.8 isopterygium bancanum 12.1 ± 9.0 pyrrobryum spiniforme 12.7 ± 12.7 chaetomitrium lanceolatum 6.5 ± 6.5 acroporium lamprophyllum 9.4 ± 9.0 table 1. the six highest coverage (cm , ± standard error) of terrestrial and arboreal bryophytes species in quadrates of quadrat (20 x 30 cm) in plot i, ii, iii, and iv. see also appendix 1. 2 bryophytes found at trunk bases (0-2 m) ranged from 11 to 28 species. each tree trunk base mostly had more liverworts (6 13 species) than mosses (5 6 species) (fig. 2). though the difference was not significant (anova, n=5, p=0.05), the number of liverworts and mosses in plot iv was slightly higher than those in other plots. on the contrary, plot ii has lower species richness than the other plots. species abundance of bryophytes that were estimated based on bryophytes coverage on the substrate showed that the most dominant species of terrestrial bryophytes (indicated by high coverage) is mostly included in the group of mosses, whereas most of arboreal bryophytes having high coverage are the liverworts species. in common, coverage of arboreal species is higher than that of terrestrial species (table 1). though species richness in the plot iv was higher than that of other plots, the coverage of species in plots iv was lower than those in other plots. the arboreal species with the highest coverage in plot iv is which covered aboutsyrrhopodon muelleri arboreal and terrestrial bryophytes communities nunik s. ariyanti .et al 86 biotropia vol. 18 no. 2, 2011 table 2. the species of arboreal and terrestrial bryophytes occurred at least 20% of sampled quadrates (n = 25) in plot i, ii, iii, and iv. see also appendix 1. arboreal bryophytes terrestrial bryophytes plot / species frekuency of occurance (%) plot / species frekuency of occurance (%) plot i plot i syrrhopodon tristichus 80 hypnodendron sp. 1 40 leucobryum javense 40 trichosteleum elegantissimum 24 bazzania vittata 40 plagiochila frondescens 36 plagiochilion oppositum 36 schistochila sci urea 28 plagiochila dendroides 24 radula javanica 20 plot ii plot ii lejeunea anisophyla 28 callyscostella papillata 20 lopidium trichocladon 24 heteroscyphus argutus 20 heteroscyphus argutus 24 spruceanthus polymorphus 24 pinnatella sp. 1 20 radula multiflora 20 plot iii plot iii lejeunea anisophyla 44 trichosteleum boschii 24 mitthyridium junquilianum 24 harpalejeunea filicuspis 24 lejeunea discreta 24 radula javanica 24 leucophanes octoblepharoides 20 mitthyri dium flavum 20 mitthyridium wallisii 20 thysananthus spatulistipus 20 plot iv plot iv leucophanes octoblepharoides 28 heteroscyphus argutus 20 heteroscyphus argutus 28 schistochila aligera 28 cheilolejeunea ceylanica 24 acroporium lam prophyllum 20 leucophanes massartii 20 plagiochila bantamensis 20 radula javanica 20 20 cm (3.3%) of the quadrats. the arboreal species with highest coverage is in plot i that covered 120 cm (20%) of the quadrates. the six most abundant species in the plot i covered more than 80 cm (13.3%) area of quadrate, whereas the six most abundant species in others plot cover less than 40 cm (6.7%) of the quadrates (table 1). distributions of species in the plots are mostly rare. most species were found in only one of 25 sampled quadrates. some species are found only twice and others more often in the quadrates (appendix 1). only two species were found in more than 10 quadrates. the common species found in at least 20% of the sampled quadrates are 2 2 2 2 syrrhopodon tristichus 87 listed in table 2. liverwort species are more numerous in the arboreal plots (16 species, of 26 species in total), whereas mosses are more numerous in the terrestrial plots (4 of 5 in total). among the species which have high frequency of occurrence is the epiphytic mosses in plot i which were found at twenty quadrates. the frequency of occurrence of the species is about 80 %. other species were often found in the plot are , , (both species are epiphytic species in plot i) sp. 1, and (both are terrestrial species in plot i and ii respectively), all of those species have 40 % of the frequency of occurrence (table 2). the bryophyte diversity at natural primary forest of the mount halimun salak national park hutan is quite high, it comprises no less than 150 species found across the arboreal (trunk bases) and terestrial habitates. the arboreal diversity is almost as high as in the submontane forest area of lore lindu national park, central sulawesi, where ariyanti . (2008) found about 169 bryophytes species on trunk bases in twelve plots. the slightly higher figure for central sulawesi is perhaps because the research in central sulawesi was conducted in three forest types (natural primary forest, secondary forest, cacao agroforest), while the present research was conducted in primary forest only. the result of arboreal species (116 species) that were inventoried from 20 sample trees was higher compared with the number of species collected from fewer tree samples and reported for primary submontane (1100 m), lower montane (1400 m) and upper montane (3250 m) forest in lore lindu park, central sulawesi, by gradstein & culmsee (2010). having the same number of tree samples, ariyanti . 2008 collected 112 species from primary submontane (1000 m) forest in lore lindu national park. the arboreal diversity would have been much higher when the whole trees would have been inventoried. sporn . (2009) collected more epiphitic species than ariyanti . (2008) from the same locations studied by ariyanti . (2008). however, sporn . (2009) sampled the whole trees (the tree trunk and canopy). when species richness of arboreal and terrestrial bryophyte are compared, the richness of terrestrial bryophytes is lower than that of arboreal species in all the plots. it is not in accordance with the richness of terrestrial bryophytes at the temperate spruce forest which species richness of terrestrial bryophyte exceeds those of arboreal bryophytes (botting . 2008). tropical rain forests are characterized by high diversity of big tree plants which offers various habitats for epiphytic bryophytes, it perhaps relates to the high diversity of arboreal bryophytes at tropical forest than those at temperate forest in which commonly dominated by several tree species. on the other hand, understorey habitat are covered by canopy forest that determined light intensity penetrating to the ground for bryophytes photosynthesis. more over, the low rate of litter decomposition in the tropical forest may affect the substrates availability for terrestrial bryophytes. the terrestrial substrates in the understorey of primary syrrhopodon tristichus leucobryum javense bazzania vittata hypnodendron taxithelium isocladum et al et al et al et al et al et al et al discussion arboreal and terrestrial bryophytes communities nunik s. ariyanti .et al 88 biotropia vol. 18 no. 2, 2011 forest in the mount halimun salak national park were restricted, since the ground was mostly covered by leaf litters, herbs and scrub plants. terrestrial bryophytes were found growing in mats or clusters at patched substrates such as rotten log, humus, or rock at slightly open area in the forest understorey. the greater richness of the arboreal habitat than the terrestrial habitat has been discussed by numerous authors (richards 1984; gradstein & pocs 1989; frahm 1990; frahm & gradstein 1991; gradstein . ; gradstein & culmsee 2010). richards (1984) mentioned that bryophytes may be absent on undisturbed soil in lowland rain forests and only locally common in montane forests. gradstein & culmsee (2010) also reported that for the arboreal species: liverworts species richness increased toward higher elevation, whereas moss richness decreased. bryophytes may represent important components of forest floor and epiphytes communities in the forest ecosystem, contributing high number of species to forest diversity. vegetation analysis of corridors forest at the mount halimun salak national park conducted by sambas and purwaningsih yield about 27 36 species of tree plants in the plot of 0.2 ha (http://www.tnhalimun.go.id/document.php/ document/article/49/38/). based on this research it appeared that the richness of bryophytes may exceed that of the tree plant. based on the species number in the plots, it is estimated that bryophytes may contribute about 46 79 species to the plants community in the forest of the mount halimun salak national park. though the difference was not statistically significant (anova, n=25, p=0.05), species richness in the plot at higher altitude (plot i, 1730 m) tend tobe lower than those at the lower altitudes (plot iv, 1000 m). the differences in species richness was also reported by gradstein and culmsee (2010) that occurred between lower montane and upper montane rain forest of lore lindu national park in central sulawesi. on the other hand, species abundance of bryophytes at higher altitude was higher than those at lower altitudes. the result is in accordance with that of tropical american forest that the coverage of bryophytes increased with the increase of the altitude (gradstein & pocs 1989) and also observed between lower montane and upper montane rain forest of lore lindu national park (gradstein & culmsee 2010) some terrestrial species that have highest coverage in the plots were the mosses species of , , dan . those three species were common species, they were often found in the plots (with the frequency of occurrence of about 20 % and they are included in the family hypnodendraceae, hookeriaceae, and sematophyllaceae, respectively. the most dominant arboreal species in terms of coverage are , (calymperaceae), , (lejeuneaceae), , , and (plagiochilaceae). the moss species of and the liverwort species of were also the most common species which occurred in 40 % of the 25 quadrate samples. this result is relevant to the bryophytes commonly found in oligophotic habitat such as the understorey forest consisting of many dendroid, feather or bracket-type of mosses and liverworts which are specific of this type habitats. these bryophytes belong to the family of hookeriaceae, pterobryaceae, neckeraceae, plagiochilaceae, and lejeuneaceae (gradstein & pocs 1989). et al 2001 hypnodendron calicostella papillata trichosteleum boschii syrrhopodon tristichus syrrhopodon muelleri, mitthyridium junquilianum spruceanthus polymorphus lejeunea anisophylla plagiochila frondescens plagiochila sciophyla plagiochila frondescens syrrhopodon tristichus lejeunea anisophyla 89 conclusions acknowledgments references bryophytes communities at primary forest of the mount halimun salak national park comprised about 150 species of liverworts and mosses found across various terrestrial and arboreal substrates. the species diversity of arboreal bryophytes at 900 1730 m is higher than those of terrestrial bryophytes. forest at the higher elevation tends to have lower species richness than that at lower elevation. in the contrary, the bryophytes abundances are higher at high elevation compared to that at lower elevation. we would like to thank prof. s. robbert gradstein (museum national d'histoire naturale, department systematique et evolution) for the valuable suggestions and review of this manuscript. indah wahyuni, dian apriani, marinda sari sofiyana, and saiful bachri assisted us with collecting samples in the field. funding for this study was provided by research project of dipa-biotrop dikti 2009. a a . a cebey c, gradstein sr, kromer t. 2003. species richness and habitat diversivication of bryophytes in submontane rain forest and fallows in bolivia. journal of tropical ecology, 18: 1-16. ndrew nr, rodgerson l, dunlop m. 2003. variation in invertebrate bryophyte communitiy structure at different spatial scales along altitudinal gradients. journal of biogeography, 30: 731-746 riyanti ns, bos mm, kartawinata k, tjitrosoedirdjo ss, guhardja e, gradstein sr. 2008. bryophytes on tree trunks in natural forests, selectively logged forests and cacao agroforests in central sulawesi, indonesia. biological conservation, 141: 2516-2527. aryanti ns, gradstein sr, sporn sg, angelika e, tan bc. 2009. catalogue of the bryophytes of sulawesi, suppplement 1. blumea, 54: 287-289. aryanti ns, gradstein sr. 2007. wallace's line and the distribution of the liverworts of sulawesi. cryptog. bryol. 28: 3-14. bartram eb. 1939. . vol. 68. manila: bureau of printing. botting rs, campbell j, fredeen al. 2008. contrasting arboreal and terrestrial macrolichen and bryophyte communities in old-growth sub-boreal spruce forests of central british columbia. the bryologist, 111(4): 607-619. eddy a. 1988. vol. 1, sphagnales to dicranales. london: british museum. eddy a. 1990. vol. 2, leucobryaceae to buxbaumiaceae. london: the natural history museum. eddy a. 1996. vol. 3, splachnobryaceae to leptostomataceae. london: the natural history museum. frahm jp, gradstein sr. 1991. an altitudinal zonation of tropical rain forest using bryophytes. journal of bryogeography. 18: 75-78. frahm jp. 1990. the ecology of epiphytic bryophytes of mt. kinabalu, sabah (malaysia). nova hedwigia, 51: 121-132. the philippine journal of science a handbook of malesian mosses. a handbook of malesian mosses. a handbook of malesian mosses. arboreal and terrestrial bryophytes communities nunik s. ariyanti .et al 90 biotropia vol. 18 no. 2, 2011 gignac ld. 2001. bryophytes as indicators of climate change. the bryologist, 104: 410 420. gradstein sr, churchill sp, salazar an 2001. guide to 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of the hattori botanical laboratory, 55: 147-163. mcgee gg, kimmerer rw. 2002. forest age and management effect on epiphytic bryophyte communities in adirondack northern hardwood forest, new york, usa. canadian journal of forest research 32: 1562-1576. menzel m. 1988. annotated catalogue of the hepaticae and anthocerotae of borneo. journal of the hattori botanical laboratory 65: 145-206. nöske n, hilt n, werner f, brehm g, fiedler k, sipman hj, gradstein sr. 2008. disturbance effects on diversity in montane forest of ecuador: sessile epiphytes vs. mobile moths. basic and applied ecology. pócs t. 1980. the epiphytic biomass and its effect on the water balance of two rain forest types in the uluguru mountains (tanzania, east africa). acta botanica academiae scientiarum hungaricae, 26:143-167. richards pw. 1984. the ecology of tropical forest bryophytes. : schuster rm. (ed.) new manual of bryology, nichinan: hattori botanical laboratory, p. 1233-1270. sillet ts. 1994. foraging ecology of epiphyte-searching insectivorous birds in costa rica. condor, 96: 863-877. sillett s, gradstein sr, griffin d. 1995. bryophyte diversity of tree crowns from cloud forest and pasture in costa rica. 98: 251-260. söderström l, gradstein sr, hagborg a. 2010. a checklist of the hornworts and liverworts of java. phytotaxa 9: 53-149. sporn sg, bos mm, keßler m, gradstein sr. 2009. vertical distribution of epiphytic bryophytes in an indonesian rainforest. biodiversity and conservation, 19: 745-760. suleiman m, akiyama m, tan bc. 2006. a revised catalogue of mosses reported from borneo. journal of the hattori botanical laboratory, 99:107-183. tan bc . 2006. mosses of gunung halimun national park, west java, indonesia. reindwartia, (12): 205-214. tan bc, engel jj. 1986. an annotated checklist of philippine hepaticae. journal of the hattori botanical laboratory 60: 283-355. tan bc, iwatsuki z. 1991. a new annotated philippine moss checklist. harvard papers botany, 3:1-64. turetsky mr. 2003. the role of bryophytes in carbon and nitrogen cycling. the bryologist, 106(3):395-409 in in ficus the bryologist, et al , , , , , . appendix 1. alphabetical list of arboreal and terrestrial bryophyte species collected in the plots. a = arboreal; t = terrestrial; + = present; = absent. species habitat plot i plot ii plot iii plot iv mosses: acanthorrhyncium papillatum a, t + acroporium diminutum a, t + + + acroporium hamulatum t + acroporium lamprophyllum a, t + + + + acroporium rufum a, t + + + acroporium sec undum a + acroporium sigmatodontium a, t + + callyscostella papillata t + + chaetomitrium lanceolatum t + dicranoloma blumii t + dicranoloma braunii a, t + + dicranoloma brevisetum a, t + + + distichophyllum schmidtii t + distichophyllum sp. 1 t + + ectropothecium sp. 1 t + + exostratum blumei a,t + + fissiden crassinervis var. laxus t + fissidens gedehensis a + fissidens hollianus a, t + floribundaria floribunda a, t + himantocladium plumula a + homaliodendron flabellatum a + homaliodendron javanicum a + hypnaceae sp. 1 t + hypnodendron sp. 1 t + isopterygium albescens a, t + + + isopterygium bancanum a, t + isopterygium minu tirameum a, t + + leucobryum bowringii a + leucobryum candidum t + leucobryum javense a, t + + + + leucobryum juniperoideum a + leucophanes massartii a, t + + + leucophanes octoblepharoides a, t + + + lopidium sp. 1 a + lopidium struthiopteris a + lopidium trichocladon a + mitthyridium flavum a + mitthyridium junquilianum a, t + mitthyridium wallisii a, t + neckera tjibodensis a + pelekium velatum a, t + pinnatella anac amptolepis a + pinnatella cf. ambigua a + pinnatella microptera a + pinnatella sp. 1 a + pogonatum macrophylum t + pterobryopsis gedehensis a + pyrrobryum medium a + pyrrobryum spiniforme a + + + 91 arboreal and terrestrial bryophytes communities nunik s. ariyanti .et al species habitat plot i plot ii plot iii plot iv symphyso dontella attenuatula a + symphysodontella cylindrica a + syrrhopodon albovaginatus a + syrrhopodon muelleri a + + syrrhopodon prolifer a + syrrhopodon tristichus a, t + + taxithelium instratum a + taxithelium is ocladum t + trichosteleum boschii t + + trichosteleum elegantissimum a, t + + trimegistia calderensis a + liverworts: acromastigum divaricatum a + + aneura pinguis t + archilejeunea planiuscula a + bazzania intermedia a + + + bazzania sp. 1 a + bazzania sp. 2 a + bazzania sp. 3 a + bazzania sp. 4 a + bazzania tridens a + + + bazzania vittata a, t + + + bazzania wallisiana a + calypogaea goebelii a + ceratolej eunea belangeriana a + cheilolejeunea ceylanica a, t + + cheilolejeunea imbricata a + + cheilolejeunea longiloba a + + chiloscyphus minor a + chiloscyphus muricatus a + cololejeunea sp. 1 t + drepanolejeunea angus tifolia a + drepanolejeunea ternatensis a + + frullania apiculata a + + harpalejeunea filicuspis a + harpalejeunea filicuspis t + herbertus dricanus a + heteroscyphus argutus a, t + + + heteroscyphus coalitus t + heteroscyphus succulenthus a, t + + heteroscyphus zollingeri t + + jubula sp. 1 t + kurzia gonyotricha a + lejeunea anisophyla a, t + + + + lejeunea discreta a + + + lejeunea eifigri a + lejeunea exilis a + lejeunea obscura a, t + + + lejeunea punctiformis a + lejeunea sordida a + + lejeunea sp. 1 a + + lepidolejeunea bidentula a + appendix 1. continued 92 biotropia vol. 18 no. 2, 2011 appendix 1. continued species habitat plot i plot ii plot iii plot iv lepidolejeunea integristipula a + lepidozia hasskarliana a + lepidozia trichodes a + lepidozia wallichiana a, t + + leujenea anisophyla a + lophocolea bidentata a, t + + lophocolea minor t + lopholejeunea eulopha a + lopholejeunea horticola a + lopholejeunea nigricans a + lopholejeunea subfus ca a + lopholejeunea zollingeri a + + mastigolejeunea auriculata a + metalejeunea cuculata a + metzgeria leptoneura a + metzgeria sp. 1 a + plagiochila bantamensis a + + plagiochila dendroides a + + plagio chila frondescens a + + + plagiochila javanica a + + plagiochila junghunhiana a, t + + + plagiochila obtusa a + plagiochila parvifolia t + plagiochila pleurata a + plagiochila propingua a + + plagiochila sciophyla a, t + + + plagiochilion oppositum a + + pticanthus striatus a + radula javanica a + + + radula multiflora a + + radula retroflexa a + + + riccardia sp. 1 t + schistochila aligera a + schistochila doriae a + schistochila sciurea a + spruceanthus polymorphus a + telenarea neesii a, t + + + thysananthus retusus a + thysananthus spatulistipus a + + trichocolea tomentella a, t + + zoopsis liukiuensis t + 93 arboreal and terrestrial bryophytes communities nunik s. ariyanti .et al microsoft word 65 biotropia no. 19, 2002 : 65 84 notes on the asteraceae of sumatera sri sudarmiyati tjitrosoedirdjo dept. of biology, faculty of science and mathematics, bogor agricultural university, jl. raya pajajaran, bogor and south east asian regional center for tropical biology (seameo biotrop) p.o. box 116, bogor, indonesia. abstract an account of the tribe composition, endemic taxa, comparison with adjacent areas and weedy asteraceae of sumatera is given. based on the records of january 2000, there are 133 species of 74 genera in 11 tribes. the tribe heliantheae is the largest, with 28% of the total number of the genera, followed by astereae with 15%, inuleae 12%, senecioneae 10%, anthemideae, eupatorieae and lactuceae 8%, the other tribes are represented by 4% or less. the most diverse genus is blumea with 14 species. other genera are only represented by 10 species or less, usually 4, or 3, or 2, and mostly by 1 species only. thirty nine or about 53% are exotic genera and the native ones are less than half of the total number of the genera. in terms of indigenous and endemic species, sumatera is richer than java. there are 1 genus, 7 species and 2 varieties of asteraceae endemic to sumatera. a number of 43 important weed species were introduced from tropical america, africa, asia and europe. among these chromolaena odorata and mikania micrantha are reported as the most noxious ones. list of the genera and species recorded in sumatera is provided in this paper. key words : asteraceae/sumatera/compositions/endemic species/distribution/weedy asteraceae introduction asteraceae is one of the largest families of flowering plants which has not been revised for the flora malesiana (ross 1993). this paper is a report on the results of the study on the asteraceae of sumatera based on the records up to 2002. fundamental work of this family was done by cassini (1817,1818, 1826-1834), and bentham (1873) and bentham & hooker (1873). bentham's tribal classification has stood the test of time, and some modifications were introduced by hoffmann (1890-1894), dalla tore and harms (1907) and melchior (1964). although the 13 tribes recognized by bentham & hoffman have been largely accepted up to the present, they are obviously in need of modification considering recent discoveries in biochemistry, palynology analysis, micromorphology, anatomy, cytology, micromolecular chemistry, and semantide analysis especially cpdna. cladistic method has been widely applied, subsequent more precise generic, and tribal concepts have been developed. it has become clear that not only quite a number of genera have been misplaced, but others require a transfer to other tribes (jeffrey 1995). 65 biotropia no. 19,2002 miquel (1856) made the first revision of the asteraceae of the malay archipelago followed by van hasselt & boerlage (1884), and boerlage (1891) gave an enumeration of the species. the latest partial revision of the asteraceae of the malay archipelago was made by koster, who elaborated the tribes eupatorieae and vernonieae (koster 1935, 1941, 1948, 1952, 1953, 1958). she treated 5 species of adenostemma, 2 of ageratum, 1 of centratherum, 2 of elephantopus, 8 of eupa-torium , 2 of ethulia, 1 of mikania. in total, 57 species were described of which 11 species are introduced. she also revised the compositae of java (koster 1965) and new guinea (koster 1966; 1970; 1976; 1979; 1980). van royen (1983) treated compositae component of the alpine flora of new guinea for the species above 3000 m. after these publications, there has been no other comprehensive one on the asteraceae of malesia. the first initial survey of the flora of sumatera began with the publication of jack in 1820. it was followed by several authors e.g. miquel (1856-1861) who published them separately in a number of publications. but after miquel there has been no attempt yet to bring together the vascular flora of sumatera. the only exception is the revision of the euphorbiaceae of sumatera by shaw in 1981. whitmore and tantra (1986) listed only those species with either a bole of at least 35 cm diameter, and/ or 20 m in height, they cited only eight out often species of vernonia reported for sumatera by koster (1935). vernonia cinerea, v. cymosa, v. forbesii, v. patula and v. vagans which were cited by whitmore and tantra as a tree are actually herbs, climbers or shrubs. vernonia is the only genus where four out of the ten species are trees up to 30 m in height: v. arborea, v. durifolia, v. patentissim and v. subdentata, while the other six species of vernonia are herbs or shrubs. other asteraceae genera consist of only herbs and shrubs. miquel (1861) treated 25 species of 20 genera in 7 tribes of asteraceae in sumatera. he described 5 species of blumea, 2 species of artemisia, and 1 species of several genera i.e. adenostemma, asteromoea, conyza, eclipta, elephantopus, emilia, erigeron., gnaphalium, gynura, lagenophora, microglossa, myriactis, sonchus, sphaeranthus, wedelia, vernonia, xanthium and youngia. boerlage (1891) in his enumeration of the asteraceae of sumatera listed 41 species of 28 genera in 8 tribes. he listed 5 species of blumea, 3 species of senecio and vernonia, 2 species of adenostemma, anaphalis, conyza, wedelia, and 1 species of several genera i.e. asteromoea, bidens, cosmos, dichrocephala, eclipta, emilia, enydra, erigeron, microglossa, myriactis, sphaeranthus, spilanthes and xanthium. koster (1935) in her treatment of the tribes eupatorieae and vernonieae in sumatera described 9 species of vernonia, 3 species of adenostemma, 2 species of ageratum and 1 species of elephantopus, mikania and eupatorium . nasution (1984) described 5 species of weedy asteraceae at the plantations of aceh and north sumatera, while soerjani et al (1987) described 25 species of asteraceae in the ricefields of sumatera. in total, there were 69 species of asteraceae in sumatera known by several authors: miquel 1861; boerlage 1891; koster 1935 and soerjani et al. 1987. the records up to december 1998 showed that 66 notes on the asteraceae of sumatera sri sudarmiyati tjitrosoedirdjo 67 genera and 122 species in 10 tribes of asteraceae in sumatera were recognized (tjitrosoedirdjo 2001). up to january 2000, the author has described 133 species of 74 genera in 11 tribes in sumatera (table 1 and appendix 1). four new taxa were recognized by tjitrosoedirdjo (2002) namely prenanthes steenisii, prenanthes sumatrana, senecio dewildeorum and one variety emilia sonchifolia var. lanceolata. in sumatera, asteraceae has been recorded in a wide variety of habitats including, montane, highlands, lowlands, fmperata-fields, open spaces of the plantations, and agricultural fields. representatives may be found in nearly every type of habitat, but few taxa are present in tropical rain forests, swampy, mangrove and aquatic areas. the species are predominantly perennial herbs and shrubs. materials and methods herbarium specimens of the asteraceae of sumatera and the surrounding islands were studied from several herbaria. the herbarium bogoriense (bo) and biotrop herbarium (biot) served as the major sources. most of the specimens at bo were collected during the colonial times, while those at biot was gathered after 1970's and some specimens were collected by the author. selected specimens were kindly obtained on loan from herbarium of andalas university ('anda') representing taxa from west sumatera and also some from frim (forest research institute malaysia's/kep) were also studied. some specimens of blumea, lactuca, and senecio were provided on loan from the national herbarium of netherlands (the former rijksherbarium:l). additional specimens were collected during the course of this study from aceh, north sumatera, riau, jambi, west sumatera, south sumatera and lampung. data and information on the morphology, habitat, and distribution were collected from the specimens, literature and field observations. results and discussions the tribe composition based on the studies up to january 2000, there are 133 species of 74 genera in 11 tribes of asteraceae in sumatera that have been recorded (table 1 & appendix 1). there is an increase compared to the records as listed in 1998, where 122 species of 67 genera are in 10 tribes (tjitrosoedirdjo 2001). the 11 tribes are anthemideae, astereae, cardueae, eupatorieae, heliantheae, inuleae, lactuceae, mutisieae, senecioneae tageteae, and vernonieae. the tribe cardueae is a new record. thirty-nine genera or about 53% are exotic (table 1), and the native genera are less than half of the total number of the genera. more than 61% of these genera 67 biotropia no. 19,2002 contain only one species, 9% have three species and 5% have 4 species. others such as vernonia and blumea have ten and fourteen species, respectively. three tribes are found in java but not in sumatera: arctoctideae, calenduleae, and helenieae. notes on distribution of selected genera of asteraceae in sumatera anaphalis is a genus with a disjunct range distribution: it has been recorded from europe, asia and north america. in sumatera it is represented by two native montane species. blumea has an african-asiatic-australian range. most of the species are found in malesia with 14 in sumatera which makes it the largest genus in sumatera. the other genera have 1 to 4, rarely up to 10 species. blumeopsis is a monotypic endemic genus of eastern asia. in malesia, it is only found in sumatera (aceh and north sumatera). carpesium is a genus of the northern temperate or subtropical europe through asia to japan. one possibly introduced species has been found in the mountains of aceh. lactuca is a cosmopolitan genus, with three species in sumatera. launaea is a new generic record for sumatera. launea sarmentosa was found during the course of this study at a sandy beach near uelele, aceh. kilian (1997) reported that the species has a scattered distribution from east africa to south china (guangdong). it is found in the andaman nicobars and in thailand. it is also found in northeast australia. in malesia, it is very rare and until now only known notes on the asteraceae of sumatera sri sudarmiyati tjitrosoedirdjo autochthonously in malesia with the sumateran collection closing the gap. its occurrence in southwest java and northwest australia concurs with the ancient sites of european sailing ships that carried sand as ballast which had been obtained at beaches in east africa or sri lanka. these initially landed in pelabuhan ratu (wijnkoopsbaai) where ballast was removed, or floundered on the shores of northwest australia thus introducing the launaea. senecio is the largest genus of asteraceae, and a very heterogeneous one. its species are found in all parts of the world. in sumatera, there are two endemic montane species. sonchus is another cosmopolitan and weedy genus. there are three species in sumatera. sonchus oleraceous is commonly found at the mountain regions in java. in sumatera, it is only found in takengon, aceh. taraxacum is a cosmopolitan genus of weedy species and an extremely complex genus. there are few regularly amphimictic species, some that are occasionally amphimictic, but a vast number appear to be exclusively apomictic, often with 69 biotropia no. 19,2002 defective or quite abortive pollen. the apomictic forms are polyploids which have presumably arisen from primary interspesific hybridization. the only species found in sumatera is tentatively named taraxacum javanicum here, but may very well represent an undescribed (micro) species. vernonia is a wide ranging genus with species in tropical america, africa, madagascar. jones (1979; 1981), followed by jeffrey (1988), has suggested that actually two main subgenera, if not genera or even subtribes may be involved, an old world one, vernonia subgen. orbivestus, and satellite taxa, and one from the new world, vernonia s.s. and satellites. if correct, this means that malesian species attributed to sect. lepidaploa actually belongs to a possibjy undescribed section. in sumatera, there are ten species of which two are endemic. endemic taxa and their distribution in sumatera the asteraceae is generally a temperate to subtropical family, yet it forms an important part of the malesian flora. being temperate, it is therefore not surprising that the mountain areas in sumatera are richer in taxa than the lowlands. of the 133 species recorded in sumatera, nearly 50% have been introduced, while there are only 7 endemic species and 2 endemic varieties (table 1). these montane endemics are temperate genera and it supports the opinion of van steenis (1933) that in the tropics such as malesia, they are restricted to the mountains and represent only small offshoots from their generic centers. when compared for instance with india which has 242 endemic taxa (out of c. 900) the number in sumatera is very low. in india too many endemics (116) are confined to the montane regions of the himalayas (rao & dart 1996). aceh and west sumatera showed the highest degree of endemism, apparently due to the considerable areas of high mountains. there are one variety and five species endemic to aceh i.e. adenostemma lavenia var. sessilifolium, senecio dewildeorum, s. sumatranus, prenanthes steenisii, p. stenolimba, and p. sumatrana which are concentrated at the mount leuser nature reserve and its surrounding areas (fig. 1). three endemic species of prenanthes: p. steenisii, p. stenolimba, and p. sumatrana, have been found in aceh above 2000 m altitude (tjitrosoedirdjo 2002). in west sumatera four endemic taxa have been found on the mountains of kerinci, merapi, sago, talamau and talang namely emilia sonchifolia var. lanceolata, senecio sumatranus, vernonia durifolia and vernonia forbesii. emilia, sonchifolia var. lanceolata a newly described variety is endemic to mount kerinci (tjitrosoedirdjo 2002). jambi, south sumatera, and lampung have only one endemic species each, while there are no records of endemics in bengkulu and riau. the two endemic species of senecio, s. sumatranus and s. dewildeorum have been found at altitudes above 2500 m. senecio dewildeorum collected by van steenis from mount goh lembuh, aceh, and by de wilde and de wilde-duyfjes from mount leuser appears to be endemic to the mountain area of aceh (tjitrosoedirdjo 2002). 70   biotropia no. 19,2002 vernonia, has two endemic species: v. durifolia and v. forbesii are new records for west sumatera. vernonia durifolia is restricted to west sumatera at the mountain regions of mount sago, mount merapi, mount malintang and mount talamau which are closely located to each other at the altitude of 2000-2600 m. a comparison on the asteraceae of sumatera with adjacent areas in terms of indigenous and endemic species, sumatera is richer than java. out of 133 species, seven species and two varieties are endemic, while java has only one endemic species (phyllocephalum frutescens) out of nearly the same number (132) of species (koster in backer & bakhuizen f. 1965). although, sumatera is larger than java, it has less number in genera: 74 compared to 107. one of the reasons is that probably sumatera is less explored than java. in addition, during the colonial times, there was more international trade in java and many attempts were made to acclimatize exotic species for horticulture (e.g. cabbage, potatoes), plantation crops (oil palm, rubber, tea, tobacco) and plants with medicinal values. the presence of the kebun raya (botanical garden) in bogor certainly played an important part in this process. cargo, packing material, contaminated grain, introduction of exotic species etc. also inadvertently included the introduction of incipient weeds. among these were of course asteraceae. there are 41 genera of asteraceae which are known in java but have not yet been recorded for sumatera. most of these were introduced in table 2. the malay peninsula has less species than sumatera. turner (1995) listed 61, species, an increase of nearly 40% as compared to ridley's (1923) list of 44 species. of these 3 are endemic: erigeron oreophilus, gynura malaccensis, and vernonia rupicola. notwithstanding the suggestive epithet of the erigeron, there are no high mountains in the malay peninsula. few are over 2100 m. it appears that previously there was a connection between the himalaya and the mountain chain in sumatera. van steenis (1933b) listed ainsliae latifolia, prenanthes scandens, and rhynchospermum verticillatum which are recorded both in the himalayas and sumatera without significant difference in altitude range, about 1300-2900 m. in general, some himalayan/malesian taxa are found only in aceh and north sumatera, while others extend to central and south sumatera, or even further to java, south sulawesi and nusa tenggara. the leuser complex is especially rich in the himalayan elements, while there is also a floristic connection over the kinabalu to luzon, and over sulawesi and seram with new guinea. it is curious to note that these high-montane species (non-asteraceae) were able to reach the leuser complex, but did not venture further down south. the fact that the complex is nonvolcanic while the bukit barisan and the mountains in java are igneous, may offer a clue to this •unsolved question. other species are apparently not hampered by the occasional devastation that occurs on volcanic summits and were able to travel south. there are three genera, known in asia and sumatera which have not been recorded in java: ainsliaea, blumeopsis, and prenanthes. other species known from 72 notes on the asteraceae ot'suniaterasri sudarmiyati tjitrosoedirdjo asia and java but not yet recorded from sumatera and borneo are gnaphalium indicum l., inula cappa and senecio araneosus. the latter species turned out to be identical with cisampelopsis volubilis. inula cappa is apparently currently called as synotis cappa (yeffrey & chen 1984) or duhaldea cappa [pi. syst. evol. 176 (1991) 104]. however, koyama (1988) did not mention java, bali, lombok. he reported that the distribution of /. cappa did not reach as far as north thailand. montane genera known from sumatera and java but which have not been recorded from borneo are : anaphalis, gnaphalium, and prenanthes (van steenis 1933). weedy asteraceae of sumatera a large number of asteraceae have been introduced and became naturalized in sumatera. most of them were introduced from tropical america and the others came from elsewhere. many of the species become an adventive weeds (table 3). the diversity of climate, soil types, altitude and other factors have favoured the establisment and spread of asteraceae weeds from many regions of the world. most of the asteraceae in sumatera were introduced during the colonial times, among these were, for instance, the most noxious and dominant chromolaena odorata and mikania micrantha. both species came from tropical america and now have spread to almost all agricultural lands and plantations. 73   biotropia no. 19,2002 among the sumateran asteraceae, the records in 1996 showed that there are 32 species considered as weeds (tjitrosoedirdjo 1996). while in this report based on the observation in 2000, there are 43 species of weedy asteraceae or thirty two percent of the total number of asteraceae in sumatera (table 3). four of these species have not been reported before for sumatera: eupatorium capilifolium, son-chus oleraceus, shagneticola trilobata. and taraxacum javanicum . eupatorium capilifolium found in payakumbuh, west sumatera has a potential to become noxious weeds. sonchus oleraceous is a native of europe, north africa and continental asia. it is now a common weed in java beween 200-2700 m altitude, especially at 800 m altitude. in sumatera it is not yet so common. so far it is only known from two collections made in takengon (aceh) and in bukit gundaling, berastagi (north sumatera) at 1200-1400 m altitude. sphagneticola trilobata, a native of central and south america, is usually called wedelia trilobata. it seems that the plant was introduced and naturalized in sumatera long time ago where it was used for ground ccrver and as an ornamental plant. it has escaped from these applications and now is a weed at low to high altitudes from aceh to lampung. although it is now common, its occurrence was not reported before or neglected by the botanist collectors. taraxacum javanicum was first found in 1996 in north sumatera at bukit gundaling, berastagi, along a road side at 1400 m altitude (tjitrosoedirdjo 310, biot). in west java, it is found in montane areas between 1200-2500 m altitude, where it was first collected in 1888. in view of the tendency in taraxacum to form local microspecies rapidly this may be an undescribed one, so that a specialist will have to look into this. for this time the name t. javanicum is employed here. according to van soest in grierson (1980), this taraxacum species does not belong to the groups of indigenous to europe as was originally assumed. taraxacum javanicum has now been found in several parts of india and sri lanka. in java, however, it occurs in tea plantations and it may be introduced through this industry, the same way as it has been introduced in sri lanka (grierson 1980) among the three new weedy asteraceae found in indonesia reported by dekker (1981): acanthospermum hispidum, calyptocarpus vialis and elephantopus mollis, only e. mollis was found firstly in north sumatera in 1980 in s. bejingkar, kisaran, north sumatera (megia 230, biot). it is also known from sukarami in west sumatera where it was found in 1987 (tjitrosoedirdjo 50, biot). this species should not be confused with elepantopus scaber. it has alternate leaves and white flowers, whereas e. scaber l. has rosulate leaves and purple flowers. ageratum houstonianum mill, can easily be mistaken for the closely related a. conyzoides especially because the two are often found in the same habitat. it is also native to tropical america. in sumatera, it was first fo'und in 1928 by lorzing (13843, bo) collected in berastagi, north sumatera at 1200 m alt. it has now spread to sukarami, solok, west sumatera at an altitude of 900 m. (tjitrosoedirdjo 45, biot) and lampung at batu keramat, between gisting and kota agung at 500 m altitude (tjitrosoedirdjo 415, biot). a. conyzoides is commonly found everywhere in sumatera. 75 notes on the asteraceae of sumaterasri sudarmiyati tjitrosoedirdjo in central and south america, the genus chromolaena has about 165 species, but c. odorata is the only species which is now pantropically distributed. in sumatera, the first collection was made in deli, lubuk pakam in 1932 (van der meer-mohr 4004, bo) in tobacco plantations. after the war for independence, it had become quite common and being very distinctive by its white violet flowers, and known as "semak merdeka" (the independence shrubs) or "putihan" (whitish flowers). it has now become an important weed throughout sumatera. clibadium surinamense is another native of south america, and was first reported for sumatera by jochems who collected the first specimen in 1932 in tobacco plantations near medan. conyza sumatrensis, which is previously known as erigeron sumatrensis, is a common weed in open places of fields and plantations, and distributed widely throughout sumatera. in indonesia crassocephalum crepidioides has been confused with erechtites valerianifolia (tjitrosoedirdjo 1987). jochem (1931) noticed it for the first time in 1926 near medan. it had probably come from africa through sri lanka and from there to sumatera (van steenis 1938). within a few years, it then rapidly spread over the whole island. in java it was introduced from sumatera by tea planters, and it spread widely. it has become even more common than erechtites hieracifolia and e. valerianifolia which were introduced much earlier. crassocephalum crepidioides was identified by backer & van slooten (1924) in their "geillustreerd handboek der javaansche thee-onkruiden (no. 233)" as e. 'valerianifolia', so that many pamphlets, books, publications refer to this species as e. valerianifolia instead of c. crepidioides. in the orient c. crepidioides is sometimes confused with e. hieracifolia, as most of the labeled specimens are actually c. crepidioides (belcher 1955). the genus mikania has about 400 species mainly in the warmer parts of the new world. the only indigenous species in asia is m. cordata ,while m. micrantha is the only new world species to have been introduced there. the latter species readily takes to disturb areas and tends to be weedy (parker 1972). it was imported from paraguay in 1949 and planted in the bogor botanical garden. in 1956 this species was introduced as a non-legume ground cover in rubber plantations. wirjahardja (1976) reported that around 1976, it occupied the greater part of rubber plantations and abandoned agricultural areas in west and east java and south sumatera. recently, in west java and sumatera, m. cordata could not be found easily since it is suppressed by m. micrantha. porophyllum ruderale, a native of mexico and south america, was first reported in bogor in 1945, a new record for malesia (tjitrosoedirdjo 1991). in 1978 the first herbarium specimens from sumatera were collected from the transmigration areas of lampung (dekker & wirjahardja 2626, biot) and south sumatera (dekker & wirjahardja 2591, biot). currently, it is commonly found in sumatera, east and central java. its occurrence has also been reported for malaysia and singapore (tan & ibrahim 1992; turner 1995). 76 notes on the asteraceae of sumaterasri sudarmiyati tjitrosoedirdjo some of the weedy species of asteraceae which is currently not known in sumatera i.e. praxelis clematidea (eupatorium catarium ) and parthenium hyster-oporus have a potential to become noxious weeds. praxelis clematidea a native of south america, has been introduced in south china and queensland, australia (veldkamp 1999). partenium hysterophorus a native of the carribean islands has spread to australia, south africa, china, the pasific islands (sastroutomo & mahyudin 1990). the importation of grass seeds, cover crop seeds, wheat, grains and cattle from australia might result to the introduction of these weeds in sumatera. we have to be aware that those species have a potential in spreading rapidly. conclusions there are 133 species of 74 genera in 11 tribes of asteraceae in sumatera, based on the records in 2000. the most diverse genus is blumea with 14 species. other genera are only represented by 10 species or less, usually 4, or 3, or 2, and mostly by 1 species only. there are 1 genus, 7 species and 2 varieties endemic to sumatera in terms of indigenous and endemic species sumatera is richer than java. the high mountain region in sumatera is shown to be richest in the general asteraceae flora as well as endemic species.. thirty nine or about 53% are exotic genera and the native genera are less than half of the total number. there are 43 important weed species, which were introduced in sumatera from tropical america, africa, asia and europe. the exotic genera and species are introduced and naturalized in sumatera. the diversity in climate, soil types, altitude and other factors have favoured the establishment and spread of asteraceae weeds from many regions of the world. acknowledgements part of the dissertation of the author is supported by tmpd (team pengem-bangan program doctor) and seameo biotrop bogor, indonesia, through funding of the dip project. the author would like to express her sincere appreciation to all her supervisors , especially dr. j.f. veldkamp (l) for his suggestions and comments. thanks are also due to the directors of bo, 'anda', kep for giving the facilities to work at their herbarium and l for the loan specimens. references backer, c.a. 1918. indische duinplanten. trop. nat. 7: 59 backer, c.a. & d.f. van slooten.1924. geillustreerd handboek der javaansche thee-onkruiden. algemeen proefstation voor thee. batavia, drukkerijen roygrok & co. 77 biotropia no. 19,2002 belcher, r.o. 1955. the typification of crassocephalum moench and gynura cass. kew bull. 3: 455-465 bentham, g. 1873. notes on classification, history and geographical distribution of compositae. j. linn. soc. bot. 13: 335-577 bentham, g. & j.d. hooker. 1873. genera plantarum. 2: 1-224. lovell reeve & co., london. boerlage, j.g. 1891. flora van nederlandsch indie (compositae). e.j. brill, leiden. cassini, h. 1816. troissieme memoire sur le synantherees, analyse de la corolle. j. phys. chim. hist. nat. arts. 82: 116-146 cassini, h. 1817. quatrieme memoire sur la familie des synantherees, contenant 1'analyse de 1'ovaire et de ses assessoires. j. phys. chim. hist. nat. arts 85: 5-21 cassini, h. 1818. composees. in g. cuvier, diet. sci. nat. 10: 131-159 cassini, h. 1826-1934.opuscules phytologiques. 3 volumes. paris dalla tore, c.g. de & h. harms. 1907. genera siphonogamarum. lipsieae, berlin. dekker, r. j. 1981. notes on new or remarkable indonesian weed species. proc. konp. higi medan. jack, w. 1820. the malayan miscellanies. bencoolen jeffrey, c. & y. l. chen 1984. taxonomic studies on the tribe senecioneae (compositae) of eastern asia. kew bull. 39, 2:319 jeffrey, c. 1986. the senecioneae in east tropical africa. kew bull. 43: 195-277 jefrey, c. 1988. the vernonieae of east tropical africa. kew bull. 43:195-277. jeffrey, c. 1995. compositae systematic 1975-1993. developments and desiderata. in advances in compositae systematics d.j. n. hind, c. jeffrey & g.v. pope (ed.). the royal botanic garden kew. vol i. jochems, s.c.j. 1931. de invoer van twee nieuwe composieten in deli (gynura sp. en clibadium surinamense). trop. nat. 20: 5-10 jones, s.b. 1979. synopsis and pollen morphology of vernonia (compositae vernonieae) in the new world. rhodora 425-447. jones, s.b. 1981. synoptic classification and pollen morphology of vernonia (compositae: vernonieae) in the old world. rhodora 83: 59-75 grierson, a.j.c. 1980 in dassanayake, m.d. revised handbook of the flora of ceylon 1 (111). amerind, new delhi. hasselt, a.l. van 1884. bijdragen tot de kennis der flora van midden sumatra. flora in veth. midden sumatra 4(2): 1:49 hoffmann, o. 1890-1894.liguliflorae-cichorieae in engler & prantl. die naturlichen pflanzenfamilien kilian, n. 1997. revision oflaunaea cass. (compositae, lactuceae, sonchinae). englera 17: 288-296 koster, j.th. 1935. the compositae of the malay archipelago. blumea 1(3): 352-538 koster, j.th. 1941. notes on malay compositae 1. blumea 4(3): 482-492 78 notes on the asteraceae of sumatera sri sudarmiyati tjitrosoedirdjo koster, j. th. 1948. notes on malay compositae ii. blumea 6(1): 264-365 koster, j. th. 1952. notes on malay compositae iii. blumea 7(1): 288-291 koster, j. th. 1953. some annotation to the compositae collected by o/beccari in borneo & sumatra. koster, j. th. 1958. notes on malay compositae iv. blumea supplement iv: 170-177 koster, j. th. 1965.compositeae. in backer and bakhuizen van den brink f. fl. of java vol. ii: 362-437 koster, j. th. 1966. the compositae of new guinea i. nova guinea bot. 24: 479-614 koster, j. th. 1970. the compositae of new guinea ii. blumea 18(10: 137-145 koster, j. th. 1975. the compositae of new guinea iii. blumea 22(2): 207-217 koster, j. th. 1976. the compositae of new guinea iv. blumea 23: 163-175 koster, j. th. 1979. the compositae of new guinea v. blumea 25(1): 249-282 koster, j. th. 1980. the compositae of new guinea vi. blumea 26(l):233-243 koyama, h. 1988. taxonomic studies in the compositae of thailand. acta phytotax. geobot. 39: 161 melchior, h.a. 1964. engler's syllabus der pflanzen familien ii. band. berlin gebruder borntragen miquel, f.a.w. 1856. flora van nederlandsch indie 2: 1-116. tweede deel. fried. fleischer, leipzig. amsterdam, utrecht. miquel, f.a.w. 1861. flora van nederlandsch indie, eerste bijvoegsel, sumatra:210-213, 535-537. c.g. van der post, amsterdam. nasution, u. 1984. gulma dan pengendaliannya di perkebunan karet sumatera utara dan aceh (weeds and their control in rubber plantations in north sumatera and aceh) pp4m-tanjung morawa (in indonesian) parker, c. 1972. themikania problems. pans 18(3): 312-315 rao, r.r. & b. datt. 1996. diversity, phytogeography of indian compositae. in proceedings compositae conference. vol. i: 445-461. ridley, h.n. 1923. the flora of the malay peninsula. l. reeve & co., ltd. london. vol. ii.: 177-197 ross, m.c. 1993. annual report foundation flora malesiana 1992. fl. malesianabuli.il(2): 131-142 royen, p. van. 1983. the alpine flora of new guinea 4: 3139-3451. j. cramer, vaduz. satroutomo, s.s. & a.a. mahyudin. 1990. partenium hysterophorus l. pest data sheet no.: 43. asean pest plant quarantine center and training institute malaysia. shaw, h.k. airy. 1981. the euphorbiaceae of sumatera. kew bull. 36(2): 239-373 steenis, c.g.g.j. van 1933. on the origin of the malaysian mountain flora. part 1. fact and statement of the problems. bull. jard. bot. buit. 13(3): 125-417 steenis, c.g.g.j. van 1938. gymtra crepidioides bth. a recently introduced weed in s.e. asia. current science vii: 285 soerjani, m, a.j.g. kostermans & g. tjitrosoepomo. 1987. weeds of rice in indonesia. balai pustaka, jalarta tan, h.t.w., ibrahim, ali bin & chua, k.s. 1992. addition to the flora of singapore i gard. bull of singapore 44: 129 79 biotropia no. 19,2002 tjitrosoedirdjo, sri s. 1987. field characterization of the confused species crassocephalum crepidioides and erechtites valerianifolia in indonesia. proceed. apwss. conf. taipei. tjitrosoedirdjo, sri s. 1991. the present status and potential of porophyllum ruderale var ruderale in indonesia. proc. apwss conf. jakarta. tjitrosoedirdjo, sri s. 1996. gulma anggota suku asteraceae di sumatera (the weedy asteraceae species of sumatera). proc. higi, bandar lampung tjitrosoedirdjo, sri s. 2001. progress on the studies of asteraceae in sumatera. taxonomy: the cornerstone of biodiversity. proceedings of the fourth international flora malesiana symposium 1998. forest research institute malaysia, p: 181-186 tjitrosoedirdjo, sri s. 2002. four new taxa of asteraceae in sumatera. reinvvardtia 12 (1): 125-128 turner, i.m. 1995. a catalogue of the vascular plants of malaya. card. bull, of singapore 47(1): 168-172 veldkamp, j.f. 1999. eupatorium catarium a new name for eupatorium clematidea griseb. non. sch. brp. (compositae), a south american species naturalized and spreading in s.e. asia and queensland, australia. card. bull. singapore 51(1): 119-124 whitmore, t.c. & i.g.m. tantra. 1986. tree flora of indonesia. checklist for sumatra. forest research and development research center, bogor, indonesia wirjahardja, s. 1976. autecological study ofmikania spp. biotrop internal report. 7p. 80 notes on the asteraceae of sumaterasri sudarmiyati tjitrosoedirdjo appendix 1 list of the genera and species of asteraceae in sumatra. accepted genera and species in bold, synonyms in italic. achillea l. achillea millefolium l. acmella rich, ex pers. acmella paniculata (wall, ex dc.) r.k. jansen spilanthespaniculata wall, ex dc. spilanthes acmella auct. non murr. acmella uliginosa (sw.) r. cass. jaegera uliginosa (sw.) baker spilanthes iabadicensis a.h. moore spilanthes uliginosa sw. adenostemma forster & g. forster adenostemma lavenia (l.) kuntze adenostemma fastigiatum (blume) dc. lavenia vastigiata blume verbesina lavenia l. adenostemma macrophyllum (blume) dc. adenostemma parviflorum (blume) dc. adenostemma ovatum miq. lavenia parviflora blume ageratum l. ageratum houstonianum mill. ageratum conyzoides l. ainsliaea dc. ainsliaea latifolia (d. don) sch. bip. ainsliaeapteropoda dc. liatris latifolia d. don anaphalis dc. anaphalis javanica (reinw. ex blume) sch. bip. antennaria javanica (reinw. ex blume) dc antennaria saxatilis dc. gnaphalium javanicum reinw. ex blume gnaphalium saxatile blume anaphalis longifolia (blume) dc. gnaphalium longifolium blume artemisia l. artemisia scoparia waldst. & kit. artemisia vulgaris l. aster l. aster novae-angliae l. symphyotrichum novae-angliae (l.) nesom virgulus novae-angliae (l.) reveal & keener aster novi-belgii l. symphyotrichum novi-belgii (l.) nesom asteromoea blume asteromoea indica (l.) blume aster indicus l. boltonia indica (l.) benth. austroeupatorium r m. king & h. rob. austroeupatorium inulifolium (kunlh) r.m. king & h. rob. eupatorium inulifolium kunth eupatorium javanicum auct. non blume eupatorium pallescens dc. bidens l. bidens biternata (lour.) merr. & sherrf ex sherff bidens chinensis (l.) willd. coreopsis biternata lour. bidens pilosa c blumea dc. blumea balsamifera (l.) dc. blumea grandis (wall.) dc. conyza balsamifera l. blumea bicolor merr. blumea clarkei hook. f. blumea lessingii merr. blumea densitlora dc. blumea hieraciifolia (d. don) dc. blumea chinensis walpers blumea subsericans elmer erigeron hieraciifolius d. don blumea intermedia j. koster blumea junghuhniana (miq.) boerl. blumea dacycoma (miq.) boerl. conyza dacycoma miq. conyza junghuhniana miq. blumea korthalsiana (miq.) boerl. conyza korthalsiana miq. blumea lacera (burm. f.) dc. blumea lactucifolia dc. blumea runcinata dc. conyza lacera burm. f. blumea laciniata (roxb.) dc. blumea crepidifolia dc. blumea javanica (blume) zoll. blumea runcinata dc. blumea sonchifolia dc. conyza javanics blume conyza laciniata roxb. blumea lanceolaria (roxb.) druce blumea laxiflora elmer blumea longifolia dc. blumea myriocephala dc. blumea spectabilis dc. conyza lanceolaria roxb. blumea riparia (blume) dc. 81 biotropia no. 19,2002 blumea semivestita dc. conyza riparia blume blumea sylvatica (blume) dc. blumea sessilifolia (blume) dc. conyza sessilifolia blume conyza sylvatica blume blumea tenella dc blumea humifusa (miq.) boerl. conyza humifusa miq. blumeopsis gagnep. blumeopsis flava (dc.) gagnep. blumea flava dc. blumeopsiafalcata auct. non merr. laggera flava (dc.) benth. boltonia l'her boltonia asteroides (l.) l' her matricaria asteroides l. carpesium l. carpesium cernuum l. centaurea l. centipeda lour. centipeda minima (l.) a. br. & aschers. artemesia minima l. centipeda minuta (g.forst.) benth. ex cb. clark centipeda orbicularis lour. cotula minima (l.) willd. cotula minuta g. forst. grangea minima (l.) poir chromolaena dc. chromolaena odorata (l.) r.m. king & h. rob. eupatohum odoratum l. chrysanthemum l. chrysanthemum coronarium l. bupthalmum oleraceum lour. chrysanthemum roxburghii (desf.) dc. glebionis coronaria (l.) tzvel. pyrethrum roxburghii desf. chrysanthemum morifolium ramat. matricaria morifolia ramat. pyrethrum sinense (sabine) dc. cissampelopsis (dc.) miq. cissampelopsis volubilis (blume) miq. cacalia volubilis blume senecio araneosus dc. senecio blumei dc. senecio walkeri auct. non arn. clibadium allamand ex l. clibadium surinamense l. conyza less. conyza japonica (thunb.) less, ex dc. escenbachiajaponica (thunb.) j. koster conyza leucantha (d. don) ludlow & raven blumeopsis falcate (d. don) merr. conyza vicidula wall, ex dc. escgenbachia vicidula (dc.) j. koster erigeron falcatum d. don erigeron leucanthum d. don conyza sumatrensis (retz.) walker erigeron sumatrensis retz. erigeron linifolius auct. non willd. coreopsis l. coreopsis grandiflora hogg ex sweet coreopsis tinctoria cav. cosmos cav. cosmos bipinnatus cav. cosmos caudatus kunth cosmos sulfureus cav. bidens. sulfurea (cav.) sell. bip. cotula l. cotula anthemoides l. crassocephalum moench. crassocephalum crepidioides (benth.) s. moore crassocephalum diversifolium hiern gynura crepidioides benth. dahlia cav. dahlia pinnata auct. non cav. dicrocephala l. her. ex dc. dicrocephala integrifolia (l.f.) kuntze cotula bicolor roth dicrocephala bicolor (roth) schlechtend. dicrocephala latifolia (pers,) dc. grangea latifolia (pers) poir. eclipta l. eclipta prostrata (l.) l. eclipta alba hassk. eclipta erecta l. verbesina prostrate l. elephantopus l. elephantopus mollis kunth elephantopus martii graham ex sch. bip. elephantopus lomentosus auct. non l. elephantopus sea her l. eleutheranthera poit. eleutheranthera ruderalis (sw.) sch. bip. eleutheranthera. prostrata (sw.) sch. bip. kegelia ruderalis sch. bip. melampodium ruderale sw. ogiera ruderalis (sw.) griseb. emilia cass. emilia coccinea (sims) g. don cacalia cocinea sims cacalia sagitata willd. emilia javanica auct. non c.b. rob. emilia sagittata dc. emilia prenanthoidea dc. emilia sonchifolia (l.) dc. cacalia sonchifolia l. 82 notes on the asteraceae of sumatera sri sudarmiyati tjitrosoedirdjo crassocephalum sonchifolium (l.) less. emilia purpurea cass. emilia rigidula dc. enydra lour. enydra fluctuans lour. meyerafluctuans (lour.) spreng. erechtites raf. erechtites hieraciifolia (l.) raf. ex dc. senecio hieraciifolius l. erechtites valerianifolia (wolf) dc. crassocephalum valerianifolium less. erechtites petio/ata auct. non benth. gymtra rosea ridl. eupatoriutn l. eupatorium capillifolium (lam.) small artemesia capillifolia lam. chrysosoma capillacea michx. eupatorium foenicoloides walt. gaillardia foug. gaillardia pulchella foug. galinsoga ruiz & pavon galinsoga parvillora cav. gerbera cass. gerbera jamesonii bolus ex adlam glossocardia cass. glossocardia leschaenaultii (cass.) veldk. chrysanthellum indicum auct. non dc. chrysanthellum leschenaultii (r. cass.) backer ex kostcr chrysanthellum procumbens auct. non l.c.m. rich. gnaphalium l. gnaphalium japonicum thunb. gnaphalium pensylvanicum willd. gamochaetapensylvanicum (willd.) cabrera gnaphalium perigrinum fern. granges adans. grangea niaderaspatana (l.) poir. artemesia maderaspatana l. gynura cass. gynura aurantiaca (blume) dc. gynura densiflora miq. gynura lyrata sch. bip. ex miq. gynura mollis sch. bip ex zoll. gynura sumatrana miq. gynura procumbens (lour.) merr. cacalia procumbens lour. cacalia sarmentosa blume gynura sarmentosa (blume) dc. helianthus l. helianthus angustifolius l. helianthus annuus l. helianthus tuberosus l. hclichrysum mill. helichrysum orientale (l.) gaertn. gnaphalium orientale l. lactuca l. lactuca indica l. pterocypsela indica (l.) c. shih lactuca laevigata (blume) dc. aracium laevigatum (blume) miq. ixeridium laevigatum (blume) pak & kawano prenanthes laevigata blume lactuca saliva l. launaea cass. launaea sarmentosa (willd.) sch. bip. ex kuntze ammoseris sarmentosus (willd.) dc. lactuca sarmentosa (willd.) dc. launaea pinnatifida cass. prenanthes sarmentosa willd. laggera sch. bip. ex koch laggera alata (d. don) sch. bip. ex oliver blumea alata (d. don) dc. conyza alata (d. don) roxb. erigeron alatum d. don lagenophora cass. lagenophora lanata a. cunn. lagenophora gracilis steetz lagenophora stipitata auct. druce lagenophora sundana miq. leucanthemum mill. leucanthemum vulgare lam. chrysanthemum leucanthemum l. microglossa dc. microglossa pyrifolia (lam.) kuntze conyzaprolifera blume conyza pyrifolia lam. mycroglossa volubilis dc. mikania willd. mikania cordata (burm.f.) b.l. rob. eupatorium cordatum burm. f. eupatorium volubile vahl. mikania scandens auct. non willd. mikania volubilis (vahl.) willd. mikania micrantha kunth myriactis less. myriactis javanica (reinw. ex blume) dc. bellisjavanica reinw. ex blume notonia dc. notonia grandiflora wall, ex dc. senecio grandiflorus (dc.) jacobsen senecio indicus backer ex heyne pluchea cass. pluchea indica (l.) less. baccaris indica l. porophyllum guett. porophyllum ruderale (jacq.) cass. cacalia ruderalis (jacq.) sw. 83 biotropia no. 19,2002 kleinia ruderalis jacq. porophyllum ellipticnm cass. prenanthes l. prenanthes scandens hook. f. ex benth. prenanthes steenisii tj. prenanthes stenolimba steenis prenanthes sumatrana tj. rhyncospermum blume rhyncospermum verticillatum reinw. ex blume senecio l. senecio dewildeorum tj. senecio sumatranus martelli senecio korintjianus boerl. sigesbeckia l sigesbeckia orientalis l. solidago l. solidago canadensis l. sonchus l. sonchus arvensis l. sonchus asper (l.) hill. sonchus malaianus miq. sonchus oleraceus l sphaeranthus l. sphaeranthus africanus l. sphaeranthus indicus l. sphagneticola o. hoffm. sphagneticola calendulacea (l.) pruski complaya chinensis (osbeck) strother telechitonia chinensis (osbeck) h. rob. & cuatrecasas verbesina calendulacea l, wedelia calendulacea (l.) less. wedelia chinensis (osbeck) merr. sphagneticola trilobata (l.) pruski complaya trilobata strother seruneum trilobatum (l.) kuntze silphium trilobatum l. thelechitonia trilobata (l.) h. rob. & cuatrecasas wedelia trilobata (l.) a. hitchc. struchiuni p. br. struchium sparganophorum (l.) kuntze ethulia sparganophora l. ethulia struchium sw. sparganophorus vailantii crantz sparganophorus struchium poir. synedrella gaertn. synedrella nodiilora (l.) gaertn. verbesina nodiflora l. tagetes l. tagetes erecta l. tagetes major gaertn. taraxacum wigger taraxacum javanicum soest. taraxacum offlcinale wigger tridax l tridax procumbens l. tithonia desf. ex juss. tithonia diversifolia (hemsl.) a. gray mirasolia diversifolia hemsl. urbanisol tagetiflora (desf.) kuntze tithonia rotundifolia (mill.) s.f. blake tagetes rotundifoliam\\\. desf. tithonia tagetiflora desf. vernonia schreb. vernonia arborea buch.-ham. gynanthemum arboreum (buch.-ham) h. rob. strobocalyx arborea (buch.-ham) sch. bip. vernonia cinerea (l.) less. conyza cinerea l. cyabthillium cinereum (l.) h. rob. senecioedes cinerea (l.) post & kuntze vernonia coronata j. koster vernonia cymosa blume vernonia durifolia j. koster vernonia forbesii s. moore vernonia patcntissima j koster vernonia patula (dryand.) merr. conyza patula dryand. cyanthillium villosum blume vernonia chinense (less.) less. vernonia subdentata j. koster vernonia vagans dc. gymnanthemum vegans dc. wedelia jacq. wedelia biflora (l.) dc. seruneum biftorum (l.) kuntze seruneum strigulosum (k. schum.) kuntze wolastonia biflora (l.) dc. wolastonia slrigulosa (k. schum.) kuntze wedelia montana (blume) boerl. seruneum montanum (blume) kuntze wolastonia montana blume verbesina montana blume wedelia urticifolia (blume) dc. seruneum urticifolium (l.) kuntze verbesina urticifolia blume wolastonia urticifolia (blume) hassk. xanthium l. xanthium indicum koenig. ex roxb. xanthium inequilaterum dc. youngia cass. youngia japonica (l.) dc. zinnia l. zinnia elegans jacq. 84 65.pdf 66.pdf 67.pdf 68.pdf 69.pdf 70.pdf 71.pdf 72.pdf 73.pdf 74.pdf 75.pdf 76.pdf 77.pdf 78.pdf 79.pdf 80.pdf 81.pdf 82.pdf 83.pdf 84.pdf 2. sepus (tropical).cdr biotropia vol. 19 no. 2, 2012: 64 79 tropical forest types in west papua, the presence of forest wallaby ( ) and human disturbancedorcopsis muelleri sepus fatem & karle v. sykora received 20 march 2012/accepted 08 october 2012 the vegetation in the nuni watershed area, part of a tropical lowland forest area in the northern part of manokwari, west papua, was classified with twinspan. the area is important as a natural habitat of the forest wallaby. four habitat types comprising 6 plant communities could be distinguished belonging to grassland, four different types of open forest and undisturbed primary closed forest. a vegetation table is presented and species composition is described. each vegetation plot the presence of trails, wallaby droppings, food remains and signs of human disturbance, i.e. logging, hunting and gardening activity, and distance to settlement areas was noted. the presence of wallabies could only be noted in grassland, open forest with only little logging activity, and in undisturbed closed forest. it is strongly correlated to distance from villages and negatively correlated to logging and hunting. the relation with food plant availability appears to be only low. the results indicate that vegetation structure, vegetation composition and food plant availability are less important than human disturbance. regulations reducing the disturbance by logging and hunting are urgently needed. wallaby, plant communities, vegetation analysis, wildlife, tropical forest, papua 1* 2 1 2 environmental and forest conservation laboratory,forestry faculty, papua state university, jl. gunung salju amban manokwari, papua barat-indonesia, 98314 nature conservation and plant science group, environmental dept, wageningen university and research centre, the netherlands abstract introduction key words: the island of new guinea (papua new guinea and west papua) occupies a phytogeographically important position between asia and west melanesia on one hand, and australia and the pacific on the other. in west papua, many ecosystems occur in a range from the coastal to the alpine zone of which the tropical forest is the dominant ecosystem. mammalian species are numerous in the tropical forest ecosystems of west papua. new guinea hosts a unique fauna of mammals due to its geological history (petocz 1989; muller 2005), * corresponding author : sepus_fatem@yahoo.com 64 differing from other areas in indonesia, but also other regions in the world (petocz 1994; flannery 1995). however, forest fragmentation, changes the environment, i.e. the physical conditions, tree species richness or tree structure (carlot 2009) and plant demography (borhidi 1998). changes in the size, shape or configuration of a habitat as a result of fragmentation has an effect on populations of various animals, such as small mammals), bird sand arboreal marsupials (connel 1978; chave 2008). menzies (1991), petocz (1994), flannery (1995) and westerman 2001) suggest that the diversity in endemic marsupials of new guinea is higher than is reflected by current classifications. their population status, habitat, vegetation structure and food composition should urgently be studied in order to better understand their ecological needs. this knowledge can be used as guidelines for ecological planning and management to avoid habitat destruction and decline of endemic marsupials. the current status and ecology of the forest wallaby ( , macropodidae) is insufficiently known. our study site manokwari (west papua) is part of its distribution area. the vegetation of manokwari has hardly been described. in this research we describe the vegetation composition of the vegetation types in the nuni watershed area in the northern part of manokwari regency, west papua. besides, we investigated the presence of the forest wallaby and its relation with vegetation, including food plant availability and human influence. our research is based on the following questions: 1. what plant communities occur in the area and how are they characterised by species composition, soil, human influence and structure? 2. what is the presence of the forest wallaby in the different plant communities? 3. how can differences in forest wallaby presence be explained?. the vegetation in the nuni watershed area, part of tropical lowland forest area in the northern part of manokwari regency, west papua provinces was studied between february 4 and april 6, 2010. for a map showing the location see appendix 1 (internet appendix). the vegetation composition and the wallaby presence was studied in 8 transects of 4 vegetation plots or relevés sized 50 x 50 m and one extra plot of 70 x 70 m, i.e. 33 plots in total. in order to have a wider range of forest types and possible densities of forest wallabies, one extra plot of 70x70 was made. the bigger size did not, however, influence the plant species composition and wallaby presence. due to the purpose for comparing, which plot more presence and covered macropodidae present. the transects were selected based on differences in forest type, hunting and logging intensity and distance from villages. average distance between the plots was 50-70 m. the survey was done in primary forest, open forest and grassland, from the river bank upward to the top of the mountain. vegetation composition was recorded using the braun-blanquet method. cover was estimated using the scale as modified by barkman, doing and segal (1964). this scale was transformed into an ordinal 9 scale (sykora 2009). species, were identified et al. ( dorcopsis muelleri materials and methods 65 tropical forest types in west papua – sepus fatem & karle v. sykora according to jhon (1997), whitmore (1997); paijmans (1976); van steenis c.g.g.j (2005); lekitto 2008); c.g.g. j van steenis (1989). most of the identification was done directly in the field and only few species were identified in herbarium of papua state university, manokwari-indonesia. the vegetation was classified using twinspan (hill & gaugh 1980; sýkora 2009; de boer . 2009). the plant communities distinguished were named using two different and/or dominant species. for each plot the presence of trails, wallaby droppings, food remains and signs of human disturbance, i.e. logging (number of tree stumps), hunting and gardening activity, and distance to settlement areas was noted. hunting frequency per week/ month/year was determined as well as the number of animals shot per hunting trip. the distance between settlement areas and the hunting areas was measured. the extent of the conversion of forests into gardens was registered by measuring the total garden area (m ) and the distance to settlement area (m). the presence of macropodidae species was recorded visually in each plot in the night, evening and morning. all strips were observed at thesame time with two people per strip.for each plot the presence and cover (%) of food plant species was recorded. the relation between vegetation composition and external variables like food avalability, anthropogenenic pressure and wallaby presence was studied using multivariate gradient analysis (detrended correspondence analysis). significance was tested by monte carlo permutation test. in this research we studied the habitat of forest wallaby ( ) and its relation to vegetation composition. the plant communities in the different habitats were described. so far the habitat of the macropodidae species has only been described in general terms (menzies 1991; petocz 1994; flannery 1995). according to these authors macropodidae, especially forest wallaby occur from sea level up to an altitude of 400 m. their habitats vary from flood plains, few gravel, bushy areas, rocky river banks to forested hills optimally to an altitude of 200 meter asl (flannery 1995). however, habitat use and territory of this species has not yet been studied in more detail. the ecology of has not been studied recently. most research only focuses on tree kangaroos. the habitat used of forest wallaby is described. a total of 258 plant species was recorded (245 angiosperms, 10 ferns allies, 3 gymnosperms, see appendix 1). after twinspan analysis, six plant communities were distinguished. only four of these plant communities appeared to be used by as habitat. an overview is given of the habitat types and plant communities in which was found. the two plant communities where no activity of could be detected are described as well. et al. et al. ( et al dorcopsis muelleri d. muelleri darcopsis muelleri, dorcopsis muelleri d. muelleri d. muelleri d. muelleri 2 results and discussions vegetation biotropia vol. 19 no. 2, 2012 66 habitats, plant communities and activityd. muelleri . grassland this habitat type consists of a small thicket with grass species in combination with small herbs, woody herbs, shrubs and trees. the canopy is formed by small trees only and covers only 10 %. the herb layer is 90 cm high and there is no moss layer. the depth of the litter layer is only 1 cm. it is represented by the and community which is characterised by 17 different species for habitat and food of forest wallaby. this habitat is characteristic of open sites: sp, sp. and sp . , sp. , and provide food for forest wallabies. mostly the grass species such as and are up to 1 m high. in papua new guinea, , and common grass species of floodplains can grow up to 1-2 m high (harkink 1987). grasslands grow all along the river basins and in small valleys, where they occur due to the fluctuating water levels resulting in temporary flooding, followed by drainage of the river banks. this grassland is presently the most common natural grassland of lowland tropical vegetation area in west papua. this community is generally found on relatively flat (< 3°), temporarily flooded, sandy soil mixed with some gravel. as it occurs close to the river the sandy soil structure is crumbly, drainage is high and organic matter is lacking (brookfield 1971; petocz 1987; bps 2009). this habitat (fig. 1) is mainly used by the forest wallaby as feeding area, but also for shelter and to drink and play. trails and food remains were found of two individuals. imperata cylindrica ipomea aquatica imperata ipomea batatas, sacharum spontaneum, neprolepsis biserata, jussiaee octavilis, malotus philipinensis, piper aduncum, macaranga mappa, mimosa pudica, muntingia callabura, mucuna novaeguinesis, puararai javanica, spondias cytherea, micania zingiber ipomea batatas zingiber muntingia callabura mimosa pudica imperata cylindrica sacharum spontaneum imperata cylindrica, sacharum spontaneum sorghum nitidum phragmites karka, 2 2 figure 1. typical river bank grassland consisting of sparse thicket, grasses and herbs. (photo: sf sepus fatem) this natural grassland, which can be very extensive (128 665 meters in one research site), is also used by animals like deer. seventeen food plants were found with a mean cover of 2.1%. tropical forest types in west papua – sepus fatem & karle v. sykora 67 open forest two communities, the sp and community, were distinguished in the open forest habitat, depending on the absence or presence of some logging activity. this habitat type, a transition between grassland and tall forest, is characterised by shrubs, climbing vines and woody vines, lianas and pioneer species combined with small trees (fig. 2). the canopy cover is 47 % and the tree diameter is medium; besides some ferns and lianas occur. the herb layer is 51 cm high. it is commonly found surrounding the closed forest. it is represented by the community and sp which is differentiated by , , , , , , , , sp, , sp. 1, , , , , , sp, sp., , , , , sp., and . furthermore, , , and were food plant wallaby in this habitat. this forest type grows on hills at an altitude of approximately 40 meter above sea level. it occurs on inceptisols i.e. new immature, still developing soils with hardly any soil horizon. the depth of the solum is less than 2 m. the rock material in these sites mostly consists of sandstone and mudstone (brookfield 1971; petocz 1989; bps 2009). the soil is covered with 2 cm of litter. the distance to the nearest village is about 6.5 km and anthropogenic pressure is low. also the hunting frequency is very low (in average 1 time/month). this forest type appears to be one of the main habitat types of due to the low human pressure and its importance as a feeding habitat. more than 5 individuals of forest wallaby were spotted and their dung and trails were observed. in average 12 (7-20) food plant species were found with a mean cover of 2.5 %. ficus robusta dendrochide community musa paradisiaca callamus longipina of ficus robusta dendrochide hornstendia scottiana pandanus dubius derris alba sterculia shillinglawi ficus japonica carica papaya orioconide nitida endospernum moluccanum rhapidophora spathodea campanulata ficus neolaleba atra durio zibethinus lancium domesticum cyatea molucanna mangifera indica dendrobium prinium callamus warbugii ananas comosus macaranga gigantea drymopholeus litigiosus cleytances ficus variegata cyatea molucanna ficus variegata, mangifera indica, sphatodea campanulata carica papaya, ficus japonica ananas commosus lancium domesticum d. muelleri open forest transitional to grassland. figure 2. transition between river bank grassland and real forest (photo : sf-sepus fatem) biotropia vol. 19 no. 2, 2012 68 open forest after logging (successional forest) figure 3. open forest after logging (photo: sf-sepus fatem) in this habitat type the process of secondary succession is clearly visible. due to selective logging several trees with big diameter are still present. it is represented by three plant communities, the community of and the community of and the (fig. 3). the community of and is differentiated by sp, and . it is characterised by plants often seen some years after logging. is a pioneer species, and are light demanding and prefer open areas. the different species group is represented by plants indicative of high anthropogenic disturbance. even though food plants like , and are present no forest wallabies were registered. trees left over after logging have a height of 10-12 m, while tree of the regrowth reach 3-7 m. as the logging intensity was only low and the number of tree species selected to be cut was limited, the vegetation already started to restore after three years time. the average canopy cover is 48 %. the undergrowth is dominated by a species rich herb layer which is 90 cm tall, and by shrubs. it grows on flat areas (average slope 7°) with an altitude between 38-45 m. asl, at a distance of about 4 km from the nearest village. the soil consits of an inceptisol, i.e. a new, still developing soil with a hardly developed soil horizon. the solum is not more than 1 m deep (brookfield 1971; petocz 1989; bps 2009). the soil surface is covered by an average litter layer of 2 cm. due to the flatness of the area, logging is easy and the forest on this site was logged some years ago. this habitat type is used as feeding area by species. four individuals of the forest wallaby and their trails, food remains and dung were noted.in average 14 (11-17) food plant species were registered with a mean cover of 2.7 %. the community of is differentiated by sp, sp., sp sp., musa paradisiaca callamus longipina, diospyros hebecarpa-lepinopsis ternatensis smilax malacensispandanus tectorius communit y musa paradisiaca callamus longipina musa paradisiaca, callamus longipina, palaquium lobbianum, planconella obofata, archidendron bogoriensis, machinlaya celebia, toona coleynea sperata, fagraia rasemosa, macaranga tesylata, pigafetta filaris, ficus tingtoria, haplolobus selebica, podocarpus blumei canarium indicum musa paradisiaca callamus longipina palaquium lobbianum musa paradisiaca canarium indicum ficus tingtoria d. muelleri diospyros hebecarpa-lepinopsis ternatensis diospyros hebecarpa, lepinopsis ternatensis, spatiostemon javensis, clerodendron gluta premna corymbosa, tetrameles nudiflora, prunus arborea, amorphopalus , corimborchis tropical forest types in west papua – sepus fatem & karle v. sykora 69 aglaia spectabilis, bambusa alectrion davallia solida, harpulia branchin redgea, lindsea repens, streblus elongate, rhapidophora duabanga molucanna, giowa horsfieldia laevigata, apostasia odorata, ficus nodosa, callamus ficus anulata, ochrosia barbonica pangium edula. d. muelleri smilax malacensis-pandanus tectorius smilax malacensis smilax malacensis, pandanus tectorius, garcinia picrorrhiza, cayratia trifoliate, mangivera minor, endiandra ficus septica, litsea ladermanii, sterculia parkinsonii, arenga microcarpa, policyas nodosa, cananga odorata, adina nerifolius, rhus taitensis, lea acualeata, pandanus polycarpa, dianella ensifolia, elaeocarpus angustifolius, syzigium versteegi, horsfeldia sylvestris, ficus simisfera, cerbera floribunda, myristica gigantea, actinodaphne nitida, pterocimbium beccari, syzigium archidendron parviflorum, ficus pubescens, ligodium circinatum, davallia hymenophy, disoxylum cyclopeltis crenata, smilax malabatricum, phacomeria speciosa, alocasia zebrine, aglaia simisifera, sononia krasipen, ficus aurantiaceae, micania micantha, nastus holtumianus, gramatophylum papuana, aserantium opositifolium, terminalia complanata, calocasia mastixiodendron pachyclados. ficus aurantiaceae, ficus nodosa, ficus septica, ficus anulata, ficus semisfera, myristica gigantea, horsfeldia laevigata, premna corymbosa, rhapidophora terminalia complanata, syzigium ficus pubescens horsfeldia sylvestris mangivera minor syzigium versteegii smilax malacensis sp., sp., sp., sp., sp., sp., and this vegetation is 10-40 m tall and mainly consists of species that remained after logging besides of newly settled trees and of some pioneer species. canopy cover ranges between 40-50 %. unlike other communities a moss layer is present. mosses are growing on the rocks present under the canopy. the average height of the herb layer is 15 cm. the soil is covered with an average litter layer of 1 cm. it is characteristically developing 15-30 years after logging and grows at an altitude of 123 m at a distance of about 400 m from village. it is frequently found on mid slopes of moderately rocky sites (6-30%) consisting of limestone outcrops. recently, this forest was intensively logged (>10 lumberjacks/day, 20 times/week). although on average 11 (5-19) food plants were registered with a mean cover of 2.4%, no trails, dung or food remains of could be observed. the community is dominated by and is further differentiated by sp., sp., sp., sp and in the last two communities about 15 species of food plant were found: sp., sp., , , and . this forest has a canopy cover of 55 % and is characterized by climbing species and lianas like . the herb layer is 40 cm high and the moss layer is 1 cm. because of logging and the nearness of a logging road, this open forest is characterised by the presence of pioneer species. it grows at an altitude of 167 meter asl on hills with slopes of 8-20°. the landscape consists of undulating plateaus with humus or karstic mounds (bps 2009). the soil is classified as an inceptisol on limestone (brookfield 1971; petocz 1989; bps 2009) and covered by 1 cm of litter. the high calcium content of the soil indicates a ph which is sufficiently high to support the nutrient availability for the plants. the nearest distance to a village is about 700 m. although on average 12 (10-15) food plant species were registered with a mean cover of 2.7 % no trail, dung and other indications of forest wallaby presence could be observed. biotropia vol. 19 no. 2, 2012 70 undisturbed (“primary”) closed forest this habitat type is dominated by trees with big diameter. the size of the trees is variable, both small and big trees occur. the vegetation is further characterized by many lianas and other climbing species. vegetation height is ranging from 5 to 40 m. the herb layer is 38 cm high. as canopy cover is high (80%), the undergrowth consists only of few small shrubs and herbs (fig. 4). this habitat is represented by the community of which is differentiated by , sp., , , sp., , sp., , , , , , , sp., , sp., sp., , sp., , and sommeria leucophylaa-paraltropis glabra sommieria leucophylla alpinia paraltropis glabra buchanania arborescens adina garcinia latisima garcinia orania palindan pterocarpus indicus ficus benyamina intsia bijuga anthocepalus chinensis paracroton pendulous licuala parasarianthes falcataria baringtonia eudia hernandia ovigera popowia ficus pincorhiza alleuritis molucanna gymnacantera farcuhariana. figure 4. undisturbed(“primary'') forest, one of habitat of wallaby (photo: sf-sepus fatem) it grows on flat valley floors with meandering rivers, at an altitude between 40-102 m asl. it is composed of plant species frequent on moderate slopes (15°) of stabile shaded ecosystems with flat topography. the soil can be classified as an inceptisol (brookfield 1971; petocz 1987; bps 2009). are food plants for the forest wallaby. eight individuals of the forest wallaby and its dung and food remains were observed. in average 10 (6-12) food plant species were counted with a mean cover of 2.34 %. plants common for open to closed forest have a wide amplitude concerning light conditions and can grow both in light open forests and below the canopy of tall trees. some have their optimum in shade while other species grow better in the presence of light. species indicative of more shady conditions below taller vegetation, include , , sp., and , (alhamid 1988; maturbongs 2001; asri 2005; arijani 2006). , sp. and and are characteristic of open woody vegetation with and without partial shade; (maturbongs 2001 and asri 2005). this represents the ecotone between open forest and closed ficus benyamina, ficus pincorhizza, intsia bijuga selaginella martensii scindapsus pietus scindapsus euscuarius, phylodendron meremia peltata asplenium nidus, korthalzia zippelii amomum aculeatum octomeles sumatrana pandanus arthocarpus altilis homalium foetidium common differentiating plants tropical forest types in west papua – sepus fatem & karle v. sykora 71 forest. this is supported by asri (2005) in west papua; harkink (1987) in papua new guinea; and meijaard . (2005) in kalimantan. sp., is the most dominant species in this habitat type, and has been found in our research to co-occur with some early pioneer species. , , , , sp. and are differentiating the primary forest, where human interference is only low or even absent. some species are commonly occurring in open to closed forest and in forest with former low logging intensity. the logged forest is restoring to later forest stages by succession. in this forest type some plant species facilitate the growth of other species by providing shade and protection. here some species like; and provide food for herbivores, like macropodidae animals (maturbongs 2001; fatem 2008). also open forest and logged forest have species in common. in these habitat types species grow fast in order to catch light, like , sp., . are food source for animals like . one of the lianas, occurs in all distinguished plant communities. it suppresses tree regeneration and increases tree mortality. lianas also influence competition between trees and thus they effect forest composition. lianas are also a valuable food source for some animals as well as for local population of people. they enable canopy to canopy access for arboreal species (bongers 2002). other species are common differentials for closed forest regenerated after logging. also in this vegetation several lianas grow as pioneer species. the vegetation is further characterized by tall trees (10-40 m), like , , , , , and this vegetation is found in the lowland forest area and close to villages and the coastal area. besides, , , and are species typical of lowland tropical vegetation as reported by jhon (1997); maturbongs (2001); meijerd (2005); kartikasari (2012). wallaby presence was observed in 4 of the 6 distinguished plant communities belonging to grassland, open forest with only little logging activity, and in undisturbed closed forest. it was however not registered from forests with clear influence of logging. appears to be very sensitive to human disturbance. according to detrended correspondence analysis (fig. 5) the presence of the forest wallaby (mac pre, tra) is strongly correlated to distance from villages (r respectively 0.69, 0.54) and negatively correlated to logging (log perr -0.79 and -0.60, cut int r -0.75 and 0.65, amostu r -0.75 and -0.65) and hunting (hunt int r -0.32 and -0.28). et al clomarippsidacae sommeria leuchophylla garcinia latisima pterocarpus indicus buchaninia arborenscens adina orania palindan musa paradisiaca ficus tingtoria et al. callamus aruensis poliyalthia freycinetia scandens arthocarpus vresianus, pometia corriacea d. muelleri meremia peltata, alstonia scholaris gnetum gnemon canarium dekamanum callamus cayensis pommetia acuminata syzigium malacensis prainea limpato. gnetum gnemon alstonia scholaris pometia acuminata canarium dekamanun et al. d. muelleri vegetation, disturbance and wallaby presence 2 2 2 2 2 biotropia vol. 19 no. 2, 2012 72 figure 5. ordination diagram showing the first two axes of a dca analysis. the arrows represent the correlation with external variables, showing both the direction and the strength of the correlation (length of the arrow). arrows in the same direction are positively correlated, opposite arrows are negatively correlated. note; hunting intensity (hunt int), altitude (alt), cutting intensity (cut int), amount of stump (amostu), logging period (log per), slope (slo), moss layer (ml), dung (dun), encountered (macrenc), feeding remnant (fee rem), present (mac pre), distance (dis), trail (tra), litter layer (ll), herb layer (hl), food plant species (foo pla), food plant cover (fp cov), canopy cover % (can). d. muelleri d. muelleri the relation between wallaby presence and food plant availability appears to be only low (r 0,14 and 0,02). as the structure and species composition of the 4 wallaby plant communities differs considerably, and food plants are present in all plant communities and as wallaby presence is highly correlated to human disturbance and has not been found in vegetation with human disturbance, our results indicate that vegetation structure, vegetation composition and food plant availability are less important than human disturbance. four plant communities are used by the forest wallaby for foraging and as their territory i.e. the community, the community of sp, the community of and and the community of representing grassland, open and closed forest. it was not detected in the community of and the community representing logged forests. even the presence of food plants did not guarantee the presence of forest wallabies. species appear to be very sensitive to human disturbance. there is a strong negative relation between the presence of this species and on the other hand logging, distance to villages and hunting. therefore, the habitat of this species 2 conclusions imperata cylindrica, ipomea aquatica ficus robusta, dendrocnide musa paradisiaca callamus longipina sommeria leucophylaa-paraltropis glabra diospyros hebecarpa-lepinopsis ternatensis smilax malacensis-pandanus tectorius d. muelleri should tropical forest types in west papua – sepus fatem & karle v. sykora 73 be protected and conserved by government regulations reducing the disturbance by logging and hunting. other stake holders should be involved to create public support. comunity based wildlife management can be used to reduce anthropogenic pressure. although this study gives a good description of the habitat and vegetation in which the wallaby has been found to forage, further more detailed research is needed to better understand the relation between wallaby presence, food preferences and human influence. our study is descriptive and consequently only shows correlations. although it clearly indicates the sensitivity of the forest wallaby for anthropogenic disturbance even if food plants are present, it is recommended to prove this relation experimentally for instance by reducing the anthropogenic influence in certain areas. references arijani, setiadi d, edi g, ibnul q. 2006.vegetation analysis of the up-stream cianjur watershed in mount gedepangrango national park's. biodiversitas. volume 7. alhamid h. 1988. studi habitat dan populasi burung cenderawasih kecil ( , shaw) di areal bekas tebangan pt. inhutani ii dalam kawasan cagar alam pegunungan arfak manokwari. 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f -2 0 b f -2 1 w f -2 4 w f -2 5 s f -3 3 s f -3 0 s f -3 1 s f -3 2 a 1 a 2 a 2 a 2 a 2 a 2 a 2 a 2 a 3 a 3 a 3 a 3 a 3 a 3 a 3 a 3 a 3 a 4 a 4 a 4 a 4 a 5 a 5 a 5 a 5 a 5 a 5 a 6 a 6 a 6 a 6 a 6 a 6 m o s s la y e r (c m ) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 1 1 1 1 0 1 0 0 1 h e rb la y e r (c m ) 9 0 5 0 6 0 4 0 4 0 9 0 3 0 5 0 5 0 4 0 5 0 5 0 3 0 5 0 1 0 4 0 2 0 9 0 9 0 9 0 9 0 1 0 2 0 2 0 1 0 2 0 2 0 1 5 2 0 5 0 5 0 5 0 5 0 l it te r la y e r (c m ) 1 2 2 2 2 1 2 2 4 4 4 2 4 4 4 2 2 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 c a n o p y (% ) 1 0 5 0 4 0 5 0 5 0 4 0 5 0 5 0 9 0 9 0 7 0 8 0 9 0 9 0 9 0 6 0 7 0 5 0 5 0 4 0 5 0 5 0 5 0 4 0 4 0 5 0 5 0 5 0 5 0 5 0 6 0 6 0 6 0 a lt it u d e (m .a .s .l .) 2 9 4 7 3 8 4 2 3 0 5 0 3 9 4 6 5 1 4 9 7 1 5 4 7 1 5 7 1 0 2 4 4 8 2 4 5 4 5 3 8 4 3 1 0 9 1 3 8 6 2 9 8 1 5 5 1 7 8 1 5 5 1 7 8 1 7 9 1 3 0 1 6 9 1 9 4 s lo p e (° ) 3 .4 3 5 .7 1 9 .0 9 5 .7 1 5 .7 1 1 2 .4 1 5 .7 1 1 4 .5 7 1 3 .5 0 1 .1 5 3 2 .6 2 1 3 .5 0 1 6 .7 0 1 1 .3 1 7 .9 7 6 .8 4 2 3 .7 5 5 .7 1 7 .9 7 4 .5 7 1 0 .2 0 1 2 .4 1 2 2 .7 8 1 9 .8 0 5 .7 1 3 2 .6 2 1 6 .7 0 1 0 .2 0 1 6 .7 0 1 6 .7 0 1 4 .5 7 1 9 .8 0 5 .7 1 d is ta n c e (m ) 5 1 4 3 7 2 6 4 5 1 2 0 6 7 2 3 6 7 3 4 6 0 0 2 6 7 4 6 6 7 5 1 8 0 8 4 8 0 5 6 6 0 1 5 6 0 4 2 6 0 6 7 8 0 9 3 5 1 2 0 8 0 3 5 5 1 2 0 4 3 2 6 4 3 4 9 4 2 3 0 4 2 6 5 4 9 9 6 1 7 4 8 2 6 0 8 7 3 7 8 9 0 7 5 5 8 9 1 8 7 6 4 1 8 5 0 8 6 7 8 a m o u n t s tu m p (i n d /c u tt in g ti m e s ) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 5 5 6 5 5 5 5 5 5 5 5 5 h u n ti n g in te n s it y (t im e s /w e e k ) 0 2 1 1 1 2 1 1 1 1 2 1 2 1 0 1 1 0 0 1 0 3 3 1 1 1 1 3 3 3 3 3 3 c u tt in g in te n s it y (t im e s /w e e k ) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 5 5 5 5 5 5 5 5 5 5 5 5 l o g g in g p e ri o d (y e a r) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 3 3 3 3 3 3 3 3 3 3 3 3 m a c ro p o d id a e e n c o u n te r (i n d ) 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 f e e d in g re m n a n t 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l e v e c o d e p f -a 1 s f -b 1 3 p f -a 2 s f -a 9 s f -a 1 0 h f -1 4 s f -a 1 1 s f -a 1 2 p f -b 7 p f -b 6 h f -1 5 h f -1 6 h f -1 7 p f -b 8 p f -a 4 p f -b 5 p f -a 3 l a -2 8 l a -2 9 l a -2 6 l a -2 7 w f -2 2 w f -2 3 b f -1 8 b f -1 9 b f -2 0 b f -2 1 w f -2 4 w f -2 5 s f -3 3 s f -3 0 s f -3 1 s f -3 2 d if fe re n ti a l s p e c g ro u p 1 . im p e ra ta c y li n d ri c a ip o m o e a a q u a ti c a g ra s s la n d im p e ra ta s p 9 1 0 0 9 n e p h ro le p s is b is e ra ta 9 1 0 0 9 9 1 1 4 3 4 1 1 1 1 3 2 5 3 8 1 7 8 im p e ra ta c y lin d ri c a 9 1 0 0 9 9 1 4 9 ip o m e a a q u a ti c a 8 1 0 0 8 s a c c h a ru m s p o n ta n e u m 8 1 0 0 8 8 1 4 8 9 1 1 9 m a lo tu s p h ili p in e n s is 6 1 0 0 6 1 1 3 3 6 7 2 3 1 2 5 0 2 ip o m e a b a ta ta s 5 1 0 0 5 j u s s ia e e o c ta v a lif is 4 1 0 0 4 5 3 2 4 5 7 4 3 5 4 3 3 4 p ip e r a d u n c u m 1 1 0 0 1 5 5 3 3 4 7 1 4 4 1 1 4 2 2 5 0 2 4 1 3 3 3 6 1 7 6 m a c a ra n g a m a p p a 1 1 0 0 1 9 8 2 4 3 6 1 2 5 1 1 1 3 3 1 3 7 3 3 5 m im o s a p u d ic a 1 1 0 0 1 m u n ti g ia c a lla b u ra 1 1 0 0 1 m u c u n a n o v a e g u in e s is 1 1 0 0 1 1 4 3 8 5 7 4 2 2 5 2 p u e ra ri a ja v a n ic a 1 1 0 0 1 1 1 4 1 1 1 1 1 s p o n d ia s c y th e re a 1 1 0 0 1 1 2 5 1 3 6 3 5 0 4 7 1 7 7 m ic a n ia s p 1 1 0 0 1 3 3 3 4 3 3 7 8 5 0 8 1 1 7 1 z in g ib e r s p 2 1 1 0 0 1 2 2 2 2 1 5 6 2 3 1 7 3 d if fe re n ti a l s p e c . g ro u p 2 : f ic u s ro b u s ta -d e n d ro c n id e s p , g ra s s la n d -f o re s t tr a n s it io n f ic u s ro b u s ta 8 2 9 3 2 7 3 1 0 0 5 1 1 1 3 3 1 1 1 5 0 1 2 1 7 2 1 1 3 4 6 7 2 d o n a x c a n if o rm is 5 2 9 8 8 9 9 1 0 0 7 3 3 4 2 3 5 6 3 2 5 5 0 4 2 1 3 3 2 1 1 7 5 0 3 d e n d ro c h id e s p 9 1 7 3 5 7 1 5 1 1 1 1 2 2 5 2 f ic u s tr a c ip is o n 5 1 5 2 3 7 1 3 3 2 5 3 1 1 7 1 h o rn s te n d ia s c o tt ia n a 7 4 1 9 4 7 1 5 2 3 3 3 3 3 7 2 5 7 1 1 3 3 1 p a n d a n u s d u b iu s 4 2 2 1 5 7 2 d e rr is a lb a 5 5 7 1 5 7 5 1 1 7 1 s te rc u lia s h ill in g la w i 1 2 2 4 3 2 1 1 1 1 1 1 7 1 1 1 7 1 f ic u s ja p o n ic a 5 1 4 4 3 3 1 2 5 1 1 1 3 3 1 c a ri y a p a p a y a 1 1 1 4 3 1 1 1 3 3 1 o ri o c n id e n it id a 1 1 2 9 1 1 1 7 1 e n d o s p e rn u m m o lu c c a n u m 1 2 2 9 2 1 2 5 1 1 1 3 3 1 1 1 7 1 r h a p id o p h o ra s p 1 3 2 9 2 s p a th o d e a c a m p a n u la ta 7 1 2 9 4 n e o la le b a a tr a 1 2 2 9 2 3 1 7 3 f ic u s s p 1 9 1 4 9 v it te x p in n a ta 8 1 4 8 1 1 1 3 3 1 1 1 7 1 1 2 3 3 2 d u ri o z ib e th in u s 7 1 4 7 l a n c iu m d o m e s ti c u m 7 1 4 7 c y a te a m o lu c a n n a 6 1 4 6 1 1 1 1 f ic u s v a ri e g a ta 3 1 4 3 1 2 5 1 c le y ta n c e s s p 3 1 4 3 1 1 1 1 p a n d a n u s c o n o id e u s 2 1 4 2 d ry m o p h o le u s lit ig io s u s 2 1 4 2 7 1 1 7 t h e o b ro m a c a c a o 2 1 4 2 m a c a ra n g a g ig a n te a 1 1 4 1 m a n g if e ra in d ic a 1 1 4 1 a n a n a s c o m o s u s 1 1 4 1 c a lla m u s w a rb u g ii 1 1 4 1 c o c o s n u c if e ra 1 1 4 1 p ri n iu m s p 1 1 4 1 s a lla c a e d u lis 1 1 4 1 k o k o o n a o c h ra c e a 1 1 4 1 1 1 1 1 d e n d ro b iu m s p 1 1 4 1 m u s a p a ra d is ia c a -c a ll a m u s lo n g ip in a , lo g g e d fo re s t d io s p y ro s h e b e c a rp a -l e p in o p s is te rn a te n s is , lo g g e d fo re s t o n s it e s w it h la rg e c o v e r o f ro c k s . s m il a x m a la c e n s is -p a n d a n u s te c to ri u s , l o g g e d fo re s t w it h ro c k s 6 im p e ra ta c y li n d ri c a ip o m o e a a q u a ti c a g ra s s la n d c o m m u n it y 2 3 4 5 f ic u s ro b u s ta -d e n d ro c n id e s p , g ra s s la n d -f o re s t tr a n s it io n s o m e ri a le u c h o p h il la -p a ra tr o p is g la b ra tropical forest types n west papua, the presence of forest wallaby sepus fatemi – et al. 76 a p p en d ix 2. d if fe re n ti al ve ge ta ti o n ta b le w it h b o th se p ar at e re le vé s an d sy n o p ti c in fo rm at io n . f o r th e se p ar at e re le vé s th e co ve rab u n d an ce is re p re se n te d b y th e o rd in al 9sc al e, sy n o p ti c in fo rm at io n co n si st s o f % p re se n ce an d ch ar ac te ri st ic co ve r (m ea n co ve r o f th e n u m b er o f o cc u rr en ce s o f a sp ec ie s in a ce rt ai n cl u st er ). d if fe re n ti a l s p e c ie s g ro u p 3 : s o m e ri a le u c h o p h il la -p a ra tr o p is g la b ra s o m m ie ri a le u c o p h y lla 7 7 9 8 9 1 6 7 7 a lp in ia s p 2 4 3 1 3 5 6 3 1 1 3 3 1 p a ra lt ro p is g la b ra 4 1 3 1 1 5 6 2 8 2 5 8 b u c h a n a n ia a rb o re s c e n s 4 4 2 3 3 3 a d in a s p 2 1 1 3 3 1 g a rc in ia la ti s im a 1 1 1 3 3 1 1 2 5 1 g a rc in ia s p 1 1 1 3 3 1 o ra n ia p a lin d a n 5 1 6 3 3 4 p te ro c a rp u s in d ic u s 1 1 4 1 3 1 1 3 3 2 f ic u s b e n y a m in a 3 1 1 3 3 2 in ts ia b iju g a 5 1 2 2 3 a n th o c e p a lu s c h in e n s is 3 1 4 3 1 3 2 2 2 p a ra c ro to n p e n d u lo u s 2 2 2 2 2 l ic u a la s p 5 1 4 5 5 1 2 2 3 p a ra s a ri a n th e s fa lc a ta ri a 1 1 4 1 2 1 2 2 2 2 1 7 2 b a ri n g to n ia s p 9 1 1 9 e u d ia s p 4 1 1 4 h e rn a n d ia o v ig e ra 2 1 1 2 p o p o w ia s p 1 1 1 1 f ic u s p ic ro rr h iz a 1 1 1 1 a le u ri ti s m o lu c a n n a 1 1 1 1 g y m n a c a n te ra fa rc u h a ri a n a 1 1 1 1 d if f e r e n t ia l s p e c ie s . d 1 s e la g in e lla m a rt e n s i 9 3 9 7 9 9 9 1 0 0 8 8 9 9 5 7 8 7 7 9 1 0 0 8 9 9 5 0 9 s c in d a p s u s p ie tu s 4 4 2 3 3 7 1 3 2 3 2 3 4 2 4 4 3 1 0 0 3 t a b e rn a e m o n ta n a s p 1 4 6 1 4 7 8 6 4 2 3 4 6 7 5 6 4 s c in d a p u s e u s c u a ri u s 1 4 4 3 5 7 3 2 4 1 3 4 4 3 7 8 3 h o m a liu m fo e ti d iu m 6 8 4 2 5 7 5 3 1 2 4 2 8 6 7 3 4 2 5 7 5 4 1 1 7 1 2 1 3 3 2 f la g e ri a in d ic a 1 3 5 2 5 7 3 1 1 1 3 3 1 2 2 5 0 2 p le o m e le a n g u s ti fo lia 3 1 2 9 2 4 3 2 2 4 3 2 5 3 b a lb it is rh y z o p h y lla 4 2 4 4 3 3 4 7 5 4 6 5 4 7 8 5 p ip e r in te rr u p tu m 1 2 1 4 3 1 2 1 2 2 1 5 6 2 1 2 2 3 6 7 2 c lo m a ri p p s id a c a e s p 2 4 2 3 5 7 3 4 2 6 1 3 6 6 7 4 1 1 7 1 d if fe re n ti a l s p e c . g ro u p 4 : m u s a p a ra d is ia c a -c a ll a m u s lo n g ip in a , lo g g e d fo re s t m u s a p a ra d is ia c a 5 1 1 4 3 2 1 1 1 1 5 2 3 8 1 0 0 5 1 1 3 5 0 2 1 1 7 1 c a lla m u s lo n g ip in a 1 5 2 9 3 2 7 4 3 3 4 4 2 3 3 1 0 0 3 p a la q u iu m lo b b ia n u m 3 1 4 3 6 9 2 4 1 5 6 4 9 8 4 7 1 0 0 7 7 5 3 3 6 1 2 3 3 2 p la n c o n e lla o b o fa ta 1 1 4 1 8 1 1 8 7 3 4 6 1 0 0 5 2 2 7 3 6 7 4 2 1 7 2 a rc h id e n d ro n b o g o ri e n s is 0 3 4 5 0 4 2 1 3 3 2 m a c h in la y a c e le b ia 0 1 1 5 0 1 1 1 7 1 t o o n a s p 1 1 1 1 7 2 5 7 c o le y n e a s p e ra ta 0 1 2 5 1 f a g ra ia ra s e m o s a 0 1 2 5 1 m a c a ra n g a te s y la ta 0 1 2 5 1 p ig a fe tt a fi la ri s 0 1 2 5 1 f ic u s ti n g to ri a 0 1 2 5 1 h a p lo lo b u s s e le b ic a 0 1 2 5 1 6 1 7 6 p o d o c a rp u s b lu m e i 1 2 2 2 2 1 2 5 1 1 1 7 1 c a n a ri u m in d ic u m 1 1 1 1 1 2 5 1 d if f e r e n t ia l s p e c ie s . d 2 s e m e c a rp u s p a p u a n a 8 1 1 3 3 3 6 3 1 7 5 3 1 1 3 3 1 1 1 7 1 d ry m o p h lo u e s o liv o rm is 2 1 4 2 4 3 7 3 3 5 2 6 3 7 5 4 1 1 7 1 f re y n e ti a s p 3 1 4 3 5 7 1 1 1 1 6 7 3 4 7 5 0 6 2 1 7 2 b u b ia s p 1 2 2 4 1 5 6 2 3 2 5 3 l a p o rt e a c a n d e n s is 1 1 2 9 1 3 8 4 2 1 5 6 4 6 2 5 6 g o n o c a ri u m lit o ra lle 3 1 3 1 4 4 2 6 6 5 0 6 1 1 1 5 0 1 c y n o m e tr a s p 2 1 1 3 3 1 5 1 5 0 3 1 1 7 1 p in a n g a ru m p h ia n a 1 7 4 1 4 4 3 2 5 5 0 4 c a n a ri u m h ir s u tu m 1 6 7 3 3 5 2 2 5 2 1 1 7 1 c a m p n o s p e rm a b re v ip e ti o la ta 4 1 4 3 3 3 1 2 5 1 g ir in o p s s p 7 1 1 7 8 2 5 8 d if fe re n ti a l s p e c . g ro u p 5 : d io s p y ro s h e b e c a rp a -l e p in o p s is te rn a te n s is , lo g g e d fo re s t o n s it e s w it h la rg e c o v e r o f ro c k s . d io s p y ro s h e b e c a rp a 3 8 1 7 2 8 3 4 l e p in o p s is te rn a te n s is 1 1 1 1 3 2 5 3 1 1 2 2 6 7 2 s p a ti o s te m o n ja v e n s is 2 6 8 9 6 7 6 4 1 7 4 c le ro d e n d ro n s p 1 1 3 5 0 2 g lu ta s p 2 8 6 5 0 5 p re m n a c o ry m b o s a 7 1 1 5 0 3 t e tr a m e le s n u d if lo ra 1 1 1 5 0 1 p ru n u s a rb o re a 1 2 3 3 2 a m o rp h o p a lu s s p 3 1 3 3 2 c o ri m b o rc h is s p 1 1 2 2 1 1 1 3 3 1 a g la ia s p e c ta b ili s 1 1 3 3 1 b a m b u s a s p 1 1 4 1 9 1 7 9 a le c tr io n s p 3 1 7 3 d a v a lli a s o lid a 3 1 7 3 h a rp u lia s p 2 1 7 2 1 1 7 1 b ra n c h in re d g e a 1 1 1 1 2 1 7 2 l in d s e a re p e n s 2 1 7 2 s tr e b lu s e lo n g a ta 1 1 7 1 r h a p id o p h o ra s p 1 1 7 1 d u a b a n g a m o lu c a n n a 1 1 7 1 g io w a s p 1 1 7 1 h o rs fi e ld ia la e v ig a ta 1 1 7 1 a p o s ta s ia o d o ra ta 1 1 7 1 f ic u s n o d o s a 2 1 4 2 1 1 1 1 1 1 7 1 c a lla m u s s p 1 1 4 1 1 1 7 1 f ic u s a n u la ta 1 1 1 1 1 1 7 1 o c h ro s ia b a rb o n ic a 1 1 7 1 p a n g iu m e d u la 1 1 7 1 biotropia vol. 19 no. 2, 2012 77 tropical forest types n west papua, the presence of forest wallaby sepus fatemi – et al. a p p en d ix 2. c o n ti n u ed d if fe re n ti a l s p e c . g ro u p 6 : s m il a x m a la c e n s is -p a n d a n u s te c to ri u s , l o g g e d fo re s t w it h ro c k s s m ila x m a la c e n s is 1 2 2 9 2 1 1 1 1 1 5 3 3 3 2 6 1 3 7 9 1 0 0 5 p a n d a n u s te c to ri u s 1 1 7 1 2 1 1 4 6 7 2 g a rc in ia p ic ro rr h iz a 1 1 4 1 1 1 1 1 1 1 3 3 1 1 1 1 1 6 7 1 c a y ra ti a tr if o lia ta 2 6 5 5 0 4 m a n g iv e ra m in o r 1 1 4 1 2 1 6 5 0 3 e n d ia n d ra s p 2 1 7 2 1 3 2 5 0 2 f ic u s s e p ti c a 4 1 2 9 3 1 1 4 5 0 2 l it s e a la d re m a n ii 5 1 1 5 1 1 7 1 1 2 2 5 0 2 s te rc u lia p a rk in s o n ii 1 1 7 2 1 4 5 0 2 a re n g a m ic ro c a rp a 2 1 4 2 1 1 1 1 1 3 1 5 0 2 p o lic y a s n o d o s a 1 1 3 5 0 2 c a n a n g a o d o ra ta 1 1 2 9 1 1 2 5 1 1 1 7 1 4 7 3 3 6 a d in a n e ri fo liu s 1 1 3 3 1 2 9 3 3 6 t e y s m a n io d e n d ro n b o g o ri e n s is 2 1 1 2 9 2 5 9 2 7 3 3 5 r h u s ta it e n s is 1 1 3 3 1 3 5 3 3 4 d ia n e lla e n s if o lia 1 1 4 1 1 6 3 3 4 e la e o c a rp u s a n g u s ti fo liu s 1 1 1 1 1 6 3 3 4 s y z ig iu m v e rs te e g i 1 1 1 5 0 1 2 3 3 3 3 h o rs fe ld ia s y lv e s tr is 1 1 1 1 2 1 7 2 1 3 3 3 2 f ic u s s e m is e fe ra 1 4 3 3 3 c e rb e ra fl o ri b u n d a 6 1 1 6 1 1 7 1 1 2 3 3 2 m y ri s ti c a g ig a n te a 1 1 7 1 1 1 3 3 1 a c ti n o d a p h n e n it id a 1 1 7 1 1 1 3 3 1 p te ro c im b iu m b e c a ri 2 1 2 2 2 1 1 7 1 1 1 3 3 1 s y z ig iu m s p 5 1 2 2 3 1 1 3 3 1 a rc h id e n d ro n p a rv if lo ru m 1 1 7 1 1 1 3 3 1 f ic u s p u b e s c e n s 9 1 7 9 l ig o d iu m c ir c in a tu m 7 1 7 7 d a v a lli a h y m e n o p h y 1 1 4 1 6 1 7 6 d is o x y lu m s p 2 1 7 2 5 1 7 5 c y c lo p e lt is c re n a ta 1 1 1 1 4 1 7 4 s m ila x m a la b a tr ic u m 4 1 7 4 p h a c o m e ri a s p e c io s a 4 1 7 4 a lo c a s ia z e b ri n a 3 1 7 3 a g la ia s im is if e ra 3 1 7 3 s o n o n ia k ra s ip e n 2 1 7 2 f ic u s a u ra n ti a c e a e 2 1 7 2 m ic a n ia m ic a n th a 2 1 7 2 n a s tu s h o lt u m ia n u s 2 1 7 2 g ra m a to p h y lu m p a p u a n a 1 1 7 1 a s e ra n ti u m o p o s it if o liu m 1 1 7 1 t e rm in a lia c o m p la n a ta 1 1 1 1 1 1 7 1 c a lo c a s ia s p 1 1 7 1 m a s ti x io d e n d ro n p a c h y c la d o s 4 1 4 4 2 1 1 2 1 1 7 1 l e a a c u a le a ta 5 1 1 5 1 1 7 1 1 1 7 1 p a n d a n u s p o ly c a rp a 7 1 1 7 1 1 7 1 d if f e r e n t ia l s p e c ie s .d 3 k o o rd e rs io d e n d ro n p in n a tu m 2 2 4 2 7 3 1 0 0 3 8 3 9 5 6 7 6 s te rc u lia m a c ro p h y lla 1 1 1 1 4 1 2 4 7 6 1 0 0 4 8 1 7 6 6 7 6 p o m e ti a c o rr ia c e a 1 2 2 2 2 8 3 3 8 9 8 3 6 8 8 9 9 9 8 1 0 0 9 p te ri g o ta h o rs fe ld ia 1 1 2 9 1 1 1 1 1 7 4 3 2 1 8 3 3 7 3 4 7 6 7 5 c ry p to c a ry a s p 6 1 4 6 1 1 1 1 1 7 1 5 0 3 1 5 7 7 2 8 3 4 l u n a s ia a m a ra 7 9 2 8 9 8 3 7 3 1 9 5 0 4 s is ip u s ju ju b a 4 1 1 4 1 2 5 1 3 1 1 5 0 2 2 3 6 2 7 8 3 4 p a la q u iu m a m b o in e n s is 1 1 1 1 1 8 9 9 9 8 3 7 7 4 3 6 6 7 5 d ra c o n tu m e lu m d a o 1 1 1 4 3 1 2 1 1 2 2 2 5 2 2 2 4 1 6 7 2 2 7 2 5 0 4 a re a n g ili s a fl a v a 2 1 2 2 2 3 3 2 7 6 7 4 6 2 3 3 4 d is o x y lu m m o lli s im u s 2 1 3 5 0 2 1 1 3 9 6 7 4 f re y n e ti a s c a n d e n s 3 1 3 3 2 1 1 2 1 6 7 1 78 m y ri s ti c a fa tu a 1 1 4 1 1 5 3 3 3 1 1 2 3 6 7 2 c a lla m u s a ru e n s is 1 2 1 5 0 1 1 1 3 5 6 7 3 g lo s id io n s p 1 1 1 5 0 1 2 2 3 3 2 p o liy a lt h ia s p 1 1 2 2 1 3 3 4 5 0 3 2 7 3 3 5 n u c le a o ri e n ta lis 4 1 3 3 3 2 7 3 5 0 4 p o liy a lt h ia s u m a tr a n a 5 2 5 5 4 3 3 3 4 2 3 3 3 3 in o c a rp u s fa g iv e ru s 1 1 1 1 1 1 7 1 2 1 3 3 2 d io s p y ro s p a p u a n a 1 1 7 1 1 1 7 1 p la ti c e ri u m b if fu rc a tu m 1 1 4 1 1 1 7 1 1 1 7 1 p y s o n ia u m b e lif e ra 1 1 7 1 1 1 7 1 r a p a n e a s p 1 1 1 1 1 1 7 1 2 1 7 2 t e rm in a la c a n ic u la ta 1 1 7 1 1 1 7 1 m o ri n d a c it ri fo lli a 1 1 4 1 1 1 7 1 1 1 7 1 p o ly a lt h ia g la u c a 4 1 7 4 4 1 7 4 a rt h o c a rp u s v re s ia n u s 1 1 7 1 1 1 7 1 d if f e r e n t ia l s p e c ie s .d 4 a ls to n ia s c h o la ri s 3 1 1 1 4 4 2 1 2 2 7 5 2 1 1 1 3 1 8 3 1 4 1 7 4 g n e tu m g n e m o d e is 7 3 1 1 2 5 6 3 2 2 5 0 2 1 2 3 3 2 1 1 5 1 1 8 3 2 c a n a ri u m d e k a m a n u m 2 1 8 7 4 4 5 7 7 1 7 5 5 4 2 3 3 3 4 3 3 3 4 l ic u a la te liv e ra 7 6 2 1 7 1 6 7 4 1 7 8 7 5 5 2 3 3 3 3 4 1 2 5 0 2 c a lla m u s c a y e n s is 1 1 1 3 3 1 2 1 5 0 2 1 1 7 5 0 3 9 1 7 9 p o m e ti a a c u m in a ta 1 1 1 1 1 2 5 1 1 1 3 3 1 3 1 7 3 s y z ig iu m m a la c e n s is 1 4 1 3 3 2 1 2 5 1 1 1 7 1 3 1 7 3 p ra in e a lim p a to 2 1 4 2 0 2 2 5 2 1 1 7 1 c o m m u n it y c r e m a in in g s p e c ie s m e re m ia p e lt a ta 2 1 0 0 2 7 5 9 4 3 8 8 6 6 4 5 5 6 2 3 6 7 4 8 9 7 7 5 8 6 3 8 4 7 8 1 0 0 6 2 2 7 2 6 7 3 g n e tu m g n e m o 4 1 1 1 5 7 2 3 3 2 1 3 6 1 1 8 9 3 2 5 1 2 1 0 0 3 1 1 3 3 1 1 2 4 4 3 3 1 0 0 3 c e lt is l a ti fo lia 1 1 2 9 1 9 1 1 6 1 5 6 4 8 3 2 5 1 0 0 5 7 8 5 5 0 7 9 9 9 8 8 3 1 0 0 8 a m o m u m a c u le a tu m 8 1 3 4 7 9 1 1 0 0 5 1 4 3 4 4 4 3 3 5 4 7 5 4 2 4 3 3 3 1 1 1 5 0 1 k o rt h a lz ia z ip p e lii 1 1 9 8 9 7 8 6 6 7 7 9 7 3 1 1 7 8 5 5 4 3 7 1 0 0 5 1 1 7 1 h o rs fe ld ia p a rv if lo ra 5 2 9 8 5 7 6 5 6 7 7 2 2 1 7 8 9 5 8 6 3 5 1 0 0 6 8 3 2 5 0 4 3 2 3 5 0 3 m e d u s a n th e ra la x if lo ra 1 1 1 4 3 1 1 7 5 3 1 5 6 3 4 1 6 8 1 0 0 5 5 7 7 5 0 6 2 1 7 2 p im e le o d e n d ro m a m b o in ic u m 1 1 2 9 1 5 2 1 1 2 1 6 7 2 4 6 3 7 1 0 0 5 8 4 8 8 8 8 3 7 1 3 7 3 7 8 3 4 p h y lo d e n d ro n s p 1 1 2 4 3 1 6 2 5 8 2 1 1 1 8 9 3 4 6 5 8 1 0 0 6 2 5 1 5 0 3 2 7 7 5 0 5 h o rs fe ld ia ir y a 1 1 4 1 2 2 1 3 3 2 3 1 3 6 1 0 0 3 2 8 3 5 0 4 1 1 3 3 1 p ip e r g ib b ili m b u m 2 1 3 7 4 7 1 3 4 3 4 5 6 9 6 7 5 5 8 5 0 7 3 3 6 4 6 7 4 6 5 3 8 8 1 1 0 0 5 b a u h in ia to m e n to s a 3 1 1 1 6 7 1 2 5 5 7 1 8 5 6 5 7 3 5 0 5 3 3 2 7 6 7 4 6 1 7 6 7 2 1 0 0 5 p o m e ti a p in n a ta 6 4 2 8 5 7 5 8 9 7 9 9 6 5 9 9 1 0 0 8 9 5 8 7 5 7 1 3 3 3 2 1 1 2 5 0 1 m a n ilt o a b ro w n o id e s 1 1 4 1 5 2 5 1 4 4 3 6 7 7 7 5 7 4 5 3 2 1 2 1 0 0 3 7 7 7 5 0 7 a rt h o c a rp u s a lt ili s 4 1 5 9 5 7 5 4 7 1 1 1 1 1 1 8 9 2 6 5 3 7 5 5 1 1 2 5 0 1 6 1 2 2 6 7 3 a g la ia o d o ra ta 1 1 1 4 3 1 3 5 5 1 7 6 1 7 8 4 4 4 5 0 4 5 1 8 7 6 7 5 2 1 2 4 6 7 2 l it s e a ti m o ri a n a 3 7 2 9 5 2 1 4 1 4 4 2 5 1 2 7 5 3 3 7 3 3 5 1 1 7 1 h y d ri a s te le rh o p a lo c a rp a 1 7 5 4 3 4 6 6 2 2 6 2 2 6 7 5 3 1 4 3 3 3 h a p lo lo b u s la n c e o tu s 1 1 4 1 2 7 6 3 3 5 4 7 2 7 5 4 1 1 2 5 0 1 1 3 3 3 2 a s p le n iu m n id u s 1 1 4 1 2 1 1 2 3 2 2 7 5 2 1 1 1 5 0 1 a re c a m a c ro c a ly x 1 2 2 9 2 3 3 5 1 1 5 6 3 3 2 8 7 5 4 1 1 7 1 1 1 7 1 z in g ib e r s p 1 4 1 2 2 1 7 1 2 1 2 2 2 2 2 2 1 7 5 2 5 2 3 3 4 2 1 1 5 0 1 p a n d a n u s s p 5 6 9 6 2 7 1 6 1 1 5 4 4 4 3 3 2 5 3 7 1 2 5 0 3 g o n io ta la m u s s p 1 1 4 1 5 1 2 2 3 1 6 7 2 7 2 5 0 5 2 3 3 3 3 2 1 7 2 c a ry o th a ru m p h ia n a 1 1 1 1 5 7 1 1 1 1 1 1 2 5 1 1 2 1 5 0 1 1 1 1 2 1 8 3 1 c h is o c h e to n s e ra m ic u s 1 4 3 9 5 7 4 9 9 6 8 7 5 6 8 8 6 5 0 7 2 1 7 2 r h o p a lo b a s te la d e rm a n ii 4 3 2 9 4 9 1 5 3 3 5 3 1 5 0 2 1 1 1 5 0 1 1 2 1 5 0 1 e u o d ia e le ry a n a 1 2 2 9 2 1 1 1 1 5 3 5 0 4 2 2 3 3 2 p a lm e ri a s c a n d e n s 2 1 4 2 3 3 5 3 3 4 3 2 5 0 3 7 1 7 7 o s m o x y lu n g lo b u la re 1 1 2 9 1 1 1 1 1 1 2 5 0 2 1 1 7 1 1 1 7 1 l ic u a la la u te rb a c h ii 3 1 2 9 2 6 7 8 3 3 7 1 7 5 0 4 1 1 7 1 3 7 3 3 5 d ry p e te s s p 1 1 4 1 6 2 2 2 4 3 1 1 5 0 2 3 1 7 3 o c to m e le s s u m a tr a n a 5 1 1 4 3 2 1 1 1 1 1 2 5 1 2 1 7 2 2 1 3 3 2 g o m p a n d ra g la b o s a 1 1 4 1 5 6 7 3 3 6 2 1 7 2 2 2 3 3 2 a n ti a ri s to x ic a ri a 1 1 4 1 1 1 1 3 3 1 3 1 7 3 2 1 3 3 2 c h is o c h e to n c e ra m ic u s 9 1 4 9 7 9 6 3 3 7 8 2 5 8 1 1 3 3 1 a g la ia s p 1 4 2 9 3 1 9 2 2 5 1 1 7 1 1 1 3 3 1 m y ri s ti c a p s e d o a rg e n te a 7 8 2 9 8 1 1 2 2 1 7 2 5 7 1 1 7 1 2 1 7 2 biotropia vol. 19 no. 2, 2012 79 3. iman rusmana page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 microsoft word 32 biotropia no. 21,2003 : 32 -44 control of aflatoxigenic aspergillus flavus in peanuts using nonaflatoxigenic a. flavus, a. niger and trichoderma harzianum okky setyawati dharmaputra seameo biotrop, p.o. box 116, bogor, indonesia and faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia asmarina s.r. putri, ina retnowati and santiambarwati seameo biotrop, p.o. box 116, bogor, indonesia abstract the effects of nontoxigenic aspergillus flavus, a. niger and trichoderma harzianum inoculated into planting media on toxigenic a. flavus infection and its aflatoxin production in peanut kernels at harvest were investigated together with (1) the moisture content of planting media before peanut planting, at the time of inflorescence, and at harvest, (2) the population of aflatoxigenic and nonaflatoxigenic a. flavus, a. niger and t. harzianum in peanut planting media before peanut planting, at the time of inflorescence, and at harvest, (3) the moisture content of peanut kernels at harvest, and (4) toxigenic a. flavus invasion in peanut plant parts (roots, stems, petioles, leaves and flowers) at the time of inflorescence. the fungal isolates were inoculated into planting media at the same time with the planting of peanut seeds. peanut plants were grown under glasshouse conditions. treated planting media were inoculated with the combined use of (1) toxigenic and nontoxigenic a. flavus, (2) toxigenic a. flavus and a. niger, and (3) toxigenic a. flavus and t. harzianum. planting media inoculated only with each fungal isolate and uninoculated planting media were used as controls. two watering treatments of peanut plants were carried out, i.e. watering until harvest and not watering for 15 days before harvest. the populations of the fungal isolates in the planting media and peanut kernels were determined using dilution method followed by pour plate method; the percentages of toxigenic a. flavus and test fungal colonizations in peanut plant parts were determined using plating method; the moisture content of planting media and peanut kernels were determined using oven method; the aflatoxin content of peanut kernels was determined using thin layer chromatography method. the results indicated that at the time of harvest the decrease in moisture contents of planting media not watered for 15 days before harvest was higher than those watered until harvest. the lowest population of toxigenic a. flavus was in planting media inoculated with the combined use of toxigenic and nontoxigenic a. flavus at the time of inflorescence and at the time of harvest. toxigenic a. flavus could invade the roots, stems and flowers of peanut plants. the lowest percentage of invasion was on the plant parts which planting media were inoculated with the combined use of toxigenic and nontoxigenic a. flavus. the moisture content of peanut kernels originated from watered plants until harvest were higher than those not watered for 15 days before harvest. the population of toxigenic a. flavus in peanut kernels derived from the plants whose planting media were inoculated with the combined use of toxigenic a. flavus and each test fungi, was lower than those inoculated only with toxigenic a. flavus. it indicated that the test fungi inoculated into planting media could inhibit toxigenic a. flavus infection in peanut kernels. aflatoxin was only detected in peanut kernels originated from one plant whose planting medium was inoculated only with toxigenic a. flavus and the plant was watered until the time of harvest. toxigenic a. flavus infection and aflatoxin production were not influenced by planting media which were not watered for 15 days before harvest. keywords: biocontrol / aflatoxigenic / nonaflatoxigenic / aspergillus flavus i aspergillus niger i trichoderma harzianum i peanuts 32 biotropia no. 21, 2003 introduction aflatoxins are potential hepatotoxic and carcinogenic metabolites produced by the fungi aspergillus flavus, a. parasiticus, and a. nomius. contamination of afla-toxins occurs when peanut kernels become infected by the three fungal species under drought stress before harvest, during the drying phase in the field, or under unsuitable storage conditions. pitt and hocking (1996) reported that 45, 33 and 22% of 215 peanut samples collected from farm storage, middlemen and retailers in bogor and yogyakarta contained aflatoxins of more than 50, 300 and 1000 ppb, respectively. levels of 1000 ppb of aflatoxins will cause liver damage in man and animals. lower levels of aflatoxins consumed from peanut products can cause liver cancer and premature death in humans, as well as reducing productivity of livestock. based on the report of the 23rd session of the joint faoavho food standards programme, held in rome, italy, 28 june 3 july 1999, codex alimentarius commissions adopted 15 ppb as the maximum level of total aflatoxins to be contained in peanuts intended for further processing. the entry of aflatoxin in the peanut plant could be from the roots, flowers, leaves damaged by insects, and from air or dust. the most important point of entry to the developing peanut, however, is directly from the soil surrounding it. so, the point of application of any biocontrol measure such as competitive exclusion should be in the soil of peanut growing fields. there are only few reports on the reduction of aflatoxins by competitive fungi. ehrlich et al. (1985) reported that by growing a. parasiticus in mixed culture with penicillium oxalicum, toxin productions of both species were reduced. to assess this effect under more practical conditions, wicklow et al. (1988) inoculated corn kernels with toxigenic a. flavus at preharvest, with or without a range of other fungi present. they found that much lower levels of preharvest aflatoxin were produced in the presence of some other fungal species. dorner et al. (1999) found that aflatoxin contamination of corn was reduced when the soil of corn plots were inoculated with nonaflatoxigenic strain of a. flavus. dharmaputra et al. (2001) reported that non-aflatoxigenic a. flavus bio 2127, a. niger bio 2129 and trichoderma harzianum bio 19130 were antagonistic to aflatoxigenic a. flavus bio 2128 in vitro. aspergillus niger was the most promising fungal antagonist, because it caused the highest percent inhibition of aflatoxin production of a. flavus bio 2128. the objectives of this study were to investigate the effects of nontoxigenic a. flavus, a. niger and t. harzianum inoculated into the peanut planting media on toxigenic a. flavus infection and its aflatoxin production in peanut kernels at harvest. investigations were also carried out on (1) the moisture contents of planting media before peanut planting, at the time of inflorescence, and at harvest, (2) the population of aflatoxigenic and nonaflatoxigenic a. flavus, a. niger and t. harzianum in peanut planting media before peanut planting, at the time of inflorescence and at harvest, (3) the moisture content of peanut kernels at the time of harvest, and (4) toxigenic a. flavus invasion in peanut plant parts (roots, stems, petioles, leaves and flowers) at the time of inflorescence. 33 control of aflatoxigenic aspergillus flavus in peanuts okky s. dharmaputra et al. materials and methods planting media, peanut variety, isolates of toxigenic a. flavus and test fungi a mixture of soil (latosol type), sand and casting (2:1:1) was used as planting medium, while peanut variety komodo was obtained from the research institute for legumes and tuber crops (rilet) in malang. four fungal isolates were used: toxigenic a. flavus bio 2128, nontoxigenic a. flavus bio 2127, a. niger bio 2129, and t. harzianum bio 19130. the latter three fungal isolates were used as test fungi. preparation of a. flavus , a. niger and t. harzianum inocula mixtures of ground rice bran and tap water (4:3) were placed in transparent polyethylene bags (125 g/bag). they were then sterilized in an autoclave for 1 h, and incubated at room temperature for one night. for the time being, each fungal isolate was grown on potato dextrose agar in petri dishes (diam 9 cm) and incubated at room temperature for 6 days. five ml of spore suspension (106 spores/ml) of each fungal isolate was inoculated into the mixture of sterilized ground rice bran and tap water. they were then incubated at room temperature for 7 days to obtain the inocula of planting media. inoculation of toxigenic a. flavus and test fungi into planting media, and seed planting pasteurized planting media were placed in black polyethylene bags (15 kg/bag). the inocula of planting media were then mixed with planting media at the time of germinated-seed planting (1 seed^ag). treated planting media were inoculated with the combined use of (1) toxigenic and nontoxigenic a. flavus, (2) toxigenic a. flavus and a. niger, (3) toxigenic a. flavus and t. harzianum. the comparison of toxigenic a. flavus and each test fungal isolate was 1:1 (one portion was equivalent with 30 g of inoculum). planting media inoculated only with each fungal isolate and uninoculated planting media were used as controls. six replicates (6 bags) were used for each treatment (including the controls). the bags with the peanut plants were placed under greenhouse conditions. two watering treatments of peanut plants were carried out, i.e. (1) watering until harvest and (2) not watering for 15 days before harvest. sterilized tap water was used for watering peanut plants. the individual plants were watered with the same volumes of sterilized tap water. sampling of planting media sampling of planting media were conducted : before peanut-seed planting, but after each treated planting medium as well as each control (except uninoculated planting media) have been mixed with fungal 34 biotropia no. 21,2003 inocula homogeneously. they were then placed in clean polyethylene bags (about 1 kg/bag/treatment or control). at the time of inflorescence and at the time of harvest. the peanut plants were pulled prior to sampling of planting media. each treated planting medium as well as each control was mixed homogeneously; they were then placed in clean polyethylene bags (about 1 kg/bag/treatment or control). each sample of planting media (about 1 kg) was divided several times manually to obtain working samples for moisture content and fungal analysis, and for reserved sample. determination of moisture contents of planting media; population of toxigenic a. flavus and test fungi in planting media before peanut seed planting, at the time of inflorescence and at the time of harvest moisture content of planting media derived from each bag was determined using oven method at 105°c for 12 hours (johnson et al. 1960). the population of each fungal isolate in planting media before peanut seed planting, at the time of inflorescence and at the time of harvest was determined using serial dilution method, followed by pour plate method (johnson and curl 1972) on pda containing chloramphenicol (100 mg/l media). sampling of peanut plants at the time of inflorescence at the time of inflorescence (25 30 days after planting), peanut plants grown on treated planting media as well as on the controls, were pulled. their roots, stems, petioles, leaves and flowers were then cut into pieces (10 pieces/plant) about 1 cm long, surface sterilized using 0.5% na-hypochloride for one minute, rinsed with sterile distilled water, dried on sterilized filter paper, and finally plated on potato dextrose agar (pda) containing chloramphenicol (100 mg/l media). the percentage of each fungal isolate colonization in various plant parts was determined using the following formula: number of plant part pieces colonized by each fungal isolate plant part pieces colonized = number of plant part pieces x 100% by each fungal isolate plated on agar media determination of moisture content of peanut kernels; population of toxigenic a. flavus and test fungi, and aflatoxin content in peanut kernels peanuts were harvested at 90 days after planting. they were then shelled manually and aseptically. peanut kernels derived from treated planting media and 35 control of aflatoxigenic aspergillus flaws in peanuts okky s. dharmaputra et ai the controls were divided using a box sample divider to obtain working samples for moisture content, fungal and aflatoxin analyses. the moisture content of peanut kernels was determined using the oven method (bsi 1995). the population of toxigenic a. flavus and test fungi was determined using serial dilution method, followed by pour plate method on pda containing chloramphenicol (100 mg/l media). aflatoxin content in peanut kernels was determined using thin layer chromatography method'(ao ac 1995). statistical analyses the experimental design used in this study was completely randomized factorial design with two factors, i.e. the kind of fungal inocula in planting media and watering of peanut plants. the kinds of fungal inocula consisted of (1) the combined use of toxigenic and nontoxigenic a. flavus, (2) toxigenic a. flavus and a. niger, (3) toxigenic a. flavus and t. harzianum, (4) toxigenic a. flavus, (5) nontoxigenic a. flavus, (6) a. niger, (7) t. harzianum, and (8) uninoculated planting media.-peanut plants (1) were watered until harvest and (2) not watered for 15 days before harvest. results and discussion moisture content of planting media before peanut seed planting, at the time of inflorescence and harvest the fungal inocula did not give any significant difference on the moisture contents of planting media at the time of inflorescence. besides, the moisture contents of planting media before peanut planting were not significantly different from the time of inflorescence. it indicated that up to the time of inflorescence, the moisture contents of planting media were always stable, although the media were inoculated with various fungal inocula. the moisture content of planting media treated with various fungal inocula and the time of watering decreased from before peanut planting up to harvest. fungal inocula and their interaction with time of watering were not significantly different with the decrease of moisture contents, while time of watering showed significant differences. table 1 shows that the decrease in moisture content of planting media not watered for 15 days before harvest (22.9%) was higher than that watered until harvest (11.1%). 36 biotropia no. 21,2003 table 1. the decrease in moisture content of planting media before peanut planting until harvest time treatment decrease of moisture content (%) watered planting media 11.09 a not watered planting media 22.85 b numbers followed by the same letter do not differ significantly according to duncan multiple range test at 95% confidence level population of toxigenic a. flavus and test fungi in planting media before peanut planting, at the time of inflorescence and at the time of harvest in the planting media, populations of toxigenic a. flavus and test fungi in-creaseed at the time of inflorescence as well as at the time of harvest. at the time of inflorescence, fungal inocula gave significant differences on the increase of toxigenic a. flavus and test fungal populations. at the time of harvest, fungal inocula and time of watering gave significant differences on the increase of fungal populations. the increase of toxigenic a. flavus population in planting media inoculated with the combined use of toxigenic and nontoxigenic a. flavus was lower compared with planting media inoculated (1) only with toxigenic a. flavus, (2) with the combined use of toxigenic a. flavus and a. niger, and (3) with the combined use of toxigenic a. flavus and t. harzianum, either at the time of inflorescence or at the time of harvest (table 2). the increase of each test fungal population in either the planting media inoculated with each test fungi or in the planting media inoculated with the combined use of toxigenic a. flavus and each test fungi were higher than the increase of toxigenic a. flavus population in the planting media inoculated only with toxigenic a. flavus and in the planting media inoculated with the combined use of toxigenic a. flavus and test fungi, either at the time of inflorescence or at the time of harvest (table 2). it indicated' that the growth of toxigenic a. flavus was more inhibited compared with the growth of test fungi, either in the planting media inoculated with each fungal isolate or in the planting media inoculated with the combined use of toxigenic a. flavus and test fungi. at the time of harvest, the increase of fungal population in the watered planting media was higher than that not watered for 15 days before harvest (table 3). it was related to the decrease of moisture content which was higher in the not watered planting media compared with the watered planting media (table 1), consequently fungal growth was inhibited. 37 at the time of harvest in the uninoculated planting media, three fungal isolates were found: nontoxigenic a. flavus, cladosporium cladosporioides and t. harzianum. it was assumed that fungal contamination originated from the air or from peanut seeds. 38 biotrop1a no. 21,2003 toxigenic a. flavus and test fungal invasion in peanut plants at the time of inflorescence pitt et al. (1991) stated that the invasion of toxigenic a. flavus in peanut plants could originate from the soils or from peanut seeds grown under greenhouse conditions. toxigenic a. flavus invasion was observed in peanut plants where the planting media were inoculated with the fungus as well as its combination with the test fungi (table 4). its percentage of invasion occurring in plant parts adjacent to the soils (root and stem) was higher than the other parts (petioles and leaves). pitt et al. (1991) reported that a. flavus could invade all plant parts, with the highest invasion occurring in plant parts adjacent to the soils. the percentage of toxigenic a. flavus invasion in plants where the planting media were inoculated with the combined use of toxigenic and nontoxigenic a. flavus, toxigenic a. flavus and a. niger was lower than that only inoculated with toxigenic a. flavus. nevertheless, the percentage of toxigenic a. flavus invasion was higher in plants where the planting media were inoculated with the combined use of the fungus and t. harzianum (table 4). in the planting media inoculated with the combined use of toxigenic a. flavus and nontoxigenic a. flavus, also the combined use of toxigenic a. flavus and a. niger, the invasion of toxigenic a. flavus were only in the roots, while in the planting media inoculated only with toxigenic a. flavus, this fungus could invade roots, stems and flowers. in the planting media inoculated with toxigenic a. flavus and t. harzianum, toxigenic a. flavus could invade all the plant parts. the percentage of toxigenic a. flavus invasions was related to the increase of toxigenic a. flavus population in the planting media (table 2). the lowest increase of toxigenic a. flavus population was in the planting media inoculated with the combined use of toxigenic and nontoxigenic a. flavus, while the highest was in the planting media inoculated with the combined use of toxigenic a. flavus and t. harzianum. in the planting media inoculated only with toxigenic a. flavus as well as those inoculated with the combined use of toxigenic a. flavus and t. harzianum, toxigenic a. flavus could also invade flowers (table 4). it is related to the position of peanut flowers, i.e. adjacent to the planting media. if the flowers come in contact with the planting media, they could be invaded by toxigenic a. flavus. toxigenic a. flavus derived from the planting media can systemically invade peanut plant parts adjacent to the soil up to the plant parts above the soil surface. the fungus can also invade young peanut plants. based on microscopic examination, little or no damage was observed in the cells of stems invaded by a. flavus (pitt et al. 1991). toxigenic a. flavus invasion did not cause any disease in peanut plants. 39 test fungi could also colonize roots, stems, petioles, leaves and flowers (table 4). some plant parts, either on planting media inoculated with toxigenic a. flavus, test fungi, or uninoculated planting media, were contaminated with other fungi such as exosporium sp., fusarium spp., gilmaniella spp., sphaeropsis sp. and tuber   biotrop1a no. 21,2003 cularia sp. (table 4). it was assumed that the fungal contamination originated from the air or peanut seeds. moisture content of peanut kernels at the time of harvest the fungal inocula and their interaction with time of watering did not give any significant difference on the moisture content of peanut kernels at the time of harvest, while only time of watering gave significant differences. the moisture content of peanut kernels derived from watered plants until harvest was higher than those of not watered plants for 15 days before harvest (table 5). the effects of nontoxigenic a. flavus, a. niger and t. hanianum on toxigenic a. flavus infection and aflatoxin production in peanut kernels from six replications derived from the planting media inoculated with toxigenic a. flavus and its combination with test fungi, in general only one replication of peanut kernels was infected by the fungus (table 6). it indicated that the capability of toxigenic a. flavus in infecting peanut kernels was relatively low. toxigenic a. flavus population in peanut kernels derived from the plants where the planting media were inoculated with the combination of toxigenic a. flavus and each test fungal isolate was lower than that of only inoculated with toxigenic a. flavus (table 6). it indicated that the test fungal isolates inoculated into planting media could inhibit toxigenic a. flavus infection in peanut kernels. aflatoxin was only detected in peanut kernels derived from the plants where the planting media were only inoculated with toxigenic a. flavus and the plants were always watered until harvest time. aflatoxin b] was produced with a concentration of 32 ppb (table 7). it indicated that toxigenic a. flavus (21 cfu/g dry weight) in peanut kernels (table 6) could produce aflatoxin. aflatoxin was not detected in peanut kernels derived from the plants where the planting media were inoculated with a combination of toxigenic a. flavus and test fungi, either in watered plants or unwatered plants for 15 days before harvest, because the test fungi could inhibit aflatoxin production of toxigenic a. flavus. inhibition of aflatoxin production caused by the test fungi could be affected by the control of aflatoxigenic aspergillus flavus in peanuts okky s. dharmaputra et al. dose of biocontrol agent inocula. according to dorner et al. (1998), aflatoxin production in peanut kernels decreased with the increase of the dose of biocontrol agents inocula (nontoxigenic a. flavus and a. parasiticus) inoculated into the soils. aflatoxin content in peanut kernels with the dose of biocontrol agents inocula 0, 2, 10 and 50 g/m were 337.6, 73.7, 34.8 and 33.3 ppb, respectively. biotropia no. 21, 2003 conclusions at the time of harvest, decrease in the moisture content of peanuts in the planting media not watered for 15 days before harvest was higher than those watered until harvest. the lowest population of toxigenic a. flavus was in the planting media inoculated with the combined use of toxigenic and nontoxigenic a. flavus at the time of inflorescence and at the time of harvest. toxigenic a. flavus could invade the roots, stems and flowers of peanut plants. the lowest percentage of invasion was on the plant parts where the planting media were inoculated with the combined use of toxigenic and nontoxigenic a. flavus. the moisture content of peanut kernels originated from plants watered until harvest was higher than those not watered for 15 days before harvest. the population of toxigenic a. flavus in peanut kernels derived from the plants where the planting media were inoculated with the combined use of toxigenic a. flavus and each test fungi, was lower than that only inoculated with toxigenic a. flavus. it indicated that the test fungi inoculated into planting media could inhibit toxigenic a. flavus infection in peanut kernels. aflatoxin b] was only detected in peanut kernels originated from one plant where the planting medium was only inoculated with toxigenic a. flavus and the plant watered until harvest. toxigenic a. flavus infection and aflatoxin bj production were not influenced by the planting media which were not watered for 15 days before harvest. 43 control of aflatoxigenic aspergillus flavus in peanuts okky s. dharmaputra et al. acknowledgement the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the research institute for legumes and tuber crops (rilet), malang, in providing peanut seeds var. komodo. we also thank ms. shanti saraswati, department of biology, faculty of mathematics and natural sciences, bogor agricultural university, and the technicians of the laboratory of pest and disease management, seameo biotrop for their valuable assistance in conducting the experiment. references aoac. 1995. natural toxins. in e. scott (ed.). official methods of analysis of natural poison. 6th ed. association of official analytical chemists, arlington. chap. 49. p. 1-5. bs1. 1995. oilseeds-determination of moisture and volatile matter content. british standard international. dharmaputra, o.s., a.s.r. putri, i. retnowati and s. ambarwati. 2001. soil mycobiota of peanut fields at wonogiri regency, central java: their effect on the growth and aflatoxin production of aspergillus flavus in vitro. biotropiano. 17: 3058 dorner, j.w., r.j. cole and p.d. blankenship. 1998. effect of inoculum rate of biological control agents on preharvest aflatoxin contamination of peanuts. biological control 12: 171-176 dorner, j.w., r.j. cole and d.t. wicklow. 1999. aflatoxin reduction in corn through field application of competitive fungi. journal of food protection 62(6): 650-656 ehrlich, k., a. ciegler, m.a. klich and l. lee. 1985. fungal competition and mycotoxin production on corn. experimentia 41: 691 693. johnson, l.f., e.a. curl, j.h. bond and h.a. fribourg. 1960. methods for studying soil microflora-plant disease relationships. burgess publishing company, minneapolis. johnson, l.f. and e.a. curl. 1972. methods for research on the ecology of soil-borne plant pathogens. burgess publishing company, minneapolis. pitt, j.i., s.k.. dyer and s. mccammon. 1991. systemic invasion of developing peanut plants by aspergillus flavus. letters in applied microbiology 13: 16-20. pitt, j.i. and a.d. hocking. 1996. current knowledge of fungi and mycotoxins associated with food commodities in southeast asia. in highley, e. and g.i. johnson (eds). mycotoxin contamination in grains. pp. 5-10. aciar technical reports 37, canberra. wicklow, d.t., b.w. horn, o.l. shotwell, c.w. hesseltine and r.w. caldwell. 1988. fungal interference with aspergillus flavus infection and aflatoxin contamination of maize grown in a controlled environment. phytopathology 78: 68 74. 44 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf microsoft word 40 biotropia no. 23, 2004 : 40 46 preservation of garut rams spermatozoon as a source of male germ plasm arief boediono'*, herdisu, and muhammad rizal' 'faculty of veterinary medicine, bogor agricultural university, bogor 16680, indonesia 2 center for the assessment and application of farming technology, agency for the assessment and application of technology, jakarta, indonesia abstract this study was conducted to examine the quality of ejaculated sperm by garut rams to be used for artificial insemination (ai) and viability of sperm that were collected from preserved cauda epididymis (4°c up to 12 days) for assisted reproductive technology. the semen was collected by artificial vagina, with the sperm motility, live sperm, acrosomal intact, and intact plasma membrane observed. sperm motility was 75%, while for the live sperm, intact plasma membrane and sperm abnormality were 91.5%, 90.0%, and 1.8%, respectively. in the other study, sperm was collected from cauda epididymis by aspiration method and diluted in different media: 1) brackett oliphant (bo) media and 2) modified phosphate buffer saline (mpbs). evaluation of sperm motility and intact plasma membrane were conducted after washing, counting and dilution of the sperm. the results of this study showed that the sperm motility and intact plasma membrane could be maintained better in bo rather than pbs medium although they were not statistically different (p>0.05). at day 12 of preservation, the motility and intact plasma membrane of sperm collected from cauda epididymis were 0.7% and 1.33% for motility and plasma membrane intact, respectively. these findings showed that the garut rams semen was qualified for ai and frozen processing; in vitro embryo production by introducing the assisted reproductive technology such as intracytoplasmic sperm injection (icsi) could be applied by using the sperm collected from preserved cauda epididymis until 12 days of preservation at 4°c. keywords : reproduction/spcrmatogenesis/inscmination/garut rams/small ruminant introduction indonesia is a country with tremendous amounts of genetic resources and biodiversity. in indonesia, the percentage of meat and wool produced by sheep as a small ruminant in west java province is approximately 46.2% (anonymous 1999). one of the ruminants is garut sheep, endemic to indonesia, and potential for meat production. moreover, they have a good performance for sheep contest. the population of garut sheep decreased from 7.6 million in 1997 to 7.2 million in 2001. in an attempt to increase the garut sheep population, several efforts should be done to maintain the population. application of reproduction biotechnology such as artificial insemination (ai) and in vitro embryo production are the solutions. artificial insemination has been routinely done in large ruminants such as cattle and * correspondence: arief boediono, lab. of embryology, dept. of anatomy, fac. of veterinary medicine bogor agriculturaluniversity, jalan agatis, ipb campus darmaga, bogor 16680, indonesia. e-mail: abl@cbn.net.id 40 biotropia no. 23, 2004 buffaloes. however, in small ruminants like sheep and goat this technique should be modified according to the small size of the reproductive tract. sperm usually was collected from ejaculation for artificial insemination procedure. collected sperms were then diluted for fresh sperm insemination, sperm preservation or sperm cryopreservation. in the case of dead ram, sperm was collected from cauda epididymis. epididymal sperm may be the last chance to ensure preservation of genetic materials after injury or death of a valuable male. studies have been conducted to determine if epididymal sperm can be used to produce viable embryos and offsprings. iwamatsu and chang (1971) have reported successful fertilization of mouse oocytes using epididymal sperm. fuller and whittingham (1996) reported the production of normal mouse fetuses after ivf using epididymal sperm that had been cooled at 4°c. sperm collected from the caudal epididymides has been used for both ivf and intracytoplasmic sperm injection (icsi) in domestic cats (pope et al. 1998). caprine epididymal sperm has also been reported for use in the production of ivf embryos to the blastocyst stage (song and iritani 1988), and production of an ivf goat offspring born from artificial insemination (slash et al. 2000). from recent studies, viable epididymal sperm can be harvested post-mortem for possible use in assisted reproductive technologies (art). most of the species evaluated so far, produce more viable sperm when the testicles are stored at 4°c, prior to harvesting the epididymal sperm. the objective of this study was to identify an alternative to collect ovine ejaculate and epididymal sperm for subsequent fertilization. materials and methods sperm collection ejaculated sperm. sperm ejaculate was collected from 5 healthy garut rams by using artificial vagina. the artificial vagina consists of a hose 20 to 25 cm in length and 5 to 7 cm in diameter, with a rubber liner. the temperature of artificial vagina was set between 42 to 46°c, and it is necessary to lubricate it with k-y jelly. at the time of the ram mounts to the ewe, his penis was gently guided inside the artificial vagina connected with a warm tube (37°c) to avoid the cold shock. after ejaculation, the tube containing the semen was removed and placed in a water bath at 30°c. testicle preservation ovine testicles were collected as pairs from mature rams from a slaughterhouse. one pair of testicle (control testicle) was processed shortly after the rams were slaughtered (day-0), while the other testicle of the pair was processed and stored at 4°c up to 12 days of preservation. collected sperms were evaluated daily. 41 preservation of garut rams spermatozoon arief boediono and muhammad rizal epididymal sperm. after storage at 4°c, each testicle was allowed to warm at room temperature (25°c) for not more than 30 minutes. during this warming period, the testicle was dissected away from the connecting tissues. sperm dilution media (bracket! and oliphant, bo and modified phosphate buffer saline, mpbs) were warmed in water bath to 37°c prior to exposure to the epididymal sperm. sperm was collected from the cauda epididymis region by aspiration method. briefly, sperm was collected by aspiration using 21g needle connected to 3 ml spuit already filled with 1 ml warmed dilution media. collected sperm was then washed by centri-fugation at 500g for 3 minutes to create sperm pellet. the supernatant was discarded and the pellet was resuspended in sperm dilution media. evaluation the volume of ejaculated sperm was recorded in each collection. microscopic evaluation of the sperm was done including the sperm concentration, motility, percentage of live sperm, intact plasma membrane, and morphology. for epididymal sperm, analysis was done on percentage of sperm motility and intact plasma membrane. an evaluation of live and dead sperm was done by eosin-negrosin staining. the appearing red sperms were considered dead, otherwise they are alive. the sperm which had an intact plasma membrane was shown by coiled sperm tail after incubation in hypo-osmotic solution at 37°c for 30 min (correa and zavos 1994). hypo-osmotic solution contained 0.032 m nacl in destilled water. data were analyzed using the simple one-way anova to make comparisons between testicle pairs and duncan's test to identify the difference in means between the treatment groups. results and discussion ejaculated sperm was successfully collected from 5 rams. the mean number of garut ram ejaculated sperm was 3463 x 106 sperm rnl"', with the average volume of semen per ejaculation of 0.7 ml (table 1). by comparison, the volume of semen per ejaculation was 1.7 ml in st. croix sheep (feradis 1999), and 1.05 ml in suffolk sheep (boland et.al. 1985). the difference had correlation with individual factors such as age, body weight, and breed (hafez and hafez 2000). the motility and percentage of live sperm of garut ram ejaculation were 75.0% and 91.5%, respectively. these-results were higher than that reported previously in st. croix (64.0% and 76.0% for motility and percentage of live sperm, respectively) (sirman and situmorang 1987). 42 biotropia no. 23, 2004 the percentage of intact plasma membrane of garut ram ejaculated sperm was 90.0%. this was higher than the percentage of intact plasma membrane of st. croix sheep ejaculated sperm i.e. 86.3% (feradis 1999). when sperm was exposed to hypo-osmotic solution, the spermatozoa that had intact plasma membrane would be swollen. if the sperm have intact plasma membrane, the water that influx into the cell could not come out from the cell. the percentage of intact plasma membrane had positive correlation with sperm motility. no single test accurately predicted fertility of a sperm sample; however, examining various physical characteristics of semen could determine greater fertility potential. processing semen for artificial insemination or sperm freezing depends on semen quality. analysis of ram semen involves obtaining maximum information about the physiology status of testicular and epididymis functions by examining one or several ejaculations. in general, the minimal requirements for semen processing (fresh or frozen semen) would include: at least 65% motility, concentration 700 x 106 sperm ml"', and less than 20% morphological abnormalities (hafez and hafez 2000). motility and intact plasma membrane of sperm collected from cauda epididymis could be found up to 24 hours of incubation in vitro both in mpbs or bo medium. at the time of collection sperm motility was 58.0% in mpbs medium and 55.7% in bo medium. they decreased gradually during the in vitro incubation up to 24 hours (0.3% in mpbs and 0.6% in bo medium) (figure 1). intact plasma membrane of sperm collected from cauda epididymis was 60.0% in mpbs and 56.7% in bo medium at the time of sperm collection. the intact plasma membrane in both media during in vitro incubation exhibited a declining pattern, as with sperm motility. however, the intact plasma membrane was higher than sperms motility after 18 hours of incubation. this was shown by some of the immotile sperm that had intact plasma membrane. in this case, spermatozoa may be used for in vitro fertilization by injection of single sperm directly into the oocyte cytoplasm called intracytoplasmic sperm injection (icsi). 43 preservation of garut rams spermatozoon — aricf boediono and muhammad rizal 1812 incubation incubation period (hours) 60.0 ■ mot mpbs q mot bo □q mpi □mpbs q figure 1. motility and intact plasma membrane of sperm collected from cauda epididymis after incubation in vitro in different medium. mot mpbs: sperm motility in mpbs medium, mot bo: sperm motility in bo medium; mpi mpbs: intact plasma membrane of sperm in mpbs medium, mpi bo: intact plasma membrane in bo medium. the sperm motility and intact plasma membrane of sperm collected from cauda epididymis decreased significantly after 2 days of preservation at 4°c. however, 0.7% motile sperm and 1.3% sperm with intact plasma membrane were found after 12 days of preservation (figure 2). according to motility and live sperm, rizal et al. (2004) reported that sperm collected from testicles after 3 days of preservation at 5°c could be used for artificial insemination or in vitro embryo production of sheep. epididymal sperm was used in a goat in vitro embryo production that resulted in cleavage and development of blastocyst, although none of the blastocysts were transfered to the recipients (song and iritani 1988). further work is needed to optimize the in vitro embryo production, through micro-fertilization procedure. the valuable sperm has the ability to fertilize the oocytes and, therefore, could increase the chances of propagating valuable genetics. intracytoplasmic sperm injection (icsi) could initiate the early embryo development to cleavage in goat oocyte (boediono 2001). moreover, cochran et al. (1998) reported that live ivf offsprings from oocyte donor mares have been produced by using icsi procedure. although there has been some success using assisted reproductive technologies, more researches are needed to improve the efficiency of the procedures. 44 conclusions these results indicate that garut rams semen was qualified for ai and frozen processing, and motile sperm can still be collected from cauda epididymis after being stored at 4°c up to 12 days. sperm cooled in the testicle at 4°c could be used in assisted reproductive technologies (intracytoplasmic sperm injection) for the untimely death of a valuable sheep and an effective tool to conserve important genetic resources. this study investigated the standard parameter of sperm analysis. it would stand to reason that further investigations with particular emphasis on chromatin damage and/or fertility are suggested. acknowledgements we would like to thank lesan putra breeding farm, bogor for providing the garut rams. this research was partly financed by seameo-biotrop fy 2003, no.l05/res/iii/2003. references anonymus. 1999. buku statistik petcmakan. direktorat jenderal pcternakan. departemen pertanian republik indonesia dan asosiasi obat hcwan indonesia. jakarta. blash s., mcllican d, and w. gavin. 2000. cryopreservation of cpididymal sperm obtained at necropsy from goats. theriogenology, 54:899-905. 45 preservation of garut rams spermatozoon arief boediono and muhammad rizal boediono a. 2001. sperm immobilization prior to intracytoplasmic sperm injection (icsi) and oocytc activation improve early development of microfertilizcd goat oocytcs. reprotech, 1:29-34. boland m.p., a.a. al-kamali, t.f. crosby, n.b. haynes 1, c.m. howlcs 1, d.l. kelleher and i gordon. 1985. the influence of breed, season and photoperiod on semen characteristics, testicular size, libido and plasma hormone concentrations in rams. animal reproduction science, 9: 241-252. cochran r, meintjes m, rcggio b, hylan d, carter j, pinto c, paccamonti d, and r.a. godke. 1998. live foals produced from sperm-injected oocytcs derived from pregnant mares. j equine vet sci, 18:736-740. correa j.r. and p.m. zavos. 1994. the hypo-osmotic swelling test : its employment as an assay to evaluate the functional integrity of the frozen-thawed bovine sperm membrane. theriogenology, 42:351-360. feradis. 1999. penggunaan antioksidan dalam pcngcnccr semen beku dan metodc sinkronisasi estrus pada program inseminasi buatan domba st. croix. disertasi doktor. program pascasarjana institut pcrtanian bogor. bogor. fuller sj and d.g. whittingham . 1996. effect of cooling mouse spermatozoa to 4°c on fertilization and embryonic development. j. rcprod. fcrtil., 108:139-145. hafez e.s. e, and b. hafez 2000. reproduction on farm animals. 7th ed. lippincott williams & wilkins. philadelphia. iwamatsu y. and m. c. chang. 1971 factors involved in the fertilization of mouse eggs in vitro. j. reprod. fcrtil., 26:197-208. pope c. e., johnson c.a. , mcrae m.a. , kellcr g.l. and b.l. dresser. 1998. development of embryos produce by intracytoplasmic sperm injection of cat oocytes. anim rcprod sci, 53:221-236. rizal m, herdis and a. boediono. 2004. viability of rams epididymal sperm after preservation in low temperature (5°c). animal reproduction 6(l):30-36. sirman p and p. situmorang. 1987. evaluasi semen domba cair. ilmu dan peternakan, 3:1-3. song h.b and a. iritani. 1988. in vitro fertilization of goat follicular oocytes with epididymal spermatozoa. korean j anim sci, 30:636-642. 46 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf fiber disruption of betung bamboo ( ) by combined fungal and dendrocalamus asper microwave pretreatment widya fatriasari *, wasrin syafii , nyoman wistara , khaswar syamsu ,1 2 2 3 bambang prasetya , s. heris anita and lucky risanto4 1 1 1research center for biomaterials, indonesian institute of sciences (lipi), jalan raya bogor km 46 cibinong, bogor 16911, indonesia 2department of forest product technology, faculty of forestry, institut pertanian bogor, bogor 16680, indonesia 3department of agro-industrial technology, faculty of agricultural engineering and technology, institut pertanian bogor, bogor 16680, indonesia 4 thnational standardization agency, manggala wanabakti building blok iv, 4 floor, jalan gatot subroto, senayan, jakarta, indonesia received 22 january 2014/accepted 3 december 2015 abstract combined microwave pretreatment is an attractive method to alter carbohydrate and lignin structure of fungal and lignocellulosic materials for improving hydrolysis process to convert these lignocellulosic materials to bioethanol. this study was conducted to obtain information on the and lignin characteristic changes after carbohydrate combined biological microwave pretreatment of amboo. based on our previous research, incubation for 30 days and betung b using 5 and 10% (w/v) inoculum loading of white rot fungi, which has better delignification trametes versicolor selectivity compared to the other incubation time, was chosen as the pretreatment prior to microwave fungal pretreatment for 5, 10 and 12.5 minutes at . the evaluation of characteristic changes after pretreatment was 330 w performed using the analysis of ftir spectroscopy, x-ray diffraction and sem. ftir spectra demonstrated that the combined change pretreatment only affected the of intensity bands of ftir spectra, without any changes in the functional groups. r unconjugated bonds of carbohydrate peaked at 1,736 cm this band intensity decrease occu red on -1 (c = 0 in xylan), 1,373 cm (c-h deformation in cellulose and hemicellulose), 1,165 cm (c-o-c vibration in cellulose -1 -1 and hemicellulose) 895 cm (c-h deformation or c-o-c stretching at β-glicosidic linkage characteristic in and -1 cellulose) the pretreatment decreased the hydrogen bond stretching of cellulose and the linkage between lignin and . carbohydrate associated with crystallinity of bamboo cellulose l. this decrease of hydrogen bond was , i lustrated by occurring structural changes. the crystallinity tended to increase slightly due to the cleavage of the amorphous index fraction. sem image illustrated that the pretreatment disrupted the fiber structure. the longer duration of microwave s irradiation, the greater the degradation level of fiber. : keywords betung bamboo, and lignin changes, biological microwave pretreatment, carbohydrate combined and ftir, sem, xrd introduction increasing concern of greenhouse gas emission and the depletion of fossil fuels have been considered as the main driving force in exploring renewable energy sources (hu & wen 2008; zhang 2008). abundant lignocellulosic materials are potential bioresources to produce liquid biofuel, such as bioethanol. however, the recalcitrance nature of biomass due to the presence of lignin and cellulose crystalline structure prevents optimum enzyme penetration during hydrolysis. effective pretreatment prior to hydrolysis stage is required to improve biomass digestibility. pretreatments are emphasized mainly to increase feedstock surface area and porosity, as well as to reduce cellulose crystallinity, lignin content and hemicellulose content (mosier . 2005; galbe & et al zacchi 200 ; wyman . 2007; cara . 2008). 7 et al et al an effective pretreatment of biomass is indicated * corresponding author : widya_fatriasari@yahoo.com biotropia vol. 22 no. 2, 2015: 81 94 81 doi: 10.11598/btb.2015.22.2.363 mailto:widya_fatriasari@yahoo.com 82 by sugar release improvement, reduced carbohydrate degradation and the lack of inhibitory by-products such as furfural, hydroxymethyl furfural (hmf) and organic acids formation ( ) kuhnel . 2011; agbor . 2011et al et al and also be cost-effective ( &yang wyman 2008; agbor . 2011et al ). bamboos are versatile fast growing species of c plant type with very efficient photosynthesis 4 ability. theoretical value of c photosynthesis is 4 approximately 8%. biomass productivity of bamboo is about 20-40 ton s/ha/year. it is ne approximately 7-30% higher than that of woody plants (kant 2010) and other energy crops such as poplar, switch grass, miscanthus, common reed and bagasse (sathitsuksanoh . 20 ; zhang et al 10 2008). bamboos are distributed in the tropics, subtropics and temperate zones ( . lobovikov et al 2007) and cover 1% of the world's forest area (kant 2010). most of bamboo population (65%) grows in asia, especially in indonesia with 160 bamboo species (widjaja 2001). this ranks third (5%) in world bamboo's population after china (14%) and india (30%) (lobovikov . 2007). et al betung amboo is considered among the most b important species in indonesia (dransfield & widjaja 1995). previous study of six indonesia's bamboo species demonstrated that fiber morphology, physical and chemical properties of betung bamboo were better than those of kuning, tali, andong, ampel and black bamboos (fatriasari & hermiati 2008). after single pretreatment, enzymatic hydrolysis in simultaneous saccharification and fermentation (ssf) can be applied to produce bioethanol. however, single pretreatment tends to produce low sugar yield, consume time and require high production cost due to enzyme requirement in saccharification process. combined biological-microwave pretreatment could improve ethanol yield of biomass via ssf method. white-rot fungi used in biological pretreatment degrade lignin polymer by secreting ligninolytic enzyme nazarpour (zhang 2007; et al. et al. 2013). an appropriate fungal strain is needed to obtain a satisfying delignification selectivity and enzymatic hydrolysis yield. delignification selectivity of betung bamboo with trametes versicolor was found to be better than that with and pleurotus ostreatus phanerochaete chrysosporium et al (fatriasari . 2011; falah . et al 2011). microwave radiation of lignocellulosic material in aqueous environment is also found promising (kheswani . 2007). this et al pretreatment method has been applied for switch grass, bagasse, rice straw, woody plants, oil palm empty fruit bunch, oil palm trunk and frond (hu & wen 2008 keshwani 2007; anita . ; et al 2012; risanto . 2012; lai & idris 2013). the et al advantages of the method include short processing time and high product yield and quality (hermiati . 2011). microwave et al pretreatment supplies direct internal heat to biomass resulted from polar bond vibration as they align with the magnetic field (kheswani .et al 2007). microwave pretreatment can increase ion production, solubilize non-polar material and hydrolyze biomass without catalyst (tsubaki & azuma 2011). in a study on biological and microwave pretreatment of etung amboo, incubation for b b 30 days in biological pretreatment resulted in high lignin removal and less cellulose loss (fatriasari et al. 2014a). furthermore, microwave pretreatment of biomass at utes330 w for 5, 10 and 12.5 min resulted in lower of the aweight loss cellulose and hemicellulose other compared to that of pretreatment conditions (fatriasari 2014b)et al. . to the best of our knowledge, no study has been reported on the changes lignin and of carbohydrate structure after combined biologicalmicrowave pretreatment. this study was conducted to obtain information on the carbohydrate and lignin characteristic changes after biological microwave combined and pretreatment of amboobetung b . materials and methods material preparation betung bamboo ( dendrocalamus asper (schult.f.)) of less than 2 years old was collected from bamboo plantation of the research center for biomaterials lipi, cibinong, indonesia. the bark of the collected bamboo was removed and the barkless bamboo was chipped before being ground into fine powder. the powder was sieved to obtain 40-60 mesh bamboo meal and then stored in a sealed plastic bag at room temperature. betung bamboo meal was subjected to biological pretreatment before microwave irradiation. biotropia vol. 22 no. 2, 2015 fiber disruption of betung bamboo ( ) by combined fungal fatriasaridendrocalamus asper and microwave pretreatment – et al. bamboo sample preparation bamboo meal was watered with ratio of 1:4 and then manually stirred until completely mixed. the wet bamboo-meal was then put in a jar and steamed for 30 minutes at approximately 100 c o and finally in an for 20 minutes sterilized autoclave at 121 c.o inoculum stock preparation fungus inoculum was cultured on t. versicolor malt extract agar (mea) slant (10.65 g of mea were diluted in 300 ml distilled ) for water 7-14 days. 5 ml at the end of incubation period, of jis (japan industrial standard) broth medium 3 g kh po , 2 g mgso .7h o, 25 g ( 2 4 4 2 glucose, 5 g pepton and 10 g malt extract diluted into 1 l of distilled water) was injected to the then scratched with loop to release slant and the mycelium from the slant agar. resulting as much as of previously prepared 5 ml fungi suspension was then poured into the remaining 95 ml of the jis broth medium and stationery incubated at 27 c for 7 days. after incubation, o 8 10 g of corn steep liquor was poured into the 100 ml inoculum and homogenized twice with a high speed waring blender (each homogenization was conducted for 20 seconds). inoculation method bamboo-meal 15 g ied ) having ( oven dr weight 7.46% moisture content was inoculated with 5 and 10% (w/v) iedinoculum of dr bamboo and incubated at 27 c for 30 dayso . microwave pretreatment microwave pretreatment was carried out in an oven microwave sharp p-360j (s) with 2,450 mhz frequency and power output of 1,100 w. as much as 1 g of oven-dried pretreated sample was inserted into a teflon tube (vessel), added with distilled water to obtain a solid-toliquid ratio (slr) of 1:30 (w/v) and stirred for 15 minutes. subsequently, the sample was exposed to microwave irradiation at 330 w for 5, 10 and 12.5 minutes. after microwaving, the pulp was removed from the oven and immediately put into iced water for 15-20 minutes to cool the pulp. the residue (solid fraction) was separated from the hydrolysate (liquid fraction) by filtration. the changes of content, morphological, cellulose and lignin characteristics chemical component determination prior to determination of chemical component of control and pretreated bamboo, the moisture content samples were measured of following the procedures of tappi t12 os-75 . free extractive bamboo-meal was prepared with ethanol-benzene (1:2) extraction for chemical component analysis. acid-insoluble lignin, acid soluble lignin, holocellulose, a-cellulose and ash content were determined in accordance with the tappi t13 os-54, , tappi tappi um 250 t9m-54, and t 15 os-tappi t17m-55 tappi 58 standards, respectively. the calculation of weight loss following method of was done the pandey pitman (2003), while selectivity value & was calculated as ratio of lignin loss to the cellulose loss (yu . 20 )et al 09 . cellulose crystallinity index determination the crystallinity was determined using index diffraction intensity data of x-ray diffraction (xrd) according to the formulation of zhou et al. (200 ). measurement was carried out with 5 shimadzu xrd-700 maximax series. ni radiation was filtered by cuk at 0.15406 nm wave α number. x-ray was operated at 40 kv of voltage, 30 ma of electrical current and scanned 2 theta ( )0 of 10-40 in 2 minute.o o per allomorphic structure of cellulose z-discriminate function 200 ) was (hult . 3et al used to differentiate allomorphic properties of cellulose crystalline structure. the function was built up by separating cellulose i and i using d-α β spaces obtained from x-ray analysis, i.e. two equatorial d-spacing: 0.59-0.62 (d ) and 0.52-0.55 1 nm (d ). z > 0 indicates bacteria algae type (i , 2 α rich triclinic structure) and z < 0 indicates cotton and flax types (predominantly i structure/ β monoclinic). crysta lite size of cellulosel the crystallite size of cellulose was determined using diffraction pattern obtained from 101 , 10-1 , 002 and 040 lattice planes ( ) ( ) ( ) ( ) of bamboo ).(zhao . 2007et al morphological structure analysis morphological structure of pretreated bamboo was analyzed through sem micrograph obtained by a jeoul/eo sem. bamboo 83 sample was installed in the sample holder (stub) using sputter canter and then scanned at 15 kv with 10 mm of working distance with 750x and 10,000x of magnification. biodegradation pattern biodegradation pattern was analyzed through fourier transform infrared spectrometry (ftir) spectrograph. to obtain ftir spectra, 4 mg of bamboo-meal was embedded in 200 mg of kbr (potassium bromide) spectroscopy grade and then pelletized at 5,000 psi. the diameter and thickness of the pellet were approximately 1.3 cm and 0.5 cm, respectively. infrared spectrum patterns (peak height and area) were analyzed by using ftir abb mb 3000. all of the spectra were recorded at a spectral resolution of 16 cm -1 with the accumulation of 5 scans per sample with absorption mode in the range of 4,000-500 cm . -1 the characteristic of carbohydrates and lignin was analyzed based on the relative band intensity change referring to the method of pandey (pandey & pitman 2003). peak height and area values of lignin associated bands were rationed compared to carbohydrate reference peaks at 1,720; 1,366; 1,180 and 879 cm to provide relative -1 changes in the composition of the structural components relative to each other determined using horizon mb software. statistical analysis all experiments were performed in triplicate. sample preparation for sem, ftir and xrd analyses has been conducted by manually mixing all triplicate treated and untreated samples. the pretreatment combination effects on chemical component changes and losses were analyzed by anova (analysis of variance) using minitab release 13.2 software. significant differences among treatment combinations were evaluated using tukey's multiple range comparisons at < p 0.05. results and discussion chemical content of pretreated bamboo the chemical component composition change of pretreated bamboo is depicted in figure 1 which shows that bamboo has high a-cellulose content. cellulose is the main source of c-6 sugar convertible to ethanol. in this study, combined biological-microwave pretreatment was utilized to reduce lignin content. the pretreatment was expected to degrade lignin and hemicellulose. removal of the c-5 hemicellulose could increase sugar fermentation by saccharomyces cerevisiae considering that the c-5 sugar of hemicellulose cannot be efficiently fermented by the yeast. 84 ds : 2.91 0.46 1.08 0.77 0.59 1.14 wl asl ail hc ac e 100% 80% 60% 40% 20% 0% control biological-microwave pretreatment 5% il for 5 min 5% il for 10 min 5% il for 12,5 min 10% il for 5 min 10% il for 10 min 10% il for 12,5 min figure 1 chemical component composition change of bamboo after biological-microwave pretreatment. components: s il (inoculum loading); wl (weight loss); asl (acid soluble lignin); ail (acid insoluble lignin); hc (hemicellulose); ac (alpha cellulose); e (ethanol-benzene extractive); ds (delignification selectivity) biotropia vol. 22 no. 2, 2015 h e m i c r o w a v e t c o m b i n e d f u n g a l pretreatment changed the compositionchemical of pretreated bamboo of (fig. 1). pretreatment 10% inoculum loading show lower weight loss ed or higher yield inoculum loading. than that of 5% longer irradiation time tended to increase weight loss. might be related more intensive this to a lignin degradation activity than carbohydrate removal in higher inoculum loading. total weight loss was approximately of 5.47-19.88% tat stical . s i analysis indicated that inoculum loading gave only significant effect to alpha cellulose and hemicellulose content, weight loss and hemicellulose loss only . on the other hand, irradiation time gave significant effect on weight loss and lignin loss . based on ukey's ( < 0.05) tp pairwise comparison, weight loss due to irradiation time differen . were significantly t i nteraction between inoculum loading and irradition time weight loss. significantly affected however, no interaction between inoculum effect loading and irradition time on the alpha cellulose loss . prolonging irradiation time for 10 was found min affected decrease of apha cellulose utes the loss for both inoculum loading. even though, the irradiation time for 12.5 min caused higher utes lignin degradation, also caused higher alphait cellulose loss. thus, greater extend of irradiation time was not required. the highest selectivity value (up to 2) was found after bamboo pretreated 5% was with inoculum loading and then irradiated for 5 was min . a higher selectivity value indicate that utes s lignin polymer is more effective than cleavage cellulose degradation. tatistical analysis s of the present results dindicate that the inoculum loading and irradiation time did not significant ly affect selectivity value ( < 0.05).p degradation of carbohydrate (alpha cellulose) also occurred during delignification activity (fig. 1). it might be due to partial hydrogen bond disruption of the lcc (lignin carbohydrate complex) (li . 2010). bet al iological pretreatment caused opening complex of the lignocellulose structure through depolymerization of lignin and brought about carbohydrates increasing accesibility. icrowave pretreatment after m biological pretreatment also help toed alter the ultrastructure of cellulose and degrade lignin and hemicellulose in lignocellulosic materials that bring about increasing susceptibility of lignocellulosic materials (binod . 2012). et al microwave heating transfers and induces heat directly into bamboo substrate, causing the depolymerization of sugar building block into oligosaccharides (ebringerova 2006). under acid pretreatment at high pressure and temperature, sugar monomer such as glucose and xylose can be further degraded in hydroxymethyl furfural and furfural (hmf) behera . 2014) ( . et al the degradation product can be released during acid pretreatment condition. more severe of microwave pretreatment condition led to decrease hemicellulosic monosaccarides in hydrolyzate and increase the formation of sugar degradation product (kuhnel 2011). variouset al. potential s edcompound in hydrolyzate consist acetic acid, formic acid, furan derivatives of (5-hmf and furfural) and phenolic compounds might be wasgenerated. the inhibitor presence not excepted the effect of limationdue to efficient process (zhang . 2011; fermentation et al talebnia . 2010). however, due to our research et al focus to observe the change of chemical was component and cellulose structure of pretreated samples, this potential inhibitor on hydrolyzate was d not observe . cellulose structure changes of pretreated bamboo ftir spectroscopy was used to investigate changes in the chemical structure of pretreated samples (fig. 2 3). slight changes occurred in and spectrum peaks of biomass were treated with both 5 and 10% inoculum loading, but no changes appeared in the functional groups during pretreatment. it might be caused by uncompleted disruption of lignin that encapsulated cellulose. the broad absorption was observed at the wave number of around 3,340 cm . this wave number -1 was assigned to hydrogen bond (o-h) stretching absorption. o-h stretching region at the wave number of 3,000-3,600 cm of pretreated -1 bamboo spectra was more identical to the o-h stretching region from cellulose i. the band at 2,700-2,901 cm is related to the c-h stretching -1 (pandey & pitman 2003). biological-microwave pretreatment affected the peak area and bandheight of 3,340 cm wave number (o-h -1 stretching) (fig. 2 and 3). it indicated a weak intra and inter molecular bond of o-h group (goshadrou . 2011).et al 85 fiber disruption of betung bamboo ( ) by combined fungal fatriasaridendrocalamus asper and microwave pretreatment – et al. ftir spectra with frequencies in the region of 1,600 and 1,510 (aromatic ring vibration), 1,470 and 1,460 cm (c-h deformations and aromatic -1 ring vibrations) can be found in the lignin structure (fengel & wegener 1992). lignin of bamboo consisting of guaiacyl (g) and syringyl (s) propane units containing one and two metoxyl groups can be clearly observed in all treatments at wave number of 1,327 cm for syringyl propane -1 units and 1,257 cm for guaiacyl propane units. -1 the higher absorbencies in the finger print of pretreatment of both 5 and 10% inoculum loading of pretreated samples compared to control was found at irradiation of 10 minutes. the higher lignin and cellulose content in this condition (fig. 2) which can be confirmed by this spectra (fig. 3). the absorbance of syringyl (1,327 cm ) was lower than that of guaiacyl -1 86 figure 2 ftir spectra of bamboo after fungal pretreatment (5% inoculum loading for 30 days) subjected to microwave pretreatment figure 3 ftir spectra of bamboo after fungal pretreatment (10% inoculum loading for 30 days) subjected to microwave pretreatment biotropia vol. 22 no. 2, 2015 (1,257 cm ) indicating a higher syringyl content in -1 control and treatments. the typical infrared band frequencies and ftir spectras of bamboo components in units of wave numbers are listed in table 1. the sharp bands around 895 cm is attributed -1 to β-glicosidic linkage between the sugar units in cellulose (nelson & o’connor 1964) which can be clearly seen in all spectra. the increasing microwave irradiation reduced band intensity of functional group (c=o) in hemicellulose (3), c-h in cellulose and hemicellulose (9), and c-o-c in hemicellulose (12). it might be attributed to the decrease of hemicellulose content after pretreatment along with lignin. sixty functional groups can be observed in six pretreatment conditions. each identified functional group can be found in all treatments, although a slight shift in wave number occurred. the treatments decreased functional groups intensity without changing functional group types. effect of combined fungal and microwave pretreatment on bamboo morphology sem micrograph of pretreated samples (fig. 4 and 5) was used to observe morphological features and surface characteristics of pretreated bamboo with increasing microwave irradiation. 87 table 1 assignments of ir band of bamboo after biological-microwave pretreatment no control* biological pretreatment assignments 5% of inoculum loading 10% of inoculum loading microwave pretreatment 5 minutes 10 minutes 12.5 minutes 5 minutes 10 minutes 12.5 minutes wave number (cm-1) 1 3,394 3,340 3,333 3,418 3,418 3,425 3,364 a strong and broad hydrogen bond (o-h) stretching absorption1 2 2,901 2,901 2,901 2,901 2,901 2,901 2,901 a prominent c-h stretching absorption1 3 1,736 1,728 1,736 1,736 1,728 1,728 1,713 unconjugated c=o in xylans 1 4 1,643 1,643 1,651 1,651 1,643 1,643 1,643 absorbed o-h and conjugated c-o1 aromatic skeletal1 5 1,605 1,605 1,605 1,605 1,605 1,605 1,605 aromatic skeletal1 6 1,512 1,512 1,512 1,512 1,512 1,512 1,512 7 1,458 1,458 1,458 1,458 1,458 1,458 1,458 c-h deformation1 8 1,427 1,427 1,427 1,427 1,427 1,427 1,427 c-h2 scissoring motion1 9 1,373 1,373 1,373 1,373 1,373 1,373 1,373 c-h deformation1 10 1,335 1,327 1,335 1,335 1,327 1,327 1,327 c-h vibration1 c1-o vibration in syringyl derivates1 11 1,257 1,257 1,257 1,250 1,257 1,257 1,250 guaiacyl ring1 c-o stretch1 12 1,165 1,165 1,165 1,165 1,165 1,165 1,165 c-o-c vibration1 13 1,111 1,111 1,111 1,111 1,111 1,111 1,111 aromatic skeletal and c-o stretch1 14 1,049 1,041 1,034 1,041 1,041 1,041 1,041 c-o stretch1 15 895 895 895 895 895 895 895 c-o-c stretching at β-glicosidic linkage or c-h deformation in cellulose2 16 833 833 833 833 833 833 833 c-h vibration3 notes: control* = data has been used in other paper (fatriasari . 2014b)et al 1 = pandey and pitman (2003) 2 = chen (2011)et al. 3 = cheng . (2013)et al fiber disruption of betung bamboo ( ) by combined fungal fatriasaridendrocalamus asper and microwave pretreatment – et al. sem images of samples treated with both 5 and 10% inoculum loading show that partial disruption occurred on the fiber structure. degraded lignin polymer and removal of hemicellulose in cell wall might responsible to be this disorganized morphology. longer microwave irradiation caused greater fiber degradation level. the cell wall morphology changes caused by lignin removal resulted in greater deconstruction of fiber surface, providing better cellulose penetration. partial degradation of lignin and hemicellulose destroyed some ether bonds in lignin and lignin-carbohydrate complex leading to the disruption of the hydrogen bond between cellulose, thus fibrillation occurred (li . 2010). et al the cellulose digestibility can be potentially enhanced by preferential cleavage of lignin (nazarpour . 2013). treatments of 5% et al 88 10 minutes (330 w) 12.5 min utes (330 w) 5 minutes (330 w) a b 10 minutes (330 w) 12.5 min utes (330 w) 5 minutes (330 w) a b figure 4 sem micrograph pretreated bamboo (5% inoculum loading for 30 days) subjected to microwave pretreatment of with (a) 750x and (b) 10,000x magnification figure 5 sem micrograph pretreated bamboo (10% inoculum loading for 30 days) subjected to microwave pretreatment of with (a) 750x and (b) 10,000x magnification biotropia vol. 22 no. 2, 2015 inoculum loading for 10 and 12.5 minutes microwave irradiation resulted in more openedup sponge-like structures providing wider surface area to increase the rate of subsequent hydrolysis reactions. cellulose structure allomorph crystalline allomorph of pretreated bamboo as result of mixing triplicate samples a observed by xrd is presented in table . in general, 2 cellulose consists of i (one-chain triclinic) and i α β (two-chain monoclinic cells), which can be determined by z-discriminant. z < 0 and z > 0 indicate the i and i allomorph types, respectively β α . monoclinic (i ) cellulose is more stable than β triclinic (i ), and tends to be the final product in α annealing treatment credou of all celluloses ( & barthelot . 2014) all treatments except for the 5% inoculum loading for 10 minutes had monoclinic structure. however, the cause of this phenomenon has not yet understood the presence of the i phase been . α was expected to improve cellulose digestibility due to its higher degradation than that of i . in β addition, this structure is meta-stable and more reactive than i ( ).β moon . 2011et al bio of ombined degradation pattern c biological-microwave p tretreatmen biodegradation patterns of bamboo during combined biological-microwave pretreatment were evaluated by ftir analysis 6 7 . (figure and ) a detailed ftir spectroscopic analysis based on pandey's analysis method was performed to calculate relative intensities of aromatic skeletal vibration against typical bands of carbohydrate on pretreated bamboo (pandey & pitman 2003). 89 table 2 crystalline allomorph of pretreated bamboo biological pretreatment microwave pretreatment crystallite allomorph crystal allomorphinoculum loading (%) power loading (w) microwave irradiation (minutes) d (101) nm d (10-1) nm z control* 0.5824 0.5349 -45.47 iβ 5 330 5 0.5987 0.5466 -28.49 iβ 10 0.6110 0.5230 13.69 iα 12.5 0.5955 0.5473 -34.50 iβ 10 5 0.5520 0.5163 -80.11 iβ 10 0.5583 0.5134 -66.92 iβ 12.5 0.5740 0.5181 -44.48 iβ note: control* = data have been used in other paper (fatriasari . 2014 )et al b fig 6 ftir spectra of bamboo after biological pretreatment (5% inoculum loading for 30 days) subjected to microwave ure pretreatment (1) 1,736 cm , (2) 1,512 cm , (3) 1,373 cm , (4) 1,165 cm and (5) 897 cm-1 -1 -1 -1 -1 fiber disruption of betung bamboo ( ) by combined fungal fatriasaridendrocalamus asper and microwave pretreatment – et al. relative changes in the intensities of aromatic skeletal in lignin peaks at 1,512 cm against four -1 unconjugated bonds of carbohydrate peaks at 1,736 cm (c=o in xylan), 1,373 cm (c-h -1 -1 deformation in cellulose and hemicellulose), 1,165 cm (c-o-c vibration in cellulose and -1 hemicellulose), 895 cm (c-h deformation or c--1 o-c stretching at β-glicos idic l inkage characteristics in cellulose) calculated by peak heights and areas are summarized in table .3 in 5% inoculum loading, increasing microwave irradiation period tended to increase the ratio of lignin/carbohydrate. it indicated that prolonged microwave irradiation decreased its lignin degrading ability of pretreated bamboo. carbohydrate degradation after pretreatment contributed to this phenomenon. crystallinity index (ci) and crystallite size of cellulose crystalline and amorphous structure of cellulose can be identified from primary peak of xrd pattern ranging from 22-23 and 0 secondary peak in the range of 16-18 (lai & idris 0 2013; liu . 2012). these peaks can be et al determined within the mentioned range for all treatments, which indicates that the crystalline and amorphous region of cellulose. the crystallinity index of pretreated bamboo has been used to interpret changes of cellulose after pretreatment (table 4). intensity transformation in hydrogen bonding of cellulose can be reflected from width variation of crystallization peak. 90 fig 7 ftir spectra of bamboo after biological pretreatment (10% inoculum loading for 30 days) subjected to microwave ure pretreatment (1) 1,736 cm , (2) 1,512 cm , (3) 1,373 cm , (4) 1,165 cm and (5) 897 cm-1 -1 -1 -1 -1 table ratio of intensity of lignin-associated band with carbohydrate bands of pretreated 3 bamboo biological pretreatment microwave pretreatment relative intensities a of aromatic skeletal vibration (i1,512) againts typical bands for carbohydrates inoculum loading (%) power loading (w) irradiation time (minutes) i1,512/i1,736 i1,512/i1,373 i1,512/i1,165 i1,512/i897 control* 1.04(1.06) 1.02(1.06) 0.98(0.95) 1.28(1.29) 5 30 5 1.24(0.83) 0.86(0.6) 0.67(0.33) 1.74(3.75) 10 1.49(1.19) 0.88(0.57) 0.64(0.34) 1.59(3.57) 12.5 1.74(1.88) 0.94(0.81) 0.58(0.48) 3.26(2.5) 10 5 1.42(1.37) 0.83(0.74) 0.59(0.48) 1.55(1.51) 10 1.28(0.75) 0.78(0.48) 0.56(0.29) 1.58(4.0) 12.5 1.36(1.33) 0.84(0.77) 0.57(0.47) 1.73(1.69) note: control* = data have been used in other paper (fatriasari . 2014 )et al b biotropia vol. 22 no. 2, 2015 the crystallinity index tended to rise along with the increasing microwave irradiation. in biomass, cellulose contains crystalline area, while lignin and hemicellulose are amorphous in nature (o'dowyer . 2007). the increasing of et al crystallinity index might be caused by solubilization of amorphous component from the fibers under pretreatment condition (kim & holtzapple 2006). this phenomenon was supported by the occurring component loss in lignin as presented in figure 1. the increase of crystallinity index after pretreatment was also described in previous research (singh 2014; et al. bak . 2009).et al crystallinity index of cellulose is among the most important properties of lignocelluloses that can be measured by ftir spectroscopy. crystallinity index changes can be studied from loi (lateral order index), defined as the absorbance ratio of a to a (oh . 2005), 1,427 895 et al obtained from ftir spectra data. increasing of microwave irradiation time tended to increase loi. it might be attributed to higher amorphous region of cellulose compared to crystalline region. this phenomenon was in line with crystallinity index measured by xrd analysis. crystallite size of cellulose in bamboo varied at lattice planes of (101), (10-1) and (002). the crystallite size ranged from 5.19 to 10.68 (table 5) . the highest crystallite size of cellulose at (002) lattice plane was found in pretreatment of 5% inoculum loading for 12.5 minutes. crystallite size at lattice planes (101) and (10-1) of several pretreated bamboo can not be calculated because s there no value of full width at half were maximum (fwhm) in this peak. the microwave irradiation duration increaed crytallite size at (002) lattice plane. the highest crystalline length 91 table 4 crystallinity index (ci) and lateral order index (loi) of pretreated bamboo biological pretreatment microwave pretreatment crystallinity index (ci) lateral order index (loi) inoculum loading (%) power loading (w) irradiation time (minutes) fc (crystaline) fa (amorf) ci a1,427 (crystalline) a897 (amorf) loi control* 0.69 2.13 24.58 0.50 0.40 1.25 5 330 5 0.99 1.48 40.19 0.98 0.51 1.92 10 1.23 1.72 41.76 1.43 0.84 1.70 12.5 1.12 1.54 42.04 0.74 0.23 3.22 10 5 1.16 1.74 39.98 1.14 0.65 1.75 10 1.13 1.63 40.96 0.75 0.40 1.88 12.5 0.93 1.34 40.84 1.20 0.63 1.91 note: control* = data have been used in other paper (fatriasari . 2014 )et al b table 5 crystallite size of pretreated bamboo biological pretreatment microwave pretreatment crystallite size (nm) inoculum loading (%) power loading (w) irradiation time (minutes) d (101) d (10-1) d (002) d (040) controla 5.46 8.71 5.59 16.52 5 330 5 ndb 6.57 5.17 20.99 10 14.95 7.07 5.74 38.80 12.5 8.69 4.86 6.19 16.51 10 5 ndb 5.32 5.74 86.33 10 5.20 5.36 5.82 22.85 12.5 10.68 ndb 5.47 36.98 notes: control = data have been used in other paper (fatriasari . 2014 )a et al b = b not detected fiber disruption of betung bamboo ( ) by combined fungal fatriasaridendrocalamus asper and microwave pretreatment – et al. at (040) lattice plane of cellulose was found in the 10% inoculum loading for 5 minutes. there was no similar trend in crystalline length changes caused by increasing of microwave irradiation between 5 and 10% of inoculum loading. conclusions the characteristic changes of lignin and carbohydrate combined of betung bamboo with biological-microwave pretreatment was evaluated. the pretreatment caused the chemical loss of the component. the 5% inoculum loading irradiated for 5 min demonstrated the highest selectivity utes value (up to 2). based on ftir spectra, there was no change in functional g roups after pretreatment also . moreover, ftir spectra demonstrated the presence of hydrogen bond stretching along with microwave irradiation exposure indicating the structural changes occur ed the r after pretreatment. cleavage of the amorphous d to the component contribute increasing crystallinity index disruption of fiber . structure due to pretreatments was confirmed by sem the longer duration of microwave , in which irradiation, the greater the degradation level of fiber. acknowledgements the authors would like to thank seameo biotrop for providing financial support as part of phd thesis of the first author through dipa 2013. moreover, the authors expressed their gratitude to dwi h restuningsih, st. and raden budi l.permana in for their support financial report and technical assistance. references agbor vb, cicek n, sparling r, berlin a, levin db. 2011. biomass pretreatment: fundamentals toward application. biotechno adv 29:675-85l . anita sh, risanto l, hermiati e, fatriasari w. 2012. pretreatment of oil palm empty fruit bunch (opefb) using microwave irradiation. in: the 3 rd international symposium of iwors (indonesia wood research society). proceedings: 2015 november 4; 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purwantoro . 2010). the main risks of plant species loss are possibly forest conversion and land use change as well as illegal logging. fluctuating microclimate conditions caused by forest clearance and possibly by climate change may alter the germination and hence, composition of forest understorey. the genus should be a priority in conservation, potentially to allow reintroduction activity. species have * corresponding author : a.satyanti@gmail.com 80 considerable economic value as ornamental plants and some have potential as medicinal plants and as a vegetable (tebbit 2005; chiew 2005, girmansyah 2008). in banten, west java, , known as 'krokot', is popular among locals as vegetable (djarwaningsih 2010). in bali, is used for curing cough (hartutiningsih 2005). in west java, the sundanese called and as hariang or asam-asam for substituting sour taste from in local dishes (purwantoro 2010; wiriadinata 2002) and it is also used for traditional medicine (priyadi 2010). a number of species are close to extinct due to habitat destruction (chiew 2005). research in is challenging as species are really under pressure in their natural habitat; research has to keep up with the pace of species loss due to habitat loss. logging can destroy populations, and limestone quarries have contributed to the extinction of those endemic to limestone hills. the vulnerability of extinction for is compounded by the fact that many are extremely local endemics (kiew 2005). in peninsular malaysia, of nearly 60 species, 26 are known from single localities and some of their populations are small (kiew 2005). therefore, conservation finds its relevance here. kebun raya eka karya in bali, an conservation location in indonesia, harbours more than 60 indigenous species (thomas 2009) and is regarded as the richest collection among botanical gardens in the world (hartutiningsih 2005). conservation efforts begin with exploration activity, which involves the inventory and collection of species, and habitat study. in addition to collection and taxonomic studies, there is a need for ecological study, in particular which environmental factors determine the existence of particular species in its natural habitat. there is very scant microclimate information available for tropical habitats, especially in the case of . therefore, this study aims at describing: (i) which species are found in two forest sites in bedugul, bali, (ii) measure environmental variables in each habitat, and (iii) look for correlation between the recorded environmental parameters and the frequency of species. observation on populations and environmental data were conducted in two locations, namely bukit tapak (08°16'00” s and 115°08'00”e) and (reboisation forest, 08°16'00” s and 115°09'00”e). bukit tapak is mainly secondary forest with somewhat heavier canopy cover which is represented by lower solar radiation lux values in general (table 1, fig. 1). most of the in bukit tapak already produce either flower or fruits whereas in reboisation forest, the observation was done along small open roads which is intensively managed by mowing, leading to shorter plants with less reproductive organs (fig. 2). b. multangula et al. b. multangula b. multangula b. robusta tamarindus indica et al. et al. begonia begonia begonia begonia ex-situ ex-situ begonia et al. begonia ex-situ begonia begonia begonia begonia hutan kontrak begonia materials and methods study site 81 microclimate preference and habitat of begonia in bedugul, bali, – annisa satyanti et al. bukit tapak, batukaru nature reserve, bali batukaru nature reserve is located in baturiti subdistrict, tabanan and sukasade subdistrict, buleleng. the topography ranges variably from flat to steep, approximately 8% to 45% slope. the area of the reserve is 8557.98 ha, which consists of 3 sections which are resort pemangkuan hutan (rph) pupuan, rph penebel, and rph candikuning (ksda-bali 2009). the access to the nature reserve is via wangaye gede village, subdistrict penebel. the dominant vegetation consists of cemara pandak ( ), cemara geseng ( ), kepelan ( sp.), juwet manting ( ), and seming ( sp.). on forest floors, several species of can be found. dacrycarpus imbricatus casuarina junghuhniana magnolia crypteronia paniculata pometia begonia figure 1. ; hutan tapak siteb. multangula figure 2. (left, middle) and (right); reboisation forest siteb. longifolia b. baliensis biotropia vol. 19 no. 2, 2012 82 reboisation forest area at “eka karya” botanical garden, bali species and data collection data analysis the reboisation area (hereafter also written as ) consists of flat, and hilly topography at an altitude of 1250-1400 m asl (purwantoro 2010). the forest comprises 22-52 years old rasamala ( ), gintungan ( ) and cempaka ( ). other trees and herbs naturally exist including lateng ( ), , sp. and spp. the submontane cool climate of bali botanical garden facilitates good growth of species. the climate is type b with precipitation rate of 2000-3000 mm/year; a 7-9 month rainy season, and 1-3 month dry season, intercepted solar radiation of 4660%, relative humidity of 78-96% and mean wind speed 7.27 km/hour (hartutiningsih 2005). the exploration is using a random purposive method, meaning that one track is taken for each site and standing from the line and observing the existing population, we decided where to establish the plot. we sampled 40 plots, 20 plots in each site. the size of the plot was 0.5 m × 0.5 m. within the plot several measurements were taken for number of individuals as well as the following microclimatic factors: temperature (°c), solar radiation (lux), air humidity (%), soil ph, soil moisture (%) and altitude (m). temperature, solar radiation, and air humidity were measured using lutron lm-8100. to measure temperature, solar radiation, and air humidity the device was located directly above individuals, at about 1.3 m above ground. soil ph and soil moisture were measured using soil tester takemura dm-5. the soil tester was placed about 10 cm (max 30 cm) from the main clumps. gps garmin 60 was used to indicate altitude and geographical coordinates. all measurements were carried out between 10 am and 3 pm. in addition, we collected plant samples within the plot, adjacent to for further identification in the lab. plant size, including diameter and height, number of seedlings, number of individuals in reproductive phase, were also measured (data not shown in this paper). differences between plots of each environmental factor, viz. temperature (°c), solar radiation (lux), air humidity (%), soil ph, soil moisture (%) and altitude (m) were observed using univariate analysis, viz. t-test performed in spss for windows (version 19.0). in addition, t-test was also performed to find whether there was difference between species abundance of the two forests, bukit tapak and . for this analysis, the species are considered as four variables: , , f. white and f. red. subsequently, separate analysis for each forest site was also conducted. within each forest site, species were treated as four ( , , f. white and f. red) or three variables ( , , and ) which will be further explained in the discussion. for the analysis in each forest, analysis of variance, onehutan kontrak altingia excelsa bischofia javanica michelia champaca laportea microstigma eugenia uniflora garcinia begonia begonia et al. begonia begonia begonia begonia begonia begonia univariate analysis begonia hutan kontrak begonia b. multangula b. baliensis b. longifolia b. longifolia begonia b. multangula b. baliensis b. longifolia b. longifolia b. multangula b. baliensis b. longifolia 83 microclimate preference and habitat of begonia in bedugul, bali, – annisa satyanti et al. way anova was planned to be employed. however, due to the nature of the data which were not normally distributed and the variance was not homogeneous, while the data remained the same upon transformation efforts, non-parametric data analysis was then carried out. kruskal wallis test was hence performed to investigate whether there was difference in species (considered as either four or three variables) within each forest. multivariate analysis was used to see the simultaneous correlation between species and the measured environmental factors across forty sites and how these variables may explain the abundance of species. pcord5 for windows was employed for detrended correspondence analysis (dca). the dca showed that the length of gradient resulted as 1.880, and based on this we re-conduct the analysis using principal component analysis (pca). to observe how species abundance is explained by environmental factors we established a graph by determining the main matrix as species abundance in each plot and the second matrix as environmental factors of each plot. as a rule of thumb, when given r2-cutoff ≥ 0.300 the environmental variables shown in the graph are considered significant enough to explain the abundance of species. however, it was not possible to get the environmental variables vectors with r2-cutoff ≥ 0.300 from our data consequently, we chose r2-cutoff 0.004 to show the vectors in the species abundance and microclimate graph. blume (fig. 1) herbs, height 100 150 cm. green stem, hairy, segment swollen, segment 10 12 cm. leaves hairy, clear venation, length 10 15 cm, width 8 10 cm, margins clearly serrate. flower white, fruit capsule, without wing, smooth. flowering whole year, distinguishable from large leaf, growth in group and abundant in certain patches. distribution: mountain forest in java, sumatra, and bali, altitude up to 2400 m asl. for nomenclatural information and a full description, refer to hughes (2008) and hughes and girmansyah (2011). blume (fig 2, left and middle) erect herb, cane like growth, 50 cm height, smooth stem. leaves surface smooth, tip either sharp or blunt, size 12 20 × 6 12 cm. flower emerges from axillary leaf, short; male flower white, tepals 4; female tepals 5 6. fruits without wing, surface with dots. commonly found on humid places, slopes, grow solitary. in indonesia, frequently found in the islands of java, sumatra, bali and sulawesi. for nomenclatural information and a full description, see kiew (2005), hughes (2008), hughes and girmansyah (2011). begonia begonia begonia begonia . begonia . b. longifolia multivariate analysis results and discussions species descriptions b. multangula b. longifolia 84 biotropia vol. 19 no. 2, 2012 begonia baliensis begonia longifolia b. baliensis b. multangula girm. b. baliensis b. multangula b. longifolia hutan kontrak per se begonia begonia baliensis smilax elaeocarpus diplazium esculentum clautylon b. longifolia homalanthus giganteus ardisia cyrtandra procris ruhlandii adiantum alyxia adiantum smilax glochidion digitaria diplazium flacourtia nephrolepis biserrata homalanthus giganteus polygonum chinense raphidopora elatostema stigosum eupatorium triplinerve diplazium pilea syzygium b. multangula meliosma peruginea digitaria (fig. 2, right) erect and cane-like, seldom branched, 15 100 cm tall, stem brownish green to reddish brown; nodes brownish green to reddish brown, swollen. leaves distant, lamina oblique, broadly ovate, asymmetric, basal lobe rounded, 3.5 7.5 cm long. inflorescences axillary, few flowered. fruits berry, 4 5 mm long, green when ripe, fleshy, globose, elongated into a fleshy beak, glabrous, 3-lobed, with one larger wing, not splitting. seeds barrel-shaped, 0.25 0.3 mm long. distribution: bali. for nomenclatural information and a full description, see girmansyah (2008). the number of individuals found and recorded in each forest is shown in figure 3, where it depicts the total number of individuals recorded across plots. , and (red and white forms), were all found in bukit tapak and . as we did not conduct association study, we will not present association for in the respective forest. instead, we will elaborate list of other species found in the plot as follows. was found with sp. (smilacaceae), sp. (elaeocarpaceae), (pterydophyta), sp. (euphorbiaceae). was recorded adjacent to (euphorbiaceae), sp. (myrsinaceae), sp. (gesneriaceae), (urticaceae), sp., sp. (apocynaceae), sp. (adiantaceae), sp. (smilacaceae), sp. (euphorbiaceae), sp. (poaceae), sp. (denst.), sp. (flacourtiaceae), (nephr.), (euph.), (polygon.), sp. (araceae), (urticaceae), (asteraceae), sp. (denst.), sp. (urticaceae), sp. (myrtaceae). was found with (sab.), and sp. (poaceae). , , in their natural habitat and their association figure 3. species average abundance across 40 plots in (20 plots) and (20 plots); error bars show 95% ci. begonia hutan tapak hutan kontrak 85 microclimate preference and habitat of begonia in bedugul, bali, – annisa satyanti et al. microclimate conditions the average value of microclimatic factors, viz. air humidity, temperature, solar radiation, soil ph and moisture between two habitats of in bedugul differed (table 1), except for soil moisture or humidity. the overview of the environmental variables or microclimate can be found in figure 4. the habitat conditions in both forests were somewhat different. was frequently disturbed by mowing or herbs cutting and received more abundant light and is relatively warmer (table 1, figure 4). in order to understand the habitat preferences of each species, with respect to microclimate, we used multivariate analysis. for the analysis, species abundance was used as main matrix whereas environmental variables recorded across forty plots were used as second matrix. in general, we found that the three species of occupy similar microhabitats, as shown by the short vectors of the environmental variables (figure 5). in other words, in each plot there was no characterizing environmental variable that distinguished species preference. other factors that were not measured, on the other hand, may determine habitat preference. such factors may refer to for example, nutrient availability, soil texture, mineral structure, root competition, litter layer, topsoil depth, allelopathy or competition with bryophytes, etc. in fact, species are able to occupy wide range of habitat type and microclimate, as in the case recorded for in thailand (phutthai 2009). figure 6 depicts the distribution and abundance of species across plots. main matrix and second matrix were both species abundance across plots. when r2-cutoff is set to higher threshold i.e. 0.600, only f. white and f. red vectors were able to show up indicating that plots significant characters the frequency of found in . when r2-cutoff reduced to 0.300, vectors produced were for f. red, f. white, and . thus, the presence of these species were able to be grouped based on plots, except for begonia hutan kontrak begonia begonia begonia begonia begonia begonia begonia et al. begonia begonia b. longifolia b. longifolia b. longifolia b. longifolia b. longifolia b. baliensis begonia table 1. microclimatic features of two natural habitats of . ttest was applied to define statistical difference of each factor between two sites begonia abiotic factor forest site mean se of mean p-value temperature (°c) bukit tapak 23.6 0.169 0.000 reboisation forest 27.1 0.475 ** solar radiation (lux) bukit tapak 472.95 107.0367 0.000 reboisation forest 2246.197 502.2649 ** air humidity (%) bukit tapak 71.86 0.4245 0.043 reboisation forest 70.175 0.6871 * soil ph bukit tapak 6.225 0.571 0.043 reboisation forest 6.02 0.793 * soil moisture (%) bukit tapak 78.057 1.652 0.801 reboisation forest 78.759 2.203 (ns) altitude (m asl) bukit tapak 1428.95 2.259 0.000 reboisation forest 1351.1 7.021 ** p-value indicates * as significant; ** as higly significant; (ns) as not significant at 95% confidence interval 86 biotropia vol. 19 no. 2, 2012 . however, from figure 5, environmental variables to characterize each plot were not found. f. white had a high correlation with axis 1, and on the other hand, f. red had a high correlation with axis 2. interestingly, within , white and red form has a tendency to be present in different plots. further investigation on how these two forms differ in occupying site shall be carried out. based on figure 6, and were relatively frequent to occupy similar plots, even though not significant (higher r2-cutoff= 0.106). , , and f. red were somewhat found to occupy similar sites, but completely apart from f. white. b. multangula b. longifolia b. longifolia b. longifolia b. baliensis b. multangula b. baliensis b. multangula b. longifolia b. longifolia figure 4. the microclimatic features of the 40 study plots (20 plots in bukit tapak and 20 plots in or reboisation forest)hutan kontrak f re q u e n c y altitude (m asl) b u k it t a p a k r e b o is a tio n f o re s t f o re s t s ite s 15 10 5 0 15 10 5 0 1325 1350 1375 1400 1425 f re q u e n c y b u k it t a p a k r e b o is a tio n f o re s t f o re s t s ite s air humidity (%) 66,0 68,0 70,0 72,0 74,0 76,0 78,0 80,0 6 4 2 0 6 4 2 0 f re q u e n c y soil ph b u k it t a p a k r e b o is a tio n f o re s t f o re s t s ite s 8 6 4 2 0 5,0 5,5 6,0 6,5 7,0 7,5 8 6 4 2 0 f re q u e n c y temperature ( c) o b u k it t a p a k r e b o is a tio n f o re s t f o re s t s ite s 12 10 8 6 4 2 0 20 22 24 26 28 30 12 10 8 6 4 2 0 f re q u e n c y soil humidity (%) b u k it t a p a k r e b o is a tio n f o re s t f o re s t s ite s 50 60 70 80 90 100 12 10 8 6 4 2 0 12 10 8 6 4 2 0 f re q u e n c y solar radiation (lux) b u k it t a p a k r e b o is a tio n f o re s t f o re s t s ite s ,0 2000,0 4000,0 6000,0 8000,0 20 15 10 5 0 20 15 10 5 0 87 microclimate preference and habitat of begonia in bedugul, bali, – annisa satyanti et al. figure 5. principal component analysis of species abundance and measured environmental factors in bukit tapak (bt) dan hutan reboisasi (hk) (r2cutoff=0.04; length of gradient axis 1=1.996, axis 2=1.763). these environmental variables were not significant (when r2-cutoff ≥ 0.300, no vectors was produced) related to plots and hence, could not explain species abundance. begonia begonia figure 6. principal component analysis of (form red and white), and (r2cutoff=0.004). correlation of , , f. red and f. white to axis 1were 0.285, 0.235, 0.243, and -0.989, respectively; whereas the correlation value of species to axis 2 were -0.263, -0.620, 0.923, and 0.04, respectively. b. longifolia b. multangula b. baliensis b. multangula b. baliensis b. longifolia b. longifolia begonia 88 biotropia vol. 19 no. 2, 2012 b. multangula begonia b.longifolia b. baliensis begonia begonia b. multangula b. baliensis b. longifolia b. longifolia b. longifolia hutan kontrak begonia et al b.multangula b. longifolia b. multangula b. robusta b. longifolia b. muricata b. bracteata b.multangula b. multangula begonia et al. b. multangula b. longifolia b. longifolia b. longifolia b. longifolia b. multangula b. baliensis b. multangula begonia b. robusta is actually known to have a wide distribution range and is present in java, sumatra and lesser sunda islands (hughes 2008). girmansyah (2008) in his study on of bali and lombok described the distribution of which extends from the himalayas (india) to south china, vietnam and through thailand, peninsular malaysia, and indonesia (sumatra, java, bali, and lombok). is endemic to bali and is known to occupy humid forest along trails at 1300 – 1800 m either in small colonies or large populations (girmansyah 2008). through univariate analysis, the difference between species abundance between the two forest types was similar (t-test, p=0.0061). subsequently, for bukit tapak the four species ( , , f. red, f. white) abundance did not differ among species (chi-square=1.913, p=0.591) neither when the two forms of was pooled together (chi-square=2.018, p=0.365). the results were similar to , species did not differ among each other, either way when considered as four (chi-square=0.556, p=0.906) or three (chisquare=0.476, p=0.788) different species. a plant diversity study in resort cidahu gunung halimun salak, west java (larashati . 2010) pointed out that is abundant in areas which intercept higher solar radiation, whereas within the same research site tended to prefer areas with heavier shade such as the forest floor. and were found to be abundant along paths, and around an open, frequently disturbed helicopter pad. furthermore, , , and were found mostly in forest under heavy shade. however, on the forest floor, was also found. it seems that has a wide range of preference and therefore it confirms our pca result (figure 6) that the correlation to axis 1 and axis 2 were the lowest amongst other species observed, which were 0.235 and -0.263, respectively. another study in gunung halimun national park by wiriadinata (2002) recorded that was very abundant. the distribution of this species is within ground cover of mountainous forests in java up to 2400 m altitude. in gunung halimun, it was always found to grow in clumps or groups at a wide range of altitudes, 900-1800 m. the flowering and fruiting period is all year round, and it prefers medium soil moisture and humidity. surprisingly, they also found that is on the contrary rare (only found in two spots in cikaniki and ciptarasa), grows solitary, and prefers very moist substrates or semi-waterlogged. was found only at 9001000 m altitude in this study. these studies did not mention any differentiation (colour) of in the forest in java. (both forms) and are cane-like, whereas is shrub like. this trait can be further analyzed using correlation with specific abiotic factors, such as light interception or shade tolerance. however, a study by shiodera and kohyama (2005) showed that in gunung halimun salak, at 1000-2000 m altitude, population of observed were found generally under medium light intensity. unfortunately, no further information refers to the extent of how light intensity determines the abundance of sp. on the other hand, its close relative, i.e. , present in similar forest, showed a tolerance to a wider lowmediumand high light intensity. further ecological studies on habitat preference should address more features of the environment such as nutrient availability, 89 microclimate preference and habitat of begonia in bedugul, bali, – annisa satyanti et al. competition, and disturbance level as many species are known for their narrow endemism, but a small number of species have a wide distributional range as well. as it has been hypothesized in several previous studies that light might determine the abundance of and , further research is needed to address this aspect in addition to that presented here. the abundance of all species between two forest types and within each forest were not significantly different. microclimate factors of two observed habitats of in bedugul were relatively equal between two forests and do not characterize habitat. however, there was a strong habitat separation between f. white and f. red. even though not statistically evident, (f. red), , and tend to occupy similar plots. unfortunately, environmental variables measured in the study did not appear significant to characterize each plot, and hence we could not able to identify which abiotic factors were able to determine the occurrence of species. several previous studies emphasized light intensity as explaining abiotic factor that distinguish the abundance in and in java, and hence, further elaborative study should address this matter. the project work was funded by the indonesian higher education department or dikti (2010-2011) research grant for “potensi lamiaceae, begoniaceae, dan lamiaceae sebagai bahan baku obat dan aspek konservasinya”. made ardaka and i wayan mastra (bali botanical gardens) are acknowledged for their technical assistance in the field, while didit okta pribadi (bogor botanical gardens) and maria hanauer (university of regensburg)for the advise on multivariate analysis. we would like to thank to an anonymous reviewer and dr. mark hughes (royal botanic garden edinburgh) for the constructive comments on the manuscript. begonia b. multangula b. longifolia begonia b. longifolia b. longifolia b. multangula b. baliensis begonia b. multangula b. longifolia conclusions acknowledgments references chiew h. 2005. begonias under threat. accessed from bgci (botanic gardens conservation international). webpage ( ) on 15 january 2011. djarwaningsih t, sulistiarini d, sunarti s, aerida ih, dewi, mahyuni r. 2010. karakterisasi tipe vegetasi dan keanekaragaman jenis flora/ jamur di cagar alam gunung tukung gede serang banten. laporan akhir program insentif peneliti dan perekayasa, lipi tahun 2010. departemen pendidikan nasional dan lembaga ilmu pengetahuan indonesia. girmansyah d. 2008. a taxonomic study of bali and lombok (begoniaceae). reinwardtia 12(5): 419-34. hartutiningsih. 2005. kebun raya bali, candikuning: upt balai konservasi tumbuhan kebun raya bali, lipi. isbn: lipi press. 797-26-2410-4. http://www.bgci.org/worldwide/news/0087/ begonia begonia 90 biotropia vol. 19 no. 2, 2012 hartutiningsih, ardaka im, siregar m. 2007. masa berbunga 22 jenis alam di kebun raya eka karya bali. biodiversitas 8(3): 192-96. hughes m. 2008. an annotated checklist of southeast asian . royal botanical garden edinburgh, uk. hughes md. girmansyah. 2011. a revision of sect. (hassk.) warb. from sumatra. gardens' bulletin singapore 62(2): 27-39. ksda-bali. 2009. cagar alam batukahu. accessed online from on 13 january 2012. kiew r. 2005. begonias of peninsular malaysia. natural history publications (borneo), sdn. bhd. kota kinabalu, sabah, malaysia. larashati, mirmanto ie, mansur h, wiriadinata h. 2010. penelitian ekologi jenis tumbuhan sebagai dasar pengelolaan dan pengembangan taman nasional gunung halimun-salak. laporan akhir program insentif peneliti dan perekayasa, lipi tahun 2010. departemen pendidikan nasional dan lembaga ilmu pengetahuan indonesia. purwantoro rs, hartutiningsih, siregar m, sudarmono, fijridiyanto ia, satyanti a. 2010. potensi lamiaceae, begoniaceae, dan lamiaceae sebagai bahan baku obat dan aspek konservasinya. laporan akhir tahun program insentif peneliti dan perekayasa lipi. departemen pendidikan nasional dan lembaga ilmu pengetahuan indonesia. priyadi h, takao g, rahmawati i, supriyanto b, nursal wi, rahman i. 2010. five hundred plant species in gunung halimun salak national park, west java: a checklist including sundanese names, distribution and use. cifor, bogor, indonesia. phuttai t, sands m, sridith k. 2009. field surveys of natural populations of l. in thailand. thai forest bulletin (bot.) special issue: 186-98. shirodea s, kohyama t. 2005. tradeoff and diversity of leaf/ shoot traits among plant life forms in non seasonal environment. in environmental conservation and land use management of wetland ecosystem in southeast asia annual report for april 2004-march 2005; core university programme between hokkaido university japan and research center for biology lipi, indonesia. sponsored by japan society for promotion of science (march 2005). tebbitt mc. 2005. . cultivation, identification, and natural history. published in association with brooklyn botanic garden. timber press, usa. thomas dc, ardi wh, hartutiningsih, hughes m. 2009. two new species of (begoniaceae) from south sulawesi, indonesia. edinburgh j bot 66 (2): 229-38. wiriadinata h, girmansyah d, hoover s, hunter j. 2002. kekayaan taman nasional gunung halimun. berita biologi 6(1) edisi khusus “biodiversitas taman nasional gunung halimun” (ii): 91-97. begonia begonia begonia sphenanthera begonia begonias begonia begonia http://www.ksda-bali.go.id/?page_id=11 91 microclimate preference and habitat of begonia in bedugul, bali, – annisa satyanti et al. 516 isrok (eimeria species).cdr eimeria species composition and factors influencing oocysts shedding in dairy farm, bandung, indonesia isrok malikus sufi , umi cahyaningsih nd etih sudarnika1* 2 2a 1 disease investigation center subang, ministry of agriculture, subang 41212, indonesia 2department of animal disease sciences and veterinary public health, faculty of veterinary medicine, institut pertanian bogor a, bogor 16680, indonesi received 15 july 2015/accepted 25 may 2017 abstract coccidiosis is one of the most widely distributed parasitic diseases of cattle throughout the world. coccidiosis infection in ruminants was caused by eimeria spp. the objective of this study was to determine eimeria species composition and various factors influencing eimeria oocysts shedding in dairy farm. this study was conducted with a cross-sectional study design in dairy farm in south bandung district from july 2014 to january 2015. samples were obtained from 400 dairy cattle (196 cattle at age < 6 months, 37 cattle at age 6 12 months and 167 cattle at age > 12 months). fecal samples were collected, examined and counted for eimeria species composition and numbers of oocysts per gram of feces (opg) using mcmaster technique. a questionnaire was completed for individual dairy cattle farmer to record information about cattle's health and husbandry. the effect of cattle's sex, age and type of pen flooring to opg values were analyzed using mann-whitney and kruskal-wallis tests. the kruskal-wallis test was performed followed by dunn test as a multiple comparison test. ten species of eimeria were identified in all infected cattle. among the eimeria identified species, eimeria bovis was found to have the highest prevalence (42.5%), followed subsequently by eimeria wyomingensis (39.1%), eimeria bukidnonensis (32.4%), eimeria pellita (26.3%), eimeria auburnensis (19.6%), eimeria zuernii (17.3%), eimeria cylindrica (3.9%), eimeria canadensis (3.9%), eimeria brasiliensis (3.4%) and eimeria alabamensis (1.1%). the numbers of oocysts shed was correlated significantly (p < 0.05) with cattle's sex and age as well type of pen flooring which influenced the infection pressure. younger calves aged less than 6 months shed the highest amount of eimeria oocysts than older cattle. many factors may cause the increasing number of opg in fecal samples. therefore, it is important to keep good sanitation and control of eimeria among dairy cattle in the kpbs pangalengan dairy farm. keywords: coccidiosis, cross-sectional, dairy farm, eimeria, opg introduction coccidiosis is a protozoic disease in cattle caused by eimeria spp more than twelve different . species of eimeria have been described in cattle ; most of which are considered harmless. of these many species, e. bovis and e. zuernii are highly pathogenic causing mortality and morbidity by disturbing absorption mechanisms (rehman et al. 2011). infection calves large occurs on caused by numbers of oocyst of e. bovis or e. zuernii may s , result in severe diarrhea with feces containing blood, fibrin and intestinal tissue. however, coccidiosis in cattle commonly occurs as subclinical disease without signs of the disease and involving great economical losses due to reduced appetite, reduced body weight, impaired feed conversion, unthriftness, diarrhea, dysentery, anemia and increased susceptibility to other diseases (abebe et al. 2008). a poorer body condition score (bcs), potentially as the result of a disease, can be connected to a lower milk production in dairy cattle (lassen 2009). c is estimated to cause annualoccidiosis economic loss in excess of us 400 million in the us d (bruhn et al. 2011). the development of clinical coccidiosis in cattle mainly depends on factors such as species * corresponding author: isrok.sufi@gmail.com 104 biotropia 4 2 7 104 113 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 2 516 of , age of infected animal, number of eimeria oocysts ingested, presence of concurrent infections, as well as type of production system and management practices (abebe . 2008). et al the aim of this present study was to determine the species composition and factors eimeria influencing the incidence of oocysts eimeria shedding in cooperation of south bandung dairy farm (kpbs) pangelangan, district of bandung, indonesia. thus, determination of species composition and evaluation of factors influencing the shedding of oocysts per eimeria gram (opg) is very useful in designing efficient control strategies. materials and methods study area the study was conducted in kpbs pangalengan, south bandung district, west java province from july 2014 to january 2015. kpbs pangalengan bandung covers 27.3 ha of highland area with an altitude of 1,000 to 1,420 m asl, 12 – o28 c mean annual temperature and 60 – 70% relative humidity. the average annual rainfall was > 2,000 mm during the study period. study design c applied based ross-sectional study design was on thrusfield (2007). a total of 400 dairy cattle (n) from a total population of 14 000 dairy cattle , distributed on 37 cooperation service area (tpk) and 225 farmers group were selected for the s study with 95% confidence interval, 50% expected prevalence (p) and 5% accepted error (l). the relevant formula for a 95% confidence interval is thrusfield 2007) based on ( : n = 4pq/l2 where: n = total of cattle p = expected prevalence q = 1 – p l = accepted error samples obtained from dairy cattle farmers covered all age groups i.e. calves (age < 6 months); weaners (6 12 months); and adults (age > 12 months). from the total of 225 farmers groups, 10 farmers groups consisted of 80 farmers were randomly selected because these farmers had complete cattle age groups. from each of these farmers, five fecal cattle samples were collected including all calves in the selected farmers, while the remainder fecal samples were taken from weaners and adults. sample collection fecal samples of 20 50 g were collected from each age group. the feces was directly obtained from cattle's rectum or immediately collected after cattle defecation. fecal samples were stored in respective plastic bag and preserved in o refrigerator at 4 c before being tested in the laboratory for the occurrence of eimeria spp. oocysts. fecal consistency was assessed immediately after sampling and classified as normal or diarrheal without any additional differentiations (bangoura et al. 2011). the plastic bags containing fecal samples were labeled with sampling information such as sample number, sampling date, sampling location, cattle's age group, and farmer's name. practices of animal health management were recorded by the participating farmers in a questionnaire. in addition, record of cattle's sex and age (in months) were documented for each cattle from which a sample was taken. counting of poocysts er gram of feces (opg) fecal samples were examined and oocysts numbers were counted using the mcmaster method. two grams of fecal material was mixed thoroughly with 28 ml of sugar-salt solution and filtered through a 200 µm mesh wire sieve or tea strainer. the suspension was equally poured into two mcmaster counting chambers (2 x 0.15 ml) and was let still for 5 minutes. the counting was carried out using light microscope with 100x magnification. the numbers of oocysts in the two chambers were multiplied by dilution factor (50) to obtain the number of oocysts per gram of feces (opg). this protocol was modified from dong et al. (2012), lucas et al. (2006) and soulsby (1986). eimeria species identification the remainder of each positive samples were centrifuged at 1,500 rpm for 5 minutes at room temperature of 25 c for identification, a o . eimeria species composition and factors influencing oocysts shedding in dairy farm – sufi et al. 105 data analysis the effect of cattle's sex and age as well as type of pen flooring to eimeria opg counts were analyzed using mann-whitney and kruskalwallis tests. the kruskal-wallis test was performed followed by dunn test as multiple comparison test. results and discussion species identification and composition of eimeria. results of this study showed that the shedding of eimeria oocysts were commonly occurred in kpbs pangalengan dairy farm in bandung. from flotation of the oocysts in sugar-salt eimeria solution was performed. oocysts were measured under ocular eye piece that was calibrated with micrometer under 40x objective lense of a light microscope. identification of species was eimeria based on the morphological features of the oocysts (size, index, shape, color and texture of oocyst's wall, presence or absence of micropyle and polar cap) with the aid of taxonomic keys (soulsby 1986; daugschies & najdrowski 2005). measurement of oocysts index value were performed by dividing the length and width of oocysts. the shape of oocysts eimeria were examined for each sample and classified as round (length/width = 1), ovoid (length/width between 1 1.5) and ellips (length/width > 1.5) (cahyaningsih & supriyanto 2007). 106 biotropia vol. 24 no. 2, 2017 (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) 10 µm10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm 10 µm figure 1 the results of eimeria species oocysts in dairy cattle in kpbs pangalengan, bandung (a) eimeria bovis; (b) eimeria wyomingensis; (c) eimeria bukidnonensis; (d) eimeria pellita; (e) eimeria auburnensis; (f) eimeria zuernii; (g) eimeria cylindrica; (h) eimeria canadensis; (i) eimeria brasiliensis; (j) eimeria alabamensis 400 fecal samples, there were 179 fecal samples (44.8% prevalence; confidence interval (ci) 95%; 40.0 49.6 ) contained eimeria oocysts. a total of 10 eimeria species i.e. e. bovis, e. wyomingensis, e. bukidnonensis, e. pellita, e. auburnensis, e. zuernii, e. cylindrica, e. canadensis, e. brasiliensis and e. alabamensis were identified from the 179 positive fecal samples collected from kpbs pangalengan dairy farm (fig. 1, table 1, table 2). our study showed that e. bovis was the most prevalent species (42.5%; ci 95%: 37.1 47.8) and had the opg highest mean opg number (538.2 ( ocysts er ram of feces); ci 95%: 191.5 -o p g 884.8) followed by (39.1; ci 95%: e. wyomingensis 33.8 44.4 and 285.0; ci 95%: 136.0 434.0) and e. bukidnonensis (32.4; ci 95%: 27.3 37.5 and 237.1; ci 95%: 128.3 345.9) the . e. zuernii had second highest mean opg counts (509.7; ci 95%: 46.1 973.3). the lowest prevalent species was e. alabamensis (1.1; ci 95%: 0.0 2.3 and 75.0; ci 95%: 0.0 392.7) (table 3). a number of authors reported that e. bovis was the most prevalent species in cattle, but clinical coccidiosis was not observed in the calves or in adults cattle (kennedy & kralka 1987; lucas et al. 2006; heidari & gharekhani 2014). the results of those studies were in close agreement with the results of our study. according to waruiru et al. (2000), the mere present of pathogenic eimeria spp. did not necessarily indicate clinical disease. eimeria species were classified as highly pathogenic (e. bovis and e. zuernii), low pathogenic (e. ellipsoidalis, e. alabamensis, e auburnensis and e. subspherica) and non-pathogenic (e. brasiliensis, e. bukidnonensis, e. canadensis, e. cylindrica, e. pellita dan e. wyomingensis) (lassen & jarvis 2009). in other tropical area such as brazil, s several species of eimeria in cattle found were also ( floriao et al. 2015). e. bovis was recorded as the highest prevalen coccidian species which s in t wa accordance with reports of arslan and tuzer (1998), lucas et al. (2006) and abebe et al. (2008). 107 table 1 oocyst morphology of eimeria species of cattle has been identified in fecal samples obtained from kpbs pangalengan, bandung length (µm) width (µm) index form color other characteristics eimeria species 26.25-33.00 18.75-22.50 1.17-1.48 ovoid greenish-brown bilayered wall, micropyle e. bovis *29.77±1.37 *21.58±0.73 *1.38±0.05 37.50-43.50 26.25-30.75 1.22-1.49 ovoid greenish-brown single layered wall, micropyle e. wyomingensis *42.67±2.04 *29.58±1.20 *1.44±0.04 47.25-49.50 33.75-37.50 1.26-1.47 ovoid yellowish-brown bilayered wall, micropyle e. bukidnonensis *48.70±0.68 *35.72±1.88 *1.37±0.08 36.00-37.50 26.25-29.25 1.26-1.37 ovoid dark brown thick wall, micropyle e. pellita *37.43±0.28 *29.16±0.51 *1.28±0.02 37.50-45.75 22.50 1.67-2.03 ellips yellowish-brown bilayered wall, micropyle e. auburnensis *38.74±2.73 *22.50±0.00 *1.72±0.12 15.75-20.25 15.00-17.25 1.05-1.35 ovoid pale yellow thin wall, no micropyle e. zuernii *19.87±1.19 *16.65±1.01 *1.20±0.08 25.50-27.00 15.00 1.70-1.80 ellips pale yellow single layered wall, no micropyle e. cylindrica *25.93±0.73 *15.00±0.00 *1.73±0.05 30.00-33.75 25.50-26.25 1.14-1.32 ovoid yellowish bilayered wall, micropyle e. canadensis *30.42±1.25 *26.00±0.38 *1.17±0.06 41.25-42.75 25.50-26.25 1.60-1.63 ellips brownish-yellow bilayered wall, micropyle e. brasiliensis *41.50±0.53 *25.67±0.33 *1.62±0.007 15.00-18.75 11.25-15.00 1.25-1.33 ovoid pale yellow smooth wall, no micropyle e. alabamensis *16.88±2.65 *13.13±2.65 *1.29±0.06 note: * mean; ± standard deviation = = eimeria species composition and factors influencing oocysts shedding in dairy farm – sufi et al. 108 biotropia vol. 24 no. 2, 2017 t ab le o o cy st m o rp h o lo gy o f s p ec ie s o f ca tt le b as ed o n r ef er en ce s 2 e im er ia sp ec ie s a cc o rd in g to s o ul sb y (1 98 6) a cc o rd in g to d au gs ch ie s an d n aj dr ow sk i ( 20 05 ) l en gt h (µ m ) w id th (µ m ) in d ex f o rm c o lo r o th er ch ar ac te ri st ic s l en gt h (µ m ) w id th (µ m ) in de x f o rm c o lo r o th er ch ar ac te ri st ic s e . b ov is 23 -3 4 17 -2 3 1. 35 -1 .4 8 o vo id g re en is h b ro w n sm o o th w al l, m ic ro p yl e 23 -3 4 17 -2 3 1. 35 -1 .4 8 o vo id b ro w n is h ye llo w b ila ye re d w al l, m ic ro p yl e e . w yo m in ge ns is 37 -4 4. 9 26 .4 -3 0. 8 1. 40 -1 .4 6 o vo id g re en is h b ro w n m ic ro p yl e 37 -4 5 26 -3 1 1. 42 -1 .4 5 o vo id y el lo w is h b ro w n si n gl e la ye re d w al l, m ic ro p yl e e . b uk id no ne ns is 47 -5 0 33 -3 8 1. 32 -1 .4 2 o va l y el lo w is h b ro w n b ila ye re d w al l, m ic ro p yl e 47 -5 0 33 -3 8 1. 32 -1 .4 2 p ea rsh ap ed b ro w n is h ye llo w b ila ye re d w al l, m ic ro p yl e e . p el lit a 36 .1 -4 0. 9 26 .5 -3 0. 2 1. 35 -1 .3 6 e gg -s h ap ed d ar k b ro w n t h ic k w al l, m ic ro p yl e 36 -4 1 26 -3 0 1. 37 -1 .3 8 o vo id d ar k b ro w n t h ic k w al l, m ic ro p yl e e . a ub ur ne ns is 32 -4 6 20 -2 5 1. 60 -1 .8 4 e lli p s y el lo w is h b ro w n sm o o th w al l, m ic ro p yl e 32 -4 6 20 -2 5 1. 60 -1 .8 4 e lli p so id y el lo w is h b ro w n b ila ye re d w al l, m ic ro p yl e e . z ue rn ii 15 -2 2 13 -1 8 1. 15 -1 .2 2 su b sp h er ic al p al e ye llo w t h in w al l, n o m ic ro p yl e 15 -2 2 13 -1 8 1. 15 -1 .2 2 su b ov o id c o lo rl es s si n gl e la ye re d w al l, n o m ic ro p yl e e . c yl in dr ica 16 -2 7 12 -1 5 1. 67 -1 .8 0 c yl in dr ic al c o lo rl es s t h in w al l, n o m ic ro p yl e 16 -2 7 12 -1 5 1. 67 -1 .8 0 e lip so id c o lo rl es s si n gl e la ye re d w al l, n o m ic ro p yl e e . c an ad en si s 28 -3 7 20 -2 7 1. 37 -1 .4 0 o vo id / el lip s y el lo w is h b ro w n sm o o th w al l, m ic ro p yl e 28 -3 7 20 -2 7 1. 37 -1 .4 0 o vo id / el lip so id y el lo w is h b ila ye re d w al l, m ic ro p yl e e . b ra si lie ns is 34 .2 -4 2. 7 24 .2 -2 9. 9 1. 64 -1 .6 5 e lli p s y el lo w sm o o th w al l, m ic ro p yl e 34 -4 3 24 -3 0 1. 65 -1 .6 7 e lli p so id b ro w n is h ye llo w b ila ye re d w al l, m ic ro p yl e e . a la ba m en si s 13 -2 4 11 -1 6 1. 18 -1 .4 4 p ea rsh ap ed c o lo rl es s t h in w al l, n o m ic ro p yl e 13 -2 4 11 -1 6 1. 18 -1 .4 4 o vo id c o lo rl es s / p al e ye llo w t h in w al l, n o m ic ro p yl e according to daugschies najdrowski (2005), and e. bovis and e. zuernii are the most pathogenic of the bovine coccidia urthermore, e. alabamensis . f causes disease at extremely large infective doses (> 10 million oocysts) only. e. alabamensis usually induces watery diarrhea without blood, dehydration, depression and reduced growth , whereas infection remains subclinical under moderate infection pressure. single or mixed infection -species caused by different pecieseimeria s in this study, mixed-species infection experienced by a single cattle hosting several eimeria species were commonly observed. the number of eimeria species in single and mixedspecies infection per examined fecal sample ranged from 1 to 5. mixed-species infection caused by 2 to 5 eimeria species were found in 55.9% of cases (ci 95%; 50.5 61.3) the . remainder fecal samples had been infected with single infection of eimeria species (44.1%; ci 95%: 38.7 49.5) (table 4). thethis finding is similar to works of kennedy kralka (1987) in canada, which and reported 5 species. the results of mixedeimeria species we infection re lower than the observation of arslan and tuzer (1998) in turkey who reported 6 species, abebe . (2008) in eimeria et al ethiopia who reported 7 species yu eimeria , et al. (2011) in china who reported 10 eimeria species and dong (2012) who recorded et al. also in china 8 species. many previous studies indicated eimeria that under natural conditions, mixed-species infection re much more common than cases we single species infection (yu 2011; dong et al. et al. 2012). the level of pathogenicity of eimeria species increased higher level due to mixedto a species eimeria, infection with other pathogenic causing mortality in cattle (daughschies & najdrowski 2005). 109 table 3 eimeria species identified, the prevalence and mean opg counts in dairy cattle in kpbs pangalengan dairy farm, bandung species dairy cattle (n = 179) mean opg (ci 95%)positive no. mean prevalence (%) (ci 95%) e. bovis 76 42.5 (37.1 47.8) 538.2 (191.5 884.8) e. wyomingensis 70 39.1 (33.8 44.4) 285.0 (136.0 434.0) e. bukidnonensis 58 32.4 (27.3 37.5) 237.1 (128.3 345.9) e. pellita 47 26.3 (21.5 31.1) 177.7 (124.7 230.7) e. auburnensis 35 19.6 (15.2 23.9) 397.1 (0.0 820.9) e. zuernii 31 17.3 (13.2 21.4) 509.7 (46.1 973.3) e. cylindrica 7 3.9 (1.8 6.02) 78.6 (42.2 115.0) e. canadensis 7 3.9 (1.8 6.02) 121.4 (51.5 191.3) e. brasiliensis 6 3.4 (1.4 5.3) 125.0 (45.4 204.6) e. alabamensis 2 1.1 (0.0 2.3) 75.0 (0.0 392.7) table 4 single and mixed infection of eimeria species no. of eimeria spp. dairy cattle (n = 179) positive no. % of positive samples (ci 95%) 1 79 44.1 (38.7 49.5) 2 59 33.0 (27.8 38.1) 3 24 13.4 (9.7 17.1) 4 15 8.4 (5.4 11.4) 5 2 1.1 (0.0 2.3) total 179 100.0 eimeria species composition and factors influencing oocysts shedding in dairy farm – sufi et al. the effect of sex, age and type of cattle's pen flooring to opg countseimeria the effect of various categorical factors to opg counts were first analyzed with the mannwhitney and kruskal-wallis tests. a mannwhitney test was used to compare mean opg between 2 groups, whereas kruskal-wallis test was used to compare mean opg among 3 or more groups. the kruskal-wallis test was performed followed by dunn test as a multiple comparison test. a p value < 0.05 was required to indicate significance. several categorical factors (cattle's sex, age and type of pen flooring) and mean opg counts are presented in table 5. a highly significant ( = 0.000) effect was p observed between different sexes of cattle . intensity of infections were shown by eimeria mean opg found in cattle. this study showed that higher opg counts were found in male cattle (855.6 opg; ci 95%: 150.9 – 1 560.4) compared , with females (144.5 opg; ci 95%: 77.7 211.4). in this study, male cattle harbored more coccidia than female cattle. this situation might be caused by less care given to the male cattle as compared to the female cattle that were deemed to produce future cows. this result was consistent with reports from other researchers showing significant correlation ( < 0.05) between cattle's p sex and coccidiosis infection (heidari & gharekhani 2014). previous studies done on cattle reported higher prevalence of in female eimeria than in male cattle. nevertheless, this could be attributed to the greater physiological stress experienced by female cattle in relation to pregnancies and breeding as compared to male cattle (rehman 2011; dawid 2012; et al. et al. alemayehu 2013; heidari 2014).et al. et al. r is shed esults of th study showed that calves much higher numbers of oocyst than s those of weaners and adults. for instance, the mean opg for the calves was 537.0 (ci 95%; 231.8 842.2), whereas the weaners and adults mean opg for were 154.1 (ci 95%; 79.8 228.3) and 22.5 (ci 95%; 11.0 33.9), respectively. analysis showed that there were statistically significant differences in opg levels among different age groups ( <p 0.05). subsequently, dunn test as a multiple comparison test showed that the opg values for adult cattle were found to be significantly correlate ( < 0.05) the weaners and calves, d p to respectively. however, weaners the did not show significant effect ( > 0.05) the calves in opg p to levels caused by coccidiosis (table ).5 age is a major risk factor in coccidiosis spreading orbidity and risk of infection , while m s s are gr ater in calves (abebe 2008)e et al. compared to other age groups. calves had significantly higher oocyst counts ( < 0.05) than adultss p . these results were et in agreement with waruiru al. et al. (2000) in kenya and dong (2012) in shanghai, china who demonstrated that age strongly influence the intensity of opg d eimeria counts in cattle. coccidiosis is a self-limiting disease. spontaneus recovery without specific treatment is common when multiplication stage of the coccidia has passed, which suggests that previous exposure may have contributed to the 110 biotropia vol. 24 no. 2, 2017 table 5 results of mann-whitney and kruskal-wallis tests followed by dunn test toward several factors and mean opg counts categorical factors no. dairy cattle (n) mean opg (ci 95%) p value sex male 80 855.6 (150.9 – 1,560.4) 0.000* female 320 144.5 (77.7 211.4) age calves (< 6 months) 196 537.0a (231.8 842.2) 0.000* weaners (6-12 months) 37 154.1a (79.8 228.3) adults (> 12 months) 167 22.5b (11.0 33.9) type of pen flooring straw 13 53.9a (0.0 162.4) 0.000* wood 70 703.6b (0.0 – 1,450.2) rubber 96 405.7b (116.9 694.6) cement 221 116.7a (52.4 181.1) development of a certain immunity level in the older cattle as compared to the younger ones that did not have previous exposure (dong 2012; et al. heidari 2014; kocis 2015).et al. et al. this study showed that w eimeriaeaners shed oocysts opg counts of 154.1 (ci 95%; 79.8with 228.3). ean opg the weaners re lower m for we than the calves ( > 0.05). in tudy, that for p this s range of were 0 opg in calves and weaners 24 450 and 0 900, respectively. opg values over , 5 000 indicate a clinical case (arslan tuzer , & 1998). esults of oocysts counts calves r eimeria for and weaners in were not different from this study previous bruhn (2012) wh study by et al. ich reported that no difference was observed ( >p 0.05) in epg and opg counts between calves in the pre-weaning and post-weaning phases. most cattle examined during this study had low opg counts, suggesting that the infections were usually subclinical. this result concurred with other cross-sectional observational studies on eimeria spp. in iran, which also did not observe cases of clinical cases of clinical coccidiosis among infected cattle, probably due to low quantities of oocysts eliminated in the cattle's feces (heidari 2014; heidari & gharekhani et al. 2014). l eimeriaevel of oocyst shedding was significant depende on different types of pen ly d flooring ( < 0.05). when the types of flooring p were compared, on straw floorcattle kept ing exhibited lower opg values (53.9; ci 95%: 0.0 162.4) than on wood those kept on flooring (703.6; ci 95%: 0.0 – 1 450.2) and rubber , on floor (405.7; ci 95%: 116.9 694.6) ( < 0.05). ing p however, straw floor significanting did not show difference on p compared to opg counts ( > 0.05) cement floor . moreover, wood floor ing ing did not show difference on significant opg counts compared to ing p rubber floor ( > 0.05). yet, eimeria p on opg counts was higher ( < 0.05) wood floor compared to cement floor . ing ing higher oocysts count was recorded in eimeria cattle ing reared on rubber floor compared to cement floor ( < 0.05) (table ).ing p 5 this finding was in agreement with a study by bangoura et al. (2011) who described a significant association between different flooring types used for rearing cattle and the level of eimeria oocysts shedding ( < 0.05). rehman et al. (2011) stated p that eimeria infection was more prevalent ( < 0.05) in non-cement flooring type compared p to partially cement flooring type. lower eimeria prevalence in cattle kept on cement flooring may be caused by the easiness of cleaning and disinfecting cement flooring, which resulted to less contaminated floor compared to non-cement floor. in this study the eimeria, lowest level of oocysts in cattle kept on straw occurred flooring than those kept on other flooring types, because solid floor (wood, rubber and cement ing types ) are ier the solid flooring dirt than straw floors if types are s not scraped and cleaned properly. traw floor type usually used by calves ing is temporarily aged 0 to 1 month to keep warm. the calves daily r al ement of ingemov and replac straw floor with a new straw resulted in lower opg set of s eimeria counts occurence of and reduced the eimeria oocyst shedding. revious studies concluded that p cleaning s an important factor in preventing wa high of oocysts (bangoura counts eimeria et al. 2011; rehman 2011).et al. was cattle clinical coccidiosis observed in pens having leadinginsufficient aeration to high concentration of ammonia, co and moisture. 2 also, clinical coccidiosis occurred in cattle pens where feces accumulated on the ground due to unsuited slatted floors (daughschies & najdrowski 2005). local temperature, humidity, ammonia concentration and ph on pen flooring affected ' et al. oocysts survival (gulliksen 2009; bangoura 2011).et al. in having intensive rearing high population density, disease transmission more readilyis occurred and oocysts are highly available within the environment. thus, in dairy calves, coccidiosis occurs more frequently and appears with greater severity (bruhn 2011). it can be et al. assumed that the in a group will be cattle living infected at the same time, or at around the same time, under field condition. moreover, continuous oocysts shed from subclinical ding infected calves contaminate the environment and the caus severe coccidiosis in cattle's hair ing highly susceptible new calves that are kept in these areas (abebe 2008; alemayehu 2013). et al. et al. different hygiene conditions and farm management, breeding, study design and animal s methods, climates and different geographical regions may be the main cause of varied results (yu 2011)et al. . 111 eimeria species composition and factors influencing oocysts shedding in dairy farm – sufi et al. conclusions e. bovis was the highest prevalent eimeria species and had the highest level of mean opg compared to other eimeria spesies. mixed infections in single cattle with 2 – 5 eimeria species were commonly observed. several species of eimeria in cattle are found in other tropical areas. various categorical factors (cattle's sex, age and type of pen flooring) influenced the number of opg found in fecal samples. eimeria has significant pathogenic potential. it is important to control the occurrence of eimeria. acknowledgements the authors thank all members of protozoology laboratory, faculty of veterinary medicine, institut pertanian bogor for facilitating this study with the necessary equipment. references abebe r, wossene a, kumsa b. 2008. epidemiology of eimeria infections in calves in addis ababa and debre zeit dairy farms, ethiopia. intern j appl res vet med 6(1):24-30. 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[retrieved on 18 may 2015]. yu sk, gao m, huang n, jia yq, lin q. 2011. prevalence of occidial nfection in attle in shaanxi province, c i c northwestern china. j an vet adv 10(20):2716-9. [internet] . docsdrive.com/pdfs/ http:// medwelljournals/javaa/2011/2716-2719.pdf. doi: 10.3923/javaa.2011.2716.2719. [retrieved on 15 february 2015]. 113 eimeria species composition and factors influencing oocysts shedding in dairy farm – sufi et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 3. etty riani.cdr page 1 page 2 page 3 page 4 page 5 6. alex (the spread).cdr 5. okky (the occurence) rev.cdr biotropia vol. 18 no. 2, 2011: 102 122 the occurrence of insects and fungi, and aflatoxin b contamination of stored sorghum in demak and wonogiri regencies, central java 1 okky setyawati dharmaputra , santi ambarwati , and ina retnowati recipient of biotrop research grant 2010/accepted 25 november 2011 the objectives of this study were to collect informations on the method of postharvest handling of sorghum and to investigate the moisture contents, insects infestation, fungal infection, and aflatoxin b contents of stored sorghum grains collected from various stages of the delivery chain in demak and wonogiri regencies, central java. in demak regency sorghum cultivation was monoculture, variety cultivated was upc-s1. in wonogiri regency sorghum cultivation was intercropping with secondary crop and cassava. sorghum varieties cultivated were kawali, numbu, zh30, mandau and hibrida hybrids. there was a difference between the method of postharvest handling of sorghum at farmer and collector levels in demak and wonogiri regencies. in general the method of postharvest handling of sorghum in demak regency was more appropriate and more advance compared to that in wonogiri regency. the moisture contents of sorghum at farmer as well as at collector level in demak regency (13.0%) and wonogiri regency (12.9%) were still lower that that of normal (safe) moisture content of sorghum. the number of insect species associated with sorghum in various distribution level in demak and wonogiri regencies was 10 and 17 species, respectively. the dominant insects species were and . the number of fungal species found in sorghum at various distribution level in demak and wonogiri regencies was 23 species, respectively. in general, the dominant fungal species were , and . in demak regency aflatoxin b contents of sorghum at farmer and collector levels were 22.50 and 15.45 ppb, respectively, while in wonogiri regency 2.27 and 10.28 ppb, respectively. insects, fungi, aflatoxin b , stored sorghum, demak and wonogiri regencies, central java 1,2 2 1 1 2 seameo biotrop, jl. raya tajur km. 6, po box 116, bogor16134, indonesia department of biology, faculty of mathematics and natural sciences, bogor agricultural university, darmaga campus, bogor 16680, indonesia sitophilus zeamais tribolium castaneum aspergillus flavus fusarium semitectum f. verticillioides abstract 1 1 1key words: 102 introduction a . t e : ● t m ● t . i s a foodstuff, sorghum ( (l) moench) is the fifth most important cereal after rice, wheat, maize and barley in the world. in many countries, sorghum is used as food and feedstuff, industrial raw materials such as for ethanol, beer, wine, syrup, glue, paint and modification of starch. compared to other foodcrops, sorghum can adapt to broad agroecology, resistant to dryness, high productivity, and more resistant to pests and diseases. apart from that, sorghum has nutritional content that is almost the same with milled rice as well as maize, consequently sorghum is good to be consumed either as alternative food or feedstuff. according to suarni (2001) protein contents in sorghum, milled rice and maize were 10.11, 9.28 and 11.02%, respectively; their lipid contents were 3.65, 1.88 and 5.42%, respectively; their carbohydrate contents were 80.42, 86.45 and 79.95%, respectively. although in indonesia the production of sorghum is still low, it is potential to be cultivated and developed, especially in marginal and dry areas (bp2aptp 2008). to anticipate food crisis caused by global warming, it is important to cultivate and to develop sorghum in indonesia. during storage grains could be infested by insects, microorganisms, mites and rats. insects are considered as the most significant cause of losses. among microorganisms, fungi are the most important cause of deterioration of stored grains. the role of insects on fungal infection cannot be disregarded. aside from injuring the grains, insects also serve as carriers of fungi. furthermore, the metabolic activities of insects produce enough heat and moisture (especially during long-term storage) which stimulate fungal growth. fungal infection in grains can cause discolouration, decrease in physical quality and nutritional contents, and mycotoxin contamination (sauer . 1992). aflatoxins are the most dangerous mycotoxins, they can cause liver cancer in human and animal. there are four kinds of aflatoxins found in food and feedstuff, i.e. aflatoxins b , b , g and g . the most toxic of the four kinds of aflatoxins is aflatoxin b (afb1) he objectives of this study wer o obtain informations on postharvest handling methods of sorghu o investigate the degree of insect and fungal attacks, and afb1 contamination of stored sorghum collected from various stages of the delivery chain in demak and wonogiri regencies, central java. the moisture contents of sorghum were also determined, because moisture content is the important environmental factor for the development of insects and fungi n demak regency, surveys and sampling of sorghum were conducted in july and august 2010, while for wonogiri regency in april and july 2010. in demak regency (demak and mijen subdistricts), surveys and sampling of sorghum were conducted at farmer and collector levels and a traditional market in the city of demak. in ime and location of survey sorghum bicolor et al 1 2 1 2 1 materials and methods t s 103 occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al biotropia vol. 18 no. 2, 2011 104 wonogiri regency (purwantoro, eromoko and pracimantoro subdistricts), surveys and sampling of sorghum were conducted at farmer level and collector level in pracimantoro subdistrict. in demak regency (demak and mijen subdistricts), surveys and sampling of sorghum were conducted in july and august 2010 at farmer and collector levels and a traditional market in the city of demak. in wonogiri regency (purwantoro, eromoko and pracimantoro subdistricts) surveys and sampling of sorghum were conducted during april and july 2010 at farmer level and at collector level in pracimantoro subdistrict nterviews were conducted during the surveys and sampling to collect information on postharvest handling of sorghum at farmer and collector levels. number of respondents from each level distribution chain in each regency could be different depending on the field conditions during the surveys. the questionnaires contained among others questions on postharvest handling carried out by farmers and collectors and problems encountered by them he number of sorghum samples collected from regency was 46 with the breakdown as follows : farmers (34), collectors (9) and retailers in traditional market (3). sorghum samples collected from regency was 38 consisting of samples from farmers (23) and collectors (15) (table 1). the samples were collected from places where the respondents stored sorghum at the time of the interview. the number of sorghum samples in each level distribution chain was determined based on the existence of sorghum at the time of interviews. as much as 2 kg of each sample was collected randomly from each respondent, it was then placed in a clean plastic bag. insects from each sample were separated using graded sieves, then they were preserved in vials containing ethanol 70%. . i . t i s s s nterviews using questionnaire ampling and method to obtain working sample demak wonogiri table 1. level of distribution chain, subdistrict origin of the sample, and the number of sorghum samples at various stages of the delivery chain in demak and wonogiri regenciess regency level of distribution chain subdistrict origin of the sample number of samples demak farmer demak and mijen 34 collector demak and mijen 9 retailer at traditional market demak 3 wonogiri farmer eromoko 13 pracimantoro 8 purwantoro 2 collector eromoko 1 pracimantoro 14 total 84 105 each sample was then divided three times using a sample box divider to obtain eight working samples for the determination of the percentage of sorghum grains infected by each fungal species, afb1 content, and a reserve sample oisture contents of sorghum grains were determined right after sample collections using moisture meter delmhorst model g-7. two replicates were used from each sample. insect species was identified using the publication of haines (1991) as the main reference. the degree of insect species infestation in each sample was determined based on its number per kg of sorghum. fungi were isolated using a plating method on dichloran 18% glycerol agar (dg18) (hocking and pitt 1980). two hundred sorghum grains were used from each sample. fungal species was identified using the publication of pitt and hocking (2009) as the main reference. in this study only afb1 content was determined, because it is the most dangerous aflatoxin compared to the other three kinds of aflatoxins (afb2, afg1 and afg2). afb1 content was determined according to thin layer chromatography method (aoac 2005). two replicates were used from each sample he results of interviews with farmers and collectors in demak regency are presented in tables 2 and 3. at level the method of sorghum cultivation was monoculture. the variety of sorghum cultivated was upc-s1. the range of land area for sorghum cultivation was 0.25-2 ha. sorghum was harvested three months after planting. the method of harvesting was by cutting its panicle using a sickle. the panicles of sorghum were sun-dried on tarpaulin or cement floor. in general sorghum grains were separated from panicles using a paddy thresher. most of sorghum was sold to collectors, although a small part was used as seeds to be replanted. sorghum was packed in polypropylene bag. in general storage duration of sorghum collected as samples was between 0-7 days. the condition of storage was dirty. no insects and fungi were found in sorghum samples, because the duration of storage was relatively short. the variety of sorghum bought by was upc-s1. sorghum was bought from farmers, collectors at village level, or from farmers and collectors at village/ subdistrict levels. if they were still not dry enough, further sun-drying was conducted on cement floor. the grains were separated from panicles using a paddy thresher and packed in polypropylene bags. sorghum samples have been stored for 0-7 days (66% of respondents) and 10-15 days (34% of respondents). the condition of storage (warehouse) was appropiate (78% of respondents) and not so appropriate (22% of respondents). sorghum was sold to big traders in wonosari (44% of . m . t d t interviews using questionnaires etermination of moisture content, insect infestation, fungal infection, and aflatoxin b conten farmer collector 1 results and discussion interviews using questionnaires in demak regency occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al 106 biotropia vol. 18 no. 2, 2011 table 2. results of interviews with on postharvest handling of sorghum in demak and wonogiri regencies farmers no. subject % respondent*) demak wonogiri 1. methods of sorghum cultivation: a. intercrop with secondary crop and cassava b. intercrop with secondary crop and cassava, and the use of fertilizer c. monoculture 0 0 100 57 43 0 2. variety of sorghum cultivated: a. kawali, numbu and zh 30 (white colour of grains) b. mandau and hibrida (red and reddish brown of grains) c. upc-s1 0 0 100 39 61 0 3. land area: a. 0.25 ha b. 0.25 ha c. 1.00 ha d. 1.50 ha e. 2.00 ha 29 44 15 3 9 48 13 39 0 0 4. harvesting time (days after planting): a. < 90 days b. 90 days 0 100 0 100 5. method of harvesting: a. by cutting the stalk of panicle using a sickle b. other method 100 0 100 0 6. method of drying(in the form of panicle): a. sun-drying on tarpaulin or cement floor b. hanging on racks c. inserted on wood located outside of the wall of the house made from zinc 100 0 0 83 13 4 7. method of threshing: a. manually b. beaten using a coconut leaf c. beaten using a of wood/bamboopiece d. beaten using a hammer e. beaten using a piece of stalk of tree f. using a paddy thresher 0 0 9 0 0 91 4 4 74 9 9 0 8. harvested sorghum were: a. sold to collector b. sold to collector and seed seller c. usedby farmers as cattle/chicken feed d. used by farmers as seeds and cattle feed e. b,d and consumed by farmers (by steaming) 0 100 0 0 0 30 57 5 4 4 9. method of storage: a. stored in plastic (polypropylene) bag b. spread out on tarpaulin 100 0 87 13 10. duration of storage of sorghum collected as sample: a. 0 -7 days b. 21 days c. 1 month d. 2 months e. 3 months 94 0 0 3 3 9 18 64 9 0 11. sanitation of storage: a. unappropriate (dirty) b. appropriate 100 0 100 0 107 respondents), big traders in other big cities (kudus, solo, yogyakarta, demak and bantul) (34% of respondents), and entrepreneur of oyster mushroom cultivation (22% of respondents) he results of interviews with farmers and collectors in wonogiri regency are presented in tables 2 and 3. in this regency, at level the method of sorghum cultivation was intercropping with cassava (57% of respondents); intercropping with secondary crop and cassava, and using fertilizer (43% of respondents). the sorghum varieties cultivated were kawali, numbu and zh 30 (zh-30 is a mutant line from batan), the colour of their grains were white (39% of respondents); while for mandau and hibrida varieties , the colour of their grains were red and brownishred, respectively (61% of respondents). the range of land area for sorghum cultivation was 0.25-1 ha. sorghum was harvested three months after planting, while the method of harvesting was by cutting its panicle using a sickle. in general, the panicles of sorghum were sun-dried on tarpaulin or cement floor. the grains were separated from panicles manually (4% of respondents) or beaten using a coconut leaf (4% of respondents), a stick of wood or bamboo (74% of respondents), a hammer (9% of respondents), and a piece of tree stalk (9% of respondents). sorghum was sold to collectors (30% of respondents), to collectors and for seeds to be replanted (57% of respondents), for feed (cattle or chicken) (5% of respondents), for seeds and cattle feed (4% of respondents); was sold to collectors, for seeds and consumption by steaming (4% of respondents). sorghum was packed in polypropylene bag (87% of respondents) and was spread out on tarpaulin (13% of respondents). the storage duration of collected sorghum for samples was 0-2 months. in general, the storage condition was dirty. during storage, insects were found (35% of respondents), while no fungi were found. the variety of sorghum bought by was kawali and numbu (53% of respondents), mandau hibrida varieties (47% of respondents). sorghum was bought from farmers (65% of respondents), from collectors at village level (12% of respondents), from farmers and collectors at village/subdistricts level (23% of respondents). panicles of sorghum were bought after sun-dried conducted by farmers (6% of respondents), after sun-dried by farmers in the form of grains (47% of respondents), after sun-dried by collector at village level in the form of panicle or grain . t interviews using questionnaires in wonogiri regency farmer collector table 2. continued no. subject % respondent*) demak wonogiri 12. insect pest was found in sorghum during storage: a. yes b. no 0 100 9 91 13. based on visual observation, fungi were found in sorghum during storage: a. yes b. no 0 100 0 100 *) number of respondent in demak regency : 34 number of respondent in wonogiri regency : 23 occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al (47% of respondents). if sorghum bought from farmers were not yet properly dried, further sun-drying was carried out on cement floor. grains were separated from panicles using a paddy thresher and packed in polypropylene bag. the storage duration of sorghum samples was 0-7 days (59% of respondents), 1 month (29% of respondents), and 3 months (12% of respondents). in general, the condition of storage was not so appropriate. most of sorghum was sold to big traders in wonosari (71% of respondents). table 3. results of interviews with on post-harvest handling of sorghum in demak and wonogiri regencies collectors no. subject % respondent*) demak wonogiri 1. variety of sorghum bought: a. kawali and numbu (white colour of grains) b. mandau and hibrida (red and reddish brown of grains) c. upc-s1 0 0 100 53 47 0 2. sorghum was bought from: a. farmers b. from collectors at village level c. from farmers and collectors at village/subdistrict level 44 34 22 65 12 23 3. sorghum was bought: a. after being dried by farmer, in the form of panicle b. after being dried by farmer, in the form of grain c. after being dried by collector at village level, in the form of panicle or grain 0 100 0 6 47 47 4. method of drying of sorghum (not properly dried) bought from farmer : a. sun-drying on cement floor b. other method 100 0 100 0 5. method of threshing : a. using a paddy threshing b. other method 100 0 100 0 6. method of storage: a. stored in plastic (polypropylene) bag b. other method 100 0 100 0 7. duration of storage of sorghum collected as samples: a. 0 – 7 days b. 10 15 days c. 1 month d. 3 months 66 34 0 0 59 0 29 12 8. conditon of the storage(warehouse): a. not so appropriate b. appropriate 22 78 82 18 9. sorghum was sold to: a. big traders in wonosari b. big trader s in other big cities (kudus, solo, yogyakarta, demak, and bantul) c. entrepreneur of oyster mushroom cultivation 44 34 22 71 24 6 *) number of respondent in demak regency: 9 number of respondent in wonogiri regency: 15 biotropia vol. 18 no. 2, 2011 108 moisture content t . he range and mean of moisture contents of sorghum collected from farmers and collectors in demak and wonogiri regencies are presented in table 4. in demak and wonogiri regencies the mean moisture contents of sorghum collected from farmers were almost the same with those collected from collectors, i.e. 13.0 and 12.9%, respectively, because in general the storage period of sorghum at farmer level was relatively short. the moisture contents of sorghum collected from retailers at traditional market in demak regency were lower than those collected from farmers as well as collectors. the moisture contents of grains were always in equilibrium with the relative humidity of the storage. the moisture content will increase with the increase of the relative humidity, consequently fungal growth will be stimulated. in this study the mean moisture contents of sorghum at farmer and collector levels were still lower than the normal moisture content of sorghum. christensen . (1990) reported, that the normal moisture content of sorghum with superior and second qualities were 13 and 14%, respectively et al table 4. range and mean of moisture content of sorghum collected from farmers, collectors and retailers at traditional market in demak and wonogiri regencies regency level of distribution chain range (mean) of moisture content (% wet basis) demak farmer 10.7 – 14.2 (13.0) collector 12.5 – 13.8 (13.0) retailer at traditional market 12.0 – 12.6 (12.2) wonogiri farmer 11.9 – 13.8 (12.9) collector 11.9 – 14.2 (12.9) insect infestation insect infestation of sorghum in demak regency insect infestation of sorghum in wonogiri regency t . t he total number of insect species associated with sorghum at farmers, collectors and retailers at traditional market was 10 species (table 5). the insect species belong to order coleoptera (8 species), order hymenoptera (1 species) and order lepidoptera (1 species). most of the insect species were primary and secondary insect pest, and one species was a parasit insect. and were the dominant insects found (fig. 1). was only found in sorghum at retailer in traditional market and it was the dominant insect he total number of insect species associated with sorghum at farmer and collector levels was 17 species (table 6). the insects species belong to order coleoptera (10 species), order lepidoptera (3 species), order hymenoptera (2 species), order hemiptera (1 species) and order psocoptera (1 species). most of the species was primary and secondary insect pests, one species as a predator, and another one was a parasitic insect. s , and were the dominant insect species found (fig. 2). another insect found in some sorghum samples was , a parasite of egg sitophilus zeamais tribolium castaneum oryzaephilus surinamensis itophilus zeamais tribolium castaneum sitotroga cerealella anisoptermalus calandrae sitophilus zeamais . 109 occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al 0 20 40 60 80 100 120 1 2 3 4 5 6 7 8 9 10 fl cl l rl fl = farmer level; cl = collector level; rl = retailer level 1. 6. 2. sp. 7. 3. sp. 8. 4. 9. 5. 10. figure 1. percentage of sorghum samples infested by insects at farmer, collector and retailer levels in demak regency ahasverus advena sitophilus zeamais carpophilus tribolium castaneum cryptolestes typhaea stercorea oryzaephilus surinamensis anisoptermalus calandrae rhyzopertha dominica ephestia cautella fl = farmer level; cl = collector level 1. 7. 13. ants 2. 8. 14. 3. sp. 9. 15. 4. sp. 10. 16. 5. 11. 17. 6. 12. figure 2. percentage of sorghum samples infested by insects at farmer and collector levels in wonogiri regency ahasverus advena sitophilus zeamais araecerus fasciculatus thaneroclerus buqueti corcyra cephalonica carpohilus tribolium castaneum ephestia cautella cryptolestes typhaea stercorea sitotroga cerealella dinoderus minutes xylocoris flavipes liposcelis entomophilus rhyzopertha dominica anisoptermalus calandrae biotropia vol. 18 no. 2, 2011 110 t ab le 5. sp ec ie s an d n u m b er o f in se ct s in so rg h u m at fa rm er s, co lle ct o rs an d re ta ile rs in tr ad it io n al m ar ke t i n re ge n cy d em ak n o in se ct st at u s n u m b er (% ) o f sa m p le s in fe st ed b y in se ct m ea n av er ag e o f n u m b er in se ct s / kg sa m p le f ar m er c o lle ct o r r et ai le r at tr ad it io n al m ar ke t f ar m er c o lle ct o r r et ai le r at tr ad it io n al m ar ke t l ar va im ag o l ar va im ag o l ar va im ag o c o l e o p t e r a 1 a ha sv er us ad ve na (w al tl ) p es t 0 0 1 (3 3. 33 ) 0 0 0 0 0 1. 47 2 c ar po ph ilu s sp . p es t 3 (8 .8 2) 2 (2 2. 22 ) 2 (6 6. 67 ) 01. 83 (0 .6 1) 00. 53 (0 .3 5) 0 0. 52 -1 .3 9 (0 .9 6) 0 0. 49 -3 .9 2 (2 .2 1) 3 c ry pt ol es te s sp . p es t 2 (5 .8 8) 2 (2 2. 22 ) 1 (3 3. 33 ) 0 8. 65 -3 6. 17 (2 2. 41 ) 0. 47 -0 .5 4 (0 .5 1) 0 0. 49 1. 47 4 o ry za ep hi lu s su ri na m en si s (l in n ae u s) p es t 0 0 2 (6 6. 67 ) 0 0 0 0 0 0. 98 (0 .9 8) 5 r hy zo pe rt ha do m in ic a (f ab ri ch iu s) p es t 2 (5 .8 8) 0 0 0 2. 88 -7 .4 5 (5 .1 7) 0 0 0 0 6 s it op hi lu s z e am ai s m o ts ch u ls ky p es t 6 (1 7. 65 ) 1 (1 1. 11 ) 3 (1 00 .0 ) 0 0. 43 -3 89 .3 6 (1 11 .7 6) 0 3. 23 0 0. 49 -2 1. 08 (8 .5 0) 7 t ri bo li um ca st an eu m (h er b st ) p es t 15 (4 4. 12 ) 6 (6 6. 67 ) 1 (3 3. 33 ) 012 .6 4 (2 .4 0) 02. 29 (0 .3 2) 06. 36 (1 .6 9) 07. 69 (1 .3 6) 0 0. 49 8 t yp ha ea st er co re a (l in n ae u s) p es t 2 (5 .8 8) 2 (2 2. 22 ) 0 0 0. 51 -0 .5 3 (0 .5 2) 0 0. 47 -1 .2 8 (0 .8 8) 0 0 h y m e n o p t e r a 9 a ni so pt er m al us ca la nd ra e (h o w ar d ) p ar as it e 2 (5 .8 8) 0 1 (3 3. 33 ) 0 52 .1 355 .7 7 (5 3. 95 ) 0 0 0 0. 98 l e p id o p t e r a 10 e ph es ti a ca ut el la (w al ke r) p es t 0 3 (3 3. 33 ) 1 (3 3. 33 ) 0 0 0. 45 -0 .8 5 (0 .5 9) 0 0. 49 0 111 occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al t ab le 6. sp ec ie s an d n u m b er o f in se ct s in so rg h u m at fa rm er s an d co lle ct o rs in re ge n cy w on og ir i n o in se ct st at u s n u m b er (% ) o f sa m p le s in fe st ed b y in se ct m ea n av er ag e o f n u m b er in se ct s / kg sa m p le f ar m er c o lle ct o r f ar m er c o lle ct o r l ar va im ag o l ar va im ag o c o l e o p t e r a 1 a ha sv er us ad ve na (w al tl ) p es t 0 1 (6 .6 7) 0 0 0 0. 56 2 a ra ec er us fa sc ic ul at us (d eg ee r) p es t 3 (1 3. 04 ) 5 (3 3. 33 ) 0 0. 42 -0 .8 1 (0 .6 4) 010 .2 6 (2 .2 0) 0. 58. 82 (3 .2 9) 3 c ar po ph ilu s sp . p es t 2 (8 .7 0) 3 (2 0. 0) 0 0. 42 -0 .8 1 (0 .6 2) 0 0. 39 -0 .5 2 (0 .4 4) 4 c ry pt ol es te s sp . p es t 5 (2 1. 74 ) 2 (1 3. 33 ) 012 .0 8 (2 .4 2) 03. 14 (1 .7 8) 00. 63 (0 .3 2) 01. 01 (0 .5 1) 5 d in od er us m in ut us (f ab ri ci u s) p es t 1 (4 .3 5) 1 (6 .6 7) 0 0. 36 0 0. 63 6 r hy zo pe rt ha do m in ic a (f ab ri ch iu s) p es t 3 (1 3. 04 ) 2 (1 3. 33 ) 0 0. 36 -2 .7 3 (1 .3 0) 0 0. 51. 25 (0 .8 8) 7 s it op hi lu s z e am ai s m o ts ch u ls k y p es t 15 (6 5. 22 ) 11 (7 3. 33 ) 025 .5 3 (3 .0 9) 034 .6 8 (6 .0 2) 03. 53 (0 .6 5) 026 .5 0 (6 .5 8) 8 t ha ne ro cl er us bu qu et i( l ef ev re ) p es t 0 1 (6 .6 7) 0 0 0 0. 52 9 t ri bo liu m ca st an eu m (h er b st ) p es t 14 (6 0. 87 ) 6 (4 0. 0) 05. 12 (1 .3 2) 02. 05 (0 .2 7) 03. 75 (1 .1 8) 01. 0 (0 .1 7) 10 t yp ha ea st er co re a (l in n ae u s) p es t 0 2 (1 3. 33 ) 0 0 0 0. 52 -0 .6 3 (0 .5 8) h e m ip t e r a 11 x yl oc or is fla vi pe s (r eu te r) p re d at o r 1 (4 .3 5) 1 (6 .6 7) 0 0. 36 0 0. 50 h y m e n o p t e r a 12 a ni so pt er m al us ca la nd ra e (h o w ar d ) p ar as it e 4 (1 7. 39 ) 0 0 0. 68 -2 .4 2 (1 .6 5) 0 0 13 s em ut p es t 1 (4 .3 5) 0 0 30 .2 6 0 0 l e p id o p t e r a 14 c or cy ra ce ph al on ic a (s ta in to n ) p es t 1 (4 .3 5) 0 3. 15 0 0 0 15 e ph es ti a ca ut el la (w al ke r) p es t 5 (2 1. 74 ) 2 (1 3. 33 ) 0. 42 -2 .2 2 (0 .9 8) 0 0. 50. 74 (0 .6 2) 0 16 s it ot ro ga ce re al el la (o liv ie r) p es t 10 (4 3. 48 ) 4 (2 6. 67 ) 017 .1 4 (2 .8 9) 012 .3 8 (2 .6 1) 0 0. 63 -3 .0 (1 .3 1) p so c o p t e r a 17 l ip os ce lis en to m op hi lu s (e n d er le in ) p es t 1 (4 .3 5) 1 (6 .6 7) 0 0. 61 0 0. 39 biotropia vol. 18 no. 2, 2011 112 sitophilus zeamais sitotroga cerealella araecerus fasciculatus, thaneroclerus buqueti, dinoderus minutus, ephestia cautella typhaea stercorea carpophilus ahasverus advena sitophilus zeamais cryptolestes ferrugineus latheticca oryzae liposcelis bostrychophilus oryzaephilus surinamensis plodia interpunctella rhyzopertha dominica sitophilus zeamais tribolium castaneum cryptolestes ferrugineus r. dominica et al aspergillus candidus a. flavus a. niger a. tamarii a. wentii cladosporium cladosporioides colletotrichum curvularia lunata c. pallescens endomyces fibuliger eurotium chevalieri e. repens e. rubrum fusarium proliferatum f. semitectum f. vercillioides lasiodiplodia theobromae penicillium citrinum pestalotiopsis guepinii cladosporium cladosporioides curvularia lunata c. pallescens fusarium proliferatum f. semitectum f. vercillioides lasiodiplodia theobromae pestalotiopsis guepinii a. flavus a. niger a. tamarii a. wentii colletotrichum curvularia lunata c. pallescens endomyces fibuliger eurotium chevalieri e. repens, e. rubrum f. proliferatum f. semitectum f. vercillioides lasiodiplodia theobromae penicillium citrinum pestalotiopsis guepinii and were the main insect pest in sorghum, while the other insect species were also the main insect pest of other foodstuff in storage. most collectors stored more than one kind of commodity, consequently some insect species found in other commodities were also found in sorghum. they were and . these insects were generally found in other commodities, such as milled rice, maize and mungbean. the existence of these insects was probably due to migration from one commodity to the other kinds of commodities. in general the number of insect species found in samples collected from collectors who stored sorghum for a longer period was relatively higher than collected from farmer. aside from sorghum, they also stored various agricultural commodities. other insects found in sorghum were sp. and . these insect species were not important, but the existence of these insects gave an indication, that the moisture content of sorghum was high, consequently sorghum could be easily infected by fungi. based on the result of inventory of insect species associated with stored sorghum at farmer and collector levels, was almost always found in all samples. as much as eight insect species was generally found in stored sorghum in taiwan, i.e. , , , , , , and . the dominant insects were and (peng 1998). according to mendesil . (2007) in ethiopia most farmers estimated the loss of stored sorghum caused by insect was up to 50%. insect infestation in sorghum was due to high temperature and unappropriate sanitation of the storage ercentage of sorghum grain infected by fungi in demak regency is presented in table 7. twenty three fungal species were isolated from sorghum collected from , i.e. , , , , , , sp., , , , , , , , , , , , , isolates 3, 7, 8 and 10. eight out of 23 fungal species belong to field fungi, i.e. , , , , , , , and . the existence of these fungi was probably due to optimum temperature or relative humidity for fungal growth before or after harvest. twenty fungal species were isolated from sorghum collected from , i.e. , , , , sp., , , , , , , , , , , , isolates 3, 8 and 10. field fungi were also still found in sorghum collected from collectors, because at farmer level sorghum was stored in a short period. it was also assumed, that the condition of storage was suitable for fungal growth. . p fungal infection fungal infection of sorghum in demak regency farmer collector 113 occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al t ab le 7. f u n ga l i n fe ct io n in so rg h u m co lle ct ed fr o m fa rm er s, co lle ct o rs an d re ta ile rs at tr ad it io n al m ar ke t in re ge n cy d em ak n o f u n gi n u m b er (% ) o f sa m p le s in fe ct ed b y fu n gi r an ge (m ea n ) p er ce n ta ge o f so rg h u m in fe ct ed b y fu n gi in in fe ct ed sa m p le s f ar m er c o lle ct o r r et ai le r at tr ad it io n al m ar ke t f ar m er c o lle ct o r r et ai le r at tr ad it io n al m ar ke t 1 a sp er gi llu s ca nd id us 2 (5 .8 8) 0 (0 .0 0) 1 (3 3. 33 ) 1. 02. 5 (1 .7 5) 0 1. 50 2 a . fla vu s 30 (8 8. 24 ) 9 (1 00 .0 0) 3 (1 00 .0 0) 0. 510 0. 0 (7 1. 7) 2. 099 .5 (7 4. 28 ) 13 .5 -8 7. 5 (6 1. 50 ) 3 a . ni ge r 26 (7 6. 47 ) 7 (7 7. 78 ) 3 (1 00 .0 0) 0. 552 .5 (6 .3 5) 1. 043 .0 (1 4. 29 ) 0. 53. 5 (2 .0 0) 4 a . ta m ar ii 17 (5 0. 00 ) 7 (7 7. 78 ) 1 (3 3. 33 ) 0. 546 .0 (7 .2 1) 1. 09. 0 (3 .5 0) 1. 5 5 a . w en ti i 7 (2 0. 59 ) 2 (2 2. 22 ) 0 (0 .0 0) 0. 52. 5 (1 .3 6) 0. 5 (0 .5 ) 0 6 c la do sp or iu m cl ad os po ri oi de s 4 (1 1. 76 ) 0 (0 .0 0) 1 (3 3. 33 ) 0. 51. 5 (0 .8 8) 0 11 .5 7 c ol le to tr ic hu m sp . 18 (5 2. 94 ) 8 (8 8. 89 ) 3 (1 00 .0 0) 0. 523 .5 (6 .5 0) 0. 513 .0 (4 .2 5) 3. 010 .0 (6 .0 0) 8 c ur vu la ri a lu na ta 19 (5 5. 88 ) 8 (8 8. 89 ) 3 (1 00 .0 0) 0. 522 .5 (7 .3 9) 4. 016 .5 (1 0. 88 ) 1. 07. 0 (4 .8 3) 9 c . pa lle sc en s 29 (8 5. 29 ) 8 (8 8. 89 ) 3 (1 00 .0 0) 1. 043 .5 (1 2. 05 ) 4. 524 .0 (1 0. 81 ) 4. 517 .0 (1 1. 17 ) 10 e nd om yc es fib ul ig er 2 (5 .8 8) 1 (1 1. 11 ) 2 (6 6. 67 ) 3. 580 .5 (4 2. 0) 5. 0 1. 04. 0 (2 .5 0) 11 e ur ot iu m ch ev al ie ri 9 (2 6. 47 ) 1 (1 1. 11 ) 3 (1 00 .0 0) 0. 512 .0 (2 .6 1) 1. 0 0. 517 .5 (7 .0 0) 12 e . re pe ns 6 (1 7. 65 ) 1 (1 1. 11 ) 1 (3 3. 33 ) 0. 52. 5 (1 .0 8) 0. 5 0. 5 13 e . ru br um 5 (1 4. 71 ) 1 (1 1. 11 ) 2 (6 6. 67 ) 0. 59. 0 (2 .6 0) 1. 5 0. 51. 5 (1 .0 ) 14 f us ar iu m pr ol if er at um 1 (2 .9 4) 2 (2 2. 22 ) 0 (0 .0 0) 17 .5 0. 51. 0 (0 .7 5) 0 15 f . se m it ec tu m 26 (7 6. 47 ) 9 (1 00 .0 0) 3 (1 00 .0 0) 1. 567 .5 (1 9. 77 ) 1. 029 .5 (1 2. 06 ) 2. 58. 0 (3 .0 ) 16 f . ve rt ic ill io id es 25 (7 3. 53 ) 8 (8 8. 89 ) 3 (1 00 .0 0) 0. 59. 5 (3 .6 8) 1. 522 .5 (6 .2 5) 1. 04. 5 (2 .8 3) 17 l as io di pl od ia th eo br om ae 20 (5 8. 82 ) 7 (7 7. 78 ) 3 (1 00 .0 0) 0. 57. 0 (1 .6 5) 0. 54. 0 (1 .7 1) 0. 51. 0 (0 .6 7) 18 p . ci tr in um 29 (8 5. 29 ) 9 (1 00 .0 0) 3 (1 00 .0 0) 0. 562 .5 (1 5. 72 ) 1. 021 .5 (6 .8 9) 3. 56. 0 (4 .5 ) 19 p es ta lo ti op si s gu e p in ii 16 (4 7. 06 ) 5 (5 5. 56 ) 2 (6 6. 67 ) 0. 55. 5 (2 .0 6) 0. 52. 5 (1 .4 0) 0. 51. 0 (7 .5 0) 20 is o la te 3 9 (2 6. 47 ) 2 (2 2. 22 ) 0 (0 .0 0) 0. 59. 5 (2 .3 9) 0. 51. 0 (0 .7 5) 0 21 is o la te 7 1 (2 .9 4) 0 (0 .0 0) 1 (3 3. 33 ) 0. 5 0 2. 0 22 is o la te 8 1 (2 .9 4) 1 (1 1. 11 ) 1 (3 3. 33 ) 0. 5 1. 0 1. 0 23 is o la te 10 8 (2 3. 53 ) 1 (1 1. 11 ) 0 (0 .0 0) 2. 08. 0 (4 .3 8) 12 .5 0 n u m b er o f sa m p le s at fa rm er le ve l: 34 n u m b er o f sa m p le s at co lle ct o r le ve l: 9 n u m b er o f sa m p le s at re ta ile r in tr ad it io n al m ar k e t: 3 biotropia vol. 18 no. 2, 2011 114 nineteen fungal species were isolated from sorghum collected from , i.e. , , , , sp., , , , , , , , , , , isolates 7 and 8. fungi which were often isolated from 34 sorghum samples collected from were (88.24% of samples), and (85.24%, respectively), and (76.47%, respectively) and (73.53%). caused highest percentage of infection (71.7%), with the range of 0.5 100.0%, while the lowest were isolates 7 and 8 (0.5%, respectively). fungi which were always isolated from 9 sorghum samples collected from were , and (100%, respectively), while fungi which were often isolated were sp., , and (88.89%, respectively), , and (77.78%, respectively). caused highest percentage of infection (74.28%), with the range of 2.0 99.5%, while the lowest were and (0.5%, respectively). fungi which were always isolated from three sorghum samples collected from were , , sp., , , , , , and (100%, respectively), while , , and (66.67%, respectively) were often isolated. caused highest percentage of infection (61.50%), with the range of 13.5 87.5%, while the lowest was (0.5%). in general, the dominant fungal species infecting sorghum grain collected from various stages of the delivery chain in demak regency were , and (fig. 3) retailer at traditional market farmer collector retailer at traditional market a. candidus a. flavus a. niger a. tamarii cladosporium cladosporioides, colletotrichum curvularia lunata c. pallescens endomyces fibuliger eurotium chevalieri e. repens, e. rubrum f. semitectum f. vercillioides lasiodiplodia theobromae penicillium citrinum pestalotiopsis guepinii a. flavus curvularia pallescens penicillium citrinum a. niger f. semitectum f. verticillioides aspergillus flavus a. flavus f. semitectum penicillium citrinum colletotrichum curvularia lunata c. pallescens f. vercillioides a. niger a. tamarii lasiodiplodia theobromae aspergillus flavus a. wentii e. repens a. flavus a. niger colletotrichum curvularia lunata c. pallescens eurotium chevalieri f. semitectum f. vercillioides lasiodiplodia theobromae penicillium citrinum endomyces fibuliger eurotium rubrum pestalotiopsis guepinii aspergillus flavus e. repens a. flavus f. semitectum f. verticillioides . 88.24 100 100 76.47 100 100 73.53 88.89 100 0 20 40 60 80 100 aspergillus flavus fusarium semitectum f. verticillioides fl cl rl fl = farmer level; cl = collector level; rl = retailer level figure 3. percentage of sorghum samples infected by , and at farmer, collector and retail levels in demak regency aspergillus flavus, fusarium semitectum f. verticillioides 115 occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al t ab le 8. f u n ga l i n fe ct io n in so rg h u m co lle ct ed fr o m fa rm er s an d co lle ct o rs in re ge n cy w on og ir i n o f u n gi n u m b er (% ) sa m p le s in fe o f ct ed b y fu n gi r an ge (m ea n ) p er ce n ta ge o f so rg h u m in fe ct ed b y fu n gi in in fe ct ed sa m p le s f ar m er c o lle ct o r f ar m er c o lle ct o r 1 a sp er gi llu s fla vu s 18 (7 8. 26 ) 14 (9 3. 33 ) 0. 599 .5 (1 7. 44 ) 0. 586 .5 (2 3. 21 ) 2 a . ni ge r 11 (4 7. 83 ) 14 (9 3. 33 ) 0. 57. 0 (2 .9 1) 0. 517 .5 (4 .7 9) 3 a . ta m ar ii 11 (4 7. 83 ) 9 (6 0. 00 ) 1. 056 .5 (9 .5 5) 1. 021 .5 (6 .7 2) 4 a . w en ti i 9 (3 9. 13 ) 14 (9 3. 33 ) 0. 519 .0 (7 .1 7) 0. 581 .5 (9 .9 6) 5 c la do sp or iu m cl ad os po ri oi de s 9 (3 9. 13 ) 0 (0 .0 0) 0. 514 .5 (3 .0 0) 0 6 c ol le to tr ic hu m sp . 14 (6 0. 87 ) 2 (1 3. 33 ) 1. 012 .0 (6 .4 3) 1. 03. 5 (2 .2 5) 7 c ur vu la ri a lu na ta 10 (4 3. 48 ) 1 (6 .6 7) 0. 513 .5 (7 .9 5) 5. 0 8 c . pa lle sc en s 19 (8 2. 61 ) 9 (6 0. 00 ) 2. 018 .0 (7 .2 6) 0. 59. 5 (3 .4 4) 9 e nd om yc es fib ul ig er 6 (2 6. 09 ) 1 (6 .6 7) 1. 089 .5 (3 0. 67 ) 87 .0 10 e ur ot iu m ch ev al ie ri 7 (3 0. 43 ) 4 (2 6. 67 ) 0. 59. 0 (3 .1 4) 2. 03. 0 (2 .5 0) 11 e . re pe ns 6 (2 6. 09 ) 0 (0 .0 0) 0. 51. 5 (0 .9 2) 0 12 e . ru br um 8 (3 4. 78 ) 9 (6 0. 00 ) 0. 560 .0 (1 3. 63 ) 0. 518 .5 (4 .8 3) 13 f us ar iu m pr ol if er at um 18 (7 8. 26 ) 11 (7 3. 33 ) 0. 546 .0 (1 5. 17 ) 2. 045 .0 (1 5. 64 ) 14 f . se m it ec tu m 22 (9 5. 65 ) 14 (9 3. 33 ) 2. 075 .5 (2 4. 48 ) 1. 029 .0 (7 .1 8) 15 f . ve rt ic ill io id es 22 (9 5. 65 ) 14 (9 3. 33 ) 1. 070 .5 (2 9. 09 ) 1. 595 .5 (5 1. 64 ) 16 l as io di pl od ia th eo br om ae 1 (4 .3 5) 2 (1 3. 33 ) 6. 5 1 (1 ) 17 p en ic ill iu m sp .1 3 (1 3. 04 ) 8 (5 3. 33 ) 2. 06. 5 (4 .5 0) 0. 547 .0 (1 4. 69 ) 18 p en ic ill iu m sp .2 4 (1 7. 39 ) 7 (4 6. 67 ) 1. 530 .5 (1 1. 00 ) 9. 549 .0 (2 8. 07 ) 19 p . ci tr in um 23 (1 00 ) 12 (8 0. 00 ) 0. 597 .0 (3 0. 24 ) 1. 590 .5 (3 2. 33 ) 20 p . is la nd ic um 2 (8 .7 0) 2 (1 3. 33 ) 12. 5 (1 .7 5) 2. 53. 5 (3 .0 ) 21 p es ta lo ti op si s gu e p in ii 21 (9 1. 30 ) 12 (8 0. 00 ) 0. 511 .1 (2 .8 6) 1. 05. 5 (2 .8 ) 22 is o la te 3 15 (6 5. 22 ) 9 (6 0. 00 ) 0. 517 .5 (4 .6 7) 0. 517 .0 (4 .2 8) 23 is o la te 7 9 (3 9. 13 ) 0 (0 .0 0) 0. 59. 5 (3 .3 8) 0 n u m b er o f sa m p le s at fa rm er le ve l: 23 n u m b er o f sa m p le s at co lle ct o r le ve l : 15 biotropia vol. 18 no. 2, 2011 116 fungal infection of sorghum in wonogiri regency percentage of sorghum infected by fungi in wonogiri regency is presented in table 8. twenty three fungal species were isolated from sorghum collected from , i.e. , , , , , sp., , , , , , , , , , , , , sp. 1, sp. 2, , isolates 3 and 7. eight out of 23 fungal species were belong to field fungi, i.e. , , , , , , , and . twenty fungal species were isolated from sorghum collected from i.e. , , , , sp., , , , , , , , , , , , sp. 1, sp. 2, , and isolate 3. field fungi were still found in sorghum collected from collectors, because the duration of storage was relatively short. it was also assumed, that the storage condition was suitable for fungal growth. (100% of samples) was always isolated from 23 sorghum samples collected from , while and (95.65%, respectively), (91.30%), (82.61%), and (78.26%, respectively) were often isolated. caused highest percentage of infection (30.67%), with the range of 1.0 85.5%, while the lowest was (0.92%), with the range of 0.50 1.50%. farmer collector, farmer aspergillus flavus a. niger a. tamarii a. wentii cladosporium cladosporioides colletotrichum curvularia lunata c. pallescens endomyces fibuliger eurotium chevalieri e. repens e. rubrum fusarium proliferatum f. semitectum f. vercillioides lasiodiplodia theobromae penicillium citrinum p. islandicum penicillium penicillium pestalotiopsis guepinii cladosporium cladosporioides curvularia lunata c. pallescens fusarium proliferatum f. semitectum f. vercillioides lasiodiplodia theobromae pestalotiopsis guepinii a. flavus a. niger a. tamarii a. wentii colletotrichum curvularia lunata c. pallescens endomyces fibuliger eurotium chevalieri e. rubrum f. proliferatum f. semitectum f. vercillioides lasiodiplodia theobromae penicillium citrinum p. islandicum penicillium penicillium pestalotiopsis guepinii penicillium citrinum f. semitectum f. verticillioides pestalotiopsis guepinii curvularia pallescens a. flavus f. proliferatum endomyces fibuliger eurotium repens 78.26 93.33 95.6593.33 95.6593.33 0 10 20 30 40 50 60 70 80 90 100 aspergillus flavus fusarium semitectum f. verticillioides fl cl fl = farmer level; cl = collector level figure 4. percentage of sorghum samples infected by and at farmer and collector levels in wonogiri regency aspergillus flavus, fusarium semitectum f. verticillioides 117 occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al table 9. aflatoxin b content in sorghum collected from farmers, collectors and retailers at traditional market in demak and wonogiri regencies 1 regency level of distribution chain number (%) of samples contaminated by afb1 range (mean) of afb1 content (ppb) in contaminated samples demak farmer 21 (61.76) 4.54-90.78 (22.50) collector 5 (55.56) 13.62-22.73 (15.45) retailer at traditional market 0 0 wonogiri farmer 1 (4.35) 2.27 collector 6 (40.0) 4.57-22.85 (10.28) fungi which were often isolated from 15 sorghum samples collected from were , , , and (93.33%, respectively), and (80%). caused highest percentage of infection (87.0%), while the lowest was (1.0%). and are field fungi, while are post-harvest (storage) fungi. in general, the dominant fungal species infecting sorghum grain collected from various stages of the delivery chain in wonogiri regency were also , and (fig. 4) ercentage of samples contaminated by afb1 in sorghum collected from farmers (61.76%) was higher than that of collected from collectors (55.56%). the mean of afb1 content of sorghum collected from farmes (22.50 ppb, with the range of 4.54 90.78 ppb) was higher than those of collected from collectors (15.45 ppb, with the range of 13.62 22.73 ppb) (table 9). this might be due to more toxigenic strains of available in sorghum collected from farmers compared to those collected from collectors. according to pitt and hocking (2009) aflatoxin production among others depend on the certain strains of and the existence of other fungal species which are antagonistic to aflatoxigenic . dharmaputra . (2001) reported that was able to inhibit growth, consequently aflatoxin production was also inhibited as much as 80% ercentage of samples contaminated by afb1 in sorghum collected from collectors (40%) was higher than collected from farmers (4.35%) (table 9). it was probably due to the duration of storage at collectors was longer than that of at farmers, or the existence of aflatoxigenic found in sorghum collected from farmers was lower that that of in sorghum collected from collectors. the main and range of afb1 content in sorghum samples at farmers was 2.27 and 2.27 ppb, respectively, while at collectors was 10.28 ppb (4.57 22.85 ppb) (table 9). da silva . (2004) reported that 59 strains were found from 10 fresh sorghum samples collected from farmers and 130 sorghum samples collected from collector a. flavus a. niger a. wentii f. semitectum f. verticillioides penicillium citrinum endomyces fibuliger lasiodiplodia theobromae fusarium semitectum f. verticillioides a. flavus, a. niger a. wentii a. flavus f. semitectum f. verticillioides a. flavus a. flavus a. flavus et al in vitro aspergillus niger a. flavus a. flavus et al a. flavus . p . p aflatoxin b content1 aflatoxin b content of sorghum in demak regency aflatoxin b content of sorghum in wonogiri regency 1 1 biotropia vol. 18 no. 2, 2011 118 collector in brazil. as much as 38 (64.4%) of 59 strains were able to produce afb +afb with the range of 12,00-3,282.50 ppb. aflatoxin production was affected among others by the kind of substrates, moisture content and relative humidity, water activity, damaged grains, concentration of oxygen and carbondioxide, temperature and the duration of storage, interaction among microorganisms and the existence of insects (diener & davis 1969). pitt and hocking (1996) stated that aflatoxin content exceeding 1 000 ppb toxic to animal and human. fao (2004) reported that in russia afb1 content in sorghum should not exceed 5 ppb, while in taiwan total aflatoxin content (afb1 + afb2 + afg1 + afg2) should not exceed 10 ppb. in indonesia there are no regulations concerning the maximum tolerable limit of aflatoxin content in sorghum. although in general sorghum at farmer and collector levels were not contaminated by aflatoxin, post-harvest handling in each level of distribution chain should be conducted appropriately to prevent aflatoxin contamination. the method of postharvest handling will affect fungal infection (including ) and aflatoxin contamination. in brazil the dominant fungi found in fresh harvested sorghum (10 samples) and stored sorghum (130 samples) were (57.1%), (42.7%), (25.1%), (21.4%) and 9 genera of other fungi with mycelia. , and were important fungi, because certain species of these fungi can produce toxin. the population range of these fungi were 1 x 10 36 x 10 , 1 x 10 295 x 10 and 1 x 10 20 x 10 /g, respectively. the most often fungi found was and . as much as 12.8% of the samples was contaminated by afb1. their range was 7 33 ppb (da silva . 2000). in nigeria nine pathogenic and saprophytic fungi were found in stored sorghum ( ) which were previously air-dried. pathogenic fungi found were , and , while most of saprophytic fungi belong to , especially , . and (ogundero 2007). was the important fungus of sorghum in argentine in 1991, 1992 and 1993. the dominant fungus was , while the most often fungi isolated were , , and (gonzales . 1997). the percentage of sorghum infected by , , , , , and , i.e. 93.75, 75.0, 62.75, 62.50, 62.50, 56.25 and 37.5%, respectively (hemanth . 2007). schroeder and boller (1973) reported that aflatoxin was found in sorghum samples collected in two of three periods in texas. in 1970, 6% of 114 sorghum samples were contaminated by aflatoxin. their range and mean of contents were 3 20 ppb and 10 ppb, respectively. in 1971, 16% of 25 sorghum samples were contaminated by aflatoxin. their range and mean of contents were 4 9 ppb and 6 ppb, respectively. in general, bandyopadhyay (2000) stated that some species of , , , , , , , and were pathogenic fungi in sorghum seeds. hemanth (2007) reported, that five ( , . , and ) fungal species often isolated were able to inhibit the germination of sorghum. aside from that, 1 2 a. flavus phoma aspergillus fusarium rhizopus fusarium aspergillus penicillium cfu aspergillus flavus fusarium moniliforme et al sorghum guineense cladosporium vignae macrophomina phaseolina helminthosporium turcicum aspergillus a. flavus a fumigatus a. niger fusarium fusarium moniliforme alternaria alternata phoma sorghina penicillium funiculosum aspergillus flavus et al alternaria alternata fusarium moniliforme colletotrichum graminicol;a acremonium strictum phoma sorghina macrophomina phaseolina pestalotia guepinii et al et al. aspergillus alternaria cladosporium diplodia fusarium curvularia phoma penicillium et al. fusarium semitectum, f. verticillioides, f. proliferatum c pallescens a. flavus 3 3 3 3 3 3 119 occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al the diversity of fungi in sorghum collected from 10 regions in karnataka, india depended on the variety of sorghum, the structure and the location of the storage. according to schroeder and boller (1973) in 1969 and 1971 the percentage of sorghum samples collected in texas and infected by was 100%, respectively, while the percentage of samples contaminated by aflatoxin was 24 and 40%, respectively. reddy (2002) reported that in india 2, 2, 0 and 2 out of 29 sorg um samples were contaminated by aflatoxin with the range of 10 29 ppb, 30 49 ppb, 50 100 ppb, and >100 ppb, respectively here was a slight difference in the method of postharvest handling of sorghum at farmer and collector levels in demak and wonogiri regencies, especially the method of threshing sorghum grain. in demak and wonogiri regencies the farmers sun-dried sorghum in the form of panicles on tarpaulin or cement floor, while the collectors sundried the sorghum only on cement floor. in demak regency most of farmers threshed sorghum using a paddy thresher, while in wonogiri regency most of farmers threshed sorghum using a stick of wood. in these two regencies collectors threshed sorghum using a paddy thresher. in demak regency farmers and collectors stored sorghum grain in polypropylene bags. only in wonogiri regency most farmers and collectors stored sorghum in polypropylene bags. the moisture contents of sorghum at farmer and collector levels in demak and wonogiri regencies were still lower compared to normal moisture content of sorghum. various insect species associated with sorghum was found at various stages of the delivery chain in demak and wonogiri regencies. the dominant insects species were and . various field and postharvest storage fungi were isolated from sorghum at various stages of the delivery chain in demak and wonogiri regencies. the dominant fungal species found were , and . generally, afb1 content in sorghum at various stages of the delivery chain in demak and wonogiri regencies were low, i.e. ≤ 22.5 ppb he authors gratefully acknowledge the financial support of seameo biotrop through dipa 2010 by contract agreement no. 050.11/psrp/spkpnlt/iii/10. sincere thanks are also due to the head of the indonesian government's regional office of agriculture in demak and the indonesian government's regional office of agricultural crop and horticulture in wonogiri; to mrs. ratnaningsih, mr. edi suryadi, ms. nijma nurfadila, ms. amanda windyarani for their assistance, and the reviewers of this manuscript h . t . t . a. flavus et al. sitophilus zeamais tribolium castaneum aspergillus flavus fusarium semitectum f. verticillioides conclusions acknowledgments 120 biotropia vol. 18 no. 2, 2011 references [aoac] association of official analytical chemist. 2005. natural toxins. horwitz w, editor. official methods of analysis of aoac international. 18 ed. ch.49, p. 11. gaithersburg : aoac. bandyopadhyay, r., butler, d.r., chandrasekhar, a. reddy, r.k., and navi, s.s. 2000. biology, epidemiology, and management of sorghum grain mold. in chandrashekar a, bandyopadhyay r., hall aj, editor. technical and institutional options for sorghum grain mold management: proceedings of an international consultation; patancheru, 18-19 may 2000. india : international crops research insitute for the semi-arid tropics (icrisat). p. 34-71. [bp2aptp] balai pengembangan perbenihan produksi tanaman pangan dan hortikultura. 2008. pengembangan teknologi dan produksi perbenihan tanaman sorghum. yogyakarta: bp2aptp. christensen cm, miller bs, johnston js. 1990. moisture and its measurement. : sauer db, editor. storage of cereal grains and their products. 4th edition. st paul: american association of cereal chemist, p. 39-54. dharmaputra os, putri asr, retnowati i, ambarwati s. 2001. soil mycobiota of peanut fields in wonogiri regency, central java: their effect on the growth and aflatoxin production of aspergillus flavus in vitro. biotropia, 17: 30-58. diener ul, davis nd. 1969. aflatoxin formation by aspergillus flavus. goldblatt la, editor. aflatoxin. scientific background, control and implications. new york: academic press. p. 13-54. fao. 2004. worldwide regulations for mycotoxins in food and feed in 2003. fao food and nutrition paper 81. rome: food and agriculture organization of the united nations. gonzalez hhl, martinez ej, resnik sl. 1997. fungi associated with sorghum grain from argentina. mycopathologia, 13 (1): 35 41. haines cp. 1991. insects and arachnids of tropical stored products : their biology and identification (a training manual). kent: natural resources institute. hemanth rm, niranjana sr, nayaka sh. 2007. health status of farmers saved paddy, sorghum, sunflower and cowpea seeds in karnataka, india. wj agricultural science, 3(2): 167 177. hocking ad, pitt ji. 1980. dichloran-glycerol medium for enumeration of xerophilic fungi from low-moisture foods. applied and environmental microbiology, 30: 448 492. mendesil e, abdeta c, tesfaye a, shumeta z, jifar h. 2007. farmers perceptions and management practices of insect pests on stored sorghum in southwestern ethiopia. crop protection, 26 (12): 1817-1825. ogundero vw. 2007. the fungal flora of post-harvest grains of sorghum guineense stapf and their importance in pathogenicity. journal of basic microbiology, 26 (6); 359-363. peng wk. 1998. insects in domestic corn and sorghum stored in steel silos in taiwan. plant protection bulletin, 40: 309-314. pitt ji, hocking ad. 1996. current knowledge of fungi and mycotoxins associated with food commodities in southeast asia. in highley e, johnson gi, editor. mycotoxin contamination in grains. paper presented at the 17 asean technical seminar on grain postharvest technology, lumut, malaysia, 25-27 july 1995. canberra: australian centre for international agricultural research. p. 5-10. pitt ji, hocking ad. 2009. fungi and food spoilage. new york: springer. reddy dvr et al. 2002. estimation of aflatoxin levels in selected foods and feeds in india. hanak e, boutrif e, fabrie p, pineiro, editor. food safety management in developing countries. proceedings of the international workshop, cirad-fao; montpellier, 11-13 december 2000. france: cirad fao. p. 14. sauer db, meronuck ra, christensen cm. 1992. microflora. in sauer db (editor). storage of cereal grains and their product. 4 ed. minnesota: american association of cereal chemistry. p. 313 340. schroeder hw, boller ra. 1973. aflatoxin production of species and strains of the group isolated from field crops. applied microbiology, 25 (6): 885 889. in in in in aspergillus flavus th th th 121 occurrence of insects and fungi, and aflatoxin b contamination okky setyawati dharmaputra .1 et al da silva jb, pozzi cr, mallozzi mab, ortega em, correa b. 2000. mycoflora and occurrence of aflatoxin b and fumonisin b during storage of brazilian sorghum. journal of agricultural and food chemistry, 48(9): 4352-4356. da silva jb, dilkin p, fonseca h, correa b. 2004. production of aflatoksin by aspergillus flavus and of fumonisin by fusarium species isolated from brazilian sorghum. brazilian journal of microbiology, 35:182-186. suarni. 2001. tepung komposit sorgum, jagung dan beras untuk pembuatan kue basah (cake). risalah penelitian jagung dan serealia. maros: balai penelitian jagung dan serealia. p. 55-60. 1 1 122 biotropia vol. 18 no. 2, 2011 cellulase production by m1 using bacillus subtilis pretreated groundnut shell based liquid state fermentation ashish vyas *, chayanika putatunda , joginder singh and deepak vyas1 1 1 2 1department of microbiology, school of biosciences and biotechnology, lovely professional university, phagwara, punjab, india 2laboratory of microbial technology and plant pathology, department of botany, dr hs gour university (a central university), sagar, madhya pradesh 470003, india received 22 january 2014/accepted 21 june 2016 abstract groundnut shell which is rich in natural cellulose was assessed as a substrate for production of cellulase enzyme by cellulolytic bacteria. in the present investigation the bacterial isolate m1 was found to be capable of bacillus subtilis producing high amount of endoglucanase and exoglucanase on alkali treated groundnut shell. the effect of some nitrogen sources, amino acids and ca ions in the medium containing pretreated groundnut shell were also evaluated. ++ it was observed that 2% substrate concentration, 1 mm calcium concentration were optimum for cellulase production. ammonium nitrate was found to be the best among nitrogen sources tested. asparagine, tryptophan and methionine were found to be stimulatory for cellulase activity. : , cellulase, endoglucanase, exoglucanase, groundnut shellkeywords bacillus subtilis introduction microbial cellulases consist of three types of hydrolytic enzymes: 1. endo-(1, 4) -d-glucanase β (also known as endocellulase, carboxymethyl cellulose and endoglucanase) which makes random cleavages at the b-glycosidic linkages of cellulose; 2. exo-(1, 4) -d glucanase (also called β cellobiohydrolases and exocellulase) which hydrolyses cellobiose units from the termini of cellulose chain; and 3. -glucosidase (synonym β cellobiase ) which releases glucose from cellobiose and short chain oligosaccharides (kim . 2008). et al the cellulases form of a very important group of enzymes that find applications in a variety of industries including textile industries, paper and pulp industries, food processing industries, wine and brewery industry etc. (kuhad . 2011). et al however, the cost of enzymes poses a problem to their large scale utilization, especially for the environmentally crucial processes like production of biofuels (brijwani . 2010). so, there is a et al need to search cheaper methods for producing these enzymes. one of the possible methods for achieving this, is by using lignocellulosic wastes for the production of cellulases. lignocellulosics offer a promising solution, since they are abundantly available and are produced in large amount as agricultural by-products. organic waste from renewable forest and agricultural residues are rich source of cellulose, hemicellulose and lignin (brauns & brauns 1960) and therefore, can be possibly used in fermentation process for the production of cellulases. groundnut ( l) is an arachis hypogea important oil seed crop of india. the pod or dry pericarp contains about 25-40% shell (dey et al. 2002). the compositional analysis of groundnut shell indicates that the shell contains cellulose (65.7%), carbohydrates (21.2%), protein (7.3%), minerals (4.5%) and lipids (1.2%) (masenda 2004). groundnut shell is used as manure (rao et al. 2009), used in mushroom cultivation (jain & vyas 2002), and in production of extracellular enzymes (vyas 2005).et al. the capacity of selected to bacillus subtilis produce and secrete large quantities of biotropia vol. 23 no. 1, 2016: 28 34 doi: 10.11598/btb.201 .2 . .6 3 1 472 * c orresponding author: ashish.vyas@lpu.co.in 28 mailto:ashish.vyas@lpu.co.in extracellular enzymes has placed them among the most important industrial enzymes producers (schallmey 2004). this study reports et al. bacillus subtilis m1 as potent producer of cellulase enzymes on pretreated groundnut shell. materials and methods organism bacillus subtilis m1 was isolated from decomposing organic waste containing cellulose. bacteria was isolated by enrichment method (hans & srinivasan 1968) for which 1 g mixture of rotting cellulosic substrates was inoculated into the isolation medium consisting of mineral salts solution supplemented with 0.1% yeast extract and strip of filter paper. after 3 to 7 days of incubation at 30 c on reciprocal shaker, a patch o of a yellow pigmented material appeared at the liquid air interface on the filter paper. as soon as the pigmented material appeared, a portion of filter paper was transferred with sterile wire and inoculated into fresh medium. the process was repeated several times to enrich aerobic and mesophilic cellulose utilizing organisms. the filter paper from the enriched culture was removed, macerated in a small amount of sterile water and streaked on the plates containing carboxy methyl cellulose agar. the bacterial colony was purified and maintained on nutrient agar medium slants at 4 c.o substrate gs (groundnut shell) was collected from local suppliers in and around sagar district, madhya pradesh, india. shell (1 kg) was dipped in water (5 l) to remove any amount of soluble sugar present in the substrate and then dried at 80 c for o 36 hours in electric oven and then chopped into small pieces (krishna 1999), which were then ground in an electric grinder and sieved through 100 µm mesh sieve and kept at room temperature. pretreatment of gs powdered groundnut shell was treated separately with alkali (naoh) and designated as sample atrt (alkali treated), while acid (hcl) treated shells were labeled as sample actrt (acid treated). dried and powdered shell (100 g) was treated for 24 hours with 0.25n naoh (500 ml) (koijam 2000) and with 0.25n hcl (500 ml). et al. after treatment the powdered shell was repeatedly washed with distilled water until it reached neutral ph 7 and then dried over night at 60 c.o inoculum preparation bacterial inoculum consisted of 2 ml of 24 hours old culture grown on nutrient broth giving absorbance of 1.2 at 660 nm. fermentation medium (submerged) mandels and reese medium (mandels & reese 1957)was used for cellulase production. chemical composition of medium was proteose peptone, 1.0; (nh ) so , 1.4; kh po 2.0; nh -co-nh 4 2 4 2 4, 2 2, 0.3; mgso .7h o, 0.3; cacl , 0.3; feso .7h o, 4 2 2 4 2 0.005; mnso .h o, 0.0016; zncl , 0.0017; and 4 2 2 pretreated lignocellulosic substrate (gs), 10 g/l; 1,000 ml distilled water; ph 5.3. cellulase production the medium (25 ml) was dispensed in 150 ml conical flask and autoclaved at 15 psi for 15 minutes. the flasks were inoculated with bacterial culture and incubated at 37 1 c under stationary + o conditions. the samples (10 ml) were withdrawn aseptically after the 6 day of incubation and th supernatant was obtained after centrifugation at 9,500 rpm for 20 minutes. the supernatant obtained was used for enzymatic activity. the control consisting of untreated gs was also run simultaneously. all tests were performed in duplicate. enzyme assay endoglucanase and exoglucanase activities were measured (mandels 1974) in terms of international unit (iu), which is micromoles (µm) of glucose released/minute/ml. results and discussion cellulases are a group of industrially important enzymes and their demand is increasing day by day with the growth of enzyme industry. groundnut shell was assessed as cheap alternative lignocellulosic substrate for producing 29 cellulase production by m1 using pretreated groundnut shell ashish vyasbacillus subtilis – et al. cellulase enzymes since it is known to possess high percentage of cellulose (masenda 2004). however, the lignin present in the groundnut shell can impede the cellulase enzymes. so, delignification was performed through alkali (naoh) and acid (hcl) pretreatment. in the present investigation, the isolate m1 was found to be potent producer of cellulase enzymes on pretreated groundnut shell. it was isolated from decomposing organic waste containing cellulose as per the enrichment method described by hans and srinivasan (1968). microorganism found was gram positive, spore producing, catalase positive rod shaped bacterium. it was identified as bacillus subtilis by imtech (institute of microbial technology), chandigarh, india. under the conditions of ph 5, temperature 50 c and an incubation period of 6 days, the o bacterial isolate m1's endoglucanase b. subtilis activity was found to be 2 fold (0.314 iu/ml) and exoglucanase activity was found to be 3.3 fold (0.043 fpu/ml) in atrt gs as compared to untreated gs. in actrt gs, 1.5 fold (0.219 iu/ml) increased endoglucanase and 1.5 fold (0.020 fpu/ml) exoglucanase activity was obtained as compared with the untreated. so, the pretreatment resulted in higher enzyme production and the alkali treatment was found to be more efficient than the acid treatment (fig. 1). alkali (naoh) treatment of gs was found to be mor superior to acid (hcl) e treatment as it modifies lignocellulosic substrate by increasing the pore size and solubilizing lignin and hemicellulose. it increases the surface area of cellulose, reducing its crystallinity (ming . et al 20 ).08 figure 1 effect of alkali and acid treatments of groundnut shell on production of cellulase by m1bacillus subtilis figure 2 effect of alkali pretreatment of different concentrations on groundnut shell on cellulase production by bacillus subtilis m1 endoglucanase activity (iu/ml) exoglucanase activity (fpu/ml) endoglucanase activity (iu/ml) exoglucanase activity (fpu/ml) 30 biotropia vol. 23 no. 1, 2016 control alkali treated (0.25n naoh) acid treated (0.25n hcl) control 0.25n naoh 0.50n naoh 1n naoh 2n naoh pretreatment of powdered gs at different alkali concentrations (0.25n, 0.5n, 1n, 2n naoh) showed that m1 produced the b. subtilis highest endoglucanase (0.381 iu/ml) and exoglucanase (0.087 fpu/ml) activities in case of 1n naoh treated gs. endoglucanase activity was increased by 2.7 fold and exoglucanase activity by 6.9 fold higher compared to untreated gs (fig. 2). the increased cellulase activity at 1n naoh concentration might be due to increased solubilization of lignin and increase in swelling of cellulose i to cellulose ii. aguiar (2001) also repor ted 1n naoh pretreatment of lignocellulosic substrates as best source for cellulase production. chemical pretreatment of lignocellulosic material followed by its hydrolysis suggested that accessible surface area is a key determinant for enhancing cellulase yield. however, sarkar and aikat (2012) reported the highest activity of cellulases by aspergillus fumigatus nitdgpka3 on rice straw pretreated with 0.5 m naoh. the amount of nutrients present in the production medium is one of the major factors impacting the microbial enzyme production. the effect of five nitrogen sources (ammonium sulfate, ammonium nitrate, potassium nitrate, peptone, urea) were tested separately (fig. 3). each nitrogen source was added in equivalent amount to the total nitrogen present in the medium (587 mg nitrogen/l medium) keeping the available nitrogen constant. ammonium nitrate was found to be the best for endoglucanase activity (0.507 iu/ml) and exoglucanase activity (0.091 fpu/ml) followed by ammonium , sulfate potassium nitrate, peptone and urea. nitrogen is the major constituent of protoplasm and building block of enzymes (proteins). sethi (2013) et al. also reported ammonium salt to be excellent source of nitrogen for cellulase production and concluded that this may be due to their direct entry of ammonium ion in protein synthesis. similarly, amino acids are nitrogen containing organic compound and therefore, have influence on cellulase synthesis, but the specific side groups present on the amino acids can also play some role in cellulase synthesis. among various amino acids added to the medium (0.2 w/v), tryphtophan (0.675 iu/ml), asparagine (0.629 iu/ml) and methionine (0.564 iu/ml) were found to be the better suited for endoglucanase activity (fig. 4). on the other hand, alanine, arginine and threonine were found to be suppressive in action. b. subtilis m1 showed maximum exoglucanase activity in the presence of asparagine (0.126 fpu/ml) followed by metheonine (0.108 fpu/ml) and tryptophan (0.095 fpu/ml). amino acids, being the building blocks of protein, have a profound influence in the cellulase synthesis of bacteria. the increased production might be due to the enhanced synthesis of cellulolytic enzymes in the presence of amino acids. asparagine and metheonine were found to be stimulatory for cellulase activity in av49 by vyas . aspergillus terreus et al (2005). figure 3 effect of nitrogen sources on cellulase production on alkali treated groundnut shell by m1bacillus subtilis endoglucanase activity (iu/ml) exoglucanase activity (fpu/ml) 31 cellulase production by m1 using pretreated groundnut shell ashish vyasbacillus subtilis – et al. control ammonium sulfate ammonium nitrate potassium nitrate peptone urea the activity and stability of various enzymes are known to be impacted by various ions. different concentrations of calcium (0.5-5 mm) had differential impact on the enzymatic activity (fig. 5). low (1 mm) concentration produced maximum endoglucanase activity (0.564 iu/ml). however, at higher concentration (2-5 mm) endoglucanase activity was almost static, yet remained higher than control. exoglucanase activity was maximum (0.085 fpu/ml) at 1 mm concentration and thereafter, it decreased up to 5 mm. metal ions are generally cellulase stimulators at low concentration, whereas inhibitory at very high concentration. ca was found to positively ++ inf luence the act iv i ty of cel lulase at concentrations less than 1 mmol/l by wang et al. (2012). in the present study, ca (1 mm) was ++ found to be stimulatory and higher concentration showed static endoand exoglucanase activities by . ca (10 mm) is reported (kim b. subtilis et al.++ 2001) to be st imulatory for cel lulase (exoglucanase) activity in trichoderma reesei. strong affinity and tightness of the enzymes would disrupt the hydrogen bonding network in the crystal lattice, ultimately collapsing the ordered structure of cellulose particles and preventing adhesion. kotchoni (2003), et al. however, reported enhanced cellulase activity at low calcium concentration (1 mm) in bacillus pumilus bpcri6. the effect of substrate concentration is also known to impact the enzyme activity. in the present investigation it was observed that with figure 4 effect of different amino acids on cellulase production on alkali treated groundnut shell by m1bacillus subtilis figure 5 effect of calcium concentration on cellulase production on alkali treated groundnut shell by bacillus subtilis m1 endoglucanase activity (iu/ml) exoglucanase activity (fpu/ml) endoglucanase activity (iu/ml) exoglucanase activity (fpu/ml) control alanine methionine threonine asparagine arginine tryptophan 32 biotropia vol. 23 no. 1, 2016 0.5 mm ca 1 mm ca 2 mm ca 3 mm ca 4 mm ca 5 mm ca increased substrate concentration, the endoglucanase activity showed increasing trend up to 2% substrate concentration (0.517 iu/ml), thereafter, no further increase in activity was observed (fig. 6). maximum exoglucanase activity was observed at 1% substrate concentration (0.090 fpu/ml). okonkwo (2014) also observed maximum activity at 1% substrate concentration in case of cellulase produced by aspergillus flavus. conclusions the present findings raise the possibility of using groundnut shell as a substrate for production of cellulase enzymes by bacillus subtilis m1. the cultural conditions as well as the type of pretreatment also seem to have profound impact on the enzyme activity. there is a need to assess the production at pilot plant scale, so that it can be used subsequently at the commercial scale in the long run. acknowledgements the first author thanked the head of botany department, dr hs gour university for providing necessary laboratory facilities during research tenure. thanks are also due to prof m.k. bhat, institute of food research, colney, norwich, england for providing relevant literature references aguiar cl. 2001. biodegradation of the cellulose from sugarcane bagasse by fungal cellulase. cienc tecnol aliment 3:117-21. brauns fe, brauns da. 1960. the chemistry of lignin covering the literature for the 1949-1958. san diego (usa): academic press san diego. brijwani k, oberoi hs, vadlani pv. 2010. production of a cellulolytic enzyme system in mixed-culture solidstate fermentation of soybean hulls supplemented with wheat bran. process biochem 45:120–8. dey r, pal kk, chauhan sm, bhatt dm, misra jb. 2002. groundnut shell decomposition potential of some cellulolytic microorganisms. ind j microbiol 42: 165-7. hans yw, srinivasan vr. 1968. isolation and characterization of cellulose utilizing bacterium. app microbiol 16:1140-5. jain ak, vyas d. 2002. yield response of on pleurotus florida wheat straw in combination with other substrates. mushroom res 11:19-20. kim dw, jang yh, kim cs, lee ns. 2001. effect of metals ions on the degradation and adsorption of two cellobiohydrolases on microcrystalline cellulose. bull korean chem soc 22:716-20. kim sj, lee cm, han br, kim my, yeo ys, yoon sh, koo bs, jun hk. 2008. characterization of a gene encoding cellulase from uncultured soil bacteria. fems microbiol lett 282:45-51. koijam b, sharma nc, gupta s. 2000. production and characterization of fungal cellulase from cellulosic waste. asian j microbiol biotechnol environ sci 4:113-20. kotchoni os, shonukan oo, gachomo we. 2003. bacillus pumilus bpcri 6, a promising candidate for cellulase production under condition of catabolite repression. afr j biotechnol 2:140-6. figure 6 effect of substrate concentration on cellulase production on alkali treated groundnut shell by m1bacillus subtilis endoglucanase activity (iu/ml) exoglucanase activity (fpu/ml) 33 cellulase production by m1 using pretreated groundnut shell ashish vyasbacillus subtilis – et al. 1% gs (w/v) 2% gs (w/v) 3% gs (w/v) 4% gs (w/v) 5% gs (w/v) krishna c. 1999. production of bacterial cellulases by solid state bioprocessing of banana wastes. biores technol 69:231-9. kuhad rc, gupta r, singh a. 2011. microbial cellulases and their industrial applications. enzyme research [internet]. [cited 2015]; article id 280696. available from: http://www.hindawi.com/journals/er/ 2011/280696/ doi:10.4061/2011/280696. mandels m, reese et. 1957. induction of cellulases in fungi in as influenced by carbon source. j trichoderma viride bacteriol 37:269-78. mandels m. [internet]. 1974. laboratory procedures, hand book. usa: us army natick laboratories. available from: http://calvin.biotech.wisc.edu/ jeffries/cellulases/mandels.html. masenda e. [internet]. 2004. groundnut shells, substrate. mushroom growers handbook i. south korea: mushworld. p 120-122. available from: www.mushworld.com/service/handbook/2004/ch apter-5-8.pdf. ming c, jing z, liming x. 2008. enzymatic hydrolysis of maize straw polysaccharides for the production of reducing sugars. carbohydr polym 71:411–5. okonkwo if. 2014. effect of substrate concentration on the activity of cellulase produced by . aspergillus flavus ind j app res 7: 32-4. rao cs, wani sp, sahrawat kl, rajasekharao b. 2009. nutrient management strategies in participatory watersheds in semi arid tropical india. ind j fert 5(12):113-28. sarkar n, aikat k. 2012. alkali pretreatment of rice straw and enhanced cellulase production by a locally isolated fungus nitdgpka3. aspergillus fumigatus j microbiol biotech res 2(5):717-26. schallmey m, singh a, ward op. 2004. developments in the use of species for industrial production.bacillus canadian j microbio 50(1):1-17. sethi s, datta a, gupta bl, gupta s. 2013. optimization of cellulase production from bacteria isolated from soil. isrn biotechnology [internet]. 2013:985685. available from: http://www.hindawi.com/ journals/isrn/2013/985685/ doi: 10.5402/2013/ 985685. vyas a, vyas d, vyas km. 2005. production and optimization of cellulases on pretreated groundnut shell by av49. j sci ind aspergillus terreus res 64:281-6. wang g, zhang x, wang li, wang k, peng f, wang l. 2012. the activity and kinetic properties of cellulases in substrates containing metal ions and acid radicals. adv biol chem 2:390-5. 34 biotropia vol. 23 no. 1, 2016 http://www.hindawi.com/journals/er/ http://calvin.biotech.wisc.edu/ http://www.mushworld.com/service/handbook/2004/ch http://www.hindawi.com/ isolation and production of extracellular enzyme from mannanolytic thermophilic bacterium originally from lampung isolation and characterization of mannanolytic thermophilic bacteria from palm oil shell and their mannanase enzyme production properties biotropia no. 25, 2005 : 1 – 10 sumardi1, antonius suwanto2,, maggy thenawidjaja3, and tresnawati purwadaria4 1department of biology, faculty of science and mathematics, university of lampung, jl. s. brojonegoro no. 1, bandar lampung 35145, indonesia 2department of biology, faculty of science and mathematics, bogor agricultural university, jl. raya pajajaran, bogor 16144, indonesia 3department of food science and human nutrition, bogor agricultural university, darmaga, 16680, bogor, indonesia 4indonesian research institute for animal production, p.o. box 221, bogor 16002, indonesia abstract a mannanolytic thermophilic bacterium (l-07) was isolated from palm oil shell after 2 days of enrichment in liquid medium supplemented with 1% palm kernel meal as mannan source. sequence analysis of 16s-rrna indicated that l-07 was similar (98%) to geobacillus stearothermophilus, a species of thermophilic aerobic bacteria. we found that g. stearothermophilus l-07 produced extracellular β-1,4-mannanases, but no β-manosidase and α-galactosidase activities. the growth of l-07 reached its maximum (3.0 x 106 cell/ml) at 12-20 hours, while the highest β-mannanase activity (0.52 u/ml) was observed in culture medium after 36 hours of cultivation at 60oc. the medium containing locust bean gum was the best for producing extracellular β-1,4-mannanases compared with kolang kaling, konjak, and palm kernel meal. sds-page and zymogram analysis demonstrated that crude mannanase complex of l-07 from locust bean gum containing medium comprised three active bands with molecular weight of 85, 73 and 50 kda. keywords : extracellular enzyme/mannanase/geobacillus stearothermophilus introduction hemicelluloses are the second most abundant polysaccharide in nature after cellulose. the major constituents of hemicellulose are the hetero-1,4-β-d-xylans and hetero-1,4-β-d-mannans (galactoglucomannan, galactomannan, and glucomannan). the heteroxylans are found mainly in grasses, cereals, and hardwoods (angiosperms). the mannans are more abundant in copra, palm, coffee, and locust bean endosperms (araujo and ward 1990). mannanolytic microbes were found in soil, compost, and animal rumen (zakaria et al. 1998). biodegradation of ß-mannans is caused by β-mannanase (1,4-β-d mannan manohidrolase [ec 3.2.1.78]) produced from bacteria and fungi. the enzyme hydrolyses the ß-(1,4) linkages in backbone of mannan polymer, ∗corresponding author : asuwanto@indo.net.id 1 producing short chain mannoligosaccharides. then, these compounds can be further degraded by the action of β-mannosidase (β-d-mannosidase [ec 3.2.1.25]) and a αgalactosidase (ec 3.2.1.22) (duffaud et al. 1997). mannan degradation from glucomannan and galactomannan produces manno-oligosaccharide, mannobiose, and mannose. mannan-degrading enzymes can be used for numerous applications in food, feed, pulp, and paper industries. biotropia no. 25, 2005 in the palm oil factory, during the composting process of palm shells containing hemicellulose the temperature rises to 65oc. at such temperature, many thermophilic bacteria are able to develop special properties to survive and prosper in the habitat. the purposes of this study were to isolate the mannanolytic thermophilic bacterium from palm shells, a solid waste of palm oil factory in lampung, and to study its ß-mannanase production. materials and methods materials palm shell as source of thermophilic bacteria was obtained from palm oil industry in natar, lampung at 65oc. locust bean gum (galactomannan) was obtained from sigma chemical company. other chemicals were analytical grade from merck industry. isolation of thermophilic mannanolytic strains one milligram of palm shells was enriched in a medium containing 0.2% yeast extract; 0.2% trypton, 1.0% palm kernel meal, 0.02% mgso4, 0.14% kh2po4, and 0.1% (nh4)2so4 at 70oc for two days with agitation (120 rpm). one hundred microliters of each enrichment culture was spread aerobically on the same medium onto agar plates containing 0.3% locust bean gum instead of palm kernel meal. thermophilic mannanolytic isolates showed a clear zone around the colony after staining with a solution of 0.1% congo red for 15 minutes and destaining through repeated washing with 1 m nacl. characterization and identification of isolate morphological properties and taxonomic characteristics of the best isolate were studied according to the methods as described in “bergey’s manual of systematic bacteriology” (holt et al. 1994). identification of the isolate was determined through 16s rrna sequence analysis. genomic dna extraction was performed using the ctab method. pcrmediated amplification of the 16s rrna was performed as described by marchesi et al. (1998) employing geneamp pcr system 2400 (perkin elmer). pcr product was purified and sequenced. cluster analysis was conducted according to a program 2 provided by european bioinformatics institute (http://www. ebi.ac.uk) and phylogenetic tree was constructed using treecon software (peer and watcher 1993). isolation and characterization of mannanolytic thermophilic bacteria – sumardi et al. extracellular enzyme production the best bacterial isolate was grown in the medium for mannanase production which contained 0.35% yeast extract, 0.35% trypton, 0.035% mgso4, 0.245% kh2po4, 0.175% (nh4)2so4, 0.2% nacl, and 0.65% locust bean gum (ph 7.0). locust bean gum was replaced with other carbon sources (kolang kaling, konjak, palm kernel meal) when the effect of carbon sources on the enzyme production was examined. a 250-ml flask containing 50 ml of the medium was inoculated with a loopful of cells taken from a stock slant and was precultured at 60oc on shaker (120 rpm) for 6-8 hours. the same volume of medium and flasks for enzyme production were inoculated with 2 ml of this culture and cultivated at 60oc for 48 h. aliquots of the culture medium were sampled at 4 h intervals to determine β-mannanase activity and viable bacterium number. intracellular enzyme extraction g. stearothermophilus l-07 cells were concentrated from 50 ml liquid medium by centrifugation at 3,200 x g for 10 min. at 4oc. the cell pellets were resuspended in 1 ml of ice-cold condition containing 50 mm phosphat buffer. the cell lysis process used sonication with a soniprep 150 (usa). the sonicator was set to 16 micron amplitude for 5 minutes. intracellular enzymes were detected specifically βmannanase, β-mannosidase, and α-galactosidase. enzyme assay β-mannosidase and α-galactosidase activities were determined by monitoring the release of p-nitrophenol from p-nirophenyl b-d-mannopyranoside or pnitrophenyl α-galactophyranoside (sigma chemical co., usa), respectively. for each assay, 0.9 ml aliquots of 1 mm substrate in 50 mm sodium phosphate buffer (ph 7.0) and 0.1 ml of enzyme were mixed and incubated at 80oc for 30 minutes and the reaction stopped by the addition of 0.1 ml solution of 0.4 m na2co3. the release of p-nitrophenyl (pnp) was measured spectrophotometrically by monitoring the changes in absorbance at 405 nm. control was prepared with the addition of enzyme after na2co3 solution. one unit of β-mannosidase or α-galactosidase activity was defined as the amount of enzyme releasing 1 μmol pnp per min. under the specified assay condition. β-mannanase activity was determined by monitoring the release of reducing sugars. the reaction mixture, containing 0.5% locust bean gum, 50 mm sodium phosphate buffer (ph 7.0), suitably diluted enzyme solution in a total volume of 1 ml, was incubated at 80oc for 30 minutes. the reducing sugar content was determined by dinitrosalisylic acid method. one unit of enzyme was defined as the 3 amount of enzyme producing 1 μmol of mannose per minute under the given assay condition. biotropia no. 25, 2005 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and zymogram sds-page was performed in 8% polyacrylamide gels. protein bands were visualized after staining with silver nitrate. zymogram was prepared using 0.1% locust bean gum co-polymerized with polyacrylamide. after separation, sds was removed by method of spindler and rapp (1997). then, the gel was equilibrated through several washings and finally immersed in 50 mm sodium acetate buffer (ph 6.0), followed by incubation at 65oc for 35 minutes, staining with 0.1% congo red for 15 minutes, and destaining in 1 m nacl. results and discussion we found 15 bacterial isolates from palm oil shell. then the bacteria were screened for mannanase activity on selected medium agar plate containing locust bean gum at 70oc with ph 7.0. only isolate l-07 showed mannanase activity. the isolate l-07 could grow at 55-70oc and showed mannanase activity as high as 3.1 u/mg. a. b. figure 1. bacterial isolate l-07. a. gram-positive rod-shaped cells were cultured at 65oc for 1 day. b. cells produced endospore in terminal position after 5 days of incubation. table 1 shows the morphological and physiological characteristics of l-07. l07 was gram-positive (figure 1.a), aerobic, catalase-positive, and endosporeforming bacterium (figure 1.b). it was non-motile and rod-shaped (1-1.2 x 5-5.7 μm). based on these characteristics l-07 belonged to the genus bacillus. 4 table 1. morphological and physiological characteristics of l07 isolate isolation and characterization of mannanolytic thermophilic bacteria – sumardi et al. characteristics strain l-07 shape rod endospora shape oval/cylindrical endospora position terminal gram stain positive catalase positive motility negative growth at 37oc negative growth 55 – 75oc optimum growth temperature 60-70oc cell size (μm) 1,2x5,7 nitrate reduction positive indole production negative urease positive voges proskauer test negative utilization as sole carbon source of glucose positive mannose positive xylose positive sucrose positive arabinose positive ramnose negative citrate negative lactose negative mannitol negative sorbitol negative adonitol negative rafinose negative hydrolysis of mannan positive hydrolysis of carboxymethyl cellulose positive hydrolysis of starch negative hydrolysis of xylan negative then, the total sequence of 1315 bp of 16s rrna gene of l-07 showed 98% similarity to that of geobacillus stearothermophilus. the constructed phylogenetic tree for l-07 is shown in figure 2. g. stearothermophilus (formerly bacillus stearothermophilus) was a highly resistant thermophilic organism. major strains of the genus geobacillus live in geothermal areas, such as the oil field subsurface and hydrothermal vents (nazina et al. 2001; and euzeby 2005). at this time, no study has reported on the presence of mannanolytic thermophilic bacteria from palm shell. as comparison aurora et al (2003) showed that bacillus pumilus dyp2, a mannanolytic mesophilic bacteria, was isolated from west sumatra copra soil sample, indonesia. another bacterium, b. stearothermophilus atcc 12016 has been previously reported to produce thermostable β-mannanase (ethier et al. 1998). 5 figure 2. phylogenetic tree construction for l-07 isolate. bootstrap values are shown at each node tree. when g. stearothermophilus l-07 produced thermophilic mannanase, the enzyme was observed primarily in culture media. the other two enzymes, α-1,6 galactosidase and β-1,4 mannosidase were not detected in extracellular enzyme preparation (table 2). these enzymes might act to provide simple sugar to geobacillus stearothermophilus l-07 growing on locust bean gum as base media. regarding the cellular localization of these three activities, we speculate that the mannan backbone was cleaved by endo-acting β-1,4 mannanase prior to the transport of smaller galactomannans to the site of the other two enzymes. the other two enzymes, exo-acting α-1,6 galactosidase and β-1,4 mannosidase, are found in the cell membrane. as comparison, duffaud et al. (1997) showed that extracellular β-1,4 mannanase of thermotoga neopalitana 5068 was sevenfold greater than the cell extract. the other two enzymes, α-1,6 galactosidase and β-1,4 mannosidase were found in the cell the extract. table 2. the specific activity of extracellular and intracellular mannanolytic enzymes from geobacillus stearothermophilus l-07 cultured at 60oc for 36 hours specific activity (u/mg) no kind of enzyme intracellular extracellular 1 β-1,4-mananase 0.02 ± 0.002 3.10 ± 0.050 2 β-1,4-manosidase 0.03 ± 0.003 0.00 ± 0.000 3 α-1,6-galactosidase 24.1 ± 0.060 0.00 ± 0.000 72 93 92 100 100 pyrococcus furiosus subsp. woesei alicyclobacillus acidocaldarius mih321 g. stearothermophylus bgsc 9a21 l-07 10% biotropia no. 25, 2005 e. coli thermus aquaticus yt-1 c. botulinun kyto-f l. delbrueckii subsp. bulgaricus atcc11842 b.subtilis wl-7 100 50 b. thermoleovorans ccr11 85 6 we also observed the growth and extracellular β-mannanase activity of g. stearothermophilus l-07 as shown in figure 3. after 12-20 hours of observation strain l-07 reached its maximum growth, while the highest β-1,4-mannanase activity was found in culture medium after 36 hours of cultivation. β-1,4-mannanase may be associated with the cell membrane. so, after g. stearothermophilus cells died β-1,4-mannanase leaked out from membrane and increased the activities. besides for mannan degradation to produce manno-oligosaccharide, this thermostable enzyme can be used in the bleaching process (ethier et al. 1998). the other β-mannanase from bacillus sp. was active at 70oc. the enzyme was stable when incubated for 1 h at ≤ 60oc (ooi and kikuchi 1995). while the βmannanase from rhodothermus marinus retained 87% of its initial activity after 1 h at 90oc (politz et al. 2000). 4,5 5 5,5 6 6,5 7 0 4 8 12 16 20 24 28 32 36 40 44 48 time (h) lo g ce ll nu m be r/m l 0 0,1 0,2 0,3 0,4 0,5 0,6 m an na na se a ct iv ity (u /m l) figure 3. growth curve and extracellular mannanase activity of geobacillus stearothermophilus l-07. strain l-07 was cultured at 60oc for 48 hours in the enzyme production medium. -■ logarithmic viable cell number and -οmannanase activity. we studied the effect on the kind of carbon source on β-1,4 mannanase production. the bacteria produced active β-1,4 mannanase when they were grown in a kind of carbon source as medium (table 3). table 3. effect of carbon sources on the mannanase production. g. stearothermophilus l-07 was cultured at 60oc for 36 hours. no carbon sources relative activity (%) 1 none 2 ± 0.01 locust bean gum 2 100 ± 0.04 kolang kaling (endosperm from arenga pinata) 46 ± 0.06 3 konjak 4 13 ± 0.05 palm kernel meal 0 ± 0.00 5 isolation and characterization of mannanolytic thermophilic bacteria – sumardi et al. 7 the enzyme activity in the medium containing locust bean gum was the highest, followed by that in kolang kaling, and konjak. no activity was observed in palm kernel meal. thus, the best induction medium consisted of locust bean gum. it may be pointed out that the mannanase activity is preferably induced by polysaccharides containing mannose or galactose as monomeric unit, and the induction is strongly increased by heterogeneous polysaccharides containing mannose and galactose. locust bean gum consists of 88% galactomannan (whistler and bemiller 1973). the composition may support the increase of mannanase production. so far, there was no accurate information on mannan composition in kolang kaling and palm kernel meal. however, they are found in palm seeds where galactomannans are major part of their dry weight. biotropia no. 25, 2005 konjak mannan consist of 50-60% glucomannan containing mannose and glucose. as comparison, torrie et al. (1990) reported that the highest mannanase production was found from mold of trichoderma harzianum e58 in the medium containing locust bean gum (26.7 u/mg), followed by konjak (glucomannan) (7.5 u/mg). zakaria et al. (1998) showed that the enzyme activity from flavobacterium sp. in the medium containing guar gum was the highest, followed by locust bean gum. in another research, hossain et al. (1996), showed that bacillus sp. kk01 in coconut meal resulted in mannanase activity of 0.04 u/mg. in non-mannan medium, 2% relative activity of mannanase was still found. in spite of non-mannan carbon source addition, the base medium might contain small amount of mannan from yeast extract. the mannan from yeast extract could induce mannanase production. there was no mannanase activity in media containing mannan from palm kernel meal. when g. stearothermophilus l-07 was isolated by enrichment in liquid medium supplemented with 1% palm kernel meal as mannan source, the bacterium might survive due to synergism with other bacteria. furthermore, g. stearothermophilus l-07 could not survive alone if it was cultured in palm kernel meal medium. according to sumardi (2005), the palm kernel meal produced inhibiting substance after sterilization using an autoclave. thus, g. stearothermophilus l-07 could neither grow nor produce β-mannanase. the enzyme from g. stearothermophilus l-07 in the locust bean gum was further run on sds-page and analyzed by zymogram assay towards locust bean gum. three bands were observed at molecular mass of approximately 85, 73, and 50 kda, respectively (figure 4). the presence of three isozymes indicated that at least three mannanase encoded genes are present in l-07. other g. stearothermophilus strains have been previously reported to produce mannanase with molecular mass of 76 kda and active at 70oc (ethier et al. 1998). 8 m cr z isolation and characterization of mannanolytic thermophilic bacteria – sumardi et al. kda 85 kda 73 kda 50 kda 94 67 43 30 20 14 figure 4. zymogram of crude mannanase from g. stearothermophilus l-07. lane m indicates the molecular size standard used in this study. lane cr indicates crude enzyme mannanase separated in sds-page. lane z indicates bands of mannanase activity detected by zymogram. conclusions we isolated g. stearothermophilus l-07 from palm shell, a solid waste of palm oil factory in lampung. the bacterium produced extracellular β-1,4mannanase in locust bean gum medium, while β-mannosidase and α-galactosidase were not detected. the optimum enzyme activity was observed after 36 hours of incubation at 60oc. the mannanase comprised three active bands with molecular weight of 85, 73, and 50 kda. acknowledgements this research was financially supported by bpps fellowship from national education department and central microbial diversity of bogor agriculture university. references araujo, a & o.p. ward. 1990. extracellular mannanases and galactanases from selected fungi. j. indust. microbiology. 6: 171-178 aurora d d, y. lestari, & a. meryandini. 2003. identifikasi bakteri penghasil mananase serta karakterisasi enzimnya. j. mikrobiol. indon. 8(1) : 31-33 duffaud,g.d, c.m. mccutchen, p. leduc, k.n. parker, & r.m. kelly. 1997. purification and characterization of extremely thermostable β-mannanase, β-mannosidase, and α-galactosidase from 9 the hyperthermophilic eubacterium thermotoga neopalitana 5068. appl. environ. microbiol. 63: 169-177. biotropia no. 25, 2005 euzeby. 2005. list of prokaryotic names with standing in nomenclature. last full update september 14, 2005. url: http://www.bacterio.net. ethier n, g. talbot, & j. sygusch. 1998. gene cloning, dna sequencing, and expression of thermostable β-mannanase from bacillus stearothermophilus. appl. environ. microbiol. 64: 4428-4432. holt jg, nr krieg, pha sneath, jt staley, & st williams. 1994. bergey,s manual of determinative bacteriology. ninth edition. baltimore : william and wilkins. hossain, h.z., j. abe, & s. hizukuri. 1996. multiple forms of β-mannanase from bacillus sp. kk01. enzyme microb. technol. 18 :95-98. marchesi, j.r.,t. sato, a.j. weightman, t.a. martin, j.c. fry, s.j. hiom, d. dymock, & w.g. wade. 1998. design and evolution of useful bacterium spesific pcr primers that amplify genes coding for bacterial 16s-rrna, j. bacteriol. 64: 795-799. nazina t n, t p tourova, a b poltaraus, e v novikova, a a grigoryan, a e ivanova, a m lysenko, v v petrunyaka, g a osipov, s s belyaev, & m v ivanov. 2001. taxonomyc study of aerobic thermophilic bacilli : description of geobacillus subterraneus gen. nov., sp. nov. and geobacillus uzenensis sp. nov. from petroleum reservoirs and transfer of bacillus stearothermophilus, bacillus thermocatenulatus, bacillus thermoleovorans, bacillus kaustophilus, bacillus thermoglucosidasius, and bacillus thermodenitrificans to geobacillus as the new combinations g. stearothermophilus, g. thermocatenulatus, g. thermoleovorans, g. kaustophilus, g. thermoglucosidasius, and g. thermodenitrificans. int. j. syst. evol. microbiol. 51: 433-446. ooi t & d. kikuchi. 1995. purification and some properties of β-mannanase from bacillus sp. world j. microbiol. biotechnol. 11: 310-314. peer & watcher. 1993. treecon: a software package for the construction and drawing of evolutionary trees. comput. applic. biosci, 9: 177-182 . politz o, m krah, kk thomsen, & r borriss. 2000. a highly thermostable endo-(1,4)β-mannanase from the marine bacterium rhodothermus marinus. appl. microbiol. biotechnol. 53: 715-721. spindler, k.d. & c. rapp. 1997. detection of chitin degrading enzymes on gels in: chitin handbook by eds. r.a.a. muzzarelli & m.g. peter. european chitin society. sumardi. 2005. isolasi, karakterisasi, dan produksi β-mananase ekstraseluler dari geobacillus stearothermophilus l-07 [disertasi]. bogor: sekolah pascasarjana, institut pertanian bogor . torrie, j.p., d.j. senior, & j.n. saddler. 1990. production of β-mannanases by trichoderma harzianum e58. appl. microbiol. biotechnol. 34: 303-307. whistler, r.l. & j.n. bemiller. 1973. industrial gum: polysaccharides and their derivates. academic press. new york. zakaria, m.m., m. ashiuchi, s. yamamoto, & t. yagi. 1998. optimization for β-mannanase production of a psychrophilic bacterium, flavobacterium sp. biosci. biotechnol. biochem., 62: 655-660. 10 http://www.bacterio.net/ introduction isolation of thermophilic mannanolytic strains characterization and identification of isolate extracellular enzyme production enzyme assay results and discussion we found 15 bacterial isolates from palm oil shell. then the bacteria were screened for mannanase activity on selected medium agar plate containing locust bean gum at 70oc with ph 7.0. only isolate l-07 showed mannanase activity. the isolate l-07 could grow at 55-70oc and showed mannanase activity as high as 3.1 u/mg. 6. erina sulistiani et al.cdr agrobacterium page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 730 bambang sulis (proposed model).cdr proposed on levels of model degraded land at merawu watershed, banjarnegara regency, central java province, indonesia bambang sulistyo1*, totok gunawan , hartono , projo danoedoro2 2 2 3and rochmat martanto 1 as 38371faculty of agriculture, universit bengkulu, bengkulu , indonesia 2 as sleman, 55281faculty of geography, universit gadjah mada, yogyakarta , indonesia 3 college 55293national land , sleman, yogyakarta , indonesia received 30 november 2016/accepted 10 june 2017 abstract conservation of degraded land in indonesia requires maps of degraded land. the maps were established based on a model developed in 1998 by the then indonesia department of forestry. the model has 2 weaknesses i.e. 1. high level of uncertainty due to vector-based data used to build the thematic maps and 2. parameters redundancy or duplication from the model. this research was aimed to build up a proposed model on levels of degraded land at merawu watershed using fully raster-based data supported with remote sensing and gis techniques. parameters analyzed were slope, erosivity (r), erodibility (k), slope length and steepness (ls), cover and management (c), support practice (p) and percentage of canopy cover. these data were presented in fully raster format. management parameter was not explicitly used in this research because management parameter was already represented by the c and p parameters. five parameters were directly obtained using fully raster format, i.e. slope, ls, c, p and percentage of canopy cover. the other 2 parameters went through spatial interpolation process before being presented as fully raster format. correlation analysis among parameters was carried out. parameters having high correlation coefficient (r ≥ 0.8) were excluded from the model to avoid redundancy. the proposed model only used parameters having low correlation coefficient. the research result showed that the determination of levels of degraded land was more accurate when using only erosion parameters, formulated as: level of degraded land (lod ) ≈ erosion ≈ r x k x ls x c x pl . keywords: degraded land, gis, raster, remodel, remote sensing introduction soil erosion management is a dilemmatic problem for developing countries such as indonesia. erosion is accelerating from year to year due to the uncontrollable pressure of population and economic growth fulazzaky & ( gany 2009; sulistyo . 2009; bohre & chaubey et al 2014; sulistyo 2015a). there are six types of land degradation i.e. water erosion, wind erosion, soil fertility decline, salinitation, water logging and the lowering of water table (fao 1994). in most of tropical countries such as indonesia, degraded land is mostly caused by water erosion resulted from high intensity of rainfall (abdurachman & sutono 2002), especially when land covers were removed. soil of the bare land would be washed away by the runoff water to the lowest places or estuaries (arsyad 2000). identification of degraded land can be done by investigating watershed in the condition of areas. indonesia, the number of critical highly s and eroded watershed areas increasing from year are to year (hidayat . 2014; kartodihardjo 2008).et al t ,o conserve the degraded land it is important to identify the objectives of the conservation and to disseminate these objectives to the stakeholders in the form of maps of degraded land (tarigan 2012; & ). these gibbs salmon 2015 maps are usually developed from a model consisted of several chosen parameters, to reduce cost et al(sulistyo . 2017). *corresponding author: bambangsulistyounib@gmail. com; bsulistyo@unib.ac.id biotropia 4 3 7 220 229 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 3 730 220 in indonesia, the model and guidelines are published through decree no. 041/kpts/v/ 1998 of 21 april 1998 by the directorate general of reforestation and land rehabilitation, depar tment of forestr y (depar temen kehutanan 1998). this decree is updated through the regulation of the director general of land rehabilitation and social forestry on guidelines for monitoring and evaluation of watersheds, number: p.04/v-set/2009, dated 5 march 2009, but still using the same concept ( ekement rian kehutanan the model implemented 2009). is nationwide. mathematically, the level of degraded land (abbreviated as lodl for further discussion) is formulated as: lodl = w slope + w erosion + w percentage 1 2 3 of canopy cover + w management .. (1)4 where w , w , w and w are weighting factors .1 2 3 4 the weighting factors are determined based on the criteria of the lodl that published and are presented in tables. determination of the lodl is initiated by computing estimated erosion using usle the ( ) developed by universal soil loss equation model wischmeier and smith (1978), and formulated as: a = r k ls c p ......................................... x x x x (2) where: a = mean annual soil erosion rate in (ton/hectare/year) r = rainfall erosivity factor (r factor) in (mj mm/ha/h/year) k = soil erodibility factor (k factor) in (ton hectare/mj/mm) ls = slope length and steepness factor (ls factor) (dimensionless) c = cover and management factor (c factor) (dimensionless) p = support practice factor (p factor) (dimensionless) by replacing erosion in equation (1) with equation (2), a new equation becomes: (3) lodl = w slope + w ( x x x x ) + w r k ls c p1 2 3 percentage of canopy cover + w 4 management ....................................... (3) some weaknesses in the model are: 1) the input data are mostly simplified vector-based thematic maps leading to high uncertainty; and 2) the existing formula or model contains redundant or duplicate parameters. demers (2008) stated that the presentation of earth information in vector data model assumes the existence of homogeneity in a mapping unit as a result of classification and simplification, so that the variation of earth information is reduced, leading to uncertainties. those uncertainties can be minimized by applying a fully-raster-based model which is able to keep the variation and objectivity of earth information ; (aronoff 1989 hadmoko 2007). the fully-raster-based model is among technological advances in remote sensing (rs) and geographical information system (gis) fields. sulistyo a studied land unit elimination (2015 ) analysis to determine lodl using vector-based format. land unit elimination causes a decrease in erosion rate, affects lodl and finally influences the recommendations for conserving degraded land. originally, usle developed in is small agricultural areas in north america having characteristics of moderate climate (low in rain intensity), slope 3 18%, with consistent cropping and management system (asdak 2007). several modifications are needed when implementing usle in loca t ions hav ing d i f f e ren t characteristics, especially modifications on parameters used to provide more reliable erosion estimation. fistikoglu and harmancioglu (2002) concluded that erosion model developed using usle was more reliable when the analysis was carried out using small raster-based data. sulistyo et al proved. (2009) that erosion modeling using fully raster-based data very provided high accuracy compared to the actual erosion. a fullyraster-based erosion model uses raster-formatted data input. the raster-formatted data input are not products of vector to raster conversion algorithm. three usle parameters (ls, c and p) can directly produce raster-formatted data, while data provided by the other two usle parameters (r and k) has to be transformed using spatial interpolation to obtain raster-formatted data. spatial interpolation is the process of using points with known values, to estimate the values of the other points (chang 2008). several parameters of usle were analyzed respectively i.e. r, c, ls, and k (sulistyo 2011a; sulistyo 2011b; sulistyo et al. 2011; sulistyo 221 proposed model on levels of degraded land at merawu watershed, indonesia sulistyo et al. 2015b). percentage of canopy cover was also analyzed (sulistyo et al. 2013). all those parameters were analyzed using raster-based data format. this study was aimed to develop alternative model for lodl at merawu watershed, using raster-based data format to avoid parameters redundancy or duplication. materials and methods this research is the ultimate research on degraded land by the first author. several preliminary studies on this subject were already published by the first author, i.e. sulistyo et al. 2009; sulistyo 2011a; sulistyo 2011b; sulistyo et al. 2011; sulistyo et al. 2013; sulistyo 2015b; sulistyo 2016 and sulistyo et al. 2017. therefore, the data, hardware, software, research areas and methods used in this research were similar to those preliminary studies. study area the study area was located at merawu watershed, geographically lied at 10941'24" – 10950'24" e and 710'12" – 722'12" s, administratively located in banjarnegara district, central java province, indonesia. merawu watershed covered an area of ±22,734 ha with 3 biotropia vol. 24 no. 3, 2017 222 figure 1 study area main rivers flowing through the area from north to south i.e. merawu, urang and penaraban rivers (fig. 1). data and methods data used were topographical map, landform + map, remotely-sensed data of landsat 7 etm , rainfall data recorded at merawu watershed and its surroundings, as well as other data and reports related to the study. gis software used were ilwis (integrated land and water information system) version 3.4 and arcview version 3.3. several equipment used for field work were binoculars, compass, hagameter, munsell soil color chart, tape, soil sample ring kit, auger soil boring tool, global positioning system tool and digital camera. data from all parameters affecting lodl were analyzed as fully-raster-based format. pixel size used for this study was 30 x 30 m. lodl model was developed using fully-raster-based data format (fig. 2). the main research stages were creating all lodl parameters, then implementing correlation analysis among parameters. to implement correlation analysis among parameters, all data had to be presented in quantitative form. to fulfil this requirement, the management factor as qualitative factor had to be excluded from equation (3). moreover, management factor were represented by c and p factors. for further analyses, equation (3) was modified to: lodl = w slope + w (r x k x ls x c x p) + w 1 2 3 percentage of canopy cover (4) ............. showedequation (4) explicitly that the number of parameters slope, r s lodl were factor, k factor, ls factor, c factor, p factor, and percentage of canopy cover . (7 parameters) creatin arametersg lodl p lodl parameters were created by similar methods as the previous studies carried out by the first author (sulistyo et al. 2009; sulistyo 2011a; sulistyo 2011b; sulistyo et al. 2011; sulistyo et al. 2013; sulistyo 2015b; sulistyo 2016; sulistyo et al. 2017). the exception was the use of ndvi to create c factor; therefore, c factor was formulated as (sulistyo et al. 2011): c = 0.6 – 0.77 x ndvi (5) ....................................... percentage of canopy cover was formulated as (sulistyo et al. 2013): % cc = 118.33 x ndvi – 17.605 (6) ..................... the ndvi can be extracted from satellite data to land cover according to obtain information on its density formulated as (silleos et al.. ndvi was 2006): ndvi = (nir – r) / (nir + r) (7) ....................... where: nir near infrared band= r red band= 223 proposed model on levels of degraded land at merawu watershed, indonesia sulistyo et al. figure 2 flow diagram of the study cor re lat ion analysis among lodl parameters in relation to the raster-based degraded land modeling, each parameter contained spatial information pixel valueor . pixel value for each parameter of the lodl was then viewed as pixel value for each band or channel in remote sensing digital image. c , as one of multi-band orrelation analysis statistics in remote sensing analysis, was applied to determine the closeness of one band toward other bands (ilwis user's guide 2002; lillesand et al. 2004). ndividual bands of a multi-spectral image i a i ying datare often highly correlated, mpl redundancy . and duplication correlation matrix was implemented t o evaluate the degree of correlation between individual bands. correlation matrix (a normali ed form of covariance matrix) z ha values in the range of -1 to 1 representing a s , strong negative correlation to a strong positive correlation respectively alues close to zero , . v represent correlation. weak bands showing the least correlation, which implied the largest amount of image variations, were chosen to be included in the multi-band composite, prior to conducting multi-spectral classification analysis. lodl parameters having the least correlation value were chosen to be included in the model. c orrelation value is the classified as “high” if correlation value is ≥ 0.8 gordon et al. 1992). ( results and discussion maps of each lodl parameters in rasterbased data format are presented in figures 3 to 9, while the correlation matrix of lodl parameters is presented in table 1. table 1 shows that almost all lodl parameters has correlation values i.e. between 0.01 (p low factor and k factor) and 0.44 (p factor and slope). percentage of canopy cover had high negative correlation value with c factor (r = -1.00), meaning that an increase in percentage of canopy cover would cause a decrease in c factor. slope had correlation value with ls factor (r high positive = 0.99), meaning that an increase in slope would cause an increase in ls factor. et al to berenard . (1997) formulated c factor calculated multiplying rior and se from p l u (plu) ercentage of anopy over assessed and p c c for different over types (cc) and surface cover c (sc). inclusion of the ercentage this meant that p of anopy over in formula s c c lodl wa redundant. slope length and steepness factor (ls factor) consist of two subfactors the slope length (l) s , i.e. and the slope gradient (s). lope length (l) s is defined as: “the distance from the point of origin of the surface flow to the point where each slope gradient (s) decreases enough for the beginning of deposition or when the flow comes to concentrate in a defined channel” wischmeier ( & smith 1978). that inclusion of slope this meant in formula redundant.lodl was based on wasthose results and reasons, it concluded that percentage of canopy cover should not be used simultaneously with c factor in determining the lodl slope and that should not be used simultaneously with ls factor. therefore, should formula to determine lodl only use parameters of usle model because usle considered all biophysical model has aspects affecting lodl. revised model the to calculate lodl is: lodl ≈ erosion ≈ x x x x (8)r k ls c p .......... the revised model is in line with the conclusion stated by abdurachman and sutono (2002) that the lodl in indonesia is mainly caused by erosion due to high amount and 224 biotropia vol. 24 no. 3, 2017 table 1 correlation matrix of lodl parameters c k ls p slope % canopy r c 1.00 0.09 -0.26 -0.32 -0.26 -1.00 -0.04 k 0.09 1.00 0.06 0.01 0.06 -0.09 0.21 ls -0.26 0.06 1.00 0.39 0.99 0.26 0.01 p -0.32 0.01 0.39 1.00 0.44 0.32 0.06 slope -0.26 0.06 0.99 0.44 1.00 0.27 0.03 % canopy -1.00 -0.09 0.26 0.32 0.27 1.00 0.04 r -0.04 0.21 0.01 0.06 0.03 0.04 1.00 225 proposed model on levels of degraded land at merawu watershed, indonesia sulistyo et al. figure 3 map of r factor figure 4 map of k factor figure 5 map of ls factor figure 6 map of c factor 226 biotropia vol. 24 no. 3, 2017 figure 7 map of p factor figure 8 map of slope figure 9 map of percentage of canopy cover intensity of rainfall. the erosion terminology is used worldwide as an indicator of degraded land occurrence. this model is identical with the level of erosion hazard (in bahasa: tingkat bahaya erosi or tbe) which is formulated in the decree no. 041/kpts/v/1998 of the then indonesia's ministry of forestry. level of erosion hazard was obtained by overlaying the erosion hazard map, derived from the usle model, with soil depth map. the average of soil depth at merawu w wa (very deep) therefore atershed s 2.16 m . , the revised model of this study lodl only co ed erosion . e s of nsider the occurrence th result this revised lodl model were simplified into 5 classes based on sturges' rule (fig. 10; table 2). table 2 shows that 2,151 ha (9.5%) of merawu watershed were areas having vegetation index ≤ 0. those areas were areas with water. merawu watershed area had 15,891 ha (70%) of very low and levels of degraded land. the other 4,689 low ha (20.6%) had and levels moderate, high very high of degraded land. conclusions percentage of canopy cover should not be used simultaneously with c factor in determining the lodl. slope should not be used simultaneously with ls factor. the best scenario was to use only erosion parameter derived from the usle model to determine the lodl model. the proposed lodl model: lodl ( ) ≈ erosion ≈ r level of degraded land x k x ls x c x p. proposed model on levels of degraded land at merawu watershed, indonesia sulistyo et al. table 2 the area of each class of the lodl at merawu watershed no level erosion (mm/year) area (ha) area (%) 1. very low 0.01 to 1 12,470 54.9 2. low 1.01 to 2 3,421 15.1 3. moderate 2.01 to 3 1,714 7.5 4. high 3.01 to 4 982 4.3 5. very high > 4 1,993 8.8 6. areas having vegetation index 2,151 9.5 total 22,734 100.0 (-) figure 10 results of the revised lodl model 227 ≤ 0 acknowledgements the authors thank the indonesian directorate general of higher education for providing grant to conduct this research. references abdurachman a, sutono. 2002. soil erosion control technology. research and development of soil and agro-climate. bogor (id): the agency for agricultural research and development. aronoff s. 1989. geographic information systems: a management perspective. ottawa (ca): wdl publication. arsyad s 2000 konservasi anah an ir ipb . . t d a . bogor (id): press. asdak c. 2007. hidrologi dan engelolaan aerah liran p d a s yogyakarta (id):ungai. gadjah mada university press. bohre p chaubey op. 2014. restoration of degraded lands , through plantation forests. 14:18-27.gjsfr chang kt. 2008. introduction to geographic information s new york (us) :ystems. mcgraw-hil l international. demers mn. 2008. fundamental of eographic g i s . new york (us):nformation ystems john wiley & sons. departemen kehutanan 1998 guideline for the . . a p l rrrangement of lanning for and ehabilitation in a w a . (id)atershed rea jakarta . f a o 1994 land ood gricultural rganization [fao]. . degradation in south asia: its severity, causes and effects upon the people world soil . rome (it): resources reports. fulazzaky ma gany aha. 2009 challenges of soil erosion , . and sludge management for sustainable development in indonesia. j environ manage 90:2387-92. fistikoglu o harmancioglu nb 2002 integration of gis , . . with usle in assessment of soil erosion. water resour manag 16:447-67. gibbs hk, salmon jm. 2015. mapping the world's degraded lands. appl geogr 57:12-21. gordon nd, mcmohan ta, finlayson bl. 1992 stream . h a i e . new york idrology: n ntroduction for cologist (us): . john wiley & sons hadmoko ds. 2007. toward gisased integrated landslide b hazard assessment: critical overview. indon j a es geogr 39:55-77. hidayat l, susanto s, sudira p, jayadi r. 2014. penilaian kinerja pengelolaan daerah aliran sungai berbasis model hidrologi elementer kasus: daerah tangkapan air waduk mrica. agritech 34:337-46. international institute for aerospace survey and earth sciences ilwis user's guide. enschede. 2002. (nl): itc-ilwis. kartodihardjo h. 2008. management of natural resources and environment. bengkulu (id): universitas bengkulu. kement rian kehutanan 2009 peraturan direktur e . . jenderal rehabilitasi lahan dan perhutanan sosial tentang pedoman monitoring dan evaluasi daerah aliran sungai, nomor: p.04/v-set/2009, tanggal: 5 maret 2009. jakarta (id). lillesand tm, kiefer rw, chipman j. 2004 remote ensing . s an mage nterpretationd i i . fifth edition. new york (us): . john wiley & sons renard kg, forster gr, weesies ga, mccool dk yoder , dc. 1997. predicting soil erosion by water: guide a to conservation planning with the revised universal soil loss equation (rusle) . washington dc (us): us d a epartment of griculture. silleos ng, alexandridis tk, gitas iz perakis k. 2006. , vegetation ndices: dvances ade in iomass i a m b e v m lstimation and egetation onitoring in the ast 30 y .ears geocarto int 21:21-8. sulistyo b, gunawan t, hartono, danoedoro p. 2009 . toward a fully and absolutely raster-based erosion modeling by using rs and gis indones j geogr. 41:149-70. sulistyo b, gunawan t, hartono, danoedoro p 2011 . . pemetaan faktor c yang diturunkan dari berbagai indeks vegetasi data penginderaan jauh sebagai masukan pemodelan erosi di das merawu (c actor f m d f v v iapping erived rom arious egetation ndices of r s d a i f e memotely ensed ata s nput or rosion odeling a . t merawu watershed) jml 18:68-78. sulistyo b. 2011 pengaruh rosivitas ujan yang iperoleh a. e h d dari umus yang erbeda terhadap emodelan rosi r b p e b r kerbasis aster (studi asus di das merawu, banjarnegara, jawa tengah) (the ffect of ain e r e g d f arosivity enerated from ifferent ormulae on r b e .asterased rosion) agritech 31:250-9. sulistyo b. 2011 the fect of hoosing ifferent ontour b. ef c d c i o a f r b e mnterval n ully asterased rosion odeling (the ase n merawu watershed, banjarnegara, c i central java) 16:258-66.. jts sulistyo b, gunawan t, hartono, danoedoro p. 2013 . pemodelan persentase tajuk di das merawu yang diturunkan dari berbagai indeks vegetasi data penginderaan jauh (modeling of ercentage of p c a c area d fanopy t merawu atchment erived rom v v i r s d . arious egetation ndices of emotely ensed ata) for geo 27:23-32. sulistyo b. 2015a. kajian perubahan tingkat kekritisan lahan sebagai akibat proses eliminasi unit lahan: studi kasus di kawasan pertambangan danau mas hitam, 228 biotropia vol. 24 no. 3, 2017 provinsi bengkulu (the study of land degradation level as a result of land unit elimination process: case study in mining region of danau mas hitam, bengkulu province). national seminar on biodiversity, university club. yogyakarta (id): university of gadjah mada. p. 828-33. sulistyo b. 2015b. pemodelan faktor k berbasis raster sebagai masukan pemodelan erosi di das merawu, banjarnegara, provinsi jawa tengah (modeling of raster-based of k factor as input for erosion modeling at merawu catchment area, banjarnegara, central java province). jurnal manusia dan lingkungan 22:240-6. sulistyo b. 2016. the effect of choosing three different c factor formulae derived from ndvi on a fully rasterbased erosion modeling. iop conf ser: earth environ sci 47(1):1-10. doi:10.1088/17551315/47/1/012030. sulistyo b, gunawan t, hartono, danoedoro p, listyaningrum n. 2017. tabsolute accuracy of he erosion model of dem-ndvi nd t s a i ' modification. ijg 13:23-34. tarigan sd. 2012. methods for delineating degraded land at citarum watershed, west java, indonesia. j tropical soils 17:267-74. wischmeier wh, smith dd 1978 predicting ainfall . . r e l a g c prosion osses: uide to onservation lanning. washington dc (us): usda agriculture handbook no. 37. 229 proposed model on levels of degraded land at merawu watershed, indonesia sulistyo et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 2. diah ratnadewi.cdr biotropia vol. 20 no. 1, 2013: 10 18 enhanced production level of quinine in cell suspension culture of cinchona ledgeriana moens by paclobutrazol 1 2 3diah ratnadewi *, dedi satriawan , and sumaryono 1department of biology, faculty of mathematics and natural sciences, bogor agricultural university, darmaga campus, bogor 16680, indonesia 2department of biology, faculty of mathematics and natural sciences, bengkulu university, kandang limun campus, bengkulu, indonesia 3indonesian biotechnology research institute for estate crops, jalan taman kencana no. 1, bogor 16151, indonesia received 10 januari 2013/accepted 22 may 2013 abstract quinine is one of the major alkaloids in cinchona spp., and it is used both as a medication and as a drink additive. the plant produces most of its alkaloids in the bark after 6-8 years of age. repeated harvests can be performed until the plant dies, but only after every 3-5 years. we tested an improved method for culturing cell suspensions of cinchona ledgeriana investigate the possibility of increasing production of quinine. the clone qrc 315 was treated with either a growth retardant (abscisic acid or paclobutrazol) or precursor feeding of ltryptophan. to generate stress, we applied mannitol at 5.3 g/l combined with sucrose at a lower concentration (20 g/l), and we used sucrose at 30 g/l as the control. paclobutrazol (7 mg/l) significantly suppressed cell growth and produced the highest level of quinine (11%) after 7 weeks of culture. l-tryptophan also reduced cell growth, but without any positive effect in the production of quinoline. the highest amount of quinine per culture flask, however, resulted from cells treated with 3 mg/l abscisic acid. key words: cinchona ledgeriana, suspension culture, quinine, paclobutrazol introduction more than 30 alkaloids have been isolated from the bark of cinchona spp. but the most economically important cinchona alkaloids are the quinolines: quinine, quinidine, cinchonine, and cinchonidine (mccalley 2002). quinine is the major alkaloid and has been used for hundreds of years as an anti-fever agent and for prevention and treatment of malaria. malaria still exists in many tropical areas of the world. the world health organization (2011) reported 36 000-655 000 deaths . . * corresponding author : diahbiologi.ipb@gmail.com 10 doi: 10.11598/btb.2013.20.1.10 between 2009 and 2010 in 106 malaria-endemic countries. apart from its use to treat malaria, quinine also has analgesic and anti-inflammatory properties; its sulfate salt is used in eye lotions as an antibacterial and anesthetic agent (taylor 2005, kurian & sankar 2007). south americans use quinine as a natural remedy for cancer, amoebic infections, heart problems, diarrhea, dysentery, varicose veins, and nocturnal leg cramps (taylor 2005). in addition, quinine salts are added to tonic drinks and beverages to achieve a bitter taste. taylor (2005) reported a commercial market of approximately 300-500 metric tons of quinine alkaloids, which are extracted annually from 5000-10000 metric tons of harvested bark. at the time of that report, half of the harvest was directed to the food industry. the bark of wild cinchona yields 7% quinine, whereas cultivated crops yield up to 15%, none of which can be obtained until after the plant is 6-12 years old (barrett 1928). quinine production from its cultivation is, therefore restricted by these growth limitations. cell suspension culture of cinchona spp. could solve the above problem. several groups of researchers have attempted enhancement of cinchona alkaloid production in cell suspension cultures by using stressing agents, precursors, elicitors, auxins, and enzymes (harkes et al. 1986, wijnsma et al. 1986, payne et al. 1987, robins et al. 1987, toruan-mathius et al. 2006). previous research on c. ledgeriana clone qrc 313 increased quinine content to 0.12%, which was 11-fold more than that in control cells (ratnadewi & sumaryono 2010). although promising, this result is still far from satisfactory. in this study, we sought to improve quinine production methods by testing a selected potential clone of c. ledgeriana in treated cell-suspension cultures. materials and method preparation of cell-suspension cultures lamina of young leaves (the second or the third leaf from the shoot tip) from the c. ledgeriana clone qrc 315 were surface-sterilized with 0.2% dithane m-45 for 10 minutes, followed by treatment with 20% commercial naocl for 15 minutes. these lamina were used as explants for callus production on semi-solid woody plant (wp) media (lloyd & mccown 1981), which contained 15 µm picloram, 2 µm benzyladenin (ba), 1 µm phloroglucinol, and was solidified with 3.5 g/l gelrite (mb cell). sucrose, at a standard level (30 g/l), was added to the media. after 8 weeks in the initiation and proliferation media, fast-growing calli were transferred to baffle flasks that contained wp liquid media, and homogenized for 1 week before being used for cell suspension culture. liquid media composition was the same as the semi-solid, except that ba was reduced to 0.5 µm. this procedure was taken from that described by sumaryono and riyadi (2005), with slight modification. one spatula (0.2-0.3 g) of cells, which had been filtered through 1000 and then 50µm filter mesh, was transferred into erlenmeyer flasks containing 20 ml liquid wp media, of which the composition was the same as for cell homogenization. this was designated as the basic media for this research.we varied the basic media by addition of certain growth regulators: either (1) abscisic acid (aba) or paclobutrazol (pbz); 11 enhanced production level of quinine by paclobutrazol – diah ratnadewi et al. and (2) a precursor, l-tryptophan (trp), in combination with sucrose (at either a standard or lower concentration) with or without mannitol. we applied the following treatments to the basic media: (1) 1 mg/l aba + 30 g/l sucrose(aba1), 3 mg/l aba + 30 g/l sucrose (aba3), 0.2 mg/l tryptophan + 20 g/l sucrose + 5.3 g/l mannitol (trp0.2-m); (2) 2 mg/l tryptophan + 20 g/l sucrose + 5.3 g/l mannitol (trp2-m); 5 mg/l pbz + 20 g/l sucrose + 5.3 g/l mannitol (pbz5-m); and 7 mg/l pbz + 20 g/l sucrose + 5.3 g/l mannitol (pbz7-m). the molarity of 30 g/l sucrose was equivalent to that of 20 g/l sucrose + 5.3 g/l mannitol. the cell suspension cultures were maintained for 7 weeks on a horizontal shaker, at 100 rpm, 26 ± 1°c, 2under light intensity of 20 µmol photon/m /second for 12 hours per day. cell growth was measured once per week until the cells were harvested. we used cell volume after sedimentation (cvs) to represent cell growth, according to the methods described by blom et al. (1992).cell volume (a ml) was then calibrated into fresh weight (g) using the following formula: {(a 0.4565) 0.3252}. quinoline extraction and analysis at weeks 6 and 7, five flasks of suspension culture were simultaneously harvested from each treatment for quinoline alkaloid content because cinhona ledgeriana cells achieve their maximum growth at week 6 (sumaryono & riyadi 2005). reversed-phase high-performance liquid chromatography (hplc; varian-usa, type 940) was used to measure the four major quinolines (quinine, quinidine, cinchonine, and cinchonidine). quinoline extraction and analysis was performed as described previously by klink (1979), with some modifications. briefly, as much as 0.5 g oven-dried cell powder was taken for the extraction and purification, boiled in 95 ml aquadest for 25 minutes, and then allowed to cool at room temperature. the remaining liquid was then adjusted to 100 g by adding aquadest. a 5-ml aliquot was taken from decanted solution and filtered using a 0.45-µm millipore filter then injected into an hplc column (pursuit xrs3 µ c-18, column length 150 cm x 4.6 mm). the column temperature was adjusted oto 30 c. aquadest, acetonitril, and glacial acetic acid (ratio 81:18:1, respectively) served as an eluent using a 0.6 ml per minute flow rate with six attenuation. standard quinoline alkaloids (sigma, germany) served as positive controls. the chromatogram was detected by uv-vis at 250 nm. data from each treatment were collected from duplo analysis. results and discussion cell growth one week after being transferred into treated liquid media, the cells grew well. treatment with aba with the normal rate of sucrose (30 g/l) demonstrated the highest growth, whereas trp0.2-m, trp 2-m, pbz5-m, and pbz7-m resulted in lower performance, even compared to the control. pbz7-m greatly restricted cell growth, whereas pbz5-m slightly reduced cell growth. in general, the highest fresh weight was reached at the sixth week in the treated media, and it decreased afterwards (fig. 1-a, 12 biotropia vol. 20 no. 1, 2013 -b, -c). this was consistent with the results of sumaryono and riyadi (2005). in previous work (ratnadewi & sumaryono 2010), the incorporation of aba and pbz into the media at the fifth week of cell culture had no negative effect on cell growth. in this research, aba treatment from the beginning of the culture even increased growth. aba can both promote and inhibit plant growth (finkelstein & rock 2002). it has a complex role in various cellular processes, such as embryo maturation and germination, vegetative growth, and tolerant response to drought stress (xiong & zhu 2003 hirayama & shinozaki 2007, hubbard et al. 2010). figure 1. cell growth in suspension culture of c. ledgeriana treated with aba (a), tryptophan (b) and pbz (c). data are expressed as mean ± sd (n = 10). c, control media/basic media; aba1, basic media treated with1 mg/l aba; aba3, basic media with 3 mg/l aba; trp0.2-m, basic media with 0.2 mg/l tryptophan + 20 g/l sucrose + 5.3 g/l mannitol; trp2-m, basic media with 2 mg/l tryptophan + 20 g/l sucrose + 5.3 g/l mannitol; pbz5-m, basic media with 5 mg/l pbz + 20 g/l sucrose + 5.3 g/l mannitol; pbz7-m, basic media with 7 mg/l pbz + 20 g/l sucrose + 5.3 g/l mannitol. enhanced production level of quinine by paclobutrazol – diah ratnadewi et al. 13 in contrast, we observed that growth inhibition of pbz, applied from the starting point of culture, reduced cell growth. we hypothesized that the inhibiting action of pbz required more time to cause an effect. by increasing the concentration of pbz to 7 mg/l, combined with 5.3 g/l mannitol and 20 g/l sucrose, the cell growth rate diminished significantly (fig. 1-c). pbz is known to be an antagonist of gibberellins. it inhibits gibberellin biosynthesis at the point when kaurene is oxidized into kaurenoic acid (graebe 1987, chaney 2004), because gibberellin and aba biosynthesis begin at the same initial pathway (i.e., from geranyl pyrophosphate),both growth regulators may be affected by pbz along their synthesis pathways. also, blockage of the terpenoid pathway causes accumulation of aba (chaney 2004) and could therefore, lead to unbalanced cellular metabolism. this, in turn, would reduce the rate of cell division (graebe 1987). chaney (2004) suggested that these cells will still divide and differentiate at the same rate but simply do not elongate. this may explain why cell growth was lower in the cultures treated with pbz. one of the precursors in the biosynthesis of quinoline alkaloids is tryptophan (facchini 2001). we used it in combination with mannitol as a partial substitute of sucrose in the culture media with the expectation that it would increase the production of quinoline in cells. tryptophan is also involved in the first step of the auxin biosynthesis pathway. in this study, cell growth rate in tryptophan-treated media, however, was less than that of untreated cells. this may be because tryptophanderived auxin either was not at an optimal level or simply auxin synthesis from tryptophan did not occur. sucrose is the main source of energy and carbon in in vitro cultures. substitution of some sucrose in media with mannitol reduces the supply of carbon and energy to cells because mannitol is an alcohol sugar that cannot replace sucrose (although it determines the osmotic potential value of media). mannitol has been used as an osmotic stressing agent in many experiments (hassanein 2004, 2010; hussein & aqlan 2011). le clere et al. (2010) confirmed that sugar concentration in in vitro-cultured maize kernels regulates the expression of the gene, zmyuc, which is involved in the biosynthesis of auxin through hydroxylation of tryptamine. reduced levels of sucrose in the culture media, along with mannitol substitution, combined with tryptophan, might explain the lower growth rate of the corresponding treated cultures, which may have used less tryptophan for the synthesis of auxin. quinine in the suspension cultures in normal culture media, cinchona cells produced quinoline alkaloids as demonstrated by robins et al. (1986) and ratnadewi and sumaryono (2010). quinine appeared to be the main alkaloid produced by the cells, occurring at levels of 4-7% in the control media (c). we found all four alkaloids at the sixth week of culture in all treatments (table 1). cinchonidine was the least abundant, followed by cinchonine, quinidine, and quinine, which was the most abundant. this percentage of quinine was further augmented 1 week later, at the seventh week of culture, which was in contrast to our results for quinidine, cinchonine, and cinchonidine. the four types of alkaloid are readily 14 biotropia vol. 20 no. 1, 2013 transformed from one to another type due to their similar molecular structure (tadeusz 2007). all the treatments applied to the suspension cultures were best at producing quinine rather than the other types of quinoline. cell suspension treated with pbz7-m presented a remarkably high percentage of quinine content (11%). this value was 1.5-fold higher than that of the untreated cultures at the seventh week. in cells treated with aba, trp-m or pbz5-m, the quinine level was either at an average level or less than that contained in the untreated cells (c). precursor feeding of tryptophan did not improve quinoline alkaloid production in cinchona cell cultures. this is consistent with the results obtained by robins et al. (1987) in c. pubescens and by payne et al. (1987) in a transformed line of c.ledgeriana. harkes et al. (1986) and robins et al. (1987) found an accumulation of β-carboline alkaloids and indole-3-aldehyde in tryptophan-fed growth media, with and without cinchona cells. according to cao et al. (2007), β-carboline alkaloids (a large group of indole alkaloids) are easily transformed from tryptophan, tryptamine, pyruvate, or acetate precursors by pictet-spengler reactions, even in foods and beverages. this indicates that the transformation of tryptophan to quinoline alkaloids needs more particular conditions in living cells. moreover, payne et al. (1987) suggested that growing cultures in darkness is more favorable than under light when attempting to enhance alkaloid accumulation. ratnadewi and sumaryono (2010) found that providing 0.2 mg/l tryptophan to the c ledgeriana clone qrc 313 increased total alkaloids (predominantly cinchonidine) 35fold more than those in untreated cultures after 6 weeks. cinchona plants naturally have variable types of alkaloids. according to taylor (2005), quinine content averages 1-3% in c. pubescens and 3-13% in c ledgeriana, whereas c. succirubra can reach 4-14% but only 6-8 years after planting. when we examined the dry weight cells collected from each culture flask and the quinine content percentage, we found that cells treated with aba which had the highest rate of cell growth produced the highest amount of quinine at 6 and 7 weeks of culture at the end, compared to any other treatment (table 2-a and -b) and aba3 had the greatest abundance of quinine (almost 3 times more than that produced by pbz7-m per flask). it remains challenging to improve quinine production in cells treated with pbz7-m. table 1. quinoline content in cells of c. ledgeriana at 6 and 7 weeks of culture (% dry weight) quinine quinidine cinchonine cinchonidine treatment 6 weeks 7 weeks 6 weeks 7 weeks 6 weeks 7 weeks 6 weeks 7 weeks c 4.60 7.12 2.61 1.40 0.69 0.42 0.40 0.18 aba1 3.97 5.7 2.40 2.32 0.57 0.56 0.27 0.20 aba3 5.08 6.83 3.11 2.19 0.46 0.37 0.51 0.15 trp0.2-m 3.72 5.12 2.45 1.48 0.80 0.49 0.29 0.22 trp2-m 4.36 6.04 1.97 1.26 0.62 0.50 0.32 0.34 pbz5-m 5.01 5.75 2.80 2.07 0.73 0.71 0.19 0.28 pbz7-m 4.15 10.90 3.66 2.39 2.90 0.65 0.28 0.30 note : these data are means of duplo analysis, collected each from 5 flasks of cell culture enhanced production level of quinine by paclobutrazol – diah ratnadewi et al. 15 conclusions the growth retardant paclobutrazol at 7 mg/l combined with 5.3 mg/l mannitol and a lower concentration of sucrose (20 mg/l) significantly increased the accumulation of quinine in the cells of c. ledgeriana clone qrc 315. quinine approached the level extracted from productive c. ledgeriana bark. precursor feeding by l-tryptophan to the cell culture media increased neither quinine nor the other major quinoline alkaloids. the way in which it transforms to quinoline alkaloids remains incompletely understood. the high growth rate of cells treated with 3 mg/l aba and sucrose at normal concentration (30 g/l) resulted in the most abundance of quinine per culture flask. acknowledgments the authors thank the iaard of the indonesian ministry of agriculture for sponsoring this research through the incentive program. table 2. production of quinine per flask of cell culture of c.ledgeriana treated with various substances after 6 (a) and 7 weeks (b) treatment means of cell dry weight/flask (g)* quinine (%)** quinine (mg/flask) c 0.122 ± 0.042 4.6 0.561 aba1 0.201 ± 0.024 3.97 0.722 aba3 0.182 ± 0.046 5.08 1.021 trp0,2-m 0.091 ± 0.018 3.72 0.338 trp2-m 0.106 ± 0.032 4.36 0.462 pbz5-m 0.116 ± 0.078 5.01 0.581 pbz7-m 0.064 ± 0.041 4.15 0.265 treatment mean of cell dry weight/flask (g) * quinine (%) ** quinine (mg/flask) c 0.115 ± 0 .045 7.12 0.819 aba1 0.175 ± 0 .032 5.70 0.997 aba3 0.206 ± 0 .043 6.83 1.407 trp0,2 -m 0.091 ± 0 .023 5.12 0.466 trp2 -m 0.111 ± 0 .030 6.04 0.670 pbz5 -m 0.097 ± 0 .064 5.75 0.558 pbz7 -m 0.050 ± 0 .037 10 .90 0.545 a b 16 biotropia vol. 20 no. 1, 2013 references barrett. 1928. the tropical crops. new york: the mcmillan. blom tjm, kreis w, van iren f, libbenga kr. 1992. a non-invasive method for the routine-estimation of fresh weight of cells grown in batch suspension cultures. plant cell rep 11:146-9. cao r, peng w, wang z, xu a. 2007. β-carboline alkaloids: biochemical and pharmacological functions. curr med chem14: 479-500. chaney wr. 2004. paclobutrazol: more than just a growth retardant. pro-hort conf. peoria il: febr 4th. department of forestry and nat res, purdue university.www.ina online.org/prohort/treegrowth. pdf. facchini pj. 2001. alkaloid biosynthesis in plants: biochemistry, cell biology, molecular regulation and metabolic engineering applications. ann rev plant physiol plant mol biol 52:29-66. finkelstein rr, rock cd. 2002. abscisic biosynthesis and response. in: the arabidopsis book. new york. amer soc plant biologists1-52. graebe je. 1987. gibberellin biosynthesis and control. annu rev in plant physiol 38: 419-65. harkes paa, de jong pj, wijnsma r, verpoorte r, van der leer. 1986. the influence of production media on cinchona cell cultures; 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[tropical plant database: file for quinine (cinchona officinalis). rainforest database.com/plants/quinine.htm. 2005]. cited 13 august 2012. toruan-mathius n, haris n, santoso j, ade-heri. 2006. effect of elicitation on growth and alkaloid quinoline production in hairy root of cinchona plant (cinchona succirubra pavon ex klotzsch). menara perkebunan 74:10-22. wijnsma r, verpoorte r, harkes paa, van vliet tb, ten hoopen hjg, svendsen ab. 1986. the influence of initial sucrose and nitrate concentration on the growth of cinchona ledgeriana cell suspension cultures and the production of alkaloids and anthraquinones. plant cell tiss org cult 7: 21-9. world health organization: world malaria report. [http://www.who.int/malaria/world_malaria_report2011/en/2011]. cited 26 august 2012. xiong l, zhu j-k. 2003. regulation of abscisic acid biosynthesis. plant physiol 133: 29-36. 18 biotropia vol. 20 no. 1, 2013 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 5. samsuri (restoration) revi... biotropia vol. 21 no. 2, 2014: 111 124 restoration priority index development of degraded tropical forest landscape in batang toru watershed, north sumatera, indonesia samsuri , i nengah surati jaya , cecep kusmana and kukuh murtilaksono , received 15 april 2014/accepted 6 september 2014 tropical forest fragmentation has triggered forest degradation and decreased forest connectivity. further, degradation of tropical rain forest area reduces forest functions as global biological resources and affects livelihood of rural communities. to regain forest functions, a landscape restoration approach is needed. the research aimed to develop a restoration index model for degraded tropical forest landscapes in batang toru watershed. the proposed restoration index was constructed by four indices i.e. forest degradation, forest connectivity, forest fragmentation, and socio economic indices. regression analysis was conducted to develop models for four indices. discriminant analysis was conducted to obtain restoration index model. the forest landscape fragmentation index increased in the period of 1989-2013, while connectivity index tended to decrease during the same period. forest connectivity index had higher weight than other indices in calculating restoration index. this indicated that connectivity of batang toru forest landscape needed to be maintained and enhanced to ensure habitat quality and reduce loss of biodiversity. the research showed that sarula sub-watershed had high restoration index value, so it should be the first area to be restored. connectivity, fragmentation, fragstats, forest ecosystem, spatial 1* 2 3 4 1 department of forestry, faculty of agriculture sumatera utara university medan, indonesia department offorest management, faculty of forestry, bogor agricultural university bogor, indonesia department of silviculture, faculty of forestry, bogor agricultural university bogor, indonesia department ofwatershed management,faculty of agriculture, bogor agricultural university bogor, indonesia 2 3 4 abstract keywords: * corresponding author : gsamsuri@gmail.com doi: 10.11598/btb.2014.21.2.5 111 introduction recently, forest management is facing various problems arising either from natural disaster or from human activities such as forest fire, landslide, illegal logging, encroachment, shifting cultivation, and forest conversion. in some areas, the condition of degraded forest has been at a critical level; hence it might threaten the sustainability of forest ecosystem. forest degradation has also become a global issue because it gives significant influence on global climate change. according to itto/iucn (2005), the landscape of world's tropical forests remains 45%, and fragmented spatially in degraded condition. degradation of tropical rain forest reduces global biological resources, and causes poverty occurrence in communities in and around the degraded forest (lamb . 2005). during the period from 1970 to 1990, the degraded forest area in indonesia was estimated to be between 0.6 1.2 million ha per year. the forest degradation rate in the period of 1986 -1997 was about 1.7 million ha per year (fwi/gfw 2002); between 1994 and 2000 it was about 2.83 ha million per year (mof 2005), and in the period of 1970-1990 it was about 1.2 million per year. according to fao (2011), forest decrease in indonesia was at an estimated rate of 0.5% in the period of 2000-2010 and 1.7% in the period of 1990-2000 (fig. 1). this decrease is higher than the average of southeastern asia's which is at the level of 1% in the period of 1990-2000 and 0.4% in the period of 2000-2010 (fao 2011). et al figure 1. deforestation rate in several south east asia countries during 1990 2010 112 biotropia vol. 21 no. 2, 2014 in north sumatera, deforestation was estimated to be 6,508,525 ha (28% of total forest area) in the period of 1985-1997 (fwi/gfw 2002). batang toru forest as one of remaining tropical forest ecosystem in sumatera has deforestation rate of about 1.17% per year from 1994 to 2009. the area decreased from 160,000 ha to 150,000 ha in 2009. batang toru gets high pressure because of the openness of access resulting from forest encroachment and forest cover change. encroachment and forest cover change are the main drivers of forest degradation (mendoza . 2005; reddy 2013). degraded forest may cause flood, erosion and landslides (cotler & ortegalarrocea 2006). on the other hand, batang toru forest landscape is habitat for 67 species of mammals, 287 species of birds, 110 types of herpetofauna and 688 types of plants. batang toru is the habitat of the orang-utan ( ). the numbers of orang-utan are about 15% of total population of orang-utan in sumatera which is estimated to be about 6,600 individuals. besides providing important habitat for orang-utan, batang toru landscapes are also home for other rare animals such as , and antelopes. furthermore, batang toru forest landscape is also a habitat for unique flora of sumatera such as and several types of orchids (perbatakusuma & kaprawi 2011). the importance of the ecosystem functions of the batang toru as a regulator of hydrologic cycle and wildlife habitat should be maintained. therefore, forest and land degradation should be reduced by regaining landscape condition of batang toru forest through the restoration of forest landscape. forest restoration is done recently by activities to obtain a balancing function between forest conservation and rural community needed (reitbergen-mccraken 2007), regaining ecological integrity function and improving human welfare (mansourian . 1986) in deforested or degraded landscapes. such efforts can provide optimal results if well adapted to the characteristics of the ecosystem or the type of disturbances. recently, the ministry of forestry has a method to determine an index of land degradation based on biophysics condition. the ecosystem functions and socio economics aspects have not been considered as priority for area restoration. in this research, ecosystem functions and socio economics aspects are considered in determining the restoration index. to obtain the index, it needs a model design that expresses the index standard used in planning the activities of forest restoration. this research aimed to develop a restoration index model for degraded tropical forest landscapes in batang toru watershed (fig. 2a). this study was conducted in 3 sub-watersheds of batang toru watershed i.e. puli, sarula, and batang toru hilir sub-watersheds. the three study areas covered three districts i.e. tapanuli selatan, tapanuli utara and tapanuli tengah. this study used landsat images (batistella 2000, apan . 2002) to classify land cover types in several years (1989, 2001 and 2013). those images were landsat tm5 image of 1989, landsat etm7 image of 2001, and landsat oli image of 2013. this study required et al et al. pongo abelii tapirus indicus, panthera tigris sumatrae pardofelis marmorata, helarctos malayanus rafflesia gadutensis et al. et al et al. et al materials and methods 113 restoration riority ndex evelopment of egraded ropical orest samsurip i d d t f – et al. several thematic maps including watershed, river network, road network, and contour maps. the classification of land cover types was done according to classification scheme used by the ministry of forestry. field surveys were conducted to verify the land cover types and to collect vegetation data. field sample plots were determined by purposive sampling method and laid out by representation of part of watershed (upstream, middle stream and downstream), land system and land cover types. the equipments used in the field survey were gps, compass, haga hypsometer, diameter tape, and questionnaires. analysis of satellite images was conducted using erdas imagine 9.1. envi 4.5. arcgis 9.x was used in the spatial analysis of thematic maps (contour, road, and river maps). fragstats ver 3.3 (mcgarigal & marks 1995; mcgarigal 2002) was used to derive necessary landscape metrics. the research consisted of 5 stages to get the restoration index, as follows: landsat images were interpreted to get the normalized difference vegetation index (ndvi) and land cover types for year of 1989, 2000 and 2013 (jaya 2009). a field check was conducted to verify the classified land cover types. in addition, vegetation data were collected in several square sample plots of 2,500 m (fig. 2b). et al. 1. forest degradation index 2 figure 2. location of study site (a) and design of sample plot (b) 114 biotropia vol. 21 no. 2, 2014 trees in different growth stages were measured in four different size quadrants namely quadrant 1, 2, 3 and 4. an analysis of the vegetation data was conducted to get the degree of forest degradation (kreb 1989, magurran 1988). forest degradation was approached using ndvi value class. ndvi was predicted using vegetation parameters namely species diversity index (x ), basal area (x ), and stand density (x ). each range of ndvi was scored using likert scale (syarifi . 2007) (table 1), then it was used to model forest degradation index through regression analysis (puspaningsih 2011, rohyani 2012). the independent variables were distance to main road (x ), distance to main river (x ) and percent of slope (x ). forest land cover types were analyzed using the fragstats software. fragstats analysis was performed to derive landscape metrics. the range length of orang-utan was used as data input in fragstats analysis. flf degree was calculated based on landscape metrics (mcgarigal & marks 1995). landscape metrics used to determine the degree of flf were patch number per area (patch density), proximity and contiguity (fahrig 2003, mcgarigal 2002). each landscape metric was classified (table 1) and scored using likert scale (syarifi 2007) indicating the degree of flf. hence, it was used to develop flf index by regression analysis (puspaningsih 2011, rohyani 2012). the independent variables were value of contiguity between patch (x ), proximity value of each patch (x ), and the number of patch (x ) 1 2 3 4 5 6 7 8 9 . et al 2. forest landscape fragmentation (flf) index et al. no sub factor value score no sub factor value score 1 ndvi <0 0 – 0.25 0.25 – 0.50 0.50 – 0.75 >0.70 5 4 3 2 1 5 connectan <20 20 – 40 40 – 60 60 – 80 >80 1 2 3 4 5 2 number of patch per area (patch density) >325 285-325 245285 205 –245 < 205 5 4 3 2 1 6 radius of gyration <200 200-400 400-600 600 – 800 >800 1 2 3 4 5 3 proximity >3.270 2.551-3.270 1.234-2.551 555-1.233 <554 5 4 3 2 1 7 employment farmer 5 labour/official employee 3 un-employee 1 4 contiguity <0.2 0.2-0.4 0.4-0.6 0.6-0.8 >0.8 5 4 3 2 1 8 salary/income (million idr/month) <0. 6 6 – 3 3 -6 6– 8 >8 5 4 3 2 1 table 1 score of each sub factors 115 restoration riority ndex evelopment of egraded ropical orest samsurip i d d t f – et al. 3. forest landscape connectivity (flc) index et al. 4. socio economics index 5. developing restoration index et al. et al flc degree was calculated based on landscape metrics indicating connectivity i.e. radius of gyration and connectan of forest area (fahrig 2003; mcgarigal 2002). each metric was scored (table 1) to determine degree of flc, then it was used to model flc index by regression analysis (puspaningsih 2011; rohyani 2012). the independent variables were value of connectan among forest patch (x ) and value of radius of gyration of each forest patch (x ). socio economics perception of people living surrounding forest area was studied by interviewing them. interview was guided by semi-structured method using questionnaires. each answer was scored using likert scale (syarifi 2007) (table 1). regression analysis was used to model socio economics index (puspaningsih 2011; rohyani 2012). the independent variables were maximum score of income in millions rupiahs earned by people's group around the forest (x ), and score of occupation type group namely farmer group; company labor, government officer and informal sector labor group, and plantation estate owners group (x ). four map indices were overlaid and samples were taken from 225 unit (4 ha per unit). then, restoration index (z) was developed using four indices i.e. forest degradation index (y ), forest landscape fragmentation index (y ), forest landscape connectivity index (y ) and socio economics index (y ). discriminant analysis was perfomed to get a restoration index model (thatam 1998; sifriyani 2012). regression analysis resulted in this study showed that the model of forest degradation was y = 1 + 2.0771x + 0.0094x 1.4675x with determination coefficient of 90%. therefore, spatial and statistical analyses were also conducted and resulted the model of forest degradation index as y = 1.10571 0.00005x 0.00006x 0.00732x with determination coefficient of 63.19%. based on the model of forest degradation index, it was found out that higher stand density value and higher basal area proved that the forest condition were less degraded. vegetation density in three sub-watersheds was low and the estimates from different plots ranged from 12 to 93 individuals per ha, other sumateran lowland mountain natural forest could reach 120 tree per ha (whitten . 1987). the current stand density value indicated that forest cover decreased and it indicated degradation (lund 2009). morever, the nearness to road and river networks trigerred forest degradation because the road and river become people's access to go inside and outside the forest. generally, river and road networks are used to enter the forest. closer distance from river and road networks to the forest means the forest has more pressure than other forest locations. the road and river can be used to estimate forest degradation risk. 10 11 12 13 1 2 3 4 1 2 3 1 4 5 6 results and discussion forest degradation index 116 biotropia vol. 21 no. 2, 2014 forest in the study area was located on relatively high elevation and rough topography. in this condition, slope could cause difficulties in accessing and disturbing forest. human tends to choose land having easier accessibility to settle down, which leads to land conversion. the model of forest degradation was used to derive forest degradation map (fig. 3). forest degradation map divided forest landscape into degradation classes: lower, low, moderate, and high. degradation of forest landscape was located in area close to road and river networks. therefore, forest landscape close to the road should receive higher priority to be restored. figure 3. forest degradation index of batang toru watershed forest landscape fragmentation (flf) index orang-utan is endemic fauna in the forest region of batang toru needing proper habitat in batang toru's forest. one main goal of batang toru landscape restoration was to provide habitat for orang-utan and other biodiversity. the orang-utan habitat characteristics were used as input data in landscape metrics analysis. the day range length of orang-utan was at least 500 m per day. this radius was used as basic calculation for landscape metrics on every 500 m. this radius area was assumed as minimum habitat of orang-utan. furthermore, the day range length of orang-utan was not more than 1,000 m per day. this radius was used as an input for forest edge length of metrics landscape (sinaga 1992). 117 restoration riority ndex evelopment of egraded ropical orest samsurip i d d t f – et al. 2 7 8 9 regression model of flf index resulted from this study was y = 0.7819 0.576x 0.0000001x +0.0552x ,withdetermination coefficient of 87.20%. based on landscape metrics value, it was known that flf degree increased from 1989 to 2013 which was indicated by the increasing of patch density (forman & godron 1986; forman 1995; fahrig 2003) per ha (fig. 4a) and by the increasing of patch proximity (fig. 4b). patch density in three sub-watersheds (puli, sarula and batang toru hilir) rose from about 0.10 in 1989 to 0.60 in 2013. the area of low flf index to medium flf index was bigger than that of high flf index. most flf index area was in the road distance of 1,000 m to 5,000 m where people could reach out and entered the forest in a day (jaya . 2007). flf index map showed that most forest landscape in batang toru watershed was in low flf index (<0.2), so it was categorized as low flf category (fig. 5a). low flf supported ecosystem function restoration because the material flow in the ecosystem still ran quite well due to low isolated forest patch which would not reduce organism mobility inside the forest. et al figure 4. forest patch density (a) and forest patch proximity (b) figure 5. forest fragmentation (a) and forest connectivity (b) of year 2013 in batang toru watershed 118 biotropia vol. 21 no. 2, 2014 forest landscape connectivity (flc) index forest landscape connectivity (flc) index in batang toru watershed decreased from 1989 to 2013. it was indicated by the decreasing of radius of gyration (fahrig 2003) from 400-700 range in 1989 to 50-200 range in 2013 (fig. 6a) and by the decreasing of connectan value from 3-9% range in 1989 to 0-2% range in 2013 (fig. 6b). regression analysis resulted from this study showed that the model for flc index was y =0.1144+0.0006x + 0.1954x with determination coefficient of 66.16%. most area of puli sub-watershed had medium connectivity index, while high connectivity index belonged to forest path in sarula sub-watershed. a small forest patch which was located in batang toru hilir sub-watershed had very low connectivity index because it was separated by other land cover types. non-forest cover area was located between small forest patches. larger non-forest cover area made small forest patches more separated from others. this will be a barrier for organism movement between forest patches. the derived map of flc index (fig. 5b) showed that puli sub-watershed had the highest flc index compared to sarula and batang toru hilir sub-watersheds. meanwhile, batang toru hilir and sarula sub-watersheds had low flc index. batang toru hilir sub-watershed was located in downstream of batang toru watershed where many lands clearing activities occurred, caused by long distance between forest patch remained. this condition was also occurred in puli sub-watershed located in upper stream of batang toru watershed. 3 10 11 figure 6. radius of gyration (a) and forest patch connectan (b) of each sub-watershed 119 restoration riority ndex evelopment of egraded ropical orest samsurip i d d t f – et al. flc map also showed that the most forest landscape of sarula sub-watershed had high connectivity index (0.5-0.6). the forest landscape with higher connectivity index was forest of sarula (0.6-0.8) located along main road through the sub-watershed. by restoring this forest, the forest landscape would be connected with the closest forest patches, so the connectivity between forest patches would be high and would improve the functional relationship (fry & tress 2007) in forest ecosystem. loss of natural connectivity of ecosystem is threat in distribution and survival of orangutan and other wildlife. as the habitat of wildlife, the flc of batang toru is important for diversity conservation. more serious attention is needed to improve the connectivity of forest landscape and habitat conservation, because landscape connectivity facilitates organism movement, genetical exchange, and other ecology of material flow (crooks & sanjayan 2006). habitat requirements for fauna and flora is the main key in biodiversity conservation, including stability and integrity of natural ecosystem (taylor . 1993; collinge 2000). therefore, it is very important to consider connectivity as a base in planning conservation and landscape change analysis. the model of socio economics index resulted from this study was y = 0.65450.043077x +0.004396x with determination coefficient of 24.78%. socio economics condition became a main key for the success of landscape restoration activities. people's perceptions on forest landscape restoration activities were different. the perceptions depended on people's level of income, education, main occupation types, age and duration of staying. the analysis showed that occupation types and level of income significantly affected people's participation possibility in forest landscape restoration. based on occupation types, employed people were highly depended on natural resources quality and therefore, they absolutely agreed and would participate in all restoration steps. farmer group was more supportive in landscape restoration compared to plantation workers or owners. the farmer group would participate in landscape restoration to have more income and land for cultivation. as well as farmers, low income people tended to participate in restoration activities to have more income (fig. 7a). people owning large estate area tended not to participate in landscape restoration. those people were worried their plantation would be disturbed, so it might reduce production of the plantation. people with high level of income disagreed and would not participate in forest landscape restoration. this condition should become consideration in determining location and determining partners in managing forest landscape restoration. forest landscape restoration programs should increase people's welfare and produce more woods from forest plantation (nawir . 2008). this study showed that the restoration index model was z = 0.133y + 0.024y + 0.939y + 0.079y with correlation coefficient of 55.7%. based on value of cannonical et al et al socio economics index restoration index 4 12 13 1 2 3 4 120 biotropia vol. 21 no. 2, 2014 discriminant coefficient, it was found out that socio economic index had less impact compared to the other four predictors, while the most influential model was connectivity index model. hit ratio value from discriminant analysis is 54.5%, which was sufficient for grouping into 3 different classes. based on test using alpha value of 0.05, the discriminant function result was accurate, because value was bigger than table value. hence, the restoration index model was used to create restoration index map (fig. 7b). the figure shows that high restoration index was located in sarula sub-watershed. it meant that when the restoration project was to be conducted, sarula sub-watershed should be the first area to be restored. based on matrix value of discriminant function, it was found out that the connectivity index had canonical coefficient value higher than other indices (selman 2006), therefore, it became the most important index followed by forest degradation index. connectivity index is important in some research because it is potential in mitigating habitat fragmentation impact (anderson & jenkins 2006). considering the landscape connectivity index would ensure the connectivity of wildlife habitat. socio economic index had less influence in the model indicating that people's participation possibility was relatively less important as consideration in restoration activity planning when the forest landscape was highly degraded. people's participation possibility should be encouraged to increase support in forest landscape restoration plan. press's q press's q x 2 figure 7. possibility participation map (a) and restoration index priority of forest landscape batang toru watershed (b) 121 restoration riority ndex evelopment of egraded ropical orest samsurip i d d t f – et al. et al et al increasing of ecosystem connectivity is one main objective of forest landscape management (saura . 2011), and also maintenance of stability and integrity of natural ecosystem (decout . 2010). forest landscape restoration is aimed to restore forest landscape as close as natural ecosystem. forest patch connectivity and forest degradation level become the most important indices, therefore, consideration to improve connectivity between separated patches and degradation levels becomes high priority. these indices will improve the current guidelines and methods for forest restoration. biophysical factor is the main consideration in planning site restoration (mof and jica, 2014). recently, land and forest biophysical characteristics are also used to determine site priority for land rehabilitation. ecologically, the connectivity index is one important factor in material flow and material cycle in forest ecosystem. in sarula sub-watershed, most forest degradation was in very high restoration index area. the index showed that sarula sub-watershed became the main priority for landscape restoration activities in batang toru watershed. restoring landscape of sarula sub-watershed will improve the whole condition of batang toru watershed because sarula sub-watershed is located in the upstream of batang toru watershed. batang toru forest landscape was heading for heavy degradation indicated by decreasing of species diversity level, increasing of medium and high ecosystem pressures, increasing of forest landscape fragmentation, declining of forest landscape connectivity. forest landscape restoration index was determined by forest landscsape connectivity index and forest degradation index. the forest connectivity index should be considered in planning site restoration to ensure wildlife habitat quality and biodiversity. the restoration index would enhance the current guidelines and methods for forest restoration. forest fragmentation was potential indicator to determine priority for site restoration. people's participation possibility gave less influence in landscape restoration index model. people needed to be encouraged to increase their involvement in forest landscape restoration activities in batang toru watershed. the map of forest landscape restoration index shows that sarula sub-watershed to be the first area restored before other sub watersheds. the restoration index needs other factors to fulfill all requirements such as land degradation index. therefore, further research is needed to design site restoration site in priority sub-watershed area. this research was part of phd thesis of the first author, funded by seameobiotrop dipa 2013. the authors extended deep appreciation to the ministry of education and culture, republic of indonesia for the scholarship and support to conclusions acknowledgements 122 biotropia vol. 21 no. 2, 2014 accomplish this paper. sincere appreciation was also extended to anonymous reviewers for corrections and comments. references anderson b, jenkins cn. 2006. new york, (us): columbia university press. apan aa, steven sr, mark sp. 2002. mapping and analysis of changes in the riparian landscape structure of the lockyer 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nazaruddin. 1987. . yogyakarta (id): gadjah mada university press. statistik kehutanan indonesia. pedoman dan tata cara restorasi di kawasan konservasi (hutan hujan tropis pegunungan dan hutan mooson tropis rehabilitasi hutan di indonesia, akan kemanakah arahnya setelah lebih dari tiga dasawarsa?. kajian spasial lahan kritis berbasis sistim informasi geografis untuk rehabilitasi kawasan koridor satwa liar dan harangan desa di kawasan hutan batang toru provinsi sumatera utara . phd thesis , the forest landscape restoration handbook (earthscan forestry library) phd thesis. . planning at the landscape scale pongo pygmaeus master thesis spatial decision support system multivariate data analysis the ecology of sumatera 124 biotropia vol. 21 no. 2, 2014 biotropia vol. 27 no. 1, 2020: 69 79 doi: 10.11598/btb.2020.27.1.1122 69 association of tree communities with soil properties in a semi deciduous forest of perlis, peninsular malaysia radhiah zakaria1*, mohd nizam mohd said2,3 and faridah hanum ibrahim4 1faculty of public health, universitas muhammadiyah aceh, banda aceh 23123, indonesia 2school of environmental and natural resource sciences, faculty of science and technology, universiti kebangsaan malaysia, 43600 bangi, selangor, malaysia 3institute of climate change, universiti kebangsaan malaysia, 43600 bangi, selangor, malaysia 4faculty of forestry, universiti putra malaysia, 43400 serdang, selangor, malaysia received 20 august 2018 / accepted 16 november 2018 abstract plant community distribution is associated with environmental factors, particularly, the soil properties of habitats. this study was conducted to determine the effect of soil properties on the association of tree communities within three distinct habitats in a semi-deciduous forest in perlis state park (psp), perlis. eighteen plots of 40 × 60 m (0.24 ha each) with sampling areas of 1.92 ha (8 plots) in setul formation, 0.96 ha (4 plots) in granite and 1.44 ha (6 plots) in kubang pasu formation (totalling 4.32 ha) were established at the psp. all trees with 5.0 cm and above diameter at breast height (dbh) were enumerated, while the top soil samples were collected from each plot for soil analyses. a total of 412 tree species, 207 genera, and 68 families were recorded; 270 tree species from 152 genera and 57 families in the setul forest; 204 tree species of 130 genera and 50 families in the granite forest; and 109 tree species from 76 genera and 31 families in the kubang pasu forest. euphorbiaceae was the most represented family at setul, granite and kubang pasu with 36, 19 and 12 species, respectively. soil properties significantly varied among the study sites. setul had loam, kubang pasu had clayloam, and granite had the sandy-loam texture. the soils were acidic and had low to high concentrations of available nutrients. ordinations using canonical correspondence analysis indicated that the soil factors play an important role in the distribution and diversity of plants in these forest habitats. keywords: canonical correspondence analysis, perlis state park, semi-deciduous forest, vegetation– environment relationship introduction in tropical rain forest, the tree species composition varies with the type of habitat (richards 1952) and their distributions are often associated with environmental factors (newbery & proctor 1984; fujii et al. 2018). studies conducted in different tropical regions had emphasized the influence of environmental factors, especially soil properties, on plant species distributions (wentworth 1981; oliveirafilho et al. 2001; nizam et al. 2013). relationships between soil variables and plant species distribution had been discovered in various habitats, such as in granite and limestone areas (nizam et al. 2013), grasslands (cachovanová et al. 2012), savannas (barruch 2005) and the tropical rain forests (silk et al. 2010; sukri et al. 2012). peninsular malaysia is predominantly of the rainforests type and comprise only a small restricted part of a semi-deciduous forest. the semi-deciduous forest in peninsular malaysia is located near kra isthmus, a transitional zone where malesian and indochinese floristic regions intersect (van steenis 1979; whitmore 1984; middleton 2003). the state of perlis, which is situated in the northernmost of peninsular malaysia, is the only part in this country with a semi-deciduous forest (faridah-hanum 2006). *corresponding author, e-mail: radhiah@unmuha.ac.id https://link.springer.com/article/10.1007/s12224-012-9131-3#auth-1 biotropia vol. 27 no. 1, 2020 70 the perlis state park (psp) exhibits a semideciduous forest with most areas consisting of limestone hill forests and a small portion of granite-based parent material. this study aimed to determine the association between the tree communities and their soil properties under different forest habitats with different rock formations. this study is timely because the increasing human pressures on limestone and granite habitats compromise their integrity, diversity and functions. limestone forests are being replaced by plantations, agricultural crops and forages to increase their productivity. materials and methods study site and tree sampling a semi-deciduous forest in psp, perlis, peninsular malaysia, was selected as the study site (latitude 634'n to 643'n, longitude 10010'e to 10013'e) (fig. 1). tree samplings were conducted in 18 plots of 40 × 60 m each, covering a survey area of 4.32 ha. due to different topography of the selected forest habitats, several plots were selectively established to avoid rocks and huge boulders. as such, the number of plots that were established also varied between forest habitats of different geological formations; eight plots (1.92 ha) in the setul, six plots (1.44 ha) in the kubang pasu, and four plots (0.96 ha) in the granite. all trees with 5 cm and above diameter at breast height (dbh) were enumerated and identified using the published keys (whitmore 1972; whitmore 1973; ng 1978; ng 1989). since the number of plots in each forest habitat was unequal as mentioned earlier, the rarefaction analysis was conducted using ecosim software program (gotelli & entsminger 2003) to produce the rarefaction curves. rarefaction allows comparison of the observed richness and diversity between sites even though sampling efforts are not equal, or samples differ in the total number of individuals (lee & chao 1994). the rarefaction analysis of the study site confirmed that although the sampling efforts between habitats were unequal, nevertheless the species richness was of the same trend with the actual observation in the survey plots (zakaria et al. 2015). perlis state park figure 1 location of the perlis state park in the state of perlis, peninsular malaysia association of tree communities with soil properties – zakaria et al. 71 soil sampling and analysis using the soil corer, the top soil samples at 0-15 cm depth were collected from 18 established plots of various habitats within the psp. five replicate samples were obtained from each plot and air dried at room temperature. the replicates were then homogenized together to represent one composite sample from each plot. root fragments and unwanted materials were removed and the soils were lightly ground and passed through a 2 mm mesh sieve before analysis. the soil samples were analyzed for physical properties, i.e., particle size distribution and organic matter (om) content as well as chemical properties, i.e., exchangeable acid cations (al3+ and h+), exchangeable base cations (k+, mg2+, na+ and ca2+) and available nutrients (phosphorus (p), potassium (k) and magnesium (mg). om content was measured using loss on ignition techniques, while the soil ph was recorded based on soil–water ratio of 1:2.5 (mclean 1967). by titration, the exchangeable acid cations were analyzed in 1 m kcl while the exchangeable base cations used the 1 m ammonium acetate extract (black 1967; shamshuddin 1981). to determine the cation concentrations, the extracts were run using the perkin-elmer atomic absorption spectrophotometer (aas) model 3300. available nutrients (p, k and mg) in the soil samples were extracted using 1 m ammonium acetate–acetic acid. the concentration of p in the extract was recorded using ultraviolet (uv) spectrophotometer, whilst k and mg concentrations were measured using the aas. data analysis the enumerated trees in the plots were investigated for overall floristic composition. the soil parameters among the study plots were tested for significant difference using t-test. association between tree communities and soil variables was analyzed using the multivariate techniques of canonical correspondence analysis (cca) (ter braak 1987; ter braak & prentice 1988; ter braak 1992), which is available in canoco for windows 4.56 (ter braak 2009). tree species with four occurrences, or less, were not included in the analysis because they weaken the uniformity when assigned to the groups (legendre & gallagher 2001; barruch 2005). the species with four occurrences or less were disregarded to limit the number of species to less than 200 and consequently increase the accuracy of the results of cca ordination diagram. thus, 161 tree species were selected for the cca. detrended correspondence analysis (dca) was first conducted on the species data to confirm their unimodality and subsequently justify the appropriateness of using cca (lepš & šmilauer 2003). soil variables selected for the cca were soil ph, available p, available magnesium (mg2+), calcium (ca2+), natrium (na+), potassium (k+) content, ammonium-n (nh4-n) and nitrate-n (no3-n). the abundance values were log-transformed prior to matrix processing because their distributions were skewed toward extremely large values (ter braak 1995). the correlation significance between matrices was tested using monte-carlo permutation test based on 499 random trials at a 0.05 significant level (lepš & šmilauer 2003). the ordination diagrams were subsequently plotted by canodraw 4.14 to illustrate the association patterns of tree communities in relation to soil properties. results and discussion tree species composition a total of 4,300 trees were recorded within the sampling area of 4.32 ha at perlis state park. the floristic composition included 412 tree species, 207 genera and 68 families. the setul habitat showed high species richness of 270 species from 1,722 trees; belonging to 152 genera and 57 tree families and the plots in granite habitat showed 204 tree species, from 1,245 trees; belonging to 130 genera and 50 families and the plots in the kubang pasu habitat contained 109 tree species from 1,333 trees, belonging to 76 genera and 31 families. in all three habitats, euphorbiaceae was the most represented family with 36 species in setul, 19 species in granite and 12 in kubang pasu. on the contrary, 11 families were represented by only one species with a single individual in setul, granite and kubang pasu habitats. these families include actinidiaceae (saurauia pentapetala), ancistrocladaceae (ancistroclados biotropia vol. 27 no. 1, 2020 72 tectorius), anisophylleaceae (anisophyllea apetala), convolvulaceae (erycibe albida), ctenolophonaceae (ctenolophon parvifolius), hypericaceae (cratoxylum formosum), magnoliaceae (magnolia elegans), oleaceae (chionanthus macrocarpus), opiliaceae (milientha suavis), oxalidaceae (sarcotheca griffithii) and rhizophoraceae (gynotroches axillaris). hence, these species were not considered in the analysis of species association with soil variables. soil properties the three habitats have different soil properties as follows: the soil in setul was loam whereas those in granite and kubang pasu were sandy and silty-loam, respectively. granite habitat had the lowest silt and clay contents but had the highest sand content at more than 69% among all study sites (table 1). the high sand content in granite is related to sandy parent material; the quartz sand derived from granitic hill was washed out and deposited a long time ago as reflected by the high sandy materials and extremely low clay and silt contents (osumi 1979; zaidey et al. 2010). in terms of om content, the granite soil had the significantly lowest om content (3.12 ± 0.08) followed by the setul soil (6.97 ± 1.96) and kubang pasu soil (10.72 ± 2.01) (table 1). the lowest om content in granite is related to the lower percentage of silt and clay in the soil; because the absence of silt and clay generally increases decomposition rates and decreases om for a particular set of environment interactions (paul & clark 1996). overall, it is apparent that the amount of om in the study sites is generally low. this low om content is due to the high decomposition rate of om in tropical rainforest soils (longman & jenik 1987). the low soil ph in granite (4.39), setul (5.64) and kubang pasu (5.98) indicated that the soils were strongly to moderately acidic. the soil ph in granite was significantly lower than those of setul and kubang pasu (table 1). granite soil has the highest acidity probably because it originated from the acidic parent rocks (juo 1981) and the decomposition of om also adds to the acidification of surface soil (zaidey et al. 2010). a similar study recorded that most soils in peninsular malaysia tropical rainforests are acidic with ph values between 4.5 and 5.5 (othman & shamshuddin 1982). table 1 summary of soil variables in setul, granite and kubang pasu habitats at perlis state park, perlis soil parameters granite (mean ± s.e.) setul (mean ± s.e.) kubang pasu (mean ± s.e.) p value organic matter (om) (%) 3.12 ± 0.08a 6.97 ± 1.96b 10.72 ± 2.01b p < 0.05 sand (%) 69.75 ± 2.12a 41.45 ± 4.68b 36.60 ± 8.52b p < 0.01 silt (%) 14.15 ± 0.77a 35.61 ± 3.89b 44.20 ± 2.17b p < 0.01 clay (%) 16.05 ± 1.42 21.42 ± 2.80 19.20 ± 2.23 ns soil ph 4.39 ± 0.12a 5.64 ± 0.35b 5.98 ± 0.30b p < 0.01 exchangeable cations (meq/100g) ca2+ 0.94 ± 0.27a 5.5 ± 2.05b 7.87 ± 2.18b p < 0.05 mg2+ 0.41 ± 0.02a 0.75 ± 0.16b 0.95 ± 0.44a p < 0.05 na+ 0.20 ± 0.04 0.13 ± 0.02 0.13 ± 0.02 ns k+ 0.32 ± 0.03a 0.50 ± 0.06ab 0.57 ± 0.12b p < 0.01 al3+ 0.60 ± 0.16 0.19 ± 0.14 0 ns h+ 0.50 ± 0.03 0.35 ± 0.04 0.34 ± 0.02 ns cec 2.92 ± 0.2a 7.58 ± 2.09b 9.87 ± 2.54b p < 0.05 available nutrients (ug/g) phosphorus (p) 9.04 ± 0.31a 7.18 ± 0.69b 7.27 ± 0.37b p < 0.05 nitrate-n (no3-n) 27.00 ± 1.5 34.39 ± 6.45 30.96 ± 4.94 ns ammonium-n (nh4-n) 3.59 ± 0.46a 18.64 ± 3.57b 30.81 ± 3.57b p < 0.01 magnesium (mg) 28.56 ± 0.51 105.35 ± 48.56 139.49 ± 66.78 ns potassium (k) 175.02 ± 8.43a 227.27 ± 32.4b 253.80 ± 26.35ab p < 0.05 note: mean values in a row with the same letter were not significantly different. association of tree communities with soil properties – zakaria et al. 73 the cation exchange capacity (cec in granite was the lowest (2.92 ± 0.20 meq/100 g,) was significantly different from those in setul (7.58 ± 2.09 meq/100 g) and kubang pasu (9.87 ± 2.54 meq/100 g). cec varies with the clay–humus content of the soil. clay soils usually have large cec, whereas sandy soils have low cec (cruickshank 1972). the sites showed various concentrations of available macronutrients, namely magnesium (mg), potassium (k) and phosphorus (p), and soluble nutrients, i.e., ammonium-n and nitrate-n (table 1). the phosphorus concentration in granite plots (9.04 ± 0.31 μg/g) was significantly higher than those in setul (7.18 ± 0.69 μg/g) and kubang pasu (7.27 ± 0.37 μg/g). the available magnesium (mg) in granite (28.56 ± 0.51 μg/g), setul (105.35 ± 48.56 μg/g) and kubang pasu (139.49 ± 66.78 μg/g) did not differ significantly. the available inorganic soil nitrogen is in the form of nitrate-n (no3-n) and ammoniumn (nh4-n). the concentrations of these two soluble nutrients showed a consistent trend; the granite habitat had lower concentration than the setul and kubang pasu habitats. ammonium-n was significantly low at granite (3.59 ± 0.46 μg/g), higher at setul (18.64 ± 3.57 μg/g) and the highest at kubang pasu (30.81 ± 3.57 μg/g). nevertheless, the concentration of nitrate-n was not significantly different among the habitats. the lowest availability of inorganic soil nitrogen in granite habitat was in line with the om in the habitat. tan (2009) mentioned that soil inorganic n content increases linearly as organic matter increases in the soil. the inorganic n was characterized by a large no3-n, which exceeds the nh4-n at all the habitats; this might be attributed to the drier conditions in the study sites (yamashita et al. 2003). relationships between tree communities and soil variables dca confirmed that the data on tree communities were unimodal with the length of gradient of the first axis at 5.758 (table 2), which was greater than 4 standard deviation (sd) before being analyzed by cca (lepš & šmilauer 2003). the eigenvalue produced by the dca axis 1 (0.758) was high, indicating an environmental gradient where most species vary essentially in their abundance (ter braak 1995). plots in granite (symbol x) were clustered together, while plots in setul (symbol ○) and kubang pasu (symbol +) exhibited the gradient that occurs from granite to limestone (fig. 2). these dca ordination plots indicated the approximate locations of the sample plots. the plots that clumped together represented the plots with relatively similar floristic attributes, while the separated plots indicate dissimilar floristic composition. the unclearly separated plots in kubang pasu and setul proved that kubang pasu formation is overlain by setul formation (basir & zaiton 2002). therefore, these habitats shared similar needs for mineral nutrient elements and other edaphic variables. table 2 summary of the detrended correspondence analysis (dca) of the vegetation data in 18 plots at the perlis state park, perlis axes 1 2 3 4 total inertia eigenvalues 0.758 0.464 0.393 0.174 6.527 length of gradient 5.758 3.879 3.012 2.509 cumulative percentage variance of species data 11.6 18.7 24.7 27.4 sum of all eigenvalues 6.527 biotropia vol. 27 no. 1, 2020 74 -1 6 -1 4 figure 2 dca ordination diagram of plots based on the abundance of tree species at perlis state park, perlis notes: x = plots in granite; ○ = plots in setul; + = plots in kubang pasu. based on the cca analysis, the species– environment correlations were high on the first and second axes with values of 0.988 and 0.974, respectively (table 3). the eigenvalue was 0.561 for the first axis and 0.444 for the second axis. additionally, the cumulative variation explained by the first three axes of the speciesenvironment relationship was 55.2%. the monte-carlo permutation test also indicated a significant difference on the eigenvalues among the ordina-tion axes (p = 0.002). the cca ordination plot showed the approximate locations of sample plots and the locations, lengths and directions of soil chemical variables (fig. 3). plots in granite (1-4) were clustered together and associated with available phosphorus. meanwhile, setul and kubang pasu plots did not cluster into the well-defined group. their plots were mostly assembled on the bottom right part of the diagram and were strongly correlated with several soil variables, i.e., magnesium (mg), potassium (k), calcium (ca), ph, available nitrate-n (no3) and available ammonium-n (nh4). the cca biplot of species and soil variables showed the species occurrence in relation to soil variables (fig. 4). most species were clumped on the centre part of the diagram and correlated with several soil variables such as available phosphorus (p) and available ammonium-n (nh4). some species such as antidesma cuspidatum (47) were clearly associated with calcium, whilst ficus annulata (102) and duabanga grandiflora (88) were strongly associated with ammonium-n (fig. 4; table 4). furthermore, hopea ferrea (34) had strong association with sodium. aglaia affinis (93) had strong association with magnesium, whilst bombax valetonii (23) was strongly associated with potassium and magnesium. however, alstonia macrophylla (19), terminalia subspathulata (30), senna timorensis (87), ficus aurata (103) and colona merguensis (155) showed a weak association with soil factors when they are farther away from all the soil gradients (fig. 4; table 4). table 3 summary of the canonical correspondence analysis (cca) of the vegetation and soil chemical properties in the 18 plots at perlis state park, perlis axes 1 2 3 4 total inertia eigenvalues 0.561 0.444 0.356 0.301 4.238 species-environment correlations 0.988 0.974 0.961 0.987 cumulative percentage variance of species data 13.2 23.7 32.1 39.2 cumulative percentage variance of species-environment relation 22.7 40.8 55.2 67.5 sum of all eigenvalues 4.238 sum of all canonical eigenvalues 2.464 association of tree communities with soil properties – zakaria et al. 75 the variations in species distribution within the study plots were strongly correlated to edaphic factors and vegetation distribution pattern. calcium and ph were some factors that strongly influenced the vegetation pattern in the limestone study area (setul and kubang pasu), whereas sodium and phosphorus strongly affected the vegetation pattern in granite area. soils which developed on granite are low in calcium and magnesium (burnham 1974), whereas soils that developed on limestone parent material such as in setul and kubang pasu are mainly high in calcium and ph (gauld & robertson 1985). -1.5 1.5 -2 .0 3 .0 php ca mg na k no3 nh4 1 2 3 4 5 6 7 89 10 11 12 13 14 15 16 17 18 figure 3 cca ordination plot showing the approximate locations of sample plots and location, length and directions of soil variables notes: x = plots in granite (1-4); ○ = plots in setul (5-12); + = plots in kubang pasu (13-18). -0.8 1.0 -1 .0 1 .0 1 8 9 12 16 19 23 30 31 34 37 40 47 56 58 62 66 75 83 87 88 90 91 92 93 101 102 103 105 109 110 115 132 134 138 143 144 147 155 157 161 php ca mg na k no3 nh4 figure 4 canonical correspondence analyses of the biplot for tree species and soil variables at the perlis state park note: numbers denote the species in the plots as listed in table 4. biotropia vol. 27 no. 1, 2020 76 table 4 list of species with code numbers referring to diagram in fig. 4 species code species species code species 1 alangium kurzii 61 sapium baccatum 2 boea oppositifolia 62 sauroupus suberosus 3 buchanania arborescens 63 trigonostemon villosa 4 dracontomelon dao 64 lithocarpus cantleyanus 5 gluta elegans 65 casearia capitellata 6 gluta velutina 66 homalium longifolium 7 parishia insignis 67 hydnocarpus castanea 8 pentaspodon curtisii 68 hydnocarpus curtisii 9 semecarpus cochinchinensis 69 hydnocarpus filipes 10 semecarpus curtisii 70 osmelia maingayi 11 swintonia floribunda 71 garcinia eugeniifolia 12 alphonsea curtisii 72 garcinia hambroniana 13 goniothalamus tenuifolius 73 garcinia nigroleniata 14 mitrephora maingayi 74 garcinia parvifolia 15 orophea cuneiformis 75 kayea kunstleri 16 polyalthia glauca 76 mesua ferrea 17 xylopia ferruginea var. oxyantha 77 stemonurus malaccensis 18 xylopia magna 78 cryptocarya ferrea 19 alstonia macrophylla 79 cryptocarya rugulosa 20 kibatalia maingayi 80 litsea machilifolia 21 ilex cymosa 81 barringtonia scortechinii 22 radermachera glandulosa 82 abdulmajidia rimata 23 bombax valetonii 83 leea aequata 24 durio lowianus 84 callerya artropurpurea 25 canarium littorale f. rufum 85 cynometra malaccensis 26 dacryodes rubiginosa 86 saraca cauliflora 27 dacryodes rugosa 87 senna timoriensis 28 lophopetalum javanicum 88 duabanga grandiflora 29 parastemon urophyllus 89 lagerstroemia floribunda 30 terminalia subspathulata 90 lagerstroemia ovalifolia 31 tetrameles nudiflora 91 memecylon caeraleum 32 dipterocarpus costatus 92 memecylon minutiflorum 33 dipterocarpus fagineus 93 aglaia affinis 34 hopea ferrea 94 aglaia argentea 35 hopea latifolia 95 aglaia simplicifolia 36 parashorea stellata 96 aglaia spectabilis 37 shorea macroptera 97 aphanamixis polystachya 38 shorea siamense 98 chisocheton ceramicus 39 vatica cinerea 99 chisocheton patens 40 diospyros andamanica 100 chukrasia tabularis 41 diospyros buxifolia 101 artocarpus dadah 42 diospyros scortechinii 102 ficus annulata 43 diospyros sumatrana 103 ficus aurata 44 diospyros venosa 104 ficus fistulosa 45 elaeocarpus rugosus 105 ficus microcarpa 46 erythroxylum cuneatum 106 ficus oligodon 47 antidesma cuspidatum 107 ficus sundaica 48 aporosa aurea 108 ficus variegata 49 baccaurea griffithii 109 streblus elongatus 50 chondrostylis kunstleri 110 streblus ilicifolius 51 cleistanthus hirsutulus 111 streblus macrophyllus 52 croton argyratus 112 streblus toxoides 53 croton cascarilloides 113 horsfieldia polyspherula 54 croton laevifolius 114 honsfieldia sucosa 55 dimorphocalyx muricatus var. minor 115 horsfieldia tomentosa 56 drypetes longifolia 116 knema laurina 57 koilodepas longifolium 117 knema patentinervia 58 macaranga andamanica 118 ardisia crassa 59 macaranga lowii 119 ardisia pachysandra 60 mallotus peltatus 120 maesa ramentacea association of tree communities with soil properties – zakaria et al. 77 tabel 4 continued species code species species code species 121 syzygium cerasiforme 141 pometia pinnata 122 syzygium glaucum 142 xerospermum noronhianum 123 ochna integerrima 143 chrysophyllum roxburghii 124 galearia fulva 144 palaquium hexandrum 125 galearia maingayi 145 palaquium microphyllum 126 xanthophyllum affine 146 eurycoma apiculata 127 xanthophyllum griffithii 147 pterocymbium javanicum 128 prunus grisea 148 pterospermum javanicum 129 aidia densiflora 149 pterospermum pectiniforme 130 diplospora malaccensis 150 pterygota alata 131 ixora pendula 151 sterculia cordata 132 morinda elliptica 152 sterculia gilva 133 psydrax sp. 7 153 sterculia parviflora 134 psydrax sp. 8 154 tetramerista glabra 135 dimocarpus longan ssp. longan 155 colona merguensis 136 guioa bijuga 156 grewia viminea 137 harpullia cupanioides 157 pentace strychnoidea 138 nephelium costatum 158 schoutenia accrescens 139 nephelium lappaceum 159 teijsmanniodendron coriaceum 140 paranephelium macrophyllum 160 vitex pinnata 161 vitex siamica conclusion the differences in floristic patterns between limestone (setul and kubang pasu) and granite habitats at psp suggested that environmental gradients influenced floristic composition. the soil properties such as organic matter, moisture, clay, silt, calcium and ph were among the factors that strongly associated with the limestone habitats, while sand and available phosphorus were strongly correlated with granite. differences in the availability of elemental mineral nutrient between the soils of granite and limestone may have also contributed to the contrasting vegetation. in relation to soil properties, the different habitats displayed varied spatial distributions among the tree species. identifying the key underlying gradients, abiotic conditions and major soil influences on the vegetation pattern is essential to gain information about the ecology of particular species and to formulate plans to protect and conserve this fragile habitat. acknowledgements the authors would like to thank the perlis forestry department, malaysia for the cooperation and support throughout the study. the gratitude also goes to mrs latifah, mr ibrahim edham, ms siti eryani, the late mr badrol and mr ahmed zainuddin ibrahim for their assistance rendered during the fieldworks and laboratory procedures. the authors also acknowledge funding from the research university grant gup-2013-056. references barruch z. 2005. vegetation-environment relationships and classification of the seasonal savannas in venezuela. flora 200:49-64. basir j, zaiton h. 2002. geomorphology and geology of the setul formation in perlis. in: latiff a, kasim o, yusoff ar, faridah-hanum i, editors. kepelbagaian biologi dan pengurusan taman negeri perlis: persekitaran fizikal, biologi dan sosial wang mu, edn 1. kangar (my): perlis forestry 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institute malaysia. p. 89-109. zaidey ak, arifin a, zahari i, hazandy ah, zaki mh, affendy h, … nik muhamad m. 2010. characteristic soil properties of lowland and hill dipterocarp forests at peninsular malaysia. int j soil sci 5(3):112-30. zakaria r, nizam ms, faridah hanum i. 2015. floristic, diversity and structure of tree communities in three distinct forest habitats of perlis state park, perlis, peninsular malaysia. j sci 5(11):1016-23. intraspecific variation on early growth of neolamarckia cadamba miq. in provenance-progeny tests in west java province, indonesia dede j. sudrajat , iskandar z. siregar , ulfah j. siregar ,1* 1 2 2,3 nurhasybi , irdika mansur nurul khumaida2,3 4 and 1forest tree seed technology research institute , , bogor 16143 indonesia 2department of silviculture ,, faculty of forestry, institut pertanian bogor bogor 16680, indonesia 3seameo-biotrop, bogor 16134, indonesia 4departmen of agronomy and ho ticulturt r e, faculty of agriculture, institut pertanian bogor, bogor 16680, indonesia received 21 october 2014/accepted 2 may 2016 abstract genetic parameters on early growth of , an indigenous species with potential as a source of neolamarckia cadamba wood timber, were estimated in open-pollinated provenance-progeny tests at two sites in west java province, indonesia. the experiment was conducted using randomized complete block design with 12 provenances, 105 families and 5 replications of 4-tree row plots. total height and root collar diameter were measured at the age of 18 months growth. significant differences among the provenances and families within provenances were observed for height and collar diameter at all sites, except for the collar diameter of among provenances at parungpanjang site. in general, garut (gsj) provenance performed better growth at the two sites than kualakencana (kkp) and nusa kambangan (nkj) provenances. the evaluation of component of variance at the two sites showed that the provenance effects (ranging from 0.5 to 1.7%) contributed more to total variance than family within provenance effects (ranging from 0.4 to 0.6%). genetic correlations between height and collar diameter were weak to moderate. heritability was low for all traits at limbangan, while at parungpanjang, the heritability was moderate. estimation of genetic gain for height and diameter by proportional selected family 0.30 was 0.13 and 0.18 for limbangan and 0.31 and 0.16 for parungpanjang. heritability measurement should be sustained to reach stable value. stable heritability combined with selection of family and selection within family will improve genetic gain. : keywords genetic correlation, genetic parameter, heritability, progeny, provenance, neolamarckia cadamba, selection introduction neolamarckia cadamba (roxb.) miq. commonly known as white jabon is a native fast growing tree species playing an important role in both commercial and traditional farming systems in several sites in indonesia (krisnawati 2011; et al. kallio . 2011). this species produces timber et al for pulp and light construction (soerianegara & lemmens 1993). various parts of the plant also have many bioactive substances, such as an t iox idant , hypog lyc emic subs tance , hypolipidemic substance, antibacterial substance, antimicrobial substance, etc. (acharyya et al. 2011; xu 2011; mishra & siddique 2011). et al. n. cadamba has a very extensive natural distribution extending from india, nepal, burma, sri lanka and malaysia across indonesia, philippines and papua new guinea (lamprecht 1989). in indonesia, is distributed white jabon almost across the entire country (soerianegara & lemmens 1993). evolutionary theory predicts that species with large population sizes, broad geographic ranges and varying climate condition will have large inter and intraspecific variation (rawat & bakshi 2011). population within plant species with wider geographic ranges has higher allozyme variation and the widely distributed species have more overall allozyme variation (hamrick & godt 1989). some studies showed that genetic variation of ha been n. cadamba detected among provenances for quantitative * corresponding author : djsudrajat@yahoo.com biotropia vol. 23 no. 1, 2016: 10 20 doi: 10.11598/btb.2016.2 . .3 1 439 10 mailto:djsudrajat@yahoo.com seed and seedling morpho-physiological parameters (sudrajat 2016), as well as among natural populations to aflp loci (sudrajat . et al 2015). given its wide ecological amplitude and a large amount of genetic variation, is n. cadamba a promising species for tree improvement program to increase its productivity and quality. tree improvement program of is n. cadamba not yet running well, so that large cultivation activities of this species is not yet supported by high quality seed. the main seed sources for all kinds of plantation activities are from unknown origin, which are collected from unimproved seed sources. however, the initiation of the tree improvement program of is very n. cadamba important as a basic for high qualified seed procurement. the success of tree improvement efforts depends upon many factors including genetic variation, heritability of desired parameters and potential gains derived (nebgen & lowe 1982; borralho 2001), so it is very important to use the genetic material from wide natural distribution of the species to establish a breeding population. in indonesia, establishment of breeding population with open pollinated progenies of plus trees is widely applied as the first step towards tree improvement for fast growing species (hashimoto 1996; soeseno 1988). it et al. might be the most reasonable and practical way to meet an immediate demand for genetically improved seed to be used in reforestation programs in indonesia. traditionally, forest genetic tests have been conducted sequentially with successive species, provenance and progeny trials. in practice, however, there is strong economic pressure to reduce the testing interval between these stages in a traditional tree improvement program. the use of combined provenance-progeny test has been advocated to reduce the testing interval between provenance and progeny stages (zheng 1994; sebbenn et al. et al. 2003; finkeldey 2005). it may be extended to combine provenance testing, progeny testing, seed production and conservation of ex situ n. cadamba in a single trial. the study investigated intraspecific variation for early growth parameters in 105 families from 12 provenances originated from seven n. cadamba indonesian islands. the objectives of this research were: i) to examine the distribution of genetic variation among and within provenances and families within provenances and ii) to determine the extent of genetic control for growth parameters in the form of provenanceprogeny test at two different locations in west java , indonesiaprovince . materials and methods open-pollinated seeds of 105 families of n. cadamba were collected from 12 natural populations distributed in sumatera, java, nusa kambangan, , celebes, kalimantan/borneo sumbawa and papua (table 1, fig. 1) from march to july 2012. the sampled trees were phenotypically average or above average with respect to stem diameter (diameter at breast height/dbh) and total height compared with surrounding trees in the population. the distance among mother trees within population was kept at a minimum of 100 m to minimize relation between seed lots. seeds were collected by climbing the tree samples. every seed lot was separated for each sample tree and information about location and number of samples was noted at every seed lot. the information was kept until the seed reached nursery stage to establish the provenance-progeny trials. seeds were sown in plastic pot filled with media consisted of sand, compost and charcoal (5:3:1, by volume). prior to sowing, the potting mixture was treated with 0.2% a fungicide to avoid any chances of fungus infection attacking newly germinated seedlings. pots were watersprayed with fine sprayer to keep them wet and were maintained in greenhouse. young four-leaf seedlings were transplanted on polythene bags (10 cm in diameter and 15 cm in height), containing media consisted of top soil, sand and compost in a ratio of 2:1:1 (volume) and were set up in nursery. seedlings were grown at 50% light intensity using shade net for 3 months. one month afterwards the seedlings were exposed at open area for hardening off. seedlings were irrigated based on the operational regime for the nursery. forty vigorous and healthy seedlings were selected from each family to be planted at two trial sites. the experiment was set up in two sites in west java , i.e. site i in limbangan, province garut (07°02'23” s, 108°00'43” e, 520 m altitude) and site ii in parungpanjang, bogor (06°20'42” s, 106°06'15” e, 52 m altitude). 11 intraspecific riation of va in provenance-progeny tests neolamarckia cadamba – et al.dede j. sudrajat 12 biotropia vol. 23 no. 1, 2016 average annual rainfall and temperature were 2,580 mm and at site i, respectively and 27 °c 2,440 mm and 28 °c at site ii, respectively. soil at site i and site ii had low level of n, p, k and corganic with ph of 5.1 and 4.2, respectively. site i was an open private land with slope ranged from 5 to 15%, having high incidence of domestic animal and was planted with irregular agricultural crops in several parts of area. site ii was an even area within state forest land covered with dense weed, that grew rapidly even after cleaning. the locations were selected based on several reasons. limbangan was selected to represent medium to high level of elevated land where many n. cadamba plantations were cultivated as dominant species in forest community areas, such as in garut district, west java province. parungpanjang was selected to represent the common site condition of forest plantation industries that are generally established in podsolic soil associated with low ph and low soil nutrients. four-month-old seedlings were planted at the fields in february 2013 (planting hole size 40 x 40 x 40 cm) administered with 3 kg manure per seedling as basic fertilizer. the spacing between planting hole was 3 x 3 m. the experiment was conducted in randomized complete block design (rcbd) with 5 replications of 4-tree row plots (fig. 2). within two months after planting, 100 g of npk fertilizer (15:15:15) was applied to each figure 1 geographic distribution of 12 populations was tested figure 2 design of planting test of at parungpanjang, bogor and limbangan, garutn. cadamba 13 intraspecific riation of va in provenance-progeny tests neolamarckia cadamba – et al.dede j. sudrajat plant. weed competition was kept to a minimum by manual weeding. all trees in each plot constituted the measuring unit in all replications. the first assessment was carried out at the age of 6 months and subsequently after 12 months. in this paper, results of 16 months growth (4 months in nursery + 12 months in field) planting have been described. the parameters assessed are height (m), collar diameter (cm) and survival percentage. data analysis the data were analyzed for each site, to determine the provenance and family effects using two-way analysis of variance (anova). before performing the analysis, data were examined for conformity to normal distribution and homogeneity of the variance assumptions. anova for all parameters was conducted using the following statistical model (falconer & mackay 1996; sebbenn . 2003):et al y + p + f(p) ( ) + rp + ijkl i j k j ij = μ + r rf(p) ( ) + e ( )ik j l ijk an extra term (across site effect) was added to the model to investigate the significance of the difference of parameters across site. thus, the model used was: y = µ + s + p + f(p) ( ) + sp + sf(p) ijkl i j k j ij ik ( ) + e ( )j l ijk where : y = the measurement on the individual of ijkl l th the family from the provenance in k j th th the replicationi th μ = the overall mean r the effect of replication ( =1, 2, 3, ..,n)i = i i th s = the effect of site ( =1, 2)i i i th p = the effect of provenance ( =1, 2, 3, …, n)j j j th f(p) = the effect of family in provenance k( j) k j th th ( =1, 2, 3, …, n)k rp = the interaction effect between ij i th j replications and provenances th rf(p) = the interaction between ik( j) i th k j replications and family within th th provenance e = the residual.l(ijk) the sas proc glm was used to obtain the coefficients of the expected mean squares for the calculation of heritability. the components of variance were calculated using varcomp proc from sas statistical program (cary 1999). heritability was estimated from the variance components as described in falconer and mackay (1996). narrow sense individual ( ) and family h i2 heritability ( ) for each parameter were h f2 estimated using equations: where: = additive genetic variance = between-family-within-provenance variance component = phenotypic variance calculated as where: = variance due to interaction between rep l i cat ion and famil y -w ithinprovenance (experimental error) = variance among individual trees within family (sampling error) = family phenotypic variance, calculated as where: k k2 3 and = respectively, coefficients for and in the expected mean squares. phenotypic ( ) and genetic correlations rp xy( ) ( ) were estimated from the component of rg xy( ) variance and covariance (falconer 1981) substituted into the standard equation for the product moment correlation coefficient: where: and = components of variance of phenotypic and genotypic products of x parameter and = components of variance of phenotypic and genotypic products of y parameter and = components of covariance of phenotypic and genotypic products of x and y parameters, respectively. genetic gain (∆) was calculated by falconer's (1981) formula: ∆g = h s = h i σ2 2 p where: i = intensity of selection taken from zobel and talbert (1984) with proportion selected 0.70, 0.50 and 0.30 σp = phenotypic standard deviation. 14 biotropia vol. 23 no. 1, 2016 results and discussion growth rate and genetic variation significant differences were observed among provenances and among families within provenances for all parameters at limbangan and parungpanjang, except for collar diameter of among provenances at parungpanjang (table 2). at the limbangan, the kkp provenance attained a maximum height followed by gsj, pkc, pgc and nkj provenances. maximum collar diameter was recorded in gsj provenance followed by kkp, pgc, rps and nkj. field survival was recorded maximum in kkp provenance, followed by krs, pgs, rps and gsj. the kkp provenance had the highest height growth and presented approximately 44% faster than the slowest growth provenance. for root collar diameter, the gsj provenance was higher and presented about 59% higher growth than the least growth provenance. the range of variation at family level of irrespective provenances was large in respect to height and root collar diameter (table 3) as proven by values of coefficient of variation. over all kkp, gsj and pgc provenances were better than others. the range of family variation (irrespective provenance) at parungpanjang was high in all growth parameters. maximum height was recorded in gsj provenance, which was closely followed by apj, nkj, kkp and bhs provenances. maximum root collar diameter was attained by bhs provenance, however, it was at par with pkc, pgc and apj provenances. gsj provenance had the highest growth for height and presented about 17% higher growth than the least growth provenance. for root collar diameter, differences among provenances means were not statistically significant and the best provenance was only 10% larger than the least provenance. ten provenances showed field survival more than 70%, except apj and blb provenances. based on the values of cv and range of the family means, a wide range of variability was observed in respect of growth parameters ( ).table 3 the height and collar diameter growth of n. cadamba were better at limbangan than those at parungpanjang. the plant growth at parungpanjang was affected by heavy growth of weeds causing suppression and competition for nutrition and growth space. the factor might have led to slow growth of the plant at the site. on the other hand, survival percentage was higher at parungpanjang. survival percentage at limbangan was reduced by disturbance of domestic animal and irregular agricultural crops in several parts of the area covering the main plants in early growth of seedlings. in general, growth performance in both sites is better than the other progeny test of conducted in n. cadamba wonogiri, central java (setyadi . province et al 2013). differences in height and root collar diameter growth were greater among families than among provenances. at limbangan, the best growing family exceeded the least growing family by 243% and 234% for height and root collar diameter, table 2 mean square for height and collar diameter in provenance-progeny tests of at two sites in west java n. cadamba province, indonesia source of variation degrees of freedom mean square limbangan, garut parungpanjang, bogor height (m) collar diameter (cm) survival (%) height (m) collar diameter (cm) survival (%) blocks 4 61.281 ** 221.693 ** 9,310.667ns 8.604 ** 35.499 ** 25,694.552** provenance 11 3.858 ** 16.933** 3,949.994ns 0.833 * 1.6748 ns 2,902.045ns fam (prov) 93 2.165 ** 8.051** 2,018.897ns 0.864 ** 3.428** 2,167.913ns block*prov 44 1.859 ** 6.208* 1,860.280ns 1.196 ** 4.758** 2,799.698ns block*fam(prov) 359 1.973 ** 7.498 ** 2,644.036ns 1.053 ** 3.897** 2,841.399ns error 1,044 0.740 3.353 2,153.968 0.413 1.681 1,862.556 notes: ** = significant at <0.01 p * = significant at <0.05p ns = not significant 15 intraspecific riation of va in provenance-progeny tests neolamarckia cadamba – et al.dede j. sudrajat respectively. at parungpanjang, the best family exceeded the least family by 57% and 122% for height and root collar diameter, respectively (data are not shown). these results showed the potential of provenance-progeny test of for n. cadamba selection. analysis of variance across the sites revealed no significant provenance against site interaction and family within provenance against table 4 growth performance (mean ± standard deviation) of various provenances of across different sitesn. cadamba provenance height (m) collar diameter (cm) survival (%) rps 2.15±0.94 4.42±2.16 70.0 krs 2.11±0.97 3.92±1.96 69.5 oks 2.25±1.19 4.19±2.10 64.8 gsj 2.50±1.22 4.77±2.27 66.9 nkj 2.35±1.16 4.35±2.16 60.7 apj 2.23±1.04 4.11±2.04 60.7 blb 1.97±0.77 3.63±1.82 58.2 ktb 2.15±0.71 4.06±2.19 67.0 pgc 2.30±1.10 4.49±2.23 70.1 pkc 2.33±1.10 4.36±2.08 63.2 bhs 2.31±1.05 4.33±2.10 61.9 kkp 2.50±0.86 4.59±1.83 61.3 f test: provenance 2.80 * 3.57 ** 1.43 ns provenance *site 2.49 ns 2.14 ns 0.70 ns family*site 1.55 ns 1.41 ns 0.84 ns notes: ** = significant at <0.01p * = significant at < 0.05p ns = not significant table 3 growth performance (mean ± standard deviation) of various provenances in provenance-progeny tests of n. cadamba provinceat two sites in west java , indonesia (number in parentheses are ranks) provenance limbangan, garut parungpanjang, bogor height (m) collar diameter (cm) survival (%) height (m) collar diameter (cm) survival (%) rps 2.34±1.20 (8) 4.78±2.77 (4) 62.5 (4) 1.97±0.58 (12) 4.06±1.59 (7) 77.5 (2) krs 2.14±1.12 (11) 3.92±2.26 (11) 63.9 (2) 2.08±0.82 (8) 3.93±1.66 (10) 75.0 (6) oks 2.46±1.48 (6) 4.45±2.53 (8) 58.6 (8) 2.04±0.78 (11) 3.94±1.54 (9) 70.9 (9) gsj 2.67±1.47 (2) 5.35±2.75 (1) 61.9 (5) 2.31±0.79 (1) 4.11±1.26 (5) 71.9 (8) nkj 2.47±1.34 (5) 4.68±2.59 (5) 50.7 (11) 2.26±0.99 (3) 4.09±1.73 (6) 70.7 (10) apj 2.17±1.10 (9) 4.08±2.31 (10) 53.2 (10) 2.28±0.98 (2) 4.13±1.78 (4) 68.2 (11) blb 2.17±1.16(10) 4.27±2.41 (9) 58.8 (7) 2.12±0.78 (6) 3.78±1.55 (12) 57.5 (12) ktb 1.89±1.30(12) 3.35±2.45 (12) 60.2 (6) 2.05±0.40 (10) 3.86±1.22 (11) 73.8 (7) pgc 2.57±1.30 (4) 4.87±2.61 (3) 63.7 (3) 2.06±0.81 (9) 4.14±1.74 (3) 76.4 (3) pkc 2.58±0.89 (3) 4.56±2.72 (6) 47.5 (12) 2.11±0.82 (7) 4.17±1.67 (2) 78.9 (1) bhs 2.42±0.75 (7) 4.49±2.07 (7) 53.8 (9) 2.21±0.92 (5) 4.19±1.78 (1) 76.3 (4) kkp 2.73±0.95 (1) 5.15±2.06 (2) 75.0 (1) 2.25±0.71 (4) 4.00±1.38 (8) 75.0 (5) family range 1.40-4.81 2.10-7.03 35-90 1.38-2.97 2.50-5.56 40-100 mean 2.42 4.49 59.1 2.14 4.08 72.7 cv (%) 35.50 40.76 30.06 31.76 notes: rps = rimbopanti nature reserve-sumatera, krs = kampar-riau-sumatera, oks = ogan komering ilir-sumatera, gsj = garut selatan-java, nkj = nusa kambangan, apj = alas purwo-java, blb = batulicin-kalimantan, ktb =kapuas-kalimantan, pgc = parangloe-sulawesi, pkc = pomalaa-sulawesi, bhs = batuhijau-sumbawa, kkp =kuala kencana-papua 16 biotropia vol. 23 no. 1, 2016 site interaction for all parameters (table 4). in across site, a significant difference was revealed by provenances for height and diameter parameters. among different provenances, the performance of the gsj and kkp provenances was found significantly superior than the others. the component of variance attributed to variation among provenances ranged from 0.5% for height at parungpanjang to 1.7% for root collar diameter at limbangan. on the other hand, component of variance attributed to variation among families within provenances were lower than component of variance among provenance, ranged from 0.4 to 0.6% at both sites (table 5). the higher values toward variation among provenance in relation to families within provenances suggested that provenances are isolated or gen flow is insufficient to overlap the effect of selection and/or genetic drift. larger genetic variation among provenances than genetic variation among families within provenances were also observed in provenanceprogeny test by zheng (1994) for et al. pinus caribaea in china, bali-uckas . (1999) for et al acer platanoides in sweden, sebbenn (2003) for et al. araucaria angustifolia et al in brazil, adinugraha . (2013) for tectona grandis in gunung kidul, and setiadi and fauzi (2015) for in araucaria cunninghamii bondowoso, indonesia. on the other hand, study based on aflp loci using 4 populations of n. cadamba showed that variation among population (27%) was much lower than variation within population (73%) (sudrajat . 2015). different et al trend was caused by different number and distribution of populationn. cadamba . genetic parameters phenotypic and genetic correlations between height and root collar diameter parameters at parungpanjang were higher than those at limbangan (table 6). this indicated that the possibility of selection in one parameter at parungpanjang is more efficient than those at limbangan. phenotypic correlations were higher than genetic correlations among height and root collar diameter parameters (table 5). high phenotypic correlation provided more support for combining these surveyed parameters as early selecting criteria. narrow sense individual ( ) and within h i2 families ( ) heritability at limbangan were poor h f2 (ranged from 0.031 to 0.055), while the table 5 components of variance and relative contribution (number in parentheses) of replications ( ), provenances ( ), σ σ2 2 r p interaction between replications and provenances ( ), families within provenances ( ), interaction between σ σ2 2 rp f(p) replications and families ( ) and individual within families ( ) of provenance-progeny tests at two sites in west σ σ2 2 rf(p) e java , indonesiaprovince components of variance limbangan, garut parungpanjang, bogor height (m) collar diameter (cm) height (m) collar diameter (cm) σ2r 0.49330 (28.0%) 1.54578 (22.9%) 0.06746 (9.1%) 0.27601 (9.5%) σ2p 0.02681 (1.5%) 0.11501 (1.7%) 0.00377 (0.5%) 0.04134 (1.4%) σ2rp 0.00465 (0.3%) 0.04640 (0.7%) 0.01546 (2.1%) 0.07356 (2.5%) σ2f(p) 0.01045 (0.6%) 0.03875 (0.6%) 0.00359 (0.5%) 0.01216 (0.4%) σ2rf(p) 0.48827 (27.7%) 1.64063 (24.3%) 0.23901 (32.2%) 0.82821 (28.4%) σ2e 0.74002 (42.0%) 3.35359 (49.8%) 0.41369 (55.7%) 1.68163 (57.7%) table 6 genetic (upper diagonal) and phenotype (lower diagonal) correlation coefficients among traits in provenanceprogeny tests of two sites in west java , indonesian. cadamba at province limbangan, garut parungpanjang, bogor height collar diameter height collar diameter height 0.29 0.56 collar diameter 0.799** 0.885** note: ** = significant at <0.01p 17 intraspecific riation of va in provenance-progeny tests neolamarckia cadamba – et al.dede j. sudrajat heritabilities at parungpanjang site were moderate and ranged from 0.093 to 0.178 (table 7). values of heritability changed with sites with the same set of genotypes. lower value of heritability of the trial in limbangan could be due to high incidence of domestic animal and irregular agricultural crops covering several parts of area causing high environmental difference. setyadi et al. (2013) n. cadamba studied 75 half-sib families explored from java island and found higher family heritability of approximately 0.32 for height and 0.38 for stem diameter at breast height. poor values of heritability in this study could be due to plant age that was still young, indicating the genetic control of the parameters was still weak. for example at parungpanjang, family heritability at 6 months old plant (0.093 for height and 0.066 for root collar diameter) was lower than family heritability at 12 months old plant (0.150 for height and 0.178 for root collar diameter), indicating heritability was not yet stable. trend of increasing heritability with age was also reported in the studies of (osario eucalyptus grandis et al. 2001; gapare 2003), et al. pinus banksiana et (weng al. 2006) e. urophylla et al. and (kien 2009). the increased heritability with age for growth parameters could also be resulted from competitive effects occurred in later age in the stand (kien 2009). according to zobel and et al. talbert (1984), the heritability values often change with age when the environment changes and when the genetic control of the characteristic changes as the trees reach mature age. therefore, activities to improve site environment conditions will be very important to optimize growth and to reach stable heritability. heritability estimated should be interpreted carefully because of unequal number of families per provenances presented in the trials. in a multiprovenance-progeny test, the family variance is averaged across different provenances, with more information provided by the better-represented provenances (kien 2009). in all parameters at et al. both sites, family heritability was found to be higher than individual heritability. some studies in other species also revealed similar trend, such as in araucaria angustifolia et al.in brazil (sebbenn 2003) and in turkey (gulcu & celik 2009). pinus brutia this result indicated that higher genetic gain can be achieved with family selection. estimation of genetic gain is determined by heritability value, selection intensity and phenotypic standard deviation. in this research, simulation to predict the genetic gain used proportional selected family of 30%, 50% and 70% and the corresponding selection intensity were 1.15, 0.79 and 0.49 (zobel & talbert 1984). the higher value of genetic gain estimation for height and collar diameter by proportional selected family of 30% was 0.13 and 0.18 for limbangan and 0.31 and 0.16 for parungpanjang (table 8). predictions of genetic gain from family table 7 estimated heritabilities in provenance-progeny tests of two sites in west java , indonesian. cadamba at province notes : category of heritability values according to and cotterill dean (1990): y of poorheritabilit <0.1 = heritability of moderate0.1-0.3 = heritability of high>0.3 = table 8 estimates of genetic gain for height and diameter by within family of in provenance-progeny tests of n. cadamba proportion selected garut parungpanjang height diameter height diameter 0.30 0.13 0.18 0.31 0.16 0.50 0.05 0.09 0.09 0.07 0.70 0.03 0.05 0.05 0.04 18 biotropia vol. 23 no. 1, 2016 selection simulation revealed that height parameter showed higher values than collar diameter parameters. it was also revealed that genetic gain of height parameters at parungpanjang was higher than genetic gain at limbangan. johnson . (1955) and seghal . et al et al (1995) pointed out that heritability estimates along with genetic gain is more useful than heritability alone, because the heritability estimates indicated only the effectiveness of selection on genotype based on phenotypic performance, but fails to indicate the genetic progress. high heritability and genetic gain indicated the additive gen action on these parameters for their expression. conclusions levels of genetic variation in were n. cadamba found to be higher for all parameters among provenances than within provenances indicating possibility to use well performing provenances as seed sources for reforestation and regeneration practices. estimated family heritability was higher than narrow sense individual heritability, indicating the possibility of greater gains with selection among families rather than mass selection. however, heritability was low to moderate and not yet stable for all parameters at both sites, indicating that possibility of genetic gains with selection is limited and selection should be done until the heritability reached relatively high and stable values. a combination of family and within family selection would be effective in improving growth of this species. improving growth environment conditions with controlling the domestic animal and weed intensively should be applied immediately and continuously to optimize the plant growth, stabilize heritability and to optimize genetic gain. acknowledgements the authors wish to acknowledge seameobiotrop, pt. arutmin indonesia (batu licin, south kalimantan), pt. dasa intiga (kapuas, centr kalimantan), pt. newmont indonesia al (batu hijau, sumbawa) for providing accommodations during genetic material exploration. the authors were grateful to the authorities of forest tree seed technology research institute, bogor, for financing the establishment of provenance-progeny trials at parungpanjang, bogor and limbangan, garut. references acharyya s, rathore ds, kumar 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bacterial).cdr optimal bacterial density and fertilizer dosage for bioremediation of oil contaminated sandy beach: a case of cilacap, indonesia 1* 2 2 yeti darmayati , harpasis s. sanusi , tri prartono , 3 1dwi andreas santosa and ruyitno nuchsin 1 lembaga ilmu pengetahuan indonesia marine microbiology laboratory, research center for oceanography, (lipi/ e ) jakarta 11048, indonesian institut of sciences , indonesia 2 institut pertanian bogor bogor 16680, faculty of fisheries and marine sciences, , indonesia 3 faculty of agriculture, , indonesiinstitut pertanian bogor bogor 16680, a received 20 july 2016/accepted 5 july 2017 abstract bioremediation, involving biostimulation and/or bioaugmentation, is a promising method to overcome oil spills in cilacap coastal waters. cilacap coastal area has high risk on oil pollution. this study investigated the stimulatory effect of nitrogen concentration, bacterial density and the composition of bacterial culture in enhancing oil degradation in this area. the applications of 4 different concentrations of slow release fertilizer (srf) and 2 different densities of bacterial cells in the form of single (rco/b/08_008) and mixed culture were employed in microcosm experiments for 28 days. the efficacy of combining bacterial culture and fertilizer application in various concentrations was also tested. oil degradation, bacterial growth and environmental parameters were monitored periodically during the experiments. the results showed that oil degradation rate was more influenced by nutrient concentration (biostimulation) than bacterial number or culture composition (bioaugmentation) added. the efficacy of biostimulation in degrading oil was better than that of bioaugmentation. biostimulation increased oil degradation up to 6.4 times higher than the control. the optimum of fertilizer concentration added was 7.5 mg n/g(c:n ratio of 1,000 : 75), which increased depletion rate both in biostimulation-only and the combination of biostimulation with bioaugmentation up to 6.4 and 7.5 times higher than the control, respectively. it is suggested that bioremediation of oil-contaminated sandy beach in cilacap would be optimal by employing a combination of slow release fertilizer at 8concentration having c/n ratio = 1,000 : 75 and rco/b/08_008 culture at density of 0.5 x 10 cells/ml (100% homology with alcanivorax sp. te-9). keywords: bacteria, bioremediation, coastal, fertilizer, indonesia, oil, optimal introduction petroleum and its derived products are the major source of energy. to ensure long-term energy security for indonesia, pertamina (the indonesian oil and gas company) plans to expand and upgrade the refinery capacity plant in dumai, cilacap and balongan. this expansion increases the pollution risk of cilacap coastal waters due to oil spill. therefore, there is a need to anticipate the impact of oil pollution in this area. bioremediation is considered to be a promising biological approach to overcome oil pollution in marine environment (swannel et al. 1996; jackson & purdue 1999; munawar et al. 2007; xu 2010). bioremediation is basically an enhancement of biodegradation rate (leahy & colwell 1990; mrozika & seget 2010). biological degradation process depends on several factors such as environmental conditions (ph, temperature, dissolved oxygen/do, degree of acclimation, accessibility of nutrients), numbers of microorganisms, types of microorganisms, cellular transport properties, chemical partitioning in growth medium and chemical structure of compounds degraded (coartes et al. 2009; lin et al. 2009). to enhance the effectivity of bioremediation, those environmental factors are modified with a p p l i e d n u t r i e n t s u p p l e m e n t a t i o n * corresponding author: yeti.darmayati@yahoo.com biotropia 4 3 7 182 191 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 3 611 182 (biostimulation) and introduction of specific c o m p e t e n t s t r a i n s o r c o n s o r t i a o f microorganisms (bioaugmentation) (mrozika & seget 2010; xu & liu 2010). few successes of implementing biostimulation and bioaugmentation strategies for remediating oil contaminated marine sediment in several tropical countries such as singapore, indonesia and india were reported (xu 2003; xu et al. et al. 2005; munawar . 2007; darmayati 2010; et al darmayati . 2015; pasumarthi & mutnuri et al 2016). field investigation in inter-tidal foreshore environment in singapore and indonesia demonstrated that fertilizer (osmocote) was able to significantly enhanced hydrocarbonoclastic bacterial growth and increased the biodegradation of aliphatics and polycyclic aromatic hydrocarbon (pah) 2010; darmayati 2010 .(xu ) osmocote is an anorganic slow release fertilizer (srf) coated by polymeric resin and consisted of water-soluble n–p–k at concentrations of 18%, 4.8% and 8.34%, respectively. petroganik, another fertilizer type, is organic nutrient having capability of stimulating soil microorganisms and enhancing oil degradation in oil contaminated surabaya coastal area (munawar 2007).et al. introduction of several selected bacteria in oil contaminated cilacap coastal area through mesocosm study was proven to increase the rate of oil degradation up to 1.6 2.2 times higher than the rate of oil degradation in the control site (darmayati . 2015).et al until now, information is lacking on the optimal bacterial density and fertilizer dosage needed to increase oil degradation rate in indonesian coastal area. this present study was aimed to study the stimulatory effect of nitrogen concentration, bacterial density and composition of bacterial culture to accelerate oil degradation in cilacap coastal area. cilacap coastal area is one of the most often areas polluted by oil spills. materials and methods preparation of bacterial culture three isolates of oil and pahs degrading bacteria collected from jakarta bay sediment were provided by marine microbiology laboratory, research center for oceanography, lembaga ilmu pengetahuan indonesia (lipi/indonesian institute of sciences). the isolates were rco/b/08-008 (ab055207 sp. te-9, alcanivorax similarity 100%), rco/b/08-004 (am905859 pseudomonas balearica, similarity 99%) and rco/b/08-015. the first two isolates were identified using partial sequencing of 16s rrna. the third isolate had not been sequenced yet; therefore, it only had a name code (hatmanti & darmayati 2009). to prepare the mix cultured inoculum, the three strains were grown separately in marine agar at 30 c for 72 hours. each strain o was harvested using driglasky stick and sterilized saline water, then transferred into sterilized tube. the density of each strain was calculated using measurement of optical density (od) and graph which correlate od and bacterial cell number. dilution method using sterilized saline water was conducted to make similar density. the mixed culture was formulated by mixing all the three strain cultures in the same volume to have a density ratio of 1 : 1 : 1. preparation of artificially oil contaminated sediment oil contaminated sediment was prepared by spiking oil mousse of arabian light crude oil (alco) into the sample of sandy sediment and seawater collected from cilacap coastal area. oil mousse is a thick foamy mixture of oil and water which is formed when petroleum products are mixed with water due to the actions of wind and waves (petropedia inc 2017). oil mousse used in this study was prepared by mixing cilacap seawater and alco in 70 : 30 proportion using sterilized mixer for 15 x 3 minutes. oil mousse of 4 mg was spiked into 10 g sample of cilacap sediment to achieve target concentration of 100,000 ppm (100 mg/g) of oil-contaminated sediment. fertilizer application gramafix® fertilizer was used in this study. this fertilizer is a granular srf fertilizer containing macro and micro nutrients (n:p:k:mg:c:s:micro element = 22:7:12:2:4:3:1). this fertilizer is commonly used for agricultural purposes. this fertilizer was selected due to their good performance in previous studies on oil 183 optimal bacterial density and fertilizer dosage for bioremediation darmayati et al. each microcosm in slurry form contained 15 ml seawater and 10 g oil-contaminated sediment having concentration of 100 mg/g. a microcosm is an experimental unit in a 50 ml sterilized falcon tube. experiments were conducted for 28 days in an incubator shaker (having shaking speed of 100 rpm) at room temperature (28 ± 2 c). o measurement of oil concentration was conducted in triplicates for each treatment at each sampling time. measurements of supporting data such as numbers of bacterial cells, environmental factors (dissolved oxygen/do, ph and salinity) and nutrient (nitrogen total) were conducted without any replications in each treatment at each sampling time. for biostimulation experiment, measurements were conducted at day-0, day-14 and day-28 after exposure. measurements of all parameters for the bioaugmentation and combination were conducted only at day-0 and day-28 after exposure. analysis of oil, bacteria and environmental parameters oil concentration was measured from the slurry of the whole content of each microcosm. the extraction of oil was carried out using centrifugation and maceration methods with a degradation rate conducted in our laboratory (unpublished data). there were 3 experiments conducted to determine the optimal fertilizer dosage for bioremediation i.e. biostimulation, bioaugmentation and combination between biostimulation and bioaugmentation. in the biostimulation experiment, the applied fertilizer dosages were 1.88 mg n/g; 3.75 mg n/g; 7.50 mg n/g; and 15.00 mg n/g, respectively (table 1). in the bioaugmentation experiment, there was no fertilizer applied (table 1). in the combination between biostimulation and bioaugmentation experiment, the applied fertilizer dosages were 7.50 mg n/gand 15.00 mg n/g (table 1). experimental design to study the stimulatory effect of nitrogen concentration, bacterial density and the composition of bacterial culture in accelerating oil degradation, three experiments were conducted in completely randomized design (table 1). the treatments applied were separate alcospiked sediment treatments amended with nitrogen using different fertilizer dosages and/or bacteria at different densities and compositions. one control treatment was set up as not having srf fertilizer and no bacterial amendment. biotropia vol. 24 no. 3, 2017 table 1 treatment performed in each different experiment experiment treatment nitrogen concentration (mg/g) single culture density (cell/ml) mixed culture density (cell/ml) biostimulation control no fertilizer and bacteria addition a 1.88 b 3.75 c 7.50 d 15.00 bioaugmentation control no fertilizer and bacteria addition a 0.5 x 108 b 1.0 x 108 c 0.5 x 108 d 1.0 x 108 combination control no fertilizer and bacteria addition a 7.50 0.5 x 108 b 7.50 1.0 x 108 c 7.50 0.5 x 108 d 7.50 1.0 x 108 e 15.00 0.5 x 108 f 15.00 1.0 x 108 g 15.00 0.5 x 108 h 15.00 1.0 x 108 184 mixture of dichloromethane: n-hexana (1 : 1) having proanalysis grade as a solvent. na so was 2 4 used to absorb the water remained in the extracted oil. oil concentration in the slurry represented the concentration of oil in the treatment. the numbers of bacterial cells on overlying water indicated the density of bacterial cells in porewater inside the microcosm. a detachment of bacteria was conducted by placing the microcosm in a vortex and shaking the vortex at 300 rpm for 5 minutes. a subsample was drawn from the shaken microcosm. counting of total bacterial cells was conducted using dilution method conducted on the subsample. acridine orange solution (0.05%, 2.29 ml) was added into the subsample. the supernatant was then filtered using polycarbonate membrane having pore size of 0.22 µm. the polycarbonate membrane was previously submerged in sudan black solution to obtain a contrast background. direct counting of total bacterial cells was conducted under epifluorescent microscope (hobbie et al. 1977). to monitor the changes of environmental condition and nitrogen concentration in this microcosm system during experiment, one set of microcosm was provided for each purpose. the microcosm set was prepared similarly with the microcosm for measuring oil concentration and counting the numbers of bacterial cells. however, the numbers of microcosms were only prepared for one replication at each sampling time in each treatment. measurements of salinity, ph and dissolved oxygen (do) were carried out for the overlying water using hand refractometer (for measuring salinity), ph meter (horiba, navi d-54) and do meter (horiba, ysi 55), respectively. concentration changes of soluble nitrogen (n) were measured by filtering, extracting and analyzing total nitrogen concentration for the whole content of microcosms. persulfate digestion method was used for extraction prior to nutrient measurement ). total (zhu et al. 2001 nitrogen concentration was determined using hach dr 800 coloridirect reading meter using hach . proprietary reagents analytical data experiments were conducted using three independent replications for oil concentration. percentage of oil depletion was calculated from oil concentration applied in the first and the third experiments after 28 days of incubation. oil degradation data were analyzed using one way anova. the mean of the data were compared by duncan multiple range test (dmrt) at p ≤ 0.05. kolmogorov-smirnov test was used to test the normality of data, while the levene statistic test was used to test data heterogeneity. statistical software used was spss 16. numbers of bacterial cells, nitrogen concentration and environmental data were provided as supporting data. these supporting data were only available for each treatment at each sampling time (without any replication). these supporting data were qualitatively analyzed. results and discussion biostimulation application of slow release fertilizer (srf) increased oil degradation rate in oil-contaminated coastal environment. however, the concentration of srf should be carefully calculated to avoid a side effect in the oil-contaminated area, such as excessive algal growth. this study showed that srf was effective to increase oil degradation. applications of srf at all tested n concentrations were able to increase degradation rate significantly (table 2). oil weight after being treated with srf (for 28 days) ranged from 0.453 ± 0.03 to 0.815 ± 0.01 g per 10 g sediment. the remaining oil in the control was at the level of 1.185 ± 0.06 g per 10 g sediment. the percentage of oil depletion in the srf treatment was between 44.2 ± 4.3% and 65.9 ± 3.4%. the percentage in the control treatment was only 13.3 ± 2.6%. there was statistically significant difference berween control and treatments. oil depletion and a numbers of bacterial cells were observed in the control. the results showed that oil degrading bacteria were available in this study site. however, the nutrients availability was insufficient for the growth of oil degrading bacteria due to the occurrence of oil spill. nutrients concentration and hydrocarbon bioavailability are the key factors affecting oil biodegradation rates in oil-contaminated sites (xu et al. 2005). other studies proved that the growth of oil degrading bacteria and thus, oil degradation rate can be strongly increased by applying fertilizers containing inorganic n and p 185 optimal bacterial density and fertilizer dosage for bioremediation darmayati et al. (swannell et al. 1996; röling et al. 2002; coulon et al. 2007; darmayati 2010). there was no guarantee that higher fertilizer dosage resulted to higher oil degradation rate. results of this study showed that excessive dosage of fertilizer inhibited the increase of oil depletion rate and bacterial numbers. biostimulation treatments applying fertilizer with nitrogen concentration of 1.88 and 7.50 mg/g showed an increase in oil depletion efficiency and in bacterial growth. the oil depletion efficiency increased in a range of 2.3 5.0 times compared to that in control. the bacterial density were in a range of 4.1 4.6 times higher compared to that in control (table 2). however, the enhancement of oil degradation was only 3.3 times when nitrogen concentration increased up to 15.0 mg/g. this result indicated that carbon/nitrogen (c/n) ratio of 1,000 : 75 and 1,000 : 18.8 provided better environment for the growth of oil degrading bacteria than the ratio of 1,000 : 150. xu 2003) mentioned that et al. ( adding 0.8% of nitrogen (c/n ratio 1,000 : 33) and 1.5% of nitrogen (c/n ratio 1,000 : 61) of slow release fertilizer (osmocote) consisting of 18% nitrogen, 4.8% phosphor and 8.3% kalium (w/w) to oil contaminated sediment were sufficient to maximize the metabolic activity of biomass and to increase biodegradation of straight chain and branch chain of n-alkane, respectively. there was high positive correlation between percentage of oil depletion and bacterial density. this was especially shown between the percentage of oil depletion obtained at day-28 and the bacterial density at day-14 (r = 0.87). coefficient of determination of 0.77 showed that 77% of variations in the percentage of oil depletion were explained by bacterial density; while the remaining 23% of variations in percentage of oil depletion might be due to other factors such as nutrients and other environmental factors. until certain level, high oil concentration and high nutrient availability may increase bacterial growth. this study showed that bacterial density in microcosm with srf application was 2.4 – 4.6 times higher than that in control at day-14 (table 2). high bacterial density in this experiment represented high abundance of oil–degrading bacteria. coulon (2007) mentioned that n et al. and p increased the abundance of oil degrading bacteria and total oil degradation. our previous study showed also that microbial biomass increased and the growth rate of oil degrading bacteria was higher than total bacteria (darmayati 2010). population of oil degrading bacteria used oil as their source of energy and nutrient. higher table 2 the impact of nitrogen addition at various concentrations (mg/g) on oil depletion efficiency and bacterial density during 28 days exposure; including average and standard deviations of oil content and oil depletion (n = 3), also bacterial number (n = 1) nitrogen concentration added (mg/g) oil content (g) oil depletion (%) 0 d 7 d 14 d 28 d 28 d 0 1.280 + 0.06 1.289 + 0.02 1.270 + 0.06 1.185 + 0.06 13.3 + 2.6 e 1.88 1.280 + 0.06 0.984 + 0.01 0.787 + 0.09 0.609 + 0.33 51.9 + 3.4 b 3.75 1.280 + 0.06 0.996 + 0.01 0.904 + 0.09 0.815 + 0.01 31.0 + 3.6 d 7.50 1.280 + 0.06 1.007 + 0.01 0.834 + 012 0.453 + 0.03 65.9 + 3.4 a 15.00 1.280 + 0.06 1.001 + 0.00 0.809 + 0.02 0.72 + 0.03 44.2 + 4.3 c bacterial cells number (cells/ml) 0 d 7 d 14 d 28 d 0 6.e+07 6.e+07 1.e+08 2.e+08 1.88 2.e+08 2.e+08 3.e+08 9.e+08 3.75 8.e+07 8.e+07 3.e+08 5.e+08 7.50 2.e+08 2.e+08 4.e+08 1.e+09 15.00 2.e+08 2.e+08 3.e+08 9.e+08 note: the same letter (a e) in the same column showed that the difference was not statistically significant (p < 0.05) 186 biotropia vol. 24 no. 3, 2017 number of oil degrading bacteria led to higher rate of oil depletion. bioaugmentation bioaugmentation was able to increase oil degradation, both in single or mixed culture t reatment . however, b ioaugmentat ion contributed lower increase of oil degradation compared to the increase caused by biostimulation. significant (p < 0.05) increase on the percentage of oil degradation caused by bioaugmentation ranged from 28.4 ± 1.9% to 34.3 ± 6.2% (table 3), while the increase of oil degradation percentage caused by biostimulation ranged from 44.2 ± 4.3% to 65.91 ± 3.4 % (table 3). this occurrence happened because the growth of oil degrading bacteria (hydrocarbonoclastic bacteria) was limited by nutrient insufficiency in the area of oil spill. c/n ratio was higher than it should be when oil spill occurred and no added nutrient administered to the oil spill area. increase of bacterial density in each treatment was observed, but the increase was in a low rate. the highest numbers of bacterial cells (8.2 x 10 8 cells/ml) was observed at day-14 after treatment in the microcosm supplemented with mixed culture. this number was only 3.61 times higher than the bacterial number in control. there was a strong positive correlation between numbers of bacterial cells and oil degradation percentage (r = 0.91) in this study. oil degradation was supported by bioaugmentation with sp. which was much more alcanivorax effective than nutrient addition (mckew . et al 2007). oil pollution in thames estuary was in less density compared to that in cilacap coastal area. the cilacap coastal area are heavily contaminated by oil due to industries and oil refinery establishments located in the area. introduction of mixed culture (alcanivorax sp. te-9, pseudomonas balearica and rco/b/08) 015 and single culture (alcanivorax sp.) did not give any significant effects on oil degradation rate in cilacap coastal area. different numbers of bacterial cells supplemented did not exhibit significant impact on oil depletion rate (table 3). treatment administering alcanivorax sp. te89te-9 having density of 1.0 x 10 cells/ml exhibited the highest oil degradation rate (34.3% in 28 days), although it was not significantly different from the other three treatments (table 3). cilacap coastal area had high diversity of oil degrading bacteria and various enzymes needed for oil degradation were available in this area. oil degrading bacteria available in cilacap coastal area were flexibacteraceae bacterium, bacillus aquamaris, b. megaterium, b. pumilis, halobacillus trueperi and rhodobacteraceae bacterium (syakti et al. 2013). combination treatment combination treatment of biostimulation and bioaugmentation exhibited the best performance of oil bioremediation. in this experiment, oil reduction was observed in the range of 13.1 ± 0.3% – 74.6 ± 3.6% at day-28 days after treatment. bioaugmentation supplemented with srf fertilizer having nitrogen concentration of 7.5 mg/g showed higher oil reduction than that in combination treatment supplemented with srf fertilizer having nitrogen concentration of 15.0 mg/g (table 4). these results occurred both in single strain or mixed culture and also in high or table 3 the impact of bacterial amendment on oil depletion rate (at 28 days) and bacterial abundance (at 14 days); including average and standard deviations of oil depletion (n = 3) treatment bacterial cell addition (cells/ml) oil depletion (% ) bacterial cells number (cells/ml) control 0 13.33 + 0.3 a 2.28e+08 single culture 0.5 x 108 28.37 + 1.9 b 7.87e+08 1.0 x 108 34.34 + 6.2 b 8.22e+08 mixed culture 0.5 x 108 30.53 + 4.8 b 7.87e+08 1.0 x 108 27.81 + 0.2 b 5.16e+08 note: the same letter (a b) in the same column showed that the difference was not statistically significant (p < 0.05) 187 optimal bacterial density and fertilizer dosage for bioremediation darmayati et al. low bacterial density. this study confirmed that c/n ratio of 1,000 : 75 was better than 1,000 : 150 for bacterial growth. excessive nutrients concentration might suppress the growth of bacteria, whereas in combination treatments, nutrients concentration was lower, but sufficient for bacterial growth. sufficient loading rates of nitrogen wil l be necessary to tr ig ger biostimulation. according to gibbs et al. (1975) approximately 4 mm of nitrogen was required to breakdown 1 mg of crude oil; phosphorus did not limit p/n ratio to a minimum of 0.02. loading rates of nutrients below critical concentration would be a waste of resources; the same as excessive use which could promote secondary impacts such as harmful algal bloom and oxygen depletion (bragg et al. 1994; jackson & pardue 1999). the combination of bioaugmentation and biostimulation approach by nitrogen amendment at moderate level was proven, in the laboratory and in the field, as the best approach for reducing oil in environment (darmayati et al. 2015; pasumarthi & mutnuri 2016). our study in cilacap coastal area using mesocosm approach confirmed that combination of bioaugmentationbiostimulation is the best strategy for cleaning up oil spills (darmayati et al. 2015). other studies in goa (india) and egypt supported this conclusion (pasummarthi & mutnuri 2016; el-borai et al. 2016). enviromental condition environmental conditions (ph, temperature, dissolved oxygen, degree of acclimation, accessibility of nutrients) are among important factors in biological degradation processes, besides numbers of microorganisms, types of microorganisms, cellular transport properties, chemical partitioning in growth medium and chemical structure of degraded compounds (coartes et al. 2009; lin et al. 2009). in this study, the impact of nutrients addition into environment could be observed obviously such as the changes of salinity and do levels during exposures time (fig. 1). salinity was shown as an important factor in bacterial growth influencing oil biodegradation in this study. the addition of srf fertilizer increased salinity depending on the added fertilizer concentration. srf application at 150 mg n/mlincreased salinity up to 65 ppt (fig. 1). the increase of salinity might be caused by gramafix which contained high concentration of m a c r o n u t r i e n t s ( n p k ) , s e c o n d a r y macronutrients (mg, s, ca) and micronutrients (zn, fe, cl, mn, b, bo, mo). the ratio of mg, ca, s and micronutrient was 2:4:3:1. high content of minerals in this srf fertilizer seemed to play an important role in increasing the catalytic activity of enzymes produced by oil degrading bacteria. 2+ 2+ cookson jr (1995) mentioned that mg and ca table 4 comparison of oil depletion percentage (at 28 days) and bacterial abundance (at 14 days) in oil contaminated sediment exposed by different treatment of n concentration and bacterial amendment for 28 days experiment treatment parameter measured fertilizer culture bacterial cell number added oil depletion (%) bacterial density (cells/ ml) control 0 13.1 + 0.3 a 2.e+08 n = 7.5 mg/g single 0.5 x 108 74.6 + 3.6 c 1.e+09 1.0 x 108 30.2 + 4.2 f 8.e+08 mixed 0.5 x 108 64.9 + 1.0 d 1.e+09 1.0 x 108 46.7 + 1.5 e 1.e+09 n =15.0 mg/g single 0.5 x 108 24.8 + 2.2 c 6.e+08 1.0 x 108 38.7 + 0.3 b 9.e+08 mixed 0.5 x 108 15.9 + 3.4 a 6.e+08 1.0 x 108 13.1 + 0.3 a 6.e+08 note: columns marked by the same letter (a to g) are not statistically significant different (p < 0.05). no replications for bacterial cells number 188 biotropia vol. 24 no. 3, 2017 are metallic ions that can function as a co-factor in the catalytic enzyme activity of microbes. in this study, the range of salinity during the experiment was 32 – 65 ppt. this high level of salinity might still be tolerable by marine oildegrading bacteria, which was shown by the bacterial growth observed at day-14 (table 3 & 4). other study focused on biodegradation of crude oil by a mixed bacterial community isolated from marine sediment with varying concentrations of sodium chloride (mille 199 ). initially, the et al. 1 amount of degraded oil increased with increasing salt concentration, to a maximum level of 0.4 mol/lnacl. thereafter, the amount of degraded oil decreased with increasing salt concentration, probably because the salt-tolerance limit of the figure 1 environmental parameters condition at day-0 and day-28 of experiments in three different bioremediation experiments 189 optimal bacterial density and fertilizer dosage for bioremediation darmayati et al. bacteria was reached. in this experiment, the salinity of seawater amended by fertilizer at nitrogen concentration of 7.5 mg/g sediment and 15.0 mg/g sediment increased up to 45 – 50 ppt and 52 – 65 ppt, respectively. the salinity value in biostimulation and combination of biostimulation-bioaugmentation treatments was higher than control (fig. 1). these experiments were conducted in the range of dissolved oxygen (do) value between 0.64 – 6.05 mg/l, ph value of 5.52 – 8.19 and salinity value of 32 – 65 ppt (fig. 1). during the experiments, do value was always getting lower along with incubation time and increased numbers of bacteria. dissolved oxygen was sufficiently available to be consumed by bacteria to metabolize oil. no aeration was provided during the experiments. the oxidation of the substrate by oxygenases during the process of aerobic respiration, for which molecular oxygen is required, occurs in the initial steps of catabolism of aliphatic, cyclic and aromatic hydrocarbons by bacteria and fungi (leahy & colwell 1990). ph value during the experiment was in the range of 5.56 – 7.99 which was optimal range for biodegradation process. bioremediation treatment used in this study provided good results for reducing oil in the marine environment. nitrogen availability in control was in natural condition, which was in the range of 7 12 mg/l. addition of fertilizer increased nitrogen availability in microcosm. however, available nitrogen observed in moderate concentration of nitrogen (7.5 mg/g) were higher than that in high concentration of nitrogen (15.0 mg/g) (fig. 1). this might be caused by excessive nitrogen which suppressed the growth of bacteria in the environment with having nitrogen concentration of 3,943 – 5,900 ppm, so that the numbers of bacterial cells were lower and oil degradation rate was slower than the combination treatment (at nitrogen concentration of 1,940 – 2,090 ppm). conclusions oil depletion rate was influenced by the numbers of bacterial cells and nutrients concentration. efficiency of oil degradation in biostimulation treatment was better than that in bioaugmentation treatment. however, the highest oil degradation rate was observed at a combination of bioaugmentation-biostimulation treatment. this treatment was able to increase oil degradation rate up to 7.5 times higher than that in control. in addition, the salinity range of 32 – 65 ppt caused by fertilizer addition might still be tolerable for marine oil-degrading bacteria. bioremediation of oil contaminated sandy beach in cilacap would be optimal by employing a combination of slow release fertilizer (g) at c/n ratio of 1,000 : 75 and rco/b/08_008 culture having bacterial density of 0.5 x 10 8 cells/ml (100% homology with sp. alcanivorax te-9). acknowledgements this research was supported by the research centre 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oilcontaminated soil: comparison of different biostimulation and bioaugmentation treatments. j hazard mater 183: 395-401. xu , obbard jp, tay etc. 2003 optimization of slowr . release fertilizer dosage for bioremediation of oilcontaminated beach sediment in a tropical environment. water sci technol 47(1):303-8. xu ym. 2010 bioremediation of crude oil-contaminated soil: comparison of different biostimulation and bioaugmentation treatments. j hazard mater 183:395-401. zhu x,venosa a, suidan mt lee k. 2001 guidelines for , the bioremediation of marine shorelines. cincinnati (us): us environmental protection agency. 191 optimal bacterial density and fertilizer dosage for bioremediation darmayati et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 biomass and carbon stock estimation inventory of indonesian bananas ( spp.) and musa its potential for nrole land rehabilitatio setyawan agung danarto* lia hapsariand purwodadi botanic garden – indonesian institute of sciences, jalan surabaya-malang km. 65 purwodadi, pasuruan, east java 6716, indonesia received 24 february 2014/accepted 30 november 2015 abstract bananas ( spp.) are widely cultivated in indonesia. they are extensively grown in backyards, home gardens, musa intercropped with short term crops and also in agroforestry sy . stem the potential of bananas to sequester carbon has been reported but there is limited knowledge on the performance of various cultivars. an inventory of biomass and c-stock estimation on banana accessions has been conducted in germplasm plots purwodadi botanic garden, musa pasuruan. estimation on biomass and c-stock have been conducted for 42 individual banana accessions, comprised 5 wild banana species and 37 cultivars using non-destructive method i.e. allometric equation for banana. the objectives ere conduct inventory on the biomass and c-stock estimation of indonesian bananas in of this study w to germplasm collection of purwodadi botanic garden, to make the projections of time average above ground for cstock of banana farming system in indonesia and to discuss the potential role of bananas in land rehabilitation. the results showed that estimation value of biomass and c-stock varied from one to another. wild banana accession musa balbisiana musa acuminataspecies had higher biomass and c-stock value than wild species. banana cultivars containing one or two “b” genome (abb and aab) were more vigorous and contributed higher biomass and c-stock than aaa and aa cultivars. among cultivars, the highest c-stock was contributed by pisang kepok bung (average of 6.92 kg c/plant) whereas the lowest c-stock was contributed by pisang rayap (average of 0.67 kg c/plant). in average, various indonesian bananas studied contributed around 2.26 kg c/ or 0.98 tonnes c/ha. plant the growing area of bananas increased from 73,539 ha in 2000 to 101,822 ha in 2010, which was decreased to 100,600 ha in 2014, contributing c-stock around 72.28 tonnes c in 2000 increasing to 100.07 tonnes c in 2010 with a decrease to 98.97 tonnes c in 2014. these numbers are still limited only to the recorded areas. anana plants in combination with b woody tree crops, are potential as important component of agroforestry r and mixed farming systems , home ga dens to rehabilitate and reforest landscape, to decrease carbon emission in atmosphere in the form of biomass and cstocks and to meet the economic needs for local surrounding community. keywords: banan stock estimation, purwodadi a, biomass, carbon (c-stock), , , musa acuminata musa balbisiana botanic garden r, ehabilitation introduction climate change is environmental issue an that has been discussed limate change is always . c caused by energy absorbed from sun as short wave then reflected in atmosphere as infrared long wave radiation. greenhouse gas absorb es effect s infrared radiation which is retained in atmosphere as heat energy causing the in of the earth's crease temperature, , efforts mitigate therefore to greenhouse gas needed saharjo & es are ( wardhana 2011 . world bank stated that ) in 2003, the co about 2earth's concentration was 27 billion metric ton, which a 9% increase from was 1 the year ipcc noted that 1990. (2014) the earth's temperature 1880 to 2012 showed from an increas from 0 65 c to 1 06 c. tudy by o oe . . a s ollivier (2014) reported countries et al. that contributing large amount of co in 2a atmosphere re china (29%), (15%), we usa european union (11%) india 4 4% , brazil , ( . ) ( . ) ( . the forests 6 2% and indonesia 2 3%). peat and fire in indonesia responsible for s were estimated the released of 2 co emission to the atmosphere a b o u t c t o c0 . 8 1 g t 2 . 5 7 g t in 1997 (page 2002). particular in borneo et al.* corresponding author : setyawan.10535@gmail.com biotropia vol. 22 no. 2, 2015: 102 108 doi: 10.11598/btb.2015.22.2.376 102 mailto:setyawan.10535@gmail.com island, the annual average carbon emission from forest fires was estimated about 0.02 to 0.06 gt c per year (kuntoro . 2015)et al . process amount 2 of decreasing co in the atmosphere through the process of plant photosynthesis is called c sequestration. in arbon the photosynthetic process, co in the 2 atmosphere a by plant, transformed to is bsorbed carbohydrate compound dispersed to alls and parts of plant. carbon sequestration describes long-term storage of co or other forms of 2 carbon to either mitigate or defer global warming and avoid dangerous climate change. it has been proposed as a way to slow the atmospheric greenhouse gases, which are released by burning fossil fuels (hairiah & rahayu 2007). therefore, it is important to conduct the study to examine ability of plant to a carbon, different species bsorb especially in the climate change mitigation efforts. agroforestry system is cultivation a practice combining trees and annual crop or other farm activities adopted by smallholders to meet their needs for food, medicine, timber, fuel, fodder and market commodities provides valuable . it also environmental services such as soil fertility replenishment, water catchment protection, carbon sequestration, conservation biodiversity and landscape restoration (garrity 2004) bananas . ( spp.) are extensively grown in backyards, musa home gardens, agroforestry system and are intercropped with short term crops. anana b s are the favorite s grown most plant in agroforestry system intercropped with other imp rtant tree o crops commodities (coffee, cacao, rubber), fruit trees timber bout 9 5% and . a . of bananas species occur in home gardens system contributing the to ecosystem services individual . only a handful of smallholder agroforestry systems store small amount of carbon per area basi , while the c systems store as much arbon as secondary c several forests (roshetko 2002). et al. the potential of bananas to sequester carbon has been reported by daphine (2014) on east african highland bananas, but there is limited knowledge on the performance of various cultivars in indonesia. being part of the primary center of origin and diversity so that has large number of bananas (musaceae) both wild seeded species and edible seedless or cultivar with many local names and synonimies (espino 1992; et al. valmayor . 2000)et al . there is no less than 200 local cultivars cultivated and developed across indonesia archipelago (nasution & yamada 2001). objectives erethe of this study w to conduct inventory on the biomass and c-stock estimation of indonesian bananas in germplasm collection of purwodadi botanic garden, to make the projections of time average above ground for c-stock of banana farming system in indonesia and to discuss the potential role of bananas in land rehabilitation. materials and methods study site the was conducted at banana collection study plots of purwodadi botanic garden – indonesian institute of sciences from . april to may 2012 purwodadi botanic garden collect has a ion of musa germplasm both wild and cultivated species varieties o ethr ugh exploration, plant exchang , grants community or personal contribution and from several regions all over indonesia, mostly from eastern indonesia. current collections in 201 accessions comprise 7 wild 4 is about 134 d species 127 cultivated varieties.and materials the materials were 4 selected banana studied 2 accessions of purwodadi botanic garden co lections comprise 5 wild species and 3 l d 7 cultivars. the diameter at breast height (dbh) of the i o wasndividual pseud stem measured at mature age (already flowering) using tape meter with of replicationsminimum two per accession. as reported by daphine (2014) that c-stock of banana plants was significantly influenced by growth stages in which maturity stage was the optimal stage to be measured (daphine 2014). biomass and c-stock estimation biomass was estimated using non-destructive method i e. allometric equation for banana . ( ) and then -stock was kurniawan . 2010 c et al estima e by its mass tot d crossing bio average value of in plants which is 0.46 (hairiah c-stock et al. 2010). the development of allometric equations has been investigated specific , based on condition species and or communit (ketterings plant ies et al. 2001; wibowo 2010):et al. biotropia vol. 22 no. 2, 2015 103 y = 0.0303 x d2.1345 z= y x 0.46 w :here y = plant biomass ( g)k d = diameter at breast high (cm) z = c-stock (kg c/plant). results and discussion biomass in was affected by interaction plants of genetic and environmental factor. in this study, the purwodadi botanic garden provides homogenous environmental condition, i.e. soil type p and . , water su plies culture practices therefore, the biomass results were mostly affected by its genetic factor. results showed the that the estimation value of biomass and c-stock tended to vary from one to banana accession another. pseudostem dbh was confirmed as the best predictor for biomass estimation in banana plants and it is recommended to be used in most carbon related studies. the more vigorous banana plants estimation value of contribute higher biomass and c-stock . (fig. 1) c stock related to photosynthesislevel is plant process its. based on photosynthesis pathway, banana c3 plant. in c3 plants are classified as plants 2, co and water from the environment are enzymatically combined with a five-carbon acceptor molecule to contribute two molecules of a three-carbon intermediate. c3 plants respond favorably to higher concentrations of carbon dioxide than c4 and cam. c4 plants include corn, sugar cane and many other tropical grasses, whereas cam plants include orchids, bromeliad and succulent plants (taiz & zeiger 2002). biomass and c-stock inventory results in wild banana species wild bananas are pioneer plants and can grow in various conditions. it commonly grows wild in the forests, road sides and river banks musa . wild balbisiana species is also being cultivated by farmers to get the leaves for various wrapping purposes he immature fruits also edible for . t are any traditional side dishes ild is . w musa acuminata rarely cultivated. there are also some other species of bananas (not studied here) such as musa velutina musa ornata musa borneensis , , etc. that are commonly cultivated for ornamentals due to its beautiful performances nasution yamada ( & 2001; hapsari . 2015a).et al among the wild studied, species musa balbisiana much species contributes higher level of biomass and c-stock estimation than musa acuminata musa balbisianaspecies (table 1). is considered to be more vigorous robust and , as well as , while musa drought and disease resistant acuminata species is slender but has attractive more morphology .et al(daniells 2001; nasution & yamada 2001). species (pisang musa balbisiana figure relation of pseudostem dbh to biomass and c-stock 1 positive cor values in banana plants 104 biomass nd carbon stock estimation inventory f ndonesian bananas ( spp.)a o i danarto and hapsarimusa – klutuk wulung) has pseudostem diameterlarge ( ) contribut21.47 cm ing around 21.09 kg/plant biomass and around 9.7 kg/plant c-stock. m . acuminata utilifes. r var with slender pseudostem (4 99 cm) contributed 0 94 . around . kg/plant biomass around . kg c/plant c-stock and 0 43 (table 1). biomass and c-stock inventory results in various banana cultivars edible banana cultivars biomass have lower and c-stock s than bananas. the genetic value wild composition of is the combination musa balbisiana of wild (donor a genome) and musa acuminata musa balbisiana (donor b genome). genomic composition can be identified using morphology (jumari & pudjoarinto 2000) and genetic (hapsari et al. 2015b). banana cultivars containing one or two “b” genome (abb and aab cultivars) are more vigorous and contribute higher biomass and c-stock than the aaa and aa cultivars. pisang the kepok bung (abb) is most vigorous cultivar with pseudostem diameter of 18.32 cm ing around 15.04 kg/plant contribut biomass and around 6.92 kg c/plant c stock. pisang slender cultivarrayap (aa) is the most with pseudostem diameter of 6.14 cm contribut ing around 1.46 kg/plant biomass and around 0.67 kg c/plant c stock. pisang triolin having aab genome biomass and contributed c-stock values in between pisang kepok bung (abb) and pisang rayap (aa) (f . . the ig 2) average value of c-stock contributed by banana plants per genome group from the highest to the lowest as follows: bb wild (7.22 kg values are c/plant), abb cultivars (2.74 kg c/plant), aab cultivars (2.11 kg c/plant), aaa cultivars (1.73 kg c/plant), aa cultivars (1.55 kg c/plant) and aa wild (0.72 kg c/plant). in traditional home gardens and in agroforestry, farmers plant various local cultivars. however, the commercial scale farmers plant bananas cultivars based on consumers' preference and agroclimate condition in an area. pisang kepok (abb) is the most favorite cultivar to be cooked, while pisang raja (aab), pisang ambon (aaa) and pisang mas (aa) are often processed for dessert. wild species and cultivars studied contributed an average of 2.26 kg c/plant cstock. t a in banana fime verage c-stock arming s ystem in indonesia banana s farming system recognize 3 different planting distance based on its canopy size s s, i.e. 6 x 6 m for wide canopy, 5 x 5 m for medium canopy and 4 x 4 m for small canopy (cahyono 1996). banana plants may contribute an average of 0.98 c tonnes/ha c-stock. this number is quite high if compared to c-stock contributed by understory of agroforestry system which only contributed 0.2-0.3 c tonnes/ha. however, agroforestry of coffea plants contributed higher c-stock than banana plants, i.e. 2.0-12.0 c tonnes/ha (forda 2010). the growing area of bananas increased from 73,539 ha in 2000 to 101,822 ha in 2010, which was decreased to 100,600 ha in 2014 (ministry of agriculture 2015), contributing c-stock around 72.28 tonnes c in 2000 increasing to 100.07 tonnes c in 2010 with a decrease to 98.97 tonnes c in 2014. these numbers are still limited only to the recorded areas. potential role of banana plants for land rehabilitation tropical forests in southeast asia are constantly changing as a result of and logging land such asconversion logging activities, complete deforestation conversion from forest , to grassland or annual crops, tree plantations and other woody perennial crops (lasco 2002; monde 2009). those vast area of degraded land are in need of rehabilitation. agroforestry system may become approach prevent deforestation by an to biotropia vol. 22 no. 2, 2015 table estimation 1 pseudostem dbh, biomass and carbon stock of wild banana species species local name pseudostem dbh (cm) biomass (kg/plant) c-stock (kg c/plant) musa balbisiana klutuk wulung 21.47 21.09 9.70 musa balbisiana klutuk ijo 15.35 10.30 4.74 musa acuminata var. alasensis pisang hutan 6.78 1.80 0.83 musa acuminata var. rutilifes pisang cici hutan 4.99 0.94 0.43 musa acuminata var. tomentosa unti darek 7.07 1.97 0.91 105 figure pseudostem dbh, biomass and c-stock of various indonesian banana cultivars 2 estimation 106 providing on-farm source trees s. agroforestry system provides better carbon storage than the usual annual crops farming system because agroforestry system intercropped trees with annual crops, continuously giving much higher biomass and litters in varied quality (utami 2003).et al. home garden as smaller level of agroforestry is species rich and tree-based system producing wood and non-wood products and therefore, producing high biomass. due to high biomass produced, this system potentially offers carbon storage. in terms of aboveground biomass, home garden contains more carbon per hectare than grasslands, cassava fields and imperata young rubber agroforestry (roshetko . et al 2002). biomass nd carbon stock estimation inventory f ndonesian bananas ( spp.)a o i danarto and hapsarimusa – biotropia vol. 22 no. 2, 2015 107 table 2 ime average c-stock in indonesia banana farming systems t in 2000-2014 planting distance number of plants/ha c-stock (c kg/ha) time average c-stock contribution year 2000 (c tonnes) year 2010 (c tonnes) year 2014 (c tonnes) wide canopy 6 x 6 m 278 628.67 46.23 64.01 63.24 medium canopy 5 x 5 m 400 905.29 66.57 92.18 91.07 narrow canopy 4 x 4 m 625 1,414.52 104.02 144.03 142.30 average 434 982.83 72.28 100.07 98.87 banana as a component of mixed agroforestry system has moderate c-stock contribution. this study showed that one hectare of banana plants store more carbon (0.98 tonnes) than cassava (0.5 tonnes) and grassland (0.7 tonnes) imperata (hairiah 1997). banana plants store much less carbon than perennial woody plants or trees, however, banana has high economic value and provide shades to tree crops commodities (coffee, cacao, rubber), fruit and timber (roshetko . 2002). also, et al banana plants produce fruit all year round which continuously contribute food to smallholder farmers in the area (hapsari 2011). the role of agroforestry in absorbing co as 2 well as in storing and maintaining carbon stocks is lower than that of natural forests, but this system can increase carbon stocks on degraded lands (widianto . 2003)et al . conclusions banana accessions contributed varied estimated per value of biomass and c-stock accessions depend on their characteristic ing s performance. pseudostem dbh was confirmed as the best predictor for biomass estimation in banana plants and it is recommended to be used in most carbon related studies. the more vigorous banana plants estimation value contribute higher of biomass and c-stock the c-stock value . ranged from / / . 0.67 kg c plant to 6.92 kg c plant in average, various indonesian bananas studied contributed around 2.26 kg c/ or 0.98 plant tonnes c/ha. the growing area of bananas increased from 73,539 ha in 2000 to 101,822 ha in 2010, which was decreased to 100,600 ha in 2014, contributing c-stock around 72.28 tonnes c in 2000 increasing to 100.07 tonnes c in 2010 and decreasing to 98.97 tonnes c in 2014. agroforestry is species rich and tree-based system producing wood and non-wood products and therefore, producing high biomass. due to high biomass produced, this system potentially offers carbon storage. anana plants in combination b with woody tree crops, are potential as important component of agroforestry r and , home ga dens mixed farming systems to rehabilitate and reforest landscape, to decrease carbon emission in atmosphere in the form of biomass and c-stocks and to meet the economic needs for local surrounding community. acknowledgements the authors would like to acknowledge ahmad masrum and lamiran for their technical guidance during the study observation in banana collection purwodadi botanic garden. field of references cahyono b. 1996. . pisang (budidaya dan nalisis saha ani)a u t yogyakarta (id): kanisius. p 80. daniells j, jenny c, karamura d tomekpe k. 2001. , musalogue: a atalogue of musa ermplasm. diversity in c g the enus musag . montpellier (fr): international network for the improvement of banana and plantain (inibap). the international plant genetic resources institute (ipgri). daphine k. 2014. arbon equestration otential f ast c s p o e a h b c i ufrican ighland anana ultivars n ganda. dissertation. 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(icraf) sea regional office. 39 .p http://aplikasi.pertanian.go.id/bdsp/hasil_ko 499 darius (identification of secretory).cdr identification of secretory structure, histochemistry and p c hytochemical ompounds of edicinal lant qm p hyptis capitata jac . darius rupa , yohana sulistyaningsih , dorly diah ratnadewi 1 2* 2 2 c. and 1 universitas department of biology education, faculty of teacher training and education, borneo tarakan, tarakan 77123, indonesia 2 t of y c of h cs and natural sciences, departmen biolog , fa ulty mat emati institut pertanian bogor, bogor 16680 a , indonesi received 29 june 2015/accepted 13 june 2016 abstract hyptis capitata jacq. (common name: knobweed or false ironwort) belongs to lamiaceae family and is among known traditional medicine. the anak dalam tribe of jambi province uses the leaves of h. capitata to cure external and internal wounds. this study was aimed at identifying and analyzing secretory structure, histochemistry and phytochemical content of h. capitata leaves. the results showed that h. capitata leaves have secretory structures in the form of peltate, capitate and uniseriate glandular trichomes on the upper and lower leaf surfaces, with idioblast cells scattered throughout the leaf mesophyll. histochemical tests indicated that the peltate trichomes have four head cells, containing alkaloids and terpenoids. this study classified capitate trichomes into two types: 1. type i has one stalk cell and two head cells, all containing alkaloids, terpenoids and lipophilic compounds; 2. type ii has long stalk cells consisting of seven to ten cells with one head cell, all containing alkaloids and terpenoids. uniseriate glandular trichomes of h. capitata leaves consist of four to eight cells containing alkaloids and terpenoids. the idioblast cells are round-shaped and contain lipophilic compounds. gc-ms analysis showed that h. capitata leaves contain terpenoid compounds assumed to serve as anti-infective agents, including l-limonene, eugenol, farnesol isomers a, d-nerolidol, hexahydrofarnesol and neophytadiene. keywords: glandular trichome, hyptis capitata, idioblast, medicinal plants, phyto-compounds introduction rapid deforestation in jambi area will cause a great loss of medicinal plants. it is, therefore, important to preserve the tribal knowledge on herbal medicine. is one of various hyptis capitata plant species used as herbal medicine by anak dalam tribe, which is native to jambi province in sumatera, indonesia. hyptis capitata an erect herbaceous plant, is native to central and tropical south america. the plant is usually abundantly found in open fields and roadsides. in indonesia, particularly in jambi province h. capitata, the leaves of (fig. 1) are used by the tribe as herbal medicine to anak dalam cure external and internal wounds. the tribe crushes or squeezes the leaves prior to using them to cure wound. the lea can be f sap consumed to treat inner injuries (t menggungu tarip, anak dalam tribe, pers. comm. 2012). this plant is also used as traditional medicine in india, jamaica and bangladesh (michell & ahmad 2006; biswas et al. 2010 phytochemical study ). a conducted by okach et al. (2013) reported that several other species of the lamiaceae family, such as (e.mey. ex benth) becium obovatum . n.e.br., leucas calostachys oliv, ocimum kilimandscharicum baker ex. gurke, plectranthus barbatus satureja and biflora (ham ex. andrews d. don) brig., contain sterols, terpenoids, alkaloids, saponins, glycosides, flavonoids and tannins. dickison (2000) reported that most medicinal plants have secretory structures that play a part in metabolite production. various chemical * corresponding author: yo_sulistya@yahoo.com 94 biotropia 4 2 7 94 103 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 2 499 compounds, such as essential oils, resins, latex, mineral salts, alkaloids and glycosides, are produced by secretory structures. werker (1993) stated that phytochemical substance of terpenoids, alkaloids and various volatile compounds are generally produced by the glandular trichomes contained in the species of lamiaceae. scientific study to identify the secretory structures which might be the site of accumulation and/or biosynthesis of metabolite and phytochemical compounds in will h. capitata improve the production of its desirable phytochemical compounds through tissue or cell culture. this study was aimed at identifying the secretory structure analyz , ing the accumulated metabolite compounds in the secretory structure and analyzing the h. phytochemical content in capitata . this research also examined the leaves efficacy of h. capitata plant, so that the natural substance contained in the plant can be scientifically justified for its pharmaceutical properties. materials and methods plant material collection the study was carried out in tropical lowland rain forest of bukit dua belas national park in jambi province, sumatera, indonesia (01°96'109" s, 102°58'630" e, 87 m asl). leaf samples were collected from areas of jungle-rubber agroforest, which is an agroforestry system that combines rubber trees with natural wild plant species grown surrounding the rubber trees. leaves were harvested in the dry season of july 2013 at average temperature of 29 °c and average relative humidity of 84%. microscopic observation and histochemical analysis were performed on leaves from the third node of the plant, with three replications. a composite of leaves taken from plant several samples was subjected to phytochemical analysis. for microscop observation leaves ic purpose, were 70% or preserved in ethanol. f observation using scanning electron microscope , (sem) the leaves were in faa solution preserved (formaldehyde, glacial acetic acid and 70% ethanol ; 1 : 1 : 18). fresh leaves were collected for the histochemi or gc-ms analysis cal analysis. f the samples were dried for three days and sunthen dried at 60 for three days oven°c . observation using light microscope paradermal leaf sections were prepared as semi-permanent slides, following a procedure suggested by sass (1951). the preserved leaves were washed with distilled water for five minutes, soaked in 50% hno solution for five minutes, 3 rinsed with distilled water for one minute, and then the adaxial and abaxial surfaces of the leaves were peeled. the samples were observed using a light microscope (olympus cx21) at 100x and 400x magnifications. identification of medicinal properties of hyptis capitata jacq. – rupa et al. 95 figure 1 h. capitata grows wildly in the jungle-rubber agroforest at bukit duabelas national park, jambi province 70% ethanol and observed using light microscope at 400x magnification. the presence of lipophilic compound was by the production of s indicated red, yellow, or orange colors in the leaf tissue, following et al ( boix . 2011). phytochemical ianalys s phytochemical components the presence of in the leaf tissue was identified using gc-ms technique. two milligrams of powdered sample was placed in the chamber of the gc-ms instrument shimadzu qp2010 . to analyze ( ) the chemical composition, the sample went through the following processes: pyrolysis temperature 00 , oven temperature , o o 4 c 50 c injector temperature 280 , inter-surface o c temperature 280 o c and ion source temperature 200 . the carrier gas was helium. the o c spectrogram mass resulting from those processes was calculated then automatically by the gc-ms instrument. the calculation was based on the similarity of mass number/charge number pattern referring to the gc-ms instrument's database nist wiley databases. / results and discussion in the leaves of h. capitata, there were two types of secretory structure observed i.e. glandular trichomes and idioblast cells. glandular trichomes were found on the upper (adaxial) surface and lower (abaxial) surface of the leaves. idioblast cells were found throughout the leaf mesophyll, from the palisade to the spongy parenchyma. there were one peltate, two types of capitate and one uniseriate glandular trichome identified in this species (fig. 2). members of the lamiaceae family, such as ocimum canum, mentha spicata as well as scutellaria linearis benth., s. baiacalensis, s. gallericulata l. and s agrestis (shang et al. 2010; venkateshapp & . sreenath 2013; oliveira et al. 2013), generally have glandular trichomes. several species have both peltate and capitate trichomes, while others have either only peltate or only capitate trichomes (huang . 2008). species of lamiaceae family et al having various types of glandular trichomes include salvia chrysophylla, egyptian ballota, isodon rubescens, pogostemon cablin benth., scutellaria agrestis and rosmarinus officinalis (kahraman et al. 2009; observation using scanning electron microscope (sem) the leaf samples were washed in caccodylate buffer for two hours using an ultrasonic cleaner (sibata su-6the, japan), pre-fixed in a 2.5% glutaraldehyde solution for two days at 4 °c, and then fixed in 2% tannic acid solution for 6 hours. the samples were then washed twice in caccodylate buffer for 10 minutes each time, rinsed with distilled water for 15 minutes, and dehydrated in a graded ethanol series of 70%, 80% and 90% for 10 minutes each, followed by twice dehydration in 100% ethanol for 10 minutes each time. the samples were then soaked twice in tert-butanol for 10 minutes each time, dried in vacuum drier for 20 minutes, coated with gold, and finally observed using a scanning electron microscope (jeol jsm 5310 lv hitachi) at 20 kv ( 200x, 750x and 1,000x magnifications). histochemical analysis for histochemical analysis, fresh leaves were transversely sectioned at 15 20 µm, using a dual purpose microtome (yamato rv-240). a small piece of fresh leaf lamina was inserted in the specimen holder. the specimen holder was then filled with water and frozen using the freezing system. the specimen embedded in the frozen water was then sectioned directly using a microtome blade. leaf sections were then treated with specific reagents to identify the presence and specific locality of terpenoids, alkaloids and lipophilic compound . terpenoid in the s presence leaf tissues was identified by soaking leaf section a in 5% cupric acetate solution, as suggested in harbone 1993 . a positive test for terpenoids ( ) was indicated by the appearance of yellow or brownish yellow color in the tissue. alkaloid presence was lamina tested by soaking leaf sections in wagner reagent. a positive result was indicated by the presence of reddish brown or yellow deposits. for negative control, the leaf sections were pre treated with 5% tartaric acid in 95% ethanol for 48 hours at room temperature, as suggested in ( furr & mahiberg 1981). the presence of slipophilic compound was tested using sliced samples washed in 70% ethanol for one , which were . minute then soaked in 0 03% sudan iv for 30 minutes at 40 c in a water bath. o the sliced sample w then washed quickly in s ere 96 biotropia vol. 24 no. 2, 2017 salmaki et al. 2009; liu & liu 2012; rusydi et al. 2013; oliveira et al. 2013; boix et al. 2011). species belong to genus having peltate and capitate hyptis glandular trichomes include andh. caespitosa h. suaveolens (rudall 2007; jelani & prabhakar 1991). has peltate glandular trichomes h. capitata in a submerged (sunken) position in the epidermal tissue. this type of glandular trichomes was also found in several species, such as oci um basilicum, oci um irvinei h. caespitosam m and (ogunkule & oladele 2000; rudall 2007). glandular trichomes peltate glandular trichomes, found on the leaves of h. capitata, consisted of four head cells and one basal cell. the head cells of peltate glandular trichomes in lamiaceae family vary widely in terms of cell numbers. some species have three to six head cells, for example those of isodon rubescens (liu & liu 2012). several species of genus ziziphora have 12 to 18 head cells, namely ziziphora clinopodioides, z. tenuior, z. taurica and z. taurica subsp. cleonioides (kaya et al. 2013). there are 4 head cells in the peltate glandular trichomes of both h. capitata and h. caespitosa (rudall 2007). capitate glandular trichomes in lamiaceae family usually consisted of one to two head cells with short or long stalks consisted of one to three cells. these are found in many species, such as lavandula pinnata, thymus lykae, isodon rubescens and rosmarinus officinalis (huang et al. 2008; marin et al. 2008; liu & liu 2012; boix et al. 2011). all three types of glandular trichome were found scattered on both leaf surfaces of h. capitata. type i of capitate glandular trichomes in h. capitata had a short stalk with one cell and one head consisted of two cells. type ii of capitate glandular trichomes had a long stalk consisted of seven to ten cells and one head cell. uniseriate glandular trichomes consisted of four to eight cells and had a tapered tip (fig. 2). peltate glandular trichomes were generally shorter than other trichomes. however, t heir head was much larger than that of capitate size glandular trichomes. the size of each trichome in the adaxial and abaxial surfaces was similar (table 1). in fact, most trichome dimensions in adaxial and abaxial surfaces showed no significant differences, except for the width of type i capitate glandular trichome which was greater in the abaxial surface than that in adaxial surface. 97 figure 2 glandular trichome types of h. capitata; images obtained using sem (a, b, c) p = peltate, c1 = capitate glandular trichome type i, c2 = capitate glandular trichome type ii and u = uniseriate (note: bar = 50 µm) table 1 size and density of glandular trichomes of h. capitata leaf trichome types length of trichome stalk (µm) trichome head (µm) density (mm-2) length width adaxial abaxial adaxial abaxial adaxial abaxial adaxial abaxial peltate 27.7±0.3 28±0.5 ns 43.6±0.3 44.4±1.2 ns 9.6±0.7 32.3±1.9 s capitate type i 7.5±0.5 7.8±0.9 s 12.8±0.5 13.3±0.9 ns 25.1±0.5 27.5±0.9 s 12.6±3.3 18.9±1.3 s capitate type ii 351±14.7 354.7±8.1 ns 27.5± 0.7 28.3±0.3 ns 19.1±0.5 19.4±0.9 ns 3.9±0.3 4.5±0.2 s uniseriate 343.7±32.6 342.7±44.2 ns 3.9±0.7 4.4±0.3 ns idioblast diameter (µm) diameter (µm) palisade 5.9±0.4 sponge 4.2±0.3 s palisade 261.2±.15.4 sponge 271.7 ±33.2 ns note: s = significant; ns = non-significant (based on t-test at α = 5%) identification of medicinal properties of hyptis capitata jacq. – rupa et al. peltate and capitate glandular trichomes were randomly scattered in the adaxial and abaxial surfaces of leaves. the density of peltate glandular trichomes was much higher in the abaxial than that in the adaxial . surface the distribution densities of both types of capitate glandular trichomes were similarly higher in abaxial than in adaxial he iseriate surfaces. t un trichomes were distributed equally in the adaxial and abaxial (table 1)surfaces . s species tudies on various showed that glandular trichomes are usually found to be more densely distributed surface of the leaf in abaxial . only in certain species that glandular trichomes are such as h. more abundant in the adaxial side, in suaveolens o. basilicumand (ogunkule & oladele 2000). the peltate and capitate glandular trichomes found in the leaves of are h. capitata more abundant surface in abaxial than in adaxial . this distribution pattern is observed in several also species belong to lamiaceae family i.e. mentha piperita, salvia chrysophylla rosmarinus officinalisand (turner . ; kahraman . 20 ; boix et al 1999 et al 10 et al 1 . 201 ). the same pattern is also observed in several other families, such as dipterocarpaceae (parashorea (gossypium hirsutum spp); malvaceae l.) and fabaceae l.) (dahlin . (phaseolus vulgaris et al 1992; noraini & cutler 2009; bondada & oosterhuis 2000). idioblast cells h. capitataidioblast cells in the leaves of were round-shape and the mesophyll of the d found in leaf, from the palisade to the spongy .parenchyma idioblast cells in the leaves of different plant species are not always found throughout the mesophyll. in some cases idioblast cells are found only in specific tissues. i litsea euosma, n litsea praecox actinodaphne trichocarpa the and idioblast cells were only present in the palisade parenchyma the leaves of , while in phoebe forrestii, they were found in the spongy parenchyma hai (gang & 1999). idioblast cells found in the palisade parenchyma of were larger than similar h. capitata cells located in the spongy parenchyma, while the cell densities showed no significant difference (table 1). species a variety of different exhibit sizes and densities of idioblast cell. for example, in , several species of machilus and persea genera various sizes and densities of idioblast cells have been previously reported i.e. the size of idioblast cells in palisade and spongy parenchyma of machilus leptophylla were 30 40 µm with a density of 3 mm n m. yunnanensis the idioblast -2; i cells were 25 50 µm with a density of 60 mm ; -2 in m. salicoides the idioblast cells were 30 40 µm with a density of 24 mm he largest idioblast -2; t cells were found in persea americana at 35 45 µm with a density of 89 mm (gang & hai 1999). -2 referring to these data the size of idioblast , cells in palisade and spongy parenchyma the of h. capitata were much smaller in size than those in machilus and persea genera, while the density was much higher than those of the two generas. histochemical analysis of glandular trichome and idioblast cells histochemical testing of type i capitate glandular trichomes gave a positive result for terpenoids, as confirmed by the yellow-colored response to a cupric acetate reagent. the presence of was indicated by alkaloids the formation of a brownish yellow deposit, when treated with wagner's reagent. type ii capitate glandular trichomes were shown to terpenoids, as contain indicated by the formation of a yellow color in the resultant mixture. this type ii also contained alkaloids brown color as indicated by response as well as s as indicated by lipophilic compound yellow-colored reaction. peltate glandular trichomes were tested positive for terpenoids and alkaloids indicated by yellow to brownish-yellow color. iseriate-type trichomes were tested un positive for terpenoids as indicated by brown color as well as for alkaloids indicated by yellow to brownish-green color (fig. ).3 various studies reported on the metabolites produced by the glandular trichomes in lamiaceae plants. nazzar et al. (2010) reported that peltate and capitate glandular trichomes in lamiaceae generally secrete essential oils in the form of terpenoid compounds. according to gang et al. (2002), terpenoids in ocimum basilicum (lamiaceae) were produced by the peltate glandular trichomes. glandular trichomes in mentha piperita and mentha spicata produced terpenoids in form of monoterpenes (fahn 1979). in salvia sclarea l., capitate glandular trichomes play a role in producing essential oil 98 biotropia vol. 24 no. 2, 2017 compounds like diterpene and monoterpenes in form of linalool and linalyl acetate compounds. peltate glandular trichomes of salvia sclarea l. produce sesquiterpene compounds (schmiderer et al. 2008). liu and liu (2012) reported that the glandular trichomes in the leaves of isodon rubescens (lamiaceae) contain a combination of phytochemical compounds, consisting of phenolic compounds, terpenoids, flavonoids, carbohydrates and alkaloids. 99 figure 3 glandular trichomes in h. capitata. histochemical tests using cupric acetate for terpenoid (a, e, i and m); sudan iv test for lipophilic substance (b, f, j and n); glandular trichome in water (control) (l); wagner test for alkaloid (c, g, k and o); negative control for alkaloid test using tartaric acid reagent (d, h and p) (note: bar = 50 µm) a b d c h e g f i j l k n p o m figure 4 histochemical results for idioblast cells in leaf transversal section; using sudan iv for lipophilic compounds (a) and water as a control (b) ep = epidermis, pl = palisade parenchyma and sp = spongy parenchyma (note: bar = 50 µm) a ep pl sp b identification of medicinal properties of hyptis capitata jacq. – rupa et al. no r time metabolite group ri relative area (%) constituent 1 2.950 alcohol 85249549 10.02 (o-d)ethenol 2 3.185 amide 113186810 13.30 acetamide, 2,2-dichloro (cas) dichloroacetamide 3 4.501 acetic acid 37932716 4.46 acetic acid (cas) ethylic acid 4 5.228 acetal 38862858 4.57 2-propanone, 1-hydroxy (cas) acetol 5 13.313 ketone 17521339 2.06 1,2-cyclopentanedione 6 15.039 phenol 13550624 1.59 phenol (cas) izal 7 15.633 terpenoid (monoterpene) 159030886 18.69 l-limonene 8 15.881 ketone 16614244 1.95 2 cyclopenten-1-one, 2-hydroxy-3-methyl (cas) corylon 9 17.108 phenol 37470245 4.40 ph enol, 4-methoxy (cas) hqmme 10 17.448 aldheida alkyl 17828416 2.10 pentanal (cas) n-pentanal 11 18.631 aromatic aldehyde 9097365 1.07 benzaldehyde, 2-methyl (cas) o-tolualdehyde 12 19.688 ester 13182646 1.55 2-propenoic acid, 2-methyl-, ethyl ester (cas) ethyl methacrylate 13 19.849 acetic acid 15135388 1.78 2,3-dihydro-benzofuran 14 21.122 aromatic heterocyclic 31783131 3.74 1h-indole (cas) indole 15 21.198 phenol 29786120 3.50 phenol, 4-ethenyl-2-methoxy 16 21.813 terpenoid 8964401 1.05 phenol, 2-methoxy-4-(2-propenyl) (cas) eugenol 17 22.552 alkaloid 29383206 3.45 1h-indole, 3-methyl (cas) 3-methylindole 18 22.705 terpenoid (sesquiterpene) 8814772 1.04 farnesol isomer a 19 22.915 terpenoid (sesquiterpene) 10811896 1.27 d-nerolidol 20 23.496 polycyclic aromatic 12729349 1.50 2h-1-benzopyran-2-one (cas) coumarin 21 26.417 terpenoid (sesquiterpene) 8734907 1.03 1-dodecanol, 3,7,11-trimethyl (cas) hexahydrofarnesol 22 27.717 terpenoid (diterpene) 24454424 2.87 neophytadiene 23 27.999 terpenoid 17043839 2.00 dodeca-1,6-dien-12-ol, 6,10-dimethyl 24 28.207 terpenoid (diterpene) 11252199 1.32 neophytadiene 25 28.671 fatty acid 13380605 1.57 tetradecanoic acid, 12-methyl-, methyl ester (cas) methyl 12methyltetrade 26 29.191 fatty acid 12112786 1.42 9-octadecenoic acid (z) (cas) oleic acid 27 29.537 vitamin 10213731 1.20 vitamin d2-7,8-diol 28 30.656 fatty acid 9972296 1.17 tricyclo[3.2.2.0e2,4]non-8-en-exo-6,exo-7-dicarboximid, 3,3-dic 29 31.789 fatty acid 19635391 2.31 hexadecanamide (cas) amide 16 30 43.507 fatty acid 17189048 2.02 2,6,10,14,18,22-tetracosahexaene, 2,6,10,15,19,23-hexamethyl (cas) squ total 850925187 100 histochemical tests conducted on the idioblast cells in the leaves of h. capitata did not detect terpenoids and alkaloids in these structures. however, these cells demonstrated the presence of indicated by lipophilic compounds, as yellow to orange colors (fig. ). found 4 such cell types were in (l.) (lamiaceae) (lersten & physostegia virginiana curtis 1998), but the idioblast cells containing such compounds are not specific to this family. similar cell types are present also in various families, such as lauraceae, asteraceae, caprifol iaceae, rosaceae, leguminosae ( ) and bixaceae (caesalpinia echinata cochlospermum rhegium et al et al) (lersten . 2006; teixeira . 2007; filho et al . . 2014) gas chromatograph mass spectrometer ( )gc ms gc-ms analysis revealed the presence of alkaloids, terpenoids, fatty acids and phenolic compounds (table 2). l-limonene, eugenol, f a r n e s o l i s o m e r s a , d n e r o l i d o l , hexahydrofarnesol, neophytadiene and dodeca1,6-dien-12-ol, 6,10-dimethyl were the predominant terpenoids found in the leaf tissue. l-limonene was terpenoid detected in the largest quantity (18.69%). the alkaloids were detected in only one substance i.e. 1h-indole, 3-methyl-(3methylindole). those compounds support the healing of wound or infection and also act as antibacterial agents. according to pei et al. (2009), 100 table 2 phytochemical compounds of h. capitata leaves using gc/ms biotropia vol. 24 no. 2, 2017 eugenol (a member of terpenoids) also acts as antibacterial agent. eugenol is able to inhibit the growth of gram-negative and gram-positive bacteria (catherine et al. 2012). antibacterial tests of eugenol proved that this substance can inhibit the growth of salmonella typhi a gram-negative , bacteria (devi et al. 2010). d'alessio et al. (2014) reported that d-limonene acts as an antiinflammatory that can signifcantly heal skin inf lammation caused by murine 12-otetradecanoylphorbol-13-acetate (tpa). this substance can also heal wounds by reducing neovascularization in mice. kusma et al. (2009) reported that essential oil of salvia sclarea leaves is used as analgesic, anti-inflammatory, antioxidant, antifungal and antibacterial agents. this oil is toxic to various pathogenic microbe including s, staphylococcus aureus, s. epidermidis, enterococcus faecalis, escherichia coli, pseudomonas aeruginosa and candida albicans fungus. curvelo et al. (2014) reported that nerolidol extracted from piper claussenianum (miq.) c. dc. is able to inhibit the development of c albicans, a fungus which causes . candidiasis. according to ragasa et al. (2009), neophytadiene can inhibit the growth of some fungi and bacteria including c. albicans, aspergillus . niger, trichophyton mentagrophytes, e coli and. p s eudomonas a e r ug ino sa . neophytad iene compounds serve as antipyretic, analgesic, antiinflammatory, antimicrobial and antioxidant agents (raman et al. 2012). leaf of h. suaveolens is the extract used as traditional medicine in brazil to treat inflammation, peptic ulcers, infections can and reduce gastric lesions in oral application in mice ( et al et aljesus . 2013). according to shaikat . (2012), he extract can cure diarrhea t leaf in mice previously treated with h. suaveolenscastor oil. contains a combination of terpenoids, consisting of eucaliptol, gamma-ellemene, beta-pynene, (+)3-carene, trans-beta-cariophyl lene and germacrene (moreira . 2010)et al . according to malar . (2012) et al h. suaveolens also contains alkaloids, glycosides, saponins, tannins and flavonoids he leaf extract of can ; t h. suaveolens inhibit the growth of various bacteria such as aeromonas formicans, aeromonas hydrophila, bacillus subtilis, e. coli, klebsiella pneumoniae and pseudomonas aeruginosa ( et al as well as malar . 2012), the growth of pathogenic fungi aspergillus flavus, a. parasiticus, a. ochraceus, a. fumigatus a. niger et aland (moreira . 2010). h. verticillata jacq. is used as traditional medicine in central america. th plant containsis lignans, triterpenes, diterpenes, sesquiterpenes, monoterpenes, flavonoids, polyphenols and alkaloids. the plant extracts and isolated crude phytochemicals play a role in antibacterial and anti-fungal treatment of infectious diseases (picking . 2013)et al . conclusions h. capitata leaves have secretory structures in the form of glandular trichomes and idioblast cells. the secretory structures produce secondary metabolite compounds. glandular trichomes generally contain terpenoids and alkaloids. idioblast cells contain lipophilic compounds. the terpenoid compounds, such as limonene, eugenol, farnesol isomers a, d-nerolidol, koumarin and neophytadiene, play roles in the healing process of wound or infection and act as antibacterial agents. acknowledgements the authors thank institut pertanian bogor (ipb/bogor agricultural universtiy), indonesia and göettingen university, germany, through the collaborative research centre (crc) 990 start up project in 2012, as well as the directorate general of higher education through boptn funding of 2013. deep appreciation goes to dr mohamad rafi for his assistance in gc-ms analysis. references biswas a, bari ma, roy m, bhadra sk. 2010. inherited folk pharmaceutical knowledge of tribal people in the chittagong-hill tracts, bangladesh. indian j tradit know 9(1):77-89. boix yf, victório cp, defaveri aca, arruda rco, sato a, lage cls. 201 . glandular trichomes of 1 rosmarinus officinalis l.: anatomical and phytochemical analyses of leaf volatiles. 145:848-56.plant biosyst bondada br, oosterhuis d. 2000. comparative epidermal ultrastructure of cotton ( l.) leaf, gossypium hirsutum bract and capsule wall. 86:1143-52. ann bot catherine aa, deepika h, negi ps. 2012. antibacterial activity of eugenol and peppermint oil in model food systems. j essent oil res 24:481-6. 101 identification of medicinal properties of hyptis capitata jacq. – rupa et al. curvelo jar, marques am, barreto als, romanos mtv, portela mb, kaplan mac, soares rma. 2014. a novel nerolidol-rich essential oil from piper claussenianum modulates candida albicans biofilm. j med microbiol 63:697-702. d'alessio pa, mirshahi m, bisson jf, bene mc. 2014. skin repair properties of limonene and perillyl alcohol din murine models. antiinflamm antiallergy agents med chem 13(1):29-35. dahlin rm, brick ma, ogg jb. 1992. characterization and density of trichomes on three common bean cultivars. econ bot 46(3):299-304. devi kp, nisha sa, sakthivel r, pandian sk. 2010. eugenol (an essential oil of clove) acts as an antibacterial agent against salmonella typhi by disrupting the cellular membrane. j ethnopharmacol 30:107-15. dickison wc. 2000. integrative plant anatomy. first edition. new york (us): academic press. 533 p. fahn a. 1979. secretory tissues in plants. new york (us): academic press. 302 p. filho scv, ferreire all, vasconcelos jm, silva ls, pereira lcs. 2014. secretory structures in cochlospermum rhegium (schrank) pilg. 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(lamiaceae). ann botlondon 71:43-50. 103 identification of medicinal properties of hyptis capitata jacq. – rupa et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 3. vincentius (primary).cdr biotropia vol. 20 no. 2, 2013: 89 103 primary productivity of jakarta bay in a changing environment: anthropogenic and climate change impacts vincentius siregar *, and alan. f. koropitan received 12 june 2013/accepted 3 december 2013 jakarta bay receives direct impact from the rapid development of infrastructure and landbased industries which contributed to the increase in pollution and nutrient, and at the same time facing climate change. this condition influenced growth of chlorophylland primary production. to investigate changes of primary production in jakarta bay due to anthropogenic and climate change impacts, a field measurement, laboratory experiment and collection of several data sets have been conducted. the study showed that impact of anthropogenic, particularly sediment load from the land to primary production is important. the intensification of primary production occurs in the middle region of jakarta bay, while the chlorophyllconcentration is high in the river mouth area. the anthropogenic impact is indicated by the land use change that has increased to 73% during the last ten years. the laboratory experiments by injecting co in the waters, as a global warming simulation, have shown a decrease in chlorophylland primary production. therefore, the combination of anthropogenic and climate change may have a double impact on the jakarta bay ecosystem. primary production, jakarta bay, anthropogenic, climate change, impact 1,2 1,3 1. 2. 3. department of marine science and technology, faculty of fisheries and marine science, bogor agricultural university, bogor, indonesia, laboratory of remote sensing and ecology, seameo biotrop, bogor, indonesia center for oceanography and marine technology surya, university, tangerang, indonesia a a a abstract introduction key words: 2 as a part of the java sea, the mass transport of water and material in jakarta bay are controlled by ocean dynamics which is generally influenced by monsoon system and tidal mixing. wyrtki (1961) showed that there are two distinct seasons over the indonesia seas: southeast monsoon and northwest monsoon. associated with the east wind from australia, southeast monsoon brings warm dried air during boreal summer (june-august). in contrast, the west wind from asia or northwest monsoon brings warm moist air during the boreal winter (december-february). thus, * corresponding author : vincents@biotrop.org doi: 10.11598/btb.2013.20.2.5 89 monsoon systems also control the dry season (northwest monsoon) and wet season (southeast monsoon) in indonesian seas where the monsoon wind patterns also affect those of surface flow. in jakarta bay, the distribution of nutrients is strongly affected by monsoon system along the year, but its spatial distribution is more pronounced, high near the coast and low at seaward (ilahude 1995). although coastal waters is an area relatively small in global seas, but it plays an important role in biogeochemical cycles. gattuso . (1998) reported that coastal water contributed 14-30% of primary productivity, 80% of organic matter decomposition, and 90% mineralized sediments. in the sea, half of the production of organic matter was provided by phytoplankton (boyce . 2010). as many shelf seas in the world, ecosystems of the java sea are under pressure due to human activities in the coastal region. the java sea covers an area of 450 000 km where some cities and industries are located along northern coast of java island. talaue mcmanus (2000) reported that most of the pollution in the java sea is caused by domestic sewage, agricultural, industrial and waste as well as solid waste that goes to the coastal region. rapid development of infrastructure and land-based industries also contributed to the pollution increaseof the coast, for example: jakarta bay and its surrounding waters are influenced by human activities of the jakarta metropolitan area (jma) which contribute to the pollution of the jakarta bay (suwandana . 2011). population in jma has been increasing year by year, as reported by the indonesian statistics that the population has increased to 30.2 % from 7.6 million in 2007 to 9.8 million in 2012 (provinsi dki 2012). this has an implication to land use change of the coastal area and its vicinities, hence affected jakarta bay. therefore, the population of jma may have direct impact (exploitation and human settlements) and indirect impact (via watersheds) to jakarta bay. anthropogenic impact due to human activities has caused an increase in the concentration of nitrates and phosphates in jakarta bay, to 5 and 15 times, respectively, in the last three decades (arifin 2005). in addition, the eutrophication condition in jakarta bay can be categorized as hyper-eutrophication, especially in estuarine waters or region near river mouth as reported by damar (2003). on the other hand, the java sea ecosystem is facing climate change impact, particularly sea surface temperature (sst) rise and marine acidification. according to the ipcc's predictions (christensen . 2007), increase of sst in the southeast asia waters will be about 1.5 3.7 c during the last 21st century. marine acidification is related with decline of ph as a result of absorbing atmospheric co by the global ocean. according to orr . (2005) marine ph has decreased to 0.1 since post-industrial era. regarding to the recent condition of jakarta bay and climate change issue, hence, this research aim was to investigate changes of primary production in jakarta bay due to anthropogenic and climate change impacts. the field measurement of some water quality parameters at 21 stations and laboratory experiments had been carried out during the period of 9 14 july 2012. in et al et al et al et al et al 2 o 2 materials and method 90 biotropia vol. 20 no. 2, 2013 addition, landsat satellite data as well as secondary data had been collected and analyzed. water samples parameters were taken including water quality, primary production and chlorophyll. water quality parameters measured were surface temperature, salinity, turbidity, using water quality checker at a depth of 0 10 m. primary production and chlorophyllwater samples were collected using water sample at a depth of 1 2 m, and the transparency was measured using a secchi disk. secondary data for nutrient concentrations analysis was obtained from damar (2003). the field stations are shown in fig. 1. laboratory experiments were set up in untung jawa island, northern part of jakarta bay using seawater collected directly in real time from the sampling area to measure chlorophylland primary production influenced by increasing temperature and co injection as treatments. two different treatments have been performed to increase the seawater temperature 1 c and 2 c of the initial temperature (29 -30 c) using tube lamps, while c gas was injected to seawater until 450 ppm and 560 ppm, respectively. gross primary production (gpp) and net primary production (npp) were measured based on dissolved oxygen in the light and dark bottles before and after incubation (3 hours incubation using halogen lamp) in field laboratory with ambient condition. dissolved oxygen was measured based on modified winkler method (strickland & parsons, 1972; apha, 1980). chlorophyll-a parameter was measured using fluorometric method. in addition, the landsat satellite data were downloaded from source : ( ) to analyze land use coverage change during 10 years (2002-2011 using color composite, total suspended solid (tss) applying formula of ambarwulan (2002) and chlorophylldistributions in jakarta bay using an algorithm (wouthuyzen 1991). a a niskin a a 2 2 0 0 0 0 o http://earthexplorer.usgs.gov/ 91 primary productivity of jakarta bay in a changing environment vincentius siregar– et al. figure 1. sampling stations and its clustering in jakarta bay results and discussion general condition of jakarta bay water jakarta bay receives material and water discharges from several rivers that cover jam and its surrounding area, such as cisadane in the western part and citarum in the eastern part. in addition, there are several rivers that drain in the central part of jakarta bay, such as: muara gembong, cikarang, marunda, muara angke, and kamal. all rivers are responsible in carrying sediment in the bay and affect tss and transparency of the water. considering the sediment distribution regime, four clusters of sampling stations were based on their distance from the coast-line as shown in table 1. 92 biotropia vol. 20 no. 2, 2013 table 1. clustering of sampling stations cluster station description 1 15, 12, 11, 4, 5 near estuary and coastline 2 16, 13, 10, 3 45 km from coastline 3 17, 14, 6, 2 8-10 km from costline 4 19, 18, 9, 1, 8, 7, 20 >10 km from coastline sea surface temperature depends on several factors such as precipitation, evaporation, wind speed, intensity of sunlight, and freshwater input from the river. in jakarta bay, distribution of the sea surface temperature horizontally decreased from near the coast to seaward (fig. 2). the vertical distribution of sea temperature showed the same pattern as those of horizontal distribution (fig. 3). figure 2. horizontal distribution of sea surface temperature july 2012 93 figure 3. vertical distribution of sea temperature of jakarta bay waters the role of river discharges in jakarta bay was represented by sea surface salinity distribution. in jakarta bay, the river discharges were relatively low as shown in figure 4 where the observed salinity distribution tended to decrease slightly from cluster 4 to cluster 1.this condition was actually influenced by level of rainfall during dry season (july 2012) at which the sampling period was carried out. in general, the sea surface salinity in jakarta bay is typical of coastal water varying between 30.4 to 32.1 psu. figure 4. sea surface salinity distribution in jakarta bay july 2012 primary productivity of jakarta bay in a changing environment vincentius siregar– et al. cluster 1 cluster 2 cluster 3 cluster 4 94 biotropia vol. 20 no. 2, 2013 the river discharges were relatively low, although the effect of human activities in the coastal region has affected the turbidity of jakarta bay, particularly in custer 1 and cluster 2 regions. turbidity consisted of colloidal particles and suspended pollutants, such as: organic materials and inorganic waste from industry, households and aquaculture. turbidity could be caused by the presence of organic materials produced by the waste industry. the turbidity in jakarta bay varied between 2.43 7.00 ntu (fig. 5) and the high turbid water was located in st.15 (muara angke), st. 11 (priok), and st. 3 and 4 (cikarang). so, the turbidity was high at the coastline due to land runoff and gradually decreases seawards. similar to the turbidity, total dissolved substance (tds) distribution had the same pattern. the dissolved substance could be carbonate and bicarbonate, chloride, sulfate, phosphate, nitrate, calcium, magnesium, sodium, organic ions, and more ions. the tds ranges between 31 32 g/l, where the highest concentration occurs in st. 15 (muara angke). the observed secchi disk depth distribution in jakarta bay showed that cluster 1 and cluster 2 were lower than cluster 3 and cluster 4. it is emphasized that the transparency of jakarta bay close to the estuary and coastline were poor. the secchi depth for each cluster was varied between 1.3 and 3.5 m (cluster 1), 1.6 and 3 m (cluster 2), 4 and 4.5 m (cluster 3), 3 and 6.2 m (cluster 4). figure 5. turbidity distributions in jakarta bay based on field measurement on july 2012 95 a a a chlorophyllis a necessary pigment of phytoplankton for photosynthesis. phytoplankton serves as primary producers in the chain of life in the sea, so its existence is very important as the basis of life on the sea. the chlorophyllconcentration in jakarta bay varied between 0.78-15.74 mg/l (figure 6), while the annual chlorophyllconcentration derived from the satellite data (2002-2012) varied from 1.09 mg/l to 6.07 mg/l and showed a stable trend , except in 2011-2012 the trend was increasing significantly close to the coastal area (cluster1,2) (fig. 7). figure 6. chlorophylldistribution in jakarta bay based on field measurement on july 2012 a figure 7. annual chlorophyllconcentration derived from satellite data(2002-2012)a primary productivity of jakarta bay in a changing environment vincentius siregar– et al. c h lo ro p h y ll (m g /l ) 96 biotropia vol. 20 no. 2, 2013 increasing chlorophyllmainly occurred in the near river mouth of st. 11 (tanjung priok) and st. 15 (muara angke) due to runoff from the jma. however, low primary production was observed surrounding the river mouth, whereas high production occurred in the centre of jakarta bay (fig. 8). this could indicate that little support of the photosynthesis in the river mouth occurred and was probably due to the high turbidity. a figure 8. npp distributions in jakarta bay based on field measurement on july 2012 anthropogenic impact the studies of anthropogenic impact and primary productivity seas were analyzed based on the ratio of chlorophylland npp. chlorophyllas major component in phytoplankton is responsible for primary production activities. increasing of chlorophyll-a concentration in the waters resulted high primary productivities. ratio of chlorophylland npp has relation to an individual's capacity to produce phytoplankton primary productivity. the higher the ratio of chlorophylland npp, the lower the capacity of phytoplankton result in the primary productivity. this indicated that abundance of chlorophylldoes not mean ability of primary productivity to increase in proportion to the amount of chlorophyll. in this case, the impact of anthropogenic pollution, can affect capacity of chlorophyll-a in generating primer productivity. to analyze the impact of land use change on primary production in jakarta bay, figure 9 shows ratio of chlorophyll/npp. the high ratio means more chlorophyllto produce primary production or the high ratio the less efficiency in photosynthesis the ratio indicates the efficiency in photosynthesis processes by phytoplankton. the average value of ratio 79.83 in cluster 1, 60.1 in cluster 2, 38.05 in cluster 3 and 20.39 in cluster 4 . thus, anthropogenic pressures were high in near river mouths or coastal region (cluster 1) and gradually decrease to the seaward (cluster 4). a a a a a a a a figure 9. distribution of ratio between chlorophylland npp in all stationsa the analysis of the land cover change is only carried out by grouping some particular class settlements, pond/swamp, rice field, and vegetation corresponding with the anthropogenic impacts of the waters. the maps of the land cover change during the last ten years are shown in figure 10. the variation of land use change in jma concluded that settlement class has increased approximately 73% in the past 10 years. it implies that the anthropogenic contribution from residential waste (household and industry) has increased year by year. as the rice fields are likely to decrease, pond or swamp tends to remain unchanged. meanwhile, vegetation cover increased due to the development of the green city program of jakarta, which stated that every area should leave at least 30% of the open green area. the land cover change from 2002 to 2011 is shown in figure 11. the total suspended sediment concentration (tss) extracted from satellite data during 2002-2011 in jakarta bay waters increased and showed higher concentration in the mouth of rivers (cluster 1-2). tss concentrations showed positive correlation with the turbidity. this similar pattern was also shown during the observation period and the concentration varied from 0.11 mg/l to> 135 mg/l. the spatial distribution of concentration of the tss tends to develop year by year in the coast seaward. this is probably due to the increase of sediment load from land as a result of significant land cover change in jma and surrounding cities. in addition, figure 12 shows ratio of compensation depth (dk) and attenuation depth (k). the compensation depth (dk) is a depth of water column where photosynthesis rate is the same as respiration rate. the compensation depth characterizes the occurrence of primary productivity capacity. attenuation depth (k) is a maximum depth for sunlight intensity in water column, to allow photosynthesis processes. in turbid water such as jakarta bay, depth attenuation can be a limiting factor in photosynthesis process. thus, ratio of compensation depth and attenuation depth characterize capacity of primary productivity. the higher ratio of dk and k, the greater capacity of primary production. the ratio of dk/k is generally high in cluster 4 and low in cluster 1. this is consistent with the high sediment load from land as shown previously in turbidity distribution (fig. 5). therefore, the land use change and sediment supply have influenced significantly to the primary production in jakarta bay. of 97 primary productivity of jakarta bay in a changing environment vincentius siregar– et al. legend cloud rice field settlements vegetation pond / swamp figure 10. land cover changes during the last ten years (2002-2011) biotropia vol. 20 no. 2, 2013 98 figure 11. land cover change from 2002 to 2011 figure 12. ratio between compensation depth(dk) and attenuation depth (k) in all stations. climate change impact the annual average of minimum surface air temperature in jakarta bay have increased during 2002-2011 (fig. 13). the lowest and the highest of minimum surface air temperature occurred in 2008 and 2011 were 25.33 c and 25.76 c, respectively. at the same period, the maximum surface air temperature tended to decrease, where the highest and the lowest occurred in 2002 and 2008, respectively. those surface air temperatures could be used as a primary measurement of climate change in jakarta bay and its surrounding areas (hansen . 2010). 0 0 et al 99 primary productivity of jakarta bay in a changing environment vincentius siregar– et al. the laboratory experiments as simulation of the effects of climate change was set up streamlined in nature, where the created conditions are closed systems. however, to achieve conditions similar to the process in nature, the location of the experiment was placed close to the environmental conditions with a relatively recent natural changes. in general, the experiment concluded that there has been a decline in chlorophyll-a and primary productivity (npp) with increasing temperature 1 c and 2 c and co gas injection (450 and 560 ppm) into the water sample, after the incubation process. in contrast, hashioka and yamanaka (2007) reported that chlorophylland npp in the sub-tropical and temperate waters will be increasing under global warming scenario, based on numerical modeling. this contradiction may suggest that marine ecosystem in tropical region has different response with those in sub-tropical and temperate regions. the summary of laboratory experiments related with climate change scenarios is presented in table 2. 0 0 2 a 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 tm 25.40 25.44 25.42 25.55 25.60 25.53 25.33 25.79 25.85 25.87 y = 0.050x + 25.30 r² = 0.599 25.00 25.10 25.20 25.30 25.40 25.50 25.60 25.70 25.80 25.90 26.00 °c figure 13. annual average of minimum (left) and maximum (right) of surface air temperature in jakarta bay during 2002-2011 (source : bmkg, jakarta, 2012) table 2. the increased influence of temperature and co towards primary productivity and chlorophyll2 a type of experiment gpp respiration npp chlorophyll-a (mgc/m3/day) (mgc/m3/day) (mgc/m3/day) (mg/m3) control before incubation 1.916 control after incubation 2.634 3.088 0.061 2.394 temperature 1 oc after incubation 2.593 3.108 0.003 1.373 temperature 2 oc after incubation 2.557 3.065 0.003 1.075 injection co2: 450 ppm 2.441 3.086 -0.131 1.391 injection co2: 560 ppm 2.392 3.257 -0.323 1.073 note :gpp = gross primary productivity/photosynthesis npp = net primary productivity biotropia vol. 20 no. 2, 2013 100 in order to evaluate the dominant parameters that affect npp in jakarta bay, principal component analysis (pca) has been performed. selected parameters for pca are nutrients (nitrite, nitrate, ammonium and phosphate) and chlorophyllas shown in figure 14. in this analysis, nutrient concentrations was obtained from damar (2003) where the chlorophyllis based on the present study. a a figure 14. pca of chlorophyll, ammonium, phosphate and nppa figure 15. ammonia concentration on the tide and low tide in estuary of jakarta bay . pca results showed that chlorophyll, ammonium, phosphate and npp have positive correlation. in this case, chlorophylland ammonium are closely associated. this linkage suggests that ammonium influence significantly on phytoplankton growth (chlorophyllconcentrations) in jakarta bay. ammonium is a kind of nutrient nitrogen species, which generally come from sources of organic nitrogen remineralization processes in form of particles, as well as dissolved. in addition, ammonium sources are also coming from industrial activities. nitrogen species such as nitrate and nitrite are generally derived from agriculture due to the use of chemical fertilizers. according to data from the environmental status report of the jma (bplhd jakarta 2011), the ammonium supply in jakarta bay is mainly by rivers where the high contribution is muara cakung and muara sunter, particularly during ebb tide (fig. 15). both river mouths are located at tanjung priok, an international marine port of jma a a a 101 primary productivity of jakarta bay in a changing environment vincentius siregar– et al. tide low tide bm 8.00 7.00 6.00 5 4 3 2 1 .00 .00 .00 .00 .00 m kam al c en gk ar en g m ang ke m kar an g m anc ol m sun te r m c ak un g m m ar un da m g em bo ng tide low tide bm assessment of anthropogenic impacts and climate change on primary production in jakarta bay, indicated that the role of human activities in jakarta is more dominant, especially in the waters close to coastal or estuarine river. for example, the efficiency in photosynthesis is controlled by sediment load, not only containing clay and sand, but also organic particles or contaminants that enter through the rivers. this condition influences the change of water quality, resource supply , habitat availability and climate (lotze . 2002). the impact of climate change in this case was significant and may have a double impact in combination with the anthropogenic change. the anthropogenic impact was clearly shown by less efficiency in primary production in jakarta bay, particularly close to the river mouths or coastline due to sediment load from jakarta metropolitan area. the anthropogenic impactwas mainly influenced by domestic waste and land use change that had increased to 73% during the last ten years. the climate change impacts based on laboratory experiments by simulation of increasing temperature and c0 gas have shown a decrease in chlorophylland primary production. therefore, the anthropogenic and climate change may have a double impact to the jakarta bay ecosystem. the financial support for this research was provided by seameo biotrop under their 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potential utility of remote sensing data acquired from earth observation satelites for monitoring in coastal zone environment. naga report volume 2 103 primary productivity of jakarta bay in a changing environment vincentius siregar– et al. 4. i made (lipid) revisi.cdr biotropia vol. 21 no. 2, 2014: 100 110 lipid accumulation by ath using palm oil mill effluent as substrate flavodon flavus i made sudiana , atit kanti , helbert , senlie octaviana and suprapedi received 3 april 2014/accepted 6 october 2014 large amount of palm oil mill effluent (pome) is generated annually. the waste would be potential for production of single cell oils (scos). the objective of this study was to evaluate lipid accumulation by fungi using pome as substrate. seven filamentous fungi were initially isolated from various biomes. the study results showed that out of these 7 fungi, five of them produced endoglucanase and accumulated lipid about 34.3-87.5% of their dry cell mass using pome as substrate. the five fungi were identified as ath, sp., sp., sp. 1 t30, and sp.2 t50. the highest lipid accumulation was obtained by ath. the profile of trans-esterified scos revealed high content of saturated and mono-unsaturated fatty acids i.e., palmitic (c16:0), stearic (c18:0) and oleic (c18:1) acids similar to conventional vegetable oils used for biodiesel production. the strain that was able to use organic substrates in pome indicated that they are promising strain for biofuel feedstock as well as for fulfilling effluent quality for wastewater discharge. fatty acid methyl ester, , oleaginous fungi, palm oil mill effluent (pome), single cell oil *1 1 1 1 2 1 2 research center for biology, indonesian institute of sciences, cibinong 16911, indonesia research center for physic, indonesian institute of sciences,komplek puspiptek tangerang selatan, banten, indonesia flavodon flavus aspergillus trichoderma fusarium fusarium flavodon flavus flavodon flavus abstract introduction keywords: as awareness on the limited stock of fossil fuel and concern on environmental hazard of petroleum fuel is growing, research on sustainable biofuel is gaining its popularity (richana 2010; fujita 2004; wraight & ramos 2005; jing 2010; mohammadi 2011). microbial based biofuel (micro-diesel) has become more attractive due to its environmental incentive and renewable properties (zheng et al. et al. et al. et al. * corresponding author : imadesudianalipi@gmail.com doi: 10.11598/btb.2014.21.2.4 100 2012). the merit of micro-diesel over petroleum fuel is clear, but our understanding on microbial diversity and physiology of oleaginous microbes is still incomplete to produce economically viable micro-diesel (zhao . 2012). current practice of biodiesel production is through trans-esterification of vegetable oils or animal fats with short chain alcohols. feedstock acquires more than 70% of total biofuel production cost, and this limit further expansion of biodiesel (wei . 2013). exploiting lipid accumulating microorganism would offer solution for feedstock generation (wang . 2011). some lipid accumulating fungi i.e. , sp., var. , sp., sp., sp. and sp. accumulate large amounts of lipids greater than 40% per dry cell weight (wei 2013; wynn . 2001) under n-limited cultivation conditions. oils derived from microbes have many advantages over plant oil due to having short life cycle, less labor required, less affection by venue, season and climate, and easier to scale up (wang . 2013). these oleaginous characters would place microbial oils as potential feedstocks for biodiesel production in the future (liu . 2012). lignocellulose waste are abundant in tropical region. several microbes use lignocelluloses hydrolysate for biofuel production (tsigie . 2011). pome contains high strength organic substances with total chemical oxygen demand (cod) about 30,000-40,000 ppm and 3% lipid (wu . 2010). utilizing these wastes for biofuel production offer manifold benefits through reducing total organic in effluent, and generate biofuel feedstock via bioconversion palm oil waste into triacyl glycerol rich microbial cell (coleman 2004). fungi would be good candidate for biofuel feedstock, since some fungi produce extracellular cellulase which break down complex polysaccharide into fermentable sugar (goyal . 1991), consume it for lipid synthesis and finally accumulate it into lipid bodies which primarily consist of triacylglicerol (rossi . 2010). this study aims to evaluate the lipid accumulation by fungi using pome as substrate. this study initially isolated 7 filamentous fungi from various biomes as listed in table 1. et al et al et al cunninghamella echinulata microsphaeropsis mortierella isabellina, m. ramanniana angulispora mucor circinelloides, phomopsis cephalosporium sclerocystis nigrospora et al. et al et al et al et al et al et al et al materials and methods fungi species used table 1. fungi used in this study name of species fungi sources sp. soil of cibinong west java ath decaying wood of south east sulawesi comp insect frass sp1 t30 sludge of wastewater treatment in west java sp2 t50 sludge of wastewater treatment in west java sp soil of cibinong west java sp. soil of cibinong west java aspergillus flavodon flavus paecilomyces lilacinus. fusarium fusarium trichoderma mucor 101 lipid accumulation by ath using palm oil mill effluent i made sudianaflavodon flavus – et al. culture conditions determination of cellulolytic fungi endoglucanase assays biomass the fungal inoculants of the 7 filamentous fungi was prepared following mulder (1989) and kimura . (2004). briefly, fungal spores that had been incubated at 30 °c for 5 days on potato dextrose a l ×10 spores/ml was inoculated into 150 ml of the seed culture medium containing (g/l): glucose, 30; yeast extract, 5; kno , 1; kh po , 2.5; znso ·7h o, 0.01; cuso ·5h o, 0.002; mnso , 0.01; mgso ·7h o, 0.5; feso ·7h o, 0.02; and cacl , 0.1. the initial ph of the medium was adjusted to 5.5 by adding 10 m naoh in a 500 ml flask and incubated at 30 °c with shaking at 125 rpm for 24 hours. then, 10 ml of the seed culture was transferred into 250 ml of growth medium containing pome obtained from pt perkebunan nusantara, malimping, west java. prepared medium was autoclaved for 20 minutes before use. the culture was incubated at 30 °c with shaking at 125 rpm for 24 hours. the ability of fungi to hydrolyze cellulose was evaluated using cmc containing media following zhou . (2004). to evaluate the effect of temperature on the endoglucanase activity, the cultures were separately incubated at 30 °c and 50 °c with shaking at 125 rpm for 5 days. enzymatic activity was assayed following zhou . (2004). briefly, the culture were placed in 50 ml centrifuge tubes, then centrifuged at 2,500 g at 4 ºc for 30 minutes activity measurements, 2% carboxymethylcellulose (cmc, sigma) was l l l s were incubated at 50 ºc for 30 minutes. reducing sugars were determined using the 3,5-dinitrosalycilic acid (dns) assay according to dinis , (2009). l utes (after adding the supernatant to the reaction mixture) up to 45 minutes l utes and immediately cooled on ice for 5 minutes. finally, l was measured at 540 nm in a spectrophotometer (uv mini 1240 shimadzu). one enzyme unit is defined as mmol glucose produced by 1 ml enzyme per hour. biomass fungal was determined according to ogbo (2010). briefly, biomass was harvested by filtration, and fungal pellet were washed with 50 ml deionized water for removal of residual nutrients, and lyophilized at -50 °c in a vacuum of 1 mbar for 12 hours and weighed. fungal biomass was expressed as grams of dry weight per liter of culture medium. et al. et al et al et al et al. gar plates (difco laboratories, detroit, mi, usa) were harvested and suspended in seed culture medium. a spore suspension (500 μ ) containing approximately 4 . supernatants were clarified by filtration through 0.45 μm nitrocellulose filters (pall). for enzymatic dissolved in 50 mm citrate buffer ph 5. enzymatic reactions contained 200 μ of supernatant, 300 μ of 50 mm citrate buffer ph 5, plus 500 μ of each substrate solution. the reaction mixture briefly, 50 μ aliquots were taken every 5 min , then mixed with 50 μ of a dns solution, boiled for 5 min 500 μ of water were added and absorbance 6 3 2 4 4 2 4 2 4 4 2 4 2 2 biotropia vol. 21 no. 2, 2014 102 lipid concentration lipid composition analysis the lipid concentration of fungi broth was determined from a standard curve obtained by plotting absorbance against the corresponding lipid concentration determined by the conventional method of acid hydrolysis followed by solvent extraction and gravimetric estimation. forty milligrams samples were extracted with 3 ml of chloroform/methanol (1/2, v/v) by vortexing (1 minute) and centrifugating at 2,500 g for 15 minutes at room temperature. the supernatants were collected and residues were re-extracted twice with 2 ml of chloroform/methanol (1/1, v/v) by centrifugation as stated above. all the supernatants were pooled together, filtered with whatman filter no. 1 (whatman, usa), and washed with 2 ml of milli-q water, followed by centrifugation at 2,500 g for 5 minutes. the lower organic phases were collected and evaporated to dryness under nitrogen and total lipid contents were determined gravimetrically (sitepu . 2013). the total lipid concentration was determined by gas chromatographic analysis of the total fatty acids directly trans-methylesterified from dried cell (liu 2008 ; kosa & ragauskas 2011). one milliliter of 10% methanolic-hcl and 0.5 ml methylene chloride were added to the dried biomass and placed at 60 °c for 3 hours for direct methyl-esterification. the reaction was stopped by the addition of 2 ml saturated nacl solution and 1 ml hexane. the resultant methyl esters recovered in the hexane layer were then applied to a gas chromatograph (gcms-qp 2010-ultra; shimadzu, kyoto, japan) equipped with a famewax capillary column (30 m× 0.25 mm i.d., gl science, tokyo, japan) under temperature programming (150-250 °c at 5 °c/minute increments). peanut oil (nacalai tesque, kyoto, japan) was trans-methylesterified and used as the reference material. overall, all fungi studied showed cmc-ase activity (fig.1), but the strains of sp., and comp grew slowly. therefore, we omitted these strains for further study. ath produced the highest cmc-ase at 96 hours incubation. other strains having slightly lower activity were sp. and comp. maximum activity attained at 96 hours, and decreased at 144 hours. the ability of cultures to produce cmc-ase indicated these fungi produced endoglucanases, ec 3.2.1.4, and played important role in cellulose hydrolyses (hasper 2002). kitcha and cheirsilp (2014) observed newly isolated fungi as being able to hydrolyze cellulose of palm by products. to gain cellulolytic character of fungi in this study, we further evaluated their ability to hydrolyze pome at 30 °c and 50 °c. et al et al. mucor paecilomyces lilacinus flavodon flavus aspergillus paecilomyces lilacinus et al. aspergillus tubingensis results and discussion lipid accumulation by ath using palm oil mill effluent i made sudianaflavodon flavus – et al. 103 endoglucanase activity at 30 °c overall, endoglucanase activity of fungi grown in pome is summarized in figure 2. the highest endoglucanase activity was obtained by ath and sp. grown at 30 °c after 96 hours cultivation. slightly lower activity was observed on sp.2 st50. generally lower cmc-ase was observed at 144 hours fermentation. flavodon flavus aspergillus fusarium figure 1. cmc-ase (endoglucanase) activity of fungi grown on cmc-medium figure 2. endoglucanase activity of selected isolates grown on pome at 30 °c aspergillus sp. flavodon flavus ath paecilomyces sp. comp fusarium sp1 t30 fusarium sp2 t50 trichoderma sp. mucor sp. aspergillus sp. flavodon flavus fusarium sp1 t30 fusarium sp2 t50 trichoderma sp. 104 biotropia vol. 21 no. 2, 2014 endoglucanase activity at 50 °c effect of growth temperature on endoglucanase activity endoglucanase activity of selected fungi grown on pome at 50 °c varies among fungal species depending on incubation period (fig.3). maximum activity was showed by ath at 96 hours incubation. lower activity was obtained after 144 hours cultivation. in general, cmc-ase activity was higher in cultures incubated at higher temperature (fig.4). almost 50% increase of cmc-ase activity was achieved by ath. fungi have been intensively explored for hydrolyses of lignocellulose materials. ath is wood decayed fungi isolated from south east sulawesi, indonesia. appears to be widely distributed lignocellulolytic fungi isolated from decaying sea grass from a coral lagoon off the west coast of india (raghukumar 1999; mtui & nakamura 2008) and produces extracellular lignin-modifying enzymes (lmes): manganese-dependent peroxidase (mnp), lignin peroxidase (lip), and laccase when grown in n-limited media (mtui & nakamura 2008). these enzyme characters would be advantageous for producing fermentable substances for triacylglicerol synthesis using lignin containing waste such as pome (duarte 2013; lam & lee 2011). we also noticed that a culture of ath having high cmc-ase or endoglucanase activity indicated that hydrolyses and product hydrolyses consumption affected enzyme synthesis (vlasenko 2010). flavodon flavus flavodon flavus flavodon flavus flavodon flavus et al. flavodon flavus et al. figure 3. endoglucanase activity of selected isolates grown on pome at 50 °c aspergillus sp.flavodon flavus fusarium sp1 t30 fusarium sp2 t50 trichoderma sp. lipid accumulation by ath using palm oil mill effluent i made sudianaflavodon flavus – et al. 105 lipid accumulation fatty acid profiles of fungal scos pome is good substrate for growing lipid accumulating fungi (fig.5), as shown by the lipid production (75% lipid per cell dry-weight). the highest lipid accumulation was attained by ath when grown at 30 °c, but less when the culture was grown at 50 °c. incubation temperature affected lipogenesis of fungi (fig.5). the suitability of scos from oleaginous fungi was evaluated and observed contained 92% (w/w) neutral lipids in its sco (wu 2011). other lipid producer, , contained lower neutral lipid fractions (18.5%, w/w), higher amount of polar lipids (35%, w/w ), and free fatty acids (32% w/w) in its sco (wynn . 2001). fatty acid profiles compositions of selected fungi were obtained through transesterification of triacyl glycerol with methanol under alkaline condition. fatty acid methyl esters (fame) was mainly composed of methyl palmitate ( c h o ), methyl cis-10-heptadecenoate (c h o ) and methyl oleate (c h o ) (table 2), indicating that the fame is closely related to palm and soybean fatty acids (ratledge & wynn, 2002). there was slight differences in concentration of lipid species among fungi evaluated (wynn 2001; khot 2012; chan 2010). the variability in fatty acid composition of oleaginous organism could be due to culture technique (chi 2011) and species dependence (gasmi . 2011). flavodon flavus cunninghamella echinulata et al. mucor circinelloides et al et al. et al. et al. et al. et al 17 34 2 18 34 2 19 36 2 figure 4. comparison of endoglucanase activity of selected isolates grown on pome at 30 °c and 50 °c aspergillus sp. flavodon flavus fusarium sp1 fusarium sp2 trichoderma sp. aspergillus sp. flavodon flavus fusarium sp1 fusarium sp2 trichoderma sp. 106 biotropia vol. 21 no. 2, 2014 figure 5. lipid accumulation by fungi grown on pome at 30 °c and 50 °c after 6-day fermentation table 2. lipid composition of fungi grown on pome cultured at 30 °c after 6 days fermentation fatty acid methyl esters (fame) trichoderma sp. flavodon flavus ath sp. fusarium sp1 t30 fusarium sp2 t50 methyl palmitate(c17h34o2) 31.39 27.05 23.62 21.36 20.21 methyl palmitoleate(c17h32o2) 29.65 26.28 21.08 21.08 20.68 methyl cis-10heptadecenoate (c18h34o2) 3.1 5.32 4.36 5.32 4.32 methyl stearate (c19h38o2 ) 4.14 4.28 5.23 4.23 3.69 methyl linoleate (c19h34o2) 5.41 6.21 5.21 4.65 6.1 methyl butanoate 6.72 11.2 7.06 6.1 9.2 methyl oleate (c19h36o2) 6.06 6.21 7.32 7.36 6.35 methyl linolenate (c19h32o2) 4.11 3.11 9.81 9.98 8.21 methyl cisvaccenate(c19h32o2) 2.38 2.32 6.8 8.69 6.98 methyl cis-12octadecenoate 2.71 4.82 5.91 4.91 8.6 methyl myristate(c15h30o2) 4.33 3.2 3.6 6.32 5.66 aspergillus sp.flavodon flavus fusarium sp1 t30 fusarium sp2 t50 trichoderma sp. lipid accumulation by ath using palm oil mill effluent i made sudianaflavodon flavus – et al. 107 conclusions acknowledgements references fungi were able to accumulate lipid in large amount using pome as substrates, indicating that introducing fungi into pome wastewater treatment plant could be proposed for future biofuel production and pollution reduction. this research was supported by lipi competitive research grant 2013-2014, and we expressed our sincere gratitude to dian nurcahyanto, yeni yuliani for laboratory asisstance. chan yj, chong, mf, law, cl. 2010. biological treatment of anaerobically digested palm oil mill effluent (pome) using a lab-scale sequencing batch reactor (sbr). j environ manage 91(8): 1738-46. chi z zheng, y, jiang, a, chen, s. 2011. lipid production by culturing oleaginous yeast and algae with food waste and municipal wastewater in an integrated process. applied biochemistry and biotechnology 165(2): 442-53. coleman r. 2004. enzymes of triacylglycerol synthesis and their regulation. progress in lipid research 43(2): 134-76. dinis, mj, bezerra, rm. f, nunes, f, dias, a. a, guedes, c. v, ferreira, l. m., m., rodrigues, m. a m. 2009. modification of wheat straw lignin by solid state fermentation with white-rot fungi. bioresource technology 100(20): 4829-35. duarte sh, de andrade, ccp, ghiselli, g, maugeri, f. 2013. exploration of brazilian biodiversity and selection of a new oleaginous yeast strain cultivated in raw glycerol. bioresoivce technology 138: 377-81. fujita y, ito, j, ueda, m, fukuda, h, kondo, a. 2004. synergistic saccharification and direct fermentation to ethanol of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme. app and environ microbiol (2): 1207-12. gasmi n, ayed, a, ammar, b, zrigui, r, nicaud, j-m, kallel, h. 2011. development of a cultivation process for the enhancement of human interferon alpha 2b production in the oleaginous yeast, . microbi cell fact 10(90): 1-11. goyal a, ghosh, b, eveleigh, d. 1991. characteristics of fungal cellulases. bioresovice technol 36(1): 37-50. hasper aa, dekkers, e, mil, m van, vondervoort, pj i. van de, graaff, l. h. de. 2002. a new endoglucanase from with major activity towards xyloglucan. appl environ microbio 68(4): 1556-60. jing q, spiertz, jhj, hengsdijk h, van keulen h, cao w, dai, t. 2010. adaptation and performance of rice genotypes in tropical and subtropical environments. 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kinetics of cornsstalk pretreated by oleaginous fungi . biores technol 102(8): 5255-58. wu ty, mohammad aw, jahim jm, anuar n. 2010. pollution control technologies for the treatment of palm oil mill effluent (pome) through end-of-pipe processes. j environ manage 91(7): 1467-90. wynn jp, hamid aa, li, y, ratledge, c. 2001. biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi and . microbiology 147(10): 2857-64. chlorella vulgaris flavodon flavus 7 flavodon flavus biodiesel feedstocks and processing technologies yarrowia lipolytica yarrowia lipolytica mucor circinelloides: beauveria bassiana bacillus thuringiensis cunninghamella echinulata mucor circinelloides mortierella alpina lipid accumulation by ath using palm oil mill effluent i made sudianaflavodon flavus – et al. 109 zhao x, peng f, du, w, liu c, liu d. 2012. effects of some inhibitors on the growth and lipid accumulation of oleaginous yeast and preparation of biodiesel by enzymatic transesterification oftthe lipid. bioprocess biosyst engi 35(6): 993-1004. zheng y, chi z, ahring bk, chen s. 2012. oleaginous yeast for biofuel production: ammonia's effect. biomass and bioenergy 37: 141-121. zhou x, chen h, li z. 2004. cmc-ase activity assay as a method for cellulase adsorption analysis. enzyme microbi tech 35(5): 455-59. rhodosporidium toruloides cryptococcus curvatus 110 biotropia vol. 21 no. 2, 2014 microsoft word 47 biotropia no. 19, 2002 : 47 56 plojdy analysis and dna content of mutant banana "pisang berangan" using flow cytometry abdelgadir rayis shadia, rofina yasmin othman and mak chai division of genetics and molecular biology, institute of biological sciences, faculty of science, university of malaya, kuala lumpur, malaysia abstract mutagens cause random changes in the nuclear dna or cytoplasmic organelles, resulting in gene, chromosomal or genomic mutations and hence, create variability. in this study, flow cytometry (fcm) was used to determine ploidy levels and dna content in gamma-irradiated variants of mutated pisang berangan (cv. intan, aaa) a local banana genotype. induced variants such as short plant stature (stunted growth), late flowering plants (late maturity) and abnormalities in bunch characters were selected to study possible changes at the dna level. the study showed that dna content of mutated plants differed from non-irradiated control and that irradiation had the most effect at high doses (40 and 60 gy). the increase of dna content in 20 gy and 30 gy treated plants was not more than that of the control plants. the values of genomic dna content of gamma-irradiation variants decreased as the dose of irradiation increased from 20 to 60 gy, indicating that the high dose of gamma-irradiation had a significant effect on the genome of the plants. the analysis further showed that phenotypic variation due to mutagenesis was reflected in the dna content of the plants. the results also showed that ploidy levels were not affected by gamma-irradiation even at high doses. keywords: musa spp./mutation breeding/ flow cytometry/ ploidy level/ dna content introduction banana is the most important fruit crop in the world (kaemmer et al. \ 997). it forms the primary food source for millions of people in many parts of the world and ranks next to rice and wheat (inibap 1992). the cultivated bananas are mostly triploid (2n=3x=33) and exhibit a marked degree of sterility. sterility is a main problem of banana breeding, which is very complex depending on meiotic irregularities (novak 1992). therefore, genetic improvement has been difficult due to lack of useful genetic variability and low level of female fertility. hence, the potential of biotechnological techniques to complement conventional musa breeding has been widely investigated (wiame et al. 2000; vuylsteke 1998; ortiz & vuylsteke 1996). these techniques involved in vitro multiplication, mutation breeding and genetic manipulation. popular clones planted in malaysia on a commercial scale are mainly cavendish bananas and pisang berangan cv. intan (aaa) (mak et al. 1995). of the total cultivated areas almost half are cultivated with pisang berangan and the cavendish types for both local consumption and export (siti hawa 1998). pisang berangan has good fruit quality, colour, texture, size and shelf-life, in addition to its ease of ripening without cold treatment. the plants are however, relatively tall with mode 47 biotropia no. 19,2002 rate yield and are very susceptible to a number of diseases particularly fusarium wilt caused by fusarium oxysporum f. sp. cubense. pisang berangan is a sterile tri-ploid banana; therefore, complementary techniques such as tissue culture and mutagenesis are of great potential. induced mutation in bananas is an attractive means to generate genetic variability. mutation induction coupled with in vitro techniques induces morphological changes and also increases variability in quantitative traits. physical mutagens, such as gamma ray, are tools for enhancing and generating genetic variations by inducing mutations at the gene, chromosome and genome level, in nuclear and cytoplasmic organelle dna (larkin 1998). somaclonal variation provides additional source of variation. off-types have been noted for plant stature (mainly dwarf), leaf foliage, pseudostem and bunch characters (small bunches, short fingers, long peduncle and elongated male buds). the dna nuclear content of various banana varieties were not explored or determined until the use of flow cytometry method for plant was introduced in the early 1980s. flow cytometric analysis of nuclear dna content provides an alternative for individual and largescale ploidy screening (dolezel 1991). the technique first developed for analysis of human cells has been adopted as a convenient tool for estimation of nuclear dna content and ploidy level constitutions in plants (dolezel 1991; dolezel et al. 1994; dolezel et al. 1997). dolezel et al. (1994) modified the technique for analysis of nuclear genome of bananas and plantains. the method that was used to screen ploidy levels of plants regenerated from in vitro culture was also used to estimate genome size and its variation in musa. this paper presents the results of a study to evaluate the variability in pisang berangan cv. intan (aaa) generated by gamma irradiation in terms of changes in nuclear dna content and chromosome level by flow cytometric analysis. materials and methods vigorous and disease-free suckers of pisang berangan (aaa) were selected from fieldgrown, true-to-type, fruit-bearing mother plants. shoot tip meristem (approximately 5x5x5 mm) was removed with a sterilized scalpel, then transferred directly to modified ms medium. subculturing was carried out after 4-6 weeks or earlier depending on blackening of explants. shoots initials developed after 3 weeks and were proliferated on induction medium with the bap concentration of 4.5 mg/l. multiplication of propagules was carried out by subdividing the newly formed shoots or bud clusters and reculturing them on fresh medium at 4-6 week intervals up to sub-culture 6. ms medium was then supplemented with 2mg/l 1aa and 5mg/l kinetin for rooting. root generation took 6-8 weeks until plantlets reached the optimal size of 4 -5 cm. the shoot-tip meristem pieces (about 1cm x 2mm) were aseptically excised from micropropagated plantlets of pisang berangan. each shoot-tip was cut longitudinally into two pieces and then transferred to sterile moist petri dishes. each 48 ploidy analysis and dna content of banana mutant — abdelgadir rayis shadia et al. petri dish contained 10 meristem pieces and was sealed with parafilm. gamma induction was then carried out at the malaysian institute of nuclear technology research (mint). the meristem pieces were irradiated in a gamma cell with a cobalt-60 (60co) source (gc 400a, 10 kci). the doses included 0 (control), 20, 30, 40 & 60 gy at a dose rate of 0.54 gy/sec. the irradiated meristem pieces were washed thoroughly with sterile distilled water and cultured on modified ms solid medium (ho & tan 1990). sub-culturing was carried out at 4 6 week intervals until generation m|v4 i.e. three sub-cultures. the explants after mivj were allowed to root in rooting medium containing ms salts. after three to four weeks in rooting medium, the plantlets were deflasked and transplanted in the nursery for acclimatization or hardening (8-9 weeks). the plantlets were maintained under 70% -80% shade (using shading net) and high humidity. these materials were generated for field evaluation at the farm. the plants were grown in rows spaced at 2.5m x 2.5m in a triangular pattern with completely randomized design. abnormal and mutant plants identified from different gamma-dose treatments (20, 30, 40, and 60 gy) were selected from in vitro plantlets, glass house-grown and field-grown plants. plant materials and sample preparation for determination of ploidy level and identification of nuclear dna content by fcm, nuclei were isolated from young leaves following -the method developed by gailbraith et al. (1983). approximately 40 50 mg of young leaves from each sample (mutated & glycine max var. palmetto) were chopped up together with a sharp scalpel blade in a glass petri dish containing 1 ml lboi lysis (dolezel 1991), supplemented with 50 ug/ml each of propidium-iodide (pi) and rnase. glycine max was used as internal reference standard for identifying the nuclear dna content by fcm. an intact interphase nucleus was released from the cut surface directly into 2-ml lysis buffer supplemented with 0.1 mg/ml of 4,6-diamidino-2-phenylindole (dapi). the homogenate-stained nuclei were filtered through 50 jam nylon mesh into the analysis tube and its fluorescence was analyzed by fcm. instrument setting and alignment the fluorescence intensity of dapi-stained nuclei was measured using a partec ca11 flow cytometer (partec gmbh, munster, germany), that uses high-pressure mercury arc lamp (hbooow/2l), as an excitation light source. these were filtered through a combination of optical filters: kgi, bg38 and ugi for excitation: tk560 as dichronic mirror, and a long-pass emission filter of rg590. autoclaved deionized water was used as a sheath fluid. sample analysis about 2 ml of the homogenate nuclei was analyzed in the cell counter analyzer cytometer (model partec cca-ii). the fluorescence was measured with a 100 w high-pressure mercury arc lamp as a light source filtered as mentioned above. 49 biotropia no. 19,2002 the dna genome size of the sample was then estimated using the ratio of gl peaks. the nuclear dna content was quantified by the following formula 2c nuclear dna content fpg] = 2.5/fluorescence ratio* * fluorescence ratio = peak mean of musa____ peak mean of glycine max symbol c is used for dna content of haploid set of chromosomes. glycine max has 2c dna content = 2.5 pg (tiersch et al. 1989). results and discussion mutagenic treatment may cause histogenic disturbances. at the molecular level, mutagens cause random changes in the nuclear dna or cytoplasmic organelles, resulting in gene, chromosomal or genomic mutations that create variability. the changes may be a result of alterations of the dna sequence of gene such as gain, loss or substitution of one base pair by another. therefore, monitoring of mutagen-induced dna damage is potentially very useful in mutation breeding studies. in this study, mutation induction was used to generate variability in pisang berangan (aaa). at high doses of irradiation, too many mutational events per cell may be induced, with increased risk that a favourable mutation is accompanied by one or more unfavourable genetic changes. however, in vegetatively propagated crops (bananas) it is impossible to separate favourable from unfavourable mutations by cross-breeding mutants among each other or by back-crossing with the original material. therefore, the evaluation of mutants is very difficult unless alternative techniques such as analysis of dna content or molecular fingerprinting are used. to a certain extent, the evaluation of mutants can also be phenotypically possible depending upon certain characteristics when compared with control plants. flow cytometry (fcm) was used in this study to determine ploidy levels and dna content in gamma-irradiated (pisang berangan) variants. the variants chosen for dna analysis include those with short plant stature (stunted growth), late flowering (or late in maturity) and abnormal bunch characteristic (table 1). the selection of short stature plants was based on two main categories, severely stunted and weak (plant height were < 75 cm) and short plants measuring 150-170 cm. while the selection of lateness in flowering was compared with control plants. the late flowering types ranged from 380 to 465 days. some plants showed more than one variant character such as short plants that were also late in maturity or even failed to bunching. the number of variants with bunch and finger abnormalities recorded was few and included those with short compressed bunch with few hands or combs (< 4), bunch with long peduncle and abundant persistent flowers at the distal end of the bunch stalk, bunch with male flowers only or bunch with gaps (missing combs), fused fingers and long and thin fingers. ploidy analysis and dna content of banana mutant abdelgadir rayis shadia et at. flow cytometric analysis for ploidy and dna content the most reliable conventional method of ploidy level analysis is the counting of chromosomes of metaphase plates by using root tips. however, the preparation and microscopic analysis is time consuming, and a difficult task as banana chromosomes are extremely small, indistinguishable and frequently few cell divisions are visible in a single root tip. this has led to the development of flow cytometric techniques for rapid routine ploidy determination in musa (dolezel et al. 1994). in addition, flow cytometry is recommended for the accurate estimation of nuclear dna content (novak 1992; dolezel et al. 1989). flow cytometry involves the analysis of intact nuclei, hence dividing cells are not required, and the analysis is not limited to meristematic tissues. moreover, a small amount of fresh leaf tissue (5 50 mg) is sufficient, that means this method could be applied to plants at the early stage of development. analysis of the relative fluorescent intensity of propidium-iodide stained nuclei yielded a histogram showing two dominant peaks corresponding to g\ and g2 nuclei of pisang berangan and glycine max cv. palmetto, respectively. the majority of cells in full-grown plants does not participate in cell division and reside in a so-called go (g is gap) stage of the cell cycle. at this stage, the nuclear dna content reflect the ploidy state of the plant. cells, which are involved in divisions, start from a comparable so-called gi state and subsequently pass through s (= dna-synthesis), g2 (= an interphase nuclear stage with a doubled dna content preceding the actual nuclear division). accordingly nuclear dna content in absolute units (genome size in picogram-pg dna) was adopted for all samples used. such estimation requires comparison with a reference standard having a known dna content. in this study, glycine max was used as an internal standard because its genome size is relatively constant (dolezel et al. 1994, vinderlov et al. 1983) and to avoid bias due to staining and instrumental changes when estimating nuclear dna 44 biotropia no. 19,2002 ploidy by flow cytometric analysis. the precision of the ratio between the standard and the sample (pisang berangan) reflects the accuracy of dna content measurements. estimation of ploidy level was done by external standardization procedure comparing the position of the g| peak on a histogram to that of a reference plant with known ploidy. the results showed no significant differences in ploidy level between all samples used (3n), indicating that irradiation at the. doses used did not have an effect on ploidy. differences were, however, found between gamma-irradiated berangan variants with respect to dna content. dolezel (1995) reported similar findings in his analysis of a large populations of nuclei (5-2000) of mutated african plantains in which the occurrence of even small subpopulations differing in ploidy levels (mixoploidy) can be detected. the applications envisaged include control of ploidy level stability and screening for novel ploidy levels. figure (1) shows flow cytometric analysis of genomic dna content of pisang berangan nuclei and from the reference (glycine max cv. palmetto) stained with propidium iodide (pi). the nuclei isolated from musa and glycine, were analyzed simultaneously. most of the nuclei were in g, phase of the cell cycle. the first peak (channel 75) represented g, nuclei of musa, while the second peak represented g, nuclei of glycine. g, peak of pisang berangan was positioned on channel 75, and the other peak of glycine max was on channel 100. a third peak also appeared at channel 200 reflecting the g2 phase of the reference internal standard nuclei. ploidy analysis and dna content of banana mutant abdelgadir rayis shadia i et al. a measurement for the resolution of the histogramscoefficient of variation (cv)shows the variation of the analysis from the control values. overall means of g] peaks of pisang berangan ranged from 1.3% to 2.34%, whereas in glycine plants, it ranged from 1.02% to 1.67%. the estimated values of peak ratio of these samples could be used to discriminate between gamma-irradiated variants and non-irradiated plants. the peak appeared in the histogram, g[ for pisang berangan and g| for glycine max, indicated that soybean has more dna content than berangan. higher cv in berangan indicated that variation due to mutagenesis occurred within these samples. the discrepancies between observed and expected values could indicate the occurrence of changes in dna content due to effects of gammairradiation. all selected variants (short stature, late flowering and bunch abnormalities) showed dna content different from control plants. to investigate the significance of differences between observed frequencies arranged in these three characters the use of contingency x2-test for independence was performed. the test showed that there is a differential effect of gamma irradiation on these characters, but the two sets of variables are independently acting. dna content of different types of variants induced by gamma-irradiation (mutants) all selected mutants (short stature, late flowering and bunch characteristics) showed variation in dna content. an effect of irradiation appears to be reflected by an increase in variation of nuclear dna content (increase of coefficient of variation of g| and g2 peaks). the mean of the distribution of g0/g, nuclei represents a 2c (presynthetic level) dna amount in relative units. the ratio of g0/ g, peak means (musal glycine) was calculated and dna content of musa was estimated according to the formula: dna content (pg) = 2.5/peak ratio (dolezel et al. 1994). the ratio of gl peak means for non-irradiated control plants was gi of glycine/ g, of musa i.e. 100/75 = 1.333. the 2c dna content of pisang berangan (aaa) was calculated using the ratio of gl peak means: 2.5/1.333=1.875 pg dna or 1.875 ± 0.02 pg. the estimated value of peak ratio of these plants did not differ from the mean value obtained by fcm analysis (1.85 pg). the estimated value of dna content (pg & mbp) for different variants chosen from each group is shown in table 2. short plant variants (gamma-irradiated at different doses) displayed a wide range of dna content (1.0% 8.0%) as compared with non-irradiated plants (control). the variants showing lateness in flowering showed moderate changes in dna content as compared to short stature variants (0.8% 2.7%). bunch abnormalities variants showed slight to moderate changes in dna content as compared to control plants with dna content differences ranging from 0.1% to 1.1%. the mann-whitney test (non-parametric and distribution-free statistics) showed that there were no significant differences between the three characters in 53 biotropia no. 19,2002 dna content at (0.005 >p >0.001). on the other hand, phenotypic variation due to mutagenesis is reflected in the dna content of plants. table 3 demonstrates that the degree of variation in dna content values increased as the dose of gamma-irradiation was increased from 20 gy through to 60 gy. the dna content at 20 & 30 gy treatments differed from that of nonirradiated plants by 2.5% and 2.8%, respectively. whereas, 40 gy and 60 gy-variants showed more changes (4.7% and 4.5% for 40 gy & 60 gy treatments, respectively) (table 3). 54 ploidy analysis and dna content of banana mutant — abdelgadir rayis shadia et al. the relationship between short stature variants and high doses of irradiation was apparent. the majority of short stature or stunted growth variants were selected from 40 & 60 gy treatments. (table 1). other variants such as late maturity and bunch-abnormalities also showed decrease in genomic dna content ranging from 2% to 4%, suggesting that the variation in dna content has phenotypic effects via its influence on cell size and mitotic cycle time (van harten 1998). the results indicate that gamma ray has a direct effect at the dna level of pisang berangan and that this variation can be effectively measured using the flow cytometric technique. the high range of phenotypic changes observed at 40 gy and 60 gy treatments might be due to broken segments lost or translocated or may originate from deletions in the nucleotide sequences in structural genes, changes in the promoter sequences, deletions of the introns, regulating gene and repressers, which may either cause frame shift mutations or lead to the production of modified gene expression. it is estimated that at least 90% of the radiationinduced mutations refers to deletions (van harten 1998). in addition, duplication may occur resulting in an increase in the amount of dna content control (insertion). this phenomenon might be due to jumping genes or double replication in dna (copied twice) induced by irradiation. this technique has immediate application for selection of potentially useful mutants for pisang berangan breeding programme. it may be expected that the number of practical applications will increase and plant breeders will even more extensively use flow cytometry. acknowledgements i would like to express my gratitude to the malaysian institute for nuclear technology research (mint) and mr. azhar mohamad for the facilities and assistance throughout the duration of experiment. also to prof. t. k. mukheraji (institute of biological science) for his advice in statistical analysis. references dolezel, j. p. binarova and s. luretti. 1989. analysis of nuclear dna content in plant cells by flow cytometry. bio. plant. 31: 133-120. dolezel, j., m.dolezelova and f. novak. 1994. nuclear dna amount in diploid bananas (musa accuminata and m.balbisiana). biol. plant. 36: 351-357. dolezel, j. m. a. lysak, i. van den houwe, m. dolezelova, n. roux. 1997. use of flow cytometry for rapid determination in musa species, infomusa. 6: 6-9. dolezel, j., 1991. flow cytometric analysis of nuclear dna content in higher plants phytochem. analysis 2: 143154. 55 biotropia no. 19,2002 dolezel, j. 1995. application of karyology and cytometry in mutation breeding of african plantain in vitro. fao/iaea research co-ordination meeting on: cellular biology and biotechnology including mutation techniques for creation of new useful banana genotypes. fao/iaea, 1995, p. 13-22. gailbraith, d. w., k. r. harkins, j. m. maddox, n. m. ayres, d. p. sharma and e. firoozabady. 1983. rapid flow cytometric analysis of the cell cycle in intact plant tissues. science, 220: 1049-1051. ho, y. w. and y. p. tan. 1990. performance of tissue cultured bananas. perak planters annual report 1990: 6775. inibap, 1992. banana and plantainfood for thought. in: annual report inibap, montpellier, france. kaemmer, d., d. fisher, r. l. jarret, f. c. baurens, a. grapin, d. dambier, j. l. noyer, c. lanaud, g. kahl and p. j. l. lagoda 1997. molecular breeding in the genus musa: a strong case for stms marker technology. euphytica 96: 46-63. larkin, p. j.. 1998. induced mutation for crop improvement. in: somaclonal variation and induced mutations in crop improvement, (eds) by jain, s. m.; d. s. brar and b. s. ahloowalia (1998). p 3 13. mak c., y. w. ho, y. p. tan and r. ibrahim. 1995. novariaa new banana mutant induced by gamma irradiation. infomusa 4: 1. novak, f.j., 1992. musa (banana and plantains) in: biotechnology of perennial fruit crops, p.449-488 (eds f.a. hammerschlag and r.e litz) wallingford: c.a.b. international. ortiz, r. and d. vuylsteke. 1996. recent advance in musa genetics, breeding and biotechnology. plant breeding abst. 1996,66: 1355-1363. siti hawa, j. 1998. commercial exploitation of the banana diversity in malaysia. proceedings of the first national banana seminar (genting highland) 23-25 nov. 1998. tiersch, t. r., r. w. chandler, s. s. watchel and s. elias. 1989. reference standards for flow cytometry and application in comparative studies of nuclear dna content. cytometry, 10: 706-710. van harten, a. m., 1998. mutation breeding. theory and practical applications, cambridge univ., p. 163-203. vinderlov l. l., i. j. christensen and n. i. nissen. 1983. standardization of high-resolution flow cytometric dna analysis by the simultaneous use of chicken and trout red blood cells as internal reference standards. cytometry 3: 328-331. vuylsteke, d. r., 1998. field performance of banana micropropagules and somaclones. in: somaclonal variation and induced mutation in crop improvement (eds) by s. m. jain; d. s. brar; and b. s. ahloowalia) p. 219-231. wiame i., r. swennen, l. sagi, plas-lhw-van-der, j. j. dons, j. vanderleyden and m-de loosse. 2000. pcrbased cloning of candidate disease resistance gene from banana (musa acuminata). in: proceedings of the xxv international horticultural congress. part ii. application of biotechnology & molecular biology and breeding, genome analysis, brussels, belgium, 2-7 august, 1998. acta-horticulturae, 2000. 521,51-57. 56 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf 53.pdf 54.pdf 55.pdf 56.pdf microsoft word 29 bio fropia no. 22, 2004: 29-39 isolation and selection of alkaline proteolytic bacteria from leather processing waste and enzyme characterization budiasih wahyuntari', nisa r mubarikand marita anggarani2 agency for the assessment & application of technology, jakarta, indonesia ²bogor agricultural university, bogor, indonesia abstract the aims of this experiment were to isolate alkaline protease producing bacteria from leather processing waste, and to study the biochemical properties of the enzyme produced by the selected bacteria. nine bacterial isolates incubated at 37"c, revealed proteolytic activity on skim milk containing media. four isolates were grown at ph 9 and another four isolates at ph 10 and only one isolate at ph 11. however, in further subculture, there were only three isolates that showed proteolytic activity, namely, d2, d7, and dl l . among the three isolates, isolate d2 was the highest protease producer. the highest protease production (36.5u/l) was reached after a 36-hr fermentation at ph 9. the optimum activity of d2 protease was observed at ph 8 and 60"c. the enzyme was stable at ph range of 7-10, and at temperature of 52-62"c. in the presence of 5mm edta or pmsf, the crude enzyme activity decreased to 7.04% and 23.29% respectively, which indicated that the enzyme might be a metal dependent serine protease. zymogram analysis revealed the molecular weight of the enzyme was about 42.8kd. keywords: leather/ waste/protease/alkaline introduction alkaline protease, along with other alkaline enzymes such as amylase and cellulose, has been used on an industrial scale. microbial akaline protease is the enzyme that dominate commercial application, mainly for laundry detergents. alkaline protease with elastolytic and keratolytic activity is used in the tannery industry for dehairing and the bating of skin and hides (taylor et al. 1987). alkaline protease from bacillus spp. was reported to be used to decompose gelatin on x-ray film for silver recovery (horikoshi 1996). other uses of alkaline protease are for medical purposes, food and chemical industries, as well as in waste treatment plants. alkaline protease has been used in industrial scales and has been an attractive option for researchers for the last three decades since the first publication concerning the enzyme by horikoshi (1971). studies on exploration of alkaline protease producing microorganisms, which produce novel enzymes have been extensively carried out by researchers all over the world (banerjee et al. 1999; singh et al. 2001; johnvesly & naik, 2001; kanekar et al. 2002, joo et al 2002; gessesse et al. 2002; moreira et al. 2003). 29 biotropia no. 22, 2004 most of the alkaline protease producing microorganisms were isolated from natural, as well as manmade alkaline environments. such microorganisms were also isolated from non-alkaline habitats, such as neutral, and acidic soil (kumar et al. 1999). this experiment aims to isolate alkaline protease producing microorganisms from alkaline waste of leather processing and to study the biochemical properties of the enzyme of the selected isolate. materials and methods isolation, screening and identification of microbial strains the samples for screening were taken from the waste of different leather processing steps including soaking (ph 10), liming (ph 13), and deliming (ph 8.5-9.0) from pt. muhara dwi tunggal laju, and pt. gunung putri, in bogor. the microorganisms were enriched on agar plates containing (gram per liter) 2 gr yeast extract, 0.4 gr (nh4)2so4, 1 gr mgso4.7h2o, 0.5 gr cacl^.2 h2o, 0.8 gr kh2po4, 40 gr agar, and 8 gr skimmed milk. the ph medium was adjusted to 8-11 using naoh. the microorganisms were incubated at 24-40°c for 3 days. the colonies which revealed proteolytic activity were then isolated and screened further, using the same composition of medium. the isolates were screened based on proteolytic index which defined as a ratio of clear zone and colony diameter. the isolates that had the highest proteolytic index were screened further for protease production in luria broth containing 1% peptone, 0.5 gr yeast extract and 0.5% nacl at ph 9, 10 and 11. in the same medium composition, they were then incubated in a shaking incubator for 19 hours at 37°c. the fermented broth was then centrifuged at 4000 rpm for 20 minutes. the protease activity was finally assayed and the isolates were selected based on the protease production. protease assay protease activity was measured using a method suggested by walter (1984). the method was based on amino acid hydrolyzed by the enzyme and was calculated as tyrosine. one unit of enzyme activity was defined as the amount of enzyme which liberate one micromol tyrosine per minute at certain condition. preparation of protease the selected isolate was cultured in 5 l fermentor containing 2.5 l enzyme media production with the same composition as mentioned above with addition of 0.2% skim milk as an inducer. after a 36-hr incubation at 37°c, the fermented broth was filtered using microfilter, and then concentrated further using 10 kd membrane ultrafilter module (minitan, milipore). 30 isolation and selection of alkaline proteolytic bacteria budiasih wahyuntari et al. effect of ph and temperature studies the effect of ph on protease activity produced by the selected isolates was determined at 37°c in 50 mm tris-hcl buffer at ph 7-8 and in glycine-naoh at ph 9-11. to determine the effect of temperature on protease activity, the assay was performed in 50 mm tris-hcl buffer (ph 8) at temperature range of 30-70°c. effect of protease inhibitors and metal ions the concentrated enzyme solution was incubated in 2 and 5mm edta or pmsf in 50 mm tris-hcl at ph 8 for 30 minutes at 5°c. the same concentration of 2 and 5 mm mgcl2, cacl2 , zncl2 , mncl2 , fecl3 and feso4 were also incubated with the enzyme at 5°c for 30 minutes. residual activity was measured under optimum ph and temperature. protease activity assayed in the absence of inhibitors and metal ions was considered as 100%. polyacrylamide gel electrophoresis and zymogram analysis zymogram method was employed to determine the molecular weight of the protease. the concentrated enzyme solution was loaded into page containing skimmed milk (0.2%) which copolymerized with 7.5% acrylamide. electrophoresis was run at 110 v, 90 ma for 1.5 hours. after completing electrophoresis, the gel was soaked in 50 mm tris-hcl containing 5 mm cacl2 then incubated at optimum ph and temperature of the enzyme. visualization of the enzyme staining activity was carried out by staining the gel in commassie brilliant blue solution. clear band on the skim milk containing electrophoresis gel indicated the protein band which had shown proteolytic activity. the standard molecular weight marker used was hmw protein standard marker (amersham). results and discussion isolation of protease-producing bacteria there were 9 isolates which showed proteolytic activity based on clear zone formation on skim milk containing media. four isolates grew well on media at ph 9m, another four isolates at ph 10, and only one isolate at ph 11. table 1 shows the physical properties, proteolytic index, and protease activity of the isolates. proteolytic activity of isolate t4 decreased in the following experiment, and therefore the isolate was excluded. isolate d2, d7 and dl l were selected based on their proteolytic index and ability to produce protease in submerged culture. biochemical characteristics of the selected isolates are displayed in table 2. based on their physical, and biochemical characteristics, it can be concluded that the isolates (d2, d7 and dll) most likely belong to bacillus spp. 31 selection of bacterial strain. further selection of the best proteolytic bacteria was based on the productivity of the isolate at die highest ph. all the isolates screened were grown at three different levels of ph (9; 10 and 11). however, all isolates did not grow well at ph 32 isolation and selection of alkaline proteolytic bacteria budiasih wahyuntari el al. 11. figures 1, 2 and 3 reveal growth and protease production rates of d2, d7, and dl l , respectively. the growth rate of isolate d2 was only slightly better at ph 9 as compared to ph 10. however, its protease production rate at ph 9 was higher than that at ph 10. the highest protease production of isolate d2 was at 36-hour fermentation with a protease concentration of 3.65 u/l. the growth rate of isolate d7 at ph 9 was slightly better than at ph 10. however, the protease production rate was higher at ph 10 after 36 hours incubation with the activity of 10.6 u/l. figure 3 shows that the growth rate and protease production of isolate dl 1 was better at ph 9 and with the highest protease production at 24 hour incubation time with enzyme activity of 5.8 u/l. the protease produced by all isolates in skim milk containing medium was much higher as compared to enzyme produced in luria broth used in early selection of isolates. the data indicated that the enzyme produced by all isolates observed were inducible ones. among the three isolates observed, isolate d2 produced the highest protease, which showed that the isolate was the most tolerable to alkaline condition. it was assumed that the enzyme belonged to alkaline protease family. therefore, t he enzyme of the isolate was selected for further study.   isolation and selection of alkaline proteolytic bacteria budiasih wahyuntari et al. effect of ph and temperature study on protease activity optimum activity of d2 isolate protease was observed at ph 8 and 60uc (figure 4). the enzyme relative activity was 70% at ph 7 and 9.5 and at 52 and 62°c. these data show that the enzyme is an alkalo and thermotolerant one. studies on isolation and characterization of alkaline protease have been ongoing for the last three decades (horikoshi 1996 and kumar and takagi, 1998). these are the most recent studies on the subject. singh et al. (2001) reported that optimum activity of bacillus sp. ssr1 was reached at the range of ph 8-11, 40°c, the optimum activity was shifted up to 45°c due to the presence of calcium ions. thermostable alkaline protease from bacillus sp. jb-99 reported by johnvesly and naik (2001) had an optimum activity at ph 11 and 70°c. this bacterium was isolated from sugarcane molasses at ph 5.8. but with a chemically defined medium, the bacterium was able to produce alkaline thermostable protease. gessesse et al. (2002) succeeded in isolating two bacterial strains from lake abjata (an alkaline soda lake in ethiopia), using chicken feather meal as the sole source of nitrogen and carbon. the bacteria were identified as nestemkonia sp. al-20 and b. pseudofirmus al-89. the optimum enzyme activities of al-20 and al-89 were observed at ph 10, 70°c and at ph 11, 50°c, respectively. the al-20 protease was calcium independent whereas the al-89 was calcium dependent. both enzymes were indicated as serine protease. bacillus, horikoshii protease had optimum activity at ph 9, 45"c, and the enzyme was stable at the ph range of 5.5-12 and temperature up to 50°c (joo et al, 2002). effect of protease inhibitor and divalent ions the presence of 5 mm phenyl methyl sulfonyl fluoride (pmsf) reduced the protease activity down to 23.3% which indicated the enzyme belongs to serine protease. five mm eot a almost totally inhibiting the enzyme with a remaining activity of 7% only, which showed that the enzyme activity depends on the presence of metal ion. the study on the effect of several metal salts revealed that calcium, zinc and manganese ion increased the activity of the enzyme up to 117.2%, 111.3%, and 114.4%, respectively. whereas, magnesium, ferry and ferrous ions at 5 mm decreased the enzyme activity down to 91.9%, 64.8%, and 71.3%, respectively. previous studies on alkaline proteases reported that for maximum activity, some serine alkaline proteases were dependent on divalent ion such as calcium, magnesium and manganese (kumar & takagi, 1998). however, gessesse et al (2002) reported that serine alkaline protease activity produced by nesternkonia sp was not dependent on the presence of calcium ion. the enzyme was not affected by pmsf, but, it was inhibited by another serine protease inhibitor 3,4-dichlorocoumarine at concentration of 100 (j.m,. 36 isolation and selection of alkaline proteolytic bacteria budiasih wahyuntari et al. control edta pmsf mg2+ ca2+ zn 2+ mn 2+ fe 3+ fe 2+ inhibitor/cation d : 2 mm, • : 5 mm figure 6. et'fecl of protease inhibitor and cation on enzyme activity molecular weight of the enzyme zymogram analysis revealed that the molecular weight of the enzyme was approximately 42.8kd (figure 7). that molecular weight of most bacillus alkaline proteases reported ranges from 15 to 45 kd (kumar & takagi, 1998). a more recent report by singh et al (2001) also stated that the molecular weight of serine alkaline protease produced by bacillus sp ssr1 was 29kd which was also lower than the previous reports . figure 7. zymogram otisolate d2 protease 37 biotropia no. 22, 2004 conclusion in this exploration of protease-producing bacteria from leather processing waste, only few bacteria that produce alkaloprotease were discovered. among the three isolates screened. isolate d2 produced the highest protease in submerged culture medium. based on physical and biochemical properties of the isolate, d2 most likely belongs to bacillus sp. the optimum activity of the enzyme produced was observed at ph 8, 60°c. the enzyme retained 70% of its activity at the range ph of 7-10. at ph 8, the enzyme also retained more than 70% of its activity at the range temperature of 52-62°c. the enzyme was identified as serine protease and its activity was affected by the presence of calcium, zink and manganese ion. the molecular weight of the enzyme as determined using zymogram analysis was approximately 42.8 kd. further investigation is needed to identify the isolate using more reliable means such as 16rrna sequencing the enzyme can be used in a condition that is slightly alkaline at around ph 8 and at 50-60°c. however, to find suitable application of the enzyme, other considerations have to be taken into account such as type of substrate of the respected process. hence, it is necessary to investigate different kinds of protein substrates such as hemoglobin, collagen, gelatin, keratin etc. selecting the best medium to produce the enzyme is also necessary, as well as more thorough characterization of the enzyme such as the stability of the enzyme toward ph, temperature, and some additives that might be used in enzyme application. references banerjee uc, rk sani, w azmi and r soni. 1999. thermostable alkaline protease from bacillus brevi.t and its characterization as a laundry detergent additive, process biochem. 35:213-219. gessesse a, hk rajni, ba gashe arid b mattiasson. 2002. novel alkaline proteases from alkaliphilic bacteria grown on chicken feather. enzyme & microbial tech. 6250:1-6. horikoshi, k, 1971. production of alkaline enzymes by alkalophilic microorganisms. part 1. alkaline protease produced by bacillus no. 221. agric. biol. chem. 36:1407-1414. horikoshi k, 1996. alkaliphiles from an industrial point of view, eems microbiol riev, 18:259-270. johnvesly b. and gr naik. 2001, studies on production of thermostable alkaline protease from thermophilic bacillus sp jb-99 in a chemically defined medium, process biochem. 37:139-144. joo hs, sg kumar, gc park, kt kirn, sr paik and cs chang. 2002. optimization of the production of an extracellular alkaline protease from bacillus horikoshii, process biochem, 38:155-159. kanekar pp, ss nilegaonkar, ss sarnaik and as kelkar. 2002. optimization of protease activity of alkaliphilic bacteria isolated from an alkaline lake in india, bioresource tech. 85:87-93. kumar cg, and h takagi 1998. microbial alkaline proteases: from a bioindustrial viewpoint, biochem adv, 17:561-594. 38 isolation and selection of alkaline proteolytic bacteria budiasih wahyuntari et al. moreira ka, ts porto, mfs teixeira, alf porto and jl lima filho. 2003. new alkaline protease from nocardiopsis sp partial purification and characterization, process biochem.oo: 1-6 . singh j, n batra, and rc sobti 2001, serine alkaline protease from newly isolated bacillus sp ssr1, process biochem, 36:781-785. taylor mm, dg bailey and sh feaiheller. 1987. a review of the use of enzymes in tannery, j. ame leather chem assoc, 82:153-165. walter he. 1984. proteinases in methods of enzymatic analysis, (ed. h.u. bergmeyer and m qrassl), verlag chemie. weinheim-deerfield, florida-basse!, vol. 5:271-276. 39 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf biotropia no. 4, 1990/1991: 49-56 a study on weed control in soybean s. tjitrosemito tropical agricultural pest biology programme, seameo biotrop, bogor, indonesia abstract two field experiments on weed control in soybeans were carried out at biotrop, bogor, indonesia from february to june, 1989. the critical period for weed control was found to be between 20 40 days after planting of soybean (c. v. wilis) grown at a planting distance of 40 x 10 cm. it did not coincide with the fastest growth in terms of trifoliate leaf number. further studies were suggested to understand the physiological growth of soybean related to weed control. pendimethalin at 6601320 g a.e./ha applied one day after sowing did not cause any phytotoxic effect to soybean and had good weed control performance. introduction with the establishment of the soybean yield gap analysis project (sygap) in indonesia (douphin et al. 1986), about 34.5 million ha were identified to be suitable for soybean planting (pasaribu & mclntosh 1986). it was assumed, therefore, that it was adequate area-wise but when yield/ha was considered, it was still very low (0.7-0.8 ton/ha; somaatmadja 1983). this low yield may be attributed to various factors like (1) poor stand, (2) poor growth, (3) weed problems, (4) excess water, (5) empty pods due to insect damage or drought (pasaribu & mclntosh 1986). fachurrozi et al. (1988) surveyed the production system of soybean at farmers level in east java (district of pasuruan). they reported that weeding was the most labour intensive operation. it took 51 % and 65% of the total work hours and 32% and 34% of the total cash production costs in two villages of oro-oro pule and sumberbanteng. however, although the cost of weeding was high, their studies indicated that yield could be increased by controlling weeds, pests and diseases. twice weeding resulted in marginal rates of return (mrr) of 118 147%, whereas mrr of controlling pests and diseases was 1012 — 1454%. these figures showed how beneficial the control of "pests" (insect, pathogens, weeds, vertebrate pests) was. recent work indicated that soybean crops planted at various planting distances from 30 x 10 cm to 40 x 20 cm could tolerate infestation of weeds up to 30% of sdr (tjitrosemito 1987). the growth of soybean was, however, not optimal as was also reported by pasaribu and mclntosh (1986). in this context, it is appropriate to emphasize that weed control (weed management) is an integral part of the whole production management. the success of weed control is to be judged also from its ability to increase yield with sufficient profit. 49 biotropia no. 4, 1990/1991 material and methods experiment 1 a field experiment was conducted at biotrop, bogor from november 1988-february 1989 to determine the critical period of weed control on soybean crops (c.v. wilis). fourteen treatments consisting of various manual control schemes were applied in plots measuring 4 x 5 m 2 replicated 3 x with randomized block design. the fourteen treatments were: 1. manual weed control from planting up to 10 days after planting (dap) 2. manual weed control from planting up to 20 dap 3. manual weed control from planting up to 30 dap 4. manual weed control from planting up to 40 dap 5. manual weed control from planting up to 50 dap 6. manual weed control from planting up to 60 dap 7. manual weed control from planting up to harvest 8. manual weed control from 10 dap to harvest 9. manual weed control from 20 dap to harvest 10. manual weed control from 30 dap to harvest 11. manual weed control from 40 dap to harvest 12. manual weed control from 50 dap to harvest 13. manual weed control from 60 dap to harvest 14. no weeding at all. the plots were manually cultivated before planting and fertilized at the rate of 45 kg n/ha, 50 kg p2o5/ha and 50 kg k2o/ha, applied by broadcasting along the planting rows. the planting distance was 40 x 10 cm. insect pest control was done by spraying azodrin 60 wsc at 2 ml/it solution; while dithane m-45 at 2 g/1 was used against pathogens. the soybean was harvested at 98 dap; and the yield was compared statistically using lsd at 5%. weeds were sampled before harvest using a quadrat of 50 x 50 cm 2x in each plot. the growth of soybean was recorded in terms of trifoliate leaf number. experiment 2 another field experiment was also conducted at biotrop from march-june, 1989. there were 12 treatments applied in plots measuring 2.5 x 10 m replicated 3x in a randomized block design. this experiment was a repetition of 50 a study on weed control in soybean-s. tjitrosemito the one of previous year, since some herbicidal treatments showed high crop mortality. the treatments were: 1. untreated plot 2. imazethapyr (i) : pre-emergence : 50 g a.e./ha 3. imazethapyr (i) : pre-emergence : 75 g a.e./ha 4. imazethapyr (i) : pre-emergence : 100 g a.e./ha 5. imazethapyr (i) : pre-emergence : 150 g a.e./ha 6. imazethapyr (i) : early post : 100 g a.e./ha 7. pendimethalin (p) : pre-emergence : 660 g a.e./ha 8. pendimethalin (p) : pre-emergence : 1320 g a.e./ha 9. p + 1 : pre-emergence : 660 + 50 g a.e./ha 10. p + 1 : pre-emergence : 1320 + 50 g a.e./ha 11. alachlor : pre-emergence : 1440 g a.i/ha 12. manual weeding : at 3 and 6 weeks : the spraying of herbicides was done using a cp 15 knapsack sprayer, calibrated to deliver 400 1 solution/ha, using yellow nozzle at high pressure. the pre-emergence spraying was done one day after planting. the samplings were carried out using a quadrat measuring 50 x 50 cm 2 . the plots were manually cultivated and fertilized with 60 kg p2o5/ha; 45 kg n/ha and 50 kg k2o5/ha at planting time, broadcast along the soybean row. to prevent damage to seedlings of o. phaseoli, furadan was utilized. the soybean was harvested at 98 days after planting. results and discussion the soybean yield from experiment i is shown in table 1. when expressed in the graph (figure 1) it indicates that the critical period of weed control is between 20 and 40 dap. lengthening the weed free period from 10 to 40 dap increased the yield i.e. from 0.62 ton/ha to 1.36 ton/ha, but further lengthening the period of control did not increase the yield any more. leaving the field unweeded for a period of 10 20 dap did not really matter, but leaving more than 20 days unweeded reduced the yield considerably i.e. from 1.54 ton/ha to 1.33 ton/ha. when weeding was done only 40 dap the yield was further reduced. the weed infestations were presented in table 2 (weed count) and table 3 (dry weight). the infestations of borreria alata, ageratum conyzoides, eleutheran 51 biotropia no. 4, 1990/1991 table 1. the mean yield of soybean at various control schemes treatment ton/ha 1. weeded up to 10 days after planting (dap) 0.62 2. weeded up to 20 dap 0.92 3. weeded up to 30 dap 1.25 4. weeded up to 40 dap 1.36 5. weeded up to 50 dap 1.36 6. weeded up to 60 dap 1.35 7. weeded up to harvest 1.50 8. weeded from 10 dap to harvest 1.50 9. weeded from 20 dap to harvest 1.54 10. weeded from 30 dap to harvest 1.33 11. weeded from 40 dap to harvest 1.26 12. weeded from 50 dap to harvest 1.05 13. weeded from 60 dap to harvest 0.85 14. no weeding at all 0.65 lsd (5%) 0.14 cv 10% figure 1. the effect of "weed free" and "weed present" periods on yield of soybean. the critical period appeared to be between 20-40 dap. 52 a study on weed control in soybean-s. tjitrosemito table 2. mean weed count/50 x 50 cm2 at various weeding schemes (average of 3 blocks) b.a. : borreria alata (aubl.) dc a.c. : ageratum conyzoides l. e.r. : eleutheranthera ruderalis sch. m.sp. : mitracarpus sp. c.r. : cyperus rotundus l. d.c. : digitaria ciliaris (retz) koel. others include : celosea argantea leucas lavandosalia stachytarpheta jamaicensis euphorbia geniculata oxalis sp. phyllantus niruri l. cleome rutidospermum dc. emelia sonchifolia tridax procumbens eleusine indica thera ruderalis, digitaria ciliaris, and to some degree also cyperus rotundus were considerable when not weeded and they constituted the main weeds in the area. the growth performance of soybean is presented in figure 2. early in the growth stage, the growth was slow especially in the first 2-3 weeks in terms of leaf number. this was the period when the presence of weeds was not felt by the crops. from 3 to 6 weeks (20 40 dap) the growth was still slow compared to the period between 40 to 60 days when growth was from 9 to 16 leaves/plant. however, at 60 dap, the crop started to shed the leaves and the number of leaves went down. 53 biotropia no. 4, 1990/1991 table 3. mean dry weight of weed grains/50 x 50 cm2 at various weeding schemes (average of 3 blocks) figure 2. the growth performance of soybean (c.v. wilis) in terms of number of trifoliate leaves. 54 a study on weed control in soybean-s. tjitrosemito probably, availability of resources in the period of 20 40 dap was necessary to support the rapid growth in the following period. as pointed out by spitters and van den bergh (1982), once nutrients or moisture were taken up by the competing weeds, they were not available anymore to the crop. further works are certainly needed to really understand the physiological growth of soybean and its relation to weed control the soybean yields in the experiment are presented in table 4. contrary to the result of the previous year (tjitrosemito 1988) soybean treated with pendimethalin did not show any phytotoxicity with the pre-emergence spraying done one day after planting. at the same time, the control of weed was better, thus facilitating a higher yield. as was reported earlier (tjitrosemito 1988) borreria alata was the main weed. pendimenthalin at 1320 g a.e./ha was good in controlling digitaria ciliaris. table 4. the mean of soybean yield (ton/ha) under various weed control treatments references dauphin, f., j.w.t. bottema and a. rachim. 1986. soybean yield constraints, socio-economics or technologic. palawija news iii (2): 1-2. fachrurrozi, q. j. laumans and r. krisdiana. 1988. production economic aspects of soybean grown on legal: a case study in pasuruan district east java. penelitian palawija 3 (2): 105-115. s omaatmadja, s. 1983. development of soybean culture in indonesia in: international symposium on soybean in the tropics and substropics. tropical research center, ministry of agriculture, forestry and fishery japan. tropical agricultural research series no. 17: 23-35. 55 biotropia no. 4, 1990/1991 pasaribu and me. intosh. 1986. soybean production in indonesia. soybean in tropical and subtropical cropping systems. proc. symp. tsukuba, japan 26 sept.1 oct. 1983. avrdc: 1-11. spitters, c.j.t. and j.p. van den bergh. 1982. competition between crop and weeds. a system approach in: holzner and numata (eds) biology and ecology of weeds: 137-148. tjitrosemito, s. 1987. threshold level of weed control in soybean crop for small farmers. proc. xlth apwss conf. i: 247-258. ______, 1988. weed control on soybean crop using imazethapyr. paper presented to the second trop. weed conf. phuket, thailand. december 6-10, 1988. 56 49.pdf 50.pdf 51.pdf 52.pdf 53.pdf 54.pdf 55.pdf 56.pdf microsoft word 40 biotropia no. 22, 2004: 40 58 medicinal herbs of pasir mayang, jambi: ethnopharmacy and toxicity screening hilman affandi*, arif nuryadin* and susilo b. prayogo* *seameo biotrop, jl. raya tajur km. 6 bogor, indonesia abstract this article presents the results of an investigation concerning the use of herbal medicinal plants by the people of pasir mayang, sub-district (municipality) of vii koto, district of tebo, jambi province, sumatera. the data collection was based on interviews with the healers and other villagers of pasir mayang who possess knowledge of the different plants and their medicinal uses. the study recorded 57 species of medicinal plants used in pasir mayang. the detailed uses of the 57 medicinal plants are given. all plant species were subjected to phytochemical analysis and toxicity tests, and the outcome of the analysis on the presence of alkaloids, saponins, steroids/terpenoids, and the level of toxicity against brine shrimp (anemia salina) are presented. a comparison with other studies reported in the literature seems to indicate that a high frequency of the use of leaves in therapy may be a part of a larger cultural phenomenon among the tropical forest tribes of southeast asia. keywords : indonesia/jarabi/pasir mayang/medicinal plants/ethuophannacy/toxicity screening introduction pasir mayang is the name of an area in the district of tebo in the province of jambi. in the beginning, the name of pasir mayang was dusun tuo. however, since the establishment of pt industries et forest asiatiques (ifa), it has used the name pasir mayang. in line with the increasing population and then later, with the influx of settlers from neighboring communities, pasir mayang developed to become several villages which were administratively included in the sub-district of vii koto. pasir mayang can be reached by road from the district of muara bungo, traversing about 20 km, then turning right at the simpang sawmill for about another 20 km on the way to the transmigration area of rimbo bujang to the village of dusun tuo. from there, the sub-district vii koto can be reached by continuing 8 km more. the access to the village of sungai karang can be reached from dusun tuo through unpaved roads traversing 3 km to batanghari river, then crossing the batanghari river by boat and taking another 15 km more of unpaved roads to the village of sungai karang. the sub-district vii koto covers an area of 112,700 ha bordering the province of west sumatera in the northern side. the southern part of the area is bounded by the sub-district of tebo ulu, and the western side by the district of bungo. meanwhile, the eastern side is bounded by the province of riau. 40 biotropia no. 22, 2004 the population of the sub-district vii koto, based on a census conducted by the office of subdistrict vii koto on april 2001, is composed of 19,249 individuals which consisted of 9,595 men, and 9,654 women, comprising over 11 villages. forest is no longer a source of income for the people of pasir mayang. the forest in the area has been used for collecting a wide range of forest fruits and medicinal plants, not to mention building materials and firewood. occasionally, people collect rattan from the forest, but even this is hard to find, because lots of rattan had been collected for a long time for commercial purposes. while the people of pasii mayang are not "primitive" forest-dwellers, they have retained a relatively simple agricultural way of life. therefore, agriculture is the main occupation of the people of pasir mayang. traditional farming (ladang) usually takes place far away from the villages and this usually involves one-quarter to one-half hectare of forest which are cleared and burned and planted with a wide variety of crops. most of their harvest, however, are adequate only to supply their own needs. the cultivation of "pohon karet" or rubber tree (hevea brasiliensis) is important in pasir mayang as one of the main sources of income in this otherwise moneyless society. rubber is planted in the forest close to the villages. at certain ages of the rubber trees, farmers tap and collect the latex, and sell them to the collectors in the district of vii koto or tebo. they also plant coffee in the forest for their own use or sell to the market. the health center, more widely known as puskesmas, is located in the sub-district of vii koto, headed by a medical doctor, assisted by another medical doctor, one dentist and eight paramedical personnel. health services are provided free of charge for those who could provide proof that they are not able to pay. a bidan (nurse) is available in each village. nurses are trained to diagnose basic illnesses and diseases and can provide a number of common medicines. these common generic medicines which are subsidized by the government with generic names, are supplied every three months from the health center in the sub-district of vii koto and are given free of charge. pembantu puskesmas (health sub-centers) exist in each village in the sub-district of vii koto, headed by a nurse and some paramedical personnel. the most prevalent illnesses are diarrhea, cholera and malaria. meanwhile, other communicable diseases include leprosy, framboesia, tetanus neonatorum, diphtheria, whooping cough, and filiariasis. no smallpox cases have been registered. due to the proximity of the area, and also on account of scarcity of medicine, doctors could not conduct regular visits to villages, which are far from the sub-district of vii koto. only when an epidemic occurs or the situation becomes really threatening medical assistance will be sent. the distance between the district and the health centers, as well as the high expenses which travel incurs are the main obstacles preventing the population from making use of the health facilities provided by the government for them. people generally accept the modern medicines that provide wide therapeutic measures for illnesses and diseases that people suffer from, except for mental illness. 41 medicinal herbs of pasir mayang, jambi hilman affandi et al. for this group of diseases, people will then ask for help directly from the traditional healers. in much the same way, if health problems persist and cannot be solved by the health center, they will then turn to traditional healers who combine herbal remedies with mystical ceremonies which, according to those interviewed, counter the influence of what was generally believed to be "evil spirits" which are responsible for the illnesses, diseases, or even accidents the citizens experience. almost all cultures have a long history of folk or traditional medicine, which includes the use of medicinal plants or herbs prescribed by local folk or traditional doctors in the neighborhood. even in ancient cultures, people methodically and systematically collected information on herbs and developed well-defined herbal pharmacopoeias. this must have also occurred to the ancient people of pasir mayang, for there are still quite a number of traditional healers who do their ritual practices in the communities. this study provides an outline of the contemporary knowledge on the use of herbal medicinal plants by the community around pasir mayang with the purpose of preserving this aspect of traditional knowledge for the generations to come. specifically, this research aims to make an inventory of herbal medicinal plants available in pasir mayang and how these are used by the community residents. it further aims to describe the herbal plants used for medicinal treatment by the indigenous people paying particular attention to the methods of preparation prior to its use, identification of the specific illness or disease that can be treated by each plant, and the effect of the treatment. phytochemical analysis and toxicity tests had also been conducted on the medicinal plants collected in the area, to determine the toxic level of the methanolic plant extracts against brine shrimp (artemia salina) and to trace the presence of specific chemical substances such as alkaloids, steroids, saponins, and flavonoids. methodology data collection of pasir mayang medicinal plants information on the traditional medicinal plants and herbs in pasir mayang was collected by a team of four workers in a duration of two months in the year 2003, in april and october. information was obtained for the series of one-hectare plots. in each case, one-strip transects (25 x 100m) were made within each plot marked out by coloured plastic ropes at both ends and in the centre. inputs were collected in the presence of local healers or other villagers by walking along the centre line. the villagers were asked to identify the local names and uses of each plant occurring within the series of transects. for every plant with indication of traditional use(s), every information was recorded and herbarium specimens of each species were collected by pressing between newsprints, stacked and placed in heavy plastic bags, treated with denatured alcohol and packed, opened only upon arrival at the seameo biotrop laboratory in bogor. small amounts of plant materials from 42 biotropia no. 22, 2004 each plant were taken to the seameo biotrop natural products laboratory for phytochemical analysis carried out at a later date. toxicity test (brine shrimp lethality bioassay) twenty grams of air-dried plant materials from each species were extracted with methanol at room temperature. the crude extracts were evaporated under reduced pressure until dried. sea water was poured into a small tank and shrimp eggs were added to the tank provided with aerator. after two days the shrimp eggs hatched and mature as naupili. each crude extract was weighed (20 mg) and dissolved with dichloromethane (2 ml). from this solution, 500, 50, 5ul were transferred to small petri dish corresponding to 1000, 100, 10 (j.g/ml (ppm), respectively (meyer et al. 1982). the solvent was evaporated by standing overnight at ambient temperature. after two days, when the shrimp larvae were ready, sea water was added to each petri dish containing 10 shrimps per dish (30 shrimps per dilution), and adjusted the volume with sea water to 5 ml/dish. the number of shrimps that survived were counted and recorded after 24 hours. the data were analyzed with finney computer program to determine lc50 values at 95% confidence intervals (hostettmann 1991). each crude extract of the sample was tested in three replicates with one control each. the controls were treated in a similar manner without the presence of the crude extracts. phytochemical analysis alkaloid testing was conducted according to procedures described by culvenor and fitzgerald (1963) : two to four leaves, fruits and/or bark of each plant of the collected species were preserved in ethanol, after being dried in the laboratory, grounded with clean sands and added with 10 ml chloroform. ammoniacal chloroform (10 ml, 0.05 m) was added, stirred and filtered, then shaken with aqueous sulphuric acid (10 drops, 2m). then the aqueous layer was tested with mayer's reagent. a dense, heavy precipitate was designated as 3+, strong precipate as 2+ and weak precipate as 1+. terpenoids/steroids and saponins were screened on the basis of liebermann-burchard and froth test, respectively, as generally described by simes et al. (1959). an amount of 2-4 g dry leaves were cut into small pieces and boiled in ethanol (25 ml, 15 min.), filtered while still hot and evaporated to dryness. the extract was triturated with ether and the insoluble ether shaken with water (ca. 5 ml) in a vi x 5-inch test tube. a foam more than 3 cm and lasting for more than 15 minutes was designated as 3+, between 2-3 cm was designated as 2+ and foam of 1-2 cm was designated as 1+. the ether soluble fraction was subjected to the liebermann-burchard test using acetic anhydride and sulphuric acid. the formation of a bright purple, red or pink coloration was considered 3+ while moderate and weak colora 43 medicinal herbs of pasir mayang, jambi hilman affandi et at. tion were designated 2+ and 1+, respectively. in the case of doubtful coloration produced, the ethereal fraction was passed through a pasteur pipette filled with active charcoal just enough to absorb chlorophyll and treated as above. results and discussion a total of 57 herbs were collected and noted according to their uses for various purposes by the healers and villagers of pasir mayang. of these, the medicinal uses which were presumed magical or superstitious in nature were considered of no interest for the present study and have been omitted for further discussions. many of these comprise common weedy species and a few cultivated ones, all of which were easily available, in those areas where this study was conducted, as well as in most other villages in pasir mayang (affandi et al. 1996). many of the plants, however, were of forest origin, growing in the secondary and little-disturbed forests in the surrounding areas. in the list, species were arranged alphabetically by family/followed by common name / plant part(s) used / and a description of their uses. these were summarized in appendix 1. medicinal uses the process to cure common maladies and illnesses among the people of pasir mayang involves accessing both traditional and modern methods. generally, the people of pasir mayang rely mostly on traditional medicines to treat minor ailments or are used as first aid towards a cure. they seek " modern " medicine only when the traditional methods have been exhausted. the most frequently utilized plant part is the leaf (table 1). this preference for leaves is apparently derived from the fact that therein, many species store high concentrations of bioactive compounds (moore 1994). in addition to efficacy, other factors that may contribute to the preference for leaves as medicine include the ease with which they may be collected, stored, and transported and the ease with which bioactive compounds may be extracted. the most frequently cited modes of preparation (table 2) were: poultice (mashed, crushed, or chopped plant part), decoctions (boiling of plant parts), and used directly from the plants (none). the most frequently encountered modes of administration are oral and topical (table 3) which may be preferred because they are believed to be the most effective means for delivering bioactive compounds into the body. 44 phytochemical analysis and toxicity tests the phytochemical analysis is a preliminary test to trace the presence of specific chemical substances such as alkaloids, steroids/triterpenes, and saponins without resulting to a biological screening. a total of 57 species from 30 families were phytochemically tested in the laboratory of natural products at seameo biotrop. the results of the tests are summarized in table 4. 45     biotropia no. 22, 2002 medicinal herbs of pasir mayang, jambi hilman affandi et al.  alkaloids  of the 57 plant species, three species gave strong positive reaction (3+ 4+) to alkaloid. those species were fibraurea sp , zizyphus sp., and uncaria sp. (table 5).        saponins  of the 57 species tested for positive reaction to saponins, 12 species gave a strong positive reaction (3+ 4+) (table 6). saponin is particularly prevalent in bridelia glauca, b. minutiflora, macaranga triloba, callophylum mucumense, litsea aurea, barringtonia racemosa, magnolia mackottii, pternandra coerulescens, ficus variegata, eugenia sp., arenga pinnata, calamus sp., miletia sp., mucuna gigantea, xanthophyllum sp., uncaria sp., euphoria malaiensis, payena acuminata, lygodium circinatum, trema orientalis, and lantana camara.   steroids/triterpenes almost all plant species collected indicated positive response to steroids/ terpenes. nine plant species showed strong reaction with 3+ or 4+ rating (table 7). le compounds are particularly prevalent in mallotus paninlatus; clochidion •borescens; litsea monopetala; dictyopteris irregularis; selaginella hieron.           toxicity tests brine shrimp (artemia salind) lethality test is a simple, fast and inexpensive oassay in the search for bioactive compounds from plant extracts. the value of ore than 1000 ppm is considered non-lethal and hence shown as negative (-ve). a tal of more than 57 samples were tested and only ten showed toxicity against brine rimps. of the ten plant species tested, three species (dictiopteris irregularis pr., intana camara linn, and gleichenia imearis clarke) indicated some potential xicity which will require further investigation (table 8).   medicinal herbs of pasir mayang, jambi hilmau affandi et al. references affandi, h., nuryadin, a., susilo.b.p and supriatno. 1996. phytochemical survey of the forest tree species of pasir mayang, jambi, sumatera, seamed biotrop, research report. culvenor, c.c.j. arid j.s. fitzgerald. 1963. a field method for alkaloid screening of plants. j. pharm. sci. 52:303304. hostettmann, k., ed. 1991. method in plant biochemistry, vol 6 : assays for bioactivity, pergamon press, new york. meyerb.n., n.r. ferrigni, j.e. putman, l.b.jacobsen, d.e. nichol and j.l. mclaughli. 1982 brine shrimp: a convenient general bioassay for active plant constituents. planta medica, 45:31-34 moore, p.o. 1994. 'trials in bad taste." nature 370:410-411. simes, j.j.h., j.g. tracey, lj. webb and dj. dunstan. 1959. australian phytochemical survey ih, saponins in eastern australian flowering plants. bulletin no. 281 c.s.i.r.o., australia, melbourne. 52             40.pdf 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf 47.pdf 48.pdf 49.pdf 50.pdf 51.pdf 52.pdf 53.pdf 54.pdf 55.pdf 56.pdf 57.pdf 58.pdf molecular characterization of begomovirus infecting yard long bean ( subsp. vigna unguiculata sesquipedalis l.) in java, indonesia s 1 1* 1ari nurulita , sri hendrastuti hidayat , kikin hamzah mutaqin and john thomas2 1department of plant protection, faculty of agriculture, institut pertanian bogor, bogor 16680, indonesia 2uqueensland alltrance for agriculture and food innovation (qaafi) u , niversity of queensland, st. lucia campus, brisbane australia received /accepted 21 may 2014 6 june 2015 abstract begomovirus was identified as one of the causal agents associated with yellow mosaic disease on yard long bean ( subsp. l.) in java. previous study reported that has infected several vigna unguiculata sesquipedalis begomovirus leguminosae in south asia. several have been reported to infect important crops in indonesia. those begomoviruses begomoviruses were characterized based on nucleotide sequences. this study was conducted to identify and characterize taken from yard long bean samples based on specific genome property of common begomovirus in java region. three main activities were conducted: i) sample collection in yard long bean fields located in central java (tegal, magelang, and klaten), yogyakarta (sleman) and west java (bogor and subang) provinces; ii) virus detection using i-elisa, pcr and sequencing; iii) molecular characterization of using software bioedit v.7.0.5 and begomovirus mega 6.06. yellow mosaic disease was found in almost all fields. infection of and was detected potyvirus begomovirus using i-elisa and pcr, respectively. both viruses were detected as either single or mixed infection samples . collected from tegal, klaten, magelang, subang and bogor were positively infected by based on specific begomovirus viral dna amplification. sequence analysis indicated that infecting yard long bean is mungbean yellow begomovirus mosaic india virus (mymiv) and it belongs to the same group with mymiv from bangladesh, india, pakistan and nepal. further analysis showed the conserved region of around common region, i.e. “tata box” begomovirus sequence, hair pin loop structure, repetitive sequence and the conserved nonanucleotide sequence taatattac were also determined. this is the first report of mymiv infection in indonesia. keywords: begomovirus, common region, dna sequencing, i-elisa, mymiv, pcr, yard long bean introduction a outbreak yellow mosaic disease was reported in yard long bean ( subsp. vigna unguiculata sesquipedalis l.) growing area in java since 2008 (damayanti 2009). disease was asso-et al. the ciat bean common mosaic ed with infection of virus (bcmv), cucumber mosaic virus (cmv) and (damayanti 2009; hidayat begomovirus et al. 2011, ; tsai . 2013). the personal communication et al at same time, similar yellow mosaic disease caused by begomovirus was first reported from pakistan (ilyas et al. et al. . 2010) and nepal (shahid 2012) begomovirus geminivirus is a member of group which is transmitted in nature by whitefly bemisia tabaci gen. (hemiptera: aleyrodidae). molecular character of its genome is very unique, istics having one or two circular single stranded dna (2.6–2.7 kb) and represented as monopartite or bipartite, respectively (hull 2002). serious crop diseases caused by infection of have begomovirus been reported in indonesia. pepper yellow leaf curl virus (pepylcv), tomato yellow leaf curl virus (tylcv), and tobacco leaf curl virus (tlcv) are considered as the major factor causing yield loss on chilli pepper, tomato and tobacco, respectively (aidawati 2005; hidayat et al. et al. 2006; 2008). damage and potential yield loss in yard long bean caused by should begomovirus be anticipated by establishing detection method and disease control strategy. to do so, basic information regarding molecular and biological characters of the virus is required.* corresponding author : srihendrastutihidayat@gmail.com biotropia vol. 22 no. 1, 2015: 53 60 doi: 10.11598/btb.2015.22.1.401 53 mailto:srihendrastutihidayat@gmail.com biotropia vol. 22 no. 1, 2015 54 m begomovirusethod for detection of is commonly based on polymerase chain reaction (pcr) technique using universal primers. degenarate primers pal1v1978/ par1c715 was first used by rojas (1993) as universal primer et al. to detect several infectedgeminiviruses from plants belong to solanaceae, leguminosae, euphorbiaceae and malvaceae from south america. since then, the primer pair has been used to identify and characterize various members of . begomovirus fragment of dna amplified by this begomovirus pair of primers covers part of al1 region (replicase gene), common region and intergenic region and part of ar1 region (coat protein gene). molecular characters of can be begomovirus determined by analyzing this dna fragment sequences due to its conserved and diversed region. common region for each is begomovirus different, except loop region on hairpin structure, 5'-taatattac-3' which is known as the conserved nonanucleotide region (lazarowitz 1987). >92% similarity on begomovirus posse sings their hairpin loop structure can be as one grouped species (fauquet 2005).et al. this paper explained the attempts to identify and characterize from yard long bean begomovirus samples based on specific genome in java property of common region. materials and methods collection of field samples samples collection was conducted in west java (bogor and subang), central java (tegal, klaten and magelang) and yogyakarta (sleman). leaf samples were collected from the plants age 6–9 wap using purposive sampling method based on yellow mosaic symptom described by damayanti et al. et al. (2009) and ilyas (2010). fresh tissue was directly subjected for virus detection and the remaining were stored as isolate collection in the laboratory -80 c.at o virus detection by indirect enzyme-linked immunosorbent assay (i-elisa) two major viruses on yard long bean i.e. potyvirus and cmv were detected from leaf samples using i-elisa following commercial kit manufacturer (protocol leibniz-institut dsmz gmbh germany) infected leaves was ground in , . coating buffer (1/10, v/v). coating buffer (ph 9.6) contain 15 mm na co (1.59 g), 2.38 mm 2 3ing nahco (2.93 g), 3.08 mm nan (0.20 g) and 3 3 h o was added to final volume of 1 l. aliquots 2 of each sample (100 µl) was dispensed to each microtitters' well, then incubated overnight at 4 c. microtitter plate was washed 8 times using o pbs-tween (0.137 m nacl (8.0 g), 1.47 mm kh po (0.2 g), 8.1 mm na hpo (1.15 g), 2.68 2 4 2 4 mm kcl (0.2 g), 3.08 mm nan (0.2 g), add h o 3 2 up to 1 l ph 7.4 and add 0.5 ml tween 20/ l). plates were dried up by tapping upside down on tissue paper. blocking solution (2% skim milk diluted in pbs-tween) was added 100 µl to each well then incubated at 37 c for 30 minutes. o blocking solution was removed and plate was tapped dry. first antibody (igg) was diluted in conjugate buffer (pbs-tween containing 2% pvp (serva pvp-15 polyvinyl pyrrolidon) and 0.2% egg albumin) according to manufacturer recommendation, then 100 µl conjugate buffer contains igg was added to each well, incubated at 37 c for 2-4 hours, then washed by pbs-tween. o second antibody (igg-ap) was diluted in conjugate buffer according to manufacturer recommendation, then 100 µl conjugate buffer containing igg-ap was added to each well, incubated at 37 c for 2 hours then washed using o pbs-tween. substrate (10 mg p-nitrophenyl phosphate) was dissolved in substrate buffer (97 ml diethanolamine, 600 ml h o, 3.08 mm nan 2 3 (0.2 g), adjust to ph 9.8 with hcl and make up to 1 l with h o) added 100 µl to each well. plate 2 was incubated in the room temperature under low light intensity and reaction was evaluated. positive reaction was qualitatively indicated by appearance of yellow color and quantitatively determined by measuring absorbance value at 405 nm wave length, i.e. two times absorbance value of negative control (healthy plant), using elisa reader (biorad 550). virus detection by pcr total viral dna was isolated from infected leaf following a procedure described by doyle and doyle (1987) with modification. fresh minor tissue (0.1 g) was ground with liquid itrogen to n powder, 500 µl of ctab buffer (10% cetyltrimethyl-ammonium (100 ml), 0.1 m bromide tris-hcl ph 8 (100 ml), 0.05 m edta (50 ml), 0.5 m nacl (126 ml), 1% β-mercapto-ethanol molecular haracterization f nfecting ard ong ean sari nurulitac o i y l bbegomovirus – et al. (10 ml and added h o up to 1 l was added and ) 2 the sap was transferred to 1.5 ml clean tube. the sap was incubated in water bath at 65 c for 1 hour, o shaked every 10 minutes to separate and lipid prote in . f ive hundred micro l i t e r o f chloroform/iso-amyl alcohol (24:1, v/v) was added o the liquid, then the tube was vortexed for t 5 minutes and centrifuged at 14,000 rpm for 15 minutes. the supernatant was pipetted to 1.5 ml clean tube, 1/10 volume of 3 m ammonium acetate and 2/3 volume of isopropanol was added, respectively. the liquid was mixed gently then incubated overnight at -20 c or 4 hours at o room temperature. after incubation, the liquid was centrifuged at 12,000 rpm for 10 minutes to precipitate dna and then was discarded flowthrough. the pellets were washed with 500 µl of 70% ethanol, centrifuged at 8,000 rpm for 5 minutes and dried in room temperature after discarding the flow through. the dried pellets containing total dna were dissolved in 50 to 100 µl of nuclease free water or te buffer, ph 8 and the dna was ready for pcr. amplification of viral dna was conducted following method described by rojas (1993) et al. to confirm geminivirus infection. pcr reaction contained 10xpcr buffer, 25mm mgc , 2.5mm l2 dntps, 10µm each of primer pal1v1978 and par1c715, polymerase (5u/µl), 1 µl of taq dna and the reaction was adjusted to 25 µl with nuclease free water. amplifications was performed in geneamp pcr system 9700 machine with 5 minutes at 94 c for pre-heating, o followed by 30 cycles of denaturation (1 minute at 94 c), annealing (1 minute at 50 c) and extension o o (3 minutes at 72 c). the last cycle was followed by o 72 c for 3 minutes and decreased at 4 c. agarose o o gel electrophoresis was used to visualize pcr products. dna sequencing viral dna fragments obtained from direct pcr amplification were sent to pt genetika science, indonesia and australian genome research facility, australia, respectively for dna sequencing. sequence data were compared with other sequences from genbank (ncbi 2013) and analysed using software programs bioedit v.7.0.5, clc sequence viewer 7, and mega 6.06. results and discussion identification of from field begomovirus samples incidence of yellow mosaic disease was very high i.e. 80% to 100% in most growing areas. infected plants were easily recognized in the field based on visual symptoms. three main symptoms of yellow mosaic disease in the field were observed i.e. 1) yellowing; 2) yellowing with green spot; and 3) mosaic vein b nding (fig. 1 and a table 1). the most common symptom found in every field was yellowing. yellowing with green spot was thought as early symptom before it developed into yellowing. further severe infection caused smaller pods and leaves. these type of symptom had also been reported as s typical symptoms of yellow mosaic disease of yard long bean in south asia (ilyas 2010). et al. begomovirus was detected in 11 infected plant samples (fig. 2) showing yellowing and yellowing with green spot symptoms from all locations. however, fragment was not begomovirus successfully amplified from samples showing mosaic vein banding from subang viral detection using i-elisa revealed the infection of potyvirus 55 figure 1. symptoms of yellow mosaic disease on yard long bean: (a) yellowing; (b) yellowing with green spot; (c) mosaic vein banding biotropia vol. 22 no. 1, 2015 56 table 1. detection of cucumber mosaic virus, and from leaf samples potyvirus begomovirus collected in wet season 2012 from various locations using i-elisa and pcra location code of isolates symptoms description i-elisab pcr cmv potyvirus begomovirus tegal tegal 1 yellowing + + tegal 2 yellowing mosaic + klaten klaten 1 yellowing + klaten 2 yellowing mosaic sleman sleman 1 yellowing + + sleman2 yellowing mosaic + + magelang magelang 1 yellowing mosaic + magelang 2 yellowing + + magelang 3 yellowing mosaic + subang subang 1 mosaic vein banding + subang 2 mosaic vein banding + subang 3 mosaic vein banding subang 4 yellowing + bogor bogor 1 yellowing + bogor 2 yellowing + figure 2. visualization of amplification from leaf samples using universal primers pal1v1978/ par1c715 on begomovirus 1 % agarose gel. m, 1 kb marker dna (thermo scientific, us); k+, dna of pepylciv; 1-15 leaf samples from fields (1, tegal 1; 2, tegal 2; 3, klaten 1; 4, klaten 2; 5, sleman 1; 6, sleman 2; 7, magelang 1; 8, magelang 2; 9, magelang 3; 10, subang 1; 11, subang 2; 12, subang 3; 13, subang 4; 14, bogor 1; 15, bogor 2) a most plants were in generative stage when samples was collected = not detected; + = detectedb but not of cucumber mosaic virus (cmv). mosaic vein banding symptom caused by potyvirus infection had been described previously by damayanti (2009). this result indicated the et al. association of with yellow mosaic begomovirus disease of yard long bean in java. analysis of sequence identity of begomovirus infecting yard long bean nucleotide sequences were obtained for begomovirus isolates from tegal, klaten, magelang, subang and bogor. the sequence of isolate from sleman was not good due to unreadable chromatogram, therefore it was not included in the further sequence analysis. analysis of their identity by comparing to sequences on the genebank showed their highest homology with mungbean yellow mosaic india virus (mymiv) from brebes and purwakarta, i.e. >92%, followed by mymiv from bangladesh, nepal, pakistan and india, i.e. >87% (table 2). their homology to mymv, another virus causing yellow mosaic disease in south asia, was only 71-76% and to other reported from indonesia was begomovirus even lower i.e. 5154%. further dendogram analysis to study their relationship showed that all 57 table 2. nucleotide sequence homology (%) of infecting yard long bean in java with other reported begomovirus begomoviruses earlier in genebank begomovirus infecting yard long bean begomovirus isolates from genebanka) 1 2 3 4 5 6 7 8 9 10 11 12 ptgl1 99.7 97.9 92.5 91.1 92.2 92.1 76.0 55.8 53.8 51.4 51.5 16.6 tegal 2 95.7 94.7 89.5 88.3 89.4 89.3 73.1 54.6 53.9 52.5 52.0 17.4 klaten 96.0 93.2 88.0 86.8 87.9 87.9 71.9 54.1 54.2 52.9 51.9 17.2 magelang 95.9 92.8 87.7 86.4 87.5 87.5 71.7 53.5 53.8 52.1 51.6 16.8 magelang 2 95.3 92.8 87.7 86.4 87.5 87.5 71.7 53.5 53.8 52.1 51.6 16.8 subang 95.5 94.7 89.5 88.3 89.4 89.3 73.0 54.6 54.0 52.5 52.0 17.4 bogor 1 95.4 94.8 89.6 88.4 89.5 89.4 73.0 54.7 54.1 52.5 52.2 17.4 bogor 2 95.1 93.8 88.6 87.4 88.5 88.4 72.6 53.7 54.1 52.7 52.1 17.5 a)1= mymiv brebes (jn368436); 2= mymiv purwakarta (jn368434); 3= mymiv india (kc852204); 4= mymiv bangladesh (af314145); 5= mymiv nepal (ay271895); 6= mymiv pakistan (am992618); 7= mymv india (kc911271); 8= pepylciv (ab246170); 9= tlciv (ab241671), 10= tolcndv (data unpublished); 11= tolcjv (189848); 12= bcmv bogor (fj653916) figure 3. phylogenetic analysis of mungbean yellow mosaic india virus based on alignment of partial nucleotide sequences of the dna-a of using mega 6.06 (algorithm neighbor joining with 1,000 bootstraps replicates)begomoviruses begomovirus infecting mungbean (mymiv and mymv) belonged to similar cluster and they were separated from infecting other crops begomoviruses (pepylciv, tlciv, tylcndv, tolcjv) (fig. 3). it indicated that mymiv from java had closer genetic relationship to mymiv from south asia than other from begomoviruses indonesia. analysis of common region sequences of begomovirus infecting yard long bean molecular characters of could be begomovirus determined by analyzing the top region of its genome covering common region and intergenic region representing the conserved and diversed region, respectively. common region for each begomovirus was different, except loop region on molecular haracterization f nfecting ard ong ean sari nurulitac o i y l bbegomovirus – et al. biotropia vol. 22 no. 1, 2015 58 figure 4. alignment of nucleotide sequences of the cr of mungbean yellow mosaic india virus (mvmiv) isolates from java with other reported mymiv in genebank. the alignment showing repetitive sequences (grey shadow), invert repeat (green shadow), tata sequences (bold letter with purple shadow) and the hairpin loop region (underlined letter with yellow shadow) with nonanucleotide sequence (bold and underlined letter with turquoise shadow) 59 hairpin structure, 5'-taatattac-3' which is known as the conserved nonanucleotide region (lazarowitz 1987). comparison of iteron and protein products variations have been used to determine of mymv and mymiv from several regionals in india (varma & malathi 2003; usharani . 2004) and et al tylcv isolates from israel, sardinia and thailand (argüelo-astorga . 1994). however, et al exception was reported on several cases, for example potato yellow mosaic virus with tylcv-sardinia and bgmv-brazil with tylcv-israel have identical iterons although they have a rather far distance relationship (argüelo-astorga 1994).et al. our analysis showed that common region of begomovirus isolates infecting yard long bean in java consists of repetitive sequence (atcggtgt), s tata box, and hair pin loop structure (fig 4). . three direct repeats (two of them are tandem repeat sequences) can be found before tata .box in all mymiv and mymv isolates (fig 4). iteron and tata box sequence always present together in cr of each begomovirus species (lazarowitz 1987). the function of iteron and tata box was described by argüelo-astorga et al. (1994) as initiator for rolling circle replication which was known as rap-specific binding sites. similar characteristics were reported for eastern hemisphere geminivirus except acmv and icmv, western hemisphere geminivirus, et al.and sqlcv-e and –r (argüelo-astorga 1994), and mymiv on soybean (usharani . et al 2004). hair pin loop structure of each isolates was identical to mymiv from bangladesh, pakistan, india, nepal, and one isolate of pepylciv, but they were different from tlciv and pepylciv (fig. 5). >92% similarity begomovirus posse sings on their hairpin loop structure could be grouped as one species (fauquet 2005; hidayat . et al. et al 2008). therefore, all isolates from this begomovirus study was the same species with mymiv from bangladesh, india, pakistan, and nepal. conclusions molecular detection and characterization confirmed the association of mymiv in yellow mosaic disease of yard long bean in java. virus isolates from java had the highest similarity (>85%) with mymiv isolates from bangladesh, pakistan, india, and nepal. further study on characters of the virus, such as host range, insect transmission and disease spread, are very important to understand disease development and control. acknowledgements this research was funded by collaboration between ausaid funded economic cooperation work program of the asean– australia–new zealand free trade agreement and department of plant protection, faculty of agriculture, institut pertanian bogor. figure 5. hairpin loop structures of several isolates. a. mymiv tegal; b. mymiv klaten; c. mymiv begomovirus magelang; d. mymiv subang; e. mymiv purwakarta; f. mymiv bogor; g. mymiv bangladesh; h. mymiv pakistan; i. mymiv india; j. mymiv nepal; k. tlciv indonesia; l. pepylciv indonesia molecular haracterization f nfecting ard ong ean sari nurulitac o i y l bbegomovirus – et al. biotropia vol. 22 no. 1, 2015 60 references aidawati n, hidayat sh, suseno r, hidayat p, sujiprihati s. 2005. identifikasi yang menginfeksi tomat geminivirus berdasarkan pada teknik polymerase chain reactionrestriction fragment length polymorphism. j mikrobiol indon 10(1): 29-32. argüelo-astorga gr, guevara-gonzález rg, herreraestrella lr, riverabustamante rf. 1994. geminivirus replication origins have a groupspecific organization of iterative elements: a model for replication. virology 203:90100. damayanti ta, alabi oj, rauf a, naidu ra. 2009. severe outbreak of yellow mosaic disease on the yard long bean in bogor, west java. hayati j of biosci 16(2):78-82. doyle jj, doyle jj. 1987. a rapid dna isolation of procedure for small quantities of fresh leaf tissue. phytochem bull 19:11-9. fauquet cm, mayo ma, maniloff j, desselberger u, ball la, editor. 2005. . eight report of virus taxonomy the international committee on taxonomy of viruses. san diego (us): viroldivint union of microb soc. hidayat sh, chatchawankanpanich o, rusli e, aidawati n. 2006. associated with pepper yellow leaf begomovirus curl disease in west java, indonesia. j mikrobiol indon 11:87-90. hidayat sh, chatchawankapanich o, aidawati n. 2008. molecular identification and sequence analysis of tobacco leaf curl virus from jember, east java, indonesia. hayati j of biosci 15(1):13-7. hull r. 2002. . london (gb): matthew's plant virology 4 edth academic press. ilyas m, qazi j, mansoor s, briddon rw. 2010. genetic diversity and phylogeography of begomoviruses infecting legumes in pakistan. j gen virol [internet]. 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[cited 2014 apr 3]; 142:145164. available on: http://onlinelibrary.wiley.com/ doi/10.1111/j.17447348.2003.tb00240.x/pdf. http://www.pubfacts.com/detail/20375225/gene http://www.ncbi.nlm.nih.gov. http://www. http://www.ndrs.org.uk/article.php http://onlinelibrary.wiley.com/ doi: 10.11598/btb.2015.22.2.422 rapd analysis to detect somaclonal variation of pineapple cultures during in vitro micropropagation ika roostika , nurul khumaida and sintho wahyuning ardie1* 2 2 1indonesian center for agricultural biotechnology and genetic resources research and development (icabiograd) bogor 16111, indonesia 2department of agronomy and horticulture, institut pertanian bogor, bogor 16680, indonesia received 7 september 2014/ accepted 20 januari 2015 abstract plant off-type becomes a concern in pineapple micropropagation. reliable methods are needed to detect and to reduce plant off-type. this research was conducted to confirm the occurrence of somaclonal variation during micropropagation of pineapple clone simadu. the culture period (longand short-period) and the regeneration methods (direct organogenesis, indirect organogenesis and somatic embryogenesis) were studied to know their contribution in bringing out somaclonal variation. rapd analysis using 10 primers was performed to confirm genetic variation. the result showed that rapd assay could be applied as early detection of somaclonal variation of pineapple, where opa primers were better to be used than opj primers. the phenotypic variation occurred in the four year-old pineapple plants and plantlets were due to genetic variation. it has proved that the long-period of cultures is the main contributor of somaclonal variation, while the regeneration method and plant growth regulator could also induce genetic variation. the new cultures showed higher level of similarity. therefore, it needs a correct strategy to apply micropropagation method of pineapple to minimize plant off-type. it was recommended to avoid the use of long-period culture as mother stock and to apply direct organogenesis method for micropropagation rather than indirect organogenesis and somatic embryogenesis. : keywords ananas comosus (l.) merr., molecular markers, plant off-type introduction conventionally, pineapple can be propagated from various plant propagules, namely crown, sucker, butt or stump, hapas, ratoons and slip. however, their reproductive time is not uniform (international board for plant genetic resources 1991; coppens d'eeckenbrugge & leal 2003). conventional propagation techniques were not appropriate for rapid mass propagation, such as in cultivar smooth cayenne, where availability of vegetative propagules are commonly limited both in the type (only crown and sucker) and the number. culture techniques may be able to in vitro solve the problem, since it can provide alternative methods for mass propagation plants with higher uniformity and at shorter times. according to smith (2003), micropropagation has been et al. used for establishment of multiplication blocks, which then provide conventional planting material for larger production blocks, because the micropropagated plants are more expensive and the growers concern about the genetic off-type. it was reported that variation may be resulted from the pre-existing variation of the explants (wakasa 1979; nwauzoma & jaja 2013), the et al. long period of the culture (koornneef 1991; masoud & hamta 2008), the high level of plant growth regulator (bairu 2006), the frequent et al. subculture (eeuwens 2002), the genotype et al. dependence (zucchi . 2002), activation of et al transposable elements (bairu 2011), and et al. hypoor hypermethylation of dna (abdellatif et al. 2012). to avoid the high level of variation and to streamline the efficiency of micropropagation method, it needs a strategy to produce uniform * corresponding author : ikatambunan@yahoo.com biotropia 2 109 119 vol. 22 no. , 2015: 109 mailto:ikatambunan@yahoo.com 110 genetic identity in the seedlings. therefore, variations in the mother stocks and regenerants need to be characterized. based on the morphological characterization of cultures in vitro and phenotypic evaluation of the seedlings in the previous research, it was suggested that those variations were caused by somaclonal variation (roostika 2012a). in order to confirm this suggestion, molecular analysis needs to be conducted. molecular markers are widely used to detect somaclonal variation and genetic fidelity during micropropagation (tawar 2008; rout et al. et al. 2009; khoddamzadeh 2010; shahid . et al. et al 2014). randomly amplified polymorphism dna (rapd) is applied because it is useful for analysis of variation at many loci requiring small quantities of dna, but not prior knowledge of dna sequence nor involvement of radioactivity (williams 1990). in the case of pineapple, et al. rapd analysis has been performed to detect genetic fidelity and variants of vara. comosus queen (soneji 2002), var et al. a. comosus amarelinho (feuser 2003), var et al. a. comosus bracteatus et al.(santos 2008) and the promising clone golden pineapple (suminar 2010). the objectives of this study were: (1) to confirm the occurrence of somaclonal variation by rapd method; (2) to know the level of variation of regenerants derived from four regeneration methods (axillaries shoot proliferation, direct organogenesis, indirect organogenesis, and somatic embryogenesis); and (3) to detect the variation and to reduce the level of variation. materials and methods plant materials the plant materials were four-year-old pineapple cultures of simadu (smooth cayenne) from subang, west java, indonesia. there were three populations used in this research. preparation of population i the cultures were established from axillary bud explants, excised from the crown of pineapple plants grown in the field. the explants were sterilized using 70% ethanol and 10.5% sodium hypochlorite. the basal medium was ms salt (murashige & skoog 1962), 3% (w/v) sucrose, 0.8% (w/v) agar, 100 mg/l myo-inositol 0.1 , mg/l thiamine-hcl, 0.5 mg/l pyridoxine-hcl, 0.5 mg/l nicotinic acid with addition of 8.84 μm benzyl adenine (ba) and 9.3 kinetin (kin). μm prior to autoclaving the medium, its ph was adjusted to 5.7±0.1. the cultures were illuminated 16 hours per day with 20 μmol photons/m /s ir rad iance provided by 2 fluorescence lights in a culture room at 25±2 c. 0 after a four-year period and more than 10 in vitro times subculture, the cultures were maintained on ms basal medium with addition of 2.21 m ba μ and 4.65 m kin. the root induction was μ conducted by planting the shoots onto the ms medium containing of 15 indole butyric acid μm (iba) and 5.37 naphthalene acetic acid μm (naa) for 3 weeks. preparation of population ii the cultures that grew normally were selected. six hundred and fifty leaf bases were isolated as sources of explants that comprised approximately 1 cm of basal portion of the leaves. the explants were regenerated through direct organogenesis (roostika 2012a) and indirect et al. organogenesis regeneration (roostika et al. 2012b). the other cultures were prepared through indirect somatic embryogenesis as described by roostika . (2012c). the root et al induction medium was the same as mentioned above. preparation of population iii population iii consisted of plants collected from field (clone simadu and non simadu), the new cultures of both clones derived from shoot proli feration and direct organogenesis regeneration. the axillary buds of crown were sterilized and the shoots were initiated on in vitro ms media supplemented with 8.84 ba and μm 9.3 kin for shoot proliferation. after 6 μm months of culture period without subculture, they were maintained on ms media with addition of 2.21 μm ba and 4.65 μm kin prior to rapd analysis. rapd assay to detect somaclonal variation the leaves of acclimated variants were used as the samples in population i. about 10 shoots of cultures were pooled to make bulk samples in population ii and iii. rapd assay was performed biotropia vol. 22 no. 2, 2015 rapd analysis to detect somaclonal variation of pineapple cultures roostika . – et alin vitro using a modification method of soneji (2002). et al. the dna samples were isolated using cetyl trimethyl ammonium bromide (ctab). subsequently, they were amplified by polymerase chain reaction (pcr) machine. each reaction mixture contained 1 µl 25 ng/l genomic dna, 1 µl primer as listed at table 1 (operon tech. alameda, usa) at 10 ng/µl, 11 µl ddh o and 12 2 µl pcr mix, in a 25 µl total reaction. the pcr consisted of pre-denaturation at 94 c for 4 0 minutes, 40 cycles at 94 c for 30 seconds 0 (denaturation), 36 c for 1 minute (annealing) and 0 1 minute at 72 c (extension), post-extension at 0 72 c for 5 minutes and a final extension at 40 c 0 0 for 4 minutes. the pcr products were resolved on 1.2% agarose (promega) and electrophoresed at a constant voltage of 50 v for 50 minutes and then the bands were stained with ethidium bromide (etbr) and visualized under a uv transiluminator. a 1 kb dna ladder was used as a molecular standard. the samples were scored based on presence (coded as 1) or absence (coded as 0) of the same size bands. to select the primers, the mother plant dna and one variant ( ) needle leaf were used. the primers that showed polymorphic bands and/or scorable bands were selected. the scored data (binary data) were analyzed using the simqual, sahn and tree programs from the numerical taxonomy and multivariate analysis system version 2.02 (ntsys-pc 2.02). the similarity degrees were calculated according to the dice coefficient. groupings were carried out using the unweighted pair group method and arithmetic average (upgma) cluster analysis. results and discussion genetic variations occurred in the longperiod cultures of pineapplein vitro in order to determine the source of phenotypic variations occurred in the micropropagated cultures and plants, molecular analysis using rapd markers was conducted. plants with normal phenotype and exhibited several phenotypic variations (dwarf posture, albino stripe leaf, narrow leaf, spines spot leaf, erect posture, wider leaf, powdery leaf, climb posture, needle leaf and ultra dwarf posture) were subjected to rapd analysis using 10 primers. opa and opj have been used to characterize the variation in orchid (chen 1998), phalaenopsis et al. a. comosus et al. var. queen (soneji 2002) and var. bracteatus et al. (santos 2008). the result showed that the primers of opa were better than opj in generating amplified bands. the two primers generated the highest number of amplified bands, namely opa13 and opa16, whereas the primer of opj11 produced the lowest number of amplified bands. the result showed that the phenotypic variation occurred in the population i (the four-year-old pineapple cultures were due to genetic variation as the rapd method revealed a high polymorphism of dna bands (fig. 1). a total of 73 bands were amplified, 70 (95.9%) of which were polymorphic (table 2), indicated the high level of variation. the ntsys analysis yielded a dendrogram where the similarity coefficient ranged from 0.32 to 0.91 with high rohlf matrix correlation value (r=0.97, very 111 table 1 the list of primers and their sequences used in the rapd assay no primer sequence 3’ to 5’ 1 opa2 tgccgagctg 2 opa3 agtcagccac 3 opa7 gaaacgggtg 4 opa9 gggtaacgcc 5 opa13 cagcacccac 6 opa16 agccagcgaa 7 opa18 aggtgaccgt 8 opa19 caaacgtcgg 9 opj11 actcctgcga 10 opj13 ccacactacc biotropia vol. 22 no. 2, 2015 112 suitable). it separated the variants at the similarity coefficient of 0.32-0.61 to the control plant (fig. 2). there were 82.3% plants with altered phenotype in the population i (roostika et al. 2012a) and all of those variants were genetically different from the control plants (plants with normal phenotype) based on rapd analysis. this present study clearly showed that the use of long-period cultures open a high risk of somaclonal variation for pineapple micropropagation. regeneration method affects the somaclonal variation of pineapple based on table 3, it seems that the opa primers generated higher number of amplified bands than those produced by opj primers. the highest amplified bands were yielded from opa2 and opa3, whereas opj primers generated the lowest amplified bands. there was also a high polymorphism obtained from population ii (fig. 3; table 3). from a total 65 amplified products, 60 bands (92.3%) of which were polymorphic (table 3). the ntsys analysis yielded a dendrogram where similarity coefficient ranged from 0.34-0.99 (r=0.98). it separated the variants at the similarity coefficient of 0.34-0.65 to the control plant (fig. 4). the percentage of polymorphism was only slightly lower than population i. this result showed that the regeneration of normal phenotype-selected cultures could only reduce a small variation. it seems that chimera might have been existed in those explants sources (population i). figure 4 also showed that picloram-derived cultures had the lowest similarity (0.34). picloram is an auxinic herbicide belong to the pyridine group and commonly used at low concentration as an inducer for callus formation during somatic embryogenesis regeneration (firoozabady & moy 2004; ahmed et al. et al. 2011; noormi 2012). beside regenerated through callus formation phase, it needs a longer time to conduct somatic embryogenesis, so that the probability of genetic changes would increase. callus is an undifferentiated structure and actively doing cell division. according to miguel and marum (2011), tissue culture systems that involve acquisition of competence for extensive cell division are usually regarded as more risky of genome instability. rani and raina (2000) reported that there was a genetic instability of seedlings derived from micropropagation via callus formation. a high polymorphism (35/84 bands) was also reported by soniya (2001) on et al. tomato, cultured by picloram containing media. several previous studies repor ted the contribution of plant growth regulator in somaclonal variation, but they put less consideration in period of cultures (feuser et al. 2003; santos 2008). our study showed that et al. the long-period of cultures is the main contributor of somaclonal variation, the regeneration method and plant growth regulator could also induce genetic variation in the in vitro culture of pineapple. table 2 the number of amplified product and polymorphic bands in population i, conducted by pcr method using 10 rapd primers no primer number of amplified bands number of polymorphic bands 1 opa2 8 8 2 opa3 7 6 3 opa7 8 8 4 opa9 7 6 5 opa13 10 10 6 opa16 13 13 7 opa18 8 8 8 opa19 6 6 9 opj11 2 1 10 opj13 4 4 total number of bands 73 70 percentage (%) 100 95.9 note: population i was derived from the four-year cultures regenerated through shoot proliferation methodin vitro 113 figure 1 profiles of dna variants of pineapple cultures in population i (regenerated through shoot proliferation method), amplified by pcr using 10 rapd markers. m = kb ladder; n = control plant from field; v13 = dwarf posture; v10 = albino stripe leaf; v8 = narrow leaf; v7 = spiness spot leaf; v21 = erect posture; v12 = wider leaf; v14 = powdery leaf; v4 = climb posture; v2 = needle leaf; and v15 = ultra dwarf posture rapd analysis to detect somaclonal variation of pineapple cultures roostika . – et alin vitro opa2 opa3 opa9opa7 opa13 opa16 opa18 opa19 opj11 opj13 114 biotropia vol. 22 no. 2, 2015 figure 2 variability of variants derived from four year-old cultures of pineapple cultivar smooth cayenne regenerated through shoot proliferation method (population i), performed by 10 rapd markers, based on (sahn)-upgma analysis (r=0.97). n = control plant from field; v13 = dwarf posture; v10 = albino stripe leaf; v8 = narrow leaf; v7 = spiness spot leaf; v21 = erect posture; v12 = wider leaf; v14 = powdery leaf; v4 = climb posture; v2 = needle leaf; and v15 = ultra dwarf posture coefficient of similarity 0.32 0.47 0.62 0.77 0.91 n v13 v10 v8 v7 v4 v15 v21 v2 v12 v14 figure 3 profiles of dna variants of pineapple cultures in population ii, amplified by pcr using 10 rapd markers. the arrows show polymorphic bands. kb = kb ladder; 1 = control plant; 2 = cultures derived from direct organogenesis; 3 = cultures derived from indirect organogenesis induced by 2,4-d 21 µm; 4 = cultures derived from indirect organogenesis induced by 2,4-d 41 µm; 5 = cultures derived from indirect organogenesis induced by 2,4-d 62 µm; 6 = cultures derived from somatic embryogenesis induced by 2,4-d; 7 = cultures derived from somatic embryogenesis induced by picloram 115 shorter period of culture could suppress the somaclonal variation of pineapple in order to further confirm our finding that culture period is the main contributor of genetic variation occurred in the culture of in vitro pineapple, the new population (population iii) was generated through shoot proliferation and direct organogenesis regeneration methods. the use of opa primers was also better than opj. it can be concluded that opa primers were more suitable for detecting pineapple off-type than opj primers. interestingly, the new population revealed a higher level of similarity. the level of polymorphism in the population iii was lower than that in population i (fig. 5; table 4). the ntsys analysis yielded a dendrogram where similarity coefficient ranged from 0.65-1.0. it separated the regenerants at the similarity coefficient of 0.65-0.85 (r=0.96) to the control plant. it showed that the grouping is shifting to right, indicated the increase of similarity degree. it can be concluded that the use of the new population was better than the old population. table 3 the number of amplified product and polymorphic bands of population ii, conducted by pcr method using 10 rapd primers no primer number of amplified bands number of polymorphic bands 1 opa2 9 9 2 opa3 9 9 3 opa7 5 5 4 opa9 8 8 5 opa13 6 6 6 opa16 5 5 7 opa18 8 8 8 opa19 5 3 9 opj11 5 4 10 opj13 5 3 total number of bands 65 60 percentage (%) 100 92.3 note: population ii was derived from the normal phenotype-selected plantlets of four-year cultures, regenerated through direct in vitro organogenesis, indirect organogenesis and somatic embryogenesis figure 4 variability of cultures of pineapple cultivar smooth cayenne in population ii, performed by 10 rapd markers, based on (sahn)-upgma analysis (r=0.98). n = control plant; bn = cultures derived from direct organogenesis; d21 = cultures derived from indirect organogenesis induced by 21 µm 2,4-d; d41 = cultures derived from indirect organogenesis induced by 41 µm 2,4-d; d62 = cultures derived from indirect organogenesis induced by 62 µm 2,4-d; esd = cultures derived from somatic embryogenesis induced by 2,4-d; esp = cultures derived from somatic embryogenesis induced by picloram coefficient of similarity 0.34 0.50 0.66 0.82 0.99 s bn d21 d41 d62 esd esp rapd analysis to detect somaclonal variation of pineapple cultures roostika . – et alin vitro table 4 the number of amplified product and polymorphic bands of population iii, conducted by pcr method using 10 rapd primers no primer number of amplified bands number of polymorphic bands 1 opa2 4 4 2 opa3 3 2 3 opa7 5 2 4 opa9 4 3 5 opa13 5 1 6 opa16 6 6 7 opa18 6 3 8 opa19 8 8 9 opj11 3 3 10 opj13 4 4 total number of bands 48 36 percentage (%) 100 75 note: population iii was derived from the new cultures, regenerated by shoot proliferation and direct organogenesis in vitro method figure 5 profiles of variants dna of pineapple cultures in population iii, amplified by pcr using 10 rapd markers. kb = kb ladder; (1) clone simadu from field; (2) clone non-simadu from field; (3) normal phenotype plant derived from shoot proliferation method; (4) cultures of clone simadu derived from shoot proliferation; (5) cultures of clone nonsimadu derived from shoot proliferation; (6) cultures of clone simadu derived from direct organogenesis; and (7) cultures of clone non-simadu derived from direct organogenesis. the arrows showed several polymorphic bands biotropia vol. 22 no. 2, 2015 116 figure 6 variability of cultures of pineapple cultivar smooth cayenne in the population iii, performed by 10 rapd markers, based on (sahn)-upgma analysis (r=0.96). s = clone simadu from field; ns = clone non-simadu from field; ni = normal phenotype plant derived from shoot proliferation method; ps = cultures of clone simadu derived from shoot proliferation; pns = cultures of clone non-simadu derived from shoot proliferation; os = cultures of clone simadu derived from direct organogenesis; and ons = cultures of clone non-simadu derived from direct organogenesis coefficient of similarity 0.32 0.49 0.66 0.83 1.00 s ni ns pns ps os ons 117 monitoring the identity of cultures is important to establish the stability of a particular trait (soneji . 2002). our result showed that the et al molecular pattern of the normal phenotype plant derived from shoot proliferation method was identical with the control plant of clone simadu from field. therefore, phenotypic screening in the individual cultures is considered to be applied as early detection of plant off-type during micropropagation. rapd assay can also be applied as early detection of somaclonal variation in the population. according to bairu (2011), et al. molecular technique enables detection of variants at the juvenile stage. furthermore, getting rid of variants also could be performed in nurseries to avoid the occurrence of off-type in mass seedlings production (mohamed 2007). generally, it can be concluded that the longperiod of cultures is the main contributor of somaclonal variation of pineapple, while the regeneration method and plant growth regulator could induce genetic variation. according to leva et al. (2012), the system by which the regeneration is induced, type of tissue, explant source, media components and the duration of the culture cycle are some of the factors that are involved in inducing variation during culture. in vitro conclusions rapd assay could detect genetic changes in micropropagated pineapple so that it can be used as early detection of off-type plant. the use opa primers were better than opj primers. the longperiod of pineapple cultures was the main contributor of somaclonal variation. the use of direct organogenesis method was more recommended than the application of the indirect organogenesis and somatic embryogenesis since it does not undergo callus formation. acknowledgements the authors acknowledged the financial support by indonesia agency for agricultural research and development, ministry of agriculture of the republic of indonesia, through kkp3n program 2011. the authors also rapd analysis to detect somaclonal variation of pineapple cultures roostika . – et alin vitro thanked prof dr g.a. wattimena and prof dr ika mariska for their supervision and critical suggestions. references abdellatif kf, hegazy ae, aboshama hm, emara ha, elshahed aa. 2012. morphological and molecular characterization of somaclonal variations in tissue culture-derived banana plants. j genet eng biotechnol 10:47–53. ahmed aba, rao as, rao mv, taha m. 2011. effect of picloram, additives and plant growth regulators on somatic embryogenesis of (l.) phyla nodiflora greene. braz arch biol techn 63: 147-73. b the effect of airu mw, fennell cw, staden j van. 2006. plant growth regulators on somaclonal variation in cavendish banana ( aaa cv. 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roostika . – et alin vitro antibreast cancer activity of nanopropolis indonesia induced mammary gland tumor by on dmba in virgin sprague-dawley rats akhmad endang zainal hasan jumali1,2 2*, d mangunwidjaja , t c unarti , o suparno a setiyonoiti andra s no gus2 2 3and 1 institut pertanian , bogor 16680, indonesiadepartment of biochemistry, faculty of mathematics and sciences, bogor 2 epartmen engineering and td t of agroindustrial technology, faculty of agricultural echnology, institut pertanian bogor bogor 16680, , indonesia 3d t of clinic, reproduction and pathology, faculty of veterinary, epartmen institut pertanian bogor, bogor 16680, indonesia received 15 march 2015/accepted 15 may 2016 abstract the objective of this study was to determine the effect of nanopropolis induced rat mammary to cure cancer on tumor . after the first tumors appearance, twenty rats were using 7,12-dimethylbenz(a)anthracene (dmba) eight divided into seven groups. group 1, 2 and 3 served as , 32 and 56 recipient of nanopropolis dosages 8 µg/ml treatments served recipient of dosage µg/ml treatment served recipient of ; group 4 as propolis of 233 ; group 5 as do orubi in treatment served recipient of treatment (control) x c ; group 6 as dmba and group 7 as normal group . the effect of nanopropolis of 32 µg m and propolis 233 µg m were similar in reducing umor dosage / l dosage of / l t size, healing the wounds caused by the tumor and it turns out that there is a relationship eliminating cancer cells. between particle size absorbent material . the study suggest that nanopropolis was very s ed with small concentration effective treat mammary gland tumors and breast cancersto rat . keywords: breast cancer nanopropolis, propolis, prague-dawley rat, s introduction cancer is one of the leading causes of death in the world, especially in developed countries and the second killer in developing countries. based on data from the hospital information system (sirs) in 2007 breast cancer ranks first until 2013, in hospitalized patients in all hospitals in indonesia (16.85%), followed by cervical cancer (11.78%) (ministry of health 2013). in accordance with opinion of , tet almuir . (2003) he cause of this disease a cover of estrogen-are dependent pathway, circulating androgen, estrogen and exposure to carcinogenic materials such as smoke, ultraviolet radiation, improper diet and stress; except for age and race that cannot be changed (bates american cancer society 2016). in the early stage, breast cancer is only in the form of a lump in the mammary tissue and in an advanced stage resulting an injury to the mammary tissue. t this disease he ways to treat are the removal of tissue, radiation therapy and chemotherapy. radiat ion therapy and chemotherapy aim to destroy cancer cells are ed and control the disease so will the main tumor be shr nk, tumor growth will be slowed and u spread of cancer cells to other tissues will be prevented. now been a lot of research esadays, there have to obtain cancer , either directly medications for the treatment of cancer to reduce theor side effects of chemotherapy. the use of medications such as doxorubicin is one to inhibit cancer mean growth and reduce the occurrence of new cancers. however, the negative impact of doxorubicin treatment is the occurrence of hair loss, heart rhythm disorders and decreased white blood cell count. there is a challenge to find cancer treatment is effective and minim which ize bad effects of the main treatment. ne effort is to o biotropia vol. 23 no. 1, 2016: 35 41 35 doi: 10.11598/btb.201 .2 . .6 3 1 473 * c orresponding author: , zainalhasan@ipb.ac.id pakzainalhasan@gmail.com mailto:zainalhasan@ipb.ac.id mailto:pakzainalhasan@gmail.com 36 find and develop from medication herbs and to do on herbal medication. medication clinical test originated from herbs must be tested through a series of studies prior to its usage in cancer therapy. the tests should not involve animals. abba sa l ip ou rka b i r . (2 010) a nd e t a l purushothaman . (2012) investigated the et al incidence of breast cancer using virgin rats of sprague-dawley strain as test animals. in-vitro showed th studies at the origin of propolis locations in indonesia from five and nanopropolis from pandeglang ere le w ab to inhibit the growth of mcf-7 cell line. the nanopropolis inhibit the growth of mcf-7 cells ed at very low concentrations compared propolis to instead of nanoparticles . the (hasan . 2014)et al purpose of this study was examineto the in-vivo antibreastcancer activity of nanopropolis on the dmba induced of virgin sprague-dawley rats. materials and methods preparation of nanopropolis nanopropolis were prepared in three stages of homogenization using high speed homogenizer . some modifications were done for preparing of nanopropolis particle as previously mentioned by aimi . (2009), bhaskar . (2009), hasan . et al et al et al (2012), chen (2006) and kim . (2008)et al. et al . mammary umor nductiont i twenty eight rats were intraperitoneally injected using a m with ixture of dmba ( concentration of /25 mg kgbody weight), olive oil and physiological saline. experimental esignd after the first tumors appearance, twenty eight rats were divided into seven groups, with three animals each group. group 1, 2 and 3 served asfor recipient of nanopropolis dosages of 8, 32 and 56 µg/ml treatments served ; group 4 as recipient of dosage of 233 µg/ml propolis treatment served recipient of ; group 5 as do orubi in treatment; served x c group 6 as recipient of treatmentdmba ; and group 7 as (control)a normal group . tumor stwo dimen ional tumor areas were calculated as an ellips (abbasalipourkabir . 2010). the e et al volume of tumor was calculated by the formula of: 2v = ab /2 w here a = the longest diameter = b the shortest diameter of tumor at sacrifice, the tumor were removed for histopathological . examination hi topathology analysiss siaafter euthana , at the end of the study, the mammary tumor masses of rats were removed. sample of tissues were fixed immediately in 10% formalin overnight, embedded in paraffin, cut into 4 m sections and stained with hematoxylin-μ eosin (he). results and discussion the results of body weight rats measurements after injection and treatment were presented in figure 1. een in figure 1 normal rat body as s , weight continued to increase with time , addition as well as other treatments. the dmba treatment showed a decrease in body weight. according to cordeiro d kaliwal (2011), an effect of dmba can reduce body weight rat caused by the of nature of its toxicity despite an increase in tumor volume. tumor progress can be determined by measuring the volume test animal tumor of (abbasalipourkabir 2010; purushothaman et al. et al et al in the study of. 2012; martic . 2011). abbasalipourkabir . (2010) volume et al , tumor was calculated by measuring the length and width of the swelling, while et al.purushotaman (2012) an calcu d the tumor et ald martic . (2011) late volume by measuring the length, width and height of the tumor section. esults in mammary tissue r tumor volume of research data using the method of abbasalipourkabir (2010) can be seen in et al. figure een in figure 2 increasing tumor 2. as s , volume in dmba treatment happened only without propolis or nanopropolis treatments. biotropia vol. 23 no. 1, 2016 weeks after treatment antibreast cancer n akhmad e z h – et al. activity of nanopropolis indonesia o induced mammary . . 37 the propolis or nanopropolis treatments performed in rats decreased tumor volume. even in the 32 and 56 nanopropolis µg/ml treatment and 233 µg/ml propolis treatment there were decline after an increase in tumor volume in the fi th week after being given treatment. when f viewed from the point of decline, the injection of 56 µg/ml treatment hadnanopropolis sharp corners the tumor size reduction process , o red rccur very quickly. this occur ence may be due to the relatively high dose of the 50(6 x ic ) ingredients in nanopropolis sufficient to that is eliminate cancer cells. influence the amount of of the active ingredient component in nanopropolis ) was in trumental in (32 and 56 µg/ml s comparison with the influence of the size particle of the repair the component nanopropolis for tissue from tumor to compare propolis at 56 µg/ml. n an o pr o po l i s t r e a t m ent w i t h t h e concentration 8 ha been done to heal of µg/ml d cell and tissue, although there are tumor to repair figure 1 dy weight of rats after induction of dmba and prior necropsy (1 = group nanopropolis 8 µg, 2 = bo s of group of nanopropolis 32 µg, 3 = group nanopropolis 56 µg, 4 = group propolis µg, 5s s of s of 233 = doxorubicin s of group , 6 = group dmba and 7 = normal control weeks after treatment figure 2 tumor volume of mice after induction by dmba prior to necropsy (1 = group nanopropolis 8 µg , of /ml 2 = group of nanopropolis 32 µg , 3 = group nanopropolis 56 µ , 4 = group propolis /ml of g/ml of 233 /ml doxorubicin of group µg , 5 = group, 6 = group dmba and 7 = normal control ) 38 biotropia vol. 23 no. 1, 2016 cells. on tre tment with a concentration of 32 a µg/ml, had occurredformation of intact tissue to washeal and subcutaneous fat formed and there were e rpr sence of intact epide mal glands and many gl bular epithelium as well as o s reversal of in ,tact tumor draining wounds. similarly nanopropolis treatment at a concentration 56 of µg/ml r to lower occu red heal much better than concentration of result nanopropolis. this show thated the amount of active ingredient content affected more to the healing rate of cancer. figure 3 showed the result of administration of nanopropolis on rat mammary tissue (32 and 56 ) which ha repair skin µg/ml d ed tissue after induced by dmba being . t issue conditions were better in he t nanopropolis concentration of fig 456 µg/ml ( . ), the formation of new tissue was much more than giving nanopropolis at concentration of and 8 32 µg/ml. d of this prove that the administration nanopropolis increasingly play a role in ed mammary tissue wounds caused by dmba induced tumor results. this result showed that the amount of active ingredient affected the healing rate of cancer. the greater the concentration nanopropolis used, the greater of the effect tumor tissue healing wounds. the on influence of different concentrations and time of administration of propolis against different cancer cells ha also been studied by d bufalo . et al (2007). presence of a tumor as a result of wound healing propolis administration s the using wa formation of epithelial tissues been that had studied by de moura . (2011), although using et al different propolis. this condition was supported by the presence of components of propolis (hasan . 2014) or mineral contents (hasanet al et al. 2013). at a concentration of the 56 µg/ml mammary were healthy and aesthetically alveoli clean in figure 4a seen the contain . it is ingalveoli blood plasma (blue arrows pointing as result of ) a mammary tissue repair. on other rats the groups (dmba or negative control) in which group nanopropolis or propolis injection was not p , erformed mammary tissue condition was still going on angiogenesis and cancer cells enters blood vessels. in this condition, cancer cell figure 3 the mammary tissue of virgin rat after induced by mba and received treatment of being d injection a. nanopropolis (32 µg/ml) b. nanopropolis (56 µg/ml) ( = skin epi = and blue arrow thelium, yellow arrow normal hair licles white arrow capillary epidermis he stainingfol , = ) ( , 200x) figure 4 a) the ammary tissue of rats sd induced by dmba and nanopropolis every seven days within m 56 mg/ml two months, b) the mammary tissue of rats sd induced by dmba and no treatment within two months after induction (black arrow blue arrow = filled blood plasma white arrow = cancer cells, = , with , alveole alveole red arrow = connective tissue) he staining, ( 200x) 39 spread in so much connective tissue and almost eve y cancer cells r tissue as is shown in figure 4b ( (white arrows) and connective tissue (red arrows) .) the physical condition of the mammary tissue healing drying can be seen when condition occur in an area that has suffered injury due to red the tumor fig 5a while the mammary tissue ( . ). was swollen , caused by the tumor the effect of dmba induced in virgin rats were large swell (fig. 5b). even one rat in this group had inflammation of the mammary tissue. his condition can be t refe red to as stage iv breast cancerr . tested activities of propolis curing cancer proved dosage 233 µg/ml c that a of an heal damage by the tumor tissue compared s caused with the positive control treatment without induction by dmba treatment. positive control (without induction by dmba) does not cure cancer develop accumulation of and even ed cancer cells and tissue damage fig 6( . ). inoue et al. (2008) showed that the inhibition of cancer growth occur ed after concentration of r propolis 320 µg/ml. , bermúdez (2006) similarly et al. reported that 10%wound healing with propolis only occur as much as only the 60% for occurrence . in breast of re-epithelialization tissue repair seen around the damaged can be tissue with (blue arrow) and re-epithelialization hair l ( for the follic es yellow arrow) in figure 6a. dmba research, induced mammary wounds were formed in the mammary tissue and there were many cancer cells fig ( . 6b). f shows drying igure 7 cancer cells condition with black spots on the new blood vessels, but there are still cancer cells expected to remain active. this is because the treatment required quite long , so that the healing process a time was still running and still not finished on was . treatment with propolis 233 µgdosage of /ml, the drying process indicated the healing in tissues affected by cancer. dosage µg/ml at of 32 nanopropolis has the ability to cure cancer which to has the same effect as much as 233 of propol is µg/ml administration. the particle size in nanopropolis is very small so active ingredient remaining in , nanopropolis can log into the network with ease, while the of propolis with 233 dosage µg/ml which was active against tumor growth is caused figure 5 a) the physical condition of the mammary tissue healing can be experienced with drying occur in an area that has s suffered injury due to tumor after injection of b) non treatment or only induction of nanopropolis 32 µg/ml, dmba c) for ( = , and condition before treatment after induction of dmba 90 days yellow arrow dry wound, white arrow mammary swelling bec use of tumor = a ) figure 6 rat mammary tumor tissue after being induced by dmba and treated with injection of (a) propolis (233 virgin μg/ml) and (b) without treatment (positive control) (blue arrow = skin epithelium, yellow arrow = normal hair follicle, white arrow = inflammation) (he staining, 200x) antibreast cancer n akhmad e z h – et al. activity of nanopropolis indonesia o induced mammary . . 40 biotropia vol. 23 no. 1, 2016 by the presence of the active components in propolis prevent tumor progression. this fact ing was caused by the presence of compounds in either nanopropolis or propolis, such as organic acids like firulic acid and acid, polyphenols caffeic and flavonoids in propolis inhibit the which proliferation of cancer cells ole of . the r flavonoids and ca fe acid is to inhibit the f ic formation of protein kinases that are used for cell proliferation result is going cell which to inhibit formation process and apoptosis induced occur (madeo . 2004). in accordance rence et al with the results of de moura (2011), the et al. healing of wounds caused by the tumor may occur due to administration of propolis. meanwhile, according to sun . (2012) crysin components et al present in propolis can decrease the volume of tumor that occurs in the mammary tissue of dmba induced . the research conducted by mice bhattacharjee . (2012) and lim . (2011) et al et al states that there is a relationship between particle size absorbent materials on the healing of cancer. conclusions the effect of nanopropolis of 32 s with dosage µ /ml with dosage of µ /ml g and propolis 233 g w the same in ere reducing tumor size, healing the wounds caused by the tumor and eliminat ing cancer cells. it turn out that there is a ed relationship between particle ize absorbent s material . the study s on the healing of cancer suggest that nanopropolis ed with small concentration (dosage of 32 µg/ml) was very effective for the treatment of rat mammary gland tumors and breast cancers. acknowledgements the authors to thank bpps fellowship wish of indonesian ministry of education and culture, and seameo biotrop for their financial supports and institut pertanian bogor for its facilities for conducting this study. references abbasalipourkabir r, salehzadeh a, abdullah r. 2010. antitumor activity of tamoxifen loaded solid lipid nanoparticles on induced mammary tumor gland in sprague-dawley rats. afr j biotech 9(43):733745. aimi m, nemori r, ogiwara k .. 2009. casien nanoparticle inventor. bates american cancer society [internet]. 2016. causes, risk factors, and prevention topics. usa: american cancer society; [cited 2016 may 31]. available from: http://www.cancer.org/cancer/ breastcancer/detailedguide/breast-cancer-riskfactors. bermúdez ic, garcía gs, piloto aa, pérez yf, valdivieso ag. 2006. effect of the cuban propolis collected in manzanillo area on the wounds healing in rats. pharmacol 3:416-21. bhaskar k, anbu , ravichandiran , venkateswarlu , rao j v v ym. 2009. lipid nanoparticles for transdermal delivery of flurbiprofen: formulation, in-vitro, ex-vivo and studies. lipids health disease 8(6):758-in vivo 76. bhattacharjee s, erchov d, fytianos k, van der gucht j, alink gm, rietjens imcm, marcelis atm, zuilhof h. 2012. cytotoxicity and cellular uptake of triblock copolymer nanoparticles with different size and surface characteristics. particle fibre toxicol 9(11):1-19. figure 7 mammary tissue (a) and skin tissue (b) dmba-induced sd rats treated with propolis at a dosage of 233 g/ml µ every 7 days within 2 months (red arrows = vein, white arrow = dead cancer cells, black arrow = hair follicle) (he staining, 200x) http://www.cancer.org/cancer/ 41 bufalo mc, candeias mgj, sforcin jm. 2007. in vitro cytotoxic effect of brazillian green propolis on human laryngeal epidemoid carcinoma (hep-2) cells. advance access pub 6(4):483-7. chen m, diao g, zhang e. 2006. study of inclusion complex of beta-cyclodextrin and nitrobenzene. chem 63:522-9. cordeiro mc, kaliwal bb. 2011. antioxidant activity of bark extract if spreng on dmba bridelia retusa induced mammary carcinogenesis in female sprague dawley rats. j ph rm 2(1):14-20.a de moura sal, negri g, salatino a, lima ldc, dourado lpa, mendes jb, andrade sp, ferreira mand, cara dc. 2011. aqueous extract of brazilian green propolis: primary components, evaluation of inflammation and wound healing by using subcutaneous implanted sponges. evid based complement alternat med 2011:1-8. hasan aez, ambarsari l, artika im, julistiono h, tarunasari d. 2013. induction resistance of candida sp. y390 to ethanol stress by kopyor coconut and virgin coconut oil. emir j food agric 25(10):790-7. hasan aez, artika im, fahri vr, sari n. 2012. potency of nanopropolis from stingless bee spp. as trigona antibacteria gents. chem progress 5(1):1-7.a hasan aez, mangunwidjaja d, sunarti tc, suparno o, setiyono a. 2014. investigating the antioxidant and anticytotoxic activities of propolis collected from five regions of indonesia and their abilities to induce apoptosis. emir j food agric 26(5):390-8. inoue k, saito m, kanai t, kawata t, shigematsu n, uno t, isobe k, liu ch, ito h. 2008. antitumor effects of water soluble propolis on a mouse sarcoma cell line and . am j chin med 36(3):625-34.in vivo in vitro kim dm, lee , aum , kim 2008. preparation of gd sh hj. propolis nanofood and application to human cancer. biol pharm bull 31(9):1704 10. lim hn, nurzulaikha r, harrison i, lim ss, tan wt, yeo mc. 2011. spherical tin oxide, sno particles 2 fabricated via facile hydrothermal method for detection of mercury (ii) ions. int j electrochem sci 6:4329-40. madeo f, herker , wissing , jungwirth , eisenber , e s h t frohlich . 2004. apoptosis in yeast. current ku opinion microbiol 7:655–60. martic k, vlacic z, rudman f, lambasa s, tomasovicloncaric c, stanec z. tumor and reast 2011. b volume ratio as a predictive factor for axillary lymph node metastases in t1c ductal invasive breast cancer: prospective observational clinicopathological study. jpn j clin oncol 41(12):1322-6. ministry of health . 2013. [internet] indonesia: ministry of health; [cited 2013 june 14]. available from: http://www.depkes.go.id/index.php/berita/pressrelease/2233-seminar-sehari -dalam-rangkamemperingati-hari-kanker-sedunia-2013.html. muir d, kanthan r, kanthan sc. 2003. male versus female breast cancers. arch pathol lab med 127(1):36-41. purushothaman a, nandhakumar e, sachdananram p. 201 . anticancer effect of shemamrithaa (a 2 phytochemical formulation) on 7,12-dimethyl benz(a)anthracene induced mammary carcinoma in rats. asian j pharm clinical res 5(1):101-7 . sun lp, chen al, hung ac, chien yh, huang js, huang cy, chen yw, chen cn. 2012. chrysin: a histone deacetylase 8 inhibitor with anticancer activity and a suitable candidate for the standardization of chinese propolis. j agric food chem 60:11748-58. antibreast cancer n akhmad e z h – et al. activity of nanopropolis indonesia o induced mammary . . http://www.depkes.go.id/index.php/berita/pressuntitled biotropia vol. 21 no. 1, 2014: 59 70 doi: 10.11598/btb.2014.21.1.6 f* r r r f r /identification of aspergillus flavus and detection of its aflatoxin genes isolated from peanut and peanut processed products kemala s. nagur1'2, namplah sukarno1 and sri listiyowati1 ' department of biology, faculty of mathematics and natural sciences, bogor agricultural university, dramaga campus, bogor 16680, indonesia 2nationalquality control laboratory of drug and food, national agency of drug and food control,jakarta 10560, indonesia received 2 may 2014/accepted 8july 2014 abstract aspergillusflavus is one of the main producers of aflatoxin. therefore, the presence of the fungus is becoming serious problem on food safety. this research was aimed to isolate and identify a. flavus from peanut and its processed products collected from several traditional markets in bogor, depok and jakarta, and to detect the aflatoxin genes. fungal identification was carried out using morphological characteristics and species specific primers of fvaviq1/flaq2 and afla-f/afla-r,while detection of aflatoxin genes employed four specific primers of apa-2 (,aflk), nor-1 (afld), ver-1 {aflm) and omt-1 (aflp). from 36 samples, the a. flavus group was only found in peanut kernels samples with viable count of specific colonies in the range of 0.01-5.52 x 104 cfu/g. eighteen isolates were identified as a.flavusbased on species specific primers, fvaviq1/flaq2 and afla-f/afla-r by producing amplicons of about 100 and 413 bp, respectively. based on aflatoxin gene analysis, all 18 isolates successfully amplified by both apa-2and nor-1,83.3% by omt-1 and 72.2 % by ver-1 primers which involved in aflatoxin production.the amplicons size of apa-2, nor-1, ver-1 and omt-1 primer pairs were about 1032,400,895and 1024 bp, respectively. keywords: aflatoxin genes,afpa, aspergillusflavus, peanut, species specific primers introduction peanut is an important agriculture commodity after rice, maize and soybean in indonesia. humidity and tropical climate make peanut kernels and processed products easily infected by fungi particularly during inadequate drying and improper storage which results in physical damage, discoloration, lower quality of nutritional content and mycotoxin contamination of the products (sauer etal.1992). * corresponding author : nampiahsukarno@yahoo.com 59 biotropia vol. 21 no. 1, 2014 mycotoxin causes serious problems to human and animal health. aflatoxin is the most toxic compound in mycotoxin group, it is carcinogenic and teratogenic (jecfa 1997). there are four naturally forms of aflatoxin, namely b, (afb,), b2 (afbj, g, (afg,), and g2 (afg^). afb, was the most hazardous because it was very carcinogenic among other form of aflatoxin and usually found at the highest concentration in contaminated food including peanut and processed peanut products (pitt 2000). aflatoxin is mostly produced by aspergillusjlavus and a.parasiticus, which belongs to the aspergillus section flavi. aspergillus parasiticus produces both afb and afg, while a.jlavus produces only afb, and afb2, but not all strains of a.jlavus are able to produce aflatoxin (pitt & hocking2009). aflatoxin quantification is commonly done using thin layer chromatography and high performance liquid chromatography but the studies on the level of fungal infection and the identification of aflatoxin producing fungi could be an alternative for was produced. >f aflatoxigenic fungi by using specific medium aspergillusjlavusparasiticus agar (afpa) developed by pitt et al. (1983). this medium is suitable and recommended for determining a.jlavus and a.parasiticusin food and feed due to its simplicity in application since the detection needs only 2 days incubation.the afpa medium, however, could not differentiate a. jlavus from a. parasiticus, therefore, further differentiation method need to be used such as morphological characteristics by microscopic and culture techniques. these techniques, however, are time consuming and may result in false positive. the afpa medium also can not differentiate between aflatoxigenic and non-toxigenic a.jlavus. several studies had been done by using polymerase chain reaction (pcr)-based methods using full length of its regions and species specific primers to identify a. jlavus. these methods are more sensitive compared to conventional method. there are several species specific primers available to identify a.jlavus such as fvaviq1/ flaq2 (sardinas etal.2011) designed from its2 rdna region and afla-f/aflar (hue et al. 2013) designed from aflatoxin biosynthesis sequences published on genbank.these studies indicated that the primers arevery specific and able to amplify a.jlavusonly. it has been reported that not all strains of a.jlavus are aflatoxin producer. it is, therefore, important to detect a. jlavus carrying aflatoxin gen for controlling the aflatoxin contamination on the products. this determination is also important to be done before the toxin is expressed in early development of the fungi. the aflatoxin biosynthesis pathway involved 25 genes that clustered in a 75-kb dna region (bhatnagar et al. 2006). there are four pairs of primers, apa-2, nor-1, ver-1 and omt-1, available to identify aflatoxin genes in a.jlavus. the apa-2 gene (ajik) involved in regulation of aflatoxin biosynthesis by controlling the expression of the nor-1 and ver-1 genes (liu & chu 1998; woloshuk et al.1994). the nor-1 gene encodes norsolorinic acid reductase and converts norsolorinic acid to averantin (chang etal.1992).the ver-1 gene encodes versicolorin a dehydrogenase, and converts versicolorin a to sterigmatocystin (skory et al. 1992). the omt-1 gene encodes sterigmatocystinomethyltransferase and is required for conversion of demethylsterigmatocystin and dehydrodemethylsterigmatocystin to sterigmatocystin and dihydrosterigmatocystin, indicating the quality of the food and feed products before the toxin several studies had been done to detect, quantify and identify species c k — \ 60 r r s r f r* identification of aspergillus jlavus and detection of its aflatoxin — kemala s. nagur et al respectively (yu et al. 1995a). all these primers had been studied and used to detect toxigenic a. jlavus in grains, foods and feeds successfully (farber et al 1997; manonmani etal 2005). aspergillusjlavus is able to grow on various nutrient sources. in indonesia, many commodities such as peanuts, maize, pepper and feed ingredients were reported to be contaminated by the fungus and caused high level of aflatoxin concentration in the commodities. about 70% of the peanut kernels samples collected from retailers in bogor, cianjur and wonogiri regencies contained more than 15 ppb of aflatoxin.the highest percentage of peanut kernels infected hy a.jlavus was found at the retails level in the traditional markets (dharmaputra etal 2005;2007). early detection of aflatoxin producer needs to be done in order to improve the quality of the commodities and to formulate strategy on prevention and control of aflatoxin contamination in the products.the aim of this study was to detect growth of aflatoxin producing fungus, a.jlavus by isolation and identification of the fungi and detection of the fungal ability to produce aflatoxin from peanut kernel and its processed products. fungal isolation was done using afpa specific medium, identification by morphology and molecular methods, and detection of aflatoxin genes involved in aflatoxin biosynthesis pathway was carried out using aflatoxin specific producer primers. materials and methods isolation of aspergillus flavusgroup thirty six samples of peanut kernels, roasted peanuts with skin pod, flour-coated peanuts, and branded bumbu pecel (dry peanut sauce) were collected from traditional markets in bogor, depok and jakarta. fungal isolation was done by dilution plating method on afpa medium (nmkl 2004). twenty five grams of each sample was grounded separately using a blender in medium speed (scale 3 out of 5) (philips), then suspended in solution containing 0.1% peptone and 0.025% tween 20 (1:10, w/v). the samples were then homogenized by stomacher for 2 min and treated into several serial dilutions 1:1000. a total volume of 100 pi of each dilution was spread onto duplicate afpa plates (oxoid) using sterile glass rod, incubated at 30±1 °c for 48±3 h. specific colonies with bright yellow orange on lower side of the plates were recorded as the number of colony forming units (cfu).the colonies were cultured and maintained on potato dextrose agar medium for further analysis. identification of aspergillusjlavusgroup total of 18 isolates were recovered from the samples. isolated a.jlavus groups were identified by morphological and molecular methods. the morphological identification was carried out using the method described by pitt & hocking (2009). molecular analysis was carried out using two species specific primers of fvaviq1/flaq2 and afla-f/afla-r developed by sardinas etal (2011) and hue etal (2013), respectively. for comparison of pcr analysis, five strains were also 61 biotropia vol. 21 no.1, 2014 used as standard cultures for positive and negative controls.the strains were a. flavus 1a1 obtained from seameo biotrop (bogor, indonesia), a.flavusn&rc 33021 and a. flavus nbrc 30107. two species, a. parasiticus nbrc 33224 and a. nomius nbrc 33223were also included in the analysis. fungal dna was extracted according to the method described by raeder and broda (1985) with some modifications by using phenol-chloroform-isoamyl alcohol (pci) and chloroform-isoamyl alcohol (cl) instead of chloroform and isopropanol. each fungal strain was grown in 100 ml flask containing 50 ml potato dextrose broth (pdb) in rotary shaker agitated at100 rpm in room temperature for 3days.at harvest, the mycelium was filtered using whatman #2 and washed with sterile distilled water. one gram of washed mycelium was grounded using a mortar and liquid nitrogen. dna concentration was measured by nanodrop 2000 spectrophotometer (thermo scientific) and kept in -20°c for further analysis. pcr reactions were performed using two pairs of species specific primers for identification of a. flavus. the primers pairs were fvaviq1/flaq2 and aflaf/afla-r with the sequences 5’-gtcgtcccctctccgg-3’ for fvaviq1 and s’-ctggaaaaagattgatttgcg-t for flaq2 to amplify a fragment of 100 bp (sardinas etal.2011); 5’-ggtggtga-agaagtctatctaagg-3' for aflafand 5'-aaggcataaagggtgtggag-3' for afla-r to amplify a fragment of 413 bp (hue et al. 2013). amplification of fungal dna was performed in a total volume of 25 pi. the reaction mixtures contained 12.5 pi pcr master mix 2x (promega),12.5 pmol of each primer, ±100 ngdna template and nuclease free water. amplification reaction was performed as follows: pre-denaturation for 5 min at 94 °c and followed by 35 cycles for 30s at 94 °c for denaturation, 60s at 58 °c for annealing, 90s at 72 °c for extension,and 7 min at 72°c for final extension by using the multigene optimax thermal cycler (labnet international, inc). the pcr products were analyzed on 1.0% agarose gel in lx tae buffer, stained with ethidium bromide solution and visualized under uv light illumination (g box syngene). a positive control (dna of a.flavus from standard cultures) and negative control (no dna target and dna of a.parasiticus nbrc 33224 and a. tf^/anbrc 33223) were included in this analysis. detection of genes involved in biosynthesis of aflatoxin all isolates showed positive result after amplification using fvaviq1/flaq2 and afla-f/afla-r species specific primers were used for further pcr analysis to determine genes involved in aflatoxin production by using 4 primer pairs of apa-2, nor-1, ver-1 and omt-1. the pcr reactions and conditions were the same as indicated in pcr amplification of using species specific primers, except the annealing was carried out at 68 °c. the sequences and the expected pcr products of each primer are presented in table1. three isolates were selected for sequence analysis using omt-1 primers based on sampling location and their ability to be amplified by the four primers tested.the pcr products were sequenced by first base services (malaysia) using the same primer. dna sequences of omt-1 gene were analyzed with the bioedit ver.7.0.0 (hall 1999) n \ s w zr ’s 62 identification of aspergillusflavus and detection of its aflatoxin-kemala s. nagur et al. r* r *f r t* f r r f r table1. spesific primers used for detection of genes involved in biosynthesis of aflatoxin primer codes targ et gen sequence (5,-̂ 3’) size products (bp) apa2-f** apa2-r afl,r tat-ctc-ccc-ccg-ggc-atc-tcc-cgg ccg-tca-gac-agc-cac-tgg-aca-cgg 1032 norl-f* norl-r afld acc-gct-acg-ccg-gca-ctc-tcg-gca-c gtt-ggc-cgc-cag-ctt-cga-cac-tcc-g 400 verl-f** verl-r aflm atg-tcg-gat-aat-cac-cgt-tta-gat-ggc cga-aaa-gcg-cca-cca-tcc-acc-cca-atg 895 omtl-f** omtl-r aflp ggc-ccg-gtt-cct-tgg-ctc-cta-agc cgc-ccc-agt-gag-acc-ctt-cct-cg 1024 references : * geisen (1996); ** shapira (1996) and aligned using clustal w (thompson etal.1994). phylogenetic tree was performed by using neighbor-joining method model kimura 2-parameter using mega 5 with 1000 bootstrap replications (tamura etal.2011).based on previous study by varga etal.(2011), sequences areno outgroup was chosen during the analysis of omt-1 gene because no available from any other aflatoxigenic species outside aspergillussection results and discussions isolation of aspergillus flavus group based on the data obtained from 36 samples of peanut kernels, roasted peanuts with skin pod, flour-coated peanuts, and branded bumbu pecel indicated that fungal colony was only shown on peanut kernels. number of fungal population based on viable count of specific colony showed that level of fungal infection varied between locations. the number of colonies were ranging from 0.01 5.52 x 104 cfu/g, in which the highest population was found on peanut kernels collected from bogor followed by depok and jakarta areas (table 2). this might be due to bogor had higher humidity and rainfall compared to the two other locations resulted in higher humidity in room storage and moisture content of peanut kernels. the relative humidity at the time of sampling in bogor, depok and jakarta areas were 84%, 82% and 81%, respectively (bmkg 2013a,b), which would correlate with the moisture content of peanut kernels. population of a.flavus in bogor obtained in our study was 5.52 x 10 cfu/g, which was 10 fold higher than reported by dharmaputra (2010) which showed the fungal population collected from the same regency but different traditional markets location was 0.49 x 104 cfu/g. the traditional market (retailers) is the last distribution chain of the commodities beforebeing delivered to consumer.long chain distribution before being delivered to consumer contributed to the possibility of broken peanut kernels which made it easier for infection of fungi including aflatoxigenic fungi. 63 biotropia vol. 21 no. 1, 2014 table 2. population of a. flavusgtowp isolated from peanut and its processed products samples number of colony (viable count, cfu x 104 /gram) jakarta depok bogor peanut kernels processed peanut products (roasted peanuts with skin pod, flour-coated peanuts, bumbu pecel (dry peanut sauce) 0.01 2.1 5.52 0 0 0 no colony was found on roasted peanuts with skin pod, flour-coated peanuts, and branded bumbupecel samples in this study. this might be caused by heating of peanut kernels as raw material during the processing which could kill the fungus. furthermore, this could also be due to standardization method applied to peanut kernels used as raw materials in factory before being processed into peanut products as the peanut kernels were usually obtained directly from the farmers. dharmaputra etal. (2013) reported that the populations of a. flavus in processed peanut products were relatively low, which was less than 1 cfu/g in fresh weight basis. aspergillus flavus infection and aflatoxin production particularly in peanut kernels were related to methods of postharvest handling from farmers up to retailers in market and the duration of storage (dharmaputra etal. 2005). fungal identification eighteen out of 28 fungal isolates isolated from peanut kernels showed orange yellow reverse coloration on afpa medium confirming that the isolates were strains of a.flavusgroup (fig.la).the 18 isolates had similar microscopic characteristics such as conidial shapes varied from spherical to elliptical, septate hyphae, and conidial heads uniseriate (phialides only) (fig. lb, c, d). the sizes of microscopic morphological structures of each isolate are shown in table 3. differentiating a. flavus from a. parasiticus based on morphological characteristics was not easy since it could give false positive result. rodrigues et al. (2007) reported that the use of scanning electron microscopy (sem) for the conidial wall ornamentation examination was the primary character analysis for separatingof the two species in morphological identification. further identification using species specific primer showed that all isolates produced amplicon size of about 100 bp for fvaviq1/flaq2 primer and 413 bp for afla-f/afla-r (fig. 2). both primers were also successfully amplified the positive control cultures of a. flavus 141, a. flavus nbrc 33021, and a. flavus nbrc 30107, but failed to amplify a. parasiticus nbrc 33224 and a. nomius 33223 used as negative control (fig 3a and 3b). molecular analysis indicated that all 18 isolates were a.flavusspecies. our results were in agreement with the finding reported bysardinas et al. (2011) and hue et al (2013) who tested the same species specific primers pairs on several species of aspergillus spp. non a. flavus. the fvaviq1/flaq2 primer pair a s ) ’t s s 64 f* r 'f r r* f— r< /> t*• r i lentification of aspergillusjlavus and detection of its aflatoxin kemala s. nagur etal. \\l 'w m 9a 10 [im 10 fim jkgsr c 1 0-+ut1 \ d conidiofor figure1. macroscopic (a) and microscopic (b, c, d) of a. jlavus group isolated from peanut kernels in bogor,depok andjakarta table 3. size of hypha, vesicle, phialid and conidium of aspergillus jlavus group ; solated from peanut kernels in bogor,depok andjakarta isolates code size (jim) size (pm)isolates conidia hypha vesicle phialid code conidia hypha vesicle phialid j1 j2 d1 d2 d3 d4 b1 b2 b3 b4 b5 3-5 3-4 3-4 3-4 3-4 3-5 3-4 3-4 3-4 3-4 3-5 5-7 3-5 5-8 6-8 6-7 6-8 5-8 5-7 5-6 6-8 6-8 11-13 11-14 14-17 15-20 16-18 14-28 14-17 11-18 15-17 16-18 14-18 6-7 6-7 7-8 6-8 5-7 6-8 5-9 5-8 5-6 5-6 5-6 b6 b7 b8 b9 bio bll b12 747* 33021* 30107* 3-4 3-4 3-4 3-4 4-5 3-4 3-4 2-4 3-5 3-5 6-7 6-8 7-8 5-7 7-8 5-6 5-6 4-8 4-6 4-7 15-18 15-17 18-21 15-19 16-19 15-17 15-19 18-26 15-22 17-21 5-6 6-7 5-6 5-7 5-6 5-6 6-7 5-7 6-8 5-6 (*) = stani lard cultures of positive ajlavus did not amplify dna of a. jlavus group such as a. tamarii, a. bombycis,a. fumigatus, ajerreus, a. niger,a. tubingensis,a.carbonarius,ajaponicusand a. ochraceus (sardinas etal. 2011), an d afla-f/afla-r did not amplify the dna of a.parasiticus,a. ory^ae,a. niger and a.candidus (hue etal.2013). detectic n of aflatoxin genes the p opulation of a.jlavusis not always correlated with aflatoxin production, as it is depenc ting on the strain whether or not the fungus carried aflatoxin producer gene in their g enome. detection of aflatoxin genes on isolated a. jlavus were carried out using 4 p rimer pairs in this study to detect genes fragment involved in the aflatoxin biosynthetic pathway (fig. 4). the two primers apa-2 and nor-1 was successfully produced amplicons from all 18 isolates and standard cultures of a.jlavus, whereas, omt-1 amolicons detected 15 isolates and 2 standard cultures, ver-1 amplicons detected 13isolate s and 2standard cultures (table 4). 65 biotropia vol. 21 no. 1, 2014 m 1 2 3 4 5 6 7 8 9 250 bp 1 10,11.12' x3 -14; .15 16 17 18 250 bp ! a 500 bp 500 bp figure 2.agarose gel electrophoresis of pcr products from 18 dna isolates using species specific primers of fvaviq1/flaq2 (a) and afla-f/afla-r (b) (m:marker1 kb;lane1-18:samples) 250 bp m 1 2 3 4 5 m 1 2 3 4 5 s3 |a =2 500 bp rr b figure3. agarose gel electrophoresis of pcr products from 5 dna isolates using species specific primers of fvaviq1/flaq2 (a) and afla-f/afla-r (b) (m: marker 1 kb; lane 1-2 : standard cultures of a.flavus,?>\ a.flavus from samples;4-5:standard cultures of a.parasiticusand a.nomius) 1000 bp 1000 bp 1000 bp > 1000 bp < ; -j 1 2 3 4 5 6 7 8 9 500 bp 500 bp 1000 bp^ 1000 bp ^ figure 4.agarose gel electrophoresis of pcr products from 18 dna isolates using specific primers, for apa-2 (a) nor-1 (b) ver-1 (c) omt-1 (d) (m: marker 1 kb; lane 1-18:samples) a.s n ** s s s-v 66 identification of aspergillusflavus and detection of its aflatoxin — kemala s. nagur et al. ** r r r f* r in previous study, a. flavus isolated from peanut from north vietnam showed that the ver1 gene was the most representative (82%) gene followed by nor1, omt1 and apa-2 genes (73, 70 and 67%, respectively) (pham & dam 2010), while in india, aflr. (apa-2) and omt genes appeared on 80% of peanut samples (somashekar et al 2004). our study indicated that all 18 isolates carried aflatoxin producer genes. the information from this study could be used as early detection to determine which strain of a.flavus that has potential as aflatoxin producer. further study is needed to analyze the aflatoxin expression genes of a. flavus. the gene expression depends on several conditions such as transcriptional regulatory factors, physiological response and environmental factors (ph, water activity (aj and temperature) (schmidt et al. 2009; abdel-hadi etal. 2010). 99 4* 98 l25836 aspergillus flavus isolate cra01-2b b2 b1 — ay510451 aspergillus flavus isolate af13 l25835 aspergillus flavus strain 70 — ay510453 aspergillus flavus isolate af70 ioo| |— d1 651ay5104!65 •ays10455 aspergillus flavus isolate af36 figure 5. the phylogenetic tree of a.flavus ¥>\^ a. flavus ¥>2 and a. flavusdl based on omt1 sequence by neighbor-joining method model kimura 2-parameter with 1000 bootstrap replications.bootstrap values (<50%) are not shown table 4. the presence of the target genes involved in aflatoxin biosynthesis of aspergillus flavusspecies code ttarget gene code target genes no of of a. flavus apa-2 nor1 ver-1 omt-1 no a. flavus apa-2 nor1 ver1 omt1 1 + + + 11 b5 + + + + 2 j2 + + + 12 b6 + + + + 3 d1 + + + + 13 b7 + + + 4 d2 + + + + 14 b8 + + + ’ + 5 d3 + + + 15 b9 + + + + 6 d4 + + + 16 b10 + + + + 7 b1 + + + + 17 bll + + + + 8 b2 + + + + 18 b12 + + 9 b3 + + + 19 747* + + + + 10 b4 + + + + 20 33021* + + + + (+) : detected; (-) : not detected; (*) : standard cultures of positive a. flavus r 67 biotropia vol. 21 no. 1, 2014 further analysis using sequences analysis for omt-1 genes which taking part at the end of aflatoxin biosynthesis pathway revealed that isolates a.flavus b1 and a.flavus b2 were closely related to a.flavus isolate cra01-2b, while isolate a. flavus d1 was closely related to a. flavus isolate af36 (fig. 5). a. flavus isolate cra01-2b has been used to study biosynthesis aflatoxin pathway while a. flavus isolate af36 is noncarcinogenic and has been used as biopesticides for controlling aflatoxin contamination in cotton seed (yu et al.1995b, ehrlich & cotty 2004). the omt-1 gene ( omtk.) was oneof manygenes involved in the aflatoxin biosynthetic pathway. conclusions eighteen isolates of a.flavus isolates were isolated from peanut kernels samples and identified based on morphological and moleculer analyses. the apa-2 and nor-1 were detected in 18 isolates, omt-1 in 15 isolates (83.3%) while ver-1 gene in 13isolates (72.2%). only 11 isolates had all the 4 genes involved in biosynthesis aflatoxin pathway. > % s 1ii acknowledgements this research was funded by i-mhere (indonesia managing higher education for relevance and efficiency) b2.1 on international standardization of ipbcc in year 2012. references v s abdel-hadi a. carter d, magan a. 2010. temporal monitoring of the nor-1 (ajld) gene of aspergillus 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of ajir, a regulatory locus foraflatoxin biosynthesis. appl environ microbiol60:2408-2414. yu j, chang p-k, payne ga, cary jw, bhatnagar d, cleveland te. 1995a. comparison of the omta genes encoding o-methyltransferase involved in aflatoxin biosynthesis from aspergillus parasiticus and aspergillusflavus.gene163:121-125. yu j etal.1995b. comparative mapping of aflatoxin pathway gene clusters in aspergillus parasiticus and aspergillus jlavus.appl environ microbiol61:2365-2371. yu j, chang pk, cary jw, wright m, bhatnagar d, cleveland te, payne ga, linz je. 1995b. comparative mapping of aflatoxin pathway gene clusters in aspergillus parasiticus and aspergillusjlavus. appl environ microbiol 61:2365-2371. si \ 3* 4 9p» 70 -/y / 1241.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 1. nur ain izza etal page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 489 (fatiman genetik).cdr genetic diversity and trait association analysis of indonesian rice (oryza sativa l.) germplasm using ssr markers fatimah*, lina herlina and tiur sudiaty silitonga indonesian center for agricultural biotechnology and genetic resources research and development, bogor 16111, indonesia received: 26 may 2015/accepted: 4 july 2016 abstract generating a new crop variety with certain desirable traits requires germplasm collection with wide genetic diversity to facilitate accelerated development of improved cultivars. this study was conducted to characterize genetic diversity of indonesian rice accessions using ssr markers and their agro-morphological characters to allow the identification of marker trait association (mta). a survey of genetic diversity among 72 traditional landraces of 15 provinces in indonesia and 24 improved varieties was conducted using four multiplex panels of 14 fluorescent-labeled ssr markers and 22 agro-morphological characters. a total of 140 alleles with an average of 10 alleles per locus and polymorphism information content (pic) values at 0.73. the population structure identified five subgroups. a total of 4 highly significant mta were identified. one character with highly significant mta was number of panicle associated with rm277. based on ssr marker, rm105 had two mtas for auricle color and ligule shape. after validation, these mtas may be deployed to improve rice productivity. keywords: genetic diversity, indonesian rice, marker-trait association, ssr introduction through various exploration missions, more than 4,000 rice germplasms consisted of landraces, elite lines, high yielding varieties, introduced variety/lines as well as 88 accessions of wild rice species are kept in indonesian center for agricultural biotechnology and genetic resources research and development (icabiograd). they have very important role as genetic resources. in breeding program to create high yielding varieties of food crops, it is important to carry out characterization and evaluation of germplasms to increase utilization of genetic resources (silitonga et al. 2001, silitonga 2010). agro-morphological characterization of germplasm accessions is fundamental in order to provide information for plant breeding programs (lin 1991, nascimento et al. 2011). systematic study and characterization of collected germplasms are not only important for utilizing the appropriate attribute based on donors, but also essential in the present era for protecting the unique rice. thus, there is a need to collect, exploit and evaluate the untapped germplasm (parikh et al. 2012). identification of cultivars with distinct morphological traits facilitates high resolution qtl mapping and validates candidate genes responsible for quantitative agronomical characters (li et al. 2010). the advantage of using fluorescent-labeled microsatellite markers for genetic diversity analysis in rice with multiple dyes allows efficient genotyping using multiplex panels of markers (blair et al. 2002, coburn et al. 2002, jain et al. 2004, thomson et al. 2007). the use of capillary electrophoresis provides additional benefits, including greater automation, ease of use and precision in fragment sizing, which makes it the preferred technology for simple sequence repeat (ssr) genotyping in rice (lu et al. 2004, garris et al. 2005, thomson et al. 2007). previous study carried out on genetic diversity and population structure of indonesian germplasm characterized 330 rice biotropia 3 2 6 105 115 vol. 2 no. , 201 : doi: 10.11598/btb.2016.2 . .3 2 489 * corresponding author: fatimahsuw@gmail.com 105 accessions, including 246 geographically-diverse indonesian landraces and 63 indonesian improved cultivars, using capillary electrophoresis with 30 fluorescent-labeled microsatellite markers (thomson et al. 2007). several researchers reported the use of agromorphological markers combined with ssr markers in the characterization and study of rice (oryza sativa l.) germplasm diversity. using 42 agro-morphological characters associated with 39 ssr markers, bajracharya et al. (2006) examined genetic relationships among 147 high altitude rice landraces from nepal. agrama et al. (2007) identified ssr markers associated with agronomically important phenotypic traits using 92 rice germplasm accessions evaluated for yield and kernel characteristics; when being genotyped with 123 ssr markers it was shown that many of the associated markers were located in regions where qtl had previously been identified. seetharam et al. (2009) characterized 30 rice genotypes comprising landraces, pure lines, somaclones, breeding lines and varieties specifically adapted to costal saline environments using ssr markers and morphological characters. the objectives of the present study were to characterize genetic variation of 96 rice accessions using ssr markers and agro-morphological characters allowing identification of marker trait association and to provide useful information to facilitate rice plant breeding programs. materials and methods seeds of 72 indonesian rice landrace accessions originated from 15 provinces were obtained from indonesian center for agricultural biotechnology and genetic resources research and development (icabiograd). twentyfour cultivated varieties were added from indonesian center for rice research, agency for agricultural research and development (iaard), indonesia ministry of agriculture. the control variety used in this study was inpari 1 for indica. inpari 1 was improved variety originated from ir64 and irbb7 which developed for resistance to leaf blight disease. it was developed by conventional breeding method, while rice variety code was developed using marker assisted selection (bustamam et al. 2002). the control variety for tropical japonica rice was kalimutu. it was rice variety introduced from kenya having drought resistance characteristic (silitonga 2010). these rice paddy varieties were first sown in five plastic boxes which contains 20 rice varieties each. fourteen days later seedlings were transplanted into a set of boxes containing natural paddy soil. rice plants were grown under greenhouse maintenance. molecular analysis dna was extracted using dellaporta (1983) method. individual polymerase chain reaction was performed in 20.0 l of a mixture containing µ 5 l (10 ng/ l) dna template of each sample, µ µ 1.2 l (5 m) ceq-compatible fluorescent-µ labeled primer pairs, 2 l of dntp mix (2.5 mm), µ 2 l 10x pcr buffer and 0.2 l roche faststart µ µ taq dna polymerase kit 5 units/ l. the pcr µ machine of dna engine tetrad2 peltier thermal cycler mj research was run in program following touchdown procedure which consisted oof 4 minutes initial denaturation step at 95 c, o13 cycles touchdown at -0.5 c increments, 45 o oseconds at 95 c, 45 seconds at 61.5 c decreased o oby 0.5 c every cycle, 30 seconds at 72 c, followed oby 27 cycles at 45 seconds at 95 c, 45 seconds at o o55 c, 30 seconds at 72 c and a final extention ostep for 5 minutes at 72 c. the automatic capillary electrophoresis was used to separate and visualize the pcr products following procedures recommended by the manufacturer (beckman 2004). data analysis the ssr fragment analysis was based on the allele sizes generated and implemented in powermarker v3.25 (liu & muse 2005) that calculated the allele frequency, gene diversity and polymorphism information content (pic). the population structure of the rice accessions was calculated using structure v3.2 (pritchard et al. 2000). the posterior probabilities were estimated assuming prior values of k between 1 and 8 using the markov chain monte carlo method (mcmc). the results of this study were based on burn-in period length of 10,000; mcmc iteration after burn-in of 100,000; with twenty replications. the degree of admixture, alpha, was inferred from the data. when alpha is close to zero, most individuals are essentially from one population or another; whereas when alpha >1, this means that 106 biotropia vol. 23 no. 2, 2016 dinu toru madha kedhi mama wangi kero sibuyung pendek padi merah pulut olan 107 genetic diversity and trait association analysis of rice germplasms using ssr – fatimah et al. most individuals are admixed (falush et al. 2003). the k value was determined by lnp(d) in structure output and delta k based on the rate of change in lnp(d) between successive k (evanno et al. 2005). both lnp(d) in structure output and its derived delta k were used to determine the k value. to determine the putative association between agro-morphological characterization and the ssr markers it was run on mixed linear model (mlm) method in tassel (bradbury et al. 2007). the p value calculated by tassel determined whether a qtl was associated with the marker. of the several methods suggested to correct false positive in association analysis, even keeping stringent p value benchmark, the most stringent cor rection method cal led ' 'bonfer roni correction'' was used in the present study. the -4 threshold was found to be 7.1 x 10 at significance level of 1% after bonferroni multiple test correction (0.01/14). agro-morphological characteristic the accessions were characterized according to descriptors established by silitonga et al. (2003). a total of 13 qualitative and 9 quantitative descriptors were used. the qualitative descriptors were: auricle color, ligule colour, ligule shape, leaf type, flag leaf attitude, collar color, culm color, culm habit, panicle exertion, panicle type, apiculus color, lemma and palea color and thresh ability. the quantitative descriptors were: plant height, total number of tiller, number of effective tiller, panicle length, number of panicle, number of fertile grain, number of sterile grain, grain percentage of sterility and 1,000 grain weight. descriptive analysis was used to calculate the quantitative descriptors. genetic variability of observed characters was calculated using anderson and bancroft (1952) formula. 2genetic variability is broad when σ ≥2(σ ) g g 2and narrow when σ <2(σ ). the qualitative g g descriptors analyzed were determined by principal coordinate analysis (pcoa) generated from ntsyspc 2.11p to create a scatter plot showing genetic relationships among accessions. results and discussion ssr fragment analysis a total of 140 alleles, with an average of 10 alleles per locus were detected at the 14 ssr loci. the number of alleles per locus ranged from 4 i.e. rm484 and rm124 to as many as 17 i.e. rm474. rm484 presented the smallest allele size range (4 bp), while rm250 had the largest allele size range (74 bp). polymorphism information content (pic) values were high for most of the microsatellites, averaging 0.73 and ranging from a low of 0.54 i.e. rm484 to a high of 0.89 i.e. rm541. rm250 presented the highest frequency allele up to 62%. rm541 and rm144 showed the lowest frequency alleles, 17% and 19%, respectively (table 1). table 1 list of indonesian rice landraces and improved varieties used in this study misik a unggul sawah gading garu kumpang emas talun bajang k.sampi hiling talun bura pontianak c umbang pulai pulut silu 108 biotropia vol. 23 no. 2, 2016 table 1 continued 109 ssr markers generate multi allelic data revealing genetic structure of a natural population consisting of diverse genotypes (remington et al. 2001). this study, using 96 indonesian rice accessions, revealed a total of 140 alleles detected from 14 ssr loci. this finding was lower compared to the study results obtained by thomson et al. (2007) who detected 394 alleles at 30 ssr loci across 330 rice accessions. however, abundant genetic variation in this study was identified with an overall gene diversity of 0.75 and average of 10 alleles per locus (tabel 2) compared to an overall gene diversity of 0.68 and average of 11.9 alleles per locus (thomson et al. 2007). polymorphism information content (pic) values, a reflection of allele diversity and frequency among the varieties, averaged 0.73 and ranged from a low of 0.54 to a high of 0.89 compared to results obtained from the study carried out by thomson et al. (2007). the pic values ranged from 0.34 to 0.88 (rm154 and rm474), with an average of 0.66. different results of this study and the study conducted by thomson et al. (2007) might be caused by different sampling strategies, due to different provinces, different numbers of provinces and different numbers of ssr markers used by the two studies). population structure fourteen ssr markers were distributed on the rice genome to assess genetic architecture and population structure. it was observed that deltak declined after k = 5 was continuously significant, suggesting the presence of five subgroups (fig. 1). at k = 5, most accessions were classified into five groups, which corresponded to indica-1 with 20 consisted of improved varieties, traditional rice sub-species japonica with 13 accessions, indica2 with 21 germplasm accessions, indica-3 with 24 germplasm accessions and indica-4 with 18 germplasm accessions and no admixture population was found. the degree of admixture (alpha) generated in this study was close to zero (0.04) which means most individuals are essentially from one population. analysis of population structure identified the highest log likelihood and delta k with the number table 2 data summary for 14 fluorescent-labeled microsatellite markers across 96 rice accessions panel dyea marker chr motifb no.of allele range major allelec picd gene diversity size (bp) frequency (%) 1 d3 rm484 10 (at)9 4 292-296 0.59 294 58 0.54 1 d4 rm105 9 (cct)6 7 100-137 0.79 133 30 0.76 1 d4 rm536 11 (ct)16 10 218-259 0.60 232 60 0.58 2 d3 rm124 4 (tc)10 4 266-272 0.63 268 51 0.56 2 d4 rm162 6 (ac)20 12 204-250 0.84 210 31 0.83 2 d4 rm277 12 (ga)11 8 112-137 0.72 121 45 0.68 3 d3 rm287 11 (ga)21 11 93-157 0.83 113 26 0.80 3 d3 rm259 1 (ct)17 14 119-179 0.79 154 34 0.77 3 d4 rm250 2 (ct)17 10 119-193 0.59 171 62 0.57 3 d4 rm514 3 (ac)12 12 240-274 0.80 249 29 0.77 4 d3 rm541 6 (tc)16 14 131-198 0.90 182 17 0.89 4 d3 rm144 11 (att)11 11 220-286 0.88 220 19 0.87 4 d4 rm474 10 (at)13 17 228-299 0.88 229 25 0.87 4 d4 rm171 10 (gatg)5 6 321-345 0.70 341 48 0.67 afluorescent dyes for ceq beckman genetic analyzer: d2 = black, d3 = green, and d4 = blue bmotif of the ssr and number of repeats as previously published (http://www.gramene.org) cmajor allele is defined as the allele with the highest frequency dpic = polymorphism information content genetic diversity and trait association analysis of rice germplasms using ssr – fatimah et al. 110 biotropia vol. 23 no. 2, 2016 of populations set at five (k = 5). these five populations corresponded to one tropical japonica population (13.5%) and four indica populations and no admixture population was identified. these four subgroups corresponded to indonesian improved varieties in subgroup 1 (20.8%), rice landraces in subgroup 3 (21.8%) and subgroup 4 (25%) and subgroup 5 (18.7%) (fig. 2). these results were quite different from previously analyzed population structure at k =2 of 330 indonesian rice varieties with 30 microsatellite markers that divided indonesian germplasms into indica and tropical japonica (thomson et al. 2007). the k = 2 setting forced all accessions into two populations, causing the more distant related groups, such as the o. rufipogon and aus accessions, to appear as admixtures between indica and japonica. khush et al. (2003) analyzed 3,670 indonesian varieties with 11 isozymes and found out that 69% of the sampled indonesian germplasms as indica, 28% as japonica and 3% as intermediates. however, it was difficult to make any distinctions between landraces and improved varieties. agro-morphological characteristic pcoa analysis classified the 13 qualitative agro-morphological characteristics into three groups, which corresponded to the traditional rice subspecies tropical japonica with 19 accessions, indica-1 with 23 germplasm accessions and indica-2 with 54 improved varieties and germplasm accessions (fig. 3). this result was different from the ssr analysis which classified the characteristics into five groups. the ssr results divided the third group in agro-morphological characteristic into three groups which separated the indica (group 1) from improved varieties and indica (group 3 and 5) from germplasm accessions. the descriptive analysis of 9 quantitative descriptors of 96 indonesian rice accessions is presented in table 3. the broad variability of quantitative morphological character was found in all 9 quantitative descriptors. genetic variability is considered figure 1 the presence of k = 5 based on posterior probability, l(k) and delta k where k ranged from 1 8 figure 2 bar plot representing population structure of five subgroups of indonesian rice in this study. red=indica-1 (improved varieties), green= japonica, blue=indica-2 (germplasm), yellow=indica-3 (germplasm), and pink=indica-4 (germplasm) 111 2 broad when σ ≥2(σ ) and narrow when g g 2 σ <2(σ ) (anderson & brancoft 1952).g g based on the culm habit there were 5 types of characteristics however we found 3 types in these rice germplasm i.e.: erect (15°), semi o erect/intermediate (20 ), and open (40°). in this study, the 96 accessions were divided into 3 characteristics i.e. erect (78%), semi-erect (21%) and open (1%). from these, the cultivated rice varieties were divided into 2 characteristics i.e. erect (83%) and semi-erect (17%) while indonesian rice landraces were divided into 3 characteristics i.e. erect (76.4%), semi-erect (22.2%) and open (1.4%). similar with rabara et al. (2014) mentioned that most of the traditional rice varieties in philippines had thick culms and an erect culm habit. regarding for improving yield, wu et al. (2011) shown that large culm rice varieties have a higher number of grains per panicle and a longer spike length. gealy et al. figure 3 the pcoa analysis based on 13 qualitative agro-morphological characteristics classified into three groups, which corresponded to the traditional rice sub-species japonica (19 accessions), indica-1 (23 accessions) consisted of germplasm accessions and indica-2 (54 accessions) consisted of improved varieties and germplasm accessions table 3 descriptive analysis of quantitative descriptors of 96 indonesian rice character min max range means sd se variance skewness kurtosis ph 84.7 195.3 110.6 129.87 28.46 2.90 809.80 0.00 -0.99 tt 4 16 12 7.95 2.49 0.25 6.20 1.33 1.72 net 4 13 9 6.82 2.08 0.21 4.32 1.10 0.99 pl 14.8 35.7 20.9 23.33 3.89 0.40 15.17 0.44 0.75 np 2 23 21 5.46 2.50 0.26 6.26 3.82 24.69 ngf 1.3 482 480.7 100.36 67.86 6.96 4,604.61 2.17 9.46 nsg 4.7 258 253.3 52.32 39.47 4.05 1,557.70 2.15 7.40 pcs 6 97.3 91.3 34.86 17.50 1.80 306.09 0.90 1.02 togw 12 37.9 25.9 21.00 4.31 0.44 18.60 0.64 1.93 notes: ph=plant height, tt= total number of tiller, net=number of effective tiller, pl=panicle length, np=, number of panicle, ngf= number of fertile grain, nsg= number of sterile grain, pcs= percentage of sterility, togw=total 1,000 grain weight genetic diversity and trait association analysis of rice germplasms using ssr – fatimah et al. 112 biotropia vol. 23 no. 2, 2016 (2006) and hussain et al. (2010) reported that the presence of erect plant types could be due to backcrossing between accessions or with commercial rice cultivar. the culm color was characterized as light gold (92%) and green (8%), while the cultivated accessions were characterized as light gold. the collar color from the 96 accessions was characterized as light green (95%) and purple (5% i.e. bulan sabit, gedangan, loneng, padi kuda, and pulut silu). the cultivated accessions were characterized as light green. the ligule color from the 96 accessions was characterized as absent (93%), purple lines (5%) and purple (2%), while the cultivated accessions were characterized as absent. the auricle color from the 96 accessions were characterized as absent (83%), purple lines (16%) and purple (1% i.e. gedangan and toliwang iv), while the cultivated accessions were characterized as absent. the flag leaf attitude from the 96 accessions were characterized as 28% erect, 49% semi erect, 3% horizontal and 20% descending, in which the cultivated accessions were in erect category. dewi et al. (2009) mentioned that the erect flag leaf was more desirable than the horizontal one due to the higher sunlight intercept and might have a positive correlation with the high yield. narayanan et al. (2007) reported that flag leaf as the major source of phloem-delivered photo assimilates during the grain-filling stage in rice and khalifa et al. (2008) showed that cutting of flag leaves result to 45% grain yield loss. there were 5 panicle types (attitude) i.e. erect (compact panicle), semi-erect (semi-compact panicle), spread (open panicle), horizontal and drooping. the 96 accessions category were 93% semi-erect and 7% spreading, in which the cultivated accessions were in semi-erect category. the means for panicle number per plant was 5.4 from 96 accessions (table 3). for the cultivated varieties, the mean panicle number was 5.1, in which inpari 11 had the highest panicle number (23). the mean panicle number for indonesian rice landraces was 5.6, in which padai agak pandak had the highest panicle number (11). the mean for panicle length was 23.3 cm from 96 accessions (table 3). for the cultivated varieties, the mean panicle length was 21, in which inpago 4 had the longest panicle length (34.7 cm). the mean panicle length for indonesian rice landraces was 24.1, in which ketan mayang had the longest panicle length (35.7 cm). peng et al. (2014) reported that panicle structure is one of the most important factors for rice yield and panicle branches and their number affects spikelet number. dense panicle structure has been a major target for improvement of rice grain yield because of its relationship with grain number. the recorded mean for plant height in 96 accessions was 179.9 cm, with a minimum height of 84.7 cm for inpari 3, 85.7 cm for madha kedhi and a maximum of 195.3 cm for bulan sabit accession (table 3). from the 96 accessions 31.3% was dwarf (80-110 cm), 17.7% was semi-dwarf (110-130 cm) and 51% was tall (130-200 cm) (fig. 4). for the cultivated varieties, the mean height was 102 cm, which the distribution were dwarf (31.3%), semi-dwarf (17.7%) and tall (51%). the differences in the heights of the accessions were possibly due to various internodal lengths. the varieties having longer internodes produced figure 4 the agro-morphological characteristics based on plant height (left) and percentage of fertility (right) on 96 accessions 113 taller plants (ashrafuzzaman et al. 2009; hussain et al. 2014) and mohammad et al. (2002) reported that plant height depends on genetic makeup of a plant and environmental conditions. there were 4 types of fertility based on the percentage of fertility of the 96 accessions i.e. sterile (5.2%), half sterile (58. 3%), fertile (31.3%) and very fertile (5. 2%) (fig. 4). the cultivated rice varieties were categorized as sterile (0%), half sterile (75%), fertile (20.8%) and very fertile (4.2% i.e. sintanur). indonesian rice landraces were categorized as sterile (6.9%), half sterile (47.2%), fertile (40.4%) and very fertile (5.5% i.e. gando, padi dewi, talun bajang and ringkak siman). kwon et al. (2015) found that a deficiency of slr1 activity causes different spikelet fertility phenotypes in two different cultivars, koshihikari and h143 which h143 exhibits partial sterility of spikelets while koshihikari has normal fertility. liu et al. (2013) mentioned that spikelet fertility is a critical yield-determining trait that is influenced by genetic background and environmental factors including rain, wind, and temperature. anther development and pollen viability particularly affect spikelet fertility. association analysis a total of 4 marker-trait associations were identified with 14 different ssr markers (table 4). based on agro-morphological characters, one character with highly significant mta was number of panicle associated with rm277 on chromosome 12. liu et al. (2008) identified qtl for primary branch number in panicle (qpbn-6) on chromosome 6 in the interval of rm3 rm162. mu et al. (2005) and liu et al. (2008) explained that the primary branch number per panicle was determined by the initial size of the reproductive apex. the floret number per primary branch was determined by the cell division activity. thicker panicle neck development and larger panicle came from the parallel development during the panicle initiation, or floret developing. grain filling were enhanced by stronger transporting process of water and nutrients based on thicker panicle neck. spikelet number was mostly determined during the panicle initiation, while the number and weight of filled grain were largely affected by the conductive tissue together with the source strength. wickneswari et al. (2012) reported that rm259 and rm208 were used to identify qtl for grains per panicle as peak markers for qgpp-1 and qgpp-2, respectively. among the mtas obtained in this study, some were similar as previously reported, indicating that these mtas were reliable. these reliable mtas were repeatable in different studies with various segregation population. other mtas had been probed for the first time, indicating that more information on qtls could be found using populations of different genetic backgrounds. this is important for an in-depth understanding of the genetic control of yield traits in rice and for future rice production using molecular marker–assisted selection in rice breeding (iqbal et al. 2003). conclusions high resolution dna banding pattern of the ceq capillary gel electrophoresis system measured genetic variation of 96 rice varieties using 14 ssr markers, through population structure analysis. taken together, the ssr markers identified in this study could be used for marker-trait association (mta) analysis of agromorphological characters. this study resulted to a table 4 marker-trait associations (mtas) identified for select agronomic traits no trait marker f value p value 1 auricle color rm105 4.4963 8.89e-05 2 collar color rm287 6.3796 1.09e-06 3 ligule shape rm105 4.8616 3.70e-05 rm536 5.0657 1.61e-05 rm514 5.3648 3.91e-06 4 number of panicle rm277 13.779 7.50e-10 genetic diversity and trait association analysis of rice germplasms using ssr – fatimah et al. total of 140 alleles with an average of 10 alleles per locus and polymorphism information content (pic) value at 0.73. the population structure analysis identified five subgroups. a total of 4 highly significant mta were identified. one character with highly significant mta was number of panicle associated with rm277. based on ssr marker, rm105 had two mtas for auricle color and ligule shape. these mtas may be deployed in improving rice productivity, after being validated. acknowledgements we 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(ebenaceae) diospyros celebica , an endemic in sulawesi island, tree species indonesia * muhammad restu, gusmiaty g. and siti halimah larekeng biotechnology and tree breeding laboratory, faculty of forestry, universitas hasanuddin, makassar 90245, indonesia received 23 november 2015/accepted 23 may 2017 abstract diosphyros celebica bakh., also known as sulawesi ebony, is an endemic to central and northern sulawesi. information about pollen dispersal patterns of d. celebica have not been previously investigated. this study was aimed to determine pollination type, percentage of selfing and outcrossing, as well as distance of pollen dispersal of d. celebica. this study was conducted at experimental forest of universitas hasanuddin, maros district, south sulawesi province, indonesia. one hundred and sixty six individuals of d. celebica consisted of adult trees and seedlings were analyzed in this study. ninety four adult trees were selected to become parent trees. the trees were mapped with gps coordinates. all samples were genotyped using four ssr markers loci. parental analysis and determination of pollen dispersal patterns were carried out using cervus 3.0.3. results indicated that the evaluated markers were effective for assigning candidate of male parents to all evaluated seedlings. donated pollens could come from male parents in any directions relative to female parent positions. pollen dispersal pattern showed outcrossing pollination among different male parents (pollen donated trees). the results indicated that seeds were produced predominatly by outcrossing. pollen dispersal reached up to 166 m. pollen related processes were linked through female parents, pollinators availability and ecological environment. simultaneous use of progeny genotyping, spatially explicit analysis of environmental variables and outcomes of plant–animal interactions were the key elements for an expanded approach to gene flow analysis considering dispersal via pollen and seeds. research on pollen dispersal of d. celebica should be carried out in other forest types, such as mixed forest and highland forest. keywords: diospyros celebica bakh., endemic species, microsatellite marker, parentage analysis, pollen dispersal introduction recent endangered status of several ebony species in natural forests are within worldwide attention (world conservation monitoring centre 1998). diosphyros celebica bakh. (ebenacea), also known as sulawesi ebony, is an endemic tree to sulawesi island, indonesia. this ebony species is currently categorized as vulnerable species according to the iucn red list of threatened species (e.t33203a9765120, world conservation monitoring centre 1998). previous study by restu (2007) indicated that genetic diversity of d. celebica is low compared to other forest plants. the study found that all five of d. celebica provenances studied showed higher homozygosity, due to probable self-pollinating or mating among related individuals. therefore, their genetic diversity was lower (95.4%) than those of other plants. species with very narrow genetic base usually produce bad genetic planting stock material due to inbreeding depression. in order to prevent sulawesi ebony from becoming endangered species, conservation strategy is needed. knowledge on pollen dispersal distance and mating system estimate are vital for developing effective conservation and management strategies of endangered species. these parameters may be directly translated into information about minimum density of individuals or minimum area required to avoid reproductive isolation (nason et * corresponding author: sitih5h.82@gmail.com biotropia 4 3 7 173 181 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 3 562 173 al. 1998). pollen flow studies involving isolated individuals can assess the role of these individuals in pollen movement dynamics within fragmented landscapes. trees increase the number of pollen donors by implementing long distance pollen dispersal and higher outcrossing rate, as was observed in the population (fuchs & cuajiniquil hamrick 2011). therefore, conservation effort should be directed to preserve such individuals. removal of these individuals may negatively impact genetic composition of the remnant populations (fuchs & hamrick 2011). molecular markers are useful in pollen dispersal studies because these markers are capable of identifying parental genotype, genetic diversity and progenies. molecular markers and parentage analysis can be used to estimate selfing and outcrossing rate (milleron et al. 2012). microsatellite markers were used to determine genetic diversity in endangered species dipterocarpus littoralis (dwiyanti et al. 2014), nothofagus sp. et al. and paeonia (sola 2016) jishanensis et al. (xu 2016). pollen dispersal of various plant species was carried out using parentage analysis . pollen (austerlitz et al. 2004) dispersal evaluations were done in quercus garryana (marsico et al. 2009), pinus koraiensis (feng et al. 2010), tectona grandis (prabha et al. 2011), phoenix canariensis et al. cocos nucifera (larekeng (saro 2014), et al. 2015b) erhytrophleum suaveolens and (duminil et al. 2016). currently, there were no information about mating system and pollen dispersal pattern of d. celebica. this study was aimed to determine pollination type, percentage of selfing and outcrossing a distance of pollen dispersal s well as in d. celebica. materials and methods study peciess d. celebica (ebenacea) is an endemic tree to sulawesi island, indonesia. in southern sulawesi, this tree species can be found in gowa, maros, pangkep, barru, sidrap, luwu and east luwu districts. this tree can also be found in western sulawesi (mamuju) and central sulawesi (donggala, parigi moutong and poso). protected forest is the main location for this species (asdar et al. 2015). highadult tree may reach up to 40 m having 10 to 26 m clear bole. trunk diameter is over 1 5 m . above root crown. the root crown can reach 4 m high the tree. has simple, alternate, long elliptical leave 12 to 35 cms. each leaf has length of , width of . and 2 5 to 7 cm brown leaf hair (trichome) at underside leaf surface which is a the specific characteristic of (riswan 2002; d. celebica kinho 2013). this species has monoecious flowers which means female and male gametes are found in a tree. pollination occurred in the same tree resulted to leither imperfect fruits or fruits that wil be aborted before reaching maturation (santoso et al. 2002a grown at the). flowers are fourth axillary (flos axillary) from twig base or at seventh the axillary flowers from twig tip. are commonly formed in the middle section of the canopy. flowering is influenced by wind, rainfall, light and temperature (santoso 2002 )b . study ites d. celebica trees used for this study were located at the experimental forest of universitas hasanuddin. this experimental forest is a nonfragmented forest. the area is administratively located mostly in limampocoe village, cenrana subdistrict (previously known as camba subdistrict), maros district, south sulawesi o oprovince, indonesia (119 44'34'' 119 46'17'' e o oand 04 58'7'' 05 00'30'' s), with altitude of 300 800 m (fig. 1). according to the administration of forestry, the area of experimental forest is part of bulusaraung forest, bengo police forest, lebbo tengae forest, forest service of maros and forest service of south sulawesi. the study site consisted of plots and blocks. each block was divided into several plots ranging from 50 to 170 ha (based on field conditions and tree types). plots were bordered using natural boundaries, such as rivers and valleys. plots within forest area were separated into subgroup plots based on tree type, age, tree density, vacant lot, natural forest and rocks. 174 biotropia vol. 24 no. 3, 2017 microsatellite analysis dna isolation was conducted using the ctab method (sambrook & russell 2001) with mod i f i ca t ion (larekeng 2015) . rna contaminants were removed using rnase treatment following standard procedures (sambrook & russel 2001). seventeen ssr markers loci generated by liang et al. (2015) were evaluated to observe their polymorphism. only four loci were selected for this study. to generate markers, pcr amplifications were conducted using the following reaction mixtures i.e. 2 µl of dna; 0.625 µl of primers, 6.25 µl pcr mix (kapa biosystem), and 3 µl ddh 0. 2 amplifications were conducted using the following steps: one cycle of pre-amplification at 94 °c for 5 minutes, 35 cycles of amplification steps at 94 °c for 15 seconds (template denaturation), annealing temperature for 15 seconds (primer annealing) and 72 °c for 5 seconds (primer extension), and one cycle of final extension at 72 °c for 10 minutes as suggested by kapa biosystem kit. the generated ssr markers were separated using superfine resolution agarose 3% (seng et al. 2013) using tae 0.5x buffer (brody & kern sampling one hundred and sixty six individuals of d. celebica consisted of adult trees and seedlings were analyzed in this study. ninety four adult trees were selected to become parent trees. eleven of the 94 individuals were used as female and male parents. female parent was a tree having seedlings grown under its canopy. the assumption was that seedlings grown right under the canopy of a tree was produced by the tree. only 11 of 94 adult trees had seedlings. two to ten seedlings from each evaluated female parent were selected. thus, the total of evaluated seedlings was 72 seedlings. leaves from adult trees and seedlings were used as dna sources. sample collection was conducted in november 2015. nly adult trees ha in the study site, o 94 d seedlings under their canopgrown ies, which might be caused by early flowering time at the collection time. alrasyid (2002) stated that flowering time of in march april d. celebica was and fruit ripening time in september was november. the location of the female and the assigned male parents were plotted in the map of adult individuals generated by arcgis 10.1 software. 175 high outcrossing rate and pollen dispersal distance of diospyros celebica restu et al. figure 1. map of study site: experimental forest of universitas hasanuddin (note: the mark in this map indicates the positions of male and female parents of d. celebica) 2004) and stained using gel red staining. the electrophoregrams were visualized over uv transluminescence table and recorded by digital camera. the recorded pictures were used to determine genotypes of the evaluated samples. the laboratory activities were done at the biotechnology and tree breeding laboratory, faculty of forestry, universitas hasanuddin, makassar, indonesia. parentage analysis each sample of the progeny arrays had a known female parent, but unknown male parent (as pollen donor). the candidates of male parent could come from any sampled adult population, including the female parent population. this study was conducted to determine the assigned male parent donating pollen to generate progeny arrays. parentage analysis using genotype of progenies, female parents and potential male parents was done using cervus version 3.0.3 software (kalinowski et al. 2007; marshall et al. 1998). the progeny and female parent genotypes were compared with those of other adult trees. the assigned male parent was selected based on the output of cervus analysis results (kalinowski et al. 2007; marshall et al. 1998). potential male parent with positive likelihood of odds score (lod score) might be the actual male parent of the progeny (marshall et al. 1998). the lod score was determined based on the matching genotype of progeny, female parent and potential male parent. simulation was conducted to determine the threshold level for 80 and 95% confidence the levels before the final parentage analysis step. ale m parents each progeny were denti assigned to i fied base on parentage analysisd (maskromo et al. 2016). analysis of pollen dispersal pattern distance between the known female parent and the assigned male parent was calculated using arcgis 10.1 software. distance between and position of both female and male parents in the generated map were then used to illustrate pattern of pollen dispersal in the location. selfpollination was defined as male parent being identical to female parent. otherwise, they were assigned as cross-pollination. results and discussion microsatellite analysis pcr amplification was done by four ssr primers obtained from diospyros kaki (liang et al. 2015), modified based on research carried out by larekeng et al. (2016). nine of 17 ssr primers were able to produce polymorphic, strong and clear alleles on the dna of sulawesi ebony grown in the experimental forest of universitas hasanuddin, i.e. maros provenance. selected primer pairs are displayed in table 1. ofdna amplification of ssr markers sulawesi e maros p wasbony rovenance an initial step o the u of specific primer for molecular f se s genome research . ssr markers on sulawesi ebony were intensively used in various molecular analyses, such as genetic diversity analysis between and among populations, genetic stability analysis in clonal plants derived from in vitro somatic embryogenesis, parents and progeny 176 locus name/ genbank accession no. repeat motif primer sequence (5’-3’) tm (oc) allele size (bp) 1430 dc588341 (gag)5 f: tca gta aag ctg cgg gca tc r: acg gtt ctc ctg atc ctc acg 56 190 –250 8917 dc591591 (at)10 f: aca cgt tca gta cca gga ggg a r: agt acc aca aac c ac cag tgg 55 166 –197 9004 dc591297 (gcagga)3 f: gcc aca aac ttc aca gag gac c r: agg cga gtg cga gta aga cga a 55 251 –272 ssrdk29 dq097497 (ccttt)8 f: atcatgagatcagagccgtc r: cacgttaacgttacggaaca 53 112 –152 source: liang et al. 2015 table 1 ssr loci used for pollen dispersal analysis of sulawesi ebony from maros provenance biotropia vol. 24 no. 3, 2017 arrays pollen dispersal analysis (larekeng as well as et al. was 2016). primer screening required to obtain strong and clear polymorphic-band pattern of pcr products. the intended pcr products were chosen from . n random primers ot all tested nucleotide primer pairs amplified produced products (positive primer) not all positive . also, primer pairs good polymorphic dna produced fragments (siregar 2008)et al. . parentage analysis parentage analysis was able to identify the male parents for 72 progeny arrays. the results of parentage analysis also indicated that male parents assigned to the 25% of progenies (18 individuals) were at least in the minimum of 95% confidence level. those assigned to the 51.4% of progenies (37 individuals) were at least in the minimum of 80% confidence level. male parents assigned to the other 23.6% of progenies (17 individuals) were at lower-than-80% confidence level. at the lower-than-80% confidence level, however, the assigned male parents had positive lod score, indicating that these male parents were the possible true parents of the progenies (marshall et al. 1998). female parents of d. celebica could reciprocally donate their pollens and thus, performed selfpollination. within the male parents assigned to the 72 progeny arrays, self-pollination occurred in 8 events (11.1%) and cross pollination occurred in 64 events (88.9%). complete scheme and pollination types identified based on results of pollen dispersal analysis are presented in table 2. larekeng et al. (2015a) showed that pollination rate in tall kalianda coconut was 98% outcrossing, while the other 2% was selfpollination. self-pollination provided higher reproductive assurance (ra) by compensating a shortage of outcross pollen. the role of selfpollination in mitigating pollen limitation in a selfcompatible population was rarely evaluated. the assay of limited pollen commonly used in selfpollination did not adequately quantify the potential fecundity advantage of ra (eckert et al. 2010). pattern of pollen dispersal in this study, parentage analysis was conducted using cervus. distance between the known female parent and the assigned male parent was calculated using arcgis 10.1 software. the results showed that range of pollen dispersal was 0 – 166 (table 2). the farthest pollen dispersal was exhibited by female parent number 35 (table 2). distance between female parents and the assigned male parents were determined based on their gps positions (fig. 2 & 3). female parent (pe 63) received 6 donated pollens from four different assigned male parents (pe 27, 34, 35, 71) (fig. 2). positions of the assigned male parents relative to the female parent (pe 63) in the study site are presented in figure 2. female parent (pe 65) received 10 donated pollens from three different assigned male parents (pe 27, 45, 71) (fig. 3). positions of the assigned male parents relative to the female parent (pe 65) in the study site are presented in figure 3. 177 high outcrossing rate and pollen dispersal distance of diospyros celebica restu et al. table 2 crossing schemes and pollination types identified based on results of pollen dispersal analysis of d. celebica progeny arrays no female parents number of seedling selfing rate (%) crossing rate (%) pollen dispersal distance for each seedlings (m) 1 2 3 4 5 6 7 8 9 10 11 7 17 22 27 35 50 54 62 63 65 67 5 2 6 8 3 8 9 4 10 10 7 0 0 0 62.5 66.7 0 0 0 0 0 28.6 100 100 100 37.5 33.3 100 100 100 100 100 71.4 50; 52; 126; 50; 126 102; 152 51; 51; 51; 102; 102; 51 93; 0; 93; 0; 93; 0; 0; 0 0; 0; 166 60; 60; 64; 51; 44; 44; 51; 60 77; 77; 77; 77; 39; 21; 8; 71; 71 43; 43; 43; 107 43; 35; 66; 66; 53; 53; 66; 53; 52; 112 92; 39; 79; 79; 79; 79; 79; 92; 39; 92 0; 81; 82; 0; 134; 134; 81 178 figure 2 pattern of pollen movement to female parent pe63 ( ) based on parentage analysis (note: the mark in this map indicates the positions of d. celebica male parents) figure 3 pattern of pollen movement to female parent pe65 ( ) based on parentage analysis (note: the mark in this map indicates the positions of d. celebica male parents) biotropia vol. 24 no. 3, 2017 179 high outcrossing rate and pollen dispersal distance of diospyros celebica restu et al. numbers of pollination events for each distance class are presented in figure 4. the assigned male parents were distributed almost evenly among different distance classes of the female parents. the 0 m distance indicated selfpollination events. a pollen dispersal study on oenocarpus bataua, an insect-pollinated palm tree species, showed that pollen dispersal could be very far, exceeding the distance to the nearest neighbor (ottewell et al. 2012). molecular microsatel l ite loci (ssrs) implemented in a study on mating system and pollen dispersal of hymenaea coubaril indicated that several events of self-pollination occurred in this tree species (carneiro et al. 2011), although it was previously believed that this tree species had obligatory crossing mating system due to a probable self-incompatibility system (dunphy et al. 2004). a study on (an endangered guaiacum sanctum tree species) showed the predominant outcrossing characteristic of this tree species (fuchs & hamrick 2011). the study also showed that the supra-annual flowering in palo verde national park (pvnp) might reduce the numbers of reproducing individuals, affecting the proportion of outcross progeny. the study further discussed that population of should preserve g. sanctum enough adults to ensure seed production even in a period when only few individuals reproduce. the same study suggested that in a fragmented population, pollen dispersal can be very far and thus, pollen is readily transported across the fragmented population or altered habitats to ensure tree species preservation. further investigations on genetic diversity of d. littoralis life history should concentrate on characteristics, mechanisms pollen/seed of dispersal, seed germination, vegetative propagation and impacts from herbivores and pathogens dwiyanti et al. 2014). imultaneous ( s use of progeny genotyping spatially explicit , analysis of environmental variables the and outcomes of plant–animal interactions key are elements for an expanded approach to gene flow analysis considering dispersal via pollen and seeds. speciesd. celebica is an endangered tropical tree and currently , only few information is available concerning reproductive biology of this tree species suggested. conservation genetics theory that significant size of population should be preserved to ensure the maintenance of genetic diversity and to reduce detrimental effects of drift and inbreeding. mating system and gene-flow distance estimates allow better predictions of minimum viable population sizes, ensuring reproductive success. conclusions pollen dispersal pattern of sulawesi ebony occurred in outcrossing pollination among its different donated trees. based on the progenies analysis, ebony female parent is a dominantly outcrossing pollination species. the results figure 4 numbers of pollination events for each distance class parentage analysis from diosphyros celebica bakh. in sulawesi island shows range of pollen dispersal was 0 – 166 m. acknowledgements the authors and thank yuni fitri cahyaningsih mirza arsiaty arsyad their assistance in the for laboratory works and manuscript writing. references alrasyid h. 2002. kajian budidaya pohon eboni. berita biologi [edisi khusus: manajemen eboni] 6(2):219 25. asdar m, prayitno ta, lukmandaru g, faridah e. 2015. sebaran, potensi dan kualitas kayu eboni (diospyros celabica bakh.) di sulawesi. j agroland 22 (2):94-105. austerlitz f, dick cw, klein ek, muratorio so, smouse pe, sork vl. 2004. using a genetic markers to estimate the pollen dispersal curve. mol ecol 13:937 54. brody jr, kern se. 2004. history and principles of c o n d u c t i ve m e d i a f o r s t a n d a r d d n a electrophoresis. anal biochem 333(1):1–13. carneiro fs, lacerda aeb, lemes mr, gribel r, kanashiro m, wadt lho, sebbenn am. 2011. effects of selective logging on the mating system and pollen dispersal of hymenaea courbaril l. 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species 1998: e.t33203a9765120. xu x, cheng f, xian h, peng l. 2016. genetic diversity and population structure of endangered endemic paeonia jishanensis in china and conservation implications. biochem syst ecol 66: 319-25. doi: 10.1016/j.bse.2016.05.003. 181 high outcrossing rate and pollen dispersal distance of diospyros celebica restu et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 1145.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 page 14 page 15 page 16 biotropia vol. 28 no. 1,2021: 54 63 doi: 10.1 1598/btb.2021.28.1 .i055 ethnobotanical study of digestive systems disorders in baduy ethnic, indonesia rida oictorida igiastini',', indria wahyuni' and irma saraswat13 'dqartment o f biology education, faculq o f teacher training and education, universitas sultan ageng tirttdyasa, kota serang, banten 42 1 17, indonesia 'center ofexcellencefor food sectin$ (icefory), universitas sultan ageng tirtqasa, serang, banten 42163, indonesia 'department ofelectrical engineering, faculo ofengineering, universitas sultan ageng tirttdyasa, ciltgon, banten 42435, indonesid received 20 april 2018/accepted 20 december 2019 abstract digestive disorders rank among the most commonly faced problems in indonesia, particularly for the baduy people in banten province. the baduy population lives along water-rich areas, yet their lack of sanitation facilities and unawareness of disease prevention methods have resulted in high morbidity and mortality rates in their communities, largely due to digestive system disorders that they continue to treat with medicinal plants. using quantitative ethnobotanical approaches this survey was undertaken to document the use of indigenous medicinal plants to treat and prevent different types of digestive system disorders among the baduy communities. ethnic knowledge on their medicinal plants were collected from 30 informants from the baduy people. quantitative approaches were used to determine the use value and informant consensus factor values of the collected data. the baduy population currently uses 54 medicinal plant species belonging to 30 families, in treating digestive system disorders. additional research is, however required, to validate the function of the medicinal plants and identity of their active compounds. keywords: baduy people, digestive disorders, medicinal plants introduction indonesia is a mega-&verse country with its flora composition particularly important in traditional medicine and agriculture and whose identity is deeply rooted among the indigenous groups throughout the indonesian archipelago. more than 1,800 plant species inhabit several forest formations across indonesia, 940 of which are used in herbal medicine by indigenous communities, yet only 300 species are used by the indonesian pharmaceutical industry (lip1 2014). indigenous knowledge is a product of generations upon generations of experience and constitutes a connection between indigenous communities and local natural resources (davies & kassler 2015). among these resources are plants that indigenous community members gather for food, medicines, religious rituals, and *corresponqng author, email: rida.khastini@untirta.ac.id other cultural activities. the indigenous baduy communities in banten province, indonesia, form part of the immense cultural diversity of humanity, and their collective knowledge, passed down from generation to generation, constitutes a source of immeasurable cultural wealth. the baduy people have traditionally used medicinal plants in the treatment of various diseases. the communities continued use of ethno medicine stems from its cost effectiveness, social acceptability, minimal side effects, and accessibihty (chawla e t al. 2013). since the baduy typically reside in mountainous areas where access to public healthcare systems remains limited, they have accumulated profound experience with treating and preventing diseases with medicinal plants, whose diversity in their environment is exceptionally high. digestive system &sorders have recently gained considerable attention among scholars in ethno medicine. such disorders are a major ethnobotany of digestive system disorder among the baduy people in indonesia khastini e t al. cause of morbidity both in indonesia and around the world, particularly among indigenous people, includmg the baduy, whose inadequate access to hygienic levels of sanitation exacerbates the transmission and prevalence of various digestive diseases. only 34% of people in low-income countries have access to adequate sanitation, and that figure is roughly 2.6 blllion people worldwide (mars e t al 2010). researches on local knowledge about medicinal plants is becoming increasingly important in defining strategies for the conservation management of biological resources (jeruto e t al 2008). fortunately, through extension activities, these ethnobotanical studes have become increasingly useful in developing healthcare and conservation programs (vandebroek e t al 2010). given those trends, valuable information about the importance of mekcinal plants and indigenous knowledge disseminated orally warrants its collection, documentation and quantitative evaluation in order to guide future researches and prevent knowledge loss and erosion during its transmission from generation to generation. although some ethnobotanical surveys have been conducted among the baduy (iskandar & iskandar 2017), there was no comprehensive report on the use of medicinal plants to specifically treat digestive system disorders. hence, this paper aims to assess the traditional uses of medcinal plants among the baduy and to compile profiles of the plants by applying quantitative methods. the information that would be generated could not only expand ethno medicinal knowledge but also support general awareness on conserving indigenous medicinal plants in indonesia. materials and methods the workflow contained the following specific steps: data collection and data processing analysis (fig. 1). study site the study was conducted at inner baduy, cibeo hamlets, i<anekes village, and leuwidamar sub district, in lebak, banten province, indonesia (figure 2). geographically, the d a g e is located at longtude 6°27'27'1 6"30'07' s and latitude 108°3'911 106°4'5" e at an altitude of 300 600 m asl. the region is located roughly at 172 krn west of jakarta, indonesia. the region experiences an average temperature of 20 oc, and the average rainfall is 4,000 mm/year. generally, three types of soil are available in the region: dark latosol, brown alluvial, and andosol. ethnographically, the baduy community inhabits 5,101.85 ha, of which 2,101.85 ha constitutes the residential area and roughly 3,000 ha of which are protected forest areas. the baduy speak their ethnic language sunda, and as indigenous people, they depend on immediate natural resources in their vicinity for their livelihood, which they supplement with production from the primary sectors such as agriculture, horticulture, and livestock. data collection data processing analysis plants for treating and figure 1 flow chart of ethnobotanical study in the baduy ethnic community biotropia vol. 28 no. 1,2021 legends gvrngksndag . . . . . . . . . . . village boundary lnner baduy e!33l study site i outer baduy footpath inner baduy hamlets 7 river outer baduy hamlets okm figure 2 map of the study site. baduy area of the ignekes village, leuwidamar subdistrict, lebak, banten province (iskandar & iskandar, 2017) data collection prior to documenting ethno pharmacological data, semi-structured interviews and focus group discussions, personal conversations with locals, and field surveys (martin 1995) were conducted in september 2016 until july 2017. a total of 30 household heads from the cibeo hamlet with age ranging from 20 to 40 years with an average age of 35 years, participated in the study as informants. all informants were ethnic baduy and were born and raised in their respective villages. the names and ages of the informants, the local names of plants and plant parts used, the method of preparation, and the medicinal uses, were recorded. prior to the field survey, several textbooks of plant diversity were consulted for identification purposes including the flora malesiana series (van steenis 1972) and flora of java (backer & van den brink 1965). medicinal plants were photographed and made into herbarium specimen for complete identification by the botanist at the laboratory of biology education, faculty of teacher training and education, university of sultan ageng tirtayasa, indonesia. the observed plant morphology included the characters of the roots, stems, leaves, flowers, seeds and fruit. analysis the use values (uv) of the medcinal plant species were analyzed based on galeano e t al. (2000) and according to the equation uv = u/ v, in whch u is the number of mentions per species, and n is the number of informants. uv is a quantitative parameter that indicates the relative importance of species known by the locals. the informant consensus factor (icf), initially developed by trotter and logan (1986), was used to identify the relative importance of medicinal plants in ailment categories in a particular culture. icf was calculated according to the equation icf = nur nt / nur 1, in which nur is the number of uses mentioned in ethnobotany of digestive system disorder among the baduy people in indonesia iulastini e t al. each category, and nt is the number of species indicated in each category. icf values range from 0 to 1; values closer to 1 indicate a higher proportion of informant consensus on plant species used against a disease category, whereas values closer to 0 indicate the opposite, a lower informant consensus on plant species used as a cure. results and discussion a total of 54 species belonging to 47 genera and 30 families were recorded for the treatment of dgestive system disorders (table 1). for each species, the scientific name with voucher number, family, vernacular name in sundanese, ailments treated, parts used, forms of preparation, and uses were documented. the reported plant families included arecaceae, fabaceae, zingiberaceae (four species each), acanthaceae, euphorbiaceae, moraceae, pipera ceae (three species each), apocynaceae, convolvulaceae, melastomataceae, menisperma ceae, poaceae, rubiaceae, solanaceae (two species each), amaranthaceae, begoniaceae, compositae, dilleniaceae, gnetaceae, lamia ceae, liliaceae, magnoliophyta, malvaceae, meliaceae, mimosaceae, myristicaceae, myrtaceae, palmae, phyllanthaceae, and verbenaceae (one species each). all the reported plants were commonly used in treating digestive system disorders by the baduy community because they are readily cultivated or grow as wild plants that can be harvested from the nearby forest where the baduy live. table 1 medicinal plants used to treat digestive system disorders in baduy communities ailments treated or parts forms of local name use value family species cultipreparation (sundanese) vated plant uses used and use (uv) acanthaceae strobilanthes n'spus b1. pecah beling c hemorrhoids leaf decoction 0.50 staurogyne elongata pi.) o.i< reundeu w stomach ache leaf edible 0.47 andrographispaniculata purm.f.) s a d o t o w diarrhea leaf decoction 0.83 wall. ex nees amaranthaceae celosia azentea l. gamet w appetite stimulant leaf crushed 0.43 apocynaceae qlophora n'ssioides b1. areuy peujit w stomach ache latex infusion 0.23 i<otok alstonia scholaris r. br. lame w intestinal worm latex infusion 0.17 infection arecaceae areca catechu l. jebug w toothache root crushed 0.60 ageratum cony~oides l. babadotan w flatulence leaf decoction 0.53 blumea balsamifera (l.) dc. capeu w appetite stimulant leaf decoction 0.33 . . cocos nucifera l. var. uiridis l<alapa hejo c flatulence bark peel decoction 0.87 begoniaceae begonia isoptera dryand areuy w stomach ache leaf decoction 0.20 bingbiringan compositae mikania cordata (burm.f.) b.l. areuy w jaunchce, stomach whole decoction 0.27 rob. caputuher ache plant convolvulaceae lepistemon binectan$mm (wall.) areuy w stomach ache leaf, resin decoction 0.40 i<untze plumpung merremiapeltdta (l.) merr. areuy w flatulence leaf decoction 0.17 palungpung dilleniaceae tetracera indica merr. areuy asahan w stomach ache bark juice drink 0.10 euphorbiaceae acabpha caturus b1. i<i lauk w appetite stimulant juvenile edible 0.53 leaf macaranga tanarius (l.) muell.ai-g. mara w gastric ulcer leaf decoction 0.13 eubhorbia hirta l. patikan kebo, w mouth ulcer latex rubbed 0.60 nanangkaan fabaceae eytbrina litho.pemza miq. dadap w stomach ache leaf crushed 0.47 abmsprecatorius l. saga w mouth ulcer leaf, root crushed 0.60 pterocapus indicus wdld. angsana w toothache resin crushed 0.77 mimosapudica duchass. & walp gehgeran, w mouth ulcer leaf crushed 0.70 putri malu gnetaceae gnetum neglecturn b1. ihsungka c intestinal worm stem juice decoction 0.67 infection larniaceae orthosiphon starnineus (blume) i<umis ucing c flatulence leaf decoction 0.80 miq. ldtaceae cordilnefisticosa (l.) a.chev. hanjuang c hemorrhoids rhizome, decoction 0.53 leaf biotropia vol. 28 no. 1,2021 table 1 (continued) ailments treated or parts forms of local name culti use value family species preparation (sundanese) vated plant uses used and use (uv) magnoliophyta villebrzmea mbescens (bl.) b1 nangsi w toothache stem juice edible 0.63 malvaceae durio +bethinus murr. icadu c toothache nectar edible 0.43 melastomataceae melastoma mlabathricum auct. harendong w diarrhea, leaf crushed 0.60 toothache, mouth ulcer bellucia amnanthera triana harendong w stomach ache fruit edible 0.43 leuweun~ meliaceae sandoricum koe$ape (burm.f.) icecapi c diarrhea leaf, bark edible 0.43 merr. menispermaceae arcangeliisaflava merr. areuy ki w diarrhea, jaundice root, bark decoction 0.20 koneng tinospora nnspa (l.) diels. martawali w jaunqce, intestinal bark, leaf decoction 0.57 worm infection mimosaceae pitbecelobim lobatum benth. jengkol c appetite stimulant juvenile edible 0.77 leaf moraceae ficus hispida l. f. bisoro w diarrhea, stomach juvenile edible 0.27 ache, jaundice leaf, fruit ficus sagittutu vahl. areuy h baok w mouth ulcer, latex decoction 0.27 stomach ache, jaundice ficzls racemosa roxb. walen w stomach ache juvenile edible 0.63 leaf myristicaceae horsfzeldiaglabra warb. i(i beo, c toothache fruit, leaf edible 0.77 icalapa ciung mvrtaceae psiditm pl~aiava l. tambu batu c diarrhea fruit. leaf edible 0.27 palmae arengapinnata (wurmb) merr. icaung w constipation root decoction 0.83 phvllanthaceae phvllanthus acidus skeels. cereme w a ~ ~ e t i t e stimulant fruit edible 0.87 piperaceae piper betle l. sereh c toothache, mouth leaf crushed 0.83 ulcer piper sulcatum b1. areuy rinu w flatulence fruit edible 0.27 pipar aduncum l. gedebong w nausea seed crushed 0.63 poaceae imperata ylindrical (i,.) p.beauv. eurih w mouth ulcer, root decoction 0.50 pharyngitis andropogon nardus l. sereh c appetite stimulant leaf decoction 0.57 rubiaceae morinda citrz@lia l. mengkudu c jaundice fruit edible 0.67 nauclea orientalis l. gempol w nausea root decoction 0.67 solanaceae physalis minima l. cecenet w intestinal worm root decoction 0.80 pbsalir angulata l. infection cecendet w mouth ulcer, gastric fruit edible 0.67 ulcer, stomach ache verbenaceae clerodendmm serratum (l.) moon singugu w intestinal worm leaf decoction 0.67 infection zingberaceae zingiber oficinale roscoe jahe c flatulence root decoction 0.93 zingiber xemmbet (l.) roscoe ex lempuyang c appetite stimulant root decoction 0.67 sm. zingiber amaricans b1. lampuyang c stomach ache leaf decoction 0.67 costus speciosus smith pacing c jaunqce stem juice decoction 0.83 different plant parts were used for the the uvs of the plants ranged from 0.1 to treatment of various digestive system disorders 0.733. the interview results revealed that the (fig. 3). the kaves were the most commonb three most selected species were zingiber oflknale used (430/0), the (150i0), fruit (0.73) that had the reatest uv among all plants, (13%), resin (loo/o), bark (~oo), stem substance followed by cocos nac@ra l. var vir;;dis and (5%), whole plant (2%), nectar (2%), and seed pblantas kdas skeels (table 1). a high uv value (2%). in most cases, the medicinal plants were prepared in the forms of juice, decoction, for a particular plant indcated a high number of infusion, paste, and powder. uses reported by informants. ethnobotany of digestive system disorder among the baduy people in indonesia khastini et al q o l leaf root fruit i43: resin 4 bark ' stem substance i whole plant nectar seed figure 3 use patterns of the documented medicinal plant parts table 2 category of ailment treated and informant consensus factor (icf) ailments treated or plant uses number of taxa number of use report icf constipation 2 26 0.96 gastric ulcer 2 12 0.91 nausea 2 11 0.90 hemorrhoid 2 8 0.86 diarrhea 6 28 0.81 intestinal worm infection 5 21 0.80 flatulence 6 25 0.79 appetite stimulant 7 25 0.75 mouth ulcer 6 18 0.71 toothache 6 16 0.67 pharyngitis 2 4 0.67 stomach ache 13 20 0.37 jaundice 7 10 0.33 icf was determined for 13 ailments or plant uses: appetite stimulant, constipation, diarrhea, flatulence, gastric ulcer, hemorrhoids, intestinal worm infection, jaundice, mouth ulcer, stomach ache, toothache, pharyngitis, and nausea. icfs ranged from 0.33 to 0.96, with a mean value of 0.733 (table 2). the ailment treated with the most use reports was constipation (0.96). the icf values for disease treatment depended upon the availability of the medicinal plant species in the area of the studied baduy community. increasing access to medicinal plants in indonesia, a country with one of the world's most highly diversified plant species, has greatly benefited the locals, especially members of indonesia's indigenous communities. many studies conducted among indigenous communities elsewhere in indonesia. for instance, among the dayak communities in west kalirnantan @iba et al. 2013) and sundanese communities in west java (roosita 2008), have documented not only the plant parts used (e.g., leaves, wood, fruits, roots, and flowers) and their processing techniques but also their beneficial effects. this current study documented that information on the uses of 54 plant species in treating 13 different aliments among the baduy people were collected from reliable informants and were based on their personal experience. the commonly used plants that the baduy have a b s t e r e d to treat and prevent digestive disorders were readily available and had healing properties. biotropia vol. 28 no. 1,2021 to obtain extracts used for medicine, the ginger has the greatest uv, its rhizomes are informants collected the fresh or dried plant materials from the entire plants or their parts. of all the plant parts used, the leaves were the most commonly prepared part as medicines (43%). leaves contain a myriad of secondary metabolites or phytochemicals that play a vital role in the treatment and prevention of various digestive disorders (i<umar et al. 2009). the baduy typically use fresh leaves by boiling these in water for decoctions, as they also d d with other plant parts used for medicinal materials. such preparation usually involved reducing the water volume by a third. studies conducted in other indigenous communities around the world have shown that leaves were also the most used plant part for medicine at rates of 51.39 o/o in the turgo hamlet of yogjakarta, indonesia (nahdi et al. 2016), 47.65% in argentina (teves et al. 2015), and 34% in the limpopo &strict of south africa (semenya & maroni 2012). the current results also supported the findings of handayani (2015), that leaves were the most frequently used parts in plant-based medcines. leaves and other plant materials, including rhizomes, were also prepared for medicines by pounding and squeezing. according to the informants, the baduy also mixed some plants with other plants depending on their intended uses. single and mixed medicinal plant preparations may have different effects, for a mixed-medicinal plant formula may contain several hundred times the natural constituents that numerous single-medicinal plants contained. quantitative analysis was performed to authenticate and project the relative importance of medicinal plant species in their traditional use. plants with high uv were also used among other indigenous communities throughout indonesia (nulfitriani et al 2013). the plants with the first three largest uv among the medcinal plants documented in baduy area were; ginger (zingiber o@cinale roscoe), coconut (cocos nucfera l.), and pkyllanthus adus skeels. used not only as a component in medical treatments but also as an additive in numerous foods and daily beverages (e.g., bandrek and b.jigur) of the baduy people (figure 4). due to its aromatic compounds, ginger is a valuable rhizome that releases a spicy, pungent, pleasant smell. the effectiveness of using ginger in the preparation of reme&es against various digestive problems, including loss of appetite, flatulence, indgestion, diarrhea, and dysentery, was also reported by sidhu & pannu (201 0). coconut (cocos nucifera l.) registered the second-greatest uv among the baduy who cultivate the plant and use all of its parts in one way or another for diverse purposes in their daily lives. the parts of the fruit, including the coconut kernel and tender coconut water, have numerous medicinal properties, including antibacterial, antifungal, antiviral, antiparasitic, and immunostimulant properties, that are effective against digestive system disorders (debmandal & mandal, 2011). the medcinal properties of coconut emanating from its constituents, include vitamin b, nicotinic acid (b3, 0.64 pg/ml), pantothenic acid (b5, 0.52 pg/ml), biotin (0.02 pg/ml), riboflavin (b2, <0.01 ng/ml), folic acid (0.003 pg/ml), and trace quantities of vitamins b1, b6, and c, as well as pyridoxine, thiamine, amino acids, l arginine, plant hormones (auxin, 1,3 diphenylurea, and cytokinin), enzymes (acid phosphatase, catalase, dehydrogenase, diastase, peroxidase, and rna polymerases), and other growth-promoting factors (solangih & iqbal, 2011). other studies conducted in several countries have also reported the use of coconut in treating digestive system disorders. people in brazil have used the extract from the husk fiber of c. nucifera to treat diarrhea (akinyele et al. 201 i), which people in ghana have treated with coconut milk (lima et al. 201 5). ethnobotany of digestive system disorder among the baduy people in indonesia khastini e t al. figure 4 the third largest uv medicinal plants used by baduy ethnic. a. zingiber oflcinale roscoe. b. cocos nxn9m l. var. viridis. c. pbylzannthxs acidas skeels. plylkantbzts acidus skeels has the third largest uv in this study. this species is not only functioned as edible fruit, but also is known to cure wide spectrum of diseases in asia, the carribean region, as well as central and south america (tan et al. 2020). several studies showed that this plant is abundant in a number of phytochemicals, such as flavonoids, alkaloids, lignans, terpenes, steroids, and essential oil (chakraborty e t al 2012; nisar et ak201 8), which have remarkable health benefits. among the study plants, tetrdcera indim, has the lowest uv. most informants were unfamiliar with the medicinal plant species and had little knowledge about its ethnobotanical uses. species in the genus twrscera have also been used as medicinal plants by the ehotile people near aby lagoon in c6te d'ivoire, africa. however, these plants containing the sought-after exudate are found only in old marshlands that are scarce and difficult to access (malan et al. 2015). according to one key informant, most of the raw materials of these medicinal plants were difficult to find along the village periphery, as the original habitats of the cultivated plants were in the wild and were located in the forest. based some areas in the forest are considered to be sacred, and entry is forbidden. forests and other types of habitat provide a wide range of medicinal plant resource-base for primary healthcare, especially in rural areas (lindborg e t al.2012). this study has identified a fairly large number of plants used to treat various ailments, particularly among the baduy community. the high diversity of species reported to be readily available for medicinal purposes could indicate not only a dependency on plants in the treatment of ailments among the baduy but also an immense amount of local knowledge about herbal medicines in the community. despite the widespread use of those plants, however, no pharmacological studies have been conducted to determine their efficacy and safe use. hence, the identity of the medicinal plants used by the baduy provided a platform for formulating further preclinical and clinical studies to determine the efficacy and safe use of herbal preparations in indonesia and elsewhere. conclusions on vegetation structure, the baduy forest lands can be divided into two categories-numa which in summary, the showed that the is developed from fields that have long been badu~ people banten indonesia, abandoned, and are covered with dominant use immediate natural resources namely, 54 plants growing on the land, and leuwet/ng kokot as plant species, both as a whole plant and in part protected areas, forbidden from entry or use as a and as fresh and dried materials, and that they field. both of these contain a high diversity of possess indigenous knowledge to treat digestive plant sources for medicinal purposes. however, system disorders in their community. biotropia vol. 28 no. 1,2021 acknowledgements the study was conducted with financial support from the islamic development bank arranged by the universitas sultan ageng tirtayasa (no. 356/un43.9/pl/2017). the authors would like to express their gratitudes to the baduy community for participating in this study as well as for sharing their indigenous knowledge and hospitality. the authors would also like to acknowledge all of the students of biology education at the universitas sultan ageng tirtayasa who contributed to this study in various ways. references akinyele ta, okoh 0 0 , akinpelu da, okoh ai. 2011. in vitro antibacterial properties of crude aqueous and n-hexane extracts of the husk of cocos nncifera. molecules 16(3):2135-45. backer a, van den brink b. 1965. flora of java (spermatophytes only). volume i. noordhoff (nl): n.v.p. groningen. international conference of masyarakat biodiversitas indonesia, bandung. 1 (6):1425-32. indonesian institute of sciences. 2014. present status of indonesian biodiversity. jakarta (id): indonesian 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the ovary is superior, producing fruits in the form of drupe which are free on the receptacles. is the type species of the genus and was described from australia (brown 1810). australia houses 38 species of (hyland 1989) and rest of the species are distributed mostly in malesian regions with very few in east asia and pacific islands and extending north to southern china. new guinea is the largest island in malesian regions and a recent study on the species of revealed that * corresponding author : debyarifiani@yahoo.com 59 there are 46 species in that island with a high number of endemic species (arifiani 2012, unpubl.). the species of occurred generally in lowland forests (from 0 600 m above sea level) and the number of species decreases at higher altitude in montane forest (above 1000 m). delimiting species of requires both vegetative and floral characters evaluation. the characters evaluated include indument in all part surfaces, leaf, inflorescence, tepal, stamen, staminal glands, staminodia and fruit. detailed observation of the characters in several specimens suggested that the specimens of coode and dockrill 32655 and womersley and vandenberg 37195 represent an undescribed species. the description of was done using herbarium specimens available at herbarium bogoriense (bo) and loan specimens from singapore botanic gardens (sing). the study was carried out following methods of rifai (2011) in species enumeration. specimen characterization was done to collect morphological data by observing the characters of all specimens under the microscope (table 1). all measurements, the number and states of the characters were noted including their position, color, fragrance, texture, density and shapes. all measurements are for dried specimens or as otherwise stated. characterization method and botanical terms used endiandra endiandra endiandra kassamensis materials and methods 60 biotropia vol. 19 no. 2, 2012 plant parts characters habit height; width twig color; indument type, orientation and density terminal bud indument; shape; size leaf arrangement; texture leaf blade shape; size; apex; base; surface midrib texture lateral veins number; angle minor venation reticulation density petiole shape; indument; length inflorescence type; position; length; indument pedicel indument; length bract shape; size receptacle depth; indument tepal opening; shape; size; indument stamens number; shape; indument; size glands number; shape; indument; size staminodes number; shape; indument; size pistil shape; indument; length fruit shape; size table 1. characters observed in the taxonomic study (kostermans 1957, rohwer 1993) followed veldkamp (1987). characters observed were compared and correlated to each other in order to assign specimens to a discrete taxon. finally, description for each species was created, including information on distribution, habitat and ecology, and notes on the specific characters important for the taxon. similar to vegetatively but has more lateral veins and coarser reticulation. the flowers of bears no staminal glands but staminodes are present. type: (holo bo; iso sing), kassam pass, kainantu, eastern highlands district, png. tree up to 43 m high, 90 cm in diameter. solid, dark brown, glabrous. conical, big, with densely pubescent with short appressed hairs. alternate; petiole thin, canaliculate above, ca. 1 cm long, glabrous; blade coriaceous narrowly elliptic to elliptic, 11-14 x 3-6 cm, glabrous on both surfaces, apex acuminate, base cuneate; midrib flat to slightly impressed above, raised below, both surfaces glabrous; lateral veins diverging, 10-11 pairs, slightly raised, glabrous on both surfaces; minor venation coarsely reticulate, prominent. paniculate, bear many flowers, up to 12 cm long, terminal or axillary, with sparse short hairs; bracts caducous; pedicels slender, ca. 3 mm long, with dense curly hairs brown (fresh), erect, 2 mm in diameter; tepals thin, soft, subequal (inner ones smaller), narrowly ovate, outer ones results and discussions endiandra fulva e. kassamensis endiandra kassamensis womersley and vandenberg 37195 twigs terminal buds leaves inflorescences flowers , . 61 endiandra kassamensis et al.(lauraceae) a new species from papua new guinea – deby arifiani figure 1. arifiani. a. habit; b. intact flower; c. flower with 2 tepals removed; d. anther; e. staminode; f. pistil ( ). endiandra kassamensis womersley and vandenberg ngf 37195 1.2-1.5 x 1-1.2 mm, inner ones 1-1.2 x 0.8, with sparse long curly hairs outside, same inside especially basal part at anthers attachment; glands none; stamens 3; anthers somewhat triangular, 1 x 0.6 mm, glabrous; filament short, 0.1 mm long; locules small, roundish; staminodia 3, pentagonal, 0.4 x 0.3 mm, with curly hairs; receptacles deep, with curly hairs; ovary ovoid, 0.6-0.9 mm long, glabrous; style ca. 0.3 mm long; stigma inconspicuous. unknown distribution eastern highlands district (png). habitat & ecology rain forest, on hillside, subcanopy; alt. 1280-1372 m. specimens examined (3 sheets) coode & dockrill 32655 (bo); womersley & vandenberg 37195 (bo, sing). notes arifiani is different from other species of in new guinea because of the composition of its flowers. the species bears no glands but interestingly staminodia are present. vegetatively, is similar to teschner but bears more lateral veins and coarser reticulation. is endemic in new guinea and has a restricted distribution, i.e. in the eastern part of new guinea (papua new guinea) and was not found further west in west papua. futhermore the species grows only in a relatively high altitude forest. according to rohwer (1993), the diversity of lauraceae species are higher in lowland forests, which is in agreement with the diversity of in general except for several species of including that was only found in higher altitude forests, in the restricted area of kassam pass, kainantu subdistrict. this fact leads to be considered a rare species. detailed studies of endemic and rare species of are important for providing information for decision makers in allocating conservation efforts. is described for the first time in this study. it is a rare species and endemic to new guinea. the first author graciously thanks prof. mien a. rifai for valuable guidance and constructive suggestion during the research and to dr. sri s. tjitrosoedirdjo for valuable discussion and support during the research and preparation of the manuscript. we acknowledge the generosity of the curator of sing herbarium for the loan materials. we would like to thank the anonymous reviewer for the important comments and to mr. subari for the botanical line drawings. fruits . endiandra kassamensis endiandra e. kassamensis e. fulva endiandra kassamensis endiandra endiandra e. kassamensis e. kassamensis endiandra endiandra kassamensis conclusions acknowledgments 62 biotropia vol. 19 no. 2, 2012 63 references arifiani d. 2012. the biosystematic study of r.br. (lauraceae) in new guinea [dissertation]. depok, indonesia: universitas indonesia. 144 p. brown r. 1810. . typis richardi taylor et socii, london. hyland bpm. 1989. a revision of (excluding ). aust syst bot 2: 135-367. kostermans ajgh. 1957. . 4: 193-256. rifai ma. 2011. asas-asas sistematika biologi. herbarium bogoriense, pusat penelitian biologi-lipi, bogor. rohwer jg. 1993. . in: kubitzki k, rohwer jg, bittrich v. editors. the families and genera of vascular plants ii. berlin: springer verlag. van der werff h, richter hg. 1996. toward an improved classification of . ann mo bot gard 83: 40918. veldkamp jf. 1987. sequence of organs and terminology of characters. in: manual of herbarium taxonomy, theory and practice, de vogel e.f, editor. unesco. endiandra prodromus florae novae hollandiae et insulae van diemen lauraceae cassytha lauraceae reinwardtia lauraceae lauraceae endiandra kassamensis et al.(lauraceae) a new species from papua new guinea – deby arifiani microhabitat influence on growth distribution pattern of ramin ( ) in siak, riau gonystylus bancanus province** didit okta pribadi* and yayan wahyu c. kusuma center for plant conservation, botanic gardens, indonesian institute of sciences, bogor, indonesia r 4eceived 12 april 2012/ accepted 31 december 201 abstract plant growth distribution patterns are influenced by habitat characteristics, ability of adaptation and association with other plant or animals. the influence of those factors, especially habitat characteristic, needs to be species defined to support plant conservation management. this study was aimed to: 1) measure plant growth dependence on their microhabitat; 2) define microhabitat variables that significantly influence the growth; and 3) develop suitable conservation measures at species level. ramin ( ) is one of major timber species that has been facing gonystylus bancanus high exploitation in indonesia. this species is usually found on specific “peat swamps” ecosystem. data were collected through primary surveys in riau province and analyzed by clustering the adult based on total height and basal area variables and describing the distribution pattern of cluster. then, discriminant function analysis the (dfa) was used to overlay the cluster with the distribution of microhabitat characteristic consist altitude, slope, ing soil humidity, soil ph, peat depth and canopy cover (measured in percentage). the results showed that distribution of microhabitat matched with 67.4% of height distribution and 78.3% of width distribution of tree basal area. altitude and canopy cover percentage had significant correlation with total height distribution (α=0.05). meanwhile, altitude, canopy cover and slope had significant correlation with basal area (α=0.1). however, peat depth variable showed an interesting pattern since shallower peat depth was followed by wider basal area. high correlation between plant growth and its microhabitat suggested that to conserve , conservation offered better strategy g. bancanus in-situ than conservationex-situ . keywords: discriminant function analysis, microhabitat, peat swamp forest, plant growth gonystylus bancanus, distribution, ramin introduction ramin ( ) wood is one of the gonystylus bancanus most valuable timbers in indonesia. it has various local names such as gaharu buaya, medang keladi or pulai miang. it is known as an expensive wood which is similar to merbau ( spp., especially intsia intsia bijuga intsia palembanica shorea and ), meranti ( spp.), bangkirai ( spp. and spp.), shorea hopea eboni ( spp.) and cendana (diospyros santalum album l.). people usually use it as material for construction, making doors and windows and producing various kinds of furniture. ramin wood is produced from genus , a gonystylus member of thymelaeaceae family and gonystyloideae subfamily (sidiyasa 2005). approximately, 30 species of are gonystylus distributed in brunei darussalam, fiji, indonesia (kalimantan and sumatera), malaysia (peninsular malaysia, sabah and sarawak), singapore, nicobar island, solomon island and the philippines (soerianegara . 1994). there are et al ten species which are found in indonesia namely g. affinis g. bancanus g. radlk., (miq.) kurz., brunnescens g. confusus g. airy shaw, airy shaw, forbesii g. keithii g. macrophyllus gilg., airy shaw, (miq.) airy shaw, hook f., g. maingayi g. velutinus airy shaw, and airy shaw (shaw g. xylocarpus 1954; purba 2002).et al. biotropia vol. 22 no. 1, 2015: 1 10 doi: 10.11598/btb.2015.22.1.284 1 * corresponding author : diditpribadi@yahoo.com **this paper was presented at association for tropical biology and conservation (atbc) conference 2010 on tropical biodiversity: surviving the food, energy and climate crisis (19-23 july 2010). sanur-denpasar, bali, indonesia mailto:diditpribadi@yahoo.com biotropia vol. 22 no. 1, 2015 2 gonystylus bancanus is the most valuable ramin wood traded in international market. it can be found in peninsular malaysia, sabah, sarawak, sumatera, bangka, kalimantan and brunei darussalam (lim 2004; sidiyasa 2005). it et al. grows in peat swamp forest having wet climate and waterlogged soil. it mostly becomes dominant tree at peat depth of 350-600 mm (istomo 2006; rostiwati . 2007).et al in the early 1980s, indonesia had become the biggest exporter of ramin followed by sarawak and peninsular malaysia (soerianegara et al. 1994). in the 1970s the annual harvested volume of ramin in indonesia was about 1.5 million m , and it decreased drastically to 131,000 3 m in the year of 2000 (lim . 2004) which 3 et al indicated that exploitation of ramin wood had exceeded their regeneration capacity (bismark et al. 2005). therefore, ramin protection and conservation efforts have been started by including spp., especially , in gonystylus g. bancanus appendix ii cites in 2004 (samedi 2005). it means that the trade of spp., especially gonystylus g. bancanus, has been restricted by certain quota every year. trade restriction is among policies which are expected to reduce ramin wood's exploitation. however, since illegal logging activities and the loss of forestland are still continuing in indonesia, it should be coupled with direct conservation measures in the field. bogor botanical garden is one of the indonesian conservation institutions having responsibility to conserve such kind of plants. therefore, this research becomes important to analyze the growth pattern of g. bancanus in its habitat, thus conservation management could be defined properly. plant growth distribution patterns are influenced by habitat characteristics, ability of adaptation and association with other plant species or animals. recently, studies on the natural distribution of plants and animals species are growing not only to assess the impact of anthropogenic effect or climate change on their habitat, but also to define appropriate conservation management of the species (guissan & thuiller 2005; sinclair . 2010). et al every species has different strength of relationship with its habitat, thus some species have endemism distribution and the others have cosmopolitan distribution. the endemicity level of a species will determine the effectiveness of conservation management effort either in ex-situ or conservation.in-situ based on the above reasons, the aims of this research were: 1) to measure growth dependence of on their microhabitat; 2) to define g. bancanus microhabitat variables that significantly influence the growth of ; and 3) to formulate g. bancanus suitable conservation measures for . g. bancanus few parts of the research had been orally presented during atbc (association for tropical biology and conservation) 2010 conference in bali. materials and methods site description the research was conducted in danau pulau besar/danau bawah wildlife sanctuary that encompasses an area of 25,000 ha. this area is located in dayun village, siak district, siak municipality, riau province. geographically, this area lies between 00 35'-00 45' n and 102 10'-0 0 0 102 19' e (ipb & uksda riau 2000). it is 0 managed by balai besar konservasi sumber daya alam, riau province. generally, this area consists of peat swamp area which has flat topography, low altitude from 2 to 6 m above sea level and slope ranging from 00 to 3 (ipb & uksda riau 2000). it is situated in 0 an alluvium area which was produced by siak river sedimentation, thus it contains high organic and inorganic matters. based on climate type of schmidt-fergusson, this area is classified into type a which has rainfall from 2,200 to 2,600 mm per year. temperature of the area is 26.2 c per year on 0 average, with maximum temperature of 32.4 c 0 and minimum temperature of 21.7 c. air 0 humidity in this area can reach 84% on average with maximum humidity of 97% and minimum humidity of 60% (ipb & uksda riau 2000). research time and location the research was conducted from 5 to 17 december 2007. the location of the study site was danau pulau besar/danau bawah wildlife sanctuary, siak municipality, riau province. microhabitat influence on growth distribution pattern of ramin ( ) didit okta pribadigonystylus bancanus – et al. survey methods field survey was conducted to collect primary data which consisted of spatial distribution of g. bancanus, total height and diameter breast height of adult tree, including microhabitat characteristics in each location of g. bancanus comprising altitude, slope, soil humidity, soil ph, peat depth and canopy cover (in percentage). to investigate the microhabitat characteristics, unbound plots of ca. 100 m were applied with at 2 least one individual of centered within g. bancanus the plot. the sample was taken based on purposive random sampling method because the research was only focused on as the g. bancanus target species. data analysis data were analyzed by clustering the adult based on total height and basal area variables. the criteria used to define the cluster were as follows:  cluster 1: more than average + standard deviation  cluster 2: average ± standard deviation  cluster 3: less than average – standard deviation furthermore, discriminant function analysis (dfa) was used to overlay the cluster with the distribution of microhabitat characteristics consisted of altitude, slope, soil humidity, soil ph, peat depth and canopy cover. dfa is one of multivariate analyses used to evaluate the accuracy of cluster formation based on several quantitative variables. in this study, cluster of growth formation based on height and width of basal area was evaluated by microhabitat characteristics. the correlation between cluster of growth (based on height and width of basal area) and microhabitat characteristics were used to estimate influence value of microhabitat characteristics on growth distribution of . in addition, dfa was g. bancanus also used to discover which microhabitat characteristics best explained differences on distribution of the species (mcgarigal . 2000). et al when correlation between the plant and its habitat is relatively high, it means that the adaptation of the plant in different habitat is relatively low. results and discussion based on height variable, was g. bancanus divided into three clusters: 1) cluster 1 consisted of 9 individuals with 32-35 m of total height; 2) cluster 2 consisted of 29 individuals with 22-30 m of total height; 3) cluster 3 consisted of 8 individuals with 15-20 m of total height. based on basal area, was divided into three g. bancanus clusters: 1) cluster 1 consisted of 7 individuals with 2,428-2,307 cm ; 2) cluster 2 consisted of 31 2 individuals with 594-1,698 cm ; 3) cluster 3 2 consisted of 8 individuals with 154-330 cm .2 each cluster of height and basal area had microhabitat characteristics that were shown in table 1 and 2, respectively. there were 6 measured microhabitat characteristics i.e. altitude, slope, soil humidity, soil ph, peat depth and canopy cover. each cluster had respective mean and standard deviation for each microhabitat characteristic. based on the above results, dfa was conducted to evaluate whether clustering of height matched with distribution of microhabitat characteristics. the result showed that distribution of microhabitat characteristics only 67.4% matched with cluster of height. however, further dfa analysis on microhabitat characteristics expressed that the individuals of g. bancanus could be grouped only in 2 clusters with detailed explanation as follows (table 3): a. nine individuals member of cluster 1 of height should be included in cluster 2 of microhabitat characteristics; b. from 29 individuals member of cluster 2 of height, 26 individuals should be included in cluster 2 of microhabitat characteristics, while the other 3 individuals should be included in cluster 3 of microhabitat characteristics; c. from 8 individuals member of cluster 3 of height, 3 individuals should be included in cluster 2 of microhabitat characteristics, while the other 5 individuals should be included in cluster 3 of microhabitat characteristics. since the correlation between distribution of height with microhabitat characteristics was only 3 biotropia vol. 22 no. 1, 2015 4 table 1. microhabitat characteristics of each cluster for total height group statistics total height mean standard deviation valid n (listwise) unweighted weighted 1 alt 23.8889 1.90029 9 9.000 slope 12.2222 6.96020 9 9.000 srh 94.4444 11.30388 9 9.000 sph 5.3556 .46934 9 9.000 pd 119.8889 26.47666 9 9.000 cc .0000 .00000 9 9.000 2 alt 23.1724 2.81665 29 29.000 slope 12.7241 5.94406 29 29.000 srh 95.7931 10.23083 29 29.000 sph 5.4069 .59036 29 29.000 pd 112.3966 29.87510 29 29.000 cc 18.2759 24.79472 29 29.000 3 alt 18.7500 4.97853 8 8.000 slope 9.6875 4.78791 8 8.000 srh 94.3750 10.50085 8 8.000 sph 5.1125 .41897 8 8.000 pd 121.0625 34.62290 8 8.000 cc 55.6250 29.20830 8 8.000 total alt 22.5435 3.55094 46 46.000 slope 12.0978 5.95410 46 46.000 srh 95.2826 10.26897 46 46.000 sph 5.3457 .54353 46 46.000 pd 115.3696 29.69824 46 46.000 cc 21.1957 28.65862 46 46.000 notes: alt = altitude; slope = slope; srh = soil humidity; sph = soil ph; pd = peat depth; cc = canopy cover 67.4%, it was predicted that there were other factors affecting plant total height distribution such as genetic factors, age differences, or even other physiological factors (ryan . 2006). et al although the influence of age differences was minimized by selecting only adult plants which had similar appearance, however, the age of the plants was not measured quantitatively (e.g. tree rings approach) study. in this another result of dfa showed that there were only two variables having significant influence on the tree height distribution consisting altitude and canopy cover (table 4). based on information from table 1, it was described that increasing altitude was followed by increasing tree total height, while decreasing canopy cover was followed by increasing total height. figure 1 shows that cluster 2 (higher tree height) was located in higher altitude and lower canopy cover, whereas cluster 3 (lower tree height) was located in lower altitude and higher canopy cover. this explained light sufficiency is important to support the height growth of . higher g. bancanus altitude and less canopy cover gave more open space to the plant for getting more light intensity. no, 5 table 2. microhabitat characteristics of each cluster for basal area group statistics basal area mean standard deviation valid n (listwise) unweighted weighted 1 alt 23.8571 2.85357 7 7.000 slope 17.8571 4.94734 7 7.000 srh 95.7143 11.33893 7 7.000 sph 5.2000 .46547 7 7.000 pd 106.0571 38.28607 7 7.000 cc 4.2857 11.33893 7 7.000 2 alt 23.1613 2.55730 31 31.000 slope 11.2903 5.74424 31 31.000 srh 96.0645 9.93960 31 31.000 sph 5.4194 .58276 31 31.000 pd 113.8097 27.25907 31 31.000 cc 15.4839 24.60877 31 31.000 3 alt 19.0000 5.31843 8 8.000 slope 10.1875 5.02805 8 8.000 srh 91.8750 11.31923 8 8.000 sph 5.1875 .42908 8 8.000 pd 129.5625 30.02075 8 8.000 cc 58.1250 24.19231 8 8.000 total alt 22.5435 3.55094 46 46.000 slope 12.0978 5.95410 46 46.000 srh 95.2826 10.26897 46 46.000 sph 5.3457 .54353 46 46.000 pd 115.3696 29.69824 46 46.000 cc 21.1957 28.65862 46 46.000 note: alt = altitude; slope = slope; srh = soil humidity; sph = soil ph; pd = peat depth; cc = canopy cover table 3. evaluation result for cluster of total height membership based on microhabitat characteristics by using dfa classification resultsa total height predicted group membership 1 2 3 total original count 1 0 9 0 9 2 0 26 3 29 3 0 3 5 8 % 1 .0 100.0 .0 100.0 .0 89.7 10.3 100.0 3 .0 37.5 62.5 100.0 notes: 1. = 67.4% of original grouped cases were correctly classifieda 2. cluster of height are represented by row, while cluster of microhabitat characteristics are represented by column no. microhabitat influence on growth distribution pattern of ramin ( ) didit okta pribadigonystylus bancanus – et al. 2 biotropia vol. 22 no. 1, 2015 6 table 4. significant influences from microhabitat characteristics on tree total height distribution tests of equality of group means wilks' lambda f df1 df2 significance level alt .748 7.236 2 43 .002 slope .964 .811 2 43 .451 srh .996 .093 2 43 .911 sph .959 .918 2 43 .407 pd .982 .386 2 43 .682 cc .627 12.772 2 43 .000 figure 1. plot of cluster of tree height based on altitude and canopy cover variables simultaneously, dfa was also conducted to evaluate whether clustering of basal area matched with distribution of microhabitat characteristics. the result showed that distribution of microhabitat characteristics only matched 78.3% with the cluster of basal area. detailed explanation of the result was as follows (table 5): a. from 7 individuals member of cluster 1 of basal area, 2 individuals should be included in cluster 1 of microhabitat characteristics, while the other 5 individuals should be entered in cluster 2 of microhabitat characteristics; b. from 31 individuals member of cluster 2 of basal area, 1 individual should be included in cluster 1 of microhabitat characteristics, 28 individuals should be included in cluster 2 of microhabitat characteristics, while the other 2 individuals should be included in cluster 3 of microhabitat characteristics; c. from 8 individuals member of cluster 3 of basal area, 2 individuals should be included in cluster 2 of microhabitat characteristics, while the other 6 individuals should be included in cluster 3 of microhabitat characteristics. note: significance level of respective variable is shown by value in the sixth column which is less than 0.05 7 distribution of microhabitat characteristics had higher correlation with variable of basal area than with variable of height. it described that 78.3% of basal area distribution were influenced by microhabitat characteristics and the rest was influenced by other factors such as genetic factors or age differences. as mentioned before, we did not measure the influence of age differences. table 5. evaluation result for cluster basal area width membership based on microhabitat characteristics by using dfa classification resultsa basal area predicted group membership 1 2 3 total original count 1 2 5 0 7 2 1 28 2 31 3 0 2 6 8 % 1 28.6 71.4 .0 100.0 2 3.2 90.3 6.5 100.0 3 .0 25.0 75.0 100.0 notes: 1. = 78.3% of original grouped cases were correctly classifieda 2. cluster of basal area are represented by row, while cluster of microhabitat characteristics are represented by column table 6. significant influences of microhabitat characteristics on basal area distribution tests of equality of group means wilks' lambda f df1 df2 significance level alt .781 6.035 2 43 .005 slope .823 4.609 2 43 .015 srh .976 .525 2 43 .595 sph .961 .870 2 43 .426 pd .942 1.319 2 43 .278 cc .623 12.995 2 43 .000 note: significance level of respective variable is shown by value in the sixth column which is less than 0.05 figure 2. plot of cluster of basal area based on altitude, slope and canopy cover microhabitat influence on growth distribution pattern of ramin ( ) didit okta pribadigonystylus bancanus – et al. biotropia vol. 22 no. 1, 2015 8 another result of dfa showed that there were three variables having significant influence on basal area distribution consisting of altitude, slope and canopy cover (table 6). based on information from table 2, the increasing altitude, slope and decreasing canopy cover were followed by increasing basal area. this could also be seen from figure 2 where cluster 1 (the widest basal area) was located in higher altitude, higher slope and lower canopy cover, whereas cluster 3 (the narrowest basal area) was located in lower altitude, lower slope and higher canopy cover. peat depth variable showed an interesting pattern since shallower peat depth was followed by wider basal area, although not significant. the above result indicated that the distribution of microhabitat characteristics had 67.4% correlation, with distribution of g. bancanus vertical growth. microhabitat characteristics which could support vertical growth of g. bancanus were higher altitude and decreasing canopy cover. this meant that needs g. bancanus sufficient light intensity to support its vertical growth. therefore, sufficient light intensity should be provided if would be g. bancanus cultivated in conservation to produce ex-situ longer log. a research by jans . (2012) showed et al that the seedling growth of was the g. bancanus highest in partial sunlight and reduced with decreasing light intensity. afterwards, big-sized tree will flourish in full sunlight (partomihardjo et al. 2008). the result also indicated that distribution of microhabitat characteristics had higher correlation with distribution of basal area than with vertical growth. the correlation of microhabitat characteristics with basal area was 78.3%. microhabitat characteristics supporting basal area growth of were higher g. bancanus altitude, increasing slope, decreasing canopy cover, while level and shallower peat depth could also support basal area growth. peat swamp ecosystem is usually developed in a basin area. therefore, the result actually indicated that would have maxim basal area if g. bancanus um they grew at the edge of the basin area which had higher altitude, higher slope, lower canopy cover and shallower peat depth. the result followed istomo (1998) and bismark . (2005) where et al deeper peat depth gave higher density, thus shallower peat depth gave lower density and higher basal area. it showed that the range of their habitat actually was relatively narrow. the distribution of adult at the edge of the g. bancanus basin area, recorded by gps, in danau pulau besar/danau bawah wildlife sanctuary could be seen in figure 3. from the correlation between microhabitat characteristics and distribution of vertical growth and basal area, it could be estimated that planting of without any treatments in the g. bancanus outside area of their habitat would not be optimal. the survival rate would be low, and if they were successfully grown, the growth rate would be very slow s . ismail in term of height and tree basal area et al. g. bancanus (2007) found that planting of in non-peat swamp area after 11 years resulted to low survival rate (52%), low average of total height (8.16 m), and low average diameter breast height (9.1 cm). therefore, even though g. bancanus can be planted in non-peat swamp areas, limiting factor on utilization will be the relatively small diameter and log length. new or existing technology that can utilize smaller size logs may solve the problem (ismail . 2007). apparently, et al planting of is not easy and its growth g. bancanus is relatively very slow. according to the above discussion, although there is a possibility to conserve in an g. bancanus ex-situ in-situ conservation area, but the role of conservation becomes more important to conserve in an optimal condition. g. bancanus therefore, massive exploitation and land use change in peat swamp ecosystem become two important challenges in conserving . g. bancanus however, a technolog breakthrough to y cultivate outside their habitat is g. bancanus still needed to strengthen the conservation of this species. as suggested by krigas . (2010) et al this kind of study can provide valuable information to facilitate the transfer from wild habitats to man-made habitats like botanical garden. this transfer is important to produce material for reintroduction and reinforcement of this species when its existence in nature becomes increasingly rare (guerrant 2004). et al. bogor botanical garden has been planting seedlings of since year 2007, but g. bancanus their growth are still insignificant despite their survival. figure 3. distribution of within the study area in riau province in 2008 g. bancanus conclusions high correlation between the distribution of tree growth and microhabitat characteristics suggested that conservation offers better in-situ strategy than conservation to conserve ex-situ g. bancanus , especially when an appropriate planting method is not yet developed. acknowledgements we thanked our colleagues (yupi isnaini, enda suhenda and m. madhari) for their assistance in various aspects of this study. we also thanked the people in dpb-dw (sutrisno, jumaat, beni ishak silalahi) for their hospitality, and bksda riau for the permission support. the study was funded by dipa center for plant conservation bogor botanical gardens-lipi. references bismark m, kalima t, wibowo a, and sawitri r. 2005. growing stock, distribution and conservation of ramin in indonesia. technical report no. 01. itto pro.87/03 rev. 2 (f). identification of gonystylus spp. (ramin) potency, distribution, conservation and plantation barrier. ministry of forestry, center for forest and nature conservation research and development in cooperation with international tropical timber organization. guerrant eo, havens k, maunder m. 2004. ex-situ plant conservation: supporting species survival in the wild. washington dc(us):island press. guisan a, thuiller w. 2005. predicting species distribution: offering more than simple habitat model. ecol lett 8: 993-1009. institut pertanian bogor, unit konservasi sumberdaya alam riau. 2000. laporan akhir rencana pengelolaan suaka margasatwa danau pulau besar/danau bawah kabupaten siak propinsi riau. bogor (id):unit konservasi sumberdaya alam riau dan fakultas kehutanan institut pertanian bogor. ismail p, shamsudin i, abdul rahman k, hashim ws, ismail h. 2007. planting of in gonystylus bancanus non peat swamp area. jtfs 19 (1): 50-6. istomo. 1998. penyebaran pertumbuhan pohon ramin ( (miq.) kurz.) di hutan rawa gonystylus bancanus gambut: studi kasus di hph pt. inhutani iii kalimantan tengah. laboratorium ekologi hutan. buletin manajemen hutan. p 33-9. istomo. 2006. kandungan fosfor dan kalsium pada tanah dan biomassa hutan rawa gambut (studi kasus di wilayah hph pt. diamond raya timber, bagan siapi-api, provinsi riau). jmht 12 (3): 40-57. jans wwp, dibor l, verwer c, kruijt b, tan s, van der meer pj. 2012. effects of light and soil flooding on the growth and photosynthesis of ramin (gonystylus bancanus) seedlings in malaysia. jtfs 24(1):54-63. krigas n, mouflis g, grigoriadou k, and maloupa e. 2010. conservation of important plants from the ionian islands at the balkan botanic garden of kroussia, n greece: using gis to link the collection data in-situ with plant propagation and cultivation. ex-situ biodivers conserv 19:3583-603. 9 microhabitat influence on growth distribution pattern of ramin ( ) didit okta pribadigonystylus bancanus – et al. lim t w, soehartono t, keong ch. 2004. framing the picture: an assessment of ramin trade in indonesia, malaysia and singapore. malaysia(my): traffic southeast asia. mcgarigal k, cushman sa, stafford s. 2000. multivariate statistics for wildlife and ecology research. new york(us): springer verlag. partomihardjo t, prajadinata, s, hidayat a. 2008. current status of ramin seeds source in sumatra. technical report itto project po 426/06 rev. 1 (f). indonesia: ministry of forestry. purba c, suparno, hariyono, zulfahmi, seventri ad. 2002. laporan studi perdagangan domestik dan internasional kayu ramin sebagai bagian dari pengumpulan informasi dasar dalam kerangka pelestarian jenis ramin di indonesia. bogor(id): forest watch indonesia/telapak. rostiwati t, murniati, hendromono. 2007. growth of ramin ( (miq.) kurz.) plantation gonystylus bancanus on various peat swamp forests in indonesia. ijfr 4 (2): 73-81. ryan mg, phillips n, bond bj. 2006. the hydraulic limitation hypothesis revisited. plant cell environ (29): 367–81. samedi. 2005. kontrol perdagangan ramin ( spp.) gonystylus internasional. dalam: semiloka nasional konservasi dan pembangunan hutan ramin di indonesia. prosiding. 28 september 2005; bogor (id): puslitbang hutan dan konservasi alam-itto. pp 60-72. shaw a. 1954. gonystilaceae. in: van steenis, cggj editor. flora malesiana i, vol. (4). jakarta(id): noordhoffkolff n. v. p 350-65. sidiyasa k. 2005. potensi botani, ekonomi dan ekologi ramin ( spp.). dalam: semiloka nasional gonystylus konservasi dan pembangunan hutan di ramin indonesia melalui regulasi perdagangan dan pemacuan alih teknologi konservasi, penanaman dan tehnik silvikultur. prosiding: 28 september 2005; bogor (id): pusat penelitian dan pengembangan hutan dan konservasi alam bekerja sama dengan itto dan ppd. p 9-34. sinclair sj, white md, newell gr. 2010. how seful re u a s d m mpecies istribution odels for anaging b f ciodiversity under uture limates? ecol soc 15(1): 8 [online] url: http://www.ecologyandsociety. org/vol15/iss1/art8/ soerianegara i, sambas en, martawijaya, sudo s, groen le. gonystylus teijsm. & binnend. in: 1994. prosea: plant resource of south-east asia 5 (1) timber trees: major commercial timber. edited by soerianegara i and rhmj lemmens. wageningen (nl):prosea, pudoc. p 221-30. 10 biotropia vol. 22 no. 1, 2015 http://www.ecologyandsociety. biotropia vol. 28 no. 2, 2021: 117 127 doi: 10.11598/btb.2021.28.2.1174 117 natural habitat of bali starling (leucopsar rothschildi) in bali barat national park, indonesia sutomo1 and eddie van etten2 1research centre for plant conservation and botanic garden, indonesian institute of sciences (lipi), candikuning, baturiti, tabanan, bali 82191, indonesia 2school of science, edith cowan university, joondalup, perth, western australia 6027, australia received 18 december 2018/accepted 26 march 2020 abstract the indonesian tropical savannas and dry forests provide habitats to various endemic wildlife. unfortunately, a few of these endemic species are now seriously threatened and are red listed in the conservation status of international union for conservation of nature (iucn). among these species, the bali starling or bali mynah leucopsar rotschildi, locally known as jalak bali, is now mostly restricted to the bali barat national park. given the high extinction risk faced by such species, conservation programs require multidisciplinary approaches that would address both the biological attributes of the species itself and their habitat requirements. regrettably, for many species, their habitat ecology remains inadequately understood. hence, this study aimed to: 1. characterize the bali starling habitat in terms of structure and floristic composition; and 2. document evidences of vegetation cover changes in the bali barat national park. analysis of remote sensing imagery and field sampling for vegetation attributes were conducted to address these objectives. normalized difference vegetation index (ndvi) was calculated from landsat imageries using red and near infrared bands. tree cover percentage data were downloaded from vegetation continuous fields (vcf) of the university of maryland’s website. results showed that forest and savanna are the dominant land cover types in the bali barat national park. however, their distribution is somewhat dynamic with changes in vegetation cover and greenness found across the years which increase the cover of woody plants is the general trend. the bali starling in the bali barat national park is mostly found at or near distinct vegetation boundaries, such as the borders between savanna and forest, savanna and cropland, savanna and shrubland, settlement and cropland and, between forest and shrubland. although cekik in jembrana, bali and brumbun bay in west bali, as the conservation sites for bali starling, are both planted with tree species providing shelter and food for bali starling, the bird has not been seen in the two areas since the 1990s. these results further confirm the importance of examining the habitat patterns of endemic birds within a landscape that are influenced by multiple factors interacting in space and time. addressing data inadequacy in habitat patterns of endemic species distribution is crucial in developing conservation management strategies. hence, evaluating the habitat remnants of the bali starling is vital for its conservation and needed reintroduction and eventual release to its natural habitat. keywords: bali starling, habitat suitability, savanna introduction tropical savannas and dry forests are important ecosystems which comprise those habitats supporting various endemic wildlife of indonesia. a few of these species are now under serious threat of extinction and, consequently, have high conservation status according to the iucn (iucn 2014), including banteng (bos javanicus) that is now mostly confined to the savanna in the baluran national park of east java province, the komodo dragon (varanus komodoensis) which is endemic to the komodo islands of east nusa tenggara province, and the endemic bali starling bird (leucopsar rotschildi) which is now mainly found in the savanna of bali barat national park (bbnp) on the northwest tip of bali province. unfortunately, the rapid and widespread habitat loss and variable management capacities in natural reserves had posed considerable risk to biodiversity (purwandana et al. 2014). *corresponding author, email: tommo.murdoch@gmail.com biotropia vol. 28 no. 2, 2021 118 the critically endangered bali starling (leucopsar rothschildi) is the only endemic bird found in bali. the first bali starling known to science was collected near bubunan, bali and was described by stresemann (1912). the bali starling is an attractive aviary bird being largely white, with black wings, tail tips and bare skin of a turquoise-blue color on the lores and behind the eye. on account of its restricted range, extremely small numbers growing in the wild and persistent pressures on the last free ranging birds, the bali starling is considered critically endangered according to the latest international union for conservation of nature (iucn) threat categories (iucn 2014). habitat destruction and bird capture for the pet trade brought the species to the verge of extinction (van balen et al. 2000). in 1998, estimates showed that less than 20 birds remained in the wild within a small area on the northwest tip of bali island within the boundaries of bali barat national park (bbnp) (collins et al. 1998). although the wild population is near extinction, bali starlings have been successfully bred in captivity (collins et al. 1998). the species’ original habitat in bali was described as ‘dry savanna and shrub woodlands’ and ‘tall and dense forest’ in the 1920s (van der paardt 1926), and until this time it was believed to be historically restricted to a narrow belt of dry monsoon climate in northern bali and east java (van balen et al. 2000). from 1920-1960, its range had shrunk to the fire-induced open shrub and savanna woodland, found below an elevation of 150 175 masl in the northeast part of the prapat agung peninsular within the bbnp (van balen et al. 2000). bird distribution and abundance patterns within a landscape are influenced by multiple factors interacting spatially and temporally (orians & wittenberger 1991). habitat structure and floristic composition, such as percentage of canopy cover, tree species diversity and the distribution of specific plant taxa, are known to have significant roles in defining the spatial occurrence of bird species (james & wamer 1982; rice et al. 1984; wiens & rotenberry 1981). with the high extinction risk faced by such species, intended conservation programs are likely to require multidisciplinary approaches that would address both the biological attributes and resource requirements of the species, such as their habitat requirements and conditions (estoque et al. 2012). therefore, habitat evaluation is important in the conservation of bali starling and its reintroduction and eventual release back to their natural habitat. moreover, knowledge and better understanding on the potential distribution and habitat suitability of the bali starling is important in selecting potential sites for future ex-situ conservation and breeding programs designed to save this endemic bird from its extinction. several studies on the bali starling have focused on the bird itself; ranging from its behavior, reproduction, breeding, genetics, taxonomy, demography and reintroduction, among others (collins & smith 1994; collins et al. 1998; de iongh et al. 1982; dirgayusa et al. 2000; seibels et al. 1997; williams & feistner 2006). however, studies on the habitat of bali starling are scarce (widodo 2014), considering that these habitats are vital to the ongoing maintenance of its viable populations, and yet are also very prone to conversion, disturbances and degradation. therefore, this study aimed to: 1. describe the current distribution of bali starling and characterize its habitat structure at the bali barat national park in terms of its plant community structure and composition, and 2. assess the cover and greenness index (ndvi) to quantify the dynamics of vegetation cover in these habitat areas; and 3), assess the degree of recent habitat changes broadly affecting the bali starling species at bali barat national park (bbnp). materials and methods the bali barat national park (bbnp), located on the northwestern side of bali, indonesia, covers around 19,000 ha (~ 5% of bali's total land area) comprising 15,588 ha of terrestrial areas and 3,415 ha of marine habitats. a seaport at gilimanuk is situated on the west of the park. bbnp is also bordered with several villages and can be reached by roads from gilimanuk and singaraja, or by using ferries from ketapang, east java. several major habitat types are found in the national park; savanna, mangroves, montane and mixed-monsoon forests, and, coral islands. bali barat was bali starling (leucopsar rotschildi) natural habitat structure in bali barat – sutomo and van etten 119 designated as a national park in 1984 based on the ministry of forestry decree (no. 096/kptsii/1984). the area is predominanly of the latosol soil type, reddish in color, weakly crumbed and sticky when wet although hardened and cracked when dry. the park is topographically varied, ranging from plains near the coast to steep hills and mountains. it has four mountains, namely prapat agung, banyuwedang, klatakan and sangiang (the highest peak at 1,414 m). off the coast, four islands are also under the bbnp jurisdiction, namely menjangan, burung, gadung and kalong islands. bbnp has a moderate climate seasoned with monthly rains, but higher rainfall occurs during the wet season in december to february. the average annual rainfall ranges from 900 1,500 mm and average temperature is at 33°c (masy'ud et al. 2008; masy'ud et al. 2007; whitten et al. 1996). reports on the bali starling population, distribution and their habitat were obtained through the published literature and also via personal communications with bbnp rangers and managers. data on its local distribution were obtained from de longh et al., (1982), whitten et al., (1996), van balen et al., (2000) and bbnp manager, wiryawan, (2014, pers.comm.). based on these studies, the bali starling occurrence data were divided into three eras of distribution, namely 1984, 1994 and 2010, considering the only 3 years of reliable and accurate surveys done on these years. an overlay analysis of these bali starling location data was done using indonesia’s topographical/ earth surface map (rupa bumi indonesia/rbi) for the year 2001 (scale 1 : 80,000) obtained from the indonesian geospatial agency (big/bakosurtanal). the 2001 land use data was the most recently available. to avoid misalignment, all the data used the same datum and the same map projection within the gis (wgs 1984-utm, zone_50). the vegetation continuous fields (vcf) collection contains proportional estimates for vegetative cover types: woody vegetation, herbaceous vegetation, and bare ground. the product is derived from all seven bands of the moderate resolution imaging spectroradiometer (modis) sensor onboard nasa's terra satellite. this continuous classification scheme of the vcf product may depict areas of heterogeneous land cover better than traditional discrete classification schemes. while traditional classification schemes indicate where land cover types are concentrated, this vcf product is best for showing how much of a land cover such as "forest" or "grassland" exists anywhere on a land surface (dimiceli et al. 2011). ndvi is an index derived from remotely sensed imagery which can differentiate between vegetation types by showing the difference between near infrared (which is strongly reflected by vegetation) and red light (which is absorbed by vegetation). ndvi is correlated to vegetation biomass, vigour and photosynthetic activity. this index exploits the reflectance patterns of ground elements in the red (r) and near-infrared (nir) bands of the electromagnetic spectrum to distinguish green vegetation from its background soil brightness, and is calculated as (nir r)/ (nir + r). ndvi values range from -1 to 1, with positive values representing vegetated areas and negative values representing non-vegetated regions (sankaran 2001). the ndvi ratio approach usually adopted for land cover change estimation is used here in preference to the more commonly employed post-classification pixel-by-pixel comparison method (lillesand et al. 2008) since it also permits identification of areas where changes in vegetative cover have been significant, but insufficient to cause change in class membership (sankaran 2001). in order to generate normalized difference vegetation index (ndvi), a number of landsat images were used. landsat images were downloaded from http://earthexplorer.usgs. gov/path 117, row 066. the chosen downloaded images are those with minimal cloud cover percentage by selected scenes with at least image quality level 9 (no errors detected, perfect scene) (table 1). biotropia vol. 28 no. 2, 2021 120 table 1 details of downloaded images for ndvi analysis images source date acquired spatial resolution image quality cloud cover 1 landsat 4 21/03/1989 30 x 30 m 9 20 2 landsat 7 12/11/1999 30 x 30 m 9 8.63 3 landsat 7 31/05/2003 30 x 30 m 9 7.53 4 landsat 8 11/06/2016 30 x 30 m 9 24.99 ndvi was generated using the ndvi feature in arcmap (arcgis 10.1) image analysis toolbar. band 1, 2, 3, and 4 were chosen for landsat 4 and 7, whereas band 2, 3, 4, and 5 were chosen for landsat 8 as input images in arcmap which represent the blue, green, red and near infrared (nir) bands. by choosing the image analysis tab, all the bands layers were merged into one composite layer and then the rgb (red-green-blue) channels were adjusted to show just the nir, red and green bands to extract the ndvi values. once ndvi images were generated, different levels of a green color scheme was applied for easier interpretation. these ndvi values must be used carefully as they represent only one time in the chosen years (due to the limited availability of good images for the chosen years) and as these will mainly reflect recent rainfall, especially in terms of groundcover. then the data points for the 1984, 1994 and 2010 bali starling locations were overlaid on the ndvi images from different years (1989, 1999, 2003 and 2016). these years were chosen because these years were the closest years to the years of bali starling location (as clear image of the exact years of bali starling location cannot be found). the mean and sd from nine pixel values surrounding the exact coordinate locations of the bali starling was then calculated from the ndvi images. changes in mean ndvi of starling locations between different years were tested for significance using anova in spss and, when significant differences were detected, a post-hoc test was performed. from 1979 to 1994, the bali starling was regularly observed in brumbun bay, west bali. from 1995 to 2009, no survey was done in the area, therefore no bali starling data were obtained from these locations. in 2010, the surveys conducted by bbnp has recorded bali starling population in this location again. at another site, cekik in jembrana, west bali, the bali starling was also observed during the period 1979-1994, but in the 2010 survey, bbnp did not find the starling in the area. fieldwork was again conducted in november 2014 in these two locations, namely cekik and brumbun, and these areas were cross-checked with a fire map based on modis burned areas produced from year 2000 to 2013 to obtain information on fire history. however, no fire occurred at these localities. the landsat imagery during this period also confirmed that no major fires occurred at bbnp. during the dry season in september to november 2014, ten sampling plots of 50 x 50 m were established randomly in each savanna sites (cekik and brumbun). in each of the 50 x 50 m plots, smaller plots of 5 x 5 m were nested randomly. inside the 50 x 50 m plot, all the tree species ≥ 10 cm diameter at 1.3.m height (dbh) were identified, measured and recorded. in the smaller-nested plots, all the groundcovers species were noted (grasses, herbaceous and ferns) and their coverages were estimated. species in each site were identified in the field where possible and a field herbarium was created for easier identification at the species level during subsequent field works. the identification was assisted by personnel from herbarium bogoriense and herbarium baliensis of the indonesian institute of sciences (lipi) who used flora books such as the “flora of java” (backer & van den brink 1963), “mountain flora of java” (van steenis 1972), “weeds of rice in indonesia” (soerjani et al. 1986), “ ecology of java and bali” (whitten et al. 1996) and “ecology of nusa tenggara and maluku” (monk et al. 2000) and names were standardized based on the plant list (www.theplantlist.org). the importance value index or ivi (kent 2011; kent & coker 1992) was calculated for each species in each plot to understand the structure and plant community composition of each savanna. ivi was used to describe the quantitative structure of the community (curtis & mcintosh 1950; kent & coker 1992). this value represents the contribution of a species to the community in terms of the number of plants within the quadrats (density), its contribution to the community through its distribution (frequency), and its influence on the bali starling (leucopsar rotschildi) natural habitat structure in bali barat – sutomo and van etten 121 other species through its dominance. the importance value index was calculated for each tree species and ground cover in each of the study sites. the differences in plant community composition between brumbun and cekik savanna were tested using abundance data (cover). the data were square-root transformed prior to constructing a resemblance matrix based on the bray-curtis similarity index (valessini 2009). a non-metric multidimensional scaling (nmds) ordination diagram was then generated based on the resemblance matrix. the difference in species composition between savannas was then tested for significance using one-way anosim (analysis of similarity). the ranosim statistic values, generated by anosim, are a relative measure of separation of the a priori defined groups. a zero (0) value indicates that no significant difference existed among the groups, and one (1) value indicates that all samples within groups are more similar to one another than any samples from different groups (clarke 1993). this multivariate analyses made use of the primer v.6 package (clarke & gorley 2005). results and discussion based on the bird observations analysis, the starling is mostly found in a relatively open vegetation (such as savanna and open shrubland) and along their boundaries with other vegetation types in the bali barat national park (bbnp) (fig. 1). this confirms the reports of (de iongh et al. 1982; dirgayusa et al. 2000). based on the bbnp manager’s report, the bird was observed at or near the ecotones between savanna and forest, savanna and cropland, savanna and shrub land, settlement and cropland, and finally, the forest and shrubland. this report is supported by the ndvi analysis for the three-year record of the species locations in 1984, 1994 and 2003 which showed a shift in the preferred habitat of the bali starling from primary forest habitat in the 1980s to more open vegetation areas including, but not limited to, dry forest/monsoon forest, secondary forest and savanna (fig. 2). these results further confirmed those of van balen et al. (2000) that the bali starling range on bali island has shrunk to the fire-induced open shrub and savanna woodland below elevations of 150-175 m in the northeast part of the prapat agung peninsular of the bali barat national park. biotropia vol. 28 no. 2, 2021 122 figure 1 overlay of bali starling occurences with the 2001 land use map of bali barat national park notes: ‘sawah tadah hujan’ refers to a rain fed paddy field. ‘sawah irigasi’ refers to irrigated paddy field. 1984 bali starling (leucopsar rotschildi) natural habitat structure in bali barat – sutomo and van etten 123 1994 2010 figure 2 bali starling habitat distribution based on their normalized difference vegetation index (ndvi) range class notes: habitat and localities are presented for 1984 (upper, aligned with ndvi image data 21/03/1989), 1994 (middle, aligned with ndvi image date 12/11/1999) and 2010 (lower, aligned with the ndvi image data 11/06/2016); ranges of ndvi: 0.2 0.3 = cropland-grassland-savanna; 0.301 0.4 = savanna shrubland-mangrove; 0.401 0.5 = dry forest-monsoon forest; >0.5 = primary forest-broad leaves evergreen forest. this ndvi classes follow siswoyo (2014). this apparent shift in the habitat preference of bali starling is likely to have been influenced by several interacting factors, primarily, the lack of fire in the bbnp. changes in fire regime has resulted in the presence of more shaded habitat as very little grasslands/savannas have become available. secondly, the increased human population and associated infrastructure and land use (dwellings, roads, croplands) in the areas adjacent to bbnp, that led to a decreasing available habitat for the bali starling. the cekikgilimanuk area has experienced a major increase in human-dominated land uses, mainly through conversion of savanna. accounts of the local inhabitants indicate that the conversion of monsoon forest to agricultural land had a negative impact on bali starlings (van balen et al. 2000). lastly, the shift in habitat preference is perhaps due to changes in plant species composition and vegetation structure (which is also related to the lack of fire) which has affected the bali starling utilization of plants for food and nesting. some 22 plant species belonging to 14 families were recorded in the two savannas. at cekik 10 species belonged to eight families, whereas at brumbun 20 species were from 12 families. significant differences were observed using the bray-curtis species similarity index biotropia vol. 28 no. 2, 2021 124 between the savanna sites (ranosim = 0.228; p<0.003) (fig. 3). eight species were present in both savannas (cekik and brumbun sites) namely chromolaena odorata, lantana camara, desmodium laxiflorum, grewia eriocarpa, bridelia stipularis, cynodon dactylon, calamagrostis australis, and ziziphus mauritiana (synonym z. jujuba). using the shannon-wiener species diversity index, no significant difference (p>0.05) was observed between brumbun and cekik, however species richness was significantly different (p<0.05) between the two savannas (fig. 3). brumbun had more species than cekik. plant species composition was categorized based on their different uses by the bali starling, namely: food, shelter (nesting) or a combination of both (table 2). as plant-food source, cekik has generally higher cover. six species used as a food source for bali starling were ziziphus mauritiana, grewia eriocarpa, schleichera oleosa, streblus asper, azadirachta indica, (tree species) and lantana camara (herbaceous). two species, borassus flabellifer (arecaceae) and acacia leucophloea (synonym vachellia leucophloea) (fabaceae), were both used for shelter. the latter (a. leucophloea) was also utilized as a food source (combined). some of the plant species used, s. oleosa and b. flabellifer, were present only in cekik whereas s. asper, a. indica and a. leucophloea were found only in brumbun. some plant species were present in both of the locations such as z. mauritiana, g. eriocarpa (tree species) and the invasive exotic climber l. camara (table 2). transform: square root resemblance: s17 bray curtis similarity locations brumbun 2010 cekik 1984 3d stress: 0,06 figure 3 non metric multi dimensional scaling (nmds) ordination based on the bray-curtis similarity index on plant species abundance and composition between brumbun and cekik savanna areas at bali barat national park table 2 plant species used by bali starling at the sampling sites (brumbun and cekik) in bali barat national park species family habitus usage found at and ivi ziziphus mauritiana rhamnaceae tree food cekik (32.5) and brumbun (32.5) grewia eriocarpa malvaceae tree food cekik (16.25) and brumbun (16) schleicera oleosa fabaceae tree food cekik (16.25) borassus flabellifer arecaceae tree nest cekik (61.25) streblus asper moraceae tree food brumbun (16) azadirachta indica meliaceae tree food brumbun (16) acacia leucophloea fabaceae tree nest, food brumbun (16) lantana camara asteraceae herb food cekik (26.14) and brumbun (9.8) the bali starling was not observed in cekik since the mid 1990s, although this area has plant species (schleicera oleosa and borassus flabellifer) that are known to provide shelter and food for the bird (widodo 2014). unlike similar areas, i.e., brumbun with its acacia leucophloea, that have bali starling (leucopsar rotschildi) natural habitat structure in bali barat – sutomo and van etten 125 been successfully re-colonized by bali starling in recent times. this is probably related to several factors like plant species diversity and richness, increases in human habitation that might have led to the decrease in plant species richnessdiversity as well as the increasing risk of poaching. plant species in brumbun is richer compare to cekik. different species of tree dominate in cekik and brumbun however, the composition of the groundcover between the two locations was relatively similar where exotic invasive species such as chromolaena odorata and lantana camara dominate the understory. however, the bali starling habitat in the late 1990s were open woodlands which were dominated by a. leucophloea trees with an undergrowth of l. camara and c. odorata shrubs, and imperata cylindrica grass, and intersected by moister and more densely forested valleys with dominant trees of grewia eriocarpa, vitex pubescens, b. flabellifer and schoutenia ovata (van balen et al. 2000). this vegetation type might, however, be sub-optimal habitat for the bali starling and the bird might have been driven there by poaching pressure (van balen et al. 2000). in west java, a relatively low bird species diversity on the southern bandung (urbanized areas) was attributable to humans (fardila & sjarmidi 2012). land use and other aspects of the environment were interrelated to such an extent with bird communities distribution in north bandung, west java (fardila & sjarmidi 2012). in other studies, bird species richness was significantly higher in natural than in urban habitats in spain (palomino & carrascal 2006). one factor in the decline of the bali starling have been the conversion of savanna and forests to non-native tree plantations, crop land and villages (collins & smith 1994). this was clearly observed in the cekik area. the absence of the bali starling in cekik might also be due to land use changes (fragmented landscape) and the increasing human presence in the area. cekik is located near gilimanuk, a busy port of bali that connects the island with java island. whereas brumbun, is located on the prapat agung peninsula, a more remote area of the national park located near the ranger’s outpost in the northern tip of the national park. in fragmented landscapes, species persistence depends on their ability to use different habitats, so that less suitable habitats may still favour the connectivity of the most suitable habitats in the landscape (calviño-cancela et al. 2012). remaining fragments of native habitats such as forests are often surrounded by a matrix of modified seminatural habitats, such as tree plantations and croplands, which can still provide habitat for species associated with natural forests (lindenmayer & hobbs 2004). in spain, fragmented land, eucalypt plantations has lower species diversity than native forest, but eucalypt plantations provide habitat for species typical of shrublands when young, but do not contribute significantly to the maintenance of the understory biodiversity associated with native forests (calviño-cancela et al. 2012). both locations, cekik and brumbun, provide suitable habitats for bali starlings, although cekik is decreasing into a savanna-forest size, it is more open and more human-populated. most of the bird’s former habitat has been converted into coconut and kapok plantations, and human settlements. cekik ability to provide habitat for the species, the relatively similar species richness with brumbun, and the distribution of specific plant taxa that influence the availability of shelter, feeding and breeding resources (ziziphus mauritiana, grewia eriocarpa, schleicera oleosa, and borassus flabellifer), distinctly contribute to their importance for bali starlings. poaching is the major threat to bali starlings. in the 1960s, the birds were trapped intensively to provide the demands of indonesian, american and european private aviculturists. in 1966, the iucn listed the species as endangered (collins & smith 1994). the indonesian government responded with the 1971 law prohibiting the hunting, capture and export of the bird (van balen et al. 2000). however, poaching has continued in spite of efforts to increase patrols by the park rangers (collins & smith 1994; dirgayusa et al. 2000). in the bali barat national park, specifically in cekik, the local rangers often encountered illegal poachers checking the handcrafted “pigeon holes” made by the bali barat management to facilitate the survival of post-captivity bred starlings that were recently released in the area. this study further confirmed the importance of examining habitat characteristics and dynamics of endemic birds within landscapes that are influenced by multiple factors interacting in space and time (fardila & sjarmidi biotropia vol. 28 no. 2, 2021 126 2012; orians & wittenberger 1991). habitat structure and floristic composition, such as percent canopy cover, tree species diversity and the distribution of specific plant taxa, are known to exhibit a significant role in defining bird species’ occurrences in space (james & wamer 1982; rice et al. 1984; wiens & rotenberry 1981). conclusion in summary, this study suggests that both forest and savanna are important land cover types and habitat for bali starling at bali barat national park considering that the changes in the relative proportions of these types, as well as availability of ecotones between them, are likely to be particularly important for this species. the increase in woody plant cover in the remaining savannas of northern bbnp, which may reflect a lack of burning in the area where the bali starling is known to currently occur, is therefore of primary concern. in cekik, it is suggested to intensify protection efforts and, where feasible, to restore the native species of bali starlings to its best or much improved conservation stautus. acknowledgments the authors would like to thank edith cowan university and rufford foundation for funding this research. special gratitude for wiryawan who had provided permission to conduct this research at bali barat national park and his tremendous assistance during the research. special thanks are also extended to i ketut sandi and i.b.k. arinasa from bali botanical garden for plant sample identification. the authors agreed that each author contributes equally to the paper. references backer ca, van den brink rcb. 1963. flora of java. leiden (nl): the rijksherbarium. calviño-cancela m, rubido-bara m, van etten ejb. 2012. do eucalypt plantations 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composition and important value index of birds in relation to study of bali starling (leucopsar rothschildi) in the bali barat national park zoo indonesia 23:21-34. wiens ja, rotenberry jt. 1981. habitat associations and community structure of birds in shrubsteppe environments. ecol monogr 51:21-41. williams tm, feistner atc. 2006. reproduction of bali starlings leucopsar rothschildi at durrell wildlife conservation trust, jersey. int zoo yearb 40:27189. 5. erina (callus).cdr biotropia vol. 19 no. 2, 2012: 103 114 callus induction and filaments regeneration from callus of cottonii seaweed ( (doty) collected from natuna islands, riau islands province kappaphycus alvarezii erina sulistiani , dinar tri soelistyowati , alimuddin , samsul ahmad yani recipient of biotrop research grant 2010/accepted 28 juni 2012 the objective of this study was to obtain the optimal medium for callus induction from thallus explants of doty and to regenerate filamentous callus from induced callus. before cultured cottonii seaweeds collected from the natuna islands (riau islands province) were acclimatized in greenhouse and in semi-sterile culture in the laboratory. sterilized explants were cultured on pes and conwy media solidified with 0.8% bacto agar. in each of these media two combinations of plant growth regulators i.e. ba+iaa and ba+naa were added. the concentrations of ba used were 0, 0.5, 1 mg/l, the concentrations of iaa were 0, 2.5, 5 mg/l, whereas the concentration of naa were 0, 0.5, 1 mg/l. the result indicated that the optimal medium for callus induction was pes solidified medium supplemented with ba 1 mg/l. types of callus formed were (a) white compact callus, (b) white filamentous callus, (c) greenish/brownish callus. regeneration of callus into clumps of filament had been done by subculturing the callus into pes solidified medium supplemented with ba 1 mg/l + iaa 2.5 mg/l. tissue culture, callus induction, filamentous callus, seaweed, 1* 2 2 1 1) 2) tissue culture-services laboratory-seameo biotrop, bogor, indonesia faculty of fisheries and marine science, bogor agricultural university, bogor, indonesia kappaphycus alvarezii ( ) , kappaphycus alvarezii abstract introduction key words: cottonii seaweed or is one type of carrageenan-producing seaweed massively cultivated in indonesia. the ministry of marine affairs and fisheries of republic of indonesia continues to attempt the development of seaweed farming and cottonii seaweed processing industry. to support the efforts to increase the production of this seaweed, continuous availability of quality seeds is required. kappaphycus alvarezii * corresponding author : esulistiani@biotrop.org 103 since cultivated in the 1970s, seeds of cottonii seaweed are obtained from vegetative propagation. repeated clonal propagation caused a decrease in genetic variability resulting in decreased growth rate, carrageenan yield and gel strength. in addition, the decrease in variability also causes increased susceptibility to disease (hurtado & cheney 2003). application of tissue culture techniques in seaweed is expected to help produce a superior clone, thereby increasing the provision of seeds required for cultivation and to produce uniform seedlings in large quantities but in a short time. generally, seaweed tissue culture stages include preparation of axenic explants, callus induction and regeneration of callus into thallus and young plantlets of seaweed. the objective of this study was to obtain the optimal medium for callus induction from thallus explants of collected from natuna islands, riau islands province, indonesia. to regenerate filamentous callus from induced callus. seaweeds were collected twice from the natuna islands, first on april 20 and the second on may 20, 2010. to keep it fresh during the journey to bogor, seaweed was inserted into cardboard boxes in a quite wet condition. every two clumps of seaweeds were placed separately from each other on the boxes, while old newspapers were used as partition.then the cardboard boxes were closed and inserted into a sack tied using raffia. to reduce mortality at the time of semi-sterile culture in the laboratory, new seaweed brought from the sea need to be acclimatized and maintained in a clean and controlled environment in greenhouse before the semi-sterile culture in the laboratory. the purpose of acclimatization was to adapt the seaweed from the sea to the new environment of the aquarium placed in greenhouse. seaweeds were maintained in the aquarium containing seawater as much as 7/8 part of the aquarium sized 30 cm wide x 35 cm high x 90 cm long. using recirculation system, seawater in the aquarium was flown into the container filter consisting layers of fiber glass wool, activated carbon and coral. seawater that had been filtered was reflown using a pump and pvc pipe to the aquarium. the aquaria also were equipped with an aerator, and shaded by 65% shading net. after 1 month, the seaweed with light green color, clean from dirt and epiphyte were acclimatized in semi-sterile culture in the laboratory. seaweed was washed using seawater which was sterilized by autoclave. the dirt was cleaned gently using a soft brush. mucus attached to the seaweed was cleaned by rinsing 2-3 times with sterilized seawater. subsequently, the seaweeds were cut into pieces ± 10 cm long and cultured in 10-liter glass jar, which already contains conwy (cw) liquid medium (liao . 1983). before use , the culture media were sterilized by autoclave at temperature of 121ºc for 1 hour. the experiment was repeated in 3 jars, each jar contained 4 liters of media and 150 g of seaweed. after 3 weeks, the weight of still alive seaweeds was k. alvarezii et al materials and methods seaweed acclimatization. 104 biotropia vol. 19 no. 2, 2012 measured again to obtain the survival rate of seaweed (%) in the semi-sterile culture system. the cultures were placed on culture rack, illuminated with fluorescent lamps at 1500 lux of light intensity with a 12:12 light and dark cycle. the room temperature was set between 22-25ºc. in the first 2 weeks, the media was replaced 2 times a week with fresh media. after 2 weeks, the media was replaced once a week, for another 3 weeks. preparations of axenic explants in this study were mostly obtained according to methodology described by reddy . (2003). the apical parts of thallus were selected from semi-sterile culture to be used as explants for callus induction. explants were taken from the cultures age 5 weeks and 1 week. thalli were cut into pieces with a length of 4-5 cm. subsequently, the explants were soaked in tween solution (5 drops in 200 ml) for 10 minutes while occasionally shaken, and then the explants were rinsed twice with sterilized seawater. explants then were soaked in 0.5% povidone iodine (betadine) (0.5 ml in 100 ml of sterilized seawater) for 3 minutes. after that, the explants were rinsed 3-4 times with sterilized seawater. explants were then dried with sterilized tissue papers and immersed in cw medium containing 3% antibiotics mixture (penicillin g 1 g, streptomycin sulphate 2 g, kanamycin 1 g, nystatin 25 mg, neomycin 200 mg in 100 ml distilled water). the cultures were shaken with a shaker for 60 hours, at room temperature of 22-25ºc and illuminated with fluorescent lamps at 1500 lux of light intensity with a 12:12 light and dark cycle. then the explants were washed with sterilized seawater, and dried with sterilized tissue papers then planted in the cw medium solidified with 0.8% bacto agar. observations were carried out to record the condition of explants and the percentage of contaminated explants. explants that had been observed for 2 weeks and did not contaminate were used as explants for callus induction. explants were cut into pieces with length of 4-5 mm. each explant then was wiped gently with sterile tissue papers to remove moisture and any mucilaginous substances exuded from the cut ends. then explants were planted on treatment media for callus induction. to investigate the optimal basic culture media, explants were cultured on provasoli enriched seawater/pes (provasoli 1968) and conwy/cw (liao . 1983) media solidified with 0.8% bacto agar. in each of these media two combinations of plant growth regulators i.e. ba+iaa and ba+naa were added. the concentrations of ba were 0, 0.5, 1 mg/l, the concentrations of iaa were 0, 2.5, 5 mg/l, whereas the concentrations of naa were 0, 0.5, 1 mg/l. each treatment was done with 10 replications. the cultures were stored in a culture room, with room temperature between 22-25ºc, and 60-70% of relative humidity. the cultures were illuminated with fluorescent lamps at 1500 lux of light intensity with a 12:12 light and dark cycle. preparation of axenic explants callus induction et al et al 105 callus induction and filaments regeneration from callus of cottonii seaweed erina sulistiani– et al. regeneration of filamentous callus seaweed acclimatization observations on callus growth were done by giving a rating (score). quantity of callus growth was determined in 6 values representing 6 categories of callus growth, namely: 0 = no callus induction, the explants were bleached ; 1 = quantity of callus growth was very low; 2 = quantity of callus growth was low; 3 = quantity of callus growth was medium, 4 = quantity of callus growth was high; and 5 = quantity of callus growth was very high. data on callus growth were analyzed with non-parametric statistical test of kruskal-wallis using spss 16.0 software. the dun's multiple comparison test was used to distinguish treatments with significant differences. after 6 weeks in treatment media of callus induction, all explants were subcultured in pes solid medium containing 1 mg/l ba + 2.5 mg/l iaa for 2 months to enhance callus growth. subsequently, all callus outgrowth were excised from the explants and subcultured separately in pes solid medium containing 1 mg/l ba + 2.5 mg/l iaa for another 2 months to regenerate filamentous callus. installation of seaweed maintenance equipment in the greenhouse, was good enough to maintain the life of seaweeds brought from the sea. cottonii seaweed harvested from natuna islands, on 20 of may 2010 had survived up to one month, with 20% of mortality rate. the highest mortality occurred in the first two weeks, where the seaweed suffered stress during travel distance from the sea to the laboratory and the influence of packaging. after 3 weeks maintained in greenhouse, the tip of thallus began to grow. it was characterized by the formation of light green buds at the tip of thallus. installation of seaweed maintenance equipment in the greenhouse is useful to adapt the seaweed to the new environment (acclimatization). it also can be used to store seaweed stock for a long time as source of explants for seaweed tissue culture studies and genetic improvement to produce quality seeds of seaweed. observations after three weeks maintained in semi-sterile culture showed that survival rate of seaweeds that came from greenhouse was 72%. in our first experiment, seaweeds brought directly from the sea were acclimatized in the semisterile culture. as a result, the survival rate of seaweeds was 0-25%. this indicated that the acclimatization of seaweed in greenhouse, succeeded to increase the survival rate of seaweed in acclimatization phase in semi-sterile culture in the laboratory. due to long distance transportation from natuna islands to bogor, which took two days one night and humid condition of packaging, cottonii seaweed produced a lot of mucus. the mucus caused the seaweed thallus to bleach and die.. in addition, the high concentration of mucus in the static culture may also be a factor causing the death of seaweed. results and discussions th k. alvarezii 106 biotropia vol. 19 no. 2, 2012 based on these results, the new seaweeds brought from the sea need to be adapted first and maintained in a cleaner and controlled environment in the greenhouse before semi-sterile culture in the laboratory. acclimatization in a greenhouse with a recirculation system could refresh the seaweed conditions which suffered stress during delivery. in addition, the mucus contained in the seaweed could be reduced by recirculation system of water management. seawater in the aquarium was flown into the filter container to clean the dirt and slime carrying bacteria, then the filtered seawater was reflown to the aquarium. acclimatization stages in laboratory was very important to be optimized, because the sterilization of explants to obtain axenic explants could be more easily obtained from explants that had been acclimatized first in laboratory than seaweed explants collected directly from the sea. in addition, rate of callus induction would be higher when thallus explants were used that had been acclimatized first in the laboratory (reddy . 2003). the number of explants that had been sterilized was 283 explants. observation data 2 weeks after planting indicated that explants which were acclimatized 5 weeks in semi sterile culture produced higher sterile explants (64.5%) than the explants that has been acclimatized 1 week (43.4%). all contaminations were caused by bacteria. however, the explants remained green during the entire section, where for 1 week old explants (44%) the percentage was higher than for 5-week old explants (27.4%). the rest changed color to brown (browning) or white (bleaching) as a sign that explants were dead, because 5-week-old explants had more young thallus with a diameter <4 mm. these thalli were the result of seaweed growth during acclimatization on semi-sterile culture which had smaller diameter than thallus of seaweed which grows in the sea (fig. 1). while for 1 week old explants grown on semi-sterile culture, thalli were originally derived from the sea, and had larger diameter between 4-5 mm. these thalli had a higher resistance to the sterilization process than <4 mm diameter of thallus explants (muñoz . 2006). et al et al preparation of axenic explants 107 figure 1. cottonii seaweed on cw medium has survived and grown up to 5 weeks after planting in semi-sterile culture. callus induction and filaments regeneration from callus of cottonii seaweed erina sulistiani– et al. concentration of pgr time required for callus induction (days) in basic medium : cw pes without pgr 16 15 bap 0 mg/l + iaa 2.5 mg/l 19 14 bap 0 mg/l + iaa 5 mg/l nc 16 bap 0.5 mg/l + iaa 0 mg/l 22 18 bap 0.5 mg/l + iaa 2.5 mg/l 24 19 bap 0.5 mg/l + iaa 5 mg/l 25 nc bap 1 mg/l + iaa 0 mg/l 26 22 bap 1 mg/l + iaa 2.5 mg/l 26 15 bap 1 mg/l + iaa 5 mg/l 25 15 bap 0 mg/l + naa 0.5 mg/l 25 15 bap 0 mg/l + naa 1 mg/l 20 25 bap 0.5 mg/l + naa 0 mg/l 22 18 bap 0.5 mg/l + naa 0.5 mg/l 15 15 bap 0.5 mg/l + naa 1 mg/l 21 21 bap 1 mg/l + naa 0 mg/l 26 22 bap 1 mg/l + naa 0.5 mg/l 19 17 bap 1 mg/l + naa 1 mg/l 16 nc average time (days) 22 18 callus induction the most rapid callus induction occurred at 14 days after planting in the media treatment, while the slowest occurred at 28 days after planting. explants which did not show the growth of callus over 28 days after planting become bleached. the growth of callus usually occurred in medullar region (fig. 2a), but also on cortical regions in some explants i.e. the skin surface of the thallus and the tip of apical thallus (fig. 2b). most explants in pes medium formed callus faster than in cw medium. the average time required for callus induction on pes media was 18 days, while for cw medium it was 22 days (table 1). 108 a b figure 2. the location where the callus began to grow on thallus explants namely: (a) in the medullar region; (b) in the cortical region and apical thallus tip. tabel 1. average time required for callus induction in pes and cw media supplemented with combination of ba + iaa and ba + naa. note: nc = no callus induction and explants bleached biotropia vol. 19 no. 2, 2012 six weeks after planting, pes medium generated an average rate of callus induction higher (35.6%) than in cw medium (31.7%) (table 2). this result was different from the research of suryati and mulyaningrum (2009) which indicated that the cw medium had a better rate of callus induction than pes medium. this difference could be due to different sources of seaweed explants used, because according to george (1993) in addition to the culture medium, there are three factors that could affect growth and morphogenesis in tissue culture, namely genotype of explants source, environment of culture and tissue-dependent factor. pes medium had been widely reported to successfully establish and regenerate callus into plantlets of young seaweeds (reddy 2003; and munoz . 2006). in addition to , pes medium had also succeeded to induce and regenerate callus on other species of seaweed such as sp, sp, sp (huang & fujita 1997), sp (collantes 2004; kumar . 2007) , and (kumar . 2007). in addition to pes medium, research of hayashi (2008) showed that the induction of callus had succeeded either by using the f/2 50 medium (seawater enriched with guillard & ryther 50% solution) and vs 50 medium (seawater enriched with 50% von stosch's solution). hurtado and biter (2007) had done plantlet regeneration of var. adik-adik by using ess (erd schreibers seawater) medium. while hurtado . (2009) and yunque (2010) had established procedures for production of tissue culture seedlings of with media ampep (acadian marine plant extract powder). the highest rate of callus induction was found in pes medium + ba 1 mg/l (70%) (table 2). explants that did not form callus generally bleached on the third week after planting. explants which mostly bleached were thallus explants with diameter less than 3 mm. the addition of iaa or naa produced a reduction on the average rate of callus induction, both on cw and pes media, as shown on most media added with auxins iaa or naa had an average rate of callus induction lower than in media without auxins. according to bradley (1991) and yokoya . (2010), some types of plant hormone naturally occur in seaweed tissue both auxin and cytokinin (endogenous auxin/cytokinin), such as iaa, aba (absisic acid), paa (phenyl acetic acid), ip (isopentenyladenin), and cz (cis-zeatin). the decrease rate of callus induction (%) due to the addition of auxin iaa/naa was probably due to hormones content in thallus explants was quite optimal to form callus, so that the addition of plant growth regulator caused in excessive concentrations resulting in decreased rate of callus induction. negative influence of iaa on callus growth was probably caused by the use of too high concentration , while suryati and mulyaningrum (2009) found that the optimal concentration was 0.4 mg/l. concentrations higher than 0.4 mg/l caused a decrease rate of callus induction. observations on callus growth on each treatment media was done by giving a rating (score). quantity of callus growth was determined in 6 values representing 6 categories of callus growth. examples of callus growth in each category were shown in figure 3. k. alvarezii et al. et al k. alvarezii grateloupi carpopeltis ptilophora gracilaria et al. et al hypnea musciformis turbinaria conoides et al et al. k. alvarezii k. alvarezii et al et al. k. alvarezii et al 109 callus induction and filaments regeneration from callus of cottonii seaweed erina sulistiani– et al. ~ table 2. callus induction rate (%) on pes and cw media supplemented with combination of ba + iaa and ba + naa. 110 concentration of pgr average rate of callus induction (%) in basic medium cw pes without pgr 40.00 20.00 bap 0 mg/l + iaa 2.5 mg/l 20.00 50.00 bap 0 mg/l + iaa 5 mg/l 0.00 40.00 bap 0.5 mg/l + iaa 0 mg/l 40.00 40.00 bap 0.5 mg/l + iaa 2.5 mg/l 30.00 20.00 bap 0.5 mg/l + iaa 5 mg/l 40.00 0.00 bap 1 mg/l + iaa 0 mg/l 30.00 70.00 bap 1 mg/l + iaa 2.5 mg/l 30.00 50.00 bap 1 mg/l + iaa 5 mg/l 30.00 30.00 bap 0 mg/l + naa 0.5 mg/l 20.00 30.00 bap 0 mg/l + naa 1 mg/l 30.00 50.00 bap 0.5 mg/l + naa 0 mg/l 40.00 40.00 bap 0.5 mg/l + naa 0.5 mg/l 10.00 50.00 bap 0.5 mg/l + naa 1 mg/l 40.00 50.00 bap 1 mg/l + naa 0 mg/l 30.00 70.00 bap 1 mg/l + naa 0.5 mg/l 60.00 10.00 bap 1 mg/l + naa 1 mg/l 40.00 0.00 average rate (%) 31.18 36.47 a cb d e f figure 3 . callus represented by 6 values of callus growth, namely: (a) 0 = no callus induction, the explants become bleached; (b) 1 = quantity of callus growth was very low; (c) 2 = quantity of callus growth was low; (d) 3 = quantity of callus growth was medium, (e) 4 = quantity of callus growth was high; and (f) 5 = quantity of callus growth was very high ( scale bar = 3 mm). biotropia vol. 19 no. 2, 2012 the data of callus growth after 6 weeks in treatment media were analyzed with non-parametric statistical test of kruskal-wallis using spss 16.0 software. the results showed that the media types, the addition of plant growth regulator ba, iaa and naa did not significantly affect quantity of callus growth of cottonii seaweed. similarly, the interaction between these factors had no significant effect on quantity of callus growth (p < 0.05). the data on pes mediated callus growth were re-tested for statistical analysis of krukall wallis for two factors, namely the concentration factor of ba and iaa, and concentration factor of ba and naa. statistical test of data on callus growth in pes medium with ba and iaa treatment indicated that both of these factors and their interactions had no significant effect on callus growth (p < 0.05). meanwhile, in pes medium with ba and naa treatment, the results of statistical tests showed the two factors also had no significant effect, but the interaction of both factors significantly affected callus growth (p < 0.05). the highest mean rank of callus growth was pes medium + ba 1 mg/l without the addition of naa. based on the results of multiple range test of dunn, the treatment was significantly different from other treatments (p < 0.05) (table 3). the addition of plant growth regulator was often used to increase the rate of callus induction (%) and callus growth in seaweeds (dawes & koch 1991; huang & fujita 1997; yokoya & handro 2004; yokoya . 2004; munoz 2006; hayashi . 2008). plant growth regulators of auxin and cytokinin were used either individually or in combination. in this study, the addition of plant growth regulator ba + naa and bap + iaa in the medium did not significantly result in increased rate of callus induction and growth of seaweed . similar results of this study were also reported by reddy . (2003). et al et al et al k. alvarezii et al table 3. effect of naa and ba concentration on the growth of callus 6 weeks after planting 111 naa concentration (mg/l) ba concentration (mg/l) 0 0.5 1 0 37.85 abc 51.00 abc 58.50 a 0.5 42.50 abc 55.45 abc 32.60 bc 1 51.35 abc 51.25 abc 29.00 c a b c figure 4 . types of callus formed after 6 weeks in callus induction media : (a) white compact callus; (b) white filamentous callus; (c) greenish/brownish callus (scale bar = 4 mm). note: numbers followed by same letter are not significantly different based on dunn's multiple range test (p < 0.05). callus induction and filaments regeneration from callus of cottonii seaweed erina sulistiani– et al. ~ the role of plant growth regulators (pgrs) on callus induction in multi-cellular algae such as seaweed is still debatable and showed no definite trend (bradley 1991; evan & trewasvas 1991; baweja . 2009; yokoya 2010). the inconsistency in responses to pgrs by seaweeds could be mostly due to lack of understanding of the physiological role of these substances in their growth and differentiation (reddy 2008). based on the observations after 6 weeks on induction medium, there were several types of callus, namely: (a) white compact callus, (b) white filamentous callus, (c) greenish/brownish callus (fig. 4). filaments are rows of cells which form long chains. generally in seaweed, the long chain of cells are not branched (unbranched filaments) except of oscillatoriaceae species (lobban & harrison 1994). after 6 weeks in the treatment media for callus induction, all of formed callus were sub-cultured into pes medium + ba 1 mg/l + iaa 2.5 mg/l in order to enhance callus growth. after 2 months in the media, the appearance of callus changed. the et al et al. et al. regeneration of filamentous callus figure 5. changes of white compact callus to greenish/brownish callus after 2 months subcultured in pes medium added with ba 1 mg/l + iaa 2.5 mg/l (scale bar = 4 mm). f f figure 6 . growth of filaments clumps (f) on greenish/brownish callus 8 weeks after subcultured in pes medium +ba 1 mg/l + iaa 2.5 mg/ (scale bar = 2 mm). biotropia vol. 19 no. 2, 2012 112 white compact callus turned into greenish/ brownish friable callus with filaments that grow on it (fig. 5). the greenish/brownish callus was isolated from thallus explants then subcultured in pes medium + ba 1 mg/l + iaa 2.5 mg/l. after 2 months, greenish/brownish filaments in large quantities grown on the callus. growth of these filaments aside from growing on the surface of the callus also grow at the bottom of the callus penetrating the culture medium (fig. 6). pes medium supplemented with ba and iaa had successfully regenerated callus into more clumps of filaments. filament formation is the initial phase of young seaweed formation (shao 2004). addition of ba and iaa also had been reported to regenerate callus into young seaweed as in the study of huang and fujita (1997) and hayashi . (2008). the procedures of callus induction and filaments regeneration from callus of cottonii seaweed ( derived from natuna islands (riau islands province) were as follows: 1. due to long distance transportation, seaweed brought from the sea has to be acclimatized in greenhouse for 4 weeks, then 1 week in laboratory before cultured in medium of callus induction 2. the most effective medium for callus induction of seaweed was pes medium supplemented with ba 1 mg/l. 3. regeneration of callus into clumps of filaments could be done in pes medium supplemented with ba 1 mg/l and iaa 2.5 mg/l. this research was funded by seameo biotrop awarded to erina sulistiani, tissue culture laboratory, services laboratory, seameo biotrop, bogor indonesia. et al. et al kappaphycus alvarezii) k. alvarezii conclusions acknowledgments references baweja pd, sahoo p, garcia-jimenez, robaina rr. 2009. seaweed tissue culture as applied to biotechnology: problems, achievements and prospects. phycol res 57: 45-58. 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effects of plant growth regulators and culture medium on morphogenesis of (rhodophyta) cultured . j appl phycol 14: 97-102. yokoya ns, west ja, luchi ae. 2004. effect of plant growth regulator on callus formation, growth and regeneration in axenic tissue culture of and (gracilariales, rhodophyta). phycol res 52: 244-54. yokoya j phycol. 46: 1198-205. yunque d, tibubos k, hurtado aq, critchley at. 2010. optimization of culture conditions for tissue culture production of young plantlets of carrageenophyte . j appl phycol. doi 10.1007/s10811010-9594-7. nd kappaphycus alvarezii eucheuma denticulatum kappaphycus alvarezii ascophyllum nodosum kappaphycus varieties kappaphycus alvarezii in in vitro kappaphycus alvarezii filament grateloupia turuturu kappaphycus alvarezii in vitro solieria filiformis in vitro gracilaria tenuistipitata gracilaria perplexa kappaphycus ns, stirk wa, van staden j, nova´k, o, turecˇkova´, v, peˇncˇı´k a., strnad m. 2010. endogenous cytokinins, auxins, and abscisic acid in red algae from brazil. biotropia vol. 19 no. 2, 2012 114 the effect of the light gradient inside a soybean leaf on the curvature factor of the light response curve and the estimation the light gradients inside soybean leaves and their effect on the curvature factor of the light response curves of photosynthesis biotropia no. 25, 2005 : 29 – 49 tania june biotrop icsea, seameo biotrop, btic building, jl. raya tajur km. 6 bogor, indonesia; email: taniajune@biotrop.org; and laboratory of agrometeorology, bogor agricultural university, bogor, indonesia abstract light gradients within leaves are not included in the model of farquhar, although a steep light gradient does exist within leaves. for a bifacial leaf, the model shows good agreement with measured data, but for an isobilateral leaf the model may underestimate photosynthesis measured by conventional gas exchange. isobilateral leaves easily developed when plants were grown in growth chambers where some light were reflected from the growth chamber metal base onto the lower surface of the leaves during growth, resulting in adjustment of the photosynthetic capacity inside the leaves. this could also happen in the field when canopy is very sparse and lower surface of leaves was exposed to reflected light from soil surface. complications occurred when fitting the light response curves of the electron transport rate, due to the interaction between the quantum yield of electron transport (a2) and the curvature factor (θ). it is suspected that there may be an interaction with the light gradient within the leaf. this manuscript discusses the effect of a light gradient inside a soybean leaf on the estimation of θ. it is shown in the manuscript how the light curves of the isobilateral leaves (at different degree) responded when measured using conventional gas exchange and how it affected the estimation of θ and the electron transport capacity, jmax. an experiment was conducted to prove the hypothesis that this “out of ordinary” estimate of θ (and hence jmax) was due to the unmatched distribution of photosynthetic capacity with distribution of absorbed light. keywords : light gradient / photosynthetic capacity (jmax) / curvature factor (θ) / gas exchange introduction anatomically, there are two types of leaves, bifacial and isobilateral. bifacial leaves have different adaxial and abaxial surfaces (i.e. top and bottom), while in isobilateral leaves these differences do not exist (kirschbaum 1986). these differences are due to the way the leaves receive irradiance. bifacial leaves, which are usually planophile (perpendicular to the stem), receive most light from the adaxial surface while isobilateral leaves receive light from both sides. the distribution of rubisco and electron transport components within these two types of leaves are different. besides these biochemical differences, lloyd et al. (1992) and syvertsen et al. (1995) found that the mesophyll of leaves exposed to different light intensities also changes, and so it might be that these two types of leaves differ anatomically as well. the optimal distribution of the photosynthetic machinery (rubisco activity and electron transport) would follow the gradient of irradiance in a theoretical bifacial 29 mailto:taniajune@biotrop.org leaf, receiving irradiance from one direction, as shown mathematically by farquhar (1989), while in isobilateral leaves a bimodal distribution would be expected as light comes from both sides of the leaves (kirschbaum 1986). any functional isobilaterality will affect the interpretation of the gas exchange data as gas exchange measurement normally was done with irradiance given to the upper side of the leaf (adaxial surface) only. biotropia no. 25, 2005 light gradients in the leaves were not included in the model of farquhar et al. (l980), although a steep light gradient does exist within leaves (terashima & saeki l983; vogelmann & björn l984). for a bifacial leaf, even without the consideration of the light gradient within the leaves, the model shows good agreement with measured data (harley et al. l985; june 2002), presumably for the reason noted above. however, for isobilateral leaves the model may not give a good representation of the relationship between capacities (e.g., rubisco and electron transport) and actual photosynthesis rate as function of irradiance, in a conventional gas exchange system. materials and methods plant materials indeterminate soybean (glycine max [l.] merr.) were grown in 12 liter plastic pots containing sand and vermiculate mixture (1:1, v/v). plants were exposed to a controlled environment in a growth chamber where relative humidity was kept constant at 60/70 % day/night and three temperatures: 20/15, 25/20 and 32/27 day/night oc with [co2] of 350 μmol mol-1. models of leaf photosynthesis leaf photosynthesis can be described by the equations developed by farquhar et al. (l980) and farquhar & von caemmerer (1982). the rate of photosynthesis is controlled by rubisco (rubp carboxylase-oxygenase), the rate of regeneration of rubp, and the relative partial pressures of co2 (ci) and o2 at the site of co2 fixation. under a given set of environmental conditions, the net co2 assimilation rate, a, is taken as being either the rubisco-limited rate, av, or the predicted rubpregeneration limited rate of photosynthesis, aj, whichever is the lower at a particular ci. (this holds for ci > γ*, the co2 compensation partial pressure in the absence of dark respiration.) a has units of μmol m-2 s-1. a j c c rj i i d= − + ⎛ ⎝ ⎜ ⎞ ⎠ ⎟ − 4 2 γ γ * * (1) 30 the light gradients inside soybean leaves and their effect on the curvature factor – tania june a v c k o k c rv c i c o i d= − +⎛ ⎝⎜ ⎞ ⎠⎟ + ⎛ ⎝ ⎜ ⎜ ⎜ ⎞ ⎠ ⎟ ⎟ ⎟ −max *γ 1 (2) a = min (aj, av) (3) where ci = partial pressure of co2 in the leaf (μbar); γ* = co2 compensation partial pressure in the absence of dark respiration (μbar); rd = dark respiration by the leaf which continues in the light (μmol m-2 s-1); o = ambient partial pressure of oxygen (mbar); kc and ko are michaelis-menten constants for carboxylation and oxygenation by rubisco (μbar and mbar, respectively); vcmax is the maximum rate of rubisco activity in the leaf (μmol m-2 s-1); and j is the actual electron transport rate (μmol m-2 s-1). the temperature dependence of kc and ko follows the arrhenius unction: f ( ) ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ ⎟⎟ ⎠ ⎞ ⎜⎜ ⎝ ⎛ + −= 273 2.2981 2.298 exp25, tr e kk c cc (4) ( ) ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ ⎟⎟ ⎠ ⎞ ⎜⎜ ⎝ ⎛ + −= 273 2.2981 2.298 exp25, tr e kk o oo (5) where r is the universal gas constant, 8.3144 j mol-1 k-1, and t is temperature in oc. ec and eo are the apparent activation energies and the 25 subscript refers to the value at 25oc. the temperature effect on the co2 compensation point of photosynthesis in the absence of mitochondrial respiration follows the equation of von caemmerer et al. (l994): ( ) ( )225036.02588.19.36* −+−+=γ tt (6) the parameters kc and ko indicate the intrinsic kinetic properties of rubisco. they are relatively constant, varying only with temperature for all c3 species (berry & björkman l980; jordan & ogren l984), and hence in this analysis the values presented by badger & collatz (l977) and von caemmerer et al. (l994) were used. the values for kc, ko and γ* (pa) at 25oc are 40.4, 24800, 3.69 and the activation energies (j mol-1) for kc and ko are 59400, 36000, respectively, assuming wall conductance gw = (von caemmerer et al. l994; badger & collatz l977). the rate of electron transport, j, follows the equation by farquhar & wong (l984): ∞ 31 biotropia no. 25, 2005 ( ) j ia j ia j ia j = + − + −2 2 2 24 2 max max maxθ θ (7) where jmax is the maximum light-saturated rate of electron transport of the leaf (μmol m-2 s-1), θ is the curvature factor of the light response curve that varies from 0 (rectangular hyperbola) to 1 (two straight lines quasi blackman), a2 is the quantum yield (in terms of incident par) of electron transport at low light and i is the light intensity (μmol m-2 s-1) incident on the leaf. results and discussions example of light response curves from the measurement of the light response curves, where the incident light ranged from 0 to 1650 μmol m-2 s-1, rd was determined by extrapolation of a linear regression at the lower end of the response curve (at i = 0 150 μmol m-2 s-1). using this interpolated rd along with γ* corrected for each temperature using equation (6), j was calculated from eq. (1) and then jmax, θ and a2 were estimated by fitting the jirradiance curve with equation (7). figure 1 shows example of the light response curves of the electron transport rate for plants grown at [co2] of 350 μmol mol-1 and air temperatures of 32/27oc and 20/15oc (day/night) and measured at the gas exchange at three different temperatures. the electron transport rate was calculated using eq. (1). 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 2 /2 7 o c (3 5 0 ) j, μ m ol m -2 s -1 i o , μm o l m -2 s -1 0 5 0 0 1 0 0 0 1 5 0 0 2 0 0 0 2 0 /1 5 o c (3 5 0 ) j, μm ol m -2 s-1 300 250 200 150 100 50 0 0 500 1000 1500 2000 0 500 1000 1500 2000 lo, μmol m-2 s-1 32/27oc(350) 20/15oc(350) figure 1. light response curves of the electron transport rate measured at three temperatures (δ: 15oc; : 25oc and ∇: 35oc). plants were grown under different conditions as indicated in the graph and measured at [co2] of 700 μmol mol-1. 32 the light gradients inside soybean leaves and their effect on the curvature factor – tania june the negative curvature factor (θ) and its effect on the estimation of jmax when these soybean plants were grown in the growth chambers, about 11 % of the incident light was reflected from the metal base of the chamber onto the lower surface of the leaf, resulting in a changed distribution of light and hence, presumably, of photosynthetic capacity inside the leaf. in other words it became partially isobilateral. when the light response curve of this leaf was measured by gas exchange, with light reaching only the upper surface of the leaf, the upper and lower surfaces of the leaf will have saturated at different “times”, i.e. at different irradiances on the adaxial surface. this means that chloroplasts near the abaxial surface continue to increase in irradiance at levels where the adaxial chloroplasts are light saturated. this continuous response at high irradiance will result in a very low fitted curvature factor, θ, as shown in the following parts. the value of rate of bending, θ, tended to be 1 (leverenz 1987; oya & laisk 1976) as the distribution of light given during the gas exchange measurement was made more nearly proportional to the distribution of the photosynthetic capacity (developed during growth). the soybean data showed that when gas exchange measurement with the light source aimed at the top part of the leaf was done, the value of θ was -1.86 at 15oc, 0.6 at 35oc and then decreased to -0.39 at 40oc (june 2002). i hypothesise that the very low value of θ was due to the proportion of light given to each surface of the leaf during measurement not being similar to what the leaf received during growth. if some levels of light reached the lower leaf surface during gas exchange measurements, then this low value of θ should increase as was suggested by oya & laisk (1976), leverenz (1987) and farquhar (l989). fitted parameters (jmax, θ, a2 ) the fitted parameters as functions of the growth and measurement temperatures are listed in table 1 (a2 and θ fitted freely) and table 2 (a2 constrained) using eq. (7) with j from eq. (1) and gw = ∞, and varying the fitting procedure as described below. the fitted parameters a2, jmax and θ in table 1 resulted from the fitting procedure where all the parameters vary freely. the estimation of θ (curvature factor) was very low, reaching -4.85 for plants grown at 32/27oc when measured at 15oc. a negative value of θ has never been observed before, as the normal values range from 0 to 1 (farquhar & wong 1984). the estimation of jmax becomes unrealistically high if θ is very low. in this case, it reached 407 μmol m-2s-1 at 15oc, which is 85 % higher than the jmax value at 25oc and 17 % higher than jmax at 35oc; this is implausible as jmax is expected to increase with temperature from 15 to 35oc, as with other growth conditions in table 1. 33 table 1. list of model parameters with + s.e, for each measurement at 3-leaf temperatures and a co2 concentration of 700 μmol mol-1. fitting of the light response curve was done using eq. (7) by letting all the parameters fit freely and assuming gw = ∞. biotropia no. 25, 2005 parameters of light response curve, measured at 700 μmol mol-1 [co2] growth condition day t/[co2] leaf temperature (oc) rd a2 jmax θ 20/350 15 0.65 + 0.25 0.25 + 0.01 268 + 101 -1.70 + 1.30 25 1.15 + 0.05 0.24 + 0.01 291 + 28 0.35 + 0.20 35 2.60 + 0.20 0.28 + 0.01 614 + 258 -0.24 + 0.76 20/700 15 0.35 + 0.25 0.27 + 0.07 185 + 22 -0.40 + 0.51 25 0.55 + 0.21 0.25 + 0.01 283 + 50 0.78 + 0.10 35 2.15 + 0.15 0.27 + 0.02 303 + 17 0.90 + 0.02 25/350 15 0.12 + 0.12 0.15 + 0.04 200 + 115 -1.00 + 1.87 25 1.01 + 0.44 0.22 + 0.01 212 + 43 0.70 + 0.05 35 1.74 + 0.06 0.27 + 0.02 284 + 42 0.84 + 0.01 32/350 15 0.00 + 0.00 0.34 + 0.06 162 + 4 -1.20 + 0.20 25 0.80 + 0.20 0.28 + 0.02 277 + 21 0.56 + 0.04 35 2.25 + 0.15 0.31 + 0.02 449 + 0.2 0.31 + 0.05 32/700 15 0.00 + 0.00 0.21 + 0.00 407 + 181 -4.85 + 3.55 25 1.00 + 0.04 0.25 + 0.04 220 + 21 0.00 + 0.49 35 2.15 + 0.07 0.24 + 0.01 347 + 31 0.76 + 0.10 leverenz (l988) discussed the strong correlation between the estimate of a2 and that of θ, which in some cases results in an unrealistically low estimate of a2 and an unusually high estimate of θ. based on table 1, there is no definite trend in a2 with increasing temperature, although there is a tendency for the value to be lower at 15 oc than at 35oc as shown by 60 % of the data. the parameter a2 (quantum yield of electron transport) has a theoretical maximum of 0.5 in red light (due to two photosystems) and 0.375 in white light. in practice, these values are usually lower (for example, by 15%, kirschbaum & farquhar l987). several studies have shown that the quantum yield of co2 assimilation decreases as temperature increases between 15 and 35oc (when measured at ambient [co2]). for example, ehleringer & björkman (1977), with encelia california; ku & edwards (1978) with triticum aestivum; ehleringer & pearcy (1983) with avena sativa; osborne & garrett (1983) with lolium perenne; leverenz & oquist (1987) 34 with pinus sylvestris. these yields are complicated by the increasing proportion of light energy used for photorespiration at higher temperatures. there are few temperature studies that have eliminated the effects of rubp oxygenation by either working at low [o2], high [co2], or by correcting the measurements using light given both from the top and bottom surfaces of the leaves. the light gradients inside soybean leaves and their effect on the curvature factor – tania june however, harley et al. (l985) working with soybean found that a2 increased from 0.16 at 15oc, 0.21 at 20oc, 0.27 at 25oc, 0.26 at 30oc, 0.25 at 35oc and down to 0.22 at 40oc. there may be some difficulties in comparing these values with ours as they used a different equation from eq. 3.7 that had no independent cuvature term. wang et al. (1996), working with scots pine, found that a2 increased from 0.18 at 6oc to 0.30 at 21oc and decreased to 0.23 by 32oc. there are no consistent differences in a2 observed between the growth treatments in this experiment. osborne & garrett (1983) and leverenz & oquist (1987) showed evidence that low temperature stress and frost hardening may change membrane properties and result in lower a2. according to wilkins et al. (l994), the difference in the values of a2 between growth treatments could reflect changes in the structure of the needle tissue, the chlorophyll content and the structure of the thylakoid membrane. with all parameters fitted freely in table 1, there was only a slight tendency for a2 to increase with short-term temperature variation from 0.24 ± 0.06 at 15oc, to 0.25 ± 0.02 at 20oc and 0.27 ± 0.02 at 35oc. the mean value is 0.26 ± 0.04. this value is slightly higher than the mean value (0.23) across all temperatures found by harley et al. (1985) for soybean, and slightly less than that, 0.28, found by kirschbaum & farquhar (1987) in eucalyptus pauciflora, at 25oc, when allowance is made for an absorptance of 0.9. it is identical to the value found by ehleringer & björkman (1977) for quantum yield in white light as a mean of seven species. in 2 % [o2] they obtained a quantum yield of co2 assimilation 0.0733. multiplying by 4ē/co2 and by an assumed absorptance of 0.9 yields a2 = 0.26. other data on quantum yield in white light and low [o2] of co2 assimilation of sets of c3 species summarised by evans (1987) are 0.296 and 0.324 [with 0.328 and 0.388 in two individual studies]. to reduce the effect of “noise” in a2 on the estimates of θ, a second set of fitting procedures was used holding a2 constant at 0.26. the resulting fitted parameters θ and jmax are shown in table 2. with a2 held constant, the effect of temperature on θ becomes more consistent. θ increases with temperature in all cases and so does the estimate of jmax, except for the data coming from plants grown at 32/27oc, 700 μmol mol-1 [co2], where jmax is still very high at 15oc associated with a probably due to the very low fitted value of θ. in most cases the value of θ was still very low, and negative in almost half of the cases. a total of 67 % of the data set shows a θ value lower than the commonly accepted value of 0.7 (farquhar & wong 1984, evans & terashima l988, evans & farquhar 1991). 35 table 2. list of model parameters with + standard error, for each measurement at 3-leaf temperatures and a co2 concentration of 700 μmol mol-1. fitting of the light response curve was done using a2 = 0.26 and assuming gw = ∞. biotropia no. 25, 2005 parameters of the light response curve, measured at 700 μmol mol-1 [co2] growth condition, day t/[co2] leaf temperature (oc) jmax θ 20/350 15 277 + 98 -2.01 + 1.14 25 331 + 11 0.002 + 0.003 35 444 + 115 0.39 + 0.26 20/700 15 190 + 18 -0.56 + 0.77 25 311 + 48 0.70 + 0.10 35 300 + 35 0.92 + 0.03 25/350 15 197 + 67 -5.75 + 3.39 25 264 + 58 0.16 + 0.02 35 274 + 18 0.84 + 0.10 32/350 15 134 + 18 -0.05 + 0.54 25 267 + 35 0.65 + 0.08 35 326 + 14 0.81 + 0.08 32/700 15 490 + 154 -9.10 + 4.20 25 236 + 25 -0.16 + 0.30 35 585 + 14 -0.04 + 0.07 however, apart from my data there is supporting evidence that the best fit of θ can be lower than 0.7, as shown by wang et al. (1996) who found that θ ranges from 0.32 to 0.66 for scots pine grown and measured at different conditions. however, in contrast to the results obtained in this worl, they found that θ decreased with increasing temperature across the whole range examined. smaller values of θ are observed when the light response curves are measured using light given solely from the abaxial rather than the normal adaxial surface (oya & laisk 1976; terashima & saeki 1985; terashima 1986). leverenz (1988) found that θ values increased as the leaf acclimated to the light environment inside an integrating sphere (measurement was done at ambient co2 concentration). the values of jmax were obtained from fitting the light response measurements at temperatures of 15, 25 and 35oc and for plants with different growth conditions using eqs. (1), (2), (7) and (8). 36 the light gradients inside soybean leaves and their effect on the curvature factor – tania june what is the meaning of a negative value of θ? what does the fitted curve look like with a negative θ? to answer these questions, potential electron transport rate, j, was calculated for values of θ ranging from 1 to -9, with i2 ranging from 0 to 4000 μmol m-2 s-1 for each θ, as shown in figure 2. it can be seen that for curves with a negative value of θ, the electron transport rate, j, does not saturate (i.e. level off) even at i2 = 4000 μmol m-2 s-1. figure 2. the effect of changing the curvature factor (θ) on the response curve of electron transport at (a) low and (b) high jmax. curves were generated using eq. (7). the value of i2 where j starts to saturate will determine the fitted value of θ, as shown in figure 3. as saturation starts earlier (at lower i2), θ will become higher. for example, when saturation started at i2 = 200 μmol m-2 s-1, the estimated θ was 0.999, while when saturation started at i2 = 1000 μmol m-2 s-1, θ was 0.77. it can be argued that the light response curves which were measured here at i up to 1650 μmol m-2 s-1 in soybean leaves did not reach saturation, in particular at 15oc, and hence the estimation of θ was too low. as an example, when the j value from the θ = 0 curve at i2 = 4000 μmol m-2 s-1 in fig. 2 (b) was increased by only 4.6 % and then refitted using eq. (7) to find θ again, the value of θ dropped from 0 to -0.36 (see top two curves in fig. 3). one interpretation of the above results is that as light becomes more available (higher i), more light will be distributed to the bottom part of the leaf where photosynthetic capacity is probably increasing. this may be due to the leaf receiving 0 1000 2000 3000 4000 0 20 40 60 80 100 -9 -4 -0.9 0 0.7 1 el ec tro n tra ns po rt ra te , μ m ol m -2 s-1 0 1000 2000 3000 4000 1 0 100 200 300 400 (b) -9 -4 j max = 400 -0.9 0 0.7 (a) j max = 100 absorbed light, mol m-2 s-1μ 37 some light from the lower metal base of the growth chamber and adjusting its photosynthetic capacity accordingly. this will increase j and, therefore, decrease the value of θ. when the value of θ becomes too small (negative), significant overestimation of jmax using eq .(7) will occur. biotropia no. 25, 2005 0 1000 2000 3000 4000 5000 0 100 200 300 400 θ -0.36 0.999 0.77 0.41 0.00 el ec tro n tra ns po rt ra te , µ m ol m m -2 s-1 absorbed light, μmol m-2 s-1 figure 3. the effect of the starting point of saturation of j on the estimated value of θ. data wer heoretical simulation in the following part i will explain the mechanisms of the negative θ by means f si e generated initially with θ = 0 as in figure 2 (b). arrows indicate point of saturation. t o mulation. in the simulation, the leaf is divided into 10 layers. division of layers is based on equal amounts of chlorophyll in each layer. the contribution of each layer to the leaf co2 assimilation rate is determined by the amount of light absorbed and the photosynthetic capacity. the photosynthetic capacity is given by jmax. according to kirschbaum (l986), who applied a modified kubelka-munk theory, the pattern of space irradiance within the leaf can be approximated by an exponential curve: ( )iccii o 21i exp −= (8) where i is the space irradiance at any layer , o is irradiance incident on the top of i ii the leaf, c1 (>1) and c2 are parameters specific for each leaf (dimensionless) and i is the layer number where i is 0 < i < d, in units of 1/d x total absorptance (d is total number of layers in leaf). note that the space irradiance in the top layer, i1, is 38 generally greater than io because of internal reflections. the space irradiance in the top layer of the leaf (i1) and in the bottom layer of the leaf (id), can be calculated (kirschbaum l986): the light gradients inside soybean leaves and their effect on the curvature factor – tania june ( ) ( ) ( )u u oo o r r rrr i i − + −+−= 1 1 11 (9) ( ) ( )l l o d r r t i i − + = 1 1 (10) here ro is reflection at the air-to-leaf interface, ru and rl are internal reflectivities at layer, i, will be aio exp(-ic2) / σ =1 w the upper and lower leaf-to-air interfaces, and r and t are total reflectance and transmissivity. values for ru, ro and rl (0.37, 0.037 and 0.37 respectively) are given by jenkins & white (l957), for a refractive index of 1.48 from calculations based on the theory of geometrical optics. using equations (9) and (10) for incident irradiance of 1200 μmol m-2 s-1, r = 0.0741 and t = 0.0143 gives i1 = 1252 μmol m-2 s-1and i10 = 37.32 μmol m-2 s-1 (june, 2002). these values are then inserted into eq. (8) taking 10 layers (d = 10) to give c1 = 1.2806 and c2 = 0.372. equation (11) says that the light absorbed by any d i 2 2 exp (-ic ) where a is absorptance of the leaf given by 1-r-t. light effectively absorbed by photosystem ii at each layer, i (i), would then be given by multiplying the above by 0.5(1-f), and the electron transport rate for each layer would become ( ) j i j i j i j i i i i = + − + −2i 2i 2 2i4 2 max( ) max ( ) max( ) θ θ . (11) in the case of light incident only on one side of the leaf (upper surface), if ( ) ( ) j j i i c i c i ki i ai ai max( ) max( ) exp exp∑ ∑ ∑ = = − =2 2 1 (12) where all summations are for i =1 to 10, then 1 (13) and ther j ∝ jmax(i) ∝ iai (farquhar 1989). in this case, the shape (θ) of the total − j k j= ∑i imax( ) max( ) efore i j would be the same as the shape for an individual layer, ji. this is true in the ideal case for bifacial leaves as shown in figure 4. 39 0 500 1000 1500 2000 2500 0 20 40 60 80 5 4 3 2 1 j i 0 500 1000 1500 2000 2500 0 4 8 12 10 9 8 7 6 0 500 1000 1500 2000 2500 0 50 100 150 200 250 layer total layer layer j max a 2 theta 1 79.59 0.109 0.7 2 54.87 0.075 0.7 3 37.82 0.052 0.7 4 26.07 0.036 0.7 5 17.97 0.025 0.7 6 12.39 0.017 0.7 7 8.54 0.012 0.7 8 5.89 0.008 0.7 9 4.01 0.006 0.7 10 2.80 0.004 0.7 total 249.99 0.342 0.7 j i i o biotropia no. 25, 2005 figure 4. simulated ji light response curves of a bifacial leaf. data were generated with jmax = 250 μmol m-2 s-1, a2 = 0.3 and θ = 0.7. it shows that the shape (θ) of the curve for each layer is the same as the shape of the total. the fitting result, represented by the solid line (eq. (11)), is shown in the inset table. for a completely isobilateral leaf with, over a period of time such as a day, light reaching the leaf from both sides equally, the time-average of light in each layer would be as shown in figure 5. 40 0 2 4 6 8 10 0 2 4 6 8 10 layer 0 2 4 6 8 10 0 200 400 600 800 1000 1200 =+ i i the light gradients inside soybean leaves and their effect on the curvature factor – tania june figure 5. theoretical light gradient inside an isobilateral leaf (right plot), which is the average of the light gradient from the upper surface (left plot) and the light gradient from the lower surface (middle plot). the average light in these leaves determines the distribution of jmax(i), where ( ) ( ) ( )( ) ( )( ) j j c i c i c i c i i i max( ) max( ) . exp exp exp exp∑ ∑ ∑ = − − + − − − − ⎛ ⎝ ⎜⎜ ⎞ ⎠ ⎟⎟0 5 11 11 2 2 2 2 ( ) ( )( ) ( ) = − + − − − ⎛ ⎝ ⎜⎜ ⎞ ⎠ ⎟⎟∑ 0 5 112 2 2 . exp exp exp c i c i c i . (14) in the case of soybean leaves, where they are not completely isobilateral (50 % light absorbed from the upper surface and 50 % from the lower surface), eq. (14) should be modified as follows: ( ) ( )( ) ( ) j j a c i b c i c i i i max( ) max( ) exp exp exp∑ ∑ = − + − − − ⎛ ⎝ ⎜⎜ ⎞ ⎠ ⎟⎟ 2 2 2 11 (15) where a is the proportion of light reaching the upper surface and b (=1-a) is the proportion of light reaching the lower surface of the leaves. the light absorbed effectively by the photosystem ii at each layer would again be 0.5 (1-f) times the total light absorbed in the layer and ji would still follow eq. (11). 41 for a leaf which has already adjusted its photosynthetic capacities (jmax) to its light growing condition (as given by the values of a and b in e biotropia no. 25, 2005 q. (15)), the light respo by the values of a and b but whic able 3. fitted parameters of simulated data generated with jmax= 50 and 250 μmol m-2s-1, a2 = 0.3 and θ = 0.7, then these parameters were fitted freely using eq. (11). χ2 shows the goodness of the max 2 nse curve of each layer will have a different shape compared to the total when light comes from only one side. the curvature factor, θ, decreases as the proportion of jmax distributed to the lower part of the leaf, b, increases. this happens because the [b exp(-c2(11-i))] term in eq. (11) gets bigger and results in increasing that part of the curve at high i0, and hence θ becomes smaller. table 3 shows the simulation results for a theoretical leaf which has a distribution of its photosynthetic capacity as shown h is given light reaching only the upper surface. as the proportion of light on the top surface of the leaf during growth (a) deviates more from the measurement condition (a = 1), the curvature factor becomes smaller. this is consistent with the hypothesis that a low value of θ indicates a mis-match of light distribution during measurement with that experienced during growth. t fitting. jmax a b j a θ χ2 1 0 5 0. 0. 0.884 0.0 34 70 50 0.9 0.1 50.0 0.35 0.64 0.001 250 0.8 0.2 49.9 0.37 0.47 0.022 0.7 0.3 49.6 0.42 0.15 0.081 0.6 0.4 49.3 0.50 -0.50 0.150 0.5 0.5 49.2 0.68 -2.09 0.187 1 0 250.0 0.34 0.70 0.788 0.9 0.1 248.4 0.35 0.66 0.027 0.8 0.2 241.6 0.35 0.59 0.176 0.7 0.3 231.8 0.36 0.51 0.432 0.6 0.4 220.8 0.37 0.39 0.778 0.5 0.5 209.3 0.38 0.20 1.184 the light resp rv the ph nthet stem has b pot sed to e in the form of a quasi-blackman response, where the curvature factor should appr onse cu e of otosy ic sy een hy hesi b oach 1.0 (leverenz 1987; leverenz l988; oya & laisk l976; terashima & saeki l985). therefore, i further simulated the j vs light response curve using a curvature factor equal to 1.0, and the result is shown table 4. 42 the light gradients inside soybean leaves and their effect on the curvature factor – tania june table 4. change in the parameters of the light response curve with changing distribution of photosynthetic capacity (as shown by the a value) at jmax total = 50 μmol m-2 s-1 and 250 μmol m-2 s-1. data were generated with a2 = 0.3 and θ = 1.0 and fitted using eq. (11). χ2 shows the goodness of the fitting jmax a b jmax a2 θ χ2 50 1 0 50.0 0.34 1 0.015 0.9 0.1 50.1 0.36 0.98 0.034 0.8 0.2 50.2 0.39 0.90 0.392 0.7 0.3 50.4 0.46 0.70 1.446 0.6 0.4 51.1 0.65 -0.003 1.958 0.5 0.5 52.7 1.74 -4.89 1.421 250 1 0 250.0 0.34 1 0.9 0.1 245.9 0.35 0.99 5.857 0.8 0.2 236.3 0.36 0.98 17.009 0.7 0.3 225.4 0.38 0.94 20.620 0.6 0.4 214.8 0.40 0.88 24.577 0.5 0.5 204.0 0.42 0.75 26.370 table 4 shows that at low jmax the biggest change in the light parameters as the proportion of light coming from the upper and the lower surfaces changes are in θ and a2, not in jmax. jmax increases by 5.4 % (at low jmax) when light coming from the lower surface increases from 0 to 50 %. when jmax is high, both θ and jmax change as the light from the lower surface increases. jmax decreases by 18 % (at high jmax) when light from the lower surface increases by 50 %. a most interesting feature is that the reduction in the apparent value of θ is greatest at low jmax, and this may explain why θ was so low at 15oc in data shown in table 1. methodology to validate the hypothesis in order to test the theory discussed above, another set of soybean plants were grown in a growth chamber at 25oc and [co2] of 350 μmolmol-1. the leaves grew with either a black or a reflective surface underneath them during their growing period. the black surface only allowed up to 1.6 % of the incident light (which is reflected from the metal base of the growth chamber) to reach the lower surface of the leaf while the reflective one allowed up to 48 %. some plants were left untreated as control plants. the control plants received 10.9 % of the incident light on the lower surface of its leaves. these three treatments enabled different light gradients inside the leaf to be compared. leaves from each treatment were then measured by gas exchange, with light reaching only the upper surface and with light reaching both the upper and lower surfaces of the leaf. 43 light response curves of the co2 assimilation rate with total light intensity from around 50 μmol m-2s-1 to around 1500 μmol m-2s-1 were measured with the following conditions: biotropia no. 25, 2005 1. at 25oc, the light response curve for one leaf from each of the three conditions (black, control and reflective surface) was measured with 100 % of the light incident on the upper surface. three replications were done on a single leaf for each growth condition. the data obtained from these measurements were then fitted using eq. (11) to obtain estimations of jmax and a2 for each growth treatment. 2. the same leaves measured as in point 1 were then measured again by giving an optimum percentage of light to the lower surface at 25oc (two replications were done for each growth condition). this optimum percentage was obtained by increasing the proportion of light given to the lower surface of the leaf stepwise until a maximum co2 assimilation rate and a further decrease with increasing proportion of light were observed. the percentage of light given where this maximum assimilation rate occurred was then used to measure the light response curve. the light intensity used to obtain this optimum percentage was i2 = jmax, where the maximum bending of the j vs light response curve occurred. jmax was estimated using eq. (11) with the data set from the earlier measurements in point 1 above. as i2 = i0 a2 , the light intensity used was i0 = jmax/a2. 3. light response curves at 15 and 35oc were then measured using the optimum proportion of light given to each side of the leaves as obtained in point 2 (at 25oc). the results show that the distribution of incident light needed for maximum photosynthesis follows closely the condition the plant experienced in the growth chamber. the leaf grown with the black surface underneath, which received the least light at the lower leaf surface in the growth chamber, needed only 7.6 + 3.0 % of the total light on the lower surface during the gas exchange measurement to reach its maximum value of co2 assimilation rate. the leaf grown with the reflective surface underneath, which had the highest proportion of light received at the lower leaf surface in the growth chamber, needed 40.0 + 10.0 % of the light intensity reaching the lower leaf surface during the measurement to reach its maximum value and the control leaves needs 23.0 + 2.1 %. directing an optimal percentage of the incident light to the lower surface of the leaf during a gas exchange measurement not only increases photosynthesis compared to when light is given to the upper leaf surface only, but it also changes the curvature factor (θ) of the light response curve (table 5, figure 6). when light was given only to the upper surface of the leaf, θ of the reflective leaf was lower than θ of the black leaf with the control leaf value in the middle. when measurements were conducted with light given optimally to both sides of the leaf, θ increased for all leaves and the value did not differ significantly between growth treatments. the magnitude of this change in θ, from measurement with upper light 44 only to measurement with upper and lower light, becomes larger as the differences in light distribution between growth and measurement conditions become smaller. hence the change in θ with incident light distribution is greater for the reflective leaf than for the black leaf. the light gradients inside soybean leaves and their effect on the curvature factor – tania june table 5. the effect of giving an optimum percentage of light to the lower surface of the leaf during gas exchange measurements on the j vs light response curve parameters (using ci) (± s.e). measurements were conducted at 25 oc and 700 μmol mol-1 [co2], with 2 replicates for each measurement. parameters θ a2 black leaf upper light only lower and upper light 0.86 ± 0.06 0.93 ± 0.01 0.3 ± 0.08 0.3 ± 0.04 control leaf upper light only lower and upper light 0.73 ± 0.04 0.90 ± 0.06 0.3 ± 0.05 0.3 ± 0.03 reflective leaf upper light only lower and upper light 0.56 ± 0.07 0.93 ± 0.04 0.3 ± 0.04 0.3 ± 0.03 figure 6 shows one example of the j vs light response curves from gas exchange measurements with light reaching the upper side only and with light given to both surfaces of the leaf, for each leaf treatment (note the change in the curvature factor). 45 figure 6. light response curve of the electron transport rate (j) of (a) black leaf, (b) control leaf and (c) reflective leaf measured with light reaching the upper surface only (triangle, fitted by segmented line) and light reaching both upper and lower surfaces (circle, fitted by solid line). measurements were done at 25oc and co2 concentration of 700 μmol mol-1. parameters of the response curves are (a) θ = 0.84, 0.94; a2 = 0.3, 0.3; jmax = 215, 207, (b) θ = 0.73, 0.95; a2 = 0.3, 0.3; jmax = 276, 223, (c) θ = 0.49, 0.96; a2 = 0.3, 0.25; jmax = 259, 210, segmented and solid line, respectively. the optimum percentage of light directed to the lower surface was 10.0 %, 20.4 % and 50.0 % for black, control and reflective leaf respectively. 0 400 800 1200 1600 0 50 100 150 200 250 c b 0 50 100 150 200 250 a j, µm ol m -2 s-1 io, µmol m-2 s-1 io , µ m ol m -2 s-1 biotropia no. 25, 2005 conclusions as found and discussed earlier, θ is partly an artefact of the distribution of light intensity in relation to the distribution of photosynthetic capacity, where θ will reach 1 if the two distributions match. during gas exchange measurements, if the proportion of light incident on the upper and lower surfaces of the leaf is similar to the light condition experienced by the leaf during its growing period, then the curvature factor (θ) of the light response curve moves closer to 1, confirming the work by leverenz (1988), although the 46 value of 0.7 has been commonly used in fitting the light response curve as first suggested by farquhar & wong (1984) and evans & terashima (1987) and used by farquhar & evans (1991). the light gradients inside soybean leaves and their effect on the curvature factor – tania june as the proportion of light given to both sides of the leaf deviates further from the leaf’s growing condition, the apparent whole leaf curvature factor becomes lower. in the case of a bifacial leaf measured with light only given to the upper surface, the upper and lower parts of the leaf would reach saturation at different light intensities causing the light response curve to bend more slowly and resulting in a lower value of the curvature factor. this finding has to be taken into consideration when modelling the effect of light on canopy photosynthesis. for example, when using the sun-shade model (de pury & farquhar l997), the curvature factors for the sun and the shade parts of the canopy would change during the time course of the day. estimation of jmax depends on the curvature factor value, and hence it is important to have a degree of certainty in this value. this experiment and associated modelling reveal the effect of a differential distribution of photosynthetic capacity throughout a leaf from the distribution of irradiance. the same concepts apply at the canopy level, which the sun-shade model addresses to an extent. references badger, m.r. and g.j. collazt. 1977. studies on the kinetic mechanism of ribulose-1,5 bisphosphate carboxylase reactions, with particular reference to the effect of temperature on kinetic parameters. carnegie institute of washington yearbook, 76: 355-361. berry, j. and o. bjorkman. 1980. photosynthetic response and adaption to temperature in higher plants. annual review of plant physiology, 31: 491-543. de pury, d.g.g and g.d, farquhar 1997. simple scaling of photosybthesis from leaves to canopies without the errors of big-leaf models. plant cell and environment, 20: 537-557. ehleringer, j., and o. bjorkman. 1977. quantum yields for co2 uptake in c3 and c4 plants:dependence on temperature, co2 and o2 concentration. plant physiology, 59: 86-90. ehleringer, j., and . w. pearcy. 1983. variation in quantum yield for co2 uptake among c3 and c4 plants. plant physiology, 73: 555-559. evans, j.r. 1987. the dependence of quantum yield on wavelength and growth irradiance. australian journal of plant physiology, 14: 69-79. evans, j.r. and g.d. farquhar. 1991. modelind canopy photosynthesis from the biochemistry of the c3 chloroplast. in “modeling crop photosynthesis-from biochemistry to canopy”. 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(crop science society of america inc.: madison). evans j. r and i. terashima. 1987. effects of nitrogen nutrition on electron transport components and photosynthesis in spinach. australian journal of plant physiology, 14: 59-68. evans j. r and i. terashima. 1988. photosynthetic characteristics of spinach leaves grown with different nitrogen treatments. plants cell physiology, 29: 157-165. 47 farquhar, g.d. 1989. models of integrated photosynthesis in cell and leaves. philosophical transaction of the royal society of london series b, 323: 357-368. biotropia no. 25, 2005 farquhar, g.d., von caemmerer, s and j.a. berry. 1980. a biochemical model of photosynthetic co2 assimilation in leaves of c3 species. planta, 149: 78-90. farquhar, g.d. and s.c. wong. 1984. an empirical model of stomatal conductance. australian journal of plant physiology, 11:.191-210. farquhar, g. d. and s. von caemmerer. l982. modelling of photosynthetic responses to environmental conditions. physiological plant 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australian national university, canberra, pp. 185. kirschbaum, m.u.f. and g.d. farquhar 1987. investigation of the co2 dependence of quantum yield and respiration in eucalyptus pauciflora. plant physiology, 83: 1032-1036. ku, s.b. and g.e. edwards. 1978. oxygen inhibition of photosynthesis. iii. temperature dependence of quantum solubility ratio. planta, 140: 1-6. leverenz, j.w.1987. chlorophyll content and the light response curve of shade-adapted conifer needles. physiologia plantarum, 71: 20-29. leverenz, j.w.1988. the effects of illumination sequence, co2 concentration, temperature and acclimation on the convexity of the photosynthetic light response curve. physiologia plantarum, 74: 332-341. leverenz, j.w. and g. oquist. 1987. quantum yields of photosynthesis at temperatures between -2°c and 35°c in a cold-tolerant c3 plant (pinus sylvestris) during the course of year. plant, cell and environment, 10: 287-295. lloyd, j., syvertsen, j.p., kriedemann, p.e. and g.d. farquhar. 1992. low conductances for co2 diffusion from stomata to the sites of carboxylation in leaves of woody spesies. plant, cell and environment, 15: 873-899. osborne, b.a. and m.k. garrett. 1983. quantum yields for co2 uptake in some diploid and tetraploid plant species. plant, cell and environment, 6: 135-144. oya, v.m. and a.k.h. laisk. 1976. adaption of the photosynthesis apparatus to the light profile in the leaf. soviet plants physiology, 23: 381-386. 48 syvertsen, j.p., lloyd, j., mccnchie, c., kriedemann, p.e. and g.d. farquhar. 1995. on the relationship between leaf anatomy and co2 diffusion through the mesophyll of hypostomatous leaves. plant, cell and environment, 18: 149-157. the light gradients inside soybean leaves and their effect on the curvature factor – tania june terashima, i. 1986. dorsiventrality in photosynthetic light response curves of a leaf. journal of experimental botany, 37: 399-405. terashima, i. and t. saeki. 1983. light environment with leaf l. optical properties of paradermal sections of camellia leaves with special reference to differences in the optical properties of palisade and spongy tissues. plant cell physiology, 24: 1493-1501. terashima, i. and t. saeki. 1985. a new model for leaf photosynthesis incorporating the gradiens of light environment and of photosynthetic properties of chloroplasts within a leaf. ann. botany, 56: 489499. von caemmerer, s. and j.r. evans. 1994. the kinetics of ribulose1,5-bisphosphate carboxylase/oxygenase in vivo inferred from measurements of photosynthesis in leaves of transgenic tobacco. planta, 195: 88-97. von caemmerer, s., evans, j. r., hudson, g.s. and t.j. andrews. 1994. the kinetics of rubisco inferred from measurements of photosynthesis in leaves of transgenic tobacco with reduced rubisco content. planta, 195: 33-47. vogelmann, t.c. and l.o. bjorn. 1984. measurement of light gradients and spectral regime in plan tissue with a fiber optic probe. physiol. plant, 60: 361-368. wang, k.y., kellomaki, s. and k. laitinen. 1996. acclimation of photosynthetic parameter in scots pine after three years exposure to elevated temperature and co2. agricultural and forest meteorology, 82: 195-217. wilkins, d., van oosten, j. j. and r.t. besford. 1994. effects of elevated co2 on growth and chloroplast protein in prunus avium. tree physiology 14: 769-779. 49 plant materials models of leaf photosynthesis example of light response curves the negative curvature factor (() and its effect on the estimation of jmax fitted parameters (jmax, (, a2 ) growth parameters microsoft word 1 biotropia no. 19, 2002 : 1 25 fungal population, aflatoxin and free fatty acid contents of peanuts packed in different bag types sonia s.p. bulaong natural sciences research institute, university of the philippines, diliman, quezon city, philippines okky s. dharmaputra seameobiotrop, p.o. box 116, bogor, indonesia and faculty of mathematics and natural sciences, bogor agricultural university, bogor, indonesia abstract shelled peanuts of gajah var. with initial moisture content of 7% were stored at 11 kg/bag in four bag types namely: jute bag, polypropylene bag, jute bag doubled with thin polyethylene (pe), and jute bag doubled with thick pe. storage was done for six months under warehouse conditions with monitoring of relative humidity and temperature. samples taken at the beginning of storage and every month thereafter were analyzed for moisture content, fungal population, aflatoxin and free fatty acid contents. statistical analyses showed that moisture content, fungal population, and free fatty acid contents were significantly higher in jute and polypropylene bags than in pe-dou,bled jute bags. no significant differences were obtained in aflatoxin contents among bag types but at the end of six months storage, toxin level in jute bag exceeded the 30 ppb limit. polypropylene had second highest toxin level at 23 ppb. the pe-doubled bags had 17 and 19 ppb total aflatoxins for thin and thick films, respectively. the results indicated that the immediate packaging of dried shelled peanuts at safe moisture level in plastic films with water vapor transmission rated of 1 g/m2/24 hr or lower is recommended. this packaging will delay critical increases in moisture content, fungal population, aflatoxin and free fatty acid contents of peanut kernels at ambient storage conditions. keywords: peanuts / bag types / fungal population / aflatoxin content / free fatty acid content. introduction peanuts (arachis hypogaea l.) is a valuable source of protein and fats for human and livestock. in 2000 the production of peanuts in indonesia was 718 000 tons (bps 2001). peanuts are subject to various types of deterioration during storage. one cause of deterioration is fungal growth which makes the material unacceptable to consumers. storage fungi can cause decrease of germination capability, loss in weight, discoloration of kernels, heating and mustiness, chemical and nutritional changes, and mycotoxin contamination (sauer et al. 1992). they can change fat quality of peanuts by hydrolytic enzymes producing free fatty acids and glycerol (pomeranz 1992). altogether, these changes lead to a lower quality or rejection of commodity biotropia no. 19,2002 as foodstuff. three fungal species, namely aspergillusflavus, a. parasiticus, and a. nomius produce aflatoxins as secondary metabolites (pitt and hocking 1997). the toxins are known to be carcinogenic, hepatotoxic and teratogenic in test animals. based on the report of the 23rd session of the joint fao/who food standards programme, held in rome, italy, 28 june 3 july 1999, codex alimentarius commission adopted the maximum level of 15 ppb for total aflatoxins in peanuts intended for further processing. the problem of fungal growth and aflatoxin contamination of foodstuffs remains, especially in developing countries where handling and storage technologies are still being developed. pitt and hocking (1996) reported that aflatoxin exceeding 50 ppb contaminated 45, 22 and 25% of 215, 81 and 94 peanut samples collected from retailers in 1990/1991 in indonesia, philippines and thailand, respectively. in line with this study lubulwa and davis (1994) reported that the total annual cost of aflatoxin contamination in indonesia, philippines and thailand was about $a 158 million. indonesia incurred 84% (= $a 132 million) of this cost, thailand incurred 13% (= $a 21 million) and philippines 3% (= $a 5 million) of the cost. moisture content is the most important factor affecting fungal growth in stored products. peanuts are stable at 70% relative humidity between 7 9% moisture content, at which conditions fungal growth is arrested (icar 1987). when the product absorbs moisture from the environment or when the relative humidity exceeds the equilibrium rh of the kernels, fungal growth occurs. for storage, therefore, the packaging material used should have a water vapor transmission rate (wvtr) low enough to minimize moisture absorption from the environment. in commercial practice, 2 types of bags are commonly used to store peanuts, i.e. jute and polypropylene bags. jute bags easily absorb moisture but allow good airflow. polypropylene is non-absorptive but tends to trap heat inside (kennedy and devereau 1994). the ability of polyethylene (pe) bags, singly or in combination with polypropylene to inhibit fungal growth and aflatoxin production in wet corn for two weeks has been reported (siriacha et al. 1990). peanut kernels packed in pe bags then stored in jute bags were found to remain viable up to 7 months of storage (reddye/fl/. 1992). the objectives of this study were to determine the effects of different bag types on fungal population, aflatoxin and free fatty acid contents of stored peanuts together with change in moisture content. the effects of storage duration on the changes in the parameters mentioned above were also determined. materials and methods methods of harvesting, drying and shelling of peanut the peanut used in this study was the "gajah" variety, a cross between schwarz and spanish lines. the plants were cultivated in the citayam farm of the research institute for food crop biotechnology, bogor, indonesia. it was harvested fungal population, aflatoxin and free fatty acid sonia s.p. bulaong & okky s. dharmaputra 100 days after sowing. the pods were stripped from plants and sundried to moisture content of about 8%. pods were shelled manually to minimize damage to kernels. before storage, shelled peanuts were fumigated with phosphine for 5 days at 2 grams/ton to control insect pests that may exist. prior to bagging, damaged or moldy kernels were hand picked from the batch. storage of peanut shelled peanuts with initial moisture content of about 7% were packed at 11 kg/bag in 0.42 m x 0.47 m bags. the bag types used were (1) jute bag (jb), (2) polypropylene bag (pb), (3) jute bag doubled with thin polyethylene (pe) bag (jb+1), and (4) jute bag doubled with thick pe bag (jb+2). the physical properties of the pe bags are given in table 1. each bag type (treatment) was replicated 3 times (4 bags per replication or stack). each stack was arranged randomly and placed upright on wooden pallets. the bags were stored under warehouse conditions for 6 months. ambient temperature and relative humidity were recorded using a wilh. lambercht thermo-hydgrograph type 252. sampling method samples were taken from each bag at the beginning of storage and subsequently every month thereafter until 6 months storage. initial samples were taken from 3 points (top, middle and bottom) in each bag using a sampling spear. every sampling, the samples were taken from the same points. about 250 grams samples from each bag in a stack were mixed homogeneously to get a 1000 gram primary sample. the primary sample was divided twice using a sample divider to obtain working samples for moisture content, fungal population, aflatoxin and free fatty acid contents. a 500 gram portion was set aside as reserved sample. samples for fungal population were refrigerated until analysis. samples for aflatoxin and free fatty acids tests were frozen until analysis. moisture content was carried out on the day of sampling. samples were analysed in duplicates. biotropia no. 19,2002 moisture content analysis moisture content of kernels was determined using oven method (bsi 1980), 2 replicates per sample. the kernels were ground and dried in an oven at 130°c for 2 hours. the moisture content was calculated using the following formula: determination of fungal population the dilution method followed with pour-plating using dichloran 18% glycerol agar (dg18) (pitt and hocking 1997) was adopted. a 25 gram sample was ground for 2 minutes and mixed with 225 ml sterile distilled water to make an initial dilution of 10"1. this initial suspension was shaken manually for 2 minutes, then settled for 30 seconds before aliquots were taken from the surface. dilutions from 10"2 and higher were shaken for 1 minute before sampling. platings were done from 10"' to 10"4 at the beginning and after 1 month of storage. later samplings were plated up to 10"5 dilution. four replicate plates were used per dilution. plates were incubated at ambient temperature for 6 days. populations of individual species were determined using the following formula: fungal population, aflatoxin and free fatty acid sonia s.p. bulaong & okky s. dharmaputra aflatoxin analysis the thin layer chromatography (tlc) method of blaney et al. (1984) was used for aflatoxin analysis. a 10 gram portion of ground peanut was mixed with 50 ml acetonitrile, 5 ml 4% kcl and 1 ml 5% hcl. the mixture was stirred for 45 minutes over a magnetic stirrer. the mixture was filtered and defatted twice with 25 ml n-hexane in a separator/ funnel. the acetonitrile phase was extracted with two (25 ml) portions of dichloromethane. water was removed by filtering through 2 gm anhydrous sodium sulphate into a round-bottom flask. dichloromethane extract was evaporated to dryness on a rotary evaporator. the residue was dissolved in 0.5 ml chloroform and spotted on tlc plate at 20 uj per spot. a series of standard solutions of aflatoxin bj, b2, gi and g2 were spotted on the same plate for comparison. the plates were developed in a chamber containing chloroform: acetone (9:1). plates were air-dried and viewed under uv 366 nm. aflatoxin concentration was calculated using the following formula (bainton et al. 1980): free fatty acid analysis the titration method of joslyn (1970) for peanut oil was adopted. peanut oil was extracted from a 30 gram sample using a soxhlet apparatus. a 10 gram portion of oil was dissolved in hot 100 ml neutralized ethanol. the solution was titrated using 0.1 n naoh solution with phenolpthalein indicator. free fatty acid was calculated as oleic acid and reported as fatty acid value (fav) or mg kom/g sample. statistical analysis the data were analyzed using a completely randomized factorial design with two factors. the first and second factors were bag type and duration of storage, respectively. biotropia no. 19,2002 results and discussion the effect of bag type and duration of storage on moisture content based on statistical analysis, type of bags, duration of storage and their interaction gave very significant differences on moisture content of peanut kernels (table 2). at 95% confidence level, the moisture contents (m.c.) in both polypropylene bag (pb) and jute bag (jb) were significantly higher than m.c. in jute bag doubled with thin pe (jb+1) and jute bag doubled with thick pe (jb+2) (table 3). the m.c. of jb and pb were not significantly different but significant difference exists between m.c. of jb+1 and jb+2. the m.c. of peanut kernels packed in jb, pb, (jb+1) and (jb+2) were 8.23, 8.26, 7.55 and 7.49%, respectively. the lack of significant difference between m.c. in jb and pb suggests that moisture transfers through both types of packaging were at similar rates. as expected, jb+2 had lower m.c. than jb+1 due to its lower wvtr. the difference between m.c. in plastic-doubled bags was only 0.06%. this slight yet significant difference might be due to the condition of the bags during storage. since sampling was done by spear method, 3 points on one side of each bag were punctured at the start of storage. these openings were covered with a plastic tape which shrank with time and did not completely sealed the holes. at these points, the kernels were exposed to the ambient. under this condition, jb+1 and jb+2 bags increased to 1.04% and 0.87% m.c., respectively during 6 months of storage (table 3). the critical m.c. for peanuts at which a. flavus grows and produces aflatoxin is at 9 -10% corresponding to water activity (aw) of 0.85 (who 1979). based on these results, jb+2 is the better packaging material since it allowed less moisture increase from the initial 7% m.c. of the kernels. bulaong (1998) reported that at 25°c the sorption isotherm of peanut kernels var. gajah approximates the isotherm of other fungal population, aflatoxin and free fatty acid sonia s.p. bulaong & okky s. dharmaputra peanut varieties. at this temperature and erh of 85%, the kernels of peanuts var. gajah have an equilibrium moisture content (emc) of about 12.5%. the m.c. in the different bags changed significantly with duration of storage (table 3). from the start of storage until month 2 the m.c. were relatively constant. significant increases were observed from month 3 until month 6. the slight insignificant decrease in m.c. during months 1 and 2 could be due to the low rh -high temperature in the warehouse during the dry season (appendix 1). the vapor pressure (v.p.) of the kernels might have been higher than the v.p. of the surrounding air inside the warehouse. upon the onset of the wet season in month 3, significant increases in m.c. were observed. the respective means of m.c. for months 0, 1, 2, 3, 4, 5 and 6 were 7.20, 7.17, 7.12, 7.60, 8.42, 8.74 and 8.97%, respectively. biotropia no. 19,2002 significant differences in the m.c. for bag type-duration of storage interactions were observed mostly at month 3. for jb, the first significant increase was observed at month 4 at 9.26%. in jb, the succeeding 2 months still had significant increases at 9.58% and 9.97%. in pb, the first significant increase in m.c. was at month 3 at 7.77%. this was followed with significant increases in months 4, 5 and 6 with 9.15, 9.51 and 9.70% m.c., respectively. for jb+1, there was a significant decrease at month 2 from the initial 7.19% m.c. in the other bag types, the decreases in month 2 were insignificant. in pb and jb which are both porous, the kernels might be equilibrating with the .ambient at such a fast rate that moisture content appeared relatively stable. in the case of jb+1, the film puts a barrier to moisture migration such that after the kernels have lost moisture to low rh environment over a period of time, the regain of moisture proceeds of a slower rate than in the porous bags. the m.c. in jb+1 for months 3 and 4 were not different at 7.71%. significantly different increases occurred in months 5 and 6 with 8.0 and 8.23% respectively. in the case of jb+2, it appeared that this was the only system where m.c. did not decrease at month 1. moisture migration into jb+2 appeared to be slow but consistent. means from month 0 to month 2 were not significantly different. a significant increase occurred at month 3. the increase in month 4 was not significantly different from month 3. moisture increases in months 5 and 6 were significantly different from month 4. 8 fungal population, aflatoxin and free fatty acid sonia s.p. bulaong & okky s. dharmaputra at month 6, the final m.c. in the bags were significantly different with jb > pb > jb+1 > jb+2, they were 9.97, 9.70, 8.23 and 7.98%, respectively (table 3). the results suggested that moisture migration through jb was faster than at pb. this may be due to the difference in pore size and permeability of the 2 bags. pb is made of synthetic material less absorbent than jute fiber. as expected, jb+1 allowed more moisture migration than jb+2 because of its higher wvtr. of all the bag types, jb+2 showed consistent but slow increase in m.c. during storage. moisture increases in jb and pb were drastic at the times rh was high. moisture content in jb+1 during these months of high rh also significantly increased at 8 and 8.23% for months 5 and 6, respectively. at the same period, jb+2 did not have significant moisture increase. with the final moisture content of 9.97%, jute bag was already within the critical moisture limit of 9 10% for peanuts. jb+2 with final 7.98% was still in save moisture level for storage up to six months. the changes in m.c. during storage in the four treatments are illustrated in figure 1. the effect of bag type and duration of storage on fungal population bag types, duration of storage and their interaction produced very significant differences on fungal population of stored peanuts (table 2). at 95% confidence level, the fungal populations during 6 months of storage were in the order of jb > pb > jb+2 > jb+1 (table 4). biotropia no. 19.2002 there was a significant decrease in fungal count at month 1. this coincided with a slight insignificant decrease in moisture content at the same month (table 3). at months 2 and 3, the fungal population increased significantly from month 1. the means between months 2 and 3 were not significantly different although at month 3, the moisture content significantly increased. at month 4 was another significant decrease in the population. this occurred despite the significant increase in the moisture content at 8.42% (table 3). at months 5 and 6 significant increases in fungal counts coincided with ncreases in moisture content. fungal counts were 32 535 and 94 856 cfu/g (db), respectively. the fungi have been described to be sensitive detectors of moisture changes in stored grains. their sensitivity was described to be comparable to sophisticated equipment (sauer et al. 1992). the decrease in fungal count at month 1 may be attributed to the significant decrease of 0.03% moisture content (table 3). with a further (insignificant) decrease of 0.03% moisture content for month 2, there was however a significant increase in the fungal population (table 4). this was caused by the growth of penicillium funiculosum which was the dominant species in all bag types at the beginning of storage (table 5). penicillium funiculosum was common in peanuts both before and after harvest (joffe 1969). the occurrence of p. funiculosum in peanuts before harvest indicated that this fungus is also a field fungus. dharmaputra and putri (1997) found that p. funiculosum was a dominant species in peanuts with different percentages of splitted kernels (0, 25, 50, 75 and 100%) stored for 2 months under laboratory conditions. its population was between 0.7x10 1.2xl03 cfu/g (wb). pitt et al. (1998) reported that p. funiculosum were found in 4% of 256 retail samples examined and 9% of all kernels examined (50 kernels/sample) in bogor and yogyakarta, indonesia.     fungal population, aflatoxin and free fatty acid sonia s.p. bulaong & okky s. dharmaputra it was assumed that the dominance of p. funiculosum was due to a little change in moisture content. according to sauer et al. (1992) some fungal species were observed to become dominant with as little as 0.2% change in moisture content. the significant decrease in the fungal population at month 4 may be attributed to a physiological change in the fungi. although there was a significant increase in moisture content at this time, the population decreased. it has been suggested that vegetative growth and secondary metabolism are two competitive processes in the life cycle of an organism (garraway and evans 1984). the onset of secondary metabolism, such as aflatoxin synthesis, requires additional physiological resources like moisture, enzymes and nutrients. hence, it competes with vegetative growth. this may account for the decrease in fungal count despite moisture increase. secondary metabolism is triggered by accumulation of primary metabolic intermediates. these intermediates could have started in the kernels from the start of fungal colonization during preor post-harvest of peanuts until month 3 of storage. secondary metabolism is viewed by some as a way for microorganisms to remove excess primary metabolites intermediates so that the primary processes, like growth, may continue in times of environmental stress (garraway and evans 1984). aflatoxins were first detected at month 4 when fungal count was lowest and the moisture content had significantly increased to 8.42%. aspergillus flavus constitutes a minor percentage of the total population, its range was from 0.001 -1.45% of population during month 4 to month 6 (table 5). it was possible that secondary metabolism occurred as well in the other fungal species. significant differences were observed in the bag type-duration of storage interactions. for jb, pb and jb+1, fungal counts were significantly different from month 0 to month 6. only at jb+2 during months 2 and 3 the fungal counts were not significantly different (table 4), despite the significantly higher moisture content at month 3 in jb+2 (table 3). it could be explained in the light of competition for moisture during secondary metabolism as discussed in earlier part. the changes in fungal population in the treatments during storage are illustrated in figure 2. for all bag types, the significant increases in fungal count was attributed to 2 fungal species, i.e. penicillium funiculosum and aspergillus penicillioides. in jb, the dominant fungus was p. funiculosum from month 0 to month 4. at month 5 and 6, the dominant species was a. penicillioides (table 5). for pb, the dominant species from month 0 to month 4 was p. funiculosum, while a. penicillioides became dominant in pb from month 4 to month 6. eurotium amstelodami was the second dominant species from month 5 to 6 in jb and pb. in jb+1 p. funiculosum was dominant from month 0 until month 6. eurotium chevalieri was the second dominant species at 1.70 and 17.6% of population for months 5 and 6, respectively. in jb+2, p. funiculosum was dominant from month 0 to month 6. eurotium chevalieri contributed 2.08 and 0.75% to total population at months 5 and 6 respectively. the changes in the fungal population in each bag type are presented in figures 3 and 4. 13   fungal population, aflatoxin and free fatty acid sonia s.p. bulaong & okky s. dharmaputra in the complex system where several fungal species exist together, the dominance of a species may not be solely due to its ability to tolerate existing moisture conditions. several types of interactions exist among fungi. aside from competition for space, moisture and nutrients, there could be inhibitory interactions as well due to release of metabolites of derailing of biochemical path ways (choudhary 1992). the sudden succession between p. funiculosum and a. penicillioides in jb and pb during the latter months of storage may be due to a combination of these interactions. a possible reason for the continued dominance of p. funiculosum in pe-doubled bags may be its ability to compete with other species when both moisture and o2 are less than normal. aspergillus penicillioides was observed in conditions of higher moisture content and o2 in the porous bags. the effect of bag type and duration of storage on aflatoxin content of peanuts two kinds of aflatoxins were detected, i.e. aflatoxin bi and b2. based on the analyses of variance there were no significant differences in total aflatoxin content among bag types and the interaction between bag types and duration of storage (table 2). the means for jb, pb, jb+1 and jb +2 were 13.1, 8.9, 8.7 and 9.2 ppb, respectively (table 6, figure 5). 15 biotropia no.19,2002 aflatoxins were first detected at month 4 at concentration of 2.73 ppb. there was a significant increase to 4.69 ppb at month 5. at month 6, the increase to 22.58 ppb was highly significant (table 6, figure 5). at month 4, jb and pb had higher aflatoxin levels than jb+1 and jb +2. the levels were 4.6, 3.6, 2.7 and 0 ppb, respectively (table 6). at month 5, jb+2 and jb+1 had higher levels than jb and pb. the levels were 8.7, 6.7, 3.3 and 0 ppb, respectively. at month 6, jb had higher levels than pb, jb+2 and jb+1. the values were 31.5, 23.1, 18.9 and 16.8 ppb, respectively. aflatoxins were not detected in the samples until month 3 of storage. it was assumed that aflatoxigenic a. flavus population in the soil of peanut planting field was low. the moisture content of the samples remained relatively stable from month 0 to month 2 at values 7.12 7.19% (table 3). this moisture content corresponds to water activity of 0.65, insufficient for aflatoxin production. other works have reported a similar absence of aflatoxins in peanuts at low moisture content conditions. at 6.9% m.c., aflatoxin in shelled spanish peanuts was not detected after 8 months stored at cold storage (wilson and jay 1976). significant increases in moisture content started from month 3. at month 4, aflatoxins were detected in 3 bag types in small amounts. the moisture contents at month 4 for jb and pb were 9.26 and 0.15%, respectively, within the 9 10% range, of lower limit for aflatoxin production in peanuts (who 1979). jb+1 at month 4 had only 7.7% moisture content yet it was positive for aflatoxin production. jb+2 also had a moisture content less than 8% during storage, and it was positive for aflatoxins (tables 3 and 6). it appears that aflatoxin synthesis could occur at moisture levels lower than normal range for a. flavus growth. 17 biotropia no. 19,2002 aspergillus flavus count did not correlate with aflatoxin production (table 7). one reason is that only a minor portion of the total fungal population is responsible for the toxin production. aflatoxin production depends among others on the strain of a. flavus (kozakiewicz 1996; pitt and hocking 1997). an interesting correlation may exist between free fatty acid contents and aflatoxins. glycerol was reported to be an inducer of bbh b2 + g2 production by a. parasiticus (abdollahi and buchanan 1981). it induced higher levels of aflatoxin than glucose, galactose, fructose and xylose at controlled conditions. sufficient levels of glycerol might have been produced in stored peanuts during hydrolysis of triglycerides into free fatty acids and glycerol. it follows that in samples with high levels of free fatty acids, glycerol would also be high and so would aflatoxin level. it also follows that after some aflatoxin synthesis, glycerol level (and free fatty acid) would be lower. such a correlation may explain the decrease in free fatty acid contents during months 5 and 6 when aflatoxin synthesis increased (tables 6 and 8). the higher increase in free fatty acid contents in jb and pb from month 0 to month 3 compared with jb+1 and jb+2 could also result to the observed differences in aflatoxin levels of the bag types (table 6). the role of seed lipases in free fatty acid production is discussed in the next section. the major role   biotropia no. 19,2002 of lipases in the kernels in lipid hydrolysis may offer explanation why aflatoxin occurred in jb+1 and jb+2 at moisture levels lower than the minimum level reported for a. flavus growth. the means of free fatty acids and final aflatoxins levels in each bag are illustrated in figure 6. fungal population, aflatoxin and free fatty acid sonia s.p. bulaong & okky s. dharmaputra the effect of bag type and duration of storage on free fatty acid contents bag types, duration of storage and their interactions gave very significant differences on free fatty acid (ffa) contents of stored peanuts (table 2). at 95% confidence level, the ffa contents in the 4 bags were significantly different. jb and pb had insignificantly different means at 0.51 and 0.52 fav, respectively. jb+1 and jb+2 had significantly different means at 0.47 and 0.43 fav, respectively (table 8). significant differences in ffa contents were observed during storage. the lowest level was at month 0 at 0.32 fav. a significant increase to 0.45 fav was observed at month 1. this was relatively unchanged until month 2 (table 8). significant increases were again observed in months 3 and 4. these were followed with significant decreases in months 5 and 6. the values for months 0, 1,2, 3, 4, 5 and 6 were 0.32, 0.45, 0.42, 0.54, 0.57, 0.56 and 0.50 fav, respectively. interactions between bag type and duration of storage resulted to significantly different ffa contents. at month 0, the 3 bag types contained kernels with approximately same ffa contents at 0.33 0.34 fav. only pb had a significantly lower level at 0.28 fav (table 8). at month 1, pb had the highest increase in ffa with 0.20 fav increase from month 0. jb had the second highest increase at month 1 with 0.18 fav increase from month 0. jb+1 had 0.12 unit increase while jb+2 had only 0.04 fav increase. the highest increase in ffa contents in jb, pb and jb+1 occurred at month 1. this was followed with a significant decrease at month 2 for the same bag types. jb+2 showed a significant increase from month 1 to month 2. from month 3 to month 4, significant increases were observed in jb and jb+1. in jb+2, the means for months 3 and 4 were not significantly different. in pb, there was a decrease in ffa at month 4. significant decreases in ffa contents were observed in jb from month 4 to months 5 and 6. in jb+1, the decrease was observed from month 5 to month 6. in jb+2, the decrease was from month 4 to month 5 (table 8). free fatty acids are released from the hydrolysis of triglycerides. the rapid formation of ffa during month 1 of storage for jb, pb and jb+1 was due to the activity of lipases in the kernels (acker 1969; pomeranz 1992). these upases are known to be active even at low water activity or moisture content levels. at month 1, moisture content was generally lower than at month 0 and the fungal population was significantly lower than month 0 (tables 3 and 4). this is another proof that the lipase activity must be largely due to seed enzymes rather than fungal enzymes at the low moisture level. at month 2, there were significant decreases in ffa contents in jb, pb and jb+1. only jb+2 has a significant increase to 0.43 mg koh/g sample (table 8). jb+2 was the only bag system where ffa contents were more consistently increasing with duration of storage. the fluctuations in month 2 in the 3 other bag types could be attributed only to the higher permeability to o2 and moisture. during months 3 and 4, increases in ffa in jb, pb and jb+1 were significant, but not in jb+2. at month 5, significant decreases were observed in jb 21 biotropia no. 19,2002 and jb+2. pb at month 5 regained significantly higher ffa contents compared with month 4 when it lost ffa. jb+1 at month 5 still had an increasing ffa content. at month 6, a significant drop was observed in both jb and jb+1. pb had a significant increase at month 6 to 0.80 fav. jb+2 had an insignificant increase at month 6 to 0.40 fav. the possible correlation between free fatty acids and aflatoxin synthesis was discussed in the previous section. essentially, analysis in this study indicates that high free fatty acid contents correspond to high glycerol levels since both are byproducts of lipid hydrolysis. since glycerol induces aflatoxin synthesis, a bag system with high free fatty acid may also produce high levels of aflatoxin. this appears to be true in the case of jb and pb which had the highest initial increases in ffa content at month 1. table 6 shows that at month 6, peanuts packed in jb had the highest aflatoxin content at 31.5 ppb. pb had the second highest content at 23.1 ppb. jb+1 and jb+2 with less increase in ffa at month 1 had 16.8 and 18.9 ppb of aflatoxin, respectively. it appears from the analysis of data that a packaging relatively impermeable to o2 and moisture would allow slower lipid hydrolysis than porous materials. in this respect, it may be concluded that immediate packing of peanuts in protective films may be a way to slow down aflatoxin production in peanuts. conclusions the use of polyethylene lining in jute bags effected lower moisture gains than either jute or polypropylene bag alone in peanut storage. in the order of decreasing moisture gains pb > jb > jb+1 > jb+2. the dominant fungal species found in peanuts during storage were aspergillus penicillioides and penicilliumfuniculosum. bag type and duration of storage caused significant differences in fungal populations both in quantity and quality. in the order of decreasing fungal populations, jb > pb > jb+2 > jb+1. fungal populations generally increased with duration of storage, except at the beginning of secondary metabolism (aflatoxin synthesis). level of free fatty acids changed significantly according to bag type and duration of storage. in the order of decreasing free fatty acid, pb > jb > jb+1 > jb+2. aflatoxin level was not significantly affected by bag type. in the order of decreasing total aflatoxin, jb > jb+2 > pb > jb+1. aflatoxin level changed significantly with duration of storage. in the order of decreasing aflatoxin level, month 6 > month 5 > month 4. aflatoxin level was formed at water activity and moisture content lower than previously reported. aflatoxin level was apparently related to free fatty acid level. 22 fungal population, aflatoxin and free fatty acid sonia s.p. bulaong & okky s. dharmaputra recommendation shelled peanuts with moisture content of 7% should be immediately packed in polyethylene bags with water vapor transmission rate of 1 gm/m2/24 hr or lower in order to preserve its quality. such packaging will delay moisture increase, minimize fungal growth, aflatoxin production and free fatty acid formation. acknowledgements the authors gratefully acknowledge the financial support of the canadian international development agency (cida) through seameo biotrop. we also thank the other scientists and technicians of the laboratory of pest and disease management, and laboratory of chemistry, seameo biotrop who have in one way or another contributed to this research; and dr. gloria l. enriquez, former deputy director for programme and marketing of seameo biotrop for her suggestions and encouragement. references abdollahi, a. and r.l. buchanan. 1981. regulation of aflatoxin biosynthesis. induction of allatoxin production by various carbohydrates. j food sci. 46: 633-635. acker, l.w. 1969. water activity and enzyme activity. food tech. 23: 27-40. bainton, s.j., r.d. coker, b.d. jones, e.m. morley, n.j. nagler and r.l. turner. 1980. mycoioxin training manual. tropical products institute, london. blaney, d.j., c.j. moore and a.l. tyler. 1984. mycotoxins and fungal damage in maize harvested during 1982 in far north queensland. austr. j. agric. res. 35: 463-471. bps. 2001. statistik indonesia 2000. badan pusat statistik, jakarta. in indonesian. bsi. 1980. methods of test for cereals and pulses. part 3. determination of moisture content of cereal.-, and cereal products (routine methods). british standards institution. isbn 0580 11 4333. bulaong, s.s.p. 1998. sorption isotherm of peanut kernels var. gajah. seameo biotroi'. unpublished. choudhary, a.k. 1992. influence of microbial co-inhibitors on aflatoxin synthesis of aspergillus flavim on maize kernels. letters appl. microb. 14: 143-147. dharmaputra, o.s. and a.s.r. putri. 1997. the relation between splitted kernels and population of storage fungi in peanuts. seameo biotrop. unpublished. fardiaz, s. 1989. food microbiology. inter university center for food and nutrition. bogor agricultural university, indonesia, p. 268. garravvay, m.o. and r.c. evans. 1984. fungal nutrition and physiology. john wiley and sons, new york. 23 biotropiano. 19,2002 icar. 1987. aflatoxin in groundnut-technologies for better crops. publications and information division, indian council of agricultural research, new delhi, india. joffe, a.z. 1969. the mycoflora of fresh and stored groundnut kernels in israel. oleagineux 27: 489-491. joslyn, m.a. 1970. acidimetry. in: joslyn, m.a. (ed.). pp. 401-446. methods in food analysis; physical, chemical and instrumental methods of analysis. academic press, new york. kennedy, l. and a. devereau. 1994. observations on large-scale outdoor maize storage in jute and woven polypropylene sacks in zimbabwe. in: highley, e., e.j. wright, h.j. banks and b.r. champ (eds.). pp. 290295. proceedings of the 6th international working conference on stored-product protection. canberra, australia, 1723 april 1994. kozakiewicz, z. 1996. occurrence and significance of storage fungi and associated mycotoxins in rice and cereal grains. in: highley, e. and g.i. johnson (eds.). pp. 18-26. aciar technical reports 37. mycotoxin contamination in grains. paper presented at the 17th asean technical seminar on grain postharvest technology. lumut, malaysia, 25-27 july 1995. lubulwa, g. and j. davis. 1994. estimating the social costs of the impacts of fungi and aflatoxins in maize and peanuts. in: highley, e., e.j. wright, h.j. banks and b.r. champ (eds.). pp. 1017-1042. proceedings of the 6th international working conference on stored-product protection. canberra, australia, 17-23 april 1994. lubulwa, g. and j. davis. 1996. completed-project economic assessment of two aciar projects on fungi and aflatoxins: a discussion of methodology issues and some estimates of potential benefits. in: highley, e. and g.i. johnson (eds.). pp. 66-98. aciar technical reports 37. mycotoxin contamination in grains. paper presented at the 17lh asean technical seminar on grain postharvest technology. lumut, malaysia, 25-27 july 1995. pitt, j.i. and a.d. hocking. 1996. current knowledge of fungi and mycotoxin assessment of food commodities in south east asia. in: highley, e. and g.i. johnson (eds.). pp. 5-10. aciar technical reports 37. mycotoxin contamination in grains. paper presented at the 17"' asean technical seminar on grain postharvest technology. lumut, malaysia, 25-27 july 1995. pitt, j.i. and a.d. hocking. 1997. fungi and food spoilage. blackie academic & professional, london. pitt, j.i., a.d. hocking, b.f. miscamble, o.s. dharmaputra, k.r. kuswanto, e.s. rahayu and sardjono. 1998. the mycoflora of food commodities from indonesia. journal of food mycology 1(1): 41-60. pomeranz, y. 1992. biochemical, functional and nutritional changes during storage. in: sauer, d.b. (ed.). storage of cereal grains and their products. amer. assoc. cereal chemists, inc. st. paul, minnesota, pp. 55-141. reddy, p.s., m.s. basu, m.a. khaleque, m.s. haque, a. ali, h. malek, h. than, t. soe, b. regunathan, b. mishra, t. murthy and s. nigam. 1992. status of groundnut research and production in south asia. in: nigam, s.n. (ed.). groundnuta global perspective. proceedings of an int'l. workshop 25-29 november 1991. icrisat center, india. icrisat. sauer, d.b., r.a. meronuck and c.m. christensen. 1992. microflora. in: sauer, d.b. (ed.). storage of cereal grains and their products. amer. assoc. cereal chemists, inc. st. paul, minnesota, pp. 313-340. siriacha, p., k. kawashima, m. saito, p. tan-boon-ek and d. buangsuwon. 1990. prevention of thai maize from the infection by aspergillus flavus and aflatoxin contamination in various packages. proc. jpn. assoc. mycotoxicology 32: 41-46. wilson, d. and e. jay. 1976. effect of controlled atmosphere storage of aflatoxin production in high moisture peanuts (groundnuts). j. stored prod. res. 12: 97-100. who. 1979. mycotoxins. world health organization, geneva. 24 appendix 1. average temperature and relative humidity in warehouse during storage of peanuts month temperature (°c) relative humidity (%) july august september october november december january 25.6 26.9 27.4 27.2 26.7 27.3 68.5 66.7 70.4 75.5 78.7 76.9 25 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf 10.pdf 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 22.pdf 23.pdf 24.pdf 25.pdf microsoft word 15 biotropia no. 22, 2004: 15-28 soil quality changes following forest clearance in bengkulu, sumatra i.p. handayani department of soil science, agriculture college, university of bengkulu bengkulu, sumatra 38371 a, indonesia abstract intense destruction and degradation of tropical forests is recognized as one of the environmental threats and tragedies. these have increased the need to assess the effects of subsequent land-use following forest extraction on soil quality. therefore, the objective of this study is to evaluate the impacts of land-use type on soil quality properties in bengkulu province, sumatra. soil samples were collected from adjacent sites including natural secondary forest, bare land, cultivated land and grassland. the results show that land-use following forest clearance lowered saturated hydraulic conductivity (85%), porosity (10.50%), soil water content at field capacity (34%),c organic (27%), n total (26%), inorganic n (37%), soil microbial biomass c (32%), mineralizable c (22%), and particulate organic matter (50%), but slightly increased water soluble organic c. specific respiration activity rates increased about 14% in cultivated soils compared to natural forest soils, indicating greater c turnover per labile c pool in the form of soil microbial biomass, thus decreased biologically active soil organic matter. forest conversion tends to reduce the c,ffg/crer for all deforested sites. all of deforested areas relatively have infertile soil, with the worst case found in cultivated field. the c^g/crd of cultivated fields was about 24% less than that of remnant forest (1.07). grassland apparently maintains only slightly higher soil c levels than the bare land. on average, degradation index of soil following forest clearance was 35% with the highest deterioration occurred in the bare land (38%). fallowing the fields by naturally growth of imperata cylindrica for about 15 yr in abandoned land after 3-5 years of cultivation did not improve the soil quality. moreover, forest clearance has an impact on soil quality as resulted in the loss of a physically protected organic matter and reduction in some labile c pools, thus declined biological activity at disturbed ecosystems. keywords: degradation index / forest / imperata cylindrica grassland / soil quality/ soil organic matter introduction the integration among the primary natural resources vegetation, soil and water, are important factors for maintaining terrestrial ecosystem functions and productivity. human poverty and a continuous decline in the amount of agricultural area per family have led to exploitation of natural resources and deforestation on secondary forest in developing countries, such as indonesia. consequently, more forest land are converted to cropland at an alarming rate (riswan and hartanti 1995). these trends have caused a need to determine the effects of forest conversion and deforestation on soil quality. subsequent land-use change following deforestation or cultivation of deforested land may rapidly decline soil quality, as ecologically sensitive indicators of the tropical ecosystem are not able to buffer the impacts of land management practices. as a result, severe degradation in soil quality may lead to a permanent loss of land productivity (handayani 2001;mcdonald et al. 2002). evaluation of soil 15 biotropia no. 22, 2004 properties upon land conversion tor varying agricultural purposes is most crucial to assess early changes in soil quality. therefore, the specific objectives of this research were:(i) to determine and compare the changes in the physical, chemical and biological soil properties of soils in response to different land-use; and (ii) to calculate the degradation index of each tropical disturbed ecosystem. materials and methods study site the study site is located around the rajo lelo forest garden (kawasan taman hutan raya rajo lelo), 15 km north of bengkulu city, sumatra. the area was originally covered with typical tropical forest speces, such as arthocarpus champede, parkia spesiosa, durio zibethinus, pithecellobium latum, cinnamomum porectum, spondias pinnata kurt, rhodomnia cinerea, areca sp. and aporosa aurita. the forest have been cleared through cutting the wood to meet the increasing demands for timber and agricultural land. for more than 25 years, people living in the surrounding forest garden have often encroached upon and cultivated agricultural crops in the deforested land without using any fertilizer and soil conservation practices. these agricultural activities have caused regeneration of existing residual vegetation on clear-land and degraded deforested areas. the climate of the area is tropical monsoon with rainy season occuring from september to march and dry season from april to august. the mean annual temperature is 26uc, while the mean rainfall is 200 mm/month. the soils are classified as very fine, mixed, isohyperthermic, typic palehumult, according to us soil taxonomy. these soils are poorly to moderately drained and occur on undulating to flat uplands. the elevation is below 50 m above sea level. soil sampling surface soil samples at the depth interval of 0 to 15 cm were collected from three sites under each of four adjacent land-use/land cover types: (1) secondary forest ('natural forest'), (2) secondary forest cleared, and soil subsequently maintained weed-free ('bare land '), (3) secondary forest cleared, burned and soil planted with annual vegetable crops for about 6 years ('cultivated land') and (4) secondary forest cleared, burned and soil cultivated for about 5 years then abandoned, thus naturally grown by imperata cylindrica for about 15 years ('grassland') (figure 1.). for each site, 16 soil cores (1.9 cm diameter each) were randomly sampled and mixed to obtain a composite sample that was sealed in a plastic bag. field-moist soil samples were gently sieved through a 2 mm mesh to remove stones, roots, and large organic residues and sealed in plastic bags to store at 4°c. soil biological analyses were carried out within 10 days of sampling after an overnight acclimatization period at room temperature. 16   biotropia no. 22, 2004 soil physical, chemical and biological analyses saturated hydraulic conductivity (ksat) was determined by the constant-head method according to klute and dierksen (1986). soil bulk density (bd) was assessed by the core method and total porosity was calculated assuming a particle density of 2.65 g cm"3. gravimetric water field capacity was measured by pressure plate apparatus method (scott et al. 1994). soil particle size analysis was conducted by the hydrometer method. soil ph was determined in 1:2.5 soil-water slurry, using a combination of glass electrode. total c (tc) and nitrogen (tn) contents were determined on finely ground air-dried soils by wet combustion according to the method of kandeler (1995). soil microbial c (smbc) was determined by the fumigation-incubation method (jenkinson and powlson 1976) with the following modifications. moist soil (30 g dry-weight equivalent) was placed in 50-ml beakers, fumigated, brought to a water potential of approximately -30 j kg"1 with de-ionized water (0.3 kg kg"'), and incubated in 1-l air-tight canning jars in the presence of 10 ml of 0.5 m koh at 26°c for 10 days. the quantity of co2-c absorbed in the alkali was determined by titration (anderson 1982). soil microbial biomass c was determined from the following equation: smbc = [mg c02-c kg¯¹ soil (10 dy)¯¹]funigatjkc where kc = 0.41 (voroney and paul 1984). mineralizable c was estimated from the quantity of coi-c and net nkt-n + no3-n, respectively, released from an unfumigated sample during 10-day incubation at 26°c and a soil water potential of -30 j kg-1 (campbell et al. 1991). specific respiratory activity of soil microbial biomass c was estimated by dividing the net potential microbial activity (i.e., mineralizable c) by the size of the smbc (campbell et al. 1991). particulate organic matter (pom-c) was determined by a modified version of the cambardella and elliot (1992) method. pom-c was isolated by dispersing 30 g of soil in sodium hexametaphosphate and passing the dispersed sample through a 53-(am sieve, which retains the pom fraction + sand and allows the passage of mineral associated soil organic matter. the sand and pom fraction was dried at 50dc, finely ground and subsampled for total organic c. water soluble organic c (wsoc) was determined by the method of mazzarino et al. (1993). soil suspensions (1:2 soil:distilled water) were shaken for 30 minutes at 150 rev/min then centrifuged for 5 minutes at 2500 rev/min and filtered through whatman no 42 filter paper that had been rinsed with distilled water. carbon in the extract was determined following nelson and sommers (1982). current method for inventory of soil organic matter is based on an estimate of the soil c stored under natural vegetation and the relative changes due to aspects of human land use. in this case, calculation of a ratio of the measured soil organic c (corg) and a reference corg value for forest (top) soils of the same texture and ph was 18 soil quality changes following forest clearance i.p. handayani needed (van noordwijk et at. 1997). the ratio of corg/cret could be used as a 'sustainability indicator'. if the value of cqrg/cref ratio is 1, this means the soil is similar to that of forest, and/or is a "fertile soil"; values towards 0 mean "infertile soil". the current equation for cref for upland soils in sumatra (excluding peat and wetlands soils as well as recent volcanic andisols) is : cref =(zs/12.5)¯0'58 exp (1.333+0.00994*clay%+0.00699*silt%-0.156*ph-kcl) where zs=soil sampling depth, cm statistical analysis one-way analyses of variance (anovas) procedures were performed to compare the effects of different land-use/ land management on physical, chemical and biological properties of soil. the lsd procedure was used to separate the means of the soil properties at the 0.05 probability level as significant. results and discussion soils under cultivation had higher bulk densities than other land-use type (table 1.), with an associated decline in porosity and saturated hydraulic conductivity. the bare and cultivated soils were slightly lower in silt and clay than adjacent soils under natural forest, most likely as a result of preferential removal of silt by accelerated erosion during the rainy season (handayani 2001). the lowest saturated hydraulic conductivity was found in bare soils and the highest occurred in natural forests. beginning tillage practices resulted in higher saturated conductivity in cultivated soils compared to bare soils and grassland. lower plants residue/ organic input probably accounts for the higher bulk density and decreased porosity under cultivation as compared to the natural forest, bare land and grassland soils (table 1.). enhanced soil water content at field capacity and saturated hydraulic conductivity in natural forest is consistent with greater input of labile c contributed by the high quality litter-fall and root exudates as indicated by higher wsoc and smbc (table 3.). during the processes of c turnover, extracellular polysaccharides were produced and with association of root extension have created better soil aggregation (elliot 1983; cambardella and elliot 1992), which caused higher soil water content at field capacity and saturated hydraulic conductivity. on the other hand, organic matter in cultivated soils is less physically protected than that of bare and grassland soils because tillage periodically breaks up macroaggregates and exposes previously protected organic matter in soil macroaggregates (gupta and germida 1988), causing more compacted soils (poorer soil aggregation). total nitrogen content of soils under cultivation were lower compared to levels in natural forest soils. however, cultivated soils have higher total n than that in bare land and grassland soils (table 2.), as might be expected in a system dominated by 19 biotropia no. 22, 2004 nitrogen fixing crops such as peanut and bean. the organic c levels were significantly higher in natural forest than those of bare land, 'cultivated land and grassland soils. the c/n ratios were wider under undisturbed ecosystems such as natural forest and grassland, indicating higher c accumulation or slower decomposition resulted in c stabilization inside soil aggregates (handayani et al. 1995; feller and beare 1997). bare land and cultivated soils had lower inorganic n content than that of natural forest and grassland. these proved that less available-n were released through mineralization under disturbed sites; because these ecosystems had lower n stock from labile n pools, such as in soil microbial biomass n and paniculate organic matter n (pom-n) (unpublished results 1999). the lower levels of total c and n in bare land and cultivated soils may have resulted from a combination of lower c inputs because of less biomass c returned and greater c losses because of aggregate disruption, increased by tillage, plant residue burning, and enhanced soil erosion during rainy season (scott et al. 1994; handayani 2000). the trends toward lower total c and n in the disturbed land is probably caused by the breakdown of aggregates (gupta and germida 1988; blair et al. 1995), and greater organic matter oxidation following deforestation (handayani et al. 2001) and continuous tillage (handayani and coyne 1999). 20 biotropia no. 22, 2004 from biomass burning on the bare land and cultivated soils could have returned base-forming cations to increase ph of surface soil. the ratios of corg/cref were almost similar in bare land, cultivated land and grassland (table 2) with value of 0.92, 0.81 and 0.92, except for natural forest is 1.07. the average corg/cret ratio of 1.07 under forest, suggesting that the soil c status of this forest has increased. forest conversion tends to reduce the corg/crct for all deforested sites. as suggested by van noordwijk et al. (1997), if the value of corg/cref ratio is 1, this means the soil is similar to that of forest, and/or is a "fertile soil"; values towards 0 mean "infertile soil". therefore, all of deforested areas relatively have infertile soil, with the worst case found in cultivated field. the c,,rg/cref of cultivated fields was about 24% less than that of remnant forest. grassland apparently maintains only slightly higher soil c levels than the bare land. as may be expected that grassland have higher total n and available n compared to bare land (table 2.). the ratio of corg/cref in the forest for this study was higher compared to the study in sumberjaya, west lampung sumatra. hairiah et al. (2002) reported the value of corg/cref under forest condition was 0.73, suggesting that the soil c content in the forest has declined from the undisturbed condition. in addition this ration in coffee farming systems was about 50% less that of remnant forest. the values of all of the measured biological properties were significantly higher in natural forest than in cultivated soils, except for mineralizable c (table 3.). mineralixable c rates did not vary significantly among sites, but tended to be somewhat higher in the soils under natural forest. high rates of mineralizable c can occur either as a result of large pool of labile c substrates or rapid oxidation of a smaller pool. thus high mineralizable c may indicate a high level of ecosystem productivity and soil bioactivity (mazzarino et al. 1993). a more clearly interpretable parameter is the rate of mineralizable c per unit smbc (specific respiratory activity of soil microbial biomass c = srac), high levels of which have been associated with ecosystem stresses (killham 1985; handayani and coyne 1999). the highest srac was found in cultivated soils (24.03%) and the lowest was in natural forest soils (19.74%) (table 4.). enhanced microbial activities in soils under natural forest are related to greater levels of substrates c, which resulted in the increase of the labile fraction of c organic. as a result, soil microbial communities under cultivated soils are less biologically active and more stressed than in natural forest. in addition, relatively higher rates of srac for the cultivated soils suggest that intense competition for the available c may favor those microorganisms which use more c energy for cell integrity and maintenance than for growth under perturbed or disturbed ecosystems. consequently, cultivated soils favor bacteria-based food webs which have low c assimilation efficiencies and faster turnover rates than the more efficient fungal-based food webs dominant in untilled or natural ecosystems (hendrix et al. 1986). a lower srac implies a more stable and mature system (turco et al. 1994), thus this calculation may be a good indicator of status of a soil and therefore, its soil quality (insam and hasselwandter 1989). the highest decline of smbc was in cultivated soils (42%) and the lowest occurred in bare land soils (22%). the effect was more pronounced on pom-c 22 soil quality changes following forest clearance i.p. handayani (50% reduction). undisturbed ecosystems (natural forest and grassland soils) have the lowest wsoc compared to cultivated and bare land soils. data in table 3 describe the variety amount of labile c pools under different land-use types, but the availability of labile c pools are shown in table 4. microbial biomass c is often limited in size by the availability of c-labile substrates and is sensitive to variations in land-use and soil management practices, so a lower proportion of smbc in cultivated and grassland soils is an indication of degradation of available pool c in soils under cultivated and grassland soils. clearing and burning tend to decrease root production in bare land and cultivated soils. lower root biomass and removal of clipping resulted in lower substrates availability, and thus lower smbc. wsoc appears to be the intermediate organic substrate for soil microorganisms (mcgill et al. 1986). turnover of smbc requires repleshnisment of wsoc supplies. repleshnisment mechanisms include desorption from soil colloids, dissolution from litter, exudation sloughing and exfoliation from plant roots, or hydrolisis of insoluble soil organic polymers (mcgill et al. 1986). changes in soil environment, soil management and cropping practices would be expected to affect the above mechanisms, thereby altering wsoc supply and both amount and activity of smbc.the lower proportion of wsoc in c org under undisturbed ecosystems indicates more soil organic matter was in protected condition, so they are not readily soluble or not readily available to microorganisms. pom-c has been closely associated with the active soil organic matter pool and has been successfully used as an assay of nutrient availability (dalai and mayer 1987). loss of pom-c is an important aspect of soil organic matter degradation. pom-c declined in newly bare soils and cultivated fields and lowest under grassland soils. our study suggests that pom-c characteristics may also serve as early indicators of soil organic matter aggradation. pom-c accumulated as soil organic matter was restored in natural forest and it is important for c and nutrient reservoir in soils (cambardella and elliot 1992). values of ratio pom-c/c org indi 23 biotropia no. 22, 2004 cate similar trend with values of pom-c which show the highest under natural forest soils and lowest in grassland soils. this implies that availability of pom-c continuously declined when the forest is converted to other land-use type (table 4.). this study gives an implication that bare land, cultivated and grassland soils have less physically protected soil organic matter compared with natural forest soils, resulting in soil deterioration over time. the calculation of degradation level determined by scaling technique (scott et al. 1994), reflects the percent changes in soil properties from their standard values under natural forest (table 1.). scaling of soil properties was used to relate the characteristics of one land-use to the same characteristics of another land-use by dividing the mean values of each soil property from the natural forest. therefore, the values from the natural forest would have a scale of 1.0 and other land-use would have a scale value less than 1.0, indicating the decline of soil quality. all scale values were averaged at each land-use, then subtracted by 1.0 and converted to percentage as indicated by degradation level. in this study, soil properties scale was made on saturated hydraulic conductivity, gravimetric soil water content at field capacity, porosity, c organic, n total, inorganic n, mineralizable c, soil microbial biomass c, and particulate organic matter (table 5). the calculated soil degradation index reflects the percent changes in soil properties from their values under forest (table 5). soils under bare land had the highest degradation index up to 38% compared to grassland and cultivated soils (33%) with an average of 35%. the data also indicated that fallowing the land by natural growth grassland of imperata cylindrica for about 15 years have not improved the soil quality compared to cultivated soils. in some cases, grassland ecosystem often created more deterioration in several soil properties (table 5.). these soil degradation indices clearly show that degradation of soil quality occurs when the forest systems are converted for agriculture without the use of appropriate soil conservation practices. 24 biotropia no. 22, 2004 conclusions clearing, burning and cultivation of tropical secondary forest lands resulted in degradation of soil quality as indicated by the changes in physical, chemical and biological properties. land-use following forest clearance decreased saturated hydraulic conductivity by 85%, porosity by 10.50%, soil water content at field capacity by 34%, c organic by 27%, n total by 26%, inorganic n by 37%, soil microbial biomass c by 32%, mineralizable c by 22%, and particulate organic matter by 50%, but slightly increased water soluble organic c. specific respiration activity rates increased about 14% in cultivated soils compared to natural forest soils. forest conversion tends to reduce the corg/cret for all deforested sites. all of deforested areas relatively have infertile soil, with the worst case found in cultivated field. the corg/clef of cultivated fields was about 24% less than that of remnant forest. grassland apparently maintains only slightly higher soil c levels than the bare land. bare land and cultivated soils had higher bulk density, lower saturated hydraulic conductivity, porosity and soil water content at field capacity. based on calculation for the ratio of corg/cref, all of deforested areas relatively have infertile soil, with the worst case found in cultivated field. degradation index of soil following secondary forest clearance was about 35% in average, with the greatest deterioration occurring in bare land. fallowing the abandoned land by natural grass has not improved the soil quality. therefore, improvement of soil properties under imperata grassland with well-adapted and fast growing vegetative species to compete with these grass is needed to gradually improve soil quality as well as regenerate degraded grassland. acknowledgements this work is a part of the research supported by the government of indonesia through the project under the department of research and technology (menris-tek) and the department of national higher education (dikti). the technical assistance of meizar and yulian hardi is gratefully acknowledged. we also thank the local government and local farmers for providing their fields for this study. special thanks are due to dr. meine van noordwijk for his help in reviewing the manuscript. references anderson, j.p.e. 1982. soil respiration, p. 837-871. in a.l.page et al. 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(ed.) defining soil quality for a sustainable environment. sssa special publication no. 35., madison, wi. van noordwijk, m., c.cerri, p.l.boomer, k.nugroho and m.bernoux. 1997. soil carbon dynamics in the humid tropical forest zone. geoderma 79:187-225. voroney, r.p., and e.a.paul. 1984. determination of kc and kn in situ for calibration of the chloroform fumigationincubation method. soil biol. biochem. 16:9-14. 28 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 22.pdf 23.pdf 24.pdf 25.pdf 26.pdf 27.pdf 28.pdf the expansion of (l.) merrill in merremia peltata fragmented forest of bukit barisan selatan national park enhanced by its ecophysiological attributes yansen*, wiryono, deselina, muhammad f. hidayat efratenta k. depari and department of forestry, university of bengkulu, bengkulu, indonesia r 4eceived 28 november 2013/accepted 28 september 201 abstract forest opening and fragmentation may affect the composition of vegetation by permitting the expansion or domination of one or two species in the area. this study found that , an invasive climbing creeping merremia peltata / species, has dominated some area of bukit barisan selatan national park (bbsnp). domination of this species was favoured by forest fragmentation, indicated by the significantly higher number of individuals of in the open m. peltata area than in intact forest. the ecophysiological characters of support the ability of this species to become a m. peltata strong invader. as the expansion of this species may negatively affect the biodiversity and vegetation health, the application of appropriate ecological approaches to control the domination of in the conservation area is m. peltata necessary. keywords: ecophysiology, forest fragmentation , population , merremia peltata size introduction the abundance of species in an area is a fundamental ecological parameter and a critical consideration in decision making for conservation (he gaston 2000). on a geographic scale, one & species might be abundant in one region but rare in other regions (brown 1984). unlike temperate forests, where a single factor may drive abundance and distribution of species (he 1997), many et al. underlying factors control these patterns in tropical rainforest ecosystems. some of those factors are intra and interspecific competition, predation, niche differentiation, disturbances and stochastic recruitment (janzen 1970 connell ; 1978; denslow 1987). disturbances may stimulate the regeneration in the tropical rainforest. the falling of large individual or group of trees is the most common cause of natural gap formation. this will create an open area inside the forest, which has different microclimate, particularly light intensity, from the surrounding shaded areas. this opening will allow more light reaching the forest floor stimulating seed germination and seedling establishment. many rainforest species of seedlings need more light to grow faster. however, large scale disturbances may negatively affect plant communities. ecosystem disturbances are important in changing the dynamic of species competition and may increase the opportunity of certain species to invade the ecosystem. however, the success of alien or local species to dominate one area is influenced by their biological attributes, environmental characteristic of the invaded area and biotic interaction with earlier communities, including interspecific competition (vila weiner 2004).& in the forest area, ecosystem disturbances may negatively affect the composition of vegetation by permitting the expansion or domination of one or two species in the area. biological invasion by invasive species is one of the major environmental problems which could threaten biodiversity, affect forest vegetation health and in the long term it could reduce the ability of forest to absorb carbon (vila weiner 2004 clavero& ; & garcia-berthou 2005; master . 2013). et al* corresponding author : y.yansen@ymail.com biotropia vol. 22 no. 1, 2015: 25 32 doi: 10.11598/btb.2015.22.1.353 25 mailto:y.yansen@ymail.com biotropia vol. 22 no. 1, 2015 26 thus, if invasive species invade a large area of forests, the ability of forest ecosystem to mitigate climate change may also be affected. on the other hand, invasive species may be better adapted to the temperature rise (hellman 2008).et al. merremia peltata (l.) merrill (convolvilaceae) is a climbing creeping plant that originally comes / from the pacific. this species has been reported to become invasive in many areas of the pacific. it grows very quickly in the open area and dominates degraded ecosystems (kirkham 2004). the development of this species has become a major threat for plant and animal biodiversity in national parks. bukit barisan selatan national park (bbsnp) is the third-largest area conservation in sumatera covering about . it is l3,568 km ocated in 2 southwest sumatera (4 31 5 57 s, 103 34 104 43 0 0 0 0'' '' e) 150 km along the barisan , which extends bukit range. the area of bbsnp spans across two provincial administrations, lampung and bengkulu. t contains some of his national park the largest tracts of lowland forest remaining on sumatera (o'bri n . 2004)e .et al the expansion of in relation to forest m. peltata fragmentation has been happening in bukit barisan selatan national park (bbsnp). the development of has dominated about m. peltata 7,000 ha of bbsnp area (master . 2013). this et al species grows very quick y in the open area and l forest edges. the invasion of this species results in significant ecological consequences. m. peltata creeps and covers the area and block the s development of seedling. it also climbs up the host trees or shrubs and may eventually kill them. master . (2013) found that area dominated byet al m. peltata has lower diversity index than area where m. peltata is not dominant. mega faunas, such as elephant, are also found to avoid area where this liana is dominant. in the long run, the expansion of this type of invasive species may contribute to the extinction of other species (clavero garcia & berthou 2005). existence of in an area affects species m. peltata conservation. therefore, ecological aspects of this species need to be studied. to date, there is lack of information on the ecological aspects and ecophysiological characters of this species. available information on this species so far includes the record on the invasive plants of the pacific (josekutty . 2002), its natural et al distribution in certain area in the pacific (kirkham 2004) and its impact on the plant biodiversity in the invaded area (master . 2013). the et al information on the ecological and ecophysiological characters may provide scientific foundation in future management of this species. therefore, this research investigates the population of in open density and size m. peltata and intact forests and its ecophysiological characters which may contribute to this species ability to invade a certain areaforest . materials and methods this study was conducted in pemerihan resort of bbsnp in the lampung province . (fig. 1) pemerihan resort is about 19,009 ha. this area was chosen as has started dominating m. peltata several sites. however, compared with other areas in the bbsnp, such as tampang way haru and resorts, the development of in the m. peltata pemerihan resort has not been as massive as in those two resorts. therefore, this research may provide insight into earlier stage of the invasion of this species. early observation indicated that the proliferation of might be enhanced by m. peltata forest opening; therefore, ecological aspects of this species were studied by comparing its population structure in open/fragmented and intact forests. intact forest is dominated by large trees, while open area is dominated by shrubs and smaller trees. these two areas have distinct microclimates. light intensity in the two areas was observed by using solar radiation meter. fifty (50) 5 x 5 m plots were established in the open area where this species was dominant. other 50 plots were placed in the intact forest next to the open area. the number of was counted and m. peltata the stem diameter was measured at the 0.5 m from the plant base. in case that plant individual has climbed and covered shrub or tree canopy, the canopy projection of the affected shrubs or trees was quantified. of light intensity the differences and the population attributes between the two sites were analysed by employing student's t-test . the xpansion f (l.) merrill n ragmented oreste o i f fmerremia peltata – et al.yansen observation of coverage to its host m. peltata when it climbs was approached by measuring the light intensity under the occupied canopy and above the canopy. the percentage of canopy coverage by to host's canopy was then m. peltata counted by employing formula: percentage of canopy coverage = 100 – (liuc/liac) x 100 where: liuc= light intensity under the canopy liac= light intensity above the canopy observed ecophysiological characters included pecific eaf rea (sla), wood density s l a and xylem anatomy. fifty (50) healthy leaves were taken from 50 individuals. leaf area was then measured by analyzing the leaf images using a computer program called imagej (national health institute usa). those leaves were then oven dried at 70 c for two days and were 0 measured for their dry weight. sla is the ratio of leaf area (cm ) and dry weight (g). wood density 2 was observed for 50 wood fractions from 50 individuals. after the volume of wood pieces was quantified, the wood samples were oven dried at 70 c for two days and were measured for their dry 0 weight. the wood density is the ratio between wood dry weight (g) and wood volume (cm ). 3 fifty wood samples from 50 individuals above were also taken for xylem observation. before they were observed, the wood samples were put in 70% alcohol. in the laboratory, the samples were transversally sectioned and then were observed under microscope. pictures of the samples were taken. diameter of the xylem vessels was measured using imagej software (national health institute usa). vessel diameters of stems were divided into 10 μm size classes. to determine the theoretical contribution of each vessel size class to the hydraulic conductance of the stem, vessel diameters were raised to the fourth power (hagen-poiseuille law) (tyree & zimmerman 2002). the hagen-poiseuille law is based on the experiments by hagen in 1839 and poiseuille in 1840 which found that water molecules are stationary on the capillary wall and move faster in the centre of the capillary. therefore, flow rate is proportional to the fourth power of the radius of the capillary (tyree & zimmerman 2002). the relative contribution of each diameter class is the proportion of the sum of all the conduits raised to the fourth power. this sum reflects the capacity of stem to conduct water, which will consequently affect the ability of plants to grow faster. figure 1. the location of bukit barisan selatan national park in southwest sumatera (inset). the study site was at the pemerihan resort ( ) 27 results and discussion the expansion of has become a real m. peltata problem for species conservation in the bbsnp area. since it established in early 1970s, was m. peltata has dominated approximately 7,000 ha of bbsnp area (master 2013). satellite image et al. showed that in 2002, dominated area of m. peltata about 6,393 ha. this area had become about 7,008 ha in 2008 (tnbbs 2011). area of bbsnp which will be dominated by to be wider m. peltata is likely in the future. in the pemerihan resort, where this study was conducted, the expansion of m. peltata has just been occurring for the last years. this few showed that this species keeps spreading to all areas of bbsnp. light intensity between the two study sites differed significantly ( = 10.571; <0.01). in the t p intact area, the light which went through the canopy and reached forest floor was about 171.26 lux. in the open area, the average light intensity was about 293.30 lux. this difference reflected the gradient of energy available for vegetation in these two different sites. the average number of in both m. peltata study sites differed significantly ( = 4.907; t p <0.01) (fig. 2). in the open area, the average number of per area was 4.46 individuals m. peltata per 25 m . in the intact area it was only 0.20 2 m. peltata m. plants per 25 m . the figure shows that 2 peltata preferred an open and fragmented area. this research indicated that forest opening enhanced the expansion of . as a fast m. peltata growing species quickly dominated the m. peltata landscape and form a dense population. the number of in the open area was 20 times m. peltata as many as that the in shaded area. the area openness provides a full sunlight, in which this condition is favoured by pioneer species (whitmore 1989). the domination of one species can also be indicated by the total basal area. this study found that total basal area of in the open m. peltata area, i.e. 8.84 cm per 25 m , was higher than 2 2 m. peltata plants in the shaded area, i.e. 0.58 per 25 m 2 ( = 4.435; <0.01) (fig. 3). this indicated that t p total accumulation of biomass of was m. peltata much higher in the open area than in the shaded area. disturbances, in this case forest clearing/ fragmentation, change the ecosystem dramatically (hooper 2004) by affecting the et al. availability of crucial resources, such as light, water and nutrients, in the area. consequently, plants may respond differently to this gradient and ultimately this will determine which species will coexist (brokaw busing 2000). the & differences in performance involve establishment, growth and survival. the success of m. peltata to dominate the area depended on their ability to exploit resources and adapt with the available environmental niches. as the development of this species was much better in the open area, the more forest fragments created more chances this species had to invade the forest area. biotropia vol. 22 no. 1, 2015 open forest intact forest figure 2. the number of individuals of per 25 m in the open and intact forest areas. error bar indicates 95% m. peltata 2 confidence intervals 28 open forest intact forest canopy of the host plants occupied by m. peltata had high canopy density. the average percentage of canopy coverage of m. peltata occupied trees/shrubs was 76.19% in the open area and 77.63% in the shaded area (table 1). in comparison, surrounding trees/shrubs which were not occupied by had only about m. peltata 30–50% canopy coverage. this showed that m. peltata had covered the host canopy severely, which subsequently might affect the growth of those trees or shrubs. figure 3. total basal area of per 25 m in the open and intact forest areas. error bar indicates 95% confidence m. peltata 2 intervals table 1. the percentage of canopy density of trees/shrubs occupied by in the two sites of observationm. peltata ind open forest intact forest canopy diameter (m) percentage of canopy coverage canopy diameter (m) percentage of canopy coverage 1 4 86.79 3 69.20 2 3 77.47 7 58.37 3 4 72.48 4 86.40 4 4 80.96 7 86.25 5 3 74.92 4 82.71 6 4 74.13 2 86.75 7 3 68.25 3 79.45 8 4 75.58 5 81.58 9 3 74.25 7 58.80 10 4 77.03 4 86.85 average 76.19 77.63 wood density of was 0.6 g/cm m. peltata 3 (table 2). in comparison, the value of water density as a standard at 4 c f was 1 g/cm . 0 3 hence, the value of wood density of m. peltata was categorized low. the value of sla of m. peltata was 160.33 cm /g (table 2). compared 2 with other climbing species, this value was very high. observation on and s freycinetia excelsa raphidophora australasica showed that their sla values erew 9.74 cm /g and 16.36 cm /g, 2 2 respectively (yansen 2012). 29 the xpansion f (l.) merrill n ragmented oreste o i f fmerremia peltata – et al.yansen sla is commonly found to have a significant negative correlation with leaf thickness (vile . et al 2005). leaves with high sla (i.e. low leaf mass area/lma), are usually thinner and have low tissue density and low leaf construction cost (osunkoya . 2010). on the other hand, tet al hicker leaves (i.e. lower sla/higher lma) may correlate to longevity of leaf life span (chabot hicks & 1982; putz . 1995 mediavilla . 2001), high et al et al; construction cost (westoby . 2002) and more et al drought tolerance (salleo lo gullo 1990& ; mediavilla . 2001). the leaf turn-over of et al m. peltata is fast; hence it can grow very quickly in a short time. the average diameter of the xylem m. peltata vessels was 292 µm (fig. 4). the vessel size class distribution showed that about 40% of vessels in m. peltata stem had a diameter greater than 300 μm. however, over 300 μm vessels in fact provided about 70% of the hydraulic conductance (fig. 5). the average vessel diameters of are also higher than several m. peltata climbing plants studied by fisher . (2002) et al and yansen (2012) (table 3). these findings indicated that the ability of stem to m. peltata conduct water was high which significantly contributed to the ability of this species to grow faster. the ecophysiological characters of m. peltata show that this species do not invest too much es on structural strength tend to grow faster. and it s this indicates that this species is categorized as ruderal (grime 2002) which dominates the disturbed areas but it will not persist in the long term when the forest area becomes more intact and shaded. therefore, an ecological engineering is needed to reduce the domination of this species in the area. biotropia vol. 22 no. 1, 2015 table 2. specific leaf area and wood density (with standard errors) of m. peltata no observed characters value + se 1 specific leaf area 160.30 + 4.80 cm2/g 2 wood density 0.60 + 0.03 g/cm3 figure 4. the transverse section through stem xylem tissue of showing its vessels (v)m. peltata 30 conclusions the differences in population attributes of m. peltata in the open and intact forests indicated that the expansion of this species was enhanced by forest fragmentation. it grew quickly in open area. the ecophysiological characters of also m. peltata supported the ability of this species to become a strong invader and competitor in a short-time. if more forest fragments were created, this species had more chances to invade the forest area. as the domination of this species had significant ecological consequences, a long term observation on population and growth dynamics essential. was therefore, the application of appropriate ecological approaches to overcome the domination of this species could be applied. acknowledgements this research was fully funded by the directorate of higher education, the ministry of education and culture, republic of indonesia, in which the authors thank the institution. ed the authors also thanked candra lumbagaol and ahmad ritonga for their assistance in the field. this project was conducted in bukit barisan selatan national park and we thank the bbsnp office that ha granted ed d permit to access the conservation area. size class (µm) figure 5. distributions of stem vessel diameters of in 10 μm size classes. the graphs show the number of vessels m. peltata of each size class as a percentage of the total vessel number (%n) and the theoretical contribution of each size class to hydraulic conductance (σd ) of the stem. the hypothetical contribution to conductance was calculated by 4 summing the diameters of vessels in each size class to the fourth power (hagen-poiseuille law) (tyree & zimmerman 2002). table 3 average diameter of vessels of several climbing rattans (1-6), climbing pandan (7) and climbing aroid (8) . us compared to current stud species (9) (fisher . 2002; yansen 2012).ly ied et al no. species average diameter of vessels (µm) 1 2 3 4 5 6 7 8 9 calamus insignis daemono ropshystrix korthalsia echinometra korthalsia rigida korthalsia rostrata plectocomia elongate freycinetia excelsa rhaphidophora australasica merremia peltata 213 243 380 532 205 458 150 170 292 31 the xpansion f (l.) merrill n ragmented oreste o i f fmerremia peltata – et al.yansen references brokaw n, busing rt. 2000. niche versus chance and tree diversity in forest gaps. tree 15: 183-88. brown jh. 1984. on the relationship between abundance and distribution of species. am nat 124: 255-79. bchabot bf, hicks dj. 1982. the ecology of leaf life spans. ann rev eco sys 13: 229-60. clavero m, garcıa-berthou e. 2005. invasive species are a leading cause of animal extinctions. trends eco evo 20: 110-15. connell, jh. 1978. diversity in tropical rainforest and coral reefs. science 199: 1302-10. denslow js. 1987. tropical rainforest gaps and tree species diversity. ann rev eco sys 18: 431-52. fisher jb, tan htw, toh lpl. 2002. xylem of attans: r vessel dimensions in climbing palms. am j bot 89: 196-202. grime jp. 2002. . new plant strategies and 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secondary hemi-epiphytic vines. phd hesist . townsville (au): james cook university . biotropia vol. 22 no. 1, 2015 32 sri-13 mei 2017-524 (sunardi invasion).cdr invasion of willd. after eruptionacacia decurrens of mount merapi, indonesia sunardi sulistijorini and titiek setyawati1*, 1 2 1department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, bogor 16680 indonesia, 2 rresearch center of conservation and ehabilitation, ministry of environment and forestry, bogor 16610 i, ndonesia received 12 august 2015/accepted 12 november 2016 abstract eruption of mount merapi in 2010 caused a dense cover of acacia decurrens willd., which is an invasive alien plant species (iaps). the dense cover happened in all areas of mount merapi national park (mmnp) in java, indonesia. this study was aimed to describe the relationship between major natural disturbance from volcanic eruption in triggering the invasion of a. decurrens in mount merapi national park. vegetation data were collected using line transect in two different sites. the first site was cangkringan which was affected by pyroclastic flow and the second site was selo which was not affected by pyroclastic flow. distribution patterns and association of a. decurrens with other species in each location was analyzed using ordination analysis of the non-metric multidimensional scaling (nmds). microclimate such as temperature, humidity, light density and soil humidity was recorded in each location. correlation between species abundance and microclimate data was assessed using canonical correspondence analysis (cca). the results showed that the population of a. decurrens was more dominant in cangkringan than in selo site. cangkringan site was impacted with pyroclastic flow during mount merapi eruption in 2010, while selo site was not affected. in cangkringan, a. decurrens was distributed in clump, while in selo the plant was randomly distributed. ordination analysis using nmds showed that there was positive association between a. decurrens and herbaceous plant. negative association was observed between a. decurrens and other tree species. cca analysis showed that temperature and light density was positively correlated with a. decurrens abundance. this study showed that the iaps invasion in mmnp was correlated with the eruption of mount merapi. keywords: acacia decurrens, autecology, eruption, invasive introduction mount merapi national park (mmnp) is a protected forest, rich with various flora and fauna. mount merapi, located in the mmnp, is the most active volcano in indonesia. the eruption of mount merapi from 26 october to 6 november 2010 was recorded as the worst disaster since its eruption in 1870 (bnpb 2011). mount merapi eruption in 2010 was characterized by pyroclastic flow (a fast moving flow containing high-density mixture of hot lava blocks, pumice, ash and volcanic gas) which reached temperature of o 400 – 600 c and speed of 130 km/h, causing vegetation destruction, among others. vegetation succession after the eruption showed a decline in the diversity of alpine vegetation. the native plant was replaced by invasive alien plant species (iaps) which became dominant in several locations affected by the eruption of mount merapi. dadap ( and pine (erythrina longifolia) pinus merkusii), which were initially the most common plant species in this area, were not recorded after the eruption. pine is not a local species in this mountain area, however, this plant might have been escaped from the neighboring plantation forest managed by state forest enterprises. invasive alien species (ias) is a combination of alien species and their invasive characteristics. an alien species is a species (at the level of species, subspecies, varieties) that had been intentionally or unintentionally introduced (as whole organism, part of the body, gametes, seeds, eggs or propagules that are able to live and reproduce in biotropia 4 1 7 35 46 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 1 524 * corresponding author: sunardi.mansyur@gmail.com 35 the new habitat) into areas outside of its natural distribution range. invasive species, either native or introduced species, become densely established and broadly affect the habitat and land uses resulting to serious environmental, social and economic impacts (cbd-unep 2014). acacia decurrens is one of serious invasive alien plant species (iaps) in mmnp. the plant became dominant in the areas affected by pyroclastic flow. this plant, originated from australia, belongs to family of fabaceae, subfamily of mimosoidae and genus of . acacia within their native distribution range, acacia decurrens is considered as fast growing pioneer species and often causes serious concern due to its potential invasion to their new habitat. iaps can establish themselves in a disturbance area and begin to dominate native vegetation. invasion by an alien species can occur in an area if the native species are unable to quickly adapt to the change of environmental conditions. iaps can significantly suppress the native plant, invertebrate and vertebrate population and communities. for example, lantana camara invaded large areas of tropical asia and australia as well as contributed to the declines of several endangered species in australia (coutts-smith & downey 2006). the invasion of in acacia nilotica baluran national park has changed the savanna into a shrub of and contributed to the acacia decreasing number of banteng ( ) bos javanicus population (setiabudi et al. 2013). plant invasion can occur due to natural disasters, climate and environment changes. the invasion can also happen in disturbed habitats. there are only a few studies focused on the impact of natural disturbance from volcano eruption to the invasion of iaps. therefore, studying biological invasions in mountains having natural disturbance is scientifically important for nature conservation. this study was aimed at determining correlation between mount merapi eruption and the invasion of in a. decurrens mmnp and at determining the autecology of a. decurrens as basic information for managing plant invasion in mmnp. it was assumed that the eruption of mount merapi with the pyroclastic flow has positive correlation with the abundance population of in mount merapi. the a. decurrens autecology study was performed to explain the characteristic, dynamic population of individual species, life cycle and ecological factors, including physical, biological and environmental factors. materials and methods study site the research was conducted from march to december 2014. the research was conducted in mount merapi national park (mmnp). the research location was divided into two sites. the first site was cangkringan located at o o 7 35.771” – 110 26.375” (954 m asl). this site was affected by pyroclastic flow during the eruption in 2010. the second site was selo located o o at 7 30.843” – 100 27.188” (1,044 m asl). this site was not affected by the pyroclastic flow. the study at each site was conducted at three different altitude zones i.e. high zone (1,400 – 1,700 m asl), middle zone (1,100 – 1,400 m asl) and lower zone (800 – 1,100 m asl). sampling design and data analysis vegetation analysis was performed using a systematic sampling with transect line along 100 m for each zone in the two study sites. each transect line consisted of 10 quadrats of 10 x 10 m (for tree species), 10 quadrats of 5 x 5 m (for sapling species) and 10 quadrats of 2 x 2 m (for herb species) (cropper 1993; krebs 2002). height and diameter data of a. decurrens trees were collected at each site. these data were collected from 15 trees that were inside the 10 quadrat plots of 10 × 10 m. environmental data collected at each site were soil physical and chemical properties, air temperature, humidity, light intensity and wind speed. importance value index (ivi) was calculated to determine the overall importance of each species in the community structure. in calculating this index, the percentage values of the relative frequency, relative density and relative dominance were summed up together . (kent & paddy 1992) species diversity was determined using shannonwiener diversity index (h), while species dominance was determined using simpson's dominance index (d). shannon-wiener diversity index (h) is an index that is commonly used to characterize species diversity in a community. shannon36 biotropia vol. 24 no. 1, 2017 ∑ = sum of the quadrat countxi ∑ = sum of the quadrat count from the total of 2 xi species in the community the deviation from random expectation can be tested using critical values of the chi-square distribution with n-1 degrees of freedom. confidence interval around 1 can be calculated by the uniform (mu) and clumped (mc) indices (krebs 2002). where: 2 x 0.0975 = value of chi-square from the table with (n-1), degree of freedom that has 97.5 % of the area to right 2 x 0.0025 = value of chi-square from the table with (n-1), degree of freedom that has 2.5 % of the area to right xi = number of individual of species in a set of quadrat n = number of quadrat standardized morisita index was calculated using the following four formulas: standardized morisita index of dispersion (ip) ranges from -1.0 to +1.0 with 95% confidence limit at +0.5. ip = 0 means that the plants are in random dispersion, ip > 0 means that the plants are in clumped dispersion and ip < 0 means that the plants are in uniform dispersion. correlation between environmental factors and abundance of a. decurrens was analyzed using canonical correspondence analysis (cca) of the past v2. software (legendre & legendre 1998; hammer et al. 2005). distribution and association between a. decurrens and other species in the community were determined using species ordination of the non-metric multidimensional scaling and calculated using primer v5. software (clarke & gorley 2015). wiener diversity index (h) accounts for both abundance and evenness of the existing species, as is shown in this formula: where: h = shannon-wiener diversity index pi = proportion of species n = number of speciesi n = number of quadrat simpson's dominance index (d) is a simple mathematical measure that characterizes species diversity in a community. the proportion of species (ni) relative to the total number of species (n) is calculated and squared. the squared proportions for all the species are summed, and the reciprocal is taken: where: d = simpson's dominance index n = number of speciesi n = number of quadrat the simpson's index ranges: 1. if d = 0 – 0.5 this means that none species were dominant 2. if d = 0.5 – 1 this means that there is a dominant species evenness index (e) can be calculated by dividing h by h (where h = ln ). equitability smax max assumes a value between 0 and 1 with 1 being complete evenness. where: e = evenness index h = shannon-wiener value s = total number of species in the community plant distribution pattern was analyzed using morisita index of dispersion (ið), as is shown by this formula: where: ið = morisita index of dispersion n = sample size 37 invasion of acacia decurrens. after eruption of mount merapi – sunardi et al. results and discussion species composition of the vegetation at cangkringan and selo sites in terms of importance value index (ivi) and morisita index of dispersion for herbs, sapling and tree for each altitude zone are shown in table 1 and 2. vegetation at cangkringan site was dominated by herbs species of imperata cylindrica, centella asiatica, impatiens balsamina and by sapling and tree species of a. decurrens (table 1). vegetation at selo site was dominated by species of , , c. asiatica eupatorium odoratum eupatorium riparium pennisetum purpureum, , pogonatum sp., and by sapling and tree species of a. decurrens vaccinium varingiaefolium albizia , and lophantha (table 2). 38 biotropia vol. 24 no. 1, 2017 table 1 vegetation observed at cangkringan site that were affected by pyroclastic flow no. species family importance value index morisita index zone 1 zone 2 zone 3 zone 1 zone 2 zone 3 herbs 1 centella asiatica apiaceae 14.46 19.46 13.59 c c c 2 ageratum conyzoides l.* asteraceae 4.59 7.88 11.88 c c c 3 bidens biternata asteraceae 10.64 6.32 8.85 c c u 4 emilia sonchifolia asteraceae 4.30 10.92 7.11 c c c 5 eupatorium odoratum* asteraceae 8.54 1.18 7.00 c c c 6 eupatorium inulifolium* asteraceae 5.46 13.70 4.37 c c c 7 eupatorium triplinerve* asteraceae 4.20 5.39 3.35 c c c 8 gynura crepidioides asteraceae 1.35 7.60 4.83 c c 9 mimosa pudica* asteraceae 10.14 3.64 8.23 c c c 10 pennisetum macrostachyum* asteraceae 6.85 4.77 5.68 c c c 11 polygala paniculata* asteraceae 11.87 1.28 10.11 c c c 12 sida rhombifolia asteraceae 1.07 5.29 0.86 c c c 13 stachytarpheta jamaicensis asteraceae 2.51 2.46 2.04 c c c 14 wedelia trilobata* asteraceae 9.84 3.45 8.23 c c u 15 impatiens platypetala balsaminaceae 6.97 10.92 6.14 c r c 16 cyperus rotundus* cyperaceae 3.86 3.73 3.14 c c c 17 erigeron sumatrensis retz gleicheniaceae 10.33 8.39 c u 18 melastoma malabathricum* melastomaceae 1.97 6.76 c c c 19 selaginella kraussiana mimosaceae 3.93 3.11 r c r 20 synedrella nodiflora oxalidaceae 10.07 8.26 c u c 21 imperata cylindrica* poaceae 31.19 59.85 26.59 c c 22 tithonia diversifolia* poaceae 1.07 1.09 0.86 c c c 23 tridax procumbens poaceae 3.15 2.08 11.24 c c c 24 gleichenia longissima polygalaceae 5.39 3.73 4.40 c c 25 oxalis corniculata* silaginellaceae 7.13 4.67 5.93 c c c 26 lantana camara* verbenaceae 5.48 6.53 4.48 c c c 27 rubus chrysophyllus miq. verbenaceae 1.97 1.99 1.56 c c c sapling 1 acacia decurrens* fabaceae 80.84 81.84 84.00 c c c 2 erythrina variegata fabaceae 57.82 35.05 c c 3 paraserianthes falcataria fabaceae 23.19 79.62 c u 4 schima wallichii theaceae 59.82 c pole 1 acacia decurrens* fabaceae 157.68 158.68 146.09 r c c 2 erythrina variegata fabaceae 28.93 30.93 21.30 c c c 3 paraserianthes falcataria fabaceae 9.64 12.64 32.61 c c c notes: = not found in the site; c = clump; r = random; u = uniform * = invasive plant based on information from global ias database (www.issg.org) herbs at cangkringan site were dominated by asteraceae family, while at selo site were dominated by asteraceae and verbenaceae families. the domination of asteraceae family occurred because this family can well adapt to the mountain slope conditions. this family also has role in ecosystem functions, such as preventing erosion and enriching soil organic matter 39 table 2 vegetation observed at selo site that were not affected by pyroclastic flow no. species family importance value index morisita index zone 1 zone 2 zone 3 zone 1 zone 2 zone 3 herbs 1 centella asiatica apiaceae 34.16 20.43 5.18 r c c 2 foeniculum vulgare apiaceae 5.05 0.0 0.00 c 3 anaphalis javanica asteraceae 6.32 10.50 16.21 u c c 4 anaphalis longifolia asteraceae 3.79 11.31 9.96 u c c 5 lactuca sativa asteraceae 4.25 6.07 19.20 c c c 6 athyrium sp. athyriaceae 7.34 6.07 6.62 c c c 7 impatiens platypetala balsaminaceae 12.16 12.16 0.00 c c 8 erigeron sumatrensis retz* compositae 5.51 9.84 0.00 c c 9 indigofera cassioides fabaceae 4.40 1.97 0.00 c c 10 eupatorium odoratum* malvaceae 5.93 9.03 27.02 c c r 11 eupatorium riparium* malvaceae 21.34 9.36 8.49 c c c 12 melastoma malabathricum * melastomaceae 5.97 3.44 5.15 c c c 13 oxalis corniculata oxalidaceae 12.38 59.80 0.00 c c 14 pennisetum purpureum* poaceae 42.09 25.40 50.56 c r r 15 pogonatum sp. poaceae 10.70 0.0 31.00 c c 16 brugmansia candida solanaceae 4.10 0.0 0.00 c 17 lantana camara* verbenaceae 7.08 5.90 11.43 c c c 18 rubus chrysophyllus miq. verbenaceae 2.83 3.44 4.78 c c c 19 rubus plicatus verbenaceae 4.55 5.24 7.72 c c r sapling 1 acacia decurrens* fabaceae 33.67 42.51 58.67 r r r 2 vaccinium varingiaefolium ericaceae 39.29 64.64 104.53 c c m 3 albizia lophantha* fabaceae 37.90 30.38 36.00 c c m 4 erythrina lithosperma fabaceae 27.29 30.95 0.00 c u 5 toona sureni meliaceae 16.52 8.13 0.00 c c 6 cinchona succirubra rubiaceae 22.90 11.69 0.00 c c 7 dodonaea viscosa sapindaceae 14.52 12.69 0.00 a 8 schima wallichii theaceae 21.90 42.51 0.00 c c pole 1 casuarina junghuhniana casuarinaceae 17.44 18.44 23.81 c c c 2 cupressus montana cupressaceae 13.04 15.04 10.48 c c c 3 vaccinium varingiaefolium ericaceae 19.60 22.60 47.86 c c c 4 acacia decurrens* fabaceae 41.94 45.94 107.38 c c r 5 albizia lophantha fabaceae 37.77 42.77 11.19 c c c 6 erythrina lithosperma fabaceae 18.39 24.39 0.00 c c 7 toona sureni meliaceae 6.52 0.00 0.00 c 8 myrica javanica myricaceae 8.06 0.00 0.00 c 9 cinchona succirubra rubiaceae 9.01 13.52 0.00 c c 10 dodonaea viscosa sapindaceae 4.98 0.00 0.00 c 11 schima wallichii theaceae 24.69 16.06 0.00 c r _notes: = not found in the site; c = clump; r = random; u = uniform * = invasive plant based on information from global ias database (www.issg.org) invasion of acacia decurrens. after eruption of mount merapi – sunardi et al. (kumolo & utami 2011). they are often classified as weed on agricultural land. one species member i.e. was reported by sunaryo e. odoratum et al. (2012) and uji (2010) for invading several locations in the mount salak and mount gede pangrango national park. based on database from invasive species specialist group (issg 2015), numbers of herbs and tree species in the two study sites were listed as invasive alien plant species (iaps). only few numbers of native plants were found after the eruption. native plants can adapt to a very low frequency of disturbance and take a long time to recover into the same population size as before the disturbance (smith & tunison 1992). a. decurrens is the most dominant and has high risk to the environment in mount merapi national park. this species densely covered the vegetation at cangkringan site. morisita index of dispersion showed that the a. decurrens was dispersed into clumped pattern (table 1 & 2). in natural ecosystem, plant distribution or dispersion was correlated with environmental condition and competition with other species in the community. random dispersion of plant in the community is rarely found in the tropic ecosystem (call & nilsen 2003). clump distribution of the plant was also caused by high seed reproduction of mature plants which seeds fall surrounding the parent plant. environmental factors such as climate, wind, soil nutrients may influence plant distribution. clump distribution of plant species affected species diversity and evenness of plant community (soerianegara & indrawan 1998). the results showed that at cangkringan site population of was clump distribution a. decurrens (table 1) with lower diversity index compared to the index at selo site (table 3). a. decurrens at selo site was randomly distributed (table 2). species diversity in a plant community was affected by density of the individual species, the larger number of the species and the spread of each species. at cangkringan site the evenness index showed that the herbs were more stable than the pole or tree plant due to the invasion of which a. decurrens densely distributed in clumps. simpson's dominance index (d) showed that some species dominated plant community both at cangkringan and selo sites. pole and tree vegetations at cangkringan site had low dominance indices than those at selo site. smaller dominance index indicated that there was a species spread and dominated the coverage of an area (krebs 2002). 40 biotropia vol. 24 no. 1, 2017 table 3 shannon-wiener diversity index (h), evenness index (e) and simpson's dominance index (d) of vegetations at cangkringan and selo sites location zone vegetation category h e d cangkringan 1 herb 2.80 0.84 0.91 sapling 1.39 0.86 0.73 pole 0.39 0.35 0.19 2 herb 2.80 0.88 0.72 sapling 0.55 0.79 0.86 pole 0.17 0.15 0.87 3 herb 2.89 0.87 0.92 sapling 1.00 1.44 0.60 pole 0.15 0.14 0.96 selo 1 herb 2.15 0.65 0.82 sapling 2.66 1.65 0.85 pole 2.02 1.84 0.53 2 herb 1.89 0.57 0.75 sapling 1.84 1.14 0.37 pole 2.02 1.84 0.76 3 herb 1.95 0.59 0.80 sapling 0.98 0.61 0.44 pole 0.99 0.90 0.52 invasion of a. decurrens, forming monoculture thickets, may reduce the abundance of native plant species. few native species found at the cangkringan site was highly invaded by a. decurrens. reduction of species diversity may be caused by competition for soil nutrients between native species and iaps. other possible cause was the ability of a. decurrens (legume family) to increase soil nitrogen content (nitrogen fixing). changes in vegetation composition and loss of diversity are considered to negatively affect wildlife through quality and quantity of ecosystem services (mc. donald et al. 2012). autecology perhutani (state forest enterprise) introduced a. decurrens into mmnp around 1980s, before the region was declared as a conservation area. a. decurrens was used as divider between plantation area with agriculture land. over time, was acacia widespread in several regions around the national park. significant high number of a. decurrens population was recorded in 2011, a few months after the eruption of mount merapi which occurred from october to november 2010. acacia has hard and thick seed coat to remain dormant in certain circumstances. also, the seed structure makes it possible for to germinate under acacia unsuitable condition caused by major disturbances. invasive species tend to have generalist pollination, mass seed production, efficient seed dispersal and persistent seed bank which can endure heat caused by fire or disturbance that triggered seed germination (gibson 2011). this study indicated that the et al. pyroclastic flow broke the dormancy of seed stored in the soil (soil seed bank). invasion acacia in mmnp was caused by major disturbances resulted from mount merapi eruption. the invasion occurred in several areas impacted by the pyroclastic flow. the pyroclastic flow contained materials that can stimulate biological production in the impacted environment. the materials can be used as fertilizer and contain many elements that can increase soil fertility. there is positive correlation between a. decurrens invasion and mount merapi eruption, accompanied by pyroclastic flow. fire is one of natural disturbances and important processes for maintaining species diversity in forest ecosystem (walker & del moral 2003). non-native plant species with an established seed bank tend to respond positively to post-fire conditions, such as increased soil temperatures, increased light levels, reduced competitions and increased available nitrogen (wagner & fraterrigo 2015). a. decurrens is known for their high flammability and has tendency to quickly recover from disturbance compared to native species (brooks et al. 2004). a. decur r enshigh population of at cangkringan site was an example showing correlation between area impacted by pyroclastic flow and invasion in the area. selo site had acacia low population of because selo site was acacia not impacted by pyroclastic flow (table 1 & 2). invasive species responded quicker to disturbance than non-invasive species (gibson et al. 2011). invasive alien plant species can widely spread, have high growth rate and have high tolerance to physical conditions such as fire, flood, drought and other natural disturbances. these physical conditions are the major factors for alien plant species to invade a new area (velde et al. 2006). there are four factors influencing the success of species invasion i.e. disturbances occurred in ecosystem (fire or natural disaster), ability to compete, availability of resources and pressure of propagules (moser 2009). et al. species invasion is the result of interactions between habitat suitability and pressure of propagules (rejmànek 2005). changes in et al. environmental conditions may affect the level of species invasion. changes in environmental conditions interfere with the balance of competition between native plants and foreign plants. among changes in the environment are limited metabolism, high temperature and presence of toxin (alpert 2000).et al. a. decurrenscluster analysis showed that has positive correlation only with herbs. high numbers of correlation were observed between a. decurrens c. asiatica e. riparium impatiens and , , platypetala pennisetum purpureum and (fig.1). negative correlations occurred between a. decurrens and native species. the results also showed that at cangkringan site there was a decrease in the number of native tree species due to high numbers of (fig. 2)a. decurrens . 41 invasion of acacia decurrens. after eruption of mount merapi – sunardi et al. being tree and woody species, a. decurrens is a competitor to other tree species. the dominance of a. decurrens presents an extreme challenge for the native plant communities because the rapid canopies growth of a. decurrens hindered other plants from obtaining light. it is very important to understand that invasion process varies along biotic and abiotic gradients within a local environment. abiotic variable is the most important factor to study the plant autecology (swamy et al. 2000). the study of abiotic gradient analysis showed that the invasion of a. decurrens correlated with available light and weather temperature (fig. 3). changes in available light, wind speed, humidity, temperature and soil moisture induced by fragmentation of forest ecosystems often add to competitive advantages of invasive species over native species (emily et al. 2004). mount merapi eruption has burned down all of the native vegetation and transform it into the open area. as the open area, automatically the light density was increased light intensity and also the increase of the temperature after the eruption. 42 biotropia vol. 24 no. 1, 2017 figure 1 nmds ordination result (2d stress = 0.01, measured from relationship between actual dissimilarities and distances) which shows species composition difference in each sample plot and change in abundance of a. decurrens figure 2 nmds ordination result (2d stress = 0.11, measured from relationship between actual dissimilarities and distances) which shows association clusters of plant community including a. decurrens at sampling locations s1 s3 c3 c2 s2 c1 figure 3 the influence of environmental factors in the two study sites towards a. decurrens population using canonical correspondence analysis (cca) notes: c = cangkringan; s = selo; 1 = zone 1; 2 = zone 2; 3 = zone 3 the increase of temperature and the light density was triggered the seed bank to germinate. iaps can endure extreme temperature which makes them competitor to the native vegetation (hellmann et al. 2008; ordonez et al. 2010; van kleunen et al. 2010). song et al. (2010) studied the effect of extreme high temperatures for the invasive wedelia trilobata, and found out that in the extreme high temperature condition, w. trilobata experienced less inhibition of relative growth rate (rgr) and biomass production than the native plant wedelia chinensis. our study compared diameter and height of a. decurrens located in area affected by pyroclastic flow (cangkringan) and those located in area not affected by pyroclastic flow (selo). diameter of a. decurrens at cangkringan site was lower than that at selo site. inverse effect observed for the height of a. decurrens (fig. 4 & 5). tree height or diameter was also affected by species composition of understory (suryanto et al. 2010). iaps also produce large amount of litter under the canopies, which may influence understory species (williams & wardle 2007). results of this figure 4 diameter of a. decurrens at cangkringan site (invaded) and selo site (uninvaded) 43 invasion of acacia decurrens. after eruption of mount merapi – sunardi et al. a x is 2 -1.8 -1.5 -1.2 -0.9 -0.6 -0.3 0.3 0.6 0.9 1.2 1.5 1.8 0.3 0.6 0.9 1.2 1.5 1.8 -0.3 -0.6 -0.9 -1.2 -1.5 -1.8 axis 1 light temperature soil humidity soil ph wind humidity xs1+c2 xs2 xs3 +c3 +c1 figure 5 height of a. decurrens at cangkringan site (invaded) and selo site (uninvaded) study revealed that there were different sizes of diameters as well as dissimilarity in native understory plant composition between those located at cangkringan site (invaded by a. decurrens) and those located at selo site (not invaded by a. decurrens). dense population at cangkringan (invaded site) would lead to intraspecific competition in obtaining resources from the environment. a. decurrens invaded from low elevation to higher elevation; therefore, it was adapted to the climate of mount merapi. the management of mmnp has to be aware of the invasive ability of a. decurrens in order to control the spreading of this plant species. among effective treatments to eradicate population are cut stump a. decurrens method, herbicide treatment and seed production decrease. we suggest conducting a study for determining the most feasible and most economical management strategy to eradicate this invasive species. among characteristics of are a. decurrens having mass seed production, seed germination triggered by high temperature, high potential to spread and ability to produce root sucker. in managing the invasion, it is not immediately apparent whether the eradication method used will be cost effective or not. therefore, it is very important to have a good understanding of the invaded site, the invasive plant species characteristics, the biological impacts and the management strategy to control invasive plant species (moore 2011).et al. conclusions mount merapi eruption triggered the spread of a. decurrens in mmnp. the importance value index showed that the invasion of a. decurrens was more dominant in cangkringan site (affected by the eruption) than in selo site (not affected by the eruption). nmds ordination showed the differences in the composition and abundance of a. decurrens between cangkringan site and selo site. a. decurrens showed a clump dispersal pattern at cangkringan site and a random dispersal patterns at selo site. environmental factors positively correlated with the abundance of a. decurrens were temperature and light intensity. acknowledgements we are thankful to unep/gef project “removing barriers to invasive species management in production and protection forest in south east asia (foris) – indonesia who funded this research. many thanks to the management of mount merapi national park for assistance in collecting field data. references alpert p, bone e, holzapfel c. 2000. invasiveness, invisibility and the role of environmental stress in the spread of non-native plants. perspect plant ecol evol syst 3(1):52-66. 44 biotropia vol. 24 no. 1, 2017 badan nasional penanggulangan bencana [bnpb]. 2011. impact of mount merapi eruption. gema bnpb. 2(1):17-20. brooks ml, d'antonio cm, richadson dm, grace jb, keeley je, ditomasso jm, hobss rj, pellant m, pyke d. 2004. effect of invasive alien plant on fire gerimes. bioscience 54: 677-88. call lj, nilsen et. 2003. analysis of spatial patterns and spatial association between the invasive tree-ofheaven ( ) and the native black ailanthus altissima locust ( ). am midl nat. 150:1-4.robinia pseudoacacia clarke kr, gorley rn. 200 . primer: plymouth routines 1 in multivariate ecological research. plymouth (uk): primer-e ltd. 129 p. convention on biological diversity-united nations environment program cbd 2014. 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1-10. 46 biotropia vol. 24 no. 1, 2017 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 biotropia vol. 29 no. 3, 2022: 213 224 doi: 10.11598/btb.2022.29.3.1583 213 the molecular approach reveals the relationship among venus clams (meretrix spp.) community in malaysia mohd hanafi idris1, abu hena mustafa kamal1, hadi hamli2*, amy halimah rajaee2,3 and abdulla-al-asif2 1faculty of fisheries and food science, universiti malaysia terengganu, 21300 kuala nerus, terengganu, malaysia 2department of animal science and fishery, faculty of agricultural and forestry sciences, universiti putra malaysia, bintulu sarawak campus, bintulu, sarawak, malaysia 3institut ekosains borneo, universiti putra malaysia, bintulu sarawak campus, bintulu, sarawak, malaysia received 12 april 2021/accepted 2 june 2022 abstract molecular study is important to detect variations and similarities among species from the same genus, in case if they do not encompass any morphological or physiological differences. the study was conducted to differentiate among species of meretrix spp. (meretrix lyrata, m. meretrix, and m. lusoria) obtained from two locations in malaysia through the phylogenetic tree. the adductor muscle tissues were used to extract dna and to perform other procedures; the samples were subjected to analyses using pcr and gel electrophoresis. the multiple sequence comparison was conducted by muscle and the phylogenetic relationships were established using maximum likelihood (ml) statistical methods with mega 6.0 statistical software. m. lyrata samples showed 99% similarity to the three accessions sequence, where m. lyrata indicated 87% similarities, and m. meretrix showed not more than 89% similarities from the deposited sequence. the nucleotide base composition sequences consisted of the mean of thiamine (t) 37.9%, cytosine (c) 15.4%, adenine (a) 27.4%, and guanine (g) 19.4%. maximum likelihood (ml) analysis was conducted using the tamura 3-parameter model to establish five major clades on meretrix spp. and two out-groups clades significantly different from the meretrix spp. these major clades were closely related to each other at the 50% evidence of bootstrap, which grouped as genus meretrix. the present study on meretrix spp. from the sarawak locality was able to differentiate coi sequences between m. lyrata, m. meretrix, and m. lusoria. m. lusoria was close related to m. meretrix with strong bootstrap supporting evidence at 96% scoring. moreover, m. lyrata was inferred as the ancestor to m. meretrix, and m. lusoria from sarawak, malaysia. keywords: bivalve, blast, borneo, mtdna, pcr, phylogenetic analysis introduction genetic variation can be characterized into two groups which are intraspecific and interspecific (arruda et al. 2009; ehlers et al. 2016; layton et al. 2016; thia et al. 2016). intraspecific is the variation within individuals from similar species such as subspecies (stevens et al. 2010). on the other hand, interspecific is the variation within individuals above the species level. genetic variation of natural populations is thought to be governed by the mutual effects of irregular genetic drift, restricted gene flow, and variance of selection pressures. intense natural selection is an important factor influencing variation among, and within groups (endler 1986). the natural selection can be stabilized the species characters such that genetic variation within the population might be reduced. disruptive selection may lead to genetic divergence, and therefore, may eventually increase genetic variation among population (backeljau et al. 2001). mitochondrial dna (mtdna) is one of the dna markers that are popular for dna identification. this marker is the maternal inheritance that supplies information absent in nuclear markers (okumuú & çiftci 2003). *corresponding author, email: hadihamli@upm.edu.my biotropia vol. 29 no. 3, 2022 214 therefore, selecting tissue from the female individual is essential to ensure information regarding the species ancestor. however, the maternal inheritance by mtdna can be restricted by the geographical with no mixing of mtdna haplogroups from the different geological regions (luttikhuizen et al. 2003; mishmar et al. 2003; layton et al. 2016). furthermore, inheritance characteristics on a particular defect gene could be recognized using an mtdna marker (wallace et al. 1999). this is significant, particularly in aquaculture, to ensure cultured species are free from any inherited diseases from the parents. moreover, mtdna could be applied for species identification and phylogeny studies due to the particular inherited gene variance. the typical mitochondria gene used for the phylogenetic study is cytochrome c oxidase subunit i (coi). this mtdna marker has been applied for genetic relationship and phylogenetic analysis for meretrix spp. from japan, korea, and china (yamakawa et al. 2008, chen et al. 2009; torii et al. 2010; kim & yoon 2014; sato et al. 2016). parentage assignment from a similar population but different parents was also successfully implemented for meretrix meretrix culture (lu et al. 2011). due to the intensive mtdna marker application, the universal primer of coi had been created based on the conserve gene sequence across the species to facilitate coi analyzing region on different metazoan species (folmer et al. 1994). the study of mollusca started from the study of morris & purchon (1981), their approach was to enlist and taxonomic study of available species; however, the story of mollusk study goes on, and malaysian scientists and academician studied a different aspect of mollusca; from taxonomy to diversity, along with the genetic aspect of this community (hamli et al. 2012a, b; 2013; 2015; 2017; 2019; 2020a, b, c; foon et al. 2017; al-asif et al. 2020; al-asif et al. 2021; idris et al. 2017a, b). genetic and molecular study of class bivalvia and gastropoda is not new in malaysian territory, including east malaysia (sabah and sarawak provinces). some previous study revealed the genetic diversity of asian green mussel (perna viridis) in the waters of sabah (lau et al. 2018), dna study of neritid (chee & mohd nor 2016), genetic diversity of razor clam in kuala selangor, malaysia (hassan & kanakaraju 2016). there is no genetic approach of venus clam (meretrix spp.) was found in malaysia, and the genetic relationship among the available venus clam species is still unknown. therefore, the present study aims to differentiate among meretrix spp. from different through the phylogenetic tree. materials and methods sample collection a total of 30 samples of meretrix lyrata, m. meretrix, and m. lusoria were collected from two areas, kuching and kabong, sarawak province, malaysia (fig. 1). adductor muscle tissues from the females were separated from the shell and preserved with 95% of ethanol (wang et al. 2010) for further analysis at the parasitology laboratory, department of animal science and fishery, universiti putra malaysia, at the bintulu sarawak campus. dna extraction about 10 to 20 mg of adductor muscle tissue from each sample was used for extraction using a tissue dna extraction kit (vivantis) following the manufacturer's instructions and stored at -20 °c. the extracted dna was then quantified through agarose gel electrophoresis. dna quantification and gel electrophoresis extracted dna was quantified following rengarajan et al. (2002) to ensure the amount of dna within 1 µl. each quantified dna was adjusted to 100 µl to ensure the total amount of dna sample used for amplification was equal. quantification of genomic dna was compared based on lambda hind iii marker (23130 bp). gel agarose concentration used for dna quantification was 0.8% in 1x tae (trisacetate-edta) buffer and the electrophoresis procedure was conducted following lee et al. (2012). a total of 5 µl of extracted genomic sample and 1 µl of dye (ez-vision one dye) were added into the well. after all the samples and markers were loaded, electrophoresis was run for 40 minutes with 120 volts of electricity. the running of electrophoresis was monitored until the red dye was moved 3/4 of the gel length, and the electrophoresis was stopped. molecular approach establishes relationship among venus clams (meretrix spp.) community – idris et al. 215 figure 1 study area of meretrix spp. for genetic study the gel was then removed from the tank and was photographed under a uv light gel imager (redtm, alpha innotech). quantification process based on appeared bands on the gel to be viewed with alpha view software and band intensity from extracted samples was compared with lambda hind iii marker for 100 ng of dna. while 1.5% of gel agarose concentration was used for the polymerase chain reaction (pcr) through 0.3 g of agarose powder into 20 ml of 1x tbe (tris-borate-edta) buffer, the mixture was swirled and heated in the microwave to ensure agarose powder was dissolved. heated mixture was monitored to make sure the mixture was not boiled. after agarose powder dissolved in the buffer, the mixture was left to cool and then was poured slowly into the prepared gel tank while ensuring that no bubble was trapped. immediately, the comb was inserted into the poured mixture, while letting the agarose mixture to be cool and solidify for 30 minutes. after the gel became solid, the comb was removed and the running buffer 1x tba or 1x tbe was poured into the gel tank until all the gel parts submerged. polymerase chain reaction (pcr) amplification amplification of dna was performed in 50 µl of pcr mixture using universal marker cytochrome c oxidase subunit i (coi) with sequence lco1490: 5’-ggtcaacaaatcata aagatattgg-3’ and hco2198: 5’taaacttcagggtgaccaaaaaatca-3’ (folmer et al. 1994). each pcr reaction consisted of 0.1 mm of reverse and forward primers, 0.2 mm of dntp, 2.0 mm mgcl2, 1x buffer, 1.25 units of taq polymerase, 100 ng of template dna, and ultrapure water to complete 50 µl. a total of 50 µl of pcr mixture were amplified in xp thermal cycler block (bioer technology co. ltd) with pre-denaturation at 95 °c for 5 minutes, then followed by 35 cycles of 1 minute at 95 °c for denaturation, 1 minute for annealing at 40 °c, 1 minute 30 second at 72 °c for extension and 72 °c for 7 minutes for final extension (folmer et al. 1994). the last cycle temperature was maintained at 4 °c. the pcr product then underwent electrophoresis. gel electrophoresis gel agarose concentration used was 1.5% in 1x tbe (tris-borate-edta) buffer. the electrophoresis procedure was carried out following lee et al. (2012). a total of 100 ng of pcr product was added into the well with 1 µl of dye (ez-vision one dye) and 1 µl of 100 bp ladder (promega) with 1 µl of dye (ez-vision one dye) at the end of the well. after all samples and markers were loaded, electrophoresis was run for 60 minutes with 80 volts of electricity. the running of biotropia vol. 29 no. 3, 2022 216 electrophoresis was monitored until the red dye was moved 3/4 of the gel length, and the electrophoresis was stopped. the gel was then removed from the tank and was photographed under a uv light gel imager (redtm, alpha innotech). the appeared band was then compared to the ladder to identify the fragment size. purification and sequencing pcr products were purified before sequencing. according to the manufacturer's instruction, purification has been done to remove excess dinucleotide triphosphate (dntp) using themo scientific genejet pcr purification kit. afterward, purified samples were sent to the first base laboratory, malaysia, for dna sequencing. both strands of pcr products and primers were sequenced using the applied biosystems bigdye terminator v3.1 cycle sequencing kit. statistical analysis the obtained sequences were inspected and assembled using bioedit version 7.0. the assembled sequence of m. lyrata, m. meretrix, and m. lusoria were then compared with all species of meretrix spp. in genbank of national center for biotechnology information (ncbi) using basic local alignment system tool (blast) 2.2.31 (national center for biotechnology information, bethesda, md, usa [http://www.ncbi.nlm. nih.gov/blast/]). the highest similarity sequence from the genbank with meretrix spp. was selected for phylogenetic analysis in the present study. the present meretrix spp. sequence, 16 accessions sequence mtdna, coi sequence, and 2 species with coi sequence as an outgroup which mercenaria mercenaria and paphia gallus (table 1) in genbank ncbi were aligned based on multiple sequences comparison by logexpectation using muscle (edgar 2004). phylogenetic relationships were developed using maximum likelihood (ml) statistical methods with mega 6.0 statistical software (tamura et al. 2013). the evolutionary history was inferred using the maximum likelihood method based on the tamura 3-parameter model (tamura 1992). the non-uniformity of evolutionary rates among sites was modeled using discrete gamma distribution (+g) with 5 rate categories. the bootstrap agreement tree inferred from 500 replicates (felsenstein 1985) was taken to represent the evolutionary history of the analyzed taxa (felsenstein 1985). branches equivalent to partitions reproduced in more than 50% bootstrap replicates were illustrated. the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test was shown next to the branches (felsenstein 1985). the heuristic search's initial tree was obtained automatically by applying neighbor-join and bionj algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood (mcl) approach and then selecting the topology with a superior log-likelihood value. evolutionary analyses were conducted in mega6 (tamura et al. 2013). table 1 list of meretrix accession sequences from the genebank (ncbi) meretrix accessions geographical locations genbank acession number citation meretrix lyrate sarawak, malaysia present study present study m. lusoria sarawak, malaysia present study present study m. meretrix sarawak, malaysia present study present study m. lyrata china kc832317.1 wu et al. (2014) m. lyrata china j n898944.1 cheng et al. (2013) m. lyrata china hm124580.1 chen et al. (2011b) m. lusoria china gq903339.1 wang et al. (2010) m. lusoria japan ab853870.1 yamakawa & imai (2013) m. lusoria japan ab853865.1 yamakawa & imai (2013) m. meretrix china gq463598.1 he et al. (2011) m. meretrix china hm124578.1 chen et al. (2011b) m. meretrix china jn043623.1 wang et al. (2011a) m. petechialis china eu145977.1 ren et al. (2009) m. petechialis china hm124582.1 chen et al. (2011b) molecular approach establishes relationship among venus clams (meretrix spp.) community – idris et al. 217 table 1 (continued) m. petechialis china hq703177.1 chen et al. (2011a) m. lamarckii china gu071281.1 wang et al. (2011b) m. lamarckii china hm124579.1 chen et al. (2011b) m. lamarckii japan ab059420.1 hamaguchi et al. (2001) m. casta india jq773441.1 ranjith et al. (2012) mercenaria mercenaria * canada hm884239.1 layton et al. (2014) paphia gallus * china hq703232.1 chen et al. (2011a) note: * = outgroup. results and discussion the coi marker produces a fragment size at 710 bp for m. lyrata, m. meretrix, and m. lusoria (fig. 2). figure 2 fragment size produced by meretrix spp. using coi marker notes: 1 = m. lyrata; 2 = m. lusoria; 3 = m. meretrix. basic local alignment system tool (blast) analysis indicated that the m. lyrata samples from the present study had 99% similarity to the three accessions sequence (kc832317.1, jn898944.1, hm124580.1) of m. lyrata compared to other accessions sequence (table 2). different accession sequences were only showed lower than 87% of similarities with m. lyrata from the sarawak locality. the present study on m. lusoria showed higher similarities with m. lyrata with accessions sequence kc832317.1 and hm124580.1, and m. meretrix with accessions sequence hm124578.1 and jn043623.1. other accessions sequence were less than 89% of similarities with the present study on m. lusoria. there was no similarity of more than 89% within m. meretrix from sarawak province with genebank ncbi accessions sequence of m. meretrix. the higher similarity sequence was represented by the accession sequence hm124580.1 of m. lyrata with 89% similarity. the nucleotide sequence for m. lyrata in the present study showed potential similarity with the accession nucleotide sequence of m. lyrata from the genbank ncbi with more than 90% similarity compared to m. meretrix and m. lusoria. some previous studies conferred the morphology feature on m. lyrata as being the easiest to distinguish from m. meretrix and m. lusoria (hamli et al. 2012b, 2015, 2016, 2017). however, the nucleotide sequence for m. meretrix and m. lusoria showed lower than 90% similarity with the accession nucleotide sequence from the genbank ncbi. hence, the blast biotropia vol. 29 no. 3, 2022 218 analysis could not confirm and distinguish m. meretrix and m. lusoria from the sarawak locality through the coi sequence. current findings were contradictory with results of the morphology and morphometric studies, which distinguished differences between m. meretrix and m. lusoria. however, wang & dunbrack (2004) reported that the application of blast only produces short and conserve fragment alignment. thus, the analysis was frequently unable to identify many relationships, particularly below 40% sequence identity. low sequence identity of fragment creates uncertainty of gaps and insertion, thus compromising confidence in overall modeling of protein related to genome sequence annotation (gan et al. 2002). m. lusoria and m. meretrix were hard to identify by rough morphological inspection due to similar features, particularly the outer shell. however, a detailed examination of the inner shell generated morphological variation of the distinct profile of the pallial sinus scar between m. lusoria and m. meretrix. yet, using the blast application to confirm the differences between the two species appeared to provide uncertain results. rather than depending on sequence identity percentage, phylogenetic relationship perhaps provides more justified evidence on the present genome characteristic of meretrix spp. to be evaluated against the accession sequence in the genbank. the current phylogenetic tree was constructed based on maximum likelihood (ml), which applied an algorithm that calculates the probabilistic approaches to build a relationship tree between compared species based upon the nucleotides or amino acid sequences (guindon & gascuel 2003). meretrix spp. from the sarawak locality indicates the close relationship between m. lusoria and m. meretrix. the variation in the coi sequence of this meretrix spp. can be distinguished, hence, we are able to clarify the intricacy conferred using the blast application. moreover, m. lusoria and m. meretrix had similar ancestors represented by m. lyrata from a similar locality. therefore, coi sequence in m. lyrata had a low level of sequence identity among meretrix spp. this was in agreement with chen et al. (2009) and wu et al. (2014), which suggested that m. lyrata has the level of sequence that portrays as the ancestor to other meretrix spp. by referring to coi and transfer rna (trna), respectively. sequence characteristics a total of 21 nucleotide sequences were analyzed from three meretrix spp. of sarawak locality, 16 accessions sequence of meretrix spp. and two outgroup species represented by mercenaria mercenaria and paphia gallus. codon positions included were 1st + 2nd + 3rd + noncoding. all positions containing gaps and missing data were eliminated. there were a total of 435 positions in the final dataset. the nucleotide base composition sequences were comprised with mean of thiamine (t) 37.9%, cytosine (c) 15.4%, adenine (a) 27.4%, and guanine (g) 19.4% (table 3). table 2 similarity percentage of meretrix spp. in the present study with 16 selected meretrix spp. mtdna accessions sequence from genbank (ncbi) accession sequence genbank species m. lyrata (%) m. lusoria (%) m. meretrix (%) kc832317.1 m. lyrate 99 90 88 jn898944.1 m. lyrata 99 < 83 < 84 hm124580.1 m. lyrata 99 90 89 gq463598.1 m. meretrix 86 86 85 hm124578.1 m. meretrix 87 90 87 jn043623.1 m. meretrix 87 90 87 gq903339.1 m. lusoria < 85 86 86 ab853865.1 m. lusoria 85 87 85 ab853870.1 m. lusoria < 85 86 85 eu145977.1 m. petechialis 86 85 85 hm124582.1 m. petechialis < 85 85 85 hq703177.1 m. petechialis < 85 86 85 gu071281.1 m. lamarckii < 85 < 83 86 ab059420.1 m. lamarckii < 85 < 83 87 hm124579.1 m. lamarckii < 85 < 83 86 jq773441.1 m. casta < 85 88 87 molecular approach establishes relationship among venus clams (meretrix spp.) community – idris et al. 219 table 3 nucleotide base composition for accession sequences species accession t % c % a % g % total meretrix lyrata sarawak 24.8 19.5 42.1 13.6 435.0 m. lusoria sarawak 24.4 20.2 41.8 13.6 435.0 m. meretrix sarawak 23.7 20.7 42.1 13.6 435.0 m. lyrata kc832317.1 41.8 14.0 23.7 20.5 435.0 m. lyrata jn898944.1 42.1 13.8 24.1 20.0 435.0 m. lyrata hm124580.1 42.1 13.8 24.1 20.0 435.0 m. lusoria gq903339.1 42.1 13.6 23.0 21.4 435.0 m. lusoria ab853870.1 23.0 20.5 43.0 13.6 435.0 m. lusoria ab853865.1 22.3 22.1 43.0 12.6 435.0 m. meretrix gq463598.1 42.8 13.1 23.0 21.1 435.0 m. meretrix hm124578.1 41.6 14.3 22.3 21.8 435.0 m. meretrix jn043623.1 42.1 13.8 24.1 20.0 435.0 m. petechialis eu145977.1 42.8 13.1 23.0 21.1 435.0 m. petechialis hm124582.1 41.8 13.8 23.7 20.7 435.0 m. petechialis hq703177.1 42.3 13.3 23.7 20.7 435.0 m. lamarckii gu071281.1 43.7 13.8 19.8 22.8 435.0 m. lamarckii hm124579.1 43.7 13.6 20.7 22.1 435.0 m. lamarckii ab059420.1 42.3 13.8 22.1 21.8 435.0 m. casta jq773441.1 42.3 14.7 23.2 19.8 435.0 mercenaria mercenaria hm884239.1 40.8 14.7 21.1 23.4 436.0 paphia gallus hq703232.1 44.4 12.0 23.0 20.7 435.0 average 37.9 15.4 27.4 19.4 435.0 phylogenetic analysis a discrete gamma distribution was used to model evolutionary rate differences among sites (5 categories (+g, parameter = 2.0104)). maximum likelihood (ml) analysis conducted using the tamura 3-parameter model has constructed five major clades on meretrix spp. in the present study with designed out-groups mercenaria mercenaria and paphia gallus. these major clades were closely related to each other at the 50% evidence of bootstrap, which grouped as meretrix genus (fig. 3). two monophyly groups were formed, represented by clade 3 and 4, while clade 1, 2, and 5 were formed, paraphyly groups. meretrix spp. from the sarawak locality was clustered in clade 1 alongside two m. lusoria of japan locality with accession sequence number ab853865.1 and ab853870.1. clade 2 comprised two m. meretrix from the china locality with accession sequence number hm124578.1 and jn898944.1. clade 3 consisted of m. lyrata from the china locality with accession sequence number kc832317.1, hm124580.1, jn898944.1, and m. casta from the india locality with accession sequence jq773441.1. meretrix lamarckii from the japan locality (ab059420.1) was clustered with m. lamarckii from the china locality (gu071281.1; hm124579.1) in clade 4. all m. petechialis (hm124582.1, hq703177.1, eu145977.1) from the china locality were clustered with m. meretrix (gq463598.1) and m. lusoria (eu145977.1) from the china locality, respectively, in clade 5. phylogenetic analysis on three meretrix from the sarawak locality is closely related to the m. lusoria from the japan locality. however, the result contrasts with m. lyrata from blast analysis which inferred a high similarity sequence with m. lyrata accession sequence. the application of ml analysis for the phylogenetic tree was calculated using the substitution rate in the sample sequence and accession sequence, which produced different inferences than blast analysis (guindon & gascuel 2003). furthermore, meretrix spp. from the sarawak locality and m. lusoria from the japan locality had a high percentage of cytosine and adenine than that of other meretrix spp. from different localities. meretrix spp. from the sarawak locality only had a relationship with other meretrix spp. localities at more than 50% of bootstrap scoring, which is inferred as under meretrix genus. inconsistent cladistic group between m. meretrix (gq463598.1) m. lusoria (gq903339.1), m. casta (jq773441.1), and m. petechialis (hm124582.1, hq703177.1, eu145977.1) from the present study indicated that the three species had a close relationship following wu et al. (2014) and chen et al. (2009). hence, chen et al. (2009) suggested that m. lusoria and m. petechialis as a junior synonym to m. meretrix. biotropia vol. 29 no. 3, 2022 220 figure 3 phylogenetic tree of meretrix accession sequence inferred from the maximum likelihood analysis using tamura three-parameter model note: only bootstrap score greater than 50% are shown. the present study on meretrix spp. from the sarawak locality was able to differentiate coi sequences among m. lyrata, m. meretrix, and m. lusoria. m. lusoria was closely related to m. meretrix with strong bootstrap supporting evidence at 96% scoring. m. lyrata became the ancestor to m. lusoria and m. meretrix with strong bootstrap evidence at 100% of the scoring. meretrix spp. from the sarawak locality is closely related to the m. lusoria of the japan locality with 100% scoring on bootstrap supporting evidence to form clade 1. meretrix spp. from the sarawak locality was closely associated with other accession sequences for meretrix spp. at bootstrap supporting evidence at 50% scoring. based on ml phylogenetic analysis, all m. lyrata with accession sequence kc832317.1, jn898944.1, and hm124580.1 formed the cladistic group with strong bootstrap evidence at 99%. all m. lamarckii with accession sequence gu071281.1, ab059420.1, and hm124579.1 also formed cladistic at 94% of bootstrap evidence. m. casta (jq773441.1) formed an individual clade which indicated this species distance from other species except for m. lyrata. other meretrix spp. accession sequence, such as m. meretrix (gq463598.1) m. lusoria (gq903339.1), and m. petechialis (hm124582.1, hq703177.1, eu145977.1) formed inconsistent cladistic groups, while m. meretrix with accession sequence hm124578.1 and jn043623.1 formed individual clade apart from other m. meretrix (gq463598.1) at 99% evidence of bootstrap scoring. mitochondria dna is known to have higher transition mutation rates, particularly synonymous sites in the nucleotide sequence that affected the rates of evolution in organisms (brown et al. 1982; caterino et al. 2000; overton & rhoads 2004). this fast rate of evolution is one of the features suitable for phylogenetic study, particularly in higher taxa. moreover, higher taxa less affected by the recombination (guo et al. 2006), uniparental inheritance (passamonti et al. 2003; passamonti & plazzi 2020), heteroplasmy with paternal leakage (bromham et al. 2003; wolff et al. 2013; mastrantonio et al. 2019), and selective sweeps (ballard & rand 2005; james et al. 2016; hill 2019). despite proper application on higher taxa, this is the contrast when nucleotide sequences at the species level take into account those which are more defected. the evolution rate is influenced by thermal adaptation, mitochondrial, nuclear interaction, and infection with wolbachia spp. (ballard & rand 2005). hence, meretrix spp. from the sarawak locality possibly has a different evolution rate in mtdna, which caused divergence from the meretrix spp. from other localities, particularly from the china regions. molecular approach establishes relationship among venus clams (meretrix spp.) community – idris et al. 221 the current phylogenetic analysis is difficult to verify that species meretrix spp. belong to the sarawak locality. bivalves had high variability in size and gene group of mtdna, which vary extensively between species from a similar genus (xu et al. 2012). meretrix spp. from the sarawak locality are separated in terms of geographical distance. the geographical distance affects the mtdna feature as described by mishmar et al. (2003) that maternal inheritance can be restricted by the geographical distance with no mixing of mtdna haplogroups from the different geological region. therefore, other environmental conditions in each region influence the organisms' mutation rate (massey & buckling 2002). ambiguous meretrix spp. identification from the sarawak locality might be possibly related to other several features, such as sample used. the mtdna marker feature is the maternal inheritance which supplies the missing information in nuclear markers (okumuú & çiftci 2003). therefore, the female individual's tissue selection is crucial during the genetic study to ensure reliable information regarding the species ancestor. however, female selections for the genetic research have not been mentioned in each previous study for the genbank accession sequence, causing the doubtness of the reliability of accession sequence in the genbank, leading to the divergence of meretrix spp. from the sarawak locality compared to the previous species sequence. moreover, the accession sequence is well doubted due to the non-intensive morphological identification for each species which only depending on the rough observation. the present study suggested that appliance on a single marker is insufficient to support species identification and species-level phylogenetic analysis. therefore, additional variance types of molecular markers such as allozyme and nuclear markers are required to create substantial evidence on species verification to ensure the intensive morphological study's genetic approachability. depending on the genetic approach, the lack of intensive morphological evidence would create ambiguous species naming in taxonomy. conclusion the molecular approach application was able to distinguish three meretrix spp. from sarawak province, malaysia. moreover, m. lyrata is inferred to as the ancestor of m. meretrix and m. lusoria from sarawak. regardless of ancestor identification and species distinctions, the coi gene in the present study was unable to verify which species meretrix spp. belonged to the sarawak locality. several disadvantages to the coi sequence that influence the phylogenetic analysis involve the consistent accession sequence from genbank as the main reference for the current study. the present study suggested that additional dna markers for species identification and phylogenetic analysis are recommended in supporting the intensive morphological identification. acknowledgments the research team would like to acknowledge the ministry of higher education malaysia f.r.g.s. research grant code, frgs/1/2018/ wab13/upm/02/2, and the department of animal science and fishery, universiti putra malaysia, bintulu sarawak campus, for the technical support. references al-asif a, hamli h, abu hena mk, idris mh, gerusu gj, ismail jb, karim nu. 2020. benthic macrofaunal assemblage in seagrass-mangrove complex and adjacent ecosystems of punang-sari estuary, lawas, sarawak, malaysia. 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(mollusca: veneridae) in the western pacific ocean. pacific sci 62(3): 385-94. preliminary study of herbal topical lotion repellent made of betel leaves ( ) and piper betle patchouli oil ( ) mixture against pogostemon cablin yellow fever mosquito ( )aedes aegypti mutiara widawati , and m. umar riandi1* 1 1ciamis unit of vector borne diseases research, national institute of health research and development, indonesian inistry of healthm , pangandaran, ciamis, indonesia r 4eceived 25 february 2014/accepted 7 december 201 abstract exploration of plants as natural repellent against yellow fever mosquito ( ) that transmits dengue virus aedes aegypti to human is still under way betel leaves ( ) could be used as repellent material. the study was aimed to test the . piper betle repellency and safety etel and patchouli oil , potency, of topical lotion repellent of b mixture against yellow fever mosquito. this study used nulliparous of 3-5 days old female yellow fever mosquito. irritation test before the study, was conducted as . five treatments with five replications was implemented to seek the most effective safety test repellent substance i.e. deet lotion for positive control, betel leaves and patchouli oil mixture lotion, betel leaves only lotion, patchouli oil only lotion and base lotion only. the substance effectiveness was determined based on the rejection of yellow fever mosquito to bite human's arms and was analyzed using protective percentage. the irritation test showed no safety concerns. protective percentage analysis showed that modified betel leaves lotion had mean protective power of over 90% for 6 hours the modified lotions had the same protective . the data showed that percentage as the deet (ratio: pp (betel+patchouli)/pp (deet) =0.98; 95% confidence interval 0.93, 1.04; -p value=0.50). this indicated that betel leaves mixed with patchouli oil is potential to be used as safe repellent against yellow fever mosquito. keywords: aedes aegypti, betel leaves, dhf, formulations, lotion, repellent, yellow fever mosquito introduction utilization of synthetic insecticide can cause a resistance of the exposed mosquitoes towards the insecticide compounds when used continuously . insecticides commonly used are available in spray, burnt, electric and lotion forms. spray, burnt, and electric insecticides have dangerous effects to health through inhalation which vapor may easily enter the respiratory system and may even enter the blood stream. various effects including nerve, liver, and respiratory disorders may happen. long-term use may cause cancer (zaim & guillet 2002). lotion repellent is one of alternative insecticides that do not harm the respiratory system because it is directly applied to the skin. the lotion is used to discourage mosquitoes from biting human's skin. generally, the marketed lotion contains active chemical substance called diethyl toluamide (deet). this substance is only allowed in indonesia at the concentration of 15%. deet has negative effects i.e. skin irritation, hives (urticaria), and even brain malfunction (encephalopathy) the use of essential oils as . direct repellents is less effective because of its volatile nature. therefore, alternative repellent form may be developed to lengthen the repellent's durability. in this study, essential oil used as fixative agent in developing mosquito repellent lotion was patchouli oil extracted from patchouli (pogostemon cablin) . lotion is a clear preparation that does not leave stain on its user. in addition, lotion is a liquid form, thus facilitating the active ingredient inside the lotion to be evenly absorbed through the skin providing protection to its user. adding fixative biotropia vol. 22 no. 1, 2015: 45 51 doi: 10.11598/btb.2015.22.1.378 45 * corresponding author : mutiara_61@yahoo.com mailto:mutiara_61@yahoo.com biotropia vol. 22 no. 1, 2015 46 agent to repellent substance increases the potency of repellent lotion to function as long as deet lotion. patchouli oil has 100% protection against yellow fever mosquito ( )a aegypti . for 2 hours under betel leaves ( laboratory condition. piper betle) are among therapeutic plants growing in indonesia. the leaves contain saponin, phenolic, alkaloid, and substance that can be used as mosquito repellent . eugenol is essential oil extracted from clove leaves proven to control mosquito larvae and adult mosquitoes or repel mosquitoes. betel leaves contain lower concentration of eugenol (30%) compared to eugenol concentration in clove leaves (71%). the objective of this study was to test the repellency, potency of topical and safety lotion repellent of betel and patchouli oil mixture against yellow fever mosquito. materials and methods rearing nulliparous mosquitoes colonies of yellow fever mosquito ( ) a. aegypti were reared in the insectarium at laboratory of health research, ciamis unit of vector borne diseases research, national institute of health research and development, indonesian inistry m of health at pangandaran, ciamis, indonesia. the hatched larvae were held in plastic trays and larval diet was added to each tray. newly emerged pupae were transferred to screen cage (size 30x30x30 cm ) and emerged as adult mosquitoes. 3 these adult mosquitoes were kept in the insectarium with ambient temperature (25-30 c) o and were provided with soaked cotton balls containing 5% multivitamin solution. production of betel+patchouli mixture lotion the first lotion base solution was produced by weighing carbopol ultrez 10, adding it to 3 g of 10 ml dispersedwater until it . stir the solution completely until there were no trapped air bubbles. finally, add trietanolamine into the 0.50 g solution. this was called the first lotion base solution. the next step was adding lycerine 2 g of g to the first lotion base while being stirred solution to achieve homogenous substance. then, the remaining trietanolamine was added. 0.25 g of this was called the second lotion base solution. the next step was diluting methyl paraben 0.10 g in water which was then added to the 10 ml of second lotion base solution and stirred to achieve homogenous mixture. this was called the final lotion base solution. afterwards, betel oil 2 ml of in 95% ethanol 0.2 ml was mixed with patchouli oil and then was added to the final in 95% ethanol lotion base solution while being stirred until the solution became homogenous to obtain the formula of anti-mosquito lotion with optimal concentration. after all of ingredients were homogenously mixed, ml distilled water was 80 added to the mixture subsequently, ph was . measured and verified at the range of 4-7. test on human subject for lotion safety tests, 20 adult male and female volunteers 25-45 years with age range of old weight 50-70 kg and body range of were recruited for the efficacy tests, other f. ive adult male volunteers with age range of 25-45 years old and body weight range of 50-70 kg who had no history of allergy to arthropod bites were recruited. before signing an informed consent form, the volunteers were interviewed and explain about the methodology, probable ed discomforts and remedial arrangements. testing the biological stability s and afety lotions were stored in closed vials for up to six months and stability of the fraction was determined in conditions at varied room temperatures. testing was lotion safety conducted on 20 volunteers. their skin was applied with 4 treatments for 4 days, one treatment each day. s (betel + the treatment were deet, modified patchouli mixture) betel lotion, lotion (2% betel mixed with lotion), patchouli lotion (0.2% patchouli mixed with lotion). lotion was applied and the reaction was observed for 6 hours after application. laboratory repellent bioassay the lotions were tested for repellency against yellow fever mosquito ( ) by a. aegypti following indonesian pesticide commission guidelines under laboratory conditions. six hundred 3 to 5 days old female nulliparrous mosquitoes were taken from a stock cage using an preliminary study of herbal topical lotion repellent made of betel leaves ( ) and mutiara widawatipiper betle – et al. aspirator and placed inside six cages. each cage had a length of 50 cm, width 35 cm and height 40 cm and was made of nylon netting, iron wire framed, with 2 holes on the front to insert hands. each cage consisted of 100 mosquitoes. the night before being exposed to the blood meal they were deprived of the multivitamin solution and supplied with water pads only. six hours prior to the blood feeding, the water soaked cotton pads were removed. he assays were done in a well-t ventilated room that is equipped with 6 tables and 6 chairs, with temperature of 26-30 c and relative o humidity around 60-80%. thirty minutes before starting the test, the left forearm of a human subject was treated with repellent there were 5 test repellents i.e. lotion . base, deet, betel+patchouli mixture, betel 2%, and patchouli 0.2%. each repellent was tested on one subject. one gram of repellent was equally applied on 650 cm of skin surface area2 between the left elbow and wrist. the area between the elbow and wrist served as negative control by applying water on the skin surface. a latex glove was worn over the hand to protect from mosquito bites. tests were conducted by placing the repellent-treated (left) forearm into a test cage for 5 minutes, and any mosquitoes that had taken a blood meal were replaced. the control (right) forearm was placed into the test cage for 5 minutes, immediately after placing the repellenttreated (left) forearm into the test cage. these tests were conducted at 60 minutes intervals for 6 hours starting from the moment when the treatment applied (hour 0: 08.00 am) was , and the number of landing mosquitoes was recorded. landing mosquitoes were blown from the arm, in an effort to stop any blood from being taken. repellency data were as protective presented percentage (pp). the whole procedure was repeated same volunteers n on the i 5 separate days. during testing, body temperature of the human as well as ambient temperature, subject, humidity and lighting were checked. each test repellent was tested on 35 separate occasions (each hour for hours, repeated in 5 0-6 days). for each test repellent, the protection percentage at each of the 35 occasions was calculated using the following formula: where: c = the number of mosquito landings on the control (right) forearm t = the number of mosquito landing on the repellent-treated (left) forearm statistical analysis to calculate confidence intervals for the pp for each hour, the percentile of confidence interval were set on a parametric bootstrap with 10,000 replications. it was assumed that the number of landings on each arm is in accordance with poisson distribution with mean determined by the sample mean from the 5 days. there was no over dispersed data in the data distribution. the pp for different test repellents was calculated using a ratio. the 95% confidence interval was determined using the percentile parametric bootstrap with 10,000 replications using similar assumptions as with the pp confidence intervals. results and discussion safety and preference subjectbased on human safety test, 10 s thought betel+patchouli did not that the lotions give any effect , 6 s s subject thought that the betel+patchouli lotion was good subject, and 4 s thought that the betel+patchouli lotion was very good. deet and lotion did not betel+patchouli show irritation . n atchouli lotion test, 1 any s i the p out of 20 skin rash subjects experienced . in the betel subjects lotion test, 2 out of 20 showed skin ras 2h (table ). efficacy experiment subjects had body temperature d range between c environmental temperature 36-36.5 , 0 was 27-28 was 89-95%, and lighting c, humidity0 was 12-87 lux. environment the conditions were considered as confounding variables because they might tamper with the condition of the mosquitoes and human subjects, and it was hard to control. efficacy experiment showed that human subjects applied with deet and lotion had stable body temperature during experiment. 47 pp = 100 × c t c according to the results, deet and modified betel lotion had a protection n estimated percentage of more than 90% from yellow fever mosquito ( ) for the 6-hour duration. a. aegypti although the number of mosquito landings decreased quite drastically during the fourth hour, since a similar finding occurred in the control group, it did not clearly influence the magnitude of protection. during the fourth hour, when the test performed after 12:00, the activity of the mosquitoes started to decrease. this might be explained by the cycle of mosquito's life, where their resting period lied after 12:00. thus, the mosquito perching started to decrease (chadee 2013). betel leave contains various chemical compounds, including saphonine, phenolic, alkaloids, and substance which can be used as repellents . betel leave (paluch . 2010)et al commonly contains 30% eugenol (bhalerao . et al 2013) which has been proven for controlling biotropia vol. 22 no. 1, 2015 48 table 1 overall impression of lotion from 20 s. betel+patchouli mixture subject subject overall impression not good regular good very good 1 + 2 + 3 + 4 + 5 + 6 + 7 + 8 + 9 + 10 + 11 + 12 + 13 + 14 + 15 + 16 + 17 + 18 + 19 + 20 + table . number of adverse events by treatment out of 20 subjects tested (with percent).2 treatment skin rash itch swollen no adverse events deet 0 0 0 20 (100%) modified (betel+patchouli mixture) lotion 0 0 0 20 (100%) betel lotion 2 (10%) 0 0 18 (90%) patchouli lotion 1 (5%) 0 0 19 (95%) 49 larvae and adult mosquitoes (eliningaya 2008). other study also reported that eugenol derived from clove leaves (70-93%) has a potential to repel mosquitoes. although the composition of betel leaves were not as eugenol-rich as clove leaves, it also has the potential to repel yellow fever mosquito ( ) after some modifications.a. aegypti betel lotion mosquitoes repellent produced in this study is targeting and antennae of palpi mosquitoes because these two structures on mosquito body are most sensitive to the aroma of modified lotions. the aroma of plant extracts could cover the scent of the human body and thus impairing the mosquito's ability to detect the presence of humans (stella et al. 2010) . based on the safety test, this preliminary test showed that our modified repellent potentially safe to be is used in repellency test. the control arms were comparable landing between the 4 subjects who got the test repellents, while the control arm of subject who got the base only had substantially fewer mosquito landings. figure 1. number of mosquito landings for the control arm (dotted lines) and the repellent-treated arm (solid lines) for each of the 5 days (different colors) preliminary study of herbal topical lotion repellent made of betel leaves ( ) mutiara widawatipiper betle – et al. biotropia vol. 22 no. 1, 2015 50 the mean protection percentage (pp) for each test repellent was presented with 95% confidence intervals. mixture of betel+patchouli lotion performed very similarly to deet at every hour, while the betel 2% only matched the deet until hour 3, by hour 4 its pp dropped substantially. at 6 hours, deet and betel+patchouli had pp around 90%. deet had pp = 92.2% (95% ci 88.1; 95.2), while betel+patchouli had pp = 90.4% (95% ci 86.7; 93.4). both deet ( = 0.29) and p betel+patchouli ( = 0.85) were not significantly p different from 90%. the “base only lotion” provided no protection, and the patchouli 0.2% provided poorer protection compared to other active repellents. the betel+patchouli had about the same protection power as the deet (ratio: pp (betel+patchouli)/pp (deet) = 0.98; 95% confidence interval 0.93; 1.04; -value = 0.50), p while betel 2% alone had significantly less protection (ratio: pp (betel)/pp (deet) = 0.78; 95% ci 0.71; 0.86; < 0.001).p ata analysis was expected to be the d uninterrupted for the by environmental aspect. comparison of pp between different test repellents, each test repellent was tested on only one subject. this is a limitation of the design. however, each subject was acting as his or her own control o systematic biases on the landing , s counts (e.g. caused by personal odor of a subject) might affect both the control and the repellenttreated arm. these biases might cancel out when pp was used. moreover, the systematic bias caused by always starting the test with the control arm at each hour. for comparing pp for different test repellents, both test repellents will have that same bias, and it may be canceled out in the comparison. deet performed well. he lotion t modified could be considered as a repellent due to potential its 90% protective power for hours and lack of 6 safety concerns. although deet protective power was high, betel lotion might work as an alternative for deet. the main problem of natural repellent is durability. several studies have shown that natural ingredients rarely fulfill the requirements of repellent effectiveness. however, the modification of formulation by adding patchouli oil as a fixative increased the potential of natural ingredients to be used as repellent. conclusions modified betel (betel+patchouli mixture) lotion had shown repellent potential for yellow fever mosquito ( ) mosquitoes with aedes aegypti estimated mean protection power of 90. % 6 4 at hours. this study has opened up the possibility for further research to assess the effects of this repellent formulation, and modifications of other natural repellent plants. figure 2. mean protection power with 95% confidence intervals (some cis were hidden, since they were not as large as the mean symbol). the points were slightly shifted at each hour so that the different test repellent points do not overlap 51 acknowledgements authors would like to thank to michael fay for statistical assistance. the author would also like to thank to infectious disease ina responds group mentors, ibu emil, ibu inge, and mr. joshi for their suggestions, member of loka litbang ciamis for their contribution in this research assays and ina respond for their assistance s in making manuscript writing workshop. this research was funded by balitbangkes as one of risbinkes project. references bhalerao sa, verma dr, gavankar rv, teli mc, rane yy, drawana vs, tri kanned a. 2013. phytochemistry, pharmacological profile and therapeutic uses of piper betle linn. an overview. j pharmacogn phytochem 1(2): 10 9.bhuiyan mni. 2012. constituents of the essential oil from leaves and buds of clove ( (l.) syzigium caryophyllatum alston). ajpp 6(16): 1260-3. badan pengawas obat dan makanan. 1985. ormularium f kosmetik indonesia. jakarta. chadee dd. 2013. resting behaviour of in aedes aegypti trinidad: with evidence for the re-introduction of indoor residual spraying (irs) for dengue control. parasites & vectors[internet].[cited 2014 01 15]; 6(1): 255. available at:http://www.pubmedcentral. nih.gov/articlerender.fcgi?artid=3847653&tool=p mcentrez&rendertype=abstract. eliningaya njk. 2008. etnobotanical study of some of mosquito repellent plants in north eastern tanzania . malar j 7:152. hemingway j. . 2004. hawkes mj, mc carrao l, ranson h the molecular basis of insecticide resistance in mosquitoes. j insect biochem molec biol 34(7): 653 65.kardinan a. 2010. potensi adas ( ) sebagai foeniculum vulgare bahan aktif lotion anti nyamuk demam berdarah ( ). bul littro 21(1): 61 8aedes aegypti . komisi pestisida departemen pertanian. 1995. metoda standar pengujian efikasi pestisida. jakarta. koreng g, matsui d, bailey b. 2003. deet based insect repellents safety implications for children, pregnant and lactating women. can med assoc j 169: 209 12.loka litbang p2b2 ciamis. 2010. standard operational procedure rearing nyamuk. ciamis. paluch g, bartholomay l, coats j. 2010. mosquito repellents: a review of chemical structure diversity and olfaction. pest management science 66(9): -925 35. stella l, olivero-verbel j, stashenko e. 2010. repellent activity of essential oils: a review. bioresour technol j [internet]. [cited 2013 12 26]; 101(1): 372 78. available at: http://dx.doi.org/10.1016/ j.biortech.2009.07.048. tawatsin a, thavara u, chansang u, cahavittumrung p, thidarat b, wongsin kongman p, bansidhi j, mulla ms. 2006. field evaluation of deet, repel care, and three plant based essential oil repellents against mosquitoes, black flies (diptera: simuliidae) and land leeches (arhynchobdellida: haemadipsidae) in thailand. j am mosq control assoc 22(2): 30613. trongtokit y, rongsriyam y, kolamisran m, apiwath nasurn c. 2005. comparative repellency of 38 essential oils against mosquito bites. phytother res 19(4): 3039. zaim m, guillet p. 2002. alternative insecticides: an urgent need. trends parasitol 18(4): 1613. preliminary study of herbal topical lotion repellent made of betel leaves ( ) mutiara widawatipiper betle – et al. http://www.pubmedcentral. http://dx.doi.org/10.1016/ microsoft word 11 biotropia vol. 13 no. 1, 2006 : 11 21 seedbank and seedling emergence characteristics of weeds in ricefield soils of the muda granary area in north-west peninsular malaysia mahfuza begum''*, abdul shukor juraimi'', syed omar bin syed rastan", rajan amartalingam^ and azmi bin man2) " faculty of agriculture, university putra malaysia, 43400 sci'dang. malaysia l, pulau pinang, malaysia abstract tlie experiment was conducted in the glasshouse of upm from march 2003 to june 2004 to determine the soil seedbank in the ricefields ot'muda rice granary area in peninsular malaysia. six soil cores of 5 cm in diameter and 10 cm depth were sampled from each of 24 fields. all samples from each individual field were bulked and placed in plastic trays of 38 x 25 x 10 cm. soil was moistened as required and emergence of weed seedlings were recorded over period of one year. after one year, remaining seeds were separated, removed and identified. the total seed bank was estimated at 1136.48 million/ha of which 62.35% (708.60 million seedlings ha"1) germinated within 12 months and 37.65% (427.88 million seeds ha"1) remained ungerminated. total of 20 taxa were recognized. based on importance value (i.v.) the five most dominant species in terms of emerged seedling were fimbristylis miliacea, leplochloa chinensis, litjwigia hyssopifolia, cyperus difformii and c. iria. of the remaining seeds the five dominant species with decreasing trend in ranking were f. miliacea, scirpus lateriflonis, monochoria vagina/is, l. hyssopifolia and l. chinensis. ranking of total seed reserves (seedlings+ remaining seeds) were similar to emerged seedling indicating that emerged seedlings reflect the actual weed flora in the muda area. among the dominant species f. miliacea accounted for 58.07% of emerged seedlings, 79.31% of remaining seeds and 66.07% of total seed bank. total seedling emergence of all species was higher in the first observation in april 2003 and cumulative seedling emergence showed no clear peaks. key words : seedbank, seedling emergence, weeds, ricefield soils, malaysia introduction the weed seedbank comprising viable seeds either on the surface or in the soil, is the principal source of annual weed infestations in field crops. the seedbank consists of new seeds recently shed by weeds and older seeds that have persisted in the soil for several years. changes in agricultural management practices alter the pattern of disturbance and produce changes in seedbank characteristics. changes in these seedbank characteristics often lead to changes in the size and species composition of the weed flora (roberts and neilson 1981; wrucke and arnold 1985; cardinal/. 1991; clementsetal. 1996). * corresponding author: mafupaz@hotmail.com 11 biotropia vol. 13 no. 1, 2006 in muda rice granary, the change in cultural practices and use of agrochemicals had led to a shift in species from broad-leaved sedge dominance weed flora to grasses (azmi et al. 1995). the building up of certain weed species with continuous use of particular herbicides may be due to inherent resistance to that herbicide or the continuous absorption of the herbicide at sub-lethal concentrations leading to gradual development of herbicide resistance. sometimes, elimination of the competitors of a particular weed favours the abundance and predominance of the weed in that particular environment (azmi and baki 1995; ho 1998). these weeds produce abundant viable seeds and generally the vast majority of seeds entering the seedbank come from such annual weeds growing on the land (roberts 1981; hume and archibold 1986), and would thus represent the potential weed flora (kott 1947; rahman et al. 1995). therefore, knowledge of the dynamics of the seedbank is necessary to determine whether weed seed population has changed and the rate of change. species composition of the flora may be sometimes more important than total number of seeds (roberts and ricketts 1979). however, weed seed populations in cultivated soils are generally composed of a few dominant species that are present in high numbers, a few others present at moderate levels, and a large variety of species present in the soil at low levels (vengris 1953; wilson and furrer 1996). knowledge of the size and species composition of this seedbank would be useful in predicting future weed infestations (carretero 1977). the fate of seeds in the soil is difficult to determine and comprehensive information on the seed pool dynamics of most species is sparse. high weed seed populations occur in tropical soils, but limited data is available on emergence patterns (zimdahl et al. 1988). most weeds show some periodicity of emergence (roberts and margaret 1980). practical knowledge of periodicity of germination is of significant importance, since it is a major factor in determining the association of weeds with cropping systems and to enable a degree of forecasting as to which weed species may occur in a seedbed. predicting potential weed emergence is fundamental in the development of integrated pest management strategies for weed control. predictions of emerged seedling densities allow estimations of weed competition, crop yield loss, need for herbicides, financial returns and weed seed production at the end of the growing season (forcella 1992). furthermore, the remaining seeds in the soil are also a major concern to understand the soil seedbank status (cardina and sparrow 1996). this study was conducted to determine the germinable seedbank, total seed reserve, species composition of the entire germinable seedbank, ranking of species and their emergence pattern in the soils of the muda rice granary area. materials and methods sample sites were located in northern parts of muda ricefields at kedah in the north-west of peninsular malaysia. soil samples were obtained from 24 fields in march 2003. six soil cores of 5 cm in diameter and 10 cm depth were sampled from 12 seedbank and seedling emergence characteristic of weeds m. begum etal. each field in a w shaped pattern. samples from each individual field were bulked and air-dried in the glasshouse. subsequently, the soil samples were passed through a 4-mm sieve to remove large debris and break up soil pods. samples from each of the 24 fields were placed in 38 x 25 x 10 cm plastic trays. each plastic tray was filled with 2.0 kg of soil. the samples were daily sprinkled with water as needed in order to keep them moist. weed seedlings that emerged were identified, counted, and removed at one-month intervals, throughout the one-year germination period. seedlings were identified using the seedling keys of chancellor (1966). weedy rice seedlings were excluded in this study because of the obvious difficulty of identifying between weedy rice and off-type rice. seedlings of questionable identity were transferred to pots and grown until maturity to facilitate identification. after the removal of each batch of seedlings, soils were air dried for 3 days, thoroughly mixed in order to expose the weed seeds to the upper layer of the soil, and rewetted to permit further germination. this process was repeated 12 times from march 2003 to february 2004. after one year any remaining seeds were separated by the method described by wilson et al. (1985). the soil in each tray was passed through a descending series of five sieves containing screens of the following sizes: 4 mm (5 mesh), 2 mm (10 mesh), 850 jim (20 mesh), 425 urn (40 mesh), and 250 urn (60 mesh). water was run through the sieves to enhance sample separation through the screens. the contents collected in each screen were removed, sun dried, and seeds were removed under a luminated magnifier. seeds from entire samples were sorted using a dissecting microscope and counted according to species. the seed counts were expressed in numbers per m2. the total of emerged seedling during one year period and the remaining seeds represent the total species composition and seed reserve of respective species. seed and seedling counts were converted to numbers per m2 within 10 cm soil depth. results and discussion seedbank density total seed reserve in the muda area was 113648 seeds m"2 equivalent to 1136.48 million ha"1, of which 70860 seeds m"2 germinated within 12 months and remaining seeds were 42 788 m"2 (table 1). the total number of buried seeds as well as germinable seeds reported here are higher compared with 29 551 viable seeds m"2 reported by pane (1997) and much lower than densities of 712228 to 930910 13 biotropia vol. 13 no. i, 2006 seeds m"2 recorded by ismail et al. (1995) in direct-seeded ricefields at kampung tandop in muda area. however, watanabe et al. (1997) noted that such abundance reported by the latter tar exceeds that usually observed in cropped land, including upland ricefields' which had two orders of magnitude lower. while in the philippines, vega and sierra (1970) reported more or less similar viable seeds as the present study, with 800 million seeds ha"' in ricefields within a plough depth of about 15 cm. species composition in seedbank a total of 20 taxa were recognized, of which 14 common weed species emerged from the soils collected in the muda area (table 1). based on dominance ranking, the highest population of seedlings that emerged were of f. miliacea followed by l. chinensis, c. difformis, c. iria, l. hyssopifolia, sphenoclea zeylanica, m. vaginalis, limnophila erecta, hedyotis diffusa, ceratopteris thalictroides, echinochloa crus-galli (complex), bacopa rotundifolia and eleocharis variegata and e. colona (table 1). pane (1997) reported 17 species in muda ricefields of which the higher population of seedlings emergence were l. chinensis, f. miliacea, c. difformis, s. zeylanica, l. octovulvis, m. vaginalis, c. iria, lindernia octovulvis, e. crus-galli, and sagittaria guyanensis. the dominant species were almost similar as in the present study. another seven species were rare and present in relatively low numbers. after the 12-month gennination period, the seeds remaining in soils comprised 13 weed species (table 1). of these weed species seven, namely f. miliacea, l. chinensis, c. difformis, c. iria, l. hyssopifolia, m. vaginalis and e. crus-galli (complex) had been recorded as seedlings during the 12month period, while seeds of 6 weed species, previously not found as seedlings, were from scirpus lateriflorus, s. juncoides, s. guyanensis, nymphoides indica, najas graminea and cleome viscosa. the non-emergence of these species may be due to the very low population of these species combined with low germination rate. ismail et al. (1995) had observed that although seeds of s. juncoides were the dominant species in volunteer seedling fields in the muda area, but its summed dominance ratio values of emerging seedlings was very small, which also indicate its low germination rate. seeds of s. lateriflorus, s. juncoides, s. guyanensis can persist in the dormant state in the soil and dormancy may be broken through straw burn and tillage (azmi 2005; pers. comm.). no remaining seeds were detected for seven weed species which had germinated during 12 months period, namely s. zeylanica, c. thalictroides, l. erecta, h. diffusa, e. variegata, b. rotundifolia and e. colona (table 1). ismail et al. (1995) also observed that although c. thalictroides, hedyotis sp., bacopa sp. emerged in direct seeded ricefields in muda, no seeds of these species was found in soil seed reserves. based on total seed reserve, the species composition in descending order was f. miliacea, l. chinensis, l. hyssopifolia, c. difformis, c. iria, m. vaginalis, s. zeylanica, s. lateriflorus, l. erecta, e. crus-galli (complex), c. thalictroides, h. diffusa, s. juncoides, s. guyanensis, b. rotundifolia, n. indica, e. variegata, n. graminea, e. colona and c. viscosa (table 1). however, 14 seeclbank and seedling emergence characteristic of weeds m. begum el al. table 1. germinable soil seedbank, remaining seed and total seed reserves in the muda rice granary area weed species seedbank germinable within 12 months (no.m"2) remaining seed reserves (no.m"2) total seed reserve (germinable seeds + remaining seeds) f. miliacea 41148 33936 75084 l. chiiienxis 7655 989 8644 c. difformis 4947 42 4989 c. iria 4530 428 4958 l. hyssopifolia 3633 1561 5194 s. zeylanica 2402 2402 m. vaginali.i 1481 1598 3079 l. erecta 1292 1292 h. diffusa 1129 1129 c. thalictroides 1045 1045 e. crus-galli 670 477 1147 b. mtundifolia 644 ' 644 e. variegata 227 227 e. colona 57 57 s. laterifloms 1613 1613 s.juncoides 943 943 s. guyanensis 742 742 n. indica 235 235 n. gramineu 201 201 c. viscosa 23 23 total 70860 42788 113648 due to the limited size of soil samples analyzed for seeds, only seeds of most predominant species were expected to be detected with consistency (forcella et al. 1992). occurrence of species frequency the frequency of occurrence of emerged seedlings and remaining seeds of individual component species in the seedbank are shown in table 2. f. miliacea, l. chinensis and l. hyssopifolia were most frequent, and found in all sampled fields (100% frequency), followed by c. difformis (95.83%), c. iria (95.83%), m. vaginalis (95.83%), s. zeylanica (91.67%), e. crus-galli (91.67%), l. erecta (83.33%), c. thalictroides (75%) and h. diffusa (58.33%) (table 2). these were found in large numbers up to 1046-41148 in"2, except e. crus-galli, which had a high frequency of occurrence but the number of seeds was 670 m"2 (table 2). watanabe et al. (1997) observed that echinochloa species were well controlled by herbicide and few adult plants, less than eight plants m"2 (mostly 0-3 plants m"2) were observed growing after rice heading. average number of spikelets/plant of e. crus-galli was 972 (watanabe et al. 1997) which was lower than other dominant species 15 biotropia vol. 13 no. 1,2006 (viz. l. chinensis, 1877 spikelets per plant). in spite of the frequency, total seed incorporation was lower than with other dominant species like l. chinensis, f, miliacea, l. hyssopifolia, c. iria, c. difformis etc. in the case of remaining seeds, the most frequent species were m. vaginalis (87.5%), f. miliacea (83.33%), l. hyssopifolia (83.33%), 5. giiyanensis (79.19%), s. lateriflorus (70.83%), e. crus-galli (complex) (58.33%) and l chinensis (54.17%). all species had large seed populations, except e. cms-galli (complex). bahtia et al. (1990) reported that seed reserves of e. crus-galli were mostly 97.7% exhausted during one season and only a small fraction (2.3%) carried over to the second season. no seed germination occurred during the third season and soil was free of viable seeds. similarly, azmi et al (1995) detected very few viable seeds of echinochloa crus-galli in the soil after six cropping seasons, suggesting seed longevity of barnyard grass in ricefields was shorter than three years. this study revealed that most frequent species appeared as a most dominant species. the percentage of occurrence of many species were high which have higher percentage viability than m vaginalis and f. miliacea (table 2). this indicates that these species are persistent in the seed bank, and hence become dominant in the above ground flora, even though there was a shift towards grassy weeds due to changes in the cultural practice from transplanting to direct seeding. dominant species in seedbank according to importance value, the five most dominant species in terms of emerged seedlings were f. miliacea (58.07%), l. chinensis (10.80%), c. difformis (6.98%), c. iria (6.39%) and l. hyssopifolia (5.13%) with another eight weed species sharing only 12.55% of the total emerged seedlings (figure la). in terms of seeds remaining in the soil after 12 months, the five most important species were f. miliacea (79.31%), s. laterifloms (3.77%), m. vaginalis (3.74 %), l. hyssopifolia (3.65%) and l. chinensis (2.31%), while other species shared only 7.22% (figure ib). the five most dominant weed species in terms of total seed reserves (seedling + remaining seeds) in the muda area were f. miliacea (66.07%), l. chinensis (7.61%), l. hyssopifolia (4.57%), c. difformis (4.39%) and c. iria (4.36%) while the rest of the species shared only 13% of the total seed reserve (figure ic). this was similar to the germinable seedlings of 12 months period, but with slight variation in ranking of species. pane (1997) reported that the predominant weed species in the muda area, with respect to emerged seedlings in the seedbank study, were l. chinensis, f. miliacea, c. difformis. s. zeylanica and l. octovulvis. the results were almost similar to the present study, but the relative percentage and ranking were slightly different. reserves of seeds in soil are typically dominated by two to four species (wilson and furrer 1996). at irri, zimdahl et al. (1988) reported that in lowland irrigated ricefields three weed species, namely c. difformis (52%), f. miliacea (26%) and m. vaginalis (18%) , covered 96% of total emerged seedlings. in this study emergence density, ungerminated seeds and total seed reserve of f. miliacea were much higher than other weeds. watanabe et al. (1996) also observed 16   note  :  fimmi‐  f. miliacea;  lepch‐  l  chinensis; cypdi‐ c. difformis; cypir‐ c.  iria  and  ludhy‐  l. hyssopifolia;  monva‐ m. vaginalis; sirla‐ s1. lateriflorus.  17  biotropia vol. 13 no. 1, 2006 higher emergence of f. miliacea seedlings in the muda area. pane (1997) reported a f. miliacea germinable seedbank of 22.9%, which was lower than this study, but was the second most dominant species after l. chinensis (26.9%), which according to ho et al. (1995) occupied 80% of above-ground weed flora. the domination of the seedbank by a single annual species, in this case f. miliacea, is not unusual. a single species often comprises over half of the soil seedbank (thompson 1986; schott and hamberg 1997; navie et al. 2004). common lambsquarters comprised more than 50% of the seedbank (clements et al. 1996). these clearly explain the huge seedbank and persistence o'f f. miliacea in the area. emergence pattern in general, weed emergence is influenced by soil disturbance, temperature, rainfall, soil moisture and radiant energy. in the present study the highest percentage of emerged seedlings was recorded in the first observation in april (20.29%). seedlings emerged with each soil disturbance at emergence rates of between 4.45-20.29. seedlings continued to emerge irrespective of the time of year, but in reduced numbers. however, periodic seedling emergence showed no clear peaks during the 12-month period (figure 2). within the first two months 32.84% of seedlings had emerged and more than 50% seedlings emerged within 5 months. watanabe et al. (1996) observed that weed seedlings emerged mostly in the first thirty to forty days after rice seeding, although l. chinensis, f. miliacea, m. vaginalis, l. hyssopifolia, and broadleaved weeds often emerged over a longer duration. zimdahl et al. (1988) observed that one third of all weed species emerged within 3 weeks of tillage, and 57% emerged within 6 weeks in upland soils at irri, whereas 38 and 51% of total emergence occurred within 3 and 6 weeks after tillage, respectively. jensen (1969) also found seedling emergence accounted for \ 18 seedbank and seedling emergence characteristic of weeds m. begum et al. only about 25% of the seeds in the soil and that most of those that did so in the first month. he found the strong correlation between immediate seedling emergence in the glasshouse and field emergence suggests only the first flush of seedling emergence need to be considered. conclusions a total 20 taxa (including seedling + remaining seeds) were found of which 14 species were germinable, while six species did not germinate during the 12-month period in this study. total seed reserve was 1136.48 million ha"' of which 708.60 million ha"' was germinable within the 12month period. previous studies had only reported the germinable seedbank in sampled soils. no information was available on dormant seeds of different species, which would reflect the actual seed reserves of the muda area. f. miliacea, l. chinensis, l. hyssopifolia, c. difformis and c. iria appeared to be the most frequent species and made up the bulk of the seed population in the soil, with more than 80% occurring as a germinable seedbank. among these important species the domination of the seedbank by a f. miliacea comprised 58.07%, 79.31% and 66.07% of germinable seedbank, ungerminated seeds and total seed reserves, respectively. the result indicates that the very large size of this seedbank is probably due to both its prolific seed production and the ability of its seeds to persist more than a year. it would be valuable to predict the size of a weed infestation before it actually occurred, and this is especially true if reliance is to be placed on pre-emergence herbicides, which must be applied before the nature and severity of the potential infestation are visible. to determine whether site-specific weed management is practical, the first criterion is to decide whether weeds (density, species) vary enough from field to field. the second step is to obtain accurate and reliable information about weed species and density on specific field. the third step matches weed management solutions with problems. acknowledgement our appreciation goes to the third world organization for women in science (twows) trieste, italy and universiti putra malaysia under the intensification of research in priority areas (irpa) (no.: 01-02-04-0778-pr0068/05-05 ) who had provided the grant of postgraduate fellowship and research facilities for this study. references azmi, m. and b.b. bald. 1995. the succession of noxious weeds in tropical asian ricefields with emphasis on malaysian rice ecosystem. in: proceeding 15* asian pacific weed science society conference, tsukuba, japan, p. 51-67. azmi, m., m. mashhor, k. itoh, and h. watanabe. 1995. life cycle and seed longevity of echinochloa crus-galli complex in direct seeded rice in malaysia. in: proceeding of 15 asian pacific weed science conference, tsukuba, japan, p. 505-511. 19 biotropia vol. 13 no. i, 2006 bhatia r.k., k..s. sandhu and t. singh. 1990. geiinination and longevity of echinochloa crus-galli l. under natural conditions. j. res. punjab agric. univ., 27 (1):17-21. carretero, /.l. 1977. estimation del contenido de semillas de malas hierbas de un suelo agricola como prediction de su flora adventicia. ann. inst. biol. caranilles, 34: 267-278. cardina, j., e. regnier and k. harrison. 1991. long-term tillage effects on seedbanks in three ohio soils. weed sci., 39: 186194. cardina, j. and d.h. sparrow. 1996. a comparison of methods to predict weed seedling populations from the soil seedbank. weed sci., 44: 46-5 1 . chancellor, r.j. 1966. the identification of weed seedlings of farm and garden. blackwell scientific publication, oxford. clements, d.r., d.l. benoit, s.d. murphy and c.j. swanton. 1996. tillage effects on weed seed return and seedbank composition. weed sci., 44:3 14-322. forcella, f. 1992. prediction of weed seedling densities from buried seed reserves. weed res., 32: 29-38. forcella, f., r.g. wilson, k..a. renner, j. dekker, r.g. harvey, d.a. aim, d.d. buhler and j. cardina. 1992. weed seedbanks of the u.s. cornbelt: magnitude, variation, emergence and application. weed sci., 40: 636-644. jensen, h.a., 1969. content of buried seeds in arable soils in denmark and its relation to the weed population. dansk botanisk arkiv., 27: 7-57. ho, n.k. 1998. the rice agro-ecosystem of the muda irrigation scheme: an overview. in: nashriah et at. (eds.) rice agrosystem of the muda irrigation scheme, malaysia. malaysian institute of nuclear technology research (mint) and mada, bangi, kajang, malaysia, p. 1-24. ho, n.k., y.m. esa, and m. abu bakar. 1995. implementation of agricultural extension programme on integrated weed management in rice: malaysia approaches and experience. rice 1pm network workshop on weed management in rice production. 19-23 june 1993. ferringhi beach hotel, penang, malaysia, p. 14. hume, l. and o.w. archibold. 1986. the influence of a weedy habitat on the seed bank of an adjacent cultivated field. canadian j. of bot., 64: 1879-1883. ismail, s., z.n. faezah and n.k. ho. 1995. weed population and their buried seeds in ricefields of the muda area, kedah, malaysia. pertanika j. trop. agric. sci., 1 8( 1 ):2 1 -28. kott, s.a. 1947. the biological properties of weedy plants and the struggle against the wecdiness of soils. ogiz-sel khozgiz. moskva. navie, s.c., f.d. panetta, r.e. mcfadyen, and s.w. adkins, 2004. germinable soil seedbanks of central queensland rangelands invaded by the exotic weed parthenium hysterophoru.i l. weed biol. and manag.,4: 154-167. pane, h., 1997. studies on ecology and biology of red sprangletop [ leptocmoa chinensis) (l.) nees] and its management in direct seeded rice. ph. d. thesis. universiti sains malaysia, pp. 41-60. rahman, a., t.k. james, n. grbavac, and j. mellsop. 1995. evaluation of two methods for enumerating the soil weed seedbank. in proceedings 4s'1' n.z plant protection conference, www.hortnet.co.nz / roberts, h.a. and m.e. ricketts. 1979. quantitative relationships between the weed flora after cultivation and the seed population in the soil. weed res., 19:269-275. roberts, h.a. and e. margaret. 1980. emergence patterns of weed seedlings in relation to cultivation and rainfall. weed res., 20:377-386. 20 seedbank and seedling emergence characteristic of weeds m. begum et al. roberts, h.a. 1981. seed banks in soils. advances appl. biol., 6:1-55. roberts, h.a. and j.e. neilson. 1981. changes in the soil seedbank of four long-term crop/herbicide experiments, i. appl. ecol., 18: 661-668. schott, g.w. and s.p. hamberg. 1997. the seed rain and seedbank of an adjacent native tallgrass prairie and old field. can. j. bot., 75:1-7. thompson, k. 1986. small-scale heterogeneity in the seedbank of an acidic grassland. j. ecol., 74 : 733-738. vega, m.r. and j.n. sierra. 1970. population of weed seeds in a lowland rice field. phillipp. agric., 54:1-7. vengris, j. 1953. weed populations as related to certain cultivated crops in the connecticut river vally, ma. weeds, 2: 125134. watanabe, h., m. azmi, and md. z. israail. 1997. emergence of major weeds and their population change in wetseeded rice fields of the muda area, peninsular malaysia. in: proceedings of 16th asian pacific weed science society conference, malaysian plant protection society, rajan, a. (ed.), kuala lumpur, pp. 246-250. wilson, r.g., e.d. derr and l.a. nelson. 1985. potential for using weed seed content in the soil to predict future weed problems. weed sci., 33:171-175. wilson, r.g. and j. furrer. 1996. 1996. where do weeds come from? university of nebraska-lincoln cooperative extension, http : // www.iam-.unl.edu /pubs /weeds / g807.htm wrucke, m.a. and w.e. arnold. 1985. weed species distribution as influenced by tillage and herbicides. weed sci., 33; 853856. zimdahl, r.l., k.. moody and r.t. lubigan. 1988. patterns of weed emergence in tropical soil. weed sci., 36: 603-608. 21 11.pdf 12.pdf 13.pdf 14.pdf 15.pdf 16.pdf 17.pdf 18.pdf 19.pdf 20.pdf 21.pdf 884-fiks.cdr 44.3 10.9 i ii iii 44.3 0.56 0.63 0.69 0.76 0.82 g1 g4 g2 g7 g10 g8 g3 g5 g9 g6 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 sri-10. 1007-layout.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 sri-13 mei 2017-507 (anjana evaluation).cdr evaluation of cytotoxicity of the aqueous leaf extract of benth.pogostemon heyneanus (java patchouli) anjana sheela* and john ernest thoppil cell and molecular biology division, department of botany, university of calicut, malappuram 673635, kerala, india received 4 july 2015/accepted 27 january 2017 abstract several plants have been recently reported to possess anticancer potential. allium cepa root tip assay is a preliminary study to assess the cytotoxic effect of plant extracts. the cytotoxic activity of plants can be correlated to their anticancer potential. cytotoxic potential of pogostemon heyneanus (lamiaceae) was evaluated using a. cepa root meristematic cells. this study was aimed at analyzing cytotoxic potential of p. heyneanus using allium cepa root tip assay. four different concentrations of the aqueous leaf extracts at three different durations were examined. distilled water was used as control. the extract was found to be cytotoxic at all tested concentrations, when compared to control. mitotic index was found to be decreasing with the increase in extract concentrations and treatment durations. the aqueous extract of p. heyneanus was found to be an effective cytotoxic agent, inducing various clastogenic and nonclastogenic aberrations such as chromosome gaps, bridges, multipolar anaphase, fragments, nuclear budding and lesions, hyperchromasia, laggards and mitotic pairing. keywords: allium cepa assay, anti-mitotic, chromosomal aberrations, cytotoxicity, pogostemon heyneanus introduction recent years have witnessed the importance of plant-derived herbal medicines against many dreadful diseases. the cytotoxic potential of plant extracts can be tested preliminarily for their anticancer activity using assay. allium cepa p. heyneanus, commonly called as java patchouli, is an aromatic species of lamiaceae family. the plant is used to make the pungent, dark amber colored patchouli oil, containing patchoulene, patchouli alcohol and eugenol as the major components. the oil possesses antibacterial activity against , , escherichia coli staphylococcus aureus streptococcus pyogenes bacterium coli bacterium typhosum, , and . it is also used in mycobacterium tuberculosis insect-repellent preparations. essential oil extracted from the leaf was reported to possess remarkable larvicidal potential against aedes albopictus (anjana & thoppil 2013). another species of the genus, has been reported to p. cablin, be having anticancer properties (jeong . 2013).et al a wide variety of secondary metabolites obtained from medicinal plants possesses the ability to treat cancer. they exhibit cytotoxic effect by interfering with cell-cycle kinetics (galimuhtasib & bakkar 2002). these medications are effective against proliferating cells. these medications produce cytotoxic effect either by damaging the cell dna during the s-phase of the cell cycle or by blocking the formation of mitotic spindle (sehgal . 2006). plant et al cytotoxic bioassays have a good correlation with mammalian test systems, validating the application of plant-based medications for genotoxic assays (jovtchev . 2002; yi & meng et al 2003; çelik & äslanturk 2006, 2007). toxicological tests using was found to allium cepa have high correlation with other test systems (mit-217 cell test with mice, rats or humans in vivo) and could be used in toxicological research (lubini . 2008).et al there are no previous reports on the cytotoxic activities of and therefore, the present p. heyneanus study was aimed at evaluating the cytotoxic and antimitotic properties of p. heyneanus. biotropia 4 1 7 28 34 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 1 507 * corresponding author: anjana.s586@gmail.com 28 materials and methods collection of plant materials plant materials of p. heyneanus was obtained from the centre of medicinal plants research, arya vaidya sala, kottakkal, kerala, india. the collected plant materials were maintained in the greenhouse and authenticated by dr a.k. pradeep, assistant professor, department of botany, university of calicut, kerala india. a voucher specimen was deposited at the calicut university herbarium (cali 123733). preparation of leaf extracts fresh leaves of p. heyneanus were collected from the plants grown in the greenhouse and were rinsed thoroughly, shade dried and then ground into powder. the crude extract was prepared by boiling the dried powdered material in distilled water for 30 minutes. the extracts were prepared in four different concentrations (0.5, 0.1, 0.05 and 0.01%). the extracts were then filtered to remove particulate matter. allium cepa root tip assay fresh and healthy bulbs of allium cepa were obtained from the local market. the bulbs were germinated over water before being transferred to the test plant extracts. when the roots were about 5 mm long, the bulbs were placed on beakers containing p. heyneanus leaf extracts of four different concentrations (0.5, 0.1, 0.05 and 0.01%), such that the roots were immersed in the extracts. the roots were immersed for 3 different durations, i.e. 2, 1 and ½ hours. the sprouted roots were also treated with distilled water, which served as control. the experimental set up had five replicates. the root tips were fixed in carnoy's fluid (1 part of glacial acetic acid: 2 parts of absolute alcohol) after the treatment. these roots were then hydrolyzed in 1n hcl for 5 minutes. after hydrolysis, the roots were washed in distilled water. root tips were cut into 1 2 mm long pieces and placed on a glass slide. a small drop of 2% aceto-orcein was placed on the root tip and the root tip was squashed. the slides were then scanned under leica dm 1000 trinocular research microscope and photomicrographs were taken. the numbers of cells, dividing and nondividing, were recorded. the degree of chromosomal aberration was expressed as the percentage of aberrant cells, which was calculated as the number of aberrant cells to the total number of cells examined. mitotic index (mi) was calculated by expressing the numbers of dividing cells as a percentage of total cells counted for each treatment and the control. numbers of aberrant cells _____________________percentage aberration = x 100 total number of cells numbers of dividing cells ____________________________mitotic index = x 100 total number of cells statistical analysis the data were analyzed using one way analysis of variance (one-way anova). means of the mitotic indices and chromosome aberrations with standard errors for each concentration of the extracts were calculated. duncan's multiple range test was performed to determine the significant differences between the different treatments made. statistical significance was accepted for p < 0.05 (auti et al. 2010). all statistical analyses were carried out using spss 17.0 statistical package. results and discussion mitosis was normal in the root tips kept as control, whereas the mitotic indices of all a. cepa roots treated with p. heyneanus leaf extracts were significantly lower than the mitotic index of control (table 1). the mitotic index rates were found to be decreasing with the increasing concentrations of the aqueous extract compared with the control (table 1). chromosome aberrations were observed in all stages of mitosis. major clastogenic abnormalities observed were nuclear budding and lesions, chromosome fragments and gaps, ring chromosomes, chromosome bridges, stickiness, hyperchromasia and non-clastogenic aberrations including stellate anaphase, ball t e l o p h a s e , e q u a t o r i a l s e p a r a t i o n o f chromosomes, chromosome lag gards, misorientation at metaphase, hyperploids and hypoploid cells, double metaphase, early ball metaphase, mitotic pairing at metaphase, cytotoxicity analysis of pogostemon heyneanus benth. – sheela and thoppil 29 biotropia vol. 24 no. 1, 2017 30 table 1 percentage of mitotic indices and chromosomal aberrations observed in allium cepa root meristems treated with p. heyneanus leaf extracts concentration mitotic index (+ se) % of aberrant cells (+ se) ½ hour 1 hour 2 hours ½ hour 1 hour 2 hours control 26.00+ 0.86a 23.14+ 0.94a 25.47 + 0.09a 0a 0a 0a 0.01% 17.11 + 3.00b 16.37 + 1.50b 16.23 + 1.39b 36.03+0.75b 41.81+0.56b 46.07+1.30b 0.05% 15.67 + 0.34b 15.10+ 0.93b 15.09 + 0.80b 39.16+1.32bc 43.64+1.82bc 48.14+0.93bc 0.1% 14.32 + 1.31b 14.30 + 1.31b 13.83 +0.50bc 39.50+1.86bc 47.98+3.07c 51.60+2.61c 0.5 % 13.39+ 0.62b 12.89+ 1.12b 12.14 + 0.52c 42.88+2.88c 48.32+1.61c 62.85+1.91d note: numbers followed by the same letters in the same column are not significantly different at p < 0.05 action on dna biosynthesis (majewska et al. 2003). nuclear buds were observed to originate from the nuclear envelope in situ at certain regions of the interphase nucleus of the treated cells and these might be a result of the excessive production of nucleic acids and proteins, induced by cytotoxicants (akaneme & iyioke 2008). stickiness (fig. 1t) might result from improper folding of chromosome fibers which made the chromatids connected by means of subchromatid bridges (hellgren & morre 1992). chromosome gaps (fig. 1d), which were distributed at random on the chromosomes, were formed by the action of mutagenic substances (mcgill et al. 1974). chromosome bridges (fig. 1b & 1t) and fragments (fig. 1c) were clastogenic effects, resulting from chromosome and chromatid breaks (fiskesjo 1997). ring chromosomes (fig. 1r) might arise due to the spontaneous breakage of chromosomal ends, followed by the joining of the raw ends of the chromosomes (singh 2003). ball metaphase (fig. 1g) might be due to the localized activity of spindle apparatus at the centre, so that the centromeres remained at the equator and arms radiated in different directions, in the form of a ball. in ball telophase (fig. 1a), the sister chromatids separated into a hollow ball of chromosomes that resulted from the early cleavage divisions in some aberrant cells (morgan 2006). separation of daughter chromosomes parallel to the equator rather than towards the poles (fig. 1h) was an acute aberrant condition that arose as multipolar anaphase, pole to pole arrangement at metaphase and vagrant chromosomes at metaphase (fig. 1). there were significant differences (p < 0.05) between treated groups and control group in mitotic index. low mitotic index observed in the treated root tips might be due to direct genotoxic effect of p. heyneanus extract. the percentage of mitotic indices and chromosomal aberrations induced by different concentrations of the extract at different treatment durations are summarized in table 1. decrease in mitotic index rates explained cytotoxicity of the aqueous extract. this might be due to the obstruction of the onset of prophase, the arrest of one or more mitotic phases, or the slowing of the rate of cell progression through mitosis (fiskesjo & levan 1993). reduction in the mitotic activity could also be due to inhibition of dna synthesis or a blocking in the g2 phase of the cell cycle, preventing the cell from entering mitosis (christopher & kapoor 1988). chromosomal aberrations are changes in chromosome structure resulting from a break or exchange of chromosomal material. most of chromosomal aberrations observed in cells are lethal, but there are many corresponding aberrations that are viable and can cause genetic effects, either somatic or inherited (sudhakar et al. 2001; akinboro & bakare 2007). aberrations observed at the interphase stage included nuclear lesions (fig. 1f) and nuclear budding (fig. 1p). the presence of nuclear lesions offers cytological evidence for the inhibitory 31 figure 1 chromosomal aberrations induced by different concentrations of aqueous leaf extracts of p. heyneanus notes: a) ball telophase; (b) chromosome bridge at anaphase; (c) chromosome fragment at metaphase; (d) chromosome gaps at metaphase; (e) double metaphase; (f) double nuclear lesion; (g) early ball metaphase; (h) equatorial separation at anaphase; (i) hyperchromasia; (j) hyperploid cell; (k) hypoploid cell; (l) laggards at anaphase; (m) misorientation at metaphase; (n) mitotic pairing at metaphase; (o) multipolar anaphase; (p) nuclear budding at interphase; (q) pole to pole arrangement at metaphase; (r) ring and vagrant chromosomes at metaphase; (s) stellate anaphase; (t) sticky double chromosome bridge at anaphase cytotoxicity analysis of pogostemon heyneanus benth. – sheela and thoppil a result of errors in the mitotic spindle assembly and dynamics (ford & correll 1992). stellate arrangement (fig. 1s) of chromosomes might be due to the clumping of daughter chromosome groups in a star like manner and might be occurring as a result of the complete disturbance of spindle (raj & rao 1972). hyperchromasia (fig. 1i) was an extremely condensed and deeply staining state of interphase nucleus induced by toxic environmental chemicals or incompatible conditions, which might be caused by progressive heterochromatinization (gernand . 2005). misorientation of et al chromosomes (fig. 1m) could be due to a distortion of the spindle apparatus or a tilt in the equator ia l organizat ion of metaphase chromosomes (selim . 1981). the occurrence et al of hyperploid (fig. 1j) cells might be attributed to spindle inhibition, lack of anaphase movement or failure of cell plate formation (meske & hartmann 1995). abnormal pole to pole orientation of chromosomes (fig. 1q) at metaphase leading to equatorial separation of chromosomes at anaphase was an acute aberrant condition that arose as a result of irregular pathways of spindle assembly and abnormal spindle activity (waters & salmon 1997). multipolar anaphases (fig. 1o) were consequences of weak c-mitosis and could lead to aneuploidy (fiskesjo 1988). the induction of vagrant chromosomes (fig. 1r) led to the separation of unequal number of chromosomes in the daughter nuclei and subsequently formation of daughter cells with unequal sized or irregularly shaped nuclei at interphase (el-ghamery . et al 2003). laggards were whole chromosomes that failed to migrate to either pole at anaphase because of possible damage to the kinetochore (asita & mokhobo 2013). presence of hypoploid cells (fig. 1k) might be due to the occurrence of multipolar mitosis or lagging chromosomes (seoana . 2000). mitotic pairing (fig. 1n) was a et al rare abnormality which might be due to an increase in rna content (wen . 1989).et al mitotic index (mi) measured the proportion of cells in the m-phase of the cell cycle. the inhibition of m-phase could be considered as cellular death or a delay in the cell proliferation kinetics (nagpal & grover 1994; ribeiro . et al 2016). the decrease in mitotic index and the presence of these chromosomal aberrations in root a. cepa tips treated with extract when p. heyneanus compared with the control might be attributed to the presence of phytochemical constituents in the crude extract of . is rich in p. heyneanus p. heyneanus volatile oils containing compounds like patchouli alcohol, nerolidol, -pinene (murugan . β etc. et al 2010). patchouli alcohol, isolated from p. cablin was found to exhibit antitumor effect against human lung cancer cell lines (lu . 2016)et al . conclusions the aqueous extract of p. heyneanus possesses a dose dependent inhibitory effect on cell division of allium cepa. further studies are required to characterize and isolate the cytotoxic agents present in p. heyneanus. acknowledgements the first author is thankful to the council for scientific and industrial research (csir), india for funding this study. references akaneme fi, iyioke iv. 2008. mutagenic potentials of the sterilizing fluid – purital on root tip mitosis of allium cepa. bio res 6:293-7. akinboro a, bakare aa. 2007. cytotoxic and genotoxic effects of aqueous extracts of five medicinal plants on allium cepa linn. j ethnopharmacol 112:470 -5. anjana s, thoppil je. 2013. chemical composition of the essential oils of four spp. and their pogostemon 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universitas nusa bangsa, bogor 16166, indonesia 2department of silviculture, faculty of forestry, institut pertanian bogor, bogor 16680, indonesia 3 department of biology, faculty of mathematics and natural sciences, institut pertanian bogor, bogor 16680, indonesia 4department of agronomy, faculty of agriculture, institut pertanian bogor, bogor 16680, indonesia received 25 october 2016/accepted 18 september 2017 abstract information on metal adaptation of plants is necessary to understand the mechanism of heavy metal, including lead (pb), remediation mediated by forest plants in contaminated land. this study aimed to find out the adaptation mechanism of jabon (anthocephalus cadamba roxb) seedlings to excessive lead level based on the tolerance index of growth performance and lead transport to plant tissue. the seedlings were exposed to lead (pb(no ) ) with the 3 2 concentrations of 0, 0.5, 1, 1.5, 5 and 10 mm in nutrient culture for 15 days. the result showed that the tolerance index (ti) of the seedlings was significantly decreased by pb exposure up to 1.5 mm, but the ti values were more than 75%. all seedlings died at pb concentration of 5 mm and up. pb accumulated in all parts of the seedlings, with the highest concentration found in the leaves (735.9 ppm) under pb concentration of 0.5 mm. the pb was found to be transported to the top portion of the seedlings indicated by transport factor (tf) that was more than 1. the results suggest that jabon can adapt to excessive pb exposure up to 1.5 mm and has the potential as a remediator plant. keywords: accumulation, jabon, lead, nutrient culture, tolerance introduction lead (pb) is considered as the second most dangerous heavy metal after arsenic and it is among the biggest pollutants in water and terrestrial ecosystems generated from a variety of activities including household and industrial works (ebrahimpour & mushrifah 2008; tangahu et al. 2011; alaribe & agamuthu 2015). in a certain concentration, pb is toxic to plants, animals, and microorganisms. when absorbed by the human body, it will produce various deleterious effects on the hematopoietic, renal, reproductive and central nervous system, mainly through increased oxidative stress (flora et al. 2012). heavy metal contamination in the soil can reduce soil fertility for long-term. high pb levels in the soil need remedial action to enable the soil to re its normal function ( . 2011). gain tangahu et al phytoremediation has become an effective, environmentally friendly and affordable technological solution through the extraction or removal inactive metals and metal pollutants of from contaminated soil (henry . 2013; et al sharma & pandey 2014). phytoremediation is based on the fact that plants have membrane active pumps that work solar energy to extract through certain heavy metals from the environment to proceed with the translocation, bioaccumulation, or contaminant degradation depend on the ing abilities of the entire plant body (rascioa & navari-izzo. 2011; tangahu . 2011).et al jabon (anthocephalus cadamba) is known as a tropical forest species with high tolerance to marginal land, and high shoot and roots biomass productions (krisnawati et al. 2011; setyaningsih et al. 2012). forest plants with deep roots * corresponding author: luluk.setya@gmail.com biotropia 5 1 8 64 71 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.1.712 64 character and great biomass have a great potential to be used as remediator plant (capuana 2010) in contaminated forest land. however, there is still limited information about the tolerance character of tropical forest plants to heavy metals and their potentcy as remediators. each plant may have a tolerant level and pattern of different adaptations to heavy metal exposure conditions. there is still little information related to tolerant level of jabon to lead, even though many other species have been extensively evaluated such as sunflower (niu et al. 2010; kim et al. 2009), canola, mustard and vetiver grass (kim et al. 2009), lantana camara (alaribe & agamuthu 2015) and pinus radiata (javris & leung 2002). therefore, the effort to learn about the tolerance and remediation capability of jabon to heavy metal pb is necessary and important. this research was conducted in nutrient culture media to determine (1) the tolerant level of jabon seedling to by observing root growth s pb and biomass, and (2) the degree jabon by which seedlings absorb pb. materials and methods s aged one month old ninety jabon seedling with 5 cm height were the obtained from tissue culture laborator of seameo y the regional centre for tropical biology ( ) in biotrop bogor, indonesia, transferred into culture and media in plastic box container equippeds with aerator tube for lead exposure experiment. these s seedlings were grown in aerator tube containing 1 s litre of liquid culture media (sopandie nutrient 1990) adapted for 4 days and then subjected to a treatment using pb for 15 days. adaptation media nutrient was 1/5 dosage of nutrient culture without pb. media treatment was 1/3 dosage of nutrient culture treated by pb (no3) with 6 2 different pb concentrations i.e. 0, 0.5, 1, 1.5, 5 and 10 mm respectively (setyaningsih 2012). , et al. during the experiment the media culture was , set to have ph 4.5-5 using naoh and hcl. composition of nutrient culture refers to the “ n s ”sopandie utrient olution (sopandie 1990) consisting of distilled water and nutrient solution w ich contained 1.5 mm ca(no ) 4h o; 1.0 mm h 3 2. 2 nh no ; 1.0 mm kcl; 0.4 mm mgso ; 1.0 mm 4 3 4 kh po ; 0.50 ppm mnso ; 0.02 ppm 2 4 4 cuso .5h o; 0.05 ppm znso .7h o; 0.05 ppm 4 2 4 2 h bo ; 0.01 ppm (nh ) mo o .4h o; 68 mm 3 3 4 6 7 24 2 feedta. during the adaptation and treatment, the aeration was supplied using electrical aerator. parameters analysed in the experiment were root and shoot growth and plant biomass to verify the response of plants to all pb treatments. pb content was analyzed only from samples of control plants (without pb), the plants with lowest pb treatment (0.5 mm), and the plant with highest pb treatment by which the plants were still alive until the end period of the treatment, to compare the accumulation capacity of plants under lower and higher pb concentration in the media. seedling growth was observed by measuring the length of root and stem , and biomass s s dry weight harvest . when the plant samples were ed the number of dead seedlings was also counted. the tolerance index (ti) was calculated at different pb concentrations using the following equation: ti (%) = 100 x (root length under metal treatment)/(root length in the control solution) (wang . 2011). if ti <50 = not tolerant; et al 50≤ti <100 = medium tolerant; ti ≥100 = tolerant (professional judgment). pb accumulation in seedling tissue was calculated by measuring the concentration of pb in the roots, stems and leaves of control plants, the plants with lowest pb treatment (0.5 mm), and the highest treatment that were still alive. the seedlings were harvested and dried in the oven with a temperature of 45 ± 2°c until its weight was constant. for seedling special preparation roots, before being dried, considted of soaking them in 1.0 mm h-edta for 30 minutes to remove pb attached on the root surface and rinsed three times in h o (javris & leung 2002). 2 pb concentration ined using atomic was determ absorption spectroscopy (aas) which previously had been calibrated with a standard solution of pb. the obtained pb concentration was then used for calculating the value of bioconcentration factor (bcf), transport factor (tf) and bioaccumulation (b) (mun et al. 2008), using the following sthe equation : bcf = c / c root soil where rootc is the concentration of pb in roots and c is pb concentration in soil soil / media t aerial rootf = c / c where aerialc is pb concentration in the shoots b = c x dw dw is (g pb/plant) where aerial tissue dry weight 65 jabon (anthocephalus cadamba roxb) potency for remediating lead setyaningsih et al. unguiculata (kopitte et al. 2007) and paraserianthes falcataria (setyaningsih et al. 2012). chlorosis and necrosis occurred presumably because photosynthetic pigment was damage as a result of decreased production of chlorophyll a and b when pb exposure increased up to 1500 ppm (wang et al. 2011). the declined production of chlorophyll occurred as a result of heavy metal disturbance that replaced magnesium (mg), an important ion in the central of chlorophyll molecules, and inhibit the synthesis of chlorophyll by inhibition of enzyme activity (wang et al. 2011; xiao et al. 2008). root damage was very likely caused by the rapid inhibition of root growth. inhibition of root growth could be caused by inhibition of cell division in root tip due to immobilization of pb ion in the cell wall (jiang & liu 2010). roots became blackish, short and fat. this phenomenon has also been reported no the roots of v. unguiculata which became shorter and had more secondary roots ( ), and hadi & aziz 2015 also the root of in response to pb on s p. falcataria exposure (setyaningsih . 2012).et al pb treatment for 15 days caused significant decrease (p< 0.05) root length, dry weight and in tolerance index (ti) of jabon seedling . root s length value range from 8.3 11 cm which d to gradual ly decreased by increasing pb concentration exposure (table 1). dry weight of seedling had value range of 1.28 1.63 g which s decreased following the pattern of open polynomial curve of y = 0.48x 0.94x + 1.63 (r² 2 = 1, p <0.05). ti of jabon seedlings rangethe d from % to 75 100% which decreased significantly with the increase pb exposure, in concentration and the maximum reduction was 25% (table 1). e the there was no significant chang on growth performance of jabon seedling exposed to s when pb up to 1.5 mm, with value concentration ti of 75% or higher. this data showed that jabon seedlings categorized as can be having intermediate ce pb the ti value of toleran to . jabon seedling is apparently smaller than to pb p. falcataria with ti value of seedling 90% when exposed to the same level of pb (setyaningsih et al. chlorophytum 2012), and also smaller than comosum value with ti of 73% even though it was -1 exposed to 1500 mg kg (1500 ppm) of pb for 90 days (wang . 2011). moreover, et al the ti of jabon was also even smaller compared to acacia farnesiana pb , known as bioaccumulator, which the study was carried out in completely randomized design for treatments of 6 different pb concentrations, i.e. p0 = without pb (0 mm), p1 = 0.5 mm of pb, p2 = 1.0 mm of pb, p3 = 1.5 mm of pb, p4 = 5 mm of pb and p5 = 10 mm of pb. each treatment was repeated 3 times. three plants were used per unit of treatment, so that the total plants were 54 plants. all the data were analysed using the microsoft excel and spss 16 to calculate the analysis of variance and to compare the means of the treatments by dmrt analysis. the analysis of regression was also carried out to recognize the typical pattern of the treatment effects (exposure of pb). results and discussion pb tolerance of jabon seedling all jabon seedlings exposed to pb up to 1.5 mm were able to survive until 15 days. however, seedlings exposed to pb of 5 mm and 10 mm showed toxicity symptoms during the second day and completely died after 5 days suggesting that 5 mm is a toxic concentration for jabon seedling. this supports the findings of hadi and aziz (2015) and wang et al. (2011). the symptom started with chlorosis and necrosis on the tip of the leaves (the leaves turned to yellow and dried nd up) on the 2 day, followed by the appearance of rd th brown spots on the 3 day. on the 4 day, most of ththe leaves rolled up and finally dried on 5 day followed by drying of the petiole and branch in the following days. toxic symptoms also appeared in the roots of the seedlings. by this reason, the measurement of pb concentration of the highest treatment was carried out from the plants treated by 1.5 mm of pb. the seedlings grown in the media without pb had fibrous root with white and transparent colour. the seedlings exposed to 0.5 1.5 mm of pb had white fibrous roots with some dark gray spost (about 1 cm) while the whole roots of seedlings exposed to 5 and 10 mm of pb had became dark indicating cell death. symptoms of toxicity due to pb began with yellowing leaves, the appearance of brown spots in almost all the leaves, leaf curling and leaf drying as well as stem drying on fol owing days with a l grayish black color of roots. these symptoms had also been reported in other plants such as chlorophytum comosum (wang et al. 2011), vigna biotropia vol. 25 no. 1, 2018 66 the ti decreased only 8% exposure to 1000 when -1 mg l pb for 60 days (amalia . 2011). ti et al the level of jabon to is better than cuttings of pb jatropha curcas has a value which ti less than 80% when exposed to 0.5 mm of pb and became only 39.2% when it was exposed to 4 mm pb (shu . et al 2011), or even lower if it was compared to the growth of annual plant such as cowpea (vigna unguiculata was recorded to be ) which hampered by only being exposed to 1 μm of pb (kopitte . et al 2007). pb concentration, distribution and bioaccumulation in jabon seedlings exposure of jabon seeldings to pb for 15 days in liquid culture caused an increase of pb concentration in seedling tissue up to 735 ppm or approximately 12.6 times higher in seedlings under control treatment. the largest average of pb concentration was found in the leaves (735.93 ppm), then in the roots (459.04 ppm) and the in the stems (438.96 ppm) (table 2). the pattern of pb concentration in response to the increasing pb exposure followed the trend of open downward polynomial curve of y = -626.79x2 + 1077.9x + 76.81 (r ² = 1, p <0.05) (figure 1). comparison of pb concentration in seedling tissues to that in media indicates the value of bioconcentration factor (bcf). bcf value of jabon was found to seedlings range from 0.5 to 2.9, and the biggest average of bcf was found in the leaves (0.6 4.6). the bcf value of jabon seedling exposed to 0.5 mm of pb was the s highest as compared to that of abon exposed to j 1.5 mm of pb or lower concentration (figure 2). scomparison of pb content in the stem and leaf tissue to that of root seedling indicates s the s the value of the transport factor (tf). the tf value of pb in jabon seedling range s was found to from to 0.4 1.63. the tf value of leaves was greater than that of stem. the tf value increased by pb exposure to 0.5 and 1.5 mm, with the highest tf value seedling exposed to observed in s 0.5 mm of pb (figure 3). in s was pb bioaccumulation jabon seedling -2observed to from to range 1.73 36.55 x 10 mg/plant. pb accumulationthe highest was -2recorded at to in roots 3.74 36.55 x 10 mg/plant. the seedling exposed to 0.5 mm of pb had the s highest bioaccumulation value ranging from 10.41 36.55 x 10 mg/plants, followed by the -2to seedling exposed 1.5 mm of pb (table 3). the s highest value of bioaccumulation occurred in the root treated by 0.5 mm of pb that was almost 10 s times higher than control, i.e. 36.55 x that of the 10 mg/plant (table 3)-2 . 67 table 1 growth of jabon seedling exposured to pb on nutrient culture during 15 days pb (mm) root length (cm) dry biomass (gram) ti (%) pb 0 11.0 ± 1.0 b* 1.63 ± 0.34 b 100 bc pb 0.5 8.8 ± 0.2 a 1.28 ± 0.14 a 80 ab pb 1 8.3 ± 0.6 a 1.38 ± 0.33 a 75 a pb 1.5 8.5 ± 0.5 a 1.30 ± 0.13 a 77 ab *note: mean ± standard deviation followed by the same letter in the same column indicates no significant difference in the error rate of 5% by dmrt test table 2 pb concentrations on parts of jabon seedlings after 15-days exposure in nutrient culture pb exposure (mm) pb concentration per g dry weight (ppm) roots stem leaves pb 0 76.81 ± 18.60 b * 27.61 ± 15.98 a 58.38 ± 34.97 a pb 0.5 459.04 ± 94.76 d 438.96 ± 206.63 c 735.93 ± 205.78 d pb 1.5 283.32 ± 1.06 c 164.34 ± 24.44 ab 360.06 ± 56.35 c * note: mean ± standard deviation followed by the same letter in the same column indicates no significant difference in the error rate of 5% by dmrt test jabon (anthocephalus cadamba roxb) potency for remediating lead setyaningsih et al. 68 biotropia vol. 25 no. 1, 2018 figure 2 bioconcentration factor of pb on roots, stem and leaves of jabon seedlings exposed to pb during 15 days (note: the dashed line showed the threshold of plant categorized as pb extractor (tangahu et al. 2011)) figure 3 transport factor of pb on stems, and leaves of jabon seedlings exposed to pb during 15 days figure 1 relation of pb concentration (▲) in root and dry biomass (ᴏ) of jabon seedling on pb exposure 69 jabon seedlings exposed to pb for 15 days in a nutrient culture led to an increase of pb concentration in seedling tissues. based on the bcf value which ranged between 0.4 4.6, and the pb exposure in the media culture led to the increase of pb concentration in the tissues of the seedlings recorded with the highest concentration at that of the 735 ppm or 12.6 times higher than control (table 2). the leaves of the seedlings exposed to 0.5 mm of pb were found to have the largest pb concentration followed by the roots and then stems, with the tf values was ranging from 0.9 1.59 (table 2, fig. 1 and 2). to this result showed that pb concentration in jabon seedlings was greater as compared to that of (390 ppm) , pinus radiata (javris & leung 2002) but lower than that in kenaf (4281 ppm) (mun . et al 2008), p. (1773 ppm) (setyaningsih . falcataria et al 2012) and (12.7 ppm) (monfared platanus orientalis et al. 2013). the data also showed that bcf value of seedlings exposed 1.5 mm of pb was lower than to that exposed pb 0.5 mm. the exposure of 1.5 to mm of pb inhibited the plants more and caused more damage than 0.5 mm , the exposure to of pb which resulted in the reduction of pb accumulation from media to the seedling tissues. another possibility was the leakage or efflux of pb and exit back to the media due to damaging effect of pb. concentration seedling roots pb in followed the polimonial pattern of open downwards, while seedling biomass (dry weight) followed the polimonial pattern of open up in response to the increased pb concentration (fig. 3). in other words, exposure 0.5 mm to pb caused the highest pb concentration in the seedling tissue and resulted in the lowest dry biomass. this suggests that the growth declining of jabon seedling is highly correlated with the levels of pb in the tissue, the higher level of pb caused decreasing of growth more indicated by lower dry biomass. such conditions was also reported by other investigator in c. comosum (wang . 2011).et al the higher tf value of seedling due to pb exposure as compared to control suggested that the transfer of pb into the shoot of seedling occurred efficiently. tf value of jabon seedlings was likely to exceed one (tf> 1) indicating that jabon seedling prefer to keep pb in the shootsed which could classify ajabon as metal accumulator (tangahu . 2011).et al pb accumulation in jabon roots was the largest which was almost 3 times of that in the stem (2.82 – 10.41 x 10 mg/plant) and 2 times of that in the -2 leaves (1.73 24.58 x 10 mg/plant). therefore, -2 the sequence of pb accumulation in jabon seedling was the roots > leaves >stem. this phenomenon has also been reported in some other plants such as kenaf which has been reported to accumulate pb up to 97% of the total bioaccumulation (mun . 2008) and the indian et al mustard which accumulated pb more than 95% (reisinger . 2008)et al . however, the pb accumulation capacity of jabon is still lower compared with some other species that have been characterized as pb accumulators. these . pb accumulators include brassica campestris l, brassica carinata a. br., brassica juncea (l.) czern. and brassica nigra (l.) koch which could accumulate -1more than 100 mg pb g dry weight (tangahu et al. 2011). pb although the concentration in the tissue was relatively small, because this plant produced high biomass with high bcf and tf , the values total pb accumulation of jabon seedling was relatively high. based on the concept that remediation effectiveness depends on the amount of contaminant accumulated by plant biomass and/or by contaminant level in plant tissues table 3 pb bioaccumulation in jabon seedlings exposed to pb during 15 days in nutrient culture pb (mm) bioaccumulation (10-2 mg/plant) roots stem leaves pb 0 3.74 ± 1.06 a* 2.82 ± 0.11 a 1.73 ± 0.56 a pb 0.5 36.55 ± 8.90 c 10.41 ± 1.15 c 24.58 ± 0.76 c pb 1.5 18.12 ± 1.98 b 7.18 ± 1.27 b 8.98 ± 0.82 b *note: mean ± standard deviation followed by the same letter in the same column indicates no significant difference in the error rate of 5% by dmrt test jabon (anthocephalus cadamba roxb) potency for remediating lead setyaningsih et al. 70 biotropia vol. 25 no. 1, 2018 (rodriguez . 2005) we conclude that jabon et al , may have potential function as pb remediator. the efficiency of metal translocation from roots to shoots is one of the key factors to distinguish whether the plant is categorized as metal hyper-accumulator or not. metal accumulation in the shoot are often limited by the binding of metal cations that is being exchanged in the cell walls of xylem (kim . 2009). et al however, the hyper-accumulator plant may have the ability to overcome this limitation by forming a negative charge or neutral formation of organicmetal complex. some aspects such as plant species, properties of medium, the root zone, vegetative uptake and addition of chelating agent are several factors which can affect the uptake mechanism of heavy metals (rodriguez et al. 2005; tangahu et al. 2011). formation of metal-organic acid complex also can be an important mechanism to increase the uptake by plants although free pb ions of metal are easier to be accessible. conclusions jabon seed l ings exh ib i ted to le rant characteristic at least up to 1.5 mm (450 ppm) of pb, based on some indicators of tolerant index. the pb was distributed to all part of the seedlings, with the highest concentration found in the leaves (735.9 ppm). however, because of higher biomass, the highest bioaccumulation was found -2 in the roots (36.55x10 mg per plant). jabon seedling can be classified as metal accumulator and may have great potential as remediator in the lead-contaminated areas. acknowledgements the author acknowledges the government of republic of indonesia through the directorate general 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et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 hal. 164-178 1114.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 microsoft word 26 biotropia no. 19, 2002 : 26 46 review on aflatoxin in indonesian foodand feedstuffs and their products okky setyawati dharmaputra seameo biotrop, p.o. box 116, bogor, indonesia; and department of biology , faculty of mathematics and natural sciences, bogor agricultural university, jl. raya pajajaran, bogor, indonesia abstract aflatoxin is a human carcinogen that could contaminate foodand feedstuffs, and hence is a major food quality problem throughout the world. afiatoxin is produced by certain strains of aspergillusjlavus and //. parasiticus. a number of studies have been carried out in indonesia on atlatoxin contamination in indonesian foodand feedstuffs and their products from 1990 up to present. they were maize, maize product, peanuts, soybean and soybean meal, black and white pepper, feed ingredients; chicken and duck feeds. samples were collected from farmers, traders (middlemen), retailers (markets), supermarkets, exporters; poultry and duck community-based farms; and feed mill industries. high levels of aflatoxins were often found in maize, peanuts, chicken feed derived from markets, and duck feed. low levels of aflatoxins were found in soybean meal and chicken feedstuff. aflatoxins were not detected in soybean, black and white pepper. other studies have also been carried out on the effect of carbondioxide (co2), phosphine, black pepper extract and antagonistic fungi on aflatoxin production of a. flavus in vitro\ and the effect of airtight storage, phosphine, ammonium hydroxide, fermentation process, bag types, and phosphine in combination with different bag types on atlatoxin contents of maize, peanuts and soybean meal. some of these methods reduced aflatoxin contents significantly. keywords: aspergillus flavus i aflatoxin / food-and feed stuffs / product introduction aflatoxin is a human carcinogen that contaminates food-and feedstuffs, produced by the common fungi aspergillus flavus and a. parasiticus. the aflatoxin problem is a worldwide phenomenon, but it is particularly severe in developing countries, where food safety and security systems are not well developed to protect consumers against unsafe food products. in more developed countries, consumers are more aware of food safety issues such as aflatoxin, and are increasingly demanding those foods meet strict regulatory standards. aflatoxin limits for some commodities by importing countries are presented in table 1. among the foodstuff, peanuts is easily contaminated with aflatoxin. based on the report of the 23rd session of the joint fap/who food standards programme, held in rome, italy, 28 june 3 july 1999, codex alimentarius commission adopted the maximum level of total aflatoxins in peanuts intended for further processing at 15 ppb. 26 biotropia no. 19,2002 this paper reviews the researches that have been undertaken in indonesia from 1990 to date on aflatoxin contamination in foodand feedstuffs and their products, as well as control ofa.flavus and aflatoxin. aflatoxin in food-and feedstuffs and'their products aflatoxin in maize and maize products in indonesia, maize is the second most important crop after rice. purwoko et al. (1991) studied aflatoxin content of 34 maize samples collected from poultry farms and poultry feedmills located around jakarta and bogor. aflatoxins were found in 91% of maize samples, and the predominant form was aflatoxin b|. the total concentration of aflatoxins in maize samples ranged from 22 to 6171 ppb, of which 91% contained aflatoxin bbh ranging from 22 to 4074 ppb, while aflatoxin b2 (71%) ranged from 11 to 3021 ppb. aflatoxin g, was present in two samples, at concentration of 101 and 528 ppb, and only one sample contained aflatoxin g2 at concentration of 144 ppb. maryam (1994) studied aflatoxin contamination of 32 maize samples derived from diagnostic samples sent to the research institute for veterinary science, 27 review on aflatoxin in indonesia food-and feedstuff's and their products okky s. dharmaputra bogor. it was reported that the percentage of maize samples contaminated with aflatoxin b,, b2, g, and g2 were 78.1, 78.1, 20.0 and 20.0%, respectively. their ranges were between 1.1 63.7, 0.5 12.6, 1.2 51.7, and 0.6 4.2 ppb, respectively (table 2). a study by dharmaputra et al. (1995) on aflatoxin content of 35 maize samples collected from farmers and traders in lampung province in november 1992 reported that all of the samples (100%) contained aflatoxin bi, and 31% contained aflatoxin b2 (table 3). eleven samples obtained from farmers contained 25.4 367 ppb of aflatoxin bbh and 2 samples contained 12.5 and 12.7 ppb of aflatoxin b2, respectively; 4 samples obtained from village traders contained 119.7 276.7 ppb of aflatoxin b! and 11.9 41.6 ppb of aflatoxin b2, 4 samples obtained from middle traders contained only aflatoxin b! (25.6 97.6 ppb); 16 samples of big traders contained 23.4 278.7 ppb of aflatoxin b, and 5 samples contained 24.2 61.2 ppb of aflatoxin b2b . 28 biotropia no. 19,2002 another study on aflatoxin content of 108 maize samples collected from farmers, and 32 samples from village traders during the dry and wet seasons in central lampung (lampung province) and kediri (east java province) regencies was conducted in 1993 1994 (dharmaputra et al. 1996a). at farmers level, the total aflatoxin b| contents was between 5.3 291.4 ppb, while at village trader level, between 9.7 115.2 ppb (table 4). 29 review on aflatoxin in indonesia food-and feedstuff's and their products okky s. dharmaputra a study by dharmaputra and putri (1996b) on total aflatoxin b, content in 11 samples of maize and maize products collected from some supermarkets in bogor, west java, reported that 1 sample of popcorn contained 15 ppb of the toxin; 1 sample of maizena flour contained 20 ppb of the toxin; 2 samples of maize for popcorn contained 40 and 40 ppb of the toxin, respectively; 1 sample of "maize chip" contained 19 ppb of the toxin. non detected aflatoxin was observed in 2 samples of popcorn, 1 sample of "marning", 2 samples of maize oil, and 1 sample of tortilla chips (table 5). biotropia no. 19,2002 aflatoxin in peanuts and peanut products peanuts is next to maize and soybeans as secondary crop of indonesia. dharmaputra et al. (1991) reported the aflatoxin contents in 35 peanut samples collected from 15 retailers located in three markets in bogor, west java, in september 1988. the aflatoxin contents were between 0 1154 ppb, while 80% of the peanut samples contained more than 30 ppb of aflatoxin. another study carried out by haryadi and setiastuty (1994) on aflatoxin contamination in 30 samples of raw and processed peanuts collected from various traders (big, medium and small) and processors during rainy and dry seasons in and around bogor (west java) and denpasar (bali), reported that aflatoxin b, was detected in 8 out of 15 samples collected during the rainy season, and in 5 samples collected during the dry season. aflatoxin b2 was detected in some samples contaminated with aflatoxin bi (tables 6 and 7). the possibility of aflatoxin contamination was higher among small traders, although big traders cannot guarantee that their products were free from aflatoxin contamination. the study also revealed that in processed peanuts, aflatoxins could still be present. 31 review on aflatoxin in indonesia food-and feedstuff's and their products — okky s. dharmaputra dharmaputra et al. (2002) reported that only one out of 15 samples of roasted pod and flour, and 14 samples of coated kernels of peanut collected from supermarkets in bogor, malang, pati and yogyakarta contained total aflatoxin of 15.54 and 20.72 ppb, respectively. table 8 shows the levels of aflatoxin found in maize and peanut samples from retailers located around bogor and yogyakarta. forty five percent of 215 peanut samples contained more than 50 ppb of aflatoxin, 33% more than 300 ppb, and 22% exceeded 1000 ppb (pitt and hocking, 1996). aflatoxin levels exceeding 300 ppb must be considered unsatisfactory, while those exceeding 1000 ppb must be considered totally unsatisfactory, capable of inducing acute toxic effects in both human and animals. an amount of 1% of maize and 17% of peanut samples, contained more than 1000 ppb aflatoxin. biotropia no. 19,2002 aflatoxin in soybean and soybean meal in indonesia, soybean ranks next to maize in importance as secondary food crops. the protein content of soybean is relatively high (42 50%), thus soybean meal is an important component of feedstuff. purwoko et at. (1991) studied aflatoxin contents of 10 soybean samples collected from some poultry farms and poultry feed mills located around jakarta and bogor in july 1990. aflatoxins were not detected in the samples. dharmaputra et al. (1997) reported that only 5 soybean meal samples were contaminated by aflatoxin from a study on aflatoxin content of 13 soybean meal samples collected from 10 feed mills factories, three bulog warehouses and one soybean crushing plant factory. the contents of aflatoxin were 7.9, 11.3, 14.5, 28.5 and 34 ppb, respectively. according to venkitasubramanian (1977), phytic acid in soybeans can bind zinc ions, while zinc ions are the important elements of aflatoxin synthesis. aflatoxin in pepper among spices, black and white pepper are important export commodities in indonesia. aflatoxins were not detected in 40 samples of black and white pepper (20 samples/commodity) collected from some retailers and supermarkets in bogor, and some exporters in bangka island and lampung province (dharmaputra et al. 1999). aflatoxin in chicken feed, duck feed, mixed duck feed, and rice bran dharmaputra and putri (1996b) reported that the total aflatoxin b[ content of 39 chicken feed samples collected from 3 markets in bogor during dry and wet seasons was between 0 — 200.73 ppb. twenty six out of 39 samples (67%) contained aflatoxins more than 30 ppb. a study by zahari and tarmudji (1995) on the aflatoxin content of 19 duck feed, 8 mixed duck feed and 8 rice bran samples collected from a duck farm in south kalimantan reported that in duck feed 100, 100, 52.6 and 15.8% of the samples were contaminated with aflatoxins b:, b2, g, and g2, respectively. their contents were between 4.0 160.0 ppb, 4.8 60.0 ppb, 0 60.0 ppb, and 0 8.0 ppb, respectively. in mixed duck feed, only one sample was contaminated with aflatoxin b,, and its content was 8.0 ppb. in rice bran, 75% of the samples were contaminated with aflatoxin b|. their contents were between 0 20.0 ppb. two out of 8 samples (25%) contained 7.2 and 72.0 of aflatoxin bb2. only one sample of rice bran was contaminated with aflatoxin g, (3.2 ppb). no aflatoxin g2 was detected in all samples. 33 review on ailatoxin in indonesia food-and feedstuff's and their products okky s. dharmaputra control of aspergillus fla vus and aflatoxin the effects of carbondioxide on mycelial growth and aflatoxin production of a. flavus in pure culture dharmaputra et al. (1992) studied the effects of co2 concentrations of 20, 40, 60 and 80% on mycelial growth and aflatoxin production of three isolates of a. flavus. as a control, these fungal isolates were maintained in air. the co2 concentrations used significantly affected both mycelial growth and aflatoxin production of a. flavus isolates. co2 at 20% started to inhibit the two parameters (figures 1 and 2). the growth of a. flavus and aflatoxin production decreased with the increase of co2 concentrations. figure 1. mycelial growth of a. flavus bio-16, bio-17 and bio-18 after treatment with different concentrations of co2 for 7 x 24 h on potato dextrose agar (dharmaputra et al. 1992). 34 biotropiano. 19,2002 the effect of carbondioxide on aflatoxin production in maize an investigation was carried out by dharmaputra et al. (1990) on the effect of co2 on aflatoxin production in stored maize. stacks of stored maize were sealed with pvc sheeting and treated with co2 for storage periods varying from 10 to 120 days. the concentration of co2 used was 2.4 kg/t. the control groups consisted of both stacks of maize sealed in plastic sheets, but not treated with co2, and stacks not sealed in plastic sheets. the aflatoxin b, content of maize, whether in plastic sheets and treated with co2 or only in plastic sheets, was lower than that of the unsheeted and untreated stacks (figure 3). the control showed that the aflatoxin content increased with the increasing duration of storage. 35 review on aflatoxin in indonesia food-and feedstuff's and their products okky s. dharmaputra the effect of phosphine on mycelial growth and aflatoxin production of a. flavus in pure culture dharmaputra et al. (1991) studied the effect of phosphine on mycelial growth and aflatoxin b, production of two a. flavus isolates. the concentrations of phosphine used were 0.5, 1.5, 2.5 and 3.5 mg/l. as a control, these fungal isolates were maintained in air. the phosphine concentrations significantly affected both the mycelial growth and aflatoxin production of the isolates of a. flavus. mycelial growth and aflatoxin production decreased with increasing phosphine concentrations (figures 4 and 5). inhibition of mycelial growth commenced at 0.5 mg/l (figure 4). although the two isolates were still able to produce aflatoxin after treatment with 3.5 mg/l of phosphine, the amounts were low (figure 5).   review on aflatoxin in indonesia food-and feedstuffs and their products — okky s. dharmaputra the effectivity of phosphine to maintain the quality of maize packed in two different bag types another study was conducted by putri et al. (1999) on the effectivity of phosphine to maintain the quality of maize packed in two different bag types. maize with initial moisture content of ± 14% was stored in two bag types, namely jute and polypropylene bags, and then laid on the wooden pallet for 130 days. phosphine fumigation was carried out for 5 days at the beginning of storage and after 95 days of storage. maize samples (treated with phosphine and untreated one) were taken just before and immediately after fumigation, and thereafter every 30 days for a period of 130 days. the results indicated that the highest total aflatoxin b, content was found in unfumigated jute bag, followed by unfumigated polypropylene bag, fumigated jute bag, and fumigated polypropylene bag (tab'le 9). the effect of phosphine on aflatoxin production of soybean meal an investigation was carried out by dharmaputra et al. (1993) on the effect of phosphine on aflatoxin production of stored soybean meal. soybean meal was stored in polypropylene bags for 190 days. four stacks were treated with phosphine (2.1 g/t) for 5 days, once at the beginning of storage and again after 95 days of storage. four untreated stacks served as control. 38 biotropia no. 19,2002 there was a significant difference in aflatoxin b, content between the treated and the untreated samples (table 10). aflatoxin b, content increased during prolonged storage in both treated an untreated soybean meal (figure 6). the results indicated that phosphine somehow inhibits aflatoxin bi production. review on aflatoxin in indonesia food-and feedstuff's and their products okky s. dharmaputra the effect of airtight storage on aflatoxin production in maize an investigation was carried out by dharmaputra et al. (2000) on the effect of airtight storage on aflatoxin production in maize. the maize was placed in polyethylene bag under airtight condition (initial o2 content was 1.4 ± 0.1%), and stored for six months under laboratory conditions. the initial moisture contents of maize were 14 ± 0.3, 17 ± 0.2 and 20 ± 0.2%. as control, maize with the same initial moisture contents was placed in polyethylene bag under normal condition (air) with o2 content of 21%. the total aflatoxin b, content on maize with the three initial moisture contents packed under normal condition was higher than under airtight condition (figure 7). the total aflatoxin b! content increased with the increase of storage duration. the effects of black pepper extract on mycelial growth and aflatoxin production of a. flavus in pure culture retnowati et al. (1998) studied the effects of black pepper extract on mycelial growth and aflatoxin production of a. flavus. the concentrations of black pepper extracts used were 0.25, 0.50, 0.75 and 1.00% v/v media. aspergillus flavus was grown on smky liquid medium containing different concentrations of black pepper. as a control, smky liquid medium did not contain black pepper extract. black pepper extract at 0.25% concentration started to inhibit mycelial growth and aflatoxin production of a. flavus. mycelial growth and aflatoxin contents decreased with the increase of the extract concentrations (figures 8 and 9). no aflatoxin was detected on smky liquid medium containing 1% black pepper extract. 40 biotropia no. 19,2002 the effect of ammonium hydroxide on aflatoxin production of maize the effect of ammonium hydroxide on aflatoxin production of maize stored under laboratory conditions was investigated by dharmaputra and ambarwati 41 review on aflatoxin in indonesia food-and feedstuff's and their products okky s. dharmaputra (1999). maize var. arjuna was treated with different concentrations of ammonium hydroxide, i.e. 0.5, 1.0, and 1.5% (v/v). the durations of the treatments were 12, 24 and 36 hours. the result showed that ammonium hydroxide decreased the aflatoxin content. the lowest aflatoxin content was detected when the maize was treated with 1.5% ammonium hydroxide for 36 hours (table 11). 42 biotropia no. 19,2002 the effect of fermentation process on the aflatoxin production of peanuts the effect of traditional fermentation process on aflatoxin production of peanuts was studied by edi et al. (1990) and fardiaz et al. (1993). peanuts were contaminated with aflatoxin-producing a. flaws, and incubated at 30°c for 6 days. the oil was extracted by hydraulic pressure, and the peanut presscake processed to make a fermented product called "oncom". to make "bla'ck oncom", the presscake was fermented with rhizopus oligosporus, while to make "red oncom" the presscake was fermentated with nenrospora sitophila. soaking of peanut presscake in water for 24 hours reduced the aflatoxin content to 48.6%, and it was reduced further to 42.9% after steaming at 95°c for 90 minutes. fermentation of the peanut presscake by r. oligosporus reduced the total aflatoxin content to 13.4%, while fermentation by n. sitophila reduced the total aflatoxin content to 41.1% of the original aflatoxin content in peanut presscake (figure 10). aflatoxin b, appeared to be the most sensitive to soaking and steaming processes. the decrease in aflatoxin content during soaking and steaming might be due to leaching, since aflatoxin is relatively resistant to heat. the effect of bag types on aflatoxin production of peanuts bulaong and dharmaputra (2002) studied the effect of four bag types namely jute bag, poly-propylene bag, jute bag lined with thin polyethylene (pe), and jute bag lined with thick pe, on aflatoxin production of peanuts with initial moisture content of 8%. storage was done for 6 months under warehouse conditions. 43 review on aflatoxin in indonesia food-and feedstuffs and their products okky s. dharmaputra no significant differences were obtained in aflatoxin levels among bag types, but at the end of six months storage, aflatoxin contents of peanuts packed in jute bag, polypropylene bag, jute bag lined with thin pe, and jute bag lined with thick pe were 31.5, 23.1, 16.8 and 18.9 ppb, respectively. based on the results, the immediate packaging of dried shelled peanuts at safe moisture level in jute bag lined with thin pe is recommended. the water vapor transmission rate of the thin pe is 1 g/m2/24 h. the effect of antagonistic fungi on aflatoxin production ofa.flavus in vitro investigation was carried out by dharmaputra et al. (2001) on the effect of fungi isolated from soil of peanut fields at wonogiri regency, central java, on aflatoxin production of a. flavus. figure 11 shows that a. niger was the most promising fungal antagonist, because it caused the highest percent inhibition (80%) of aflatoxin production of a. flavus isolate 102, followed by non-toxigenic a. flavus isolate 612 (61%), a. tamarii (60%) and non-toxigenic a. flavus isolate 36, (59%). conclusions only very limited analyses and surveys on the extent of aflatoxin contamination in indonesian foodand feedstuffs and their products have been carried out. high levels of aflatoxins were often found in maize and peanuts. consequently, 44 biotropia no. 19, 2002 researches have been concentrated on the effects of postharvest handling and fumigation on fungal infection and aflatoxin production in those two commodities. in soybean meal, aflatoxin contents were relatively low. no research has been carried out on aflatoxin contamination in relation with human health, cultural practices, peanut trading and industry. it is essential that a rigorous assessment of the critical points of entry of aflatoxin into the food and feed chains should be determined to more clearly assess the importance of pre-versus post-harvest management strategies. references bulaong, s.s.p. and o.s. dharmaputra. 2002. fungal population, aflatoxin and free fatty acid contents of peanuts packed in different bag types. biotropia no. 19 : 1 25. dharmaputra, o.s., h.s.s. tjitrosomo, f. aryani and m. sidik. 1990. the effect of carbondioxide on storage fungi of maize. proceedings of the 5"' international working conference on stored-product protection, bordeaux, france, 9-14 september 1990,291-301 dharmaputra, o.s., h.s.s. tjitrosomo, h. susilo and sulaswati. 1991. aspergillusflavus and aflatoxin of peanuts collected from three markets in bogor, west java, indonesia. proceedings of the 12"' asean seminar on grain postharvest technology, surabaya, indonesia, 29-31 august 1989, 110 -123. dnarmaputra, o.s., h.s.s. tjitrosomo, t.s. wardani and h. halid. 1992. the effect of carbondioxide on some biological aspects of aspergillus flavus. proceedings of the 13"' asean seminar on grain postharvest technology, bandar seri begawan, brunei darussalam, 4-7 september 1990, 280-293 dharmaputra, o.s., h.s.s. tjitrosomo, m. sidik and r.c. umaly. 1991. the effects of phosphine on some biological aspects of aspergillus flavus. aciar proceedings no. 36. proceedings of an international conference on fungi and mycotoxins in stored products, bangkok, thailand, 23-26 april 1991,244-248 dharmaputra, o.s., i. retnowati, m. sidik and h. halid. 1993. the effects of phosphine and length of storage on fungi, aflatoxin and protein content of soybean meal. proceedings of the 15'1' asean seminar on grain postharvest technology, singapore, 8-11 september 1992, 125-136 dharmaputra, o.s., i. retnowati, sunjaya and s. ambarwati. 1995. aspergillus flavus population and aflatoxin content in maize collected from farmers and traders in lampung province, south sumatra. proceedings of the 12"' national congress and scientific seminar, indonesian society for phytopathology, yogyakarta, indonesia, 6-8 september 1993. vol. 1, 560-566. (in indonesian). dharmaputra, o.s., i. retnowati, h.k. purwadaria and m. sidik. 1996a. surveys on postharvest handling, aspergillus flavus infection and aflatoxin contamination of maize collected from farmers and traders. paper presented at the 17"' asean technical seminar on grain postharvest technology, lumut, perak, malaysia, 25-27 july 1995. aciar technical reports 37, 38-53. dharmaputra, o.s., and a.s.r. putri. 1996b. aspergillus flavus population and aflatoxin content in maize, maize products and chicken feed. paper presented at the national microbiology seminar and annual scientific meeting, malang, indonesia, 12-13 november 1996. (in indonesian). dharmaputra, o.s., h. susilo and m. sidik. 1997. population of storage fungi and aflatoxin content of soybean meal. proceedings of the 14"' national congress and scientific seminar, indonesian society for phytopathology, palembang, indonesia, vol. 2,241-249. 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(in indonesian with abstract in english). dharmaputra, o.s. and s. ambarwati. 1999. detoxification effects of ammonium hydroxide on aflatoxin and total nitrogen content of maize. hayati 6(2): 25-28 dharmaputra, o.s., 1. retnowati, m. amad and s. ambarwati. 2000. airtight storage of maize: its effect on fungal infection and aflatoxin production. proceedings of the 19"' asean and 1st apec seminar on postharvest technology, ho chi minh city, vietnam, 9-12 november 1999, 474-482 dharmaputra, o.s., a.s.r. putri, i. retnowati and s. ambarwati. 2001. antagonistic effect of soil mycobiota on aflatoxin-forming aspergillus flavus in vitro. paper presented at the 20"' asean and 2"d apec seminar on postharvest technology. chiang mai, thailand, 11-14 september 200 1 . dharmaputra, o.s., a.s.r. putri and lilieanny. 2002. population of storage fungi and aflatoxin content in peanut products. internal report. seameo biotrop. (in preparation). edi, s. fardiaz and d. fardiaz. 1990. production of aflatoxin by aspergillus flavus and its destruction during fermentation of peanuts by neurospora sitophila. buletin penelitian ilmu dan teknologi pangan 1(1): 18-31. (in indonesian). fardiaz, s., s. wulan and b. setiyadi. 1993. effect of fermentation by rhizopus oligospoms on aflatoxin production by aspergillus flavus in peanut presscake. proceedings of the 5'1' asean food conference, akarta, indonesia, february 1992, 858-864 haryadi, y. and e. setiastuty. 1994. characterisation of aflatoxins bi, bb2, d and g2 in groundnuts and groundnut products. proceeding of the 6"' international working conference on stored-product protection, canberra, australia, vol. 2, 996-998. maryam, r. 1994. kontaminasi asam siklopiazonat dan aflatoksin pada jagung. makalah dibawakan pada kongres nasional perhimpunan mikologi kedokteran manusia dan hewan indonesia i dan tcmu ilmiah. bogor, 21-24 juli 1994, 289-293 pitt, j.i. and a.d. hocking. 1996. current knowledge of fungi and hiycotoxins associated with food commodities in southeast asia. mycotoxin contamination in grains. aciar technical reports 37,5-10 purwoko, h.m., b. hald and j. wolstrup. 1991. aflatoxin content and number of fungi in poultry feedstuff's from indonesia. letters in applied microbiology 12: 212-215 putri, a.s.r., o.s. dharmaputra, sunjaya and m. sidik. 1999. the effectiveness of phosphine to maintain the quality of maize packed in two different bag types. proceedings of the 7'1' international working conference on stored product protection, beijing, p.r. china, 14-19 october 1998, 274-279 retnowati, i., o.s. dharmaputra, h. susilo and h. affandi. 1998. inhibitory effects of black pepper and turmeric extracts onf the growth and aflatoxin production of aspergillus flavus . proceeding of the national microbiology seminar and annual scientific meeting, bandar lampung, indonesia, 14-15 december 1998, 210-220. (in indonesian with abstract in english) ventikitasubramanian, t.a. 1977. biosynthesis of aflatoxin and its control. in rodricks, j.v., c.w. hesseltine and m.a. mehlman (eds.). mycotoxin in human and animal health. pathotox publishers inc., part forest south, illinois, 83-98 zahari, p. and tarmudji. 1995, aflatoksikosis pada ternak itik alabio di kalimantan selatan. presiding seminar nasional teknologi veteriner untuk meningkatkan kesehatan hewan dan pengamanan bahan pangan asal ternak. cisarua, bogor, indonesia, 22-24 maret 1994, 408-41 1 46 26.pdf 27.pdf 28.pdf 29.pdf 30.pdf 31.pdf 32.pdf 33.pdf 34.pdf 35.pdf 36.pdf 37.pdf 38.pdf 39.pdf 40.pdf 41.pdf 42.pdf 43.pdf 44.pdf 45.pdf 46.pdf potential yield of acid-adaptive soybean promising lines in ultisols of tanah laut regency, south kalimantan province, indonesia heru kuswantoro indonesian legume and tuber crops research institute indonesian agency for agricultural research and development, malang 65101, indonesia received 20 november 2015/accepted 26 may 2016 abstract soybean is an important food commodity after rice and corn in indonesia. until now soybean demand cannot be fulfilled by domestic production, although the chances of fulfillment can be attempted. one of the attempts to increase domestic production is soybean planting in acid dry land that is widely available in indonesia. the research aimed to obtain soybean lines that are adaptive in acid dry land. a total of 10 soybean promising lines and two check varieties (wilis and tanggamus) were grown in acid dry land in tanah laut regency, south kalimantan province, indonesia. experimental design used was randomized complete block design with four replications. the data were analyzed using analysis of variance and followed by least significant different test. the results showed that the promising lines of sc5p2p3.5.4.1-5 and sc5p2p3.23.4.1-5 achieved production of 1.51 t/ha and 1.48 t/ha, respectively. these yields were higher than wilis and tanggamus varieties that reached 1.41 t/ha and 1.13 t/ha, respectively. these two promising lines had the potency to be developed in tanah laut regency, south kalimantan or other areas with similar soil characteristics. : acid dry land, soybean, tanah laut regency, ultisolskeywords introduction soybean is an important food crops for indonesia and is ranked third after rice and corn. soybean self-sufficiency in 2017, announced by the indonesian government, needs to be followed up with efforts in the area of soybean planting in accordance with soybean growth requirements. soybean has the ability to grow in areas with optimal up to suboptimal soil types. soybean can be planted in all soil types such as grumusols, inceptisol, ultisols and others. therefore, various efforts have been and are being carried out for soybean planting in various agroecology such as paddy fields, dry land, acid dry land and tidal swamp land. opportunity of soybean development in acid dry land is very high, not only because of soybean ability to grow in acid dry land, but also because of the wide area of acid dry land. acidic dry land area in indonesia is 108.8 million ha or approximately 69.4% of the total dry land in indonesia (bbsdlp 2012). dry land with ultisols and oxisols soil types occupy the largest area in indonesia (hidayat & mulyani 2002). acidic dry land area in south kalimantan province is 2,189,535 ha (mulyani & syarwani 2013). ultisols soil type located in south kalimantan province is included in soil suborder of udults, namely ultisols that develops in areas with udic moisture regime (nugroho & istianto 2009). soybean ability to adapt in ultisols soil types can be reflected on soybean improvement efforts through plant breeding programs as well as the amelioration of the land that suitable for soybeans growth requirements. usually, soybean improvement for acid dry land adaptation in breeding program is conducted by crossing, followed by the selection of segregating populations, yield trials and adaptation trials. adaptation trial should be conducted in the target area in order to obtain the soybean promising biotropia vol. 23 no. 1, 2016: 52 57 doi: 10.11598/btb.2016.2 . .3 1 561 * c orresponding author: herukusw@yahoo.com 52 mailto:herukusw@yahoo.com lines that can adapt in this area. in this study, the target area was acid dry land in tanah laut regency, south kalimantan province having ultisols soil type. the research aimed to obtain soybean lines that are adaptive in acid dry land. materials and methods study site the study was carried out in tanah laut regency, south kalimantan province (3°41'31.9" s; 114°44'00.8" e) having ultisols soil type with soil ph (h o) of 5.5; exchangeable al and h of 2 0.67 me.100/g and 1.53 me.100/g, respectively. other soil properties are shown in table 1. plant materials ten promising lines of sc2p2.99.5.4.5-1-6-1 (g1), sc2p2.151.3.5.1-10 (g2), sc5p2p3.5.4.1-5 ( g 3 ) , s c 5 p 2 p 3 . 2 3 . 4 . 1 3 2 8 3 ( g 4 ) , sc5p2p3.23.4.1-5 (g5), sc5p2p3.48.31.1-10 (g6), sj-5/msr.99.5.4.5-1-6-1 (g7), msr/sj5.21.3.7-3-27-1 (g8), msr/sj-5.23.4.1-3-28-3 (g9) and msr/sj-5.23.4.1-5(g10) and two check varieties tanggamus (g11) and wilis (g12) were used in this experiment. the ten promising lines were derived from crossing of mansuria and sj-5 genotypes. tanggamus is an acid-adaptive soybean variety, while wilis is a broadly-adaptive soybean variety. soil preparation to obtain optimal soil structure condition for ideal growth of the soybean, soil tillage was carried out by plowing the soil twice until 20 cm depth and then flatting by harrowing the soil. drainage canals were made every 4.5 m to prevent water logging in case of hard rainfall. design and planting the experimental design applied was randomized completely block design with four replications. plot size was 2.8 × 4.5 m and plant spacing of 40 × 15 cm with two plants per hill. fertilizers applied were 75 kg urea, 125 kg sp36 and 75 kg kcl per ha, which were spread before planting. control of weeds, pests and diseases were performed optimally by monitoring scheme. observation and data analysis observations were conducted on days to 50% flowering (then stated as days to flowering) and days to 95% maturing (then stated as days to maturing), plant height, number of reproductive nodes per plant, number of filled and unfilled pods per plant, 100 grains weight and grain yield. data were analyzed using analysis of variance (anova) and followed by least significant difference test (lsd) at = 0.05.α table 1 soil properties soil properties value ph (h2o) 5.05 ph (kcl) 4.15 n (%) 0.13 co (ppm) 1.48 p2o5 (ppm) 8.08 so4 (ppm) 42.30 fe (ppm) 48.90 mn (ppm) 245.00 cu (ppm) 6.13 zn (ppm) 4.20 k (ppm) 0.48 na (me/100g) 0.21 ca (me/100g) 5.11 mg (me/100g) 2.06 ktk (me/100g) 34.00 aldd (me/100g) 0.67 hdd (me/100g) 1.53 53 potential yield of acid-adaptive soybean promising lines in ultisols of tanah laut regency heru kuswantoro – results and discussion in this study, there were three genotypes having early days to flowering namely sc5p2p3.5.4.1-5, msr/sj-5.23.4.1-3-28-3 and wilis with days to flowering of 34 days. on the other hand, there were four genotypes having days to flowering of 40 days or higher namely sc5p2p3.23.4.1-5, sc5p2p3.48.31.1-10, sj5/msr.99.5.4.5-1-6-1 and msr/sj-5.23.4.1-5 (fig. 1). different days to flowering are affected by the genotypes, where they are affected by genes that repressing (cao . 2015) or inducing et al (na . 2013) as the response to the environ-et al mental parameters such as temperature (lee . et al 2005; xia . 2012), photoperiod and other et al environmental stimuli (xia . 2012).et al days to maturing of the tested genotypes varied. trend of days to maturing is similar to trend of days to flowering. genotype having longer flowering days also showed longer maturing days and vice versa. however, there is one genotype (wilis) having early flowering day and longer maturing days. days to maturing is important in soybean because different maturing day response to the yield. soybean is a photoperiod-sensitive and self-pollinated species. days to flowering (dtf) and maturing (dtm), duration from flowering-to-maturing (dftm) and plant height (ph) are crucial for soybean adaptability and yield (zhang . 2015). et al therefore, temperature is related to the maturity of soybean (kumagaia & sameshima 2014). plant height varied among the tested genotypes. the highest plant height was sc5p2p3.23.4.1-5 and the lowest plant was sc5p2p3.5.4.1-5. the check varieties tanggamus and wilis were included as having medium plant height in this study. plant height is influenced by the genotypes and environment. genes in the genotypes works to perform plant height supported by the environment. in rainy season soybean plant height is higher than in dry season (kuswantoro & zen 2013). in physiological point of view, plant height is also affected by solar radiation. zhang . (2014) stated that solar et al ultraviolet radiation exclusion increases soybean plant height due to the internodes elongation. plant height has relationship to grain yield as stated by many authors (lee . 2015; liu 2013).et al the lowest number of reproductive nodes was achieved by sc5p2p3.5.4.1-5. the highest number of reproductive nodes was achieved by sc2p2.99.5.4.5-1-6-1, in which sc2p2.99.5.4.51-6-1 was also included as having higher plant height. number of reproductive nodes had similar pattern to plant height (fig. 2). higher plant height leads to higher number of reproductive nodes, because higher plant allows more branches to grow than lower plant. in this case, the length of the main stem is the most important factor for plant height. at the r5 growth stage, nodes development on the main stem and on the branches reach the maximum (egli . 1985). number of reproductive nodes et al is affected by number of branches. h branch igher figure 1 days to flowering and days to maturing of acid-adaptive soybean promising lines in tanah laut regency, south kalimantan province (rainy season 2012) 54 biotropia vol. 23 no. 1, 2016 dry matter per plant affecting more branch nodes and branch reproductive nodes indicates that number of branches and number of reproductive nodes have a close relationship (carpenter & board 1997). the highest number of filled pods was achieved by sc5p2p3.23.4.1-5, msr/sj-5.23.4.13-28-3 and tanggamus, while the lowest was achieved by sc5p2p3.5.4.1-5. this indicated that the tested soybean lines varied in pods number trait. numbers of filled pods in this study were lower than other study with the same soybean lines, although the soil ph is higher. this phenomenon might be due to the lower water availability (kuswantoro & zen 2013). low water availability at pod filling period may accelerate senescence and shorten pod filling period (de sousa 1997).et al. the pattern of number of filled pods was not similar to the pattern of number of unfilled pods (fig 3). sc5p2p3.5.4.1-5 had the highest number of unfilled pods and had the lowest number of filled pods. tanggamus was the soybean variety that achieved the highest number of filled pods and unfilled pods. this difference indicated that there is no relationship between these two characters. genetic constitution has important role in expressing the number of unfilled pods against environmental effects. it is indicated by the broad-sense heritability of unfilled pods achieving 93.1% (sahay 2005).et al. figure 2 plant height and number of reproductive nodes of acid-adaptive soybean promising lines in tanah laut regency, south kalimantan province (rainy season 2012) figure 3 number of filled pods and number of unfilled pods of acid-adaptive soybean promising lines in tanah laut regency, south kalimantan province (rainy season 2012) 55 potential yield of acid-adaptive soybean promising lines in ultisols of tanah laut regency heru kuswantoro – the largest grain size were shown by sc5p2p3.23.4.1-5 and sc5p2p3.48.31.1-10, while the smallest were shown by sj5/msr.99.5.4.5-1-6-1 and msr/sj-5.23.4.1-3-28-3 (fig. 4). grain size of the check varieties were in the middle rank of the tested genotypes. grain size is one of the main components affecting grain yield. grain size can decrease (kuswantoro . et al 2014) or increase (kuswantoro 2015) grain yield through modification of environmental factors such as soil acidity. therefore, grain size is more affected by environmental factors rather than by genetic constitution (liu 2010). this is et al. supported by hakim . (2014) who found et al medium heritability with value of 46.3%. the line of sc5p2p3.23.4.1-3-28-3 achieved production of 1.51 t/ha which was higher than that of wilis and tanggamus varieties which reached production of 1.41 t/ha and 1.13 t/ha, respectively. other line that achieved relatively higher grain yield was sc5p2p3.23.4.1-5 with grain yield of 1.48 t/ha. tanggamus is the check variety for wide adaptation variety. in this study wilis had lower grain yield than that of tanggamus, the soybean check variety for adaptability to acid soil. grain yield is a complex character and affected by other components. the most important characters in supporting grain yield are grain size and number of grain yield. some authors reported that there were significant correlation (malik 2011) and genetic et al. correlation (arshad . 2006) between grain yield et al and 100 grains weight; and a positive direct effect between 100 grains weight and grain yield (elbadawy 2012). similar to number of grain size, some authors also reported the relationship between grain yield and number of pods (arslan et al. 2005), and positive direct effect with number of pods per plant (valencia-ramírez & ligarretomoreno 2012). conclusions the promising lines of sc5p2p3.5.4.1-5 and sc5p2p3.23.4.1-5 achieved production of 1.51 t/ha and 1.48 t/ha, respectively, which were higher than the production of tanggamus and wilis varieties. the yield of these two promis ing l ines was a l so suppor ted by number of filled pods. promising lines of sc5p2p3.5.4.1-5 and sc5p2p3.23.4.1-5 has potency to be developed in tanah laut regency, south kalimantan or other areas with similar soil characteristics. acknowledgements this study was funded by the korean government through asian food and agriculture cooperation initiative (afaci) project 2012. references arshad m, ali n, ghafoor a. 2006. character correlation and path coefficient in soybean (l.) glycine max merrill. pakistan j bot 38:121-30. figure 4 100-grain weight 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hauser ba, boote kj. 2014. solar ultraviolet radiation exclusion increases soybean internode lengths and plant height. agr forest meteorol 184:170 8. 57 potential yield of acid-adaptive soybean promising lines in ultisols of tanah laut regency heru kuswantoro – biotropia vol. 28 no. 1,2021: 29 37 doi: 10.11598/btb.2021.28.1.907 dominance, association and distribution pattern of tree species in burnt forest in east kalimantan subekti rahayu', agus priyono iozrton02, sambas basuni2 and agus hii(mat2 ' worldagraforesbty, jalan c@r, situ gede, sindang barang, bogor 16680, indonesia 'forest resources conseruntion and ecotourism department, faculg $foresty, institut pertanian bogor, kavzpzrs ipb dramaga, bogor 16680, indonesia received 4 october 2017/accepted 17 february 2020 abstract repeated forest fues remarkably impact species composition. pioneer species colonize the burnt forest and widely develop up to 30 years after a fire but late-succession species regenerate gradually or even disappear owing to direct impact of fires or other ecological consequences related to fires. hence, forest restoration through assisted natural regeneration needs some information about the state of post-fire species composition. to better evaluate tree species composition after repeated fires, the species dominance as an indicator of species composition was used in this research, with additional information on the species association and distribution patterns. a 1.8-hectare plot, divided into 180 subplots with size of 10 x 10 m, was established in a secondary forest in samboja research forest, east icalimantan. the sample plot was burnt in 1982/1983 and 1997/1998. all trees above 10 cm dbh were measured and leaf specimens were collected for species identification at the herbarium bogoriense, cibinong, west java. for comparison, the 1981 data from the indonesian institute of science were used. analysis of the importance value index used the species dominance data. a 2 x 2 matrix based on the presence-absence of species for each subplot was used to analyse the association index among species. variance and average value ratio of certain species present in each subplot were expressed in a dispersion index. a chi-square was used to test the significance between the association and dispersion index. thirteen years after a second fire, pioneer species of macarangagigantea were most dominant, followed by vernonia arborea, a sub climax species. this indicated that the forest was in an early succession process. pholidocarpw mqadtm was consistently dominant before and after the fire. a total of 38 pairs of species were significantly positively associated and 4801 pairs negatively associated. about 60% of species association, both negatively and positively, were among the 'native species' (species that existed before the fire events) and 'non-native species' (new comer species that regenerated after the fire) in the plot sample. a non-native species, vernonia arborea, associated negatively with the non-native species tabernaemontana sphaerocarpa, and native species, oncospem homdum, palaquizlm das_yph_vllzm and endiandra mbescens. the distribution pattern of four native species, artocarpzls anisoph_vlhs, cananga odorata, croton laev~olius and macarangagigantea, changed after repeated fires, from uniform to clumped. keywords: association, distribution pattern, dominance, east icalimantan, forest fire introduction samboja research forest is a remnant, lowland, mixed, dipterocarp forest ecosystem in east icalimantan, indonesia, in whch the commercial and high-quality timber species grow, such as meranti (shorea sp.) and ulin (ezlsideroydon ~agem) (delmy 2001). fifty-five (55) species of dipterocarpaceae were preiously *corresponding author, email: s.rahayu@cgiar.org growing in the area (i<artawinata et al. 2008). however, the forest was disturbed by major fires during the dry seasons of 1982/1983 and 1997/1998, leading to a decline in its tree species richness, from 254 to 148 species in 1.65 hectares (simbolon 2005). fires dramatically increased the canopy openness and light penetration owing to drastic declines in aboveground biomass immediately after the fires (slik et al. 2008). the high light environment was associated with an abundance of light biotropia vol. 28 no. 1,2021 demanding, light wood, large-leaved, small-seed spatial distribution of species and inter-species and long-seed dormancy species which are association are fundamental information for typically characteristics of early succession tree understanding species structures and coexistence species (slik e t al. 2010; swaine & whitrnore in communities &i e t al. 2014). however, inter 1988). specific association (between species) and spatial dominant pioneer species mostly occurred in distribution pattern of species in secondary the first decade of forest succession and then growth after fire disturbances have been rarely followed by climax species commonly studied. characterized-by the shade-tblerant species (~l ik e t al. 2008). species interactions during the succession process are of central importance in the ecology of a species because a number of biotic and abiotic factors influence the species interaction. in addition, inter-specific association plays a role in controlling species' diversity and composition (ludwig & reynolds 1988; condit e t a/. 2002). positive association will support the forest regeneration process because species will develop together, but negative association may lead to forest regeneration failure owing to the objectives of this research therefore, were (1) to analyze species' dominance affected by repeated fires; (2) to analyze associations between species and spatial distribution patterns after repeated fires; and (3) to provide recommendations for future restoration planning based on current conditions in ecosystem succession. materials and methods competition among species (wright 2002). however, species have natural mechanisms to study area avoid competition both from other individual trees and from other species through developing spatial distribution patterns (janzen 1970). restoration of degraded forest, instead of focusing only on preservation and protection of intact systems, has emerged as a conservation effort in the past few decades (dobson e t a/. 1997). traditional restoration efforts focusing on re-establishing abiotic conditions and relying on succession of biotic communities that remained resilient in degraded forest are faced with many challenges such as changes in landscape connectivity, loss of native species, shift in species dominance, trophic interaction and/or exotic invasion, and the effects of changes in the biogeochemical processes. identifying the constraints is a critical step toward providing recommendations on addressing these challenges (sudng e t dl. 2004). a shift in species dominance due to repeated fires was the constraint faced in samboja research forest (simbolon 2005; slik e t al. 2008). shifting dominance as a result of fire disturbances may affect the recovery interactions between existing species and future species composition. understanding the condition of dominant species, species association between species and spatial distribution patterns is important in forest-restoration planning. the a permanent 1.8 ha. plot was established amidst the 3,504 hectares of samboja research forest, icutai icertanegara and penajam paser utara district, east iealimantan, indonesia with location coordinates of 0'59'23"-0'59'27" s and 1 16'57'31"-1 16'57'51" e (fig. 1). the study site was a remnant lowland mixed dipterocarp forest that was affected by repeated fires in 1982/1983 and 1997/1998. methods the 1.8 hectare (120 x 150 m), divided into 180 subplots with size of 10 x 10 m, was established in the secondary forest of samboja research forest in 2011. the plot was affected by major fires in 1982/1983 and 1997/1998. trees above 10 cm diameter at breast height (dbh) were recorded from the sampling units of 10 x 10 m, with their dbh recorded and positions geo-mapped. leaf specimens were collected for species identification at the herbarium bogoriense, bogor, west java, indonesia. the collected data were then compared with those published by the indonesian institute of sciences pis) in 1981 (icartawinata e t al. 2008) and in 2003 (simbolon 2005). dominance, associakion and &stxibudon pattern of tree species in bum fo~est in east w a n t a n rahayu @ s/. figure mamboja research forest, east kalimantan, indonesia source: balitek ksda samboja khdtk sarnboja data analysis the association between species was the importance value index (ivl) of each analyzed by measuring the degree of association based solely on the presence or absence species was computed from the relative of species in the sampling units of 10 x frequency, relative density and relative dominance of the species (curtis 1959). higher 10 m subplots established in the sample plot m value indicates more dominant species (ludwig & reynolds 1988). presence and absence of single species in the sampling unit compared to others. was presented in binary data of 2 x 2 contingency (table 1). table 1 association index between two species species b presence absence species a presence a absence b where: a = number of subplots where both species, a and b, are present. b = number of subplots where species a is present, but species b is absent. c = number of subplots where species b is present, but species a is absent. d = number of subplots where both species, a and b, are absent. n = number of subplots. biotropia vol. 28 no. 1,2021 expected value of a species associated with other species was calculated using: (a + 6) (a + c) e (a) = n and association index (ai) is a e(a). chi-square test was used to test the significance of the association, using yates correction factor: degree of freedom for association index is r statistic software was used in this data analysis. there were two types of association: 1. positive, if a > e(a), that is, the pair of species occurred together more often than expected if independent. 2. negative, if a < e(a), that is, the pair of species occurred together less often than expected if independent. index of dispersion od) used to express the species distribution pattern based on the variation to mean ratio (ludwig & reynolds 1988), was computed as: where: o2 is the variance and p is the mean the three types of dispersion index are namely; (1) random, if o2 = p, (2) clumped, if o2 > p and (3) uniform, if o2 < p. chi-square statistics used to test significantly different id, was computed as: degree of freedom (db) is n-1. results and discussion species dominance the five most dominant species observed in the sample plots of the 10.5 hectare undisturbed ulin and dipterocarp forest in samboja research forest in 1981 were sborea laevis, pbolidocarpw mdjadun, dio@yros borneensis, ezlsideroylon zpvagem' and scapium macropopdurn (icartawinata et al. 2008). during the 1981 observation, three most dominant species were found in the smaller plot of 1.8 ha of undisturbed pbolidocarpas majadam) dio@yros borneensis and eztsideroylon pagem'. further analysis was focused on the smaller sample plots in the 1.8 ha. five years after the second fire of 1997/1998, three most dominant species based on stem basal area were found in the smaller plots, namely; pbolidocarpas myadan) easideroylon pagem' and dipterocarpas cornatas (simbolon 2005). these three species had individual trees that survived the repeated fires. the surviving individual trees were recognised from tree mapping analysis through comparing coordinate position and stem diameter of 1981 and 2011 data. large surviving trees having > 40 cm diameter of easideroylon pvagem' and dipterocarpas cornatas) as well as the palm species, pbolidocarpas mdjadan, having 20 30 cm diameter contributed both high basal area and ivi. other species that started to establish during the period had contributed to small ivi. the burnt area was dominated by trees having < 5 cm diameter and 10 15 cm tree diameter (sirnbolon 2005). thirteen years after the second fire (2011 observation), the three most dominant species dramatically changed from the climax species of ezasideroylon pagem' and dipterocarpas cornata~ to pioneer species of macaranga gigdntea and sub climax species of vernonia arborea. pbolidocarpus mdjadan was consistently in the third place fable 1). the ten most dominant species were mostly pioneer, except ezlsideroylon ?wagem' and dipterocarpw cornatas. this clearly indicated that 13 years after a fire disturbance, pioneer species colonized the open area. however, surviving species continued to grow among the pioneer species. the surviving individuals of pbolidocarpas mdjadan provided hgh levels of seed for the regeneration process. this endemic palm in icalimantan, mostly growing in swamp forest in the study area, was unaffected by the fires (simbolon 2005). moreover, mortality of this palm was relatively low, reaching 10% in the burnt forest, probably due to the physiological characteristics of its stem vascular structure (van nieuwstadt & shiel2005). dominance, association and distribution pattern of tree species in burn forest in east icalimantan rahayu e t al. table 1 species with the 10 highest m at 13 years after the second fire (2011 observation) no. family species local name importance value index 1 euphorbiaceae macaranga gigantea merkubung 35.29 2 asteraceae vernonia arborea merambung 30.54 3 arecaceae pholidocarpus majadum liran 12.98 4 moraceae artocarpus anisoph_ylhs mentawa 12.27 5 rutaceae melicope glabra sampang/terutup 12.19 6 euphorbiaceae croton laevfoli,vs lasa-lasa/belanti 7.46 7 lauraceae eusideroylon .ywagen' ulin 7.20 8 verbenaceae peronema canescens sungkai 6.71 9 annonaceae cananga odorata icenanga 5.73 10 dipterocarpaceae dipterocarpw cornutus keruing gajah 5.62 easideroqlon pagem' is another surviving species that had a slight decrease in m from 9.3 before to 7.2 after the fire. e. ?wagem' has the capacity to re-sprout from damaged trees after fire (delmy 2001), even from stumps and roots (van nieuwstadt & shiel 2005). this species is categorized as heavy wood, ranging 0.88 1.19 g/cm3, on average 1.04 g/cm3 wartawijaya et al. 1992). heavy wood species (> 0.8 g/cm3) have the capacity to survive fire (van nieuwstadt & sheil 2005; brando et al 2012). high density wood produces some extractive material, such as cellulose, hemicelluloses, and lignin, and other properties, that reduce the fire distribution index and ignition time (brando et al. 2012). before fire, macaranga gigdntea was a minor species in the permanent plot of samboja research forest (icartawinata e t a/. 2008). after repeated fires, the number of trees increased rapidly from 35 in 2003 (simbolon 2005) to 167 in 2011. three years after the fire, macaranga gigantea was the most dominant species in the twice-burned forest, (slik et al. 2008) and was consistently dominant at 5, 7 and 13 years. this species was regularly found as one of the most dominant species after 10 20 years in regenerating forest (silk e t al. 2008). however, in samboja research forest, m. ggigdntea was the most dominant species three years after the fire. the level of disturbance after the fire might have affected the early establishment of macaranga gigdntea. macaranga is an early succession genus that prefers a frequently disturbed habitat that is characteristic of a highly-disturbed forest (slik et al. 2003). m. gigdntea traits of being a pioneer and light demanding species are suitable for growth in burnt forest where high exposure to sunlight occurs. its typically orthodox seeds are able to lie dormant in the soil and germinate as soon as the forest canopy opens up (suita & nurhasybi 2009; susanto et al 201 6). vemonia arborea, a sub-climax species (desitarani et al. 2015), was not found in the sample plot before the fire events but developed rapidly and became the second dominant species in 201 1. however, ths species was found in an undisturbed forest located at 1 2 krn from the sample plot (jgisnawati et al. 201 1, atmoko e t al 2015). the small seeds of v: arborea can be dispersed by wind. moreover, this species was a co-dominant in the once-burned forest of sungai wein but not in the twice-burned samboja (slik e t al. 2008b). increasing a13+ after fires probably affected v. arborea colonization in burnt areas. a study in samboja lestari, east icalimantan documented that al+ content in the soil significantly increased four years after a fire and i/. arborea was associated with high a13+ content (j'asir e t al. 2010). high a13+ content in the soil becomes a limiting factor of root growth (mossor-pietraszewska 2001) for common species, but not for v. arborea whch is adapted to high a13+. in samboja, the v. arborea population developed widely after 2003 (five years after the second fire). the occurrence of pioneer and sub-chax species as the most dominant, together with establishing climax species in the burned forest has indicated that the succession process was in a competition phase (clement 191 6). in order to address restoration management goals, the assisted natural regeneration and species enrichment activities can be applied in the forest succession phase (elhot e t al. 2013; lamb & gilmour 2006). assisted natural regeneration should focus on species priorities and enrichment, particularly for sub-climax and climax species, to improve the forest function and therefore, conserve biodiversity. biotropia vol. 28 no. 1,2021 species association negative association occurred more frequently thirteen years after the second fire, an analysis of the 191 established species in the sample plots of secondary forest showed that 18,336 pairs of species pairing with themselves, consisted of 1,220 (7%) pairs with positive association, 17,115 (93%) pairs with negative association and a pair of species as neutral. chi square test found 38 pairs with significantly positive association and 4,801 pairs with negative association. t h s positive association rarely occurred but negative association was common, particularly, among 'non-native' species (icuebbing & nunez 2015). non-native and native species in this research refers to the existence of species before and after the fires. non-native species were found in the plot only after the fires but native species were found before and after the fires. however, positive association is important in establishing management priorities (icuebbing & nunez 2015). it is critical in community structure or habitat modification, in which one individual or species alters the condition of the local environment, often making a stressful habitat more hospitable to other individuals or species (stachowicz 2001). furthermore, positive association between species in mutual interaction will enhance each other's survival probabilities (ludwig & reynolds 1988). positive interactions between different species are of particular interest because of their potential to 'cascade' throughout the com munity, with a major effect on the ecosystem structure and function (stachowicz 2001). some 53% positive association and 60% negative association in the sample plot occurred between species that existed before the fire ('native') and newcomer species that regenerated after the fire ('non-native'). in this research, the between 'native7 and 'non-native' rathe; than among 'non-native'. the more frequent occurring association among the 'non-native' species indicated that 'non-native' species indirectly provide environmental support (flory & bauer 201 4). improving performance of the 'non-native' species after fires is associated with natural resources availability or changes in disturbance regime (daehler 2003). however, light competition as an indirect impact of fire disturbance was an important factor affecting interaction among individual trees or species (icunstler e t al. 2012). vernonia arborea, a 'non-native' species, was positively associated with one 'native7 species anthocephalm chinensis but negatively associated with other 'native7 species, oncospem homa'um, palaquium daqpblhm, e didndm mbescens and 'non-native7 species, tabernaemontana sphaerocapa (table 2). a. chinensis, a surviving individual tree provided a suitable environment for sub-climax species of v. arborea seedlings. anthocephal,w chinensis grows in the swampy area of the plot unaffected by fires (simbolon 2005). v. arborea generally grew in the upper elevations of the sample plot. only a few individual trees grew on the swamp area close to anthocephalzls chinensis. as a sub-climax species, v. arborea probably took advantage of a. chinensis' canopy for shade during early regeneration. negative association between i/. arborea with tabernaemontana sphaerocapa, oncospem hom'dum, palaquium daqpbllzm and endiandra mbescens was probably because of different habitat requirements. tabernaemontana sphaerocarpa, oncospem bom'dum, palaquium daqpkyllum and endidndm mbescens grew on lower elevations, in the swampy part of the sample plot. table 2 ten species' pairs with highest yates chi-square value pair of species yates x2 critical association a ea df correction value type knema laterigia neonauclea cabcina 2 0.07 15 32.24 25.00 + calopbylhm soulattri di0.pyro.r pilosanthera 2 0.08 8 25.59 15.51 + anthocephalus chinensis vernonia arborea 4 2.92 12 24.31 21.03 + knema cinerea pobalthia mmphii 2 0.09 3 23.80 7.82 + pobalthia m p h i i q p g i m brachyrachis 2 0.09 3 23.80 7.82 + tabernaemontana sphaerocarpa vernonia arborea 0 0.83 2 1861.84 5.99 tabernaemontana hauilandii vitex pinnata 0 0.01 2 129.56 3.84 oncosperma homdum vernonia arborea 0 0.83 2 127.65 3.84 palaquium dasyphyllzm vernonia arborea 2 2.50 2 91.47 25.00 endiandra mbescens vernonia arborea 0 0.83 2 68.78 3.84 notes: a= number of subplots where both species, a and b, are present; ea= expected value of a species associated with other species. dominance, association and distribution pattern of tree species in burn forest in east icalimantan rahayu et al. v. arborea generally grew in the upper elevations of the sample plot. only a few individual trees grew on the swamp area close to anthocephazas chinensis. as a sub-climax species, v. arborea probably took advantage of a . chinensis' canopy for shade during early regeneration. negative association between v. arborea with tabernaemontana sphaerocarpa, oncosperma hom'dam, paiaqaiam dagpbiiam and endiandm rabescens was probably because of chfferent habitat requirements. tabernaemontana .phaerocarpa, oncospema homzmzd~m, pazaqaiam dagtp&lzam and endidndm mbescens grew on lower elevations, in the swampy part of the sample plot. species distribution pattern species with significantly distributed clumped (table 3). the changing distribution pattern of artocarpzls anisopbizus, cananga odo~ata, croton zaevfolizls and macaranga gigdntea from uniform to clumped, after repeated fires, was due to various factors, such as tree survival in the cooler site and their stem size (davis et al. 2005). the survivor trees in the cooler site functioned as seed sources when most trees were killed, while their small stem size tended to clump. the nature of its fruits and seeds had also affected their distribution pattern after the fire. artocarpas anisop&zlzs has a compound fruit. it contains many 1.7 x 1.0 cm seeds that are difficult to chsperse without a dispersal agent, such as a big mammal. the swampy area was suitable for cananga odorata and croton zaevfohas. swampy in the 1981 observation, the species distribution in the 1.8-hectare sample plot of area depletion and droughts after the fires unchsturbed forest was a balance between became a limiting factor for c. odorata and c. uniform (48%), where the distance between iaevfohas growing in the sample plot. macaranga neighbouring inchviduals is maximised due to gigdntea, a minor species and distributed competition of resources, and random (52%) uniforml~, changed dramatically to dominant where the spacing between individual is and clumped. after biomass burning, open areas unpredictable, usually occurring in the habitat were associated with pioneer species, such as m. with consistent environmental condition. gigdfftea, due to hgh light as an repeated fires affected the species' distribution pattern. analysis of 191 species in the 1.8 hectare secondary growth forest indicated that 69 species (369'0) were distributed randomly, 28 species (14'/0) were clumped and 95 species (50%) were uniformly distributed. clumped dispersion of species was found in the secondary growth forest after repeated fires. further statistical testing using the chi-square found six source during the colonization process. clumped pattern of peronema canescens might affect the planting of this species in a forest rehabilitation program. during the forest rehabilitation activities in samboja researh forest in 1990s p. canescens was one of the species planted in the area for fire breaks and boundary area signs to community land. then, it has spread out after the repeated forest fire. table 3 dispersion index of six species in the 1.8-hectare secondary forest after repeated fires in samboja research forest species name dispersion variance average index d f critical value adocarpus anisap~llz.r 1.97 0.37 5.28 348.74 66 85.98 cananga odorata 0.21 0.1 3 1.66 36.61 22 32.67 croton laevfolizls 0.31 0.1 8 1.77 54.86 31 44.98 macaranga gigantea 1.37 0.93 1.48 245.95 166 197.06 peronema canescens 0.33 0.1 8 1.80 57.48 32 46.19 vernonia arborea 1.29 0.75 1.73 231.32 134 162.01 biotropia vol. 28 no. 1,2021 conclusion the dominance of macaranga gigantea indicated a high level of forest disturbance in that part of samboja research forest affected by two fire events. co-dominance of vemonia arborea indicated the changing environmental conditions, such as increasing a13+, whch was limiting to the species growth. occurrence of pioneer, sub-climax and climax species indicated that the forest succession was in a competition phase. assisted natural regeneration and species enrichment would be possible restoration activities for biodiversity conservation. improving non-native species performance in the twice-burned area might occur due to changing natural resources availabihty and &sturbance regime after a fire. lastly, the changing environmental conditions, species characteristics and human activities affected the distribution pattern of species. references atrnoko t, yassir i, sitepu bs, mukhlisi, widuri sa, muslim t, mediawati i, ma'mf a. 2015. keanekaragaman hayati hutan rintis wartono icadri: hutan tropis igmantan di iwdtic samboja. balikpapan (id): balai penelitian teknologi konservasi sumber daya alam. 104p. brando pm, nepstad dc, balch ji<, bolker b, christman mc, coe m, putz fe. 2012. fire-induced 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biodiversitas 17(1):192-9. swaine md, whitmore ct. 1988. o n the definition of ecological species groups in tropical forests. plant ecology 75(1):81-6. mossor-pietraszewska t. 2001. effect of aluminium on plant growth and metabolism. acta biochim pol 48(3):676-86. van nieuwstadt mgl, sheil d. 2005. drought, f ~ e and tree survival in a borneo rain forest, east kalimantan, indonesia. j ecol93:191-201. wright sj. 2002. plant diversity in tropical forests: a review of mechanisms of species coexistence. oecologia 130: 1-1 4. yassir i, van der kamp j, buurman p. 2010. secondary succession after fire in imperata grasslands of east kalimantan, indonesia. agric ecosyst environ 137:172-82. 1229.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 1. fg dwiyanti (population) r... 1. kali jati solok bokong s o lo k d e w a ta 2. solok besek biotropia vol. 28 no. 1,2021: 64 73 doi: 10.11598/btb.2021.28.1.1082 omega-3 content of black soldier fly prepupa (hefmetia illucens) fed with marine fish offal and tofu dreg ramadhas1 eig4 putra'>z*, yenyen fatmalasari', ida i(inasih3, agus dana permana', mia rosmiatil and elisa nurma riana2 'school oflife sciences and technology, institut teknologi bandzmg, jalan ganesha 10, bandung 40132, indonesia 'dtpan%ent o f biolog, institut teknologi sumatera, jalan temsan rjacudu, lmpzlng 35365, indonesia 'dtpar;tment ofbiology, uniuersitas islam negeri sunan gunung djati bandzmg, cibim, bandung 40615, indonesia received 10 june 2018/accepted 9 december 2019 abstract one of the most promising materials with great potential as a nutrition source of animal feed is the black soldier fly prepupae (bsfp) (hemetia illucens) which is fed with organic wastes. this study was designed to observe the accumulation of omega-3 from the organic wastes of marine fish offal and tofu dregs converted as the biomass of the harvested bsfp. a total of 150 g food consisted of marine fish offal and tofu dreg with different proportions of fish offa1:tofu dreg at 10:90, 25:75, 50:50, and 100% tofu dregs were fed to black soldier fly larvae (bsfl) for 21 days. at the end of the feeding period, all bsfl were harvested, weighed, and then analyzed for omega-3 fatty acids content using gas cromatography with flame ionization detector (gc-fid). results showed that bsfl reared with 25% marine fish offal produced the highest biomass (8.1 g/50 larvae) with the least development time (19 days). the total omega-3 recorded from the application of oo/o, 10°/o, 25%, and 50% of marine fish offal was 0.02%, 0.87%, 2.16% and 2.61% in 100 g of dry weight, respectively. these results showed the possibility of producing specific nutrients from bsfp biomass converted from organic wastes, thereby providing baseline knowledge for further formulation of specific animal feed from bsfp. keywords: black soldier fly, fish offal, omega-3, tofu dregs introduction organic waste is the most dominant waste produced in indonesia (zulfikar & chaerul 2010). most of these end up as piles of wastes as the collection and waste management efforts could only cover about 50 70% of wastes produced by the population (cointreau 1994; rushbrook & pugh 1999). common methods applied to manage these wastes include the costly sanitary landfill and composting which produce compost of low economic value, thereby making both methods less desirable in low income regions (cointreau 1994). another alternative in managing organic waste is its conversion into useful biomass using saprophages through the natural decomposition *corresponding author, email: ramadhani@sith.itb.ac.id process (beard & sands 1973; boushy 1991; barnard et al. 1998; ndegwa & thompson 2001; ramos-elorduy et al 2002; barry 2004; diener et al. 2009; manurung et al. 2016; supriyatna e t al. 2016). latest developments include the increasingly becoming popular trend of using insect larvae as organic wastes decomposting agents, in producing a proteinand lipid-rich biomass (diener et al. 2009). among these, the black soldier flies larvae (bsfl) (hermetid i h c e n s ) has been considered to be the best canddate due to their great ability to convert various types of organic wastes into high protein-rich biomass which could be high quality sources of protein and lipid for animal feed, as well as, potential sources of biodiesel (newton et al. 1977; newton et al. 2005a, b; st hilaire et al. 2007a; myers et al. 2008; rachmawati et al. 2010; li et al. 201 1; muin et al. improvement of omega-3 content of black soldier fly prepua (hemetin illzcen~) . . ... eka putra et al. 2017; renna et al. 2017; spranghers e t al. 2017; ignasih et al. 201 8). other benefits of using h. illzcens as organic wastes conversion agent, especially in tropical regions like indonesia, are: 1. the species is adapted to tropic and warm subtropical region (tomberlin et al. 2002); 2. able to suppress the population of house flies (musca domestica) through predation and competition for nutrition (furman e t al. 1959; newton et al. 2005a, b); 3. able to reduce pathogens like escberichia cob and salmonella entericd (erickson et al. 2004); 4. has short development time (about 2 to 3 weeks) (newby 1997); 5. adult flies do not eat fresh material and females do not oviposit their eggs directly on food source of larvae which made them less likely to tranfer disease (leclercq 1997); and 6. adult flies are not attracted to human habitats or food (van huis et a/. 2013). moreover, ths insect has digestive system rich with amylase, lipase, protease, a-galactosidase, p galactosidase, a-mannosidase, and a-fucosidase allowing them to digest almost any organic materials (jam et al. 2011). like other insects, their digestive system also allows them to accumulate certain organic molecules (st-hilaire et al. 2007b). all of these benefits can be harnessed to solve one of the main obstacles of food security in indonesia, particularly protein insufficiency, or the limited availability of protein rich yet affordable feed stock. among organic wastes produced in large volumes in indonesia, wastes with relatively unchanged organic matter properties during its production are tofu dreg and marine fish offal. about 731,501.5 ton of tofu dreg is produced annually (bisnis ui(m 2009), while 25 30% of marine fish catch ended up as wastes (ici<p 2016). valuable organic wastes such as tofu dreg is rich in carbohydrate and protein (li et al. 2012; li e t al. 2013), while the marine fish tissues are rich in beneficial unsaturated fatty acids (sahena e t al. 2009). the nutritional content of bsfl biomass highly depends on quantity and quality of the feeding material (nguyen e t al. 2015; oonincx et al. 2015). moreover, the fat content showed more variation (6.6 to 39.2%) than protein (37.0 to 62.7%) (barragan-fonseca e t al. 2017) suggesting the strong effect of the larval dietary nutrient to protein and fat content. one main purpose of bsfl production is to manufacture an alternative source of feed material for several livestock, such as poultry (leiber et al. 2017; ssepuuya et al 2017) and cultured fish species (webster et al. 2015; stadtlander et al. 2017). a growing number of studies showed the possibility of modifying the fatty acid profile of bsfl, improving the fat nutrient value (i.e., healthier fats such as omega-3) of local livestock product through manipulation of feed for better valuation of the product (stoneham et al. 2018). the lipid content of insects is largely dependent on their diet (stanley-samuelson & dadd 1983) and many of them have natural long-chain unsaturated fatty acids in their biomass (thompson 1973). these omega-3 content of bsfl could be enhanced by allowing them to feed on fish waste from rainbow trout processing plant (st-hilare et al. 2007b). unlike rainbow trout, most marine tropical fishes have low omega-3 content. in h s study, the combination of both marine fish offal and tofu dreg were utilized as feeding material of bsfl to enhanced the omega-3 content of its biomass. the main objective of this study is to produce bsfl biomass with high omega-3 content at laboratory scale as a basis for large scale production of an omega-3 rich alternative source of livestock feed converted from organic wastes. materials and methods animal specimen the larvae of the black solder fly were obtained from the eggs that were purchased from bsf farm in sumedang, west java. all eggs were kept in the substance made of commercial chicken feed (60% moisture) and kept at a constant temperature (28 "c, 70% rh) in a container (50 x 25 x 10 cm) at the laboratory of enviromental toxicology, school of lfe sciences and technology, bandung, indonesia. animal treatment the marine fish offal originally from local fish market and tofu dreg, produced as waste of the local tofu industry of sumedang, west java, biotropia vol. 28 no. 1,2021 table 1 proximate analysis of the feeding material proportion of fish offal to protein lipid carbohydrate tofu dreg ("10) ("10) ("10) 0% 19.59 6.35 6.07 were used as the growth medium in ths study. all materials were washed with chlorine-free water. four combinations of "marine fish offal: tofu dreg", at 10:90; 50:50; 25:75, and 100% tofu dreg, were prepared as food for the black soldier flies larvae and kept frozen for 24 hours before treatment. the proximate analysis was conducted on all feed (table 1). fifty individuals of two-day old larvae were placed inside plastic cups (with three replicates for each treatment) and fed every 3 days with 150 mg (wet weight, 60% moisture content) of the food prepared according to the treatment. the larvae were initially placed onto the prepared and defrosted food inside the plastic cup. larval weight and weight of remaining food were measured every 3 days until 40% of all larvae metamorphed into prepupae when feedmg was also stopped (tomberlin et a/. 2002). all prepupae were removed daily from each container and weighed, then placed in plastic container and prepared for extraction. all experiments were conducted at room temperature of 30 32 "c, relative humidity of 65 85%, and 12 hour photoperiod. sample preparation for fatty acid analysis extraction all prepupae were cleaned by fresh water and dried at 60 oc for 12 hours. about 0.15 g of dried sample was ground then mixed with 3 ml solvent (2 part chloroform and 1 part methanol). the mixture was then homogenized for 30 min, centrifuged at 5,400 rpm at 4 "c for 5 to 15 min and the supernatant was then collected. homogenization and centrifugation processes were repeated 3 times. the collected supernatant was then evaporized to remove the organic solvent. the crude lipid sample was mixed with 4 ml naoh 0.5 m with methanol solvent. about 0.024o/o butylated hydroxytoluene (bht) was added, and then the solution was agitated for 8 hours at 55 "c. samples obtained after the esterification process were added with 300 ml methanol and 1 2.5% hzs04 (v/v) in meoh, and then agitated for 1 hour. the n-hexane and nacl (1 : 1) solution was added to the samples and then centrifugated for 10 min (4,000 g) at room temperature. nazs04 was then added in order to remove the excess water from the samples, and the supernatant was collected and kept in a separate container. gc-fid analysis for omega 3 content the omega 3 content analysis was conducted using gas chromatography-flame ionzation detection (gc-fid). gc column used was db-5 30 m x 0.25 mm id x 0.25 pm with nz and hz as the carrier gases. the applied injector temperature was 200 "c and the detector temperature was 300 "c. the oven temperature which started at 140 "c was increased to 270 "c, at a rate of 4 "c/min, and maintained for 7.5 min. the fatty acid was quantified with external standard method based on the formula: as x ces cs = aes where: cs = sample concentration as = sample area aes = external standard area ces = external standard concentration data analysis the final weight of harvested prepupae were compared across all diet composition using analysis of variance, with significance level set at p < 0.05, followed by the tukey-igamer hsd test when significant differences were detected. all analyses were done using spss version 22.0. improvement of omega-3 content of black soldier fly prepua (hemzetia illucens) . . ... eka putra et al. results and discussion component has altered the intake of other the black soldier fly larvae were able to recycle a proportion of fish waste which consists of high protein and fat. the 10% of fish offal fed to the larvae has slightly improved the weight of harvested prepupae. however, the ad&tion of more fish offal at 50% showed negative correlation to harvested weight, although not significant table 2). the high mortality of larvae when fed with higher fish offal concentration was probably caused by: 1. lack of balance in the protein: carbohydrate combination of the feeding material. both of these macromolecules are very important for growth, reproduction and survival of insects (agulla e t al. 2013). proximate analysis showed the protein : carbohydrate level of 28.54 : 1 (table 1). studies showed that high protein content of the feed material produced high mortality rate in bsfl population (cammack & tomberlin 2017; barragan fonseca e t al. 2019; danieli e t al. 2019); 2. the production of juice through natural decomposition of the fish offal. this juice increased the moisture of feeding material, reduced the amount of available oz required for growth of insect larvae, and induced the molting process (harrison e t al. 2006; iaok & harrison 2009; harrison & haddad 2011; callier & nijhout 201 1). since bsfl were subjected to specific diet restrictions, the consumption of one &et components. since larval weight is highly associated with carbohydrate content (le gall & behmer 201 4; barragan-fonseca 201 9) this probably explains the presence of heavier larvae at 25% fish offal. larvae fed with 25% and 50% fish offal required less development time to metamorph into the harvested prepupae (table 1). the shorter development time of bsfl reared on high protein diet in the current study agrees with other results reported by oonincx e t al (2015b) and cammack & tomberlin (2017). the development time of larvae in this study was even lower than those of other studies (fatchurochim e t al. 1989; myers e t al. 2008; diener 2009; nguyen e t al. 2013; oonincx e t al. 2015a, b; st-hilaire e t al. 2007b; cammack & tomberlin 2017). this is probably related to the higher rearing temperature used in the current study. however, this result indicated the potential applicability of this method in tropical region like indonesia. the growth pattern showing weight gain was stopped at dfferent times among the groups. larvae at groups of 25% and 50% fish offal, stopped gaining weight at lbth day while those of groups 0% and 10% fish offal at 19th day (fig. 1). the peak weights among groups were similar indicating that variations in the food composition did not affect the critical weight for prepupae development. table 2 final weight and development time of harvested prepupae proportion of fish offal: fresh weight per 50 prepupae development time dry weight per 50 prepupae tofu dreg* (yo) (8, (day) k) note: * = percent combination of fish offal: tofu dreg biotropia vol. 28 no. 1,2021 7 10 13 16 19 days 0% 010% 25% m50% figure 1 changes in the weight of black soldier fly larvae fed with various combination of fish offal and tofu dreg notes: data on 50% fish offal are not shown as more than 40% of larvae already metamorphosed into prepupae; * = significant at p < 0.05. most of the fatty acid content of bsfp biomass consisted of saturated fatty acid (fig. 2). similar results of highly saturated fatty acid content of black soldier fly prepupae were also obtained by other studies (st-hilarie 2007b; finke 2013; ramos-bueno e t al. 2016; surendra e t al. 2016; renna e t al. 2017; schiavone e t al. 2017; spranghers e t al. 2017). the feeding material rich in carbohydrate but low in protein produced more crude fat (barragan-fonseca 2019) which is related to lipogenic activity and incorporation of lipid as body reserve (nestel & nemny-lavy 2008). the higher saturated fatty acid content in 10°/o fish offal group indicated the possibhty of bsfp converting the unsaturated fatty acid into saturated fatty acid while hgher fat content of the 50% fish offal group maybe responsible for the higher content of unsaturated fatty acid (danieli e t al. 201 9). further analysis on each component of fatty acid showed high content of c12:o (lauric acid) (table 3). this conformed with other studies on the bioconversion of organic wastes by black soldier fly larvae (st-hilarie 2007b; sealey e t al. 201 1; leong e t al. 201 5; spranghers e t a/. 2017). the increase of lauric acid content associated with the decreasing amount of tofu dreg indicated the possible accumulation of lauric acid from fish offal as tropical fish tissues are rich in saturated fatty acid (gopakumar & nair 1972). the high content of this fatty acid might be related to the antibacterial function of insect larvae (urbanek e t al. 2012). richness in c12 : 0 found in bsf favor its inclusion in poultry feed since ths fatty acid is better absorbed and metabolized than the long chain fatty acids (lcfa) for nutrition and for antibacterial activities (skrivanova e t al. 2006; kim & rhee 2016). however, hgher saturated fatty acid content of the harvested prepupae could also increase the saturated fatty acid content of meat when it was applied as feed, a condition which is undesirable for modern consumers who prefer healthier meat and meat products (schiavone e t al. 2017). improvement of omega-3 content of black soldier fly prepua (hemzetia illucens) . . . .. eka putra e t al. 0% fish offal 10% fish offal 25% fish offal 50% fish offal 6 5 x 2 4 .w lc 3 0 figure 2 proportion of saturatted and unsaturatted fatty acid of harvested prepupae (% dry matter basis) table 3 fatty acid compostion of black soldier fly prepupe (% dry matter basis) 5.61 diet combination lipid acid 0% fish offal 10% fish offal 25% fish offal 50% fish offal 2.59 p 8 2 e 1 0 0 c lo:o c 12:o c 140 c 16:l c 16:o c 18:o c 18:3n-3; ala c 20:5 c 20:5n-3; epa c 22:6n-3: dha unsaturated fatty acid 4.23 2.6 total omega-3 0.02 0.87 2.16 2.61 1.69 1.57 omega-3 consists of three main lipid acids, ala, epa, and dha. the amount of fish offal added to the diet was positively correlated with the amount of these molecules inside prepupae biomass. in general, the total amount of epa and dha of prepupae in this study was higher than simdar study by st-hillaire e t al. (2007b) (fig. 3). low concentration of omega 3 in prepupae might be related to the low omega 3 content of tropical fish offal (osman e t al. 2001; mohanty e t al. 201 6; alhazzaa e t al. 201 8). combining fish wastes with other types of wastes could change the molecular properties of the biomass produced. higher nutritional value of tofu dreg compared to cow manure which has much lower nutritional value might explain the difference of the results between this study and that of st-hdlaire e t al. (2007b). this study showed that fatty acids content of the black soldier fly prepupae could be increased and manipulated in order to produce desirable "healthy" fatty acids such as ala, epa, and dha by feeding the larvae with more tropical marine fish waste (barroso e t al. 2017; danielli et al. 2019). although the application of this 1.07 method as an omega-3 source for human consumption would still require further studies, ths method is already applicable in the production of a high-quality animal-grade foodstuff that is a suitable replacement for fish meal, fish oil, or vegetable oils in animal diets through the recycling of organic wastes. furthermore, the application of omega-3 rich feed material may change the fatty acid profile of the meat into a much healthier meat that is rich with unsaturated fatty acid (saito e t al. 1996; li e t al. 2016). the fatty acid composition of bsf lipid highly depends on the rearing substrate (makkar e t al. 2014; oonincx e t al. 2015b; leong e t al. 2016). therefore, further research is suggested to assess how much fatty acid can be improved through variation in substrate composition due to differences in fatty acid requirement for growth rocher 2010). when the fatty acid profile of bsf wdl be equal to the animal diets that contained fish meal/oil it will reduce the pressures on global marine fisheries and will also maintain environmental health (tomberlin e t al. 2015). biotropia vol. 28 no. 1,2021 tofu dreg 10% fish offal 25% fish offal 50% fish offal and 90% tofu and 75% tofu and 50% tofu dreg dreg dreg c. 3.5 3.5 x oala h epa i3 oha total x e 3 e p . 2.5 p -2.5 m 4 d m 2 2 2 1.5 1.5 m m z 1 1 0.5 m 0.5 0 p n. cow manure 10% fish offal 25% fish offal 50% fish offal and 90% cow and 75% cow and 50% cow manure manure manure oala hepa bdha .total ii$ f n * o n figure 3 percentage of a-linolenic acid (ala), eicosapentaenoic acid @pa), and docosahexaenoic acid @ha) on a total dry matter basis for prepupae feed diets containing different proportions of fish offal and tofu dreg (left) and results of sheppard et al. 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vhesis]. bandung (id): institut teknologi bandung. 6. okky (postharvest).cdr biotropia vol. 19 no. 2, 2012: 115 129 postharvest quality improvement of sorghum ( (l.) moench) grainssorghum bicolor okky setyawati dharmaputra , santi ambarwati , and ina retnowati received 29 june 2012/accepted 26 october 2012 the objectives of this study were (a) to investigate the effect of postharvest handling (threshing and storing) methods on the quality of sorghum ( (l) moench) grains variety numbu, in terms of the percentages of damaged grains and seed germination, population growth of , and ; fumonisin b and carbohydrate contents, and the percentage of weight loss during storage. the change of moisture contents of sorghum grains was also recorded. threshing was conducted using wooden stick and a paddy thresher. sorghum grains were packed in hermetic plastic bags. the conditions inside of the bags were airtight and normal. each bag with different conditions inside was infested with 10 pairs of (1-14 days old). sorghum was stored for one, two and three months under warehouse conditions. the results showed, that the moisture contents of sorghum were lower than its standard safe moisture content (<14%) during storage. at the beginning of storage, the percentage of damaged grains caused by threshing using wooden stick was higher than that of using a paddy thresher. the increase of percentage of damaged grains was caused among others by increase population under normal oxygen concentration inside of the bag (about 21%), consequently the percentage of weight loss was also increased. the percentage of seed germination of sorghum threshed using wooden stick was lower than that of threshed using a paddy thresher. the percentage of seed germination decreased with the increase of storage duration. population of and . decreased with the increase of storage duration. fumonisin b content of sorghum threshed using wooden stick was higher than that of using a paddy thresher during one, two and three months of storage. fumonisin b contents were considered low. in general, carbohydrate content of sorghum threshed using either wooden stick or paddy thresher from the beginning up to three months of storage were not significantly different. threshing using a paddy thresher was better in comparison to threshing using wooden stick. postharvest quality, sorghum, 1,2* 1 1 1 2 seameo biotrop, jl. raya tajur km. 6, bogor, 16134, indonesia department of biology, faculty of mathematics and natural sciences, bogor agricultural university, dramaga campus, bogor 16680, indonesia sorghum bicolor sitophilus zeamais fusarium proliferatum f. verticillioides s. zeamais s. zeamais f. proliferatum f verticillioides sorghum bicolor abstract 1 1 1 key words: * corresponding author : okky@biotrop.org 115 introduction as foodstuff, sorghum ( (l) moench) is the fifth most important cereal after rice, wheat, maize, and barley in the world. sorghum can adapt to broad agroecology, gives high production, and is more resistant to drought, pests and diseases, compared to other food crop. it has also high nutritional content which is consequently good to be used either as alternative food or feedstuff. according to suarni (2001) sorghum has carbohydrate content of 80.42% as compared to 86.45% in milled rice and 79.95% in maize. in terms of protein, sorghum has slightly higher content (10.11%) than milled rice (9.28%), but slightly lower than maize (11.02%). sorghum's lipid content value (3.65%) is between milled rice (1.88%) and maize (5.42%). the use of sorghum as foodstuff in the form of flour is more beneficial, because it is easier to be processed into food products such as cake, cookies, bread and noodle. the capability of sorghum flour substitution upon wheat flour varied, i.e. for cookies, cake, bread and noodle was 50-75%, 30-50%, 20-25% and 15-20%, respectively (suarni 2004). sorghum grains could substitute maize for feed stuff, because the nutritional content of sorghum is not so different from that of maize, (sirappa 2003). since long time ago sorghum plant has been recognized by farmers in indonesia, especially in java, west nusa tenggara and east nusa tenggara. nevertheless, the production of sorghum is still low. although in indonesia the production of sorghum is still low compared to other countries such as india, china and united states, it is equally important to pay attention to the postharvest handling of this commodity to maintain its good quality during storage. cultivation and development of sorghum are important to anticipate food crisis caused by global warming. in indonesia, the low production of sorghum grain is due to (a) the nonavailability of appropriate technology for sorghum handling and processing after harvest, (b) the limited knowledge on how to process sorghum grain into various food products, (c) the lower price of sorghum grains compared to milled rice, maize and peanuts. the postharvest handling (drying, threshing and storing) can affect the quality of sorghum. dharmaputra . (2010) reported that based on the surveys conducted in demak and wonogiri regencies in 2010, the postharvest handling of sorghum practiced by farmers and collectors was not carried out appropriately, therefore sorghum grains were easily attacked by insects. the dominant insect species found were and . the most common fungi found in sorghum were , and . during storage, sorghum could be infested by insects, microorganisms, mites and rats. insects are considered the most significant cause of postharvest losses. among microorganisms, fungi are the most important cause of deterioration of stored grains. the role of insects in fungal infection cannot be disregarded. aside from injuring grains, insects also serve as carriers of fungi. furthermore, the metabolic activities of insects produce heat and moisture (especially during longterm storage) which stimulate fungal growth. fungal infection in grains can cause discoloration, decrease sorghum bicolor et al sitophilus zeamais tribolium castaneum aspergillus flavus fusarium semitectum f. verticillioides 116 biotropia vol. 19 no. 2, 2012 in germination, physical quality and nutritional contents, and also mycotoxin contamination (sauer 1992). fumonisins are important mycotoxins, produced mainly by and in several agricultural products worldwide, especially in maize and sorghum (marasas . 1988; abdel-hafez . 1990; da silva . 2000). the former name of is . according to desjardins (2006) there are four kinds of fumonisins occurring at significant levels in naturally contaminated grain, i.e. fumonisin b , b , b and b . the most toxic among these four kinds of fumonisins is fumonisin b which can cause leukoencephalomalacia in equines (marasas . 1988 ) and rabbits, pulmonary edema in swine (bucci . 1996; harrison . 1990) and it has been reported as a probable cause of esophageal cancer in humans (marasas 2001). a high incidence of esophageal cancer has been observed in certain geographic areas and ethnic groups in africa, asia and latin america. in indonesia, no research has been done on fumonisin level in sorghum. the method of postharvest handling is one of the factors that can affect the quality of sorghum. however, the postharvest practices in indonesia are still inadequate, it is important to conduct research on the effects of different postharvest treatments on the quality of sorghum grain. this study is a continuation of the research conducted last year. the objectives of the research were: (1) to investigate the effects of postharvest handling (threshing and storing) on the quality of sorghum grains, in terms of the percentages of damaged grains and seed germination, population growth of , . and . , fumonisin b and carbohydrate contents, and the percentage of weight loss; (2) to record the change of moisture contents of sorghum grains during storage, and (3) to recommend proper postharvest handling methods to ensure the quality of sorghum grains during storage. the research results should provide recommendations to farmers, collectors, food and feed industries on the most adequate postharvest handling practices (threshing and storing) for sorghum to maintain good quality of grain during storage the variety of sorghum used in this study was numbu, cultivated by pt tri fondasi indonesia in balaraja subdistrict, tangerang regency, west java. the numbu grains were harvested 95 days after sowing. sorghum was harvested by cutting the panicle from the standing stalk using a sickle. sorghum in the form of panicles was sun-dried on tarpaulin. drying was conducted up to moisture content of about 13%. threshing was done using wooden stick and a paddy thresher. after threshing, grains were separated from dirt and chaff by winnowing. sun-drying, threshing and winnowing were conducted in the location et al. f. proliferatum f. verticillioides et al et al et al f. verticillioides f. moniliforme et al et al et al s. zeamais f proliferatum f verticillioides 1 2 3 4 1 1 materials and methods variety of sorghum methods of harvesting, drying, threshing and winnowing . 117 postharvest quality improvement of sorghum okky s. dharmaputra– et al. where sorghum was cultivated. sorghum grains were then transported to seameo biotrop, bogor, by a car equipped with air conditioning. prior to packaging, sorghum grains were: (a) fumigated with phosphine for five days at 2 grams/ton of sorghum to control insect pest that may exist, (b) checked for the percentage of damaged grains. damaged grains included cracked and broken grains caused by threshing. the grains were packed using hermetic bags under airtight conditions, i.e. oxygen concentration inside was about 18%, and under normal conditions (oxygen concentration inside was about 21%). each bag type of packaging material contained 2 kg of sorghum grains. characteristics of plastic packaging material used to pack sorghum grains are presented in table 1. in three replicates, inside of each bag type with different oxygen concentrations were treated as follows: (a) infestation with ten pairs of adult (1-14 days old) , (b) different method of threshing, and (c) different storage duration. the bags designated as “control” were not infested with . z s, but were also subjected to different methods of threshing as well as storage durations. sorghum grains were stored for one, two and three months under warehouse conditions. bags containing sorghum were placed on wooden shelves randomly. the temperature and relative humidity of the storage were recorded using a thermohygrograph. grain samples were collected from each bag before storage and subsequently every month thereafter until three months of storage. in samples infested with . , the insects were separated from the sorghum grains using a sieve, they were then preserved in vials containing 70% ethanol. after that, each sample was divided three times using a box sample divider to obtain working samples for the determination of moisture content, percentages of damaged grains and seed germinations, population of and ; fumonisin b and carbohydrate contents, and a reserve sample. the following quality parameters were determined in the experiments: moisture content, percentages of damaged grains , seed germinations, populations of , and , fumonisin b and carbohydrate contents, percentage of weight loss. methods of packaging and storing sampling methods determination of quality parameters sitophilus zeamais s eamai s zeamais f. proliferatum f. verticillioides s. zeamais f. proliferatum f. verticillioides 1 1 118 biotropia vol. 19 no. 2, 2012 statistical analyses moisture content of sorghum (based on wet basis) was determined as soon as samples were collected using delmhorst model g-7 moisture meter. this instrument has been caliberated and cross-checked using oven method. two replicates were used for each sample. damaged grains were collected after one, two and three months of storage including cracked, broken and damaged grains (damage caused by . z or fungi). the number of grains used for the determination of damaged grain percentage was 300 per sample. percentage of seed germination was determined based on blotter method (mathur and kongsdal 2001). population growth of was determined based on the number of adult insects per kg of each sample. and were isolated and enumerated using a serial dilution method followed by pour plate method on dichlorane chloramphenicol peptone agar (dcpa) (pitt & hocking 2009). fumonisin b and carbohydrate contents were determined using liquid chromatography-mass spectrophotometry (lc-ms) (zöllner & mayer-helm 2006) and sni (1992) methods, respectively. the percentage of weight loss was determined at the end of storage. grain samples contained in each bag were weighed before and at the end of storage. grain samples were taken periodically from each bag and weighed. the data were analyzed using completely randomized factorial design with four factors. the first, second, third and fourth factors were the method of threshing, oxygen concentrations inside of the bags, presence or absence of , and storage duration, respectively. s eamais s. zeamais fusarium verticillioides f. proliferatum s. zeamais 1 119 table 1. laboratory analyses of plastic packaging material* parameter test condition of sample unit test method test result uncertainty at 95% confidence level, k=2 wvtr (water vapour transmission rate) good g/m2 day astmf 124920062 2.8946 ± 0.3619 temperature = 37.8 oc rh = 100% thickness of sample = 82.5 μm *analysed by laboratory of test and calibration, the agency for chemical and packaging, jakarta, indonesia postharvest quality improvement of sorghum okky s. dharmaputra– et al. / results and discussions moisture content percentage of damaged grains moisture content of grains is one of the important factors that affected the deterioration of grains during storage. high moisture content will give an opportunity for fungal growth. sni (1992) defines 14% as the maximum moisture content of sorghum grains during storage. method of threshing, duration of storage and their interaction gave very significant differences in moisture content of sorghum grain. moisture content of sorghum threshed using wooden stick at the beginning of storage, subsequently after 1, 2 and 3 months of storage was significantly different from samples threshed using a paddy thresher during the same periods. the lowest moisture content (13.30%) was found in sorghum grain threshed using wooden stick before storage and after two months of storage (table 2). the highest moisture content (13.72%) was found in sorghum grain threshed using wooden stick after three months of storage. it was significantly different from that threshed using wooden stick before storage or after two months of storage. according to christensen . (1992) moisture content is always in equilibrium with the relative humidity of storage room. bala (1997) reported, that moisture content is also affected by the temperature of storage room. in this study, the range and mean of the temperature of storage room during storage were 26.8 29.3 c (28.1 c), while the range and mean of relative humidity of storage room were 47.5 70.8% (64.5%) (table 3). lacey and magan (1991) reported that among the microorganisms which colonize grains, fungi are the most tolerant to low relative humidity, consequently fungi has an important role in the deterioration of grain. method of threshing, duration of storage and their interaction gave very significant differences (at 99% significance level) in the percentage of damaged grain, while the interaction among method of threshing, infested with insects, oxygen concentration and duration of storage affected less significantly (95% significance level) the percentage of damaged grain. at the beginning of storage, the percentage of damaged grain of sorghum threshed using a wooden stick was higher and significantly different from that threshed with a paddy thresher. the percentage of damaged grain in each treatment combination increased and was significantly different during three months of storage. the sorghum grains threshed either using wooden stick or a paddy thresher, not infested with insects, packed under low or normal oxygen concentrations did not gave any significant difference on the percentage of damaged grains after three months of storage (table 4). the lowest percentage of damaged grain was found in sorghum threshed using a paddy thresher, not introduced with insect and stored under low oxygen concentration (ms0h) at the beginning of storage (5.00%), while the highest was found in sorghum et al o o 120 biotropia vol. 19 no. 2, 2012 threshed using wooden stick, infested with insect and stored under normal oxygen concentration (ks1n) after three months of storage ( 21.47%) (table 4). according to dobie (1991) is an important insect pest in stored grain. in indonesia could be a dominant insect species in milled rice and maize. the adult can live for several months up to one year. their eggs, larvae and et al. s. zeamais s. zeamais 121 table 2. moisture content of sorghum grains threshed using wooden stick and a paddy thresher during storage duration of storage (month) moisture content (%) wooden stick paddy thresher mean ± std mean ± std 0 13.30 ± 0.08 a 13.38 ± 0.05 b 1 13.55 ± 0.05 c 13.67 ± 0.15 de 2 13.30 ± 0.09 a 13.52 ± 0.12 c 3 13.72 ± 0.07 e 13.64 ± 0.09 d note: numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level table 3. range and mean values of temperature and relative humidity during storage duration of storage (month) range and mean of temperature (oc) range and mean of relative humidity (%) 0 1 27.4-29.3 (28.3) 54.0-70.8 (59.8) 1 2 26.8-28.9 (28.3) 47.5-68.6 (57.7) 2 3 27.2-28.2 (27.8) 48.7-58.6 (57.9) table 4. percentage of damaged grains of sorghum caused by various treatments treatment duration of storage (months) 0 1 2 3 ks0h 9.90 ± 0.00 fegd 10.63± 2.63 fcegd 12.08 ± 2.23 cebd 20.20 ± 0.08 a ks0n 8.44 ± 1.66 fhgi 11.62 ± 0.16 fcebd 14.60 ± 0.00 b 18.48 ± 2.53 a ks1h 7.66 ± 1.26 hjgi 12.53 ± 2.80 cbd 13.33 ± 1.54 cb 19.73 ± 1.62 a ks1n 9.38 ± 0.35 fhegd 10.03 ± 3.03 fegd 11.99 ± 2.95 cebd 21.47 ± 0.83 a ms0h 5.00 ± 0.22 j 8.98 ± 0.22 fhegi 9.76 ± 0.19 fegd 10.45 ± 0.00 fcegd ms0n 5.04 ± 0.36 j 5.92 ± 0.12 ji 7.79 ± 0.82 hjgi 9.45 ± 0.45 fhegd ms1h 6.20 ± 0.45 hji 9.01 ± 0.00 fhegi 8.93 ± 2.06 fhegi 10.05 ± 0.05 fegd ms1n 5.07 ± 0.47 j 7.74 ± 4.46 hjgi 8.91 ± 1.63 fhegi 9.87 ± 0.56 fegd note: numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level k = wooden stick ; s0 = not infested with ; n = normal oxygen concentration (±21%) m = a paddy thresher ; s1 = infested with ; h = low oxygen concentration (± 18%) s. zeamais s. zeamais postharvest quality improvement of sorghum okky s. dharmaputra– et al. pupae are found inside of grain. dharmaputra . (2010) reported, that the dominant insect species of sorghum stored at farmer and collector levels in wonogiri and demak regencies were and . the existence of damaged grain before storage was due to the different threshers. during storage, the increase of damaged grain in sorghum infested with insects could be caused by insect and fungal attacks, while that not infested with insects may be due to fungal infection. effects of threshing and storage duration was highly different in the percentage of seed germination. percentage of seed germination in sorghum threshed using wooden stick (91.04%) was lower and significantly different from that of sorghum threshed using a paddy thresher (93.33%) (table 5). it was stronger correlated with the percentage of damaged grain in sorghum threshed using wooden stick than in grain threshed with a paddy thresher. percentage of seed germination during storage decreased with the increase storage duration (table 6). percentage of seed germination at the beginning of storage (93.88%) was significantly different from that after one (91.96%), two (91.88%) and three months of storage (91.04%). percentage of seed germination after one, two and three months of storage was not significantly different. according to neergard (1979) fungal infection in the embryo of seed is a factor causing the decrease of seed germination during storage. percentage of seed germination after three months of storage was more than 90%, thus the percentage is still higher than the minimum standard germination of seed, i.e. 70% (directorate general of food crops 1984). the capability of seed to germinate is affected by the atmosphere composition of storage room, but its effect is lower compared to the moisture content and the temperature of the storage ( priestley 1986). et al s. zeamais t. castaneum percentage of seed germination 122 biotropia vol. 19 no. 2, 2012 table 5. percentage of seed germination in sorghum threshed with wooden stick vs a paddy thresher thresher seed germination (%) a wooden stick 91.04 a paddy thresher 93.33 b note : numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level table 6. percentage of seed germination in sorghum grains during storage duration of storage (month) seed germination (%) mean ± std 0 93.88 ± 4.07 a 1 91.96 ± 2.20 b 2 91.88 ± 2.47 b 3 91.04 ± 3.22 b note : numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level population of andf. proliferatum f. verticillioides population of f. proliferatum s. zeamais f. proliferatum f. proliferatum f. proliferatum f. proliferatum f. prolliferatum population of f. verticillioides f. verticillioides f verticillioides f. verticillioides f. verticillioides fusarium aspergillus, eurotium penicillium f. proliferatum the duration of storage gave very significant difference (99% significance level) to the population of , while the interaction between the infestation by insects and the duration of storage, and the interaction among the four factors (method of threshing, presence of , oxygen concentration and duration of storage) gave significant difference (95% significance level) to the population of . population of in sorghum subjected to different postharvest treatments differed in the samples examined immediately after harvest but no clear trend could be detected. in contrast, no significant difference could be detected between treatments after 1, 2 and 3 months in storage (table 7). the population of in sorghum subjected to various treatments and storage duration fluctuated considerably resulted to very high standard deviation. the fluctuation was probably due to the existence of other fungal species which were antagonistic or synergistic to the growth of . the population of in grain subjected to various treatments decreased with the increase of storage duration. f. verticillioides the duration of storage gave significant difference in population. population of decreased significantly with the increase of storage duration (table 8). the population of . at the beginning of storage (1370 cfu/g) was significantly higher than that after three months of storage (433 cfu/g). the decrease of population was probably due to the existence of other fungal species which were antagonistic to the growth of . according to neergaard (1979) there are two groups of fungi infecting grains, i.e. field fungi and postharvest fungi. belongs to field fungi, while and belong to postharvest fungi. during storage, the activity of field fungi often stops, because they need high relative humidity (≥ 90%). 123 table 7. population of (cfu/g) in sorghum grains related to various treatments fusarium proliferatum treatment duration of storage (month) 0 1 2 3 ks0h 7333.33 ± 10540.44 ba 433.33 ± 133.50 c 289.00 ± 329.02 c 311.00 ± 371.55 c ks0n 1855.67 ± 650.07 c 933.33 ± 851.25 c 133.33 ± 88.44 c 77.67 ± 134.52 c ks1h 755.67 ± 680.58 c 578.00 ± 943.70 c 1444.67 ± 1924.89 c 333.33 ± 296.52 c ks1n 2677.67 ± 2117.54 bc 377.67 ± 77.36 c 533.33 ± 695.72 c 300.00 ± 185.71 c ms0h 1700.00 ± 1010.79 c 411.00 ± 416.61 c 500.00 ± 433.35 c 266.33 ± 57.74 c ms0n 9155.67 ± 8592.37 a 167.00 ± 0.00 c 113.00 ± 72.11 c 255.33 ± 385.09 c ms1h 1516.50 ± 118.09 c 1167.00 ± 707.11 c 2033.50 ± 2781.05 c 233.50 ± 47.38 c ms1n 1333.25 ± 585.33 c 358.50 ± 421.22 c 933.25 ± 968.68 c 1025.00 ± 1540.18 c note : numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level postharvest quality improvement of sorghum okky s. dharmaputra– et al. fumonisin b content carbohydrate content 1 1 1 1 1 1 1 1 1 method of threshing and duration of storage, and their interaction gave very significant differences in fumonisin b content. fumonisin b content in sorghum threshed using wooden stick was higher and significantly different from that of sorghum threshed with a paddy thresher after one, two and three months of storage (table 9). fumonisin b content in sorghum threshed using wooden stick fluctuated and showed significant differences at various points in time during storage. the content in sorghum threshed with a paddy thresher also fluctuated, but showed no significant differences between sampling times. the fluctuation of fumonisin b content in sorghum was probably due to the existence of certain strains of which can produce fumonisin b . the lowest fumonisin b (1.58 ppb) was found in sorghum threshed using wooden stick at the beginning of storage, while the highest in sorghum also threshed using wooden stick after one month of storage (27.55 ppb). fao (2004) reported the maximum allowable fumonisin b content in some countries. in france the maximum fumonisin b content allowed in cereals and their processed products is 1000 ppb, in cuba for maize and milled rice 1000 ppb, in switzerland for maize 1000 ppb. the existence of f and other toxigenic species of , and fumonisin production in the field and their products are determined by environmental factors in the field, during transportation and storage (gamanya & sibanda 2001). the duration of storage and the interaction between method of threshing were very significant (99% significance level). the carbohydrate content in sorghum before storage (67.33%) was not significantly different from that after two (68.64%) and three months (67.02%) of storage, but it was significantly different from that after one month of storage (70.81%) (table 10). the carbohydrate content in sorghum threshed using wooden stick and a paddy thresher after one and two months of storage was significantly different, while the content at the beginning of storage and after three months of storage was not significantly different. the lowest carbohydrate content was found in sorghum threshed using a paddy thresher at the beginning of storage (65.98%), while the highest content was found in sorghum threshed using a paddy thresher after one month of storage (74.11%). f. verticillioides . moniliforme fusarium biotropia vol. 19 no. 2, 2012 table 8. population of during storagefusarium verticillioides duration of storage (month) population of f. verticillioides (cfu/g) mean ± std 0 1370.88 ± 1320.68 a 1 1293.04 ± 2202.69 ba 2 1157.38 ± 1491.77 ba 3 433.00 ± 590.17 b numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level 124 suarni (2001) reported that carbohydrate content in sorghum was 80.42%. in this study the range of carbohydrate contents in samples that had been threshed using wooden stick was 66.69 71.28% during storage, while the content in sorghum threshed using a paddy thresher was 65.98 74.11% during storage (table 11). the difference of carbohydrate contents in this study and that of suarni (2001) was probably due to the difference of sorghum variety and the method of cultivation. garraway and evans (1984) reported, that carbon source needed for fungal growth were (among others) the carbohydrates (e. g. monosachharides). the capability of fungi to use sugars could be different among fungal species, even sometimes among different strains of the same species. the method of threshing and duration of storage differ very significant by (99% significance level) in the population of adult , while the interaction between the two factors gave a significant difference (95% significance level). population of in sorghum threshed using wooden stick after one and three months of storage was higher and significantly different from that of samples threshed with a paddy thresher (table 12). in general population either in sorghum threshed using wooden stick or a paddy thresher increased during storage. according to dobie (1991) the actual length of the life cycle of depends also upon the type and quality of grains being infested, i.e. in different varieties of maize, mean development periods of at 27°c and relative humidity 70% have shown to vary from 31 to 37 days. the duration of storage gave very significant difference (99% significance level) in weight loss of sorghum. the percentage of weight loss increased and differed significantly with the increase of storage duration (table 13). the weight losses of sorghum at the beginning of storage, subsequently after one, two and three months of storage were 1.62, 1.92, 2.21 and 2.30%, respectively. the increase of weight loss was due among others by the increase of the percentage of damaged grain (table 4) and population of (table 12) during storage. sitophilus zeamais population percentage of weight loss s. zeamais s. zeamais s. zeamais et al. s. zeamais s. zeamais s. zeamais duration of storage (month) wooden stick paddy thresher mean ± std mean ± std 0 1.58 ± 0.81 a 1.94 ± 1.47 a 1 27.55 ± 12.87 d 3.22 ± 2.71 a 2 20.43 ± 7.56 c 4.65 ± 4.66 a 3 12.57 ± 12.25 b 2.43 ± 3.32 a numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level table 9. fumonisin b content (ppb) in sorghum grains threshed using wooden stick and a paddy thresher during storage 1 125 postharvest quality improvement of sorghum okky s. dharmaputra– et al. biotropia vol. 19 no. 2, 2012 table 10. carbohydrate content in sorghum grains during storage duration of storage (month) carbohydrate content (%) mean ± std 0 67.33 ± 3.33 b 1 70.81 ± 5.57 a 2 68.64 ± 4.05 b 3 67.02 ± 1.45 b note : numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level table 11. carbohydrate content (%) in sorghum grains threshed using a wooden stick and a paddy thresher during storage duration of storage (month) wooden stick paddy thresher mean ± std mean ± std 0 68.68 ± 4.18 ab 65.98 ± 1.32 a 1 67.51 ± 3.57 a 74.11 ± 5.34 c 2 71.28 ± 2.28 b 66.00 ± 3.71 a 3 66.69 ± 1.48 a 67.36 ± 1.41 a note : numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level table 12. population of adult (number/kg) in sorghum grains threshed using wooden stick and a paddy thresher during storage tests sitophilus zeamais duration of storage (months) wooden stick paddy thresher mean ± std mean ± std 0 10.00 ± 0.00 a 10.00 ± 0.00 a 1 20.38 ± 3.37 c 13.62 ± 1.81 b 2 15.98 ± 3.73 b 15.29 ± 1.97 b 3 21.11 ± 2.40 c 15.02 ± 1.16 b note : numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level table 13. percentage of weight loss of sorghum grains during storage duration of storage (months) weight loss (%) mean ± std 0 1.62 ± 0.44 a 1 1.92 ± 0.54 b 2 2.21 ± 0.51 bc 3 2.30 ± 0.49 c note : numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level 126 conclusions acknowledgments 1. the moisture content of sorghum grain subjected to various postharvest treatments was lower than that of safe moisture content of sorghum for storage (< 14%). 2. at the beginning of storage the percentage of damaged grains of sorghum threshed using wooden stick was higher than that of threshed using a paddy thresher. the percentage of damaged grain in each treatment increased with the increase of storage duration. after three months of storage the highest percentage of damaged grain was found in sorghum threshed using wooden stick, infested with , and stored under normal oxygen concentration (± 21%). 3. the seed germination percentage of sorghum threshed using wooden stick was lower than that of samples threshed with a paddy thresher. during storage the percentage of seed germination decreased with the increase of storage duration. 4. populations of and decreased with the increase of storage duration. fumonisin b content in sorghum threshed with wooden stick was higher than that of samples threshed with a paddy thresher during one, two and three months of storage. fumonisin b contents were considered low. in general carbohydrate content in sorghum threshed using either wooden stick or a paddy thresher from the beginning of storage up to three months of storage was not significantly different. after three months of storage, population either in sorghum threshed using wooden stick or a paddy thresher was higher and significantly different from that of at the beginning of storage. the percentage of weight loss of sorghum increased with the increase of storage duration. after three months of storage the percentage of weight loss was higher and and significantly different compared to that at the beginning of storage or any other sampling time before three months. the two different oxygen concentrations inside of the bags did not produce any significant difference in the quality of sorghum. threshing using a paddy thresher was better in comparison to threshing using a wooden stick. the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to pt tri fondasi indonesia for providing sorghum grains, to the agency for chemical and packaging, jakarta for analyzing plastic packaging material, to pt angler biochemlab, surabaya for analyzing fumonisin b content, to indonesian center for agricultural post harvest research and development for analyzing carbohydrate content; to mr. edi suryadi, ms. nijma nurfadila, ms. lia amelia, ms. dwi ayu kencana ungu, for their assistance. we would like also to express our appreciation to the reviewers of this manuscript. sitophilus zeamais fusarium proliferatum f. verticillioides s. zeamais 1 1 1 127 postharvest quality improvement of sorghum okky s. dharmaputra– et al. references abdel-hafez sii, moubasher h, shoreit m, ismail m. 1990. fungal flora associated with combine harvester wheat and sorghum dusts from egypt. j basic microbiol 30: 467-9. bala bk. 1997. drying and storage of cereal grains. new hampshire: 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al. biotropia vol. 28 no. 2, 2021: 173 180 doi: 10.11598/btb.2021.28.2.1296 173 environmental stress on the reproduction of non-human primates mashitah shikhmaidin*,1,2, nur hafizah mohammed1 and ahmad ismail1 1department of biology, faculty of science, universiti putra malaysia, 43400 upm serdang, selangor, malaysia 2institute of tropical agriculture and food security (itafos), universiti putra malaysia, 43400 upm serdang, selangor, malaysia received 9 january 2020/accepted 29 may 2020 abstract this paper aims to review some highlights on the effects of environmental stresses on the non-human primate population, particularly, climate change and food limitation that may have resulted in their poor reproductive performance. the international union for conservation of nature (iucn) lists more than a third of the world’s primates as critically endangered or vulnerable. non-human primates, which are the closest biological relatives of humans, are threatened with extinction from human activities and environmental stress. deforestation is the main problem that intercalates with climate change. either, indirectly or directly, those extinction factors could interrupt the physiological basis of reproduction among non-human primates. researches on other species showed that high ambient temperature causing heat stress had harmed the reproductive performance by interfering with the hypothalamic-pituitary-gonadal axis. therefore, the survival, conservation and sustainability of nonhuman primates growing in captivity and in the wild, require more works and researches to be done. keywords: climate change, conservation, heat stress, primate, reproductive introduction the non-human primate populations are increasingly endangered as they experienced warnings and threats from abrupt climate changes that slowly result in their population extinction. the intergovernmental panel on climate change (ipcc) has warned about the impacts of the escalating global temperatures. moreover, global warming will continue even though it is not regionally uniform and the nations’ potential for adapting to this situation is limited in terms of protecting the natural systems. this situation is more complex than just rising temperature because climate change is an environmental stress causing changes in the body physiological metabolism in a short-term to long-term fight-or-flight response (schulte 2014). the intense increases in humidity and ambient temperature had resulted in animals and plants being exposed to adverse circumstances that threaten their survival, and ultimately, their biodiversity. climate change would directly or indirectly cause reduction and shifting of wildlife populations and habitats, including the nonhuman primates, thereby eventually inhibiting the survivability and sustainability of the species. most primates are widely distributed throughout the tropical and subtropical regions of africa, america and asia. the majority of primates live in tropical areas that are rainfed and rain forested (reed & fleagle 1995), but they also exist in tropical dry forests, mangrove vegetation above high-tide levels, savannas, grasslands, inland wetlands and rocky areas (mittermeier et al. 2013). habitat for this group of animals is widespread but some of the areas are no longer able to accommodate these primates’ populations, as the areas have been mostly disturbed by human activities. about 80% of the malaysian rainforests are degraded *corresponding author, email: mashitah@upm.edu.my biotropia vol. 28 no. 2, 2021 174 by logging and largely covered with oil palm plantations (sophie 2013). logging had threatened the biodiversity of those areas, including the nonhuman primate populations. deforestation is also a result of human overpopulation happening in some places in the african and asian countries. the populations of nonhuman primates are disturbed by human population growth, economic expansion and other human activities, such as those in the agricultural industry. the significant thermal impact that obviously elevated the effects of climate change has led to phenological changes in the wildlife habitats. extreme irregularities in phenological events have also resulted in shifting food sources that has changed many natural cycles of pollinators and plants, predators and prey, and of primate and their food sources (visser 2008; both et al. 2009; butt et al. 2015). all these disturbance factors threatening the nonhuman primate species have forced them to move out of their habitat range to get clean water, food, and to raise their offspring. these external factors, such as climate change, demanded an adaptive response from the animals to a new environment (altmann et al. 2002; beehner et al. 2006a; estrada et al. 2017; fairbanks et al. 2011). the critical challenge facing the global population of nonhuman primates is therefore, survival or extinction, because as animals lose the ability to control their environment (for examples, habitats lost and climate change), their physiological structure ultimately change. moreover, it could cause adverse effects on their reproduction and development. this paper briefly reviews and discusses the impact of physical disturbances such as feed restriction and climate change on the reproductive physiology as these factors slowly create stress affecting reproduction and eventually, the survival of the nonhuman primates. review does climate change affect the reproductive performance of nonhuman primates? in the last few decades, the effects of environmental stress to animals, mainly climate change, has become a major concern. however, still sparse works are done on the sustainability of wildlife, particularly the nonhuman primates, in this complex stress phenomenon. many researches showed that continuous increase of the ambient temperature resulting in heat stress had significantly affected the reproductive physiology of animals, including nonhuman primates (beehner et al. 2006ab; sharpe, 2010; rylander et al. 2013; wang et al. 2014). an acute increase of ambient temperature generally affect the breeding patterns of animals through physiological changes in the brain, the center of the nervous system. studies among livestock animals showed that extremely high temperatures strongly affected the hypothalamic-pituitary-gonadal axis (shelton & huston 1968; wolfenson et al. 2000; santolaria et al. 2014). these deleterious effects of heat stress could lead to disruptions in the reproductive performance of nonhuman primates. about 419 primate species, such as lemurs, lorises, tarsiers, monkeys and apes, would experience 10% more warming than the global average (taylor 2016). meanwhile, some primates would experience increases of more than 1.5 oc in annual average temperature for every degree in global warming. therefore, primates that show high sensitivity to little changes on ambient temperature could experience more intense effects in their reproduction as compared to other primates. the consequence of climate change on the reproductive performance of nonhuman primates not only occurs in females but also in males. a 43 ˚c heat stress of about 30 minutes could induce apoptosis of germ cells in the testis of macaca fascicularis, a non-human primate, (zhang et al. 2005). this situation was described in studies where suppression of testicular function under heat stress had led to a decrease in fertility in ruminants (hamilton et al. 2016), and humans (garolla et al. 2013; rao et al. 2015). those testis exposed to high ambient temperature could constantly effect the dna fragmentation of sperms; thus, reduces the quality of sperms. an elevated surrounding temperature causing a rise in testicular temperature result in a reduction in sperm output, low sperm motility and an increase of morphologically abnormal spermatozoa (hansen 2009). germ cell depletion and increase in dna damage are induced by heat stress (lue et al. environmental stress on reproduction of nonhuman primates – shikhmaidin et al. 175 1999; paul et al. 2008). oxidative stress clearly disturbs the functionality of sperm and leads to sperm damage, deformity, and eventually, male infertility (halliwell & gutteridge 2007; mahfouz et al. 2010). animals vulnerable to environmental changes, particularly heat stress, also exhibited changes in their mating behavior (deutsch et al. 2008; mcfarland et al. 2014). in female, heat stress had significant adverse effects on the reproductive performance particularly, delayed estrus cycle, lower embryo survivability, slow fetus development and early pregnancy abortion (beehner et al. 2006b; hansen 2009; wiederholt et al. 2011). documented information on the effects of these environmental stressors towards reproduction patterns of the female and male nonhuman primates is still sparse. hence more studies are needed on understanding the impact of heat stress or climate change particularly on reproductive-physiology and reproductive behavior. identifying anthropogenic stressors on nonhuman primates is also essential for conservation planning and management of these primates (graham et al. 2016). importance of nutrition and reproduction the overall functions of the reproductive system are largely controlled by the hormonal interactions of the hypothalamic-pituitaryovarian axis, but the final output also depends on extrinsic factors such as nutrition. the diet that is easily digested and rich in fibre, carbohydrates and lipid from fruits, leaves, buds and insects, constitute an immense food source for most nonhuman primates (strier 2007; lambert 2010; moges et al. 2014; kassim et al. 2017). however, habitat fragmentation and climate change could harm food availability. captive orangutans fed with high-quality feeds have shorter inter-birth intervals as compared to wild orangutans (kuze et al. 2012). therefore, with a good quality food sources, the orangutan population will result in an increased reproductive performance and eventually increased population density. food availability and nutritional composition are also important to the population density of nonhuman primates (hanya & chapman 2013). two factors that contribute to pregnancy failure in females are the inadequate food intake and increased energy expenditure. it is important for pregnant females to meet the basic metabolic requirements mainly during early pregnancy to avoid negative energy balance. studies done on the nutritional status and reproduction of laboratory and livestock animals recorded that underfed animals experienced delayed puberty period, decreased embryo survival rate, suppressed spermatogenesis and reduced number of sperm motility. moreover, females that were deprived of food or underfed were less fertile and this reduces the chances of fertilisation; thus, affecting the population birth rate and eventually causing a decrease in population. this probably occurs severely among subordinate females of a nonhuman primate. dominant females of marmoset monkey produced more offsprings as compared to subordinate females in a healthy environment (abbot 1987). in the physiological level, dominant females can defeat the reproductive performance of subordinate females. the impact of a threatening activity, such as forest abuse, can damage the individual survival of nonhuman primate species, decrease fertility, and will shrink the population through time. climate disaster also leads to the extinction of plants and animals. a study on wild baboons documented that pregnancy failure was high if the females conceived after the drought (beehner et al. 2006b). during the 1990s, the decline in the orangutan population by at least 30% of its total population was caused by fires and drought in the bornean forests (gould et al. 1999). in addition, wildfires in mexico also affected the nonhuman primates, whereby the habitats of new world monkeys were destroyed by drought and el nino. reduction of food availability as a result of el nino event caused the population size reduction of atelines one year after the event (wiederholt & post 2010). extreme temperatures can decrease the food production and drinking water availability of the existing nonhuman primates. high ambient temperature could decrease the concentration of fibre and protein contents of leaves due to high atmospheric carbon dioxide. in addition, a rising level of heat and carbon dioxide could make the leaf or green resources less nutritious (gray & brady 2016) and could also affect the plant physiology resulting in changes in the number and size of leaves (taylor et al. 2003; dermody et biotropia vol. 28 no. 2, 2021 176 al. 2006). higher temperatures also increased the plant matter toxicity resulting in reduced leaf sizes (moore et al. 2015). decreasing food source could also increase the competition within the population. in the long run, this indirect effect might impact the evolutionary and ecological processes of a certain population. obviously, several factors contribute to food limitation in nonhuman primate habitats, such as forest exploitation, forest clearing for agriculture and climate change. as mentioned earlier, nutritional restriction can interrupt the breeding pattern, fertility, puberty development, embryo growth and development, and can increase susceptibility to disease and predation, intensify mortality of infants and mothers, and can delay reproductive maturity to a much later age (korstjens & hillyer 2016; chaves et al. 2019; laver et al. 2020). the synchronization of reproductive events of primate to fit the availability of the resources was also affected (van schaik et al. 1993; post 2013). climate change occuring in the african regions has cause significant changes in fruit production, as it was lower between 1988 and 1993 than between 1994 and 2003 (erhart and overdorff 2008). this event negatively affected the red-fronted lemurs (eulemur fulvus rufus), specifically their reproductive patterns, sex ratio, group sizes and population density. early embryo wastage, one of the key factors towards species extinction, is a particularly critical issue for nonhuman primates,. the nonhuman primates in captive management may need to refine the feed intake that would satisfy the dietary needs and take into account to have an optimal reproductive functions. reproductive and stress hormones on reproduction it is well-known that progesterone is the main reproductive hormone that is essential to maintain pregnancy in animals, including in nonhuman primates and humans. meanwhile, testosterone is important in mating behaviour and confounded with dominance rank in male primates (dixson 1998; wallen & zehr 2004). expressions of reproductive hormones depend on maternal responsiveness with the environments and differ between young and experienced female primates (rangel‐negrín et al. 2009; saltzman & maestripieri 2011). the understanding of neuroendocrine and primate maternal behaviour has increased, but of the physiology mechanism underlying the effects is still limited. during early pregnancy in nonhuman primates, progesterone secretion is solely from corpus luteum and it is replaced by the placenta during mid-pregnancy (beehner et al. 2006a). an in vitro study on macaque recorded the direct actions of progesterone and oestradiol on primate pre-antral follicle development (ting et al. 2015). androgens appeared to be a survival factor but hindered antral follicle differentiation; oestradiol appeared to be a survival and growth factor at the pre-antral and early antral stages, whereas progesterone may not be essential during early folliculogenesis in primates. macaque and baboon are nonhuman primate models used for studies related to human reproductive health, such as contraception, reproductive aging, infertility, ovarian function, and reproductive tract disorder (wandji et al. 1997; d'hooghe et al. 2004; ting et al. 2015). maternal stress during pregnancy that is controlled by the hypothalamus–pituitary– adrenal (hpa) axes could produce disruptive effects on embryo and fetal growth and development (del giudice 2012; laver et al. 2020). among wild baboons, food limitation due to drought contributed to fetal loss in the amboseli population (beehner et al. 2006b). a study on orangutans in southeast asian tropical forests showed that solitary lifestyle is relative to late weaning which is a consequence of their ecological environment (vogel et al. 2015). faecal glucocorticoid (fgc) is a survival probability indicator of many nonhuman primates, such as ring-tailed lemurs and redbellied lemurs. it shows high mortality rate due to habitat degradation that would cause reduction in fruit availability (pride 2005; tecot 2013; balestri et al. 2014). the non-invasive technique was used to measure reproductive hormones and cortisol through urine and faeces during a normal reproductive cycle (rangel-negrín et al. 2009). chaves et al. (2019) used non-invasive biomarkers, fecal glucocorticoid metabolites to assess the physiological stress of adult wild brown howlers and the food availability in brazilian atlantic forest fragments, and found environmental stress on reproduction of nonhuman primates – shikhmaidin et al. 177 that nursing females are highly nutrition demanding for their pregnancy and lactation it shows that hormones obsess and affect the appetite throughout pregnancy, explaining the consistent association between undernutrition and reproductive failure. studies on brain activity via hypothalamic-pituitary-ovarian axis and stress hormones on embryo and fetal losses are still scarce. we suggest that researches need to focus on this because as observed, maternal hormonal patterns and external factors are important in sustaining embryo survivability. conclusion and future perspectives this article has focused on how environmental stress, mainly climate change and poor dietary intake, can lead to reproductive stress and failure among nonhuman primates. these factors, known to be either directly or indirectly link to the hypothalamic-ovarian axis, harm the reproductive sites. furthermore, the anthropogenic stressors on primate populations also contribute towards species extinctions, particularly in animals with slow reproductive rates. all these factors are closely related to the extinction of this vulnerable species. primates are a flagship species for entire ecosystems, so its conservation also present important consequences for many other species. the success of its conservation and preservation programs are dependent on the ability of the species to reproduce successfully and to minimize offspring loss. thus, important information on the fitness and success of its reproduction provides an understanding of the physiological-environment interactions of the nonhuman primates reproduction. moreover, the successful reproduction is fundamental to the survival and evolution of all species. 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expression of hsp105 and hsp60 during germ cell apoptosis in the heat-treated testes of adult cynomolgus monkeys (macaca fascicularis). front biosci 10(1):3110-21. biotropia vol. 28 no. 2, 2021 180 799 ho soo ying (soil character) revisi tek.cdr soil characteristics under intensified shifting cultivation for upland rice cultivation in upland sabal, sarawak, malaysia *ho soo ying , mohd effendi bin wasli and mugunthan perumal department of plant science and environmental ecology, faculty of resource science and technology, universiti malaysia sarawak, 94300 kota samarahan, sarawak, malaysia received16 january 2017/accepted 12 september 2017 abstract shifting cultivation practices are regarded as the most important agricultural land-use in the marginal uplands of sarawak for the livelihood of the rural communities. in response to various socio-economic consequences, previous practices on traditional form of shifting cultivation were altered into much sedentary farming practices. soil productivity to sustain rice yield under current intensified forms of shifting cultivation should be of prime importance for food security among the local community at the marginal uplands of sarawak. therefore, this study was conducted to characterize soil properties influenced by the current intensified shifting cultivation practices at sabal upland area, sarawak. along with the use of agrochemicals, shifting cultivation at sabal area was conducted through single cultivation practices (10 to 15 years of fallow period and a cycle of rice cultivation) and multiple cultivation practices (5 to 7 years of fallow period and two cycles of rice cultivation). before burning practices, soil ph was highly acidic (ranged from 4.28 to 4.72) in single cultivation sites; while multiple cultivation sites showed less acidic nature (soil ph ranged from 4.98 to 5.23) with relatively high secondary macronutrient contents, especially soil exchangeable mg and ca. no significant observation was found after burning practice in total c and total n of the soils at both sites. the level of soil exchangeable bases (k, mg and ca) and available p increased after the burning practices in single cultivation farmlands; while multiple cultivation sites showed lower to no increase in exchangeable bases and available p, partly attributed to the limited supply of nutrients from the aboveground biomass. after the harvesting of rice, soils at both sites tended to be more acidic and lower in macronutrient contents (k, mg and ca), primarily ascribable to crop uptake during the cultivation period. the average yield of rice in single cultivation sites and multiple cultivation sites were 721 kg/ha and 391 kg/ha, respectively. our findings suggested that the usage of agrochemicals, particularly fertilizers in multiple cultivation sites are necessary for sustaining rice yield to restore the depleted macronutrients (especially k, mg and ca) after successive cultivation. keywords: intensive, macronutrients, shifting cultivation, soil characteristics, upland rice field, upland sarawak introduction shifting cultivation is an old, primitive and predominant land-use system in remote mountainous areas of many regions across the globe. for decades, the dayak tribe, including the ibans' and the bidayuhs' in sarawak practices shifting cultivation of upland rice as their source of staple food (kleinman et al. 1995). teng (1993) reported that the annual rate at which land is cleared by the local people in sarawak ranges from 75,000 to 150,000 ha or 0.6 to 1.2% of the total land areas; while 2.7 x 10 ha or 22% of the state 6 land is either underused or had been used at least once for shifting cultivation. such agricultural system could be regarded as ecologically sound and sustainable under the condition of low population density, poor soil fertility and inaccessibility to market (fox et al. 2000; mertz 2002; ickowitz 2006; nielson et al. 2006). in recent decades, scarcity of arable land resources due to population expansion and urban development resulted in gradual alteration of traditional shifting cultivation practices. it is said that current form of shifting cultivation practice is intensified with the shortening of fallow length, longer cultivation period and incorporation of agrochemicals, especially fertilizers in their * corresponding author: sherylsooying@gmail.com biotropia 5 1 8 72 83 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.1.799 72 farming practices (ichikawa 2004; kendawang et al. et al. 2005; wasli 2009). in reality, some farmers continuously cultivate upland rice at the same land for few cycles. shortened fallow period caused insufficient recovery of vegetation and soils for the next cultivation, indirectly resulting to the decreasing crop productivity, increasing pest and disease cases and declining of soil fertility. additionally, applications of agrochemicals especially fertilizers and pesticides are widely practiced by the local farmers partly due to subsidy scheme provided by local government agencies. as the modification in the current intensified shifting cultivation practice remained unapprehended in sarawak especially in the aspect of soil agroecology, there is a need to obtain fundamental information on the suitability of current state of modified shifting cultivation practices toward sustaining the livelihood and subsistence of the local farmers in the upland agroecosystem of sarawak. few studies were conducted mainly on the effect and impacts of shifting cultivation to soil ecosystems (cramb 1989; funakawa . 1997; et al kendawang . 2004; tanaka . 2004; et al et al et al et al et etsuko . 2004; kendawang . 2005; bruun al. et al. 2009; wasli 2011). some researchers reported on the current practices involving shorter fallow period and continuous cultivation for few cycles in sarawak (kendawang . 2004; et al bruun e . 2006; wasli . 2009). in addition, t al et al dalle and de blois (2006) pointed out that reduction of fallow cycles caused a decline in noncrop plant resources especially in pioneer species and an increase in short-lived weedy species. however, none of their studies discussed the outcome of recent intensified shifting cultivation, with special reference to soil physicochemical aspects under different stages of shifting cultivation cycles. present study is necessary to obtain the baseline information on soil fertility as one of the primary indicator of sustaining upland agriculture productivity, soil resources, selfsubsistence and livelihood of the upland communities in sarawak. thus, this study was aimed to characterize soil physicochemical properties under current intensified shifting cultivation practices at sabal upland area, sarawak. previously, kendawang (2004) studied soil et al. fertility in this area based on conventional shifting cultivation practices. they concluded that soils at sabal area were naturally infertile and difficult for the farmers to sustain rice production through traditional form of shifting cultivation practices under such soil condition. materials and methods study area the study was conducted at sabal upland area (01°04'24.6” n, 110°58'08.6” e), sarawak, malaysia as shown in figure 1. the study area consisted of mainly undulating hills which extended to steep and mountainous klingkang range where it meets the border of kalimantan, indonesia. sabal is located about 110 km from south-east of kuching city and is accessible by kuching-sri aman central road. local communities resided in the study area are mainly of iban origin for approximately 100 years. the climate condition of the study area is tropical climate with a mean annual temperature of approximately 32.5 °c and negligible monthly variation throughout the year (meteorological department 2014). the average mean precipitation is about 3,585 mm obtained from the nearest weather station located at sri aman airport (meteorological department 2014). soils in the study area were derived from non-calcareous sedimentary rock consisting of fine and whitish sandstone which is further c l a s s i f i ed i n to oxyaqu i c o r spod i c q u a r t z i p s a m m e n t s b a s e d o n u s da classification system (butt 1983; soil survey staff 1999). most of the villagers are farmers cultivating subsistence crops such as upland rice, fruits and vegetables as well as other cash crops i.e. rubber, pepper and oil palm. site selection and agronomic practices in relation to upland rice cultivation field survey was conducted from august 2013 to march 2014. prior to soil sampling, community survey via rapid rural appraisal (rra) approach was conducted to collect baseline information on family background, land-use history and crop management practices. desirable sites were selected based on the availability of farmlands with shifting cultivation i.e. single cultivation and multiple cultivation practices. single cultivation practice refers to the long fallow period with short cultivation period. multiple cultivation practice 73 soil characteristics under intensified shifting cultivation ying et al. cultivation or in multiple cultivation practices. fertilizers were applied under single cultivation practices. unlike research results reported previously in tanaka (2007a) at mujong river et al. area, previous conventional single cycle of shifting cultivation involved no fertilizers application. compound n-p-k (17.5-15.5-10.0) and urea (46%) were commonly used in both cultivation practices. the application rate of fertilizers generally varied among the household with an average application rate of 132 kg/haper cycle for single cultivation sites and 190 kg/ha per cycle for multiple cultivation sites, giving rise to average n-p-k (40-10-7) kg/ha/year and n-p-k (64-13-8) kg/ha/year for single and multiple upland rice cultivation, respectively. in addition, herbicides and pesticides were commonly applied in both cultivation practices during land preparation and rice farming period. based on the farmers' experience, agrochemicals requirements under multiple cultivation sites were generally higher compared to single cultivation sites due to factors such as weeds and pest infestation. thus, multiple cultivation sites were regarded as intensified in terms of reduction of fallow age, laborious farm work maintenance and higher rate of agrochemicals application. refers to the short fallow period with long cultivation period. farmers' perception on site suitability for rice cultivation is based on their traditional knowledge, typically through soil observation and vegetation composition of the desired lands. accessibility and location of the farmlands were particularly important in farmer's decision for site selection. overall, a total of 8 sites for both single cultivation farmlands (n = 4) and multiple cultivation farmlands (n = 4) were selected after interviews. the slope gradients for all study sites ranged from 12 to 20° with elevation ranged from 23 to 88 m above sea level. land-use history of all study sites was illustrated in table 1. single cultivation sites were fallowed for 11 to 15 years; while multiple cultivation sites had shorter fallow period of 5 to 7 years with continuous cultivation for two cycles, thus intensified. farmlands size usually depended on the numbers of family members and availability of manpower in managing the farmlands. at sabal area, the farmland sizes ranged from 0.47 to 3.82 ha with an average of 1.86 ha grochemicals . a such as fertilizers and pesticides subsidized by government agencies were widely used in the current farming practices, either in single biotropia vol. 25 no. 1, 2018 74 figure 1 map of the study area soil sampling and analysis soil samples were collected at 3 different phases of a typical shifting cultivation cycle, notably before burning (bb) of dried vegetation after land clearing (august), after burning (ab) of the dried vegetation (september) and after harvesting (ah) of upland rice (february) in 2013 and 2014, respectively. the yield of upland rice in each farmland at single and multiple sites were recorded as well. composite soil samples were collected within two weeks' period of each phase at soil depth of 0 – 10 cm and 30 – 40 cm from three random points within the same location at the farmland area. on the other hand, core samples (100 cm ) were collected from the same 3 depth in triplicates for soil bulk density determination. composite soil samples collected were air-dried and crushed to pass through sieves with 2 mm mesh for the determination of soil physicochemical properties. soil ph (h o) was determined in water. soil 2 ph (kcl) in 1m kcl was determined in a soil to solution ratio of 1:5 using glass electrode method. electrical conductivity (ec) was measured after the ph (h o) measurement using ec meter. total 2 c is determined by loss on ignition method (dean 1974). total n was determined by kjeldahl acid digestion and measured by colorimeter method. contents of exchangeable bases and the cation exchange capacity (cec) were measured respectively, after successive extraction using 1m ammonium acetate adjusted to ph 7.0 and 10% nacl. contents of exchangeable bases were deter mined using atomic absorption spectrophotometry (aas) for ca, mg, k and na (coleman et al. 1959). exchangeable al and h were extracted with 1m kcl from ph (kcl) filtrates. exchange acidity (al+h) was determined using titration method with 0.01m naoh. content of exchangeable al was determined with 0.01m hcl. content of exchangeable h was calculated as the difference between the values of the exchange acidity and exchangeable al. available p was quantified using bray ii method (kuo 1996). in bray ii method, soil samples were extracted with extracting solution consisted of 1m nh f and 0.5m hcl. 4 color developing reagent was then added into the extract. available phosphorus was determined by a b s o r b a n c e m e a s u r e m e n t w i t h u v spectrophotometer at wavelength of 710 nm (bray & kurtz 1945). particle size distribution was determined using pipette method with the assistance of soil textural class (gee & bauder 1986); while soil hardness was measured by yamanaka-type push cone penetrometer. data analyses all statistical analyses were performed using spss version 22 (spss inc.) for comparison of soil physicochemical properties between single and multiple cultivation practices using student's t-tests. relationships among yield and fallow period, fertilizers applied as well as soil physicochemical properties were computerized using pearson's correlation coefficients. 75 soil characteristics under intensified shifting cultivation ying et al. table 1 land-use history of the selected study sites note: r = upland rice cultivation; = fallow period 76 biotropia vol. 25 no. 1, 2018 results and discussion soil physicochemical properties under shifting cultivation at the study area table 2 presented the average values of surface and subsurface soil physicochemical properties at different stages of shifting cultivation cycle in relation to single cultivation and multiple cultivation practices at sabal area. at all stages, no substantial differences were observed for average surface and subsurface soil properties between single and multiple cultivation sites. it is noteworthy that the discussion of current study was mainly focused on the surface soil layer (0 – 10 cm) rather than on the subsurface soil layer (30 – 40 cm). the effect of burning practice was not distinct at the subsurface layer (kumada et al. 1985; kyuma et al. 1985; tanaka et al. 2001; kendawang et al. 2004; kendawang et al. 2005). however, the properties of the subsoil layer should be obtained to clarify if any s ignif icant changes in ter ms of soi l physicochemical properties were observed under current intensified multiple cultivation practices adapted by the local farmers at the study area. et al.previous study by kendawang (2004) reported that the soils at sabal area were characterized by relatively sandy texture and low nutrient contents. soil organic matter (som) in the surface soils was visible, mainly composed of coarse fragments of plant debris; while clay mineral composition was dominated by kaolin minerals, and to a lesser extent by quartz (kendawang 2004).et al. soil ph (h o) under single cultivation sites 2 were acidic (ranging from 4.28 to 4.72) before burning practices; while soil ph (h o) value 2 showed an increase to the range from 4.54 to 5.23 after burning practices (table 2). later, soil ph (h o) decrease slightly to the range of 4.40 to 5.16 2 after harvesting of the upland rice. in contrast, soil ph (h o) under multiple cultivation sites 2 showed gradual decrease with the average of 5.11 at before burning to 4.83 after burning and further decrease to 4.65 after harvesting stages. before burning practice significantly lower soil ph (h o) 2 values in single cultivation sites due to the vigorous uptake of basic cations by the existing vegetation during fallow period (tanaka et al. 1997; wasli et al. 2011). multiple cultivation sites with lower fallow period were less acidic indicating the remnant ash effects from previous burning practices and fertilizers residues from previous year. after burning practices, soil ph (h o) 2 showed an increase from 4.56 to 4.90 at single cultivation sites compared to multiple cultivation sites, mainly caused by direct effect of ash addition into the surface soil. various studies reported that burning practice significantly increases soil ph due to acid neutralizing capacity of ash (nye & greenland 1964; kyuma et al. 1985; armando et al. 1996). in contrast, such effect was not observed in multiple cultivation sites after the burning practice. shortening of fallow period in multiple cultivation sites has led to poor vegetation regrowth, which in turn limiting the ash input to soils during burning to alleviate soil acidity (wasli et al. 2009). soil ph (h o) was 2 further acidified after the harvesting phase in both cultivations. soil acidity was mainly caused by the removal of basic cations in the soils from the uptake of the upland rice crops (tulaphitak et al. 1985; gafur et al. 2000; biswas et al. 2012). however, it can be observed that soil acidity was more pronounced in multiple cultivation sites due to the depletion of nutrients after the second cycle of upland rice cropping. in our study, soil total c and total n varied widely among the stages under both types of cultivation practices. in the surface layer, total c of the soil ranged from 14.2 g/kg to 40.3 g/kg; while total n of the soil ranged from 0.97 g/kg to 3.00 g/kg. present studies showed no remarkable changes in total c and total n of the soil after burning practice at both single and multiple cultivation sites. in fact, the contents of total c and total n in most single and multiple cultivation sites, decreased after burning practices which is in agreement with results of previous findings (ewel et al. 1981; andriesse & schelhaas 1987; hölscher et al. 1997; gafur et al. 2000; tanaka et al. 2001). unlike those reported by various researchers (nye & greenland 1964; kyuma et al. 1985; tulaphitak et al. 1985), significant increase in total c and total n of the soil were usually found after burning practices. several sites (both single and multiple) showed a slight increase of total c and total n after burning. in their study at northeast thailand, kyuma and pairintra (1983) investigated that burning resulted in partially carbonized litter added into the soil. however, our study showed that the c/n ratio in all both single and multiple 77 soil characteristics under intensified shifting cultivation ying et al. table 2 surface and subsurface soil physicochemical properties under different stages of shifting cultivation cycle in single cultivation and multiple cultivation farmlands note: data in this table are presented as mean ± standard deviation * = significant (p < 0.05) differences between single and multiple cultivation practices at different stages of shifting cultivation cycle a b c d sc = single cultivation; mc = multiple cultivation; ecec = effective cec, sum of exchangeable bases and exchangeable al; base sat = sum of eexchangeable bases in percent of cec; al sat = exchangeable al in percent of ecec f hardness was measured using yamanaka-type push cone penetrometer cultivation sites tended to either remain unchanged or decrease after burning, indicating that limited or no fresh (partially carbonized) plant materials were added into the soils. as reported by kendawang et al. (2004), in sandy textured soils, fragments of som was not absorbed onto or closely associated with soil particles, but occurred as mineral particles which might be washed away during the rainfall after the burning practices. our study was supported by this observation as the sand contents were negatively correlated with the amount of total c (r = -0.767, p < 0.05) and total n (r = -0.848, p < 0.01) (fig. 2). 78 biotropia vol. 25 no. 1, 2018 from our field observation, the amount of incompletely burnt, charred branches and stems of young and mature trees left distributed over the field in single cultivation sites were observable (fig. 3). young trees store relatively more nutrients in leaves and branches, whereas mature trees store more nutrients in the stem and large branches (kyuma 1985). the branches and stems from et al. the felling did not burn completely, suggesting that some of the nutrients which stored in the plant materials were not released completely into the soils. such condition resulted in no remarkable changes in the levels of c and n contents in single cultivation sites, although the fallow period was longer as compared to multiple cultivation sites. additionally, decreasing total c and total n of soil could be ascribed to the lost total c and n of soil through volatilization to the atmosphere when large quantities of fuel were consumed at the surface layer (giardina 2000). therefore, et al. soil organic n might be lost to the atmosphere during the thermal oxidation of organic matter in the form of oxidized n gases and n , resulting to 2 lower total n levels in the soils even after burning practices (raison 1979). andriesse and schelhaas (1987) also pointed out that c disappears faster than n under high temperature. as the levels of soil total c and total n did not show significant increase after burning practices, it can be predicted that the source of n for rice growth mainly supplies through the inorganic form from chemical fertilizers, rather than the biomass from the burnt vegetation. after harvesting of crops, total c and n generally decreased at the end of the cultivation cycle at both single and multiple cultivation sites. much of the decline in soil total c and total n may be attributed to the decomposition of unhumified materials (nye & greenland 1960), though erosion and crop uptake decrease the level of soil total c and total n as well as the end of the harvesting cycle. content of available p at the surface soil in multiple cultivation sites were generally higher with a wide variation as compared to single cultivation sites. one of the multiple cultivation sites showed an extremely high value of available p which was 113.5 mg p/kg before burning practice. such condition could be attributed to the unused portion of p added by ashes or the soil samples might have been collected from the points where large amounts of burnt biomass accumulated or chemical fertilizers had been applied (wasli et al. 2009). similar to soil total n, soil available p did not show remarkable changes after burning practices at single cultivation sites. on the other hand, multiple cultivation sites showed decrease of soil available p levels after burning practices. contrary to studies reported by other researchers (nakano 1978; tulaphitak et al. 1985), available p of soil did not peak after burning phase in both single and multiple cultivation farmlands. as charred plant biomass was left distributed in the field, the p nutrient was not sufficient to release from the burning process. subsequently, levels of available p of the soil decreased after the harvesting of rice crops at the end of the cycles partly attributed to crop uptake and loss through leaching as well as erosion. in multiple cultivation sites, the contents of available p in surface soil were relatively higher after the second cycles, suggesting that excess and immobile p from the fertilizers might be accumulated in these farmlands under figure 2 relationship among sand and total c (g/kg) and total n (g/kg) after burning at the surface soils 79 soil characteristics under intensified shifting cultivation ying et al. continuous cultivation practices. as for subsurface layer, the soil available p contents in both single and multiple cultivation sites did not vary from each other after harvesting practices, generally within the range of 1.7 mg p/kg to 4.0 mg p/kg. before the burning practice, multiple cultivation sites showed higher levels of exchangeable bases (k, mg and ca), generally ranged from 0.05 to 1.25 cmol /kg, primarily due c to remnant ash effects and nutrients from the previous cultivations. after burning practice, the soil exchangeable bases (k, mg and ca) were increased to a range of 0.11 to 1.55 cmol /kg in c single cultivation practice from a range of 0.01 0.70 cmol /kg (before burning practice) due to the c release of nutrients from dried vegetation biomass during burning. burning usually leads to accumulation of potash which contributes to the release of macronutrients needed by plants, i.e. potassium, calcium and magnesium (nye & greenland 1964; driessen . 1976; kyuma et al et al. 1985). multiple cultivation sites showed little variations in exchangeable bases (k, mg and ca) after burning practices. levels of exchangeable bases (k, mg and ca) slightly increased at several sites compared to single cultivation sites. herbaceous plant indicating infertile land such aslalang (imperata cylindrica) usually predominates after a short fallow period of less than 3 years (tanaka et al. 2007b; wasli et al. 2009), resulting in the restricted supply of nutrients into the soils from the burning of the aboveground biomass. after the harvesting of rice crops, the levels of exchangeable bases decreased rapidly, suggesting that the input of bases through ash effect and subsequent fertilizers application was depleted at both farmlands. several nutrients might be lost into the environment through volatilization, runoff and leaching throughout the cycle after burning practices, the remaining ones were used by the rice crops during cultivation. at the end of the cycle (ah), single cultivation sites showed higher levels of bases content as compared to the soils at the stage of before burning practice (bb). although fertilizers input might affect the levels of bases at the end of the cycle, another reason could be ascribable to the existing of remnant ash effects during cultivation period (nye & greenland 1964; juo & manu 1996; etsuko et al. 2004). conversely, the levels of surface and subsurface soil exchangeable bases at some multiple cultivation sites were lowered at the end of the cycle (ah) as compared to before burning practices (bb). such condition indicated that the available nutrients might not be sufficient for next cultivation after two consecutive cycles of cultivation. thus, the farmer should consider whether to move to another farmland or increase the amount of fertilizers application as the amount of nutrient supply by the aboveground biomass could be limited after two cycles of cultivation. rice productivity under current intensified practice of shifting cultivation in general, the yield of both single and multiple cultivation practices varied widely among the study sites. the yield of single cultivation sites ranged from 225 kg/ha to 1,145 kg/ha with an average of 721 kg/ha. multiple figure 3 charred branches of young and old vegetations scattered over the single and multiple cultivations upland rice farms cultivation sites ranged from 99 kg/ha to 652 kg/ha with an average of 391 kg/ha. teng (1993) reported that the yield from traditional shifting cultivation practice of upland rice in sarawak ranged from 460 kg ha to 1,184 kg ha with an / / average yield of 738 kg ha. thus, study / current showed lower yield as compared those reported to by teng (1993). it was noticeable that no major relationship was found between rice yield and soil properties the study area (data not shown). in figure 4(a) showed the relationship between upland rice yield and the period under fallow current single and multiple cultivation practices. in single cultivation sites, a yield of upland higher rice observed under fallow length was longer from 19 to 25 years yield of upland rice . lower was observed under the fallow length of 11 to shorter 12 years. ice yield under multiple cultivation sites r with period of 5 to 7 years almost fallowing was similar to single cultivation sites with fallowing period of 10 to 15 years. on the other hand, figure 4(b) showed the relationship between yield and fertilizers applied per cycle under single and multiple cultivation sites. although the rate of fertilizers application under multiple cultivation sites were slightly higher than single cultivation sites, the upland rice production under multiple cultivation sites was still lower than that of single cultivation sites. single cultivation required minimal amount of fertilizers i.e. less than 100 kg/ha to produce the yield of more than 1,000 kg/ha, considering the fallow period of more than 20 years. such suggest that condition leads to ions agrochemicals, especially application re fertilizers a crucial to sustain rice production under short fallow period of multiple cultivation practices. as finding new farmlands with long fallow period is rather difficult, multiple cultivation practices with the appropriate use of agrochemicals could be one of the options to sustain the rice production on the study area. additionally, som plays an important role as nutrient reservoir in determining soil productivity, further investigations should highlight the dynamics of som and soil microbial ecological aspects under present intensified multiple cultivation practices, especially after the burning practices. on the other hand, comprehensive nutrient management plan involving both chemical and organic fertilizers with suitable soil conservation practices can be introduced into current intensified shifting cultivation towards sustaining the rice production at the study area. conclusions current shifting cultivation has been intensified through multiple cycles cultivation with the shortening of fallow period and use of agrochemicals at upland sabal area. there were no major significant differences between soil properties among single and multiple cultivation sites at all stages of shifting cultivation cycles within the surface and subsurface layer, except for soil ph (h o). surface soil ph (h o) under single 2 2 cultivation sites increased after the burning practice and decreased after the harvesting of the upland rice. surface soil ph (h o) under multiple2 cultivation sites were acidified from the beginning until the end of the cycles. total c and soil total n of the soil did not significantly different figure 4 relationship among upland rice yield and (a) fallow period and (b) fertilizers applied per cycle of cultivation, under single and multiple cultivation sites biotropia vol. 25 no. 1, 2018 80 between one another at both single and multiple cultivation sites. the amount of exchangeable macronutrients (k, mg and ca) at the surface and subsurface soils at multiple cultivation sites showed decreasing trends of nutrients contents after subsequent cycles of cultivation as compared to single cultivation sites. continuous cultivation without proper nutrient management plan could result in depletion of soil and exhaustion available macronutrient contents. the function of the fallow period has been substituted by the agrochemicals such as fertilizers and herbicides in sustaining the crop yield. appropriate usage of agrochemicals could be an option to sustain the rice production for the local people under multiple cultivation practices. acknowledgements this study was partially funded by grant-in-aid of japan malaysian association (jma), mybrain15 (mymaster), ministry of education malaysia, as well as zamalah graduate scholarship and special fund for phd students (grant no: f07(dpp55)/1334/2016(2)) from universiti malaysia sarawak (unimas). we thank the local people at sabal area, especially mr ekin empati for the hospitality and kind assistance during field visitation. references andriesse jp, schelhaas rm. 1987. a monitoring studies on nutrient cycles in soils used for shifting cultivation under various climatic conditions in tropical asia. iii. the effects of land clearing through burning on fertility level. agric ecosyst environ 19:311-32. armando snl, darwin wa, jackie om. 1996. fine root mineralisation, soil organic matter and exchangeable cation dynamics in slash and burn agriculture in the semi-arid northeast brazil. agric ecosyst environ 59:191-202. biswas a, alagmir m, haque sms, osman kt. 2012. study on soils under shifting cultivation and other land use categories in chittagong hill tracts, bangladesh. j forest res 23:261-5. bray rh kurtz lt. 1945. determination of total, organic , and available forms 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s, kendawang jj, abdu a, lat j. 2011. soils and vegetation condition of natural forest and secondary fallow forests within batang ai national park boundary, sarawak, malaysia. kuroshio sci 5:67-76. soil characteristics under intensified shifting cultivation ying et al. 83 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 684 goutam banerjee (effect of culture).cdr effect of culture parameters on protease and cellulase production by two bacterial strains, corynebacterium alkanolyticum bacillus licheniformisath3 and cbh7 isolated from fish gut goutam banerjee , ankita nandi and arun kumar ray 1,2 1 1,* 1department of zoology, visva-bharati university, santiniketan 731235, india 2 department of biochemistry, university of calcutta, kolkata 700019, india received 7 august 2016/accepted 22 july 2017 abstract microbial protease and cellulase are in high demand by different industries due to their minimal cost and availability. this study was aimed to maximize the production of protease and cellulase using two bacteria, corynebacterium alkanolyticum ath3 and bacillus licheniformis cbh7, isolated from fish gut. this study demonstrated the effect of different culture parameters in protease and cellulase production using two different bacterial strains. results of this study clearly indicated the importance of different parameters such as moisture content, ph, incubation temperature, incubation period, inoculum size, carbon sources and nitrogen sources in enzyme production. the most critical parameters affecting the enzymes production were ph, temperature, carbon and nitrogen sources. further investigations are required to enhance the enzymes production using genetic engineering. keywords: bacterial strains, culture parameters, product maximization, protease and cellulase introduction protease catalyzes the hydrolysis of protein and cellulose forming amino acids, while cellulase catalyzes glucose (ray et al. 2012a). protease and cellulase are biotechnologically very important and mainly isolated from different living organisms such as plants, bacteria and fungi (saddler 1993; gupta et al. 2002; banerjee et al. 2015b). gut bacteria are proven to produce various substances such as vitamin k, riboflavin, and various enzymes (e.g. protease, amylase, cellulase, lipase, phytase) (ray et al. 2012a). proteases isolated from microbial source are preferred over the enzymes isolated from plant and animal sources because they possess all the characteristics required for their biotechnological application (rao et al. 1998; beg & gupta 2003). proteases constitute 50 – 65% of the global industrial enzymes market (banerjee et al. 2015b). proteases from microbial sources are widely used in different industries such as food, textile and beverages (pastor et al. 2001). proteases are useful for preparing high quality functional feeds through bioconversion of low-cost feed materials (esakkiraj et al. 2007). among several bacterial strains, genus bacillus is considered to be the most important commercial enzyme producers (ray et al. 2012a; banerjee & ray 2016). cellulose, a polymer of glucose, is the primary structural component of most plant cell walls. although this polysaccharide is the most common carbohydrate on earth, relatively few animals are able to utilize this resource efficiently (goodenough & goodenough 1993; he et al. 2015). utilization of cellulose as a nutrient source requires enzymes that cleave beta-1, 4 glycosidic bonds between constituent sugars. the enzyme referred to as cellulases, are required to split beta1, 4 glycosidic bonds in the polymer to release glucose units (barr et al. 1996). although a large number of microorganisms are capable of degrading cellulose, only a few of these microorganisms produce significant quantities of extracellular enzymes capable of completely hydrolyzing crystalline cellulose in vitro. fungi are * corresponding author: aray51@yahoo.com biotropia 4 3 7 192 201 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 3 684 192 the main cellulase-producing microorganisms, though a few bacteria and actinomycetes are also recently reported to be good producer of cellulase (ray et al. 2012a; lugani et al. 2015; banerjee & ray 2016). microbial enzymes have the advantage of large scale production using established fermentation techniques. a successful fermentation process requires favorable environmental and nutritional conditions for the microorganisms (ray . et al 2007). information on the optimum conditions for protease and cellulase production using fish gut bacteria is limited (esakkiraj 2007; ray . et al. et al 2007, 2012b). this study was aimed to optimize the environmental and nutritional parameters to enhance protease and cellulase production using two bacterial strains, corynebacterium alkanolyticum ath3 and cbh7, isolated bacillus licheniformis from the gut of climbing perch, anabas testudineus and walking catfish, , respectively.clarias batrachus materials and methods bacterial strains and culture medium two bacterial strains cor ynebacterium alkanolyticum ath3 (genbank accession no. jx656749) and bacillus licheniformis cbh7 (genbank accession no. hq005269) were isolated from the gut of two species of indian airbreathing fish, i.e. climbing perch, anabas testudineus and walking catfish, clarias batrachus, respectively (banerjee & ray 2013; banerjee et al. 2015a). both isolates were cultured in tryptone soya agar broth for 24 hours at 30 oc to increase the viable count. the bacterial culture medium was prepared according to ray et al. (2012b). enzyme assay extracellular protease activity was assayed following the method of walter (1984). the amount of tyrosine released in the assay medium was measured at 273 nm. cellulase activity was estimated according to the method of denison and kohen (1977). the production of reducing sugar (glucose) from cmc substrate through cellulolytic activity was measured at 540 nm. one unit of specific activity is defined as amount of product released per milligram protein per mil l i l i ter enzyme per minute. protein concentration was determined according to lowry . (1951) using bovine serum albumin as et al a standard. effect of different physical parameters on enzyme production moisture content maximization in order to determine the optimum moisture content, bacteria were cultured in protease (peptone-gelat in) and cel lulase media (carboxymethylcellulose), which were prepared by moistening the media with basal salt (elaborate basal salt) solution. moisture content of the fermentation medium ranged from 5 to75%. effect of ph of the medium optimization of suitable ph for enzymes production was determined by culturing the bacterial strains in tryptone soya agar broth at different ph ranging from 5.0 10.0. incubation temperature maximization optimum temperature of incubation for protease and cellulase production was determined by culturing the bacterial strains at increasing temperature ranging from 20 to 50 c with 10 c o o intervals. incubation period maximization incubation period is one of the most important parameter in fermentation process. to determine the optimum incubation period, enzymes activity was measured up to 120 hours at an interval of 24 hours. effect of inoculum size for this purpose, fermentation media were seeded with 1%, 2%, 3%, 4%, 5% and 6% seed culture in tryptone soya agar broth. effect of different carbon sources in order to optimize the suitable carbon source for enzyme production, fermentation media were prepared with different carbon sources such as glucose, maltose, sucrose, lactose, arabinose, fructose and starch. these alternative carbon sources at 0.2% level were used to replace the original carbon source in the media. effect of different nitrogen sources to optimize the appropriate nitrogen source, the fermentation media were supplemented with both organic (arginine, l-asparagine, tryoptone, beef extract, tyrosine, gelatin) and inorganic 193 effect of culture parameters on enzyme production banerjee et al. metabolic process requirements to supply sufficient energy for maximum biomass production (ray et al. 2012b). cellulase production reached the maximum level using two bacterial strains bacillus subtilis and bacillus circulans at 10% moisture content (ray et al. 2007). protease production reached the optimum level using bacillus cereus at at 120% moisture content (vijayaraghavan et al. 2014). moisture content was a crucial factor for producing bacterial extracellular enzyme, because water was required for growth, metabolic activities and biochemical process. thus, optimum moisture content was required during fermentation process. ph of the culture medium was another important factor controlling enzyme production and activity. in the present study, protease production exhibited by . ath3 c alkanolyticum was maximum at ph 7.5 (3.81±0.096 u), whereas b. licheniformis cbh7 showed the highest production at ph 8.0 (2.22±0.204 u (table 2). on the other hand, cellulase production exhibited by these two bacterial strains was recorded to be the highest at ph 7.5 (8.41±0.83 u in c. alkanolyticum ath3 and 6.27±0.216 u in b. licheniformis cbh7, table 2) and thereafter declined rapidly. cellulase production using bacillus subtilis cy5 isolated from the gastrointestinal (gi) tracts of common carp, cyprinus carpio and using b. circulans tp3 isolated from the gi tracts of tilapia, oreochromis (ammonium chloride, ammonium sulphate, sodium nitrate and potassium nitrate) nitrogen sources at 0.2% level replacing the original nitrogen source in the media. statistical analysis the analysis of variance for different values was carried out in microsoft excel 2007. one way anova followed by duncan's multiple range test (duncan 1955) was conducted at significance level of p < 0.05. results and discussion protease and cellulase production using the two bacterial strains at different moisture content (5 75%) are tabulated in table 1. protease production using the two bacterial strains was the highest at 10% moisture content (3.79± 0.155 u in corynebacterium alkanolyticum ath3 and 2.16±0.132 u in bacillus licheniformis cbh7). maximum cellulase activity was recorded at 15% moisture content (8.34±0.144 u in c. alkanolyticum ath3 and 6.17±0.115 u in b. licheniformis cbh7). medium maximization was the first and foremost step to enhance enzymes production in laboratory experiments and in fermentation industries. each and every element should be in a proper amount to fulfill the biotropia vol. 24 no. 3, 2017 194 table 1 effect of moisture content on enzyme production moisture content (%) specific activity of protease (u )1 specific activity of cellulase (u 2) 5 10 15 20 25 30 50 75 ath3 cbh7 ath3 cbh7 2.79± 0.110b 3.79± 0.155a 2.63± 0.098c 1.22± 0.045d 0.71± 0.051e 0.42± 0.06f 0.33± 0.036f 0.29± 0.018g 1.24± 0.045c 2.16± 0.132a 1.71± 0.055b 0.63± 0.08d 0.58± 0.043d 0.49± 0.034e 0.66± 0.036d 0.42± 0.062e 6.91± 0.183c 7.56± 0.266b 8.34± 0.144a 4.25± 0.158d 2.45± 0.062e 2.29± 0.034e 2.04± 0.055f 1.38± 0.045g 4.18± 0.183d 5.13± 0.098b 6.17± 0.115a 4.69± 0.238c 4.31± 0.091d 3.45± 0.111f 3.88± 0.135e 2.77± 0.078g note: data are presented as mean±sem. n = 3 replications. numbers followed by the same letter did not differ significantly at p < 0.05. 1 u = µg of tyrosine liberated/mg protein/minute. 2 u = µg of glucose liberated/mg protein/minute. mossambicus was higher in optimum ph range of 7.0 to 7.5 (ray et al. 2007). protease activity was maximum at ph range of 6.0 to 6.5 using two bacterial strains, bacillus licheniformis bf2 isolated from the foregut of bata fish (labeo bata) and bacillus subtilis bh4 isolated from the hindgut of bata fish (labeo bata) (ray et al. 2012b). increasing ph beyond optimum level could interfere with amino acid composition of the enzyme leading to the significant decrease of the enzyme activity (esakkiraj et al. 2007). optimum protease production using b. subtilis and the highest protease activity were reached at ph 10.0 (pant et al. 2015). sethi et al. (2013) reported optimum ph required for producing cellulase using several soil bacterial species. in a detailed investigation, fagade and bamigboye (2012) also optimized culture media parameters for cellulase production. the results of their study showed that ph 7.0 was optimum for bacterial strains pseudomonas putida, bacillus subtilis and bacillus licheniformis i, however, ph 5.5 was recorded to be the best for cellulase production using bacterial isolate bacillus licheniformis ii. incubation period is an important parameter for enzyme production by the microorganisms. some microorganisms produce maximally in their exponential growth phase, while other 195 effect of culture parameters on enzyme production banerjee et al. table 2 effect of ph on protease and celluase production ph specific activity of protease (u1) specific activity of cellulase (u2) 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 ath3 cbh7 ath3 cbh7 0.49± 0.055f 0.58± 0.043f 1.74± 0.13d 2.78± 0.193c 3.45± 0.16b 3.81± 0.096a 1.61± 0.052d 0.86± 0.021e 0.31± 0.01f 0.28± 0.052f 0.71± 0.07e 1.13± 0.052d 1.56± 0.085c 1.73± 0.088b 1.94± 0.134b 2.22± 0.204a 1.77± 0.160b 0.95± 0.052d 2.09± 0.075f 3.82 ± 0.17 e 4.90± 0.262d 5.77± 0.145c 7.55± 0.124b 8.41 ± 0.83 a 4.13± 0.410e 2.68± 0.461f 1.43± 0.104g 2.39± 0.249e 2.95± 0.301d 3.48± 0.153d 4.16± 0.202c 5.20± 0.215b 6.27± 0.216a 6.09± 0.705a 5.10± 0.306b 4.49± 0.514c note: data are presented as mean±sem. n = 3 replications. numbers followed by the same letter did not differ significantly at p < 0.05. 1 u = µg of tyrosine liberated/mg protein/minute. 2 u = µg of glucose liberated/mg protein/minute. table 3 effect of fermentation period on enzymes production fermentation period (hours) specific activity of protease (u )1 specific activity of cellulase (u 2) 24 48 72 96 120 ath3 cbh7 ath3 cbh7 1.98± 0.121d 2.72± 0.141b 3.06± 0.206b 3.95± 0.206a 2.32± 0.225c 0.36± 0.062d 0.88± 0.141c 1.57± 0.262b 2.32± 0.113a 1.61± 0.347b 3.87± 0.326d 5.23± 0.248c 8.53± 0.655a 8.07± 0.370a 6.29± 0.266b 2.14± 0.141e 2.99± 0.283d 4.58± 0.643c 6.42± 0.471a 5.47± 0.481b note: data are presented as mean±sem. n = 3 replications. numbers followed by the same letter did not differ significantly at p < 0.05. 1 u = µg of tyrosine liberated/mg protein/minute. 2 u = µg of glucose liberated/mg protein/minute. microorganisms exhibited maximum production in their stationary growth phase. bacterial strains, c alkanolyticum b. licheniformis. ath3 and cbh7 exhibited the highest protease activity (3.95±0.206 u and 2.32±0.113 u, respectively) at 96 hours fermentation period (table 3). cellulase production exhibited by .c alkanolyticum ath3 was maximum at 72 hours of incubation period (8.53±0.655 u). b. licheniformis cbh7 exhibited maximum cellulase production at 96 hours (6.42±0.471 u) (table 3). singh and kaur (2012) optimized the culture condition for cellulase production using js1 bacillus sphaericus and recorded maximum cellulase production at 48 hours of incubation period. in the year 2013, suganthi . (2013) also optimized the culture et al condition of and stated that the b. licheniformis maximum protease production occurred at 24 hours of incubation period. the optimization of incubation period is crucial for obtaining maximum product and thus, several investigations have been conducted in this direction (shafee . et al 2005; ray . 2007; sethi . 2013). bacterial et al et al growth physiology (lag, log, stationary and decline phase) in liquid medium depends on incubation period. in general, maximum enzymes are produced during the log phase and then decreased rapidly due to interference of secondary metabolites. inoculum size is a crucial factor in fermentation process, as the duration of bacterial lag phase is highly dependent on inoculums quantity. the culture used to inoculate the fermentation medium must be in a healthy condition. in this present experiment, a wide range of inoculums size (1 to 5%) was used to get the maximum yield. protease production by .c alkanolyticum b. licheniformis ath3 and cbh7 was the highest when the inoculum size was 2% (4.04±0.132 u) and 3% (2.39±0.243 u), respectively (table 4). cellulase activities exhibited by . c alkanolyticum ath3 (8.58±0.340 u) and cbh7 b. licheniformis (6.51±0.473 u) were found to be the highest at 3% inoculum size (table 4). inoculum quantity normally used is between 3% and 10% of the medium volume (lincoln 1960; meyrath & suchanek 1972; hunt & stieber 1986). lincoln (1960) stressed that bacterial inocula should be transferred in the logarithmic growth phase when the cells were still metabolically active. shafee et al. (2005), however, reported that 4% is the optimum inoculum size for protease production in b. cereus strain 146. similarly, goyal . (2014) obtained et al the highest cellulase activity produced by bacillus sp. at 1% inoculum size using rice straw as substrate. in recent years, the effects of inoculum sizes on protease and cellulase production have been investigated and published by several researchers (suganthi . 2013; agarwal . et al et al 2014; lugani . 2015).et al temperature plays a critical role in maintaining normal body physiology in all living organisms, including bacteria. maximum protease yield by b. licheniformis cbh7 was observed when incubation temperature was 40 c (2.47±0.461 u), while .o c alkanolyticum ath3 exhibited the highest protease production at 45 c (4.18±0.57 u) (table 5) o . 196 biotropia vol. 24 no. 3, 2017 table 4 effect of inoculum size on enzymes production inoculum size (%) specific activity of protease (u1) specific activity of cellulase (u2) 1 2 3 4 5 ath3 cbh7 ath3 cbh7 2.98± 0.208b 4.04± 0.132a 3.10± 0.384b 0.87± 0.079c 0.21± 0.069d 0.82± 0.06c 1.43± 0.2b 2.39± 0.243a 0.98± 0.124c 0.27± 0.036d 6.23± 0.317c 7.16± 0.416b 8.58± 0.340a 3.23± 0.245d 2.01± 0.13e 3.58± 0.26c 5.63± 0.70b 6.51± 0.473a 3.42± 0.205c 2.22± 0.246d note: data are presented as mean±sem. n = 3 replications. numbers followed by the same letter did not differ significantly at p < 0.05. 1 u = µg of tyrosine liberated/mg protein/minute. 2 u = µg of glucose liberated/mg protein/minute. on the other hand, the highest cellulase activity exhibited by these strains was observed at 40 c o (8.66±0.762 u for . ath3 and c alkanolyticum 6.63±0.34 u for cbh7) (table 5). b. licheniformis in this direction, ray . (2007) observed and et al stated that 40 c is the most effective o temperature for cellulase production using b. subtilis b. circulans cy5 and tp3. transport of enzymes to extracellular medium is lower at low temperature compared to that at high temperature (rajoka 2004). however, at higher temperature, the high maintenance of energy requirement for cellular growth is needed due to thermal denaturation of enzymes of the metabolic pathway (aiba . 1973), resulting in maximum et al production. recently, pant . (2015) reported 44 et al oc as the most effective temperature for protease production using . on the other bacillus subtilis hand, sethi . (2013) optimized the cellulase et al production using different soil bacterial strains and reported that 40 c as the optimum o temperature. rasul . (2015) also isolated et al different bacterial strains from soil and sugar industries and optimized their cellulase production. microorganisms grow slowly at a temperature below or above the normal growth temperature due to a reduced rate of cellular production (ray . 2007). therefore, it is et al important to maintain ambient temperature to obtain maximum production. effect of various carbon sources on protease and cellulase production were presented in figure 1a and 1b, respectively. among seven carbon sources, fructose and sucrose were detected to be the most suitable carbon sources for protease production using .c alkanolyticum b. ath3 (4.25±0.52 u) and licheniformis cbh7 (2.53±0.28 u). on the other hand, glucose was recorded to be more effective for cellulase production using . c alkanolyticum ath3 (8.71±0.78 u) and cbh7 b. licheniformis (6.66±0.61 u). it is well established that carbohydrates induce most of the cellulolytic enzymes in bacteria and fungi (morikawa . et al 1995). rajoka and malik (1997) used cellulose, cellulosic residues, xylan, cellobiose and cmc as carbon sources for the enhanced production of cellulases using strain. juhász . cellulomonas et al (2005) reported that high cellulolytic activity could be enhanced using steam-pretreated corn stover as carbon source. carbon sources are very important for bacterial protease and cellulase production (sethi . 2013; suganthi . 2013; et al et al pant . 2015).et al nitrogen sources are the building block of amino acids and considered to be the most important parameter in enzyme production. among various inorganic and organic nitrogen sources, tryptone was detected to be the most important nitrogen source in both protease (4.33±0.388 u and 2.64±0.17 u) and cellulase (8.77±0.66 u and 6.72±0.38 u) production using c alkanolyticum b. licheniformis. ath3 and cbh7 (fig. 2a & 2b). recently, lugani et al. (2015) tested the effect of different nitrogen sources (peptone, yeast 197 effect of culture parameters on enzyme production banerjee et al. table 5 effect of different temperature on enzymes activity different temperature (oc) specific activity of protease (u1) specific activity of cellulase (u2) 20 30 35 40 45 50 ath3 cbh7 ath3 cbh7 0.73± 0.081e 1.45± 0.105d 2.23± 0.246c 3.19± 0.42b 4.18± 0.42a 1.99± 0.19c 0.46± 0.043c 0.79± 0.19c 1.51± 0.173b 2.47± 0.158a 0.88± 0.131c 0.45± 0.072c 2.87± 0.182d 5.13± 0.228c 7.18± 0.55b 8.66± 0.762a 5.03± 0232c 1.89± 0.122e 2.72± 0.25d 3.79± 0.255c 4.92± 0.56b 6.63± 0.34a 4.42± 0.482b 2.34± 0.381d note: data are presented as mean±sem. n = 3 replications. numbers followed by the same letter did not differ significantly at p < 0.05. 1 u = µg of tyrosine liberated/mg protein/minute. 2 u = µg of glucose liberated/mg protein/minute. 198 biotropia vol. 24 no. 3, 2017 figure 1 effect of different carbon sources on enzyme production (note: glu = glucose; mal = maltose; sucro = sucrose; lac = lactose; arabi = arabinose; fruc = fructose; star = starch. data 1 2are presented as mean±sem. a = protease activity; b = cellulase activity. u = µg of tyrosine liberated/mg protein/minute; u = µg of glucose liberated/mg protein/minute) figure 2 effect of nitrogen sources on protease and cellulase production (note: 1 = arginine; 2 = beef; 3 = tryptone; 4 = aspergine; 5 = tyrosine; 6 = nh cl; 7 = nano 8 = (nh ) so ; 9 = kno 4 3; 4 2 4 3; 110 = gelatin. a = protease activity; b = cellulase activity. data are presented as mean±sem. u = µg of tyrosine liberated/mg 2protein/minute; u = µg of glucose liberated/mg protein/minute) 199 effect of culture parameters on enzyme production banerjee et al. extract, ammonium sulphate, ammonium chloride and ammonium nitrate) on cellulase production from bacterial source. results of their investigation reported peptone as the effective nitrogen source in culture medium of bacillus sp. y3. in this direction, ray et al. (2007) stated that organic nitrogen sources exert better result compared to inorganic nitrogen sources in cellulase production using bacillus subtilis cy5 and b. circulans tp3. in recent years, the effects of nitrogen sources on bacterial protease production have also been reported by several researchers (ray et al. 2012b; suganthi et al. 2013; nisha & divakaran 2014; pant et al. 2015). in the present study, inorganic nitrogen sources and other amino acids such as l-aspergine and tyrosine were the poor nitrogen sources for cellulase production. on the contrary, spiridonov and wilson (1998) found that nh compounds are the most 4 favorable nitrogen sources for protein and cellulase synthesis. bacterial flora existing in digestive tract represents a very important and diversified enzymatic potential (ray . 2012a; banerjee . et al et al 2013; banerjee & ray 2016). extracellular enzymes production using bacterial flora not only helps in digestion, but also has great application in different industries (saha . 2006; roy . et al et al 2009; banerjee & ray 2016). ringø et al. 2012; among several enzymes, microbial protease and cellulase have high demand in different biotechnological industries such as food, leather, beverage and detergent (banerjee . 2015b). et al production of these different types of extracellular enzymes largely depends on medium compositions and different physical factors (ray et al. 2012b). medium optimization is the most important step to enhance the production in laboratory experiments and in fermentation industries. until now, reports on optimization of enzymes produced by gut bacteria are scanty, and thus, extensive research should be conducted to fulfill the demand. conclusions this study emphasized on production maximization of bacterial protease and cellulase due to their important contribution in several biotechnological industries. bacterial strains corynebacterium alkanolyticum bacillus ath3 and licheniformis cbh7 as active producer of extracellular protease and cellulase, might be useful in different industries like bakery, dairy, meat, like textile, food, laundry, paper, biofuel and brewing sectors. acknowledgements we are very much thankful to university grants commission (ugc), new delhi, for financial support. we are also grateful to department of zoology, visva-bharati university, india, for providing such vast support. references agarwal t, saxena mk, chandrawat mps. 2014. production and optimization of cellulase enzyme by pseudomonas aeruginosa mtcc 4643 using sawdust as a substrate. int j sci res 4:1-3. aiba s, humphrey ae, millis nf. 1973. biochemical engineering. second edition. new york (us): academic press. p. 92127. banerjee g, ray ak, askarian f, ringø e. 2013. characterization and identification of enzymeproducing autochthonous bacteria from the gastrointestinal tract of two indian air-breathing fish. benef microbes 4:277–84. banerjee g, dan sk, nandi a, ghosh p, ray ak. 2015a. autochthonous gut 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production from a halotolerant isolated from saltern bacillus licheniformis sediment. j genet eng biotechnol 11:47-52. vijayaraghavan p, lazarus s, vincent sgp. 2014. de-hairing protease production by an isolated bacillus cereus strain at under solid-state fermentation using cow dung: biosynthesis and properties. saudi j biol sci 21:27–34. walter he. 1984. methods of enzymatic analysis. weinheim (de): verlag chemie. p. 238. 201 optimal bacterial density and fertilizer dosage for bioremediation banerjee et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 microsoft word 1 biotropia no. 23, 2004 : 1 12 optimization of cellulase production with penicillium nalgiovense sll grown on pretreated wheat pollard tresnawati purwadaria1, agnes t. kumalasari2, tutiharyati1, pius p. ketaren' and arnold p. sinurat1 1 indonesian research institute for animal production, po box 221, bogor 16002, indonesia 'department of chemistry, faculty of science and mathematics, bogor agricultural university, jl. raya pajajaran, bogor, indonesia abstract the cellulase production with penicillium nalgiovense s11 on wheat pollard was enhanced using substrate pretreatments, i.e.: (i) mechanic process by wiley milling, (ii) reducing sugars removal by water soaking, and (iii) chemical pretrcatment by 0.5% naoh soaking at 100°c. the enzyme production stated as enzyme activities of all prctrcated substrates were higher than the untreated substrate. although soaking with water showed significant increase in enzyme activities, the highest cmcase (ec 3.2.1.4), fpase (filter papcrase) and pglucosidase (ec 3.2.1.21) were observed on naoh pretreated pollard. the naoh prctreatment also enhanced the enzyme production by increasing substrate concentration from 2 to 4%. the optimal incubation time in the cellulase production on 4% naoh-pretrcated pollard was observed on the fifth day. addition of 250 ppm glucose also increased the enzyme activities. the optimal treatments increased the specific activities of cmcase, fpase, and |3-glucosidase into 60, 4, and 198 times, respectively, as compared to the specific activities on 2% unpretreated pollard. keywords : cellulase production/ pollard pretreatments/ water soaking/ naoh soaking introduction most poultry feedstuff such as corn and soybean meals, rye, barley, rice bran, wheat bran, coconut meal, and palm kernel cake from plants contain fibers including hemicellulose, cellulose, and lignin. in contrast with ruminants, poultry digestive track does not digest hemicellulose and cellulose. recently, it was reported that fermentation occurred in the caecum of poultry due to microbial activity (josefiak et al. 2004), the fermentation could break down cellulose in a small quantity. the addition of cellulases and hemicellulases in the poultry feed had been reported to increase body weight, efficiency of feed utilization, the apparent metabolizable energy, and dry matter digestibility (campbell and bedford 1992; friesen et al. 1992; marquardt et al. 1996). the effectiveness of the enzymes was related to the adequate activity of enzyme added. cellulase production has been studied since thrichoderma reesei was isolated by reese in 1950. however, the enzyme activity was limited by its low specific activity. penicillium nalgiovense sll was isolated from the nest of termites glyptotermes montanus (nurbayti 2002). the mold had the ability to produce cellulase that digested high crystalline cellulose (filter paper), the most common components in natural cellulose. the enzyme could be produced in the submerged culture containing 2% wheat pollard. increasing the substrate into 3% concentration 1 biotropia no. 23, 2004 reduced the enzyme activity due to limited oxygen availability and increasing the repression effect caused by soluble carbohydrate, such as starch and reducing sugars (gong and tsao 1979). the repression effect from the pollard could be omitted by reducing the soluble sugars using water soaking. in addition, the production of enzyme could be enhanced by mechanic treatment on the substrate using milling and extrussion, and chemical treatment using phosphoric acid, hydrochloric acid, sodium hydroxide (naoh), and ammonia (klyosov 1986; marsden and gray 1986). among the chemical pretreatments, naoh was the most commonly used. the naoh treatment swells the crystalline regions of cellulose and extends the amorphous regions. the amorphous regions are more digested than the crystalline regions. recently, shin et al. (2000) found that the naoh pretreatment at room temperature extracted the short molecules in the amorphous region of tenzel, a cellulose fiber produced from wood pulp into the open-up structure. this condition enhanced the cellulase hydrolysis activity. in this experiment the cellulase production of p. nalgiovense s11 was optimized by substrate pretreatments i.e.: (i) mechanic process by wiley milling; (ii) removal of reducing sugars by water soaking; and (iii) chemical treatment by naoh soaking at 100°c. the activities of carboxymethylcellulase (cmcase) and filter paperase (fpase) in hydrolyzing the backbone of amorphous and crystalline cellulose, as well as p-glucosidase that cleaves the cellooligomers into glucose were determined as parameters for enzyme production. materials and methods pretreatment of wheat pollard wheat pollard was obtained from pt bogasari wheatmill and used as substrate for p. nalgiovense s11 (riap collection) in producing cellulase. the wheat pollard was pretreated before being used as substrate i.e.: (i) wheat pollard without pretreatment as a control referred as pollard; (ii) milling; (iii) water soaking; and (iv) naoh soaking, referred as water and naoh solution pretreated, respectively. in the wiley mill, the pollard was ground to a fine powder of 0.5 mm. for the water treatment, 25 g pollard was soaked for one hour in 500 ml of water at room temperature, then the water was squeezed out using cotton cloth, the solid material was dried in the 40°c oven, and ground with wiley mill (0.5mm). the method for naoh pretreatment was carried out following purwadaria (1988). the same amount of pollard (25g) was soaked in 500 ml of 0.5% naoh solution (w/v) for 1 hour at 100°c. then the pollard was filtered through cotton cloth and washed under running tap water until the wash water was neutral to ph paper. excess water was squeezed out, dried in the 40°c oven, and ground with wiley mill. ccllulase production of penicillium nalgiovense si 1 tresnawati purwadaria et al. enzyme production enzymes were produced by p. nalgiovense s11 in the mandels medium containing yeast extract 3 g/1, minerals in g/1 (nh4)2so4 1.4, kh2po4 2.0, mgso4 0.3, urea 0.3, and cacl2 0.3 and'in ppm feso4 5, mnso4 16, znso4 14, and cocl2 20 and pollard (purwadaria et al. 2003a). the concentration of pollard and pretreated pollard was 2, 3, or 4%. molds were cultivated in 50 ml medium in a 250 ml erlenmeyer flask at 29°c using reciprocal shaker (150 rpm), after inoculation with 2 ml of spore suspension from five day pda culture slant (15x10*'spores/ml). the incubation time was carried out for three, four, or five days. sodium azide was added at 0.2% final concentration. the culture was then centrifuged (12 000 rpm, 20 min, 4°c) and supernatant was collected for enzyme assays. enzyme activities the activity of carboxymethylcellulase (cmcase) was assayed by determining the reducing sugars produced from cmc as glucose or mannose, respectively (haggett et al. 1979). one unit was defined as amount of enzyme which liberates one umol glucose per minute. the p-dglucosidase and (3-d-cellobiosidase were assayed using p-p-d-nitrophenylglucoside and p-p-dnitrophenylcellobioside (from sigma company) as substrates and one unit was defined as enzyme which liberates one umol nitrophenol per minute (ide et al. 1983; deshpande et al. 1984). filterpaperase (fpase) was determined by using filter paper whatman no 1 (1 cm x 6 cm) as substrate following mandels and sternberg (1976). the production of 2 mg glucose in the reaction was stated as 0.185 unit of fpase. all assays considered the controls prepared as samples, but without incubation time. specific activity of all enzymes was calculated in units/mg protein (u/mg). determination of protein, reducing sugar, and fiber component concentrations protein concentration was determined by bradford method (1976) and bovine serum albumin was used as standard. reducing sugar concentration in pollards was determined after water extraction using dns method (miller 1959). the concentrations of fiber components (cellulose, hemicellulose, lignin and silica) of pollard before and after pretreatment were calculated as neutral and acid dietary fiber according to van soest and robertson (1968). results and discussions every pretreatment of the pollard enhanced the enzyme production (activities) of p. nalgiovense s11 (table 1). milling increased the ratio of surface area to volume resulting to better mold growth and enzyme production. the results are in agreement with cellulase production of bacillus subtilis cbtk 106 on banana biotropia no. 23, 2004 wastes (krishna 1999). it was reported that the increase of the bacterial enzyme activities (cmcase, fpase, and p-d-glucosidase) was parallel with particle reduction. however, the optimum reduction was observed at particle size of 0.4 mm. in the case of p. nalgiovense s11, compared with untreated pollard, milling at particle size of 0.5 mm increased fpase and p-d-glucosidase, but slightly decreased cmcase activities. in this condition, oligosaccharides and the size reduction of crystalline and amorphous celluloses might induce more fpase and p-d-glucosidase, but repressed the cmcase production. table 1. ccllulaso production of p. nalgiovense si 1 on pretrcatcd substrate pollard pretreatments [protein] (ug/ml) activity(u/ml) specific activity (u/mg protein) cmcase fpase p-gluc cmcase fpase p-gluc none (control) 322±34 0.10+0.05 0.90+0.02 0.004+0.001 0.31+0.16 2.82+0.33 0.014+0.006 milling 334±55 0.06+0.02 1.89+0.18 0.024±0.012 0.19+0.02 5.8h1.35 0.070+0.021 water soaking 181+ 4 1.02+0.02 1.77+0.07 0.091+0.004 5.61+0.03 9.78+0.50 0.504+0.017 naoh soaking 229+30 2.96+0.11 1.93+0.07 0.199+0.031 13.08+1.38 8.56+1.41 0.870+0.080 fpase was filter paperase, p-gluc was p-glucosidase milling also slightly increased extracellular (soluble) protein. therefore, the ratio of specific activities (u/mg protein) of each enzyme between milling and non-treatment was similar with the activities (u/ml). fpase and p-d-glucosidase of specific and enzyme activities were higher in milling, while cmcase was lower than those of untreated pollard. in the case of water pretreatment, soaking decreased the reducing sugar content (table 2) that reduced the repression effect. some starch and pectin were solubilised and rinsed with water resulting in the decrease of the viscosity of the medium (pawlik et al. 1990). then, the mold had better growth and enzyme production than in untreated pollard due to better oxygen transportation and substrate homogen-ization. although, all enzyme activities increased in water pretreatment, extracellular protein was decreased than in untreated pollard. these indicated that the protein in the water pretreatment process might be only related to enzyme production as the soluble protein from pollard was entirely rinsed. the increase of enzyme activities and the decrease of protein concentration resulted in the increase of the specific activities of cmcase, fpase, and p-d-glucosidase to 18, 3, and 35 times, respectively, than those in untreated substrate, while the activities were only 10, 2, and 23 times higher than those in untreated substrate, respectively. ccllulase production of penicillium nalgiovense s11 trcsnawati purwadaria et al. table 2. the fiber and reducing sugar contents of pollards before and after treatment pretreatments [reducing sugars] (%) [fiber] (%) hemiccllulosc cellulose lignin none (control) 0.16 23.0 7.4 1.5 milling 0.17 23.0 7.4 1.5 water soaking 0.13 37.8 12.0 2.0 naoh soaking 0.01 49.5 26.9 7.1 the highest enzyme activity was observed in naoh pretreatment. in this process milling, water soaking and naoh action in the fiber, all together changed the pollard composition (table 2) and altered the substrate into more digestible fibers (khrisna 1999, shin et al. 2000; and devrije et al. 2002). among pretreated pollard, naoh pollard had the highest hemicellulose, cellulose, and lignin contents (table 2) due to the solubilisation of some dry materials including amino acids, starch, pectin, and short oligosaccharides. although its fiber content was the highest, more digestible fiber in naoh treatment resulted better growth and enzyme production. since the reducing sugars of naoh pretreated pollard was very low, it was possible that s11 used only glucose or energy to grow from cellulolytic hydrolysis. the less repression effect of small amount of reducing sugars and the need for energy highly induced the enzyme production and increased the extracellular protein content (table 1). the naoh pretreatment also altered the fiber structure into more swelling materials as a result it can be more submerged in the culture. before treatment, the pollard was more floating. these conditions affected better substrate homogenization and oxygen transferred in the culture, which is important for the mold growth. the high protein content in the naoh pretreated pollard resulted in lower specific activity of fpase than of the water pretreatment. however, other enzyme activities and protein content were much higher than in the water pretreated pollard. therefore, naoh pretreatment was applied for further experiment. in the following experiment, the concentration of naoh pretreated pollard increased from 2 to 3 and 4%. increasing the substrate content changed the enzyme production in the course of incubation time (figure 1 and 2). the repression effect in the enzyme production by increasing the untreated substrate from 2 to 3% (nurbayti 2002) was not observed in this experiment. the highest production of cmcase for digesting amorphous cellulose was observed at 4% pollard. the optimum cmcase activity in 2% pollard was observed at three days incubation time and thereafter was decreasing, while in 3% pollard reached the optimum activity at four days, and in 4% pollard was still increasing even at five days incubation time (figure 1a). compared with cmcase activity, fpase showed different patterns (figure ib). for biotropia no. 23, 2004 every substrate concentration, the optimum activity was reached at 4 days incubation time and thereafter it was declining. the highest fpase activity was observed in 4% pollard that also had the highest reduction at five days incubation time. the production of hydrolytic enzymes was generally parallel with the growth curve of microorganisms, the activity was increasing until optimum incubation time and then was stable or decreasing (sachslehner et al. 1998). the optimum incubation time for enzyme production was related to substrate concentration. the higher substrate concentration of coconut meal in submerged culture of eupeniciuium javanicum resulted in longer lag phase or longer optimum incubation time for p-mannanase production (purwadaria et al. 2003b). in the case of cmcase production with p. nalgiovense sll, the optimum activity in 2% substrate concentration was reached at three days incubation time, after that, due to limited nutrient concentration, the enzyme production ceased. in the higher substrate concentrations, cmcase reached the optimal activities after longer incubation time, four days for 3% pollard, and at least 5 days for 4% pollard. in the more concentrated substrate, the culture needs longer incubation time due to less oxygen transfer. longer optimum incubation time in the higher substrate concentration rarely occurred in the soluble substrate. increasing xylobiose from 0.05 to 1.00% for the production of pmannanase with sclerotium rolfsii resulted to similar optimum incubation time at 15 hours (sachslehner et al. 1998). in this case, concentration higher than 1% might repress the enzyme production. the reduction of fpase on 4% naoh pretreated pollard at five days incubation time showed that unlike the cmcase, the fpase was not produced anymore. it is difficult to explain why the cmcase was still produced, while fpase stopped. the reasonable explanation for the reduction of fpase was enzyme instability. nurbayti (2002) reported the reduction of fpase activity from culture filtrate of sll stored at room temperature. the highest reduction of fpase in the 4% substrate might be due to the highest reducing sugar concentration produced from the hydrolytic action of cellulase in the substrate, which inhibited the activities of the enzyme especially the fpase. the curves of specific activities for both cmcase and fpase (figure 2) were similar to curves of enzyme activities (figure 1) reflecting those which were not affected by the protein concentration. this result confirmed the data from pretreatment comparison (table 1), where the extracellular protein produced in naoh pretreated pollard was more for cellulase, but not for other proteins. the optimum substrate concentration to produce cmcase and fpase was 4%, however, the optimum incubation time for cmcase and fpase were five and four days; respectively. the high fpase is important for feed supplement application, since most natural celluloses contain crystalline structure. however, in the fifth day the increase of cmcase was much higher than the decrease of fpase. the cmcase was also needed for the synergistic hydrolytic activity on sigmacell 20, the microcrystalline cellulose (purwadaria 1995). therefore, the fifth day incubation in 4% substrate was selected for further experiment in exploring the effect of glucose addition. cellulase production of penicillium nalgiovense s l l tresnawati purwadaria et al. the addition of glucose at 250 ppm in 4% naoh pretreated pollard (containing 50 ppm reducing sugars) increased cmcase and fpase, while higher glucose concentration reduced the activities (figure 3). however, the reduction of cmcase and fpase in the addition of glucose at 1000 rpm were only 6 and 9%, respectively. p. nalgiovense sll is quite resistant to catabolite repression. in another experiment the isolate still produced cellulases in 3% naoh pretreated pollard added with 10,000 ppm (1%) glucose, but the activity was much lower than that without glucose addition (sanjaya 2003). 250 500 750 1000 glucose concentration addition(ppm) figure 3. the relative specific cellulase activities from cultures added with glucose p-glucosidase and cellobiohydrolase activities were determined in the optimum fpase and cmcase production with the addition of glucose at 250 ppm. compared to the p-glucosidase and cellobiohydrolase produced in 4% pollard without glucose addition, those were 0.87 and 0.14 u/ml, respectively, p-glucosidase was increased to 1.10 u/ml, while cellobiohydrolase was reduced into 0.07 u/ml. similar to cmcase and fpase, p-glucosidase was enhanced by the addition of glucose at 250 ppm, but the condition repressed the cellobiohydrolase. the cellobiohydrolase might be the least resistant enzyme to catabolite repression. the cellulase production was induced by short oligosaccharides including disaccharides, such as cellobiose, xylobiose and sophorose, while pure or natural polysaccharides induced more cmcase, xylanase, and mannanase (sachslehner et al. 1998). in the low concentration of monosaccharides such as glucose, galactose, and mannose, the low constitutive cellulase could be produced (gong and tsao 1979; sachslehner et al. 1998). therefore, in the low glucose addition (250 ppm), biotropia no. 23, 2004 the cellulase production of p. nalgiovense si 1 increased, since glucose was used by the mold to start growing. the low addition of glucose (500 ppm) in 1% gum locust bean was also reported to increase the production of mannanase by e. javanicum (haryati et al. 1995). generally in the presence of high reducing sugars, the cellulase is produced after the reducing sugars are used (sachslehner et al. 1998). the same authors also reported that although one dose of cellobiose 3.0 mm (0.5%) could induce the cellulase production of s. rolfsii, the ten times incorporation at 0.3 mm at every 2.5 hours induced 2.5 times higher enzyme. in the case of our experiment, the addition of glucose at 250 ppm increased cmcase, fpase, and p-glucosidase, but reduced cellobiohydrolase. detailed experiment to observe the regulation mechanism of s11 has not yet been carried out. it could be concluded from this experiment that naoh pretreatment in wheat pollard, increasing substrate concentration from 2 to 4% and addition of glucose at 250 ppm at 5 days incubation are the optimum conditions to produce cellulase of p. nalgiovense sll based on its specific activities of cmcase, fpase and p-glucosidase. the expense of naoh pretreatment and the gain of enzyme activities has to be economically evaluated. acknowledgment the authors appreciate drh. sulistyani phd. from chemistry department, fmipa-bogor agricultural university for sharing her comments on the result of the experiment. references bradford, m.m. 1976. a rapid and sensitive method for the quantitation of microgram quantities of protein 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induction of mannanasc, xylanase and endoglucanase activities in sclerotium rolfsii. appl. environ. microbiol. 64: 594-600. sanjaya, s. 2003. produksi selulase penicillium nalgiovense sll hasil radiasi dengan ultra violet (cellulase production of penicillium nalgiovense sl l mutants by ultra violet radiation). thesis. chemistry department. fmipa pakuan university, bogor, indonesia. 11 biotropia no. 23, 2004 shin, y. k.. son, and d.i. yoo. 2000. structural changes in tencel by enzymatic hydrolysis. j. appl. polym. sci. 76: 1644-1651. van soest, p.j. and j.b. robertson. 1968. system of analysis for evaluating fibrous feeds. jn w.j. pigden ed. standardization of analytical methodology for feed. cent. canada, idrc. 134e. 12 1.pdf 2.pdf 3.pdf 4.pdf 5.pdf 6.pdf 7.pdf 8.pdf 9.pdf 10.pdf 11.pdf 12.pdf sri-13 mei 2017-480 (balachandran pollinator).cdr pollinator diversity and foraging dynamics on monsoon crop of cucurbits in a traditional landscape of south indian west coast c. balachandran, m.d. subash chandran, s. vinay, naik shrikant and t.v. ramachandra* energy and wetlands research group, centre for ecological science, indian institute of science, bangalore, karnataka 560012, india received 13 april 2015/accepted 11 october 2016 abstract studies on insect pollinator ecology and dynamics are very rarely carried out in traditional indian agriculture landscapes. indiscriminate landscape changes in the rural areas and tendencies towards crop monocultures can have significant effects on pollinator habitats and effectiveness. this study was aimed at observing insect pollinators, their visitation frequencies and timings on monsoon cucurbit crops such as cucumis sativus l., c. pubescens willd., momordica charantia l., trichonsanthes anguina l. and luffa acutangula l. (roxb.), in a coastal karnataka village. this study was also aimed at covering the significance of the surrounding landscape elements in sustaining pollinator elements. bees, such as apis dorsata, a. cerana and trigona sp., were major visitors on all cucurbits, except snake gourd which was pollinated mainly by lepidopterans. insect species were found to partition floral resources of any given crops between them by minimal overlapping in their visitation timings. natural elements of the landscape around, mainly a village forest and rocky savanna furnished habitats for bees and lepidopterans. prolifically blooming monsoon herbs on lateritic plateaus, by providing nectar resources for pollinators, presumably play key role in making the case study village well known for monsoon vegetables. keywords: agroecosystem, crop pollination, cucurbits, foraging behavior, landscape, laterite introduction zoophilous pollination by diverse animal pollinators is very important in flowering plants. about 87 of 115 important food crops of the world depend on animal pollination (kevan 1999; steffan-dewenter & westphal 2008). insects pollinate an estimated 70% of flowering plants (schoonhoven et al. 1998), of which the bee community (family apidae) is the most important and efficient pollinators (danforth et al. 2006). increased flower visitation rate by insects causes more pollen deposits on the stigma, benefitting higher seed setting and better seed quality (engel & irwin 2003). naturally, peak flowering seasons correspond with the highest densities of pollinator taxa (wolfe & barrett 1988). studies from an ecological perspective reveal landscape level habitat heterogeneity as having strong bearings on sustaining insect pollinators (verhulst et al. 2004; roschewitz et al. 2005; fahrig et al. 2011). linkages within pollination guilds can be disrupted in crop areas of habitat patches reduction causing increasing distances between nesting and foraging areas (steffan-dewenter et al. 2006; pauw 2007). most pollinators require a reliable supply of nectar within natural pollinator specific foraging distances to provide sufficient pollinator services including gene flow. honeybees are known to forage in 1 3 km radius from the colony (within 1 km for apis florea fabricius, 1.5 km for a. cerana fabricius and 3 km for a. dorsata fabricius), although pollination efficiency is at the best between 183 – 275 m (free 1993; abrol 2012). landscape maintenance focusing on better plant-pollinator interactions and on pesticide-free farming will have much significance in ensuring adequate food supplies for the world (palma et al. 2015). the more we know about pollinators, plant pollination services biotropia 4 1 7 16 27 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 1 480 * corresponding author: cestvr@ces.iisc.ernet.in 16 and the interactions between agro-ecosystems and pollination management, the more we can understand how to conserve them and manage them to maintain biodiversity, ensure ecosystem health and improve human livelihoods (fao 2016). cucurbitaceae (118 genera, 825 species), members known as cucurbits, is an important family of food crops used as fruits (melons), salads (cucumber, gherkins, long melon), sweets (ash gourd, pointed gourd), pickles (gherkins), desserts (melons), etc. although widely distributed, cucurbit diversity is more concentrated in tropics and subtropics with hotspots in southeast asia, west africa, madagascar and mexico (schaefer & renner 2011). india is rich in cucurbits and their wild relatives (31 genera, 94 species including 10 endemics). cucurbits genera with the highest numbers of species are trichosanthes (22 species), cucumis (11 species), momordica (8 species) and zehneria (5 species) (renner & pandey 2013). honeybees are considered the most efficient pollinator for cucurbit (grewal & sidhu 1979). this study on cultivation of mixed crop of cucurbits in the monsoon period conducted in utttara kannada, district of south indian west coast, is important because it relates to high production of pesticide free, good quality and wholesome gourds from a relatively small area by indigenous farmers. the undulating terrain borders the foothills of the western ghat mountain ranges. the cultivators are indigenous subsistence farmers of halakkivokkal community which depends on traditional farming techniques, cattle manure and leaf manure. the rural landscape has rich biodiversity despite being in the vicinity of the densely populated town kumta and nearby villages. although cucurbits are grown year-round in india, studies on pollination during monsoon are needed in southwest india when cucurbit cultivation is at the peak. the study was aimed at documenting diversities of pollinator insects, their visitation timings and frequencies on cucurbits i.e. cucumis sativus cucumis pubescensl. (cucumber), willd. (mangalore gourd), l. (bitter momordica charantia gourd), l. (snake gourd) and trichonsanthes anguina luffa acutangula l. (roxb.) (ridge gourd). it also aimed at the role of diverse elements of a traditional landscape in sustaining cucurbit pollinators and the synchronism of the pollinators with the varying flowering schedules of respective crops. materials and methods study area tannirkuli village (14.45876 ºn and 74.42694 ºe), in the hegde panchayat of kumta taluk of uttara kannada in the south indian west coast was chosen for the study. tannirkuli is dominated by halakkivokkal community farmers who are skilled growers of vegetables using traditional agricultural techniques. during the intense coastal monsoon, the farmers mainly grow cucurbits in a 10 ha well-drained ground, while rice is cultivated in the flooded fields. groundnut, vegetables and cucurbits constitute the second crop in the postmonsoon fields when the hill slopes are too dry for cultivation (fig. 1). pollinator diversity and foraging dynamics on monsoon crop of cucurbits – balachandran et al. 17 figure 1 map of study area: tannirkuli village, uttara kannada district, karnataka state, india flowering and continued at weekly intervals until flowering nearly stopped (belavadi & ganeshaiah 2013). numbers of visitor insects were counted species-wise. the frequency of visitation by a species was categorized as very frequent (>50% of the floral visits); frequent (>20% and <50%); and rare (<20%). insect specimens were collected using a sweep-net and identified using sampling design the preliminary studies were carried out from july to early august 2013. observations on insect visitations on flowers were conducted from midaugust to the end of september. the crops studied were cucumber, bitter gourd, snake gourd, ridge gourd and mangalore gourd (fig. 2). the observations on pollination were started at 10% biotropia vol. 24 no. 1, 2017 18 figure 2 flower and fruit of cucurbits plants notes: a & b = male flower of cucumis sativus, c & d = female flower of cucumis sativus; e & f = male flower of momordica charantia, g & h = female flower of momordica charantia; i = male flower of luffa acutangula, j = female flower of luffa acutangula, k = delias eucharis on trichosanthes anguina, l = fruit of cucumis pubescens, m = fruit of trichosanthes anguina, n = fruit of luffa acutangula, o = fruit of momordica charantia, p = fruit of cucumis sativus taxonomic keys (michener 2007; belavadi & ganeshaiah 2013). the specimens were examined using stereo microscope for the presence of pollen and segregated as pollinators and nonpollinators. the pollen grains were matched with the host pollen separately collected before ascertaining the visitor insect as a pollinator of a specified crop, in accordance with belavadi and ganeshaiah (2013). the insect specimens were maintained in the kumta field station of centre for ecological sciences, indian institute of science. foraging dynamics insect foraging behavior was studied between 06:00 and 22:30 hours, at hourly intervals during species-specific blooming times. the observation protocols and frequency of visitors were measured using a hand tally counter and stopwatch following free (1993). sampling units 2of 1 m (approximately covering 20 30 flowers) were selected and the visitor insects were documented. each observation period for any crop selected was for 5 minutes, at 3 replications per hour. the foraging patterns were assessed based on the dominant groups of visitor insects and peak foraging times. landscape analysis landscape elements of 3 km radius around the study area were deciphered using remote sensing data from google earth and landsat-8 imageries of 2014. the time series spatial data acquired from landsat series multispectral sensor and thematic mapper sensors were downloaded from http://glcf.umiacs.umd.edu/data. land use (lu) analysis involved preprocessing, classification and accuracy assessment. land use classification was done using supervised pattern classifier-gaussian maximum likelihood algorithm through open source gis i.e. grassgeographic resource analysis support system downloaded from http://ces.iisc.ernet.in/grass (fig. 3). the classifications were based on derived signatures (training polygons). classifier performance was assessed considering reference pixels using kappa (κ) statistics (ramachandra et al. 2013). figure 3 method followed for land use assessment satellite data pre processing acquisition false colour composite land use classification accurancy assessment ground data, toposheets, bhuvan, google eart 19 pollinator diversity and foraging dynamics on monsoon crop of cucurbits – balachandran et al. results and discussion diversity and abundance of insect visitors on cucurbits table 1 lists the spatial extent of landscape elements in the study area. about 25.1% of the area was used for agricultural fields (mainly paddy cultivation), 12.3% was used for horticulture (coconut, areca), 4.4% for forest area, 0.8% for built-up area and 0.5% for vegetable gardens. the overall accuracy of the classified data was 85.32% with kappa of 0.813 (table 1). male flowers opened earlier than the female ones in all species. in bitter gourd, cucumber and mangalore gourd anthesis commenced before sunrise, in ridge gourd towards sunset and in snake gourd after sunset (table 2). a total of 24 insect species (22 genera, 14 families, 4 orders), visited cucurbit flowers (table 3). table 1 elements of agricultural landscapes with beneficiary resource for pollinators land use classification area (0-3 km radius) % f n ha built-up residential area, industrial area, mixed pixels with built-up area 23.7 0.8 open area open lands, quarries 212.1 7.5 open wetland aquaculture and wetlands 96.4 3.4 water river, drainages 308.8 10.9 agriculture agriculture fields current fallow and sown (paddy) 709.3 25.1 cashew cashew plantations in scrub jungle 269.6 9.5 + + forest evergreen forests, deciduous and mixed forests 123.7 4.4 + + horticulture areca gardens, coconut plantations 349.2 12.3 + laterite laterite quarry, laterite open lands 318.5 11.3 + laterite acacia acacia auriculiformis plantation in laterite plateaus 193.8 6.9 acacia acacia auriculiformis plantation 210.8 7.5 vegetable farm cucurbit varieties with inter crops like ladies finger 12.8 0.5 + total area (ha) 2,828.7 100 overall accuracy of classification: 85.32%; kappa statistics: 0.813 notes: n = nesting habitats; f = foraging habitats; + = present table 2 anthesis time observed in cucurbits studied (in the monsoon months) species common name male flower female flower starting time full bloom starting time full bloom momordica charantia bitter gourd 5:20 a.m 5:40 a.m 5:40 a.m 6:30 a.m cucumis sativus cucumber 6:10 a.m 6:40 a.m 6:10 a.m 7:10 a.m cucumis pubescens mangalore gourd 6:30 a.m 6:55 a.m 7:00 a.m 8:20 a.m luffa acutangula ridge gourd 5:20 p.m 6:10 p.m 5:25 p.m 6:30 p.m trichosanthes anguina snake gourd 8:20 p.m 8:50 p.m 8:30 p.m 9:25 p.m 20 biotropia vol. 24 no. 1, 2017 with 8 species of butterflies and 3 species of moths from 7 families, the lepidopterans had the highest numbers of pollinators. hymenopterans followed with 6 species of bees and 3 species of wasps. bees were the most frequent flower visitors. the dipterans (flies) and coleopterans (beetles), with 2 species each, were infrequent visitors and had no notable role in pollination as specimen examination revealed general absence of pollen. hymenopterans were observed as having the highest visitations on ridge gourd, followed by mangalore gourd, cucumber and bitter gourd, respectively. lepidopterans were dominant on snake gourd and frequent on bitter gourd. bitter gourd was also visited by bees. coleopterans and dipterans were infrequent on all cucurbits, except snake gourd. crop-wise abundance of insect visitors is summarized in figure 4. a. dorsata had the highest abundance of visits on ridge gourd (70.1%) followed by cucumber (66%), mangalore gourd (56%) and bitter gourd (38%). a. dorsata was negligible on snake gourd. a. cerana was most abundant on mangalore gourd (39.6%) and cucumber (31.6%). ridge gourd and bitter gourd also had good dependence on a. cerana. trigona sp. more frequently visited bitter gourd than others. butterflies and moths were predominant table 3 insect species found on cucurbit flowers during the monsoon period family species common name c . s at iv us (c uc um b er ) c . p ub es ce ns (m an ga lo re g o ur d) m . c ha ra nt ia (b it te r go ur d) l . a cu ta ng ul a (r id ge g o ur d) t . a ng ui na (s n ak e go ur d) hymenoptera apidae apis dorsata fabricius giant honeybee + + + + apis cerana indica fabricius indian honeybee + + + + trigona jurine stingless bee + + + + ceratina latreille small carpenter bee + + + + xylocopa latreille carpenter bee + halictidae halictus latreille sweat bee + + vespidae vespa cincta degeer yellow banded wasp + + vespa l. common wasp + polistes latreille paper wasp + lepidoptera nymphalidae hypolimnas bolina l. great eggfly + papilionidae papilio polytes l. common mormon + + graphium agamemnon l. tailed jay + + pieridae catopsilia pomona fabricius common emigrant + + eurema hecabe l. common grass yellow + delias eucharis drury common jezebel + + papilionidae pachliopta hector l. crimson rose + hesperidae borbo cinnara wallace rice swift + + + + sphingidae cephonodes hylas l. bee-hawk moth + + + + arctiidae amata bicincta kollar handmaiden moth + + + geometridae dysphania percota swinhoe blue tiger moth + + coleoptera chrysomelidae aulacophora dejean leaf beetle + + + coccinellidae henosepilachna li & cook ladybird beetle + + diptera tabanidae tabanus l. horsefly + muscidae musca domestica l. housefly + + + note : + = present 21 pollinator diversity and foraging dynamics on monsoon crop of cucurbits – balachandran et al. on snake gourd. beetles and flies were the least important pollinators on all crops and did not visit snake gourd. in regards of pollinators visitation on cucumis spp., 11 insect species visited and 10 c. pubescens species visited (table 3). was c. sativus a. dorsata observed dominant visitor on spp., 66% cucumis on mangalore gourd and 56% on cucumber. a. cerana had 39.6% visitation on mangalore gourd and 31.6% on cucumber in dharwad, . neighboring district of uttara kannada, prakash (2002) recorded 27 species of insects pollinated cucumber during post-monsoon season, where honeybees ( and ) as a. dorsata, a. cerana a. florea well as smith were the trigona iridipennis dominating visitors, validating our findings on honeybees as prime pollinators of spp.cucumis bitter gourd was visited by 13 species in which a. dorsata trigonaand sp. constituted 80.2%. nidagundi and sattagi (2005) recorded 10 insect visitors on bitter gourd in dharwad i.e. 8 hymenopterans and 2 lepidopterans. hymenopterans t. iridipennis, halictus gutturosus vachal and were important pollinators of a. florea bitter gourd (subhakar 2011). in bangalore, et al. 78.09% of pollinators on ridge gourd were bees, mainly and a. cerana, a. florea tetragonula iridipennis (kuberappa 2008). in our study, the wild bee et al. a. dorsata had the prime role as pollinator (70.1%). the rest of pollinators consisted of other bees, butterflies, flies, beetles and hawk moths. foraging dynamics insect forage is a collective process of individuals, as well as of the group (traniello 1989). crop-wise foraging times in our study (trichosanthes excluded) by different insects are presented in figures 5 to 8. a. dorsata and a. cerana, visited cucumbers from 06:30 to 16:30 hours, until the flowers wilted (fig. 5). a. dorsata peak visit of was during 10:30 hours (18.87 visits/m /5 minutes) and 11:30 hours 2 (20 visits/m /5 minutes). the visits were lesser 2 earlier and later (0.2, 0.4 and 1.2 visits/m / 2 5 minutes at 07:30, 08:30 and 16:30 hours, respectively). peak foraging by in a a. dorsata dharwad study was between 11:00 to 12:00 hours (pateel & sattagi 2007). conner-michigan (1969) reported that 10:00 to 15:00 hours as the most effective time for cucumber pollination in ohio, in which a flower required at least 8 to 10 visits by bees for satisfactory fruit set. the maximum foraging activity of in our study nearly a. cerana overlapped with that of . although the a. dorsata flowers bloomed at sunrise, the highest visitation frequency of was at 9:30 hours (8.8 a. cerana visits/m /5 minuntes) and 10:30 hours (9.73 2 visits/m /5 minutes). visitations declined from 2 11:30 hours (7.71 visits/m /5 minutes), reached 2 minimum at 15:30 hours (0.13 visit/m / 2 5 minutes). sp. was only a minor forager of trigona cucumber. other infrequent visitors like butterflies, moths and beetles foraged mainly figure 4 bar diagram of insect abundance on different cucurbit plants 22 biotropia vol. 24 no. 1, 2017 100% 75% 50% 25% 0% ridge gourd cucumber mangalore gourd bitter gourd snake gourd flies beetles moths butterflies wasps other bees a b u n d an ce figure 6 foraging patterns of diverse insect visitors on cucumis pubescens between 07:30 to 10:30 hours. insect foragers on mangalore gourd were observed from 06:30 to 17:30 hours (fig. 6). the highest foraging 2frequency of 13.6 visits/m /5 minutes was observed at 12:30 hours by apis dorsata and the least was observed at 07:30 hours. apis cerana had 2peak visitations at 13:30 hours (11.2 visits/m /5 minutes), the lowest was at 08:30 and 17:30 hours 2(0.21visits/m /5 minutes). trigona sp. foraged 2maximum at 11:30 hours with 2.1 visits/m /5 minutes and was not to be found from 15:30 hours. figure 5 foraging pattern of diverse insect visitors on cucumis sativus 23 pollinator diversity and foraging dynamics on monsoon crop of cucurbits – balachandran et al. 2 n o. o f in d iv id u al s / m / 5 m in u te s 2 n o. o f in d iv id u al s / m / 5 m in u te s apis cerana apis dorsata trigona sp other bees beetle moth flies straight lines = primary axis dot lines = secondary axis apis cerana apis dorsata trigona sp beetle butterfly moth straight lines = primary axis dot lines = secondary axis insects foraged on bitter gourd (momordica charantia) from 06:30 to 14:30 hours (fig. 7). the stingless bee trigona sp. as the major pollinator had peak visitations at 09:30 hours 2 (6.88 visits/m /5 minutes); lesser at 07:30 hours 2 (6.75 visits/m /5 minutes). by midday, trigona sp. shifted to other cucurbits, mainly to cucumis spp. visitations of a. dorsata nearly overlapped with that of trigona sp. in the 2 morning, peaking at 07:30 hours (7 visits/m /5 2 minutes) and 08:30 hours (6.88 visits/m /5 minutes). a. cerana succeeded reaching 2 maximum at 08:30 hours (3.14 visits/m /5 minutes). the butterflies were infrequent on bitter gourd, the highest at 09:30 hours (1.75 2 visits/m /5 minutes). as bitter gourd flowers bloomed very early in the morning, few moths could be found at 07:30 hours. study of subhakar et al. (2011) in tirupati also showed that trigona (iridipennis) was the most abundant and frequent 2 visitor on bitter gourd (10.83 visits/m /5 minutes). ridge gourd luffa acutangula is an important vegetable in coastal uttara kannada. foraging was observed from 17:30 to 22:30 hours (fig. 8). honeybees were major foragers. a. dorsata made 2 13.67 visits/m /5 minutes at 18:30 hours, while 2 a. cerana had 4.67 visits/m /5 minutes at 17:30 figure 7 foraging patterns of diverse insect visitors on m. charantia figure 8 foraging patterns of diverse insect visitors on luffa acutangula 24 biotropia vol. 24 no. 1, 2017 2 n o. o f in d iv id u al s / m / 5 m in u te s apis cerana apis dorsata trigona beetlebutterfly moth straight lines = primary axis dot lines = secondary axis 2 n o. o f in d iv id u al s / m / 5 m in u te s 6:30 7:30 8:30 9:30 11:3010:30 14:3012:30 13:30 apis cerana apis dorsata trigona sp beetle other bees moth straight lines = primary axis dot lines = secondary axis wasp hours. beetles, wasps and other bees were rare visitors. a. cerana, a. florea and tetragonula iridipennis were reported as main pollinators of luffa sp. in bangalore (kuberappa et al. 2008). shrivastava (1991) reported sphingid moth as an important pollinator of luffa sp., an evening bloomer. for snake gourd, a night bloomer with white flowers, moths were the main visitors during night, while butterflies frequented after daybreak. the bee-hawk moth had the highest visitations between 20:30 and 22:30 hours. the rainy nights of southwest monsoon hampered further observation in the midnight hours. elements of the landscape insect pollinators are known to rely on diverse elements of landscapes (habitats) for resources like nectar, pollen, nesting materials and speciesspecific nesting sites. landscape element analysis of the study area (with 3 km buffer) was done using remote-sensing data and field data (fig. 9). forests provide nesting and foraging sites for insect pollinators, ensuring their permanence in the landscape. high proportions of natural or semi-natural habitats close to agricultural area are known to benefit bee diversity, mutualistic interactions and insect foraging movements (hagen & kraemer 2010). in the 3 km radius around the vegetable growing area, there were forest, cashew trees (in scrub jungle), horticulture, laterite plateaus and vegetable farm, which provided food for the insects. only forest and cashew trees in scrub jungle were the main nesting areas for the insects. the presence of almost 100 ha forest patch, within the 3 km radius in the halkar village, was of high significance, especially in the vicinity of kumta, a municipal town. the forest has been under careful protection by the village community for generations and is acclaimed as one of the best examples of community managed forests in india (chandran 2001). the low laterite plateaus characteristic of uttara kannada plays as the key role in providing foraging resources for honeybees and lepidopterans (balachandran 2014). the et al. forests and scrub on laterite hills provided habitats for changes in landscape a. dorsata. structure are considered to be the primary cause of limitation in pollination services in agricultural systems (viana . 2012).et al in retrospect, plants can escape competition by utilizing different pollinator species or guilds, due to species-specific floral morphology and blooming times, favoring resource partitioning, conferring mutual benefits to both hosts and pollinators (pleasants 1980; schoonhoven et al. 2005). bees are prominent among cucurbit pollinators. the foraging schedules on any given species showed minimal overlapping between different foragers. took advantage of a. dorsata, the early morning blooming of bitter gourd, congregated on it from 06:30 to 09:30 hours, and shifted to cucumber and mangalore gourd as the day progresses, although it continued to forage in lesser frequencies until the flowers ceased by midday. after completing their visits on cucumis spp. during the rest of the day, extended a. dorsata their foraging activity into the night on ridge gourd, although in lesser frequencies, up to 22:30 hours. the knowledge on foraging activity and local climate is important for carrying out multispecies cropping of gourds and cucumbers to obtain maximum crop production. figure 9 land use elements in the landscape (with 3 km radius surrounding) 25 pollinator diversity and foraging dynamics on monsoon crop of cucurbits – balachandran et al. conclusions insect pollinators can enhance production without additional inputs other than organic manure. bees, mainly a. dorsata, a. cerana and trigona sp. were the major pollinators on all cucurbits, except snake gourd. traditional landscapes of coastal uttara kannada had all the necessary elements for sheltering and feeding of pollinator guilds which co-existed harmoniously with the cropping schedules of the local farmers. the rare survival of a small scale but highly productive, traditional mixed farming system in the densely populated central indian west coast, paying prominence to the conservation of natural habitats that support rich guilds of pollinators in the very vicinity of cultivation areas, calls for promotion of inclusive agricultural development taking forward the valuable knowledge systems of traditional cultivators. acknowledgements we are greatly indebted to the nrdms division, ministry of science and technology, government of india. we are grateful to the envis division, ministry of environment, forests and climate change, government of india as well as to indian institute of science for the financial and infrastructure supports. references abrol dp. 2012. pollination biology: biodiversity conservation and agricultural production. new york (us): springer dordrecht heidelberg. 792 p. balachandran c, chandran mds, ramachandra tv. 2014. keystone food resources for honeybees in south india west coast during monsoon. curr sci 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pollination services? j pollinat ecol:31-41. wolfe lm, barrett sch. 1988. temporal changes in the pollinator fauna of tristylous , an pontederia cordata aquatic plant. canadian j zool 66(6):1421-24. 27 pollinator diversity and foraging dynamics on monsoon crop of cucurbits – balachandran et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 biotropia vol. 28 no. 3,2021: 231 238 doi: 10.11598/btb.2021.28.3.1344 growth inhibition of fungal plant pathogens by antagonist bacteria using dual culture assays yan ramona* ztchool $biology, facalg of mathematics and sciences, uniuersitas udqana " integrated laborat09 for biosciences and biotechnology, uniuersitas udqana received 6 april 2020 /accepted 26 july 2020 abstract there has been excessive use of chemical-based pesticides globally resulting in significant environmental consequences with adverse effects on human health. therefore, more sustainable environment friendly alternative solutions are needed to counter such environmental and health challenges. development of biocontrol method is a sustainable solution to these challenges. the main objective of this research was to investigate the efficacy of potential antagonist bacteria to inhibit important fungal plant pathogens in uitro by applying dual culture assays on potato dextrose agar fda) or trypticase soya agar (tsa) media, with a view to isolate and screen potential antagonists for development of future biocontrol agents. the target pathogens were fasariam o y p o r m , ceratoystis sp., apergilasj7au.w and apergillz~s niger, commonly found to infect horticultural plants in bedugul village in north bali island. the antagonist bacteria were isolated from various sources such as soil of rhizosphere zone and roots of lettuce plants and mature compost. the potential of antagonist candidates was screened on the basis of inhibitory activity against targeted fungal pathogens. bacterial antagonists with highest zone of inhibition were identified up to genus level using biochemical tests, and the results were matched with those specified in the bergey's manual of determinative bacteriology. fifteen bacterial isolates were successfully isolated from various sources, and 60% of these isolates showed antagonistic activity in vitm against fungal pathogens with various degree of inhibition. this indicated the initial potential to develop as biocontrol agents. based on preliminary identification, genera of baciiias and psezldomonas were found to be the predominant isolates and in addition genus acinetobacter was also identified in this study. keywords: antagonistic, a.pergiilas $au.us, aspergiiias niger, ceratoystis sp., dual culture assay, fmrizlm 0 ~ ~ p 0 m / m introduction chemical-based pesticides have been used to control plant pathogens in farming practices for decades, and its application in agricultural sector into the future wdl continue in the short to the medium term (bueno e t al. 2017), despite the pollution challenges. these chemical-based solutions such as pesticides are used to protect agricultural crops from attack by fungi, insects, weeds, and rodents as well as lengthen the lifespan of farming products or prevent spoilage (i<umar e t al. 2012). application of chemical pesticides is also common in non-agricultural *corresponding author, email: yan-ramona@unud.ac.id sectors, such as sport facilities, shampoos for animals, and boats (nicolopoulou-stamati e t al. 2016). long term application of chemical based pesticides in agricultural and non-agricultural sectors has resulted environment and health impact in everyday life. the residue of such harmful compounds is now found in soil, waterways, agricultural crops, milk and meat causing human health challenges (kim et al. 2016). the presence of these chemical contaminations in soil also frequently kills beneficial microbiota of soil, such as mycorrhiza and plant growth promoting rhzobacteria beyond the target pathogens. furthermore, increase in resistance of plant pathogens may occur as a result of excessive application of such biotropia vol. 28 no. 3,2021 chemical compounds in farming practices (lucas e t al. 2015). this may also result in increased dose or innovations in new pesticides that are needed to control the same pathogens (fernandes et al. 2010). other studies have also reported negative impacts of pesticide exposure to the consumer and community (andersson 2014). in terms of health impacts pesticides studies indicate neurological effects, diabetes, respiratory diseases, fetal diseases, genetic ksorders and cancer in humans (andersson 2014; iom et al. 2017). methyl bromide is an example of a widely used broad spectrum pesticide which was phased out in developed countries without an alternative (unep 2014). in addition it is claimed that these chemicals contribute to greenhouse gas emission (heimpel et ai. 2013). although above serious challenges exist, pesticides use in farming practices will continue and totally phasing out of such compounds will take time until better sustainable non-toxic alternatives are found (bueno et al. 2017). biological based sustainable technologies are being intensively researched towards the development of alternative methods to control plant pathogens worldwide. integrated pest management (ipm), including the use of biological control agents to control pests and pathogens is one of the most favorable and sustainable solution paths globally. in bali, particularly in bedugul village, crop rotation in -farming practices has been-applied for years. horticultural crops planted in this village include tomato, lettuce, cabbage, potato, and broccoli. at the time of sample collection, ths farm was planted with lettuce plants, and therefore, the bacterial antagonists were isolated from soil in the rhizosphere zone and roots of lettuce plants from this area. in addition to these sources, we also isolated antagonists from vermi compost collected from a composting site in denpasar city where earth worms were used as starter in the process of composting. previous literatures for example in gehan e t a/. (2018) have reported that mature composts (including vermi compost) are rich in antagonists of fungal pathogens. based on the above rationale, the goal of ths study focused on isolation, screening, and identification of potential of bacterial biocontrol agents to inhibit plant pathogens, with a view toward the development of biocontrol agents, so that chemical-based fungicide use in the future can be reduced. the primary objective of ths study was to investigate the effectiveness of bacterial antagonist isolates to inhibit the in vitro growth of several fungal plant pathogens (fasam'am oqporam, cerato ystis sp., aspepiiias flavas, and aspegjills niger), commonly found to infect horticultural plants (including lettuce plants) in bali, particularly in bedugul area (suryanti et al. 2013) and neighboring villages of bedugul, including i<embang merta village (wulansari et al. 201 5). materials and methods fungal pathogens fungal pathogens (fasam'am oypomm, cerato ysth sp., a.pe@i/as yavzls, and ape@iias niger) were obtained from stock culture collections of the mycology laboratory, school of biology, universitas udayana, bali. these fungal isolates were identified in our previous study from samples collected in bedugul village (suryanti et ai. 2013). for regular use, these pathogens were subcultured on fresh pda. for long-term storage, they were grown for 48 hours at ambient temperature on potato dextrose agar, cut into 1 x 1 cm2, placed in sterile distilled water, and stored at 4 "c. sample collection and isolation of potential bacterial antagonists antagonists of plant pathogens were isolated from several sources, namely mature vermi compost (a composting site in denpasar city), soils sampled from rhizhosphere in lettuce farm in bedugul, bali, and rhizoplane (roots) of lettuce plants from bedugul, bali. isolation of bacterial antagonists from these sources was conducted by applying serial dilution and plating method on potato dextrose agar (pda) or trypticase soya agar (tsa). samples weighing 10 g were dissolved in saline solution (0.85% w/v nac1) and volume was adjusted to 100 ml to obtain dilution rate of lo-', and stomached for 20 minutes to release attached microbes on particles of soil or other samples. these samples were then further diluted in saline solution up to and 0.1 ml of sample from each dilution growth inhibition of fungal plant pathogens by bacterial antagonists yan ramona was then spread onto either potato dextrose agar (pda) or trypticase soya agar (tsa), followed by incubation at ambient temperature for 24 -72 hours. colonies that could be distinguished appeared on media (at dilution rates of 10" following 48 hours incubation at ambient temperature and were streaked for single colony isolation to obtain pure culture isolates. these pure cultures were then suspended in trypticase soya broth (tsb) or potato dextrose broth (pdb) with 30% glycerol in the medium, and stored at -80°c (as stock cultures) until needed for further experiments. in vitro dual culture assay for screening potential antagonists isolates of bacteria obtained previously were spot inoculated in triplicates onto the periphery of pda or tsa plates (corresponding to the medlum on which they were isolated). plugs (1 cm2) of fungal pathogens were then placed in the center of the plates followed by incubation at ambient temperature or approximately at 25 "c for 1 to 7 days, after which time the inhibition zone between the antagonists and pathogens were measured from three different angles using a vernier calipers, and then averaged. three replications were made in the experiment to obtain representative data. isolates showing inhibition zone were subcultured on fresh pda or tsa and stored at 4 oc for further study. for long term storage, these isolates were stored in tsb with 30% glycerol and stored at -80 "c. preliminary identification of biocontrol agent candidates bacterial isolates showing in vitro antagonistic activity against tested plant fungal pathogens were preliminarily identified up to genus level on the basis of their morphological characteristics and specific selected tests (for soil bacterial identification) and the results were matched with those indicated in bergey's manual of systematic bacteriology (krieg & holt 1984; holt e t al. 1994). the tested characteristics of bacterial antagonist candidates were: gram stain and cell morphology, oxidative/fermentative properties, spore stain, motility, flagella stain, oxidase, urease, and catalase production, ability to hydrolyze casein, uv fluorescence, levan production, gelatin hydrolysis, casein hydrolysis, and starch hydrolysis. the procedures (preliminary identification of soil bacteria up to genus level) of all tests followed the steps specified in ramona (2003), suryanti e t al. (2013) and wulansari e t al. (201 5). results and discussion a total of fifteen (15) distinguishable bacterial isolates were successfully isolated from this study. of these, 60% of the isolates showed antagonistic activity in vitro against one or more tested fungal pathogens with various degree of inhibition (fig. 1 and table 1). rhizosphere zone of lettuce plants appeared to be the best source of potential antagonists. most bacterial antagonists isolated in this study were obtained from such zone and they showed various degree of in vitro inhibition of plant pathogens (table 1). surprisingly, only 2 isolates from lettuce roots inhibited the plant pathogen in vho. bacterial isolates obtained from vermi-compost inhibited the in vitro growth of all fungal pathogens used in the study with various degree of inhibition (table 1). it can be seen in table 1 that isolates a4, a5, ti, and t2 which were identified as baciiizts or p~eztdomonas (table 2 and 3) appeared to have the widest spectrum of in vitro inhibition. these isolates inhibited the growth of all tested fungal pathogens with various degree of inhibition, depending on the species of pathogens. these results provided us with initial indication of antagonists with potential to be developed as biocontrol agents. t h s can now be targeted for further greenhouse or field trial scale studies for validation and effective application. the inhibition zone produced by these bacterial antagonists against the targeted fungal pathogens varied between 0.86 f 0.11 cm and 3.20 f 0.00 cm (table 1). isolate a1 whch was identified to belong to genus of pseudomonas only inhbited the growth of fzt~aiztm o.yqbormm, with inhibition zone of 1.83 f 0.15 cm (table 1 and 2). the o r i p of this isolate was the rhzosphere zone of lettuce plants. the limited number of isolates obtained in ths study (table 1, 2, and 3) was partly due to the primary focus on budding overall knowledge and basic tools related to in uitro cultivation of biotropia vol. 28 no. 3,2021 beneficial antagonistic microbes against fungal pathogens of horticultural crops and from this foundation then subsequently build a wider screening system for extensive isolation of biocontrol agents. previous reports indicated that an optimal optimized screening system is essential for in vitro cultivation from wider substrates (weller 1988) and this study advanced this objective. our results from this initial study were in line with malleswari (2014) and suthar e t al. (2016) who reported similar numbers of antagonist isolates from soil samples. in synthetic media, such as tsa or pda (considered as general media for bacteria and fungi, respectively) genera of bacillus, pmdomonas, and some genera belonging to enterobacteriaceae commonly grow well on such media and dominate during culturing. bacteria or fungi with specific or unidentified need will not show growth response on such media and has been reviewed previously by pham and kim (2012) and as suggested only 1% of soil bacteria may be culturable on synthetic media. therefore, there is a need for further improvement of methods as well as content and quality of synthetic media so that wider range of soil bacteria become culturable and therefore increase the probability to obtain potential isolates for biocontrol development. pham and igm (2016) proposed a novel method for bacterial cultivation by applying polycarbonate permeable membrane of trans well plates in liquid cultures with common synthetic media to mimic the actual environmental conditions. by applying such method they increased numbers of culturable isolates by more than 20%. such methods will be built on the foundations of this study in future strategies. the dual culture assays applied in our study (following isolation of antagonist candidates) appeared to be very convenient to initially indicate antagonism of bacterial antagonists against plant fungal pathogens. these results will have to be evaluated and correlated for their effectiveness in further greenhouse or field scales experiments. overall, this is a classical method and has been used effectively to isolate and initially screen bacterial antagonists of fungal plant pathogens in vitro. other studies recently applied this method to obtain bacterial antagonist candidates and subsequently identified the same to target fungal pathogens (suthar e t al. 2016; wang e t al 2019); and manandhar e t al. 2019). this indicates that dual culture method is still a reliable approach to initially screen and identify antagonistic activity of a bacterial isolate against fungal or bacterial pathogens. digwe 1 some examples of ia .uitw antaganism between bacterial isolates obtained in this study and the tested pathogens. (a) ps8~hmow sp. vs c ~ ~ k o p i f sp., @) ps81domona sp. vs aspergdihs niggt", (c)~ b~ilzfs sp. vs rfpqdii~@aas and (d). b(dn'&s sp. vs ffian'itrn o x ~ p m r n note: arrowheads show the inhibition zones between the bacterial antagonists and tested fungal pathogens. growth inhibition of fungal plant pathogens by bacterial antagonists yan ramona table 1 average diameter of in vitro inhibition zones (on pda or tsa media) of bacterial antagonist candidates on several plant pathogens (fmvium oq~orzlm, ceratoystis sp., a.pergilusj7auas, and a.pegiiias niger) diameter of inhibition zones on fungal pathogens (cm)* isolate codes source of antagonists f. oq.porzlm cerato ystis sp. a. j7auu.s a. niger a1 (pda) lettuce rhizosphere 1.83 f 0.15 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 a4 (pda) lettuce rhizosphere 1.06 f 0,06 2.20 f 0.10 1.50 f 0.10 1.60 f 0.11 a5 (pda) lettuce rhizosphere 3.20 f 0.00 2.46 f 0.1 1 1.33 f 0.11 1.30 f 0.10 a6 (pda) lettuce rhizosphere 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 ti (pda) vermi-compost 0.96 f 0.06 1.80 f 0.06 1.13 f 0.11 1.13 f 0.11 t2 (pda) vermi-compost 0.86 f 0.11 1.83 f 0.06 1.26 f 0.06 0.90 + 0.10 s1 (tsa) lettuce rhizosphere 1.63 f 0.06 0.00 f 0.00 3.36 f 0.30 0.00 f 0.00 s2 (tsa) lettuce rhizosphere 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 s3 (tsa) lettuce rhizosphere 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 1.13 f 0.15 f1 (tsa) roots of lettuce 1.50 f 010 0.00 f 0.00 2.70 f 0.10 0.00 f 0.00 f2 (tsa) roots of lettuce 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 f3 (tsa) roots of lettuce 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 r1 (tsa) roots of lettuce 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 r2 (tsa) roots of lettuce 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 0.00 f 0.00 r3 (tsa) roots of lettuce 0.00 f 0.00 0.00 f 0.00 1.60 f 0.17 0.00 f 0.00 notes: * values in table 1 f standard deviation are averages of triplicates; pda (potato dextrose agar) and tsa (trypticase soy agar) were media used in the dual culture assays. the relative average inhibition zones shown in table 1 varied from 0.0 to 3.36 cm, and these apparently were affected by types and the origin of the isolates. the zone of inhibition was also determined by species of fungal pathogens tested. generally, bacterial isolates obtained from rhizosphere of lettuce plants was found to be antagonistic against one or more fungal pathogens tested in this study (table 1). similar results were also reported by ramona (2003) and wang et al. (2019) who found abundant antagonist isolates in the rhizosphere zones of various plants. this indicates that rhizosphere area can still be considered as a reliable source of bacterial antagonists of plant pathogens for development of biocontrol agents. in addition to rhizosphere zone, antagonists can also be isolated from many sources, such as mature compost or surface of plant roots (rhizoplane) (table 1). some researchers have also often isolated these antagonists from suppressive soil (schlatter e t al. 2017), suppressive compost (gehan eta/. 2018), or from contaminated media in the laboratory (ramona 2003). the origin of the antagonist isolates is not the most important aspect in the development of biocontrol agents. the effectiveness of these antagonists in protecting horticultural plants from attack by certain pathogens either in greenhouse or field applications is apparently more important consideration than their origins. to increase the effectiveness of antagonist isolations, ideally these antagonists should be isolated from places where these agents are to be applied, as they are already well-adapted in such areas of plant systems (tvtupps 2001; ganeshan & i<umar 2005; dukare e t al. 201 8). spectrum of fungal pathogen inhibition in vitro of the bacterial isolates obtained from this study varied among species (table 1). some isolates were able to control only one tested fungal pathogen, while the other controlled two or more pathogens (table 1). generally, bacterial antagonists isolated from verrni compost and rhizosphere zone of lettuce plants appeared to have wider spectrum of fungal pathogen inhibition in vitro when compared to those isolated from other sources. surprisingly, isolates obtained from rhizoplane (hair-root surfaces) of lettuce plants were found to be less effective in inhibiting the four fungal pathogens in vitro than isolates obtained from other sources (table 1). this phenomenon was contradictory to that reported by wei et a/. (1996) and walia et al. (2013), but in line with that reported by oka (2004). although some isolates in table 1 showed a wide spectrum of fungal pathogen inhibition, these isolates will have to be further biotropia vol. 28 no. 3,2021 tested to study their effectiveness when applied in glasshouse or field trials. ramona (2003) reported that a positive correlation between the results of in vitro bioassay and those of glasshouse or field trials may not be necessary. the reasons for these were extensively reviewed by whipps (2001) and o'brien (2016) and wider testing is essential following the screening for practical application. although observable inhibition zones were clearly indicated in the in vitro bioassay fable 1 and fig. i), the mechanisms by which bacterial antagonists may inhibit the fungal pathogens (for examples: antibiosis, siderophore production, or other mechanisms) was still inconclusive. besides that, the effect of this antagonism (whether it was lethal or fungi static) has not yet been answered and w d be considered in future studies. bacterial isolates with abllity to inhibit the growth of at least one tested fungal pathogen were initially identified up to genus level, based on the method of soil bacterial identification specified in ramona (2003) and suryanti e t al. (2013). the results are shown in table 2 and table 3. based on these results, the genus name of the isolates was determined. one of the pseadomonas isolates showed fluorescence properties under uv light, in addition to the results of other tests generally used in the initial preliminary identification of soil bacteria, and therefore, it was identified as p. flzlorescence (table 2). among the 9 bacterial antagonists, 5 isolates belong to baciiias sp. as all of them produce endospore and showed characteristics of genus baciiias as shown in table 3. p r e h n a r y identification of the isolates obtained from this study showed that genera of baciiias and pseadomonas were predominant bacterial isolates contained in all samples (table 2). this is in line with that reported by icumari and iulanna (2016) and soare e t al. (2019). the presence of these two bacterial genera in the soil or other samples is relatively more abundant than others and is partly due to simpler requirements for growth of these groups when compared to others (raaijmakers e t al. 2010). the ability of baciiius sp. to produce endospore also contributes to the survival of this genus in adverse environmental conditions (icumari & iulanna 201 6). besides ban'iias and pseadomonas, genus of acinetobacter was also isolated in this study (table 2). table 2 preliminary identification of gram-negative bacterial antagonists isolate gram flagella uv starch levan gelatin preliminary o/f motility oxidase pigment codes stain position fluorescence hydrolysis production hydrolysis identity a1 negative 0 + polar + + pseudomonas sp. a4 negative 0 + polar + + + p.~uorescence ti negative 0 + polar + + pseudomonas sp. s1 negative 0 nd + creamy acinetobacter sp. note: o/f: oxidative/fermentative. table 3 preliminary identification of gram-positive bacterial antagonists isolate gram flagella starch casein preliminary o/f motility catalase pigment spore urease colony codes stain position hydrolysis hydrolysis identity a5 positive 0 + polar + + + + + dry baciiius sp. t2 positive 0 + polar + + + + + dry baciiius sp. s3 positive 0 + polar + + + + dry ban'iius sp. f1 positive 0 + + + + dry baciiius sp. r3 positive 0 + polar + + + + + dry baciiius sp. note: o/f: oxidative/fermentative. growth inhibition of fungal plant pathogens by bacterial antagonists yan ramona conclusion nine out of 15 (60 yo) bacterial isolates showed antagonistic activities against several tested fungal pathogens in the in vitro dual culture assay, although the mechanisms by which these biocontrol candidates inhibit the fungal pathogen is still inconclusive, and therefore further study is needed to specifically elucidate the mechanisms (such as antibiosis, production of siderophore, etc.) of biocontrol. in the initial and preliminary identification of these antagonists, it was found that genera of baciiius and p~eudomonas were predominant isolates found in all samples. besides these, genus of acinetobacter was also isolated and identified in this study. acknowledgements the author wishes to acknowledge laboratory of mycology, school of biology, universitas udayana for the provision of cultures of fungal pathogens. the laboratory of illicrobiology, school of biology, universitas udayana, and the integrated laboratory for biosciences and biotechnology, universitas udayana should also be acknowledged for the provision of facilities and equipment during this project. special gratitudes also go to prof i<alidas shetty (ndsu-usa) for his proofreading and criticism on this manuscript. references andersson h, tago d, treich n. 2014. pesticides and health: a review of evidence on health effects, valuation of risks, and benefit cost analysis. advances in health economics and health services research. p.1-61. bueno adf, carvalho ga, dos santos ac, 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special issue):487-511. wulansari n t, ramona y, proborini mw. 2015. identification of xantbomonas campestns isolated from rhizosphere zone of broccoli farm at icembang merta village, tabanan-bali. jurnal metamorfosa vol ii(1):29-33. 2. churiyah.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 01. 1379 okky setyawati dharmaputra et al..cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 sri-09. 1152-layout.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 840 dewi yuniati (growth performance) revisi.cdr growth performance and enzyme activit in ies catfish fed water [pangasianodon hypophthalmus] with hyacinth-based diet * dewi yuniati , nur bambang priyo utomo, mia setiawati and alimuddin department of aquaculture, faculty of fisheries and marine science, ,institut pertanian bogor bogor 16680, indonesia received 26 april 2017 / accepted 14 august 2017 abstract an alternative subtitution of pollard as an imported feed ingredient is a neccesity and one of the potential ingredients is water hyacinth (eichhornia crassipes). this study was conducted to evaluate growth performance and enzyme activity in catfish (pangasianodon hypophthalmus). diet with five different levels of water hyacinth subtitute of pollard (0%, 25%, 50%, 75%, and 100%) were fed to catfish for 60 days. seventy (70) catfish fry with average initial body weight of 2.45±0.15 g were maintained in 100cm x 80cm x 60cm aquaria. fish fed at satiation level three times daily at 8 am, 12 pm, and 4 pm. with dietary of 25% water hyacinth, growth performance and protease activity similar to 0% treatment. feed intake, protein digestibility, feed efficiency, protein efficiency ratio, and protease and amylase enzyme activities decreased (p<0.05) in those fed with more than 25% water hyacinth. catfish fed 25% water hyacinth showed significantly (p<0.05) higher daily growth rate, feed efficiency and protein digestibility than those with other treatments. based on the growth performance and enzyme activity, we can conclude that the optimum dietary level of water hyacinth subtitute pollard for fry catfish is 25%. keywords: catfish, growth performance, pollard, water hyacinth introduction feed is the main component to meet the energy needs of the fish. has a high pollard digestibility and bioavailability as carbohydrate source in fish feed (suprayudi et al. 2010). munir et al. 2015) ollard contain ( report that p s 53.40% nfe, lipid14.78% protein, 7.77% , 9.78% crude fibre, and 4.34% ash owever , h an alternative subtitution of pollard as an imported feed ingredient is a neccesity. research on the use of ingredients of fish feed has been done ulva ; lactuca havea brasiliensis lemna, singgle cell protein , , ( et al.mahasu 2016; inara 2011; bag 2011; utomo et al ). the research among others, focused . 2007 on the efforts of using local ingredients and creating efficiency of feed cost. one of the potential ingredients to substitute pollard in fish feed is water hyacinth (eichhornia crassipes). water hyacinth has lower cost than pollard, water hyacinth cost rp 2000/kg while pollard cost . . s with base on therp 4800/kg and change time dollar rate. water hyacinth is a south american native plant that has spread to more than 50 countries, including indonesia. water hyacinth is one of the aquatic plants that grow in abundance in rivers, rice fields, and lakes or dams. its characteristics include rapid growth and the ability to compete with other aquatic plants (shu et al. 2014). water hyacinth can cover the surface of water bodies, thereby reducing the amount of sunlight and air penetrating into the water. in addition, the plant can cause shallowing and increase evaporation due to evapotranspiration. in a dense population, water hyacinth can impede water flow and block irrigation channels (sotolu & sule 2011). therefore, the presence of water hyacinth in water bodies is more often regarded as harmful weeds that can negatively impact the aquatic ecosystem (tellez et al. 2008).* corresponding author: dewiyun25@gmail.com biotropia 5 2 8 140 147 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.2.840 140 in previous study, water hyacinth has been used as fish feed in tilapia (khalil et al. 2015; bag et al. 2011; hontiveros et al. 2015; muchtaromah et al. 2012), cyprinus carpio (mohapatra 2015), clarias gariepinus (sotolu & sule 2011), and labeo rohita (saha & ray 2011). the use of water hyacinth flour as a substitute of fish meal for clarias gariepinus feed for 26.72% and 31.63% showed a lower digestibility rate (sotolu & sule 2011). however, according to sotolu and sule (2011), it is still possible to use the plant for fish feed. hence, water hyacinth was chosen to be the raw material of fish feed in this research. furthermore, chemical analysis of water hyacinth shows that the plant contains nutrients similar to that of pollard, 30.81% nfe, 12.40% protein, 4.72% lipid with digestibility of water hyacinth leaves reaching up to76.4% (chang et al. 2013; hontiveros & serrano 2015). the utilization of nutrients in fish feed is closely linked to enzyme activity in fish. the use of different feeds in fish can affect their enzyme activities. marzuqi (2015) revealed that milkfish given different contents of carbohydrates experience different amylase enzyme activities in line with the increasing contents of carbohydrates in the feed. according to tibin et al. (2012) the use of water hyacinth indicated a decrease in feed utilization in tilapia fish. there is still gap in knowledge about the use of water hyacinth as a source of plant material in catfish feed. thus, the present research was carried out to evaluate the use of water hyacinth flour in fish feed as a source of carbohydrate in the growth performance and enzyme activity of catfish pangasianodon hypophthalmus, an important farmed fish in southeast asia. materials and methods water hyacinth flour the water hyacinth flour made from leaves and stems used in this research was taken from the sukabumi centre for freshwater aquaculture. the water hyacinth was first cleaned and then 0dried in oven at 60 c for 24 hours. the dried water hyacinth was then grinded into flour. before being used as the ingredient for the feed, a test of heavy metal content was conducted to determine the presence of lead (pb), cadmium (cd) and mercury (hg). the heavy metal content analysis was done using the atomic absorption spectrophotometry method (apha 2012). the test results showed that no heavy metal contents of lead, cadmium, and mercury were detected in the water hyacinth flour. experimental diets diet containing the five different levels of water hyacinth (0%, 25%, 50%, 75% 100%) and as pollard substitute. the premix used were commercial product called lagantor from kalbe. 1note: nitrogen free extract 2gross energy, 1 g protein = 5.6 kcal, 1 g lipid = 9.4 kcal, 1 g nfe = 4.1 kcal (takeuchi 1988). -1 table 1 composition and nutrients contained of the experimental diets (% kg dry matter) 141 water hyacinth-based diet for pangasianodon hypophthalmus – yuniati et al. webster and lim (2002); protein and lipid retention were calculated based on takeuchi (1988). after the final weighing, three pieces of fish were randomly taken from each aquaria for a proximate analysis. water content analysis was done by drying the sample in an oven at the temperature of 105 110 c for 6 hours; analysis 0 – of crude fibre was carried out with dissolving method using strong acids and bases and heating; protein analysis with kjeldahl method, lipid analysis of dried samples with soxhlet method, lipid analysis of wet samples with folch method, and ash content analysis by heating sample in a furnace at 600 c (takeuchi 1988). 0 enzyme activity analysis the enzyme activities analysed included those of amylase, protease, and cellulase. the enzyme activities were observed at the end of the experiment. amylase enzyme activity was measured by the method of worthington (1993). cellulase enzyme activity was measured by the method of kader and omar (1998). protease enzyme activity was measured using bradford's method (bradford 1976). digestibility test the effect of water hyacinth used in feed digestibility was measured using the indirect method by adding chromium oxide cr o as an 2 3 indicator in the feed. digestibility test was conducted after growth performance tests using the same fish. the fish were fed three times a day at satiation at 8 am, 12pm, and 4 pm. faecal wastes were first to be collected on the fourth day after giving the feed containing cr o . the faeces were 2 3 collected 30 minutes after feeding. the collected faeces from each aquaria were stored in a freezer. the collection of the faeces was done for 25 days. once collected, the faeces were dried in the oven 0 at 110 c for 4-6 hours. the faeces sample was analysed for its cr o and protein contents. the 2 3 total digestibility value and protein digestibility were calculated using formula of takeuchi (1988). the ingredients used were weighed according to their composition and mixed . in a mixing machine after getting evenly mixed, the feed was moulded and heated in the oven at 60 c for 4 hours. the 0 composition of the feed and the nutrients contained in it are presented in table 1. fish rearing the catfish pangasianodon hypopthalmus used originated from cibanteng, bogor, indonesia. the fish were maintained in the aquatic laboratory of the southeast asian regional centre for tropical biology (seameo biotrop). they were reared in 100cm x 80cm x 60cm aquaria. the aquaria were cleaned and disinfected using 30 ppm chlorine before use. each of the aquaria was filled with 200l of water. fish acclimatization was done in two weeks before the rearing period. during the acclimatization, the fish were fed with 50% treatment feed at satiation level. after acclimatization, the fish were weighed, and the average weight was 2.45±0.15 g. the fish were then placed randomly into the 15 aquaria with a stocking density of 70 fishes/aquaria. the rearing of the fish was done for 60 days. feeding was done at satiation for three times daily at 8 am, 12 pm, and 4 pm. the growth was measured by sampling every fifteen days. every three days 70% of the water was replaced to maintain the quality of the water during the rearing. each aquaria was equipped with aeration 0 and heater regulated at 28 c. the temperature of the water was measured daily, while the water quality was measured three times during the farming process, with parameters including dissolved oxygen, ph, and ammonia. the water quality during farming was at the optimum range for catfish, with a temperature ranging from 0 -1 28–29 c, dissolved oxygen of 5.6–6.3 mg l , ph -1 7.6–7.8 and ammonia 0.0005–0.02 mg l . at the end of the rearing period, the fish biomass in each of the aquaria was measured. the parameters of survival rate, the amount of feed intake, feed efficiency, and daily growth rate were calculated based on halver and hardy (2002); protein efficiency ratio was calculated based on 142 biotropia vol. 25 no. 2, 2018 statistical analysis the design employed in this research was the completely randomized design with five treatments and three replications. data were analysed using spss software ver. 22.0. to find the effects of treatments, data were tested with anova and followed by duncan test with a 95% confidence level. the difference between treatments was found with a significance value of p<0.05. results and discussion growth performance the results showed that the use of water hyacinth of more than 25% in diet caused a decrease in feed intake, daily growth rate, protein digestibility, protein efficiency ratio, and daily growth rate. lipid retention and protein retention in the 75% and 100% treatments were lower compared to those in other treatments, where in the 100% treatment, the retention rates were 7.85% and 19.87%, respectively (table 2). the use of water hyacinth in fish feed at certain levels gave different results in the feed intake (fi) of the catfish. in this research, the use of more than 25% water hyacinth in feed caused a decreased in fi. the decreases of fi in the 50%, 75% and 100% treatments compared to 0% treatment were 11.83%, 38.89%, and 45.88% respectively. in tilapia, the use of water hyacinth in feed did not affect feed consumption, indicating that tilapia could accept feed containing water hyacinth better than pangasianodon hypopthalmus (hontiveros et al. 2015). the difference in the feed intake levels in each treatment was assumed to be caused by a difference in the feed palatability. palatability is a response to certain feed, which is influenced, among others, by feed characteristics. in addition, the acceptance of fish to raw material is different for each species (setiawati et al. 2014). venero et al. (2008) mentioned that the use of plant materials can reduce feed acceptability and feed palatability. palatability is linked to attractability that will influence responses in feed searching, intake, and ingestion or acceptability (inara 2011). the decreased rate of fi was followed by a decreased in digestibility rate. digestibility is the process of breaking down feed into a simpler form that is readily absorbed by intestinal walls and into the blood vessel system. there was no difference in the total digestibility of feed in all treatments (p>0.05). this is in contrast to protein digestibility that experienced a decrease in line with the increasing amount of water hyacinth in feed (p<0.05). diet contain 0% and 25% water ing hyacinth showed similiar result 82%, whilst in of tabel 2 final biomass (bt), feed intake (fi), total digestibility (td), protein digestibility (pd), protein retention (pr), lipid retention (lr), protein efficiency ratio (per), daily growth rate (dgr), feed efficiency (fe), survival rate (sr) of catfish (pangasianodon hypophthalmus) fed with feed containing water hyacinth note: *) the values in the same rows with different superscript letters indicate significant differences (p<0.05). 143 td water hyacinth-based diet for pangasianodon hypophthalmus – yuniati et al. -1dgr (%day ) the 50%, 75%, and 100% treatments, it decreased to 79.92%, 74.57%, and 75.96% respectively. the factors affecting digestibility are, among others, the treatments before and after feed making, material sources, particle size, fish size, and nonprotein components in the feed (usman 2002). the decrease protein digestibility value 50-in of 100% treatment in this study were caused by the increasing amount of crude fibre that can disturb feed utilization and growth. mohapatra (2015) reports that the use of water hyacinth significantly increase the crude fibre and affect the feed s s utilization of cyprinus. in the study the current , crude fibre content of 0–100% treatments were at the range of 3.47–13.06%. the higher the fibre content in the feed, the lower digestibility of protein. this result is the in accordance with report of suprayudi et al. (2010). the end products of the consumed and digested feed are nutrients that can be used and then stored in the fish body. the levels of protein and lipid retentions in the 0–50% treatments were the same, and they declined in the 75% and 100% treatments. the different protein and lipid retention levels were caused by differences in the amount of feed intake and feed digestibility value. carbohydrate, protein, and lipid are the three components that contribute the highest amount of energy for the fish. if low amounts of nutrients are absorbed, their potential to be stored in the fish will also be low, because the nutrients will be first used for the fish activities. this also applies to growth: if a small amount of energy is received by the fish, then the energy for its growth will be even much less. the use of certain levels of water hyacinth in feed gave positive effect on the growth of (mohapatra 2015). in this research, the cyprinus use of more than 25% water hyacinth negatively impacted the growth of the catfish. compared to 0% treatment the percentages of daily growth in , the 50%, 75% and 100% treatments decreased. the decreased of daily growth in the 50%, 75%, and 100% treatments were 27.1% day , 55.6% day-1 1 -1 , and 69.9% day , respectively. the lowering growth rate in the fish can be attributed to the unfulfilled needs of nutrients in the fish and the inability of the fish to use the energy and materials in their feed (usman 2002). feed intake in the 0% and 25% treatments were better than that in the other treatments, hence the amount of the ingested feed was greater in the 0% and 25% treatments. the difference presumably caused more energy to be received in 0% and 25% treatments, resulting in better growth. in this research, the crude fibre content in the feed increased along with the increasing proportion of water hyacinth flour in the feed. the crude fibre derived from vegetable materials is in general difficult to be digested by fish. in addition, one of the components of cell walls in plants is cellulose that is intractable (inara 2011). the better growth in the 0% and 25% treatments reflects feed efficiency value. this value indicates that the antinutritional substances and the high fibre content in the 0% and 25% treatments did not disturb the use of feed by the catfish. the survival rate ranged from 97.14–98.57%, indicating that water hyacinth did have any harmful effects on the fish. enzymes ctivitiesa similar protease enzyme activities were found in the 0% and 25% treatments, thus have different result compared to the 50%-100% treatments (fig. 1). similar cellulase enzyme activities were found in all treatments (fig. 2). different amylase enzyme activities were observed in all treatment (fig. 3). enzymes have an important role in digestion process. the protease enzyme activities in the 0% and 25% treatments were greater than those in the 50–100% treatments. the declining protease enzyme activities in the 50–100% treatments were assumed to be caused by the anti-nutritional substances in the feed. water hyacinth contains anti-nutritional substances such as tannin and phytic acid of 0.98% and 0.42%, respectively. anti-nutritional substances such as tannin can affect growth and enzyme profiles of fish. tannin impedes protease enzyme activity and therefore can reduce protein digestibility, while phytic acid can reduce protein and mineral bioavailability (saha & ray 2011). in addition, the declining enzyme activities can also be caused by the amount of the substrates present feed in the intake. in this study, fish fed diet containing 50–100% water hyacinth showed lower feed intake than those fed 0% and 25% water with hyacinth, which was directly proportional to the amount of substrates for the enzyme in their digestive systems. the higher feed intake and protein digestibility in the 0% and 25% treatments compared to those in other treatments had an impact on the high value of protein 144 biotropia vol. 25 no. 2, 2018 figure 1 protease enzyme activities in catfish fed with feed containing different levels of water hyacinth figure 2 cellulase enzyme activities in catfish fed with feed containing different levels of water hyacinth figure 3 amylase enzyme activities in catfish fed with feed containing different levels of water hyacinth 145 water hyacinth-based diet for pangasianodon hypophthalmus – yuniati et al. efficiency ratio in these treatments, namely 2.30 and 2.37, respectively. the cellulase enzyme activities observed were not different in all treatments. this is attributed to the low amount of cellulase enzyme in the fish that worked maximally in all treatments, although there were differences in the amounts of feed intake and water hyacinth in each feed. the amylase enzyme activities were found to be different in all treatments. the amylase enzyme activities in the 50–100% treatments were lower than those in other treatments. amylase enzymes break down starch in feed. the amylase enzyme activites decreased with increasing level of water hyacinth assumed to be caused by the low carbohydrate content as the level of water hyacinth increased. conclusion the pangasianodon hypopthalmus fed diet containing 25% water hyacinth subtitute pollard showed similar growth performance and protease activity with 0% treatment. the use of water hyacinth of more than 25% showed lower growth performance than other treatments. water hyacinth flour can be used for up to 25% or 6.25% in the diet of pangasianodon hypopthalmus. acknowledgements we would like to express our gratitude to the southeast asian regional centre for tropical biology (seameo biotrop) for the opportunity to conduct our research in its aquatic laboratory, and our gratitude also goes to the centre for freshwater aquaculture, sukabumi, for providing water hyacinth flour for the study. references american public health association (apha). 2012. standard method for examination of water and wastewater, 22th edition. 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[single cell protein as substitution for fish meal in juvenile common carp (cyprinus carpio) diets]. jurnal perikanan 9(2):188-93. venero ja, davis da, lin c. 2008. use of plant protein sources in crustacean diets. new york (us): the howort press. p. 163–203. webster cd, lim ce. 2002. nutrient requirements and feeding of finfish for aquaculture. new york (us): cabi publishing. p. 4-5. worthington v. 1993. worthington enzyme manual. new jersey (us): worthington biochemical corporation. 147 water hyacinth-based diet for pangasianodon hypophthalmus – yuniati et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 317 (andes modeling).cdr modeling of tree growth after forest fire in mount ciremai national park, indonesia 1* 2 andes hamuraby rozak and tedi setiadi 1 cibodas botanic gardens, indonesian institute of sciences, cianjur 43253, indonesia 2bird conservation society, bandung 40184, indonesia received 12 july 2013/accepted: 14 october 2016 abstract forest fire is a massive threat towards tropical forest causing various negative impacts to nature and human being. forest fire often leads to alteration of forest structure and its functions. this study of tree growth after forest fire was conducted using a model simulation. the model was performed at the individual level of plant community and built to analyze the potential of tree growth and its scenario for post-fire recovery. five important tree species from montane forest of mount ciremai were chosen to build the model based on four main parameters i.e. plant growth rate, diameter at breast height (dbh), tree-to-grass competition and tree-to-tree competition. the scenario of postfire recovery was performed by replanting similar species with 5 cm dbh seedling. prediction from our model showed that most of the chosen species would recover to its pre-fire condition after 37 50 years. considering the limitation of competition after re-planting, it was suggested to minimize tree to tree competition and applied silvicultural treatments to maximize tree growth and tree community recovery. keywords: forest fire, mount ciremai national park, tree growth model, tropical montane forest introduction nowadays, forest fire is a serious threat towards tropical land including tropical forest, plantation and other forestry land. fire used to be extremely rare in tropical forests, leaving ample time for forests to regenerate to pre-fire conditions. moreover, undisturbed old-grown forests were difficult to burn due to their air humidity and high moisture content in soil and litter, even after prolonged droughts (slik et al. 2010; knox & clarke 2012). however, nowadays, tropical forest fires occur more frequently and at larger spatial scales than in the previous decades (van nieustadt & sheil 2005; laurance 2007; slik et al. 2008). in recent years, forest fire occurs every year in dry season and had become the characteristic of disturbed tropical forest ecosystem (stolle et al. 2003). several reasons can be identified as main causes of catastrophic fire i.e. population growth, changes in land-use practices and short-term climatic oscillations commonly known as el nino (kinnaird & o'brien 1998; le page et al. 2008). forest fire causes several negative impacts to nature and human being. it affects public health as the net forest emissions may have released carbon which equivalent to 41% of worldwide fossil fuel used in 19971998 (cochrane 2003). the fire itself led to local extinctions of some plants and animals. furthermore, a country may suffer economic losses in forestry, non-timber forest products and agriculture. fire is able to deteriorate forest structure, tree species diversity, tree species composition, aboveground biomass and forest soil (flannigan et al. 2000; mcintosh et al. 2005; knox & clarke 2012). several studies related to forest fire and its recovery had been conducted in tropical forest of indonesia. slik et al. (2002) recorded that tree density (diameter >10 cm) and canopy closure in borneo were able to recover within 10 to 15 years. other studies had also been conducted focusing on recovery of the habitat and species diversity after forest fire in borneo (slik 2004; watanabe et al. 2009; slik et al. 2010). however, tree ingrowths during this initial phase of regeneration consist mostly of early successional, fast-growing tree biotropia 3 2 6 65 73 vol. 2 no. , 201 : doi: 10.11598/btb.2016.2 . .3 2 317 * corresponding author: andes.hamuraby.rozak@lipi.go.id 65 species with low wood densities. successful recovery of forest aboveground biomass, therefore, depends strongly on the regeneration of the pre-fire species composition (slik et al. 2008). in addition, tree community recovery after forest fire was also determined by several conditions i.e. growth rate, resprouting rate, competition and survival rate. study on post-fire recovery using ecological modeling as a prediction tool has never been applied in indonesian forest, specifically in tropical montane forest. in this study, we have modeled the recovery of five dominant trees in mount ciremai national park (mcnp) after forest fire. the model was developed using the growth model of menaut et al. (1990). however, tree biomass calculation was modified by replacing the estimation of volume from diameter and height function (botkin's equation; botkin et al. 1972) to allometric equation of biomass calculation (basuki et al. 2009). the biomass equation was applied due to the importance of biomass re-growth rate in post-fire forest recovery (minchella et al. 2009). the potential growth of the selected trees was predicted for fifty-year projection. the selection of tree species was based on plant community data in mcnp by rozak (2007). recent publication showed that mcnp experienced at least four forest fire events in 2011, especially in long dry season (syachera 2011). therefore, this study was aimed to: predict the potential growth of selected trees before forest fire event (normal growth) and predict the best scenario for replantation based on tree to tree competition. the results of the study were expected to provide information related to post-fire forest recovery which is important for mcnp management. materials and methods study site and tree community data mount ciremai national park (mcnp) is located in west java province ca. 270 km eastward from jakarta, the capital of indonesia. geographically, the mcnp is situated at 108 20' – º 108 40' e and 6 40' – 6 58' s. the highest º º º elevation reaches 3,078 m asl. making this mountain to be the highest mountain in west java. topographically, around 26.52% of the area has 0 8 slope. the rest of the area has slope more º º than 8 or equal to 73.48% of the area (rozak & º gunawan 2015). the annual mean of precipitation is 2,000 mm and the annual temperature is 22 c (kuningan government º 2011). the tree community data were collected in 2007 from cigugur-darma resort located at the eastern part of the mcnp. tree community data were collected from twenty quadrat plots (10 x 10 m) which were placed in parallel to mountain topography. within each plot, all live trees taller than 1.3 m were identified and dbh greater than 10 cm were measured. five tree species with the highest important value index (ivi) were chosen for model development and the name of those tree species were standardized through the plant list website (www.theplantlist.org). the important value index were generated from three parameters i.e. dominance index, abundance index and frequency index. we selected the dbh from engelhardtia spicata blume, ficus ribes reinw. ex blume, trema orientalis (l) blume, saurauria pendula blume and lithocarpus pallidus (blume) rehder in the study site, as the reference size of initial dbh before forest fire event (table 1). 66 species ivi dbh h dmax hmax engelhardtia spicata lechen ex blume 32,482 35 22 60 25 ficus ribes reinw. ex blume 28,469 15 8 30 15 trema orientalis (l.) blume 21,582 34 16 60 25 saurauia pendula blume 19,891 17 7 30 17 lithocarpus pallidus (blume) rehder 18,041 12 9 35 21 table 1 the important value index (ivi) of five dominated tree species in mount ciremai national park (mcnp) biotropia vol. 23 no. 2, 2016 notes: ivi = important value index; dbh = diameter at breast height (cm); h = actual height of tree (m) measured by hagameter; d = maximum diameter (cm); h = maximum height (m). value of d and h were derived from max max max max hanum and van der maesen (1997) as well as van der vossen and wessel (2000) parameters d and h were collected from field data. parameters d and h were according to max max hanum and van der maesen (1997) as well as van der vossen and wessel (2000). the results obtained based on equation 2 were negative and therefore, unrealistic. subsequently, the original equation from botkin et al. (1972) was applied as in equation 3. combining equations (1) and (3) to become equation (4): the following equation was developed using equation (1) and (3) to combine normal growth rate of tree community and to take into account the tree-to-tree competition (c ):tt basuki et al. (2009) had developed more accurate equations to predict tree biomass 2 (b, kg/tree) using allometric model (with r = 0.963) of the tree species found in mixed tropical forest based on diameter at breast height (dbh). the equation is expressed by: which means: where: c = the intercept (= -1.201) α = the slope coefficient (= 2.196) of basuki et al. (2009) equations. plant growth is considered as the changing of biomass over time (replacing the volume equation (5) to biomass equation (7)). therefore, formula to measure the next year biomass is: modeling tree growth after replantation forest fire caused all tree community perished in the study area and the replanting was done using selected tree species which is important for the area. the importance of these tree species was represented by the high important value indices shown for these tree species. modeling process a model to explore the growth trend of tree structure which suffered from forest fire in the tropical mountain forest was developed. the model follows a gleasonian approach, integrating the biology and the fate of all individuals throughout their life cycle (menaut et al. 1990). the model was based on two conditions of selected tree growth and estimated size of each tree. these conditions were: 1. normal growth which means that there was no forest fire. in this condition, the biomass of the tree at initial condition (year 0), at the next 25 years and at the next 50 years were counted; 2. plant growth after plantation which means that there was forest fire occurred; the selected trees were planted accordingly. therefore, the model was based on the growth and estimated biomass of the replanted trees. the time of each tree species reaching the same tree size at normal condition (without fire) was recorded. the modeling was focused on the components of replanted tree competition with its potential competitors i.e. grass (especially for the early stage of the growth) and other tree species. the model of tree growth was developed in “r software”. the inputs of the model were: 1. an initial tree community structure from field data survey; 2. allometric equation consisted of diameter, height and biomass; 3. plant growth rate; 4. parameter of competition, both tree to grass and tree to tree competitions. modeling the normal tree growth the used approach in this study was developed by menaut et al. (1990) who expressed potential tree growth as a function of growth rate 3 (g) multiplied by volume (v, m ) of tree: menaut et al. (1990) adopted volume equation from botkin et al. (1972) which is: where: d = diameter at breast height in cm; d = maximum diameter at breast height in cm;max h = height in m; h = maximum height in m.max 67 modeling of tree growth after forest fire – rozak and setiadi dv dt g v v d2 1 d h dmax maxh v d2 1 d h d hmax max dv dt g d2 1 d h d hmax max v t 1 v t g d2 1 d h d hmax max ctt ln(b) ln(dbh) b ec (dbh) b t 1 b t g e 1.201 dbh 2.196 ctt c ............................... (6) ............................... (7) ..... (5) .................... (4) ....................... (3) ......................................................... (1) ............................. (2) ......... (8) after forest fire, in the succession process of the community, there were interactions and competitions from the pioneer species, such as competition within pioneer species to get light, nutrition, etc. according to menaut et al. (1990), the competition component encompassed two kinds of competitive interactions i.e. tree-tograss competition (c ) and tree-to-tree tg competition (c ). based on menaut et al. (1990), tt the c value used was 0.99 for a tree reaching 2 m tg high and c value used were 0.3, 0.5 and 0.7.tt the change of biomass from one year to the next year based on its potential growth (equations 7 & 8) limited by competitions (c and c ) would tt tg be: data interpretation the projection of tree community in mount ciremai national park (mcnp) for 50 years was examined. tree growth was modeled to get the pre-fire condition, represented by the value of total aboveground biomass between the normal growth projection (equation 8) and after replantation (equation 9). one important parameter to determine the speed of tree species growth is the value of tree to tree competition. the results of low, medium and high c were tt compared, i.e. c = 0.3 (three other tree canopies tt overlapped with replanted tree canopies), c = 0.5 tt (two other tree canopies overlapped with replanted tree canopies) and c = 0.7 (only one tt tree canopy overlapped with replanted tree canopies). overlapped tree canopies were used to characterize the tree-to-tree competition. other 1.201 important coefficient is c which reflected the tg intensity of competition from grass (and other herbaceous layer) towards the newly planted trees. results and discussion plant growth model provided the projection of future size of the plant. in this study, the model was developed based on several parameters i.e. plant growth rate, competition and initial biometric of the measured tree. furthermore, the model tried to predict the future state of tree community after being suffered from forest fire and had been replanted. the replantation was an effort to accelerate the succession of the tree community in order to shorten the recovery period. previous publications mentioned that tropical forest regeneration to pre-fire condition took a long time (viedma et al. 1997; kinnaird & o'brien 1998). four of five replanted species reached the prefire condition within 37 50 years (fig. 1 to 5). based on the comparison among three after-fire models and pre-fire model, the fastest model to reach the pre-fire condition/initial biomass (table 2) was the model with lower value of treeto-tree competition (c ). the value of c was tt tt generated from the total number of possible overlapped canopies among neighboring trees (menaut et al. 1990). in general, model having one overlapped tree canopy provided the fastest period to reach the pre-fire condition. exceptionally, s. pendula did not reach the expected value within the 50 years period. table 2 biomass projection from five tree species without fire event in mount ciremai national park (mcnp) species dbh initial biomass (kg) biomass-25 (kg) biomass-50 (kg) engelhardtia spicata 35 742 1,060 1,538 ficus ribes 15 115 186 304 trema orientalis 34 696 949 1,311 saurauia pendula 17 152 222 331 lithocarpus pallidus 12 115 296 788 notes: biomass 25 = expected biomass after 25 years biomass 50 = expected biomass after 50 years 68 biotropia vol. 23 no. 2, 2016 bnew t 1 bnew t g e dbh 2.196 ctg ctt ... (9) biomass growth after replantation for each tree engelhardtia spicata lechen ex blume biomass growth model for single tree species e. spicata (fig. 1) predicted that in the next 50 years the tree would reach 1,315 kg (with c = 0.7), tt 345 kg (with c = 0.5) and 87 kg (with c = 0.3). tt tt the model also predicted that e. spicata would reach the same biomass as initial/pre-fire biomass after 44 years of planting when based on c = 0.7. tt however, for c = 0.5 and c = 0.3, the model tt tt predicted that the biomass would not reach the initial/pre-fire biomass after 50 years of planting. ficus ribes reinw. ex blume based on the normal growth model, total biomass for f. ribes would be 304 kg in 50 years. meanwhile, the initial/pre-fire total biomass based on this model was 115 kg (table 2). based on the results generated from the application of three different values of tree-to-tree competition on the model for plant recovery, it was predicted that the initial/pre-fire biomass would be achieved by c = 0.7 in 37 years after replantation. tt this prediction was the fastest period compared to the c = 0.5 and c = 0.3, which provided tt tt prediction of above 50 years after planting to reach the initial/pre-fire biomass (fig. 2). figure 1 biomass growth projection for engelhardtia spicata figure 2 biomass growth projection for ficus ribes 69 modeling of tree growth after forest fire – rozak and setiadi trema orientalis (l.) blume based on the normal growth model, the initial/pre-fire biomass for t. orientalis was 696 kg. in 50 years projection, the total biomass would reach 1,311 kg (table 2). subsequently, c = 0.7 tt was predicted to reach the initial/pre-fire condition within 50 years after planting (fig. 3). meanwhile, the c = 0.5 and c = 0.3 were tt tt predicted to provide lower biomass at all projection periods. saurauia pendula blume the initial biomass of predicted by s. pendula the normal growth model was 152 kg. within 50 years after planting, the total biomass of s. pendula was predicted to reach 331 kg (table 2). however, the given value within 50 years period from the post-fire model did not reach the initial biomass value obtained from the normal growth model. moreover, all results of the post-fire model considering three different values of treeto-tree competition were lower than the results from the normal growth model. for instance, in the next 50 years, tree biomass would be 145 kg (for c = 0.7), 69 kg (for c = 0.5) and 33 kg (for tt tt c = 0.3) (fig. 4).tt figure 3 biomass growth projection for trema orientalis figure 4 biomass growth projection for saurauia pendula 70 biotropia vol. 23 no. 2, 2016 figure 5 biomass growth projection for lithocarpus pallidus lithocarpus pallidus (blume) rehder according to the pre-fire model, the initial biomass of l. pallidus was 115 kg and total biomass for 50 years after planting was predicted to be 788 kg (table 2). interesting results were shown on l. pallidus which had three possible values of tree-to-tree competition within 50 years after replanting. for c = 0.7, the total initial/pre-tt fire biomass was predicted to be reached in 19 years after replanting. for c = 0.5, the total tt initial/pre-fire biomass was predicted to be reached in 27 years after replanting, while c = 0.3 tt was predicted to reach the total initial/pre-fire biomass in 43 years after replanting (fig. 5). consequently, the number of neighboring trees can be different to get expected plant growth within certain period. for instance, l. pallidus can grow with its canopy overlapped with the canopies of two other trees. factor controlling tree growth other contributing factor to overall results was the initial biometric of tree species i.e. diameter at breast height (dbh) and growth rate. the application of intensive silvicultural treatments is required to increase tree growth rate (peña-carlos et al. 2008). among applicable silvicultural treatments to increase tree growth rate are: 1. reducing the amount of vegetations competing with tree seedlings (minimizing c and c ); 2. tt tg early-fertilizing; 3. thinning; and 4. pruning (gonzales-ochoa & de las heras 2002; gonzalesochoa et al. 2004; zald et al. 2008). model improvement consideration based on the results obtained from the developed models, it is important to redevelop the model because at some point (for example c tt = 0.7, lithocarpus pallidus, between year 30 50, fig. 5) the biomass growth seemed to be unrealistic compared to the expected normal growth. the possibility is to develop allometric equation using logistic model. the tree-to-tree competition value should also be remeasured to include site specific factor, because the menaut's data only included the savannah ecosystem. conclusions the models developed in this study predicted normal and after-replantated tree growth. in general, this study provided further information regarding tree growth after replantation in tropical montane forests. acknowledgements we are grateful to the cibodas botanic gardens (indonesian institute of sciences) and mount ciremai national park (ministry of 71 modeling of tree growth after forest fire – rozak and setiadi forestry, republic of indonesia) for funding and granting permission to conduct research in the mount ciremai national park. we thank dr william cheung for constructive suggestions on our work and ayu s. nurinsiyah for improving the text. references basuki tm, van laake pe, skidmore ak, hussin ya. 2009. allometric equations for estimating the aboveground biomass in tropical lowland dipterocarp forests. for ecol manage 257:1684-94. botkin db, janak jf, walis jr. 1972. some ecological consequences of a computer model of forest growth. j ecol 60(3):649-872. cochrane ma. 2003. fire science for rainforests. nature 421:913-9. flannigan md, stocks bj, wotton bm. 2000. climate change and forest fires. sci total environ 262:221-9. gonzales-ochoa a, de las heras j. 2002. effects of post-fire silviculture practices on pachyrhinus squamosus defoliation levels and growth of pinus halepenses mill. for ecol manage 167:185-94. gonzales-ochoa a, lopez-serrano fr, de las heras j. 2004. does post-fire forest management increase tree growth and cone production in pinus halepensis? for ecol manage 188:235-47. hanum if, van der maesen ljg, editors. 1997. plant resources of south-east asia no. 11: auxiliary plants. leiden (nl): backhuys publisher. p.252-5. kinnaird mf, o'brien tg. 1998. ecological effects of wildfire on lowland rainforest in sumatra. conserv biol 12(5):954-6. knox kje, clarke pj. 2012. fire severity, feedback effects and resilience to alternative community states in forest assemblages. for ecol manage 265:47-54. kuningan government. 2011. mount ciremai national park. available online on www.kuningankab.go.id. thaccessed on 14 february 2011. 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http://www.theplantlist.org/ st(accessed 1 january 2013). van der vossen ham, wessel m, editors. 2000. plant resources of south-east asia no. 16: stimulants. leiden (nl): backhuys publisher. p.128-9. 72 biotropia vol. 23 no. 2, 2016 van nieustadt mgl, sheil d. 2005. drought, fire and tree survival in a borneo rain forest, east kalimantan, indonesia. j ecol 93:191-201. viedma o, melia j, segarra d, garcia-haro j. 1997. modeling rates of ecosystem recovery after fires by using landsat tm data. remote sens environ 61:383-98. watanabe nm, suzuki e, simbolon h. 2009. reestablishment of rattans after forest fire in east kalimantan, borneo. tropics 18(1):13-21. zald hsj, gray an, north m, kern ra. 2008. initial tree regeneration responses to fire and thinning treatments in a sierra nevada mixed-conifer forest, usa. for ecol manage 256:168-79. 73 modeling of tree growth after forest fire – rozak and setiadi page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 1099.cdr ( )% 100 x 1.724 : a c d c (%)carbon ofpercent ÷ ö ç è è æ = avicennia spp. sonneratia spp. ceriops spp. aegiceras spp. rhizophora spp. bruguiera spp. lumnitzera spp. excoecaria agallocha xylocarpus spp. heritiera sp. terminalia cattapa page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 736 nguyen tan chung (biochemical).cdr biochemical characteristics of fluvisol thionic linked to land use types in souhtern vietnam nguyen tan chung , sy danh chung and hoang van thoi1* 2 2 1department of biotechnology, nong lam university, ho chi minh city 70000, viet nam 2 department of centre for fruit tree research, southerm institute of fruit tree reseach, my tho 203, viet nam 3 department of forest ecology and environment, forest science institute of south viet nam, ho chi minh city 70000, viet nam received 10 december 2016/accepted 25 may 2017 abstract thionic fluvisols soil in southern vietnam is like typical acid sulfate soil in the tropics and is severely polluted due to human activities. salinity intrusion and industrial wastewater contamination are the main cause of environmental degradation in soil ecosystem. this research was aimed to determine a link between biochemical soil properties and land use types to provide suitable solutions for afforestation and soil restoration. soil sampling was conducted in five different land use types at four soil layers (o, ab, bj and cp). the five land use types were sugarcane crop; melaleuca plantation; 2-year acacia plantation; 5-year acacia plantation; and control (grass-covered land). the results showed that soil in those five land use types were very acidic (ph ≤ 4) having poor-nutrient condition with range of orthophosphate content of 378 640 mg/kg, n-nh of 586 999 mg/kg and n-no of 830 1,112 mg/kg. 4 3 concentration of toxic ions was very high with large variation among land use types and soil depths i.e. 1,799 – 12,403 23+mg so /kg; 22 1,645 mg exchangeable fe/kg and 34 88 mg al /kg soil. the lowest concentration of exchangeable 4 3+ 2-fe and so ions were found in sugarcane and melaleuca plantations, respectively. twenty-three sulfur-oxidizing 4 bacteria and two iron-oxidizing bacteria were identified. all these bacteria were initially identified as thiobacillus sp. sugarcane and melaleuca plantations exhibited the most diverse thiobacillus species which linked to reduction of 2-exchangeable fe and so concentrations in these two land use types. this study indicated that thiobacillus sp. could 4 grow well in the thionic fluvisols. it is proposed that melaleuca and sugarcane species could reduce iron and sulfur contents in thionic fluvisols in the tropics. keywords: acacia, acid sulfate soil, melaleuca; sugarcane, thionic fluvisols, thiobacillus sp. introduction thionic fluvisols are acid sulfate soils 2containing high so content, having very low ph 4 (3 4) and containing high concentration of 3+ 3+ 2exchangeable al and fe . high content of so 4 accumulating in the soil mainly originates from an oxidation of potential minerals containing sulfur to form sulfuric acid (h so ) (sokolova & 2 4 alekseeva 2008). while acid sulfate soil derives from sulfides oxidation containing minerals such as organic matter, sulfate, iron and aluminum, it could be formed in brackish water soil or other highly potential sulfate soil areas with a participation of microorganisms (dao & hoang 2005). potential acid sulfate soil is a large source of heavy metal which causes pollution when being exposed to oxygen due to natural phenomena or artificial drainage, oxidizing pyrite to create h so and thus, decrease soil ph 2 4 (breemen 1993; nguyen et al. 2004). generally, iron and aluminum are often considered as toxic elements in the soils and greatly affect plant growth and development because excess of these elements can be toxic to plants (nguyen et al. 2004; dao & hoang 2005). concentrations of 2+ 3+ 3+ exchangeable fe (fe and fe ) and al exceeding 500 and 135 mg/kg, respectively, are toxic to rice (le et al. 2000). when soil ph is less than 4.5, macro-nutrient ions (n, k, ca, mg, p) have relatively low concentration in acid soils. in contrast, al and fe are normally abundant in the soil with low ph (brady & well 2002). the more *corresponding author: ntchung@hcmuaf.edu.vn biotropia 4 3 7 246 261 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 3 736 246 acidic the soil is, the higher concentrations of these two ions are. according to dao and hoang (2005), 2+ 3+ 2concentrations of fe , al and so ions in acid 4 sulfate soil are much higher than concentrations that can be tolerated by plant. sulfur and sulfate are two main compounds involved in the formation process of acidic soil. total sulfur in two layers of jarosite (bj) and pyrite (cp) is an index to distinguish between an acid sulfate soil and other soil types. major forms of sulfur in the 22-acid soil are fes, fes , h s, so , and so . 2 2 3 4 2-concentration of so ions greatly varies among 4 2-different types of acidic soils. so ions are toxic 4 to plants and are obstacles for land reclamation. in southern vietnam, total sulfur in the acidic soil ranges from 1 4% and can reach 5 6% when the acidic soil experiences a long-term flood (dao & hoang 2005). metabolism and transformation of sulfur forms are complex with participation of sulfur-reducing bacteria in anaerobic conditions ( .lamers et al. 2012; pester et al. 2012) a total area of potential acid sulfate soil in southern vietnam was 2,415,727 ha (in 2005) (ho et al. 2010). this area had active acidic soil area of 279,946 ha in 2005, which increased to 1,078,169 ha in 2011 (pham 2011). concentrations of et al. fe and s ions increased while concentrations of k , al , total organic carbon (toc), n and p + 3+ tended to decrease between year of 1975 and 2005 (ho 2010). kyuma (1976) and le ( 2003) et al. showed that toc content in the acidic soil located in the mekong delta was pretty high, ranging from 1.0 to 4.8% for soil having high concentration of sulfur ions and ranging from 3.2 to 5.2% for soil having low concentration of sulfur ions. due to high content of toc, the acidic soil also contains high concentration of total nitrogen (tn) (0.15 0.25%). however, concentration of total phosphorus (tp) (0.01 0.05%) and total potassium (tk) (0.03 0.09%) were very low in the acidic soil. 3+ 3+ 2-contents of al , fe , so and other 4 nutrients in acid sulfate soil greatly vary among soil layers and seasons (le et al. 2000; le 2003). soil in the mekong delta had average organic layer depths (o) of 20 23 cm, transitional layer (ab) of 23 46 cm, jarosite layer (bj) or sulfur forms producing layer of 47 88 cm and pyrite layer (bp) greater than 89 cm (duong et al. 2010). total organic carbon (toc) and total nitrogen (tn) in layer o of acidic soil are 5.47% and 0.26%, respectively (ngo 2010). other studies in southern vietnam conducted by nguyen et al. (2011) showed that soil ph, toc and tn in topsoil layer (o) were respectively 3.38 3.75; 2.24 4.04%; and 0.15 0.27%. concentrations of 3+ 3+ 2-al , fe and so ions not only varied due to 4 seasonal changes, but also depended on depths of soil layers (do & nguyen 1999; tran et al. 2011). tran et al. (2011) showed concentrations of exchangeable fe ions were seasonally fluctuated in layer o, which could reach a peak of 11,780 mg/kg at the beginning of the rainy season, but dramatically decreased to 521 mg/kg at the end of 3+the rainy season while al concentration greatly varied among soil layers, fluctuated from 0 to 749 mg/kg in soil layer a and 104 to 1,109 mg/kg soil in soil layer cp. other study measuring 2+ 3+ 2-concentrations of fe , al and so in layer bj 4 of different acid sulfate soils in southern vietnam 2showed that so concentration was less than 4 0.15%, 0.11 0.25%, and 0.27 0.74% for acidic soils having less, medium and high activity of 2+ 3+ sulfur forms; while fe and al concentrations ranged from 29 166 mg/kg and 73 111 mg/kg, respectively (ngo 2002). vegetation density and composition reflect soil biological property and fertility depending on soil location, type and depth (dang 2009). at et al. soil depth of 0 22 cm, microbial density could reach 232 x 10 cfu/g, however, vegetation 5 density was much lower at soil depth higher than 55 cm (araragi & tangcham 1979). hitomo & naoto (2005) identified four species of thiomonas genus that could reduce sulfur from paddy rice in sado island of japan, including t. cuprina, t. intermedia, t. perometabolis t. thermosulfate. and these microorganisms could transform the concentration of thiosulfate ions from 1.51 to 1.60 mm from initial thiosulfate concentration of 1.61 mm used for the cultural media (hitomo & naoto 2005). gram negative thiobacillus isolated from paddy soil in india is highly capable of reducing sulfur forms (rajagopal & sridar 2007). results of other study conducted in acid sulfate soil in binh chanh district of ho chi minh city showed that a total number of sulfurreducing microbes at soil depths of 0 20 and 20 40 cm ranged from 1.6 x 10 to 8.9 x 10 cfu/g 5 5 soil (nguyen & phan 1992). the study also identified sp. which mainly thiobacillus contributed to oxidize sulfate ions and pointed out that the presence of sp. thiobacillus 247 biochemical characteristics of thionic fluvisol linked to land use types chung et al. biotropia vol. 24 no. 3, 2017 248 therefore, threatens biodiversity of the ecosystem leading to the decrease in crop production. it is important to use every possible effort to rehabilitate and restore degraded acid sulfate soil quality based on soil biochemical characteristics. biological approach is among efforts to restore the degraded acid sulfate soil. this study was aimed to determine biochemical characteristics of acid sulfate soil in relation to different vegetation types grown on the soil and to select appropriate crops to renovate degraded acid sulfate soil. materials and methods several steps were conducted to achieve the research objectives i.e. 1. measuring nutrients concentrations (c, n, p, k) and toxins (exchangeable fe , al , so ) of acid sulfate soil 3+ 3+ 24 in southern vietnam; 2. isolating and identifying microorganisms that are capable of reducing sulfur and iron; and 3. evaluating potential relationship between biochemical characteristics and land use types. study site this study was conducted on five different land use types located in binh chanh protected forest, binh chanh district of ho chi minh city. this location represented thionic fluvisols soil (acid sulfate soil) having high concentration of 2-so . this area was highly polluted due to 4 anthropogenic activities and industrial wastewater. the study site was surrounded by a canal system to water several species of plants growing in the area. land use types five land use types were selected for conducting sample collections i.e. sugarcane plantation, 2-year plantation, 5-year acacia acacia plantation, 10-year plantation and melaleuca grass-covered land (as control; the grass was dominated by sp. and sp.)eleocharis cyprus . statistical analysis this study was designed using completely randomized design with six replications for each treatment. the treatments were the five land use depended on land use types and cultivation time of crops. in order to improve acidic status of sulfate soil, some researchers planted different species on sulfate soil (le 1999; wathinee 2015; et al. et al. kogawara 2006). watanabe (1997) et al. et al. conducted trials on adaptability of melaleuca cajuputi melastona marabathricum and on acid sulfate soil in thailand and observed that these species grew well. was capable of melaleuca cajuputi accumulating high aluminum concentration in its root system, while melastona marabathricum accumulated al in its foliage. satoshi (2006) et al. showed that in a greenhouse condition having lack of oxygen and acidic ph of 5.8, m. cajuputi grew better than . le (1999) e. camaldulensis et al. and thai (2009) planted and melaleuca leucadendra melaleuca cajuputi on acid soil located in the mekong delta and observed that melaleuca leucadendra melaleuca cajuputi and could adapt to the mekong delta region with survival rate greater than 75%. pham and pham (2009) and pham and vu (2014) planted sp. and sp. on eucalyptus melaleuca seasonally inundated soil in the mekong delta and observed that ,eucalyptus camaldulensis eucalyptus tereticormis melaleuca leucadendra melaleuca viridiflora , , and could grow very well on the melaleuca cajuputi acid sulfate soil. 3+ melaleuca sp. can fix al ions into its root system through symbiosis activities of soil microorganisms associated with the root which can release organic acids to neutralize al ions 3+ (tran 2012). this capability of sp. leads melaleuca to the role of sp. in reclamation of melaleuca sulfate-affected soil in the mekong delta. in addition to highly adaptable sp. in melaleuca the mekong delta (nakabayashi 2001; et al. duong 2005), there are many native plant et al. species grow on acid sulfate soil in the mekong delta (dang 2009; pham 2014). nine et al. et al. species dominating sulfate soils are melaleuca cajeputi, polygonum tomentosum, nypa fruticans, eleocharis dulcis, nymphaea pubescens, sonneratia caseolaris, melastoma , cryptocoryne ciliata sp. and annona glabra. there are several indigenous species adapting to seasonally flooded sulfate soil such as , calophyllum inophyllum thespesia populnea and (dang 2009; pham gluta velutina et al. et al. 2014). environmental quality of acid sulfate soil in southern vietnam is seriously degraded and 249 types i.e. sugarcane plantation, 2-year acacia plantation, 5-year plantation, 10-year acacia melaleuca plantation and grass-covered land (as control; the grass was dominated by sp. eleocharis and sp.). anova of this experimental cyprus design including the tukey's test for determining the significant differences were computed using minitab 17 software at < 0.05. sigmaplot 12 p software was used to develop charts. soil samples collection six different soil sampling points were randomly selected in each land use type to collect soil samples and to determine soil profiles. soil samples were collected from four soil layers i.e. o (organic layer), ab (transitional layer between a and bj), bj (jarosite layer) and cp (pyrite layer) (duong et al. 2010). soil samples were air-dried for two weeks, then were sieved using 2-mm mesh size sieve. soil samples were analyzed at the soil laboratory of forest science institute of south vietnam. macronutrient elements and toxins were analyzed from soil samples (tcvn 1995). different batches of soil samples for isolating and identifying microbes capable of transforming or oxidizing iron and sulfur were also collected from o and ab soil layers of the same soil sampling points in each land use type (araragi & tangcham 1979; nguyen & phan 1992). figure 1 study sites in the mekong delta, vietnam table 1 analytical methods used to measure soil nutrients and toxins criteria analytical method ph soil: ddi water = 1 : 5 total nitrogen (tn) kjeldahl method total phosphorus (tp) dry-ashed and 6n hcl total potassium (tk) dry-ashed and 6n hcl total organic carbon (toc) walkley – black nh4 + soil: 2m kcl = 1 : 10 no3 soil: 2m kcl = 1 : 10 orthophosphate mehlich 3 al3+ mehlich 3 so4 2turbidimetric method fe3+ mehlich 3 biochemical characteristics of thionic fluvisol linked to land use types chung et al. 250 biotropia vol. 24 no. 3, 2017 for inganalytical methods applied measur + macronutrients (c, n, p, k, nh , no , 4 3 orthophosphate) and toxic ions (exchangeable 3+ 3+ 2fe , al , so ) re presented in table 1.4 a isolation and identification of sulfuroxidizing microbes isolation of sulfur-oxidizing microbes was carried out based on methods described by rajagopal and sridar (2007), using the starkey and thiosulfate culture media. these culture media had initial ph of 8.0. the total number of sulfurreducing microbes was determined using method described by tcvn (2005) and computed using equation 1 developed by tcvn (2005): where: a = numbers of bacteria cells in 1 g soil (colony forming units or cfu/g) n = total colony count n = number of disks having colony growth at i each dilution v = amount of sample solution in each disk f = dilution concentration in each diski in order to screen microbes capable of sulfur oxidation and/or transformation, the isolated microbes were continually cultured in thiosulfate media supplemented by three different concentrations of na s o i.e. 5, 10 and 15 g/l 2 2 3 culture media. once screened, these microbes were tested for their biochemical characteristics such as catalase, voges-proskauer indole, citrat, gram, and (vp), nutritional types using methods described by nguyen et al. (1978) and nguyen (2005a; 2005b). the biochemical characteristics test were carried out prior to classifying and identifying these microbes. total numbers of microbes where determined afterward. isolation and identification of fe-oxidizing microbes iron-oxidizing microbes were isolated using culture media containing . this 2+1% of fe ion method was described by . suparna et al. (2014) total number of fe-reducing microbes was determined using methods described by tcvn (2005) and computed using equation 1. the isolated microbes were continually cultured in 2+ culture media 3% and 6% of fe containing ion. these media were used t iron-toleranto screen microbes capable of iron as well as microbes oxidation. lassification and dentification of the c i s microbes were based on creened carried out growth probability test in culture media having four levels of ph i.e. . biochemical 3, 5, 7, 9 characteristics test was also carried out prior to classifying and identifying the microbes. the biochemical characteristics test included catalase, g m ram, obilization ability of colonies and assimilation total ability of organic carbon. numbers of microbes where determined afterward. results and discussion soil ph soil ph is an important indicator as it relates directly to crops development, microbial activity and biochemical reactions occurring in soils. the study results showed that soil ph of the five land use types were less than 4.0 (table 2). soil ph differed among land use types within each soil layer of o, ab and bj (table 2). although sugarcane plantation and grass-covered land (control) had higher ph than the 2-year acacia plantation, 5-year acacia plantation and melaleuca plantation, the ph difference was not significant. table 2 soil ph of five land use types within each soil layer soil layer sugarcane plantation 2-year acacia plantation 5-year acacia plantation melaleuca plantation grass-covered land (control) o 3.8ab 3.3b 3.5ab 3.2b 3.9a ab 3.6ab 2.9c 3.4bc 3.5ab 4.0a bj 3.5abc 3.0c 3.2bc 3.6ab 4.0a cp 3.3ns 3.3ns 3.3ns 3.8ns 3.7ns note: numbers within a row (soil layer) followed by the same letter did not differ significantly at p < 0.05 ns = non significantly different 251 concentrations of n and p among land use types total nitrogen (tn) is an important criteria used to evaluate soil fertility depending on the content of soil organic matter. nitrogen in soil is produced by microbial decomposition of soil organic matter and is a result from fixation of nitrogen gas by rhizosphere in plant root system. nitrogen mainly accumulated in topsoil layer (o layer) and ab layer for all land use types (fig. 1). tn concentration in the topsoil layer was higher compared to those in deeper soil layers within each land use types. tn concentration ranged from 2.3 g/kg (in sugarcane plantation) to 5.2 g/kg (melaleuca plantation) (table 3). tn concentration in topsoil layer (o layer) was slightly different from that in ab layer, however, it was significantly different from tn concentration in bj and cp layers, with exception for melaleuca plantation and grass-covered land (control). -concentration of no ranged from 830 1,112 3 +mg/kg soil, while concentration of nh ranged 4 from 586 999 mg/kg soil (table 3). +concentrations of tn, no , nh decreased 3 4 with increasing soil depth (fig. 1). the topsoil layer of melaleuca plantation always contained the -highest concentrations of tn (5.2 g/kg), no 3 +(1,112 mg/kg) and nh (999 mg/kg soil) (table 4 3). sugarcane and 2-year acacia plantations -showed the lowest concentrations of no and 3 + nh (table 3).4 concentrations of total phosphorus (tp) and orthophosphate in topsoil layer (o layer) were low for all land use types (table 3; fig. 2). range of tp and orthophosphate concentrations in topsoil layer (o layer) were 785 958 mg/kg and 378 640 mg/kg, respectively (table 3; fig. 2). low concentration of orthophosphate might be due to partial binding of orthophosphate with al and fe to form al-p and fe-p complex 3+ 3+ (bertsch 1996; brady & well 2002). under acidic condition (ph < 4.0) the increase of al and fe 3+ 3+ concentrations resulted to a reduction of orthophosphate ions through al-p and fe-p complexation (brady & well 2002). tp concentrations in topsoil layer (o layer) were not significantly different among the five land use types. the highest orthophosphate concentration was observed in the topsoil layer (o layer) of the 5-year acacia plantation (table 3; fig. 2). table 3 variations of n and p concentrations among land use types criteria soil layer sugarcane crop 2-year acacia plantation 5-year acacia plantation melaleuca plantation grass-covered land (control) p value tn (g/kg) o 2.28b 2.37b 2.50b 5.18a 2.36b 0.002 ab 1.68ab 1.96a 2.14a 2.00a 0.98b 0.012 bj 0.86b 1.12ab 1.19ab 1.84a 0.72b 0.003 cp 0.82ns 1.98ns 0.96ns 1.12ns 0.50ns 0.091 nh4 + (mg/kg) o 611.7bc 585.7c 861.8ab 998.7a 775.2bc 0.001 ab 448.5b 441.2b 736.5a 467.3b 386.1b 0.003 bj 133.8b 171.1b 504.6a 526.5a 266.6b < 0.001 cp 106.6ns 136.9ns 219.0ns 152.0ns 227.8ns 0.078 no3 (mg/kg) o 830.0bc 625.1c 1070.1ab 1111.8a 991.4ab < 0.001 ab 656.1abc 586.1bc 898.4a 850.7ab 436.6c 0.001 bj 263.4c 268.0c 599.9ab 706.5a 447.3b < 0.001 cp 218.1b 218.1b 294.4ab 395.1a 216.2b 0.012 tp (mg/kg) o 784.6ns 800.6ns 957.9ns 871.7ns 810.9ns 0.118 ab 578.9bc 737.7abc 758.0ab 535.4c 939.8a < 0.001 bj 553.2bc 687.2b 671.3b 457.9c 905.1a < 0.001 cp 603.9b 628.6b 591.1b 519.3b 855.7a 0.004 po4 3 (mg/kg) o 493.7ab 462.4b 640.0a 395.5b 377.7b 0.001 ab 307.2b 417.3ab 461.0a 297.3b 303.0b 0.002 bj 280.5ab 362.2a 349.8a 274.3ab 219.5b 0.013 cp 337.3a 289.0ab 315.6a 249.3ab 156.5b 0.012 note: numbers within a row (soil layer) followed by the same letter did not differ significantly at p < 0.05 ns = non significantly different biochemical characteristics of thionic fluvisol linked to land use types chung et al. 252 biotropia vol. 24 no. 3, 2017 variation of toc and tk among land use types toc concentration within each land use type decreased with increasing soil depth (table 4). in each of land use types, toc concentration in the topsoil layer was higher than those in deeper layers. within topsoil layer, the lowest toc concentration (2.98%) was observed in sugarcane plantation, whereas the highest toc concentration (5.29%) was observed in melaleuca plantation. differences of toc concentration were significantly different (table 4). toc concentrations in all land use types observed in this study were slightly lower than the average toc concentration observed in previous study conducted in the mekong delta i.e. 5.5% (ngo 2010). the lowest tk concentration was observed in melaleuca plantation ranging from 0.494 to 0.564%. other land use types showed similar tk concentrations, ranging from 0.641 to 0.769%, which was higher than tk concentration in melaleuca plantation. differences in tk concentration, however, were not significant. tk concentrations showed in this study were within the results of previous study conducted in the mekong delta (ngo 2002). although five land use types did not show a significant difference of tk concentrations in their topsoil layer, they were significantly different among vegetation types within taking into account of each other lower layers (ab, bj, cp) (table 4). concentrations of tn, tp, toc and tk were significantly lower in topsoil layer (o layer) than figure 2 concentrations of total and bioavailable n and p in five land use types 253 those in deeper soil layers (ab, bj, cp) within each land use type (table 3 & 4). bioavailability of fe, al and s elements in soil concentration of exchangeable fe ions was particularly very low topsoil layer of sugarcane plantation (20 mg/kg soil), 2-year acacia plantation (48 mg/kg soil) and 5-year acacia plantation (37 mg/kg soil). concentration of exchangeable fe ions was highly accumulated in cp soil layer of all land use types ranging from 684 mg/kg (in sugarcane plantation) to 2,645 mg/kg ( in g rass-covered land) . the h ighest concentration of exchangeable fe ions was observed in the ab, bj and cp soil layers of melaleuca plantation.the lowest concentrations of ions were observed in the grass-3+ 2al and so4 covered land (control) and melaleuca plantation (table 5). concentration of ion in all soil layers of 2-so4 melaleuca plantation was not significantly different, ranging from 3 to 4 g/kg soil. the lowest concentration of was observed in 2-so ion4 the soil layers of ab, bj and cp melaleuca 2-plantation so 4. the highest concentration of ion 2-year acacia plantation was observed in the and 5-year acacia plantation for all soil layers, ranging from .2 .4 / . the concentration 9 to 12 g kg differences within the same soil layer were not significant. 3+ concentration of al ion was similar within the same soil layer among five land use types. table 4 variations of total organic carbon (toc) and total potassium (tk) among land use types soil layer sugarcane crop 2-year acacia plantation 5-year acacia plantation melaleuca plantation grass-covered land (control) p value toc (%) o 2.98b 3.08b 3.44b 5.29a 3.21b < 0.001 ab 2.38bc 2.96ab 3.66a 3.57ab 1.39c < 0.001 bj 1.29b 1.88b 1.72b 3.08a 1.71b 0.001 cp 1.83 ns 2.50 ns 2.06 ns 1.89 ns 2.22 ns 0.903 tk (%) o 0.662a 0.650a 0.641a 0.494b 0.660a 0.006 ab 0.691a 0.659a 0.652a 0.511b 0.751a < 0.001 bj 0.769a 0.702a 0.696a 0.514b 0.696a < 0.001 cp 0.694ab 0.675b 0.759a 0.564c 0.654b < 0.000 note: numbers within a row (soil layer) followed by the same letter did not differ significantly at p < 0.05 ns = non significantly different 3+ 3+ 2-table 5 variations of al , fe and so concentrations among land use types4 criteria soil layer sugarcane crop 2-year acacia plantation 5-year acacia plantation melaleuca plantation grass-covered land (control) p value al3+ (mg/kg) o 37.9ns 49.7ns 38.8ns 40.7ns 34.3ns 0.101 ab 49.9ns 56.0ns 61.8ns 59.0ns 43.8ns 0.105 bj 63.4ab 61.2ab 74.0a 52.8bc 37.5c 0.001 cp 84.9a 63.2ab 68.5ab 70.8a 46.2b 0.003 exchangeable fe (mg/kg) o 22.4c 44.6c 38.7c 223.6b 455.4a < 0.001 ab 16.0c 51.0c 35.6c 1462.6a 958.0b < 0.001 bj 62.1d 1052.8b 489.4c 1269.3ab 1365.4a < 0.001 cp 684.6b 1721.0a 1692.4a 1612.5a 2144.9a < 0.001 so4 2 (g/kg) o 1.80c 10.04a 7.65b 2.38c 3.86c < 0.001 ab 3.24b 9.57a 9.00a 3.33b 3.45b < 0.001 bj 4.50b 12.62a 4.99b 2.70b 3.37b < 0.001 cp 10.92a 10.93a 8.11b 3.94c 8.66ab < 0.001 note: numbers within a row (soil layer) followed by the same letter did not differ significantly at p < 0.05 ns = non significantly different biochemical characteristics of thionic fluvisol linked to land use types chung et al. 254 biotropia vol. 24 no. 3, 2017 3+ concentration of al ion increased with 3+increasing soil depth. range of al ion concentrations was 38 85 mg/kg for all soil layers among the five land use types. concentrations of 3+ al ion were significantly different between o soil layer and cj soil layer within each land use type (table 5; fig. 3). bioavailability of exchangeable fe ions and al ion fluctuated with soil ph differences. 3+ grass-covered land having higher soil ph exhibited lower concentration of al ion and 3+ higher concentration of exchangeable fe ions (dao & hoang 2005). 3+ 2-figure 3 concentrations of soil exchangeable fe ions, al ion and so ion among land use types4 255 isolation of sulfurand iron-oxidizing microbes soil samples taken from o and ab soil layers were used to isolate microbes capable of reducing sulfur and iron content in the soil. total numbers of microbes where determined afterward. total number of sulfur-oxidizing microbes varied from 0.75 x 10 to 172 x 10 cfu/g soil 5 5 (table 6). total numbers of sulfur-oxidizing microbes isolated from the o soil layer were not significantly different among the five land use types. total numbers of sulfur-oxidizing microbes isolated from the ab soil layer were significantly different among the five land use types. the highest number of sulfur-oxidizing microbes was recorded in soil samples taken from the ab soil layer of the plantation (172 x 10 5melaleuca cfu/g), followed by the -year plantation acacia (134 x 10 cfu/g). these results indicated that 5 species richness and species density of sulfuroxidizing microbes were quite high among the five land use types. total number of iron-oxidizing microbes (range of 1.0 x 10 4.1 x 10 cfu/g for all land 4 4 use types) was much lower than those of sulfuroxidizing microbes (table 6). italicized numbers within brackets in table 6 indicated the numbers of sulfuror iron-oxidizing microbial species isolated from soil samples. these microbes survived in the sulfuror iron-enriched culture media. there were 23 different microbial species capable of growing well and metabolizing sulfur in sulfur-enriched culture media. most of these microbial species were isolated from o layer. the highest numbers of sulfur-oxidizing microbes were found in the o layer of sugarcane plantation (8 species), followed by those found in the o layer of plantation (6 species). other species melaleuca were found in the o layer of 2-year acacia plantation (3 species), in the o layer of 5-year acacia plantation (3 species), in the ab layer of sugarcane plantation (1 species), in the ab layer of 5-year plantation (1 species) and in the acacia ab layer of grass-covered land/control (1 species). in the starky's sulfur-enriched culture media, colonies of these microbes had yellow, orange or dark pink color with smooth surface, which are similar to the results of studies conducted by and ) and hitomo naoto (2005 rajagopal and sridar (2007). two microbia l spec ies capable of metabolizing iron in iron-enriched culture media were found in the ab layer of sugarcane plantation (1 species) and in the ab layer of 5year plantation (1 species). diameter of acacia these iron-oxidizing microbes colonies ranged from 2 to 4 mm. the colonies grew closely to the surface of the culture media and had goldenwhite color. identification of sulfur-oxidizing microbial genera ten of the 23 sulfur-oxidizing microbial species performed high capacity of sulfur oxidation and thus, reduced ph of the starkey culture media and thiosulfate from 8.0 to 5.0 (table 7). ph 8.0 is the initial ph of the starkey and thiosulfate media. all of these ten microbial species was able to transform more than 50% of na s o content in 2 2 3 culture media supplemented with 5 mg na s o /l (table 7).2 2 3 table 6 total numbers of sulfurand iron-oxidizing microbes isolated from soil samples soil layer sugarcane plantation 2-year acacia plantation 5-year acacia plantation melaleuca plantation grass-covered land (control) total sulfur-oxidizing microbes (cfu/g soil) o 11.8 x 105 (8) 16.8 x 105 (3) 3.9 x 105 (3) 0.75 x 105 (6) 2.1 x 105 (0) ab 31.3 x 105 (1) 134 x 105 (0) 12.5 x 105 (1) 172 x 105 (0) 11.4 x 105 (1) total iron-oxidizing microbes (cfu/g soil) o 1.9 x 104 1.4 x 104 2.4 x 104 1.4 x 104 1.3 x 104 ab 1.7 x 104 (1) 1.5 x 104 4.1 x 104 (1) 1.0 x 104 1.1 x 104 note: italic numbers in the brackets are the twenty-three different species of sulfur-reducing microbes and two different species of ironreducing microbes isolated from soil samples biochemical characteristics of thionic fluvisol linked to land use types chung et al. biotropia vol. 24 no. 3, 2017 256 g en er a is o la te d fr o m su lf ur – en ri ch ed cu lt ur e m ed ia p h in c ul tu re m ed ia af te r m ic ro b es is o la ti o n (o ri gi n al p h = 8 ) r ea ct io n s n ut ri ti o n al ty p e c o n ce n tr at io n o f n a 2 s 2 o 3 su p p le m en te d in to th e t h io su lf at e cu lt ur e m ed ia 5 g/ l 10 g /l 15 g /l st ar ke y t h io su lf at e c at al as e v p in do l c it ra t g ra m g c st r (% ) g c st r (% ) g c st r (% ) su ga -g en 1 5 8 p o s n eg n eg p o s n eg au to tr o p h ic + + 61 .1 0 0 su ga -g en 2 5 5 p o s n eg n eg p o s n eg h et er o tr o p h ic + + 52 .9 0 0 su ga -g en 3 4. 5 5 p o s n eg n eg p o s n eg h et er o tr o p h ic + + 53 .3 0 0 su ga -g en 4 4. 5 8 p o s n eg n eg p o s n eg au to tr o p h ic + + + 98 .0 + + + 93 .5 0 su ga -g en 5 5 8 p o s n eg n eg p o s n eg h et er o tr o p h ic + + 64 .5 0 0 su ga -g en 6 4. 5 5 p o s n eg n eg p o s n eg au to tr o p h ic + + 70 .6 0 0 m el -a -g en 7 5 5 p o s n eg n eg p o s n eg h et er o tr o p h ic + + + 84 .8 0 0 m el -a -g en 8 5 6 p o s n eg n eg p o s n eg h et er o tr o p h ic + + 73 .7 0 0 m el -a -g en 9 4. 5 8 p o s n eg n eg p o s n eg h et er o tr o p h ic + + + 97 .1 + 33 .3 0 g ra -a b -g en 10 4. 5 8 p o s n eg n eg p o s n eg au to tr o p h ic + + + 96 .1 + + + 95 .9 0 t ab le 7 r es ul ts o f b io ch em ic al a n al ys es o f th e te n s ul fu rox id iz in g m ic ro b es n o te : su ga -g en 1 = g en us 1 o f t hi ob ac ill us s p. is o la te d fr o m t h e a la ye r o f s ug ar ca n e p la n ta ti o n h av in g h ig h ly t ra n sf o rm ed s ul fu r; su ga -g en 2 = g en us 2 o f t hi ob ac ill us s p. is o la te d fr o m t h e a la ye r o f s ug ar ca n e p la n ta ti o n h av in g h ig h ly t ra n sf o rm ed s ul fu r; su ga -g en 3 = g en us 3 o f t hi ob ac ill us s p. is o la te d fr o m t h e a la ye r o f s ug ar ca n e p la n ta ti o n h av in g h ig h ly t ra n sf o rm ed s ul fu r; su ga -g en 4 = g en us 4 o f t hi ob ac ill us s p. is o la te d fr o m t h e a la ye r o f s ug ar ca n e p la n ta ti o n h av in g h ig h ly t ra n sf o rm ed s ul fu r; su ga -g en 5 = g en us 5 o f t hi ob ac ill us s p. is o la te d fr o m t h e a la ye r o f s ug ar ca n e p la n ta ti o n h av in g h ig h ly t ra n sf o rm ed s ul fu r; su ga -g en 6 = g en us 6 o f t hi ob ac ill us s p. is o la te d fr o m t h e a la ye r o f s ug ar ca n e p la n ta ti o n h av in g h ig h ly t ra n sf o rm ed s ul fu r; m el -a -g en 7 = g en us 7 o f t hi ob ac ill us s p. is o la te d fr o m t h e a la ye r o f m el al eu ca p la n ta ti o n h av in g h ig h ly t ra n sf o rm ed s ul fu r; m el -a -g en 8 = g en us 8 o f t hi ob ac ill us s p. is o la te d fr o m t h e a la ye r o f m el al eu ca p la n ta ti o n h av in g h ig h ly t ra n sf o rm ed s ul fu r; m el -a -g en 9 = g en us 9 o f t hi ob ac ill us s p. is o la te d fr o m t h e a la ye r o f m el al eu ca p la n ta ti o n h av in g h ig h ly t ra n sf o rm ed s ul fu r; g ra -a b -g en 10 = g en us 1 0 o f t hi ob ac ill us s p. is o la te d th e a b la ye r o f g ra ss -c ov er ed la n d/ co n tr o l h av in g h ig h ly t ra n sf o rm ed s ul fu r; p o s = p o si ti ve r ea ct io n ; n eg = n eg at iv e re ac ti o n ; g c = g ro w th c ap ac it y; s -t r ( % ) = p er ce n ta ge o f tr an sf o rm ed s ul fu r; = n o g ro w th ; + = n o rm al g ro w th ; + + = g o o d gr ow th ; + + + = ve ry g o o d gr ow th 257 three species were able to transform more than 96% of na s o in thiosulfate culture media 2 2 3 supplemented with 5 mg na s o /l i.e. sug-a-2 2 3 gen4 mel-a-gen9 gra-ab-gen10, , and (table 7). these three species were able to decrease ph of the starkey media from 8.0 to 4.5, however, these species were not able to decrease ph of thiosulfate media (table 7). these three species were the only species survived and grew well in thiosulfate culture media supplemented with 10 mg na s o /l, however, they did not survive in 2 2 3 thiosulfate culture media supplemented with 15 mg na s o /l (table 7). these three microbial 2 2 3 species were found in the o and ab layers of sugarcane plantation, plantation and melaleuca grass-covered land/control, and thus, would be able to oxidize sulfur in the soil of these three land use types (fig. 3). results of biochemical testing for the ten sulfur-oxidizing microbial species are presented in table 7. all of these species had negative reaction toward gram, vp and indol tests, however, they had positive reaction toward catalase and citrat tests. nutritional test showed that the ten sulfur-oxidizing microbial species consisted of three autotrophic species from sugarcane plantation, three heterotrophic species from sugarcane plantation, one autotrophic species from grass-covered land/control and three heterotrophic species from melaleuca plantation (table 7). autotrophic microbes are microbes that can synthesize inorganic carbon sources for their growth without the presence of organic carbon compounds. heterotrophic microbes are microbes that can only grow well in media containing high organic carbon source and cannot grow without organic carbon compounds (brady & well 2002). identification of these ten sulfur-oxidizing microbial species was conducted based on the results of biochemical characteristics analyses, results of studies conducted by kantachote and innuwat (2004) and handbook of bacterial identification written by kelly and harrison (1989). the identification indicated that the ten sulfur-oxidizing microbial species belong to genus .thiobacillus identification of iron-oxidizing microbial genera results of biochemical testing for the two iron-oxidizing microbial species are presented in table 8. two iron-oxidizing microbial species were isolated from the five land use types. these two microbial species showed negative reaction toward gram test, however, showed positive reaction toward catalase test. these two iron-oxidizing microbial species were initially isolated in culture media containing 1% fe . the two iron-oxidizing microbial species 2+ grew well in iron-enriched culture media having 3 and 6% fe concentrations with culture media 2+ ph ≤ 5.0. these two microbial species showed negative reaction toward glucose, sucrose and maltose tests, indicating that these two microbial species were autotrophic. identification based on biochemical characteristics analyses, results of studies conducted by kantachote and innuwat (2004) and handbook of bacterial identification written by kelly and harrison (1989) indicated that these two iron-oxidizing microbial species belong to genus .thiobacillus biochemical characteristics of thionic fluvisol linked to land use types chung et al. biotropia vol. 24 no. 3, 2017 258 t ab le 8 r es ul ts o f b io ch em ic al a n al ys es o f tw o h ig h ly ir o n -o xi di zi n g p er fo rm ed m ic ro b es g en er a is o la te d f ro m ir o n -e n ri ch ed c ul tu re m ed ia r ea ct io n s a b ili ty t o c o lo n y p ro du ct io n a b ili ty t o co lo n y m o b ili za ti o n g ro w th w it h ir o n su p p le m en t h av in g co n ce n tr at io n o f g ro w th w it h s up p le m en t o f o rg an ic ca rb o n s o ur ce s g ro w th in c ul tu re m ed ia h av in g di ff er en t p h c at al as e g ra m 3% 6% g lu co se su cr o se m al to se 3 5 7 9 su ga b -g en 1 p o s n eg + + + + + a ca -a b -g en 2 p o s n eg + + + + + n o te : su ga -g en 1 = g en us 1 o f t hi ob ac ill us s p. is o la te d fr o m th e a b la ye r o f s ug ar ca n e p la n ta ti o n h av in g h ig h ly tr an sf o rm ed ir o n ; a ca -a b -g en 2 = g en us 2 o f t hi ob ac ill us s p. i so la te d fr o m th e a b la ye r o f 5ye ar a ca cia p la n ta ti o n h ig h ly tr an sf o rm ed ir o n ; p o s = p o si ti ve re ac ti o n ; n eg = n eg at iv e re ac ti o n ; + = n o rm al g ro w th ; = n o g ro w th 259 conclusions the five land use types had very acidic soil with soil ph of ≤ 4.0, low concentrations of phosphorus and potassium. total organic carbon (toc) in o layers in all five land use types was higher than that in deeper soil layers. melaleuca plantation had the highest concentration of toc in the o layer. concentrations of exchangeable 3+ 3+ 2fe , al , so ions varied among the five land use 4 2-types. the lowest concentration of so was 4 observed in the grass-covered land (control) and melaleuca plantation. ten sulfur-oxidizing microbial species were isolated from sugarcane plantation, melaleuca plantation and grass-covered land/control and were identified as belong to genus . thiobacillus two iron-oxidizing microbial species were isolated from sugarcane plantation and 5-year acacia plantation and were also identified as belong to genus .thiobacillus it is proposed that there is a strong relationship between sp. and sugarcane plantation thiobacillus which indicates that sp. isolated from thiobacillus sugarcane plantation can oxidize sulfur and iron in the polluted acid sulfate soil. acknowledgements the authors thank ho chi minh people committee's department of forestry and forest science institute of 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vietnam standard and quality insitute, directorate for standards, metrology and quality. vietnam standard and quality insitute. 2005. tcvn 4884:2005. microorganisms in livestock food, methods in determination of total microbial number on cultural media, and technique on counting microbial colony. hanoi (vn): vietnam standard and quality insitute, directorate for standards, metrology and quality. 261 watanabe t, osaki m, tadano t. 1997. response of plants adapted in low ph soils to aluminum. plant nutr for sustainable food prod environ 78:459-60. wathinee s, nuntawoot s, waravooth s. 2015. growth and aboveground biomass of plantation in m. cajuputi trat province-thailand. thai j for 34:57-64. biochemical characteristics of thionic fluvisol linked to land use types chung et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 page 14 page 15 page 16 706 yuzammi (the peculiar).cdr the peculiar petiole growth of calluses amorphophallus titanum e (becc.) becc. x arcang and its implications for conservation effortsex situ yuzammi , kartika ning tyas and tri handayani * cent for plant conservation bogor botanic gardens, indonesian institute of sciences (lipi), bogor 16122, indonesiaer received 06 october 2016/accepted 06 october 2017 abstract amorphophallus titanum is a plant species endemic to sumatera. land degradation and illegal capture of hornbills (a distributor of a. titanum seeds) is leading to potential extinction of a. titanum in the wild. in order to conserve the species and save it from extinction, there is an urgent need to develop methods to propagate it both in situ and ex situ. the aim of this research was to discover environmental factors triggering callus growth from petiole cuttings of a. titanum in its natural habitat in sumatera and to determine the viability of callus pieces as a propagation material. a completely randomized design with a single factor, i.e., callus size, was employed on five callus replicates. each 2replicate consisted of four callus samples. the treatments consisted of three callus piece sizes i.e. 0.5 x.0.5 cm , 1 x 1 2 2cm , 2 x 2 cm . for each replicate, the following parameters of growth were assessed: the time of appearance of shoots; the shoot height when the first leaf fully opened; the petiole diameter; the diameter of the leaf lamina; and the number of young shoots. the results of our field observations showed that environmental factors such as temperature, humidity, and soil influence the formation of the callus in the wild. the size of the callus affected the shoot and root growth. the best result was obtained from callus 2 cm², which could produce 2–3 shoots with an average height of 18.8 cm at the time of first fully opened leaf. the collection of petiole calluses of a. titanum that formed in its natural habitat are recommended instead of carrying the tuber.this ex vitro calluses can be used as propagation materials and then it could be planted in the botanic gardens as one of ex situ conservation effort. keywords: amorphophallus titanum, ex vitro propagation, ex situ conservation, petiole callus, sumatera introduction the genus amorphophallus belongs to the araceae family which comprises more than 220 species, ranging from the subtropical eastern himalayas through subtropical and tropical asia into the tropical western pacific and northeastern australia (boyce et al. 2012; yuzammi et al. 2017). currently, 26 species (11.81 % of all amorphophallus species) are found in indonesia, of which 17 species (65.38 % of all indonesian amor phopha l l u s spec i e s ) a r e endemic (hetterscheid & ittenbach 1996; yuzammi et al. 2014; yuzammi et al. 2017). one of the indonesian endemic species of amorphophallus a. titanum is . it is only found in sumatera, the home of the largest inflorescence in the world. this endemic species is distributed throughout al l sumateran rainforests . unfortunately, the conservation status of a. titanum as a vulnerable species is excluded from both the international union for conservation of nature (iucn) and world conservation monitoring centre (wcmc) due to inadequate comprehensive population data on it. the population of a. titanum in the wild decreased significantly in recent times. therefore, population studies of in its natural a. titanum habitat are urgently needed. several factors such as illegal logging, land degradation, illegal capture of hornbills (a distributor of seeds) and a. titanum destruction of the plant due to belief of local people based on a myth are leading to potential extinction of the species in the wild. local people in kapahiang regency believe that within the petiole of a , there is a 'sacred stone' that . titanum can cure many kinds of diseases and even counter act effects of poisoning which encourages them * corresponding author: yuzammi@yahoo.co.id biotropia 5 1 8 56 63 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.1.706 56 to harvest the plant in the wild. moreover, there is an additional belief by local people that the species is a predator of human beings due to the petiole pattern of a. titanum resembles a 'snake pattern', which leads them to destroy the species when they find it growing in their fields. based on several fields observation a. titanum , is likely to go extinct in the wild unless action is taken on many fronts. increasing public awareness and properly educating local people about a. titanum will play an important role in conservation efforts. another approach to conserving the species and saving it from extinction is through improvement in techniques for propagating the species. several propagation techniques of a. titanum have been applied such as from seeds, tuber and cutting. other propagation technique could involve the of petiole calluses which are naturally formed in the wild this peculiar petiole growth . phenomenon is one of the plant's natural attempts to regenerate. the main objectives of this study were to investigate whether environment factors that trigger callus growth in its natural habitat and to determine the viability of callus pieces after growing them out as in ex situ or in botanic gardens. materials and methods field study several field studies have been carried out over the past decade i.e., in april 2006, january 2013 and september 2015. the field studies were located in bukit daun protection forest and in selected farmers' fields in tebat monok village, kapahiang regency, bengkulu province, indonesia, where the material for research ex situ was taken. the kepahiang regency is about 40 km to northeast from bengkulu city (fig.1). ex situ propagation the research experiment was conducted in the nursery of the centre for plant conservation, bogor botanic gardens –indonesian institute of sciences (lipi), indonesia from january to august 2013. research materials were obtained from calluses which emerged on the chopped petiole of natural growing a. titanum. a completely randomized design with a single factor, i.e. callus size, was employed on five callus replicates. each replicate consisted of four samples. the treatments consisted of three callus piece sizes i.e. 0.5 x.0.5 cm, 1 x 1 cm, 2 x 2 cm. calluses raised from a chopped petiole were cut up according to the appropriate treatment size. however, the calluses tended to be irregular in shape therefore the callus-pieces were generally not perfect cubes. the callus pieces were planted on a mixed growing medium of 1:1 mix of chopped fern root with charcoaled rice husks. the following parameters were assessed: time to appearance of shoots; shoot height when the first leaf fully opened; diameter of the emergent petiole; diameter of the lamina; and number of young shoots. all the data were analized using 57 peculiar petiole calluses growth of amorphophallus titanum (becc.) becc. ex arcang yuzammi et al. figure 1 left – indonesian map (above) and sumatera island, bengkulu province (in red box, below); right – the location of fieldworks in kapahiang regency, tebat monok village and selected farmers'fields hidayat and yuzammi (2008) reported that the distribution pattern for a titanum tends to be . clustered, which may increase the risk from threats in the wild. moreover, unregulated trading by underpaid locals who collect large amounts of the species from the wild for transport to japan and korea (hetterscheid & ittenbach 1996) may lead to extinction in the future. in addition, land degradation, disappearance of the giant hornbill birds as seed distributors and the prevalence of destructive myths are all contributing to the species becoming vulnerable. nevertheless, a titanum has its own ability to . regenerate from its petioles in the wild. this peculiar growth from the petiole of a. titanum occurs when it is chopped and left naturally (fig. 2 2 2 c, d, e). usually, the chopped petiole is best left in a standing position for calluses to form, the petiole being attached to the ground directly. it should not be far from a river for this to occur. if chopped and left in a prostrate position, the petioles did not form calluses during our observations in the field. it is assumed that after cutting of the petiole, the temperature and the environment humidity must be right the for petiole not to decay. water and nutrients must continue to flow to the leaves in order that the leaves can still carry out photosynthesis. according to mr. holidin (pers. com.), one conservation-minded local community member, it takes more than six months to develop a new individual plant from a piece of chopped petiole in the forest (fig. f g).2 , 2 he hejnowicz and barthlott (2005) stated that t petiole of a titanum is composed of . a single layer of epidermis, a thin parenchymatous cortex, a thick layer of collenchyma strand embedded in compact pharenchyma and a core of aerenchyma (fig. 3). this petiole , produces exudates when cut initially appears in epidermis layer 2 2 and (fig. a, b) covered the damage zone. it is presumed that this exud e stimulates the formation of callus in the at damage zone. moreover, ikeuchi et al. (2013) mentioned that the callus form is associated with wounding. this callus can be produced from a single differentiated cell, many cells are totipotent, and those are able to regenerate. plant development is usually induced by many kinds of hormone growth, notably cytokinin and auxin. mostly cytokinin is produced within the sas software. when several roots appeared from the new young shoot, it was separated from the callus immediately. this induced a new shoot from the callus. a cross section of a fresh petiole from the seedling of a. titanum is made to demonstrate that the calluses are generated from epidermis tissue using a light microscope with 40 x magnification. results and discussion growth of a. titanum in natural habitat (in situ) based on our field study in sumatera, a. titanum is often found in coffee and cocoa plantations, sometimes growing in extreme habitats such as on limestone outcrops and in places of heavy soil erosion. most specimens of the species are found growing on steep areas with slope gradient ranging from 30% to 60% and mostly close to flowing water such as streams or rivers. the sites where a. titanum usually thrives have a temperature of 25˚–27˚c, atmospheric humidity of 85% – 95% and soil moisture of 65% – 90% which is relatively high and appears to be optimal for the growth of the species. the soil type is a dull yellowish-brown to dark brown forest soil, based on color charts (oyama & takehara 1976) and the soil ph is 5.1–6.4. in addition, hetterscheid and ittenbach (1996) have mentioned that the species is also found in semi-open spaces in young secondary forest from 0–1200 m altitude. the species has high tolerance of, and quickly adapts to, sur rounding environments. however, the species prefers to have good soil drainage and balance of soil nutrients for better growth. a titanumthe presence of . in its natural determinedhabitat is not only by climate and by soil nutrients but also the integrity of all aspects of the biotic components of the habitat, including human the support of the local community and government regulation. in of addition, international are conservation agencies expected to support the of the preservation species in the wild. biotropia vol. 25 no. 1, 2018 58 roots naturally (taiz & zeiger 2010). sometimes the plant can also produce both auxin and cytokinin in the leaves as well in the roots (srivastava 2002). therefore, when the petiole is cut, the transportation of cytokinin from the roots to the shoots is suddently stopped but the auxin and cytokinin are still delivered from the leaves to the damage zone of the petiole, polarly. subsequently, the ratio of auxin and cytokinin may change in the damaged area of the petiole and needs to be appropriate for the formation of callus. the callus then is stimulated to grow until 59 peculiar petiole calluses growth of amorphophallus titanum (becc.) becc. ex arcang yuzammi et al. figure petiole cross section (a); exudate from the petiole (b); a part of a petiole forming callus naturally (c); callus 2 appears with roots (d); part of a rachis forming a callus (e); seedling growth from a piece of planted petiole in the forest (f); a chopped section of petiole planted in the forest by a conservation-minded local community member (g) covering the entire damage petiole surface irregularly. the appearance of the callus is probably initiated from the epidermis of the damage petiole zone (fig. 3). the ratio of auxin to cytokinin will influence callus and root formation. root tip elongation requires a high ratio of auxin to cytokinin while a somewhat lower ratio of auxin to cytokinin will induce callus formation (gaba 2005). the callus develops a clump and then turns to producing small tubers. at this stage, the small tubers still adhere to each other . and some are produced roots a tuber starts to enlarge and young shoots emerge before separating eventually. in the juvenile stage, one tuber can generate two or three young shoots. production outside the natural of a. titanum habitat (ex situ) the study explored improving a propagation technique for to help address its a. titanum extinction problem . p in its natural habitat etiole calluses a. titanum to the bogor of were brought botanic gardens to examine their ex situ propagation potential . (fig. 4b) a. titanumthe petiole of resembles a tree trunk and its anatomy is typical of monocot stems ( ).fig. 4a in appropriate condition, chopped petiole of developed ex vitro calluses a. titanum naturally, which can be used as propagation material. this is supported by totipotency theory that one cell can grow and develop to be a new 60 biotropia vol. 25 no. 1, 2018 figure 3 cross section of petiole from the seedling of amorphophallus titanum (40 x) using light microscope; the emergence of the callus is probably initiated from the epidermis of the damage petiole zone figure 4 the petiole of a. titanum resembling a tree trunk in its natural habitat (a), cross section of a part of a petiole forming calluses initiated from the epidermis (b) a b 61 peculiar petiole calluses growth of amorphophallus titanum (becc.) becc. ex arcang yuzammi et al. plant (gahan 2007). these calluses then will emerge a new plant into in appropriate environment. the appearance of young shoot and roots depend on the ratio of auxin and cytokinin hormones. the inducement of shoot growth as a result of cytokinin content higher than auxin.is meanwhile, the emerge of roots is caused the nce auxin content is higher than cytokininwhich (gaba 2005). during the propagation experiment, all petiole calluses were able to produce young shoots, however the callus size significantly affected all measured parameters (table 1). cytokinin is known to be involved in shoot initiation (davies 2004; srivastava 2002) and our data showed that the induction of shoot growth may be proportional to the cytokinin content of a callus piece. in calluses of 0.5 cm² size, the first young shoot emerged 46.8 ± 2.9 days after planting, while for calluses of size 1 cm² and 2 cm², emergence appeared 52.4 ± 3 and 52.3 ± 3 days after planting, respectively. furthermore, the number of shoots that emerged was also influenced by callus size. the callus 0.5 x 0.5 cm² size only produced one shoot with a plant height of 5.2 ± 0.2 cm (at the time of the first fully opened leaf). on the other hand, the calluses 1 x 1 cm² and 2 x 2 cm² in size generated one, two or three shoots, and plant height at the time of the first fully opened leaf was 11.4 ± 0.3 cm and 18.8 ± 0.3 cm, respectively (table 1). this finding suggests that bigger calluses retain greater amount of stored energy that is required for shoot and root formation. lower energy available for shoot formation will produce a smaller plant. smaller calluses are therefore expected to take a longer time to reach the same size in the young shoot compared to calluses 2 x 2 cm² in size. since the young shoot which emerged from the callus had several roots, the new plant was separated quickly from the callus. the purpose of separating the new shoot from the callus was to stimulate the formation of more new shoots in the callus (fig. 5). according to gahan and george (2008), when a shoot emerges then it is likely that auxin production will inhibit further shoot formation. this means that all the stored energy of the callus will be used just for table 1 the effect of a. titanum callus size on shoot emergence, shoot height, leaf diameter, petiole diameter and number of shoots callus size (cm²) shoot emergence (days) shoot height (cm) leaf diameter (cm) petiole diameter (mm) number of shoots 0.5 x 0.5 46.8 ± 2.9 b 5.2 ± 0.2 c 6.8 ± 0.5 c 2.41 ± 0.13 c 1.0 ± 0.0 b 1 x 1 52.4 ± 3.1 a 11.4 ± 0.3 b 10.1 ± 0.6 b 3.64 ± 0.21 b 1.2 ± 0.2 b 2 x 2 52.3 ± 3.2 a 18.8 ± 0.3 a 14.4 ± 0.1 a 5.69 ± 0.34 a 2.4 ± 0.7 a note: the shoot height, leaf and petiole diameters are measured when first leaf is fully opened (values for a particular parameter followed by the same letter are not significantly different at p<0.05) figure 5 the a. titanum shoot emerging from the callus (a) and the shoot separated from the callus (b) 62 biotropia vol. 25 no. 1, 2018 growing this one shoot. this experiment showed that a callus with 0.5x0.5 cm² size appeared to be totally depleted of its stored energy in forming a single shoot with its roots. therefore, not more than one shoot was produced. for calluses of 2x2 cm² size, separation of an emergent shoot with its roots from the callus stimulated new shoot formation, because the inhibiting factor (auxin presumably) had been removed. this callus can then produce another one or even two shoots. this research shows that natural calluses of a. titanum can be used as propagation material without plant growth regulator and provides a great potential to ensure its conservation in nature by local people. besides, the result can also be applied by botanic gardens, as an ex situ conservation, to imply collecting the material of a. titanum from the fields. using natural calluses as propagation material has several advantages namely maintains the population of is a. titanum in its habitat, provides more plant materials, easy to carry, less damage compared to carry the tuber, easy to grow, requires less space and will produce plenty of seedlings in short time. conclusions calluses that arise naturally on erect, chopped petioles of a. titanum attached directly to the ground are able to form new shoots. the appearance of these calluses in its natural habitat is supported by environmental factors such as water supply, good soil drainage and balance of soil nutrients for better growth. ex vitro callus development also needs a day temperature of 25˚–27˚c, atmospheric humidity of 85% – 95%, soil moisture content of 65% – 90%, and soil ph of 5.1 – 6.4. when a callus is grown ex situ on an appropriate medium, the number and size of the shoots formed are influenced by the size of the callus. the optimum callus size for propagation is 2x2 cm², which can produce 2-3 shoots and 18.8 cm high at the time of first fully opened leaf. the difficulty to find a. titanum in its natural habitat was due to the over exploitation and destruction of this species. nevertheless, the occurrence of natural calluses on its petiole cutting ensures its in situ and ex situ conservation. ex vitro calluses of a. titanum is recommended as one of propagation materials in the future, notably for ex situ conservation e.g. in botanic gardens. acknowledgements we would like to express our thanks to the director of the center of plant conservation, bogor botanic gardens – indonesian institute of sciences for supporting and funding our expeditions to bengkulu province. we express our sincere gratitude to dr graham eagleton and prof dr i made sudiana for proof reading and correcting the english in the manuscript.we also thank ms. rani yudarwati, m.si, for providing the maps and all members of the bengkulu expedition teams for accompanying us during the field study. special thanks are due to mr. holidin and his brothers for their generous company while we were in the field. references boyce pca, sookchaloem , hetterscheid wla, gusman d g, jacobsen ng, idei t, du v 2012 araceae fl. n . . . thailand 11(2):1–221. davies pj 2004 the plant hormones: their ature, . . n o f . i : ccurrence and unctions n davies pj, editor. plant ormones iosynthesis, ignal ransduction, h b s t a . . ction london (uk): kluwer academic publisher p. 1-35. gaba vp 2005 plant growth regulators in plant tissue . . culture and development n trigiano rn, gray , . i : dj editors. plant evelopment and iotechnology. d b new york (us): crc press. p. 87-99. gahan pb 200 totipotency and the cell cycle. n jain sm, . 7. i : h ä g g m a n , e d i t o r s . p r o t o c o l s f o r h m w tt ficropropagation of oody rees and ruits. dordrecht (nl): springer. .p. 3-14 gahan pb, george 2008 adventitious egeneration n ef. . r . i : george ef, hall , de klerk , editors. plant ma gj p t c 1ropagation by issue ulture vol. . the background. dordrecht (nl): springer .. p. 355-402 hejnowicz z barthlott w. 2005. structural and mechanical , pecualiarities of the petioles of giant leaves of amorphophallus (araceae). 92:391-403.j bot hetterscheid wla, ittenbach 1996 everything you s. . always wanted to know about , but amorphophallus were afraid to stick your nose into!!!!! aroideana 19:7-131. hidayat s, yuzammi 2008 investigation on the natural . . population of (becc.) becc: amorphophallus titanum a case study in the forest areas of bengkulu. buletin kebun raya 11(1):9–15. ikeuchi m, sugimonto k, iwase a. 2013. review, lant p c i rallus: mechanisms of nduction and epression. plant cell 25:3159 73.63 peculiar petiole calluses growth of amorphophallus titanum (becc.) becc. ex arcang yuzammi et al. oyama m, takehara h. 1976. revise standard soil color charts. tokyo (jp): research council for agriculture, forestry and fisheries, ministry of agriculture and forestry. srivastava lm 2002 plant growth and development, . . hormones and environment. london (uk): academic press . 772 p. taiz l, zeige . 20 plant physiology. new york (uk): r e 10. the benjamin/cummings publishing co. inc. 672 p. yuzammi, witono hetterscheid 2014 jr, wla. . conservation tatus of s amorphophallus discophorus backer & alderw. (araceae) in java, indonesia. reinwardtia 14(1):27–33. yuzammi, kurniawan a, asih nps, erlinawati i , hetterscheid w. 2017. the amorphophallus of indonesia. center for plant bogor (id): conservation botanic gardens, indonesian institute of sciences (lipi). page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 biotropia vol. 28 no. 2, 2021: 92 101 doi: 10.11598/btb.2021.28.2.1078 92 growth of black soldier fly larvae (hermetia illucens) fed with pak choi (brassica chinensis) and carp (cyprinus carpio) residues agus dana permana1, ramadhani eka putra1,2*, auliya nurulfah2, mia rosmiati1, ida kinasih3, and dian anggria sari2 1school of life sciences and technology, institut teknologi bandung, bandung 40132, indonesia 2department of biology, institut teknologi sumatera, lampung, indonesia 3departement of biology, universitas islam negeri sunan gunung djati bandung, bandung 40614, indonesia received 8 june 2018/accepted 12 february 2020 abstract one main drawback of the local animal industry is the inavailability of affordable and sustainable protein supply for the livestock. insect larvae, such as the black soldier fly (hermetia illucens) larvae (bsfl), have been considered as a protein source which can be produced at a large scale using low cost organic wastes as feeding material. this study was designed to determine the response of bsfl to various waste combinations of vegetable and animal remains, pak choi (brassica chinensis) residues (s) and carp (cyprinus caprio) fish offal (i)). a total of 540 bsfl were fed with 100 mg/larvae/day combination of vegetable wastes: animal waste 70%: 30% (s > i), 50%: 50% (s = i), and 30%: 70% (s < i). among the feed combinations, the s < i group showed the best results as it produced the significantly highest weight of bsfl at 122.8 mg/larvae and approximate digestibility of 62.01%, with the least pupae mortality rate at 4.29%. keywords: bioconversion, biomass, brassica chinensis, cyprinus carpio, hermetia illucens introduction as the communities develop and increase in size, the amount of waste generated by the human population also increases. in the year 2000, about 49% of the total world population lived in cities and generated more than three million metric tons of daily waste (e.g., household items, food waste, packaging, ash) and this number is predicted to double in 2025 (hoornweg et al. 2013). in 2007, the amount of food waste generated worldwide as the result of economic activities, from production to consumption, was estimated at 1.6 gtonnes (fao 2013). these wastes are taking up space in landfills, as the most common waste management practice, thereby contributing to the spread of pathogens, production of noxious odors, and a significant amount of co2 (zhang et al. 2019). for several decades, researchers worldwide have developed a method to process organic matter away from landfills using biotic decomposer such as black soldier fly larvae (bsfl), earthworm, house fly, and mealworm (beard & sands 1973; el boushy 1991; ndegwa & thompson 2001; ramos-elorduy et al. 2002; elissen et al. 2006; diener et al. 2009), in which bsfl is considered as the best candidate. the black soldier fly is of neotropic origin and now spread in all warmer regions through natural and human-mediated dispersal (callan 1974; marshall et al. 2015). this species can colonize a wide range of organic wastes, including agricultural wastes (manurung et al. 2016; supriyatna et al. 2016), animal and human remains (tomberin et al. 2005; pujol-liz et al. 2008), fish offal (st-hilaire et al. 2007a), food waste (diener et al. 2011; nguyen et al. 2013; *corresponding author: ramadhani@sith.itb.ac.id growth of black soldier fly larvae fed with pakchoi (hermetia illucens) and carp (cyprinus carpio) remains – permana et al. 93 oonincx et al. 2015a), as well as human and livestock feces (myers et al. 2008; banks et al. 2014; oonincx et al. 2015b). due to its biological characteristic and being easily mass-produced (sheppard et al. 2002), this species has been studied to recycle the nutrients found in organic wastes to be converted into protein-rich and fatrich biomass (sheppard et al. 1994; diener et al. 2009; li et al. 2011; surendra et al. 2016). through the bioconversion process, the species is applied as a feed ingredient for aquaculture, livestock, and poultry industries (newton et al. 1977; st-hilaire et al. 2007b; li et al. 2016; magalhaes et al. 2017; renna et al. 2017; schiavone et al. 2017). however, the heterogeneity of available organic material created a challenge to the optimization and implementation of this system, especially in the municipal areas. restaurant waste, for example, containing animal and plant matters which are rich in carbohydrate and a similar amount of protein and fat, while mixed fruits and vegetables are rich in carbohydrate with significantly low-fat content. in indonesia, most organic wastes are produced through economic activities in the traditional and modern markets which are dominated by vegetables and animal remains. applying these heterogeneous resources as diet for black soldier fly would affect the development, productivity, some life-history traits, and chemical composition of the biomass (tomberlin et al. 2002; oonincx et al. 2015a; tschirner & simon 2015; cammack & tomberlin 2017). this study was designed to imitate the real condition in indonesia as a model for other similarly developed tropical countries in which different organic wastes are produced. the objectives of this experiment were 1) to compare the consumption efficiency of bsfl to diet combination of vegetable waste and animal residues, and 2) to determine the effects of diet composition on its growth, development time, pupae survival, and on the adult sex ratio. the results of this study could be used as the basis for diet manipulation in optimizing waste reduction, converting organic materials to insect biomass, and sustainability of the bioconversion system using municipal organic wastes. materials and methods animal specimen this study used the seven-day old larvae of the black soldier fly that were obtained from eggs purchased from a bsf farm in sumedang, west java. all the eggs were kept on the substance made of commercial chicken feed (60% moisture) and kept at constant temperature (28 oc, 70% rh) in a container (50 x 25 x 10 cm) at the laboratory of environmental toxicology, school of life sciences and technology, bandung, indonesia. treatment each treatment (with nine replicates) contained 60 larvae fed with 100 mg/day/larvae (wet weight, 60% moisture content) of a diet combination of vegetable wastes (brassica chinensis) and carp (cyprinus caprio) fish offal. the treatments were composed of diet combination ratios of fish offal: vegetable wastes, namely; 30 : 70 ( s > i), 50 : 50 (s = i), and 70 : 30 (s < i), and replicated 3 times. the seven-day old larvae were initially placed into a plastic cup (with a height of 12 cm, upper diameter 7 cm, lower diameter 5 cm) filled with feeding material, and covered with a black sheet. the lid of the cup contained holes to allow air circulation. to prevent oviposition of other flies and parasitoids, a round dark cloth with diameter 0.01 mm was clamped between box and lid. the diet for larvae was prepared, weighed, and kept frozen 24 hours before the treatment to prevent the decomposition process. all cups were kept in a shady area. sampling and feeding were conducted every three days (diener et al. 2009; lalander et al. 2019) during which period the remaining larvae were transferred into another glass already filled with the next feed. the residual material of the previous glass was dried at 60 oc for dry mass determination. feeding was conducted until more than 40% of all larvae metamorphosed into prepupae (tomberlin et al. 2002; lalander et al. 2019) while weighing was conducted until all larva metamorphosed into prepupae. all prepupae were removed daily from each container and weighed, then placed in a plastic container for biotropia vol. 28 no. 2, 2021 94 further rearing process into an adult. prepupae and pupae were held in the same incubator in which the larvae were reared and monitored for adult emergence daily (cammack & tomberlin 2017). data analysis larvae growth rate and productivity future production of insect larvae through the bioconversion method highly depends on the larvae growth rate. in this study, the growth rate of each larva was determined by daily biomass change (waldbauer 1968), with the following formula: growth rate = 𝐵/t (1) where: b = weight gain (mg) t = development time (days) on the other hand, the productivity of determined by formula: productivity = [dry weight of larvae/(t x v)] (2) where: t = larvae rearing period (day) v = volume of rearing container (dm3) in this study, the volume of reactor applied was 0.414 dm3. consumption ability the ability of larvae to consume diet was determined by ad (approximate digestibility), ecd (efficiency of conversion of digestedfeed), wri (waste reduction index), and proportion of diet used for metabolism, converted into biomass, and undigested. ad parameter was used to determine the effectiveness and larvae ability to digest the diet which could be measured by the formula: ad = (i-f)/i x 100% (3) where: ad = approximate digestibility i = initial weight of diet (mg) f = weight of residue (undigested food + excretions) (mg) the ability of larvae to digest each diet composition was measured by ecd based on the formulae of scriber and slansky (1982) and modified by dienar et al. (2009): b = (i – f) – m0 (4) ecd = b/(i – f) (5) where: b = the total amount of food use for growth i = total amount of food offered during the experiment f = total amount of residue (undigested food + excretory food) m = amount of food metabolized by larvae (calculated by mass balance) dry weight was used in all calculations. to measure overall material reduction, the time of larvae development was required to reduce the amount of food included in calculation along with overall degradation (d) of waste. all of those variables were defined as waste reduction index (wri) which was determined by the formula: wri = [(i-f)/i x 100]/t (6) where: i = initial weight of diet (mg) f = total amount of residue (undigested food + excretory), and larvae rearing period (day) mass balance mass balance is one approach to design the biomass production system and to predict the digestibility of the diet. in this approach, the total amount of feed consumed by larvae was divided into three outputs: the mass of diet material that is used to maintain homeostasis of larvae, the mass of undigested diet material, and the harvested biomass (fig. 1). statistical analysis one way anova (p ≤ 0.05) with subsequent tukey hsd tests were applied to detect the difference of the means among all treatments. growth of black soldier fly larvae fed with pakchoi (hermetia illucens) and carp (cyprinus carpio) remains – permana et al. 95 figure 1 mass balance model of organic waste bioconversion into body biomass of the bsfl results and discussion black soldier fly larvae growth the pattern of larval growth among treatments was relatively similar as all prepupae reached pupal stage on day 21 for all treatments. moreover, the larval weight of group s < i was the highest, followed by s = i and s < i. on average, the final weight of harvested prepupae was 122.80 mg for group s < i which was significantly higher than group s = i (113.88 mg) and s > i (106.89 mg) (fig. 2). diet quality affects the growth and development time of bsfl (furmant et al. 1959; myers et al. 2008; diener et al. 2009; oonincx et al. 2015a,b). the development time to reach the prepupae stage could range from two weeks, under optimal condition, to more than 3 months if the food is limited (table 1). in this study, the development time of bsfl to reach the prepupae stage was shorter than most of the other studies (tabel 1). higher protein and fatty acid content in the fish offal might have provided the necessary nutrients for the larval growth and metabolism (cammack & tomberlin 2017). on the other hand, those nutrients might have also encouraged the diversity of bacterial species, some of which might be associated with bsfl and promote larval growth and development (dong et al. 2009; yu et al. 2010, 2011; jeon et al. 2011; zheng et al. 2013). furthermore, other bacteria unassociated with bsfl, such as escherichia coli, salmonella enterica, and pseudomonas marginalis, could have been killed by the antimicrobial substance produced by bsfl and then used as a source of nutrition (erickson et al. 2004; liu et al. 2008; park et al. 2015). figure 2 growth pattern of bsfl biotropia vol. 28 no. 2, 2021 96 table 1 comparative data on development time and efficiency of conversion of digested-feed (ecd) for black soldier fly larvae on various substrates reference substrate development time (days) ecd (%) may (1961) housefly medium 18 myers et al. (2008) dairy manure 28-30 diener et al. (2009) chicken feed 16-42 24.4 38.0 sealey et al. (2009) dairy manure 120 li et al. (2011) dairy manure < 31 gobbi et al. (2013) meat meal 33 gobbi et al. (2013) hen feed 15 manurung et al. (2016) rice straw 38-52 5.69 10.85 supriyatna et al. (2016) cassava peel 20-54 12 21 abduh et al. (2017a) rubber seed 12.5 25.9 abduh et al. (2017b) pandanus tectorius 6.3 27.4 this study combination of vegetables and fish offal 21 17.33 22.53 productivity among all treatments, larvae of group s < i had the significantly highest productivity (11.15 mg/larvae/day/dm3) compared to group s > i (9.69 mg/larvae/day/dm3) and group s = i (9.42 mg/larvae/day/dm3) (fig. 3). the larva of group s < i which contained more protein has produced significantly larger prepupae (anova, p < 0.05). this conformed with earlier studies of diener et al. (2009), cammack & tomberlin (2017). furthermore, the higher moisture content might have also contributed to the higher prepupae biomass of group s < i. consumption ability the consumption-ability of the larva was determined by waste reduction index (wri), efficiency of conversion of digested-feed (ecd), and approximate digestibility (ad). among treatments, the group s < i produced the significantly highest wri (3.13) which indicated a higher preference of larvae to richer proteinand-lipidcontaining diet (fig. 4). the level of waste reduction was similar to chicken feed (diener et al. 2009) however, it was higher than rice straw feed (manurung et al. 2016) and rubber seed feed (abduh et al. 2017a) which indicated the effect of diet composition to the level of consumption by bsfl. the effectiveness of larvae to convert digested diet into biomass was measured by ecd. in this study, the ecd ranged between 17.33 to 22.53% with group s < i showing the lowest ecd (17.33%) while also recording the significantly highest ad at 62.01% (table 2). figure 3 productivity of bsfl note: * = significant at p < 0.05). growth of black soldier fly larvae fed with pakchoi (hermetia illucens) and carp (cyprinus carpio) remains – permana et al. 97 figure 4 waste reduction index of diet note: * = significant at p < 0.05). table 2 ecd and ad among different diet regimes diet (100 mg/larva/day) ecd (efficiency of conversion of digested-feed) ad (approximate digestibility) s = i (50 : 50) 22.53% 49.46% s > i (70 : 30) 21.56% 45.45% s < i (30 : 70) 17.33% 62.01% the ecd level recorded in this study was relatively higher than most of the previous studies (table 1). ecd decreased when the quality of diet decreased (higher proportion of undigested material, such as cellulose and hemicellulose). however, in this study, the larval group that consumed the protein has showed lower ecd than the group that consumed a cellulose-rich diet. higher ecd manifested by bsfl receiving an inferior diet could be a strategy to obtain more nutrients by increasing the consumption rate (couture et al. 2016). on the other hand, as shown by ad, a diet with higher protein content was more readily digested by the larvae which compensated for its lower ecd. this compensation allowed the s < i group to produce heavier harvested prepupae. mass balance of bioconversion process around 4.45 to 6.66% of the substrate was transformed into biomass, 40.99 to 55.35% was used for metabolism, and 37.99 to 54.55% was undigested. the highest transformation rate of the substrate into biomass was recorded on the group s < i (fig. 5). high transformation rate of the substrate to biomass in group s < i indicated the importance of food digestibility in the production of more biomass. pupae mortality and adult sex ratio the level of pupae mortality was low at all groups, around 4.29 to 10.71%. the highest mortality was recorded at group s > i (10.71%) while the lowest in group s < i (4.29%) (fig. 6). the level of pupae mortality in this study was lower than those of tomberlin et al. (2002) and gobbi et al. (2013) and of cammack and tomberlin (2017). generally, the larvae of black soldier fly are fed on a wide variety of substrates on the larval stage and accumulate a large store of fat to reduce and/or eliminate the need for the adult to feed (sheppard et al. 2002). for this reason, the quality of diet plays a key role in the development of adults during the pupae stage. to produce high quality food, the larvae of insects tend to consume a balanced diet that is optimum for its growth and development (gobbi et al. 2013). this study showed that larvae reared with the high protein diet combined with complex carbohydrates (from vegetable wastes) could significantly reduce the level of pupae mortality. furthermore, the juice produced by the degradation of fish offal maintained the diet moisture which highly influenced pupae mortality, particularly for fly species (cickova et al. 2012; cammack & tomberlin 2017). biotropia vol. 28 no. 2, 2021 98 figure 5 proportion of digested feed used for metabolism, converted into biomass, and left undigested (residue) note: * = significant at p < 0.05). figure 6 percentage of pupae mortality note: * = significant at p < 0.05). figure 7 proportion of adult female and male more female adults were produced than the males for all groups although the proportion was more balanced in s < i group with strong differences manifested by the group receiving the balanced feed (fig. 7). the female-biased sex ratio showed in this study confirmed some earlier studies (tomberlin et al. 2002; zarkani & miswati 2012; gobbi et al. 2013). however, other studies reported a more male-biased adult proportion when larva were fed with artificial feeds (ma et al. 2018; meneguz et al. 2018). variables like ph of the substrate, nutritional variability, and feeding regime were probably the factors that govern the sex ratio of adult flies growth of black soldier fly larvae fed with pakchoi (hermetia illucens) and carp (cyprinus carpio) remains – permana et al. 99 (quezada-garcia et al. 2014; ma et al. 2018; meneguz et al. 2018). strong differences on the sex ratio related with substrate differences in this study are against some previous studies reporting the insignificant effect of diet quality and content to the sex ratio of black soldier fly (tomberlin et al. 2002; gobbi et al. 2013; diener et al. 2015). however, this result agrees with the study of zarkani & miswati (2012) in the tropical region which provides some evidences of the effect of environmental factor to the final stage of the developmental period of bsfl. conclusions diet combination consisting of a higher proportion of protein and lipid has resulted in a higher weight of harvested prepupae and the lowest mortality of the black soldier fly which was probably due to a higher consumption rate and approximately higher digestibility. to improve the sustainable production of the feed, further studies are suggested on the rearing environment of larvae particularly, on reducing the mortality rate of pupae brought about by the bioconverted waste that is high in protein and lipid; on the effect of various waste materials to biomass production and sustainability; and on the composition of biomass. acknowledgments this study was partly and equally supported by riset itb 2013 and hibah kompetensi 2018 granted to the corresponding author, p3mi funding granted to the first author, and dipa boptan uin sunan gunung djati bandung granted to the last author. references abduh my, jamilah m, istiandari p, syaripudin, manurung r. 2017a. bioconversion of rubber seeds to produce protein and oil-rich biomass using black soldier fly larva assisted by microbes. j entomol zool stud 5(4):591-7. abduh my, manurung r, faustina a, affanda e, siregar irh. 2017b. bioconversion of pandanus tectorius using black soldier fly larvae for the production of edible oil and protein-rich biomass. j entomol zool stud 5(1):803-9. banks ij, gibson wt, cameron mm. 2014. growth rates of black soldier fly larvae fed on fresh human faeces and their implication for improving sanitation. trop med int health 19(1):14-22. barnard dr, harms rh, sloan dr. 1998. biodegradation of poultry manure by house fly (diptera: muscidae). environ entomol 27(3):600-5. barry t. 2004. evaluation of the economic, social, and biological feasibility of bioconverting food wastes with the black soldier fly (hermetia illucens) [dissertation]. texas (us): university of north texas. beard rl, sands dc. 1973. factors affecting degradation of poultry manure by flies. environ entomol 2(5):801-6. callan em. 1974. hermetia illucens (diptera: stratiomyidae), a cosmopolitan american species long established in australia and new-zealand. entomol mon mag 109:232-4. cammack ja, tomberlin jk. 2017. the impact of diet protein and carbohydrate on select life-history traits of the black soldier fly hermetia illucens (l.) 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histopathological figure of eye and brain of tilapia fish (oreochromis mossambicus) dwinna aliza1*, nazaruddin1, aya sofia abda2 and annisa asri2 1pathology laboratory, faculty of veterinary medicine, universitas syiah kuala, banda aceh 23111, indonesia 2veterinary medicine study program, faculty of veterinary medicine, universitas syiah kuala, banda aceh 23111, indonesia received 8 august 2021/accepted 26 january 2022 abstract mercury pollution brings harmful effects to aquatic animals, the environment and eventually to human health. mercury accumulates in the liver, kidney, eye lens and brain of fish, resulting in organ damage. this study aimed to determine the effect of hgcl2 exposure on anatomical pathology and histopathology of tilapia fish eye and brain. a total of 36 male tilapia fish were allotted into 4 treatment groups with 3 replications. fish were exposed to 0.00, 0.25, 0.50, and 0.75 ppm of hgcl2 for 10, 20, and 30 days. subsequently, the anatomical pathology was observed followed by histopathological examination. anatomical pathology examination of fish eye on day 30 showed white membrane on the eye lens surface, pupil diminution, and sunken eyes. the brain demonstrated hemorrhage, necrosis, discolorations, and granulated area. the retina showed necrosis, retina pigmentation flexiform layer widened, and cone cell atrophy. brain depicted structural and cellular damage such as degeneration necrosis and vacuolation. hgcl2 exposure changes the anatomical pathology and histopathology of tilapia fish eye and brain. keywords: cellular damage, fish brain, fish eye lens, histopathology, mercury chloride introduction heavy metals polluted the waters of the sea and rivers. among heavy metals pollutants is mercury (hg). mercury has volatile properties, takes the form of liquid at a certain temperature, capable of dissolving various metals and very toxic element to all living things (gworek et al. 2020; wang et al. 2017). mercury in nature comes in two forms, namely inorganic mercury such as mercury chloride (hgcl2) and mercury oxide (hgo), and organic/organomercury consisting of methylmercury (ch3hg+) and ethylmercury (c2h5hg+). both types are toxic, however the natural and anthropogenic emissions occur as inorganic form (zulaikhah et al. 2020; macirella et al. 2016). mercury chloride in sediments on the seabed and rivers can be converted by microorganisms into organic metal mercury (r-o-hg) compounds, which remain soluble in water and accumulate in sediments. high hg concentrations can cause death in fish, whereas sublethal concentrations lead to disruption of organ function (saturday 2018; rauf et al. 2018). mercury accumulated in the body of aquatic animals will damage or stimulate the enzymatic system, which resulting in the decreased adaptability of the animal concerned to the polluted environment (mehouel et al. 2019; lee & wendy 2017). mercury enters the tissues of living organisms through several ways, namely the respiratory, digestive, and penetrating through the skin (dogan & canli 2018; ismail & mahboub 2016). mercury that enters the body of aquatic organisms cannot be digested, however, it can be dissolved in fat. the fat-soluble metal is able to penetrate the cell membrane, so that eventually the mercury metal ions will accumulate in the cells and other organs. the highest accumulation is usually in the detoxification organs (liver) and excretory *corresponding author, email: dwinna.aliza@unsyiah.ac.id biotropia vol. 29 no. 2, 2022 104 organs (kidneys) (macirella et al. 2016; jyotsna et al. 2014; moustafa et al. 2016). in fish, the organs that accumulate the most heavy metal of mercury are the kidneys, liver, brain, and lens of the eye (sobhanardakani et al. 2018; gupta & kumar 2006; pereira et al. 2014; kimakova et al. 2018). severe sublethal toxicity affects the morphology of tissue histology, physiological conditions such as metabolism, growth and development of fish morphology, biochemistry (blood chemistry and enzyme activity), behavior (neurophysiology) and fish breeding (korbas et al. 2017; nazaruddin et al. 2020). according to morcillo et al. (2017), exposure to hg can produce highly variable effects which depend on the magnitude and duration of exposure. this phenomenon was proven by nazaruddin et al. (2020) which found the most severe histopathological condition on male gonad organs of tilapia fish exposed to the longest time period of hgcl2. korbas et al. (2017) stated that fish eye has the potential for hg absorption since it has a wide surface of exposure and continuously contact with external media and thus, becomes an effective absorption route of hg. high accumulation of mercury in the eye lens causes ocular anomalies such as cataracts or opacity, as previously reported in humans affected by hg (lemire et al. 2010). pereira et al. (2014) also stated that eye is the often-neglected organ in accumulation of heavy metals mercury studies. a thoroughly histopathological study on changes of fish eyes exposed to hg has never been reported. in the brain, mercury will accumulate in the cerebrum cortex and cerebellum where it will be oxidized to a form of mercuric (hg++). mercury ions will bind to sulfhydryl from enzyme and cellular proteins that interfere with enzyme function and cell transport (morcillo et al. 2017), leading to degeneration of nerve cells in cerebellum of the brain (berntssen et al. 2003). although there is a considerable amount of information available on toxicity of mercury to fish tissues and cells, which is among the most sensitive biological responses due to mercury exposure. furthermore, this study focusing on eye and brain which proved to be the most impacted organs physiologically affected by mercury pollution. materials and methods fish and experimental design this study used 36 male tilapia (oreochromis mossambicus) fish within similar sexual maturity stage and body weight of 250-300 g. fish were obtained from farmer’s ponds at cadek village, banda aceh, indonesia. fish were apparently healthy and free from skin lesions or external parasites. the fish were maintained in glass aquaria (80 x 60 x 40 cm capacity) having 100 l of dechlorinated tap water. each aquarium was equipped with aerator. fish were acclimatized for 7 days to the laboratory environment and fed three times daily. this experimental study implemented a completely randomized design. fish were allotted into 4 groups (n = 9) with 3 replications. group 1 (k1) was negative control, while group 2, 3, and 4 (k2, k3 and k4) were treated with hgcl2 0.25, 0.5 and 0.75 ppm. the treatments were exposed to hgcl2 for 10, 20, and 30 days. histopathological examination the fish were dissected to collect the eye and brain then subsequently fixed with davidson solution for 24 h. the fish tissues were then dehydrated in an ascending series of alcohol, cleared in xylene and embedded in paraffin wax. section of 5 µm thick were cut, processed and stained with haematoxylin and eosin (h&e). the observations of the slides were done using olympus light microscope and the histopathological changes of the tissues were documented using the photo microscope. results and discussion the anatomical pathology of tilapia fish eye the observation of the anatomical pathology of tilapia fish showed that fish eye in the control group (k1) was normal indicated by clear, fresh, and intact eye ball and lens (fig. 1). the observation did not show any changes of eye performance of the fish exposed to hgcl2 on day 10 and 20, however on day 30 fish in group k2 and k3 demonstrated soft and transparence white layer on the surface of eye ball. this layer became thicker and wider, which almost cover mercury chloride exposure changes the histopathological figure of eye and brain of tilapia fish – dwinna aliza et al. 105 all surface of the eye ball, thus, looks like cataracts. furthermore, the diminution of the eye pupil and sunken eyes was also clearly observed (fig. 1). normal fish eye is bright, intact, transparent, and no mucos layer on the eye ball surface. fish eye, although modified a lot, is basically similar to other vertebrates, consisting of the cornea, corneal epithelium, lens, retina, choroid, optic nerve and iris (takashima & hibiya 1995). fish eye anatomy is rather horizontal in the anterior part, with the intention that the convex lens almost touches the cornea which is an important transparent part of the eye ball scleroid coat. the lens has a layer that resembles a layer of onions, the layer on the lens is transparent, nonnucleated and inelastic. not like the lenses in mammals which are elastic and can be modified by the contraction of the eye muscles, the lenses in fish are not elastic, thus they must be pulled inward toward the retina with the retractor lentic muscles to accommodate changes in vision (azab et al. 2017). anatomical pathology changes occured in the form of white membranes in the eye ball surface might be caused by physical and systemic factors. physical factors occur due to direct contact between hgcl2 and the surface of the eye mucosa, while systemic factors occur through inhalation or ingestion of toxic compounds which are then carried by blood circulation and reaches the eye organ resulted in nerves and balance disorders such as blindness and swim abnormally. this happens since the eyes are connected to the optic lobe of the brain through the optic nerve (rajeshkumar et al. 2017). furthermore, korbas et al. (2013) stated that fish eyes have wide surface which continuously contact with external media leading to an effective absorption route of hg. in addition, fish eyes appeared sunken, indicated high accumulation of hgcl2 in the eye tissue which lead to contraction and sunken of the eye ball. according to pereira et al. (2013), sunken eyes are caused by physiological reaction and fish resistance to exposure of pollutants, bacterial infections or viruses which changes the morphology of fish eyes such as sunken and cloudy. in this study cataracts were also found due to hgcl2 exposure. figure 1 anatomical pathology of tilapia fish exposed to hgcl2 on day 30 notes: k1 = control group; k2 = 0.25 ppm hgcl2; k3 = 0.50 ppm hgcl2; k4 = 0.75 ppm hgcl2; a = eye pupil; b = white layer on eye ball surface; c = sunken eye. k3 k1 k4 k2 biotropia vol. 29 no. 2, 2022 106 histopatology of tilapia fish eye lens and retina histopathology of tilapia eye lens in control group (k1) showed lens layer was transparent and pink in color. similar results were observed on tilapia fish lens in group k2 on observation days 10 and 20. in contrast, on day 30, k2 showed a change in the form of hemorrhage in the iris but the lens was still intact. k3 treatment showed a severe necrosis area in the lens of the eye as well as in the k4 treatment. moreover, in k4 groups a change in the color of the lens that was pink turned into purple in the middle of the lens and became granulated were observed (fig. 2). normal tilapia retina were shown in the control group (k1). the structure of the tissue showed very clear layers and there were no changes of the structure, color and size. the fundamental layers of the retina were still complete and remain unchanged. the structure of the retina layers from inner to outer layer consists of the optic nerve layer, the ganglion layer, the inner flexiform layer, the inner nucleus layer, the outer flexiform layer, the outer nucleus layer, cone cells and stem cells, the retinal pigment cell layer, and choroid (genten et al. 2009) (fig. 3). the histopathological examination of tilapia fish eye retina exposed by hgcl2 revealed necrosis in inner and outer layer of nucleus in k2 and k3 on day 10 observation, while k4 showed cell necrosis in outer layer of nucleus, cone cell and rod cell atrophy which resulted in unarranged of both cell structure and widened inner flexiform layer. moreover, on day 20, fish eye retina in k2 showed cell necrosis in the inner and outer layers of nucleus, while in k3 cell necrosis of inner nucleus layer and cone cell necrosis leading to widened rod cell was revealed. fish in k4 group showed cell necrosis in inner and outer nucleus layer and cone cell atrophy, furthermore, retina pigment cell started to widened (fig. 3). severe damages of the eye retina tissue were observed on day 30. k2 demonstrated inner nucleus cell necrosis, cone cell and rod cell atrophy, and widened retina pigment. fish in k3 groups showed cell necrosis in almost all layers, thus the borders between layers disappeared. severe condition was shown in k4 group, eye retina was damaged which resulted in total changes of all layers. all cells were necrosis causing the borderless condition between the layers and became one-piece tissue. in additon, hyperplasia of the cells also found in k2, k3, and k4. the highest hgcl2 concentration and the longest exposure time caused cells degeneration and lost of their function which end with cell death. this observed in day 20 and 30 observation, which cell damages were clearly seen in each component of retina layers. figure 2 histopathology of tilapia fish eye lens exposed to hgcl2 on day 30 notes: k1 = control group; k2 = 0.25 ppm hgcl2; k3 = 0.50 ppm hgcl2; k4 = 0.75 ppm hgcl2; a = eye lens; b = necrotic area; c = haemorrhage. (he 40x). k1 k2 k3 k4 mercury chloride exposure changes the histopathological figure of eye and brain of tilapia fish – dwinna aliza et al. 107 histologically, eye lens is composed of collagen produced by the epithelium cells of the eyepiece, forming a layer that resembles a layer of onions, transparent, thin, has no core, and not elastic (mumford et al. 2007). results of this study indicated that exposure to hgcl2 for 30 days has the potential to cause damage to eye tissue and high concentration of hgcl2 also show obvious histopathological damage. this is in accordance with the statement of mela et al. (2013) that the longer exposure and the higher concentration of hg can produce severe and varied forms of effects. for example, purple discoloration of fish eye lens was caused by a chronic hgcl2 exposure since the discoloration was only observed on the 30th day of observation. other lesion found was hemorrhage that occurred in the iris of fish eye due to the accumulation of hgcl2 in the blood leading to blood vessels to rupture. when hemorrhaging occurs, the intake of nutrients and other substances to the eye will stop, thus the cells will experience the lack of nutrients which eventually cause cell necrosis followed by lost in its function (pereira et al. 2013). in this study most necrosis was found in the retina layer which was marked by the loss of cell parts and appeared empty (vacuole). this is in accordance with takashima & hibiya (1995) that necrosis describes a condition where a decrease in tissue activity is characterized by the loss of several parts of the cell, one by one from one tissue, so that in a short time the cell will be dead. jeevanaraj et al. (2016) also reported that there was necrosis in retinal tissue due to chronic mercury exposure in h. malabaricus traira fish. the observations also showed hyperplasia of retina cells as a response of cell adaptation to the stimulation of toxic compounds. it was found that cellular proliferation caused the retina layer to undergo hyperplasia and caused the boundary layer components to be irregular. this can affect the function of the retina which is very sensitive to light (azab et al. 2017). similar mechanism of proliferation occurred to the brain cerebellum tissue causing the lost of layers boundaries of the tissue. atrophy occurred in the cone and rod cells of fish eye in group k2, k3, and k4 on day 10, 20 and 30. athrophy is suspected to be caused by inhibited vascularization activity caused blood intake to the eye is inadequate. cone cells and rod cells are receptor cells in the retina that are very sensitive to light. fish tend to use their vision to adapt to light when looking for food, because according to hunt et al. (2015) cone cells have the ability in terms of sensitivity to light and sharpness of vision. the damage of the cone cells or rod cells will affect the behavior of fish related to the sharpness of vision, axis of vision, and maximum visibility in fish (pereira et al. 2014). histopathology of the brain microscopic examination of the brain cerebellum of tilapia fish in control group demonstrated that the fish brain cerebellum consists of two parts namely cortex (gray matter) dan medulla (white matter). cortex consists of three layers, the outer layer called molecular layer, middle layer (ganglioner), and inner layer called granular layer (periventriculare). every layer has specific cells. satelite cell, neuroglia cell and basket cell were found in molecular layer, while purkinje cell was found in ganglioner layer, and granuler cells and golgy cells were found in granuler layer. cerebellum tissue of tilapia fish in group k1, k2, and k3 showed relatively similar figure between 10, 20, and 30 days of treatment that the molecular layer was widened, while the ganglioner layer was thinner compared to control group (fig. 4). the highest the mercury concentration was exposed the widest the molecular layer observed. the widening of molecular and granular layers of brain cerebellum was caused by cell proliferation of one layer to the others. in contrast, ganglioner layer was thinner along with the higher dosage of hgcl2 exposure. granular layer showed different patterns compared to the two previous layers. in the low dose of hgcl2 exposure the granular layer was thinner than normal tissue, however, in higher dose of exposure the granular layer became thicker. the ganglioner and granulare layer disappeared after being exposed to hgcl2 for 30 days. other than structural damage of cerebellum tissue caused by cell proliferation, cellular changes were also observed such as cloudy swelling degeneration, cell swelling, necrosis and vacuolation. biotropia vol. 29 no. 2, 2022 108 figure 3 histopathology of tilapia eye retina exposed to hgcl2 on day 30. notes: a = cell necrosis; b = cone cell atrophy; c = widened of eye retina pigment; d = widened of inner fleksiform layer. (he 400x). the tilapia fish brain cerebellum exposed to hgcl2 also showed the presence of vacuolation in all treatment groups. similar results were reported by berntssen et al. (2003) in atlantic salmon (salmo salar) fish exposed to chronic dietary mercury. vacuolysis has characteristics such as a round, hollow hole that occurs because of the accumulation of fat. the contributing factors of vacuolations are the accumulation of toxic substances, oxygen damage and excess fat consumption (chamarthi et al. 2013). vacuolysis will lead to disruption of cell metabolism process which eventually resulted in cell lysis (lakshmaiah 2017). these vacuoles are seen more in brain samples with the highest dose of mercury exposure. this is consistent with the statement of rajeshkumar et al. (2017), that the degree of pathological damage is highly dependent on the dose and duration of exposure. under conditions of higher mercury exposure the vacuole turned into expanding empty spaces. similar results are reported by lakshmaiah (2017), in catfish exposed by various concentrations of glyphosate herbicides which is neurotoxic, characterized by severe degeneration, edema and vacuole changes into large empty spaces. the vacuolation that occurred in this study might share similar effect as the herbicide since mercury is also neurotoxic. cell swelling or vacuole degeneration is reversible. thus, cell returns to normal when the exposure to toxic substances is terminated. bose et al. (2013) reported that increasing the dose of heavy metal exposure causes vacuolation in the molecular layer of cerebellum. k4 k3 k1 k2 mercury chloride exposure changes the histopathological figure of eye and brain of tilapia fish – dwinna aliza et al. 109 figure 4 histopathological feature of tilapia fish brain cerebellum notes: a-b = control group; c-d = k3 group; a = medula; b = cortex; c = molecular layer; d = ganglioner layer; e = granulare layer; f = proliferative purkinje cell; g = proliferative neuroglia cell; h = proliferative granulare cell. (he 40x, 400x). cell changes mainly occurred in neuroglia cells in the molecular layer, purkinje cells in the ganglioner layer and granulare cells in the granular layer. it is clearly seen that necrosis were observed on purkinje cells and granulare cells. the cell swelling was shown by purkinje cells as well as neuroglia cells. swelling of purkinje cells was reported by chamarthi et al. (2014) in the cerebellum of the teleost catfish induced by benzene petrochemical. the neuroglial cell swelling was reported by erhunmwunse et al. (2014) in tilapia exposed to glyphosate herbicide. conclusion hgcl2 exposure changes anatomical pathology and histopathological of tilapia fish eye and brain. the higher the hgcl2 concentration and the longest time of exposure, the most severe the changes of tilapia fish eye and brain tissues. acknowledgments the authors thank mrs. denny irmawati hasan from the department of pathology, universitas syiah kuala for her tremendous support in histopathological and staining method processes. this study was a laboratory grant project supported by the faculty of veterinary medicine, universitas syiah kuala. references azab am, shoman hm, el-deeb rm, abdelhafez hm, samei sea. 2017. comparative studies on the histology of eye retina in some nile fishes with different dial activities. ejhm 68:815-23. berntssen mhg, aatland a, handy rd. 2003. chronic dietary mercury exposure causes oxidative stress, brain lesions and altered behavior in atlantic salmon (salmo salar) parr. aquat toxicol 65(1): 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histopathological figure of eye and brain of tilapia fish – dwinna aliza et al. 111 planktonic biomass of the river ravi, pakistan. trjfas 19(10): 857-64. saturday a. 2018. mercury and its associated impacts on environment and human health: a review. j environ health sci 4(2):37-43. sobhanardakani s, hosseini sv, tayebi l. 2018. heavy metals contamination of canned fish and related health implications in iran. trjfas 18:951-7. takashima f, hibiya t. 1995. an atlas of fish histology. normal and pathological features. 2nd edition. tokyo (jp): kondansha ltd. wang lk, chen jp, hung yt, shammas nk. 2017. heavy metals in the environment. new york (us): crc press. zulaikhah, st, wahyuwibowo j, pratama aa. 2020. mercury and its effects on human health: a review of the literature. ijphs 9(2):103-14. biotropia vol. 28 no. 3,2021: 214 220 doi: 10.1 1598/btb.2021.28.3.1333 effectiveness of indigenous endomycorrhizal biofertilizer prototype o n organic salak leaves and fruits i n bali i nyoman rai i*, i i(etut suada', i wayan wiraatmajal and ni icomang alit astiari2 'stuaj program ofagroecotechnology, faculty afagriculture, uniuersitas udgana, kampzls unud bukit jimbaran, kuta selatan, badung 80361, bali, indonesia * s tuh program ofagrotechnology, faculty ofagriculture, uniuersitas warmadewa, denpasar 80235, bali, indonesia received 14 february 2020 /accepted 1 july 2020 abstract organically cultivated salak (salacca xalacca) on dry land has limited production in bali. typically, fertilization is carried out using leaf litter or other organic fertilizers because soil fertility is low for salak plantations. the present study analyzed the effectiveness of an indigenous endomycorrhizal biofertilizer on the nutrient and total carbohydrate content of salak leaves and fruits. the study used a randomized block design with nine replicates. the treatment consisted of three levels, i.e., 1. fertilization with leaf litter only, as is practiced by the farmers and as control (c); 2. fertilization with indigenous endomycorrhizae biofertilizer prototype (p); and 3. combined fertilization with leaf litter and indigenous endomycorrhizae biofertilizer prototype (pm). spores of indigenous endomycorrhizae from salak plantations were used for making a biofertilizer prototype. the results showed that p and cp treatments provided beneficial results such as: 1. significantly increased the chlorophyll, relative water content of leaves, as well as the number and weight of fruits per tree; 2. improved fruit quality by increasing sweetness and weight per fruit; and 3. had a positive effect on water uptake and nutrient absorption as indicated by high n and p of leaf tissue and high carbohydrate content of leaves. keywords: biofertilizer, endomycorrhizae, organic, prototype, salacca xalacca introduction bali province is a major tourist destination in indonesia. increasing the number of domestic and international tourists leads to an increased demand for fruits, including organic salak fruits. salak cultivation in bali has been carried out organically since the 1500s and is hugely beneficial to the farmers; however, the quantity, quality, and continuity of organic salak fruit produced is low (rai e t al. 2014). this low productivity can be attributed to the nature-dependency of the farmers, who have not yet implemented adequate agricultural practices. typically, fertilization only uses leaf litter, while irrigation relies only on rainfall. thus, the ferulization of organic salak renders low soil fertility thereby decreasing the plant productivity *corresponding author, email: rainyoman@unud.ac.id over time (rai e t al. 201 4). rai e t al (201 0) found that the nutrient content of n, p, and i< in the leaf tissue of organic salak in bali was low; also, the soil fertility was low as indicated by the levels of c-organic and the content of n, p, and k in the soils. in order to maintain the soil fertility and environmental sustainability for high organic salak productivity, the cultivation should be carried out with indgenous endomycorrhizae biofertilizer based on the fact that the diversity of indigenous endomycorrhizae fungal species in nature is very large paslam e t al. 201 1; proborini 2013; suamba e t al 2014; sarah & ibrar 2016; invam 20 1 7). igm e t al. (2017) established a close correlation between soil, plants, and endomycorrhizae. indigenous endomycorrhizae isolated from plants in certain locations, will be more effective if applied directly to the target plant concerned where it is taken as compared effectiveness of indigenous endomycorrhizal biofertilizer prototype on organic salak leaves rai et all to the endomycorrhizae brought from outside (non-indigenous endomycorrhizae) . the arbuscular mycorrhzal symbiosis between plants and fungi exerts a positive impact on improving the soil structure, expanding the water absorption areas, increasing the plant tolerance to biotic and abiotic stresses, increasing the nutrient uptake, and increasing the plant growth and yield (nikhat 2014; olagunju et al. 2014; soka & ritchie 2016). in addition, endomychorrizae increases plant tolerance to various biotic and abiotic stresses, including alkalinity and metal toxicity (abbasi et al. 2015; bitterlich et al. 2018). the endomycorrhizae fungus whch is also called arbuscular mycorrhizae is beneficial to the host plants as it plays a role in increasing nutrient absorption through the endomycorrhizal structures that form large root surface areas so that the plant roots can absorb the nutrients (baslam et al. 2011; sasvari 2012; brundrett & tedersoo 2018). endomycorrhizae plays a role in increasing plant resistance to root pathogen attack through antibiotics produced during symbiosis with plant roots that weaken and also kill the pathogenic bacteria, viruses, and fungi (brundrett 2017; sasvari, 2012). furthermore, the diversity and activity of indigenous endomycorrhizae in plant roots are mainly determined by the type of the host plant (ningsih et al. 2013; sadhana 2014), environmental growth factors, such as climate and soil moisture/drought (hernadi 2012; quiroga et al. 2017; mathimaran et al. 2017; i<ehri et al. 2018), and the level of soil fertility (tahat & sijam 2012; kavitha & nelson 2013; mo et al. 2016). rai et al. (2019a) explored and identified morphologically and genetically the indigenous endomycorrhizae from salak root areas in three regencies in bali: icarangasem, gianyar, and tabanan. in our study, two genera of indigenous endomycorrhizae in salak were identified i.e., glomzls and entrophospora. the genus glomzls consisted of three species (glomzls cabens, glomzls czlstos, and glomzls indicum) , while genus entrophospora consisted of only one species (entropbospora-sp-sh 197095.06fu). after propagation, the spores of these species were formulated into prototypes of biological ferulizers/bioferulizer with carrier medla zeolite, quartz sand, sea sand, and volcanic sand. the most effective method to be applied in salak seedling is a prototype composed of a mixture of 100 spores from glomzls species in 500 g volcanic sand carrier media. this phenomenon was shown by the optimal growth of salak seedling and the maximum (100%) colonization of endomycorrhizae at the roots (rai et al, 2019b). positive test results on salak seedling growth need to be followed up by evaluating the efficiency of the prototype of indigenous endomycorrhizae biofertilizer on organic salak plantations in the field. thus, the present study aimed to determine the effect of prototype indigenous endomycorrhizae biofertilizers on organic salak production and its effect on the nutrient and carbohydrate content of the leaves. materiaj3 and methods the study was conducted from february to december 2019 in organic salak farms owned by farmers in sibetan village, bebandem district, icarangasem regency. the study used 15-year old salak trees and a randomized block design with nine replicates. three levels of fertilization were applied as follows: 1. fertilized with leaf litter only, as is practiced by the farmers and as control (c); 2. fertilized with prototype indigenous endomycorrhizae biofertilizer 0; and 3. combination of leaf litter and prototype indigenous endomycorrhizae bioferthzer (cp) . therefore, 27 salak trees with a total plot size of 216 m square were needed. the spores of indigenous endomycorrhizae were taken from salak roots in sibetan village. the isolation of the spores was carried out using the wet filtering technique, followed by centrifugation as described by brundrett (2017). these spores were propagated according to the method described by rai et al. (201 8), using corn as a host plant with water stress treatments. topping or shoot cutting was carried out at the age of 4 weeks. watering to field capacity was carried out from the beginning of seed planting to 1 week of age, followed by watering to 50% of field capacity until 4 weeks of age. after topping at the end of the 4th week, watering was stopped so that the plant experienced stress. then, the plant was dismantled to harvest the spores from the multiplication at the end of the 5th week. subsequently, the resulting spores were placed on volcanic sand: 100 spores/500 g sand. biotropia vol. 28 no. 3,2021 fertilization was conducted by making a hole, 30 cm wide and 20 cm deep, around the tree at a distance of 40 cm from the base of the tree. the salak trees in treatment c were fertilized with 4 kg leaf litter of salak/tree. the leaves were cut into 20-cm-long pieces, placed in a hole, and covered with soil. on the other hand, the trees in the p treatment were fertilized with prototype indigenous endomycorrhizae made from a mixture of 100 spores of three glomas species (glomas cabense, glomtls czlstos, and glomas indicum) in 500 g volcanic sand carrier media per plant. the prototype of the biofertilizer was evenly spread around the tree and covered with soil. interestingly, the cp treatment was carried out by spreading a prototype of the biofertilizer made from a mixture of 100 spores of three glomzts species in 500 g of volcanic sand carrier media per plant, followed by spreading the salak leaf litter on it evenly around the tree. the chlorophyll content and the relative water content (rwc) of the leaves, the percentage of fruit set, the number and weight of fruits per tree, weight per fruit, fruit diameter, fruit sweetness level, n and p content of the leaves, and total sugar, reducing sugar (r-sugar) and sucrose content of the leaves, and root colonization by indigenous endomycorrhizae were recorded. the colonization of roots by indigenous endomycorrhizae was observed by the slide method of giovannetti and mosse (1980) using the following formula: percentage of colonized roots = the number of colonized roots/the total number of roots observed x 100%. the percentage of fruit set was calculated as follows: the number of flowers developed into fruits/the number of flowers x 100%. the leaf chlorophyll content measured three times by chlorophyll meter spad-502 in april, june, and august, and the average was calculated. the total sugar was analyzed by anthrone method, r sugar by nelson-somogyi method, and the sucrose content was calculated by subtracting the value of total sugar content from that of r sugar, and multiplied by 0.95. the rwc was measured from matured leaves. after being cut from the tree, the mature leaves were then immediately wrapped in aluminium foil, stored in an icebox, and transported to the laboratory for further analysis. thirty pieces of leaf samples (10 pieces from the tip of the leaf, 10 pieces from the middle, and 10 pieces from the bottom), with a diameter of 1 cm, were taken from leaf sheaths using a round punch and weighed ( w i ) . these leaf samples were immersed in water and irradiated with 40 w fluorescent light at room temperature for 5 h. then, each piece of leaf sample was carefully dried with paper towel, and weighed (wz), followed by oven-drying at 70 oc for 24 h before being estimated (w3) . the value of rwc was calculated by the formula: p ~ w z ) / (w3-wz) x 100%. the nutrient content of n and p leaves was analyzed by kjeldahl method and the p-availability was analyzed by olsen method. the number and weight of fruits per tree were calculated cumulatively at the end of the study. moreover, the weight of each fruit was calculated as follows: total weight of fruit per tree/the number of fruits per tree. these data were analyzed using analysis of variance with spss (statistical product and service solution) software version 26. if the f test showed a significant difference, the least significant difference (lsd) test was performed to differentiate the average value among tretaments. results a n d discussion the results of the analysis of variance (anova) showed that the fertihzation treatment significantly affected all the observed variables. the weight and number of fruits per tree (790.80 g and 16.81 fruits, respectively) in the treatment of indigenous endomycorrhizae biofertilizer prototype (100 spores/500 g volcanic sand carrier me&a) were sipficantly hgher than those fertilized with leaf litter/control (653.81 g and 14.94 fruits, respectively), but were not significantly different from cp (790.14 g and 16.56 fruits, respectively) (table 1). the quality of salak fruit in the p and cp treatments was significantly improved as compared to that in treatment c, as shown by the increased sweetness of the fruit (table 2) and the weight per fruit (table 1). the high number and weight of fruit per tree, as well as the increased fruit quality due to the administration of indigenous endomycorrhizae biofertilizer prototype, elevated the n and p content of the leaves as compared to the control (table 2); also, the chlorophyll content and rwc of the leaves were increased (table 1). visually, the trees treated with c and cp treatments had dark green and fresh leaves as compared to the control. effectiveness of indigenous endomycorrhizal biofertilizer prototype on organic salak leaves rai e t al: table 1 effect of prototype indigenous endomycorrhizae biofertilizers on the leaf chlorophyll content, rwc of leaves, number and weight of fmits per tree, weight per fruit, and fruit sweetness in salak plants leaves chlorophyll leaves relative number of weight of weight per fruit treatments content water content fruit per tree fruit per tree fruit sweetness (spad) ("0) (unit) 0 0 (% brix) lsd 5% 0.67 0.98 1.59 96.34 7.27 0.51 note: numbers followed by the same letter in the same column did not differ significantly in the lsd 5% level. higher production and yield quality in p and cp treatments as compared to control (c) were related to hgh leaf chlorophyll content (table 1) and the ability of salak tree to absorb nutrients and water (table 2). the high p and n content in the leaves resulted from the p and cp treatments was significantly associated with root colonization as compared to the c treatment. table 2 displayed that root colonization by indigenous endomycorrhizae in the p and cp treatments was 100%, but only 23.14% in treatment c. the high root colonization in p and cp revealed a mutualism symbiosis between indigenous endomycorrhizae and salak trees, which in turn, increased the p and n content and the rwc in the leaves. the endomycorrhizae infects the roots of plants but does not cause injury, whch in turn causing mutual reciprocal processes. host plants obtain nutrients from endomycorrhizae, while endomycorrhizae obtain carbohydrates or food from host plants (hernadi e t al. 2012; zasvari et al. 2012). h~gh nutrient content and rwc in the leaves resulted from the p and cp treatments increased the chlorophyll content, which improved the photosynthesis as indicated by elevated total sugar, r-sugar, and sucrose content in the leaves resulted from the p and cp treatments as compared to that in c (table 2). the present study showed that indigenous endomycorrhizae fungus isolated from root of salak trees is suitable to be used as a biofertihzer. the administration of indigenous endomycorrhizae resulted in the absorption of nutrients and water through the endomycorrhizae structure that enlarges the surface area of salak root, so that the plant roots can absorb the nutrients and water (baslam e t al. 201 1; zasvari 2012; brundrett & tedersoo 2018, bitterlich e t al. 201 8). spore density and root colonization of host plants are largely determined by the compatibility of endomycorrhizae with host plants, environmental factors, and interactions between endomycorrhizae and chemical compounds produced by host plants (beltrano e t al. 2013; sarah & ibrar 2016). thus, a correlation between indigenous endomycorrhizae biofertilizers prototype and salak trees can be suspected, which increases the yield, the quality of the yield, and the physiological processes of salak trees. the results of this efficiency test are in accordance with those of previous studies wherein endomycorrhizal fungi biofertilizer can increase the growth, production and quality of pineapple yield (nurhandayani e t al. 2013), chill (tanwar e t al. 2013), teak (proborini, 2013), tea (nepaleon e t al. 2012) and apple (fediala e t al. 2018). endomycorrhizae plays a role in increasing plant resistance to drought or lack of water in the dry season because the root of the plants possess mycelium that can reach water in the wider rhizosphere area of the soil and adsorb water despite h t e d availabihty (sasvari 2012). furthermore, it is involved in producing various growth regulators such as auxins, cytokmins, and gibberellins and vitamins that can increase the growth of plant organs and roots that do not rapidly age and hence can function effectively in the absorption of nutrients and other solutes (baslam e t al. 201 1; tanwar e t al 2013). the endomycorrhizae also improve the soil structure because the mycelium on the outside of the roots of plants (covering soil grains) produces polysaccharide gels that increase the stability of soil aggregates (io-uger 2011; sasvari 2012; sadhana 201 4; jansa e t al. 201 6; i<lm e t al. 201 7). biotropia vol. 28 no. 3,2021 table 2 effect of prototype indigenous endomycorrhizae biofertilizer application on the n and p nutrient, total sugar, r-sugar, and sucrose content of the leaves and root colonization on salak plants total sugar r-sugar n content p content of of sucrose content root content of treatments of leaves leaves of leaves colonization leaves ("4 ("/o) leaves ("10) ("10) ("10) lsd 5% 0.03 0.01 0.51 0.22 0.79 3.16 note: numbers followed by the same letter in the same column did not differ significantly in the lsd 5% level. although c was not given indigenous endomycorrhizae bioferulizer, root colonization occurred albeit with significantly lower intention as compared to that in p and cp treatments. the data showed that indigenous endomycorrhizae were naturally present in salak root area; however, for a positive influence on the production and quality of salak, the population needed to be increased by giving indgenous endomycorrhizae biofertihzer for the optimal production of the plants. the indigenous endomycorrhizal fungi, also known as arbuscular mycorrhizal fungi, in nature is diversified (baslam e t al. 2011; proborini 2013; suamba e t al. 2014), with a positive effect on the salak trees (juliadewi e t a/. 2014). indigenous endomycorrhizae is naturally associated with plant roots without human intervention and has a high potential for extensive colonization because it recognizes the host plant and has a higher tolerance to environmental conditions, such as high stress (sadhana 2014; quiroga e t al. 2017). based on these characteristics, it can be speculated that indigenous endomycorrhizae isolated from salak roots has great potential to be developed as biofertilizer because it adapts to host plants to be fertilized (sasvari 2012; sadhana 2014; hohmann & messmer 2017). conclusions acknowledgements our gratitude goes to universitas udayana for financial support through udayana invention grants in 2019. the authors also thank the directorate general of higher education, ministry of research, technology and higher education for financial support through the higher education applied grants 2018. references abbasi, hisamuddin, akhtar h, sharf r. 2015. vesicular arbuscular mycorrhizal (vw fungi: a tool for sustainable agriculture. amer j plant nutr fert techno 5(2):40-9. baslam m, garmendia i, goicoechea n. 2011. arbuscular mycorrhizal fungi (amf) improved growth and nutritional quality of greenhouse grown 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gupta a. 2013. arbuscular mycorrhizal inoculation and super phosphate application influence plant growth and yield of capsicum annuum. j soil sci plant nutr 13(1):55-66. biotropia vol. 29 no. 3, 2022: 244 253 doi: 10.11598/btb.2022.29.3.1711 244 role of mycorrhiza helper bacteria on mycorrhizal colonization and nematode pratylenchus coffeae infection reginawanti hindersah1*, elena f. l. lilipaly2, imam mudakir2, iis nur asyiah2 and rita harni3 1faculty of agriculture, universitas padjadjaran, sumedang 45363, indonesia 2biological education study program, faculty of teacher training and education, universitas jember, jember 68121, indonesia 3indonesian centre for estate crop research and development, sukabumi 43357, indonesia received 21 december 2021/accepted 3 august 2022 abstract the coffee nursery is susceptible to endoparasitic pratylenchus coffeae. application of biological method in the nursery is suggested to control the nematode population and maintain the seedling health. the objectives of this study were to observe the ability of arbuscular mycorrhiza fungi (amf) glomus spp. and liquid inoculant of mycorrhiza helper bacteria (mhb) consortium pseudomonas diminuta and bacillus subtilis for increasing amf colonization and reducing the infection p. coffeae in arabica coffee seedling and their growth. a pot experiment was conducted using a completely randomized block design with four treatments and five replications. the treatments were glomus spp. spore inoculation without and with two concentrations of mhb. the control treatment did not receive glomus spp. and mhb. the seedlings were growing in the greenhouse for three months. the results indicated that glomus spp. and mhb consortium significantly reduced the nematode total number in soil and roots by approximately 30%; and infection degree of p. coffeae by 50%. the application of glomus spp. significantly increased root colonization by mycorrhizal fungi, but mhb inoculation did not affect the mycorrhizal colonization. seedlings treated with mhb had higher shoot length compared to the plant without mhb and control; but the leaves number and shoot dry weight of seedlings were not affected by all treatments. even though the root fresh weight was reduced after mhb treatment, the lateral roots growth of mhb-treated seedling visually was improved. the experiment demonstrated that mhb was efficient to reduce p. coffeae infection of arabica seedling. keywords: glomus spp., infection degree, nematode population, root colonization, seedling growth introduction arabica coffee is the valuable agriculture commodity in global market. brazil is still the first coffee producer worldwide while indonesia is the 4th largest producer and exporters of coffee beans. in 2020, the coffee beans production in indonesia was 12.0 million of 60-kg bags. in growing coffee many challenges occur and have to be dealt with endoparasitic pratylenchus coffeae which well adapted to warm tropical and subtropical agroecosystem (thiep et al. 2018; tarno et al. 2021). pratylenchus are root lesion nematode; mainly infecting the cortical parenchyma which inhibit the water and nutrients absorption and thereby, cause serious root damage and limit plant growth (yu et al. 2012). in general, this problem was resolved by applying chemical and soil disinfectant as well as shading. nowadays, the use of arbuscular mycorrhizal fungi (amf) is suggested in order to reduce the harmful effect of chemicals on the agricultural environment. the amf is well known as biofertilizer, which inoculation become a prominent way to save phosphorous fertilizer and increase coffee tree growth in tropical agroecosystem (sewnet & tuju 2013; hernández-acosta et al. 2018). to date, researcher have reported that other advantage of mycorrhizal formation in roots is to reduce nematodes in coffee tree seedlings as *corresponding author, email: reginawanti@unpad.ac.id role of mycorrhiza helper bacteria on mycorrhizal colonization – hindersah et al. 245 well as replanted coffee fields (asyiah et al. 2015; pham et al. 2020). moreover, biocontrol strategy using amf may protect the plant form nematode pathogen and induce plant resistance against this pathogen (veresoglou & rillig 2012; poveda et al. 2020). among the amf species, the natural occurrence of glomus spp. in soil and rhizosphere of coffee tree are reported (prates júnior et al. 2019). the effect of amf on coffee growth were recorded. introducing amf on coffee nursery resulted in increment of height, foliar area and root volume arabica coffee seedling (hernandez-acosta et al. 2018). coffee tree inoculated by amf spores combined with chemical fertilizer showed better mycorrhizal colonization and tree growth (daras et al. 2015). certain soil bacteria have been reported to develop positive interaction with amf, which bacteria are known as mycorrhiza helper bacteria (frey-klett et al. 2007). the bacteria increase fungal spore germination and mycelium growth in soil and root-mycelium recognition (deveau & labbé 2016). the ability of gramnegative proteobacteria pseudomonas and a grampositive bacillus to help mycorrhizal establishment has been reviewed (frey-klett et al. 2007; labbé et al. 2014). both bacteria are well known plant growth promoting rhizobacteria (pgpr) which induce plant growth by the mechanisms of phytohormones production, biocontrol of soilborne diseases, arbuscular mycorrhiza stimulation and acquired systemic resistance induction (jankiewicz & kołtonowicz 2012; patel & saraf 2017; hashem et al. 2019). we have developed mycorrhiza helper bacteria (mhb) liquid inoculant of bacillus as well as pseudomonas. in previous research, single application of b. subtilis and p. diminuta suppressed the population of nematode p. coffeae in coffee seedlings by 71.3% and 64.2%, respectively compared to the untreated seedlings (asyiah et al. 2015). nonetheless, the ability of mixed cultures of both bacteria to induce mycorrhizal formation and repress p. coffeae infection have not been studied. controlling coffee pest in the nursery is an important step to enhance tree growth and coffee production in the field. improving the quality of coffee seedlings is essential to support the sustainability of coffee plantation. the objectives of this study were to determine the ability of mycorrhiza glomus spp. and the liquid formula of mhb consortium to enhance amf colonization and reduce p. coffeae infection, as well as to observe the effect of glomus spp and liquid formula of mhb consortium on the growth of coffee seedlings. materials and methods the experiment was carried out in the greenhouse of coffee and cocoa research institute (ccri) at jember, east java province, indonesia which is located in the tropical area at 60 m asl. the range of annual temperature and humidity in year 2019 were 23.0 29.9 oc and 67 86%, respectively. preparation of mhb and p. coffeae inoculants was conducted at the microbiology laboratory of universitas jember. the spores of glomus spp. (zygomycetous fungi) were provided by laboratory of mycology at the faculty of agriculture, universitas gadjah mada, while the arabica coffee seedlings were obtained from ccri. table 1 major essential nutrient composition of soil before experiment parameters methodsa value phh2o potentiometry 5.6 organic c walkey and black 2.39% total n kjeldahl 0.24% c to n ratio calculation 9.95 available p spectrophotometry 14.65 mg/kg available k spectrophotometry 79.82 mg/kg note: a = based on aoac proximate analysis method (2012). the top soil of inceptisols with loam texture (prastowo et al. 2013) was collected from the agricultural field at ccri. the soil properties showed that the soil was average in organic carbon (c) and total nitrogen (n), low in available phosphor (p) and high in potassium (k) (table 1). the c to n ratio of soil was low indicating the nitrogen was available for plants. preparation of glomus spp. and mhb inoculants arbuscular mycorrhizal fungi propagation was carried out in zeolite-based media with corn as host plant. a 150-g sterilized zeolite were poured into 250-ml transparent cup and biotropia vol. 29 no. 3, 2022 246 saturated with 0.5% nacl solution. then 100 glomus spp. spores were spread evenly on the surface of zeolite prior to sow two corn seeds and covered with 50 g of zeolite. substrate humidity was maintained by adding 30 ml water per day. the first fertilization was carried out a week after sowing by applying 30 ml of liquid mixed fertilizer at a concentration of 1 g/l; similar fertilization was done twice a week. two months later watering was reduced gradually in order to induce spore formation. the corn plants were eliminated and the growth substrates containing glomus spp. spores were used as amf inoculant. the production of bacterial liquid inoculant was carried out using molasses-based broth. molasses containing 19.58 c/n ratio, 0.18% p2o5, and 0.39% k2o was obtained from probolinggo sugar factory. each mhb species was grown separately in 2% molasses-based broth for 2 days at a temperature of 30 oc, then two volumes of b. subtilis were mixed with three volumes of p. diminuta. the liquid bacterial consortium was then put on a shaker at room temperature for three days. the final concentration of mixed mhb liquid inoculant was 109 colony forming unit (cfu)/ml. experimental design and set up the pot experiment was conducted in completely randomized block design composed of four treatments and six replications. the arabica coffee seedlings were treated with glomus spp. inoculant without mhb consortium (mhb-0) and with 108 cfu/ml mhb inoculant (mhb-1) and 109 cfu/ml mhb inoculant (mhb-2). control plants received neither glomus spp. nor mhb inoculant. the mature p. coffeae was applied to all treatments. plant substrate consisting of air-dried soil, sand and commercial compost in balanced composition was sterilized at 115 oc for 30 min and placed overnight at room temperature. as much as 5 kg plant substrate was put into opaque polyethylene pot with 5 drainage holes on its bottom and stored at the greenhouse. an 8-cm depth planting hole with 5 cm diameter was made in each pot prior to growing 3-month old coffee seedlings. the average shoot height and leaves number of seedlings at planting time were 11.2±0.08 cm and 4.2±0.05, respectively. soon after transplanting, 1 g of urea fertilizer was applied into 2-cm depth hole with a distance of 5 cm away from the stem seedlings. then, the holes were covered with the growth media. a week after transplanting, 50 mature p. coffeae was spread on 4-cm depth circular band with a distance of 5 cm from plant stem and then covered with thin layer of substrate. subsequently, 9.1 g glomus spp. inoculant containing 100 spores were spread over similar circular band and covered with substrates. inoculation of mixed mhb were carried out by pouring the liquid inoculant evenly around the seedling stem. the mhb-treated plants were inoculated with 1 ml of bacterial inoculant which was diluted in 99 ml distilled water, while control plants were watered with 100 ml distilled water. all seedlings on potted soil were grown in the greenhouse for 3 months. parameters measurement and statistical analysis plant height and leaves number were measured once a month until 3 months after treatment. fresh weight of shoot and root as well as shoot dry weight were measured at the end of the experiment. the root dry weight was not analyzed since the roots were used for nematode enumeration. the shoot dry weight was determined after being oven-dried at 70 oc for two days. after 3 months of treatment, the number of nematodes in root and soil were counted by extracting the nematodes from 10 g of soil sample and 100 ml of root extract following the method of baermann funnel (van bezooijen 2006). the soil and roots extracts samples were passed through a different set of filters with an opening size of 40 mesh and followed by the 325 mesh before counting the nematodes under a light microscope at 100x magnification. determination of root colonization degree (rc) by amf was carried out following the method of kormanik dan mc graw (1982). the roots were cut into 2-cm length root segments and subsequently soaked in 2% koh at 80 oc, followed by staining the roots with acid fuchsin. a total of 10 stained root segments were placed in object glass and covered with cover glass. the cover glass then pressed manually for flatting the roots and removing excess liquid. the number of infected roots was observed using a light microscope at 400 x magnification. role of mycorrhiza helper bacteria on mycorrhizal colonization – hindersah et al. 247 the infected roots were determined by the hypha, vesicle and arbuscule occurrence in root segments. the rc (%) was calculated by dividing the number of infected root segments by number of observed root segments. at the end of experiment, the infection degree (id) for estimating disease severity due to p. coffeae was calculated using a scale of five classes (table 2) by using the townsendheuberger formula described by scalzo et al. (2012): id (%) = σ1i (ni.vi) ............................................ (1) (n.v) where: vi = infection class; ni = number of seedlings in one class; n = total seedling number; v = the highest class; i = the number of classes. table 2 the classes for scaling p. coffeae infection based on leaves and roots conditions class description 0 healthy seedling, the roots are normal without color alteration; leaves are green without yellow spots. 1 less than half leaves had a yellow spot but the leaves number was similar to healthy seedling; roots had altered their color to yellow and brown. 2 at least half of leaves number showed yellowing symptom and seedlings had some fallen leaves; roots are not healthy with rottenlateral roots. 3 the seedling is dominated by yellow leaves and the number of intact leaves were less than half of healthy seedling; their root system was not fully developed and rotted. 4 seedling had 1-2 yellow leaves and their roots were badly damaged indicated by brown in color and rotten-lateral roots. all data were subjected to analysis of variance (p ≤ 0.05). if the effect of treatment on the parameter was significant then the least significant difference (lsd) test was carried out at p ≤ 0.05. statistical analysis was performed using spss 16 statistical program. results and discussion root colonization and infection degree without glomus spp. inoculation, the roots did not show any root colonization (rc) by amf (fig. 1a), while inoculation of glomus spp. resulted in rc higher than 80%. the result verified that seedlings without mhb showed similar rc compared to seedlings treated with lower concentration of mhb inoculant (mhb-1). furthermore, seedlings inoculated with higher concentration of mhb (mhb-2) caused significant rc reduction up to 12.4%. low nematode infection degree (id) was demonstrated by seedlings grown with the application of glomus spp. inoculation compared to control (fig. 1b). the application of mhb combined with glomus spp. inoculation enabled to decrease id up to 50% compared to the control. seedlings inoculated with mhb containing 108 cfu/ml (mhb-1) demonstrated lower id even though it was not significantly different from the results showed by the inoculation of mhb-2 (109 cfu/ml) based on lsd test. microscopic observation revealed that nematode p. coffeae infected roots of arabica coffee at 3 months after treatment (fig. 2). moreover, mycorrhizal colonization in the root cell was clearly showed by arbuscular and vesicular formations. figure 1 effect of mycorrhiza helper bacteria consortium on (a) root colonization by amf and (b) infection degree caused by p. coffeae on coffee seedlings treated with glomus spp. at 3 months after treatment note: control = without glomus spp. and mhb inoculations. c c a b a a 120 100 80 60 40 20 0 120 100 80 60 40 20 0 c b b a biotropia vol. 29 no. 3, 2022 248 figure 2 mature nematode in coffee root cell of coffee seedling infected by (a) p. coffeae and (b) amf colonization resulted in vesicular formation in the root cells note: photo source: elena f.l.lilipaly, using olympus digital camera. the glomus spp. inoculation with and without mhb lowered the nematode population compared to the control even though the differences were not statistically significant (table 3). regardless of statistical analysis, lower nematode population in roots was shown by seedlings with glomus spp. and mhb. the lowest nematode population in soil was shown by seedlings treated with low concentration of mhb inoculant even though this decline was not statistically different based on lsd test. in general, the significant reduction of total nematode count was performed by seedlings treated with 108 cfu/ml and 109 cfu/ml of mhb liquid inoculants. results of this experiment found that glomus spp. inoculation with or without mhb increased root colonization by amf. the soil was sterilized before experiment and considered free from indigenous amf. therefore, only exogenous glomus spp. colonized the roots. this result verified that mhb did not induce mycorrhizal formation in roots and mhb slightly reduced root infection. microscopic observation showed that the presence of external hyphae, arbuscular and vesicular were only detected in glomus-treated roots. the lower rc in mhb-treated seedling shown in this experiment disagrees with the induction of amf spore germination, mycelium growth, and rootmycelium recognition proposed by deveau & labbé (2016). the failure of mbh to increase rc might be related to the inability of mhb to perform those roles. the arabica coffee seedlings were responsive to glomus spp. inoculation which relates to soil acidity and available p in soil. before experiment the soil acidity was 5.6. in general, soil fungi showed optimal mycelial growth at ph of 4.5 to 5.5. certain glomus spp. species produce the highest number of spores in soil ph of 6.5 (costa et al. 2013). the acidic and slightly acidic soil condition was suitable for the growth of glomus spp. and hence, their colonization in roots (rohyadi et al. 2004). table 3 effect of mycorrhiza glomus spp. and mycorrhiza helper bacteria consortium on p. coffeae population in roots and soils at 3 months after treatment treatments nematode number nematode total reduction (%)a root soil total control 221±29.6a 214±54.1a 435±72.0a glomus spp. without mhb 281±60.8a 104.4±4.4a 385.4±66.0a 11.4 glomus spp. + mhb-1 200.6±77.5a 74.4±12.5a 275±77.6a 36.7 glomus spp. + mhb-2 181.8±27.9a 112.2±18.1a 294.2±37.6a 32.4 note: values followed by the same letter in the same column are not significantly different according to lsd test at p ≤ 0.05. role of mycorrhiza helper bacteria on mycorrhizal colonization – hindersah et al. 249 amf colonization in roots treated with glomus spp. is promoted by low available p content in soil. before experiment, the soil contained as low as 14.65 mg/kg of available p. the growth media composed of soil-sandcompost without inorganic p fertilizer. high p in soil and high rate of p fertilizer can inhibit am colonization (linderman & davis 2004). in contrast, adding only moderate inorganic p fertilizer enhanced the root colonization and diversity of amf communities (higo et al. 2020). the experiment found that inoculation of glomus spp. combined with mhb resulted in the significant reduction of nematode population up to 32 36% and id by 43 56%. both mhb produce phytohormone that play an essential role to activate the mechanisms of plant defence response against unfavourable condition (egamberdieva et al., 2014). chitinase is probably produced by mhb pseudomonas and bacillus as described by some researchers (zhong et al. 2015; amar et al. 2016). this chemical substance might limit nematode pathogen development. in the root knot of nematode, chitinase produced by paenibacillus illinoisensis was capable to lysis nematode eggshell and inhibit egg hatching (jung et al. 2002). however, the mechanism of chitinase to repress p. coffeae infection is remained unclear. increased root colonization by amf in glomus-treated seedlings might be related to low id of p. coffeae. mycorrhiza can suppress the population of p. coffeae through symbiont competition on space, carbon and nitrogen (bell et al. 2021) and induce the lignification of root endodermic cells that possibly decreased nematode pathogen infection (linderman 1994; elsen et al. 2001). the decrease of nematode population in our experiment agrees with the 68% of pratylenchus population reduction in soil of apple seedling after glomus intraradices inoculation (ceustermans et al. 2018). plant growth glomus spp. inoculation with or without any concentration of mhb liquid inoculant did not affect plant height and number of leaves at one and two weeks based on lsd test (fig. 3a & 3b). the application of glomus spp. and mhb enhanced plant height at 3 months after treatment, with decreasing number of leaves compared to control plants. this experiment showed that mixed inoculation of glomus spp. and mhb significantly increased plant height up to 22% compared to seedlings inoculated with glomus spp. without mhb (fig. 3a). the highest number of leaves was demonstrated by seedlings inoculated by glomus spp. without mhb (fig. 3b). the fresh and dry weight of shoots had not altered due to mhb inoculation (table 4). in contrast, growing coffee seedlings with glomus spp. but without mhb inoculation resulted in higher root weight over mhb-treated seedlings. inoculation of mhb clearly increased plant height at 3 months after treatment (fig. 3) but their dry weight was not different. figure 3 effect of mycorrhiza helper bacteria inoculation on plant height and number of leaves of coffee seedlings treated with glomus spp. at 1, 2 and 3 months after treatment note: control = without glomus spp. and mhb inoculations. a b a a a b b a b a 30 25 20 15 10 5 0 16 14 12 10 8 6 4 2 0 biotropia vol. 29 no. 3, 2022 250 table 4 effect of mycorrhiza glomus spp. and mycorrhiza helper bacteria inoculation on weight of shoot and root of coffee seedlings at 3 months after treatment treatments shoot weight (g) root fresh weighta dry weight fresh weight control 0.44 ± 0.25a 1.60 ± 1.09a 0.43 ± 0.28b glomus + without mhb 0.49 ± 0.26a 1.93 ± 1.11a 0.36 ± 0.26b glomus + mhb-1 0.44 ± 0.05a 1.64 ± 0.60a 0.11 ± 0.04a glomus + mhb-2 0.42 ± 0.08a 1.49 ± 0.36a 0.14 ± 0.08a note: values followed by the same letter in the same column are not significantly different according to lsd test at p ≤ 0.05. based on visual observation, the performance of seedlings treated with glomus spp. was better than that of the control plant (fig. 4). despite lower root fresh weight, seedlings inoculated with glomus spp. combined with mhb liquid in general had more rigorous lateral roots than plants without being inoculated with glomus spp. and mhb (fig. 5). moreover, the tap root of control seedlings and seedlings without mhb are likely thicker than roots of seedlings inoculated with mbh. significant increase in shoot height was observed in the seedlings inoculated with glomus spp. combined with mhb (b. subtilis and p. diminuta) at 3 months after treatment. shoot elongation is determined by several growth factors including the regulation of cell division and enlargement that affected by sufficient nutrient and plant growth substances. in the photosynthate partitioning, the stems are the sink that demands the energy (sugar) to elongate, while leaves are the source that produces the starch through photosynthesis. the sugar allocation from the leaves and essential ion taken by the roots were not suffice for shoot development. a plant growth hormone called auxin influences and integrates in plant growth (sachs 2005). figure 4 shoot performance of arabica coffee after being infected by p. coffeae and inoculation by glomus spp. and mhb note: control = without glomus spp. and mhb inoculations. figure 5 root performance of arabica coffee after being infected by p. coffeae and inoculated with glomus spp. and mhb note: control = without glomus spp. and mhb inoculations. role of mycorrhiza helper bacteria on mycorrhizal colonization – hindersah et al. 251 plants produce auxin in the shoot tissues and has an essential role in initiating root growth and inhibiting primary root development (sachs 2005; alarcón et al. 2019). visual observation showed better growth of the seedlings’ lateral roots growth inoculated with glomus spp. combined with mhb compared to roots without glomus spp. and mhb inoculations. (fig. 3). mhb liquid inoculant of p. diminuta and b. subtilis consortium contained 4.01 mg/l of iaa, a group of auxin hormone that is capable of improving lateral root growth and subsequently facilitating better nutrient uptake and shoot growth. the results of this experiment agree with a study showing an increase in cucumber rooting after the application of low concentrations of exogenous auxin (balliu & sallaku 2017). the intensive growth of lateral roots in mhb-treated seedlings might cause low fresh weight of root because the primary roots become thin. in relation to root colonization by glomus spp., better root growth provides more physical contact establishments between fungi and roots. the mycorrhizal fungi are obligate symbiont characterized by forming arbuscular and/or vesicular in root cells. both particular and functional structure are found in glomus-treated coffee seedlings. this experiment found that glomus spp. are compatible with coffee seedlings as host plant and possibly build a positive interaction with the community of indigenous amf. both pseudomonas and bacillus in this experiment are not endophytic bacteria, so they do not compete with amf for infection sites. the precise mechanisms by which p. diminuta and b. subtilis decrease the infection degree of p. coffeae and repress nematode population in the presence of amf have to be studied. conclusion mixed liquid formula of mycorrhiza helper bacteria (mhb) consisted of p. diminuta and b. subtilis combined with spore of arbuscular mycorrhiza fungi glomus spp. significantly reduced the total number of p. coffeae in roots and soil by 30%. both microbial inoculations decrease the infection degree (id) of p. coffeae. introducing 108 cfu/ml and 109 cfu/ml mhb cells to the arabica coffee seedlings increased the id up to 50%. mhb has no significant role on the enhancement of root colonization (rc) by glomus spores in root seedlings inoculated with glomus spp. slightly but significant reduction of rc was shown in roots treated with mhb. a combination of mhb and glomus spp. increased shoot height at 3 months after treatments without affecting its dry and fresh weight. the decrease of root weight was observed in seedlings inoculated with mhb and glomus spp., with thinner primary roots and more intensive lateral roots. we suggest to introduce the inoculation of glomus spp. spores and mhb at the coffee nursery in order to minimize p. coffeae infection. acknowledgment the research was funded by the indonesian agricultural research and development agency of the ministry of agriculture. we would like to thank the director of the coffee and cocoa research institute for providing greenhouse and coffee seedlings. references andrade cc, young ki, johnson wl, villa me, buraczyk ca, messer wb, hanley ka. 2016. rise and fall of vector infectivity during sequential strain displacements by mosquito-borne dengue virus. j evol biol 29: 2205-18. alarcón mv, salguero j, lloret pg. 2019. auxin modulated initiation of lateral roots is linked to pericycle cell length in maize. front plant sci 10: 11. amar b. taha c, zaghloul i, el-mahdy ar, el-massry mh. 2016. effect of ph and temperature on bacillus sp. r2 chitinase activity and stability. proc technol 22: 471-7. asyiah in, harni r, wiryadiputra s, fauzi i. 2015. populasi pratylenchus coffeae (z.) dan pertumbuhan bibit kopi arabika akibat inokulasi pseudomonas diminuta l. dan bacillus subtilis (c.). 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[cited 2019 june 6]. available from http://www.nematologia.com.br/files/tematicos/ 5.pdf veresoglou sd, rillig mc. 2012. suppression of fungal and nematode plant pathogens through arbuscular mycorrhizal fungi. biol let 8: 214-7. yu y-t, liu h-l, zhu a-g, zhang g, zeng l-b, xue sd. 2012. a review of root lesion nematode: identification and plant resistance. adv microbiol 2012(2): 411-6. zhong w, ding s, guo h. 2015. the chitinase c gene pschic from pseudomonas sp. and its synergistic effects on larvicidal activity. genet mol biol 38(3): 366-72. short com 805 azimah haji (assessment).cdr assessment of seedling abundance, survival and growth of two dipterocarp species in peat swamp forests of brunei darussalam hazimah haji mohd din, nor basirah bakiri, rahayu sukmaria sukri and faizah metali* environmental and life sciences programme, faculty of science, universiti brunei darussalam, gadong be1410, brunei darussalam received 31 january 2017 / accepted 21 march 2018 abstract dryobalanops rappa becc. and shorea albida sym. are bornean endemics of high conservation value and increasingly threatened by anthropogenic disturbances. in-situ study of seedling abundance and growth performance of these dipterocarp species was conducted in two selected peat swamp forests of brunei darussalam, following a mast fruiting event in march–may 2014. within six 6 x 6 m plots at each forest site, d. rappa seedlings at the anduki peat swamp forest and s. albida seedlings at the badas peat swamp forest were measured for abundance at the initial census in september 2014, as well as survival and relative growth rates (rgr) after a period of 5 months, with the final census in february 2015. we found significantly higher seedling abundance for d. rappa (1885 ± 208) than s. albida (160 ± 71). significantly higher percentage survival was recorded for d. rappa seedlings (90.8 ± 2.2%) in comparison to s. albida -1 -1seedlings (81.7 ± 2.2%). s. albida seedlings (0.24 ± 0.02 mm mm month ) showed significantly greater rgr in stem -1 -1diameter than d. rappa seedlings (0.18 ± 0.02 mm mm month ), however, there were no significant differences in the rgrs based on seedling height, leaf number and biomass between d. rappa and s. albida seedlings. in terms of seedling abundance and percentage survival, d. rappa seedlings appeared to be more successful in regeneration and may potentially be used for rehabilitation of degraded tropical peat swamps and other forest types. our results suggested that greater conservation efforts of peat swamps must be made to protect the bornean endemic plant species, in particular s. albida. keywords: borneo, dipterocarpaceae, dryobalanops rappa, relative growth rates, shorea albida introduction peat swamp forest is a unique ecosystem that represents the second major forest type in brunei darussalam, accounting for 15.6% (or 90,884 ha) of brunei's forest (wong et al. 2015; forestry department 2016). it is mainly located in the belait district, interconnecting with the peat swamps of the baram basin in sarawak. bornean peat swamp forests are ombrogenous and have six 'phasic communities', each consisting of distinct plant communities (anderson 1963). over the years, there has been a significant reduction in peat swamps in brunei darussalam mainly due to infrastructure development and forest fires (pg harun 2015; wong 2016). peat swamps house unique flora, some of which are endemic and increasingly threatened in their natural ranges due to anthropogenic disturbances such as legal or illegal logging, forest fires and land-use changes (yule 2010; posa et al. 2011). most tree families in lowland dipterocarps are found in peat swamps, but many peat swamp species are restricted and physiologically adapted to this extreme forest environment (ng & ibrahim 2001). in brunei darussalam, extensive stands of dryobalanops rappa becc. or 'kapur paya' exist on shallow peat overlying sand in lumut and anduki, while pure stands of shorea albida sym. occur in the 'padang alan' and 'alan bunga' peat swamp forests of badas, both in the belait district (anderson 1964; ashton et al. 2003; wong et al. 2015). the anduki peat swamp forests experienced massive forest fires during the * corresponding author: faizah.metali@ubd.edu.bn biotropia 5 2 8 148 154 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.2.805 148 1997–1998 el niño drought (wooster et al. 2012), resulting in fragmented stands of intact d. rappa existing within an otherwise developed area. low regeneration rates have been observed in natural d. rappa populations (wong & kamariah 1999), and this species is increasingly threatened from illegal logging (phillips 1998). the badas peat swamps consist of gregarious stands of s. albida that are decreasing in population density and have been showing few signs of natural regeneration for the past 30 years (kobayashi 1998). the iucn red list classifies s. albida as endangered due to rapid population loss within its natural forest ranges, coupled with a regeneration status that is reported to be virtually non-existent (ashton 1998). furthermore, s. albida is endemic to north and west borneo (ashton 1998) and so the remaining stands in the badas peat swamps in brunei are likely to be one of the few remaining intact stands of s. albida in borneo and globally (forestry department 2014). both d. rappa and s. albida are important timber tree species that rely on irregular mass flowering and mast fruiting episodes as a means of their natural regeneration (appanah & turnbull 1998). although several studies have been conducted on the survival and growth of dipterocarpaceae seedlings in bornean forests (turner 1990; delissio et al. 2002; nakagawa et al. 2005; shimamura et al. 2006; takeuchi & nakashizuka 2007; daisuke et al. 2013), only a few have focussed specifically on peat swamp species (ibrahim 1996; gavin & peart 1997; saito et al. 2005; jans et al. 2012), and no study has been conducted on s. albida seedlings to date. following a small mass flowering and a mast fruiting event in the badas and anduki peat swamps in march–may 2014, the abundance, survival and growth of d. rappa and s. albida seedlings at the understorey of these two peat swamp forests were assessed in brunei darussalam. our study provides preliminary findings on two native dipterocarp species that can potentially be utilised in efforts to naturally regenerate and rehabilitate disturbed peat swamp forests. materials and methods the study was conducted in the anduki and badas peat swamps located in the belait district, brunei darussalam, north-west borneo, at distances of ca. 14 km from each other by road. a dominant dipterocarp species (dipterocarpaceae) in each peat swamp site was selected. the anduki peat swamp (4°37'39.00"n, 114°22'1.14"e) is located in the coastal areas of belait district within the anduki forest reserve and is dominated by dryobalanops rappa becc. the badas peat swamp (4°34'9.12"n, 114°24'40.08"e) is located further inland, south of seria and is mainly populated by shorea albida sym. a total of six plots (6 m x 6 m or 2 36 m each) plots were set up: three plots of shorea albida at badas and three plots of dryobalanops rappa at anduki. plots were located ca. 50 m away from each other and each plot was set up with a mother tree of the respective species located at the centre of the plot. abundance of d. rappa seedlings in anduki and s. albida seedlings in badas were quantified per plot at the initial census in september 2014, ca. 3–5 months after the mass flowering and mast fruiting events. within each plot, 40 seedlings of either d. rappa (at anduki) or s. albida (at badas) were randomly chosen and tagged in september 2014. the seedling censuses were conducted twice, in september 2014 and after 5 months, in february 2015. the tagged seedlings were measured for percentage survival, stem height (measured from the point of measurement painted white on soil surface to the top leaf bud), stem diameter and number of leaves (counted fully expanded leaves, excluding heavily eaten leaves i.e. approximately 70% of lamina eaten and dead leaves) at both censuses. a total of 10 seedlings of each species were randomly chosen from outside each of the three study plots per census and harvested to provide estimates of initial and final dry biomass. at both harvests, seedlings were washed with distilled water, oven-dried at 60 c for 48 h and weighed. o the relative growth rate (rgr) based on stem height (rgrh), stem diameter (rgrd), number of leaves (rgrl) and biomass (rgrb) per 149 seedling abundance, survival and growth of two dipterocarp species – mohd din et al. recorded higher survival and rgr for d. rappa seedlings planted in lowland mixed dipterocarp forest (sukri 2010) and degraded heath forest (w. h. tuah, unpubl. data). natural disturbance within the understorey may influence the survival of tree seedlings (appanah & turnbull 1998) and can be an explanation for the low percentage survival of s. albida. yamada (1997) reported that badas peat swamp has higher incidences of fallen trees and presence of buttress roots on its forest floor compared to anduki peat swamp. during the rainy season between december 2014 – january 2015 in badas, s. albida seedlings may not survive when hit by fallen trees and branches, thus decreasing its survival percentage. through the mast fruiting period, the s. albida seeds may fall on the false forest floor in badas, which is formed from litter accumulation on buttress roots. this may lead to s. albida seedlings mortality as the false floor may fall to the peat when disturbed. seedling predation and microenvironment also influence seedling survival, and it has been recorded elsewhere in borneo that late fruiting of shorea species increased the possibility of seed losses to predation (curran & webb 2000). anduki peat swamp has different physiological environment compared to badas peat swamp forest in terms of its natural disturbance. fragmentation of the anduki forest due to rapid forest fires may cause its forest floor to develop shallow litter and peat overlying sandy soil (yamada 1997). the anduki forest reserve is dependent on waterlogged conditions to maintain the peat bog (yussof 2015) and d. rappa seedlings are well-adapted to waterlogged peat (yamada & suzuki 2004). survival and growth of s. albida seedlings have been found to be negatively affected by low light intensity (kobayashi 1998), water shortage during dry spells (kobayashi 1998) and excessive water during rainy period (dixon et al. 2013), which may be factors that explain their low survival rates in badas. d. rappa is a light-demanding species (ashton et al. s. albida 2003), while seedlings have been recorded as shade-intolerant (kobayashi 1998; pers. obs.). regardless of the light environment, light-demanders tend to possess larger specific leaf area and higher photosynthetic capacity, which result in greater carbon gain, and potentially higher survival, compared to shadetolerant species (ghazoul & sheil 2010). light has seedling over the five-month census period were calculated following hunt (1982): rgr = (log we 2 – log w ) / (t – t ), w and we 1 2 1 2 1 where are stem height, stem diameter, total number of leaves or biomass and . to compute rgrb, t – t is 5 months2 1 seedlings were paired by ranked values of seedling biomass at the initial and final harvests. between-species differences seedling abundance, percentage seedling survival and rgr values were determined using t-tests in r 2.15.2 (r core team 2014). all data were first explored to confirm the normality of residuals and homogeneity of variances, and where necessary, data was log -transformed, with the exception of 10 percentage survival, which was arcsinetransformed. results and discussion using the initial census survey, d. rappa seedlings at the anduki peat swamp forest showed 2 significantly higher mean abundance per 36 m plots compared to s. albida seedlings at the badas peat swamp (d. rappa: 1885 ± 208 vs. s. albida: 160 ± 71, p< 0.01). this is a probable reflection of the higher density of adult d. rappa trees in anduki, compared to the more sparsely populated adult s. albida trees in badas (pers. obs.). additionally, the higher d. rappa seedling abundance may be due to higher abundance of flowering mother trees, as almost all mature d. rappa trees flowered at anduki whilst not all mature s. albida trees flowered at badas during the same mass flowering event in 2014 (pers. obs.). other studies have similarly documented a positive relationship between the number of mother trees and seedling density, with the highest seedling densities found in areas with high adult abundance (itoh et al. 1997; webb & peart 1999; backlund 2013). mean percentage survival of tagged d. rappa seedlings over the period of 5 months was significantly greater than s. albida seedlings (90.8 ± 2.2% vs. 81.7 ± 2.2%, p< 0.05). all relative growth rates (rgrs) data of both species showed similar growth rates (fig. 1) that there were no significant differences in all relative growth rates (rgr) except for rgr in stem diameter (rgrd), in which d. rappa seedlings had significantly lower rgrd than those of s. albida seedlings (0.18 ± 0.02 vs. 0.24 ± 0.02 mm mm month ; p< 0.01; -1 -1 fig. 1b). other studies in brunei darussalam have biotropia vol. 25 no. 2, 2018 150 been recognised to significantly influence survival and growth of dipterocarp seedlings (scholes et al. 1996; whitmore & brown 1996). for example, d. rappa s. albidaand saplings were planted in the open in ongoing reforestation trial plots of degraded waterlogged heath forests ( kerapah forests) in brunei darussalam, and have performed successfully in terms of its survival and growth rates over a period of more than a year (w. h. tuah, unpubl. data). it was found that s. albida seedlings had significantly higher rgrd compared to d. rappa seedlings. this suggests that in the shaded understorey of peat swamp forests, s. albida seedlings allocated more resources to stem diameter growth than height growth. tropical tree species that preferentially invest resources into stem diameter growth and wood density typically exhibit traits that defend against herbivory and pathogens, as well as for the figure 1 differences in relative growth rates (rgr) for seedlings in anduki and seedlings in dryobalanops rappa shorea albida badas: (a) rgrh (stem height; cm cm month ), (b) rgrd (stem diameter; mm mm month ), (c) rgrl -1 -1 -1 -1 (number of leaves; leaf number month ) and (d) rgrb (biomass; g g month ). rgr data were determined for -1 -1 -1 the 40 tagged and seedlings, except for rgrb which was determined for the 10 seedlings d. rappa s. albida harvested outside each plot at each census. the asterisk sign within each rgr denotes significant differences of means between species (marked with red dots) using t-tests (*, < 0.05; **, <0.01; ***, < 0.001). ns=non-p p p significant. 151 seedling abundance, survival and growth of two dipterocarp species – mohd din et al. development of their root system (kitajima 1994), all of which decrease investment into height growth. the badas peat swamp forest has a flat, densely crowded canopy (yamada 1997), which restricts light entry to the understorey and may further inhibit height growth of s. albida seedlings. these shade-intolerant s. albida seedlings (kobayashi 1998) require ample light to grow in height. higher growth rates of s. albida in the open areas than in the closed canopy forest was recorded in the ongoing rehabilitation trial plots in the kerapah forests of brunei darussalam (w. h. tuah, unpubl. data). both the dryobalanops rappa peat swamp forest in anduki as well as the shorea albida peat swamp forest in badas have been identified as critical habitats that are of high conservation value (forestry department 2014). peat swamps are highly vulnerable to fires, particularly during the dry season and when the water table have been lowered (yule 2010). changes in drainage and hydrological conditions significantly increase repeated fire events (posa et al. 2011). repeated fires in anduki and badas, especially during drier periods and drought events, coupled with slow seedling growth rates, will undoubtedly result in further losses of both d. rappa and s. albida populations. in the case of s. albida, this loss will be further exacerbated by the low number of survived seedlings and mother trees flowering at the same time (pers. obs.). conclusion this study recorded significant findings on dryobalanops rappa seedlings in anduki and shorea albida seedlings in badas. d. rappa seedlings were more abundant than s. albida seedlings, and also showed higher percentage seedling survival. s. albida seedlings, however, appeared to invest more in stem diameter growth, as their relative growth rate in terms of stem diameter was significantly higher compared to d. rappa seedlings. we suggest that d. rappa seedlings may be suitable as a species that can be used to rehabilitate degraded peat swamp forests, but detailed, longer term studies are needed to further support this conclusion. finally, given that peat swamps forests are fast disappearing due to natural and anthropogenic disturbances, their protection and conservation efforts should be prioritised to protect the bornean endemic plant species, in particular s. albida. acknowledgements the authors thank the forestry department, ministry of primary resources and tourism (mprt), brunei darussalam for granting permission to conduct research in the anduki and badas peat swamps (no. 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[cited 2015 oct 3]. available from: http://borneobulletin.com.bn biotropia vol. 25 no. 2, 2018 154 page 1 page 2 page 3 page 4 page 5 page 6 page 7 3. oratai sukcharoen et al.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 900-fiks.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 5. dita maria virginia et al.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 896-fiks.cdr pop1 pop2 pop3 pop8 pop12 pop18 pop5 pop17 pop11 pop7 pop10 pop13 pop14 pop18 pop8 pop9 pop15 pop4 pop19 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 724 siti sofiah (flora diversity) revisi.cdr flora diversity, composition and ecology in besiq bermai tropical forest of damai district, east kalimantan * siti sofiah , destario metusala, trimanto and siti nurfadilah purwodadi botanic garden indonesian institute of sciences pasuruan , , (lipi), 67163 indonesia received 17 november 2016 / accepted 16 november 2017 abstract besiq bermai forest is part of kalimantan forests known for vast plant diversity. the present study aimed to investigate flora diversity, composition, and ecology in besiq bermai forest to support the management of biodiversity and forest conservation. thirteen plots were established with different sizes of plots (100 m x 20 m plots for trees; 40 m x 5 m plots for saplings; and 5 m x 5 m plots for understory). data recorded included plant species name and individual number of each plant species. data analysed were shannon-wiener diversity index, relative density, relative frequency, relative dominance and important value index. the principal component analysis (pca) was performed to determine relationship between edaphic components and flora occurrence. the results showed that there were 93 species of trees (belonging to 48 genera and 22 families), 112 species of saplings (belonging to 62 genera and 43 families), and 48 species of understory (belonging to 28 genera and 20 families). shannon-wiener diversity index (h') were 6.05, 6.25 and 3.26 for tree, saplings and understory, respectively. the most common family for tree and saplings in the forest ecosystem in this area was dipterocarpaceae (shorea spp). species of tree with the highest importance value index were dillenia excelsa, syzygium sp. and shorea parvifolia. the highest importance value index for species of saplings were macaranga triloba and shorea parvifolia; and for species of understory were phrynium jagorianum. ecological (edaphic) factors affecting the occurence and establishment of flora in bermai forest were total n and c/n ratio. the present study has implication for the management of biodiversity and forest conservation. keywords: composition, ecology, flora diversity, kalimantan, tropical forest introduction tropical forest is one of the hotspots-regions of biodiversity, with high plant diversity, complex ecosystems, and greatest variety of flora and fauna (richards 1952; whitmore 1988; de bruyn et al. 2014). borneo is recognised as one of the biodiversity hotspots regions, along with 24 other hotspot regions in the world (marchese 2015). east kalimantan, located in the eastern part of borneo, possess thousands of potential plants in the flora diversity of indonesia. tropical forest along indonesia-malaysia (indo-malay region) is known for its largest tropical forest ecosystem in the world (gaither & rocha 2013). inventory to investigate plant diversity and floristic composition in natural forests is important to the level of adaptation to the environment and their ecological significance (reddy . 2011), and is absolutely essential et al understanding the forest ecosystem dynamics (reddy . 2008). it plays an important role in the et al management of biodiversity as essential bioresources and in the management of conservation of species and forest ecosystem (malouin . 2015; lukác . 2013; meng et al z et al et al et al. 2011; nurfadilah 2015; you . 2016; huang . 2016). plant diversity and floristic et al composition in several tropical forests addressed for the management of biodiversity and ecosystem conservation have been widely studied accross the world, in costa rica (gilman . et al 2016), in china (meng . 2011), in ghana et al (addo-fordjour . 2009), in ethiopia (kebede et al et al et al. 2016), in tanzania (giliba . 2011), in bangladesh (feroz 2016).et al. despite its great diversity, tropical forests have experienced the highest diversity loss. tropical * corresponding author: sofie2291@yahoo.com biotropia 5 2 8 85 94 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.2.724 85 forests in indonesia, especially in kalimantan, is amongst experiencing higher diversity loss those (bruhl & eltz 2010; margono 2014; hansen et al. et al. 2009). approximately 57 % of low land rain forests in kalimantan conservation area (hutan lindung) has disappeared 1885-2001 between ( et al. ; stibig et al. 2007).gaveau 2013 exploitation of tropical forests, especially in kalimantan, has occured for a long period (gaveau 2016). et al. generally, the loss of forests in kalimantan has been caused by the land use change into agricultural areas (busch 201 ), & ferretti-gallon 7 plantation of oil palms, and mining. restoration should be conducted for the recovery of forest ecosystems. restoration is vital for recovery of tropical deforestation and degraded land, and for conservation of biodiversity (ashton 2014; et al. kettle 2012). in the management of restoration, biodiversity and forest conservation, analysis of the flora diversity and composition, and combined understanding of ecology and environmental factors that influence the diversity in forests is important. the present study aimed to investigate the flora diversity, composition and ecology in besiq bermai forest damai district of in east kalimantan. the study site was coal mining area and targeted for reforestation restoration after mining (post-mining). the results of the present study on the flora diversity, composition, and ecology of forests is important for the rehabilitation of coal mining program areas. 86 biotropia vol. 25 no. 2, 2018 figure 1 a map of study site area in besiq bermai forest, east kalimantan, indonesia materials and methods the study was conducted in low land rain tropical forests of village besiq bermai of damai 0 district in east kalimantan, gps coordinate 00 0 51'.02.6”s, 115 26'30.7”e (fig. 1). this forest area was a primary forest that started undergoing degradation and becoming a secondary forest. in the present study, nested plots were established, with stratified random sampling method. thirteen plots were made with different sizes of plots, out of the total forest area of about 2000 ha; 100 m x 20 m plots for trees, 40 m x 5 m plots for saplings, and 5 m x 5m plots for understory. category of trees and saplings were determined by the size of diameter at breast height (dbh) of woody plants; tree (dbh > 30 cm) and saplings (dbh between 5 cm 30 cm). understory are groundcover plants growing on the forest floor which are typically herbs. all plant species within the plots were recorded. to investigate the floristic composition, some parameters including shannon-wiener diversity index, important value index, density (d), relative density (rd), frequency (f), relative frequency (rf), dominance (c) and relative dominance (rc) for each species, were analysed. number of individuals of a taxon x 100rd = total number of individuals number of plots containing a taxon x 100rf = total frequencies of all taxa and the establishment of flora in their habitats, the principal component analysis (pca) method with minitab 16 software was used. results and discussion flora diversity the study showed that the flora diversity in besiq bermai forest, east kalimantan was relatively high. an index of plant diversity of greater than 3 (>3) is regarded as high-value area (khan et al. 2012). plant diversity index indicating the degree of diversity of plants at the study site in the study was high, with the shannon-wiener diversity index (h') for trees, shrubs and understory in this area being 6.05, 6.25 and 3.26, respectively (fig. 2). comparatively, the diversity index at the study site was higher than that of other parts of kalimantan or borneo, such as in lubuk kakap, west kalimantan with h' for tree being 3.54 (budiharta 2010) and in a lowland dipterocarp forest of north borneo with h' for tree was less than 3.5 (godoong et al. 2014). the difference in the degree of diversity at the study site which is located in east kalimantan and other parts of kalimantan/borneo might be due to the difference in regions within borneo. guhardja et al. (2012) reported that the diversity varied within kalimantan/borneo, with one of the highest diversity found in east kalimantan. furthermore, kurniawan and parikesit (2008) reported that plant diversity and composition in an area depends on some factors including altitude, humidity, nutrient availability, illumination, topography and soil types. basal area of a taxon x 100rc = total basal area of taxa h'= -∑ pi ln pi ; pi= ni/ ni = number of individual from species-i n = total number of individual to analyse edaphic factors that influence the growth and the establishment of flora at the study site, soil samples were collected. soils were collected in each plot for 3 replicates. the collection of soils for soil physical and chemical profiles were collected from the depth of 0 cm 30 cm, and 30 cm 60 cm and 3 replicates for each plot. the soil physicochemical properties were assessed in the soil science laboratory of agriculture research institute of mulawarman university, samarinda and were analysed through the drying stage temperature of 105°c. soil physical factors analysed included soil texture (sand, silt and clay), while chemistry factor s included soil acidity (ph), organic c, c/n ratio, organic matter (om) and n-total. soil texture analysis was conducted by separating sand, silt and clay particles by quantitative method through mechanical analysis process. this process consists of spreading aggregate soil into single grains, followed by sedimentation. soil acidity (ph) was measured in soil and water mixture extracts with a ratio of 1: 5, c content was analysed by walkley and black (1934) method, while total n was determined by kjeldahl (188 ) method. to 3 investigate ecological factors focussing on edaphic variables that influenced the occurence 87 flora diversity, composition and ecology in besiq bermai tropical forest – sofiah et al. figure 2 shannon – wiener diversity index (h') of tree, sapling and herb in forest of besiq bermai trees 6.05 7 6 5 4 3 2 1 0 6.247 3.264 saplings habitus s h an n on -w ie n er d iv er si ty in d ex ( h ’) herbs floristic composition of trees this study showed that, there were 93 species of trees from 48 genera and 22 families in besiq bermai forest. some of the most important species for tree included dillenia excelsa, syzygium sp. and (fig. 3). , shorea parvifolia dillenia excelsa was the most important species in terms of basal area based on rc, however, it had the lowest number of plants. sp. was the second syzygium most important species in terms of species frequency, which was often found in plots at the study site. ranked number shorea parvifolia three in ten most important species, it had the largest number of plants (fig. 3). genus of shorea dominated the study site with nine species, and three of the species were in the list of ten most important species in terms of their abundance. the present study also demonstrated that the most important plant family for trees was dipterocarpaceae, followed by lauraceae and myrtaceae (fig. 4). dipterocarpaceae also had the largest number of species (17 species) including the genera shorea, hopea and dipterocarpus; followed by lauraceae with 12 species (i.e genera of eusideroxylon, litsea and actinodaphne), burseraceae with seven species (i.e. genera of canarium, dacryodes and santiria), leguminosae with seven species (i.e. genera of sindora, koompassia and dialium), and myrtaceae with six species in a single genus of syzygium. 88 figure 4 ten most important plant families in the category of trees in besiq bermai forest. family important value is the sum of important value index of all species contained in a single family. biotropia vol. 25 no. 2, 2018 figure 3 ten most important tree species in besiq bermai forest (note: rd = relative density; rf = relative frequency; rc = relative dominance; ivi=important value index) the results of this study was similar with other studies that showed that the most important family in other forests within borneo/kalimantan was dipterocarpaceae. kartawinata (2008) et al. reported that a family with the highest important value index in the lowland dipterocarp forest at wanariset saboja, east kalimantan, was dipterocarpaceae. arbainsyah (2014) also et al. reported that the most abundant seedling of the species in the primary forest in labanan east kalimantan was dipterocarpaceae. budiharta (2010) also showed that dipterocarpaceae dominated the biodiversity protection area of lubuk kakap, west kalimantan. saner (2012) et al. also reported that tropical lowland dipterocarp rain forests in sabah, malaysian borneo, was also dominated by dipterocarp species. the dominance of dipterocarpaceae in the forests might be in part due to its mast fruiting, the synchronous production of fruits in multi-years cycle at intervals of several years. this irregular fruiting may enhance pollination efficiency, seed survival, and seedling establishment (visser et al . 2011; rodriguez et al. 2013; oshima et al 2015). . the predator satiation hypothesis stated that starvation of seed predators in time of no production of fruits decreases seed predation and therefore, increase tree regeneration (visser et al . 2011). floristic omposition of saplingsc revealed the present study also the floristic composition of saplings. the most important species for saplings was in terms macaranga triloba of basal area, followed by . shorea parvifolia syzygium . xylopia malayana sp and (fig. 5). the pattern of the most important species for trees 89 figure 5 ten most important species for sapling in besiq bermai forest (note: rd=relative density; rf=relative frequency; rc=relative dominance; ivi=important value index) figure 6 ten most important plant families in besiq bermai forest in the category of saplings. family important value is the sum of important value index of all species contained in a single family. flora diversity, composition and ecology in besiq bermai tropical forest – sofiah et al. 90 and saplings in study was different. budiharta this (2010) also showed variation of pattern of the flora composition between tree and saplings in west kalimantan that might be due to the difference in mast flowering and fruiting frequencies between species which influence the survival of seedlings. the most important family for saplings was dipterocarpaceae, followed by euphorbiaceae and annonaceae (fig. 6). dipterocarpaceae also had the highest number of species (10 species) including the genera shorea and hopea; followed by euphorbiaceae with 9 species (i.e genera of xylopia and monocarpia). floristic omposition of understoryc the results of the study also showed the flora composition for understory. the most important species for understory was calathea sp., followed by pandanus sp., and alpinia sp. (fig. 7). analysis of compositional diversity of understory flora in the study showed that the understory flora was dominated by calathea sp. that belongs to the plant family marantaceae (fig. 7). the dominance of calathea in the shaded forest canopy with low light intensity in the forest floor in this study may be related to its low tolerance of light intensity and its capacity to grow in shaded areas with low figure 7 ten most important species for understory (note: rd=relative density; rf=relative frequency; ivi=important value index) biotropia vol. 25 no. 2, 2018 figure 8 ten most important families for understory ble ch nu m o rie nt al e 91 light intensity. matlaga (2008) reported that clonal offspring of calathea grew and survived well in shaded understory. the most important family for understory in besiq bermai forest was marantaceae, followed by zingiberaceae and pandanaceae (fig. 8). in terms of the number of species, marantaceae and zingiberaceae were high with six (6) species from the genera calathea, phrynium and stachyphrynium. zingiberaceae at the study site consisted of 6 species from the genera zingiber, amomum and alpinia. ecology of flora in besiq bermai forest the establishment of flora in their habitats showed the adaptation of the flora to their environment. the diversity and distribution of many plant species are influenced by the ecological factors including edaphic factors (heineman et al. 2015; vleminckx et al. 2015). through (pca),principal component analysis environmental (soil) factors consisting of five soil variables (ph, organic c, total n, organic matter, and c/n ratio) and environment variables (temperature, humidity, light intensity and elevation) could be classified into two principal components. the two components principal explain 68.8% of all edaphic factors measured. s the first component explained 35.4% of all edaphic factors measured, while the second component only explained 33.4%. this showed that the first component provided more information than the second component in describing and identifying environmental conditions in besiq bermai forest in which many plant species occured. c/n ratio of soils was the most influential variable towards the factors in the first component (pc1), while total n was the most influential variable towards the factors in the second component (pc2) (table 1). the model of the habitat of flora in besiq bermai forest with its edaphic factors can be formulated based on table 1. pc1 = 0.27 ph + 0.35 oc + 0.17 tn+ 0.36 om + 0.37 c/n r; pc2 = -0.35 ph + 0.26 oc+0.46 tn + 0.27 om -0.10 c/n r. each soil variable in pc 1 and in pc2 was not strongly correlated (table 1). there are many soil factors (variables) that influence the diversity and distribution of plant species, such as organic matter, exchangable cation, soil types and other soil factors. these soil factors form the characteristics of habitats and can be the determinants of the distribution and diversity of flora in their habitats (peña-claros et al. 2012; vleminckx et al. 2015; zhang et al. 2016). some studies have shown that species diversity and composition in some ecosystems were related to the site characteristic including edaphic factors (gautam et al. 2016; khan et al. 2017). gautam et al. (2016) showed that the most important factors influencing tree diversity in the north indian moist deciduous forests were exchangeable base cations and phosphorous. furthermore, peñaclaros et al. (2012) showed that in the dry forest in bolivia, correlation between soil characteristics and plant diversity was more related to ca, organic matter, n, mg and na. in this study, the first factor that mostly influenced the diversity and establishment of flora in besiq bermai forest was total n in the soil. nitrogen is an essential element for the growth and development of plants. the availability of n in the soil affect the growth, development, and establishment of flora in besiq bermai forest. table 1 eigen value of soil elements in besiq bermai forest pc1 (component factors 1) pc2 (component factors 2) eigen value 5.11 2.92 proportion 0.3 0.17 cumulatif 0.3 0.47 variabels: ph (ph) 0.27 -0.35 organic c (oc) 0.35 0.26 total n (tn) 0.17 0.46 organic matter (om) 0.36 0.27 c/n ratio (c/n r) 0.37 -0.10 flora diversity, composition and ecology in besiq bermai tropical forest – sofiah et al. 92 the second factor that influenced the occurence and establishment of flora in besiq bermai forest was c/n ratio. c/n ratio shows the rate of decomposition of organic matter and mineralisation of nutrient elements in soils (tisdale 1985). ross et al. (2011) showed the relationship between the establishment of plant species and c/n ratio indicating that c/n ratio influenced the diversity of plant species in their habitat . the c/n ratio indicates the decomposition of organic matter and in relation to the nutrient provision in soils plays key roles in the function of forest ecosystem for the establishment of the flora. implications for conservation the present study has shown the flora diversity, compositon, and ecology in besiq bermai forest. the selected study site was a coal mining area. with the obligation and responsibility to rehabilitate and restore sites after the coal is mined, the results of the present study is required for the reconstruction of the sites and reverting the flora diversity and composition, and ecology of the forest. the present study contributes to support the management of biodiversity and forest conservation after coal mining. the study is also required for rehabilitation program of degraded post mining lands. the soil chemistry that most influences the existence of species of the shorea family is c/n ratio and total nitrogen. the c/n ratio can explain the speed of overhauling organic matter in the form of chemical-bound nutrient decomposition and mineralization in the form of complex compounds. when planting at post-mining areas, it's advisable to use additional organic materials, such as compost, manure, or the addition of calcium, depending on the deficiency in organic matter and acidity level of the soil. there are several species included in the iucn red list, with critically endangered category, namely , and shorea leprosula dipterocarpus cornutus hopea ferruginea. the dominance of the various shorea plants that are included in the iucn redlist, as well as other dominant local species should be given priority as the species used in the restoration of post-mining areas due to their ecological suitability with forest ecosystems in the study sites. conclusion the diversity of plants in besiq bermai forest was relatively high, with shannon-wiener diversity index (h') 6.05, 6.25 and 3.26 for tree, sap l ing and unders tor y, respect ive ly. dipterocarpaceae was the most commmon plant family for tree and saplings, while marantaceae was the dominant family for understory. edaphic factors that had the highest influence on the occurence and establishment of flora in besiq bermai forest were total n and c/n ratio. the present study has an implication to support management of biodiversity and forest conservation. acknowledgements we would like to thank the team of exploration for the assistance in the field. this research was supported by research program on plant diversity and habitat in coal mining exploration areas of pt. bharinto ekatama and pt. indo tambangraya megah tbk branch, west kutai, east kalimantan. references addo-fordjour p, obeng s, anning ak, addo mg. 2009. floristic composition, structure and natural regeneration in a moist semi-deciduous forest following anthropogenic disturbances and plant invasion. int j biodivers conserv 1(2):021-037. arbainsyah, de longh hh, kustiawan w, de snoo gr. 2014. structure, composition and diversity of plant communities in fsc-certified, selectively logged forests of different ages compared to primary rain forest. biodivers conserv 23(10):2445-72. ashton ms, gunatilleke cvs, gunatilleke iaun, singhakumara bmp, gamage s, shibayama t, tomimura c. 2014. restoration of rain forest beneath pine plantations: a relay floristic model with special application to tropical south asia. forest ecol manag 329:351–9. bruhl ca, eltz t. 2010. fuelling the biodiversity crisis: species loss of ground-dwelling forest ants in oil palm plantations in sabah, malaysia (borneo). biodivers conserv 19(2) 519-29.: budiharta s. 2010. floristic composition at biodiversity protection area in lubuk kakap, district of ketapang, west kalimantan biodiversitas . 11(3):151-6. biotropia vol. 25 no. 2, 2018 93 busch j, ferretti-gallon k. 2017. what drives deforestation and what stops it? 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h, jin h , khaldi a , kwak m , lee t , khaine i , y h j y k woo s 2016. plant diversity in different y. bioclimatic zones in tunisia. j asia pac biodivers 9 :56-62.(1) zhang c, li x, chen l, xie g, liu c, pei s. 2016. effects of topographical and edaphic factors on tree community structure and diversity of subtropical mountain forests in the lower lancang river basin. forests 7 222(10): . 94 biotropia vol. 25 no. 2, 2018 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 881-fiks.cdr adult young seedling number of individual 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 adult young seedling number of individual 0 10 20 30 40 50 60 70 80 90 100110 120130 140 adult young seedling number of individual 0 10 20 30 40 50 60 70 80 90 100 110120 130 140 adult young seedling number of individual 0 10 20 30 40 50 60 70 80 90 100 110120 130 140 adult young seedling number of individual 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 adult young seedling number of individual 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 biotropia vol. 28 no. 2, 2021: 149 155 doi: 10.11598/btb.2021.28.2.1277 149 agronomic traits of local wetland rice varieties in jambi province julistia bobihoe*, jumakir, araz meilin, and endrizal balai pengkajian teknologi pertanian jambi (jambi assessment institute for agricultural technology/aiat), jambi 36128, indonesia received 26 june 2019/accepted 23 july 2019 abstract indonesia’s swamplands are among areas earmarked for future agricultural development. as a type of wet lands, swamplands are inundated and have soil properties that are uniquely different from other agroecosystems. in indonesia, some of these areas a r e currently u s e d fo r r i c e cultivation of the country’s very diverse genetic resources of local rice varieties. most o f t h e farmers continue to plant and cultivate the local rice swampland varieties because of the abilities to adapt to extreme environments. this study on the agronomic traits of the local swampland rice varieties was carried out to evaluate their agronomic characters and identify varieties having superior quality traits. the research was carried out from april to october 2016 using a single plot method at the rantau kapas mudo village, batanghari regency, jambi province. eleven (11) rice varieties were planted in 10 x 5 m single plots, with a spacing of 25 x 25 cm and 1 m distance between plots. these 11 genetic resources of the local swampland rice varieties, include the serendah halus, rimbun daun, karya, serendah bawang, sereh aek, botol, pontianak, semut, dawi, ketan itam and di. the observed characters consisted of the plant height at harvest, number of productive tillers, age of harvest, number of grains per panicle, number of filled grains per panicle, number of empty grains per panicle, weight of 1,000 grains, seed shape and the production volume. there were differences among the local swampland rice varieties with the highest production volume of 3.32 tonnes/ha obtained from the rimbun daun variety, followed by 2.86 tonnes/ha from the dawi variety. these two varieties had shown potential to become the leading regional swampland rice varieties. keywords: agronomic traits, local rice, swampland introduction swampland is a very unique agro-ecosystem with characteristically inundated soils. in indonesia, it is one of those areas designated for future agricultural development (noor & fadjry 2008). swampland is also distinguished by the presence or absence of the influence of a surrounding river. those inundated by the surrounding river is called the river swamp, while the free or unaffected swampy area is called a caged or half-caged swamp (kosman & jumberi 1996). increasing rice production in swampland had increased farmers' income, and had supported food self-sufficiency (djafar 1992). however, the main obstacle in swamp rice cultivation is the uncontrolled water flow that during the rainy season the entire area is inundated quite deep for quite a long time. this makes it difficult for farmers to estimate the rice planting period. puddles that are too high during the vegetative phase due to flooding and heavy rains, occur after the seedlings are moved into the field. these are growth constraints that result in low production of even the low-yield rice variety. almost synchronously, the risk of crop failure due to drought can also occur if there is no rain when the rice plants bloom (swamp research center 2008). hence, farmers generally plant rice varieties that are genetically adapted to the local growing environment in swamplands. germplasm is the fourth natural resource in addition to water, land and air resources which need to be conserved. hence, preservation of *corresponding author, email: julistia117@gmail.com biotropia vol. 28 no. 2, 2021 150 germplasm as a genetic source will affect the success of agricultural development programs. the desired food sufficiency will depend on the regions’ own germplasm diversity, because in reality superior varieties, which have been, are being, or will be produced are a collection of specific genetic diversity that is expressed in the desired superior qualities. the regional genetic resource is the starting point in the formation of plant varieties that produce high yield, are pests and diseases resistant, and are tolerant to environmental stress (situmeang 2015). agricultural genetic resources (agrobiodiversity) are one of the most important germplasm to be protected from extinction and potential genetic erosion (diwyanto & setiadi 2008). in reality, these resources have been and will continue to be utilized for the survival and welfare of the community, at the local, regional, national, and global levels. as a mega-biodiverse country, indonesia is currently thought to be rich in germplasm collection. however, indonesia actually has inadequate collection of germplasm for producing superior varieties. genetic diversity is an economic, tourism, health and cultural resource. however, it is not evenly distributed in each region as it depends on the regional ecosystem (wardana 2002). natural resources utilization, accompanied by preservation of region-owned diversity and uniqueness, can be directed toward human welfare and carried out continuously from one generation to the next. the archipelagic nature of indonesia with its various tribes and cultures, is closely related to genetic resource utilization which may vary between regions and agroecology. cultural diversity together with agricultural genetic resources diversity will produce diversified community knowledge on resource utilization for food, shelter, clothing, medicines and industrial raw materials (diwyanto & setiadi 2008). among the various wetland agroecosystems (irrigation, rainfed and swamp) in jambi province, the swampland agroecosystems are the widest, comprising some 137,132 ha with the swampland area occupying 25,157 ha currently planted with local rice varieties (cpm 2016). these varieties are potential alternative food resources that need to be inventoried and conserved for the production of local superior varieties. local rice varieties are those varieties that have long been adapted in certain areas and are generally consumed as food in its original form (hajoeningtijas & purnawanto 2013). these local varieties need to be preserved as regional genetic resources and as a source of breeding material for future varieties improvement programs (arda 2013; rais 2004). the characteristics of these local rice varieties are largely not well identified, so knowledge on their potential and development opportunities as superior local rice varieties is still lacking. in the field, their local population still looks diverse, particularly the plant height, ripening or harvest age, shape, and grain color. the local varieties have adapted to certain environmental conditions and were characterized based on their low production rate, high and strong trunk, chronic unresponsiveness to input or fertilization, diverse physical characteristics, delicious taste, popularity among many consumers, and high market price. this will affect farmers’ production of rice. seeds of local rice varieties used by farmers are also of low quality because they are continuously obtained from farmers’ rice crops and inherited from generation to generation (bobihoe 2014). the purpose of this study is, therefore, to determine the characteristics of local rice in swampland and to discover the varieties that have superior qualities based on their agronomic characteristics. these results are expected to help expand opportunities to produce novel rice varieties with better qualities thereby improving rice productivity in the swamplands. materials and methods the research activities were carried out from april to october 2016 at rantau kapas mudo village, batanghari regency, jambi province. eleven (11) local varieties of swampland rice were studied, namely serendah halus, rimbun daun, karya, serendah bawang, sereh aek, botol, pontianak, semut, dawi, ketan itam, and di. using the single plot method, each variety was planted in a 10 x 5 m single plots with 25 x 25 cm spacing and 1 m distance between plots. the study location was classified as low-lying, lower topography with a pool of water between agronomic traits of local wetland rice varieties in jambi province – bobihoe et al. 151 50 and 100 cm deep during a period between three and six months. the swampyland is generally muddy and has a medium to high soil fertility (waluyo & suparwoto 2016). the observed parameters included those at the vegetative and reproductive phases. the vegetative characters consisted of the plant height at harvest (measured from the base of the stem to the highest tip of panicle), the number of productive tillers (calculated at the time of harvest). the generative characters were measured after harvest time and consisted of the harvest age (calculated from the number of days after planting), number of grains (counted number of grains) per panicle, number of filled grain (calculated number of filled grains) per panicle, number of empty grains (calculated number of empty grains) per panicle, weight of 1,000 grains (weight of 1,000 grains of grain content), and yield (tonnes/ha). plant data were observed agronomically and tabulated for 10 clumps of rice plants. results and discussion eleven (11) local swamp rice varieties characterized in terms of agronomical traits, consisted of serendah halus, leaf rimbun, karya, serendah bawang, serehaek, bottle, pontianak, semut, dawi, ketanitam, and di (table 1). these were chosen from the farmers’ fields located at rantau kapas mudo village, muaro tembesi district, jambi province, indonesia. vegetative characteristics of the rice plants plant height the height of the tested local rice varieties ranged between 86.2 and 147.6 cm with serendah bawang being the tallest at 147.6 cm, and botol the shortest at 86.2 cm (table 1). the crops were classified into three categories based on their height, i.e., short (<110 cm), medium (110 to 130 cm), and tall (>130). the local rice varieties were generally tall (>130 cm) (scale 5) except botol, dawi, and di (<110 cm) (scale 1). the plant heights were measured at the start of the harvest. medium-height plants (110 130 cm) more likely developed more productive tillers than taller crops, as tall crops use up photosynthates for vegetative growth, making photosynthesis less efficient. the results showed that the rice cultivation system in the swampland produced taller plants than that in the upland cultivation system. rice plant heights can be used as a parameter of rice growth, but taller plants do not guarantee greater yields (kristia & iskandar 2018). plant height is one component that influences a plant’s weight. the desired characteristics in the development of superior rice varieties are stems that are short and stiff, considering that plants with this characteristic will be resistant to fall, be responsive to fertilization, and have a more balanced proportion between grain and straw. another characteristic is the plants’ absorption of n; the higher the performance of a plant, the higher the likelihood of its vulnerability (kristia & iskandar 2018). table 1 vegetative characteristics of the local swampland rice varieties no. variety name plant height* (cm) number of productive tillers** (tiller) 1. botol 86.2 10 2. pontianak 139 10 3. semut 120.8 10 4. dawi 91.3 13 5. ketan hitam 111.4 9 6. di 104.7 10 7. serendah halus 141.4 7 8. rimbun daun 132.9 10 9. karya 140.6 9 10. serendah bawang 147.6 6 11. sereh aek 143.6 9 notes: * = average plant height at harvest (cm); ** = number of productive tillers at harvest (tillers). biotropia vol. 28 no. 2, 2021 152 among the selection criteria for rice plants, height is related to the length and shortness of the panicles and to the shedding resistance of plant stems. plants tend to be shorter at locations higher than sea level (simanulang 2001). although plant height is one selection criteria for rice plants, nevertheless, taller stems do not influence the level of production (suprapto & dradjat 2005). high plant growth does not guarantee high plant productivity. optimal plant growth has a large influence on the relationship between panicle length and yield. plants that grow well absorbs large quantities of nutrients. the availability of these nutrients in the soil influences photosynthetic activity in a way that plants can increase crop growth and yield (aribawa 2012). number of productive tillers the number of productive tillers varied from 6 to 13 with the highest number produced in the karya variety (13 tillers) and the lowest number in serendah bawang (6 tillers). this low number of tillers is probably due to the submersion of the initial seedling for about a week, thus inhibiting the formation of saplings (icr 2016). high and low plant growth and yield are also influenced by internal factors which include genetic traits and plant derivatives, and the external or environmental factors, such as soil regime and biotic factors (cepy & wayan 2011). the different number of tillers produced by each variety is thought to be due to the influence of these factors. the number of tillers and plant height differs because the genetic properties of each variety also differ (manurung & ismunadji 1988). productive tillers directly affect the high and low yields of grain as it greatly determines the number of panicles (simanulang 2001). more productive tiller means more number of panicles. in this study, a correlation exists between the number of panicles and yield; the higher number of panicles resulted in higher yield of the plants. generative characteristics of the rice plants in the generative growth phase, the characteristics observed included the harvest age, number of grains, number of filled grains, empty grains, weight of 1,000 grains, age of harvest and yield (table 2). harvest period (number of days after seedling) generally, the cultivation ages for rice crop are categorized into early age (about 110 days) and old age (more than 120 days). the local rice varieties are generally old at >151 days after seedling stage, while high yielding varieties mature at 105 124 days after seedling stage (icrr 2016). the local swamp rice cultivars are generally cultivated within 150 days, while the local variety is cultivated at an early age (120 days for karya). obviously, shorter harvest age is preferred by farmers because it means shorter harvest time gaps and more frequent harvest periods. agronomic traits of local wetland rice varieties in jambi province – bobihoe et al. 153 table 2 yield components of the local landraces of swampland no. variety name harvest age1 (dap) number of grain per panicle2 (grain) number of filled grains per panicle3 (grain) number of empty grain per panicle4 (grain) weight of 1,000 grains5 (g) volume production6 (tonnes/ha) 1. botol 150 132 74 58 22 2.32 2. pontianak 150 122 88 34 23 2.24 3. semut 150 113 49 64 14 1.26 4. dawi 150 172 80 92 16 2.86 5. ketan hitam 150 136 82 54 21 2.05 6. di 150 132 78 54 23 2.42 7. serendah halus 150 151 61 90 21 1.77 8. rimbun daun 150 173 86 87 24 3.32 9. karya 120 137 47 90 19 1.97 10. serendah bawang 150 170 103 67 25 2.04 11. sereh aek 150 165 73 83 20 2.37 notes: 1 = average age of harvest (day after planting); 2 = number of grain per panicle (grain); 3 = number of filled grains per panicle (grain); 4 = number of empty grain per panicle (grain) (empty grains were generally found at the base of panicles); 5 = weight of 1,000 grains (g); and 6 = volume production (ton/ha) of local rice swamp varieties. number of grains per panicle (grains), number of filled grains per panicle (grains), number of empty grains per panicle (grains). the dawi and rimbun daun varieties produced the highest number of grains per panicle, 172 and 173 grains per panicle, respectively, while the semut variety produced the least numbers of grains per panicle at 113 grains/panicle. moreover, serendah bawang variety had the highest grain content at 103 filled grains, while semut variety had the least grain content at 42 filled grains. the lowest number of empty or unhulled grains was produced in the pontianak variety at 34 grains while the highest number was found in dawi variety at 92 grains. this high number of empty grains showed the inability of those plants to fill their own grains. such a low yield can be due to genetic or environmental factors. empty grains affect the rice yield; the higher the percentage of empty grains, the lower the rice yield would be. consequently, higher proportion of empty seeds would result in lower rice production. the number of filled grains per panicle correlates with rice yield, hence, it is one of the criteria for a high yielding rice variety. although the number of filled grains is markedly correlated with rice crop yield, the latter variable is still strongly influenced by the number of empty grains. likewise, the weight of grain contents also determines the weight of the yield. empty seeds also affect the rice yield; the higher the percentage of empty grains, the greater the effect on rice yields. new types of rice varieties with high yield potential, generally possess the following characteristics, namely (1) the number of grains per panicle is between 150 and 250 grains; (2) the percentage of filled grains is between 85% and 95%; and (3) the percentage of empty grains is between 5 and 15% (abdullah 2008). generally, the number of grains per panicle is positively correlated with panicle length. the longer the panicle is formed, the more amount of grain is accommodated by the concerned panicle. meanwhile, the number of filled grains and the weight of 1,000 grains formed in one panicle is highly dependent on the photosynthesis (seed filling) of the plants during their growth and on the genetic properties of the cultivated rice plants (ariwibawa 2012). weight per 1,000 grains (g) and weight of yield (tonnes/ha) the weight per 1,000 grains of each variety varied between 14 g and 25 g. the highest weight per 1,000 grains was produced in the serendah bawang variety at 25 g, while the lowest was found in the semut variety at 14 g. weight per 1,000 grains indirectly describes the biotropia vol. 28 no. 2, 2021 154 size or enormity of a rice grain. the higher the weight per 1,000 grains of a rice variety, the larger is its grain size, and vice versa. the grain size is affected by its genetic properties and adaptability to its growing environment. during the dry season, the highlands with their low temperatures, affect the weight of the 1,000 grains. the difference in weight per 1,000 grains of plants determines the ability of a variety to produce a lot of grains which is often inversely proportional to its ability to produce large and heavy grains, nevertheless, a high production rate can still be achieved with a large number of grains, even though the grain size is smaller (simanulang 2001). weight of the 1,000 grains indirectly illustrates the grain size of a rice variety. strains/varieties with large grains produce higher weights of the 1,000 grains and vice versa. grain size is influenced by genetic traits and adaptability to the growing environment. hence, rice yield is also affected by the the weight of the 1,000 grains. finally, of all the varieties tested, rimbun daun produced the highest yield at 3.32 tonnes/ha. this result coincides with that of the number of grains per panicle where the rimbun daun variety produced the highest yield of 173 grains, while the lowest yield was at 1.26 tonnes/ha in the semut variety. this result also confirmed the effects of both the lowest number of filled grains per panicle and the lowest weight per 1,000 grains on the production volume of the semut variety. plant yield is determined by its components’ yield. hence, an imbalance in the components will greatly influence the expected potential result. similarly, rice yield is determined by its yield components, such as the number of filled grains per panicle and the weight per 1,000 grains. correlation of the plant’s tangible yield with its weight per 1,000 grains and its number of filled grains per panicle is one of the criteria for selecting the rice variety which can produce a high yield (manurung & ismunadji 1988). the yield per hectare of the eleven tested local swamp rice varieties planted on lebak swamp showed that two local varieties produced the two highest yields of more than 2.5 tonnes per ha, namely; rimbun daun at 3.32 tonnes/ha and dawi at 2.86 tonnes/ha. these two varieties have shown some potential for becoming the superior local rice varieties. finally, with the use of appropriate cultivation technology, these two local rice swamp varieties will produce their expected potential yield. conclusion in summary, two local rice varieties have exhibited high yield potential of above 2.5 tonnes per hectare, namely; rimbun daun at 3.32 tonnes/ha, and dawi at 2.86 tonnes/ha. these two varieties have shown some potential for large scale production and prospects of becoming the leading rice varieties in the swampy areas. acknowledgments the author would like to thank the technicians ms. hasniarti, ms. kusningsih, and mr. joko supriyanto; batanghari district extension instructor sp, muhammad; jambi bptp worker yatmi; and driver dendy; who helped in the implementation of the local rice characterization activities in batanghari regency, jambi province. references abdullah b, tjokrowidodo s, sularjo. 2008. the development and prospect of assembling new types of rice in indonesia. agricultural research and development journal 27:1-9. agricultural research and development agency (arda). 2013. inventory and/or collection of plant genetic resources in indonesia. center for biotechnology research and development and agricultural genetic resources. agricultural research and development agency. ministry of agriculture. ariwibawa ib. 2012. effect of planting systems on increasing rice productivity in wetland wetlands paper presented at the national seminar on food sovereignty and energy. faculty of agriculture, university of trunojoyo madura, june 2012. bobihoe j, hernita d, salvia e, muliyanti k, hayanti sy, supriyanto j, hasniarti, … , endrizal. 2014. management report on genetic resources. agricultural technology assessment center (bptp) jambi. center for agricultural technology agronomic traits of local wetland rice varieties in jambi province – bobihoe et al. 155 research and development (bbp2tp). agricultural research and development agency. ministry of agriculture. cepy, wayan w. 2011. growth and yield of rice plants (oryza sativa l.) in vertisol media and entisol in various water management techniques and types of fertilizers. crop agro journal 4(2): 49-56. cpm. 2016. paddy production in jambi province. agricultural survey. jambi (id): central statistics agency (cpm). diwyanto k, setiadi b. 2008. germplasm in the management of utilization and conservation of sources agricultural genetic power. jakarta (id): national commission on germplasm. djafar zr. 1992. the potential for swampland to achieve and preserve food self-sufficiency. proceedings of the national seminar: utilizing the potential of swampland for achieving and preserving food self-sufficiency. palembang (id): faculty of agriculture, universitas sriwijaya. hajoeningtijas od, purnawanto am. 2013. diversity of local rice in banyumas regency, central java. agritech. xv (2):69-77. icrr. 2016. research institute for rice planting in sukamandi. agricultural research and development agency. kodir ka, yuana j, triyandara. 2016. inventory and characteristics of swamp local rice morphology in south sumatra. germplasm bulletin 22 (2):101108. bb biogen. agricultural research and development agency. kosman e, jumberi a. 1996. display of farming potential in swampy land. in b. prayudi et al. (eds.). proceedings of the sut technology seminar on swamp and dry land. book i. balittra banjarbaru. kristia a, iskandar l. 2018. growth and production of several kalimantan local rice cultivars. agroforti bulletin 6 (2):260-70. manurung so, ismunadji m. 1988. rice: morphology and physiology of rice. agricultural research and development agency. bogor (id): food crop research and development center. noor m, fadjry. 2008. opportunities and constraints of agricultural development in the swampy agroecosystem: the case of primatani village in south kalimantan. proceedings of the national workshop on the acceleration of the application of science and technology and technological innovations in supporting food security and revitalization of agricultural development. jambi, 11–12 december 2007. jambi aiat, food security guidance board jambi province. bbp2tp. research and development agency. silitonga ts, somantri ih, daradjat aa, kurniawan h. 2003. guide to rice crop characterization and evaluation systems. national germplasm commission. agricultural research and development agency. agriculture department. simanulang za. 2001. selection criteria for agronomic and quality properties. training and coordination of participatory breeding programs (shuttle breeding) and multilocation tests. sukamandi, 9– 14 april 2001. research institute for rice, sukamandi. suprapto, dradjat a, 2005. plasma nutrition bulletin 11(1). swamp research center. 2008. increased productivity of lowland land through rice planting tolerant to drought and drought. http://balittra.litbang. deptan.go.id. waluyo, suparwoto. 2016. the role of new superior rice variety in increasing productivity and income of wetland farmers in oganilir regency, south sumatra. proceedings of the national seminar on the 53rdanniversary of the faculty of agriculture, sriwijaya university, palembang, 14 september 2016. isbn 978-979-8389-24-5. wardana hd. 2002. utilization of germplasm in natural herbal and cosmetics industries. germplasm bulletin 8(2): 84-5. biotropia vol. 29 no. 1, 2022: 56 68 doi: 10.11598/btb.2022.29.1.1626 56 incorporation of sodium hyaluronate and nyamplung (calophylum inophyllum) cake extract to improve bioplastic characteristic rini umiyati1*, chusnul hidayat2, ria millati2 and teguh ariyanto3 1department of food technology, faculty of engineering, universitas pgri semarang, semarang 50125, indonesia 2department of food and agricultural product technology, faculty of agriculture technology, universitas gadjah mada, yogyakarta 55281, indonesia 3department of chemical engineering, faculty of engineering, universitas gadjah mada, yogyakarta 55281, indonesia received 1 september 2021 / accepted 25 february 2022 abstract the cross-linking agent plays an important role in bioplastic mechanical properties. this study aimed to determine the effect of sodium hyaluronate (soha) as a synthetic cross-linking agent and the addition of nyamplung cake extract (nce) as an antimicrobial agent in the manufacture of bioplastic with hydroxypropyl starch (hps) as based ingredient using the thermo-compression method. the novelty of the study was thiocyanate (scn) formation in zone 3 (2,161.66-2,162.02/cm) and cyanate (c-n=o) in zone 6 (1,411.57 1,412.61/cm) of (1, 2 and 3%) soha bioplastic and cyanate formation in zone 6 and 7 (1,411.37-1,558.59/cm) of (1, 2 and 3%) soha-20% nce combined bioplastic originating from acetanilide group in soha and amide group in nce. the formation of scn and c-n=o in 2 and 3% soha bioplastic improved its sensitivity against gram-positive bacteria (staphylococcus aureus) indicated by 0.6 mm and 0.45 mm inhibition zone, respectively. c-n=o formed in (1, 2 and 3%) soha-20% nce combined with bioplastic had 3.25 mm average inhibition zone against gram-positive bacteria (s. aureus), 2.75 mm against gram-negative bacteria (escherichia coli), and 0.71 mm against fungi (aspergillus niger). the analysis of mechanical properties showed that an addition of 3% soha was able to increase tensile strength and modulus of elasticity while reducing elongation, water solubility and water vapor permeability. addition of (1, 2 and 3%) soha-20% nce resulted in a reverse effect. keywords: acetanilide, amide, cross-linking agent, cyanate, thiocyanate introduction natural polymers, such as starch, have various potential applications to replace commercial market-dominant petroleum-based plastics due to their abundant availability, degradability, renewability and low price (samsudin & hani 2019). the most critical property of plastic is its ability to be adjusted for specific purposes. the increasing utilization of starch for commercial use, such as to produce biodegradable thermoplastic, is expected to reduce problems caused by the petroleum-based polymer in solid waste disposal (manoi & rizvi 2010). as a renewable polymer source with filmforming ability, starch fulfills several requirements as an alternative for plastic, such as having abundant availability, high extraction yield, cheap, biodegradable and biocompatible. starch-based bioplastic is also odorless, tasteless, colorless, non-toxic, and semi-permeable to carbondioxide, temperature, oxygen, as well as fat and aroma components (shah et al. 2016). starch-based bioplastic as an environmentally friendly package has various disadvantages related to the hydrophilic and hygroscopic properties of starch. in this study, hydroxypropyl starch (hps) as a modified starch was used as the main material, because hps is more feasible to be processed into bioplastic due to its lower solubility temperature, with more transparent and flexible plastic properties (woggum et al. 2015). therefore, the mechanical properties of bioplastic can be improved and *corresponding author, email: riniumiyati@upgris.ac.id incorporation of sodium hyaluronate and nyamplung (calophylum inophyllum) – rini umiyati 57 adjusted by adding a plasticizer or cross-linking agent. a plasticizer is a group of small molecules that are able to blend and interact among polymer chains by intermolecular force, thus reduces glass transition temperature of a material to improve malleability, flexibility, extension and to soften the texture (wypych 2017). on the other hand, cross-linking mechanism works by formating bridges of immediate intermolecular bond by chemicals known as cross-linking agent. cross-linking means polymer molecules are interconnected by bond (canisag 2015). cross-linking agent is substances that are able to form permanent chemical bond among polymer chains, to increase material rigidity, firmness, strength, less flexible and increase glass transition temperature (tg). cross-linking agent might affect permeability property, thermal stability, glass transition temperature and the rate of wear (frost et al. 2013) due to formation of covalent or ionic bond connecting a polymer chain to the others which enhance polymer strength. cross-linking process is critical to modify starch properties by enhancing the starch’s intra and inter-molecular bonds in random patterns. the bonds tend to inhibit starch interaction to water while bringing structural integrity on starch-based biodegradable materials during hydraulic pressure and high humidity. crosslinking process can be done by starch treatment, both in semi-solid or pulp form, using reagent with ability to form ether or esther bond among hydroxyl (-oh) in starch molecule (manoi & rizvi 2010). cross-linking agents are able to form cross bond that enhances matrix density and tensile strength, and even permanent chemical bond among polymer chains, thus generate more rigid, strong, less flexible material and increase glass transition temperature (tg). polyfunctional chemicals such as phosphor oxychloride (pocl3), sodium trimetaphosphate (stmp), sodium tripolyphosphate (stpp), epiclorohydrine (epi), a mixture of adipic, acetate anhydride, and mixture of succinic anhydride and vinyl acetate are some of the common cross-linking agents for starch. in this study, we used sodium hyaluronate (soha) as a cross-linking agent. soha is a polymer of disaccharides, consisting of d-glucuronic acid and d-n-acetyglucosamine, linked by β-1, 4 and β-1, 3 bonded by glycosidicbonds, into polymer compounds that do not have uv absorbing chromophores (ruckmani et al. 2013). hyaluronate supports the structure of connective tissue by acting as a magnet to maintain fluidity and form viscous liquids with lubrication properties. soha has low toxicity and no report of negative effect on human (spec-chem ind. 2014). soha is also suitable as cross-linking agent and encapsulates various active ingredients (contipro 2018). this study used nyamplung cake extract (nce) as antimicrobial agent because of the ability of nce against gram-positive bacteria (staphylococcus aureus) and gram-negative bacteria (escherichia coli). bioplastic is more sensitive against gram-positive bacteria compared to gram-negative bacteria. bioplastic has the largest inhibition zone of 30 mm against staphylococcus aureus and that of 23 mm against escherichia coli. the abiltiy of bioplastic as antimicrobial agent might be related to the content of the bioplastic extract which serves as natural cross-linking and antimicrobial agents (umiyati et al. 2019). the purpose of this study was to determine the effect of soha as synthetic cross-linking agent and nyamplung cake extract (nce) as antimicrobial agent in the manufacture of bioplastic with hps as basic ingredient using thermo-compression method. this study demonstrated that the features of bioplastic produced showed better mechanical properties, having antimicrobial ability compared to the conventional bioplastic. materials and methods materials nyamplung cake (calophyllum inophyllum l.) was obtained from biodiesel industry in purworejo, central java, indonesia. nyamplung cakes were sorted and dried until reaching moisture content below 10-15%. the dried cakes were finely ground and sifted using a 120mesh sieve to obtain nyamplung cake powder. subsequently, 20 g of the nyampung cake powder was extracted by using 120 ml of 96% ethanol and placed on a hot plate stirrer at 80 °c for 1 h. the mixture was filtered using whatman filter paper no. 1. based on the optimal method biotropia vol. 29 no. 1, 2022 58 by chana-thaworn et al. (2011), the extracted liquid was then evaporated and ovendried at 50 °c for 24 h. other materials used in this study were hydroxypropil starch (hps), sodium hyaluronate (soha), glycerol 20%, etanol 96% and distilled water. instruments thermo-compression machine, ftir (thermo scientific nicolet is10), scanning electron microscopy (jeol jsm 6510) and texture analyzer (brookfield usa) were used in this study. bioplastic preparation various concentrations of soha (1%, 2% and 3% b/b) were mixed with 10 g of hps, 2 g of nyamplung cake extract, 1.5 g of glycerol and 1 g of distilled water, then mixed at 21,000 rpm for 45 min until homogen. the mixture was then made into bioplastic using thermocompression mold. the mixture was then placed in the middle of a 0.5 mm-thick aluminum frame sized 10 × 10 cm that was put between the two previously heated aluminum plates, molded at 140 °c and 250 kg/m2 pressure for 6 min in heated hydrolic pressure machine (rasheed et al. 2015). fourier transform infrared spectroscopy (ftirs) analysis is a method to identify structural changes in starch chains due to interaction among extract, glycerol, naha, and hps molecules (bilal et al. 2015). ftir spectra of bioplastic was measured by thermo scientific nicolet is10, equipped with smart atr diamond at an area of 650-4,000/cm using 32 scanning and resolution of 8/cm. scanning electron microscopy (sem) was used to analyze microplastic structure of the bioplastic. sample of bioplastic was stored in a desiccator with p2o5 absorbent for two weeks to ensure no moisture left in the bioplastic sample. for cross-section observation, the bioplastic sample was frozen in n2 liquid for cryofracture preservation (espinel et al. 2014). all samples were placed in bronze stub and covered with gold sheets before imaging. micrograph of the surface of bioplastic and fracture was obtained using sem (jsm-6510la) at 10-300,000x magnification and 1-10 nm resolution. characterization of bioplastic physical properties mechanical properties such as tensile strength (σ), elongation (ε), modulus of elasticity (y), water solubility (ws) and water vapor permeability (wvp) were measured based on the methods in astm d638. prior to mechanical properties analysis, the bioplastic sample was conditioned at rh of 50 ± 5 and 23 °c for 48 h in humid chamber. tensile strength at room temperature was measured using brookfiled usa analyzer texture. bioplastic specimen was cut according to the method by astm d1708 at a constant pull rate of 5 mm/minute. solubility measurement of bioplastic sample in water was conducted by drying the bioplastic sample at 105 °c and then weighed as m3. subsequenty, the bioplastic sample was soaked in 50 ml distilled water at 24 °c for 6 h, then ovendried at 105 °c for 24 h and weighed as m4 (hassannia-kolaee et al. 2016; kumari et al. 2017), before calculation using formula below (1): ws = m3 – m4 x 100 ..….……………….. (1) m4 where: m3 = weight of bioplastic sample before drying m4 = weight of bioplastic sample after drying water vapor permeability (wvp) was analyzed using standard method in astm (1996) e96. the bioplastic sample was placed as a cover on circle permeation cells sized 4 cm in diameter containing silica gel (0% rh) and placed in a desiccator which was previously filled with saturated sodium chloride liquid (75% rh), then stored at 30 °c. water vapor permeability rate was measured by permeation cell weighing at a frequency of every 30 min to obtain several points. the wvp was counted using a formula (2) developed by wirawan et al. (2012): wvp = wptr x m ….…………….. (2) ps (rh1 – rh2) where: wvp = water vapor permeability (g.mm/ kpa.s.m2) wvtr = mass increase (g) ps = pressure of saturated water vapor (pa) incorporation of sodium hyaluronate and nyamplung (calophylum inophyllum) – rini umiyati 59 rh1 = relative humidity in desiccator rh2 = relative humidity in permeation cell m = average thickness of bioplastic layer (m) characterization of bioplastic antimicrobial properties disk agar method was used for analyzing the antimicrobial activity of bioplastic sample using distilled water as solvent. as much as 0.1 ml of 10% bacteria and fungi suspension was inoculated on mueller hinton agar and on potato dextrose agar media, respectively. bioplastic sample of 1 cm diameter was placed on a sterile disk sized 6 mm diameter and let to dry. subsequently, the bioplastic sample was placed on the surface of culture media in a petri dish that was previously inoculated with tested microorganisms, then incubated at 37 °c for 1-2 days for bacteria and at 30 °c for 2-3 days for fungi, based on optimum condition reported by chanwitheesuk et al. (2007). results and discussion bioplastic characteristics mechanical properties comparison of mechanical properties in the form of tensile strength (σ), elongation at break (ε) and modulus of elasticity (y) of bioplastic prepared with (1, 2 and 3%) soha, combination of (1, 2 and 3%) soha-20% nce compared to that prepared by using 20% nce and control is presented in figure 1. those mechanical parameters depend on microstructure characteristics (aguirre et al. 2013). bioplastic thickness ranged from 0.24 to 0.32 mm (data not shown). results of this study showed that the control bioplastic had significant increase of tensile strength (σ) compared to 3% soha bioplastic and 20% nce bioplastic, by 23.7% and 50.8%, respectively, while that of (1, 2 and 3%) soha20% nce bioplastic had significant reduction by an average of 62.4% (fig 1). in terms of elongation at break (ε), the (2 and 3%) soha bioplastic showed significant reductions compared to the control bioplastic by an average of 32.4%, while the (1, 2 and 3%) soha-20% nce bioplastic had a significant increase of 224.2%. for modulus of elasticity (y), there was significant difference between the control and (1, 2 and 3%) soha, (1, 2 and 3%) soha-20% nce combination, and 20% nce. this indicated that soha addition to 3% could function as effective cross-linking agent to improve bioplastic mechanical properties. soha works to facilitate esterification of starch oh groups in overcoming the poor polymer compatibility by incorporating compatible substances (ortega-toro et al. 2014). figure 1 effect of soha addition and combination of soha-nce on the mechanical properties of hps bio-plastic notes: bar indicates mean ± standar deviation. different letters in the same column indicate significant differences (p < 0.05). biotropia vol. 29 no. 1, 2022 60 soha is a widely distributed natural mucopolysaccharide polymer, which in mammals is known as connective tissue. crosslinking process is done by soha to increase the in vivo retention time, so that the hydroxyl groups within the polymer chains bind to one another. the cross-linking reaction in soha is carried out in a homogeneous aqueous alkaline solution which can enhance anti-biodegradation ability by increasing the degree of cross-linking and extending the in vivo retention time (xuejun et al. 2015). utilization of the modified hps starch as basic material may also serve to improve bioplastic mechanical properties due to its high amylose content of modified starch. therefore, hps is feasible as bioplastic raw material, has mechanical strength and functions as good oxygen inhibitors (woggum et al. 2015). concentration of cross-linking agent, ph, treatment temperature and storage period might determine cross-linking bond. reaction condition can be varied according to the type of cross-linking agent (canisag 2015). utilization of soha-nce combination reduced σ and y. higher concentration of soha on nce resulted in a lower σ. these effects were probably due to the loss of amide group in nce bioplastic (20% extract) by the combination. on the other hand, soha-nce combination increased ε, which probably due to h2o formation from reactions involving soha and hydroxyl groups from hps starch, glycerolsoha and amide groups of nce-soha. c-n=o is an intermediate product of the first stage of thiocyanate hydrolysis, which was then hydrolyzed into ammonia and bicarbonate (doble & kumar 2005). stronger interaction of hps-soha-glycerol reduces y and maximum tension as well as tension-when-separate resulted in a more flexible bioplastic, and therefore, showing the function of soha as cross-linking agent (contipro 2018). modified starch was reportedly a more stable raw material for making bioplastic compared to native starch (gutiérrez et al. 2015). bioplastic preparation might also play a role as thermo-compression utilizes high temperature and hydroulic pressure. there was also the possibility of maillard reaction which can form more compact networks (leceta et al. 2013). hps as raw material might affect bioplastic mechanical properties due to the effect of hydroxypropylation that interfere the interand intra-molecular hydrogen bond of the starch chain. the effect can enervate starch granule while increasing starch chain movement in an amorph area and enhancing hps swelling power compared to native starch with increasing molar substitusion (woggum et al. 2015). soha addition brought significant increase on σ at ≥ 3%, but nce addition in soha bioplastic did otherwise. water solubility (ws) soha and soha-nce combination affected bioplastic water solubility (ws) in terms of bioplastic integrity in watery media. high solubility of bioplastic indicates low water resistency (gutiérrez et al. 2015). ws is defined as the ratio of dry matter in water, such as bioplastic soaked in distilled water (hassanniakolaee et al. 2016). results of this study showed that ws of bioplastic made by using soha, combination of soha-nce and nce were significantly lower than that of control (fig. 2). soha bioplastic when compared to control had an average of lower ws values by 6.6%, while bioplastic made by using soha-nce combination had an average of lower values by 15.9%, then bioplastic made by using nce had an average of lower values by 20.95%. insignificant ws decrease was observed in 2% soha compared to 3% soha bioplastics and vice versa, as well as in 1% soha-20% nce combination compared to nce bioplastics (fig. 2). this showed that nce addition affected the molecular structure of soha bio-plastic. during bioplastic preparation, phosphor of stmp (as a cross-linking agent in making modified starch) reacted with hydroxyl (-oh) group of starch to form phosphate distarch (cross-bond) and other phosphate derivates as indicated by the ft-ir results. cross-linking process enhances granule structure starch and inhibit both water absorption and starch solubility, thus limit the mobility of starch chain in amorph area (manoi & rizvi 2010). bioplastic prepared by hps-soha had lower solubility compared to control, with increasing concentration of cross-linking agent. this incorporation of sodium hyaluronate and nyamplung (calophylum inophyllum) – rini umiyati 61 figure 2 effect of soha addition and combination of soha-nce on the water solubility of hps bio-plastic notes: bar indicates mean ± standar deviation. different letters in the same column indicate significant differences (p < 0.05). finding was comparable to previous study conducted by (manoi & rizvi 2010) which indicated that the formation of phosphate distarch act as cross-linking agent might limit swelling and hydration of starch granules. hyaluronate is known as a hydrophilic polymer derivative polysaccharide that has ability as an enhancer of percutaneous penetration by changing composition of cells of tightly arranged materials into more tenuous so that permeability is increased. permeability has an inverse relationship with ws so that the addition of soha can decrease ws of bioplastics (djajadisastra et al. 2014). ws is an important property of bioplastic for food prevention application, particularly in high water activity, or when bioplastic must be in contact with water, such as during food processing. generally, high solubility indicates lower water endurance, though high solubility might be advantageous for some applications (chana-thaworn et al. 2011). according to (spec-chem ind. 2014), soha can function as a lubricant and film maker, where soha is a high molecular weight polymer which is a strong lubricant and film maker. soha as cross-linking agent can be observed from its ability to reduce the ws of bioplastic. it was contrary to the results obtained from soha-nce that increased ws due to water formation from reaction between hps, soha and nce, showing the hydrophilic property of bioplastic. water vapor permeability (wvp) wvp is a bioplastic ability to withold penetrating water vapor and provides information on water vapor transmission through bioplastic as a critical property of food packaging (leceta et al. 2013). bioplastic permeability is determined by the difference of water vapor concentration between one-side to the other side of bioplastic, with higher difference indicating faster mass transfer and also affected by bioplastic thickness. the results of wvp analysis showed that the control bioplastic had significant difference compared to bioplastic prepared by using 3% soha, sohance combination and nce. increasing concentration of 3% soha and 20% nce bioplastic was able to decrease wvp by an average of 3.66% and 3.89%, respectively, while (1, 2 and 3%) soha-20% nce combination on the contrary increased wvp by an average of 8.25% (fig. 3). biotropia vol. 29 no. 1, 2022 62 figure 3 effect of soha addition and combination soha-nce on the wvp of hps bioplastic notes: bar indicates mean ± standar deviation. different letters in the same column indicate significant differences (p < 0.05). the results of wvp analysis showed that the control bioplastic had significant difference compared to bioplastic prepared by using 3% soha, soha-nce combination and nce. increasing concentration of 3% soha and 20% nce bioplastic was able to decrease wvp by an average of 3.66% and 3.89%, respectively, while (1, 2 and 3%) soha-20% nce combination on the contrary increased wvp by an average of 8.25%. previous study conducted by gutiérrez et al. (2015) reported a tendency of increasing wvp and ws in bioplastic containing native starch, which indicated higher rate of hydrophilic property. another possibility for increasing wvp in (1, 2 and 3%) soha-20% nce bioplastic was by implementing higher hydrophilic property in the (1, 2 and 3%) soha20% nce bioplastic compared to (1, 2 and 3%) soha and 20% nce bioplastic. water holding capacity of soha is very high compared to other moisturizers. the constanta of soha moisture evaporation rate is lower than that of other moisturizers. this shows that soha has strong water retention properties, with moisture evaporation rate constanta of 8.0 ± 0.1x100-2/min (spec-chem ind. 2014). the main function of bioplastic or edible film is to inhibit vapor transfer from the surrounding environment to the food covered by the bioplastic, or between two different components of food products. therefore, wvp value needs to be as low as possible (chana-thaworn et al. 2011). wvp is also determined by bioplastic thickness as well as by the glycerol and starch concentrations. thickness of hydrophilic film might determine wvp, with higher thickness resulted in higher resistance against mass transfer and increasing partial water pressure in equilibrium of film inner surface, thus enhance water vapor permeability of film hydrophilic property with higher thickness (gutiérrez et al. 2015). the enhancing wvp is caused by the changes of partial water vapor pressure of the exposed inner surface of the film. separate utilization of soha and 20% nce was able to reduce wvp, indicating their function as cross-linking agent due to their ability to inhibit water vapor penetrating the bioplastic. on the other hand, different results occurred in bioplastic made from a combination of (1, 2 and 3%) soha-20% nce due to deteriorated cross-linked bond from water formation which subsequently caused bioplastic became hydrophilic. fourier transform infrared spectroscopy (ftir) analysis the recent development of material technique has resulted in demand increase for fast, reliable and non-destructive analytical methods to control preparation process and physicochemical characterization. ftir provides general information on chemistry of the surface and overall characters as well as their functions in polymer (ricci et al. 2015). ftir spectra of control, (1, 2 and 3%) soha, (1, 2 and 3%) soha-20% nce and 20% nce bioplastic was presented to measure the results of reaction involved in bioplastic preparation made from hps with soha as cross-linking agent, as well as combination of soha-nce (fig. 4). incorporation of sodium hyaluronate and nyamplung (calophylum inophyllum) – rini umiyati 63 figure 4 ftir spectra of control, (1, 2 and 3%) soha, (1, 2 and 3%) soha-20% nce and 20% nce bioplastics differences in several peaks of bioplastic made by using 20% nce compared to (1, 2, and 3%) soha and combination of (1, 2 and 3%) soha-20% nce (fig 4; table 1). zone 1 representing bioplastic made by using soha at 3,285.91-3,288.09/cm; soha-nce at 3,287.46 3,297.68/cm; and nce at 3,282.13/cm showed oh bond vibration, related to bound, free, interand intra-molecular hydroxyl group (bilal et al. 2015). zone 2 representing bioplastic made by using soha at 2,926.41-2,927.94/cm; sohance at 2,927.28-2,927.61/cm; and control at 2,924.49/cm indicated stretching vibration of ketones (ch3-co). zone 3 representing bioplastic made by using soha at 2,161.66 2,162.02/cm and 1% soha-20% nce showed stretching vibration of scn (thyocianates). scn can be derived from cyanide-rich plants, such as cassava, sweet potato, corn, sugar cane, sorghum and linseed (chandler & day 2012). scn presence in (1, 2, and 3%) soha bioplastic was probably derived from corn starch as hps raw material. scn functions as host defense and as cyanide detoxification product. scn is a preferable substrate for lipid peroxidation (lpo), for catalytic reduction caused by hydrogen peroxyde (h2o2) into hypocyanate acid (hoscn) (chandler & day 2012). eighty percent (80%) scn in the body comes from cyanide absorbed from food which transformed into scn in metabolism (simeonova & fishbein 2004). in addition, the presence of scn vibrations may originate from reaction between acetanilide group in soha and sulfate compounds from hps. zone 4 representing (2% and 3%) soha and (1, 2, and 3%) soha-20% nce bioplastic showed oh-p=o bond area, which arguably indicated cross-linking activity through transformation of starch granule by bifunctional or multifunctional reagents which were able to form ether or ester bond with hydroxyl group of starch (gui-jie et al. 2006). in zone 8 (1,411.37 1,558.59/cm) of (1, 2 and 3%) soha-20% nce bioplastic and zone 9 (1,411.571,412.61/cm) of (1, 2 and 3%) soha bioplastic, cyanate (c-n=o) was formed. whereas, in the 20% nce bioplastic at 1,704.17/cm, amide group was present. this indicated that nce combined with (2 and 3%) soha removed scn vibration. besides hydroxyl group and glycerol, amide group from nce and acetanilide from soha also played important roles in determining bioplastic mechanical properties as well as cyanate (c-n=o) formation observed in peak (1,558.46-1,558.59) (fig. 4; table 1). there was a higher presence of c-n=o in (1, 2 and 3%) soha-20% nce compared to that in (1, 2 and 3%) soha bioplastic which probably caused by nce addition, in dosedependent manner with the decreasing cyanide concentration due to oxidation. cyanide can be photocatalytic oxidized into c-n=o, biotropia vol. 29 no. 1, 2022 64 20% nce bioplastics incorporation of sodium hyaluronate and nyamplung (calophylum inophyllum) – rini umiyati 65 particularly in alkali media (destanoğlu & gümüş-yılmaz 2016). the photocatalytic oxidation was shown by increasing the rate of c-n=o formed in (1, 2 and 3%) soha-20% nce bioplastic due to nce addition, which low ph was caused by hydrolyzed tannin dominated by gallic acid. in zone 10, 13 and 14, both soha and soha-nce bioplastics, ether group was formed as an indication of crosslinking process between hydroxyl group of hps and gallic acid or amide from hydrolyzed tannin. moreover, peaks dominated by ether and aromatic groups also found at peaks 15, 16, 18 and 19. utilization of (1, 2 and 3%) soha in hps bioplastic preparation caused scn and c-n=o formation, while 20% nce addition caused increasing c-n=o concentration that arguably determined bioplastic mechanical properties and antimicrobia activity. scanning electron microscopy analysis micrograph resulted from the scanning electron microscopy (sem) analysis was used to study the tensile surface fracture of the eight bioplastic samples and microstructure changes caused by the addition of (1, 2 and 3%) soha and combination (1, 2 and 3%) soha-20% nce. the control bioplastic had homogenous surface microstructure with cracks in several parts. the microcracks formation was possibly caused by the absence of cross-linking agent in bioplastic (ortega-toro et al. 2014). addition of cross-linking agent can be detected by white spots of agglomeration on bioplastic surface made by applying addition of (2 and 3%) soha. the spots appeared as a side effect of uneven mixing or lack of solvent to dissolve crosslinking agent. different to bioplastic microstucture made by using (1, 2 and 3%) soha, those made by using (1, 2 and 3%) soha-20% nce combination had a very significantly different result compared to control, (1, 2 and 3%) soha and 20% nce. addition of 20% nce resulted in aggregated formation on bioplastic surface, hence more flexible compared to other bioplastic samples. the flexibility was as also indicated by higher ε of bioplastic made by using (1, 2 and 3%) soha-20% nce compared to the other bioplastic samples (fig. 5). sem micrograph of the eight bioplastic samples showed significantly different results, with (2 and 3%) soha bioplastic had agglomeration as indication of cross-linking agent addition, whereas (1, 2 and 3%) soha bioplastic had more flexible texture from water formation because the final reaction product rendered hydrophilic property of bioplastic. nce contains organic acid having plasticizing effect that contributes to inhibiting starch partial recrystalization (ortega-toro et al. 2014). control 1%soha 2%soha 3%soha 1%soha 20%nce 2%soha 20%nce 3%soha 20%nce 20%nce figure 5 sem micrographs of the control, (1, 2, and 3%) soha, (1, 2, and 3%) soha-20% nce and 20% nce bioplastics biotropia vol. 29 no. 1, 2022 66 antimicrobial analysis antimicrobial capabilities from control bioplastic was compared to that of (1, 2 and 3%) soha, (1, 2 and 3%) soha-20% nce and 20% nce bioplastics by determining the inhibition zone diameter against gram-positive (s. aureus) and gram-negative (e. coli) bacteria, as well as fungi (a. niger). the results showed that control and 1% soha had no antimicrobial activity, although the ftir spectrum analysis showed that there is a cyanate suspected as an antimicrobial agent. the lack of antimicrobial activity was probably due to the small concentration of the cyanate. the 2 and 3% soha had inhibition zone indicating antibacteria activity against grampositive bacteria (s. aureus) with diameter of 0.6 mm and 0.45 mm, respectively (table 2). the presence of antimicrobial activity was probably caused by the presence of scn and c-n=o in the soha addition during bioplastic preparation, or in the soha addition within the hps which generated scn and c-n=o groups. scn can be generated from catalysis of cyanide (fawell et al. 2007). other study conducted by (destanoğlu & gümüş-yılmaz 2016) mentioned that scn is a chronic exposure of low dose of cyanide. besides, scn is utilized in various industrial process, such as photofinishing, production of herbicide and insecticide, colorant, production of acrylic fiber, thio-urea production, metal separation and coating, as well as soil sterilization and corrosion inhibition (doble & kumar 2005). nce addition to (1, 2 and 3%) soha-20% nce bio-plastic, was able to improve antimicrobial properties against gram-positive (s. aureus) and gram-negative (e. coli) bacteria as well as fungi (a. niger). antimicrobial activity of (1, 2 and 3%) soha20% nce bioplastic was considered better than the control, (1, 2 and 3%) soha and 20% nce, which probably due to the presence of c-n=o that was predictably increased with the increasing nce. the existence of c-n=o is probably derived from acetanilide group found in soha and amide group contained in nce. acetanilide is the first derivative of aniline having analgesic and antipyretic properties (khan et al. 2016). c-n=o concentration can be gradually increased with time, in line with the reduction of cyanide due to oxidation (destanoğlu & gümüş-yılmaz 2016). the analysis results showed that (1, 2 and 3%) soha-20% nce bioplastic was more sensitive against s. aureus than e. coli or a. niger (table 2). this indicated that s. aureus was more sensitive to c-n=o in (1, 2 and 3%) soha-20% nce bioplastic. different results were obtained by 20% nce bioplastic containing amide, toward which e. coli had higher sensitivity than s. aureus, with no antimicrobial activity against a. niger. results of this analysis also indicated that gram-positive bacteria (s. aureus) were more sensitive toward scn or c-n=o in both 3% soha and (1, 2 and 3%) soha-20% nce bioplastics compared to gram-negative bacteria (e. coli) and fungi (a. niger). however, c-n=o compounds provided antimicrobial activity for (1, 2 and 3%) soha-20% nce bioplastic against s. aureus, e. coli and a. niger. on the other hand, amide content in 20% nce showed higher sensitivity against e. coli than s. aureus, with no antimicrobial activity against fungi (a. niger). table 2 inhibition zone obtained in the antimicrobial analysis no sample inhibition zone against e. coli s. aureus a. niger 1 hps-0 0a 0a 0a 2 hps-0-soha-1 0a 0a 0a 3 hps-0-soha-2 0a 0a 0a 4 hps-0-soha-3 0a 0.45 ± 0.26b 0a 5 hps-20-soha-1 3.17 ± 0.29b 3.57 ± 0.55c 0.77 ± 0.12b 6 hps-20-soha-2 2.67 ± 0.06c 3.23 ± 0.31c 0.80 ± 0.00b 7 hps-20-soha-3 2.40 ± 0.17d 2.97 ± 0.42d 0.57 ± 0.06c 8 hps-20 5.07 ± 0.20e 4.32 ± 0.25e 0a incorporation of sodium hyaluronate and nyamplung (calophylum inophyllum) – rini umiyati 67 conclusion addition of (1, 2, and 3%) soha as crosslinking agent and combination of (1, 2, and 3%) soha-20% nce had significant effects on mechanical properties and antimicrobial activity of hps bioplastic. soha at 2 and 3% in hps bioplastic improved mechanical properties in form of σ and y increase while reducing ɛ, ws and wvp. the reduction was caused by the presence of scn and c-n=o which probably derived from acetanilide group found in soha causing antimicrobial activity against grampositive bacteria (s. aureus). on the contrary, combination of (1, 2 and 3%) soha-20% nce degraded mechanical properties by reducing σ and y followed by ɛ, ws and wvp decrease, which probably due to water formation from reactions involving hps, 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'use of starch in food packaging'. in starch-based materials in food packaging. academic press. p.229-56. https://doi: 10.1016/b978-0-12-809439-6.00008-x. shah u, naqash f, gani a, masoodi fa. 2016. art and science behind modified starch edible films and coatings: a review. compr rev food sci food saf 15:568-80. https://doi.org/10.1111/1541-4337. 12197 simeonova fp, fishbein l. 2004. hydrogen cyanide and cyanides: human health aspects. geneva (ch): world health organization. spec-chem ind. 2014. sodium hyaluronate. spec-chem, china. umiyati r, millati r, ariyanto t, hidayat c. 2019. physical and antimicrobial properties of hydroxypropyl starch bio-plastics incorporated with nyamplung (calophyllum inophyllum) cake extract as an environmentally friendly food packaging. e3s web of conferences 125, 07011. international conference on energy, environment, epidemiology and information, universitas diponegoro. woggum t, sirivongpaisal p, wittaya t. 2015. characteristics and properties of hydroxypropylated rice starch based biodegradable films. food hydrocoll 50: 54-64. https://doi.org/ 10.1016/ j.foodhyd.2015.04.010 wypych g. 2017. typical methods of quality control of plasticizers. a handbook of plasticizers (3rd edition). toronto (ca): chemtec publishing. pp.85-109. https://doi.org/10.1016/b978-1895198-97-3.50003-2 xuejun c, xiaobin z, zou, y. 2015. cross-linked sodium hyaluronate gel for tissue filler for plastic surgery and preparation method thereof 1(19): 1-4. united states: 14/432 (a patent). biotropia vol. 28 no. 3,2021: 204 213 doi: 10.1 1598/btb.2021.28.3.1330 understanding natural regeneration in burned tropical peatland: a strategy to accelerate the forest recovery process dwi fuji lestary, taryono darusman, fransisicjs a. harsanto, desra arriyadi and ginanjar katingan-mentqa project) pt rimba makmur utama) jalan kantin no. 8, bogor 16121, indonesia received 11 february 2020/accepted 17 june 2020 abstract the 2015 massive forest fires across central icalimantan left large areas of burned peatlands that need to be restored, demanding substantial resources. to understand natural regeneration of burned peatland and how planting might accelerate its recovery process, the regrowth of burned peatlands was measured with different fire frequencies. three transects were established. each transect consisted of five 20 x 20 m plots with 30 m intervals. all woody species were recorded and classified into three categories as new regrowth, regrowth, and remnant trees that survive from the last fire. in addition, data from fifteen 2 x 2 m permanent natural regeneration plots and evaluation on survival rate of 2017 planting were also analyzed. research results suggested that the absence of remnant trees due to frequent or severe fires does not always impede the emergence of new recruitments, although diversity of forest regrowth is likely to be affected by its proximity to forest remnants. the floristic composition also showed a domination of pioneer species, giving evidence that forest recovery is initiated. our study indicated that the combination of fire frequency, fire intensity, and proximity to remnant forest will produce different degrees of forest recovery, and the result will be unique for each site. it is concluded that to support the recovery process through planting activity, the successional stage of the designated sites should be determined first. the common planting method on large areas with mixed climax-high valuable trees was not beneficial unless the restoration sites had reached the later stages of succession. keywords: natural regeneration, peatland £ire, restoration, succession, tropical peatland introduction indonesian peatlands account for 14.91 milhon ha and contribute to more than 35% of the world's peatlands (osala e t al. 2016). however, although peatlands store a substantial amount of carbon compared to other land uses and provide important hydrological services to the surroundmg areas, their presence is threatened by human disturbance, especially due to the need to clear vegetation for agricultural lands (page e t al. 2009). osaki e t al. (2016) stated that the agricultural activity on peatlands in indonesia has a long and complex historical substance, with fires playing an important role in *corresponding author, email: dwi.puji@ptrmu.com this story, although fire is not likely naturlly occurred on peat swamp forest ecosystem. fires have been commonly used to clear peatland forests, and h s such burning become massive in areas whenever a long drought occurs, such as in commonplace during el nino climatic phases (page e t al. 2009; shiodera e t al. 2016). from 1990 to 2015, about 61% of indonesia's peatland forests were lost or damaged, with only 6% of virgin peatland forest remaining by 201 5 (graham e t al. 201 7). the last massive forest fire in indonesia was in 2015, whch burned 2.6 millions ha of lands, where 33% of them were peatlands (glauber & gunawan 2015). noxious haze and tonnes of greenhouse gases (ghgs) were released, catching national and international attention as well as raising awareness. understanding natural regeneration in burned tropical peatland: a strategy to accelerate lestari et al this disaster spurred the needs for better efforts in peatland restoration and fire prevention. unfortunately, planting on such a remote and wide landscape requires a substantial amount of capital, ranging from 500 to 3500 usd per hectare (giesen & sari 2018). on the other hand, relying on natural regeneration unlikely to be enough as it may result in low diversity (blackham et al. 2014). moreover, unlike numerous studies on dryland or tropical forests, studies on the recovery process of peatland after fire are less common or still at their early stage (graham et a/. 2017; page et al. 2009; shiodera et al. 2016). this results in considerable uncertainties around the effectiveness of current peatland restoration practice. the hypothesis was that the result of forest recovery over times w d vary depenchg on the fire frequency, fire intensity, and proximity to remnant forest. to assess the vegetation recovery process and succession on recently burned peatland, their natural regeneration on sites which have dfferent fire history and proximity to the nearest remnant forest was measured. species composition and diversity after fires were compared, and the effects of the current practice of tree planting on peatland restoration was investigated. specifically, this study aimed to investigate whether the common practices on peat forest revegetation were parallel with natural process of forest succession. moreover, ths study was part of an ongoing vegetation survey focuses on peat swamp forest succession after fire. this study only examined vegetation or floristic component of peatland restoration, whde restoration related to the hydrological function and other components of biodiversity are not covered. materials and methods study location the study was conducted along the big canal on the southern area of the ecosystem restoration concession of pt rimba makmur utama, also known as the icatingan-mentaya project, ieatingan district, central icalimantan located at 2o32'36.8" s to 3o01'43.6" s and 113°00'29.7" e to 113°18'57.4" e (fig. 1). the study location is a typical degraded peatland forest mostly damaged by logging activities in the late 1970s to early 2000s, and subsequent canal drainage mainly for agricultures and transportation network, as well as forest fires. peat depth on the study location ranged from 300 cm to 450 cm, with annual precipitation of about 2820 mm (information was collected from the weather station at haji assan sampit airport by rossita e t al. (201 8). in the late 1990s, the public works agency (dinas pekejaan urnurn) constructed a 24 km long canal to connect icotawaringin timur and icatingan district. nowadays, about 6 km of the canal cuts across the restoration concession and has become the main cause of the surrounding peat drainage. before the concession was granted in 2013, fires occurred almost annually along the banks of the canal. vegetation along the canal is dominated by ferns and shrubs, with a few clumps of pioneer species. biotropia vol. 28 no. 3,2021 study location legend n ~0n-m bdudanes canal figure 1 map of the study location on the ecosystem restoration concession note: the map was displayed using planet lab satellite image year of 2017. plot observation and data analysis nata~a l saccession plots diameter at breast height (dbh, if plant height is 1.3 m or more). local names were identified on the field by a well-trained local botanist and their three transects within the natural succession scientific names were identified using the guide observation site (fig. 1) were developed in april book of pt rimba makmur utama. during the 2018 on the southeast part of the canal to observation, all transects were covered by observe natural regeneration of peatland forest shrubs and ferns with patchy pioneer tree after fires. the first transect (transect fb, species (fig. 2). both ferns and shrubs are frequently burned) was located on an area that typical species that emerge on peat ecosystem was close to the canal (0.5 krn distance). it was after fire such as stenochlaenapaiastm's and cypems burned three times in the period of 2010-2015 rotgndzls with a height of more than 2 m. and was far from the forest edge (1.5 km analysis was then conducted by dividing all distance). the second transect (transect fbf, woody plants into three classes: 1. new frequently burned, close to forest edge) was also regrowth, which included all woody plants with burned three times in between those years but was located far from the canal (1 km distance) and near to the forest edge on the west side of the transect. the last transect (nb, newly burned) has never been burned before 2014 and only caught fire once in 2015. this transect was also isolated from the nearest canal and forest edge. before the attack of frequent fires, the land cover of the three transects was a secondary forest. details regarding the fire history of the study location can be seen in table 1. five 20 x 20 m plots were established on each transect (fig. 2). each plot was located 30 m away from each other. in total, 15 observation plots (0.6 ha) were measured in dus study. within each plot, all woody plants were counted and measured in terms of their bole diameter (if plant height is less than 1.3 m) or height i 150 cm that were assumed to emerge later after fire; 2. regrowth, which included all woody plants with height > 1.5 m and dbh < 10 cm that were assumed to emerge soon after the fire in 2015; 3. survivor, which included the remaining trees surviving from the last fire with dbh 2 10 cm. species composition, density, species richness (shannon's diversity index), and species evenness (pielou's evenness index) were analyzed to examine the structure and composition of the existing natural regeneration and stage of the succession process. bray-curtis dissidarity index and nmds ordination were also calculated to understand the pattern of species composition among transects and acquire the notable species within each study site. all analyses were performed using r version 3.4.0 with vegan package 2.4-3 (oksanen etal. 2017). understanding natural regeneration in burned tropical peatland: a strategy to accelerate lestari e t all table 1 fire history on the three transects from 2010 to 2018 fire history (4 symbol means there was fire in the conspecific year) years transect fb transect fbf transect nb notes: fire and hotspot data were analyzed from landsat 5, 7, 8 and sentinel 2 satellite images. each image on each year were displayed on composite mode using similar band combination of swir, nir and green, and hotspot historical data acquired from national institute of aeronautics and space of indonesia (lapan) website were overlaid. n a t ~ r a l regeneration plots the typical species that appear after fires on peatland forest. in total, fifteen 2 x 2 m permanent plots were established within the ecosystem restoration concession area of pt runba makrnur utama, which was distributed on the southern part of the canal (fig. 1, natural regeneration plots). plots were located on degraded peatland that burned almost annually before 2015. the last fire incidence was in 2014. in 2015, these plots were established, and all seedlings less than 1.5 m in height were recorded every six months. the trend of natural regeneration composition from year to year was then analyzed to examine tree plantings on degrdded peatland as an ecosystem restoration concession, the concession is responsible for planting activity on their areas, and the result is monitored periodically. in january 2017, in total, 19,670 seedlings of local tree species were planted. about 2.5% of seedlings were monitored and the survivor rate was calculated in 9 months and 17 months after planting. biotropia vol. 28 no. 3,2021 results and discussion species composition on different sites fires that attacked the study area resulted in the low density of remaining trees, as shown in figure 3. transect fb likely received a higher degree of fire incidence as only 5 trees/ha were left on this site, while transects fbf and nb had hgher density and more diverse remnant tress (fig. 4). the study results suggested that the proximity to main canal influenced the intensity of fire, where fires normally started from the surroundmg canal. transect fbf had the highest density and more surviving tree species, possibly due to the lower severity of fire on ths site, as well as its proximity to the forest edge. in addition, based on our observation, limited number of surviving trees indicated that those native climax-species were mostly not equipped with natural mechanism to survive under fires, as fire was not a natural phenomenon in tropical peat swamp environment unlike in dry sclerophyll forests where fires could occur naturally. the only protection was the wet and inundated peat environment that mostly absent when peat was drained. fb fbf n b -new regrowth .regrowth survivor figure 3 the average density of woody species on different locations notes: transect fb: frequently burned, close to canal, far from forest; transect fbf: frequently burned, far from canal, close to forest edge; transect nb: only burned once in 2015, far from forest and canal, divided by three size classes: new regrowth, regrowth, and survivor. error bars indicated standard error. php new regrowth .regrowth .survivor figure 4 number of species on different locations notes: transect fb: frequently burned, close to canal, far from forest; transect fbf: frequently burned, far from canal, close to forest edge; transect nb: only burned once in 2015, far from forest and canal, divided by three size classes: new regrowth, regrowth, and survivor. understanding natural regeneration in burned tropical peatland: a strategy to accelerate lestari et al: it is likely that the availability of survivor recruitments after disturbance is often trees does not guarantee the emergence of new recruitments, although cleary and priadjati (2005) stated that the presence of remnant trees might be important to accelerate the succession process. three years after the last fire incidence in 2015, recruitments of woody species were abundant on the three transects. even though only a small number of remaining trees were present on transect fb, recruitments on this site were very dense (in total 800 recruitments/ha appeared after the 2015 fire) compared to transect fbf and transect nb. on the other hand, although higher recruitment density can be found on transect fb, the density itself is not parallel to the species diversity, which is relatively low at transect fb. the 800 recruitments/ha on &is transect were composed of 21 species only, while transects fbf and nb contained 28 and 24 species of recruitments, respectively. this indicates that the density of the remnant determined by features of its local landscape. bray-curtis dissimilarity index calculation among the three transects also indicated that transect fb was the least similar compared to the other two transects, while transects fbf and nb share more similarities (table 3). the nmds ordnation displayed clearer segregation by showing that transect fb tends to segregate from the rest of the transects. this transect was characterized with more long-lived pioneer species, such as melalezlca leacadendron, melicope lanzl-ankenda, jyggiam sp., and macarangapminosa, whde transects fbf and nb were also rich with other generalist and late successional species, such as alstonia scholaris, ficzls spp., and nepheliam mangd_yi. unhke other researchers who reported that burned peatland forests in icalimantan were normally dominated by pioneer species especially combretocapzls and cratoxylm species (blackham et al. 2014; graham et al. 2017; shiodera e t al. 2016), there were no indications about the existence of these trees after fire might not affect the emergence of species in our study sites. however, it is new recruitments on peatland forest, as a source confirmed that our study sites were still at the of seeds might come from various sources. seedbanks were likely absent due to frequent fires, but sources of seeds were possibly supplied from the nearest sites by their dispersal agents. therefore, proximity to the remnant forest is expected to play an important role to increase diversity of these recruitments. this is supported by higher shannon' diversity index fable 2) on transect fbf compared to other transects. this is consistent with chazdon (2008) who stated that the nature of forest early stage of forest succession as most species that were supposed to be present in undisturbed peat swamp forests as mentioned by mirmanto (201 0) were absent. moreover, mrmanto (201 0) also reported that at least 2,000 trees/ha with more than 30 species could be found within 0.25 ha area of burned peatland. this indicated that the density of regrowth on our study site was still relatively low although the species richness (especially on fbf) demonstrated a valuable sign of recovery. table 2 shannon's diversity index (h') and pielou's evenness (e3 on different locations site new regrowth regrowth survivor h' fb fbf nb all sites e' fb fbf nb au sites notes: transect fb: frequently burned, close to canal, far from forest; transect fbf: frequently burned, far from canal, close to forest edge; transect nb: only burned once in 2015, far from forest and canal, divided by three size classes: new regrowth, regrowth, and survivor. biotropia vol. 28 no. 3,2021 table 3 bray curtis dissimilarity index among the three transects bray curtis dissimilarity index fb fbf fbf 0.610 nb 0.607 0.467 figure 5 non-metric multidimensional scaling (nmds) ordination with stress value < 0.2, showing that this ordination displays a fair representation of species composition on each plot. plots 1-5 are plots on fb transect, while plots 6-10 and 11-15 are located on fbf and nb transects, respectively moreover, our study indicated that fires frequencies are not the only determining factor on forest recruitment on this study site, although according to shiodera e t al. (2016), intense and repeated fires reduced the ability of forests to regenerate. the combination of fire frequency, fire intensity, and proximity to remnant forest will produce different degrees of forest recovery, and the result will be unique for each site (graham e t al. 2017). our study showed that frequent fires might not impede new recruitments. however, proximity to the nearest forest edge might impact the diversity of regrowth. this is because proximity to forest remnant plays an important role in producing seeds to ensure the continuous emergence of recruitments. for example, despite receiving frequent fires in the last nine years, transect fbf recruits more diverse regrowth compared to transect nb which was burned just once in 2015. another study on ex mega rice project in central i(alimantan also stated that natural regeneration on isolated degraded peatlands resulted in slow and patchy regrowth with low diversity (blackham e t al. 201 4). pioneer species, which seeds were mostly dispersed by wind or birds, or sourced from dormant seedbank within the peat layer. only few resproutings were found and mostly appeared from ficzls spp. it is supported by chazdon (2008) that initial succession is normally composed of long-lived pioneer species that change slowly over times. moreover, table 4 also displayed a phenomenon that most recruitments were not conspecific to the remaining trees that survive after fire. for example, the presence of a few dipterocarp trees on transect nb was not followed by the emergence of seedlings from these species. once again, our study showed that abundant mother trees would not give a substantial advantage on recruitments unless they are able to regenerate. the presence of climax species such as shorea spp., would not likely support initial forest recovery as these species are not able to produce continuous seeds for regeneration due to h t e d pollination (ghazoul 2005), and if they are able, seedlings of climax species might find it hard to survive due to extreme heat and sun radiation on a typical open peatland. vegetation recoveries over times and the forest recovery process starts with impact on peatland restoration colonization (chazdon 2008), the key to table 4 shows that new recruitments after peatland vegetation recovery after fires is to fires on the three transects were dominated by enable vegetation colonization as soon as understanding natural regeneration in burned tropical peatland: a strategy to accelerate lestari et al. table 4 the four most dominant species within each transect transect fb transect fbf transect nb new regrowth alstonia scholaris melicope lung-ankenda campnospemza coridceum macaranga przlinosa regrowth melicope lunu-ankenda melaleuca sp. campnospem coriaceum alstonia scholaris survivor campnosperma coriaceum campnosperma coridceum alstonia scholaris ficzs spp. ntphelium mangqi elaeocarpus acmocarpus macaranga prtlinosa campnosperma coriaceum tetractomia o bouata tetractomia o bouata alstonia scholaris campnospema coriacem elaeocarpus acmocarpus alstonia scholaris caqbnosperma coriaceum melicope lunu-ankenda jjpgium spp. 2 alstonia scholaris melicope lunu-ankenda ficus spp. sypgium spp 2 myristica iners shorea te_ysmanianna shorea ulginosa tetractomia obovata possible, and this depends on the availability of this finding is supported by our observation regeneration sources (seeds or resproutings). on 15 of the 2 x 2 m permanent plots of natural unfortunately, heavily degraded peatlands are regeneration (fig. 6). these plots were burned commonly dominated by high and dense ferns almost at an annual basis. with the last fire and shrubs that impede other woody species to incidence being in 2014. from this figure, grow (page et 2009)' given this several pioneer species (meialeuca ieucadendmon, only pioneer species are able to grow and supply meiicope iunu-ankenda, s_yggiam sp.) dominated the continuous seeds for further colonization (hapsari et al. 201 8; shiodera et al. 2016). only whole study area. however, in the third year, late when ths condtion is achieved, late successional and generalist species successional species might then be able to (caqfloqe~ma cokacer*ni, aaonia ~cboiahj, emerge dispersed by birds or bats, and bring the cte~o~@~~flpa~~zfo~aj) started to appear, although recovery process to the next stage. pioneer species still dominated. melole~~ca ieucadendron cope iunlt-ankeda -campno sperma coriaceum --.c ctenolophon parvifolius melaleuca leucadendmn -melicope lunu-ankeda --f syzygium sp. -ampelocis sus sp. -esyzyigrurn sp. cf campanulaturn ~ a l s f o n r a scholaris figure 6 trend of recruitments on the smaller natural regeneration permanent plots, observed from 2015 to 2018 note: the last fire attack was in 2014, and before that year, all plots were almost annually burned. biotropia vol. 28 no. 3,2021 the evidence that only pioneer species are able to secure the stand initiation process asks a question to the common technique of vegetation restoration on degraded peatland forest in indonesia. common practice normally involves line or blanket planting on a large area with a mix of pioneer and climax species regardless of their ability to produce continuous seed sources and resprouting ability for rapid colonization. planting also normally prioritizes high economic value species or rare species which are beneficial only when they are purposed for enrichment planting after the first stage of the successional phase (stand initiation) is achieved. a paleocological study by hapsari et al. (2018) stated that floristic composition in degraded peat-swamp forest in sumatra can passively recover, and this is marked by initial domination of rapidly generating trees such as gnetzam, calopkyllum, sapotaceae, and ficus to assure tree colonization and finally enable other late successional species to naturally establish either dispersed by bats or birds. our examination in 9 and 17 months after planting various mixed species, showed that pioneer species had a much higher survival rate and so dominated the revegetation (including species such as alstonia) combretocarpu~, and syugium), and only a few late successional-high valuable tree species (such as dyera, dio.$yros) and shorea) were found (figure 7). moreover, our findings showed higher overall survival rates compared to another trial planting experiment by tata (2017), and slightly lower rates compared to a study by lampela e t al. (2017). our study suggested that planting late successional as well as high valuable tree species, without examining first which the successional stage the site is in, is unnecessary. although some species with high commercial value are able to grow on the initial phase of forest recovery, however, as previously mentioned, it will not give any beneficial value if those species are not able to produce continuous regeneration for stand initiation process. however, late successional or climax species could sull be incorporated in the initial planting but with smaller number compared to the pioneer species. again, these climax species are beneficial for enrichment planting only, where the planting purpose is to increase species diversity on a site that has passed the first phase of the successional process. therefore, to increase the effectiveness of the forest recovery process on recently burned peatland, planting rapidly regenerating or pioneer species to ensure stand colonization is highly recommended. figure 7 survival rate of tree seedlings calculated in 9 months after planting q?+9m) and 17 months after planting (p+ 17m) understanding natural regeneration in burned tropical peatland: a strategy to accelerate lestari et al: conclusion to ensure the forest recovery process, forest colonization with species that can produce continuous species accumulation, either by sprouting or producing seed sources, needs to be addressed first. the common method of restoration practice using expensive species that cannot guarantee continuous self-regeneration is unbeneficial unless the restoration sites have reached the later stage of succession. the successional stage of the designated area should be determined first, as planting should focus on species that meet the needs of the successional stage on the designated sites. to ensure forest recovery, vegetation restoration on peatland should be parallel with hydrological restoration and fire prevention. acknowledgements we wish to acknowledge all field staff of pt rimba makmur utama who helped the authors undertake routine data collection in the field, and local communities who enrich our insight on the peatland forest ecosystem with their important-original knowledge. references blackham gv, webb el, corlett rt. 2014. natural regeneration in a degraded tropical peatland, central iwmantan, indonesia: implications for forest restoration. forest ecology and management, 324, 8-15. https://doi.org/lo.1016/ j.foreco.2014.03.041 chazdon rl. 2008. chance and determinism in tropical forest succession. in: carson kt, schnitzer sa, editors. tropical forest community ecology. london: willey-blackwell. p. 384-408. cleary dfr, priadjati a. 2005. vegetation responses to burning in a rain forest in borneo. plant ecology, 177(2), 145-163. https://doi.org/l0.l007/s11258 005-2107-0 ghazoul j. 2005. pollen and seed dispersal among dispersed plants. biological reviews, 80(3), 413 443. https://doi.org/10.1017/s1464793105006731 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6/j.foreco.2016.12.004 mirmanto e. 2010. vegetation analyses of sebangau peat swamp forest, central icalimantan. biodiversitas, journal of biological diversity 11 (2):82-8. https://doi.org/l0.l3057/biodiv/dl10206 oksanen aj, blanchet fg, friendly m, igndt r, legendre p, mcglinn d, . . . szoecs e. 2017. package 'vegan'. osaki m, nursyamsi d, noor m, wahyunto, segah h. 2016. peatland in indonesia. in: osaki m. tsuji n, editors, tropical peatland ecosystem, tokyo up):: springer. p. 33-48. page s, hosciko a, wosten h, jauhiainen j, silvius m, rieley j, . . . limin s. 2009. restoration ecology of lowland tropical peatlands in southeast asia: current knowledge and bture research directions. ecosystems, 12(6):888-905. https://doi.org/ 10.1007/~10021-008-9216-2 rossita a, witono a, darusman t, lestari dp, rlsdiyanto i. 2018. water table depth fluctuations during e n s 0 phenomenon on different tropical peat swamp forest land covers in i<atingan, indonesia. iop conference series: earth and environmental science, 129(1). https://doi.org/l0.1088/1755 1315/129/1/012001 shiodera s, atikah t, apandi i, seino t, haraguchi a, rahajoe j, icohyama t. 2016. peat-fire impact on forest structure in peatland of central kalimantan. in: osaki m. tsuji n, editors, tropical peatland ecosystem, tokyo (jp): springer. p. 197-212. tata hl. 2017. native species for degraded peat swamp forest rehabilitation. jurnal silvikultur tropika, 7(3):s80-2. 1131.cdr prevention methods of a. hydrophila and p. fluorescens infection in tilapia page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 685 siti mariam (light-color).cdr light-color-induced changes in fatty acid biosynthesis in chlorella sp. strain ks-ma2 in early stationary growth phase 1 2 3 4siti-mariam osman , tse seng chuah , saw hong loh , thye san cha and aziz ahmad 1* 1 school of fundamental sciences, universiti malaysia terengganu, kuala terengganu 21030, terengganu, malaysia 2 school of science and food technology, universiti malaysia terengganu, kuala terengganu 21030, terengganu, malaysia 3 school of marine and environmental sciences, universiti malaysia terengganu, kuala terengganu 21030, terengganu, malaysia 4institute of marine biotechnology, universiti malaysia terengganu, kuala terengganu 21030, terengganu, malaysi a received 08 august 2016/accepted 24 june 2017 abstract optimization of light supply remains a critical issue in microalgae biotechnology. the impacts of light color on fatty acid production and biosynthesis in microalgae are poorly understood. the aim of this study was to determine the effect of light color on growth and fatty acid content in chlorella strain ks-ma2. cells were cultured on f/2 medium and incubated under blue, green, red or white light. the cells' growth, fatty acid composition and the expression levels of the ketoacyl synthase 1 (kas-1), omega-6 desaturase (ω-6 fad) and omega-3 desaturase (ω-3 fad) genes were measured at the early stationary growth phase. results of this study indicated that light color affected cell density and fatty acid profile produced by chlorella sp. strain ks-ma2. cells cultured under blue, red and white light had higher cell density than those cultured under green light. palmitic acid (38.62 ± 3.29% of biomass dry weight) and linolenic acid (7.96 ± 0.88% of biomass dry weight) were highly accumulated under white light. stearic acid was dominant under blue light (11.11 ± 0.14% of biomass dry weight), whereas oleic acid was dominant under red light (30.50 ± 0.14% of biomass dry weight). linoleic acid was highly produced under green and blue light (28.63 ± 1.36% and 26.00 ± 0.81 % of biomass dry weight, respectively). kas-1 and ω-6 fad were highly expressed under blue light, whereas ω-3 fad was highly expressed under green light. the production of particular fatty acids of interest from chlorella could be achieved by shifting color of light used during the incubation of the cell cultures. blue-light is the most suitable light color for producing biomass and stearic acid by chlorella strain ks-ma2. keywords: light spectrum, linoleic acid, linolenic acid, oleic acid, palmitic acid, stearic acid introduction demand for microalgae as food, animal feed supplements, feedstock and biofuel is increasing tremendously (wacker et al. 2016). the success of algae-based industrial products depends on high biomass productivity and quality with low production cost (abu-ghosh et al. 2016). biomass productivity is closely interrelated with the amount of light energy received, which is processed and stored in chemical compounds (chen et al. 2015; liu et al. 2012). sunlight is the most cost-effective energy source for microalgae production, but it has drawbacks, including changes in weather, day and night cycles, and seasonal changes, which affect light intensity and spectrum (abu-ghosh et al. 2016). optimization of light supply remains a critical issue in microalgae biotechnology. varying light intensity might reduce the photosynthetic rate, hindering microalgae growth due to shortage of energy (ge et al. 2013). light intensity also contributes to temperature changes and affects the accumulation of polyunsaturated fatty acids on * corresponding author: aaziz@umt.edu.my biotropia 5 1 8 33 42 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.1.685 33 microalgae (wacker et al. 2016; piepho et al. 2011). cell division is an energy-consuming process and microalgae tend to accumulate sufficient energy before proliferation (zachleder et al. 2016) the compounds of energy storage, such as lipids and starch in particular, tend to accumulate in the algal cells under stress conditions, when cell growth halts (chen et al. 2015). in addition, light intensity can affect microalgae physiological characteristics, including pigment and chemical composition, growth rate, ion transport (kwon et al. 2013) and lipid classes (wacker et al. 2016; liu et al. 2012). sun et al. (2014) showed that the total lipid content of neochloris oleoabundans increases when the light intensity is increased from 50 to 200 µmol/m /s, 2 but decreases with light intensity higher than 200 µmol/m /s. the rate of photosynthetic activity 2 declines during limited light, but linearly increases with increasing light availability (choi et al. 2013). generally, light intensity refers to the number of photons (energy) per unit area and unit time. the energy of each photon is inversely proportional to the wavelength of the associated electromagnetic wave i.e. the shorter the wavelength, the more energetic the photon. for example, the energy of a photon of blue light ( = λ 450 nm) is 2.76 ev, while the energy of a photon of red light ( = 700 nm) is 1.8 ev. white light λ contains a spectrum of visible light with different wavelengths. microalgae usually absorb photons in the range of 400 to 700 nm for photosynthesis; the wavelengths absorbed are species-specific (kim . 2014). therefore, light color has been et al shown to have a significant impact on microalgae productivity. for example, de mooij . (2016) et al showed that the highest productivity of chlamydomonas reinhardtii is obtained under yellow light, whereas the lowest is obtained under blue and red light. the lowest biomass of spirulina platensis culture is obtained with blue led, and the highest is obtained with pink and red led (markou 2014). on the other hand, ge et al. (2013) found that red light enhances biomass production of . however, the chlorella vulgaris impacts of light color on fatty acid production and expression of fatty acid biosynthesis genes, particularly in microalgae, are poorly understood. previous research has focused on the photoautotrophic production of microalgae oil using white light as energy source. however, no explicit data are available concerning the differences in cell proliferation and energy accumulation with different wavelengths of light. the objectives of this study were to determine the effects of different-colored light on growth, fatty acid composition, total oil content and expression of genes for fatty acid biosynthesis proteins ketoacyl-acp synthase-i ( ), kas-1 omega-6 desaturase ( ) and omega-3 ω-6 fad desaturase ( ) in strain ks-ma2. ω-3 fad chlorella kas is required for the addition of malonyl-coa in the elongation of butyryl-acp from a 4to 14carbon chain (kainou 2006). fad enzymes et al. ( fad and fad) are involved in the ω-6 ω-3 insertion of double bonds at the and ω-6 ω-3 positions of pre-formed fatty acid chains in reactions requiring oxygen, nadh and cytochrome and reducing equivalent (zhang b5 et al. . 2016) materials and methods a stock culture of mangrove-isolated chlorella strain ks-ma2 was obtained from the microalgae stock culture collection of the universiti malaysia terengganu, malaysia and used in this study. the chlorella cells were maintained on f/2 medium (guillard & ryther 1962) agar plates, which were prepared using natural seawater with salinity of 30 mg/l. medium was solidified with 10 g/l of bacteriological agar and sterilized by autoclaving at 121 °c for 20 minutes. a single colony was streaked onto a sterilized agar plate and subsequently incubated in a growth chamber at 25 o with 55 c under 24 hour white-light illumination 70 µmol photons/m/s light intensity with constant aeration. this culture was considered the chlorella stock culture and was re-streaked onto a newly prepared agar plate every two months to maintain the cells in the institute of marine biotechnology, universiti malaysia terengganu. light color treatments 6 chlorellaa total of 5 × 10 cells/ml of strain ks-ma2 was aseptically transferred into 2.5 l freshly prepared f/2 medium. the salinity level was adjusted to 30 mg/l. the cultures were incubated at 25 ± 1 c, with illumination for 24 o hous per day at 55 70 µmol photons/m/s light intensity and constant aeration. the light colors used during incubation were white (control; λ = 400 750 nm), red (λ = 650 nm), blue (λ = 475 nm) 34 biotropia vol. 25 no. 1, 2018 35 and 350 ml/minute, respectively. the temperatures were programmed as follows: an oinitial temperature of 35 c for 0.5 minute; o oincreased to 195 c at a rate of 25 c/minute; osubsequently increased to 205 c at a rate of 3 oc/minute; and finally increased to 320 o oc/minute at a rate of 8 c/minute and held for 6.64 minutes. the fatty acid components were identified by comparing their retention time and fragmentation pattern with established standards. the supelco 37 component fame reference standard mix (sigma-aldrich) was used to identify and quantify the percentage of cisand trans-fatty acid isomers in the samples. examination of gene expression levels by real-time pcr total rna was isolated with gf-1 total rna extraction kit (vivantis) according to the manufacturer's instructions and treated with dnase i (fermentas) to remove contaminating dna. dna-free rna was confirmed by pcr amplification of the 18s rdna gene using the rna as templates. subsequently, 1 μg of the rna was reverse transcribed with iscript reverse transcription supermix (bio-rad) in accordance with the manufacturer's instructions. the cdna generated was directly used for quantitative realtime pcr. the real-time pcr was performed in a myiq single color real-time pcr detection system (bio-rad) using sybr green real-time pcr master mix (bio-rad) according to the manufacturer's instructions. the pcr mixture consisted of cdna (50 ng, 1 l), 0.4 μ μm final concentration of each forward and reverse primer (table 1), 10 l 2× iq sybr green supermix μ and nuclease-free water to the final volume of 20 μl. after heating at 95 °c for 15 seconds, the real or green (λ = 510 nm) provided by a fluorescent lamp source covered with colored cellophane. cell densities were measured every two days until a constant reading was obtained, indicating the cells had attained early stationary phase, and the cells were later harvested. three replications were prepared for each treatment. at harvest, cell numbers, biomass as dry weight, and total oil and fatty acid content were determined. the cell numbers and absorbance at 600 nm were measured on a 10-fold serial dilution of culture using a haemocytometer (neubauer) and biophotometer (bio-rad), respectively, in triplicate. cell density was calculated based on plotted calibration curve at 600 nm. quantification of oil and fatty acid contents oil extraction and esterification of fatty acids to methyl esters (fames) was carried out according to the modified methods of cha et al. (2011). oil was extracted from a dried sample of chlorella cells. a 500 mg of sample was vortexed in 10 ml of hcl (concentrate) for 3 minutes and subsequently boiled using double-boiling technique for 30 minutes. after cooling to room temperature, the oil were extracted with 25 ml hexane for 1 minute and repeated twice with 15 ml hexane. extracts containing fames were combined, hexane residue was removed using vaporization with a rotavapor r-210/215 o(buchi). the extracted oil was incubated at 80 c until a constant weight was achieved. fames were analyzed using a gas chromatograph (gc-6890, agilent) equipped with a flame ionization detector fitted with a capillary column (db-225ms, supelco). the injector temperature was set at 250 oc, and the flow rate of carrier gas (he) was 2.4 ml/minute. the flow rate for h gas and air were 2 primers name primers sequence (5' 3") size of amplicon (bp) 18s f1 5'-cct gcg gct taa ttt gac tca aca cg -3' 172 18s r2 5'-tag cag gct gag gtc acg ttc g -3' kas1 f5 5'-cca tga ttg gtc att gct tgg gag c -3' 151 kas1 r5 5'-gct ctt gct tca tgt ttg gga cca c-3' o6d f3 5'-ctt cac cca cga agg cac agg c -3' 129 o6d r3 5'-cct gca cac tgc tgg gaa cg-3' o3d f2 5'-cat gtt gag aac gac gag tcc tgg tat c-3' 162 o3d r2 5'-gtc aaa gtg gga gcc agt ctt gc-3' 35 fatty acid biosynthesis in chlorella sp. strain ks-ma2 osman et al. table 1 forward and reverse primers for quantitative real-rime pcr b a b time pcr amplification was programmed for 40 cycles of 95 °c for 35 seconds, 64.2 °c for 35 seconds and 72 c for 30 seconds. specificity of all o pcr amplifications was verified by a melting curve at the completion of each run from 55 to 95 °c at 0.5 °c increment. the gene expression data were analysed using the 2 method (schmittgen −δδct & livak 2008; livak & schmittgen 2001). the data obtained were represented as the fold change in the target gene in the treatments relative to the control and normalized to the expression level of the 18s rrna reference gene. statistical analysis differences in total oil and fatty acid content among the light-color treatments of the chlorella strain were statistically analysed by one-way anova using spss version 16.0. significant differences among means were identified by tukey's test at p = 0.05. (www.ibm.com/software/ analytic/spss). results and discussion growth of chlorella results of this study showed that light color did not affect the cell growth of chlorella strain ks-ma2. nevertheless, light color changed fatty acid composition. increased cell proliferation led to the increase of cell densities and biomass obtained. cell densities varied among light treatments (fig. 1). cells cultivated under blue light reached early stationary phase after 16 days of culture. cells cultivated under green, white and red light reached early stationary phase after 18, 20 and 22 days of culture, respectively (fig. 1a). comparatively, cells incubated under red light took longer to attain stationary phase compared to those under other light colors. cell densities in red-, whiteand blue-light treatments did not differ significantly (p > 0.05). cell numbers recorded were fluctuated from 2.08 10 cells/ml 8 to 1.72 10 cells/ml. biomass dry weight 8 36 biotropia vol. 25 no. 1, 2018 figure 1 effect of light colors on: (a) cell density and biomass at the early stationary phase and (b) growth of chlorella strain ks-ma2 on f/2 medium (note: bars represent cell densities and lines represent biomass dry weight (mean ± sd; n=3); values followed by the same letter were not significantly different according to tukey's test with p = 0.05 significance level) obtained also did not significantly differ (p > 0.05; fig. 1b). cell proliferation is dependent on the available energy in forming atp or nadph. the available energy is derived from photosynthetic activity (zachleder et al. 2016). in addition, photosynthetic efficiency is associated with photon energy, which is inversely proportional to light wavelength (kim et al. 2014), i.e. the shorter the wavelength, the more energetic the photon (choi et al. 2013). thus, blue light is more efficient for photosynthesis and lipid biosynthesis (markou 2014). however, blue light appeared to cause photo-inhibition, inducing cell damage and reduced the time to attain early stationary phase. blue light has been reported to up-regulate the lhcx1 protein and zeaxanthin epoxidase, a proteins involved in photoprotection in phaeodactylum tricornutum (costa et al. 2013). on the other hand, red light has longer wavelength than blue light and thus, might prevent photoinhibition (george et al. 2014; xu et al. 2013). therefore, the highest cell density and biomass were obtained in the red-light treatment (fig. 1b). similar findings have been reported for scenedesmus sp. (kim et al. 2013) and c. vulgaris (xu et al. 2013; yan et al. 2013). however, different algal classes have different light requirements for growth and photosynthesis (kwon et al. 2013; cheirsilp & torpee 2012). total oil content and fatty acid composition total oil content (fig. 2) and fatty acid composition (fig. 3 & 4) varied among the light color treatments (p < 0.05). cells under blue-light and white light treatments contained the highest percentage of total oil, up to 22.6% of total dry weight (fig. 2). this was also 3.4and 3.7-fold higher than that of cells under redand greenlight treatments, respectively. saturated fatty acid accumulation did not differ significantly ( > 0.05) p in the treatments of white-, blueand green-light (fig. 3a), but it was higher than the red-light. meanwhile, monounsaturated fatty acids were highly accumulated under the red-light treatment (30.9%) (fig. 3b). this accumulation was 1.19-, 1.44and 1.47-fold higher than under white, blue and green light, respectively. in contrast, cells cultured under green light produced higher proportions of polyunsaturated fatty acids (pufas: 34.5%) compared to those under white (25.7%) or red (30.3 %) light (fig. 3c). however, this value did not significantly differ ( > 0.05) p from the blue-light treatment (32.2%). figure 4 shows the relative amount of the five major fatty acids i.e. palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid, in chlorella strain ks-ma2 cultured under different light colors. palmitic acid (38.6%) and linolenic acid (7.9%) were highly accumulated ( < 0.05) p under white-light conditions (fig. 4a & 4e) compared to the other treatments. meanwhile, the accumulation of stearic acid (11.1%) was the highest under blue-light treatment, 2.3-fold higher than that under white light (fig. 4b). conversely, oleic acid (30.5%) was highly accumulated under red light (fig. 4c), and linoleic acid (28.6%) was highly accumulated under green light (fig. 4d). results of this study are in line with previous reports stating that different light colors change the total oil accumulated (markou 2014; liu et al. 2012; marchetti et al. 2012) and fatty acid profile (wacker et al. 2016; piepho et al. 2011). light with higher 37 fatty acid biosynthesis in chlorella sp. strain ks-ma2 osman et al. figure 2 effect of light color on total oil (mean ± sd) (note: values followed by the same letter were not significantly different according to tukey's test with p = 0.05 significance level) 38 biotropia vol. 25 no. 1, 2018 energy, such as blue or white light, might increase the atp and nadph available for triacylglycerol synthesis (fig. 2). addition of carbon in the elongation of fatty acid chains requires nadph, derived from photosynthesis (chen et al. 2015). moreover, blue light is used for enzyme activation and regulation of gene transcription (das et al. 2011). high transcription of kas-1 under bluelight treatment (fig. 5a) might be due to the increased photosynthetic activity. a b c figure 3 effect of light color on: (a) saturated fatty acid; (b) monounsaturated fatty acid; and (c) pufa content at the early stationary phase of chlorella strain ks-ma2 cultured on f/2 medium (mean ± sd; n=3) (note: values followed by the same letter were not significantly different by tukey's test with p = 0.05 significance level) 39 therefore, more carbon flux generated from photosynthesis is channelled to lipid biosynthesis (liu 2012), involving the elongation of fatty et al. acid chains; thus, resulting in higher accumulation of unsaturated fatty acids (fig. 3a), the palmitic acid (fig. 4a) and stearic acid (fig. 4b) under high energy light. in addition, higher total oil production is important for light-stress adaptation (choi 2013). the lipid layers et al. function as a light filter to reduce irradiation on the cell components, to prevent photo oxidative damage and to reduce water loss (liu 2012)et al. . fatty acid biosynthesis in chlorella sp. strain ks-ma2 osman et al. a b e c d figure 4 effect of light color on: (a) palmitic acid; (b) stearic acid; (c) oleic acid; (d) linoleic acid; and (e) linolenic acid content at early stationary phase of chlorella strain ks-ma2 cultured on f/2 medium (mean ± sd; n=3) p(note: values followed by the same letter were not significantly different according to tukey's test with = 0.05 significance level) 40 biotropia vol. 25 no. 1, 2018 expression levels of kas-1, -6 fad and -3 ω ω fad results of this study showed that kas-1 (fig. 5a) and ω-6 fad (fig. 5b) were highly transcribed under blue light. it was 5.3and 2.2-fold of the reference gene, respectively. the ω-3 fad was more highly transcribed under green light (2.8fold of the reference gene; fig. 5c). this was 3.8-, 1.8and 2.3-fold higher than that under white light, respectively. these genes exhibited the lowest transcription levels under red-light treatment. fatty acid desaturation is important for microalgae tolerance toward strong light (choi et al. 2013; solovchenko et al. 2008). pufas are a b c figure 5 effect of light color on mean fold change in relative expression of: (a) kas-1; (b) ω-6 fad; and (c) ω-3 fad of chlorella strain ks-ma2 (mean ± sd; n=3) (note: values followed by the same letter were not significantly different according to tukey's test with p = 0.05 significance level) 41 necessary for the maintenance of photosynthetic membrane function. pufas also play an important role in acclimation to low light conditions (solovchenko 2008). high pufa et al. content obtained in blueand green-light was mainly contributed by linoleic acid (fig 4d). the -α linolenic (fig. 4e) produced in white-light was the least i.e. 8 % of biomass dry weight. blue light also induced the expression of acyl-lipid desaturase (kis 1998), which might be et al. responsible in higher accumulation of -linolenic α (fig. 4e) and transcription levels of (fig. ω-6 fad 5b). on the other hand, higher transcription levels of under green-light (fig. 5c) did not ω-3 fad contribute to the accumulation of -linolenic acid. α the increase of and transcription kas-1 ω-6 fad levels might be associated with photo-protection against higher light intensity (yoshioka 2012). et al. nonetheless, knowledge on the post translation of these genes is remained unknown. this study showed that there was inconsistent requirement of light in production among microalgae pufas species (wacker et al. 2016). this species-specific l ight acclimation strategy ref lected to photosynthetic factors and differences in light spectra. conclusions light color played a major role in the proliferation of chlorella strain ks-ma2 and its fatty acids profile. mass production of chlorella biomass could be obtained under blue-light conditions, whereas total oil was better obtained under whiteor red-light conditions. higher production of specific fatty acids could be achieved using different light color during the cultivation of cells i.e. white light for palmitic acid and linolenic acid, blue light for stearic acid and red light for oleic acid. linoleic acid accumulation was not influenced by light color. the transcription level of kas-1 and ω-6 fad were highly activated by blue-light, while the transcription level of ω-3 fad was highly activated by green-light. acknowledgements this study was funded by science fund project under the ministry of agriculture of malaysia (grant number sf-05-01-12-sf0007). references abu-ghosh s, fixler d, dubinsky z, iluz d. 2016 flashing light in microalgae biotechnology. bioresour technol 203:357-63. cha ts, chen jw, goh eg, aziz a, loh sh. 2011. differential regulation of fatty acid biosynthesis in two species in response to nitrate chlorella treatments and the potential of binary blending microalgae oils for biodiesel application. bioresour technol 102:10633-40. cheirsilp b, torpee s. 2012. enhanced growth and lipid production of microalgae under mixotrophic culture condition: effect of light 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melia1*, amrozi amrozi2, muhammad agil2 and iman supriatna2 1faculty of veterinary medicine, universitas syiah kuala, banda aceh 23111, indonesia 2faculty of veterinary medicine, institut pertanian bogor, bogor 16680, indonesia received 5 april 2017 / accepted 16 july 2019 abstract this study aimed to find out the characteristics of gayo horses and to identify the population of gayo horses. data collection was conducted through a selection process from more than 100 local horses distributed in central aceh subdistricts, gayo lues and bener meriah. the selection process resulted in 30 gayo horses having varying ages. detail observation was performed on six gayo horses consisting of 3 male horses and 3 female horses. the observation comprised morphological observation and morphometry. data collection of gayo horse population was based on the annual report from the local animal husbandry and fishery office of central aceh subdistrict over the last 5 years. results of this study showed that gayo horses have a straight cranial shape with smaller size, thick and stiff mane, and ears akin to donkey’s ears. the study also showed that gayo horse’s body height ranged between 113-120 cm with a body weight range of 215-280 kg. gayo horse is agile despite living in mountainous areas and able to carry heavy loads. the population of gayo horses have been declining since 20102014. gayo horse is included in a large pony category. conservation efforts are essential to save gayo horse from extinction. keywords: characteristics, gayo horse, hallmarks, population introduction indonesian local horses are among livestock commodities supporting national development, especially in the animal husbandry subsector. gayo horse is one of the indonesian native horse germplasms (soehardjono 1990). this has been written in the ministry of agriculture decree no. 1054/kpts/sr.120/10/2014 which stated that the gayo horse is an indonesian native horse strain. gayo horse population is spread all over gayo highland in the central aceh subdistrict, including its autonomous regions called gayo lues and bener meriah. central aceh subdistrict is known by various other names such as “nation above the clouds”, “gayo highland” and “negeri antara”. in this region, the annual gayo traditional horse race was held to commemorate the proclamation of independence of the republic of indonesia and the anniversary of the central aceh subdistrict. many owners of gayo horses have the intention to breed gayo horse with a thoroughbred horse, creating a new type of horse known as “astaga horse” (australia-gayo). nowadays, pure gayo horse is hard to find, which indicated that there might have been a very drastic decline of the gayo horse population. the attempt to conserve the gayo horse as one of the indonesian native horse germplasms must be done immediately to save the gayo horse from extinction. the objective of this study is to describe gayo horses’ characteristics and to identify the number of gayo horses’ population, which is crucial for future research concerning the reproduction of gayo horses. materials and methods research procedure data collection was carried out by means of direct identification at the gayo horses’ habitat. additionally, the past and present information on gayo horses was obtained through direct *corresponding author, email: julimelia@unsyiah.ac.id biotropia vol. 29 no. 1, 2022 2 interviews with several owners of gayo horses and with officials of the local animal husbandry and fisheries office of the central aceh subdistrict. one hundred local horses were selected from areas surrounding the central aceh subdistrict and its autonomous regions called gayo lues and bener meriah. in the initial phase, the identification process was conducted according to the criteria written in the ministry of agriculture decree no. 1054/ kpts/sr.120/10/2014. of the 100 selected local horses, 30 horses were identified as gayo horses having varying ages. a more thorough identification process was conducted on 6 gayo horses consisting of 3 male horses and 3 female horses. gayo horse morphology and morphometry the morphological examination was carried out visually and documented by slr camera (nikon d3200, af-s dx zoom-nikkor 1855mmf/3,5-5,6g ed ii). the morphological examination included observation on body shape, eye shape, mane condition, head shape, ear shape, neck shape, nape and tail. morphometry examination included measurement on body height, girth and body length. body height was measured from the tip of the front leg up to its withers. the girth was measured from the lower abdomen from the fossa olecranon perpendicular with os sternum until above os vertebrae. body length was measured from the connection of cartilago and os scapula horizontally until behind the horse’s os pelvis. body weight measurement is predicted by using the schoorl formula: (body weight = (chest circumference + 22)2/100) and measured in kilograms (kg). population data data on gayo horses’ population were collected from the annual reports of the years 2010-2014 published by the local animal husbandry and fisheries office of central aceh subdistrict and its autonomous regions, i.e., gayo lues and bener meriah. data analysis gayo horses’ morphology and morphometry data were descriptively analyzed. the population data were analyzed and depicted in graphs. the prediction of gayo horses’ population extinction was analyzed exponentially by using excel 2016 software. results and discussion gayo horses are included into large ponies for having a body height of not more than 1.47 m. on the other hand, miniature ponies have body height ranging from ≤ 86 cm (a type) up to 88.6 cm (b type) (campbell 1992). according to ensminger (1962), a pony’s height is less than 1.45 m while standing, with 250-450 kg body weight, and usually are descendants of lightweight horses. being included as large ponies, gayo horses have different body sizes compared to foreign horses. the size differences are often influenced by environmental factors, such as topography and climate (ohsawa et al. 2008; steinheim et al. 2008; kosoma & purzyc 2009). ponies are often used for recreation purposes (rogers et al. 2006), and for therapy programs (burke 2002). generally, gayo horses have similar characteristics to other local horse types in indonesia. however, gayo horses have several uniqueness, such as having straight head shape with smaller body size, thick and stiff mane, and having ears similar to donkey’s ears (table 1; fig. 1). table 1 the morphology of gayo horses morphology shape color body short black, dun, chestnut, grey, white eye small black gaze sharp mane thick and stiff head straight ear similar to donkey nape long throatlatch wide tail medium to long similar to body color, mix biological characteristics of indonesian gayo horse – juli melia et al. 3 figure 1 gayo horse appearance notes: a. female; b. male. gayo horses have body height of 113-120 cm, chest circumference of 136-139 cm, body length of 102-105 cm, and body weight of 215280 kg (table 2). a pictured guide for easy identification of gayo horse in the field is presented in figure 2. table 2 the morphometry of gayo horses morphometry height (cm) chest circumference (cm) body length (cm) weight (kg) male 118-120 (119.00±1.00) 137-139 (138.00±1.00) 103-105 (104.33±1.15) 225-280 (256.67±28.43) female 113-115 (114.33±1.15) 136-138 (137.00±1.00) 102-105 (103.67±1.53) 215-250 (233.33±17.56) figure 2 a pictured guide for easy identification of gayo horses in the field a b biotropia vol. 29 no. 1, 2022 4 several distinctive marks indicating the temperament and intelligence of local indonesian horses, which also exist in gayo horses, are the condition and shape of hair whorl, i.e., 1. pusar cekak; hair whorl under the jaw, indicating that the horse is always being preyed upon by tiger; 2. pusar terbang; hair whorl by the front feet around the left and right knees area, indicating that the horse is suitable as a racehorse due to its fast speed; 3. pusar dada; hair whorl by the chest, indicating that the horse can spin fast and not fall in a sharp turn during a race; 4. pusar gedung; hair whorl by the flank or below the flank, indicating a mild or tame temperament and is suitable as brood; 5. pusar turun tangis; hair whorl under both eyes, indicating temperamental personality, sometimes being tame and sometimes being wild; 6. pusar ruk; hair whorl by the hind legs behind the knees, indicating a wild and hard to control personality; 7. pusar lipan; hair whorl around the throat and under the ear, indicating a hard to control or wild personality. gayo horses have several positive characteristics, such as power and agility, having capabilities to carry heavy loads, having good temperament, having good endurance, having high adaptivity toward various environmental conditions, having survival capabilities with minimal feed availability, and having easy maintenance, which makes gayo horses very economical for its owner. judging from various aspects, gayo horses have considerably high strategic values. based on cultural aspects, the gayo horse symbolizes social status. usually, a boy’s manhood is determined from the moment a boy can ride a gayo horse without a saddle in a race, which custom is still practiced nowadays. from an economical aspect, the gayo horse is used as a source of animal protein for the local people who consume horse meat. gayo horse is often sold to other regions such as north sumatra to be consumed. from the utilization aspect, the gayo horse is used as a plow puller in rice fields and as a freight horse to transport agricultural products. based on data obtained from the local animal husbandry and fisheries office of the central aceh subdistrict, the population of gayo horses drastically decrease in 2010-2014 (fig. 3). prediction analysis indicated that gayo horse will extinct in 2037 (fig. 4). main reasons that might cause the decrease of the gayo horse population: 1. crossbreeding; 2. uncontrolled culling; 3. existence of modern machinery. figure 3 gayo horses population at central aceh subdistrict biological characteristics of indonesian gayo horse – juli melia et al. 5 figure 4 prediction of gayo horse extinction the upkeeping of gayo horses is usually done conventionally. after the agricultural harvest season, the gayo horses are released to the mountainous area and rice fields. the mating system of the gayo horse is akin to that of primates. only superior stallions may mate with the female horses around the area where the stallion is kept. if a stallion is defeated in a fight before mating, the defeated stallion will be moved to the other group of horses, which causes inbreeding leading to the decrease of body size, height, and weight of gayo horses. nowadays, the upkeeping system of gayo horses has separated the stallion from the mare gayo horses, even though it is still conventionally conducted. the mare horse is brought to the stallion only at the breeding time. the body maturity of gayo horses occurs at 12-15 months of age, while sexual maturity starts at 12-18 months of age (personal communication with owners). usually, the mare of the gayo horse has 21 days of the estrus cycle. all information obtained from owners was proven through complete research on the reproduction status of gayo horses. determination of reproduction status is among the most important factor of animal nurturing and breeding management, including for horses, because reproduction status has a close link with animal’s basic reproductive physiology. a number of techniques can be utilized, such as ovarium dynamic observation by using ultrasonography (amrozi et al. 2004; cuervoarango & newcombe 2008; derar & hussein 2011; melia et al. 2014), hormonal analysis (agil et al. 2008) and histology examination on reproductive organs. hormonal analysis in a horse is generally conducted using the horse’s blood samples (bollwein et al. 2002; utt et al. 2007; ginther et al. 2010). all of these data can be used as the basic reproductive physiology data of gayo horses, which will support the application of reproductive technology as one of the means to save endangered the population of gayo horses from extinction. conclusion gayo horse is included in a large pony category. conservation efforts are essential to save gayo horses from extinction. acknowledgments the authors thank the directorate of research and community services, directorate general of research development, technology, and higher education, the ministry of research technology and higher education for providing research funding through doctor dissertation research scheme number: 025/sp2h/lt/ drpm/ii/2016 dated 17 february 2016. special gratitudes are also delivered to the central aceh office of animal husbandry and biotropia vol. 29 no. 1, 2022 6 fishery for assisting the authors during data collection process. references agil m, supriatna i, purwantara b, candra d. 2008. assessment of fertility status in the male sumatran rhino at the sumatran rhino sanctuary, way kambas national park, lampung. hayati journal of bioscience 15:39-48. amrozi, kamimura s, ando t, hamana k. 2004. distribution of estrogen receptor alpha in the dominant follicles and corpus luteum at the three stages of the estrous cycle in japanese black cows. j vet med sci 66(10):1183-8. bollwein h, mayer r, weber f, stolla r. 2002. luteal blood flow during the estrous cycle in mares. theriogenology 65:2043-51. burke d. 2002. australian miniature ponies. [accessed 2013 april 06]. available at: http:// burkesbackyard.com.au/2002/archives/2002/road tests/horse_breeds/australian_miniature_ponies campbell me. 1992. selected aspects of miniature horse reproduction. proceeding of the annual meeting of the society for theriogenology. p. 89-96. cuervo-arango j newcombe jr. 2008. repeatability of preovulatory follicular diameter and uterine edema pattern in two consecutive cycles in the mare and how they are influenced by ovulation inductors. theriogenology 69:681-7. derar ri, hussein ha. 2011. ovarian follicular dynamics during the estrous cycle in jennies in upper egypt. veterinary medicine international article id 860518, 6 p. https://doi:10.4061/2011 /860518. ensminger me. 1962. animal science. animal agriculture series. 5th ed. danville (us): printers & publishers. inc. ginther oj, almamun, shahiduzzaman akm, beg ma. 2010. disruption of the periovulatory lh surge by a transient increase in circulating 17β-estradiol at the time of ovulation in mares. j anim r sci 117:178-82. kosoma m, purzyc h. 2009. konik and hucul horses: a comparative study of exterior measurements. j anim sci 87:2245-54. melia j, amrozi, tumbelaka li. 2014. ovarian dynamics of endometritis cows treated with a combination of intrauterine infusion of gentamicin, flumequine, and pgf2α analog. j ked hewan 8(2):111-5. ohsawa t, saito y, sawada h, ide y. 2008. impact of altitude and topography on the genetic diversity of quercus serrata populations in the chichibu mountain central japan. j anim sci 203(3):187-96. rogers cw, gee ek, hangoor e, firth ec. 2006. preliminary survey of congenital and reproductive disorders in the new zealand miniature horse population. proceedings of the new zealand society of animal production 66:274-8. soehardjono o. 1990. kuda. jakarta (id): yayasan pamulang. steinheim g, odegard j, ådnoy t, klemetsdal g. 2008. genotype by environment interaction for lamb weaning weight in two norwegian sheep breeds. j anim sci 86:33-9. utt md, acosta tj, wiltbank mc, ginther oj. 2007. acute effects of prostaglandin f2α on systemic oxytocin and progesterone concentrations during the mid-or late-luteal phase in mares. j anim r sci 97:63-73. biotropia vol. 29 no. 3, 2022: 185 192 doi: 10.11598/btb.2022.29.3.1393 185 postharvest quality improvement of nutmeg (myristica fragrans) okky setyawati dharmaputra*, santi ambarwati, ina retnowati and nijma nurfadila phytopathology laboratory, science innovation and technology department, seameo biotrop, bogor 16134, indonesia received 11 august 2020/accepted 19 september 2021 abstract nutmeg (myristica fragrans) or fragrant nutmeg is an important commodity that has been used in the food and pharmaceutical industries, hence its quality should be monitored. the objectives of this study were to: 1) identify critical control points (ccp) in nutmeg’s postharvest handling process and prepare nutmeg haccp (hazard analysis and critical control point) system and 2) provide a recommendation on ghp (good handling practices) of nutmeg in order to maintain its quality in relation to food safety issue which is very important for international trade. ripe fruits of nutmeg were collected after the fruits had reached maturity and fallen from their trees. a paranet was placed under each nutmeg tree to prevent the ripe nutmeg fruits from falling on the ground. the subsequent processes were taking out the nutmeg seeds from the fruits and separating the nutmeg seeds from the pulps and maces. after that, the nutmeg seeds underwent the drying process by using the smoke and oven-dried methods until the moisture content of the nutmeg seeds was reduced by 10%. subsequently, the nutmeg seeds were divided into two parts, prior to the storing process. the first part was fumigated by using phosphine (2 g/m3) for eight days and the second part was not fumigated. the sampling of nutmeg seeds was conducted at the beginning of storage and after four months of storage. the parameters observed were moisture content, percentage of damaged kernels, the population of each fungal species, and aflatoxin content. the results showed that moisture content, fungal population, aflatoxin b1, and total aflatoxin contents of nutmeg kernels having been dried by using the smokeand oven-dried methods with and without fumigation still complied with the requirements related to food safety, although the nutmegs were stored for four months. the results of this research could also determine the critical control point (ccp) in the postharvest handling process of nutmegs, i.e., 1) choosing only ripe nutmeg fruits to be harvested; 2) harvesting method by preventing the ripe nutmeg fruits from falling on the ground; 3) drying process of nutmeg seeds should be conducted immediately after separating the nutmegs from the maces by using the smokeor oven-dried methods; and 4) nutmeg seeds were stored with the shells. keywords: nutmeg, postharvest, quality, shells, storage introduction nutmeg (myristica fragrans) or fragrant nutmeg is an important commodity widely used in the food and pharmaceutical industries, hence its quality should be monitored (punnathara 2011). nutmeg is native to the moluccas islands of indonesia, but nowadays nutmeg is also grown on penang island in malaysia, in the caribbean (particularly grenada), in the southern state of kerala in india, and on the island of zanzibar. djaelani (2018) reported that north moluccas is the largest nutmeg producer in indonesia. according to cbi (2015), indonesia and grenada dominate nutmeg production and export to european countries with world market shares of 75% and 20%, respectively. india, malaysia, papua new guinea, sri lanka, and caribbean islands, such as st. vincent are also producers and exporters of nutmeg. during the postharvest period (including storage), nutmeg could be infested by insects and microorganisms. among microorganisms, fungi are the most important cause of stored *corresponding author, email: okky@biotrop.org biotropia vol. 29 no. 3, 2022 186 foodstuffs deterioration. fungal infection in foodstuffs can cause discoloration, a decrease in physical quality and nutritional contents, and mycotoxin contamination. aflatoxins are toxins produced by aspergillus flavus and a. parasiticus. aflatoxins are considered dangerous due to their association with various diseases in humans and animals, such as aflatoxicosis and liver cancer. there are four naturally occurring aflatoxins in many commodities, i.e., aflatoxins b1, b2, g1, and g2. the most toxic aflatoxin is aflatoxin b1 (basappa 2009). according to fao (2004), european union has determined the maximum tolerable limits (mtl) of aflatoxin b1 and total aflatoxins in nutmeg as 5 and 10 ppb, respectively. dharmaputra et al. (2015) reported that the postharvest handling method of nutmeg conducted by farmers and collectors in north sulawesi province was not appropriate. as the postharvest handling method of nutmeg can affect the quality of nutmeg, it is important to conduct research on the effect of some methods of postharvest handling on the quality of nutmeg, especially on fungal infection and aflatoxin contamination. the objectives of the research were to: 1) identify critical control point (ccp) in the nutmeg postharvest handling process and prepare the nutmeg haccp (hazard analysis and critical control points) system and 2) provide a recommendation on ghp (good handling practices) of nutmeg in order to maintain its quality in relation to food safety issue which is very important for international trade. materials and methods time and location of research collection of nutmeg fruits and drying of nutmegs with their shells were conducted in the location where nutmeg trees were cultivated, i.e., in kauditan subdistrict, north minahasa regency, north sulawesi province. storage of nutmeg took place in a warehouse located in bitung municipality, north sulawesi province. the determination of moisture content, percentage of damaged kernels, the population of each fungal species infecting kernels, and aflatoxin content were conducted at the food and feed as well as the phytopathology laboratories, seameo biotrop, bogor. collecting nutmeg fruits, nutmegs drying and fumigation ripe fruits of nutmeg were collected one week after they had fallen from nutmeg trees. a paranet was placed under each nutmeg tree to prevent the ripe nutmeg fruits from falling on the ground. the paranet was placed at 1 m above ground (fig. 1). figure 1 a paranet installed under each nutmeg tree to catch the ripe nutmeg fruits that naturally fell from the nutmeg trees the pulps and maces of nutmeg fruits were then separated from the whole nutmeg seeds. nutmeg seeds were dried until the moisture content was reduced by 10%. the drying process was conducted by using: 1) smoke-dried and 2) oven-dried methods (fig 2). subsequently, the nutmegs were divided into two parts prior to storage, i.e., 1) fumigated by using phosphine (2 g/m3) for 8 days to prevent the occurrence of insect infestation during storage and 2) not fumigated. the drying methods and fumigation were replicated three times. paranet postharvest quality improvement of nutmeg (myristica fragrans) – dharmaputra et al. 187 (a) (b) figure 2 drying of nutmeg using: (a) smoke-dried and (b) oven-dried methods packaging and storing of nutmeg the fumigated and non-fumigated nutmegs in the shell were packed in gunny bags. each bag contained 5 kg of nutmegs-in-shells and was stored for four months under warehouse conditions (fig. 3). in three replicates, each bag containing nutmegs was treated as follows: (a) drying methods; (b) fumigated and not fumigated; and (c) storage durations, i.e., at the beginning of storage and four months of storage. the sampling of nutmegs was conducted at the beginning of storage and after four months of storage. the number of experimental units was 24, i.e., 2 drying methods x 2 fumigated and not fumigated x 2 storage durations x 3 replications. the temperature and relative humidity of the storage were recorded using a thermohygrograph. sampling and obtaining working samples the sampling of nutmegs was conducted at the beginning of storage and after four months of storage. insects found in nutmeg were separated from nutmeg using a sieve. the insects were then preserved in vials containing 70% ethanol. each sample of nutmeg seeds was mixed homogeneously. nutmegs-in-shell were then shelled using a hammer to get nutmeg kernels. after that the nutmeg samples were separated into eight parts i.e., two parts were used for determining the percentage of damaged kernels, while the other six parts were used for determining the moisture content, fungal population, and aflatoxin content. subsequently, the six parts were ground using mill powder tech model rt 04 and mixed homogenously on a plastic tray (40 x 30 x 5 cm). the ground nutmeg was then divided into eight parts to be used as working samples, i.e., one part for determining moisture content, three parts for determining fungal population, and four parts for determining total aflatoxin content. (a) (b) figure 3 condition at the outside (a) and the inside (b) of the warehouse used for storing nutmeg seeds for four months biotropia vol. 29 no. 3, 2022 188 determination of moisture content, percentage of damaged kernels, fungal population, and aflatoxin content the moisture content of nutmeg kernels (based on a wet basis) was determined based on iso 939, i.e., the distillation method (sni 2015). two replicates were used for each sample. damaged kernels included shriveled, cracked, broken kernels, moldy and insect-damaged kernels. the percentage of damaged kernels was determined using the following formula: weight of damaged kernels (g) x 100% weight of working sample used for damaged kernel analysis (g) fungi were isolated using the serial dilution method, followed by the pour plate method on dichloran 18% glycerol agar (dg18) (pitt & hocking 2009). each fungal species was identified following pitt and hocking (2009) and samson et al. (2010). aflatoxin contents were determined using high-performance liquid chromatography (hplc) method (vicam 2007). two replicates were used for each sample. statistical analysis the data were analyzed using a completely randomized block factorial design with three factors, i.e., the drying methods, fumigated and not fumigated nutmeg seeds, and storage durations, respectively. results and discussion moisture content one of the important factors causing the deterioration of foodstuff during storage is moisture content. sni (2015) determined 10% as the maximum moisture content of nutmeg seeds during storage. based on the analysis of variance, there were no significant differences in moisture content among the applied treatments and their interaction (drying methods, fumigation, and storage duration). ranges of moisture content of nutmegs with various treatments at the beginning of storage and after four months of storage were 7.4 7.6% and 7.1 7.7%, respectively. those percentages were lower than the maximum limit of moisture content determined by sni (2015) (table 1). table 1 moisture content of nutmeg caused by various treatments during storage treatment moisture content (%) storage duration (months) 0 4 smoke-dried and fumigated 7.4 ± 0.3a 7.4 ± 0.3a oven-dried and fumigated 7.5 ± 0.3a 7.1 ± 0.3a smoke-dried and unfumigated 7.6 ± 0.1a 7.7 ± 0.3a oven-dried and unfumigated 7.5 ± 0.3a 7.3 ± 0.4a moisture content is always in equilibrium with the relative humidity of a storage room. the humidity of the storage environment will be absorbed by foodstuff stored in the storage room having high relative humidity. on the other hand, foodstuff will lose its humidity if it is stored in a storage room having low relative humidity. moisture content is also affected by the temperature of a storage room. in this study, the mean and range of temperature and relative humidity of the storage room decreased after four months of storage. the mean and range of temperature and relative humidity of the storage room at 0 4 months of storage were 27.9 ± 1.6 oc (23.2 32.8 oc) and 73.7 ± 4.2% (59.0 84.6%). percentage of damaged kernels sni (2015) determined damaged kernels including damages caused by insects and fungal attacks, cracked, broken, and shriveled kernels. based on the analysis of variance, storage duration contributed to the significant differences in the percentage of damaged kernels, while drying methods, fumigation and their interactions did not contribute any significant differences. the percentage of damaged kernels of nutmeg increased after four months of storage (table 2). table 2 percentage of damaged kernels of nutmeg during storage storage duration (months) damaged kernels (%) 0 40.5 ± 5.3a 4 45.7 ± 3.8b postharvest quality improvement of nutmeg (myristica fragrans) – dharmaputra et al. 189 in this study, the damaged kernels were arguably caused by the occurrence of insects in nutmeg during storage. there were three insect larvae in the fumigated nutmegs. as many as 20 adult insects and 7 insect larvae were found in nutmeg that were not fumigated. these findings indicated that some insects were resistant to phosphine. according to gautam et al. (2016), phosphine resistance in stored product insects occurs worldwide and is a major challenge to the continued effective use of this fumigant. phosphine resistance is present in tribolium castaneum and plodia interpunctella populations in california almond storage and processing facilities. dharmaputra et al. (2018) reported that the dominant insect in nutmeg after being stored for four months was araecerus fasciculatus. haines (1991) and rees (2004) also reported that a. fasciculatus is the most important insect infesting spices, including nutmeg. according to childers and woodruff (1980), a. fasciculatus is a primary insect pest in stored products, such as nutmegs in north and south america, africa, asia, australia, and europe. total fungal population as many as eight fungal species were isolated in nutmeg in this research. yeast was the dominant fungal species and was often isolated in nutmeg samples at the beginning of storage (table 3). in all treatments, the population of each fungal species (except yeast) was relatively low (< 10 cfu/g wet basis). aspergillus flavus was not found. these findings indicated that the postharvest handling method conducted in this research, from harvesting up to storing was appropriate to ensure good quality of nutmegs. based on analysis of variance (data transformed in log (x+1)), the interaction between drying methods and fumigation contributed to significant differences in total fungal population in nutmeg, while storage duration contributed to very significant differences. fumigated nutmeg, dried using smokeand oven-dried methods did not show any significant differences in total fungal population. on the other hand, the total fungal population in unfumigated nutmeg, dried using the smokedried method was lower than that of using the oven-dried method (table 4). the total fungal population in nutmeg after four months of storage was lower than that of the population at the beginning of storage (table 5). it was assumed that the yeast did not grow in nutmeg having moisture content suitable for storing (< 10%) during four months of storage. table 3 population of each fungal species treatment fungi fungal population (cfu/g) storage duration (month) 0 4 smoke-dried and fumigated aspergillus flavus 1 0 a. chevalieri 0 3 a. ochraceus 1 0 cladosporium cladosporioides 4 1 penicillium citrinum 0 1 p. islandicum 0 1 yeast 12 0 smoke-dried and not fumigated c. cladosporioides 2 1 p. citrinum 1 0 yeast 2 0 oven-dried and fumigated a. niger 1 0 c. cladosporioides 1 1 p. citrinum 0 1 yeast 23 0 oven-dried and not fumigated c. cladosporioides 0 2 p. citrinum 0 2 yeast 4,660 0 biotropia vol. 29 no. 3, 2022 190 table 4 total fungal population in nutmeg drying method total fungal population (cfu/g wet basis) fumigated unfumigated smoke-dried 13 ± 9b 3 ± 3a oven-dried 14 ± 16b 2,332 ± 5,395c table 5 total fungal population in nutmeg during storage storage duration (months) total fungal population (cfu/g wet basis) 0 1,178 ± 3,832a 4 4 ± 4b according to dharmaputra et al. (2015), the dominant fungal species in nutmeg collected from farmers and collectors in north minahasa regency were penicillium citrinum, a. niger, eurotium repens, a. flavus, and endomyces fibuliger. ichinose et al. (2006) reported that eurotium spp. was the predominant fungi found in 12 powdered nutmeg samples collected from retailers in indonesia. aspergillus flavus (1.0 x 102 cfu/g) was detected in one sample. according to mandeel (2005), peeled seeds of nutmeg imported from india, sri lanka, indonesia, and brazil were found to be infected by aspergillus niger, a. flavus, and rhizopus stolonifer. the predominant species was a. flavus. toma and abdulla (2013) reported 20 fungal and one yeast species isolated from 16 samples of spices and herbal medicines in shekalla market, erbil city, iraq. five of the 20 fungal species that contaminated nutmeg were a. flavus and a. niger (1 x 103 cfu/g, respectively), a. ochraceus (2 x 103 cfu/g), a. versicolor (6 x 103cfu/g), and a. wentii (2 x 203 cfu/g ). aflatoxin content aflatoxin b1 and total aflatoxin of nutmeg in various treatments were lower than the limit detection determined by hplc (< 0.92 µg/kg). these findings indicated that the postharvest handling implemented in this research was appropriate to ensure the good quality of nutmeg seeds on the occurrence of fungi and aflatoxin contamination during four months of storage. studies by dharmaputra et al. (2018) found that: a) the total aflatoxin content of nutmegs originating from hand-picked ripe fruits was lower than that from ripe fruits fell on the ground; b) total aflatoxin content in nutmegs with shells was lower than that in nutmegs without shells; and c) total aflatoxin content in nutmegs was lower for those dried using the smokeand oven-dried methods compared to those dried using the sun-dried method. the smoke-dried method could prevent toxin production produced by toxigenic a. flavus (uraih & ogbadu 1982). tabata et al. (1993) reported that aflatoxin was found in 3,054 of foodstuffs and their processed products, among others in nutmeg. the highest aflatoxin contamination was found in nutmeg (80%), while aflatoxin b1 was found in pistachio (1,382 ppb). takahashi (1993) also reported that in 1986 1991, as much as 29 (43%) of 67 samples of nutmeg collected in japan were contaminated with aflatoxin. according to martin et al. (2001), three nutmeg samples contained aflatoxin b1 from 1 to 5 ppb, three other samples 6 20 ppb, and 2 samples with 54 and 58 ppb, respectively. aflatoxin is also detected in spices, aromatic herbs, and medicinal herbs collected from common markets, supermarkets, shops, and warehouses in italy in the period of 2000 2005. nutmeg was one of the six spices which were analyzed for aflatoxin content. one of the three nutmeg samples was contaminated with aflatoxin. the contents of aflatoxin b1 and b2 in nutmeg were 2.27 and 0.47 ppb, respectively, while aflatoxin g1 and g2 were not detected (romagnoli et al. 2007). as many as 52 samples of nutmeg were imported from india and indonesia. twenty-two samples were heat treated, while the other 30 samples were not heat-treated. the heat-treated samples were less contaminated by aflatoxin than those in the untreated samples. nutmeg in powder form had more contamination than that of the whole nutmeg. of the powdered nutmeg subjected to steam treatment, 72.5% of samples were positive for total aflatoxin contamination, with a range of 0 17.2 ppb (pesavento et al. 2016). conclusion the moisture content, fungal population, aflatoxin b1, and total aflatoxin contents in nutmeg dried using smokeand oven-dried methods, fumigated and not fumigated, still complied with requirements related to food postharvest quality improvement of nutmeg (myristica fragrans) – dharmaputra et al. 191 safety, although they were stored until four months. the results of this research could also determine the critical control point (ccp) in the postharvest handling process of nutmegs, i.e., 1) choosing only ripe nutmeg fruits to be harvested; 2) harvesting method by preventing the ripe nutmeg fruits from falling on the ground; 3) drying process of nutmeg seeds should be conducted immediately after separating the nutmegs from the pulps and maces by using the smokeor oven-dried methods; and 4) nutmegs were stored with the shells. the ccp can be used as a recommendation for farmers, collectors, and exporters concerning appropriate postharvest handling methods (good handling practice) to ensure the good quality of nutmeg during storage. acknowledgments the authors gratefully acknowledge the financial support of the government of indonesia. thanks are due to the cv multi rempah sulawesi for the cooperation in conducting the preparation of the research, storing, and sampling of nutmeg; to mr. sunjaya, mr. edi suryadi, mrs. ratnaningsih and mrs. syifa fauzia for their assistance. references basappa sc. 2009. aflatoxins; formation, analysis, and control. new delhi (in): narosa publishing house. cbi. ministry of foreign affairs. 2015. cbi product factsheet: nutmeg in europe. the hague (nl): cbi market. childers cc, woodruff re. 1980. a bibliography of the coffee bean weevil araecerus fasciculatus (coleoptera: anthribidae). bull entomol soc america 26 (3): 384-94. dharmaputra os, ambarwati s, retnowati i, nurfadila n. 2015. fungal infection and aflatoxin contamination in stored nutmeg (myristica fragrans) kernels at various stages of delivery chain in north sulawesi province. biotropia 22(2): 129-39. dharmaputra os, ambarwati s, retnowati i, nurfadila n. 2018a. determining appropriate postharvest handling method to minimize fungal infection and aflatoxin contamination in nutmeg (myristica fragrans). int food res j 25(2): 545-52. dharmaputra, os, sunjaya, retnowati i, nurfadila n. 2018b. keanekaragaman serangga hama pala (myristica fragrans) dan tingkat kerusakannya di penyimpanan. [diversity of insect pest and percentage of damaged kernels in stored nutmeg (myristica fragrans)]. jurnal entomologi indonesia 15(2): 57-64. djaelani ki. 2018. enam provinsi penghasil pala terbesar di indonesia bertemu di ternate. http:// kieraha.com/6-provinsi-penghasil-pala-terbesar-diindonesia-bertemu-di-ternate [26 october 2018]. fao. 2004. worldwide regulations for mycotoxins in food and feed in 2003. fao food and nutrition paper 81. rome (it): food and agriculture organization of the united nations. gautam sg, opit gp, hosoda e. 2016. phosphine resistance in adult and immature life stages of tribolium castaneum (coleoptera: tenebrionidae) and plodia interpunctella (lepidoptera: pyralidae) populations in california. j economic entomol: 1-19. doi: 10.1093/jee/tow/221 haines cp. 1991. insects and arachnids of tropical stored products: their biology and identification (a training manual). kent (gb): natural resources institute. ichinoe m, takahashi h, ikeda n, kanai y, kiraku y. 2006. occurrence of aspergillus flavus and storage fungi in nutmeg. proceedings 2nd international symposium on mycotoxicology. bangkok, thailand, 13-14 december 2006. mandeel qa. 2005. fungal contamination of some imported spices. mycopathologia 59: 291-8. pesavento g, ostuni m, calonico c, rossi s, capei r, lo nostro a. 2016. mycotic and aflatoxin contamination in myristica fragrans seeds (nutmeg) and capsicum annum (chili), packed in italy and commercialized worldwide. j prev med hye 57: e102-e109. pitt ji, hocking ad. 2009. fungi and food spoilage. new york (us): springer. punnathara cj. 2011. campaign launched to check aflatoxin in spices. http://www. peppertrade.com. br/vernoticia08b1g09.php?idn=2062. [6 jun 2011]. rees d. 2004. insects of stored products. collingwood (au): csiro publishing. romagnoli b, menna v, gruppioni n, bergamini c. 2007. aflatoxins in spices, aromatic herbs, herb-teas and medicinal plants marketed in italy. food control 18: 697-701. samson ra, houbraken j, thrane u, frisvad jc, andersen b. 2010. food and indoor fungi. cbs laboratory manual series. utrecht (nl): cbsknaw fungal biodiversity centre. biotropia vol. 29 no. 3, 2022 192 sni. 2015. pala. sni 0006-2015. jakarta (id): badan standardisasi nasional. tabata s, kamimura h, ibe a, hashimoto h, iida m, tamura y, nishima t.1993. aflatoxin contamination in foods and foodstuffs in tokyo: 1986-1990. j aoac int 76(1): 32-5. takahashi t. 1993. aflatoxin contamination in nutmeg: analysis of interfering tlc spots. j food sci 58: 197-8. toma fm, abdulla f. 2013. isolation and identification of fungi from spices and medicinal plants. res j environ earth sci 5(3): 131-8. uraih n, ogbadu l. 1982. influence of woodsmoke on aflatoxin production by aspergillus flavus. european j appl microbiol biotechnol 14: 51-3. vicam. 2007. aflatest instruction manual for hplc. watertown (us): vicam. p 14. biotropia vol. 28 no. 1,2021: 1 10 doi: 10.1 1598/btb.2021.28.1.623 reproductive biology of freshwater clam pokea (ba tissa violacea var. celebensis, von marten 1897) (bivalvia: corbiculidae) i n pohara river, kendari, southeast sulawesi province, indonesia bahtiarl, la anadi1, wa nurgayahl, m hamzah* and udhi e. hernawanz 'faczllty offideries and marine science, huluoleo university, jalan hea mokodompit no. 1 maluka andzlonohzl, kendari 93232, indonesia 2reseurch centerfor oceanogr@h_y, indonesidn institnte ofsciences, jalun pan' putih i , ancol timur, jakarta 14430, indonesia received 15 february 2016/accepted 23 december 2019 abstract the freshwater clam, locally known as pokea, (butissa violaceu var celebensis, von martens 1897; bivalvia: corbulidae) is a popular and widely consumed food in icendari, southeast sulawesi. despite its popularity, basic information required for conservation management, such as reproductive biology, is lacking. hence, this study aims to examine the reproductive biology of the clam obtained from pohara river, icendari, southeast sulawesi province, indonesia. pokea samples were collected monthly from february 2012 to january 2013. its reproductive biology, including sex ratio, stage of gonadal maturity, gonadosomatic index (gsi), fecundity, and size of the first mature gonad from each sample were recorded. data were analyzed using chi-square test and linear regression in the package sigma plot v.6.o. pokea population in pohara river was male-biased. the population spawns throughout the year and the peak spawning season was in august-september. mature gonads were found at small shell size (indicating early sexual maturity). this gonadal development in pokea that might have been influenced by food availability, is a very relevant baseline information for the conservation of pokea population in the pohara river. keywords: clams, freshwater, gonadal maturity, spawning introduction the freshwater clam, locally known as pokea, ( b a t k uiolacea var celebensis, von martens 1897, bivalvia: corbulidae) is a popular and widely consumed food in icendari, southeast sulawesi (fig. 1). geograplucally, the genus batissa is widely distributed in the western and southern pacific (from malaysia, philippines, papua new guinea, western australia to fiji) (dudgeon & morton 1989). b. uiolacea was known to be distributed in southeast asia and northern australia (sastrapradja 1977). in indonesia, this species occurs in some of the big islands, incluchng sumatra (putri 2005), java *corresponding author, email: udhiehernawan@gmail.com (sastrapradja 1977), papua (djajasasmita 1977) and sulawesi (i<usnoto 1953). in southeast sulawesi, the clams are found in some of the big rivers in the southeast peninsular, such as pohara river, lasolo river, roraya river and laeya ever (bahtiar 2005). ecologically, this species occupies the substrate surface or lives beneath the substrate in the river estuary (sastrapradja 1977; djajasasmita 1977; bahtiar 2007a). pokea can be found in a variety of sediment types, from gravel to clay (bahtiar 2007b; bahtiar et al. 2008). this clam species lives in small and big colonies (bahtiar 2007a). these clams were believed to have different reproductive characteristics from other species of freshwater bivalves. this is because individuals of the clams are unisexual, while other species are mostly hermaphrodite. biotropia vol. 28 no. 1,2021 figure 1 the freshwater clam pokea (batha violacea var cehbensis) notes: a: width; b: length. very likely due to a combination of uncontrolled harvesting and sand mining in its habitat, the pokea populations in pohara river have been declining. this is manifested by the declining trend of both the number and size of the sampled pokea. in addition, localized extinction has been reported in the mining areas. this situation calls for some form of conservation management intervention of this species (bahtiar e t al. 2012). despite its high demand for consumption, coupled with its declining populations, some basic information required for effective conservation management, such as reproductive biology, is lacking (bahtiar 2012). accurate information on the reproductive biology of this species is critically relevant for several conservation practices, particularly in determining the harvesting season and minimum clam size for sustainable harvesting (cantillaneza e t al. 2005). hence, this research aims to examine the reproductive biology of the pokea clam in pohara river, kendari, southeast sulawesi. material and methods sample and data collection 122023'556" e), from february 2012 to january 2013 (fig. 2). during the first week of each month, a total of 120 individual clams were collected, then separated based on the gender. male individuals have milky white colored gonad, while female ones have brown colored gonad. the recorded parameters for reproductive biology, included sex ratio, fecundity, stage of gonadal maturity, gonado somatic index, and size of the first mature gonad from each samples. gonadal development was divided into 5 levels: inactive (stage i), early development (stage 11), final development (stage 111), mature (stage iv), spawning/post-spawning (stage v). this gonadal development was examined based on the gonad morphology and histology (bahtiar 2012). the gonado somatic index (gsi) was measured based on illanes e t al. (1985) and wolff (1987) methods. fecundity was assessed based on gravirnetric method, using egg weight, total gonad weight, and body weight (eiffendi 2002). size of the first mature gonad was screened from all class sizes (arocha & barios 2009). the phytoplankton abundance was also recorded at the sampling sites. all laboratory works were conducted at the faculty of pokea samples were collected monthly from fisheries, haluoleo university, icendari, the estuary of pohara river (03"58'551m s and southeast sulawesi province, indonesia. reproductive biology of freshmter dam pokea in p o h m river bahtiar d al. '4 0 160 3201(m c v-1 s e a 0 345mwm d mars di-t i i n e g u l i . . i west uyrroeiol(met r o r i e a . 0 66 1m km tmm! ,a&=s 'ns%w€ 1-re figure 2 map of the sampling sites at pohara river, kendari, southeast sulawesi province data analysis the significant difference of the sex ratio was determined using the chi-square (x? test where: (mzighani 2005), using the following formula: = fecundity number where: " (oi ei)' x2 = c i=l oi n = number of eggs from sampled gonads g = total gonad weight g = weight of the sampled gonads oi = the number of males and females the relationship between fecundity and the observed at i shell width was analyzed using regression ei = the expected number of males and analysis (steel & torrie 1981). the probability females at i of mature gonad was calculated using nonlinear regression functions with a logarithmic curve gonado somatic index (gsi) was calculated (arocha & barios 2009), using the following using the formula described by sastry (1979): formula: bg gsi = ~ 1 0 0 % bt where: gsi = gonado somatic index bg = gonad weight (g) bt = body weight includmg gonad (g) pokea's fecundity was calculated based on the gravimetric method (effendi 2002), using the following formula: where: y = probabihty of mature gonad (yo) e = exponential function a = intercept b = slope x = width at i (cm) biotropia vol. 28 no. 1,2021 the relationshp between the gsi and gonadal development represents the phytoplankton abundance was analyzed using reproductive processes that occur until the the pearson correlation. all statistical analyses spawning event. the processes were divided were done using sigma plot v.6.o. into five stages, from inactive phase to the spawning phase. the parallel gonadal stages of both male and female pokea observed in this results and discussion study confirmed the studes of bahtiar (2012). sex ratio the number of male individual clams was higher than that of the female individuals at each sampling month, at an average male/female ratio of 65/35 (table 1). the sex ratio was significantly male biased, indicated by the chi square test value = 0.0003). this male-biased sex ratio conforms with previous studies by bahtiar (2005) and bahtiar (2012). however, sex ratio in bivalve population is not always male biased but varies with different species. for example, the clam gam' elongata population exhbited a slightly female-biased sex ratio (nabuap & campos 2006). the male-biased sex ratio might be the consequence of the fact that (i) pokea reproduces by external ferohzation, and (ii) males of pokea are generally smaller in size than females. in external fertilization, the sperms are subjected to greater mortality in the aquatic environment. therefore, to increase the probability of a successful fertilization, the number of male individuals should be higher than that of female indviduals (bahtiar 2012). table 1 sex ratio of the freshwater clam poliea furthermore, the peak periods of spawning events were also similar. partial spawning was also observed based on the changes in the egg dameter. egg diameters at gonadal stage iv consisted of two size groups, with median values of 21.60 pm and 159.16 pm (bahtiar 2012). at the post-spawning stage or), three size groups were reported, with median value of 22.80 pm, 172.06 pm and 262.82 pm (fig. 4). t h ~ s condition implied that the eggs reached maturity at different periods, indicating that the pokea spawns throughout the year. therefore, pokea can be classified as a partial/multiple spawner, although it has a peak period of spawning. partial/multiple spawner is common in bivalves; for example the clam venw veracosa spawns all year long, however the spawning event peaks in august. some populations spawn in december firado etal. 2003), or the clam donax tmnc~lzls shows two peaks of spawning periods (march and august) with gametogenesis cycle starts in late november and finishes by the end of august (gaspar et al. 1999). month male (%) female (o/o) march 2012 april 201 2 may 2012 june 2012 july 2012 august 2012 september 2012 october 2012 november 2012 december 2012 january 201 3 f e b m a ~ 201 3 total 65.0591 34.9409 reproductive biology of freshwater clam pokea in pohara river bahtiar et al. male loo 1 c, c e a 20 0 0 ? a g m u female -0 0 40 20 " i o 2012 20 13 mar apr may jun jul aug sep oct nov dec jan feb figure 3 stages of gonadal development in male and female pokea notes: stage i (inactive); early stage i1 (development); stage i11 (final development); stage iv (mature); and stage v (spawning/post-spawning). i gonad stage 1 v gonad stage v gonadal stage egg diameter ( lm) egg diameter (pm) figure 4 bhattacharya analysis on the distribution of egg diameter on gonadal stage iv and v (modified from bahtiar 2012) gonado somatic index (gsi) from 1.46 to 15.69, and peaked in july. after the both male and female pokea showed similar peak event in july, the gs1 value temporal patterns of gsi. averaged gsi ranged declined in august-september 2012 (fig. 5). biotropia vol. 28 no. 1,2021 female male 0 i i i i i i i i i i i i i 2012 20 13 mar apr may jun jul aug sep oct nov dec jan feb observation month figure 5 mean gsi of male and female h k e a in each observation month gsi indicates annual reproductive cycle and it links with the stages of gonadal maturity. therefore, a concordance of temporal pattern existed between gsi and gonadal stages, in whch gsi peaked in july, followed by the peak of spawning event in august-september. bahtiar (2012) also reported a similar pattern. reproductive cycles in bivalves varies with different species. for example, corbicula australis spawns at the end of september and october (bryne 2000), while corbicula puminea spawns twice a year in early may or june and late september (mouthon 2001). this intraspecific variation in the reproductive cycle is probably related with different geographical locations. however, this is not conclusive as evidences or studies on the reproductive biology of pokea are still lacking. gonadal maturity is determined based on egg diameter changes in each month, as egg size represents reproductive growth in the gonad (haggertyet al 2005). when the gsi values were compared, changes in egg diameter showed a similar temporal pattern. this is because the gsi value is determined by egg size (bahtiar 2012) (fig. 6). a similar pattern was also observed in the species megalonaias nervosa showing the number and size of oosit increased at the end of june (mature gonad) unul the end of september (spawning), then decreased in early october (inactive phase) (haggerty et al 2005). fecundity the relationship between fecundity (i?) with the median class of shell width (l) was determined from a simple regression equation, f = 36815"l-81706 with a determination coefficient (r) = 0.97. an individual with a minimal shell width of 2.97 cm contained a total of 4652 eggs, while another one with a width of 5.93 cm contained 130,465 eggs (fig. 7). numbers of eggs foundin this study were relatively smaller compared with the findings of bahtiar (2012) ranging from 4,950 to 1,007,384. the small number of eggs observed in this study might be a consequence of the environmental disturbances resulted from the sand mining activities. the dsturbances might have triggered the early maturity of the clams resulting in the decreased number of eggs. compared with other species, the egg number recorded in ths study was much lower. for example, the unionid mussels (unionidae) contained 200,000 to 17,000,000 eggs (vaughn 2005) and the macker mussel actinonaias hgamentina contained 80,616 to 1,561,224 eggs (moles & layzer 2008). biotropia vol. 28 no. 1,2021 shell width (cm) figure 7 linear relationship between fecundity and shell width in pokea o male 4 female 0 2 4 6 8 shell width (cm) figure 8 size of the first mature gonad in male and female pokea relationship between the reproductive the bivalve reproductive performance. the parameters and environmental factors important role of food availability in triggering simple h e plots showed a concordance between the reproductive performance of pokea (gsi) and environmental conditions (phytoplankton abundance). this pattern was further examined in a simple linear regression showing that the relationship has a coefficient of determination, r2 = 69.05% (fig. 9). as pokea feeds on phytoplankton, the increase of phytoplankton abundance in july might be related with gonadal maturity in pokea, as it reached a spawning peak in august-september. hence, environmental condition has influenced sexual maturity has been well documented, particularly in bivalve farming. the increased gsi in a m e l t e n iradians was stimulated by an external factor that increased availability of food in terms of organic matter (gosling 2002). the increase of gsi in gam' elongata occurred following the increased of food supply, again in terms of organic matter (nabuab & campos 2006). other environmental factors, such as ph, temperature, organic matter, and seston, might also be important pahtiar 2012). reproductive biology of freshwater clam pokea in pohara river bahtiar et al. f + phytoplankton abundance i apr may jun jul aug sep oct nov dec jan feb observation month figure 9 relationship between gsi and phytoplankton abundance conclusion (tetraptzlw albidus) from the western central atlantic. aust j mar freshwater res 43:393-402. the pokea (batissa violacea var celebensis) bahtiar. 2005. icajian populasi pokea (batissa violacea var. population in pohara &ver was male-biased. celebensis, von martens, 1897), 1897 di sungai the population spawned throughout the year pohara, icendari, sulawesi tenggara. vhesis]. bogor (id): institut pertanian bogor. and the peak spawning season was in august september. mature gonad was found at small bahtiar. 2007a. iconservasi populasi pokea (batissa violacea var. celebensis von martens, 1897), di sungai pohara, shell size (indicating early sexual maturity). food icendari, sulawesi tenggara. laporan hibah availability might have influenced the gonadal bersaing. jakarta (id): dp2m-dikti. development in pokea. t h s is a very relevant baseline information for the conservation management of pokea population in the pohara river. acknowledgment the authors would like to thank all the field assistants during the sampling period and the dean of the faculty of fisheries and marine sciences, haluoleo university, i<endari, southeast sulawesi, indonesia for the support and suggestions for the completion of this study. references arocha f, barios a. 2009. sex ratio, spawning seasonality, sexual maturity, and fecundity of white marlin bahtiar. 2007b. preferensi habitat dan lingkungan perairan pokea (batissa violacea celebensis martens, 1897) di sungai pohara, icendari, sulawesi tenggara. aqua hayati 5(2):81-7. bahtiar, yulianda f, setyobudiandi i. 2008. icajian aspek pertumbuhan populasi pokea (batissa violacea var. celebensis, von martens 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environment and forestry, palu 94111, indonesia received 27 sept 2022 / revised 22 sept 2023 / accepted 30 oct 2023 abstract forest and land fires in indonesia need attention in mitigation efforts, specifically in conservation areas. lore lindu national park (llnp) is a conservation area that plays a role in preserving endemic plants and animals and their habitats. forest fires often occur in the lore lindu national park area, especially in the savanna area. research on the factors that influence forest fires in llnp needs to be carried out to protect and prevent the risk of forest fires to protect the ecosystem. this study aims to analyze the relationship between rainfall and land cover on the number of hotspots as an indicator of forest fires in llnp in 2017-2021. thus, preventive strategies can be carried out as early as possible to prevent forest fires. this research was conducted by spatially and statistically analyzing 2017-2021 rainfall data in sigi and poso regencies, hotspot data from the terra/aqua, snpp, and modisnasa satellite in 2017-2021 in llnp, and llnp land cover data. in statistical analysis, rainfall data for hotspots was analyzed using bivariate pearson, and land cover data for hotspots was analyzed spatially using arcmap with categorical analysis. the results showed 55 hotspots in llnp from 2017-2021. the bivariate pearson correlation test results between rainfall and hotspots are -0.028. the relationship between rainfall and hotspots shows a nondirectional correlation. the lower the rainfall intensity, the more hotspots are found. spatially, the highest number of hotspots occurred in areas with the land cover of secondary dryland forest types, with a total of 29 spots. llnp will endeavour to increase security patrols in certain seasons and cover types that have the potential for forest and land fires. keywords: forest fires, hotspots, land cover, lore lindu, rainfall introduction forest fires in indonesia havebecome national and global environmental issues (ananda et al. 2022). forest fires affects air quality, landscapes and ecosystems, soil and water quality, and causes major problems with greenhouse gas emissions (badri et al. 2018). forest fires occur due to climatic or artificial factors (saputra et al. 2021). in addition, forest fires in indonesia happen almost every year. in 2015, indonesia's forest fires were the largest after forest fires in 1997 and 1998 (endrawati et al. 2017). the government is still trying to prevent and deal with forest fires in indonesia. the government campaigns with the community to prevent forest fires in indonesia, (muzaki et al. 2021), strengthens policies, and takes concrete actions to protect forest areas. one of the government's policies in protecting forest and land areas is through the forestry and other land uses (folu) net sink 2030 program. folu net sink is a condition where the amount of carbon absorbed by forests equals or exceeds the carbon emissions produced. efforts to prevent deforestation and forest fires are one of crucial mitigations in maintaining the earth cycle and the climate crisis so that emissions released by forests can be greater in 2030. another effort to control forest and land fires is monitoring by remote sensing. remote sensing is an approach to obtaining various information such as areas, objects, and data analysis without direct contact with the areas, objects, and studies being analyzed (utomo et al. 2017). one of the remote sensing methods to detect forest fires is *corresponding author, email: maariffadjri@gmail.com biotropia vol. 31 no. 1, 2024 2 to use satellite image data, such as hotspot data. hotspots can be used as indicators of forest fires with the modis (moderate resolution imaging spectroradiometer) sensor (giglio et al. 2016). the satellite data used to detect hotspots is terra/aqua, snpp, and nasa satellite with the modis sensor (indradjad et al. 2019). data related to forest fires can be accessed through the directorate general of climate change (ppi) website of the ministry of environment and forestry via https://sipongi.menlhk.go.id. the directorate general of ppi has also launched the si pongi application to monitor real-time hotspots in indonesia. forest fires are generally caused by human activities (dini et al. 2022). cochrane (2013) generally described how human activities cause forest fires through deforestation, agriculture, and animal husbandry. forest fires affect the condition of land cover, structure, density, composition, and forest productivity (fitria et al. 2021). moreover, deforestation, land cover changes, population growth, and climate deviations cause forest fires (harrison et al. 2009). in addition to human factors, natural factors also affect forest fires, one of which is climate (putra & husni 2021). this is because the climate (temperature, humidity, rainfall, and wind speed) can affect the dryness of surface fuels, the amount of oxygen, and the spread of fire (syaufina 2008). low rainfall causes a prolonged dry season which impacts drought. this condition increases the level of vulnerability to forest fires. other factors that influence the occurrence of forest fires are land cover conditions, soil types, and other biophysical environmental factors (yusuf et al. 2019). rainfall is the height of rainwater that falls on a flat surface with conditions that do not evaporate, seep, and flow (mulyono 2016). the unit of rainfall in indonesia is expressed in millimeters (mm), which means that one square meter area on a flat surface accommodates one millimeter of water or one liter of water (prawaka et al. 2016). rainfall conditions play an important role in the hydrological cycle on the earth's surface. low rainfall impacts a prolonged dry season and can trigger forest and land fires. lore lindu national park (llnp) is one of the conservation areas in central sulawesi. geographically, llnp is located at coordinates 119o 90’ 120o 16’ east longitude and 1o 8’ 1o 3' south latitude. administratively, llnp is located in sigi regency and poso regency and is surrounded by 71 buffer villages. the area of llnp is 215,733.70 ha and has 1,532 hectares of a savanna ecosystem, which is prone to forest fires. llnp is important in preserving endemic plants and animals and their habitats. forest and land fires often occur within the llnp area. these fires generally occur during the summer with low rainfall and in open areas. efforts to protect and prevent the risk of forest fires must be carried out so that the ecosystem that becomes the habitat of wild plants and animals can be maintained. many researchers have investigated the level of forest fire susceptibility, including examining the relationship between hotspots and rainfall in aceh barat regency (saharjo & nasution 2021); and analyzing forest fire susceptibility in various climates (rainfall) and vegetation (land cover, biomass, and humidity) (jaya et al. 2007). however, research on the level of forest fire susceptibility in llnp has never been published. therefore, this research needs to be carried out to analyze the relationship between rainfall and land cover on the number of hotspots as an indicator of forest fires in llnp in 2017-2021 so that preventive measures in preventing forest fires can be carried out as early as possible. materials and methods this research was conducted from july to september 2022 in the llnp. this study used stationaries, laptops with arcmap gis 10.5 software, microsoft office word, microsoft excel, and spss 22. the materials used hotspot data from the terra/aqua satellites, snpp, and modis-nasa (with a medium (30 79%) and high (80 100%) confidence level) for the period 2017 to 2021 in llnp, monthly rainfall data for sigi district (gimpu and sungku stations) and poso district (doda, north lore, lore piore, and maholo stations) for the 20172021 period from the meteorology, climatology, and geophysics agency (bmkg), and llnp land cover data which is the result of llnp's proprietary land cover analysis and in collaboration with a third party. the initial stage in the data analysis process classifies hotspot data for the 2017-2021 period the relationship between rainfall and land cover on the number of hotspots – fadjri maarif and cesar mappatoba 3 based on time (month and year). the distribution of hotspots using terra/aqua satellites, snpp, and modis-nasa data accessed via https://sipongi.menlhk.go.id. hotspot data in central sulawesi province is overlaid with the shapefile map of the llnp area using the arcmap gis ver 10.5 application. furthermore, the hotspot data in central sulawesi province is processed with a map shapefile of the llnp area through the intersect function so that only hotspot data remains in the llnp. rainfall data for 2017-2021 is processed with a map of the llnp area through the inverse distance weighted (idw) function in the arcmap gis ver 10.5 application. the radius used is a standard radius with a value of five times the output raster cell size. this stage is carried out to determine areas that have the potential for high, medium and low rainfall in the llnp area. rainfall classification refers to the classification level of the meteorological, climatological and geophysical agency (bmkg). hot spot data in llnp is overlaid (intersect) with a map of rainfall conditions in llnp to determine the suitability between areas with certain rainfall conditions and the distribution of hotspots within them. next, the hotspot data in the llnp is overlaid with the shapefile of the land cover map in the llnp. this stage is carried out to determine the distribution of hotspots based on land cover in llnp. in statistical data analysis, hotspot data and rainfall data analyzed using bivariate pearson with spss ver 22 application. this step was carried out to determine the correlation value between hotspots and rainfall in llnp. the bivariate pearson correlation equation is as follows: 𝑟 = 𝑛 ∑ 𝑥𝑦 − ∑ 𝑥 ∑ 𝑦 √𝑛 ∑ 𝑥2 − (∑ 𝑥)2. √𝑛 ∑ 𝑦2 − (∑ 𝑦)2 where: r = correlation coefficient x = independent variable y = dependent variable n = number of data results and discussion hotspots distribution a hotspot is a term for a pixel with a temperature value above a certain threshold from the interpretation of satellite imagery, which can be used as an indication of forest and land fires (the ministry of environment and forestry 2016). hotspots observed as indicators of forest fires are generally recordings from the modis sensor. data from the terra/aqua, snpp, and modis-nasa satellites show hotspots recorded by sensors within the llnp. the data used with a medium (30 79%) and high (80 100%) confidence level) based on the results of monitoring hotspots in llnp from 2017 to 2021, the number fluctuates yearly (table 1). table 1 the number of hotspot in tnl year number of hotspot 2017 6 2018 9 2019 16 2020 13 2021 11 total 55 the number of hotspots from 2017 to 2021 is 55 spots. during the last five years, hotspot patterns increased from 2017 to 2019, with 6, 9, and 16 spots, respectively. the most hotspots occurred in 2019, with a total of 16 spots. then, it decreased in the next two years with 13 and 11 spots, respectively (figure 1). https://sipongi.menlhk.go.id/ biotropia vol. 31 no. 1, 2024 4 figure 1 the number of hotspots each month from 2017 to 2021 the hotspot pattern occurring every month from 2017 to 2020 showed a low intensity at the beginning of the year, but in 2021 it showed a high intensity. the number of hotspots every month in the last five years showed an increasing pattern from july to november. at the end of the year, the hotspot decreased until the beginning of the following year. the highest number of hotspots occurred in september 2019, with seven spots. meanwhile, in february, may, and june, no hotspots were found from 2017 to 2021 because of the changes in rainfall intensity, which were higher from the beginning to the middle of the year than at the end of the year. rainfall is one of the factors that affect the emergence of hotspots. according to (prabowo et al. 2020), the condition of high rainfall is caused by the appearance of convective clouds that cause rain, thus preventing the emergence of hotspots. the relationship between rainfall and hotspots the meteorology, climatology, and geophysics agency (bmkg) divides the classification of monthly rainfall into three categories: dry season (0-100 mm/month), humid season (100-200 mm/month), and wet season (>200 mm/month). based on the observations of rainfall data from 2017 to 2021, the average rainfall in sigi and poso provinces overall is 162,45 mm/month in the category of medium-level rainfall or humid conditions. the highest rainfall was recorded in 2017, with an average of 177 mm/month. the number of hotspots in 2017 was recorded at six spots, the year with the least hotspots in five years. rainfall recorded in five years from june to september has a lower intensity than the average rainfall in other months. on the other hand, hotspots that occur from july to october have a fairly high number compared to other months. therefore, the higher the intensity of rainfall, the fewer the number of hotspots found (figure 2). 0 0 0 3 0 0 0 1 0 2 0 00 0 0 0 0 0 0 5 2 2 0 00 0 0 0 0 0 2 4 7 0 0 3 0 0 3 1 0 0 0 1 1 2 5 0 2 0 4 1 0 0 0 0 0 3 0 1 0 1 2 3 4 5 6 7 8 jan feb mar apr may jun jul aug sep oct nov des 2017 2018 2019 2020 2021 the relationship between rainfall and land cover on the number of hotspots – fadjri maarif and cesar mappatoba 5 figure 2 graph of the relationship between rainfall and the number of hotspots in llnp from 2017 to 2021 based on the distribution map of the hotspots to rainfall, the presence of hotspots is more widely distributed in areas with categories of humid and dry season. the number of hotspots in the dry season is 14 spots (22.45%), 39 spots in the humid season (70.91%), and 2 spots in the wet season (3.64%) (figure 3). the lower rainfall intensity causes this condition in poso regency (average 130.50 mm/month) than the rainfall intensity in sigi regency (average 226.34 mm/ month). the number of hotspots distributed in areas with dry and humid conditions is estimated to be influenced by the el nino phenomenon (hadiyani & nurhayati, 2022), which inhibits the growth of clouds that cause rain so that air and soil conditions become drier. it shows that high and low rainfall intensity in an area can control the number of hotspots in that area. figure 3 distribution of hotspots based on rainfall in llnp 0 1 2 3 4 5 6 7 8 0 50 100 150 200 250 300 350 400 450 ja n '1 7 f eb m a r a p r m a y ju n ju l a u g s ep o ct n o v d es ja n '1 8 f eb m a r a p r m a y ju n ju l a u g s ep o ct n o v d es ja n '1 9 f eb m a r a p r m a y ju n ju l a u g s ep o ct n o v d es ja n '2 0 f eb m a r a p r m a y ju n ju l a u g s ep o ct n o v d es ja n '2 1 f eb m a r a p r m a y ju n ju l a u g s ep o ct n o v d es rainfall (mm/month) hotspots biotropia vol. 31 no. 1, 2024 6 the bivariate pearson correlation test results between rainfall and hotspots are -0.028, which shows a negative correlation in the relationship between rainfall and the number of hotspots. it means that the two have a non-directional correlation. thus, the higher the rainfall intensity, the smaller the number of hotspots found, and vice versa. it is in line with the results of syaufina & sukmana (2008), that the increase and decrease in the number of hotspots in a certain month are related to the increase and decrease in rainfall intensity. the value of -0.028 in the analysis of rainfall and the number of hotspots interprets the two as having a very low correlation. the p-value between rainfall and hotspot shows 0.833. this value is above the 0.05 significance value limit (5% error probability), so rainfall cannot be used as the only factor/parameter that affects the number of hotspots (table 1). syaufina & hafni (2018) described the p-value is higher than the maximum error limit value because the hotspot is not normally distributed, and there is a difference in the time of occurrence. table 1 the results of the statistical test of the correlation of rainfall with the number of hotspot in llnp rainfall hotspot rainfall pearson correlation 1 -.028 sig. (2-tailed) .833 n 55 55 hotspot pearson correlation -.028 1 sig. (2-tailed) .833 n 55 55 according to aflahah et al. (2019), rainfall is closely related to temperature. temperature changes can affect rainfall patterns which impact the length of the dry season, thereby allowing the potential for forest and land fires to occur. the rainfall factor does not significantly influence the number of hotspots in llnp, so it is necessary to analyze other climatic factors such as temperature, humidity, and others. the relationship between land cover and hotspots land cover is a physical form of an object that covers land and is natural (utomo et al. 2017). forest fires can occur due to the influence of the land cover type on the surface of an area. different types of land cover affect the characteristics of the material on it to burn. the hotspots mapping to land cover in llnp (data from the llnp and third-party analyses) shows that hotspots were recorded in five types of land cover: 29 spots of secondary dryland forest (52.73%), 16 spots (29.09%) of savanna, 8 spots (14 .55%) of plantations, 1 spot (1.82%) of scrubland and dry riceland, respectively (figure 4). figure 4 distribution of hotspot based on land cover in llnp the relationship between rainfall and land cover on the number of hotspots – fadjri maarif and cesar mappatoba 7 secondary dryland forest is the most extensive type of land cover in the llnp area, with an area of 174,411.20 ha. the condition of the secondary dryland forest that covers most of the llnp area causes the hotspots to be more widely distributed. according to sinaga & darmawan (2014), secondary dryland forest is the appearance of lowland, mountainous, and hilly forests that have shown the occurrence of former logging activities. it indicates that the secondary dryland forest type land cover is an area where human activities utilize forest products. figure 4 shows that the type of secondary dryland forest land cover is close to the settlements around the llnp area, so human activities possibly cause the hotspot for this type of land cover. like secondary dryland forests, land cover types of plantations and dry riceland are also areas that local residents widely use. plantation activities have a close relationship with land clearing so forest fires can occur intentionally by humans. savanna and scrubland are the areas recorded as having a total of 16 and 1 hotspot, respectively. these two types of land cover have the characteristics of areas with no forest and vegetation. this condition causes higher temperatures, more flammable surfaces, or low rainfall during the dry season. based on the distribution pattern of hotspots in the llnp, the number of hotspots on the edge of the llnp is higher than the center of the llnp area. this is because the hotspots recorded in the llnp are mostly caused by human activities related to plantation activities by local residents. this has an impact on the incidence of forest fires which are more often found around areas that are easily accessible by local residents. whereas in the central area of the area no hotspots were found because the area is far from the reach of human activity so no hotspots were found in the middle of the llnp area. based on rasyid (2014), forest fires are generally caused by the human factor. some of the causes of forest fires due to human activities include: (1) traditional farming system, (2) forest clearing, and (3) structural causes. impact on biodiversity forest fires have an impact on biodiversity in these ecosystems. burning forests will damage ecosystem conditions and are difficult to restore. ecosystem conditions that experience forest fires will cause loss of vegetation on the land surface and the occurrence of open areas. the opened area in an ecosystem has an impact on the loss of several species of plants and animals in that area. the loss of these species also threatens the existence of species that have a high conservation status of rarity so that some species are threatened with extinction. several species of important and rare value generally grow in forest areas with dense vegetation, so if an opened area occurs in an ecosystem it will threaten the extinction of these species. lore lindu national park is a nature conservation area that is home to several protected and endangered species. there are endemic species such as tarsier, anoa, maleo, sulawesi hornbill, and several other exotic flora. forest and land fires are a threat to the existence of these species, so efforts to protect and preserve these species in their ecosystems need to be increased, especially in certain seasons and in certain types of land cover. conclusion the 55 hotspots in llnp were recorded from 2017 to 2021. the most hotspots were in 2019, with a total of 16 hotspots. the hotspot pattern fluctuates yearly, with hotspot conditions increasing from july to october. the number of hotspots each year is influenced by conditions of rainfall intensity. the two variables show a nondirectional correlation, meaning that the higher the rainfall, the lower the hotspot. the distribution pattern of hotspots is more common in poso regency, with an average rainfall intensity lower than the average rainfall intensity in sigi regency. the distribution of hotspots by type of land cover shows that the number of hotspots is more in the secondary dry land forest area, as many as 29 points. the condition of hotspot distribution based on land cover occurs around the settlements in llnp and is indicated to occur artificially by humans. rainfall and land cover can be used as factors that affect the presence of hotspots in llnp. however, in addition to rainfall, it is necessary to correlate it with other climatic factors such as humidity and temperature for a more comprehensive study result. this research is limited to spatial analysis of the relationship between hotspot data and land cover. the next recommendation is to analyse biotropia vol. 31 no. 1, 2024 8 how socio-economic factors affect the needs of the community to get their needs in the forest, 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dengan kelas tutupan lahan menggunakan data citra satelit landsat (studi kasus: kabupaten pati) [analysing the relationship between land surface temperature variation and land cover class using landsat satellite image data (case study: pati district)]. jurnal geodesi undip, 6(2), 71-80. yusuf a, hapsoh, siregar sh, nurrochmat dr. 2019. analisis kebakaran hutan dan lahan di provinsi riau [analysis of forest and land fires in riau province]. dinamika lingkungan indonesia, 6(2), 67-84. doi: 10.31258/dli.6.2.p.67-84. 911.cdr 159 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 1124.cdr liquid (commercial) liquid powder page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 sri-13 mei 2017-550 (mohd khalizan isolation and characteriz).cdr isolation and characterization of a molybdenum-reducing and phenolicand catechol-degrading enterobacter sp. strain saw-2 mohd khalizan sabullah 1,2* 2 2, mohd fadhil rahman , siti aqlima ahmad , mohd rosni sulaiman , mohd shukri shukor , azlan jualang gansau , 3 1 1 nor aripin shamaan and mohd yunus shukor5 2 1faculty of science and natural resources, universiti malaysia sabah, kota kinabalu 88400, sabah, malaysia 2 department of biochemistry, faculty of biotechnology and biomolecular sciences, universiti putra malaysia, serdang 43400, selangor, malaysia 3 faculty of food science and nutrition, universiti malaysia sabah, kota kinabalu 88400, sabah, malaysia 4snoc international sdn bhd, lot 343, inland port 71800, negeri sembilan, malaysia 5faculty of medicine and health sciences, islamic science university of malaysia, kuala lumpur 55100, malaysia received 21 october 2015/accepted 10 august 2016 abstract molybdenum is an emerging pollutant worldwide. the objective of this study is to isolate molybdenum-reducing bacterium with the ability to grow on phenolic compounds (phenol and catechol). the screening process was carried out on a microplate. the bacterium reduced molybdenum in the form of sodium molybdate to molybdenum blue (mo-blue). the bacterium required a narrow ph range for optimal reduction of molybdenum, i.e. between ph 6.3 and o6.8, with temperature between 34 and 37 c. molybdate reduction to mo-blue was best supported by glucose as the carbon source. however, both phenol and catechol could not support molybdate reduction. other requirements for molybdate reduction included sodium molybdate concentrations between 15 and 30 mm, and phosphate concentration of 5.0 mm. the bacterium exhibited a mo-blue absorption spectrum with a shoulder at 700 nm and a maximum peak near the infrared region at 865 nm. the mo-reducing bacterium was partially identified as enterobacter sp. strain saw-2. the capability of this bacterium to grow on toxic phenolic compounds and to detoxify molybdenum made it a significant agent for bioremediation. keywords: catechol, enterobacter sp., molybdenum, molybdenum blue, phenol introduction heavy metals are toxic to organisms. some microorganisms are able to detoxify heavy metals by employing mechanisms such as biosorption, sequestration or chelation, active pumping and reduction (batta et al. 2013; oves et al. 2013; banerjee et al. 2016). microorganisms having capability to reduce heavy metals such as chromium, molybdenum mercury into less toxic forms were documented. molybdenum (mo) is essential to organisms as it is a cofactor to many important enzymes such as nitrogenase, sulfite oxidase, aldehyde oxidase and xanthine oxidoreductase (daniels et al. 2008; leimkühler et al. 2011). molybdenum is not generally toxic to human. however, molybdenum is very toxic to ruminants, such as sheep and cattle at concentrations as low as several parts per million (ppm). an elevated level of molybdenum in ruminants causes a disease known as hypercuprosis (ward 1978). recent data showed that molybdenum is toxic to spermatogenesis and embryogenesis in animals, including catfish and mice (meeker et al. 2008; bi et al. 2013; zhai et al. 2013; zhang et al. 2013). toxic effect of molybdenum to these animals warrants removal of molybdenum from the environment. heavy usage of molybdenum in various steel products and lubricants resulted in high concentration of molybdenum reaching hundreds of ppm in the water bodies of the black sea and tokyo bay biotropia 4 1 7 47 58 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 1 550 * corresponding author: ejanz_sart2@yahoo.com 47 (davis 1991; neunhäuserer et al. 2001). terrestrially, in the mine tailings of a molybdenum mine in new mexico, as high as 2,000 ppm of molybdenum concentration were documented (runnells et al. 1976). together with heavy metals, oil, grease and phenolics are hydrocarbon which are reported to be the number one scheduled waste (yadzir et al. 2016). phenol and phenolic compounds (fig. 1) are not only toxic to human, but also to many other organisms (shukor et al. 2008a; shukor et al. 2008b; rahman et al. 2009; yunus et al. 2009). the presence of multiple pollutants requires microorganisms with multiple detoxification ability. thus, the objective of this study is to isolate such a microorganism. in this study, we reported on the isolation of a molybdenumreducing bacterium with the ability to grow on phenolic compounds (phenol and catechol). materials and methods chemicals chemica ls such as na moo .2h o, 2 4 2 mgso .7h o, (nh ) .so nacl and na hpo 4 2 4 2 4, 2 4 were purchased from sigma aldrich (st. louis, mo, usa) and were of analytical grade while glucose and yeast extract were purchased from fisher (malaysia). isolation of molybdenum-reducing bacterium molybdenum-reducing bacterium was isolated using two kinds of media, i.e. low phosphate molybdate (lpm) and high phosphate molybdate (hpm) media. lpm medium consists of the following composition: mgso .7h o (0.05% 4 2 w/v), (0.206% or 10 mm), yeast na moo .2h o2 4 2 extract (0.05% w/v), (nh ) .so (0.3% w/v), 4 2 4 glucose (1% w/v), nacl (0.5% w/v) and na hpo (0.071% w/v or 5 mm). the lpm 2 4 medium was adjusted to ph 7.0. agar having concentration of 1.5% (w/v) was added to the lpm solid media. bacterial cell harvesting from the lpm medium could not be carried out, as blue aggregates formed in the lpm liquid media. hence, the phosphate concentration was increased to 100 mm which made the medium became hpm (ghani 1993). soil materials et al. for the isolation of molybdenum-reducing bacteria were taken from topsoil depth of 5 cm in a polluted area in kuching, sarawak (1.6077° n, 110.3785° e) in april, 2011. a cell suspension containing 1 g of soil and 10 ml of sterile distilled water was prepared. soil suspension of 0.1 ml aliquot was immediately spread on an agar plate containing lpm media. the plates were incubated for 48 hours at room temperature (27 c). several white and blue colonies appeared o afterwards. colony with the strongest blue intensity was restreaked on lpm agar until a pure culture was obtained. hpm media was utilized to prepare for the resting cells of the molybdenumreducing bacterium. growth of the bacterium was carried out in a 1 l culture volume, shaken using orbital shaker, set at 120 rpm, and incubated for 48 hours at room temperature (27 c). cells o were harvested via centrifugation (10,000 xg, for 10 minutes). distilled water was utilized to wash the bacterial pellets after centrifugation twice. the pellets were then resuspended in 10 ml of lpm media (5 mm) with the glucose omitted. preparation of resting cells preparation and use of resting cells in a microtiter format were carried out based on shukor and shukor (2014). cell suspension prepared previously (180 ml) was transferred to each well of a sterile microplate. sterile glucose (20 ml) from a 10% (w/v) stock solution was 48 biotropia vol. 24 no. 1, 2017 (a) phenol (b) catechol (c) 2-chlorophenol figure 1 chemical structure of some toxic phenolic compounds results and discussion bacterial molybdenum reduction to mo-blue is a phenomenon that has been described for more than one hundred years and is a prospective bioremediation tool (shukor et al. 2014). this phenomenon was initially observed in 1896 in e. coli bacterium (levine 1925). comprehensive research about this phenomenon was only started in 1985 in the e. coli k12 bacterium (campbell et al. 1985). later on, sodium molybdate reduction into mo-blue by the thiobacillus ferrooxidans bacterium, a chemolitotroph, was reported by sugio et al. (1988) without mentioning the works of campbell et al. (1985). this suggested the scarcity of publications on this phenomenon. enterobacter cloacae strain 48 (ec 48) is the first bacterium isolated from the malaysian soils with the capacity to reduce molybdate (ghani et al. 1993). the first molybdenum-reducing bacterium reported with the ability to detoxify other xenobiotics is the sds-degrading klebsiella oxtoca (halmi et al. 2013). isolation of more xenobiotics-detoxifying molybdenum-reducing bacterium can expand the capability of existing isolates for bioremediation of sites containing molybdenum, together with other xenobiotics. partial identification of the molybdenumreducing bacterium the bacterium was gram-negative, rodshaped and motile. the biochemical test results using the abis online software showed that bacterium having the highest homology (90%) and the highest accuracy (97%) was enterobacter cloacae (table 1). the bacterium is tentatively named enterobacter sp. strain saw-2. identification of the bacterium up to the species level can only be carried out through the addition of several more polyphasic methods, such as16s rrna gene sequencing, dna–dna hybridization determination of genomic dna g+c content and fatty acid profile (ahmad et al. 2013). two molybdenumreducing bacteria from this genus, i.e. enterobacter cloacae strain 48 (ghani et al. 1993) and enterobacter sp. strain dr.y13 (shukor et al. 2009) were previously reported. a bacterium from this genus has been reported for having ability to tolerate high concentrations of heavy metals such as nickel, cadmium and lead at levels of up to 700, mixed with the cell suspension. the microplate was covered with a sterile sealing tape (corning® microplate). the microplate was incubated at o room temperature (27 c) for 48 hours. mo-blue production was determined at 750 nm in a microtiter plate reader model no. 680, (biorad, richmond, ca). the specific extinction -1 -1 coefficient of 11.69 mm cm was utilized to determine mo-blue concentration (shukor et al. 2003). heavy metals such as lead (ii), arsenic (v), mercury (ii), silver (i), chromium (vi) copper (ii) and cadmium (ii) on mo-blue production were obtained from atomic absorption spectrometry (aas) calibration solutions (merck chemical co., germany). the capability of phenolics to act as electron donors was tested using the microplate format. the phenolics tested were phenol, 2,4d in i t rophenol , pentach lorophenol , 2 chlorophenol, 4-chlorophenol, catechol, salicylic acid, 4-nonylphenol, p-hydroxybenzoic acid, benzoate and 2-napthol. the phenolics replaced glucose and were tested at 200 mg/l, but in a volume of 50 ml (arif et al. 2013). if the phenolics could be used as electron donors, moblue production will increase. on the other hand, ability of the above phenolics to support the growth of this bacterium independently from molybdenum-reduction was carried out in hpm media minus molybdenum. the increase of bacterial growth after 48 hours of incubation at room temperature, which was an indication of phenolics assimilation, was measured at 600 nm. identification of the molybdenum-reducing bacterium standard biochemical tests according to the bergey's manual (holt et al. 1994) was used to identify the molybdenum-reducing bacterium. in addition, a software-based method (abis online system) was utilized to interpret the results (costin & ionut 2015). briefly. briefly, the standard methods included gram staining, detection of motility via the hanging drop method, and various biochemical tests. statistical analysis data analyses were carried out using graphpad prism v 6.0 (www.graphpad.com). the student's t-test or anova with tukey's test as the post hoc analysis was carried out to compare means of groups. 49 isolation and characterization of enterobacter sp. strain saw-2 – sabullah et al. 900 and 1,100 ppm, respectively (banerjee et al. 2015). this phenomenon suggested that being heavy metal tolerant and conducting heavy metal reduction are part of the strategies employed by bacteria from this genus to combat heavy metal toxicity. nickel was detected to be more toxic (700 ppm), followed by cadmium (900 ppm) and lead (1,100 ppm). optimal ph that supported molybdenum reduction was between 6.5 and 6.8 (fig. 2), in temperature range of 34 37 °c (fig. 3). 50 biotropia vol. 24 no. 1, 2017 table 1 biochemical tests for enterobacter sp. strain saw-2 notes: + = positive result; − = negative result; d = indeterminate result 0.0 0.5 1.0 1.5 2.0 5.5 6.0 6.5 7.0 7.5 8.0 ph a b s 7 5 0 n m figure 2 effect of initial ph on molybdenum reduction by enterobacter sp. strain saw-2 note: error bars represent mean±standard deviation (n = 3) high-throughput method using a microplate format was employed in this study. this method can accelerate characterization works, while acquiring more information as opposed to the regular shake-flask method (iyamu et al. 2008; shukor & shukor 2014). the use of resting cells for studying mo-blue production was initiated in ec 48 (ghani et al. 1993). resting cells were also used in studying heavy metals reduction such as in chromate (llovera et al. 1993), selenate (losi & jr 1997) and xenobiotics biodegradation such as diesel (auffret et al. 2015) and phenol (sedighi & vahabzadeh 2014). molybdenum absorbance spectrum the scanning absorption spectrum of the resultant mo-blue from enterobacter sp. strain saw-2 showed a maximum peak at 865 nm, and a characteristic shoulder at about 700 nm (fig. 4). 51 0.0 0.5 1.0 1.5 2.0 2.5 20 30 40 50 60 temperature ( o c) a b s 7 5 0 n m figure 3 effect of temperature on molybdenum reduction by enterobacter sp. strain saw-2 note: error bars represent mean±standard deviation (n = 3) 0.0 0.5 1.0 1.5 2.0 600 650 700 750 800 850 900 950 wavelength (nm) a b s 8 h 24 h 32 h 48 h figure 4 scanning absorption spectrum of mo-blue from enterobacter sp. strain saw-2 at different time intervals isolation and characterization of enterobacter sp. strain saw-2 – sabullah et al. it was observed that the resulting spectrum resembled the spectrum of the mo-blue produced by the phosphate determination method (clesceri et al. 1989). spectrum produced by phosphate determination exhibited a maximum absorption around 880 to 890 nm. a characteristic shoulder was seen around 700 to 720 nm (hori et al. 1988). all of the mo-blue spectra from previously isolated molybdenum-reducing bacteria showed similar spectra. based on this knowledge, a hypothesis was developed for this study i.e. molybdenum reduction should proceed via the intermediate phosphomolybdate (shukor et al. 2007). another basis for the hypothesis was the confirmation of the presence of phosphomolybdate species through spectrophotometric analysis of the resultant absorption spectrum (sims 1961; yoshimura et al. 1986; hori et al. 1988). aside from the demonstration of the unique spectroscopic profile of the blue product, the presence of mo-blue by itself can visually prove that molybdenum reduction has taken place. reduced phosphomolybdate or mo-blue has a fractional oxidation state between 6+ and 5+ (sidgwick 1951; kazansky & fedotov 1980). other metals, such as mercury and chromate, show no visible color when experiencing reduction through bacterial process (suzuki et al. 1992). therefore, confirmation of reduction has to be carried out through epr-electron paramagnetic resonance (suzuki et al. 1992). the presence of an intermediate species is not unique to mo-blue production, as this is also reported in bacterial chromate reduction, such as bacteria pseudomonas ambigua (suzuki et al. 1992) and shewanella putrefaciens (now known as s. oneidensis) (myers et al. 2000). in these bacterial reduction processes, an intermediate species cr5+ was observed (myers et al. 2000). effect of electron donor on molybdate reduction molybdate reduction to mo-blue was best supported by the sugar glucose. this was followed by sucrose, maltose, l-rhamnose, raffinose, d-mannose, lactose, mucate, cellobiose, d-mannitol, d-adonitol, melibiose, glycerol, d-sorbitol and l-arabinose in descending order (fig. 5). mo-blue production by other sugars such as dulcitol, salicin and myo-inositol were found to be not significantly different ( > 0.05) from p control based on anova . with tukey's test previous works demonstrated that molybdenumreducing bacteria prefer simple assimilable sugars, such as sucrose, glucose and fructose (campbell et al. et al et al 1985; ghani . 1993; rahman . 2009; yunus . 2009; et al shukor et al. 2009; ahmad et al. 2013; othman et al. 2013; abo-shakeer et al. 2013). the metabolic pathways glycolysis, kreb's cycle and electron transport chain were used to convert these sugars to nadh and nadph. both reducing equivalents are electron donating substrates for the molybdenum reducing-enzyme (shukor et al. 2014). 52 biotropia vol. 24 no. 1, 2017 0.0 0.5 1.0 1.5 2.0 2.5 d -a do ni to l lara bi no se c el lo bi os e d ul ci to l g ly ce ro l d -g lu co se m yo -in os ito l la ct os e m al to se d -m an ni to l d -m an no se m el ib io se m uc at e r af fin os e lr ha m no se sal ic in d -s or bi to l suc ro se c on tro l a b s 7 5 0 n m figure 5 effect of different electron donor sources (1% w/v) on molybdenum reduction note: error bars represent mean±standard deviation (n = 3) effect of phosphate and molybdate concentrations to molybdate reduction the optimal concentration of phosphate supporting optimal molybdenum reduction occurred at 5 mm. concentrations higher than this dramatically ceased production of mo-blue (fig. 6). high phosphate concentrations inhibit the stability of phosphomolybdate, of which upon reduction, it is converted to mo-blue glenn & crane 1956; sims 1961; shukor et al. 2000). molybdenum-reducing bacteria isolated previously are also strongly inhibited by phosphate concentration higher than 5 mm (shukor & syed 2010; lim et al. 2012; ahmad et al. 2013; halmi et al. 2013; othman et al. 2013; aboshakeer et al. 2013; khan et al. 2014; shukor et al. 2014). enterobacter sp. strain saw-2 can tolerate and reduce molybdenum at sodium molybdate concentrations as high as 60 mm, but the production of mo-blue was severely inhibited. the optimal concentrations of molybdate which supported the reduction, were between 15 and 30 mm (fig. 7). the other molybdenum-reducing 53 0.0 1.0 2.0 3.0 0 10 20 30 40 50 phosphate (mm) a b s 7 5 0 n m figure 6 molybdenum reduction by enterobacter sp. strain saw-2 at various phosphate concentrations note: error bars represent mean±standard deviation (n = 3) 0.0 0.5 1.0 1.5 2.0 2.5 0 20 40 60 80 molybdate (mm) a b s 7 5 0 n m figure 7 molybdenum reduction by enterobacter sp. strain saw-2 at various sodium molybdate concentrations note: error bars represent mean±standard deviation (n = 3) isolation and characterization of enterobacter sp. strain saw-2 – sabullah et al. bacteria isolated to date, require optimal molybdate concentration between 10 and 80 mm (shukor, rahman . 2010; shukor & syed 2010; et al lim . 2012; ahmad . 2013; halmi . et al et al et al 2013; othman . 2013; abo-shakeer . et al et al 2013; shukor . 2014). reduction at these very et al high concentrations is an advantage for molybdenum bioremediation in an area having high concentration of this metal. for instance, in new mexico where molybdenum concentration as high as 2,000 ppm or about 20 mm was reported (jacobs et al. 2014). effect of heavy metals bacterial molybdate reduction to mo-blue was inhibited by other heavy metals, which included cadmium (ii), mercury (ii), copper (ii) and silver (i) at 2 ppm with inhibition levels of 45.5, 36.8, 28.4 and 24.4%, respectively. these inhibition levels were compared to control, which was assigned as having 100% activity (fig. 8). molybdate reduction to mo-blue in many of the previously isolated molybdenum-reducing bacteria were inhibited by similar toxic heavy metals (shukor & syed 2010; lim et al. 2012; othman et al. 2013; shukor et al. 2014). in hexavalent chromate reduction to the trivalent state by bacillus sp. (arutchelvan et al. 2006) and enterobacter cloacae strain h01 (rege et al. 1997), the metal ions of mercury and copper were strong inhibitors. the supposed target of these metals was chromate reductase. this enzyme utilized the electron donors nadh and nadph to convert soluble toxic chromium 6+ to the insoluble less toxic chromium 3+. mercury binds strongly to sulfhydryl groups. additionally, mercury also binds, with variable strength, to carboxyl, amide, phosphoryl and amine groups of protein. this is the reason why mercury is one of the most toxic metal ions known to human. silver also binds to the sulfhydryl group of enzymes. copper preferentially binds to cysteine, histidine and methionine residues of enzymes (camakaris et al. 1999). the toxicity of these metal ions can be remedied through the addition of chemical additives including phosphate, calcium carbonate, manganese oxide and magnesium hydroxide (hettiarachchi et al. 2000; deeb & altalhi 2009). pheno l i cs as carbon sources f o r molybdenum reduction and independent growth preliminary screening works on phenolics as carbon sources supporting molybdenum reduction failed to give positive results (data not shown). however, the bacterium was able to grow on the phenolic compounds (phenol and catechol) (fig. 9). 54 biotropia vol. 24 no. 1, 2017 0 20 40 60 80 figure 8 the effect of other heavy metals on mo-blue production by enterobacter sp. strain saw-2 note: error bars represent mean±standard deviation (n = 3) biodegradation of phenol and phenolic compounds by microorganisms has long been an object of research. bacteria that could degrade phenol and phenolic compounds include pseudomonas species (folsom et al. 1990; tomasi et al. 1995; aravindhan et al. 2014; hasan & jabeen 2015), bacillus brevis (arutchelvan et al. 2006), alcaligenes sp. (bai et al. 2007), ochrobactrum sp. (kiliç 2009), acinetobacter sp. (ahmad et al. 2011; yadzir et al. 2016) and rhodococcus species (arif et al. 2013). conclusions enterobacter sp. strain saw-2 showed the novel ability to reduce heavy metal molybdenum to mo-blue. this bacterium can also grow on the phenolic compounds (phenol and catechol). the identity of this bacterium is not completely robust; therefore, molecular identification is needed to further identify this species. this bacterium demonstrated a narrow ph and temperature ranges for optimal reduction. glucose was the most effective electron donor for optimal molybdenum reduction. the bacterium needed a critical phosphate concentration of 5.0 mm. the optimal molybdate concentrations were between 15 and 30 mm. the absorption spectrum of the mo-blue generated indicated that it was a reduced phosphomolybdate. cadmium (ii), mercury (ii), copper (ii) and silver (i) inhibited the process of molybdenum reduction to mo-blue. presently, efforts are ongoing to purify the molybdenumreducing enzyme and to characterize phenolics biodegradation studies in greater detail. from bioremediation point of view, this reported bacterium might be an important bio-tool in reducing environmental pollutants like molybdenum. acknowledgements this project was funded by the research university grant scheme (rugs), project no. 0501-09-0750ru/f1. references abo-shakeer lka, ahmad sa, shukor my, shamaan na, syed ma. 2013. isolation and characterization of a molybdenum-reducing bacillus pumilus strain lbna. j environ microbiol toxicol 1:9–14. ahmad sa, shukor my, shamaan na, mac cormack wp, syed ma. 2013. molybdate reduction to molybdenum blue by an antarctic bacterium. bio med res int 2013, ar t ic le number 871941. doi:10.1155/2013/871941. ahmad sa, syed ma, arif nm, shukor mya, shamaan na. 2011. isolation, identif ication and characterization of elevated phenol degrading acinetobacter sp. strain aq5nol1. aust j basic appl sci 5:1035–45. 55 0.0 0.2 0.4 0.6 0.8 1.0 c on tro l phe no l 2, 4d in itr op he no 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in water by gel-phase colorimetry with molybdenum blue. anal chem 58:591–4. 58 biotropia vol. 24 no. 1, 2017 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 biotropia vol. 30 no. 2, 2023: 220 231 doi: 10.11598/btb.2023.30.2.1896 220 diversity, ecology and conservation status of nepenthes in west sumatra province, indonesia muhammad mansur1,2*, andi salamah3*, edi mirmanto2 and francis q. brearley4 1postgraduate biology study program, faculty of mathematics and natural sciences, universitas indonesia, depok, west java, 16404, indonesia. 2research center for ecology and ethnobiology, indonesian national research and innovation agency (brin), jalan raya jakarta-bogor, km 46, cibinong science center, cibinong, west java, 16911, indonesia. 3cellular and molecular mechanisms in biological system (cembios) research group, department of biology, faculty of mathematics and natural sciences, universitas indonesia, depok, west java, 16404, indonesia. 4department of natural science, manchester metropolitan university, chester street, ,,,,jmanchester, m1 5gd, uk received 14 february 2023 / revised 11 april 2023 /accepted 12 april 2023 abstract nepenthes is the largest carnivorous plant genus present in indonesia. there are 39 species of nepenthes pitcher plants recorded in sumatra from lowland to montane forests, and 34 of them are endemic; this represents the greatest species diversity of nepenthes after borneo. field studies were conducted in 2021 and 2022 to increase our knowledge of the diversity, habitats and distributions of nepenthes in west sumatra province. twenty-three species of nepenthes were recorded from the province, consisting of 15 highland species, 4 mid-elevation species and 4 lowland species. ecophysiological studies conducted at bukit malalak showed clumped distributions of n. bongso, n. dubia, n. eustachya and n. rhombicaulis. foliar and pitcher fluid nutrient concentrations were found to be similar to those cited in other recent studies although growth rates were slightly more rapid than at gunung talang. bukit malalak is a new locality for two threatened species, namely n. dubia (cr) and n. rhombicaulis (vu), enlarging their extents of occurrence. in total, nine species from west sumatra are threatened and conservation actions are urgently needed for these and other nepenthes species remaining on the island. keywords: associations, carnivorous plants, diversity, ecology, foliar nutrients, indonesian nepenthes, montane forest, red list, west sumatra introduction nepenthes is the only genus in the nepenthaceae family (phillipps & lamb 1996; clarke 2001). nepenthes are climbing lianas that produce characteristic fluid-filled pitchers extending from tendrils at the end of leaf-like phyllodes. the pitchers have a range of adaptations to attract, catch, retain and digest mostly insect prey (moran & clarke 2010). the indonesian archipelago, with a land area of 1,919,440 km2, is in the centre of the distribution of nepenthes (which extends from madagascar to new caledonia). in 2021, 80 species, or approximately 44 % of all nepenthes (181), were recorded from indonesia (mansur et al. 2021), with diversity concentrated in borneo and sumatra. over the past few decades, many new species have been described, particularly in indonesia, malaysia and the philippines. in the last three years alone, three new species have been described in sumatra: n. putaiguneung al farishy, metusala & jebb in kerinci seblat national park (metusala et al. 2020), n. longiptera victoriano in aceh (victoriano 2021) and n. harauensis hernawati, r. satria & chi.c.lee in west sumatra (hernawati et al. 2022b). there are now 39 nepenthes species recorded in sumatra (hernawati et al. 2022a). mansur et al. (2022a) recently documented the 22 species found in the province of north sumatra, but information from other provinces remains poor. this is particularly true of the provinces through which the barisan mountains run, as these are likely to harbour the highest diversity of *corresponding author, email: mansurhalik@yahoo.com/ salamah@sci.ui.ac.id mailto:mansurhalik@yahoo.com mailto:salamah@sci.ui.ac.id diversity, ecology and conservation status of nepenthes in west sumatra province, indonesia – mansur et al. 221 nepenthes due to their topographic variation (nerz 2005). furthermore, there are few ecophysiological studies of nepenthes species endemic to sumatra, in contrast to those in borneo (but see pavlovič et al. 2009, 2010; moran et al. 2012; mansur et al. 2022b). west sumatra has a diverse topography, ranging from lowlands (< 500 m asl) to mountains (> 2000 m asl), with a broad range of associated microclimates and geologies. these conditions allow this province to support a high diversity of nepenthes species. the estimated forest cover in west sumatra is 1,897,911 ha, of which 41.7% is protected forest, 15.8% is production forest and 42.5% is within nature reserves and nature conservation areas (dinas kehutanan sumatra barat 2018). the aim of this study was therefore to determine the species diversity, distribution, population, habitat and ecology of nepenthes in west sumatra province. materials and methods field studies were conducted in october 2021 and october 2022 (totalling four weeks) in agam regency (bukit malalak and gunung singgalang), limapuluh kota regency (air putih nature reserve, batu karang-harau, kelok sembilan and palupuah), solok regency (gunung talang) and around the city of padang (figure 1). exploration was conducted to inventory the species of nepenthes in each study location. further study of literature, along with examination of herbarium specimens at herbarium bogoriense-cibinong, took place in may and june 2022 to add information about the species of nepenthes in west sumatra; pasaman regency was only studied through a review of literature and herbarium specimens. we further focused on at bukit malalak (00°09 s; 100°23' e) where three plots of 0.09 ha each (10 m × 90 m) were established to determine the abundance of nepenthes and the tree species growing in their habitat. each species of nepenthes in the nine subplots (10 × 10 m each) and their positions (x and y) were recorded. trees (ø ≥ 5 cm) in each subplot were identified and their trunk diameter, total height and positions (x and y) were measured. all data collected was processed and analysed according to the mueller-dombois & ellenberg method (1974) to obtain values for basal area (ba), relative frequency (rf), relative density (rd), relative dominance (rdo), and importance value index (ivi). a few individuals of each nepenthes species were numbered with small aluminium tags, and the top leaf of each individual was punched with a small hole using a paper hole-punch. after 12 months, the plants still living were re-censused and the stem length and number of leaves of each individual were re-recorded. figure 1 map of six study sites in four regencies/cities in west sumatra province (study site 1: around padang city (00°60' s; 100°23' e); study site 2: gunung talang (00°59' s; 100°41' e); study site 3: gunung singgalang (00°24' s; 100°20' e); study site 4: bukit malalak (00°09' s; 100°23' e); study site 5: kelok sembilan (s: 00°04' s ; 100°42' e), air putih nature reserve and palupuah (00°03' s; 100°41' e); study site 6: batu karang-harau (00°03' s; 100°44' e). biotropia vol. 30 no. 2, 2023 222 interspecific associations between plant species were calculated using a 2 × 2 contingency table (ludwig & reynolds 1988) on species that had an ivi > 10 % (zulkarnaen et al. 2017). the calculated χ2 value was then compared with the χ2 table at the 5 % test level. if the value of χ2 was greater than that in the χ2 table at the 5 % test level, then there was an association between the two species, whereas if the value of χ2 was less than that in the χ2 table, then there was no association (mueller dombois & ellenberg 1974). the strength of the associations were then calculated using the ochiai (1957) index with values ranging from 0 to 1, wherein the stronger the association between the two plant species, the closer the index value is to 1 (ludwig & reynolds 1988). equations are presented in more detail in mansur et al. (2022a). finally, to determine the spatial distribution of each nepenthes species (i.e. regular, random or clumped), morisita’s (1959) index of dispersion was calculated using a quadrat size of 10 m × 10 m. laboratory procedures analysis of nutrient concentrations was carried out on samples of n. bongso korth., n. dubia danser and n. rhombicaulis sh.kurata from bukit malalak. leaf samples were dried in an oven at 60 °c for three days and then ground with a pestle and mortar. two ml of mixed acid (sulphuric acid, nitric acid and perchloric acid) was added to 0.2 g of leaf material and heated on a hotplate at 170 °c until the solution was clear; samples were then diluted to a final volume of 10 ml before analysis. phosphorus concentration was determined using a colorimetric method, in which 1 ml of sample was added to 3 ml of distilled water and then reacted with 1 ml of p dye; the yellow colour of the sample was measured using a shimadzu biospec-mini 1240 uv-vis spectrophotometer at 450 nm. nitrogen concentration was also determined by a colorimetric method, where 2 ml of the sample reacted with 4 ml of sodium phenoxide and 4 ml of 5% naocl; the blue colour of the sample was measured using a spectrophotometer as above but at a wavelength of 636 nm. other elements were determined with atomic absorption spectrophotometry using a shimadzu aa-6800. pitcher fluid was analysed as above after being filtered to remove any debris. results and discussion diversity in total, 23 species of nepenthes were recorded from west sumatra province, comprising 18 species documented during the field surveys, two species represented by herbarium specimens (n. jamban chi c.lee, hernawati & akhriadi: bo.1979938 and n. lingulata chi c.lee, hernawati & akhriadi: bo.1979937), two species documented from the region in literature records, i.e., n. izumiae troy davis, c.clarke & tamin (clarke et al. 2003) and n. jacquelineae c.clarke, troy davis & tamin (clarke 2001), and one species also indicated for the region via a credible secondhand source, namely n. naga akhriadi, hernawati, primaldhi & m.hambali (putra-rsn nepenthes nursery, pers. comm.; table 1). the majority of species documented are considered highland species (15), with four species each of mid-elevation and lowland species. seven species are restricted to west sumatra, 11 are found across other provinces of sumatra (in addition to west sumatra) and five are also found on other indonesian islands. nine species have been assessed as having a threatened status (2 × vu, 2 × en, 5 × cr) against the international union for conservation of nature (iucn) 3.1 criteria (iucn 2001) (clarke et al. 2000a, b, c; clarke 2014; hernawati & clarke 2014; hernawati et al. 2014; cross et al. 2020). limapuluh kota regency had the greatest number of species (9), followed by pasaman regency (8 species), solok and agam regencies (7 species each) and then padang city, with just 2 species. we also found four natural hybrids: n. talangensis nerz & wistuba × n. bongso and n. talangensis × n. inermis danser at gunung talang, and n. eustachya miq. × n. albomarginata w.lobb ex lindl., and n. gracilis korth. × n. eustachya in air putih nature reserve. diversity, ecology and conservation status of nepenthes in west sumatra province, indonesia – mansur et al. 223 table 1 habitat, distribution, elevation and conservation status of the 23 nepenthes species recorded from west sumatra province. species in bold were recorded from the field survey nepenthes habitat regency elevation distribution iucn red list category ag lm pd ps sl adnata tamin & m.hotta ex schlauer forest ● m/h ws en [d] albomarginata w.lobb ex lindl. forest/shrubland ● ● m/h indo lc ampullaria jack shrubland ● l/m indo lc bongso korth. forest ● ● h s lc dubia danser mossy forest ● h ws cr [b1+2e] eustachya miq. forest/shrubland ● ● ● m s lc gracilis korth. shrubland ● ● ● l indo lc harauensis hernawati, r.satria & chi.c.lee forest ● h ws inermis danser mossy forest ● h s lc izumiae troy davis, c.clarke & tamin forest/shrubland ● h ws lc jacquelineae c.clarke, troy davis & tamin mossy forest ● h ws cr [b2ab(v) c2a(i)] jamban chi c.lee, hernawati & akhriadi: mossy forest ● h s cr [b2ab(v) c2a(i)] lingulata chi c.lee, hernawati & akhriadi: forest ● m s cr [b2ab(v)] longifolia nerz & wistuba forest/riparian ● m s lc mirabilis (lour.) druce shrubland ● l indo lc naga akhriadi, hernawati, primaldhi & m.hambali forest ● h s vu [d2] pectinata danser forest ● ● ● h s lc reinwardtiana miq. shrubland ● ● l/m indo lc rhombicaulis sh.kurata forest ● h s vu [d2] singalana becc. forest ● h s lc spathulata danser forest/mountain peak ● ● h s lc talangensis nerz & wistuba forest/mossy forest ● h ws en [c2b] tenuis nerz & wistuba forest ● h ws cr [a2] notes: ag = agam, lm = limapuluh kota, pd = padang city, ps = pasaman, sl= solok; l = lowland, m = midelevation, h = highland; ws = west sumatra only, s = sumatra only, indo = other islands in indonesia (in addition to sumatra); lc = least concern, vu = vulnerable, en = endangered, cr = critically endangered, = not yet assessed against iucn red list criteria. in our survey, conducted across five regencies in west sumatra, we recorded 23 species of nepenthes, 18 of which are endemic to the island of sumatra. this number is comparable to our survey in north sumatra (22, including two taxa yet to be formally described; mansur et al. 2022a), with 12 species common to both surveys. nepenthes eustachya, n. gracilis and n. pectinata danser are three species whose distribution is quite broad, as they are found in three regencies/cities; indeed, n. gracilis is widespread not only in western indonesia, but also in peninsular malaysia and southern indochina. other species, however, are more limited, notably n. talangensis, which is found only on gunung talang (solok regency; nerz & wistuba 1997), n. jacquelineae in pasaman regency (clarke 2001) and n. harauensis in limapuluh kota regency (hernawati et al. 2022b), all three of which are the type localities for those species, while n. jamban, n. lingulata and n. naga are found not only in pasaman regency (west sumatra), but also in mandailing natal regency (north sumatra), on the border between west sumatra and north sumatra. population nepenthes population measurements were only carried out at bukit malalak. in the study plots, n. bongso generally grew as an epiphyte under a shady canopy with a small population (plot 2), while in open areas (mountain peak), it grew terrestrially with a larger population (plot 1). in a total plot area of 0.27 ha, seven individuals of n. biotropia vol. 30 no. 2, 2023 224 dubia were found: four in plot 1 and three outside of the plot in mossy forest (mountain peak), growing terrestrially under a slightly open canopy (table 2). nepenthes eustachya was abundant in plot 3 and grew terrestrially in shady areas. thirteen individuals of n. rhombicaulis were found in plot 1 and ten were found in plot 2, where they grew terrestrially in shady areas. morisita’s index was greater than 1 for all species in all plots, indicating a clumped distribution; the exception was of n. rhombicaulis in plot 2, which had a value of 0.80, indicating a more random distribution. these clumped distributions are in keeping with earlier studies (adam 2002; damit et al. 2017), which is likely due to similar habitat requirements for light or sufficiently moist soil, for example. more advanced statistical techniques, such as those based on ripley’s k, are likely to be more informative regarding spatial patterns if sufficient individuals are measured (brearley et al. 2023). table 2 number of individuals of four nepenthes species in three plots each of 0.09 ha at bukit malalak, agam regency (sumatra). nepenthes elevation (m) plot 1 (1586 m asl) 2 (1329 m asl) 3 (1186 m asl) total bongso 1300–1600 64 6 0 70 dubia 1550–1600 4 0 0 4 eustachya 1150–1300 0 0 50 50 rhombicaulis 1300–1600 13 10 0 23 figure 2 nepenthes distributions in three 0.09 ha plots on bukit malalak, agam regency (sumatra). open circles show locations of each tree species with larger circles indicating trees of larger stem diameter. coloured circles indicate locations of nepenthes individuals of different species diversity, ecology and conservation status of nepenthes in west sumatra province, indonesia – mansur et al. 225 habitat at the bukit malalak study site, nepenthes plants grew in primary forest with very steep sloping. the forest’s condition was intact, with few signs of human disturbance. in the total plot area of 2700 m2, there were 416 individual trees (ø ≥ 5 cm) recorded, comprising 34 species, 29 genera and 20 families. myrtaceae was the most species-rich family, with eight species, five of which were syzygium. the five dominant tree species in the nepenthes habitat were ternstroemia gymnanthera (wight & arn.) bedd. (ivi = 42.1) followed by syzygium zeylanicum (l.) dc. (28.5), myrsine avenis (blume) a.dc. (24.0), rhodoleia championii hook. (22.1) and leptospermum javanicum blume (19.1; table 3). table 3 list of tree species and their abundance in 0.27 ha of forest plots containing nepenthes species at bukit malalak, agam regency (sumatra) species family ba (m2) rd (%) rf (%) rdo (%) ivi (%) ternstroemia gymnanthera (wight & arn.) bedd. pentaphylacaceae 1.330 22.1 6.04 14.0 42.1 syzygium zeylanicum (l.) dc. myrtaceae 1.100 11.5 5.37 11.6 28.5 myrsine avenis (blume) a.dc. primulaceae 0.720 10.3 6.04 7.60 24.0 rhodoleia championii hook. hamamelidaceae 0.860 7.69 5.37 9.02 22.1 leptospermum javanicum blume myrtaceae 1.170 3.37 3.36 12.3 19.1 syzygium sumatranum (miq.) widodo myrtaceae 0.760 4.33 6.04 8.01 18.4 castanopsis costata (blume) a.dc. fagaceae 0.500 4.33 6.04 5.26 15.6 syzygium bankense (hassk.) merr. & l.m.perry myrtaceae 0.440 4.09 4.70 4.65 13.4 gaultheria heterophylla var. latifolia (blume) kron & p.w.fritsch ericaceae 0.360 3.85 5.37 3.80 13.0 pittosporum ferrugineum w.t.aiton pittosporaceae 0.350 2.16 5.37 3.71 11.2 carallia eugenioidea king rhizophoraceae 0.210 3.61 4.70 2.17 10.5 eriosolena composita (l.f.) tiegh. thymelaeaceae 0.310 3.13 3.36 3.29 9.77 neolitsea cinnamomea (ridl.) kosterm. lauraceae 0.200 1.92 3.36 2.06 7.34 tristaniopsis merguensis (griff.) peter g.wilson & j.t.waterh. myrtaceae 0.200 1.92 2.68 2.11 6.72 heptapleurum sp. araliaceae 0.060 1.44 3.36 0.58 5.38 dacrydium elatum (roxb.) wall. ex hook. podocarpaceae 0.160 0.96 2.68 1.68 5.32 adinandra dumosa jack pentaphylacaceae 0.150 1.68 2.01 1.54 5.24 pterophylla fraxinea d.don cunoniaceae 0.170 0.96 2.01 1.76 4.74 syzygium acuminatissimum (blume) dc. myrtaceae 0.100 1.68 1.34 1.07 4.1 calophyllum teysmannii miq. calophyllaceae 0.070 0.96 2.01 0.75 3.73 polyosma ilicifolia blume escalloniaceae 0.050 1.20 2.01 0.49 3.71 archidendron bubalinum (jack) i.c.nielsen fabaceae 0.020 0.96 2.01 0.18 3.15 symplocos adenophylla wall. ex g.don symplocaceae 0.020 0.72 2.01 0.25 2.99 schima wallichii (dc.) korth. theaceae 0.020 0.72 2.01 0.21 2.95 elaeocarpus mastersii king elaeocarpaceae 0.020 0.72 2.01 0.20 2.93 litsea sp. lauraceae 0.050 0.48 1.34 0.51 2.34 wendlandia densiflora (blume) dc. rubiaceae 0.020 0.72 1.34 0.26 2.32 lithocarpus conocarpus (oudem.) rehder fagaceae 0.020 0.72 1.34 0.23 2.29 myrica javanica blume myricaceae 0.040 0.48 1.34 0.39 2.21 vaccinium lucidum (blume) miq. ericaceae 0.010 0.24 0.67 0.09 1.00 rhodamnia cinerea jack myrtaceae 0.010 0.24 0.67 0.07 0.98 syzygium antisepticum (blume) merr. & l.m.perry myrtaceae 0.010 0.24 0.67 0.06 0.98 garcinia lateriflora blume clusiaceae 0.004 0.24 0.67 0.04 0.95 timonius flavescens (jacq.) baker rubiaceae 0.003 0.24 0.67 0.03 0.94 grand total 9.517 note: ba = basal area, rd = relative density, rf = relative frequency, rdo = relative dominance, ivi = importance value index biotropia vol. 30 no. 2, 2023 226 interspecific associations the ochiai index showed that n. bongso was positively associated with n. dubia and strongly positively associated with ternstroemia gymnanthera in plot 1 (table 4). in plot 2, n. rhombicaulis was positively associated with n. bongso, but they were weakly associated in plot 1. nepenthes rhombicaulis was also strongly positively associated with five species with a very high ochiai index in plot 2 and, in plot 3, n. eustachya had positive associations with seven species with a very high ochiai index (table 4). adam (2002) also found positive associations between n. × kinabaluensis sh.kurata and n. villosa hook.f. and leptospermum recurvum hook.f. (myrtaceae) in montane forest in northern borneo, so it would be valuable to determine the traits of the trees that nepenthes tend to associate with. furthermore, understanding which habitats and which particular tree species or communities certain nepenthes species associate with may provide further insight into where these nepenthes species may be found in other localities. table 4 chi-square (χ2) and ochiai index (oi) values showing inter-specific associations between nepenthes bongso (plot 1), n. rhombicaulis (plot 2), and n. eustachya (plot 3) and other nepenthes species and the ten tree species with the greater ivis at bukit malalak, agam regency (sumatra) species plot 1: association with n. bongso plot 2: association with n. rhombicaulis plot 3: association with n. eustachya chi-squared (χ 2) ochiai index (oi) chi-squared (χ 2) ochiai index (oi) chi-squared (χ 2) ochiai index (oi) nepenthes dubia + 0.61 (high) absent absent absent absent nepenthes bongso na na + 0.74 (high) absent absent nepenthes rhombicaulis 0.25 (low) na na absent absent ternstroemia gymnanthera + 0.88 (very high) + 0.88 (very high) + 0.67 (high) syzygium zeylanicum absent absent 0.44 (low) + 0.94 (very high) myrsine avenis + 0.50 (high) + 0.88 (very high) + 0.88 (very high) rhodoleia championii 0.25 (low) + 0.77 (very high) + 0.82 (very high) leptospermum javanicum + 0.53 (high) absent absent absent absent syzygium sumatranum absent absent absent absent + 1.00 (very high) castanopsis costata absent absent + 0.80 (very high) + 0.56 (high) syzygium bankense absent absent (low) + 0.75 (very high) gaultheria heterophylla + 0.89 (very high) + 0.66 (high) + 0.75 (very high) pittosporum ferrugineum absent absent 0.00 (very low) + 0.82 (very high) diversity, ecology and conservation status of nepenthes in west sumatra province, indonesia – mansur et al. 227 growth rates leaf production rates and stem growth rates were quite variable within species. there were no significant differences in the rates among the four species measured over one year (only one individual of n. bongso could be measured, because the others died; table 5). the mean stem growth rate was significantly greater on bukit malalak (42.1 ± s.e. 4.8 cm yr-1) than on gunung talang (29.9 ± 3.6 cm yr-1) (t = 2.06, p = 0.05), although the leaf production rate was not (9.8 ± 1.2 leaves yr-1 versus 7.3 ± 0.6 leaves yr-1, respectively; mansur et al. in review); this could be due to the lower elevation of the plants studied on bukit malalak or simply the fact that different species were measured at each site (with the exception of n. bongso). it would be interesting to further examine the possible tradeoffs between leaf and stem production rate and plant survival (mansur & brearley 2008), as has been done in tropical tree seedling studies (brearly et al. 2016). nutrient concentrations macronutrient concentrations in the leaves were greater than those in the nepenthes pitcher fluid, and there were no significant differences in nitrogen or potassium concentrations between species, but phosphorus was greatest in the leaves of n. dubia and least in n. rhombicaulis. the concentrations of foliar magnesium were not significantly different between the three species; however, calcium was greatest in n. bongso and sodium was greatest in n. dubia and least in n. bongso. pitcher fluid concentrations were variable, and there were few significant differences among species, although n dubia had notably lower p, k and mg concentrations (table 6). our ecological studies on bukit malalak showed that the habitat of nepenthes was an infertile latosol (unpubl. data), indicated by the presence of a large number of syzygium species and tristaniopsis merguensis (griff.) peter g.wilson & j.t.waterh. this compares with more fertile volcanic andosols as found on other mountains in the region, such as gunung singgalang and gunung talang. however, these differences were not reflected in the foliar n, p or k concentrations when compared with material from gunung talang (mansur et al. in review), although n was greater and k was lower than the species from north sumatra (mansur et al. 2022b). in contrast, less mg was noted in the leaves from bukit malalak than those from gunung talang (but more than those from north sumatra) and less ca but more na was found in the nepenthes leaves from gunung talang and north sumatra. if the plants obtain sufficient nutrients from their insect prey, this may be one reason nutrient concentrations in the sampled leaves do not differ between locations despite contrasting soil fertilities. indeed, we earlier showed that foliar n concentrations of nepenthes did not vary along elevation gradients, although there was an impact on foliar p concentrations (mansur et al. 2022b). determinants of foliar nutrient concentrations will differ by species and locality. how they influence the performance of different nepenthes species would be valuable further research. table 5 growth rates of four nepenthes species in their natural habitat at bukit malalak, agam regency (sumatra) over an twelve-month period (oct 2021 to oct 2022). also shown are the environmental conditions and survival rates. values are mean ± standard error nepenthes plot conditions no. of individuals survival (%) no. of new leaves produced length of new stem produced (cm) bongso 2 slightly shaded 1 33 5.0 24.6 dubia 1 fairly open 3 100 9.7 ± 0.3 48.9 ± 7.8 eustachya 3 shaded 3 100 13.3 ± 2.9 52.2 ± 8.0 rhombicaulis 2 slightly shaded 4 100 8.5 ± 1.6 33.7 ± 7.6 biotropia vol. 30 no. 2, 2023 228 table 6. nutrient concentrations in leaves and pitcher fluid of three nepenthes in bukit malalak, agam regency (sumatra). values are mean ± standard error with letters indicating significant differences (at p < 0.05) with a tukey’s test element leaves (%) pitcher fluid (mg l-1) bongso dubia rhombicaulis bongso dubia rhombicaulis n 1.09 ± 0.02 a 1.01 ± 0.01 a 1.36 ± 0.22 a 0.97 ± 0.22 a 3.52 ± 1.44 a 1.09 ± 0.04 a p 0.10 ± <0.01 ab 0.14 ± 0.02 b 0.08 ± 0.01 a 5.04 ± 0.03 a 0.18 ± 0.05 a 4.51 ± 2.35 a k 1.32 ± 0.02 a 1.08 ± 0.18 a 1.10 ± 0.05 a 721 ± 363 a 6.72 ± 1.23 a 283 ± 163 a ca 0.16 ± 0.02 b 0.05 ± 0.01 a 0.09 ± 0.02 a 491 ± 27 b 294 ± 4.6 ab 172 ± 86 a mg 0.14 ± 0.01 a 0.12 ± 0.01 a 0.13 ± <0.01 a 92 ± 11 c 4.9 ± 0.2 a 50 ± 5.6 b na 0.42 ± 0.04 a 0.92 ± 0.14 b 0.70 ± 0.02 ab 0.85 ± 0.03 a 1.48 ± 0.07 a 516 ± 296 a conservation status of the 23 species reported from west sumatra, nine have threatened conservation status, namely n. dubia (cr), n. jacquelineae (cr), n. jamban (cr), n. tenuis (cr), n. adnata tamin & m.hotta ex schlauer (en), n. talangensis (en), n. tenuis nerz & wistuba (en), n. naga (vu) and n. rhombicaulis (vu). at nearly 40 % of the known species in the province, this is a worryingly high proportion. our research extends the ranges of n. jamban and n. lingulata from their type locality in north sumatra (lee et al. 2006) to west sumatra based on herbarium collections deposited at herbarium bogoriense (bo) by wewin tjiasmanto and that of n. naga through personal communication (putra). previously, n. dubia was only known to grow on gunung talakmau, pasaman and west pasaman regencies, at an elevation of 1800 to 2700 m asl (clarke 2001), and mandailing natal regency, north sumatra (sahal, pers. comm.). our research also extends the known range of n. dubia to include bukit malalak, thereby increasing the size of the small, originally documented population and its extent of occurrence (eoo). this is positive, as species with larger ranges and population sizes are less prone to extinction; this species is currently listed as critically endangered, satisfying the red list criteria b1+2e, i.e. it has a small geographic range and small population size (clarke et al. 2000a; cross et al. 2020). the eoo of n. rhombicaulis is also extended, having been formally documented only from gunung pangalubao near lake toba in north sumatra, about 300 km distant. this species is currently assessed as vulnerable under the red list criterion d2, i.e. it has a restricted area of occupancy (clarke et al. 2000b). cross et al. (2020) listed n. tenuis as cr under criteria a2, i.e. a greater than 80% reduction in population over the last 10 years. they further considered that it might be extinct; however, we found this species growing in the batu karang-harau area, albeit with a very small population of about 20 individuals. despite extending the range and population of these species, they are still threatened, as we discuss below. the other six species are listed as threatened because of their restricted ranges and an observed decline in their population in the cases of n. jacquelineae, n. jamban and n. talangensis (clarke et al. 2000c; cross et al. 2020). nepenthes harauensis has only recently been described (hernawati et al. 2022b) and has not yet been formally assessed for the iucn red list; nevertheless, the restricted range of this species coupled with the rapid rate of environmental change in sumatran ecosystems strongly suggests that it is likely to be threatened in its natural environment and would likely fall under the endangered category if formally assessed (although we would need a better estimate of of its populations’ size and trajectory). other species that might be found in west sumatra province include n. putaiguneung, which was recently described from an unknown location in the extensive kerinci seblat national park (metusala et al. 2020), as well as n. aristolochioides jebb & cheek, which is known diversity, ecology and conservation status of nepenthes in west sumatra province, indonesia – mansur et al. 229 from three locations in the park where it is under threat from collectors (mcpherson 2009), underlining the problems of plant conservation even in formally protected areas (linkie et al. 2010). with the exception of the two latter species, most of the other species described from sumatra (hernawati et al. 2022a) and not recorded from the north (mansur et al. 2022a) or west sumatra provinces (in this study) are found in the very north of the island in aceh or in the more central jambi province. although rates of land-use change are very high in indonesia (margono et al. 2012; austin et al. 2019), many rare and often endemic species remain in the sumatran mountains due to the protected status of some localities, combined with their poor accessibility. many of these species are not in ex-situ cultivation and a stochastic environmental event or agricultural invasion could rapidly cause the extinction of the entire global population of some of these locally endemic species. conservation needs and responsibilities are therefore extremely high. further exploration and formal records would add to the quality of existing assessments. beyond assessing their conservation status, the key threats to the majority of nepenthes in indonesia are land-use change and collection for the horticultural trade (clarke et al. 2018; cross et al. 2020), so practical conservation methods need to address these challenges that will only be successful with the support of local communities and governments. these fascinating restricted range and threatened nepenthes species in sumatra need further study, especially regarding their distribution and population status. a more detailed exploration of the mountainous regions of sumatra may well find further populations, as we have done here. figure 3 nepenthes species recorded from west sumatra: (a) n. adnata (lower pitcher), (b) n. albomarginata (lower pitcher), (c) n. ampullaria (rosette pitcher), (d) n. bongso (lower pitcher), (e) n. dubia (upper pitcher), (f) n. eustachya (lower pitcher), (g) n. gracilis (upper pitcher), (h) n. harauensis (lower pitcher), (i) n. inermis (upper pitcher), (j) n. izumiae (upper pitcher), (k) n. jacquelineae (upper pitcher), (l) n. jamban (upper pitcher), (m) n. lingulata (upper pitcher), (n) n. longifolia (upper pitcher), (o) n. mirabilis (upper pitcher), (p) n. naga (upper pitcher), (q) n. pectinata (rosette pitcher), (r) n. reinwardtiana (upper pitcher), (s) n. rhombicaulis (upper pitcher), (t) n. singalana (upper pitcher), (u) n. spathulata (upper pitcher), (v) n. talangensis (upper pitcher), (w) n. tenuis (upper pitcher). photos a & p by putra, photos b–i, n, o, q–s, v & w by m. mansur, photos j–m by sahal, photo t by sunardi, photo u by yusran e. ritonga. authorities are noted in table 1 biotropia vol. 30 no. 2, 2023 230 conclusion our research documented 23 species of nepenthes in west sumatra province through direct observation (18 species) and the remainder through herbarium specimens, literature review and second-hand information. of that total, 18 species are endemic to sumatra, nine of which are threatened with extinction (2 × vu, 2 × en and 5 × cr). we examined nepenthes at one of our study sites (bukit malalak) in more detail and compared ecological patterns and processes with those occurring elsewhere. nepenthes species require further observation, especially regarding their distribution and population, and additional exploration on poorly known sumatran mountains will lead to new discoveries in this fascinating plant genus. acknowledgments this research was funded by grants from beauval zoo (france) and chester zoo (uk). thanks to phillip esseen, paul leach, nicolas leroux, johanna rode-margono and stuart young for help and support of this research. special thanks to sunardi, heru hartantri, yusran e. ritonga, putra, rhima and daniel ferdinan for their assistance during the fieldwork. we thank the reviewers for their comments that improved the manuscript. references adam j. 2002. demographic study of nepenthes species (nepenthaceae) recorded along the trail to the summit of mount kinabalu in sabah, malaysia. pakistan journal of biological sciences 5(4): 419-426. doi: 10.3923/pjbs.2002.419.426 austin kg, schwantes a, gu y, kasibhatla ps. 2019. what causes deforestation in indonesia? environmental research letters 14(2): 024007. doi: 10.1088/1748-9326/aaf6db brearley fq, mansur m, eichhorn mp. 2023. spatial patterning of 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https://doi.org/10.2331/suisan.22.526 https://doi.org/10.1093/aob/mcp121 https://doi.org/10.1093/aob/mcp121 https://doi.org/10.1007/s11099-010-0028-1 https://doi.org/10.14203/reinwardtia.v20i1.3932 https://doi.org/10.14203/reinwardtia.v20i1.3932 https://doi.org/10.20956/jal.v8i16.2985 biotropia vol. 30 no. 2, 2023: 116 127 doi: 10.11598/btb.2023.30.2.1038 116 removal efficiency of nitrogen, phosphorus and heavy metals associated with swine wastewater using aquatic macrophytes nguyen tan chung* and nguyen van ha department of environmental biotechnology, faculty of biological sciences, nong lam university ho chi minh city, vietnam received 23 march 2018 /revised february 26, 2023 /accepted 3 march 2023 abstract wastes from breeding farms have globally increased greenhouse gases and caused a serious pollution to aquatic environments. biogas treatment polymer bags could significantly reduce organic compounds; however, they could not effectively treat other pollutants in animal wastewater. the objective of this study was to assess removal efficiency of salinity and pollutants associated with pig wastewater using aquatic macrophytes. four macrophytes namely acrotichum aureum, eleocharis dulcis, typha domengensis, and limnophyton obtusifolium and a soil control without vegetation were randomly assigned into fifteen mesocosms (1.2 x 0.7 x 0.6m) with 3 replicates for each treatment. pig wastewater was filled continuously into input chambers of mesocosms in every three day with 5 liters. water samples were collected from output chambers with 60 and 120 days after treatment while soil and vegetation samples were collected at the beginning and the end of the experiment. the results showed that e. dulcis, t. domengensis, and l. obtusifolium were dominant in removal of n, p, cu and zn and suspended solids as well; e. dulcis and t. domengensis significantly increased the dissolved oxygen; whereas the treatment of l. obtusifolium species showed the best efficiency in salt-ion removal. pollutants of n, p, cu and zn tend to accumulate more in the macrophyte roots than in their leaves. accumulation of n, p, cu and zn in the l. obtusifolium’s biomass is the highest compared with other treatments. from findings, it is suggested that a combination of three aquatic macrophytes including e. dulcis, t. domengensis, and l. obtusifolium could establish a constructed wetland system to directly treat pollutants of livestock wastewaters keywords: aquatic macrophytes, nitrogen, phosphorus, salinity, swine wastewater treatment introduction rapid growth of poultry and livestock sectors have caused seriously adverse effects on environmental quality and natural ecosystems and threatened human health (mawdsley et al. 1995, honda et al. 2000, nguyen 2010). wastes from poultry and livestock activities annually contribute to a large number of greenhouse gases which result in the global warming (fao 2014). throughout the vietnam, there are more than 10 million different households and farms with approximately 27 million pigs that consume annually about 18 million tons of industrial feed (nguyen 2009). the livestock sector discharges about 73 million tons of wastes per year, in which wastes of pig farms count for 33.4% (xuan 2009). this could not only reducing the quality of products but also increasing pollutants and heavy metals in wastes. major pollutants associating with the swine wastes include organic matters, nitrogen, phosphorus, copper, and zinc and other trace metals (cang et al. 2004, truong 2010, dang et al. 2014). according to the surveyed results about status of waste treatment of pig farms from eight different regions throughout vietnam, nguyen (2010) showed that 74% of pig farms have applied biogas plants to treat pig wastes while about 26% of the households and farms have been directly discharged the wastes into the environment. although treatment polymer bags have effectively been removed organic carbon but they could not reduce pollution of nitrogen (n), phosphorus (p) and other trace metals. nguyen & pham (2012) who evaluated the treatment effectiveness of pig wastewater removal by biogas treatment polymer bags at a *corresponding author, email: ntchung@hcmuaf.edu.vn removal efficiency of nitrogen, phosphorus and heavy metals associated with swine wastewater – chung and văn hà 117 level of households showed that biogas plants could reduce up to 85% cod (chemical oxygen demand) and 76% bod5 (biological oxygen demand in five days) but only 12% and 7% for total nitrogen (tn) and total phosphorus (tp). other study which assessed the wastewater treatment effectiveness by biogas plants at 12 pig farms in the provinces of red river delta in the northern part of vietnam, vu et al. (2008) pointed out that biogas reduced 6570% cod, 75-81% bod5, and 10-28% tn. meanwhile, biogas treatment polymer bags did not impact on reducing heavy metals in pig wastewater. in recent decades, the use of constructed wetlands to treat different polluted sources has achieved a very high efficiency (vymazal 2011, shelef et al. 2013). four major factors determining a successful application of constructed wetlands include type of the aquatic plants, retention time of wastewater staying on the treatment system, the depth of the mud, and the depth of water column (usepa 2000). cattail and eleocharis species are two species of aquatic plants that have a wide distribution throughout the world and have a high performance in pollutant treatment (marois et al. 2015, mitsch et al. 2015). particularly, eleocharis species is recently used to remove n and p because of its high treatment efficiency in a short time (pham 2013). suhendrayatna et al. (2012) used cattails (typha sp) and reed species (phragmites communis) to treat municipal wastewater and showed that cattailspecies were more resilience to the wastewater and removed more bod, cod and tss (total suspended solid) higher reed species. in the other research, lam & ngo (2013) pointed out that cattail species was capable of absorbing 0,17g n and 0,09g p/m2/day in wastewater treatment of intensive catfish ponds with closed recirculation, whereas absortion ability of heavy metals was 456 1549 mg/kg (fe) and 1.2 to 7.6 mg/kg (pb) (anning et al. 2013). the main goal of this research was to identify some species of aquatic plants that peform a high efficiency in handling salinity and majority pollutants in pig wastewater. to achieve the research objectives, a series of examinations were conducted : (i) assessing capability to remove bod, cod, n, p, cu, zn and salinity in pig wastewater between treatments, (ii) determining the ability to absorb and accumulate n, p, cu and zn in soil and biomass of plant species. research results will provide a scientific basis for a suitable choice of aquatic plant species in handling organic pollutants, salinity and heavy metals for wastewater in general, particularly pig wastewater. materials and methods experimental design four plant species consisting of typha domengensis, eleocharis dulcis, acrostichum aureum, limnophyton obtusifolium and one control without plant were assigned a completely random into 15 composite barrels with 1.2m (length) x 0.6m (width) x 0.7m (depth) with 3 replications for each treatment (figure 1). a layer of soil was placed at the bottom of each barrel with a depth of 30cm. plants were grown with an initial density of 6 plants/experimental barrels, exception to the control. water level in each treatment was gradually raised to plant height after plantation, and water column-maintained depth of 30 cm from the mud surface by an effluent system with pvc ф-34 pipes. the composite barrels were buried into the soil with a depth of 30 cm equal to the soil depth inside each barrel. raw wastewater collected from pig farms was contained in a wastewater tank and filled into the barrels with frequency of every 3 days and 5 liters per time at an inlet zone of each treatment through the influent wastewater system as described in figure 1. based on the volume of water column, the surface area of barrel and the amount of wastewater entered (adelaja et al. 2014; dong et al. 2022), a retention time of wastewater stayed in each treatment system was estimated approximately 14 days. determination of soil and water temperature in order to examine if temperature affects efficacy of pollutant removal, hoboware device was used to measure temperature in water column and soil of treatments during experimental time. fifteen hoboware gauges (figure 2a) were deployed into the middle of soil layer and water column within each treatment and established to automatically recorded temperature in every 30 minutes. the gauges were mounted on pvc ф-27 pipes and fasten by biotropia vol. 30 no. 2, 2023 118 figure 1 a) experimental design with a treatment system; b) plant growth after 3-months handling plastic tubes. every two gauges were arranged on the same pvc pipe and aligned in the middle of water column and soil layer of each treatment. measurement of salinity, do, and ph to measure salinity among experimental treatments, electrical conductivity (ec) was measured, and then be converted ec to salt concentration (rhoades et al. 1999, nrw service centre 2007). dissolved oxygen (do), ec, and ph in effluent were measured at the outlet zone of each effluent system in every 2 weeks from 9:00 am to 10:00 am using ysi650 mds machine (figure 2b). ysi’s readers were deployed into water column with a depth of 10cm at the outlet zone to measure do, ec and ph. the readers were set up stabilizing in 60 seconds before recording and displaying results. to ensure qa/qc, the machine was calibrated in one hour before conducting the measurement and checked right after measurement by following the user manual’s standard operating procedures (ysi incorporated 2009). figure 2 a) hoboware; b) ysi650 mds machine effluent system wastewater tank influent system e. dulcis t. domengensis l. obtusifolium a. aureum a b a b removal efficiency of nitrogen, phosphorus and heavy metals associated with swine wastewater – chung and văn hà 119 collection and analysis of effluent samples to evaluate effectiveness of pollutant removal among experimental treatments, samples of effluent water were collected after two and four months. collection of effluent samples was conducted at the outlet zone of effluent system in the morning by using a mini pump and collected in one day before putting the wastewater. effluent water was directly pumped into a 2-liter plastic bottle and kept inside an ice cooler before sending to nlu’s research institute of biotechnology and environment on the same collection day for analyzing bod5, cod, n, p, zn, and cu. methods applied to analyze for these components are presented in table 1. table 1 methods used to analyze samples sample type criteria analytical methods water bod5 tcvn 6001:2008 cod sweww 5220c:2012 tn tcvn 6638:2008 tp tcvn 6020:2008 cu2+, zn2+ aciar-aas 007-2007 soil tn tcvn 6498 – 1999 tp tcvn 8940 – 2011 cu, zn aciar-aas 019-2007 plant tn 10tcn 451 – 2001 tp 10tcn 453 – 2001 cu, zn aciar-aas 0192007 accumulation of pollutants in soil and plant soil and vegetation samples were collected in one day before wastewater was initially filled into barrels corresponded with one month after planting, and at the end of experiment to determine contents of n, p, cu and zn in soil and plant biomass. soil samples were taken at a depth of 5cm from soil surface by a soil core. in each treatment barrel, soil was randomly collected at three different positions, mixed them together, then placed it into a ziplock bag and kept it in an ice cooler before sending to laboratory for analysis. similarly to soil sampling, plant sample was collected from three plants in three different locations of each barrel. the sample was distinctly divided into leaves and roots. these samples were cut into 2-cm pieces, arranged neatly in paper bags, and dried in oven at 65oc for 7 days before sending for analysis. analytical methods of n, p, cu, and zn applied for plant and soil are presented in table 1. an increasing accumulative rate of pollutants in biomass computed by the following equation: h (%) = 100 x (cb-final– cb-initial)/cb-final where: cb-initial = concentration of pollutants in plant biomass (mg/kg) collected in one day before putting wastewater cb-final = concentration of pollutants in plant biomass (mg/kg) collected in one day before putting wastewater at the end of the experiment. n:p ratio = tn/tp, where concentrations of n and p accumulated in the plant biomass (mg/kg). statistics and anova analysis method this one-factor experiment was completely randomized factor with three replications for each treatment. software of minitab 17 was used for anova analysis to evaluate significantly differences among treatments, and for tukey’s multiple comparision method (α = 0.05). charts and figures were created by using the software of sigma plot 12. results and discussion concentrations of influent pollutants table 2 shows that concentrations of untreated pollutants in raw pig wastewater were very high. bod5 and cod were 2687.0 and 31516.0 mg o2/l, whereas concentrations of total nitrogen (tn), total phosphorus (tp), zn2+ and cu2+ were 2207.0; 1335.0; 23.4; 15.4 mg/l respectively. especially the pig wastewater contained a high electrical conductivity (ec) of 8.3 ms/cm, but a very low do content of 1.8 mg o2/l. concentrations of these pollutants were much higher than those of previous studies (chang & liu 2002, chung et al. 2004, nguyen & pham 2012). the reason for that was because wastewater samples were taken from tunnels of pigsty which directly received waste from pig manures and pigsty washing, and have not been decomposed with a condensed concentration. based on concentration of the untreated pollutants in the raw wastewater, loading rate established for each treatment, and retention time of wastewater in each treatment, initial influent concentrations of cod, bod5, tn, tp, zn and cu were 806.1; 9455.0; 662.2; 400.6; 7.0; and 4.6 mg/l respectively. biotropia vol. 30 no. 2, 2023 120 table 2 concentration of influent pollutants parameter unit raw wastewater initial average influent* ph 7.2 ec ms/cm 8.3 do mg/l 1.8 bod5 mg/l 2687.0 806.1 cod mg/l 31516.0 9455.0 tn mg/l 2207.0 662.2 tp mg/l 1335.0 400.6 zn2+ mg/l 23.4 7.0 cu2+ mg/l 15.4 4.6 * concentration estimated by basing on the raw wastewater concentration and retention time of the wastewater temperature variation in soil and water column temperature is an important ecological factor and directly affects respiration of microorganisms in a treatment system. variation of daily average temperature in soil and water among treatments were showed in figure 3. in general, the temperature in soil and water fluctuated from 24 – 31oc. in the first stage of development, there was a temperature difference. there was also a temperature difference among treatments in soil due to young plants, but no difference of those in water. in the final growth period, there was no different temperatures among the treatments in both soil and water. temperatures of t. domengenis and l. obtusifolium showed lower than those of other treatments. variations of temperatures among treatments could impact on removal efficiency of pollutants (ab-halim et al. 2016; alisawi, 2020; shatat and al-najar, 2011). figure 3 temperature fluctuation of treatments following the treated time: a) soil; b) water a b 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 removal efficiency of nitrogen, phosphorus and heavy metals associated with swine wastewater – chung and văn hà 121 do, ec and removal efficacy of salt dissolved oxygen (do) in water is dependent on photosynthesis processes of aquatic plants and a microbial respiration in water column and soil. in the respiration, microorganisms consume a large amount of do to decompose organic compounds. an amount of do used for the microbial respiration are often supplemented by photosynthesis of aquatic vegetation. the low level of dissolved oxygen in water is not only a sign of contamination and is but also an important factor in determining water quality and pollution (bozorg-haddad, 2021; kulkarni, 2016). in freshwater ecosystems, most of aquatic animals require amount of dissolved oxygen higher than or equal to 3 mg o2/l for survival. for example, fish cannot survive for long in water with dissolved oxygen less than 5 mg/l (bozorg-haddad, 2021). table 2 shows that the pig wastewater contained a large amount of organic compounds with a low do of 1.8 mgo2/l. althought do concentrations of each treatment fluctuated in over the experimental periods, two treatments of t. domengenis and e. dulcis generally improved the do (figure 4). do concentration of these two treatments was generally ranged from 5 to 9 mg o2/l. most aquatic plants and animals require oxygen to survive; fish, for instance, cannot survive for long in water with dissolved oxygen less than 5 mg/l. the low level of dissolved oxygen in water is a sign of contamination and is an important factor in determining water quality, pollution control and treatment process. the do in a saturated solution varies with the water temperature and elevation. figure 4 changes of do, ec and salt ion contents following the treated time among treatments biotropia vol. 30 no. 2, 2023 122 electrical conductivity (ec) shows concentration of ions which exist in water is usually derived from the soluble salt ions such as na+, k+, cl-, so4 2-. a high content of soluble ions would negatively influence and inhibit a growth of aquatic organisms. salinity in fresh water normally ranges from 0.01 to 0.50‰. the pig wastewater contained a large amount of soluble salt ions with ec of 8.3 ms/cm (table 2) which corresponded to salinity content of 4.6‰, ten times higher than the normal range of salinity in natural freshwater ecosystems. due to no vegetation, ec in control treatment accumulatively increased following the treated time resulted in a gradual accumulation of the salt content during the experiment (figure 4). species of t. domengenis, l. obtusifolium, and e. dulcis performed a high absorption of salt ions which resulted in a very low ec and salinity. salinity of these treatments was always lower than 0.50‰ over 14 weeks of the treated experiment (figure 4). exception for e. dulcis, since this species grew very quickly and had its shorter growth cycle than those of others, about 10 weeks, when this species died and returned its turnovers into water, and decomposed turnovers quickly (marois et al. 2015, mitsch et al. 2015). this resulted in an increasing of ec and salinity for this treatment in the last stage of the experiment. removal efficacy of bod5, cod, n, p, zn and cu as showed in table 2, the pig wastewater contained a high concentration of organic matters with cod of 31,516 mg o2/l and biochemical oxygen demand (bod5) of 2,687 mg o2/l. based on the input load, the retention time and the total volume of each treatment system, initial influent concentrations of cod and bod5 in all treatments were 9455.0 and 806.1 mg o2/l respectively (table 2). sixty days after treated, three treatments of l. obtusifolium, e. dulcis, and t. domengensis removed a large amount of organic matters, and as a result, the cod contents of these treatments reduced to 55.5, 36.0, and 176.0 mg o2/l respectively (figure 5). but in 120 days after treated, the cod content of l. obtusifolium treatment continued dropping to 46 mg o2/l, whereas the cod of e. dulcis treatment went up to 560 mg o2/ l, which was explained by due to the 10-month growth cycle of e. dulcis species. similar to effectiveness of cod handling, the treatments of l. obtusifolium, and t. domengensis showed the best efficacy of bod5. pig wastewater was polluted with high n and p, where the concentrations of tn and tp are respectively 2207.0 and 1335.0 mg/l, while their influent concentrations at each treatment were 662.0 mg/l (tn) and 400.6 mg/l (tp) (table 2). decomposition of organic pollutants by microorganisms associated with treatments of l. obtusifolium, e. dulcis, and t. domengensis reduced the bod and cod. this resulted in decomposition and transformation of organic n and p compounds into inorganic compounds for plant uptakes. these three treatments showed the best performance of n and p removal after 60 days treated. the tn contents of l. obtusifolium, e. dulcis, and t. domengensis were respectively 2.4, 10, and 3.3 mg/l. but after treated in 120 days, tn contents of all treatments increased more than doubled in comparison with those at the treated time of 60 days. similarly to total n, the content of total p (tp) was also removed significantly after 60 days and increased after 120 days, except for two treatments of l. obtusifolium and t. domengensis (figure 5). cu and zn ussualy supplements to food of livestocks, especially in pig foods, but pollution of cu and zn in pig wastewater is paid less attention. concentrations of these two heavy metals in the pig wastewater are so high, approximately 23.4 and 15.4 mg/l for cu and zn. based on input load and retention time on the treatment system, the initial average concentration of cu and zn were 4.6 and 7.0 mg/l (figure 5). removal capabilities of cu and zn are very different among treatments. the treatment of l. obtusifolium removed completely cu and zn due to plant uptake. as a consequence, concentrations of cu and zn in this treatment was nearly equal to zero (non detection) after 60 days treated. in addition to the l. obtusifolium treatment, the a. aureum also showed a higher efficiency of cu and zn removal than others. removal efficiency of nitrogen, phosphorus and heavy metals associated with swine wastewater – chung and văn hà 123 figure 5 changes of bod5, cod, n, p, zn and cu following the treated time among treatments accumulation of n, p, zn, cu in soil according to results of anova analysis shown in table 3, the initial n and p contents in soil were not different from the treatments (p = 0.186 for tn, p = 0.669 for tp). concentration of initial soil tn ranged from 284 to 323 mg n/kg dried soil, whereas the initial tp concentration in soil ranged from 62 to 67 mg p/kg dried soil. baseline concentrations of tn and tp used for the experiment were very low. however, both n and p concentrations in the soil were increased for all treatments. due to without vegetation, the control treatment accumulated the lower than those of three other treatments because of its ability of low n transformation and decomposition in water, while the treatment of l. obtusifolium had a the highest accumulation of tn and tp contents in soil with 504 mg n/kg and 210 mg p/kg, respectively. this result were corresponded with the lowest tn and tp concentrations of l. obtusifolium treatment in water. initially baseline cu and zn concentrations in the soil utilized for the experiment were very low and did not differ among treatments with the cu concentration under non-detection zero and zn concentration ranged from 3.1 to 3.9 mg/kg (p = 0.822) (table 3). at the end of the experiment, although accumulation of cu content in soil increased in all treatments, approximately 6 mg cu/kg, there was no significant difference (p = 0.157). in contrast to cu accumulation in soil, zn concentrations were significantly different from the treatments (p < 0.000). the treatment of l. obtusifolium showed the highest zn accumulation with 11.4 mg zn/kg. this is resulted from the high zn removal efficiency of l. obtusifolium treatment in water which assisted by organic matter breakdown associated with microorganisms in this treatment. treated time (day) biotropia vol. 30 no. 2, 2023 124 table 3 accumulation of n, p, cu and zn in soil/sediment parameter treated time (day) concentration of treatments (mg/kg dry soil weight) (mean ± se) t. domengensis e. dulcis a. aureum l. obtusifolium control tn 0 313 ± 22 301 ± 5 323 ± 3 284 ± 31 304 ± 15 120 467 ± 21 409 ± 13 386 ± 48 504 ± 9 636 ± 16 tp 0 65 ± 8 63 ± 2 67 ± 1 62 ± 1 63 ± 5 120 164 ± 8 123 ± 24 140 ± 2 210 ± 19 267 ± 8 cu 0 nd nd nd nd nd 120 6.0 ± 0.5 6.0 ± 1.0 6.2 ± 0.7 6.1 ± 0.8 7.4 ± 0.5 zn 0 3.8 ± 1.9 3.9 ± 0.9 3.3 ± 0.2 3.1 ± 0.3 3.4 ± 0.5 120 5.2 ± 2.2 4.1 ± 0.8 4.3 ± 1.0 11.4 ± 2.6 14.6 ± 1.4 nd: none detection contents of n, p, cu and zn in plant biomass since the initial n baseline content in foliages and roots are significantly different among treatments (table 4). the treatment of l. obtusifolium contained the highest n concentrations in root (0.97% ± 0.15) and foliage (1.90% ± 0.10). this difference could be explained that because in first month after planting (not input the wastewater to treatments), the l. obtusifolium species grew faster than others treatments resulting in higher accumulation of n content in its root and foliage. this corresponded with the lowest initial n content of l. obtusifolium treatment in the soil (table 3). after treated in 120 days, root and foliage n concentrations of all treatments significantly increased in comparison with their initial baseline concentrations (table 4). the increasing percentage of n absorption rate by vegetation treatments within root and foliage were presented in figure 6. all treatments showed a high n absorption rate in roots with greater than 50%, but there was no a significant difference of the rate among the treatments. the highest n absorption rate in roots reached 67% for the a. aureum treatment while the lowest n absorption rate was 50% for the l. obtusifolium treatment. on the other hand, the l. obtusifolium treatment showed the highest foliage n absorption with the 28.9%, followed by the treatment of e. dulcis (19.6%), from which the rates of two these treatments were significantly different from two other remaining treatments. if combined with the root and foliage absorbed n contents, the treatment of l. obtusifolium performed the highest capacity to accumulate nitrogen in its biomass. generally, under a normal condition, all treatments accumulated lower n in roots than in foliages, but with the addition of n increasing amount from pig wastewater, n tended to be accumulated more in roots than in foliage in order to create a balance of n content in their biomass. table 4 concentrations of n, p, cu and zn in plant biomass treatment bio-mass type one day before treated 120 days after treated tn (%) tp (%) zn (mg/kg) cu (mg/kg) tn (%) tp (%) zn (mg/kg) cu (mg/kg) t. domengensis root 0.75 ± 0.05 0.182 ± 0.005 77.1 ± 20.4 19.9 ± 4.7 2.27 ± 0.30 0.907 ± 0.113 154 ± 15.0 19.5 ± 10.5 leaf 1.65 ± 0.09 0.240 ± 0.025 nd nd 1.71 ± 0.02 0.263 ± 0.015 nd nd e. dulcis root 0.55 ± 0.05 0.254 ± 0.048 19.5 ± 5.0 13.7 ± 7.9 1.54 ± 0.24 0.607 ± 0.014 93.5 ± 13.5 26.0 ± 1.0 leaf 1.25 ± 0.15 0.314 ± 0.003 nd nd 1.59 ± 0.41 0.477 ± 0.021 nd nd a. aureum root 0.95 ± 0.05 0.213 ± 0.061 32.1 ± 13.5 26.8 ± 11.4 2.09 ± 0.17 0.689 ± 0.031 45.0 ± 17.0 34.0 ± 16.6 leaf 1.85 ± 0.06 0.105 ± 0.005 nd nd 1.97 ± 0.18 0.272 ± 0.037 nd nd l. obtusifolium root 0.97 ± 0.15 0.329 ± 0.004 73.2 ± 1.2 5.3 ± 0.4 2.00 ± 0.26 1.078 ± 0.017 154 ± 43.8 80.0 ± 6.0 leaf 1.90 ± 0.10 0.339 ± 0.094 nd nd 2.68 ± 0.17 0.486 ± 0.017 nd nd nd: none detection removal efficiency of nitrogen, phosphorus and heavy metals associated with swine wastewater – chung and văn hà 125 similar to n status accumulated in biomass, the initial root and foliage p concentrations were very different among treatments. the treatment of l. obtusifolium always performed the best ability to uptake p into its biomass, from which the initial p baseline concentration were 0.329% ± 0.004 in root and 0.339% ± 0.094 in foliage and that the p concentration accumulated in its roots and foliage after 120 days were respectively 1.078% ± 0.017 and 0.486% ± 0.017 (table 4). in contrast to n absorption results, phosphorus mainly accumulated in roots of treatment’s plants after the 4-month handling period. if it takes into account of absorbed p rate in biomass, the treatment of t. domengensis showed the best performance of p absorption rate in its biomass, about 68% in root and 62% in foliage (figure 6). the n/p ratio in plant biomass is an ecological indicator, which is used to assess a level of excess or deficiency of n or p content provided for growth of trees in different ecosystems. n/p ratio of 14-15 reflects a balance between n and p contents in an ecosystem. the n/p ratios in biomass of all treatments in this study were rather low, approximately 3 in root and 8 in foliage for treatments (figure 6). these results indicated that the pig wastewater was more n polluted than p in a term of n/p ratio for plant uptake. according to the analyzed results presented in table 4, both cu and zn were only accumulated in roots of treatments. cu and zn concentrations in foliage were very low and even under detectable in some treatments. at the same rate of initial zn and cu influent loading, two treatments of t. domengensis and l. obtusifolium accumulated the highest zn content in their roots, 154.0 ± 15.0 and 154.3 mg/kg ± 43.8 respectively (table 4), where only the treatment of l. obtusifolium showed the highest cu absorption in its roots with cu concentration of 80.0 mg/kg ± 6. figure 6 n and p absorption rate and n:p ratio in plant biomass among treatments root foliage biotropia vol. 30 no. 2, 2023 126 conclusion three species of t. domengenis, l. obtusifolium, and e. dulcis performed a high removal efficacy of pollutants accociated with the pig wastewater including salinity, cod, bod, n, p, cu and zn. with its ecological 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6-series multiparameter water quality sondes. biotropia vol. 28 no. 3,2021: 181 192 doi: 10.1 1598/btb.2021.28.3.1047 overexpression of ga! gene increases growth and hyposaline tolerance in kappaphycus aalvarezii transgenic plantlets erina sulistiani1*, suharson02, ence darmo jaya supena2 and miftahudin2 'seanizeo biotrop, jalan rdya tdurkm 6, bogor 16134, indonesia 'department of biolo~, faculiy ofniathematics and natural sciences, institzit pertanian bogor, bogor 16680, indonesia. received 4 april 2018/accepted 27 june 2020 abstract g proteins are membrane proteins that play roles in signal transduction in living organisms. they consist of a, p and y subunits. the g protein a subunit (ga) plays a role in plant resistance toward biotic and abiotic environmental stresses. transgenic plantlets of kappaph_yczis aluare+i carrying the ga gene (derived from soybean) have been successfully obtained through agrobacterium tnmefaaciens-mediated transformation. the present study aimed to: 1. compare the growth of non-transgenic and transgenic plantlets of k. alvareei in vitro using provasoli enriched seawater (pes) medium with normal salinity and hyposalinity and 2. analyze the expression level of the ga gene in transgenic plantlets using quantitative polymerase chain reaction (qpcr). the results showed that all transgenic plantlets (six clones) had significantly higher daily growth rate (dgr, %/d) than that of non transgenic under the condition of normal salinity (30 ppt) and hyposalinity (15 and 20 ppt) for 5 weeks of observation. at 15 ppt, transgenic plantlets were more tolerant than non-transgenic ones, as most thalli of transgenic plantlets remained brown in color, whereas most thalli of non-transgenic plantlets were bleached. the results of the qpcr analysis showed that the expression of the ga gene in transgenic plantlets increased by 6.43 8.03 times compared with that of non-transgenic plantlets. the result of pearson correlation analysis showed that relative expression of ga gene had a strong correlation, both with dgrs in normal salinity and hyposalinity of transgenic plantlets (correlation coefficient > 0.7). the correlation was linearly positive, where increased expression of the ga gene was strongly associated with an increase in dgrs. keywords: ga gene overexpression, hyposaline tolerance, k@aph_ycus alvareei, transgenic plantlet introduction kappapbctls alvareei doty is a commercially important species of red algae (rhodophyta) that is known in the trade as 'cottonii seaweed'. this species is the main source of kappa (x) carrageenan, which is widely used in the pharmaceutical, food and cosmetics industries. k alvare?? is widely cultivated in coastal waters of indonesia and philippines. however, in the cultivation of k. alvareei in coastal waters, major problems may occur such as salinity change and other frequent pronounced fluctuations in environmental factors in coastal waters. k al'vare?? grows at a salinity of 28 34 ppt (part *corresponding author, email: esulistiani@biotrop.org per thousand) (parenreng et al. 201 1). however, in rainy season with heavy rainfall, the salinity of seawater can decrease up to below 28 ppt. the decrease of salinity subjects the seaweed to hyposaline stress. the stress can cause the occurrence ice-ice dsease in seaweed, which symptoms include slow growth, bleaching and decay of some or all of the thalli (parenrengi e t al. 2011). to overcome the impacts of ice-ice disease in seaweed, a genetic transformation needs to be done to obtain k ahare?? that is tolerant to hyposaline stress. the heterotrimeric g proteins are membrane proteins consisted of the a, p and y subunits (ga, gp and gy) (urano e t al. 201 3). they play a role in signal transduction in a living organism as mediators that send extracellular signals from biotropia vol. 28 no. 3,2023 receptor molecules in the cell membrane to materials and methods effector molecules. in plants, gcc proteins play a role on cell growth, cell proliferation, defense against hsease, stomata1 movement, channel regulation, sugar sensing and some hormonal responses (urano et al. 2013). moreover, according to chakraborty et al. (2015), ga proteins play a role in plant resistance to biotic and abiotic environmental stresses. seaweeds respond to changes in salinity by adjusting the balance of osmotic potential between internal and external cells via pumping of inorganic ions such as i<+, na+ and c1into or out of the cells (karsten 2012). the influx of external ca2+ into the cytoplasm facilitates the maintenance of i<+ and na+ homeostasis during salinity stress because ca2+ plays a role in activating the na+/hf salt-overly-sensitive1 (sos1) antiporter and the cation/ht nhx antiporter (hasegawa 201 3). in hyposaline condition, ca2+ also plays a role in reducing the permeability of the plasma membrane to other ions, thereby reducing ion loss that occurs during water influx (hurd et al. 2014). in addition, ca2+ plays a role in the regulation of aquaporin, which is a water channel protein (gilliham et al. 201 1) recently found in algal cell plasma membranes as well (anderberg et al. 201 1). gcc protein signaling plays a role in ca2+ influx to the cytoplasm, owing to the abhty of ga protein in regulating ca2+ channels in plasma membranes (gelli et al. 1997; aharon et al. 1998; thuleau et al. 1 998; zhang et a/. 201 1). based on the above description, overexpression of the ga gene in k alvareei is expected to increase ca2+ influx to the cytoplasm and increase the tolerance of ths seaweed to hyposaline stress (< 28 ppt). transgenic plantlets of k. alvareei carrying the ga gene (derived from soybean) have been successfully obtained through agrobacte~am tamefdciens-mediated transformation (sulistiani et al. 2019). the study aimed to: 1. conduct growth performance test of non-transgenic and transgenic plantlets of k. alvare~i in an in vitro culture medium with normal salinity and hyposalinity and 2. analyze the expression level of the ga gene in non-transgenic and transgenic plantlets using qpcr method. growth performance test of non transgenic plantlets in hyposaline condition non-transgenic plantlets were regenerated from callus which induced from apical thallus of k alvare@i (collected from takalar, south sulawesi, indonesia). the callus was grown into plantlets by culturing the plantlets in provasoli enriched seawater (pes) medium (provasoli 1968) for 6 months (sulistiani & yani 2014). the experiment was conducted to observe the growth of non-transgenic plantlets in pes medium with normal salinity and hyposalinity. the plantlets (about 3 g) were grown in an aerated culture for 12 weeks with five treatments, i.e., pes with 5 different salinity levels: 15, 20, 25, 30, and 35 ppt (1,000 ml/bottle). each treatment was conducted in three replicates. the cultures were continuously aerated with an air blower and placed in a culture room at temperature of 22 24 "c, lighting of 1,500 lux and a 12 : 12-h light and dark cycle. the medium was replenished weekly. the natural seawater used in this study had a salinity level of 32 ppt. the lower-than-32-ppt salinity level was prepared by adding distilled water to the natural seawater and stirring the solution using a magnetic stirrer until the desired s a h t y level was reached. in contrast, to obtain salinity level of more than 32 ppt, nacl was added to the natural seawater and stirred unul reaching the desired salhty level. salinity was measured using a refractometer. the plantlets were weighed weekly, and the daily growth rates (dgrs, %/d) were calculated using the formula of dawes et al. (1 994): ln (final weight) ln (initial weight) dgr = .o. o f d q x 100 seawater with salinity level causing significant stress and decreased dgrs in the non transgenic plantlets was used in the growth performance tests of transgenic plantlets in a hyposaline medium to select transgenic plantlets with tolerance to hyposalinity. overexpression of gcl gene in kappapbycu~ aluareei transgenic plantlets sulistiani et al. tolerance test of transgenic plantlets in hyposaline condition an experiment was conducted to determine the tolerance of transgenic plantlets in pes medium with hyposaline condition, which exerted stress on the non-transgenic plantlets. six clones of transgenic plantlets (ll, e2, e5, e6, e7 and e8) were selected from a previous study. pcr analysis was used to confirm that the transgenic plantlets carried the ga gene with a constitutive promoter camv 35s (sulistiani et al. 2019). transgenic plantlets were regenerated from genetically transformed callus using the same method as that used for the non transgenic plantlets. the transgenic plantlets (about 0.3 g) were grown in 500 ml of pes mehum with three hfferent salinity: 30 ppt (normal salinity), 20 ppt and 15 ppt (hyposalinity). for comparison (control), non-transgenic plantlets were also grown in the same salinity treatments. the experiment was conducted for 5 weeks, with each salinity treatment being performed in three replicates. the plantlets were weighed weekly and the dgrs were calculated. one sample of each of the non-transgenic and transgenic plantlets (each about 4 g) was taken for calcium content analysis. the method of analysis was based on sni 2354.5: 2011 (bsnnational standardization agency of indonesia 201 i), and the calcium content was measured using an atomic absorption spectrophotometer (aas). quantification of gly gene expression in transgenic plantlets using qpcr total rna was extracted from two clones of putative transgenic plantlets and one clone of non-transgenic plantlets of k. alvare?? (6 months old in an aerated culture) using trizolb reagent (invitrogen), and cdna was synthesised using the iscripttm cdna synthesis h t (bio-rad). extractions of total rna and synthesis of cdna were performed with three biological replicates for each clone. quantification of ga gene expression in transgenic and non transgenic of k ahare?? was performed using quantitative polymerase chain reaction (qpcr) applied biosystems steponeplusm real-time pcr system. the ga cdna was amplified using the specific primer ga-f 5'cac gag gtt gct ttc tag ttt c -3 'and ga-r 5' atg agc ttc acg aaa acg ac -3'. the actin gene of k. ahare@' was used as an internal control of gene expression with the following specific primers: icact-f: 5'-ccg tcc cga tit acg agg gtta-3' and k-act-r: 5' gca tga gga gct tcg cca tcc-3' (rajamuddin 2016). the ga cdna and actin cdna were amplified in two replicates for each biological replicate. qpcr for ga cdna and actin cdna was performed in a 10 pl reaction volume containing 200 ng cdna, 2.5 pmol forward primer, 2.5 pmol reverse primer, 5 pl of s y b r ~ ~ select master m x and nuclease-free water. the cdna ga gene was amplified as follows: pre-denaturation at 95 "c for 3 min, followed by 40 cycles of denaturation at 94 "c for 10 s, and annealing at 57 "c for 40 s. the amplification of actin cdna was performed using the same protocol as that followed for ga cdna, except that annealing was performed at 55 "c. quantification of the expression level was based on the expression of ga cdna relative to that of the reference gene (actin gene of k. ahare@) using the 2-mct method. the calculation was performed by comparing the value of ct (cycle threshold) from the expression of the two genes. relative gene expression was represented by 2"act, where m c t = act transgenic act non-transgenic and act = ct target gene ct reference gene (livak & schmittgen 2001). statistical analysis statistical analyses of dgr data were determined using analysis of variance (anova) and duncan's multiple range test. the statistical analyses were performed in ibm spss statistics 19.0 for windows. results and discussion effect of salinity on growth and development of non-transgenic plantlets the results of statistical analysis showed that the salinity of the culture mehum significantly affected the dgrs of k. ahare?? plantlets. the highest dgrs upon cultured for 12 weeks in pes mehum were obtained at s a h t y of 25 30 ppt (1.9 2.4o/o/d; fig. 1). based on the biotropia vol. 28 no. 3,2021 literature, the seaweed k. alvareei grows well in coastal waters with a salinity of 28 34 ppt (parenrengi e t al. 201 1). the results of this study showed that k. ahare?? plantlets cultured in pes medium with a salinity of 25 ppt still grew optimally, showing no significant difference from the dgrs of plantlets at a salinity of 30 ppt. at a salinity of 20 ppt, the plantlets still survived, but their dgrs were lower than dgrs of plantlets at a salinity of 30 ppt. this suggests that k. ahare?? plantlets that were regenerated from callus through somatic embryogenesis were tolerant to hyposalinity in vitro up to 20 ppt salinity. in hyposaline condtion, the na' concentration outside seaweed cells is lower than that inside the cells. therefore, water flows enter the cells following the osmotic gradient, whch in turn increases the volume of cells and their turgor. under these conditions, the cells perform an osmotic adjustment mechanism by pumping inorganic ions such as k+, na+ and c1 in and out from the apoplast to cytoplasm until the conditions return to the normal turgor pressure and homeostasis of i<+/na+ is achieved (hurd e t al. 2014). the decrease of dgrs upon exposure to hyposaline stress is due to the seaweed cells allocating most of their metabolic energy to the osmotic adjustment process, which in turn reduces the amount of metabolic energy used for growth (karsten 2012). the growth decline is also due to the reduction of oxygen species (ros) during hyposaline stress (liu e t al. 2012). at 15 ppt sah ty , dgrs of k. ahare@ plantlet were sipficantly lower than those at 20 35 ppt salinity levels (fig. 1). the weekly data indicated that the dgrs of plantlets cultivated in 15 ppt salinity level decreased during the first week of experiment (fig. 2). at that time, the most of thallus plantlets changed color from reddish brown to white (bleaching), whch indcated that most of the thallus cells had died. the lowest dgr occurred in the third week (-o.lo/o/d), in which some of the bleached thalli began to soften and brittle, which reduced the plantlet weight compared to that at the beginning of the experiment. hayashl et al. (2010) also cultured pieces of r alvareqi thallus originating from coastal waters in culture media with 15 ppt salinity level, the thallus experienced bleaching and ded withn three days. the osmotic adjustment mechanism was unsuccessful with extreme hyposaline stress (15 ppt), resulting in damage to the membranes, organelles and enzymes and ultimately seaweed cell death (hurd et a/ 2014). in contrast, at 20 ppt salinity level, the decrease in the dgrs was not as severe as that at 15 ppt salinity level, because the thalli did not undergo bleaching. these results suggested that non-transgenic plantlets of k. ahare?? were not resistant or photosynthesis efficiency and photosynthetic sensitive to hyposaline stress at 15 ppt salinity fixation caused by the formation of reactive level. figure 1 daily growth rates (dgrs) of kappaplyczls alvare+' non-transgenic plantlets for 12 weeks in pes medium with 15 35 ppt salinity note: numbers followed by the same letter are not significantly different, based on duncan's multiple-range test at p < 0.01, n = 3. overexpression of ge gene in kz$$apbyccz/s alvamei transgenic plantlets sulistiani et al. 15 ppt m20 ppt weeks figure 2 daily growth rates @grs) of non-transgenic plantlets in pes medium with at 15 and 20 ppt salinity levels for 1 5 weeks of rearing at 15 ppt, a small part of the thalli of non transgenic plantlets was still brown, especially at its base and tip (fig. 3a). the brown part of the thallus grew thicker, which caused the dgrs to increase again at the fourth week (fig. 2). at the 7h week, in the brown part of the thallus, many buds grew, but thallus branches did not grow until the 12& week of maintenance at 15 ppt salinity, with the thallus buds remaining short (fig. 3b). when the plantlets were subcultured in pes medium with normal salinity (30 ppt), the thallus buds began to form thallus branches, whereas the bleached part of the thallus did not grow (fig. 3c). after 2 months, the regenerated part of thallus began to separate itself from the bleached thallus and grew normally. at a salinity of 20 35 ppt, all of the plantlet thalli were still reddish brown. under hyposaline condtion (20 ppt) (fig. 4a), the plantlets formed more thallus branches than those under normal salinity (25 and 30 ppt) (figs. 4b and 4c), but the thallus branches were short. in contrast, in hypersaline conditions (35 ppt), plantlets formed fewer thallus branches than those under normal salinity (fig. 4d), and the thallus plantlets grow elongated. the results of this study indicated that the condition of the thallus branches can be used as an indicator of whether k. ahare?? grows in coastal waters with appropriate salinity seawater, hyposalinity or hypersalinity. development of k. alvanei non-transgenic plantlets in pes medium with 15 ppt salinity: (a) most part of the thallus was bleached at the second week; (b) growth of thdus buds at the brown part of the thallus at the seventh weeks; (c) growth of thdus branches after plantlets had been subcultured in pes medium with normal salinity (30 ppt) note: scale bars = 2 mm. biotropia vol. 28 no. 3,2021 figure 4 comparison of thallus branches of non-transgenic plantlets after 12 weeks in pes medium with different salinity: (a) 20 ppt; (b) 25 ppt; (c) 30 ppt; and (d) 35 ppt note: scale bars = 10 mm. tolerance of transgenic plantlets in hyposaline conditions based on the experimental results obtained in non-transgenic plantlets, six transgenic plantlets of k alva~eei (l7, e2, e3, e5, e6, e7 and e8) and one nontransgenic (n) plantlet were grown on pes medium only with a salinity of 15, 20 and 30 ppt for 5 weeks to test its tolerance to hyposaline stress. the result showed that at normal salinity, all of the transgenic plantlets had a significantly higher dgr (2.1 3.4% / d) than that of non-transgenic ones (1.4% / d) (fig. 5). at 20 ppt salinity, all transgenic plantlets had higher dgrs (1.7 2.3% / d) than that of non-transgenic ones under normal salinity (1.4% / d) pig. 5), whereas non-transgenic plantlets had lower dgrs (0.8% / d) than that of non-transgenic ones in normal salinity. at 20 ppt salinity, non-transgenic and transgenic plantlets still survived, the color of the thalli was still brown (fig. 6), but in the non transgenic, most of the thallus brown color has faded (fig. 6a). this shows that the tolerance of transgenic plantlets to 20 ppt salinity was higher than non-transgenic plantlets, because they can still grow faster than normal in these salinity, whereas the growth of non-transgenic plantlets was stunted or lower than the growth in normal salinity conditions. these results showed that overexpression of the ga gene increased the dgrs of transgenic plantlets under the condition of normal salinity and 20 ppt. this is because the ga protein plays a role in regulating cell proliferation and promoting growth as had been studied in arabid0psi.r plants (ullah 2001; chen e t al 2006; colaneri et ak. 2014) and rice (izawa 2010). figure 5 daily growth rates (dgrs) of non-transgenic (n) and transgenic (i. and e) plantlets under normal salinity (30 ppt) and hyposalinity (15 and 20 ppt) for 5 weeks observation. note: columns followed by the same letter are not significantly different based on duncan's multiple-range test at p <0.01, n = 3). overexpression of gcl gene in igppaph_ycz/s alvareei transgenic plantlets sulistiani et al. figure 6 development of non-transgenic and transgenic thalli after 5 weeks at 20 ppt salinity. (a) non-transgenic; (b) transgenic note: scale bars = 5 rnm at 15 ppt salinity, all transgenic plantlets showed higher dgrs (0.8 1.2% / d) than those of non-transgenic plantlets (0.0% / d) (fig. 5). in the first week, dgrs of non transgenic plantlets decreased into -0.8% / d at 15 ppt salinity (fig. 7). most parts of the non transgenic plantlet thalli (86% / d) was bleached after 3 days in pes medium with 15 ppt salinity (fig. 8a), this caused the dgr of non transgenic plantlets to fall below 0% / d during the first 2 weeks. whereas, most parts of the transgenic plantlet thalli were still brown, with only 3 7% of the th& being bleached, until the 5& week of the experiment at 15 ppt salinity (fig. 8b). therefore, all of the transgenic plantlets still grew even with a lower dgrs (0.8 1.2% / d) than non-transgenic ones under normal salinity (1.4% / d). the effect of hyposaline stress on the growth of transgenic plantlets began to be seen in the second week of the experiment. the dgrs of transgenic plantlets was still high in the first week of the experiment (1 3.5% / d) (fig. 7). based on these results it is expected that, if high rainfall were to occur and seawater salinity were to drop to 15 ppt in coastal waters for 1 week, k. alvare~z' transgenic seaweed would grow normally. moreover, under conditions of heavy rainfall for 2 5 weeks, transgenic seaweed would survive even with lower dgr than under normal rainfall conditions. this study showed that overexpression of the ga gene could increase tolerance to hyposahne stress of up to 15 ppt. the mechanism of osmotic adjustment still occurs within the transgenic plantlet cells at 15 ppt salinity. this is because the ga protein plays a role in regulating ca2+ influx to the cytoplasm through ion channels in the plasma membrane (gelli et al. 1997; aharon et al 1998; thuleau et al 1998; zhang et al. 2011). in tomato plants, a constitutively active form of tgal has been shown to increase the probability of ca2+ channels being open (aharon et al 1998). ca2+ is a central regulator in plant cell physiology and plays an important role in the response to abiotic stresses including hyposaline stress (pei & gilroy 2008). figure -1.0 i 1 week 0 2 weeks a3 weeks a4 weeks a5 weeks 7 weekly dgrs of k alvareei non-transgenic and transgenic plantlets in pes medium with 15 ppt salinity ca2+ plays a role in activating the sos1 antiporter and the nhx antiporter. the sosl antiporter mediates the movement of na+ in both directions from the apoplast to the cytoplasm through secondary active transport, driven by the h+ gradient across the plasma membrane. meanwhile, the nhx antiporter mediates the movement of naf in both directions from the vacuole to the cytoplasm (rodriguez-rosales e t al 2009). the overexpression of the ga gene increases the ca2+ influx to the cytoplasm in hyposaline condition, so that activation of sosl and nhx antiporter also helps to maintain a balance of na+ concentration between that inside and outside the cells under extreme hyposalinity (1 5 pp~). the results of calcium content analysis showed that transgenic plantlets under normal salinity conditions had higher calcium content (2.49%) than that in non-transgenic plantlets (2.06yo) (fig. 9). this indicated that overexpression of the ga gene increased the ca2+ concentration in the cytoplasrna. the transgenic plantlets in hyposaline conditions had higher calcium content (5.97% and 9.27%) than that under normal salinity (2.49%). moreover, the calcium content of non-transgenic thalli in hyposaline conditions was lower than that under normal salinity, which was due to most of the non-transgenic thalli experiencing bleaching and being brittle in hyposaline conditions, especially at 15 ppt. ca2+ is a structural component of cell walls and membranes in seaweed (hurd e t a/ 2014). increased ca2+ concentrations in the cytoplasm caused the thalli of transgenic plantlets to thicken and become more rigid at salinity level of 15 ppt (fig. 10b) than at 30 ppt (fig. 10a). according to hepler (2005), ca2+ plays a crucial role in determining the structural rigidity of the cell wall, high content of ca2+ should increase the rigidity and decrease the plasticity of cell walls. ca2+ ions also play a role in regulating the flow of water into plant cells, both through apoplast and symplast (gilliham e t al. 2011). increased rigidity of the k al'vare@i transgenic thalli at 15 ppt salinity is an attempt to inhibit excessive influx of water into the cytoplasm under hyposalin conditions. ion ca2+ regulates the flow of water into the apolast by affecting the structure of the cell wall. increased concentration of ca2+ ions in the cytoplasm stimulates the synthesis of cell wall precursors (cellulose and nonselulose) in the cytoplasm, then these precursors are released into the apolast to form thicker or rigid cell walls (hawkesford e t al. 2012). in the flow of water through the symplast, ca2+ ions affect the opening of aquaporin which regulates the flow of water entering through the plasma membrane into the cytoplasm. a low concentration of ca2+ ions in the cytoplasm will open aquaporin, while a high concentration of ca2+ ions in the cytoplasm will close the aquaporin (gilliham e t al. 2011). therefore, an increase in tolerance of the transgenic plantlet to hyposalin may be due to an increase in ca2+ ions in the cytoplasm which has closed aquaporin in the plasma membrane so as to prevent excessive inflow of water into the cytoplasm during hyposalin stress. all of the transgenic plantlet in 15 ppt were subcultured in pes medium with normal salinity. after 2 months, the entirety of transgenic plantlets thallus grew numerous thallus branches and regenerated normally (fig. 10c). in contrast, in non-transgenic plantlets, only small part of each thallus regenerated normally, i.e., at its base and tip (fig. 3c). overexpression of ga gene in k;tppqbh_yms alvare@ transgenic plantlets sulistiani et al. q non-transgenic transgenic 30 p p ~ 20 p p ~ 15 p p ~ figure 9 calcium content of k. adu~re@i non-transgenic and transgenic plantlets after 5 weeks of culture in pes medium with normal salinity (30 ppt) and hyposalinity (15-20 ppt) figure 10 thallus morphology of k alvare@i transgenic plantlets after 5 weeks of culture at : (a) 30 ppt, (b) 15 ppt and (c) 15 ppt + 8 weeks at 30 ppt note: scale bars = 7 mm. quantification of goc gene expression in transgenic plantlets using qpcr. two clones of transgenic plantlets (35 and eb), for which tolerance to hyposahity had already been proved, and one clone of non transgenic plantlet (n) were used for expression analysis of the ga gene using qpcr. the expression analysis was performed to determine the change in ga gene expression in transgenic of k. aluarev? into which the ga gene with a constitutive camv 35s promoter had been introduced, compared with that in non transgenic plantlets. the changes in gene expression were analyzed by 2-mct method. the result showed that the expression of the gagene in the e5 transgenic plantlets increased by 6.43 times compared with expression level in non transgenic plantlets, whereas in e6 transgenic plantlets, it increased by 8.03 times (fig. 11). the level of transgene expression in transgenic plants is influenced by many factors, especially by the location of the integration of transgene in plant genomes, gene silencing, and promoters that accompany transgene (page & minocha 2004). the camv 35s promoter has been used as a strong and constitutive promoter for expressing transgenes in many different plants, although with different levels of efficiency depending on the species. in dicot plants, the camv 35s promoter is a very strong constitutive promoter, causing high levels of gene expression. the results of this study showed that camv 35s promoters could increase expression level of the ga gene in transgenic plantlets of k. aluare?? (6.43 8.03 times) to a level hgher than that in transgenic tobacco plantlets (4.96 times) (fajri 2015). the expression of transgenes using this promoters in transgenic plantlets of k. ahare?? has also been reported for the gfp marker gene (rajamuddin e t al. 2014) and the h-carrageenase gene (rajamuddin 2016). the camv 35s promoters have also been successfully used to express transgenes in other species of red algae, such as the gus reporter gene in porpbyra yexoensis (cheney e t al. 2001) and the lac2 reporter gene in gradarid gracilis (huddy e t al. 20 12). in microalga chlamydoomonas reinhardtii, the camv 35s promoter had also successfully expressed the gus reporter gene (p glucuronidase) (pratheesh e t al. 2012), and in microalga dzlnaliella s a h , the promoter has successfully expressed the gfp gene reporter (srinivasan & gothandam 201 6). biotropia vol. 28 no. 3,2021 1 0 n e5 e6 figure 11 relative expression of (2-mct) gcc gene in kappaph_ycz/s ahare@ transgenic plantlets (e5 and e6) that were tolerant of hyposalin stress, to non-transgenic (n) plantlets that were intolerant of hyposalin stress pearson correlation analysis (rumsey 2010) was performed on the data of relative ga gene expression (re) (fig. 11) and dgrs of nontransgenic and transgenic plantlets in normal salinity (30 ppt) and hyposalinity (20 ppt and 15 ppt) (fig. 5). the analysis showed that re of ga gene had a strong correlation, both with dgrs in normal salinity and hyposalinity because the correlation coefficient was more than 0.7 (rumsey 201 0). the correlation coefficient between re of ga gene and dgrs at 30 ppt salinity was significant at 1% test level, whereas with dgrs at 20 and 15 ppt salinity was significant at 5% test level (table 1). the correlation between re of ga gene and dgrs was linearly positive, where increased expression of the ga gene was strongly associated with an increase in dgrs (fig. 12). results of correlation analysis showed that the increase in daily growth rate of transgenic plantlets, both in normal salinity and hyposalinity (fig. 5) was strongly associated with an increased ga gene expression in transgenic plantlets (fig. 11). this proved that over-expression of the ga gene had increased the growth of k. ahare?? transgenic. as reported in arabidopsis and rice plants, goc protein was a positive modulator of cell division processes (ullah e t al. 2001; chen et al 2006; colaneri e t al 201 4; izawa e t al. 201 0). table 1 results of pearson correlation analysis between the relative expression of the ga gene and the daily growth rate (dgrs) in normal salinity (30 ppt) and hyposalinity (20 and 15 ppt) dgr significance value correlation coefficient 30 ppt salinity 0.009** 0.806 20 ppt salinity 0.020* 0.749 15 ppt salinlty 0.028* 0.724 notes: ** = correlation coefficient is significant at 1% test level. * = correlation coefficient is significant at 5% test level. -1 ' relative expression figure 12 correlation between the relative expression (re) of the ga gene with the daily growth rate of non-transgenic and transgenic plantlets on culture media with normal salinity (30 ppt) and hyposalinity (20 ppt and 15 ppt) overexpression of gcc gene in kappapbycm alvareei transgenic plantlets sulistiani et al conclusion transgenic plantlets of k. alvare~z' had a significantly hgher daily growth rate than that of non-transgenic plantlets in pes medium with normal salinity (30 ppt) and hyposalinity (15 and 20 ppt). the transgenic plantlets showed tolerance to hyposalinity at 15 ppt. the transgenic plantlets which a high daily growth rate and tolerance to hyposalinity had a higher expression level of the ga gene than that in non transgenic plantlets. acknowledgements we thank seameo biotrop southeast asian regional center for tropical biology for the support of materials and facihties for mediated by the heterotrimeric g protein complex. bmc plant biol 14:129. dawes cj, lluisma ao, trono gc. 1994. laboratory and field growth studies of 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regulation by g-proteins, the cytoskeleton and second messengers. curr opin plant biol 1 :424-7. ullah h, chen jg, young jc., im i=, sussman mr, jones am. 2001. modulation of cell proliferation by heterotrimeric g protein in arabidopsis. science 292:2066-69. urano d, chen jg, botella jr, jones am. 2013. heterotrimeric g protein signalling in the plant kingdom. open biol. 3: 120186. zhang w, jeon bw, assmann sm. 2011. heterotrimeric g-protein regulation of ros signalling and calcium currents in arabidqbsis guard cells. j of exp bot 62(7):2371-9. bic>tropiiz vnl. 28 no. 3,2021: 193 203 doi: 10.1 153x/htb.2021.28.3.1325 assessment of the quality of akaj3ica coffee beans from three processing methods and two types of packaging mkterials** oktcy setyawat1 dl-] arm,4putraw, santi ahlbxrfyati, in a remowrati and n i p m pu'urfrzdila rcccjvcd 14january 22c)20/acccptcd 27 april 2020 abstract in sr~utheast 'isla, indonesia is the second h ~ g l ~ e s t producing country nf coffee beans after vietnam. cn~~sequenrip, indonesia comperes with other countries in producing good q u n l i ~ coffee 1,cans. hou,wer, not many pcaplc havc sufficjcnt skills in tackling problems relatcd to the postharvest handling of coffee bcans. thc objectire o f thjs study uras tr) asscqs die quality ( ~ f hrabica cnffee (c.i~ff~o omri~icu) beans in terms o f moisture contcnt. fungal infcction (cspccially ochmtosin ,i or oti% producing fungi), cjta contamination, and the tastc of the coffee during str~rilge. the diree processing mmctl~r)ds used were dry, wet, and scrni-wet mclthods. the i,eans were packed using nvo types of packagng marcrials, i.e., kantmq s~r?/orhigh p s garrrcr and pol>+prop~*lene bags (4 kg/bag). they were then stored under warehouse conditions for 4 months o f storage. thc rnolsturc content of cofl'ec i~cans prncesscrl using thc thrcc mcthnris and paclccd using polypmpylcnc hagswwas higher than that of crlffee beans packed using kamtflq .l'rmclr high pas barrier; however, ~t was still i.n\ver than the safe moisture content for coffee determined by the tndonesran national standard (12.j0/o). / i ~ f ~ i / h n i p was fotlnd in coffee processed using the tl~rec methods and packed using a kqntoq .smor iiigh -has bnrj1r.r. its populatir~n was relativelv lour (< 0.1 x 10 ch/g wet basis). /ispemi//u.~ ocjrrrttz~.r was found in coffee processed using dry nnd wet methods at the beginning of storzgc. i t s prrpulatiun \vas also relatively low (< o..? s 10 cfu/g %-.i>. ot11 content was not detected in all coffer samples, herausc it mns lower than the dctcction limit of tlic instrument uscd (< 1.85 ppl,). a t tlic beginning of rhc storagc, all coffee sarnplcs wcrc dnminated i>y yeast with the population of 1.9 x 1112 1.2 s 10' c f ~ / ~ w.h. the taste of coffee in r-arious trcatmcnts during 4 months of storagc was still above tlw total standard scorc for specialty firarlc 2 80. thc l~iglicst total scorc (84) was fot~nd in coffee beans processed using a dry rnetl~od and packed in kmionq! .srfllar high gas harricr. the thrcc processing mei-hocls and thc two types r)f packaging materials can l ~ e uscd tn maintain the cluality c ~ f ct~ffec beans for 4 months of starage. keywords: arabica coffce bcans, processing methods, quality, types of packaging materials introduction around 70 countrics worldwide are coffee bean producers, tvjth the overwhelming majority of the supplrr coming from developing countries like draxil, vietnam, colombia, indonesia, and ethiopia i\valton 201 8). tndonesia is the fourth largest producer in the world and the second largess in southeast asia after vietnam. two *c:orresponding aut l~c~r, ernajl: oklrv@hiotrt~p.clrg " this paper iws prescntetl at the international conference science based ingrcclients: the future for fnncl in asia; jakarta 3 5 october 2018, tndonesia idnds of coffee renowned in indonesia are robusta coffee (cofea c~nepboraj and arabica coffee (6 nrabica). rnhusta and arabica coffee contribute around x3"/11 and 14'yn, respectively, to the total coffee production in indonesia (gaeki 201 8). as one of the largest producers, indonesia competes with other countries in producing good quality coffee beans, hence, stringent rules must be imposed to maintain the qualitc. of exported beans. however, not many people have sufficient s m s in tackling problems related to the postharvest han&ng of coffee beans. biotropia vol. 28 no. 3,2021 in indonesia, there are three processing methods of coffee beans, i.e., dry, wet, and semi-wet processing (kementerian pertanian ri 2012). dry (natural) processing method is a process which consisted of sun-drying the harvested ripe coffee berries for 14 days until the moisture content attained & 10°/o, followed by shelling of the husks and hulls of the coffee berries. wet (fully washed) processing method is a process which consisted of shelling the husks of ripe coffee berries to become wet green beans with hulls. the wet green beans were then fermented for one night and washed using water to eliminate the mucus, followed by sun-drying for 7 days until reachng moisture content of & 10% (dried green beans). the hulls of the beans were then shelled ('green beans). semi-wet (semi-washed) processing method is a process whch consisted of shelling the husks of ripe coffee berries. the wet green beans were fermented for one night and washed to eliminate the mucus, followed by sun-drying for one day unul the moisture content attained & 40%. the hulls were then shelled to obtain green coffee. after shelling, the beans were further sun-dried for 5 days until the moisture content attained & 10%. according to dharmaputra et al. (2019), polypropylene bag is a type of packaging material used by farmers, collectors, and big traders in tana toraja and north toraja regencies to store coffee beans. in makassar, exporters generally use jute bags doubled with polyethylene hermetic bags to store coffee beans. during storage, coffee beans may be infested by insects, microorganisms, mites, and rats. among microorganisms, fungi are the most dominant cause of deterioration in stored grains or seeds. fungal infection in grains can cause discoloration, musty odor, weight loss, reduction in nutritional contents, and mycotoxin contamination. ochratoxin a (ota) contamination in coffee beans becomes a very important subject as some consumer countries have imposed the maximum tolerable limits (mtl) of ota. bpom r i (2018) has determined the mtl of ota in powdered coffee and kopi sangmi (roasted coffee) to be as much as 5 ppb, while that in instant coffee to be as much as 10 ppb. in brazil, the maximum limit allowed for ota in coffee is 10 ppb (brazil 201 1). ota is a potent nephrotoxic mycotoxin that has been linked to hdney problems in both livestock and human populations (cabaiies et al. 2010). it also has carcinogenic, genotoxic, and immunotoxin properties. ota has been reported in temperate and tropical countries to naturally occur mainly on cereals and their products. according to bui-iuimke and wu (2015), in tropical regions, ota is mainly produced by a.pe@llus carbonarius, a. niger, and a. ocbraceas, while in sub-tropical regions it is produced by penin'llium vemcosum. dharmaputra et al. (2019) reported that in tana toraja regency, north toraja regency, and makassar municipality, indonesia, penin'llium citrnam was the dominant fungus found in coffee beans collected from farmers, collectors, and traders. meanwhile, a. niger wais the dominant fungus found in those beans from the exporters. at trader level, 46% of the samples were infected by a. ocbraceus and a. niger. at exporter level, 44% of the samples were infected by a. ocbraceus, while 78% of the samples were infected by a. niger (dharmaputra et al. 2019). rosavani and harada (2019) reported that in 2019, nine of ten samples of green coffee beans collected from argopuro mountain areas, jember, east java province, indonesia were infected with ota, in which the highest concentration was 0.4319 ppm and the lowest concentration was 0.0146 ppm. good handling processing (ghp) in coffee beans is not only useful in preventing ochratoxigenic fungal growth but also useful in providmg good taste and aroma. according to sunarharum et al. (2018) wet fermentation and controlled mechanical drying were found to yield a better coffee sensory profile than natural and semi-wet fermentation. other research, evangelista et al. (201 5) explained that mgeropma caribbica and hanseniaqora uvamm were dominant yeasts in coffee beans fermented using wet method from lavras, brazil; while tomlaqora delbmeckii was dominant yeast in coffee beans from monte carmelo. tripetch and borompichaichartkul (2019) reported that high density polyethylene (hdpe) bag was better than a jute sack for green coffee beans for 15 months of storage, because the polyethylene bag has the capability assessment of the quality of arabica coffee beans dharmaputra et al of preserving the moisture content, color and materials and methods chlorogenic acid of the coffee beans. postharvest handling methods affect the source of green coffee beans quality of coffee beans. consequently, they also arabica green coffee beans grade 1 (grade affect the price to one) were from cv frbsa agrolestari. foreign trade. the objectives of this study were ne coffee was cdtivated by a big coffee to: 1. assess the quality of arabica coffee beans located in pangalengan subdistrics in terms of moisture content, fungal infection bandung regency, west java. the coffee (especially ota producing fungi), ota plantation has an altitude of +1,200 m above contamination of of fee beans, and the taste of sea level. ripe coffee berries were harvested in the coffee during storage and 2. recommend one day to obtain a homogenous sample. they appropriate processing methods and the type of were then processed using three different packagmg materials to ensure the quality of methods, i.e., natural (fig.l), wet (fig. 2), and arabica coffee beans during storage. semi-wet methods (fig. 3). figure 2 drying of wet green beans with h d s processed using the wet method biotropia vol. 28 no. 3,2021 ( 4 (b) figure 3 drying of wet green beans with h d s (a) and further drying of green beans using the semi-wet method @) packaging and storing methods green coffee beans (10% moisture content) were packed using two types of packaging materials i.e., polypropylene and fintmzg s e m high gas barrier bags (4 kg/bag). they were then stored under warehouse conditions for two and four months (fig. 4). the temperature and relative humidity of the storage were determined using thermo-hygrometer haar-synth hygro. sampling methods samples of coffee beans were subsequently taken from each bag at the beginning, after 2 months, and after 4 months of storage (k 1 kg/bag). each sample was then divided three times using a box divider to obtain working samples for moisture content, fungal population and ota content determinations, and cupping test. figure 4 coffee beans packed in kantong s e m high gas barrier (a) and polypropylene (b) bags under warehouse conditions assessment of the quality of arabica coffee beans dharmaputra e t al. determination of moisture content, fungal population, and ota content moisture content of green coffee beans (based on wet basis) was determined using oven method (sni 2008). fungi were isolated using a serial dilution method, followed by the pour plate method on dichloran 18% glycerol agar (dg18) (pitt & hocking 2009). each fungal population was determined based on the number of fungal colonies on dg18 in a certain dilution factor. each fungal species was identified using pitt and hocking (2009) and samson e t ul (2010) as the main reference. ota contents were determined using high performance liquid chromatography (hplc) method (aoac 2012). two replicates were used for each sample. ground coffee was test of coffee laboratory of pt icemenady industri mandiri, bogor. statistical analysis the data were analyzed using completely randomized factorial design with three factors. the 1", 2nd, and 3rd factors were the processing methods, the types of packaging materials, and the duration of storages, respectively. three replications were implemented for each treatment. thus, the numbers of unit experiment were 3 x 2 x 3 x 3 = 54. results and discussion moisture content extracted using moisture content of coffee beans resulted bicarbonate (70:30). sample extract was diluted from ,nous treatments at fie be$ing of using o.o1o'o tween 20, and it was then storage, followed by 2 and 4 months of storage, 'luted using ochra test irnmunoaffiity is presented in table 1. the range and mean of column. detection was determined using temperature and relative hufidity of the hplc-fl detector. warehouse during storage are presented in table 2. the moisture content of coffee beans cupping test processed using the three methods and packed cupping test of the samples was based on using polypropylene bags was higher than that standard cupping protocol issued by the coffee of coffee beans packed using kantong semar hgh quality institute and specialty coffee gas barrier bags. however, it was still lower than association of america (scaa 2015). the the safe moisture content for coffee determined panelists have a certificate from cqi q grader. in 2008 by the indonesian national standard, cupping test was conducted at the cupping i.e., 12.5%. table 1 moisture content of coffee beans resulted from various treatments at the beginning, after 2 months, and after 4 months of storage moisture content (o/o wet basis) 0 month 2 months 4 months processing method kantong kantong kantong semar polypropylene semar high polypropylene high gas polypropylene semar high gas barrier bag gas barrier bag barrier bag natural method 9.5 be 9.6 bc 9.8 ca 10.4 d 9.8 c 11.2 h wet method 8.7 a 9.2 ef 8.8 a 10.3 d 8.9 ag 11.2 h semi-wet method 8.8 ab 9.6 bc 9.1 fg 10.3 d 9.2 ef 11.2 h notes: numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level; 0 month = coffee beans at the beginning of storage; 2 months = coffee beans after 2 months of storage; 4 months = coffee beans after 4 months of storage. biotropia vol. 28 no. 3,2021 table 2 range and mean of temperature and relative humidity in the warehouse for storing coffee beans during storage duration of storage range and mean of temperature range and mean of relative humiditj (month) pc) ("0) at the beginning of storage, the moisture fungal population and ota content content of coffee beans processed using the dry method was hgher than that of wet and semi wet methods. in the dry processing method, the drying of coffee beans could not reach the maximum drying level because the beans were still protected by the pulp of beans. aeration inside of polypropylene bags was higher than that of kantong semar high gas barrier bags; consequently, the moisture content of coffee beans packed in polypropylene bags was higher than that packed in kantong semar high gas barrier bags. the research results showed that aspe~iiizas ochracezls (ota producing fungus) was found only in coffee beans processed using dry and wet methods at the beginning of storage. its population was relatively low, i.e., 1 and 3 cfu/g w.b (table 3). the population of a. niger (ota producing fungus) in coffee beans processed using the three dfferent methods was relatively low (< 10 cfu/g w.b) (table 4). the hghest population of a . niger was found in coffee beans processed using the semi-wet method (table 3). table 3 fungal population of coffee beans resulted from various treatments at the beginning, after 2 months, and after 4 months of storage treatment fungal population (cfu/g wet basis) fungi 0 month 2 months 4 months fh aspel-gllusjlauus a. cbeualieri a. niger a. ochracezls a. penicilloides a. tamarii cladosponztm cladosporioides ftlsdmzlm auenacem fusarium sp. penia'iliam n'tnnum wallemia sebi yeast a yeast b yeast b2 sh aspe@llusjlauus a. cbeualien' a. niger a. penicilloides cladospon'zlm cladosporioides fasariurn sp. peninllium citrinm yeast a yeast b yeast b2 n h aspe@llusjlauus a. cbeualieri a . niger a. ocbraceus a. penicilloides a. tamarii assessment of the quality of arabica coffee beans dharmaputra et al. table 3 (continued) treatment fungi fungal population (cfu/g wet basis) 0 month 2 months 4 months cladosporium cladosporioides fzlsarium avenaceum penicillm citrinum yeast a yeast b aspergillus flavus a. chevalien' a. niger a. ochraceus a. penicilloides a. tamarii cladosporium cladosporioides fusarium avenaceum fusarium sp. penicillium citnnunz yeast a yeast b yeast d sp aspergiiius chevalieri a. niger a. penicillioides cladosporimm cladosponoides penicillm citnnm yeast a yeast b yeast b2 yeast d np aspergillus flavus a. chevalieri a. niger a. penin'llioides a. tamarii cladospo~um cladosponoides fusarium avenaceum penicillium citr;kum yeast a yeast b yeast d notes: n = coffee beans processed using the natural method; f = coffee beans processed using the wet method; s = coffee beans processed using the semi-wet method; h = coffee beans packed in kantong semar high gas barrier bags; p = coffee beans packed in polypropylene bags. table 4 population of a. nigerin coffee beans resulted from various processing methods processing method population of a. niger (cfu/g wet basis) natural method 1 (1 2) b wet method 2 (1 3) ab semi-wet method 4 ( 2 9 ) a note: numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level. biotropia vol. 28 no. 3,2021 according to pitt et al. (2000), ota is mainly produced by aspergiiius carbonamhs, a. niger, and a. ocbraceus in tropical regions, while in sub tropical regions it is produced by penin'lliwm vemcosm and p. nordicum. batista et al. (2009) reported that genus of a.pe@dus section circumdati and section n&m' were found in coffee berries and and green beans when they were harvested and during processing. a.pe@iius section circumdati was found in coffee berries and green beans, i.e., 80% and 4lo/o, respectively, but their population decreased after the first stage of processing. as many as 33% of 12 samples of coffee beans processed using the wet method was contaminated by aspergiiius section circumdati. as much as 73% aspeqiiius section nigm' was found in coffee berries and 25% of it was found in the processed coffee beans. de fatima et al. (2013) reported a number of fungi were isolated from 30 samples of arabica coffee. as many as 20 samples of coffee beans were collected by using conventional cultivation system, and 10 samples were collected by using organic cultivation system. as many as 480 fdamentous fungal species were isolated. they belong to genus a.pe?gdu.r, group circumdati, and nigri. ota producing fungi isolated were a. auricoumu~, a. ocbraceu~) a. ostianzls, a. niger, and agregate of a . niger. the most frequent ota producing fungi species was a. ocbracezas which corresponded to 89.55% of samples. ota contents in all samples during storage were lower than the detection limit of the instrument (< 1.85 ppb). samples were dominated by yeast at the beginning of storage. its population was 1.9 x 10' 1.2 x lo3 cfu/g w.b (table 5). it was assumed that the yeast inhibited the growth of ota producing fungi; consequently, it prevented ota production. accordmg to masoud et al. (2005), volatile compounds such as ethyl acetate, isobutyl acetate, 2-phenyl ethyl acetate, ethyl propionate, and isoamyl alcohol which are produced during coffee processing by picbia anomala) p. kiyveri, and hanseniaqora uvam, inhibited ota producing fungi (a. ocbraceus) and prevented the ota production. the total of yeast population decreased during storage (table 5). it was assumed that yeast competed with filamentous fungi. filamentous fungi (except a. niger and a. ocbraceu~) were isolated from coffee beans, i.e., a. jlavzas, a. cbevalieri, a. penin'ilioides, a. tamarii, cladospom'um cladosporioides, fzlsdtium avenaceum, fusam'um sp., penicillum citrnum, waiiemia sebi (table 3). munyendo et al. (2017) reported that fungal infection in coffee beans processed using the dry method was higher than that processed using the wet method. table 5 total yeast population of coffee beans resulted from various treatments total population of yeast (cfu/g wet basis) 0 month 2 months 4 months processing method kantong jemar kantong semar kantong semar high polypropylene polypropylene polypropylene high gas high gas gas barrier bag barrier bag barrier bag natural method 97 152 0 f 0 f 0 f 0 f (30-174) abcd (40-370) abcd wet method 262 1 185 534 24 39 0 f (33-533) a (397-1,683) abcd (213-1,143) ab (3-60) def (10-57) bcd semi-wet method 187 442 170 25 2 0 f (120-283) abcd (136-986) abc (40-387) abcd (17-37) cde (0-7) ef note: numbers followed by the same letter do not differ significantly according to duncan's multiple range test at 95% confidence level. assessment of the quality of arabica coffee beans dharmaputra et al. cupping test the flavor of coffee beans handled using the three processing methods which were then specialty coffee beans are not accepted when packed in fintong semar high gas barrier bags the cupping test score is lower than 80 (scaa and polypropylene bags and stored for 4 months 2015)' accor&ng to atmawinata (2002)' in was still above the total score of specialty grade general, coffee is not consumed because of its 80 the highest total score was nutritional value, but because of the flavor and obtained by the coffee beans processed with the physiological causing people natural ~rocessinp method which were then awake, have improved moods, feel fresh, have less fatigue condition, and to feel enthusiatic. saepudin (2005) reported that the value of coffee beans is not only determined by physical quality but also by the value of the flavor. therefore, cupping test is one of the methods to determine the quality of coffee in importing countries. the qualification of coffee beans based on the cupping test fmal scores was divided into 4 categories, i.e., outstanding (90 loo), excellent (85 89.99), very good (80 84.99), and below specialty quality (below 80) (scaa 2015). coffee beans with outstanding, excellent, and very good categories with the final score of 80 100 belong to specialty qualification. on the contrary, coffee beans with below specialty category do not belong to specialty qualification, but they may still properly be consumed. according to the panelists, coffee beans could not be consumed when the final score was 5 30. i 0 packed in kantong semar high gas barrier bags (84). borem e t al. (2013) reported that kantong semar high gas barrier bags are also impermeable to water and gases including co2, 0 2 , and n2. ribeiro et al. (201 1) explained that hermetic bags are able to maintain the sensory qualities of materials, such as volatile compounds, textures, and colors. it is suitable for certain products requiring the manipulation of atmospheric conditions. therefore, kantong semar high gas barrier bags are able to maintain the sensory quality, especially the aroma of packaged material. murthy and naidu (2011) found that the taste of coffee beans processed by using the wet processing method was better than that processed by using the dry processing method because the aroma of volatile taste in roasting of coffee beans processed using the wet method was better than those processed using the dry method. table 6 results of cupping test of coffee beans processed by various treatments after 2 and 4 months of storage score attribute fh fp sh sp nh np 2 rnnt 4 rnnt 2 rnnt 4 rnnt 2 rnnt 4 rnnt 2 rnnt 4 rnnt 2 rnnt 4 rnnt 2 rnnt 4 rnnt fragrance flavor aftertaste acidity body balance uniformity clean cup sweetness overall total score defect final score notes:laboratory of cupping test: pt icemenady industri mandiri, bogor; 0 = not present; 1 = unacceptable; 2 = very poor; 3 = poor; 4 = fair; 5 = average; 6 1 good; 7 = very good; 8 = excellent; 9 = outstanding; 10 = exceptional; specialty grade 2 80; fh = full wash (wet method) using kantong semar high gas barrier bag; fp = full wash (wet method) using polyprophylene bag; sh = semi-wet method using kantong semar high gas barrier bag; sp = semi-wet method using polyprophylene bag; nh = natural method using kantong semar high gas barrier bag; np = natural method using polyprophylene bag. biotropia vol. 28 no. 3,2021 conclusion agrolestari and pt kemenady industri mandiri moisture content of coffee beans processed using the three processing methods and then packed using polypropylene bags was hgher than those packed using kantong semar hgh gas barrier bags. however, the moisture content was still lower than the safe moisture content for coffee determined by the indonesian national standard (1 2.5%). a.pe~ills niger (ota producing fungus) was found in coffee beans processed by using the three processing methods and packed using kantong semar high gas barrier bags. the fungal population was relatively low (< 0.1 x 10 cfu/g wet basis). a.pergiilus ocbraceus (ota producing fungus) was found in coffee beans processed by using dry and wet methods at the beginning of the storage. the fungal population was also relatively low (< 0.3 x 10 cfu/g w.b). ota content was not detected in all coffee samples, because it was lower than the detection limit of the instrument used (< 1.85 ppb). at the beginning of storage, all coffee samples were dominated by yeast with the population of 1.9 x 102 1.2 x lo3 cfu/g w.b. the taste of coffee in various treatments during the 4 months of storage was still above the total standard cupping test score for specialty grade 2 80. the highest total score (84) was found in coffee beans processed by using the dry processing method and packed in kantong semar high gas barrier bags. the three processing methods and the two types of packaging materials can be used to maintain the quality of coffee beans during 4 months of storage. future research concerning the use of other types of packaging materials is needed. acknowledgements the authors would like to acknowledge seameo biotrop for providing financial support through ddftdr isidn pelaksanaan anggdran (dipa) 2017. special gratitude goes to mr tony igistianto juwono from pt multi panelindo margawi, semarang for allowing us using the kantong s e m r high gas barrier bags in our study. thanks are also due to cv frinsa for their assistance during the research. references aoac official method 2004.10. 2012. ochratoxin a in green coffee. 19th (ed.). in: latimer gw, editor. official methods of analysis of aoac international. gaithersburg (us): aoac international. vol 11, chapter 49:67-9. atmawinata 0. 2002. peranan uji citarasa dalam pengendalian mutu kopi f h e role of cupping test in controlling the quality of coffee]. lecture note presented in training on cupping test of coffee. jember (id): indonesian research institute of coffee and cacao. badan pengawas obat dan makanan ppomi. 2018. peraturan badan pom nomor 8 tahun 2018 tentang batas maksimum cemaran kimia dalam pangan olahan pegulation of the head of the indonesian agency for food and drug control number 8 of 2018 concerning chemical contamination in processed food]. jakarta (id): badan pom republik indonesia. batista lr, sm chalfoun, cf silva, m cirillo, ea varga, rf schwan. 2009. ochratoxin a in coffee beans (coffea arabica l.) processed by dry and wet methods. food control 20:784-90. borem fm, riberio fc, figueiredo lp, giomo gs, fortunato va, isquerdo ep. 2013. evaluation of the sensory and color quality of coffee beans stored in hermetic-packaging. j stored product res 52:l-6. brazil. 201 1. rules on maximum permitted limits for mycotoxins in foods and beverages. resolution rdc no. 7 of february 18, 2011. brasilia (br): brazilian ministry of health. bui-i(limke tr, wu f. 2015. ochratoxin a and human health risk: a review of the evidence. crit rev food sci nutr 55(13):1860-9. doi: 10.1080/ 10408398.2012.724480 cabaiiee fj, bragulat ma, castella g. 2010. ochratoxin a producing species in the genus penin'ilium. toxins 2:1111-20. doi: 10.3390/toxins2051111 dharmaputra os, ambarwati s, retnowati i, nurfadila n. 2019. fungal infection of stored arabica coffee (cofea arabica) beans in tana toraja regency, south sulawesi province, indonesia. biotropia 26(2):127-35. de fatima re, borges jg, cirillo ma, prado g, paiva lc, batista lr. 2013. ochratoxigenic fungi associated with green coffee beans (coffea arabica l.) in conventional and organic cultivation in brazil. braz j microbial 44 (2):377-84. doi: 10.1590/51517 83822013 0002 00006. evangelista sr, da cruz pedroso miguel mg, silva cf, pinheiro acm, schwan rf. 201 5. microbiological assessment of the quality of arabica coffee beans dharmaputra et all diversity associated with the spontaneous wet method of coffee fermentation. international journal of food microbiology 210:102-112. gabungan eksportir icopi indonesia [gaeiu] . 201 8. areal dan produksi kopi [areal and coffee production. jakarta (id): gaei(i; [cited 2018 oct 311. available from: http: //gaeki.or.id/areal-dan produksi. icementerian pertanian republik indonesia. 2012. peraturan menteri pertanian ri nomor 52/permentan/ot.140/9/2012, tanggal 4 september 2012 tentang pedoman penanganan pascapanen kopi [regulation of the ministry of agriculture, the republic of indonesia number ot.140/9/2012 concerning guidelines of coffee postharvest handling]. jakarta (id): icementerian pertanian republik indonesia. [cited 2019 nov 131 available from: ditjenpp.kemenkumham.go.id/ arsip/bn/2012/bn909-2012lamp.pdf masoud w, poll l, jakobsen m. 2005. influence of volatile compounds produced by yeasts predominant during processing of coffea arabica in east africa on growth and ochratoxin a (ota) production by aspergiiiz~zls ochracezls. yeast: 22: 1 133-42. munyendo lm, icoskei r, mburu m fnternet]. 2017. molds and ochratoxin a associated with green coffee (coffea arabica) beans processed by dry and wet methods in nyeri country. iosr journal of environmental science, toxicology and food technology (iosr-jestfq eissn: 2319-2402, p-issn: 2319-2399, vol 11, issue 6 ver. i (june 2017), pp. 66-72. [cited 2017 oct 151. available from www.iosrjournals.org. doi: 10.9790/2402 1106016672 murthy, ps, naidu mm. 2011. improvement of robusta coffee fermentation with microbial enzymes. european appl sci 3(4):130-9. pitt ji, basilico jc, abarca ml, lopez c. 2000. mycotoxins and toxigenic fungi. medical mycol38:41-6. pitt ji, hocking ad. 2009. fungi and food spoilage. (3'* ed.). new york (us): springer. ribeiro fc, borem fm, giomo gs, lima rrd, malta mr, figueiredo lp. 2011. storage of green coffee in hermetic packaging injected with c02. j stored product res 47:341-8. rosavani ma, harada h. 2019. presence of ochratoxin in a green coffee beans processed naturally by smallholder farmers in jember. novel techniques in nutrition and food sci 9(3):270-1. saepudin a. 2005. evaluasi faktor-faktor yang mempengaruhi citarasa kopi arabika dengan menggunakan manova dan analisis profil. (studi kasus: hasil uji citarasa kopi arabika igntamani tahun 2004). [skripsi]. bogor (id): fakultas matematika dan ilmu pengetahuan alam ipb. available from http://repository.ipb.ac.id/handle/ 123456789/12968 samson ra, houbraken j, thrane u, frisvad jc, andersen b. 2010. food and indoor fungi. cbs laboratory manual series. utrecht (nl): cbs i m a w fungal biodiversity centre. scaa. 2015. scaa protocols; cupping specialty coffee. specialty coffee association of america. [cited 2017 nov 161. available from https:// www.scaa.org/pdf/resources/cupping-protoco1s.pdf standar nasional indonesia. 2008. biji kopi [coffee beans]. jakarta (id): badan standardisasi nasional. sunarhamm wb, yuwono ss, nadhiroh h. 2018. effect of different post-harvest processing on the sensory profile of java arabica coffee. advances in food science, sustainable agriculture and agoindustrial engineering 1 (1):9-13. tripetch p, borompichaichartkul c. 2019. effect of packaging materials and storage time on changes of colour, phenolic content, chlorogenic acid and antioxidant activity in arabica green coffee beans (coffea arabica l. cv. catimor). journal of stored products research 84:101510. walton jx fnternet] 2018. countries produce most coffee. new york (us): investopedia; [updated 2019 feb 09; cited 2018 oct 311. available from https://www.investopedia.com/articles/investing. sri-13 mei 2017-556 (iyan predicting).cdr predicting the impact of climate change on the distribution of f. muell.flindersia pimenteliana in indonesian papua and papua new guinea iyan robiansyah * center for plant conservation bogor botanic garden, indonesian institute of sciences, bogor 16003, indonesia received 12 december 2015/accepted 12 august 2016 abstract population of flindersia pimenteliana (maple silkwood) in indonesian papua and papua new guinea is severely fragmented and experiencing a continuing decline due to habitat destruction and illegal logging. this species is very susceptible to environmental changes and at greater risk of extinction due to its small and fragmented geographic ranges and low abundance. using maximum entropy (maxent) method, the present study predicted the impact of climate change on the distribution of the species across its native distribution area. elevation and 19 bioclimatic variables commonly used in species distribution modeling were used as predictors. the prediction model of the 2current potential distribution identified a total area of 156,214 km in indonesian papua and papua new guinea (18% of total land area) as suitable habitat for f. pimenteliana. elevation and precipitation of the wettest, coldest and warmest quarters contributed most to the model. based on the average of hadgem2-es and miroc-esm models, potential distribution projections under rcp8.5 scenario suggested a habitat gain of 16% for 2050 and 8% for 2070 in the species distribution. whereas under rcp4.5, an average habitat gain of 7% was predicted for both 2050 and 2070. the newly suitable habitats were predicted to be found mainly in southern and western highland of papua new guinea. protection of these areas from habitat destruction and land use change is needed to assist f. pimenteliana find the most suitable climate for its survival. keywords: climate change, distribution models, flindersia pimenteliana, maxent, prediction introduction climate is one of the most important factors influencing plant distribution. at population scale, seed germination, growth and survival of plants are strongly influenced by temperature and precipitation (walck et al. 2011). thus, the rapidly changing climate for the coming decades will undoubtedly alter local environment where plants grow and consequently change their abundance and distribution (ipcc 2013). plants have only two alternatives in dealing with future climate change in order to survive i.e. either adapting in situ or dispersing to find the most suitable climate for their survival. failing to find the most suitable environment will result in the extinction of the plants. in this context, understanding and predicting the response of plant species and possible distribution alternatives under climate change condition are important to develop proactive strategies to reduce the impact of climate change on plant species diversity. species distribution models (sdms) is an important tool in ecology and biodiversity conservation, both for understanding the factors that affect species distribution and for predicting the response of species to climate change (peterson et al. 2011; franklin 2013; guisan et al. 2013; guillera-arroita et al. 2015). these models correlate species occurrence data and environmental variables to estimate species distribution using statisticalor machine-learning procedures (phillips et al. 2006; roberts & hamann 2012). among available models, maximum entropy (maxent) (phillips et al. 2006) is one of the best sdms for analyzing the presence-only data in terms of ability to distinguish between areas where a species is biotropia 4 1 7 59 70 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 1 556 * corresponding author: iyan.robiansyah@lipi.go.id 59 present, versus those where it is absent (elith et al. 2006). with more than 1,000 applications since 2006, maxent is also one of the most popular sdms, mainly due to the user-friendliness of the software and its high predictive accuracy compared to other sdms (merow et al. 2013). flindersia pimenteliana f. muell. (rutaceae) (maple silkwood) is a tree native to queensland (australia), indonesian papua and papua new guinea (australian tropical rainforest plants, http://keys.trin.org.au). the species is usually found in well developed rain forests and in various sites, but reaches its best development in upland and montane rain forests. evaluation on population status of in indonesian f. pimenteliana papua and papua new guinea revealed that the tree is categorized as according to endangered the international union for conservation of nature (iucn) redlist category and criteria (eddowes 1998). illegal logging is identified as major threat for this species, leading to severe population fragmentation and decline in the population size. such species is very sensitive to environmental changes and at greater risk of extinction. in this study, maxent was used as a tool to predict the impact of climate change on the distribution of across its native f. pimenteliana environment. this study was aimed to: (1) predict current potential distribution of in f. pimenteliana indonesian papua and papua new guinea; (2) identify the environmental factors associated with habitat distribution of and (3) f. pimenteliana estimate the impact of climate change to the future potential distribution of the tree. the results of this study may serve as a basis in developing long term adaptation strategies for assisting in dealing with climate f. pimenteliana change. materials and methods target species and occurrence data flindersia pimenteliana f. muell. (synonym: f. chrysantha merr. & l.m. perry and f. mazlini f.m. bailey) (rutaceae) is a tree having height up to 40 m. this plant species can be a canopy or subcanopy tree. its habitat ranges from near sea level to 1,300 m asl. the tree has a straight, cylindrical trunk which can be unbranched for up to 20 m and its diameter can reach 100 cm (conn & damas 2006). f. pimenteliana is heavily exploited from the wild due to its good quality timber, which is suitable for wood craft, furniture, moldings and interior construction purposes (purnawati 2013). there had been several efforts to plant this tree in order to minimize timber harvesting from the wild. however, due to the trunk and canopy large sizes, it is economically impossible to grow this tree in plantation until reaching the same good timber quality as it is from the wild (australian tropical rainforest plants, http://keys.trin.org.au). based on population assessment in indonesian papua and papua new guinea, the iucn classifies f. pimenteliana as endangered species, without considering population of this species in queensland (australia). occurrence data of f. pimenteliana were obtained from the global biodiversity information facility (gbif) (www.gbif.org), an open access data portal that provides rich information about the known presence of organisms. a total of 74 records with geographical coordinate were initially obtained from gbif. this low numbers of occurrences were mainly due to lack of occurrence information from indonesian papua. the data were then filtered for duplicate and autocorrelated occurrence points using spatially rarefy occurrence data tool in sdmtoolbox (brown 2014). the tool removes spatial cluster of localities according to climate heterogeneity. for this purpose, the tool was set as having two classes, natural breaks classification type and 10 km minimum distance. the resulting data were composed of 23 unique distribution records which were then used for building the model. this filtering method can maximize the number of spatially independent localities and can enhance model performance by removing over-fit of the models towards environmental bias (veloz 2009; hijmans 2012; boria et al. 2014). creation of bias file maxent typically selects background points from a large area and compares them with the present data to differentiate between the suitable and unsuitable environmental conditions. van der wal et al. (2009) showed that model 60 biotropia vol. 24 no. 1, 2017 this purpose, pairwise pearson correlation coefficient of current bioclimatic data was calculated using sdmtoolbox and r ≤ ±0.9 was used as a cut-off threshold to determine the exclusion of highly correlated variables (table 1). the resulting variable set was composed of 9 predictors (table 2). variables commonly used in ecological studies and best represented the original input of climate data (bio1, bio2, and bio12) as well as those with the least correlation to others were retained in the model. together with elevation data, these variables were considered to be the representatives of predictor candidates and were assessed through maxent's jackknife test. as a consequence, variables that contributed < 1.0% were eliminated and the final explanatory variables obtained were then used to build maxent models for (table 2).f. pimenteliana future climate projections to pred ic t fu ture d i s t r ibut ion of f. pimenteliana, downscaled and calibrated global circulation model (gcm) of hadgem2-es (hadley centre global environment model, version 2-earth system) and miroc-esm (model for interdisciplinary research on climate-earth system models) for 2050 (average for 2041 2060) and 2070 (average for 2061 2080) were used (hijmans 2005; et al. http://www.worldclim.org). these data were among the most recent gcm climate projections that were used in the fifth assessment intergovernmental panel on climate change (ipcc) report. each of these climate models had 30 arc-seconds resolution and projected two representative concentration pathway (rcp) emission scenarios, namely rcp4.5 and rcp8.5. while the rcp4.5 represented low emission scenario, the rcp8.5 was a scenario of high greenhouse gas emissions and represented the worst case scenario of climate model simulation in the fifth assessment ipcc report (see riahi (2011) for more et al. details of the scenarios). final models for future habitat prediction were obtained by averaging results from hadgem2-es and miroc-esm future climate models. performance was lower when background points were selected from a large area. this happened because the background points selected, which were very distant from all presence points, were more likely to show environmental conditions that were very different from those for the presence data (anderson & raza 2010; barbetmassin et al. 2012). thus, the larger the study area, the higher proportion of less informative background points is included in the model. to overcome this problem, a bias file was created in the present study to restrict background point selection and hence, increase the model performance. using buffered minimum-convex polygon tool in sdmtoolbox, the bias file was built with maximum radial distance of 10 km from the occurrence points. selection of environmental variables the elevation and current bioclimatic data set was obtained from the shuttle radar topography mission (srtm) global elevation data (http:// srtm.csi.cgiar.org/) and worldclim 1.4 database (hijmans et al. 2005; http:// www.worldclim.org), respectively. the current bioclimatic data (nix 1986) consisted of 19 variables and were derived from monthly rainfall, while the temperature data were obtained from weather stations across the globe within the period of 1950 2000. these data showed annual trends, seasonality and extreme environmental factors; and were frequently used in predicting species distribution. hence, a total of 20 environmental variables were initially considered for model building. all the environmental layers used in the model had 30 arc-seconds or ≈1 km resolution. these layers were clipped to the indonesian papua and papua new guinea political boundaries and then converted to ascii raster files using esri arcmap 10.1. due to the limitation of data availability, the present study did not include other important variables influencing the distribution of plant species, such as soil characteristics, distance from water bodies and groundwater table. to reduce model overfitting and minimize high co-linearity, highly correlated bioclimatic variables were removed from the model. for 61 impact of climate change on flindersia pimenteliana – robiansyah 62 biotropia vol. 24 no. 1, 2017 b io 1 b io 2 b io 3 b io 4 b io 5 b io 6 b io 7 b io 8 b io 9 b io 10 b io 11 b io 12 b io 13 b io 14 b io 15 b io 16 b io 17 b io 18 b io 2 -0 .6 1 b io 3 -0 .1 7 0. 31 b io 4 -0 .1 1 0. 01 -0 .9 0 b io 5 0. 98 -0 .4 9 -0 .2 5 b io 6 0. 99 -0 .7 0 -0 .1 5 -0 .1 5 0. 95 b io 7 -0 .5 4 0. 89 -0 .1 6 0. 45 -0 .3 8 -0 .6 5 b io 8 0. 99 -0 .5 8 -0 .2 0 -0 .0 8 0. 98 0. 98 -0 .5 0 b io 9 0. 99 -0 .6 3 -0 .1 2 -0 .1 6 0. 96 0. 99 -0 .5 9 0. 97 b io 10 1. 00 -0 .6 1 -0 .2 3 -0 .0 5 0. 99 0. 98 -0 .5 1 0. 99 0. 98 b io 11 1. 00 -0 .6 0 -0 .0 9 -0 .2 0 0. 97 0. 99 -0 .5 7 0. 99 0. 99 0. 99 b io 12 -0 .0 7 0. 08 0. 13 -0 .0 4 -0 .0 6 -0 .0 5 0. 02 -0 .1 2 0. 01 -0 .0 7 -0 .0 7 b io 13 0. 07 0. 04 0. 00 0. 02 0. 10 0. 07 0. 04 0. 03 0. 12 0. 07 0. 07 0. 87 b io 14 -0 .1 6 0. 05 0. 25 -0 .1 3 -0 .1 7 -0 .1 2 -0 .0 8 -0 .2 0 -0 .0 7 -0 .1 7 -0 .1 4 0. 90 0. 63 b io 15 0. 23 -0 .0 4 -0 .4 1 0. 26 0. 27 0. 17 0. 16 0. 26 0. 14 0. 25 0. 20 -0 .5 9 -0 .2 0 -0 .8 3 b io 16 0. 05 0. 04 -0 .0 3 0. 06 0. 08 0. 04 0. 06 0. 01 0. 09 0. 05 0. 04 0. 88 0. 99 0. 63 -0 .1 9 b io 17 -0 .1 3 0. 03 0. 22 -0 .1 1 -0 .1 5 -0 .0 9 -0 .0 7 -0 .1 8 -0 .0 4 -0 .1 4 -0 .1 2 0. 93 0. 66 0. 99 -0 .8 1 0. 66 b io 18 -0 .2 4 0. 34 0. 14 0. 01 -0 .1 9 -0 .2 5 0. 28 -0 .2 3 -0 .2 3 -0 .2 4 -0 .2 3 0. 76 0. 70 0. 63 -0 .3 4 0. 71 0. 64 b io 19 -0 .0 5 -0 .1 0 0. 09 -0 .0 3 -0 .0 7 0. 00 -0 .1 5 -0 .1 2 0. 06 -0 .0 5 -0 .0 4 0. 89 0. 73 0. 87 -0 .6 5 0. 73 0. 89 0. 45 t ab le 1 p ai rw is e p ea rs o n c o rr el at io n c o ef fi ci en t o f 19 b io cl im at ic v ar ia b le s us ed fo r p re di ct in g p o te n ti al d is tr ib ut io n o f f lin de rs ia p im en te lia na n o te s: v al ue s ≥ ± 0. 9 ar e sh ow n in b o ld m ea n in g o f ea ch v ar ia b le c an b e re fe rr ed t o t h e w o rl dc lim w eb si te ( h tt p :/ /w w w .w o rl dc lim .o rg ) 63 t ab le 2 p er ce n ta ge c o n tr ib ut io n o f en vi ro n m en ta l v ar ia b le s to th e m ax en t m o de ls fo r f lin de rs ia p im en te lia na p o te n ti al d is tr ib ut io n . v ar ia b le s ex cl ud ed fr o m th e fi n al m o de l a re a ls o lis te d an d th us h av e p er ce n t co n tr ib ut io n o f 0. a ls o s h ow n f o r ea ch v ar ia b le a re m ea n s an d st an da rd d ev ia ti o n s va lu es f o r cu rr en t an d fu tu re c lim at e (2 05 0 an d 20 70 ) un de r r ep re se n ta ti ve c o n ce n tr at io n p at h w ay (r c p ) 4 .5 a n d r c p 8 .5 s ce n ar io s v ar ia b le s p er ce n t co n tr ib ut io n c ur re n t 20 50 20 70 r c p 4. 5 r c p 8. 5 r c p 4. 5 r c p 8. 5 p re ci p it at io n in t h e w et te st m o n th (b io 13 ) 37 .6 35 2. 5 ± 8 5. 5 37 0. 1± 6 9. 3 37 2. 2 ± 9 6. 0 37 4. 9 ± 9 8. 2 37 8. 8 ± 1 02 .5 e le va ti o n 28 .8 55 0. 6 ± 7 91 .4 55 0. 6 ± 7 91 .4 55 0. 6 ± 7 91 .4 55 0. 6 ± 7 91 .4 55 0. 6 ± 7 91 .4 p re ci p it at io n in t h e co ld es t qu ar te r (b io 19 ) 14 .3 64 2. 4 ± 3 37 .1 62 4. 4 ± 3 48 .9 63 8. 6 ± 3 56 .1 65 7. 4 ± 3 5. 3 65 2. 8 ± 3 72 .9 p re ci p it at io n in t h e w ar m es t qu ar te r (b io 18 ) 9. 9 83 6. 4 ± 1 86 .9 79 4. 4 ± 1 93 .5 76 5. 3 ± 1 94 .0 79 9. 8 ± 2 01 .9 76 6. 8 ± 2 26 .3 is o th er m al it y (b io 3) 7. 2 83 .2 ± 5 .7 82 .8 ± 6 .0 82 .6 ± 5 .5 82 .0 ± 6 .2 81 .9 ± 6 .2 m ea n d iu rn al r an ge in t em p er at ur e (b io 2) 2. 2 86 .4 ± 1 2. 9 86 .7 ± 1 3. 9 90 .6 ± 1 5. 3 85 .7 ± 1 3. 2 87 .3 ± 1 5. 0 a n nu al m ea n t em p er at ur e (b io 1) 0 24 0. 8 ± 3 8. 9 25 7. 6 ± 3 8. 9 26 1. 1 ± 3 8. 8 26 2. 7 ± 3 8. 6 27 2. 6 ± 3 8. 7 t em p er at ur e an nu al r an ge ( b io 7) 0 10 3. 4 ± 1 4. 6 10 4. 4 ± 1 6. 5 10 9. 5 ± 1 9. 3 10 4. 5 ± 1 2. 3 11 0. 3 ± 2 1. 3 a n nu al p re ci p it at io n ( b io 12 ) 0 30 69 .2 ± 8 62 .5 30 58 .7 ± 8 94 .6 27 34 .4 ± 7 90 .6 33 50 .6 ± 9 90 .4 30 54 .6 ± 9 80 .7 p re ci p it at io n s ea so n al it y (c o ef fi ci en t o f va ri at io n ) (b io 15 ) 0 25 .4 ± 1 4. 3 29 .3 ± 1 5. 9 31 .2 ± 1 5. 4 28 .9 ± 1 6. 2 31 .3 ± 1 6. 7 impact of climate change on flindersia pimenteliana – robiansyah the potential range of loss and gain of f. pimenteliana was calculated for the year of 2050 and 2070. to determine whether the species assemblages would be constant or change in the future compared to the current potential habitats, the turnover rate ( ) was t calculated using the following formula (hu et al. 2010): where: t = species turnover rate g = species gain l = species loss sr = current species potential distribution t value ranges from 0 to 100, where value of 100 indicates that the species assemblages are different, while value of 0 indicates that the species assemblages are similar with previous conditions (trisurat 2011).et al. model development and evaluation the maximum entropy method, as implemented in maxent (version 3.3.3k), was used to model the potential distribution range of f. pimenteliana. to validate and calibrate the model, the maxent modeling was run through spatially jackknife tool of sdmtoolbox. model validation was done using geographically structured threefold cross-validation method. furthermore, the tool tested different combinations of five model feature class types (1=linear; 2=linear and quadratic; 3= hinge; 4=linear, quadratic, and hinge; and 5=linear, quadratic, hinge, product, and threshold) to optimize the model performance. each of these model parameter classes was run in five replicates. finally, the best model was automatically selected by evaluating the omission rate, area under the curve (auc) and model feature class complexity of each model. to measure the importance of environmental variables, the procedure was used spatially jackknife which created response curves. the best model selected was used to run the final model using all of the occurrence points. the model was also projected to the future climates of 2050 and 2070 using rcp4.5 and rcp8.5 emission scenarios. maximum training sensitivity plus specificity logistic threshold was used to convert the continuous suitability index into suitable and unsuitable areas for f. pimenteliana. the predicted suitable areas were then clipped by 25 m resolution palsar-2/palsar forest cover of 2015 obtained from the japan aerospace exploration agency (www.eorc.jaxa.jp). results and discussion model performance and environmental variable responses although there are some drawbacks attributed to maxent, such as overfitting, model complexity dependency and independent evaluation data requirement (radosavljevic & anderson 2014), its user-friendliness has attracted many application in sdms. furthermore, maxent has been shown to perform better than other models for analyzing presence-only data. the model also has the best predictive power even with very low sample size (wisz et al. 2008). in the present study, using 23 occurrence data, the maxent model developed for predicting the potential distribution of f. pimenteliana was significantly better than random expectations. the average test auc for the replicate runs was 0.710 (sd = 0.043). peterson et al. (2011) argued that the auc value of 0.7 0.9 indicated moderate performance of the model. the present study was the first attempt on building sdms to predict potential current and future distribution of f. pimenteliana in indonesian papua and papua new guinea. after removing highly correlated bioclimatic variables and those with < 1% contribution, there were 6 variables identified as being important in creating model fit for (table 2). f. pimenteliana precipitation-related variables (bio13, bio18 and bio19) had the most influence to f. pimenteliana distribution. these variables together contributed 61.8 % to the model. on the other hand, temperature-related variables (bio3 and bio2) together contributed only 9.4% to the model. furthermore, elevation was also important variable for distribution with 28.8% f. pimenteliana contribution. the significant role of precipitation on tree species distribution in tropical forests was well documented (baltzer 2008; brenes-et al. arguedas 2009; baltzer & davies 2012). soil et al. water availability affected by precipitation is a direct determinant of tropical trees distributions in both local and regional scales (engelbrecht et al. 2007; toledo 2012)et al. . 64 biotropia vol. 24 no. 1, 2017 t = 100 × g+ l sr + g response curves of the top four of highly contributed variables for f. pimenteliana were shown in figure 1. habitats with elevation between 400 – 1,500 m had high potential suitability for the species. furthermore, habitats with precipitation of the wettest month between 160 300 mm (bio13) had high suitability for sustaining f. pimenteliana. in addition, the species preferred habitat having precipitation of the coldest quarter (bio19) and the warmest quarter (bio18) between 100 400 mm and 360 760 mm, respectively. habitat suitability of f. pimenteliana decreased with the increasing value of the precipitation-related variables. this pattern is understandable as, according to schuur (2003), increased precipitation in humid tropical forests is known to have negative effect on plant growth and net primary productivity. the author argued that high rainfall in humid ecosystem may reduce plant growth and productivity by decreasing radiation inputs, increasing nutrient leaching or reducing soil oxygen availability. prediction of current distribution using maximum training sensitivity plus specificity logistic threshold (0.396), model prediction of the current distribution identified a 2 total area of 156,214 km (18% of total land area) as suitable habitat for f. pimenteliana. liu et al. (2013, 2016) suggested that for presence-only data, maximum training sensitivity plus specificity logistic threshold can be used confidently for threshold selection as it produced higher sensitivity compared to other methods. most of the suitable habitats were observed in mountainous regions and southern coastal areas of the main island where the precipitation intensity was relatively low (fig. 2). in indonesia, potentially suitable habitats for f. pimenteliana were mostly located along mountain chains of indonesian papua, extending from the mountain ranges in sorong regency to the jayawijaya mountains in pegunungan bintang regency. there were also relatively separated suitable 65 figure 1 response curves of the top four of highly contributed variables for maxent model of flindersia pimenteliana. the red center line represents the mean values derived from the cross-validation runs, while the blue curve delineates the standard deviation. variable definitions: bio13, elevation (m), bio19 and bio18 represent precipitation (mm) of the wettest month as well as the coldest and warmest quarters, respectively impact of climate change on flindersia pimenteliana – robiansyah l o g is ti c o u tp u t (p ro b a b il it y o f p re se n ce ) 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 100 200 300 400 500 600 700 800 bio13 elevation 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 bio19 bio18 400 600 800 1000 1200 1400 1600 18002000150010005000 regions for such as in waigeo island f. pimenteliana in raja ampat regency, fakfak mountains in fakfak regency, kumawa mountains in kaimana regency and coastal areas in the southern region of merauke regency. for papua new guinea, most of the potentially suitable habitats were observed to be along mountain regions in eastern highlands and owen stanley range. furthermore, suitable habitats were also detected in southwestern corner of the western province, coastal areas of east sepik province, east part of new britain island and new ireland island. very little is known about the habitat distribution of f. pimenteliana in indonesia as study examining this species is very scarce. recent study conducted by purnawati et al. (2012) and purnawati (2013) reported that the species was observed in teluk cendrawasih national park, teluk wondama regency, which was also included as a suitable habitat in the present model prediction. further surveys in other predicted distribution areas of the plant are needed to validate the prediction of the present study. for papua new guinea, some of the predicted areas agree with areas mentioned in the specimen records of f. pimenteliana held by the royal botanic garden of sydney (http://plantnet. rbgsyd.nsw.gov.au). these areas included many localities in morobe province (more than 53 records), eilogo in central province, bewani in west sepik province, mount obree and toma village in northern province, mount kilkerran and rabaraba in milne bay province and goroko and aiyura in eastern highlands province. further surveys are required in other predicted areas to validate the model prediction, especially in western province where specimen record of f. pimenteliana is absent. in addition, new surveys in previously known areas are also considered necessary as most of the records mentioned above are very old (some records are dated back to 1923). protected areas are one of the most important tools in plant conservation. in the present study, most of the predicted suitable habitats of f. pimenteliana were located outside the existing protected areas (fig. 3). only 24,393.9 km 2 (15.6%) of these habitats were currently covered by terrestrial protected areas in indonesian papua and papua new guinea. as can be seen in figure 3, protected areas with high coverage of predicted suitable habitats for are mostly f. pimenteliana found in indonesia, including tambrauw mountain nature reserve, lorenzt national park, jayawijaya nature reserve, enarotali nature reserve and kumawa mountain nature reserve. to protect and conserve , expansion f. pimenteliana of existing protected areas or establishing the new one is necessary to cover the predicted suitable areas as much as possible. this action is especially needed in papua new guinea where only small percentage of the predicted suitable habitats is covered by the existing protected areas. although the present study was able to predict suitable habitats of with high f. pimenteliana success rate, care should be taken when implementing the results in the field-based conservation programs. since maxent estimates the fundamental niche of a species, the predicted distribution presented in this study might be 66 biotropia vol. 24 no. 1, 2017 figure 2 predicted potential distribution of flindersia pimenteliana for current (2016) and future climate conditions (2050 and 2070) under rcp4.5 and rcp8.5. future habitat predictions were obtained by averaging results from hadgem2-es and miroc-esm future climate models overestimated (pearson 2007). the model does not consider other factors influencing the distribution of plant species such as dispersal process, anthropogenic influences, biotic interactions or geographic barriers (pearson 2007; soberón 2007). prediction of future distribution future distribution of f. pimenteliana predicted by hadgem2-es and miroc-esm model generally had low agreement in papua new guinea compared to that in indonesian papua. standard deviation between the two models was 67 figure 3 current (2016) potential distribution of flindersia pimenteliana and existing protected areas in papua and papua new guinea according to world database on protected areas (wdpa, http://www.protectedplanet.net/) figure 4 standard deviation of predicted probabilities of flindersia pimenteliana occurrence from hadgem2-es and miroc-esm future climate models. black dots are occurrence data used in modeling impact of climate change on flindersia pimenteliana – robiansyah higher in papua new guinea, especially in regions where no sample data were recorded (fig. 4). based on the average value of the models, the suitable habitat for f. pimenteliana was predicted to decline by 3% for rcp4.5 and 2% for rcp8.5 in the year 2050. the suitable habitat gains for the two rcps were 7% and 16%, respectively. hence, the percentage turnover in 2050 was estimated to be 41% and 54% for rcp4.5 and rcp8.5, respectively (table 3). for 2070, the suitable habitat for the species was predicted to decline by 4% for both rcp4.5 and rcp8.5, whereas the habitat gains were 7% and 8%, respectively. therefore, the percentage turnover in 2070 was estimated to be 44% for rcp4.5 and 48% for rcp8.5 (table 3). under rcp4.5, the species was predicted to gain suitable habitats in southern and western highland of papua new guinea in 2050. this gain was relatively stable and still could be observed in 2070. higher suitable habitat gain was observed under rcp8.5 in 2050. this gain, however, was not stable and greatly decreased in 2070 (fig. 2). in contrast, the suitable habitat along jayawijaya mountains, coastal area of merauke regency and east sepik province, and the most east of owen stanley range in papua new guinea were predicted to disappear in 2050. these habitat losses were similar for both rcps and relatively stable until 2070 (fig. 2). since the species had turnover rate of more than 30%, major shift in distribution was predicted to occur in the future for both rcps. while some of predicted suitable habitats in southern coastal regions disappeared, mountainous areas located in the middle of papua new guinea were predicted to become suitable habitats for f. pimenteliana in 2050 and 2070. changes in timing and seasonality of rainfall in the future may be responsible for this range shift. australian bureau of meteorology and csiro (2011) predicted rainfall pattern change in papua new guinea with more than 15% increase in annual and seasonal rainfall by 2090. this range shift of geographic distribution towards higher elevation is also observed in many terrestrial organisms and is commonly linked to increased growth and dispersal success due to warmer climate (chen et al. 2011). although the temperature was predicted to increase at 0.11 °c per decade over papua new guinea (australian bureau of meteorology and csiro 2011) and in the range of 0.2 0.3 °c per decade over indonesian papua (boer & faqih 2004), the present study, however, was unable to detect this correlation as all temperature-related variables had little effect to the model. conclusions it is likely that f. pimenteliana will benefit from climate change, as environmentally suitable ranges for this species are projected to increase by 2050 and 2070. the present study predicted major range shift of geographic distribution for the species towards higher elevation. the newly suitable habitats were predicted to be found mainly in southern and western highland of papua new guinea. these are the areas where the protection of the species might be the most feasible and cost-effective in the future. thus, protection of these areas from habitat destruction and land use changes is needed to make sure that f. pimenteliana can adapt to the climate change. further field surveys are required to validate the prediction of the present study. furthermore, it is recommended to obtain additional occurrence data and include other important predictors (e.g. edaphic factors) to minimize uncertainty in climate projections. 68 biotropia vol. 24 no. 1, 2017 table 3 predicted gain, loss and total suitable habitats, and turnover rate of flindersia pimenteliana for future climate conditions (2050 and 2070) under representative concentration pathway (rcp) 4.5 and rcp 8.5 2050 2070 rcp4.5 rcp8.5 rcp4.5 rcp8.5 suitable (km2) 195,058 275,956 188,578 190,397 gain (km2) 64,516 140,551 64,818 71,940 loss (km2) 25,671 20,810 32,454 37,757 turnover (%) 41 54 44 48 references anderson rp, raza a. 2010. the effect of the extent of the study region on gis models of species geographic distributions and estimates of niche evolution: preliminary tests with montane rodents (genus nephelomys) in venezuela. j biogeogr 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91 doi: 10.11598/btb.2021.28.1.1159 harvesting time and viability of ixora coccinea 'dwarf red coccinea' pollen saowaros phanomchai', i(itt1 bodhipadmai, sowoch noichindai, luepol punnai(anta2 and david w.m. leung3* 'division ofagro-industrial technology, facalg *plied science, king mongkat's university o f technology no& bangkoh bangsue, bangkok 10800, thailand 2faculty o f environment and resource stua'ies, mahidol universig, salaya, nakhon pathom 73 170, thailand 'school ofbiological sciences, universig o f canterbuy, christchurch 8 140, new zealand received 1 december 2018/accepted 14 february 2020 abstract the cultivated variety of the non-native i. coccinea, dwarf red coccinea @rc), is most popular and widely spread all over thailand. however, knowledge about its pollen morphology and fertility for plant breeding purposes, is limited. this study aimed to investigate the quantity, viability and germinability of pollen grains collected from the flowers of drc at different times on a summer day, particularly from 8 am to 4 pm. pollen quantity was determined using a haemacytometer while its viability and germinability were examined after staining with 1% acetocarmine and allowing the pollen to germinate on a modified agar-gelled germination medium. the pollen collected at 10 am had the highest pollen density (53.3x104 pollen/ml) and viability percentage (72.05'/0). when these pollen were allowed to germinate on an artificial medium supplemented with various sucrose concentrations, the highest in vitro pollen germinability was found at the medium containing 10% sucrose. hence, the best time to collect the i. coccinea, cv. 'dwarf red coccinea' pollen was at 10 am. however, further investigations are recommended on the effects of daily or hourly environmental changes particularly, ambient temperature and humidity, on the quantity and quality of harvestable pollen as well as on the pistil phenology, to develop a more complete breeding strategy for the ixora species. keywords: plant breeding, pollen fertility, rubiaceae introduction ixora is one of the pantropical genera in the rubiaceae farmly comprising at least 500 species (mouly e t al 2009). among the 28 cultivated varieties, the non-native i. coccinea or dwarf red coccinea (drc) is the most popular and widely grown all over thailand. it is used as flower bed border, living fence, pot plant or individual shrub (puff et al 2005; mouly e t al: 2009; chamchumroon 2014). drc has a superior character as a year-round and non-stop bloomer. its weak characters include a requirement of full sudght to partial shade and excellent drainage in pots. both the quantity and quality of pollen collected from flowers are important in plant breedmg (ashman et al 2004; colling et al *corresponding author, e-mail: david.leung@canterbury.ac.nz 2004). both the abiotic (humidity and temperature) and biotic (pollinator) components of the environment also influence the pollen amount and viability of the flowering plant (aronne 1999; de luca e t al. 2013). hence, it is of fundamental interest to study pollen. few studies have focused on pollen morphology of ixora spp. (de block & robbrecht 1998; sreekala et al 2003). however, the pollen quantity and quality of i. coccinea, cv. dwarf red coccinea, have never been described. thus, the objective of this study was to determine the morphology, and changes in viabhty, density, and germinability of drc pollen collected at different times of the day when the flowers were in full bloom. under a light microscope, the pollen of this species has a generally prolate shape which is different from that of i. congesta and i. arborea. ixora pollen morphology and fertility phanomchai et al, materials and methods plant material inflorescences were collected from the blooming ixora coccinea, cv. dwarf red coccinea (drc) plants at the garden of king mongkut's university of technology, north bangkok at 2 hour intervals from 8 am to 4 pm on a sunny day in three consecutive weeks of march, the summer season in thailand. pollen size, shape and viability the drc flowers were randomly selected at different times of the day (8 am, 10 am, 12 midday, 2 pm and 4 pm). the pollen grains were released by holding each flower upside down over a glass slide and by tapping (fig. 1). overall, pollen from 20 anthers from 5 different plants of different populations were placed on a glass slide for observation of pollen size and shape under a light microscope. the pollen viability was then investigated by staining with 1% (w/v) acetocarmine. the unstained pollen grains were non-viable whde the red stained grains were considered viable. before and after pollen staining, its shape was clarified by using the p/e ratio (punt e t al. 2007; hesse e t al. 2009). all pollen grains were randomly selected with 50 and 30 replications to determine pollen size and viability, respectively. pollen density the density (or quantity) of drc pollen grains was investigated at different times (8 am, 10 am, 12 am, 2 pm and 4 pm) using a modified method based on bunderson et al. (2012). pollen grains from ten flowers were placed in a microcentrifuge tube. some 60 pl glycerol and 40 pl distilled water were then added into the tube and were mixed for 30 s using a vortex mixer. then, 8 pl of the pollen suspension was placed in a haemacytometer (improved neubauer rulings, boeco, germany) using a micropipette. the pollen grains were then covered with a glass and spread over the grid which was divided into nine large squares. only the pollen grains in the grid number 1, 2, 3 and 4 were counted (fig. 2). those pollen grains that touched the line on the bottom and right of the grid were omitted from counting. figure 1 ixora coccinea, cv. 'dwarf red coccinea' flower notes: a = top view (an arrow pointed at an anther); b = side view (bar = 1 cm). figure 2 grid layout of haemacytometer illustrating the position of number 1, 2, 3 and 4 pollen grains that were counted (modified from legresley and mcdermott, 2012) notes: the formula for pollen density calculation (legresley and mcdermott, 2012) is as follows: the average number of pollen per ml = [@ollen number in chamber 1+2+3+4)/4] x lo4 = average count per large square x lo4; data from this experiment were collected from 6 replications. biotropia vol. 28 no. 1,2021 pollen germination results and discussion in vitro pollen germination test was carried out using the modified mercado e t al. (1994) medium which consisted of 0.1 mm boric acid, 1 mm calcium chloride and various sucrose concentrations [o, 5, 10 and 20% (w/v)]. the medium was adjusted to ph 5.7, gelled with 0.9% (w/v) agar and sterilized at 121 oc, 15 psi for 20 min. later, pollen grains from ten flowers were brushed over the surface of the germination medium. the grains were incubated at 25k5 "c for 24 h in a dark room. pollen grains were considered to have germinated when pollen tube length was twice longer than the diameter of pollen grain. percentages of germination data were averaged from 20 replications. scanning electron microscope analysis pollen samples were collected at 10 am and sent to the scientific and technological research equipment centre, chulalongkorn university for morphological analysis using a scanning electron microscope (sem-eds, model jsm-6610lv, jeol ltd., tokyo, japan). data analysis anova of all data were carried out and the mean values in pollen size, viability, density and germination were compared using the duncan test at p < 0.05. one of the characters used to describe and identify a pollen grain of a flowering plant is its size and shape. for this purpose, measurements of the polar axis q and equatorial axis q have been broadly used (groppo et a/. 2010; chwil 2015). in the case of i. coccinea, cv. dwarf red coccinea (drc), most pollen grains were probably shed from the anther before 4 pm as there was insufficient number (less than 10 grains per flower) by that time. therefore, pollen at this time was excluded in the experiments. under the light microscope, the drc pollen had polar axis longer than equatorial diameter and the p/e ratio was around 1.80 to 1.94 before staining (table 1). pollen with a polar axis longer than equatorial diameter or p/e ratio of 1.33-2.00 was described as prolate punt e t al. 2007; hesse et al. 2009). moreover, the largest diameter was generally used for specifying the size of pollen. pollen diameter between 26 and 50 pm was categorized as a medum size (hesse e t al. 2009). thus, the drc pollen from 8 am to 2 pm was generally prolate in shape and had a me&um size before staining (fig. 3; table 1). when compared with other ixora species observed under the light microscope, the shape of drc pollen was dissimdar to those of ixora conge& (suboblate) and i. arborea (oblate spheroidal) (ibrahim et al. 2012; prabhakar & ramakrishna 2014). table 1 pollen size and shape of ixora coccinea, cv. 'dwarf red coccinea' (under a light microscope) at different times before staining length of the axis (pm) time p/e ratio shape p e 8 am 39.4 k 0.210b 21.8 k 0.238a 1.8 k 0.012b prolate 10 am 39.6 k 0.261b 22.0 k 0.178a 1.8 k 0.008b prolate 12 am 39.1 k 0.169b 20.2 k 0.130~ 1.9 k 0.011a prolate 2 pm 40.2 k 0.135a 21.0 k 0.176b 1.9 k 0.014a prolate notes: values are means of 50 replications k se. data marked by the same letter in a column are not significantly different (p < 0.05). ixora pollen morphology and fertility phanomchai et al, figure 3 pollen morphology of ixora coccinea, cv. 'dwarf red coccinea' before staining note: bar = 20 ym. to examine more closely, the drc pollen were also investigated under a scanning electron microscope. the polar shape was tri-lobulate and the equatorial shape of this monad pollen was prolate (fig. 4). the grain had tricolpate aperture and psilate-perforate sculpturing. this was different from i. congesta whose pollen had suboblate shape, microreticulate sexine ornamentation, pericolpate aperture and quadrangular o u h e (ibrahim et al. 201 2). these results suggested that pollen from the genus ixora may have divergent forms in different species. pollen staining with acetocarmine is one of the most widely used technique in estimating pollen viability (malayeri e t al. 2012). after the drc pollen grains were hydrated with 1% (w/v) acetocarmine, the shape of the dry pollen changed from prolate to spheroidal (fig. 5) and the diameter was around 32.6 36.7 pm (table 2). moreover, the viable pollens, as manifested by those exhibiting acetocarmine staining, were most numerous at 10 am (72.05%) (table 2). in another study, (sreekala et al. 2003) the anther dehscence in ixora agastlyamaiqana occurred at 7 am to 1 pm and the peak anther dehiscence was also at 10 am. in the current research, the experiment was done until 4 pm when there was a noticeable decline in pollen quantity and quality. figure 4 polar (a) and equatorial (b) views of ixora coccinea, cv. 'dwarf red coccinea' pollen under a scanning electron microscope biotropia vol. 28 no. 1,2021 figure 5 pollens of ixora coccinea, cv. 'dwarf red loccmea arter stamng wtn 170 (w/ v) acetocarmine notes: pollens were collected at different times: a) 8 am, b) 10 am, c) 12 noon; and d) 2 pm. bar = 20 pm. table 2 pollen density, diameter and viability after staining of ixora coccinea, cv. 'dwarf red coccinea' at different times time diameter' ('pm) viability2 (oh) density3 (pollen per rnl) 8am 36.4 f 0.5a 61.8 f1.7b 8.9x104f 4.6x103c 10 am 36.7 + 0.3a 72.0 + 1.0a 5 3 . 3 ~ 1 0 ~ + 0.2x103a 12 am 36.6 + 0.3a 60.7 + o.6b 18.7x1o4 + 3.8x103b 2 pm 32.6 + o.lb 36.0 + 1 . 0 ~ 7.7x104+ 1.9x1o3c notes: 'values are means of 50 replications f se; 2values are means of 30 replications fse; 3values are means of 6 replications fse. data marked by the same ietter in a column are not significantly different (p < 0.05). although the thai rubiaceae plants are mainly dependent on animal-assisted pollination (puff etal. 2005), the best time to collect highly viable pollen of i. coccinea, cv. 'dwarf red coccinea7 for artificial breeding would be at 10 am. this was also the time of the day that the highest number of pollen per ml (53.292~10~) was collected (fig. 6; table 2). possibly, the pollen density was reduced after 10 am because of the continuous shedding of pollen from the anther to the external environment. pollen numbers are effectively counted using a haemacytometer (godini 1981; kelly e t al. 2002; bunderson e t al. 2012), and in the current study, a neubauer improved haemacytometer was used to successfully determine the pollen density. density of pollen collected at different times throughout the day was rarely investigated. considering the findings of the present study, it is possible that not only different amounts of pollen are produced at different flowering seasons (piotrowska 2012; peel e t al. 2014), but also different quantities of pollen may be obtained at different times of the day. hence, further studies are recommended to investigate the effects of environmental conditions particularly, ambient temperature and humidity of pollen shedding/collection at different times of the day. figure 6 pollen density of ixora coccinea, cv. 'dwarf red coccinea' on a neubauer improved haemacytometer notes: pollens were collected at different times: a) 8 am, b) 10 am; c) 12 noon; and d) 2 pm; all figures from grid a. ixora pollen morphology and fertility phanomchai et al. since little information is available on the germination potential of i. coccinea, cv. 'dwarf red coccinea' pollen, the pollen collected at 10 am was used in this study to determine the effect of different sucrose concentrations on pollen germination on an agar medium (modified based on mercado e t al. 1994). the highest percentage of pollen germination (about 34%) occurred at the medium containing 10% sucrose (table 3). at higher sucrose concentrations (20% sucrose) fewer than 10% of pollen germinated. this is consistent with other studies showing that sucrose concentration is an important factor for in vitro pollen germination (fig. 7; table 3). the ixora pollen may lose their viability quickly after collection. the fresh pollen exhibited 72% viability as revealed by acetocarmine staining. however, during in vitro germination, more pollen could have lost viability. another possibility is that sucrose may not be the only factor to enhance ixora pollen germination. to increase the germinability, other factors (for example, boric acid concentrations, fragdah et al 2019) could also be studied in the future. the optimal sucrose concentration for in vitro pollen germination appears to depend on the plant species. for example, cannonball tree pollen needed a high level of sucrose (20% w/v), while two forms of day-blooming native thai waterlily responded very well on a low sucrose concentration (5% w/v) (bodhipadma e t al 2013; 2016). in contrast, the germination percentage of drc pollen was sharply reduced at sucrose concentrations that were lower and higher than 10%. table 3 percentage of ixora coccinea, cv. 'dwarf red coccinea' pollen germination at 10 am on modified mercado et all (1994) medium supplemented with different sucrose concentration sucrose concentrations germination (yo) 0.0 + o.od 3.2 f 0 . 5 ~ 33.8 f 1.7a 6.6 f 0.56b notes: values are means of 20 replications + se. data marked by the same letter in a row do not significantly differ (i? < 0.05). figure 7 ixora coccinea, cv. 'dwarf red coccinea' pollen germination on modified mercado et al. (1994) medium notes: a = 0%; b = 5%; c = 10%; d) 20% (w/v) sucrose. bar = 50 pm. biotropia vol. 28 no. 1,2021 conclusion the quantity of i. coccinea, cv. 'dwarf red coccinea' (drc) pollen produced was related to the different production times during the day. the number of drc pollen grain peaked at 10 am which also exhbited the hghest estimated viability based on 1% (w/v) acetocarmine staining and density measurement with a neubauer improved haemacytometer. this is probably related to the varying environmental conditions like ambient temperature and hurni&ty at different times of the day of pollen collection. moreover, the optimal sucrose concentration of 10% (w/v) was essential for the drc pollen germination. since other studies on ixora pollen were m a d y focused on pollen morphology, it would be of interest to study the effect of sucrose level on the germination of pollen from other ixora spp. another implication of the present study is that for the breeding of ixora coccinea 'dwarf red coccinea', the critical time to collect pollen would be 10 am. to develop a more complete breeding strategy, further study is recommended on the pistil phenology of this species and other related species, and on the effect of temperature and humidity on ixora. references aronne g. 1999. effects of relative humidity and temperature stress on pollen viability of cistas incanw and mjrtus communis. grana 38:364-7. ashman tl, iolight mi,, steets ja e t al. 2004. pollen limitation of plant reproduction: ecological and evolutionary causes and consequences. ecology 85:2408-21. bodhipadma i<, noichinda s, thaiyanto p, leung dwm. 2013. morphology, viability, and germinability of pollen from two forms of nymphaea nouchali var. uersicolor: a day-blooming waterlily. science asia 39:214-8. bodhipadma i(, noichinda s, permchalad k, changbandist s, phanomchai s, punnakanta l, leung dwm. 2016. a study of cannonball trees in thailand: hood staminodes are larger than ring stamens, but only germination of stamina1 ring pollen can be stimulated by exogenous sucrose. i w t n b : ijast 9:167-73. bunderson dl, water w, wells h, levetin e. 2012. predicting and quantifying pollen production in janipems ashei forests. phytologia 94:417-38. chamchumroon v. 2014. two new species of ixora (rubiaceae) from thailand. tfb (botany) 42535-90. chwil m. 2015. micromorphology of pollen grains of fruit trees of the genus pmnus. acta sci pol-hortoru 14:115-29. colling g, reckinger c, matthies d. 2004. effects of pollen quantity and quality on reproduction and offspring vigor in the rare plant scoqonera hz~milis (asteraceae). am j bot 91 :1774-82. de block p, robbrecht e. 1998. pollen morphology of the pavetteae (rubiaceae, ixoroideae) and its taxonomic significance. grana 37:260-75. de luca pa, bussie're lf, souto-vilaros d, goulson d, mason ac, vallejo-marl'n m. 2013. variability in bumblebee pollination buzzes affects the quantity of pollen released from flowers. oecologia 172:805-16. fragallah sada, lin s, li n, ligate ej, chen y. 2019. effects of sucrose, boric acid, ph, and incubation time on in vitro germination of pollen and tube growth of chinese fir (cunnighamial lanceolata l.). forests 10:102. godini a. 1981. counting pollen grains of some almond cultivars by means of an haemocytometer. in : grempa colloque 1980, ciheam-options mediterrantennes strie etudes 1981 -i. paris (fr) : institut agronomique mtditerranten de zaragoza (iamz). p.83-6. groppo m, da cruz-barros mav, da silva correa am. 2010. pollen morphology of species of hortia (rutaceae). rev bras bot 33:13-20. hesse m, halbritter h, weber m et al. 2009. pollen terminology: an illustrated handbook. new york (us): springer wein. ibrahim if, balasundram sk, abdullah nap, alias ms, mardan m. 2012. morphological characterization of pollen collected by apis dorsata from a tropical rainforest. int j botany 8:96-106. kelly ji<, rasch a, i m s z s. 2002. a method to estimate pollen viability from pollen size variation. am j bot 89:1021-3. legresley m, mcdermott g. 2012. counting chamber methods for quantitative phytoplankton analysis haemocytometer, palmer-maloney cell and sedgewick-rafter cell. in: icarlson b, cusack c, bresnan e, editors. microscopic and molecular methods for quantitative phytoplankton analysis (ioc manuals and guides, no. 55). paris (fr): unesco. p.25-30. malayeri be, noori m, jafari m. 2012. using the pollen viability and morphology for fluoride pollution biomonitoring. biol trace elem res 147:315-9. mercado ja, fernandez-muzor r, quesada ma. 1994. in vitro germination of pepper pollen in liquid medium. scientia horticulture 57:273-81. ixora pollen morphology and fertility phanomchai e t al. mouly a, razafimandimbison gs, ichodabandeh a, bremer b. 2009. phylogeny and classification of the species-rich pantropical showy genus ixora (rubiacrae-ixoreae) with indications of geographical monophyletic units and hybrids. am j bot 96:686-706. peel rg, 0rby pv, skj0th ca, kennedy r, schliinssen v, smith m, sommer j, hertel 0. 2014. seasonal variation in diurnal atmospheric grass pollen concentration profiles. biogeosciences 11:821-32. piotrowska k. 2012. meteorological factors and airborne h e x l. pollen concentration in lublin. acta agrobot 65:45-52. prabhakar r, ramakrishna h. 2014. palynodiversity in boath mandal forest division of adilabad district, telangana state, india. int j pharm life sci 5:3685-93. puff c, chayamarit i<, chamchumroon v. 2005. rubiaceae of thailand: a pictorial guide to indigenous and cultivated genera. bangkok ph): the forest herbarium, department of national parks, wildlife and conservation. punt w, hoen pp, blackmore s, nilsson s, le thomas a. 2007. glossary of pollen and spore terminology. rev palaeobot palyno 143:l-81. sreekala ai<, rajkumar g, pandurangan ag. 2003. studies on floral biology of ixora agastbamaiayana sivadasan & mohanan a rare southern western ghats endemic. zoos print j 18:1041-2. biotropia vol. 30 no. 2, 2023: 232 241 doi: 10.11598/btb.2023.30.2.1902 232 use of biological organic fertilizers and pesticides to improve potato cultivation in slope andisols tamad1*, loekas soesanto2 and akhmad rizqul karim3 1department of soil science, faculty of agriculture, jenderal soedirman university, purwokerto, central java, 53123, indonesia 2department of phytopathology, faculty of agriculture, jenderal soedirman university, purwokerto, central java, 53123, indonesia 3department of agribusiness, faculty of agriculture, jenderal soedirman university, purwokerto, central java, 53123, indonesia received 21 february 2023 / revised 13 march 2023 / accepted 13 march 2023 abstract in the 1990s, potato yield in the andisols of dieng, central java, indonesia, was approximately 30 t ha–1, but this value decreased rapidly to 12–15 t ha–1 in recent years. this rapid decline could be attributed to the use of unbalanced organic and chemical fertilizers, without the application of conservation techniques. therefore, this study aimed to sustainably improve the local potato cultivation pattern of farmers on andisols using biological organic fertilizers and pesticides (bofp). a randomized block design was used with two factors, namely: 1) 20 t bofp, 300 kg urea, 500 kg sp 36, 300 kg kcl, and 200 kg lime ha–1, and 2) comparison with the pattern of farmers, consisting of 20 t of chicken manure, 1–t npk, and 250 kg za ha–1. the potato plant mounds were tilted 10% to the contour direction, and each treatment was carried out with 16 replications. granola seeds were used to plant potato during the rainy season from march-june 2022. the results showed that the plants cultivated using the local pattern of farmers were affected by wilt from fusarium spp, while the use of bofp decreased the incidence of the disease by 80%. furthermore, the bofp pattern significantly increased andisols organic-c from 1.78% to 3.83% and total soil p from 5.20% to 11.34%, compared to the pattern of farmers. it also increased potato yields from 12.31 t ha–1 to 22.93 t ha–1 and the r/c from 0.85 to 1.23, compared to the pattern of farmers. based on the results, the use of bofp pattern decreased wilt attacks by fusarium spp, improved the productivity of andisols, as well as increased potato production and profits of farmers. keywords: beneficial microbes, farmer income, manure, potato, soil productivity introduction farmers on the andisols slopes have been cultivating potato using unbalanced organic and chemical fertilizers, without applying conservation techniques. furthermore, potato plant mounds are often prepared toward the hill (figure 1), which causes a decrease in soil fertility due to nutrient imbalances and intensive erosion. this planting pattern has also led to a decrease in potato production of 10–15 t ha–1 compared to the volume recorded in the 1990s. the decline was due to a reduction in the productivity of andisols and an increasingly intensive potato plant disease. dieng, central java, indonesia, is situated on andisols land, which is known to have a high adsorption capacity for phosphorus (p), leading to low availability of p for plants. this high adsorption capacity is due to the presence of aluminol groups (al–oh and al–oh2 +), ferrihydrite, and al-humus complex (pizarra et al. 2008; delfim et al. 2018). the application of a large amount of p fertilizer to the andisols does not necessarily increase the availability of p to plants, as the efficiency of p fertilization is low, ranging from 10% to 20% of applied p (sacrc 2004; elásquez et al. 2016). however, the available p can be increased through the use of organic fertilizer enriched with phosphate microbial (pm), organic matter ameliorant (humic-fulvic), and signal quorum sensing (qs), as a regulator of the effectiveness of biological agents. *corresponding author, email: tamad_1965@yahoo.com; tamad@unsoed.ac.id mailto:tamad_1965@yahoo.com mailto:tamad@unsoed.ac.id potato cultivation in slope andisols – tamad et al. 233 figure 1 the potato cultivation system with mounds in the direction of the slopes in dieng, central java, indonesia pm, such as pseudomonas trivialis, p. putida, and p. fluorescens, has the potential to increase p availability (arcand and schneider 2006; tian et al. 2021; yadav et al. 2021) by dissolving inorganic–p and mineralizing organic–p. in andisols, these microbes have been found to increase the dissolution of inorganic–p by 535– 575%, mineralization of organic–p by 336– 387%, and reduced adsorbed–p to 15–30% (tamad et al. 2021). intensive disease infestation, including late blight, potato bacterial wilt, tuber rot, fusarium wilt, and dry spot caused by phytopthora infestans, ralstonia solanacearum, colleotrichum coccodes, fusarium sp., and alternaria solani, can contribute to low potato production. an alternative for controlling potato disease is the use of bio p60, a biological pesticide containing crude secondary metabolites of bacterial antagonist p. fluorescens p60 (soesanto et al. 2011). another critical factor that must be considered during cultivation in andisols is the need for soil amendments. humic materials include humin, humic acid c10h12o5n, and fulvic acid c12h12o9n. humic acid is brownish-black, resistant to degradation, and contains a negative charge that is dependent on ph. several studies showed that humic-fulvic had the potential to be used for soil amendment (stevenson 1994; baveye and wander 2019). however, the effectiveness of andisol amendments is greatly influenced by soil microbial activity. the biochemical process that regulates population density-dependent microbial behavior through crucial signaling molecules is known as quorum sensing (qs) (teplitski et al. 2011; ma et al. 2018; coquant et al. 2020; ward et al. 2001). gram-negative bacteria typically produce n-acyl–homoserine lactones (ahl) as a qs signal (ward et al. 2001; muras et al. 2020). a previous study also showed that plant root extracts could serve as a source of n–ahl (tamad et al. 2020). the enrichment of organic fertilizers with microbes and signal quorum sensing as biological organic fertilizers effectively increases soil fertility. tamad et al. (2020a) stated that using biological organic fertilizer (bof) of up to 20 t ha–1 increased potato yields by 2 t ha–1 compared to the cultivation pattern of farmers in kaligua, central java, indonesia. the use of bof in potato cultivation also increased r/c from 1.13 to 1.53, and profits rose by idr 29.995.573 ha–1 compared to the practice of farmers. therefore, this study aims to improve the local potato cultivation pattern of farmers on andisols using bofp. the results are expected to increase potato production, sustainably improve the quality of andisols, and increase the income of farmers. materials and methods this study was carried out in andisol dieng, central java, where the soil had slightly acidic ph h2o, medium organic–c, high k2o, high p2o5, and medium n of 5.63, 2.39%, 0.4%, 3.20%, and 0.34%, respectively. furthermore, the region had an area of 3000 hectares and an altitude of 1,000–2,500 m above sea level, which was very suitable for potato cultivation. this study utilized biological organic fertilizers and pesticides, which were applied using conservation techniques in partnership with local farmer groups. bofp formulation the bofp formula was produced using chicken manure inoculated with phosphate solubilizing microbial and bio p60 (107 cfu g–1) biotropia vol. 30 no. 2, 2023 234 at a concentration of 0.025% v w–1, followed by the addition of humic-fulvic acids and 1% v w–1 qs signal. the bofp components were then mixed with the granulator under moist conditions, as shown in figure 2. subsequently, the product obtained was incubated for four weeks with 15–20% moisture content. figure 2 the formulation of biological organic fertilizers pesticides (bofp) preparation of phosphate microbial (pm) culture pm consortium of pseudomonas trivialis, p. putida, and p. fluorescens was grown on molasse culture for one week (end of log phase) (tamad et al. 2020). the standard plate count (spc) method was then used to determine pm at a minimum population of 107 cfu ml–1. production of bio p60 culture bio p60 was an isolate of p. fluorescens p60 cultured in snail broth for one week (soesanto et al. 2013). the spc method was utilized to assess the microbial population at a minimum of 107 cfu ml–1. humic-fulvic acids extract humic-fulvic acids were obtained from harvested top potato plant waste. furthermore, extraction was carried out by soaking the waste in 1 m technical naoh for two weeks (modified tan 1998; dulaquais et al. 2018). n-ahl extract potato plant root waste was used as a source of quorum-sensing signals for microbes (tamad et al. 2020). n-ahl compound, a quorum sensing signal, was obtained by soaking the waste in 5% acetonitrile for two weeks (rani et al. 2011). bofp application this study used a randomized block design with two factors, namely: 1) the application of 20 t bofp, 300 kg urea, 500 kg sp 36, 300 kg kcl, and 200 kg lime ha–1, and 2) comparison with the application pattern of the farmers, consisting of 20 t of chicken manure, 1–t npk, and 250 kg za ha–1, with 16 repetitions. the potato granola plant mounds were tilted at a 10% contour coverage rate per ha, as shown in figure 3. the seed potato was 800 kg ha–1, and the plant was spaced at 30 cm x 70 cm interval total obtain a total population of 47,000 ha–1. fertilization was performed every 20 days: a) at 20–40 days after planting (dap) when tuber growth begins; b) at 40–60 dap when tuber enlargement begins; c) at 60-90 dap (plant optimal enlargement); and d) at 90–110 dap after all the leaves dry up. furthermore, the variables observed included soil parameters, growth and yield components of potato (10% of the population were sample plants), and farming analysis (revenue and total cost). the variable of ph was measured with a ph meter, ec was assessed with an ec meter, c was analyzed with walkley and black, and p was evaluated with the hcl extraction method. figure 3 the design of potato mounds cutting a 10% sloping slope in dieng, central java, indonesia statistical analysis variance analysis was carried out on the data on soil, potato growth, and yield using fisher's test (p<0.05). subsequently, significant differences were determined by calculating the mean value using duncan's multiple range test. the statistical analysis was performed using the costat ver 6.451 application. the difference in the effect of the treatments on the economic value was carried out by assessing the production costs, income, and profits (r/c ratio). potato cultivation in slope andisols – tamad et al. 235 results and discussion soil, potato growth, and potato yield the growth of potato appeared normal up to 60 dap, as shown in figure 4. however, at 80 dap, the bofp pattern demonstrated better growth compared to farmers’ pattern, which was infested by diseases, as indicated by the falling and withering of leaves in figure 5. the plant often faced several challenges during the rainy season, including falling of leaves and intensive wilt disease. figure 4 the optimal growth of potato plant at 60 the day after planting (dap) in andisols dieng, central java, indonesia, with mound tilt of 10% contour direction figure 5 the differences in potato growth at 80 dap in andisols between farmers’ pattern and bofp pattern treatments this study showed significant improvement in soil, potato, and economic variables in bofp pattern compared to farmers’ pattern, as shown in table 1. it was observed that the plant was susceptible to fusarium infection, which caused wilt symptoms (thanaa et al. 2018). furthermore, fusarium is one of the most important genera of phytopathogenic fungi, causing potato wilt in the field (azil et al. 2021). the results showed that the application of bofp and conservation techniques to andisols improved the growth of potato plants and reduced fungi attack. p. fluorescens as bio p60 microbes produced secondary metabolites, which helped to inhibit fusarium sp wilt (soesanto et al. 2011; deveau et al. 2016). a previous study explored the antifungal activity of the extracellular metabolites of the most effective isolates of fusarium species infecting potato (trabelsi et al. 2016). table 1 the effect of bofp pattern and farmers’ pattern treatment in andisols on soil and potato variables average no variable n0a n1b 1 soil ph h2o 5.56 a 6.04 b 2 soil ph kcl 4.68 a 5.30 b 3 soil electric conductivity/ec (ms cm–1) 409.11 a 324.29 a 4 soil organic–c (%) 1.78 a 3.83 b 5 soil total–p (%) 2.27 a 4.95 b 6 plant height (cm) 54.88 a 63.19 b 7 leave number (pieces) 60.38 a 116.69 b 8 fall leave age (days) 85 a 100 b 9 fusarium spp attack (%) 80 20 10 tuber number 5.2 a 6.0 a 11 tuber diameter (mm) 51.24 a 56.74 b 12 tuber length (mm) 68.56 a 73.92 b 13 tuber volume (mmh3) 631.88 a 723.75 b 14 tuber weight (g plant–1) 301.69 a 561.81 b 15 tuber yield (t ha–1) 12.31 a 22.93 b 16 tuber p uptake (%) 0.23 a 0.23 a 17 tuber p (g plant–1) 5.46 a 8.81 b 18 r/c ratio 0.85 1.23 note: an0 = farmers’ pattern treatment is 20 t chicken manure ha–1, 1 t npk ha–1, and 250 kg za ha–1 bn1 = bofp pattern treatment is 20 t bofp ha–1, 300 kg urea, 500 kg sp 36, 300 kg kcl, and 200 kg calcite ha–1 biotropia vol. 30 no. 2, 2023 236 enrichment of chicken manure with bio p60 led to a significant reduction in the intensity of the fusarium wilt attack on potato plants, thereby increasing their productive age. bio p60 is a secondary metabolite formula derived from the antagonist bacterium p. fluorescens strain p60, which has been tested to treat various plant diseases. furthermore, p. fluorescens produced secondary metabolites that contain several bioactive compounds. it also played an essential role in inhibiting microbial growth in the soil, including fusarium sp., the cause of wilt in potato (soesanto et al. 2011; deveau et al. 2016). p. fluorescens is a ubiquitous soil bacterium that promotes plant health, and some of its strains produce secondary metabolites, such as 2,4– diacetyl phloroglucinol (dapg), phenazines, and hydrogen cyanide, thereby inhibiting soilborne pathogens (neidig et al. 2011). the strain p. fluorescens p60 inhibited fusarium wilt of shallots (santoso et al. 2007) and tomatoes (soesanto et al. 2011), stem rot of peanuts, chili virus, tomato microbe wilt (soesanto et al. 2013), and stem base rot of dragon fruit (hamarawati et al. 2018). the results showed that bofp pattern (n1) increased the soil ph (h2o) from acid to slightly acid and decreased the electrical conductivity compared to the farmers’ practice (n0) (figure 6). furthermore, it significantly increased soil ph (kcl), organic–c, and total–p. the application of the bofp pattern was also shown to improve the quality of andisol by enhancing some chemical characteristics. the application of bofp improved the chemical characteristics of andisols. furthermore, the enrichment of chicken manure with pm and balanced chemical fertilization improved the soil ph, organic-c, and total–p. based on the results, the inoculation of phosphate-solubilizing microorganisms increased soil biomass and microbial activity (wang et al. 2022). pm had been reported to mediate the bioavailability of p by mineralizing organic–p and dissolving the inorganic variant. the soluble p in andisols was affected by inorganic–p solubility, mineralized organic–p, and adsorption–p (tian et al. 2021). phosphate solubilizing microorganisms as a component of the entire soil community can be manipulated to be more effective for plant nutrition (raymond et al. 2021). these microbes can increase phosphate availability through the dissolution of inorganic– p and mineralization of organic–p. the dissolution of inorganic–p was often carried out through the production of organic acids, such as sulfuric acid, nitric, and carbonate. the mineralization of organic–p occurred due to the production of extracellular enzymes, such as phosphatase, phytase, phosphonatase, and c–p lyase by pm. the phosphatase enzyme converts high molecular weight organic phosphate into low molecules by hydrolyzing the bonds with the release of ions (prabhu et al. 2018). figure 6 the effect of the farmers’ pattern (n0) and the bofp pattern (n1) treatments on the andisols variables potato cultivation in slope andisols – tamad et al. 237 pm significantly increased inorganic–p solubility and organic–p mineralization, while significantly reducing adsorption–p in andisols. the ability of pm ability to dissolve p was influenced by the type of species, p compounds, organic acids released, and microbe population. furthermore, its capacity to mineralize organic– p depended on the activity of phosphatase and phytase (tamad et al. 2021). the results showed that pm increased p availability, improved plant growth, and restored soil fertility. phosphate-solubilizing microorganisms have enormous potential as biofertilizers because they can increase the bioavailability of p for plants, offer sustainability, improve soil fertility, and increase crop yields (timofeeva et al. 2022). the pm isolates pseudomonas trivialis, p. putida, and p. fluorescence released citrate, lactate, malonic, oxalic, and acetic acid of 156.25 mg kg–1. the phosphatase and phytase yields obtained from phosphate microbe ranged from 12 to 47 mg po4 3– dm–3 h–1. the amount of p dissolved by pm was higher between 147.66 and 194.61 mg p kg–1 compared to the control (31.06 mg p kg–1). furthermore, pm inoculation produced mineralized organic–p of 63.69 mg p kg–1, compared to the control with 23.7 mg p kg–1 (tamad et al. 2021). it also increased the dissolution of p up to 300% compared to the control (tamad et al. 2020). based on these results, pm can be applied to plants to promote growth or increase p availability, while reducing the dependence on phosphate-based fertilizers and restoring soil fertility (yadav et al. 2021). bofp pattern significantly increased plant height, number of leaves, and age of potato plants, as well as decreased fusarium wilt attack compared to farmers’ pattern, as shown in figure 7. bofp pattern significantly improved tuber quality in terms of diameter, length, and volume, as shown in figure 8. it also significantly increased the number of potato plants and yield per ha compared to the farmers’ practice. the yield obtained in the rainy season from farmers and bofp patterns was 12 t ha–1 and 23 t ha–1, respectively. bofp pattern treatment significantly increased the total p of andisols (figure 6), which supported the uptake of p by potato grown on the soil. the results showed that p uptake in potato tubers was significantly higher in bofp pattern. however, the p content obtained from both patterns was the same, as shown in figure 9. bofp was found to significantly increase potato yield plant–1, as shown in figure 8. figure 7 the effect of farmers’ pattern (n0) and bofp pattern (n1) treatments on potato variables in andisols biotropia vol. 30 no. 2, 2023 238 figure 8 the effect of farmers’ pattern (n0) and bofp pattern (n1) treatments on the potato variables in andisols figure 9 the effect of farmers’ pattern (n0) and bofp pattern (n1) treatments on the potato p variables and the r/c ratio in andisols the application of bofp in andisols improved p_absorption, the quality of tubers, and the yields of the plant. biological organic fertilizer (bof) is a chicken manure enriched with phosphate bacteria, humic–fulvic acid, and n–acyl homoserine lactone to effectively increase p solubility and increase yield. furthermore, potato yield in kaligua, central java, indonesia, using a bof dose of 20 t ha–1 increased tuber yield to 2 t ha–1 compared to chicken manure (tamad et al. 2020). the application of bopf technology to potato cultivation in andisol during the rainy growing season significant;y increased production and farmers' profits. profitability of potato cultivation farming based on the profitability analysis of potato farming, bofp pattern increased the r/c ratio compared to farmers’ pattern, as shown in table 2 and figure 9. based on the results, the total cost of cultivation with bofp pattern was greater, namely idr 5 million ha–1, which was 4% higher than farmers’ pattern. the total revenue for cultivation was 51% greater than farmers’ pattern. bofp potato cultivation provided a profit of idr 31.4 million ha–1, while farmers’ pattern led to a loss of idr 19.5 million ha–1, as shown in table 2. potato cultivation in slope andisols – tamad et al. 239 table 2 the difference in potato farming between farmers’ pattern and bofp pattern on revenues, total cost profits, and efficiency per hectare no. differences farmers’ pattern bofp pattern 1 revenue (idr) 109.030.000 165.096.000 2 total cost (idr) 128.536.667 133.700.667 3 profits (idr)a –19.506.667 31.395.333 4 efficiencyb 0.85 1.23 note: aprofits = revenue – total cost befficiency = revenue / total cost farming efficiency was calculated based on revenues and total expenses (anggraeni et al. 2021). potato cultivation with bofp pattern had better efficiency compared to the farmer’s pattern, as shown in table 2. the r/c value of 1.23 in bofp pattern showed that one unit of the cost used to cultivate potato provided 23% of the profit from the cost. meanwhile, farmers’ pattern showed that farming was inefficient, as indicated by r/c < 1. the results showed that the profits of farmers increased after the application of bofp. the increase in yield of potato cultivated with bofp pattern was shown to exceed the additional cost required. the profit obtained for the use of this pattern also exceeded that of farmers' pattern. during the rainy season, the use of farmers’ pattern led to losses due to rain factors and intensive fusarium wilt disease. the calculation of potato farming efficiency was aimed at comparing the results with the cost of using resources. potato cultivation with bofp pattern can provide better results than farmers’ pattern. the average profit from environmentally friendly farming within a year was also shown to be higher compared to conventional methods (saem et al. 2016). the yield quantity played a significant role in determining the significant gain from potato farming with bofp. the application of biofertilizers significantly correlated with increased outcomes (khoiriyah et al. 2018). conclusion the application of bofp pattern on potato cultivation in andisols decreased fusarium spp wilt attack, improved soil characteristics, enhanced vegetative component, and increased yields from 12.32 to 22.93 t ha–1. during the rainy season, farmers’ pattern caused a loss of idr 19.9 million, while bofp yielded a gain of idr 31.4 million. furthermore, bofp pattern increased the r/c ratio from 0.85 to 1.23, as well as andisols’ productivity, potato production, and farmers’ profits. acknowledgments the authors are grateful to the ministry of education, culture, research, and technology (kemdikbudristek) as well as the educational fund management institution (lembaga pengelola dana pendidikan/lpdp) of 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marilyn s. combalicer* department of forest biological sciences, college of forestry and natural resources (cfnr), university of the philippines los baños (uplb), college, laguna 4031, philippines received 8 september 2021/accepted 10 december 2021 abstract previous studies on the embryo structure of legumes species had resulted in the division of the fabaceae family into two great subfamilies based on embryo axis curvature. research on seed morphology and anatomy adds to the knowledge of taxonomy, evolution and ecology. this study determined the seed developmental anatomy, pod and seed morphology as well as germination characteristics of the observed variants (t1 small prickles; t2 medium prickles; t3 long prickles) of pterocarpus indicus willd. f. echinatus locally known as prickly narra in the mount makiling forest reserve (mmfr). based on the anatomy of the root (radicle) and shoot apex, the formation of the leaf primordium in t2 seeds after radicle protrusion was more progressive. it was observed that the germination rate and the percentage were the highest in t2, where the apical dome was welldeveloped. the germination, pod and seed morphological characters as well as seed anatomical characters were proven to be systematically informative by showing significant differences among the variants. keywords: fabaceae, faboidae, meristem, pod, radicle introduction the fabaceae family consisting of 686 genera with more than 18,000 species is the third largest flowering plant family after asteraceae and orchidaceae (mabberley 1997). the study of de candolle (1825), resulted in the division of fabaceae into two great subfamilies based on embryo axis curvature (i.e., curvembriae and rectembriae). the first subfamily contains the faboideae, while the second one contains the caesalpinioideae and mimosoideae. even though embryo axis curvature is currently not considered as the best character for primary divisions in the family, it may be one of the seed characters (especially hilar characters) used to divide the faboideae from the other subfamilies (oliveira & paiva 2005). faboideae is the largest of the fabaceae subfamilies, with about 440 genera and 12,000 species (polhill 1981). certain seed characteristics are very helpful for faboid generic identifications i.e., aril, endosperm, radicle concealment by the cotyledons, cotyledon lobes over the radicle, overall radicle shape, radicle tip shape, and radicle length relative to that of the cotyledons (kirkbride et al. 2003). capitaine (1912) stated the importance of legume seed morphology in legume classification and identification at the tribal, generic and specific levels. in the last 30 years, there has been a revival of interest in seed morphology and about 225 publications on the subject have been documented (kirkbride et al. 2003). research on seed morphology and anatomy adds to the knowledge of taxonomy, evolution, and ecology of angiospermae species (cortez & carmello-guerreiro 2008). however, it is essential to emphasize that seed morphology generally presents little phenotypic plasticity. conversely, embryological characters, typically *corresponding author, email: mscombalicer@up.edu.ph developmental morpho-anatomy and germination of pterocarpus indicus variants – kate c. capilitan et al. 125 constant in the genera, serve as an important indicator of taxonomic affinity (von teichman & van wyk 1991). according to oliveira and paiva (2005), there are only few descriptive and ontogenetic studies on seed structure causing difficulties in building the hypothesis of evolutive trends affecting seeds. knowledge on fabaceous seeds is mainly related to hard seeds (baskin & baskin 1998; baskin et al. 2000) while studies on seeds are limited, with the majority of such studies concentrating on species of agricultural interest, mainly soybean and bean (qutob et al. 2008). basic knowledge about seed characterization has proven its importance in dealing with problems in the field of seed technology and is essential in activities designed to maintain biodiversity and germplasm conservation. external morphology characterization of seeds has been used by various authors in species identification (youngberg et al. 1998; cappers & bekker 2013). characters mostly used include shape, weight, diameter, color and texture. populations of pterocarpus indicus or narra over the natural range of distribution has declined over the years due to unselective cutting and overall habitat loss. therefore, this species has been categorized under endangered category (iucn v. 2021-1) while being categorized as critically endangered under the philippine national red list for plants (denr-dao 2017-11). to conserve the remaining population, intensive research on the species have been conducted (gazal et al. 2004; krishnapillay et al. 1994; xu et al. 2016). there are two recognized forms of narra (p. indicus) namely smooth narra (pterocarpus indicus forma indicus) and prickly narra (p. indicus f. echinatus). however, some authors have recognized the presence of intermediate forms (jøker 2000; duke 1983). this may be attributed to the high degree of cross-pollination in p. indicus. there have been a few studies on morphology and field germination of the species, but there remains a dearth of information on the anatomy of the seeds, especially of p. indicus f. echinatus. this study determined the developmental anatomy, pod and seed morphology as well as germination characteristics of the observed variants of p. indicus f. echinatus collected from mmfr to assess the extent of variation in this form. materials and methods study site and seed collection seeds were collected in the mt. makiling forest reserve (mmfr) (14o8’ n and 121o12’ e) in 2018. flowering of p. indicus f. echinatus starts in january and peaks around april and may. regular fruiting season starts from january to july or september to november. selection of mother trees was based on characters that distinguish p. indicus f. echinatus trees from the smooth form; leaflets distinctly more obovate with acuminate apex and inner bark relatively whitish to yellowish. however, the observed variation in prickles length was confirmed during pod maturity, usually four months after fruit bud formation. pods were collected and air-dried. seeds were extracted from the pod using scissors and forceps. pods are disc-shaped, flat and have winged margins or samara. unlike most legumes, the pterocarpus pod is indehiscent and is wind dispersed. the pod also floats in water and can be water dispersed. around 5 cm across, the pod has a central woody corky bulge containing 1 3 seeds. the seeds have very thin seed coats (orwa et al. 2009). morphological measurement pod and seed characters were described and compared among the three variants. for the pod morphology, different variants of p. indicus f. echinatus were categorized not only by the length of the prickles but also by the shape and size of the pods, number of seeds per pod, and number of prickles. for the seed morphology, seed length and width were determined using a ruler (mm). the mean for every characteristic was calculated. anatomical measurement permanent sections of the germinated seeds were obtained using a modified histological paraffin technique by johansen (1940). the seeds were fixed using formalin: acetic acid: alcohol (faa) solution for two weeks, dehydrated using different percentages of alcohol and infiltrated with paraffin wax (tables 1 and 2). the seeds were then embedded in paraffin wax and mounted on wooden blocks measuring 2 x 2 x 2 cm. the samples were sectioned using a rotary microtome with a biotropia vol. 29 no. 2, 2022 126 thickness of 10 to 15 µm. the resulting paraffin ribbons were then mounted on glass slides, decerated, stained with safranin and counterstained with fast green. drops of entellan were added over the stained sections prior to the addition of cover slips and were then air-dried. photomicrographs of the seed’s embryo were obtained using optika microscope under 400x magnification. the thickness of different tissues (root apical meristem, shoot apical meristem, procambium, ground meristem, protoderm and leaf primordium) were measured using the optika software. the development of the embryo was observed starting from the ungerminated seeds until the radicle protrusion and elongation of the embryo. haupt (1953), fahn (1967), bell (2008) and shipunov (2020) were followed to describe the developmental anatomy of the seed. table 1 paraffin schedule (fixation, dehydration, infiltration and embedding) solution procedure per day duration in solution (hours) fixation (faa-a and faa-b mixture) one week 50% etoh day 1 1st dehydration 1 hour 50% etoh day 1 2nd dehydration 1 hour 50% etoh day 1 3rd dehydration 1 hour 50% etoh day 1 4th dehydration 1 hour j1 day 1 2 hours j2 day 1 overnight j3 day 2 2 hours j4 day 2 2 hours j5 day 2 2 hours j6 day 2 (in warm place; vial uncorked) overnight j6 day 3 (3 tba changes every 2 hours) 6 hours tba (1) day 3 (uncorked at room temperature) (fumehood) 1 4 hours tba + paraffin pellets day 3 uncorked at 65 oc) 3 to 4 hours table 2 staining schedule for pterocarpus indicus samples solution time (minutes) xylene 15 tba 15 absolute ethanol 15 95% ethanol 15 50% ethanol 15 h2o 3 safranin 30 h2o 3 to 4 rinses 50% ethanol 15 95% ethanol 15 fast green 5 95% ethanol 15 absolute ethanol 15 tba 30 xylene 30 developmental morpho-anatomy and germination of pterocarpus indicus variants – kate c. capilitan et al. 127 germination viability test was conducted to p. indicus f. echinatus seeds via flotation method (dayan & reaviles 1995). viable seeds were washed with running water and were arranged in sterilized petri dishes containing filter paper with water. the set-up was done in the microtechnique laboratory of the department of forest biological sciences, college of forestry and natural resources, university of the philippines los baños (dfbs, cfnr, uplb). the seeds are considered germinated when visible protrusions of plumule is observed. for the germination percentage (equation 1) and germination rate (equation 2) (awasthi et al. 2016), the following formulae are used: germination percentage (%) = number of total germinated seeds x 100 (1) total number of seeds tested germination rate = number of germinated seeds + + number of germinated seeds (2) day of first count day of final count experimental design and analysis the germination experiments used a simple complete randomized design (crd) with three treatments having four replicates of 50 seeds each. the experiment included three variants (as treatments) of p. indicus f. echinatus, namely: t1 = small prickles; t2 = medium prickles; and t3 = long prickles. the one-way analysis of variance (anova) and duncan’s multiple range test (dmrt) were used to test for significance of the mean differences in terms of pod and seed morphological characters (pod diameter, length of prickles, number of prickles, seed length and seed width) and germination characteristics of p. indicus f. echinatus. analyses were performed using r studio version 4.1 (r studio team 2020). results and discussion morphological features certain characters (pod diameter, length of prickles and number of prickles) displayed variation among variants and were found to be potentially informative, whereas other characters (pod shape, presence of prickles and seed shape) were observed to be similar in all variants studied. all p. indicus f. echinatus pods found in this study were thin, papery-winged and disc-shaped. generally, the pods had bulge at the center containing the seeds. pod usually has a diameter of 5 8 cm, but all have their own unique characteristics. pods of t1 variant contained up to four (4) seeds, while only 1 3 seeds were found in t2 and t3 variants. on the other hand, the pod diameter had nearly significant variations among the three variants (p = 0.0535), while both the length of prickles (p = 0.000) and the average number of prickles (p = 0.000) were significantly differentiated among the variants. t2 had numerous prickles (95.095±0.461) per pod compared to those of t1 and t3. lastly, t3 had soft and the longest prickles (8.764±0.027) while t2 and t1 had hard prickles and shorter prickles (table 3). table 3 pod morphological characteristics of the pterocarpus indicus f. echinatus variants collected from mmfr morphological characteristic variant t1 t2 t3 pod shape disc-shaped disc-shaped disc-shaped pod diameter (cm) 6.450±0.136b 6.746±0.071a 6.524±0.047ab no. of seeds 1 4 1 3 1 3 length of prickles (mm) 6.248±0.026c 7.230±0.028b 8.764±0.027a number of prickles 62.095±0.809b 95.095±0.461a 55.524±0.562c stiffness of prickles hard hard soft notes: t1 = small prickles; t2 = medium prickles; t3 = long prickles. biotropia vol. 29 no. 2, 2022 128 table 4 seed morphological characteristics of the pterocarpus indicus f. echinatus variants collected from mmfr morphological character variant t1 t2 t3 seed shape falcate falcate falcate seed color orange-brown orange-brown to reddish-brown reddish-brown to brown seed width (mm) 5.167±0. 122ns 4.905±0. 127ns 5.095±0. 159ns seed length (mm) 13.571±0.316ns 13.333±0.349ns 12.905±0.325ns note: ns = no significant difference among the variants at p < 0.05. seed color of variants varied from orange brown to reddish-brown. seed width (p = 0.380) and seed length (p = 0.354) showed no significant variations among the three variants (table 4). in this study, the seeds of p. indicus f. echinatus were flat, falcate-shaped and had almost similar width and length ranging from 4.905 to 5.167 mm and 12.905 to 13.571 mm, respectively. the pod description of p. indicus f. echinatus in this study is consistent with the reports of orwa et al. (2009), thomson (2006), francis (2002) and flores et al. (2021) having indehiscent discshaped and flat pod with winged margins. about 5 cm across, it has a central woody-corky bulge containing several seeds. dayan and reaviles (1995) reported p. indicus f. echinatus pod length of 5 6 cm including the wing (1.5 2.5 cm), while rojo (1977) and duke (1983) reported 4 7 cm and 4 6 cm pod diameter, respectively. in this study, 5 8 cm pod diameter was observed. flores et al. (2021) confirmed that p. indicus f. echinatus has pod size ranging from 5 8 cm. light environment and soil moisture influence fruit quality including fruit size and color (kozlowski & pallardy 1997; raina 2003). different literatures reported various number of seeds per pod, such as francis (2002) reported 1 4 seeds per pod, orwa et al. (2009) reported 1 3 seeds per pod, while jøker (2000) and duke (1983) reported 1 2 seeds per pod. in this study, the t2 and t3 variants of p. indicus f. echinatus contained 1 3 seeds per pod while t1 has 1 4 seeds per pod. according to kelly (1984), the number of seeds per seeding plant is one of the basic parameters necessary for a description of the population dynamics of species which does not reproduce vegetatively. moreover, it was emphasized that in situations where the number of seeds per fruit was found to vary within years or among treatments, there seemed to be a stable relationship between fruits per plant and seed numbers. in terms of prickles, t2 was found to have numerous hard prickles, while t1 and t3 were found to have a lesser number of prickles (table 3), which is in agreement with the study of flores et al. (2021). kellogg et al. (2011) defined prickles as outgrowths of epidermal tissues and can provide a simple developmental system for the study of the control of cell proliferation and growth. prickles constitute one of the many types of plant defense against vertebrate herbivore (janzen & martin 1982; cooper & owen-smith 1986; milewski et al. 1991 as cited by ronel & lev-yadun 2012). for seed morphology, seed width and seed length in this study were not significantly different among p. indicus f. echinatus variants. jøker (2000) and thomson (2006) reported 6 8 mm seed length for p. indicus with brown papery testa. according to harper (1977) and silvertown (1989) as cited by chacon et al. (1998), seed size is a life history trait that may affect the fitness of the parent’s plants and the population regeneration process. large seeds tend to have a positive effect on germination. in this study, t2 had a significantly similar germination percentage to that of t1, but different from that of t3 (table 5). the morphology distinction of angiosperm seeds and the relative consistency of seed structures in narrow taxonomic units allow the use of seed characteristics in taxonomic research (esau 1977). the most significant seed morphological characters are shape, size, testa surface, the position of hilum and the presence or absence of specialized structures such as aril, caruncle or elaiosomes. the differences in bristle-like prickles or spicules in terms of length and number as well as number of seeds per pod as observed in p. indicus f. echinatus may support the argument of jøker (2000) and duke (1983) that intermediate forms may occur. developmental morpho-anatomy and germination of pterocarpus indicus variants – kate c. capilitan et al. 129 anatomical features in the root apex, the root cap had become more developed after protrusion, consisting of 7 10 layers of cells. for the germinated seeds, the root tip of t2 was more round compared to the root tips of t1 and t3 (fig. 1). the procambium became more evident after radicle protrusion and was the thickest in t3. the ground meristem also increased in thickness after radicle protrusion. the shoot apical meristem (sam) of the embryo was domeshaped which was observed in the embryo of all p. indicus f. echinatus variants. well-developed leaf primordium was observed in all of the embryos after radicle protrusion. on the other hand, the formation of leaf primordium in t2 seeds after radicle protrusion was more progressive (fig. 2). sam is essentially a dome-shaped structure with undifferentiated cells at the tip, surrounded by a differentiating peripheral zone that participates in leaf formation. a well-developed apical dome is directly related to high germination frequency (corredoira et al. 2002). a less developed apical dome would mean lesser germination frequency. in this study, welldeveloped leaf primordium can be observed in all of the embryos after radicle protrusion (fig. 2). figure 1 longitudinal section of the root apex of the variants of pterocarpus indicus willd. f. echinatus collected from mmfr notes: a-c = before protrusion; d-f = after protrusion. a and d = t1 root apex; b and e = t2 root apex; c and f = t3 root apex. rc = root cap; ram = root apical meristem; pc = procambium; pd = protoderm; gm = ground meristem. scale bar = 200 µm. figure 2 longitudinal section of the shoot apex of the variants of pterocarpus indicus willd. f. echinatus collected from mmfr notes: a-c = before protrusion; d-f = after protrusion. a and d = t1 root apex; b and e = t2 root apex; c and f = t3 root apex. sam = shoot apical meristem; pc = procambium; pd = protoderm; lp = leaf promordium. scale bar = 200 µm. biotropia vol. 29 no. 2, 2022 130 seed anatomical characters showed value in verifying taxonomic relationships (esau 1977). p. indicus f. echinatus was found to have a root tip cap of 7 10 layers of cells. in this study, the root tip cap of t2 variant was found to be round, while those of t1 and t3 were pointed. roue et al. (2020) proved in their study that root tip cap structure affects apex penetration and reorientation, in which rectangular-shaped root tip cap showed enhanced penetration abilities compared to the pointed root tip cap. kumpf and nowack (2015) mentioned that modern plant biology has unraveled that many of the functions that darwin attributed to the root tip are actually accomplished by the root tip cap, which is a multi-layered dome of spindleshaped parenchyma cells that overlies the growing root tip (iijima et al. 2008). the root tip cap surrounds and protects the meristematic stem cells at the growing root tip. in addition, the root tip cap shows a rapid turnover of shortlived cells regulated by an intricate balance of cell generation, differentiation and degeneration. in arabidopsis thaliana, the root tip cap cells are actively killed and degraded on the root surface, while a limited amount of short-lived ‘borderlike’ cells are released into the rhizosphere (durand et al. 2009; fendrych et al. 2014). based on the classification of kumpf and nowack (2015), p. indicus f. echinatus can be considered to have a closed meristem structures which form their cell lineages from specific stem cells, including the root tip cap lineage, which shows a defined root cap stem cells and a layered root cap structure. while new root cap cells are constantly produced by root cap stem cells in an indeterminate fashion, the size and cell number of the root cap are determinate (barlow 2003). the procambium in this study became more evident after radicle protrusion and was the thickest in t3. the ground meristem also increased in thickness after radicle protrusion. the procambium provides the basis for the differential modulation of long-distance transport capacities and plant body stability (jouannet et al. 2015). germination results of germination percentage and germination rate showed significant differences for the three variants. germination percentage and germination rate were both the highest in t2 with 82.67% and 42.86, respectively (table 5; fig. 3). table 5 mean germination percentage and rate ± se of the pterocarpus indicus f. echinatus variants collected from mmfr parameter variant t1 t2 t3 germination percentage (%) 60±1.15ab 82.67±3.53a 50.667±0.667b germination rate (no. of seeds per nth day) 16.73±1.15b 42.86±1.95a 23.46±1.79b note: numbers followed by the same letter are not significantly different based on duncan test at p < 0.05. figure 3 seeds showing germination up to radicle protrusion notes: a = t1; b = t2; c = t3. day 0 to day 3 from left to right. scale bar = 1 cm. developmental morpho-anatomy and germination of pterocarpus indicus variants – kate c. capilitan et al. 131 in this study, seeds began to germinate 3 4 days after sowing, which is similar to the observation of thomson (2006). germination usually commences with the uptake of water by the dry seed through imbibition and is completed when the radicle extends to penetrate the structures that surround it (bewley 1997). in the study of de sedas et al. (2019) the osmotic effect due to salinity was the main inhibitory factor that reduced germination of inland neotropical tree species such as minquartia guainensis, apeiba menbranacea, ormosia ccoccinea and ochroma pyramidale. high germination percentage and germination rate in t2 (table 5; fig. 3) can be attributed to various factors. in the study of vozzo (2003) as cited by luna et al. (2014), tropical species that benefit from reagent, such as hydrogen peroxide, included albizia species and camphor tree seeds. valio and scarpa (2001) proved that the seeds germination percentage and rate of seven tropical pioneer species in brazil (cecropia hololeuca, c. pachystachya, c. glazioui, solanum gracillimum, s. granuloso-leprosum, s. tabacifolium and miconia chamissois) were significantly higher in the irradiated condition than in shaded condition. on the other hand, seed germination rate of peltophorum dubium varied with water potential treatment (daibes & cardoso 2020). conclusion the germination, pod and seed morphological characters and seed anatomical characters proved to be informative by distinguishing significant differences among the observed variants of p. indicus f. echinatus. the variants can be differentiated by the average number of prickles, length of prickles, stiffness of prickles, pod diameter, number of seeds per pod and seed color. the formation of the leaf primordium in t2 seeds after radicle protrusion was more progressive. the observed variants did not differ in their germination characteristics. differences of shoot apical meristem can be observed based on the development of the apical dome of each variant, which is related to the germination percentage of each variant. this study on p. indicus f. echinatus is limited to mmfr only and examines seed only and does not include other parts of the tree. also, this study does not include parameters such as seed coat and fruit anatomy. it is therefore recommended that further research, such as molecular and dna analyses, be conducted in the future to shed light on the observed variation in this study. characters such as seed coat anatomy can add more knowledge on the separation of the variants being studied. acknowledgments the authors wish to thank the department of forest biological sciences and the makiling center for mountain ecosystems (mcme), cfnr-uplb for allowing the use of their facilities to conduct this study. this study is partially supported by the dost-pcaarrdfunded project “germplasm conservation of selected indigenous forest trees in mmfr”. references abud hy, goncalves nr, pereira mds, pereira dss, reis rdge, bezerra ame. 2012. germination and morphological characterization of the fruits, seeds and seedling of pilosocereus gounellei. rev braz bot 35(1):11-6. awasthi p, karki h, vibhuti bk, bargali ss. 2016. germination and seedling growth of pulse crop (vigna spp.) as affected by soil salt stress. curr agric res j 4(2):159-70. barlow pw. 2003. the root cap: cell dynamics, cell differentiation and cap function. j plant growth regul 21:261-86. baskin cc, baskin jm. 1998. seeds: ecology, biogeography and evolution of dormancy and germination. san diego (us): academic press. 666 p. baskin jm, baskin cc, li x. 2000. taxonomy, anatomy and evolution of physical dormancy in seeds. plant species biol 15:139-52. benedict jc, smith sy, collinson me, skornickova jl, specht cd, marone f, xiao x, parkinson dy. 2015. seed morphology and anatomy and its utility in recognizing subfamilies and tribes of zingiberaceae. am j bot 102(11):1814-41. cappers rtj, bekker rm. 2013. a manual for the identification of plant seeds and fruits. gröningen (nl): barkhuis & university of gröningen library. 272 p. biotropia vol. 29 no. 2, 2022 132 chacon p, bustamante ro, henriquea c. 1998. the effect of seed size on germination and seedling growth of cryptocarya alba (lauraceae) in chile. rev chil hist nat 71(2):189-97. cervantes e, martin jj, saadaoui e. 2016. updated methods for seed shape analysis. scientifica [internet]. 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[cited 2019 may 31]; 10: 706. doi: 10.3389/ fpls.2019.00706 xu cx, zeng j, cui tc, chen qd, ma yp. 2016. introduction, growth performance and ecological adaptability of hongmu tree species (pterocarpus spp.) in china. j trop for sci 23(3):260-7. youngberg h, hannaway d, mosley a. 1998. 4-h plant and seed identification and crop judging. lecture guide. corvallis (us): oregon state university extension service. 20 p. 7. himmah rustiami & lulut dwi sulistyaningsih.cdr page 1 page 2 page 3 page 4 1233.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 sri-03. 933-layout.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 1107.cdr a d e b c page 1 page 2 page 3 page 4 page 5 page 6 biotropia vol. 30 no. 3, 2023: 253 262 doi: 10.11598/btb.2023.30.3.1812 253 post-siege genotoxic hazards in lake lanao, philippines by micronucleus assay fema abamo1,2*, annabella villarino2 and ed lucille mae ferranco2 1 mamitua saber research center, mindanao state university (msu), marawi city, 9700 philippines 2biology dept, college of natural sciences and mathematics, msu, marawi city, 9700 philippines received 3 september 2022 / revised 27 july 2023 / accepted 7 august 2023 abstract massive war pollutants brought by marawi siege in the philippines have contaminated the city environs and the surrounding ancient lake lanao. munition residues including heavy metals are reportedly genotoxic hence this study was conducted to determine the post-siege genotoxic hazards posed by the munition pollutants through micronucleus (mn) assay on the slow-moving edible gastropod snails vivipara angularis muller (locally known as “suso”) thriving in the lake. mn, a biomarker of genotoxicity was examined and counted in the hemocytes of exposed juvenile and adult snails. results revealed that mn frequencies (no. of mn/1000 hemocytes) were significantly higher in snails sampled in lake lanao lakeshores than in the reference site lake dapao. among adult samples, there was a decreasing trend of mn frequencies with increasing distance from lakeshore fronting warzone ‘ground zero’ where the battle was heaviest (site a) to sampling sites away from it: sites b, c, and d (lake dapao) which are 8.15 km, 24.41 km, and 34.45 km, respectively. moreover, varied patterns of micronucleation were observed between age groups and sites, i.e., in all sites except c, adults had greater mn counts than juveniles which were significant in site a only. it is a generally recognized observation that mn frequency increases with age; unexpectedly juveniles displayed significantly higher mn counts than adults in site c. the elevated mn frequencies in the snail hemocytes exposed to war pollution could be attributed to genotoxic munition residues eroded and washed into the lake water. moreover, heavy metals which are common components of weaponries were also detected in the snail muscles, although at concentrations within safe levels but continued consumption may be cautioned to avoid biomagnification. other genotoxins must be present in site c other than munition residues predisposing the juvenile snails. the results are baseline data on the mn frequencies in v. angularis exposed to war pollutants in lake lanao which need further investigation. keywords: genotoxicity, lake lanao, marawi siege, micronucleus assay introduction the devastating marawi siege, an armed conflict that lasted for five months in 2017 was the longest urban warfare in the philippines, polluting the air, soil, and water environment and destroying biodiversity. adding to the insurmountable damage are the numerous illhealth effects that could be suffered by the inhabitants, especially humans. high-tech ammunitions utilized both by the terrorist and the government troops included bombs, mortars, grenades such as rocket-propelled grenades (rpg), hand-held, anti-tank grenade launchers, and the likes aside from the conventional rifles, improvised explosive devices (ieds), and carpet, and aerial bombs (kapoor, 2017, pareño, 2017, & miller, 2017). these are depots of toxic pollutants such as high-melting explosives (hmx), hexahydro-1,3,5-trinitro-1,3,5triazine (rdx), trinitrotoluene (tnt), and heavy metals with potential genotoxic threats such as lead (pb), arsenic (as), nickel (ni), copper (cu) and zinc (zn), strontium (sr), magnesium (mg), and barium (ba), mercury (hg), cadmium (cd) antimony (sb), iron (fe) and chromium (cr) (iarc 2021, broomandi et al. 2020, skalny et al. 2021, chatterjee et al. 2017). known components of weaponries listed in iarc (2021) are established mutagens and carcinogens causing cancers affecting the respiratory tract, liver, bone, blood, and other parts of the human body. the magnitude of weaponries used during the siege *corresponding author, email: fema.abamo@msumain.edu.ph biotropia vol. 30 no. 3, 2023 254 heavily contaminated the city environs and its surrounding lake with genotoxic pollutants posing health hazards to the local inhabitants as well as to the organisms thriving in the second largest lake in the country and one of the ancient lakes in the world, that is, lake lanao, in the province of lanao del sur (lds). genotoxicity is the ability of chemicals to damage the genetic information of the cells thereby resulting in mutations and eventual carcinogenesis (seukep et al., 2014). one reliable method to assess the genotoxic potential of some substances (such as heavy metals, pesticides including gamma radiation), is the micronucleus (mn) assay and it is used in various test organisms including man, mice, land and aquatic snails, mussels, and fishes (silva et al. 2011, da silva 2013, bolognesi & fenech 2012, gentile et al. 2012, luzhna & kovalchuk, 2013, beedanagari et al. 2014, da rocha et al. 2016, hayashi 2016, el safty et al. 2018, nikdehghan et al. 2018, and de vasconcelos lima 2019, fenech et al. 2020). the present study made use of the freshwater gastropods vivipara angularis muller (locally known as “suso”) in the mn assays instead of b. glabrata because they are abundant in lake lanao and utilized as food by the meranaw (people of the lake). the strategic use of the snails in mn assays is anchored on their sedentary lifestyle crawling on soil and rock substratum. they use their gills as filter feeding apparatus and respiratory organs and are the first barrier to potential contaminants and the ideal target for genotoxic studies. they are tolerant to heavy metals and bio-accumulate them (baroudi et al., 2020). importantly, snails are less mobile compared to other organisms like fishes, thus they cannot easily escape from toxic substances present in their habitat. this study therefore primarily aimed to estimate the post-siege genotoxic hazards posed by munition residues in the lake through micronucleus assay using the v. angularis muller thriving in the lake. it also examined the micronucleation pattern among exposed snails sampled in lake lanao and nonexposed snails in the reference site, lake dapao, lds. factors like the distance of the sampling sites from the war zone and the age groups of the sampled snails were considered in the mn assays. materials and methods study design and sampling sites descriptive cross-sectional study design and the purposive sampling collection (accessibility of the sites and age groups of snails) were conducted in assessing the genotoxicity of the war residues by mn assay in the hemocytes of the exposed freshwater gastropods v. angularis of lake lanao. figure 1 shows the map of the philippines and the sampling sites, location coordinates, and the distance between sites. the first site was the lakeshore fronting marawi city marked as ground zero (site a) where the battle was fiercest and where residues of war were substantially deposited; the second (b) and third (c) sites were 8.15 km and 24.41 km away from site a. these three sites represent munition residues exposed sites. site d is the reference lake (non-exposed site), lake dapao, lds, 34.45 km from site a. the coordinates were determined using garmin(r), gpsmap 64x, and the actual sites were generated by plotting the coordinates through google earth. the distance of the sites from site a was calculated through movable type scripts (https://www.movable-type.co.uk/ scripts/latlong.html). https://www.movable-type.co.uk/%20scripts/latlong.html https://www.movable-type.co.uk/%20scripts/latlong.html post-siege genotoxic hazards in lake lanao, philippines – abamo et al. 255 figure 1a) map of the philippines showing the location of marawi city and lake lanao in the red inset. b) sampling sites (balloons) in lake lanao shores and their corresponding coordinates: red, site a, ground zero in marawi city; yellow, site b; blue, site c; and green, reference site (lake dapao) test organisms, collection, and identification the test organisms were v. angularis muller (class gastropoda, phylum mollusca, fig. 2) color, description, and identification of the snails were based on the work of pagulayan & cepillo (1991), moneva et al. (2012), and torres et al. (2014). sub-adult and adult v. angularis have 4 whorls with shell colors ranging from dark greenish brown or greyish yellow. it is striated but not with a hammer pattern. immature or juvenile vivipara is usually up to 14-16 mm while adult length ranges from 18-31 mm. the height of the shell is 25–35 mm. the width of the shell is 20– 26 mm. the shell has 5.5-6 weakly convex whorls. the last whorl is relatively large compared to that of other vivipara species. the umbilicus is narrow. vivipara snails were randomly collected within 100 m along the lakeshores and 5-50 m perpendicular to the shores. at least 20 juveniles and 20 adult snails were purposively handpicked, systematically washed to remove mud, placed separately in containers with lake water, and transported to research laboratory, biology department, mindanao state university, marawi campus. they were temporarily kept in the aerated aquaria with lettuce leaves. micronucleus assay proceeded immediately as soon as they were brought to the laboratory. figure 2 vivipara angularis muller snails collected from lake lanao (a); adults (b); and juvenile (c) samples biotropia vol. 30 no. 3, 2023 256 micronucleus assay twenty (n=20) v. angularis, i.e., ten (n=10) juvenile and ten (n=10) mature gastropods from each site, including the reference site, were randomly selected, and processed for mn assay following the work of da silva et al., 2013, de vasconcelos lima, 2019. to collect 0.1 ml of hemolymph, the operculum of the snail was gently tapped by a micropipette tip triggering the snails to retract and release hemolymph. the hemolymph was smeared into clean slides and air-dried for 10 min, followed by adding 0.1 ml of ringer’s solution, and 0.1 ml of the 10 mm edta solution. the smears were placed in a moist chamber for 30 min before fixing with glutaraldehyde for 5 min, rinsed with ringer’s solution, stained with giemsa solution (giemsa set, medic) for 7 min then finally washed with distilled water and air-dried. each slide was viewed for blind scoring of mn under a phase contrast microscope (olympus, ckx53). blind scoring was employed. with the help of a handheld counter, 1000 hemocytes (cells) were examined per individual gastropod, such that, (10 adults + 10 juveniles) x 1000 cells = 20,000 cells were examined per site. only hemocytes with an intact nucleus and cellular distinction were considered and the mn was identified and counted when its diameter was smaller than onethird the size of the nucleus, its color and texture should be similar to that of the nucleus and not in direct contact with the main nucleus. a digital camera (xcam1080phb, toup tek, china) was used to aid in counting the mn in the monitor and in taking photomicrographs. detection of heavy metals in the snail muscles muscles from adults and juvenile snails (150 g x 3 replicates) from each sampling site were harvested and immediately analyzed for heavy metals utilizing atomic absorption spectrophotometry (aas) for cd, cr, and pb, silver diethyldithiocarbamate-colorimetric (modified) for as and cold vapor aas for hg. statistical analysis the resulting mn frequencies between exposed and non-exposed sites and between juvenile and adult samples were analyzed statistically using statistical package for the social sciences (spss) for windows. using the shapirowilks test, the means of the mn frequencies for sites and age groups were found not normally distributed, hence kruskal-wallis and the mannwhitney u tests were used to analyze the differences of the mn frequencies in various sites and age groups. results and discussion micronucleation by sampling sites micronucleation (figure 3) was observed in the hemocytes of v. angularis in all sampling sites. table 1 shows the mean frequencies of the mn per site and the variation was highly significant by the kruskal wallis test, [p=.000, h(3, n=80)=28.0326]. indeed, mn frequencies were significantly higher in lake lanao sites compared to the reference site, lake dapao. the highest mean was recorded in site a, the ground zero where the encounter was heftiest, and the lowest in site d, the reference lake. a pairwise comparison of the mean ranks by the mannwhitney u test illustrated that site a was significantly higher than other sites (p<0.5) except for site c (p=0.37346). conversely, the reference site was significantly lower than the rest of the sites (p<.05). post-siege genotoxic hazards in lake lanao, philippines – abamo et al. 257 figure 3a-b photomicrographs of v. angularis hemocytes showing micronuclei (yellow arrows) in adult and juvenile samples. c, hemocytes without micronuclei are shown in c. scale bars, 5µm table 1 mn frequencies in the hemocytes of adult and juvenile v. angularis sampled from the different sites in lake lanao and in lake, dapao, philippines sampling sites mn frequency* mean sd juvenile adult total juvenile adult total site a 3.6 10 6.8 1.776 6.6 5.736 site b 2.4 2.4 2.4 2.319 1.506 1.903 site c 6.8 3.1 4.95 3.938 2.885 3.859 site d 1.5 0.8 1.15 1.65 0.422 1.226 total 3.58 4.08 3.82 3.21 5.025 4.197 *frequency values (mn observed per 1000 hemocytes) biotropia vol. 30 no. 3, 2023 258 spontaneous micronuclei formation can happen in any eukaryotic cell as it divides. it is often brought about by a broken chromosome fragment or rarely an entire chromosome that lags and remains outside the nucleus after cell division (beedanagari et al. 2014). these are extranuclear bodies that contain damaged chromosome fragments or whole chromosomes that are not incorporated into the nucleus after cell division (luzhna & kovalchuk 2013). it can be induced by defects in the cell repair machinery and accumulation of dna damages and chromosomal aberrations. this explains why mn was also observed in the reference lake although the count was significantly lower than the exposed sites. as mentioned earlier, mn frequencies were shown to increase in cells of various organisms that were exposed to carcinogens and genotoxins, thus, mn assay is one of the most widely used genotoxicity biomarkers, providing an efficient measure of chromosomal dna damage. the elevated mn counts in site a could be attributed to genotoxic chemicals most probably from war pollutants washed into the lake water where the snail samples were thriving. it must be noted that site a is fronting “ground zero” in marawi where the encounter was densest. iarc (2021) provided a list of eroded components of munitions that are carcinogenic including heavy metals, such as as, hg, cd, cr, and pb. these heavy metals were detected in the muscle tissues of v. angularis from sampling sites (table 6), and jalova et al. (2021) also reported heavy metals in commonly consumed fishes sampled from lake lanao, though the levels detected were mostly lower than the standard limits (who/fao 2010, 2011, factor et al. 2012, perelonia et al. 2017, and gbogbo et al. 2017). a closer look at table 2 revealed that a higher concentration of ar, (0.27 mg/l) from snail muscles was detected in site a, which was higher than the national acceptable limit in drinking water set at 0.010 mg/l, although the safe limit for mollusk as food is set at 1mg/kg. nevertheless, this detection may call for some concerns due to the continued consumption of the snails by the locals because this trace metal, aside from being highly toxic and carcinogenic (apostol et al. 2022), can biomagnify along with other toxic pollutants, that is, to increase in concentration in the living organisms successively at higher levels in the food chain (hepp 2017). the study by sanderson et al., (2017), reported that hmx and trace levels of rdx explosives compounds were found in the fiddler crabs from the live impact area (lia) in the island of vieques in puerto rico which also recorded higher levels of some metals such as arsenic. moreover, the study by dong et al. (2019) also published that arsenic-exposed human populations through drinking contaminated water had increased micronucleus formation from the buccal cells, lymphocytes, and urothelial cell samples. moreover, gbogbo et al. (2017) detected levels of arsenic in freshwater fish and shellfish from 0.2 to 2.2 mg/l in ghana rivers and recommended in their study that the maximum quantities of the organisms considered safe for consumption ranged from 375 to 5250 g per week. table 2 mean concentration of as, cd, cr, pb, hg (mg/l) detected in muscle tissues of v. angularis collected from the different sampling sites sampling sites heavy metals (mg/l) arsenic (as) mercury (hg) cadmium (cd) chromium (cr) lead (pb) site a 0.27 ≤ 0.01* ≤ 0.025* ≤ 0.025* 0.350 site b ≤ 0.025* ≤ 0.01* ≤ 0.025* 0.893 0.647 site c ≤ 0.025* ≤ 0.01* ≤ 0.025* 0.92 0.507 site d ≤ 0.025* ≤ 0.01* ≤ 0.025* ≤ 0.025* 0.54 *machine reporting limit post-siege genotoxic hazards in lake lanao, philippines – abamo et al. 259 micronucleation by age groups and increasing distance from ground zero figure 1 shows the relative distance of the sampling sites from site a where the fiercest fight occurred. looking at figure 4, adult snails obviously displayed a decreasing trend of mn frequencies with increasing distance from site a but not among the juvenile samples. comparing the mn counts between age groups, it is clearly seen that the exposed adults in site a marked significantly high mn frequencies compared to their juvenile counterparts (table 3). these results implied that the genotoxic contaminants in these sites affected the adult snails more than the juvenile samples. it makes sense knowing that the average life expectancy of freshwater snails is 3 to 5 years (https://foliargarden.com/ lifeexpectancy-of-a-freshwater-snail/), and some of the adult samples must have already been at the site during the siege and thus were the most exposed to war pollutants at the highest magnitude which may have resulted in dna damage as indicated by higher mn frequency compared to the juveniles. figure 4 profile plots of the estimated marginal means of mn frequencies in different sites and age groups showing a downward trend among adults from sites a, and b to d, except in site c which showed an unexpected increase table 3 pairwise comparison of mn frequencies between age groups at different sites by mann-whitney u sites comparison of mn frequencies between juvenile and adult sig. level remarks site a 0.011 significantly different (adult has higher count) site b 0.853 not significantly different site c 0.023 significantly different (adult has lower count) site d 0.579 not significantly different biotropia vol. 30 no. 3, 2023 260 on the other hand, in site c, which is 24.41 km away from site a, a different mn pattern was observed wherein juveniles displayed higher mn counts than adults. these results were unexpectedly alarming and need further studies. this must be attributed to some genotoxic substances other than the munition residues due to war which particularly predisposed the young snails. one possible explanation is the nearby brassware industry which utilizes brass, a metal alloy primarily composed of cu and zn. copper alloy with a higher amount of cu is known to cause ‘contact killing’, possibly via redox cycling and production of reactive oxygen species (ros) groups that can cause lethal damage to cell membranes and oxidative damage to dna (grass et al. 2011 and bille et al. 2013). dna has a higher affinity to cu2+ which may affect the integrity of dna and the normal process of dna replication and transcription (govindarajua et al. 2013) and consequently micronucleus formation (nikdehghan et al. 2018)substantial evidence has shown the possible genotoxicity of cu and zn in aquatic organisms. moreover, looking back at table 2, cr was detected highest in site c although it was closely followed by site b. the study by el safty et al. (2018) demonstrated that crand ni-exposed workers of the electroplating industry had increased mn signifying cytogenetic damage caused by these heavy metals. the observed mn frequencies in this study are comparable to the works of silva et al. (2011) and de vasconcelos lima (2019) who tested the genotoxicity of gamma radiation and heavy metals and domestic sewage sludge, respectively against freshwater snails b. glabrata using the mn assay. the assay was also utilized by da silva et al. (2013) when they tested tobacco leaves in the land snails helix aspersa. their results considered mn assays as a reliable parameter to measure genotoxic hazards in the environment using snails. although the mn frequencies in the present study are baseline data to approximate genotoxicity in the munition-laden lake lanao using v. angularis. further studies must be conducted to verify the genotoxic chemicals in crucial sites. conclusion post-siege genotoxic hazard in lake lanao, lds, philippines brought by marawi siege in 2017 was assessed through mn assay using v. angularis snails sampled from different sites in lake lanao and in the reference site lake dapao. results demonstrated a higher mn frequency in snails sampled from munition-exposed sites in lake lanao lakeshores than in the reference site, lake dapao. among the adult samples, there was a decreasing trend of mn frequencies with increasing distance from lake shore fronting ground zero at marawi city where the siege was heaviest. a remarkable difference was also found in the mn frequency patterns between the age groups. in all sites, except site c, adults had higher mn frequencies than juveniles and the difference was significant in site a. moreover, it is generally accepted that mn frequency increases with age, but unexpectedly, mn frequencies were significantly higher among the juvenile samples than adult samples in site c. the post-siege genotoxic hazards possibly posed by the war pollutants in lake lanao near ground zero cannot be overlooked. local leaders must be informed of the results for possible interventions to protect the health and well-being of the populace around marawi and lake lanao. acknowledgement the authors are truly grateful to the philippine council for health research and development department of science and technology (pchrd-dost) grants-in-aid program for the generous financial support for this project. references alborz a. 2013. environmental characteristics and prevalence of birth defects among children in postwar iraq: implications for policies on rebuilding the iraqi education system. med confl surviv 29(1):2644. https://doi.org/0.1080/13623699.2013.765197 apostol glc, valenzuela s, seposo x. 2022. arsenic in groundwater sources from selected communities surrounding taal volcano, philippines: an exploratory study. earth 3: 448-459. https://doi.org/10.3390/earth3010027 https://doi.org/10.3390/earth3010027 post-siege genotoxic hazards in lake lanao, philippines – abamo et al. 261 baroudi, f., al-alam, j., fajloun, z., & millet, m. 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(2010). joint expert committee on food additives (jecfa) meeting (72nd: rome, italy), pp. 16. https:// apps.who.int/iris/handle/10665/44520 who/fao (2011). jecfa meeting (73rd: switzerland). evaluation of certain food additives and contaminants: technical report series 960: 228. https://apps.who.int/iris/handle/ 10665/44515 https://monographs.iarc.who.int/list-of-classifications https://monographs.iarc.who.int/list-of-classifications https://monographs.iarc.who.int/list-of-classifications https://doi.org/10.46989/001c.24939 https://doi.org/10.1007/s10661-016-5491-0 https://doi.org/10.1016/j.envres.2021.111568 https://apps.who.int/iris/handle/10665/44520 https://apps.who.int/iris/handle/10665/44520 https://apps.who.int/iris/handle/10665/44515 https://apps.who.int/iris/handle/10665/44515 biotropia vol. 29 no. 2, 2022: 181 184 doi: 10.11598/btb.2022.29.2.1709 181 short communication ageing and the amount of dna extracted from bawean deer (axis kuhlii) feces adi nugroho*1, denni susanto1 and sena adi subrata2 1department of bioresources technology and veterinary, vocational college, universitas gadjah mada, yogyakarta 55281, indonesia 2department of forest resource conservation, faculty of forestry, universitas gadjah mada, yogyakarta 55281, indonesia received 4 december 2021/accepted 15 march 2022 abstract noninvasive genetic sampling is the most reliable approach to collect endangered species which are typically rare and elusive. however, the approach is hampered by poor quality and quantity of dna amount, and therefore, a pilot study is required. our current short communication article reports the first noninvasive genetic sampling approach using feces of critically endangered bawean deer (axis kuhlii) to test the effect of aging on the amount of dna extraction. fecal samples of the bawean deer were collected from the bawean deer captive unit in sangkapura village, bawean island, east java. the samples were grouped into two different categories, namely: 1) new samples: for feces that had just been expelled for less than a day and 2) old samples: for feces that were estimated to have been expelled for more than a day. there were 11 new samples and 16 old samples. the samples were extracted using the qiagen mini stool kit. the dna quantification was carried out by using a fluorometer. the results of the extraction between the two categories were analyzed by the kruskal wallis test. the results showed that the mean rank value of the new samples was better (15.27) than the old samples (13.13). the p value of the kruskal wallis test, however, was higher than the asymp significant value, indicating that there was no meaningful differences (p = 0.49) between the two samples categories. the work described in this short communication article is a preliminary result and an important step in the study of bawean deer population genetic. keywords: bawean deer, biodiversity, critically endangered, dna, noninvasive sampling introduction conservation strategies for endangered species require genetic information (whiteley et al. 2015) that can be difficult to obtain using a traditional method such as invasive sampling and destructive sampling. in small and elusive wildlife populations, noninvasive dna sampling was successfully employed for obtaining genetic data (kohn & wayne 1997). however, the effective use of noninvasive samples for genetic analyses of wildlife populations is hampered by typically poor quality and low quantity of the dna obtained (taberlet & waits 1999). in large animals, dna samples can be obtained from fluids, hair and feces gathered in the field. however, these types of samples usually provide dna of low quantity, quality, and integrity, causing the difficulties in analyzing some molecular markers (venegas et al. 2020). dna of the depositor is present in sloughed intestinal epithelial cells (waits & paetkau 2005). dna degradation in fecal dna is enhanced by ageing of the samples and by the action of variable environmental conditions (e.g., temperature, humidity, exposure to sun or rain) (nsubuga et al. 2004). the effectiveness of genetic analysis using fecal samples would be improved if some of the factors affecting dna yield could be identified and used as guidelines in sample collection. recent study has analyzed the effect of ageing and environmental condition on the amount of dna extraction from deer feces *corresponding author, email: adi.adinugroho@ugm.ac.id biotropia vol. 29 no. 2, 2022 182 (brinkman et al. 2010; ebert et al. 2021; gupta et al. 2018). however, these studies were conducted on temperate climate conditions. to our knowledge, such study has not been conducted in tropical environments which have different uv radiation from sunlight and humidity. our current short communication article reports the first noninvasive genetic approach using feces of critically endangered bawean deer (axis kuhlii) to test ageing on the amount of dna extraction. materials and methods sample collection the bawean deer feces were collected from the bawean deer captive unit at bawean island, east java. the fecal samples were categorized as new for feces that has probably been deposited for less than one day and old for feces that has been deposited for more than 1 day. the two categories of feces were differentiated by evaluating its appearance and by monitoring the depositor on the captivity. the feces were collected using a feces container added with silica granules and labeled. the fecal samples were collected on august 2021. the fecal dna were immediately extracted at the laboratory of bioresources and veterinary technology, ugm vocational college. dna extraction the dna extraction was carried out by using qiamp dna stool mini kit (qiagen) following the manufacturer’s protocol. the results of the extraction (double stranded dna only) were measured by a fluorometer (quantus fluorometer, promega). results and discussion the 27 collected bawean deer feces were categorized as the new and old feces. the new category is characterized by the moist and glittering appearance, while the old category is relatively dry and harsh in appearance (figs. 1 & 2). figure 1 bawean deer feces categorized as new with the moist and glittering appearance ageing and the amount of dna extracted from bawean deer feces – adi nugroho et al. 183 figure 2 bawean deer feces categorized as old with the dry and harsh appearance the dna extraction showed different quantities of dna at different sample conditions as shown in table 1. table 1 quantification of dna extraction from bawean deer feces new samples (ng/µl) old samples (ng/µl) 0.615 0.73 0.993 1.245 2.061 0.805 1.512 0.73 0.079 1.106 2.857 1.655 12.224 6.352 2.613 8.82 5.845 0.066 7.123 0.207 3.179 7.727 1.189 3.057 1.972 2.743 3.379 kruskall wallis test showed that the new feces samples resulted to better dna quantification compared to the old feces samples (mean rank = 15.27). however, there is no significant difference between the two categories (p = 0.477). table 2 kruskall wallis analysis sample n mean rank quantity new 11 15.27 old 16 13.13 total 27 table 3 kruskal wallis test (p > 0.05) quantity kruskal-wallis h 0.477 df 1 asymp. sig. 0.490 a. kruskal wallis test b. grouping variable: sample this finding is in line with the work of agetsuyama-hanigara et al. (2017) showing that it is recommended to use fecal samples not older than 3 days old for genetic analysis of the sika deer (cervus nippon yakushimae). this study shed the light on the importance of collecting the new fecal samples for studying bawean deer genetics in a natural population with a noninvasive sampling approach. however, the finding is only limited to dna quantity which can come from bacteria, fungi, or any other genetic sources in the feces. further research is required to explore the optimum condition for successful species-specific bawean deer dna amplification. biotropia vol. 29 no. 2, 2022 184 conclusion the data showed that the new fecal samples provided better results of dna extraction than the old fecal sample. nevertheless, the differences are not significant. acknowledgments the author would like to thank the research directorate of universitas gadjah mada for supporting this study through the young lecturer grant no. 2458/un1/ditlit/ditlit/pt/2021. references agetsuma-yanagihara y, inoue e, agetsuma n. 2017. effects of time and environmental conditions on the quality of dna extracted from fecal samples for genotyping of wild deer in a warm temperate broad-leaved forest. mammal res 62(2):201-7. doi: 10.1007/s13364-016-0305-x brinkman tj, schwartz mk, person dk, pilgrim kl, hundertmark kj. 2010. effects of time and rainfall on pcr success using dna extracted from deer fecal pellets. conserv genet 11(4): 1547-52. doi: 10.1007/s10592-009-9928-7 ebert c, sandrini j, welter b, thiele b, hohmann u. 2021. estimating red deer (cervus elaphus) population size based on non-invasive genetic sampling. eur j wildl res 67(27):1-13. doi: 10.1007/s10344-021-01456-8 gupta sk, kumar a, angom s, singh b, ghazi mgu, tuboi c, hussain sa. 2018. genetic analysis of endangered hog deer (axis porcinus) reveals two distinct lineages from the indian subcontinent. sci rep 8(1):1-12. doi: 10.1038/s41598-018-34482-9 kohn mh, wayne rk. 1997. facts from feces revisited. trends ecol evol 12(6)l223-7. doi: 10.1016/s0169-5347(97)01050-1 nsubuga am, robbins mm, roeder ad, morin pa, boesch c, vigilant l. 2004. factors affecting the amount of genomic dna extracted from ape faeces and the identification of an improved sample storage method. mol ecol 13(7):2089-94. doi: 10.1111/j.1365-294x.2004.02207.x taberlet p, waits lp, luikart g. 1999. noninvasive genetic sampling: look before you leap. trends ecol evol 14(8):323-7. venegas c, varas v, vásquez jp, marín jc, de genómica l, de ciencias f, bio-bío u. 2020. non-invasive genetic sampling of deer: a method for dna extraction and genetic analysis from antlers. gayana 84(1):75-82. waits l, paetkau d. 2005. noninvasive: genetic sampling tools for wildlife biologists: a review of applications and recommendations for accurate data collection. j wildl manage 69(4):1419-33. whiteley ar, fitzpatrick sw, funk wc, tallmon da. 2015. genetic rescue to the rescue. trends ecol evol 30(1):42-9. doi: 10.1016/ j.tree.2014.10.009 biotropia vol. 29 no. 2, 2022: 112 123 doi: 10.11598/btb.2022.29.2.1637 112 dragonflies diversity and land cover changes in the batubolong river, west lombok district muhammad zulhariadi1*, raden dedi irawan2, aulia zulfaeda3, nurul hidayani4 and frendi irawan5 1,2,3biology education department, faculty of tarbiyah and teaching, universitas islam negeri mataram, mataram 83116, indonesia 4remote sensing and gis department, vocational college, universitas gadjah mada, yogyakarta 55281, indonesia 5indonesia dragonfly society, yogyakarta 55272, indonesia received 19 august 2021/accepted 13 october 2021 abstract west lombok district is the second largest district in lombok islands. the diversity of dragonflies (order odonata) as a bioindicator of environmental quality has not been widely studied in the west lombok region. this study aimed to determine the species diversity of dragonflies (order odonata) found in batubolong river, west lombok district and its relation to the occurring land cover changes. this study was carried out by using a descriptive explorative method, where the sampling technique was done by means of line transects. maps of study and sampling locations as well as land cover changes were made using arcgis 10.4.1 software based on primary and secondary data. the results showed that there were 11 species of dragonflies with a shannon-wiener diversity index value (h') of 2.212 (medium diversity), a population density index (c) of 0.126 (low dominance), and a species evenness index (e) of 0.923 (high uniformity). our study also found two rare species of dragonfly in lombok island i.e., 3 individuals of nososticta emphyla (lieftinck, 1936) with 9% relative abundance and 1 individual of drepanosticta berlandi (lieftinck, 1939) with 3% relative abundance. the discovery of d. berlandi in lombok is the third time after the findings in 1896 and the 19th century. analysis of satellite data around the sampling area within the period 2013-2020 showed that there has been an increase in land cover of 6,149.29 m2. the increase in land cover changes may have caused the disappearance of several odonata species in the sampling location. keywords: batubolong river, diversity, dragonfly, land cover, odonata introduction west lombok district is among districts in west nusa tenggara province, indonesia, located at 115°49.12'04”-116°20'15.62”e and 8°24'33.82"-8°55'19"s and is directly adjacent to mataram city, central lombok and north lombok districts and the indian ocean (bps statistics lobar 2021). the west lombok district has a large area with relatively dense population and settlements in the west nusa tenggara province (ntb). biodiversity in this district is similar to that in other cities/districts in the province. the west lombok district has several forests and extensive beaches, rivers and a nature tourism park (taman wisata alam/twa) managed by the provincial office of the natural resources conservation center of the ntb (rahmawati et al. 2019). several tree species as birds’ habitat as well as rice fields and plantations in several areas of the district still exist. there are also various types of dragonfly species in various areas of west lombok district. dragonfly (odonata) is a good bioindicator of water quality (dolný et al. 2013; koneri et al. 2020). the damselfly (suborder zygoptera) and the common dragonfly (suborder anisoptera) are members of the order odonata of the phylum arthropoda. various types of dragonflies are identified in various spots in west lombok district. the habitats of the damselfly and the dragonfly are scattered at various points, ranging from forests, rice fields, areas close to housings to areas far from s ettlements, hot and open areas to shady and closed areas. several study *corresponding author, email: zulhariadi@uinmataram.ac.id dragonfly diversity and land cover changes in the batubolong river – muhammad zulhariadi et al. 113 locations regarding dragonflies have been reported in the west lombok area, namely the batubolong river and pusuk area (kosterin 2014), kerandangan twa (rahmawati et al. 2019) and suranadi twa (ilhamdi et al. 2021). changes in land cover and forest land conversions contribute to the decreasing diversity of various fauna species, especially several types of dragonflies that are vulnerable to environmental changes. several types of dragonflies inhabit several areas of west lombok regency which are still beautiful because dragonflies choose to live in a safe and comfortable place for their reproductive activities, especially in the life of their nymphs (ansori 2009; dolný et al. 2011). the batubolong river is a river that has its headwaters in lembah sari village, batulayar district and passes through batubolong hamlet, west batulayar village and has an estuary on the batubolong beach, batulayar district. this study aimed to observe the diversity of dragonflies (order odonata) in the batubolong river, west lombok district and to determine the impact of the recent changes in land cover around the river area toward dragonfly diversity. the results of this study will be compared with previous studies that have taken data in the batubolong river, west lombok. materials and methods this study was carried out by using descriptive explorative method for identifying the types of dragonflies (suborder anisoptera) and damselfly (suborder zygoptera) in the batubolong river. sampling was carried out for 4 months from april 2021 (rainy season) up to july 2021 (dry season) in the middle part of the batubolong river (fig. 1). the purposive sampling technique was used with line transects. environmental parameters observed were water ph and temperature, total dissolved solids (tds), electrical conductivity (ec), altitude, wind speed, air temperature and humidity. documentation of dragonflies was carried out by using a camera, while the identification of dragonflies was conducted by using references from irawan & rahadi (2018), kosterin (2014), steinmann (1997) and gbif.org. the relative species abundance, species diversity index, population density index and species evenness index were then calculated based on the data obtained from the field. maps of study and sampling locations as well as land cover changes were developod by using arcgis 10.4.1 software based on primary and secondary data. figure 1 map of the study location in the batubolong river, batulayar barat village, batulayar subdistrict, west lombok district biotropia vol. 29 no. 2, 2022 114 the relative species abundance was calculated using the formula: rsa = ni x 100% n where: rsa = relative species abundance ni = number of individual dragonflies i n = number of individuals of all (total) types of dragonflies the shannon-wiener diversity index (h') was calculated using the formula: h' = σ (pi ln pi) where: h' = shannon-wiener diversity index pi = ratio of the number of individuals of one species to the total number of individuals in the sample within the plot (n/n) diversity index criteria: h' < 1 = low diversity 1 < h' < 3 = medium diversity h' > 3 = high diversity population density/dominance index (c) was calculated using the formula: where: c = dominance index ni = number of individuals of one species n = total individuals of all species environmental community criteria based on dominance index: 0.00 ≤ c ≤ 0.30 = low dominance 0.30 ≤ c ≤ 0.60 = medium dominance 0.60 ≤ c ≤ 1,00 = high dominance the evenness index of species at the sampling point was calculated using the formula: e = h' ln s where: e = evenness index h' = shannon-wiener diversity index s = number of species environmental community criteria based on evenness index: 0.00 < e < 0.50 = small uniformity/depressed community 0.05 < e < 0.75 = moderate uniformity/ unstable community 0.75 < e < 1.00 = high uniformity/stable community results and discussion landscape and canopy in the batubolong river sampling area batubolong river is a type of river with small to large granite rocks adorning the river (fig. 2). various types of canopy plants over the riverbanks providing shady and beautiful ambiance are dominated by various types of bamboo, ivory mahogany (dysoxylum gaudichaudianum), cembirit/kumbi (tabernaemontana sphaerocarpa), pacific walnut (dracontomelon dao), tamarind (tamarandus indicus), mahogany (swietenia mahagoni), acacia (acacia sp.), kapok (ceiba pentandra), mango (mangifera indica), sugar palm (arenga pinnata), cashew (anacardium occidentale), black rosewood (dalbergia latifolia), and sengon (paraserianthes falcataria). several types of shrubs and medium-sized trees growing along the riverbanks include lantana (lantana camara), siam weed (chromolaena odorata), quickstick (gliricidia sepium), largeleaf rosemallow (hibiscus macrophyllus), various types of bananas (musa sp.), bandicoot berry (leea aequata l.) sparrow mango (buchanania arborescens), taro (araceae), grasses (graminaceae), vines such as spurred butterfly pea (centrosema virginianum), sweet leaf (sauropus androgynous), papaya (carica papaya), ferns (pteridophyta), and turmeric (curcuma sp.). dragonfly diversity and land cover changes in the batubolong river – muhammad zulhariadi et al. 115 figure 2 landscape area at the dragonfly observation point on the batubolong river notes: a & b = river view during rainy season (4 april 2021); c & d = river view during dry season (6 july 2021). source: personal data (2021). most of the batubolong river stream is far from the residential area. only the downstream is close to the residents' villages. the canopy cover area on the batubolong river is in the class i-iv category (class i: 0-25% canopy cover, class ii: 26-50% canopy cover, class iii: 51-75% canopy cover, and grade iv: 76-100% canopy cover) (buckley et al. 2018; hendriks 2020). topographical and water physicalchemical parameters in the batubolong river table 1 presents the results of topographical and water physical-chemical parameters measurement in the batubolong river. in the topographical aspect, the batubolong river which passes through the area around the batulayar barat village is still at a low altitude (20 40 masl) with the highest air temperature of 36.1 oc and the lowest is 25.1 oc at the sampling time. the neutral water ph (6.5) combined with low tds (tds = 66 ppm), and low ec values (ec= 136 µs/cm) indicate that river water quality was still within the range of standard water quality (khairunnas & gusman 2018). the river water is also free of total dissolved solids and low in electrical conductivity; thus, it is safe as drinking water (afrianita et al. 2017; gasim et al. 2015). the standard of total dissolved solids for drinking water is below 1,000 ppm (who 1996). the water temperature (25 28 oc) of the batubolong river is still in the moderate category which is very good for the development of nymph larvae of several types of dragonflies. in the water bodies, medium to small fish and small crabs are found which can be a threat to dragonfly nymphs. biotropia vol. 29 no. 2, 2022 116 table 1 measurement of topographical and water physical-chemical parameters in the batubolong river no. topographical and water physicalchemical parameters measurement results observation date 1. altitude (masl) 20 40 6 july 2021 2. air temperature (oc) 30.4 36.1 25.1 28.9 4 april 2021 (10.56 13.53 wita) 7 june 2021 (10.07 12.13 wita) 3. humidity (%) 74 86 57 65 4 april 2021 (10.56 13.53 wita) 7 june 2021 (10.07 12.13 wita) 4. water ph 6.5 4 april 2021 and 7 june 2021 5. soil ph 5.8 4 april 2021 6. total dissolved solid (tds) (ppm) 66 7 june 2021 7. electrical conductivity (ec) (µs/cm) 136 7 june 2021 8. water temperature (oc) 27 28 25 4 april 2021 (10.56 13.53 wita) 7 june 2021 (10.07 12.13 wita) 9. wind speed (knot) 0.0 1.6 0.0 0.5 4 april 2021 (10.56 13.53 wita) 7 june 2021 (10.07 12.13 wita) dragonfly (odonata) diversity in batubolong river the observed dragonflies (odonata) at various points on the batubolong river is presented in table 2. the distribution map of dragonflies (order odonata) found at the sampling location on the batubolong river is presented in figure 3. table 2 dragonflies (odonata) distribution in the batubolong river no. suborder and species name total observation time coordinate point perch and brightness altitude (masl) air temperature (oc) / humidity(%) a. suborder zygoptera 1. euphaea lara lombokensis (mclachlan, 1898) 5 10.50 wita, 04/04/2021 and 06/07/2021 -8.497485, 116.065951 on a rock, on a small branch 200-300 lux (shady) 20 30 / 86 2. pseudagrion pilidorsum declaratum (lieftinck, 1936) 5 10.50 wita, 04/04/2021 and 06/07/2021 -8.497485, 116.065951 on a rock, on a small branch 200-300 lux (shady) 20 30 / 86 3. nososticta emphyla (lieftinck, 1936) 3 13.53 wita, 04/04/2021 and 06/07/2021 -8.497350, 116.066083 -8.496795, 116.067063 on the leaves and twigs of the lantana (lantana camara) and kirinyuh (chromolaena odorata) trees 200-1500 lux (shady bright) 40 36.1 / 74 4. drepanosticta berlandi (lieftinck, 1939) 1 11.01 wita, 06/07/2021 -8.496795, 116.067063 under the leaves and stems of trees on the river 200 lux (shady) 40 28.7 / 66 dragonfly diversity and land cover changes in the batubolong river – muhammad zulhariadi et al. 117 table 2 (continued) b. suborder anisoptera 5. orthetrum testaceum soembanum (forster, 1903) 5 11.11 wita, 04/04/2021 and 06/07/2021 -8.497503, 116.065891 on a wooden branch < 2,000 lux (hot) 20 31.5 / 81 6. neurothemis ramburii (brauer, 1866) 3 11.32 wita, 04/04/2021 and 06/07/2021 -8.497597, 116.065791 on a wooden branch < 2,000 lux (hot) 20 30.4 / 85 7. trithemis festiva (rambur, 1842) 3 11.35 wita, 04/04/2021 and 06/07/2021 -8.497597, 116.065791 on the rock < 2,000 lux (hot) 20 30.4 / 85 8. orthetrum glaucum (brauer, 1865) 2 11.45 wita, 04/04/2021 and 06/07/2021 -8.497597, 116.065791 on the rock < 2,000 lux (hot) 20 30.4 / 85 9. agrionoptera insignis insignis (rambur, 1842) 1 11.39 wita, 06/07/2021 -8.495512, 116.068244 on the taro leaves 2,000 lux 20 28.9 / 57 10. orthetrum sabina (drury, 1773) 1 11.04 wita, 06/07/2021 -8.496763, 116.067334 on the branches of the bush < 2,000 lux (hot) 20 34.5 / 45 11. diplacodes trivialis (rambur, 1842) 2 11.41 wita, 06/07/2021 -8.495515, 116.068239 on stones and twigs < 2,000 lux (hot) 20 28.9 / 57 figure 3 distribution map of dragonfly (order odonata) at sampling locations on the batubolong river note: several species have the same location point. biotropia vol. 29 no. 2, 2022 118 sampling was carried out in the rainy (april) and dry (july) seasons. more dragonfly species were found in april (rainy season) than in july (dry season). dragonfly species found both in the rainy and dry seasons were: euphaea lara lombokensis (mclachlan, 1898), pseudagrion pilidorsum declaratum (lieftinck, 1936), nososticta emphyla (lieftinck, 1936), orthetrum testaceum soembanum (forster, 1903), neurothemis ramburii (brauer, 1866), trithemis festiva (rambur, 1842), and orthetrum glaucum (brauer, 1865). species of dragonflies (order odonata) that were only found during sampling in july (dry season) were drepanosticta berlandi (lieftinck, 1939), agrionoptera insignis insignis (rambur, 1842), orthetrum sabina (drury, 1773), and diplacodes trivialis (rambur, 1842). during the rainy season, the river water is abundant so that dragonflies have more opportunities to reproduce because water is a medium for dragonflies to lay eggs and develop larvae. the canopy and the light intensity also determine the types of dragonfly species existing around the river. dragonflies which prefer hot areas in the batubolong river are: orthetrum sabina (drury, 1773), diplacodes trivialis (rambur, 1842), orthetrum testaceum soembanum (forster, 1903), neurothemis ramburii (brauer, 1866), trithemis festiva (ramburi, 1866). 1842), and orthetrum glaucum (brauer, 1865). this findings is in agreement with the work of buchori et al. (2019). on the other hand, dragonflies which prefer shaded to moderate light areas in the batubolong river are: euphaea lara lombokensis (mclachlan, 1898), pseudagrion pilidorsum declaratum (lieftinck, 1936), nososticta emphyla (lieftinck, 1936), drepanosticta berlandi (lieftinck, 1939), and agrionoptera insignis insignis (rambur, 1842). relative species abundance, species diversity, population density and species evenness indices table 3 presents the calculation results of relative species abundance, species diversity, population density and evenness indices. euphaea lara lombokensis had the highest relative abundance of 21%, followed by pseudagrion pilidorsum declaratum and orthetrum testaceum soembanum of 15%, respectively, indicating that these species were able to breed and adapt well to the batubolong river. these three species are easy to find in rivers or streams having clean water with water ph close to neutral. the calculation results on the shannonwiener diversity index (h') of 2.212 (medium diversity category) indicated that the dragonfly species found in the sampling area were relatively few. the medium diversity may be caused by the crowded activity of the population around the river, especially in the downstream. at several points in the sampling area, there were logged trees and cowsheds owned by residents. therefore, it is necessary to expand the sampling area, especially in the upstream part which is still in the form of primary forest, considering that some dragonflies are very sensitive to community activities and environmental changes. table 3 calculation of relative species abundance, species diversity, population density and evenness indices no. species name total pi rsa ln pi h' c e 1. euphaea lara lombokensis (mclachlan, 1898) 7 0.212 21% 1.551 0.329 0.045 2. pseudagrion pilidorsum declaratum (lieftinck, 1936) 5 0.152 15% 1.887 0.286 0.023 3. nososticta emphyla (lieftinck, 1936) 3 0.091 9% 2.398 0.218 0.008 4. drepanosticta berlandi (lieftinck, 1939) 1 0.030 3% 3.497 0.106 0.001 5. orthetrum testaceum soembanum (forster, 1903) 5 0.152 15% 1.887 0.286 0.023 6. neurothemis ramburii (brauer, 1866) 3 0.091 9% 2.398 0.218 0.008 7. trithemis festiva (rambur, 1842) 3 0.091 9% 2.398 0.218 0.008 8. orthetrum glaucum (brauer, 1865) 2 0.061 6% 2.803 0.170 0.004 9. agrionoptera insignis insignis (rambur, 1842) 1 0.030 3% 3.497 0.106 0.001 10. orthetrum sabina (drury, 1773) 1 0.030 3% 3.497 0.106 0.001 11. diplacodes trivialis (rambur, 1842) 2 0.061 6% 2.803 0.170 0.004 total 33 1 100% 28.615 2.212 0.126 0.923 notes: pi = ratio between the number of individuals of a species with the number of individuals of all species (n/n); rsa = relative abundance; h' = shannon-wiener diversity index; c = dominance index; e = evenness index. dragonfly diversity and land cover changes in the batubolong river – muhammad zulhariadi et al. 119 nososticta emphyla is a beautiful and rare dragonfly with only 3 species found in the sampling location (rsa = 9%). this dragonfly has a characteristic of having a metallic purple combined with black colors on the thorax. the last 3 segments at the end of the abdomen have a bright purple color. lieftinck (1953) described an adult male of n. emphyla dragonfly found on flores island has a deep reddish-purple color on the head. kosterin (2014) described n. emphyla dragonfly as having purple color gradually changing to citron-yellow on the thorax on the abdominal segments 1-3 with purple and brilliant cobalt blue on segments 8-10. this dragonfly is classified as rare on lombok island and is only found in the batubolong river area. according to the iucn redlist, n. emphyla is in the data deficient (dd) category and its presence is only recorded in the nusa tenggara islands, namely lombok island, sumbawa island and flores island (dow 2020). n. emphyla is found in the batubolong river at coordinates -8.497350, 116.066083 and -8.496795, 116.067063. when found, this dragonfly were flying across the riverbank and then perched on the nearby lantana (lantana camara), kirinyuh (chromolaena odorata) and bamboo trees. these dragonflies tend to be shy and will fly up into taller trees when caught. drepanosticta berlandi (lieftinck, 1939) was the rarest needle dragonfly found at the sampling point because only one male was found in immature condition. according to the iucn redlist, this species is endemic to the island of lombok and is recorded 2 times, namely in 1896 at an altitude of 2,000 masl as many as 2 males and in the 19th century as many as 1 male (kalkman 2009). the latest data indicated that in 2016, d. berlandi was also found on sumba island, east nusa tenggara in forest areas in the manupeu tanah daru and laiwangi wanggameti national parks, with high humidity, wet vegetation and dense canopy (irawan & rahadi 2018). when found in the batubolong river, d. berlandi was perching on the leaves under a shady tree. d. berlandi is classified as a long needle dragonfly with a body length of 5.96 cm. d. berlandi is categorized as dd (data deficient) in the iucn red list or the distribution and population data are still lacking so that the risk of extinction of this dragonfly is still unclear (kalkman 2009). several quite rare dragonfly species of the suborder anisoptera (large dragonflies) were found in the batubolong river. dragonfly orthetrum testaceum subsp. soembanun (foerster, 1903) is one of the large dragonflies that is quite sensitive to environmental changes and is only found in rivers or streams that are still clean. according to a report from gbif.org (20122019), the dragonfly orthetrum testaceum subsp. soembanun is only recorded in the islands of nusa tenggara (lombok island, sumbawa island, flores island, alor island and rote island). in addition, the dragonfly orthetrum testaceum subsp. soembanun is also reported to be in the batubolong river on lombok island (kosterin 2014) and in the manupeu tanah daru and laiwangi wanggameti national parks on sumba island (irawan & rahadi 2018). this dragonfly mating behavior can be seen during copulation and oviposition. at the time of copulation, the male and female dragonflies form a tandem formation, namely the female attaches the end of the abdomen (appendage) to the male secondary genitalia (segments 1–2 on the ventral abdomen), while oviposition is when the female lays her eggs on the substrate with an ovipositor (loiola & de marco 2011). the uniqueness of oviposition behavior in dragonflies orthetrum testaceum subsp. soembanun, happens when the female lays her eggs in the water, the males fly over them to guard them (irawan & rahadi 2018). at the sampling time of our study in the batubolong river, the male orthetrum testaceum subsp. soembanun dragonflies were at the river, while the female orthetrum testaceum subsp. soembanun perched on tree branches at the top of the river. another rare large dragonfly found in the batubolong river is agrionoptera insignis subsp. insignis (rambur 1842). according to gbif.org data between 1997-2020, this dragonfly is only found in 3 locations in indonesia, namely java island, kalimantan island and papua island. another report states that this dragonfly has been found in the batubolong river (lombok island) (kosterin 2014) and in the manupeu tanah daru and laiwangi wanggameti national parks on sumba island (ntt) (irawan & rahadi 2018). at young age, this dragonfly is similar to lathrecista asiatica, especially in terms of the biotropia vol. 29 no. 2, 2022 120 pattern on the thorax and abdomen. if you look closely, the two species have different thorax patterns. agrionoptera insignis insignis has a thinner (flat) abdomen with a slightly thicker septum than l. asiatica. at the end of agrionoptera insignis insignis abdomen there are 3 black segments, while in l. asiatica only the last 2 segments are black. land cover changes around the sampling locations on the batubolong river the analysis results of land cover changes using satellite imagery data for the period 20132020 around the dragonfly sampling locations on the batubolong river is presented in figure 4, figure 5 and table 4. figure 4 map of land cover changes around the sampling locations for the period 2013-2020 dragonfly diversity and land cover changes in the batubolong river – muhammad zulhariadi et al. 121 figure 5 land cover changes in area surrounding the sampling locations notes: spatial analysis using gis applications obtained from: 1) high resolution satellite map of ntb province from lapan (2021); 2) map of ntb provincial rtrw administrative data from pupr ntb province. table 4 land cover area around the batubolong river no. year land cover area (m2) 1 2013-2015 24,532.95 2 2016 25,064.20 3 2017 25,689.16 4 2018 25,689.16 5 2019 26,340.81 6 2020 30,682.24 notes: spatial analysis using gis applications obtained from: 1) high resolution satellite map of ntb province from lapan (2021); 2) map of ntb provincial rtrw administrative data from pupr ntb province the largest land cover of 30,682.24 m2 occurred in 2020 (table 4), or an increase in land cover area of 6,149.29 in the period of 2013-2020. the occurring land cover changes may cause changes in the existing biodiversity, especially in the dragonfly population (kosterin 2014). previous studies of kosterin (2014) reported several dragonflies species found in the middle of the batubolong river in february 2014. the research data regarding dragonflies obtained by kosterin (2014) is quite diverse. there are differences in the dragonfly species data previously obtained by kosterin (2014) compared to the data in our study in 2021. the data difference is regarding the dragonflies diversity on the batubolong river. the data comparison is presented in table 5. table 5 data comparison of odonata research on the batubolong river (middle part) between 2014 and 2021 no. aspect kosterin (2014) our study (2021) 1. month of research februari april – juli 2. number of odonata species found 11 11 3. the odonata species found by kosterin (2014) and in our study (2021) 1. euphaea lara lombokensis (mclachlan, 1898) 2. pseudagrion pilidorsum declaratum (lieftinck, 1936) 3. nososticta emphyla (lieftinck, 1936) 4. orthetrum testaceum soembanum (forster, 1903) 5. trithemis festiva (rambur, 1842) 6. orthetrum glaucum (brauer, 1865) 7. agrionoptera insignis insignis (rambur, 1842) 8. orthetrum sabina (drury, 1773) 4. the odonata species found by kosterin (2014), but not found in our study (2021) 1. pantala flavescens (fabricius, 1798) 2. trithemis lilacina (förster, 1899) 3. rhyothemis phyllis (sulzer, 1776) 5. odonata species found in our study (2021), but not found by kosterin (2014) 1. drepanosticta berlandi (lieftinck, 1939) 2. diplacodes trivialis (rambur, 1842) 3. neurothemis ramburii (brauer, 1866) biotropia vol. 29 no. 2, 2022 122 the numbers of odonata dragonflies found in 2014 and 2021 are the same, but of different species. the difference may have been caused by: 1) the difference in study period and 2) the land cover changes occurring around the middle of the batubolong river. in 2014, it rained almost every month, except in september. in february 2014 the rainy season started with 111 mm of rainfall and 11 rainy days (bps-statistics lobar 2015). meanwhile, april 2021 was the rainy season, where river water was overflowing. in july 2021, it was the beginning of the dry season which dried up the batubolong river. in the middle of august 2021 it rained intensively. in 2021, as much as 34.8% of indonesia's territory experienced above normal dry conditions (wetter dry season, namely the dry season rainfall is higher than the climatological average) (bmkg 2021). the weather changes indicates that there has been a climate change that occurring between 2014 and 2021 which can affect the diversity of various kinds of invertebrates, including the order odonata (brook et al. 2006; clausnitzer et al. 2009). changes in the environment around the forest due to human disturbances can cause changes in the diversity of the order odonata (buchori et al. 2019; dolný et al., 2013; & šigutová et al., 2019). species drepanosticta berlandi (lieftinck, 1939) was found at the beginning of the dry season, at an altitude of 40 masl. according to harabiš & dolný (2010), the distribution of dragonfly is determined by several ecological factors, one of which is the altitude range. d. berlandi is a rare dragonfly recorded only on the lombok island and is a new species found on sumba island (irawan & rahadi 2018). meanwhile, trithemis lilacina was first discovered on sumbawa island by förster in 1899, by lieftinck in 1936 and 1953 on lombok island (steinmann 1997). based on data from gbif.org, t. lilacina was recorded only on islands of lombok, sumbawa, flores and timor (bánki et al. 2021). conclusion there are differences in dragonfly research data between those obtained by kosterin (2014) and the ones found in our study in 2021, in terms of different species found. our study discovered a rare needle dragonfly, namely drepanosticta berlandi (lieftinck, 1939) which has never been reported before in the batubolong river. satellite data showed that there has been a change in land cover in the period 2013-2020 around the batubolong river. the government and residents are expected to continually preserve the environment around the batubolong river by wisely utilizing the natural resources to support the sustainability of the existing rare dragonfly species. acknowledgments the authors sincerely thank the faculty of tarbiyah and teaching, universitas islam negeri mataram for funding this study. special gratitudes are also extended to the students for their assistance in collecting field data and to my beloved wife for developing the map of land cover change. references afrianita r, edwin t, alawiyah a. 2017. analisis intrusi air laut dengan pengukuran total dissolved solids (tds) air sumur gali di kecamatan padang utara. 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7(1):16-25. doi: 10.33394/ bjib.v7 i1.2381 šigutová h, šipoš j, dolný a. 2019. a novel approach involving the use of odonata as indicators of tropical forest degradation: when family matters. ecol indic 104(7):229-36. doi: 10.1016/ j.ecolind. 2019.05.001 steinmann h. 1997. world catalogue of odonata i. in world catalogue of odonata i. doi: 10.1515/9783110824438 [who] world health organization & international programme on chemical safety. 1996. guidelines for drinking-water quality. vol. 2. health criteria and other supporting information, 2nd edition. world health organization. https:// apps.who.int/iris/handle/10665/38551. biotropia vol. 30 no. 2, 2023: 195 205 doi: 10.11598/btb.2023.30.2.1842 195 molecular characterization of aspergillus flavus toxigenicity in agricultural commodities in indonesia anidah1*, winiati p. rahayu2,3, siti nurjanah2,3 and ina retnowati1 1seameo biotrop, jalan raya tajur km. 6, bogor 16134, indonesia 2department of food science and technology, ipb university, darmaga campus, bogor 16680, indonesia 3south-east asia food and agricultural science and technology (seafast) center, ipb university, darmaga campus, bogor 16680, indonesia received 7 november 2022 / revised 20 june 2023 /accepted 20 june 2023 abstract toxigenic aspergillus flavus is a primary producer of aflatoxin in indonesia, and its presence can lead to the contamination of agricultural commodities. this contamination poses a risk to export-targeted commodities, potentially resulting in their rejection. therefore, this study aims to characterize the molecular profile of native a. flavus isolated from several indonesian agricultural products, with a major focus on its toxigenicity and toxin production. a total of 18 a. flavus collections were isolated from nutmeg, ground peanut, cacao, coffee bean, corn, white pepper, and soil peanut plantation. species identification was carried out using molecular and morphological approaches. the toxigenicity of isolates was characterized based on the amplification of aflatoxin gene clusters, while toxin production was assessed through growth simulation on a 10% coconut broth media followed by hplc quantification. the result showed that all isolates were confirmed as a. flavus based on the morphological and sequence analysis of the its region. a total of 11 isolates (61%) were confirmed as toxigenic and produced 1-2 types of aflatoxin, in varying concentrations of high, moderate, or low levels of afb1. high levels of afb1 produced by seven isolates namely bio3313, bio33212, bio3361, bio33404, bio3338, bio3352, and bio3344, had concentration levels ranging from 76.78 to 2241.06 µg/kg, while three isolates (bio3314, bio3312, and bio3381) produced afb1 below 1 µg/kg. twenty-nine pairs of aflatoxin gene-specific sequences were successfully amplified as a single band, while some produced non-specific patterns in several low toxigenic and non-toxigenic isolates. based on the results, it was concluded that completed gene clusters and variations of gene deletion were observed in both toxigenic and non-toxigenic isolates. however, no specific target gene could effectively distinguish the two groups. two non-toxigenic isolates namely bio3393 and bio33403 exhibited a large deletion and could be potential candidates for biocontrol agents. keywords: aflatoxin, aspergillus flavus, its, pcr, toxigenic introduction aflatoxin contamination in agricultural products is a food safety concern in indonesia and worldwide. this carcinogenic mycotoxin is produced mainly by toxigenic aspergillus flavus and a. paraciticus during various stages of agricultural production including pre-harvest, harvest, post-harvest, transportation, and storage. the prevalence of aflatoxin is particularly high in tropical and subtropical regions due to optimal humidity and temperature conditions for toxin production (bhat et al. 2010). in indonesia, aflatoxin contamination has been reported in agricultural products such as nutmeg, corn, peanuts, pepper, and cocoa (dharmaputra 2002). this contamination poses a significant challenge for export-targeted commodities, leading to economic losses for farmers and exporters. for instance, upon arrival in the importing country, nutmeg was found to have a high level of aflatoxin due to the growth of toxigenic a. flavus (anidah et al. 2020). the growth was concluded to have *corresponding author, email: anidah@biotrop.seameo.id biotropia vol. 30 no. 2, 2023 196 occurred during transportation based on the low levels of the toxin detected before shipping. the activities of regulatory proteins and enzymes encoded by more than 25 genes cluster play an essential role in aflatoxin production (yu et al. 2002). non-toxigenic a. flavus was reportedly associated with the deletions of a part or the entire aflatoxin genes cluster (chang et al. 2007). however, the molecular mechanisms underlying the loss of the aflatoxin production ability in a. flavus are currently not well understood (rao et al. 2020; schmidt-heydt et al. 2008). pcr detection for afld (nor1), aflp (omta), aflm (ver1), and aflr genes have been applied as a marker to differentiate toxigenic and non-toxigenic isolates in different studies. these include isolated native to indonesia (anidah et al. 2020), animal feed in iran (davari et al. 2014), aspergillus species in korea (kim et al. 2011), maize in italy (degola et al. 2006), and an herbal product in italy (criseo et al. 2001). the results showed various deletion patterns for nontoxigenic isolates which could be distinguished from toxigenic ones having 4 complete genes or no deletion. however, it was observed that the toxigenic isolate exhibited variations in deletion patterns when amplified using a complete set of aflatoxin biosynthetic genes, rendering the use of 4 genes as a marker irrelevant. the characterization of deletion patterns within all the aflatoxin biosynthetic gene clusters has been elucidated in non-toxigenic isolates to find and assess the biocontrol agent candidate (rao et al. 2020; wei et al. 2014; donner et al. 2010; yin et al. 2009; chang et al. 2005). however, the genetic variability assessment in toxigenic isolates has not been widely carried out, despite its significance in distinguishing between toxigenic and non-toxigenic isolates. a molecular approach that characterizes the presence or absence of aflatoxin biosynthetic genes can indicate the functional status of the biosynthesis pathway gene (tran-dinh et al. 2014). this approach could serve as an effective marker for the identification of toxigenic isolates in food commodities. therefore, this study aims to characterize the molecular profiles of toxigenic and no-toxigenic a. flavus isolates from agricultural commodities in indonesia by using an aflatoxin biosynthetic genes cluster. materials and methods a. flavus isolates the a. flavus isolates used in this study (table 1) were from the phytopathology laboratory of seameo biotrop. these isolates were sourced from various regions in indonesia and included samples from nutmeg, ground peanut, cacao, coffee bean, corn, white pepper, and soil peanut plantation. all collected samples were grown in the potato dextrose agar/pda (himedia, india) with parafilm oil as a preservative, and were reinoculation in pda at 25oc for seven days before use. table 1 a. flavus isolates from agricultural products isolates commodities (origin) bio3376 nutmeg (manado – north sulawesi) bio33211 bio33212 bio33403 bio33404 bio3313 ground peanut (bogor – west java) bio3381 bio3334 ground peanut (wonogiri – central java) bio3338 bio3342 bio3344 bio3312 cacao (south sulawesi) bio3314 coffee bean (jember – east java) bio3393 coffee bean (toraja – south sulawesi) bio3382 corn (bogor – west java) bio3383 white pepper (bogor – west java) bio3361 soil (wonogiri – central java) bio3352 morphological characterization the morphological characterization of the isolates was carried out according to samson et al. (2004) and pitt & hocking (2009). czapek yeast autolysate (cya) agar was used for macromorphological observation. each isolate was inoculated at two points, and incubated at temperatures of 25 and 37oc in the dark for seven days. the colony color was observed, and the diameter was measured. meanwhile, the micromorphological observation was conducted by preparing microscopic mounts in lactic acid with cotton blue from cya colonies. the excess conidia and air bubbles were removed by adding a drop of 70% ethanol, then conidia morphology and presence, size of sclerotia, and head seriation were analyzed. molecular characterization of aspergillus flavus toxigenicity in agricultural commodities in indonesia – anidah et al. 197 dna extraction the mycelia from a 3-day-old culture in 50 ml potato dextrose broth/pdb (himedia, india) were harvested and rinsed using sterile distilled water and then dried using filter paper. the sample was crushed using a mortar and pestle with continuous addition of liquid nitrogen until fine mycelia powders were obtained. dna was extracted using dneasy plant kit (qiagen, germany) according to the manufacturer’s instructions and the quality was assessed through electrophoresis in 1% agarose gel (invitrogen, usa) with sybrsafe dye (invitrogen, usa) in tris-acetate-edta (tae) buffer. the absorbance was measured at 260 nm wavelength for quantification, and dna was stored at -20 oc until further use. species identification molecular species identification was carried out using its 1 (tccgtaggtgaacctg cgg) and its 4 (tcctccgcttattgata tgc), as forward and reverse primers against all isolates and sequencing of the its region (white et al. 1990). the amplification reaction was carried out by mixing 100 ng of dna template, 0.2 m of each primer, and 1x gotaq hot start pcr master mix (promega, usa), in a total volume of 40 µl. the amplification program was performed in the genamp pcr system 9700 (abi, usa) thermal cycler with specific conditions including pre-denaturation for 5 minutes at 95oc, followed by 35 cycles of denaturation at 94oc, annealing at 5oc, and extension at 72oc for 30 seconds each. an additional final extension at 72oc for 7 minutes was also included. the amplified product was visualized by 1.2% agarose gel electrophoresis, followed by sequence analysis in 1st base (singapore). the sequences were analyzed for similarity using blast (http://blast.ncbi.nlm.nih.gov/blat.cgi) and submitted to the ncbi database. the neighborjoining method with 10000 bootstrap replicates in mega x software was used for tree construction (kumar et al. 2018). differentiation of toxigenic and nontoxigenic isolates toxigenic and non-toxigenic isolates were determined by growth simulation on an aflatoxin-inducing media (davis et al. 1987). total aflatoxin was extracted from a ten-day inoculated culture in 10% (v/v) coconut broth media/cbm, according to the aoac method 991.31 (aoac, 2000). for extraction, a 25 ml filtrate of the culture was mixed with 5 g nacl and 125 ml of methanol: water (70:30; v:v). the mixture was filtered through a glass microfiber filter after 2 times dilution with purified water. aflatoxins b1, b2, g1, and g2 from the extract were selectively isolated using the aflatest affinity column (vicam, usa). furthermore, the content of the extract was determined using hplc with post-column derivatization (vicam 2007). all the extraction chemicals and solvents used were from merck, germany, while the aflatoxin standard was from sigma, usa. pcr amplification using aflatoxin biosynthesis genes pcr amplification using aflatoxin biosynthesis genes was carried out according to chang et al. (2005). the isolated dna was amplified by pcr to detect the presence or absence of aflatoxin biosynthesis gene fragments using 29 pairs of primers as shown in table 2. table 2 list of aflatoxin biosynthesis primer sequences primer pair sequence aflu (norbcypa) f: gtgcccagcatcttggtcca r: aggacttgatgattcctcgtc aflt f: atgacatgctaatcgacgag r: aggcgcatgctacggatc aflc (pksa) f: actttgagggcgttctgtgc r: ctttcggtggtcggtgattc afld (nor1) f: agcacgatcaagagaggctc r: gatctcaactcccctggtag afla (fasa/hexa) f: tcctatccagtccacctcgta r: cacatctttgtcttgcccgc aflb (fasb/hexb) f: acaatcgaatgacaacactgc r: ccaccgaatccactacctaca aflr f: atggtcgtccttatcgttctc r: ccatgacaaagacggatcc afls f: cttcaacaacgacccaaggtt r: agatgagatacactgccgca aflh (adha) f: cctcgtgggagagccaaatc r: ggagcaagaaggttacagcg aflj (esta) f: cgatgggactgacggtgatt r; accacgccgctgactttat afle (nora) f: gtgttcgtgtgtcgccctta r: gtcggtgcttctcatcctga aflm (ver1) f: catcggtgctgccatcgc r: cctcgtctacctgctcatcg afln (vera) f: ccgcaacaccacaagtagca r: aaacgctctccaggcacctt aflg (avna) f: gcgatagaactgacaaaggca r: gaatgagtctccaaaggcgag afll(verb) f: ttcagtgacaaaggtcttcgc r: ggcagcgtt attgagcatct biotropia vol. 30 no. 2, 2023 198 afli (avfa) f: attcaaatcctcgttcggtcg r: tagcccgttggttgtgttcc aflo (omtb) f: acagacgatgtgggcaaacg r: acgcagtccttgttagaggtg aflp (omta) f: caggatatcattgtggacgg r: ctcctctaccagtggcttcg aflq (orda) f: aaggcagcggaatacaagcg r: acaagggcgtcaataaagggt aflk (vbs) f: aacgagcagcgtaagggtct r: tcagccagagcatacacagtg aflv (cypx) f: ggagcctaccattcgcaaca r: ggctttgacgaacagattccg aflw (moxy) f: tgctactggaacgaagaccg r: cgacgacaaccaaacgcaa aflx (ordb) f: gctgctactggaatgaagacc r: atgcgacgacaaccaaacg afly (hypa) f: cgcaagacggcagagatact r: gctccttcagttccacacca nada f: tgacgaggcctgcgagctgt r: aagcctcttcagaacggtca hexa f: tgtcctcacctctggcgtat r: agaccaaccactcttatgggc glca f: agacacagtcatcgcctgtt r: ggtgcgaataggtgcaggta sugr f: tcagctgaagcgctcgagag r: gtattgccgcactatgtatg c4 f: atcgtgcagacaggaacac r: ggtgccttggcctatgcgct a total of 20 µl of the reaction mixture was prepared for each isolate by mixing 50 ng dna, 0.2 µm each primer pair, 1x gotaq hot start pcr master mix (promega, usa), and molecular grade water. pcr amplification was performed in a thermal cycler geneamp pcr system 9700 (abi, usa) with specific conditions of pre-denaturation for 5 minutes at 94oc, followed by 30 cycles of denaturation at 94oc, annealing at 55oc, and extension at 72oc for 1 minute respectively. an additional final extension was carried out at 72oc for 6 minutes. the pcr products were visualized by electrophoresis on a 1.5% agarose gel in tae buffer, with sybrsafe dye (invitrogen, usa). the presence or absence of amplicon was observed, and the results were scored for the presence (1) or absence (0) of aflatoxin genes. to construct the phylogenetic tree, cluster analysis was performed using the unweighted pair-group method with arithmetic mean (upgma) functionality in ntsyspc 2.1 software (department of ecology and evolution, state university of new york, ny, usa). results and discussion morphological and molecular identification of a. flavus all isolates were identified as a. flavus based on macro and micromorphology observations, following the guidelines of pitt & hocking (2009). the colony diameter range was 3.8 5.6 cm and 4.2 6.4 cm when grown on cya agar at 25 and 37oc, respectively. the colony colors were greyish-green, yellow-green, and oliveyellow, while the reverse side was uncolored to reddish-brown. conidia were observed as smooth to finely rough with biseriate conidial heads, globose to subglobose, and 3 5 µm in diameter. moreover, the stipes were hyaline, smooth, variable in length, mostly 350-600 µm, and the diameter just below vesicles was 3 8 µm. the vesicles were globose to sub-globose, and 10-30 µm in diameter. isolates grew well at 25 and 37oc and no sclerotia were observed as shown in figure 1. molecular characterization of aspergillus flavus toxigenicity in agricultural commodities in indonesia – anidah et al. 199 figure 1 colonies of the 18 a. flavus on cya (7 days at 25 oc) notes: a = bio3313; b = bio33212; c = bio3361; d = bio33404; e = bio3338; f = bio3352; g = bio3344; h = bio3334; i = bio3314; j = bio3312; k = bio3381; l = bio3382; m = bio3383; n = bio3376; o = bio33211; p = bio3342; q = bio33403; r = bio3393. j k l m n p q o r b a c d e f g h i biotropia vol. 30 no. 2, 2023 200 species identification at the molecular level was conducted by amplifying the its region as the official locus for fungal dna barcoding. amplification using its 1 and its 4 yielded a single amplicon of 600 bp in all isolates. the amplified products were sequenced and analyzed for similarity using blast (http:// blast.ncbi.nln.nih.gov/blast.cgi), and all the generated sequences were submitted to the ncbi database (table 3). afterward, a phylogenetic tree was constructed using mega 6, which included sequences from genbank for a. flavus (nr111041.1), a. paraciticus (nr151784.1), a. niger (nr111348.1), and a. fumigatus (nr121481.1). all isolates were found to have 100% homology to a. flavus (figure 2). the neighbor-joining method with 10000 bootstrap replicates in mega x software was used for tree construction. bootstrapping was translated as the accuracy value of the phylogenetic tree against randomization of 10000 repetitions (tamura et al. 2007). the morphological and molecular analysis, both confirmed all isolates identified as a. flavus (figures 1 and 2). toxigenic and non-toxigenic isolate differentiation toxigenic a. flavus was induced to produce aflatoxins in the cbm containing 10% coconut milk due to the presence of fat and fatty acids (lin & dianese 1976). during incubation, a. flavus mycelia grew on the media’s surface, releasing the toxin in the solution. the aflatoxin content produced was extracted from the cbm filtrate, followed by identification and quantification by hplc compared to respective standards as afb1, afb2, afg1, and afg2. a total of 11 isolates from a. flavus (61%) produced aflatoxin with varying concentrations, while seven (39%) did not, as shown in table 3. the toxigenic isolates yielded two forms of aflatoxins with a combination of afb1-afb2 (three isolates), and afb1-afg1 (three isolates), while the remaining five only produced afb. however, none of the toxigenic isolates produced afg2 as shown in table 3. figure 2 phylogenetic tree of a. flavus isolates using neighbor-joining method by mega x software (10000 x bootstrap) http://blast.ncbi.nln.nih.gov/blast.cgi http://blast.ncbi.nln.nih.gov/blast.cgi molecular characterization of aspergillus flavus toxigenicity in agricultural commodities in indonesia – anidah et al. 201 table 3 aflatoxin contents from 18 isolates of a. flavus a. flavus isolate accession number aflatoxin (µg/kg) b1 g1 b2 g2 bio3313 on619457 2241.06 nd 66.8 nd bio33212 on619471 702.72 nd nd nd bio3361 on619464 607.67 1081.15 nd nd bio33404 on619473 255.27 nd nd nd bio3338 on619460 217.34 1486.52 nd nd bio3352 on619463 126.96 200.06 nd nd bio3344 on619462 76.78 nd 2.60 nd bio3334 on619459 7.68 nd 0.14 nd bio3314 on619458 0.62 nd nd nd bio3312 on619456 0.31 nd nd nd bio3381 on619466 0.10 nd nd nd bio3382 on619467 nd nd nd nd bio3383 on619468 nd nd nd nd bio3376 on619465 nd nd nd nd bio33211 on619470 nd nd nd nd bio3342 on619461 nd nd nd nd bio33403 on619472 nd nd nd nd bio3393 on619469 nd nd nd nd notes: nd = not detected; below the loq (b1 = 0.0202; b2 = 0.0171; g1 = 0.0220; g2 = 0.0183) in µg/kg all toxigenic isolates exhibited the capacity to produce afb1 in various concentrations, ranging from low, medium, to high levels. a total of seven toxigenic isolates had a high concentration of afb1 ranging from 76.78 to 2241.06 µg/kg, while one isolate had a moderate concentration of 7.68 µg/kg. furthermore, three other toxigenic isolates produced afb1 below 1 µg/kg as shown in table 3. the presence of toxigenic isolates in food commodities poses a significant threat due to their ability to produce aflatoxins. the maximum value in food commodities permitted by regulations in indonesia is 15 µg/kg afb1 (bpom 2012). based on the results, the potential of a. flavus to produce aflatoxin varied significantly among both strains. in nature, the percentage of the non-toxigenic strain varies from 0 to more than 80% worldwide (rao et al. 2020; yin et al. 2009; chang et al. 2007). molecular profile of toxigenic and nontoxigenic a. flavus isolates although the species identification by morphological and molecular approaches confirmed the same result, they could not distinguish between the toxigenic and nontoxigenic isolates. the toxigenicity of a. flavus was determined by the presence of genes encoding aflatoxin biosynthesis which was detected by pcr (yu et al. 2002). the pcrbased molecular technique offers the advantage of rapid diagnosis with a high level of sensitivity and specificity, compared to the conventional method (mamo et al. 2017). based on the results, twenty-nine pairs of primers were successfully amplified in 18 a. flavus isolates. the majority of the isolates produced single amplicons, while some yielded non-specific amplicons as multiband patterns in several low toxigenic and non-toxigenic isolates (figure 3). the presence of multiband amplicons indicates the formation of nonspecific product and suggested that the absence of aflatoxin production may be due to base-pair substitution mutations, resulting in the formation of non-functional gene products (levin 2012; criseo et al. 2001). the amplification results of 3 toxigenic isolates with high to low levels of afb1 showed no deletion of the amplicon. bio3313 which exhibited high afb1 production, as well as bio3334 and bio3381 with moderate and low afb1 production, respectively, had the complete aflatoxin gene cluster. similarly, the complete amplicons were also found in bio3382 as a non-toxigenic isolate. this result was consistent with previous studies which showed a complete set of genes in the nontoxigenic a. flavus (wei et al. 2014; kim et al. 2011; criseo et al. 2001). although amplification using dna-based pcr confirmed the presence of the target gene in the genomic dna, it was unable to explain the level of gene expression. this suggested the need for a real-time pcr as a biotropia vol. 30 no. 2, 2023 202 more proficient method but the gene expression result strongly depends on the reference stain, culture condition, and media used to induce the mrna expression (rao et al. 2020). figure 3 profile of all amplicons of aflatoxin biosynthesis genes in toxigenic and non-toxigenic isolates notes: * = non-toxigenic isolates; a = bio3313; b = bio3338; c = bio3352; d = bio3361; e = bio3376; f = bio33211; g = bio33212; h = bio33403; i = bio33404; j = bio3312; k = bio3314; l = bio3334; m = bio3342; n = bio3344; o = bio3381; p = bio3382; q = bio3383; r = bio3393. afld/nor1 aflu/norb a aflt aflc/pksa afla/fasa aflb/fasb aflr afls aflh/adha aflj/esta afle/nora aflm/ver1 afln/vera aflg/avna afll/verb afli/avfa aflo/omtb aflp/omta aflq/orda aflk/vbs aflv/cypx aflw/moxy aflx/ordb afly/hypa nada hexa glca sugr c4 a b c d e* f* g h* i j k l m* n o p* q* r * molecular characterization of aspergillus flavus toxigenicity in agricultural commodities in indonesia – anidah et al. 203 variations in the deletion of aflatoxin biosynthetic genes were observed in both toxigenic and non-toxigenic isolates. the deletion involved one to a maximum of four genes in toxigenic isolates, except for bio3312 which exhibited a low level of afb1 production below 1µg/kg and experienced nine genes deletion, with some appearing as non-specific amplicons. meanwhile, non-toxigenic isolates showed one to 26 gene deletions. two nontoxigenic isolates, namely bio 33403 (90% deletion) and bio3393 (79% deletion) exhibited significant gene deletions, providing scientific data to address the safety issues in the application of biological control. according to a previous study, non-toxigenic isolates with extensive gene deletions are potential candidates for biocontrol agents (donner et al. 2010). moreover, extensive aflatoxin gene deletion is preferred to prevent adverse genetic recombination (chang et al. 2005). molecular characterization of non-toxigenic a. flavus using partial or completed aflatoxin biosynthetic genes has been carried out. pcr detection using afld (nor1), aflp (omta), aflm (ver1), and aflr as partial target genes were used in several studies to discriminate non-toxigenic isolates (anidah et al. 2020; davari et al. 2014; degola et al. 2006; criseo et al. 2001). in this study, afld, aflp, and aflr genes were found to be deleted in toxigenic isolates, while the aflr gene appeared in several non-toxigenic isolates. similar results were observed by rao et al. (2020), suggesting that these partial gene detections may not serve as reliable markers. genetic variations of non-toxigenic a. flavus isolates based on completed aflatoxin biosynthetic genes cluster have also been reported. chang et al. (2005) amplified 25 genes from 38 non-toxigenic a. flavus isolates in the united states and found eight deletion patterns. yin et al. (2009) analyzed the presence of 11 genes and found five deletion patterns from 11 non-toxigenic isolates in china. furthermore, donner et al. (2010) examined 21 non-toxigenic isolates in nigeria by amplifying 21 genes and found nine deletion patterns. wei et al. (2014) also found 25 deletion patterns by amplifying 29 genes from 76 non-toxigenic isolates in china. all the aforementioned studies focused on the variation of deletion patterns in non-toxigenic isolates with a major emphasis on their use as biocontrol agents in the field. the results indicated that the genetic variability from the aflatoxin biosynthetic gene cluster is diverse in non-toxigenic isolates. a comparison of the deletion patterns might contribute to a better understanding of specific markers for differentiating isolates based on toxigenicity. a cluster analysis was performed to identify the similarities among the toxigenic and nontoxigenic isolates based on the presence of aflatoxin genes. the genetic similarity coefficients (gsc) ranged from approximately 0.28 to 1.00. the phylogenetic tree assembled using the upgma grouped the 18 isolates into two main clusters. one group had the largest number of isolates (16 isolates) including toxigenic as well as non-toxigenic isolates. another group had 2 non-toxigenic isolates with large deletion, namely bio3393, and bio33403. this indicates that no specific genes could distinguish between toxigenic and non-toxigenic isolates (figure 4). biotropia vol. 30 no. 2, 2023 204 figure 4 dendrogram illustrating genetic relationships among 18 isolates of a. flavus, generated by the upgma cluster analysis (ntsys) calculated from 29 primers of aflatoxin biosynthesis genes notes: ** = highly toxigenic isolates; * = low toxigenic isolates. a. flavus isolates were identified at the species level through morphological and molecular analysis, (figures 1 and 2), while the toxin analysis was used to distinguish between toxigenic and non-toxigenic isolates (table 3). molecular characterization results based on aflatoxin biosynthetic genes provided an overview of the diversity profile at the molecular level (figures 3 and 4). the results also contributed to a better understanding of toxigenicity mechanisms. the genetic variations observed in each toxigenic and non-toxigenic isolate are important to explore the specific markers candidate for detection purposes and the development of biological agents. conclusion all isolates involved in this study were confirmed as a. flavus based on morphological and molecular identifications, while hplc assays confirmed their ability to produce aflatoxins. twenty-nine pairs of primers were successfully amplified, and the majority produced a single amplicon in toxigenic isolates. however, certain low toxigenic and nontoxigenic isolates produced non-specific amplicon patterns. deletion variations in several target genes were found in both isolates but no specific target gene could distinguish the toxigenicity based on the genetic similarity data. this study provides an overview of the molecular profiles of toxigenic and nontoxigenic a. flavus isolates. two non-toxigenic isolates with significant gene deletion were characterized and identified as potential candidates for biocontrol agents. the information about the molecular characterization of a. flavus based on toxigenicity could support the development of effective biological control strategies to prevent aflatoxin contamination. acknowledgments the authors would like to acknowledge seameo biotrop for providing financial support through daftar isian pelaksanaan anggaran (dipa) 2021. thanks to prof. dr. okky setyawati dharmaputra from fitophatology laboratory of seameo biotrop for their a. flavus collection isolates. references anidah, rahayu wp, nurjanah s. 2020. screening of toxigenic aspergillus flavus strains and aflatoxin content from agricultural commodities in indonesia. in: sitanggang az, rahayu wp, antara ns, santoso u, giyatmi, ardiansyah, haryono a, eds. proceedings of the 16th asean food conference-16th afc, isbn 978-989-758-467-1, p. 221-6. doi: 10.5220/ 0009981202210226 molecular characterization of aspergillus flavus toxigenicity in agricultural commodities in indonesia – anidah et al. 205 aoac. 2000. section 49.2.18 (aoac method 991.31) for corn, raw peanut, and peanut butter. in official method of analysis. 17th edition. gaithersburg (us): aoac international. bhat r, rai rv, karim aa. 2010. mycotoxins in food and feed: present status and future concerns. compr rev food sci food saf 9: 57-81. 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microbiol 107: 1857-65. yu j, bhatnagar d, ehrlich kc. 2002. aflatoxin biosynthesis. iberoam micol 19: 191-200. hal. 190-201 cover single.cdr biotropia vol. 27 no. 2, 2020: 134 142 doi: 10.11598/btb.2020.27.2.1182 diversity and vertical distribution of vascular epiphytes on a malaysian mangrove island** shahrudin rohanp, fei lin lee and abdul shukor yusoff vacuity science and marine environment, universiti malaysia terengganu, 21030 kuala nerus, malaysia received 01 january 2019 / accepted 07 february 2019 abstract many studies have attempted to explain the diversity and abundance of epiphytic plants in major ecosystems, worldwide. however, investigations on the abundance of epiphytic plants in mangroves have remained rare. the aim of this research was to study the diversity and vertical distribution of vascular epiphytes in a mangrove forest in peninsular malaysia. the sampling was done over a 0.1 hectare of pulau telaga tujuh, a mangrove island in terengganu, malaysia. trees with vascular epiphytes were divided into three strata: basal, trunk and canopy. the vascular epiphytes were identified, and the number of individuals in each stratum was recorded. in total, 8 species of vascular epiphytes from 6 genera and 4 families were recorded. pulau telaga tujuh mangrove forest exhibited a relatively low diversity of vascular epiphytes (ff = 1.43). the dominance of hydnopytum formicarum significantly contributed to the diversity of vascular epiphytes in this forest. the highest abundance of epiphytes was observed on the trunks of the host trees. the vertical distribution pattern observed in this study is associated with the adaptation of the epiphytic plants to stresses in the mangrove ecosystem, particularly, drought and salt spray. pulau telaga tujuh had a high density of vascular epiphytes but lower diversity compared with other ecosystems. keywords: epiphyte ecology, mangroves, setiu wedands, spatial distribution introduction epiphytes are plants that germinate and live on trees without taking nutrients from the soil or the host tree. vascular epiphytes such as orchids and ferns are able to adapt to an aerial environment, resulting in their morphological variations. diversity of vascular epiphytes cannot be overemphasized as it contributes to vascular plants diversity constituting roughly 9% of all the vascular plants (zotz 2013). since the 18th century, studies of vascular epiphytes have been conducted around the world, particularly in tropical regions known for their rich biodiversity (zotz 2016). vascular epiphyte diversity plays an important role in forest ecosystems especially of those epiphytes living in the canopy zones as these plants help to regulate the cycling of nutrients (coxson & nadkarni 1995). dead ^corresponding author, e-mail: rohanishahmdin@umt.edu.my **this paper was presented at the 3rdinternational conference on tropical biology 2018, 20-21 september 2018, bogor, west java, indonesia vascular epiphytes decompose and create organic matter on the ground or form humus on tree branches or trunks (coxson & nadkarni 1995). this organic matter provides nutrients and increases the biomass of the ground, especially in primary forest (nadkarni et al. 2004). vascular epiphytes also provide a variety of food resources for many organisms such as birds and insects. for example, 59% of 33 species of birds in a tropical rain forest in costa rica were observed foraging epiphytes resources (nadkarni & matelson 1989). some epiphytes exhibit a positive association with ants. ants help to disperse seeds for epiphytes, and the plants provide a nest for the ants (kaufmann et al. 2001; dejean et al. 2003). the distribution of vascular epiphytes tends to be affected by the availability of substrate and the distance of dispersal of seeds rather than the mode of dispersal (nieder et al. 2000). the microhabitat (e.g., light intensity, proximity to soil or exposure to wind and precipitation) is associated with the availability of substrate and therefore, influence species richness and the 134 diversity and vertical distribution of vascular epiphytes — rohani et al abundance of vascular epiphytes in each stratum and host trees (quaresma & jardin 2014; woods et al 2015; getaneh & gamo 2016; wang et al 2016). vascular epiphytes also tended to colonize zones with similar microhabitats indicating that vascular epiphytes may be specific to a particular strata (woods 2013). strata specificity shed light on the pattern of vertical distribution of vascular epiphytes (kersten et al 2009). the tendency of epiphytes to occupy a similar microhabitat can also stem out from their resource-utilizing mechanisms. since major portion of epiphytes do not attach to the soil, epiphytes show several strategies to obtain nutrients and water supply from their microenvironment (benzing 1990). epiphytes get nutrients from internal and external inputs; internally from leaves leachate from host-tree and nitrogen fixing bacteria (hietz et al 2002) and externally through dry deposition and precipitation (fiirnkranz et al 2008; zotz 2016). epiphytes also receive nutrients input from throughfalls and stemflow (awasthi et al 1995; chuyong et al 2004). moreover, different epiphytes use different nutrient uptake mechanisms, hence, the co-occurrence of different assemblage of epiphytes (cardelus & mack 2010). to adapt to drought environment in the canopy, epiphytes developed morphological adaptations such as succulent leaves and water absorbing structures (benzing 1990; hietz et al 1999). physiological adaptation such as crassulacean acid metabolism (cam) photosynthesis also occurs among many epiphytes (silvera et al 2010). cam photosynthetic plants, like many epiphytes, open their stomata during night time to capture co2 and close the stomata during daylight. many studies have focused on documenting the richness of mangrove flora yet, little attention has been given to epiphytic plants in this forest type. epiphytes are perhaps the most susceptible to mangrove destruction because they depend on having large trees as their hosts. recently, the epiphyte hydnophytum formicarum was found to be remarkably abundant in pulau telaga tujuh, malaysia (rohani et al 2017). this finding motivated the authors to investigate the occurrence of other vascular epiphytes on the island. hence, this study aimed to determine the species diversity and vertical distribution of vascular epiphytes in a mangrove ecosystem in the setiu wetlands in the east coast of peninsular malaysia. materials and methods study site the research was conducted in pulau telaga tujuh, setiu wetlands, terengganu, malaysia (05°41.800’ n; 102°41.770’ e). the setiu wetlands is an ecosystem with nine interconnecting habitats of beach, sea, mudflat, fresh and brackish water, river, islands, coastal and mangrove forests (jamilah et al 2015). this unique ecosystem, gazetted as state park in may 2018, support a myriad of biodiversity and are equally important to local people’s livelihoods. the study site was one of the small islets in the setiu lagoon (5.42 ha), situated side by side with 13 other islands. data collection ten plots of 10 m2 each (1,000 m2 in total) were randomly established along the island. every tree in each subplot was inspected for the occurrence of vascular epiphytes. the abundance of all vascular epiphytes, the strata and the host characteristics (e.g., stem diameter and height) were recorded. the host’s strata were classified into three: 1) canopy first branch to the tip of the tree; 2) trunk 1.3 m above the ground to the first branch; and 3) basal ground up to 1.3 m high in the host tree. for trees with an enlarged stem the base was 1.3 m above the ground (getaneh & gamo 2016). all of the vascular epiphytes were included in the census except for the seedlings that were difficult to identify up to the species level. the epiphytes that grew in clusters that were difficult to count individually were counted as an independent individual (getaneh & gamo 2016). binoculars were used to observe the epiphytes that were located high in the trees. the names of the epiphyte species and host plants were referred to a catalogue of the vascular plants of malaya (turner 1995). voucher specimens were deposited at the universiti malaysia terengganu herbarium (umtp). 135 biotropia vol. 27 no. 2, 2020 data analysis vascular epiphyte diversity was analyzed using two diversity indices; shannon diversity index (h’) and simpson’s index (d) (magurran & mcgill 2011). to check the similarity between the epiphyte communities of each host tree, a cluster analysis was carried out using the jaccard similarity index based on the number of individuals of each vascular epiphyte species on each host tree. the data were analyzed using past version 3.15 software (hammer et al. 2001). results and discussion species diversity a total of 929 individuals belonging to 8 species, 6 genera and 4 families were recorded in this study (table 1) (fig. 1). the density of vascular epiphytes in the plots was, on average, 0.93 individuals/m2. the shannon diversity index of this plant was low (h’ = 1.43). meanwhile, the simpson’s index (d) was 0.31, indicating the dominance of one species in the species richness. mangrove forests in southeast asia exhibit at least 28 species of epiphytic plants (giesen et al. 2007). in other words, 28.6% of mangrove epiphytic species can be found in pulau telaga tujuh. in comparison, an earlier epiphytic study in a malaysian mangrove forest found 16 species from 8 families (japar sidik et al. 2001). apocynaceae, with 3 species, was the wellrepresented family in terms of species number. hydnophytum formicarum was the species with the largest number of individuals in the study site. in many previous studies on epiphytes, species with dust-like seeds have been found to be dominant. orchidaceae was recorded as the dominant epiphyte family in the natural forests as well as in rubber plantations in malaysia (kiew & anthonysamy 1987). most species from orchidaceae have small seeds which facilitated their dispersal by wind. the rank-abundance curve (fig. 3) revealed an unequal proportion of common species and rare species. the steep line in the graph was derived from the dominance of hydnophytum formicarum. this plant, which has a close association with ants (hence sometimes called the “ ant plant” ), was also reported as a common species and more abundant than other non-antassociated species in ulu endau, johor, malaysia (kiew & anthonysamy 1987). a high abundance of ant-associated epiphytic species was also found in the amazonian lowland rainforest (nieder et al. 2000). this high abundance of the ant plants in one ecosystem may be due to the ants helping to cultivate the seeds. this phenomenon, called ant farming, was proposed by chomicki and renner (2016) after their observation of the evolutionary history between ants and epiphytic plants. the ants collected the seeds and deposited these in the bark fissures, where the seeds germinated. this situation might also have occurred in hydnophytum formicarum in pulau telaga tujuh as their individuals were distributed in a clumped pattern (rohani et al. 2017). table 1 list of vascular epiphytes in pulau telaga tujuh, terengganu, malaysia species family number of individuals dendrobium crumenatum orchidaceae 8 dischidia nummularia apocynaceae 150 hoya coronaria apocynaceae 51 hoya verticillata apocynaceae 5 hydnophytum formicarum rubiaceae 462 pyrrosia piloselloides polypodiaceae 127 taeniophyllum glandulosum orchidaceae 125 dendrobium sp. orchidaceae 1 136 diversity and vertical distribution of vascular epiphytes — rohani et al. > b ^xa * m r\\ xv 1*.el*3» \vi-i s vi £»7. a r ’x: * t •<r i •isa i,n ai:rv ft v, r ^ \; mc y.y« * 0 9 v xy.x i5 v. £e ;^3rv ^> i\ ar.s'-. \ i* a i ii hi -* b % ra, a, <*?? figure 2 photos of vascular epiphytes found in pulau telaga tujuh, terangganu notes: a = dendrobium cnminatum\ b = hydnophylum formicarum\ c = dischidia nummularia\ d = hoya verticillatcr, e = pyrrosiapiloselloides\ f = taeniophyllum glandulosum; g = dendrobium sp. 137 biotropia vol. 27 no. 2, 2020 c # o o a © £ 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 0 \ •\ \ \.\ 2 4 6 8 10 rank figure 3 rank-abundance of the 8 vascular epiphyte species in pulau telaga tujuh, terengganu, malaysia a total of 152 trees were occupied with vascular epiphytes (table 2). these host trees are consisted of nine families, 11 genera and 13 species. host trees that were occupied by the large number of epiphytes included heritiera littoralis, xylocarpus granatum and ceriops\ippeliana\ meanwhile pandanus fascicularis was the least occupied (table 3). cluster analysis grouped the host species into three major groups based on the composition of epiphytic species on the host trees (fig. 4). thirty percent of the vascular epiphytes of planchonella obovata were similar to those of other host trees that shared at least 50% similar vascular epiphytes. epiphyte species richness is positively correlateed with the host’s stem diameter (hayasaka et al 2012; woods 2013; wang et al. 2016; sousa et al 2017). however, the hosts with a high degree of epiphyte species richness were inland trees. this finding was confirmed in the clustering analysis where the third group comprised of trees that grow at mid-intertidal to landward zones. another study highlighted the important role of inland trees as hosts to many epiphytic ferns in the mangroves of thailand (hayasaka et al. 2012). the trees in the inland zone tend to be mature and larger. this situation was also true in this study because the largest tree in the study plots, xjjlocarpus granatum, has a dbh of 60 cm. table 2 host tree species with vascular epiphytes in pulau telaga tujuh, terengganu host tree family number of individual epiphytes hvicennia marina avicenniaceae 3 bruguiera cylindrica rhizophoraceae 7 bruguiera gymnorrhiga rhizophoraceae 4 casuarina equisetifolia casuarinaceae 5 ceriops tagal rhizophoraceae 9 ceriops gippeliana rhizophoraceae 30 excoecaria agallocha euphorbiaceae 4 heritiera littoralis sterculiaceae 34 hibiscus tiliaceus malvaceae 3 pandanus fascicularis pandanaceae 2 planchonella obovata sapotaceae 3 kjjigophora apiculata rhizophoraceae 13 xylocarpus granatum meliaceae 35 138 p. obovata h. tiliaceus p. fascicularis a. marina c. equisetifolia b. gymnorrhiza e. agallocha b. cylindrica c. decandra r. apiculata x. granatum c. tagal h. littoralis h os t tr ee s p ec ie s similarity (jaccard) 1.0 0.9 0/8 0.7 0.6 0.5 0.4 0.3 diversity and vertical distribution of vascular epiphytes — rohani et al. phorophytes characteristics such as bark structure can also influence the distribution of the epiphytes (wyse & burns 2011; sayago et al. 2013; wagner et al. 2015). three phorophytes with the highest number of epiphytes were observed sharing a common trait, having a generally rough bark surface. xylocarpus granatum possess a flaky and dippled bark, meanwhile hiritiera littoralis and ceriops decandra have fissured barks. these rough bark structures were able to trap much humus that was deposited as substrates for epiphytes (nurfadilah 2015). rough and fissured barks are able to accumulate litter and debris and to retain moisture from water trapped in between their crevices (reyes et al. 2010; zytynska et al. 2011). these structures will serve as microsites for epiphytes attachments aside from acting as water catchment and accumulating nutrients (wagner et al. 2015). rough surfaces also provide better mechanical support for attachments with better gripped surfaces for frictions, thereby logically explaining why plants species with smooth surface structure such as nypa fruticans was not chosen as phorophytes even though they are quite numerous in pulau telaga tujuh. table 3 abundance of the vascular epiphytes on each host tree species in pulau telaga tujuh, terengganu number of individuals per epiphyte species on the host treehost tree species dc dn hc hv hf pp tg ds total avicennia marina 5 2 0 0 9 0 0 0 16 brugueira cylindrica 0 1 0 0 6 15 2 0 24 brugueira gymnorrhiya 0 7 0 0 4 14 0 0 25 casuarina equisetifolia 0 0 0 0 10 6 0 0 16 ceriops tagal 0 3 1 3 3 2 7 0 19 ceriops yippeliana 1 22 7 0 52 14 53 0 149 exoecaria agallocha 0 26 0 0 10 11 0 0 47 heritiera littoralis 0 38 11 2 158 28 42 1 280 hibiscus tiliaceus 0 1 0 0 3 0 0 0 4 pandanus fascicularis 1 11 0 0 4 2 0 0 18 pallaquim obovata 0 0 2 0 7 0 0 0 9 rhiyophora apiculata 1 9 2 0 23 25 20 0 80 xylocarpus granatum 0 30 28 0 173 10 1 0 242 notes: epiphytes: dc — dendrobium crumenatum\ dn — dischidianummularia\ hc — hoja coronaria\ hv — hoya verticillata\ hf = hydnophytumformicarum\ pp = pyrrosiapiloselloides\ tg = taeniophyllumglandulosung ds = dendrobium sp. i i i i i i _i t figure 4 cluster analysis according to the number of individuals per vascular epiphyte of each host tree species using the jaccard similarity index 139 biotropia vol. 27 no. 2, 2020 vertical distribution the “ trunk” stratum was most frequently found to harbor epiphytes, followed by “ canopy” and “ basal” (table 4). this type of vertical distribution was more or less consistent with many epiphytic studies (quaresma & jardin 2014; getaneh & gamo 2016; mohamed et al 2017). the preference of epiphytes in certain strata is influenced by their ability to adapt with various levels of photon flux density (pfd) and humidity (benzing 1990). this acclimation process is prominent in stressful environment in mangrove forests as epiphytes are highly exposed to drought and salt spray. to adapt to drought conditions, the epiphytes in pulau telaga tujuh probably undergo the cam photosynthesis as physiological adaptation to survive in the stressful environment (table 5). the relationship between cam strategy and the epiphyte preferences for certain strata in this study is consistent with other observation that cam species tend to clump in areas of higher light intensity and exposure (zotz 2004). most cam species are characterized by thick leaves, thereby reducing water loss (barrerazambrano et al 2014), which was also observed in all species found in pulau telaga tujuh. having schlerophyllous leaves also helps the epiphytes fight against the high salinity environment. most epiphytes do not become halophyte to adapt with the saline environment (zotz & reuter 2009). hence, the epiphytes’ low preference for sites at the basal area. furthermore, permanent inundation of the basal region is not favourable for plant growth because a stable substrate is important for epiphyte establishment (nieder et al 2000; hayasaka et al. 2012; woods 2013). similarly, a low concentration of epiphytes was observed at the lower strata of the host trees in our study. table 4 the presence of epiphytic plants on each stratum in pulau telaga tujuh vascular epiphyte species number of individual epiphytes per stratum basal trunk canopy dendrobium crumenatum 6 2 0 dischidia nummularia 22 82 46 hoya coronaria 4 41 6 hoya verticillata 0 4 1 hydnophytum formicarum 22 289 151 pyrrosia piloselloides 31 74 22 taeniophyllum glandulosum 45 59 21 dendrobium sp. 0 0 1 total 130 551 248 table 5 mode of photosynthesis for each vascular epiphytes in pulau telaga tujuh extrapolated from winter et al. (1983) and hew & yong (2004) epiphyte species mode of photosynthesis dendrobium crumenatum cam dischidia nummularia cam hoya coronaria cam hoya verticillata cam hydnophytum formicarum likely to vary between c3-cam pyrrosia piloselloides cam taeniophyllum glandulosum cam dendrobium sp. more likely to be cam than c3 140 diversity and vertical distribution of vascular epiphytes — rohani et al. conclusion the diversity of vascular epiphytes in the mangrove forest of pulau telaga tujuh was relatively low (shannon index, it = 1.43) and the most abundant species was hydnophjtum formicarum. the vertical distribution of the epiphytes, being mostly situated on the trunk stratum, depicted the adaptations of these plants to stresses in the mangrove ecosystem, such as drought and salt spray. these findings can help support the conservation efforts directed towards this less appreciated plant group. acknowledgements the authors would like to thank the ministry of education of malaysia for funding 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in muara gembong estuary, bekasi, west java province, indonesia raden indarjani* and siti nurhayati 1department of biology, faculty of science and technology, universitas islam as-syafiiyah, jakarta 17411, indonesia received 13 september 2021/accepted 24 march 2022 abstract the study of macrozoobenthic in seaweed culture in muara gembong estuary, bekasi district, west java province, was conducted in may to july 2018. the study was aimed at understanding the roles of macrozoobenthic organisms as ecosystem engineering in seaweed habitat by identifying macrozoobenthic community structures using various biological indices. sampling sites were conducted at three selected intertidal ponds used for seaweed culture at different distances and perpendicular to the coastline. samples of macrozoobenthic organisms were collected using an ekman grab during low tide periods. the study results showed that the macrozoobenthic community from the three ponds were consisted of 9 major benthic families and 14 genera with a total of 139 individuals. the results also showed that gastropod of the genus cerithiidae was the dominant taxa found in every pond which contributed to 42.45% of the total macrozoobenthic found in the three ponds and became the main contributing taxa to the macrozoobenthic community structure. in addition, genus platynereis of the polychaeta class was found to be another important taxon which contributed to 14.39% of the total macrozoobenthic found in the three ponds. the genus platynereis were mostly found in the second pond with muddy coarse sandy sediment substrate containing more silt compared to the other two ponds. the rare taxon was the genus lithophaga from family mytilidae represented by 1 individual. our study concluded that the macrozoobenthic community structure in the three ponds was categorized as poorly diverse indicating that the pond system was unstable. the shannon-wiener diversity index (h’) was only 0.87 on average with the highest diversity index (h’=1.47) was found in the third pond located at the farthest area of the coastline. meanwhile, the average of evenness index was 0.34 indicating that the distribution of the taxa was uneven with a tendency of being dominated by certain taxa. keywords: biological indices, community structure, macrozoobenthic, seaweed aquaculture introduction indonesia is the among the largest global producers of seaweed and supplies around twothird of the global demand (deswati & luhur 2014). gracilaria sp. and eucheuma cottonii are the two popular species of seaweed out of the 50 cultured seaweeds species in indonesia (deswati & luhur 2014). west java provincial authorities claimed that seaweed is one of the primadonna of the aquaculture export commodities with bekasi district contributing 10,000 tonnes of dried seaweed, in which 7,000 tonnes was produced from the muara gembong seaweed culture area (mujiyanto et al. 2014). seaweed is often cultivated in polyculture system with milkfish (chanos chanos) and/or tiger shrimp (penaeus monodon). these practices cause multiplying effects to the environment, economy growth and food security, if this practice is done properly. a case study in the gulf of castelamarre, italy reported valuable polyculture practices as a reliable economic tool through increasing fish production and sustainable environmental management (zenone & sara 2007). as an ecological engineering practice, seaweed polyculture can reduce the impact of wastes from fish cultivation through recycling of particulate and dissolved matters and enhancing the total productivity for both commodities (troell et al. 2013). *corresponding author, email: indarjani61@gmail.com mailto:indarjani61@gmail.com macrozoobenthic community structures in seaweed culture pond at muara gembong estuary – indarjani and nurhayati 135 the growth of gracilaria sp. mostly depends on the availability of nutrients as this species need nutrient uptake of around 0.0082 -0.0149 ppm/day which in turn can eliminate the excessive fish food (komarawidjaja & kurniawan 2008). thus, water quality parameters such as the level of nutrient content, suspended organic matter as well as weedsand diseases-free environment are important requirements for culturing gracilaria sp. to attain maximum growth (reksono et al. 2012; mujiyanto et al. 2014). in aquatic ecosystem, nutrients availability was maintained by macrozoobenthic organisms. the macrozoobenthic community has a complex structure consisting of a wide range of invertebrate animals inhabiting the bottom of aquatic ecosystem. macrozoobenthic have critical roles in maintaining energy flow and mineralization cycles ensuring that the aquatic ecosystem is functioning at an optimum level (indarjani 2003). macrozoobenthic are mostly found as sessile animals, crawling, burrowing and digging the sediment, permanently or temporarily. macrozoobenthic’s live is associated with substrates. macrozoobenthic is favorable as bioindicator organisms due to its capacity to record the environmental changes. the distribution of macrozoobenthic is determined by physical, chemical and biological parameters which will influence the abundance and composition of the macrozoobenthic as well as the producers, herbivores and predators (snelgrove 1998; indarjani 2003). seaweed and macrozoobenthic perform a mutualistic relationship referring to nutrientcycling and food web, even though the magnitude and mechanism of this relationship is still limited. thomsen et al. (2013) reported that the increase biomass of seaweed gracilaria vermiculophylla is associated with the abundance of benthic invertebrates because the root system of the seaweed facilitated a creation of a 3d mosaic structures and interstitial spaces that provide spaces for different sizes of invertebrates to live and grow. in addition, cummin et al. (2004) suggested that seaweed enteromorpha intestinalis influences the structure of macrobenthos communities compared with unvegetated plot in an australian estuary. the thickness of algal and oxygen concentration at the bottom layer explained the best structure of macrozoobenthic community and enhanced the species richness (lauringson & kotta 2006). muara gembong is located at the north of bekasi district adjacent to jakarta bay. in 2008, the gracilaria sp. polyculture system was introduced and became one of the potential local commodities for the livelihood of the local community and strengthening the food security in that area. the muara gembong estuary is around 13,205,702 ha and has been developed intensively causing impact that may influence the macrozoobenthic communities (pirzan & pong-masak 2008). looking at the potential of increasing the productivity of seaweed culture, this study aimed to gain understanding on: 1) the roles of macrozoobenthic organisms as ecosystem engineering in the habitat of gracilaria sp. by identifying macrozoobenthic community structure using several biological indices and 2) the effect of physicochemical variables on the types of macrozoobenthic community. materials and methods study site and time the study was conducted from may to july 2018, in an urban estuarine called muara gembong, that located under the administration of muara gembong subdistrict which lies at latitude 6º10’53”-6º30’6” s and 106º48’28”107º27’29” e. sampling site determination the location of sampling site was pantai sederhana village, one of the six villages under the administration of muara gembong subdistrict (fig. 1). in this location, seaweed culture was carried out in intertidal ponds with varying substrates of the pond bottom. the ponds have separate inlet and discharge sluice gates and water canals surrounded by various densities of mangrove trees, i.e., avicennia sp., sonneratia sp., rhizophora spp. and bruguiera sp. biotropia vol. 29 no. 2, 2022 136 figure 1 sampling sites located in pantai sederhana village, muara gembong estuary, bekasi district, west java province, indonesia the first pond was located at 100 m from the coastline and has an area of approximately 1.3 ha surrounded by low density of mangrove tress. at this location, the gracilaria seaweed seemed to be well maintained. visually, the substrate type of the pond bottom was fine sandy sediment. the second pond was at 250 m from the coastline with an area of 3.7 ha. the gracilaria sp. seaweed seemed to be poorly grown. the substrate type of the pond bottom was muddy coarse sandy sediment. mangrove trees with low density were planted around the second pond. the third pond of around 4.0 ha was located at 500 m from the coastline and was close to the fishermen’s residential area. the gracilaria sp. seaweed in the third pond seemed to be better maintained when compared with the seaweed in the second pond. due to the closeness of the third pond’s location with the fishermen’s residential area, household wastes may overflow to the third pond during high tide. the third pond may also receive less seawater compared to the other two previous ponds because of its distance from the coastline. the substrate of the third pond bottom was fine sandy sediment. a high density of mangrove tress was found around the third pond. sample collections samples of macrozoobenthic organisms were collected from each seaweed culture pond during low tide. five replicates of macrozoobenthic samples were taken at each pond (a total of 15 samples) by using the ekman grab sampler having a diameter of 152 x 152 x 152 mm, which was suitable for fine sediment (putro 2011). the samples were then stored in airtight plastic bags and preserved in 10% buffered formalin before being transported to the laboratory. in the laboratory, the samples west java administrative areas city regency capital west java map (source: www.google.com) map of muara gembong, bekasi (source: www.google.com) pantai sederhana village (source: www.google.com) macrozoobenthic community structures in seaweed culture pond at muara gembong estuary – indarjani and nurhayati 137 were washed through a 0.5 mm mesh sieve. the subsequent identification of macrozoobenthic organisms was carried out up to the genus level, referring to indarjani (2003) and bremmer (2005). after the identification process, the macrozoobenthic samples were preserved in 70% alcohol. water quality parameters such as ph, dissolved oxygen, water temperature, water clarity and salinity were recorded in situ. ph was measured using ph meter, salinity using refractometer, water clarity using secchi disk, dissolved oxygen using do meter. the water sample collection were also done in five replications. data analysis data analysis was conducted using descriptive statistics to describe and summarize the data. the data analysis may show certain patterns of macrozoobenthic distribution. to examine ecological function of macrozoobenthic community, several biological indices were calculated, as suggested by pakpahan et al. (2013) such as: 1) shannonwiener diversity index (h’) to calculate the taxa diversity (odum 1971); 2) evenness index (e) to examine the distribution of taxa (elliott 1971); 3) simpson dominance index (d) to observe the dominance of taxa related to the macrozoobenthic community (odum 1971); and 4) abundance relative index (r) to examine the population degree of certain taxa that support to the total abundance of macrozoobenthic community. the total abundance (n) was then standardized by dividing the total of biota of each pond to densities per m2 resulted in a value in number of abundances of each taxon per m2 (cox 1995). results and discussion water quallity parameters one of the most important factors in marine culture fisheries is the quality of seawater which act as growth medium for benthic organisms, both flora and fauna. results of water quality paramaters measurement are presented in table 1. the results showed that water quality among the culture ponds was quite varied. data of temperature, salinity and ph were considered to be suitable for culturing graciliaria sp., even though water clarity and dissolved oxygen was found not suitable (mujiyanto et al. 2014). data of water clarity in all ponds were lower than the suitable range. the low water transparency may have been caused by the period of low tide when the measurement was carried out. during the measurement, the secchi disk may have been covered by seaweed plants, while the pond water may have been murky due to high suspended organic matter from the fish food wastes, fertilizers, floating debris from the surrounding mangrove trees and household wastes. the lower dissolved oxygen value could be explained as a result the shallowness of the pond, high decomposition process, disturbed water, residue of organisms’ respiration during the night, and relatively high temperature during the day when water sampling was conducted. data of abiotic water quality parameters showed that temperature was around 31.6-31.8 oc, which nearly reached the highest temperature for suitable range of seaweed culture. the depth of each culture pond was around one meter due to dry season when the sample collection was conducted. in addition, mujiyanto et al. (2014) stated that the muara gembong estuary was classified as having shallow waters. however, in regard to the existence of macrozoobenthic communities, the water quality parameters were within the acceptable range (marpaung et al. 2014; syamsurisal 2011). macrozoobenthic compositions our study found 4 phyla, 5 classes, 6 ordos, 9 families and 14 genera of macrozoobenthic with a total number of 139 individuals in muara gembong seaweed culture ponds (table 2). previous research conducted by wijaya and indriatmoko (2019) reported that 44 genera of benthic organisms was identified from the muara gembong waters and dominated by the class polychaeta that made up 52.50% of macrozoobenthic community. the high yield of macrozoobenthic organisms obtained presumably due to large area of sampling sites, while in this study the macrozoobenthic organisms were collected from three selected seaweed culture ponds. in addition, the abundance of macrozoobenthic organisms and taxa richness were relatively different among the three ponds which could influence the macrozoobenthic community structures in this area. biotropia vol. 29 no. 2, 2022 138 table 1 average values of water quality parameters at each seaweed culture pond no abiotic water quality parameters first pond second pond third pond suitability range of water quality parameters for seaweed culture 1 temperature (oc) 31.8 31.6 31.6 29-32 2 secchi disk transparency (cm) 16.3 17.7 16.7 75-153 3 salinity (‰) 20.8 20.6 19.4 16-25 4 ph 7.6 7.5 7.8 7.5-8.5 5 do (mg/l) 3.20 3.20 3.25 6.5-8.5 table 2 composition of macrozoobenthic assemblages collected during the study no genus first pond (individuals) second pond (individuals) third pond (individuals) total (individuals) 1 telescopium 2 0 0 2 2 cerithiidae 43 2 14 59 3 cerithideopsilla 0 1 1 2 4 cerithium 0 1 5 6 5 tarebia 0 0 9 9 6 sermyla 0 1 5 6 7 terebralia 1 1 4 6 8 melanoides 0 0 4 4 9 hydrobia 0 0 5 5 10 lithophaga 0 1 0 1 11 platynereis 5 9 6 20 12 notomastus 0 0 2 2 13 sipuncula 0 0 1 1 14 litopenaeus 1 3 12 16 total (individuals) 52 19 68 139 results of our study indicated that the existing macrozoobenthic communities in seaweed culture ponds were mostly dominated by marine gastropods cerithiidae (59 individuals or 42.45%) which was extremely abundant in the first pond. the second dominant taxon was platynereis of the class polychaeta (20 individuals or 14% of the total macrozoobenthic found in this study) which was abundant in the second pond. in addition, marine bivalve of lithophaga and unsegmented marine worm sipuncula could be reckoned as rare taxa. only one lithophaga was found in the second pond and only one sipuncula was found in the third pond. the second pond inhabited by 19 individuals of 8 taxa or 14% of the total macrozoobenthic organisms seemed to be an unfavorable habitat for macrozoobenthic compared with the other two ponds. conversely, the platynereis was found quite many in the second pond where the seaweed culture was poorly maintained. in contrast, the third pond inhabited by 68 individuals of 12 taxa or 49% of the total macrozoobenthic found in this study was considered as a favorable habitat for macrozoobenthic where the seaweed culture was well maintained and surrounded by dense mangrove trees. meanwhile, the first pond which was situated adjacent to the coastline had the lowest taxa richness (52 individuals of 5 taxa) with gastropods cerithiidae as the most abundant macrozoobenthic organisms (82%) in the first pond. different compositions of macrozoobenthic communities may have been caused by the types of substrates and the microenvironment factors where the macrozoobenthic organisms live. based on visual (qualitative) observations, the first pond has the fine sandy sediment substrate which is relatively similar to the type of substrate in the third pond. the second pond has the muddy coarse sandy sediment substrate. wiyaningtiyah et al. (2014) stated that fine sandy sediment has a better capability in accumulating organic materials compared with coarse sandy sediment. the different types of substrates influence abundance and types of macrozoobenthic community structures (hadiyanto 2018). community structure of macrozoobenthic assemblages the macrozoobenthic community structure was assessed using various biological indices, i.e., the shannon-wiener diversity index, the evenness index and the dominancy index. the calculated indices indicated that the macrozoobenthic community structure was categorized as poorly diverse which suggested that the community system was unstable (fig. 2). macrozoobenthic community structures in seaweed culture pond at muara gembong estuary – indarjani and nurhayati 139 figure 2 macrozoobenthic community structure showing irregular trends the average of shannon-wiener diversity index (h’) was 0.87 with the highest index of 1.47 was found in the third pond which was the farthest pond from the coastline. snelgrove (1998) predicted that species diversity is mainly controlled by the fluctuations of environmental factors and tends to be low in physically controlled environment, such as in seaweed culture pond. meanwhile, the evenness index value ranged from 0.25 to 0.55 with an average of 0.34. the low value of evenness index indicated the uneven distribution of the taxa in which the community tends to be dominated by certain taxa. in our study, the cerithiidae dominated the macrozoobenthic community structure in the first pond, located adjacently to the coastline. the existence of cerithiidae contributed significantly to the macrozoobenthic community structure in the muara gembong seaweed culture ponds. this finding is in agreement with the work of ludwig and reynolds (1988) which indicated that the evenness index should be maximum when all species in the samples were equally abundant. the evenness index can drop to zero when the relative density of species diverges away from the evenness. referring to the values of the calculated biological indices, the third pond was considered as the favorable habitat for macrozoobenthic organisms, having the highest score of the shannon-wiener diversity index (h’ = 1.46) and the evenness index (e = 0.55). the low dominancy index (c = 0.31) indicated that macrozoobenthic organisms had relatively similar opportunity to grow and the population was distributed accordingly. the third pond occupied by seaweeds and surrounded by dense mangrove trees with fine sandy sediment substrate may have been contributed to the richness of macrozoobenthic taxa. seaweed root system might facilitate a creation of a 3d mosaic structure and interstitial spaces of substrate so that different sizes of invertebrates organisms could live and grow (thomsen et al. 2013). the curve linearity of the macrozoobenthic species living in a densely algal environment has higher increasing slope compared with macrozoobenthic assemblages in a poorly vegetated area (lauringson & jonne 2006). in addition, mangrove debris around the pond might become potential food resources for the macrozoobenthic functioning as wastes decomposer (palanisamy & khan 2013). dominant taxa examinations the first pond in this study was dominated by gastropods cerithiidae and had density of 8 individuals per meter square with relative abundance of 82%. the gastropods cerithiidae is considered as the characterized macrozoobenthic assemblages of seaweed culture in muara gembong. previous research reported by ardli et al. (2015) and kamaluddin (2017) showed that the distribution and density of cerithiidae were influenced by types of substrates and other various supporting factors such as dietary factors, water conditions, predators and competition. those factors also influenced the abundance of cerithiidae in our study which were varied among the three ponds. thus, in this case, the cerithiidae can be reckoned as a cosmopolitan taxon and has a good adapting capability to live and grow in the fine biotropia vol. 29 no. 2, 2022 140 and muddy sandy sediment substrates. the fine sandy sediment may be favorable for the digging and burrowing macrozoobenthic organisms, like gastropods cerethiidae (schirjvers et al. 1998). besides, the fine sandy sediment substrate is effective in trapping organic materials that stimulate the optimum growth of macrozoobenthic organisms. another important taxon in our study that contributed to the macrozoobenthic community structure was platynereis of the polychaeta class. this organism was found in every pond with the highest relative abundance of 47.37% and became the main contributing taxa to the macrozoobenthic community structure in the second pond. the type of substrate in the second pond may have been more suitable for platynereis than for other taxa. taqwa et al. (2014) confirmed that class polychaeta can easily be found in muddy as well as fine sandy substrates, which is relatively similar with the second pond’s substrate type. according to cambi et al. (2000) platynereis dumerii as mezograzer species prefers the brown seaweed sphacelaria. our study showed that the density of platynereis in every pond were not much different indicating that the environment of the three ponds was suitable for platynereis to grow. conclusion the study of macrozoobenthic community inhabiting seaweed culture ponds in muara gembong estuary showed that the macrozoobenthic community structure was characterized with low diversity, low evenness and high dominance indices, which indicated that the environmental condition was unstable and the macrozoobenthic community structure was dominated by certain taxa. marine gastropod cerithiidae seemed to be the main contributing taxa to the macrozoobenthic community structure as this organism was found in every pond. the second abundant macrozoobenthic organisms was platynereis. this study also showed that the third pond located at the farthest distance from the coastline seemed to be the favorable habitat for macrozoobenthic organisms. seaweed in this pond was grown luxuriant and surrounded by dense mangrove trees, with a higher diversity index, high taxa richness, and high evenness index, indicating that organisms in this pond have similar opportunities to grow. the first pond located adjacent to the coastline was considered favorable for gastropode genus cerithiidae, even though this pond had the lowest taxa richness, lower evenness index and high dominance index values. the second pond can be seen as the “transition” pond between the first and the third ponds considering its biological indices values were in between. references ardli er, widyastuti a, yani e. 2015. study of bioecological changes in mangrove ecosystem restoration in segara anakan. cilacap, central java province [kajian perubahan bioekologi pada restorasi ekosistem mangrove di segara anakan, cilacap]. biosfera 32(1): 19. bremmer j. 2005. assessing ecological functioning in marine benthic communities. 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[thesis]. retrived from universitas hasanuddin, faculty of marine and fisheries, dicipline of marine ecology. makassar (id): universitas hasanuddin. taqwa n, muskananfola r, ruswahyuni. 2014. study of relationship between substrate and organic content with macrozoobenthos in sayung river estuary, demak district [studi hubungan substrat dasar dan bahan kandungan organik dalam sedimen dengan kelimpahan hewan makrobentos di muara sungai sayung, kabupaten demak]. maquares 3(1):125-33. doi: https://doi.org/10.14710/ marj.v3i1.4429 thomsen ms, staehr p, nejrup lb. 2013. effect of the invasive macroalgae gracilaria vermiculophylla on two co-occuring foundation species and associated invertebrates. j aquat invasions 8(2):133-45. doi 10.339/ ai.2013.8.2.02 troell mh, neori c, chopin a, buschmann t, kautsky ah, yarish n. 2003. integrated mariculture: asking the right questions. aquaculture 226:69-90. wijaya d, indriatmoko. 2019. komposisi dan sebaran makrozoobentos di perairan muara gembong [composition and distribution of macrozoobenthic in muara gembong waters]. book chapter. management startegy of ecology coastal resouces of muara gembong, jakarta bay. krismono, pranowo ws, editors. jakarta (id): amafradpress. 234 p. wiyaningtiyah aa, setiobudiandi s, taurusman aa. 2014. keterkaitan struktur komunitas makrozoobentos antara habitat mangrove, lamun dan reef crest di pulau kelapa dua, kepulauan seribu]. [connectivity of macrozoobenthos community structure along gradient mangrove, seagrass and reef crest habitats in pulau kelapa dua, kepulauan seribu, jakarta]. bonoworo wetlands 4(1): 37-48 doi:10 1 3057/bonorowo/w040103 zenone a, sara g. 2007. polyculture as a tool to increase the economic income: a study case in the gulf of castellamare. j animal sci 6 (suppl 1):837-48. https://doi.org/10.1007/s10750-005-1009-4 https://doi.org/10.1007/s10750-005-1009-4 http://dx.doi.org/10.1186/2046-9063-9-15 https://ejournal.undip.ac.id/index.php/sm/article/view/7912 https://ejournal.undip.ac.id/index.php/sm/article/view/7912 https://doi.org/10.14710/marj.v3i1.4429 https://doi.org/10.14710/marj.v3i1.4429 biotropia vol. 29 no. 2, 2022 142 biotropla vol. 28 no. 1,2021: 38 45 doi: 10.11598/btb.2021.28.1.983 increasing risk of heavy metal contamination i n silvofishery ponds endah dwi hastutt, rini budihastuti and sri darmanti biology department, faczllo $science and mathematics, diponegaro universig, jalan prof: soedarto, sh tembalang, semarang 50275, indonesia received 18 january 201 8/accepted 17 april 2020 abstract this research aimed to determine the concentration of organic matter, pb and cd found in a silvofishery pond, to assess the toxicity level, to analyze the changes in their concentration within a year's period and to analyze the correlation between concentration and changes. the research was conducted by doing five field observation activities and laboratory analysis from may 2016 to july 2017. data were analyzed using anova and correlation tests. the concentration of pb and cd in the organic matter, was increasing in all five observations. throughout the research, the concentration level ranged at 1.60 3.30 mg/kg for organic matter, 3.130 8.230 mg/kg for pb, and 1.089 2.820 mg/kg for cd. in all observations, the toxicity level of cd concentration in the sediment exceeded the standards recommended by us epa (i 1.0 mg/kg) and anzecc & armcanz (5 1.5 mg/kg), while pb concentration was within the safe range (5 21 mg/kg and 5 50 mg/kg, respectively). moreover, the concentration and accumulation of pb and cd were strongly correlated, which indicated the possibility of having the same pollutant sources. therefore, a better management plan is recommended to avoid heavy metal accumulation in silvofishery ponds, a plan that would include the arrangement of mangrove plants in inlet canals and their periodic pruning to prevent the heavy metal from returning to the environment through the litter fall. keywords: aquatic toxicology, bioremediation, cadmium, heavy metal, sediment chemistry, silvofishery introduction silvofishery has supported the aquaculture activities through its emphasis on environ mental services (suwarto e t al 2015). the silvofishery system integrates mangrove plants into ponds to minimize environmental pollution caused by its effluent (hastuti & budihastuti 2016). the mangrove ecosystems has also improved the natural primary productivity because of nutrient cycling pudihastuti e t al 201 3). mangrove plays an important role in controlling the pollutants in the coastal ecosystem. its root ecosystem can trap the organic matter sediments which contain the heavy metal pollutants (yunus e t al. 201 1). thus, reducing the concentration of pollutants in the aquatic system. mangrove plants can also act as 'corresponding author, email: endah-pdil@yahoo.com bio-remediators that are capable of absorbing pollutants from the environment and accumulating these in its body parts (pakzadtoochaei 201 3). thus, mangroves have dual functions of controlling pollutant distribution, and remediating its growing environment. mangrove roots trap the polluted sediments and these sediments are then suspended around the tree. thus, as pollutants accumulate, their concentration below the mangrove stands increases (kathiresan e t al. 2014). however, by absorbing the pollutant, the mangroves would decrease its concentration in the sediments (selanno e t al. 2015). thus, the accumulation and diminishing rates of pollutants are affected by the balance between the deposition of pollutants and the absorption capacity of mangrove plants. heavy metal is one of the most dangerous types of pollutants that could be consumed and accumulated in plants and animals organs, increasing risk of heavy metal contamination in pond dwi hastuti e t al: resulting in toxicity (reis e t al. 2010; asati e t al. 2016). thus, food chain is the most important aspect in the accumulation of heavy metal toxicity (hajeb e t al. 2014). contamination of lower level organisms would lead to the accumulation of heavy metal in higher level organisms. obviously, the risk of heavy metal contamination may increase in a polluted environment. among the heavy metal contaminants, pb and cd are the most common aquatic pollutants produced from daily activities. sources of pb include volcanic explosions, forest fires, lead emissions from industry and transportation, metal processing, and manufacturing (zhang e t al. 2015). anthropogenic sources of pb pollution include paints and glazed budding materials (walraven e t al. 2016). the main sources of cd in the sediment are the industrial and mining wastes (yu e t al. 2010; donovan e t al. 2016). although still debatable, the application of p ferulizer was also considered as an additional source of cd in the agricultural soil (roberts 201 4). in order to secure environmental safety against pollutant contamination, some countries had developed environmental quality standards which differ in their applications. us epa applies a maximum value of 1 mg/kg for cadmium (cd) and 21 ppm for lead (pb), while anzecc & armcanz recommends 1.5 mg/kg for cd and 50mg/kg for pb (hubner e t al. 2009). in view of the environmental standards imposed by many agencies, the threat of heavy metal contamination is still very evident. moreover, heavy metal does not appear as single element but is mostly attached to organic matter in the environment. the accumulation of heavy metal is related to the accumulation of organic matter (yang e t al. 2010). hence, studies on the trend of organic matter and heavy metal accumulation are equally important. silvofishery has been considered as a "good management system" applied in conservative aquaculture (jonell & henriksson 2014). in most cases, however, the real application is different from the proposed model. for instance, plantations are set in the middle of ponds without any separating dikes whch are supposed to prevent direct contact between the pool and the mangrove. thus, the risk of pollutant contamination to the livestock cultivars might increase. silvofishery is mostly applied in degraded ponds (yunus e t al. 2015), making the heavy metal accumulation an additional risk to its already poor environmental quality. however, little concern has been directed towards organic matter and heavy metal accumulation in silvofishery. hence, this research was conducted to study the concentration of pb and cd in a silvofishery pond, and its toxicity level, to analyze the changes in their concentration level within a year's period, and to analyze the correlation between these concentrations and changes. materials and methods the research was conducted from may 2016 to july 2017 in a silvofishery pond in mangunharjo village, tugu district, semarang city, central java, indonesia. twenty-seven sampling points were selected and dstributed throughout the pond area (fig. 1). data were collected during the five sampling periods in may, july, and september 2016 and in may and july 2017. generally, the weather was bright with warm temperature during the sampling period. since the sampling was carried out during the dry season, the fresh water dominated the supply to the silvofishery pond. data collection was carried out to determine the concentration of organic matter, lead (pb) and cadmium (cd) in the sediment across a one-year period. samples obtained from the sedment accumulated below the mangrove stands were then analyzed in the laboratory. as much as 500 g sediment samples were taken from the surface to a depth of 30 cm using scoop and dpper. the sediment was readily washed. therefore, ekman grab could not be used for the sediment sampling. the water was filtered before the sediment was moved into the plastic bag. laboratory analysis was done at the wahana laboratorium, semarang. the organic matter was analyzed using the ashing method, whde total heavy metal content was analyzed using atomic absorption spectrophotometry (aas) method. biotropia vol. 28 no. 1,2021 inlet tunnel outlet tunnel ~ o o o o , o o o p o o o o o o ' 8 , o ~ o ' q o o o o p o ~ o o \ o o o o o p o o o o o \ /\\ -. \ / i \ ! ' / effluent \ \ / sampling points figure 1 schematic diagram of sampling points changes in organic matter and heavy metal concentration at each sampling point were also computed and analyzed. analysis of variance was performed on the mean values of organic matter and heavy metal taken from different sampling periods, as well as their changes over those periods. pearson correlation analysis was performed to determine the relation between periodic average of organic matter, pb and cd concentrations and their changes during the study period. results a n d discussion i pond decreased from the first to the second observation. among the sampling periods, the second period showed the most significant increase in the concentration of pb and cd (table 1). concentration of heavy metals indicated that pb was still within the safe range (table 1). the maximum pb concentration (8.090 mg/kg), was far below the critical value proposed by us epa (21 mg/kg) and anzecc & armcaz (50 mg/kg). thus, the silvofishery pond in mangunharjo vdlage was safe from pb contamination. however, the cd concentration has exceeded the recommended limit. the variations existed in the concentration of minimum concentration of cd in the first (1.089 organic matter, pb and cd in the silvofishery mg/kg) to third (1.460 mg/kg) observations pond over the five different sampling periods. exceeded the us epa limit of i 1 mg/kg. in data showed that there were increases in the fourth and fifth observations, the minimum average concentrations and decreases in range, concentration of cd even exceeded the limit set indicating a trend towards stabhation. the by anzecc & armcaz with the expected concentration of organic matter, pb and cd concentration of 5 1.5 mg/kg. table 1 concentration level of organic matter, pb and cd in the silvofishery pond sediments observation period no. parameters i i1 111 lv v 1. organic matter (%) a range 1.89-3.30 1.71-3.12 1.60-2.70 1.65-2.62 1.90-2.83 b average 2.23 + 0.28~ 2.21 f 0.31~ 2.27 + 0.27~ 2.29 + 0.25~ 2.38 f 0.20~ c averape chan~e -0.04~ 0.05x 0.02" 0.ogx " " 2. pb (mg/kg) a range 3.130-8.230 4.120-7.230 4.353-8.090 4.501-7.930 5.130-7.449 b average c average change " u 3. cd (mg/kg) a range 1.089'-2.4094 1.1 1 8'-2.4404 1.460'-2.8204 1.5109-2.7104 1.5834-2.7724 b average 1.8874 f 0.353p 1.8514 k 0.344 2.1104' k 0.339s 2.1204' k 0.3364 2.1714 f 0.2584 c average change -0.026x 0.259~ o.01ox 0.052" notes: e = exceeded the standard value of us epa, i$ = exceeded the standard value of us epa and anzecc & armcaz; pg 4, x, y = different letter across the same row indicates significant differences. increasing risk of heavy metal contamination in pond dwi hastuti e t al: the analysis of variance (anova) of organic matter did not show any significant difference among the mean values and concentration changes of the five sampling periods. t h s indicated that the management of organic matter concentrations and changes in the silvofishery pond was effective. however, the anova of pb and cd concentrations and changes showed that there was a significant difference among the different sampling periods. there were two groups of mean concentration: the first group which consisted of the first and second observations, and the second group which consisted of the third to fifth observations. this indicated that a significant increase in pb and cd accumulation happened in the second period. analysis of the concentration changes showed that a sipficant change occurred between the second period and the other periods. the sipficant increase in pb and cd concentration in the second period indicated the dominance of heavy metal accumulation in certain sampling periods. another important concern manifested by these findings is the increasing accumulation of pb and cd. in the first period, the concentration of both heavy metal elements was relatively low, but in the second to fourth periods it tended to increase. this indicated the increasing risk of heavy metal pollution in the silvofishery pond, which might trigger the contamination of the livestock cultivars. the concentration of cd alone was sufficient proof that the pond was contaminated. thus, a more effective pond management plan should be formulated in order to avoid the danger of continuous deposition of heavy metal pollution and subsequent contamination. heavy metal accumulation in the sediment is related to the sedimentation process. the sediment carries various substances including organic matter and heavy metal. thus, the accumulation of heavy metal might be interrelated with that of any other substances. this research also evaluated the correlation between heavy metal and organic matter (table 2). although the correlation coefficients were high, the organic matter concentration was not related to pb and cd concentration, as well as with concentration changes (table 2). however, the concentrations and changes of pb and cd correlated with each other. this indicated that the source of organic matter was not only sediment accumulation. low correlation level between concentration changes indcated the difference in the accumulation rate between the organic matter of pb and cd. the high and significant correlation between pb and cd concentrations and changes showed that both heavy metal elements came from the same source, i.e., the contaminated sediment. the silvofishery pond in mangunharjo village has accumulated organic matter and heavy metal elements, as indicated by the increasing concentration of these substances in the sediment. mangrove vegetations in the pond provide fine sedment trapping by slowing down the current; thus, resulting in an increase in the sedimentation rate (adame e t ul. 2010). an increase in heavy metal concentration in ponds is hghly related to the water sources. coastal ponds are supplied by both sea and river water. however, river streams are considered as the main source of pollutants in the coastal area since these are the channels of anthropogenic and industrial waste disposal (icumar e t ul. 2015). table 2 correlation among om, pb, and cd concentration and changes in a silvofishery pond correlation (sig.) average concentration om pb cd average concentration change om ---0.394 (0.606) 0.484 (0.516) pb ---0.995 (0.005)** cd --- notes: * = correlation is significant at the 0.05 level; ** = correlation is significant at the 0.01 level; some cells are left blank intentionally due to similar correlation items biotropia vol. 28 no. 1,2021 various inland activities produce heavy metal contaminated wastes which then enter the aquatic system (zhang e t al. 2010). when the freshwater flows to the estuaries, the heavy metal is dispersed throughout the surrounding ecosystem (ruzhong e t al. 2010), such as the ocean and ponds. most heavy metal pollutants are bound to fine sediments (zhang et al. 2014). when the water enters vegetated ecosystem, the flow would slow down, increasing the chance of sedimentation (turgut et al. 2015). thus, the accumulation of heavy metal occurs along with sediment deposition. the increasing heavy metal concentration in the silvofishery pond indicated that the water sources were polluted. the source could be from the freshwater stream or the seawater. the semarang rivers, as well as the city's coastal areas, have been known to be polluted due to high industrial activities (hastuti 2015). thus, there is a severe risk of pollutant contamination in the silvofishery ponds. moreover, the heavy metal concentration is expected to continually increase due to sediment trapping by mangrove roots in the ponds. although the mangroves are known as bioremediators of heavy metal pollutants (kdnnan e t al. 2016), their capacity is limited. the mangrove trees could accumulate heavy metal elements in their various parts but the elements would be returned to the environment through litter fall and the decomposition processes (martuti et al. 2017). thus, the accumulation rate of heavy metal pollutants would depend on the supply rate from the sources and the uptake rate by the mangroves. the increasing trend of pollutants accumulation showed that the mangrove uptake rates could not match the pollutant supply rates. this requires the development of more effective management plans to prevent the increasing risk of heavy metal contamination and to reduce the toxicity level of the livestock cultivars. the extreme increase of organic matter and heavy metal concentration in the second period (from the 2nd to 31d observations) manifested the high input rate of sediment during that period. this might have occurred due to frequent rains that flooded the pond (gharbi e t al. 2016). and carried hgh amount of sediments from the river stream to the ponds. the ponds which generally occupy a single canal as water inlet and outlet would trap incoming water, thus increasing the chance of sediment deposition. cd concentration in the silvofishery pond indicated an unsafe environment. although heavy metal concentration in the sediment is not directly related to water quality, this may affect the availability of dissolved heavy metal in the water (hassaan e t al. 2016). us epa recommends a maximum concentration of 1 mg/kg of cd in the sediment, wme anzecc & armcanz7s standard is hgher at 1.5 mg/kg (hubner et al. 2009). however, in a l l observations, the average concentration of heavy metal exceeded both standards, indicating a high possibility of a polluted environment since the beginning. therefore, such increases in the concentration rates need thorough consideration in the management of silvofishery ponds. this research showed that pb and cd were accumulating in the silvofishery pond sediment. however, only cd exceeded the recommended value, while pb was still within the safe range. such condition increases the alarming possibihty of heavy metal contamination of cultivated fish (icumar et al. 2011). any toxic elements in the aquatic system can be easily transported from one organism to another through the food chain. heavy metal pollutants would be absorbed by a low trophic level organism such as phytoplanktons, which are then consumed by zooplanktons and finally by fish (hassaan et al! 2016). thus, the risk of contaminated fish consumed by humans increases as well. through the food chain phenomena, heavy metal elements will bioaccumulate in living organisms; plants, animals, and humans. the toxicity in a plant is usually shown by several symptoms, such as changes in leaf color or abnormal plant growth (nagajyoti et al. 2010). unfortunately, toxicity in mangrove is usually unnoticeable as they have a high level of tolerance due to their bioaccumulative capability (icannan etal. 2016). thus, the effect of toxicity may expand to other aquatic animals including crabs, shells, and cultured fishes. some animals are also known to be tolerant to heavy metal toxicity, such as shells (shirneshan & bakhtiari 2012). however, the animals with low tolerance level may show toxicity symptoms, such as declining growth rate and damaged liver and kidney tissues (jayakumar et al. 2016). increasing risk of heavy metal contamination in pond dwi hastuti e t al: the bioaccumulative capability of aquatic organisms increases the chance of heavy metal consumption and subsequent accumulation in the human body. consuming a small amount of contaminated fish would not affect the human body in a short period of time. however, the effects of heavy metal toxicity would be noticeable when the concentration is high enough. various symptoms caused by cd toxicity include kidney damage, osteoporosis, cardiovascular diseases, hypertension, diabetes, modification of several organs, infertility, and many others (bernhoft 2013). to avoid cd toxicity, humans can only take a maximum of 1 pg/kg per day (usepa 1998). the analysis shows that the concentration and accumulation of pb and cd correlated with each other indicating that a common factor had caused the accumulation of both elements. sedment input from contaminated sources was considered as one possible factor. thus, both heavy metal elements were deposited along with the sediments (zhang et al. 2014). the insignificant correlation between the heavy metal and organic matter concentration and accumulation indicated that organic matter had further decomposed to nutrients. this research shows that even though silvofishery provides various beneficial environmental services, some negative impacts might arise after a considerable period of time. various heavy metal elements might accumulate over time, thereby resulting in their increasing concentration. after certain periods of time, the accumulated heavy metal may exceed the safe limit and cause environmental toxicity leading to environmental hazards, especially for aquaculture activities related to human livelihood. in order to maintain environmental quality by avoiding heavy metal toxicity in silvofishery ponds, a more effective management action plan is urgently needed. decreasing the heavy metal concentration in the pond sediments must be the main objective, especially in mangunharjo village. however, the process may take a long time, and controlling the heavy metal concentration in sediments is a gradual process and cannot be done instantly. an important management intervention to decrease the sediment input from the pond is by separating the mangrove plantation. dikes need to be constructed around it, mangroves should be planted in the inlet as a canal formation, thereby filtering the water input and sediment deposition before entering the pond. in so doing, the risk of heavy metal accumulation in the pond is decreased. another strategy to decrease heavy metal concentration in the ecosystem is by pruning the mangrove branches and leaves. thus, heavy metals absorbed and accumulated in the plant organs, particularly the leaves and branches, could be totally removed and prevented from reentering the ecosystem through litter falls. the pruning also stimulates the development of fresh branches and leaves, thereby providing more space for heavy metal absorption in the ecosystem. in this way, the concentration of heavy metal in the silvofishery pond would gradually decrease. the combination of both techniques may improve the effectiveness of heavy metal removal from the silvofishery pond ecosystem, thus, minimizing the risk of heavy metal toxicity. conclusion the silvofishery pond has accumulated organic matter rich with heavy metallic elements pb and cd, indicating an increased risk of environmental toxicity. the pond was contaminated with cd whose concentration has exceeded the recommended level by both us epa (i 1.0 mg/kg) and anzecc & armcanz (5 1.5 mg/kg). this excessive concentration might affect the aquatic organisms in the ecosystem. the increasing accumulation rate during the last periods of observation indicated an imbalance between pollutant supply and mangrove uptake rates. the concentration and accumulation rates of pb and cd show a hgh degree of correlation, indicating a possibility that both metallic elements come from the same sources. the recommendations for better management plans and actions include changing the pond setting by planting mangroves in the inlet canals, developing dikes to avoid direct rapid water flow from the mangroves to the pond, and pruning the mangrove leaves and branches to decrease heavy metal recycling. such pruning would also promote the development of fresh leaves and biotropia vol. 28 no. 1,2021 branches which can increase heavy metal absorption. acknowledgement this research was supported by the pnbp dipa university of diponegoro funding source batch 2017 no. 275-066/un7.5.1 /pg/2017. references adame mf, neil d, wright sf, lovelock ce. 2010. sedimentation within and among mangrove forests along a gradient of geomorphological settings. estuar coast shelf sci. 86(1):21-30. asati a, pichhode m, nikhil k. 2016. effect of heavy 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water, soils and plants in riparian wetlands in the pearl river estuary, south china. procedia environ sci 2(5):1344-54. zhang r, wilson vl, hou a, meng g. 2015. source of lead pollution, its influence on public health and the countermeasures. int j heal anim sci food saf 2(1):18-31. 890-fiks.cdr a pulau katang variety cultivar b c hutan jati variety cultivar gunung batur variety cultivar a 0 1 2 3 4 5 6 7 8 1.5% 3.0% 4.5% 6.0% 7.5% 3000 2500 2000 1500 1000 500 0 hydrolysis time (h) r ed u ci n g su ga r ( g m l -1 ) m 0 1 2 3 4 5 6 7 8 1.5% 3.0% 4.5% 6.0% 7.5% 60000 50000 40000 30000 20000 10000 0 hydrolysis time (h) t ot al s u ga r ( g m l -1 ) m b a 0 1 2 3 4 5 6 7 8 1:5 1:2 1:1 1:10 6000 5000 4000 3000 2000 1000 0 hydrolysis time (h) r ed u ci n g ( g m l -1 ) m 0 1 2 3 4 5 6 7 8 1:5 1:2 1:1 1:10 50000 45000 40000 35000 30000 25000 20000 15000 10000 5000 0 hydrolysis time (h) r ed u ci n g ( g m l -1 ) m b liquid oligosaccharides powder oligosaccharides c ba tlc page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 biotropia vol. 28 no. 2, 2021: 165 172 doi: 10.11598/btb.2021.28.2.1297 165 karamunting (melastoma malabathricum) extracts on white shrimp (litopenaeus vannamei) maturity ahmad ridwan1 and awaludin2 1school of life sciences and technology, bandung institute of technology, bandung, indonesia 2department of aquaculture, faculty of fisheries and marine science, university of borneo tarakan, indonesia received 4 september 2019/accepted 30 january 2020 abstract white shrimp litopenaeus vannamei is one of the prime shrimp commodities cultivated in indonesia. as such, the discovery of more efficient seed production techniques for this species is deemed necessary. karamunting (melastoma malabathricum) extract contains the cholesterol precursor called lanosterol, a phytosterol which is used by crustaceans to form the animal steroid hormone that is very crucial in their reproduction. hence, this research aimed to determine the ovary development of mature l. vannamei individuals injected with the karamunting ethanol extract. the experiment was carried out in several stages. firstly, injecting the white shrimp at the base of the 5th leg, every 3 days for 15 days with variable control dosage 0 (c), 10 mg/kg bw (t1), 7.5 mg/kg bw (t2), 5 mg/kg bw (t3), 2 mg/kg bw (t4) and 1 mg/kg bw (t5), where bw is body weight. secondly, isolating the white shrimp parent ovary. thirdly, measuring the progesterone level in the ovary using the radioimmunoassay (ria) method. fourthly, observing the histology of white shrimp parent ovary and, finally, analyzing the data. measurements of the increase in progesterone levels showed that the administration of karamunting ethanol extract significantly affected the progesterone production (p˂0.05). histology observations of gonadal development in the control, t5 and t4 showed that the cells developed to previtellogenesis oocytes whereas in treatment t1, t2 and t3 ovary cells developed into endogenous vitellogenesis oocytes and only in t1 did the ovarian cells develop to form exogenous vitellogenesis oocytes. karamunting extract significantly increased the oocyte sizes (p˂0.05). at the start of the experiment, the average oocyte sizes were at 15.57 ± 3.15 µm at the end of the experiment, the control was at 25.29 ± 2.69 µm and the ovarian treatments produced the following oocyte sizes; t1 at 65.65 ± 2.64 µm, t2 at 63.98 ± 3.06 µm, t3 at 39.12 ± 6.01 µm, t4 at 28.08 ± 0.84 µm and t5 at 27.65 ± 0.71 µm. the extract produced oocyte sizes greater than at the beginning of maintenance and control. apparently, the lanosterol in the karamunting extract had increased the hormone progesterone resulting in an accelerated gonadal maturity and enlargement of oocyte sizes in the parent individuals of the white shrimp. keywords: gonadal maturity, histology, melastoma malabathricum, litopenaeus vannamei, progesterone, reproduction introduction one of the important world-wide aquaculture industries is shrimp farming which accounted for the global 2.9 million tons of shrimp farm production in 2016 (fao 2017), of which 75% came from asia-pacific. indonesia contributed about 390 tons. one of the shrimp commodities cultivated in indonesia is the white shrimp (litopenaeus vannamei). for better cultivation, more efficient techniques are needed for white shrimp production. several ways were tried to increase its seed production, among others; eye ablation, hormone injections, and high protein feeding. however, some of these methods have not been able to significantly improve shrimp reproduction. gonad maturity in white shrimp is influenced by two antagonistic hormones, namely gonad inhibitng hormone (gih), which is synthesized in the x sinus organ glands (xo-sg) in the eye and gonad stimulating hormone (gsh) produced by the brain and chest ganglion. moreover, there is the involvement of other hormones in regulating reproduction in crustacean animals, such as the progesterone, follicle stimulating hormone (fsh), and *corresponding author, email: ridwan@sith.itb.ac.id biotropia vol. 28 no. 2, 2021 166 luteinizing hormone (lh). the availability of cholesterol strongly influences the development of crustacean ovaries. in the endocrine system, cholesterol is a precursor of steroid hormones that function for the reproduction and maturation of the gonads (wouters et al. 2001). cholesterol is a sterol compound which is a precursor of steroid hormones and molting hormones (sheen 2000). this is one of the chemical compounds that cannot be synthesized by crustaceans (kanazawa et al. 1988) but is very much needed by the parent shrimp. therefore, to meet the cholesterol requirements, crustaceans obtain it from outside the body through feed intake. cholesterol sources can be obtained from plants that form secondary metabolites. one plant that contains cholesterol is karamunting (melastoma malabathricum) which is widely known for its medicinal uses particularly its secondary metabolic compounds that consist of saponins, tannins, triterpenoids/steroids, flavonoids. karamunting extracts obtained by using ethanol solvents contained lanosterol, the cholesterol commonly found in plants (ridwan et al. 2015). karamunting plants also contained sitosterol α and β amyrin from the hexane fraction (nuresti et al. 2003). mangrove crabs (scylla serrata) supplemented with serotine cholesterol had experienced an accelerated ripening of the parent ovaries (pattiasina et al. 2010). until recently, the use of synthetic cholesterol is still the foundation for accelerating the maturity of shrimp gonads, while the use of natural cholesterol from plants is still very rarely done. therefore, this research was conducted to prove whether or not the administration of karamunting extract can increase the amount or content/quantity of hormone progesterone and accelerate the maturation of white shrimp gonads. materials and methods materials the materials used in this study include karamunting (melastoma malabathricum) ethanol extract, mature individuals of white shrimp (litopenaeus vannamei), feed, formalin, xylol, 100% ethanol, distilled water, paraffin, picric acid, and eosin. experimental design the shrimp broodstock, with shrimps weighing 32 35 g, was placed in a 30 x 30 x 60 cm aquarium. the shrimps were fed with fresh worms and oysters at a dosage of 15% of body weight per day. feeding was carried out 5 times a day at 04.00 h, 07.00 h, 13.00 h, 18.00 h, and 23.00 h. this study was conducted using a completely randomized design (crd) with 6 treatments and 5 repetitions. using a 1 ml tuberculin syringe, the karamunting extract was injected at the base of the fifth leg of the white shrimp following the method of tarsim et al. (2007) at extract dosages of 10 mg/kg bw (t1); 7.5 mg/kg bw (t2); 5 mg/kg bw (t3); 2.5 mg/kg bw (t4); 1 mg/kg bw (t5); and control or without extract (c). the parameters that were used included the increase in the progesterone content by using the radioimmunoassay (ria) method and in the ovary development using the histology staining method. at the end of the 15-day observation period, the shrimp gonads were isolated to measure the level of gonad maturity. all data obtained were statistically analyzed by using anova. results and discussion karamunting ethanol extract on the white shrimp progesterone hormone the injection of estradiol-17β into the female white shrimp had stimulated the development of the gonads to the level of gonad maturity (lgm) i (tarsim et al. 2007). whereas white shrimp with human chorionic gonadotropin, progesterone, or a combination of both in the form of feed and injection, had stimulated the ovaries to lgm iv in the 11-month-old shrimp weighing 80 120 g and lgm ii in the 5.5month-old shrimp weighing 60 80 g (ismail 1991). the progesterone content at the start of the experiment was at 0.03 ± 0 ng/ml. after the 15day experiment the contents were measured as follows:, control (c ) was at 0.03 ± 0 ng/ml, t1 at 0.082 ± 0.050 ng/ml), t2 at 0.058 ± 0.023 ng/ml, t3 at 0.034 ± 0.005 ng/ml, t4 at 0.03 ± 0 ng/ml and t5 at 0.03 ± 0 ng/ml). an increase in the hormone progesterone was observed in karamunting (melastoma malabathricum) extracts on white shrimp (litopenaeus vannamei) maturity – ridwan and awaludin 167 the shrimp applied with karamunting extract t1 and t2 (fig. 1.). this shows that karamunting extract containing lanosterol had increased the content of the cholesterol hormone, resulting in the acceleration of gonadal maturity of the parent white shrimp. similar results were also observed among the tiger shrimps treated by eye ablation and serotonin hormone injection (wongprasert et al. 2006). histology gonad development was observed not only morphologically but also histologically. histological observation was intended to see the development of oocyte cells in the gonads of the parent white shrimp (l. vannamei) as influenced by the karamunting ethanol extract. the development of oocyte in shrimp were classified as previtellogenic, endogenous vitellogenic oocytes, and exogenous vitellogenic oocytes (wilder et al. 2010). based on observations of oocyte development after exposure to the karamunting ethanol extract, oocyte development results were obtained at the beginning of the observation and the end of the observation (fig. 2). figure 1 the average progesterone content of white shrimp l. vannamei injected with karamunting m. malabathricum ethanol extract notes: a = initial; c = control; t1 = 10 mg/kg bw; t2 = 7.5 mg/kg bw; t3 = 5 mg/kg bw; t4 = 2 mg/kg bw; t5 = 1 mg/kg bw; a, b = means with different letters differ significantly from each other at p<0.05. biotropia vol. 28 no. 2, 2021 168 figure 2 histology of litopenaeus vannamei gonads notes: a = initial; c = control; t1 = 10 mg/kg bw; t2 = 7.5 mg/kg bw; t3 = 5 mg/kg bw; t4 = 2 mg/kg bw; t5 = 1 mg/kg bw; a, b = means of different letters significantly differ from each other at p<0.05 using anova; o: oogonia; pre: previtellogenic oocytes; en: endogenous vitellogenic oocytes; ex: exogenous vitellogenic oocytes; 20x enlargement. ovarian development among shrimps is characterized by the formation of cortical rods in the oocytes after the accumulation of yolk (clark et al. 1980). at the beginning of the treatment, the gonads were generally not developed yet (fig. 2.a), these gonads were still in the accumulation of oogonia. after the 15 days, the control and all the treatments exhibited gonad cell development. in the control (fig. 2.c), the gonads developed into oocytes at the previtellogenic oocytes stage, and part of the ovary was still oogonia. whereas in t4 (2 mg/kg bw) and t5 (1 mg/kg bw) the development of gonads did not differ from that of the control which was still at the previtellogenic oocytes stage. in ti (10 mg/kg bw) (fig. 2.t1), the oocyte development was observed in the endogenous phase of vitellogenic oocytes, and several parts of the gonad have formed exogenous a o pre t4 pre t5 pre t2 en t1 ex en t3 en pre pre o c karamunting (melastoma malabathricum) extracts on white shrimp (litopenaeus vannamei) maturity – ridwan and awaludin 169 vitellogenic oocytes and have a larger oocyte size as compared to control (fig. 3). in t2 (7.5 mg/kg bw) (fig. 2.t2), the oocyte development was in the endogenous phase of vitellogenic oocytes as compared with that of the control which was in the previtellogenic oocytes phase. in t3 (5 mg/kg bw) (fig. 2.t3), the ovary development was in the endogenous phase of vitellogenic oocytes and some still have the previtellogenic oocytes as compared with that of the control which was also in the previtellogenic oocytes phase. the mean oocyte size at the beginning of the experiment, at 15.57 ± 3.15 µm, was smaller than that of the control at 25.29 ± 2.69 µm (fig. 3). this indicates that after the 15-day maintenance, the ovary developed in both the control and the treatments. furthermore, the size of the oocytes in t1 at 65.65 ± 2.64 µm, t2 at 63.98 ± 3.06 µm, t3 at 39.12 ± 6.01 µm, t4 at 28.08 ± 0.84 µm and t5 at 27.65 ± 0.71 µm were also significantly bigger than that of the initial size of the oocyte and control. the control had the smallest oocyte at the end of the observation while t1 had the largest. the application of karamunting ethanol extracts significantly increased the oocyte diameter sizes among the treatments (p>0.05) with the largest diameter at t1 and t2 indicating that karamunting has compounds that can accelerate gonad maturity. in another study, the injection of estradiol 17β has also increased the diameter size of oocytes in l. vannamei (tarsim et al. (2007). karamunting ethanol extract on gonad somatic index (gsi) the application of karamunting ethanol extract also increased the gonad somatic index (gsi). the high increase in individual gsi indicated gonadal development manifested by gonads growing bigger (fig. 4). the use of karamunting ethanol extract has shown that it can meet the average gsi requirements in t1 (1.39 ± 0.04), t2 (1.26 ± 0.03) and t3 (1.01 ± 0.08) higher than the control (0.84 ± 0.02). at t4 (0.91 ± 0.03) and t5 (0.83 ± 0.04), the gsi increases were not significantly different from the controls. figure 3 oocyte average diameter size of the parent litopenaeus vannamei injected with melastoma malabathricum ethanol extract notes: a = initial; c = control; t1 = 10 mg/kg bw; t2 = 7.5 mg/kg bw; t3 = 5 mg/kg bw; t4 = 2 mg/kg bw; t5 = 1 mg/kg bw; a, b, c, d, e = means with different letters significantly differ from each other at p<0.05. biotropia vol. 28 no. 2, 2021 170 figure 4 average somatic gonad index of parent white shrimp litopenaeus vannamei injected with karamunting melastoma malabathricum ethanol extract notes: c = control; t1 = 10 mg/kg bw; t2 = 7.5 mg/kg bw; t3 = 5 mg/kg bw; t4 = 2 mg/kg bw; t5 = 1 mg/kg bw; a, b = means with different letters significantly differ from each other at p< 0.05. based on the gas chromatography mass spectrometry (gcms) test, the karamunting extract contained high lanosterol α and β amirin levels. in another study, karamunting plants also contain sitosterol α and β amyrin (nuresti et al. 2003). moreover, natural feed such as blood worms and squid had also stimulated the ovary maturation in shrimp (wouter et al. 2011). blood worms and squid are animals that contain high cholesterol (saidin 2000). increases in the gsi values of the treatments were significantly different from the control (p˂0.05). in t1 (10 mg/kg bw) and t2 (7.5 mg/kg bw), the increase in gsi was higher than that of the control (value?) at p <0.05. the t3 (5 mg/kg bw) increase in gsi was not significantly different from that of the control. a study showed that gsi increase was closely related to protein and lipid levels in the gonads, which also reflected high feed levels (rodriguezgonzalez et al. 2009a). the application of cholesterol had also optimized the increase in the ovarian weights of mangrove crabs (pattiasina et al. (2010). furthermore, the injection of serotonin on mature female individuals of the freshwater shrimp macrobrachium rosenbergii had stimulated an increase in gsi (meeratana et al. 2006). increasing the dosage of karamunting plant extract in white shrimp had impacted ovarian development. irrevocably, crustaceans need cholesterol which functions as precursors of steroid hormones in the process of gonadogenesis, maturation, and (wouters et al. 2001). gsi of t4 (2 mg/kg bw) and t5 (1 mg/kg bw) did not significantly differ from that of the controls. this is probably due to the small dosage that was not enough to produce the optimum hormonal increase in the parent white shrimp. the parent shrimp has an optimum level of protein requirements, where increasing the level of protein above the optimum level will not affect the nutritional condition (idris et al. 2011). the recommended protein levels could produce the highest spawning rate of 30% (rodriguez-gonzales et al. 2006a). water quality the optimum qualities for the maintenance medium of the white shrimp is at 28 32 oc, salinity 27 40 ppt, ph 6.5 8.3, and do 4 6 mg/l (farhan 2006). in this study, the water quality parameters were measured every day to monitor and maintain stable environmental conditions (table 1). changes in water quality on broodstock maintenance media will create stress on the shrimp which in turn will disrupt the gonadal maturation process. the growing environment was maintained at optimum conditions for the mature female white shrimp. karamunting (melastoma malabathricum) extracts on white shrimp (litopenaeus vannamei) maturity – ridwan and awaludin 171 to maintain stability of the water quality in the growing media, certain maintenance activities were carried out every day before feeding. firstly, squeezing the base to remove feces and the rest of the feed; secondly, the water media was replenished every three days at a turnover rate of 30 percent new water. table 1 qualities of the aquatic environment no parameter avarage range morning afternoon evening 1 salinity 33 34 34 2 temperature 28 29 28 3 ph 7.6 7.8 7.8 4 do 6.56 6.01 6.57 conclusion the injection of karamunting melastoma malabathricum ethanol extract on the mature female individuals of white shrimp litopenaeus vannamei had increased its hormone progesterone and accelerated its gonad maturity indicating that the extract can hasten the reproduction time thereby increasing production volume over a shorter period. references clark w h, lynn jw, yudin ai, persyn ho. 1980. morphology of the cortical reaction in the eggs ofpenaeus aztecus. the biological bulletin, 158(2):175-86. doi:10.2307/1540929 farchan m. 2006. teknik budidaya udang vannamei. serang. in: bappl-stp. food and agriculture organization. 2017. globefish highlights. a quaterly update on world seafood markets: (fao) of the united nations. huberman. 2000. shrimp endocrinology: a review. aquaculture 191:191-208. idris m. 2011. energy pathway on two repeated spawning of australian red claw crayfish (cherax quadricarinatus) [dissertation]. bandung (id): institut teknologi bandung ismail a. 1991. pengaruh rangsangan hormon terhadap perkembangan gonad individu betina dan kualitas telur udang windu (penaeus monodon farb). 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[thesis]. bandung (id): institut teknologi bandung. 792 pahrian (system dinamics) revisi.cdr system dynamics modeling of land use change in west kalimantan, indonesia 1* 2 3 pahrian ganawira siregar , jatna supriatna , raldi hendro koestoer 4 and djoko harmantyo 1 phd student, school of environmental science, multidisciplinary graduate program, universitas indonesia, jakarta 10430, indonesia 2 program study of biology, faculty of mathematics and natural sciences, universitas indonesia, depok 16424, indonesia 3 school of environmental science, multidisciplinary graduate program, universitas indonesia, jakarta 10430, indonesia 4 program study of geography, faculty of mathematics and natural sciences, universitas indonesia, depok 16424, indonesia received 23 december 2016 / accepted 02 january 2018 abstract the main effects of human activities on the environment result in land use and land cover changes. land over exploitation and development activities in west kalimantan of indonesia necessitated the focus of this research which aims to analyzing and predicting land use changes in west kalimantan. the results of such a study assist researchers, planners and policy makers to formulate suitable land use policies in the future in order to balance economic development and natural resource conservation. moreover, it makes indonesia shift from middle incomes to become a developed country in 2030. methodology employs field observation, key informant interviews, focus group discussions and system dynamics modeling. the field observation covered communities in several locations in the study site to identify pattern of land use. the system dynamics was applied to analyze the land use change system and estimate the extents of land cover change in the future. the study showed several outcomes: (i) the main leverage factors in the land use change system in west kalimantan were the desire to reach the expected economic growth and the increased per capita consumption of edible oil globally; (ii) in the business as usual modeling, the increasing global demand for edible oil will lead to significant increment of oil palm plantation area, even the total area of plantation could be wider compare to that of the remaining forest area by 2030; (iii) key interventions that need to be considered in the future is to conduct reforestation (with reforestation rate of at least 0.5% per year) and limited oil palm plantation development to maximum of 50% of developed area. keywords: agroforestry, land use, oil palm development, system dynamics, west kalimantan introduction this paper is about the insufficiency of land use policy in indonesia, particularly in west kalimantan province. the current policy has accelerated deforestation in the province with average forest conversion rate of 2.9%/year during the period of 1989 to 2008 (carlson et al. 2012). deforestation is one of the key human transformations globally (williams 2000) and land use/cover change in tropical regions is widely accepted as an important component of global change (houghton 2005; laurance 1999). land use change has considerable impacts on worldwide biodiversity, global and local climate, biogeochemical cycles, soil degradation, hydrology, food security, soil quality and human well-being (foody 2002; ipcc 2007, 2014; lambin et al. 2003; manandhar et al. 2009; sala et al. 2000; trimble & crosson 2000). nonetheless, land cover/land use changes do not only have negative effects, as some changes are related to positive increase of food and fiber yields for peoples' health and wealth (lambin & geist 2006) particularly in developing countries deforestation also contributes to job creations * corresponding author: pahrian@gmail.com biotropia 5 2 8 103 111 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.2.792 103 (rist et al. 2010) and economic growth (naidoo 2004). indonesia includes west kalimantan province, which needs to maintain economic growth to shift from middle income to becoming a developed country by 2030. meanwhile, it also needs to maintain and conserve the remaining forests and biodiversity. indonesia relies on forest resources for its economic development for more than four decades. the forestry sector became the second highest contributor of foreign exchange to indonesian economy after oil and gas sector in the 1980s. the estimated forest cover of 170 million hectares around 1900 decreased to less than 100 million hectares by the end of the 20th century due to over-exploitation in the sector. along with forests loss, indonesia also lost its biodiversity, wood supply, income, and various ecosystem services (barber et al. 2002). underlying causes of deforestation in indonesia are complex, and cover various aspects of market failure, inappropriate policy implementation in relation to forest management, lack of governance capacity at central and district levels, and other, broader socioeconomic and political issues (resosudarmo et al. 2012). forest cover decline continues in west kalimantan, as shown in fig. 1. like other parts of kalimantan, large-scale deforestation and degradation in west kalimantan was initially caused by the extraction of the commercially important dipterocarp tree species for durable hardwood, but in recent years the conversion of degraded forest to monoculture plantations, such as: oil palm plantation, fiber plantation (pulp and paper) and rubber plantation, has played a greater role (gaveau et al. 2014; abood et al. 2015). in west kalimantan, oil palm plantation began in 1980s and reached 1,060,251 hectares in 2012. the government of west kalimantan released the target for oil palm plantation in this region of 1.5 million hectares in 2025 (dinas perkebunan kalimantan barat 2014). global demand of palm oil predicted will be increased in future to meet growing global vegetable oil demand, both for food and for biodiesel (mccarthy & lei 2010; miettinen et al. 2012). this situation will lead to more deforestation in west kalimantan, because oil palm plantation development has traditionally increased by the conversion of primary or secondary forest (sheil et al. 2009). recent literature largely discussed the drivers or impact of deforestation, but there is a minimum attention on how much forest land needs to be reserved for sustainable growth and what maximum area of oil palm plantation could be allowed to develop within the landscape.this research tried to address these issues and therefore aimed to (1) identify the leverage factors of land use systems, (2) predict the land use change in future based on business as usual and other possible scenarios, and (3) identify better land use policies interventions in the future in order to balance economic development and sources: ministry of forestry (1985-2012) figure 1 forest cover condition in west kalimantan (1985-2012) 104 biotropia vol. 25 no. 2, 2018 may 2016. the information gathered was used as input for designing causal loop diagram in system dynamics modeling. system dynamic modeling land use and forest cover in the study area occurred through dynamic complex causes and factors. observing these conditions, the system dynamics mode l ing was s e l ec t ed to understanding land use change system, especially to identify leverage factors and predict changes in the future. system dynamics is a methodology used to understand how systems change over time (martin 1997) and a method for studying complex systems, based on the theory of non-linearity, dynamics and feedback control (sterman 2000). modeling of system dynamics is a cycle process, starting from the identification of problems of the system, the translation of the problems to the structure (from story to structure), modeling through stock flow diagram, simulation and validation, as well as sensitivity and policy analyses. validation was conducted with the deforestation data in west kalimantan which occurred between 1985-2012. system dynamics modeling was conducted for provincial level due to limitation of time series data. the conceptual framework of the model was: “increasing human population and global market demand for palm oil will encourage deforestation. otherwise, afforestation can be conducted through reforestation and restoration initiatives. the declining of forest condition and function would result in externalities affecting the economy, which in turn will affect the poor. poverty itself is identified as one of driving factor natural resource conservation. this research was conducted in west kalimantan, indonesia for several reasons. first, this province is the fifth largest area and one of the fastest growing oil palm plantations in indonesia (dinas perkebunan kalimantan barat 2014). secondly, this area is an important catchment area of kapuas river system with approximately 1.5 million people depending on this river system (hatta et al. 1997). thirdly, most forested land in this area is also a part of borneo heart which is identified as important forest area for the island (wwf & pwc 2011). materials and methods data for system dynamic modeling the reference data in the modeling was land use data of west kalimantan based on satellite images released by the department/ministry of forestry (environment and forests), especially for forest cover, oil palm plantation, and other non-forest category. the period of observation in this study was from 1992 to 2012 and simulation period was conducted from 2013 to 2030. the reason for this simulation until 2030 was to synchronize with government planning cycle and end of the sustainable development goals (sdgs) period. field observation, key informant (village and local leaders) interviews and focus group discussions with several local stakeholder groups (local community organizers, researchers and local government officers) in west kalimantan were conducted to obtain information on the trend of land use pattern in december 2015 to figure 2 conceptual framework of system dynamics model system dynamics modeling of land use change – siregar et al. 105 of forest conversion. forest conversion was conducted for pursuing economic growth. on the contrary, the high growth in regional economy will also hasten deforestation.” the scheme of conceptual framework of the model can be seen in fig. 2. system dynamics modeling was conducted with powersim studio 10 (license number psstudio-103365-1c4b1). . results and discussion land use change pattern forest conversions within the study area were found to be mostly initiated by turning them into rain-fed paddy field (ladang), although there were some areas of forest converted directly into rubber or oil palm plantation. mostly direct conversion of forests into oil palm plantation occurred within the palm oil concession where the potential timber resources have been exploited. the transformation of rain-fed paddy field into other forms of land use was depended on the financial capital capacity of the land owner and legal status of the land itself. the legal status was referred to government for approval as a forest zone and non-forest/ development zone. the pattern, change direction and intensity of land use change within the study area can be seen in fig. 3. the rain-fed paddy field was located in non-forest zone and not within active plantation concessions, if the landowner has adequate financial capital, the land was more likely to be converted into palm oil or rubber plantations. however, if not, the land will be used as rain-fed paddy field and gradually planted with perennial trees, such as fruit and timber trees. hence, after some time it will transform into agroforestry land and will not be used for unirrigated agricultural field. there were also many rain-fed paddy fields which later turned into scrubland, as neglected by their owners or damaged due to fires. the study has revealed that landowners transformed their rain-fed paddy fields, agroforestry land, scrubland or rubber plantation into oil palm plantation when they successfully accumulated enough financial capital or obtain loan from financial institutions, such as: credit union. oil palm plantations became the final land use within the research site. the study found it was rarely the case that, oil palm plantation turned into other land uses, even unproductive oil palm plantations have been replanted with new plant species. in the forest zone, local communities tend to use their land for rubber plantation or agroforestry, after being used as ladang (rain-fed paddy field). they knew that oil palm plantation is not allowed in the forest zone. in some places, it was found that some communities maintained figure 3 pattern, change direction and intensity of land use change at community level in west kalimantan biotropia vol. 25 no. 2, 2018 106 their ladang due to cultural reason, in order to be eligible to participate in gawai, a yearly cultural event to celebrate paddy harvesting. the products from ladang and agroforestry were mostly consumed and traded locally. timber from the forest were processed into sawn timber or plywood in the local industries, then traded locally or exported. meanwhile, fresh fruit bunches (ffb) of palm oil and rubber sheets/ lumps were processed into intermediate materials for industries located near the production area. the intermediate materials, such as: crude palm oil (cpo), palm kernel oil (pko) and ribbed smoke sheet (rss) are exported to fulfill global demand. system dynamics modeling results land use pattern information was used in developing causal loop diagram (cld) of the system dynamics modeling. the cld of the model is shown in fig. 4, with 2 balance loops and 1 reinforce loop. the first balance loop (b1) consists of the forest-environment, protectioneconomic, growth-poverty-need of landdeforestation-non forest-reforestation-forest. the reinforce loop (r2) consists of forestdeforestation-non forest-reforestation. meanwhile, the second balance loop (r3) consists of forest-deforestation-economic growthpoverty-needs of land-deforestation-non forest-reforestation-forest. the table and graph results of system dynamics modeling (fig. 5a) were evaluated in two steps, namely visual and statistical validation. first in visual evaluation, the results showed similar behavior with the reference data. then trough statistical validation, the model was valid because the mean absolute percentage error (mape) of the third reference data was less than 30% (forest: %; forest: %; and oil palm 4 non 3 plantation: %). the leverage points 18 of the system are variables that had great effects on a system or environment with little effort to change. to identify s , leverage point of the system some variables in the model were changed to observe the effects on the results. after several trial and errors exercises, it was concluded that, the leverage points were two variables, namely: national economic growth and global edible per capita. the simulation oil consumption results on identifying leverage points are shown in fig. 5b. in the next step of the system dynamics modeling, simulation with business as usual scenario was conducted to determine future condition by 2030. the simulation results showed that, forest cover will continue to decline to about 5,118,367 hectares or 35% of the area, otherwise non forested area will be expanded to 9,515,087 hectares or 65% of the total area. as predicted by some researchers, edible oil consumption will increase in the future resulting from the expansion of market distribution/consumer expansion, consumption behavior changes and biodiesel expansion (obidzinski et al. 2012; fitzherbert et al. 2008; united states department figure 4 causal loop diagram of system dynamics modeling 107 system dynamics modeling of land use change – siregar et al. of agriculture 2013). to anticipate this situation, simulation was conducted with different consumption rate, namely: the current level of consumption (0,025 tons/person/year), consumption increased by 40% (0,035tons/ person/year), consumption increased by 60% (0.04 tons/person/year), consumption increased by 100% (0.05 tons/person/year), and consumption increased by 150% (0,075 tons /person/year). the simulation results on business as usual scenario is also shown in fig. 5c. based on business as usual scenario, it appeared increasing of edible oil demand in the global market will stimulate palm oil development in the observed location. moreover, the palm oil expansion in the future can be greater than the remaining forest, especially when consumption increased by 100% or 150% (twice or triple of current consumption levels). this situation would negatively affect the existing environmental capacity. simulation was again conducted, with national economic growth of 7% per year instead of 3.12% per year which is the average rate for the last twenty years. the 7% per year is a rate that indonesia needed to move from middle incomestatus. the results showed forest cover in 2030 will be less than 139,094 hectares. it was concluded that, the condition of business as usual is not satisfactory and potentially threatens the sustainability conditions. it is therefore necessary to develop alternative and suitable interventions to mitigate deforestation. two interventions which are necessary to achieve better condition in future were identified, namely: (1) increase reforestation activities with reforestation rate of 0.5% per year; and (2) limiting palm oil development with a maximum of 50% of non-forest zone (assuming no change of legal status). the first intervention was inspired by the phenomenon of community forests in java for the last 20 years that successfully forested 800,000 hectares land between 1993-2003 (bpkh region xi & fgmfp ii 2009). the target of 0.5% per year can be achieved through supportive government policies and capacity building activities to landowners. the simulation result of intervention is shown in (a) validation between deforestation data and simulation result (b) simulation to define leverage in the system c simulation of business as usual condition figure 5 simulation of graph results biotropia vol. 25 no. 2, 2018 108 ( ) (d) simulation of suggested intervention fig. 5d, it appears there will be additional forest cover of 515,334 hectares by 2030 compared to business as usual condition. nonetheless, this area is still less than palm oil plantation area when consumption increased by 100% or 150%. to anticipate negative effect due to dominance of oil palm plantation, the second intervention must be implemented. the limitation of palm oil development in non-forest zone can determine the bearing capacity that will be used as a key device for measuring productive limit of a particular landscape. this condition is in line with lane (2010), which emphasizes the need to define the limit of something massive to ensure sustainability in land use planning. the dominance of land use that occurred should be avoided, in order for landscapes to remain productive. based on the simulation results of the system dynamics modeling, the alternative composition of land use in west kalimantan by 2030 is as follows: 1. business as usual condition with edible oil consumption rate on the current conditions, i.e.: forest cover of 5,118,367 hectares, oil palm plantations of 946,517 hectares and other non forest area of 8,568,570 hectares. 2. business as usual condition with edible oil consumption per capita increased by 150% and the national economic growth rate of 7%, i.e.: forest cover of 4,979,274 hectares, oil palm plantations of 6,744,308 hectares and other non forest area of 2,909,872 hectares. 3. intervention condition (reforestation of 0.5% per year and limits the development of oil palm to a maximum of 50% development zone), i.e.: forest cover of 5,633,701 hectares, oil palm plantation of 3,241,159 hectares and other non forest area of 5,758,594 hectares. future land use development direction limitation of palm oil development will make local governments in the observed site unpopular due to politics and economic reasons. it needs strong policy endorsement from the central government of indonesia. the implementation of limiting palm oil development needs to support the development of palm oil based advance processing industries within/ near production sites, in order to be able to withstand the added value and also to absorb labor force. based on input-output analysis in 2010, the palm oil industry in west kalimantan only has a forward linkage of 1.00 (badan pusat statistik 2012), this value is still lower than the forward linkage palm oil processing industry in malaysia, which is now about 2.39 (jaafar et al. 2015). the opportunity of palm oil downstream industry development in west kalimantan is very prospective since the government promoted biodiesel used as one of alternative energy source in future. the ministry of energy and mineral resources number 12 year 2015 is concerned with the provision, utilization, and procedures of commerce with biofuel as other fuel encouraged the use of biodiesel to about 30% of total national supply by 2025. the economic productivity within the existing oil palm plantation needs to increase and can be improved by way of: (1) intensifying palm oil production, and (2) development of other economic activities within the existing oil palm plantation area. the former can be achieved through increased knowledge and capacity building for the oil palm plantation farmers. the results of the study showed that, the palm oil productivity (ffb: 11.32 tonnes/hectare) in the observed site is still lower compared to average national productivity (16.39 tonnes/hectare) or malaysia (19.14 tonnes/hectare) (dinas perkebunan kalimantan barat 2014). meanwhile, the development of other economic activities within the existing oil palm plantation area can be achieved through the planting of timber trees or livestock integration within oil palm plantation. the government actually has encouraged the cattle and palm oil integration program since 2007, but the program has not received full support from the companies and the local farmers. only 15 out of 1,500 (1%) companies followed and implemented this program (sawit indonesia 2014). the program is expected to improve food security, through increased production of local cattle, and reduce production costs, through the provision of fertilizer, which is derived from wastes produced in the farms. it's also considered that, there is a need to encourage the development of land use which provides good economic returns and environmentally friendly, such as: agroforestry. the agroforestry with combination of some fruit and timber plants can be an alternative land use development within the observed site. planting timber plants in agroforestry programs can be a 109 system dynamics modeling of land use change – siregar et al. step to anticipate the scarcity of timber in the future. small industrial processing of agroforestry produce, such as fruit processing, are also important to develop and support the use of the added value of the products. suitability and capabi l i ty of fruit-based ag roforestr y development to empower local community economy has been identified by several previous studies (withrow-robinson & hibbs 2005; roshetko & purnomosidhi 2013). in addition, agroforestry is part of the forest garden system, which has genuine landscape utilization and has been implemented in kalimantan (salafsky 1994). conclusion the main leverage factors in the land use change system in west kalimantan were the desire to reach the expected economic growth and the increased per capita consumption of edible oil globally. in the business as usual modeling, it was found that, the increasing global demand of edible oil would lead to significant increment of oil palm plantation area; even the total area of plantation could be wider compare to that of the remaining forest area by 2030. meanwhile, the key interventions that need to be considered in the future are to conduct reforestation (with reforestation rate of at least 0.5% per year) and limitations to oil palm plantation development to a maximum of 50% of developed areas. we suggested future strategies for land use development in west kalimantan should only be not focused on the extent of oil palm plantation. it should be change to develop palm oil based advance processing industries within/ near production sites, and also introduce other economic activities within the existing oil palm plantation area and expansion of agroforestry. acknowledgements this research was partially supported by forum orangutan indonesia (orangutan indonesia forum). we thank our colleagues from s c h o o l o f e n v i r o n m e n t a l s c i e n c e , multidisciplinary graduate program, universitas indonesia, who provided insight and expertise that greatly assisted the research, although they may not 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10.1007/s10457-004-6720-z wwf, pwc [internet]. 2011. roadmap for a green economy in the heart of borneo: a scoping study. available from: http://awsassets.panda.org/ downloads/pwc_report_green_economy_roadma p_1.pdf. 111 system dynamics modeling of land use change – siregar et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 619 tri ratna (payment for enviro).cdr biotropia 4 2 7 127 139 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 2 619 * corresponding author: trsdewi74@yahoo.com 127 128 biotropia vol. 24 no. 2, 2017 129 payment for environmental services approach to reduce flood in ciliwung watershed – saridewi et al. figure 1 research location 130 biotropia vol. 24 no. 2, 2017 table 1 change of land use pattern in ciliwung watershed land use pattern year 1989 year 2000 year 2010 year 2014 ha % ha % ha % ha % water body 366.16 0.95 308.95 0.80 320.67 0.83 292.21 0.76 built-up areas 25,433.93 65.97 25,673.99 66.59 26,066.72 67.61 27,957.00 72.52 grassland areas 1,929.61 5.01 1,975.02 5.12 2,017.55 5.23 1,815.91 4.71 open areas 945.08 2.45 1,004.81 2.61 926.69 2.40 996.86 2.59 dryland farming areas 4,476.15 11.61 4,290.66 11.13 3,933.61 10.20 2,272.64 5.89 forest areas 4,614.06 11.97 4,521.74 11.73 4,390.38 11.39 4,241.21 11.00 paddy field areas 788.22 2.04 778.05 2.02 897.59 2.33 977.37 2.54 total 38,553.21 100.00 38,553.21 100.00 38,553.21 100.00 38,553.21 100.00 source : landsat satellite image 1989, 2000, 2010 and 2014 131 table 2 land use pattern, precipitation and river water level on each subwatershed year location water body (ha) built-up areas (ha) grassland (ha) open area (ha) dryland farming (ha) forest (ha) grassland (ha) precipitation (mm) water level (cm) 1989 upstream 35.9 6,269.7 124.1 188 3,257 4,614.1 725.8 5,143 110 2000 upstream 78.1 6,334.7 116.9 276.5 3,159 4,521.7 728.2 1,201 110 2010 upstream 96.7 6,665.1 148.9 186.3 3,009 4,390.4 718 840 116 2014 upstream 64.1 7,844.2 113.1 224.7 1,914 4,241.2 813.1 1,024 128 1989 midstream 74.5 13,700.3 1,526.6 496.1 1,017 0 62.4 534 250 2000 midstream 78.3 13,808.6 1,549.1 461.1 929.4 0 49.8 652 250 2010 midstream 61 13,861.9 1,563.5 456 754.3 0 179.6 1,142 400 2014 midstream 68.,8 1,4547 1,409.2 498.7 188.4 0 164.3 1,383 450 1989 downstream 255.8 5,463.9 278.9 261 202.6 0 0 1,588 685 2000 downstream 152.5 5,530.7 309 267.2 202.6 0 0 1,625 853 2010 downstream 162.9 5,539.7 305.2 284.3 170 0 0 2,360 717 2014 downstream 159.3 5,565.8 293.6 273.4 170 0 0 2,907 784 source: landsat satellite image 1989, 2000, 2010 and 2014; the ministry of public work 1989, 2000, 2010 and 2014 payment for environmental services approach to reduce flood in ciliwung watershed – saridewi et al. 132 biotropia vol. 24 no. 2, 2017 figure 2 systems thinking of the optimum spatial planning model tabel 3. land use patterns change and the change direction in upstream areas between 1989 and 2014 2014 water body forest built-up area dryland farming grassland paddy field open area grand total 19 89 water body 7.53 27.98 0.43 35.94 forest 4,241 85.91 149.10 131.35 6.42 4,614.00 built-up area 6,205 6,269.70 dryland farming 1,555 1,661.30 1.45 38.36 3,257.10 grassland 11.02 113.10 124.12 paddy field 89.96 635.86 725.82 open area 8.05 179.90 187.97 grand total 4,241 7,953 1,849 113.10 769.09 224.70 15,214 source: landsat satellite year 1989 and 2014, analyzed run off policies environmental services land cover change wtp wtapopulation value of farm land 133 figure 4 runoff submodel figure 3 submodel for land use change payment for environmental services approach to reduce flood in ciliwung watershed – saridewi et al. 134 biotropia vol. 24 no. 2, 2017 figure 5 value of farmland submodel figure 6 upstream subsidy policy submodel 135 figure 7 population dynamics submodel figure 8 environmental services submodel payment for environmental services approach to reduce flood in ciliwung watershed – saridewi et al. figure 9 description of land use pattern versus water level biotropia vol. 24 no. 2, 2017 136 figure 10 simulation of water level resulted from the four scenarios 137 payment for environmental services approach to reduce flood in ciliwung watershed – saridewi et al. table 4 land use pattern in years 2014 and 2023 land cover year 2014 year 2023 difference (ha) (ha) (%) (ha) (%) water body 299.85 0.78 317.88 0.82 18.03 forest 4,241.21 11.00 4,142.50 10.74 -98.71 built up area 27,957.0 72.52 27,116.55 70.34 -840.45 dryland farming 2,272.64 5.89 3,144.65 8.16 872.01 grassland 1,808.27 4.69 1,914.65 4.97 106.38 paddy field 977.37 2.54 920.60 2.39 -56.77 open area 996.86 2.59 996.38 2.58 -0.48 total 38,553.20 100 38,553.20 100 biotropia vol. 24 no. 2, 2017 138 139 payment for environmental services approach to reduce flood in ciliwung watershed – saridewi et al. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 doi: 10.11598/btb.2015.22.2.487 soil seed bank of an exotic sp. plantation acacia and an adjacent tropical heath forest in brunei darussalam adrian lee rahman suhaili , kushan u. tennakoon 1 2 and rahayu sukmaria sukri1* 1environmental and life sciences programme, faculty of science, universiti brunei darussalam, jalan tungku link, be 1410, brunei darussalam 2environmental and life sciences programme, faculty of science and institute for biodiversity & environmental research (iber), universiti brunei darussalam, jalan tungku link, be 1410, brunei darussalam received 28 april 2015/accepted 18 november 2015 abstract acacias are some of the most successful invasive plants in the tropics, causing significant negative impacts on the biodiversity and ecosystem services of invaded habitats. their successful invasiveness is partly attributed to the ability to accumulate large soil seed banks in the areas that they invade. seedling emergence and soil seed bank composition were compared under an plantation and an adjacent tropical heath (kerangas) forest in acacia mangium the andulau forest reserve, brunei darussalam. soil samples were collected from ten 20 x 20 m plots set up in three contrasting habitats: the plantation, the adjacent heath forest and the transition zone in between. soil samples acacia were subjected to smoke and heat treatments, following seedling emergence which was observed daily over a 12-week period. in a parallel investigation, variations in species richness, seed density and seed viability of the soil seed banks of the ten plots were investigated. seedling emergence was the highest in the plantation and the lowest in the heath forest plots, respectively. however, no significant differences among treatments and no significant treatment-habitat interactions were detected. species richness, seed density and seed viability in the plantation plots were significantly lower than those in the transition zone and intact heath forest plots. seeds were not recorded in the acacia mangium heath forest soil seed banks, but were detected in the plantation and transition zone plots. lower native plant species richness, seed density and viability in the plantation could imply higher regeneration potential for the a. mangium heath forest habitat if severe habitat destruction was to occur in this forest reserve. it is suggested that proper plantation management practices and close monitoring of soil seed banks are the best practices that could be adopted to minimize the gradual spread of invasive acacias into tropical heath forests of borneo. keywords: borneo, invasive alien plants, kerangas, seed extraction, seed viability, smoke treatment introduction invasive plants are a recognized as major threat towards biodiversity globally (davies & sheley 2007; simberloff 2009; corlett 2010). the genus acacia (fabaceae) is one of the most destructive invasive plants in the tropics, with 23 species listed as top invaders worldwide (richardson & rejmanek 2011). globally, the introduction of australian acacias began in the 1700s for aesthetical uses and production of woody pulp, fuel wood and timber (carruthers . 2011). the et al negative impacts of australian acacias are broad, often resulting in transformations to the biodiversity and services of invaded native ecosystems (le maitre . 2011). particularly in et al southeast asia, adverse impacts of acacia invasion, such as displacement of natives through competition, changes in ecosystem processes and allelopathy, have been reported (osunkoya . et al 2005; ismail & matali 2014; padmanaba & corlett 2014). the invasion successes of acacias are enhanced by their ability to accumulate large soil seed banks (richardson & kluge 2008). invasive acacias also alter the biotic resistance of resident * corresponding author : rahayu.sukri@ubd.edu.bn biotropia 2 140 150 vol. 22 no. , 2015: 140 mailto:rahayu.sukri@ubd.edu.bn seed bank communities, promoting their own spread and the secondary spread of other alien species (richardson & kluge 2008; gioria et al. 2012). invasive acacias reduce species richness, diversity and composition of soil seed banks of invaded ecosystems, thus changing the vertical structure of above-ground vegetation and community composition of the invaded ecosystems (gioria & osborne 2010). furthermore, -enriched soil seed banks act acacia as reservoirs that allow for the persistence of invasive behaviour (gioria . 2012). the success et al of acacias in the soil seed banks of invaded areas are often bolstered by anthropogenic disturbances, such as forest fires, which alter environmental conditions of temperature, light intensity and humidity in favour of germination of these exotics (richardson & kluge 2008; le maitre 2011).et al. australian acacias ( willd., acacia mangium acacia cincinnata acacia auriculiformis f. muell. and benth.) were introduced to brunei darussalam in the 1990s to be used in timber plantations and as roadside plantings (osunkoya . 2005). in the et al twenty years following this initial introduction, a. mangium a. auriculiformis and are now regarded as invasive in brunei darussalam (osunkoya . et al 2005). their spread appears to be most intense along roadside embankments and in disturbed heath (kerangas) forests along fire-prone coastal areas (osunkoya . 2005), where trees et al acacia create almost monospecific habitats. in addition to invading these coastal heath forests, acacias also appear to invade further inland in brunei, particularly from roadsides and the original plantations into neighbouring natural habitats. tropical kerangas forests account for less than 1% of all forests in brunei darussalam and are rapidly under threat from development (wong & kamariah 1999; din . 2015; wong . 2015). et al et al plants in heath forests are well adapted to nutrient deficiencies caused by dry, nutrient-poor, acidic soils (proctor 1999; fujii 2014). kerangas soils in brunei darussalam are relatively low in nutrient levels, especially low nitrogen concentrations and low ph (moran . 2000; metali . 2015). due et al et al to its ability to fix atmospheric nitrogen, acacia effectively outcompetes native heath forest species in this nutrient-poor environment (osunkoya . 2005). as such, the invasions of et al acacias are a threat to these rare and vulnerable tropical ecosystems in borneo. a first step towards testing invasion severity is to determine whether the invasive species impact negatively upon native species in its introduced habitat (vilà . 2011). knowledge on soil seed et al bank dynamics can, therefore, help predict possible habitats susceptible to invasion according to specific attributes of invasive species (cordell 2002). the specific objective of this et al. preliminary study was to investigate the negative impacts of invasive acacias by exploring the soil seed bank composition and seedling emergence patterns of an plantation and a acacia neighbouring heath forest in the andulau forest reserve (andulau fr), brunei darussalam. the effects of different treatments with fire-related cues on seedling emergence and germination from the soil seed banks of the plantation and heath forest were also investigated. three hypotheses were proposed: 1. seed density and viability is the highest acacia in the soil seed bank of the plantation acacia and the lowest within the soil seed bank of the neighbouring heath forest; 2. in the seedling emergence study, seedling diversity of native species is the highest within soils from the heath forest and the lowest in the plantation soils;acacia 3. germination of seeds is higher in acacia response to fire-related cues (smoke and heat treatments) when compared to native heath forest species. materials and methods study sites and soil sampling a. mangiumthe study was conducted in an plantation and an adjacent heath forest in the belait district of brunei darussalam (4.4167° n, 114.5833° e). the plantation, covering a total area of 1 km , is located in an intact heath 2 (kerangas) forest within compartment 8 of the andulau forest reserve (fr). it was established by the brunei forestry department in the early 1990s as a commercial plantation for timber and harvested in 2010 for manufactured wood products (joffre ali ahmad, brunei forestry department, personal communications). since this harvest, the plantation has not been actively managed and saplings are now seen to a. mangium regenerate naturally. the adjacent heath forest is biotropia vol. 22 no. 2, 2015 141 soil eed ank f n xotic sp. lantation nd n djacent ropica suhailis b o a e p a a a t l . heath forest – a et alcacia also located within compartment 8 of the andulau fr and is separated from the acacia plantation by a 5 m firebreak. this firebreak comprised of layers of sand and gravel with ditches on either side of it, allowing drainage. a single transect line was established from the acacia plantation into the adjacent intact heath forest in a north to south direction. along this line, a total of ten 20 x 20 m plots were set up (fig. 1): (1) four plots were established in the heath forest (hf; plots 1–4); (2) two plots along the transition zone between the plantation and the adjoining heath forest (tz; plots 5–6) and (3) four plots in the abandoned plantation (p; plots acacia 7–10). plots within each habitat were located at 100 m from each other. within each plot, soils at five random points were sampled to a depth of 15 cm and bulked per plot for seedling emergence treatments. at three other random points in each plot, soils were separately sampled to 5 cm depth and bulked for the seed extraction study. seedling emergence for the seedling emergence experiment, soil samples were subjected to heat and smoke treatments. for the heat treatment, soil samples (n = 2 replicates per plot) were placed in an oven at 80 °c for 10 minutes (hanley & fenner 1998; read . 2000). to conduct smoke treatment, et al methods outlined by et al.dixon (1995) and read et al oil samples . (2000) were modified as follows: s (n = 2 replicates per plot) were placed in smoke a tent and exposed to smoke generated from the combustion of fresh leaf litter collected from the hf plots. soil samples were then allowed to saturate with smoke for 90 minutes at 40 °c. heator smoke-treated soil samples from each plot were separated into 500 g sub-samples and evenly spread out over sterilized potting mixture in 30 cm by 25 cm seedling trays (price . 2010). et al the potting mixture was first sterilized by autoclaving at 121 °c and 15 psi for 20 minutes (darbar & lakzian 2007). control trays containing only sterilized potting mixtures, but without any added soil samples were maintained for the duration of the experiment to monitor for contaminations during the observation period. all seedling trays (n = 60) from the three treatments were subsequently placed in a closed plant house at universiti brunei darussalam (ubd) and watered daily. mean of minimum and maximum temperatures recorded within the plant house were 26.5±0.4 °c and 31.6±0.3 °c, respectively, mean relative humidity of the plant house was 83.3±1.8% and mean of photosynthetically active radiation (par) was 449.1 µmol photons/m /s.2 seedling emergence, expressed as the number of germinating seedlings, was recorded for each tray over a period of two weeks. all emerging figure 1 schematic diagram illustrating the placement of the plots in relation to the plantation and the acacia mangium andulau forest reserve: four plots were established in the heath forest (hf; plots 1–4); two plots in the transition zone between the plantation and the adjoining heath forest (tz; plots 5–6); and four plots in the plantation acacia (p; plots 7–10). distances between plots in each habitat were 100 m. each plot was 20 by 20 m in size 100 m 10 30 m 8 9 7 450 m 100 m 100 m 30 m 5 3 1 6 4 2 acacic mangium plantation transition zone heath forest plantation edge key: line transect fire break (5 m width) 142 biotropia vol. 22 no. 2, 2015 seedlings were identified using leaf morphological characters (de vogel 1980; ng 1991). unidentified seedlings were transferred to separate pots to allow them to grow further for eventual identification using herbarium facilities. once seedling emergence had slowed or stopped, soils in the seedling trays were disturbed by stirring with a small spade to mix soil and further encourage the growth of remaining seeds (price . 2010). et al monitoring of seedling emergence in the treatment and control trays was completed after 12 weeks, at which point no seedling germination had been observed in any of the seedling trays for 2 consecutive weeks. seed density and viability to determine seed density in the seed bank, seed extraction methods described by ball and miller (1989) and price . (2010) were followed. et al subsamples of 100 g of soil were subjected to floatation, so that seeds could be extracted using a solution of 10 g sodium hexametaphosphate, 5 g sodium bicarbonate and 25 g magnesium sulphate diluted in 200 ml of water. this solution facilitated the density separation of seeds and organic matter from the soil mineral fraction. organic matter and seed fractions that remained after separation were washed with water through 150 µm and 75 µm sieves, respectively to remove remaining soil and debris. organic matter and seed fractions were oven-dried at 60 °c overnight and dried samples were placed in envelopes for storage until they were sorted and counted. seed sorting and counting was performed using a 10x magnification dissecting microscope (meiji techno co ltd, japan). seeds were a. mangium identified by cross checking the morphology with positively identified seeds collected from a fruiting a. mangium tree. seed density was quantified as the total number of seeds extracted from the soil sample. seed viability was determined using the forceps method (ball & miller 1989; price . 2010), et al whereby a pair of forceps was used to apply a gentle pressure onto the seed. if the seed resisted this pressure, it was considered as viable, however, if the seed did not resist this pressure then it was considered as non-viable. seed viability was quantified as the mean number of viable seeds identified from the soil sample. data analysis statistical analysis was done using r version 3.0.2 (r core team 2013). one-way anova was used to determine differences between plots in heath forest (hf), transition zone (tz) and plantation (p) for the following parameters: mean seedling emergence, mean species richness, mean seed density and mean viability of the seeds extracted. assumptions of heterogeneity of variances and normality were not violated. significant differences detected from one-way anova were further analysed using tukey's hsd test. results and discussion variation of seedling emergence in -acacia present and -absent habitatsacacia over the 12-week period, a total of 118 seedlings emerged. seedling emergence was observed in all plots, except for hf plots 1 and 4. overall, seedlings emerged at a much quicker rate for heat-treated soils compared to untreated and smoke-treated soils. the p plots had the highest mean seedling emergence (0.613), while the hf plots had the lowest mean seedling emergence (0.030) over the 12 week period (fig. 2). the oneway anova indicated that seedling emergence showed significant differences between habitats (f = 14.93, < 0.001), and was significantly the p highest in the p plots compared to the hf ( < p 0.001) and tz plots ( < 0.001). seedling p emergence did not differ significantly between hf and tz plots ( > 0.05; fig. 2)p . the significantly higher seedling emergence in the p plots compared to the tz and hf plots was possibly due to the higher proportion of a. mangium seeds, as well as seeds of pioneer species, such as grasses, that were recorded from the seed banks of the p plots. seeds of pioneers are better adapted for disturbance and harsh environmental conditions such as high light availability and relatively high temperatures (swaine & whitmore 1988; khurana & singh 2006), therefore, germinate faster in the open microhabitat conditions of the abandoned plantation. in acacia contrast, seedlings of non-pioneer trees which are more abundant in the heath forest sites, for 143 example , , agrostistachys longifolia madhuca curtisii syzygium caudatilimbum gymnostoma nobile and (maidin ., unpublished data), germinate slower et al as they are generally less tolerant to high irradiance and temperature (swaine & whitmore 1988; khurana & singh 2006). a total of 11 different seedlings species were recorded during the seedling emergence study, but the only species that could be positively identified was . the remaining 10 species were a. mangium monocotyledons ( ) and dicotyledons n = 4 species (n = 6 species). all of the monocotyledons recorded were identified as grasses. a. mangium seedlings were recorded emerging from soil samples collected in tz and p plots, but were not recorded from samples from hf plots. the p plots had the highest mean number of a. mangium seedling emergence (0.110) as compared to the tz plots (0.036). the mean number of a. mangium seedlings emerged were significantly higher in the p plots than the hf plots (f = 8.293, < 0.001), p but were not different between hf and tz plots or between tz and p plots (f = 8.293, > 0.05).p the significantly lower diversity of native seedlings recorded emerging from the soil seed banks of the p plots is consistent with findings on tree species diversity conducted by maidin . et al (unpublished data) using the same plots in our study, where the lowest tree diversity was recorded in the plantation, with most of the a. mangium trees comprising tropical pioneer species such as fagraea splendens macaranga conifera macaranga , and gigantea a. mangium, in addition to . seed bank composition is highly influenced by the diversity and composition of above-ground vegetation (dalling & denslow 1998), therefore, the lower tree diversity in the p plots appeared to have resulted in similarly lower diversity in their seed banks. additionally, the absence of late successional tropical species in the acacia plantations plots may also likely be due to the absence of specific environmental triggers, such as suitable temperature, light intensity and moisture levels, that are required for seed germination of such species in the p plots (baskin & baskin 2014). we had hypothesized that seeds would acacia be recorded in the hf plots, albeit in lower densities. this is because of the close proximity of the plantation to the hf plots acacia (separated from each other by less than 1 km), which had been expected to generate high propagule pressure (lockwood . 2005; et al colautti . 2006) that would facilitate et al acacia invasion into the intact heath forest. in contrast, results of this study revealed the absence of acacias in the soil seed banks of the hf plots. it was suggested that despite the potentially high figure 2 differences in the mean number (± se) of emerging seedlings among three different habitats: heath forest (hf), transition zone (tz) and plantation (p). different letters above the bars indicated a significant difference (at α = 0.05) in the mean number of seedlings among the three habitats compared 144 soil eed ank f n xotic sp. lantation nd n djacent ropica suhailis b o a e p a a a t l . heath forest – a et alcacia biotropia vol. 22 no. 2, 2015 propagule pressure, the spread of into a. mangium the hf plots may be affected by the lack of suitable dispersers. is known to be a. mangium bird-dispersed (gibson . 2011), which should et al allow for seed dispersal over long distances. it is possible that seeds from the plantation a. mangium were not adequately dispersed into the intact heath forests, perhaps because the birds that feed on seeds do not venture into intact forests. acacia however, it was difficult to ascertain this as further studies are needed to assess bird dispersal of seeds from the plantation into the a. mangium nearby heath forest. there was a lack of significant treatment effect on seedling emergence, although it had been expected that germination of seeds a. mangium would be especially stimulated by heat and/or smoke treatments (portlock . 1990; willis & et al read 2002; kulkarni . 2007). it was argued that et al our results likely indicated that the heat and smoke treatments employed in this study were insufficient to break dormancy and stimulate seed germination, and that further investigations using different heat and/or smoke treatments may result in a significant treatment effect. in this study, mean temperatures during the smoke treatment remained at ±40 °c and soil samples were exposed to smoke treatment for only 90 minutes. no reports are currently available on the optimum environmental manipulations required to enhance seed germination in the a. mangium tropics. however, auld (1986) reported that exposure to high temperatures (between 6080 °c) over any duration, or to extremely high temperatures (between 80-100 °c) for durations up to 1 hour, was needed to break seed dormancy in . these temperatures vary acacia suaveolens greatly between different species acacia depending on their respective degrees of heat tolerance (auld 1986; wahid . 2007). smoke et al and heat are complementary in stimulating seedling emergence from soil seed banks (read et al et al. 2000; zuloaga-aguilar . 2011), and studies have found that seeds typically require acacia smoke and/or heat as cues for germination (kulkarni . 2007; zuloaga-aguilar . 2011; et al et al rawson . 2013).et al a complicating factor in this study was inhibition of seedling emergence by fungal growth, which was observed in some of our heath forest soil samples. fungi are ubiquitous in soil and may increase seed mortality thus inhibiting seed germination (crist & friese 1993; wagner & mitschunas 2008). the presence of fungi in heat and smoke treated soils from the heath forest may indicate that these soils were subjected to very little temperature flux that could have otherwise eliminated soil fungal spores (neary . 1999; et al barcenas-moreno . 2009).et al soil seed bank composition a total of 31 different seed types were collected from the ten plots sampled, of which only seeds were successfully a. mangium identified. seeds were present in soil a. mangium samples from tz and p plots, but absent in the hf soil samples. hf plots registered the highest mean seed species richness, while p plots had the lowest (11.20 vs. 3.50, f = 41.81, < 0.001; fig. p 3). there was no significant difference (f = 41.81, p > 0.05) between tz and p plots in terms of species richness. comparable observations have been reported by wang (2009) where species richness in soil et al. seed banks of established exotic and a. mangium eucalyptus exserta plantations in south china were lower than those of the adjacent natural forest habitats. studies on -invaded areas of acacia saligna the south african fynbos (natural shrubland or heathland vegetation) have also revealed reduced native seed bank richness when compared to adjacent natural forest habitats (holmes & cowling 1997; gioria . 2014). low species et al richness in the soil seed bank of a plantation directly reflects the relatively low species richness in the standing vegetation of a plantation itself (wang . 2009). plantations typically comprise et al of monocultures with one dominant species being planted over a large area (evans & turnbull 2004), and this same pattern was evident in the andulau plantation in this study. a. mangium maidin (unpublished data) has clearly shown et al. that native heath tree species richness in the standing vegetation of the same a. mangium plantation plots were significantly lower than plots located in the heath forest. our observations, therefore, concurred with the hypothesis proposed by holmes and cowling (1997), where species richness of both the standing vegetation and seed banks tend to decrease with increasing duration of invasion by exotic species such as acacia. 145 figure 3 differences in mean species richness (± se) of seeds extracted from soil samples collected in plots from the three habitats: heath forest (hf), transition zone (tz) and plantation (p) different letters above the bars indicate a . significant difference (at α = 0.05) in the mean species richness between the two compared habitats figure 4 differences in seed density the mean number of seeds extracted from soil samples (± se) collected in , defined as plots from the three habitats: heath forest (hf), transition zone (tz) and plantation (p) different letters above . the bars indicate a significant difference (at α = 0.05) in the mean number of seeds extracted between the two compared habitats seed density was significantly the highest in soil samples from tz plots and the lowest in soil samples from p plots (83.50 vs. 6.83; f = 15.54, < 0.001; fig. 4). there was no significant p difference (f = 15.54, > 0.05) in seed density of p hf and tz plots. mean number of viable seeds was highest for hf plots, and lowest for p (46.0 vs. 2.5; f = 22.79, < 0.001; fig. 5). there was no p significant difference (f = 22.79, > 0.05) p between the hf and tz plots seed viability. exotic species are known to significantly reduce seed density in the soil seed banks of areas in which they establish and invade, and over time, further decrease in seed densities are typical (bossuyt & hermy 2003; gioria . 2014). thus, the et al conversion of parts of the andulau tropical heath forest to an exotic plantation for a. mangium commercial purposes have resulted in a subsequent decrease in native seed density in the soil seed bank of the plantation and its immediate surroundings. 146 soil eed ank f n xotic sp. lantation nd n djacent ropica suhailis b o a e p a a a t l . heath forest – a et alcacia the highest seed viability was detected within soils sampled from the heath forest and the transition zone, and the lowest was in the plantation plots. this may partly be due to the lower seed density from plantation plots, compared to the heath forest and plots in the transition zone. however, differences in seed viability in the soil seed banks among the different habitats may also indicate varying abilities of these habitats to recover after severe disturbance events (ashton et al et al. 1998; ghebrehiwot . 2012). the proportion of viable seeds present in the soil seed bank at the time of disturbance is important in determining initial floristic composition following a disturbance (hopkins & graham 1984). the appearance of early successional species after a disturbance event is related to the presence of viable seeds often long buried at the site (livingston & allesio 1968; luzuriaga . 2005). it is, therefore, suggested that et al if the study area was to experience severe disturbances, the heath forest would potentially be better equipped to recover through natural regeneration, than the plantation.a. mangium spread of acacia mangium a. mangium seeds were not observed in any of the plots within the heath forest habitat about 120 m away from the edge of the plantation. however, a. mangium seeds were found in one of the plots within the transition zone between these two contrasting habitats. our own observation within the study site in andulau fr was of extensive spread of into the open, disturbed a. mangium areas immediately surrounding the acacia plantation itself, thus indicating has a. mangium indeed escaped from the original plantation site. however, several factors may have allowed the adjacent intact heath forest to be protected from the spread. invasive alien plant species a. mangium may not be able to successfully establish in intact tropical forests due to light inhibition on the ground from the closed canopy and a thick litter layer with a low proportion of bare soil (pauchard & alaback 2004). canopy coverage in particular may be crucial in slowing down the spread of invasive seeds into the heath forest as a. mangium acacias are shade-intolerant plants (martin . et al 2008). low light availability, therefore, may limit the establishment of these shade-intolerant alien invasive species in a tropical rainforest, possibly conferring some resistance to intact forests from acacia et al invasions (fine 2002; osunkoya . 2005). this current study was preliminary investigation and exploratory that it only investigated seedling emergence potentials and seed bank compositions of an intact acacia plantation, an adjacent heath forest and the transition zone between these two contrasting habitats. however, the results of this study were an important first step towards understanding the potential of invasive acacias and their spread into the soil seed bank of an adjoining rare and vulnerable heath forest habitat in brunei. it is suggested that an important next step would be to conduct a comprehensive survey on soil seed bank composition, seed viability, seedling figure 5 differences in seed viability the mean number of viable seeds extracted (± se) from soil samples , defined as collected in plots from the three habitats: heath forest (hf), transition zone (tz) and plantation (p) different . letters above the bars indicate a significant difference (at α = 0.05) in the mean number of viable seeds between the two compared habitats biotropia vol. 22 no. 2, 2015 147 emergence patterns of all other natural habitats vulnerable to invasion in brunei acacia darussalam. a comparison of these factors and the composition of standing vegetation would provide a clear insight on the invasiveness of exotic acacias in brunei darussalam. conclusions comparisons of the soil seed banks between an plantation and the adjacent heath a. mangium forest have shown higher seed density, viability and species richness in the heath forest than in the a. mangium plantation. heath forests may have a higher chance of natural regeneration following future disturbance events due to their richer soil seed banks. clearing of intact forest for acacia plantations has a detrimental effect on seed bank diversity and composition, which can have negative knock-on effects on native plant regeneration. though is a known a. mangium invasive species, due to its shade intolerant nature, it appears to have some difficulty of spreading into intact tropical forests. it is, therefore, recommended close monitoring of escaping acacia seedlings from the intact parent plantations to mitigate further spread of acacias into natural habitats. acknowledgements the authors thank the forestry department, ministry of primary resources and tourism, brunei darussalam for granting permission and entry permit to work in the andulau 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levels i (24 m), i1 (20 m), and i11 (16 m). the heights of the 108 trees were used to build the si functions, and the three truncated trees were used to examine the possibilities of the si functions. research results showed that the appropriate baseline age of acacia hybrid plantations at dong nai province is 8 years. moreover, in order to improve the effectiveness of acacia hybrid plantation businesses, the owners should focus on growing plantations at site index levels of i or ii. keywords: acacia hybrid plantations, curve of site index, dominant trees, site index introduction climate change has negatively affected human and environmental health, particularly forest ecosystem health (ipcc 2000). tropical rainforest ecosystems are one of the most important carbon sinks on the earth, playing a very critical role in balancing the global carbon cycle and co2 concentration (chaiyo et al. 2011). as such, on forest biomass and carbon sequestration researches are very relevant in forest management and planning, as well as biomass energy use (brown 2000 & 2002; zianis et al 2005). moreover, biomass changes are associated with increases in forest ecosystem growth and rates of carbon absorption and emission (fa0 2009). a forest population ratio of biomass to carbon intensity depends on an age and site index (nguyen & tran 2016). an *corresponding author, email: ntchung@hcmuaf.edu.vn accurate estimation of forest biomass is an important factor in assessing the global carbon cycle (chavt et al. 2005). several studes are interested in determining the mechanisms for worldwide carbon sequestration in different environments (bouman e t al. 1999). it is therefore, necessary to develop appropriate methods for surveying and evaluating forest biomass and carbon stocks (chambers et al. 2001; brown 2002, chavi et al: 2005). several studes also developed mathematical models for the estimation of forest productivity (dong 1974; nguyen & dao 1999; vu 2005 & 2012) and biomass functions (vien 2008: bao 2010; vu & vo 201 1; nguyen 2012; dang 2014; nguyen & tran 2016) for timber trees and different types of natural forests. although a few studies have focused on the site index curves of acacia mangizlm wdld (knsnawati etal. 2010; lumbres et al. 2018), very limited information are available on biomass estimation of acacia hybrid determining an appropriate age for estimating site index of amcia &bed ... thi ngoan and tan chung plantations at various tree ages and soil site some researchers have studied the planting indices. therefore, it is essential to predict acacia hybrid plantation biomass to determine some functions based on different site indices. the forest soil site index or soil productivity is a criterion for evaluating the site suitability for forest productivity (nguyen & dao 1999). soil site quality (or productivity of a soil site) reflects the productivity capacity of a forest, which might change due to the effects of over exploitation, fire, ferulizer application and/or soil erosion (clutter e t al. 1983). site quality is determined through forest growth and yield, mean annual increment (mai) and periodic annual increment (pai) by site index (si) and growth intercept (gi) methods (clutter e t al. 1983; larsen 1999). si can be estimated dlrectly based on the average height of dominant trees (ho) associated with a baseline age (ao) (monserud 1984). si values also depend on tree species and is determined as functions of ho or gi (monserud 1984; larsen 1999; vu 2005). the selection of a baseline age for a tree species is dependent on the life cycle (choosing a period at which population growth does not depend on species density) or business cycle (monserud 1984; larsen 1999). the site index is determined by three different methods (monserud 1984; larsen 1999). the first method involves the construction of a site index population curve, which has been commonly used since the 1940s. this method is based on the related function of ho = f(a0) to construct a site index curve. the second method is based on selecting pairs of ho and age and defining the function ho = f(a), where a is the age of a forest. therefore, site index curves have different shapes by applying different functions. the third method involves truncation of individual trees, using site index curves based on results of tree truncation. this method was created in the 1980s and has been widely applied ever since. in general, each method gives a unique interpretation result (monserud 1984; larsen 1999; vu 2005 & 2012). the si curve converts ho at a basehe age (ao) to ho at a matured age of trees (a). si curves are usually constructed as tree growth functions related to mature ages; in other words, they are based on data pairs of ho/a. a suitable si curve must be fitted by mathematical, statistical methods and must be chosen based on statistical tests (larsen 1999; vu, 2005 & 2012). and breeding of acacid hybrid species in vietnam (le 2000). the mature age of acacia hybrid plantations in southern vietnam is eight years (nguyen e t al 2006). nguyen et al. (2020) records the total area of acacid hybrid plantations in dong nai province as approximately 23,557 ha. plantations are mainly distributed in the dstricts of vinh cuu, xuan loc, and dinh quan. as of writing this manuscript, very limited information is avdable on the classification of site indices for acacia hybrid plantations in dong nai province. the objectives of h s study, therefore were; (i) to determine a baseline age of acacia hybrid plantations to establish their site indices; and (ii) to develop site index curves for acacid hybrid plantations in dong nai province. dong nai province was selected for the study site because many acacia hybrid plantations have been planted in this area with different climatic conditions, topography and soil types. currently, the total area of acacid hybrid plantations in dong nai province is 23,000 ha (institute for forest ecology and environment 2017). the results of this study will provide the scientific basis for the application of silvicultural methods to effectively manage and use acacia hybrid plantations. materials and methods description of the study site this research was conducted on one to 10 year old acacia hybrid (mention the hybrid type or parental species) plantations from in the districts of vinh cuu, dlnh quan, xuan loc, tan phu, long thanh, and bien hoa in dong nai province. these sites have three main soil types formed on basalt, shale and silt soil foundations in dong nai and all located between 1 o0 30' 03"1 1 ' 34' 57" in the northern latitudes and 106' 45' 30"107' 35' 00" in the eastern longitudes, near the equator at 50-350 m above sea level. the rainy season occurs in may to october, and the dry season occurs in november to april. the average temperature, rainfall, and humidity are 22 "c, 2100 mm, and 80°/o, respectively. biotropla vol. 28 no. 1,2021 experimental design and sample collection the site index (si) for acacia hybrid (acacia aaticahyormis or acacia mangk) plantations was determined based on the dominant tree height (ho) at a baseline age (ao). based on a preliminary survey of the age dstribution of acacia hybrid plantations in the study locations, standard sampling plots were established to measure growth criteria for each age group. based on secondary data, three standard plots were established for each of the acacia hybrid plantation age group, and each of those plots represented a typical kind of soil, as mentioned above. the areas of each standard plot varied due to plantation density at each study location; however, the number of trees per plot was consistently 39. within each plot, three sample trees representing different growth status (vietnam standard 11 567-1:2016, 201 6) were cut for tree truncation; one tree of good growing status, one of medum growing status, and one of poor growing status. the remaining thirty-six trees were used to measure tree growth criteria. thus, a total of 30 standard plots were established, in which 108 trees were measured to estimate the growth criteria and nine trees were used to measure truncation for each acacid hybrid plantation age group. the truncated trees were not included in the estimation of tree growth criteria. an average height (ho) for each age group (from 1 to 10 years) was determined by the tree truncation method. the trees used for truncation were cut at 10 cm above the ground, and the diameter at breast height (dbh or d) and stem height (ho) were determined before carrying out the tree truncation steps. each trunk was cut into segments 1.0 m in length, except for the top trunk section, which was 0.5 m. annual rings were counted at 0.0 m, 1.0 m, 1.3 m, 2.0 m, 3.0 m, 4.0 m and so on. at each truncated dameter of the tree, annual rings of the tree were counted to determine a tree age corresponding to the tree height reached within the truncated section. the rings within each tree were assembled and then labelled accrodingly for each truncated tree. the statistical characteristics described for ho, such as the mean height (ho), standard deviation (sd), coefficient of variation (cv), minimum height (ho, -), maximum height (ho, ,,,) and dfference of ho,,,, ho,-, were calculated by using results of statistical analyses. determination of a site index function the site index for acacia hybrid plantations was constructed based on functions described by schumacher (1 939). the observed empirical data of the 108 sample trees in each age group were used to fit the functions. the consistency of each fitted function (equation (3)) was evaluated by considering statistical factors, such as coefficient of determination (r2), standard deviation (sd), mean absolute error (mae) and mean absolute percent error (mape). ho = a*exp(-b/aac) (1) or ln(h0) = ln(a) b/aac (2) ln(h0) =bo+bl/aac (3) ho = exp(bo + bl/aac) (4) bo = ln(h0) bl/aac (5) where: ln(a) = bo and b = b~ from equations (4) and (5), a function of site index, si = f (a), was defined by equation (6), and parameters of si function, such as boy were defined by equation (7). ln(si) = bo + bl/aoac (6) bo = ln(si) bl/aoac (7) by substituting equation (7) into equation (3), equations (8-10) were obtained. equation (10) is described as the function of site index, with ho at a certain baseline age (ao). ln(ho) = ln(s1) bl/aoac + bl/aac (8) ln(s1) = ln(h0) bl/aoac + bl/aac (9) si = exp(ln(ho) bl(1 /aac -1 /aoac)) (10) determination of a baseline age (&) and parameters of site index curve an appropriate baseline age (ao) was chosen at the time when si was used to convert ho at a0 to ho at a certain age (a) with the smallest regression sum of squares (ssr-). the appropriate baseline age was tested for only acacia hybrid plantations with age groups of 6 10 years (a = 6-10 years). the si value was defined based on the fluctuation of ho at ao (ho,~,, ho,,,~,). the slope (parameter bl) remained the same across the site index functions. to obtain an si curve, a value of bl from equation (4) was first calculated and then substituted into equation (10) along with the value of ho at ao. the si curves were validated by using empirical data measured from the truncated trees. determining an appropriate age for estimating site index of amcia &bed ... thi ngoan and tan chung results and discussion of acacid hybrid plantations for age groups from 1 to 10 years, was defined (equation 11). statistical characteristics of acacia hybrid plantation the average ho values of the dominant trees varied from 2.6 m (year 1) to 22.4 m (year 10) (table 1). the different ranges between ho, ,,, and ho, ,,, were from 2.1 to 3.3 m (year 1) and from 15.7 to 29.0 m (year 10). coefficients of variation (cv) fluctuated from the highest 23.9% (year 2) to the lowest 14.7% (year 10). in general, ho values exhibited large variations with age and site con&tions. therefore, a categorization of acacia hybrid plantations in dong nai province into different site levels is necessary. selection of a site index function ho = exp (3,65344 2,76734/aa0,70746) (1 1) with 2 = 83.4%; mae = 2.2; mape = 16.2%. ho values were fitted by substituting the values of age groups into equation 10 (table 2). the annual periodc growth of height (zho) increased gradually for the first year (2.4 m/ year), reached a peak at 4.7 m/year in year 2, and then gradually decreased unul age 10 (0.9 m/year). the annual average height growth (aho) also increased gradually starting at year 1 (2.4 m/year), reached the highest at year 3 (3.6 m/year), and then gradually decreased unul age 10 (2.2 m/year). height growth rate (pho) decreased rapidly from 100% in year 1 to 14.1% based on the result of the regression and in year 5 and decreased again to 4.1% in year 10. correlation analyses, the function ho = f (a) that thus, at age 2 the acacia hybrid plantations expresses a relationship between height and age transitioned from rapid to slow growth. table 1 statistical characteristics of the tree heights in different age groups (n = 108) a (year) ho, observed (m) cv ("/o) ho, min (m) ho, max (m) ho, max ho, min (m) 1 2.6 15.4 2.1 3.3 1.2 2 6.7 23.9 4.0 9.1 5.1 3 11.0 17.3 7.1 14.2 7.1 4 14.0 17.1 9.4 17.9 8.5 5 15.9 18.2 10.5 20.4 9.9 6 18.0 15.0 12.2 22.7 10.5 7 18.5 20.0 12.3 24.4 12.1 8 20.3 18.7 13.6 26.8 13.2 9 21.9 15.1 14.8 27.6 12.8 10 22.4 14.7 15.7 29.0 13.3 notes: a = age; ho = height of dominant tree; cv = coefficient of variation; ho, fin = minimum height; and ho, ,,, = maximum height. table 2 fitted values of periodic annual and average annual height growths and growth rate notes: zho = periodic annual height growth; a h 0 = average annual height growth; and pho = height growth rate. biotropia vol. 28 no. 1,2021 zho (m) -----ah0 (m) figure 1 periodic annual and average annual height growth of acacia hybrid plantation by age baseline ages (&) of acacia hybrid plantations baseline ages (ao) of the acacia hybrid plantations were chosen at a year when the functions si = f(a) were fitted with the smallest regression sum of scpares (ssr,i,). in this study, the ages of acacia hybrid plantations ranged from 1 to 10 years; therefore, determination of an appropriate baseline age was tested at ages 6, 7, 8, 9 and 10 years. predicted ho values based on equation (10) and differences between observed ho and predicted ho values are presented in table 3. the highest and lowest values of ssr were 2.87 and 0.86 at the ages of 7 and 8, respectively. thus, the appropriate baseline age used for constructing si curves was 8 years. age 8 is consistent with the mature age for acacia hybrid plantations in southern vietnam (nguyen e t al. 2006). table 3 predicted ho at ao and ssr associated with ho for ages 6-10 predicted ho (m) at a. (year) ssr values associated with the predicted ho total sum of ssr 1.14 2.87 0.86 1.84 0.98 determining an appropriate age for estimating site index of amcia &bed ... thi ngoan and tan chung levels of site index at the selected baseline age the dfference between ho, ,, and ho, ,,in (ho, ,,, ho, mi, = 26.8 m 13.6 m) at the selected baseline age of 8 is 13.2 m (rounded to 13.0 m) (table 1). at the age of 8, the average height ho value for acacia hybrid plantations was 20.4 m (rounded to 20.0 m). the measured error of height was usually from k0.5 to kl.o m. if the ho value at the age of 8 (13.0 m) is divided into three levels, a range of each level is equal to 4.3 m (rounded to 4.0 m). the &stance between two levels of adjacent site indices (4.0 m) is four to eight times higher than the measured error of height. therefore, the acacia hybrid plantation was divided into three levels of site in&ces (i, 11, 111) based on the ho values in which the distance between two levels of adjacent site indices was 4.0 m. the si values of the three levels are; 24 m (level i), 20 m (level 11) and 16 m (level 111) at the baseline age of 8. the si values midway between levels i and i1 and midway between levels i1 and i11 were 22 m and 18 m, respectively. similarly, the si value at the lower margin of level 111 was 14 m, while the upper margin of level i was 26 m. table 4 functions of site index (si) at selected levels. selected site index level curves in this study, the slope @i) of si curves was the same for all three si levels. the results of the regression analysis showed that the slope value (bl) was 2.76734 and l/aoac was equal to 1/8"0.70746 = 0.22967. the values of these parameters were substituted into equation 9, and the si curves were fitted (table 4). there were no significant differences in the growth intercepts between functions of si = f (a) among the three site index levels and functions of ho = f (a), with p-values of 0.239, 0.285 and 0.261 for levels i, i1 and 111, respectively. similarly, the slopes of the site index functions were not sigmficantly different from those of the functions of ho (p-value = 0.570 for level i, p-value = 0.61 1 for level i1 and p-value = 0.380 for level 111). these results proved that functions 11 to 17 can be used to construct site index curves for acacia hybrid plantations as represented by the predicted values of site indices for each age group (table 5) and the fitted site index curves of height values by years of acacid hybrid plantations at levels of iupp,,, i, 1-11, 11, 11-111, 111, and 111~0,,, (fig. 2). si levels fitted functions of si iupper si = exp (ln (26) 2.76734*(1/aa0.70746 0.22967)) i si = exp (ln (24) 2.76734*(1 /aa0.70746 0.22967)) i1 i si = exp (ln (22) 2.76734*(1 /aa0.70746 0.22967)) i1 si = exp q n (20) 2.76734*(1 /aa0.70746 0.22967)) i1 i11 si = exp (ln (18) 2.76734*(1 /aa0.70746 0.22967)) i11 si = exp (ln (1 6) 2.76734*(1 /aa0.70746 0.22967)) iiilowcr si = exp (ln (14) 2.76734*(1 /aa0.70746 0.22967)) table 5 predicted values of site index levels for age groups from 1 to 10 years predicted ho (m) at different site index levels a (year) i,,,, i i i1 i1 i1 i11 i11 iiilo,,, biotropia vol. 28 no. 1,2021 si curve 30 5 6 a (year) figure 2 curves of siteindices associated with ho at different age groups conclusion brown s. 2002. measuring carbon in forests: current status and future challenges. journal of environmental pollution 116:363-72. acacid hybrid plantations in dong nai province could be divided into three site index chaiyo u, garivait s, wanthongchai k. 2011. carbon storage in above-ground biomass of tropical levels namely; level 1 (24 m height), i1 (20 m) deciduous forest in ratchaburi province, thailand. and i11 (16 m). for these three site index levels world academy of science, engineering and the appropriate baseline age of the plantations technology 5(10):495-500. was at age 8. the site index curves for each level chambers jq, santos js, ribeko rj, hlguchi n. 2001. 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mencuccini m. 2005. biomass and stem volume equations for tree species in europe. monographs 4. 63 p. biotropia vol. 29 no. 3, 2022: 234 243 doi: 10.11598/btb.2022.29.3.1705 234 physical and chemical qualities of corn with different moisture levels supplemented with mold inhibitor catootjie l. nalle*, max a. j. supit, angga m. akbar, angriana so’o and emiliana langodai animal husbandry department, politeknik pertanian negeri kupang, kupang-85228, indonesia received 2 december 2021/accepted 7 july 2022 abstract corn grain is used as the main energy source in poultry diet formulation. the quality of corn is easy to deteriorate during storage because of insect, fungal, and mycotoxin contamination. efforts should be made to maintain the quality of corn during storage. the present study aimed to evaluate the physical and chemical qualities of different moisture levels of corn supplemented by a mold inhibitor. a total of 750 kg of corn grains was used in the present study. a commercial mold inhibitor was used with a dose of 0.045%. the experimental design used was a 3 x 2 factorial complete randomized design. the first main factor was the different moisture levels (ml) of corn (≤ 10%, 10.0-10.9%; 11.0-11.9%), while the second main factor was mold inhibitor (mi, or +). thus, there were six treatment combinations, and each treatment comprised five replications. the results showed that ml, mi, and ml x mi interaction significantly (p < 0.05 to 0.001) affected the percentage of grain damage and fungal grain but not (p > 0.05) the moisture level of corn during 90 days of storage. except for crude protein content, the ml did not affect (p > 0.05) the proximate composition (pc) and gross energy (ge) content of corn. except for dry matter (dm), the pc and ge content of corn were not affected (p > 0.05) by mi. ml x mi interaction did not affect (p > 0.05) the pc and ge content. the aflatoxin b1 (afb1) content was similar (p > 0.05) among all treatments. except for histidine and lysine contents, the amino acid contents of corn were not affected by ml, mi, or ml x mi combination. in conclusion, the supplementation of mi in corn with different ml improved the physical quality, dm, ash, and ge content of corn grain during the storage; mi maintained the dm content but did not reduce the afb1 content of corn. except for histidine and lysine, the supplementation of mi in corn with different ml did not affect the amino acid content of corn. keywords: corn, moisture levels, mold inhibitor, quality introduction corn as a feed ingredient is the dry seeds of zea mays l that have been removed and cleaned from the cobs (national standardization agency 2013). furthermore, it was explained that based on the color, corn kernels are classified into two types, namely white corn, and yellow corn. both yellow and white corn is the main energy source in poultry diet formulation in indonesia. the proportion of corn used in poultry and monogastric diets ranges from 50 to 60%. the quality of corn as a feed ingredient is determined based on its nutrient content and the presence or absence of unwanted materials (national standardization agency 2013). corn is categorized into two levels of quality. the first and second qualities of corn contain maximum 14 and 16% moisture content, respectively (national standardization agency 2013). corn grain having high moisture content is often associated with aflatoxin content. in indonesia, freshly harvested corn usually has a high moisture content, approximately 31.28%, so that if it is not immediately and properly dried, it will very quickly become contaminated with various fungi including aspergillus flavus and fusarium (hausufa & rusae 2018; mukkun et al. 2018). the growth of aspergillus flavus is influenced by various factors such as initial moisture content, relative humidity, temperature, *corresponding author, email: catootjienalle@gmail.com physical and chemical qualities of corn with different moisture levels supplemented – nalle et al. 235 atmospheric gases, light, oxygen, carbon dioxide, ph, mechanical damage, contamination, and competitive effects of other molds (kumar et al. 2021; muga et al. 2019; mukkun et al. 2018). aspergillus flavus can live well at a temperature range of 20 35 oc, ph 4-6, relative humidity 80 to 90%, aerobic atmosphere conditions, and 18% water content (talanca & mas'ud 2009; muga et al. 2019). daou et al. (2021) reported that the optimal temperatures for aspergillus flavus to grow and to produce toxins were 35 and 33 oc, respectively. the pathogenic fungal species infesting corn during the storage will utilize corn nutrients for their growth and development, leading to a decrease in physical and chemical qualities of corn (elsamra et al. 2012; talanca & mas'ud 2009). the decrease in the physical quality of corn can be identified through odor, color, and texture (talanca & mas'ud 2009). the decrease in chemical quality of corn was identified through the decrease in nutrient content and the presence of aflatoxin. aflatoxins, which are the most potent fungal toxins (mycotoxins), are carcinogenic and teratogenic. the aflatoxins are produced during the infection and growth of aspergillus flavus and aspergillus parasiticus in several food/feed ingredients, such as maize and beans (fountain et al. 2015). aflatoxins also adversely affect the growth of livestock and humans. in terms of livestock, the toxicity of aflatoxin depends on the level of aflatoxin in the feed and the impact is different for each type of livestock. chickens are the most resistant to acute aflatoxicosis compared to other poultry (monson et al. 2015). when poultry is exposed to aflatoxins it does not cause mortality or morbidity, but considerable losses are experienced by the poultry industry because of hepatotoxicity. strategies to suppress the growth and development of pathogenic fungi is a major concern, which should lead to serious efforts in maintaining the physical and chemical quality of corn during storage. the efforts should also suppress the production of fungal toxins produced by corn so that the danger of aflatoxicosis can be reduced or even eliminated. several strategies can be applied, such as immediately drying the harvested corn, the use of antagonistic microbes such as neurospora sp. and rhizopus sp., the use of chemicals (mold inhibitors) such as ammonia and propionic acid, and natural materials such as clove powder (elsamra et al. 2012; talanca & mas'ud 2009; wang et al. 2019; oliveira et al. 2020). the results of research by elsamra et al. (2012) showed that the use of clove powder as a natural fungal inhibitor can suppress the growth of aspergillus flavus and also reduce the crude fat content of corn. the same research also proved that the use of fix-a-tox is also effective in suppressing the growth of aspergillus flavus and reducing the content of some amino acids in corn. the adverse effects of aflatoxin and strategies for preventing and eliminating aflatoxins are still a global issue. indonesia's tropical environmental conditions strongly support the growth of pathogenic fungi, such as aspergillus spp. according to weinberg et al. (2008), the harvested grains are vulnerable to molding leading to rapid decline of quality under humid and warm conditions. therefore, it is very important to find the appropriate strategy to prevent the growth activity of these pathogenic fungi. based on these considerations, a study has been conducted to evaluate the physical and chemical qualities of corn grains having different moisture contents supplemented by commercial mold inhibitor during storage. materials and methods feed ingredients the yellow corn grains having different moisture content (< 10%, 10.0 10.9%, and 11.0 11.0%) and a commercial mold inhibitor was used in this study. the yellow corn grains were obtained from farmers in south central timor regency. the mold inhibitor product was provided by a feed producer. the product contains 57% propionic acid, 30 mg/kg lead, and 0.54 mg/kg arsenic. the dose used in this study was 450 g/ton of feed. experimental design this study was designed using a factorial completely randomized design with a 3 x 2 factorial pattern with 3 levels of corn moisture content (< 10%, 10-10.9%, and 11.0-11.9%) and 2 levels of fungal inhibitors (-, +), resulting to six treatment combinations altogether. each biotropia vol. 29 no. 3, 2022 236 treatment consisted of five replications (25 kg of corn per replication). the treatments were: corn (<10% moisture content) corn (<10% moisture content) + mold inhibitor (0.045%) corn (10.0-10.9% moisture content) corn (10.0-10.9% moisture content) + mold inhibitor (0.045%) corn (11.0-11.9% moisture content) corn (11.0-11.9% moisture content) + mold inhibitor (0.045%) experimental procedure the initial moisture content of corn was measured with a grain moisture meter. then, the corn grains with different moisture contents (< 10%, 10.0-10.9%, 11.0-11.9%) without mold inhibitors were put into polyethylene bags (5 bags per treatment; 25 kg corn per bag). meanwhile, the treatments with mold inhibitor were conducted as follows: corn grains were added with mold inhibitor 0.045%, mixed, and then put into a polyethylene bag (25 kg/bag). all treatment bags were then placed on pallets and stored for three months in a feed storage room which had been cleaned and sanitized. a thermo-hygrometer was placed on the wall to control the temperature and humidity. on day 90, the moisture content of the corn grains was measured using a grain moisture meter. the sampling of corn was carried out using the cone and quartering method (campos-m & camposc 2017) and was followed by sample reduction using a seed sampler to obtain laboratory samples. laboratory samples were packed in sealed plastic bags, labeled, and sent to the laboratory for chemical analysis. chemical analysis the dry matter, crude protein, crude fat, and ash contents were determined using the aoac official method (aoac 2005). gross energy (ge) level was determined using an automatic bomb calorimeter (ika c2000). the analysis of aflatoxin (b1, b2, g1, and g2) content of corn grains was conducted at the food and feed laboratory of seameo biotrop in bogor using thin layer chromatography (tlc) (bainton et al. 1980). the limit of aflatoxin detection with tlc was 3.01 ppb for afb1, 3.50 ppb for afb2, 0.54 for afg1, and 1.0 ppb for afg2. the amino acid content of corn samples was analyzed using high-performance liquid chromatography (hplc, ici instrument/ shimadzu scl-10a/shimadzu cbm 20a) with four main steps, namely the manufacture of protein hydrolyzate, drying, derivatization, and injection into hplc. the analysis procedure was as follows: corn sample was hydrolyzed with 10 ml of 6 n hcl at 100 oc for 24 hours. the results of the hydrolysis were transferred to the evaporator flask and rinsed with 2 ml of 0.01 n hcl. this process is done 2 3 times. then the sample was dried using a rotary evaporator for 15 30 minutes to convert cysteine into cystine. the dried sample was added with 5 ml of 0.01 n hcl, then filtered. the derivatization solution was prepared by adding potassium borate buffer ph 10.4 to the sample in a ratio of 1:1. a total of 50 ml of the sample was put into an empty vial and added with 250 ml of orthoflaaldehyde, left for one min, and then filtered. subsequently, 5 ml of the sample was injected into the hplc and then made a standard chromatogram using ready-to-use amino acids that underwent the same treatment as the sample. measurements 1. insect-damaged seed (%): corn grains from each plastic bag was sampled and reduced several times to get to 1.5 kg of samples by using the cone and quartering method (campos-m & campos-c 2017). then, the insect-damaged seeds were taken from the reduced sample and weighed. the percentage of insect-damaged seeds was then calculated using the following formula (nyarko et al. 2021): % insect-damaged seeds = insect-damaged seeds (g) x 100% total weight of corn sample (g) 2. moldy seeds (%): the sampling procedure to quantify the moldy seeds was similar to the sampling method for insect-damaged seeds. the moldy seeds were characterized by color change (shahbazi & shahbazi 2018). the percentage of moldy seeds was calculated by the formula: % moldy seeds = moldy seeds (g) x 100% total weight of corn sample (g) physical and chemical qualities of corn with different moisture levels supplemented – nalle et al. 237 statistical analysis the data obtained were analyzed by using the two-way analysis of variance (anova) following the general linear model procedure of sas (sas ondemand of the sas system). significance was determined at p < 0.05 and the duncan test was then conducted to determine the significant differences between mean values. results and discussions effect of mold inhibitor on physical quality of corn physical and chemical damages to corn kernels during storage can be caused by various factors, such as the initial moisture content of corn during storage, the temperature, humidity, corn variety, and warehouse pests (mutungi et al. 2019; mukkun et al. 2018; muga et al. 2018; li et al. 2014; suleiman et al. 2013). table 1 shows the effect of treatments on the physical quality and moisture content of corn stored for 90 days. the results showed that a significant (p < 0.05) interaction was found between the moisture level (ml) and mold inhibitor (mi) in the percentage of insect-damaged seeds and moldy seeds. on the other hand, no significant interaction (p > 0.05) between the moisture level (ml) and mold inhibitor (mi) was observed in the moisture content of corn harvested on day 90. table 1 shows that corn with different moisture contents (10.1 10.9% and 11.0 11.9%) supplemented with mold inhibitor had a significant lower percentage of damaged seeds and moldy seeds (p < 0.05) compared to the group of corn with the same moisture contents without mold inhibitor. the reduction of the damaged seeds and moldy seeds ranged from 1.46% to 77.9% and 16.9% to 80.5%, respectively (table 1). the percentage of reduction increased in the group of corn with a higher moisture content supplemented by mold inhibitor. this phenomenon indicates that the mold inhibitor works more effectively to prevent physical damage to corn with high moisture content (figs. 1 & 2). table 1 the effect of treatments on the physical quality and the moisture content of corn during 90 days of storage moisture level (ml) mold inhibitor (mi) insect-damaged seed (%) moldy grain (%) moisture content (%) < 10.0% 2.73c 1.24b 10.38 + 2.69c 1.03b 10.42 10.0-10.9% 8.86b 2.92a 10.42 + 5.05c 1.58b 10.48 11.0-11.9% 19.25a 3.94a 10.38 + 4.25c 0.77b 10.30 sem 0.828 0.457 0.533 main factors moisture level (ml) ≤ 10.0% 2.71c 1.14c 10.40b 10.0-10.9% 6.96b 2.25b 10.45a 11.0-11.9% 11.75a 2.35a 10.34c sem 0.585 0.323 0.037 mold inhibitor (mi) 10.28a 2.70a 10.39 + 3.99b 1.13b 10.40 sem 0.478 0.264 0.307 probability p > f ml *** * ns mi *** *** ns ml × mi *** * ns notes: different superscripts in the same column indicate significant differences (p < 0.05); * = significantly different at p < 0.05; *** = significantly different at p < 0.001; ns = not significantly different (p > 0.05); sem = standard error of mean. biotropia vol. 29 no. 3, 2022 238 figure 1 stored corn grains without mold inhibitor figure 2 stored corn grains with mold inhibitor results of the present study was in agreement with those conducted by elsamra et al. (2012) who reported that the addition of mold inhibitor reduced the percentage of damaged seeds (80%). damaged corn is generally characterized by the appearance of holes and maize weevil (sitophilus zeamais). cannepele et al. (2003) stated that the damage of cereal grains due to sitophilus zeamais has an impact on weight loss, decreased physical and chemical qualities of seeds, and reduced germination. bhusal and khanal (2019) reported that the presence of maize weevil leads to the increase of aspergillus flavus infestation in corn. mukkun et al. (2018) reported from their experiment that aspergillus flavus, a. niger, a. fumigatus, fusarium spp., penicillium spp., rhizophus spp., and mucor spp. were the fungal species identified in corn during the storage. the mold inhibitor used in the present study contains some active compounds, namely propionic acid (≥ 57%), lead (≤ 30 mg/kg), arsenic (≤ 0.54 mg/kg) which can inhibit fungal growth. choojun and yoonprayong (2011) stated that propionic acid is able to inhibit the growth of fungi (having antifungal activity), such as aspergillus spp., rhizopus sp., penicillium sp., and zygosaccharomyces rouxii. telaumbanua (2019) in his literature review stated that propionic acid can inhibit the respiration process of grains and the metabolic activity of grain microorganisms. yun and lee (2016) in their literature review explained that the mechanism of propionic acid (ch3ch2cooh) in killing fungi is through mitochondrial apoptosis (programmed cell death). regarding the lead (pb) compound, amari et al. (2017) reported that lead (pb) binds into the cell wall or cell membrane of fungi causing damages to the plasticity of the cell wall of fungal cell membrane so that the mitotic activity of fungal cells is reduced. meanwhile, the arsenic (as) compound changes the ph of the corn medium to acid so that fungi cannot grow (ceci et al. 2020). results of the present study are in agreement with those carried out by nahm (1991) who reported that mold inhibitor was effective in preventing the growth of fungi and reducing the percentage of moldy seeds. the insignificant differences in moisture content of corn can be attributed to the temperature and humidity factors during the 90-day storage remains stable. these findings agreed with those conducted by telaumbanua et al. (2019). effect of mold inhibitor on proximate composition and gross energy content of corn the effect of treatments on the proximate composition and gross energy content of corn grains stored for 90 days is presented in table 2. the results showed that the interaction between moisture level (ml) and mold inhibitor (mi) did not affect (p > 0.05) the proximate composition and gross energy of corn during the experiment. however, the content of dry matter, ash, and gross energy of corn tended to be lower in a group of corn without a mold inhibitor supplementation. on the other hand, the crude protein and crude lipid contents tended to decrease in a group of corn added with mold inhibitor. physical and chemical qualities of corn with different moisture levels supplemented – nalle et al. 239 table 2 the effect of treatments on the proximate composition and gross energy content of corn during 90 days of storage moisture level (ml) mold inhibitor (mi) dry matter crude protein crude lipid ash gross energy ……………..% (as fed)………………. (kcal/kg dm) <10.0% 89.02 7.01 4.96 0.922 2914 + 89.70 6.94 4.74 0.935 3004 10.0-10.9% 89.10 7.71 4.93 1.082 2971 + 89.77 7.35 4.69 1.250 2994 11.0-11.9% 89.33 7.38 4.74 1.067 3002 + 90.05 7.04 4.91 1.250 3014 sem 0.166 0.195 0.103 0.137 995 main effects moisture level (ml) <10.0% 89.36 6.97b 4.85 0.929 2959 10.0-10.9% 89.43 7.52a 4.81 1.166 2983 11.0-11.9% 89.67 7.21a 4.82 1.159 3008 sem 0.117 0.138 0.072 0.097 703 mold inhibitor (mi) 89.15b 7.36 4.87 1.024 2962 + 89.83a 7.11 4.78 1.145 3004 sem 0.095 0.112 0.059 0.079 574.2 probability p> f ml ns * ns ns ns mi *** ns ns ns ns ml × mi ns ns ns ns ns notes: different superscripts in the same column indicate significant differences (p < 0.05); * = significantly different at p < 0.05; *** = significantly different at p < 0.001; ns = not significantly different (p > 0.05); sem = standard error of mean. except for crude protein content, the first main effect of moisture level (ml) did not affect (p > 0.05) the proximate composition and gross energy content of corn over the 90-day storage period. significant differences (p < 0.05) in crude protein (cp) content were observed between the 10% ml and the other two moisture content (10.0 10.9% and 11.0 11.9%). the low cp content of corn with ml < 10% was an unexpected result. the second main effect of mold inhibitor was that the mold inhibitor affected (p < 0.001) the dry matter (dm) content, but it had no significant effect (p > 0.05) on the content of crude protein, crude fat, ash, and gross energy of corn during the 90-day storage period. corn supplemented with mold inhibitor had a higher dm content (p < 0.05) than those without mold inhibitor supplementation. the present results indicated that mold inhibitor supplementation was effective in inhibiting the growth of fungi and maize weevil (sitophillus zeamais) so that the dry matter was not used by these living organisms to propagate. the insignificant difference in crude protein content was in agreement with the finding of telaumbanua et al. (2019). however, the insignificant effect of crude lipid, ash, and gross energy did not agree with those found by telaumbanua et al. (2019). the difference was probably due to the difference in the method applied, especially the duration of the experiment and the type and dose of mold inhibitor used. effect of mold inhibitor on aflatoxin content of corn according to negash (2018), there are six types of aflatoxin, involving aflatoxin b1, b2, g1, and g2, m1 (a metabolite of b1), and m2. among all types of aflatoxin, aflatoxin b1 (afb1) is the most dangerous type of aflatoxin, which can cause several harmful effects in humans and animals, such as enlarged liver, liver cancer, and hepatitis b virus infection (nalle et al. 2021; benkerroum 2020). in addition, nalle et al. (2021) also reported that even at the low level, the afb1 reduced the fat digestibility and feed efficiency, and changed the liver color. thus, it is important to minimize the adverse effect of afb1 in poultry feed ingredients. biotropia vol. 29 no. 3, 2022 240 table 3 the effect of treatments on the aflatoxin content of corn during 90 days of storage moisture level (ml) mold inhibitor (mi) afb1* afb2** afg1*** afg2**** …..……….……………..ppb……….……………....... ≤10.0% 1.72 nd nd nd + 1.15 nd nd nd 10.0-10.9% 1.72 nd nd nd + 1.72 nd nd nd 11.0-11.9% 1.15 nd nd nd + 1.15 nd nd nd sem 1.462 main effects moisture level (ml) ≤10.0% 1.43 nd nd nd 10.0-10.9% 1.72 nd nd nd 11.0-11.9% 1.15 nd nd nd sem 1.033 mold inhibitor (mi) 1.52 nd nd nd + 1.34 nd nd nd sem 0.844 probability p> f ml ns ns ns ns mi ns ns ns ns ml × mi ns ns ns ns notes: ns = not significantly different (p > 0.05); sem = standard error of mean; * = the limit of detection of afb1 was 3.01 ppb; ** = the limit of detection of afb2 was 3.50 ppb; *** = the limit of detection of afg1 was 0.54 ppb; **** = the limit of detection of afg2 was 1.0 ppb. table 3 depicts the effect of treatments on the content of aflatoxins b1, b2, g1, and g2 in corn stored for 90 days. the results proved that the interaction between the level of moisture (ml) and mold inhibitor (mi) did not significantly (p > 0.05) affect the aflatoxin content (b1, b2, g1, and g2) of corn during the trial period. the inefficacy of mold inhibitor in reducing the aflatoxin content of corn presumably because the aflatoxin level in corn was too low. however, it seems that the addition of mold inhibitor in corn grain with < 10% ml reduced the afb1 level during the storage. the main effect of moisture level (ml) or mold inhibitor (mi) had no significant effect (p > 0.05) on the content of aflatoxins (b1, b2, g1, and g2). however, the group of corn supplemented with mold inhibitor had lower afb1 concentration (1.34 ppb) compared to those that were not added with a mold inhibitor (1.54 ppb). the numerical reduction in aflatoxin level was in agreement with the findings of telaumbanua et al. (2019) who found that the aflatoxin level in the group of corn grains supplemented with mold inhibitor (propionic acid) was lower than that of control treatment. effect of treatments on the amino acid content of corn amino acid is the building block of protein and plays an important role in protein cell synthesis in animal and human beings. table 4 describes the effect of treatments on the indispensable amino acid content of corn stored for 90 days. the results showed that except for histidine and lysine, the interaction of moisture level (ml) and mold inhibitor (mi) did not significantly (p > 0.05) affect the indispensable amino acid content of corn during the 90-day storage period. the comparison was difficult to be made due to the difficulties in finding the references which conducted similar research. physical and chemical qualities of corn with different moisture levels supplemented – nalle et al. 241 table 4 the effect of treatments on the indispensable amino acid content of corn during 90 days of storage moisture level (ml) mold inhibitor (mi) arginine histidine isoleucine leucine lysine methionine phenylalanine threonine valine ………..……………….…………………% as fed…………………………………………………. <10.0% 0.710 0.545 0.480 1.200 0.510 0.300 0.595 0.245 0.265 + 0.710 0.565 0.485 1.210 0.560 0.365 0.580 0.300 0.370 10.0-10.9% 0.690 0.510 0.440 1.190 0.510 0.355 0.575 0.235 0.300 + 0.710 0.530 0.495 1.200 0.505 0.295 0.575 1.065 0.265 11.0-11.9% 0.710 0.620 0.445 1.190 0.535 0.345 0.590 0.205 0.310 + 0.680 0.525 0.455 1.195 0.520 0.345 0.615 0.240 0.255 sem 0.008 0.013 0.023 0.014 0.006 0.002 0.020 0.338 0.043 main effects moisture level (ml) <10.0% 0.710 0.555a 0.482 1.205 0.535a 0.332 0.587 0.273 0.317 10.0-10.9% 0.700 0.520b 0.467 1.195 0.507b 0.325 0.575 0.650 0.282 11.0-11.9% 0.695 0.572a 0.450 1.192 0.527a 0.345 0.602 0.222 0.282 sem 0.006 0.009 0.016 0.010 0.004 0.015 0.014 0.239 0.031 mold inhibitor (mi) 0.703 0.558 0.455 1.193 0.518 0.333 0.587 0.228 0.292 + 0.700 0.540 0.478 1.202 0.528 0.335 0.590 0.535 0.297 sem 0.005 0.007 0.013 0.008 0.004 0.012 0.011 0.195 0.025 probability p> f ml ns * ns ns * ns ns ns ns mi ns ns ns ns ns ns ns ns ns ml × mi ns *** ns ns *** ns ns ns ns notes: different superscripts in the same column indicate significant differences (p < 0.05); * = significantly different at p < 0.05; *** = significantly different at p < 0.001; ns = not significantly different (p > 0.05); sem = standard error of mean. the main effect of moisture level (ml) significantly affected (p < 0.05) the content of histidine and lysine, but it did not affect (p > 0.05) the other indispensable amino acid content of corn during the trial period. the histidine and lysine content of corn with 10.1 10.9% ml was lower (p < 0.05) than the histidine and lysine content of two other treatments. the main effect of mold inhibitor (mi) did not affect (p > 0.05) all indispensable amino acid content of corn during the experimental period. this proves that the use of mold inhibitor with a dose of 0.045% did not have an adverse impact on the indispensable amino acid content of corn. table 5 shows the effect of treatment on the dispensable amino acid content of yellow shelled corn stored for 90 days. the results of the analysis of diversity showed that the interaction of moisture level (ml) and mold inhibitor (mi) did not significantly (p > 0.05) affect the essential amino acid content of dry shelled yellow corn which was stored for 90 days. table 5 the effect of treatments on the dispensable amino acid content of corn during 90 days of storage moisture level (ml) mold inhibitor (mi) alanine aspartic acid cysteine glycine glutamic acid proline serine tyrosine ………………………..………..…........% as fed..…...………………….………………... ≤10.0% 0.825 0.625 0.205 0.805 1.900 0.200 0.585 0.290 + 0.845 0.620 0.205 0.815 1.900 0.205 0.630 0.350 10.0-10.9% 0.835 0.590 0.215 0.835 1.535 0.205 0.595 0.295 + 0.890 1.120 0.200 0.820 1.855 0.215 0.605 0.290 11.0-11.9% 0.855 0.605 0.200 0.805 1.920 0.230 0.620 0.300 + 0.855 0.620 0.195 0.805 1.865 0.230 0.615 0.305 sem 0.023 0.217 0.010 0.012 0.150 0.019 0.010 0.018 main effects moisture level (ml) <10.0% 0.835 0.622 0.205 0.810 1.900 0.225 0.607 0.320 10.0-10.9% 0.862 0.855 0.207 0.827 1.695 0.210 0.600 0.292 11.0-11.9% 0.855 0.612 0.197 0.805 1.892 0.230 0.617 0.302 sem 0.016 0.154 0.007 0.008 0.106 0.013 0.008 0.013 mold inhibitor (mi) 0.838 0.606 0.207 0.815 1.785 0.232 0.600 0.295 + 0.863 0.787 0.200 0.813 1.873 0.212 0.617 0.315 sem 0.013 0.125 0.006 0.007 0.087 0.010 0.006 0.010 probability p> f ml ns ns ns ns ns ns ns ns mi ns ns ns ns ns ns ns ns ml × mi ns ns ns ns ns ns ns ns biotropia vol. 29 no. 3, 2022 242 conclusion the supplementation of mold inhibitor (mi) in corn with different moisture levels (ml) is effective to maintain the physical quality, the dry matter (dm), ash and energy content of corn grains during the 90-day storage period. the concentration of crude protein and crude lipid tended to decrease in the group of corn with different moisture level supplemented with mold inhibitor. the addition of mold inhibitor maintained the dm content but was not effective to reduce the afb1 content of corn during the storage. except for histidine and lysine, the supplementation of mi in corn with different ml did not affect the amino acid content of corn. further research is needed to evaluate the mold inhibitor-treated corn on the growth performance of broilers and other poultry. acknowledgments the author would like to thank the state polytechnic of agriculture kupang that has provided funding for this research (contract number: 03/p3m/dipa.023.18.2.677616/ 2021). we are grateful of the in-kind contribution from pt japfa comfeed tbk in the form of mintai feed anti-mold product. the assistance provided during the research by maria wonda, geti pahnael, and suhartini salih was highly appreciated. references abass a, fischler m, schneider k, daudi s, gasper a, rüst j, ..., kabula e. 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brinkhoff et al. 2012). among the reported myxobacteria collected, those from marine samples were novel, including haliangium ochraceum, h. tepidum (fudou et al. 2002), enhygromyxa salina (iizuka et al. 2003a), plesiocystis pacifica (iizuka et al. 2003b) and pseudenhygromyxa salsuginis (iizuka et al. 2013). myxobacteria in general are characterized by their ability to form fruiting bodies and their gliding motility on solid surface. the fruiting bodies are formed after exhaustion of the food supply and contain dry resistant myxospores. colonies can spread into an unoccupied area. this spreading * corresponding author: siti.meliah@lipi.go.id biotropia 5 2 8 121 129 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.2.796 121 movement is called swarming behavior. the shape, size, color, or arrangement of vegetative cells, swarms, fruiting bodies, and myxospores are important in determining genus of myxobacteria. in contrast to most bacteria, myxobacteria are also capable of lysing living cells. for this reason, they are called predators. based on the specialization of myxobacteria in degrading biomacromolecules, they are divided into bacteriolytic myxobacteria that lyse whole cells of other microorganisms and cellulolytic myxobacteria that efficiently decompose cellulose instead of living cells (singh 1947; hou et al. 2006). myxobacteria are known for their enormous potential in producing secondary metabolites with various biological activities. for certain reasons, their utilization as potential secondary metabolite producers is limited. they are relatively difficult to isolate and purify. in addition, only some of their species are easily grown in liquid culture during fermentation. thus, they are often overlooked by industrial sectors. however, due to the emerging of multiple drugs resistance in pathogenic microorganisms, the need to find new alternatives of potential bioactive compounds from natural resources, including myxobacteria is inevitable. myxosporin, myxovalargin, and myxothiazol are some of the secondary metabolites produced by myxobacteria with antimicrobial activity. these compounds are extracted from typical bacteriolytic myxobacteria, myxococcus fulvus (dawid 2000). the genus myxococcus is also reported to produce myxovirescin (williams & mcgill 1990) and myxalamides (gerth et al. 1983; konovalova et al. 2010). thuggacin antibiotics active against mycobacterium tuberculosis; the causative agent of tuberculosis, were reported to be chondromyces crocatus (buntin et al. 2010). another secondary metabolite, epothilone that acts upon cancer cells was extracted from cellulolytic myxobacteria, sorangium cellulosum (gerth et al. 1996). the myxobacteria were isolated from various samples and places, such as seawater and sediment samples from shandong province in china (li et al. 2002); marine sediment from santa barbara in us, texel in netherlands, and bokum in germany (schäberle et al. 2010); and also soil samples from yunnan, qinghai, hebei and yuenan in china (zhang et al. 2003); india (singh & singh 1971); and kiritimati island, republic of kiribati (mohr et al. 2016). there is no sufficient record of this bacterial group that has been isolated in indonesia despite their medical importance, except a report on a comparative analysis of predation of myxococcus xanthus isolated from sulawesi in 2010 (morgan et al. 2010). however, studies on diversity and composition of prokaryotic communities in sumatera using culture independent methods revealed that fungi-like myxobacteria (sorangium and haliangium) were detected in the soils samples and were slightly more abundant in the managed soils than in rainforest soils (schneider et al. 2015). sumba and papua islands are some of the outermost islands of indonesia. the geography and ecology of these islands are different from each other. however, like most indonesian areas, they are assumed to be a biodiversity rich habitat, particularly for microbes. therefore, this research is conducted to isolate, characterize, and identify indigenous myxobacteria in order to record their occurrence in indonesia and is a preliminary step to exploit their potential as a natural producer of anti-infective. materials and methods materials and sampling methods materials used in this research are listed in table 1. these samples were collected from two different locations of indonesia in april 2016. soils were sampled using composite method. soil and limestone samples were air dried overnight to reduce the growth of untargeted microorganisms. in total, the number of samples used in this study was 37. isolation of cellulolytic and bacteriolytic myxobacteria the isolation of cellulolytic and bacteriolytic myxobacteria was conducted using methods described by reichenbach and dworkin (1992) with some modif icat ions. cel lulolyt ic myxobacteria were isolated by placing a drop of soil sample, limestone sample, and a fragment of decay wood onto a piece of whatman no. 1 filter 2paper sized 1 cm on stan 21 agar media (a mix of a solution: 1 g k hpo , 0.02 g yeast extract, 10 g 2 4 agar in 700 ml distilled water; and b solution: 1 g kno , 1 g mgso .7h o, 1 g cacl .2h o, 0.2 g 3 4 2 2 2 fecl , 0.1 g mnso .7h o in 300 ml distilled 3 4 2 122 biotropia vol. 25 no. 2, 2018 cyanocobalamin, supplemented with 25 µg/ml cycloheximide using sterilized syringe needle. the transferring process was conducted several times until pure cultures of myxobacteria were obtained. pure cultures were stored in 10% gycerol stock solution supplemented with 0.1% cacl .2h o at -80 c.o 2 2 phenotypic characterization the isolates obtained were morphologically observed with the help of dissecting (olympus sz) and binocular (olympus bx43) microscopes. several characters were observed including fruiting bodies that emerge days after incubation, vegetative cells, and also the pattern and the color of swarm colonies. the fruiting bodies were crushed to examine the myxospores. each of the pure isolates was subjected to gram staining using crystal violet, iodine, and safranin reagents. molecular identification based on 16s rrna gene analysis molecular identification was conducted based on 16s rrna gene analysis. genomic dna was extracted using a set of processes started by rinsing bacterial cells with 500 µl te buffer ph 8.0. after centrifugation at 13,000 rpm for 5 minutes, pellet obtained was resuspended with 50 µl te buffer ph 8.0 and 300 µl extraction buffer consist of tris-hcl, edta, sodium dodecyl sulfate, and nacl. this suspension was homogenized using vortex mixer for 5 minutes. an amount of 150 µl 3m sodium acetate was added to the suspension and incubated for 10 minutes at room temperature. after incubation, the suspension was centrifuged at 13,000 rpm for water). these solutions were autoclaved separately. solution a and b were combined and t h e n s u p p l e m e n t e d w i t h 2 5 µ g / m l cycloheximide. this medium was designated as st21cx. the samples in st21cx media were o incubated at 30 c for 2-4 weeks. bacteriolytic myxobacteria were isolated using baiting technique with escherichia coli. e. coli cells were cultured on luria broth (lb) medium (10 g/l tryptone, 10 g/l nacl, 5 g/l yeast extract suspended in 1l distilled water) for 24 hours. e. coli suspension was then centrifuged for 10 minutes at 10,000 rpm. the bacterial pellet was resuspended with cycloheximide solution 25 µg/ml enough to make a thick slurry of bacterial cells. e. coli cells were then cross striked on a water agar (wcx) medium (1 g/l cacl .2h o, 15 g/l 2 2 ag a r , supp l emen ted w i th 25 µg/ml cycloheximide). in the center of the cross, a peasized amount of soil and limestone sample was inoculated. the samples in wcx media were then o incubated at 30 c for 2-4 weeks. purification of myxobacteria myxobacteria obtained from both methods were transferred using sterilized syringe needle to a fresh wcx medium cross striked with autoclaved e. coli to purify them. dissecting microscope was used to recognize the myxobacteria so that this direct purification technique can be done. the bacteria were then o incubated at 30 c for 1-3 weeks. the fruiting bodies or swarm cells that produce clear zones around the dead were e. coli transferred to a modified vy/2cx medium consisting of 5 g/l baker's yeast fermipan, 1 g/l cacl .2h o, 15 g/l agar, 0.5 µg/ml 2 2 table 1 sampling locations and materials used to isolate myxobacteria sampling location altitude (masl) ph t (oc) samples number of samples wanggameti national park , sumba, east nusa tenggara e 120o 15.360’ s 10o 04.696’ – e 120o 16.703’ s 10o 03.496’ 983-1164 7 23-36 soil 13 limestone 3 decay wood 10 tambraw, west papua e 132o 15’ 05.2” s 00o 45’ 0.99” – e 132o 44’ 05.8” s 00o 52’ 0.94” 443-891 6.5 26.5-32 soil 7 decay wood 4 isolation, characterization and identification of myxobacteria – meliah and lisdiyanti 123 another 5 minutes. the supernatant obtained was transferred to a new microtube and gently mixed with isopropanol in the same volume. this mixture was centrifuged for 10 minutes at 13,000 rpm. the pellet obtained was suspended with 70% ethanol and centrifuged for 1 minute at 13,000 rpm. the dna or pellet was air dried and resuspended with 50 µl te buffer ph 8.0. the quality and quantity of genomic dna was examined by biospec-nano micro-volume uvvis spectrophotometer (shimadzu). universal eubacterial primers 27f (5'agagtttgatcctggctcag-3') and 1492r (5'-ggttaccttgttacgactt-3') were used to amplify 16s rrna gene (lane 1991). gene amplification was performed under the following o conditions: pre denaturation at 94 c for 2 minutes, subsequently followed by 35 cycles of o denaturing at 94 c for 15 seconds, annealing at 55 o o c for 30 seconds, elongation at 72 c for 1 o minute, and final extension at 72 c for 10 minutes in mastercycler gradient (eppendorf). pcr products were checked on 1% agarose gel stained with ethidium bromide solution and observed under uv transilluminator. these dna fragments were sequenced by macrogen inc. (south korea) using 27f and 1492r primers in abi 3730xl dna analyzer. the sequences of the isolates obtained were analyzed using bioedit program (hall 1999). the identification of phylogenetic neighbors was initially carried out by the blastn (altschul et al. 1997) program against the database containing type strains with validly published prokaryotic names and representatives of uncultured phylotypes (kim et al. 2012). the top thirty sequences with the highest scores were then selected for the calculation of pairwise sequence similarity using global alignment algorithm, which was implemented at the eztaxon server (http://www.ezbiocloud.net/eztaxon). the 16s rrna gene sequences in this study were submitted to genbank ncbi under these following accession numbers; mg561397 (smdw06.2), mg561394 (smcv05.1), mg561395 (smcv05.2), mg561396 (smcv05.3), mg561388 (ps3.1), mg561389 (ps3.2), mg561390 (ps3.3), mg561398 (sms05.1), mg561399 (sms05.2) and mg561400 (sms05.3). all the identified isolates were also deposited in indonesian culture collection (inacc) using the number smdw06.2 (inacc b1220), smcv05.1 (inacc b1221), smcv05.2 (inacc b1222), smcv05.3 (inacc b1223), ps3.1 (inacc b1224), ps3.2 (inacc b1225), ps3.3 (inacc b1226), sms05.1 (inacc b1227), sms05.2 (inacc b1228) and sms05.3 (inacc b1229). phylogenetic analysis multiple alignment of all the dna sequences were performed by muscle program (edgar 2004). type strains and their sequences were collected from a list of prokaryotic names with standing in nomenclature (lpsn) website ( w w w. b a c t e r i o . n e t ) a n d g e n b a n k (www.ncbi.nlm.niv.gov). kimura 2-parameter model was selected to calculate the distance matrices between sequences (kimura 1980). phylogenetic tree was constructed using neighbor-joining method (saitou & nei 1987) with 1,000 replicates of bootstrap. all these programs are implemented in mega version 6 (tamura et al. 2013). desulfovibrio desulfuricans acc. no. m34113 which belongs to deltaproteobacteria, was used as out-group in constructing the phylogenetic tree. . results and discussion taxonomically, the fruiting gliding myxobacteria belong to the phylum of proteobacteria, subphylum delta-proteobacteria, order myxococalles, and consist of 3 suborders (cystobacterineae, nannocystineae, sorangiineae). they are characterized by rod and yellow, orange, or red pigmented cells. in this study, yellow and orange pigmented swarms and fruiting bodies emerged on whatman no. 1 filter paper on st21cx and on streaked e. coli on wcx agar media, after 2-3 weeks of incubation. visible clear zones also appeared around the streaked e. coli on wcx media after 5-7 days of incubation. it is known that bacteriolytic myxobacteria consume other living bacteria or yeasts as their nutrient source. hence, these clear zones were an indication of predatory activity of bacteriolytic cells. the predatory activity of myxobacteria on other bacteria and yeasts is supported by the production of extracellular lytic enzymes and antibiotics (xiao et al. 2011). this mechanism also plays an important role in competing with their natural opponents in a habitat. 124 biotropia vol. 25 no. 2, 2018 under a dissecting microscope, swarms and fruiting bodies grown on samples were transferred to a new wcx media with dead e. coli as a nutrient source and subsequently transferred to vy/2cx agar using a sterile syringe needle. this purification method was able to produce a more uniform colony appearance (fig. 1). from a total of 37 samples using st21cx and wcx medium as isolation media, only 10 myxobacterial isolates were successfully recovered and purified. compared to common bacteria, purification of myxobacteria is relatively tricky. they easily carry contaminates, such as fungi, other bacteria, and soil amoeba, because they produce slime to help them move on a solid medium. a number of purification techniques have been developed to increase proportion of pure cultures. improved methods, such as purification with crystal violet and second baiting technique subjected to fruiting body can improve the proportion of pure culture obtained up to 42.5% and 69.7%, respectively (zhang et al. 2003). all the 10 bacterial isolates were gram negative bacteria. they were rod shaped with slight differences in size. morphologically, the isolates obtained vary in color, shape and swarming pattern. these isolates showed conspicuous characteristic of myxobacteria by producing fruiting bodies. round or spherical myxospores were observed under the microscope from crushed fruiting bodies. these characteristics were identical to morphology of myxobacteria described in bergey's manual (reichenbach 2005). based on their morphology, these isolates were grouped into three morphological groups (table 2). morphological identification based on vegetative cells, swarms, fruiting bodies, and myxospores is still valid for some myxobacteria genera. however, these morphological characters are not stable and may change or be lost under artificial growth condition despite expressed by their genotype. hence, identification and classification of myxobacteria remains challenging for most of the genera and species (garcia et al. 2010). this is also partly due to the fact that some recently isolated myxobacteria strains do not usually have typical myxobacterial morphological characteristics. therefore, molecular identification is needed to confirm the identity of the isolates. analysis on 16s rrna gene sequences of 10 isolated bacteria revealed that these isolates were member of myxobacteria group. they belong to the genera and with more myxococcus melittangium than 99% similarities (table 3). and myxococcus melittangium genera were often found in soil samples throughout the world, from tropical rain forests, centra l european forests, to mediterranean regions and so far, to our a b c figure 1 morphological appearance of purified myxobacterium collected from sumba sms05.1 (a), smdw06.2 (b), and papua ps3.1 (c) on vy/2cx agar medium after incubation for 5-10 days. fruiting bodies appeared as yellow/orange cells aggregate. table 2 isolates grouping based on morphological appearance morphological characteristics of colony myxospores total isolates orange, centrifugal pattern, orange fruiting body spherical 3 yellow swarms, orange fruiting body spherical 4 orange swarms, short stalked orange fruiting body spherical 3 125 isolation, characterization and identification of myxobacteria – meliah and lisdiyanti knowledge this is the first record of the three species occurrence in indonesia. based on morphological observation supported by molecular identification, diversity of myxobacteria obtained from this work was not numerous. some isolates might not form typical fruiting bodies at the time of observation, hence overlooked during transferring process. isolation techniques, incubation time, and media used to isolate myxobacteria still unable to culture various species occur in a habitat in this study. every method would be a compromise, as different myxobacteria species would grow well on one medium, others would grow less well and show little tendency to form fruiting bodies (dawid 2000). hence, the variation on isolation technique, purification technique, and enrichment media are expected to increase the probability to obtain more diverse myxobacteria. phylogenetic analysis of the 10 myxobacteria sequences showed that all the isolates are placed in suborder cystobacterineae (fig. 2). taxonomically, genus myxococcus along with aggregicoccus, corallococcus and pyxidicoccus are classified into the family myxococcaceae in the suborder cystobacterineae. on the other hand, the genus melittangium along with angiococcus, archangium, cystobacter, hyalangium, stigmatella and newly described genus vitiosangium (awal et al. 2017) are c lass i f i ed in to the fami ly cystobacteraceae also in the suborder cystobacterineae. table 3 molecular identification of isolated myxobacteria 126 biotropia vol. 25 no. 2, 2018 figure 2 phylogenetic tree constructed on the basis of 16s rrna gene sequences using neighbor-joining method. isolates from sumba (sm) and papua (ps) islands were placed in the suborder cystobacterineae among myxobacteria species. bar means 1 substitution per 200 nucleotides. numerals at branch points indicate the bootstrap value as percentages derived from 1000 replications. only values greater than 60% are shown. 127 isolation, characterization and identification of myxobacteria – meliah and lisdiyanti conclusion a total of 10 myxobacterial isolates was successfully isolated from soil, limestone and decay wood samples collected from two outermost islands of indonesia, sumba and papua. they were isolated using filter paper and baiting with e. coli methods. characterization of sumba and papua isolates revealed that they were gram negative and rod shaped. their colonies were yellow and orange in pigmentation, and produce fruiting bodies. molecular identification showed that they are members of myxobacterial species myxococcus fulvus, myxococcus stipitatus and melittangium lichenicola. to our knowledge, this is the first record of the occurrence of the three species in indonesia. all the isolates were deposited in indonesian culture collection (inacc) under the name myxococcus fulvus inacc b1220, myxococcus fulvus inacc b1221, myxococcus fulvus inacc b1222, myxococcus fulvus inacc b1223, myxococcus stipitatus inacc b1224, myxococcus stipitatus inacc b1225, myxococcus stipitatus inacc b1226, melittangium lichenicola inacc b1227, melittangium lichenicola inacc b1228 and melittangium lichenicola inacc b1229. acknowledgements this research was financially supported by dipa 2016 lipi. we wish to thank maman rahmansyah and everyone involved in ekspedisi widya nusantara (e-win) and nkri expedition during the samples collection. we also thank tri ratna sulistiyani, rinatu siswi and riesca martdiyanti for their help during the 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methods of isolation and purification of myxobacteria and development of fruiting body formation or two strains. j microbiol methods 54(1):21-7. zhang yq, li yz, wang b, wu zh, zhang cy, gong x, … zhang y. 2005. characteristic and living patterns of marine myxobacterial isolates. appl environ microbiol 71(6):3331-6 . 129 isolation, characterization and identification of myxobacteria – meliah and lisdiyanti page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 1031.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 705 ridesti (the growth strategies).cdr the analysis growth strategies of ten woody plant species for effective revegetation ridesti rindyastuti nd retno peni sancayaningsih 1* 2 a 1 , (lipi) ,purwodadi botanic garden indonesian institute of sciences , pasuruan 67163 indonesia laboratory of ecology and conservation, faculty of biology, gadjah mada,2 department of biology, universitas yogyakarta 55281 indonesi, a received 05 october 2016/accepted 26 may 2017 abstract the growth strategies of plant species show the ecological role which is reflexed by their adaptation to environments and competitiveness. those are essential in the study of the revegetation effectiveness. however, the growth strategies of plant species in various types of habitats have not yet been fully investigated. the objective of this study was to investigate the growth strategies of ten woody plant species which were naturalized from mangrove to lowland habitats in relation to their effectiveness for revegetation program. the seedling's growth was recorded during 4 months in purwodadi botanic garden-lipi from october 2014 to february 2015. complete randomized design with plant species as a treatment using 3 replications was carried out to examine the plant's relative growth rates (rgrs), their components, leaf nitrogen productivity and growth strategies.the study showed that rgrs of ten woody plants species varied across species. based on the pearson correlations, the plant's net assimilation rates (nar) and two ecological traits related to the root trait i.e. nitrogen productivity and specific root length (srl) were strongly correlated with the rgrs. heritiera littoralis, diospyros discolor, antidesma bunius, schleichera oleosa, madhuca longifolia and syzygium cumini have high rgrs but low specific leaf area (sla). b. asiatica has relatively low rgrs and sla, while dracontomelon dao have high rgrs and sla. it showed that most of plant species studied, except d. dao achieve growth rates and competitiveness by developing strategies through forming fine roots to maximize its ecological function in nutrients uptake. most of woody plant species are adaptive to dry lowland habitat and only d. dao potentially occupy the ecosystem. furthermore, d. discolor and s. oleosa are highly recommended for revegetation in degraded tropical lowland areas. keywords: growth strategy, revegetation, rgr, root trait, woody plants introduction natural tropical forests are being destroyed at a rapid high rate, therefore the success of forest restoration continues to be a challenge. a restoration program could help reestablish a forest vegetation eventhough it is difficult to guarantee the successful recovery of its biodiversity (elliott et al. 2013). adequate knowledges on biology and ecology of plant species related to their adaptation to habitat conditions enhance their use for decission making in reforestation programs. especially in the early stage, plant species commonly develop the strategies for using limited resource in their environments, adapting to stresses, competing and associating with others species in order to preserve their existences in their distribution ranges. the growth strategies show the ecological role and contribution of plant species to an ecosystem and imply direct result of the evolutionary control by genetic (kolb et al. 1990)s . growth strategies can be identified by analysing and comparing the growth rates, leaf and root traits since these two organs are essential in photosynthesis and nutrient uptake (wright & westoby 2000). in a constant state and similar environment, plant species were reported to have interspecific differences in growth rate that indicate different results of ecological strategy of plant species (lambers et al. 1998; pugnaire & valladers 1999; laughlin et al. 2010). in plants, the survival depends on growth rate that can not be acurately measured only by observing plant size. * corresponding author: ride17@gmail.com biotropia 5 1 8 43 55 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.1.705 43 thus, relative growth rate (rgr) and its components were modeled to understand, compare and evaluate plant growth rate. rgr and its components such as nar, sla, lwr, and lar (described in table 2) tend to reveal more of the species traits than predict plant growth rate according to its habitat (lambers et al. 1998; pugnaire & valladers 1999). growth strategy is especially related to leaf and root trait reflected in sla (leaf area per which is unit leaf dry mass) and in srl (root length per unit root dry mass). sla as an aboveground seemed to be easier to investigate than srl. srl has been suggested as the belowground analogue to sla, and is considered as a morphological index of belowground species competitiveness (cornelissen . 2003). shipley (2002) reported et al that rgr of either woody and herbaceous plant s have strong and positive correlation with nar and have weak and negative correlation with sla. according to lambers . (1998) pugnaire et al and and valladers (1999), variation in rgr is determined mainly by sla. the opposite result is partly caused by differences of irradiance level used in the experiments. in general, the contribution of rgr's component is influenced by irradiance level. at low irradiance, sla becomes the most determinant factor of rgr , and nar is the main determinant of rgr at high irradiance. thus, plant species possess that different shade tolerance will show different s response in growth rate. in general, higher sla is s the main growth atribute support plant which s invasiveness in other type of habitat (shipley s 2002; lambers . 1998; pugnaire valladers et al & 1999), with an exception, the invasive woody species successful in mediterranean which are climates. this invasive woody species has a greater biomass allocation to roots than less-invasive species (grotkopp rejma´nek 2007).& the growth strategies of plant species adapted in tropic regions with various types of habitats and shade tolerances have not yet been fully investigated. on the other hand, plant interspecific variation in rgr and its components a r e impor t an t to s tudy the spec i e s competitiveness for particular purposes such as land restoration, carbon sequestration, species invasiveness etc. local plant species which possess high growth rates and are widely distributed in certain ecological range are highly recommended for restoration of degraded areas (cairns 1995). moreover, to grow the plant species from other types of habitats, the revegetation effectiveness and plant invasiveness should be managed by understanding plant ecological traits including growth rate, growth strategy related to sla, carbon gain and physiological performance of exotic species compared to native plants (pattinson . 1998; et al grotkopp & rejma´nek 2007). for this study, ten woody plant species were investigated because they originated from various types of habitats but were developed and planted for revegetation programs in tropical dry lowland areas. the effectiveness of these species for revegetation in dry lowland habitats should be investigated in terms of their growth strategies. this study could provide valuable insights on the variation among plant species and explain the ecological success and competitiveness of plants in certain habitats. thus, the objectives of this study were to: 1) determine the interspecific variation in rgrs and its components among plant species in similar irradiance level ; 2) find s out the ecological trait of ten woody plant species; and 3) identify the growth strategies of ten woody plant species and discuss their ecological trait related to their effectiveness for revegetation in dry low land habitat. materials and methods plant material the seedlings used in this study were from the plant collections of purwodadi botanic gardenlipi. all species used in this study are c3 woody plant species (rindyastuti hapsari 2017). the & botanic garden is located in pasuruan, east java, at 300 m asl. it has a semi-deciduous climate with monthly rainfall of 246.57 mm in the wet season and 38.2 mm in the dry season. the annual temperature ranges from 20.4 to 30.6 c. three o seedlings were used for each treatment. sixteenmonth old seedlings of ten local woody plant species used in this study are described in table 1 . 44 biotropia vol. 25 no. 1, 2018 at 16 months were harvested to obtain w and the 1 seedlings were harvested 4 months later at 20 months to obtain w . all seedlings were divided 2 into three parts: leaf, stem and root. plant materials were dried in an oven with temperature of 80 c to constant weight (crescente gratani o & 2013). rgr was calculated using the following equation ( :hoffmann & poorter 2002) rgr = (ln w -ln w )/(t -t )2 1 2 1 where: ln = natural logaritm 1t = time one 2 t = time two 1w = dry weight of plant in time 1 2w = dry weight of plant in time 2 the components of rgr observed in this study are listed and described in table 2. growth conditions, experimental design and rgrs measurement seedlings of ten woody plant species were grown in horticultural pots under controlled and favourable conditions in a glasshouse located at purwodadi botanic garden-lipi, pasuruan, east java which has a temperature range of 26.2-30.8°c in the dry season and 28.7-34.6°c in the rainy season. seedlings were watered every 2 days without fertilizer treatment. seedlings were grown in a mixture of silica sand and compost with a ratio of 1:1. the experiment was caried out in complete randomized design using 3 replications with variation of species as its treatment. all seedlings were grown until 16 months then were transferred into bigger polybags with new plant media to record maximum resource uptake for their growth. three seedlings for each replication 45 growth strategies analysis of ten woody plant species rindyastuti and sancayaningsih table 1 list of species, habitat, type of plant and shade tolerance of ten woody plant species observed in this study species habitat type shade tolerance barringtonia asiatica (lemmens et al. 1995) mangrove, tropical coast evergreen woody intermediate to tolerant dracontomelon dao (orwa et al. 2009) monsoon forest (deciduous and semi-deciduous) semi-deciduous, hardwood tolerant heritiera littoralis (lemmens et al. 1995) mangrove, tropical coast evergreen, hardwood tolerant diospyros discolor (verheij & coronel 1991; lemmens et al. 1995) dry lowland tropic evergreen hardwood tolerant calophyllum inophyllum (sosef et al. 1998; orwa et al. 2009) coast-lowland tropic evergreen hardwood intolerant antidesma bunius (sosef et al. 1998; orwa et al. 2009) highland tropic evergreen hardwood tolerant schleichera oleosa (kundu & schmidt 2011) dry low-highland mix deciduous forest deciduous hardwood tolerant syzygium cumini (verheij & coronel 1991; orwa et al. 2009) dry and moist tropicaldeciduous forest evergreen hardwood tolerant madhuca longifolia (sikarwar 2002) dry lowland tropics evergreen-semievergreen, hardwood tolerant adenanthera pavonina (orwa et al. 2009) evergreen-deciduous forest evergreen-deciduous, woody tolerant leaf area measurement leaf area were used as a component of parameters listed in table 2. leaf area was calculated by comparing the proportion of an area 2 of 16 cm paper weight with the weight of the entire paper using comparison formula as follows: where leaf area l2 = l1 = area of sample paper w2 = paper weight w1 = weight of sample paper. analysis of nitrogen productivity leaves of the ten woody plants were oven o dried at temperature of 80 c for 48 hours. five g of leaf sample were grinded and analyzed using the kjeldahl method (bremmer 1965). the analyses were performed at the soil chemistry laboratory, faculty of agriculture of brawijaya university in malang. nitrogen productivity was reflected by the value of lnp (rate of dry mass increase/unit leaf nitrogen/unit time) which was calculated using the formula as follow: seed mass seeds of the ten woody plant species were collected from the plant collection at purwodadi botanic garden-lipi, pasuruan, east java in march 2016. ten fresh seeds weight were measured for each woody plant species including the seed coat. the seed mass show plant strategies which were illustrated by l-h-s (leaf trait, height and seed mass) scheme in several growthforms (westoby 1998). this variable was used to determine plant strategies scheme of species studied, whether l-h-s scheme or others. data analyses the normality and randomity of data were tested using kolmogorov-smirnov and run test. the normal data were analyzed using variance analysis (anova) at confidence level of 95%. tukey's advance analysis was carried out for data grouping. the relationship among variables which have random data were analyzed using pearson correlation test at confidence level of 95% (p<0.05), while relationships between unrandom data were analyzed using spearman rank correlation test. results and discussion interspecific variation in rgr based on the normality test, the mean of rgr and its components of the ten woody plants were normal. based on the variance analyses, there was a significant difference in rgr (p=0.014) and sla, lar, swr, lwr and rwr with p values 0.0001, 0.0001, 0.0002, 0.0002, and 0.0001, 46 biotropia vol. 25 no. 1, 2018 table 2 list of abbreviations used in the text with definitions and units of measure abbreviations definitions units rgr relative growth rate g g-1day-1 lar leaf area ratio (leaf area/plant dry mass) cm2 g-1 sla specific leaf area (leaf area/foliage dry mass) cm2 g-1 lwr leaf weight ratio (foliage mass/plant dry mass) g g-1 swr stem weight ratio (stem mass/plant dry mass) g g-1 rwr root weight ratio (root mass/plant dry mass) g g-1 nar net assimilation rate g cm -2 day-1 srl specific root length (root length/root dry mass) cm2g-1 lnp leaf nitrogen productivity (dry mass increase/unit leaf nitrogen/unit time) g g-1 day-1 leaf nitrogen productivity (lnp) = dry mass increase leaf nitrogen/time respectively (significant if p<0.05) among the ten woody plant species observed in this study. mean of rgr ranged from 4 x 10 g g day for -3 -1 -1 -3 -1 -1 cc. inophyllum and 10.7 x 10 g g day for s. umini (figure 1). mean of initial sla of the ten woody plant species ranged from 90.53 for h. littoralis to 298.56 for d. dao (figure 2). during the seedling growth, the mean of sla increased from 105.89 for h. littoralis to 321.58 for a. pavonina. the mean of initial lwr ranged from 0.094 for a. pavonina to 0.46 for d. discolor, while mean of lwr increased from 0.52 for s. cumini to 0.53 for d. discolor. the differences between species belonging to the lowest ratio and highest ratio of sla and lwr show the differences of succulence level and leaf dry matter content among species (wilson et al. 1999). the mean of initial swr ranged from 0.14 for d. discolor to 0.49 for s. cumini. the mean of swr increased from 0.29 for d. discolor to 0.61 for s. cumini in the end of observation. less mass was stored in the stem of d. discolor than in its other parts. mass allocations in the root was reflected by rwr (figure 3). mean of initial rwr ranged from 0.29 for h. littoralis to 0.54 for a. bunius. after 4 months of growth, the mean of rwr increased from 0.18 for d. discolor to 0.38 for b. asiatica. other than rwr, root trait was also reflected by srl (figure 3). srl ranged from 1.28 for b. asiatica to 7.2 for d. discolor in the initial growth and ranged from 1.52 to 10.86 at the end of the experiment. 47 figure 1 the mean of rgr and nar of ten woody plant species note: rgrs and nar were significantly different among species based on the variance analysis (p<0.05) growth strategies analysis of ten woody plant species rindyastuti and sancayaningsih b. a sia tic a d. d ao h . li ttr ora lis d. d isc olo r c. in op hy llu m a . b un ius s. o leo sa s. c um ini m . lo ng ifo lia a . p av on ina 48 biotropia vol. 25 no. 1, 2018 figure 2 the comparison between mean components of rgr related to leaf trait (lar, lwr and sla) of ten woody plant species (note: the component of rgr related to leaf trait were significantly different among species based on the variance analysis (p<0.05)) b. a sia tic a d. d ao h . li ttr ora lis d. d isc olo r c. in op hy llu m a . b un ius s. o leo sa s. c um ini m . lo ng ifo lia a . p av on ina 49 figure 3 the mean of components of rgr related to stem and root traits represented by swr, srl and rwr of ten woody plant species (note: the components of rgr related to stem and root traits were significantly different among species based on the variance analysis (p<0.05) growth strategies analysis of ten woody plant species rindyastuti and sancayaningsih b. a sia tic a d. d ao h . li ttr ora lis d. d isc olo r c. in op hy llu m a . b un ius s. o leo sa s. c um ini m . lo ng ifo lia a . p av on ina 50 biotropia vol. 25 no. 1, 2018 correlation among components of rgr correlation tests were performed to reveal the relationships between plant rgr and its components. the mean of rgrs of the ten woody plant species was strongly correlated with nar (p value 0.0001) but not with other components (figure 1, table 3). the correlation explained the importance of nar in determining rgr of woody plant species. variations in other components did not explain the differences in -4 -2 -1 rgr. nar ranged from 1.2 x 10 g cm day for -3 -2 -1 c. inophyllum to 1.9 x 10 g cm day for s. oleosa. the nar values of two species which are known as fast growing species i.e. m. longifolia and a. pavonina were more than a fifteen-fold difference with slow growing plants (figure 1). interspecific variation in rgr and its components among species showed variation of ecological traits of plant species. the ecological traits can be shown by above ground trait supported by leaf trait or below ground trait supported by root traits which serves as a chosen strategy scheme of plant species in the habitat they occur (laughlin et al. 2010; grime 1979). in several previous studies, plant strategies were illustrated by l-h-s (leaf trait, height and seed mass) scheme. seed mass showed dependence with other growth component between different growth forms (westoby 1998). for example, seed mass dependence of woody c3 was different from those of graminoids (laughlin et al. 2010). in line with the previous study, the seed mass of ten woody plant species which included in woody c3 were not correlated with the height or leaf traits of plant species. therefore, it indicated that plant strategy across species within similar growth form were not supported by seed mass but by leaf or root trait. the plant size can not literally support the mass investment to plant structure because the value of rgr is mainly supported by dry matter content especially various lignin component (laughlin et al. 2010). the larger plant could show lower rgr maybe because of the high level of water content in the plant body. the larger plant sometimes consist of abundant water than its dry matter content, for example and . b. asiatica a. pavonina thus, the growth properties of plant species could not be observed only by the plant size thus, the study of growth rate and its components proves to be very important. the study result showed that the mean rgr of fast growing plants was more than two-fold difference from the slowest growing plant while nar was a five-fold difference from the slowest growing plant. rgr of woody plants showed different pattern from those of non-woody species. according to poorter and remkes (1990), in c3 non-woody species, rgrs of the fastest growing species was three-fold more than those of the slowest growing species, while nar of the fastest growing species was two-fold more than those of the slowest growing species. fast growing species obtain an ecological advantage of a high rgr related to high competitiveness and rapid occupation (poorter r mkes 1990). & e therefore, the difference in nar between the fast and slow growers seemed to be a trustworthy evidence of its contribution to differing rgr across plant species. a strong correlation between rgrs and nar of woody plant species showed how the high rgrs in fast growing species were achieved. theoritically, rgr is the product of lar and nar. however, lar did not seem to contribute to rgrs variation. while it is clear that two other growth components related to leaf formation (sla and lwr) did not show its association with growth rate, this could be concluded that there was other more important growth plant behavior in carbon gain than to invest more mass to the leaf. nar largely reflects the rate of photosynthetic carbon gain minus carbon released by respiration. loveys . (2002) et al reported that correlation between rgr and nar is more stable than the correlation between rgr and sla. the effect of sla to the variations in rgrs is more sensitive than nar because it showed greater response in slight changes of radiance level. moreover, sla is an important plant functional trait as species which have higher sla can obtain a more extensive foliage display that captures more light for biomass investment (crescente gratani 2013). & the mean of rgrs was correlated with srl and lnp but not with sla. nitrogen productivity showed an important role in root trait of plant growth. when rgrs only correlated with root length and nitrogen productivity, this means that the woody plants in this study could achieve more mass through root trait than leaf trait (wilson . 1999). it also explains how the et al 51 fast growth of species studied can be achieved and how different rgrs across species are formed. the fast growing species have high rgrs and tend to allocate more mass to the leaves than to the roots. therefore, the species studied have greater mass allocations to the roots and low rgr. hunt and cornelissen (1997) investigated that mean of rgrs of 59 temperate herbaceous species range from 0,1260 g g day for to -1 -1 brachypodium pinnatum 0,2741 gg day for . it indicated -1 -1 epilobium hirsutum that all woody plants studied were slower growing plant compared to the most herbaceous species. variation in seed mass seed mass was one of physiological plant trait that being included into l-h-s (leaf-heightseed) scheme proposed by westoby (1998). l-h-s scheme is a plant ecological strategy which defined by leaf trait, heigh and seed mass. seed mass data of ten woody plant were not normal and not random with a p value of normality test 0,01 (p>0.05), while the p value of a run test was p 0.0001 (p>0.05). thus, seed mass of ten woody plant species were analysed and grouped descriptically. seed mass of is the b. asiatica highest while those of is the lowest a. bunius (figure 4). seed mass can be grouped into big and small seed. clasifiedpoorter and rose (2005) the weight of small seeds is <0.1 g and large seed is >0.1 g. therefore, the seed of all species studied were clasified as big seeds. furthermore, there is no variation in seed mass across species studied that support interspesific variation in rgr. nitrogen productivity leaf nitrogen was reflected in leaf n concentration and lnp (leaf nitrogen productivity). leaf n probably correlated with above or below-ground traits of plant species (wright westoby 2000). the mean of n & concentration of ten woody plant ranges from 1.59 % for to 3.03 % for . h. littoralis a. pavonina mean of lnp value ranges from 0.035 gg day for -1 -1 -1 -1 c. inophyllum s. cumini to 0.24 g g day for . based on the variance analysis, both total n and lnp were significantly different among species. it showed that n productivity vary amongs species and probably contribute to variation in rgrs. based on the correlation test, lnp is correlated with rgrs and sla (p<0.05) (table 3). it showed that n productivity contribute to the variation in rgrs and leaf area. the mean of lnp of ten woody plant species are described in figure 5. the mean of rgr is strongly correlated with srl but is not correlated with sla while the mean of rgr is strongly correlated with lnp (table 3). it indicated that the growth of ten woody plants is achieved through the growth of root length and high leaf nitrogen productivity. the strong correlation between root length and rgrs showed an important role of root function in plant growth. two species which have high srl during the growth are and while d. discolor s. oleosa the species which has the lowest srl is b. asiatica. d. discolor s. oleosa and are considered native to dry lowland habitat while is a mangroveb. asiatica figure 4 seed mass of ten woody plants species (note: there was no variation in seed mass across species studied that support interspesific variation in rgr based on normality test (p>0.05) and run test (p>0.05)) growth strategies analysis of ten woody plant species rindyastuti and sancayaningsih b. a sia tic a d. d ao h . li ttr ora lis d. d isc olo r c. in op hy llu m a . b un ius s. o leo sa s. c um ini m . lo ng ifo lia a . p av on ina species that is likely to poorly survive in dry area. sla is strongly correlated with lnp. among the studied species, the highest sla was found in d. dao a. pavonina and . these two species are known as semi-deciduous species (table 1). leaf or root trait rgrs of ten woody species were correlated with srl but were not correlated with sla, while mean of rgrs was correlated with lnp. the strong correlation between rgrs with root length and leaf nitrogen productivity explained that most of woody plant in this study could achieve more mass through root trait than leaf trait (wilson . 1999). moreover, related to the et al growth properties of plant species, most species allocated greater mass to the leaves than to roots, but slow growing species with low rgr commonly partitioned greater proportion of new biomass to the roots rather than to the leaves (wright westoby 2000). therefore, most of & 52 biotropia vol. 25 no. 1, 2018 figure 5 the mean of lnp of ten woody plants species (note: lnp were significantly different among species based on the variance analysis (p<0.05)) table 3 pearson correlation coefficients between growth components (p<0.05) components p value correlation rgr and nar 0.0001 strongly correlated rgr and lar 0.64 not correlated rgr and sla 0.2 not correlated rgr and lwr 0.57 not correlated rgr and swr 0.88 not correlated rgr and rwr 0.48 not correlated rgr and srl 0.000 strongly correlated rgr and lnp 0.000 strongly correlated nar and lnp 0.056 not correlated sla and lnp 0.000 strongly correlated lwr and lnp 0.2 not correlated lar and lnp 0.1 not correlated swr and lnp 0.267 not correlated rwr and lnp 0.302 not correlated srl and lnp 0.58 not correlated seed mass and rgr 0.87 not correlated seed mass and lwr 0.67 not correlated seed mass and swr 0.97 not correlated seed mass and rwr 0.47 not correlated seed mass and srl 0.45 not correlated seed mass and height 0.28 not correlated b. a sia tic a d. d ao h . li ttr ora lis d. d isc olo r c. in op hy llu m a . b un ius s. o leo sa s. c um ini m . lo ng ifo lia a . p av on ina species studied were clasified as slow growing species. the species which develop the root traits tend to be clasified as slow growing plant. based on the correlation among the rgr components, most of species studied were clasified as slow growing species which develop root trait in achieving mass for plant growth. among these species, d. discolor and are good examples for this growth s. oleosa strategies. in these two species, the highest rgr and srl were found, yet the sla was relatively low. m. longifolia a. pavonina and were known as fast growing woody plants. the rgr of m. longifolia was as high as those of other species while rgr of was low. has low rgr a. pavonina a. pavonina although it has the highest sla while m. longifolia has a high rgr yet low sla. because species which are confirmed as a fast growing species should show high rgr and sla, it is difficult to confirm that these two wellknown species are fast growing species. moreover, both species had among the largest increase in lar through time and largest decrease in rwr. a. pavoninathe which has high sla and high increase of lar yet large decrease of rwr may exhibit different ecological strategies from other species studied. this species tend to develop leave trait than root trait. in addition, in most of species studied, the increase of srl and the decrease of rwr were found. the formation of long root which is combined with the low roots dry mass could mean that plants in this study tended to form more fine roots to reach a wider range of water and nutrients. it showed a pattern of growth to form long root inspite of large foliage display to capture more light. the fine roots could clearly explain the growth strategies through root trait of the ten species studied. implication to land revegetation because of the massive biodiversity loss in recent years, forest restoration should be established in the framework of conservation to restore forest biodiversity and its ecological functions. cairns (1995) recommended native and local plant species to restore destroyed forest ecosystem. native and local species tend to have relatively slow growth and limited leaf expansion. on the other hand, fast growing plants which have wide ecological range distribution are highly recommended to restore degraded areas because of their ability for rapid establishment and coverage. however, non-native fast growing plants have greater potential to invade areas because of their ecological advantage of having a high rgr for high survival, competitiveness and rapid occupation (poorter remkes, 1990; & haggar . 1998; mahari giday 2014).et al & invaders are more likely to have higher rgrs, leaf area and tissue construction costs (daehler 2003), with an exception of woody plants thriving well in mediterranean climates which have greater allocation to roots than less invasive species (grotkopp rejma´nek 2007). further, higher & sla is strongly correlated to plant invasiveness (lambers et al. 1998; pugnaire valladers 1999). & most of the woody plant species in this study obtained their rgrs by developing root trait which showed that these species are less invasive and recommended for regenerating forest vegetation in dry lowland areas. dry tropical climate is a condition with relatively high temperatures and low water availability compared to other tropical climates such as rainforest areas. in dry tropical areas, plants are adapted to have mechanisms to reduce water loss through low transpiration and reach the water balance by absorbing water from the soil. root traits is a competitive advantage with respect to the dry climate with high air temperature and low water availability. adaptive roots have more fibers which can reach water in wider and deeper soil. because root function was correlated with the mean of rgrs, several species which have high rgrs yet relativelly low sla are considered to be well adapted to dry climate such as , , , h. littoralis d. discolor a. bunius s. oleosa m. longifolia, s. cumini, and . several species are known as native plants to dry lowland habitat. native species are preferred to be reintroduced in degraded areas to fix damaged soil by restoring soil fertility and enhancing microbe population which provides sufficient nutrients for plants (álvarez-sánchez 2009). based on this study, et al. native species are considered to have high rgrs which supported by root trait thus they are highly recommended for restoration programs in degraded areas. of the species studied, d. discolor and which are native to dry lowland s. oleosa, areas yet have high rgr and developed fine 53 growth strategies analysis of ten woody plant species rindyastuti and sancayaningsih roots, are highly recommended for revegetation in dry lowland habitat. b. asiatica is a native species to mangrove habitat which has relatively low rgr and sla, while which is adapted to monsoon forest d. dao has high rgr and sla this indicates that . b. asiatica exhibits relatively low competitiveness and occupation potential compared to other species. for being introduced to other types of habitat, non-native species are better of having low competition so that the species do not occupy the plant community in new habitat (bazzaz 1979). hence, can be more b. asiatica recommended for planting program in dry lowland habitat than . based on the d. dao d. daohigh rgr and sla, expands larger foliage to capture more sunlight. therefore, this species has the potential to occupy the ecosystem. other species which have high sla but low rgr, i.e. may develop a. pavonina, growth strategies through leaf trait but do not potentially invade the ecosystem. for further study, it is suggested to investigate whether d. dao release allelopathy, provide ground water and co-occur with other species and growthform to reveal the ecological suitability to new habitat in order to examine the effect of plant growth to soil and plant community. the ecosystem services of a species relating to root trait should be highlighted to maximize the advantage of restoration such as for hydrological services in degraded landscapes (bruijnzeel 2004; hall . 2011)et al . conclusions there are variations in rgrs across the ten woody plants species as determined by nar, nitrogen productivity and root trait. to achieve the growth rates and competitiveness, the woody plant species studied tend to develop strategies through forming fine roots to maximize its ecological function in nutrient uptake except d. dao. most of the woody plant species are adaptive to dry lowland habitat and only d. dao has the potential to occupy the ecosystem. d. discolor and s. oleosa are highly reccomended for revegetation programs in degraded tropical lowland areas. acknowledgements the authors would like to thank kemenristek dikti scholarship which support the whole project of ecophysiological study of woody plants and dr titut yulistyarini as co-promotor. we like to thank mr. riyadi and mr. roif marsono for their technical contributions during experiment in purwodadi botanic garden-lipi. we also like to thank ms. samantha tesoriero for writing consultation. references álvarez-sánchez j, sánchez-gallen i, guadarrama p. 2009. analyses of ecophysiological traits of tropical rain forest seedlings under arbuscular mycorrhization: implications in ecological restoration. in: varma a, kharkwal ac, editors. symbiotic fungi: principles and practice. berlin (de): springer-verlag berlin heidelberg. p. 293-305. bazzaz fa. 1979. the physiological ecology of plant succession. annu rev ecol syst 10:351-71. bremmer jm. 1965. 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australian oody pecies. funct ecol 14(1):97-107.w s westoby m. 1998. a leaf-height-seed (lhs) plant ecology strategy scheme. plant soil 199:213 27. 55 growth strategies analysis of ten woody plant species rindyastuti and sancayaningsih page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 biotropia vol. 28 no. 1,2021: 11 20 doi: 10.11598/btb.2021.28.1.820 dynamics of microbial community during tempeh fermentation rahmadina radital, antonius suwantol*, norio i(urosawa2, aris tri wahyudi' and iman rusmana' 'department ofbiolo8, faculij ofmathematics and natural sciences, institat pertanian bogor, dramga, bogor 16680, indonesia 'department o f science and engineeringfor sustainable innovation, faculg ofscience and engineerin& soka universig, hachioji, tokyo 192-8577, japan received 27 february 2017/accepted 14 february 2020 abstract tempeh is a sliceable soybean-cake fermented by the bacteria rhixopzls olgo@orus. various bacteria are detected during tempeh cultivation, yet limited information is available about their origin or sources. the present study aimed to examine the source/s of bacterial community in tempeh by combining metagenomics analysis and cultivation technique. samples were obtained from a number of steps in the tempeh production using the double-boiling method for the soybean (wjb tempeh production). all samples were plated on enterobacteriaceae and lactic acid bacteria media. total dna were extracted directly from tempeh for metagenomics analysis, applying the high-throughput sequencing (hts) and cloned 16s rrna genes. firmicutes and proteobacteria were the predominant and second dominant bacteria that existed in the fresh tempeh (ft) obtained by metagenomics analysis. however, cultivation technique showed that proteobacteria was the predominant phylum, suggesting that most of the firmicutes were not culturable. ft was dominated by lactobadus and acetobacter. both ft and water-soaked tempeh (sw) were dominated by the same species of lactobacillus, i.e., l. delbreuckii and l mucosae, indicating that sw was probably the source of bacterial community that was established in the final fermentation product. predominant bacteria in the starter culture (sc), acinetobacter, was not detected in ft, indicating that the bacteria in sc did not play a significant role in the ft bacterial community development. keywords: firmicutes, high-throughput sequencing, proteobacteria, starter culture introduction the indonesian traditional food tempeh is made from soybean fermented by the mold ll3ippu.r olzg0spom.r. however, the microbial composition of tempeh also included bacteria, as the "contaminants" in addition to the mold (mulyowidarso e t al. 1990; barus e t al. 2008; efriwati, e t al. 201 3; nurdmi e t al. 201 5). among the producers in indonesia, the tempeh production procedures usually differ and depend on the area, experience, and resources. however, the general procedure for tempeh production has few similarities, such as boiling the soybean, dehulling, soaking, mixing with inoculum, and incubation for two days. the producers usually "corresponding author, e-mail: asuwanto@indo.net.id &ffer in boiling the soybean for the second time after soaking (barus e t al. 2008; efriwati e t al. 2013; nurdini e t al. 2015). four out of five tempeh producers in bogor, did not re-boil the soybean after soaking. however, wjb producers applied two-times or double-boiling of soybean (barus e t al. 2008). of all the methods used for tempeh processing, the initial source of the bacteria in the final product fermented has not yet been verified. tempeh production in indonesia is usually done in "open" fermentation using unsterile wooden equipments, where other microbes are probably involved in the production processes. hence, various bacteria are found in tempeh, such as lactic acid bacteria (lab) and enterobacteriaceae (barus e t al. 2008; efriwati e t al. 2013; nurdini e t al. 2015). lactic acid bacteria biotropia vol. 28 no. 1,2021 are present at the beginning of the fermentation process (water soaking of soybean) until the end of the fermentation process (efriwati e t al. 2013). the population of lactic acid bacteria varies according to the stage of fermentation process. its presence is usually shown by the decrease in ph as a consequence of natural acidification, especially during the soaking processes (efriwati etal. 2013). however, recent studies showed that laru which waj used as a starter culture in wjb productions, did not contain any enterobac teriaceae nor lactic acid bacteria (nurdini e t al. 2015). bacterial community determination in tempeh was only limited to the culture dependent method (barus e t al. 2008; nurdini e t al. 2015), t-rflp (terminal restriction fragment length polymorphsms) from culturable bacteria (efriwati e t al. 2013), and arisa (amplified ribosomal intergenic sequence analysis) (seumahu e t al. 2013). for the starter culture, the method used was only culture-dependent (nurdini e t al. 2015). these approaches however, provided only limited information and, therefore, could not reflect the composition of microbial communities. to overcome this limitation, the 16s rrna gene was used to represent the microbial community in a metagenomics analysis. t h s study attempted to determine the origin of the bacterial community in tempeh ecosystem by applying the metagenomics approaches derived from cloned 16s rrnas gene sequences from high-throughput sequencing using the total 16s rrna gene. materials and methods sampling and sample for analysis the samples used in this research were obtained from wjb (warung jambu) tempeh industries (barus e t al. 2008), namely raw soybean (rs), fresh water soybean (fw), water soaked soybean (sw), pre-inoculated dehulled soybean (bi), commercial lam as starter culture (sc), and fresh tempeh (ft). each sample was taken three times from wjb tempeh production and transported in ice boxes for direct analysis. microbiological analysis one gram of sc was inoculated into 25 g sterile water-soaked soybean in a 250 ml erlenmeyer. incubation was done for 2 days before doing the serial dilution. twenty-five grams each of rs, bi, sc, ft, or 25 ml of fw and sw, were added into 225 ml 0.85% (w/v) nacl (merck, usa) for serial dilution. a 100 yl aliquot of suspension from each dilution were spread on eosin methylene blue agar (emb) (merck, usa) to determine the total count of enterobacteriaceae and on man-rogosa and sharpe agar (mrsa) (merck, usa) with 0.2% of sodium azide (plengvidhya e t al. 2007) for enumeration of lactic acid bacteria. each analysis was done in two replicates and incubated at 30 "c for 3-5 days for mesophilic bacteria? and enterobacteriaceae; and 5-7 days for lactic acid bacteria. dna extraction for metagenornics analysis another one gram of sc was inoculated into 25 g sterile soaked soybean in a 250 ml erlenmeyer and incubated in 30 "c for 48 days to make a sterile tempeh. a hundred gram of sterile tempeh, bi, and ft were homogenized separately in 300 ml of pbs (phosphate buffer saline) for 1 rnin, following the procedure described in seumahu e t al. (2012). twenty-five grams of rs sample were soaked overnight into 225 ml sterile water in the shaker. a hundred liters of fw and water-soaked rs; and 30 rnl of sw were extracted using power water microbial dna isolation-igt (mobio, usa) according to the protocol described by the manufacturers. dna products were initially visualized by electrophoresis using 1% (w/v) agarose gel for further analysis. pcr amplification for 16s rrna gene the predominant colonies from each medium and dna extracted for metagenome analysis were then amplified by using b27f and u1492r (sakai & icurosawa 2016). the pcr reaction contained the 25 pl of final solution consisting of 12.5 yl of emerald amp max pcr master mix (taicara, japan), 1 yl of 10 pmol of each primer, and 1 yl of dna extract. samples were amplified in the pcr machine with the following conditions: 94 "c for 3 min; 30 cycles of 94 "c for 30 seconds, 58 "c for 30 seconds, 72 "c for 1 min 30 seconds; and continued with 72 "c for 2 min. the pcr products (about 1,500 bp) were purified by dynamics of microbial community during tempeh fermentation radita e t al. using illustra gfx pcr dna and gel band 5 min. sequencing of the pcr products was purification kit (ge healthcare, ui<). pcr done using the custom service provided by the products of predominant colonies were bioengineering lab (japan) and fastmac (japan). sequenced by custom service provided by eurofin genomics (japan). data analysis cloned 16s rrna gene analysis the 16s rrna gene fragments obtained by pcr were cloned into pt7 blue t-vector (novagen, germany). the recombinant plasmid were transformed into eschem'chia coli dh5a (tai<ara, japan) plated onto luria-bertani (lb) plates including 100 pg/rnl ampicillin (wako) and 40 pg/rnl x-gal (tai<ara, japan). the inserted 16s rrna gene in the plasmid was amplified by pcr using 1 pl of the culture as template with the primers t7p-f and t7u-r with the following conditions; 94 "c for 3 min; 35 cycles 94 "c for 30 seconds, 51 "c for 30 seconds, 72 "c for 2 min; followed by 72 "c for 5 min (nishiyama et al. 2013; sakai & i<urosawa 201 6). raw fastq files were demultiplexed, quality filtered, and analyzed with qiime 1.6.0. the bacterial 16s rrna gene sequences clustered using the qiime subsampled reference otu pichng pipeline with uclust-reference against the greengenes 16s rrna database, were clustered at 97% pairwise identity (bokuhch & millis 201 3). shannon-wiener index (h3, simpson index @), and the equitability (e) were calculated to describe community diversity, similarity between species, and relative importance of each operational taxonomic unit (otu) withn the entire assemblages (pangastuti et al. 2010). results and discussion identification of 16s rrna gene clones culturable enterobacteriaceae and lactic all the cloned 16s rrna gene and acid bacteria predominant colonies were checked for chimeric proteobacteria, represented by enterobac sequences using the ver. 11.0.1 teriaceae, were predominant in the ft and sc software homologl: search program (japan). samples (fig. 1). enterobacteriaceae presented sequences with similarity of 98% and above in the raw material rs was 3.6 105 c f u / ~ , and were grouped together into the same p ~ y ~ ~ ~ y p ~ decreased to 2.4 x 10' cfu/g in bi after being (watanabe e f 2008). sequences were boiled twice, then reached its maximum value in submitted for blastn database7 searching for ft with 2.9 107 cfu/g klebr f i pneumoniae 6s rrna gene for (blast, were the proteobacteria in ft http://www.ncbi.nlm.nih.gov/blast/). samples, while previous study reported high-throughput sequencing analysis for the amplification of the v4 domain in bacteria 16s rrna, the primers 515f and 806r were modified to contain the miseq illumina adapter region. pcr reaction using tai<ara extaq hs (tai<ara, japan) with pcr condition consisting of an initial 94 'c for 2 rnin followed by 20 cycles of 94 "c for 30 sec, 50 "c for 30 sec, 72 "c for 30 sec, and final extension of 72 "c for 5 rnin. the pcr product was purified with ampure xp beads (belkman counter, japan) before applying the tailed-pcr using 15-index2 and 17-index1 primers. the pcr reaction consisted of an initial 94 "c for 2 min, followed by 8 cycle of 94 "c for 30 sec, 55 "c for 30 sec, 72 "c for 30 sec, and final extension of 72 "c for ~seudomonm putida and acinetobacter sp. as the predominant bacteria in the wjb tempeh (barns et al. 2008). on the other hand, lactic acid bacteria reached its maximum value in sw, lo7 cfu/g, where the natural acidification occurred with the presence of lactic acid bacteria (efriwati et al. 201 3) and in addition of 1.4 x 10' cfu/g of enterobacteriaceae. previous study reported similar number of lactic acid bacteria detected in water-soaked soybean at 6.0 x lo5 cfu/g and fresh tempeh from wjb producer at 4.3 x lo5 cfu/g (barus e t al. 2008). whereas in this current study, lactic acid bacteria were 7.7 x lo4 cfu/g in the ft sample predominantly the ldctobdci/l~~ /actis, l. fermentam, and weissella confusa. biotropla vol. 28 no. 1,2021 figure 1 number of total bacteria populations based on culture-dependent technique from wjb production notes: rs = raw soybean; fw = fresh water soybean; sw = water-soaked soybean; bi = soybean before inoculation; sc = starter culture; ft = fresh tempeh. l.e+08 l.e+07 l.e+06 l.e+05 l.e+04 i 4 lactic acid bacteria i 1 high-throughput sequencing data processing sequencing of the amplicon pool resulted in 971,785 paired-ends assembled reads after the assembly and sample index-based demulti plexing, which were reduced to 804,136 cleaned reads after being sorted for quality issues (homopolimers, length), chimera sequence, and miss amplified taxa (chloroplast, mitochondria, archaea, or unassigned kingdom) with average length of 340 bp. all of these sequences were used for taxonomy-based analysis, which consisted of kingdom, phylum, class, ordo, family, and genus level. indices regarding l.e+03 l.e+02 l.e+01 l.e+00 i diversity parameters were measured with the number of otus (operational taxonomic unit) showing generally high values, from a minimum of 303 in rs to a maximum of 2,705 in fw, thus indicating the richness of the samples fable 1). shannon-wiener index, simpson index, and equitability also showed similar patterns among the samples. fw and enriched sc had the highest values among the indices, indicating that the number of individual species in each otu was even. however, the bi samples had the lowest sirnpson index for the predominant bacteria obtained in greater number than other species. 4 enterobacteriaceae i i i table 1 number of sequences, observed diversity richness (otus), and diversity estimates of bacteria rs fw sw bi sc ft shannonsirnpson equitability samples total reads clean reads total otus wiener index index (hi) @) q notes: rs = raw soybean; fw = fresh water soybean; sw = water-soaked soybean; bi = pre-inoculated soybean; sc = starter culture ; ft: fresh tempeh. dynamics of microbial community during tempeh fermentation radita et al. bacterial communities structure from wjb production taxa present in 1% or more in at least one sample are identified and listed, while rarer taxa were grouped under the "other" label (fig. 2). hts data analysis showed that wjb tempeh was produced by only four phyla, including firmicutes, proteobacteria, actinobacteria, and bacteroidetes. the latter mentioned only showed in the fw sample, with its participants in other samples less than 1%. firmicutes and proteobacteria were the predominant and second dominant bacteria that existed in the wjb tempeh. in the raw material used for tempeh production, firmicutes was dominant in rs (93%) while proteobacteria was dominant in fw (73%) and sc (96%) with proportion from each clean reads. firmicutes were also the predominant bacteria in ft (88%) and sw (960/o), while the dehulled-soaked and pre inoculated soybean, bi, was dominated by proteobacteria (98%). firrnicutes population in ft presumably originated from rs and/or sw. lactobacillales was the predominant ordo in all samples populated by firmicutes, namely; rs, sw. and ft. while medominant orders double boiling before the inoculation of the soaked soybean, resulted in the decrease of its species richness and decline of its number (efriwati et al: 2013). rhodospirilales vastly increased as the predominant ordo detected in the bi sample with 97% abundance, while lactobacillales significantly diminished from the soaking process. however, rhodospirilales abundance was decreased in ft until 11% and the bacterial predominance shifted to lactobacilalles (88%). unlike the result shown by culturable technique, the sole ordo signifying proteobacteria in sw was rhodospirilales (1.60/0), while enterobacteriaceae occurrence itself was less than 1%. other predominant proteobacteria orders that occurred were enterobacteriales (34%) and pseudomonadales (36%) in the sc samples. as expected, neither of these orders occurred more than 1% in the final fermentation product, ft, due to its small amount of administration for tempeh fermentation. enterobacteriales, which harbored r pnezlmoniae as vitamin biz producer in tempeh (ashok et al 2012), was detected in ft with 0.05% from the entire clean reads. with 340 bp representing ~roteobacteiia varied in their average length sequence, it was not adequate number in other samples (figs. 3 & 4). with enough to identify enterobacteriales until genera 57% abundance, burkholderiales was level, unlike the other orders (humblot & guyot predominant in fw, but these microbes 2009; roh e t al 2010). thus, information about vanished once the soybeans were soaked and the presence of r pneiwzoniea in ft was only was not detected in the next step. however, the achieved by culture technique. others bacteroidetes actinobacteria firmicutes proteobacteria figure 2 phyla of bacteria present in the wjb sample analyzed with total 16s rrna sequence notes: phyla acquired in samples less than 1% was put under "others" label. rs = raw soybean; fw = fresh water; sw = water-soaked soybean; bi = pre-inoculated soybean; sc = starter culture; ft = fresh tempeh. biotropia vol. 28 no. 1,2021 100% h others 90% h xanthomonadales 80% h sphingomonadales 70% . saprospirales h rhodosp~rilalles 60% h rhodocyclales 50% h pseudomonadales 40% lactobacillales 30% h flavobacterales h enterobacteriales 20% h clostridiales 10% burkholderiales 0% a h cytophagales rs f w s w bi sc ft figure 3 major orders detected from wjb samples applied with total gene 16s rrna sequences analysis notes: major orders that occurred in less than 1 % of samples were put under "others" label. rs = raw soybean; fw = fresh water; sw = soaked water soybean; bi = pre-inoculated soybean; sc = starter culture; ft = fresh tempeh. twelve major genera from predominant orders were detected from the wjb samples (fig. 4). major lactobacillales genera attained from rs was different from sw's and ftys predominant genera. clostr;idm (43% abundance) and ldctococcgs (28% abundance) dominated the rs sample. yet, these bacteria were not detected in the next step. both sw (97%) and ft (92%) were populated by solely ldctoban'ii~~s. being the major bacteria in both samples, l6ctoban'iiu.r abundance was actually increased in ft, after vanishing in bi until 272 sequences of bi total reads (0.5%). these indicated that some ldctoban'iius were still alive during the second-boiling of soybean, thus they dominated once more in the final fermentation product. the second boiling of the soybean did not only shift the bacterial community presence in bi, but also slightly decreased its total number (efriwati e t al. 201 3), especially lact0baciii.s genera. apparently, the tempeh fermentation environment was very suited for ldctoban'iius growth, since it remarkably increased during the two days fermentation, 800 times higher than in its original number in bi. changes in bacterial community through the fermentation process was expected, since bacterial communities within ecosystem respond to the environmental conditions. the shifting of bacteria community also happened to food based fermented product, such as &chi (park e t al. 201 2) and dongchimi (jeong e t al. 201 3), and now in the soybean soaking process in tempeh production. abundance of some environmental bacteria which might have contributed to the ecology of the initial fermentation has shifted to lactic acid bacteria as affected by the accompanying ph decreases and temperature increase (yan e t al. 2013). moreover, lactic acid bacteria could produce antimicrobial compound aside from the organic acid that could eliminate pathogen and spoilage bacteria (trias et al. 201 0). predominant ordo in bi sample was solely acetobacter from rhodospirildes with 99% relative abundance. these bacteria already existed in the sw processing with 1.5% abundance. its existence was also reported when using the cultivation technique in water-soaked soybean in different tempeh production (barus et al. 2008). acetobacterwas known as acetic acid producer from alcohol substrate in the presence of excess oxygen (punvoko 2009). these bacteria also existed in the pre-inoculated dehulled-soybean without the second boiling of soybean, even though its relative abundance and number were lower, yet the species richness was higher than bi from wjb production (data not shown). this is probably because the bi from dynamics of microbial community during tempeh fermentation radita e t al. 100% pseudomonas 90% novosphingobium 80% limnohobitons 70% lactococcus 60%. lactobacillus 50% ¤ janthinobacterium 40% hydrogenophaga enterococcus 30% clostridium 20% bacillus 10% acinetobacter l . acetobacter rs fw sw bi sc ft figure 4 predominant genera attained from wjb samples, employing total 16s rrna gene analysis notes: major genera that occurred less than 1% in all samples were put under "others". rs = raw soybean; fw = fresh water; sw = water-soaked soybean; bi: soybean before inoculation; sc = starter culture; ft = fresh tempeh. wjb production was not washed with water after the second boiling process. moreover, the second boiling process used the soaking water, not the fresh water. thus, excessive ethanol from heterofermentative lactic acid fermentation in the soaking process was used as the substrate for acetobacter growth (purwoko 2009). the bacteria itself presumably originated from the environment where the tempeh production occurred. however, these bacteria later were reduced to 65% of its original number in ft sample, indicating that tempeh fermentation condition was not suited for acetobacter growth, thus consequently shifting to ldctobaciiizas. in another food fermentation process, acetobacter was responsible for the fermentation of vinegar (wu et al. 2010), nata (jagannath et al. 2010), and chocolate (papalexandratou e t al. 201 i), with different acetobacter species. pseudomanadales was the predominant bacteria in the sc (35%), while enterobac teriales was the second predominant bacteria in sc with its sole family, enterobacteriaceae (34%). due to its inaccurate identification data, enterobacteriaceae cannot be identified further until genera level, thus the predominant genera that existed in sc remained unknown. pseudomonadales was represented by acinetobacter (37%) and psezldomonas (23%) as the predominant genera in sc used by wjb. only 1 g of sc was added to 250 g of bi, thus the abundance of predominant bacteria in sc were detected in ft as less than 1%. sc contained ldctobaciiizas with 0.4% abundance. however, the involvement of ldctobaciiizas from sc to establish lactobaciiizls as predominant bacteria in ft was ignored, since its abundance in sc was less than 1%. bacterial community in the fermented product was not affected by bacterial community in the starter culture (park et al. 2012; polka e t al. 2015). most probably many of bacteria contained in starter culture did not play an ecological role in tempeh fermentation. some identified genera, such as acinetobacter was identified as contaminant in rye and wheat sourdough (ercolini e t al 2013); psezldomonas was commonly found in fermented shrimp (park et al. 20 1 i), while novospbingobizlm was frequent in chinese traditional sourdough (zhang & he 2013). cloned 16s rrna gene analysis for better assessment of the bacterial species involved in the bacterial community in tempeh products, cloned 16s rrna genes were used from the same pool of dna as previously analyzed. a total of 126 clones was obtained biotropia vol. 28 no. 1,2021 from six samples and divided into two major phyla, firmicutes and proteobacteria (table 2). the predominant genera obtained through ths method coincided with previous result (fig. 2), yet contradicted with the results of the cultivation technique (fig. 1). both sw and ft were dominated by the same species lactobacillas, l. delbmeckii and l. mzlcosae. the similar sequences probably indicated that predominant bacteria in ft probably originated from sw processes. moreover, lactobacill~~s, enterococcas and pediococczls genera were also present in the wjb tempeh analyzed using t-rflp (terminal restriction fragment length polymorphisms). but their position in the tempeh dominant bacteria were not reported (nurdini 2015). the possibility of drawbacks using pcr-based community analysis technique, such as biases from dna extraction and amplification; and the presence of amplified dead cells (yan et al. 2013; jeong e t al. 2014) were eliminated during the hts analysis. as previously mentioned, the abundance of lactoban'llzls genera in sw decreased after the second boiling of soybean, but it vastly increased again after 48 hours' incubation process in the tempeh fermentation. thus, lactoban'llzls sequences amplified by both methods were not a bias and not dead cells. knowledge on the variations in the bacterial community structures that occur during tempeh production, including the order of abundant and rare phylotypes, are important in understanding how bacterial community composition in tempeh production are affected by each step in the processes involved. in another study, the tempeh produced by wjb was preferable and had less bitter taste than the tempeh produced by other producers in bogor, was probably due to its lower bacterial number (barus et al. 2008). however, the lower bacterial number also corresponds to lower lactic acid bacteria which is an imuno-stimulant (soka et al. 2014), lower enterobacteriales which represent k. pnemoniae as vitamin bu producer (ashok e t al. 2012), and no bifidobacterales which is an isoflavone aglycone producer (raimondi et al. 2009). consequently, the double bohng of soybean was good to lessen the bitterness in tempeh, yet it also decreased the nutritive value that could be obtained from tempeh. limited information is available about bacterial community composi tion in food production, even though the diversity and composition of microbial com munities in food are thought to have a direct influence on the human guts' microbial con sortia (hehemann e t al. 201 0; park et al. 201 2). the dissimilar result occurred between cultivation techniques and metagenomics analysis in this study. most of the predominant bacteria that existed in tempeh products were not detected by the cultivation technique, particularly the lactic acid bacteria. t h s indicated that tempeh ecosystem dominated by bacterial communities could not be cultured by traditional methods, thus a bias occurred in favor of the predominant bacterial community found in tempeh. consequently, the results obtained by cultivation technique could not reflect the real microbial communities in tempeh (aslam et al. 2010). using metagenomics technique without the cultivation technique was neither preferable. metagenomics employing table 2 cloned bacterial 16s rrna sequences during tempeh production sample species similarity (%) phylum no. accsession rs clostridizlm beyerinckii 99 firmicutes kj957170 lactococzls taiwanensis 96 firmicutes nr-114327 fw acidovorax sp. 98 proteobacteria jx177703 cudacter sp. 99 proteobacteria jn679217 sw lactobaciiizls delbmeckii 9 8 firmicutes fj915697 lactobaciiizls mzlcosae 99 firmicutes jq805650 bi acetobacter indonesiensis 99 proteobacteria aj419841 acetobacter aceti 99 proteobacteria i(r261398 sc acinetobacter zlrsingii 99 proteobacteria lc014147 psezldomonas aermginosa 100 proteobacteria klj866890 ft lactobaciilzls fernenturn 98 firmicutes jn188384 lactobaciiizls delbmeckii 9 8 firmicutes fj915697 lactobaciilzls mucosae 99 firmicutes jq805650 notes: rs raw soybean; f w = fresh water; sw = water-soaked soybean; bi = pre-inoculated soybean; sc = starter culture; ft = fresh tempeh. dynamics of microbial community during tempeh fermentation radita e t ul pcr-based community analysis might be references selective for some population over others, due to the selective extraction of nucleic acids, selective amplification of 16s rrna gene, and the presence of dead cells (yan et al 2013; jeong e t al. 2014). however, up to date, metagenomics analysis employing hts was the most extensive sequencing tool to study the microbial community in food production processes, even for minor population of bacteria. metagenornics analysis of bacterial community in tempeh improves the understanding of bacterial ecology in tempeh which was limited in the classical knowledge of the tempeh ecosystem. conclusion firmicutes and proteobacteria were the predominant and second dominant bacteria that existed in the wjb fresh tempeh as indicated by the metagenomics analysis. however, the cultivation technique showing the significantly opposite results, might have under-estimated the firmicutes population. double bohng of soybean applied in the wjb products resulted in ashok s, sankaranarayanan m, ico y, jae ke, ainala sk, icumar v, park s. 2013. production of 3-hydroxypropionic acid from glycerol by recombinant klebsieiia pnegmoniae adbutayqbd which can produce vitamin b12 naturally. biotech bioeng 110(2):511-24. aslam 2, yasir m, iulaliq a, matsui i(, chung yr. 2010. mini review: too much bacteria still unculturable. biosci 1:59-60. barns t, suwanto a, wahyudi at, wijaya h. 2008. role of bacteria in tempeh bitter taste formation: microbiological and molecular biological analysis based on 16s rrna gene. microbiol indones 2:17-21. bokulich na, mills da. 2013. facility-specific "house7' rnicrobiome drives microbial landscape of artisan cheesemaking plants. appl environ microbiol 79(17):5214-23. ercolini d, pontonio e, de fillipis f, minervini f, la storia a, gobbetti m, di cagno r. 2013. microbial ecology dynamics during rye and wheat sourdough preparation. appl environ microbial 79:7827-36. efriwati, suwanto a, rahayu g, nuraida l. 2013. population dynamics of yeasts and lactic acid bacteria (lab) during tempeh production. hayati j biosci 20(2):57-64. the low number of bacteria existing in the fresh heheman jh, correc g, barbeyron t, helbert w, czjzek tempeh (ft) which was dominated by m, michel g. 2010. transfer of carbohydrate ldctobacillus. both ft and water-soaked soybean active enzymes from marine bacteria to japanese gut microbiota. nature 464:235-7. 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world j wcrobiol biotechnol 29(10):1969-74. zhang g, he g. 2013. predominant bacteria diversity in chinese tradition sourdough. j food sci 78(8):1218-23. biotropia vol. 29 no. 3, 2022: 254 262 doi: 10.11598/btb.2022.29.3.1764 254 trees physiological responses to air pollution in taman margasatwa ragunan and ui depok campus diana selvilia hamid1, ratna yuniati1,2* and afiatry putrika1,3 1department of biology, faculty of mathematics and natural sciences, universitas indonesia, depok 16424, indonesia 2reseacrh group of metabolomics and chemical ecology, universitas indonesia, depok 16424, indonesia 3research group of wildlife biology and sustainable landscape, universitas indonesia, depok 16424, indonesia received 15 june 2022/accepted 24 september 2022 abstract air pollution is a common environmental problem. planting trees can minimize the adverse effects of air pollution. plants can absorb and accumulate air pollutants through stomata. biochemical changes in the leaves will appear as a physiological response of plants to air pollution that can be known by calculating the apti (air pollution tolerance index) value. this study aimed to analyze the differences in physiological responses of five tree species in taman margasatwa ragunan (tmr) south jakarta and universitas indonesia (ui) depok campus as well as to find out the proper tree species planted in areas with high levels of air pollution. the leaves of five species (hevea brasiliensis, manilkara kauki, artocarpus heterophyllus, ficus septica, and mangifera indica) were used to examine the effect of air pollution. biochemical parameters (relative water content, leaf extract ph, total chlorophyll content, and ascorbic acid content) were observed from each species. the value of each parameter was calculated into the apti equation. h. brasiliensis, f. septica, and m. indica were categorized as moderately tolerant plants, m. kauki were included as intermediate plants, and a. heterophyllus was a sensitive plant to air pollution in both locations. the highest apti values were observed in m. indica in both locations. thus, the recommended species planted in a polluted area was m. indica. keywords: apti, ascorbic acid content, leaf extract ph, relative water content, total chlorophyll content introduction air pollution is one of the environmental problems faced by many countries in the world (shaddick et al. 2020), especially in developing countries (mannucci & franchini 2017). air pollution comes from natural factors and anthropogenic factors (humans) (susanto 2020). the use of motor vehicles is the largest contributor to pollution in the air. the use of low-quality fuel can worsen air quality in the environment (uka et al. 2019). pollutant gases produced include carbon monoxide (co), sulfur dioxide (so2), nitrogen oxides (nox), hydrocarbons, volatile organic compounds (vocs), and particulate matter (pm) (uka et al. 2019). developing countries account for 50 80% of the no2 and co gases in the air from motor vehicles (adeyanju 2018). poor air quality can cause diseases in humans, such as cough, asthma, respiratory diseases, lung cancer, to cardiovascular disease (manisalidis et al. 2020). plants can absorb and accumulate various air pollutants by absorption through stomata on the leaves (uka et al. 2019). based on research by roshintha and mangkoedihardjo (2016) mentioned that samanea saman can absorb co2 of 3,252.1 g/hour, swietenia macrophylla 3,112.43 g/hour followed by bauhinia purpurea 1,331.38 g/hour, alstonia scholaris 1,319.35 g/hour, and ficus benjamina 1,146.51 g/hour. this proves that plants can absorb and accumulate air pollutants, such as co2. plants exposed to air pollutants will show morphological, anatomical, biochemical, and physiological responses (uka et al. 2019). *corresponding author, email: ratnayuniati@sci.ui.ac.id trees physiological responses to air pollution – hamid et al. 255 changes in biochemical content in the leaves, namely rwc (relative water content), leaf extract ph, total chlorophyll content, and ascorbic acid content will occur when pollutant gases enter leaf tissue (uka et al. 2019). this happens in response to plant physiology to air pollution. the level of plants tolerance to air pollution can be determined by calculating the value of the air pollution tolerance index (apti) based on changes in biochemical content that happens in the leaves (zhang et al. 2016). plants having a good tolerance level can play a role in controlling air pollutants in the environment (ogunkunle et al. 2015). taman margasatwa ragunan (tmr) south jakarta and ui depok campus are green areas located in urban areas (taman margasatwa ragunan 2021; universitas indonesia 2021) with a high level of air pollution. however, conditions in both locations are assumed to be different. the intensity of vehicles in the ui depok campus is higher than that in the tmr. hence, the study aimed to: 1) analyze the difference in physiological responses of five tree species in tmr and ui depok campus based on the tolerance index and 2) find out the right tree species to plant in areas with high levels of air pollution. materials and methods study area the study was conducted in november 2021 located at taman margasatwa ragunan (tmr) south jakarta and ui depok campus (intersection area near the rectorate building). sampling points were depicted in figure 1. five tree species observed in this study were hevea brasiliensis, manilkara kauki, artocarpus heterophyllus, ficus septica, and mangifera indica. the coordinate points of the five tree plant species in both locations were recorded based on gps (global positioning system) (table 1). figure 1 location of sampling points in taman margasatwa ragunan (left) and ui depok campus (right) (intersection area near the rectorate building) notes: plants shown are: 1 = h. brasiliensis, 2 = m. kauki, 3 = a. heterophyllus, 4 = f. septica, and 5 = m. indica. table 1 coordinate points of five tree species at tmr and campus ui depok no. species tmr ui 1. hevea brasiliensis 06°18.385' s 106°49.165' e 06°20.923' s 106°49.609' e 2. manilkara kauki 06°18.492' s 106°49.206' e 06°21.998' s 106°49.571' e 3. artocarpus heterophyllus 06°18.602' s 106°49.158' e 06°22.017' s 106°49.628' e 4. ficus septica 06°18.658' s 106°49.142' e 06°21.996' s 106°49.580' e 5. mangifera indica 06°18.699' s 106°49.239' e 06°21.993' s 106°49.567' e biotropia vol. 29 no. 3, 2022 256 environmental parameters soil temperature, relative humidity, and soil ph were measured in november 2021 at both study locations. temperature and relative humidity were measured over the 4 weeks at both study locations starting from the first sampling activity. monthly average rainfall data were obtained from bps kota jakarta selatan (2021) for south jakarta and from the study conducted by said and widayat (2014) for depok area. soil samples were collected from 3 different points at each location. the measurement of soil ph was based on a method described by nadgórska-socha et al. (2017) using a digital ph meter with a ratio of soil and distilled water weight of 1 : 2.5. sampling and preparation leaf samples came from five species, i.e., h. brasiliensis, m. kauki, a. heterophyllus, f. septica, and m. indica. the samples came from a tree with a height of at least 1.5 m (kaur & nagpal 2017) which faced toward sunlight and roads (zhang et al. 2016). samples were collected three times from the same individual. the samples were taken to the laboratory for cleaning, and then weighed according to the required leaf weight for each test. subsequently, the samples were stored in a freezer having temperature of -20 oc until the sample were ready to be tested (zhang et al. 2016). relative water content (rwc) rwc measurement was carried out following the method described by ghafari et al. (2020) with modifications. as much as 5 g of leaves samples were soaked in distilled water for 24 hours. then, the samples were re-weighed and recorded as turgid weight. subsequently, the samples were oven-dried at 50 oc until reaching a constant weight and the weight was recorded as dry weight. the rwc values were expressed in percent and calculated based on the formula: rcw (%) = fw – dw x 100 tw – dw where: fw = fresh weight (g); tw = turgid weight (g); dw = dry weight (g). leaf extract ph the ph measurement of leaf extract was conducted following the method described by kaur and nagpal (2017) with modifications. a total of 5 g of leaves samples were extracted with 50 ml of distilled water. the extract was filtered and measured using a digital ph meter. total chlorophyll content measurement of total chlorophyll content was carried out following the method described by manjunath and reddy (2019). a total of 0.5 g of leaves samples were extracted and homogenized with 10 ml of 80% acetone. the extract was centrifuged at 2,500 rpm for 3 minutes. supernatant volumes were measured and absorbed at wavelengths of 663 nm and 645 nm using uv-visible spectrophotometers. the total chlorophyll content was calculated based on formula as follows (bharti et al. 2018): chlorophyll a (mg/g) = 12.7 (a663) 2.69 (a645) x v x w 1,000 chlorophyll b (mg/g) = 22.9 (a645) 4.68 (a663) x v x w 1,000 total chlorophyll (mg/g) = chlorophyll a + chlorophyll b where: a663 = absorbance at wavelength 663 nm; a645 = absorbance at wavelength 645 nm; v = supernatant volume (ml); w = sample weight (g). ascorbic acid content measurement of ascorbic acid content were carried out following the method described by patel and kumar (2018) using titration. the leaves samples solution was extracted with 100 ml of 4% oxalic acid. the extract was centrifuged at 2,500 rpm for 3 minutes. the sample solution and blanko were titrated using a dye solution until the color turned pink. the ascorbic acid content was calculated based on the formula: ascorbic acid (mg/100 g sample) = 0.5 mg x v2 ml x 100 ml x 100 v1 ml 5 ml w where: w = sample weight (g); v1 = volume of blanko solution; v2 = volume of sample solution. trees physiological responses to air pollution – hamid et al. 257 air pollution tolerance index (apti) the value of each parameter is calculated into an equation described by zhang et al. (2016): apti = a (t + p) + r 10 where: a = ascorbic acid content (mg/100g sample); t = total chlorophyll content (mg/g); p = leaf extract ph; r = relative water content (rwc). the values obtained are categorized based on the category of plant responses described by sahu et al. (2020): a) apti < mean apti – sd : sensitive (s) b) mean apti – sd < apti < mean apti : intermediate (i) c) mean apti < apti < mean apti + sd : moderately tolerant (mt) d) apti > mean apti + sd : tolerant (t) air pollution data collection the daily average of air pollutants (pm2.5, pm10, co, hc, no2, o3, and so2) in both study locations (tmr and ui depok campus) was obtained from the ispunet klhk ver 1.4.5 application. the data were recorded in the morning at 7 am, noon at 12 pm, and in the afternoon at 5 pm. data were collected from early october to the end of november 2021. the established range of ispu (air pollutant standard index) values is as follows: a) good: 1 50, b) medium: 51 100, c) unhealthy: 101 200, d) very unhealthy: 201 300, e) dangerous: > 301 (kementerian lingkungan hidup dan kehutanan republik indonesia 2020). data analysis data were analyzed descriptively. environmental and biochemical parameters as well as apti values were calculated for the average values. the average values of those parameters at the two study locations were then compared. data were presented in the form of tables and bar charts. results and discussion the average air temperature in ui depok campus was relatively higher than that in tmr (table 2). both study locations are green areas that have similar environmental conditions. higher air temperatures can reduce the humidity (utami et al. 2020). our study found out that the average air temperature in tmr was lower with higher relative humidity than that in ui depok campus. within the period of our study, the average rainfall in south jakarta was 235.96 mm per month (bps kota jakarta selatan 2021) and in depok was 278 mm per month (said & widayat 2014). table 2 comparison of environmental parameters in tmr and ui depok campus in november 2021 no. environmental parameters tmr ui depok campus 1. average temperature (c) 30.350.53 30.750.64 2. average relative humidity (%) 57.503.51 55.503.11 3. average soil ph 7.450.77 7.210.88 based on the ispu data, the range of apti value for all types of pollutants was 0.00 80.72 (fig. 2). the lowest index was observed on hc in depok (0.00) and the highest on pm2.5 in jakarta (80.72). both were observed in october 2021. in october 2021, the pm2.5, co, hc, no2, and o3 indices were observed to be higher in jakarta, while so2 was higher in depok. meanwhile, similar indices in both regions were observed in pm10. furthermore, in november 2021, the pm2.5, pm10, no2, o3, and so2 indices were observed higher in depok, while co and hc remained high in jakarta. the high so2 and pm2.5 indices in depok was presumably due to the high level of vehicle traffic on the highway, especially during peak hours. visibility will decrease along with the increased levels of pm2.5 and pm10 in the air (kementerian lingkungan hidup dan kehutanan republik indonesia 2021) because the air is filled with fine dust that can be inhaled by the respiratory system. the main sources of pm2.5 and pm10 pollutants come from combustion activities, such as the use of vehicles, construction activities, up to coal-fired power plants (haryanto et al. 2016). biotropia vol. 29 no. 3, 2022 258 figure 2 average air pollutant index in jakarta and depok in october and november 2021 h. brasiliensis, m. kauki, f. septica, and m. indica are the three tree species which have higher scores of rwc in the ui depok campus than that in tmr (fig. 3a). meanwhile, the rwc value of a. heterophyllus in both locations had the similar lowest rwc values. water plays a role in maintaining the physiological balance of plants under environmental stress. humidity and temperature in the environment affect the rwc values of the leaves. rwc values tend to decrease in plants that are exposed to high temperature and drought environment (rowshanaie et al. 2014; zhang et al. 2016). meanwhile, tolerant plants usually have a high rwc value (bahadoran et al. 2019). rwc values are related to plant tolerance levels to air pollution. the high rwc value of a species indicates that plants have a good tolerance to air pollution (zhang et al. 2016; kaur & nagpal 2017). based on rwc values in our study, m. indica, f. septica, and h. brasiliensis are more tolerant to air pollution. meanwhile, the lowest rwc values were observed in a. heterophyllus at both locations. this is presumably because a. heterophyllus cannot adapt well to an environment that tends to be dry (centre for agriculture and bioscience international 2019). leaf extract ph of the five species in ui depok campus was higher than that in tmr (fig. 3b). leaf extract ph is related to the sensitivity of plants to air pollution (kaur & nagpal 2017). plants exposed to air pollutants, especially so2, tend to produce a large amount of h+ cellular fluid. the h+ will react with so2 to form h2so4 which cause a decrease in leaf extract ph. the high leaf extract ph indicates that the plant can well absorb so2 and nox (zhang et al. 2016). trees having low leaf extract ph values indicate that the trees are more sensitive to air pollution compared to those having high leaf extract ph, which are more tolerant to air pollution (bahadoran et al. 2019; uka et al. 2019). f. septica and h. brasiliensis in our study have high leaf extract ph value compared to other species and are thought to be more tolerant to air pollution. the total chlorophyll content of three species namely h. brasiliensis, f. septica, and m. indica was observed to be higher in tmr than in ui depok campus. on the other hand, m. kauki and a. heterophyllus have higher total chlorophyll in ui depok campus than that in tmr (fig. 3c). chlorophyll is the main component of green coloring in plants (kaur & nagpal 2017). air pollutants entering the leaf tissue through the stomata cause chlorophyll to degrade. so2 in the air affects the total chlorophyll of the leaves. high concentration of so2 cause a decrease in total chlorophyll content (zhang et al. 2016). total chlorophyll in the leaves is also affected by high temperatures, dry environments, salt stress, and light intensity (zhang et al. 2016). other factors, such as plant species and leaf age, also affect total chlorophyll content (kaur & nagpal 2017). the observed higher so2 index value in depok area is suspected to be the cause of the low total chlorophyll content in tree species in ui depok campus. the higher average air temperature, trees position, and high light intensity are also suspected to be the cause of the low total chlorophyll content. meanwhile, m. kauki and a. heterophyllus showed lower total chlorophyll content in tmr, which is attributed to the location of trees that are more exposed to sunlight, thus having high light intensity. october 2021 november 2021 90.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 80.00 70.00 60.00 50.00 40.00 30.00 20.00 10.00 0.00 trees physiological responses to air pollution – hamid et al. 259 the ascorbic acid content of h. brasiliensis, m. kauki, and a. heterophyllus was higher in tmr. meanwhile, f. septica and m. indica in ui depok campus had a higher ascorbic acid content than that in tmr (fig. 3d). ascorbic acid is an antioxidant that plays an important role in maintaining the stability of cell division and cell membranes while in environmental stress. the ascorbic acid plays an important role in the synthesis of the cell wall. ascorbic acid in plants is related to plants responses to environmental stress such as air pollution, heavy metals, drought, and high temperatures (gallie 2013; zhang et al. 2016). high ascorbic acid content indicates a good tolerance to so2 in the air (uka et al. 2019). plants having high ascorbic acid content are more tolerant to air pollution compared to those having low ascorbic acid content (zhang et al. 2016). m. indica and f. septica in ui depok campus had a higher ascorbic acid content than those in tmr, which indicated that both species had a good tolerance to so2 gas that was observed to be higher in depok. h. brasiliensis in tmr was also observed to have the highest ascorbic acid content compared to other species, which indicated that the three tree species (m. indica, f. septica, and h. brasiliensis) were more tolerant plants. air pollution tolerance index (apti) values illustrate the level of plants tolerance to air pollution. based on our study, the apti values of h. brasiliensis, m. kauki, f. septica, and m. indica in ui depok campus were higher than that in tmr. meanwhile, apti value of a. heterophyllus were higher in tmr compared to that in ui depok campus. based on the categories of plant responses to air pollution described by sahu et al. (2020), each species has the same responses in both locations (table 3). the higher the apti value, the higher the tolerance of plant to air pollution. plants that are sensitive to air pollution tend to have low values of rwc, leaf extract ph, and ascorbic acid content. meanwhile, plants that are more tolerant of air pollution tend to have high values of rwc, leaf extract ph, and ascorbic acid content. there is a link between the leaf extract ph and ascorbic acid content. the high leaf extract ph increases the efficiency of converting hexose sugar into ascorbic acid which leads to an increase in the ascorbic acid content (bakiyaraj & ayyappan 2014). conversely, the low leaf extract ph decreases the efficiency of converting hexose sugar to ascorbic acid, so the ascorbic acid content tends to be low (kaur & nagpal 2017). figure 3 biochemical parameters of five tree species in tmr and ui depok campus notes: a = relative water content; b = leaf extract ph; c = total chlorophyll; and d = ascorbic acid. a c d b biotropia vol. 29 no. 3, 2022 260 table 3 biochemical parameters, apti values, and response categories of five tree species in tmr and ui depok campus species rwc (%) leaf extract ph total chlorophyll (mg/g) ascorbic acid (mg/100g sample) apti response categories tmr ui tmr ui tmr ui tmr ui tmr ui tmr ui hevea brasiliensis 93.450.58 98.130.57 7.040.41 7.100.32 1.380.03 1.290.04 0.290.00 0.100.00 9.590.50 9.890.57 mt mt manilkara kauki 85.111.33 88.612.68 5.800.20 5.840.07 1.000.09 1.070.14 0.170.03 0.100.00 8.620.15 8.930.27 i i artocarpus heterophyllus 72.664.35 72.224.12 6.300.29 6.420.18 1.360.01 1.400.02 0.120.03 0.100.00 7.350.43 7.300.41 s s ficus septica 95.521.10 96.380.38 7.310.00 7.330.01 1.120.04 1.100.07 0.080.03 0.100.00 9.620.12 9.720.04 mt mt mangifera indica 97.510.61 98.390.27 5.860.18 5.920.15 1.130.02 0.970.05 0.230.02 0.260.03 9.910.06 10.020.02 mt mt notes: mean (n = 3); s = sensitive, i = intermediate, mt = moderately tolerant, t = tolerant. plants tolerance levels studied in tmr can be sequenced as follows: m. indica > f. septica > h. brasiliensis > m. kauki > a. heterophyllus. meanwhile, the sequence of tolerance level in ui depok campus is as follows: m. indica > h. brasiliensis > f. septica > m. kauki > a. heterophyllus. in addition, the study results showed that apti values in tmr and ui depok campus had similar values, which was presumably due to the current condition of the covid-19 pandemic. during the covid-19 pandemi, the number of vehicles passing through the ui depok campus area become fewer, so the pollutants produced are also less compared to the conditions before the covid-19 pandemic. the environmental conditions in both locations tend to be the same. plants having low apti values can act as environmental bioindicators, as they are more sensitive to air pollution. meanwhile, plants having higher apti values can be used as bioaccumulators of air pollution because they have a good ability to absorb and accumulate various pollutants scattered in the air through leaf stomata. plants which are tolerant and moderately tolerant to air pollution can be grown in environments having high levels of air pollution, such as in urban areas, areas with heavy traffic, and industrial areas (kaur & nagpal 2017; uka et al. 2019). a. heterophyllus has the lowest apti values in both locations and is categorized as a sensitive plant to air pollution. the plant has a high total chlorophyll content and low leaf extract ph as well as ascorbic acid content. meanwhile, m. indica has the highest apti values in both locations. the total chlorophyll content and leaf extract ph in m. indica is quite low when compared to other species. however, m. indica has a high ascorbic acid content and the highest rwc value compared to other species. therefore, m. indica is the best plant to be grown in areas with high levels of air pollution. other than that, m. indica has a large tree stature, a large and wide canopy, and a large number of leaves (febrianti & sulistyantara 2020). m. indica is also a native plant of indonesia (centre for agriculture and bioscience international 2022) and resistant to strong winds, so it is not easily uprooted (plants for a future 2022). f. septica and h. brasiliensis can be the alternative choices because these two tree species have moderate level of tolerance as m. indica to air pollution. conclusion h. brasiliensis, m. kauki, a. heterophyllus, f. septica, and m. indica had the same responses in taman margasatwa ragunan south jakarta and ui depok campus. h. brasiliensis, f. septica, and m. indica are categorized as moderately tolerant, m. kauki includes intermediate plant, and a. heterophyllus is a sensitive plant to air pollution based on air pollution tolerant index (apti) values. based on apti values and physical characteristics of the tree, the right tree species to plant in an area with high levels of air pollution is m. indica. acknowledgments we would like to thank hibah publikasi terindeks internasional (puti) prosiding 2020 no.: nkb-3584/un2.rst/hkp.05.00/2020 for financial support provided during this research. trees physiological responses to air pollution – hamid et al. 261 references adeyanju aa. 2018. effects of vehicular emissions on human health. j clean energy technol 6(6): 411-20. bahadoran m, mortazavi sn, hajizadeh y. 2019. evaluation of anticipated performance index, biochemical, and physiological parameters of cupressus arizonica greene and juniperus excelsa bieb for greenbelt development and biomonitoring of air pollution. int j phytoremediation 21(5): 496-502. bakiyaraj r, ayyappan d. 2014. air pollution tolerance index of some terrestrial plants around an industrial area. int j mod res rev 2(1): 1-7. bharti sk, trivedi a, kumar n. 2018. air pollution tolerance index of plants growing near an industrial site. urban clim 24: 820-9. bps kota jakarta selatan. 2021. kota jakarta selatan dalam angka 2021 [south jakarta in numbers 2021]. jakarta (id): bps kota jakarta selatan. centre for agriculture and bioscience international [internet]. 2019. artocarpus heterophyllus (jackfruit). wallingford, uk: centre for agriculture and bioscience international; 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present address: faculty of agriculture, tokyo university of agriculture, kanagawa 243-0034, japan received 06 may 2016/accepted 17 july 2017 abstract amorphophallus variabilis blume, a member of araceae, is a fleshy perennial tuber crop endemic in java island, indonesia. the plant produces white edible corm; and it was used as food during famine time before 1960s. rapid ecological changes and land fragmentations in java in recent times threaten populations of a. variabilis. here, compound microsatellite markers were developed in order to develop conservation strategies in the populations. twelve primers pairs produced high polymorphism ranging from 5 to 22 alleles per locus. the observed and expected heterozygosities ranged from 0.191 to 0.851 and 0.380 to 0.943, respectively. this high allelic diversity indicates that these markers are suitable for the study on population genetic structure. cross-amplification on related and nonrelated species was performed. application of the markers on populations from dramaga conservation forest revealed high allelic richness, high diversity within and among populations. genetic distance among populations increased with an increase of geographic distance. present study suggested that, it is important to study population of a. variabilis in java in order to understand the population genetic structure and develop effective in situ conservation programs. keywords: araceae, genetic population, ssr, tuber crop, white iles-iles introduction amorphophallus variabilis blume, synonym brachyspatha variabilis (blume) schott, an araceae, is a diploid endemic perennial tuber crop native to java. its distribution in indonesia is exclusively in java, kangean and madura islands (jansen . et al 1996; yuzammi 2000). the plant naturally grows in less disturbed land under partial trees shading and at gap of forest trees at low altitude up to 700 to 900 m above sea level (jansen . 1996; et al sugiyama & santosa 2008). is locally a. variabilis called white (safii 1981; wiyani 1988) iles-iles reflecting the white color of the tuber. it is easily d i s t i n g u i s h e d m o r p h o l o g i c a l l y f r o m amorphophallus muelleri blume (sugiyama & santosa 2008) known as yellow iles-iles. the plant produces a single underground corm with several cormlets; corms and cormlets exhibit dormancy during dry season (jansen . et al 1996). the corm was utilized as staple food in java particularly during famine situation before 1960s. corm of contains high glucomannan a. variabilis (ca. 35% on a dry weight basis) (ohtsuki 1968; safii 1981; wiyani 1988). glucomannan is used as an important material in beverage, food and pharmaceutical industries (jansen . 1996; et al alonso-sande . 2009).et al botanically, a large genetic diversity has been observed within a. variabilis plants, including inflorescence (santosa et al. 2004) and leaf morphologies (sugiyama & santosa 2008). according to yuzammi (2000), the petioles vary from pure green to dark brown with different color and pattern of spots. therefore, a. variabilis * corresponding author: edisang@gmail.com biotropia 5 1 8 22 32 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.1.652 22 germplasm could be used as breeding materials (sugiyama & santosa 2008). according to zhang et al. (1998), several amorphophallus species could be hybridized. the monoecious unisexual inflorescence produces unpleasant odor during anthesis (kite & hetterschieid 1997) to attract pollinators. red bright mature berries develop after crosspollination by nitidulidae insects (santosa . et al 2004). seeds are dispersed long distance by birds and clonally propagules i.e. cormlets were dispersed nearby mother plants (sugiyama & santosa 2008). thus, in an undisturbed population, both ramets and genets co-exist. recently, the sliced corms of are a. variabilis used as feeds for swine (santosa . 2004; et al sugiyama & santosa 2008). people collect the corms without considering the conservation. on the other hand, land fragmentations and agricultural intensifications in agroforestry system for cash crops (santosa . 2005) disturbed the et al populations. consequently, the populations of a. variabilis in java are in danger. high exploitation has been known causing population decline and loss of genetic diversity in many species (leimena et al. et al. 2007; nuryanto & susanto 2010; santosa 2010). therefore, in order to develop effective program on genetic conservation of , it a. variabilis is important to evaluate population size using reliable genetic markers, such as microsatellite. microsatellite or simple sequence repeat (ssr) is widely used to develop conservation program in many species (abdul-muneer 2014; nachimuthu et al. 2015). in this study, we developed microsatellite of using compound a. variabilis isolation method for the first time. many methods have been developed for microsatell ite isolations e.g. based on sequence data (lian . 2001; fatimah & sukma et al 2011), cloning, enrichment library and dualsuppression methods (lian & hogetsu 2002; lian . 2006). in the present study, et al compound microsatellites are isolated according to procedure of lian (2006). the objective et al. of this study was to develop microsatellite markers from and its application for a. variabilis genetic population study. materials and methods plant material plant materials were collected separately for microsatellite development and population study i.e. from bogor botanical garden (bbg) and dramaga conservation forest (dcf), respectively with permission from the authorities. according to yuzammi (2000), bogor area including the sampling sites, is the center of a. variabilis diversity. to develop microsatellite markers, 2 5 g fresh and healthy leaves of were collected a. variabilis during rainy season in 2005 at the bbg, bogor, west java province, indonesia. the leaves were cleaned using moist tissue paper and put into a plastic bag containing silica gel, then stored at -30 oc until being used in genomic dna extraction. microsatellite development was conducted in the laboratory of forestry, asian natural environmental science center, university of tokyo, japan from 2009 to 2010. for population study, accessions a. variabilis were sampled and characterized in the dcf (244 m asl), bogor, west java province, indonesia from 2010 to 2011. dcf is managed by the indonesia ministry of environment and forestry. around 40 of the 60 ha dcf area is dedicated for conservation forest, locally known as the cifor forest (cifor = center for international forestry research). dcf is located about 8 km northwest of bogor botanical garden, 1 km west of situ gede lake and 1 km north of cisadane river. three populations were selected for this study, i.e. a (-6.5509798, 106.7497507,17z), b (6 .5519391,106.7494932,17z) and c (6.5548596,106.7507271,17z) (fig.1). in each population, plants were sampled from an area of about 0.5 to 1 ha. all plants having pseudo stems thicker than 2 cm at 10 cm above soil surface and were spaced more than 1 m apart were collected. plants spaced less than 1 m apart were considered as a ramet (similar genet) and not sampled. petiole colors of accessions were characterized (table 1). initially, 35, 25 and 38 samples were collected, and after optimizing the dna results a set of 11, 21 and 15 23 isolating microsatellite from amorphophallus variabilis and its application santosa et al. (novagen, usa) according to the manufacturer's instructions. the positive clones were amplified using the u19 and m13 reverse primers, and then sequenced using a thermo sequenase pre-mixed cycle sequencing kit (amersham biosciences) plus the t7 or u19 primer labeled with texas red (sigma-aldrich) on sq-5500e sequencer (hitachi, tokyo). a primer (ip1) was designed from the sequenced region flanking to the ssr. the ip1 and corresponding microsatellite primers were used as the marker. in addition to microsatellites developed from a. variabilis, twenty microsatellite markers which were developed from by santosa a. paeoniifolius et al. (2007) and stored in genbank were evaluated for its suitability toward genotyping. a. variabilis in order to extend usefulness of the microsatellite markers, the developed markers from a. variabilis were evaluated on several araceae members and other root crops species. pcr amplification and detection pcr reaction was carried out in a reaction mixture (10 µl) containing 5 10 ng dna, 0.2 mm of each dntp, 1 pcr buffer (mg free, 2+ accessions were used for further analysis from a, b and c populations, respectively. the other samples were excluded from analysis because they failed to amplify, or produced multiple and unclear bands. microsatellite development a modified cetytrimethyl ammonium bromide (ctab) method was adopted for conducting genomic dna extraction (zhou . 1999). et al dna from 1 g dry leaves was dissolved in a final volume of 200 µl water and stored in -30 c until o use. isolation of codominant compound microsatellite markers was performed according to the method of lian . (2006). in brief, the et al hae iii blunt-end restriction enzyme was used to digest dna sample. the digested dna was then ligated to an adaptor using a dna ligation kit (takara shuzo, japan). fragments flanked by a microsatellite region at one end were amplified from the constructed dna library using ssr primers, (ac) (ag) or (tc) (ac) and an adaptor 6 5 6 5 primer, ap2 (5´–ctatagggcacgcgt ggt–3´). the pcr products were subcloned using a pt7 blue perfectly blunt cloning kit biotropia vol. 25 no. 1, 2018 24 a10 a9 a8 a12 a11 a1 a3 a4 a6 a5 a2 pop. a b2 b3 b6 b7 b8 b4 b5 b9 b10 b11 b15 b18 b17 b19 b25 b26b27 b21 b20 b22 b24 b23 b28 b16 b12 b13 b14 pop. b c1 c13 c14 c15 c16 c18 c12 c7 c11 c8 c10 c6 c4 c20 c21 c22 c17 c19 pop. c indonesia java n cluster i cluster ii cluster iii mt salak bbg dcf bogor area 5 m 5 m 5 m road 2 km figure 1 site and accession positions within populations in dramaga conservation forest (dcf), bogor, indonesia (note: lines represent distance and visibility between two accessions; accessions without lines represent blockage by dense trees; color represents cluster membership; bar represents 5 meter) applied biosystems), 2.5 mm mgcl , 0.25 u of 2 ampli gold (applied biosystems, usa), 5 taq 0. μm of each ip1 primer and the corresponding ssr primer (labeled with texas red). the pcr thermal cycler (applied biosystems) were used with cycling profile: 9 minutes at 94 ºc, followed by 40 cycles of 30 seconds at 94 ºc, 30 seconds at the locus-specific annealing temperature (table 2) and 1 minute at 72 ºc, and finally a 5 minutes extension at 72 ºc. the pcr products were electrophoresed on a 6% polyacrylamide gel using an sq-5500e sequencer, and then analyzed with fraglys ver. 3 software (hitachi, tokyo). data analysis characteristics of microsatellite markers developed in the present study were tested on a. variabilis across all populations (n = 47). two bands from an individual were considered as different alleles if the differences in molecular weight were bigger than 3 base pair (bp) for the microsatellite containing trinucleotide repeat and 2 bp for the microsatellite containing dinucleotide repeat; these bp different are matter of technical procedure not related to nucleiotide repeat motif (santosa 2017). the numbers of alleles, observed (h ) and expected heterozygosities (h ) and o e polymorphic information content (pic) were calculated using cervus ver. 3.0.3 software (marshall et al. 1998). hardy-weinberg equilibrium (hwe) and linkage disequilibria between loci were tested using genepop version 4.0 software on the web (rousset 2008). presence of null allele was estimated using cervus ver. 3.0.3 software (marshall et al. 1998). number of migration (nm) was estimated using genalex software. population code petiole color population code petiole color a a1 pure green b b20 brown with small white spot a a2 pure green b b21 brown with large white and green spots a a3 brown with white spot b b22 brown with large light green spot a a4 green with brown spot b b23 brown with white spot a a5 green with white spot b b25 green with large white spot a a6 green with brown spot b b26 dark brown-black with white spot a a8 pure green b b27 brown with small white spot a a9 brown with black spot b b28 brown with small dark brown spot a a10 brown with dark brown spot c c1 pure green a a11 brown with black spot c c4 brown with white spot a a12 dark green with brown spot c c6 green with grey spot b b2 grey with brown spot c c7 light green with brown spot b b3 brown with green spot c c8 brown with pink spot b b4 grey with green spot c c10 pure green b b5 brown with green spot c c11 light green with grey spot b b6 brown with white spot c c12 pure green b b7 green with small white spot c c13 brown with white and black spot b b8 light green with black and white spot c c14 brown with light brown spot b b9 light green c c16 brown b b10 brown with brown and black spot c c17 dark brown with pink spot b b11 pure green c c20 pure green b b14 light brown with white spot c c21 dark brown with pink and black spot b b16 dark green with white spot c c22 light green with black spot b b18 brown with black spot table 1 characterization of a. variabilis accessions obtained from dramaga conservation forest, bogor, indonesia note: large spot = spot width 10mm> small spot = spot width 2.5mm< otherwise mentioned = spot size was medium spot was measured from 10 cm above soil surface to middle petiole length 25 isolating microsatellite from amorphophallus variabilis and its application santosa et al. analysis of molecular variation (amova) within (f ) and among population (f ) was is st determined from a, b and c populations separately using genalex software in 999 permutations. cluster analysis was performed using ntsyst spc 2.11p (exeter software, setauket usa). presence of allele at each locus was coded in binary form 1 (presence) or 0 (absence). individual across populations was clustered in upgma dendrogram using jaccard similarity coefficient. results and discussion polymorphism test from twenty-two microsatellite loci isolated from a. variabilis, twelve loci were polymorphic and codominant. polymorphic information content (pic) ranged from 0.351 to 0.932 (table 2) . the obser ved (h ) and expected o heterozygosities (h ) ranged from 0.191 to 0.851 e and from 0.380 to 0.946, respectively. the full length of amplified region from a. variabilis microsatellite had been deposited at genbank for public access (www.ncbi.nlm.nih.gov/genbank). two of 12 loci isolated from a. variabilis, i.e. avar01 and avar07, could deviate from hwe proportion (p 0.001) by having an excess of heterozygosities (table 2). linkage disequilibria analysis indicated that the loci had high linkages (p ≤ 0.001), i.e. between avar02 and avar04, avar04 and avar08, avar07 and avar10, and avar07 and ampa10. excluding loci avar04 and avar07, the other 10 loci could be used in population genetic study. according to li et al. (2009), a set of 10 15 or more loci was desirable for genetic population study. furthermore, two microsatellite primers from a. paeoniifolius, i.e. ampa10 and ampa15, produced clear bands in a. variabilis accessions. therefore, in total, fourteen polymorphic microsatellite loci could be used for a. variabilis genotyping. this study is the first work on the development of polymorphic codominant microsatellite markers in . microsatellite markers a. variabilis developed in the present study could be used to identify the polymorphism based on the standards proposed by lian . (2006), i.e. clearness of et al bands, common annealing temperature and different allele sizes. in the present study, annealing temperature was set at 58 and 62 c. o allele sizes among several loci had large differences in base pair size (table 2). therefore, loci avar01 and avar09, avar05 and avar06, and avar03 and avar10 could be mixed together in pcr reaction, to speed up pcr preparation and allele analysis. the loci developed from cross-a. variabilis amplified in other species (table 3), indicating that the loci could provide a useful tool for genotyping in other species. indeed, microsatellite primers are well known for its species-specific (lian & hogetsu 2002; csencsics et al. 2010), however, cross-amplifications sometimes exist (santosa . 2007; fatimah & et al sukma 2011). successful cross-amplification is probably due to the presence of homologous loci among them. santosa (2007) have reported et al. that microsatellites isolated by dual-suppression from amplified in related species, a. paeoniifolius while fatimah sukma (2011) reported that and microsatellite markers obtained from sequence data could amplify across genus. phalaenopsis however, amplification pcr products did not mean produce polymorphic allele in present study. it needs further investigation on the usefulness of the developed loci for genotyping other speciesa. variabilis . population and genetic diversity of dcf the average numbers of alleles per locus was 6.5 for 14 loci. the numbers of alleles per locus varied from 3 to 11 in population a, from 3 to 16 in population b and from 3 to 14 in population c (table 4). locus ampa15 produced the smallest number of alleles, whereas locus ampa10 produced the largest number of alleles across dcf populations. several accessions failed to amplify, i.e. locus avar02 for a12 and c20 accessions, locus avar04 for c6 and c10 accessions, locus avar06 for b9 and b11 accessions, locus avar11 for b23 and c17 accessions, locus ampa10 for c13 accession and locus ampa15 for a8 and c7 accessions. these accessions probably had null allele for particular loci. point mutation in the primer annealing sites may lead to the occurrence of null alleles causing the primer failed to amplify (lian et al. 2006). 26 biotropia vol. 25 no. 1, 2018 t ab le 2 c h ar ac te ri st ic s o f tw el ve m ic ro sa te lli te m ar ke rs is o la te d fr o m a . v ar ia bi lis l o cu s g en b an kz r ep ea t m o ti f p ri m er s eq ue n ce 5 ’ t o 3 ’ t a ( °c ) n a a lle le ( b p ) h o h e p ic a va r0 1* m f 52 72 50 (c t ) 6 (g t ) 7 f : c t t g t t c g g a c c a c c t t c t t g a c a a t c r : ( a c )7 (a g )3 62 5 12 013 0 0. 48 9 0. 54 8 0. 49 2 a va r0 2 m f 52 72 51 (c t ) 1 2( g t ) 7 f : c t c a a a a t c g a a t c t t c t c c a t t t t a c r : ( a c )7 (a g )3 62 14 12 415 4 0. 33 3 0. 87 7 0. 85 4 a va r0 3 m f 52 72 52 (c t ) 8 ..( c t t ) 1 1. .( c t ) 5 (g t ) 7 f : c a c t c t t c c a c a c t c c c c c t g t t a c a c r : ( a c )7 (a g )3 62 5 12 814 0 0. 21 3 0. 38 0 0. 35 1 a va r0 4 m f 52 72 53 (c t ) 1 3. ... (c t ) 4 (g t ) 7 f : c t t c c c t a t g c a g g t g a g t c r : ( a c )7 (a g )3 58 8 11 315 5 0. 33 3 0. 42 7 0. 39 6 a va r0 5 m f 52 72 54 (g t ) 5 (g a ) 7 f : g t g a a g g a g g t g g g c g t t t t g r : ( t c )7 (a c )3 58 5 17 718 7 0. 46 8 0. 52 0 0. 46 0 a va r0 6 m f 52 72 55 (g t ) 1 0( g a ) 7 f : c t a a c g a c t a a g g a c t t a a g c r : ( t c )7 (a c )3 58 22 63 -1 41 0. 51 1 0. 94 3 0. 92 8 a va r0 7* m f 52 72 56 (g t ) 1 0( g a ) 7 f : c g c t g a t g t a c t t g t t g a c a t t g r : ( t c )7 (a c )3 58 11 13 917 1 0. 72 3 0. 76 1 0. 71 9 a va r0 8 m f 52 72 57 (g t ) 8 (g a ) 7 f : c a t g g t c c a t g g g t t t a g c t t t g c r : ( t c )7 (a c )3 62 20 16 623 6 0. 74 5 0. 93 3 0. 91 8 a va r0 9 m f 52 72 58 (g t ) 5 (g a ) 7 f : g g t g t a c g a c t a g a g t t t t g t c g r : ( t c )7 (a c )3 62 7 17 518 3 0. 42 6 0. 67 6 0. 62 0 a va r1 0 m f 52 72 59 (g t ) 8 (g a ) 7 f : g a t g t c a t t c t c c g c c a c c g a g t a g r : ( t c )7 (a c )3 62 10 19 622 8 0. 85 1 0. 76 9 0. 72 6 a va r1 1 m f 52 72 60 (g t ) 6 (g a ) 7 f : g a t a c t g c t a t a c c g a g t g t c c t a t g r : ( t c )7 (a c )3 62 7 15 318 5 0. 20 0 0. 65 5 0. 60 7 a va r1 2 m f 52 72 61 (t c ) 8 g (c t ) 2 (g t ) 5 (c t ) 5 (g t ) 7 f : g g a a c a c t a g c g a g t a c a a t g t a t c r : ( a c )7 (a g )3 62 5 13 514 9 0. 19 1 0. 78 6 0. 74 1 n o te : = g en b an k re p o si to ry c o de o f p ri m er s an d fr ag m en ts s eq ue n ce z * = s ig n if ic an t de vi at io n f ro m h ar dy –w ei n b er g e qu ili b ri um ( < 0 .0 1) p t = a n n ea lin g te m p er at ur e a n a = n um b er o f al le le h o = o b se rv ed h et er o zy go si ty h e = e xp ec te d h et er o zy go si ty p ic = p o ly m o rp h ic i n fo rm at io n c o n te n t e ac h r ev er se p ri m er ( r ) w as t ai le d w it h a n a dd it io n al 1 9 nu cl eo ti de ( u 19 ) to t h e 5' e n d 27 isolating microsatellite from amorphophallus variabilis and its application santosa et al. 28 t ab le 3 c ro ss -s p ec ie s am p lif ic at io n o f tw el ve m ic ro sa te lli te p ri m er s de ve lo p ed f ro m a . v ar ia bi lis n o sp ec ie sz f am ily o ri gi n o f th e sp ec im en l o ci a v a r 01 02 03 04 05 06 07 08 09 10 11 12 1 a gl ao ne m a pi ct um ( r ox b. ) k un th . a ra ce ae b b g + + + + + + + + + + + + + + + + + + 2 a lo ca si a al ba s ch o tt . a ra ce ae b b g + + + + + + + + + + + + + + + + + + 3 a nt hu ri um s p p. a ra ce ae b b g + + + + + + + + + + + + + + + 4 a m or ph op ha llu s ko nj ac k . k o ch a ra ce ae t an as h i f ar m , u t + + + + + + + + + + + + + + + + + + + + 5 a m or ph op ha llu s m ue lle ri b lu m e a ra ce ae ip b + + + + + + + + + + + + + + + + + 6 a m or ph op ha llu s pa eo ni ifo liu s (d en n st .) n ic o ls o n a ra ce ae k un in ga n , i n do n es ia + + + + + + + + + + + + + + + + + + + + + + + + 7 a m or ph op ha llu s tit an um ( b ec c. ) b ec c. e x. a rc an g a ra ce ae b b g + + + + + + + + + 8 c al ad iu m b ico lo r (a it o n ) v en t. a ra ce ae ip b + + + + + + + + + + + + + + + + + + 9 c ol oc as ia e sc ul en ta ( l .) s ch o tt a ra ce ae ip b + + + + + + + + + + + + + + + 10 d ie ffe nb ac hi a fo ur ni er i n .e . b r. a ra ce ae b b g + + + + + + + + + + + + + + 11 d ra co nt iu m g ig as ( se em .) e n gl . a ra ce ae b b g + + + + + + + + + + + + + + 12 h om al om en a pe nd ul a (b lu m e) b ak h . f . a ra ce ae b b g + + + + + + + + + + + + + + + + + + + 13 ip om oe a ba ta ta s (l ) l am . c o nv o lv ul ac ea e b b g + + + + + + + + + + + + + + + 14 m an ih ot e sc ul en ta c ra n tz e up h o rb ia ce ae b b g + + + + + + + + + 15 n el um bo s p p. a ra ce ae t an as h i f ar m , u t + + + + + + + + + + + + + + + + 16 sc hi sm at og lo tis c al yp tr at e (r ox b. ) z o ll. & m o ri tz i a ra ce ae b b g + + + + + + + + + + + + + + + + + + + + 17 sp at hi ph yl lu m c an na ef ol iu m s ch o tt a ra ce ae b b g + + + + + + + + + + + + + + + + + + + 18 so la nu m ly co pe rs icu m l . so la n ac ea e t an as h i f ar m , u t + + + + + + + + + + + + + + 19 so la nu m tu be ro su m l . so la n ac ea e c ia n ju r, i n do n es ia + + + + + + + + + + + + + + + + + + + 20 t ac ca s p p. a ra ce ae b b g + + + + + + + + + + + + + + + 21 x an th os om a sa gi tif ol iu m ( l .) s ch o tt . a ra ce ae b b g + + + + + + + + + + + + + + + + + + + + + 22 z am io cu lca s za m iif ol ia e n gl . a ra ce ae b b g + + + + + + + + + + + + + + + + + z n o te : = o n e in di vi du al p er s p ec ie s b b g = b o go r b o ta n ic al g ar de n u t = u n iv er si ty o f t o ky o -j ap an ip b = i n st it ut p er ta n ia n b o go r '– ' = n o a m p lif ic at io n '+ ' = a m p lif ic at io n s in gl e b an d '+ + ' = m ul ti p le b an ds a m p lif ic at io n b an d di d n o t a lw ay s p ro du ce m ic ro sa te lli te a lle le biotropia vol. 25 no. 1, 2018 number of effective alleles of a, b and c populations were 5.43, 7.36 and 6.71, respectively. b population showed higher allelic richness as compared to a and c populations. more than one specific allele per locus existed in each population. out of 147 alleles generated by 14 loci across populations, 61 alleles were specific to particular population. a, b and c populations had 16 (26.2%), 27 (44.3%) and 18 (29.5%) specific alleles, respectively. h value was the highest in a o population, while h value was the highest in c e population. fixation indices (f ) averaged for is each population across all loci had high values ranging from 0.240 to 0.294 (table 4). amova analysis showed low f values both st within and among populations. f value across all st populations was 0.026, whereas f values among st a-b, a-c and b-c populations were 0.022 ( = p 0.009), 0.034 ( = 0.003) and 0.025 ( = 0.001), p p respectively, suggesting that each population was slightly conserved. on the other hand, variation among individuals within population was high (64.0%). according to jansen (1996) and et al. yuzammi (2000), has large phenotypic a. variabilis variations in leaf size, petiole and inflorescence color. the populations exhibited low genetic distance based on nei (1972), i.e. 0.151 for population a to population b, 0.210 for population a to population c and 0.149 for population b to population c. individuals across all populations were clustered in three groups (fig. 2). group i composed of accessions from a and c populations. group ii composed of accessions from a, b and c populations. group iii contained only one accession from population c, i.e. c22. population b was exclusively clustered under subgroup ii. in each population, with exception for b28, c1, c4, c6 and c7, accessions clustered in the same group were generally located close to each other (fig. 1). accessions a10, a11 and a12 from a population were clustered in subgroup ii with those from b population. these accessions were geographically close to each other (fig. 1). this suggests that genetic exchange via pollen occurred between accessions located at the peripheral zone of a population, as stated by pasquet . (2008). sugiyama dan santosa (2008) et al stated that nitidulidae insect becomes important pollinator in .a. variabilis morphological variations in petiole (table 1) was unlikely correlated with microsatellite profiles. accessions with green petiole without spot, i.e. a1, a2, a8, b11, c1, c10, c12 and c20 were clustered into two different groups, i.e. group i and group ii. this finding was in disagreement with the results of santosa et al. (2012), where flower size, leaf size and the presence of petiole spot were tightly linked with aflp grouping. the discrepancy between the result of santosa et al. (2012) and this study could be due to the fact that aflp is a dominant marker. in a. paeoniifolius, petiole color, shape and color of spot and petiole roughness are affected by soil fertility (sugiyama & santosa 2008). in the previous work, santosa et al. (2004) grouped a. variabilis accessions from west java province, indonesia based on inflorescence morphology into four groups. therefore, it is important to conduct further study to investigate whether petiole and peduncle colors in a. variabilis are also affected by soil fertility. table 4 description for three amorphophallus variabilis populations collected from dramaga conservation forest, bogor, indonesia population number of allele ho he fis effective richness specific a (n=11) 5.43 3.56 1.14 0.481 0.645 0.240 b (n=21) 7.36 4.58 1.93 0.464 0.667 0.282 c (n=15) 6.71 4.20 1.29 0.465 0.681 0.294 note: f = fixation index calculated using formula: (h h ) / h or 1 (h / h )is e o e o e n = number of sample, evaluated using 10 microsatellite loci from a. variabilis and two loci from a. paeoniifolius 29 isolating microsatellite from amorphophallus variabilis and its application santosa et al. conservation strategy three populations evaluated in dcf exhibited unique genetic feature. according to an interview with administrative staff members of dcf, there was no intercropping program, harvesting or any man-made disturbance on the a. variabilis populations. unexpectedly, during field survey, several a. paeoniifolius plants were found at dcf. according to local officer, the plants were introduced from yogyakarta by farm laborers in year 2000s. many bushes, weeds and tree seedlings grew densely inside dcf, forming shady condition and blocking more than 75% of sunshine. according to sugiyama and santosa (2008), amorphophallus species grow well under shading up to 75% shading level. it was possible that heavy shading disturbed the growth of , resulting to a. variabilis low density of the studied-population in dcf. sugiyama and santosa (2008) also stated that single mother corms of produced 14.3 a. variabilis cormlets per year. cormlets were detached from mother corm after dormancy release. two to seven cormlets usually grew close (0 13 cm) to mother corms, unlike seedlings from seeds which usually grew apart (> 60 cm) from the mother plant. considering that mature plant of a. variabilis is able to produce 25 280 seeds (sugiyama & santosa 2008), the population density could increase steadily yearly in undisturbed condition. it is interesting to conduct further study on population dynamic of a. variabilis in isolated condition, such as in dcf. in the present study, several accessions failed to amplify resulting to low number of sampling size in each population (table 4) that might cause an underestimation of population genetic. according to li (2009), the numbers of et al. effective alleles were determined by the number of sampling size and the numbers of loci used. a set of 40 samples per population is necessary for population study. during dna extraction, several leaf samples contained large amount of glucomannan. thus, it is important to improve 30 figure 2 dendrogram of upgma based on jaccard similarity index of a. variabilis constructed using microsatellite data obtained (note: a, b, and c codes represent a, b and c populations, respectively) biotropia vol. 25 no. 1, 2018 dna extraction method in the near future, to enhance the accuracy of population genetic evaluation. genetic distance between a and c populations was significantly larger than those of other population pairs. however, it was still unclear why these populations were distantly separated in terms of genetic profile. geographically, a and c populations were separated by about 200 meters. the large genetic distance suggested that exchange of genetic materials among a and c populations was restricted. interestingly, within c population, c22 seemed to be out of group (fig. 2). this might be caused by the fact that c22 was an introduced seed from other distant populations by birds as primary agent for long-distance seeds dispersal of (sugiyama & santosa a. variabilis 2008). the number of estimated genetic exchange or migration among a-b, a-c and b-c populations were 11.23, 7.21 and 9.81, respectively, and were considered as high. average number of estimated migrant (nm) based on the number of specific alleles across all populations was high, i.e. 9.39. smaller degree of genetic differentiation (f ) st among a and b, and b and c populations, indicated populations located closely to each other mostly underwent intense genetic exchange through cross-breeding. finding in dcf indicated that the a. variabilis existed as a meta-population. santosa (2012) et al. has suggested to conserve single large population in conservation strategy of , however, a. variabilis it should be further clarified by evaluating accessions from distant populations across java island using codominant microsatellite primers. many researchers, students and professional workers stated that a large number of a. variabilis plants existed in various agroecological condition in west java and yogyakarta provinces. therefore, it is interesting to evaluate population a. variabilis from different sites in java island to develop conservation strategy for a. variabilis. conclusions microsatellite primers developed in the present study were applicable for the population study of a. variabilis. three a. variabilis populations in dramaga conservation forest exhibited low genetic diversity among populations, but high genetic distance within a population. increasing number of specific allele with the increasing geographical distances among populations implies the importance to study genetic population from the larger geographical range in java island to determine the best conservation strategy for a. variabilis. acknowledgements part of this work was supported by grant-inaid from the japan society for the promotion of science (jsps), university of tokyo, japan and institut pertanian bogor (ipb), indonesia. we thank mr yuzammi from bogor botanical garden, indonesia, dr kazuhike nara and dr kimura megumi from laboratory of forest science, and dr o new lee from 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(in indonesian) yuzammi. 2000. a taxonomic revision of the terrestrial and aquatic (araceae) in java [thesis]. retrieved aroid from school of biological science, faculty of life science, university of new south wales. 359 p. zhang sl, liu py, sun ym. 1998. artificial adjustment of flower period and hybridizing techniques of amor phophalus konjac . acta bot yunn 0. suppl(5):62–6. zhou z, miwa m, hogetsu t. 1999. analysis of genetic structure of a population in a suillus grevillei larix kaempferi stand by polymorphism of inter-simple sequence repeat (issr). new phytol 144:55–63. 32 biotropia vol. 25 no. 1, 2018 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 1041.cdr 350.0 300.0 250.0 200.0 150.0 100.0 50.0 0.0 -1 o sm o la li ty ( m o sm o l k g ) 24 + 2 ppt 29 + 2 ppt 34 + 2 ppt 39 + 2 ppt 44 + 2 ppt treatment b a ab d c 24 + 2 ppt 29 + 2 ppt 34 + 2 ppt 39 + 2 ppt 44 + 2 ppt treatment t o ta l c o u n ts o f c o lo em o cy te s c el ls 4 -1 (x 10 c el ls m l ) 4000.0 3000.0 2000.0 1000.0 0.0 ab ab b a a t o ta l co u n ts o f p h ag o cy ti c c el ls 4 -1 (x 10 c el ls m l ) 250.0 200.0 150.0 100.0 50.0 0.0 24 + 2 ppt 29 + 2 ppt 34 + 2 ppt 39 + 2 ppt 44 + 2 ppt treatment a b b a a page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 biotropia vol. 30 no. 3, 2023: 282 296 doi: 10.11598/btb.2023.30.3.1901 282 orangutan (pongo pygmaeus ssp. wurmbii ) range pattern in punggualas, sebangau national park, central kalimantan, indonesia adventus panda1,2*, ari meididit3, okta simon3, wayan tunas artama4, dwi priyowidodo5 and tjut sugandawaty djohan6 1 doctorate study program, faculty of biology, gadjah mada university, yogyakarta 55281, indonesia 2 department of biological science, faculty of mathematics and natural science, university of palangka raya 73111, indonesia 3 forum orangutan kalimantan tengah (forkah kalteng), palangka raya 73112, indonesia 4 one health colaborating centre, gadjah mada university, yogyakarta 55281, indonesia 5 department of parasitology, faculty of veterinary medicine, gadjah mada university, yogyakarta 55281, indonesia, 6laboratory of ecology and conservation, faculty of biology, gadjah mada university, yogyakarta 55281, indonesia received 3 march 2023 / revised 2 september 2023 / accepted 5 september 2023 abstract punggualas is a logged-over forest in sebangau national park (snp), serving as a primary stronghold for orangutan (pongo pygmaeus ssp. wurmbii) population. therefore, this study aimed to evaluate orangutan range patterns and distribution within punggualas forest. the ranging data were collected inside the area of interest (aoi) from february 2015 to december 2019 (p1) and march 2020 to may 2022 (p2). the minimum convex polygon (mcp) area and kernel utilization distribution (kernelud) of orangutan were estimated using the adehabitathr package in rstudio 4.2.3, with the kernel density (kd) tool in arcgis 10.5 used for visualization. the results showed that the largest range size, measuring 259.6 ha, was observed in the mother-infant pairs throughout the investigation, surpassing the flanged male home range (hr) size of 250 ha per year reported at the natural peat laboratory, cimtrop-upr. the range pattern also varied significantly between the two study periods. during p1, the distribution area was positioned in the northern part of the aoi, shifting to the southeastern during. this indicated that p. p. wurmbii range pattern in punggualas moved to the southeastern part of the grid trails, as shown by mcp and kernelud. the probability of orangutans using random points across the study area, specifically for food, was adequate from the total basal area (m2ha-1) and tree species composition. the compression effect became evident as a potential determining factor contributing to rapid changes in range patterns during the study periods. keywords: epidemiology triad, kernel density estimation, minimum convex polygon, punggualas, sebangau national park introduction the bornean orangutan (pongo pygmaeus) is comprised of three sub-species, namely p. p. pygmaeus (west kalimantan and sarawak, malaysia), p. p. wurmbii (west kalimantan and central kalimantan) and p. p. morio (east kalimantan, extending north to include sabah, malaysia) (ancrenaz et al. 2016). however, the conservation status of this charismatic wildlife on the iucn red list has reached the critically endangered (cr) level, indicating a severe and continuous population decline over the past 45 years. the contributing factor to this decline is habitat loss, which leads to habitat fragmentation and increases the number of poachers and killings of orangutan (santika et al. 2017b; m. voigt et al. 2018; wich et al. 2012) and forest fires (erb, barrow, hofner, utami-atmoko & vogel 2018). during the 1970 to 2000 period, sebangau forest experienced logging concessions, with an influx of illegal activities in various forest areas, particularly in the katingan catchments, including punggualas, until early 2005 when the government intervened with law enforcement *corresponding author, email: apanda@mipa.upr.ac.id orangutan (pongo pygmaeus ssp. wurmbii) range pattern in punggualas – panda et al. 283 actions. both authorized logging concessions and illegal logging activities have resulted in orangutan habitat loss and fragmentation in the sebangau peat forest ecosystem. these past activities are evident in the form of semipermanent railways and canal networks used to transport logs out of the forest, which are still visible in the areas or through high-resolution satellite imagery. during the logging concessions, the railways were used to access the targeted timber, making frequent return visits to log secondary species. however, canal is currently being used for alternative purposes such as granting access to non-timber forest products including jelutong tap, medicinal plants, or fishing harvesting in the interior forests. these developments have created the orangutan-human interface in sebangau national park (snp). orangutan has the ability to adapt to humandominated landscapes (rayadin & spehar 2015). however, these conditions are ideal for pathogenic organisms such as parasites, bacterial, and virus transmission, posing a threat to orangutan and human (herrera, chakraborty, rushmore, altizer & nunn 2019; nurcahyo, konstanzová & foitová 2017; philippa & dench 2019; rondón et al. 2017). several studies stated that the source of pathogenic transmission was from tourists, researchers, the local community (woodford et al. 2003), and orangutan release, including translocation (kilbourn et al. 2003; mul et al. 2007). furthermore, anthropogenic activities are increasingly implicated as drivers of pathogenic potential (allen et al. 2017; bengis et al. 2004; daszak, cunningham & hyatt 2000; han, kramer & drake 2016; jones et al. 2008). the hypothesis of maria voigt et al. (2018) suggested that the absence of an explanation for reported orangutan deaths in their natural habitat, made it necessary to consider the existence and spread of deadly infectious diseases, as discovered in the group of apes in africa. this leads to the consideration of the factors that determine the transmission of pathogenic agents such as parasites, bacteria, and viruses in wild orangutan. bordes, morand, kelt and van vuren (2009) reported that the important determinants of parasite transmission were host mobility and home range size. mammals, such as orangutan, are host to a wide array of gastrointestinal parasites, often acquired through contact with infectious stages present in soil, feces, or vegetation, suggesting the influence of range behavior on their spread (nunn, thrall, leendertz & boesch 2011). the details on how range use behavior might influence the spread of fecally transmitted parasites are also described, specifically the term of ‘‘territoriality benefits’’ and the “fecal exposure” hypothesis. furthermore, several field parasitologists have identified the important roles of home range in parasite dynamics (foitová, huffman & wisnu 2009; labes et al. 2010). in this study, burt home range definition as “that area traversed by the individual in its normal activities of food gathering, mating and caring for young” was used to prevent any misinterpretation (burt 1943). these activities depend on the life stage, sex, and habitat of animals. home range estimated by studies that are not very intensive, short, or restricted to a small area is considered to represent core areas (singleton & carel p. van schaik 2001). this indicates that the core area of home range is the most frequently used by an animal. a previous study showed that orangutans had fixed home range, spanning from 100 to 900 ha for females and up to 2,500 ha for adult males (singleton, knott, morrogh-bernard, wich & schaik 2009). the peat swamp forests of suaq balimbing, aceh, sumatra, and tanjung puting national park, central kalimantan, have the highest recorded home range. for example, a single female orangutan may require at least 1500 ha for self-support and their offspring, while males need more than 4000 ha. these ranges include a variety of habitats, offering unique benefits due to different fruiting patterns (cattau, husson, & cheyne, 2015; saputra, perwitasarifarajallah, suci utami-atmoko, ariyanto, & van noordwijk, 2017). several investigations have identified that the male range is significantly greater compared to females due to the food availability. despite the increase in deforestation, the orangutan population has shown the opposite trend (santika et al. 2017a; maria voigt et al. 2021, 2018; wich et al. 2012). the investigation into orangutan daily range size and pattern in punggualas has been initiated biotropia vol. 30 no. 3, 2023 284 by rukmawardani & imron (2021). the results study suggested understanding pattern and range size positively benefit future forest rehabilitation. however, there is no information regarding the potential overlap between the vast area of orangutan and the subsistence activities of communities surrounding punggualas. therefore, this study aimed to estimate orangutan range size, pattern, and the determining factors influencing the range pattern shift in punggualas forest, particularly in relation to food availability. materials and methods study locations this study was conducted in punggualas, a logged-over forest, covering approximately 1.320 ha, located in the western part of snp, as presented in figure 1. this location is an active site for orangutan studies since 2010 and is allocated as a focal point of eco-tourism development for orangutans. from january to december 2018, tourist visit data to punggualas reached a total of 260 visitors (kartini, peh tn sebangau: pers. comm.). range observations range data within the area of interest (aoi) was collected from feb 2015 – dec 2019 (p1) and aug 2020 – may 2022 (p2). due to the covid19 pandemic lockdown, the observations in 2020 were only conducted from august to september and continued from may to december 2021. a standardized method was used for orangutan studies, indicating the orangutans follow protocol (h. morrogh-bernard, husson & mclardy 2002). behavioral data were collected during nest-tonest focal animal follows, using two-minute instantaneous sampling to record activity and feeding. all observed behavior was recorded in 2minute intervals and completed as the focal built night nest by a team of 2-3 personnel. observations were terminated when individuals moved beyond the boundaries of the observation grid. subsequently, names were assigned to individuals when consecutive days extended beyond four days, with their sex-age class as presented in the datasheets. figure 1 map of the study area in snp:(a) borneo island; (b) snp; and (c) orangutan observation grid in punggualas orangutan (pongo pygmaeus ssp. wurmbii) range pattern in punggualas – panda et al. 285 range data were collected using a handheld gps unit (garmin gps78) to record the location of the focal at 30-minute intervals. regarding range size, the focus was extensively on nest-tonest follows, without specifying additional criteria such as a minimum of 1000 hours followed for estimations. subsequently, orangutan position points data were extracted, analyzed, and nest-tonest observation of 21 orangutan were completed, as representative of three sex classes, consisting of two adolescent females (fa), five flanged-male (fm), and 13 mother-infant (mi), respectively. table 1 describes individual estimated age, home range (hr) in (ha), total hours followed, and habituation status. forest block sampling a rapid assessment of the habitat structure was made to determine the comparativeness between the north and south forest block, based on range results. the point quarter centered quadrat (pcq) method was used to calculate basal tree area and important value index for each forest block during focal follows. a total of 20 points in the northern forest were assessed, while another 22 points were found in the southern points. the assessment commenced when the focal spent ≥ 10 minutes in a patch tree, as one observer continuously recorded the focal behavior, while the other two operated the pcq method (mitchell 2010). the patch tree was tagged during the assessment, followed by the documentation of canopy height and connectivity within a 10 m radius from the focal latest positions. data analysis all range points, updated in each 30-minute interval, were conbined in the recapitulation matrix. minimum convex polygon (mcp) and kernel utilization distribution (kernelud) were calculated using the adehabitathr package in rstudio 4.2.3 (posit 2023), where the core area was derived from kernelud with a certain probability. since this study did not infer specific focal, the matrix excluded range estimates for individuals per annum or seasonal. the distribution was visualized in arcgis 10.5 with kernel density (kd) tool, which allowed the differentiating of various parts of the animal range according to the intensity of use (wartmann, purves & van schaik 2010). vegetation data and calculation were carried out using microsoft® excel 2019, while graphical figure and color correction were performed using coreldraw®2021. to identify the items associated with the highest loaded score for each parameter calculated from the pcq method, principal component analysis (pca) was carried out using rstudio 4.2.3 (posit 2023). table 1 orangutan id, estimated age, estimated home range (ha), total hours followed, and their habituation status in the study periods phase sc est_age orangutan id estimated hr (ha) hours followed remark phase-1 mi 24 marlenda martinus 241,82295 216 habituated phase-1 mi 21 jane jack 192,84975 176 habituated phase-1 fm 29 brown 149,0408 152 habituated phase-1 fm 22 coded 123,5825 176 habituated phase-1 mi 23 vina vino 123,565 144 habituated phase-1 mi 21 yulia yani 108,43875 104 habituated phase-1 mi 22 nani nina 92,56 192 habituated phase-1 mi 21 ibu anak 84 26 habituation phase-1 mi 26 rere reno 51,1972 120 habituated phase-1 fm 21 damang 37,16015 44 habituated phase-1 fm 20 kadir 8,49585 6 habituated phase-1 mi 26 anita_anton 9 32 habituated phase-2 fm 24 brown 3 95 habituated phase-2 af 16 adolescent_001 5 23 habituation phase-2 af 18 adolescent_002 31,216686 99 habituation phase-2 mi 25 ibu_anak 71,4011712 25 habituation phase-2 mi 23 ibu_anak001 80,034119 83 habituation phase-2 mi 20 ibuanak003 51,125365 92 habituation phase-2 mi 23 ibu_anak_2020 26,548 44 habituation phase-2 mi 20 ibu_anak_001 23 76 habituation phase-2 fm 21 unk_flanged_male01 23,65411 34 habituation biotropia vol. 30 no. 3, 2023 286 results and discussion range size mcp area was calculated using a value of 80% to include the area used beyond the core home range. this analysis yielded the estimated home range, in phases 1 and 2, as shown in figure 2, with the range size of each individual varying from 20 to > 250 ha. despite the most representative sex classes, the mother-infant had the highest average range size, approximate (p1), exceeding 250 ha (p2). a flanged male individual named brown yielded 149.04 ha (p1) and less than 5 ha (p2). the mother-infant had a greater range size than the flanged-male in the same study periods, namely kadir (fm) in phase-1 and unk_flanged_male01(fm) in phase-2. the varying results were attributed to the female behavior and range, which depended on reproductive state, particularly mating and caring for the young. the babysitting lasts for 6 to 8 years before the offspring become independent at the age of twelve (scott, knott & susanto 2019). in these years, the mother imparts and mediates essential skills for the immature offspring (noordwijk, sauren, morrogh-bernard, atmoko & schaik 2003). secondly, the range use may reflect the patch sources, as fully discussed in the subsequent section. the analysis showed relatively low range estimates for flanged-male but was not significantly small compared to previous studies. the results showed that male ranges are often larger than females, exceeding the grid trails of the study area (wartmann et al. 2010). this indicated the tendency of female orangutans to stay close to their natal location (so-called philopatric), while male orangutan typically dispersed from this area. based on the dataset, which showed the dyadic in most observations in both phases, also confirmed the co-existence of the philopatric. figure 2 home range size as per sex-classes (sc) in phase-1 (2015-2019) and phase-2 (2020-2022) derived from minimum convex polygon calculations; names provided as appeared in gps-code; mother-infant (mi), flangedmale (fm), and adolescents female (fa) orangutan (pongo pygmaeus ssp. wurmbii) range pattern in punggualas – panda et al. 287 figure 3 shows that the range overlap predominantly among mother-infant sex classes. as shown in figure 3a, the 12 individuals in phase-1 from 2015 to 2019 were concentric, while in figure 3b, observed individuals in phase2 presented that the polygon was dispersed. individual range that overlaps tend to oppose each other, as indicated by kernelud, using the ‘getverticehr’ function for those in figures 4a and 4b. figure 3 mcp per study periods; p1(a) and p2 (b) figure 4 kernel ud visualization using ‘getverticehr’ function for each observed individual during p1(a) and p2 (b) an overlap is a common occurrence among p. p. wurmbii in the natural habitat (wartmann et al. 2010). previous investigations showed that apart from mother–infant dyads, p. p. wurmbii were semi-solitaire animals occupying highly overlapping home range. female home range is assumed to be affected by ecological factors, reflecting the distribution of food sources. meanwhile, male range use is a response to the distribution of female (singleton et al. 2009; suci et al. 2009), with the compression effect leading to orangutan favoring the fine-scale habitat (helen c. morrogh-bernard, husson, harsanto & chivers 2014; helen celia morrogh-bernard, morf, chivers & krützen 2011). according to individual levels, each subset was plotted based on their range size, as presented in figures 5a and 5b, to determine the residency patterns, including 1) residents, who are consistently present for many years in a particular area, 2) commuters, observed regularly for several weeks or months each year for an extended period. this implied that commuters tended to result in home range with two separate core areas, namely multinuclear, and 3) wanderers, observed infrequently (or once) for at least 3 years, without returning to the area (singleton & carel p. van schaik 2001). based on this criterion, the only individual in punggualas, who met the requirements is brown, the 29-year-old flanged male. however, the identification of commuters and wanderers was not possible, particularly in phase-2, where new dyadic pairs were encountered during each sampling period. the identification was not carried out because 1) the observation area was relatively small, and 2) a longer study period was required, indicating the need to determine home range stability. the datasets engage more dyads, with varying range sizes, among the two phases. this indicated that the use of distribution calculated showed the probabilities of where an animal might have been biotropia vol. 30 no. 3, 2023 288 found at any randomly selected time (powell and mitchell 2012). furthermore, it showed that the greater the range size, the more likely an individual has been followed. the problem of home range stability can be solved through a long-term study in an area within defined boundaries set by the movements of the subjects (singleton & carel p. van schaik 2001). figure 5 kernelud as per individual orangutans; (a) phase-1: 2015 – 2019; and (b) phase-2: 2020 -2022 a b orangutan (pongo pygmaeus ssp. wurmbii) range pattern in punggualas – panda et al. 289 figure 6 maps constructed from the orangutan point positions, overlap with the kernel density estimate: (a) 2015-2019; and (b) 2020 – 2022; north vs south delineated by punggualas tributaries range pattern during p1, the distribution area was located within the northern part of the aoi and changed to the southeastern part in p2, as shown in figure 6. each red area showed the most overlap in forest areas, indicating a higher probability of hosting various individual normal activities such as food gathering and care for the young. in p1, the most overlap areas were shown by three zones, while a single red area dominated p2. however, a change in pattern was observed, indicating that the orangutan movement corresponded to the tree composition as a determining factor. the mean basal area (m2), trees/ha, and total basal area (m2/ha) were calculated using vegetation data from pcq. the result presented in figure 7 showed that each forest block varies slightly, while the southern part tended to have a bigger mean basal area (m2), total number of trees/ha, and total basal area (m2/ha). this implied that the southern part of the aoi, featured a greater number of individual trees per ha, particularly cryptocarya crassinerva, bluemeodendron tokbrai, and campnospermae coriaceum. however, the total basal area (m2/ha) showed the same trend in each aoi, namely shorea sp1. biotropia vol. 30 no. 3, 2023 290 figure 7 mean basal area, number of trees/ha, and total basal area (m2/ha) per tree species in the north and south forest blocks figure 8 (a) the coefficient correlations mean ba, trees/ha, and total ba (m2/ha) as per dimension; and (b) biplot principal component analysis, depicts factor loadings and cluster a b orangutan (pongo pygmaeus ssp. wurmbii) range pattern in punggualas – panda et al. 291 the first principal component was strongly correlated with a total ba (m2/ha), followed by trees/ha and mean ba, respectively (figure 8a). this indicated whether the north and south forest blocks yielded, trees/ha, and whether mean ba contributed to the total ba (m2/ha). pc1 can be considered a measure of total ba (m2/ha), while pc2 coefficient value of mean ba (0.8388) was perpendicular to the total ba (m2/ha) and opposed trees/ha. this showed that a higher mean ba value corresponded to a decrease in the contribution to the total variance of trees/ha and total ba (m2/ha). as for trees/ha, the results produced bias due to only the measurement of each quadrant tree representative. consequently, when standardized vegetation plots were deployed, different pca loading biplots would be obtained. trees composed of different species such as campnospermae coriaceum (anacardiaceae), blumeodendron tokbrai (euphorbiaceae), garcinia sp2 (clusiaceae), cryptocarpa crassinerva (lauraceae) and diospyros sp1 (ebenaceae), were placed beyond elliptical. the result of the principal component analysis in figure 8 showed that total ba (m2/ha) loading had the highest eigenvalue. however, mean ba and trees/ha loadings were pointed out in other directions and the probability of orangutans using any random point over the grid trails was adequate from tree species composition. figure 9 showed data uniformity despite the differences among mean ba, trees/ha, and total ba (m2/ha), and variation in tree species, the relative density, coverage, frequency, and important value. using habitat classification (morrogh-bernard et al. 2014a), it was discovered that the orangutan used tall connected a (tca) and medium-connected (mc). tca was characterized by the high stem density of large trees (>20 m tall) connected with high canopy (>75%). meanwhile, mc was characterized by a few tall trees with a high stem density of mediumsized trees and a connected canopy at 10 – 20 m. the interlocked canopy was inadequate to support the range pattern-shifting idea. recent observations have shown that natural tree falling has been spotted in a regularly visited grid in the northern and southern forest block of punggualas. figure 9 relative density (%), coverage (%), frequency (%), and important value index (%) as per tree species derived from point-centered quarter method biotropia vol. 30 no. 3, 2023 292 in addition to tree composition, dietary preferences were also explored as another argumentation. in peat forests, orangutan spent half of the active time feeding compared to those in a mixed-dipterocarp forest. observations showed that the focal being followed was frequently feeding on young leaves, flowers of mezzetia leptopoda (annonaceae), and immature fruits of nephelium lappaceum (sapindaceae). moreover, recent observations also indicated that the orangutan diet included the flower of madhuca motleyana (sapotaceae), inner bark, termites, and liana. the fruits from willughbeia sp1 and cf. lucinia sp1, commonly woody lianas found in punggualas, was observed from several dyad mother-infant and flanged-male. punggualas phenology data showed that the camnospermae coriaceum generally had two fruiting periods yearly, first from october/november to february/march, followed by may to june. the general dietary pattern of orangutans during the study period did not always describe the fruit as their dietary preference because seeds, flowers, leaves, and bark were also included as fallback foods. studies of the nutritional content and its relationship with the energy budget of orangutans in tuanan, central kalimantan, found that young leaves had a higher protein content than fruits (vogel et al. 2017). basedon the results, confirm that variations in energy absorption were strongly influenced by the range of fruiting periods in the sebangau habitat. a diet that depended on fibrous vegetation and bark confirmed that p. p. wurmbii shapes their mandibulae for dietary preferences (traylor-holzer et al. 2009). detail observations in ketambe study areas by (hardus, lameira, menken & wich 2012) regarding liana-derived resources such as fruit and leaves indicate the critical role of liana in the orangutan diet, specifically for orangutan population in the primary forest. table 2 list of observed tree/sapling, liana, and ficus species showing parts eaten by orangutan during p1 and p2 species family fruit seed flower leaves bark campnosperma coriaceum anacardiaceae ☐ ☒ ☐ ☐ ☐ xylopia fusca annonaceae ☒ ☒ ☐ ☐ ☐ xylopia malayana ☒ ☒ ☐ ☐ ☐ dyera polyphylla apocynaceae ☒ ☒ ☒ ☒ ☒ licania splendens chrysobalanaceae ☒ ☒ ☐ ☒ ☐ callophylum hosei clusiaceae ☒ ☐ ☐ ☒ ☐ callophylum sclerophyllum ☒ ☐ ☒ ☐ ☐ callophylum sp3 ☒ ☒ ☐ ☐ ☐ garcinia banana ☒ ☐ ☐ ☒ ☐ diospyros confertiflora ebenaceae ☒ ☒ ☐ ☐ ☐ diospyros evena ☒ ☐ ☐ ☐ ☐ diospyros pseudomalabrica ☒ ☐ ☒ ☐ ☐ diospyros siamang ☒ ☒ ☐ ☐ ☐ elaeocarpus mastersii elaeocarpaceae ☒ ☒ ☒ ☒ ☐ neoscortechinia kingii euphorbiaceae ☒ ☐ ☒ ☒ ☐ castanopsis foxworthyii fagaceae ☒ ☒ ☐ ☒ ☐ lithocarpus conocarpus ☒ ☒ ☐ ☐ ☐ litsea cf. resinosa lauraceae ☒ ☒ ☐ ☒ ☐ phoebe zsp se cf. grandis ☐ ☐ ☐ ☒ ☐ koompassia malaccensis leguminosae ☐ ☐ ☐ ☐ ☒ magnolia bintulensis magnoliaceae ☒ ☒ ☒ ☒ ☒ dactylocladus stenotachys melastomaceae ☐ ☐ ☒ ☐ ☐ aglaia rubiginosa meliaceae ☒ ☐ ☐ ☒ ☐ parartocarpus venenosa moraceae ☒ ☐ ☒ ☐ ☐ horsfidelia crassifolia myristicaceae ☒ ☐ ☒ ☐ ☐ myristica sp2 ☐ ☐ ☐ ☐ ☒ ardisia sp2 myrsinaceae ☒ ☒ ☐ ☐ ☐ syzygium sp1 myrtaceae ☒ ☒ ☐ ☐ ☐ pittosporum sp1 pittosporaceae ☐ ☐ ☐ ☒ ☐ nephelium lappaceum sapindaceae ☒ ☐ ☐ ☐ ☐ nephelium maingayi ☒ ☐ ☐ ☐ ☐ orangutan (pongo pygmaeus ssp. wurmbii) range pattern in punggualas – panda et al. 293 table 2 (continued) species family fruit seed flower leaves bark madhuca motleyana sapotaceae ☒ ☐ ☒ ☒ ☒ palaquium cochlearifolium ☒ ☐ ☒ ☐ ☐ palaquium pseudorastratum ☒ ☒ ☒ ☐ ☐ palaquium sp ☒ ☐ ☐ ☐ ☐ palaquium sp2 ☒ ☐ ☐ ☐ ☐ microcosm sp tiliaceae ☒ ☐ ☐ ☒ ☐ willughbeia sp1 apocynaceae ☒ ☐ ☐ ☒ ☐ ziziphus angustifolia rhamnaceae ☒ ☐ ☐ ☒ ☐ cf. lucinea sp1 rubiaceae ☒ ☐ ☐ ☒ ☐ ficus sp moraceae ☒ ☒ ☐ ☐ ☐ ficus sp8 ☒ ☒ ☐ ☐ ☐ notes: recorded fruits, are grouped by maturity stages, e.g., immature, semi-mature, or mature fruit. the observation made in tuanan, central kalimantan, showed the importance of liana in adolescent orangutan (saputra et al. 2017) during the absence of fruiting. the determining factor of liana density in lahg-cimtrop upr sebangau was highly correlated with tree crown size, shape, and height (schofield 2015) due to the light penetration to the forest floor. another determining factor was found to be crowding, also known as compression. this occurred when orangutan was exposed to the disturbance in a portion of their range, mostly using parts that had not been logged or so-called mosaic, and began to use their home range differently. since orangutan home range overlaps, many individuals made more biased use of the home range, resulting in population crowding into areas of undisturbed habitat, or 'refuges.' this phenomenon made male orangutans travel further away from the observation grid. to address this issue, additional perspectives were incorporated concerning anthropogenic activities in snp. potential overlap between orangutan range and subsistence activities nontimber forest products, known for their aromatic fragrance, so-called gaharu, were mainly produced by the family thymelaeaceae, specifically aquilaria beccariana van tiegh, and aquilaria filaria (oken.) merr (giesen 2015; paoli, peart, leighton & samsoedin 2001; sitepu, santoso, siran & turjaman 2011). these products were harvested from the aquilaria genus that had been infected by a particular fungus. this study also documented the activities of villagers, mostly from karuing, moving backforth and to the forest, collecting gaharu. meanwhile, their harvesting methods varied, including the use of a 1,20 m long iron stick pushed into the peat surface to detect the particular sound produced as the stick hit gaharu. after locating the suspected wood, the soil was dug, lifted out of the surface, and taken to the village to be weighed and exchanged for money. the aromatic essence of gaharu was economically beneficial in raw materials, crafted as the bracelet, and occasionally, tasbih. recent studies found that some of this aromatic wood was obtained from gonystylus bancanus (miq.) kurz, of thymelaeaceae. the wood is expensive and most wanted during logging concessions in sebangau ecosystem. verification has been made for a product claimed as gaharu (nordahlia 2017), indicating that a product other than the plant genus aquilaria is called pseudo-gaharu. although the aromatic properties and mechanical structure, the wood are not similar to truly gaharu trees, aquilaria beccariana van tiegh, and aquilaria filaria (oken.) merr. this study showed the importance of considering some factors such as distance of access and number of days spent in the interior forest should be considered. some villagers use the observation grid and also encounter the orangutan. according to local sources, these activities were held during the covid-19 pandemic and continued unto the study in phase2 (p2). whether considered a coincidence or not, there was sufficient proof that access to natural resources in the interior forest of snp contributed to the orangutan range pattern. biotropia vol. 30 no. 3, 2023 294 consequently, the park authority has now prohibited this gaharu harvesting out of the forest. recommendations for future study incorporate detailed observations on the orangutan-human interface, including the habitat structure profile, orangutan parasites burden, and the community access into the interior forest. conclusions in conclusion, this study showed that p. p. wurmbii range pattern in punggualas moved to the southeastern part of the grid trails, as indicated by mcp and kernelud. the probability of orangutans using any random point over the area, specifically for food was adequate from tree species composition and total basal area (m2/ha). additionally, the 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(2012). understanding the impacts of land-use policies on a threatened species: is there a future for the bornean orangutan? plos one, 7(11). doi: 10.1371/ journal.pone.0049142 742 alimuddin (resistance) revisi.cdr resistance against aeromonas hydrophila infection and growth of (f2)second generation african catfish selected [clarias gariepinus] using molecular markers alimuddin , fadhila maharani putri , dinamella wahjuningrum ,1* 1 1 2 2 1dian hardiantho , ade sunarma and sri nuryati 1 department of aquaculture, faculty of fisheries and marine science, institut pertanian bogor, bogor 16680, indonesia 2 a main center for freshwater aquaculture, sukabumi 43114, indonesi received 15 december 2016 / accepted 01 august 2017 abstract aeromonas hydrophila is a pathogenic bacteria that causes mass mortality in catfish. in previous studies, specific pathogen resistant (spr), a. hydrophila-resistant african catfish first generation (f1) has been cultivated by marker assisted selection using the major histocompatibility complex (mhc) 1 as a molecular marker. in this study, growth performance, inheritance of the mhc dna marker in the second generation (f2) of catfish and disease resistance against a. hydrophila infection were observed. the f2 progenies were produced by crossing f1 fish between themselves. nursery was performed in 80-l glass aquaria, 4 replications for each cross, at the same initial density, for 2 months of rearing. the results showed that daily growth rate of f2 progenies from the spr broods was significantly higher than those from broods without the marker. results of the pcr analysis showed that average number of f2 progenies from spr broods carrying the mhc marker was about 91% higher than that of control. after the fish 6reached about 12 cm body length, they were challenged by intramuscularly injecting of 0.1 ml a. hydrophila (ld : 10 50 -1cfu ml ) for 7 days. results of challenge test showed that survival of f2 offspring from the crosses of spr broods (77.2%) was about two times higher than those from brood without mhc marker (38.3%). differential leukocyte count supported the high resistance of f2 progenies from f1 broods having mhc i marker against a. hydrophila infection. in conclusion, african catfish farming carrying mhc marker potentially have higher productivity and reduces fish lose due to infection by a. hydrophila. keywords: aeromonas hydrophila, african catfish, disease resistance, growth, mhc i introduction african catfish (clarias gariepinus) is one of indonesia's leading freshwater aquaculture species (mmaf 2016). efforts to increase african catfish production is continuously done through aquaculture intensification. however, farmers often suffer loss from low survival rate, whether during nursery or growing phase. low survival is usually caused by aeromonas hydrophila bacterial infection which can result in 90% mortality in catfish (zhang et al. 2016). genetically specific pathogen resistant african catfish cultivation can be an alternative in preventing loss caused by the infection of this disease. cultivating disease resistant catfish can be done through conventional selection, however, this method takes relatively long time; for example, dropsy-resistant kasnodar common carp (cyprinus carpio) development took 9 generations (kirpichnikov 1999). the alternative effort is molecular marker assisted selection. this method has been successfully utilized to develop majalaya variety of common carp resistant against khv infection (alimuddin et al. 2011), and khv resistant common carp variety has passed variety evaluation test and spread to farmers in 2015. in our previous research through challenge test, differences of endurance against a. hydrophila infection was found in african catfish (azis et al. 2015a). pcr analysis with specific primer for * corresponding author: alimuddin@apps.ipb.ac.id biotropia 5 2 8 95 102 vol. 2 no. , 201 : 10.11598/btb.2018.25.2.742 95 major histocompatibility complex i (mhc i) gene showed that there are differences between fishes that lived or died post a. hydrophila bacterial infection (azis et al. 2015a). mhc's role in the immune system is to represent antigen. with this result, we suggested that mhc i can be used as a molecular marker of a. hydrophila infection resistant african catfish. afterward, challenge test with a. hydrophila against the first generation (f1) of crossing between fishes exhibiting molecular marker showed that the resistance can be inherited. the survival rate is about 2.2 times higher than f1 of fish with no molecular marker. mhc i molecular marker can be inherited by f1 generation with 62.5-83.4% percentage, while for control its only 25.0% (azis et al. 2015b). in this research, second generation (f2) of african catfish was produced by crossing f1 fishes exhibiting mhc i markers. the purpose of this research s to evaluate marker inheritance, wa resistance a. hydrophila against and growth during nursery. materials and methods identification of f1 broods carrying mhc marker identification of fish having the mhc i marker was conducted by following azis et al. (2015a) method. a total of five pairs matured broods were randomly taken from the pond. their genomic dna was extracted from fin tail tissue using dna isolation kit (puregene, minneapolis, usa) by following manufacturer instruction. dna was diluted with 50ml sterile distilled water (sdw). purity and dna content was measured using spectrophotometer (gene quant) at 260 nm and 280 nm wavelength. pcr amplification was performed at 25 ml final reaction volume. the solution of reaction consisted of 2.50 l 10x pcr buffer, 2 l dntp μ μ mix, 2 l of forward and reverse clmhah-01 μ primers, 0.25 l dna polymerase (kapa μ taq biosystems), 1 l genomic dna and 17.25 l μ μ sdw. pre-denaturation of pcr process was performed at 94°c for 3min, 35 cycles of amplification with denaturation at 95°c for 30 sec, annealing at 68°c for 20 sec, and extension at 72°c for 30 sec, and a final extension at 72°c for 5 min. pcr products were separated by electrophoresis 1.5% (w/v) agarose gel, at 70 on volts for 90 min. dna was visualized using red gel (biotum inc. california, us) and uv transilluminator. production and maintenance of f2 generation three pairs of f1 african catfish broodstock carrying mhc i marker and sexually mature were chosen. ovulation and spermiation were induced by injecting ovaprim (syndel laboratories ltd) -1 with 0.2 mlkg dosage. artificial fertilization was done by mixing eggs with sperm in a plastic container. f2 progeny was also developed by mating catfish broodstocks that did not exhibit mhc i marker as a control. eggs were incubated in 80 liter aquarium. african catfish were reared for 2.0 months, at the same initial density. fishes were given tubificid as live food from 3 days until 30 days after hatching (dah), ad libitum. in 28-32 dah, in addition to tubificid, fishes were also fed with pf-1000 commercial feed until 60 dah. afterward the fishes were fed hi-provit commercial feed until end of research. commercial feed was given three times daily, at satiation. to keep good water quality, aquarium water replacement was conducted for 80% of total amount every two days. water quality was measured before and after water replacement. oxygen and ammonia concentration was measured by titration method (apha 2005), whereas temperature and ph were directly measured by using thermometer and phmeter. analysis of marker inheritance and growth performance f2 progenies having the mhc i marker identification was carried out by pcr method as described above. mhc i marker identification was conducted on 20 dah f2 fishes. a total of 30 individual f2 progenies were randomly taken from each cross to determine mhc i marker inheritance. growth performance test on f2 progenies was conducted for two months. a total of 30 fishes (body weight 0.11±0.03 g) were taken from each cross which carried and did not carried mhc i marker. three replications were taken from each cross. weighing were conducted every two weeks. 96 biotropia vol. 25 no. 2, 2018 survival we . . of a, b and c cross re 76 7±7 6%, 80 0± 5 0% and 75 0±5 0%, respectively. . . . . meanwhile, negative control fish from every cross injected with pbs all survived. this showed that fish mortality was a result of a. hydrophila bacterial infection. our previous research has produced f1 afican catfish through mhc i molecular marker based selection (azis et al. 2015b). in this research, f2 african catfish were challenged against aeromonas hydrophila infection. f2 african catfish resistant was consistent with the f1 fish, in which the offsprings of broodstock exhibiting mhc i marker have higher resistance compared to fishes whose broodstocks did not (table 1). the high survival of f2 fish post a. hydrophila challenge was consistent with the percentage of individuals carrying the marker, which was higher compared to fishes whose parent did not carry mhc i marker. the number of f2 fish from marker exhibiting parent cross was approximately 2 times higher compared to those whose parent did not exhibit mhc i marker (table 1). the inheritance of marker in f2 fishes (77.0-97.0%) was relatively similar to what was reported in f1 fish by azis et al. (2015b), which is 62.6-83.4%. this suggests that the mhc i marker was passed on to f2 offsprings. similar pattern has also been found in the inheritance of cyca-dab1*05 mhc ii marker in indonesian common carp resistant to pathogen infection; 70.0% and 83.3% in the f1 and f2 generation, respectively (decree of marine and fishery minister no.24/kepmenkp/2015). mhc consists of many genes and is polymorphic (rakus 2008). the oligonucleotide primer used in this study generates three dna bands of pcr amplification product (azis et al. 2015b). these three dna pcr products are suspected to be different genes of the mhc i group (azis et al. 2015a). the selection performed on african catfish has not yet utilized one of those genes, thus the inheritance of the mhc i gene marker did not follow mendelian segregation pattern. in addition, f2 fish from parents without mhc i marker were also found to be positive for the dna marker. the number of f2 fish suffering from ulceration was fewer than in fishes whose parent did not exhibit mhc i marker. the same occurrence was found in f1 fish (azis et al. 2015b). dna analysis by pcr revealed that 76% the number of fish was counted at the end of the research to determine survival rate. chal lenge test against aeromonas hydrophila aeromonas hydrophila isolate was obtained from fish health laboratory, department of aquaculture, ipb. a total of 20 fishes (total body length 11-12 cm) of each cross were challenged by intramuscularly injecting 0.1 ml of a. hydrophila 6 -1 (ld : 10 cfu ml ; this was obtained from our 50 preliminary study). negative control fishes were injected with 0.1 ml phosphate buffer saline. the fish were kept in 80-l volume glass aquaria and each cross had three aquariums as replications. observations during challenge test treatment involved erythrocyte count, leucocyte count and differentiation, hematocrit, clinical signs, and mortality. blood samples of nine fishes from each cross were taken before and after challenge. the number of erythrocytes and leukocytes were analyzed by using the method of blaxhall and daisley (1973), and hematocrit by anderson and siwicki (1995). data analysis growth rate, survivability and blood count were analyzed with anova with the help of microsoft excel 2011 and spss version 21.0 program with 95% confidence interval. data that were significantly different was analyzed further by duncan multiple range (dmr) test. mhc i marker inheritance data on f2 generation, and quality of water rearing were analyzed by descriptive statistics. results and discussion mhc i marker inheritance and post challenge survivability utilizing method introduced by azis et al. (2015b), mhc i marker inheritance percentage from marker exhibiting broods obtained values ranging between 77.0-97.0%, whereas broods tock without marker (control) had 46.6% (table 1). challenge test result showed that of f2 survival african cat from mhc i marker exhibiting fish broods s higher (p<0.05) compared to tocks wa control without marker (cross d: 38 3±7 6%). the . . growth and resistance of f2 african catfish against aeromonas hydrophila – alimuddin et al. 97 of dead fish from parents not exhibiting mhc i marker (n=25) did not carry mhc i marker (data not shown). this showed a strong correlation between the presence of mhc i marker with fish survival against a. hydrophila infection. blood count and clinical symptoms blood count analysis result is presented in table 2. generally, erythrocyte count, leucocyte count and hematocrit from fishes of all cross before challenge were similar. erythrocyte count of african catfish before challenge ranged between 2.13-2.83 (×10 cell mm ), leucocyte 6 -3 count was about 2.93-4.35 (×10 cell mm ) and 4 -3 hematocrit around 35.80-39.40%. after the challenge, leucocyte count between challenged crosses was similar, and each lower (p<0.05) compared to control that were not challenged by a. hydrophila or only injected by pbs (ka, kb, kc, and kd). meanwhile, hematocrit level of all cross challenged by bacteria suffered decrease compared with pbs injected control (table 2). leucocyte counts of fishes injected by pbs (ka, kb, kc and kd) were lower compared to a, b, c and d cross that were challenged (table 2). test obefore the challenge , bl od count analysis showed that f2 african catfish were hea thy. it l was shown by the er throcyte count, leucocyte y count, and hematocrit which s within the range wa of healthy catfish (table ). the er throcyte 2 y count range of healthy catfish is 2.0-3.0 (×10 6 cellmm ) (hastuti & subandiyono 2015), total -3 leucocyte count ranged 1.83-4.22 (x10 cell/mm ) 4 3 (triyaningsih 2014) and hematocrit value et al. between 30.8-45.5% (yanto 2015). mmune et al. i response against bacterial of f2 fish a. hydrophila infection can be inferred from complete blood count parameter. after challenge, er throcyte y count decreased to 1.50-1.82 (×10 cellmm ), and 6 -3 hematocrit value to 20.2-25.0% (table ). ulcer in 2 infected fish caused blood vessel to burst and decrease er throcyte count. ul eration s the y c wa result of bacterial hemolysin toxin on fish's body surface (del coral . 1990). in contrast, as et al shown in table , after the challenge, leucocyte 2 count increased to 6 11-7 04 (×10 cellmm ). 4 -3 . . this increase is the fish's response to defending table 2 erythrocyte count, leucocyte count, and hematocrit of second generation african catfish from parents exhibiting mhc i marker before and after aeromonas hydrophila injection treat ment erythrocyte (x106 cell/mm3) leucocyte (x104 sel/mm3) hematocrit (%) prechallenge postchallenge prechallenge postchallenge prechallenge postchallenge a 2.38 ± 0.09ab 1.58 ± 0.11a 4.35 ± 0.09a 6.97 ± 0.37 a 35.80 ± 1.02a 23.20 ± 3.22a b 2.42 ± 0.43ab 1.82 ± 0.79a 3.66 ± 0.38 a 6.27 ± 0.87 a 37.20 ± 1.95ab 25.00 ± 0.69 a c 2.13 ± 0.25a 1.50 ± 0.33a 3.87 ± 0.25 a 6.11 ± 0.84 a 36.60 ± 0.65ab 22.50 ± 3.21 a d 2.64 ± 0.44ab 1.74 ± 0.11a 3.95 ± 0.96 a 7.04 ± 0.72 a 37.60 ± 2.17ab 20.20 ± 4.27 a ka 2.63 ± 0.38ab 3.09 ± 0.17b 4.07 ± 0.64 a 3.27 ± 1.06b 37.70 ± 1.14ab 33.30 ± 3.89b kb 2.61 ± 0.33ab 3.27 ± 0.21b 3.75 ± 0.26 a 3.05 ± 0.33 b 37.70 ± 2.29ab 38.80 ± 1.89 b kc 2.58 ± 0.30ab 3.32 ± 0.11b 3.32 ± 0.11 a 3.26 ± 0.44 b 38.90 ± 0.93b 40.60 ± 2.82 b kd 2.83 ± 0.16b 2.93 ± 0.13b 2.93 ± 0.13 a 2.98 ± 0.04 b 39.40 ± 0.55b 38.80 ± 2.65 b note: a= cross of r xr african catfish broods exhibiting mhc i marker, b= cross of r xr african catfish broods exhibiting mhc i marker, c= cross 1 1 2 2 of r xr african catfish broods exhibiting mhc i marker, d= cross of african catfish control group that did not exhibit mhc i marker. ka, kb, 3 3 kc, and kd respectively are fish from a, b, c, and d crossing injected by phosphate buffer saline (pbs). different superscript letter in the same coloumn showed a significant difference (p<0.05) biotropia vol. 25 no. 2, 2018 98 table 1 percentage of mhc i marker inheritance and survival of african catfish second generation post aeromonas hydrophila bacterial infection parameter crosses a b c d fish carrying the marker (%) 93.0 77.0 97.0 46.7 survival rate (%) 76.7 ± 7.6b 80.0 ± 5.0b 75.0 ± 5.0b 38.3 ± 7.6a note: a= cross of r xr african catfish broods exhibiting mhc i marker, b= cross of r xr african catfish broods exhibiting mhc i marker, c= cross 1 1 2 2 of r xr african catfish broods exhibiting mhc i marker, d= cross of african catfish control group that did not exhibit mhc i marker. different 3 3 superscript letter in the same row showed a significant difference (p<0.05). against pathogen infection (li . 2013; hastuti et al & subandiyono 2015). leucocyte differentiation was only observed post-infection (table 3). lymphocyte count of fishes challenged by a. hydrophila bacteria was lower compared to those injected by pbs (p<0.05). lymphocyte count of d cross fish (62.00± 2.52%) challenged by a. hydrophila was lower (p<0.05) compared to a cross (72.00 ± 2.08%), b cross (75.00 ± 6.00%), and c cross (79.00 ± 1.53%). neutrophyl count of fish injected with pbs (4.00-7.00 %) was lower compared to those challenged by a. hydrophila (p<0.05). between challenged crosses, d cross fishes (26.00 ± 2.52%) had higher neutrophyl count compared to a cross (18.00 ± 4.51%), b (16.00 ± 3.79%), and c cross (12.00 ± 3.00%). monocyte count was also higher in fishes challenged by a. hydrophila bacteria (p<0.05), and monocyte count between all crosses were similar (p>0.05). significantly different leucocyte profile between f2 fish from broods exhibiting tock marker and those from broods that did not are leucocyte differentiation, especially lymphocyte and neutrophyl (table ). the decrease of 3 lymphocyte in fish whose parents exhibiting mhc i marker post bacterial infection is lower compared to those whose parent did not exhibit mhc i marker. the lymphocyte decreased in count after challenge in f2 fishes whose parent exhibit mhc marker s lower compared to wa those whose parent did not exhibit mhc i marker. in contrast, the increase in neutrophyl count of their progenitor did not exhibit mhc i marker was higher compared to those whose parent exhibit mhc i marker. the difference in lymphocyte and neutrophyl count indicated the potential of using leucocyte differential parameter to identify fishes resistant to pathogenic bacterial infection. in addition, difference in lymphocyte and neutrophyl count showed their role in a. hydrophyla bacteria phagocytosis that resulted in f2 fish exhibiting mhc i marker showing higher (hastuti survival & subandiyono 2015). lymphocyte count in healthy catfish ranges between 80.0-90.0%, monocyte less than 15.13%, and neutrophyl about 6.0-8.0% (andayani et al. 2014). the lymphocyte count of fish challenged by bacteria (62.0-79.0%) s less compared to wa unchallenged fish (88.0-93.0%). this is in line with what was reported by ( ), martins et al. 2008 that lymphocyte percentage decreased when an individual is suffering from disease . infection neutrophyl of fish that were not challenged (4.07.0%) s lower compared to fishes that were wa challenged (12.0-16.0%). neutrophyl has an active role in first phase of inflammation. neutrophyl would be produced around the first 6 hour after inflammation (kuby 1997). thus, neutrophyl count would increase if inflammation caused by aeromonas hydrophila bacterial infection occurs. f2 fish percentage with inflamation was a bit lower compared to fish whose parent did not exhibit mhc i marker. this support the lower neutrophyl count compared to fish whose parent did not exhibit mhc i marker. monocyte count of fishes that were challenged was higher (9.014.0%) compared to monocyte count of fish injected by pbs (2.0-5.0%). the increase in monocyte count showed non-specific immune table 3 lymphocyte, neutrophil, and monocyte percentage of second generation african catfish from parents exhibiting mhc i marker challenged by aeromonas hydrophila bacteria treatment leucocyte differentiation (%) lymphocyte neutrophyl monocyte a 72.00 ± 2.08b 18.00 ± 4.51c 10.00 ± 3.46cd b 75.00 ± 6.00bc 16.00 ± 3.79bc 9.00 ± 2.89bc c 79.00 ± 1.53c 12.00 ± 3.00b 9.00 ± 3.79bcd d 62.00 ± 2.52a 26.00 ± 2.52d 14.00 ± 3.61d ka 88.00 ± 3.61d 7.00 ± 1.53a 5.00 ± 3.06a kb 93.00 ± 1.53d 4.00 ± 0.58a 4.00 ± 1.15ab kc 91.00 ± 2.08d 5.00 ± 0.33a 2.00 ± 1.00a kd 91.00 ± 0.58d 6.00 ± 0.01a 3.00 ± 0.58a note: a= cross of r xr african catfish broods exhibiting mhc i marker, b= cross of r xr african catfish broods exhibiting mhc i marker, c= cross 1 1 2 2 of r xr african catfish broods exhibiting mhc i marker, d= cross of african catfish control group that did not exhibit mhc i marker. ka, kb, 3 3 kc, and kd respectively are fish from a, b, c, and d crossing injected by phosphate buffer saline (pbs). different superscript letter in the same column showed a significant difference (p<0.05). 99 growth and resistance of f2 african catfish against aeromonas hydrophila – alimuddin et al. response from catfish's body against h a. ydrophila pathogen. clinical symptoms observation was conducted 24-96 hours post challenge. observed clinical symptoms were ulceration on skin and dropsy (edema) by the under belly of african catfish. from visual observation, about 40% of f2 offsprings of broods exhibiting mhc i marker suffered ulceration, however all control fish suffered ulceration. other studies support the assertion that f2 fish from parents carrying the mhc i marker has a higher resistance than that of from parents without the marker. clinical symptoms shown by f2 catfish were similar with those reported by previous research. clinical symptoms can be observed in 12-96 hours post infection. clinical symptoms commonly observed in 24 hours post infection were swelling skin and inflammation around injection area of a. hydrophila. afterward, fish would show dropsy, followed by ulceration (wulandari et al. 2014). the behavior of fishes during challenged test were lowering appetite and irregular swimming pattern. fishes tend to hang floating in the surface or passively at the bottom of the aquaria. most of the fishes died with damaged fin and exuded mucus. according to rey et al. (2009), fish infected by aeromonas hydrophila will show dropsy or liquidfilled swelling belly, damaged meat or ulcer indicated by skin injury, and loss of blood. infected fish will also show clinical symptoms such as irregular swimming or fish damage. weight gain and survival rate during nursery f2 fish body weight gain and survival rate during nursery are presented in table 4. the initialbody weight of fishes reared for growth rate test was 0.11±0.03 g. the results showed that daily growth rate (dgr) from two mhc i marker exhibiting f2 cross (cross b: 7.87% day and c: -1 8.32% day ) was higher (p<0.05) compared to -1 control which did not exhibit the marker effect (d cross: 7.15% day ). however, dgr of fish cross -1 a was the same as cross d (p>0.05). meanwhile, survival rate of all treatment fish was the same (p>0.05). the water quality during nursery was 24.7-30.30c temperature, 7.40-7.60 mg l-1 dissolved oxygen, 0.02-0.95 mgl ammonia and -1 6.4-7.70 ph. growth and determine aquaculture survival productivity level. in this research, f2 generation of african catfish was nursed in aquar with ia relatively good water quality. in that condition, f2 african catfish from parents with mhc i marker (crosses b and c) had higher weight gain, while the was the same with african catfish survival with those that did not exhibit the marker (table 4). thus, cultivation of african catfish exhibiting mhc i marker has the potential in having higher productivity. however, weight gain vary among crosses, necessary to choose parent therfore, its that produces higher growth. in addition, it is most likely that no link between weight gain and presence of mhc marker in parent. furthermore, a. hydrophilaif bacterial infection exist, farmers that rear african catfish exhibiting mhc i marker has greater probability in getting higher harvest compared to those with fish without mhc i marker. the statement is supported by the result of this research, through a. hydrophila survival challenge with 2 higher compared to f2 fish whose parent did not exhibit the mhc i marker. this result is relatively consistent with what was reported by azis . et al (2015b) in f1 generation of african catfish. this result also showed that mhc i marker expressing african catfish's is stable. in resistance table 4 daily growth rate (dgr) and survival rate of second generation african catfish that exhibited mhc i marker and that did not exhibit mhc i marker, in relation to their endurance against aeromonas hydrophila bacterial infection while kept in 80-l volume aquaria for 2.0 months parameter crosses a b c d dgr (%/day ) 7 .53 ± 0.57ab 7 .87 ± 0.28bc 8.32 ± 0.60c 7 .15 ± 0.18a survival rate (%) 90 .00 ± 3.33 a 98 .30 ± 3.33a 95 .80 ± 3.85a 96 .70 ± 3.30a note: a= cross of r xr african catfish broods exhibiting mhc i marker, b= cross of r xr african catfish broods exhibiting mhc i marker, c= cross 1 1 2 2 of r xr african catfish broods exhibiting mhc i marker, d= cross of african catfish control group that did not exhibit mhc i marker. different 3 3 superscript letter in the same row showed a significant difference (p<0.05). biotropia vol. 25 no. 2, 2018 100 accordance to varieties from breeding in indonesia, at least generation required to test f3 is the performance consistency of mhc i marker exhibiting african catfish before it could be released in fact khv resistant for public use. the common carp strain developed through mhc ii marker based selection (alimuddin . 2011) has et al pass releas as a new fish strain at third generation in 2015. conclusion second generation african catfish from parents exhibiting mhc i marker is known to have higher growth rate and resistant against aeromonas hydrophila bacterial infection compared to fish whose parents did not exhibit mhc i marker. mhc i marker is inherited by f2 generation, similar percentage to what is found in f1 generation. further study is needed to observe whether the oligonucleotide primers anneals to another gene that having the same nucleotide at 3', and the pcr products are the same gene as that found in f2 fish from parents carrying the mhc i marker. acknowledgements this research was partially supported by psu research grant no.: 627/it3.11/ pn/2016 from ministry of research and higher education, indonesia. references alimuddin, mubinun, santika a, carman o, faizal i, sumantadinata k. 2011. identification of the majalaya common carp strain resistance to khv infection using cyca-dab1*05 allele as a marker. indonesian aquaculture journal 6(2):157-63. american public heatlh association (apha) [internet]. 2005. standard methods for the examination of water and wastewater. [cited 2016 aug 28]. available from: http://www.mwa.co.th/download/file_ upload/smww_1000-3000.pdf andayani, marsoedi s, sanoesi e, wilujeng ae, suprastiani h. 2014. profil hematologis beberapa spesies ikan air tawar budidaya. [hematology profile of freshwater fish species]. national conference – green technology 3. p. 363-5. anderson dp, siwicki ak. 1995. basic hematology and serology for fish health programs. manila (ph): fish health section, asian fisheries society. azis, alimuddin, sukenda, zairin mjr. 2015a. identifikasi kandidat marka mhc i pada ikan lele (clarias sp.) tahan infeksi aeromonas hydrophila. [identification of mhc i marker candidate in catfish (clarias sp.) resistance to aeromonas hydrophila infection]. jurnal riset akuakultur 10(2):261-9. azis, alimuddin, sukenda, zairin mjr. 2015b. mhc i molecular marker inheritance and first generation catfish (clarias sp.) resistance against aeromonas hydrophila infection. pak j biotechnol 12(2): 131-7. blaxhall pc, daisley kw. 1973. routine hematological methods for use with fish blood. j fish biol 5(6):771-81. del coral f, shotts eb, brown j. 1990. adherence haemagglutination and cell surface characteristics of motile aeromonads virulent for fish. j fish dis 13(4):255–68. hastuti s, subandiyono. 2015. catfish (clarias gariepinus burch) well-being which maintained with biofloc technology. ijfst 10(2):74-9. kirpichnikov vs. 1999. genetics and breeding of common carp. paris (fr): inra editions. kuby j. 1997. immunology. california (us): w.h freeman and company. li c, wang r, su b, luo y, terhune j, beck b, peatman e. 2013. evasion of mucosal defenses during aeromonas hydrophila infection of channel catfish (ictalurus punctatus) skin. dev comp immunol 39(4):447-55. martins ml, mouriño jlp, amaral gv, vieira fn, dotta g, jatobá amb, … pereira-jr g. 2008. haematologica l changes in nile t i lap ia experimentally infected with enterococcus sp. braz j biol 68(3):657-61. ministry of marine affairs and fisheries republic of indonesia (mmaf) [internet]. 2016. mmaf developed environmental friendly method in catfish farming with biofloc. [cited 2016 aug 28]. available from: http://www.djpb.kkp.go.id/arsip/c/451/ kkp-kembangkan-teknologi-budidaya-ikan-leleramahl ingkungan-metode-b iofok-untukkeberlanjutan/?category_id= rakus lk. 2008. major histocompatibility (mh) polymorphism of common carp link with disease resistance [thesis]. retreived from wageningen university. rey a, verjan n, ferguson hw, iregul c. 2009. pathogenesis of aeromonas hydrophilla strain kj99 infection and its extracelullar products in two species of fish. vet rec 164(16):493-9. doi: 10.1136/vr.164.16.493 101 growth and resistance of f2 african catfish against aeromonas hydrophila – alimuddin et al. triyaningsih, sarjito, prayitno sb. 2014. pathogenicity of aeromonas hydrophila isolated from catfish (clarias gariepinus) derived from boyolali. jamtech 3(2):117. wulandari a, prayitno sb, sarjito. 2014. pathogenicity of k14 isolate which isolated from catfish (clarias gariepinus) derived from demak. jamtech 3(2):1439. yanto h, hasan sunarto. 2015. studi hematologi untuk h, diagnosa penyakit ikan secara dini di sentra produksi budidaya ikan air tawar sungai kapuas kota pontianak. [early fish disease diagnose through haematological analysis in main center for freshwater aquaculture, kapuas river, pontianak]. jurnal akuatika 6(1) 11-20.: zhang d-h, shoemaker experimental d, xu c. 2016. induction of motile in channel aeromonas septicemia catfish ( ) by waterborne challenge ictalurus punctatus w aeromonas hydrophila. ith virulent aquaculture reports 3 18 23. : biotropia vol. 25 no. 2, 2018 102 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 biotropia vol. 31 no. 1, 2024: 23 33 doi: 10.11598/btb.2024.31.1.2011 23 antibacterial and diastase enzyme activities of honey apis mellifera from indonesia rara annisaur rosyidah, akhmad endang zainal hasan* and dimas andrianto department of biochemistry, faculty of mathematics and natural sciences, ipb university, bogor 16800, indonesia received 29 april 2023 / revised 30 may 2023 / accepted 30 may 2023 abstract the quality of monofloral honey from apis mellifera as an antibacterial can be influenced by the activity of the diastase enzyme and the secondary metabolites from the nectar source. therefore, this study aimed to compare the activity of the diastase enzyme and the effectiveness of monofloral honey derived from apis mellifera bees as a natural antibacterial agent against staphylococcus aureus and escherichia coli. sampling for monofloral honey was carried out from nine different nectar sources, namely acacia carpa, calliandra calothyrsus, nephelium lappaceum, melaleuca laucadendron, ceiba pentandra, mangifera indica, coffea robusta 1, coffea robusta 2, and hevea brasiliensisensi. furthermore, diastase content was determined using uv-vis spectrophotometry and the bacterial inhibition zone using the disc diffusion method. the principal component analysis (pca) was used to analyze the clustering of diastase enzyme and antibacterial activity. the results showed that the highest diastase activity in monofloral honey was mangifera indica, nephelium lappaceum, and coffea robusta 2 at 20.00 dn. this was followed by nephelium lappaceum, ceiba pentandra, and hevea brasiliensisensisensi at 10.00 dn, acacia carpa at 6.67 dn, coffea robusta 1 at 5.00 dn, and calliandra calothyrsus 4.00 dn. the clear zones for staphylococcus aureus on coffea robusta 2, acacia carpa, nephelium lappaceum, coffea robusta 1, ceiba pentandra, hevea brasiliensisensisensi, nephelium lappaceum, calliandra calothyrsus, and mangifera indica were 19.47, 18.53, 17.73, 17.03, 16.12, 16.10, 16.03, 15.73, and 14.73 mm, respectively. additionally, the clear zones for escherichia coli on ceiba pentandra, coffea robusta 2, acacia carpa, coffea robusta 1, melaleuca laucadendron, mangifera indica, hevea brasiliensisensisensi, calliandra calothyrsus, and nephelium lappaceum were 27.93, 26.13, 24.60, 24.53; 24.53, 24.07, 21.90, 21.60, and 21.53 mm, respectively. in conclusion, clustering analysis was conducted based on nectar sources to evaluate antibacterial and diastase activity. the clusters identified are cluster 1 consisting of hevea brasiliensisensi, cluster 2 including mangifera indica, and nephelium lappaceum. others are cluster 3 consisting of acacia carpa and calliandra calothyrsus, and clustergroup 4 including nephelium lappaceum, ceiba pentandra, coffea robusta 1, and coffea robusta 2. therefore, it was necessary to carry out antibacterial testing of other bacteria, specifically salmonella typhi, and determine the minimum inhibitory concentration (mic) of honey with the best antibacterial activity in various concentration variations. keywords: antibacterial, clustering, diastase enzyme, monofloral honey introduction the quality of honey can be affected by several factors, such as during harvest, water content, color, taste, and aroma of honey. harvesting honey necessitates careful timing when it is ripe and the bees are starting to close the nectar cells. the water content of honey also affects the quality by containing approximately 17-21% water. honey is rich in simple carbohydrates because the enzymes diastase and invertase are added when the nectar is sucked and regurgitated. the diastase enzyme is produced by bees during the ripening process and is used to assess the quality of honey (sukmawati et al. 2015). based on data from riskesdas (2018), the prevalence of diarrhea in indonesia is 37.88% and has increased in 2019 to 40% in toddlers (ri ministry of health 2020). furthermore, the age group with the highest prevalence of diarrhea was 1 to 4 years (11.5%) and infants (9%). according to the who (2018), gastroenteritis in the general population has a mortality rate of 525,000 deaths per year in children under 5 years with an *corresponding author, email: pakzainalhasan@gmail.com mailto:pakzainalhasan@gmail.com biotropia vol. 31 no. 1, 2024 24 incidence rate of 1.7 million. this condition makes gastroenteritis the second leading cause of death due to infectious diseases. acute gastroenteritis is diarrhea that lasts up to 14 days and is characterized by an increase in stool volume, frequency, and water content. the most common causes of diarrhea are infections in the form of viruses, bacteria, and parasites. it is often caused by an infectious disease from staphylococcus aureus and escherichia coli bacteria, which is still a major problem in public health (muziburrahman et al. 2022). staphylococcus aureus is a round gram-positive bacteria arranged such as clusters of grapes and is quite active in its division, which is commonly found in burns and hospitals (yuliati 2017). on the other hand, escherichia coli is a gram-negative rod-shaped bacterium and can cause various diseases, such as diarrhea, which has an incubation period of 10-24 hours before showing symptoms (pratiwi 2018). antibacterial compounds have been widely used to treat diseases caused by bacteria. it is imperative to adopt preventive measures to hinder the escalating resistance of bacteria to drugs, as over time, these pathogenic bacteria can develop resistance to antibacterial compounds (panjaitan et al. 2018). one way to prevent the emergence of bacterial resistance to synthetic antibacterials is the use of natural ingredients having antibacterial activity, including honey (hanifa et al. 2020). the production of honey occurs through the transformation of nectar, a process that involves the reduction of water content within both the bee's body and the beehive. the various types of honey that exist and their potential to be used as an antibacterial agent promote this study. some of the compounds present in honey are proteins, flavonoids, and low ph allowing for the inhibition of bacterial growth. according to nayik and nanda (2015), light-colored honey contains less phenol than dark-colored. the phytochemical content of each honey varies depending on the origin of the nectar from the honey. this content is useful as an antioxidant, antibacterial, anti-inflammatory, antiviral, and antidiabetic agent, as well as affects the human body's metabolism properly, hence, they have the potential to improve health and prevent various diseases (liu et al. 2013). honey is classified as monofloral (one type of plant) or multi floral (several types of plants) depending on the origin of its nectar (soares et al. 2018; zhang et al. 2019). presently, monofloral honey from apis mellifera bees from indonesia has antibacterial compounds. the effectiveness of suppressing bacterial growth varies in relation to the content of active antibacterial compounds found in honey, as well as the extractive substances derived from nectar. a total of 9 honey specimens of the monofloral species apis mellifera from indonesia were characterized in this study. therefore, this study aimed to determine the activity of the diastase enzyme and the effectiveness of monofloral honey derived from apis mellifera bees as a natural antibacterial agent against staphylococcus aureus and escherichia coli. materials and methods materials the materials used in this study included 9 specimens of apis mellifera honey, distilled water, starch, iodine, nacl, acetate buffer, staphylococcus aureus atcc6538 and escherichia coli atcc8739 bacterial cultures, nutrient broth (nb) and mueller hiton agar (mha) media, ciprofloxacin, and aquabides. sample preparation samples of monofloral honey from indonesia, as shown in table 1, were stored in the laboratory of the biochemistry master’s study program, faculty of mathematics and natural sciences, bogor agricultural university. antibacterial and diastase enzyme activities of honey apis mellifera from indonesia – rara annisaur rosyidah et al. 25 table 1 origin of monofloral honey samples used species nectar plant city province apis mellifera acacia carpa calliandra calothyrsus nephelium lappaceum melaleuca laucadendron ceiba pentandra mangifera indica coffea robusta 1 coffea robusta 2 hevea brasiliensisensi tanjung jabung sukabumi subang sragen pati pasuruan malang jember jember jambi west java west java central java central java east java east java east java east java water content the water content of monofloral honey was measured using a refractometer according to the indonesian national standard (2018). after dripping one drop of honey sample onto the prism, it was closed and pointed towards the light, and the result was read from the blue line on the white scale. diastase enzyme activity analysis of the diastase enzyme activity of monofloral honey was performed according to the indonesian national standard (2018). a total of 5 g honey was added to 2.5 ml acetate buffer solution ph 5.3 and 1.5 ml of 0.5 m nacl solution, homogenized into a 25 ml volumetric flask, and adjusted to the mark by adding distilled water. subsequently, 10 ml of the mixture was heated in a water bath at 40°c. after 15 minutes, 5 ml of starch solution was added and homogenized. every 15 minutes, 1 ml of the sample mixture was added to 10 ml of 0.0007 n iodine and homogenized, and then 25 ml volume of distilled water was added. the absorbance value was measured at λ 660 nm and the initial limit of reaction time starts from the addition of the iodine indicator. furthermore, the collection of the solution was continued until the value of a <0.235 was obtained within a certain time interval. diastase enzyme activity (dn) was calculated by the following formula: dn = 300 t (1) where: dn = diastase enzyme activity t = time used to reach the value of a<0.235 antibacterial activity the antibacterial activity of monofloral honey was analyzed according to the method of devi (2022) with modifications. furthermore, bacterial cultures of staphylococcus aureus and escherichia coli rejuvenated in 50 ml nutrient broth (nb) media were used as tested strains for antibacterial testing. each strain was incubated for 24 hours at 37ºc and the culture that grow were subjected to the disk diffusion test. the mueller hiton agar (mha) media used in the test were sterilized using an autoclave at 121˚c for 15 minutes. furthermore, the mha media was then poured into a sterile petri dish until solidified and the bacterial culture was streaked on the mha media with a known od value. sterile discs were left overnight to absorb the honey sample to be tested at a concentration of 100% and then dried in an oven at 45˚c for 2 hours. the prepared discs were placed on a bacterial culture scratch on mha and incubated at 37˚c for 24 hours. furthermore, the clear zone formed around the disc was measured with a vernier caliper. ciprofloxacin at a concentration of 100 ppm (0.1%) and 25 µl of blank distilled water was used as the positive and negative control, respectively. data analysis each experiment was carried out in triplicate, and the diastase enzyme activity and antibacterial activity resulting in each replicate were written as the mean ± standard deviation (n=3). the oneway anova test was used to analyze data on all honey samples with a 0.05 level of significance (α) using spss version 25. a p-value <0.05 indicated that the data were significantly different, and should be further tested. duncan's test was used to determine the effect of significant differences in each monofloral honey. furthermore, the biotropia vol. 31 no. 1, 2024 26 classification of monofloral honey based on diastase enzyme and antibacterial activity was determined by the principal component analysis (pca) chemometric method with minitab 16. results and discussion water content among the 9 samples analyzed, the water content of honey adhered to predetermined standards. coffea robusta 2 honey had the lowest water content at 17.50%, while calliandra calothyrsus had the maximum at 22.00% determined by sni. the moisture content of nephelium lappaceum, melaleuca laucadendron, ceiba pentandra, mangifera indica, coffea robusta 1, acacia carpa, and hevea brasiliensisensi were 21.50%, 20.50%, 20.50%, 19.50%, 19.00%, 18.50%, and 18.00%, respectively. the anova statistical analysis supported this result, showing a significant difference in the water content of the 9 monofloral honeys (p-value <0.05). this showed that differences in plants as a source of nectar had an impact on the water content of honey. the results of duncan's test (α=0.05) on the water content of 9 monofloral honeys showed 8 groups, namely (1) calliandra calothyrsus, (2) nephelium lappaceum l., (3) melaleuca laucadendron l. and ceiba pentandra (l.) gaertn, (4) mangifera indica, (5) coffea robusta 1, (6) acacia carpa, (7) hevea brasiliensisensi m., and (8) coffea robusta 2. these groups were categorized based on the proximity of their water content, as shown in figure 1. the water content in honey determines the stability of fermentation because it allows easy fermentation over time. therefore, honey needs to be subjected to heat treatment to prevent fermentation by sugar-tolerant yeasts and maintain its liquid state (singh and singh 2018). relative humidity (rh) in indonesia ranges from 60–90%, indicating that the water content in honey is approximately 18.3–33.1%. honey is hygroscopic, and must be stored in a place that is not permeable to air. according to pujiarti et al. (2021), signs of a fermentation process are the foam and gas produced by honey. hasan et al. (2020) reported that the antibacterial activity of multiflora honey from riau rhu4 has the lowest water content of 16.45% and rhi2 honey of 20.56%. figure 1 the water content of monofloral honey note: different lowercase letters show statistically significant differences (α=0.05). 1 8 ,5 0 f 2 2 ,0 0 a 2 1 ,5 0 b 2 0 ,5 0 c 2 0 ,5 0 c 1 9 ,5 0 d 1 9 ,0 0 e 1 7 ,5 0 h 1 8 ,0 0 g 0 5 10 15 20 25 a ca ci a c a rp a c a ll ia n d ra ca lo th yr su s n ep h el iu m la p p a ce u m m el a le u ca la u ca d en d ro n c ei b a p en ta n d ra m a n g if er a i n d ic a c o ff ee r o b u st a 1 c o ff ee r o b u st a 2 h ev ea b ra si li en si w at er c o n te n t ( % ) antibacterial and diastase enzyme activities of honey apis mellifera from indonesia – rara annisaur rosyidah et al. 27 the fermentation process in yeast can be significantly influenced by the high water content in honey, leading to a decline in its overall quality. moreover, the shelf life of honey is affected by its water content and a higher water content results in a relatively short shelf life. according to hasan et al. (2020), honey can absorb up to 33% water by weight in moist air. good-quality honey has a low water content, indicating that thicker honey has a lower water content (swari et al. 2019). honey harvested at an older age contains less water than at a younger age. generally, at harvest ages <14 days, the water content of honey is relatively high. according to nasharuddin et al. (2022), indonesian honey is often fermented due to its high water content of >26%. the indonesian national standard (2018) reported that the quality requirement of water content in honey was a maximum of 22%. a previous study showed a correlation between the water and sugar content of honey, and the honey crystallization process was natural (nasharuddin et al. 2022). diastase enzyme activity the average results of diastase enzyme activity in monofloral honey were shown in figure 2. the highest diastase enzyme activity in mangifera indica, nephelium lappaceum, and coffea robusta 2 was 20.00 dn, while the lowest was in calliandra calothyrsus at 4.00 dn. furthermore, the diastase enzyme activity of melaleuca laucadendron, ceiba pentandra, and hevea brasiliensisensi was 10.00 dn, whereas acacia carpa and coffea robusta 1 were 6.67 dn and 5.00 dn, respectively. these results were supported by anova statistical analysis which showed a significant difference between the 9 monofloral honey (pvalue <0.05) based on their diastase enzyme activity. this showed that differences in plants as a source of nectar affected it. it also showed the impact of the differences in plants as a source of nectar. the results of duncan's test (α=0.05) on the activity of the diastase enzyme from 9 monofloral honey showed 5 groups, namely (1) mangifera indica, nephelium lappaceum l., and coffea robusta 2, (2) melaleuca laucadendron l., ceiba pentandra (l.) gaertn, and hevea brasiliensisensi m., (3) acacia carpa, (4) coffea robusta 1, and (5) calliandra calothyrsus m. these monofloral honey awerere categorized based on their proximity to the activity of the diastase enzyme they possess, as shown in figure 2. the diastase enzyme is one of many different types found in every variety of honey and contributes to its nutritional value. bees introduce the diastase enzyme during the ripening of the honey. this enzyme breaks the glucosidic bonds in starch and other oligoand polysaccharides into simple sugars. according to singh and singh (2018), adequate heating and storage are crucial to retaining the market value of honey since the diastase enzyme is extremely sensitive to heat. figure 2 the diastase enzyme activity value of monofloral honey note: different lowercase letters show statistically significant differences (α=0.05)/. 6 ,6 7 c 4 ,0 0 e 2 0 ,0 0 a 1 0 ,0 0 b 1 0 ,0 0 b 2 0 ,0 0 a 5 ,0 0 d 2 0 ,0 0 a 1 0 ,0 0 b 0 5 10 15 20 25 a ca ci a c a rp a c a ll ia n d ra ca lo th yr su s n ep h el iu m la p p a ce u m m el a le u ca la u ca d en d ro n c ei b a p en ta n d ra m a n g if er a in d ic a c o ff ee ro b u st a 1 c o ff ee ro b u st a 2 h ev ea b ra si li en si d ia st a se n u m b er ( d n ) biotropia vol. 31 no. 1, 2024 28 according to ichsan et al. (2022), in the chemical test with the diastase enzyme, 17 out of 22 samples were found to have good quality because they met the sni requirement. the diastase number (dn) value ranged from 0.64 to 23.11, while for fake honey, the value of dn was 0, indicating that it was undetectable. the absence of the diastase enzyme in a honey sample can serve as an indicator of potential adulteration, suggesting that the sample may be classified as fake honey. such samples solely consist of sugars without the presence of the diastase enzyme. furthermore, tulandi (2019) showed that storage temperature greatly affected the quality of honey. there was a significant difference in diastase enzyme activity in each treatment at storage temperature. the best storage temperature for honey was 26˚c (room temperature) compared to hot (50˚c) and cold (±5˚c) temperatures. the activity of this enzyme decreased with storage and heating time, as indicated by its measurement through dn. it is observed that diastase activity decreases as the temperature rises, and the decline becomes more significant with higher temperature increments. the diastase activity decreases quickly during isothermal heating at a constant temperature but increases once again as the number of heating cycles increases (singh and singh 2018). according to harjo et al. (2015), diastase activity in rubber and rambutan honey was 11.89 dn and 11.58 dn, respectively. the presence of the diastase enzyme in honey can be attributed to the bee's ability to produce saliva. consequently, a higher value of the diastase enzyme indicates a greater concentration of bee saliva contained in honey. a decrease in the value of the diastase enzyme activity can occur in the storage process before testing. therefore, it is advisable to conduct diastase enzyme testing shortly after the honey is harvested to ensure accurate assessment. the activity of the diastase enzyme is an indicator of the quality of honey because it can indicate its processing and storage time. according to pujiarti et al. (2021), storage can significantly affect the inactivation of enzymes. honey products usually do not meet requirements for commercialization after they have been heated, reheated, or stored for a long time, because the diastase activity is below the quality requirements. according to the indonesian national standard (2018), the quality requirements for the value of honey diastase activity are more than 3 dn. antibacterial activity the smallest clear zone for staphylococcus aureus was 14.73 mm in mangifera indica, while the largest was 19.47 mm in coffea robusta 2. the clear zones for acacia carpa, melaleuca laucadendron, coffea robusta 1, ceiba pentandra, hevea brasiliensisensi, nephelium lappaceum, and calliandra calothyrsus were 18.53 mm, 17.73 mm, 17.03 mm, 16.12 mm, 16.10 mm, 16.03 mm, and 15.73 mm, respectively, as shown in figure 3. the smallest clear zone for escherichia coli was 21.53 mm in nephelium lappaceum, while the largest was 27.93 mm in ceiba pentandra. the values for coffea robusta 2, acacia carpa, coffea robusta 1, melaleuca laucadendron, mangifera indica, hevea brasiliensisensi, and calliandra calothyrsus were 26.13 mm, 24.60 mm, 24.53 mm, 22.53 mm, 22.07 mm, 21.90 mm, and 21.60 mm, respectively, as shown in figure 4. the positive control on both bacteria showed that a clear zone was formed. this is consistent with the anova statistical analysis, showing that the 9 monofloral honey were significantly different (p-value <0.05) based on their antibacterial activity. the results of duncan's test (α=0.05) on the inhibition zone of staphylococcus aureus bacteria from 9 monofloral honey showed 6 groups, namely (1) coffea robusta 2 and acacia carpa, (2) acacia carpa and melaleuca laucadendron l., (3) melaleuca laucadendron l. and coffea robusta 1, (4) coffe robusta 1, ceiba pentandra (l.) gaertn, and nephelium lappaceum l., (5) ceiba pentandra (l.) gaertn, hevea brasiliensisensi m., nephelium lappaceum l., and calliandra calothyrsus m., and (6) mangifera indica. these monofloral honey were grouped based on the proximity of their inhibition zones, as shown in figure 3. furthermore, duncan's test at α=0.05 on the inhibition zone of escherichia coli bacteria showed that 4 groups, namely (1) ceiba pentandra (l.) gaertn, (2) coffe robusta 1, (3) acacia carpa and coffea robusta 2, and (4) melaleuca laucadendron l., mangifera indica, hevea brasiliensisensi m., calliandra calothyrsus m., and nephelium lappaceum l. they were groups based on the proximity of their inhibition zones, as shown in figure 4. antibacterial and diastase enzyme activities of honey apis mellifera from indonesia – rara annisaur rosyidah et al. 29 figure 3 the antibacterial activity of monofloral honey against staphylococcus aureus note: different lowercase letters show statistically significant differences (α=0.05). figure 4 the antibacterial activity of monofloral honey against escherichia coli note: different lowercase letters show statistically significant differences (α=0.05). the clear zone value is determined by subtracting the diameter of the disc used from the measured clear zone diameter. the effectiveness of bacterial inhibition by honey is influenced not only by physicochemical characteristics but also by its bioactive compounds. furthermore, the content of bioactive compounds is obtained from nectar sources that are taken and processed by honey bees (hasan et al. 2020). the monofloral honey antibacterial test was carried out on two test bacteria, namely staphylococcus aureus (gram-positive) and escherichia coli (gram-negative). furthermore, antibacterial activity was determined based on the diameter of the clear zone formed after incubation. this test used the antibiotic ciprofloxacin (100 ppm) as the positive control. according to the clinical laboratory standard institute (clsi) (2013), antibacterial activity is classified as weak or lacking when the resulting inhibition zone is ≤12 mm. similarly, it is classified as medium and strong when the inhibition zone is 13-17 mm and ≥18mm, respectively. based on this classification, the test results showed that monofloral honey had moderate to strong antibacterial activity against staphylococcus aureus. this was because the diameter of the resulting inhibition zone was in the range of 14.73 mm and 19.47 mm. the antibacterial activity of monofloral honey against escherichia coli was classified as strong because the diameter of the resulting inhibition zone was greater than 18 mm, ranging from 21.53 mm to 27.93 mm. according to hasan et al. (2020), the smallest inhibition zone of staphylococcus aureus for multi1 8 ,5 3 b ,c 1 5 ,7 3 f, g 1 6 ,0 3 e, f 1 7 ,7 3 c, d 1 6 ,1 2 e, f 1 4 ,7 3 g 1 7 ,0 3 d ,e 1 9 ,4 7 b 1 6 ,1 0 e, f 2 7 ,7 7 a 0 5 10 15 20 25 30 35 a ca ci a ca rp a c a ll ia n d ra ca lo th yr su s n ep h el iu m la p p a ce u m m el a le u ca la u ca d en … c ei b a p en ta n d ra m a n g if er a in d ic a c o ff ee ro b u st a 1 c o ff ee ro b u st a 2 h ev ea b ra si li en si c ip ro fl o xa c in c le ar z o n e s . a u re u s (m m ) 2 4 ,6 0 d 2 1 ,. 6 0 e 2 1 ,5 3 e 2 2 ,5 3 e 2 7 ,9 3 b 2 2 ,0 7 e 2 6 ,1 3 c 2 4 ,5 3 d 2 1 ,9 0 e 2 9 ,5 3 a 0 5 10 15 20 25 30 35 a ca ci a ca rp a c a ll ia n d ra ca lo th yr su s n ep h el iu m la p p a ce u m m el a le u ca la u ca d en … c ei b a p en ta n d ra m a n g if er a in d ic a c o ff ee ro b u st a 1 c o ff ee ro b u st a 2 h ev ea b ra si li en si c ip ro fl o xa ci n c le ar z o n e e . co li ( m m ) biotropia vol. 31 no. 1, 2024 30 floral honey from riau was 7.84 mm for 20% rhi1 honey, while the largest for rhi2 80% honey was 19.25 mm. the smallest inhibition zone of escherichia coli in rhi1 honey with a concentration of 40% was 1.22 mm, while the largest inhibition zone in plw1 60% honey was 4.58 mm. according to kaligis et al. (2020), the minimum inhibitory concentration (mic) of forest honey toward staphylococcus aureus, escherichia coli, and pseudomonas aeruginosa is 12.5%, 12.5%, and 25%, respectively. in this study, there was no minimum bactericidal concentration (mbc) in either forest or black honey. furthermore, the antibacterial effect of forest honey is superior to that of black honey by a small margin. gram-negative bacteria tend to be more sensitive to the antibacterial substances contained in honey than gram-positive. they also have hydrophilic groups that easily bind to polar compounds, making the antibacterial activity of honey more effective against gram-negative bacteria than gram-positive (wadi 2022). furthermore, the study conducted with monofloral honey from indonesia showed that escherichia coli was the most influential bacteria. this can be attributed to osmosis, facilitated by the high sugar content of honey, causing water to flow out of the cells, such that the resulting pure and undiluted honey prevents the growth of germs. according to almasaudi (2021), dehydration also causes cells to shrink, which hinders their ability to survive in a hypertonic sugar solution, thereby reducing the antibacterial potential. the vast majority of microbes normally grow in neutral environments with ph values between 6.5 and 7.5. one of the most distinguishing characteristics of the antibacterial qualities of honey is its acidity, which ranges between ph 3.2 and 4.5. its acidity is caused by the presence of a significant organic acid (gluconic) that is present in the sample at a concentration of 0.5% (w/v). the natural glucose oxidase enzyme converts glucose into glycogen acid, which is a particularly effective antibacterial agent. furthermore, low ph in pure and undisturbed honey can add to its antibacterial capabilities, when diluted in food or other body fluids (almasaudi 2021). cianciosi et al. (2018) reported that flavonoids and phenolic acids were among the broad group of compounds known as polyphenols and distinguished by their phenolic structures. these biological components are converted from nectar to honey and play a significant role in the health benefits of honey. the presence of numerous phenolic chemicals in honey provides a plausible explanation for its antibacterial properties (gunes et al. 2017). several studies explored the antibacterial activity of phenolic acids in honey. among these acids, caffeic was found to induce oxidative stress and chlorogenic acid increases membrane permeability, resulting in cytoplasmic and nucleotide leakage (gorniak et al. 2019). furthermore, gallic acid induces intracellular leakage as a result of the breakdown of cell membranes and increased hole formation (shi et al. 2016). p-coumaric acid disrupts cell membranes and bacterial dna brushing, and syringic acid causes cell membrane dysfunction (borges et al. 2013). the antibacterial mechanisms of flavonoids found in honey include apigenin and kaemferol, which can inhibit dna gyrase. galanin inhibits peptidoglycan synthesis from ribosomes and luteolin inhibits fas-i in mycobacteria and inhibits dna helicase dnab and recbcd. on the other hand, myricetin inhibits dna b helicase, pinocembrin induces cell lysis (collins et al. 2019), and catechins contribute to the generation of h2o2 (das et al. 2015). honey clusterization the results of the score plot on the antibacterial activity of staphylococcus aureus and escherichia coli and diastase activity show 4 main clusters of monofloral honey from indonesia with different nectar sources (figure 5). the first cluster (figure 5a) consists of hevea brasiliensisensi from jember. the second cluster (figure 5b) consists of mangifera indica from pasuruan and nephelium lappaceum from subang. the third cluster (figure 5c) consists of acacia carpa from tanjung jabung and calliandra calothyrsus from sukabumi. finally, the fourth cluster (figure 5d) consists of melaleuca laucadendron honey from sragen, ceiba pentandra from pati, coffea robusta 1 from malang, and coffea robusta 2 from jember. antibacterial and diastase enzyme activities of honey apis mellifera from indonesia – rara annisaur rosyidah et al. 31 figure 5 score plot results on antibacterial and diastase enzyme activity table 2 taxonomy of the nectar source plants used cluster species genus family ordo class phylum kingdom 1-1 hevea brasiliensisensi m. hevea euphorbiaseae euphorbiales dicotyledoneae spermatophyta plantae 2-1 mangifera indica mangifera anacardiaceae sapindales dicotyledoneae spermatophyta plantae 2-2 nephelium lappaceum l. nephelium sapindaceae sapindales magnoliopsida spermatophyta plantae 3-1 calliandra calothyrsus m. calliandra fabaceae fabales magnoliopsida magnoliophyta plantae 3-2 acacia crassicarpa acacia fabaceae fabales magnoliopsida magnoliophyta plantae 4-1 melaleuca laucadendron l. melaleuca myrtaceae myrtales magnoliopsida magnoliophyta plantae 4-2 ceiba pentandra (l.) gaertn ceiba malvaceae malvales magnoliopsida magnoliophyta plantae 4-3 coffea robusta coffea rubiaceae rubiales magnoliopsida magnoliophyta plantae 4-4 coffea robusta coffea rubiaceae rubiales magnoliopsida magnoliophyta plantae the clustering of monofloral honey derived from 9 nectar-sourced plants in indonesia was determined using the multivariate pca method. this analysis considered both the antibacterial activity and diastase enzymes. the results showed that there are groups with similarities and some exhibit dissimilarities. the pca method is used to simplify data and enhance readability by transforming the data linearly to form a new coordinate with maximum variance (ghani et al. 2016). the arrangement of taxonomy is based on phylogenetic and chemotaxonomy. in this context, plants that share a close kinship tend to possess similar types and homologies of secondary metabolite content. the identified chemical compounds have antibacterial activity. for example, cluster 1 is the genus hevea, which contains flavonoids (evahelda et al. 2017). cluster 2 is of the sapindales order, and it contains acetophenone, butanoic acid, and benzyl alcohol (rohman 2017). cluster 3 belongs to the fabaceae family, containing protocatechuic, gallic, caffeic, and ascorbic acid (obisitioiu et al. 2021). cluster 4 belongs to the magnoliopsida class, which is characterized by aromatic compounds, such as sesquiterpenes, isoflavonoids, and benzylisoquinone alkaloids (chassagne et al. 2021). the differences in honey characteristics, such as taste, aroma, color, and composition, can be attributed to the diverse range of plants whose nectar is collected by bees for honey production. these variations subsequently influence the antibacterial activity of honey. furthermore, several factors contribute to the antibacterial activity of honey, namely the types of flowers that take their nectar from different areas and their high sugar content (arumsari et al. 2019). conclusion in conclusion, the resulting diastase activity in monofloral honey includes mangifera indica, nephelium lappaceum, coffea robusta 2 at 20.00 dn, nephelium lappaceum, ceiba pentandra, and hevea brasiliensisensi at 10.00 dn, acacia carpa at 6.67 dn, coffea robusta 1 at 5.00 dn, and calliandra calothyrsus 4.00 dn. the inhibition zones for staphylococcus aureus on coffea robusta 2, acacia carpa, nephelium lappaceum, coffea robusta 1, ceiba pentandra, hevea brasiliensisensi, nephelium lappaceum, biotropia vol. 31 no. 1, 2024 32 calliandra calothyrsus, and mangifera indica were 19.47, 18.53, 17.73; 17.03, 16.12, 16.10, 16.03, 15.73, and 14.73 mm, respectively. furthermore, the inhibition zones for escherichia coli on ceiba pentandra, coffea robusta 2, acacia carpa, coffea robusta 1, melaleuca laucadendron, mangifera indica, hevea brasiliensisensi, calliandra calothyrsus, and nephelium lappaceum were 27.93, 26.13, 24.60, 24.53, 24.53, 24.07, 21.90, 21.60, and 21.53 mm, respectively. in this study, clusterization analysis was based on the antibacterial and diastase activity, namely cluster 1 consisting of hevea brasiliensisensi, cluster 2 comprising mangifera indica and nephelium lappaceum, cluster 3 containing acacia carpa and calliandra calothyrsus, as well as cluster 4 involving melaleuca laucadendron, ceiba pentandra, coffea robusta 1, and coffea robusta 2. therefore, it is necessary to carry out antibacterial testing of other bacteria, specifically salmonella typhi, and determine mic of honey with the best antibacterial activity in various concentration variations. acknowledgments the authors are grateful to the department of biochemistry, ipb university, for the support provided during this study. authors’ contribution rara annisaur rosyidah designed and conducted all the experiments, analyzed the data, performed the data visualization, and wrote the manuscript. akhmad endang zainal hasan and dimas andrianto assisted in 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[updated 2017 may 2; cited 2023 jan 22]. available from: https:// www.who.int/en/news-room/fact-sheets/detail/ diarrhoeal-disease. yuliati. 2017. test the effectiveness of honey solution as an antibacterial agent in the growth of staphylococcus aureus and pseudomonas aeruginosae using the disk diffusion method. med professional j 11(1): 7-15. zhang yz, chen yf, wu yq, si jj, zhang cp, zheng hq, hu fl. 2019. discrimination of the entomological origin of honey according to the secretions of the bee (apis cerana or apis mellifera). food res int 116: 362-369. https://www.who.int/en/news-room/fact-sheets/detail/diarrhoeal-disease https://www.who.int/en/news-room/fact-sheets/detail/diarrhoeal-disease https://www.who.int/en/news-room/fact-sheets/detail/diarrhoeal-disease 732 agustinus mardjoko (recovery of residual).cdr recovery of residual forest ecosystem as an impact of selective logging in south papua: an ecological approach agustinus murdjoko , djoko marsono , ronggo sadono 1* 2 2 and suwarno hadisusanto3 1 faculty of forestry, papua university, manokwari 98314, indonesia 2faculty of forestry, gadjah mada university, yogyakarta 55281, indonesia 3 afaculty of biology, gadjah mada university, yogyakarta 55281, indonesi received 7 december 2016/accepted 3 june 2017 abstract papua has been experiencing heavy logging activity in its forests for decades. however, only several studies focused on the effect of logging in the forest ecosystem. this research was aimed to analyze recovery processes of the forest ecosystem. the research was conducted in the logged tropical rainforest in south papua using ecological approach which used tree communities as biotic and soil condition as abiotic indicators. data were collected in the logging area of pt tunas timber lestari located in the tropical rainforest of south papua. there were five groups of forests used in this research i.e. unlogged, one year post selectively-logged, five years post selectively-logged, ten years post selectively-logged and fifteen years post selectively-logged forests. thirty nested plots were laid on each forest group. canonical correspondence analysis (cca) was applied to analyze the understory and upperstory plant communities. understory and upperstory plant communities formed different patterns due to logging. plant communities in the ten and fifteen years post-selectively logged forests were not similar to those in the unlogged forest. soil organic matter (som) content in the selectively logged forests was lower than that in the unlogged forest. these occurrences indicated that the selectively logged forests were still recovering and required more than fifteen years to be fully recovered. keywords: canonical correspondence analysis, edaphic factor, logged tropical forest, plant community, soil organic matter introduction tropical rainforests play an important role in ecosystem services, such as logging production (whitfeld et al. 2014; putz & romero 2014). the process of production mechanism in the tropical rainforest has a significant impact on abiotic and biotic elements (zambrano et al. 2014). those conditions result in the change in the tropical rainforest as an ecosystem and some circumstances of the secondary successional process take place as a response to ecological alterations. furthermore, most of the tropical rainforests are experiencing the alterations and the selective logging has a significant impact on ecological factors (corrià-ainslie . 2015; et al flores . 2014). hence, the logged tropical et al rainforests are counting on the ability of forest recovery itself. most indicators to analyse forest recovery are based on tree density, basal area (whitfeld . 2014; rutten . 2015) and et al et al growth rate of residual trees (do . 2016; et al hoang . 2011; west . 2014; sist . et al et al et al 2014; susanty . 2015) in the logged forests. et al however, the recovery of disturbed forests should not only be considered based on sustainable timber production, but the ecological elements such as soil conditions and residual trees should also be taken into account as forest recovery indicators. some areas in lowland tropical forests in south papua were intended as logging concession for decades (kuswandi & murdjoko 2015; murdjoko *corresponding author: agustinus.murdjoko.papua@gmail.com biotropia 4 3 7 230 245 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 3 732 230 2013; kuswandi 2014) . few studies concerning the effects of logging in papua logged forests were conducted. some studies focused only on damages, changes in basal area (gandhi & mitlöhner 2014), population dynamics of remaining trees (murdjoko 2013 ; kuswandi & murdjoko 2015; murdjoko 2016b) and et al. biomass stock change (hendri et al. 2012). therefore, it is necessary to analyze forest recovery using the ecological approach in south papua. in this analysis, the primary forest was considered as a stable forest ecosystem (pennington et al. 2015). ecological approach took tree communities as biotic factors where many processes such as tree associations, ecological responses of the tree to ecological change as well as successional development can be analyzed based on patterns of tree communities. besides that, soil condition alters after selective logging (hattori . 2013) et al mainly the amount of soil properties decrease such as nitrogen content (asase . 2014), soil et al organic matter (som) (prasetyo . 2015) and et al other nutrients (duah-gyamfi . 2014; wasrin et al & putera 1999; edwards . 2014; imai . et al et al 2012). consequently, the edaphic conditions were considered as abiotic indicators to support the explanation of the change in tree communities. this research was aimed to analyze recovery process of selectively logged tropical rainforest ecosystem in south papua using ecological approach. our hypotheses were: 1. tree communities in a selectively logged tropical rainforest were considered to be recovered when tree communities in the rainforest were similar to those in the primary forest; 2. the selectively logged tropical rainforest was considered to be recovered when the edaphic indicators in the rainforest were similar to those in the primary forest. materials and methods study area research was conducted in the logging area of pt tunas timber lestari located in the tropical rainforest of south papua with geographical position between 140 21` – 140 59` e and 05 50` o o o – 06 42` s (fig.1). the annual rainfall was between o 231 recovery of residual forest ecosystem: impact of selective logging murdjoko et al. figure 1 study area in logging concession of pt tunas timber lestari (murdjoko et al. 2016c) biotropia vol. 24 no. 3, 2017 232 were compared to the unlogged forest to observe the recovery process. the selective logging was carried out by selectively cutting commercial trees having diameter of ≥ 40 cm. sampling and data collection samples were collected in each forest group using systematic sampling plots. the first plot was placed at 200 m from the main road to avoid edge effect. the plots were rectangular with various sizes i.e. 1. 20 x 20 m for trees (d) having dbh (diameter at breast height) of ≥ 20 cm; 2. 10 x 10 m for poles (c) having dbh of 10 to < 20 cm; 3. 5 x 5 m for saplings (b) having height of > 1.5 m and dbh of < 10 cm; and 2 x 2 m for seedlings (a) having height of < 1.5 m. the four plots were set as nested plot (fig. 2a). thirty 3,000 and 4,000 mm with daily moisture range of 75 85 %. the edaphic condition was typified as lowland forest with almost flat topography with soil formed by alluvial process (petocz 1989). the vegetation was dominated by trees belong to dipterocarpaceae lauraceae myrtaceae, and families et al. 2015). (gandhi & mitlöhner 2014; kuswandi several other plants such as lianas, rattans, ferns, palms, herbs, orchids and pandanus grew and interacted with trees in this forest (murdjoko et al. 2016a). five groups of forests were used in this research i.e. unlogged, one year post selectivelylogged, five years post selectively-logged, ten years post selectively-logged and fifteen years post selectively-logged forests. the unlogged forest was taken as a primary forest which was a stable forest ecosystem. the selectively logged forests figure 2 nested plots to measure individual plant in both unlogged and selectively-logged forests note: a = plot for seedlings; b = plot for saplings; c = plot for for poles; d = plot for trees; (a) distance between plots = 100 m; (b) the 30 nested plots were laid on each forest group (unlogged, one year, five years, ten years and fifteen years post selectively-logged forests) 233 recovery of residual forest ecosystem: impact of selective logging murdjoko et al. nested plots were laid in each forest (fig. 2b) making a total of 150 nested plots for the 5 forest groups (unlogged, one year, five years, ten years and fifteen years post selectively-logged forests). seedlings and saplings were sampled as understory, while poles and trees were sampled as upperstory in both unlogged and selectively logged forests. data collected from seedlings, saplings, poles and trees consisted of numbers of individuals, diameter of individuals for those having dbh ≥ 10 cm and species name of individuals. species identification was carried out by two herbarium technicians. unidentified samples were set as voucher specimens and sent to the herbarium of "balai penelitian dan pengembangan lingkungan hidup dan kehutanan (bp2lhk) manokwari" and herbarium manokwariense (man) pusat penelitian keanekaragaman hayati universitas papua (ppkh-unipa), manokwari. validation of the species names of the individuals was checked online at http://www.theplantl ist .org/; http://plants.jstor.org and www.ipni.org/ipni/. soil samples were taken from the center and four corners of the 20 x 20 m plot. the litterfall samples were collected from each plot by making 1 x 1 m rectangular subplots in each plot. the soil and litterfall samples were sent to the laboratory of balai pengkajian teknologi pertanian yogyakarta for determining the content of soil organic matter (som) for soil samples as well as carbon (c) content, nitrogen (n) content and dry weight for litterfall samples. data and statistical analysis canonical correspondence analysis (cca) was applied to show the relationship among tree species using stem density and environmental factors (som, c, n contents and dry weight of litterfall) (ter braak 1987; ter braak 1986; khairil et al. 2014). plants communities were grouped as: a) understory consisted of small individuals (seedlings and saplings); and b) upperstory consisted of large individuals (poles and trees). tree communities were formed as a result of interaction among tree species, som, c content, n content, dry weight of litterfall and forest groups (unlogged, one year, five years, ten years and fifteen years post selectively-logged). the cca was computed using r softwarestatistical version 3.3.1. (r core with vegan package team 2014; oksanen 2013). the tree et al. communities were grouped using euclidean distance among tree species the euclidean . distance among tree communities was calculated as the average and confidence interval of 95%. results and discussion tree communities total tree species in the study area were 163 species and classified as understory (159 species) and upperstory (127 species) (table 1). within tree species, there were 106 species consisted of both understory and upperstory. table 1 understory (a) and upperstory (b) tree communities formed due to logging activities note: pf = unlogged forest; x1lf = one year post selectively-logged forest; x5lf = five years post selectively-logged forest; x10lf = ten years post selectively-logged forest; x15lf = fifteen years post selectively-logged forest; all = present in all forest groups; non_ac = not associated 234 biotropia vol. 24 no. 3, 2017 table 1 continued 235 recovery of residual forest ecosystem: impact of selective logging murdjoko et al. table 1 continued 236 biotropia vol. 24 no. 3, 2017 table 1 continued 237 recovery of residual forest ecosystem: impact of selective logging murdjoko et al. table 1 continued 238 biotropia vol. 24 no. 3, 2017 table 1 continued those species existed in each forest group (unlogged, one year, five years, ten years and fifteen years post selectively-logged). patterns of tree communities were formed for each forest group, especially for understory mostly occurred after logging activities. upperstory were mainly recruited from understory of remnant trees. several upperstory species were present before logging activities occurred in the forests. our study presented the results of understory and upperstory communities influenced by logging activities and edaphic conditions. there were three patterns established in our study i.e. 1. tree species formed a tree community in a forest group; 2. tree species present in all forest groups; and 3. tree species did not form a community. presence of certain tree species as understory in all forest groups was facilitated by ecological alterations, including logging activities. several tree species existed in all forest groups indicating that those tree species were not influenced by ecological alterations. distribution of understory tree community was depicted using cca having 55.34% of the variation for two axes; variation for axis 1 was 30% and variation for axis 2 was 25.34% (fig. 3; table 2). anova showed that the model was significant with < 0.05.p 239 recovery of residual forest ecosystem: impact of selective logging murdjoko et al. table 1 continued figure 3 understory of four tree communities formed due to logging activities symbolized as grey (species grown in pf), green (species grown in x1lf), yellow (species grown in x5lf) and blue (species grown in x10lf-x15lf) note: pf = unlogged forest; x1lf = one year post selectively-logged forest; x5lf = five years post selectivelylogged forest; x10lf = ten years post selectively-logged forest; x15lf = fifteen years post selectivelylogged forest; som = soil organic matter (%); lf_c = carbon content in litterfall (%); lf_n = nitrogen content in litterfall (%); lf_dw = dry weight of litterfall (g) canonical correspondence analysis (cca) grouped the understory tree species into four tree communities i.e. 28 species in the unlogged forest; 21 species in the one year post selectively-logged forest; 21 species in the five years post selectivelylogged forest and 17 species in the ten and fifteen years post selectively-logged forest (table 1a). distribution of upperstory tree community was shown of having variation of two axes of 58.26% with 31.24% variation for axis 1 and 27.03% variation for axis 2 (fig. 4; table 3). the cca model was significant at p < 0.05. edaphic factors interactions among som, c content, n table 2 summary of canonical correspondence analysis (cca) for understory tree community importance of components axes total inertia cca1 cca2 eigenvalue 0.2152 0.1818 0.7175 proportion explained 0.3 0.2534 cumulative proportion 0.3 0.5534 figure 4 upperstory of four tree communities formed due to logging activities symbolized as grey (species grown in pf), green (species grown in x1lf), yellow (species grown in x5lf) and blue (species grown in x10lf-x15lf) note: pf = unlogged forest; x1lf = one year post selectively-logged forest; x5lf = five years post selectivelylogged forest; x10lf = ten years post selectively-logged forest; x15lf = fifteen years post selectivelylogged forest; som = soil organic matter (%); lf_c = carbon content in litterfall (%); lf_n = nitrogen content in litterfall (%); lf_dw = dry weight of litterfall (g) table 3 summary of canonical correspondence analysis (cca) for upperstory tree community importance of components axes total inertia cca1 cca2 eigenvalue 0.1961 0.1697 0.6277 proportion explained 0.3124 0.2703 cumulative proportion 0.3124 0.5826 content, dry weight of litterfall and forest groups (unlogged, one year, five years, ten years and fifteen years post selectively-logged forests) were analyzed using cca to figure out the fitting edaphic factors as the indicators of logged forest recovery. results of cca showed that som tended to be higher in the unlogged forest, dry weight of litterfall tended to be higher in the five years post selectively-logged forest and c content of litterfall was higher in the one-year post selectively-logged forest (fig. 3 & 4; table 4). based on this analysis, the ten and fifteen years post selectively-logged forests were still in the recovery process, indicated by lower som content in those two logged forests compared to biotropia vol. 24 no. 3, 2017 240 the unlogged forest. in contrast, dry weight of litterfall tended to be higher in all logged forests. these results were not in line with research results obtained from logged bornean rainforest, in which one year post-logged forest produced less litterfall compared to that in the bornean primary forest. the amount of litterfall in bornean primary forest was similar to those in the bornean five years post-logged forest (prasetyo et al. 2015). this condition suggested that responses of logged forests were depended on ecological circumstances. furthermore, specific silvicultural treatments should be designed carefully by considering forest condition. ecological changes as a response to selective logging tree communities in the unlogged forest were different from those in the logged forests. the differences were due to ecological changes caused by logging activities resulted in alteration of species composition (arbainsyah . 2014; et al verburg & van eijk-bos 2003; lozada . 2012), et al tree density (decocq . 2014), tree growth rate et al (murdjoko 2016b) and association patterns et al. among biotic factors, light availability, ambient moisture, temperature, soil properties and litterfall stock as abiotic factors (murdjoko et al. 2016c). tree communities were formed as responses of each tree characteristics toward different ecological circumstances in logged forests. understory and upperstory tree communities had different reactions toward ecological changes (murdjoko et al. 2016a; zhu . 2015b). therefore, there were understory et al and upperstory tree communities consisted of the same species. tree communities consisted of seedlings and saplings stages that required more light (karsten . 2014; flores . 2014). et al et al this is the reason why logged forests had altered tree compositions compared to those in the primary forest. each logged forest has different species composition of the understory tree community. species composition of the understory tree community was different among the logged forests. understory tree community in the one year post selectively-logged forest had very different species composition compared to those in the unlogged forest (fig. 3). understory tree community in the five years post selectivelylogged forest had very different species composition compared to those in the ten and fifteen years post selectively-logged forests (fig. 3). these differences in species composition were influenced by changes in environmental conditions (corrià-ainslie . 2015; schnitzer & et al walter 2013; duah-gyamfi . 2014). et al the cca showed that understory tree community in the one year post selectively-logged forest was mainly influenced by carbon content of litterfall. understory tree community in the five years post selectively-logged forest was formed as a response toward dry weight of litterfall. the nitrogen content of litterfall affected the establishment of understory tree community in the ten and fifteen years post selectively-logged forests. understory tree community in the unlogged forest was influenced by som content. alterations of soil characteristics in the logged forests were caused by the change of microclimate conditions (asase et al. et al. 2014; imai 2012). logging activities were responsible for the widening canopy gap leading to the increase of light availability toward understory tree community (schwartz 2016). logging activities were also responsible for the decrease of tree density causing the changes in tree growth rates (verburg & van eijk-bos 2003; cannon . 1998; do . 2016). these et al et al table 4 anova of cca to analyze interactions among som, c content, n content, dry weight of litterfall and forest groups (unlogged, one year, five years, ten years and fifteen years post selectively-logged forests) edaphic factors df sums of square mean square f.model r2 p som 1 0.746 0.74644 2.438868 0.01442 0.001 * lf_c 1 0.692 0.6916 2.259688 0.01336 0.001 * lf_n 1 0.543 0.54259 1.772822 0.01048 0.005 * lf_dw 1 0.795 0.79469 2.596517 0.01536 0.001 * residuals 161 49.27566 0.30606 0.94638 total 165 52.05166 1 note: *= significant at p < 0.05 recovery of residual forest ecosystem: impact of selective logging murdjoko et al. 241 conditions triggered space and light competitions among tree species, especially in the seedlings and saplings stages(laurans . 2014).et al upperstory tree community had different patterns from the understory tree community. in the unlogged forest, species composition of understory was different from that of upperstory tree community. conspecific association occurred in the unlogged forest. not all species grown in the understory tree community grew in the upperstory tree community of unlogged forest (murdjoko et al. 2016a). ecological condition occurred in the upperstory tree community was similar to that in the understory tree community. trees in tropical forest experienced more diameter growth in the upperstory tree community (zhu et al. 2015a). upperstory tree community in the unlogged forest had very different species composition compared to those in the five years post selectively-logged forest (fig. 4). however, similar species composition was observed among upperstory tree communities in the unlogged forest, one year post selectivelylogged forest, ten and fifteen years post selectively-logged forests (fig. 4). tree species located in the five years post selectively-logged forest was the results of species competition caused by the change of ecological conditions. thus, the current species were not the same as the previous species because of the duration of the ecological process. upperstory tree community in the logged forests showed a dynamic establishment of tree community. each species had different growth rate as a response to logging impact (murdjoko et al. 2016b). some species had higher population growth rate than others leading to higher survival rate (murdjoko 2013; zuidema et al. 2009). although recovery process was seen to be happening in the ten and fifteen years post selectively-logged forests, the process still requires more time to reach the fully recovered stage. implication of ecological approach for sustainable forest management this study proposed an ecological approach to determine whether logged forests were recovered in fifteen years. existing tree communities and edaphic factors, especially som, in the unlogged forest were used as a reference of logged forest reaching recovered condition. som plays an important role to support nutrient absorption in soil (mutiso 2013). the soil of south papua et al. is mainly classified as ultisols, so the characteristic of soil is infertile (marshall & beehler 2012). selective logging activities did not seem to totally change ecological condition. the logged forest was declared to be fully recovered when its conditions had reached similar condition as those in unlogged (primary) forest, especially in terms of ecological aspects such as the content of som, stem density and species composition. therefore, it is imperative to set permanent sample plots in the unlogged (primary) and logged forests, to conduct intensive and persistent monitoring of ecological conditions and tree growth (krisnawati & wahjono 2010; ruslandi et al. 2017a; ruslandi et al. 2017b). the monitoring results would be valuable as basic information to further evaluate the silviculture protocol. useful modifications could be designed by taking ecological perspective into account. conclusions understory and upperstory tree communities formed different patterns due to logging activities. species composition existed in the tree communities in the ten and fifteen years post selectively-logged forests were not similar to that in the unlogged forest, meaning that the logged forests were still in the recovery process. som content in the logged forest was lower compared to that in the unlogged forest, indicating that the logged forests were not fully recovered. these occurrences indicated that it took more than fifteen years for the logged forests to be fully recovered. long-term studies are necessary to continuously monitor the ecological process in the logged forest in reaching the recovery stage. the recorded influential ecological factors obtained from this study can be used as indicators for logged forest recovery. acknowledgements the research was funded by beasiswa pendidikan pascasarjana dalam negeri (bppdn) 2014, manokwari regency and west papua province. the authors are thankful to the biotropia vol. 24 no. 3, 2017 242 following people for assisting in fieldwork and species vegetation identification as well as providing useful inputs: nithanel m. h. benu (balai penelitian dan pengembangan lingkungan hidup dan kehutanan manokwari), dr purnomo (faculty of biology, gadjah mada university), prof dr ir suryo hardiwinoto, m. agr. 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1907-770x 391 article higlights • camellia dalatensis and camellia capitata are critically endangered species. • habitat loss and deforestation threaten the survival of these camellia species. • vegetative propagation aids conservation of critically endangered camellia species. • rooting success depends on cutting types, growth regulators, and substrate mix. • research aids conservation efforts for vietnam’s rare and vulnerable flora. article information received 20 june 2024 revised 16 august 2024 accepted 10 september 2024 reviewer: eucharia chizoba ezigbo & anonymous *corresponding author, e-mail: lehongen@gmail.com research article conservation status and propagation of camellia dalatensis and camellia capitata by using cuttings le hong en1*, do van duong1, nguyen pham doan1, nguyen van phuc1, ngo van cam1, le thi thuy hoa1, nguyen ba trung1, ho si hung1, ngo giang phi1, nguyen thanh nguyen1, hoang thanh truong1, truong quang cuong2 1forest science institute of central highlands and south of central vietnam, da lat city 66000, lam dong province, vietnam. 2bidoup nui ba national park, da lat city 66000, lam dong province, vietnam abstract camellia dalatensis (v. d. luong, ninh & hakoda) and camellia capitata (orel, curry & luu) are classified as critically endangered (cr) by the iucn. conservation solutions for these two species have not yet been fully implemented, while wild populations are declining. this research on vegetative propagation examined the efficacy of indole-3-butyric acid (iba in solution or in powdered activated charcoal), cutting types (terminal shoot or stem cuttings), and substrates for the growth of cuttings (sand-coir dust mixes) on rooting efficiency. the highest rooting of c. dalatensis was observed in terminal shoot cuttings at 1,000 ppm iba, and 25% sand and 75% coir dust substrate. maximum values obtained were survival 95.6%, rooting percentage 88.9%, number of roots per cutting 6.9, root length 6.2 cm, and rooting index 42.9. for c. capitata, superior rooting was obtained with semi-hardwood cuttings, at 1,500 ppm iba, and equal parts of sand and coir dust as substrate. camellia capitata was more difficult to root, with maximum values of 65.6% survival, 52.2% rooting, 2.8 roots per cutting, 2.9 cm root length, and rooting index of 8.1. the results of this study can be used to support further propagation and conservation of these two endangered species. keywords: camellia capitata, camellia dalatensis, endangered species, iba, rooting, vegetative propagation introduction camellia l., the largest genus in the theaceae family, is widely distributed from bhutan, northeast india, china, and japan to indonesia and the philippines (chang & bartholomew 1984; ming & bartholomew 2007). vietnam has a high diversity of camellia and many species have been recently recorded, such as c. hiepii, c. hoaana, c. hoabinhensis, c. maianhii, c. pyriformis, and c. vanlangensis (nguyen et al. 2023; trinh et al. 2023; yang et al. 2024; trinh & hoang 2024; quach et al. 2024). camellia dalatensis was described in 2012 (tran & luong 2012) and c. capitata in 2014 (orel et al. 2014). these two species are classified as critically endangered (cr) by the iucn, with low regeneration potential due to habitat changes under the impacts of climate change and human impacts, such as deforestation from shifting cultivation and overexploitation. therefore, more quantitative information on the status and https://doi.org/10.11598/btb.2024.31.3.2284 biotropia vol. 31 no. 3, 2024 392 conservation strategies of these threatened camellia is needed (rivers & luu 2018; orel et al. 2014). propagation is one of the important components to provide seedlings for species conservation and development programs. each propagation method has its advantages and disadvantages. propagation from seeds is easy to implement. however, these two species have disadvantages in implementing seed propagation, i.e., these two species have a small number of individuals, poor fruiting ability, and seasonal seed availability. propagation via tissue culture has the potential to produce a large number of plants, but this technique requires high technology, high costs, and a long time to conduct. a cheaper alternative approach is to use cuttings for propagating these two camellia species, in which this technique has the advantage of preserving the genetic characteristics of the mother trees. propagation by using cuttings has been successful for other indigenous camellia species in vietnam, including c. chrysantha, c. flava, c. petelotii, and c. tamdaoensis (nguyen et al. 2017; nguyen et al. 2021). the aims of this study were: 1) to assess the conservation status of c. dalatensis and c. capitata in the field and 2) to determine the effects of auxin, cutting types, and substrates on root formation of the cuttings. the research results will contribute to supporting the propagation and conservation of these two threatened species of camellia. materials and methods assessment of conservation status a survey of c. dalatensis and c. capitata was undertaken using the snowball sampling approach with 15 respondents (5 forest managers, 5 ethnic people living near the forest boundary, and 5 forest planters). before completing the interview, all respondents were freely consented. the interview questionnaire had three questions: (1) have you ever seen these two plant species?; (2) where did you see these two plant species?; and (3) how many people know about these two species at this location? following the interviews, we established 5 survey transects per species, each ranging in length from 2 to 3 km, to locate mature trees. the program http://geocat.kew.org/editor (royal botanic garden 2024) and the iucn red list criteria and classification guidelines (iucn 2022) (http://www.iucnredlist.org/documents/ redlistguidelines.pdf ) were used to calculate the extent of occurrence (eoo) and area of occupancy (aoo). cutting experiments source of mother trees c. dalatensis was obtained from natural forest in tram hanh commune, da lat city, lam dong province, vietnam. c. capitata was gathered from natural forests in phuoc cat commune, cat tien district, lam dong province, vietnam. sample collection and processing the branches of c. capitata and c. dalatensis were gathered in january and may of 2022, respectively. branches with a diameter of 0.5-0.8 cm were chopped into 50-60 cm long sections, placed in foam crates, moistened with wet towels, and then transported to da lat city. the stems were rinsed multiple times in clean water before being cut around 1 cm from the node into 10-15 cm long portions for the research trials. location of propagation experiments the propagation experiments were set up in a greenhouse at the forest science institute of central highlands and south of central vietnam (fsih), da lat city, lam dong province, vietnam (11o56’35” n, 108o24’23” e). the altitude of the nursery was 1,504 m, the average annual temperature was 18-25 oc, the average annual rainfall was about 2,200 mm, and the wet season happened from may to november. the misting mode in the greenhouse was set up at 20 seconds every 2 hours. experimental design experiment 1 effect of iba on root formation the experiment was undertaken with 2 types of indole-3-butyric acid (iba) in solution and iba powder combined with activated charcoal. five concentrations of iba solution were prepared (0; 500; 1,000; 1,500; and 2,000 ppm) in water. the cuttings were submerged in the iba solution for 15 conservation status and propagation of camellia dalatensis and camellia capitata en et. al. 393 minutes. iba-activated charcoal treatments were 0, 0.5, 1.0, 1.5, and 2.0%. the iba was dissolved in a small volume of alcohol and then mixed with activated charcoal to form a slurry. the slurries were dried to evaporate the alcohol and then were ground and passed through a sieve (0.35 mm mesh size). the cuttings were briefly dipped into the powder. experiments were carried out using washed river sand in rectangular plastic drainage baskets with dimensions of 35 cm (length) x 26 cm (width) x 10 cm (height). rooting development was assessed on day 120. experiment 2 effect of cutting types on root formation the experiment compared two treatments of cutting types, i.e., terminal shoot and semi-hardwood stem cuttings. sand substrate was used in combination with the optimal result of iba in experiment 1. rooting development was assessed on day 120. experiment 3 effect of substrates on root formation four treatments of substrates were applied, i.e., 75% sand and 25% coir dust, 50% sand and 50% coir dust, 25% sand and 75% coir dust, and 100% coir dust. rooting development was assessed on day 90. data analysis the experiments were conducted in a randomized block design with three replications. the data measured were survival percentage (%), rooting percentage (%), number of roots, root length (cm), and rooting index (number of roots multiplied by root length). the data were analyzed using the duncan test in spss 26.0.0 software (statistical package for social sciences version 26). results and discussion conservation status camellia dalatensis has a narrow distribution in phat chi village, tram hanh commune, da lat city, lam dong province. the number of mature individuals in the natural habitat was 205 plants. the extent of occurrence (eoo: 0.464 km2) and area of occupancy (aoo: 8,000 km2) for critically endangered (cr) c. dalatensis are illustrated in figure 1 (upper panel). camellia capitata occurs in cat tien national park, an area managed by lam dong province (cat tien district). there were 5 mature plants in the natural habitat. c. capitata is categorized as critically endangered (cr) with eoo of 0.050 km2 and aoo of 4,000 km2 (figure 1; lower panel). both c. dalatensis and c. capitata were shown to have a very small distribution area (fig.2), which were threatened by logging, deforestation, planting of industrial crops, such as cashew, rubber and coffee, and the harvesting of non-timber forest products (beech et al. 2017; orel et al. 2014). recently, the forests are being exploited for medicinal herbs and ornamental plants with high polyphenol contents and beautiful flowers, including these two camellia species (fig. 3) (trinh 2022). effect of iba on root formation of c. dalatensis and c. capitata asexual propagation by using cuttings is the optimal solution for preserving genetic characteristics from mature plants when propagation by using seeds is limited by low fecundity. there are many factors affecting rooting, including the environment, the types of cuttings used for propagation, and the use of plant growth regulators (pgrs) (bhupathireddy et al. 2022; dewi & sabhara 2022; eed & burgoyne 2014; gautam et al. 2022; griffin et al. 1998; ray & ali 2017). cutting propagation efficiency is often increased by using exogenous pgrs, including iba treatments (bhupathireddy et al. 2022; gautam et al. 2022). however, the optimum concentrations and pgr forms (solution or solid form) can differ markedly between species. for example, 1,000 ppm iba in solution gave the best results in lycium barbarum l., but 1% iba was optimal for cornus mas l. (çelik & çetin 2021). in this study, 1,000 ppm iba was suitable for c. dalatensis, but c. capitata required a higher concentration of 1,500 ppm iba for obtaining the highest rooting percentage. biotropia vol. 31 no. 3, 2024 394 figure 1 extent of occurrence (eoo) and area of occupancy (aoo) of camellia dalatensis (upper panel) and c. capitata (lower panel) figure 2 distribution map of camellia capitata and c. dalatensis in lam dong province, vietnam conservation status and propagation of camellia dalatensis and camellia capitata en et. al. 395 figure 3 appearance of camellia capitata and c. dalatensis notes: a. seedlings of c. dalatensis in their natural habitat; b. flower of c. dalatensis; c. flower of c. capitata; d. seedlings of c. capitata in their natural habitat. differences in pgr requirements for the rooting of cuttings not only vary between plant species, but also depend on the position in the plant where the cuttings are sourced. cuttings taken from mature trees often have reduced endogenous auxin content compared to cuttings taken from juvenile plants. hence, it is necessary to undertake experiments on cuttings responses toward pgr dosages to ensure that optimal pgr dosages are being used for the correct cutting type. also, at very high concentrations, the rooting efficiency can be significantly reduced, such as in prunus laurocerasus l. at 2 g/l iba (sulusoglu & cavusoglu 2010). thus, depending on the type of tree and the age of the tree, the rooting auxin treatment needs to be adjusted to the appropriate concentration, and it is necessary to have specific studies for each tree species. the effectiveness of using iba in propagation of threatened camellia species are shown in tables 1 and 2. there were significant (p < 0.05) effects of iba rates on survival percentage, rooting percentage, number of roots, root length, and rooting index. for c. dalatensis, the survival percentage ranged from 65.56% to 80%; the rooting percentage ranged from 14.45% to 45.56%; the number of roots ranged from 0.57 to 3.80; the root length ranged from 1.67 cm to 4.23 cm; and the rooting index ranged from 1.12 to 16.32. the most effective treatment was 1,000 ppm iba. for c. capitata, the survival percentage ranged from 28.89% to 40.00%; the rooting percentage ranged from 7.78% to 31.11%; the number of roots ranged from 0.30 to 1.90; the root length ranged from 0.40 cm to 2.13 cm; and the rooting index ranged from 0.15 to 4.09. the most effective treatment was 1,500 ppm iba. biotropia vol. 31 no. 3, 2024 396 table 1 effect of iba concentration on camellia dalatensis root formation treatment survival percentage (%) rooting percentage (%) number of roots per cutting root length (cm) rooting index 500 ppm 74.45±2.22ab 32.22±2.94bc 1.73±0.35bcd 2.43±0.15b 4.29±1.11c 1,000 ppm 77.78±1.11ab 45.56±2.94a 3.80±0.32a 4.23±0.38a 16.32±2.76a 1,500 ppm 76.67±1.93ab 37.78±4.01ab 3.73±0.90a 2.90±0.75b 11.72±4.83ab 2,000 ppm 76.67±1.93ab 31.11±2.94bc 2.50±0.61ab 2.80±0.45b 6.85±2.06bc 0.5% 72.22±2.94b 22.22±2.94de 1.33±0.12bcd 2.87±0.15b 3.85±0.50c 1.0% 80.00±1.92a 35.56±1.11b 2.10±0.35bc 2.83±0.38b 5.73±0.65bc 1.5% 77.78±1.11ab 25.55±2.22cd 2.07±0.19bc 2.57±0.46b 5.29±1.10bc 2.0% 65.56±1.11c 20.00±1.92de 0.63±0.29cd 1.70±0.32b 1.12±0.67c not using pgrs 74.44±1.11ab 14.45±2.22e 0.57±0.42d 1.67±0.22b 1.12±0.91c mean 75.06±0.93 29.38±1.95 2.05±0.25 2.67±0.18 6.25±1.10c p-value 0.001 0.000 0.000 0.012 0.001 notes: *mean values in columns followed by different letters are statistically significantly different with p ≤ 0.05 using duncan’s multiple range test. table 2 effect of iba concentration on camellia capitata root formation treatment survival percentage (%) rooting percentage (%) number of roots per cutting root length (cm) rooting index 500 ppm 38.89±1.11ab 16.67±1.93c 0.83±0.90bcd 1.67±0.12c 0.99±0.21bc 1,000 ppm 40.00±1.92a 25.55±2.22b 1.37±0.12ab 1.53±0.12bc 2.10±0.25b 1,500 ppm 40.00±1.92a 31.11±1.11a 1.90±0.15a 2.13±0.24a 4.09±0.70a 2,000 ppm 31.11±1.11bcd 25.57±1.11b 0.87±0.09bcd 1.23±0.27c 1.05±0.23bc 0.5% 36.67±3.85abc 13.33±1.93c 0.57±0.09cd 1.23±0.12c 0.68±0.06bc 1.0% 32.22±2.94abcd 14.44±1.11c 1.00±0.47bc 1.80±0.15ab 1.94±1.05b 1.5% 32.22±4.01abcd 14.44±2.94c 1.40±0.21ab 1.23±0.90c 1.71±0.23b 2.0% 26.67±1.93d 11.11±1.11cd 0.97±0.15d 1.00±0.21c 1.02±0.35bc not using pgrs 28.89±2.22cd 7.78±1.11d 0.30±0.26 0.40±0.12d 0.15±0.11c mean 34.07±1.16 17.78±1.52 1.02±0.54 1.30±0.10 1.53±0.25 p-value 0.009 0.000 0.001 0.000 0.001 notes: *mean values in columns followed by different letters are statistically significantly different with p ≤ 0.05 using duncan’s multiple range test. for c. dalatensis and c. capitata, the liquid growth regulator was more effective compared to the charcoal powder in promoting rooting. results on survival percentage and rooting percentage are compared in figure 4. the most effective treatment, whether the activated charcoal-contained powder or liquid plant growth regulator. effect of cutting types on root formation cutting types are divided into softwood (nonlignified) cuttings, semi-hardwood (partly lignified) cuttings, and hardwood (lignified portion) cuttings (griffin et al. 1998; islam et al. 2010). most propagators of woody plants use semi-hardwood cuttings for higher rooting efficiency compared to softwood and hardwood cuttings (alkaç et al. 2022). in each cutting type, the cutting position, whether terminal shoot or stem cuttings, also has a great impact on rooting efficiency. in most species, liquid iba solutions are easy to prepare, while the phloem and xylem of cuttings have better access to exogenous hormones than hormones combined with powders. however, powder containing activated charcoal is an effective adsorbent for many pollutants (organic, inorganic, microbial, and biological) (mohammad-khah & ansari 2009). therefore, when the activated charcoalcontained powder is dry-mixed with a plant growth regulator, the dry mix can reduce infection. therefore, in this research, we sought to identify conservation status and propagation of camellia dalatensis and camellia capitata en et. al. 397 figure 4 comparisons on survival and rooting percentages for cuttings dipped in liquid iba or iba in charcoal for camellia dalatensis (a) and c. capitata (b) notes: dd = iba solution, dr = iba powder, dc = no iba. cuttings from terminal shoot have higher rooting efficiency. however, the use of appropriate plant growth regulators can affect the rooting efficiency of stem cuttings and ensure propagation efficiency with the number of rooted cuttings higher than that of the terminal shoot cuttings (solikin 2019). choosing the right cuttings for propagation table 3 effect of cutting types on camellia dalatensis root formation treatment survival percentage (%) rooting percentage (%) number of roots per cutting root length (cm) rooting index tc 90.00±1.92 58.89±1.11 5.10±0.59 4.50±0.12 23.08±3.20 sc 67.78±2.94 31.11±2.94 1.97±0.15 2.33±0.20 4.62±0.62 mean 78.88±5.21 45.00±6.39 3.53±0.75 3.42±0.50 13.85±4.38 p-value 0.003 0.001 0.007 0.001 0.005 notes: *mean values in columns followed by different letters are statistically significantly different with p ≤ 0.05 using duncan’s multiple range test; tc = terminal shoot cuttings; sc = stem cuttings. table 4 effect of cutting type on camellia capitata root formation treatment survival percentage (%) rooting percentage (%) number of roots per cutting root length (cm) rooting index tc 45.56±1.11 25.56±1.11 1.80±0.06 1.87±0.19 3.36±0.37 sc 37.78±2.22 34.45±2.22 1.50±0.06 2.00±0.10 3.00±0.19 mean 41.67±2.06 30.00±2.28 1.65±0.08 1.93±0.10 3.18±0.20 p-value 0.035 0.023 0.021 0.056 0.436 notes: *mean values in columns followed by different letters are statistically significantly different with p≤0.05 using duncan’s multiple range test; tc = terminal shoot cuttings; sc = stem cuttings. and increasing the efficiency of propagation are necessary solutions. for some camellia spp., it is common to choose semi-hardwood cuttings because this cutting type provides the best results. in this study, semihardwood cutting was used and divided into terminal shoot cuttings and stem cuttings (tables 3 and 4). biotropia vol. 31 no. 3, 2024 398 based on experiment 1, the iba concentrations used were 1,000 ppm for c. dalatensis and 1,500 ppm for c. capitata. the parameters of survival percentage, rooting percentage, number of roots, root length, and rooting index were significantly different for c. dalatensis (p < 0.05). terminal shoot cuttings provided better results compared to stem cuttings with the survival percentage of 90.00% compared to 67.78%, the rooting percentage reaching 58.89% compared to 31.11%, 5.10 roots per cutting compared to 1.97 roots per cutting, the root length reached 4.50 cm compared to 2.33 cm, the rooting index reached 23.08 compared to 4.62. similar results have been reported by wazir (2014) in propagating c. japonica by using terminal cuttings with 1,000 ppm iba where the highest percentage of rooting was 84.96%, and the longest root length was 18.3 cm. for c. capitata, the parameters of survival percentage, rooting percentage, and number of roots per cutting were significantly different (p < 0.05). however, root length and rooting index were not statistically significantly different (p > 0.05). the survival percentage of terminal shoot cuttings compared to stem cuttings was 45.56% compared to 37.78%. however, the rooting index for terminal shoot cuttings was about 9% lower than that of stem cuttings (25.56% versus 34.45%), and the number of roots per cutting was 1.80 compared to 1.50. the root length ranged from 1.87 cm to 2.00 cm (average 1.93 cm) and the rooting index range was 3.00 to 3.36 (average 3.18) for both cutting types. comparing the rooting parameters, the effective cutting type for c. capitata propagation was semihardwood stem cuttings. at the time of gathering cuttings in the field, the c. capitata had begun to sprout. therefore, the terminal shoot cuttings were very immature and weak, so they were easy to be infected by fungi. besides, we collected the stem cuttings at the same time that the cuttings were partially lignifying, to have better auxin synthesis; thereby, the rooting percentage of stem cuttings was higher than that of terminal shoot cuttings in c. capitata. effect of substrates on root formation the growing substrate environment should have good porosity to support smooth water draining and provide a suitable temperature and humidity regimes for root development. the substrate media can be combined with other factors, such as plant growth regulators and cutting types, to bring the highest efficiency to the propagation process (johnson et al. 2005; silva et al. 2012). the selected growing substrate for propagation in this study was a mixture of sand and coir dust. sand ensures good drainage, and coir dust improves water retention in the rooting environment. in this experiment, 1,000 ppm iba was used for semihardwood cuttings of c. dalatensis, while 1,500 ppm iba was used for semi-hardwood cuttings of c. capitata. rooting results are shown in table 5 (c. dalatensis), table 6 (c. capitata), and figure 5. table 5 effect of substrate type on camellia dalatensis root formation treatment survival percentage (%) rooting percentage (%) number of roots per cutting root length (cm) rooting index a1 94.44±1.11 75.56±1.11c 4.93±0.30c 4.60±0.06c 22.73±1.65c a2 95.56±2.94 83.33±1.93b 5.13±0.18bc 5.33±0.15b 27.41±1.47bc a3 95.56±1.11 88.89±2.22a 6.87±0.27a 6.23±0.15a 42.88±2.71a a4 95.56±2.94 81.11±1.11b 6.10±0.50ab 5.60±0.20b 34.26±3.59b mean 95.28±0.10 82.22±1.61 5.76±0.27 5.44±0.19 31.82±2.53 p-value 0.978 0.003 0.011 0.000 0.002 notes: *mean values in columns followed by different letters are statistically significantly different with p ≤ 0.05 using duncan’s multiple range test; a1 = 75% sand and 25% coir dust; a2 = 50% sand and 50% coir dust; a3 = 25% sand and 75% coir dust; a4 = 100% coir dust. conservation status and propagation of camellia dalatensis and camellia capitata en et. al. 399 table 6 effect of substrate type on camellia capitata root formation treatment survival percentage (%) rooting percentage (%) number of roots per cutting root length (cm) rooting index a1 64.45±2.22 47.78±2.22 2.03±0.09b 2.33±0.12b 4.72±0.05b a2 65.55±2.22 52.22±2.94 2.83±0.20a 2.87±0.17a 8.12±0.69a a3 65.56±2.94 53.33±1.93 2.57±1.19a 1.83±0.07c 4.73±0.50b a4 66.66±3.33 52.22±2.22 2.57±0.33a 1.77±0.09c 4.54±0.28b mean 65.56±1.84 51.39±1.19 2.50±0.11 2.20±0.14 5.53±0.49 p-value 0.952 0.406 0.026 0.001 0.001 notes: *mean values in columns followed by different letters are statistically significantly different with p ≤ 0.05 using duncan’s multiple range test; a1 = 75% sand and 25% coir dust; a2 = 50% sand and 50% coir dust; a3 = 25% sand and 75% coir dust; a4 = 100% coir dust. the effects of media types and composition on rooting efficiency have been reported in many plants (eed & burgoyne 2014). air content and oxygen diffusion rate in media are important for rooting (ercisli et al. 2002). the characteristics of the species also influence rooting success. in particular, camellia impressinervis grows well in high moisture and well drained soil (tran 2018), in which their cuttings had the best rooting results in a growth medium of 100% sand with high air content and oxygen diffusion capacity (tran et al. 2020). figure 5 rootings of camellia cuttings notes: a. c. dalatensis terminal shoot cuttings with 1,000 ppm iba in a substrate of 25% sand mixed with 75% coir dust; b. c. capitata semi-hardwood stem cuttings with 1,500 ppm iba in a substrate of 50% sand mixed with 50% coir dust. for c. dalatensis, there was no statistically significant differences in survival percentage (p > 0.05) between treatments, with a mean value of 95.28%. however, rooting percentage, number of roots, root length, and rooting index were significantly different (p < 0.05). rooting percentage ranged from 75.56% to 88.89% (average 82.22), number of roots per cutting ranged from 4.93 to 6.78 (average 5.76), root length ranged from 4.60 cm to 6.23 cm (average 5.44 cm), and rooting index ranged from 22.73 to 42.88 (average 31.82). the treatment with the best results for c. dalatensis was 25% sand:75% coir dust, which may be related to the wet environment where this species occurs. c. dalatensis was distributed in dalat city which has high humidity and high annual rainfall. for c. capitata, there was no significant difference between the survival and rooting percentages (p > 0.05), the average survival percentage was 65.56% and the average rooting percentage was 51.39%. the number of roots per cutting ranged from 2.03 to 2.83 (average 2.50), the root length ranged from 1.77 cm to 2.87 cm (average 5.44 cm) and the rooting index ranged from 4.54 to 8.12 (mean 5.53). treatment with the best results for c. capitata was the treatment with equal parts of sand and coir dust. c. capitata occurs in cat tien district which has lower humidity and rainfall compared to that in dalat city. therefore, this species was able to grow better in the medium which had similar conditions to their natural habitat. biotropia vol. 31 no. 3, 2024 400 conclusion camellia capitata and c. dalatensis have very limited distributions area. the declining natural populations face the risk of extinction due to fragmentation, deforestation, illegal logging, and clearing for agriculture. vegetative propagation protocols are essential to help domesticate these species and conserve genetic diversity. for propagation by using cuttings, root formation was effective using liquid iba at 1,000 ppm for c. dalatensis and at 1,500 ppm for c. capitata. rooting formation was the highest for terminal shoot cuttings of c. dalatensis and semi-hardwood stem cuttings of c. capitata. sand and coir dust substrates with a ratio of 25:75% for c. dalatensis and 50:50% for c. capitata were the best substrates for root formation of cuttings. acknowledgments the authors would like to thank bcgi (botanic gardens conservation international) for their financial support and the southern institute of ecology, forest science institute of central highlands and south of central vietnam, cat tien national park, and lam vien special-use forest management board created the most favorable conditions for us to complete this study. references alkaç os, öcalan on, güneş m. 2022. effect of silver nanoparticles treatments on some characteristics of “santander” lily cultivar. turkish journal of agriculturefood science and technology (turjaf) [internet]; 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(streblidae), meristaspis spp., spinturnix spp. (spinturnicidae), and ticks (ixodidae). the total prevalence of infested bats was 51.7%, with females tending to have a higher intensity compared to males. bats species with the highest prevalence of infestation were rousettus leschenaultii and myotis muricola. meanwhile, this study found no ectoparasites on macroglossus sobrinus and scotophilus kuhlii. keywords: bats, bats flies, bogor, ectoparasite, indonesia introduction bats are known to play an essential role in maintaining the ecological balance of ecosystems, serving as seed transmitters, pollinators, and natural insect control (suyanto 2001). despite their significant ecological functions, these animals also serve as the natural reservoir of various pathogens. previous studies showed that bats had unique immune systems, thereby enabling them to carry various zoonotic viruses per host species compared to rodents (irving et al. 2021; luis et al. 2013). several emerging infectious diseases have also been linked to these animals bats as a source of zoonotic pathogens, including ebola virus, sars coronavirus, nipah virus, and hendra virus (brook & dobson 2015; calisher et al. 2006). furthermore, studies conducted in indonesia showed their association with potential zoonotic diseases (diptyanusa et al. 2021; tsang et al. 2021). this indicates that the study of bats and their potential to transmit diseases to humans or other animals demands thorough investigation. one of the health issues affecting bats is ectoparasites infestation, which warrants further investigation to comprehend the ecology and health of these creatures. the commonly associated ectoparasites belong to the class insecta (bats flies and bats flea) and arachnida (ticks and mites). the species within the class insecta are known to belong to the family nycteribiidae, streblidae, hippoboscidae, and ischnopsyllidae. meanwhile, those from class arachnida are often members of the family argasidae, ixodidae, and spinturnicidae (fajri et al. 2018; azhar et al. 2015). previous studies showed that these ectoparasites in bats are vectors of pathogens, such as bartonella spp., rickettsia spp., and certain viruses with the ability *corresponding author, email: supriyono84@apps.ipb.ac.id biotropia vol. 30 no. 3, 2023 366 to cause diseases in humans or animals (szentiványi et al. 2019; reeves et al. 2016). several studies have been carried out on bat ectoparasites in several southeast asia countries, including malaysia (azhar et al. 2015), singapore (lim et al. 2020), and the philippines (alvarez et al. 2015). based on previous findings, studies related to these ectoparasites in indonesia remains significantly limited, despite the presence of over 230 distinct bat species in this region (maryanto et al. 2020). existing reports in indonesia primarily focused on recording ectoparasite infestation in different locations and species, especially megabats (nangoy et al. 2021; sauqi et al. 2021; fajri et al. 2018). based on these findings, there is a need to conduct further studies to understand the characteristics of ectoparasites, including their specific host and potential role in transmitting diseases. dramaga is a subdistrict located in bogor, indonesia, which has been extensively explored due to bats species diversity in dramaga. several studies have also been carried out in and around the ipb dramaga campus area and its surrounding villages. according to mustari et al. (2014) and mustari (2020), at least 10 species of bats were found in ipb dramaga campus. another report showed the presence of 11 species in villages around the area (sumirto 2013). due to the role of these animals in maintaining ecosystem balance, there is a need to carry out further investigations on their ecology and health problems, such as ectoparasites infestation. therefore, this study aims to identify the diversity of ectoparasites on bats in dramaga, bogor, indonesia. materials and methods study sites and sampling techniques bats used in this study were obtained from the seven locations in dramaga, bogor, indonesia from november 2021 to february 2022. the habitats selected as sampling locations included arboretums, agricultural land areas, and rural areas. furthermore, reconnaissance surveys were conducted in the area before the study began to identify the location where bats were likely to congregate. the survey targeted the regions where bats were expected to fly, such as fruiting trees or congregation sites of insects, to determine the appropriate sampling locations. the procedures for sample capturing in this study referred to the guidelines for bats reservoir data collection published by the national institute of health research and development, ministry of health, indonesia. the samples were captured at night using mist-net techniques. the mist nets were installed in determined locations and placed approximately 5 meters above the ground. the installation was carried out for four hours from 18.00 to 22.00 and monitored every hour. bats were collected as detected trapped in the mist net, placed into a cotton bag, and transported to a nearby station for further identification (balitbangkes 2015). bats identification and ectoparasites collection captured bats were identified based on morphological features and morphometries using the species identification key from the field guide of bats in indonesia (suyanto 2001). the identified samples were then observed to determine the presence of ectoparasites infestation. the collection procedure for ectoparasites was carried out using small tweezers. subsequently, the parasites obtained were placed in a labeled vial containing 70% alcohol (balitbangkes 2015), and bats were released back to the initial location. ectoparasites identification the initial grouping of ectoparasites was carried out based on their morphological characteristics. bats flies were distinguished by their large size resembling a fly with or without wings, while mites and ticks were distinguished by their relatively small size compared to bat flies. subsequently, the collected ectoparasites were identified using a microscope. bats flies were identified using the publications of maa (1971, 1968, 1962) and theodor (1967, 1959), while mites were identified using the publication of baker & delfinado (1964) and delfinado & baker (1963). ticks ectoparasites were identified using the publication of hoogstraal (1955). data analysis data on captured bats were analyzed descriptively by making a table that included the species, the number of captured bats, the number diversity of ectoparasite on bats in dramaga, bogor, indonesia – kedang et al. 367 of bats infested with ectoparasites, the prevalence of ectoparasites infestation, and the intensity of ectoparasites. data on the collected ectoparasites were analyzed descriptively by making a table containing the types of ectoparasites, hosts, sex, and the number of collected samples per host. results and discussion bats diversity a total of 56 bats were obtained in this study, belonging to the suborders megachiroptera and microchiroptera. the samples represented nine species, namely cynopterus brachyotis, cynopterus sphinx, cynopterus titthaecheilus, macroglossus sobrinus, rousettus leschenaultii, myotis muricola, nycteris javanica, pipistrellus javanicus, and scotophilus kuhlii. the results showed that the most captured species was cynopterus brachyotis, with a total of 20 samples (35.7%). the least caught species was scotophilus kuhlii, with only one sample (1.8%), as shown in table 1. all bats species found in this study had a conservation status of least concern, except nycteris javanica, which was considered vulnerable. nycteris javanica was an endemic bats and was only found in java and timor (waldien & wiantoro 2021). this study found a total of 29 samples that were infested with ectoparasites, accounting for 51.7% of the total population. the species with the highest prevalence (80%) of infestation were rousettus leschenaultia and myotis muricola. meanwhile, there was no infestation on macroglossus sobrinus and scotophilus kuhlii, as shown in table 1. the prevalence and intensity of ectoparasites in each bat sex are presented in table 2. the results showed that the number and intensity of ectoparasites varied widely among the samples’ sex and species. female rousettus lechenaultii species had the highest intensity of infestation. female bats tended to have a higher intensity of ectoparasites compared to males, but their population was smaller. table 1 bats species and prevalence of ectoparasites infestation in bats bats species number of captured bats number of infested bats prevalence (%) megachiroptera (megabat) cynopterus brachyotis 20 11 55 cynopterus sphinx 5 2 40 cynopterus titthaecheilus 11 6 54.5 macroglossus sobrinus 4 0 0 rousettus leschenaultii 5 4 80 subtotal 45 23 51.1 microchiroptera (microbat) myotis muricola 5 4 80 nycteris javanica 2 1 50 pipistrellus javanicus 3 1 33.3 scotophilus kuhlii 1 0 0 subtotal 11 6 54.5 total 56 29 51.7 table 2. intensity of ectoparasites based on bats species and sex bats species infested bats/captured bats total ectoparasites intensity of ectoparasites male female male female male female cynopterus brachyotis 10/19 1/1 20 2 2.0 2.0 cynopterus sphinx 1/3 1/2 3 1 3.0 1.0 cynopterus titthaecheilus 4/6 2/5 5 2 1.25 1.0 rousettus leschenaultii 2/3 2/2 8 24 4.0 12.0 myotis muricola 4/5 0/0 27 0 6.75 0 nycteris javanica 1/2 0/0 1 0 1.0 0 pipistrellus javanicus 1/3 0/0 2 0 2.0 0 macroglossus sobrinus 0/4 0/0 0 0 0 0 scotophilus kuhlii 0/1 0/0 0 0 0 0 total 23/46 6/10 66 29 2.87 4.83 biotropia vol. 30 no. 3, 2023 368 figure 1 bats species captured in dramaga, bogor: (a) cynopterus titthaecheilus with ectoparasites (white circle), (b) macroglossus sobrinus, (c) rousettus leschenaultii, (d) nycteris javanica this was the first study to record the diversity of ectoparasites on bats in dramaga, bogor. the results could be used to monitor the status of bats species in dramaga, bogor, in line with conservation efforts. this study also presented the first record of ectoparasites infestation on vulnerable endemic bats species, namely nycteris javanica. the species was endemic to java and timor islands and not commonly found in nature due to its declining population and conservation status being vulnerable (waldien & wiantoro 2021). due to the absence of ectoparasites on some species, further studies must be carried out to determine the possibility of infestation on bats. this study found no infestation in macroglossus sobrinus and scotophilus kuhlii, possibly due to the lack of representative samples. therefore, future reports must explore ectoparasites that could affect these species. further studies were also needed due to the inability of this current study to cover all bat species reported by mustari (2020), such as kerivoula hardwickii, rhinolophus affinis, and hipposideros diadema. ectoparasites diversity ectoparasites found in this study were from insecta and acarina classes, as shown in table 3. a total of 95 samples were collected representing three groups, namely bats flies (family nycteribiidae and streblidae), mites (family spinturnicidae), and ticks (family ixodidae). nycteribiidae bats flies were found on five microbats and megabats, namely myotis muricola, cynopterus brachyotis, cynopterus sphinx, cynopterus titthaecheilus, and rousettus leschenaultii. streblidae species was only discovered on nycteris javanica. mites were discovered on rousettus leschenaultii, myotis muricola, and pipistrellus javanicus, while ticks were only found on myotis. furthermore, rousettus leschenaultia had the highest ectoparasites infestation compared to others in dramaga. the differences in the number of infestation were due to the variations in number of captured individual bats in each species. diversity of ectoparasite on bats in dramaga, bogor, indonesia – kedang et al. 369 table 3 collected ectoparasites found on bats in dramaga, bogor no ectoparasites species number of ectoparasites total c.b c.s c.t m.sb r.l m.m n.j p.j s.k bats flies 1 basilia spp. 0 0 0 0 0 10 0 0 0 10 2 eucampsipoda sp. 0 0 0 0 21 0 0 0 0 21 3 leptocyclopodia ferrarii 22 4 7 0 0 0 0 0 0 33 4 raymondia sp. 0 0 0 0 0 0 1 0 0 1 mites 1 meristaspis spp. 0 0 0 0 11 0 0 0 0 11 2 spinturnix spp. 0 0 0 0 0 16 0 2 0 18 ticks 1 ixodidae 0 0 0 0 0 1 0 0 0 1 total 22 4 7 0 32 27 1 2 0 95 notes: c.b: cynopterus brachyotis, c.s: cynopterus sphinx, c.t: cynopterus titthaecheilus, m.sb: macroglossus sobrinus, r.l: rousettus leschenaultii, m.m: myotis muricola, n.j: nycteris javanica, p.j: pipistrellus javanicus, s.k: scotophilus kuhlii. ectoparasites from the family nycteribiidae were the most collected ectoparasites in this study. furthermore, nycteribiid bats flies were ectoparasites commonly found in megabats from the genus cynopterus and rousettus (nangoy et al. 2021). species from the genus cynopterus were reported to be the primary hosts of leptocyclopodia flies (maa 1975). several studies collected leptocyclopodia ferrarii from cynopterus brachyotis in indonesia (nangoy et al. 2021; sauqi et al. 2021). eucampsipoda bats flies were common on megabats, and they had infested eonycteris spelaea and rousettus leschenaultii in southeast asia countries (lim et al. 2020; fajri et al. 2018; azhar et al. 2015). basilia ectoparasites were commonly obtained in megabats and microbats, and poerwanto et al. (2020) reported their presence on miniopterus schreibersii in yogyakarta, indonesia. ectoparasites from the genus raymondia included streblidae bats flies found in microbats (azhar et al. 2015; maa 1962). these species could be distinguished from nycteribiidae samples by the presence of the wings (azhar et al. 2015). this study only found raymondia sp. on nycteris javanica. this result was the first record of ectoparasites infestation by the genus raymondia on nycteris javanica. figure 2 ectoparasites collected from bats in dramaga, bogor: (a) basilia sp. collected from myotis muricola, (b) eucampsipoda sp. collected from rousettus leschenaultii, (c) leptocyclopodia ferrarii collected from cynopterus spp., (d) raymondia sp. collected from nycteris javanica, (e) mite meristaspis sp. ♀ and (f) meristaspis sp. ♂ collected from rousettus leschenaultii, (g) spinturnix sp. collected from myotis muricola and pipistrellus javanicus, and (h) ixodid tick (ixodidae) collected from myotis muricola biotropia vol. 30 no. 3, 2023 370 mites and ticks were found in the genus rousettus, myotis, and pipistrellus. mite ectoparasites infesting bats in indonesia were from the spinturnicidae family, while the ticks were from the ixodidae and argasidae families (fajri et al. 2018). several studies reported ticks and mites ectoparasites infestation on bats in indonesia (poerwanto et al. 2020; fajri et al. 2018). the results of meristaspis sp. and spinturnix sp. mites from the family spinturnicidae and ticks larvae from the family ixodidae were consistent with previous studies on arachnid infestation on bats. bat-ectoparasites interaction ectoparasites in bats spent almost their entire life cycle on the host through blood consumption (hiller et al. 2019; bordes et al. 2008). the intensity and prevalence of ectoparasites were affected by several factors, including hostspecificity, habitat, nesting, gender, diet, and social behavior (nangoy et al. 2021; hiller et al. 2019; ramanantsalama et al. 2018). therefore, their distribution and abundance were associated with the distribution and abundance of the host (putra, 2014; ter hofstede & fenton, 2005). the colony size of each bat species could also influence social behavior, leading to differences in the prevalence of ectoparasites infestation. species that shared the same roosting site in larger groups increased the possibility of ectoparasites transmission between the bats (putra 2014). bats in genera cynopterus, rousettus, and pipistrellus were generally known to roost from medium to large colonies (garg et al. 2015; kumar et al. 2015; gay et al. 2014). different roosting behavior was found in macroglossus species, which tended to roost alone or in small colonies occupying different sites (putra 2014; gould 1978). the absence of ectoparasites infestation on macroglossus bats in this study was allegedly related to the behavior of the species. several reports showed that ectoparasites infestation in bats had an association with sex. in this study, females tended to have a higher intensity of ectoparasites compared to males, but they had few infested individuals due to the small population. the results were consistent with nangoy et al. (2021) on pteropodid bats in sulawesi, where females had a higher intensity due to their high susceptibility caused by several factors. fluctuations in the reproductive cycle, such as pregnancy and lactation, could suppress immunity and increase susceptibility to parasites. during this period, the female species tended to spend more time in roosting sites, increasing contact with ectoparasites or the other infested bats (tai et al. 2022; nangoy et al. 2021; webber et al. 2015). a previous study reported that extended stay in roosting sites also increased ectoparasite exposure (lim et al. 2020). compared to the results of this study, lim et al. (2020) found that male cynopterus brachyotis and eonycteris spealea had a higher level of infestation. this was because they spent more time in the roost site due to defense. grooming activities commonly carried out to expel parasites also affected infiltration among sexes since males spent more time in grooming activities related to ectoparasites consumption compared to females (ramanantsalama et al. 2018). meanwhile, godinho et al. (2013) found no association between grooming activity and the number of parasites. due to the variation in literature, further studies must be carried out to investigate ectoparasites infestation in relation to sex. ectoparasites in bats generally had certain specific hosts, and could only be found in some species. bats flies were obligate and specialized parasitic organisms found on the fur and wing membranes. furthermore, this study found a genus-specific host pattern in ectoparasites. leptocyclopodia ferarri bats flies obtained had specific hosts from the cynopterus spp. bats. these findings were consistent with several studies, where species from the genus cynopterus were primary hosts for leptocyclopodia spp. (lim et al. 2020; azhar et al. 2015). based on the results, meristaspis mites tended to infest megabats, while spinturnix mites infested microbats. the results were in line with previous studies, which recorded the infestation of meristaspis spp. mites on megabats from the genus macroglossus and rousettus (fajri & armiani 2021; fajri et al. 2018). meanwhile, the study by zania et al. (2022) reported the spinturnix mite infestation on rousettus bats in banyuwangi. this variation in findings suggested that the mites did not have a genus-specific pattern. diversity of ectoparasite on bats in dramaga, bogor, indonesia – kedang et al. 371 the presence of ectoparasites in bats contributed to the spread of pathogens between individual members in the colony. some of the parasites had various hosts and could be vectors for certain pathogens. several studies found bartonella spp. bacteria in leptocyclopodia bats flies collected from the megabats in malaysia and the philippines (low et al. 2022; morse et al. 2012). bartonella spp. was a bacteria that caused bartonellosis and was considered zoonosis (chomel & kasten 2010). bacterium bartonella spp. and rickettsia spp. were also found in mites from the genus spinturnix collected from myotis myotis bats in poland (szubert-kruszyńska et al. 2019). feng et al. (2017) found the khaeng koi virus in eucampsipoda sundaica from rousettus leschenaultia bats in china. the results of bats ectoparasites in dramaga proved that ectoparasites infestation was a major health problem in bats. the ability of ectoparasites to act as vectors of various pathogens demands further studies on their distribution in each bats species, as well as their public health importance. conclusion in conclusion, ectoparasites were one of the causes of health-related problems in bats with health importance due to their ability to act as vectors of various pathogens. ectoparasites affecting bats in dramaga were from the family nycteribiidae, streblidae, spinturnicidae, and ixodidae. the results showed that the total prevalence of infested samples in the study location was 51.7%. furthermore, the female samples tended to have a higher intensity of ectoparasites compared to males. the species with the highest prevalence of infestation were rousettus leschenaultii and myotis muricola. this study found no ectoparasites infestation on macroglossus sobrinus and scotophilus kuhlii. based on these findings, further studies were needed as some species of bats required large sample sizes. references azhar i, khan faa, ismail n, abdullah mt. 2015. checklist of bat flies (diptera: nycteribiidae and streblidae) and their associated bat hosts in malaysia. check list 11(5): 1777. alvarez jd, lit jr il, alviola pa. 2015. bat flies (diptera: nycteribiidae) from mount makiling, luzon island: new host and distribution records, with a checklist of species found in the philippines. check list 11(1): 1509. baker ew, delfinado md. 1964. spinturnicidae of south east asia and the pacific region. pacific insects 6(4): 571-91. balitbangkes. 2015. pedoman pengumpulan data reservoir (kelelawar) di lapangan. 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[case report of spinturnix spp. infestation on fruit bats (rousettus spp.) in banyuwangi]. media kedokteran hewan 33(3): 233-43. biotropia vol. 28 no. 2, 2021: 109 116 doi: 10.11598/btb.2021.28.2.1162 109 interleukin levels in the zingiber cassumunartreated mice nurkhasanah*, nanik sulistyani, yuni arum handayani, qanita kamila and annisa candra nur isnaini faculty of pharmacy, universitas ahmad dahlan, yogyakarta 55164, indonesia received 6 december 2018/accepted 12 january 2020 abstract the protein compound, cytokine, is responsible for the body’s immune system. several cytokines acting as key regulators of infection include il-10, il-12, and il-14. the chemical content of zingiber cassumunar shows potential immunomodulatory effects. this study aimed to determine the effect of the zingiber cassumunar ethanol extract (eezc) on the expressions of il-10, il-12, and il-14. the test animals, balb/c mice which were treated for 21 days, were divided into five groups, i.e., normal group (untreated), negative control group (treated with 10% of tween 80), and three treatment groups that respectively received 1.25 mg, 2.5mg, and 5mg/20g bw of eezc. on the 22nd day, the mice were induced with lipopolysaccharide (lps) intraperitoneally (except for the normal group). the interleukin expression was observed by immunohistochemistry using specific antibodies, and the expressed cells were counted under a microscope. the 21-day administration of eezc at doses of 1.25 mg, 2.5mg, and 5mg/20g bw significantly increased the expression of il-10, il-12, and il-14 in proportion to the dose thereby suggesting the potency of the extract to induce both innate and adaptive immunity. this activity may be attributable to curcumin as the active compound of the extract. keywords: curcumin, immunomodulator, interleukin, zingiber cassumunar introduction the immune system which is responsible for protecting the host from various pathogenic microorganisms also controls the immune responses and prevents over-reaction of the body’s own cells (saraiva & o’garra 2010). a decrease in the immune system can affect the body's strength to fight infections or other diseases. therefore, the presence of immunomodulator compound that can improve the immune response to diseases or infections is a vital component of the immune system. immunomodulator is a substance, which can stimulate, suppress or modulate any of the components of the immune system including both the specific and nonspecific immune system (das et al. 2014). modulation of the immune system is marked by induction, expression, amplification or inhibition of certain parts in the immune signaling and response mechanism. thus, the immunomodulator substance is used as immune stimulant for its effect on the immune system. the anti-inflammatory cytokine interleukin10 (il-10) plays a crucial role in preventing inflammatory and autoimmune pathogens. it can both impede pathogen clearance and ameliorate the immunopathology process. several types of cells can produce il-10, with the major source of il-10 varying in different tissues or during acute or chronic stages of the same infection (couper et al. 2008). il-10 plays a central role during infection by limiting the immune response to pathogens and thereby preventing damage to the host. the production of il-10 was associated with the regulatory t (treg) cells. (saraiva & o’garra 2010). interleukin-12 cytokine, produced mainly by the antigen-presenting cells (apc) which include the macrophages and dendritic cells that respond to microbes, is also vital in the immunoregulation process (abbas et al. 2017). *corresponding author, email: nurkhas@gmail.com biotropia vol. 28 no. 2, 2021 110 during the immune response, il-12 is produced as a reaction to stimuli of various compounds (including lipopolysaccharide/lps). interleukin-14 (il-14) cytokines, produced by the immune system, particularly by the activated b cells and t cells, regulates the b-cell proliferation (leca et al. 2008). il-14 can increase antibody responses to vaccinations causing autoimmunity and contribute to b-cell lymphoma formation (shen et al. 2006). zingiber cassumunar of the zingiberaceae family, known locally as bengle (javanese, indonesia), has been traditionally used for treating various diseases. z. cassumunar contains terpenoids, essential oil, and curcuminoids. several studies on z. cassumunar include its performance as anticancer (varalakshmi et al. 2008), antioxidant (vankar et al. 2006) (bua-in & paisooksantivatana 2009) and immunomodulator (nurkhasanah et al. 2017; rahmawati 2013). this plant exhibited an immunomodulatory activity by increasing phagocytic activity in vitro (chairul et al. 2009), increasing nitric oxide (no) and reactive oxygen species (ros) (nurkhasanah et al. 2017) and decreasing malondialdehyde products in plasmodium berghei-infected mice (nurmasari et al. 2014). this study documents the activity of zingiber cassumunar ethanol extract (eezc) in stimulating the immune response in vivo as observed from its effect on interleukin-10, -12 and -14. it focuses on the immune responses of these cytokines on both the innate and adaptive body immunity. the expression il-10 and il-14 are closely related to activation of adaptive immunity, while the il-12 is important in cell communication between the macrophage and t cells. this study was conducted for 28 days through oral administration on balb/c male mice. this in vivo experiment provides evidence for the higher effectiveness of eezc in increasing the immune responses, a very vital information for the development of z. cassumunar as an immunomodulatory product. materials and methods materials the zingiber cassumunar rhizome, collected from a local yogyakarta market, was identified at the biology laboratory, universitas ahmad dahlan. the test animal was obtained from the animal house of the integrated research and testing laboratory, universitas gadjah mada (lppt ugm) yogyakarta, indonesia. extraction the rhizome was selected, washed, sliced and finally oven-dried at a temperature of 50 oc. the dried rhizome was then blended or ground into powder. the extraction was carried out by maceration method using 96% ethanol as the solvent. the maceration lasted for 24 hours, and the yield were evaporated in a vacuum rotary evaporator to obtain the concentrated extract (eezc) which was used as the treatment. thin-layer chromatography (tlc) analysis of the extract the thin-layer chromatography (tlc) analysis was used to identify the active eezc compound. a total of 100.0 mg of eezc was dissolved in 10.0 ml of absolute ethanol. this procedure used curcuminoids (sigma) that was dissolved in ethanol as a standard. each 2 μl of the extract and curcuminoid were applied on silica gel gf 254 as the stationary phase and eluted with the mobile phase of chloroform : ethanol : glacial acetic acid (94 : 5 : 1). the detection of eezc was done under daylight and uv 254 nm. animal treatment the procedure of the study and the use of test animal were ethically approved by the research ethics committee of ahmad dahlan university on february 9, 2016, with reference no. 011601011. the test animals, 8 week-old balb/c mice, were acclimatized for a week before the treatment. the mice were divided into five (5) groups, namely; the normal group, negative control group which were treated with the solvent tween 80 at 10% concentration, and the 3 treatment groups (1.25 mg/20g bw; 2.5 mg/20g bw; 5 mg/20g bw; bw is abbreviation of body weight). the administration of eezc was carried out once a day, orally for 21 days (3 weeks). on the 22nd day, the mice were sacrificed using co2 gas. following sacrificing, lipopolysachcharide (lps) (sigma) with dose of interleukin levels in the zingiber cassumunar treated mice – nurkhasanah et al. 111 0.01 mg/20 g bw was injected into the peritoneal cavity area. after 1 hour, the macrophage was isolated and the expressions of interleukin-10, -12 and -14 were observed using the immunohistochemistry method with the specific antibodies of il-10, il-12 and il-14. macrophage isolation following the 21-day treatment, the mice were injected with lps in the intraperitoneal cavity. the mice were then dissected by opening the skin in the peritoneal area. as much as 10 ml of roswell park memorial institute (rpmi) (sigma) medium was injected into the stomach. the stomach was massaged, then the rpmi medium was drawn again. the medium was centrifuged for 10 minutes, and the supernatant was removed. the macrophage was washed with the medium and incubated for 24 hours. after overnight incubation, the macrophage was harvested. immunohistochemistry assay the immunohistochemistry assay was based on the method reported in nurkhasanah (2015), an indirect method using specific primary antibody that was conjugated with secondary antibody and chromogen. the expressed browncolored interleukin was the product of dimethyl amino benzidine (dab) chromogen detected under the light microscope. the cultured macrophage, which was previously fixed with 1 ml of methanol that was later removed, was washed in pbs (phosphate buffer saline) for 5 minutes. the fixed macrophage was then immersed in peroxidase blocking solution at room temperature for 10 minutes. the macrophage was then washed with running water and then re-washed with pbs. a total of 50 µl of blocking serum was added to the preparation which was then incubated in a humid temperature for 10-15 minutes. the 100 µl (with dilution 1:100) of specific antibodies (antiil-10, anti-il-12, and anti-il-14, murine recombinant, biovision) was then added to the preparation and incubated on a moist tray at room temperature for 1 hour. after the incubation, the preparations were washed with 1 ml of pbs. a total of 50 µl (with dilution 1 : 100) of anti-mouse biotin secondary antibody (biovision) was added to each preparation, which was incubated at a humid temperature for 20 minutes and then re-washed with pbs. the preparations were incubated with 50 μl of the streptavidin-peroxidase enzyme for 10 minutes, washed with pbs, and re-incubated with 50 µl of dimethyl amino benzidine (dab) chromogen (peroxidase substrate solution). the preparations were then washed with pbs and incubated with mayer’s hematoxylin as the counterstain and then rewashed with pbs in preparation for the microscopic observation at 400x magnification. the macrophages that expressed interleukin manifested brown stains. the observation was carried out from several fields of view (fov) of the microscope. the number of expressed positive cell was compared with the total number of observed cell and presented as percentage value. the results of the treated groups were statistically compared with that of the control group to analyze the effect of treatment. statistical analysis the quantitative percentages of il-10, il-12 and il-14 expressions were analyzed statistically for normality and homogeneity and then further analyzed using anova and followed by lsd analysis among the treated group. results and discussion extraction the zingiber cassumunar ethanol extract (eezc) was dark brown with a specific odor, exhibited thick consistency, and has slightly bitter taste. the extraction process produced 25.55% yield which has met the standard of the indonesian herbal pharmacopoeia (depkes ri 2008). curcumin was found to be the major content in the eezc extract, h however, the other curcumin derivates (i.e., demethoxycurcumin and bisdemethoxycurcumin) were not detected (fig. 1). curcumin reportedly showed immunomodulatory activities (varalakhmi et al. 2008). after the treatment, significant increases of il12 levels were observed among the curcumintreated animals on day 10 and 20. curcumin was biotropia vol. 28 no. 2, 2021 112 also found to induce generation of reactive oxygen species (ros) which are important in the immune responses (varalakhmi et al. 2008). curcuminoids (cassumunin a and cassumunin b) isolated from z. cassumunar were observed to have a protective effect on living cells suffering from oxidative stress (nagano et al. 1997). besides curcumin, essential oil was also reported as one main compound in zingiber cassumunar rhizome. the high essential oil content was responsible for the specific odor of z. cassumunar rhizome and extract. several studies on the phytochemical compounds and biological activities of z. cassumunar roxb had reported the main component of z. cassumunar rhizome essential oil as triquinacene 1,4-bis (methoxy), (z)-ocimene and terpinen-4-ol (buain & paisooksantivatana 2009). previous studies on its rhizome also found several phenylbutenoid compounds, curcuminoid, and sesquiterpene (zerumbon) (nakamura et al. 2009). expression of il-10 indirect immunocytochemistry was used to detect the interleukin expression in the macrophage (fig. 2). the specific antibody of il-10 interacted with interleukin-10 in the cells and attached itself to the secondary antibody. during the detection process, the secondary antibody attached itself to dimethyl amino benzidine (dab) as the chromogen, and the expression appeared as brown stains on the cytoplasm area, while the cells with negative expression appeared as blue stains as the result of counterstaining. the percentage of the il-10 expression is shown in table 1. figure 1 the tlc profile of curcuminoids standard (a) and zingiber cassumunar ethanolic extract (b), detected in daylight (a) and uv 254 nm (b). a b interleukin levels in the zingiber cassumunar treated mice – nurkhasanah et al. 113 figure 2 immunocytochemistry of il-10 expression in macrophage cells after beign treated with ethanol extract of zingiber cassumunar which was observed with 400x magnification: (a) macrophage with no expression of interleukin; and (b) macrophage with positive expression of interleukin table 1 percentage of il-10 expression on the macrophage cells of balb/c mice treated with ethanol extract of zingiber cassumunar groups mean ± sd normal negative control treatment dose of 1.25mg/20gbw treatment dose of 2.5mg/20gbw treatment dose of 5mg/20gbw 45.65 ± 1.92%* 51.86 ± 1.42% 55.91 ± 3.07% 63.68 ± 2.93%* 68.65 ± 4.42%* notes: * = significant difference with negative control (p<0.05); bw = body weight. the treatment of zingiber cassumunar ethanol extract (eezc) has increased the expression of il-10, affirming its potential as an immunomodulator. il-10 was expressed by macrophages and other dendritic cells (dc) as a response to microbial infection. the increase of il-10 expression may be attributable to the activation of extracellular signal-regulated kinase 1 (erk1) and erk2 (saraiva & o’garra 2010). such an increase will activate the specific response of the immune system and inhibit the nonspecific response. il-10 has been identified as an inhibitor of the synthesis of inflammatory mediators and pro-inflammatory cytokines that play a role in modulating fever and sickness (harden et al. 2013). the present study also found that the expression of il-10 in the negative control group significantly increased as compared to the normal group, which could be caused by the tween 80 effect. another study also reported that polysorbate (tween) 80 could increase the immune response (maggio 2012). the increased expression of il-10 was proportional to the administered dose. the higher the treatment dose, the higher the expression of il-10. however, an extremely high level of il-10 can inhibit chemokine production and prevent its role in directing lymphocytes to the lymph nodes, as manifested in mycobacterial infection, resulting in a failure to recruit and induce th1 cell differentiation (couper et al. 2008). therefore, il-10 has both immunosuppressive and immunostimulatory properties (acuner-ozbabacan et al. 2014). regulation of the il-10 expression involved the enhancement or silencing of il10 transcription and is performed by certain transcription factors activated by discrete signaltransduction pathways. following transcription, the post-transcriptional mechanisms existed and involved many of the molecular events leading to il-10 expression (saraiva & o’garra 2010). some molecules of zingiber cassumunar were involved and had affected the transcriptional biotropia vol. 28 no. 2, 2021 114 process of il-10 and resulted in the increasing il-10 levels. expression of interleukin-12 interleukin-12 (il-12) is a pro-inflammatory cytokine that induces the production of interferon-γ (ifn-γ), leading to the differentiation of t helper 1 (th1) cells and connecting the link between innate and adaptive immunity. dendritic cells (dcs) and macrophages produce il-12 in response to pathogens and infection (trinchieri 2003). production of il-12 is strongly regulated by positive and negative regulatory mechanisms. microorganism products including bacteria, intracellular parasites, fungi, double-stranded rna, bacterial dna and oligonucleotides are strong inducers of il-12 production by macrophages, monocytes, neutrophils and dcs. in this study, the lps was used to activate the macrophage production of the il-12 expression after the administration of eezc and was analyzed quantitatively (table 2). the study found out that the il-12 expression in the negative control group was not significantly different from the normal group, indicating that the solvent (tween 80) did not affect the immune response. tween 80 can stimulate the immunogenicity (maggio 2012) as also shown by the increased il-10 levels in the present study. however, the dosage applied in this study was not enough to increase the il-12 expression. eezc treatment at a dose of 1.25mg/20g bw resulted in an il-12 expression lower than the negative control. hence, the lower the dose, the less effective is the eezc active compound in increasing the il-12 expression. when the dose was increased, the il-12 expression was also heightened. the eezc immunomodulatory effects might have been due to the presence of curcumin, the active compound known to increase the immune response of the cells (nagano et al. 1997; nurkhasanah et al. 2017). curcumin treatment also elevated the il-12 expressions in mice (varalakhmi et al. 2008). the increasing level of il-12 in the treatment was caused by the capacity of curcumin in increasing reactive oxygen species (ros) and nitric oxide (no) (nurmasari et al. 2014; rahmawati 2013). ros is known to regulate the il-12 generation. curcumin was also found to stimulate the t cells, b cells, neutrophil, nk cell, and dendritic cell (nurmasari et al. 2014). the essential oil, which emitted a special odor, was also identified in the eezc (bhuiyan et al. 2008). these eezc essential oils were also reported to boost the body immune response, including the phagocytic activity of macrophages (chairul et al. 2009; nakamura et al. 2009). the active compounds from the volatile oil are the phenilbutenoids which were successfully identified as immunomodulatory (chairul et al. 2009). the eezc treatment increased the il-12 expression, thereby activating the t cells and stimulating the production of ifn-γ, which led to macrophage activation and secretion of reactive oxygen species (ros) that eliminate infections (abbas et al. 2017). furthermore, this treatment has intensified the phagocytic activity of macrophages (nurkhasanah et al. 2017). expression of interleukin-14 eezc treatment also increased the il-14 expressions after lps induction (table 3). il-14 was the first known high-molecular-weight bcell growth factor, originally identified as a b cell growth factor (shen et al. 2006). as produced by t cells and b-cells, the il-14 binds and signals through a 90-kda receptor that promotes b-cell proliferation (akdis et al. 2016). high levels of il-14 can enhance b-cell proliferation and can expand a subpopulation of memory b cells (leca et al. 2008), and if followed by the secretion of antibody, it can also eliminate the invader. table 2 il-12 expression in the macrophage cells of balb/c mice treated with ethanol extract of zingiber cassumunar (eezc) groups mean ± sd normal 64.63% ± 9.763 negative control 66.39% ± 1.603 treatment dose of 1.25mg/20g bw 51.56% ± 4.528* treatment dose of 2.5mg/20g bw 70.62% ± 3.469 treatment dose of 5mg/20g bw 77.00% ± 5.110* note: * = showed significant difference from the negative control (p<0.05). interleukin levels in the zingiber cassumunar treated mice – nurkhasanah et al. 115 table 3 interleukin-14 expressions in mice treated with ethanol extract of zingiber cassumunar groups expressions (x ± sd) normal 59.19 ± 3.07% negative control 61.24 ± 1.51% treatment dose of 1.25 mg/20g bw 57.02 ± 1.94%* treatment dose of 2.5 mg/20g bw 67.41 ± 6.60% treatment dose of 5 mg/20g bw 71.07 ± 1.30%* note: * = showed significant difference with the negative control (p<0.05). previous researches studied the increase of il-14 expression by using some medicinal herbal extracts (nurkhasanah 2015). the treatment of anthocyanin-rich rosella extract increased both the il-10 and il-14 expressions in vitro. the present research also found that the treatment of eezc increased the il-10, il-12, and il-14 expressions after lps induction. this induction stimulated the immune response as lps was recognized as an endotoxin, consisted of a lipid and a polysaccaride, found on the outer membrane of gram-negative bacteria. eezc’s active role in increasing the il-10, il12 and il-14 exhibited its potency in inducing both the innate and adaptive body immunity. studies on zingiberaceae family, including curcuma mangga, kaempferia angustifolia, and zingiber cassumunar recorded that zingiber cassumunar displayed the highest immunomodulatory activity (chairul et al. 2009). this is probably due to the active curcumin compound and its essential oil which has the phenyl butanoic substance. furthermore, a toxicity study confirmed that z. cassumunar extract has no observable adverse effect and it is well-tolerated for both acute and chronic toxicity studies (koontongkaew et al. 2014). conclusion the zingiber cassumunar ethanol extract (eezc) exhibited immunomodulatory activities by increasing the levels of il-10, il-12 and il14 cytokines in the treated mice. this study also suggested the extract’s potency to induce both the innate and adaptive body immunity. acknowledgment the 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indonesian rice varieties susiyanti1,5*, rusmana1,5, yeyen maryani2,5, sjaifuddin3,5, nanang krisdianto4,5 and mohamad ana syabana1,5 1department of agroecotechnology, faculty of agriculture, universitas sultan ageng tirtayasa, serang 42123, indonesia 2department of chemical engineering, faculty of engineering, universitas sultan ageng tirtayasa, serang 42435, indonesia 3department of science education, universitas sultan ageng tirtayasa, serang 42117, indonesia 4department of agribusiness, faculty of agriculture, universitas sultan ageng tirtayasa, serang 42123, indonesia 5 indonesia center of excellent for food security, universitas sultan ageng tirtayasa, serang 42123, indonesia received 15 march 2018 / accepted 20 december 2018 abstract rice has different varieties, with each variety possessing diverse physical and chemical characteristics. the objective of this study was to analyze the physicochemical properties of several indonesian local rice varieties. the experiment was conducted from march to april 2017 at the agriculture applied technology laboratory of the faculty of agriculture, universitas sultan ageng tirtayasa and at the laboratory of food analysis services in the department of food and technology, ipb university. nine local rice varieties from several areas in indonesia were used as samples, namely jalahawara, ciherang, pandan wangi, rojolele, sokan, bendang pulau, batang piaman, cisantana and sidrap. their physicochemical characteristics were analyzed based on some criteria such as: physical quality (weight, length, width, form and percentage of chalkiness), chemical content, water content, ash content, fat content, protein content, carbohydrate content, crude fiber content, starch, amylose and amylopectin content. data obtained were analyzed by one-way anova using a randomized block design. jalahawara has the highest percentage of chalkiness. based on the ratio of length and width, sidrap and ciherang were categorized as medium type and the others were oval/round. the heaviest and lightest based on the 1000grain weight of rice were ciherang and bendang pulau, respectively. the water content was about 2-4% for all samples. the highest and lowest amount of ash and fat content were found in sidrap and sokan, respectively. the highest and lowest amount of protein content were found in batang piaman and sokan, respectively. the highest and lowest starch content were observed in pandan wangi and ciherang. the content of amylose and amylopectin were the highest in batang piaman. the rice samples were categorized into two groups of low and medium levels of amylose. the low level of amylose were observed in cisantana, ciherang, pandan wangi and sidrap, while the medium level of amylose were observed in jalahawara, sokan, bendang pulau, batang piaman and rojolele. keywords: indonesian local rice, physicochemical properties introduction food, a basic need of human beings, is a source of energy for humans to do their activities and survive in life. the quality of both husked and un-husked rice becomes the priority of rice cultivation which is related to consumer satisfaction. more than half of world population consumes rice as the main daily source of calories and protein (tonini & cabrera 2011; rohit & parmar 2011). industrial development has affected the determination of rice taste and quality. a dominant factor in determining the taste of rice is amylose content in the rice (setyowati & kurniawati 2015). rice quality is a combination of its physical and chemical (physicochemical) characteristics particularly needed by consumers. rice tastiness is closely related to rice quality and its complex physicochemical characteristics, namely amylose content, starch, gel consistency, gelatin temperature and protein level. rice quality is also related to stickiness, sweetness, transparency and palatability. *corresponding author, e-mail: susiyanti@untirta.ac.id biotropia vol. 27 no. 1, 2020 42 palatability (good taste) is a characteristic of rice related to quality, determined by smell, appearance, taste and texture. in addition to genetic factors, such as those in the synthetic processing of starch and protein, rice quality is affected by environmental conditions, such as water availability, temperature, fertilizer utilization, drought and salinity stresses (chen et al. 2012). the physical and chemical properties of rice depend on the variety used (rolando et al. 2013). consumers have the right to information about physical characteristics and chemical content of consumable goods, including rice. sitaresmi et al. (2013) stated that a large amount of germplasm of good quality local rice variety had been identified as vulnerable and tolerant to biotic and abiotic stresses. the indonesian agricultural research and development agency had released tungro-resistant varieties, such as tukad unda, tukad petanu, tukad balian, bondojudo, kalimas, inpari 7 lanrang, inpari 8 and inpari 9 elo (suprihatno et al. 2009; mejaya et al. 2014). these variety had not been fully adopted by farmers because of its limited seeds availability, undesirable taste and poor quality which affected the selling price (muliadi et al. 2015). however, ciherang varieties have advantages in terms of its productivity, tastiness, market segments and relatively early maturity. new superior varieties are difficult to develop and market, if they do not have the previously mentioned potential characteristics. indonesia possesses a very wide rice diversity comprising around 17,000 germplasm accessions including several wild varieties (maulana et al. 2014). indonesia is the centre of rice origin. many places in indonesia still have good potential sources of the local rice seeds. each variety has different and unique characteristics, namely unique taste, color, nutrients and chemical composition (yang et al. 2010). hence, it is necessary to gather basic data related to the physicochemical properties of these local rice varieties, particularly related to plant cultivation processes that affects rice quality. the objective assessment of rice quality includes its physical and chemical characteristics, while the subjective assessment focuses on the individual and community preferences. the objective of this study was to analyze the physicochemical properties of several indonesian local rice varieties. materials and methods plant genetic materials nine varieties of the indonesian local rice were used as samples, namely java varieties (jalahawara, ciherang, pandan wangi, rojolele), sumatra varieties (sokan, bendang pulau, batang piaman) and sulawesi varieties (cisantana, sidrap). the research was conducted from march to april 2017 at the agriculture applied technology laboratory of the faculty of agriculture, universitas sultan ageng tirtayasa and food analysis laboratory, department of food technology, ipb university. methodology milled rice grains from 9 local rice varieties from several places in indonesia were used as research samples. their quality was identified based on the following criteria: physical quality, water content, ash content, fat content, protein content, carbohydrate content, crude fiber content, starch, amylose and amylopectin content. the data acquired were then analyzed using the one-way analysis of variance (anova) and a randomized block design for all the physicochemical observations. the mean values were compared using duncan's new multiple range test (dnmrt) to confirm the differences among the varieties. physical quality of rice physical quality of rice was analyzed according to utami et al. (2019). the weight of un-husked rice was measured by measuring the weight of 1,000 undamaged grains using analytical scales of each sample. moreover, the length and width of 10 rice grains from each sample were measured by using calipers. the rice form was determined by the ratio of length and width of the rice grain. furthermore, the percentage of chalkiness was obtained by observing at the opacity proportion of the endosperm. water and ash content the water and ash content were analyzed using the gravimetric method (longvah & prasad 2020). for water content, it was measured by comparing the weight of rice physicochemical properties of indonesian rice – susiyanti et al. 43 before and after being heated. the difference is assumed as the decreased water content in the local rice after being heated at 105 oc, above the boiling point of water. for ash content, an empty cup was heated in the oven and then cooled in the desiccator for 30 min. the cup was then filled with 5-gram rice of each sample. each sample was weighed and then heated in the furnace at 550 °c for 2-3 h until it turned into ash. the cup was then cooled in the desiccator and weighed subsequently. the percentage of ash content was calculated as follows: ash content (%) = [ash weight (g)/sample weight (g)] × 100%. fat content the fat content was measured using soxhlet method (sultana et al. 2018; choi et al. 2015). the fat was extracted using the non-polar fat solvents (hexane). the solvent was then distilled and evaporated; the fat extract stayed in the tube and was measured as fat weight. protein content the protein content was measured using the kjeldahl method (wang et al. 2016), assuming that all ns in the sample are protein. the observation process was done in three stages: first, destroying the sample in acid atmosphere to free the n from the sample then binding n in the form of ammonium sulfate, while c and h were oxidized into h2o and co2; second, distilling the sample to break the ammonium sulfate into ammonia based on the different boiling points; third, titration was done to measure the n content in the sample (legowo 2005). carbohydrate analysis carbohydrate analysis was done using “by difference” method (shakappa & talari 2016). the following is the equation used in calculating carbohydrate levels: carbohydrate content (%) = 100% (% water content + % ash content + % protein content + % fat content). analysis of crude fiber using the soxhlet method (sultana et al. 2018; choi et al. 2015), a 0.4 g of fat sample was extracted and then filtered with filter paper and wrapped. the filter washed with acid and alkali solvents (0.3 n naoh and 1.5 n h2so4) and placed in boiling water and was finally cleaned with distilled water. afterwards, the filter was washed again with solvent and heated to reach its boiling point, and then put in the oven for 1 hour at 105 °c, and finally, the crude fiber content was calculated. starch, amylose and amylopectin contents the starch content was analyzed according to the luff schoorl method (aoac 1995), using reagent luff schoorl from 0.1 g of ground rice sample which was then measured by the titration process. the starch content was measured based on the difference between titration on the blank paper and sample titration. sugar content was reduced after inversion (after hydrolysis using hcl 25%) inside the compound, which could be found in the table of standards. the starch content of the compound was calculated from the difference between the sugar content before and after inversion, then multiplied with 0.9. furthermore, the measurement of amylose content was conducted through iodometry based on the reaction between amylose and iodine compound which produced the blue color (juliano & villareal 1993). each sample was taken from 100 mg of rice dissolved with 1 ml of 95% ethanol and 9 ml of naoh 1 m and boiled for 10 min with water boiling at 95 c until it turned into a gel. then, 100 ml of water was added into the gel and stirred. five ml of sample solvent was homogenized with 1 ml of acid acetate 1 n, 2 ml of iodine solvent 0.01 n (step by step) and aquadest, then heated at 30 c for 20 min and measured with spectrophotometer uv-vis in 620 nm wavelength. amylopectin content was calculated by reducing the starch content with amylose, as starch basically consists of amylose and amylopectin. results and discussion determining the rice physical quality grain characteristics such as weight, length and width are prominent criteria in determining rice quality (table 1). the width, length, and shape of rice grain affect the ease of the grinding biotropia vol. 27 no. 1, 2020 44 process. jalahawara has the highest percentage of chalkiness compared to other rice and has small grains with a less transparent appearance. on the other hand, although its nutrients disappear after cooking and do influence the food quality, rice with transparent grain are still preferred by consumers. most rice consumers in principle, choose uniform and translucent grains (xi et al. 2014). rice consumers also prefer a product of satisfactory appearance even though most of the nutrients have disappeared (abbas et al. 2011). the genetic and environmental factors influenced the appearance of the rice grain which is assessed as the percentage of grain chalkiness. chalkiness is an unwanted trait that negatively affects the milling, cooking, eating, and grain looks and that reflects the main concern in many producing areas of rice (siebenmorgen et al. 2013). nevame et al. (2018) stated that the happening of the chalkiness in rice is linked to both the genetic and environmental element, notably high temperature. the highest percentage of chalkiness was found in jalahawara rice and the lowest in pandan wangi. based on its length, the local rice was classified into extra-long grain at >7 mm (cisantana, ciherang, sidrap), long grain at 6.06.99 mm (jalahawara, sokan, pandan wangi, rojolele) and medium grain at 5.5-5.99 mm (bendang pulau). based on the ratio of length and width, sidrap and ciherang rice were included in the medium size category, while the other types were categorized as short grain rice with oval or round shaped. most people in india prefer long grain, but people in south east asia prefer medium grain (jewell et al. 2011). furthermore, the heaviest 1000-grain weight was that of ciherang variety, followed by jalahawara rice, while the lightest weight was that of the bendang pulau rice. the weight of the 1000-grains of rice determined the amount of rice production. the weight of rice is affected by the environmental and atmospheric factors at the moment of grain harvesting. patindol et al. (2015) said that the environmental elements (such as air temperature, atmospheric carbon dioxide, light, water, and soil nutrients) are important for plant growth and reproduction, temperature is the most explored environmental aspect in relation to rice production and grain quality. table 1 chalkiness, grain weight, length, width and the ratio of length/width of indonesian rice varieties no rice sample % chalkiness 1,000 grains weight (g) length (cm) width (cm) ratio of length/width 1 jalahawara 89 a 24.715 b 0.655d 0.255 f 2.57 d 2 cisantana 25 f 19.575 f 0.769 a 0.261d 2.95 c 3 ciherang 62 c 25.098 a 0.742 c 0.221 i 3.36 a 4 sokan 35 e 16.890 h 0.617 g 0.254 g 2.43 e 5 bendang pulau 14 h 11.554 g 0.532 h 0.258 e 2.06 h 6 batang piaman 22 g 21.693 d 0.618 g 0.302 a 2.05 i 7 pandan wangi 5 i 19.596 f 0.624 f 0.266 c 2.35 f 8 rojolele 72 b 21.013 e 0.644 e 0.282 b 2.28 g 9 sidrap 56 d 21.995 c 0.744 b 0.222 h 3.35 b note: means with different letters inside a column are significantly different (p < 0.05, dmrt). physicochemical properties of indonesian rice – susiyanti et al. 45 figure 1 the appearance of indonesian local rice grains the opaque areas in the endosperm of the rice grains formed the starch and protein particles that break easily at grinding (fig. 1). compared to rice grains with transparent endosperm, their selling point is low. the different colors of rice are arranged genetically as a result of genetic differences of aleuron color, endosperm color and starch composition in the endosperm. white rice has white and transparent colors because it has only a little aleuron color and amylose content of about 20%. the outermost layer of the endosperm consists of aleurone cells and starch inside (ishimaru et al. 2015). the aleurone layer accumulates lipids, while the starchy endosperms mainly accumulate starch. during the ripening stage, the degree of starch accumulation is known to be out of sync, depending on the location of the starchy endosperm. water content based on the indonesian national standard no. 6128:2015 (bsn 2015), the maximum water content of premium rice is 14%, while that of medium rice is 15%. the water contents of the different rice varieties varied between 12% and 14%, and only jalahawara had the water content of above 14% (table 2); so almost all of the local rice samples are included under the category of premium rice. one of the benefits of water content standardization of the local rice is the prolonged rice storage time. if the rice has low water content, the development of microbes in the rice can be slowed down. moreover, moisture also plays an important role in determining the rice quality (tamrin et al. 2017). water content affects the physical and chemical appearance of rice. if the water content is high then the rice will turn yellow with an unpleasant aroma and cannot be stored for a long period. table 2 level of water, ash, fat, protein and carbohydrate contents of indonesian local rice varieties no rice sample water content (%) ash content (%) fat content (%) protein (%) carbohydrate (%) 1 jalahawara 14.44 a 0.54 g 0.24 h 9.41 e 75.36 h 2 cisantana 12.35 f 0.88 b 1.15 b 8.79 g 76.83 d 3 ciherang 12.68 d 0.80 c 0.66 f 9.98 b 75.88 e 4 sokan 13.01 c 0.45 i 0.21 i 7.32 i 79.01 a 5 bendang pulau 12.25 g 0.57 f 0.35 g 8.21 h 78.63 b 6 batang piaman 12.10 h 0.52 h 0.74 e 10.93 a 75.71 f 7 pandan wangi 13.71 b 0.72 e 1.06 c 9.68 c 74.84 i 8 rojolele 12.17 i 0.74 d 0.96 d 8.82 f 77.32 c 9 sidrap 12.38 e 1.00 a 2.12 a 9.57 d 74.94 g note: means with different letters inside a column are significantly different (p < 0.05, dmrt). biotropia vol. 27 no. 1, 2020 46 ash content the lowest ash content was found in sokan (0.45%), while the highest was in sidrap (1.00%). based on indonesian national standard no. 3549:2009 (bsn 2009), the maximum ash content of rice starch is 1%; hence, all the samples have fulfilled the indonesian national standard. ash content is related to the mineral content of rice because when rice is heated until 1,000 oc, only the mineral is not oxidized. ash content is also influenced by the environment where the rice grows. the composition of rice varieties differs according to the soil in which rice is grown, together with manuring (abbas et al. 2011). the minerals most frequently found in rice husk ash are p, mg, and k. in addition, it may consist of ca, cl, na, si and fe. the minerals are distributed over the outer layer of the rice grain, and the concentration decreases as they move towards the internal part of the rice. oko and ugwu (2011), said that the mineral compositions in rice are: n (0.1-1.08%), p (0.52-0.54%), k (0.15-0.20%), na (0.13-0.17%), ca (0.07-0.11%) and mg (0.19-0.26%). fat content the fat content varied from 0.21% to 2.12%. the large amount of fats (above 0.5%) was found in cisantana, ciherang, batang piaman, pandan wangi, rojolele and sidrap rice. the study results differed from that of tarigan and kusbiantoro (2011), which recorded a fat content between 0.2-0.3%. the amount of fat content in rice is influenced by the environment where the rice plant grows. kang et al. (2011) stated that the main component of fatty acid in rice was oleic acid (18:1), linoleate (18:2), and palmitate (16:0), and the composition of those three components reaches 90% of the total fatty acid in rice. protein content the lowest protein content was observed in sokan (7.32%) and the highest in batang piaman (10.93%). the highest amount of amino acids found in the rice protein were aspartate and glutamate, while the acids with the smallest amount were cysteine and tryptophan (kang et al. 2011). protein content has a close relationship with palatability or taste quality rice (song et al. 2012). protein also supports rice texture by making the barrier which results in less leaching of rice component during the cooking process. variation in protein content causes differences in the viscosity of rice paste due to its effect on the expansion of starch granules and on the character of its stiffness. protein plays an important role in rice quality based on a water-producing agent that occurs during cooking rice and that increases the stickiness of cooked rice. carbohydrate content carbohydrate was the most abundant nutrient in all the samples. the lowest (74.84%) was found in pandan wangi and the highest (79.01%) in sokan. carbohydrate content in rice is mostly in the form of starch, consisting of amylose and amylopectin. fiber is also found in rice, including hemicellulose (food fiber) and lignin (crude fiber). the shape and size of starch granules are affected by the source and the condition around the growing zone (omar et al. 2016). fiber content adequate amounts of dietary fiber are also found in rice grains (aune et al. 2011), thus rendering rice a healthy food (thomas et al. 2015). the crude fiber content varied from 8.91% to 24.51% among the rice varieties. the crude fiber content is influenced by the variety of rice. fiber is a plant component and a type of carbohydrate. fiber is not a nutrient and cannot be digested by the human system. based on its structural characteristics, fiber consists of two kinds, food fiber, and crude fiber. food fiber cannot be digested by amylose enzyme, while crude fiber can be hydrolyzed by acid and strong alkaline such as cellulose, hemicellulose, and lignin. starch, amylose and amylopectin content the starch content in the rice sample largely contributed (72.92% to 88.18%) to its dry weight (table 3). starch is composed of amylose and amylopectin (bertoft 2017). starch is one kind of carbohydrate in plants that functions as an energy reserve. starch in rice and other seeds is the major carbohydrate, so that the amount of starch is more prominent than other kinds of physicochemical properties of indonesian rice – susiyanti et al. 47 carbohydrate. the histochemical investigation of the caryopsis indicated that starch was mainly accumulated in the endosperm (yu et al. 2014). amyloplasts in endosperm contained many starch granules that were spherical during the early stages but turns polyhedric at the later stages. structurally, the starch arrangement of monosaccharide units is like glucose. glucose molecules are bonded with each other, producing two kinds of starch, amylose and amylopectin. amylose has a linear chain structure bound by α-(1,4)-d-glycoside bonds, while amylopectin has a branching structure linked by α-(1,6)-d-glycoside bonds that comprise around 4% to 5% of the total weight. the starch content varied depending on the rice variety and the environment where the rice grew. the highest amount of starch was found in pandan wangi (88.18%), and the lowest was in ciherang (72.92%). the amylose content was around 17% to 24%, and amylopectin was around 54% to 70%. the ratio of amylose/amylopectin is stated only in amylose content because amylopectin can be obtained from the reduction of starch by amylose. the ratio of amylose/amylopectin determines the texture of rice, whether hard or not, getting hard easily or not, sticky or not. the smaller the content of amylose or the higher the content of amylopectin, the stickier the rice will be. for example, when sticky or glutinous rice is cooked, physically it is really sticky because the amylose content is very low, around 1% to 2% (in waxy/glutinous rice). however, the amylopectin is very high. this study showed that the rice samples were not glutinous. non-sticky rice are classified into 4 groups based on its amylose content: 1) rice with high amylose content (25% to 33%); 2) rice with medium amylose content (20% to 25%); 3) rice with low amylose content (9% to 20%); and 4) rice with very low amylose content (2% to 9%) (masniawati et al. 2018). based on this classification, the rice samples in this study belonged to 2 groups; that is rice with low amylose content for cisantana, ciherang, pandan wangi and sidrap rice; and rice with medium amylose content for jalahawara, sokan, bendang pulau, batang piaman and rojolele. comparison between those two groups of starch (amylose and amylopectin) determines the color of rice (transparent or not) and also the texture of rice (sticky, soft, hard or really hard). sticky rice is dominated with amylopectin so that it is really sticky, while hard rice (‘pera’ rice) has an amylose content of more than 25% resulting in the seeds of rice not sticking to each other when they are well cooked. the main component of rice is starch, while the minor components are protein (4.5% to 10%) and fat (0.5%). amylose content determines the physicochemical characteristics of rice starch. starch granule which has high amylose is strong and stiff and will result in hard rice (‘pera’ rice). on the other hand, if the starch granule is soft, it can result in soft and tasty rice when it is well cooked (li et al. 2017). table 3 amounts of crude fiber, starch, amylose and amylopectin no rice sample crude fiber content (%) starch (%) amylose (%) amylose criteria amylopectin (%) 1 jalahawara 11.65 d 78.88 e 20.89 e medium 57.99 e 2 cisantana 24.51 a 77.39 g 18.31 h low 59.07 d 3 ciherang 18.67 b 72.92 i 18.48 g low 54.44 i 4 sokan 12.62 c 85.90 c 23.29 b medium 62.61 c 5 bendang pulau 8.91 h 87.39 b 23.19 c medium 64.20 b 6 batang piaman 9.39 g 79.25 d 24.61 a medium 54.63 h 7 pandan wangi 9.53 f 88.18 a 17.74 i low 70.45 a 8 rojolele 9.72 e 77.67 f 21.78 d medium 55.90 g 9 sidrap 11.73 d 76.74 h 19.30 f low 57.44 f note: means inside a column with different letters are significantly different (p < 0.05, dmrt). biotropia vol. 27 no. 1, 2020 48 conclusion the jalahawara variety was moderately transparent and had a high percentage of chalkiness compared to other varieties. the ratio of the length to width of rice grain for sidrap and ciherang varieties were considered mediumsized, and the other types as short/oval/round size. ciherang is the heaviest rice based on the 1,000-grain weight, and the lightest rice was bendang pulau. the water content in the sample of the rice was between 12-14%. the lowest ash content was found in sokan (0.45%) and the highest was in sidrap (1.00%). the fat content varied from 0.21% (sokan) to 2.12% (sidrap). the lowest protein content was found in sokan (7.32%), and the highest fat content was in batang piaman (10.93%). the highest starch content was observed in pandan wangi (88.18%), and the lowest is in ciherang (72.92%). amylose content among the varieties was around 17% to 24%, and amylopectin was around 54% to 70%. the studied local rice varieties were not sticky rice. the amylose content categorised the rice varieties into two; the first group included the varieties with low amylose content (cisantana, ciherang, pandan wangi, and sidrap); and, the second group included those varieties with medium amylose content (jalahawara, sokan, bendang pulau, batang piaman, and rojolele). acknowledgments the authors would like to express their gratitude to the islamic development bank who gave financial support as part of the research grant of the indonesia center of excellence for food security of universitas 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by scanning electron microscopy. plant prod sci 17(4):285-90. yang ds, lee ks, kays sj. 2010. characterization and discrimination of premium-quality, waxy and black pigmented rice based on odor-active compounds. j sci food agric 90(15):2595-601. doi: 10.1002/ jsfa.4126 yu x, zhou l, xiong f, wang z. 2014. structural and histochemical characterization of developing rice caryopsis. rice sci 21(3):142-9. biotropia vol. 31 no. 1, 2024: 34 43 doi: 10.11598/btb.2024.31.1.2020 34 response of amaranthus viridis plant functional traits to npk 12:12:17 and npk 15:15:15 fertilizers ker ning chew, jin zhe tan, hoe yin yik and pei sin tong* department of agricultural and food science, faculty of science, universiti tunku abdul rahman, kampar 31900, malaysia received 8 may 2023 / revised 23 january 2024 / accepted 21 february 2024 abstract a paradigm shift from the prevailing reliance on chemical methods to alternative weed-control approaches is necessary to achieve sustainable weed management. however, the understanding of weed biology explaining “how” and “why” remains insufficient in facilitating this shift. this study employed a trait-based approach — examined the number of leaves, number of inflorescences, and height — to investigate the growth and developmental patterns of amaranthus viridis, a weed species in the tropics, in response to npk fertilization. the experiments were carried out in three sets of weeds — wild population (untreated and not transplanted; n = 6), npk 15:15:15 (transplanted and fertilized with npk 15:15:15 from march 2020 to september 2020; n = 30), and npk 12:12:17 (transplanted and fertilized with npk 12:12:17 from may 2021 to september 2021). the npk treatment sets comprised five treatments, including one untreated control, with six replications for each treatment. pearson’s correlation coefficient (r) and linear regression (r2) in three models were estimated using leaves, inflorescences and height as dependent and independent variables. in model 1, the number of leaves was the dependent variable and plant height was the independent variable; model 2 included the number of inflorescences as the dependent variable and the number of leaves as the independent variable, whereas the number of inflorescences as the dependent variable and number of leaves and height as the independent variables were used in model 3. all models exhibited a significantly positive correlation and r2 (p < 0.01). specifically, model 3, examining the interactions of inflorescence with leaf numbers and plant height, demonstrated higher values for both r and r2. in conclusion, this study reveals the distinct patterns of functional traits in a. viridis in response to fertilizers and within wild populations, providing predictive models applicable to diverse data types, with implications for understanding inherent growth and responses of weed species for sustainable weed management practices, particularly in collaboration with smallholder farmers. keywords: amaranthus viridis, herbicides, plant functional traits, weed biology, weed management introduction weeds are recognized as the primary pests hindering crop establishment and growth, resulting in varying yield losses depending on the crop type (gharde et al. 2018), constituting the principal constraint in crop production (orke 2006), causing an average yield loss of 28% (vilà et al. 2021). therefore, weed management is critical for ensuring food security and environmental sustainability (yaduraju & rao 2013). the emergence of weed science as a distinct discipline is relatively recent, dating back less than 100 years, compared to other disciplines, such as plant pathology (timmons 2005). the evolution of the weed science discipline stemmed from the discovery of the first synthetic herbicide, 2,4-dichlorophenoxyacetic acid (2,4-d), in 1941 by pokorny, which subsequently underscored the importance of weed management. tillage and herbicides stand as the primary methods for weed removal from farms (maclaren et al. 2020). however, the overwhelming success of herbicides has shaped the weed science discipline into a “herbicidecentered” discipline. this shift not only poses challenges for diversifying research in weed management but also imposes several concerns related to the environment, such as *corresponding author, email: tongps@utar.edu.my effect of fertilizers on the functional traits of amaranthus viridis – ker ning chew et al. 35 environmental hazards, contamination of natural resources, and the emergence of herbicideresistant weeds, as well as consumer health. herbicide residue contaminations are detected in soil as well as in surface and groundwater (allinson et al. 2017; sun et al. 2017). another major concern associated with “herbicidecentered” weed management is food safety. numerous studies have highlighted herbicide residues in fruits and vegetables, raising apprehensions among importing countries and consumers (amjad et al. 2013; matt et al. 2013). kim et al. (2017) revealed the existence of various direct and indirect routes of chemical exposure. therefore, the persistent use of herbicides is also an alarming concern for toxic exposure to humans and other non-target organisms. these environmental and health-related challenges underscore the need for a broader perspective in weed management beyond a reliance on herbicides, such as a paradigm shift towards vegetation management on farms by understanding weed ecology, biology, and genetics (chauhan et al. 2017; clements et al. 2014). furthermore, several aspects of weeds and their interactions remain poorly understood (jordan et al. 2016). for example, the effects of fertilization on weed growth and development remain elusive (little et al. 2021). fertilizers act as a selection pressure in the competitive dynamics between crops and weeds. therefore, strategic weed management is crucial to ensure crops outcompete weeds. desired timing for weed control is when weeds and crop are still in symmetrical competition level, and not in asymmetrical competition where weeds outcompete crops (little et al. 2021). despite the urgency for effective weed management, the lack of detailed information on weed biology has resulted in weak predictions of weed species on farms; specifically, the “how” and “why” aspects have not been fully explored (ward et al. 2014). these knowledge gaps limit the science-based strategies in the application of weed management practices, particularly in developing countries (chaney & baucom 2012; chauhan et al. 2017). several weed biology articles specifically have been published in canada and australia since the 1970s (cavers et al. 2013; groves & panetta 2014); however, the species information is limited to comprehensive literature reviews, offering limited new quantitative information (ward et al. 2014). additionally, understanding the growth and development of weed species can serve as a basis for integrating different weed control methods (chaney & baucom 2012). the parameters determining plant growth include plant height, stem thickness, and biomass, whereas parameters such as the number of leaves and number of inflorescences determine plant development (dambreville et al. 2015). the amaranthaceae family consists of 70 species, of which a few are vegetables and some are weeds. slender amaranth (amaranthus viridis l.) is widely distributed, being native to 33 countries and an introduced species in 98 countries (powo 2022). amaranthus viridis is an annual broadleaf weed with a c4 photosynthetic pathway and is found on farms and in open habitats, such as roadsides. the weed species utilizes seed propagation mechanisms. amaranthus viridis has the second highest mean field density and high relative abundance among other weed species found in smallholder maize farms in malim nawar, malaysia, per field surveys conducted in 2017, 2018, and 2020 (tong & lim 2022). the high mean density and relative abundance of this weed species reflect the degree of difficulty in its control (de mol et al. 2015). this study aimed to investigate the functional relationships between traits (number of leaves, number of inflorescences, and plant height) of a. viridis in wild populations and their growth responses to npk 15:15:15 and npk 12:12:17. the present study demonstrates how enhanced knowledge of weed biology could lead to practical advances benefiting farmers; thus, our study will provide a foundation to initiate the development of science-based weed management technologies. materials and methods experimental design the present study evaluated wild a viridis. a total of 30 seedlings (5–20 cm high) from a single location in kampar, malaysia was used to ensure similar batch of seeds (hanzawa & kalisz 1993; kirkpatrick 1984). the seedlings were transferred to polybags (20 cm × 15 cm × 30 cm; l × w × h) filled with soil from the site from where a. viridis were obtained, with one seedling planted in biotropia vol. 31 no. 1, 2024 36 a polybag. the soils used were tailing parent material from the site where seedlings were found, which was a pale brown to brown upper layer of 30–70 cm. the soil composition included sandy clay to clay, weak fine subangular blocky, friable texture, overlaying brown silty clay to clay, olive-brown to brown coarse sandy clay loam with some clay balls. seedlings were planted in a randomized complete block design (rcbd) with six blocks for five treatments in a shade house, maintained at a relative light intensity of 50% in the northsouth direction. each treatment comprised six replicates. the five treatments included a control without fertilization and four sets fertilized with different dosages (1, 2, 4, and 8 g per polybag) of behn meyer nitrophoska® green npk 15-1515+2s, produced by eurochem agro (the manufacturer’s details are not known). fertilizer was applied one month after transplanting and subsequently on a weekly basis throughout the study period from march to september 2020. additionally, six plants in the wild population were studied without any treatment and were not transplanted. the number of leaves, number of inflorescences, and height (cm) of each plant were recorded weekly. plant organs were considered to reflect the functional characteristics of growth, competitive ability, and reproduction (garnier & navas 2012). the experiment was repeated from may to september 2021 for five months with another set of 30 plants. the fertilizer was changed to agrobridge npkmg 12-12-17-2+te, a muriate of potash (mop) based fertilizer imported from europe (the manufacturer’s details are not known). the duration of both experiments was similar to the maize planting cycle or longer to understand plant responses to fertilizer treatments. these fertilizers were chosen based on their common use by smallholders in maize planting, as reported in weed surveys conducted by tong & lim (2022). statistical analyses the shapiro–wilk normality and q-q plot tests were performed to determine whether a variable had a normal distribution. the null hypothesis of normal population distributions was accepted when p > 0.05 and rejected otherwise. if the normality assumption was met, the pearson correlation coefficient (r) and linear regression (r2) were assessed to analyze the relationship between the number of leaves, number of inflorescences, and plant height. three models were analyzed to estimate the correlation between the traits: model 1, where the number of leaves was used as the dependent variable and plant height as the independent variable; model 2, where inflorescence was the dependent variable and the number of leaves served as the independent variable; and model 3, where the number of inflorescences was the dependent variable and the number of leaves and plant height were the independent variables. the gradient (β) was tested for significance. a significant relationship was observed when the gradient was not zero (p < 0.001). spss version 20.0 was used for statistical analyses. results and discussion the null hypothesis of population normality was retained for a. viridis in wild populations and under the fertilizer treatments npk 15:15:15 and 12:12:17 when p > 0.05. these values were normally distributed. model 1: dependent variable: number of leaves, independent variable: plant height the number of leaves and plant height were significantly positively correlated (r = 0.679) for a. viridis under npk 12:12:17 (b = 21.821, seb = 5.900, β = 0.679, t = 3.698, p = 0.001; figure 1a). the slope coefficient for the leaves was 21.82, suggesting that a 1 cm increase in plant height increases the number of leaves by 21.82. the r2 value was 0.461, indicating that 46.1% of the variation in the number of leaves could be explained by the model containing only plant height. effect of fertilizers on the functional traits of amaranthus viridis – ker ning chew et al. 37 (a) npk 12:12:17 (b) npk 15:15:15 (c) wild plants figure 1 amaranthus viridis studied under (a) npk 12:12:17, (b) npk 15:15:15 and (c) wild plants with the number of leaves as the dependent variable and plant height as the independent variable pearson’s correlation coefficient (r = 0.646) revealed a significant (b = 36.184, seb = 10.383, β = 0.646, t = 3.485, p < 0.001) positive relationship between the number of leaves and plant height for a. viridis under npk 15:15:15 (figure 1b). an increase in height by 1 cm led to an increment of 36.18 leaves, and r2 suggested that 41.7% of the variation in the number of leaves was accounted for by height. concordantly, a positive relationship between plants and the number of leaves (r = 0.975) was observed for the wild population (figure 1c). plant height significantly predicted the number of leaves (b = 18.422, seb = 2.015, β = 0.975, t = 8.864, p = 0.001). the model explained 95.2% of the variance in the number of leaves, and a 1 cm in height increment speculated to increase the number of leaves by 18.23. model 2: dependent variable: number of inflorescences, independent variable: number of leaves assessment using model 2 revealed a positive correlation (r = 0.888) between the numbers of inflorescences and leaves for a. viridis under fertilizer npk 12:12:17 (figure 2a). linear regression analysis revealed a significant relationship (b = 0.270, seb = 0.035, β = 0.888, t = 7.706, p < 0.001). the slope coefficient for the leaves was 0.270, which indicated an increase in the number of inflorescences by 0.270 for each leaf. the model revealed that leaves accounted for 78.8% (r2 = 0.788) of the variance in inflorescences. 20.00 30.00 40.00 50.00 .00 200.00 400.00 600.00 800.00 1000.00 height l e a v e s r = 0.679 r² = 0.461 y = -469.736+21.821*x 25.00 30.00 35.00 40.00 45.00 .00 200.00 400.00 600.00 800.00 1000.00 1200.00 height l e a v e s r = 0.646 r² = 0.417 y = -835.561+36.184*x 25.00 27.50 30.00 32.50 35.00 37.50 .00 50.00 100.00 150.00 200.00 250.00 300.00 height l e a v e s r = 0.975 r² = 0.952 y = -446.237+18.230*x biotropia vol. 31 no. 1, 2024 38 (a) npk 12:12:17 (b) npk 15:15:15 (c) wild plants figure 2 amarantus viridis studied under (a) npk 12:12:17, (b) npk 15:15:15 and (c) wild plants, with inflorescence as the dependent variable and leaves as the independent variable for treatments under npk 15:15:15, a significant (b = 0.109, seb = 0.009, β = 0.949, t = 12.708, p < 0.001) positive correlation (r = 0.949) was observed (figure 2b). the slope coefficient for inflorescences was 0.109, signifying that the number of inflorescences increased by 0.109 for each additional leaf. the r2 value indicated that 90% of the variation in inflorescences could be explained by the number of leaves. the correlation coefficient (r = 0.975) between leaves and inflorescences in wild plants was robust and statistically significant (b = 1.003, seb = 0.016, β = 0.975, t = 8.745, p = 0.001; figure 2c). specifically, each additional leaf corresponds to a notable increase of 0.137 inflorescences. the proportion of variance in the number of inflorescences explained by the number of leaves was 95%. model 3: dependent variable: number of inflorescences, independent variable: number of leaves and plant height the scatterplot revealed a strong linear relationship between the number of inflorescences and leaves and height combined, with an r-value of 0.911 (figure 3a), which was statistically significant [b = 0.212 (leaves), 2.763 (height), seb = 0.044 (leaves), 1.415 (height), β = 0.696 (leaves), 0.282 (height), t = 4.810 (leaves), 1.952 (height), p < 0.001]. specifically, each inflorescence is associated with an increase of 0.212 leaves and 2.763 cm in height. furthermore, the combined effect of leaves and height accounts for 83.1% of the variance in inflorescences. .00 200.00 400.00 600.00 800.00 1000.00 .00 50.00 100.00 150.00 200.00 250.00 300.00 leaves in fl o r e s c e n c e s r = 0.888 r² = 0.788 y = 3.310+0.270*x .00 200.00 400.00 600.00 800.00 1000.00 1200.00 .00 25.00 50.00 75.00 100.00 125.00 leaves in fl o re s c e n c e s r = 0.949 r² = 0.900 y = 8.394+0.109*x .00 50.00 100.00 150.00 200.00 250.00 300.00 .00 10.00 20.00 30.00 40.00 leaves in fl o r e s c e n c e s r = 0.975 r² = 0.950 y = 1.003+0.137*x effect of fertilizers on the functional traits of amaranthus viridis – ker ning chew et al. 39 (a) npk 12:12:17 (b) npk 15:15:15 (c) wild plants figure 3 amaranthus viridis studied under (a) npk 12:12:17, (b) npk 15:15:15 and (c) wild plants with inflorescence as the dependent variable and leaves and height as the independent variable the pearson correlation coefficient of 0.960 indicated a strong linear relationship between inflorescence and both height and the number of leaves (figure 3b), with a significant relationship (b = 0.096 (leaves), 0.437 (height), seb = 0.010(leaves), 0.540 (height), β = 0.910 (leaves), 0.074 (height), t = 9.935 (leaves), 0.809 (height), p < 0.001). for each inflorescence, the number of leaves was increased by 0.096 and the height by 0.437 cm. both leaves and height collectively explained 92.2% of the variation in the number of inflorescences. additionally, a robust correlation of 0.979 was observed between inflorescences and both leaves and height (figure 3c). our findings affirmed that the number of leaves and plant height significantly predicted the number of inflorescences [b = 0.078 (leaves), 1.359 (height), seb = 0.085 (leaves), 1.650 (leaves), β = 0.449 (leaves), 0.557 (height), t = 0.820 (leaves), 1.609 (height), p = 0.008], with 95.9% of the variance in the number of inflorescences was accounted for by leaves and height. plant height is an indicator of organ growth, growth rate, and leaf number (dambreville et al. 2015; kunstler et al. 2016) and indicates reproductive maturity in herbaceous plants (garnier & navas 2012). members of amaranthus can grow up to a maximum height of 2.2 m (martínez-núñez et al. 2019). concordant with our findings, those of previous studies have demonstrated that a. retroflexus shows a linear increase in height throughout its life cycle (li et al. 2015; little et al. 2021). leaves play a crucial role in resource acquisition, while inflorescences are pivotal for reproduction. a higher leaf number indicates better growth rates and exhibits a strong correlation with other plant traits (he et al. 2020). the number of inflorescences is a reliable indicator for plants using seed production strategies, which facilitates the prediction of seed numbers (chaney & baucom 2012). each -50.00000 .00000 50.00000 100.00000 150.00000 200.00000 250.00000 .00 50.00 100.00 150.00 200.00 250.00 300.00 leaves and height in fl o re s c e n c e s r = 0.911 r² = 0.831 y = -84.891+0.212*x+2.763*x 20.00000 40.00000 60.00000 80.00000 100.00000 120.00000 .00 20.00 40.00 60.00 80.00 100.00 120.00 leaves and height in fl o r e s c e n c e s r = 0.960 r² = 0.922 y = -2.437+0.096*x+0.437*x .00000 10.00000 20.00000 30.00000 40.00000 .00 10.00 40.00 30.00 20.00 leaves and height in fl o re s c e n c e s r = 0.979 r² = 0.959 y = -26.766+0.060*x+1.359*x biotropia vol. 31 no. 1, 2024 40 inflorescence of a. viridis contained an average of 347 seeds, with a seed length of 1.25 ± 0.15 mm. growth and developmental parameters exhibit interconnectedness throughout the plant life cycle (dambreville et al. 2015; he et al. 2020). the scatterplots generated using models 1, 2, and 3 revealed strong positive relationships between the dependent and independent variables. the independent variables in these three models significantly predicted the dependent variables, with the latter demonstrating an increase corresponding to the rise in the independent variable(s). moreover, weeds in wild populations exhibited growth and development patterns mirroring those of weeds studied under npk 15:15:15 and npk 12:12:17. this finding contradicts the hypothesis of lavorel and garnier (2012), which posited distinct trait responses for wild plants and farm weeds. wild and arable weeds largely share ecological strategies, including reproduction (bourgeois et al. 2019). previous studies have shown that traitbased approaches can overcome the challenges posed by the heterogeneity within and between weed species (gaba et al. 2017; lavorel & garnier 2002). in this study, the analysis of three models aimed to explore the functional relationships between traits revealed that the findings could guide the selection of the model for a more comprehensive understanding and accurate prediction of other weed species while considering data availability for certain traits. fertilizers exert the primary selection pressure on arable weeds. the positive correlations observed in this study indicate the cooptimization of a. viridis. under favorable conditions, such as nutrient abundance, weeds strategically enhance both vegetative and reproductive aspects, consequently increasing plant fitness (li et al. 2015; little et al. 2021). an increase in vegetative components, such as height and leaves, promotes the competitive ability of weed species (hegazy et al. 2005). this study discerned that continuous resource availability promotes co-optimization between traits. weeds continue to grow and produce inflorescences until senescence (hegazy et al. 2005). the response-effect framework can clarify patterns that reflect plant functional responses and aid in grouping species at the community level, such as facilitating the upscaling of individual plant responses to fertilizers (lavorel & garnier 2012). the hub trait (i.e., inflorescence) interacts with other traits and has a higher degree of correlation, whereas the mediator trait indicates the betweenness of the two traits which is a single spectrum of traits (he, et al., 2020), suggesting model 3 was a hub trait, and models 1 and 2 were mediator traits. in this study, an assessment of these traits on farms provided insights into aboveground growth and development patterns. analyzing the growth development patterns in individual plant behavior at the trait level represents the initial phase for species comparison and broader generalization, marking the commencement of a systemic approach to weed control in practice (little et al. 2021). weeds undergo evolutionary adaptations within the temporal and spatial dimensions of the farm environment. to gain a comprehensive understanding, research efforts could be expanded to include other weed species within the same farm, investigating whether their fundamental biology aligns with that of a. viridis or similar species from different locations. effective weed management practices can be implemented by acquiring detailed information on the growth and development parameters of weed species on farms. moreover, the review of post-emergence herbicide application, encompassing considerations such as dosage, timing, and frequency at specific heights to minimize seed numbers, could be an integral component of vegetation management on farms. biological-intensification weed management involving holistic and sustainable approaches is complex and requires careful consideration and handling of several factors (gaba et al. 2017; little et al. 2021). research efforts using this framework should actively engage smallholders in validation processes and joint efforts with smallholders to review their herbicide applications are essential. to conclusively demonstrate growth and development patterns, a follow-up program combining trait and response-and-effect approaches is necessary (gaba et al. 2017; garnier & navas 2012). experimental studies have suggested a timeframe of four to six years in the fields, taking into account the pressures the smallholder farmers face from weeds, which significantly influence their decisions and practices (gaba et al. 2016). therefore, facilitating the understanding of the smallholders regarding the basic concepts and appropriate technologies effect of fertilizers on the functional traits of amaranthus viridis – ker ning chew et al. 41 through continuous learning is crucial (terlau et al. 2019). furthermore, research on farmer participation is pragmatic for the development of sustainable weed management (hall et al. 2000). although chemical herbicides remain a central control method, refining their use by adjusting the dosage and application timing of fertilizers can contribute to reducing overall herbicide usage (bastiaans et al. 2000). limitations this study has some limitations in its examination of the germination rate, vegetative stage from germination to the emergence of the first inflorescence, and overall life span from germination to senescence under different environmental conditions (i.e., wild populations, npk 15:15:15, and npk 12:12:17). to ensure practical feasibility, the growth and development of a. viridis treated with fertilizers under farm conditions were not compared. furthermore, this study focused on a single species with a singledensity approach instead of investigating intra and inter-specific weed competition. another challenge in studying weeds is their plasticity within specific habitats and at different densities. conclusion this study demonstrated the patterns of functional attributes of a. viridis in response to fertilizers and wild populations. traits such as the number of leaves, number of inflorescences, and plant height explained individual plant responses. models 1, 2, and 3, focusing on the interactions among these traits, revealed significant r and r2. specifically, model 3 of the inflorescence to leaf numbers and height showed higher r and r2 values. this study predicted that a. viridis would respond to selection pressures such as fertilizers. these three models serve as valuable tools to predict the dependent variables based on the types of available data. the applicability of these models could be extended to broadleaved species with seeds as the primary reproduction mechanism, offering insights into how the traits of a weed species in the wild 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icushairi et al. 2010). hence, improvement on the efficiency of this technology on oil palm is important for its mass production. plant growth and development in tissue culture are regulated by various environmental factors, wherein light is one of the most important. undoubtedly, light is necessary for photosynthesis and photomorphogenesis, with certain specific wavelengths playing important roles in plant tissue culture efficiency (fujiwara & i<ozai 1995). the lighting system generally used for maintaining plant tissue culture is the tubular fluorescent lamps (tfl). however, tfl has a wide spectrum and contain unnecessary wavelengths for plant growth. the light-emitting diode (led), an alternative light source for plant tissue culture, has several advantages in comparison to other lighting systems. led has wavelength specificity, durabhty, longer operating lifetime, has the ability to select spectral composition, has a smaller size, and emits less heat (gupta & jatothu 2013). the disadvantage of led is the establishment cost. however, led can still be profitable in the long run, even though it may require a high capital investment, because the long-term operating costs of led are generally lower than other lighting systems. the led lighting system using various light qualities in plant tissue culture has been applied to a variety of species, such as cymbidizm (tanaka et al. 1998), strawberry (nhut et al 2003), grape (poudel et al. 2008), antbam'zm (bucharto 2010), gosgpizlm birsatam (li et a/. 2010), onn'diam (mengxl et al. 201 i), brassica napas (li et al 2013), banana (vieira et al. 2015), 'corresponding author, email: biotechnology@sinarmasvanilla planfoolid (belle-bell0 et al. 201 6) , and agri.com sugarcane (ferreira et al 2017). the effect of biotropia vol. 28 no. 1,2021 light quality varied depending on the plant species yet little is known about led utilization with different light qualities in oil palm shoot. hence, the influence of various light qualities on in vitro growth and development of oil palm shoot was evaluated in this study. materials and methods plant materials and light quality treatments the in v h o oil palm shoots used in ths study were obtained through indirect somatic embryogenesis from young leaf explants following the wong et al (1997) method. the different lights used were white tfl (phillips t5 tch086 ev 28w, white 6500 i<), white led (vanq vq-glt8020w-27, white 10000 i(), red led (vanq vq-glt8020w-27, wavelength: 660 nm), blue led (vanq vq-glt8020w-27, wavelength: 460 nrn) and red blue led (vanq vq-glt8020w-27, wavelength: 660:460 nm (3:l)). effect of light quality on shoot development the 4-6 cm shoots, which have more than two leaves were selected and transferred onto a modified murashige & skoog (1962) medium without plant growth regulator. all cultures were maintained in a culture room at a temperature of 28 + 2 "c, relative humidity 50 f 10°/o, and with 16/8 light/dark photoperiod under various light qualities. after two months of culture, the fresh weight, the number of new leaves, the shoot height, and its root formation were observed, and then whole shoot samples from each treatment were randomly collected for chlorophyll, sugar, and endogenous hormone analyses. the whole shoots were ground in liquid nitrogen and stored at -80 "c prior to analyses. chlorophyll content analysis for chlorophyll a and at 645 nm for chlorophyll b. the chlorophyll content was calculated according to lichtenthaler (1 987). sugar content analysis the sugar content was measured, according to the method described by dubois e t al. (1951). the 2 g sample was homogenized in 10 ml of distilled water and kept in water bath at 95 "c for 5 min. the sample was then stored overnight at 70 "c. subsequently, the sample was centrifuged at 5,000 rpm for 10 rnin and 2 ml of the supernatant was transferred into another tube then added with 0.02 ml of 80% phenol and 5 ml of sulfuric acid. the mixture was then incubated at 30 "c for 30 min. the optical density was measured using a spectrophotometer (biotek epoch microplate) at 490 nm. fructose and glucose were used as the standard. endogenous hormone content analysis the endogenous hormone content was measured, according to i<elen et al (2004), with some modifications using ultra performance liquid chromatography (yuater uplc). auxin, gibberellic acid (ga), and abscisic acid (aba) were extracted from 5 g of sample and soaked overnight in 30 ml 80% methanol at 4 "c. the 5 g extracted sample was dissolved with 30 ml 0.1 m phosphate buffer (ph 8.5) and then partitioned with ethyl acetate two times. after removal of the ethyl acetate, the ph of the aqueous phase was adjusted to 2.5 with 1 n hc1. the sample was partitioned again with ethyl acetate 2 times and passed through anhydrous sodium sulphate. the ethyl acetate was evaporated in rotary evaporator at 50 "c. the dry residue containing hormones was dissolved in 2 ml of methanol and filtered into a uplc vial using a 0.2 pm millipore syringe filter. an injection volume of 1 pl was used for each analysis. the column used was water acquity uplc beh c18 1.7 pm (2.1 x 50 mm). the mobile phases constituted of acetonitrile: water (26:74) ph 4 with a flow rate of 0.2 ml/min and retention time of 3 minutes. the the chlorophyll content was extracted from signal of the compounds was monitored by a the sample using 10 ml of 100% acetone. the photodiode array (pda) detector at 208 nm. optical density was measured using a indole acetic acid (iaa), ga3, and aba (100 spectrophotometer (hitachi u-2900) at 662 nm ppm) were used as the standard compounds. growth and development of oil palm shoots under different light qualities patricia manoh e t al. for cytokinin isolation, 1 g of sample was extracted using 20 rnl 70% methanol for 4 hours at 4 "c. the extracted sample was filtered and injected into a uplc column as previously mentioned. the mobile phases constituted of water: methanol (2030) ph 2 with a flow rate of 0.35 ml/min and retention time of 0.6 minutes. the signal of the compounds was monitored by a pda detector at 210 nm. zeatin (100 ppm) was used as the standard compound. statistical analysis this experiment used randomized complete block design with three block and six treatments. data were analyzed statistically using analysis of variance (anova), and the differences among the treatment means were tested using duncan's multiple range test (dmrt) at p = 0.05. statistical analyses were conducted using sas version 9.1.3. results and discussion shoot growth and development the growth and development of oil palm shoots were influenced by the light quality (table 1). compared with other treatments, the no light (darkness) treatment gave the lowest growth. when under darkness, the number of new leaves and fresh weight were significantly lower than those under light treatments, while the results among different light treatments did not significantly differ from each other. however, the highest shoot height and fresh weight were observed in shoots growing under the combination of red and blue light-emitted diode (led). meanwhile, the highest number of new leaves was observed in shoots under white tubular fluorescent lamp (tfl) (table 1). white tfl is the most widely used light in plant tissue culture. however, whte tfl produces a wide range of wavelengths that contain unnecessary wavelengths for plant growth and development, whde led can emit light at specific wavelengths (gupta & jatothu 201 3). different wavelengths generally bring about different responses on plant growth and development. in this study, the red blue led gave the greatest growth compared to other led colors (table 1 & fig. 1). red and blue lights remarkably impacted plant growth, probably because these are the major energy sources for photosynthesis and photomor phogenesis. red and blue lights, either alone or in combination produce different responses in plant growth. red light induces shoot elongation (nhut e t al. 2003; cybularz-urban et al. 2007; poudel et al. 2008), while blue light induces chlorophyll and stomata formation (poudel et al. 2008; muneer et al. 2014). the combination of these two lights may have produced higher growth rate, due to the combined advantages of red and blue lights for inducing plant growth and development. the combination of red and blue lights also enhance the growth of other plant species, such as strawberry (nhut et al. 2003), cotton (l et al. 2010), ginseng (nhut etal. 2015), and vanilla (bello-bello et al. 201 6). table 1 effect of different light qualities on growth and development of oil palm shoots treatment number of new leaves fresh weight increase (mg) shoot height increase (cm) dark 0.51 f 0.03 white tfl 0.78 + 0.03 a white led 0.69 + 0.03 a red blue led 0.76 + 0.03 a blue led 0.76 f 0.04 a red led 0.70 + 0.03 a notes: values are mean + se for n = 330. different superscripts on the same column indicate significant differences at p = 0.05 by dmrt. biotropia vol. 28 no. 1,2021 figure 1 representative of oil palm shoots under different light qualities notes: d = dark w ?m = %bite tm;, w led = white led; rb led = combination red blue led; b led = blue led; r led = red led;, light quality and endogenous hormones light quality affects plant gzowth and development by regulating the synthesis of endogenous hormones: c y t o k h ~ ~ gibberellic acid (ga), auxin and abscisic acid (am). the endogenous hormone in the oil palm shoot showed varied responses to different light qualities (table 2). cytokinitl, ga and aba contents differed significantly from a c h other when exposed under the different hght quality treatments, indicating their different photo responses to certain wavelengths. the highest cytokinin content was observed under the white led and lowest in the darkness. gibberellic acid (ga] content was higher when the shoots were cultured under red, blue, or red and blue led when compared with white light. aba content was the highest under red blue led, and the lowest in dark vestment. auxin did not respond to any of the light quality treatments. althaugh the auxin content did not differ signifimdy among treatments, the highest was observed in red led treatment. interaction berween endogenous hormones and shoot growth synthesis of hormone in plant is regulated by plant photoreceptors, which include phpchromes, ~ to&, ;ome , and phototropin (de wit ~t .ti 20 1 6). the interaction between hght quality and endogenous hormone plays an important role in plant growth and development. ga and auxin are key players in plant elongation. the stem increment and ga content of norway spruce seedlings under red light were higher than under blue light, whereas u a content under red light were lower (ouyang t.t al', 2015). in contrast, ga eontent in petunia plants were higher under bhe light compared to red light (fukuda e t 8 2016). blue light enhances shoot elongation and red light inhibites shoot elongation in petunia plants. in this current study8 ga and auxin contents in oil palm shoots under led light were not significantly different, but ga content in shoots under blue led was higher than those under other lights. the. resulting differences may be attributed to plant species1 stage of plant growth, and other environmental factors. table 2 endogenous hormones cootent in oil palm shoots under different light qudties treatment cpkinin (mg/@ ga @/g) auxin @g/@ kg/$$ dark 0.48 f 0.04 3.39 f 0'33 3.51 f 0.68 8.13 f 5.63e white tfl 0.61 f 0.04 2.50 + 0.32 b 3.83 f 0.55 35.81 f 10,41 " wlhite led 0.70 f 0.03 2.24 -t 0,35 b 4.59 +. q.65 a 29.15 f 1034 * red blue led 0.64 f 0.02 ab 2.89 f 0.09 ah 4.37 1: 0.60 a 42.08 f 12.1 1 blue led 0.64 k 0.06 ab 3.06 f 9.26 3.99 f 0.78 a 19.77 f 9.70 bc red led 0.67 f 0.03 " 2.67 f 0.29 *b 4.79 + 093 a 30.40 f 8.51 * nates: values are mean f se f ~ r pr = 6. different letters in the same column indicate significant difference at p = 0.05 by d m growth and development of oil palm shoots under different light qualities patricia manoh e t al: shoot elongation seems to positively correlate with the interaction of endogenous hormones. in this study, shoot elongation under red led was higher than those under other lights (table 1) and this might be correlated with the higher auxin content under red led (table 2). ga and auxin are known to stimulate plant elongation through different mechanisms. auxin affects cell elongation by promoting the release of hydrogen ions from the plant cell and whch in turn reduces the stress on the cell wall. ga requires cell wall plasticity and cytoplasmic protein synthesis for cell elongation. to achieve this, cooperation with other hormones, such as auxin is required p a n g & irving 201 1). cytokinin has a different effect as it inhibits plant elongation by inhibiting auxin-promoted extension growth (cohen et al. 1991). cytohnin and blue light induced inhibitory effects on hypocotyl growth of cucumber (cohen e t al. 1991). in this study, cytokinin content and shoot elongation under light treatments were not significantly different. t h s results showed that cytokinins might not be correlated with the elongation of oil palm shoots. auxin also plays a key role in root initiation. in this study, auxin content in the oil palm shoots increased under the red led (table 2) and it induced the root initiation in the shoots (fig. 2). the process of root formation is considerably complex and involves interaction with other hormones. auxin regulates root growth by modulating the effect of ga-induced destabilization of della protein growth repressor (fu & harberd 2003). in the presence of ga, della is degraded and thereby modulates other plant hormones, such as auxin. light quality and photosynthesis light quality also influences photosynthesis activity. in this study, the effect of light quality on photosynthesis was determined by the accumulation of photosynthetic pigments (chlorophyll a and b) and primary photo synthesis products (fructose and glucose). chlorophyll and sugar content of the oil palm shoots were significantly lugher under light treatment than those under dark treatment (figs. 3 & 4). light is an important factor that regulates chlorophyll biosynthesis. the shoots under white, red blue, and blue light accumulated higher chlorophyll, compared to those under the red or dark treatment (fig. 3). several studies showed that blue light is a good light for chlorophyll synthesis and that red light decreases chlorophyll content (tanaka et al. 1998; li e t al. 2010). simdarly, in this study, the chlorophyll content in oil palm shoots decreased under red led, and increased under white and blue led (fig. 3). dark white white red blue blue red led tfl led led led figure 2 percentage of rooting in shoots under different light qualities notes: vertical bars indicate k se of the means for n = 6. different letters indicate significant differences at p = 0.05 by dmrt. biotropia vol. 28 no. 1,2021 blue light is also important in chlorophyll synthesis. blue light stimulates chlorophyll synthesis by inducing the expression of genes encoding the enzyme for two early step of chlorophyll biosynthesis, glutamate-1 semialdehyde aminotransferase and 5 aminolevulinic acid dehydratase (matters & beale 1995). chlorophyll content were also correlated to cytolilnin content in oil palm shoots. chlorophyll content under light treatments was significantly higher than that in dark treatment (fig. 3) and ths may be stimulated by high cytokinin content under light treatments (table 2). cytokinin affected chlorophyll biosynthesis by promoting the synthesis of 5-aminolevulinic acid, a precursor of chlorophyll synthesis, and enhancing activity of protochlorophyllide oxidoreductase, an enzyme in chlorophyll synthesis (cortleven & schmulling 201 5). sugar content in the oil palm shoots under different light treatments did not significantly &ffer (fig. 4). apparently, different light qualities did not affect sugar content because the in vitro shoots are mainly heterotrophic, where the shoots were grown in culture media with sugar. sugar is the main carbon source for heterotrophic growth (yasseen et al 2013). the presence of sugar in culture media might reduce the need for sugar production and limits photosynthetic activity. mg/g chlorophyll a r chlorophyll b dark white white red blue blue red led tfl led led led figure 3 chlorophyll content in oil palm shoots under different light qualities notes: vertical bars indicate k se of the means for n = 6. different letters indicate significant differences at p = 0.05 by dmrt. mg/g fructose f glucose dark white white red blue blue red led tfl led led led figure 4 sugar content in oil palm shoots under different light qualities notes: vertical bars indicate ? se of the means for n = 6. different letters indicate significant differences at p = 0.05 by dmrt. growth and development of oil palm shoots under different light qualities patricia manoh et al. conclusions light quality influenced the growth and development of the oil palm shoots. the different wavelengths produced different effects on the root and shoot activities. root initiation and shoot elongation were well-produced under red light, while chlorophyll and sugar contents were better produced under the white and blue light. although the results were not significantly different, the combination of red and blue led resulted in higher growth than the other light treatments. besides the white light, the red and blue light combination provides 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developing a model of plant vt, tung ht, vinh bt, luan tc. 2015. lighthormone interactions. plant signal behav 6(4):494 emitting diodes and their potential in callus 500. growth, plantlet development and saponin wong g, tan cc, soh ac. 1997. large scale propagation accumulation during somatic embryogenesis of of oil palm clones experiences todate. acta hort panax vietnamensir ha et grushv. biotechnol 447: 649-58. biotechnol equip 29(2):299-308. yaseen m. ahmad t. sablok g. standardi a. hafiz ia. ouyang f, mao jf, wang j, zhang s, li y. 2015. 2013. review: role of carbon sources for in transcriptome analysis reveals that red and blue vitro plant growth and development. mol biol rep light regulate growth and phytohormone 40:2837-49. cover single.cdr biotropia vol. 27 no. 2, 2020: 171 178 doi: 10.11598/btb.2020.27.2.1203 soil properties below exotic tree plantations at the saithong silvicultural research station in prachuap khiri khan province, thailand** kanokwan urairak1*, roongreang poolsirp and san kaitpraneet2 1faculty of forestry, kasetsart university, bangkok 10900, thailand department of silviculture, faculty of forestry, kasetsart university, bangkok 10900, thailand received 14january 2019 / accepted 29 march 2019 abstract often, exotic trees grow very fast in their new environments and sometimes even faster than the indigenous ones. these exotics seem to be tougher and live through harsher conditions. they may be drought resistant, and they can survive long periods with little to no rainfall. these trees are fast growing and are resistant to pests and diseases which tend to damage the locally growing trees. moreover, these trees can help improve soil physical and chemical properties. hence, the objective of this study was to compare the physical and chemical properties of soil under different tree plantations, namely fucalyptus urophylla, acacia crassicapa and acacia aulacocarpa at the saithong silvicultural research station. three soil composite samples were collected from soil depths of 0 10, 10 30, and 30 50 cm, in the three stands aged 27-year old and planted at a spacing of 2 x 2 m. soil type in these stands was sandy loam. soil moisture percentage (12.85%) was significantly the highest in the e. urophylla plantation. phosphorus (18.2 mg/kg dw), magnesium (6.98 cmolc/kg dw), and potassium (6.98 cmok/kg dw) contents were also the greatest in the e. urophylla plantation. organic matter content was the highest in the a. crassicapa plantation (7.59%), while nitrogen (0.04%) and calcium (36.17cmolc/kg dw) contents were the highest in the a. aulacocarpa plantation. significant differences were also observed in the soil bulk density and porosity attributable to structure, tillage, cropping practices, soil depth and compaction. characterization of soil pore system is equally important in understanding soil physical and parent material composition, which has a direct impact on soil chemistry and fertility. parent material rich in soluble ions (calcium, magnesium, potassium and sodium) is desirable, as these chemicals are easily dissolved in water and readily available for plants. for soils with poor nutrient content found in warm and humid tropical climates with low water supply, e. urophylla, a. crassicapa and a. aulacocapa have the potential to enhance the aboveground stand production. undoubtedly, when planted, they can help restore the soil nutrients faster and therefore, can be used in forest plantations or in agroforestry farms. keywords: exotic tree plantation, saithong silvicultural research station, soil properties introduction soil properties are important tree growth factors and as such, maintaining soil quality is paramount for tree health. soil degradation, on the other hand, involves the destruction of soil structure through loss of organic matter, due to factors related to topography, climate, and poor soil management (ozdemir 1993; haynes 2000) ^corresponding author, e-mail: kanokwan.urai@ku.th; k_urairak@hotmail.com **this paper was presented at the 3rd international conference on tropical biology 2018, 20-21 september 2018, bogor, west java, indonesia often, exotic tree species possess more useful attributes than the native species, such as, a faster growth rate, fewer pests and reduced competition, all leading to a higher economic value (kalinganire 1996; tavares et al. 1999; lott et al. 2000; takaoka 2008; anglaaere et al. 2011; tefera et al. 2014). acacia aulacocarpa and acacia crassicarpa not only have shown potential for reclaiming deforested areas, but also for shelterbelts, coastal sand dunes fixation, soil improvement and land rehabilitation on a wide range of degraded sites, as a result of their rapid growth, ability to suppress weeds and abundant skhiyobium nodulation (doran & gunn 1987). 171 biotropia vol. 27 no. 2, 2020 eucalyptus is also one of the most commonly used species for reforestation in commercial plantations, with the added benefit of being an exotic species in study site which can promote the establishment of a stable forest cover in degraded sites (farias et al. 2016). eucalyptus spp. and acacia spp. can grow rapidly in poor soils (yang et al. 2009; chen et al. 2011). planting exotic trees in tropical countries is becoming an increasingly important forestry activity as many tropical countries that depended on wood supply from natural forests are now recognizing the need to establish plantations to augment supplies from the dwindling and unsustainable natural forests. the total area of tropical forest plantations increased from about 6.7 million ha in 1965 to 109 million in 2005. though most species used for tropical plantations are already fast growing, their growth rate can still be improved substantially through appropriate silvicultural practices, such as site-species matching, site nutrient management, and the use of hybrid species. sandy soil types are well aerated, thereby, allowing suitable seeds to germinate easily and the roots to penetrate properly. however, such soil types have an inherent disadvantage of being nutrient hungry as nutrients are easily leached away due to excellent soil drainage. plants that are grown on a sandy loam soil need frequent irrigation and fertilization to maintain a healthy growth. the best way to improve a sandy loam soil is to mix organic matter into the soil. applying a 2to 4-inch thick layer of compost or peat moss over the area can significandy improve the ability of a sandy loam soil to hold nutrients. many eucalyptus plantations are grown on usually nutrient deficit sandy soil types. hence, attempts were made to plant eucalyptus in semi-arid regions on sandy soil types. in this study, leguminous trees can be planted to improve the soil nitrogen content, this can change the litter quality and soil biology. for a sustained fast-growing exotic tree plantation, it is important to manage sandy soil types in order to increase the soil organic matter content. silvicultural practices, which help increase the organic matter content in eucalyptus spp. and acacia spp. plantations on sandy soil types, are also essential for their sustainability. moreover, one of the major interests in acacia is the compounds present in the plant parts that can be used as natural herbicides. evidence of the role of allelopathy in weed control has been cited in many publications (chou 1995; rice 1995; rizvi & rizvi 1992; waller 1987). the introduction of exotic plants may alter the nutrient cycle of the system either direcdy, by modifying the quality and quantity of litter entering the soils beneath, or indirectly, by altering the physico-chemical site properties below their canopy. moreover, the planting of native species on degraded land are highly inappropriate and of high risk due to drought, insect pests and low soil fertility. as such, exotic species are planted to improve the poor soil condition in such an area. hence, the objective of this study is to compare the soil physical and chemical properties of three plantations of eucalyptus urophylla, acacia crassicarpa and acacia aulacocarpa on a site with sandy soil types and rain all year round to be an idea for soil develops in the future., to help increase the productivity of exotic tree plantations planted on sandy soil types, as was present in the study area. materials and methods the study was conducted at the saithong silvicultural research station, prachuap khiri khan district, thailand located at 11°17’04” n latitude and 98°50’25” e longitude (fig. 1). the area receives a mean annual rainfall of 1,175.9 mm, with temperatures ranging from a minimum of 16.3 °c in february to a maximum of 42.9 °c in july, with an annual mean of 28.4 °c. its soil is primarily of the dark brown to brown (7.5yr4/4) loam to sandy loam (sadao series) which has moderately fine to medium texture and very strongly acid (field ph 4.5). using a completely randomized design, three soil samples were collected from soil depths of 0 10, 10 30, 30 50 cm at the e. urophylla, a. crassicarpa and a. aulacocarpa stands, aged 27year old and planted at a spacing of 2 x 2 m. the soil physical and chemical properties were determined as follows; the bulk density using the core method (jalota et al. 1998), particle density by the pycnometer method (jalota et al. 1998) using an air comparison pycnometer, soil texture by the hydrometer method, ph in 1:1 soil: water suspension by a ph meter, organic matter using the walkley and black’s rapid titration method 172 soil properties below exotic tree plantations at saithong silvicultural research station — urairak et al. kfiiaii:io]:mguivi toul glnrny?fli -t*t ?̂nycnn bi ix 6 col:i m mmm mam [s**, mt fmm wm&sb fcajbmmbak 0 95 190 380 570 780 azt 1 sqmra kkonwtan 1:10,000 figure 1 saithong silviculture research station, prachuap khiri khan province, thailand (walkley & black 1934), total nitrogen using the dumas or dry combustion method (jackson 1965) using a chns analyzer perkinelmer series 2400 series ii chns/o elemental analyzer, the available phosphorus (p) by bray ii method (bray & kurtz 1945) using a spectrometer, and the exchangeable potassium (k), calcium (ca) and magnesium (mg) in ammonium acetate (neuoac) 1 n ph 7.0 by using an atomic absorption spectrometer. variance analyses of the experimental data were done using the spss statistic program at a significance level of p < 0.05. to monitor soil changes induced by tree growth, the soil properties, organic matter accumulation and decomposition, as well as nutrient cycling are measured on several sample plots over the full rotation of a plantation (lundgren 1978). in this study, soil sampling was carried out in the e. urophylla, a. crassicarpa and a. aulacocarpa plantations aged 27-year old located near each other on the same soil type. results and discussion soil physical properties the three stands were of a sandy loam type and experienced rain all year round. soil moisture percentage was the highest in e. urophylla plantation (12.85%) (table 1). the potassium (18.2 mg/kg), mg (6.98 cmok/kg dw) and p (6.98 cmolc/kg dw) contents were the highest in the e. urophylla plantation. (table 2). the organic matter content was the highest in a. crassicarpa plantation (7.59%). calcium (36.17cmolc/kg dw) content was the greatest in a. aulacocarpa plantation (table 2). these results revealed that leguminous species are instrumental in increasing soil organic matter content, invariably with the presence of the nitrogen-fixing bacteria (turnbull 1984; bernhard-reversat 1993; bernhard-reversat et al. 1993; higa & higa 2000). however, the leguminous species can also cause soil acidification (binkley 1992; yamashita et al. 2008; 173 biotropia vol. 27 no. 2, 2020 table 1 soil physical properties in the 27-year old e. urophylla, a. crassicarpa and a. aulacocatpa plantations at the saithong silvicultural research station, prachuap khiri khan province, thailand soil parameters depth (cm) e. urophylla a.crassicarpa a. aulacocarpa f-value particle density (g/cm3) 0-10 2.55 2.53 2.58 0.49ns 10-30 2.58 2.53 2.71 2.47ns 30-50 2.66 2.61 2.59 0.48ns mean 2.60 2.56 2.63 bulk density (g/cm3) 0-10 0.89b 0.91ab 0.92a 7.36* 10-30 0.89b 0.91a 0.90ab 5.75* 30-50 0.88b 0.91a 0.90a 16.88* mean 0.89 0.91 0.91 porosity (%) 0-10 33.84 34.82 34.70 0.70 ns 10-30 33.52 34.93 32.49 2.30 “ 30-50 32.05 33.80 33.90 1.84 ns mean 33.14 34.52 33.70 soil moisture (%) 0-10 12.76a 10.28ab 8.63b 7.36* 10-30 12.16a 9.96b 10.52b 5.81* 30-50 13.63a 10.25b 10.83b 17.26* mean 12.85 10.16 9.99 sand (%) 0-10 74.89 76.35 74.65 0.10ns 10-30 73.68 76.35 75.68 0.35ns 30-50 74.23 74.77 74.35 0.01ns mean 74.27 75.82 74.89 silt (%) 0-10 13.45 10.67 11.33 0.28ns 10-30 12.00 9.33 10.00 0.27ns 30-50 6.79 6.67 8.67 2.50ns mean 10.74 8.89 10.00 clay (%) 0-10 11.65 12.99 14.03 3.05ns 10-30 14.32 14.32 14.32 0.00ns 30-50 18.99 18.56 16.99 0.19ns mean 14.99 15.29 15.11 notes: * = significant difference at p < 0.05; superscripts a and b in the same row indicate significant differences at p < 0.05 using duncan’s multiple range test; ns = not significant. kasongo et al. 2009; koutika et al. 2014). differences in soil bulk density and porosity was caused by structure, tillage practices before and after harvest, cropping practices, soil depth and soil compaction. heavy machinery can cause soil compaction, as well as feet stomps caused by animal passage. characterization of soil system is important for understanding the composition of soil physical and parent materials or drift deposits, as these have a direct impact on soil chemistry and fertility. parent materials rich in water soluble ions, such as ca, mg, k, and sodium (na), are easily dissolved in water and readily available for absorption by plants. the mean bulk density values were generally higher for a. crassicarpa (0.91 g/cm3) and a. aulacocarpa plantations (0.91 g/cm3) (table 1). in the upper 0-10 cm of the soil profile, the values indicated a greater soil compaction for the a. aulacocarpa plantation (2.63 g/cm3) compared to the deeper soil layers. soil in the plantations was mainly of a sandy loam type. at the study sites, the data showed no evidence of textural change as a result of reforestation with e. urophylla, a. crassicarpa and a. aulacocarpa plantations. at the time scale involved, a comparatively stable property like soil texture is unlikely to undergo drastic changes as a result of planting the e. urophylla, a. crassicarpa and a. aulacocarpa trees. the porosity mean was higher in the a. crassicarpa plantation (34.52%) and the mean of soil moisture was higher in the e. urophylla plantation (12.85%) (tablet). moisture retention is an important factor governing soil fertility. however, sand is porous and has a low water retention capacity therefore, soil types in the study sites are of poor quality as they are of the sandy texture. 174 soil properties below exotic tree plantations at saithong silvicultural research station — urairak et at soil chemical properties table 2 soil chemical properties in the exotic tree plantations of e. urophylla, a. crassicarpa, and a. aulacocarpa, at the saithong silvicultural research station, prachuap khiri khan province soil parameters depth (cm) e. urophylla a. crassicarpa a. aulacocarpa f-value ph 0-10 4.20a 3.87b 3.89b 10.52* 10-30 4.64a 4.03b 4.10b 5.89* 30-50 4.71 4.23 4.39 0.08 ns mean 4.52 4.04 4.13 organic matter (%) 0-10 0.73 1.18 2.46 1.36 ns 10-30 2.01 2.52 5.83 1.60 ns 30-50 6.69 3.89 6.24 0.76 ns mean 3.14 7.59 4.84 n (%) 0-10 0.03b 0.04b 0.06a 9.80* 10-30 0.02 0.03 0.03 1.00 ns 30-50 0.02 0.03 0.02 1.50 ns mean 0.02 0.03 0.04 p (mg/kg) 0-10 2.55 1.59 1.73 2.28 ns 10-30 1.68 1.28 1.25 0.19 ns 30-50 1.24 1.04 0.59 0.95 ns mean 1.82 1.30 1.19 k (cmolc/kg dw) 0-10 9.05 8.28 9.03 0.25 ns 10-30 5.67 4.89 5.69 1.26 ns 30-50 6.21 6.16 5.94 0.27 ns mean 6.98 6.44 6.72 ca (cmolc/kg dw) 0-10 31.57b 36.45b 54.90a 8.04* 10-30 25.82 24.02 27.96 0.31 ns 30-50 29.99 25.15 25.64 0.32 ns mean 29.13 28.54 36.17 mg (cmolc/kg dw) 0-10 9.05 8.28 9.03 0.25 ns 10-30 5.67 4.89 5.69 1.26 ns 30-50 6.21 6.16 5.94 0.27 ns mean 6.98 6.44 6.72 notes: superscripts a and b, across row, indicate significant differences at p < 0.05 using duncan’s multiple range test; ns = not significant. soil in all three plantations was generally acid. the ph values were higher in e. urophylla plantation (ph 4.52) (table 2), although the difference was not significant at a depth of 3050 cm at any sites. organic matter content was the highest in the a. crassicarpa plantation (7.59%) and its level was lower at a depth of 0-10 cm. total nitrogen content was the highest in the a. aulacocarpa plantation (0.04%) and was maximum at a depth of 0 10 cm. in a similar study, soil n improved significantly under the canopy of acacia sieheriana trees in songa pastures, rwanda (mugunga & mugumo 2013). acacia has high rates of nitrification and high nitrogen availability for plants (marchante et al. 2008). soil nitrogen and nitrogen produced from the fallen leaves are tightly associated with ecosystem processes that support aboveground biodiversity, the living biomass above the soil including the stem, stump, branches, bark, seeds and foliage (clark & tilman 2008; hautier et al. 2009; dickson & foster 2011). p level was the highest in the e. urophylla plantation (6.98 mg/kg) and it was maximum at a depth of 0-10 cm (table 2). nutrient quantities, organic matter and exchangeable cations (ca, mg, and k) expressed in cmok/kg dw, were obtained from soil profiles of the e. urophylla, a. crassicarpa, and a. aulacocarpa plantations (table 2). the nitrogen (0.04%) and ca (17cmolc/kg dw) contents were the highest in the a. aulacocarpa plantation (table 2). as indicated by the exchangeable cation analyses, k content was the greatest in the e. urophylla plantation (6.98cmok/kg dw) and its level was maximum at a depth of 0-10 cm. mg content was the highest in the e. urophylla plantation (6.98 cmok/kg dw) and was 175 biotropia vol. 27 no. 2, 2020 maximum at a depth of 0-10 cm, in all the plots. ca content (36.17 cmolc/kg dw) was highest in the a. aulacocarpa plantation and was maximum at a depth of 0-10 cm. soil chemical properties were higher at the surface layer of 0 10 cm depth compared to those in the subsoils layers at 10 30 and 30 50 cm depths. this can be attributed to a higher concentration of organic matter in the upper soil layer, a condition that commonly occurs in most tropical soils (obatolu & ibiremo 1999). results of soil analyses on physical properties show that the soil texture remained unchanged under the three vegetation types. most soils in thailand, being sandy, tend to have poor mineral absorption, low acidity and low absorption. the presence of exotic tree plantations was expected to improve the sandy soil quality. the introduction of exotic plants could have altered the nutrient cycle of the system either directly, by modifying the quality and quantity of litter entering the soils beneath, or indirectly, by altering the physical-chemical site properties below their canopy. in sandy soils, the ability of a plant's root or trunks in making food is lowered, making their growth slower and inefficient. generally, the encroachment of exotic plant species poses a major threat to the biodiversity and ecosystem stability, however, little attention is given to the potential impacts of these invasions on soil nutrient cycling. the differences between exotic and native species do not show trends in other components of the nutrient cycles, for example, the size of soil pools of carbon and nitrogen. in some cases, a given species can have different effects on different sites, suggesting that the new composition can positively affect the invaded community and its environment such as the soil type, thereby influencing the direction and magnitude of the impact at an ecosystem-level. exotic plants can alter the soil nutrient dynamics particularly in terms of biomass and productivity, tissue chemistry, plant morphology and phenology. as such, research may focus on these issues and integrate these in evaluating the impacts of invasive species. individual species have affected the various components of the carbon and nutrient cycles, including the pools of aboveground and belowground carbon, nitrogen, and other elements; the net primary productivity and plant growth rates; the chemical quality and rates of litter fall; and the nutrient and carbon mineralization rates. these evidences strongly suggest that when a community undergoes species composition changes due to the invasion and spread of exotic species, subsequent changes in the nutrient cycling processes are expected. conclusion the adoption of exotic trees in agroforestry systems or forest tree plantations can significantly impact the soil nutrient content as a result of their rapid leaf turnover and nutrient release through decomposition leading to significant increases in soil fertility. in this study, the e. urophylla, a. crassicarpa and a. aulacocarpa trees helped improve soil fertility which most likely could enhance the herbage productivity, subsequently improving the agroforestry systems. integrating these species in agroforestry systems and preserving these trees in arid and semi-arid areas could help maintain and enhance the sustainability of these ecosystems. eucalyptus spp. and acacia spp. not only grow on soils with poor quality, but can also grow rapidly and survive under long periods without rainfall. the acacia and eucalyptus spp. in the present study played a significant role in improving the soil structure and the availability of essential nutrients. acknowledgements this research was supported by saithong silvicultural research station for study site and the laboratory of forest soil, department of silviculture, faculty of forestry, kasetsart university, thailand. references anglaaere lcn, cobbina j, sinclair fl, mcdonald ma. 2011. the effect of land use systems on tree diversity: farmer preference and species composition of cocoa-based agroecosystems in ghana. agrofor syst 81:249-65. 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and imperata cylindrical grassland soils in south sumatra, indonesia. for ecol manage 254:362-70. yang l, liu n, ren h, wang j. 2009. facilitation by two exotic acacia: acacia auriculiformis and acacia mangium as nurse plants in south china. for ecol manage 257:1786-93. 178 sri-06. 1102-layout.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 biotropia vol. 29 no. 1, 2022: 81 89 doi: 10.11598/btb.2022.29.1.1662 81 neuston diversity and density as bioindicator for water quality imam safir alwan nurza*, jesslyn gitta vania, muhammad khatami reynaldi, zaki gunawan rasyid and ratna komala department of biology, faculty of mathematics and natural sciences, universitas negeri jakarta, jakarta 13220, indonesia received 9 september 2021 / accepted 12 december 2021 abstract lakes and waterfalls are freshwater ecosystems having important roles in ecology, tourism and economic aspects. among living organisms existing in lakes and waterfalls is neuston. neuston lives on the surface and below the surface of the waters. neuston can be used as a bioindicator for water quality due to the neuston’s high level of sensitivity toward pollutants. the purpose of this study was to determine the density and diversity of neuston as a bioindicator for water quality in the lake and waterfall. the methods used were survey and observation. the study was carried out by using purposive sampling at two locations, namely the lake and waterfall of situ gunung, sukabumi, with a sampling area of 1 x 1 m2. the samples obtained were put into bottles containing 70% alcohol to be identified in the laboratory. environmental parameters measured were air and water temperature, water ph, water depth, turbidity, water flow velocity, dissolved oxygen, substrate and weather conditions. the results obtained indicated that the lake and waterfall of situ gunung, sukabumi had highest neuston densities were shown by gerris lacustris and dineutus assimilis. the lowest neuston densities were shown by metrobates hesperius, gerris comatus, aquarius remiges and trepobates pictus. this study showed that the environmental parameters of the lake and waterfall of situ gunung, sukabumi can still support the survival of the existing neustons. keyword: density, diversity, neuston introduction freshwater ecosystems are divided into lentic/flooded freshwater ecosystems and lotic/flowing freshwater ecosystems. the examples of lentic freshwater ecosystems are lakes and swamps, while that of lotic freshwater ecosystems are waterfalls and rivers (diantari et al. 2017; ramadhan et al. 2016). the existence of the lake is considered important because it has an ecological, social, and economic roles for the surrounding environment (postel & carpenter 1997; chen et al. 2020; heino et al. 2021; robert et al. 2020). the lake also acts as a habitat for several living organisms, such as neuston (asnil et al. 2013; ramadhan et al. 2016). according to mulyono (2018), neuston floats on water (epineuston) or below the water surface (hyponeuston). hyponeuston lives at a depth of about 0 10 cm (rumnasih 2016). neuston has a level of sensitivity to several contaminants so neuston can be used as a direct indicator for water contamination (oktarina 2015) and as a bioindicator for water quality. the upper part of the waters is the most susceptible part to environmental exposure, which in turn will impact the neuston. pollution often causes the formation of artificial stratification in lakes, where a body of water (hypolimnetic zone) is isolated. however, the hypolimnetic zone caused the contaminants trapped within the bottom of the waters and sediment masses (sutrisno & hamdani 2013; williams 2001; winton et al. 2019; perron et al. 2014; donyinah 2019). waterfalls are a form of lotic freshwater ecosystem and are generally used as natural tourist objects (siswantini & mulyana 2017). the waterfall and lake in the situ gunung area, sukabumi have an important role as one of *corresponding author, email: imamnurza@gmail.com biotropia vol. 29 no. 1, 2022 82 tourist attractions in kadudampit district, sukabumi, west java province, indonesia (soetopo 2011). situ gunung as a tourist object cannot be separated from tourists activities which will affect the surrounding environment, including the neuston community. therefore, the purpose of this study was to determine environmental factors influencing the density and diversity of neuston as water quality bioindicator in the lake and waterfall in situ gunung, sukabumi. materials and methods the study was conducted in june 2021 at situ gunung, gunung gede pangrango, kadudampit district, sukabumi. the methods used were survey and observation. the tools used were rope, wooden stick, measuring tape, thermometer, turbidimeter, do meter, loop, field guide as an identification guide, stopwatch, plastic bottle, sample bottle, sample plastic, camera, net 1 x 1 m2, identification key book and stationery. the materials used were 70% alcohol, labeling paper and universal ph indicator paper (1 14). the study was conducted in two study locations, namely lake and waterfall in the shallowest part. each location was represented by three observation stations based on differences in environmental conditions. sampling was carried out three times at each station. neuston collection was carried out at locations and stations that were purposively determined as a pick-up point sized 1 x 1 m2. neuston samples were taken by using a 1 x 1 m2 net which was placed on the water surface. the net captured neustons which went through the filtration following the horizontal movement of the surface water in the lake and waterfall. subsequently, the neustons sample was put into a bottle containing 70% alcohol. samples were identified in the field and photographed for documentation. environmental parameters measured were air and water temperature, water ph, water depth, water turbidity, water flow velocity, dissolved oxygen, substrate and weather conditions. the air temperature was measured using a calibrated air thermometer. water temperature measurements were carried out using a do meter. the ph of the water was measured using universal ph indicator paper (1 14). the water depth of the lake and waterfall was measured using a long wooden stick. the mark of the water depth was then measured by using a measuring tape. water turbidity was measured by using a calibrated turbidity meter. water flow velocity was measured by using floating method from the edge of the lake and waterfall. a rope was tied to a bottle and a 1-meter-long wooden stick. the bottle was floating at the water surface. the time taken from the moment the bottle was tied up to the wooden stick until the rope was stretched straight, was recorded by a stopwatch. the water flow velocity is expressed in m⁄s unit. the formula for calculating the water flow velocity is as follows: flow velocity = rope length (m) time (s) dissolved oxygen was measured three times using a do meter. the substrate is the surface on which an organism lives. the substrate sample of the lake was taken using the ekman grab, while the substrate of the waterfall can be seen directly because the water depth is quite shallow. the substrate was determined by looking at its composition. the types of water substrates are sand, mud, rocks, lime, and others. weather was determined by looking at the weather situation at the lake and waterfall environment. there are three kinds of weather conditions, namely sunny, cloudy, and rainy. data analysis was carried out by determining the simpson index using the formula as follows: 1. density b = t x p a x s where: t = quadrant area (1 m2 = 10,000 cm2) p = area of the taking transect (m2) a = number of individual species s = number of taking transects 2. diversity ds = 1 – d  ds = 1 –  ni (ni – 1) n (n – 1) where: ds = simpson's diversity index neuston diversity and density as bioindicator for water quality – imam safir alwan nurza et al. 83 d = dominant index ni = number of individuals species n = total number of individuals results and discussion there were six species of neuston found in the lake and waterfall of situ gunung. four species were existed in the lake, namely gerris lacustris, metrobates hesperius, gerris comatus, and aquarius remigis (fig. 1), while two species were found in the waterfall, namely dineutus assimilis and trepobates pictus (fig. 2). neuston species found in the lake and waterfall are different, depending on the habitat. gerris lacustris and gerris comatus are found in lakes due to their lentic habitat preferences. according to ye et al. (2017), gerris species inhabit several lentic habitats such as ponds, lakes and backwaters of streams. the habitat preference of gerris lacustris is a high level of water depth, covered with vegetation, which is in agreement with a study conducted by olosutean and ilie (2013) who found that gerris lacustris is correlated with water depth and have adaptation benefits from the presence of vegetation cover. the surface of the lakeside of situ gunung is covered with aquatic plants and the depth is relatively higher (58 93 cm) compared to the water depth of the waterfall (25 64 cm). in addition, gerris comatus is a characteristic organism for lentic water. gerris comatus is often found together with gerris marginatus and gerris buenoi (damgaard et al. 2014; pintar & william 2020). however, the last two species mentioned are not found in the lake. figure 1 neuston species found in the lake of situ gunung notes: a. gerris lacustris; b. metrobates hesperius; c. gerris comatus; d. aquarius remigis figure 2 neuston species found in the waterfall of situ gunung notes: a. trepobates pictus; b) dineutus assimilis a b dc a b biotropia vol. 29 no. 1, 2022 84 aquarius remigis were found in the lake of situ gunung, which fact is different from the statement of ye et al. (2017) that aquarius species are mostly confined to lotic habitats, such as water springs and rivers. aquarius species can live in two different kinds of freshwater, both lentic and lotic. aquarius species are semi-aquatic insects and opportunistic predators that live on the water surface of lakes, rivers, and the border between rivers and lentic habitats (ditrich & papáček 2016; guterres et al. 2019). metrobates hesperius were found in the lake because there are aquatic plants on the surface of the lakeside of situ gunung. metrobates hesperius lays eggs on the leaves of the floating aquatic plants (taylor 2009; ikawa et al. 2012; finet et al. 2018). the lake as a habitat supports the survival and distribution of these species. dineutus assimilis and trepobates pictus were found on the surface of a waterfall. dineutus assimilis prefers both lotic and lentic waters, such as river surfaces, lakeshores and lakes (gustafson & miller 2015; maclean 2013; webster & demerchant 2012). habitat preferences of trepobates pictus were waters with slow water flow with eutrophic, muddy and rocky conditions in lotic waters (naranjo et al. 2010; taylor & mcpherson 2006; wooden 2019; chordas 2017). the diversity index value (ds) of neuston species obtained in the lake and waterfall for each station in situ gunung is presented in table 1. diversity index of 0.50 means low diversity, diversity index value of 0.50 to 0.75 means moderate diversity index, while diversity index of 0.75 to 1 means high diversity index (nento et al. 2018). table 1 neuston diversity and total individuals in the lake and waterfall of situ gunung location station 1 station 2 station 3 ds ti ds ti ds ti lake 0.00 4 0.11 18 0.70 5 waterfall 0.47 10 0.00 6 0.48 7 notes: ds = diversity, ti = total individuals table 1 shows that neuston diversity index value (ds) varies among stations. the highest neuston diversity index in the lake occurred at station 3 with a value of 0.70 (moderate) with a total of 5 inviduals and 3 neuston species, namely gerris lacustris, gerris comatus and aquarius remigis. neuston diversity index value in the lake at station 2 was 0.11 (low) with a total of 18 individuals and 2 neuston species, namely gerris lacustris and metrobates hesperius. the lowest neuston diversity index in the lake occurred at station 1 with a value of 0.00 (low). in station 1, only one neuston species was found, namely gerris lacustris with a total of 4 individuals. the highest neuston diversity index in the waterfall occurred at station 3 with a value of 0.48 (low), where 2 neuston species were found, namely dineutus assimilis and trepobates pictus with a total of 7 individuals. the lowest neuston diversity index in the waterfall occurred at station 2 with a value of 0.00 (low), where 1 neuston species was found, namely dineutus assimilis with a total of 6 individuals. the neuston diversity index in the waterfall at station 1 was 0.47 (low) with a total of 10 individuals and 2 neuston species, namely dineutus assimilis and trepobates pictus. the different values of diversity index with total individuals obtained was due to dominant species in the lake and waterfall of situ gunung, namely gerris lacustris and dineutus assimilis. table 2 neuston density in the lake and waterfall of situ gunung location neutston species neuston density (individu/m2) lake gerris lacustris 0.24 metrobates hesperius 0.01 gerris comatus 0.01 aquarius remigis 0.01 waterfall dineutus assimilis 0.18 trepobates pictus 0.05 the highest neuston density neuston in the lake was shown by gerris lacustris with a value of 0.24 individu/m2. the three other neuston species, namely metrobates hesperius, gerris comatus, and aquarius remigis showed the same density value of 0.01 individu/m2 (table 2). on the lake surface of situ gunung at stations 1 to 3, there are aquatic plants. gerris lacustris prefers to live on aquatic plants available on the lake surface of situ gunung. according to yee (2016), gerris lacustris spends most of its life on the surface of the water. in the lake of situ gunung there were two species of gerris, namely gerris lacustris and gerris comatus. gerris species can be found in almost all aquatic habitats from water springs to tropical seas (yurtseven et al. 2016). at the waterfall of situ gunung, the neuston diversity and density as bioindicator for water quality – imam safir alwan nurza et al. 85 highest neuston density was shown by dineutus assimilis with a value of 0.18 individu/m2 and trepobates pictus with a value of 0.05 individu/m2. the common aquatic insects found in indonesian freshwater are of the order odonata, coleoptera, trichoptera, hemiptera, ephemeroptera, plecoptera and lepidoptera (mahajoeno et al. 2001; candra et al. 2014). in the lake and waterfall of situ gunung, we found orders hemiptera and coleoptera. every species found in the lake of situ gunung belongs to gerridae family. according to dwitawati et al. (2015), gerridae family is classified as having a low tolerance for pollutants. meanwhile, gerris lacustris, gerris comatus, metrobates hesperius and aquarius remigis belong to the order hemiptera. hemiptera insects often move rather than being settled in an unwanted location (mercer et al. 2017; peterson et al. 2017; wooden 2019). the predominant presence of hemiptera insects indicates that the lake is relatively less polluted (majumder 2013). the pollution that contaminates the lake of situ gunung is thought to come from household wastes and human recreational activities. aquatic insects are a good bioindicator for detecting pollution. aquatic insects are also useful for fish food and biocontrol agents (dalal & gupta 2016; ito et al. 2017). therefore, long-term monitoring of aquatic insects is needed to evaluate water quality. table 3 shows that the ph values in the lake of situ gunung for all stations are relatively constant, namely 6 (acid). the standard ph for clean water quality ranges from 6.5 to 9.0. the ph value in the lake of situ gunung which is below the clean water standard is suspected to have been polluted by household wastes due to the closeness with the mainland, hence acidic ph value. the acidic ph value is not within the suitable environment for the neuston's life. neuston can develop well in the ph range of 6.8-8.5 (gundo 2010; pratami et al. 2018). ph affects dissolved oxygen levels. the lowest dissolved oxygen level for the lake of situ gunung location was found at station 2, which was 3.28 mg/l. the acidic ph causes an increase in toxic substances in the water and decreases dissolved oxygen levels (pratami et al. 2018). dissolved oxygen plays an important role in the respiration process of most aquatic organisms (hariyani et al. 2017; pratami et al. 2018). the average of dissolved oxygen level in the lake of situ gunung was 3.62 mg/l. dissolved oxygen levels also indicate the level of pollution waters. dissolved oxygen levels below 5 ppm mean low pollution levels (hariyani et al. 2017). the low dissolved oxygen level shown in the lake of situ gunung indicated that the pollution level was low enabling neuston organisms such as water insects to obtain the needed oxygen for the respiration process. the lake of situ gunung showed the highest water turbidity value at station 1, which was 11.97 ntu. the high value of water turbidity is presumably caused by the existing fishing activities by humans and aquatic plants on the lake surface at station 1. penetration of sunlight into the lake waters can be hindered by the high value of water turbidity (pratami et al. 2018). the lowest value of water turbidity was shown at station 2, which was 4.44 ntu. the different value of water turbidity at station 2 compared to the other two stations is presumably caused by the utilization of station 2, which function is docking place for boats. also, only a few of aquatic plants were existing in station 2. the measurement of environmental parameters of the waterfall of situ gunung showed air and water temperatures of 20.7 oc and 17.9 °c, respectively, indicating a cold-water habitat for insects (table 4). in the environment of the lake of situ gunung, the air and water temperatures of 29 oc and 28 °c, respectively, indicating a warm-habitat for insects (table 3). according to mujiono et al. (2019), the higher the place is, the colder the temperature. the average of water ph of the waterfall of situ gunung is 6 (acidic) with average of dissolved oxygen value of 7.77 mg/l. dissolved oxygen levels are high in the waterfall because the waterfall is located on a hill surrounded by natural forests and has low pollution levels. meanwhile, the lake water is murky indicating water pollution that affects dissolved oxygen levels. in addition, the murky water can cause the increase of oxygen uptake from the air into the water causing the increase of dissolved oxygen in the water (diantari et al. 2017; mujiono et al. 2019; irby et al. 2015). biotropia vol. 29 no. 1, 2022 86 table 3 environmental parameters obtained from the lake of situ gunung environmental parameter station mean 1 2 3 water ph 6 6 6 6 air temperature (oc) 29 29 29 29 water temperature (oc) 28.03 27.93 27.93 27.97 water depth (cm) 58 71 93 74 water turbidity (ntu) 11.97 4.44 11.85 9.42 water flow velocity (m/s) 0.009 0.022 0.0085 0.013 dissolved oxygen (mg/l) 4.24 3.28 3.33 3.62 substrate mud and rocky mud and rocky mud and rocky weather cloudy and rainy cloudy and rainy cloudy and rainy table 4 environmental parameters obtained from the waterfall of situ gunung environment station mean 1 2 3 water ph 6 6 6 6 air temperature (oc) 20.8 20.8 20.5 20.7 water temperature (oc) 17.9 17.9 17.9 17.9 water depth (cm) 25 53.33 64.33 47.56 water turbidity (ntu) 0.41 0.46 0.51 0.46 water flow velocity (m/s) 0.49 0.58 0.13 0.40 dissolved oxygen (mg/l) 7.54 7.71 8.05 7.77 substrate rocky rocky rocky weather sunny sunny sunny the water flow of the stations at waterfall of situ gunung showed an average of water flow velocity of 0.40 m/s which affects the presence of swimming-type aquatic insects. due to the high value of water flow velocity and the presence of rocky substrate, the water insects hide behind rocks (leba et al. 2013). according to diantari et al. (2017), the rocky substrate can affect the water flow velocity in a stream. the lake water's flow velocity of 0.01 m/s causes the lake to be predominated by neustons with floating ability. the average water turbidity of the waterfall of situ gunung was 0.46 ntu with an average water depth of 47.56 cm. meanwhile, the lake showed an average turbidity of 9.42 ntu with an average water depth of 74 cm (table 3). our study showed that sunlight easily penetrated the waterfall. however, the murky lake water hindered the sunlight penetration into the lake. small value of water turbidity can support filterfeeder organisms and affect the activities of the existing orders hemiptera and coleoptera (ebenebe et al. 2016; marpaung et al. 2014). conclusion six neuston species were identified in situ gunung, namely four species in the lake of situ gunung and two species in the waterfalls of situ gunung. the four neuston species in the lake namely gerris lacustris, metrobates hesperius, gerris comatus and aquarius remigis, while the two neuston species waterfalls namely dineutus assimilis and trepobates pictus. the highest neuston densities in the lake and waterfalls were represented by gerris lacustris and dineutus assimilis, respectively. neuston diversity in the lake and waterfalls of situ gunung was low. environmental parameters of the lake and waterfalls of situ gunung were still in the normal range, except low water ph indicating polluted waters environment. therefore, longterm monitoring at the waters of situ gunung is needed for evaluating water quality parameters that support the life of aquatic organisms. neuston diversity and density as bioindicator for water quality – imam safir alwan nurza et al. 87 acknowledgments the author would like to thank the assistant of the ecology laboratory of universitas negeri jakarta who assisted us in analyzing water quality parameters of our study. special gratitudes are also delivered to the officials of the taman nasional gunung gede 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[thesis]. baton rouge (us): louisiana state university. yee kl. 2016. occurrence of aquatic macroinvertebrates from paleik''in'', sintkaing township. yadanabon university research journal 7(1):1-12. ye z, zhen y, damgaard j, chen p, zhu l, zheng c, bu w. 2018. biogeography and diversification of holarctic water striders: cenozoic temperature variation, habitat shifting and multiple intercontinental dispersals. syst entomol 43(1): 19-30. yurtseven i, serengil y, pamukçu p. 2016. seasonal changes in stream water quality and its effects on macroinvertebrate assemblages in a forested watershed. appl ecol environ res 14(1):175-88. biotropia vol. 29 no. 1, 2022: 28 38 doi: 10.11598/btb.2022.29.1.1584 28 characterization and polyphasic identification of novel rhizobacteria strain isolated from sand dunes ecosystem ketut arte widane1, annisaa widyasari2 and endah retnaningrum3* 1undergraduate student, faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia 2graduate student, faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia 3microbiology laboratory, faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia received 9 april 2021 / accepted 3 december 2021 abstract the coastal sand dune ecosystem at the parangtritis coast of yogyakarta, indonesia has unique characteristics such as low moisture sandy soil, high salinity and low nutrient content. fimbristylis cymosa is one of the plant species having the capability to survive in that unique ecosystem. in this study, rhizobacteria isolated from the rhizosphere of f. cymosa were isolated to be further analyzed on their phosphate solubilizing and antagonistic properties against fusarium oxysporum which cause the wilt disease. the isolates of phosphate solubilizing rhizobacteria (psr) having the most potential capabilities were then polyphasically identified based on phenotypic and genotypic characters followed by 16s rdna sequencing. the results showed that four psr isolates (i8, i11, i12 and i24) have high phosphate dissolution indices. the highest indices were observed in isolates i11 (3.08) and i12 (3.44), respectively. analysis of the dual plate experiments for psr i11 and psr i12 isolates against the growth of f. oxysporum also showed quite high inhibitory activities, i.e., isolate psr i11 was 42.40%, while isolate psr i12 was 42.08%. the two isolates were polyphasically identified as burkholderia dolosa. this study clearly showed that psr i11 and psr i12 isolates are very potential and prospective to be used as marginal land inoculants and as providers of phosphorus. this study also showed that the isolates are useful as biocontrol agents against f. oxysporum in plants. keywords: inhibitory activity, phosphate dissolution index, phosphorus, polyphasic identification, sandy soil introduction coastal sand dunes are aeolian landforms commonly found in coastal areas at all latitudes in the world. indonesia has coastal sand dunes located in the southern part of java island, extending from the southern coast of west java province to yogyakarta province. the most significant sand dunes formation occurs in the parangtritis coastal area, located on the southern coast of yogyakarta province. the sand dunes ecosystem has a high temperature, relatively little vegetation, strong winds, high salt content and very low groundwater content, making it a dry and infertile area (mahdavi & bergmeier 2016; campos et al. 2020). in general, plants are difficult to grow in dry and less fertile areas. however, there are several groups of plants that can survive in the sand dunes habitat. among plants that survived the sand dunes habitat on yogyakarta’s southern coast is fimbristylis cymosa, a family of cyperaceae, which grows well and is widespread along with the coastal sand dunes habitat. the growth and development of f. cymosa in the coastal sand dunes habitat are strongly influenced by the availability of essential soil macronutrients. one of the main macronutrients supporting the growth and development of f. cymosa is phosphorus. phosphorus is among minerals needed at 0.2% dry weight of f. cymosa, as a component of nucleic acids (dna and rna) for conserving energy. plants absorb phosphorus in the form of phosphate anions (po4 or po4 2-) which are dissolved in groundwater. however, phosphate anions have *corresponding author, email: endahr@ugm.ac.id novel rhizobacteria strain isolated from sand dune ecosystem – ketut arte widane et al. 29 a high tendency to bind with calcium, aluminum, and iron in the soil, which makes them insoluble and easily settled so that they cannot be absorbed by plants, including f. cymosa (yang et al. 2021). undissolved phosphorus in the soil can be dissolved by the phosphate solubilizing bacteria (psb) group. a study conducted by liu et al. (2015) in calcareous soil found psb genera bacillus, pseudomonas, rhizobium and acinetobacter. pastore et al. (2020) reported psb genera burkholderia, bacillus, and pseudomonas in forest soils. in the mangrove forest rhizosphere, teymouri et al. (2016) observed the existence of psb genera bacillus, pseudomonas and acinetobacter. rasul et al. (2019) obtained psb genera pseudomonas and bacillus from rhizospheric paddy field soil, while the psb genus paenibacillus was found from wheat rhizosphere (cherchali et al. 2019). in soil environments, there are also soilborne pathogens causing plants infection. one species of a soil-borne pathogen is fusarium oxysporum causing the fusarium wilt disease in various types of agricultural crops within the solanaceae family, such as tomatoes, potatoes, and chilies (ávila et al. 2019; srinivas et al. 2019; chowdhury et al. 2020). the wilting phenomenon that occurred in those crops due to fusarium wilt disease resulted in a significant reduction in crop yields and has been a major problem for agriculture worldwide. the use of chemical compounds for treating the wilt disease has adverse effects on non-target organisms. therefore, it is pertinent to find biocontrol agents which is considered safer for the environment. in soil environments, phosphate solubilizing bacteria (psb) are reported to be more dominantly existing in the rhizosphere. in addition, psb from the rhizosphere is known to produce active metabolites compared to other sources and is able to increase plant growth and development. psb originating from extreme environments such as sand dunes, might have specific characteristics as a biocontrol agent against f. oxysporum (ahluwalia et al. 2021; rasool et al. 2021) therefore, this study aimed to: 1. isolate and characterize phosphate solubilizing bacteria (psb) from the rhizosphere of f. cymosa living in sand dunes habitat; and 2. screen the capability of isolated psb as a biocontrol agent against f. oxysporum. materials and methods rhizosphere soil samples and isolate of f. oxysporum rhizosphere soil samples from the planting area of f. cymosa were taken and collected from 8 different sampling points along with the coastal sand dune habitat in the parangtritis coastal area, yogyakarta. the roots of the f. cymosa were carefully extracted with a shovel. soil attached to the roots was collected in sterile plastic containers as rhizosphere soil samples. the samples were then stored at 4 oc for further isolation of phosphate solubilizing bacteria (psb). the isolate of fusarium oxysporum was obtained from the culture collection of the microbiology laboratory, faculty of biology, universitas gadjah mada, yogyakarta, indonesia and was grown in potato dextrose agar (pda) medium at 30 oc for 10 days. psb isolation and phosphate dissolution index analysis ten (10) g of rhizosphere soil samples were poured into a 50 ml erlenmeyer and suspended in 90 ml of sterile distilled water, then shaken using a vortex at a speed of 200 rpm for 30 min. the sample suspension was diluted in a series of up to 10-4 dilutions. a total of 0.1 ml of the respective 10-3 and 10-4 diluted sample suspensions was inoculated on the national botanical research institute's phosphate (nbrip) agar medium by means of the spread plate method, then incubated at 30 oc for 48 72 hours. the nbrip medium consists of 1% glucose, 0.5% ca3(po4)2, 0.5% mgcl2, 0.01% (nh4)2so4, 0.0.5% mgso4.7h2o, and 0.02% kcl (nautiyal 1999). psb isolates having a clear zone around the colony were then re-isolated as psr, and purified on an nbrip medium. in order to measure the phosphate dissolution index, the psr isolates were recultured in nbrip liquid medium and incubated at 30 oc for 48 h. forty (40) μl of the incubated culture was diluted until reaching a colony having cell number 108 cfu/ml. the liquid culture was then poured into a petri dish containing nbrip agar medium by means of the pour plate method and incubated at 30 oc for 48 h. biotropia vol. 29 no. 1, 2022 30 the formed clear zone and the size of the bacterial colony were calculated. the phosphate dissolution index was determined with the following formula developed by haile et al. (2016): psi = (hzd + cd) cd where: psi = phosphate solubilization index hzd = halo zone diameter (mm) cd = colony diameter (mm) antagonistic activity of psr against f. oxysporum in vitro test of psr antagonist activity against f. oxysporum was carried out by using the dual plate experiments method according to zhang et al. (2017) with minor modifications. potato dextrose agar (pda) medium was prepared on a 9 cm diameter petri dish, followed by making a 5-mm diameter well in the pda medium at the center of the petri dish by using a cork borer. the other four 5-mm diameter wells were made at a distance of 2 cm from the well located at the center of the petri dish and from each other. f. oxysporum mycelium with a diameter of 5 mm was taken from the outermost part of the 10day-old fungal colony and was inoculated into the well at the center of the petri dish containing pda medium by using an inoculation needle. a total of 8 µl of psr isolate suspension with a cell number of 109 cfu/ml was inoculated into the other four wells in the same petri dish and then incubated at 30 °c for 10 days. the control agar plates were then prepared with the same agar medium without inoculating the psr isolates suspension. at the end of the incubation time, the diameter of f. oxysporum colonies was measured diagonally, vertically and horizontally. the inhibition percentage of psr isolates against f. oxysporum was determined using the following formula of royse and ries (1978): ip = (r1 r2) x 100% r1 where: ip = inhibition percentage r1 = diameter of f. oxysporum colonies on the control plate r2 = diameter of f. oxysporum colonies on the treated plate polyphasic identification of psr isolates polyphasic identification of psr isolates was determined based on a combination of phenotypic and genotypic characters. phenotypic characters of the observed isolates, including the morphological and biochemical characters, were determined by referring to bergey's manual of systematic bacteriology (brenner et al. 2005). the morphological characters were observed based on cell and colony morphologies. the cell morphology of psb isolates was observed based on the shape and characteristics resulting from the gram staining procedure. meanwhile, colony morphology of psb isolates was observed based on the shape, color, margins, elevation, internal structure and optical features of the colony. the biochemical characters were determined based on several tests, such as the motility, catalase, oxidase tests, glucose, sucrose, lactose fermentation tests, urease and voges-proskauer (vp) tests. the isolates were then characterized genotypically based on the 16s rdna nucleotide sequence. total genomic dna of psb isolates was extracted using the quickdnatm fungal/bacterial miniprep kit (zymo research, usa). pcr amplification of the 16s rdna gene of the psb isolates was analyzed by using 27f primers (5’-agagtttgatcct ggctcag-3’) and 1492r (5’ggttacctt gttacgactt-3’) (hossain et al. 2020). the pcr amplification was performed using a dna thermal cycler (bio-rad, usa) with the following program: pre-denaturation at 95 °c for 30 min, followed by 30 cycles consisting of denaturation at 95 °c for 30 sec, annealing at 57 °c for 1 min, extension at 72 °c for 1 min, and final extension at 72 °c for 10 min. the pcr products were purified and sequenced. the nucleotide sequences were then compared with dna sequence databases (genbank) through the blast program (blastn) at the national biotechnology information center (ncbi). a phylogenetic tree was constructed through the maximum likelihood method using molecular evolutionary genetics analysis x (mega version x). statistical analysis data were statistically analyzed using the oneway variance analysis (anova) followed by the duncan multiple range test (dmrt) approach for testing the mean differences. at p < 0.05, novel rhizobacteria strain isolated from sand dune ecosystem – ketut arte widane et al. 31 the differences were considered significant. results are expressed as means of three replicates ± standard deviation. the software used was ibm spss statistics version 21. results and discussion psr isolate and phosphate solubilization index a total of 26 bacteria were isolated from f. cymosa rhizosphere soil samples in the sand dunes habitat of parangtritis coastal area, yogyakarta, indonesia. of the 26 rhizobacterial isolates, 4 rhizobacterial isolates were selected as psr based on the clear halo formation around the colony in the nbrip medium containing tricalcium phosphate. the clear halo was formed due to the production of organic acids or polysaccharides as a result of the phosphatase enzyme activity of the psr isolate (paul & sinha 2013; behera et al. 2017). the phosphate solubilization indices of each psr isolate after 10 days of incubation are presented in table 1 (widane et al. 2018). the highest phosphate solubilization index of 3.44 was obtained from psb i12. meanwhile, the lowest phosphate solubilization index of 3.00 was obtained from psb i24. a previous study by teymouri et al. (2016) reported the phosphate solubilization index of 3.5 which was obtained from bacillus sp. found in the rhizosphere of mangrove forest. mardad et al. (2013) reported the phosphate solubilization index of 3.5 which was obtained from enterobacter hormaechei found in the phosphate rock deposits. in contrast, several other researchers reported a lower-than-three phosphate solubilization index, such as those obtained from pseudomonas (2.6) and acinetobacter (2.0) found in the rhizosphere of forest (teymouri et al. 2016). the paenibacillus genus from the wheat rhizosphere was reported to have a very low phosphate solubilization index of 1.32 (cherchali et al. 2019). pseudomonas and serratia obtained from the rhizosphere of allium cepa l. were also reported to have low phosphate solubilization indices of 2.0 and 2.1, respectively (blanco-vargas et al. 2020). table 1 phosphate solubilization index of psr isolates obtained from the f. cymosa rhizosphere after 10 days of incubation no psr isolate phosphate solubilization index 1 i8 3.04 ± 0.51bcd 2 i11 3.08 ± 0.38cd 3 i12 3.44 ± 0.19d 4 i24 3.00 ± 0.43abcd notes: the index is presented as mean ± sd; numbers followed by the same letter in a column do not show a significant difference according to the duncan test (dmrt) at p < 0.05. different values of phosphate solubilization index among psr isolates are closely related to organic acids production, causing a ph decrease in bacterial cells and their environment, leading to proton substitution in the phosphate mineral and p release (ludueña et al. 2018; rasul et al. 2021). organic acids produced by several psrs include gluconic, glycolic, oxalic, malonic and succinic acids. gluconic acid is an organic acid having a role as a phosphate solvent (joe et al. 2018; santos-torres et al. 2021), which is useful in providing p minerals needed for plant growth (valetti et al. 2018; sahandi et al. 2019). psr i11 and psr i12 isolates have a high ability to dissolve phosphate in vitro. therefore, these two isolates are the potential to be developed as agents for providing p for plants. antagonistic activity of psr isolates against f. oxysporum psr i8, psr i11, psr i12, and psr i24 isolates were tested in-vitro for their antagonistic activity against f. oxysporum by using the dual plate technique. the results showed a significant inhibitory effect on the growth of f. oxysporum mycelium (fig. 1), which was clearly seen by comparing the inhibitory effect of the four isolates with the control against the growth of f. oxysporum. the four psr isolates clearly caused a smaller colony size of f. oxysporum compared to that in the control treatment. the colony diameter of f. oxysporum, however, was different among the four psr isolates, i.e., 44.75 mm was observed in psr i11 isolate, 45.00 mm in psr i12, 53.00 mm in psr i24 and 62.50 mm in psr i8 (widane et al. 2018). biotropia vol. 29 no. 1, 2022 32 a b c d figure 1 antagonistic activity of psr isolates against f. oxysporum observed in-vitro after 14 days incubation period notes: a = psr i8; b = psr i12; c = psr i11; d = psr i24. in addition, our study showed that psr isolates also caused thinner f. oxysporum mycelium growth compared to that in the control treatment. this explains the inability of f. oxysporum to grow well in the presence of psr isolates. the graph of the inhibition percentage of psr isolates against f. oxysporum is presented in figure 2 (widane et al. 2018). the dual culture method applied in this study allows direct in-vitro interactions between psr isolates and f. oxysporum. these interactions can be in the form of competition for space or for nutrients from the growth medium, leading to growth inhibition of f. oxysporum. growth inhibition can also be caused by metabolite compounds produced by psr isolates. several metabolite compounds are reported to have the most effective antagonistic activity in inhibiting the growth of plant pathogens in the form of antibiotics, siderophores and bacteriocins (khedhera et al. 2021). bacteria can produce hydrolytic enzymes such as protease, lipase, chitinase and glucanase which can lyse fungal cells so that they have antagonistic activity against plant pathogenic fungi (cui et al. 2019; lau et al. 2020). our study also showed that the psr i11 isolate was the most effective at inhibiting the growth of f. oxysporum with an inhibition percentage of 42.40%, while the psr i12 isolate had an inhibition percentage of 42.08% which is not significantly different from that of psr i11. lower inhibition percentages were observed for psr i24 and psr i8, i.e., 31.82% and 19.58%, respectively (fig. 2). figure 2 inhibition percentage value of psr isolates against f. oxysporum after 14 days of incubation period novel rhizobacteria strain isolated from sand dune ecosystem – ketut arte widane et al. 33 several studies have also reported that bacteria isolated from the rhizosphere are able to protect plants from fungal pathogens (babu et al. 2015; panda et al. 2016). bacillus amyloliquefaciens from the rhizosphere of apple trees were reported to have inhibition capability against f. oxysporum with an inhibition percentage of 35% (guleria et al. 2016). meanwhile, research conducted by jangir et al. (2018) showed that bacillus sp. from the rhizosphere of tomato plants had antagonistic activity against f. oxysporum with higher inhibition percentage (70%) and therefore, is potential as a biocontrol agent against fusarium wilt disease in tomato plants. karthika et al. (2020) also isolated bacillus cereus from the rhizosphere of a tomato plant, which has an inhibition percentage of 66%. our study further showed that psr i11 and psr i12 isolates have the capability of phosphate solvent and have high antagonistic activity against f. oxysporum. polyphasic identification of psr isolates observation of the morphological characters of psr i11 and psr i12 showed different colony morphology forms, i.e., circular and irregular, respectively (table 2). however, the two isolates have the same colony color, elevation, internal structure, optical features and shape, such as cream, entire, convex, smooth, translucent and rod-shaped (table 2). biochemical properties of the two isolates, psr i11 and psr i12 showed similarities in terms of motility, production of catalase and oxidase enzymes and positive reaction toward the voges-proskauer test. different reactions occurred when being tested in a medium containing urea, showing that psr i11 isolate is able to produce urease, while psr i12 isolate is not able to produce urease (table 3). comparing the results from the identification of the morphological and biochemical characters with the characters described in bergey’s manual of determinative bacteriology, it is concluded that psr i11 and psr i12 isolates belong to the genus burkholderia (brenner et al. 2005). table 2 morphological characters of psr isolates from rhizosphere of f. cymosa no morphological characters psr isolate i11 i12 1. colony morphology shape circular irregular color cream cream margin entire entire elevation convex convex internal structure smooth smooth optical feature translucent translucent 2. cell morphology shape rod-shaped rod-shaped gram properties negative negative tabel 3 biochemical characters of two psr isolates from rhizosphere of f. cymosa psr isolate no biochemical characters i11 i12 1 motility test + + 2 catalase test + + 3 oxidase test + + 4 glucose fermentation 5 sucrose fermentation 6 lactose fermentation 7 urease + 8 voges-proskauer (vp) test + + biotropia vol. 29 no. 1, 2022 34 burkholderia spp. is reported of having the ability to dissolve phosphate and at the same time has antagonistic activity against f. oxysporum. simonetti et al. (2018) isolated burkholderia ambifaria from the rhizosphere of barley having a role as phosphate solvents and antagonistic properties against f. oxysporum. burkholderia cepacia and burkholderia contaminans with similar capabilities were isolated from the rhizosphere of maize (zhao et al. 2014; tagele et al. 2018). burkholderia sp. was also obtained from the rhizosphere of juçara palm (euterpe edulis mart) (de castilho et al. 2020). in addition to the plant rhizosphere, burkholderia spp. with the same properties is also obtained from the root nodules of fenugreek (trigonella foenumgraecum l.) (kumar et al. 2017). b. contaminans were also existed at the nodule of the common bean (phaseolus vulgaris) (tapia-garcía et al. 2020). apart from dissolving phosphate and having antagonistic properties against f. oxysporum, b. contaminans also has nitrogenase properties (silva et al. 2012). both psr isolates were also genotypically identified using the 16s rdna genetic marker. based on the 16s rdna gene sequencing, the psr i11 and psr i12 isolates had similarities with burkholderia dolosa strain lmg 18943b with a similarity index of 99.51% and 99.44%, respectively (table 4). the two psr isolates were categorized in the same species because the similarity percentage in the 16s rrna gene sequences was both ≥ 99% (schlaberg et al. 2012). phylogenetic trees based on 16s rdna sequences of psr i11 and psr i12 isolates were reconstructed by using comparative sequences with the in-group and out-group categories obtained from ncbi (table 4). some of the relatively close sequences of burkholderia spp. included burkholderia dolosa strain lmg 18943 (yarza et al. 2013), burkholderia latens strain r-563 (vanlaere et al. 2008), burkholderia multivorans strain struelens (bauernfeind et al. 1999), burkholderia vietnamiensis strain lmg 10929 (lipuma et al. 1999), burkholderia vietnamiensis strain tvv75 (viallard et al. 1998), burkholderia metallica strain r-16017 (vanlaere et al. 2008), burkholderia territorii strain lmg 28158 (de smet et al. 2015) and burkholderia seminalis strain r-2419 (vanlaere et al. 2008) as the in-group. meanwhile, alcaligenes faecalis subsp. parafaecalis strain g 16s was used as the out-group because the species is a classlevel classification group betaproteobacteria with burkholderia spp. in addition, alcaligenes sp. has the ability to dissolve phosphate and antibiosis properties against f. oxysporum (rasool et al. 2021). reconstruction of the phylogenetic tree was carried out using the maximum likelihood (ml) method with a substitution model (tamura-nei 93, g: gamma-distributed) and a bootstrap of 1,000 replications (fig. 3). this phylogenetic tree shows that both strains form clusters with burkholderia dolosa strain lmg 18943 in 83% bootstrap replications; therefore, reconstruction of these phylogenic trees can be trusted (hillis & bull 1993). the phylogenetic tree reconstruction of the two isolates was also in accordance with the blast results, which indicated that the two isolates could be identified as burkholderia dolosa. in addition, we already submitted the 16s rdna sequences of both strains (b. dolosa i11 and b. dolosa i12) to the genbank, with accession numbers of ok083732 and ok083731, respectively https://www.ncbi.nlm.nih.gov). tabel 4 identify sequences of psr isolates from the rhizosphere of f. cymosa based on the genbank data by using blast isolate accession number species of psr homolog identity query cover reference psr i11 nr_104973.1 burkholderia dolosa strain lmg 18943 99.51 % 99% yarza et al. (2013) nr_042632.1 burkholderia latens strain r-5630 99.30 % 99% vanlaere et al. (2008) nr_029358.1 burkholderia multivorans strain struelens 99.09 % 99% bauernfeind et al. (1999) nr_041720.1 burkholderia vietnamiensis strain lmg 10929 99.09 % 99% lipuma et al. (1999) nr_118872.1 burkholderia vietnamiensis strain tvv75 99.22% 98% viallard et al. (1998) nr_042636.1 burkholderia metallica strain r-16017 98.95% 99% vanlaere et al. (2008) nr_136496.1 burkholderia territorii strain lmg 28158 98.95% 99% de smet et al. (2015) nr_042635.1 burkholderia seminalis strain r-2419 98.74% 99% vanlaere et al. (2008) psr i12 nr_104973.1 burkholderia dolosa strain lmg 18943 99.44 % 99% yarza et al. (2013) nr_042632.1 burkholderia latens strain r-5630 99.16 % 99% vanlaere et al. (2008) nr_029358.1 burkholderia multivorans strain struelens 99.09 % 99% bauernfeind et al. (1999) nr_041720.1 burkholderia vietnamiensis strain lmg 10929 99.09 % 99% lipuma et al. (1999) nr_118872.1 burkholderia vietnamiensis strain tvv75 99.43% 98% viallard et al. (1998) nr_042636.1 burkholderia metallica strain r-16017 98.95% 99% vanlaere et al. (2008) nr_136496.1 burkholderia territorii strain lmg 28158 98.88% 99% de smet et al. (2015) nr_042635.1 burkholderia seminalis strain r-2419 98.81% 99% vanlaere et al. (2008) novel rhizobacteria strain isolated from sand dune ecosystem – ketut arte widane et al. 35 figure 3 phylogenetic tree based on 16s rdna sequences constructed by the maximum likelihood method note: numerals at the nodes indicate the bootstrap value (%) derived from 1,000 replication. conclusion psr i11 and psr i12 bacterial isolates obtained from the rhizosphere of f. cymosa in the sand dunes habitat of parangtritis, yogyakarta, indonesia showed the capability to dissolve phosphate. the two isolates also showed an inhibition percentage of more than 40%, and therefore, can be used as biocontrol agents against f. oxysporum. psr i11 and psr i12 isolates were polyphasically identified as burkholderia dolosa. since the two isolates were obtained from f. cymosa living in sand dunes habitat, which is an extreme environment, this study can be further developed into determining the possibility of obtaining these two isolates in other marginal lands, to be used as phosphate solvent and 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(2018) reported that seaweed contains many nutrients like nitrogen, phosphate, potash, plant growth hormones, and other trace elements. the use of seaweed as organic fertilizer consequently improves soil fertility and enhances plant growth. it can be applied in several different forms, including refined liquid extract and dried, pulverized organic material (raghunandan 2019). sulu province is along the southern part of mindanao, philippines, and the northern boundary of the celebes sea. it has a total area of 167,376 hectares, with 824,731 household population, and is the fifth largest island in the philippines (psa 2015). sulu has 94,500 hectares of agricultural land, wherein the municipality of patikul is the largest with 15,750 hectares. the province is predominantly agricultural with farming and fishing as the main sources of livelihood. its fertile soil and ideal climate can grow a variety of crops and exotic fruits seldom found elsewhere in the country. peanuts (a. hypogaea l.) are commonly planted by local farmers and consumed as boiled, peanut butter, roasted peanuts, peanut bars, oils, and candies. it is considered a vital source of nutrients, calories, minerals, antioxidants, and vitamins essential for optimum health. this *corresponding author, email: fatimasheirenemarajuli@gmail.com, juliet.bangi@msumain.edu.ph mailto:fatimasheirenemarajuli@gmail.com mailto:juliet.bangi@msumain.edu.ph fermented seaweed extract efficiency as foliar fertilizer – fatima sheirene m. marajuli and juliet c. bangi 77 commodity is usually intercropped with coconut and banana and is the alternative to cassava in other cropping seasons (da-saad 2020). several studies report that seaweed liquid extract boosts plant growth and yield, develops tolerance to environmental stress, increases nutrient uptake from soil, and improves antioxidant properties. it also contains major and minor nutrients, amino acids, vitamins, cytokinin, auxin, and growth-promoting substances similar to abscisic acid. by engaging with important metabolic processes including protein synthesis and nucleic acid metabolism, seaweed extract significantly contributes to the internal mechanism of plant growth (rathore et al. 2008). with the vast seaweed production in sulu province, several waste materials can be utilized as organic foliar fertilizers, given the nutrient composition of seaweeds. these natural resources can promote organic farming and able to reduce the cost of inputs in peanut production. considering the reported benefits of the use of seaweed extract, this present study was conducted to determine the effect of fermented seaweed extract (fse) as fertilizer at different frequencies and concentrations on peanut production. this study would benefit the farmers in sulu by reducing the costs of chemical inputs and dependence on synthetic fertilizers (awal et al. 2015). materials and methods this study was conducted in the experimental field of mindanao state university-sulu, college of agriculture, patikul extension demo farm in patikul, sulu. the field had a total area of 189 m², and the experiment lasted for five months (january may 2022). the experiment is a 3 x 5 factorial in split-plot randomized complete block design (rcbd). the different frequency applications of fse represent factor a (main plot) while the different concentration rates of seaweed extract represent factor b (subplots). all treatment combinations were replicated three times (table 1). soil sampling was done to analyze the nutrient status of the soil. collection of samples was randomly taken in the experimental area before plowing and the composite samples were airdried, pulverized, sieved (2 mm wire mesh), and submitted to the regional soils laboratory bureau of soils, port area, zamboanga city for soil analysis. the field was prepared by plowing, followed by harrowing to pulverize the soil. the area was leveled and laid out in a split-plot experimental design. plots were subsequently covered with plastic mulch to prevent weeds from growing. furrows were measured to have a proper distance for planting. then, the seeds were soaked in water before planting to attain good germination. the seeds were sown 30 cm between rows and 40 cm between hills with a planting depth of 2 to 3 cm and covered with fine soil in a row-hill method of planting. the seaweed extracts of kappaphycus alvarezii, a red alga belonging to the family solieraceae and the common species grown in the locality, were used in the study. the collected samples and other materials were washed several times using tap water to remove dirt. the samples were homogenized and fermented using molasses (only) for about a month. lacto-fermentation was used in the study as the usual method in the fermentation process according to the review done by ahmad et al. (2020) and wang et al. (2021). the ratio used in the fermentation is 1:1, or 1 kg of ground seaweed added with 1 liter of molasses based on the study by pascual et al. (2020). after fermentation, the extract was strained with a fine mesh cloth to separate the solid from the liquid extract. the fse was applied through foliar spray according to treatment concentration and frequency of application. fse was diluted with water and applied to the crop up to the peak of the peanut’s last flowering period (8-10 weeks) (figure 1). table 1 the 3 x 5 factorial treatments of the study using the fermented seaweed extract (fse) in peanut production, msu-sulu, barmm, philippines factor a (frequency application) factor b (concentration of application) a1 – once a week b1 – control/ no fertilizer a2 – twice a week b2 – 5% seaweed extract concentration a3 – once a month b3 10% seaweed extract concentration b4 15% seaweed extract concentration b5 20% seaweed extract concentration biotropia vol. 31 no. 1, 2024 78 figure 1 fermented seaweed extract (fse) preparation and application in peanut plants monitoring of insect pests and diseases was done every week and the control measures depended on the pests’ infestation. harvesting was done 113 days after planting, and the harvested pods were separated according to treatments. the data collected were the following: plant height (cm)per week, number of branches per plant per month, number of days to flower (about 50% plants per plot flowering), number of flowers per plant, number of pods per plant, number of seeds per pod, yield per hectare (tons/ha) by using the formula (1), and the cost and return analysis based on the return on investment (roi) formula (2). 𝑌𝑖𝑒𝑙𝑑 𝑝𝑒𝑟 𝐻𝑒𝑐𝑡𝑎𝑟𝑒 ( 𝑡𝑜𝑛 ℎ𝑎 ) = yield per plot (kg) plot size (m2) × 𝑡𝑜𝑛 1,000 𝑘𝑔 × 10,000 𝑚2 ℎ𝑎 (1) 𝑅𝑒𝑡𝑢𝑟𝑛 𝑜𝑓 𝐼𝑛𝑣𝑒𝑠𝑡𝑚𝑒𝑛𝑡 (𝑅𝑂𝐼) = net income cost of production × 100 (2) the data were analyzed using analysis of variance (anova) in split-plot randomized complete block design (rcbd) through spss. treatment means with significant differences were compared using tukey's honestly significant difference (hsd) test. results and discussion plant height (cm) the responses of peanuts in the fse application showed remarkable differences according to concentration and frequency (table 2). this proves that fse contains macronutrients that influence the growth of the test plants. based on the fse analysis, it contains enough phosphorus (0.15% p2o5) and potassium (2.46% k2o) but less nitrogen (0.30% n), which influenced the bio-responses of peanuts. plant height is a crucial characteristic that reflects overall growth and is widely used to predict biomass or final yield. in the first and second weeks, peanut plant height varies according to the treatment combinations. early plant development average height ranged from 6.56 cm to 6.94 cm with fse sprayed once or twice a week at concentrations of 10-15% (table 2). fse influenced the growth of peanuts, which conforms with the study conducted by rathore et al. (2008). the plants were tall and robust compared with plants that received a monthly application of the same dose. vigorous plants were produced as a result of the continuous application of seaweed extract during the third until the tenth week at a concentration fermented seaweed extract efficiency as foliar fertilizer – fatima sheirene m. marajuli and juliet c. bangi 79 of 10-15% applied once and twice a week. however, no statistical difference was found in fse applied twice a week at 510% concentration with 55.44 cm plant height. shorter plants were observed from control or unapplied plots with fse. the findings of the study revealed the influence of seaweed extracts having macroand micronutrients including growth hormones, which revealed a similar effect to the study conducted by prasad et al. (2010) and rathore et al. (2008). fse was reported to have enough phosphorus of about 34 ppm, magnesium and calcium of more than 450 ppm (rathore et al. 2008), and micronutrients, which made a significant impact on legumes. also, seaweed extracts have been used in several horticulture studies and are widely used as a solidifying agent in the nutrient media of plant tissue culture. the flowering stage of peanuts started in the fourth week after planting showing yellow flowers that emerged around the basal portion of the plant (table 3). the treatment combination of twice-a-week application at 10% fse concentration (a2b3) initiated early flowering at 30.82 days. still, it did not differ significantly with the treatment combination having twice the application of 5% fse concentration (a2b2) with 31 days of flowering. the late flowering of peanuts was observed in control plots (a1b1 and a2b1) without fse application at 32.98 and 32.62 days, respectively (table 2). the results showed that frequent application of seaweed extract at 5-10% concentration boosted the early flowering of peanuts. plants sprayed with fermented seaweed extract once a month have a longer number of days to flower. it can be inferred that a lesser amount of fse nutrients is received during plant growth and development (table 3). these findings conform with the soybean experiment conducted by rathore et al. (2009) applied with seaweed extract. this was possible due to the high phosphorus content of seaweed with 33.99 mg/l. it can be assumed that the early flowering of the peanut is due to the phosphorus content which is essential for plant flowering initiation and/or reproductive stage. lalog (2011) mentioned that seaweed extract is rich in macroand micronutrients that enhance the early flowering of peanut plants. it is environment-friendly, helps the growth of various crops and vegetables while improving the table 2 plant height (cm) of peanut (arachis hypogaea l.) taken every week as influenced by the different seaweed extract concentrations and frequency of application in msu-sulu, barmm, philippines fermented seaweed extract (treatments) plant height (cm) per week * frequency application (factor a) concentration (%) (factor b) 1st 2nd 3rd 4th 5th 6th 7th 8th 9th 10th a 1 once a week (1x / wk) b1 0 5.15d 11.55d 14.66cd 16.65d 20.73c 28.14d 41.74c 47.61f 51.04d 53.52b b2 5 5.44bc 11.86cd 13.66e 15.52e 18.63d 28.21d 41.04d 48.59d 51.90c 53.80b b3 10 6.58a 13.02a 15.95b 17.05cd 19.77cd 29.17bc 40.42e 49.01cd 50.88d 54.53a b4 15 6.94a 13.28a 15.79b 19.28a 20.77bc 29.67a 43.77b 49.99a 53.07b 53.92b b5 20 5.16d 11.63cd 14.15d 16.32d 20.50c 29.84a 39.82f 50.04a 53.17ab 53.53b a2 twice a week (2x / wk) b1 0 6.40ab 12.88a 14.59cd 16.61d 19.27d 28.20d 41.31cd 48.42d 50.50de 52.29d b2 5 6.65a 13.05a 14.98c 18.30b 20.63c 29.65a 44.38a 49.85a 53.79a 55.44a b3 10 6.84a 13.26a 16.61a 18.93a 23.53a 29.50a 39.70f 48.17de 50.63d 54.59a b4 15 5.39c 11.82c 14.55cd 17.38c 21.43b 29.24b 39.12g 49.41bc 50.94d 54.10b b5 20 6.56a 13.00a 14.70cd 18.54ab 23.13a 28.82c 39.22fg 49.42b 53.61a 54.81a a3 once a month (1x / mo) b1 0 6.35b 12.80a 14.33d 18.42ab 21.83b 28.63cd 38.19h 47.61fg 49.44f 53.15c b2 5 6.38b 12.82a 14.58cd 17.58c 20.17c 28.89c 42.24c 47.92ef 52.24c 54.54a b3 10 6.30b 12.64b 14.49cd 17.74bc 20.20c 28.58cd 38.64g 47.21g 50.07e 53.50b b4 15 5.79b 12.08c 14.31d 17.61c 21.50b 28.75c 43.24b 49.19c 50.84d 53.70b b5 20 5.98b 12.12c 14.90cd 17.07cd 20.47c 28.30cd 39.53f 48.07e 50.18e 53.36c anova (treatments) * * * ** ** * ** ** ** ** cv (%) 11.13 5.45 5.46 4.04 4.29 2.28 1.71 1.28 1.20 1.19 *mean of the same letter/s within a column is not significantly different based on tukey’s honestly significant difference (hsd) test number of days to flower and number of flowers per plant biotropia vol. 31 no. 1, 2024 80 quality of soil, and increases crop growth and yield. gomonet and cagasan (2020) revealed similar results in peanut number of days to flowering ranging from 30 to 33 days. most of the flowers were found in leaf axils on primary and secondary branches. accordingly, several flowers can originate from each node and the number of flowers continued to increase until the plant reached peak bloom at about 60 to 70 days after emergence, and then flower development will begin to decline. the number of flowers of peanut per plant ranged from 5 to 7 as shown in table 3. the treatment combination of once-a-week application with 10% concentration (a1b3) of fse had the highest number of flowers per plant with a mean of 6.29 but did not differ statistically with twice-a-week application of fse concentration at 5 -20%. a low number of flowers were observed in plants without fse application (control). the findings can be substantiated by the report of lalog (2011) and the nutrient content of seaweed with a greater amount of phosphorus. in addition, the once-aweek application treatment of seaweed extract with 20% concentration (a1b5) got the lowest number of flowers with a mean of 5.18 (table 3). the results of the study were inconsistent with the outcome of other treatments with 20% concentration. this shows other factors most likely affected the flowering of peanuts in the treatment combination. thus, it cannot be presumed that the higher concentration of seaweed extract will have a lesser effect on the flowering of peanuts. high temperature, low humidity, and excessive rainfall affect the response of plant flowering which most likely limit the number of flowers produced and reduce flower pollination. this can result in reduced yield and delayed pod set (awal et al. 2015). number of branches per plant the number of branches per plant was initially fewer in all treatments for the first month (table 3). it sharply increased as the growth progressed to the third month after sowing as shown in figure 2. the frequency of application and concentration of fse played a significant role in the branch formation of peanuts for three consecutive months. the number of branches per plant was consistently higher in the twice-aweek application at 10% concentration (a2b3) from the first to the third month with a mean of 6.33, 10.00, and 14.67, respectively. however, in the third month, there were no significant differences among the treatment combinations having 10% concentration applied once a week of fse (a1b3) in terms of the number of branches with 14.33 (table 3 & figure 2). table 3 the average number of days to flower, number of flowers per hill, and number of branches per plant of peanut (arachis hypogaea l.) as influenced by the different seaweed extract concentrations and frequency of application in msu-sulu, barmm, philippines. fermented seaweed extract (treatments) number of days to flower* number. of flowers per hill* number of branches per plant* frequency of application (factor a) concentration (%) (factor b) 1 mo 2 mo 3 mo a1 once a week (1x / wk) b1 0 32.98a 5.29c 4.33d 7.33d 10.67f b2 5 31.07d 5.64b 5.00cd 8.00c 11.67d b3 10 31.04d 6.29a 5.67b 9.33b 14.33a b4 15 31.6cd 6.02ab 5.00cd 7.67d 10.67f b5 20 31.31cd 5.18c 4.67d 8.00c 11.00ef a2 twice a week (2x / wk) b1 0 32.62ab 5.29c 5.00cd 8.00c 11.00ef b2 5 31.00de 6.13a 5.67b 8.00c 12.00cd b3 10 30.82e 6.24a 6.33a 10.00a 14.67a b4 15 31.04d 5.69b 5.67b 8.33c 12.00cd b5 20 31.58cd 6.07a 5.33bc 8.33c 12.33c a3 once a month (1x / mo) b1 0 32.29b 5.27c 4.33d 7.67d 11.00ef b2 5 31.4cd 6.20a 4.67d 8.00c 11.67d b3 10 31.71c 6.24a 6.00ab 9.00b 13.67b b4 15 32.07bc 5.93ab 5.00cd 7.33d 10.67f b5 20 31.02d 6.13a 4.33d 7.67d 11.00ef anova (treatments) * * * ** ** cv (%) 2.40 7.57 12.25 7.90 6.02 *mean of the same letter/s within a column is not significantly different based on tukey's honestly significant difference (hsd) test fermented seaweed extract efficiency as foliar fertilizer – fatima sheirene m. marajuli and juliet c. bangi 81 the minimum number of branches per plant was observed in control plots or without application of fse and in treatment combinations with once-a-week and once-amonth application and 15% and 20% concentration (a1b4 & a3b4) with 10.67, (a1b5 & a3b5) and 11.00 branches, respectively. the rest of the treatment combinations did not vary in the number of branches per plant ranging from 11 to 13.67 throughout the growing season (table 3 & figure 2). yield parameters a. number of pods per plant the yield of peanuts as influenced by different frequency applications and concentrations of fse are presented in table 4. peanuts with fse applied twice a week with 10% concentration (a2b3) produced the highest number of pods with 26.13, while plants with once-a-week application of fse at 5% concentration (a1b2) had 25.3 pods but without significant differences with the once-a-month application of fse at 20% concentration (a3b5) with 24.66. the lowest number of pods per plot were from control plots (a2b1) with 15.72 and 15.75, respectively. there was no variation in the number of pods among treatments with once-amonth application with 0 to 15% concentration ranging from 17.29 to 19.90 pods per plant (table 4 & figure 3). the results of the study revealed peanut pod bearing was efficient in once and twice-a-week applications of fse at 5% to 10% concentration. the findings of the study illustrate that it only requires a lower concentration of fse to produce a higher number of pods, considering the ability of peanuts as a legume to form an association in the biological nitrogen fixation. the study by gomonet and cagasan (2020) revealed the average number of pods ranged from 18 to 37 per plant, which is comparable to the result of the study conducted. it is most likely the fse contains growth hormone that influences the physiological processes of peanuts at lower concentrations. seaweed extracts contain higher phosphorus and complement peanuts’ ability for symbiotic n fixation with rhizobia (smith et al., 1987). rathore et al. (2008) reported that fse contains enough phosphorus (of about 34 ppm) and a greater amount of ca and mg that affect the flowering and pod bearing of legumes. moreover, the ability of peanuts to produce pods is directly affected by management practices and the micro climatic condition of the growing area. b. number of seeds per pod the number of seeds per pod is shown in table 4. the treatment combination of twice-aweek application fse with 10% concentration (a2b3) got the highest number of seeds per pod with a mean of 2.30 but did not vary with other treatment combinations like twice-a-week application at 5% concentration (table 4 & figure 3). the lowest number of seeds per pod was from plants with once-a-month application at 20% fse (a3b5) with a mean of 2.00 seeds per pod, which is similar to control plots (a1b1) or without fse applied. 5,29 5,64 6,29 6,02 5,18 5,29 6,13 6,24 5,69 6,07 5,27 6,2 6,24 5,93 6,13 10,67 11,67 14,33 10,67 11,00 11,00 12,00 14,67 12,00 12,33 11 11,67 13,67 10,67 11 32,98 31,07 31,04 31,6 31,31 32,62 31,00 30,82 31,04 31,58 32,29 31,4 31,71 32,07 31,02 0 5 10 15 20 25 30 35 a1 b1 a1 b2 a1 b3 a1 b4 a1 b5 a2 b1 a2 b2 a2 b3 a2 b4 a2 b5 a3 b1 a3 b2 a3 b3 a3 b4 a3 b5 no. of flowers / hill no. branches per plant (3 mos) no. of days to flowering figure 2 number of days to flower, number of flowers per hill and number of branches per plant (3 mos after) of peanut (arachis hypogaea l.) as affected by the different seaweed concentration and frequenc biotropia vol. 31 no. 1, 2024 82 table 4 the average number of pods per plant, number of seeds per pod, yield per plot (kg) and total yield tons per hectare of peanut (arachis hypogaea l.) as affected by the different seaweed extract concentrations and frequency of application fermented seaweed extract (treatments) number of pods per plant* number of seeds per pod* yield per plot (kg)* total yield (tons/ha)* frequency of application (factor a) concentration (%) (factor b) a1 once a week (1x / wk) b1 0 15.75c 2.02d 0.70d 1.13d b2 5 25.30a 2.23a 1.16a 2.31ab b3 10 16.08c 2.15bc 0.73cd 1.18d b4 15 18.20c 2.17b 0.82bcd 1.39d b5 20 21.23ab 2.15bc 0.96abc 1.83bc a2 -twice a week (2x / wk) b1 0 15.72c 2.15bc 0.71d 1.20d b2 5 24.83ab 2.28a 1.21a 2.49a b3 10 26.13a 2.30a 1.12a 2.21ab b4 15 24.13ab 2.20ab 1.03ab 1.92ab b5 20 22.31ab 2.25a 0.95bc 1.78bc a3 once a month (1x / mo) b1 0 19.00c 2.18b 0.82bcd 1.48d b2 5 19.90bc 2.23a 0.86bcd 1.54cd b3 10 17.29c 2.22a 0.74cd 1.25d b4 15 19.39c 2.10c 0.84bcd 1.46d b5 20 24.66ab 2.00d 1.06ab 2.06abc anova (replication) * * * * cv (%) 30.85 4.40 32.06 43.79 *mean of the same letter/s within a column is not significantly different based on tukey's honestly significant difference (hsd) test the result of the study was greatly influenced by the frequent application of fse and its concentration at 5% to 10%. there was inconsistency in the result like the treatment applied once a week but showed similar results in the amount of concentration applied for both twice-a-week and once-a-month applications. it is noteworthy to consider that the frequent application (twice a week) of fse increased the seed per pod. the amino acids and other phytohormones are believed to have influenced the seed formation of peanuts. this conforms with the study of other researchers like prasedya et al. (2022) but is much better than the study conducted by gomonet and cagasan (2020) in which the number of seeds per pod ranged from 12 seeds only. this is quite lower than the result of the present study, which ranges from 2-3 seeds per pod. the fse nutrient composition influenced the increase in the seed formation of a peanut since it contains sufficient phosphorus, very high calcium, and magnesium, including micronutrients like zinc and iron necessary for seed formation (prasedya et al. 2022; lalog, 2011; mosaic compan, 2013). c. yield per plot (kg) and total yield (ton/ha) the yield of peanuts was affected by the frequency and concentration of fse application (table 4 & figure 3). plants applied twice a week with a 5% concentration of fse (a2b2) produced heavier pods per plot with a mean of 1.21 kg, followed by plots with once-a-week application at a 5% concentration of fse (a1b2) with 1.16 kg per plot. twice spraying of fse every week with 10% concentration (a2b3) resulted in 1.12 kg per plot, while once-a-month application with 20% concentration of fse (a3b5) produced 1.06 kg per plot. the rest of the treatment combinations have no variation in the yield per plot (table 4). the lowest yield per plot is from the control plots (a1b1) or without application of fse with a mean of 0.70 kg per plot (table 4 & figure 3). in terms of peanut yield per hectare, the highest yield was recorded from plants applied with fse twice a week at 5% concentration (a2b2) with a mean of 2.49 tons per hectare. however, it did not differ much with those plants applied once a week at 5% concentration (a1b2) with 2.31 tons/ha. whereas peanuts sprayed twice a week at 10% concentration (a2b3) fermented seaweed extract efficiency as foliar fertilizer – fatima sheirene m. marajuli and juliet c. bangi 83 produced 2.21 tons/ha while those applied once a month at 20% concentration (a3b5) got a lower yield of 2.06 tons/ha (table 4 & figure 3). however, the result of this study reveals the obtained yield of peanuts is much higher than the reported average yield in the country of about 800 to 1,000 kg per hectare based on the report of billen et al. (2015) as cited by gomonet and cagasan (2020). it can be attributed to the positive effect of using a twice-a-week application with a 5% concentration of fse to stimulate pod formation, enhance the quality and quantity of pods, and thus increase harvest in tons per hectare. based on the study of prasedya et al. (2022), fermented brown seaweed positively contains nutrients n, k, ca, mg, and b, which make it a potent fertilizer. the result implies that fse contains plant nutrient amendments with sufficient amounts of macroand micronutrients that will increase crop yield (rathore et al. 2008; lalog 2011). cost and return analysis table 5 shows the cost and return analysis using the return-on-investment (roi) computation of fse in peanut production with different frequency applications and concentrations. plants applied once a week with a 5% concentration of fse (a1b2) have the highest return on investment (roi) of 176.46%. however, it did not differ significantly with peanuts applied twice a week with a 5% concentration of fse (a2b2) at 170.15% returns. the lowest roi was found in peanuts applied twice a week with a 20% fse concentration of 74.79%. the rest of the treatment combinations have roi ranging from 80% to 156% (table 5 and figure 4). variation in roi results was due to the differences in yield and cost of production, specifically the cost of fermented seaweed extract at different concentrations. the study shows that using fse for peanut production can give better economic returns for the local farmers and can benefit by recycling those wastes into organic fertilizers, thereby reducing the cost of farm inputs. this can save farmers from total dependence on the use of synthetic chemical fertilizers. 0,7 1,16 0,73 0,82 0,96 0,71 1,21 1,12 1,03 0,95 0,82 0,86 0,74 0,84 1,061,13 2,31 1,18 1,39 1,83 1,2 2,49 2,21 1,92 1,78 1,48 1,54 1,25 1,46 2,06 0 0,5 1 1,5 2 2,5 3 0 5 10 15 20 25 30 a1 b1 a1 b2 a1 b3 a1 b4 a1 b5 a2 b1 a2 b2 a2 b3 a2 b4 a2 b5 a3 b1 a3 b2 a3 b3 a3 b4 a3 b5 p ea n u t y ie ld / p lo t (k g ) an d t o n s p er h ec ta re n u m b er o f p o d s p er p la n t treatments yield per plot (kg)* total yield (tons/ha)* number of pods per plant* number of seeds per pod* figure 3 number of pods per plant, number of seeds per pod, yield per plot (kg) and total yield tons per hectare of peanut (arachis hypogaea l.) as affected by the different seaweed extract concentrations and biotropia vol. 31 no. 1, 2024 84 table 5 cost and return analysis using return on investment (roi) computation on peanut production with different frequencies and concentrations of fse, msusulu fermented seaweed extract (treatments) total sales php total cost php roi (%) frequency of application (factor a) concentration (%) (factor b) a1 once a week (1x / wk) b1 0 111 113.54 97.76 cd b2 5 226.83 128.55 176.46 a b3 10 115.75 143.55 80.63 d b4 15 136.33 158.55 85.99 d b5 20 179.17 173.55 103.24 cd a2 twice a week (2x / wk) b1 0 117.5 113.55 103.48 cd b2 5 244.25 143.55 170.15 a b3 10 216.33 173.55 124.65 c b4 15 188.17 203.55 92.44 d b5 20 174.67 233.55 74.79 e a3 once a month (1x / mo) b1 0 145.33 113.55 127.99 b b2 5 150.83 117.3 128.59 bc b3 10 122.83 121.05 101.47 cd b4 15 142.92 124.8 114.52 c b5 20 201.75 128.55 156.94 ab anova (replication) ** ** cv (%) 43.79 42.35 *mean of the same letter/s within a column is not significantly different based on tukey's honestly significant difference (hsd) test 97,76 176,46 80,6385,99 103,24 103,48 170,15 124,65 92,44 74,79 127,99 128,59 101,47 114,52 156,94 0 20 40 60 80 100 120 140 160 180 200 r et u rn o n i n v es tm en t (% ) treatments a1 b1 a1 b2 a1 b3 a1 b4 a1 b5 a2 b1 a2 b2 a2 b3 a2 b4 a2 b5 a3 b1 a3 b2 a3 b3 a3 b4 a3 b5 figure 4 cost and return analysis in peanut production applied with different frequency and concentration of fermented seaweed extract in msusulu, barmm, philippines fermented seaweed extract efficiency as foliar fertilizer – fatima sheirene m. marajuli and juliet c. bangi 85 conclusions the results of the study indicate that fermented seaweed extract (fse) at various frequencies and concentrations significantly affects the growth and yield response of peanuts due to nutrient components of fse with sufficient amounts of both macroand micronutrients and phytohormones present. the application of fermented seaweed extract once and twice a week at 5% and 10% concentrations as liquid fertilizer was found best for peanut production by having a greater number of pods and consequently higher yield. in terms of return on investment, a once-a-week application with 5% seaweed extract concentration gave the highest profit due to lower costs of expenses and less labor incurred during peanut production. it is necessary to conduct further study on the effect of long-term application and higher concentration of fermented seaweed extract as foliar fertilizer on peanut production. given the effects of fse, it is necessary to determine the beneficial microorganisms (effective microorganisms) present in the fermented seaweed extract as potential microorganisms in the manufacture of bio-fertilizer. further study can be conducted to determine the influence of fse at higher concentrations or greater than 20% on peanut and other commodities like cereals (rice and corn) and vegetable 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hendrayanti1, nurul laksmi winarni1,2, eliza pricilia purba1, sarah tsabitudinillah1 and nunik sri ariyanti3 1department of biology, faculty of mathematics and natural science, universitas indonesia, depok 16424, indonesia 2research group of wildlife biology and sustainable landscape, universitas indonesia, depok 16424, indonesia 3department of biology, faculty of mathematics and natural science, ipb university, bogor 16680, indonesia received 25 march 2023 / revised 15 august 2023 / accepted 11 september 2023 abstract bryophytes are nonvascular plants that have simple structures that are sensitive to environmental changes, and they can, therefore be used as indicators of air quality. the presence of bryophytes in disturbed urban ecosystems, such as residential and industrial areas, indicates that their structures have adapted to survive in such areas. the objective of this study was to compare the bryophyte diversity and air quality indices between a residential area and an industrial area in jakarta. the research was conducted in the bona indah residential area in south jakarta and the jakarta industrial estate pulogadung (jeip) urban forest. sampling was carried out using the transect method in the residential area and the quadratic method in the urban forest on three different substrates, namely rocks or concrete, soil and tree trunks. the percentage of the epiphytic bryophyte cover was measured using a 10 × 10 cm subplot. voucher specimens were stored at the herbarium ui dep and herbarium ipb. twenty-one species of moss and three species of liverwort were found in the two locations. bryophytes were found on all the substrates in the residential area, but in the urban forest, they were found only on tree trunks and rock/cement substrates. based on the shannon–wiener index, although both locations had moderate bryophyte diversity, the residential area’s bryophyte diversity was higher than that of the urban forest. the index of atmospheric purity in the residential area was 4.3, indicating a high level of pollution, and it was 0.3 in the urban forest, showing that it was also very polluted. keywords: atmospheric purity, bryophyte, diversity index, urban area introduction an urban area is a region with high population density dominated by human activities such as trade, industry and education (freedman 2018). transportation and industry produce emissions such as carbon dioxide and nitrogen oxides that contribute to air pollution in urban areas. pollution causes ecological effects such as the greenhouse effect, harm to plant and animal health and temperature stress (kanawade et al. 2010). it is damaging to urban biodiversity because several species have become more sensitive to environmental stress from pollution and habitat fragmentation (uttara et al. 2012; szlavecs et al. 2011). jakarta is one of indonesia’s largest and most populous cities (permatasari et al. 2019). according to worldpopulationreview.com, in 2023, its population was estimated at 11 million. the city’s open green spaces have been reduced in area since 2008, leading to the urban heat island phenomenon (putra et al. 2021). an industrial estate is a built-up area with relatively high pollution levels due to transportation and industrial combustion (putra et al. 2021; lestari et al. 2022), while residential areas consist of buildings and vegetation and have less air pollution than industrial areas (putra et al. 2021). the air quality problem in jakarta causes *corresponding author, email: salamah@sci.ui.ac.id biotropia vol. 30 no. 3, 2023 356 environmental stress, harming species sensitive to high temperatures, drought and air pollution, such as bryophytes (oishi & hiura 2017; oishi 2019). bryophytes are non-vascular plants that are sensitive to environmental changes. the structure of bryophytes is simple; they have a thin cuticle layer so that water and minerals can be absorbed directly through the surface through diffusion. this causes the water content of bryophytes to depend on ambient humidity, making them poikilohydric. to survive in a dry environment, bryophytes develop some morphological adaptations for water retention and transportation (vanderpoorten & goffinet 2009; proctor & tuba 2002; bahuguna et al. 2013; he et al. 2016). based on these characteristics, bryophytes are suitable as air quality indicators, particularly in urban environments. epiphytic species such as bryophytes and lichens are known to be drought-sensitive organisms. they have been reported to be absent in urban ecosystems, although certain species are known to be drought tolerant (pescott et al. 2015; bahuguna et al. 2013). this suggests that epiphytic bryophytes can be indicators of air quality through the index of atmospheric purity (iap). correlations between species distribution, iap and pollutant concentrations can reflect the level of air pollution in a region (jiang et al. 2020; bahuguna et al. 2013). since the middle of the twentieth century, urban bryophyte-related air quality has been studied in subtropical regions, such as in european and asian (japan, china, india) countries and america and australia. the study of air quality monitoring is carried out using a survey and a phytosociological approach, namely iap, which was first used in 1970 by le blanc and rao (govindapyari et al. 2010). to show air quality, iap is identified by the frequency and coverage of epiphytic bryophytes and the species resistance factor (dymitrova 2009; vanderporrten & goffinet 2009; govindapyari et al. 2010). research on urban bryophytes in tropical regions, including in indonesia, is mostly based on inventories and community structures. studies of urban bryophytes related to air quality have never been conducted in indonesia. this study aims to compare bryophyte diversity and determine air quality in a residential area and an industrial urban forest area of jakarta using the iap. materials and methods study site the research was conducted from july to december 2020 in komplek taman bona indah, a residential area in south jakarta, and the jiep urban forest, an industrial area in east jakarta (figure 1). species identification was carried out at the taxonomy laboratory of the biology department in the faculty of mathematics and natural sciences, universitas indonesia. the voucher specimen was deposited in the herbarium ui-dep. figure 1 sampling locations. (a) taman bona indah residence, (b) jiep urban forest a b bryophyte diversity in urban area, jakarta indonesia – putrika et al. 357 methods bryophytes were collected from three substrate types, namely tree trunks, soil and rock or cement in both the taman bona indah residential area and the jiep urban forest. samples from the residential area were collected from six sites on a 50 m linear transect at the main roadside and from three sites on a 50 m linear transect in a park. each transect was divided into 30 × 2 m plots. the bryophytes in the urban forest were collected from eight 25 × 25 m plots. in each plot, five trees with a minimum of 10 cm breast-high diameter were selected. epiphytic bryophytes were sampled from four cardinal directions with an area of 10 × 10 cm2 from 0–2 m above ground level. the host tree species and bark type were recorded in a datasheet. in each plot, terrestrial bryophytes were collected from quadrats of 10 × 10 cm2, and their coverage was estimated using the same quadrats of 10 × 10 cm2. voucher bryophyte samples were identified using a stereo and light microscope at the herbarium ui-dep, where the voucher specimens were stored. the bryophyte diversity was analysed using the shannon–wiener index and the important value index. the shannon–wiener diversity index is calculated using the following formula: h′ = − ∑(𝑝𝑖 ln 𝑝𝑖) 𝑆 𝑖=1 , where: h’ = the shannon–wiener diversity index, s = number of species, pi = 𝑁𝑖 𝑁 , ni = relative cover species i, n = relative cover number of s species. the important value index was measured to analyse species’ dominance in each site, as follows (jiang et al. 2020): important value index = relative cover + relative frequency 2 atmospheric pollution was analysed using iap according to the following formula (le blanc 1971; jiang et al. 2020): iap = ∑ 𝑄 𝑥 𝑓 10 𝑛 𝑖 where: iap = index atmospheric purity, q = ecological index of species (average number of species that coexist with other species), f = frequency-coverage scale. results and discussion species composition a total of 24 bryophyte species were recorded in the residential area and the urban forest (three species of liverwort and 21 species of moss). based on the locations, the species richness in the residential area was higher than in the urban forest. there were three species of liverworts and 15 species of mosses in the residential area, while there were 11 species of mosses and no liverworts in the urban forest (table 1). the number of moss species in the urban areas was higher than liverworts (giordano et al. 2004; szűcs et al. 2017; godovićová et al. 2020; fastanti & wulansari 2021). most moss species are known to be drought tolerant, so they are commonly found in open areas (vitt et al. 2014). according to mukhia et al. (2019), a high abundance of epiphytic liverworts is related to low light intensity, ranging from 460–1900 lux. outside this light intensity range, liverwort abundance is significantly reduced. consequently, the abundance and species richness of liverwort in urban areas is lower than in natural habitats because of the difference in canopy coverage density and relative humidity. the families pottiaceae, calymperaceae and bryaceae were found in both sampling sites, with pottiaceae having the highest number of species (figure 2). there were eight species of pottiaceae in the urban forest (barbula consanguinea, barbula pseudo-ehrenbergii, hyophila apiculata, hyophila beruensis, hyophila involuta, hyophila javanica, trichostomum brachydontium and weissia edentula). in the residential area, there were five species of pottiaceae (barbula javanica, hyophila apiculata, hyophila beruensis, hyophila involuta and trichosteleum singapurense) (table 1). on the other hand, calymperaceae was represented by more species in the residential area than in the urban forest. bryaceae was only represented by one species in each of the sampling sites (figure 2). biotropia vol. 30 no. 3, 2023 358 table 1 bryophyte species composition in the residential area and the urban forest no. species family location residential area urban forest a. marchantiophyta 1 haplomitrium blumei haplomitriaceae √ 2 lejeunea cocoes lejeuneaceae √ 3 lejeunea eifrigii lejeuneaceae √ b. bryophyta 1 bryum apiculatum bryaceae √ 2 calymperes crassinerve calymperaceae √ 3 calymperes motleyi calymperaceae √ √ 4 calymperes tenerum calymperaceae √ √ 5 leucophanes octoblepharioides calymperaceae √ 6 fissidens atroviridis fissidentaceae √ 7 fissidens biformis fissidentaceae √ 8 isopterygium bancanum hypnaceae √ 9 isopterygium minutirameum hypnaceae √ 10 barbula consanguinea pottiaceae √ 11 barbula javanica pottiaceae √ 12 barbula pseudo-ehrenbergii pottiaceae √ 13 hyophila apiculata pottiaceae √ √ 14 hyophila beruensis pottiaceae √ √ 15 hyophila involuta pottiaceae √ √ 16 hyophila javanica pottiaceae √ 17 trichosteleum singapurense sematophyllaceae √ 18 trichostomum brachydontium sematophyllaceae √ 19 weissia edentula pottiaceae √ 20 taxithelium nepalense sematophyllaceae √ 21 splachnobryum indicum splachnobryaceae √ number of species 18 11 total number of species 5 figure 2 family composition of bryophytes in the residential area and the urban forest bryophyte diversity in urban area, jakarta indonesia – putrika et al. 359 pottiaceae is a family of mosses that is present in open and dry environments, particularly in urban areas (da costa 2015; jiang et al. 2020). they have a small size, erect stem or acrocarpous with a short turf life-form and dark green gametophore (figure 3). the members of this family have lanceolate leaves, which become crisped when dry to reduce transpiration due to environmental heat stress. in addition, some species of pottiaceae have ornamentation on the leaf cell surface, such as papillae. the interstices between papillae are necessary to increase water conduction on the leaf surface (zander 1993; kou et al. 2014; glime 2017). furthermore, they can prevent water loss, making them more successful in a drought. calymperaceae is an epiphytic moss that was often found in both sampling locations. some species of this family have gemmae at the apex of the leaves as asexual reproductive organs, a strategy that helps them survive in unfavorable urban environments. the presence of cancellinae regions (hyaline basal leaf cells) in calymperaceae is another strategy of this family to retain more water in dry environments (figure 4) (glime 2017). lejeuneaceae, a cosmopolite family, were the only liverworts in the sampling sites found on the exposed roots of large palm trees 10–20 cm above the ground. this tree root substrate had a low light intensity at around 250 lux, causing a more humid microhabitat. liverworts generally grow in moist and shady habitats, while epiphytic liverworts grow on large or old trees (vanderpoorten & goffinet 2009; mukhia et al. 2019). figure 3 pottiaceae. (a) acrocarpous and short turf life-form. (b) papillae on the leaf surface figure 4 calymperaceae. a. the gemmae on the leaf apex. b. cancelinae a b biotropia vol. 30 no. 3, 2023 360 the type of substrate with the highest species richness was rock/cement (figure 5). cement paths and paving blocks were common kinds of rock and cement substrate in both sampling locations. according to floyed and gibson (2012), cement pavement always has crevices and cracked structures that provide a sheltered area for bryophyte colonisation. these surfaces trap soil, water and dust from the atmosphere to support bryophyte growth, provide shelter from mechanical disturbance and retain moisture for a longer period than surrounding surfaces. this substrate was dominated by pottiaceae, including hyophila and barbula. those genera are common in disturbed areas, such as urban ecosystems (zander 1993). terricolous bryophytes were found only in the residential area and absent in the urban forest (figure 5). the soil type in the latter may not have supported bryophyte growth. according to lubis et al. 2013, the jiep urban forest has a podosol soil type with low moisture and nutrient content. in addition, piles of plastic waste were found in some of the sampling plots. andayaningsih et al. (2013) also reported finding terrestrial pteridophytes in this location, indicating that jiep’s environmental conditions are unsuitable for plants sensitive to dry conditions, such as terrestrial bryophytes and pteridophytes. the number of epiphytic bryophytes in the residential area was higher than in the urban forest (figure 5). air pollution, humidity, temperature and phorophyte variation were limiting factors. the minimum frequency of epiphytic moss in urban areas can be determined from the percentage of host trees covered with epiphytic moss. twenty-four percent of phorophytes (trees used as hosts of bryophytes) were used as hosts in the residential area. however, only 8% of the urban forest was used by bryophytes as hosts (figure 6), which shows that industrial areas are more vulnerable to the growth of sensitive bryophyte species than residential areas. urban forests are surrounded by dense industrial activity and have low vegetation density, which affects microclimate conditions, causing the comfort index in the area to be lower than usual (syihabuddin et al. 2020). figure 5 number of bryophyte species on three substrates in the residential area and the urban forest bryophyte diversity in urban area, jakarta indonesia – putrika et al. 361 figure 6 presentation of phorophytes with epiphyte bryophytes the characteristics of the host trees also influenced the presence of epiphytic mosses. eusideroxylon zwageri was the only tree species colonized by bryophytes in the urban forest. in the residential area, there were three tree species hosting bryophytes, mangifera indica, swietenia mahagoni and roystonia regia. these trees had rounded canopies and a fissured bark texture, and the ph of the bark varied between 6 and 6.8. the fissured texture had hollow cracks, resulting in a more stable surface for epiphytic bryophytes. it provided a more humid and shaded habitat by retaining water and dust from the atmosphere (vanderpoorten & goffinet 2009). meanwhile, because of the palm trees’ smooth bark, there were no epiphytic bryophytes on their bark surface, although they were found on the exposed palm root surfaces near the soil, since these were more humid and shaded than the trunk. community structure and index of atmospheric purity based on the type of substrate, the bryophyte diversity index in both the residential area and the urban forest ranged from 0 to 2.06. this shows that the bryophyte diversity was very low. no bryophytes were found on the soil substrate in the urban forest, and the lowest diversity index was found in the epiphytic bryophyte community in that location (table 2). saxicolous bryophytes on rock/cement substrate in both the residential area and the urban forest had a higher diversity index than those growing on other substrates. this shows that in urban environments, saxicolous bryophytes are more tolerant than the bryophytes on other substrates. the iap in the residential area was 4.3, and in the jiep urban forest, it was 0.3 (table 2). according to the iap criteria of le blanc and slover, the air pollution in both locations was found to be highly polluted, in the range of 1–5. although the level of specific pollutants was unknown, it can be estimated by iap that the urban forest was more polluted than the residential area in south jakarta. air pollution and microclimate have a negative impact on sensitive species that can reduce iap values (jiang et al. 2020), which was shown by the lower number of epiphytic bryophytes in the urban forest. epiphytic bryophytes are not found in urban areas with high pollution levels (fudali & żołnierz; 2019: 73; jiang et al. 2020). one of the factors affecting epiphytic bryophyte diversity is traffic density. the absence of liverworts in the urban forest also indicates their sensitivity to industrial environments. the results showed that the epiphytic mosses with the highest important value index were calymperes motley and c. tenerum, while the saxicolous bryophytes with the highest important value index were hyophylla javanica and h. involuta in both locations. in addition, the terricolous bryophyte with the highest important value index was fissidens biformis (table 3). 0% 5% 10% 15% 20% 25% 30% residential area industrial area urban forest % p h o ro p h y te location biotropia vol. 30 no. 3, 2023 362 table 2 diversity index and index of atmospheric purity in the residential area and the urban forest location residential area urban forest tree trunk soil rock or cement tree trunk soil rock or cement h' 1.73 1.19 2.06 0.52 – 1.59 iap 4.3 0.3 table 3 the highest important value index of bryophytes substrate residential area urban forest species important value species important value tree calymperes motleyi 22.02 calymperes motleyi 31.25 calymperes tenerum 10.71 calymperes tenerum 26.69 calymperes crassinerve 7.74 rock or cement substrate hyophila javanica 37.67 hyophila involuta 15.54 hyophila involuta 21.06 hyophila javanica 12.50 hyophila apiculata 19.22 trichostomum brachydontium 5.26 soil fissidens biformis 35.39 fissidens atroviridis 14.97 isopterygium bancanum 10.10 some species of calymperes have also been reported as dominant bryophytes in other urban areas, such as at the universitas indonesia campus (putrika et al. 2017) and an urban agroforest in nigeria (ezukanma et al. 2019). this genus is also commonly found in home yards and oil palm plantations in the giam siak biosphere reserve (fastanti et al. 2013). from this information, we can assume that some species of calymperes are sun epiphyte bryophytes, since they always grow in open areas and belong to droughttolerant species. the highest important value index in both locations was dominated by acrocarpous species, turf and cushion life-forms, which were small, at about 0.4–7 mm. drought-tolerant moss species are commonly small and have erect stems (printanakul & jampetong 2020). in addition, turf and cushion life-forms tend to have dense leaf arrangements, which allows them to optimize water movement and storage as well as prevent excessive transpiration through a dry environment (spitale et al. 2020). turf and cushion life-forms are commonly found growing terrestrially in open habitats with high light intensity (kürschner 2004: 105; glime 2017: 4, 5, 7). according to the abiotic data, in the residential area and urban forest, the daily temperature and relative humidity ranges were 29–32°c and 50%– 61%, respectively. this condition may explain why the diversity of bryophytes in both sampling sites was low. the optimum temperature for bryophyte growth is about 15–25°c (frahm 2003; he et al. 2016). some bryophyte species in tropical lowland forests cannot survive at 27– 30°c. temperatures above 30°c can inhibit bryophyte photosynthesis (he et al. 2016). furthermore, only specific bryophyte species can survive in urban areas. conclusion bryophyte diversity in both the residential area and the jiep urban forest was dominated by acrocarpous mosses such as calymperes and hyophila. the residential area had higher bryophyte diversity than the urban forest. the iap in the urban forest was lower than that in the residential area. however, both locations fell into the category of very heavily polluted areas. further research is needed to compare the iap values to the air pollutant quantity to confirm the pollution levels in both study sites. acknowledgment we thank to research and community service (rpm) of faculty mathematics and natural science universitas indonesia for financial bryophyte diversity in urban area, jakarta indonesia – putrika et al. 363 support through riset penugasan fmipa grants under contract number 025/un2.f3.d/dppm.00.02/2022. references andayaningsih d, chikmawati t, sulistijorini. 2013. keanekaragaman tumbuhan paku terestrial di hutan kota dki jakarta. 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[cited 2022 october 10]; 483:11. available from: https://doi.org/10.1016/ j.foreco.2020.118767 https://bmcecol/ https://www.sciencedirect.com/science/article/abs/pii/s016920461730172x https://www.sciencedirect.com/science/article/abs/pii/s016920461730172x https://link.springer.com/article/10.1007/s11355-018-0356-z https://link.springer.com/article/10.1007/s11355-018-0356-z https://cmuj.cmu.ac.th/cmu_journal/cmu_journal.php https://cmuj.cmu.ac.th/cmu_journal/cmu_journal.php https://www.sciencedirect.com/science/article/pii/s1470160x19307939?via%3dihub https://www.sciencedirect.com/science/article/pii/s1470160x19307939?via%3dihub https://ojs/ https://doi.org/10.7872/cryb/v38.iss2.2017.153 https://doi.org/10.1016/j.foreco.2020.118767 https://doi.org/10.1016/j.foreco.2020.118767 05. 1675 rao m. and dhadse sharda.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 sri-01. 726-layout.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 the southeast asian journal of tropical biology vol. 31 no. 3, 2024: 422 431 doi: 10.11598/btb.2024.31.3.2320 issn: 0215-6334 | e-issn: 1907-770x 422 article higlights • the study first reported taxonomic study of dillenia sp. in malita, davao, occidental philippines. • this is the first reported findings of the bacterial activity of the bark extract of dillenia sp. against escherichia coli. article information received 8 august 2024 revised 22 september 2024 accepted 7 october 2024 reviewer: eucharia chizoba ezigbo & budi eko wahyudi *corresponding author, e-mail: leonellumogdang@gmail.com research article taxonomic description and antibacterial activity of dillenia sp. against escherichia coli shiena mae c. martina1, gene patrish c. cabalquinto1, leonel p. lumogdang2* 1institute of teacher education and information technology, southern philippines agri-business and marine and aquatic school of technology (spamast) fishing village, malita, davao occidental province 8012, philippines. 2microbiology laboratory, research and laboratory services center (rlsc) southern philippines agri-business and marine and aquatic school of technology (spamast) fishing village, malita, davao occidental province 8012, philippines introduction the world health organization (who) highlights a global health threat, i.e., the increasing prevalence of multi-drug-resistant escherichia coli strains (who 2023). this bacterium, commonly found in human or animal waste, causes severe symptoms, such as diarrhea, vomiting, stomach pains, and cramps (colina 2021). escherichia coli is a part of the human gut microbiota and a leading cause of various infections, from urinary abstract the study aimed to provide a taxonomic description of dillenia sp. and evaluate its antibacterial activity against escherichia coli. the taxonomic classification was carried out by using taxonomic keys and field guides. the antibacterial activity of dillenia sp. was screened using the kirby-bauer disk diffusion assay. taxonomic description resulted from this study confirmed dillenia sp. to be an angiosperm flowering seed plant native to the philippines, characterized by its round-shaped growth and evergreen foliage. the antibacterial activity of dillenia sp. bark extracts against e. coli was assessed using different concentrations (100 ppm, 500 ppm, and 1,000 ppm) and different extraction methods (decoction, ethanolic, and aqueous). significant differences in the antibacterial activity observed among treatments within the decoction extracts were observed, indicating varying antibacterial activities. post-hoc analysis revealed that concentrations of 100 ppm and 500 ppm were significantly more effective in inhibiting bacterial growth compared to concentration of 1,000 ppm within the decoction treatment. each treatment showed distinct patterns of antibacterial activities, with ethanolic and aqueous extracts displaying relatively consistent activities across different concentrations, while the decoction extract exhibited concentrationdependent antibacterial activities. the study provided evidence of the antibacterial potential of dillenia sp. bark extracts against e. coli, with implications for further research and potential practical applications in combating bacterial infections. recommendations include exploring lower concentration ranges, considering different treatment formulations, investigating combination treatments, and assessing antimicrobial mechanisms to enhance efficacy and guide the development of novel antibacterial drugs. keywords: antibacterial activity, dillenia, endemic, philippines tract infections to more severe systemic conditions. the emergence of multi-drug-resistant strains emphasized the urgent need for new antibacterial agents (mueller & tainter 2023). dillenia sp., a plant endemic to the philippines, has emerged as a potential source of novel antibacterial compounds. this interest stems from the presence of diverse phytochemicals within the plant, known for their activity against various bacterial strains (dante et al. 2019). however, there remain uncertainties https://doi.org/10.11598/btb.2024.31.3.2320 taxonomy and antibacterial activity of dillenia sp. against e. coli martina et. al. 423 regarding the exact taxonomic classification of dillenia philippinensis, highlighting the need for further investigation. the bark of d. philippinensis presents a particularly intriguing avenue for exploration due to its rich phytochemical profile (sabandar et al. 2017). furthermore, the first description of a dillenia sp. in the philippines is attributed to rolfe (stuart 2020). interestingly, there are currently no documented occurrences of dillenia within malita municipality, davao occidental province. despite the traditional use of dillenia sp. for its anti-microbial, anti-inflammatory, analgesic, and antidiabetic properties, there was a limited systematic exploration into the taxonomic classification and antibacterial activity of dillenia sp. against clinically significant bacteria like escherichia coli (patra 2012). the study of ragasa et al. (2009) reported that the air-dried leaves of dillenia philippinensis, commonly known as “katmon” contain betulinic acid and 3-oxoolean-12-en-30-oic acid based on assessment by using silica gel chromatography. those 2 acidic compounds exhibited moderate activity against the fungus candida albicans and slight activity against the bacteria e. coli, pseudomonas aeruginosa, staphylococcus aureus, and bacillus subtilis. the antibacterial activity of dillenia sp. leaves have been reported, but studies on the antibacterial properties of the bark extract are still lacking. therefore, this research aimed to elucidate the taxonomic identity of dillenia sp. and evaluate its ability to inhibit e. coli bacteria. the specific focus was on the potential of dillenia sp. bark extract as a source for developing new and effective antibacterial agents (stuart 2020). materials and methods in this study, the descriptive aspect focused on the taxonomic classification of dillenia sp. the experimental aspect evaluated the antibacterial activity of dillenia sp. against e. coli. plant extracts were obtained from the collected samples and subjected to antimicrobial disk susceptibility testing using standard laboratory techniques. research locale samples of dillenia sp. were collected at sitio maylaya, barangay kilalag, malita municipality, davao occidental province (6°21” n; 125°29” e). sitio maylaya had a total land area of 20 ha and is a remote area in malita municipality, located along the upland area of the municipality, and was chosen due to the occurrence of mature dillenia sp. in the area. figure 1 base map of malita municipality, davao occidental province, philippines pre-implementation phase necessary permits for taking samples of dillenia sp. were secured from pertinent authorities and landowners. approval from the department of environment and natural resources (denr), research and laboratory services center (rlsc), landowner, barangay captain, and the dean of the institute of teacher education and information technology (iteit) were also secured. collection and identification of dillenia sp. samples of dillenia sp. bark were collected from mature trees located in sitio maylaya. the samples were carefully excised from healthy bark sections using sterilized tools, ensuring minimal damage to the plant. the collected bark samples were clearly labeled with the date, location, and any other relevant information. subsequently, the collected bark samples were washed thoroughly to remove any dirt or contaminants and then allowed to air dry. the air-dried bark samples were then ground into fine powder by using a blender. biotropia vol. 31 no. 3, 2024 424 measurements of dillenia sp. tree were taken to determine the taxonomy of the plant, which include tree height, growth form, trunk description, leaf sizes, leaf shapes and arrangement, leaf margins, leaf venation and petiole, flower color and symmetry, fruit color, fruit classification, fruit type, and roots. these measurements were used to make comparisons in relation to the taxonomic keys. a field guidebook on native trees within the quirino forest landscape (carig 2020), published literature by stuart (2020), and published literature on philippine dillenia dillenia philippinensis (orate & lafrankie 2011) were used to determine dillenia sp. taxonomy. preparation of culture media and processing of dillenia sp. bark ls broth medium of 17.8 g was dissolved in 500 ml distilled water and mixed thoroughly using magnetic stirrer. six test tubes were each filled with 10 ml of the prepared broth. the test tubes were autoclaved at 121 °c for 15 minutes and then allowed to cool. in the meantime, alcohol lamp, wire loop, and contaminated water were prepared and placed in a biosafety cabinet. the wire loop was heated in the alcohol lamp until turning red, then was allowed to cool for 5 seconds before being placed in the contaminated water. the wire loop with contaminated water was then placed inside the test tube that had been filled with ls broth medium. this procedure was repeated for the other test tubes. these test tubes containing samples were placed in an incubator at 37 °c for 48 hours. dillenia sp. bark was powdered using a mortar and pestle and prepared 100 g powdered bark sample. three methods of bark extraction were then carried out: (1) decoction, (2) aqueous, and (3) ethanolic. to prepare bark decoction, 40 g of bark were used for every 200 ml of water which was boiled for 20 minutes. after boiling, bark decoction was allowed to cool, and the extract was filtered using filter paper. subsequently, the extract was evaporated using the wash bath and allowed to cool before further analysis. in preparing the aqueous extract, 40 g of powdered bark was mixed with 200 ml of distilled water for one and a half minutes (1:30 min). the mixture was then simmered on low heat for 15-20 minutes until the desired concentration was achieved. afterward, the bark extract was evaporated using water bath and allowed to cool slightly before further processing. the ethanolic extract was prepared by pouring 200 ml of ethanol over 40 g of powdered bark until completely soaked. the bark was allowed to soak in ethanol for 24 hours to 72 hours, with gentle agitation of the container from time to time to facilitate extraction. the ethanol extract was filtered with filter paper to remove plant debris, and the filtered extract was collected in a clean vial. subsequently, the extracted solution was stored in a clean erlenmeyer flask and allowed to evaporate in a water bath. antibacterial assessment escherichia coli, as test organism, was cultivated by using standard protocol following ogodo et al. (2022). mueller-hinton agar was used to cultivate e. coli. the mueller-hinton agar was prepared following the supplier’s instructions (tankeshwar 2013). there were 4 treatments of dillenia sp. bark extracts concentrations: 1) 100 ppm; 2) 500 ppm; 3) 1,000 ppm, and 4) control group, with 3 replicates for each treatment. antibacterial petri dishes were divided into four parts and labeled according to concentration, replicates, and treatment. the antibacterial petri dishes were then soaked in the aqueous, ethanolic and decoction solutions with a specific concentration of the dillenia sp. extract, allowing the disks to air dry in a sterile environment. the plates were then incubated at 37 °c for 24 hours, allowing the bacteria to grow and potentially be inhibited by the extract. zones of inhibition were observed after incubation. ethical guidelines ethical guidelines were practiced in obtaining the samples of dillenia sp. from the research taxonomy and antibacterial activity of dillenia sp. against e. coli martina et. al. 425 locale by securing the necessary permits from the denr, barangay captain and the landowners. decontamination protocol in laboratory was strictly adhered to in preparing the test organism and the antibacterial assessment assay. data analysis the zone of inhibition was calculated using the formula following bhargay et al. (2016): πr² where: r = radius of the inhibition zone statistical analysis mean, standard deviation, minimum, maximum, and quartiles for each concentration level were used in the study. one-way anova and post-hoc test were used to interpret statistical significance of the different treatments. the statistics were computed using the spss software. results and discussion taxonomic study on dillenia sp. taxonomic study conducted in this research showed that dillenia sp. is a flowering seed plant with two cotyledons (dicotyledons), classified within the division of angiosperms. dillenia sp. grows as a small tree, reaching heights between 6 m and 17 m. the tree is autotrophic with rounded shape, contributing to its aesthetic appeal. dillenia sp. is native to the philippines and thrives in terrestrial habitats within tropical climate zones. dillenia sp. is a tree reaching a height of 6 m to 15 m, smooth or nearly so (stuart 2020; carig 2020). the trunk of dillenia sp. exhibits a stout and sturdy demeanor, often reaching considerable heights in its natural habitat (fig. 2). bark of dillenia sp. appears as shallow fissures and showcases hues ranging from greyish-brown to reddish-brown tones (fig. 3). the bark not only provides a protective layer for the tree’s internal tissues, but also contributes to its aesthetic appeal. the shallow fissures add texture to the bark, giving it a rugged yet visually intriguing appearance. the leaf structure of dillenia sp. is characterized by a leathery texture, imparting a robust and durable quality to withstand various environmental conditions. their surface exhibits a glossy sheen, adding to their visual appeal and potentially serving functional purposes, such as water repellency or light reflection. the shape of dillenia sp. leaf ranges from ovate to elliptic or oblong-ovate, showcasing a broad yet elongated form that enhances their efficiency in capturing sunlight for photosynthesis, with lengths typically spanning from 12 cm to 25 cm (fig. 4). along the margins of the leaf, coarse teeth are present, providing a serrated appearance. dillenia sp. tree produces bisexual flowers with radial symmetry (fig. 5). the flowers exhibit a variety of colors, including red and white. their large size, ranging from 6 cm to 8 cm in diameter, commands attention and makes them a prominent feature in the surrounding foliage. the petals of dillenia flowers are soft and fleshy to the touch. this softness not only adds to the tactile allure of the flowers, but also underscores their ephemeral beauty. mature fruit of dillenia sp. exhibits a vibrant green coloration, signaling their ripeness and readiness for dispersal (fig. 6). the fruit is classified as simple, fleshy fruits, possessing a single-seeded structure encapsulated within a soft and pliable outer layer. the morphology of the studied dillenia sp. collected in malita municipality, davao occidental province, philippines, and the dillenia sp. reported by stuart (2020) shows a similarity in terms of tree height of 6 m to 15 m. the leaves are leathery, shining, ovate, elliptic or oblong-ovate, with 12 cm to 25 cm leaf length, and coarsely toothed at the margins. the flowers are white, large, soft, fleshy, and green, with diameter of 6 cm to 8 cm, having large fleshy sepals tightly enclosing the true fruit. based on the morphology, these characteristics are key features of dillenia sp. stuart (2020) further corroborated its taxonomic classification and distinctive characteristics. biotropia vol. 31 no. 3, 2024 426 figure 2 tree of dillenia sp. figure 3 trunk and bark of dillenia sp. taxonomy and antibacterial activity of dillenia sp. against e. coli martina et. al. 427 figure 4 leaf structure of dillenia sp. figure 5 flower structure of dillenia sp. biotropia vol. 31 no. 3, 2024 428 antibacterial activity of dillenia sp. bark extract the highest zone of inhibition (zoi) observed in this study was 42.65 mm² at 100 ppm by using decoction extraction method, while the lowest was 22.96 mm² at 1,000 ppm by using ethanolic figure 6 fruit of dillenia sp. extraction method (table 1). the lower the concentration of the extract, the larger the zone of inhibition observed in this study. this variation indicated a concentration-dependent response, with higher concentrations of dillenia sp. bark extract generally leading to smaller zoi. table 1 zone of inhibition (zoi) of different concentrations of dillenia sp. bark extract against escherichia coli concentration zone of inhibition (zoi) of each bark extract concentration and extraction method replicates ethanolic (mm²) aqueous (mm²) decoction (mm²) control (mm²) 100 ppm mean of zoi r1 r2 r3 40.69 60.79 8.01 36.50 42.99 50.24 13.50 35.58 50.24 63.59 14.13 42.65 21.23 58.06 11.30 30.20 500 ppm mean of zoi r1 r2 r3 33.17 50.24 10.99 31.47 38.47 50.24 13.82 34.18 50.24 50.24 13.19 37.89 52.78 60.79 14.13 42.57 1,000 ppm mean of zoi r1 r2 r3 19.63 39.19 10.05 22.96 38.47 50.24 10.99 33.23 50.24 55.39 13.19 39.61 50.24 38.47 12.56 33.76 taxonomy and antibacterial activity of dillenia sp. against e. coli martina et. al. 429 according to the study of wani et al. (2022), microorganisms have an extremely short generational span, are able to quickly adapt to survive in high levels of antimicrobials and are able to pass the resistance around in a population. this suggests that dillenia sp. bark extract has a doseindependent antibacterial effect on e. coli. analysis conducted in this study suggested that multiple factors influence the zoi. the lower concentrations tended to exhibit larger zoi, possibly due to increased active molecule presence inhibiting bacterial growth. additionally, methods of extraction (ethanolic, aqueous, decoction) may affect extract potency, influencing the zoi. moreover, other factors, such as bacterial growth phase, culture medium, and measurement method (e.g., agar well diffusion assay) could also be an influencing factor. the study of gajic et al. (2022) demonstrated a similar concentration-dependent relationship between plant extract concentration and antibacterial activity. furthermore, garcia et al. (2021) highlighted the impact of extraction solvent on extract potency, aligning with our observations. statistical analysis indicating significant differences among zones of inhibition (zoi) of different concentrations of dillenia sp. extracts against e. coli is presented in table 2. table 2 statistical analysis indicating significant differences among zones of inhibition of different concentrations of dillenia philippinensis extracts against e. coli extracts concentration (ppm) mean p value interpretations decoction 100 42.65a 0.00 significant 500 37.89a 1,000 39.61b aqueous 100 35.58 0.99 not significant 500 34.18 1,000 33.23 ethanolic 100 36.50 0.74 not significant 500 31.47 1,000 22.96 control 100 30.20 0.80 not significant 500 42.57 1,000 33.76 note: numbers having the same superscripts has no significant difference at p value 0.05. the analysis of variance (anova) results indicated significant differences among different concentrations of dillenia sp. bark extract within the decoction extraction method. this suggested that at least one extract concentration had a significantly different mean affecting the zone of inhibition compared to the others. these subsequent post-hoc test results confirmed that the antibacterial activity indicated by zone of inhibition varied significantly with different concentrations of the extract. specifically, the lower concentrations (100 ppm and 500 ppm) in the decoction extraction method produced significantly larger zones of inhibition compared to the 1,000 ppm treatment. this finding suggested that the antibacterial activity of the extracts is concentration-dependent, with 100 ppm and 500 ppm exhibiting stronger antibacterial effects against e. coli than the 1,000 ppm treatment. this suggested a non-linear relationship between concentration and antibacterial activity, wherein higher concentrations may not necessarily yield stronger inhibition. the plausible explanations for this phenomenon could include saturation effects, where the antibacterial agents may reach a plateau in their effectiveness at higher concentrations or even potential cytotoxicity at excessively high doses. biotropia vol. 31 no. 3, 2024 430 according to the study of aladejana et al. (2024), the antibacterial activity of dillenia sp. could be attributed to the presence of bioactive compounds, such as tannins, flavonoids, alkaloids, and phenolic compounds. these compounds possess antimicrobial properties that inhibit the growth of bacteria through various mechanisms. tannins are known for their ability to precipitate proteins, disrupting the cell membranes of bacteria and leading to cell death. similarly, flavonoids and phenolic compounds exhibit antimicrobial effects by interfering with bacterial enzymes and metabolic processes crucial for their survival and replication. some alkaloids found in dillenia sp. likely contributed to its antibacterial activity by disrupting bacterial cell membranes or inhibiting specific cellular processes. moreover, the antibacterial properties of dillenia sp. are inherent in this genus. previous study conducted by yakop et al. (2020) reported that dillenia suffruticosa contains diethyl ether and ethyl acetate extracts exhibited by higher total phenolic content (tpc) and total flavonoid content (tfc), as well as superior antioxidant activities compared to other fractions. furthermore, the methanol extract and its fractions displayed antibacterial activity against staphylococcus aureus, with the diethyl ether fraction demonstrating comparable efficacy to the standard antibiotic streptomycin. in contrast, inhibition against bacillus subtilis was observed only in certain fractions, while no inhibition was detected against e. coli and pseudomonas aeruginosa (yakop et al. 2020). in a study by ilori et al. (2022), on dillenia indica and ficus exasperata extracts, it was found that both species exhibited antibacterial activity against e. coli, s. aureus, and streptococcus pyogenes. however, the fact that dillenia sp. demonstrated antibacterial activity against e. coli at different concentrations, suggesting its potential as a source of bioactive compounds, was first reported in this study. conclusion this study confirmed that dillenia sp. is a flowering plant indigenous to the philippines, elucidating key characteristics, such as morphology and habitat. dillenia sp. investigated in this study was identified as dillenia philippinensis, which bark extracts possess antibacterial activity against escherichia coli, with varying effectiveness depending on concentration and treatment method. lower concentrations generally exhibited a larger zone of inhibition (zoi), indicating a concentration-dependent response. statistical analysis revealed significant differences among different concentrations of bark extract in the decoction extraction method, indicating varying mean effects. post-hoc test further confirmed that concentrations of 100 ppm and 500 ppm were significantly more effective than 1,000 ppm in the decoction extraction method, suggesting an optimal range of effectiveness between 100 ppm and 500 ppm. the study highlighted the potential of dillenia sp. bark extracts as antibacterial agents against e. coli, with concentrations between 100 ppm and 500 ppm. the findings from this study support the utilization of dillenia sp as a source of alternative medicine by the indigenous people in the philippines. acknowledgment the authors wish to extend their sincere gratitude to the advisory committee led by jea farida guroalim, joeper abella, and professor rommel cabalquinto for thoroughly reviewing the manuscript throughout the entire duration of the study. moreover, the authors are also thankful to the language editor, professor mae fritz moranta, for reviewing the grammar and 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http://www.stuartxchange.org/katmon.html http://www.stuartxchange.org/katmon.html https://microbeonline.com/mueller-hinton-agar/ https://microbeonline.com/mueller-hinton-agar/ _goback 1200.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 page 14 page 15 1. imas sukaesih sitanggang et al.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 biotropia vol. 28 no. 2, 2021: 141 148 doi: 10.11598/btb.2021.28.2.1274 141 biocontrol potential of endophytic aspergillus spp. against fusarium verticillioides ron patrick cuagdan campos* and james kennard sanz jacob department of biological sciences, college of arts and sciences, isabela state university, echague 3309, isabela, philippines received 15 june 2019/accepted 8 september 2019 abstract the soil-borne fungus fusarium verticillioides is the causal agent of ear, stalk and root rot of maize resulting in severe reduction in yields and quality of infected products. endophytic fungi have been reported as potential candidates in controlling pathogens since they are considered strong plant mutualists that confer disease resilience to their host. the present study was carried out to determine the in vitro antagonistic activity and biocontrol potential of endophytic aspergillus spp. associated with plectranthus amboinicus leaves against f. verticillioides. three fungal endophytes from the genus aspergillus, namely; a. tamarii, a. terreus and a. niger were isolated and identified from the leaves of p. amboinicus,. the fungal isolates were tested for their antagonism against f. verticillioides in dual culture plates. results indicated that the aspergillus endophytes can restrict the growth of f. verticillioides through varying mechanisms of antagonism. a. niger inhibited f. verticillioides by 47.37%, followed by a. tamarii (41.02%) and a. terreus (27.91%). dual culture observations revealed that a. tamarii and a. niger antagonized the growth of f. verticillioides via overgrowth mechanism while a. terreus employed antibiosis to restrict the pathogen. these varying degrees of antagonism by the aspergillus endophytes exhibited their potential as biocontrol agents and source of bioactive compounds. keywords: aspergillus, biocontrol, endophytic fungi, fusarium, rot introduction fusarium verticillioides is one of the most commonly reported soil-borne fungal pathogens infecting maize (abbas et al. 1998; bacon & hinton 1996). it is the causal agent of ear, stalk and root rot of maize. this fungus also produces secondary metabolites such as fumonisins that accumulate in maize kernels, consequently causing severe reductions in yields and quality of the products (leyva-madrigal et al. 2017; chu & li 1994). in order to maintain the abundance and quality of agricultural products world-wide, these plant pathogens need to be controlled and managed appropriately (pal & gardener 2006). currently, chemical fungicides are the most effective agents in preventing the infection of f. verticillioides. however, efforts to control postharvest diseases employing synthetic chemical control agents pose danger to the environment as they affect soil microorganism diversity (cardoso et al. 2010). endophytic fungi are microorganisms that inhabit internal plant tissues, usually involving metabolic interactions without apparent symptoms (bacon & white 2000; petrini 1991; wilson 1995). growing evidences from several studies indicate that endophytes are found in all plants and are extremely abundant and diverse (arnold et al. 2000). endophytic fungi are believed to be strong plant mutualists which can produce increased resilience against pests and plant pathogens (carroll 1988). since these microorganisms are systemically distributed throughout the host via metabolic translocation, they are noteworthy candidates for biological control (rai et al. 2007). studies have been done on the antagonistic mechanisms and actions, particularly the efficacy, of many endophytic fungi for their biocontrol potential against different plant pathogens (rahman et al. 2009). furthermore, considering their long-lasting effects and not requiring repeated periodic *corresponding author, email: rpcampos023@gmail.com biotropia vol. 28 no. 2, 2021 142 application, the use of fungi for biocontrol has more advantages than chemical fungicides (okigbo & ikediugwu 2000). several studies recorded that aspergillus species acted as biological control agents against fungal pathogens (fusarium oxysporum, pythium spp. and sclerotinia sclerotiorum) through competition, mycelial lysis, mycoparasitism and antibiosis via the synthesis of volatile and/or non-volatile metabolites (gomathi & ambikapathy 2011; daami-remadi et al. 2006; bhattacharyya & jha 2011). hence, the present study was carried out to determine the in vitro antagonistic activity and biocontrol potential of endophytic aspergillus spp. associated with plectranthus. amboinicus against f. verticillioides. materials and methods collection of plant material mature and healthy leaf samples of plectranthus amboinicus were collected in may 2018 from echague, isabela (16.6701° n, 121.7171° e). the plant materials were authenticated based on taxonomic characters through the assistance of an experienced botanist. samples were transported in sterile polypropylene bags and processed within 6 hours of collection. isolation of endophytic fungi plant samples were surface sterilized and the endophytic fungi were isolated using the method described by kusari et al. (2009) with minor modifications. leaves of p. amboinicus were washed and rinsed with running tap water and cut into 10 mm (length) by 5 mm (width) segments. each segment was then surface sterilized by sequential immersion in 75% ethanol for 2 minutes, 1% sodium hypochlorite (naocl) for 3 minutes, and then once again in 75% ethanol for 1 minute. the leaf segments were finally rinsed three times in sterile distilled water to remove excess sterilant and blot dried in sterile filter paper. the leaf segments were later inoculated onto potato dextrose agar (pda) plates supplemented with streptomycin (1 ml/l) to suppress bacterial growth. four (4) leaf segments were equidistantly placed on each amended pda plate. the plates were then sealed with parafilm and incubated at 28 °c until the growth of endophytic fungi was detected. the hyphal tip of each endophytic fungi growing out from the leaf segments were separately transferred into new amended pda plates and routinely maintained. identification of endophytic fungi the endophytic fungi were identified according to their macroscopic and microscopic characteristics, particulalrly the fruiting structures and spore. colony morphology of the endophytes was observed on coconut water agar (cwa), potato dextrose agar (pda) and malt extract agar (mea). to ascertain the identification of species, a microscopic examination of morphological structures was conducted using the agar block technique. the identification of fungi was done using the dichotomous keys and descriptions provided by quimio and hanlin (1999) and samson et al. (2014). source of test fungus pure cultures of the pathogenic fungus f. verticillioides were obtained from the maintained cultures of mycology laboratory, college of arts and sciences, isu-echague, isabela. the cultures were transferred into sterilized potato dextrose agar (pda) plates and incubated at ±30 °c to allow growth of the mycelia for seven (7) days. in vitro antagonistic activity the in vitro biocontrol potential and antagonistic activity of the endophytic fungi were tested against the pathogenic fungus f. verticillioides using the dual culture method described by matroudi et al. (2009) and john et al. (2010). agar plugs of each endophytic fungi and pathogen were obtained from the edge of 7-day-old pure cultures using sterile cork borer. the plugs were aseptically transferred 20 mm apart respectively on the center of 90 mm mea plates. control plates were inoculated with the pathogenic fungi alone. plates were incubated at 37 °c for 15 days. the interactions exhibited by the co-cultures were monitored daily and the diameter growth of both endophyte and pathogen were recorded at 5, 10 and 15 days, respectively. all control and test plates were biocontrol potential of endophytic aspergillus spp. from mexican thyme – campos and jacob 143 conducted in triplicates. percentage inhibition was calculated as compared to control (gaspar et al. 2004). the growth inhibition was calculated by using the formula: statistical analysis each of the tests was carried out using completely randomized design (crd) with three replicates for each treatment. all means were treated statistically using one-way analysis of variance (anova) and compared by tukey’s honest significant difference test at p < 0.05 using ibm™ spss v25. results and discussion identification and characterization of endophytic fungi three fungal isolates belonging to aspergillus species were selected and their morphology and growth characteristics on cwa, pda and mea were recorded for the determination of their biocontrol potential (table 1). the colony color of a. tamarii endophytes ranged from yellow to olive green which turned dark green with age (fig. 1a). the margin and form are both filamentous and elevation is slightly raised. production of colorless exudates and brown-black sclerotia was observed in various plates after extended incubation. the colonies of a. terreus showed cinnamon-brown color with floccose white mycelia which eventually turns brown to yellow-brown consisting of a dense felt of conidiophores (fig. 1b). reverse morphology was brownish to orange in color, indicating the secretion of metabolites into the medium. sclerotia was absent. meanwhile, a. niger has a distinct blackbrown colony (fig. 1c). it was both filamentous on margin and form and has umbonate elevation. the colonies initially grow with feltlike yellow to white hyphae, turning black with the formation of conidia. formation of black sclerotia and black exudate beads was also observed. the microscopic structures of the endophytes were observed in a microscope through the agar block method. the vesicles of a. tamarii have sub-globose shape, while a. terreus and a. niger have pyriform and globose shaped vesicles, respectively (fig. 1d-1f). the asexual conidia of a. terreus are smooth and hyaline, while a. tamarii conidia are finely roughened compared to the conidia of a. niger which are echinulate (fig. 1g-1i). the endophytic fungi also exhibited some common morphological structures, such as biseriate conidial heads and septate hyaline hyphae. the obtained microscopic descriptions of the aspergillus endophytes coincide with the keys and descriptions provided by samson et al. (2002). table 1 macroscopic and microscopic characteristics of the aspergillus endophytes macroscopic characteristics microscopic characteristics endophytic fungi culture media colony color reverse color colony density shape of vesicle texture of conidia seriation a. tamarii cwa browngreen white abundant subglobose smooth/finely roughened biseriate pda parrot green white luxuriant mea yellow light yellow abundant a. terreus cwa beige tan sparse pyriform smooth biseriate pda cinnamon brown sparse mea cream yellow yellow orange abundant a. niger cwa grey white luxuriant globose echinulate biseriate pda black cream abundant mea brownblack black luxuriant biotropia vol. 28 no. 2, 2021 144 figure 1 colony morphology of (a) a. tamarii; (b) a. terreus; and (c) a. niger; conidiophore of (d) a. tamarii; (e) a. terreus; and (f) a. niger; conidia of (g) a. tamarii; (h) a. terreus; and (i) a. niger in vitro antagonistic activity of endophytic fungi results of this study showed that the endophytes can variably restrict the growth of f. verticillioides (table 2). among the three fungi, a. niger produced the largest inhibition on the mycelial growth of f. verticillioides by 47.37%, followed by a. tamarii (41.02%) and a. terreus (27.91%). f. verticillioides recorded a mean radial growth diameter of 69.91 mm on the control plate. in comparison, the pathogenic fungi produced radial growth of 49.85 mm, 60.92 mm and 44.48 mm in the dual culture with a. tamarii, a. terreus and a. niger, respectively. the smaller radial growth on the dual culture plates indicated the presence of antagonism between the pathogen and the endophytes. c b a d e f g h i biocontrol potential of endophytic aspergillus spp. from mexican thyme – campos and jacob 145 table 2 radial mycelial growth and percent inhibition of f. verticillioides by three endophytic strains of aspergillus after 14 days of incubation fungal antagonists f. verticillioides radial mycelial growth (mm) percent inhibition (%) a. tamarii 49.85±10.64a 28.69% a. terreus 60.92±11.51b 12.86% a. niger 44.48±4.39a 36.38% control 69.91±16.31b notes: values are means of three replications. means in the same column with different superscript indicate significant difference at p ≤ 0.05. all the fungal antagonists inhibited the mycelial growth of f. verticillioides through different antagonistic mechanisms (fig. 2). observations on the dual culture plates of a. tamarii and f. verticillioides indicated that the endophyte antagonized the pathogen through overgrowth mechanism. the overgrowth is achieved when a fungus exhibits a higher growth rate, tolerance against metabolites produced, and a higher capacity of antibiotic production (mathiavanan et al. 2000). a noticeable change in the morphology of a. tamarii in all co-culture plates was also observed wherein the isolates became highly floccose and conidia were rarely present (fig. 2a). this suggested that the a. tamarii isolates were adapting and responding to the presence of f. verticillioides. aspergillus species are known to grow via the formation of a floccose mycelium, producing aerial hyphae that are capable of enhanced oxygen absorption and increased rates of respiration (rahardjo et al. 2005). isolates of a. niger outgrew those of f. verticillioides implying that the antagonism involved is overgrowth mechanism. macroscopic observation also revealed that the a. niger has a mutually intermingled growth with f. verticillioides without any zone of inhibition, indicating the failure of the production of antibiotics either by the pathogen or by the antagonist. a. terreus zone of inhibition was clearly observed in which there was a conspicuous space between the antagonist and the test fungus (fig. 2e). the inhibition zone is observed in all co-culture plates of a. terreus and f. verticillioides and mycelial growth of both fungi is either stunted or severely decreased in the region. the formation of a zone of inhibition is an indication of the production of antibiotic substances either by the pathogen against antagonistic fungi or vice versa (gomathi & ambikapathy 2011). the capacity of aspergillus species to inhibit fusarium isolates has been reported by several authors. a. niger initiated lysis of f. oxysporum mycelium through antibiosis (patibandn & sen 2007; dwivedi & enespa 2013). a. niger, a. tamarii and a. terreus successfully controled the growth of fusarium sambucinum and phytophthora erythroseptica (abdallah et al. 2015). a. niger was also reported as one of the best antagonists for several soil-borne, seed-borne and foliar plant pathogens (kamil et al. 2009; ahmed & upadhyay 2009). moreover, certain atoxigenic strains of aspergillus have been reported to competitively exclude aflatoxin-producing strains during crop infection and thereby reduce aflatoxin contamination. one of these, af36, has been registered as a biological control for the competitive exclusion of aflatoxin producing fungi from cottonseed (cotty 2018). aspergillus species have diverse adaptations and responses to cellular stress which allows them to be resilient in the presence of other organisms (abdallah et al. 2015). these include the deployment of biophysically diverse compatible solutes and functionally diverse protein‐stabilization proteins; hyperaccumulation of melanin in the cell wall; oxidative stress responses; ability to resist high temperatures; production of extracellular polymeric substances (eps) and formation of biofilms; and the ability to compete with other microbes. biotropia vol. 28 no. 2, 2021 146 figure 2 antagonism of f. verticillioides with a. tamarii (a, d), a. terreus (b, e) and a. niger (c, f); microscopic hyphal interactions of a. tamarii and f. verticillioides (g) and a. niger (h) with f. verticillioides conclusion the three aspergillus endophytes, namely a. tamarii, a. terreus and a niger isolated from the foliar segments of plectranthus amboinicus restricted the growth of fusarium verticillioides and the mechanisms involved overgrowth and antibiosis. the capacity of these endophytes to restrict the growth of f. verticillioides implied that these organisms can be exploited as possible alternatives to chemical control agents. acknowledgements the authors acknowledge the provision of materials and facilities by the department of biological sciences of isabela state university, echague, philippines. we also gratefully acknowledge dr helen c. ramos and dr florenda b. temanel for their assistance in checking this manuscript. c b a f e d endophyte endophyte endophyte endophyte endophyte endophyte pathogen pathogen pathogen pathogen pathogen pathogen g h f. verticillioides hyphae a. tamarii hyphae a. niger hyphae f. verticillioides hyphae biocontrol potential of endophytic aspergillus spp. from mexican thyme – campos and jacob 147 references abbas hk, mirocha cj, meronuk ra, pokorny jd, gould sl, kommedahl t. 1988. mycotoxins and fusarium spp. associated with infected ears of corn in 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phangnga forestry research station, southern thailand jetsada wongprom1*, roongreang poolsiri2, sapit diloksumpun2, chatchai ngernsaengsaruay3, samita tansakul2 and wasan chandaeng1 1forestry research center, faculty of forestry, kasetsart university, bangkok 10900, thailand 2department of silviculture, faculty of forestry, kasetsart university, bangkok 10900, thailand 3department of botany, faculty of science, kasetsart university, bangkok 10900, thailand received 3 august 2021 / accepted 20 december 2021 abstract litterfall and litter decomposition play important roles in the maintenance of nutrient cycling and rehabilitation of degraded lands. litterfall, litter decomposition and nutrient return were investigated in a 27-yearold acacia mangium plantation on sandy and clay sites, and in a mixed plantation at the phangnga forestry research station, phangnga province, thailand. additionally, secondary and primary forests were investigated and compared with the values obtained from the acacia mangium and the mixed plantations. the results indicated that litter production in a. mangium plantation on sandy and clay sites, and in mixed plantations (15.47, 11.68 and 7.89 t/ha/yr, respectively) was higher than that in the secondary and primary forests (6.34 and 6.92 t/ha/yr, respectively). the rate of litter decomposition was the greatest in the secondary forest (3.01/yr) and the lowest occurred in the primary forest (1.15/yr). the decomposition rate of the mixed leaf litter between native trees and a. mangium in plantations was higher than that of only a mangium leaf, except in the mixed plantations. a high initial nitrogen concentration in a. mangium could accelerate litter decomposition and improve litter quality in the mixed litter. in addition, the nutrient return in plantations was higher than that in the secondary and primary forests, especially for n. increased litter production, high decomposition rate and nutrient return from a. mangium plantation had important roles in nutrient cycling, suggesting that a mixed plantation consisting of a. mangium and native trees should be considered for the reclamation of mining land. keywords: litter decomposition, litterfall, mining rehabilitation, nutrient return, tropical forest introduction mining operations have been undertaken on a global scale as the raw minerals obtained play an important role in economic and infrastructure development. a high level of mineral production has been reported in asian countries (reichl et al. 2017). the ill-effects of mining cause severe impacts on the ecosystem, including soil degradation, water and air pollution, and loss of wildlife habitat. the vegetation richness and soil properties are destroyed by mining activities, resulting in the degradation of the ecosystem (bell & donnelly 2006; tripathi et al. 2016). improving environmental conditions in mining areas using natural methods takes a long time (oktavia et al. 2015). barriers to natural regeneration include environmental factors such as water, soil ph and soil properties (thaiutsa & rungruangsilp 1990; tripathi et al. 2016). forest plantations have been established on degraded lands to improve the soil properties and ecosystem function (singh et al. 2004; zhang et al. 2014; oktavia et al. 2015). rehabilitation in such areas by planting nitrogen-fixing trees has been recommended and they have been planted on many sites as *corresponding author, email: fforjdw@ku.ac.th mailto:fforjdw@ku.ac.th biotropia vol. 29 no. 1, 2022 70 they grow well and have a high aboveground biomass and litter production (dutta & agrawal 2003; singh et al. 2004). acacia mangium is the most preferred species for reclamation efforts in mining land (martpalakorn 1990; oktavia et al. 2015) and other degraded lands (kamo et al. 2008). soil properties and microclimate under such plantations are improved, resulting in the accelerated natural regeneration of native forest species (parrotta 1999; inagaki et al. 2010). litter production and decomposition play important roles in nutrient cycling and the improvement of soil fertility in terrestrial ecosystems (goma-tchimbakala & bernhardreversat 2006; paudel et al. 2015). on degraded lands, litterfall and litter decomposition are keys to restoring the ecology and soil properties because the litter is a nutrient source (lugo 1992; parsons & congdon 2008). nutrient contents of plants are closely related to tree species, especially nitrogen which is abundant in nitrogen-fixing trees (parrotta 1999; singh et al. 2004). tree composition and forest type are the main factors influencing the quality of the litter produced (parrotta 1999; zhou et al. 2006; tang et al. 2010; paudel et al. 2015). in addition, climatic factors can significantly affect litterfall (zhou et al. 2006; scherer-lorenzen et al. 2007; triadiati et al. 2011). litter fractions that fall onto the forest floor are decomposed by organisms living in the soil, and nutrients are subsequently released to the forest floor, resulting in the improved soil properties and a better forest community, which maintain forest function (parrotta 1999). litter quality and microclimate are related to the litter decomposed by soil microbes (parsons & congdon 2008; cizungu et al. 2014; zhong et al. 2017). furthermore, litter decomposition varies depending on the tree species, successional stage, and forest type (lugo 1992; parsons & congdon 2008; tang et al. 2010). the efficiency of plantations in degraded land rehabilitation not only builds productivity and improves forest structure but also increases forest function such as nutrient cycling (lugo 1992; parrotta 1999). a high litter decomposition rate and nutrient return encouraged natural succession and increased soil nutrients (lugo 1992; celentano et al. 2011), which reduced the time required for the restoration process. therefore, the objective of the current study was to evaluate the potential of a. mangium plantation for mining rehabilitation, focusing on litter production, decomposition and nutrient return in comparison to secondary and primary forests. an effort has been made to compare the rehabilitated plantation area to secondary and primary forests. materials and methods study sites this study was carried out in an abandoned tin mining area, in the phangnga forestry research station, phangnga province, southern thailand (8˚46′5″n, 98˚16′7″e) (fig. 1). exotic trees, such as acacia mangium and eucalyptus camaldulensis were commonly planted for tin mining reclamation in thailand. the mining was operated by the gravel pumping method. landform after mining was divided into clay, sand, and gravel areas. soil nutrients were observed to be very low (thaiutsa & rungruansilp 1990). the experimental plots were established in a 27-year-old a. mangium plantation, planted in a sandy soil area (ams), in a clay soil area (amc), and mixed plantation (mp). the planted trees in mp consisted of a. mangium, eucalyptus camaldulensis and dipterocarpus alatus. litterfall, litter decomposition and nutrient return of rehabilitated mining areas – jetsada wongprom et al. 71 figure 1 location of study sites in phangnga province, southern thailand in addition, the experimental plots were established in a secondary forest (sf, located at 8˚39'28″ n, 98˚24′35″ e) and a primary forest (pf, located at 8˚51' 11″ n, 98˚20′ 5″ e) as reference sites. the sf plot was in an approximately 30-year-old site abandoned after shifting cultivation. the pf plot was classified as a low tropical rainforest. dominant native trees in the 27-year-old plantations and the secondary forest were pioneer tree species including carallia brachiata, aporosa planchoniana, bridelia tomentosa, vitex pinnata, microcos paniculata and eurya acuminata. meanwhile, dominant trees in pf were swintonia schwenckii, dipterocarpus kerrii, mesua ferrea, hopea griffithii and gluta elegans. tree composition and dominant trees in ams, amc, mp, sf and pf were reported by wongprom et al. (2020). climatic conditions during the study were recorded at the climate station in the takuapa district. the rainfall was 3,260.10 mm with the rainy season occurring from april to november and the dry season happening from december to march. the mean relative humidity was 83% and the mean temperature was 27.58 °c. data on rainfall, mean temperature and relative moisture recorded during the study are shown in figure 2. figure 2 climatic conditions at takuapa district, phangnga province during the study phangnga province bangkok takuapa district pf pfrs sf primary forest (pf) secondary forest (sf) phangnga forestry research station (pfrs) n biotropia vol. 29 no. 1, 2022 72 litter production three experimental plots (each 40 x 40 m) were established in each ams, amc, mp, sf, and pf for estimating the amount of litterfall production. five litter traps (each 1 x 1 m) with a 2 mm mesh size were placed 1 m above the ground in each plot. litterfall in the traps was collected monthly for 1 year. litterfall samples were sorted into leaf, branches, reproductive parts and miscellaneous. a. mangium litter was separated from that of the other tree species. all samples were oven-dried at 80 °c for 48 h until reaching constant weight. the samples (leaf, branches, reproductive parts and miscellaneous) were analyzed during both the rainy and dry seasons. the organic debris obtained from each site was analyzed as a single sample, as it was difficult to identify the components as they were very small and had a similar texture. litter decomposition and nutrient return nylon bags (50 x 50 cm) with a 2 mm mesh size were used for investigating litter decomposition. leaf having the top five importance value index (ivi) trees in ams, amc, mp and sf, and the top seven ivi trees of pf were selected to investigate litter decomposition. litter decomposition was divided into two classes: 1) mixed leaf litter in ams, amc, mp, sf and pf; and 2) pure a. mangium leaf litter in ams, amc, and mp. in each case, 30 g samples of air-dried leaf litter were filled into the nylon bags according to the ivi ratio of the dominant trees for each site. the ivi of trees in ams, amc, mp, sf and pf was reported by wongprom et al. (2020). the litter water content was determined by first, ovendrying subsamples of air-dried litter at 80 °c for 48 h to obtain the initial dry mass. twelve litter bags from each treatment with three replications were randomly placed in each plot. three samples of both mixed leaf litter and pure a. mangium leaf litter were retrieved monthly. the leaf litter remaining in each bag was brushed to remove any soil particles and roots. the remaining litter was oven-dried at 80 °c for 48 h until a constant weight was recorded. the annual litter decomposition rate (k) was calculated according to the negative exponential decay model (olson 1963) given as k = ln (x/x0)/t, where x0 is the initial dry weight, x is the dry weight remaining at the end of the study, and t is the time period in years. nutrient return (n, p, k, ca, and mg) to the forest floor (kg/ha) was estimated by multiplying the amount of litter production by the nutrient concentration of litter for each site. chemical analysis of litter nutrient concentrations of nitrogen (n), phosphorus (p), potassium (k), calcium (ca) and magnesium (mg) of leaf, branches, reproductive parts and miscellaneous obtained from the study sites were determined during the rainy (april to november) and dry (december to march) seasons. samples of each litter type in the rainy and dry seasons were mixed and analyzed. the concentration of n was measured based on dry combustion using a cnhs analyzer, while the concentrations of p, k, ca, and mg were determined by wet washing with nho3-hclo4 acid (hno3: hclo4; 5: 2). the concentration of p was analyzed using the vanadomolybdate yellow color method with a spectrometer at a wavelength of 440 nm, while the concentrations k, ca, and mg were analyzed using atomic absorption spectrometry. data analysis nutrient concentrations of each litter sample during the rainy and dry seasons were averaged to calculate the amount of nutrients return. the annual litter production, nutrient return and litter decomposition rate among sites were analyzed using a one-way anova and the means were compared with the tukey hsd test at a 5% probability level. results and discussion litter production the annual litter production was significantly different among sites (p < 0.05). ams had the highest litterfall followed by amc, mp, pf, and sf (table 1). litterfall, litter decomposition and nutrient return of rehabilitated mining areas – jetsada wongprom et al. 73 table 1 litter production (leaf, branches, reproductive parts and miscellaneous) (t/ha/yr) of a. mangium (am) and native trees and planted trees (npt) in the 27-year-old a. mangium plantation in sandy soil area (ams), clay soil area (amc), mixed plantation (mp), secondary forest (sf), and primary forest (pf) site leaf branches reproductive parts miscellaneous total am npt am npt am npt ams 5.90a 2.85b 0.74 1.67 2.86a 0.57 0.88a 15.47a amc 3.56ab 3.83ab 0.56 1.20 1.60b 0.50 0.43b 11.68b mp 1.52b 2.86b 0.59 1.06 0.84b 0.51 0.51ab 7.89c sf 4.12ab 0.76 0.74 0.72ab 6.34c pf 4.72a 0.89 0.72 0.59ab 6.92c f value 11.59** 8.03** 1.83ns 0.87ns 16.55** 2.02ns 4.58* 27.21** notes: * = significant difference; ** = very significant difference; ns = non-significant difference; a c = different superscripts in the same column indicate significant differences at p < 0.05. litter production peaked in the dry season (december to march) (fig. 3), as a response to the water stress, which was similar to that of other tropical forests (triadiati et al. 2011; cizungu et al. 2014). leaf component was the main litter falling onto the forest floor in ams, amc, mp, sf and pf, and made up around 54 to 62% of the total litter. however, the amount of reproductive parts in ams and amc was relatively high compared to branches, especially the reproductive parts of a. mangium as it fell almost all year. a large amount of litter from a. mangium in ams was resulted from the high tree density and crown cover, while a. mangium in amc and mp had a low crown cover due to the high mortality rate. litters from other trees in ams, amc, and mp came from native tree species. planted trees in mp such as d. alatus and e. camaldulensis were also a main source of litter. leaf litter is a significant contributor of annual primary production and nutrient capital in a terrestrial ecosystem (cizungu et al. 2014; paudel et al. 2015). figure 3 total monthly litter production (t/ha/yr) of the 27-year-old a. mangium plantation in sandy soil area (ams), clay soil area (amc), mixed plantation (mp), secondary forest (sf), and primary forest (pf) biotropia vol. 29 no. 1, 2022 74 this study showed that the amount of litter production in ams and amc was higher than that in both sf and pf. a forest plantation with fast-growing trees is highly effective at building litter production compared to a natural forest (goma-tchimbakala & bernhard-reversat 2006) and secondary forest (lugo 1992; kamo et al. 2008). post mining reclamation should focus on increasing the aboveground biomass and litter production to improve the ecosystem. a. mangium is one species recommended for degraded land restoration. acacia grows well and has high aboveground biomass compared to native trees (martpalakorn 1990; kamo et al. 2008). however, the total litter production in mp was similar to that in pf and sf, although a. mangium had less litter production. the canopy cover of a. mangium can promote a high litterfall. fast nitrogen-fixing tree plays important role in the early stage by rapid growth to create a canopy in order to suppress the weeds and shrubs that could obstruct the establishment of native tree understory vegetation and to increase nitrogen availability (ingaki et al. 2010; lanuza et al. 2018). however, the litterfall in ams, amc and mp was not only sourced from planted trees but also native trees through natural succession. old plantations have high tree diversity and richness (lugo 1992; koonkhunthod et al. 2007), as litterfall is contributed to by native trees. native tree species are usually pioneer trees having high growth rates (chazdon 2014; chen et al. 2017) and their litter production increases with succession age (zhou et al. 2006; feng et al. 2019). litterfall production plays an important role in forest functions such as carbon and nutrient cycling (moura et al. 2016; paudel et al. 2015; lanuza et al. 2018) because litter is a source of nutrient pools, which releases nutrient to the forest floor via litter decomposer activities (lugo 1992; fisher & binkley 2000), suggesting that a. mangium should be recommended for reclamation in a mining area due to the capability of a. mangium in bringing out high litter production. litter production in the sf and pf study sites was not different, resulting from pioneer trees producing litter in natural succession. forest structure and tree composition of succession forests affected litter biomass (lanuza et al. 2018). long-lived pioneer trees and high tree diversity were observed in sf. the litter production in the sf and pf study sites was similar to that in many evergreen forests in thailand (6.42 to 7.85 t/ha/yr) (bunyavejchewin 2001; glumphabutr et al. 2007). however, litter production in our study was lower than that in other tropical forests (8.30 13.67 t/ha/yr) (anderson et al. 1983; tang et al. 2010; triadiati et al. 2011; paudel et al. 2015). litter decomposition litter decomposition of pure litter and mixed litter was significantly different among treatments (p < 0.05). the litter decomposition rate (k) ranged between 1.15 and 3.01/yr, which was classified as medium to high levels. the decomposition rate in sf was the highest, indicating litters were rapid litter decay, but not different in amc (2.91/yr) (table 2). the dominant trees in sf and amc were pioneer tree species and were of similar tree composition (wongprom et al. 2020), having thin and flexible leaves without a prominent skeleton, and so could decay easily. on the other hand, a low rate of decomposition was found in climax forest trees due to their thick, tough leaf and prominent midribs and veins. the leaf morphology of trees (leaf thickness, roughness, toughness) significantly affects the rate of litter decomposition (liao et al. 2006; cizungu et al. 2014). in addition, the chemical properties of leaf litter, including high n, n/p, low c/n and low lignin, are generally positive contributors to decomposition (xuluc-tolosa et al. 2003; liao et al. 2006; cizungu et al. 2014; rai et al. 2016). similarly, leaf litter in sf, ams, amc, and mp had a high nitrogen content, which could explain their faster rate of decomposition. leaf litter in ams, amc, and mp was rich in nitrogen due to the litter produced by a. mangium. in addition, leaves of dominant pioneer trees in sf, ams amc and mp have thin and less rigid leaves leading to a high decomposition rate compared to pf. however, the litter decomposition of pf was of moderate level (1.15/yr), which was similar to other climax tropical forests (hättenschwiler et al. 2011). litterfall, litter decomposition and nutrient return of rehabilitated mining areas – jetsada wongprom et al. 75 table 2 decomposition rate of pure a. mangium and the mixed leaf in the 27-year-old a. mangium plantation in sandy soil area (ams), clay soil area (amc), mixed plantation (mp), secondary forest (sf), and primary forest (pf) site type of leaf k (constant) ams pure a. mangium leaf 1.56bc mixed leaf 1.64bc amc pure a. mangium leaf 1.88bc mixed leaf 2.91a mp pure a. mangium leaf 2.05b mixed leaf 1.87bc sf mixed leaf 3.01a pf mixed leaf 1.15cd f value 7.905* notes: * = significant difference; a d = different superscripts in the same column indicate significant differences at p < 0.05. climate conditions and soil microbials significantly affect litter decomposition (berg & mcclaugherty 2008). this study indicated that rainfall and relative humidity in the study sites were of similar conditions. however, soil microbials could be different because soil properties in ams, amc, and mp were of poor soil compared to that in sf and pf, especially in terms of total n and om (wongprom et al. 2020), leading to low diversity and abundance of soil microbials (zhang et al. 2016). soil nutrients, n and p and organic matter (om) are significantly correlated with soil microbials (zhang et al. 2016; ngugi et al. 2020). low decomposition rate of litter was found in the pf study site. microclimate conditions may slightly affect litter decomposition. litter quality of the restored forest may significantly determine litter decomposition. meanwhile, litter decomposition rate changes depending on successional stage and tree composition (moura et al. 2016; lanaza et al. 2018). a study conducted by xuluc-tolosa et al. (2003) indicated that pioneer trees in the early succession phase has a higher decay rate than tree species in the late succession phase because pioneer trees have a high initial n concentration and low c/n. pioneer tree species have high nutrient resorption efficiency and high leaf nutritional quality, especially n concentration (gomes & luizão 2012). our study showed that a litter mixture of a. mangium and native trees generally resulted in a faster decay than the decay of pf litter. this result could be affected by synergistic effects of mixed litter with high litter quality and a high initial n concentration. mass loss is positively correlated with initial n concentration (liu et al. 2016). thus, poor litter quality is improved. mixing litters from various trees accelerate the mass loss, enhance the nutrient release and nutrient cycling (liu et al. 2016; trogisch et al. 2016; cizungu et al. 2016) and different litter components may change the litter chemical components and the decomposer community (gartner & cardon 2004). nutrient concentration in litter and nutrient return nutrient concentration in litter produced by a. mangium in this study varied among components (leaf, branches and reproductive parts). however, the n concentration was greater than that of p, k, ca, and mg (fig. 4). nutrient concentration in the leaf and branches litters of a. mangium in all sites followed the order of n>ca>k>mg>p, except for the reproductive parts, where the k concentration was higher than that of ca. biotropia vol. 29 no. 1, 2022 76 figure 4 nutrient concentration of pure a. mangium leaf, branches and reproductive parts of the 27-year-old a. mangium plantation in sandy soil area (ams), clay soil area (amc) and mixed plantation (mp) n concentration in the litter of other trees (native tree species in ams, amc, mp, sf and pf, and planted trees in mp) was the highest for all litter components, and was especially high in the litter miscellaneous component. the p concentration was relatively high in the miscellaneous and reproductive parts components, while the concentrations were similar for k, ca and mg in the leaf, branches, reproductive parts and miscellaneous components. the nutrient concentration of leaf, branches, reproductive parts and miscellaneous for the other tree species in ams, amc, mp, sf, and pf are shown in figure 5. figure 5 nutrient concentration of mixed leaf, branches, reproductive parts and miscellaneous litter of native tree species in the 27-year-old a. mangium plantation in sandy soil area (ams), clay soil area (amc), secondary forest (sf), and primary forest (pf) and planted trees and native tree species in mixed plantation (mp) litterfall, litter decomposition and nutrient return of rehabilitated mining areas – jetsada wongprom et al. 77 the nutrient return of n, p, k, ca and mg from litters to forest floor was significantly different among sites. n contributed the highest nutrient return to the forest floor in all sites. the large amounts of n, p, k, ca and mg returned in ams, amc, and mp were mostly from the litter of the planted trees, especially a. mangium (table 3). a. mangium is the dominant tree with a large size and crown cover, although the tree density is low, particularly in the amc and mp sites (wongprom et al. 2020). litters from planted trees in a plantation are still the main nutrient source to the forest floor, although the vegetation composition are shifted after restoration. leaf litter was the main source of nutrient return in all sites. however, nutrient return from miscellaneous and reproductive parts of a. mangium was relatively high. the annual nutrient return of ams, amc and pf followed a pattern of n>ca>k>mg>p, while that of mp and sf followed a pattern of n>k>ca>mg>p (table 4). the annual nutrient return of n, p, ca and mg to forest floor in ams and amc was significantly higher than that in sf and pf, especially for the nitrogen. meanwhile, the annual nutrient return of n, p, ca, and mg in sf was similar to that in pf. forest community on a restored site and in a secondary forest can accelerate litter decomposition and nutrient cycling because the litter quality is improved. nutrients returned to the forest floor can promote natural regeneration and tree growth, resulting in a complex forest structure in the long term, especially as n is a significant nutrient in developing a forest community and establishing seedlings and saplings during mining restoration and natural succession in a degraded land (zhao et al. 2013; lei et al. 2015). fast litter decomposition of restored sites resulted in high nutrient depositions, especially n. in the current study, n was the major nutrient return in ams, amc and mp, and was significantly higher than that in sf and pf. the high n return may be resulted from a. mangium due to the acacia being a nitrogen-fixing tree. moreover, the return of p, k, ca and mg to the soil of the rehabilitated sites was greater than that in pf. soil nutrients of a. mangium plantation in an abandoned mining area are higher compared to that in an abandoned mining area without the a. mangium plantation, especially in terms of n and soil organic matter (wongprom et al. 2020). therefore, the study result indicated that a. mangium improves soil chemical properties through nutrient cycling processes. n flux of nitrogen-fixing tree plays an important role in the early succession (moura et al. 2016). table 3 nutrient return (kg/ha/yr) of a. mangium (am) litter and native trees and planted trees (npt) litter in the 27-year-old a. mangium plantation in sandy soil area (ams), clay soil area (amc), and mixed plantation (mp) site n p k ca mg am npt am npt am npt am npt am npt ams 18.96a 7.12 0.94a 0.49 2.07a 1.48b 2.92a 2.14ab 1.91a 1.20b amc 10.97a 6.52 0.68a 0.52 1.37a 2.93a 2.08ab 2.64a 1.41a 1.94a mp 5.47b 6.42 0.30b 0.49 0.85b 2.73a 1.11b 1.91b 0.62b 1.56ab f value 26.37** 1.09ns 18.39** 0.37ns 14.95** 29.01** 14.48** 6.19* 15.66** 12.36** notes: * = significant difference; ** = very significant difference; ns = non-significant difference; a b = different superscripts in the same column indicate significant differences at p < 0.05. table 4 nutrient return (kg/ha/yr) of litter to the forest floor in the 27-year-old a. mangium plantation in sandy soil area (ams), clay soil area (amc), mixed plantation (mp), secondary forest (sf), and primary forest (pf) site n p k ca mg ams 26.09a 1.43a 3.56b 5.06a 3.12a amc 17.49b 1.19a 4.30a 4.72a 3.36a mp 11.89c 0.79b 3.58b 3.03b 2.18bc sf 8.26c 0.63b 4.21ab 2.72b 1.74bc pf 7.47c 0.61b 2.77c 3.20b 1.28c f value 53.60** 33.78** 15.87** 22.75** 34.63** notes: ** = significant difference; a c = different superscripts in the same column indicate significant differences at p < 0.05. biotropia vol. 29 no. 1, 2022 78 fast-growing tree plantations are commonly planted for wood production. however, they are also planted for forest restoration in degraded lands. high litter production and litterfall are significant parts in restoration processes (parrotta 1999). the n nutrient returned to the forest floor from nitrogen-fixing tree plantations was significantly higher than that from a nonnitrogen-fixing tree plantations and for other nutrients (bernhard-reversat 1996). the litter decomposition of nitrogen-fixing trees positively affect the acceleration of nutrient release and nutrient deposition to promote vegetation development and improve soil properties (lugo 1992; ruiz-jaén & aide 2005; lanuza et al. 2018), suggesting that a. mangium plantation should be used for improving nutrient cycling in mining reclamation. nutrient cycling is a key process for mining rehabilitation, relating to vegetation succession and soil development in the mining area. conclusion a. mangium plantation is an effective way for mining rehabilitation compared to the reference sites. a. mangium plantation showed relatively high litter production and litter decomposition rate which could modify the litter quality in the litter mixture. mixed litter sourced from nitrogen-fixing trees and native trees in plantations can enhance litter decomposition, which can result in higher levels of soil improvement and revegetation. ams, amc and mp had high levels of nutrient return compared to that in sf and pf, leading to contributing high levels of soil nutrients to forest floor, particularly n. the litter mixture in mp decomposed more rapidly compared to that in pf with improved the litter quality. a mixed plantation consisting of a. mangium and native tree species could be considered for reclamation efforts in mining area and other similar degraded lands. acknowledgments financial support for this study was provided by the kasetsart university research and development institute (kurdi) in bangkok, thailand. we are grateful to the staff at the phangnga forestry research station for their assistance during the field work. references anderson j, proctor mj, vallack hw. 1983. 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biology vol. 32 no. 2, 2025: 143 150 doi: 10.11598/btb.2025.32.2.2299 issn: 0215-6334 | e-issn: 1907-770x 143 the potential of syzigium aromaticum l. to control leaf spot disease (cercospora capsici) in cayenne pepper (capsicum frutescens l.) yenny muliani1, dwi susanto2*, ida adviany1, n tita nursinta1, debby ustari1 1department of agrotechnology, faculty of agriculture, universitas islam nusantara, bandung 40286, indonesia 2department of agrotechnology, faculty of science and technology, universitas halim sanusi, bandung 40116, indonesia article higlights • in preliminary lab tests, clove leaf extract clearly slowed c. capsici growth. • field trials assessed its potential to reduce leaf spot disease in cayenne pepper. • applying concentrations above 15 g/l tended to lower the observed disease intensity. • the same concentration range was also linked to an increase in leaf number. • however, low field disease incidence limited detectable yield responses. article information received : 12 july 2024 revised : 26 march 2025 accepted : 10 may 2025 *corresponding author, e-mail: dwsus123@gmail.com research paper abstract cayenne pepper (capsicum frutescens l.) is a spice plant with good potential for development in indonesia due to its high market demand. however, several challenges hinder the increase in its production, one of which is plant disease. a major disease affecting cayenne pepper is leaf spot, caused by the fungus cercospora capsici heald & f.a. wolf. although chemical pesticides can effectively control this disease, they have negative effects on the environment. the clove tree (syzygium aromaticum l.) is commonly cultivated for its dried flower buds, which have various uses. additionally, due to its chemical contents, the leaves of the clove tree have the potential to reduce the intensity of leaf spot disease. therefore, the aims of this study were: (1) to determine the effects of clove leaf extract on reducing the intensity of c. capsici infection in cayenne pepper, and (2) to determine the most effective concentration of the extract for disease reduction. the experiment used a randomized block design (rbd) with five treatments and five replications, namely: a = control (water only), b = 10 g clove leaf extract/l water, c = 15 g clove leaf extract/l water, d = 20 g clove leaf extract/l water, and e = 25 g clove leaf extract/l water. the results showed that the application of s. aromaticum leaf extract reduced the intensity of leaf spot disease caused by c. capsici, although it did not significantly affect the yield of cayenne pepper. the lack of effect on yield was attributed to the overall low disease intensity across all treatments, including the control. keywords: biopesticides, cayenne pepper, cercospora capsici, leaf spot disease, syzigium aromaticum introduction chili contains bioactive compounds such as capsaicinoids, phenols, flavonoids, and vitamin c (kusnadi et al. 2019). the capsaicin content in chili peppers is beneficial for relieving headaches, reducing joint inflammation, and promoting overall health (winarto & wisnuwati 2020). cayenne pepper (capsicum frutescens l.), a type of chili, is commonly used in indonesia as a cooking spice, an ingredient in sambal (chili paste), or even consumed fresh. the demand for cayenne pepper in indonesia tended to increase from 2018 to 2022, according to data from the central agency of statistics (armavillia 2023). in addition to meeting local needs, cayenne pepper is also exported to countries such as malaysia and singapore (budi et al. 2020). to meet this high demand, production must be increased. although the harvest area and production of cayenne pepper in indonesia fluctuated during 2018 2022, both showed an overall upward trend. however, this level of production has not fully met domestic and industrial needs. several obstacles hinder cayenne pepper farmers from increasing production, including unpredictable weather, fluctuating prices, and the presence of pests and diseases (rismayanti et al. 2022). one of the most common diseases affecting cayenne pepper plants is cercospora leaf spot, caused by the fungus cercospora capsici heald & f.a. wolf, which can result in yield losses of 30 40% (lestari & aini 2021). https://doi.org/10.11598/btb.2025.32.2.2450 biotropia vol. 32 no. 2, 2025 144 symptoms of c. capsici leaf spot disease typically appear as circular lesions (up to 1 cm in diameter) on the leaves, brown to black in color, with small white spots (approximately 0.5 1.0 mm) at the center that gradually enlarge. infected chili plants develop necrosis and are sometimes accompanied by chlorosis on the leaves (adedire et al. 2019). clove leaves contain active antibacterial and antifungal compounds, making them potentially useful for controlling plant diseases (lambiju et al. 2017). djaenuddin et al. (2018) reported that clove leaf extract had a positive effect in inhibiting the development of bipolaris maydis under laboratory conditions. similarly, hizrianti et al. (2021) found that clove leaf oil at a concentration of 0.065%, dissolved in pda medium, completely inhibited the mycelial growth of alternaria solani after 7 days of incubation at room temperature, with an inhibition rate of 100%. however, the use of clove leaf extract to control cercospora capsici heald & f.a. wolf, the causal agent of leaf spot on cayenne pepper (capsicum frutescens l.), has not yet been tested. therefore, the objectives of this study were: (1) to evaluate the effects of clove leaf extract on reducing the intensity of c. capsici infection in cayenne pepper, and (2) to determine the most effective concentration of the extract for reducing disease intensity. materials and methods the main materials used in this experiment were cayenne pepper seeds of the sonar cultivar, goat manure, npk fertilizer, clove leaves, detergent (used as a biopesticide adhesive), and water. the study employed a randomized block design (rbd) consisting of five treatments with five replications each. the biopesticide application treatments were: a = control (water only), b = 10 g clove leaf extract/l water, c = 15 g clove leaf extract/l water, d = 20 g clove leaf extract/l water, and e = 25 g clove leaf extract/l water. preliminary test preliminary tests consisted of isolating and identifying fungus, simple postulate koch test, and concentration test. isolating and identifying fungus the isolation and identification of the fungus causing leaf spot disease in cayenne pepper plants were conducted using the following steps. first, leaves showing symptoms of infection by pathogenic fungi were washed under running water to remove any adhering dirt. the leaves were then cut into pieces measuring 2 × 2 cm. surface disinfection was carried out by immersing the leaf pieces in 5% chlorine solution for 1 minute, followed by 70% alcohol for 30 seconds, and then rinsing them three times with sterile water. the sterilized leaf pieces were dried on filter paper and subsequently cultured on pda (potato dextrose agar) medium. after 7 days of incubation, fungal growth was observed on the culture. the pathogen was then identified based on its macroscopic and microscopic characteristics. macroscopic identification of the disease was conducted by observing the color of the fungal colony, as well as the direction and shape of its growth. for microscopic identification, a small portion of the pure culture was placed on a microscope slide containing a drop of sterile distilled water. a cover slip was then carefully placed over the sample to avoid the formation of air bubbles. the slide was subsequently observed under a microscope to examine the morphological characteristics of the pathogen. simple postulat koch test the pathogenic fungus isolated from infected cayenne pepper leaves was inoculated onto healthy cayenne pepper plants. koch’s postulates were applied to assess the phytopathogenic characteristics of the obtained isolate. the fungal isolate was first grown in pure culture, then inoculated onto healthy plants and observed for the development of disease symptoms. once symptoms appeared, the pathogen was re-isolated from the infected plant tissue using the same method described previously (sudiartini et al. 2021). concentration test concentration tests were conducted to determine the most effective clove leaf extract concentration for reducing the intensity of cercospora capsici heald & f.a. wolf leaf spot disease. these concentrations were later used as treatments for applying clove leaf extract to cayenne pepper plants in vivo. the concentrations tested were 10 g/l, 15 g/l, 20 g/l, 25 g/l, and a control for comparison. the concentration test was performed following the method described by andriyani and purwantisari (2019). clove leaf extract at each concentration was added to sterile petri dishes containing pda medium that had been previously the potential of syzigium aromaticum l. to control leaf spot disease – muliani et al. 145 sterilized and melted, using a ratio of 1 ml of extract to 10 ml of medium. the mixture was then gently shaken to ensure even distribution of the extract in the medium. a purified culture of c. capsici was obtained using the cork borer method and placed at the center of each petri dish. the dishes were then incubated at room temperature. the diameter of the fungal colonies was measured daily over a period of six days to assess the effect of each extract concentration. variables observed three variables were observed in this research, namely disease intensity, number of leaves and plant yield. yield total yield was weighed from five harvests. this variable measures crop productivity and reflects the effects of the treatments. results and discussion preliminary tests based on macroscopic and microscopic observations in the laboratory, the pathogen was confirmed to be cercospora capsici, as shown in figures 1 and 2. these figures indicate that the hyphae of c. capsici after 7 days of isolation were whitish-dark in color, branched, and septate, with a somewhat rough mycelial structure. according to wakhidah et al. (2021), the conidia of cercospora sp. are rod-shaped, measuring 27.5 90 µm in length and 2.5 3.75 µm in width. cercospora sp. also has dark-colored conidiophores and conidia, with three or more septa. the results of the koch postulate test are shown in figure 3. the symptoms of cercospora capsici heald & f.a. wolf leaf spot disease appeared as brown, circular spots with a whitish center, resembling a frog’s eye. these observations are consistent with sucianto and abas (2019), who reported that c. capsici causes small, round leaf spots measuring 0.25 0.5 cm in diameter, with necrotic centers that dry out and dark gray coloration, while the edges remain brown. the fungus also produces long, club-shaped conidia measuring 60 200 µm × 3 5 µm, with diameters of 3 12 µm, and short, septate conidiophores measuring 1 3 µm. i = n  y n  y 𝑥𝑥 100% where: i = disease intensity (%); n = the number of plant samples; y = the value of score; n = the number of plant samples observed; y = the highest score used. assessing disease intensity allows us to determine the severity of the disease on the plants and to compare its effects across different treatments. number of leaves the number of leaves was counted before and after the application of the biopesticide at oneweek intervals. this variable is important, as leaf count reflects the plant’s photosynthetic capacity and helps evaluate the effects of the treatment. figure 1 macroscopic characteristic of fungus c. capsici coloni of c. capsici biotropia vol. 32 no. 2, 2025 146 branched conidiophore septate hypha figure 2 microscopic characteristics of fungus c. capsici notes: a. septate hypha; b. branched conidiophore. figure 3 leaf spot caused by c. capsici symptom of leaf spot of sercospore concentration test the concentration test conducted on pda medium demonstrated that clove leaf extract could inhibit the growth of c. capsici in the laboratory (fig. 4). based on these preliminary results, it can be tentatively concluded that clove leaf extract inhibits the growth of the pathogen, and its effectiveness in controlling c. capsici will be further evaluated in this study. a b figure 4 inhibition of c. capsici by different concentration of clove leaf extract notes: (a) = 0 g/l; (b) = 10 g/l; (c) = 15 g/l; (d) = 20 g/l; and (e) = 25g/l. the potential of syzigium aromaticum l. to control leaf spot disease – muliani et al. 147 table 1 leaf spot disease intensity caused by c. capsici in cayenne pepper treatment (concentration of clove leaf extract) leaf spot disease intensity caused by c. capsici (%) before application after application (days after planting/ dap) 14 21 28 35 42 49 56 63 70 a (0 g/l water) 2.47b 2.40b 7.87a 8.67a 9.67a 10.13abc 11.13a 11.20a 15.20a b (10 g/l water) 6.93a 9.20a 8.40a 8.20a 5.33ab 12.13ab 8.87a 6.60abc 10.87ab c (15 g/l water) 4.80ab 5.27ab 5.87a 3.13b 3.00b 4.27c 3.87b 3.67c 6.27b d (20 g/l water) 6.07ab 7.13ab 6.20a 6.67ab 4.93ab 13.93a 10.73a 9.80ab 9.00ab e (25 g/l water) 6.20ab 6.80ab 4.20a 5.93ab 4.93ab 5.73bc 7.07a 5.27bc 7.07b note: values followed by the same letter are not significantly different according to duncan’s multiple range test (dmrt) at the 5% significance level. disease intensity observations of leaf spot disease intensity on cayenne pepper (capsicum frutescens l.) were conducted before and after biopesticide application at weekly intervals. observations began once the plants exhibited symptoms of leaf spot disease caused by the fungus c. capsici. the results are presented in table 1. at 14 and 21 weeks after planting (wap), disease intensity varied among the treatments. this variation occurred because no treatments had been applied earlier, so the existing disease and its development could not be controlled. at 21 wap, disease intensity ranged from 2.40% in the control to 9.20% in treatment b. by 28 wap, no significant differences were observed among the treatments, which may be due to the time required for the clove leaf extract to affect leaf spot disease. according to faqy and rustam (2019), eugenol is a phenolic compound that evaporates easily, so multiple applications are necessary to observe its effects. at 35 days after planting (dap), the average leaf spot disease intensity began to show a noticeable difference, with treatment c exhibiting significantly lower intensity compared to the other treatments and the control. similarly, at 42 dap and subsequent observations, treatment c consistently showed significantly lower disease intensity than the other treatments. this is likely due to the antifungal effect of the clove leaf extract after multiple applications. šernaitė et al. (2020) reported that the eugenol content in clove can inhibit plant pests and diseases. during the observation period, treatment a (control) consistently exhibited the highest disease intensity compared to the other treatments. this indicates that clove leaf extracts can control leaf spot disease, with effectiveness varying depending on the extract concentration. according to salam et al. (2022), cercospora leaf spot disease thrives in environments with temperatures below 28 ºc, humidity above 92%, and soil ph between 5 and 6. the disease becomes more severe during wet weather or periods of high air humidity. observations at 70 dap showed that leaf spot disease intensity tended to increase during the rainy season and under high humidity conditions. symptoms typically appeared first on older leaves and then spread to younger leaves. advanced lesions can cause leaves to become hollow, with white or pale centers and darkened edges. severe infections may result in yellowing and premature leaf drop (devianto et al. 2023). among the treatments, the clove leaf extract concentration of 15 g/l was most effective in reducing disease intensity, resulting in an average intensity of 6.27% at 70 dap. number of leaves similar to disease intensity, the effects of c. capsici on the number of leaves before treatment application (14 and 21 dap) could not be controlled and varied among individual plants. at 28 dap, the number of leaves differed, with treatment e showing the highest and treatment b the lowest values, as the treatments had not yet taken effect. the application of the biopesticide influenced an increase in the number of leaves, likely due to the essential oils in clove leaves, which are known to promote leaf development. the essential oil content in clove leaves has been reported to enhance leaf growth (harni et al. 2018). according to jirovetz et al. (2006), clove leaf oil exhibits antioxidant activity, with eugenol as the major compound (76.8%), followed by β-caryophyllene, α-humulene, and eugenyl acetate. the number of chili plant leaves is presented in table 2. at 35 dap, the average number of leaves had increased but did not differ significantly among biotropia vol. 32 no. 2, 2025 148 the treatments. this indicates that the treatments were beginning to take effect in reducing the disease, particularly for treatments b and c, which previously had the lowest leaf numbers. from 42 dap to the end of the observation period, treatments c, d, and e showed effective concentrations of biopesticides in controlling cercospora leaf spot disease compared to the control, resulting in a continued increase in the number of cayenne pepper leaves. according to thabet and khalifa (2018), clove oil contains compounds such as eugenol, acetyl eugenol, iso-eugenol, and caryophyllene, which have antibacterial and antifungal properties against various plant diseases. additionally, al-askar and rashad (2010) reported that clove extract at a concentration of 4% significantly reduced disease caused by rhizoctonia solani on pea. these findings suggested that higher concentrations of clove leaf extract promote leaf growth and help minimize leaf drop caused by c. capsici leaf spot disease. observations from 49 dap to 70 dap showed that all treatments tended to increase the number of leaves. similarly, deden and umiyati (2017) noted that increasing the concentration of biofungicides enhanced the number of leaves in shallot plants (allium ascalonicum l.). yield cayenne pepper fruits were harvested five times, from 107 dap to 119 dap. the data indicated no significant differences in yield among the treatments, as shown in table 3. data from table 1 revealed that disease intensities across all treatments, including the control, ranged from 2.47% to 13.93%, which is categorized as low intensity. consequently, the reduction in yield due to c. capsici was minimal and did not differ significantly among the treatments. the intensity of leaf spot disease tends to increase with higher rainfall intensity (heald & f.a. wolf ). under severe infection, cayenne pepper leaves become damaged and fall off prematurely. leaf loss disrupts the plant’s photosynthetic process and can lead to reduced crop yields. according to inaya et al. (2022), cercospora sp. leaf spot disease initially appears as small necrotic spots on the leaf surface, which later develop into irregular lesions and produce abundant conidia. when the disease intensity caused by this fungal pathogen becomes severe, it can significantly impair photosynthesis and negatively affect both the quantity and quality of chili production. syafiruddin and hilda (2023) also noted that pests and diseases attacking cayenne pepper plants are major factors contributing to yield reduction. table 2 effects of c. capsici on number of leaves of cayenne pepper treatment number of leaves before application after application (dap) 14 21 28 35 42 49 56 63 70 a (0 g/l water) 6.6b 12.0ab 23.0ab 33.9a 41.7b 57.3a 62.7c 84.3ab 89.5a b (10 g/l water) 8.2a 9.9b 20.7b 35.5a 44.7ab 58.7a 68.3bc 74.9b 82.5a c (15 g/l water) 7.9a 9.9b 21.7b 35.3a 50.5ab 67.3a 94.3a 117.9a 112.2a d (20 g/l water) 7.6ab 11.3b 26.3ab 40.1a 57.5a 68.5a 84.1a 106.4ab 112.3a e (25 g/l water) 8.7a 14.2a 28.9a 44.7a 57.4a 73.5a 75.7abc 105.5ab 117.8a note: values followed by the same letter are not significantly different according to duncan’s multiple range test (dmrt) at the 5% significance level. table 3 yield of cayenne pepper per plant treatment yield per plant (g) a (0 g/l water) 74.0a b (10 g/l water) 61.4a c (15 g/l water) 70.2a d (20 g/l water) 62.0a e (25 g/l water) 74.8a note: values followed by the same letter are not significantly different according to duncan’s multiple range test (dmrt) at the 5% significance level. the potential of syzigium aromaticum l. to control leaf spot disease – muliani et al. 149 conclusion the application of clove (syzygium aromaticum l.) leaf extract biopesticide reduced the intensity of leaf spot disease caused by cercospora capsici heald & f.a. wolf on cayenne pepper plants. disease intensity was low across all treatments, including the control. no significant differences in yield were observed among the treatments. references adedire om, pitan a, farinu ao, ogundipe wf. 2019. the biocontrol of soil transmitted cercospora capsici with lactobacillus plantarum. j adv microbiol 18(3)1-8. doi: 10.9734/jamb/ 2019/v18i330173 al-askar aa, rashad ym. 2010. efficacy of some plant extracts against rhizoctonia solani on pea. j plant prot res 50(3):239-43 doi: 10.2478/v10045-010-0042-0 andriyani f, purwantisari s. 2019. uji potensi ekstrak daun suren dalam menghambat pertumbuhan jamur colletotrichum capcisi secara in vitro [potential test of suren leaf extract in inhibiting the growth of colletotrichum capcisi fungus in vitro]. jurnal akademika biologi. 8(1):359. armavillia ke. 2023. angka konsumsi cabai rawit tahunan di indonesia meningkat [the annual consumption rate of cayenne pepper in indonesia increases]. [internet]. accessed on 8 may 2024. available from:. https:// data.goodstats.id/statistic/angka-konsumsi-cabai-rawittahunan-di-indonesia-meningkat-fatrk. budi p, kurniati n, marwan e. 2020. analisis usahatani cabai rawit di kecamatan sungai rumbai kabupaten mukomuko [analysis of cayenne pepper farming in sungai rumbai district, mukomuko regency]. jurnal agribisnis 13(2):1592-98. deden d, umiyati, u. 2017. pengaruh inokulasi trichoderma sp dan varietas bawang merah terhadap penyakit moler dan hasil tanaman bawang merah (allium ascalonicum l) [effect of inoculation of trichoderma sp. and shallot varieties on moler disease and yield of shallot plants (allium ascalonicum l)]. jurnal kultivasi 16(2):340-48. devianto y, dwiasnati s, sukowo b, fauzi a, baihaqi ka. 2023. application of the technique for order performance by similarity to ideal solution (topsis) to diagnose chili leaf spot disease. indonesian journal of machine learning and computer science. 3(2):136-42. djaenuddin n, suriani s, talanca ah. 2018. kombinasi biopestisida formulasi bacillus subtilis bnt8 dan pestisida nabati untuk pengendalian penyakit hawar daun bipolaris maydis pada jagung [combination of biopesticide formulated with bacillus subtilis bnt8 and botanical pesticide to control bipolaris maydis leaf blight in corn]. jurnal penelitian pertanian tanaman pangan, 2(1):43-9. doi: 10.21082/jpptp.v2n1.2018.p43-49 faqy rc, rustam r. 2019. uji beberapa konsentrasi tepung bunga cengkeh (syzygium aromaticum (l.) merr. and perry) untuk mengendalikan hama sitophilus zeamais m. pada biji jagung di penyimpanan [assessment of several concentrations of clove flower flour (syzygium aromaticum (l.) merr. and perry) to control sitophilus zeamais m. on corn kernels in storage]. unri conference series: agriculture and food security 1. doi: 10.31258/ unricsagr.1a9 harni r, taufik e, samsudin. 2018. effect of plant oils and extracts on uredospres of hemileia vastatrix germanition and attack intensity. jurnal tanaman industri dan penyegar 5(2):67-76. hartati sy. 2018. efikasi formula fungisida nabati terhadap penyakit bercak daun jahe phyllosticta sp. [efficacy of biovegetable fungicide formula against ginger leaf spot disease phyllosticta sp.] buletin penelitian tanaman rempah dan obat 24(1):42-8. hizrianti sd, natawijaya d, saepudin a. 2021. uji daya hambat minyak daun cengkeh dan ekstrak daun pepaya terhadap cendawan alternaria solani (ell. & mart.) sorauer pada tomat secara in vitro [assessment of the inhibitory power of clove leaf oil and papaya leaf extract against the fungus alternaria solani (ell. & mart.) sorauer on tomatoes in vitro]. media pertanian 6(1):30-44. doi: 10.37058/ mp.v6i1.3011 inaya n, meriem s, masriany m. 2022. identifikasi morfologi penyakit tanaman cabai (capsicum sp.) yang disebabkan oleh patogen dan serangan hama lingkup kampus uin alauddin makassar [identification of the morphology of chili plant diseases (capsicum sp.) caused by pathogens and pest attacks within the uin alauddin makassar campus]. filogeni, jurnal mahasiswa biologi 2(1):8-14. doi: 10.24252/filogeni.v2i1.27092 jirovetz l, buchbauer g, stoilova i, stoyanova a, krastanov a, schmidt e. 2006. chemical composition and antioxidant properties of clove leaf essential oil. j agric food chem 54(17):6303-7 doi: 10.1021/jf060608c. kusnadi j, andayani dw, zubaidah e, arumingtyas el. 2019. ekstraksi senyawa bioaktif cabai rawit (capsicum frutescens l.) menggunakan metode ekstraksi gelombang ultrasonik [extraction of bioactive compounds from cayenne pepper (capsicum frutescens l.) using the ultrasonic wave extraction method]. jurnal teknologi pertanian 20(2):79-84. lambiju em, wowor pm, leman ma. 2017. uji daya hambat ekstrak daun cengkih (syzygium aromaticum (l.)) terhadap bakteri enterococcus faecalis [the inhibitory power test of clove leaf extract (syzygium aromaticum (l.)) against enterococcus faecalis bacteria]. jurnal e-gigi, 5(1):79-83. doi: 10.35790/eg.5.1.2017.15547 lestari d, aini lq. 2021. pengujian konsorsium bakteri antagonis untuk mengendalikan penyakit bercak daun cercospora dan virus kuning pada tanaman cabai merah besar (capsicum annum l.) di kecamatan dampit kabupaten malang [assessment of antagonist bacterial consortium to control cercospora leaf spot disease and yellow virus on large red chili plants (capsicum annum l.) in dampit district, malang regency]. jurnal hpt 9(3):107-14. rismayanti, sumarsih e, nuryaman h, djuliansah d. 2022. risiko produksi usahatani cabai rawit (capsicum frutescenes l.) panen hijau dan panen merah [production risks of cayenne pepper farming (capsicum frutescenes l.) green harvest and red harvest]. jurnal ilmiah mahasiswa agroinfo galuh 9(3):1412-23. https://data.goodstats.id/statistic/angka-konsumsi-cabai-rawit-tahunan-di-indonesia-meningkat-fatrk https://data.goodstats.id/statistic/angka-konsumsi-cabai-rawit-tahunan-di-indonesia-meningkat-fatrk https://data.goodstats.id/statistic/angka-konsumsi-cabai-rawit-tahunan-di-indonesia-meningkat-fatrk biotropia vol. 32 no. 2, 2025 150 salam r, kripalini n, singh rki, supriya l, bumpy k. 2022. studies on leaf spot of chilli caused by cercospora capsici and its management in manipur. the pharma innovation journal 11(7):4363-74. šernaitė l, rasiukevičiūtė n, valiuškaitė a. 2020. the extracts of cinnamon and clove as potential biofungicides against strawberry grey mould. plants 9(5):613. sucianto et, abbas m. 2019. jenis, frekuensi kemunculan dan persentase penyakit cendawan pada tanaman sayuran [types, frequency of occurrence and percentage of fungal diseases in vegetable crops]. majalah ilmiah biologi biosfera 36(1):1-9. sudiartini npr, wirya gnas, sudarma im. 2021. identifikasi jamur penyebab penyakit utama pada tanaman selada (lactuca sativa l.) hidroponik [identification of the fungus that causes important disease in hydroponic lettuce (lactuca sativa l.)]. jurnal agroekoteknologi tropika 10(3):308-23. syafiruddin, hilda l. 2023. pemanfaatan tanaman sebagai pestisida hayati untuk pengendalian hama dan penyakit tanaman cabai dalam rangka mendukung pertanian berkelanjutan [utilization of plants as biopesticides to control pests and diseases of chili plants in order to support sustainable agriculture]. jurnal pengabdian mayarakat 2(3):22-34. thabet m, khalifa w. 2018. antifungal activities of clove oil against root rot and wilt pathogens of tomato plants. american-eurasian j agric & environ sci 18(3):105-14. doi: 10.5829/idosi.aejaes.2018.105.114 wakhidah n, kasrina, bustaman h. 2021. keanekaragaman jamur patogen dan gejala yang ditimbulkan pada tanaman cabai merah (capsicum annuum l.) di dataran rendah [diversity of pathogenic fungi and symptoms caused by red chili plants (capsicum annuum l.) in the lowlands]. konservasi hayati 17 (2):63-8. winarto b, wisnuwati. 2020. pembuatan makanan dan minuman herbal yang menyehatkan [making healthy herbal foods and drinks]. yogyakarta (id): deepublish. biotropia vol. 28 no. 3,2021: 239 252 doi: 10.1 1598/btb.2021.28.3.1362 antagonistic effect of yeast, acetic acid bacteria and mangosteen rind extract o n aflatoxigenic aspergifis f laws i n unfermented cocoa beans nijma nurfadila'", sutrisno~, usman ah mad^ and samsudin~ 'seameo biotrop, jalan raya tajzlr km. 6, bogor 16134, indonesia 2 s t ~ 3 pmgram of postharvest technology, faczllty ofagricultural techno lo^, instit~lt pertdnian bogor, bogor 16680, indonesia 3balai penelitan tanaman industri dan penyegar (balitttu), pamngkuda, st&abumi, indonesia 43357 received 22 may 2020 /accepted 6 january 2023 abstract yeasts and bacteria are two of common biocontrol agents to control mycotoxigenic fungi. meanwhile, the mangosteen rind extract contains xanthone and gartanin compounds for antioxidant, antiproliferation, antiinflammation, antimicrobial, and anticancer. the objectives of this research were to test the effects of yeasts, acetic acid bacteria (aab), and mangosteen rind extract on the aflatoxigenic a.ybegillz1sj7av.w growth and aflatoxin production in unfermented cocoa beans. four yeast isolates, i.e., i~satcbenkia orientalis (lo) bio 211291, 286 and 288, and endomyces fbulger (e! bio 132219; one bacteria isolate of acetobacter aceti (aa) fncc0016; and mangosteen rind extract (mre) were tested for their capabilities in inhibiting an aflatoxigenic a.javus (afj bio 3361/747 growth using the well method (in vitro). two types of yeast (10 bio 211291 and 288) were combined with a a and mre in cocoa beans (in vivo). aflatoxin production was analyzed using thin h y e r cbromathograph_v (tlc). the results showed that interaction of l o bio 211291 and 288, and efbio 132219 on aflatoxigenic af were interaction with inhibition zone > 2 mm (type d), while the interaction type of l o bio 211286 on afwere mutual intermingling growth, where both fungi grew into each other without any macroscopic sign of interaction (type a). the best treatment in agar media (in vitro) was io bio 211288 + a a on potato dextrose agar + 12 g/l mre. the highest l o population was 5.88 log cfu/g on cocoa beans inoculated by i o bio 211291 + mre in 1 day after inoculation, while the highest a. aceti population was 4.74 log cfu/g on cocoa beans with l o bio 21 1291 + bio 211288 + a a in 3 days after inoculation. two best treatments were i o bio 211288 + a a + mre and i o bio 211291 + bio 211288 + a a + mre, because there were no a.j7avuspopulation since 3 until 11 days after inoculation. aflatoxin in all samples treatment was lower than limit detection b1 (< 2.20 ppb), b2 (< 3.50 ppb), (gi < 0.54 ppb), dan (g2 < 1.00 ppb). keywords: aflatoxin, antagonistic, aspergillusj7avus, mangosteen rind, yeasts introduction indonesia is the yd cocoa beans exporter countries after ivory coast and ghana (dickson 201 8). agricultural department (201 0) reported as much as 93% of cocoa beans in indonesia was processed without fermentation (only sun dried) conducted by farmers, while the remaining 7% of cocoa beans was processed by fermentation. thompson et al (2013) explained that cocoa beans fermentation process involves *corresponding author, email: n.nurfadila@yahoo.co.id; nijma@biotrop.org some microorganisms, i.e., yeasts, acetic acid bacteria (aab), lactic acid bacteria (lab), baciiiz~s and several other bacteria, as well as filamentous fungi. according to nurhansyah (201 1) some importer countries such as malaysia and united states of america reduced the amount of cocoa beans import from indonesia, because the physicochemical quality of indonesian cocoa beans were lower than those from africa. the decreasing of cocoa beans quality was caused by unappropriate handhg during the harvesting, fermentation, drying, storing, and packaging processes that facilitates fungal contamination. biotropia vol. 28 no. 3,2021 according to asrul (2009) unfermented cocoa beans and mycotoxigenic fungi contamination, especially aflatoxigenic a.pe%iiius javus, were problems in indonesia. some f m g were isolated from cocoa beans in central sulawesi, i.e., aspergiiius javzls, a. niger, a. fumigatzls, peniciilium s p ., fusarium s p ., trichodema sp., t. virile, rhi~0pu.r sp., mucor sp., ven'iciilium sp., and geotricbm sp. a.pe@iius fiavu~ was isolated in cocoa beans after being harvested by farmers (7.2 x 108 cfu/ml), collector (4.5 x lo5 cfu/ml), and exporter levels (4.1 x lo3 cfu/ml). aflatoxin b1 content in cocoa beans after being harvested by farmers (104.80 ppb), collectors (61.31 ppb), and exporters (47.74 ppb). copetti et al. (2011) reported the occurrence of aflatoxigenic fungi and the presence of aflatoxin in 226 cocoa samples collected in brazilian farms. the aflatoxigenic fungi isolated in cocoa beans were a . javus, a. parasiticus and a. nomius. a considerable increase in numbers of these species was observed during drying and storage processes. pires et al. (2019) explained that the total aflatoxins in two cocoa bean samples from their study, from bahia, from par6 and from rond6nia were 13.2, 16.3, 11.7, and 30 pg/kg, respectively. according to basappa (2009) aflatoxin is a kind of toxins produced by a. flavus and a. parasiticus that causes liver cancer in human and animals. the types of aflatoxins found in foodstuffs and processed products are bi, b2, gi, and gg but the most dangerous for human health is aflatoxin b1 (afb1). one of the problems in unfermented cocoa beans is aflatoxigenic fung contamination. therefore, formulating and concocting a combination treatment to inhibit aflatoxigenic fungi is necessary. combinations of yeast, acetic acid bacteria, and mangosteen rind extract were used for testing the effectiveness in decreasing aflatoxigenic a. javm population and aflatoxin production. dharmaputra et al. (201 8a) reported that imctchenkia orientalis could inhibit 100°/o of ochratoxigenic a. ochraceus. it means that i. orientalis can be used as biocontrol agent, eventhough there is no one research explains that i. orientah can inhibit aflatoxigenic a . javus. sabahannur and ralle (2018) reported not only yeast, but acetobacter aceti also supposed to maintain shelf life of food stuff. meanwhile yatman (2012) explained that mangosteen can be used as medicine, because it contains xanthone for antioxidant, antiproliferation, antiimflammatory, antitumor, and anticancer. research conducted by akao et al. (2008) showed that xanthone cc-mangostin compound of mangosteen rind exract could inhbit 50% of colon cancer cells growth. the inhibitive nature of the compound is similar with commercially available anticancer drugs, i.e., 5-fu, actinomycin d, and campotechn. aisha et al. (2012) reported that xanthone extract, cc mangostin, and y-mangostin inhibited 50% of cancer cell in 6.5 rf: 1.0 mg/ml, 5.1 rf: 0.2 pg/ml, and 7.2 ? 0.4 pg/ml. therefore, combinations between yeast, acetic acid bacteria (aab) and mangosteen rind extract (mre) is expected to produce biocontrol agent on aflatoxigenic a. fiavus growth and decrease aflatoxin production in unfermented cocoa beans to be safe for next processing of chocolate products. the objectives of this research were to test the effects of yeasts, acetic acid bacteria (aab), and mangosteen rind extract on the aflatoxigenic a. flavzls growth and aflatoxin production in unfermented cocoa beans. it is expected that the research result would show a potential combination of yeast, acetic acid bacteria (aab), and mangosteen rind extract (mre) as biocontrol agent to inhbit aflatoxigenic a.flavus to improve food safety in unfermented cocoa beans. materials and methods yeast isolates, acetic acid bacteria, matoxigenic fungus, unfermented cocoa beans, and mangosteen rind extract as many as four yeast isolates were used in interaction types and antagonistic test, i.e., 1 isolate of endomycesjbuhger (efj bio 132219 and three isolates of issatcbenkia orientalis (lo) bio 211286, bio 211288, and bio 211291; only two yeast isolate were used in cocoa beans (in vivo) that showed the highest percentage of inhibition on a. fiavus in vitro, i.e., i. orientalis bio 21 1291 and bio 211288. aflatoxigenic aspetgiiiusjavus bio 3361/747 were obtained from phytopathology laboratory culture collections, antagonistic effect of yeast, acetic acid bacteria, and mangosteen rind extraction nurfadila e t al seameo biotrop; and 1 acetic acid bacteria isolate (acetobacter aceti fncc0016) was obtained from food and nutrition study centre, universitas gadjah mada, yogyakarta. as much as 62 kg of unfermented cocoa beans were obtained from sumedang regency, west java province. as much as 2 kg of mangosteen rind extracts was obtained from rj herbal, surabaya, east java province. interaction types test between yeast and atlatoxigenic a. flavus four yeast isolates were tested on aflatoxigenic a. jlavzls bio 3361/747 using direct opposition method (dharmaputra et al 2018b) (fig. 1). this method was used to determine the interaction types between the yeasts and aflatoxigenic a. jlavzls. aflatoxigenic aspevgillas jlavas bio 3361 /747 was inoculated after 4 days of each yeast inoculation in the middle of potato dextrose agar (pda) media in observation on the interaction types was conducted macroscopically between the yeast and aflatoxigenic a. jlavas (wheeler and hochng 1993). the interaction types were shown in table 1. the mathematical equation for calculating the percentage of inhibition between the yeast and aflatoxigenic a. flavas is as follows: notes: % i = percentage of inhibition, j1 = diameter of a. jlavus near to petridish, jz = diameter of a. javas near to yeast. petri dishes (a dameter of 9 cm) with a &stance of 3 cm between each other. the petri dishes figure 1 scheme of antagonisms test between yeast isolate and toxigenic a. jlavus; a= toxigenic a. with each fungal and yeast were then incubated flauus, b = yeast isolate, j1 = diameter of a. at room temperature (27 & 2 "c) for 7 days. five flauus near to petridish, jz = diameter of a.jlauas replicates were used for each isolates. the near to yeast table 1 interaction types between two fungal colonies (wheeler & hocking 1993) tvpe of interaction description of classification a mutual intermingling growth, where both fungi grew into each other without any macroscopic signs of interaction mutual inhibition on contact or space between colonies small (< 2mm) inhibition of one species on contact, the inhibited species continued to grow at a significantly reduced rate, while the inhibitor species grew at a slightly reduced rate or unchanged mutual inhibition at a distance (> 2 mm) e inhibition of one species on contact, the inhibitor species continuing to grow at a reduced rate through the inhibited colony f inhibition of one species on contact or at a distance, the inhibitor species then continuing to grow at an unchanged rate through or over the inhibited colony a notes: a: inhibitor fungi, b: inhibited species. biotropia vol. 28 no. 3,2021 obtaining of unfermented cocoa beans and antagonistic test of yeast, acetobacter mangosteen rind extract acetl; and mangosteen rind extract f ipe lindak cocoa (bulk cocoa) fruits with combinations on aflatoxigenic a. ffavus in yellow color were harvested using sterile scissors vitro from the trees. the cocoa beans were were then cut open using a knife and separated into parts of pods and cocoa beans with pulp. the next process were washing of cocoa beans from pulp and drying using sun-drying for 1 day (9 hours) to become unfermented cocoa beans. obtaining of mangosteen rind extract is shown in figure 2. as much as 12 g/l of mangosteen rind extract (mre) was used in in vitro stage, while 12 g for 500 g of cocoa beans used in in vivo stage. according to kusumaputri (2011), dr. berna eliya as a phytochemist in universitas indonesia, explained that many people consume a glass of boiled water containing mangosteen rind extract. they usually use 60 g of fresh mangosteen rind equal to 12 g of mangosteen rind extract for 1 day. the doses of mangosteen rind extract (mre) was obtained based on consumer's doses in mre capsule product, as follows: antagonistic test between yeast, acetobacter aceti and mangosteen rind extract on aflatoxigenic a. flavas bio 3361/747 was conducted using the well test method (dharmaputra et al 2016 with modification). acetobacter aceti fncc0016, issatchenkia orientah bio 211291, bio 211288, bio 211286, and endomycesjbaliger bio 13221 9) were cultured on malt extract agar (mea) media and incubated for 7 days, while the acetobacter aceti fnccool6 was cultured on peptone glucose yeast extract agar (pgya) media and incubated for 3 days at room temperature (27 k 2 oc). aflatoxigenic aspe~illusflavzzs bio 3361/747 was cultured on potato dextrose agar (pda) media and incubated for 7 days at room temperature (27 k 2 oc). five pieces (in 5 mm diameter) of pure culture of each yeast were placed into 25 ml of nutrient yeast dextrose broth (nydb) media in an erlenmeyer flask (100 ml volume), whle the acetobacter aceti isolate was placed into 25 ml of nutrient broth (nb) media. they were then incubated at 27 k 2 6~ for 7 days, and were shaken for 1 hour every 24 hours for 5 days. the conida cells of aflatoxigenic a.flavzls (5 x 106 cells/ml) were obtained by adding 15 ml of distilled water. then the a. flivas was scratched notes: de = doses of mangosteen rind extract (g), ji< = j on the upper surface using sterile inoculation number of capsules in 1 package of mangosteen rind extract product, be = weight of mangosteen needle. the conidia cells were filtered by sterile rind extract in 1 capsule (mg), and bi< = number gauze on the funnel of erlenmeyer flask (100 ml of capsules to be cdnsumed &i 1 day. volume). \i/ grinding of mangosteen rind to become sun-drying for 3 days mangosteen rind extract (mre) (7 hours/day) harvesting of ripe mangosteen fruits i packaging of 2 kg mangosteen rind extract sending of 2 kg mre separating of fruits and pericarp of mangosteen figure 2 stages of obtaining of mangosteen rind extract (mre) antagonistic effect of yeast, acetic acid bacteria, and mangosteen rind extraction nurfadila et al. the yeast, acetobacter aceti, and a. flavtls cells were precipitated by centrifugation using a centrifuge at 7,000 rpm fixed angle rotor for 15 minutes and rinsed using twice sterile disulled water, and they were then resuspended in sterile distilledwater until the concentration reached 5 x 10' cells/ml (yeast cells) and 5 x lo6 cells/ml (acetobacter aceti and aflatoxigenic a. flavm). yeasts, acetobacter aceti and a s p e ~ ~ ~ t l s f l a v a s cells were counted using a hemocytometer. a well (5 mm dameter) was prepared using a cork borer in the center of potato dextrose agar (pda) meda containing 15% cocoa beans juice and pda media containing 15% cocoa beans juice and 12 g/l of mangosteen rind extract depending on the treatments into a petri dish (9 cm diameter). as much as 20 yl of 5x10' cells/ml yeast cells suspension were placed into the well. the petri dishes were left for 30 minutes to allow penetration of cells suspension into the well. next, as much as 20 yl of 5 x 106 cells/rnl acetobacter aceti was inoculated, then a. flavas was also inoculated into the well after 1 hour. each yeast control was only inoculated with yeast cells suspension. each treatment and controls were made in 3 replicates (= 5 petri dishes), which were then incubated at room temperature (27 k 2 "c). the growth of the aflatoxigenic a.flavzls, yeasts, and acetobacter aceti in each petri dish was observed after 7 days of incubation. total unit experiment for in vitro process was (4 yeast isolates x 2 within or without acetobacter aceti x 2 kmds of media x 3 replicates) + ((4 yeast control + 1 a. flavtls controlj x 3 replicates) = 63. the radius of aflatoxigenic a. flavzas colony in each petri dish was measured before the colony reached to petri dish. mathematical equation for the percentage of fermentor inhibition to aflatoxigenic a.flavzls is: notes: o/o i = percentage of inhibition, dl = diameter of a. jlauzls control (mm), dz = diameter of a. flavzrs in each treatment (mm). testing of combination treatments to inhibit aflatoxigenic aspergilius ffavus in vivo as many as two yeast isolates with the hghest percentage of inhibition in vitro stage, acetobacter aceti fncc00 1 6, and aspergiiias flavas bio 3361/747 were used in vivo stage. the steps in the testing of the combination treatments to inhbit aflatoxigenic a. jlavtls in vivo were similar to in vitro. each treatments included positive and negative controls using 500 g of unfermented cocoa beans for each replicates. as much as 10 ml of 10' cells/ml yeast cells suspension were placed into 500 g of unfermented cocoa beans depending on the treatments. each samples was then inoculated or not inoculated by 10 ml of lo6 cells/ml a. aceti (depend on the treatments) after 30 rnin, then as much as 10 ml of lo6 cells/ml aflatoxigenic a.flavas was inoculated into the samples depending on the treatments after 1 hour. each cells inoculation was conducted sequentially to allow the cells penetration. total unit experiment for in vivo process was 12 x 4 x 2 = 96 (12 = types of combination including positive and negative controls; 4 = days after inoculation (1,3, 6 and 11); 2 = replication). determination of issatchenkia orientafis, acetobacter aceti, and aflatoxigenic aspergillus ffavus populations, and aflatoxin production yeast, acetobacter aceti, and a. jlavtls were isolated using serial dilution method (10.' up to followed by pour plate method on potato dextrose agar (pda) and incubated for 7 days incubation at 27 k 2 u (ins 2008). aflatoxin contents were determined using thin layer chromatoghraphy (tlc) (baiton et al. 2006). results and discussion interaction types between yeast and aflatoxigenic a. ffavus as many as four yeasts (one isolate of endomycesjbtllger bio 13221 9 and three isolates of issatchenkia orientalis bio 21 1286, bio 211288, and bio 211291) were used in testing the mechanisms of antagonism on aflatoxigenic a. flavtls bio 3361 /747 using drect opposition method. the interaction type of antagonism mechanism between yeast and i. orientalis bio 211291 (49.58%) and bio 211288 (35.79%), and e. jbaiger 132219 (49.29%) with aflatoxigenic a . flavtls was d (fig. 3a, 3c, 3d; table 1). this interaction type showed that there biotropia vol. 28 no. 3,2021 was a mutual inhibition with the inhibition zone of > 2 mm similar with wheeler and hocking (1 993). the interaction type of i. orientalis bio 21 1286 with a.javvzls bio 3361 /747 was a (fig. 3b) where the percentage of each inhibition was 15.630/07 respectively (table 1). this interaction showed mutual intermingling growth, where both fungi grew into each other without any macroscopic signs of interaction (fig. 3; table 1). based on the percentage of the inhibition of a.3avu-r growth using direct opposition method, three isolates (i. orientalis bio 21 1291, e. jbulger 13219, and i. orientalis bio 211288) were higher than i. orientah bio 211286. it means that i. orientah bio 211291 and bio 21 1288, and e. jbulger bio 13219 were prefer as potential component inhibitor for aflatoxigenic a. j2avu-r in cocoa beans. nevertheless, the three yeast isolates should be combined with additional materials to increase the ability of inhibition on aflatoxigenic a. javu-r in unfermented cocoa beans. however, this result was strengthened in the next step using the well in vitro method. figure 3 mechanism of interaction between four yeast isolates (issatchenka orientalis): (a) bio 211291; (b) bio 211286; (c) e~domycesjbahger bio 133219; and (d) i. orientalis bio 211288); aflatoxigenic a.pe~iiasjavt/s on potato dextrose agar (pda) media after 7 days of incubation at room temperature (28 + 2 oc) table 1 interaction types and the percentage of inhibition between yeast on aflatoxigenic a.perg'iiusjaut/s based on direct opposition method mean of radius (rnrn) isolate % inhibition interaction type interaction figure 11 12 issatchenkia orientah bio 211291 vs a.jlauas od i. orientalis bio 21 1288 vs a. jlauas od i. orientalis bio 21 1286 vs 24 20.25 15.63 c a. jlaul~s endomycesjbahger 28 14.2 49.29 a d bio 13221 9 vs a.jlauas od antagonistic effect of yeast, acetic acid bacteria, and mangosteen rind extraction nurfadila et al. antagonistic test in combination of yeast, acetobacter aceti and mangosteen rind extract on matoxigenic aspergirrus flavus in vitro according to richard and prusky (2002) yeast has some unique charactheristics such as fast growth, the ability to colonize surface of fruit, and the ability to join in nutrition competition with a pathogen, therefore it can be a biocontrol agent. yeast also has an important role in fermentation, because it can convert glucose and maltose through anaerobic respiration. the well method is a method for determining on the percentage of inhibition between yeast and pathogenic fungi. all treatments except yeast controls were not inoculated by aflatoxigenic a. flavzts. widiyanto et al. (2013) reported that unfermented cocoa beans has no any glucose, because the pulp has been removed. it means that additional material is needed to maintain beneficial microorganisms survival in unfermented cocoa beans. according to maligan et al. (2018) mangosteen rind extract (mre) contents in 100 g are 82.50% carbohydrate, 6.45% fats, 3.02% proteins, 5.87% water, and 2.10% total glucose. the nutritional content of mre may increase the yeast survival. mre also has xanthone for anticancer, antihyperglicemic, and antioxidant for human health. other research, acetobacter is one of bacteria that can oxidize glucose to gluconic and other organic acid in the same time to maintain the shelf life of foodstuff (simanjuntak et al. 201 6). therefore, in this research, each of four yeast isolates were combined with acetic acid bacteria and mre in vitro stage to test the effectiveness of those treatment in different media (pda + 15% cocoa beans juice with and without 12 g/l mre). the highest percentage of inhibition on aflatoxigenic a. flavzls bio 3361/747 was 100%, in treatment with issatchenkia orientalis bio 211288 + acetobacter aceti fnccool6 on potato dextrose agar (pda) + 15% cocoa beans juice + 12 g/l mangosteen rind extract (mre) media (fig. 4a; table 2). it means those treatment more effective than other treatments to against the aflatoxigenic a.flauus in vitro. the 2nd highest percentage of inhibition was 51.98%, in treatment i. orientalis bio 211291 on pda + 15% cocoa beans juice + 12/g mre (fig. 4b; table 2). hafsari (2011) explained that the differences in fungal diameter indicated that yeast growth is faster than that of fungi and it also obtained more nutrition than pathogenic fungi. the stunting growth of fungi was shown from the diameter of colony that was lower than the control (+). according to janisiewicz and korsen (2002) the mechanism of space and nutrition competitions could happened if the yeast's effort was higher than pathogenic fungi to get nutrition and space. the similar research from golubev (2006) explained that the capability of yeast antagonism would increase on other microorganism from different habitat, because the fungi are a new competitor that should be defeated to become a dominant in the available space and nutrition. af blo 3361 did not grow lo bio 211291 figure 4 (a) issatchenkia orientalis bio 211288 and (b) i. orientalis bio 211291 vs toxigenic a.jauzis bio 3361/747 with acetobacter aceti on potato dextrose agar + 15% cocoa beans juice + 12 g mre at room temperature (27 + 2 oc) after 7 days of incubation biotropia vol. 28 no. 3,2021 table 2 effect of different combination and media treatments on the percentage of inhibition and growth of yeast, acetic acid bacteria, and aflatoxigenic a.ji'auzrs treatment diameter (mm) inhbition combination meha yeast acetobacter aceti aspepxiiius flavus yo) issatcbenkia orientah 291 + a.flavus 15.45 37.20 20.00 i. orientalis 288 + a.flavus pda + 15% cocoa 27.35 30.15 25.56 i. orientalis 286 + a.flavus beans juice nd 40.00 9.03 endomycesjbul&er 21 9 + a.flauus nd 42.10 9.46 i. orientah 291 + a.flavus pda + 15% cocoa 25.50 34.17 26.52 i. orientalis 288 + a.flavus beans juice + 12 g/l 34.47 25.23 45.74 i. orientalis 286 + a.flavus mangosteen rind nd 39.79 14.43 e. fibulzker 21 9 + a. flavus extract nd 38.13 18.00 i. orientalis 291 +acetobacter aceti + 11.21 nd 30.60 34.1 9 aspergilasflavus pda + 15% cocoa i. orientalis 288 +a. aceti + a.fiauus beans juice 30.01 nd 24.57 47.16 i. orientah 286 +a. aceti + a. flavus nd nd 37.92 18.45 e.jibuliger 21 9 + a. aceti + ~ . ~ a v u s nd nd 39.44 15.18 i. orientah 291 +a. aceti + a.flavus pda + 15% cocoa 29.98 5.26 22.33 51.98 i. orientalis 288 +a. aceti + a.flauus beans juice + 12 g/l 34.45 18.62 nd 100.00 i. orientah 286 +a. aceti + a . flauus mangosteen rind nd nd 40.87 12.11 e.-fibuker 21 9 + a. aceti + a. flauus extract nd nd 39.77 14.47 i. orientalis 291 (control) 35 0 i. orientah 288 (control) 40 pda + 15% cocoa 0 i. orientalis 286 (control) 15 beans juice 0 e$buhger 21 9 (control) 11 0 alflavus (control+) 46.50 0 notes: (-) = not inoculated; nd = not detected; 0 = no inhibition (control). based on different yeast isolates, i. orientalis bio 21 1291 and i. orientalis bio 211288 were potential as a component inhibitor for a . j a v u s bio 3361/747 in vitro. based on different agar media, pda + 15% cocoa beans juice + 12 g/l mre media was more effective than pda + 15% cocoa beans juice meda to inhibit aflatoxigenic a. flavus in vitro. it means that mre influenced on decreasing of aflatoxigenic a. javus growth in vitro. yatman (20 12) reported that xanthone of mangosteen rind extract can be used as antimicrobial, antioxidant, antifungi, and anticancer. populations of issatchenkia orientalis, acetobacter acetl; and aspergigus ffa vus in vivo two yeast isolates with the highest percentage of inhibition on aflatoxigenic a. javus in vitro were ismtcbenkia orientalis bio 21 1291 and bio 211288. the yeast isolates were used in vivo stage. the highest population of yeast i. orientalis of cocoa beans in 1 day after inoculation was 5.88 log cfu/g, found in samples inoculated by with i. orientalir bio 211291 + mangosteen rind extract (mre) + a.javus. the highest i. orientalis population in 3 and 6 days after inoculation were 4.67 and 3.75 log cfu/g, found in samples inoculated by i. orientalis bio 211291 + bio 211288 + a. aceti + mre + a. javus. the highest i. orientalir population in 11 days after inoculation was 2.82 log cfu/g, found in samples inoculated by i. orientalis bio 211291 or bio 211288 + mre + a. flavus fable 3). the negative and positive controls, sample with acetobacter aceti + mangosteen rind extract + a. flavus were not inoculated by i. orientah. based on dfferent days after inoculation, commonly i. orientalir population in 3 day after inoculation was higher than 1, 6 and 11 days after inoculation. yeast population in all samples with mre were not sipficant dfferent with samples without mre. jamili et al. (2016) reported the dominant yeasts were found in cocoa beans during fermentation, i.e., 1 isolate candida kmsei, 3 isolates c. tropicalis, 1 isolate saccbaromycopsis jbuhgera, 1 isolate kloeckera sp., and 1 isolate saccbaromyces cerevisiae. according to ren et al. (2020) many microorganisms including bacteria, non-toxigenic fungi, and yeast strains have been investigated as potential biocontrol agents against to aflatoxigenic fungi. lee et al(2008) reported that peroxisomal 3-ketoagl-coa tbiolase biotropia vol. 28 no. 3,2021 influence the ability of a. j a v m growth during could be used to determine the ability of fungal storage are temperature, relative humidity, and growth. not only temperature, relative humidity moisture content. norlia et al. (2019) also and water activity, but also co2 levels influence reported that relative humidity and water activity the fungal growth (giorni e t al 2018). (a,) in foods are interrelated to each other and table 3 issatchenkia orientah, acetobacter aceti and a.pe@iizls yauzls populations in unfermented cocoa beans with combination treatments since 1 until 11 days after inoculation issatchenkia orientah population acetobacter aceti population aspe@izlsjlauzls population treatment 0% cfu/g) 0% cfu/g) 0% cfu/g) day after inoculation day after inoculation day after inoculation negative control (without 3.97 inoculation of microorganisms) i. orientalis 291 + i. orientalis 288 + 2.52 3.80 2.90 2.73 2.52 2.48 0.48 0.48 mre +a.flautls i. orientalis 288 + 4.38 4.30 3.20 2.82 mre + a. flauus i. orientalis 291 + 5.88 3.75 2.82 2.82 mre + a . flautls *i . orientalis 291 + i. orientalis 288 + acetobacter aceti + 3.12 4.67 3.75 2.52 4.22 3.30 2.75 3.12 3.75 nd nd nd mre +a.-flautls i. orientah 291 + i. orientalis 288 + acetobacter aceti + 3.43 4.12 2.78 1.94 3.52 4.75 3.30 2.14 0.48 1.48 1.23 nd a. jyauus i. orientalir 288 + acetobacter aceti + 2.00 4.12 2.43 nd 2.43 2.48 2.88 2.64 0.48 0.48 3.08 2.22 a. fl.uus i. orientalis 291 + acetobacter aceti + 3.07 4.64 2.12 nd 3.26 4.73 2.37 2.37 2.74 nd 1.00 1.52 mre +a.flauus *i . orientah 288 + acetobacter aceti + 3.22 4.12 2.12 nd 3.75 3.56 2.12 2.00 0.48 nd nd nd mre +a. flauus i. orientalis 291 + acetobacter aceti + 3.56 3.60 2.67 nd 2.12 2.48 2.87 2.80 2.75 2.00 2.94 3.48 a. flauzls a. aceti + mre + a. flnutls positive control (a. flauzls) > " , notes: mre = mangosteen rind extract; nd = not detected; (-) = not inoculated. antagonistic effect of yeast, acetic acid bacteria, and mangosteen rind extraction nurfadila e t al. 0 0 0 9 9 9 e 3 v v v 0 0 0 9 9 9 3 3 3 v v v 0 0 0 9 9 9 3 3 3 v v v 0 0 0 9 9 9 3 3 3 v v v * * o 2 2 r q i v v s f 4 a, 0 * * o o 2 2 0 3 v v s l ; 0 * * m a 2 2 0 9 v v s h " a, a, d 2 2 2 2 v v m i i 6 0 0 0 . l? m x x " a, v v v g lo 0 o o m l? l ? : 1 1 m m v v v 8 . .a 0 0 0 g x x x o v v v : i i 0 0 0 x 2 x 6 u v v v g 6 0 o w 0 2 9 & v v x g 0 v v 0 0 0 , j h! " v 2 2 2 v r i i u cd li u 0 0 3 x 2 : a -g g 5 4 2 % q & g 9 7 3 s + g g p : y 1 1 " -2 2 8 2 4 5 + " 2 sp: ;, g .. .% + $ .* $ :g g biotropia vol. 28 no. 3,2021 range of temperature and relative humidity i. orientalis bio 21 1288 + a. aceti + mangosteen in the storage room after cocoa beans rind extract, because a . j a v a ~ was not grown in inoculation with various treatments were 27 the samples for 3 until 11 days after inoculation. 28 oc dan 78 80%. water activity and co2 based on aflatoxins content, all combination levels were not determined in this research. suttajit (2014) reported that the optimum temperature for fungal growth was 25 40 oc. aflatoxins content in positive control were aflatoxin b1 (74.01 ppb), g1 (54.05 ppb), and total aflatoxin (128.06 ppb), while all sample treatments included negative control contained aflatoxin b1 were lower than the limit detection, i.e., bi (< 2.20 ppb), bz (< 3.50 ppb), gi (< 0.54 ppb), and g2 (< 1.00 ppb) (table 4). there were no significant differences between combination treatments on aflatoxin production. it means that all unfermented cocoa beans with combination treatments within or without mangosteen rind extract could be processed into chocolate products, because the aflatoxins were relatively safe. according to mazumder and sasmal (2001) the maximum tolerable limit for aflatoxin in cocoa beans, cocoa butter, and cocoa powder in bulgaria are 5 ppb, while in uruguay and malaysia the limits are 10 ppb. according to scott and pryzbylski (2020) the range of aflatoxins in raw cocoa beans from trinidad and ghana was 8 35 pg/kg. maciel et al. (2018) reported as much as 38% cocoa beans in southern region of bahia, brazil were contaminated by aflatoxin in the range < lod 17.795 pg/kg, 25% and 18% of total samples were contaminated by afbl and ochratoxin a in the range of <lod274.90 &/kg. conclusion there were 2 kinds of interaction types obtained from the antagonisms test between 4 yeasts (i. orientah bio 211291, bio 211288, bio 21 1286, and endomycesjl;bahger bio 132219) and aflatoxigenic a. javas, i.e., type d and a. the best combination treatment in vitro was found in unfermented cocoa beans with i. orientalis bio 21 1288 + acetobacter aceti + a. javas on potato dextrose agar with 15% cocoa beans juice and 12 g/l mangosteen rind extract media. two best treatments in unfermented cocoa beans in vivo were found in samples inoculated by i. orientah bio 211291 + bio 21 1288 + a. aceti + mangosteen rind extract and treatments were relatively safe to be processed, because its very low. acknowledgments the authors gratefully acknowledge the financial support through daftdr isidn pelaksanaan angaran (dipa) 20 1 9 and seameo biotrop. thanks are due to director of seameo biotrop and research laboratory supervisor for giving permission on using of laboratory facilities, to ms. okky setyawati dharmaputra and to mrs. ina retnowati for her suggestion, mrs. ratnaningsih and mrs. sylfa fauzia for their assistance. references agricultural department fnternet]. 2010. pencanangan gerakan nasional fermentasi kakao untuk mendukung industri dalam negeri (the declaration of the national movement for cacao fermentation to support the domestic in dust^); [updated 2010; cited 201 8 nov 11. available from: www.deptan.go.id. aisha afa, abu-salah i=, ismail 2, majid amsa. 2012. in vitro and in vivo anti-colon cancer effects of garcinia mangostana xanthones extract. compl and alternative med 12:l-10. akao y, nakagawa y, linuma m, nozawa y. 2008. anti cancer effects of xanthones from pericarps of mangosteen. int j of mol sci 9:355-70. ardhana m, fleet g. 2003. the microbial ecology of cocoa bean fermentations in indonesia. int j of food microbiol 86(1):87-99. asrul. 2009. populasi jamur mikotoksigenik dan kandungan aflatoksin pada beberapa contoh biji kakao (theobromae cocoa) (mycotoxigenicfnngip~uiation and aflatomn content in several examples af cacao beans) (theobromae cocoa). j agroland 16(3):258-67. baiton sj, coker rd, jones bd, morley em, nagler mj, turner rl. 2006. mycotoxin training manual. london (gb): tropical products institute. basappa sc. 2009. aj5'atommns; formation, anabsis and control. new delhi (in): narosa publishing house. copetti mv, iamanaka bt, pereira jl, fungaro mh, taniwaki mh. 2011. aflatoxigenic fungi and aflatoxin in cocoa. int j of food microbiol 148(2):141-4. antagonistic e f fect o f yeast, acetic acid bacteria, and mangosteen rind extraction nurfadila et al. dharmaputra o s , retnowati i , nurfadila n . 2018a. potency o f yeast as biocontrol agent o f ochratoxin a producing fungi and its e f fect o n the taste o f arabica cof fee . research report. bogor (id): s e a m e o biotrop. dharmaputra o s , sudirman li , misnawati mm. 2016. potensi khamir sebagai agen pengendalian hayati colletotrichum cqsici, cendawan penyebab antraknosa pada buah cabai (poteng of yeast as biological control agent of colletotrichnm caps& fungal causing anthracnose in chill). j hort indonesia 7(2):89 100. dharmaputra o s , sudirman i , ratnaningsih e a . 2018b. mikobiota pada buah pisang kultivar lampung untuk pengendalian hayati fusarium semitectum (mycobiota on bananafmlit h p u n g cultiuarfor biological control offusarium semitectm) . jfi 14(1) :30-8. dickson pnternet]. 2017. sepuluh negara penghasil kakao terbesar di dunia (the bzjgest o f ten producing cocoa beans countries in the world). 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holothuria atra: stocking density and time retno hartati1*, ambariyanto1, muhammad zainuri2, and widianingsih1 1department of marine science, faculty of fisheries and marine science, universitas diponegoro, kampus tembalang, semarang 50275, indonesia 2department of oceanography, faculty of fisheries and marine science, universitas diponegoro, kampus tembalang, semarang 50275, indonesia received 28 december 2018/accepted 28 january 2020 abstract strong market demand, uncontrolled exploitation and/or the inadequate fisheries resource management have caused the overexploitation of sea cucumbers. hence, sea ranching is suggested as an intervention to overcome, if not minimize, this problem. since stocking density is the most important consideration in sea cucumber rearing, this study is aimed at discovering the best stocking density for the ranching of holothuria atra. h. atra individuals were taken from the panjang island, jepara waters and reared in cages at the bottom of teluk awur waters, jepara with a density of 30, 20, or 10 individuals per cage measuring 2 × 2 × 1.8 m (with bottom area of 4 m2). the stocking of h. atra was carried out three times, starting from the time of cage installation, then at the second, and finally at the third months after installation. characteristics of the bottom sediment (i.e., chlorophyll a, b, phaeophytin, and total carotene) of the sea cucumber habitat and water quality in the cages were also measured monthly. this study showed that the growth of h. atra fluctuated. the low stocking density yielded a higher weight gain per individual than the high stocking density. the highest weight gain was observed at the stocking density of 10 individuals/cage in the second stocking month. the highest survival rate was recorded at stocking density of 30 individuals/cage (93%) in the third stocking month, considering that these sea cucumbers were only reared for three months. the highest mortality occurred at stocking density of 20 individuals/cage. low survival rate of 45% occurred at the first stocking time or after the fifth month of rearing. fission was observed among h. atra reared in the cages, resulting in smaller organisms. among the water quality parameters, the concentration of chlorophyll a, b, phaeophytin, and carotene in the sediment fluctuated with the rearing duration due to the feeding and bioturbation of sea cucumbers. the results of this study suggested that low stocking density of h. atra during the second stocking month yielded a higher growth rate. keywords: fission, growth, sea cucumber, sediment, survival introduction utilization of invertebrate fisheries resources have recently expanded in catch and value, worldwide (anderson et al. 2011). one increasingly harvested marine invertebrate species is the sea cucumber, locally known in indonesia as “teripang”, “trepang", “timun laut”, or “gamat” (hartati et al. 2015). strong market demand resulting in uncontrolled exploitation, and/or inadequate fisheries management have led to the overfishing of many marine species, including the sea cucumber (ambariyanto 2017). hence, to overcome this problem, sea ranching was suggested. sea ranching is essentially an effort to release/grow cultured or wild juveniles into the natural habitat and harvest them when they reach optimum commercial size (purcell et al. 2012a, b). some advantages of sea ranching include nominally lower inputs, as the processes between release and harvest are largely left to food availability in the habitat, and the minimum level of care in sea cucumber rearing. these practices were able to yield a marketable size of the sea cucumber. however, published studies corresponding author, email: retnohartati.undip@yahoo.com holothuria atra stocking density and stocking time – hartati et al. 129 on sea ranching of sea cucumber in indonesia is very limited. attempts at culturing sea cucumber is mainly grow-out practices, especially for h. scabra (pangkey et al. 2012). similar studies on the ranching of sea cucumber had been done in several countries mainly for h. scabra, such as; in philippines (juinio-meñez et al. 2012), australia (bowman 2012), and papua new guinea (hair et al. 2016). studies for other species were also done in ecuador and mexico for isostichopus fuscus (mercier et al. 2012) and portugal for h. arguinensis (domínguez-godino et al. 2015). consequently, to avoid the overexploitation of the natural populations of h. atra, further research on its sea ranching prospect is necessary. such study would also provide knowledge to ensure a better understanding and application of programs in marine conservation, population genetics, and connectivity patterns. a new trend emerging among indonesian fisherfolks is the cultivation of sea cucumbers in sea pens. sea ranching practices provide a way to restore damaged fisheries without having to legalize no-take zones or establish fishing rights for sea cucumbers. the sea ranching of sand fish h. scabra sea cucumber has given the fisherfolks additional activity i.e., rearing small sea cucumbers harvested from the wild in sea pens until they reach a marketable size (juiniomeñez et al. 2012; hair et al. 2016). among the recent published and unpublished data on sea cucumber from mariculture programs in the indo-pacific providing hatchery production, the use of juveniles (for experimental sea or pond farming, sea ranching, and/or stock enhancement reasons), and proponents information, very little came from indonesia (purcell et al. 2012a,b). this might be due to a lack of international publications on sea cucumber research in indonesia. moreover, for conservation purposes, no published work is available on the sea ranching of sea cucumbers in indonesia. with the declining natural stock of sea cucumbers (hartati et al. 2015), sea ranching technology has become urgent for the continuity of its production and conservation considering that h. atra, locally known as “teripang hitam”/loly fish, provides a source of protein for human consumption as well as bioactive molecules for marine pharmaceuticals. the species is also ecologically important for their sediment bioturbation and remineralization, and also demonstrate a specific reproduction scheme (i.e., asexual reproduction through natural fission). since stocking density plays a very critical role in aquaculture growth, the objectives of this study, therefore, was to determine the best stocking density and stocking time on the rearing of sea cucumber h. atra, and their specific effects on the species performance and characteristics of the bottom sediment in the cages used. materials and methods some 180 individuals of h. atra, weighing 100 150 g, were taken from panjang island, jepara waters. bottom cages measuring 2 × 2 × 1.8 m were installed in teluk awur waters, jepara with a geographic position of 06o37'43.8" s and 110o38'31.7" e. the habitat has a muddy sand substrate with sea grasses and seaweeds and is known to be a suitable location for the rearing of h. atra (hartati et al. 2017a). the stocking densities applied were 30, 20, and 10 individuals/cage (equivalent to 7.5, 5, and 2.5 individuals/m2), with three different stocking times i.e., when the cages were first installed, then at the second and the third months after installation. the stocking density applied in the study is based on the works of lavitra et al. (2010a) on h. scabra and xing et al. (2012) on apostichopus japonicas. stocking density is affected by behavioral performance due to competition for area and food. the sea cucumber weights were measured monthly to determine their growth performance and their numbers were counted at a determined time interval to calculate the survival rate. samples of bottom sediment were taken monthly to measure the biomass of the micro phytobenthic organisms through their photosynthesis pigment (chlorophyll a, b, phaeophytin; and carotene). biomass of microphytobenthos were measured as concentration of chlorophyll a, b based on jonge et al. (2012) and kuczynska et al. (2015), phaeophytin (method of montani et al. 2012) and carotene (method of androuin et al. 2018). water quality parameters (e.g., temperature, salinity, ph, and dissolved oxygen) biotropia vol. 28 no. 2, 2021 130 were also measured using a water quality checker in situ. the growth rate (weight gain) was calculated following xing et al. (2012): weight gain = w2 − w1 (1) w1 = weight of h. atra at tn-1 (grams) w2 = weight of h. atra at tn (grams) the survival rate was calculated as follows: sr (%) = (nt/n0) × 100% (2) nt = number of h. atra alive at the end of the experiment n0 = number of h. atra stocked at the beginning of the experiment results and discussion the growth and survival rates of h. atra weights of h. atra were measured at the beginning of the study and every month thereafter, until the study was completed. weight gains were influenced by both the stocking density and time. notably, high stocking density affected the weight of the reared sea cucumber. likewise, stocking on the second month after installation of the bottom cage also resulted in higher weight gains than at other time of stocking (fig. 1). the growth of h. atra was calculated based on weight gain at the end of the study. the low stocking density yielded a greater weight gain than did the higher stocking densities. the highest increase was observed at the density of 10 individuals/cage at the second stocking. the lowest weight gain was recorded at the stocking of 30 individuals/cage at the first stocking (fig. 2). stocking density remarkably affected the aquaculture growth. at all times of stocking, the lower stocking density (i.e., 10 and 20 individuals/cage) produced higher weight gains than the higher stocking density (e.g., 30 individuals/cage). the highest increase in weight (19.69 g) occurred at the stocking of 10 individuals/cage and the lowest (2.1 g) occurred at the stocking of 30 individuals/cage. stress due to high stocking density greatly affected the growth performance of the sea cucumbers h. arguinensis and h. mammata (domínguezgodino & gonzález-wangüemert 2018). stocking density had also adversely affected the growth of sea cucumber apostichopus japonicas, not by decreasing the water quality or by food competition, but rather through the role of density as an environmental stress factor (pei et al. 2012). this impact of high density led to greater variations in body weights, which also occurred in this study. stress caused by crowding or overcrowding can stimulate the endocrine system of smallsized individuals, increasing cortisol levels in coelomic fluid and, in turn, accelerating energy consumption, modifying the energy budget model, and ultimately playing a negative role in the growth and composition biochemistry of these individuals (pei et al. 2012). in this study, the growth was also lowered by the effects of fission, which made the body size smaller. at high enough densities, sea cucumbers stop growing. the density value at which sea cucumbers can no longer grow is called the critical biomass value (cbv) (lavitra et al. 2010a). the cbv for h. scabra was 650 g/m2, whereas that for juvenile h. atra is 250 g/m2 to 350 g/m2. cbv is strongly influenced by the availability of food. h. atra is a deposit feeder (asha et al. 2015; hartati et al. 2019b), so these organisms digest organic materials largely from sediments, which continue to decrease during maintenance. sea cucumbers mainly eat microphytobenthos (algal epiphyte) (paga´njime´nez et al. 2019) and bacteria (lavitra 2010b) and the presence of sunlight reaching the bottom of the waters will support rapid growth of microphytobenthos which in turn will support sea cucumber growth (mactavish et al. 2012). in this study, overcrowding caused fission which in turn slowed the growth. hence, this study suggests the use of optimum stocking density to prevent problems of overcrowding, which will result in low growth and/or variations in body size, deformation of body shape, and the phenomenon called "rotten stomach" (thickened stomach wall) (seeruttun et al. 2008). the stocking time generally influenced the growth of h. atra which for the first, second and third stocking periods, were reared for five, four, and three months, respectively. longer rearing of five months led to less weight gain of 2.1 12.52 g versus the second stocking time of holothuria atra stocking density and stocking time – hartati et al. 131 four months which recorded 13.1 19.69 g weight gain. the second stocking provided an opportunity for the cage sediments to be overgrown with the microphytobenthos thereby preparing the sediments for sea cucumber habitation. at the third stocking, the maintenance duration of three months presented an average weight increase ranging from 5.43 g to 8.86 g. b a biotropia vol. 28 no. 2, 2021 132 figure 1 average weight of h. atra reared at different stocking times and densities notes: a = stocking at month 0 from the start of installation of the bottom cages; b = month 1 after cages were installed; and c = month 2 after cages were installed. figure 2 weight growth of h. atra reared at different stocking times and densities notes: a = stocking at month 0 from the start of installation of the bottom cages; b = month 1 after cages were installed; and c = month 2 after cages were installed. the number of live h. atra differed according to stocking density. remarkable fluctuations occurred in the number of sea cucumbers calculated as survival rates that registered 100% at the 10 individuals/cage stocking density at the third stocking (fig. 3). this likely occurred because of the fission phenomenon (asexual reproduction) among the sea cucumbers reared in cages. the highest survival rate of h. atra occurred in the 30 individuals/cage stocking density, including 93% in the third stocking, which meant that sea cucumbers can be maintained for only three months (fig. 4). the lowest survival rate of 45% occurred at the density of 20 individuals/cage in the first stocking or in the fifth rearing month. during their rearing period in cages, the survival rates of sea cucumbers fluctuated. survival rate at the 30 individuals/cage stocking density was higher than that at the other stocking densities. the third stocking time also provided a high survival rate because rearing took place for only three months. the high survival rate might be caused by the existence of a fission process that increased the number of living individuals (fig. 3), but resulted in smaller physical sizes (fig. 2). c holothuria atra stocking density and stocking time – hartati et al. 133 figure 3 survival rates of h. atra reared at different stocking times and densities notes: a = stocking at month 0 from the start of installation of the bottom cages; b = month 1 after cages were installed; and c = month 2 after cages were installed. a b c biotropia vol. 28 no. 2, 2021 134 figure 4 survival rate of h. atra reared at different stocking times and densities notes: a = stocking at month 0 from the start of installation of the bottom cages; b = month 1 after cages were installed; and c = month 2 after cages were installed. table 1 number of h. atra fissions during cage rearing stoking time stocking density 30 20 10 month 0 8 11 2 month 1 8 9 8 month 2 8 10 5 total fissions 24 30 15 during its rearing period in cages with different stocking densities and stocking times, the sea cucumbers did participate in asexual reproduction or fission across all conditions (table 1). although most of these phenomena were not directly observed, the organisms that are results of fission can be distinguished from the original organisms (fig. 5). sea cucumbers naturally have a great potential to regenerate after the process of evisceration (expulsion of their internal organs). evisceration occurs due to physiological changes or in response to various external factors. during evisceration, sea cucumbers secrete a large portion of their internal organs and, within a certain period of time, such will grow back and continue to live normally (hartati et al. 2016). regeneration also occurs after the process of natural asexual reproduction by fission as well as during fission stimulation (hartati et al. 2016). this process produces new individuals or regeneration in the posterior or anterior part. fission is a naturally occuring process (fig. 5); first is the narrowing of the part that will divide into two portions (fig. 5a). after splitting (body fission), the new individual closes its wound in the division section (fission plane) (fig. 5b) and the initial regeneration appears as a bulge in the fission plane. then, regeneration of the body part happens (fig. 5c). ultimately, this fission process contributed to high survival rates but produced smaller individuals of sea cucumber. generally, in this study, the more densely populated cages experienced more fission than did the non-dense ones, but stocking time did not significantly affect the number of fission organisms (table 1). fission is a phenomenon that is highly dependent on population density (asha & diwakar 2015; asha et al. 2015; hartati et al. 2019a). in nature, fission is vital in maintaining populations of some types of holothurians to compensate for their mortality and migration (dolmatov 2014). holothuria atra stocking density and stocking time – hartati et al. 135 figure 5 sea cucumbers that are undergoing fission (a); those that have just undergone fission, where the cleavage wound has been closed (b); and the regenerated missing part of the body (c) note: → arrow mark k is the position of fission/fission plane and regeneration the biomass of the microphytobentic food sources of h. atra were measured as concentration of chlorophyll a, b, phaeophytin, and total carotene levels in the sediment (figs 6 & 7). the concentration trends of chlorophyll a, b, phaeophytin, and total carotene were almost the same in all cages; high in the first month of rearing, decreasing in the second and third months of rearing, increasing in the fourth month, and then decreasing again in the last or fifth month (figs 6 & 7). microphytobenthic or benthic microalgae are groups of photoautotrophic microorganisms that inhabit the surface layers of shallow aquatic ecosystems sediments such as diatoms, cyanobacteria, and other chlorophytes (paga´njime´nez et al. 2019). in shallow coastal waters, the microphytobenthos significantly contribute to the metabolic systems of primary producers (hardiso et al. 2013). these microphytobenthos include important food sources for meiofauna such as sea cucumbers (mfilinge & tsuchiya 2016; hartati et al. 2017a,b; paga´n-jime´nez et al. 2019). holothurians can assimilate low content of organic matter as live diatoms, bacteria and detritus by passing sediment through their gut system (tolon et al. 2015), thereby causing fluctuations in the concentrations of microphytobenthos (hartati et al. 2019b). the microphytobenthos biomass is measured as chlorophyll content in the sediments (du et al. 2017) and the ratio of total chlorophyll c and b to chlorophyll a can be a good indicator of the amount of diatomic and green algal biomass (cartaxana et al. 2016). a b c https://www.researchgate.net/profile/paulo_cartaxana?_sg%5b0%5d=6lvu8fypltbuqidrhjgvtjfryd4-ilb81vuxj9myqegscm11utvueceurbrvun2sdkjhbwc.ojkg9uwa7kzscjnmj2vdjbtfvtf2tppfhfytdgi7yspm2jmnfz1d25t1ynaujhb27zl5bmngbhtp-rea9nloag&_sg%5b1%5d=ab-ekfl8kpt3ciboo-qvandlajy_mpx_kep7-wpas5dkhrkavanhfjxz9drcregd55z-eou.wgm2xnnwemrdtdo_xkgoowamilljsiz3ipjjrpitsa0inmevjeusidps4llpjumcmxkmi5yw9vqueqguuicsia biotropia vol. 28 no. 2, 2021 136 figure 6 concentration of chlorophyll-a (a) and chlorophyll-b (b), in the cage sediments of h. atra reared at different densities figure 7 concentration of phaeophytin (a) and total carotene (b) in the cage sediments of sea cucumber h. atra reared at different densities bottom sediment is one of the basic components influencing sea cucumber habitat preferences. hence, studies of its characteristics are primarily important (slater & jeffs 2010). rocky sediments should be avoided because sea cucumbers live in mud or sand and remain in the organic material for its lifetime. h. atra is a deposit feeder that swallows sediment along with organic matter (robinson et al. 2013; asha et al. 2015) and the microbial composition in the intestine of these adult holothurians constituted a greatly diverse microorganisms, such as bacteria, viruses, protozoa, and fungi colonizing their intestines (paga´n-jime´nez et al. 2019). studies have also shown that sea cucumbers swallow marine animal feces, including even their own feces (barrio & tuya 2013). in shallow waters (<10 m depth), h. atra prefers sea grass habitats with sediments of 15% to 25% gravel and coarse sand (0.7 1.2 mm), but not mud (dissanayake & stefansson 2012). h. atra was also found mainly in sand-dominated habitats (hartati et al. 2017a). its preference for specific habitat characteristics seems to be related to the feeding and security needs of h. atra (hartati et al. 2017a,b). water quality parameters (e.g., temperature, salinity, ph, and dissolved oxygen) were also measured during the rearing of h. atra (fig. 8). water temperature is one most important parameter affecting growth and development of sea cucumbers as well as their distribution in the sea. temperatures in seawaters tend to be stable at 28 30 °c throughout the study period (fig. 8). the temperature between cages were not significantly different because the cages were close to one another. since h. atra is a tropical species, they perform optimally in the higher temperature environments between 23 31 oc (buccheri et al. 2019). the aestivation process (adaptation to high temperatures) has not been widely studied in h. atra, but the species was a b a b holothuria atra stocking density and stocking time – hartati et al. 137 observed to usually cover its body with sand during the day, which is thought to be a response to higher temperatures (hartati et al. 2019a). sea cucumbers are stenohaline invertebrates that are unable to tolerate a wide range of salinity. therefore, their settlement in river mouths, estuaries, and other bays where salinity falls below 10‰ during the monsoon season should be avoided. although there is a small river that goes into the teluk awur waters, it did not greatly affect the salinity around the cages which were located 500 m from the river mouth. salinity levels in the teluk awur waters ranging from 27.5‰ to 30.5‰ were still suitable for the sea cucumbers (fig. 8). besides temperature and salinity, the water ph can also affect h. atra. fluctuations in ph occur seasonally, in that the dry season is characterized by high water temperatures and low water movements/currents resulting in low ph values between 7.3 and 7.5, while ph increases to between 8.4 and 8.6 during the rainy season because of high water influx (gullian & preciat 2017). generally, vertebrates have the ability to regulate osmotic pressure and create very good acid-base conditions and therefore, are able to cope with changes in ph better than invertebrates which have a lower regulatory capacity (wittmann & portner 2013). however, information about the effects of ph on sea cucumber is still limited. figure 8 average temperature, salinity, ph, and dissolved oxygen of the sea water surrounding the cages of h. atra reared with different stocking densities sea cucumbers respire with their tentacles, skin, and respiration trees by consuming a significant amount of water and absorbing the dissolved oxygen inside. the hypoxia (minimum amount of oxygen) limit of h. leucospilota was 3 mg o2/l, wherein the sea cucumber released its cuverian tube to lower its energy needs in this aesthetic process (zamani et al. 2018). furthermore, the normal conditions of dissolved oxygen levels at >6 mg/l provide a biotropia vol. 28 no. 2, 2021 138 100% survival rate. for a. japonicas, dissolved oxygen was maintained at >5 mg/l (xie et al. 2013). during the rearing period in this study, the dissolved oxygen content in the cages ranged from 6 mg/l to 7 mg/l (fig. 8) and no signs of h. atra evisceration due to hypoxia was observed. sea cucumber rearing in bottom cages has been carried out previously for both h. scabra and stichopus hermannii with no food addition (purcell & agudo 2013; hartati et al. 2016). the maintenance of sea cucumbers with feeding greatly affected their growth performance (i.e., sea cucumbers grow faster when given additional feed) (yokohama 2013) and mahmoud et al. 2017). in this study, however, the h. atra were not given additional feed considering that the sediment/substrate would provide ample feed for the species. in sea ranching, the current sea cucumber management practices do not apply additional input so the existence of sea cucumbers depends largely on the availability of natural feed (purcell et al. 2012). therefore, choosing the right location for the rearing of h. atra is very crucial. conclusion h. atra reared at low stocking densities of 10 individuals/cage at the bottom cages of the teluk awur waters of jepara, exhibited higher growth and survival rates. stocking time in the second month after the cages were installed also resulted in higher growth, as the food organisms for the sea cucumber were already developed and thus, were made available. moreover, the chlorophyll a, b, phaeophytin, and carotene levels fluctuated during the time of sea cucumber rearing due to their feeding and bioturbation activities. acknowledgments the authors would like to thank seameobiotrop for partly funding this present work through the dipa biotrop 2018 of phd grant and universitas diponegoro for also funding the chemical analyses through the international publication research (rpi) grant of other than apbn lppm rkat universitas diponegoro year 2018-2019. our gratitude also goes to robertus tm, elsa la, fabian pa and mustagpirin for their help during the field work. references ambariyanto. 2017. conserving endangered marine organisms: causes, trends and challenges. in iop conference series: earth and environmental science, 55(1): 012002. iop 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single.cdr biotropia vol. 27 no. 2, 2020: 125 133 doi: 10.11598/btb.2020.27.2.1176 soil properties and tree composition in a 27-year old acacia mangium willd. plantation on abandoned mining area at phangnga forestry research station** jetsada wongprom1', roongreang poolsiri2, sapit diloksumpun2 and chatchai ngernsaengsaruay3 1faculty of forestry, kasetsarf university', bangkok 10900, thailand 2f)epartment ofsilviclture, faculty of forestry, kasetsart university, bangkok 10900, thailand department of botany, faculty of science, kasetsart university, bangkok 10900, thailand ^corresponding author, e-mail: fforjdw@ku.ac.th received 19 december 2018 / accepted 04 april 2019 abstract in order to validate the important role of acacia mangium plantation in mining rehabilitation, a study was conducted on the soil properties and tree composition in a 27-year-old a. mangium plantation growing on sandy (s27) and clay (c27) soil types, as well as a mixed plantation (mp) growing on clay soil type. the results were compared with those grown on an abandoned mining area (ab), a secondary forest (sf) and a primary forest (pf), at the phangnga forestry research station, thailand. three 40 x 40 m permanent plots were established and soil samples were randomly collected from depths of 0-10, 10-20, 20-30, and 30-50 cm, in each of the s27, c27, mp, ab, sf, and pf. the bulk density values in the s27, c27, and mp were lower than that in ab and was similar to those in sf and pf, particularly, the top soil. total nitrogen, available phosphorus, organic matter, exchangeable potassium, and magnesium contents in the s27, c27, and mp were higher than that in ab, but were lower than those in sf and pf, indicating that the soil development in the s27, c27, and mp was slower than in the sf and pf. this lower bulk density values and higher soil nutrient contents were positively contributed by the a. mangium plantation. the shannon-wiener index obtained for s27 (1.43), c27 (2.51), and mp (2.77) were lower than that for the sf (3.86). the similarity indices of the tree species found in s27, c27, mp, and pf were low, ranging from 5.83 8.00, indicating that the development of the forest community was slow compared to sf (31.03). enrichment planting with poorly dispersed shade tolerant trees has increased the diversity and improved the forest structure in the mined out areas and other similarly degraded lands. keywords: acacia mangium, mining area, soil properties, tree composition introduction ever since mining has been widely operational all over thailand, the local environment and ecosystem conditions have been severely impacted by the mining activities (macdonald et al. 2015). vegetations were destroyed and soil properties were changed from ^corresponding author, e-mail: fforjdw@ku.ac.th **this paper was presented at the 3rd international conference on tropical biology 2018, 20-21 september 2018, bogor, west java, indonesia the originally rich to currently poor soil. soil textures have become unsuitable for planting (oktavia et al 2015). in some areas, the soil has turned extremely acidic and the soil moisture went very low during the dry season (tripathi et al. 2016). as a result, soil improvement and forest restoration in mining areas has taken a long time. invariably, changes in such important factors as soil properties like soil texture, organic matter, ph, nitrogen, phosphorus and potassium have negatively affected the development of a forest community in 125 biotropia vol. 27 no. 2, 2020 previously mined areas (zhao et al. 2013; lei et al. 2015). oftentimes, nitrogen limits the growth and reestablishment of forest seedlings. as such, natural regeneration using native trees was applied to accelerate plantation establishment. however, restoration of several mined out areas is costly and is usually a long term process (oktavia et al. 2015). the popularly known nitrogen fixing tree, acacia mangium is a multipurpose fast-growing tree which plays an important role in restoring the soil quality of degraded lands. this species has been widely introduced in southeast asia (nambiar & harwood 2014). its survival, growth rate and the aboveground biomass it produces are relatively high compared to other fast growing and native tree species found in abandoned mining areas (martpalakorn 1990). a. mangium plantation has been established in degraded lands for wood production, forest restoration, as well as soil improvement (wang et al 2010). degraded land restoration using plantation is one approach for catalyzing the natural succession process and increasing the tree species diversity (tripathi et al. 2016). since native tree species have been introduced into plantations, resulting in an increased tree species richness and increased forest structure complexity, the restoration of previously mined areas using nitrogen fixing trees has been focused on soil improvement (bohre & chaubey 2014). generally, after plantation establishment, the soil physical and chemical properties will improve (zhao et al 2013; bohre & chaubey 2014), simultaneously with the ecosystem services such as litter supply, nutrient cycling, and biodiversity (parrotta 1999; celentano 2011). the objectives of this study were, firstly; to evaluate the soil properties, tree composition, and diversity of a 27-year-old a. mangium plantation located in phangnga forestry research station, thailand and compare these with an abandoned mining area (ab), secondary forest (sf) and primary forest (pf) and secondly, to recommend trees, like a. mangium, that are suitable for this site restoration programs and for other mined out areas. materials and methods study area the experiment was conducted on an abandoned tin mine at phangnga forestry research station, takuapa district, phangnga province, thailand. the area also includes a 27year-old a. mangium plantation grown on sandy (s27) and, clay (c27) soil types, as well as mixed plantation (mp) of eucalyptus camaldulensis, a. mangium, and diptercapus alatus grown on a clay soil type. phangnga forestry research station is approximately 3 km away from a primary forest (pf) and is surrounded with rubber and oil palm plantations. the area receives a mean annual rainfall of 3,668.80 mm, with rainy season occurring from april to november and dry season from december to march. its relative humidity is around 83% and its mean annual temperature is at 27.1 °c (wongprom et al 2013). in addition, secondary forest (sf), primary forest (pf), and abandoned mining area (ab) were established as reference sites. the sf plot is approximately a 30-year-old protected forest, a tropical rainforest that was disturbed in the past by shifting cultivation. soil properties soil samples were collected from the s27, c27, mp, ab, sf and pf sites at four soil depth series, 0 10, 10 20, 20 30 and 30 50 cm. three soil pits were randomly established, while soil samples were collected from each plot using a split tube sampler for the soil physical and chemical analyses. for the chemical properties, the samples within each plot were mixed thoroughly to form a soil composite. soil bulk density was analyzed by the core method, while the soil texture was analyzed by the hydrometer method. soil ph was examined by a ph meter with 1:1 soil: water ratio. total nitrogen (n) was measured using the dumas method (jackson 1965) by chns analyzer. organic matter content (om) was analyzed by the walkley and black rapid titration method. available phosphorus (p) was extracted using the bray ii method and was analyzed using a spectrometer. the exchangeable potassium (k), calcium (ca) 126 soil properties and tree composition in a 27-year old acacia mangium willd. plantation — wongprom el al. and magnesium (mg) were extracted with ammonium acetate (nhuoac) 1 n ph 7.0 and analyzed by an atomic absorption spectrometer (estefan et al. 2013). tree composition three permanent plots of 40 x 40 m were established in each of the s27, c27, mp, sf, and pf sites. each sample plot was divided into sixteen subplots of size 10 x 10 m plot. at the permanent plots, all tree species with diameter at breast height (dbh) > 4.5 cm were identified and recorded. the dbh and total height of a tree was measured using a diameter tape and haga altimeter, respectively. plant specimens were collected and identified, and the unidentified species were compared with the herbarium specimens deposited at the forest herbarium, natural parks, wildlife and plant conservation department. data analyses soil physical and chemical properties, including bulk density, porosity, soil ph, total n, available p, om, exchangeable k, ca, and mg were analyzed using a one-way analysis of variance (anova) and the means were compared using tukey’s test at 5% probability level. the importance value index (ivi), tree density, and basal area were also calculated. the ivi value was obtained as a relative density (rd), relative frequency (rf), and relative dominance (rdo) of a given tree species.the tree species diversity for each site was calculated using the shannon-wiener index (shannon & weaver 1949) using the formula: n h' =-s (pi*ln pi) n=l where, h? is the shannon-wiener index, pi is the proportion of each species in the sample, in pi is the natural logarithm of this proportion. species evenness (e) was calculated as follows (pielou 1966): e = h’ / in s where, h’ is the shannon-wiener index and s is total number of species in the sample. the similarity of plant composition was calculated using the sorensen similarity index (sorensen 1948) by the formula: s = 2c / (a + b) x 100 where, s is the sorensen similarity index, a is the number of species found in site a, b is the number of species in site b, and c is the number of species shared by the two sites. shannonwiener index and the sorensen similarity index are the most widely used tools to evaluate plant community (barrantes & sandoval 2009; martinez-ruiz & fernandez-santos 2005; zhang et al. 2014). results and discussion soil physical properties at the top soil (0 1 0 cm), the soil bulk density and soil particles were significantly different among the sites but not for porosity (table 1). however, at soil depths of 10 20, 20 30 and 30 50 cm the bulk density, soil particles, and porosity of the subsoil significantly differed among the different sites. the bulk density was the highest (1.31 g/m3) in ab, while bulk densities of s27, c27 and mp were similar to sf and pf. the bulk density can be relatively lower in mining areas having a forest plantation (bohre & chaubey 2014). soil development at ab was slow compared to those at s27, c27 and mp. plantations have been known to markedly improve the physical properties of soil, vegetation cover and natural regeneration in post mining sites (zhao et al 2013; lei et al. 2015). the variations in soil bulk density, specific gravity, porosity, water holding capacity and soil aggregation in a reclaimed mine are mainly caused by variations in organic matter. as such, soil physical properties are largely related to the organic matter content (charman & roper 2000; zhao et al 2013). 127 biotropia vol. 27 no. 2, 2020 table 1 soil physical properties on abandoned mining in the phangnga forestry research station and the reference sites depth site . soil particle (%) bulk density (g/m3) porosity (%)(cm) sand silt clay soil texture 0-10 ab 91.23a 3.99c 4.77c sandy 1.31a 56.99 s27 89.33a 2.24c 8.42bc loamy sand 1.04ab 59.67 c27 17.70c 33.31a 48.99a clay 0.94b 59.43 mp 20.15c 33.47a 46.38a clay 0.90b 60.40 sf 31.45c 25.35ab 43.20a clay 0.96ab 60.38 pf 67.73b 8.71bc 23.56b sandy clay loam 0.92b 65.93 f-value 110.16* 16.72* 29.38* 4.08* 1.24ns 10-20 ab 91.14a 3.33c 5.56b sandy 1.78a 32.86b s27 87.97ab 2.77c 7.76b loamy sand 1.48b 44.92a c27 22.56c 26.93b 50.51a clay 1.2l 53.46a mp 9.03c 38.95a 52.01a clay 1.1lc 49.08a sf 18.09c 21.26b 60.65a clay 1.09c 55.69a pf 67.1lb 7.24c 25.65b sandy clay loam 1.22c 54.55a f-value 64.30* 64.89* 30.72* 45.57* 13.91* 20-30 ab 90.06a 3.37b 6.56c sandy 1.71a 30.28c s27 84.33a 6.93b 10.07bc loamy sand 1.69a 35.62bc c27 28.37bc 27.98a 43.65ab clay 1.30b 46.12ab mp 17.70c 26.05a 56.25a clay 1.17b 49.87a sf 26.37bc 19.19ab 54.44a clay 1.21b 46.97ab pf 63.85ab 5.38b 30.77ab sandy clay loam 1.33b 43.78ab f-value 13.13* 8.37* 10.29* 22.56* 7.97* 30-50 ab 90.57a 3.21c 6.22d sandy 1.58a 38.14b s27 81.71ab 4.86c 13.43“ * loamy sand 1.55ab 41.45ab c27 31.23cd 26.12ab 42.65ab clay 1.12c 53.63a mp 8.44d 33.52a 58.04*b clay 1.05c 52.14a sf 18.42d 18.93b 62.65* clay 1.12c 52.78a pf 55.89bc 9.12c 34.98bc sandy clay loam 1.30bc 42.92ab f-value 31.88* 53.83* 19.99* 17.34* 6.51* note: * = different superscripts along the same icolumn indicate significant differences at p < 0.05. comparison between the rehabilitated sites, moderately to slightly acidic. these results s27, c27 and mp, and the reference sites, sf indicate that the a. mangium plantation had a and pf, indicate that the bulk density and significant influence in reducing soil ph, porosity were not significantly different, an observation similar to that of yamashita especially at the top soil. invariably, the forest et al (2008). amounts of exchangeable plantation had a significant role in improving the bulk density. however, the restoration of soil texture at these sites to that of the levels of pf, and in particular the sandy area with a poor soil quality, can take a long time (oktavia et al 2015). while soil bulk density and porosity in sf were similar to that in pf, degradation of the soil structure due to shifting cultivation in sf may be lesser in extent compared to that in the mined out area. soil chemical properties k and mg were significantly different among the sites at depths of 0 10, 10 20, 20 30, and 30 50 cm. mining rehabilitation with a. mangium plantation resulted in increased k and mg levels which were higher than those in ab. nevertheless, k and ca of s27, c27 and mp were lower than those in sf and pf. the levels of k, ca and mg were relatively high in the topsoil. similarly, the available p, total n and om were the highest at a depth of 0 10 cm, but was decreasing with the increasing soil depth. the topsoil was rich and higher in soil soil ph was extremely acidic in s27, c27, mp and sf, while in pf it was strongly to moderately acidic. however, soil ph in ab was nutrient content, total n, om, the available p, k, ca and mg in s27, c27 and mp than those in ab. in mining restoration, the soil nutrients 128 soil properties and tree composition in a 27-year old acacia mangium willd. plantation — wongprom et al. table 2 soil chemical properties in abandoned mining in the phangnga forestry research station and the reference sites dth site(cm) n t i exchangeable (mg/kg) available p total n om ph k ca mg (mg/kg) (%) (%) 0-10 ab 5.9a 6.33d 10.69 2.92c 5.90b 0.0l 0.67c s27 4.5bc 14.38cd 18.45 10.26bc 12.91b 0.11b 1.62bc c27 4.4c 35.37bc 23.98 33.56ab 15.16b 0.13ab 2.14b mp 4.7b 38.93b 27.46 35.34a 9.42b 0.10b 1.66bc sf 4.2c 82.27a 29.06 24.27abc 33.32a 0.17a 2.42ab pf 4.8b 40.25b 29.56 26.72abc 50.78a 0.17a 3.55a f-value 68.53* 28.60* 1.40ns 6.57* 70.22* 26.06* 15.38* 10-20 ab 6.0a 7.46b 11.17 3.21c 4.34c 0.0e 0.33b s27 4.7bc 9.76b 9.27 3.77bc 7.10bc 0.04c 0.91b c27 4.6bc 20.42b 11.81 15.91abc 7.67bc 0.04c 0.57b mp 4.8bc 24.07b 11.13 26.93a 5.32bc 0.04c 0.86b sf 4.4c 64.34a 12.83 17.38a 6.25bc 0.11b 1.49ab pf 4.9b 29.57b 12.00 16.34ab 14.52a 0.15a 2.53a f-value 35.03* 19.40* 1.45ns 11.08* 15.31* 73.57* 6.65* 20-30 ab 5.8a 5.18c 10.26 3.13b 3.22b 0.0l 0.25b s27 4.9b 8.17c 9.58 4.74b 7.32ab 0.02c 0.71b c27 4.6b 9.97bc 9.76 18.23b 7.36ab 0.03bc 0.36b mp 4.9b 19.07abc 9.59 35.42a 4.86ab 0.04bc 0.47b sf 4.6b 33.14a 14.21 9.87ab 5.65ab 0.07b 1.21ab pf 5.6a 24.08ab 10.59 16.45b 9.15a 0.13a 2.29a f-value 31.28* 11.44* 1.01ns 11.94* 4.81* 31.29* 6.84* 30-50 ab 6.1a 5.01c 9.12 2.85c 3.05 0.01b 0.19b s27 5.1b 6.19c 7.86 2.49c 5.54 0.01b 0.48b c27 4.8bc 10.75bc 12.41 18.53ab 7.12 0.02ab 0.54b mp 4.9bc 18.68ab 12.50 31.51a 2.62 0.02ab 0.41b sf 4.6c 20.12ab 9.94 5.94bc 3.59 0.04a 0.87b pf 5.7a 20.76a 5.03 17.23b 7.08 0.04a 2.16a f-value 43.13* 12.70* 3.21ns 15.41* 5.95ns 7.37* 8.95* notes: * = significant difference; ns = non-significant difference; and a — c — different superscripts along a column indicate significant differences at p < 0.05. and organic matter contents usually increased with the age of the stand (zhao et al 2013; bohre & chaubey 2014). soil nutrients rapidly increased in plantations using a nitrogen fixing tree while natural succession increased gradually (oktavia et al 2015). however, soil nutrients such as exchangeable k, available p, total n and om levels at s27, c27 and mp were lower than those in both sf and pf at a depth of 0 10 cm. overall, the soil properties in sf particularly, available p, total n and om contents were improved faster than those in s27, c27 and mp. although a., mangium was dominant in s27, c27 and mp, the level of total n was relatively low, particularly in the sandy area. these results indicate that mining activities highly impacted the soil properties and the restoration may take a longer time. tree composition and ecological characteristics there were 21, 34, 40, 81 and 94 tree species, 19, 29, 31, 62 and 68 genera, and 14, 26, 25, 33 and 38 families in the s27, c27, mp, sf and pf, respectively. the basal area, density and species diversity index in pf (3.91) was the highest, in contrast with those in s27 (1.43), which had the lowest density and species diversity index (table 3). soil characteristics showed a significant influence on the density, species diversity index, and natural regeneration (lei et al 2015), especially in the s27. soil texture in the area being sandy, has a low water holding capacity, and thus, has resulted in insufficient water supply during the dry season, a phenomenon causing high seedling mortality in the tropical rain forests (li et al 2011). soil properties play important roles on revegetation 129 biotropia vol. 27 no. 2, 2020 table 3 ecological characteristics of the rehabilitated sites, s27, c27 and mp in the phangnga forestry research station, thailand and the reference sites, sf and pf ecological characteristics sites s27 c27 mp sf pf number of species 21.00 34.00 40.00 81.00 94.00 basal area (m2/ha) 35.49 20.91 23.09 22.20 41.27 density (stems/ha) 1,010.42 1,229.17 1,393.75 1,200.00 1,497.92 shannon wiener index (ft) 1.43 2.51 2.77 3.86 3.91 evenness of species 0.46 0.71 0.74 0.88 0.86 of native trees and forest community (zhao et al. 2013; lei et al. 2015). the number of species, tree diversity and density was significantly lower in s27 and c27, than those in mp, which consisted of a. mangium, e. camaldulensis and d. alatus. a mixed plantation of many tree species can facilitate a successful seedling establishment in the understory and native trees were also found more abundantly than in pure plantations, resulting in a high tree diversity (wang et al. 2019). however, the rehabilitated sites in s27, c27 and mp had a low tree diversity compared to sf. the number of native tree species found in s27, c27 and mp were relatively less than that in sf, resulting in a low recovery of original tree species, forest structure, and forest function. in addition, species richness and evenness in sf was larger than those in s27, c27 and mp, resulting in high tree diversity. tree diversity is related with species richness and evenness (strong 2016). according to the shannonwiener index of the old teak (tectona grandis) plantation was higher when the evenness value of the plot was high (koonkhunthod et al 2007). in addition, the shannon-wiener index in s27 (1.43), c27 (2.51) and mp (2.77) was lower than that in a 34-year-old a..mangium plantation (3.22) (marod et al. 2013). a. mangium plantation can be highly effective to introduce native trees species (van et al 2005). in this study, the native species were mostly shade intolerant trees. therefore, accelerating the natural succession by enrichment planting with poorly dispersed shade tolerant trees, such as swintonia floribunda, dipterocarpus kerrii, canarium patentinervium, xanthophyllum virens, mesua ferrea, hopea gnffithii and gluta elegans, may be considered in s27, c27 and mp for improving the forest structure and increasing the tree diversity. the high tree diversity and number of trees in sf could be due to various factors such as proximity to the forest fragment, rich soil nutrient content and organic matter. the vegetation composition and richness decrease with the increasing distance from the primary forest (van et al 2005; ruiz-jaen & aide 2005). therefore, the distance from a natural forest and the surrounding mining area with oil palm and rubber plantations had largely obstructed on the process of natural forest succession in the s27, c27 and mp. this study, we showed top seven ivi in s27, c27, mp, sf and pf because they play ecological importance in ecosystem with high relative frequency, density and basal area. the dominant native trees in s27, c27 and mp included vitex pinnata, aporosa planchoniana, carallia brachiata, melicope lunu-ankenda and bridelia tomentosa (table 4). most native species were identified as pioneers because of their various life forms such as very fast growth, frequent year-round flowering, production of a large number of small seeds, lighting demand for germination, and low wood density are often shade intolerant trees (goosem & tucker 2013; elliott et al 2013). similarly, the dominant trees in sf were also classified as pioneers, such as eurya acuminata, microcos paniculata, and vitex pinnata. these trees are mostly found in disturbed areas (sinbumroong 2009) and old plantation (koonkhunthod et al. 2007). pioneer trees are tolerant to adverse environmental conditions; therefore, these species can be considered for restoring other degraded areas. twenty seven trees recorded were in both sf and pf, among which are barringtonia macrostachya, diospyros wallichii, canarium patentinervium, and garcinia cowa. however, these were only few small trees. revegetation in sf was faster than in s27, c27 and mp, resulting in a complex forest structure and high tree diversity. the similarity index between pf and sf was 31.03%, pf with mp was 8.00%, pf 130 soil properties and tree composition in a 27-year old acacia mangium willd. plantation — wongprom et al. with c27 was 6.98%, and pf with s27 was 5.83% (table 5). the similarity index between the rehabilitated sites in the mining area and pf was low suggesting that sf may be in a midsuccessional status and the rehabilitated sites at s27, c27 and mp were in early successional status, as indicated by their similarity index (habich 2001). table 4 importance value index (ivi), relative density (rd), relative frequency (rf) and relative basal areas (rdo) of the top seven trees in s27, c27, mp, sf and pf site tree species family rd rf rdo ivi s27 acacia mangium fabaceae 63.85 35.60 95.15 194.60 aporosa planchoniana phyllanthaceae 16.45 15.15 1.21 32.81 carallia brachiata rhizophoraceae 4.33 9.85 0.54 14.72 bridelia tomentosa phyllanthaceae 3.25 7.58 0.38 11.21 vitex pinnata lamiaceae 1.95 6.06 0.74 8.75 eitsea grandis lauraceae 1.52 4.55 0.22 6.29 morinda coreia rubiaceae 1.73 3.03 0.38 5.14 other species 6.92 18.18 1.38 26.48 c27 melicope lunu-ankenda rutaceae 18.19 15.11 26.59 59.89 acacia mangium fabaceae 38.83 6.22 6.55 51.60 aporosa planchoniana phyllanthaceae 5.65 7.11 17.73 30.49 carallia brachiata rhizophoraceae 8.74 9.33 11.56 29.63 vitex pinnata lamiaceae 5.61 8.89 6.17 20.66 ilex cymosa aquifoliaceae 1.92 8.00 6.36 16.28 fagraea fragrans gentianaceae 2.93 11.11 1.93 15.97 other species 23.11 34.23 18.13 75.47 mp eucalyptus camaldulensis myrtaceae 10.23 9.45 47.12 66.80 acacia mangium fabaceae 15.27 8.54 20.78 44.59 fagraea fragrans gentianaceae 15.44 10.06 5.50 31.00 ilex cymosa aquifoliaceae 15.10 9.45 3.22 27.77 dipterocapus alatus dipterocarpaceae 6.21 6.71 1.73 14.65 vitex pinnata lamiaceae 2.18 3.96 8.56 14.70 carallia brachiata rhizophoraceae 4.19 6.10 1.47 11.76 other species 31.38 45.73 11.62 88.73 sf eurya acuminata pentaphylacaceae 12.24 7.07 13.79 33.10 gmelina arborea lamiaceae 5.60 4.08 20.90 30.58 microcos paniculata malvaceae 5.39 4.62 10.21 20.22 barringtonia macrostachya lecythidaceae 5.81 6.25 1.63 13.69 vitex pinnata lamiaceae 3.11 2.99 4.86 10.96 diospyros ivallichii ebenaceae 4.77 4.08 1.94 10.79 garcinia cowa clusiaceae 2.28 2.72 4.27 9.27 other species 60.80 68.19 42.40 171.39 pf swintonia floribunda anacardiaceae 5.93 4.56 19.72 30.21 dipterocapus kerrii dipterocarpaceae 4.17 4.36 13.64 22.17 canarium patentinervium burseraceae 7.37 4.77 2.05 14.19 vanthophyllum virens polygalaceae 4.17 4.56 5.26 13.99 mesua ferrea calophyllaceae 2.04 2.70 8.17 12.91 hopea grijfithii dipterocarpaceae 4.97 4.77 2.11 11.85 gluta elegans anacardiaceae 3.69 3.11 4.07 10.87 other species 67.66 71.17 44.98 183.81 table 5 sorensen similarity index of tree species among s27, c27 and mp compared with sf and pf site sf pf s27 17.48 5.83 c27 22.61 6.98 mp 23.53 8.00 sf 100.00 31.03 pf 31.03 100.00 131 biotropia vol. 27 no. 2, 2020 conclusion the a. mangium plantation in the abandoned tin mining area played a key role in the soil improvement particularly, the top soil. the bulk density and porosity of soil under the plantation remarkably improved. the level of soil nutrients, particularly, organic matter and total n, increased as a result of the presence of a. mangium trees. however, these quantities were lower than those measured in the secondary and primary forests. the tree diversity and number of species were low in the sandy soil type in s27 (1.43), clay soil type in c27 (2.51), and mixed plantation in mp (2.77). moreover, the tree similarity indices in s27, c27, and mp, as well as in primary forest pf were relatively low (5.83 8.00). these results showed that plant development in s27, c27, and mp was slow and the dominant trees in the rehabilitated mining sites and the sf were mostly identified as belonging to the pioneer species group, suggesting that enrichment planting with poorly dispersed shade tolerant trees should be considered in improving tree diversity and forest structure and eventually, the ecosystem processes in these areas. nonetheless, the complexity of the forest structure and tree community in the rehabilitated area was a result of the a. mangium plantation, particularly the mixed plantation (mp). these results indicate the potential of a. mangium trees in restoring previously mined areas. meanwhile, pioneer trees such as vitex pinnata, carallia brachiata, microcos paniculata, and iiurya acuminata can also be selected for restoring other degraded lands. acknowledgements the authors are grateful for the financial support provided by the kasetsart university research and development institute (kurdi), thailand and for 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dynamic changes in soil and vegetation during varying ecological recovery conditions of abandoned mines in beijing. ecol eng 73:676-83. 133 the southeast asian journal of tropical biology vol. 31 no. 3, 2024: 316 329 doi: 10.11598/btb.2024.31.3.2201 issn: 0215-6334 | e-issn: 1907-770x 316 biotechnological and environmental education potential of inga edulis martius: seeds collected in the amazon, brazil andré vilhena de oliveira1, 2, maurício kersting2, adilson ben da costa1, and rosana de cassia de souza schneider1* 1graduate program in environmental technology, santa cruz do sul university-unisc, santa cruz do sul, cep 96815900, brazil. 2federal institute of amazonas–ifam, manaus, cep 69075-351, brazil article higlights • inga edulis offer potential for biotechnological and environmental applications. • high protein and carbohydrate content in seeds support diverse agroindustrial uses. • i. edulis seeds enhance environmental education via forest nurseries schools. • biotechnological potential of i. edulis seeds extends to ecosystem services. abstract inga edulis martius is a native species of the amazon forest with great potential for urban afforestation in the cities of the brazilian amazon and widely used for the recovery of degraded areas. it is commonly cultivated by the amazonian population for its edible fruit, quality wood, and excellent agroforestry components. this study aimed to select the matrices and obtain information about the behavior of the seeds in terms of drying, biometry, purity and germination, and chemical composition. carbohydrates were analyzed by using high-performance liquid chromatography, proteins by using elemental analysis, and inorganic composition by using inductively coupled plasma-optical emission spectroscopy. the seeds collected in the environmental education action were recognized about potentialities of use for biotechnological products and environmental protection. the exploitation of this species’ seeds is appealing due to their inorganic composition (n 20.4 g/ kg, p 1.71 g/kg, and others) and organic content (20.1% protein and 58% carbohydrates), as well as their potential for forest integration. the seeds exhibited a quality suitable for cultivation in nurseries, including in school settings. inga edulis is recognized in the literature for its various uses, although these are not directly related to seed exploitation. i. edulis seedlings in the schools contribute to environmental education as a sustainable practice with biotechnological potential for the amazon region. keywords: agroforestry, biotechnology, germination, inga edulis, seeds article information received 25 february 2024 revised 26 june 2024 accepted 25 july 2024 *corresponding author, e-mail: rosana@unisc.br reviewer: erlia narulita, lucélia hoehne, and ricardo vinicio abril-saltos research article introduction the brazilian fruit industry holds a prominent position nationally and internationally, owing to the country’s vast variety of exotic species. in brazil, only a few native fruit species have commercial significance, with some having regional or local importance. in contrast, many unexplored or underexplored species possess potential importance and can pose a challenge to sustainable commercialization (batista et al. 2019). the ingá-cipó (inga edulis), belonging to the fabaceae family, is a tree species also known as angá, ingá, ingá-comum, ingá-de-macaco, ingáde-metro, ingá-doce, ingá-macarrão, ingá-rabo-demico, ingá-timbó, ingá-vermelho, ingá-verdadeiro, and ingazeiro (cruz 2021). in brazil, i. edulis is found in the states of acre, amazonas, amapá, bahia, espírito santo, mato grosso, minas gerais, pará, paraná, paraíba, pernambuco, rio de janeiro, rondônia, roraima, santa catarina, and são paulo (the brazil flora group 2021). it can also be found in peru (rollo et al. 2020), ecuador (abril-saltos et al. 2018), argentina (correa et al. 2021), and others. https://doi.org/10.11598/btb.2024.31.3.2201 biotechnological and environmental education potential of inga edulis seeds oliveira et. al. 317 this species can be found in almost all central and south american countries, occurring in nearly all regions of brazil, providing ecosystem services and attracting floral visitors. additionally, i. edulis contains bioactive compounds with antioxidant and antiproliferative activities (tauchen et al. 2016; lima et al. 2020). according to lima et al. (2017) and gomes et al. (2023), the i. edulis is of medium to tall stature, with rapid growth, reaching heights of 15-20 m in open areas and up to 40 m in the forest. the flowering and fruiting stage start at three years of age in open areas. the inflorescences are axillary, sometimes terminal, grouped, with 4 to 5 spikes in the leaf axils. the flowers are white, attractive, and fragrant. the fruit is a long pod of variable size, indehiscent, green, cylindrical, thick, with multiple longitudinal sutures, and can reach up to 2-m long. seeds of i. edulis are black, with a variable number per fruit, ellipsoid in shape, smooth and glabrous, measuring 2-3 cm in length and 1-1.5 cm in width, covered with a white, soft, fibrous, fluffy, succulent, sweet, and edible aril. i. edulis aged 3-4 years can produce 20,000 to 100,000 flowers and 200 to 800 fruits. one of the most important characteristics of this species is its ability to tolerate acidic soils. according to urruth et al. (2022), the need for tropical forest conservation and the strengthening of environmental policies to mitigate anthropogenic land use promote a need for environmental management with native species. therefore, there is a need for high-quality seeds and seedlings. desiccation tolerance is one of the most essential properties of the seeds of i. edulis. desiccation tolerance is a necessary phenomenon in the plant’s life cycle and serves as an adaptation strategy that ensures seed survival during storage under stressful environmental conditions, thus ensuring species dissemination. regarding flowers, inga species and amazonian legumes, in general, are practically unexplored in characterizing volatile compounds, which are important for pollinating fragrance (marinho et al. 2014) and their biological activities. this feature potentially opens doors for biotechnological exploration. similarly, the leaves of i. edulis martius can also be used due to biological activity of their composition, such as anti-inflammatory properties. polyphenols have this effect and can also be associated with treating cardiovascular, neurodegenerative, and cancer-related diseases (silva et al. 2007). results from more than 15 years of research have already identified gallic acid, catechins, quercetins, and other phenolic compounds in i. edulis leaves, as well as flavonoids in the roots (dias et al. 2010). due to its native status and benefits to the ecosystem, the i. edulis tree is suitable for reforestation and restoration of degraded areas impacted by various human activities in its native regions, such as the amazon (martins et al. 2022). consequently, it is understood that this species is important. among the potential uses in its natural habitat, the flowers, leaves, and seeds of i. edulis can be investigated to develop new products. therefore, there is a search to recognize the potential use of seeds for developing bioproducts or biofuels based on their composition and variability and the potentials described in the literature for the seeds of this species or similar ones. producing a species with high seed productivity and natives to a region incentivizes agro-industrial exploration (rodrigues et al. 2021). this approach avoids the introduction of exotic species to produce food or bioinputs and diversifies products, creating new market opportunities. promoting the planting of species like i. edulis in the amazon region is essential for biodiversity conservation, ensuring ecosystems’ healthy functioning and local communities’ well-being. this approach also prevents agricultural movements from introducing exotic species associated with ecological imbalance. the inga tree is a species that thrives in brazil and can have low production costs, integrated with forest conservation. it warrants a study of the biotechnological potential and utilization of its seeds for homogeneous batches for commercial-scale production (rodrigues et al. 2021). incorporating i. edulis into agroforestry systems can increase agricultural biodiversity and improve the resilience of productive systems. this contributes to food security and agriculture sustainability (kittur et al. 2024). additionally, i. edulis can act as a shade tree for coffee cultivation, biotropia vol. 31 no. 3, 2024 318 leading to higher productivity and contributing to carbon stocks (solis et al. 2020), assisting in soil recovery, and helping to maintain local biodiversity (domínguez-núñez 2022). likewise, there is a recognition of the potential for seedling production to encourage the establishment of forest nurseries in schools, promoting environmental education and reforestation with a native species of exploitable biotechnological potential integrated with the environment. materials and methods the research was conducted according to a diagram presented in figure 1. samples from five different matrices were separated, with 50 seeds (ten seeds from each matrix) used for characterization. these seeds underwent procedures for biometric observation (drying, weighing, and measuring), moisture content determination, and purity analysis. some collected and analyzed seeds were stored and preserved in a dry chamber to form germplasms in the plant germplasm bank of the federal institute of amazonas (ifam). figure 1 research diagram of i. edulis exploitation to biotechnological and environmental education approach analyses were conducted to identify the seed composition’s main inorganic and organic components. selected seeds were planted to produce seedlings. these seedlings were donated or used to establish an experimental forest nursery at the school. the formation of the plant germplasm bank and the establishment of the nursery contributed to the teaching and learning process, aiming to contextualize and assimilate the potential of the species under study. seed sampling and treatment fruits from five different matrices were collected in the first semester of 2022 according to the coordinates shown in the map (fig. 2). the collection method involved manual and ladderassisted techniques, as some matrices were large. students from two schools involved in the research participated in this stage. seeds were extracted from the fruits manually. subsequently, the pulp surrounding the seeds was removed, and the seeds were washed under running water. then, the seeds were placed on cotton fabric and stored individually in a cold chamber (5-7 °c) until biometric measurements were taken. biotechnological and environmental education potential of inga edulis seeds oliveira et. al. 319 in the first biometric assessment, the seeds were weighed with their impurities. they were then sun-dried for 8 hours to 12 hours and reweighed. afterward, they were dried in a laboratory oven at 40-45 °c for 60 minutes. their size was measured using calipers and a measuring tape. the flesh was removed after submerging the fruits in water for 12 hours to 24 hours. the softened pulp was macerated and separated through a sieve. the seeds, still in the sieve, were washed under running water. in a water tank, residual materials, empty and deteriorated seeds floating, and fruit remnants were removed through flotation, while seeds in good condition sank. after this process, the seeds were ready for drying (scremin dias et al. 2006). seeds were stored in a dry chamber to preserve and form the germplasm bank. seeds with fungi during this storage stage were discarded. the purity level (p) was calculated for batches of 50 seeds and presented in relation to viable seeds (%). the moisture content (%) of the 50 seeds was calculated in relation to moisture loss in an oven. figure 2 location map of the collected i. edulis matrices source: georeferencing laboratory at unisc. physiological analysis of the seeds was carried out using a binocular microscope (physis) to observe the structure, consisting of internal seed coat, external seed coat, hilum, hypocotyl, embryonic region, endosperm, and radicle. the seeds were also assessed using a uvb transilluminator (loccus do brasil, ltb-20x20 sti). chemical analysis of seeds representative seed samples from five matrices (m1 to m5), stored in a dry chamber, were ground in a knife mill and analyzed for inorganic composition, macro and micronutrients, structural composition of carbohydrates, and elemental analysis of c and n. the inorganic composition was determined based on ash content obtained by using gravimetry method conducted in a muffle furnace at 575 °c for 12 hours (de souza et al. 2020). the ash content (%) was reported based on dry biomass. inorganic macro and micronutrient contents in the ground dried seeds (mixed matrices) were biotropia vol. 31 no. 3, 2024 320 determined using inductively coupled plasmaoptical emission spectroscopy (icp-oes/perkin elmer, optima 8300 model). carbohydrate content and the monosaccharide profile were determined using the concentrated acid hydrolysis method recommended by the national renewable energy laboratory of the united states (nrelusa) (sluiter et al. 2010). weighed samples were treated with 72% h2so4 solution (4%, v:v) and autoclaved for one hour, followed by the analysis of monosaccharide composition in the hydrolysate using high-performance liquid chromatography (hplc/shimadzu, qp2010 plus). an elemental analyzer (flash ea1112, thermo electron corporation, milan, italy) was used to determine the elemental composition of c and n. protein content was calculated by multiplying the n (%) by a factor of 6.25. establishment of the experimental forest nurseries the experimental forest nurseries were established at two schools. the first occurred at ceti (centro educacional de tempo integral) áurea pinheiro braga, located on brasil avenue, s/n – compensa iii, cep: 69036-660, manaus/ am (geographical coordinates: 3.0932137,60.0596746,15). the second school was iv cmpm (colégio da polícia militar), located on açaizeiros avenue, s/n – gilberto mestrinho, cep: 69086-485, manaus/am (geographical coordinates: -3.0756539,-59.9277989,15). at both schools, participating students were between 10 and 17 years old. activities included presenting aspects of the cultivation of i. edulis, its main characteristics, seed storage conditions, seedling preparation, planting, transportation, and seedling donation. statistical analysis the experimental results of the physical evaluation of seeds were obtained in at least three replicates and analyzed for variance (anova) with a confidence level of 95% using graphpad prism 10.3.1 software. differences among the data means were tested using tukey’s multiple test. prospects for biotechnological utilization of i. edulis a bibliometric analysis was conducted to assess the biotechnological potential of i. edulis seeds. a discussion was held based on experimental results and the potential of similar seeds found in the literature. the bibliometric analysis was conducted based on documents selected from the databases available in the capes journals portal for the last five years. for some terms, it was necessary to investigate documents from the last 20 years. the documents were analyzed using vosviewer 1.6.18 software. the bibliometric search was conducted using the terms: “inga,” “inga edulis,” “seed,” “sugar,” “antioxidant,” and “carbohydrate.” results and discussion seed quality seed quality is an essential factor in recognizing its potential utilization. seeds not viable for germination are also seeds with low utilization potential. on average, the purity level of the seeds was 42.5±11.72% (table 1). this information is also crucial for establishing experimental nurseries in schools in the amazon region, as it is the viable seeds that students would be planting. the moisture content parameter of seeds in this research was higher than 46%, consistent with rodrigues et al. (2021), indicating that the seeds had a high water content. table 1 seeds biometric measurements, purity, and moisture content of seeds collected parameters m1 m2 m3 m4 m5 width (cm) 0.87±0.08a 0.96±0.1b 0.99±0.12c 1.27±0.18d 1.12±0.18e length (cm) 3.67±0.16a 4.1±0.49b 3.79±0.24a 4.54±0.17b 4.1±0.32b diameter (cm) 3.9±0.13a 4.37±0.33b 4.54±0.42b 5.84±0.42c 4.51±0.32b purity (%) 53.80±12.73a 39.63±8.80b 37.52±9.33b 39.89±16.77b 41.67±10.99b moisture content (%) 46.20 60.37 62.48 60.11 58.33 notes: values are mean±sd for n = 10. different superscripts on the same line indicate significant differences (p < 0.05); m1 to m5 = matrices. biotechnological and environmental education potential of inga edulis seeds oliveira et. al. 321 after analyzing the physiology and morphology of seeds from five matrices, a portion of viable seeds was sent for seedlings production. despite the first matrix showing higher purity and lower physiological water loss (p < 0.05), it was found that the fourth matrix (m4) yielded the best results in terms of formation and potential use as seed plantation. according to mata (2009) and santos et al. (2016), germination is associated with morphological and physiological changes that occur during seed maturation. seed vigor, which is crucial for successful seedling emergence, is influenced by seed size; thus, larger seeds generally exhibit greater vigor. seed germination allowed for identifying structures, such as endosperm, embryo, hilum, hypocotyl, cotyledon, and radicle. seeds collected in this research exhibited the necessary structures for proper functioning, growth, and development during germination and seedlings production. subsequently, these seedlings were cultivated and donated to establish experimental forest nurseries in schools in the amazon region. establishment of experimental nurseries in the activities carried out in schools in the amazon region, students observed the time of root protrusion and seedling formation. most of the seeds germinated within three days, and there were differences among the matrices. despite having higher purity, even the matrix 1 was not recommended for planting. matrix 4 contributed to most of the seedlings as it produced more seeds with complete structures. in addition to learning about seedling and preservation, students also learned how to prepare substrate for seedling production in 1-kg bags using a special substrate (npk and limestone or chicken manure and burnt rice husk). subsequently, this special substrate was mixed with vermiculite – a mineral that retains water in the substrate and aids in aeration. a drainage layer was added to the bottom of the bags using the husks from the fruits of i. edulis. the produced seedlings are shown in figure 3. figure 3 seedlings of inga edulis martius seed analysis for the assessment of biotechnological potential inorganic composition the ash content was determined to assess the inorganic composition. ash in a plant sample is the inorganic residue from the combustion of organic matter lost through volatilization or combustion (veloso et al. 2004). the ash content of i. edulis ranged from 28.3% to 32.3%, with an average of 29.58±1.60%. it showed lower values than the same species (44%) from another region, as reported by aguiar (2021). in the analysis of macro and micronutrients in biomass, it was observed that there were elements that serve as nutrients for both human and animal consumption. the main elements found in i. edulis seeds as well as ni, cd, na, pb, and si were also analyzed, and no concentrations above the detection limit were found (table 2). the elements contained in the seeds are relevant for nutrient replenishment in the soil, either naturally or through the formulation of biotechnological products. i. edulis is among the species of nitrogen-fixing trees and may even have the ability to enhance the production of neighboring trees (nichols & carpenter 2006). the elements analyzed are present in various seeds of amazonian fruit trees. however, their concentrations vary according to the species and edaphoclimatic conditions. in the case of i. edulis seeds, the values observed across the five matrices indicated a potential for these seeds to be utilized in biotechnological applications, owing to the presence of these elements in their biomass. nitrogen, phosphorus, potassium, calcium, biotropia vol. 31 no. 3, 2024 322 magnesium, sulfur, and the micronutrients boron, copper, iron, manganese, zinc, and molybdenum are essential for metabolism and are crucial in the tissue composition in organisms that contribute to biomass growth (andrews et al. 2024). these trace elements can be particularly important in processes involving microorganisms, such as anaerobic digestion, where molybdenum, nickel, and iron may be vital for maintaining process stability and influencing biogas production (yu et al. 2016). another example of the importance of inga seeds biomass composition lies in their potential as a biofertilizer. plants, like rapeseed (eggert & von wirén 2016), require these elements, as these nutrients can affect the behavior of microorganisms, enzymatic activities, and metabolic byproducts (soltan et al. 2019). table 2 ash composition obtained from the combustion of i. edulis seed samples elements unity values elements unity values nitrogen g/kg 20.40 boron mg/kg 5.48 phosphorus g/kg 1.71 copper mg/kg 9.72 potassium g/kg 8.67 iron mg/kg 47.61 calcium g/kg 3.01 manganese mg/kg 32.90 magnesium g/kg 1.39 zinc mg/kg 35.14 sulfur g/kg 2.46 molybdenum mg/kg 1.74 organic composition the organic macromolecules commonly present in seeds are proteins and carbohydrates. in the analyzed samples, the average protein content found was 17.6%. this value is close to forage legumes, considered a source of plant protein, such as beans with up to 28% protein content (naeem et al. 2022). proteins are complex molecules of carbon, hydrogen, oxygen, nitrogen, and other elements. they can be analyzed by chns elemental analysis due to their composition of amino acids linked together by peptide bonds (ordóņez 2004). the presence of proteins in seeds is paramount for the organism that consumes this bioproduct because proteins provide essential amino acids that the human body cannot synthesize (leone et al. 2016). additionally, proteins can be important for utilizing the seed biomass in food composition due to their texture (ribeiro & seravalli 2007). the protein fraction varies with the plant’s maturity (pinheiro et al. 2005). it is associated with enzymatic activity responsible for metabolism (e.g., lipases and lipoxygenases, enzymes involved in lipid biosynthesis) and biochemical cycles that function to form structural components of cells and complex organisms (damodaran 2017). like other seeds, it can be a source of plant protein, even if it is not the main component of the seed (ullmann et al. 2023). carbohydrates, on the other hand, are organic compounds produced in the photosynthetic cells of plants and are widely distributed, being present in both animal and plant tissues (ordóņez 2005). glucose, fructose, and sucrose, which are responsible for the sweet taste in various foods (ribeiro & seravalli 2007), belong to this group. in the analyzed samples, carbohydrate content averaged 59.95%, making it an excellent source of carbohydrates, rich in glucose polysaccharides (table 3). table 3 the carbohydrate content of the analyzed i. edulis matrices matrices glucose xylose and arabinose carbohydrate (%) protein (%) average sd average sd m1 55.26a 0.73 2.83a 0.20 58.10 17.5 m2 58.18b 0.62 2.83a 0.36 61.01 17.8 m3 56.00a 0.19 2.78a 0.36 58.79 20.1 m4 59.35c 0.22 2.82a 0.15 62.16 16.6 m5 57.09d 0.15 2.59b 0.17 59.68 15.9 average 57.18 0.38 2.77 0.25 59.95 17.6 notes: different superscripts in the same column indicate significant differences (p < 0.05). biotechnological and environmental education potential of inga edulis seeds oliveira et. al. 323 potential biotechnological use of i. edulis seeds bibliometric approach when conducting a literature review for the last five years (2019-2023) , 23 research articles with “i. edulis” in their titles were found. these documents indicate potential plant uses (fig 4). figure 4 research profile on i. edulis in the last five years (2019-2023) according to databases available in the capes periodicals portal among the studies shown in figure 4, there is a commitment to studying amazonian species to obtain compounds with antifungal activity. dib et al. (2019) purified and characterized a trypsin inhibitor from i. edulis seeds with potential application against candida spp., including candida buinensis and candida tropicalis. lima et al. (2020) obtained extracts with high antioxidant activity from the seeds due to the content of anthocyanins. they isolated an anthocyanin and a mixture of three anthocyanins. lima et al. (2022) identified several compounds with biological activity, including 16 compounds among terpenes, phenolic acids, flavonoids, and anthocyanins. these authors highlighted the high content of phenolic compounds, which are important antioxidants and inhibit rheumatoid arthritis. seed applications related to using polysaccharides have not been observed in journal databases for the last 20 years, which is surprising considering that the seeds contain 59.95% carbohydrates. given the production profile of i. edulis, there is great potential for using polysaccharides, although it is not documented. many products extracted from seeds that could receive biotechnological use can be investigated for their chemoprotective and anticancer properties. those products could become innovative, active principles in pharmaceutical formulations, cosmetics, and food. the products present preventive and inhibitory functions on the growth of cancer cells, with anti-inflammatory, gastroprotective, antioxidant, antimicrobial, and other effects, as shown for the seeds of syzygium cumini skeels (kumar et al. 2022), luffa operculata (silva et al. 2022), eugenia stipitata mc vaugh, and myrtaceae (neri-numa et al. 2013). according to dib et al. (2019), seeds provide plant survival and perpetuation activities associated with chitinases, glucanases, lectins, and thionins. according to cotabarren et al. (2020), i. edulis seeds contain a serine protease inhibitor. a serine protease inhibitor implies a powerful tool to regulate critical biological processes, prevent damage caused by excessive protease activity, and develop therapies for various diseases. this inhibition process has applications in biotechnology, such as in the production of food products, where proteases need to be regulated to control foods’ texture and other properties (tavano et al. 2018). it can also have biotropia vol. 31 no. 3, 2024 324 applications as a bioinsecticide (abd el-latif 2014) and prevent immune responses by regulating proteases (carvalho et al. 2023). according to de moura martins et al. (2020), some species of inga have already been studied for their phytochemical composition. the biological activity of interest to the pharmaceutical industry has been published for inga marginata willd (álvarez et al. 1998), inga goldmanii, inga umbellifera (lokvam & kursar 2005), and inga laurina kunitz (macedo et al. 2011). i. edulis contains small peptides that act as inhibitors of proteolytic activity (serine), which can be a practical approach to deactivate proteases associated with various human diseases, such as arthritis, pancreatitis, hepatitis, cancer, aids, thrombosis, emphysema, hypertension, and muscular dystrophy (cotabarren et al. 2020). starch stands out among the metabolites stored in i. edulis seeds, followed by proteins (rodrigues et al. 2021). starch and proteins are important for i. edulis seeds’ biotechnological application. extracts from i. edulis seeds inhibited 85% of multiple myeloma cell proliferation, demonstrating the significant potential of these seeds for developing new therapeutics (ferro et al. 2022). another aspect to highlight is that some uses of i. edulis are not directly related to the seed itself. i. edulis stands out for its economic potential in reforestation, phytotherapy, energy production, and food. its bark, pulp, and seeds are used in indigenous natural medicine and the landscaping of some cities (pritchard et al. 1995; bilia et al. 2003; caramori et al. 2009). the scientific and technological exploration of the potential of these seeds is quite limited, with only folk knowledge about them. research is crucial to driving biotechnological development. this is corroborated by the high germination rate and extensive seed and viable fruit production throughout the year, as well as the fact that the populations of the amazon region highly appreciate its edible fruits. this is why its cultivation is widespread in agroforestry backyards of local communities. in addition to consuming its pulp, in indigenous communities (brazil and ecuador), roasted seeds are used for food (lojka et al. 2010; kinupp et al. 2021). experimental approach our research found more than 58% glucose content in these carbohydrates, indicating a high starch content, which would be relevant for fermentation processes. glucose content facilitates ethanol production using yeast (bahlawan et al. 2022). additionally, pentoses (xylose and arabinose) can be converted into ethanol using a zimomonas mobilis strain, as khounani et al. (2019) demonstrated for hydrolyzed monosaccharides from safflower seeds. in biotechnological processes, biotransformation involves complex processes that utilize various elements. in the seeds of i. edulis, iron was found at 47.61 mg/kg, which aids enzymes that drive metabolic reactions associated with biotechnological processes. zinc (35.14 g/kg) plays a role in dna synthesis and glucose metabolism, while magnesium is crucial for photosynthesis (ahmed et al. 2024). these elements may be necessary depending on the biotransformation process employed, allowing inga seed biomass containing polysaccharides and proteins to serve as a substrate for new products. biotechnological processes that assist agriculture involve other elements found in the seeds, such as n, p, ca, b, cu, s, mn, and mo, which may be important for biofertilization. additionally, molecules from the polysaccharides hydrolysis and proteins can have biostimulant functions (ugolini et al. 2015). protein content in the samples ranged from 15.9% to 20.1%, which may have biotechnological applications, as recognized by dib et al. (2019), given that the proteins of i. edulis exhibit antimicrobial activity. the proteins and carbohydrates found in i. edulis seeds, when hydrolyzed into extracts by chemical, enzymatic, or biological agents, such as fungi or bacteria, generate monosaccharides and amino acids, respectively (do prado et al. 2021). these compounds play a crucial role as biostimulants in germination, developing new products that can be produced from seed cake. in a biotechnological development approach utilizing the biomass from i. edulis seeds, there biotechnological and environmental education potential of inga edulis seeds oliveira et. al. 325 is potential for separating proteins followed by carbohydrates. the proteins possess added value for health-related products, and subsequently, the carbohydrates can be used to develop bioinputs, biofuels, and other products. environmental approach seeds collecting, seedlings production, and trees planting activities provide a significant environmental approach in schools and the involved community. these activities allow students and the community to understand the natural and artificial environment. furthermore, the activities encourage acquiring knowledge, values, behaviors, and practical skills, enabling active participation in the prevention and solution of environmental management challenges (dias & salgado 2023). pedagogical practices carried out in schools with the establishment of experimental forest nurseries have proven to be suitable for environmental education. they seek to instill new social values, knowledge, skills, attitudes, and competencies for sustainable, student-centered environmental practices. the changes in natural environments in the amazon, including those highlighted in the media from 2019 to 2022, have been intense, degrading biodiversity-rich areas and other natural resources. there is a need to involve students in the production of forest nurseries because of the risk of water shortages, wildfires, the advance of extensive livestock farming, and predatory and irregular mineral exploitation. these issues contribute to the extinction of flora and fauna in the amazon rainforest (luis val & wood 2022). in the activities conducted in this research, an interaction was promoted between students from the technical course in environmental management at ifam and the school community in the amazon region. the activity aimed to create a sense of belonging and scientific understanding of such a meaningful action. this included the preservation of native species from the region and the empowerment of students to plant, consequently helping to address the environmental issues in the amazon. furthermore, the popularization of i. edulis planting can help in soil recovery due to the leaf litter formed and its potential to survive in degraded soils, fixing nutrients (nichols & carpenter 2006). our environmental education work with i. edulis becomes more relevant when we understand that this species has been chosen for planting in degraded soils due to its high survival rate, good canopy development in the early years after planting, and ease of cultivation in nurseries, making it suitable for agroforestry systems (fernandez barrancos et al. 2022). the concern for environmental education related to amazonian fruit trees stems from the importance of raising awareness about biodiversity conservation and the significant role these trees play in addressing the socio-economic needs of socially marginalized communities and food insecurity (vieira & panagopoulos 2024). during school activities, the educational focus was on utilizing these fruits for children’s nutrition as an alternative to traditional fruits. nurseries are responsible for highlighting the importance of these fruits for nutrition and the care required for their seeds to ensure successful planting. cultivating and ensuring food security in urban environments using native local species is a beneficial practice that is gradually forgotten as urbanization progresses. environmental education is crucial in revitalizing the connection with forest food species (albrecht & wiek 2021). therefore, this activity was based on the premise that the school environment is one of the first steps in raising environmental awareness among future citizens, engaging students with a healthier coexistence with nature (de sousa et al. 2011). we also consider that activities involving students can promote initiatives beyond the school environment. certain species with potential for agroforestry can be studied for conscious biotechnological exploitation, balancing scientific and technological progress with ethical values and environmental concerns to promote human wellbeing and global sustainability. biotropia vol. 31 no. 3, 2024 326 conclusion in the biotechnological context, this study unveiled the composition of the primary components of i. edulis seeds, with matrix m4 showing the most favorable results in terms of physiological development. results of this study highlighted the potential for further research into using this species, especially considering its richness in carbohydrates and proteins. this discovery brings innovation to integrated forest production without causing destruction. it was also found that the fruit-bearing i. edulis is a suitable alternative for promoting environmental education in schools, engaging a larger number of people in preserving the natural resources of the amazon biome. these results demonstrate the importance of valuing and sustainably utilizing the region’s resources and promoting practices that encourage conservation and environmental awareness. acknowledgments national council for scientific and technological development cnpq – protocol 306216/2022-1, and coordination for the improvement of higher education personnel – code 001 and 88881.853970/2023-1. references abd el-latif ao. 2014. in vivo and in vitro inhibition of spodoptera littoralis gut-serine protease by 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[physicochemical characterization of acerola juice]. rev biol ciênc terra 4(2). . vieira ta, panagopoulos t. 2024. urban agriculture in brazil: possibilities and challenges for santarém, eastern amazonia. land use policy 139:107082. doi: 10.1016/j. landusepol.2024.107082 yu d, li c, wang l, zhang j, liu j, wei y. 2016. multiple effects of trace elements on methanogenesis in a two-phase anaerobic membrane bioreactor treating starch wastewater. appl microbiol biotechnol 100(15):6631-42. doi: 10.1007/s00253-016-7289-y. cover single.cdr biotropia vol. 27 no. 2, 2020: 153 161 doi: 10.11598/btb.2020.27.2.1198 limited seed dispersal may shape genetic structure of hydnophytum formicarumjack. populations in mangrove ecosystem** abdul shukor yusoff, wan bayani wan omar and shahrudin rohanf faculty of science and marine environment, universiti malaysia terengganu, 21030 kuala nerus, terengganu, malaysia received 03january 2019 / accepted 22 january 2019 abstract hydnophytum formicarum is an epiphytic plant, commonly distributed throughout southeast asia. however, its distribution is currently declining due to forest fragmentation and the subsequent habitat changes which may have also consequently affected the genetic structure of h. formicarum populations. hence, this study aimed to understand the genetic variation and differentiation within and among populations of this species. using random amplified polymorphic dna (rapd) markers, the genetic variation and genetic differentiation among three populations were assessed in order to identify potential conservation management strategies for this species in the mangrove ecosystem in malaysia. ten highly reproducible primers were used in the population analysis, resulting in a total of 221 discernible fragments. genetic variation among populations was high at 98% identified polymorphic fragments. amova showed significant genetic differentiation among the populations (opt = 0.554, p 0.001), with pairwise genetic distances between the populations ranging from 0.495 0.589. pcoa clustering analysis separated the populations according to their geographical locations. the high genetic variation within a population, high genetic differentiation between populations and clear separation in the cluster analysis indicated a restricted seed dispersal of the species. keywords: amova, epiphyte, myrmecophyte, rapd, setiu wetlands introduction epiphytes are plants growing on other plants without directly harming their hosts (zotz 2016). it is one major plant groups contributing largely to worldwide plant diversity. vascular epiphytes alone constitute about 10% of overall global vascular floristic composition (brown et al. 2015) and are distributed extensively across the world. some epiphytic species have been recorded as myrmecophytes due to their association with ants (chomicki & renner 2015). epiphyte studies have also revealed that these plants are particularly sensitive to environmental and habitat changes. hydnophytum formicarum jack is one epiphyte of the rubiaceae family that is identified as a **this paper was presented at the 3rd international conference on tropical biology 2018, 20-21 september 2018, bogor, west java, indonesia myrmecophyte due to its close association with ants as nesters and dispersers (hosoishi et al. 2018). hydnophytum formicarum is a species among 93 others in the genus hydnophytum, under subtribe hydnophytinae, which also considered as the world’s most species-rich ant plant group (chomicki & renner 2015). this species is distributed natively in southeast asia (huxley 1978). notably, the species is widely distributed in the tropical region, and no record can be found on this species in the temperate region. this species favours coastal trees, mangroves and lowland swamp savannahs and hill savannahs (huxley 1978). giesen et al. (2006) have described this species as a mangroveassociated species as its abundance is prominent in this ecosystem. in singapore, h. formicarum has been listed as critically endangered, with few in numbers currently present and no other surviving myrmecophytic epiphytes of the rubiaceae family (lok & tan 2009). 153 biotropia vol. 27 no. 2, 2020 the association of rubiaceous epiphytes including hydnophytum with their ant occupants is called symbiosis or mutualism (huxley 1980). notably, ants of the iridomyrmex spp. and crematogaster spp. were found dwelling in the epiphytes domatia (huxley 1980). ant’s occupation on the epiphytes were highly observed in parts of the plants including the stem, leaf or root of domatia (chomicki & renner 2015). ants also act as dispersers for their host epiphytes. for instance, the ant species philidris nagasau (dolichoderinae) was doing an obligate farming of some epiphytes from genus squamellaria in fiji (chomicki & renner 2016). however, the epiphytes and ants mutualism can also be affected when other changes occur such as habitat shift and also the morphological changes of the host (chomicki & renner 2017). habitat deterioration and fragmentation has affected the distribution and genetic structure of the epiphyte species crepis triasii (asteraceae) in the mediterranean islands (mayol et al. 2012) and hedyotis chrysotricha (rubiaceae) in china (yuan et al. 2012). along the east coast of peninsular malaysia, aggressive coastal development has affected the coastline (muslim et al. 2011; ahmad et al. 2014). in that same region, in the state of terengganu, an abundance of h. formicarum has been observed in the coastal islands with high proclivity to be affected by the aforementioned changes. hence, this fragmentation threat has directed the assessment of the genetic structure of h. formicarum on island populations along the coasts. studies on the genetic structure of plant’s island population have discovered that their spread is in accordance with the species’ geographical distribution (oiki et al. 2001; zhang et al. 2018). however, when there is intermixing of genetic groups, dispersers were found to normally play their roles (godoy & jordano 2001; schidegger et al. 2012). geographical barriers and isolation by distance (ibd) of populations separated by bodies of water or oceanic barriers have been thoroughly studied (dias et al. 2016; levy et al. 2016) as these factors will eventually contribute to species’ gene flow (lee & thomas 2011). one study on the genetic structure of plant populations in an island ecosystem (hufford et al. 2013) (discovered a strong genetic differentiation within and among island populations and apparent ibd between islands. a high variation within island populations and significant genetic differentiation among populations were also observed in the epiphytic orchids (mallet et al. 2014). similarly, the geographical isolation has limited the seed and pollen dispersal of banksia arborea in terrestrial islands (nistelberger et al. 2015). as island populations are relatively smaller in comparison to mainland populations, the genetic variation in island populations should be less (frankham 1996), a theory that was substantiated by hufford et al. (2013) and laukkanen et al. (2014). since no study using molecular markers has yet been conducted for h. formicarum, this study aimed to investigate the genetic variation and differentiation within and among populations for this species as these will provide basic information for management plans focusing in the conservation of this species. specifically, this study will determine and compare the genetic variation of this species in pulau telaga tujuh, pulau layat and pulau redang populations. materials and methods plant sampling leaves were sampled from 21 individual plants (one leaves per individual), of the 7 individual plants from each of the three different island mangrove forests in terengganu on the east coast of peninsular malaysia. pulau telaga tujuh (pt) and pulau layat (pl) are nearby small mangrove islands, while pulau redang (pr) is a tourism island with a fragmented mangrove ecosystem located in the district of kuala terengganu, approximately 30 km away from setiu wetlands (fig. 1). each h. formicarum leaf was collected from different phorophytes. leaves were preserved in zip-lock bags with silica gel and then frozen in -20 °c conditions prior to genetic material extraction. molecular analysis dna was extracted from the frozen leaf samples using modified ctab method (doyle & doyle 1990) after leaf grinding into lyophilized form using liquid nitrogen. the dna concentration was estimated using 154 genetic structure of h. formicarum populations — yusoff et al. biodrop (denville scientific) spectrophoto¬ meter. ten out of 20 screened universal rapd (random amplified polymorphic dna) primers by operon technologies were chosen in this study (table 1). the reproducibility of the selected primers was ensured by replicating the amplification process during optimization. genomic dna was amplified in a reaction volume of 20 pl consisting of template dna, ix reaction buffer (promega), 2 mm mgch (promega), 0.1 mg/ml bsa (promega), 0.25 mmdntps (promega), 0.25 pm of each primer (opa, 1st base) and 0.5 units of taq polymerase (promega). the dna was amplified using thermocycler (applied biosystems) at 30 and 45 cycles for 180 sec of initial denaturation and then 30 sec of denaturation at 95 °c, 30 sec annealing temperature at 32.6 °c to 37.7 °c, 60 sec of extension and then 420 sec of final extension at 72 °c. the pcr products were electrophoresed in 1.5% agarose gel and tbe buffer and ran for 90 min at 60 v. the gel was then stained using diamond® nucleic acid dye (promega) and was finally visualized and photo¬ graphed using the bio rad gel documentation system. data analyses the amplified fragments were scored as 1 if present and 0 if absent and then assembled into a data matrix. the principal coordinate analysis (pcoa) cluster analysis based on genetic distance was performed using past 3 software (hammer et al. 2001). analysis of the molecular variance (amova) (excoffier et al. 1992) was used to partition the total genetic diversity between and among populations. the popula¬ tion genetic differentiation indicator, pairwise opt of amova, was also calculated. amova and pairwise opt were both performed using genalex v6.1 (peakall & smouse 2006). pulau redang south c hina sea pulau layat i pulau tclaga tujuhrv . i figure 1 map of the hydnophytum formicarum populations in terengganu table 1 primer names and sequences chosen for pcr no. primer name primer sequence 5’ to 3’ nucleotide length c+g content (%) 1 opa-01 caggcccttc 10-mer 70.0 2 opa-03 agtcagccac 10-mer 60.0 3 opa-05 aggggtcttg 10-mer 60.0 4 opa-07 gaaacgggtg 10-mer 60.0 5 opa-09 gggtaacgcc 10-mer 70.0 6 opa-11 caatcgccgt 10-mer 60.0 5 opa-13 cagcacccac 10-mer 70.0 8 opa-15 ttccgaaccc 10-mer 60.0 9 opa-17 gaccgcttgt 10-mer 60.0 10 opa-19 caaacgtcgg 10-mer 60.0 155 biotropia vol. 27 no. 2, 2020 results and discussion genetic variation a total of 221 discernible fragments (fig. 2) from the 21 individual plants of h. formicarum were produced at an average of 22.1 fragments per primer. the amplified fragment sizes ranged from 150 to 2500 base pairs (bp). using the primer opa17, the fragments showed 98% polymorphism, with pulau telaga tujuh scoring the highest percentage (89.5%), followed by pulau redang (78.11%) and pulau layat (60.11%) (table 2) indicating that pulau telaga tujuh has the highest genetic variation since a high percentage of polymorphism in a population means a high genetic variation (oiki et al. 2001; boneh et al. 2003). moreover, genetic variation is positively correlated with population size (frankham 1996). this high genetic variation was also found within the island population of epiphytic orchids (mallet et al. 2014). the principal coordinate analysis (pcoa) further showed that the individuals from the three different populations were genetically different (fig. 3). furthermore, the rapd marker confirmed the genetic variation among the plants’ population (monaghan & halloran 1996). the percentage of polymorphism in pulau redang, an island located at sea, was 18% higher than pulau layat even though pulau layat was located just meters away from pulau telaga tujuh, which had the highest polymorphism percentage. the relatively low genetic variation in pulau redang compared to pulau telaga tujuh is possibly due to its oceanic location, which may have limited the gene flow from the mainland resources (maki 2001). islands that are closer to mainland, like pulau telaga tujuh are more likely to act as allelic sink (curto et al. 2017). presumably, pulau layat is a newly established population, considering its low genetic variation and possibly genetic diversity (hagen & hamrick 1998). since the lowest polymorphism percentage was in this population, it is considered as newly founded (ouborg et al. 1999). however, in this study, no further investigation was done to validate the reasons for the low genetic variation in pulau layat. the pcoa cluster analysis exhibited that the individuals were grouped in accordance with the populations where the samples were collected. the results from this study suggest that gene flow between populations was highly restricted. geographical barriers that have formed may have impeded the dispersal of seeds, ultimately preventing the gene flow from occurring in a wider region (monaghan & halloran 1996). however, the epiphytic ferns of hawaiian islands were generally not genetically distinct between island populations, possibly due to high interisland gene flow (ranker 1992). in contrast, the epiphytic orchids sampled from island populations in puerto rico, dominican republic and cuba, had patterns of genetic variation more similar within the islands than with populations of other islands (ackerman & ward 1999). a study on the jumellea epiphytic orchid also showed that the genetic variation between two islands populations differed significantly (blambert et al. 2016). >500bp jooobp isoobp looobp — i kbp pt1 pt2 pt3 pt4 pt5 pt6 pt7 pl1 pl2 pl3 pl4 pl5 pl6 pl7 pr i pr2 pr3 pr4 pr5 pr6 pr7 c loobp figure 2 photograph of diamond® stained agarose gel of rapd fragments using primer opa 17 dna samples from pulau telaga tujuh (pt), pulau layat (pl) and pulau redang (pr) note: lanes 1 kbp and 100 bp are the markers, while lane c is the control. 156 genetic structure of h. formicarum populations — yusoff et al. table 2 genetic variability estimates for populations of h. formicarum from rapd analysis no. code locality n n no. of loci no. of polymorphic loci % of polymorphism 1. pt pulau telaga tujuh > 3000 7 137 123 89.05 2. pl pulau layat > 100 7 101 65 60.11 3. pr pulau redang 7 7 121 99 78.11 notes: n = estimated population size; n = number of sampled individual plants of h. formicarum. o <d 1-ot 1 2 principal coordinates (pcoa) 0.3i 0.20.1-odt -0.2 -0.1 -0.1 -0.2 -0.3 -0.4j 0.1 coordinate 1 0.2 a a a 4a 0.3 0.4 a pulau telaga tujuh pulau layat o pulau redang figure 3 pcoa plot based on rapd variations amongst populations genetic differentiation between populations the analysis of molecular variance (amova) performed on the three different populations showed that the percentage of variations amongst populations (55%) were higher than the percentage of variation within populations (45%) with phipt = 0.554 (p = 0.001; 999 permutations) (table 3). based on the pairwise phipt statistics (modified version of wright’s f), the highest variation was observed between population pulau layat and pulau redang (0.589), followed by that between pulau layat and pulau telaga tujuh (0.584) and finally that between pulau telaga tujuh and pulau redang (0.495) (table 4). greater phipt values indicated greater variation. table 3 analysis of molecular variance (amova) within and among the three populations of h. formicamm source of variation df ss ms est. var. % p among populations 2 387.52 193.76 24.82 55% 0.001 within populations 18 359.42 19.96 19.96 45% 0.001 total 20 746.94 44.78 100% table 4 pairwise population phipt values (opt) pulau telaga tujuh pulau layat pulau redang 0.000 0.584 0.495 pulau telaga tujuh 0.584 0.000 0.589 pulau layat 0.495 0.589 0.000 pulau redang 157 biotropia vol. 27 no. 2, 2020 amova values signified high genetic differentiations, both between and within populations, with opt values higher than 0.25. genetic differentiations also existed among indian epiphytic orchids (nagananda et al. 2012). similarly, a study on epiphytic bromeliads vriesea sealaris and 1vriesea simplex, showed high genetic differentiations between and within populations of v. scalaris (neri et al. 2017). the high genetic differentiation between populations and clear separation in cluster analysis indicated a restricted seed dispersal of h. formicarum (nieder et al. 2000; mucciarelli et al. 2014), and this restricted seed dispersal could lead to fine scale genetic differentiation and restricted gene flow (theim et al. 2014). in our study, the geographical distance and isolation may have affected the gene flow (ouborg et al. 1999; rousset 2000). however, a low genetic differentiation among three different island populations of cephalantera rubra (brzosko & wroblewska 2003) was possibly due to short distances between the islands. hence, no barrier for gene flow existed, as the populations probably originated from the same source. the low genetic differentiation among populations of the hemi-epiphytic species ficus pumila l. in southern china, including several islands, suggested the success of pollinators in dispersal (liu et al. 2015). the relationship between hydnophytum formicarum and ants is considered a facultative mutualistic type, where ants take charge of protecting the plants from phytophagus animals and other abiotic and biotic threats, while plants provide them with a food source, nesting space or sometimes both (heil 2010). once the plant is disturbed, the ants will swarm and fend off the threats. many ant plant species have adapted their caudexes or tubers and provided shelter and protection for the ant colony associated with them (heil 2010; richards & coley 2012; gonzalez-teuber et al. 2014). this mutualism has limited the seed dispersal of h. formicarum almost exclusively to ants as dispersers. the pattern of seed dispersal indeed can shape the genetic structure and composition of plant populations (hamrick et al. 1993). on top of the geographical barrier and the isolation separating the populations, conceivably, ants were unable to cross the water between islands, over time limiting the seed dispersal of h. formicarum. higher genetic variation found within populations suggests that long distance dispersal by migrating birds has contributed to the variation (zarek 2009). however, even if some flying birds were able to consume the fruit of h. formicarum (lok & tan 2009), the number and frequency of such incidents are still low because of the ants that protect the species, making it difficult for the birds to feed on the species’ fruit. the relationship between h. moseleyanum and philidris ants however, proved that ants’ behavior has resulted in the population structure changes of h. moseleyanum (maeyama & matsumoto 2000). territorial philidris ants which aggressively attack ants from different colony could possibly result in the dispersal of h. moseleyanum seeds restricted only to a colony. a similar phenomenon could have happened in the restricted seed dispersals of h. formicarum in this study and may have eventually shaped its genetic structure. seeds dispersal by ants generated a local spatial genetic structure (peakall & beattie 1995). in other studies, the genetic structure of some plants were also found to be promoted by ants (yao 2010; perez-collazos et al. 2015). conclusion seed dispersal was limited due to geographical barriers, yet the role of ants as agents of dispersal, shed light on the changes of h. formicarum^s genetic structure and its high abundance in three mangrove forests. seeds movement by ants have structured the genetic population of h. formicarum in these 3 islands. the limitation of ants as being agents of dispersal, if not halted, curbed the gene flow occurrence among populations. this study served as the first step in evaluating conservation strategies for this species and in assisting conservation efforts particularly, on the genetic variation of the h. formicarum populations. acknowledgements this research was supported by niche research grant scheme (nrgs) (vot no. 53131/12) from ministry of education (moe), malaysia. 158 genetic structure of h. formicarum populations — yusoff et al. references ackerman jd, ward s. 1999. genetic variation in a widespread, epiphytic orchid: where is the evolutionary potential? syst bot 24(2):282-91. ahmad mf, yaacob r, mamat m, muslim am, husain ml. 2014. coastline changes in vicinity of runway platform of sultan mahmud airport, kuala terengganu: comparative analysis of oneline model versus satellite data. j appl sci 14(19):22-34. blambert l, mallet b, humeau l, pailler t. 2016. reproductive 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genetic structure of h. formicarum populations — yusoff et al. zarek m. 2009. rapd analysis of genetic structure in four natural populations of taxpis baccata from southern poland. acta biol cracov bot 51(2): 67-75. zhang z, xie w, zhang j, zhao x, zhao y, wang y. 2018. phenotypeand ssr-based estimates of genetic variation between and within two important ulymns species in western and northern china. genes 9(147):1-18. zotz g. 2016. plants on plants the biology of vascular epiphytes. basel (ch): springer. 161 sri-04. 999-layout.cdr 2500 2000 1500 1000 500 0 # i n d iv id u a ls seedling life stage juvenile mature/productive post �lowering 2211 current expon. (current) expon. (plot i) expon. (plot ii) plot i plot ii 2 6 4 528 535 63 57 137 147 31 71 a treatments weeded unweeded 3.5 3 2.5 2 1.5 1 0.5 0 n u m b e r o f le av e s b treatments weeded unweeded 20 18 16 14 12 10 8 6 4 2 0 h e ig h t in cr e m e n t (c m ) page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 biotropia vol. 28 no. 3,2021: 274 277 doi: 10.1 1598/btb.2021.28.3.1276 beardless barb cyclocheilichthys apogon (vazenciennes, 1842) (cypriniformes: cyprinidae) i n madura island, indonesia veryl hasanl*, soemarn02, maheno sri widod03 and dewa gede r a i a wiadnya4 'department offisb health management and aquaculture, uniuersitas airlanga, surabaya 601 15, indonesia 2agmecotecbnology department, agriczilture faculty, uniuersitas bramjaya, malang 65145, indonesia 'aquatic resources management department, fisheries and marine science facult), uniuersitas brawqaya, malang 65145, indonesia 4fisheries and marine resources utilixation department, fisheries and marine science facalty, universitas brawjqa, malang 65 145, indonesia received 25 june 2019/accepted 11 december 2019 abstract the freshwater fish beardless barb cyclocbeilichtbys apogon (valenciennes, 1842) is a native species of southeast asia, including western indonesia area (borneo, sumatra and java). previously, the species was found only in the mainland of java island. this paper provides the first record of c. apogon in the lembung river, one of the major rivers in madura island, indonesia, thereby extending the species distribution up to 150 krn northeast from the place of earlier record. the specimens of c. apogon were characterized as follows: dorsal fin rays 12; anal fin rays 8 9; pectoral fin rays 17 18; lateral line scales 34 35. a detailed description of the morphological characters of the specimen is provided in this manuscript. keywords: cyprinid, distribution, freshwater fish introduction in the last glacial era, southeast asia and western indonesia (sumatera, borneo and java) were still connected as a single area called sundaland, where many blocth rivers were connected to each other, extendmg from indochina to the java sea (voris 2000). major rises in sea level in the south china sea and java sea that occurred in that era had chided the sundaland into several archipelagos (pubellier & morley 201 4). this geographical change has resulted in the isolation of several freshwater fishes (hubert e t al. 2015), one of which was the beardless barb cyclocheilichtt5y.r apogon (weber & de beaufort 19 1 6). c. apogon (class: actinopterygii; order: cypriniformes; family: cyprinidae) is a freshwater fish native to southeast asia and western indonesia (rainboth 1996; icottelat 'corresponding author, email: veryl.hasan@fpk.unair.ac.id 2001; i<enthao & jearranaiprepam 2018). the species is distributed widely in rivers across mainland java, including east java, central java and west java (weber & de beaufort 1913; roberts 1993). as reported in this paper, c. apogon was recently sighted in lembung river, madura island, thereby widening the previously known distribution range of this species. materials and methods nineteen (19) live specimens of c. apogon were obtained from local fishermen during a fieldwork carried out on 22 23 march 2019 in lembung river (7o02'21" s, 113°46'35" e) (fig. 1). administratively, the site is located on madura island, sumenep regency, east java province, indonesia. the fishng gear used by the fishermen was a small hook fastened with molluscs as the baits (stein eta/. 2012). cyclocheilichtbys apogon on madura island hasan et al. figure 1 lembung river, madura island where the cyclocheilichthys qbogon were collected on 23 march 2019 to ensure the identity of c. apogon, its of the skin above the upper lip. other morphological characters were analysed using morphological characters of the 5 preserved the weber and de beaufort's (1916) model. specimens are as follows: 12 dorsal fin rays; 8 9 anal fin rays; 17 18 pectoral fin rays; 34 35 results and discussion the nineteen (19) live specimens of c. apogon had total lengths between 7.8 and 16.1 cm and total weights between 55.5 and 204 g. five (5) of these were preserved in 96% alcohol solution (hasan e t al. 2019) and deposited as laboratory specimens at the hydrobiology laboratory, universitas brawijaya, malang, indonesia (ub.0002). the remaining fourteen (1 4) were transported in polyethylene bags with oxygen and were kept as livestocks at the fish reproduction laboratory, brawijaya university, malang, indonesia. identification qclocheikichth_ys apogon was distinguished from the other species of qclocheidichth_ys by having no barbels in the snout but with conspicuous folds lateral line scales; head pointed, lips swollen, with both lips evenly curved; dorsal deeply concave, origin of dorsal is opposite to 13th scale of lateral line and nearly in the middle of a line, connecting the end of the snout and the end of the shortest caudal rays, the location of which is nearer to snout in young specimens, far behind the origin of ventrals; anal concave, with a rather weak third spine that is longer than half head; pectorals reach the ventrals; ventrals are about equal to the pectorals, reaching or surpassing the anal; caudal deeply incised, while the lobes are rounded. coloration in fresh specimen: yellowish brown, upper parts are dark brown, and each scale has a dark spot at the base; vertical fins have darkish colors, the others more or less hyahe. a blotch was also found at the end of the lateral line. all of these characters were found in every specimen collected from lembung river, madura island (fig. 2). biotropla vol. 28 no. 3,2021 figure 2 specimen of cyclocheilichth_ys ajbogon captured on 23 march 2019 from lembung river, madura island distribution the discovery of c. apogon in the lembung river, madura island, is the first record of this species beyond its type locality (mainland java: batavia, buitenzorg, ngawi, surabaya and pasuruan) (weber & de beaufort, 1916), and represents the easterly extension of its previously known distribution that is about 150 km away (fig. 3). this new record of native species cyclocbeilichth_ys apogon in the lembung river, madura island, whch is its first outside mainland java is an important contribution in understanding species diversity and biogeography (souto-santos 2019). this new record of c. apogon has improved one's knowledge and understanding of the species particularly on its extended distribution range toward further northeast. several studies on indonesian freshwater fishes were focused on single rivers. crossocheilzls obsczlrms was recorded in sumatra (tan & i<ottelat 2009), and on batang hari basin (tan & kottelat, 2008). c. obscnm also observed on musi basin where the &stance between the location of the first and the second recording was more than 250 km (iqbal e t a l (2017). the presence of c. apogon on madura island is probably due to the previous connection of lembung river to east sunda river during the last glacial era (hanebuth et al. 2000; sathamurthy & voris 2006). the connection was then being cut off and lembung river became isolated due to rising sea levels. besides geological factors, the spread of freshwater fishes outside the mainland could occur due to human induced factors. figure 3 known distribution of cyclocheidchthys apogon. black square are the previous records of the species based on weber and de beaufort's (1916) observation, mainland java. the black triangle is the recent record on lembung river, madura island cyclocheilichth_ys qbogon on madura island hasan et al conclusion cyclocbeilicht&r upogon is a southeast asian native freshwater fish that is not only found in mainland java, but also in the island of madura which is at the eastern end of java. the existence of c. upogon in such a remote area is an added information on the distribution of freshwater fishes in indonesia. references hanebuth t, stattegger i<, grootes pm. 2000. rapid flooding of the sunda shelf: a late-glacial sea level record. science 288(5468):1033-5. hasan v, tamam mb. 2019. first record of the invasive nile tilapia, oreocbromis niloticus (linnaeus, 175 8) (perciformes, cichlidae), on bawean island, indonesia. check list 15(1):225-7. https://doi.org/ 10.15560/15.1.225 hubert n, icadarusman, wibowo a. 2015. dna barcoding indonesian freshwater fishes: challenges and prospects. dna barcodes 3:144-69. https://doi.org/l0.l515/dna-2015-0018 iqbal m, yustian i, indriati w, setiawan d, setiawan a. 2017. crassocheihs obscums tan & kottelat, 2009 (teleostei, cyprinidae): distribution extension and first record for musi basin, south sumatra, indonesia. check list 13(6):1121-4. https://doi.org/l0.15560/13.6.1121 kenthao a, jearranaiprepam p. 2018. morphometric variations and fishery unit assessment of cyclacheilichth_ys qbogon (actinopterigii: cyprinidae) from three-different rivers in north-eastern thailand. pakistan journal zoology 50(1):111-22. icottelat m. 2001. fishes of laos. sri lanka &i<): wildlife heritage trust. rainboth wj. 1996. fishes of the cambodian mekong. f a 0 species identification field guide for fishery purposes. italy (it): fao. pubellier m., morley ck. 2014. the basins of sundaland (seasia): evolutionand boundary conditions: marine and petroleum geology 58:555-78. roberts tr. 1993. the freshwater fishes of java, as observed by kuhl and van hasselt in 1820-1823. zoologische verhandelingen 285:l-94. sathiamurthy e, voris hk. 2006. maps of holocene sea level transgression and submerged lakes on the sunda shelf. the natural history j chulalongkorn university 2:l-44. souto-santos ica, ferraro ga, jennings wb, vergara gls, buckup pa. 2019. geographic distribution of phallocems eigenmann, 1907 (cyprinodontiformes, poeciliidae) in the ilha grande bay hydrographic region, rio de janeiro, brazil. check list 15(1):181-92. https://doi.org/10.15560/15.1.181 stein ja, shultz ad, cooke sj, danylchuk aj, hayward i<, suski cd. 2012. the influence of hook size, type, and location on hook retention and survival of angled bonefish (albula uzllpes). fisheries research 113:147-52. voris hk. 2000. maps of pleistocene sea levels in southeast asia: shorelines, river systems and time durations. journal of biogeography 27(5):1153-67. weber mcw, de beaufort lf. 1916. the fishes of the indo-australian archipelago 111, ostariophysi: i1 cyprinoideaapodes, synbranchi. netherlands (nl): brill. the southeast asian journal of tropical biology vol. 31 no. 2, 2024: 291 307 doi: 10.11598/btb.2024.31.2.2023 issn: 0215-6334 | e-issn: 1907-770x 291 tropical palms and factors influencing their distributions and diversity, with a focus on pinanga diversity in southeast asia mohamad farid abdullah1, salwana md jaafar2, norhayati ahmad1,2, rahayu sukmaria sukri2 1environmental and life sciences program, faculty of science, universiti brunei darussalam, bandar seri begawan, brunei darussalam. 2institute for biodiversity and environmental research, universiti brunei darussalam, bandar seri begawan, brunei darussalam article higlights • the palm genus pinanga is the most diverse in southeast asia. • limited ecological studies on pinanga show the influence of environmental factors. • high pinanga endemism in borneo requires conservation action. article information received: 11 may 2023 revised: 13 july 2023 accepted: 14 july 2024 *corresponding author, e-mail: rahayu.sukri@ubd.edu.bn review article abstract palms represent some of the keystone species in tropical forests, providing numerous ecosystem services. they are widely studied by botanists because of their iconic and majestic appearance, although ecological studies of palm abundance, distributions, and diversity have only recently begun to gain attention. the abundance and diversity of palms at different spatial scales can be influenced by various factors, such as climate, soil properties, hydrology, topography and forest structure; understanding these influences is essential for conservation. this review details four abiotic factors (climate, soil chemistry, hydrology and topography) and one biotic factor (forest structure) affecting palm distribution and diversity. the genus pinanga, one of the most diverse genera of palms, is discussed in terms of its distribution and diversity in southeast asia. ecological studies on pinanga diversity in the region are examined, revealing the influence of microand macro-scale variation in environmental factors, such as litter depth and thickness, canopy openness and crown cover, elevation, slope, aspect, light intensity, humidity and air temperature. the existing knowledge gaps on pinanga ecology in southeast asia are discussed, and the need for more studies on population structures, functional traits and determinants of pinanga diversity and distributions within different lowland forest types in the region are highlighted. the high endemism shown by pinanga necessitates a more comprehensive ecological understanding of this genus to better inform its conservation and protection. keywords:  borneo, environmental factors, monocots, palmae, plant distributions introduction palms (arecaceae or palmae) are among the most abundant and important plant life in tropical rainforests (dransfield  et al. 2008; browne & karubian 2016; elias  et al. 2019). they represent one of the oldest surviving monocot families, maintaining their presence in tropical rainforest-like biomes over geological timespans (renninger & phillips 2016). palms occur in tropical and subtropical regions worldwide and are found in a wide range of ecosystem types, including rainforests, montane regions, dry forests, savannas and desert oases (martins et al. 2014; abdo 2017). in tropical forests, palms are remarkably majestic and can form either dense monodominant palm communities or speciesrich communities with many forms of growth (thonhofer et al. 2015; elias et al. 2016; balslev et al. 2016; mehmud & roy 2021). palms display a variety of growth forms, including small shrubs, lianas and large trees (muscarella et al. 2020). it is estimated that 40% of palm species are capable of growing stems of at least 10 cm diameter at 1.3 m above the ground (kissling et al. 2019). palms can take the form of tall trees entirely by primary growth because they lack secondary growth and vascular https://doi.org/10.11598/btb.2024.31.2.2023 biotropia vol. 31 no. 2, 2024 292 cambium (goodman et al. 2013). some palm trees form thick and shallow root systems that provide better support than those of deep-rooted palm trees (muscarella et al. 2020).  palms are important to tropical ecosystems because they offer various ecosystem services (cámara-leret  et al. 2017a; levis  et al. 2017; muscarella et al. 2020; montoya et al. 2021) and play an important role in forest structure and function (boukili & chazdon 2017). within tropical ecosystems, palms are notable for their high biomass, effects on plant recruitment, interactions with other plants as well as dispersers, and pollinator and nutrient turnover (montúfar et al. 2011; sørensen 2012).  palms are often considered keystone species in tropical forests due to the dependence of many animals on their flowers and fruits for food (onstein et al. 2017; macedosantana  et al. 2021). palms have also functioned as a model system for geographical ecology and rainforest evolution (kissling et al. 2012). with more than 2,500 recognized species in 183 genera worldwide, palms are one of the most diverse groups of plants (eiserhardt et al. 2011; baker & couvreur 2012; kissling et al. 2012; göldel et al. 2015; baker & dransfield 2016; cámara-leret et al. 2017a; levis et al. 2017; muscarella et al. 2020). palms are common and characteristic elements of tropical forests across the equatorial region (dransfield et al. 2008; svenning et al. 2008; baker & couvreur 2013; reichgelt et al. 2018; bogotaangel et al. 2021). palm diversity is the highest in tropical asia, followed by the americas and africa (blach-overgaard  et al. 2010; muscarella  et al. 2020). approximately half of all palm species (c. 1,200 species in 57 genera) have been recorded in tropical asia, most of which are found in malesia, with almost 1,000 species in 50 genera (dransfield  et al. 2008; renninger & phillips 2016). although malesia is known as the greatest palm diversity hotspot, there is a differentiation in species richness and distribution patterns across the region (baker & couvreur 2012). high palm species richness was recorded in both western (302 species in borneo, 162 species in the philippines) and eastern malesia (243 species in new guinea; baker & couvreur 2012). in contrast, lower species richness has been recorded in wallacea, which comprises sulawesi, the moluccas and the lesser sunda islands, with estimates of at least 62, 40 and 6 species, respectively (baker & couvreur 2012). palm abundance and diversity can have significant effects on tropical forest ecosystem functions as well as carbon sequestration (muscarella  et al. 2020). palms are widely accepted as suitable model organisms for understanding the influence of high tropical biodiversity and geographical variation (eiserhardt et al. 2011; couvreur & baker 2013). therefore, understanding the diversity, distribution and dispersal of palms is integral to their conservation (dransfield et al. 2008; eiserhardt et al. 2011; bacon et al. 2013; abdo 2017). however, quantitative analyses of the distribution patterns and factors associated with palm abundance and diversity are still lacking (muscarella et al. 2020). among the palms, the genus pinanga is one of the most diverse (govaerts et al. 2020) and has recently been gaining attention in scientific studies (mooney 2020; haji ramlan 2020; abdullah 2021). this review focuses on the factors influencing palm abundance and species diversity, as well as the diversity of pinanga palms in southeast asia. factors influencing palm abundance and species diversity global patterns in palm distributions are mainly limited by low temperatures (moraes et al. 2014). an increase in temperature can extend palm distributions to higher elevations where precipitation can then exert a significant positive effect on palm species richness (renninger & phillips 2016). several studies have reported that palm abundance and species richness are strongly correlated with environmental factors (kahn 1987; cintra  et al. 2005; eiserhardt  et al. 2011; kristiansen et al. 2011; manggat 2012; emilio et al. 2014; scariot 2015; benchimol  et al. 2017; elias et al. 2019), and palm species distributions on continental and regional scales are associated with changes in climate, soil properties and dispersal (blach‐overgaard  et al. 2010). at smaller scales, palm distributions are associated with topography, hydrology, vegetation structure, species interactions and dispersal, but the relative importance of these factors differs between studies (vormisto et al. 2004; blach‐overgaard et al. 2010; eiserhardt et al. 2011; sørensen 2012; alvaro-segura et al. 2012). here, we review four abiotic factors (climate, soil chemistry, hydrology and topography) and one biotic factor (forest structure) that influence palm abundance and species diversity (eiserhardt et al. 2011). tropical palms and factors influencing their diversity, with a focus on pinanga in southeast asia abdullah et al. 293 climate palm species distribution studies have primarily focused on the role of the environment, with climate often regarded as the main range-limiting factor, especially at large spatial scales (blach‐ overgaard et al. 2010). climate plays a crucial role in global palm distribution (muscarella et al. 2020; bogota-angel  et al. 2021), and the distribution of individual palm species and patterns of palm species richness and diversity across different vegetation types are strongly associated with climate (svenning  et al. 2008; eiserhardt  et al. 2011; sevegnani et al. 2016). previous studies also observed that climate is important in determining plant distribution and diversity (pearson & dawson 2003; kreft & jetz 2007). palm diversity hotspots, such as the sunda region, offer beneficial conditions for diversification in palms due to the prolonged periods of climatic stability, while poor palm diversity can be observed in the lesser sunda islands due to their moderately dry, seasonal climate (baker & couvreur 2012). palm diversity is strongly correlated with climatic factors, especially water (bjorholm  et al. 2005), with precipitation having the strongest positive effect on palm species richness (renniger & philips 2016). similarly, climate has the greatest influence on the distributions of palms, which appear to be more significantly influenced by water-related variables than by temperature (blach‐overgaard  et al. 2010; sørensen 2012). additionally, palm species richness is influenced by annual precipitation or the number of wet days (sørensen 2012). this supports a broad-scale study of species richness conducted by hawkins  et al. (2003), who concluded that water-related factors, such as rainfall and precipitation, are excellent drivers of plant richness in tropical and subtropical areas. blach-overgaard et al. (2010) concluded that climate was more important than habitat and human impact in determining the distribution of 25 out of 29 african palm species, with most of the palm species studied preferring humid climates, and few being influenced by temperature. in addition, palm species distributions within subtropical and temperate regions are often restricted due to low temperatures (gatti et al. 2008). therefore, palms are most diverse in warm and humid regions (eiserhardt et al. 2011). studies focusing on the influence of climatic variables on palm species richness and diversity are limited (dransfield et al. 2008), with the influence of climatic factors on palm species distributions within tropical regions evaluated for african palms only (blach-overgaard  et al. 2010). temperature seasonality has been observed to limit palm distributions and emphasizes climate as the main range determinant at large spatial scales (pearson & dawson 2003). however, it is increasingly understood that palm species distributions are sensitive to climate at multiple spatial scales (blach‐ overgaard et al. 2010), and therefore, more studies at both local and global scales are recommended (eiserhardt et al. 2011).  soil properties most tropical plant species show strong associations with edaphic conditions, and soil fertility can influence tree community composition (john et al. 2007; sukri et al. 2012). palms also show habitat associations governed by soil properties (vormisto et al. 2004; andersen et al. 2010; viana  et al. 2021). soil fertility is the second most important abiotic factor influencing palm species richness (bjorholm  et al. 2006). for example, the diversity of palm subfamilies arecoideae and calamoideae tends to increase with increasing soil fertility (bjorholm et al. 2006). similarly, the link between soil properties and palm community composition in lowland rainforests is well studied (vormisto  et al. 2004; andersen  et al. 2010), and palm community compositions are linked with soil properties, such as exchangeable base concentration, aluminum concentration and soil texture (cámara-leret et al. 2017b). studies have shown that soil chemistry can affect palms species’ abundance and richness (ruokolainen & vormisto 2000; svenning 2001; bjorholm et al. 2006; poulsen et al. 2006; eiserhardt et al. 2011; muscarella et al. 2020). the distribution and abundance of several tropical american palm species are correlated with soil factors, such as nutrient concentrations, aluminum content and clay (svenning 2001). palm species in the western amazon prefer either nutrient-poor or nutrientrich soils (ruokolainen & vormisto 2000), and the composition of amazon palm communities is significantly correlated with soil chemical properties, such as exchangeable cations and aluminum content (poulsen et al. 2006; dalling et al. 2016; viana  et al. 2021). macronutrients, such as exchangeable bases (calcium, potassium and magnesium) and phosphorus, were found to influence species abundances in palm communities in non-flooded amazonian forests more biotropia vol. 31 no. 2, 2024 294 significantly than micronutrients did (cámara et al. 2017b). similarly, exchangeable bases, phosphorus and nitrogen concentrations have been linked to the distribution of palms in various lowland tropical rainforests (andersen et al. 2010; baribault  et al. 2012; condit  et al. 2013). soil bulk density can also influence the composition of palm communities (andersen et al. 2010; viana et al. 2021), and palms can show variation across gradients of soil nutrient availability (viana et al. 2021). palm species composition can also vary with soil texture on local scales, likely due to a soil texture correlation with drainage (kristiansen et al. 2012; sørensen 2012). in contrast, some studies have also found that differences in soil properties may not be important in influencing individual palm species’ distributions at continental scales, and not all studies support a significant role for soil fertility in palm community turnover (normand et al. 2006; blach-overgaard et al. 2010). therefore, the strength of soil effects on palm community composition is variable and may depend on the spatial scale, ecosystem and habitat (eiserhardt et al. 2011; viana et al. 2021). moreover, soil chemistry often interrelates with other environmental factors, such as topography, hydrology and vegetation structure, which requires further study (eiserhardt et al. 2011). hydrology plant species distributions and diversity are also affected by hydrology, particularly through flooding and drainage (eiserhardt  et al. 2011). as precipitation is one of the most important predictors of palm species richness (kissling et al. 2012), this correlates with a higher abundance of palms in continually wet sites (viana et al. 2021). the importance of hydrology for palm species distribution has been well demonstrated for amazonian palms (kahn 1987). several studies have assessed the effects of flooding and drainage on the distribution and abundance of individual palm species (svenning 2001; montúfar & pintaud 2006). most palms are linked with either well-drained or poorly-drained soils (henderson et al. 2019), while some palm species prefer swampy areas (dransfield et al. 2008). for instance, most of the african palms occupy humid rainforests or swamplands, and are often in the riparian zone or areas where the water table is locally high, while very few species are found in dry and open habitats, such as savannas or deserts (blach‐overgaard  et al. 2010). palm species abundance in french guiana is correlated with a change in soil drainage along topographic transitions (salm  et al. 2015). additionally, salm  et al. (2015) found that the abundance of the six most common palm species was correlated with humidity gradients from floodplains to terra firme, with palm distribution from the most flood-tolerant to the least floodtolerant palm species.  differences in soil moisture and drainage have been shown to affect palms individually (svenning 2001). for example, five out of 23 palms responded individually to poor drainage in the amazonian terra firme forest (svenning 1999). significant differences in palm community composition between well-drained and poorly-drained soils have been recorded (balslev et al. 1987; kahn 1987; normand et al. 2006). for instance, palms in the terra firme (dry land) consisted of 17% of individuals and 11% of the basal area, compared with 30% of individuals and 19% of the basal area in the floodplain forest in ecuador (balslev et al. 1987). palm species richness in seasonally flooded land and terra firme forests in the amazon basin was found to be three times higher on well-drained soils (terra firme) than on seasonally flooded soils (kahn & de castro 1985). similarly, in the western amazon, higher species richness was recorded for palm communities in terra firme forests than in wetland forests (eiserhardt  et al. 2011), and in ecuador, palm species were found to be significantly more abundant in terra firme forests than in floodplain forests (balslev  et al. 1987). although flooding and drainage have strong effects on palm distribution on local to landscape scales, as well as on palm species richness on a landscape scale (eiserhardt et al. 2011), the degree to which these effects are influenced by other factors, such as vegetation structure and light availability, requires further investigation (svenning 2000). topography topography strongly affects the distribution of tropical plant species (sukri et al. 2012; wang et al. 2015; limin et al. 2022) and is known as a key influencing factor for vegetation patterns at the landscape and regional scales (sanders & rahbek 2012; moeslund et al. 2013). plant species diversity is often related to topographic heterogeneity at broader scales (kreft & jetz 2007). topography indirectly affects plant distributions by regulating other abiotic factors, such as soil conditions, wind exposure, hydrology, temperature, climate and forest structure (svenning 2001; balslev  et al. 2011; eiserhardt et al. 2011; lueder 2020). tropical palms and factors influencing their diversity, with a focus on pinanga in southeast asia abdullah et al. 295 palm species abundance, richness and composition have a significant relationship with altitude (rodrigues  et al. 2014). palms decrease in abundance with increasing soil moisture and decreasing elevation, indicating that elevation and soil moisture influence palm community patterns (bonetti  et al. 2017; lueder 2020). kreft  et al. (2006) found that rare palm species were more strongly influenced by topographic complexity, while climatic factors were strongly correlated with species richness for widespread species. at landscape scales, topography shows a correlation with palm abundance (eiserhardt  et al. 2011). for instance, the abundance of canopy palms in costa rica and wet amazonian forests was the highest on crests and steep slopes than at lower altitudes (kahn 1987). in a seasonally dry forest in southern amazonia, palm abundance decreased with elevation (salm et al. 2007), while in costa rica, palms are abundant at lower elevations (approx. 2,500 m altitude; lieberman et al. 1996). eiserhardt et al. (2011) suggested that high palm abundance in steep and rugged terrain was due to a high number of canopy gaps that were favorable for palm recruitment, while the lower abundance of palms on steep slopes compared with crests may be due to the high water overflow causing those sites to be extremely dry for palms (kahn 1987). additionally, salm  et al. (2007) showed that the high abundance of palms in low-lying areas was due to high moisture availability. at a local scale, palm species abundance differed independently of topographic position in both amazonian lowland rainforests and seasonally dry forests (kahn 1987; salm et al. 2007). in amazonian ecuador, 10 out of 23 palm species were related to specific topographic positions, while altitude and inclination tended to be less important; the opposite pattern was seen in the wet lower montane forest in ecuador, where altitude and aspect strongly influence palm species distributions (svenning 1999, 2001). in terms of species richness, both palm species richness and genus richness decrease significantly with elevation in new guinea and its adjacent islands (bachman et al. 2004). however, topography may be less influential on palm species richness at a continental scale in the americas (bjorholm et al. 2005; kreft et al. 2006). on a landscape scale, palm species richness tends to decline with elevation in the amazon rainforest (salm  et al. 2007) and along an altitudinal transect in costa rica (lieberman  et al. 1996). in contrast, poulsen  et al. (2006) found that the species richness of palms was the highest at the highest elevations in terra firme amazonian rainforest. overall, topography is important at local scales but may be less important in influencing palm species richness at broader spatial scales (bjorholm et al. 2005; kristiansen et al. 2011). topography only influences palm species distributions and community composition indirectly; thus, its effects will vary for local scale differences depending on the geographical features of each location (vormisto et al. 2004). it is also suggested that the relationship between diversity and elevation depends fundamentally on the interaction of environmental variables (rahardjo 2020). consequently, it is important to measure other environmental factors, such as soil water availability, soil nutrient conditions, hydrology, wind exposure and light availability, which are factors that directly underlie topographic gradients (svenning 2001; eiserhardt et al. 2011), and to determine how topographic variables can influence the population dynamics of palms (pasion  et al. 2022). forest structure forest structure reflects the role of plants and specific trees as ecosystem engineers that can influence environmental factors, such as light availability and litter fall (svenning 2001). the surrounding vegetation and canopy gaps can influence microclimate and light availability (carson & schnitzer 2011), and light influences species distribution through competition for light and through shade tolerances (carson & schnitzer 2011). in palm communities, several palm species favor an open canopy due to light availability (eiserhardt  et al. 2011). for example, rattan occurrence is positively correlated with the presence of treefall gaps (siebert 2005). in addition, svenning (2000) found that several palms prefer gaps and are distributed non-randomly with canopy openness. the importance of light on palms may differ between ontogenetic stages (svenning 2001). previous studies have suggested that large palms can reach the adult stage only in treefall gaps due to their light requirements increasing at the adult stage (eiserhardt et al. 2011) there are no strong relationships between canopy heterogeneity or light availability and palm community composition (eiserhardt  et al. 2011). for example, canopy openness and the presence of non-palm trees in amazonian terra firme rainforest does not significantly influence palm species richness (cintra et al. 2005). biotropia vol. 31 no. 2, 2024 296 however, the community composition of palms is associated with forest structure (browne & karubian 2016), where adult palm abundance and species richness tend to increase with canopy height, while juvenile abundance decreases with canopy openness. juvenile species richness was also found to increase with distance to the forest edge and decrease with canopy openness (browne & karubian 2016). hilário and toledo (2016) observed that forests with taller and larger trees, a closed canopy and an open understory contain higher palm abundance. however, eiserhardt  et al. (2011) argued that the influence of vegetation structure on the species richness of palms may be weak, as only a few palm species respond to canopy openness or light availability, likely due to speciesspecific preferences. genus pinanga the genus  pinanga  blume (1838) is a genus of palms in the family arecaceae (syauqina  et al. 2017; randi  et al. 2019) and is one of the most diverse genera of palms (govaerts  et al. 2020). arecaceae are persistent climbers, bushes and trees (mainasara  et al. 2019). the growth forms of  pinanga  are varied, including shrubs and tree-like or stemless plants (dransfield et al. 2008). the genus shows significant differences in morphological and quantitative characteristics, such as size, form, stem color, crown shaft, leaves and pollen (randi et al. 2019). pinanga palms can be distinguished from other palms by the entire margins of the blades of their leaves or leaflets, and by their inflorescences, flower arrangements and fruits (ang et al. 2010). pinanga can be found in a variety of habitats because each species typically prefers different environments (dransfield 1980; fernando et al. 2020). for example, pinanga simplicifrons (miq.) becc. is found in shaded and swampy areas (ang et al. 2010), while pinanga javana blume prefers hill slopes (zulkarnaen et al. 2019). some pinanga species prefer higher elevations (syahida-emiza et al. 2018; fernando et al. 2020). local communities often use pinanga palms as tools and sources of food. for example, the stalk of pinanga auriculata becc. is used to make fish traps, while edible fruit from pinanga patula blume var. microcarpa becc. can be eaten with betel (haji ramlan 2020). in bali (indonesia), pinanga arinasae witono (locally known as nyabah or jabah) leaves, fruits as well as stems are culturally important for balinese people (sutomo et al. 2018). local people mostly use p. arinasae for ornamental purposes (syauqina et al. 2017; sutomo et al. 2018), such as the production of traditional umbrellas called ‘cukup’ from the p. arinasae leaf sheath (sutomo et al. 2018). the extract from pinanga limosa ridl. may function as a potential anticancer agent, although its efficacy requires verification (mainasara et al. 2019). distribution of pinanga species currently, a total of 145 species of pinanga have been recorded worldwide (powo 2024) and their distributions range from the himalayas and south china to new guinea, with high diversity at the sunda shelf, while they are poorly represented in papuasia (coode et al. 1996; randi et al. 2019). within southeast asian tropical rainforests, pinanga  typically displays prominent levels of endemism and high species diversity (dransfield et al. 2008). the southeast asian region is the center of pinanga diversity worldwide, with 140 species of pinanga recorded (govaerts et al. 2020; zulkarnaen  et al. 2022a). the highest pinanga species richness has been recorded in malaysia and indonesia (61 and 49 species, respectively), while cambodia and laos are the least speciesrich countries (1 species each; fig. 1). no records of pinanga species have been published to date for timor-leste. these differences in species richness of pinanga in the southeast asian countries partly reflect the larger amounts of sampling effort and studies that have been conducted in malaysia and indonesia compared to countries, such as cambodia, laos and timor-leste. as studies on palm communities have gained increasing attention in the past ten years,  pinanga  palms have been discovered in various locations in southeast asia during this period (ang et al. 2010; loo et al. 2014; syauqina et al. 2017; randi  et al. 2019; fernando  et al. 2020; lim  et al. 2022). in singapore, ang et al. (2010) rediscovered p. simplicifrons, which was widely regarded as nationally extinct in singapore before this rediscovery. p. simplicifrons  was also rediscovered near lornie trail at the edge of macritchie reservoir, singapore (lim et al. 2022). these rediscoveries in specific localities have led to the assessment of p. simplicifrons as ‘critically endangered’ in singapore (ang et al. 2010; loo et al. 2014; lim  et al. 2022), although it remains unassessed in the iucn red list (iucn 2024). in malaysia, the pinanga genus was the third most dominant palm genera recorded in the dered tropical palms and factors influencing their diversity, with a focus on pinanga in southeast asia abdullah et al. 297 figure 1 variation in species richness of pinanga across southeast asia notes: the total number of pinanga species recorded in each country was compiled from coode et al. (1996), ang et al. (2010) and powo (2024). krian national park after the genera calamus and daemonorops, while pinanga chaiana j.dransf., pinanga crassipes becc. and pinanga jambusana c.k.lim were found scattered along trails in this national park (syauqina  et al. 2017). during an expedition to long banga, sarawak, at least 11 palm species were found at higher ridges, including an unidentified species of  pinanga  (syahidaemiza  et al. 2018). in indonesia, p. arinasae was found in the pilan forest in bali (wahyuni  et al. 2017), although the species was previously suggested to be present only in mt. tapak bedugul, bali (witono et al. 2002; sutomo et al. 2018). pinanga schwanerensis randi, hikmat & heatubun was newly discovered in kalimantan, growing on undulating grounds and steep slopes (randi  et al. 2019). pinanga  lepidota rendle was rediscovered on palawan island in 2020, increasing the number of pinanga  species recorded in the philippines to 27 (fernando et al. 2020). in vietnam, pinanga spiralis a.j.hend. & n.q.dung was discovered by henderson and dung (2017). within borneo, a total of 41 species of pinanga have been recorded (table 1), mostly from sarawak, brunei darussalam and sabah (odufuwa 2019; govaerts et al. 2020; powo 2024). fewer records of pinanga have been found in kalimantan, indonesian borneo, despite the larger land area and greater habitat diversity (randi et al. 2019; odufuwa 2019). the higher species richness in malaysian borneo likely partly reflects greater sampling efforts compared to kalimantan. notably, despite having the smallest land area within borneo, 23 species of pinanga have been recorded in brunei darussalam. almost all of the pinanga species recorded in borneo are endemic to the island (powo 2024), with the exception of three species (p. auriculata, p. patula and p. simplicifrons). biotropia vol. 31 no. 2, 2024 298 table 1 checklist of pinanga species recorded in borneo no. species brunei sabah sarawak kalimantan 1 pinanga albescens becc.   x x x 2 pinanga angustisecta becc. x x x   3 pinanga aristata (burret) j.dransf. x x x x 4 pinanga arundinacea ridl.   x x   5 pinanga auriculata becc. x x x   6 pinanga borneensis scheff. x x x x 7 pinanga brevipes becc. x x x x 8 pinanga capitata becc. x x x x 9 pinanga chaiana j.dransf. x x x   10 pinanga crassipes becc.   x x   11 pinanga cucullata j.dransf.     x   12 pinanga decora l.linden & rodigas   x x   13 pinanga dumetosa j.dransf. x x x   14 pinanga gracillima merr.   x x   15 pinanga jambusana c.k.lim     x   16 pinanga keahii furtado   x x x 17 pinanga lepidota rendle x x x x 18 pinanga ligulata becc.   x x x 19 pinanga limbangensis c.k.lim     x   20 pinanga minuta furtado. x x x   21 pinanga mirabilis becc. x x x x 22 pinanga mooreana j.dransf. x   x   23 pinanga pachyphylla j.dransf.     x   24 pinanga patula blume x x x x 25 pinanga pilosa (burret) j.dransf.   x x   26 pinanga ridleyana becc. x x x x 27 pinanga rivularis becc. x   x   28 pinanga rupestris j.dransf.     x   29 pinanga salicifolia blume x x x x 30 pinanga schwanerensis randi, hikmat & heatubun       x 31 pinanga sessilifolia furtado x x x x 32 pinanga simplicifrons (miq.) becc. x x x x 33 pinanga stricta becc.   x x   34 pinanga subterranea randi & w.j.baker     x x 35 pinanga tenacinervis j.dransf.     x   36 pinanga tenella (h.wendl.) scheff. x x x x 37 pinanga tomentella becc. x x x x 38 pinanga trichoneura becc.   x x   39 pinanga variegata becc. x x x x 40 pinanga veitchii h.wendl. ex h.j.veitch x   x   41 pinanga yassinii j.dransf. x         total number of species 23 29 39 19 notes: presence of each palm species in a country or state in borneo was indicated by an ‘x’. all species are bornean endemics, with the exception of three species highlighted in bold. tropical palms and factors influencing their diversity, with a focus on pinanga in southeast asia abdullah et al. 299 ecological studies on pinanga in southeast asia in southeast asia, most published studies on pinanga palms have focused on checklists and descriptive notes on  the pinanga  species found within a country (ang et al. 2010; henderson et al. 2010; adorador et al. 2020; fernando et al. 2020). despite increased attention on documenting pinanga diversity within southeast asia, studies focusing on pinanga ecology remain limited. similarly to other palms, pinanga distributions are often shaped by both microand macro-scale variation in environment. within southeast asia, studies have shown the influence of a variety of environmental factors, such as litter depth and thickness, canopy openness and crown cover, elevation, slope, aspect, light intensity, humidity and air temperature (syauqina et al. 2017; astuti et al. 2018; randi et al. 2019; zulkarnaen et al. 2019, 2022a,b; iryadi & sutomo 2020; yudaputra et al. 2021). a study by yudaputra et al. (2021) investigating the ecology of endemic p. arinasae in bali showed that litter depth, canopy openness, elevation, slope and aspect were the most important environmental factors influencing this species’ occurrence. in addition, microclimatic conditions, such as light intensity, humidity and air temperature, were recorded as significant influencing factors, with light intensity considered the main factor influencing the distribution and abundance of p. arinasae (iryadi & sutomo 2020). similarly, the population structure and habitat preferences of p. javana  at mt. slamet (central java, indonesia) were investigated (zulkarnaen et al. 2019). the study showed that the population of p. javana was dominated by adult palms on the southern slope of mt. slamet and that slope, litter thickness and crown cover influenced the species’ density. pinanga palms are also frequently found in humid areas covered by high canopy cover, near small rivers and in soil with thick litter layers, as well as in limestone areas (syauqina et al. 2017; randi et al. 2019). another study by zulkarnaen et al. (2022b) found that mt. slamet was dominated by p. javana and pinanga coronata blume, with p. javana found above waterfall areas or on hill slopes. the growth of p. javana in mt. slamet appears to require specific abiotic factors, such as slope, litter thickness and canopy cover (astuti  et al. 2018).  in contrast, p. coronata individuals were recorded under the heavy shade of the canopy (zulkarnaen et al. 2022a). in mt. ungaran (central java), p. javana was found figure 2 selected pinanga species found in brunei darussalam: a. pinanga aristata (burret) j.dransf.; b. pinanga aff. brevipes becc.; c. pinanga mooreana j.dransf.; d. pinanga lepidota rendle; e. pinanga veitchii h.wendl. ex h.j.veitch; f. pinanga mirabilis becc. and g. pinanga chaiana j.dransf. a b c d e f g biotropia vol. 31 no. 2, 2024 300 to be strongly correlated with a stable and humid microclimate, similarly to the p. javana found in mt. slamet (zulkarnaen et al. 2019, 2022b). this suggests that humidity levels can significantly influence p. javana populations. furthermore, p. javana  seems to prefer locations with steep to very steep slopes (zulkarnaen et al. 2022a). in kalimantan, randi et al. (2019) found that p. schwanerensis  grows on uneven terrain and slopes between 150-550 masl (meters above sea level), and prefers a humid area, high forest cover and thick leaf litter, as well as locations near streams. other studies, such as those in the philippines, vietnam and singapore, provide little information on the ecology of pinanga palms, typically only including information on their altitude occurrences and locality (ang  et al. 2010; henderson & dung 2017; fernando et al. 2020; lim et al. 2022). for instance, on palawan island, p. lepidota is found in dipterocarp forest at an elevation of 600 m, while p. lepidota in borneo can be found up to an elevation of 1,500 m (fernando et al. 2020). p. spiralis in vietnam is found in lowland rainforests at elevations between 200-550 m (henderson & dung 2017). p. simplicifrons  favors an open area caused by tree falls, and under high canopy cover (lim et al. 2022), and can also survive in swampy areas with rich alluvial soil (ang et al. 2010). in brunei darussalam, a total of 23 species of  pinanga  have been recorded (fig. 2), and most of these were recorded in ulu temburong national park (coode et al. 1996; haji ramlan 2020). these pinanga species are found in different habitats, from gentle to steep slopes, sandy to clay soil, and at various altitudes; additionally, most of the species are shrubs and are found near running streams (coode et al. 1996). this indicates that pinanga may have specific preferences for soil and environmental conditions.  several recent studies have focused on the ecology of pinanga  palms in brunei darussalam. the distributions of pinanga species in brunei darussalam are shaped by many factors, such as topographic positions, vegetation structure and soil properties. shapcott et al. (2022) investigated the populations of five co-occurring  pinanga  species at kuala belalong, ulu temburong, and found that the population sizes of most of these species were maintained 20 years later, with two species (p. brevipes  and  p. aristata) declining in overall abundance. they attributed the decline of understory pinanga palms to the possible influence of climate change due to increased rainfall fluctuations at kuala belalong (shapcott et al. 2022). also at kuala belalong, mooney (2020) investigated the landscape distribution of pinanga and concluded that one group of  species (p. mooreana and p. minuta) was strongly associated with ridges, while a second group (p. aristata, p. brevipes, p. dumetosa and p. veitchii) was more strongly associated with valleys. the complex topography of the kuala belalong area (sukri et al. 2012) appears to have created a range of microhabitats that enabled pinanga species to coexist within the landscape. at two contrasting mixed dipterocarp forest sites in brunei darussalam (belalong and teraja), haji ramlan (2020) recorded the influence of soil properties on pinanga species richness, with higher pinanga species richness recorded in belalong due to higher concentrations of soil nutrients (total p, mg, ca, total and exchangeable k) and higher soil organic matter content, soil ph and soil gravimetric water content at this site. abdullah (2024) recorded distinct pinanga community composition between heath and mixed dipterocarp forest types in brunei darussalam, with five pinanga species (p. aristata, p. chaiana, p. mooreana, p. variegata var. hallieriana and p. veitchii) more strongly linked with the mixed dipterocarp forest sites and two pinanga species (p. lepidota and p. salicifolia) more strongly linked with the heath forest sites. focusing specifically on the foliar nutrient content of  pinanga  species, abdullah (2021) examined its variation in three pinanga species (p. lepidota, p. mooreana and p. mirabilis) with contrasting habitats and leaf formations. the study found that those foliar nutrient concentrations differed between the three  pinanga  species and differed between different leaf formations of the same species. however, further investigations are required to relate the relationship between foliar nutrient concentrations and soil variables to further examine the ecology and habitat adaptations of these three pinanga species. knowledge gaps on pinanga ecology in southeast asia most studies of the genus pinanga in southeast asia have focused on descriptive notes (dransfield 1980; coode et al. 1996; witono et al. 2002; zulkarnaen et al. 2019; fernando et al. 2020). although descriptive notes on pinanga species are abundant (dransfield 1980, 1991; keat et al. 1998; henderson et al. 2010; fernando et al. 2020; kuhnhäuser et al. 2023), these typically include tropical palms and factors influencing their diversity, with a focus on pinanga in southeast asia abdullah et al. 301 limited information, focusing on taxonomic descriptions and brief notes on, for example, locality and habitat. studies that focus on population structures of pinanga species are still lacking, and to date, only two studies in southeast asia are known to have focused on this. in indonesia, yudaputra et al. (2021) studied the population structures of p. arinasae in bali island and found that the population comprised many seedlings, but a much lower abundance of juvenile and mature individuals. meanwhile, zulkarnaen et al. (2019, 2022a) studied the population structure of p. javana at two sites, mt. ungaran and mt. slamet, java, indonesia. they found that p. javana populations in mt. ungaran mainly comprised individuals at the seedling stage (zulkarnaen et al. 2019), while those at mt. slamet mainly comprised adult individuals with stem heights of 6.1-8.1 m and stem diameters of 7-8.9 cm (zulkarnaen et al. 2022a). in brunei darussalam, one of the first studies of pinanga was done on five pinanga species (p. aristata, p. brevipes, p. dumetosa, pinanga tenella var. tenella, and p. veitchii) by shapcott (1999). the study compared the population genetics and densities of these five pinanga species at kuala belalong, brunei, and showed that all species of pinanga palms studied had quite prominent levels of genetic diversity, which were correlated with population density. shapcott (1999) further reported that the densest species had less genetic variation than less-dense species, with p. dumetosa exhibiting the greatest gene flow between its populations and p. brevipes exhibiting the lowest.  the lack of studies on pinanga population structure in southeast asia underscores the need for increased ecological investigation into the populations of these species, particularly since the region holds the highest diversity of pinanga species globally. understanding the population biology of pinanga species, particularly those that are endemic to borneo, is of priority to better inform and design management plans and conservation efforts for these palms. although environmental factors and soil properties have been shown to influence palm communities at different spatial scales (eiserhardt et al. 2011), their influence on pinanga palm communities are only beginning to be understood. descriptives notes provide anecdotal evidence to demonstrate that pinanga species display habitat preferences (dransfield 1980; coode et al. 1996; witono et al. 2002; fernando et al. 2020), but the significant influence of environmental factors and soil conditions still requires investigation. although a few studies have statistically shown that pinanga species appear to exhibit habitat specificity, these remain few and should be expanded to the various forest types that are known to occur throughout borneo and in the wider southeast asian region. to complement investigations of drivers of pinanga diversity in different forest types, an increased understanding of functional traits of pinanga species, such as their growth forms, stem traits, fruit traits and leaf traits (kissling et al. 2019), which could explain their habitat specificity, should also be aimed for. it is notable that only three pinanga species that are recorded as bornean endemics have been assessed and listed in the iucn red list: p. chaiana (near threatened), p. mooreana (least concern) and p. patula (least concern) (iucn 2024). pinanga palms are often prized by illegal collectors and also face threats from deforestation and forest fragmentation (bellot et al. 2022). the threats faced by pinanga populations in borneo and southeast asia highlight a need for more studies of these palms to guide the conservation management of this genus throughout the region. conclusion it is widely accepted that palm abundance is influenced by various factors, such as climate, soil chemistry, hydrology, topography and vegetation structure. currently available studies on the relationship between palm abundance and species richness are still lacking because not all determinants have been quantitatively studied at all scales and because some studies involved more comprehensive investigations than others. palm species richness has been comprehensively studied only at a continental to global scale, and studies on community abundance are often conducted at local scales but less frequently at landscape to regional scales, being especially limited at a continental to global scale. therefore, it is crucial to assess the relationship between palm abundance and species richness with biotic and abiotic factors at all spatial scales. for pinanga palms, there is still insufficient ecological understanding of the factors that act as determinants of their abundance and species richness. studies to date have shown that pinanga palms typically prefer humid conditions, thick litter layers, high canopy cover and locations near water sources, such as rivers or small streams. pinanga species abundance and richness, as well as its foliar biotropia vol. 31 no. 2, 2024 302 contents, can be influenced by soil properties. detailed studies focusing on different lowland forest types with contrasting environmental conditions, and on how these factors affect pinanga species abundance and richness, coupled with studies investigating their functional traits, are needed. several pinanga species are endemic to borneo, and this highlights the need to investigate their populations and habitat preferences, particularly for the conservation of these iconic palms. acknowledgements the authors would like to thank universiti brunei darussalam for research funding (grant reference number: ubd/rsch/1.13/ficbf(b)/2022/033) and the ministry of education, brunei darussalam for the lead author’s graduate scholarship. we thank the brunei forestry department for field and herbarium support for pinanga surveys in brunei darussalam. we also thank alison shapcott and sidonie bellot for advice on pinanga species in brunei darussalam. references abdo me. 2017. a floristic study of halmahera, indonesia focusing on palms (arecaceae) and their seed dispersal [dissertation]. retrieved from florida international university. abdullah mf. 2021. variation in foliar nutrient contents of three pinanga species with contrasting habitats and leaf formations [dissertation]. retrieved from universiti brunei darussalam archive. abdullah mf. 2024. differences in diversity and communities of pinanga palms in contrasting lowland tropical forests in brunei darussalam, borneo [dissertation]. retrieved from universiti brunei darussalam archive. adorador jt, meneses zd, fernando es. 2020. pinanga gruezoi (arecaceae), a new slender clustering palm from the philippines with notes on an amended description of p. samarana. phytotaxa [internet]. 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[cited 2022 nov 18]; 20(3):7128. doi: 10.13057/biodiv/d200314 zulkarnaen rn, nisyawati n, witono jr. 2020. the growth and distribution pattern of endemic java palm (pinanga javana blume) in mt. slamet, central java, indonesia. in: aip conference proceedings (vol. 2231, no. 1). aip publishing. _hlk133429614 _goback _hlk168869484 _hlk171543452 _hlk168917891 _hlk168869576 _hlk170552839 _hlk170242498 _hlk168922927 _hlk171700550 _hlk171541272 _hlk171541240 _hlk170552588 _hlk124963370 _hlk171538806 _hlk134209183 _hlk133419042 _hlk168927439 _hlk168925764 _hlk168927400 the southeast asian journal of tropical biology vol. 32 no. 1, 2025: 69 86 doi: 10.11598/btb.2025.32.1.2416 issn: 0215-6334 | e-issn: 1907-770x 69 existing condition of gastropods community in coastal areas affected by nickel overburden in pomalaa district, southeast sulawesi province, indonesia muhammad fajar purnama1,2*, slamet budi prayitno3, max rudolf muskananfola3, and suryanti suryanti3 1doctoral program of aquatic resources management, faculty of fisheries and marine science, diponegoro university, semarang 50275, indonesia 2aquatic resources management, faculty of fisheries and marine science, halu oleo university, kendari 93232, indonesia 3aquatic resources management, faculty of fisheries and marine science, diponegoro university, semarang 50275, indonesia article higlights the present investigation provides substantiated empirical evidence demonstrating that nickel extraction operations have exerted considerable detrimental effects on the ecological integrity of the pomalaa coastal region in southeast sulawesi. environmental degradation and disruption of ecological equilibrium have been observed as direct consequences of persistent overburden waste disposal. the introduction of mining overburden into the pomalaa coastal ecosystem has resulted in the extirpation of critical gastropod taxa and significant population reductions among persisting species when compared with control sites featuring undisturbed substrate conditions. the documented diminished gastropod population densities within nickel mining-affected zones serve as biological indicators of profound environmental deterioration attributable to these industrial activities. article information received : 21 december, 2024 revised : 24 january, 2025 accepted : 12 february, 2025 *corresponding author, e-mail: muhammadfajarpurnama@students. undip.ac.id; muhammadfajarpurnama@uho.ac.id research article abstract pomalaa district is the largest existing nickel mining center in southeast sulawesi province, indonesia. pollutants from various anthropogenic activities, especially nickel mining overburden, end up in the pomalaa coastal area. this research aimed to determine the structure of the gastropods community in the impacted area and reference site. this research was carried out in the pomalaa coastal area, kolaka regency, from june to october 2023. research stations were determined using purposive sampling method and simple random sampling techniques. on-site collecting gastropods samples were carried out manually (handpicking method). impacted area and reference site showed significant differences in the number of species (16 and 21 species), presence, and density of gastropods, even though their respective ecological index categories were relatively the same (h› = 1.92 & 2.72; r = 2.93 & 3.41; e = 0.55 & 0.78; c = 0.001 & 0.04). in the impacted area, the density of gastropods was very low (1 3 ind./m2), whereas at the reference site, the density reached 10 30 ind./m2. gastropods inhabiting rocks or hard substrates, were relatively unaffected by the overburden, proven by stable population with a high density of 45 115 ind./m2. nickel overburden input flowing to the pomalaa coastal area greatly influenced the gastropods community in terms of species composition and density. on the other hand, the habitat (substrate) of gastropods as benthic organisms was covered and degraded by overburden piles up to 15 30 cm deep, which was still increasing over time. on the other hand, in the reference site area, the condition was inversely proportional. keywords: ecological index, gastropods, mining, overburden introduction grouping and classifying organisms specifically refers to the main structural forms or properties, such as dominant species, life forms (indicators), physical habitat of the community, and functional characteristics or traits. under natural conditions, a community unit will be formed based on dynamics (anomalies) ranging from unstable to stable and constantly fluctuating depending on the quality of the influencing environment (bantayan et al. 2023; bravo et al. 2021; ranjan & babu 2016; subagio & muliadi 2014; udayantha & munasinghe https://doi.org/10.11598/btb.2024.31.2.1741 mailto:muhammadfajarpurnama@students.undip.ac.id mailto:muhammadfajarpurnama@students.undip.ac.id mailto:muhammadfajarpurnama@uho.ac.id biotropia vol. 32 no. 1, 2025 70 2009). community structure or ecological index is a concept that studies and examines in detail and systematically the composition of species and their abundance in a community (ates et al. 2023; babushkin et al. 2023; čejka et al. 2023; carobene et al. 2023; degamon et al. 2023; de necker et al. 2023; fitria et al. 2023; montesinos-navaro et al. 2018). there are three approaches commonly used to describe the ecological index of a community of organisms, namely species diversity, species interactions, and functional interactions (schowalter 2000; hau et al. 2021; kudratov et al. 2023; keerthana et al. 2023; liu et al. 2023; lewin et al. 2023; medeiros et al. 2023; mansingh et al. 2021; maria 2020). the linkage of habitat characteristics with ecological indices (composition, size, and species diversity) is the main principle in studying community structure. changes in habitat even on a small scale will greatly influence community structure because they affect the species level down to the individual as the smallest component that makes up the population in the community (putro 2017; pérez-estrada et al. 2023; ramón et al. 2023; rubal et al. 2023). one of the largest communities of organisms that make up bottom aquatic (benthic) ecosystems are gastropods (vilenica et al. 2024; thilakarathne et al. 2024; ebadzadeh et al. 2024; aouissi et al. 2024; isfaeni et al. 2024; ernawati et al. 2024; presley & willig, 2023; pérez-estrada et al. 2023; ramón et al. 2023; rubal et al. 2023; sun et al. 2022; vian et al. 2022; yadav et al. 2019; susintowati et al. 2019; nybakken & bertness 2005). gastropods are the largest class of the mollusca (80%) with 100,000 living species and spread across various aquatic habitats (mukhopadhyay et al. 2024; seinor et al. 2024; curren et al. 2024; hertika et al. 2024; prayudi et al. 2024; guan et al. 2023a; guan et al. 2023b; ruppert et al. 2004; bouchet et al. 2008; strong et al. 2008; davis et al. 2015). the condition of gastropods communities in waters is greatly influenced by the quality of their living environment, especially those originating from anthropogenic activities. disturbance or input of pollutants in any forms directly impacts the dynamics of gastropods community structure. one of the water areas inhabited by gastropods communities and disturbed by anthropogenic activities is the pomalaa coastal area (purnama et al. 2024a; 2024b; 2024c). pomalaa district is administratively the largest existing nickel mining area in southeast sulawesi province (hamzah et al. 2015; hamzah 2009; zubayr 2009), which has been operating since 1968 until now. one of the impacts of nickel mining that has been going on continuously for a long time is the input of nickel overburden into the waters which covers the benthic zone causing the loss of gastropods community and other benthic organisms (timm et al. 2001; chakrabarty & das 2006). these occurrences were found in the research conducted by purnama et al (2024a; 2024b; 2024c; 2024d) in pomalaa mangrove ecosystem, southeast sulawesi province. in that research, there were 39 gastropods species with high diversity and species richness found in the area with minimal disturbance (reference site), while there were only 7 gastropods species with low diversity and species richness found in the impacted area or mangrove area which was exposed to nickel overburden. there were also evident that certain gastropods species were dominant. in other words, the impacted area has unstable ecological conditions due to disturbances originating from nickel overburden from the nickel mining activities. several coastal areas of pomalaa, particularly those near nickel mining activities (impacted areas), face pollution from nickel metal which affects the water, substrates, and biota. the concentration of nickel tends to be high in aquatic substrates and fluctuates around the standard quality threshold in water and in samples of the gastropods species t. sulcata (purnama et al. 2024a). additionally, there are physical threats due to nickel overburden input. pomalaa coastal area affected by nickel overburden is dawi-dawi village. the nickel overburden in dawi-dawi estuary flows from the upstream area where nickel is being extracted (hamzah 2009; zubayr 2009). in contrast, pomalaa district also has regions that remain unaffected by nickel overburden and serve as reference sites. these reference sites are untouched by the nickel overburden because the river basin does not connect to the mining areas. one of the reference sites is the coastal area of totobo village, which is lined with mangrove vegetation that helps protect the area from surrounding anthropogenic activities. the dawi-dawi and totobo coastal areas differ significantly in their ecological characteristics, particularly in the benthic zones and the supporting existing condition of gastropods community in pomalaa coastal areas 71 ecosystems around the areas. this includes variations in environmental disturbances caused by human activities and the corresponding gastropods communities inhabiting these areas. therefore, comparative study of gastropods communities inhabiting the impacted area (affected by nickel overburden) and the reference sites (not affected by nickel overburden) in pomalaa coastal area is very important to carry out. prior studies on this subject have never been carried out in southeast sulawesi province, in particular and indonesia, in general. this study aimed to determine the structure of gastropods communities in the impacted area and in the reference site. materials and methods time and location a comparative study of gastropods communities inhabiting the impacted area (dawi-dawi village) and the reference site (totobo village) in pomalaa coastal area was carried out from july to october 2023. the determination of these two locations as research locus were based on their close distance (± 3 km) from the existing center for nickel mining exploitation in the mountainous area (upstream) and the nickel industrial area (factory) in pomalaa. the research locations are presented in figure 1. dawi-dawi village receives direct flow of nickel overburden from the nickel exploitation area, while totobo village represents an area with minimum impact from the nickel mining area. totobo village was chosen as the reference site because the village borders the affected dawi-dawi village as a nickel overburdened area. the research took place in the coastal area of pomalaa district, kolaka regency, southeast sulawesi province (figs. 2 & 3). figure 1 map of research locations notes: is = impacted site; rs = reference site; nmea = nickel mining existing area source: google earth (2025) biotropia vol. 32 no. 1, 2025 72 figure 2 coastal areas exposed to nickel overburden (dawi-dawi village, as impacted area) figure 3 coastal areas without exposure of nickel overburden (totobo village, as reference site) research stations determination research stations and sampling points (substations) were determined in detail using the purposive sampling method as well as simple and random sampling techniques to obtain adequate samples having actual conditions related to gastropods communities in the impacted areas and reference sites. each research station covered an area of 2,500 m², which corresponded to the area of the sampling zone. the stations were located 50 to 70 m apart from each other. the distance between the impacted area and the reference site ranged from 350 to 400 m. the transect size used for collecting samples in the field was 1 m². the 1-m² transect was further divided into 16 smaller square transects, each measuring 25 cm², to observe small gastropods and those with infaunal tendencies. these transects were placed randomly throughout the sampling area in an alternating pattern. characteristics observed locations in this research were divided into 8 stations based on 2 categories, namely the impacted area and the reference site. these eight stations had ecological characteristics that were strongly influenced by the presence of coastal ecosystems, such as mangrove ecosystems (table 1). existing condition of gastropods community in pomalaa coastal areas 73 table 1 ecological characteristics of each research station no. research station ecological characteristics category coordinate 1 is 1 on shore with fine sand substrate, without mangrove forest, seagrass, and coral reef impacted area (affected by nickel overburden) 4º09’57.61” s 121º36’30.21” e 2 is 2 on shore (breakwater area), without mangrove forest, seagrass, and coral reef impacted area (affected by nickel overburden) 4º09’58.99” s 121º36’20.53” e 3 is 3 on shore with sand substrate, without mangrove forest, seagrass, and coral reef impacted area (affected by nickel overburden) 4º10’10.53” s 121º36’21.34” e 4 is 4 rocky shore with sand substrate, without mangrove forest, seagrass, and coral reef impacted area (affected by nickel overburden) 4º10’18.37” s 121º36’19.90” e 5 rs 5 on shore (fine sand substrate), with mangrove forest, without seagrass and coral reef reference site 4º09’26.54” s 121º36’41.22” e 6 rs 6 on shore (fine sand substrate), with mangrove forest, without seagrass and coral reef reference site 4º09’02.20” s 121º36’50.17” e 7 rs 7 on shore (fine sand substrate), without mangrove forest, seagrass, and coral reef reference site 4º08’36.41” s 121º36’51.53” e 8 rs 8 rocky shore, without mangrove forest, seagrass, and coral reef reference site 4º08’17.29” s 121º36’55.22” e sampling and identification gastropods samples were collected manually using hands (handpicking). gastropods species were identified using the latest and the most credible identification keys, namely: worms “world register of marine species” (https:// www.marinespecies.org), molluscabase (https:// www.molluscabase.org), collection of worldwide seashells (collection of worldwide seashells (idscaro.net)). nickel content analysis nickel content was analyzed from water, sediment, and biota (specifically, the gastropods). as much as 250 ml water sample from each station was collected and placed into sample bottles that had been rinsed with a 5% nitric acid solution. sediment sampling was performed using a plastic hand scoop to collect 0.5 l of sediment from each station. each sediment sample was placed in a scott glass bottle that had also been rinsed with a 5% nitric acid solution. for the gastropods samples, the specimens were washed with deionized distilled water for 2 hours, then dried on filter paper or tissue. the cleaned gastropods were transferred to a petri dish and stored in a refrigerator. from the gastropods specimens, 1 g of selected gastropods was digested using concentrated sulfuric and nitric acids at a temperature of 95 °c. nickel content was analyzed using an atomic density = number of individuals of species i total number of observation plots absorption spectrophotometer (aas) following the sni 6989.18-2009 test method for water samples and the aas flame method for sediment and biota samples (smoley 1992; akagi & nishimura 1991; pennuto et al. 2005). data analysis gastropods density was calculated using the following formula (khouw 2016): h’ = -∑ �� ni n � x ln ni n ��� i=1 s diversity of gastropods species was calculated using the shannon-wiener diversity index (odum 1993): where: h’ = diversity index ni = number of individuals of the ith species n = total number of individuals diversity index are divided into 3 criteria (wilhm 1975), namely: h’ < 1.0 = low species diversity 1.0 < h’ < 3 = medium species diversity h’ > 3 = high species diversity biotropia vol. 32 no. 1, 2025 74 evenness index was calculated using the following formula (odum 1993): e = h' ln s where: e = evenness index h’ = diversity index s = number of species the criteria for evenness index value are as follows: e < 0.3 = low level of species evenness 0.31 > e > 1 = medium level of evenness e > 1 = high level of species evenness the species richness index (margalef index) was calculated with the following formula (ludwig & reynolds 1988): r = (s 1) ln n where: s = number of species n = number of individuals the criteria for the species richness index (margalef index) value are as follows: r < 2.5 = low level of species richness 2.5 > r > 4 = medium level of richness r > 4 = high level of wealth type the dominance index was calculated using the following formula (odum 1993): c = ni n∑ � � 2 table 2 ecological index of gastropods in the impacted area of pomalaa coastal area no. ecological index grade category 1 diversity index (h’) 1.92 medium 2 species richness index (r) 2.93 medium 3 evenness index (e) 0.55 medium 4 dominance index (c) 0.001 there is no particular type that dominates table 3 ecological index of gastropods in the reference site of pomalaa coastal area no. ecological index grade category 1 diversity index (h’) 2.72 medium 2 species richness index (r) 3.41 medium 3 evenness index (e) 0.78 medium 4 dominance index (c) 0.04 there is no particular type that dominates ecological index for the impacted area showed values close to the low category (h’ = 1.92; r = 2.93). on the other hand, the reference site showed ecological index having values close to the high category (h’ = 2.72; r = 3.41). nevertheless, ecological index for both the impacted area and the reference site fell in to the medium category. the evenness index for both the impacted area and the reference site was similar both in value and category, i.e., eimpacted area = 0.55; ereference site = 0.78. there were no species dominance shown in the impacted area and in the reference site (cimpacted area = 0.001; creference site = 0.04). however, there are significant quantitative differences in the number of individuals contributing to these indices. uneven distribution of individual gastropods was observed in the impacted area (area exposed to nickel overburden). this area was dominated by several species that are less affected by nickel overburden, specifically, gastropods inhabiting hard substrates and rocky habitat, such as littoraria scabra, littoraria undulata, littorina littorea, and monodonta labio (fig. 4). meanwhile, gastropods inhabiting sandy substrates in the impacted area were found with low individual numbers, namely 1 3 ind./m2, such as nassarius pullus, polinices mamilla, and several other gastropods species. on the other hand, the representation of gastropods community within the reference site was balanced. ecological index in the impacted areas was influenced by a significant presence of groups of gastropods inhabiting hard substrates, along with where: ni = number of individuals i n = total number of individuals of all species the criteria for the dominance index value are: 0 < c < 0.5 = no type dominates 0.5 < c < 1 = there is a dominant type results and discussion ecological index presented for gastropods community inhabiting the impacted area and the reference site were diversity index (h’), species richness index (r), evenness index (e), and dominance index (c) (tables 2 & 3) existing condition of gastropods community in pomalaa coastal areas 75 multiple individual species coexisting in the same ecological space. this combination brought the ecological index values of the impacted areas closer to those observed at reference sites. however, there was a notable contrast in regards to the stability of gastropods community in the impacted area and in the reference site. the impacted area showed a decline of gastropods community up to only 1 to 3 individuals per m2, while the reference site exhibited relatively stable condition (fig. 5). this finding is similar to the study results conducted by kaliu and fitra (2019) on the pomalaa coastal area, where several areas having different habitat conditions apparently have the same ecological index category, although quantitatively the ecological index values at these stations are quite different, i.e., ranging from low values to approaching the medium to high category, but not exceeding the high ecological index value category, especially in terms of diversity and species richness. this phenomenon is caused by the presence of certain gastropods community with distinctive niches and high species abundance (dominating species), hence, their existence supports the areas and directly increases the ecological index value of certain stations having degrading environmental quality conditions. in our study, the presence of typical rock gastropods supported the ecological interaction system in the heavily impacted shelf area. the occurrence of gastropods inhabiting rocky habitat and other hard substrates, such as concrete, was resulted from a succession process in a habitat which had artificially changed from its original state. the reference site exhibited a fairly uniform representation of individual species within the population, but it could not be characterized as diverse or rich in species. this limitation was likely due to the absence of mangrove vegetation at several sampling stations within the reference site (fig. 6). figure 4 gastropods inhabiting hard substrates (rocks) biotropia vol. 32 no. 1, 2025 76 figure 5 comparison of the habitat of gastropods species nassarius spp. in the impacted area (a) and the reference site (b) of the pomalaa coastal area figure 6 several sampling stations in the reference site notes: a and b = sampling stations in the front zone of mangrove ecosystem; c and d = sampling stations without mangroves or ex-mangrove areas. as a result, environmental pressures caused by anthropogenic activities, such as sedimentation, directly affected the coastal area. in contrast, other areas within the reference site, which were ecologically protected by the surrounding mangrove ecosystem, showed a significantly higher population density. the increased density was a result of nutrient supply from the mangroves, as sedimentation from land did not reach these beach areas. instead, the sedimentation was trapped within the mangrove ecosystem. furthermore, variations in habitat quality among stations in the reference site influenced community dynamics in coastal areas, particularly regarding the presence of certain species and the number of individual gastropods representatives. this was especially true existing condition of gastropods community in pomalaa coastal areas 77 in locations where ecological disruptions, such as pollution from nickel overburden had persisted for an extended period (purnama et al. 2024b; 2024c). gastropods community has ecological value as one of the key species in aquatic ecosystems, where some of them are involved in the food chain cycle, namely as a food source for other animals. apart from that, gastropods can also be used by humans as a source of animal protein (cappenberg 2006). previous studies found that the quality of aquatic environment influences the structure of gastropods populations and communities (yuniarti 2012; hau et al. 2021; kudratov et al. 2023; keerthana et al. 2023; liu et al. 2023; lewin et al. 2023; medeiros et al. 2023; mansingh et al. 2021; maria 2020). the representation of individuals in a population is strongly influenced by differences in substrate and habitat characteristics as well as anthropogenic activities (hamzah 2009; zubayr 2009; presley & willig 2023; pérez-estrada et al. 2023; ramón et al. 2023; rubal et al. 2023; sun et al. 2022; vian et al. 2022; yadav et al. 2019). one of which is nickel content that accumulates in waters and organisms carried from or originating from nickel mining areas as nickel overburden. table 4 presents nickel content in water, sediment/substrate, and organisms (gastropods) obtained from the research stations at the impacted and the reference sites, showing significant differences of nickel content in gastropods, water, and sediment samples in the impacted area and reference site in pomalaa coastal waters. overall, nickel content in the water did not exceed the quality standard threshold (0.0022 0.0122 ppm), with the highest level was found in the impacted area (0.0122 ppm). in contrast, nickel content in gastropods exceeded the threshold in the impacted area (0.0702 0.0864 ppm). nickel content in gastropods at the reference site (0.0026 0.0034 ppm) and transition site (0.0112 ppm), however, remained within the quality standard threshold. sediment across all locations showed elevated nickel content (13.50 92.87 ppm), with the highest nickel content was found in the impacted area (57.81 92.87 ppm). nickel content in gastropods, water, and sediment reflected the sites’ classifications, with the impacted site having the highest concentrations due to nickel overburden and acid sludge from long-term nickel mining, in which the upstream outlet of nickel overburden also located in the impacted area. meanwhile, the reference and transition sites received diluted input of nickel overburden from the impacted area, which influenced by tidal and wave activity. all locations shared the same ecoregion, contributing to similar hydro-oceanographic conditions. water color in the impacted area differed significantly from the reference and transition sites. within the impacted area, the water color appeared to be reddish-brown (fig. 2), a typical soil color having nickel content, while within the reference site the water color was clear or translucent (fig. 3). water analysis showed that the turbidity value in the impacted area was 500 pt.co indicating severe turbidity due to nickel overburden. on the other hand, turbidity value in the reference site was 60 pt.co, which was close to marine tourism standard of 30 pt.co. research conducted by zubayr (2009) and hamzah (2009) highlighted that pomalaa coastal waters was polluted by nickel from ferronickel mining, particularly from tailings and waste rock. tailings consist of silica and other minerals, while waste rock includes low-mineral acid mine drainage. table 4 nickel content in gastropods, sediment, and water no. location nickel (ni) content threshold value of nickel content in water, sediment, and organismgastropods (ppm) water (ppm) sediment (ppm) 1 impacted site 1 0.0702** 0.0036ᴥ 92.87** “0.05 ppm” (according to the decree of the minister of environment of the republic of indonesia number 51 year 2004 concerning sea water quality standards) 2 impacted site 2 0.0864** 0.0122ᴥ 57.81** 3 transition site 0.0112ᴥ 0.0031ᴥ 36.93** 4 reference site 1 0.0034ᴥ 0.0023ᴥ 18.64** 5 reference site 2 0.0026ᴥ 0.0022ᴥ 13.50** ᴥ = did not exceed the threshold ** = exceeded the threshold biotropia vol. 32 no. 1, 2025 78 up to today, there is limited research conducted on nickel content measured in organisms and sediments, making this study crucial for understanding the water current effects on the health of aquatic organisms and environment. ecologically, the greatest impact of nickel mining overburden comes from sedimentation, leading to erosion upstream and causing shallowing process in the estuaries, resulting in the degraded gastropods habitats. the resulting loss of living space and food caused by the nickel overburden disrupts the gastropods community, potentially leading to the decline of gastropods population (wali et al. 2020; hamzah et al. 2015). nickel is one of the essential metals. however, excessive exposure to nickel can cause health issues, including systemic, immunological, neurological, and reproductive disorders, as well as developmental problems and even death. nickel is also a carcinogenic metal which can disrupt physiological functions of an organism, resulting to the death of an organism, particularly in sessile biota like gastropods, when nickel content exceeds a certain threshold. previous research showed that high nickel (ni) content in sediments correlated with the disappearance of sensitive gastropods species, with only about 20% of gastropods population was able to adapt. results of our research provided concrete comparisons that have been empirically clarified based on density data and ecological indices of the gastropods community in the impacted area and the reference site. nickel overburden in the impacted area was massive and caused systemic impact on the structure of the gastropods community in the waters of pomalaa coasteal area. the impacted area and the reference site showed very significant differences in terms of species and the number of individuals in each sampling area. this is an indication of the degradation of gastropods habitat in the pomalaa coastal area, especially in the areas exposed to nickel overburden (the impacted area). pollution causes the decline in abundance and biodiversity of aquatic organisms, one of which is the mollusk community (zahidin 2008). riniatsih and kusharto (2009) added that the diversity of gastropods species was very low in polluted waters. this statement is in line with the research results of nugroho et al. (2012) which stated that the waters of genuk, semarang, which was polluted by surrounding industrial activities, had a significant influence on the abundance of gastropods. furthermore, prabuning (2010) emphasized that in the northern coastal waters of tanjung emas port, semarang, from the east canal flood area to the west canal flood area, semarang, the waters was polluted, causing a negative influence on the life of the gastropods community in the waters. our study indicated that the substrate of the waters played a crucial role in the sustainability of the gastropods community. this finding was supported by the data, showing that the number of gastropods individuals in areas unaffected by nickel mining waste (the reference site) was significantly higher than in areas impacted by nickel overburden (the impacted area), which were supported by other studies. munandar et al. (2016) highlighted that the abundance of macrozoobenthos, including gastropods, was influenced by habitat conditions such as bottom sediments and water quality. gastropods distribution is affected by local environmental factors, food availability, predation, and competition, including sediment texture, temperature, salinity, ph, organic matter, and oxygen content (ruswahyuni & nata 2008). mollusks, particularly gastropods, exhibit high species diversity across marine habitats, but their survival is linked to water quality and habitat characteristics, including pollutants (pratiwi & ernawati 2016). research conducted by mardatila et al. (2016) in diatas lake, west sumatra province showed a diversity index (h’) of 1.07, which was categorized as medium. the diversity index of a population is influenced by the number of species and the evenness of each species. the low diversity in the waters of kelabat bay was caused by uneven distribution of the gastropods found (abdullah et al. 2021). this finding is supported by gray (1981) in mardatila et al. (2016) which stated that low diversity index values at a station can be caused by an uneven number of individuals of each species. research carried out by rahmasari et al. (2015) indicated that the total abundance of mollusk species at mertasari beach was 124.5 ind./m², significantly higher than the 0.17 1.83 ind./m² found by pratiwi and ernawati (2016) in nusa lembongan, where low dissolved oxygen levels (2.60 6.90 mg/l) were a factor. in contrast, laharjana (2016) reported an abundance at serangan beach of 106.6 ind./m², also higher than existing condition of gastropods community in pomalaa coastal areas 79 the finding in nusa lembongan, was correlated with a better oxygen level (4.16 5.26 mg/l). according to syamsurisal (2011), higher dissolved oxygen supported greater benthic populations. the gastropods diversity index at mertasari beach was classified as medium, indicating a fairly balanced ecosystem (wahyuni et al. 2015). this finding contrasts with research findings of wahyuni et al. (2015) in menaming dam, which reported a lower diversity index (0.55 1.05) due to several factors, such as polluted substrates and food source availability (rachmawaty 2011). mertasari beach hosts 15 mollusk families compared to 3 families found at menaming dam. in their research, rahmasari et al. (2015) noted that gastropods diversity was influenced by the bottom substrate. nybakken and bertness (2005) stated that particle size of substrate affected the distribution and abundance of organisms, influenced by water retention and suitability for digging or the easiness for organisms burrowing. cappenberg (2006) also found that substrate type significantly affected mollusk species, especially gastropods. the diversity index for gastropods varies by location, with bengkal beach showing the highest index at 2.4398, followed by talang siring beach at 2.0988, and jumiang beach with the lowest at 1.6200. the ecological index conditions in several places are similar to those in this study, where ecologically, ecosystem conditions with high levels of disturbance can reduce ecological integrity, and vice versa (rahmasari et al. 2015). comparisons of average gastropods species density in the impacted area and reference site in pomalaa coastal area are presented in figures 7 and 8. the number of species, presence (number of individuals), and species density of gastropods in the impacted area and the reference site showed differences, although the respective ecological index categories were relatively the same (tables 2 & 3). the number of species found in the impacted area were 16 species, while in the reference site there were 21 species (tables 5 & 6). figure 7 average species density of gastropods in the impacted area biotropia vol. 32 no. 1, 2025 80 figure 8 average gastropods species density in the reference site table 5 presence of gastropods species at each station in the impacted area (dawi-dawi village) no. gastropods species research station of impacted area (is) is 1 is 2 is 3 is 4 1 nassarius dorsatus + 2 nassarius olivaceus + 3 nassarius pullus + + 4 polinices mamilla + 5 polinices flemingianus + 6 neverita didyma + 7 littoraria scabra + + 8 littorini littorea + + 9 littoraria undulata + + 10 monodonta labio + + 11 drupella margariticola + 12 semiricinula muricoides + 13 tenguella granulata + 14 semiricinula fusca + 15 nerita lineata + + + 16 nerita undata + + + notes: + = existed; = did not exist existing condition of gastropods community in pomalaa coastal areas 81 table 6 presence of gastropods speies at each station in the reference site (totobo village) no. gastropods species research station of the referene site (rs) rs 5 rs 6 rs 7 rs 8 1 nassarius dorsatus + + + 2 nassarius olivaceus + + + 3 nassarius pullus + + + 4 nassarius (plicarcularia) globosus + + + 5 nassarius (nassarius) coronatus + + + 6 nassarius (plicarcularia) elegantissimus + + + 7 polinices mamilla + + + 8 polinices flemingianus + + + 9 neverita didyma + + + 10 vexillum vulpecula + + + 11 vexillum funereum + + + 12 littoraria scabra + 13 littorini littorea + 14 littoraria undulata + 15 monodonta labio + 16 drupella margariticola + 17 semiricinula muricoides + 18 tenguella granulata + 19 semiricinula fusca + 20 nerita lineata + 21 nerita undata + notes: + = existed; = did not exist in the impacted area, species density was very low (13 ind./m2), whereas in the reference site the density reached 10 30 ind./m2 (ratio = 1 : 10). species density in the reference site was high, especially for gastropods living in hard substrates or rocky habitat which were relatively unaffected by nickel overburden even tough their surroundings were exposed to the overburden. high species density of 45 115 ind./m2 with stable species composition was found in gastropods community inhabiting hard substrate or rocky habitat, such as littoraria scabra, littorini littorea, littoraria undulata, monodonta labio, drupella margariticola, semiricinula muricoides, tenguella granulate, semiricinula fusca, nerita lineata, and nerita undata (fig. 4). results of our study were identical to the research resuts of purnama et al. (2024a; 2024b; 2024c; 2024d) who found the same condition, where gastropods community in a mangrove area affected by overburden experienced a very drastic decrease in the number of species and individuals due to the loss of ecological space that was the niche of the mangrove gastropods community in the pomalaa mangrove ecosystem. gastropods inhabiting rocky habitat have a very high density and form groups in boulders. in addition, the gastropods community is not influenced by nickel overburden because rocks, gravel, and other hard substrates are not able to capture or store sediment, hence, when high tide occurs, the sediment carried in the overburden flow will be washed away by the water flow and settle to the bottom of the water. rocky habitat is part of the coastal ecosystem. interactions between gastropods species inhabiting rocky habitat or hard substrates and gastropods species living in other types of habitat in coastal areas occur in intersecting ecological spaces. gastropods species that inhabit substrates or bottom of waters tend to use rocks as shelter from the currents and waves in coastal areas and as hiding place from predators. apart from that, the hard substrate also becomes a feeding ground (grazing) area for gastropods because the hard substrate is overgrown with algae (purnama et al. 2024a, 2024b, 2024c). biotropia vol. 32 no. 1, 2025 82 the diversity and abundance of gastropods species are influenced by the bottom substrates of their habitats. on the south coast of pamekasan madura regency, the total diversity index of gastropods is 3.0075, indicating high diversity. the most abundant species is nassarius distortus, followed by littoraria scabra and nassarius leptospirus, with relative abundances of 11.21%, 9.09%, and 8.03%, respectively. jumiang beach has a lower diversity index of 1.6200, classified as moderate, with 8 gastropods types and 105 individuals. this beach is affected by human activities, which may impact biodiversity. the dominant species here is also nassarius distortus. talang siring beach’s diversity index is 2.0988, with 12 gastropods types and 123 individuals. the most common species is nassarius jacksonianus. bengkal beach has a diversity index of 2.4398, hosting 16 gastropods types and 245 individuals. this beach benefits from a nearby mangrove ecosystem, supporting a variety of species, with littoraria scabra and cerithium corallium being the most dominant. overall, environmental conditions significantly influence gastropods diversity across these locations (kurniawan et al. 2024; rahmasari et al. 2015). jumiang beach features a sand-type substrate, resulting in only eight types of gastropods compared to other beaches (rahmasari et al. 2015). nybakken and bertness (2005) explained that the instability of sandy substrates limits the settlement of larger organisms. research by hawari et al. (2014) indicated that gastropods at pandan beach also exhibit lower diversity on sandy substrates compared to muddy ones. the lack of vegetation on jumiang beach further reduces gastropods species, as they rely on detritus from plants (rahmasari et al. 2015). in contrast, talang siring beach, with its sandy clay substrate, supports 12 types of gastropods (rahmasari et al. 2015). bengkal beach, with a clayey silt substrate, hosts 16 types of gastropods, the highest among the locations studied (rahmasari et al. 2015). this is attributed to the presence of mangrove forests, which provide optimal conditions for gastropods (kurniawan et al. 2024; rahmasari et al. 2015). nybakken and bertness (2005) noted that mangrove ecosystem promotes minimal water movement, allowing fine sediment to accumulate. the organic material from decomposed mangrove debris enriches the sediment, providing a vital food source for gastropods, as reported by onrizal et al. (2009). several research results in several areas above showed that the species composition found on the coast, especially with fine sand substrate characteristics, is relatively the same (kurniawan et al. 2024; rahmasari et al. 2015). conclusion the nickel overburden impacted area and the reference site showed differences in the number of species, i.e., 16 species in the impacted area and 21 species in the referece site, as well as differences in the presence (number of individuals) and density of gastropods. even though the ecological index categories were the same, the magnitude of the value of index were tends to be different (h’ = 1.92 & 2.72; r = 2.93 & 3.41; e = 0.55 & 0.78; c = 0.001 & 0.04, for impacted area and reference site, respectively). in the impacted area (on short) the density of the gastropods community was very low (1 3 ind./m2), whereas at the reference site, the density reached 10 30 ind./m2. gastropods living on rocky habitat or those living on hard substrates 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[thesis]. bogor (id): faculty of graduate studies, bogor agricultural university. 127 p. https://doi.org/10.1080/13235818.2019.1644701 https://doi.org/10.1080/13235818.2019.1644701 the southeast asian journal of tropical biology vol. 32 no. 1, 2025: 24 37 doi: 10.11598/btb.2025.32.1.2319 issn: 0215-6334 | e-issn: 1907-770x 24 effect of anti-mold and mycotoxin binder on corn quality and broiler performance catootjie lusje nalle*, max arthur j. supit, johanis a. jermias, d. kusumaningrum, melani kefi and ariska benu 1department of animal husbandry, state polytechnic of agriculture, kupang 85228, indonesia article higlights • the anti-mold used effectively maintained low aflatoxin levels in 13% moisture corn. • synthetic mold inhibitors effectively decreased aflatoxin levels in corn during storage and maintained some nutritional quality • mycotoxin binder supplementation did not improve broiler performance. • broiler performance declined as aflatoxin concentrations increased. article information received : 8 august, 2024 revised : 23 january, 2025 accepted : 12 february, 2025 *corresponding author, e-mail: catootjienalle@gmail.com research article abstract the quality of animal feed is determined by high-quality ingredients and appropriate feed additives. this study aimed to assess: 1) the nutrient and aflatoxin total (at) content of corn treated with an anti-mold (a) and 2) broiler performance fed with aflatoxin b1 (afb1)-contaminated diets supplemented with a mycotoxin binder (mb). two experiments were carried out to achieve the objectives. experiment 1 was set up with a 2 x 2 factorial completely randomized design (fcrd) with two factors: moisture content (mc at 13 and 15%) and anti-mold (a, -/+). meanwhile, experiment 2 was set up with a 3 x 2 factorial completely randomized design with two factors: the afb1 (< 100, 165, 222 µg/kg) and mb (-/ +). the mc and a interaction was significant (p < 0.01) on the aflatoxin total of corn throughout the 2-month assay. the utilization of the anti-mold in afla-corn with different moisture levels did not influence (p > 0.05) the corn’s nutrient content. the mc x a interaction was significant (p < 0.05) in the valine and glycine content of the stored corn. in the second experiment, no interaction of afb1 x mb (p > 0.05) was observed in the bird’s performance during the study. the afb1 concentration (ac) of corn decreased significantly (p < 0.05 to 0.001) in feed intake, body weight gain, and feed efficiency of birds. our study concluded that the anti-mold effectively maintained low aflatoxin levels in 13% moisture corn. also, the anti-mold did not affect the nutrient profile of corn during storage. our study also showed that mycotoxin binder supplementation did not improve broiler performance and broiler performance declined as aflatoxin concentrations increased. keywords: caflatoxin, mold inhibitor, nutrient, performance, toxin binder introduction mycotoxins, toxic compounds produced by pathogenic fungi, can infect animal feed and cause various negative effects on livestock. aflatoxin, ochratoxin, zearalenone, and fumonisin are examples of mycotoxins. aflatoxin was classified into two strains, namely large (l) and small (s) strains, where the most dangerous strains were the small strains (norlia et al. 2019; almatakeez 2020). according to mohammed et al. (2021), the l-strain aflatoxin produced sclerotia with a size of > 400 µm, whereas the s-strain had a sclerotia with a size of < 400 µm. grains such as corn and peanuts are favorable media for the growth of aspergillus spp. the presence of aspergillus flavus in corn could be identified by the bright greeny-yellow fluorescence and black light test (stack & carlson 2003). the growth of aspergillus spp. in corn kernels depends on several factors, such as water activity, ph, and relative humidity (shehu & belo 2011; norlia et al. 2019). according to shehu and bello (2011), the favorable temperature and relative humidity for aspergillus spp. to grow well were 30 35 oc and 85 100%, respectively. https://doi.org/10.11598/btb.2024.31.2.1741 different moisture levels of corn supplemented with mold inhibitor 25 the growth of fungi involves two forms: yeast-like cells and mycelial growth. during their development, fungi need nutrients, such as carbohydrates (simple and complex sugars), proteins (for c, h, o, and n sources), and certain minerals (liu et al. 2016; barzee et al. 2021). all nutrients are taken from the substrates on which the fungi are growing. grains, such as corn, infested by fungi, such as aspergillus spp., may experience a decrease in nutrient components due to the utilization of corn’s chemical components by fungi. according to liu et al. (2016), saccharides and proline are utilized by aspergillus flavus for its mycelium propagation and the making of aflatoxin b1. the presence of aflatoxin in corn not only deteriorates corn quality but also triggers diseases and death in animals when being consumed. aflatoxins are mutagenic, teratogenic, and carcinogenic compounds (okechukwu et al. 2023). the amount of aflatoxin in the diet, the type of birds, and age of bird influence the harmfulness level of aflatoxin. ducks, turkeys, and chickens are resistant to aflatoxicosis, with ducks being the most resistant species (wu et al. 2021; murcia & diaz 2020). according to diaz and murcia (2019), ducks produce the highest aflatoxin b1-dihydrodiol, causing the acute toxic effect of aflatoxin b1. several efforts to control the growth and spread of toxin-producing fungi are by drying the corn immediately after harvest, by storing the corn on top of the pallets in a storage room, by using clean feed materials, and by using fungal inhibitor agents, such as charcoal and organic acids. charcoal as an inhibitor agent works by binding to mycotoxins and excreting them through chicken excreta, thus reducing the amount of toxin absorbed in the body. the application of certain commercial fungal inhibitors in feed succeeded in suppressing the a. flavus development but changed certain amino acid profiles in corn (elsamra et al. 2012). supplementation of 0.045% mintai feed anti-mold effectively protected the nutritional characteristics of low-moist corn (nalle et al. 2022). regarding the use of mycotoxin binder, nalle et al. (2021) proved that mycosorb, as a toxin binder product, did not augment the productivity of birds given a low-dose afb1 diet. on the contrary, fernandes et al. (2022) claimed that commercial toxin binders effectively increase the feed efficiency of birds that received aflatoxin diets. this indicated a contradictory result in terms of using mycotoxin binders in feed. feed security is still a worldwide issue, so it is important to intensively evaluate the strategy to maintain corn quality, especially using mold inhibitors (synthetic or natural). in ethiopia, for example, a study found that 94% of poultry feed samples were contaminated with aflatoxins with a range of levels of 18 µg/kg to 190.18 µg/ kg, exceeding the fda’s regulatory limit of 20 µg/ kg in 72.75% of samples (kassaw et al. 2022). in northern pakistan, 92.5% of poultry feed samples were found positive for aflatoxins, with grower feeds exhibiting the highest contamination levels (naveed et al. 2022). these findings highlight the widespread aflatoxin contamination in broiler feed across different regions. the economic impact is considerable, as aflatoxins can impair poultry health, leading to reduced growth rates, lower feed conversion efficiency, and increased mortality, thereby affecting overall productivity and profitability in the poultry industry. aflatoxins in poultry products pose a risk to human health, potentially leading to aflatoxicosis upon consumption. therefore, monitoring and controlling aflatoxin levels in poultry feed are crucial to mitigate these adverse effects. considering the above problems, two experiments were conducted. in the first experiment, mold inhibitors were applied to corn with different moisture content (13% and 15%). meanwhile, the mycotoxin binder was added to the aflatoxin b1 diets in the second experiment. materials and methods experiment i: trial on mold inhibitor primary materials the primary materials of this experiment were shelled corn and anti-mold. the shelled corn used complies with the quality requirements of corn as feed (indonesian national standard board 2013), as presented in tables 1 and 2. biotropia vol. 32 no. 1, 2025 26 table 1 initial quality assessment of corn used in experiment i moisture level whole seed broken seed moldy seed aflatoxin total (µg/kg)……………………………………%................................................. 13 91.98 2.09 4.85 29.5 15 89.90 2.20 5.00 52.4 table 2 quality standards for corn used as animal feed no parameter unit requirements grade i grade ii 1 moisture level (max) % 14 16 2 crude protein (min) % 8 7 3 aflatoxin (max) µg/kg 100 150 4 damaged seed (max) % 3 5 5 moldy seed (max) % 2 5 6 broken seed (max) % 2 4 7 foreign objects % 2 2 the anti-mold product contains active compounds including propionic acid (57%), lead (0.003%), and arsenic (0.0054%). the antimold dose of 45g per 100 kg of feed was applied according to the manufacturer’s recommendation. the concentration of lead and arsenic contained in the mold inhibitor used in this experiment were below the maximum permitted levels (2 ppm in feed material) by the ec directive 2002/32/ec of the european parliament and of the council of 7 may 2002 on undesirable substances in animal feed to minimize the contamination risk of these toxic substances. this regulation is in place to prevent the accumulation of harmful metals in the food chain, which can cause serious health issues, such as organ damage and cancer in animals and humans (zwolak 2020). experimental design and statistical analysis the experiment was performed in a 2 x 2 factorial completely randomized design with 2 moisture levels (13% and 15%) and anti-mold (-, +), resulting in four treatments altogether. every treatment consisted of five replicates, each containing 20 kg of corn. a two-way analysis of variance (anova) was used to analyze the data using sas application software (as ondemand). the significant difference between each treatment was further analyzed using fisher’s least significant difference test. sampling procedure corn grains (with and without mold inhibitor) were stored from day 1 to day 60. on day 60, the corn grains were sampled using the quartering method, referring to gerlach et al. (2002), followed by a seed sampler to obtain the representative sample. the laboratory samples were then chemically analyzed according to the measured parameters. chemical analysis the proximate and gross energy content was analyzed using the official method of aoac (aoac 2005). the thin layer chromatography tool was operated to determine the aflatoxin total contained in the corn samples (bainton et al. 1980). the analysis principle used was that aflatoxins in the sample were extracted with methanol and defatted with n-hexane. the clean-up process was carried out using chloroform, and aflatoxins were identified through the tlc method. the detection limits for tlc were 3.01 ppb for afb1, 3.50 ppb for afb2, 0.54 ppb for afg1, and 1.0 ppb for afg2. the amino acid concentration was identified and quantified using a chromatogram method. different moisture levels of corn supplemented with mold inhibitor 27 experiment ii: performance trial using mycotoxin binder agreement letter on laboratory animal ethics the approval from the animal ethics committee of the veterinary medicine faculty at the university of nusa cendana, kupang-indonesia, became the basis for conducting this study. the approval was stated in a formal agreement letter, number 002 keh/sk/viii/2023, signed on 7 august 2023. animals and rearing management a total of 216 one-day-old broiler chicks of the lohmann strain, each with an average body weight of 40.7 ± 0.2 g, were placed in 24 experimental pens, with a density of 9 chicks per pen. the chicks remained in the pens for 28 days, during which the feed and water were provided ad libitum. each pen measured 70 cm in length, 70 cm in width, and 70 cm in height, with the floor covered by 5 cm of paddy husk litter. a 75-watt electric bulb was provided in each pen for heating, while two 45watt bulbs were hung on the ceiling for lighting. the chicks were exposed to continuous light for the first five days to acclimate to a temperature range of 32 °c to 34 °c. after this acclimation period, light was provided only at night to help gradually reduce the temperature to the standard range of 22 °c to 24 °c. thermo-hygrometers were used to monitor temperature and humidity in the housing, with measurements taken in the morning and afternoon. primary materials the primary materials used in this study were corn contaminated with aflatoxin b1 (afb1) and alvitoxtm bio supplied by a local poultry feed mill. alvitoxtm bio works as an inhibiting and toxinbinding agent. each 100 g of alvitoxtm bio contains mannan oligosaccharides 4.0 g, hydrated sodium calcium aluminosilicate 80 g, propionic acid 1g, acetic acid 1g, benzoic acid 0.80 g, activated charcoal 2g, and calcite. the recommended dosage of alvitoxtm bio for industry is 0.05 to 0.1%. the dose of alvitoxtm bio used in the experimental diet was 0.07%. the laboratory sample of aflatoxin-b1contaminated corn (13% and 15% water content) was obtained through the quartering sampling procedure, followed by a seed divider (retsch pt 100). the quartering sampling procedure started by arranging the corn grains in a cone shape and then dividing them into quarters. two opposite quarters were discarded, and the remaining two quarters were combined. this process was repeated several times until the desired sample size was achieved. in the retch seed divider sampling method, corn grains were placed into the hopper of the divider, and the sampler used a rotating mechanism to evenly distribute the grains into small, uniform portions across eight separate bottle containers. lastly, the seeds from each container were ground with a disk mill and laboratory sample mill (foss ct 193 cyclotectm, 0.5 mm screen size). subsequently, the ground corn was analyzed to get the initial concentration of aflatoxin b1 using thin layer chromatography. the initial afb1 concentration of the corn was < 60 ppb. after getting the initial afb1 content (< 60 ppb), the corn grains were stored for one year to increase the afb1 content naturally. after one year, the corns were sampled, reduced, ground, and analyzed to evaluate the change in the afb1 content of the corns. the aflatoxin concentration of corn obtained after one year of storage was 132 ppb to 504 ppb. experimental diets eight experimental diets were formulated using aflatoxin-b1-contaminated corn (table 6). the experimental diets (table 3) in mash form were supplied to the birds for four weeks. the formula to compute the afb1 concentration in each experimental diet was as follows: volume1 x concentration1 = volume2 x concentration2 biotropia vol. 32 no. 1, 2025 28 table 3 the experimental diets for assay purposes (g/100 as fed) feed ingredients without alvitoxtm bio with alvitoxtm bio shelled corn 60.07 60.00 sbm* containing 44% protein 25.36 25.36 mbm** 5.00 5.00 local fish meal 2.50 2.50 palm oil 5.00 5.00 l-lysine hcl 99% 0.20 0.20 dl-methionine 98% 0.30 0.30 limestone powder 0.50 0.50 dcp*** 0.40 0.40 nacl 0.25 0.25 nahco3 0.12 0.12 vit-min supplement 0.30 0.30 alvitoxtm bio + total 100 100 calculated analysis ame**** (kcal/kg) 2950 2950 crude protein (g/kg) 207 207 cellulose+hemicellulose+lignin 20.0 20.0 lysine (g/kg) 12.1 12.1 methionine + cysteine (g/kg) 8.5 8.5 calcium (g/kg) 9.1 9.1 non phytate phosphorus (g/kg) 4.3 4.3 ratio of calcium:phosphor 2.1 2.1 notes: sbm = soybean meal; mbm = meat and bone meal; dcp = dicalcium phosphate; ame= apparent metabolizable energy experimental design and statistical analysis a 3 x 2 factorial completely randomized design consisting of afb1 concentration (ac) and mycotoxin binder (mb, alvitoxtm bio) factors was applied to this experiment. the ac of < 100, 165, and 222 µg/kg were used in this study. the parameters observed were the intake of feed (g/ bird), the gain of body weight (g/bird), and the ratio of feed conversion (g/g). below are the explanations of each parameter: 1. feed intake (g/bird): feed intake is the difference between the amount of feed provided and the remaining feed, measured weekly. the results are adjusted to account for any spilled feed. 2. body weight gain (g/bird): body weight gain is determined by subtracting the initial weight from the final weight. the measurements were taken weekly. 3. feed conversion ratio (g/g): the feed conversion ratio (fcr) is calculated by comparing feed intake to body weight gain (bwg), with adjustments made for dead weight. the detailed treatment combinations are as follows: code dietary treatment a aflatoxin b1 < 100 µg/kg b aflatoxin b1 < 100 µg/kg + mb (0.07%) c aflatoxin b1 165 µg/kg d aflatoxin b1 165 µg/kg + mb (0.07%) e aflatoxin b1 222 µg/kg f aflatoxin b1 222 µg/kg + mb (0.07%) all data were arranged and analyzed using two-way analysis of variance (anova) using sas application software (sas ondemand). the fisher’s least significant difference test was performed when the anova test appeared significant at a p value less than 0.05. different moisture levels of corn supplemented with mold inhibitor 29 results and discussion experiment i changes of aflatoxin total in corn table 1 describes the initial quality of corn used for the experiment. as shown in table 1, the broken seeds were observed at ≤ 2.20%. the moldy corn seeds were at ≤ 5%, and the aflatoxin total of 13% moisture content (mc) corn grains were 29.5 ppb and 56.6 ppb for 15% mc corn seeds before the trial. the at of corn increased after 60 days of the test; however, a substantial increase was detected in the group without a mold inhibitor and with the higher mc (table 4). the present result indicated that mold inhibitor (mi) decreased the at of corn with ml 13% and 15%. the percentage of at reduction was 285.3 ppb and 144.6 ppb in corn, with 13% and 15% ml, respectively. the results suggested that the active compound of the mold inhibitor product worked effectively to constrain the metabolism of pathogenic molds. the at of corn with 15% ml was higher (2,310 ppb) than that of the at level of ml 13% corn. the result indicated that the use of mi in 15% ml aflatoxin corn was not valuable since the at still increased beyond the maximum standard requirement of bsni (2013, table 2). the outcome of this trial did not align with nalle et al. (2022) due to methodological variations, particularly in moisture content and the initial corn condition. the study conducted by nalle et al. (2022) used freshly harvested corn with low moisture content (< 12%), while the current study used aflatoxin-b1-contaminated corn with a higher moisture content (> 12%). the corn grains’ final aflatoxin total (at) is presented in figures 1, 2, and 3. statistical analysis shows that moisture content (mc), anti-mold (a), and the mc x a interaction affected (p < 0.001) the corn at for the period of the experiment. as can be seen in figure 1 (the interaction between mc and a), the aflatoxin total (at) of 13% and 15% mc corn treated with the antimold product was less (p < 0.05) compared to the 13% and 15% mc corn without anti-mold supplementation. it is interesting to note that the 13% ml corn that had been contaminated with aflatoxin (29.5 ppb, table 3) before the experiment remained low in aflatoxin total (at) (38.7 ppb, table 3) after 60 days of the experiment (table 4) due to the addition of anti-mold 0.07%. these corn grains still fulfilled the maximum aflatoxin level required by the indonesian national standard board (2013), which was 50 ppb for the first quality and 100 ppb for the second quality of corn as feed. this low at level of corn after the experiment suggested that the anti-mold product used in this assay effectively prevented the further growth of aspergillus spp. that had already existed in corn seed before the experiment. as a consequence, the production of aflatoxin was also kept low. on the other hand, the aflatoxin total of 15% mc corn grains added with mi increased from 56.6 ppb to 237.4 ppb after the experiment, which was beyond the maximum level (100 ppb, second quality of corn as feed) required by the indonesian national standard board (2013). thus, these corn grains cannot be used as feed ingredients in the feed industry for complete feed making. regarding the moisture content (mc) effect, the aflatoxin total (at) of 15% mc corn was higher (p < 0.05) than that of the 13% mc corn (fig. 2). this result contradicted the findings of nalle et al. (2022), likely due to the difference in methodology. the findings suggested that storing corn kernels with high moisture content that have already been contaminated with aspergillus spp. and aflatoxin is risky, as the fungi can rapidly multiply and produce high levels of aflatoxin. figure 1 moisture content (mc, 13% and 15%) and anti-mold (a, -, +) interaction on aflatoxin total (ppb); notes: p < 0.0001; sem = 65.92. biotropia vol. 32 no. 1, 2025 30 regarding the mold inhibitor effect (fig. 3), anti-mold (a) significantly reduced (p < 0.05) the at of shelled corn, which was in contrast with the findings of nalle et al. (2022). the discrepancies may be attributed to differences in methodology. nalle et al. (2022) used shelled corn with a moisture content of less than 12%, whereas this study used dried shelled corn with a moisture content ranging from 13% to 15%. additionally, nalle et al. (2022) worked with freshly harvested corn free of aflatoxin contamination at the start, while this experiment used aflatoxin-b1-contaminated corn from the outset (table 1). the result indicated that it is important to add mold inhibitors to prevent the physical and chemical deterioration of corn seeds. the reduction in aflatoxin levels due to the addition of mold inhibitors is attributed to the action of active compounds contained in the mold inhibitor, such as propionic acid, lead, and arsenic acid. amari et al. (2017) and yun and lee (2016) highlighted that propionic acid eliminates fungal cells through a distinct mechanism that involves apoptosis signaling mediated by mitochondria. regarding the effect of lead on fungal cell destruction, amari et al. (2017) explained that lead (pb) binds to the fungal cell wall or membrane, impairing their plasticity and disrupting processes like cell division and elongation. the plasma membrane, which is the first to encounter lead, is particularly affected, as pb interferes with membrane function and alters lipid compositions. arsenic (as) compounds may lower the ph of the corn medium, creating an acidic environment that inhibits fungal growth. according to ceci et al. (2020), arsenic acid can alter the ph of the medium, which in turn affects fungal metabolism and potentially influences ph changes during fungal growth. the present experiment proved that the amount of aflatoxin in corn grains increased during the 60-day storage compared to the initial at content before the experiment (tables 1 and 4). however, the increase of aflatoxin in corn treated with the mold inhibitor was not as high as in the treatment groups without mold inhibitor addition. safe and healthy animal feed must be produced so that animals can consume good-quality feed. therefore, the responsibility of the feed industry is to formulate and produce complete feed using good-quality raw ingredients. one of the quality requirements of complete feed is that the feed must be free from mycotoxin contamination. fungal inhibitor implementation is one strategy for maintaining the quality of feed materials at some stage in storage. concerning the aflatoxin, popescu et al. (2022) explained that aflatoxin b1 is the most harmful aflatoxin due to its carcinogenic effect on humans and animals. the present study indicated an increase in aflatoxin production in corn after storage for 60 days. figure 2 moisture content (mc, 13% and 15%) effect on aflatoxin total (ppb) notes: p < 0.0001; sem = 46.98. figure 3 anti-mold (a, -, +) effect on aflatoxin total (ppb) notes: p < 0.0001; sem = 46.98. different moisture levels of corn supplemented with mold inhibitor 31 the proximate and gross energy profile of corn grains table 4 describes the chemical content of corn samples after 60 days of storage. the statistical analysis discovered an insignificant interaction (p > 0.05) between moisture content (mc) and antimold (a) on the parameters observed. the main effect of mc was not significant (p = 0.0501 and 0.067) for moisture and dry matter parameters but significant (p < 0.01) for the other proximate parameters and ge. the cp and ash content of corn with 15% mc was superior (p < 0.05) to 13% mc corn. the ge content of corn with 15% mc was poorer (p < 0.05) than the ge concentration of corn with 13% mc. concerning the anti-mold factor, the anti-mold did not produce any changes (p > 0.05) in the concentration of proximate and ge content of corn. the increased protein concentration of 15% mc corn after 60 days of storage was probably caused by the decrease of ether extract, leading to protein improvement. the other possibility was the presence of protease and crude protein of mycelial fungi (aspergillus sp.) in corn. prawira et al. (2015) reported that aspergillus flavus could produce alkaline protease. this finding was partly comparable to nalle et al. (2022). for the anti-mold factor, the insignificant effect of mi on dry matter content was not in line with nalle et al. (2022). this discrepancy was most likely a result of the disparity in methodology, especially the mc and storage duration differences. table 4 proximate composition and gross energy content of corn after 60 days of storage moisture content (mc) anti-mold moisture content dry matter crude protein ether extract ash gross energy (ge) (kcal/kg)….……% as feed…………… 13% 11.98 87.98 7.87 5.98 2.05 3996 + 12.00 88.00 7.65 5.69 1.99 4004 15% 12.22 87.86 9.02 4.69 2.22 3950 + 12.37 88.95 8.94 5.07 2.20 3932 sem 0.197 0.126 0.081 0.324 0.036 14.59 main effects moisture content (mc) 13% 11.99 87.99 7.76b 5.83a 2.02b 4000a 15% 12.29 88.40 8.98a 4.88b 2.21a 3941b sem 0.114 0.089 0.057 0.229 0.025 10.32 anti-mold (a) 12.10 87.92 8.44 5.33 2.13 3973 + 12.18 88.47 8.29 5.38 2.09 3968 sem 0.114 0.089 0.057 0.229 0.025 10.32 probability p > f mc 0.0501 0.067 <.0001 0.0036 0.0021 0.0033 a 0.3501 0.3021 0.3920 0.3686 0.4563 0.7563 mc × a 0.0890 0.0912 0.1362 0.1563 0.1833 0.09754 notes: different superscripts specify a significant difference at a p value less than 0.05; mc = moisture content; a = anti-mold. biotropia vol. 32 no. 1, 2025 32 the main effect of mc was not significant (p = 0.0501 and 0.067) for moisture and dry matter parameters but significant (p < 0.01) for the other proximate parameters and ge. the cp and ash content of corn with 15% mc was superior (p < 0.05) to 13% mc corn. the ge content of corn with 15% mc was poorer (p < 0.05) than the ge concentration of corn with 13% mc. concerning the anti-mold factor, the anti-mold did not produce any changes (p > 0.05) in the concentration of proximate and ge content of corn. the increased protein concentration of 15% mc corn after 60 days of storage was probably caused by the decrease in ether extract, leading to protein improvement. the other possibility was the presence of protease and crude protein of mycelial fungi (aspergillus sp.) in corn. prawira et al. (2015) reported that aspergillus flavus could produce alkaline protease. this finding was partly comparable to nalle et al. (2022). for the anti-mold factor, the insignificant effect of mi on dry matter content was not in line with nalle et al. (2022). this discrepancy was most likely a result of the disparity in methodology, especially the mc and storage duration differences. the amino acid content of corn table 5 represents the amino acid profile of corn with different moisture content (mc) added with mold inhibitor (mi). the result showed that the mc x a interaction was significant (p < 0.05) only in valine and glycine (table 5). the valine content of corn (15% ml without mi) was inferior (p < 0.05) to the other treatments. the glycine content of corn (ml 13% and 15% added with mi) was greater (p < 0.05) compared to that of 13% and 15% mc corn without mi addition. these results were not consistent with nalle et al. (2022). the discrepancies were likely caused by differences in research methodology, particularly the initial quality and moisture content of the dried-shelled corn used. regarding the main effect of mc, the result showed that the leucine content of corn was affected (p < 0.05) by the mc. the leucine of 13% mc corn was greater (p < 0.05) than that of 15% mc corn. the present result was in contrast with nalle et al. (2022), who found that moisture content did not affect the leucine content. the difference was probably due to the different research methodology. for the anti-mold effect, it was revealed that except for valine, the amino acid content of corn was not affected by the anti-mold used, which partly agreed with elsamra et al. (2012) and nalle et al. (2022). elsamra et al. (2012) found that anti-mold produced lower threonine, aspartic acid, cysteine, valine, isoleucine, and leucine content in corn. the disparity between the present result and the results of elsamra et al. (2012) and nalle et al. (2022) was perhaps attributable to the differentiation in methodology, particularly the initial condition of corn, type, and dosage of mold inhibitor. amino acids are organic compounds composed of amino and acid groups (wu 2013) and play a crucial role in the animal and human body for protein synthesis. payne & hagler (1983) reported from their experiment that some amino acids, especially asparagine, proline, methionine, and tryptophan, stimulated aflatoxin production. proline and asparagine were responsible for a. flavus development and aflatoxin production. the mechanism(s) by which proline and asparagine stimulate toxin production in culture is unknown. the present experiment indicated that the amino acids of corn that were responsible for a. flavus development and aflatoxin production remained stable after the experiment. overall, synthetic mold inhibitors effectively decreased aflatoxin levels in corn during storage and maintained some nutritional quality. however, due to the potential residue of harmful chemicals, such as arsenic acid and lead in animal products, it is preferable to use natural mold inhibitors. consequently, it is necessary to develop natural anti-mold feed additives to avoid those health risk problems in animals and humans. different moisture levels of corn supplemented with mold inhibitor 33 table 5 profile of indispensable amino acids (% as feed) in shelled corn following dietary treatments moisture content anti-mold leu lys meth thre valine cys gly 13% 1.260 0.525 0.305 0.170 0.335a 0.135 0.780b + 1.270 0.535 0.255 0.170 0.325a 0.145 0.810c 15% 1.185 0.520 0.340 0.135 0.280b 0.160 0.795b + 1.200 0.525 0.355 0.170 0.300a 0.145 0.855a sem 0.014 0.006 0.027 0.009 0.019 0.007 0.011 moisture content (mc) 13% 1.265a 0.530 0.280 0.170 0.330 0.140 0.795 15% 1.192b 0.522 0.347 0.152 0.290 0.152 0.825 sem 0.011 0.005 0.019 0.006 0.016 0.005 0.007 anti-mold (a) 1.222 0.522 0.322 0.152 0.307 0.147 0.787 + 1.235 0.530 0.305 0.170 0.312 0.145 0.832 sem 0.011 0.005 0.019 0.006 0.016 0.005 0.007 p value mc 0.0044 0.2729 0.0771 0.2338 0.1011 0.1106 0.0559 a 0.1908 0.5060 0.8864 0.5546 0.0299 1.0000 0.0554 mc × a 0.8621 0.4640 0.3902 0.0773 0.0303 0.2519 0.0057 notes: different superscripts specify a significant difference at a p value less than 0.05; leu = leucine; lys = lysine; meth = methionine; thre = threonine; cys = cysteine; gly = glycine; mc = moisture content; a = anti-mold. experiment ii feed intake table 6 shows the feed consumption of bird fed dietary treatments during 28 days of the experiment. the ac x mb interaction was significant (p < 0.05) on fi of broilers only in the fourth week of trial, but not in the first to the third week, partly coherent with prahara et al. (2023), who found that toxin binder addition did not affect the feed intake of broilers. the lsd test indicated that the fi of bird fed diets containing corn 15% mc (with and without mi) was lower than other treatment combination diets. the low fi in broilers could be due to the moldy smell of diets containing high aspergillus spp. according to hell and roth (2019), the moldy smell of grains occurred due to fungal contamination when the moisture content of grains was high. on the other hand, aflatoxins did not produce a bad taste and odor, but some grains, such as groundnut kernels, had a bitter taste when contaminated with aflatoxins (hell & roth 2019; mijena & ijara 2024). the mycotoxin binder supplementation in the aflatoxin diets did not appear to improve the fi, as approved by prahara et al. (2023). however, other researchers revealed the contrary results (fernandes et al. 2022). the lack of consistency may be owing to the differences in factors, such as bird strain and age, the dosage and active compounds of mycotoxin binder used, exposure time of aflatoxin, and the aflatoxin level exposure. kolossova et al. (2011) reported that the effectiveness of mycotoxin binders varies based on the chemical properties of the mycotoxin binder and mycotoxin. furthermore, it was explained that the total charge and charge distribution, the pore size, the accessible surface area, divergence, appearance, and solubility play a notable role in determining the effectiveness of the adsorbent. for the first main factor, the ac had a highly significant effect (p < 0.01 – 0.001) on the fi of broiler diets on days 7, 14, 21, and 28. the least significant different test showed that on days 7 and 21, consumption of broiler rations in the treatment < 100 ppb and 222 ppb was higher (p < 0.05) than in the treatment group 165 ppb. on day 14, the fi of birds in the 165 ppb diets was lower (p < 0.05) than in the < 100 ppb and 222 ppb treatment groups. the present result was similar to those observed by zhu et al. (2023), biotropia vol. 32 no. 1, 2025 34 who found that even at the lower afb1 content (< 80 ppb), the feed intake of laying hens decreased significantly. the tendency of birds to eat less in the aflatoxin diet group was possibly due to several factors, such as: 1) the change in the unpleasant flavor and odor of feed by aflatoxin-producing mold (hell & roth 2019; mijena & ijara 2024); and 2) the change in morphology and histology of the hemorrhaging condition of the bird’s intestine disturbing the nutrients digestibility. as a result, the nutrient digestion and emptiness of the intestine will take longer than usual, leading to a lower feed intake. monson et al. (2015) stated that the response and capability of birds to metabolize the aflatoxin in their diets depend on the strain of birds, the individual bird, age, and the dosage of aflatoxin. regarding the second main factor, the nonsignificant effect (p > 0.05) of mycotoxin binder in feed intake did not have the same result as mendoza et al. (2022) and alayande et al. (2023). the discrepancies were caused by methodological dissimilarity, particularly in the type of mycotoxin binder used. the present study used alvitoxtm bio (powder form), while mendoza et al. (2022) used a liquid toxin binder solution body weight gain the afb1 levels x mycotoxin binder interaction were comparable (p > .05) on body weight gain (bwg) of broilers during the study, disagreeing with previous researchers (fernandes et al. 2019; zabiulla et al. 2021). the insignificant effect might be because of the non-significant effect of the mycotoxin binder on feed intake. it is generally accepted that feed intake has a linear correlation to body weight gain. nazarizadeh & pourreza (2019) revealed the effectiveness of a commercial mycotoxin binder (mycosorb) in improving the bwg of birds exposed to high dosages of aflatoxin b1 (2 ppm and 4 ppm). moreover, fernandes et al. (2019) and zabiulla et al. (2021) concluded that the mycotoxin binder added to the aflatoxin diet ameliorated the bwg of broilers. the dissimilar result was possibly because of the dissimilar in mycotoxin binder product and dosage used and the level of aflatoxin in the diet. regarding the main effect of aflatoxin b1 concentration (ac), the statistical analysis proved that except for the seventh-day trial, the afb1 level significantly affected (p < 0.01 – 0.001) the body weight gain (bwg) of birds (table 6). the results of the least significant difference (lsd) test showed that the bwg of broiler chickens in the 165 µg/kg and 222 µg/kg afb1 treatment group was lower (p < 0.05) than in the < 100 µg/ kg treatment. the decrease in bwg was related to the reduced feed intake of birds. on the contrary, al-shawabkeh et al. (2009) reported that as the aflatoxin diet increased, the feed consumption would increase, but the bwg of birds decreased. the present result also did not agree with nalle et al. (2021), who reported that the level of afb1 did not change the bwg of birds. the differences were probably due to the differences in methodology, especially the level of afb1 and the type and level of mycotoxin binder product used. feed efficiency table 6 presents the feed conversion ratio (fcr) of birds that received dietary treatment. it can be seen from table 6 that, except for the 28th day, the afb1 level was significant (p < 0.05 to p < 0.001) on the fcr of birds, which partly concurred with nalle et al. (2021). this experiment showed that the fcr of birds fed a diet containing a higher afb1 tended to have higher fcr. the high fcr indicated that there might be a reduction in the nutritional quality of high aflatoxin feed. studies have shown that fungi use feed nutrients to multiply and produce toxins (liu et al. 2016; barzee et al. 2021). mycotoxin binder addition did not improve the fcr of birds (p > 0.05), as opposed to fernandes et al. (2022) and zabiulla et al. (2021) who reported an improvement in feed efficiency of broilers fed aflatoxin diets added with mycotoxin binder commercial. the difference in efficacy of mycotoxin was probably caused by the difference in mycotoxin binder type and dosage, as well as the aflatoxin level in the experimental diets. the interaction between afb1 levels and mycotoxin binder was insignificant (p > 0.05) on the fcr of broilers. different moisture levels of corn supplemented with mold inhibitor 35 ta bl e 6 g ro w th re sp on se o f b ro ile rs fe d afl at ox in -c on ta m in at ed d ie ts su pp le m en te d w ith a c om m er ci al fe ed a dd iti ve b in de r a fb 1 co nc en tr at io n (a c , µg /k g, a s f ee d) m yc ot ox in b in de r (m b ) fi (g /b ir d) b w g (g /b ir d) fc r (g /g ) 7 da ys 14 d ay s 21 d ay s 28 d ay s 7 da ys 14 d ay s 21 d ay s 28 d ay s 7 da ys 14 d ay s 21 d ay s 28 d ay s <1 00 13 8 43 6 89 4 12 98 a 78 .1 23 0 36 4 48 8 1. 76 7a 1. 98 5 2. 51 6 2. 69 0 + 13 2 43 9 87 1 12 29 ab 80 .3 21 7 37 4 52 5 1. 65 5a b 2. 03 8 2. 26 8 2. 34 5 16 5 11 9 36 5 70 3 10 03 c 75 .5 19 5 31 1 42 3 1. 58 6b 1. 88 0 2. 26 0 2. 42 7 + 12 7 40 4 73 4 10 68 c 77 .2 20 9 34 4 47 1 1. 65 4a b 1. 93 7 2. 13 3 2. 28 2 22 2 13 9 43 9 82 4 11 79 b 78 .0 20 4 33 4 45 1 1. 79 7a 2. 15 5 2. 41 5 2. 57 6 + 14 6 46 2 85 7 12 13 ab 79 .4 20 2 32 7 43 3 1. 83 4a 1. 74 4 2. 62 2 2. 65 1 se m 4. 76 7 17 .1 36 .2 35 .3 2. 17 8 7. 12 11 .4 20 .2 0. 06 13 0. 09 9 0. 13 4 0. 14 1 m ai n eff ec ts af b1 c on ce nt ra tio n (a c , µ g/ kg ) < 10 0 13 5a 43 8a 88 2a 12 63 a 79 .2 22 3a 36 9a 50 7a 1. 71 1a b 2. 01 2b 2. 39 2a b 2. 51 8 16 5 12 3b 38 5b 71 8b 10 36 b 79 .4 20 2b 32 8b 40 7b 1. 62 0b 1. 90 8b 2. 19 6b 2. 35 4 22 2 14 3a 45 1a 84 0a 11 96 a 78 .7 20 3b 33 1b 44 2b 1. 81 5a 2. 21 8a 2. 51 9a 2. 61 4 se m 3. 04 2 12 .1 25 .6 24 .9 1. 54 0 5. 03 8. 05 14 .3 0. 04 3 0. 07 0 0. 09 5 0. 09 9 m yc ot ox in b in de r ( m b) 13 2 41 3 80 7 11 60 77 .2 21 0 33 6 45 4 1. 71 7 1. 99 3 2. 39 7 2. 56 4 + 13 5 43 5 82 1 11 70 78 .9 20 9 34 8 47 6 1. 71 4 1. 90 6 2. 34 1 2. 42 6 se m 2. 15 1 8. 56 18 .3 17 .6 1. 08 9 3. 55 9 5. 69 10 .1 0. 03 1 0. 04 9 0. 06 7 0. 07 0 p va lu e ac 0. 00 08 0. 00 30 0. 00 04 <. 00 01 0. 57 50 0. 00 86 0. 00 05 0. 00 08 0. 00 37 0. 00 62 0. 01 29 0. 05 37 m b 0. 72 27 0. 56 12 0. 23 96 0. 23 54 0. 09 08 0. 86 54 0. 43 37 0. 61 99 0. 09 78 0. 95 28 0. 24 48 0. 14 47 ac × m b 0. 08 80 0. 15 79 0. 10 83 0. 01 90 0. 76 98 0. 31 40 0. 25 60 0. 11 01 0. 02 82 0. 33 78 0. 26 82 0. 53 01 n ot es : d iff er en t s up er sc rip ts in th e sim ila r c ol um n de no te si gn ifi ca nt d iff er en ce s a t a p v al ue < 0 .0 5; f i = f ee d in ta ke ; bw g = b od y w ei gh t g ai n; f c r = f ee d c on ve rs io n r at io ; a c = a fb 1 c on ce nt ra tio n; m b = m yc ot ox in b in de r. biotropia vol. 32 no. 1, 2025 36 conclusions the anti-mold effectively maintained low aflatoxin levels in 13% moisture in corn without affecting the nutrient profile of corn during storage. mycotoxin binder supplementation did not improve broiler performance. broiler performance declined as aflatoxin concentrations increased. acknowledgments the authors express their sincere gratitude to kupang state agricultural polytechnic for funding this research with contract number 01/p3m/ sp dipa-023.18.2.677616/2023. the authors are grateful and appreciate the contribution of pt. japfa comfeed tbk. for supplying anti-mold products for this research. appreciation is extended to maria wonda, geti pahnael, and suhartini salih for their assistance. references alayande l, bawa gs, rano nb, ogundipe so. 2023. response of broiler chickens fed with aflatoxin maizebased diets supplemented with various levels of mycotoxin binder. slovak j anim sci 56(1):17-29. doi: 10.36547/ sjas.822 almatakeez a. 2020. introducing an indigenous non-toxigenic aspergillus flavus strain isolated from iraqi corn grains as a bio-control agent to reduce aflatoxin contamination in corn grains. 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j, wang y, wang y. 2023. effects of moldy corn on the performance, antioxidant capacity, immune function, metabolism and residues of mycotoxins in eggs, muscle, and edible viscera of laying hens. poult sci 102:102502. doi: 10.1016/j.psj.2023.102502 zwolak i. 2020. the role of selenium in arsenic and cadmium toxicity: an updated review of scientific literature. bio trace elem res 193:44-63. doi: 10.1007/s12011-01901691-w cover single.cdr biotropia vol. 27 no. 2, 2020: 162 170 doi: 10.11598/btb.2020.27.2.1199 site index of siamese rosewood { dalbergia cochinchinensis pierre) in plantations of thailand** taengmoo phunchaisri1, chongrak wachrinrat'*, ponthep meunpong1, suwan tangmitcharoen2 and nawaphong kuasakun1 1department of silviculture, faculty of forestry, kasetsart university, bangkok 10900, thailand 2forest research and development bureau, royal forest department, bangkok 10900, thailand received 07 january 2019 / accepted 16 may 2019 abstract currendy, decreased productivity of siamese rosewood (dalbergia cochinchinensis pierre) is a problem due to illegal logging and the guidelines to manage this prevention being may be insufficient. forest plantations should be promoted to achieve sustainable wood production for this species. site index (si) is a measure that can be used to predict forest productivity to identify appropriate sites for planting siamese rosewood. the objective of the current study was to predict the si for siamese rosewood in thailand. a sample of 78 temporary plots at 26 sites in 16 provinces of thailand was selected to construct a site index curve with a base age of 30 years. the results showed that the mean age of siamese rosewood was 28 years (between 13 and 55 years), the mean diameter at breast height (dbh) was 20.03 cm (between 7.99 and 33.80 cm), and the mean dominant height was 20.79 m (between 7.32 and 24.82 m). the si equation was: si = e(kifu-12.476(a4-ab-1)) with an r2 value of 0.406 at p < 0.0001 (chosen as 30 years in the present study). the equation indicated that 14, 18, 22, 26 and 30 m trees belonged to site classes i, ii, iii, iv and v, respectively. although in natural forest, siamese rosewood is distributed in dry evergreen and mixed deciduous forests, in the present study, several sites in areas under both in situ and ex situ conservation were in the good and very good site categories. thus, both areas can be used to plant siamese rosewood and increase its productivity. keywords: siamese rosewood, site index, temporary plot introduction siamese rosewood ( dalbergia cochinchinensis pierre) is generally found in the dry evergreen and mixed deciduous forests (veesommai & kavduengtain 2004) at elevations between 100 and 775 m above sea level (eiadthong & tangmitcharoen 2015). this species is native to thailand, cambodia, laos, and vietnam. over the years, this species has been severely affected by illegal logging, due to the quality of its hardwood and high durability, leading to a gradual reduction in its numbers (cadena 2014). it was under the ‘vulnerable” category and red listed as a threatened species by the international union for conservation of nature ^corresponding author, e-mail: fforcrw@ku.ac.th *whis paper was presented at the 3rd international conference on tropical biology 2018, 20-21 september 2018, bogor, west java, indonesia (iucn) (iucn 2008), leading to a high demand of its wood and high price (forest research and development bureau 2010). this has been the prime reason affecting the numbers of siamese rosewood trees. according to a survey by the royal forest department of thailand (2015), the price of siamese rosewood heartwood was usd 30,000/m3 on the black market. such a high price provided sufficient motivation for illegal logging in both natural forest and plantations. in 2014, 1,823 m3 of siamese rosewood was seized by the thai government. the lawsuits filed for illegal logging of siamese rosewood have increased while productivity has decreased every year in thailand (department of national parks, wildlife and plant conservation 2015). a similar situation exists for other threatened species with similar hardwood characteristics, such as dalbergia oliveri gamble ex prain and tterocarpus 162 site index of siamese rosewood ('dalbergia cochinchinensis pierre) in thailand — phunchaisri et al macrocarpus kurz (internal security operations command 2014). the diminishing number of individual trees of such species is of grave concern and needs to be dealt with urgently. plantations can play an important role in the success of reviving these species. several economically viable species, including siamese rosewood, have been promoted in certain areas by the thai government. two distinct procedures have been applied: 1) in situ conservation areas (conservation of ecosystems and natural habitats and maintenance and recovery of the population that allows the species to exist in the natural environment and in the case of domesticated or cultivated plant species in an environment in which those species have developed special properties); and 2) ex situ conservation (conservation of plant genetic resources outside the natural habitat). the current methods being used for selecting areas where this planting siamese rosewood need to be improved and implemented in the future (tangmitcharoen 2013). site index (si) is one of the measures used to predict forest productivity, wood volume, and the potential rate of growth (hanson et al. 2002). the relationship between dominant height and age was used to construct a site index curve by mayhew & newton (1998). there are several techniques for determining si such as: (1) the proportion curve or guide curve method; (2) the parameter prediction method; and (3) the different equation method (clutter et al. 1983). in thailand, the proportion curve method has been used for assessing and predicting the site quality of tectona grandis l.f. (forestry research center 1996) and eucalyptus camaldulensis dehnh. plantations in the lower northeastern parts (insaun et al 2009) and acacia mangium willd. plantation in trat province (jumwong 2006). siamese rosewood research has also focused on other dimensions such as inventory, ecology, growth and yield. the prediction of si for siamese rosewood is yet to be undertaken. thus, the objective of the current research was to summarize the growth characteristics and develop a si for siamese rosewood found in plantations in thailand. materials and methods study site and data collection thailand is a tropical country located between 5°37m n and 20°27m n latitude and 97°22m e and 105°37m e longitude. the annual rainfall ranges between 1,200 and 1,600 mm and the mean annual rainfall is 1,587 mm. the mean annual temperature is around 27° c (thai meteorological department 2014). the study was conducted in siamese rosewood plantations owned by the royal forest department of thailand (rfd) and the forest industry organization (fio). twenty-six sites were selected from 16 provinces distributed throughout thailand (table 1 and fig. 1). three temporary sample plots were selected per site (78 temporary sample plots in total). each temporary plot was 0.16 ha in size and the diameter at breast height over bark (dbh) at 1.30 m above ground and total height (h), of all trees in the plot, were measured. meteorological data from the thai meteorological department for the sample sites are shown in table 2. table 1 summary of 26 siamese rosewood study sites in thailand used in the study no plantation province region code latitude longitude 1 kam phaeng phet silvicultural research station kamphaengphet n kpp1 16° 33’ 99° 30’ 2 kam phaeng phet silvicultural research station kamphaengphet n kpp2 16° 34’ 99° 30’ 3 kam phaeng phet silvicultural research station kamphaengphet n kpp3 16° 33’ 99° 30’ 4 phit sa nu lok silvicultural research station phitsanulok n pnl 16° 50’ 100° 53’ 5 in tha khin silvicultural research station chiangmai n itk 19° 9’ 98° 56’ 6 tha turn silvicultural research station surin ne tt 15° 18’ 103° 45’ 7 nong khu silvicultural research station surin ne nk 14° 41’ 103° 45’ 8 huai tha silvicultural research station sisaket ne ht1 14° 52’ 104° 26’ 9 huai tha silvicultural research station sisaket ne ht2 14° 52’ 104° 26’ 10 pha nok khao silvicultural research station khon kaen ne pnk 16° 50’ 101° 56’ 11 loei forestry and forest products research center loei ne loei 16° 52’ 101° 56’ 163 biotropia vol. 27 no. 2, 2020 table 1 (continued) 12 udon tha ni forest nursery center udon thani ne ud 16° 50’ 102° 56’ nakhon 13 mu si silvicultural research station ratchasima ne msi 14° 30’ 101° 25’ nakhon 14 mu si silvicultural research station ratchasima ne ms2 14° 30’ 101° 25’ 15 ka la sin silvicultural research station kalasin ne kls 16° 28’ 103° 24’ 16 dong lansilvicultural research station khon kaen ne dl 16° 49’ 101° 59’ 17 huai rang plantation trat e hr 12° 26’ 102° 33’ 18 tha kum noboru umeda plantation trat e tk1 12° 22’ 102° 40’ 19 tha kum noboru umeda plantation trat e tk2 12° 23’ 102° 39’ 20 tha kum noboru umeda plantation trat e tk3 12° 23’ 102° 39’ 21 rat cha bu ri silvicultural research station ratchaburi c rbr 13° 34’ 99° 44’ 22 central silvicultural research center kanchanaburi c pk1 14° 24’ 98° 55’ 23 central silvicultural research center kanchanaburi c pk2 14° 24’ 98° 55’ 24 thong pha phum silvicultural research station kanchanaburi c tpp 14° 42’ 98° 39’ 25 southern silvicultural research center songkhla s pt 7° 1’ o 0 prachuap khiri 10° 59’ 99° 27’26 sai thong silvicultural research station khan s st note: regions as identified by the thai meteorological department are: n” is north, “ ne” is northeast, “ e” is east, “ c” is central, and “ s” is south. table 2 meteorological data for the study sites average average relative no. station name temperature (°q humidity , (%) (nrim/year) study site 1 phitsanulok 28.1 74.2 1,399 pnl 2 kamphaengphet 28.5 76.8 1,372 kpp1, kpp2, kpp3 3 chiangmai 27.2 71.3 1,158 itk 4 pakchong agromet 26.4 73.3 1,206 msi, ms2 5 thatom 27.9 74.1 1,313 tt 6 surin 27.7 72.4 1,454 nk 7 sisaket agromet 27.7 74.8 1,420 ht1, ht2 8 loei 26.9 73.2 1,310 loei 9 kalasin 27.2 72.0 1,340 kls 10 khon kaen 27.7 70.4 1,196 pnk, dl 11 udonthani 27.5 71.1 1,338 ud 12 trat 27.9 81.4 4,988 hr, tk1, tk2, tk3 13 ratchaburi 28.6 78.1 1,067 rbr 14 kanchanaburi 29.0 70.4 1,104 pk1, pk2 15 thong phaphum 28.1 78.5 1,827 tpp 16 prachuap khiri khan 28.8 76.3 1,056 st 17 hatyai 28.3 79.4 1,793 pt determination of si equation the height of the tree is important in the creation of the si but the mean height of a stand is usually sensitive to age, site, class and stand density. thus, the dominant height is normally used to represent a stand and specifically mean height of the 100 tallest trees/ha is most widely used to construct an si (alder 1980). in this case, the si equation was constructed using the top 16 tallest trees per plot (plot size of 0.16 ha). there are several techniques for determining the si. however, the current study only had access to data derived from temporary plots and consequently, the guide curve method was used (alderl980) based on equation (1) and the si equation was constructed using equation (2). in (hdo) = inb0+bia-k (1), si = e (in (hdo)+bi (a-hay1)) (2), where hdo is the dominant height, in is the natural logarithm, bo and bi are regression coefficients, a is the stand age, k = 1, si is the site index, e is the natural exponent, and ab is 164 site index of siamese rosewood ( dalbergia cochinchinensis pierre) in thailand — phunchaisri et al. the base age (chosen as 30 years in the present study). based on equation 2, 5 site classes were used (i, ii, iii, iv, and v) indicating very poor, poor, moderate, good, and very good sites, respectively. the upper and lower bounds for each si class were determined using simple interpolation and extrapolation in the excel software package (microsoft corp.; redmond, ca, usa). results and discussion the growth characteristics of siamese rosewood, as determined from 26 sites, are presented in table 3. the mean age was 28 years (between 13 and 55 years), mean dbh was 20.03 cm (between 7.99 and 33.80 cm), mean h was 16.48 m (ranging between 6.18 and 23.20 m), and the mean hdo was 20.79 m (ranging between 7.32 and 24.82 m). the mean dbh, h and hdo of the tt plantation were lowest (see table 1 for identification of the plantation abbreviations used). both the highest mean dbh and mean h were in the kls plantation and the highest mean of hdo was in the pnl plantation. the mean dominant heights for kls and pnl were similar (24.18 and 24.82 m, respectively) despite their stand ages being rather different (42 and 27 years, respectively). this could be interpreted as the younger stand age alone indicating site suitability which is not necessarily correct. thus, the methodology for assessing the appropriate site using the site index had to decrease the bias associated with the different-aged stands. «00000 1200000 i § scale 1:8,000,000 0 75 150 300 450 600 i kilometer! 600000 1200000 figure 1 location of siamese rosewood study sites in thailand table 3 growth characteristics of the siamese rosewood in plantations located at the 26 sites in thailand no site code age (years) dbh ± sd (cm) h ± sd (m) hdo± sd (m) 1 tt 13 7.99 ± 3.94 6.18 ± 2.65 7.32 ± 2.22 2 rbr 22 23.42 ± 7.48 14.06 ± 3.30 14.55 ± 3.00 3 nic 25 13.23 ± 5.66 10.78 ± 3.85 16.03 ± 2.40 4 ht1 25 18.38 ± 6.06 14.58 ± 3.56 17.55 ± 1.49 5 ht2 25 21.02 ± 8.27 16.64 ± 4.13 17.43 ± 3.03 6 pnk 54 31.59 ± 7.16 22.64 ± 4.83 22.92 ± 4.70 7 loei 55 18.34 ± 6.07 17.42 ± 4.68 22.14 ± 2.43 8 ud 20 14.18 ± 5.73 13.82 ± 4.95 17.50 ± 4.13 165 biotropia vol. 27 no. 2, 2020 table 3 (continued) 9 kpp1 23 18.43 ± 7.30 16.72 ± 3.93 20.62 ± 1.90 10 msi 28 21.15 ± 6.15 16.74 ± 3.18 20.02 ± 1.16 11 ms2 28 15.41 ± 6.29 13.96 ± 4.63 20.72 ± 2.38 12 hr 30 20.49 ± 7.92 16.21 ± 5.57 22.72 ± 2.61 13 tk1 32 25.06 ± 10.62 17.58 ± 6.16 23.53 ± 2.52 14 tk2 35 22.21 ± 8.48 15.82 ± 5.36 22.04 ± 1.74 15 tk3 36 25.40 ± 9.18 15.09 ± 5.25 21.38 ± 2.01 16 kls 42 33.80 ± 9.92 23.20 ± 5.02 24.18 ± 4.94 17 pk2 18 18.39 ± 7.17 15.69 ± 4.64 20.71 ± 1.74 18 pk1 21 16.99 ± 7.65 15.09 ± 4.98 20.72 ± 1.53 19 kpp2 23 21.11 ± 7.07 19.47 ± 5.13 23.86 ± 2.38 20 kpp3 23 20.65 ± 9.55 17.54 ± 6.68 24.35 ± 1.96 21 pt 26 20.29 ± 8.14 18.24 ± 5.45 23.08 ± 0.90 22 pnl 27 15.97 ± 6.83 17.93 ± 5.45 24.82 ± 1.22 23 tpp 27 25.55 ± 11.14 20.01 ± 5.26 24.30 ± 1.28 24 st 28 19.18 ± 7.67 19.64 ± 4.62 24.42 ± 1.15 25 dl 16 16.66 ± 5.68 16.06 ± 3.42 19.89 ± 1.92 26 itk 20 15.85 ± 6.89 17.30 ± 5.16 23.60 ± 2.16 the si equation for siamese rosewood category. in this study, most of the measured obtained using the proportional curve method at sites were in either the moderate or good site a base age of 30 years was si == e(lnhdoclasses. even though th<s growth rate based on 12.476(a~'-ab~')) with anri2 value of 0.406 and dbh, h, and hdo of kls and pnl were p < 0.0001. five stratification levels were used satisfactory, the results showed that dl and based on site classes i through v for h values of 14, 18, 22, 26 and 30 m, respectively. figure 2 shows that a 30 years siamese rosewood tree with a dominant height of 22 m belonged to site class iii. this stand was predicted as a moderate site for planting. likewise, we also determined the best site planting (class v) for which trees had to be 30 m tall at age 30 years. in contrast, trees on poorer sites (site i) were only 14 m tall at age 30 years. the number of study sites in the itk were very good sites for siamese rosewood compared at the base age of 30 years. kliangpibool (2012) reported the suitable age for siamese rosewood logs was between 18 and 25 years at the price was usd 530 to 1,000/ha. however, the valuation of siamese rosewood does not depend only on the quantity as the economic valuation of hardwood is important too. kuasakun (2017) determined that the heartwood percentage of siamese rosewood at the mu si silvicultural research station (ms) classifications were 1, 6, 9, 8, and 2 for si classes was 68.30% while the si for msi and ms2 were i, ii, iii, iv, and v, respectively (table 3). the lower and upper bounds of a very poor site class were 12.00 and 15.99 m, respectively, thus, the tt plantation was in this category. on the other hand, the lower and upper bounds of a very good site were 28.00 and 32.00 m, respectively, and the dl and itk plantations were in this moderate site. in contrast, the heartwood percentage of siamese rosewood at in tha kin silvicultural research station (itk) was 21.97 while the si of itk in the present study indicated it was a very good site; nevertheless, the increasing heartwood should result with increasing age as well (visaratana et al. 2016). 166 site index of siamese rosewood ('dalbergia cochinchinensis pierre) in thailand — phunchaisri et al. 40 1 35 loo si 25 '53 h 20a i 15 i 10 ' q 5 0 0 figure 2 si curves for siamese rosewood in thailand note: dotted lines indicate the upper and lower bounds for each class based on simple interpolation/ extrapolation. table 3 site class, si and bounds at base age 30 years of siamese rosewood in thailand, where the last column indicates the class according to site quality site class si number of study sites lower limit (m) upper limit (m) site code i (very poor) 14 1 12.00 15.99 tt ii (poor) 18 6 16.00 19.99 rbr, nic, ht1, ht2, pnk, loei iii (moderate) 22 9 20.00 23.99 ud, kpp1, msi, ms2, hr, tk1, tk2, tk3, ills iv (good) 26 8 24.00 27.99 pk2, pk1, kpp2, kpp3, pt, pnl, tpp, st v (very good) 30 2 28.00 32.00 dl, itk iv iii ii i 10 20 30 40 age (year) 50 60 800000 1000000 1200000 g e g g gi o g o o g o g >lhnk gel •kl o msi g m s scale 1:3,500,000 0 30 60 120 160 240 kilometers o 0 o 800000 1000000 1200000 750000 900000 g gi **/ < § g o ft t-akh k ix1r ‘ *• gtk3tk 2 scale 1:2,000,000 0 15 30 60 90 120 kilometen 750000 900000 figure 3 in situ study sites of siamese rosewood in northeastern and eastern thailand 167 biotropia vol. 27 no. 2, 2020 the natural distribution of siamese rosewood is in the dry evergreen and mixed deciduous forests where located northeastern and eastern thailand (fig. 3). both regions have in situ conservation areas in which were located the study sites hr, tk1, tk2 and tk3 in the eastern, and tt, nk, ht1, ht2, pnk, loei, ud, msi, ms2, kls and dl in the northeastern regions. several sites in the in-situ conservation areas were not as appropriate as plantation sites as in the ex-situ conservation areas. the better ex situ sites were pk1, pk2, kpp1, kpp2, pt, pnl, tpp, st and itk were determined to be in the good and very good site classes (iv and v, see fig. 4). suanpaga & boonyuan (2016) compared the growth of eight appropriate tree species for planting in the songkhla province, southern thailand (an ex-situ conservation area for siamese rosewood). based on their results, they recommended siamese rosewood and three other species ('dipterocarpus alatus roxb, xylia xylocarpa (.roxb.) taub. var. kerrii (craib & hutch.) i. c. nielsen and ayadirachta excelsa (jack) jacobs). illegal logging has continued (sudsakorn 2012) and so plans to increase sustainable productivity need to consider prevention and promotion. chaipunya (2016) considered three aspects to maintain sustainable production from current stands: preventing illegal rosewood logging, preventing transportation, and preventing export of logs or wood products. in addition, visaratana et al. (2016) presented a case to promote both forest management and silvicultural techniques via plantations located on good sites to provide increasing productivity and economic value. siamese rosewood can fix nitrogen through rhiyobium bacteria that can increase the health of ecological systems by fixing nutrients for use also by other species (mungklarat 2001) and to improve soil fertility for reforestation purposes (so et al. 2010). with regard to the current study, several sites in the ex-situ conservation area belonged to either the good or very good site classes. thus, both ex-situ and in-situ conservation areas can be used for plantation purposes to increase productivity. 400000 600000 " !i -» l s itk stpp hm pk2pkpp si sfiit* 1:3,500,000 0 so «0 120 iso 2 seal*1:4.000,000 45 90 180 21 -+! < " ii i ' figure 4 ex situ study sites of siamese rosewood in northern, central, and southern thailand 168 site index of siamese rosewood (dalbergia cochinchinensis pierre) in thailand — phunchaisri et al. conclusion a site index curve and equation are necessities for thailand forestry to support siamese rosewood plantations dating back more than 50 years; however, extensive research commenced only recently. as a result, knowledge is still lacking of the site requirements for plantations of this species. based on the growth characteristic of siamese rosewood from the royal forest department of thailand (rfd) and the forest industry organization (fio) plantations at 26 sites represented the age ranges between 13 and 55 years, the dbh ranges between 7.99 and 33.80 cm, the h ranges between 6.18 and 23.20 m and the hdo ranges between 7.32 and 24.82 m. in addition, five si classes were constructed with most sites in the moderate site class. the si equation developed in this study can be applied to help avoid planting of siamese rosewood on inappropriate lower quality sites and place more emphasis on identifying higher quality sites for plantation establishment. several sites in the exsitu conservation area belonged either to the good or very good site classes. thus, both ex-situ and in-situ conservation areas can be used for plantation purposes to increase productivity. acknowledgements this research was supported in part by a graduate program scholarship from the graduate school, kasetsart university, bangkok, thailand. we thank the royal forest department of thailand (rfd) and the forest industry organization (fio) for helping with the supply of data and assistance with fieldwork. references alder d. 1980. forest volume estimation and yield prediction vol. 2 yield production. italy: food and agriculture organization of united nation. cadena a [internet], 2014. 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through reforestation an example of ajgeiia xylocarpa (kruz.) craib and dalbergia cochinchinensis pierre in cambodia. pacific conserv biol 16:101-16. suanpaga w, boonyuan s. 2016. growth and aboveground biomass of eight tree species in species trial for appropriate planting in had yai district, songkhla province. in: forcons proceeding 2016. p. 136. sudsakorn c. 2012. educational guidelines for preventing and combating an illegal logging of pa yung (.dalbergia cochinchinensis) [dissertation]. retrieved from joint war college. tangmitcharoen s. 2013. siamese rosewood: conservation and management of genetic resources. in: royal forest department proceedings 2013. forestry meeting on production and forest research towards sustainable development. thai meteorological department [internet]. 2014. the climate of thailand; [cited 2018 sep 24]. available from: https:/ /www.tmd.go.th/info/climate_of_thailand2524-2553.pdf veesommai u, kavduengtain p. 2004. wild trees in thailand 1. bangkok (th): h.n. group co., ltd. visaratana t, pinthong a, hutasangchai c. 2016. estimation heardwood of dalbergia cochinchinensis pierre in the plantation. in: forcons proceeding 2016. p. 72. 170 cover single.cdr biotropia vol. 27 no. 2, 2020: 143 152 doi: 10.11598/btb.2020.27.2.1197 effects of fire on structure and composition of vegetation community in pine forests, nam nao national park, thailand** kobsak wanthongchai1* and voradet tarusadamrongdet2 1 department ofsilviculture, faculty of forestry, kasetsart university, bangkok 10900, thailand department of national parks, wildlife and plant conservation, bangkok 10900, thailand received 08 january 2019 / accepted 14 august 2019 abstract anthropogenic forest fires, including those occurring in the pine forests of thailand, have been widely reported. although pine forests are fire dependent, too frequent burning could be unhealthy for the forest as it might result in nutrient depletion and ecosystem degradation. hence, this study aimed to examine the effects of forest fire on the vegetation structure and composition of a degraded pine forest (pf) and in a mixed pine-oak forest (o-pf) of phu kum khao, nam nao national park, phetchabun province, thailand. the effects of fire in one year were studied using three 50 x 50 m experimental plots established in each forest type. results showed that fire events caused a significantly higher tree mortality rate in the degraded pine forest than in the mixed pineoak forest. moreover, the seedling ratio (number of sprout to number of seedling ratio) in the mixed pine-oak forest was higher than in the degraded pine forest. although the sapling and seedling diameters were slightly different between the sites, no significant effect of burning was observed on the sapling and seedling density, diameter and height. this might be due to the high variation of fire behavior in each plot. since forest fires are a potential factor that might cause forest degradation in the study area, further studies on the effects of fire frequency on vegetation structure and composition is needed to provide sufficient fire control measures for efficient forest fire management to prevent further ecosystem degradation. keywords: burning, degraded pine forest, nam nao national park, pine-mixed oak forest introduction forest fire is a critical factor that shapes the ecosystem structure of many forested areas (pyne 2001). too frequent forest fires have caused ecosystem damage in many areas including tropical pine forests (goldammer & penafiel 1990; wanthongchai & goldammer 2011). the presence of the flammable forest floor consists mainly of grasses and pine needles, and the frequent anthropogenic burning activities or agricultural residual burning around the forest, the pine forests of phu kum khao, nam nao national park, thailand experiences fire events almost every year (tarusadamrongdet 2013). although the physical characters of pine ^corresponding author, e-mail: fforksw@ku.ac.th **this paper was presented at the 3rd international conference on tropical biology 2018, 20-21 september 2018, bogor, west java, indonesia trees such as thick bark and needle-protected buds make them resistant to fires, fire events are still a threat to the national park (kutintara 2008; brown & smith 2000). a preliminary survey in this area showed that the plant community was dominated by widely scattered large pine trees at the top canopy, while no other species were found below the canopy layer. in addition, the forest floor was covered by dense grasses, a condition that indicated some limitations to the natural regeneration process. this forest structure could be associated with annual forest fires. evidences of other external pressures causing plant mortality and degradation, such as lightning and pine resin collection were also observed in this forested area. forest fires, natural mortality and anthophogenic pressures negatively impacted the natural regeneration process and nutrient cycling which could eventually cause forest 143 biotropia vol. 27 no. 2, 2020 + i r 1 i 1: " " , phclchabun province a/ road forest guard unit x head quater lj studty area figure 1 location of nam nao national park and study area degradation. if the situation persists for so long without proper management, the pine forest community might be replaced by a savanna ecosystem (cochrane 2009). although pine forests are fire dependent ecosystems, the fire resistant ability of the vegetation depends on suitable burning regimes such as; the fire frequency, fire intensity and fire severity of the burning area, which are critical factors determining the presence of pine trees in the ecosystem must be compatible with the ecological and biological processes of the plant community (cochrane 2009). this study aimed to examine the effects of fire on vegetation structure and composition, and natural regeneration within one year after burning occurred in a degraded pine forest and a mixed oak-pine forest of phu kum khao, nam nao national park, thailand (fig. 1). materials and methods the study was conducted in a degraded pine forest (pf), a savanna-like community, which is mostly covered by grass with very few pine trees and a mixed oak-pine forest (o-pf) in phu kum khao, nam nao national park, phetchabun, thailand (kutintara 2008). three 50 x 50 m experimental plots were set up in each forest type. within each plot, there were four 25 x 25 m sub-plots. fire breaks were constructed around all the experiment plots (fig. 2). the structure and composition of trees (dbh > 4.5 cm) were recorded in each sub-plot. furthermore, a 4 x 4 m (for sapling) and 2 x 2 m (for seedling) plots were established in each sub¬ plot, for the measurement of their diameter at trunk base (do), dbh and height for all saplings and seedlings (fig. 2). the individual canopy area and height of the burning scar at the tree trunk were also measured. the crown cover of the vegetation community was photographed using a digital camera with 18 mm fish eye lens, set at 1 m from the ground. burning experiments using the head fire burning technique were conducted in march 2012 by the fire control officers of petchabun province. prior to burning, a firebreak was setup around the 50 x 50 m plot and an ignition point was started based on the wind direction. species and the number of trees (woody plant that has 144 fire effects on the vegetation of thailand pine forests — wanthongchai and tarusadamrongdet 50 m seedling1is ee£i sapling 4 m 50 m& 4 m tree25 m 2 mm ff2 m 25 m fire break 10 m figure 2 plot layout for vegetation measurement dbh > 4.5 cm), saplings (woody plant that has dbh < 4.5 cm, height > 1.30 m), seedlings (woody plant that has height < 1.30 m) and ground flora (herbaceous, grass and climber) were re-examined 12 months after burning. fuel characteristics and fire behaviors during the burning experiment were also recorded (wanthongchai eta/. 2013). crown cover was calculated using the hemiview version 2.1 program. vegetation density, basal area, diameter class distribution, and shannon-wiener diversity index were also calculated (wanthongchai eta/. 2014). effects of fire on the structure and composition of plant community were then compared between the degraded pine forest and mixed oak-pine forest, during the pre-burn and post-burn period. results and discussion structure of plant community before the burning experiment before the experimental fire event (pre-burn), 30 and 25 tree species were found in the pf and the o-pf, respectively. the disturbance regime that has degraded the pine forest may have resulted in higher number of species. vertically, the pf vegetation structure consisted of 2 canopy layers. the dominant trees were finns kesiya with heights over 30 m, and widely scattered in the study area. the second dominant species were hill evergreen tree species with heights less than 10 m, such as craibiodendron stellatum and terminalia chebula. the vertical structure of o-pf consisted of 3 canopy layers, more than 30 m high where the p. kesiya dominated the top canopy. the middle canopy was dominated by uthocarpus grandifolius,quercus auricoma, dipterocarpus obtusifolius and aporosa villosa with height of about 15 20 m. the lower canopy included the smaller trees of the top and middle canopy and some other small tree such as fhyllanthus emblica, ardenia sootepensis and memecylon scutellatum. pf has a tree density of 304 trees/ha, while o-pf had four times higher tree density (table 1). seedling density in the pf was lower than in the mixed oak-pine forest. the degraded pine forest has a sapling canopy cover of 37.45% and was slightly higher than in the mixed oak-pine forest (31.48%). the higher percentage of sapling canopy cover and lower seedling density could be due to the annual fire disturbance. pre-burning survey of species diversity found a total of 47 tree species in these study plots (table 1). number of tree species was higher in the pf than in the o-pf. both forests showed similar number of saplings and seedlings diversity (shannon-wiener index = 2.81 & 2.83 and 2.08 & 2.44 for pf and o-pf, respectively). 145 biotropia vol. 27 no. 2, 2020 table 1 pre-burn vegetation structure and composition in the degraded pine forest (pf) and the mixed oak-pine forest (o-pf) at phu kum khao, nam nao national park, thailand structurea pf o-pf tree density (ind./ha) 304a (43.50) l ,327b (242.40) basal area (m2/ ha) 11.10a (0.53) 17.40b (1.96) canopy area (m2/ ha) 5,943.10a (630.16) 12,450.60b (1,210.14) number of species 30 25 shannon-wiener index (h) 2.81 2.08 sapling density (ind./ha) 6,667a (1458.30) 4,583a (1102.40) diameter at trunk base: do (cm) 2.45a (0.34) 2.38a (0.89) diameter at breast height: dbh (cm) 0.97a (.26) 1.23a (0.41) height (m) 1.87a (0.17) 1.98a (0.28) canopy cover (%) 37.45a (4.48) 31.48a (11.30) number of species 11 13 shannon-wiener index (h) 2.22 2.39 seedling density (ind./ha) 50,833a (16729.20) 70,833a (16414.80) diameter at trunk base do (cm.) 0.50a (0.04) 0.44a (0.13) height (m) 0.52a (0.05) 0.36a (0.04) canopy cover (%) 20.79a (11.13) 44.52a (16.14) number of species 22 19 shannon-wiener index (h) 2.83 2.44 notes: adifferent superscript (a, b) indicate significant differences (/-test, p < 0.05) of vegetation structures in the two forest types; standard deviation values are presented in the parentheses. effects of burning on the structure and composition of the plant community fire effects on tree mortality and density the data collected one-year after the burning experiment showed that mortality rates of trees in pf (4.59%) was significantly higher than in o-pf (1.15%). the tree species that died after the fire in the o-pf were q. auricoma, d. obtusifolius, g. sootepensis and p. kesiya. the tree species that died after the fire in the pf consisted of c. stellatum, pterocarpus macrocarpus, albfia odoratissima, s. ivallichii, protium serratum and p. kesiya. the d. obtusifolius trees which died after burning at the o-pf had smaller diameter than the average diameter of the same species. although the burning caused the death of some species in the experimental plots, the average tree densities in the pf before and after the burning experiment did not significantly differ. in contrast, the tree density in the o-pf before and after the fire significantly differed and reduced from 1,327 to 1,313 individual trees/ha (table 2). this indicated that tree species in degraded forests, where burnings frequently occur, had adapted to fire environment and therefore could tolerate fire events. table 2 tree density during the pre-burn and the one-year post-burn experimental period plot/forest types tree* density (trees/ha) pre-burning post-burning mortality (%) pf-1 375 356.25 5.00 pf-2 312.5 293.75 6.00 pf-3 225 218.75 2.78 average-pf 304.2a (43.5) 289.6a (39.7) 4.59a (0.95) o-pf-1 1,662.5 1,643.75 1.13 o-pf-2 856.25 843.75 1.46 o-pf-3 1,462.5 1,450 0.85 average-o-pf l,327.1a (242.4) l,312.5b (240.9) 1.15b(0.18) notes: adifferent superscript (a, b) across a row indicate significant differences (/-test, p < 0.05) of tree density before and after the burning experiments in each of the forest types; standard deviation values are presented in the parentheses; *tree (dbh > 4.5 cm). 146 fire effects on the vegetation of thailand pine forests — wanthongchai and tarusadamrongdet fire effects on saplings and seedlings density, diameter and height after the fire, sapling density reduction in the pf was higher than in the o-pf (table 3). the saplings that survived after the burning experiment tended to be taller and larger in diameter than the saplings before the fire. the mean dbh range was 0.6 2.4 cm and mean height range was 1.5 2.4 m. the statistical analysis suggested that fire did not significantly affect the vegetation structures of saplings (table 3). the estimated diameter and height growth rates were 0.4 0.8 cm/year, and 0.2 0.4 m/year, respectively. the density per area and canopy area of seedlings after burning were higher in both degraded pine forest and mixed oak-pine forest, while the diameter at trunk base and height of seedlings were lower. only the seedling height in the mixed oak-pine forest significantly decreased after the fire. however, no significant difference existed between the forest for all the other parameters. the high fire intensity variations during the burning experiment could account for the insignificant difference. the lower height of seedlings could be due to new seedlings replacement after the fire. fire effects on diameter classes of saplings and seedlings the number of post-burn saplings in each diameter class in the pf was lower than before the burning experiment (fig. 3a). no saplings were found in the large diameter class in the pf site during both preand post-burn period (fig. 3a). on the other hand, no clear pattern was found in the o-pf as the number of post¬ burn saplings in small diameter class (1.1 3.0 cm) was lower than those of pre-burn, while the number in several other diameter classes, particularly in the medium diameter class (3.1 7.0 cm), was higher in the post-burn. these increases could be attributed to the low fire intensity which minimized sapling mortality (fig. 3b). the seedling distribution was spread to more class sizes in both the pf (fig. 3c) and o-pf (fig. 3d). in the o-pf, number of seedling in smaller diameter-classes was higher than that in the larger diameter-classes. overall, the mean number of seedlings in the o-pf was lower in the post-burn period except for the diameter class 0.3 0.6 cm (fig. 3d). in pf, the number of seedlings in the small and medium diameter classes in the post-burn was higher than in the pre-burn. the disappearance of grass just after the fire might be due to forest floor opening that also stimulated new seedling recruitment. nevertheless, if the fire event occurs again the following year, these seedlings could be destroyed by the subsequent burning. table 3 preand post-burn structure of saplings and seedlings in the degraded pine forest (pf) and the mixed oak-pine forest (o-pf) at phu kum khao, nam nao national park, thailand structure a pf o-pf pre-burn post-burn pre-burn post-burn sapling density (ind./ha) 6,666.70a 2,500.00a 4,583.30a 3,541.70a (1,458.30) (360.80) (1,102.40) (1,041.70) diameter at trunk base: do (cm) 2.45a (0.34) 2.23a (0.55) 2.38a (0.89) 3.00a (0.72) diameter at breast height: dbh (cm) 0.97a (0.26) 1.00a (0.25) 1.23a (0.41) 1.67a (0.54) height (m) 1.87a (0.17) 2.13a (0.09) 1.98a (0.28) 1.90a (0.26) canopy cover (%) 37.45a (4.48) 28.57a (7.05) 31.48a (11.30) 31.72a (14.73) seedling density (ind./ha) 50,833.30a 74,166.70a 70,833.30a 79,166.70a (16,729.00) (20,531.10) (16,414.80) (14,529.70) diameter at trunk base:do (cm) 0.50a (0.04) 0.48a (0.05) 0.44a (0.13) 0.34a (0.07) height: ht (m) 0.52a (0.05) 0.38a (0.01) 0.36a (0.04) 0.28b (0.05) canopy cover (%) 20.79a (11.13) 72.18a (20.18) 44.52a (6.15) 49.32a (10.90) notes: ^different superscript (a, b) across a row indicate significant differences (/-test, p < 0.05) of the pre and post burning structures of sapling and seedling in each of the forest types. 147 biotropia vol. 27 no. 2, 2020 b 3 15 pf pre-burn pf post-burn o-pf pre-burn o-pf post-burn <1.0 1.1-3.0 3.1-5.0 5.1-7.0 do size class (cm ) >7.1 <1.0 1.1-3.0 3.1-5.0 5.1-7.0 do size class (cm ) >7.1 50 45 40 35 30i. o 20 g 15 10 5 0 50 d, iiihi-t ~a l t <0.3 0.3-0.6 0.7-10 1.1-1.4 do size class (cm ) >1.4 <0.3 0.3-0.6 0.7-1.0 1.1-1.4 do size class (cm ) >1.4 figure 3 diameter class distribution of saplings trunk base (do) during the pre-burn and one year post-burn period in the degraded pine forest (pf) (a) and the mixed oak-pine forest (o-pf) (b), and diameter class distribution of seedlings during the pre-burn and one year post-burn period in the degraded pine forest (pf) (c) and the mixed oak-pine forest (o-pf) (d) fire effects on seedling katio seedling ratio, which is a measure of the coppicing ability, is the proportion of total number of new sprout and number of seedling stumps in the experimental plots. rapid coppicing ability is one critical characters which enable plants to adapt to the fire conditions. high seedling ratio indicates that the plants have high coppicing potential. the seedling ratios in the pf and o-pf forests during the pre-burn were 1.1 and 2.5, respectively. changes in seedling ratio during the post-fire event in both study sites, however, did not significandy differ (fig. 4). the ratio of the number of newly sprouted shoots, or suckers, to the number of individual seedlings (seedling ratio) in the pf were significantly lower than the ratio in the o-pf. the more frequent burning in the pf might have resulted in the more frequent tree coppicing which consumes the stored energy. therefore, this reduction of stored energy could have resulted in the reduction of seedling ratio in the subsequent fire events (wanthongchai 2008). on the other hand, the forest fire in the o-pf was less frequent than that in the pf, which saved more stored energy for coppicing as a result of higher seedling ratio. however, if the fire events remain too frequent, the coppicing ability for many seedlings will decrease and seedlings eventually die. the capacity for vegetative propagation of the trees (as indicated by ratio of sprout to seedlings) increased on the less frequently burned sites after the burning experiment in the dry dipterocarp forest (wanthongchai et al. 2008). in contrast, on the site that burned too frequently, the number of sprouts decreased after the fire. 148 pf 3 2.5 2 1.5 1 0.5 0 se ed lin g ra �o o-pf pre-burn 1-yr post-burn fire effects on the vegetation of thailand pine forests — wanthongchai and tarusadamrongdet l figure 4 pre-burn and one-year post-burn seedling ratios of the vegetation in the degraded pine forest (pf) and the mixed oak-pine forest (o-pf) pine forests are fire dependent ecosystems. fire regime, consisting of fire types, fire frequency, fire intensity, fire season and fire severity (chandler et al 1983), is a critical factor affecting the growth process, development and natural regeneration of pine forest trees (kutintara 2008). this study, particularly, demonstrated that a fire event had different impacts on the plant communities in the degraded forest and in the mixed oak-pine forest. effects of fire characteristics/behavior on the burned sites most fires that occur in the pine forests of thailand affect the saplings and seedlings more than the large trees as the fires with low to medium intensity usually burn only the surface fuel. the fire characteristics had significantly greater impact in the pf stand than in the o-pf stand (wanthongchai et al 2013). burning at the o-pf and the pf were classified as low-intensity (48 kw/m), and medium-intensity (627 kw/m), respectively (table 4). during the burning experiment, the surface soil temperatures at all sites were higher than 250 °c. however, fire did not cause temperature changes in the deeper soil layers. several tree species in the study area were resistant to the fire because of their physical characteristics, such as, thick bark and fast coppicing ability. nevertheless, these pine characteristics are not yet developed in the sapling and seedling stages, but are likely to show when they are mature. therefore, fire resistance is positively correlated with tree age (whelan 1995). tree mortality observed in the pf was 5% and in the o-pf was 1%. tree mortality could be an indicator of fire intensity. higher mortality in the degraded pine forest could imply higher fire intensity as the dense grass covering the forest floor was highly inflammable. the more frequent fire events in the degraded pine forest have prevented natural regeneration, which may lead to a probable replacement of pine forest by a savanna ecosystem. therefore, an effective forest fire management that only allows occasional fire events is critical for the conservation and restoration of this degraded pine forest. the “ fire-free period” must be allowed long enough for natural regeneration to take place. for example, the appropriate burning frequency to maintain vegetation structure and site fertility in dry dipterocarp forest was 2 3 times per decade (wanthongchai et al. 2008). fire caused a reduction in the number of saplings in both degraded pine forest and mixed oak-pine forest, however, saplings reduction in the degraded pine forest was higher than in the mixed oak-pine forest, a condition which might be due to fire intensity. in a previous study, the fire intensity and flame height in a degraded pine forest (627 kw/mand 1.4 m) was higher than in the mixed oak-pine forest (48 kw/m and 0.4 m) (wanthongchai et al 2013). this higher fire intensity in the degraded pine forest also accounted for the decrease in sapling diameter in all the diameter classes. on the other hand, the low fire intensity in the mixed oak-pine forest could account for the increase in diameter of saplings in the larger diameter classes as the fire intensity was not high enough to kill the 149 biotropia vol. 27 no. 2, 2020 table 4 quantitative average of fire characteristics, flame temperature and soil temperature in degraded pine forest (pf) and pine-oak forest (o-pf) (wanthongchai et al 2013) fire characteristic13 stand pf o-pf head-fire rate of spread (m/min) 4.5a (±1.0) 0.9b (±0.2) back-fire rate of spread (m/min) 0.8a (±0.1) 0.3b (±0.1) average flank-fire rate of spread (m/min) 1.2a (±0.2) 0.5b (±0.01) head-fire flame height (m) 2.5a (±0.2) 0.6b (±0.04) back-fire flame height (m) 1.2a (±0.3) 0.2b (±0.1) average flank-fire flame height (m) 0.6a (±0.03) 0.2b (±0.1) flame length (m) 1.4a (±0.1) 0.4b (±0.1) fireline intensity (kw/m) 626.6a (±129.9) 47.9b (±23.0) heat release per unit area (kj/m) 288.6a (±22.9) 85.8b (±40.7) flame temperature (°c) 20 cm aboveground 586.5a (±29.1) 389.7b (±55.8) 50 cm aboveground 471.6a (±45.6) 301.2a (±64.5) maximum soil temperature (°c) soil surface 484.9a (±146.6) 255.2a (±105.4) 2 cm beneath the soil surface 24.1a (±1.3) 25.9a (±0.1) 5 cm beneath the soil surface 20.2a (±0.1) 23.6b (±0.3) 10 cm beneath the soil surface 19.2a (±0.3) 21.7b (±0.2) notes: "different superscripts (a, b) across a row indicate significant differences (p < 0.05) in the average fire behavior characteristics, flame temperature and soil temperature between degraded pine forest and pine-oak forest; flame length and fireline intensity were calculated using byram’s formula (byram 1959) ; standard errors are given in parentheses. saplings but stimulated their growth instead. the decrease in the number of saplings did not always imply sapling mortality as the new shoots grew but their sizes were not big enough to be considered as saplings. the decrease in sapling height could also be attributed to this phenomenon. for the seedlings, both diameterclass distribution and numbers were higher after the fire. this is probably due to the post-burn grass mortality, which provided space for the seedlings to germinate and grow. however, if subsequent fires occur the following year, these seedlings could also be killed by the fire. effects of burning regimes fire frequency is one ajor factor that greatly affects plant community structure and composition. excessively frequent fire occurrence will likely cause ecosystem degradation (kimmins 1997), a situation that is more commonly found in degraded pine forests than in mixed oak-pine forests. degraded pine forests are composed of scattered big trees, and low sapling and seedlings density, due to tall and dominant grasses (wanthongchai et al. 2013) the main fuel proportion in the pf stand was 45% grass and 44% litter whereas in the o-pf stands 55% was leaf litter. in the dry season, these grasses and litter dry up and become flammable fuel and increase forest fire intensity, destroying the trees, saplings and seedlings. this situation could further encourage the presence of fast growing grasses and weeds, especially the xerophytic species, and, hence, ready to ignite. if this grass-fire cycle persists, the degraded pine forest will eventually be succeeded by a savanna grassland (whelan 1995). this succession process was already observed in the degraded pine forest of phu kum khao. high fire frequency and intensity could also interfere with the natural regeneration process as they cause the death of saplings and seedlings. in the degraded pine forest of this study, p. kesiya grew very tall and continuously got struck and killed by lightning, a condition that will also contribute to ecosystem succession by a savanna grassland. therefore, an immediate fire protection protocol is needed to assist the natural regeneration and allow the pine seedlings and saplings to grow since they need time to adapt to the fire dependent environment (royal forest department 2008). in addition, as the degraded pine forest floor is dominated by grass that prevent seedlings from germinating and growing, it is also important to eliminate the grass and weeds. long term fire management to 150 fire effects on the vegetation of thailand pine forests — wanthongchai and tarnsadamrongdet secure the natural regeneration of seedlings and saplings is equally important. a suitable fire frequency protocol which will facilitate natural reproduction should be further studied. the post-burn fuel loads one year after the fire in the degraded pine forests were still lower than the pre-burn level indicating that a degraded pine forest at nam nao national park requires more than one year of fire-free period to recover back to the pre-burn conditions (wanthongchai et al. 2013). conclusion fire did affect the vegetation structure and composition of the degraded pine forest and the mixed oak-pine forest. fire caused a significantly higher tree mortality in the degraded pine forest than in the mixed oak-pine forest of phu kum khao, nam nao national park, thailand. the fire effects on the natural regeneration density, diameter and height was, however, not significant. this could be due to the highly varied fire behaviour in each experimental plot. however, the number of seedlings increased after the burning experiment probably due to more open spaces for the seeds to germinate and grow. diameter of seedlings and saplings were only slightly different while the seedling ratio (sprout: seedling ratio) in the mixed oakpine forest was higher than in the degraded pine forest. acknowledgements this study was supported by the thailand research fund (trf) and the office of the higher education commission, thailand. the authors would like to thank the national park, wildlife and plant conservation department for granting the permission to conduct this study in the nam nao national park, petchabun, thailand. the authors are also grateful to all the staff of the nam nao national park, and staff of the petchabun forest fire control station for their valuable assistance throughout the fieldwork. special thanks are also accorded to the department of silviculture, faculty of forestry, kasetsart university, thailand, for providing a vehicle and equipment and to the students assisting in the fieldwork and to miss orathai pongraktham for editing the english draft of this manuscript. references brown jk, smith jk. 2000. wildland fire in ecosystems: effects of fire on flora. gen tech rep rmrsgtr-42-vol.2. ogden (us): usda forest service. byram gm. 1959. combustion of forest fuels. in: davis kp, editor. forest fire: control and use. new york (us): mcgraw-hill. p. 61-89. chandler c, cheney p, thomas p, trabaud l, williams d. 1983. fire in forestry, volume i. forest fire behavior and effects. new york (us): john willey & sons, inc. cochrane ma. 2009. tropical fire ecology: climate change, land use, and ecosystem dynamics. berlin (de): springer. goldammer jg, penafiel rs. 1990. fire in the 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pseudomonas aeruginosa b031 isolated from a hydrocarbon phytoremediation field 1* 2 endah retnaningrum and wahyu wilopo 1faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia 2 department of geological engineering, faculty of engineering, universitas gadjah mada, yogyakarta 55281, indonesia received 31 january 2017 / accepted 29 january 2018 abstract the biosurfactants are used by several industrial sectors such as petroleum, agriculture, food production, chemistry, cosmetics, and pharmaceuticals. because of their hydrophobic and hydrophilic moieties, they have potency to reduce surface tension, interfacial tension between water-hydrocarbon systems, and low micelle concentration. their characteristics strongly depend on the producer strain as well as on the medium composition, such as carbon and nitrogen sources. this study was conducted to investigate the influence of different sources of carbon (n-hexadecane, glycerol and glucose) and nitrogen (urea, nh cl and nano ) for the production of biosurfactants by a new strain of 4 3 pseudomonas aeruginosa b031 isolated from a rhizosphere of paraserianthes falcataria l. nielsen, a hardwood plant species at a phytoremediation field. the biosurfactant characteristics of the strain were evaluated, particularly its surface-active properties and potential to remove hydrocarbon. glycerol was found to be the optimum carbon source, with rhamnose concentration, emulsification index, and critical micelle concentration (cmc) of 718 mg/l, 37%, and 35 mn/m, respectively. sodium nitrate (nano ) was observed as the optimum nitrogen source, with rhamnose 3 concentration, emulsification index, and cmc of 290 mg/l, 30%, and 24 mn/m, respectively. these biosurfactants efficiently reduced surface tension of culture broth from 42 mn/m to 31 mn/m for the glycerol treatment and from 37 mn/m to 24 mn/m for the sodium nitrate treatment. the crude biosurfactants from the glycerol and sodium nitrate treatments also removed 87.5% and 84%, respectively, of crude oil from sand. these rates were higher than those of the chemical surfactants (sds and triton x-100). these findings indicate that the biosurfactants produced by the strain from both glycerol and nano treatments can efficiently decrease the interfacial tension of culture broth 3 dilution and have a high emulsion index, thus hold promise in hydrocarbon bioremediation application. keywords: bioremediation, biosurfactant, glycerol, nano , optimum3 introduction biosurfactants are amphiphatic compounds produced by a wide variety of microorganisms that either adhere to cell surface or are excreted extracellular in the growth medium (al-bahry . et al 2013; geetha . 2018). they contain et al hydrophobic and hydrophilic moieties that reduce surface tension, interfacial tension between waterhydrocarbon systems, and low micelle concentration (zdziennicka & jańczuk 2018). as such, biosurfactants are used by several industrial sectors such as petroleum, agriculture, food production, chemistry, cosmetics, and pharmaceuticals (lai . 2009; pacwa-et al plociniczak . 2011). they could be used also to et al remove hydrocarbon contamination (mnif & ghribi 2015; ma . 2018).et al biosurfactants are composed of lipopeptides, glycolipids, phospholipids, fatty acids, neutral lipids, and polymeric biosurfactants (moya et al. 2015). often produced during the stationary phase of bacterial growth, they exhibit considerable substrate specificity (calvo et al. 2009). their characteristics strongly depend on the producer strain as well as on the medium * corresponding author: endahr@ugm.ac.id biotropia 5 2 8 130 139 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.2.808 130 composition, such as carbon and nitrogen sources (janek et al. 2010; liu et al. 2015). microbial strains belonging to species bacillus sp. (barakat . 2017), (rufino et al candida lipolytica et al rhodococcus et al. 2011), sp. (shavandi . 2011), pseudomas et al et alsp. (thavasi . 2011; sakthipriya . 2015) and (burgos-diaz sphingobacterium detergens et al. 2013) have been reported to produce biosurfactants. of these, the rhamnolipids biosurfactants produced by sp. is of pseudomonas interest in that it could remove hydrocarbon contamination (deepika . 2016; ma . 2016; et al et al ma . 2018). due to economic considerations in et al the industry, biosurfactants are commonly applied as whole-cells culture or crude biosurfactant. as reported by patowary . (2018), purification et al process of biosurfactants consumed almost 60% of the total production costs. a phytoremediation field in balikpapan, east kalimantan, indonesia, uses paraserianthes falcataria l. nielsen, a hardwood plant species, for hydrocarbon removal. previous studies show that plants for phytoremediation could remove hydrocarbon pollutants through mechanisms such as biodegradation, phytovolatilization, accumulation, and transformation (kong . et al 2016; guo . 2017). when soil hydrocarbon et al concentration in the phytoremediation field was measured in may 2014, it was found that the plants removed 50% of the hydrocarbon pollution within 3 months. in comparison, other hardwood plants, such as and , tectona grandis gmelina arborea are reported to remove only 10% and 15%, respectively (agbogidi . 2007; yenn . 2014). et al et al the differences in the hydrocarbon removal ability are influenced by the plant species, which create unique habitats for the soil microbial population (cristaldi . 2017). that is, the soil et al microbial population is strongly influenced by the plant species used for phytoremediation. the process of removing hydrocarbon in a phytoremediation field results from the complex interaction between roots, rhizosphere and soil microorganisms (khan . 2013; fatima . et al et al 2018). these association induce the particular physiology and biochemistry of plant roots, resulting in the process of hydrocarbons removal. as such, phytoremediation processes can make nutrients available, such as carbon and nitrogen (hou . 2015). glucose, a carbon source, is et al produced during photosynthesis, and is exuded into the soil by plant roots (khan . 2013). on et al other hand, the degradation process of hydrocarbon contamination in the soil environment induces n-hexadecane and glycerol, which are carbon sources also (varjani 2017). during the phytoremediation process, adding n fertilizer (e.g., urea, nh cl, nano ) enhances 4 3 hydrocarbon degradation because nitrogen becomes available to both plant and soil microorganism. a strain of pseudomonas aeruginosa b031, which was isolated from the rhizosphere of paraserianthes falcataria l. nielsen, probably has unique characters, as shown by thavasi et al. (2011) and sakthipriya et al. (2015), especially in terms of biosurfactant production. in this regard, it is impor tant to invest igate the medium compositions involving variations in carbon sources (n-hexadecane, glycerol, and glucose) and nitrogen sources (urea, nh cl and nano ) for 4 3 the production of biosurfactants by pseudomonas aeruginosa b031. the characteristics of the strain were evaluated, particularly its surface-active properties and potential to remove hydrocarbons. materials and methods biosurfactant-producing bacterial strain a biosurfactant-producing bacterial strain belonging to pseudomonas aeruginosa b031 was isolated from a hydrocarbon phytoremediation field in balikpapan, east kalimantan, indonesia. the strain was isolated and screened using methods described by hassanshahian and emitazi (2008). it was identified following the methods described in bergey's manual systematic of bacteriology (brenner et al. 2005). this bacterial isolate was then sub-cultured on luria bertani medium agar tubes as a stock culture before use as inoculum. inoculum propagation p. aeruginosathe strain stock culture of b031 was inoculated into a plate containing luria bertani and incubated at 37ºc. after 24 h, one loop of culture was inoculated to 50 ml of stone mineral salt solution (smss) medium in a 250 ml erlenmeyer flask. the culture was then incubated at 37ºc for 18 h and agitated at 200 rpm in a shaking incubator (thorsen . 2013). at the et al end of the incubation period, a culture sample 131 biosurfactants produced by pseudomonas aeruginosa b031 – retnaningrum and wilopo prepared using different concentrations of rhamnose (vinogradov et al. 2016). emulsification index (e24) the emulsification index of the culture samples was determined using the method described by panjiar et al. (2015). two ml of hexadecane and 2 ml of the cell free supernatant were mixed in a test tube and homogenized in a vortex at 3,500 rpm high speed for 2 min. the emulsification stability was measured after 24 h. the emulsification index was calculated as follows: e24 = x 100 height of emulsion formed (cm) total height of solution (cm) surface tension measurement the surface tension of the cell-free supernatant was measured in a k6 tensiometer (shimadzu), using the du nouy ring method. the values reported are the mean of three measurements (joshi & shekhawat 2014). critical micelle concentration (cmc) cell-free culture media of different carbon and nitrogen sources were diluted into various extents with sterile medium to analyze for surface tension (1/128, 1/64, 1/32, 1/16, 1/8, ¼, ½, and 1). the dilutions at which a surfactant begins to aggregate and had no further significant reduction in surface tension were assessed as cmcs. these measurements were estimated graphically by plotting the surface tension values versus the culture broth dilution values (cheng 2013). hydrocarbon removal ability the potential of the crude biosurfactant to remove hydrocarbons from contaminated sand was evaluated using the methodology described by aparna et al. (2012). sandy soil samples were collected from the parangtritis beach, indonesia; the samples were air dried and then sieved using a 2-mm mesh. furthermore, samples were mixed with tap water up to the moisture content 19 % (v/w). soil samples (20 g each) were polluted by 2 g of crude oil (pertamina, balongan, cilacap, indonesia), then transferred to 250-ml erlenmeyer flasks. the flask was added with 40 ml of the cell-free fermented broth and then was taken and adjusted with sterile smss medium in order to obtain a cell suspension of 0.7 od ( o p t i c a l d e n s i t y ) , d e t e r m i n e d b y a spectrophotometer. the number of bacterial cells corresponded to an inoculum of 10 cfu/ml. 7 the smss medium used for these experiments consisted of the following (g l ): 0.05% kh po , -1 2 4 0.1% k hpo , 0.05%, mgso .7h o, 0.01% kcl, 2 4 4 2 and 0.001% feso .7h o; the ph was adjusted to 4 2 7.0 by 1.0 m hcl (ph 7.2). optimization of medium and cultivation condition the influence of carbon and nitrogen sources on the biosurfactant yield and its properties was studied in the smss medium supplemented with 2% (v/v) of their sources. the carbon sources were n-hexadecane, glycerol, and glucose. the nitrogen sources were urea, nh cl and nano . 4 3 a 2% cell suspension of 0.7 od at 600 nm was inoculated into a 500-ml flask containing 100 ml smss medium. the culture was then incubated at 37ºc under agitation of 200 rpm. after 96 hours of incubation, culture samples were collected and further evaluated for bacterial biomass and biosurfactant characteristics. biomass and biosurfactant characteristics the culture samples collected previously were centrifuged at 36,000 g, temperature of 4ºc for 1 h. the pellet cells were dried overnight at 105ºc and then weighed to measure the pseudomonas aeruginosa b031 biomass. the crude biosurfactants in the cell-free culture medium obtained were charac ter ized in te r ms of rhamnose concentration and surface-active properties, including emulsification index (e24), surface tension, and critical micelle concentration (cmc). rhamnose concentration rhamnose concentration in the cell-free culture medium was measured using the phenolsulphuric method (seedevi et al. 2018). one ml of the cell-free culture broth was mixed with 0.5 ml of 80% phenol and 2.5 ml of concentrated sulphuric acid. after the mixture was incubated for 10 min at room temperature, the absorbance w a s m e a s u r e d a t 4 9 0 n m u s i n g a spectrophotometer. the rhamnose concentration was then calculated using a standard curve 132 biotropia vol. 25 no. 2, 2018 incubated at 37ºc under agitation of 200 rpm in a shaking incubator. after 18 h incubation, the sample was added wi th 120 ml of dichloromethane as the extracting solvent, then centrifuged at 10,000×g for 15 min. the amount of residual hydrocarbon in the sample was determined by gravimetric analysis (villalobos et al. 2008). for comparison, the hydrocarbon removal ability of sds, triton x-100 and distilled water (control) was also analyzed under the same conditions. statistical analysis all statistical analyses were performed using stat view for windows (sas institute, cary, nc, usa) based on p<0.05. the effects of the treatments and their differences were assessed using analysis of variance (anova) and duncan multiple range test (dmrt) method, respectively. results and discussion bacterial strain identification table 1 lists the morphological, cultural, and biochemical characteristics of the bacterial strain selected for this study. this isolate developed pale green pigmentation on asparagine medium and released a sweet grape-like odor. its colony was small, rough, and convex. the isolate was gram negative and showed motility under microscope investigation. the biochemical analysis showed that it had abilities on denitrification, gelatin liquefaction, and starch hydrolysis. it showed positive results in the oxidase and catalase tests. it could use glucose, fructose, and mannitol as carbon source to ferment, resulting in acid production. in addition, the isolate could grow in a temperature range of 30°c to 42°c and in a ph range of 5-9. according to bergey's manual of systematic bacteriology, along with the results of the analyses, the isolate was identified as pseudomonas aeruginosa et al . (brenner . 2005) effect of carbon on strain biomass and biosurfactant properties table 2 presented the bacterial biomass and characteristics of the biosurfactant produced by pseudomonas aeruginosa b031 using different carbon sources, such as glycerol, glucose and ncharacteristic observation* morphology colony color pale green colony type small, rough, convex cell shape rods gram staining negative motility + biochemistry denitrification + gelatin liquefaction + starch hydrolysis + oxidase test + catalase test + carbon utilization glucose + fructose + mannitol + culture temperature 30˚c + 37˚c + 42˚c + ph 5 + 7 + 8 + 9 + table 1 morphological, biochemical and cultural characteristics of the selected strain b031 * _note: + indicates growth of strain, indicates no growth of strain table 2 effect of carbon source on cell growth, rhamnolipid concentration, emulsion index, surface tension and final ph during biosurfactant production by pseudomonas aeruginosa b031 carbon source biomass (g/l)* ph* rhamnose (mg/l)* emulsion index (%) surface tension (mn/m)* n-hexadecane 0.3±0.01b 5.1±0.03a 449±19c 20±0.10.3b 37±0.3c glycerol 0.5±0.02a 7.0±0.02c 718±15d 37±0.2a 31±0.2b glucose 0.1±0.01c 3.4±0.01b 180±11a 10±0.01c 44±0.1a control 0±0d 7.2±0.01d 0±0b 0±0d 63±0d *note: data in this table are presented as mean ± standard deviation. their values with different superscripts in the same column are significantly different (p < 0.05). 133 biosurfactants produced by pseudomonas aeruginosa b031 – retnaningrum and wilopo hexadecane. as observed, the strain was able to use all the evaluated carbon sources to grow and produce biosurfactants. all the carbon sources were found to have significantly influenced bacterial biomass, ph, rhamnose concentration, emulsion index and surface tension. among the tested carbon sources, glycerol was found to be the optimum for biosurfactant production of the strain. its yield was higher than those reported by previous researchers who also used pseudomonas sp and obtained rhamnolipids (amani et al. 2013; sodagari et al. 2018). the highest cell biomass and rhamnose concentration among the observations were 0.5 g/l and 718 mg/l, respectively. these results were expected since glycerol as a carbon source is taken up more easily than the others. on other hand, the use of nhexadecane, which is a very complex and heterogeneous carbon source, resulted in the lowest biomass and biosurfactant yield. moreover, the biosurfactant yield was lower than the results obtained by varjani and upasani (2016) who reported that the pseudomonas aeruginosa ncim 5514 strain produced 1.450 mg/l of rhamnolipid when glycerol was added at 3% w/v as carbon and energy source. further, the biosurfactant produced by the strain using glycerol had the highest significant reduction in surface tension and formed a stable emulsion on cultivation media compared with nhexadecane and glucose (p<0.05). the surface tension and emulsion index values were 31 mn/m and 37 %, respectively. the use of glycerol also stabilized the ph condition, whereas the use of glucose and n-hexadecane resulted in a decreased ph. this lower ph value was probably due to the production of secondary metabolic acid (müller et al. 2012). critical micelle concentration (cmc), which indicates the efficiency of a surfactant, is another important characteristic to consider. to measure the cmcs of the biosurfactant under study, culture broths of three treatments (n-hexadecane, glycerol, glucose) were diluted to various extents with a sterile medium and analyzed for surface tensions (fig. 1). the point of inflection of the curve, which was obtained by measuring the surface tensions of the various dilutions, corresponds to the surfactant concentration equal to cmc. analysis of the cmcs values of the glycerol, glucose, and n–hexadecane treatments were 35 mn/m, 44 mn/m, and 41 mn/m, respectively (p<0.05). the lower cmc value of glycerol treatment indicates that glycerol is the most efficient carbon source among the three sources tested (kłosowska-chomiczewska . 2017). et al monteiro . (2018) reported that glycerol could et al be obtained abundantly and cheaply as a byproduct of biodiesel from animal fats and vegetable oil production. glycerol could be obtained also from trans-esterification of vegetable oils and fossil sources (petroleum, natural gas, coal). therefore, glycerol is a promising and abundant substrate for biosurfactant production. figure 1 changes in surface tension in culture broths of pseudomonas aeruginosa b031 at various dilutions using three carbon sources. arrows correspond to the dilution values that are equal to cmcs. vertical bars indicate ± standard deviation of means (n=3). 134 biotropia vol. 25 no. 2, 2018 effect of nitrogen sources on strain biomass and biosurfactant properties sodium nitrate was found to be significantly more effective than urea and ammonium chloride (p<0.05) (table 3). the data obtained imply that the use of sodium nitrate as nitrogen sources is better for supporting the growth of the strain and stabilizing the ph of the cultivation medium. these conditions induced a significantly higher surface tension reduction and the formation of a stable emulsion on the cultivation media using sodium nitrate treatment compared with the urea and ammonium chloride treatments (p<0.05). the results were in accordance with previous studies (ma et al. 2016). according to kryachko et al. (2016), the bacteria cell uses nitrates, ammonia, and amino acids as nitrogen sources to produce biosurfactant. during these processes, the no 3 have to be reduced to no and then to nh . the 2 3 nh can be assimilated either by glutamate 3 dehydrogenase to produce glutamate or, with glutamine, by glutamine synthetase to produce glutamine. glutamine and α-ketoglutarate are then transformed to glutamine by l-glutamine 2oxoglutarate aminotransferase. therefore, in -comparison with nh , the assimilation of no as 3 3 a nitrogen source is slower, simulating a nitrogenlimiting condition that is favorable to biosurfactant production (rizzo et al. 2017). the evaluation of the cmcs of the culture broths of three treatments (sodium nitrate, urea, and ammonium chloride) shows significant differences among the treatments (p<0.05) (fig. 2). the cmc values of the treatments were 24 mn/m for sodium nitrate, 35 mn/m for urea, and 43 mn/m for ammonium chloride. the reduction in surface tension and cmc of the biosurfactants is used as primary criterion for se l ec t ion of b iosur fac tant -produc ing microorganisms (thavasi 2011). this research demonstrated that the crude biosurfactant produced by the strain using glycerol and sodium nitrate as substrates decreased surface tension of the cultivation media by more than10 mn/m, thus they can be considered good surfactants (vijayakumar & saravanan 2015). as presented in fig. 1 and 2, surface tension decreased from 42 mn/m to a minimum value of 31 mn/m for the glycerol treatment and from 37 mn/m to 24 table 3 effect of nitrogen source on cell growth, rhamnolipid concentration, emulsion index, surface tension and final ph during biosurfactant production by pseudomonas aeruginosa b031 nitrogen source biomass (g/l)* ph* rhamnose (mg/l)* emulsion index (%)* surface tension (mn/m)* urea 1.8±0.01a 6.8±0.01a 220±19c 28±0.06a 35±0.04a nh4cl 1.0±0.01b 4.8±0.02b 90±15d 17±0.2b 42±0.5b nano3 2.5±0.02c 7.3±0.01c 290±11a 30±0.1c 24±0.1c control 0±0d 7.2±0.01c 0±0b 0±0d 63±0d figure 2 changes in the surface tension of culture broths of pseudomonas aruginosa b031 at various dilutions using three nitrogen sources. arrows correspond to the dilution values that are equal to cmcs. vertical bars indicate ± standard deviation of means (n=3). 135 note: *data in this table are presented as mean + standard deviation. their values with different superscripts in the same column are significantly different (p< 0.05). biosurfactants produced by pseudomonas aeruginosa b031 – retnaningrum and wilopo mn/m for the sodium nitrate treatment. these capacities are comparable to the outcomes of similar previous research wherein surface tension was reduced to a minimal value of 27-28 mn/m (petrikov . 2013). other studies had been et al carried out also on biosurfactants by . , p fluorescens for which the minimal value of surface tension was 27.5 mn/m (i̇kizler . 2017). therefore, et al this study showed that both glycerol and sodium nitrate are efficient and promising substrates for biosurfactant production from pseudomonas aruginosa b031. application of biosurfactants in hydrocarbon removal an increasing number of sites have been polluted by hydrocarbons become a seriously effect to ecosystem and human health critical. for removing these pollutions, environmentally and low-cost technology was highly required. because oil has low water solubility, which increase its sorption by soil particles, researchers have been exploring the use of biosurfactants to accelerate hydrocarbon removal from the contaminated sites (arslan . 2017).et al the use of crude biosurfactant to remove hydrocarbon contaminants is common in bioremediation technologies. therefore, this study investigated the application of the biosurfactant from b031 in its crude p. aeruginosa form, without prior costly extraction or purification steps. the experiment used cell-free supernatant containing the crude biosurfactant and chemical surfactants (sds and triton x-100) to verify the former's capacity to remove crude oil from sand samples. the biosurfactant produced b031, p. aeruginosa sds, triton x-100, and distilled water (control) showed significantly different (p<0.05) capabilities in removing crude oil from contaminated sand (table 4). the crude biosurfactants from both glycerol and sodium nitrate treatments yielded higher values (87.5% and 84%, respectively), but no significant difference (p<0.05) was observed between them. the removals obtained by sds and triton x-100 as synthetic surfactants at the same concentration were lower (73% and 71.6%, respectively), which that of distilled water (control) was only 30.2%. the results imply that the cell-free supernatant containing the crude biosurfactant was practically as effective as the isolated biosurfactant in removing crude oil, indicating the possible use of the unpurified biosurfactant to minimize production costs. the ability of biosurfactants to remove crude oil can be explained as follows: biosurfactants promote the transport of hydrophobic contaminants toward an aqueous phase through some particular interactions, resulting in emulsification and micellization, thus leading to their removal (costa . 2010). the results of et al the experiments indicate that these biosurfactants are more effective in oil recovery than the chemical surfactants (sds or triton x-100). these results were in accordance with the results of lai . (2009) who observed that et al biosurfactants such as rhamnolipids and surfactin are more efficient in removing hydrocarbon than tween-800 and triton x-100. therefore, these biosurfactants indicate good prospects for applications, especially for bioremediation. conclusion the biosurfactant produced by pseudomonas aeruginosa b031 was found to produce the highest biomass and the best surface-activities and emulsification properties when glycerol and sodium nitrate were used as carbon and nitrogen sources, respectively. the crude biosurfactants table 4 comparison of the hydrocarbon removal ability of the crude bacterial biosurfactant, sds, triton x-100 and distilled water (control) sample crude oil removal (%) crude bacterial biosurfactant from glycerol treatment 87.5±7d crude bacterial biosurfactant from nano3 treatment 84.±a 6d sds 73.0±3b triton x-100 71.6±2c distilled water (control) 30.2±2a note: * data in this table are presented as mean ± standard deviation. their values with different superscripts in the same column are significantly different (p < 0.05). 136 biotropia vol. 25 no. 2, 2018 produced by the strain were able to efficiently decrease the interfacial tension of culture broth dilution. these biosurfactants were capable of efficiently removing crude oil from sand samples, performing better than chemical surfactants, thus hold promise for hydrocarbon bioremediation application. acknowledgements we acknowledge financial support from boptn 2015 research grant of faculty of biology, universitas gadjah mada, yogyakarta, indonesia (contract number: ugm/bi/2460/ um/03/02). authors also thank ms sofianingtyas for her help in preparing the strain. references agbogidi mo, dolor ed, okechukwu em. 2007. evaluation of (linn.) and (roxb.) tectona grandis gmelina arborea for phytoremediation in crude oil contaminated soils. agric conspec sci 72(2):149-52. al-bahry sn, al-wahaibi ym, elshafie ae, al-bemani as, joshi sj, al-makhmari hs, al-sulaimani hs. 2013. 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retnaningrum and wilopo page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 biotropia vol. 28 no. 3,2021: 253 262 doi: 10.1 1598/btb.2021.28.3.1416 tree community structure and aboveground carbon stock of sacred forest i n pasaman, west sumatera santhyami'x*, ad1 basui(rtadi2, mufti petala patria' and rochadi abdulhadi' 'department ofbiolo~ edzlcation, faculg o f teacher training and education, universitas muhammad&ah szlrakarta, szlkoharj.~ 57162, indonesia 'department of biology, fa cult^ @mathematics and natzlral sciences, uniuersitas indonesia., depok: 16424, indonesia 3division ofbotay, herbarium bogoriense, research centerfar biology, lembaga ilmu pengetahaan indonesid, bogor 1691 i , indonesia received 20 august 2020 /accepted 7 september 2021 abstract this is an analysis of the composition and structure of tree community of bukit badindiang sacred forest in nagari simpang, pasaman, west sumatera province, indonesia. the study aimed to: 1. to obtain a representative account of the structure and composition of tree community of the sacred forest; 2. to estimate the aboveground carbon stock (c-stock) accumulated on the tree community. a one-hectare plot was divided into 25 subplots of 20 x 20 m each for tree and debris data collection. in each subplots, there were a 5 x 5 m sapling subplots. planting of 1 x 1 m seedling, understorey plant and litter subplots were nested inside. a total of 446 trees were recorded, representing 139 species from 49 families with a total basal area of 38.59 m2. the most dominant tree species was campnospemza anriculata pmportance value (iv) of 19.1 91. the other prevalent species were ficus benjamina (iv ~ 1 6 . 5 0 ) and mallotzls caudatus (iv ~14.78) . a total of 62 species (44.6o/o) was considered locally rare with density of 1 tree/ha. mallotzls candatus had the highest density (37 trees/ha) and ficus benjamina had the highest basal area p a ) (5.61 m2 = 14.51% of the total). euphorbiaceae (iv= 37.40) was the dominant family. the richest families were euphorbiaceae (10) and lauraceae (10). the total estimated aboveground c-stock was 190.62 mgc/ha, with the highest c was contributed by trees (178.85 mgc/ha or 93.8% of total). trees with diameter class of 10 69.99 cm stored 62% of total tree carbon. the species richness, tree density and c-stock of bakit badindiang sacred forest were relatively higher than those in several disturbed lowland forests in sumatera. keywords: carbon stock, community structure, sacred forest, tree species diversity, west sumatera introduction local wisdom has always been involved in preserving biodiversity in indonesia, shown in numerous forms such as sacred forest (hutan larangan) (wadley & colfer 2004), cultural landscape (christiawan 2015) and traditional harvesting right (boedhihartono 2017). west sumatera province is among regions in indonesia in which sacred forest is still practiced. this paper will enlighten the potential of bukit badindiang sacred forest, a customary forest (hutan adat) of nagari sirnpang, simpang alahan mati (simpati) district, pasaman region in west sumatera province, indonesia. *corresponding author, email: san91 s@ums.ac.id sacred forest is one of the oldest forms of natural forest conservation by traditional community. generally, sacred forest is a part of the natural forest ecosystem with high level of trees and animals diversity. in some cases, some prominent features such as water spring, fountain, and even oldest tree could be found in a sacred forest. they provide habitats and space for infrequent and threatened species of trees and animals and have a vital conservation value despite of their characteristically small areas (boraiah et al. 2001). some scholars have studied the h k between 'the sacred' and biodiversity preservation of natural forests all over the world (negi 2015; allendorf et al. 2013; ormsby et a l 2010). for instance, sacred groves become a refuge spot for mammals, birds, plants, and biotropia vol. 28 no. 3,2021 other forest-dwelling animals (basu 2000; chandran & hughes 1997; sinha 1995). sacred forest is also considered as carbon sink (pradhan e t all. 2016; pala et all. 2012). on the contrary, local community also depends on sacred forest for various products used in daily life (burman 2003). the aims of this study were: 1. to obtain the fundamental descriptive account of floristic structure and composition of bukit badindiang sacred forest under the management of customary law in nagari simpang, simpati district, pasaman, west sumatera province in conserving tree species diversity and 2. to estimate the capacity of sequestering carbon. an improved knowledge of basic ecological data, including floristic composition and c-stock potential of the forest is required for the development of natural ecosystem conservation and forest management. to date, there are relatively few detailed descriptions and quantitative assessments and estimation of customary and sacred forest floristic and c stock in indonesia qndrajaya 2013; bpk 2012; soendjoto et al. 2009). bukit badindiang is one of the forested areas in pasaman known as 'walled hill' as collecting timber from this site is restricted. bukit badindiang belongs to i<oto tribe who establishes this forest as 'sacred' area as the water fountain, a source of clean water for the whole village, is located in this forest. sacred means: 1. public (outside of i<oto members) may only collect non-timber forest products such as firewood, fruits, honey, medicine, and resin; 2. timber can only be harvested by or as the permission of koto leader for the community mosque construction and emergency needs; 3. not to fell 'kayzi t z i n ~ a babeleang' means it was forbidden to cut down the low abundance trees. analysis of species inventory data collected in a one-hectare plot in this paper will be limited to the representation of the forest with reference to the key structural parameters such as pattern of relative abundance, species richness, and family structure and estimated aboveground c-stock. materials and methods sampling location and sampling samphg was carried out in simpati district, located at 100°08' 100°12' e and 00"04' n 00"03' s with an altitude of 100 890 m above sea level (fig. 1). the topography of nagari simpang is a plain land surrounded by hills of natural forest. temperature ranges from 27 30 "c with a mean annual rainfall of 3,891 mm/year. nagari simpang is situated in the wet region belonging to the rainfall type a climatically (schmidt & ferguson 1951). the monthly rainfall ranges from 132 mm to 400 mm and the mean rainy days range from 4 days to 20 days. the soil of the area belongs to the litosol and red yellow podzolic categories (bps pasaman 201 1). the study site was a hilly area in the northeast part of simpati district. figure 1 a. west sumatera province; b. pasaman region; c. study site (source: google maps 2018) tree community structure and aboveground carbon stock of sacred forest santhyami e t al. data collection was conducted using quadrat biology, lip1 in cibinong, indonesia. the methods (cox 1967; mueller-dombois & identification and nomenclature of each tree ellenberg 1974). the design and size of plot species followed the tree flora of indonesia: implemented in this study followed bsni (2011) checklist for sumatera (txktmore & tantra using normative references based on cop-15 1986) and flora malesiana (steenis e t al. 1948 decisions on redd + methodology guidance 2013). (dec. 4/cp-15), ipcc 2006 guidelines for national greenhouse gas inventories and ipcc data analysis 2003 good practice guidance for land use, land use changes and forestry, as this study was also aimed to estimate the aboveground c-stock. a nested plot design of one hectare was established subjectively in the most accessible area of bukit badindiang. the plot was further divided into 25 trees and debris subplots of 20 x 20 m each. the size of the plot and subplots were as follows: seedlings (woody tree species with leaves more than two strands and a height of < 1.5 m); understorey layer species (non-ligneous and ligneous herbs, ferns, shrubs and tree species with heights d . 5 m); and litter with a minimum area of 1 m2, saplings (dbh < 10 cm and a height of > 1.5 m) with a minimum area of 25 m2 and trees (dbh 210 cm) and debris with a minimum area of 400 m2. (dbh = diameter at breast height). samples collection the entire tree individuals in the 400 m2 plot and saplings in 25 m2 plots were numbered and collected. dbh of all trees and saplings were measured. seedlings and understorey layer species in the 1 m2 plot were all cut and mixed. wet weight was measured. then as many as 300 g of sample were taken to measure the dry weight. litter was collected in 1 m2 subsize. au of the collected litters were filtered with a 2 mm sieve and then ovendried at 70 "c until reaching constant weight. to determine the biomass of debris, the diameter and the length of all debris in the 400 m2 plots were measured. herbarium specimen collection and identification collected herbarium specimens of all species w i t h the plots were identified at the herbarium bogoriense, research center for the total of density, frequency, and dominance can be used to signify the importance of a species. it is expressed in terms of relative values. relative density (rd), relative frequency r ) and relative dominance (rdo) were then calculated based on cox (1967) and mueller-dombois and ellenberg (1974). the index of species diversity (h3 was calculated by using shannon-wiener index (swi) (shannon & wiener 1963). species dominance (cd) was calculated following the index by simpson (1949). aboveground c-stock in one type of ecosystem or habitat is expressed in mg/ha. the total biomass in the plot (mg/ha) is converted to c-stock (mgc/ha). aboveground biomass of tree was estimated by using specific allometric equation based on dbh. the allometric equations used in this study was developed by icetterings e t al. (2001) specifically for woody trees on sumatera secondary forest. also, debris biomass was calculated by using equation by nascimento and laurance (2002). results and discussion species composition and structure in one hectare plot, there were 466 trees recorded, representing 139 species and 49 families with a total basal area of 38.59 m2. each of 62 species (44.6%) had only a density of one tree/ha (fig 2). h' was 3.13 and species dominance (cd) was 0.64. table 1 shows that campnosperma aumculata, ficus beyamina, mallotus caudatw and a?-tocaqus iancezfolius were four most important species, with iv > 10. biotropia vol. 28 no. 3,2021 1 2 3 4 5 7 8 9 10 13 14 17 18 19 21 37 number of individuals figure 2 number of species and number of individuals of trees in a one-hectare plot of forest in simpang, alahan mati, west surnatera province. tabel 1 the relative density (rd), relative frequency (rf), relative dominance (rdo) and importance value (iv = rd +rf+ rdo) of the ten most important species based on iv no species family rd (?/o) rf (yo) rdo (0'0) iv (?lo) campnosperma auriculata ficus bevamina mallotus caudatzls a7itocaqu.r lancefolius sjqygium newosum castanopsis tungumt sy~g ium aquem quercus argentata mallotus mollissimzis reinwardtiodendron humile anacardiaceae moraceae euphorbiaceae moraceae myrtaceae fagaceae myrtaceae fagaceae euphorbiaceae meliaceae species density, dominance and distribution density and frequency. there were only four within plot varied. nine out of ten species with species with f > 30%. euphorbiaceae was the highest density were also species with the the most important family followed by highest frequency, but they were in different anacardiaceae and moraceae (table 3). orders (table 2). m. cazldatz~ had the highest tabel 2 ten species with highest frequency (f), density @) and basal area (ba) species group species a ten species with highest mallotzls caudattl~ (37), s_y.ggim neruosum (21), campnospem aumi.ulata (19), s_y.ggium density (number of aqueum (1 8), artacarpw lanceifolius (1 7), mallotzrs mollissimus (14), castanopsis tungurrut tress/ha) (1 3), reinwardtiodendron hamile (1 o), barringtonia conoidea (9), meio~ne uirgata (9) b ten species with highest mallotus caudatus (0.72), q ~ g i u m neruosum (0.52), artocalpus bnceifolius (0.4), sy.ggium frequency (yo ) aqueum (0.4), mallotzls mollissirnus (0.32), reinwardtiodendron hamile (0.32), castanopsis tzmgzumt (0.28), meio~ne uirgata (0.28), quercus argentata (0.28), campnospem auriculata (0.28) c ten species with highest ficus benjamina (5.6), campnospemza auriczlata (5.27), artocalpw lancefolius (1.54), basal area (m2) quercus argentata (1.39), castanopsis tuugurrut (1.29), antidesma leucopodum (0.88), styrax benxoin (0.82), mang@rafoetidd (0.8), nephelium juglandifolia (0.791, mallotus caudatus (0.68) d ten species with highest campnospem auriculata (1 9.1 9), ficus benjamina (1 6.5), mallotzrs caudatus (14.78), importance values (yo) artacarpw lanceifolius (1 0.47), syggium nervosum (9.42), castanopsis tungumt (8.12), q.ggium aqueum (7.85), querczls argentata (7.50), mallotus mollirsmus (6.02), reinwarntiodendron humile (5.79) tree community structure and aboveground carbon stock of sacred forest santhyarni e t al. tabel 3 summary of family characteristics at tree level characteristics family eleven families with number of euphorbiaceae (lo), lauraceae (lo), meliaceae (8), malvaceae 0, moraceae (7 , species > 5 myrtaceae (7), dipterocarpaceae (6), phyllanthaceae (6), anacardiaceae (5), annonaceae (5), fabaceae (5) ten families with highest euphorbiaceae (85), myrtaceae (53), anacardiaceae (42), meliaceae (291, moraceae density (tress/ha) (27, fagaceae (26), lauraceae (25), annonaceae (1 6), malvaceae (16), styraxaceae (14) ten families with highest basal moraceae (7.65), anacardiaceae (6.40), fagaceae (2.76), euphorbiaceae (2.05), area (m2) meliaceae (2.04), dipterocarpaceae (1.63), lauraceae (1.62), myrtaceae (1.38), styraxaceae (1.35), phyllanthaceae (1.22) ten families with highest euphorbiaceae (37.40), anacardiaceae (33.21), moraceae (31.26), myrtaceae importance values (iv) (24.56), meliaceae (1 8.291, fagaceae (1 7.81), lauraceae (1 5.77), malvaceae (1 0.83), dipterocarpaceae (10.68), styraxaceae (9.33) 60 al .so 41 40 & 30 n 5 2018 z 10 10 0 i i o : basal area class (m2) diameter class (cm) figure 3 a. number of species according to the ba classes and b. the number of trees according to the diameter classes the total basal area for all species was 38.59 m2, with the mean ba of 0.28 m2. f. beyamina had the highest ba (14.51% of the total). a total of 106 species (76.3%) had ba of < 0.3 m2 (fig. 3a). a hgh proportion of trees in the samples was represented by small indviduals. trees with dbh < 30 cm had the highest number, i.e., 377 indviduals (72.3%) (fig. 3a). this study represents an ecosystem of a lowland rainforest under the customary conservation sense in west sumatera province. it presents a collected data set systematically that can be compared to similar plot inventories of other tropical rainforests in indonesia. table 4 compares tree species diversity of this study with other natural forests in sumatera. the species richness and tree density in customary forest of nagari simpang, pasaman was higher than those in several lowland forests in sumatera, but lower than one on batang gadis national park (np) north sumatera, considering that the ecological status of batang gadis np as undisturbed protected forest (kartawinata et al 2004). heretofore, the only other floristically quantitative description of natural forest in pasaman was published by yusuf e t al. (2005) in rimbo panti. tree density in ths study was higher compared to rimbo panti. lowland forest variations are related to terrain, topography, and degree of natural or anthropogenic dsturbance. tree density on the ridge is relatively higher than on the flat location wme the basal area is lower (whtmore 1986). the topography of b b o panti forest was relatively flat compared to the steep buht badindiang with average slope of 34.2". another factor is human pressure. it should be noted that although h m b o panti status was as a nature reserve, the forest was sull experiencing the pressure from the human. there were sull found some open canopy due to logging and replaced by secondary tree species such as o m a h n t b a a e and macaranga tanam'as. species with the highest iv was c. aam'calata. it is a mid-canopy tree in undisturbed mixed dipterocarp and freshwater swamp forests. species in this genus are generally strongly light biotropia vol. 28 no. 3,2021 demanding even when young, and regenerate more abundantly in more open or disturbed habitats. on the study site, they were found mostly on relatively flat spots on the top of the hill. it can be assumed that those species grew great in the past throughout the phase when canopy gaps were established in consequence of natural disturbances and human activities. the land-use histories of bukit bandindiang showed that before 1970 this forest was used to be a shortcut to get to bonjol subdistrict. i<oto people planted many hnds of economically valuable trees such as parkia speciosa, dario ebenthinas, and archidendron paaciflomm on the opened flat area. the existence of these trees is proof that some parts of this forest were disturbed. another interesting species highlighted is m. caadatw. although it was only the third rank in terms of iv, these trees were recorded as species with the highest density and frequency on the sampling unit. it means that m. caadatus was the species with the highest number of inkviduals and could be evenly found on most plots at the same time. generally, m. caadatzls can be found on understorey of forest, riverine forest, kerangas forest, selectively logged forest, mainly on undulating to steep land and ridges of m s ; soil yellow to brown sand or red clay. the fruiting and flowering seasons of it is throughout the year (welzen & sierra 2006). it explains why this species could be easily found on the study site. the higher value of the diversity indices represented the greater dversity and the abundance of tree species (adekunle et al. 2013). swi for tree species diversity in this study was 3.13. this values showed a relatively high number of tree species diversity based on the diversity index criterion by soegianto (1 994). there are sixty species or more than 40% of the total species found on the site had a density of one tree/ha. this number is relatively high compared to a similar study on buht duabelas np, jambi that only have forty-four species (30% of total species). this result was strongly related to the local wisdom that prohibits people to cut trees with low abundance. furthermore, among sixty species recorded, there were five species included in the iucn red list, i.e., apomsa sp., lophopetalm wightidnm, para~horea bcida, pmnm arborea, and xanthopbllzlm sp. the forest at the family level was dominated by euphorbiaceae. the result is similar to the prior study conducted at h b o panti. in the tropical region, the family of euphorbiaceae is found to be superior to other families in most cases, especially in disturbed forest. species on this family are known to have good adaptability to any type of forest (whitmore 1989). another interesting family is dipterocarpaceae. despite a low number of individuals found on the site, dipterocarpaceae was on top ten f a d e s with the highest iv because of their averagely wide basal area. the two most significant species of dipterocarpaceae found were dipterocarpas baadii and shorea hopeifoh. both are included in iucn red ls t as vulnerable and critically endangered criteria, respectively. the forest investigated in this study was characterized by a high abundance of small trees with dbh < 30 cm. t h s result is similar to other disturbed forests in sumatera (yusuf et al. 2005; hadi et al 2009; rahmah et al. 2016). the relatively small number of large trees can be explained by two main causes. there was a possibility that there might be a restricted number of species that authentically matured to these diameters (hartshorn 1980) and their seedlings require to meet optimal circumstances of environment or locations for growth, to out compete other species, the fast-growing one particularly. another cause was the abundance of certain big tree species (i.e., dipterocarpaceae) could have been already reduced by selective logging for human needs. as the timber harvesting in buht badindiang was restricted to only a few concerns, we consider natural causes also to play a significant role in restricting the number of big trees in our study area. this assumption is based on the existence of fallen decaying big trees on the forest floor and tree stumps and other wood remains resulting from natural cutting. it is also shown in relatively high percentage of estimated debris biomass found on the plots fable 5). tree community structure and aboveground carbon stock of sacred forest santhyarni e t al. tabel 4 comparison tree diversity in several natural forest in sumatera locality methods area of number number number source plot (ha) of trees of species of families west surnatera province nagari simpang, pasaman, quadrat i 466 139 49 present study west sumatera province rimbo panti natural forest, quadrat 323 114 yusuf et al. 2 pasarnan, west sumatera province 41 (2005) siberut, west sumatera belt province transect hadi e t al. (2009) other forest on sumatera belt 4 hatan adat kampar, riau transect rernina (201 7) 5 bukit duabelas np, jambi quadrat 414 113 rahmah e t al 38 (2016) mt. leuser np, north quadrat 687 110 samsoedin & sumatera 34 heriyanto (2010) batang gadis np, north 1 583 205 iwtawinata e t al. quadrat sumatera 41 (2004) aboveground c-stock the total aboveground c-stock estimated was 190.62 mgc/ha (table 5), with the highest c was contributed by trees. f. benjamina, c. auriczlata and a. lanceifohus were the top three species that contributed c (44.6% of total carbon from trees). tree diameter class of 150 199.9 cm stored the highest amount of carbon (42.54 mgc/ha) p ig 4). tree with diameter class of 60 69.99 cm placed the second rank with c stock of 39.04 mgc/ha. this class was filled by 14 species from a varied family with a dominant c accumulator was c. aum'culata. it accumulated nearly 33% of total c-stock in that class. trees with a diameter class of 10 69.99 cm stored 62% of total tree carbon in a hectare, followed by trees with a diameter class of 150 199.99 cm (24%) and 70 100 cm (14%). tabel 5 summary of total aboveground c-stock based on carbon pool total tree total carbon carbon pool biomass @%/ha) biomass (mgc/ha) tree 357.70 178.85 sapling 5.72 2.86 seedling 0.46 0.23 litter 1.24 0.62 debris 16.13 8.07 total 381.25 190.62 d 9 6 ' 6 ' > 8 , . b ~ o o d o \ d o , 9 6 ' 9 @ o \ > s, s, $%, jsg "99 9, 9, "99 9, 9, 9, 9, 9, ??9, "99, diameter class (cm) figure 4 distribution of tree carbon biomass based on diameter class biotropia vol. 28 no. 3,2021 estimated c pools in several forest types can be referred to in taking decisions about c management within forests. parameters to measure the carbon dioxide binding level on a type of land-use are tree biomass (mg/ha) and carbon biomass (mgc/ha). it was estimated that the sacred forest of nagari simpang pasaman stored 190.62 mgc/ha. the value estimated on this study is relatively comparable to other c stock assessment of sumatera natural forest. van noordwijk e t al. (2002) assessed secondary forests in sumber jaya, lampung with an average estimated carbon of 195 mgc/ha. laumonier e t al. (2010) have also estimated aboveground c-stock land-scape of several dipterocarp forests of sumatera, amounting 180.5 mg/ha (ranging between 270 480 mg/ha). the forest can act as a source and a sink based on the level of succession, specific type disturbance, or level of human intervention (masera e t al 2003). on this study site, the small to medium trees contributed more agb than large trees. 62% of carbon was stored by trees with chameter class of 10 69.99 cm. as on this stage, the tree grow vigorously, accorbgly with the prompt accumulation of c-stock. however, the conservation of mature large trees with chameter >i50 cm is highly suggested considering 24% of carbon on ths forest was accumulated on these trees. bukit badindiang forest is preserved by local communities for conservation and religious purpose. local government established bukit badindiang as a customary forest. the result of this study can serve as a baseline for the implementation of carbon-based project activities. community-based sacred conservation management of this forest attempts to avoid deforestation and encroachment. forest area on this district is under severe threat from the construction of gold mining projects. the surrounding community depends on forests mostly to meet their needs for timber and non timber forest products. conservation of bukit badindiang forest will lead to increased exploitation of surroundmg unprotected forests if conservation strategies are not proceed and implemented in those areas. conclusion in one hectare plot, we recorded 466 trees, representing 139 species and 49 families with a total basal area of 38.59 m2 with estimated aboveground c-stock of 190.62 mgc/ha. it can be concluded that the sacred forest of bukit badindiang was a disturbed forest structurally and floristically with heterogeneous species composition. it is reflected by low frequency and density of the species. the species richness and tree density were relatively higher than those in several other disturbed lowland forests of sumatera. the mixed composition of the forest studied provides numerous important ecological niches and emphasizes the outstanding contribution of this forest to the biodversity of the region. bukit badinchang forest can provide models for sustainable uthzation of forest resources in the region. further research on structure and composition of forests of other community-based conservation over a more comprehensive area in the region are needed. acknowledgements this study has been supported by a grant from kemristek dikti. thanks to iceltan and gapoktan member and staff in both pasaman and padang pariaman for the access and permit to make plots and to the following people for field data collection and analysis: erna, robi, nita, aldo and isul. we also thank universitas negeri padang for providing some tools used during fieldworks. references adekunle vaj, olagoke ao, akinele so. 2013. tree species diversity and structure of a nigerian strict nature reserve. tropical ecology 54:275-89. allendorf td, yang j. 2013. the role of ecosystem services in park-people relationships: the case of gaoligongshan nature reserve in southwest china. biol conserv 167:187-93. badan pusat statistik (bps) pasaman. 2011. statistic of simpang alahan mati subdistrict. available from: https://pasamankab.bps.go.id/publication.html accessed february 14,201 8 pndonesia]. tree community structure and aboveground carbon stock of sacred forest santhyami e t al. badan standar nasional indonesia (bsni). 2011 biomass in mixed secondary forests. for ecol measurement and calculation of c-stocks field manag 120:199-209. measurements for forest c-stock estimation (ground-based forest carbon accounting). jakarta (id): bsn. 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[indonesian]. 7. norman arie prayogo et al.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 biotropia vol. 29 no. 3, 2022: 225 233 doi: 10.11598/btb.2022.29.3.1669 225 cadmium, nickel, and lead concentration of municipal dumpsite in western samar, philippines mariss bostrillo varona1, judy-ann ligo amistoso1 and pearl aphrodite bobon-carnice12* 1department of natural sciences, eastern visayas state university, tacloban city 6500, philippines 2office of research and development, eastern visayas state university, tacloban city 6500, philippines received 21 september 2021/accepted 8 march 2022 abstract heavy metal is one of the major problems due to its accumulation from the soil to the food chain, wherein dumpsites are the primary sources of heavy metal pollution. this study aimed to determine the presence of heavy metals in the soil of santa rita, western samar dumpsite and to quantify them to obtain knowledge on the possible high contamination that may affect the surrounding areas. this study focused on the presence and concentrations of heavy metals cd, ni, and pb. eighteen (18) soil samples were acquired within the three sampling sites: shoulder slope, main dumpsite, and foot slope. each sampling site has three sampling points with a depth of 0 30 cm and 30 60 cm. analysis showed that all heavy metals are present in the dumpsite, and the concentrations ranged from 0 0.1 mg/kg, 0.09 3.7 mg/kg, and 0.09 3.7 mg/kg for cadmium, lead, and nickel, respectively. in comparing heavy metals within the sampling sites and depths, only cadmium has a significant difference, while ni and pb have no significant difference. compared with who standards, all heavy metals tested still fall within the standard limit. therefore, the dumpsite is still at a safe level. however, residents should take measures to maintain the soil quality since heavy metal contamination in dumpsites is likely to exacerbate. keywords: cadmium, lead, municipal dumpsite, nickel, soil pollution introduction one of the global issues is improper waste management. open dumping and open burnings are the most implemented waste treatment and final disposal systems, mainly available in lowincome countries (ferronato & torretta 2019). the increased population and the rising demand for food and other essentials equate to increased waste generated daily in each household, wherein this waste eventually dumps in dumpsites and impacts the soil and the surface environment (mekonnen et al. 2020). uncontrolled disposal results in severe heavy metal contamination (vongdala et al. 2019). further, dumpsite operation is a source of heavy metal pollution that can affect the biosphere (sakawi et al. 2013). heavy metals naturally occur in the earth's crust with high atomic weight and density at least five times greater than water (tchounwou et al. 2012). heavy metal is one environmental contaminant affecting aquatic and terrestrial ecosystems (sakawi et al. 2013). heavy metals possess a severe risk because they tend to bioaccumulate, which means an increase in chemical concentration in a biological organism over time compared to the chemical concentration in the environment (helmenstine 2021). sources of heavy metals include mining and plating, fertilizers and pesticides, sludge dumping, and municipal waste (mudgal 2010). the liquid that exudes and percolates solid wastes eventually transfers to the soil. heavy metals, pesticides, and hydrocarbons are threatening substances that bind in liquid and constantly contaminate soil and water (adelekan & alawode 2011). dumpsite of santa rita is located at sitio canonay of barangay rosal, santa rita, western samar. the dumpsite is already 20 years old, and all kinds of waste are accepted from all *corresponding author, email: pearl.carnice@evsu.edu.ph biotropia vol. 29 no. 3, 2022 226 barangays of santa rita, western samar. the municipal government of santa rita owns the entire area of the dumpsite. the main dumpsite is 45 m from the nearby residences and 22 m away from the rice plants in sitio canonay. this study aimed to determine and quantify the concentrations of heavy metals present in the santa rita, western samar dumpsite soil and provide knowledge about the possible high contamination of heavy metals that may affect the surrounding areas. materials and methods study site the study was conducted in santa rita, western samar, where the dumpsite is located at sitio canonay of barangay rosal (fig. 1). the dumpsite was divided into three sampling sites: shoulder slope, main dumpsite, and foot slope, wherein three sampling points in each sampling site were randomly selected. the dumpsite comprises verdant hills covered with grassland and forest vegetation on the shoulder slope, a bulk of wastes that covers the main dumpsite with ipomoea aquatica (water spinach) plantation that surrounds some parts of the area, and a rice field that could be found at the foot slope of the dumpsite. sample collection, preparation, and heavy metal analysis a descriptive analysis of research was utilized in this study. each sampling point was dug up to at least 60 cm depth. then, two (2) composite samples in every sampling point were collected according to their depth (0 30 cm and 30 60 cm), wherein 18 soil samples were acquired within the three sampling sites. after sampling, all soil samples were air-dried, crushed using a mortar and pestle, sieved through a 2-mm sieve, and stored in a clean, resealable, properly labeled bag. figure 1 location map showing the sampling points cd, ni, and pb concentration of municipal dumpsite in western samar, philippines – varona et al. 227 soil samples were digested using the dtpa acid extraction solution. the digested soil samples' cd, ni, and pb concentrations were analyzed using atomic absorption spectrophotometry (aas) equipped with pb hollow cathode lamp (varian spectraaa 220 fs with sips pump unit and autosampler sps-5). standard solutions of the heavy metals were utilized in the preparation of the calibration curve. the following formula was used to compute pb, cd, and ni concentration in the dumpsite soil. heavy metals in soil (ppm) = ml of extraction solution x conc. (ppm) grams of soil where: ml of extraction solution = volume of extraction solution grams of soil = weight of soil sample in grams conc. (ppm) = result of aas concentration in mg/l statistical analysis two-way analysis of variance (anova) was used in this study to determine the significant variation in the mean concentrations of identified heavy metals in soil concerning sampling depth and sampling site. natural logarithm was used to obtain the assumption of anova in which all data should be in a normal distribution and have homogenous variance. shapiro wilk's test was used for analyzing the normality, and levene's test was used to determine the homogeneity. in comparing the concentrations of identified heavy metals with standard, a two-tailed t-test was used in this study. all statistical computation was run in the program r project where in to run the statistical computing in r software, packages used were dplyr, car, rstatix, tidyverse, and ggpubr (r core team 2021). acceptance and rejection of the hypothesis were set at a 0.05 level of significance. results and discussion heavy metal contents of dumpsite soil cd, ni, and pb in different sampling sites at different depths were found in the study site (table 1). the environment and its components in santa rita have been seriously polluted by heavy metals, which have compromised the ability of the environment to foster life and take its intrinsic values. heavy metals commonly occur naturally on earth, but anthropogenic activities result in large quantities of different environmental components (masindi & muedi 2018). the presence of nickel could be attributed since nickel is a metal with widespread distribution in the environment, including minerals. it is an essential constituent with many industrial and commercial uses (iyaka 2011). nickel can exist in soils in several forms, such as inorganic crystalline minerals or precipitates, complexed or adsorbed on organic cation surfaces, or inorganic cation exchange surfaces, among others (cempel & nikel 2006). nickel is a ubiquitous pollutant and carcinogen toxic metal found in many hazardous waste sites, especially in dumpsite that receives different types of waste, such as municipal solid waste, clinical waste, and industrial waste. industrial waste materials, lime, fertilizer, and sewage sludge contribute to the significant nickel sources in the soil (das et al. 2018). table 1 concentrations of heavy metals in dumpsite soil at different sampling sites and depths sampling sites sampling points gps coordinates cd (mg/kg) ni (mg/kg) pb (mg/kg) 0 30 cm 30 60 cm 0 30 cm 30 60 cm 0 30 cm 30 60 cm shoulder slope point 1 n 11o28’9.52364” e 124 o57'7.20043" trace trace 3.717 3.054 0.246 0.327 point 2 n 11o28’10.09024” e 124o57’6.34467” trace trace 0.494 0.378 trace trace point 3 n 11o28’10.32682” e 124o57’5.89446” trace trace 2.715 3.679 0.795 0.566 mean 2.308667 2.370333 0.5205 0.4465 main dumpsite point 1 n 11o28’9.17942” e 124o57’6.63555” 0.0114 0.1094 0.446 1.078 1.51 58.642 point 2 n 11o28’8.81745” e 124o57’7.07731” 0.0014 trace 2.154 1.91 0.318 0.846 point 3 n 11o28’8.4058” e 124o57’6.42071” trace 0.0504 1.939 1.065 0.7 11.578 mean 0.0064 0.0799 1.513 1.351 0.842667 23.68867 foot slope point 1 n 11o28’9.05403” e 124o57’2.92525” 0.0024 0.001 0.167 0.175 2.291 1.893 point 2 n 11o28’8.94994” e 124o57’4.08035” trace 0.001 0.448 0.409 1.438 1.525 point 3 n 11o28’8.17159” e 124o57’12.86853” trace 0.0023 0.085 0.147 1.469 1.56 mean 0.0024 0.001433 0.233333 0.243667 1.732667 1.659333 biotropia vol. 29 no. 3, 2022 228 on the other hand, lead was found at all sampling sites and depths within the dumpsite of santa rita, western samar. lead could be found in the dumpsite because it has some unique physical and chemical properties from historical times and has become a common environmental pollutant. lead forms different complexes with soil elements. it only takes a small piece of the lead as these complexes within the soil are phytoavailable (pourrut et al. 2011). lead has remained in the ground for thousands of years, not biodegradable (epa 2019). it poses a severe environmental risk due to its continued usage in every part of the world due to the abundance of gasoline, industrial processes, lead-based painting, lead-containing pipes, and lead-acid batteries (wani et al. 2015). lead consumption has increased due to increased demand for lead-containing products, such as batteries, electronic products, cathode ray tubes, pvc stabilizers, and lead pigments. these leadcontaining products increase lead-containing waste (european commission dg env 2002). moreover, cadmium was also found within the dumpsite of santa rita. cadmium can be released into the environment through anthropogenic activities and can be used in many industrial uses, such as plastic, pigment, enamels, ceramics, and steel plating. in addition, industrial processes produce cadmium as a byproduct (hasan et al. 2019). results of our study showed that nickel is found at all sampling points and depths. lead was present in trace amounts at the shoulder slope point 2 and other sampling points and depths. cadmium was mainly found at the foot slope at 30 60 cm depth, sparsely found at the main dumpsite, and was present only in trace amounts at the shoulder slope. the mean concentrations of cadmium, nickel, and lead in soil ranged from 0 0.1 mg/kg, 0.09 3.7 mg/kg, and 0.09 3.7 mg/kg for cadmium, nickel, and lead, respectively. mean concentrations were 0.0084±0.0030 mg/kg, 1.3517±0.4382 mg/kg, and 1.0959±0.2475 mg/kg, respectively, for 0 30 cm depth and 0.0137±0.0122 mg/kg, 1.3217±0.4322 mg/kg, and 1.1195±0.2560 mg/kg respectively for 30 60 cm depth. the results agree with the result from orodu et al. (2017), who reported a range of 0.09 1.10 mg/kg, 0.19 1.84 mg/kg, and 0.47 14.33 mg/kg for cadmium, nickel, and lead, respectively with means of 0.22±0.08 mg/kg, 1.03±0.16 mg/kg, and 5.17±5.04 mg/kg. results of our study also agree with amostautua et al. (2014), which determines the concentration of heavy metals in the dumpsite and cadmium. they reported a range of < 0.000.1±0.01 mg/kg, while amadi et al. (2012) studied different heavy metals, cadmium, and lead, resulting from a mean concentration of 0.15 ppm and 16.0 ppm, respectively. essien et al. (2019) reported a range of 0.92 1.4 mg/kg, 1.8 3.7 mg/kg, and 0.79 0.98 mg/kg taken during the wet season while 0.124 1.4 mg/kg, 1.72 2.98 mg/kg and 0.94 4.12 mg/kg taken during the dry season for nickel, lead and cadmium, respectively. it can be seen that heavy metal concentrations followed the order ni > pb > cd, which is in agreement with the results from orodu et al. (2017), amadi et al. (2012), essien et al. (2019), and makuleke & ngole-jeme (2020). figure 2 shows the mean concentrations of the metals from each sampling site at different depths. the trends observed at a sampling depth of 0 30 cm were generally the same as those taken at 30 60 cm. differences in heavy metal concentration can be attributed to the amount of waste in the dumpsite carrying the different heavy metals. for example, nickel having the highest levels could be attributed to more nickelcontaining wastes being dumped, such as buttons, zips, coins, household appliance tools, and other consumer products (asemave & annwange 2013). extensive industrial use of nickel has led to widespread environmental pollution (das et al. 2018). on the other hand, cadmium generally has the lowest concentration. this could be due to the decrease in demand for cadmium-containing products in the industry. specifically, the use of cadmium for pigments, pvc stabilizers, and plating has been phased out. as an impurity in zinc and fertilizers, turnover of cadmium has also been significantly decreased due to refining and changes in raw materials (european commission dg env 2002), resulting in a decrease in cadmium-containing products that end up in waste. cd, ni, and pb concentration of municipal dumpsite in western samar, philippines – varona et al. 229 figure 2 comparison of (a) cd, (b) ni, and (c) pb concentration means per sampling site and depth comparison of heavy metal contents according to sampling site and depth table 2 presents the statistical analysis results of comparing concentrations of cadmium, nickel, and lead at the different sampling sites and depths. significant differences are indicated by p ≤ 0.05. in case of significant differences, the results of post-hoc tukey's honest significant difference (hsd) are indicated in table 3. the mean values of cd, ni, and pb for all sampling sites were 0.008 mg/kg, 1.351 mg/kg, and 1.096 mg/kg, respectively, for 0 30 cm depth. while for 30 60 cm depth, the mean values of cd, ni, and pb were 0.033 mg/kg, 1.322 mg/kg and 1.119 mg/kg, respectively for all sampling sites (table 2). the computed p values showed no significant differences between the two sampling depths for all heavy metals tested. in addition, the computed p values of cd, ni, and pb in each sampling site for both sampling depths are 0.001, 0.003, and 0.012, respectively. it means that all sampling sites differed and suggested that dumpsite soil lacks the capacity to impede the downward migration of leachate from topsoil to subsoil (amadi et al. 2012). moreover, only cadmium had a depth-site interaction wherein two independent variables should be tested whether the effect of one independent variable is dependent or affects the other. biotropia vol. 29 no. 3, 2022 230 table 2 differences in cadmium, nickel and lead concentrations between different sampling sites and depths sampling depth heavy metals mean concentration (mg/kg) p value (α = 0.05) interpretation 0 30 cm 30 60 cm cd 0.0084 0.03282 0.9421 not significant ni 1.3517 1.32166 0.8945 not significant pb 1.095875 1.1195 0.9320 not significant sampling site heavy metals mean concentration (mg/kg) p value (α = 0.05) interpretation shoulder slope main dumpsite foot slope cd 0.04565 0.001675 0.0006 significant ni 2.3395 1.432 0.2385 0.0026 significant pb 0.4835 0.8435 1.13067 0.0115 significant depth sampling site interaction heavy metals p value (α = 0.05) interpretation cd 0.0207 significant ni 0.9767 not significant pb 0.9505 not significant table 3 presents results for multiple pairwise comparisons using tukey's honest significant difference test for the indicated heavy metals. this is employed to identify which groups are significantly different from each other. results was found that cadmium levels were approximately 3 times higher in the main dumpsite than in the foot slope. for nickel and lead, only the main effect of the sampling site was found to be significant. nickel concentrations at both the main dumpsite and shoulder slope are approximately 2 times higher than concentrations at the foot slope (table 3). this suggested a stronger upward lateral leaching (raji & adeoye 2017), leading to more nickel accumulation at the shoulder slope than at the foot slope. in the case of lead, levels at the foot slope were found to be 1.3 times higher compared to the shoulder slope, suggesting a slightly higher downward lateral leaching (raji & adeoye 2017). differences in leaching strength can be attributed to differences in soil properties (asemave & anhwange 2013; makuleke & ngole-jeme 2020). to better visualize the interaction between the depth effect and sampling site effect for cadmium, an interaction plot is presented in figure 3. an interaction effect is the simultaneous effect of two or more independent variables on one dependent variable. their combined effect is significantly greater (or substantially less) than the sum of the parts. the presence of interaction effects in survey research is essential because it tells researchers how two or more independent variables impact the dependent variable. table 3 tukey's hsd results for nickel, lead, and cadmium heavy metal sampling site difference p value (α = 0.05) interpretation ni main dumpsite foot slope 1.8365680 0.0092866 significant shoulder slope foot slope 2. 1108135 0.0035699 significant pb shoulder slope foot slope -1.3433094 0.0104619 significant cd main dumpsite foot slope 2.9373 0.0006842 significant depth sampling site interaction difference p value (α = 0.05) interpretation 60 cm main dumpsite 30 cm foot slope 3.4320323 0.0103995 significant 30 cm main dumpsite 60 cm foot slope 2.1559770 0.0191866 significant 60 cm main dumpsite 60 cm foot slope 4.0298647 0.0018821 significant 60 cm main dumpsite 30 cm main dumpsite 1.8738877 0.0421485 significant cd, ni, and pb concentration of municipal dumpsite in western samar, philippines – varona et al. 231 cadmium concentration at the main dumpsite is 3 times higher than the foot slope (fig. 3). however, differences can be observed due to the interaction between depth and sampling site effects. specifically, cadmium concentration at the main dumpsite (0 30 cm) is 2 times higher compared to the foot slope (30 60 cm), 3 times higher at the main dumpsite (30 60 cm) than the foot slope (0 30 cm), and 4 times higher at the main dumpsite (30 60 cm) compared to foot slope (30 60 cm). comparison of heavy metal contents with standard permissible limits in soil table 4 presents the results for comparison of heavy metal concentrations with the maximum permissible limits set by the world health organization (who). the permissible limits of heavy metals in soil are 0.02 0.5 (mg/kg), 0.1 5 mg/kg, and 0.3 10 mg/kg for cadmium, nickel, and lead, respectively (who 2001). a soil sample is polluted with a particular heavy metal if the concentration exceeds the upper limit of the acceptable range (ogundele et al. 2015). therefore, to determine whether soil samples from santa rita, western samar dumpsite was polluted with the indicated heavy metals, the right-tailed t-test was employed to compare the sample's mean concentration with the maximum permissible. figure 3 depth sampling site interaction plot for cadmium table 4 t-test results for comparison of mean concentrations with who standard heavy metals sampling sites sampling depth (cm) mean concentration who standard p-value (α = 0.05) interpretation cd shoulder slope 0 30 n/a 0.05 n/a n/a 30 60 n/a 0.05 n/a n/a main dumpsite 0 30 n/a 0.05 n/a n/a 30 60 0.0799 0.05 0.2479 no significant difference foot slope 0 30 n/a 0.05 n/a n/a 30 60 0.0014 0.05 1 no significant difference ni shoulder slope 0 30 2.3087 5 0.9471 no significant difference 30 60 2.3703 5 0.9391 no significant difference main dumpsite 0 30 1.513 5 0.9885 no significant difference 30 60 1.351 5 0.9971 no significant difference foot slope 0 30 0.2333 5 0.9997 no significant difference 30 60 0.2437 5 0.9998 no significant difference pb shoulder slope 0 30 0.5205 10 0.9908 no significant difference 30 60 0.4465 10 0.9960 no significant difference main dumpsite 0 30 0.8427 10 0.9993 no significant difference 30 60 23.689 10 0.2606 no significant difference foot slope 0 30 1.7327 10 0.9994 no significant difference 30 60 1.6593 10 0.9999 no significant difference biotropia vol. 29 no. 3, 2022 232 the results obtained showed that all heavy metal concentrations were not significantly higher than the maximum permissible limits set by the who (table 4). as such, they still fall within the permissible limits of who. it means that the concentrations of all indicated heavy metals are still at safe levels. heavy metals in the dumpsite soil should not threaten anyone, particularly the surrounding susceptible environment. however, serious measures should still be taken to maintain soil quality since heavy metal contamination in dumpsites will likely exacerbate in the future (orodu et al. 2017). conclusion identified heavy metals are all found at different sampling sites and depths of santa rita western samar dumpsite soil. the mean concentrations (mg/kg) of ni is the predominant, followed by pb, and the least is cd. no significant differences were observed in the concentrations of pb and ni in different sampling sites and depths, but significantly different in the concentration of cd. in addition, no significant differences were observed between the concentrations of identified heavy metals in all sampling sites at different depths and the standard permissible limits. therefore, all concentrations of identified heavy metals were within the permissible limits in soil. acknowledgments the authors acknowledge prof fe t. piedad, prof adorne y. madera, and prof grechelle n. socias for their essential contributions during the final polishing of this manuscript. references adelekan ba, alawode ao. 2011. contribution of municipal refuse dumps to heavy metals concentrations in soil profile and groundwater in ibadan nigeria. j appl biosci 40: 2727-37. amadi n, olasehinde p, okosun e, okoye o, okunlala a, baba a, dan-hassan m. 2012 . a comparative study on the impact of avu and ihie dumpsites on soil quality in southeastern nigeria. am j chem 2(1): 17-23. amos-tautua bmw, onigbinde ao, ere d. 2014. assessment of some heavy metals and physicochemical properties in surface soils of municipal open waste dumpsite in yenagoa, nigeria. afr j enviro sci technol 8(1): 41-7. asemave k, amhwange, ba. 2013. evaluation of heavy metals in waste dumpsites. germany: lap lambert academic publishing. cempel m, nikel g. 2006. nickel: a review of its sources and environmental toxicology. pol j environ stud 15(3): 375-82. das kk, reddy rc, bagoji ib, das s, bagali s, mullur l, ..., biradar ms. 2018. primary concept of nickel toxicity-an overview. j basic clin physiol pharmacol 30(2): 141-52. environmental protection agency. 2019. learn about lead. 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[updated 2020 dec 14; cited 2020 jan 23]. available from: https://www.who.int/fact-sheets/detail/standardfor-the-contents-of-heavy-metals-in-soils http://www.who.int/fact-sheets/detail/standard-for-the-contents-of-heavy-metals-in http://www.who.int/fact-sheets/detail/standard-for-the-contents-of-heavy-metals-in the southeast asian journal of tropical biology vol. 32 no. 2, 2025: 228 241 doi: 10.11598/btb.2025.32.2.2486 issn: 0215-6334 | e-issn: 1907-770x 228 highlights • traditional knowledge plays a crucial role biodiversity conservation. • in teluk bintuni district, local regulations support the integration of traditional knowledge, promoting collaboration for sustainable resource management. • the study highlights the need for community engagement, capacity building, and education to effectively utilize traditional ecological. • successful conservation initiatives require collaboration to balance economic growth with environmental stewardship and social inclusivity, ultimately fostering sustainable development. abstract traditional knowledge is vital for wildlife management and forest conservation in tropical regions and is passed down through generations. thus, this research was designed to identify the challenges and opportunities encountered in implementing traditional ecological knowledge (tek) during regional development, particularly in relation to natural resource management in teluk bintuni district. the data were collected through interviews with local people and a focus group discussion (fgd) involving government officials, non-governmental organizations (ngos), and local figures. the results revealed that the government had enacted regional regulation number 1 of 2019 to recognize the customary rights of seven tribes, i.e., the kuri, wamesa, irorutu, sebyar, simuri, sougb, and moskona tribes, thereby protecting traditional communities. the regulations emphasize the prioritization of people's rights in development, supported by the establishment of lma tujuh suku (a traditional community institution of seven tribes). the tribes have been utilizing tek to deal with social and environmental issues, integrating the issues into development procedures to foster conservation and economic growth. the challenges, such as poor infrastructure, political instability, and low community engagement, hinder effective implementation. the strengths include accessible natural resources and international funding for conservation. the study highlighted the importance of education, stakeholder collaboration, and technology in documenting and incorporating tek into conservation planning through participatory research, capacity building, and adaptive management. keywords: conservation, indigenous people, natural resources, traditional knowledge, tropical rainforest integrating traditional ecological knowledge in teluk bintuni district: opportunities and challenges for sustainable resource management nimrod agustinus andyratnah rumayomi1, agustinus murdjoko2,4,*, antoni ungirwalu2, mulyadi3, nithanel mikael hendrik benu5, dony aristone djitmau2 1doctoral program of environmental science, papua university, manokwari 98314, indonesia; 2faculty of forestry, papua university, manokwari 98314, indonesia; 3faculty of animal husbandry, papua university, manokwari 98314, indonesia; 4research centre for biodiversity, papua university, manokwari 98314, indonesia; 5center for implementation of environmental and forestry instrument standards, manokwari 98311, indonesia article information received : 10 march 2025 revised : 16 april 2025 accepted : 21 april 2025 *corresponding author, e-mail: agustinus.murdjoko.papua@gmail.com https://doi.org/10.11598/btb.2025.32.2.2486 integrating traditional ecological knowledge in teluk bintuni district rumayomi et al. 229 introduction traditional knowledge has been part of local people for generations and is crucial for effective wildlife management and biodiversity preservation in tropical countries, as the tropical forests have the richest species of trees (cámaraleret et al. 2020; liang et al. 2022) and fauna (de souza amorim et al. 2022; holt et al. 2013; oliver et al. 2022). the knowledge encompasses the holistic understanding, practices, and beliefs of indigenous communities concerning their environment, passed down over generations. this knowledge system is particularly relevant in tropical countries where biodiversity is rich, but threatened by anthropogenic pressures like deforestation, climate change, and urbanization (popradit et al. 2015; reyes-garcía et al. 2016). indigenous people often possess detailed ecological knowledge about local species, ecosystems, and sustainable practices, which can enhance conservation strategies by integrating local ecological dynamics into broader management frameworks. cultural relevance is another advantage of incorporating traditional knowledge in conservation programs. indigenous people are more likely to support efforts when actively designing and implementing conservation initiatives (asamoah et al. 2024; ford & martinez 2000). traditional practices, such as shifting cultivation used by indigenous farmers, promote sustainability and biodiversity conservation. by recognizing these practices within conservation programs, tropical countries can leverage existing local expertise to promote sustainable development (van der sande et al. 2016; whitfeld et al. 2014). the inclusion of traditional knowledge in policy-making processes ensures that the voices of indigenous people are heard in discussions about natural resource management and environmental protection, leading to more comprehensive and effective policies. natural resources, such as forests, are vital for providing ecological, economic, and social benefits. in developing countries, forests serve as a critical source of livelihood, food security, energy needs, and cultural identity for millions of people (berkes et al. 2000; carson et al. 2018; sierrahuelsz & kainer 2018). however, sustainable utilization of forest resources remains a pressing challenge due to deforestation, land degradation, and competing economic pressures (popradit et al. 2015; tsujino et al. 2016). local knowledge, often referred to as traditional ecological knowledge (tek), is pivotal in fostering sustainable forest management and utilization practices. local communities possess intricate knowledge of forest ecosystems, enabling them to employ sustainable harvesting techniques, such as selective logging, rotational grazing, and controlled burning, thereby promoting the conservation of these ecosystems. they also have an awareness of the medicinal, nutritional, and ecological value of various plant and animal species, such as sacred groves or areas within forests holding cultural or spiritual significance (reyes-garcía et al. 2016; zhao et al. 2016). local knowledge also equips communities with strategies to cope with environmental changes, such as climate variability and resource scarcity. in addition, local communities have developed expertise in identifying, harvesting, and processing non-timber forest products (ntfps), which offer an alternative income source that reduces dependence on timber extraction and large-scale deforestation (nunes et al. 2020; thomaz sandroni 2023). however, integrating local knowledge into formal forest management strategies faces several challenges, including marginalization of local communities, erosion of traditional practices, conflicts between modern and traditional approaches, and lack of documentation (joa et al. 2018; lawer & ishaq 2024). to harness the full potential of local knowledge for sustainable forest utilization in developing countries, strategies can be employed, such as participatory forest management, strengthening land tenure rights, documenting and disseminating traditional practices, integrating local knowledge into national policies, and providing economic incentives for sustainable practices. teluk bintuni district, one of the developing districts, has been part of forest utilization for decades, particularly for logging concessions and local use (rumayomi et al. 2024). recently, the primary development targets have been the infrastructure and empowerment of local people by enhancing their capability. despite its progress, teluk bintuni faces challenges that require careful attention. issues, such as the unequal distribution of development benefits, limited access to technology, and the need for greater investment in human capital, remain pressing concerns. maintaining social harmony in a culturally diverse region also demands inclusive policies that respect local traditions and values. looking ahead, the biotropia vol. 32 no.2, 2025 230 future development of teluk bintuni will depend on sustainable practices that integrate economic growth with environmental stewardship and social inclusivity. collaborative efforts between government agencies, private companies, local communities, and international organizations will be essential to achieving long-term success. furthermore, traditional knowledge, which balances the economic and ecological perspectives, is the primary driver of regional management, such as natural resource and forest utilization. hence, their collaboration can be a “win-win solution” by understanding the traditional knowledge and regional development goals. however, the undesirable conditions of the collaboration can be reduced by regular monitoring. thus, the challenges and opportunities presented by traditional knowledge during regional development, particularly in natural resources management, in teluk bintuni district, must be investigated. this study aimed to describe the potential use of traditional knowledge from the tribes in the teluk bintuni district and to integrate this knowledge into development initiatives. by identifying the stakeholders in the district, the possibility of implementing traditional knowledge can be proposed. moreover, the results of this study could contribute to the development program in the district, particularly about sustainable forest management. materials and methods study site teluk bintuni district, located in the west papua province of indonesia, was selected as the study area for this study. the administrative districts that make up this region are located on three sides of teluk bintuni, which is a gulf that divides the bird's head peninsula and the bomberai peninsula, with a total area of 18,637 km2 (fig. 1). this study was conducted in the administration area of teluk bintuni district (1°57'50" 3°11'26" ls and 1.9.32°44'59" 134°14'49" e) covering the living area of seven tribes as stated in the regional regulations number 1 year 2019 on the recognition and protection of traditional legal communities in teluk bintuni district, in which the seven tribes are kuri, wamesa, irorutu, sebyar, simuri, sougb, and moskona. the main study area is dominated by lowland forests, except mangroves. the lowland forest is dominated by primary forests, some of which are utilized for shifting cultivation, resulting in secondary forests. the forest consisted of lifeforms like ferns, herbs, lianas, shrubs, trees, palms/screw palms in the primary and secondary forests in the study area where the primary forest had a higher diversity, with 246 species found in the primary forest. in comparison, 162 species were found in the secondary forest. in their study, rumayoni figure 1 teluk bintuni district is shown as the area marked with a red box on the main map. integrating traditional ecological knowledge in teluk bintuni district rumayomi et al. 231 et al. (2024) found that 98 out of 310 species were shared between the two forests, with 36.3% exclusive to the primary forest and 15.7% exclusive to the secondary forest. data and interview data concerning regional regulations in teluk bintuni district, in the context of socio-cultural and natural resource management, were the focus of this study. the implementation of traditional knowledge, primarily traditional ecological knowledge (tek), was incorporated into the natural resources management conducted by local people. data collection was conducted through interviews with respondents using a non-random process, which considered the perspective of the research goal (cox 2015). the interview method was carried out using the guidance to collect social data, particularly with tribal leaders (7 respondents), heads of villages, and local people (30 respondents), while a focus group discussion (fgd) was performed by the stakeholders like the central government (2 respondents), local government (27 respondents based on the list on chapter iii in the regional regulation of teluk bintuni district number 7 year 2016 on the formation and composition of regional organization in teluk bintuni district), non-governmental organizations/ngos (2 respondents), religion leaders (5 respondents), and representation of women (1 respondent). the interview process was conducted in informal situations to allow flexibility for the respondent in addressing the questions (maryudi & fisher 2020). another interview with the government was conducted using the list of institutions obtained from initial talks with the government's representatives. the heads of villages and local people were selected using the purposive method, which required an understanding of socio-cultural conditions through preliminary observation. to enrich the data, the local traders of wildlife meat and crops were also interviewed to learn about the process of hunting, traditional cultivation, and their perspective regarding the role of the ecosystem in their livelihoods. in order to collect data from local people, this study employed purposive sampling to select respondents, followed by the snowball method to gather additional information. before conducting the interview, we observed the social conditions of the local people by gathering information from some tribal leaders and village heads. the observation was used to determine the starting point of a purposive sample, where the researcher’s judgment was employed to select the initial respondent as the expert respondent based on their ability to identify other potential respondents. once the expert respondent was identified, other respondents were selected based on the guidance provided by the expert respondent (drané et al. 2018; goodman 1961). data analysis as this study collected qualitative data, we conducted a narrative analysis by presenting data on socio-cultural and natural resources management regulations in teluk bintuni district. furthermore, the data of tek was expressed by describing various interactions between local people and the natural resources. we hypothesized that the more local people interact with natural resources, the more dependent they are on these resources. for example, the use of vegetated areas as a traditional cultivation and hunting system implemented by the local people. the data were presented with illustrations using authentic documentation as strong evidence obtained during the study. to set the strategies for integrating tek into regional development, we performed the strengths, weaknesses, opportunities, and threats (swot) analysis by describing the condition of teluk bintuni district. the four factors were derived based on information collected from the focus group discussion (fgd) and interviews, which were presented using the eurequip matrix (ansoff, 1980; weihrich, 1982), followed by a narrative presentation of the strategic issues. the swot analysis may be used to guide potential approaches by using the strengths and opportunities as positive factors, while minimizing the undesirable conditions of weaknesses and threats (bull et al. 2016; navarro-martínez et al. 2020). results and discussion regulation consisting of the local people's contents teluk bintuni district has accommodated the customary rights of the seven tribes by gazetting the regional regulations number 1 of 2019 concerning the recognition and protection of traditional legal communities in teluk bintuni district. this regulation supported the position of local people, as mentioned in the seven tribes: biotropia vol. 32 no.2, 2025 232 kuri, wamesa, irorutu, sebyar, simuri, sougb, and moskona. moreover, to legally support the regulation, the regent issued regulation number 22 of 2020 concerning guidelines for identification, verification, and determination of traditional legal communities in teluk bintuni district. according to the guidelines, the teluk bintuni district has legally prioritized the rights of local people during development. furthermore, the establishment of the indigenous people’s institution (in bahasa indonesia, lembaga masyarakat adat tujuh suku [hereafter referred to as lma tujuh suku]) as the office (fig. 2a; 2b) and the monument has been built to show the seven tribes (fig. 2d). other regulations have also been made to strengthen the position of local people in teluk bintuni, i.e., 1) decision of the regent of bintuni bay number 188.4.5/064/2023 concerning recognition of the yen clan's traditional legal community in the moskona tribe in bintuni bay district and 2) regent's decree number: 188.4.5/ c-8 year 2021 concerning the formation of a committee for indigenous people in teluk bintuni district, indicating that the district considers the local people during the development, so this condition can be seen as a strong opportunity to include traditional knowledge of tribes in this district. recently, the government of teluk bintuni district implemented these regulations by issuing several guidelines to the committee to identify the customary rights of the tribes. the process is related to the activities of the private sector, such as the oil and gas sector and logging concessions, which require large areas to impact forest conversion or secondary succession. interactions with natural resources in the administrative area of teluk bintuni district, there are seven tribes legally acknowledged. the tribes have been living in this figure 2 institutions to support conservation and acknowledgement of customary right notes: (a) the office of lma tujuh suku of teluk bintuni district; (b) the organizational structure of lma tujuh suku is displayed in the office of lma tujuh suku; (c) the office of natural resources conservation centre (balai konservasi sumber daya alam/bksda seksi konservasi wilayah iii bintuni) in charge of conservation program; (d) the monument of tujuh suku (seven tribes) as the recognition of customary rights. (photo credit: nimrod agustinus andyratnah rumayomi) integrating traditional ecological knowledge in teluk bintuni district rumayomi et al. 233 area for generations and have been implementing traditional knowledge. many studies indicated that the tribes still perform the cultural processes in their livelihood, which vary due to their living areas, ranging from coastal to terrestrial, as the tribes of kuri, wamesa, irorutu, sebyar, and simuri live and have customary rights near the shoreline, while sougb and moskona tend to inhabit inland areas. the tribes actually understand and agree with the customary rights belonging to each tribe, where each tribe consists of a clan, as stated legally in the appendix of regional regulation number 1 of 2019. in the appendix of the regulation, the names of clans are written as the result of several stages during the process, based on discussions and consolidation between the local government and the tribal leaders in which the traditional knowledge is developed based on the agreement and understanding of local people transferred among generations verbally and by practice. hence, to implement and manage traditional knowledge, the tribes rely on tribal leaders, village heads, or older people. the tribes have been practicing traditional knowledge by conducting traditional cultivation, as well as implementing hunting systems and fishing (fig. 3). in certain conditions, some tribal members are allowed to rent out areas to be used by others, such as outsiders (not from the seven tribes). during a legal trade, such as in areas where local people are involved, they must agree with the owner of customary rights before obtaining the certificate. the lma usually issues the agreement as a recommendation from the tribal representation during the trade. figure 3 illustrates various types of interactions and dependencies between local people and natural resources. figure 3 the dependency of local people in teluk bintuni on the natural resources notes: (a) the traditional cultivation; (b) the post-burning process during the shifting cultivation where they collect the plant debris and make a pile, then burn far away from the border to avoid forest fire; (c) the souvenir made of molluscs as a result of barter process and can be used as dowry; (d) the traditional selling of wild boar meat in the traditional kiosk; (e) products of traditional crops, like sweet potato; (f ) papaya flowers and leaves as vegetables; (g) sweet potato and bananas; (h) durian. (photo credit: nimrod agustinus andyratnah rumayomi) biotropia vol. 32 no.2, 2025 234 strengths, weaknesses, opportunities, and threats we have asked and proposed the conditions that determine the aspects of traditional ecological knowledge (tek) implementation in the teluk bintuni district area during a focus group discussion (fgd). hence, in this study, we described the factors impacting tek implementation during the development program of teluk bintuni district, whether they were inhibiting or supporting elements. the factors were grouped into strengths, weaknesses, opportunities, and threats (swot) to describe the extent to which traditional knowledge was integrated into the development programs of teluk bintuni district. the use of tek is related to the sustainable management of natural resources, like forests. thus, the harmonization between conservation and exploitation can be addressed during exploitation. the implementation of ecological and economic perspectives during forest utilization is conducted by local people and the private sectors, which is considered as the primary goal. as a result, conservation programs in developing districts often face unique challenges and opportunities, particularly when integrating conservation programs with tek, encompassing the cumulative knowledge, practices, and beliefs of indigenous and local communities passed down through generations and are deeply rooted in the sustainable management of natural resources. while leveraging tek in conservation programs offers immense potential, it also requires a careful assessment of strengths, weaknesses, opportunities, and threats (swot) to ensure effective implementation and long-term success (fig. 4). strengths as stated in the regional development plan of teluk bintuni for 2021-2026 and regional regulation number 1 of 2019, this district has committed to implementing sustainable development programs. moreover, this area is part of the west papua province, which has declared a conservation area of approximately 70% and aims to protect the indigenous people, as stated in the manokwari declaration (cámara-leret et al. 2019). moreover, many regulations have been issued regarding the recognition and protection of local rights, indicating that the government prioritizes the local people during development. the local people's structure has been described as comprising seven tribes within this district administration, each with traditional knowledge that has been passed down for generations, particularly concerning sustainable management. many studies showed that teluk bintuni district's natural resources consist of vegetation with high biodiversity value (kasihiw et al. 2023, 2024; figure 4 eurequip matrix showing the swot description of the potential integration of traditional ecological knowledge (tek) during the development programs in teluk bintuni district and the potential strategies integrating traditional ecological knowledge in teluk bintuni district rumayomi et al. 235 rumayomi et al. 2024; sillanpää et al. 2017). moreover, the natural resources in this district have been exploited, which could provide financial support in addition to the regular funds. weaknesses the infrastructure aspect of teluk bintuni district is being developed to balance accessibility. however, some areas in the district have not yet been well developed due to many developing districts in papua now prioritize road development (engert et al. 2024; gaveau et al. 2021). furthermore, the political system, like the election, would impact the position of stakeholders in the government. the atmosphere that focuses on the development mission mainly relies on personal leaders, impacting the integration of the conservation program and tek. opportunities we described global financing and policies as opportunities to implement tek in the development program of teluk bintuni district. the global perspective prioritizes the local communities, like conservation programs and natural resources management, particularly in the tropics (bong et al. 2024; erbaugh et al. 2020; kumar et al. 2021). teluk bintuni district is the location of several non-governmental organizations (ngos) that work as government partners. additionally, information technology, supported by an internet connection, has been accessible to the resident areas, facilitating the distribution of information and data storage related to the development program, including tek implementation. as the government has stated, infrastructure development is now a priority, including road expansion, which leads to improved accessibility. threats being a developing area, accessibility to remote areas in teluk bintuni district is low, resulting in some people living in marginal conditions. the marginal area primarily covers lowland tropical rainforests with some coastal areas dominated by mangrove ecosystems (kasihiw et al. 2024; rumayomi et al. 2024; sillanpää et al. 2017). potential threats of deforestation and forest degradation are likely to take place as this area is a part of the location of some companies. the deforestation and forest degradation lead to biodiversity loss, which has already happened in many tropical rainforests (spracklen et al. 2015; laurance 1999; li et al. 2022). moreover, incoming people inhabiting the teluk bintuni district will affect socio-culture, resulting in acculturation that may impact social and psychological factors. proposing the potential strategies to integrate tek into the development program in teluk bintuni district, the integration between traditional ecological knowledge (tek) and regional planning and development is hindered by limitations and threats, as the district is prioritizing infrastructure construction to improve accessibility in the areas. despite political instability, supporting variables are identified as strengths and possibilities that would achieve an optimistic aim. companies that have invested over the years have exploited natural resources in this district. conservation programs, like the carbon fund, are supported by international funds. due to their high biodiversity and wide distribution areas, conservation areas are associated with varied traditional knowledge, making biodiversity and cultural conditions fascinating topics to study. international rules may support sustainable management, as this district has also incorporated ngos into biodiversity and social conservation projects as part of its sustainable program for regional development. the implementation requires information technology, supported by an internet connection, to distribute and communicate data across different locations. our study proposes strategies formulated from the eurequip matrix that can be used to integrate traditional knowledge and regional programs. collecting information on tek using education and research education and awareness programs are essential for increasing biodiversity conservation awareness among local communities. cultural education should be incorporated into local education systems. workshops and training sessions should be organized to educate community members about conservation techniques (carson et al. 2018; gouwakinnou et al. 2019). collaboration with stakeholders, including ngos and government agencies, is essential. monitoring and evaluation mechanisms should be established, such as community-based monitoring and feedback systems (guibrunet et al. 2021; lawer & ishaq 2024). biotropia vol. 32 no.2, 2025 236 integrating traditional ecological knowledge (tek) into conservation programs in developing districts presents a promising pathway toward sustainable resource management and biodiversity preservation. however, its success depends on addressing inherent weaknesses and mitigating potential threats through strategic planning and collaboration. research must be conducted by enhancing collaboration with universities and research boards to obtain data on traditional ecological knowledge (tek) in teluk bintuni district. hence, the local government must set up systematic research planning and promote partnerships with the researchers. the study must have scientific content and be published by a scientific publisher. hence, these studies can serve as a fundamental basis for setting policies in this district. furthermore, the monitoring and evaluating of the implementation of the regulations must be regularly carried out to enhance the process of achieving the development goals. so, both infrastructure and social development can be balanced in this development. as a result, this condition could mitigate the social conflict between the government and local people. to maximize the strengths of tek, conservation programs must prioritize community engagement, capacity building, and documentation efforts while fostering partnerships between traditional knowledge holders and scientific experts. policymakers must also play a crucial role by creating supportive legal frameworks that protect intellectual property rights and ensure equitable benefit-sharing. increasing the collaboration between multisectors and stakeholders information technology (it) can support administrative processes, including tek implementation, which can be used to connect and store databases on tek implementation for each tribe. moreover, the technology enhances communication among stakeholders and the circulation of information, such as using digital maps that can be integrated with the traditional boundaries of customary rights. information technology supports the transparency of many activities (toivonen et al. 2019). with information technology, monitoring the implementation would be technically easy, as some studies have used such technology to monitor wildlife (moßbrucker et al. 2016; scotson et al. 2017). for instance, anyone can access the regulations, and most people can understand the updated regulations. collaboration can be performed to strengthen the implementation among stakeholders. for example, a study was conducted to identify the tek and local wisdom among tribes, and the results can be provided online, where the local agreement of tribes can be written using the proper methods. conservation programs in developing districts should involve local communities during the planning and implementation of the programs (börner et al. 2016; chen et al. 2020; donaldson et al. 2017) by building trust, incorporating local knowledge, as well as providing training and resources to empower the human resources related to the programs. integrating tek into conservation planning involves conducting participatory research, mapping resources, and developing adaptive management plans. sustainable practices like agroecology, wildlife management, and restoration projects should align with scientific research and tek. for instance, the teluk bintuni district is part of an area managed by the natural resources conservation centre (balai konservasi sumber daya alam/bksda seksi konservasi wilayah iii bintuni) in terms of vegetation and wildlife management. therefore, coordination and identification are necessary to involve local people in the conservation program. as part of west papua province, the manokwari declaration has stated that forests should be conserved and sustainable development should be run (cámara-leret et al. 2019). hence, this district is getting support for planning and implementation, such as policies and financial assistance. teluk bintuni district also acknowledges the tribes as part of the government, as stated in local regulations. thus, this potency can be put into the planning and development of this district so that the government (regent and legislative board) can run the program following the roadmap. improving strategies of a sustainable program traditional ecological knowledge (tek) refers to the understanding and insights indigenous and local communities have developed over generations regarding their environment, ecosystems, and sustainable resource management. tek also encompasses a holistic view of the relationship integrating traditional ecological knowledge in teluk bintuni district rumayomi et al. 237 between people and nature, integrating cultural beliefs, practices, and ecological knowledge. leveraging tek in teluk bintuni district can significantly enhance conservation efforts by aligning them with local customs and practices. the conservation program supports the goals of sustainable development programs, which can be used to balance economic and ecological goals. many studies have shown that the role of local people significantly influences the development process (alamgir et al. 2020; ellis et al. 2017; mayhew et al. 2019). therefore, several practices can be recommended. community engagement and participation are considered as the primary strategies for conservation programs in developing districts to engage local communities actively in the planning and implementation processes by establishing relationships based on mutual respects between conservation organizations and local communities, incorporating local knowledge, and utilizing tek to inform decisions about land use, species management, and habitat restoration. capacity building is implemented by providing training and human resources to empower local communities to participate actively in conservation efforts. tek can be integrated with conservation planning by conducting participatory research and engaging community members in data collection about local biodiversity, ecosystem services, and traditional practices. mapping resources can be carried out using community knowledge to create maps identifying important ecological areas, culturally significant sites, and resource distribution. developing adaptive management plans can be built to create flexible management strategies that can be adjusted based on ongoing feedback from the community. achieving economic and social visions it is interesting to note that promoting sustainable practices conservation programs should promote sustainable practices that align with both scientific research and tek, like agroecology, encouraging agricultural methods that are informed by traditional practices while also incorporating modern sustainable techniques; wildlife management by respecting traditional hunting practices while ensuring species conservation; and restoration projects by utilizing native plant species identified through tek for habitat restoration projects to ensure ecological compatibility. another crucial factor is the implementation of education and awareness programs on the importance of biodiversity conservation among local populations. it can be conducted through cultural education by incorporating teachings about the significance of biodiversity into local education systems, using culturally relevant materials and workshops, and organizing training sessions and events where community members can learn about conservation techniques alongside traditional practices. during the planning and implementation of regional development, collaboration with stakeholders, such as partnerships with ngos specializing in conservation, can provide additional resources, expertise, and government involvement, engaging government agencies to support policies that recognize and incorporate tek into formal conservation frameworks. for example, successful conservation programs require collaboration among various stakeholders, such as ngos and government agencies. community-based monitoring systems empower local communities to monitor biodiversity changes and provide continuous feedback on the success or challenges in implementing conservation strategies (dirhamsyah 2013; gouda et al. 2021; rocca & zielinski 2022). overall, conservation programs should prioritize building trust, incorporating local knowledge, and promoting community engagement in planning and implementation. to track the progress of the integration in planning and implementation, it is essential to conduct regular monitoring and evaluation. monitoring and evaluation can be carried out through community-based monitoring systems, empowering local communities to track biodiversity changes using their traditional knowledge systems. the feedback can be built by creating channels for continuous feedback from community members regarding the successes or challenges faced in implementing conservation strategies. biotropia vol. 32 no.2, 2025 238 conclusion teluk bintuni district has acknowledged the customary rights of seven tribes by issuing regional regulations number 1 of 2019 to protect traditional communities. the acknowledgement is recognized by prioritizing people's rights during development, supported by additional regulations, and by establishing the lma tujuh suku involving kuri, wamesa, irorutu, sebyar, simuri, sougb, and moskona tribes who have lived in the area for generations, by practising traditional ecological knowledge (tek), having the primary goals of socio-economic and ecological factors. the tek is incorporated into development programs as a method to achieve conservation and economic growth goals. several challenges, such as limited infrastructure, political instability, and the need for community engagement, hinder effective implementation. strengths include the availability of natural resources and international funding for conservation efforts. our study emphasized the importance of education, stakeholder collaboration, and the use of technology to document and share tek. strategies for incorporating tek into conservation planning, such as participatory research, capacity building, and adaptive management, are vital. ultimately, effective conservation programs must prioritize community-based involvement, trustbuilding, and the incorporation of local knowledge to achieve sustainable development goals in this district. acknowledgments the authors would like to thank the government, 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measurements? plos one 11:1-16. doi: 10.1371/journal.pone.0152061 biotropia vol. 30 no. 2, 2023: 206 219 doi: 10.11598/btb.2023.30.2.1850 206 preliminary study: feeding ecology and daily activity of three colored langur (presbytis chrysomelas ssp cruciger thomas, 1892) in danau sentarum national park nyoto santoso, sutopo*, lorenzo elton meo, natasya nurul fauziah and alfatheya margwita diva department of forest resources conservation and ecotourism, faculty of forestry and environment, ipb university, bogor, 16680, indonesia received 13 october 2022 / revised 8 may 2023 / accepted 8 may 2023 abstract three colored langur (presbytis chrysomelas ssp cruciger thomas, 1892) is a primate that has been recognized as a critically endangered species in accordance with the international union for conservation of nature, however still not protected and lacks substantial information about the bio-ecology of their natural habitat. the habitat plays a determining factor not only in space utilization but also the daily activities of the three colored langurs (p c cruciger). the aim of this preliminary study is to collect information regarding their habitat characteristic, feed species, daily activity, and canopy stratum utilization. this research was conducted between july and august of 2021 at bukit semujan, lupak mawang resort, danau sentarum national park. the method implemented was to collect the habitat characteristic by plot samples, and their daily activity data by scan sampling with consecutive recording. the study demonstrated that langur inhabited both primary as well as mixed forests (swamp, cultivation land, and secondary forest). there were 27 species as feeds of langurs and the most preferred types of feed are gita susu (willughbeia coriacea), merepat (unidentified), and karet (hevea brasiliensis). the most preferred feed compositions consisted of leaves (50%), fruits (30%), and seeds (20%). the most frequently utilized stratum for activities was stratum c (70,49%) and b (27.87%). the highest daily activities were categorized into three parts of time, the morning was dominated by social (44,26%), the afternoon was dominated by rest (59,7 7%), and the evening was dominated by social (73,68%). the highest social activities shown by three colored langurs were agonistic (48,48%), followed by vocalization (39,39%), playing (10,61%), exploring (1,52%), and sexual (0%). keywords: daily activities, feeding ecology, presbytis chrysomelas, ranging pattern, three colored langur introduction three colored langurs locally known as lutung sentarum (presbytis chrysomelas ssp cruciger thomas 1892) and referenced as langur for this paper is one of the endemic primates of bornean island and danau sentarum national park, a place used as their natural habitat. nevertheless, it has been categorized as critically endangered species by the international union for conservation of nature (iucn), however it is yet to be registered as the protected species in accordance with the regulation of animal protection in indonesia. this can be attributed to the absence of adequate ecological information on the species, namely, habitat characteristics and daily activities. so, far there has been no research that specifically examines the ecology of the feed and space utilization for the langur habitat. the distribution of p c ssp cruciger was found at sungai pelaik sub-village but only with encounters notes, and do not provide comprehensive information on the ecology (rifki et al. 2019). research that has been done previously is still very finite on topics regarding habitat characteristics (musyafa and santoso 2020) and population estimates that still need to be repeated (aripin et al. 2019). sources *corresponding author, email: nyotosa@apps.ipb.ac.id mailto:nyotosa@apps.ipb.ac.id preliminart study: feeding ecology and daily activity of three colored langur – santoso et al. 207 indicate that this species are also inhabitants of the north borneo island additionally known in sabah, sarawak (malaysia) and brunei darussalam, however there is not adequate information available to understand the comprehensive ecology of this species in their natural habitat (nijman 2020). areas that are practiced as natural habitat of langur in danau sentarum national park can be found in the forest of bukit semujan. administratively, these habitats scope at lupak mawang resort and at government administration including the selimbau and jongkong district, kapuas hulu regency. the condition of feed resources in the habitat of the three colored langur also affects their daily activity pattern. habitat impacted the canopy utilization for distinct activities too, namely, moving, foraging, and social recreations (watanabe 1981; hadi et al. 2012). primates such as three colored langurs usually exhibit further characteristics of their activity with vocalization or agonism from intraspecies interactions (singh et al. 2011; houle et al. 2006). this study aims to identify ecological characteristics in the form of habitat and feed, as well as daily activity, and ranging patterns of three colored langurs. materials and methods study site and time this research was conducted at bukit semujan, lupak mawang resort, danau sentarum national park, west kalimantan, indonesia with geographic coordinates of 00°45'–01°02' n and 111°55'–112°26' e. primary data collected namely, group size, habitat characteristic, feeds, and daily activity were observed between july and august of 2021. bukit semujan has topography that ranges from flat (elevation of 60 above sea level) and continues to be wavy at an elevation of 60–80 and high cliff at an elevation of 80–300 above sea level. land cover condition in accordance with the on ground truthing and rupa bumi indonesia classification of kapuas hulu regency encompasses the primary forest at the top of the hills and mixed forest that includes secondary forest surrounding the sub-hills, swamp forest, and small area of the cultivation land. following figures exhibit the study area (figure 1a and 1b). figure 1a land cover condition at bukit semujan captured by an aerial photo taken by a drone biotropia vol. 30 no. 2, 2023 208 figure 2b map of the research location at bukit semujan, lupak mawang resort field data of habitat characteristic implemented the plot sample with purposive sampling for every single land coverage and ecotone areas (soerianegara and indrawan 1988). total sampling comprised of 12 plots, located in swamp forest, secondary, and mixed forest. plot sample size were prepared in blocks of 20 x 20 m for trees habitus, 10 x 10 m for pole habitus, 5 x 5 m for sapling habitus, and 2 x2 m for herbs and seedling habitus, and these samples were further divided into 4 plots at three habitats. these plots were distributed perpendicular to each hill gradient from swamp forest, mixed and primary forest. in order to analyze the habitat of three colored langurs, the established methods included vegetation analysis and the land coverage classification in accordance with the ministry of forestry and environment (2021) that consisted of three main habitat types namely, secondary forest, swamp forest, and mixed forest. the vegetation analysis was further categorized into relative density (2), relative frequency (4), relative dominance (6), and the importance value index (inp) (4, 5) on each growth level of vegetation. meanwhile, the availability of langur feed in the two ecosystems was determined based on the relative density value of each species. these values were utilized to establish the dominant species of a habitat (parmadi et al. 2016). density (k) =  ind.species_i ...................... (1) areas of plot sample relative density (kr) = density a species x 100% ................... (2) density all number species frequency (f) = number of plot sample that was found ...................... (3) total number of all plot sample relative frequency (fr) = frequency a species x 100% ................... (4) frequency all the species dominance (f) = basal areas species_i ...................... (5) areas of plot sample relative dominance (dr) = dominance a species x 100% ................... (6) dominance of all species preliminart study: feeding ecology and daily activity of three colored langur – santoso et al. 209 inp herbs, seedling, and sapling = kr + fr inp pole and trees = kr + fr + dr to illustrate the structure of habitat langurs the profile diagram by sexifs software (hardja and vincent 2008) was implemented. data collected to create the structure profile using this software consist x: the x position of the tree base (m), y: the y position of the tree base (m), species: the species label, if the label is match with the one in the species list, then it will be linked, otherwise new species definition will be created, dbh: the diameter at breast height of the tree (m), height: the height of the tree (m), cr depth: crown depth (m), cr curve: crown curve (m), cr radius: crown radius in vertical projection, can be more than one value separated by semicolon (m), rotation: a rotation of the vertical projection of the crown geometry (degree), cp: crown position index (0 1), cf: crown form index (0 1). this illustration will exhibit the vertical model of the ranging pattern by three colored langurs. the tree canopy stratum used by three colored langurs was further classified into several strata, namely stratum a (>30 m), stratum b (20 30 m), stratum c (4 20 m), stratum d (1 4 m), and stratum e (0 1 m) (soerianegara and indrawan 1988). method to identify the daily and social activities was use scan sampling with 1 minute interval time to record each activity of an individual in a group at a certain time cumulatively (hepworth & hamilton 2001). to study the behavior pattern of the three colored langurs, continuous recording was practiced. each behavior pattern was categorized into eating (take and eat foods to the mouth), moving (movements from one to another site using their quadrupedal), resting (off from all their activity and it can be indicated by the closed eyes or sleeping time), and social activity (napier and napier 1967). the social behavioral pattern was further classified into agonistic, vocalization, playing, exploring, and sexual. each behavior was calculated as a percentage of its frequency and duration. results and discussion group size of langur the field observation recorded three groups of langurs with total 16 encounters. each group consisted of 21 24 individuals and was widespread in several parts of bukit semujan. the first group recorded nine individuals, including an infant and a baby in the south hill.eanwhile, on the west side (second group) of the hill five individuals were observed. third group with seven individuals was recorded on the east of the hill, including one sole male with direct encounter who was observed more than two times always ranging alone at the surrounding camp research. this individual langur always observed around the group 2, so we notes the appearance at group 2. we have assumed that the alone male langur is a subadult individual and was rejected by the original group. it can be indicated and visible from body-size and genitals that are not too big and clearly visible. following is the group composition of langurs inhabiting the study site during the preliminary study (table 1). table 1 composition and time of appearance of three colored langurs in bukit semujan. group time of appearance 1. 12/7/2021 16/7/2021 18/7/2021 10 10 12 2. 10/7/2021 11/7/2021 12/7/2021 22/7/2021 24/7/2021 25/7/2021 26/7/2021 27/7/2021 5 2 5 1* 3 4 2 1* 3. 18/7/2021 7 notes: *alone male biotropia vol. 30 no. 2, 2023 210 habitat characteristic the habitat of the three colored langurs in bukit samujan, lupak mawang resort has been distinguished in two distinct ecosystems, namely, the primary forest, and the mixed forest that occurs between swamp and sub-hill forest. based on the results of vegetation identification in both ecosystems, there were 27 plant species that were found directly consumed as langur feeds. there are three species for each growth level that have the highest value index in both ecosystems, which signifies the cruciality of these species and its relevance in langur feeds for their future regeneration. the types of vegetation in the primary forest with the highest index value have been depicted in table 2 below. habitat characteristics of primary forest is that it has thicker and firmer tree canopy than mixed forest. these characteristics not only play a significant role as a feed source, but also functions as a sleeping tree and aids in easier movement during their locomotion (febriyanti 2008). arboreal primates will choose sleeping trees based on the proportion and thickness of branches (giovana 2015). three colored langurs use the lush and tall canopy to acquire shelter from the sun during the day. the mixed forest has a good continuity canopy but is divided into several segments. it restricts langurs’ movements to lower planes and constraints them to rotate to reach other tree crowns. table 2 highest index value of vegetation in primary forest growth level/habitus local name primary forest mixed forest scientific name inp (%) local name scientific name inp (%) seedling resak garcinia lateriflora 73,93 blaban bukit syzygium rostratum 54,87 keratih bukit cleistanthus sumatranus 43,50 keranji bukit garcinia rostrata 41,77 kretih shorea sp. 28,73 ubah bukit syzygium laxiflorum 12,87 sapling kemerawan d. rappa 22,71 ubah bukit syzygium laxiflorum 29,59 blitan fordia splendidissima 18,89 blaban pepah ptychopyxis bacciformis 21,85 resak garcinia lateriflora 18,89 kebesi memecylon myrsinoides 20,54 pole resak garcinia lateriflora 59,11 engkupak ptychopyxis bacciformis 45,97 masam unidentified 30,11 pau pimelodendron griffithianum 41,31 keranji tikus xerospermum norohianum 30,08 sikup bukit gardenia sp. 38,04 tree medang drepananthus havilandii 35,84 kelangsau dryobalanops lanceolata 59,67 mengkirai dryobalanops lanceolata 32,15 cempedak air artocarpus teysmannii 26,07 resak bara cleistanthus sumatranus 22,77 belaban whiteodendron moultonianum 18,41 figure 3 canopy projection of three colored langur’s habitat (a) mixed forest (b) primary forest preliminart study: feeding ecology and daily activity of three colored langur – santoso et al. 211 species and feed availability based on the results, there were 9 feed species that were recognized to be directly consumed by langurs in the study area during the observation, and the remaining 18 species were acquainted with the local community information. for the preliminary study, this is a big potential for diverse information and data about three colored langur’s feed species, especially during fruiting season. the following table contains the species list of langur’s feed and their respective fragments that were consumed during the preliminary study (table 4). the myrtaceae family tree was found to be the chief feed resource among the tree species. previous research found 19 species of feed trees in the same location. the dominant feed belonged to clusiaceae, moraceae, anacardiaceae, and euphorbiaceae (musyaffa 2020). the types of feed highly preferred were gita susu (willughbeia coriacea), merepat (unidentified), and karet (hevea brasiliensis). based on these feed data, there is some unusual feed like h brasilliensis. h brasiliensis was present a long time before the study areas have become a national park, and previous status as a nature and wild reserve from 1981–19831. h brasiliensis was planted by the local community that has indigenous land surrounding the bukit semujan. nevertheless, consumption of rubber seed during the preliminary study was staggering, however, field observations confirmed that langurs not only eat the seeds but also the young leaves of the rubber tree. however, langurs can only consume the already planted ones now due to the new regulations set by the national park that restrict further planting of rubber trees in the protected land area. table 4 feed species and their consumed parts no. local name scientific name habitat habitat type parts eaten leaves fruit seed 1 tekam padi* polyalthia insignis annonaceae swamp √ 2 buah gita susu* willughbeia coriacea apocynaceae mixed forest √ √ 3 resak* garcinia lateriflora clusiaceae mixed forest √ 4 keranji bukit garcinia rostrata clusiaceae mixed forest √ 5 kenarin diospyros sp. ebenaceae swamp √ 6 karet* hevea brasiliensis euphorbiaceae cultivation land √ √ 7 temau cratoxylum grauncum hypericaceae swamp √ 8 putat barringtonia sp. lecythidaceae swamp √ √ 9 engkurung* grewia paniculata malvaceae swamp √ √ 10 empakan durio kutejensis malvaceae mixed forest √ 11 kebesi pternandra galeata melastomaceae mixed forest √ √ 12 ara* ficus spathulifolia moraceae secondary forest sub-hill forest √ 13 tenggelam timbul syzygium havilandii myrtaceae swamp √ 14 engkuni baccaurea parviflora phyllanthaceae swamp √ √ 15 sikup pantai gardenia sp. rubiaceae swamp √ 16 entangis ixora sp. rubiaceae swamp √ √ 17 sibau* nephelium unicatum sapindaceae mixed forest √ 18 kemerawan lempung* dipterocarpus rappa dipterocarpaceae mixed forest √ 19 merepat* unidentified unidentified mixed forest √ √ 20 tawun unidentified unidentified mixed forest √ 21 insubal bukit unidentified unidentified mixed forest √ 22 pregi bukit unidentified unidentified mixed forest √ 23 terap artocarpus odoratissimus moraceae mixed forest √ 24 merbemban xanthophyllum affine polygalaceae swamp √ 25 masung syzygium claviflora myrtaceae swamp √ √ 26 peregi unidentified unidentified mixed forest √ √ 27 jijab syzygium sp. myrtaceae mixed forest √ *based on primary observation data, and the local name refers to the melayu language by community within the study site 1 zonation books of danau sentarum national park, 2014 (not published) biotropia vol. 30 no. 2, 2023 212 figure 3 several documentations of feed species that are eaten by lutung sentarum such as a. grewia paniculata; b. bark from rubber seed (hevea brasiliensis), c. bark from buah gita (willughbeia coriacea). the feed part for consumption included leaves, fruits, and seeds. three colored langurs primarily consumed leaves (50%) in comparison to other parts, such as fruits (30%), and seeds (20%). the genus prebytis is one of the primates that eat fruits and leaves, however mainly prefers leaves parts (sumarni 2016). presbytis chrysomelas ssp cruciger is the same as their relative species in one genus, namely p. comata and p. hosei (ruhiyat 1983; mitchell 1994). the type of feed consumed by three colored langurs was dominant from tree species (89%) and lianas (11%). three colored langurs chiefly consumed leaf of rice tekam (polyalthia insignis). some leaf species have a complete source of nutrients including protein, carbohydrates, fat, tannins, and water (zulfahri and pohan 2016). the species that consumed the fruit were gita susu (willughbeia coriacea), sibau (nephelium unicatum), and karet (hevea brasiliensis). three colored langurs usually consume fruits that are small to adequately sized with range diameter 0,5 – 5 centimeter and lightly colored. primates did like fruits with hard skin, cracked, and yellow to brown color (leighton & leighton 1983). in addition to the leaves and fruits, three colored langurs also consume the seeds of several types of feed species. seeds of karet (hevea brasiliensis) are a rich source of forage for langurs. the water, protein, fat, and crude fiber content is beneficial for the optimum metabolism required for the growth of langur (syamsunarno & sunarno 2019). the potential of feed availability in both habitats can be determined through the relative density of each species. it was influenced by physical and biotic habitat factors and disturbances from destructive activities (violita et al. 2015). the relative density of a plant species will determine the dominance of that species in a community (putri & sudrajat 2017). the results of the relative density of forage plant species in the primary forest are shown in table 5. table 5 relative density of feed plant species in primary and mixed forest growth level type of ecosystem name species relative density (%) seedling primary forest resak garcinia lateriflora 36,4 kebesi memecylon myrsinoides 22,4 sibau nephelium unicantum 0,7 mixed forest keranji bukit garcinia rostrata 31,2 kenarin diospyros sp. 1,9 engkurung grewia paniculata 1,1 sapling primary forest kemerawan d. rappa 18,3 resak garcinia lateriflora 10,2 karet hevea brasiliensis 2 keranji itea macrophylla 2 resak batu cleistanthus sumatranus 2 mixed forest kebesi bukit pternandra galeata 11 kebesi m. myrsinoides 6,7 engkunik barringtonia macrostachya 6,1 kenarin diospyros sp. 6,1 keranji bukit garcinia rostrata 5,5 engkurung grewia paniculata 0,6 preliminart study: feeding ecology and daily activity of three colored langur – santoso et al. 213 pole primary forest resak garcinia lateriflora 30 keranji tikus xerospermum norohianum 10 keranji bukit garcinia rostrata 10 keranji itea macrophylla 5 mixed forest keranji bukit garcinia rostrata 10,3 engkurung grewia paniculata 9,6 kenarin diospyros sp. 9,4 sikup bukit gardenia sp. 8,2 engkunik dehaasia caesia 5 tree primary forest resak garcinia lateriflora 10,9 keranji itea macrophylla 2,7 terap tidak diketahui 1,3 keranji bukit garcinia rostrata 1,3 mixed forest engkurung grewia paniculata 4,7 sikup rimba garcinia rostrata 4,7 sikup bukit gardenia sp. 4,7 karet hevea brasiliensis 2,3 engkunik dehaasia caesia 2,3 the relative density of feed plants in the primary forest was 9 species of seedlings, 20 species of saplings, 11 species of poles, and 23 species of trees. the seedling and sapling density could be an indicator of feed availability in the future, while the pole and tree level indicate the current availability of feed (shankar 2001). the feed species with high regeneration capacity in a primary forest is resak (garcinia lateriflora). this species has a high relative density at every growth level. on the other hand, keranji (itea macrophylla) has an overall high relative density at all growth stages, except the seedling level. meanwhile, the relative density of mixed forest was 16 species of seedlings, 16 species of saplings, 13 species of poles, and 18 species of trees. the feed species with high regeneration capacity is engkurung (grewia paniculata). the other species with good regeneration and inadequate growth rates were keranji bukit (garcinia rostrata) and kenarin (diospyros sp.). then, the species that did not have good regeneration were sikup rimba (garcinia rostrata) and karet (hevea brasiliensis). the species like g panculata, g rostrata and diospyros sp with high regeneration need to increase, and species with low regeneration either. daily activity lutung sentarum begins activity at dawn (06.00) until early evening (18.00), adding the total time for their activity to 320 minutes. this diurnal primate will give signal communication to a movement for their group, called a morning call. this behavior exhibits in other species too such as p. thomasi, which emits a morning call from a sleeping tree (wich et al. 2002). during midday between 12.00–13.00, the three colored langurs will tend to find a place for rest after foraging in the morning. foraging in the morning would increase body temperature with air temperature (prayogo 2006). their activities were divided into foraging, moving, resting, and social. the percentage of each activity is divided into three parts of times, namely morning (06.00–10.00), afternoon (10.01–14.00), and afternoon (14.01– 18.00) in figure 3. biotropia vol. 30 no. 2, 2023 214 figure 4 daily activities of three colored langurs at different time three colored langurs allocate more time to do certain activities, especially social behavior and foraging in the morning. some activities usually coincide with another activity. in addition, the langurs will keep moving until they find suitable feed trees that the group needs (figure 4a). the movement gesture of langurs was rarely quadrupedal, walking using all four legs and arms. this movement could be down and up or to move to other trees by jumping. an adult male as the group leader always led the group movement. however, in some cases, it has been found that adult females with babies would lead the group movement. in all circumstances, juvenile or young individuals are restricted from leading the group (nursal 2001). lutung sentarum will rest more during the day after foraging (figure 4b). this can be attributed to several factors, ranging from the process in the body’s metabolism after eating to the influence of air temperature that affects body condition (alikodra 1990; prayogo 2006). lutung sentarum spent 1–2 hours resting and sleeping during the day. the rest location usually chosen had a sturdy branch or a dense canopy, such as mengkirai (dryobalanops lanceolata) species. the resting position of the lutung sentarum includes hunched sitting with one hand holding the trunk or branch of a nearby tree. it is also found in other primates, namely trachypithecus auratus. the positions found were bending over with the head tucked into the stomach between the two knees of the legs, the soles of the feet overlapping each other and the hands holding the branches (giovana 2015). preliminart study: feeding ecology and daily activity of three colored langur – santoso et al. 215 figure 5 three colored langurs’ behavior (a) moving (b) resting the activity of the lutung sentarum in the afternoon dominates by social behavior, namely a collection of behaviors carried out by two or more individuals and interconnected to survive. this activity could be carried out with other species. these activities are divided into agonistic and affiliative (sajuthi et al. 2016). agonistic is a form of negative response to something like fights and coalitions. as for affiliation, it is a form of positive response such as grooming, playing, vocalization, and sexual activity. based on the analysis, the social activity amount to 66 times with a total duration of 72 minutes. agonistic is the most active behavioral pattern (48.48%) of social activities. this behavior is a gesture of response to becoming steady and grinning position. the group leader (alpha male) would exhibit this position with vocalizations. this posture aims to display objects that make langur feel threatened and to repel. usually, this position can be sitting or bending the body toward other species to make the langur look big. chimpanzees (pan troglophytes) also exhibited it by straightening their body hair to make their posture look bigger and more dangerous (van hooff 1973). vocalization is an expression by making sounds for other interspecies and environments (irawan 2011). the vocals of three colored lutung sentarum were identified into two categories, its sounds with loud and long characteristics aimed at disturbance as a form of threat. then the voice with a shorter duration is addressed to group members as a form of disturbance warning. both types of vocalizations belong to loud calls, vocalizations that are loud and prominent in the primate vocal repertoire (delgado 2006). in addition, there are many functions of vocalization, namely as a marker of social status, efforts to maintain food sources, maintaining territory between groups, and maintaining cohesion between group members (wich et al. 2002, wich et al. 2003, wich &nunn 2022, chiarello 1995, riley 2005). the playing activities most carried out by young males, included chasing and wrestling each other. playing will train the motor nerves to avoid predators, protect themselves, and attract partners (spinka et al. 2001). then for grooming, it is usually done by adult female individuals to their babies. this activity shows proximity to each individual in groups. it is done by treating and searching for lice on the ears, neck, shoulders, and back with the hands, feet, teeth, and tongue (napier & napier 1985). grooming is also good for health maintenance (zamma 2002). activities that were not found in this research were sexual. the fundamental of living things was to obtain and maintain populations of their species according to the maturity level of an individual. lutung sentarum are polygamous and practices polygynous mating system, so mating does not refer to the mating season like other species, namely macaca fascicularis (setiawan 2002). stratum use on daily activity based on the encounters with three colored langurs, their distribution comprehends several areas of primary and mixed forest. this biotropia vol. 30 no. 2, 2023 216 movement has been influenced by factors of feed, shelter, and a safe place for langur groups. during afternoons, three colored langurs tend to move to the hills because of the tall trees that are more than 30 meters in height with lush canopy. using the stratum of three colored langurs in the primary and mixed forest had different purposes based on their activities. trees for three colored langurs as arboreal animals are important, especially for rest, selfprotection, foraging, mating, and socializing. the results showed that most of the canopy proportions used by three colored langurs were stratum c (70.49%) and stratum b (27.87%). it was similar to the previous study in which three colored langurs used stratum c (78%) dominantly (musyaffa 2020). the stratum utility is presented in figure 5. stratum c was a place for foraging activity that included seven encounters with the langur group there. the longest resting time is about 103 minutes. mengkirai (dryobalanops lanceolata) is one of the sleeping trees, and the langur group was found at an altitude of 30 meters on the mengkirai tree. in addition, there was a case in this study that lutung sentarum would descend to the stratum e (ground) to pick up fallen food. the moving activity of three colored langurs has been carried out in stratum b and c for supply needs that avoided disturbance. the canopy has a function to make it easier for the lutung sentarum to move through connected tree branches. other primates, such as the javan gibbon, use a dense canopy to support their cruising range (zanuansyah 2013). figure 5 stratum utility based on three colored langurs’ activities figure 6 stratum projection in ranging pattern (a) mixed forest (b) primary forest preliminart study: feeding ecology and daily activity of three colored langur – santoso et al. 217 in addition, the social activities mostly had a similar stratum to other activities. the langur appearance on stratum c and b amounted to 19 times and five times during the research respectively. vocalization was the most common activity; the sounds were louder and echoed further. other species, such as the trachypithecus auratus will choose trees with upper-middle crowns for vocalization so that the sound echoes further and clearer (oktaviani 2009). the use of ranging patterns by three colored langurs in primary and mixed forest habitats is exhibited in figure 6. in addition, this habitat is inhabited by other species, especially in the primate class. according to ecological theory, the concepts of niche and resource competition are central to the coexistence of sympatric species (gause 1934; tokeshi 1999). conservation implication for conservational efforts, feeding ecology is crucial including the type and composition of feed in its natural habitat not only for better understanding but also to conduct habitat enrichment, security and further research related to bio-ecology and conservation of langur in the natural habitat by the long-term national park managers and interested parties. as we are aware, primates do not receive the required research attention and are often neglected due to arduous and restricted collection of primary data in their habitat. this preliminary study is a progressive step forward toward future research and to draw the attention of researchers and observers to conduct numerous studies on primate species that are currently lacking the bio-ecology data. conclusion the habitat of the three colored langurs in bukit semujan, lupak mawang resort was discovered in both primary as well as mixed forests. the feed species for three colored langurs in both habitats amounted to 27 species, and the highly preferred are gita susu (willughbeia coriacea), merepat (unidentified), and karet (hevea brasiliensis). its feed existed in study areas because of prior sowing by the local community before the land was distinguished as part of the national park. meanwhile, the highly preferred feed compositions were leaves (50%), fruits (30%), and seeds (20%). the highly preferred stratum for their activities was stratum c (70.49%) and b (27.87%). the identification of vegetation species for resting or sleeping was mengkirai (dryobalanops lanceolata). the highest daily activity of three colored langurs is divided into three parts of time, the morning dominated by social activity (44.26%), the day dominated by resting 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http://dx.doi.org/10.1163/15685390252902292 https://link.springer.com/journal/265 https://link.springer.com/journal/265 https://doi.org/10.1007/s00265-002-0541-8 https://doi.org/10.1007/s00265-002-0541-8 https://doi.org/10.1046/j.1439-0310.2003.00837.x https://doi.org/10.1007/bf02629575 https://doi.org/10.1007/bf02629575 the southeast asian journal of tropical biology vol. 31 no. 2, 2024: 134 145 doi: 10.11598/btb.2024.31.2.1741 issn: 0215-6334 | e-issn: 1907-770x 134 carbon sequestration in agrosilviculture agroforestry systems: preliminary results from three villages in uttaradit province, northern thailand chattanong podong1*, krissana khamfong1, supawadee noinamsai2 and sukanya mhon-ing3 1department of environmental science, faculty of science and technology, uttaradit rajabhat university, thailand 53000. 2department of environmental science, faculty of science and technology, phibulsongkarm rajabhat university, thailand 65000. 3department of biology, faculty of science and technology, uttaradit rajabhat university, thailand 53000. article higlights • agroforestry boosts carbon storage, reducing greenhouse gas emissions significantly. • diverse tree-based farming enhances biodiversity, soil health, and climate resilience. • carbon sequestration in agroforestry supports sustainable agriculture and environmental balance. • agroforestry practices mitigate climate change by storing carbon in trees and soil. • combining trees with crops provides multiple ecological and economic benefits. abstract one of the processes for compensating greenhouse gas emissions is atmospheric carbon removal and storage in the terrestrial biosphere. agricultural systems to which trees are returned for careful management alongside crops and animals are thought to be substantial co2 sinks. people are increasingly realizing the importance of agroforestry because it is good for the environment and farming. in this study, total carbon pools from the aboveground biomass carbon (abgc), forest floor carbon (ffc), and soil organic carbon (soc) were investigated and carbon storage data for some agroforestry practices native to uttaradit in northern thailand were analyzed. the role of these carbon pools in reducing co2 concentrations in the atmosphere was also discussed. the results showed differences in the total carbon stock sourced from traditional agroforestry (taf), applied agroforestry (aaf), and developed agroforestry (daf). the total carbon store (abgc + tffc + soc) of taf, aaf, and daf was 267.05 mg c/ha, 226.48 mg c/ha, and 324.70 mg c/ha, respectively. soc contributed 47.64%, 54.26%, and 44.81% and abgc contributed 22.75%, 19.79%, and 23.90% to the total carbon stock in taf, aaf, and daf, respectively. the co2 adsorption was 979.27 mg co2/ha, 830.50 mg co2/ha, and 1,190.6 mg co2/ha in taf, aaf, and daf, respectively. it is clear that agroforestry systems serve as carbon sinks in terrestrial ecosystems. although the comparison of agroforestry practices and other land use types is important for carbon mitigation and the implementation of the “land use, land use change, and forestry” concept for co2 sinks, it is also crucial to compare the potential of carbon sequestration in different co2 pools. keywords: agroforestry, agrosilviculture, carbon sequestration, carbon stock, thailand introduction land use change is one of the most important sources of global human carbon emissions (ipcc 2014) and is intricately tied to biodiversity and the biogeochemical cycle (romshoo 2004; nagendra et al. 2013). human caused climate change has a negative impact on a wide variety of industries and populations, most notably those that rely on rain-fed agriculture. according to the world meteorological organization (2007), increased atmospheric concentrations of greenhouse gases (ghgs) are the primary cause of climate change. the increasing concentration of the ghg carbon dioxide (co2) in the atmosphere has a substantial effect on the global climate (malhi et al. 1999). article information received 19 january 2023 revised 12 may 2024 accepted 22 may 2024 *corresponding author, e-mail: chattanong.pod@uru.ac.th research article https://doi.org/10.11598/btb.2024.31.2.1741 carbon sequestration in agrosilviculture agroforestry systems podong et. al. 135 the current concentration of co2 in the atmosphere is at least 400 parts per million. the enormous increase in the co2 concentration has resulted in an increase of 0.17 °c every decade in the earth’s average temperature. deforestation and forest degradation are the primary sources of ghg emissions in the majority of tropical countries. thus, the terrestrial ecosystem’s role in the global carbon cycle has roused the interest of researchers and policymakers alike. the exchange between the atmosphere and vegetation is bidirectional, with co2 fixation into biomass by photosynthesis roughly balanced by co2 emission via decomposition and burning. each year, around 60 pg of carbon is transferred (both ways) between terrestrial ecosystems and the atmosphere, resulting in a net terrestrial uptake of 0.710 pg c (lasco 2002). as widely known, the near-surface air and oceans of the earth have been warming in recent years and are likely to continue to do so in the future (asako 2007). a critical method of mitigating climate change aims to reduce the concentrations of ghgs, notably co2, in the atmosphere. this is accomplished via the carbon sequestration process (nair 2011), in addition to lowering emissions at their source. carbon sequestration is possible in terrestrial ecosystems, such as forests and agroforests, as well as woodland and scrubland. agroforestry is the practice of combining trees with crops or pasture (nair et al. 2010). it is a treebased agricultural approach adopted over many years in a variety of countries, including thailand. over the last four decades, agroforestry has gained recognition as an integrated strategy for sustainable land use due to its productivity and environmental benefits. it has received increased attention as a result of its recent certification as a kyoto protocol global climate change mitigation strategy (nair et al. 2009). the majority of carbon in trees and shrubs is stored as aboveground biomass, with 50% of the total biomass acting as a carbon sink (aklilu et al. 2015). the aboveground carbon (abgc) stock is the estimated quantity of carbon that makes up 50% of the total vegetative biomass (icraf 2006; lal 2005). belowground biomass, the fraction of vegetation occurring belowground as roots, accounts for around 25-30% of the aboveground biomass, depending on the species, root structure, and ecological conditions (icraf 2006; lal 2005). kumar and nair (2011) defined total biomass carbon as the accumulation of both aboveand belowground carbon in vegetation. agroforestry is an umbrella term that refers to a diverse range of systems and approaches in which parklands are one of the practices. typically, parklands are defined as landscapes of cultivated or recently fallowed fields interspersed with mature trees (abdelkadir & bishaw 2003) or as the coexistence of woody plants and grasses in subtropical and tropical savanna habitats (bayala et al. 2006). individual trees and shrubs are planted across vast swaths of cropland, while understory crops are cultivated. some of these trees were left behind after the natural forest was converted to other land uses; others regrew after farmers cleared the land and still others are intentionally retained or planted on farms (icraf 2006) to provide a range of products and services, including soil structure enhancement, local temperature regulation, erosion risk reduction, and carbon sequestration. agroforestry systems increase smallholder farmers’ resilience by enhancing water efficiency, the microclimate, soil productivity, and nutrient cycling, facilitating the management of pests and diseases, increasing agricultural production, and diversifying and increasing farm income, all the while sequestering carbon (lasco et al. 2014). carbon pools can be managed more effectively by better understanding the aboveground biomass of trees and soil organic carbon reserves. however, additional research is necessary before agroforestry systems are considered for inclusion in global carbon sequestration agendas (nair et al. 2010). agroforestry technologies and techniques help to address a range of economic and environmental concerns. to date, the majority of research on agroforestry systems has been on their spatial design, food production, soil fertility management, and system interactions, with little emphasis on their ecosystem services, such as biodiversity protection and carbon sequestration (negash 2013). biotropia vol. 31 no. 2, 2024 136 uttaradit province is an important agroforestry area in the northern region of thailand. agroforestry is implemented in three districts, mueang, laplae, and tha pla. most of the agroforestry areas are located on high mountains. the highest elevation of the agroforestry sites is 700 masl, with a slope of 5-100%. one of the factors influencing the growth in agroforestry in thailand is the change in the agricultural system from subsistence to commercialization due to population increase. the cultivation of cash crops, such as durian and longkong, has increased after the transformation of the original forest for planting fruit trees, thus causing the forest area to rapidly decrease. the ecological transition from the original natural ecosystem to an agricultural ecosystem causes land degradation due to continuous monocultures and horticultural crops, resulting in lower crop yields year after year and higher production costs. this study collected and documented critical data on the significance of parkland agroforestry for the uttaradit province’s climate change mitigation efforts through carbon sequestration. this study will contribute to the understanding of how to conserve these unique agroforestry systems and their ecosystem services to local communities, such as food production and income, soil and water protection, and soil fertility maintenance, as well as other services, such as carbon sequestration for climate change mitigation and conservation of biodiversity (negash 2013). the purpose of this study was to ascertain the carbon storage capacity of agroforestry parklands in uttaradit province, northern thailand. it evaluated the carbon stored in the study area’s various agroforestry types, aboveground and belowground biomass, and soil carbon pools and assessed the tree species with the highest carbon stock potential in the study area. materials and methods site description the study area was located in uttaradit province, thailand, and consisted of three sites: the mae phun subdistrict, laplae district (lpaf); the ban dan na kham subdistrict, mueang uttaradit district (dk-af); and the nang phaya subdistrict, tha pla district (np-af). the study areas are shown in figure 1. figure 1 the locations of the study areas in the three villages investigated in uttaradit province, thailand carbon sequestration in agrosilviculture agroforestry systems podong et. al. 137 traditional agroforestry (taf), age 20 years traditional agroforestry is the indigenous understanding of agroforestry practices passed down through generations of farmers. in taf, the plants thrive in a variety of climates and terrains. it is easy to practice and requires minimal capital, especially in inhabited or abandoned regions. the inhabited land is tended by weeding undesired plants in specific locations and sowing seeds (in rows or non-rows) of desired plants. because these plants do not require much maintenance and care to develop shoots and root systems, their chances of survival are greater than those of grafted plants or cuttings. unwanted plant species that are introduced or purposefully left to compete for growth can also be eventually harvested and used, yielding benefits comparable to those of “ladang” in indonesia, where these plants are used to construct dwellings and animal shelters and for fuel. applied agroforestry (aaf), age 18 years the aaf system enhances the structure and quality of production to meet market demands under current circumstances. this type of agroforestry is sometimes a continuation of taf. the canopies of indigenous cultivars grown from seed can be converted to be similar to those of other popular cultivars by altering persistence, bark, branches, shoots, etc. these alterations are considered to conserve the original species at the same time, because when the altered part is cut off, the relevant component of the original species grows to replace it. the garden’s cross-sectional structure is diminished as a result of this alteration. harvesting is simple. revenue may increase since the goods can be sold at a higher price because the canopy has been changed to a variety that the market requires. developed agroforestry (daf), age 15 years daf is a modern agroforestry system that combines current techniques and approaches to forest management with agroforestry. for example, the planting of the garden may start with digging, grading, and setting up and drilling holes in rows. then, good plant material obtained as cuttings or grafts are planted in the prepared soil in alternate rows, alternating strips, or a combination of the two. this form of agroforestry plantation has a structural profile of less than 20 meters. the canopy layer and the age layer are very similar. an irrigation system and a production control scheme may be necessary depending on needs. sampling and data collection methods a survey of the plants in the different agroforestry regimes was carried out from december 2020 to may 2021 using the plant community analysis method. using stratified random sampling, 15 sample plots, each of 0.16 ha (40 m × 40 m), were established on the summit, shoulder, and foot slopes of the site. the altitudinal range of these plots was 700-900 masl. each plot was divided into 16 subplots (10 m × 10 m). the data collected included measurements of stem girth over bark at breast height (1.3 m above the ground) and the heights of all tree species taller than 1.5 meters, as well as of seedlings, undergrowth, climbers, other vegetation, and standing dead trees. in addition, litter from all plants growing on the surface was collected and weighed. litter samples were ovendried at 80 °c for 48 hours or until reaching a constant weight and then weighed. tsutsumi et al. (1983) used allometric equations to determine the amount of plant biomass of each agroforestry system. they used the biomass equations for mixed forests based on the mixed deciduous-dry evergreen forest of chai ya phum province, as follows: ws = 0.0509 (d2h)0.919 r2 = 0.978 wb = 0.00893 (d2h)0.977 r2 = 0.890 wl = 0.0140 (d2h)0.669 r2 = 0.714 wr = 0.0313 (d2h)0.805 r2 = 0.981 where: ws = tree stem biomass (kg) wb = branch biomass (kg) wl = leaf biomass (kg) wr = root biomass (kg) d = tree stem diameter over bark at 1.30 m above ground (cm) h = tree height (m) biotropia vol. 31 no. 2, 2024 138 to estimate soil organic carbon (soc), soil samples were collected from a depth of 0-15 cm and of 15-30 cm at five random locations in each 50 m × 50 m plot. after air drying and crushing, the soil samples were sieved through a 2-mm mesh sieve, removing roots, other plants, and garbage. the soc content was determined using the walkley and black technique (walkley & black 1934). the bulk density of the soil was determined using undisturbed soil samples. the abgc store per unit area (mg c/ha) and the biomass content of all plants were measured. the amount of carbon stock was calculated by multiplying the aboveground and belowground biomass by 0.47 (ipcc 2006). sequestered carbon dioxide the absorption rate of co2 can be calculated as the carbon stock in biomass multiplied by the weight of a molecule of carbon. co2 is composed of one molecule of carbon and two molecules of oxygen. the atomic weights of carbon and oxygen are 12.00 and 15.99, respectively. therefore, the weight of co2 is 44.01. the ratio of co2 to c is 44.01/12.00 = 3.67. to determine the weight of co2 sequestered in the carbon stock, the weight of carbon must be multiplied by 3.667 (mcpherson 1998). the equation to calculate co2 sequestration is as follows:  sequestered co2 = cs × 44.01/12.00 where: cs = carbon stock (t/ha) c = atomic weight of carbon evaluation of carbon stock in different parts of the agroforestry system the carbon stocks in different parts of trees and in soil were determined with the adjusted equations for carbon stock in plants (vashum & jayakumar 2012), litter, and trees (zheng et al. 2008) as follows: where: cst = carbon stock (t/ha) = stem carbon stock = branch carbon stock = leaf carbon stock = groundcover plant carbon stock = litter carbon stock = soc of the agrosilviculture agroforestry system results and discussion characteristics of agrosilviculture types in practice the diameter at breast height (dbh) of trees in the agroforestry system appeared to be higher than those in other forest types in general. taf, aaf, and daf systems in uttaradit province had a dbh of 12.16±.84 cm, 86.89±6.81 cm, and 27.60±7.38 cm, respectively (table 1). the average dbh of tropical moist deciduous forest individuals was found to be lower; sal-dominated forests had a dbh of 18.4 cm (ranged from 5.09 cm to 86.62 cm), whereas tropical moist deciduous forests had a dbh of 18.25 cm (ranged from 5.09 cm to 93.63 cm) (manas et al. 2020). type density (tree/ha) mean slope (%) mean elevation (m) diameter at breast height (cm) min max mean ± sd taf 532 30 321 1.11 26.12 12.16±7.84 aaf 387 32 445 10.18 38.12 26.89±6.81 daf 350 31 683 13.30 36.11 27.60±7.38 note: taf = traditional agroforestry; aaf = applied agroforestry; daf = developed agroforestry. the association between average dbh and tree density showed that due to increased competition between trees for resources, such as nutrients, space, and sunlight, the trees growth rate can be slower in dense forests (ram et al. 2020). similarly, takahashi et al. (2018) also noted the large dbh and low tree density of evergreen conifers in the shizumo forest reserve. table 1 agroforestry plots in types of agrosilviculture agroforestry in practice carbon sequestration in agrosilviculture agroforestry systems podong et. al. 139 species richness of a degraded forest is the result of the responses of different species to disturbances (sagar et al. 2003). the majority of plant species observed on site were similar to those found in uttaradit province’s general agroforestry and mixed deciduous forest. in our study, durio zibethinus l., nephelium hypoleucum kurz, adenanthera pavonina l., and garuga pinnata roxb. were examples of taf trees. lansium domesticum corrêa, aglaia dookkoo griff., durio zibethinus l., and tetrameles nudiflora r.br. were examples of aaf trees.durio zibethinus l., bombax ceiba l., lansium domesticum corrêa, and albizia odoratissima (l.f.) benth. were examples of daf trees. according to the community land allocation project, the agroforestry system in chiang mai province has 243 plant species, of which 144 are woody perennial tree species and 99 are cultivars of agricultural crops (pongpichai et al. 2019). taf methods revealed the presence of the east kalimantan natives langsat (lansium domesticum corrêa) and durian (durio zibethinus l.) in kampung birang and kampung merabu, respectively (hartoyo et al. 2016). aboveground and belowground biomass in types of agrosilviculture agroforestry in practice the carbon stock of parkland agroforestry methods differed significantly between agroecological regions, as biomass was influenced by stand age, tree species and structure, management approaches, diversity, and composition (chave et al. 2004). the proportion of aboveand belowground carbon stocks in agrosilviculture agroforestry of varying practice types is shown in table 2. agrosilviculture agroforestry systems in uttaradit province, northern thailand, stored the most carbon in daf at 101.58 mg c/ha, followed by 79 mg c/ha in taf and 58.76 mg c/ha in aaf. the carbon content of total tree organic carbon in daf, taf, and aaf was 96.27 mg c/ha, 71.74 mg c/ha, and 52.54 mg c/ha, respectively. biomass and carbon stock estimation showed 192.99 mg c/ha in aboveground biomass and 96.50 mg c/ha carbon stock in trees, herbs, and shrubs in the nambor wildlife sanctuary in assam’s golaghat and karbi anglong districts (krishna et al. 2019). in this study, taf had the highest total carbon stock in the undergrowth at 3.54 mg c/ha, whereas aaf had the lowest at 3.14 mg c/ha. forestry sector ghg inventories require accurate estimations of leaf litter and dead debris carbon (ipcc 2006). taf had the largest carbon stock in litter at 3.72 mg c/ha, followed by aaf at 3.08 mg c/ha and daf at 2.76 mg c/ha (table 2). while preservation of pasture/ grassland after slash-and-burn cultivation resulted in a progressive reduction in total system carbon stocks, agroforestry systems, such as bush fallows and agroforests, accumulate approximately 60% of initial forest carbon stocks in approximately 30 years (patrick et al. 2005). although each line of trees can create a significant quantity of litterfall and root biomass, hedgerows or windbreaks may not contribute much to the buildup of soil carbon at the field level due to the comparatively small fraction of area covered by trees. according to rao et al. (1998), boundary tree plantings have a maximum effect of 10 meters on both sides of the boundary. when applied to this scenario, a 50% increase in carbon stocks along a 100-meter tree line results in a 10% increase in carbon per acre. on the other hand, boundary plantings can help enhance soil conditions and, therefore, promote carbon sequestration by enhancing crop yield and minimizing soil loss due to erosion. table 2 carbon stocks (mg c/ha) aboveand below ground in agrosilviculture agroforestry type cs cb cl cr ttc ugc lc ffc abgc tffc taf 39.42 8.07 6.01 18.24 71.74 3.54 3.72 7.26 60.76 79 aaf 28.18 5.62 4.81 13.93 52.54 3.14 3.08 6.22 44.83 58.76 daf 53.57 11.14 7.59 23.98 96.27 2.55 2.76 5.31 77.61 101.58 notes: cs = stem carbon; cb = branch carbon; cl = leaf carbon; cr = root carbon; ttc = total tree organic carbon (cs + cb + cl + cr); ugc = carbon stock of undergrowth, seedlings, and saplings combined; lc = carbon in litter; ffc = forest floor carbon (ugc + lc); abgc = aboveground carbon (cs + cb + cl + ffc); tffc = total aboveground carbon stock; taf = traditional agroforestry; aaf = applied agroforestry; daf = developed agroforestry. biotropia vol. 31 no. 2, 2024 140 soil organic carbon stock agroecosystems contribute significantly to the global carbon cycle, accounting for roughly 12% of all terrestrial carbon (smith et al. 1993; dixon et al. 1994; dixon 1995). soil degradation associated with land use change is a significant source of carbon loss and co2 accumulation in the atmosphere. shifting cultivation, pasture management through paddy culture, nitrogen fertilization, and animal production are all agroforestry activities that contribute to ghg emissions (dixon 1995; le mer & roger 2001). as soil depth increases, the percentage of soil organic carbon tends to decrease. at a depth of 0-15 cm, the highest average soil organic carbon was 2.32% in daf, 2.20% in taf, and 2.09% in aaf, with soil organic carbon percentages ranging from 1.23% to 2.64%. at a depth of 15-30 cm, the highest average soil organic carbon was 2.21% in daf, 1.94% in taf, and 1.78% in aaf, with soil organic carbon percentages ranging from 1.62% to 2.65%. at a depth of 30-60 cm, the highest average soil organic carbon was 2.09% in daf, 1.71% in aaf, and 1.65% in taf, with soil organic carbon percentages ranging from 1.45% to 2.37% (figure 2). this biomass carbon is decomposed by microbial population into leaf litter and soc (ramachandran et al. 2007). in many tropical locations, regular pruning and root turnover have resulted in the accumulation of soil organic matter and nutrient stocks in the soil (lehmann et al. 1998; rao et al. 1998; kumar et al. 2001). in a 12-year hedgerow intercropping trial on nigerian alfisols, g. sepium with leucaena leucocephala increased surface soc by 15% (2.38 mg c/ha) compared to solitary crops (kang et al. 1999). a 12% increase in soc (0.23 mg c/ha) was also seen after five years of hedgerow intercropping with inga edulis in typic paleudult soils in peru (alegre & rao 1996). the average soil ph of aaf, taf, and daf was 7.06±0.07, 6.88±0.05, and 6.95±0.05, respectively. the average soil electrical conductivity (ec) of taf, aaf, and daf was 38.45±1.56 µs, 37.26±1.33 µs, and 37.12±2.08 µs, respectively. the average soil bulk density (bd) of aaf, taf, and daf was 1.14±0.09%, 1.10±0.05%, and 1.08±0.04%, respectively. the soc content of daf, taf and aaf was 2.20±0.12, 1.93±0.28, and 1.86±0.20, respectively. daf had the highest average soil carbon stock, at 145.51±0.55 mg c/ ha, whereas taf and aaf had 127.29±0.96 mg c/ha and 122.89±0.84 mg c/ha, respectively (table 3). soil ph in the chao phraya basin, thailand, was in the range of 5.9 to 6.5 compared to that in the traditional home garden agroforestry system. the majority of the soil phosphorus content is in soluble forms that are available to plants within this range. this indicated that the fertility and ph conditions of home garden soils were good for agricultural purposes. 0.00 0.50 1.00 1.50 2.00 2.50 3.00 traditional applied developed so il or ga ni c ca rb on ( % ) 0-15 cm 15-30 cm 30-60 cm figure 2 soil carbon stock in agrosilviculture agroforestry systems categorized by practice type table 3 soil properties and soil organic carbon in agrosilviculture agroforestry systems type ph ec (µs) bd (%) soc (%) soc (mg c/ha) taf 6.88±0.05 38.45±1.56 1.10±0.05 1.93±0.28 127.29±0.96 aaf 7.06±0.07 37.26±1.33 1.14±0.09 1.86±0.20 122.89±0.84 daf 6.95±0.05 37.12±2.08 1.08±0.04 2.20±0.12 145.51±0.55 notes: mean ± standard error; ec = electrical conductivity; bd = soil bulk density; soc = soil organic carbon; taf = traditional agroforestry; aaf = applied agroforestry; daf = developed agroforestry. carbon sequestration in agrosilviculture agroforestry systems podong et. al. 141 fruits, leaves, young shoots, bark, flowers, and other non-timber goods were some of the products of the traditional home garden agroforestry system. the traditional home garden agroforestry system is not completely harvested, which guarantees that the system’s nutrient export is kept to a minimum (gajaseni & gajaseni 1999). total carbon stock and sequestration the total carbon stock (abgc + tffc + soc) was 267.05 mg c/ha in taf, 226.48 mg c/ha in aaf, and 324.70 mg c/ha in daf. the contribution of soc to the total carbon stock was 47.64% in taf, 54.26% in aaf, and 44.81% in daf. abgc contributed 22.75%, 19.79%, and 23.90% to the total carbon stock in taf, aaf, and daf, respectively (table 4; figs. 3 & 4). co2 adsorption was 1,190.6 mg co2/ha in daf, 979.27 mg co2/ha in taf, and 830.50 mg co2/ ha in aaf (fig. 3). the results showed that abgc, bgc, soc, and total carbon were all positive. in minjar shenkora, the average carbon stock of parkland was 59.65 mg c/ha (reta et al. 2021). in the teak smallholder compensation scheme implemented by inpang agroforestry, which was started by the inpang registered teak carbon bank activity, covering about 300 hectares, the baseline c determined for registered land is 44,801 mg co2 (samek et al. 2011), with each hectare storing about 149 mg co2. the amount of carbon sequestered is mostly determined by the agroforestry system in situ, which forms and functions are influenced by environmental and socioeconomic factors. tree species and system management are elements that also influence carbon storage in agroforestry systems. the carbon storage capacity of agroforestry systems in southeast asia is estimated to be 39-195 mg c/ha in dry lowlands and 12-228 c/ha in humid tropical areas (alain & serigne 2003). if croplands and pastures were rehabilitated by converting to tree-based systems, net abgc sequestration would likely occur, as well as an increase in belowground carbon in the case of farmland conversions. over a 25-year period, carbon sequestration could range from 10 to 70 mg c/ha in vegetation and 5 to 15 mg c/ha in the soil (murdiyarso et al. 2000; palm et al. 2000; hairiah et al. 2001). table 4 total carbon stock and soil organic carbon (mg c/ha) in agrosilviculture agroforestry systems type abgc tffc soc tc taf 60.76 79 127.29 267.05 aaf 44.83 58.76 122.89 226.48 daf 77.61 101.58 145.51 324.70 notes: abgc = aboveground carbon; tffc = total aboveground carbon stock; soc = soil organic carbon; tc = total carbon; taf = traditional agroforestry; aaf = applied agroforestry; daf = developed agroforestry. figure 3 the proportion of carbon stock and carbondioxide adsorption in agrosilviculture agroforestry categorized by practice type notes: taf = traditional agroforestry; aaf = applied agroforestry; daf = developed agroforestry. biotropia vol. 31 no. 2, 2024 142 figure 4 carbon stock in agrosilviculture agroforestry, classified by practice type agroforestry categorized by practice type the potential for rapid carbon sequestration in the humid tropics is predominantly in the vegetation, and to a lesser extent in the topsoil, based on the observed magnitude of changes in carbon stocks. however, less is known about the possible changes in carbon stocks in soil at greater depths. most estimates of changes in carbon stocks have failed to account for root biomass of trees in forests or agroforestry systems, owing to the technical difficulty of precise measurement. agroforestry systems have the ability to sequester some carbon in the root system. in the upper 0-50 cm level of soil, roots in agroforestry systems have been shown to have a time-averaged carbon stock ranging from around 6 mg c/ha for shifting cultivation to about 20 mg c/ha for tree fallows (woomer & palm 1998). rubber agroforests and secondary vegetation also show higher emissions, implying that agroforestry systems may not always minimize soil co2 emissions. in order to measure the net c balance of these systems, emissions must be clearly compared to belowground carbon allocation (patrick et al. 2005). correlation analysis and matrix plot of abgc, total aboveground carbon stock (tffc), soc, total carbon, density, and basal area showed that abgc was positively correlated with density (r = 0.975, p < 0.05), soc (r = 0.948, p < 0.05), and tffc (r = 0.895, p < 0.05) (table 5; fig. 5). notes: cs = stem carbon; cb = branch carbon; cl = leaf carbon; cr = root carbon; lc = carbon in litter; soc = soil organic carbon. abgc tffc soc density basal area abgc 1 tffc 0.895 1 soc 0.948 0.953 1 density 0.975** 0.971 0.853 1 basal area -0.070 -0.054 0.250 -0.291 1 note: ** p value < 0.05. table 5 pearson correlation matrix of aboveground carbon (abgc), total aboveground carbon stock (tffc), soil organic carbon (soc), total carbon (tc), density, and basal area carbon sequestration in agrosilviculture agroforestry systems podong et. al. 143 sequestration is ultimately determined by the fate of the carbon stockpiles that have been increasing over time. carbon sequestration in agroforestry systems is a dynamic process that can be broken down into stages. many systems in a facility are likely to be emitters of ghgs (due to loss of carbon and nitrogen from vegetation and soil). in the next stages, tonnes of carbon are stored in the soil as well as in tree boles, stems, and roots within an extremely short amount of time. a portion of the carbon will return to the atmosphere when the trees are harvested and the land is returned to cultivation (sequential systems) (dixon 1995). if the initial store of carbon has a positive net carbon balance after several decades, then effective sequestration can be evaluated (feller et al. 2001). perennial agroforestry systems (perennial-crop combinations, agroforests, and windbreaks) that allow full tree growth and where the woody component represents a significant portion of total biomass are the only systems that allow carbon storage in plant biomass. in these systems, sequestration can continue even after the wood is harvested. if boles, stems, or branches are transformed into long-lasting items, their carbon storage life can be extended significantly (roy 1999). conclusion agrosilviculture agroforestry practices in uttaradit province, northern thailand, have resulted in a wide range of carbon sequestration rates; that is, the potential for sequestering carbon and other nutrients in daf is high, while the potential for sequestering these nutrients in taf and aaf is much lower. according to these findings, carbon loss in northern thailand’s agroforests is linked to the removal of aboveground biomass, the organic matter in the soil, and even fine root carbon. ratios such as these, are the commonest in advanced forms of forestry; on the other hand, the ratios for taf and aaf are much lower. as a result, if a switch is made from taf to modern agroforestry, the carbon loss from aboveground biomass would outweigh the loss from other carbon pools. similarly, in agroforestry practices, top soil (0-15 cm) has the highest potential for carbon sequestration in soc content. it is clear that agroforestry plays an important role in terrestrial ecosystems as carbon sinks. although understanding the potential of carbon sequestration in different co2 pools is important, it is also crucial to compare agroforestry practices to other land use types because the information gained would be significant for carbon mitigation and the implementation of the “land use, land use change, and forestry (lulucf)” concept for co2 sinks. figure 5 pearson correlation matrix of aboveground carbon (abgc), total aboveground carbon stock (tffc), soil organic carbon (soc), total carbon (tc), density, and basal area biotropia vol. 31 no. 2, 2024 144 references abdelkadir a, bishaw b. 2003. agroforestry and community forestry for rehabilitation of degraded watersheds in the ethiopian highlands. intern symp contemporary devel iss ethiopia 78:52-75 adisti pph, iskandar zs, supriyanto, lilik bp, ida t . 2016. biodiversity, carbon stocks and community monitoring in traditional agroforestry practices: preliminary results from two investigated villages in berau, east kalimantan. procedia environ sci 33:376-85. aklilu b, mikrewongel t, yitebitu m, agena a. 2015. estimation of carbon stored in agroforestry practices in gununo watershed, wolayitta zone, ethiopia. j ecosys ecograph 2015(5):1 alain a, serigne tk .2003. carbon sequestration in tropical agroforestry systems. agric ecosyst environ 99:15-27. alegre jc, rao mr. 1996. soil and water conservation by contour hedging in the humid tropics of peru. agric ecosyst environ 57:17-25. asako t. 2007. carbon sequestration potential of agroforestry systems in the west african sahel: an assessment of biological and socio-economic feasibility. 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0215-6334 | e-issn: 1907-770x 228 a new species of the odontomachus infandus species group (hymenoptera: formicidae) from pangasinan, philippines, with notes on species ecology darrell c. acuña1,2,4* and decier oliver r. mapile1,3 1philippine arachnological society, inc., paco, manila 1007, philippines. 2research center for natural and applied sciences, university of santo tomas, manila 1008, philippines. 3philippine science high school ilocos region campus, department of science and technology, ilocos sur 2728, philippines article higlights • new species of odontomachus • distribution and ecological notes • updated species key article information received 4 october 2023 revised 17 march 2024 accepted 6 may 2024 *corresponding author, e-mail: drrllcn@gmail.com research article abstract a new species of odontomachus latreille, 1804 named o. ampipitbaybay sp. nov. is illustrated and described from specimens collected along the coastal area of alaminos city, pangasinan, luzon island, philippines, with notes on its species ecology. the new species is diagnosed and compared against closely related species from luzon. a distribution map of selected philippine odontomachus species is provided, and a modification to the key to the philippine odontomachus is proposed. keywords: beach habitat, hundred islands, luzon, philippine ants introduction philippine ants are of great interest due to their high richness brought by the geologic history and diverse ecological features of the archipelago. however, only about half of the estimated 1,000 species of philippine ants have been discovered and described thus far, and much of their biology is still unknown (general & alpert 2012). unfortunately, the inherent challenges brought by the country’s archipelagic setting and the few myrmecologists in the field coupled with low funding opportunities produce a poor picture of ant diversity and distribution in the philippines (general 2021). being still understudied, the philippine ant fauna offers many opportunities to researchers. one notable group of ants, especially in rural areas, are the trap-jaw ants from the genus odontomachus latreille, 1804, locally termed ampipit by the ilocano-speaking pangasinenses. these are large ground-foraging ants that are easily recognized by their elongated mandibles, often seen wide open when threatened or ready to capture their prey. this genus comprises 74 valid extant species (antweb 2024; bolton 2024). general and alpert (2012) listed 11 species in the philippines. in sorger and zettel’s (2011) review of this genus, they provided a key for the philippines that contains a different list of 11 species that includes 2 unnamed species. the most recent additions to the philippine fauna are the new species, o. ferminae general, 2018, from sibuyan island (general 2018) and o. pangantihoni zettel & sorger, 2023 from panay island (zettel & sorger 2023). at present, there is a total of 13 valid species in the philippines. in their review, sorger and zettel (2011) distinguished two sets within odontomachus. the first set contains three unrelated species from three species groups, namely o. simillimus, o. malignus, and o. rixosus. the other set includes the infandus species group composed of regionally endemic and often forest-dwelling species. https://doi.org/10.11598/btb.2024.31.2.2083 https://orcid.org/0000-0002-2958-8763 https://orcid.org/0009-0000-8933-2690 new odontomachus species description and ecology from pangasinan, philippines – acuña & mapile 229 before this study, the infandus species group was represented in the philippines by eight described species: odontomachus alius sorger & zettel, 2011, odontomachus banksi forel, 1910, odontomachus ferminae general, 2018, odontomachus infandus smith, 1858, odontomachus pangantihoni zettel & sorger, 2023, odontomachus philippinus emery, 1893, odontomachus schoedli sorger & zettel, 2011, and odontomachus scifictus sorger & zettel, 2011 (bolton 2024; antweb 2024). after a brief collecting trip in a coastal forest fragment in alaminos city in the northwestern part of pangasinan, luzon island (fig. 1), several specimens of trap-jaw ants that were superficially similar to the highland o. schoedli were collected. further examination revealed that these specimens constitute a new species, herein described, belonging to the infandus species group. this discovery is a testament to the rich diversity of philippine ants and terrestrial arthropods in general, eliciting more research in understudied habitats, especially in threatened areas. materials and methods the specimens described herein were collected from a small undisturbed beach adjacent to bolo beach, on coastal limestone aggregates with a small forest fragment near the hundred islands national park. foraging worker specimens were manually collected and pinned on cardboard mounts. specimens were examined, measured, and imaged using an olympus sz61 stereomicroscope with touptek camera attachment (model xcamtop4k8mpa) and toupview software version x64. the morphology of o. infandus and o. schoedli followed sorger & zettel (2011) and additional data of types were acquired from antweb (2024). measurements were given from holotype followed by paratype mean (paratype m; n = 7) and the paratype minimum (min) and maximum (max) values in parenthesis: holotype (paratype m, min–max). all measurements were given in millimeters rounded to the nearest 0.01 mm. measurements and indices followed sorger and zettel (2011) and general (2018), including the additional index (pti). measurements: tl total length. length of the entire ant measured in dorsal view with head stretched out, from anterior margin of the mandible to apex of gaster, excluding sting. hl head length. maximum length of the head in full-face view, excluding mandibles, measured from anteriormost point of clypeal margin to posterior-most point of head vertex, parallel to midline. hw head width. maximum width of head in full-face view (including eyes when surpassing head outline). mdl mandible length. maximum length of the mandible in frontal view of head measured from mandibular insertion to apex. msl mesosoma length. maximum length of mesosoma, measured in lateral view, diagonal from cervical shield to the posterolateral propodeal edge. pnw pronotum width. maximum width of pronotum in dorsal view. pth petiole height. maximum height of petiole, measured in lateral view as a straight line from the bottom edge of the petiole, perpendicular to petiolar apex. ptl petiole length. measured in lateral view along the dorsal outline of petiole from small antero-apical tooth to apex. ptw petiole width. maximum width of petiole in dorsal view. sl scape length. maximum length of antennal scape in dorsal view excluding basal constriction. gl gaster length, maximum length from base of first gastral tergite to the apex of gaster, excluding sting, measured in lateral view. indices: ci cephalic index. hw / hl × 100 mdi mandible index. mdl / hl × 100 si scape index. sl / hw × 100 pti petiole index. pth / ptl × 100 depositories: pasi philippine arachnological society, inc. reference collection, manila pnm philippine national museum of natural history, manila biotropia vol. 31 no. 2, 2024 230 materials examined: odontomachus ampipitbaybay sp. nov., holotype worker, pasi hym0001: philippines: luzon, pangasinan, alaminos city, brgy. telbang, near bolo beach, 16°10'47.6" n 120°03'11.2" e, 29.vi.2023, dc acuña & chv alpez leg.; paratypes, 7 workers, pasi hym0002–0008: same label as holotype; pasi (to be deposited in pnm). odontomachus infandus smith, 1858, 15 workers, pasi hym0009–hym0023: philippines: luzon, pangasinan, sison, brgy. labayug, southwest part of cordillera central range, 20 results and discussion taxonomy family formicidae latreille, 1809 subfamily ponerinae lepeletier, 1835 genus odontomachus latreille, 1804 type species: formica haematoda linnaeus, 1758, by monotypy odontomachus ampipitbaybay sp. nov. urn:lsid:zoobank.org:pub:2f531215-a6ff4fbd-9dd0-4b9c5e21584f urn:lsid:zoobank.org:act:a93dd1b4-6ca64d4b-b7e4-33d3eca8da44 (figs. 2, 3, 4a–c, & 5a–c) type material: holotype, worker, pasi hym0001; paratypes, 7 workers, pasi hym0002-0008 etymology: the specific epithet comprises the ilocano nouns ‘ampipit’ which means trap-jaw ant, and ‘baybay’ which means beach, forming the word ampipit-baybay which translates to ‘trap-jaw ant of the beach’; noun in apposition. figure 1 distribution map of selected odontomachus species showing records of o. ampipitbaybay sp. nov. (red), o. schoedli (green), and o. infandus (blue) based on this study and sorger & zettel (2011), including unverified records from antweb (2024) and general & buenavente (2017) (question mark) inset: satellite image of the type locality. map generated by dor mapile using qgis 3.16. https://en.wikipedia.org/wiki/pierre_andr%c3%a9_latreille https://en.wikipedia.org/wiki/am%c3%a9d%c3%a9e_louis_michel_le_peletier,_comte_de_saint-fargeau new odontomachus species description and ecology from pangasinan, philippines – acuña & mapile 231 diagnosis (worker): member of the infandus species group following the treatment of sorger and zettel (2011). workers are different from other members of the infandus species group and similar to odontomachus schoedli sorger & zettel, 2011 and odontomachus infandus smith, 1858 in having posterior dorsum of head mainly striate (figs. 3a, 4a) and mesopleuron with complete transverse striation (fig. 3c). workers are also similar to o. schoedli in being unicolored head to petiole, having a reddish-brown color in life and a dark gaster (figs. 5a-5c). they can be distinguished from o. schoedli and o. infandus (i) in having a smooth area on the ocular ridge, medially (fig. 4a, arrow); (ii) in having a relatively shorter scape with si < 134.15 (table 1); (iii) in possessing a slightly curved and not s-shaped petiolar spine (fig. 4b) (almost straight in o. schoedli and more curved backward and s-shaped in o. infandus; fig. 4e; sorger & zettel, 2011); (iv) in having a stout subpetiolar process with base reaching less than half of petiole, ventrally (fig. 4c) (reaching more than half for the latter two species and broader on o. schoedli; fig. 4f; see sorger & zettel, 2011, p. 145); (v) in having a distinctly depressed dorsum of propodeum on lateral view ( fig. 3c, arrow); and (vi) in having a smaller body size with maximum tl of 13.45 (up to 16 mm on the latter two species; see table 1). workers further differ from o. schoedli in having a pronotum with mostly longitudinal striation (fig. 3c, 3d). figure 2 habitus of o. ampipitbaybay sp. nov. holotype worker, pasi hym0001, (a) lateral and (b) dorsal view notes: photos by dc acuña. scale = 5 mm. biotropia vol. 31 no. 2, 2024 232 they also differ from o. infandus in having a finer striation on the head and mesosoma and in possessing a lighter color in life which is reddish brown from head to petiole (figs. 5a-5c) (blackish brown in o. infandus; figs. 4d-4f; sorger & zettel, 2011). description (worker): measurements: tl 13.18 (13.18, 12.84-13.45), hl 3.17 (3.20, 3.093.40), hw 2.19 (2.23, 2.11-2.46), mdl 1.96 (1.97, 1.85-2.11), msl 4.97 (4.97, 4.89-5.03), pnw 1.77 (1.77, 1.75-1.80), pth 1.24 (1.24, 1.23-1.27), ptl 1.37 (1.37, 1.34-1.39), ptw 0.40 (0.40, 0.38-0.42), sl 2.80 (2.87, 2.64-3.30), gl 3.23 (3.23, 3.19-3.26). indices: ci 69.09 (69.65, 68.30-72.35), mdi 61.83 (61.50, 59.87-62.06), si 127.85 (128.77, 125.12-134.15), pti 90.51 (90.90, 91.37-91.79). head longer than wide, widest at eye level, posterior margin concave, and with relatively long mandible and short scape (figs. 2a-b, 3ab, 4a; table 1). mandible with acute apical and subapical teeth (broken tip on the holotype). dorsum of propodeum depressed in lateral view (fig. 3c, arrow). petiole smooth, spine stout and slightly curved (fig. 4b). subpetiolar process stout, subtriangular, pointing posteriorly, with margination, and with base reaching less than half of ventral petiole (fig. 4c). gaster smooth with elongated pit at tergite i medially. legs long and slender. sculpture: dorsum of the head mainly striate, radiating to posterior margin, with a smooth area on ocular ridge and around median suture posteriorly, consistent in all workers examined herein (figs. 3a-b, 4c). gena and genal furrow are mainly smooth (figs. 3b, 4a). head ventrally smooth. striation of pronotum mainly longitudinal laterally and dorsally with longitudinal ovoid loops creating transverse striae on the anterior and posterior dorsum (figs. 3c, 3d). striation of mesonotum and propodeum entirely transverse (figs. 3c, 3d). mesopleuron completely striate with very fine transverse striation (fig. 3c). striation of mesosoma transverse ventrally. overall striation is very fine. pilosity: setation on the head and mesosoma is short and sparse. setation on mandibles, petiole, and gaster is short and very sparse. color in life: head, antennae, mandibles, mesosoma, and petiole are reddish brown. gaster is mainly dark brown and anteriorly brown. legs are yellow. (figs. 5a-5c) gyne and male: unknown. distribution: philippine endemic, known from the type locality, luzon island: alaminos city, brgy. telbang (adjacent to bolo beach, near hundred islands national park). odontomachus ampipitbaybay sp. nov. workers were found on an undisturbed beach adjacent to bolo beach (fig. 6). they were discovered foraging among leaf litter at the coast (figs. 5a-5c). nests were built on clay loam soil situated on a narrow platform of coastal limestone aggregates elevated at approximately 2 masl and 2 m away from the tidal zone (fig. 6b). like its congeners within the infandus species group, o. ampipitbaybay sp. nov. may present another regionally endemic species. it is currently known from the forested coastal karst area of alaminos city, pangasinan. its unique ecology deviates from the usually forest-dwelling species of the infandus species group (sorger & zettel 2011). interestingly, its seaside habitat is reminiscent of the malignus species group containing o. malignus and o. litoralis that inhabit and venture into intertidal zones (wang et al. 2020). current observations do not support o. ampipitbaybay sp. nov. as an intertidal species, owing to the elevated coastal limestone aggregates. however, further observations are necessary. table 1 total length and indices of the workers of o. ampipitbaybay sp. nov. and closely related species. data on o. infandus and o. schoedli are acquired from sorger & zettel (2011) species tl ci mdi si pti o. ampipitbaybay sp. nov. 12.84-13.45 68.30-72.35 59.87-62.06 125.12-134.15 90.51-91.79 o. infandus 12.00-16.57 68.00-73.00 52.00-58.00 138.00-152.00 82.99-99.15 o. schoedli 12.75-16.25 72.00-73.00 55.00-59.00 137.00-146.00 96.73-105.3 new odontomachus species description and ecology from pangasinan, philippines – acuña & mapile 233 figure 3 odontomachus ampipitbaybay sp. nov. holotype worker, pasi hym0001 (a-d). head (a) lateral and (b) dorsal view. mesosoma (c) lateral and (d) dorsal view notes: arrow pointing to the depression. photos by dc acuña. scales =1 mm. biotropia vol. 31 no. 2, 2024 234 figure 4 head and petiole structures (a-f ). o. ampipitbaybay sp. nov. holotype worker, pasi hym0001, (a) head, dorsal view, (b) petiole and (c) subpetiolar process, lateral view. o. infandus, pasi hym0009, (d) head, dorsal view, (e) petiole, and (f ) subpetiolar process, lateral view notes: arrows pointing to the smooth area; red lines show the basal length of subpetiolar process. photos by dc acuña. scales = 1 mm (figs. a, b, d, & e), 0.5 mm (figs. c & f ). new odontomachus species description and ecology from pangasinan, philippines – acuña & mapile 235 the small forest fragment habitat of o. ampipitbaybay sp. nov. at the beach is nearly encroached by man-made fish ponds (fig. 1 inset). interestingly, it was absent from the nearby bolo beach, a tourist area. the encroachment of coastal forests and disturbances along beaches due to tourism and aquaculture activities could pose conservation challenges for this species. notably, the type locality is situated close to the hundred islands national park, a group of islets legislated as a national park. surveying the hundred islands national park may reveal additional colonies of this new species and provide insights into the terrestrial arthropods inhabiting the islets. however, our ability to conduct extensive fieldwork was constrained by a lack of funding. given the species’ potentially limited range, determining its area of occupancy and extent of occurrence would be beneficial for informing potential conservation efforts. closely related species of o. ampipitbaybay sp. nov., the o. infandus and o. schoedli, are known to occur on secondary to primary forest floors, with o. schoedli notably found at higher elevations of the central cordillera range (mt. province & benguet; sorger & zettel 2011). o. infandus is more widespread with records from the islands of luzon and mindoro, with two recent reports from mindanao island (general & buenavente 2017; antweb 2024). o. infandus was reported to occur in mt. hamiguitan, davao oriental, mindanao island (general & buenavente 2017) and tinago falls, buru-in [=buru-un], iligan city, lanao del norte, mindanao island (antweb 2024; fig. 1). however, we were unable to verify the identity of both records. we noticed that the record from iligan city seems to be misidentified and could represent a new species. a series of images of a worker (casent0650353) available on antweb (2024) shows the head with a large smooth area around the median furrow and lighter body color, contrary to o. infandus which has a head almost fully striated and darker in color. the existence of other distinct morphological characters will support heterospecificity. figure 5 odontomachus species from northern luzon in their natural habitat (in situ). (a-c) o. ampipitbaybay sp. nov., types, in alaminos city, pangasinan. (d & e) o. schoedli, in la trinidad, benguet notes: photos by dc acuña (a-c) and dj cachero (d & e). biotropia vol. 31 no. 2, 2024 236 figure 6 habitat of odontomahcus ampipitbaybay sp. nov. (a & b) undisturbed beach habitat near bolo beach inset: collection site on the coastal limestone aggregates with leaf litter and clay loam soil. note: photos by dc acuña. new odontomachus species description and ecology from pangasinan, philippines – acuña & mapile 237 conclusion odontomachus ampipitbaybay sp. nov. is unique based on its ecology and the distinct structures of its head, mesosoma, and petiole. the description of its reproductive members and genetic analysis will improve its taxonomic placement. this species is the only member of the infandus species group whose habitat is in the coastal area. this species raises the number of odontomachus in the philippines to 14 valid species. extensive work on this poorly studied taxon, especially in central philippines, may reveal rich diversity for the philippines. modification on the sorger and zettel (2011) key, proposed herein, couplet 8 is modified to give way for the additional couplet 11: 8 head, mesosoma, and petiole unicolorously reddish brown. petiole stout. ..................................................11 – head distinctly lighter than mesosoma. petiole less stout......................................................................9 11 petiole with an almost straight spine. striation of pronotum mainly transverse. subpetiolar process broad, with base extending more than half of petiole. species from the mountains of northern luzon…............................... odontomachus schoedli – petiole with a slightly curved spine. striation of pronotum mainly longitudinal. ocular ridge smooth medially. sub-petiolar process stout, with base extending less than half of petiole. scape short. species from the coast of north-western pangasinan...............………….... odontomachus ampipitbaybay sp. nov. acknowledgment we are very grateful to the department of environment and natural resources regional office 1 in san fernando city, la union, for granting wildlife gratuitous permit no. 2022-004, and to the city of alaminos and municipality of sison, pangasinan, for giving consent to collect arthropod specimens. we are also thankful to mr. leonardo a. guevarra jr. of the university of santo tomas for his consent to use a microscope for imaging, to mr. dan jones cachero of baguio city for providing images and data of o. schoedli, to ms. christine hazel alpez of pangasinan for helping us with field sampling and proofreading the early draft of the manuscript, to ms. emerald r. padaong of philippine science high schoolilocos region campus for her english editing service, and to the reviewers, dr. herbert zettel of natural history museum, vienna and dr. rijal satria of universitas negeri padang, whose comments greatly improved this paper. references antweb. 2024. antweb version 8.103.2. california academy of science, usa; [accessed 2024 january 29]. available from: https://www.antweb.org bolton b. 2024. an online catalog of the ants of the world. [accessed 2024 january 29]. available from: http://antcat. org general dem, alpert g. 2012. a synoptic review of the ant genera (hymenoptera, formicidae) of the philippines. zookeys 200:1-111. doi: 10.3897/zookeys.200.2447 general dem, buenavente pac. 2017. checklist of the ants of mt. hamiguitan, mindanao island, philippines (hymenoptera: formicidae). halteres 8: 92-102. doi: 10.5281/zenodo.894187 general dem. 2018. odontomachus ferminae, a new philippine species of the infandus species group (hymenoptera: formicidae). halteres 9: 157-62. doi: 10.5281/zenodo.1434108 general dem. 2021. a preliminary checklist of the ants (hymenoptera: formicidae) of the mt. pantaron range, bukidnon province, mindanao island, philippines. halteres 12: 4-14. doi: 10.5281/zenodo.5371745 sorger dm, zettel h. 2011. on the ants (hymenoptera: formicidae) of the philippine islands: v. the genus odontomachus latreille, 1804. myrmecol news 14:14163. wang wy, yamada a, yamane s. 2020. maritime trapjaw ants (hymenoptera, formicidae, ponerinae) of the indo-australian region: redescription of odontomachus malignus smith and description of a related new species from singapore, including first descriptions of males. zookeys 915:137-74. doi: 10.3897/zookeys.915.38968 zettel h, sorger dm. 2023. another new trap-jaw ant (hymenoptera, formicidae, odontomachus latreille, 1804) from the philippines. linzer biologische beiträge 55/1:381-5. _goback 10. 1915 liwayway acero.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 biotropia vol. 30 no. 2, 2023: 158 170 doi: 10.11598/btb.2023.30.2.1778 158 identification of the moluccan megapode (eulipoa wallacei) natural habitat in haruku island, indonesia and its vegetation composition handy erwin pier leimena1*, achmad sjarmidi2 and tati suryati syamsudin2 1biology department, faculty of mathematics and natural sciences, pattimura university, m. putuhena street, pattimura university campus, poka, ambon, 97233, indonesia 2school of life science and technology, institut teknologi bandung, ganesa street no. 10, bandung, west java, 40132, indonesia received 7 july 2022 / revised 9 april 2023 /accepted 11 april 2023 abstract the characteristics of island vegetation greatly influence the activities of endemic birds in island areas, such as the moluccan endemic megapodes (eulipoa wallacei) on haruku island. therefore, it is essential to identify the specific location of the habitat utilized by individual birds for their daily activities on haruku island and to analyze the composition of the vegetation and the variety of plant species. identification of bird habitat locations using radio-tracking on four newly hatched chicks and four adult birds. a total of 330 individual tracking points were recorded during the study period. the vegetation sample used a total of 420 plots for seedlings, saplings, poles, and trees which were then analyzed for importance value index (ivi), diversity, evenness, and similarity. we found that the tanjung maleo forest was their nesting habitat, while the marunimei and lalean forests were their daily habitats. a total of 91 plant species and 60 plant families were discovered with the vegetation diversity value of the three habitats was moderate (h’mean = 3.07) and tended to be dominated by air plant (kalanchoe pinnata), sword fern (nephrolepsis exalta), cogongrass (imperata cylindrica), coco-grass (cyperus rotundus), indian camphorweed (pluchea indica), and lanzone (lansium domesticum) (emean = 0.88), and have a relatively low level of species similarity between habitats (simean = 38.30%). we found that the daily habitat of the moluccan megapode on haruku island was around their nesting sites and has a complex structure because it was composed four vegetation strata. therefore, for conservation purposes, habitat management must prioritize preserving forest habitats around the bird nesting sites. keywords: diversity, evenness, moluccan scrubfowl, momoa, maleo, similarity, vegetation composition introduction the maluku islands, indonesia, located in the wallacea region, support endemic bird species, such as the moluccan megapode, through various habitat types (yuni & yuda 2020). tropical forests provide complex habitats rich in composition and structure of vegetation for shelter, foraging, roosting or sheltering, and breeding (baldeck et al. 2012; bu et al. 2014; velasques-trujillo et al. 2021). therefore, the composition and diversity of habitat vegetation support the diversity of endemic, rare, threatened, and endangered wild birds (sodhi et al. 2011; rocha et al. 2015; devenish‐nelson et al. 2019; feng et al. 2020). the structure of a bird’s habitat, which includes vertical structure and floristic composition, is an essential component of its habitat. vegetation structure and floristic composition have been used to predict the presence and diversity of bird species for a long time. some bird taxa are influenced by vegetation structural factors (e.g., the complexity of stand configuration or architecture), while natural habitat of eulipoa wallacei and its vegetation composition – leimena et al. 159 others are influenced by vegetation composition factors (e.g., vegetation type) (tews et al. 2004; tassicker et al. 2006; gao et al. 2014; rutten et al. 2015; melo et al. 2020; moudry et al. 2021). island bird species in the tropics are threatened by several environmental challenges, including habitat change and destruction (duncan & blackburn 2007; taylor & kumar 2016; radley et al. 2020; dri et al. 2021). habitat destruction causes changes in the structure and composition of vegetation in bird habitats, thus affecting the survival and reproduction of bird populations (ruiz-gutierrez et al. 2008; matthews et al. 2015; tulloch et al. 2016; tchoumbou et al. 2020; atikah et al. 2021). one of the native and endemic bird species in the maluku islands is the moluccan megapode (eulipoa wallacei), which is only found in northern maluku and central maluku. like other megapodes, the moluccan megapode is super-precocial and classified as a terrestrial bird (colar et al. 1994; argeloo & dekker 1996; dekker et al. 2000; heij & rompas 2011). moluccan megapodes inhabit coastal areas through tropical rain forests of more than 750 m asl (dekker et al. 1995; heij et al. 1997; heij 2001; heij & rompas 2011; birdlife international 2022). the occurrence of the birds in coastal areas is related to their nesting behavior, which involves digging holes in the sand substrate to lay their eggs (dekker et al. 1995; jones et al. 1995; dekker et al. 2000; heij et al. 1997; heij & rompas 2011). the population of moluccan megapodes has been decreasing due to habitat destruction and egg harvesting (colar et al. 1994; argeloo & dekker 1996; dekker et al. 2000; heij & rompas 2011), so it has been designated as a vulnerable species by the iucn since 1994 (birdlife international 2016). studies in 1997 by heij et al. (1997) confirmed that habitat destruction caused the moluccan megapode to become rare on several large islands, such as seram island and bacan island, and smaller islands, such as ambon island and ternate island. likewise, several nesting sites have been abandoned and are no longer used by birds due to the destruction of their nesting habitat. the effect of habitat destruction on the decline of the moluccan megapode indicates that habitat quality and composition play an essential role in the bird populations. adult moluccan megapodes use vegetation around their nesting site for perching before and after nesting activities. meanwhile, newly hatched chicks require vegetation cover as shelter from predators (heij et al. 1997; heij & rompas 2011). in central maluku, one of the largest nesting sites of the moluccan megapode is tanjung maleo in haruku island (heij et al. 1997; heij & rompas 2011). until recently, information about the habitat used by moluccan megapodes on haruku island has been scarce and limited to habitats around bird nesting sites (heij et al. 1997; heij & rompas 2011; sjafani et al. 2015), while daily habitat use studies have never been reported. better knowledge of the specific habitats used by moluccan megapodes allows conservation managers to carry out appropriate engineering to protect bird habitats from expanding settlements near nesting habitats or opening new agricultural fields in their daily habitats. habitat protection engineering can be carried out by delimiting habitat boundaries, maintaining the composition of vegetation strata, or providing corridors for the distribution of chicks and pathways for adult birds to and from their nesting habitat in tanjung maleo. this study investigated habitat use by moluccan megapodes on haruku island. we also analyze the composition of the vegetation and the diversity of plant species in these habitats to understand the influence of habitat on birds’ daily activity patterns. materials and method study area the study was conducted on haruku island in central maluku district (3.567os, 128.483oe) (figure 1). haruku island has an area of 274 km2 and a topography of mountains and hills, with the highest point reaching 554 meters above sea level. the climate on haruku island is locally influenced by tropical marine climate and monsoon climate (aldrian & dwi susanto 2003; wirjohamidjojo & swarinoto 2010) with relatively high rainfall of 224.58 mm per year in 2021 (bps propinsi maluku 2022). biotropia vol. 30 no. 2, 2023 160 figure 1 study sites used to identify natural habitats and analyze the composition and diversity of the vegetation of the moluccan megapode (eulipoa wallacei) habitat in haruku island, indonesia [dotted line boxes indicate the location of daily bird habitats]. identification of bird habitat bird habitat identification was conducted by using radio-tracking. during 40 days of observation from december 2020 until january 2021, 330 points of bird presence were recorded. individual females and chicks were tracked from their nesting sites to collect data on the relationship between nesting habitats and daily foraging habitats. this study did not use adult males because only adult females carried out nesting activities at nest sites in tanjung maleo, while adult males remained in their daily habitat (heij et al. 1997; heij & rompas 2011). adult male birds were challenging to catch in their daily habitat because of their camouflage amidst the surrounding understory vegetation. eight birds were utilized, including four newly hatched chicks and four adult females. chicks and adult females were caught using a hand capture technique (de beer et al. 2001; bloom et al. 2007; whitworth et al. 2007; busse & meissner 2015) during nesting and when the chicks emerged from their nesting holes. for tracking purposes, the sirtrack v8009 transmitter was utilized for chicks, and the sirtrack p04347 transmitter for the adult birds. using loctite cyanoacrylate (bowman et al. 2002; diemer et al. 2014), the transmitter was attached to the base of the wing. the movement and position of individual birds were tracked using biotrack sikka vsr 04, receiver, yagi antennae, and gps garmin 64s. the birds were tracked every day on foot (ground tracking). the position of the universal transverse mercator coordinate for each bird was plotted on a map of the study site to determine the area used by birds using arcgis 10.3 and arcmap 10.3. natural habitat of eulipoa wallacei and its vegetation composition – leimena et al. 161 vegetation sampling intensive floristic inventory and plot-based vegetation survey (krebs 1998; colwell 2009) was conducted at forest sites on haruku island that were used by individual moluccan megapodes, specifically in tanjung maleo, marunimei, and lalean. vegetation sampling was carried out at the same time as bird habitat identification. twelve transect lines measuring 500 and 1000 meters were utilized, and the distance between transects was 250 meters. the distance between plots was 100 meters. the plot sizes were 1 m x 1 m (seedlings), 5 m x 5 m (saplings), 10 m x 10 m (poles), and 20 m x 20 m (trees) (barbour et al. 1987; woodward et al. 2009; rahman et al. 2016; kusmana 2017; peng et al. 2018). the observed variables for each stage were as follows: (i) seedling stage: germinated seeds to < 1.5 m in height, (ii) sapling stage: height >1.5 m to a diameter at breast height (dbh) < 10 cm, (iii) pole stage: 10 cm < dbh < 20 cm (barbour et al. 1987; woodward et al. 2009; kusmana 2017). there were 84 plots in tanjung maleo, 168 in marunimei, and plots in lalean. vegetation parameters included species name, number of species, number of individual species, and diameter at breast height (dbh). plant species were identified using a collection of identification guides (whitmore 1978; soerianegara & lemmens 1994; lemmens et al. 1995; llamas 2003; gunawan et al. 2019). data analysis all coordinate positions of bird presence points were plotted onto the haruku island land cover map to obtain the moluccan megapode habitat land cover type. the land cover map of the bird habitat area was determined using a 2021 haruku island land cover map combined with 2021 google earth imagery data, a 1:50,000 scale map of haruku island in 2021, and a 2020 landsat 5 tm image (composite color band 4 band 3 band 2) with a spatial resolution of 30 meters. land cover types on bird habitats were created using arcmap 10.3. the land cover classification was based on the indonesian national land cover standards agency (national standardization agency of indonesia 2010). vegetation structure and floristic composition were determined based on the analysis of the importance value index (ivi) (bendre & kumar 2010), species diversity (shannon – wiener index) (shannon & weaver 1963; odum 1983), species evenness (pielou index) (odum 1983), and species similarity (sorensen index) (sorensen 1948). the importance value index (ivi) is the sum of relative frequency, density, and dominance values. the species diversity was calculated by using the shannon-wiener index: h’ = -∑ pi ln pi where: pi = proportion of individuals of the i-th species to individuals of all species found. the species evenness index was calculated by using the pielou index: e = h’/ln (s) where: h’ = shannon-wiener diversity indeks; s = total number of species in the sample; e = 1 if all species are represented equally in the sample, and e is close to zero if one species predominates strongly. the species similarity index (si) between sites was calculated using the sorensen index: si = (2c/(a+b)) x 100%, where: c = standard number of species at the two sites; a = number of species at site a; b = the total number of species at site b. the f test was conducted using one-way anova to compare the composition of vegetation growth strata between bird habitat forests. result and discussion habitat of moluccan megapode the radio-tracking record of individual chicks and adult females of moluccan megapodes on haruku island indicated that bird activity occurred in forest areas near their nesting sites. birds were active in their nesting biotropia vol. 30 no. 2, 2023 162 site at tanjung maleo, marunimei, and lalean forests. marunimei forest is ± 1.60 km northeast of tanjung maleo, and lalean forest is ± 2.50 km southeast of tanjung maleo. the moluccan megapodes habitat was classified as moor-farm-field and dryland forest based on the land cover type. the type of land cover around the bird’s nesting site in tanjung maleo was classified as a development area because it was adjacent to a residential area. however, around the bird nesting site in tanjung maleo, there were still various types of vegetation specially guarded by the community as part of the bird’s nesting habitat. meanwhile, the marunimei forest, as the bird’s daily habitat, was classified as a moor-farm field, while the lalean forest was classified as a dry land forest (figure 2). these results confirm previous research indicating that moluccan megapodes could survive and thrive in various habitat types ranging from secondary forests to open beaches, lowland forests, and tropical rain forests (dekker et al. 1995; heij et al. 1997; heij 2001; heij & rompas 2011). in contrast to their diverse daily habitats, bird nesting habitats were always found in coastal areas, specifically open beach areas surrounded by forest areas (dekker et al. 1995; jones et al. 1995; dekker et al. 2000; heij et al. 1997; heij & rompas 2011). megapode species in island areas are known to be more active in tropical forests and use coastal areas for breeding. therefore, both habitat types are essential for survival (goth & vogel 1995; jones et al. 1995; dekker et al. 2000; pangauadam & brodie 2019; paguntalan et al. 2021). the daily activities of the moluccan megapode within a radius of ± 2.50 km from their egglaying locations in tanjung maleo indicated that most of their daily activities are not far from their egg-laying locations. several studies of other bird species also show that most of the birds' daily activities occur in areas not far from their nesting sites (ryan & jamieson 1998; yaremych et al. 2004; bosch et al. 2010; rechetelo et al. 2016). adult female moluccan megapode used the forest in tanjung maleo for perching before starting nesting activities or as a perch after nesting, before flying away from the nest site (heij et al. 1997; heij & rompas 2011). for chicks, the forest around their nesting site in tanjung maleo was used as a shelter from the moment they hatch (heij et al. 1997; heij & rompas 2011) because of their super-precocial nature (colar et al. 1994; argeloo & dekker 1996; starck & ricklefs 1998; dekker et al. 2000). consequently, vegetation around the nesting site was crucial to the chick’s survival. it has been reported that moluccan megapode chicks immediately moved to nearby vegetation after hatching (heij et al. 1997; heij & rompas 2011). the use of vegetation around the hatchery as a shelter for chicks has also been reported among the australian brush-turkey chicks (alectura lathami) (goth & jones 2001; goth & vogel 2002; goth & vogel 2003; goth & evans 2005). natural habitat of eulipoa wallacei and its vegetation composition – leimena et al. 163 figure 2 the point of presence of individual moluccan megapodes (eulipoa wallacei) in haruku island based on radiotracking results. vegetation structure and floristic composition of the three forest locations used by the moluccan megapodes, the marunimei forest has the highest number of plant species (48 species). in contrast, the tanjung maleo forest has the most diminutive plant species (14) (table 1). the number of plant species in the tanjung maleo forest is currently fewer than the study results in 1997. in 1997, approximately 20 species were found in the tanjung maleo forest (heij et al. 1997; heij & rompas 2011). these results indicated that in the twenty-four years since 1997, the number of plant species in the tanjung maleo forest has decreased. because tanjung maleo's forest was close to the villages, it was believed that the expansion of the residential area was responsible for the decline in species. the number of plant species in the maluku megapod habitat on haruku island from this study was higher than the results of a survey conducted by ahmad (2014) around bird nesting sites on halmahera island, which only found as many as 13 to 17 plant species. this study also found that the highest number of plant families was found in the marunimei forest area (30 families), and the lowest was in the tanjung maleo forest area (14 families) (table 1, figure 3). no dominant plant species families were found in the tanjung maleo forest; each family only consisted of one species. in contrast, in marunimei and lalean forests, members of the fabaceae family dominated the vegetation. the current number of plant families in the tanjung maleo forest is the same as the 1997 study by heij et al. (1997) and heij & rompas (2011), but in 1997 the fabaceae family had more than one species. table 1 the number of species, individual species, and plant families in forest habitats utilized by moluccan megapodes (eulipoa wallacei) in haruku island forest areas species (n) individual species (n) family (n) tg. maleo 14 49 14 marunimei 48 1401 29 lalean 28 345 16 total 91 1795 60 biotropia vol. 30 no. 2, 2023 164 figure 3 comparison of the number of plant species per family in three forest areas used by moluccan megapodes (eulipoa wallacei) in haruku island (a) tanjung maleo, (b) marunimei, and (c) lalean table 2 comparison of vegetation structural composition in three forest areas used by moluccan megapodes (eulipoa wallacei) in haruku island forest areas vegetation growth stratum tree pole sapling seedling number of individuals (n) percentage (%) number of individuals (n) percentage (%) number of individuals (n) percentage (%) number of individuals (n) percentage (%) tg maleo 34 69.39 4 8.16 2 4.08 9 18.37 marunimei 605 43.18 226 16.13 165 11.78 405 28.91 lalean 109 31.59 69 20.00 14 4.06 153 44.35 average 249 100 60 189 the strata of vegetation growth in the three moluccan megapodes habitat forest areas included trees, poles, saplings, and seedlings (table 2). the tree strata dominated the forest area in tanjung maleo (63.39%) and marunimei (43.18%), while the seedling strata dominated the lalean forest area (44.35%). the f test showed that the vegetation's composition significantly differs between habitat forests (fcount = 8.82 > ftable = 4.26). this study showed that the forest regions surrounding bird nesting sites were classified as having complex vegetation because they contain four growth strata: trees, poles, saplings, and seedlings. the moluccan megapode habitat on halmahera island also consists of four growth strata (ahmad 2014). studies of several megapode species show that their habitat tends to be dominated by tree strata compared to other growth strata (goth & vogel 1995; khairuddin & yamin 2019; pangau-adam & brodie 2019; paguntalan et al. 2021; dhafir et al. 2022 ). however, several species of megapodes, such as the australian brush turkey (alectura lathami) (goth & vogel 2003), the wattle brush turkey (aepypodius arfakianus) (pangau-adam & 0 1 2 3 4 5 acathaceae amaranthaceae annonaceae burseraceae crassulacea lamiaceae meliaceae myristicaceae anacardiaceae apocynaceae lauraceae malvaceae myrtaceae arecaceae asteraceae fabaceae number of species per family 0 1 2 3 4 5 6 acathaceae amaranthaceae apocynaceae burseraceae crassulacea cyperaceae elaeocarpaceae flacoutiaceae meliaceae myristicaceae nephrolepidaceae oxalidaceae phyllanthaceae plantaginaceae poaceae rubiaceae rutaceae santalaceae selaginellaceae annonaceae arecaceae lauraceae moraceae asteraceae lamiaceae malvaceae myrtaceae anacardiaceae fabaceae number of species per family 0 1 2 anacardiaceae annonaceae apocynaceae burseraceae calophyllaceae clusiaceae convolvulaceae crassulacea lauraceae lecythidaceae malvaceae moraceae nephrolepidaceae sapindaceae number of species per family (b) (c) natural habitat of eulipoa wallacei and its vegetation composition – leimena et al. 165 brodie 2019), and the orange-footed scrubfowl (megapodius reindwartii) (khairuddin & yamin 2019) are also found in a sapling or seedling strata. complex vegetation contributes to the survival of wild species populations by providing a suitable microclimate, shelter, and a place to rest and reproduce (baldeck et al. 2012; bu et al. 2014; bergner et al. 2015). in tanjung maleo, the percentage of tree strata is currently higher than in 1997. in this study, the tree strata in the tanjung maleo forest reached 70 percent, whereas the 1997 study found only 67 percent (heij et al. 1997; heij & rompas 2011). likewise, tree strata dominated the marunimei forest. on the other hand, the seedling layer dominated the lalean forest. tree strata in the tanjung maleo forest function as perching for birds when they come to the nest or leave their nesting sites, while the strata of seedlings and saplings serve as protection for the chicks after hatching (heij et al. 1997; heij & rompas 2011). observations in the study area show that the moluccan megapodes also use trees in their habitat as perching to move between habitats or escape from predators. individual birds used the tree layers in their habitat as perches, resting spots, and shelters. moluccan megapodes' activity primarily occurs on the forest floor (mackinnon & wind 1980; del hoyo et al. 1994; strange 2012) and causes variations in vegetation strata to be essential for the survival of individual birds (bucklin et al. 2015; walther & pirsig 2017; velasques-trujillo et al. 2021). species composition and diversity based on the importance value index (ivi), the dominant species in each forest area were different (table 3). the dominant species in the tanjung maleo forest were air plants (kalanchoe pinnata) (ivi = 67.96) and sword ferns (nephrolepsis exalta) (ivi = 61.47). marunimei forest was dominated by cogongrass (imperata cylindrica) (ivi = 36.80) and coco-grass (cyperus rotundus) (ivi = 32.59), and the lalean forest was dominated by indian camphorweed (pluchea indica) (ivi = 24.23) and lanzones (lansium domesticum) (ivi = 18.30). the dominant plant species in the tanjung maleo forest differed from the dominant species around the moluccan megapodes nesting site on halmahera island in north maluku. the vegetation around the nesting site at galela on halmahera island was dominated by more varied species, namely bay table 3 the vegetation composition of the nine most abundant plant species in three forest areas used by moluccan megapodes (eulipoa wallacei) in haruku island no forest areas tanjung maleo marunimei lalean species name % inp species name % inp nama ilmiah % inp 1 kalanchoe pinnata 21.93 imperata cylindrica 12.27 pluchea indica 8.08 2 nephrolepsis exaltata 20.34 cyperus rotundus 10.86 lansium domesticum 6.10 3 planchonia valida 9.91 nephrolepsis exaltata 8.49 amaranthus spinosis 6.07 4 cananga odorata 7.58 selaginella doederleinii 4.04 tectona grandis 5.61 5 alstonia scholaris 6.08 ficus benjamina 3.84 leucaena leucocephala 4.82 6 ipomoea pes caprae 5.72 lannea grandis 3.51 durio zibethinus 4.58 7 garcinia bancana 5.34 durio zibethinus 3.23 calameae sp 4.56 8 eusideroxylon zwageri 4.06 cocos nucifera 3.21 myristica fragrans 4.55 9 mangifera indica 3.69 artocaphus heterophyllus 3.07 cocos nucifera 3.94 10 others (6 species) 15.36 others (39 species) 47.47 others (19 species) 51.70 total percentage 100.00 100.00 100.00 hops (ipomoea pescrapae), clover (marsilea creanata), mangrove (rhizophora sp), and tropical almond (terminalia catapa) (sjafani et al. 2015). these results indicated that plant species around bird nesting sites did not significantly affect bird arrivals for nesting. dominant plant species in both forest areas adjacent to nesting sites (marunimei and lalean forests) act as shelters and support the daily activities of moluccan megapodes on haruku island. the highest diversity of plant species in the three forest areas around the moluccan megapodes site was in the marunimei forest (h’ = 3.87), while the lowest was in the tanjung maleo forest (h’ = 2.27) (table 4a). in terms of diversity, the plant species diversity in biotropia vol. 30 no. 2, 2023 166 the three forest regions was categorized as moderate. in addition to its diversity, species abundance distribution in the three forest areas, as measured by an evenness index, was close to one. the lalean forest had the most notable species distribution equality (e = 0.92) (table 4a). the evenness index indicates that the distribution of plant species abundance in the three locations tends to be relatively even and does not indicate the dominance of one or more species. nevertheless, according to the importance value index, each type of bird habitat contains several dominant plant species. tanjung maleo forest has a greater variety and more even distribution of plant species than halmahera island’s forest (sjafani et al. 2015). the diversity and evenness of plant species in the moluccan megapode habitat were higher than the orangefooted scrubfowl habitat on moyo island, west nusa tenggara (h’ = 2.42 dan e = 0.86) (khairuddin & yamin 2019). table 4 comparison of ecological index values between the three forest areas used by moluccan megapodes (eulipoa wallacei) in haruku island a. species diversity and evenness index between forest areas forest areas shannon-wiener index (h’) pielou index (e) tg. maleo 2.27 0.86 marunimei 3.87 0.86 lalean 3.06 0.92 b. similarity and dissimilarity between forest areas forest areas similarity (%) dissimilarity (%) tg. maleo – marunimei 25.81 74.19 tg. maleo – lalean 28.57 71.43 marunimei – lalean 60.53 39.47 average 38.30 61.70 this study found that from the three forest areas, the similarity of plant species between tanjung maleo forest, a bird nesting site, with the other two forests was low (is = 25.81% and is = 28.57%). on the other hand, the similarity of plant species between marunimei and lalean forests was high (is = 60.53%) (table 4b). therefore, the dissimilarity of plant species between the bird’s nesting site in tanjung maleo and the other two forest areas indicates the difference in vegetation between the bird’s nesting and daily habitats. the difference in species diversity between the forests surrounding bird nesting sites on haruku island and halmahera island indicates that the habitat types of moluccan megapodes were diverse and not influenced by specific plant species. the low average similarity between the three forests (simean = 38.30 percent) reflects the diversity of vegetation in the forest surrounding the haruku island bird nesting site. however, the similarity index between the three bird habitats revealed vegetation type differences between nesting habitats and the habitat of the moluccan megapodes on haruku island. conclusion this study enhances our understanding of the habitat conditions of moluccan megapodes on indonesia’s haruku island. it was determined that the moluccan megapodes on haruku island were active in the forest area approximately 2.50 kilometers from their nesting sites. based on the distance from the nesting site, the forest area in tanjung maleo serves as a bird nesting habitat. in contrast, the other two forests (marunimei and lalean) are a bird’s daily habitat. haruku island’s bird habitat consists of four layers of vegetation that support the activities of chicks and adult birds during nesting, foraging, and perching. variations in plant species diversity among the three habitats indicate moluccan megapodes’ ability to occupy diverse habitat types. regarding plant species diversity, nesting habitats and daily habitats of natural habitat of eulipoa wallacei and its vegetation composition – leimena et al. 167 birds differ by more than 70% of plant species, indicating differences in vegetation between nesting habitats and daily habitats of birds. an important finding from this study was that conservation efforts for the moluccan megapodes on haruku island should prioritize nesting and daily habitats in the forest area around the bird's nest sites. acknowledgments special thanks to d. usemahu and kailolo village for their assistance and cooperation during fieldwork, r. huda, and international animal rescue indonesia foundation for providing a tracking receiver and a yagi antenna and school of life science and technology, institut teknologi bandung for supporting facilities during the study. this research was funded by the directorate general of higher education, ministry of education, culture, research, and technology of the republic of indonesia as a scholarship and doctoral research grant to the first author. references ahmad z. 2014. selection strategy for laying eggs by mamoa bird (eulipoa wallacei gray, 1980) in galela. 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(a) the southeast asian journal of tropical biology vol. 31 no. 2, 2024: 349 358 doi: 10.11598/btb.2024.31.2.2214 issn: 0215-6334 | e-issn: 1907-770x 349 in vitro plant regeneration through protocorm-like bodies derived from stem thin layer of anubias barteri var. nana petite lê thị kiều giang, nguyễn văn tính, trần thị kim ngân, đặng huỳnh thúy vy, nguyễn vũ phong* faculty of biological sciences, nong lam university, ho chi minh city 72820, vietnam article higlights • effective induction of plbs from stem thin layer and shoot regeneration using specific bap and auxin combinations. • significant role of potato extract in enhancing shoot regeneration. • highest shoot regeneration rate achieved with 3 mg/l bap concentration. article information received 5 march 2024 revised 1 april 2024 accepted 3 april 2024 *corresponding author, e-mail: nvphong@hcmuaf.edu.vn research article abstract anubias barteri var. nana petite, a highly valued ornamental aquatic plant within the araceae family, encounters challenges in vegetative propagation due to its slow multiplication rate. this study aimed to identify the optimal concentrations of plant growth regulators and potato extract for the micropropagation of this species through shoot tips and protocorm-like bodies (plbs). initially, shoot tips were sterilized and cultured on the murashige and skoog medium (ms) with varying concentrations of 6-benzylaminopurine (bap) to induce growth and multiplication. furthermore, the induction of plbs and shoot regeneration was facilitated using bap in combination with auxins, including naphthaleneacetic acid (naa), indole-3-acetic acid (iaa), or indole-3-butyric acid (iba), in various combinations. the study also explored the impact of potato extract (pe) on shoot regeneration and the significant role of iba in root development. results indicated that the highest shoot regeneration rate from shoot tips was achieved at a bap concentration of 3 mg/l. effective induction of plbs from stem explants, followed by shoot regeneration, was achieved with 1.5 mg/l bap combined with 0.5 mg/l naa or 0.5 mg/l iaa, and 3 mg/l bap with 1.0 mg/l iba. the inclusion of 50 g/l potato extract significantly enhanced shoot proliferation, and 0.5 mg/l iba proved vital for root development. the plantlets acclimatized successfully in aquariums with a 100% survival rate. future research will aim to further enhance plb multiplication. the developed micropropagation protocol offers a promising approach for the mass production of a. barteri var. nana petite, effectively overcoming the limitations of natural propagation methods. keywords: anubias barteri var. nana petite, plant growth regulators, protocorm-like bodies (plbs), shoot induction, shoot regeneration introduction anubias barteri var. nana petite holds significant commercial importance as a highly sought-after decorative aquatic plant within the araceae family. this cultivated variant, renowned for its slow growth and cluster formation, typically ranging from 1 cm to 5 cm in height, has garnered popularity in both aquatic and ornamental settings (george et al. 2015). despite its aesthetic appeal, the natural propagation of a. barteri has proven inefficient, leading to numerous studies on micropropagation techniques, including in vitro multiplication (george et al. 2015; huang et al. 1994; kanchanapoom et al. 2012; rittirat et al. 2021), in vitro organogenesis (surendra et al. 2019; rittirat et al. 2023), and hydroponic culture (sholichah et al. 2020). however, these efforts have resulted in an average shoot production ranging between 2 shoots to 5 shoots from a single shoot. to address this limitation, the utilization of protocorm-like bodies (plbs) presents a promising approach for mass production because of their rapid formation, uniform characteristics, diseasefree nature, and sustainability (cardoso et al. 2020; sheelavanthmath et al. 2005). noteworthy success biotropia vol. 31 no. 2, 2024 350 has been achieved in the utilization of plbs for the proliferation and multiplication of ornamental plants within the araceae family. for example, anthurium andreanum cv. cancan exhibited 97.8% rate of plb induction, averaging 120 plbs for each explant over a period of 50 days in cultivation (gantait et al. 2012; yu et al. 2009). similarly, tdz was shown effective for inducing plbs from leaf explants of cattleya tigrine with 17.5% induction rate, and ga3 significantly increases shoot regeneration from plbs with approximately 220 developed plantlets per culture flask (fritsche et al. 2022). despite these successes, there is currently no documented report on regeneration from plbs on anubias barteri var. nana petite. given the documented success of plbs in various plant species, this study aimed to identify the optimal concentrations of plant growth regulators and potato extract for propagating this species through shoot tips and plbs. the development of this micropropagation protocol provides a methodical approach to augment the propagation of anubias barteri var. nana petite, effectively overcoming the limitations of natural propagation methods. materials and methods shoot sterilization shoots of anubias barteri var. nana petite were subjected to surface sterilization using soap for 15 minutes, followed by exposure to an antifungal agent (mancozeb, india) for 10 minutes, washed with running water, and further rinsed with 70% alcohol for 30 seconds. subsequently, explants were soaked in a commercial bleach solution (5% sodium hypochlorite) at concentrations of 10%, 20%, 30%, and 40% (v/v) for 15 minutes, followed by a 30-minute soak with an antibiotic solution (1 mg/ml). after disinfection, 3-5 mm shoot tip explants were isolated and placed on ms (murashige and skoog, 1962) basal medium. shoot multiplication from shoot tips shoot tips free of microbial infection were cultured in ms media supplemented with (0, 1.0, 2.0, 3.0, 4.0 mg/l) bap for 6 weeks for shoot induction. in a subsequent experiment, a single shoot (approximately 0.8 cm in height) was transferred to ms medium supplemented with various bap concentrations (2.0, 3.0, 4.0 mg/l) for multiplication over 4 weeks. shoot multiplication parameters, including the number of shoots, shoot height, number of leaves per shoot, and leaf length were recorded. protocorm-like bodies (plbs) induction, proliferation and shoot regeneration for plb induction, in vitro stems were separated from the cluster, leaves were removed, and the stem was cut into 1-2 mm transverse slices. these slices were placed on ms medium containing 10% (v/v) coconut water (cw), 2% (w/v) sucrose, and complemented with (0.5-3.0 mg/l) bap along with (0.5-2.0 mg/l) naa. the percentage of plb induction and the number of plbs per explant were recorded after 6 weeks of dark culturing. plb morphology and counting were performed at 2x to 4x magnification under a stereo microscope (olympus sz51, japan). secondary plb induction and shoot regeneration for plb proliferation, plb clusters were separated into individuals and placed on ms medium containing 10% (v/v) cw, 2% (w/v) sucrose, added (0.5-1.5 mg/l) bap and (0.2-0.5 mg/l) iaa. after 3 weeks, the secondary plb number and diameter, indicating plb growth speed, were recorded. to regenerate shoots from plbs, tuber-shaped plbs were separated and transferred to ms medium containing 10% (v/v) cw, 2% (w/v) sucrose, 1.0 g/l activated charcoal, 2 g/l peptone, and 1-3 mg/l ba and 0.5 mg/l iba. after 3 weeks, shoot height was measured, and shoot quality was determined by leaf color, stem diameter, and leaf length. effect of potato extract on shoot proliferation to assess the impact of potato extract on shoot proliferation, plbs were cultured on ms medium complemented with 1.5 mg/l bap and 0.5 mg/l naa ranging from 10 g/l to 50 g/l of pe. data on shoot number and individual shoot height were collected after the designated culture period. rooting in vitro plantlets (1-1.5 cm) regenerated from plbs were cultured on ms medium supplemented with 0.1% (w/v) activated carbon, 10% (v/v) cw, and indole-3-butyric acid (iba) at different concentrations (0.5, 1.0, 1.5, 2.0 mg/l). after 4 weeks, the number of roots per shoot, root length, shoot height, and number of leaves per shoot were recorded. in vitro plant regeneration of anubias barteri var. nana petite giang et al. 351 experimental design and statistical analysis all experiments were conducted in a completely randomized design (crd) with three repetitions for each treatment, and each repetition included five jars, each containing at least one explant. the data underwent basic statistical analysis and were examined using anova. mean values were compared using duncan’s method at 5% probability level. the data were presented as the average followed by the standard deviation (m±sd). results and discussion explants sterilization to prepare explants, decontamination was performed using various concentrations of commercial bleach (5% sodium hypochlorite) for a 15-minute disinfection. explants were crucially transferred to clean water for at least one week before sterilization to avoid a high infection rate. a 10% bleach solution yielded a 40% disinfection rate, but higher concentrations (up to 40%) were necessary to achieve a 100% sterilization rate, ensuring successful decontamination. the shoot tips, retaining their green color, initiated new shoots. shoot multiplication in this experiment, varying concentrations of bap were added to the ms medium to promote shoot formation and multiplication (table 1). during the shoot induction stage, shoot tips showed distinct responses to different bap concentrations after 10 days of culture. pgr-free ms media resulted in minimal shoot formation (1.13 shoots/explant), significantly increasing with higher bap concentrations. the maximum shoot yield (5.33 shoots/explant) was observed after 6 weeks on a medium with 3 mg/l ba, showcasing superior quality in terms of average shoot height (1.01 cm), leaves per shoot (4.92), and leaf length (1.03 cm) (table 1; figs. 1a-c). subsequent stages confirmed the efficiency of 3 mg/l bap in shoot multiplication, with clusters displaying large, green leaves, and long roots (figs. 1d-f ). the response to bap concentration observed in this study aligns with previous research on araceae species, such as a. barteri var nana (kanchanapoom et al. 2012; rittirat et al. 2021), cryptocoryne wendtii (rittirat et al. 2020), and anubias heterophylla (rittirat et al. 2021). these findings highlight the significant impact of bap concentration on shoot regeneration in a. barteri from shoot tips. however, adaptation to high pgr concentrations is not universal. for successful shoot regeneration from basal buds of a. barteri var. nana, it is necessary to use a minimal concentration of bap (0.2 mg/l), and shoot elongation was obtained on pgr-free ms basal medium (sheeja et al. 2015). other studies within the araceae family, utilizing lower bap concentrations, resulted in fewer shoots (george et al. 2015). in general, ba, with or without auxin supplementation, serves as the primary plant growth regulator in shoot induction and multiplication. ms medium supplemented with 3 mg/l bap has proven suitable for maintaining long-term in vitro cultures. shoot quality remained stable after four subculture cycles. protocorm-like body (plb) induction after 10 days of culture, transverse slices exhibited callus formation. after four weeks of cultivation, plbs began to emerge, initially appearing opaque white and assuming a tuberlike shape (table 2; fig. 2). the anova and subsequent post hoc analysis demonstrated a highly significant influence of bap and naa, and their interaction, on explant responses. for bap, three distinct groups revealed concentrations that induced significant plb formation. similarly, specific naa concentrations significantly impacted explant responses, particularly in plb induction. after 6 weeks of culture in media containing either 0.5 or 1.5 mg/l bap combined with 0.5 mg/l naa (p1, p3) resulted in underdeveloped buds on the browned callus, whereas 1.5 mg/l bap with 1.0 or 1.5 mg/l naa (p9, p15) induced callogenesis without plbs (figs. 2a-b). conversely, treatments p2, p5, p8, p11, p12, p14, p19, p20, p21, and p23 led to significant plb induction, showing variations in leafy plantlet, globular callus, and adventitious roots. treatments p4, p6, p10, p13, p16, p17, p18, p22, p24 resulted in tiny plb germination (figs. 2c-e). specific combinations of bap and naa concentrations (1.0 mg/l bap + 0.5 mg/l naa; 3.0 mg/l bap + 1.5 mg/l naa; 1.0 mg/l bap + 0.5 mg/l naa) significantly enhanced plb formation and leafy plantlet development (figs. 2 f-h). however, concentration-dependent effects were observed, leading to callusing or limited plb growth as certain concentrations. notably, a bap concentration of 1.5 mg/l, combined with 0.5 mg/l naa, showed a tendency to promote a robust plb induction rate. biotropia vol. 31 no. 2, 2024 352 table 1 effect of bap added to culture media on shoot formation after 6 weeks of culture bap (mg/l) number of shoots shoot height (cm) number of leaves leaf length (cm) 0.0 1.13d± 0.12 0.89b± 0.09 4.1c± 0.55 0.93d± 0.04 1.0 2.07c± 0.12 0.89b± 0.05 4.33bc±0.31 0.94cd± 0.02 2.0 3.87b ±0.12 0.92b± 0.02 4.63ab ±0.30 0.96bc± 0.03 3.0 5.33a ±0.31 1.01a±0.06 4.92a ±0.30 1.03a± 0.01 4.0 4.20b± 0.20 0.93b± 0.03 4.71ab ±0.21 0.98b± 0.03 notes: means followed by different letters indicate significant differences (duncan's test, p = 0.05). figure 1 shoot induction from shoot tips (a-c) and multiplication (d-f ) in response to varying bap concentrations after 6 weeks notes: (a, d) = 2 mg/l ba; (b, e) = 3 mg/l ba; (c, f ) = 4 mg/l ba. table 2 effect of bap combined with naa on callus/plbs/shoot formation after 6 weeks of culture treatment bap (mg/l) naa (mg/l) plb induction rate (%) plbs/explant morphogenesis of explant p0 0.0 0.0 0 0±0 callusing p1 0.5 0.5 100 5.89bc±0.81 tiny buds undeveloped, callus brown p2 1.0 0.5 100 6.56ab±0.85 plbs, leafy plantlets p3 1.5 0.5 100 7.78a±0.93 tiny buds undeveloped, callus brown p4 2.0 0.5 100 3.44de±0.62 tiny plbs sprout p5 2.5 0.5 100 2.56def±0.53 plbs, leafy plantlets p6 3.0 0.5 100 3.67def±0.64 tiny plbs sprout p7 0.5 1.0 100 4.56cd±0.71 globular callus, adventitious buds in vitro plant regeneration of anubias barteri var. nana petite giang et al. 353 treatment bap (mg/l) naa (mg/l) plb induction rate (%) plbs/explant morphogenesis of explant p8 1.0 1.0 100 4.44cd±0.70 plbs sprout p9 1.5 1.0 0 0g±0 callusing p10 2.0 1.0 100 3.00def±0.58 tiny plbs sprout. p11 2.5 1.0 100 1.22fg±0.37 plbs, leafy plantlets p12 3.0 1.0 100 3.00cdef±0.58 plbs, leafy plantlets p13 0.5 1.5 100 3.00def±0.58 small globular plbs, adventitious roots p14 1.0 1.5 100 3.22de±0.60 plbs sprout p15 1.5 1.5 0 0g±0 callusing p16 2.0 1.5 100 1.78efg±0.44 small plb p17 2.5 1.5 100 1.00efg±0.33 tiny plbs sprout. p18 3.0 1.5 100 1.11efg±0.35 plbs p19 0.5 2.0 100 2.33def±0.51 plbs, adventitious roots p20 1.0 2.0 100 2.00efg±0.47 plbs sprout p21 1.5 2.0 100 3.78de±0.65 plbs, adventitious roots p22 2.0 2.0 100 1.56def±0.42 tiny plbs sprouted. p23 2.5 2.0 100 2.44defg±0.52 plbs, leafy plantlets p24 3.0 2.0 100 2.67def±0.54 tiny plbs sprouted, adventitious roots. notes: means followed by different letters indicate significant differences (duncan's test, p = 0.01). figure 2 morphogenesis of transverse slices after 6 weeks of culture in the presence of different concentrations of bap and naa notes: a = underdeveloped buds on the browned callus; b = callogenesis without plb; c-e = plb induction with various patterns, i.e., leafy plantlet, globular callus, adventitious roots, and tiny plb germination; f-h = plb formation and leafy plantlet development. biotropia vol. 31 no. 2, 2024 354 research has showcased the efficacy of utilizing both naa and bap in stimulating plb induction across different species within the araceae family. rittirat et al. (2021) demonstrated the number of shoots per explants (3.60±0.24) was formed in the culture medium containing 1.0 mg/l naa in combination with 1.0 mg/l bap, followed by culture medium with 3.0 mg/l bap (2.40±0.24). mondal et al. (2014) found that a combination of 2 mg/l naa and 2 mg/l bap was most effective for plb induction in doritis pulcherrima lindl. sarma and tanti (2017) further supported these findings, showing that a combination of 3.0 mg/l bap and 1.0 mg/l naa was most successful in inducing shoot formation in aristolochia saccate. these studies collectively demonstrated that a combination of naa and bap is a popular approach for promoting plb induction and plantlet regeneration in araceae species. secondary plb induction and shoot regeneration plbs were subcultured on ms media with varying bap and iaa concentrations, leading to secondary plb (splb) formation (table 3). at 0.5 mg/l bap and 0.2 mg/l iaa, a 25.0% plb induction rate was observed, with a mean diameter of 0.61 cm. however, the quantity of splb generated by each explant was 0.33, indicating moderate development. increasing bap to 1.0 mg/l whereas maintaining 0.2 mg/l iaa resulted in a notable decline in the plb induction rate (0.00%), along with a decrease in both the number of secondary plbs and their diameter, suggesting a dose-dependent negative effect of bap on plb formation at this concentration. interestingly, at 1.0 mg/l bap with an elevated iaa concentration of 0.5 mg/l, the plb induction rate rebounded to 33.3%, with an increase in both the number of splb and their diameter, suggesting a potential synergistic effect between bap and iaa. at the highest concentrations of both bap (1.5 mg/l) and iaa (0.5 mg/l), the plb induction rate reached 68.1%, demonstrating substantial improvement. the number of splbs increased to 1.55, and the diameter of the plbs reached 0.67 cm, indicating a robust response to the combined higher concentrations of bap and iaa. these results highlight the significant influence of the interplay between bap and iaa concentrations on plb induction and subsequent development. the optimal combination for enhanced plb formation appears to be 1.5 mg/l bap and 0.5 mg/l iaa, offering valuable insights for optimizing micropropagation protocols for this specific plant. the impact of different concentrations of bap and iba on shoot regeneration from plbs is presented in table 4. at 1.0 mg/l bap and 0.5 mg/l iba, shoots reached a height of 0.65 cm. increasing bap to 1.5 mg/l whereas maintaining iba at 0.5 mg/l resulted in a slightly improved shoot length of 0.68 cm and enhanced shoot quality. similarly, at bap concentrations of 2.0 mg/l with 0.5 mg/l iba, shoot length remained consistent at 0.68 cm. at higher bap concentrations (2.5 mg/l) and 0.5 mg/l iba, the shoot length increased to 0.83 cm. notably, increasing iba to 1.0 mg/l with bap at 1.0 mg/l significantly improved shoot length to 1.23 cm, with good quality, indicating vigorous shoot regeneration. for bap ranging from 1.5 mg/l to 3.0 mg/l with 1.0 mg/l iba, shoot lengths varied from 0.94 cm to 1.57 cm, and the shoot quality was good. these results emphasized the crucial role of the interaction between bap and iba concentrations in shoot regeneration potential from plbs. the combination of bap and iaa is effective in promoting shoot proliferation and elongation in quercus suber l. (romano et al. 1992). this combination also enhances shoot growth and proliferation in aegle marmelos (ajithkumar & seeni 1998). in the production of plbs in orchids, bap is the most effective growth regulator, followed by kinetin, naa, iaa, 2,4-d, and gibberellic acid (ga3) (saiprasad et al. 2002). in the case of pointed gourd, bap has been found to induce callus formation, whereas the addition of iaa has been shown to enhance rooting (komal 2011). these findings suggested that the combination of bap and iaa may have a positive effect on plbs multiplication and shoot regeneration in araceae, but further research is needed to confirm this. impact of potato extract on shoot regeneration the impact of varying potato extract (pe) concentrations on shoot regeneration from plbs is shown in table 5. without pe, an average of 2.33 shoots was noted for each explant, accompanied by an average shoot length of 1.21 cm. however, at 10 g/l, 20 g/l, and 30 g/l pe, the number of shoots per explant nearly doubled to 4.19, 4.37, and 4.62, and the shoot height increased to 1.72, 2.08, and 2.36 cm. the most significant enhancement in shoot regeneration occurred at 40 g/l and 50 g/l pe concentrations. at 40 g/l, shoots per explant increased to 5.52, and the shoot height reached in vitro plant regeneration of anubias barteri var. nana petite giang et al. 355 2.74 cm. the highest concentration, 50 g/l, yielded the most favourable outcome, with 6.55 shoots per explant and a maximum shoot height of 2.80 cm (fig. 3). this highlighted a positive correlation between pe concentration and both the quantity and quality of shoots regenerated from plbs, suggesting a potential role of specific compounds within the pe in promoting shoot development. studies indicate that plantlet regeneration from protocorm-like bodies in orchids is significantly enhanced by the application of pe. rahman (2004) found that pe enhanced plantlet regeneration and growth, with the optimum concentration being 100 ml/l. this finding was further supported by islam et al. (2012), who reported that pe enhances the germination of seeds and the growth of seedlings of vanda roxburgii orchids. the beneficial impact of pe on the growth and development of plbs was also demonstrated by lee (2003) in cypripedium formosanum (lee & lee 2003). collectively, these studies suggest that pe can be a valuable supplement in the medium for regenerating of plantlets from plbs in orchids. effects of iba on rooting and plantlet growth the impact of iba on the root and plantlet development of a. barteri after a 4-week culture period is shown in table 6. with iba of 0.5 mg/l, all parameters showed significant improvement. the number of roots increased to 6.19, with a longer average root length of 2.50 cm. shoot height and leaf number also increased to 4.72 cm and 6.81, respectively. at 1.0 mg/l iba, whereas the number of roots slightly decreased to 4.81, both root length (2.14 cm) and shoot height (4.80 cm) remained relatively higher compared to the control, with a leaf number of 6.11. further increases in iba concentration to 1.5 mg/l and 2.0 mg/l led to a gradual decline in the number and length of roots, shoot height, and leaf number. the results indicated that iba positively influences root and plantlet development in a. barteri var. nana petite with an optimal response observed at 0.5 mg/l iba. higher concentrations, beyond 0.5 mg/l, showed diminishing returns, suggesting a dose-dependent effect. table 3 effect of ba and iaa added to secondary plb induction after 4 weeks of culture plant growth regulators (mg/l) splb induction rate (%) splbs/explant diameter of plb (cm) ba iaa 0.5 0.2 25.0bc±14.4 0.33bc±0.33 0.61abc±0.10 1.0 0.2 0.00d±0.00 0.00c±0.00 0.50c±0.06 1.5 0.2 8.33cd±8.33 0.33bc±0.33 0.51bc±0.01 0.5 0.5 25.0bc±14.4 1.00ab±0.00 0.56abc±0.03 1.0 0.5 33.33b±8.33 1.00ab±0.00 0.65ab±0.03 1.5 0.5 68.06a±3.67 1.55a±0.29 0.67a±0.16 notes: means followed by different letters indicate significant differences (duncan's test, p = 0.05). table 4 effect of bap combined iba on shoot formation from protocorm-like bodies bap (mg/l) iba (mg/l) shoot height (cm) shoot quality 1.0 0.5 0.65b±0.09 poor 1.5 0.5 0.68b±0.09 fair 2.0 0.5 0.68b±0.09 fair 2.5 0.5 0.83b±0.08 fair 3.0 0.5 0.74b±0.14 fair 1.0 1.0 1.23ab±0.20 excellent 1.5 1.0 0.94ab±0.27 fair 2.0 1.0 1.00ab±0.24 fair 2.5 1.0 0.91ab±0.09 good 3.0 1.0 1.57a±0.55 fair notes: means followed by different letters indicate significant differences (duncan's test, p = 0.05). biotropia vol. 31 no. 2, 2024 356 table 5 impact of potato extract on the quantity and quality of shoots potato extract (g/l) shoots/explant shoot height (cm) 0 2.33e±0.11 1.21e±0.06 10 4.19d±0.13 1.72d±0.13 20 4.37cd±0.06 2.08c±0.07 30 4.62c±0.17 2.36b±0.11 40 5.52b±0.23 2.74a±0.07 50 6.55a±0.19 2.80a±0.04 notes: means followed by different letters indicate significant differences (duncan's test, p = 0.05). figure 3 the effect of potato extract on the growth of plbs derived plantlets notes: a = 0 g/l; b = 10 g/l; c = 20 g/l; d = 30 g/l; e = 40 g/l; f = 50 g/l. table 6 effects of iba on root and plantlet after 4 weeks of culture iba (mg/l) no. of roots root length (cm) shoot height (cm) no. of leaves 0.0 4.22bc±0.29 1.64c±0.33 4.12b±0.31 5.22bc±0.40 0.5 6.19a±0.56 2.50a±0.11 4.72a±0.14 6.81a±0.45 1.0 4.81b±0.39 2.14ab±0.19 4.80a±0.19 6.11ab±0.51 1.5 3.33cd±0.22 1.74bc±0.07 4.19b±0.07 4.74cd±0.17 2.0 2.70d±0.17 1.05d±0.02 3.94b±0.09 4.00d±0.22 notes: means followed by different letters indicate significant differences (duncan's test, p = 0.05). in vitro plant regeneration of anubias barteri var. nana petite giang et al. 357 conclusion this study successfully developed a micropropagation protocol for anubias barteri var. nana petite, enabling rapid and efficient multiplication. through precise sterilization and optimized growth conditions, including the use of 3 mg/l benzyl adenine (bap), the protocol demonstrated high rates of shoot regeneration. additionally, the induction of protocorm-like bodies (plbs) from stem explants and subsequent shoot regeneration highlighted the effectiveness of 1.5 mg/l bap combined with 0.5 mg/l naa or 0.5 mg/l iaa; 3 mg/l bap plus 1.0 mg/l iba. the study also demonstrates the impact of 50 g/l of potato extract on shoot proliferation and 0.5 mg/l of indole-3-butyric acid (iba) in root development. this improved method presents a promising approach for the mass production of a. barteri var. nana petite, addressing the challenges associated with natural propagation and meeting market demands for this valued ornamental aquatic plant. references ajithkumar d, seeni s. 1998. rapid clonal multiplication through in vitro axillary shoot proliferation of aegle marmelos (l.) corr., a medicinal tree. plant cell reports. 17: 422-6. doi: 10.1007/s002990050418 cardoso jc, zanello ca, chen jt. 2020. an overview of orchid protocorm-like bodies: mass propagation, biotechnology, molecular aspects, and breeding. int j mol sci 21(3):985. cui j, liu j, deng m, chen j, henny rj. 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biotropia vol. 30 no. 2, 2023: 147 157 doi: 10.11598/btb.2023.30.2.1765 147 production optimization, partial purification, and thrombolytic activity evaluation of protease of bacillus cereus hsfi-10 ainutajriani ainutajriani1, sri darmawati1, dewi seswita zilda2, muhammad ardi afriansyah3, ragil saptaningtyas3 and stalis norma ethica1* 1magister of clinical laboratory science, postgraduate program, universitas muhammadiyah semarang, central java, 50273, indonesia 2research center for deep sea, earth sciences and maritime research organization, national research and innovation agency (brin), jl. pasir putih raya pademangan, north jakarta city, jakarta, 14430, indonesia 3department of medical laboratory technology, faculty of nursing and health sciences, universitas muhammadiyah semarang, central java, 50273, indonesia received 17 june 2022 / revised 2 march 2023 /accepted 11 march 2023 abstract cardiovascular disease is the primary cause of mortality in the world due to the formation of blood clots or thrombi in blood vessels. bacterial proteases commonly function as thrombus dissolver agents in the pharmaceutical industry. bacterial isolate hsfi-10 (holothuria scabra fermented intestine-10) previously isolated from rusip fermented sea cucumber had demonstrated thrombolytic activity. this study aimed to produce crude protease of hsfi-10 strain at an optimized incubation time and determine the thrombolytic activity of crude and dialysate proteases on a, b, ab, and o blood types. isolate hsfi-10 was first molecularly identified and found to be bacillus cereus with a homology level of 99.80% with bacillus cereus strain st06. the optimum crude enzyme was obtained after 48-h incubation with an activity of 222.52 u/ml, which increased to 438.84 u/ml after ammonium sulfate precipitation and dialysis. clot lysis activity of crude enzymes was measured based on the gravimetry method on blood in the abo system, showing results that ranged from 68.99% to 69.76%, while the dialysate ranged from 81.16% to 82.52%. in conclusion, partial purification of bacterial protease could increase both its specific and thrombolytic activities on human blood in the abo system, with only 1% activity variability between a, b, ab, and o blood types. keywords: bacillus cereus hsfi-10, blood system, clot lysis, partial purification, thrombosis introduction thrombosis is the leading cause of global death in cardiovascular disease (cvd) (scheres et al. 2018). cardiovascular is a group of diseases related to the heart and blood vessels. according to the world health organization (who), this disease causes 31% of death worldwide. who predicts that by 2030, cardiovascular disease will continue to increase to more than 23.6 million people. the number is two times higher than the death rate from cancer (who 2017). thrombosis causes blood to clot (forming a thrombus), so blood vessels in the heart and brain will be blocked (martina et al. 2019). thrombus occurs in the area of the injured vascular wall, thereby stimulating platelet adhesion in that direction. the attached platelets are activated and release adenosine diphosphate (adp) and thromboxane a2 (txa2), which causes other platelets to stick to the activated platelets. platelet aggregation is strengthened with the help of clotting factors in the form of fibrin threads to form a hemostatic plug (bartinelli et al. 2013; durachim and astuti, 2018). thrombosis therapy can use anticoagulant agents, anti-platelet drugs, fibrinolytic/thrombolytic enzymes, and surgery. however, these drugs have side effects such as headaches, urticaria (allergic reactions), increased clotting time, nausea, vomiting, bleeding complications, low fibrin specificity, and *corresponding author, email: norma@unimus.ac.id biotropia vol. 30 no. 2, 2023 148 relatively high price (saxena et al. 2015; krishnamurthy et al. 2018; sharma et al. 2019). research conducted by hidayati et al. (2021) successfully isolated protease-producing bacteria from the digestive organs of the sand sea cucumber coded hsfi-10 to -12 (holothuria scabra fermented intestine-10). the crude enzyme of bacterial isolate hsfi-10 showed higher thrombolytic ability than the positive control nattokinase (a commercial antithrombosis agent). however, the bacterial isolates were only known for their microscopic and macroscopic characteristics. in addition, the thrombolysis test does not explicitly mention blood groups a, b, ab, and o. in contrast, antigens a and b in the abo system blood group impact hemostatic balance and respond differently to thrombolytic therapy (separham et al. 2020; mehic et al. 2020). identification was done to determine bacterial species by amplifying the 16s rrna gene using polymerase chain reaction (pcr). this molecular diagnostic method is more sensitive, specific, efficient, and faster than manual identification (shahi et al. 2018). bacteria secrete proteases in the production medium. each bacterium has a different enzyme production time depending on the type of bacteria (sharma et al. 2015)—partial purification of the protease enzyme bacillus sp. hsfi-10 can be done by precipitating enzymes using ammonium sulfate. this salt has a fairly high ionic strength, does not cause damage to proteins, and has a high solubility in water (mukherjee, 2019). the remaining ammonium sulfate salt from the enzyme precipitate is removed by dialysis (razzaq et al. 2019). this study aims to determine the type of bacteria based on the 16s rrna gene sequence using the pcr method, to determine differences in protease enzyme activity based on the optimization of production time for 24, 48, and 72 hours and to determine the description of a, b, ab, and o blood smears after the addition thrombolytic protease enzymes of bacillus sp. hsfi-10. materials and methods bacterial origin bacterial strain hsfi-10 was previously isolated in an earlier study from sand sea cucumber h. scabra captured from its captivity at marine bio industry office (bbil), the indonesian institute of sciences (lipi), kodek gulf village, lombok, west nusa tenggara (figure 1). the isolate was then subcultured, and the purified colonies were further analyzed to identify and confirm its fundamental characteristics. figure 1 location of the captivity of sand sea cucumber h. scabra, which intestine was the source of hsfi bacteria including hsfi-10 strain: (red dot, coordinates 8°24'15.1"s 116°04'47.2"e) (google map 2022) thrombolytic protease of bacillus cereus hsfi-10 – ainutajriani et al. 149 procedures bacterial subculture and crude protease production bacterial subculture was done by cultivating a loop-full of hsfi-10 bacterial single colony nutrient agar (na) medium and then incubated at 37℃ for 24 hours. a single colony of isolate hsfi-10 obtained was then tested to confirm its proteolytic activities by streaking it on skim milk agar (sma) media and re-incubated for 24 hours at 37°c. the clear proteolytic zone formed around the colony's growth was observed (fuad et al. 2020; hidayati et al. 2021). bacterial molecular identification by cloning 16s rdna bacterial colonies of hsfi-10 were inoculated into the bhib 1 ml medium. after incubation at 37°c for 2 × 24 hours. bhib media was centrifuged for 1 min at 12000 rpm. dna genome was extracted with a dna extraction kit from geneaid, namely presto™ mini gdna bacteria kit. the amplification process used go tag green master mix (promega). the universal primers used were 27f (5'-aga gtt tga tcc tgg ctc ag-3') and 1492-r (5'-ggt tac ctt gtt acg act t-3'). amplicon was visualized using the major science uv transilluminator (darmawati et al. 2015). cloning of the 16s rrna gene was done as previously reported by darmawati (2015) using plasmid vector pta2 transformed in e. coli zymo 5α competent cells. recombinants were screened using the blue-white method (green & sambrook 2021). plasmids were isolated according to the previously reported method, with the results of recombinant dna checked with electrophoresis on 0.8% agarose gel. sequencing was carried out using abi sequence programs with primers t7 and t3 5'taatacgactcactataggg-3' and 5'ccctttagtgagggttaatt-3' as previously reported (darmawati 2015). next, bioinformatics dna sequencing results are analyzed using bioinformatics devices, then processed manually and matched with data in www.ncbi.nih.gov through the blast program (ethica et al. 2018). optimization of bacterial crude protease production colony of bacillus sp. hsfi-10, which has proteolytic abilities inoculated in minimally synthetic medium (msm) (nacl 0.1%, k2hpo4 0.1%, ammonium sulfate 0.7%, mgso4.7h2o 0.01%, yeast extract 0.05%, skim milk 1%) and incubated at 37°c for 24,48 and 72 h (farooq et al. 2021). the extraction of thrombolytic protease enzymes is done by bacterial culture centrifugation at 10,000 rpm, where supernatant was regarded as a crude enzyme. it further measured the enzyme activity of the bergmeyer and grab method (1983) using a spectrophotometer at λ 600 nm. partial purification of bacterial protease partial purification was initiated by precipitation using 70% ammonium sulfate, as much as 400 g added slowly in 1 l of crude enzymes as previously described (natsir et al. 2015). cellophane bags were initially soaked in hot water at 60°c for 2 mins, then replaced with 0.2% sulfuric acid as previously reported. enzymes obtained from the deposition of ammonium sulfate were put in cellophane bags. both ends of the cellophane bag are tied, then the cellophane bag is soaked with a 500-700 ml phosphate buffer of 0.05 m ph 7 at ± 4°c for 2 h. the soaking buffer is replaced every 2 hours until all salts are separated (abbas et al. 2018). specific enzyme and clot lysis activity assay the protease-specific activity was measured using the modified bergmeyer & grab (1983) (natsir et al. 2015; si & jang 2018). in vitro, blood clot lysis tests were conducted on blood from 4 volunteers, each with blood types a, b, ab, and o, in 6 of the 1.5 ml microtubes previously weighed. each tube was coded 1-6, tube 1 (negative control), tube 2 (positive control), tube 3 (blood type a), tube 4 (blood type b), tube 5 (blood type ab), and tube 6 (blood type o), and filled with 600 μl blood of each. as previously reported, gravimetry determined clot lysis percentage (fuad et al. 2020; hidayati et al. 2021; prasad et al. 2006). proteolytic activity test a single colony of bacillus sp. bacteria. hsfi10 obtained from the results of bacterial purification in na media tested its proteolytic abilities. bacteria that grow on na media were streaked on skim milk agar (sma) media and incubated for 24 h at 37°c then the clear zone biotropia vol. 30 no. 2, 2023 150 formed around the growth of the colony was measured (fuad et al. 2020; hidayati et al. 2021). optimization of crude enzyme protease production time from bacillus cereus culture colony of bacillus sp. hsfi-10 with proteolytic abilities was inoculated in minimal synthetic medium (msm) incubated at 37°c for 24, 48, and 72 h. extraction of thrombolytic protease enzymes was done by centrifugation at 10,000 rpm for 20 mins at 4°c. the obtained supernatant was regarded as a crude enzyme, and its activity was further measured by bergmeyer & grab method (1983) using a spectrophotometer at λ=600 nm (farooq et al. 2021). partial purification of crude protease 70% of ammonium sulfate (400 g) was inserted slowly in 1 l of crude enzymes in cold conditions until dissolved for 2 hours. the enzyme solution was kept in the refrigerator to precipitate the enzymes overnight. the enzyme solution was concentrated at 4°c at 10,000 rpm for 30 mins, and the formed pellet was kept. the pellet was then rushed with a 15 ml tris-cl buffer of 0.05 m ph 8 (natsir et al. 2015). cellophane bags were prepared by soaking in hot water and 0.2% sulfuric acid solution before use. enzymes obtained from the deposition of ammonium sulfate are put in cellophane bags. dialysis was conducted as previously described until all salts were separated (abbas et al. 2018). enzyme-specific activity assay the protease-specific activity was measured using the modified bergmeyer & grab (1983) folin reagent method (natsir et al. 2015; si and jang 2018). clot lysis (thrombolysis) activity in vitro assay in vitro blood clot lysis tests before and after enzyme purification were conducted on blood taken from 4 volunteers, each with blood types a, b, ab, and o. six 1.5-ml-microtube tubes that had been weighed were prepared. each tube was coded 1-6, tube 1 (negative control), tube 2 (positive control), tube 3 (blood type a), tube 4 (blood type b), tube 5 (blood type ab), and tube 6 (blood type o). the percentage of % blood clots was determined following the previously reported method and was conducted in duplicates (prasad et al. 2006; fuad et al. 2020; hidayati et al. 2021). the effect of blood clot lysis was also observed microscopically by the eustrek (removal) technique according to the may grunwald-giemsa mixed method (geneser 1994). results were observed with magnification under a dino-lite digital microscope and documented with a camera (ethica et al. 2018). results and discussion bacterial subculture and morphology characteristics bacillus sp. hsfi-10 has a circular shape, edge (entire), size (3 mm), milk-white color, convex elevation, and smooth consistency (figure 2a)— bacillus sp. hsfi-10 has a short life in na media (figure 2b), a colony life of approximately 4 days of storage at cold temperatures, characterized by colony colors beginning to fade and cannot grow back on the new na medium. figure 2 characteristics of bacillus sp. hsfi-10 in agar nutrient (na) (a) colonies after 24-h incubation, (b) after stored for 4 days. c. on skim milk agar media with lugol-staining (clear zone showing proteolytic activity) thrombolytic protease of bacillus cereus hsfi-10 – ainutajriani et al. 151 figure 3 similar characteristics of bacillus sp. hsfi-10 on blood agar plate media. (a) compared to (b) b. cereus indicates β-hemolysis (milojevic et al. 2019) gram staining showed the rod shape was gram-positive, lined, and had spores. b. cereus could produce a clear zone on the 7th day of cultivation on skim milk agar (sma) media with a diameter of 36 mm. sma contains casein as a protease enzyme substrate. bacterial growth in blood agar plate (bap) media indicates a βhemolysis pattern, characterized by an area of clear zone around the colony (figure 3) (bottone 2010; lu et al. 2018). as seen in figure 3, the macroscopic characteristics of bacteria on bap media were evaluated by the shape, color, size, edge, and elevation of the colony as well as discoloration in the media (e. g. hemolysis in the medium for blood) (pitt et al. 2012). the result aligned with the mogrovejo et al. (2020) study reporting that bacillus cereus can disintegrate red blood cells (forming a β-hemolysis pattern on bap). b. cereus hsfi-10 morphology and properties reported in this study following the results reported by hidayati et al. (2021). the proteolytic ability test aims to determine bacteria that have the potential to produce proteases, characterized by the formation of a clear zone around the bacterial colony (assaf et al. 2020). the clear zone produced by proteolytic bacteria occurs due to protease activity which breaks the peptide bonds of casein in skim milk medium by breaking the co-nh peptide bond with the entry of water into the molecule, thereby releasing amino acids (baehaki et al. 2011; ethica et al. 2018; artha et al. 2019). bacterial molecular identification dna extract of bacillus sp. hsfi-10 has a concentration of 232.6 ng/μl and a purity of 1.82. the purity of extracted dna was high if the absorbance ratio (λ260/λ280) was 1.83. if the absorbance ratio is less than 1.8, proteins still contaminate dna. if greater than 1.8, the dna is contaminated with rna (gupta 2019). the genomic 16s rrna gene cloning technique obtained the full-length 16s rrna gene sequence (green & sambrook 2021). the cloning process was done by ligating, transforming, and isolating recombinant dna from transformants (green & sambrook 2021). recombinant dna was then transformed in escherichia coli zymo 5α bacterial cells and grown on a solid medium. the success of cloning could be seen through white colonies (clones carrying recombinant dna). the white colony indicates the lacz region on the plasmid is inactive because it has been inserted by the inserted gene so that the galactose present in the media cannot be hydrolyzed by the -galactosidase enzyme which, if active, will form blue colonies (sambrook et al. 1989; darmawati 2015)— results of the 16s rrna gene cloning of bacillus sp. hsfi-10 is displayed in figure 4, with an amplicon size of 1517 bp. biotropia vol. 30 no. 2, 2023 152 figure 4 the complete sequence of the 16s rrna bacillus sp. hsfi-10 obtained from cloning and sequencing 16s rrna gene the consensus was made on the forward and reversed 16s rdna sequences using the bio edit program (hall 2004). the 16s rrna gene sequence data saved in fasta format were then analyzed and matched with the data available in the gene bank basic local alignment search tool (blast). the results of blast analysis of bacillus sp. hsfi-10 16s rrna gene fragments showed a homology level of 99.80% with bacillus cereus strain st06 (acc. no.: mh475925.1). optimization of crude production time for protease enzymes from bacterial culture crude enzyme production was optimized with various incubation times of 24, 48, and 72 hours. the results of this study indicate that bacillus cereus hsfi-10 can produce the most optimum crude enzyme at 48 hours with an enzyme activity of 222.52 u/ml. it has been reported that protease production was proportional to bacterial growth. in the early stages of the growth curve, bacteria produced few proteases and optimum production in the stationary phase (ahmadpour and yakhchali 2017; pagarra et al. 2020; suleiman et al. 2020). partial purification of protease and specific activity assay the protease activity of b. cereus hsfi-10 was calculated using the equation from hidayati et al. (2021): y = 0.0019 x + 0.0092. y = absorbance, while x = enzyme activity (u/ml). enzyme activity was expressed as the number of tyrosine amino acids released by the casein substrate per unit of time under test conditions (zainuddin et al. 2020). results of specific activity assay on crude and dialysate protease of b. cereus hsfi-10 are shown in table 1. table 1 absorbance and specific activity data of crude and dialysate protease of b. cereus hsfi-10 protease extract absorbance at λ=600 nm at incubation time (h)*: enzyme activity (u/ml) at incubation time (h)*: 24 48 72 24 48 72 crude 0,264 0,433 0,346 134,105 222,526 146,000 dialysate 0,843 438,842 *all tests were conducted in duplicate thrombolytic protease of bacillus cereus hsfi-10 – ainutajriani et al. 153 based on data in table 3, it could be inferred that ammonium sulfate precipitation followed by dialysis could increase the enzyme-specific activity by almost doubling from 222.526 u/m to 438.842 u/ml. these results are to the research of mothe and sultanpuram (2016), reporting that enzymes purified by dialysis had high activity compared to crude enzymes, where the level of enzyme purity could reach 2.23 times higher. clot lysis activity assay on crude and partially purified protease crude and dialysate enzyme b. cereus hsfi-10 could lyse abo blood clot better than nattokinase as control (figure 5). a higher percentage of clot lysis characterized it. the % blood clot lysis of each sample is shown in table 2. nattokinase is a serine protease enzyme with fibrinolytic and antithrombotic activity. this ability can be used for cardiovascular treatment. several studies have stated that nattokinase can thin the blood and dissolve blood clots in experimental animals and humans (gallelli et al. 2021; chen et al. 2022). this study shows that crude enzymes and dialysate can lyse blood clots better than nattokinase (figure 5). hence, the protease enzyme from b. cereus hsfi-10 bacteria can be used as a substitute for nattokinase. as seen in table 2, part of the limitation of this preliminary study is that the thrombolysis ability test was conducted only in duplicates with the objective of screening. one of the reasons is that the gravimetry test, first described by prasad et al. 2006, is semi-quantitative. the qualitative part of the assessment lies in whether the lyse blood is still present before weighing the step of the method. thus, the more accurate thrombolysis test should be confirmed with the in vivo one involving precise crude and dialysate bacterial protease dosage followed by statistical analysis (dewi et al. 2022). the effect of clot lysis on o blood cells before and after adding crude and dialysate enzymes was also observed under a microscope with 400× magnification, with results displayed in figure 6. figure 5 crude protease enzyme thrombolysis test and dialysate on blood type o table 2 results of the thrombolysis ability test for crude protease enzymes and dialysates in abo blood type blood type initial clot weight (g)* final clot weight (g)* % clot lysis* nk crude dialysate nk crude dialysate nk crude dialysate a 0.358 0.358 0.299 0.141 0.111 0.055 60.614 68.994 81.605 b 0.333 0.334 0.282 0.133 0.101 0.051 60.060 69.760 81.914 ab 0.290 0.290 0.276 0.116 0.088 0.052 60.000 69.655 81.159 o 0.369 0.368 0.349 0.145 0.113 0.061 60.704 69.293 82.521 *all measurement was conducted in duplicates nk: nattokinase biotropia vol. 30 no. 2, 2023 154 figure 6 the observation results on protease clot lysis activity on o blood cells using a dino-lite digital microscope. (a) negative control, (b) positive control, (c) crude enzyme, (d) dialysate enzyme as seen in figure 6, platelets undergoing aggregation and erythrocytes experience changes in shape and overlap (figure 6a), indicating the occurrence of blood clots. while figures 6b, c, and d show that platelets did not undergo aggregation, erythrocytes did not change in shape and are evenly distributed. the platelet condition was due to favorable control treatment, crude, and dialysate, which can lyse blood clots. these results align with the research of fuad et al., 2020, where crude protease enzymes produced by staphylococcus hominis hsft-2, s. saprophyticus hsft-11, and bacillus aryabhattai hsft-5 caused erythrocytes not to create, spread evenly and platelets did not aggregate. our results showed that partial purification by ammonium sulfate precipitation and dialysis on produced bacterial crude protease is very beneficial. the partial purification could increase bacterial protease-specific activity from 222.52 u/ml to 438.84 u/ml. it could also increase clot lysis activity based on the gravimetry method on blood in the abo system from 68.99% 69.76% (crude protease) to 81.16% (crude 82.52% (dialysate protease). the next step will be enzyme purification techniques using various chromatography-based methods to maximize protease's specific and clot lysis activity from b. cereus hsfi-10 (westphal & van berkel 2021). in this study, bacterial proteases had a similar effect on the lysis of abo blood clots regardless of their different types of agglutination. despite the previously reported data that blood type did affect the formation of blood clots, it has been known that individuals with non-o blood type have been shown to have a higher risk of thrombus formation than individuals with blood type o. this was associated with levels of coagulation factors, especially von willebrand factor (vwf), vwf levels 30% higher in individuals with non-o blood type compared with blood group o (holle et al. 2020; mohamed et al. 2020; separham et al. 2020; pendu et al. 2021). conclusion the optimum production of crude enzyme of thrombolytic protease-producing bacterium, b. cereus hsfi-10, resulting in the highest specific activity, was at 48-h incubation. partial purification of bacterial protease increased both its specific and thrombolytic activities in human blood of the abo group system with only 1% activity variability between a, b, ab, and o blood types. acknowledgments the authors would like to express their sincere gratitude to the center for research and community service (lppm) universitas muhammadiyah semarang for financially supporting this research through an internal grant in 2021. thrombolytic protease of bacillus cereus hsfi-10 – ainutajriani et al. 155 references abbas, n., siddique, h.n., masood, f., shehzadi, a., abbas, z. and ali, s., 2020. production of protease enzyme from bacillus subtilis using skimmed milk. science international (lahore), 32(2), pp.211-214. 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https://www.who.int/en/news-room/factsheets/ detail/cardiovascular diseases(cvds) sri-13 mei 2017-621 (romnick ecological) rev layout.cdr ecological services of agroforestry landscapes in selected watershed areas in the philippines and indonesia romnick s. baliton , christine wulandari , leila d. landicho , rowena 1 2 3* esperanza d. cabahug , roselyn f. paelmo , reynaldo a. comia , roberto g. 3 4 3 visco , pitojo budiono , susni herwanti , rusita and arnold karl sa. castillo 1 5 6 6 3 1 institute of renewable natural resources, college of forestry and natural resources, university of the philippines los banos, college, laguna 4031, philippines 2 graduate program of forestry, universitas lampung, bandar lampung 35141, indonesia 3 institute of agroforestry, college of forestry and natural resources, university of the philippines los banos, college, laguna 4031, philippines 4 institute of crop science, college of agriculture and food science, university of the philippines los banos, college, laguna 4031, philippines 5 faculty of social science and politic, universitas lampung, bandar lampung 35141, indonesia 6forestry department, faculty of agriculture, universitas lampung, bandar lampung 35141, indonesia received 7 february 2016/accepted 18 january 2017 abstract this article argues that the practice of agroforestry provides ecological contributions to the smallholder farmers cultivating in the watershed areas. specifically, this farming system provides contribution to carbon sequestration potential of the woody perennials and the biodiversity conservation of the other components of the system. this argument is based on the research conducted in molawin-dampalit sub-watershed, mt. makiling forest reserve in the philippines and way betung watershed in indonesia. the research involved an interview session of 106 and 261 smallholder farmers and an assessment of 27 and 14 agroforesty plots for carbon stock assessment and biodiversity assessment, respectively. results indicated that the total carbon found among the crop components was 52.32 mgc/ha in molawin-dampalit sub-watershed and 244.26 mgc/ha in way betung watershed, which suggested the high carbon sequestration potential of the woody perennials and understory crops in an agroforestry system. the farm lots being cultivated by the smallholder farmers were found to contribute to biodiversity conservation having a moderate biodiversity index of 2.59 and 2.53, respectively. with these findings, promotion of desired agroforestry systems in suitable portions of the watershed areas should be intensified and heightened to contribute to ecological balance across the landscape. agroforestry should always be an integral part of all initiatives toward ecological restoration with the cultivators/smallholder farmers as potential partners. the agroforestry system should consider all the technical and socioeconomic considerations toward having diverse components and ensure food security among the smallholder farmers throughout the year. keywords: agroforestry, biodiversity index, carbon stock, molawin-dampalit sub-watershed, way betung watershed biotropia 4 1 7 71 84 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 1 621 * corresponding author: ldlandicho@gmail.com 71 introduction southeast asia is among regions enlisted as biologically rich and diverse. three countries, including malaysia, philippines and indonesia, are in fact cited as mega-diverse countries in the region, being the homes of a number of plant and animal species. at present, however, southeast asia's biodiversity is highly threatened. sodhi et al. (2004) highlighted that the international union for the conservation of nature and natural resources (iucn) listed three plant and eight animal species as already extinct in the region. furthermore, the authors emphasized that the number of threatened species in southeast asia including in the iucn categories of critically endangered (ce), endangered (en) and vulnerable (vu) ranges from 20 (ce) to 686 (vu) 72 biotropia vol. 24 no. 1, 2017 crops, woody perennials and/or animals or aquatic resources for the twin purpose of production and conservation. tolentino et al. (2010) highlighted that the diversity of plants used in agroforestry provides multiple benefits at different times of the year. these diverse combinations can help buffer its practitioners from the risk of income loss due to price variability, crop failure and other unanticipated problems. ecologically, agroforestry helps enhance biodiversity of the environment. it is expected that as the diversity of agroforestry farms increases, farmers would have the opportunity to make use of the flora-fauna interaction to control pests and diseases, improve microbiology and nutrient cycling. all these are requisites for survival and improved plant growth. this article highlights the respective biodiversity and carbon sequestration potentials of selected agroforestry landscapes in the molawin-dampalit sub-watershed in the philippines and way betung watershed in indonesia. materials and methods study site way betung watershed has an area of 5,260 ha, 51% of which is classified as the forest park area and the remaining 49% is classified as agricultural areas (fig. 1). most of the upland farmers are engaged in agroforestry, particularly species for vascular plants, 6 91 species for fish, 0 23 amphibian species, 4 28 reptile species, 7 116 bird species and 5 147 mammal species (sodhi et al. 2004). biodiversity loss in southeast asia is attributed to a number of factors, including deforestation or clearing of the forest cover; conversion of agricultural lands to other economic purposes; natural calamities such as el niño, extreme weather events, climate change, forest fires; continued dependence on forest resources as livelihood of the growing population; and, invasive species. these all boil down to the rapid ecological, social and economic changes that the world faces. most often, the forest dwellers are accused as culprits of the biodiversity loss because of their continued dependence on the forest resources for economic and livelihood activities. two of the most popular watersheds in southeast asia are the molawin-dampalit subwatershed located inside the mt. makiling forest reserve (mmfr) in the philippines and the way betung watershed in indonesia. besides being the habitat of different flora and fauna, both watersheds have similar conditions such that they are both forest reserves, and therefore, should be free from human occupancy. while policies for the protection, preservation and conservation of these two watersheds are being imposed by their respective governments, these areas continue to be the homes of a number of upland farmers and migrants. agroforestry is a land use management system which combines the production of agricultural figure 1 map of way betung watershed, lampung, indonesia 73 ecological services of agroforestry landscapes in watershed areas – baliton et al. figure 2 map of the mmfr highlighting the molawin-dampalit sub-watershed as the study site in the philippines combining the major fruit tree species such as durian, mangosteen, jackfruit, integrated with cacao, coffee, rubber and other forest trees such as mahogany, alstoria and other forest species. the mmfr in the philippines, on the other hand, is a 4,244-ha multiple use forest reservation area (fig. 2). according to sargento (1995), mmfr is a protection forest and a watershed reserve, specifically used for training and research laboratory, water source for surrounding communities, biological sanctuary and gene pool of many plant and animal species. this reserve, however, has become a home to a number of farmers and migrants who are engaged in farming. these farmers practice agroforestry, particularly the multi-storey system which combines the production of coconut, fruit trees and other agricultural cash crops. socioeconomic characterization the characterization was carried out using a pre-tested sur vey quest ionnaire. this quest ionnaire captured socioeconomic information, views and perceptions about agroforestry practices. a total of 106 respondents in molawin-dampalit sub-watershed and 261 respondents in way betung watershed were selected using random sampling. results of the socioeconomic survey were analyzed using descriptive statistics, such as frequency counts, percentages and weighted scores. biodiversity assessment the assessment was conducted by measuring the following parameters of biodiversity, i.e. population density or the number of individual species per unit area; frequency of species distribution; dominance value based on frequency, diameter or biomass; relative and importance values based on density and frequency; and diversity and evenness indices based on the relative and importance values. importance value (iv) was computed to determine the dominant species for each site. the iv is the sum of the relative density, relative frequency and relative coverage. these values were computed using the following formula: total area sampled total number of tree individuals counted per species density = 100* relative density total number of all species total number of tree individuals counted per species = species dominance 2 (dbh)*(0.7854)= relative dominance 100* dominance of a species total dominance of all species = 100* total number of plots number of plots species occur =species frequency 100* total frequency of all species frequency of a species relative frequency = importance value =�relative density + relative coverage +�relative frequency 74 biotropia vol. 24 no. 1, 2017 s h= ∑ (pi * ln p i) i=1 h j = ______ ln s the measures of biodiversity were obtained using the shannon-wiener diversity index (h) (magurran 2004) calculated using formula as follows: the pielou's evenness index (j) was calculated using formula: where: h = shannon-wiener diversity index j = pielou's evenness index p = fraction of the entire population made up of i species i s = total numbers of species encountered ∑ = sum from species 1 to species s note: the power to which the base e (e = 2.718281828.......) must be raised to obtain a number is called the natural logarithm (ln) of the number the index was calculated by dividing the number of individuals of each species found in the sample by the total number of all species (represented by p), multiplied by the fraction of its natural log (p * ln p ). this procedure was 1 1 repeated for all of the different species. the sum of all the (p * ln p ) represents the value of h. 1 1 physical evidences of the movement of wildlife in the agroforestry matrix were noted in terms of frequency, duration and kind of species. carbon stock assessment the carbon stock of different agroforestry systems was measured using the biomass estimation method. tree biomass was calculated using the allometric equation of brown (1997) (equation 1). this was done by measuring the standing aboveground biomass of the woody perennials or live trees with diameter at breast height (dbh) of 5 cm and above. the total tree biomass density and carbon stored in various agroforestry systems were calculated using equation 2. carbon stock of herbaceous (living non-perennial crops) and litter found in the soil surface was calculated to get the total aboveground biomass of each agroforestry system (equation 3). belowground biomass of trees and other perennials was obtained using the default value proposed by delaney (1999) i.e. 15% of the aboveground biomass. equation 1: tagb= exp(2.134+2.530 ln(dbh)) where: tagb = total aboveground biomass in kg/tree exp {…} = “raised to the power of ” ln = natural log of {…} dbh = diameter at breast height in cm equation 2: c stored (mgc/ha) = tree biomass density *c content tree biomass density = tree biomass (mg)/sample area in hectare equation 3: total fresh weight (kg) * subsample dry weight (g) 2 ______________________________________total dry weight (kg/m ) = 2 subsample fresh weight (g) * sample area (m ) c stored (mgc/ha) = tree biomass density *c content results and discussion socioeconomic and biophysical characteristics of the study sites way betung watershed and molawindampalit sub-watershed are considered as watershed and forest reserves, which are inhabited by a number of people. results of the socioeconomic characterization indicated that most of the farmers in the two study sites were male as represented by 73% of the total number of respondents (table 1). this finding validated previous research which concluded that, in general, farming had become a male-dominated activity (landicho et al. 2014; landicho, 2015). majority (86%) of them were married with an average household size of 5. majority (51%) of the participants had family members ranging from 4 6 members. concurrent with other research, this finding implied the availability of family labor and that the farm households in the upland communities were mostly big. table 1 also highlights that the age of farmers in the two study sites were entirely different. the age range of the farmers in molawin-dampalit watershed was from 51 to 60. this data suggested that despite their age, the farmers were able to maintain their current farming systems. however, this finding also presented threat on the sustainability of their farming system, especially considering that not all family members 75 table 1 socioeconomic characteristics of the farmer-respondents in molawin-dampalit sub-watershed and way betung watershed socioeconomic characteristic study site total % molawin-dampalit way betung frequency % frequency % sex male 63 59 204 78 267 73 female 43 41 57 22 100 27 subtotal 106 100 261 100 367 100 civil status single 9 9 5 2 14 4 married 80 75 237 90 317 86 separated 2 2 1 1 3 1 widow/er 14 13 18 7 32 9 no answer 1 1 0 0 1 0 subtotal 106 100 261 100 367 100 household size 1-3 40 38 87 33 127 35 4-6 41 39 147 56 188 51 > 6 23 22 27 10 50 14 no answer 2 1 0 0 0 total 106 100 261 100 367 100 average 5 5 age < 30 8 32 40 11 30-40 12 93 105 29 41-50 24 83 107 29 51-60 29 42 71 19 > 60 33 11 44 12 subtotal 106 100 261 100 367 100 average 54 43 number of household members involved in farming 1-3 101 96 164 63 265 72 4-6 4 4 37 37 41 27 > 6 1 1 0 0 1 00.27 subtotal 106 261 367 100 income source farming 44 41 214 82 258 70 off-farm 0 0 27 10 27 7 non-farm 2 2 20 8 20 5 farming+off-farm 2 2 0 0 2 1 farming+non-farm 52 49 0 0 52 14 farming +off-farm+ non-farm 5 5 0 0 5 2 subtotal 106 100 261 100 367 100 ecological services of agroforestry landscapes in watershed areas – baliton et al. table 2 biophysical characteristics of the farms being cultivated by the farmer-respondents in molawin-dampalit subwatershed and way betung watershed biophysical characteristic study site total % molawin-dampalit way betung frequency % frequency % farm size < one hectare 51 44 149 57 200 54 1-3 45 46 111 42 156 43 3.1-5 6 6 1 1 7 2 > 5 4 4 0 0 4 1 total 106 100 261 100 367 100 status of farm ownership owned 9 8 0 0 9 2 tenant 19 18 17 7 36 10 rented 1 1 0 0 1 1 in public lands 70 66 244 93 314 85 no answer 7 7 0 0 7 2 total 106 100 261 100 367 100 farm topography rolling 48 45 132 51 180 49 steep 10 9 57 22 67 18 flat 31 29 72 27 103 27 flat to rolling 16 15 0 0 16 15 no answer 1 1 0 0 1 1 total 106 100 261 100 367 100 source of water for crop irrigation spring 12 11 60 23 72 19 river/creek 5 5 35 13 40 11 rainfed 85 79 166 640 251 68 others (e.g. irrigation) 6 5 0 6 2 total 108 100 261 100 369 100 76 biotropia vol. 24 no. 1, 2017 were trained to develop and maintain their farms. on the other hand, the farmers in way betung were still young and most probably in their productive age, as majority of them fell within the age range of 31 40 years old. this finding suggested that these farmers could already be the second-line farmers. these young farmers might have already been trained by the older farmers, and/or farming may have just started recently in way betung watershed. furthermore, this data indicated that these young farmers would have higher opportunities for improving their farms. there were only 1 3 members of the family that were engaged in farm development activities. in most cases, though, only the husband and the wife concentrated in farming. it could be that their children were still young; busy in their schooling, or not interested in farming at all. while farming was the major source of income of most (70%) of the farmer-respondents, there were also households whose members were engaged in non-farm activities as an additional source of income. in general, farm income was relatively low with most of the respondents having an estimated farm income of less than usd 200 and usd 200 500 in the philippines and indonesia, respectively. the low farm income could be attributed to the biophysical conditions of their farm as well as the scope and orientation of their agricultural production. in general, farm lots in the two study sites were cultivated by smallholder farmers. this was because majority (54%) of the farmerrespondents in the two study sites cultivated lands which were less than a hectare (table 2). this farming practice provided them with an estimated annual income of less than usd 200 (table 1). table 3 agricultural production systems being employed by the farmer-respondents in molawin-dampalit subwatershed and way betung watershed production system frequency total % molawin-dampalit way betung frequency % frequency % cropping system monocropping 7 7 1 0.40 7 2 crop rotation 3 3 0 0 3 1 relay cropping 4 4 0 0 4 1 multiple cropping 43 42 31 12 74 20 agroforestry 45 43 229 87.6 274 77 forest plantation 2 1 0 0 2 1 total 106 100 261 100 367 100 crop components vegetables 54 16 0 0 52 9 rice 1 0.30 0 0 1 0.17 corn 14 4 0 0 14 2 root crops 56 16.7 0 0 56 9 fruit trees 101 30 192 73.56 293 49 herbs 2 12 0 0 2 0.33 ornamentals 40 12 0 0 40 7 forest trees 65 19 69 26.44 134 23 total 333 100 261 100 594 100 77 understandably, these farmers could not cultivate big farm sizes primarily because they were cultivating in the public/state lands. thus, they were bound with certain rules and policies in their agricultural production. most (85%) of the farmer-respondents did not own the lands that they cultivated, and therefore, agricultural expansion was not possible. smallholder farmers are described as those who cultivate less than three hectares of land area (esfim ). by this definition, farmers in 2017 the two study sites are categorized as smallholder farmers. while their production orientation was for subsistence, the surpluses were sold in the market for their additional household income. besides being smallholder farmers, the biophysical characteristics of their farms were characterized as marginal. the topography was generally rolling (49%) and some with steep slopes (18%) and, therefore, the risk of soil erosion was high. however, the risk was being controlled with the practice of sustainable farming system, such as agroforestry. crops are generally dependent on rainfall as the main source of water/irrigation. thus, any drastic changes in rainfall and temperature patterns greatly affect their agricultural production. agroforestry practices in the two study sites with the prevailing biophysical and socioeconomic conditions, the farmerrespondents were observed to maximize the land use of their farms. most of the farmerrespondents were engaged in agroforestry (77%) and multiple cropping (20%) across the landscapes in the two study sites (table 3). the practice of agroforestry was noted in the high-elevation areas, while multiple cropping was highly observed in relatively lower elevation across the two landscapes. this was because the study sites were mostly dominated by forest and fruit trees. thus, opening of areas to give way for the production of agricultural crops was not permitted. farmers whose farms were located within the upper stream of the reserves/ watershed planted other woody perennials with smaller canopy, root crops and other shadeecological services of agroforestry landscapes in watershed areas – baliton et al. 78 biotropia vol. 24 no. 1, 2017 tolerant crops as understory. on the other hand, farmers cultivating in open areas having lower elevation had higher opportunities for raising short-term and medium-term crop species. farmer-respondents in molawin-dampalit sub-watershed planted a variety of crop components compared with the farmerrespondents in way betung watershed who planted only fruit trees (table 3). among crop components included vegetable crops (16%), fruit trees (30%), root crops (17%), cereals like rice and corn (4.34%), forest trees (19%) and ornamentals (19%). farmers in the molawin-dampalit subwatershed might have enough open spaces where they could plant short-term crops, while farmers in way betung watershed might have shaded spaces, which might not be suitable for cultivating short-term agricultural crops. furthermore, almost 100% of the farmlands in way betung watershed were considered as public lands, which made the farmers bound with policies and regulations on crop cultivation (table 2). farmlands in molawin-dampalit sub-watershed were bound for forest reserve. farmlands located in the lowland ecosystems were still suitable for appropriate agricultural production. biodiversity assessment species composition in the two watersheds a total of 35 tree species with at least 5 cm dbh were found across the 27 sampling plots in molawin-dampalit sub-watershed (table 4). these identified species consisted of 333 individuals belong to 19 tree families. the data revealed that fabaceae had the highest number of species (6), followed by moraceae (5) and meliaceae with three (3) species. annonaceae, th malvaceae and sapindaceae ranked 4 having two (2) species each, while the remaining families had one (1) species each. in terms of the total number of individuals, the dominant families recorded were meliaceae with a total of 90, followed by musaceae (84), sapindaceae (50) and fabaceae (30). four families namely euphorbiacea, lamiaceae, oxalidaceae and sapotaceae ranked the least with only one individual recorded across the sampling areas. in way betung watershed, there were 14 families and 26 species, with 548 individuals found (table 5). the highest number of species belongs to family fabaceae having six (6) species, followed by myrtaceae, meliaceae and arecaceae. family table 4 summary of existing tree families with corresponding number of species and individuals in molawin-dampalit sub-watershed family name number of species number of individuals rank # of species # of individual anacardiaceae 1 8 5 8 annonaceae 2 7 4 9 arecaceae 1 9 5 7 bignoniaceae 1 2 5 12 caricaceae 1 6 5 10 euphorbiaceae 1 1 5 13 fabaceae 6 30 1 4 fagaceae 1 2 5 12 lamiaceae 1 1 5 13 lauraceae 1 2 5 12 malvaceae 2 16 4 5 meliaceae 3 90 3 1 moraceae 5 13 2 6 musaceae 1 84 5 2 oxalidaceae 1 1 5 13 rubiaceae 1 5 5 11 rutaceae 3 5 3 11 sapindaceae 2 50 4 3 sapotaceae 1 1 5 13 total 35 333 --79 table 5 summary of existing tree families with corresponding number of species and individuals in way betung watershed family name number of species number of individuals rank # of species # of individual anacardiaceae 1 2 4 10 arecaceae 3 9 2 9 euphorbiaceae 1 161 4 1 fabaceae 6 27 1 5 gnetaceae 1 35 4 4 lauraceae 1 16 4 7 malyaceae 2 80 3 3 malvaceae 1 154 4 2 meliaceae 3 12 2 8 myrtaceae 3 27 2 5 rubiaceae 1 21 4 6 rhamnaceae 1 2 4 10 sapindaceae 1 1 4 11 sapotaceae 1 1 4 11 total 26 548 - - euphorbiaceae had the highest number of individuals (161), followed by malvaceae and malyaceae. these findings indicated a higher general species composition in molawin-dampalit subwatershed compared to that in way betung watershed. however, the number of individual species in molawin-dampalit sub-watershed was lower compared to that in way betung watershed. this could be explained by the fact that the molawin-dampalit sub-watershed was one of the area severely hit by typhoon glenda (international name rammasun) in 2014. this could explain why, at the time of the study, the floral components were still on their regeneration stage, mostly below 5 cm dbh. importance value of identified plant species in agroforestry landscape across the sampling plots of molawindampalit sub-watershed, banana (musa sapientum) was found to be the most dominant having the highest importance value (iv) of 62.07%. it was followed by rambutan (nephelium lappaceum) and lanzones (lansium domesticum) with iv of 36.47% and 32.59%, respectively. other species having high iv were chico (manilkara sapota) – 25.66%, mangga (mangifera indica) – 21.58% and mahogany (swietenia macrophylla) – 18.79% . table 6 shows the summary of seven (7) dominant plant species with the highest iv. the dominance of banana, rambutan and lanzones in molawin-dampalit sub-watershed table 6 top seven dominant species across sampling plots in molawin-dampalit sub-watershed, mt. makiling forest reserve, philippines species name scientific name importance value (iv) (%) saging musa sapientum 62.07 rambutan nephelium lappaceum 36.47 lanzones lansium domesticum 32.59 chico manilkara sapota 25.66 mangga mangifera indica 21.58 mahogany swietenia macrophylla 18.79 durian durio zibethinus 12.00 ecological services of agroforestry landscapes in watershed areas – baliton et al. 80 biotropia vol. 24 no. 1, 2017 indicated the farmers' preference for cultivating these crops, primarily because of their economic value. meanwhile, this research found out that the dominant tree species in way betung was rubber tree (hevea brasiliensis) with iv of 70.36%, followed by durian, cacao, melinjo, petai, avocado and coffee. based on the iv, the dominance level of a species in a community can be known (indriyanto 2006). table 7 shows the top seven dominant species across the sampling plots in way betung watershed. rubber tree is one of the major high value crop that is being cultivated in indonesia, malaysia and thailand because of its economic potential. this explains why this species was frequently found in way betung watershed. shannon-wiener diversity index (h) and pielou's evenness index (j) across sampling plots in molawin-dampalit sub-watershed was 2.59 and 0.45, respectively (table 8). sampling plots in way betung watershed recorded shannon-wiener diversity index (h) of 2.53 and pielou's evenness index (j) of 0.41. based on the h value, diversity of the agroforestry landscape in molawin-dampalit sub-watershed and way betung watershed was considered to be moderate (fernando et al. 1998) (table 9). the computed shannon-wiener diversity index (h) indicated that employing an agroforestry practice/system in a landscape may increase the diversity of the landscape compared table 7 top seven dominant species across sampling plots in way betung watershed, indonesia species name scientific name importance value (iv) (%) rubber hevea brasiliensis 70.36 durian durio zibethinus 59.72 cacao theobroma cacao 48.07 melinjo gnetum gnemon 22.54 petai parkia speciose 12.46 avocado persea americana 12.14 coffee coffea robusta 8.87 table 8 shannon-wiener diversity index and pielou's evenness index across sampling plots in the two study sites main plot molawin-dampalit sub-watershed way betung watershed circular plot: 8.9 m radius h 2.59 2.53 j 0.45 0.41 table 9 classification scheme of shannon-wiener diversity index (fernando et al. 1998) relative value shannon-wiener diversity index (h) very high 3.50 and above high 3.00 – 3.49 moderate 2.50 – 2.99 low 2.0 – 2.49 very low 1.99 and below 81 to employing monoculture type of farming system. meanwhile, low value of computed pielou's evenness index (j) indicated that the number of individual per species in the agroforestry landscape was not evenly distributed. this finding was validated by noble and dirzon (1997) who highlighted that agroforestry is increasingly being identified as an integrated land use that can directly enhance plant diversity while reducing habitat loss and fragmentation (brent et al. 2006). khanal (2011) also contends that traditional agroforestry practices contribute to the conservation of biodiversity in the western hills of nepal through in-situ conservation of tree species on farms, reduction of pressure on remaining forests, and the provision of suitable habitat for a number of plants on farmland. this contention was supported by meta-analysis on the effects of agroforestry, biodiversity levels and ecosystems services conducted by torralba et al. (2016). torralba et al. (2016) argued that agroforestry can enhance biodiversity and ecosystem service provisions related to conventional agriculture and forestry in europe. furthermore, agroforestry can help the flow of wild plants’ genes as well as increase fauna population size and diversity in protected area corridors, if it is tried as a buffer to connect patches of natural forests to facilitate habitat interconnectivity on a larger scale (baguinon et al. 2007). carbon stock assessment biomass density of agroforestry landscape a mean total of 116.26 mg/ha was recorded in the agroforestry landscape of molawin-dampalit sub-watershed (table 10). the computed mean total biomass density in this study site was higher than the overall mean of agroforestry (102.80 mg/ha) in the philippines (lasco & pulhin 2003). another study of zamora (1999) on biomass density of narra (pterocarpus indicus) + cacao agroforestry system in makiling (191.6 mg/ha) was also comparable with the results obtained in this study. it can be noted that sampling plots in way betung watershed were mostly forest and fruit trees which in turn contributed much on the biomass density with a mean total value of 542.80 mg/ha. this was comprised mostly of 86.12% table 10 biomass density (mg/ha) of agroforestry landscape in molawin-dampalit sub-watershed and way betung watershed item biomass density (mg/ha) aboveground belowground mean total trees and other perennial herbaceous litter trees and other perennial molawin-dampalit minimum 1.98 0.27 0.34 0.30 116.26 maximum 401.83 2.31 7.45 60.27 mean (µ) 97.87 (84%) 1.33 (1%) 2.38 (2%) 14.68 (13%) standard deviation (±) 91.76 0.73 2.07 13.76 # of sample plots (n) 27 27 27 27 way betung minimum 168.74 1.58 1.78 25.31 542.80 maximum 1,160.17 2.31 4.22 174.03 mean (µ) 467.47 (86.12%) 1.81 (0.33%) 3.40 (0.63%) 70.12 (12.92%) standard deviation (±) 242.10 0.17 0.59 36.31 # of sample plots (n) 14 14 14 14 note: values shown inside the parenthesis are the percentage compositions of different carbon pools ecological services of agroforestry landscapes in watershed areas – baliton et al. 82 biotropia vol. 24 no. 1, 2017 item carbon density (mgc/ha) aboveground belowground mean total trees and other perennials herbaceous litter trees and other perennials molawin-dampalit minimum 0.89 0.12 0.15 0.13 52.32 maximum 180.82 1.04 3.35 27.12 mean (µ) 44.04 (84%) 0.60 (1%) 1.07 (2%) 6.61 (13%) standard deviation (±) 41.29 0.33 0.93 6.19 # of sample plots (n) 27 27 27 27 way betung minimum 75.93 0.71 0.80 11.39 244.26 maximum 522.08 0.96 1.90 78.31 mean (µ) 210.36 (86.12%) 0.82 (0.33%) 1.53 (0.63%) 31.55 (12.92%) standard deviation (±) 108.94 0.08 0.27 16.34 # of sample plots (n) 14 14 14 14 table 11 carbon stored in agroforestry landscape of molawin-dampalit sub-watershed and way betung watershed note: values shown inside the parenthesis are the percentage compositions of different carbon pools from the trees and other perennials, while the lowest was recorded in herbaceous and litter with less than 1%. these results were consistent with the previous study conducted by wulandari (2013) in watershed areas in indonesia. biomass density of the agroforestry landscape varied considerably in all carbon pools measured as indicated by high values of standard deviation. huge variation in the biomass density could be attributed to the differences of the components of sampled agroforestry or farming systems. based on the characterization of the farms, there were some farmers who cultivated fruit trees as their main crop which contributed much to the biomass density. other farmers planted only few fruit trees. in addition, farming practices influenced the amount of biomass density of the herbaceous and litter pool, such as weeding and composting. these practices reduced the amount of herbaceous/undergrowth biomass in the area. some farmers were doing these practices, while others were not. carbon stock of agroforestry landscape the aboveground tree and other perennial crops (84%) ranked first in terms of percentage contribution to mean total carbon density of the area. it is followed by belowground tree and other perennial with 13%, then litter (2%) and herbaceous plants provided the least percentage contribution with 1% (table 11). the computed mean total carbon stock (52.32 mgc/ha) was comparable to the overall mean carbon density of secondary forests in the philippines (59.0 mgc/ha) as reported by lasco and pulhin (2003). a study of palma and carandang (2014) reported a higher mean carbon stock (92.78 mgc/ha) of an agroforestry system in misamis oriental. results of these studies already included soil carbon content in the analysis, while this research only focused on the total above and belowground biomass. inclusion of the soil carbon pool could significantly increase the carbon stock due to high concentration of carbon in the soil. expectedly, the computed mean total carbon density of way betung watershed was higher than that of the molawin-dampalit sub-watershed. carbon density in way betung was 244.26 mgc/ha, which was almost four (4) times bigger than that in molawin-dampalit (52.32 mgc/ha). the huge difference could be directly attributed 83 to the abundance of tree in agroforestry systems in indonesia. therefore, this research also validates the argument that agroforestry is a costeffective strategy for climate change mitigation, particularly the tree-based farming systems. conclusions agroforestry farms and practices contribute to the conservation and protection of the way betung watershed and molawin-dampalit subwatershed. diverse crop components in the agroforestry farms, including their interaction, promote biodiversity conservation in these watershed areas, both yielding a moderate level of diversity index. woody perennials, herbaceous crops and litter components of the agroforestry farms contribute to carbon sequestration by having carbon stock of 244.26 mgc/ha in way betung watershed and 52.32 mgc/hain molawindampalit sub-watershed. these ecological services are significant contributions of agroforestry to climate change mitigation. implications and recommendations agroforestry farming practices provide ecological contributions, particularly in carbon sequestration and biodiversity conservation. these contributions of agroforestry practices already offer potentials in addressing environmental degradation in many upland communities in southeast asia. it is necessary to promote the use of agroforestry as a production technology of the government and/or non-government programs on sustainable forest management and upland development. such programs or policies should put emphasis on the use of fruit tree-based agroforestry system to avoid further opening or clearing of forested areas in higher and midelevation areas. the use of fruit tree-based system can enhance the use of soil and water conservation measures and other supportive technologies to control soil erosion and degradation particularly in high-elevation areas. results of carbon stock assessment of various agroforestry systems can provide information on national green house gas (ghg) inventory which is mandated to the committed parties including the philippines in the united nations framework convention on climate change (unfccc). future research should dwell on analyzing the effects and contribution of each of the different types and variants of agroforestry systems to biodivers i ty conser vat ion and carbon sequestration. this kind of research would generate empirical data that would guide researchers, extension workers and policy makers the most appropriate type of agroforestry system that should be scaled-up among the upland farming communities. references baguinon nt, lasco rd, macandog dm, pasicolan pn, villancio vt. 2007. agroforestry and land use in the philippines. bogor (id): world agro-forestry centre. 236 p. brent s, boffa jm, scherr sj. 2006. the potentials of agroforestry to contribute to the conservation and enhancement of landscape biodiversity. chapter 11. world agroforestr y in the future. http://www.worldagroforestry.org/downloads/pu blications/pdfs/bc06146.pdf. retrieved on 2 february 2017. p. 95-101 . brown s. 1997. estimating biomass and biomass change of tropical forest: a primer. fao. forestry paper 134. rome (it): food agriculture organization of the united nations. 55 p. delaney m, roshetko j. 1999. field test of carbon monitoring methods for home gardens in indonesia. field tests of carbon monitoring methods in forestry projects. arlington (us): forest carbon moni tor ing prog ram, winrock international. p. 45-51. esfim [internet]. 2017. empowering smallholder farmers in markets. available from: www.esfim.org/wp/ ph i l ipp ines_es f im_paper1_rev jan29 .doc. philippines country report. retrieved on march 17 2017. fernando es, balatibat jb, perlas jr, jumawid rjj. 1998. resource inventory and assessment of biodiversity in subic bay metropolitan authority (unpublished report). indriyanto. 2006. ekologi hutan. edisi pertama. pt bumi aksara. jakarta. 210 p. khanal s. 2011. contribution of agroforestry in biodiversity conservation and rural needs fulfillment: a case study from kaski district. ms thesis. pokhara (np): institute of forestry, tribhuvan university. available from: http://www. forestrynepal.org/images/thesis/thesis_ ecological services of agroforestry landscapes in watershed areas – baliton et al. 84 biotropia vol. 24 no. 1, 2017 suman_khanal.pdf. retrieved on 27 february, 2017. landicho ld, paelmo rf, cabahug rd, visco rg, abadillos mg. 2014. prospects and dilemmas of promoting organic agriculture in the upland farming communities in the philippines. paper presented during the international conference on food security and nutrition, 30 march 1 april 2014, shanghai, china. ipcbee volume 67:60-5. landicho ld. 2015. development pathways towards sustainability of upland farming communities in the philippines. phd dissertation. laguna (ph): university of the philippines los banos. 230 p. lasco rd, pulhin fb. 2003. philippine forest ecosystems and climate change: carbon stocks, rate of sequestration and the kyoto protocol. ann trop res 25(2):37-51. magurran ae. 2004. measuring biological diversity. carleton (au): blackwell publishing. 264 p. available from: http://www2.ib.unicamp.br/ profs/thomas/ne002_2011/maio10/magurran %202004%20c2-4.pdf. retrieved on 31 january 2017. noble, i.r. and r. dirzo. 1997. forests as humandominated ecosystems. science 277:522-525. in: brent s, boffa jm, scherr sj. 2006. the potentials of agroforestry to contribute to the conservation and enhancement of landscape biodiversity. chapter 11. world agroforestry in the future. http://www.worldagroforestry.org/downloads/ publications/pdfs/bc06146.pdf. retrieved on 2 february 2017. p. 95-101. palma ra, carandang wm. 2014. carbon density of bagras ( blume) in smallholder eucalypotus deglupta tree-based agroforestry systems in northern mindanao, philippines. philipp j agrofor 1(1):73-80. sargento jo. 1995. participation of major stakeholders in the conservation of the makiling forest reserve. phd dissertation. laguna (ph): university of the philippines los banos. sodhi ns, koh lp, brook bw, ng pkl. 2004. southeast asian biodiversity: an impending disaster. trends ecol evolut 19(12):654-60. available from: http://www.savethefrogs.com/threats/palm/imag es/2004-sodhi-se-asia-tree.pdf. retrieved on 23 january 2017. tolentino ll, landicho ld, de luna cc, cabahug rd. 2010. chapter 20. case study: agroforestry in the philippines. in. constance lt, editor. handbook of climate change and society. oxford (uk): routledge publishing. p. 317-331. torralba m, fagerholm n, burgess pj, moreno g, plieninger t. 2016. do european agroforestry systems enhance biodiversity and ecosystem services? a meta-analysis. agric ecosyst environ volume 230:150-61. available from: http://www. sciencedirect.com/science/article/pii/s016788091 6303097. retrieved on 3 february 2017. wulandari c. 2013. pendugaan karbon di lahan agroforestry register 19 (carbon prediction on agroforestry area in register 19). bandarlampung (id): lampung university. zamora d. 1999. carbon dioxide (co ) storage potential of 2 multistory agroforestry systems in mt. makiling. msc thesis. laguna (ph): university of the philippines. page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 page 14 02. 1386 akmad endang zainal hasan et al..cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 biotropia vol. 29 no. 3, 2022: 203 212 doi: 10.11598/btb.2022.29.3.1569 203 diversity and status of butterflies in awasian water forest reserve, mt. hilong-hilong, philippines mary cris g. abao1, kevin c. quiňonez1, lea jane g. elegio1, mark freddie d. suarez1, alma b. mohagan3,4 and arturo g. gracia jr.2 1college of teacher education, surigao del sur state university-main campus, tandag city, surigao del sur 8300, philippines 2department of natural sciences and mathematics, college of arts and sciences, surigao del sur state university-main campus, tandag city, surigao del sur 8300, philippines 3department of biology, college of arts and sciences, central mindanao university, bukidnon 8310, philippines 4zoological section, university museum, central mindanao university, musuan, bukidnon 8310, philippines received 22 march 2021/accepted 14 october 2021 abstract butterflies are deemed as an essential faunal group in the ecosystem due to their ecological services. however, continuous habitat loss leads to the decline of its population. thus, this study was conducted to assess its diversity and status in one of the watersheds of mt. hilong-hilong. sweep netting (336 man-hours) and butterfly trapping (200 trap days) were carried out to document the species. fifty-seven species of butterflies were recorded with the family nymphalidae as the most represented group (n = 30). species diversity (h') was relatively higher in dipterocarp (h' = 1.49) forest than agroecosystem (h' = 1.39), a result primarily influenced by favorable ecological support like food availability. endemicity was 31%, which comprised mostly of rare species. noteworthy findings are the listing of globally and nationally rare species but locally assessed as common. based on the results, the area harbors an array of butterfly species and various rare species that requires an effective management plan to conserve the organisms. keywords: butterflies, diversity, lepidoptera, mt. hilong-hilong, richness introduction lepidoptera is one of the largest families in the insects' realm, where butterflies well represent most of the species. its occurrence and diversity are considered good indicators for any terrestrial biotopes (kunte 2000; aluri & rao 2002; thomas 2005; arya & dayakrishna 2014), which also denote environmental quality changes and served a vital role in agricultural landscapes (munyuli 2012; nacua et al. 2015a). lepidoptera are excellent pollinators that ensure the reproduction and survival of plants used by other organisms as sources of food, reproductive areas, and medicine (mohagan & treadaway 2010). despite the high diversity, sociological and ecological functions of butterflies, the taxa are still not spared from gradual extinction due to overexploitation, illegal trading, and habitat loss which are the manifestations of uncontrollable anthropogenic activities (myers et al. 2000; brook et al. 2008; serengil et al. 2010; parria et al. 2017). owing to habitat destruction for developmental activities in an urban environment and unscientific management of natural resources, most of our native butterflies are fast disappearing. at present, their survival is under threat (nair et al. 2014) and these threats are highly observable in the philippines, making the country one of the hottest hotspots (bisson et al. 2003). hence, determining the diversity, level of endemism, and distribution of species is necessary for it serves as bases and guidelines for the formulation of conservation measures (ehrlich & hanski 2004; pyke & ehrlich 2010). awasian water forest reserve is one of the watersheds of mt. hilong-hilong, a key *corresponding author, email address: artzgracia@gmail.com mailto:artzgracia@gmail.com biotropia vol. 29 no. 3, 2022 204 biodiversity area (kba) in the philippines located in the northeastern mindanao. mt. hilong-hilong itself has been considered as an ideal abode for various forms of flora and fauna (pef 2008). however, ecological information is observed to be fragmented and limited, with scientific studies mainly focused on vertebrates and the western part of the ecosystem (agusan provinces). in contrast, invertebrates, especially butterflies and the eastern part (surigao provinces), are relatively poorly known, with only one accessible data set on butterflies from the study of ramirez and mohagan (2012). the area is also considered a vulnerable habitat under the criterion of very high due to highly observed anthropogenic pressures (birdlife international 2020), especially on the aspect of the rapid growth of human population in the uplands, mining, agricultural expansion, and road expansion and development (haribon 2017). for these reasons, the study was conducted to address the scarcity of ecological information on butterflies in the area and provide information that can be utilized for effective environmental management planning. the study generally aimed to assess the diversity through the determination of species composition and richness of butterflies across habitat types, as well as the evaluation of its status in comparison with the global and national assessments. materials and methods duration and description of the study area the study was conducted at awasian water forest reserve, mt. hilong-hilong in tandag city surigao del sur, philippines located at 9.07579 n and 126.14006 e (fig. 1). the study was carried out on 1 9 october 2017, covering nine days of sampling. the site's topography is generally plain, rolling, and gently sloping. the area can be reached for approximately two hours by walking from the nearest human settlements. the climatic condition in the area falls under the type ii climate condition of the philippines. it has rainfall distributed throughout the year, with a negligible short dry season (mpdo 2004). the area has two vegetations, the agroecosystem and dipterocarp forests. figure 1 location and spot map of the study site (birdlife international 2020) butterflies of awasian water forest reserve – abao et al. 205 establishment of study stations and habitat assessment two transect lines with a length of 1,000 m were established in the study area with an aerial distance of 200 m from each transect. the reason for such aerial distance is to avoid or lessen possible bias in documenting the species. the first transect line was laid across the human trail in the agroecosystem and labeled as transect 1 (t1), while the second transect line was laid across the dipterocarp forest and labeled as transect 2 (t2). habitat assessment was carried out through the documentation of the plant community and composition, canopy cover, nearness of water bodies, distance from the nearest human settlements, and temperature. sampling techniques collection and capturing the butterflies was carried out primarily through sweep netting. the activity was actively performed from 9:00 am to 3:00 pm, for these are the hours the butterflies are highly active. a total of 336 man-hours of sampling effort was spent for the entire duration of the study. wherein 168 man-hours were spent per transect or habitat. as for this technique, the researchers wore brightly colored clothes to attract butterflies. also, 25 pieces of classical butterfly traps containing muscovado sugar solutions were deployed along the first 500 m of the transect lines. each trap was placed with a 20 m interval from each other. the traps were hung in the place which was convenient for butterflies feeding like open fields. identification, preservation, and data analysis preliminary identification of butterflies was carried out using taxonomic keys. other references, such as books, journals, and photographs (mohagan & treadways 2010; treadway 2012; ramirez & mohagan 2012) of the previously identified specimens, were also used. after the initial identification, the samples were sent to the zoological section of the university museum of central mindanao university (cmu), a state-governed research university for verification. the collected species of butterflies were pinned and preserved using naphthalene balls and powder. data analyses that include the computation of species rarefaction and diversity indices were analyzed using biodiversity professional (biopro) software version 2.0 (mcaleece 1997). the butterfly's global status assessment was based on the international union conservation for nature (iucn 2020), while the established national and local assessments by treadaway (1995) as well as mohagan and treadaway (2010) were adopted. results and discussion taxonomic composition and overall richness fifty-seven (57) species of butterflies were recorded in the study area. these species were classified into 43 genera belonging to 5 families. among the five families, family hesperiidae was the least represented with three species observed, followed by family papilionidae (n = 5), pieridae (n = 8), lycaenidae (n = 11), and nymphalidae (n = 30) (table 1). the low representation of species under hesperiidae was attributed to a generally thicker canopy in the area. the thick canopy makes the habitat shadier, which is not favorable for the hesperiidae, which species used to inhabit an open place and near the shrubs (braby 2016). hesperiids also prefer to feed on various weedy plants, including pigweeds and lamb's quarter (hilty 2013), which is not present in any vegetation types of the current sampling area. biotropia vol. 29 no. 3, 2022 206 table 1 species list, endemicity, and status of observed butterflies in awasian water forest reserve, mt. hilong-hilong taxon assessment endemism local national global family hesperiidae 1 hasora moestissima moestissima r c ne 2 tagiades japetus titus r c c ne 3 tagiades trebellius martinus r c c pe family lycaenidae 4 allotinus pallax apsecus c c ne 5 arhopala abseus abseus c c ne 6 caleta angola angola r c ne 7 cheritra orpheus orpheus r c pe 8 eooxylides neduna neduna c 9 hypolycaena sipylus tharrytas c c ne 10 jamides alecto manillana c r pe 11 jamides celeno lydanus c r r ne 12 jamides philatu osias c r ne 13 nacaduba borenice leei c 14 prosotas nora semperi r c ne family nymphalidae 15 acrophtalmia albofasciata r r me 16 acrophtalmia leto ochine c c ne 18 amathusia phidippus pollicaris r c c ne 19 cirrochroa tyche tyche c c ne 20 cyrestis maenalis c c ne 21 danaus melanippus c c ne 22 elymnias beza beza r c me 23 euploea amulciber mindanensis c c ne 24 euploea euniceleucogaris r 25 faunis phaon leuces c c ne 26 junonia hedonia ida c c c ne 27 lassipa pata semperi r r ne 28 lexias panopus miscus r c ne 29 milanitis boisduvalia r r pe 30 mycalesis micromede micromede c r ne 31 mycalesis federi federi r r pe 32 mycalesis mineus philippina c c r ne 33 mycalesis tagala semiraza c r ne 34 neptis mindorana pseudosoma c pe 35 neptis pampanga boholica r r c ne 36 pantaporia dama commixta c c c pe 37 pantoporia cyrilla cyrilla r c pe 17 pantoporia sp. r 38 phaedyma columella messogai c c ne 39 phalantha phalantha phalantha c c ne 40 ptychandra schadenbergi r r pe 41 ragadia melindena mindeninse c r pe 42 tanaecia leucotaenia acquamarina r c ne 43 tarattia cosmia cosmia c pe 44 ypthima sempera chaboras r r pe family papilionidae 45 atrophaneura semperi r r r pe 46 graphium argamemnon argamemnon r c ne 47 melenaides deiphobus rumanzovia r c ne 48 melenaides helenus hystaspes c c c me 49 pachliopta mariae mariae c c pe family pieridae 50 appias nepheleelis r r ne 51 cepora aspasia orantia r c ne 52 eurema blanda valli volans c c c ne 53 eurema hecabeta miathis c c ne 54 eurema sarilalas arilata c r r pe 55 gandaca harina mindanensis c c ne 56 leptosia nina terantia r c ne 57 pareronia boebera trinobantes c c ne total number of families 5 total number of genera 43 total number of species 57 notes: c = common; r = rare; ne = non-endemic; pe = philippine endemic; me = mindanao endemic. butterflies of awasian water forest reserve – abao et al. 207 the family nymphalidae was observed to be the most represented group. this finding is attributed to the study site's general characteristics as a forest. nymphalids are perceived to be dominant in a forested area, particularly in tropical regions (sarkar 2011; harsh et al. 2015). its abundance is attributed to the availability of food resources from the variety of host plants and favorable microclimate conditions (widhiono 2015). at the course of the conduct, various plants were flowering and fruiting. among these plants are the dominant species in the area like shorea spp. and other trees like lansium dosmesticum, artocarpus odoratissimus, and durio zibithenus, which make the condition suited to the requirements of the butterfly group for their feeding behavior. this observation agrees with opler et al. (2017) claim that nymphalids' feeding behavior depends on the nectar, sap flows, and rotting fruit, wherein the food availability from one vegetation type influences the butterfly composition (toledo & mohagan 2011). the total richness observed in this study is comparatively higher compared to some of the faunistic studies conducted in the philippines. zapanta et al. (2016) at bulusan, bulakan, only recorded 21 species, whereas the lepidopteran assessment carried out in lipa, batangas documented only 25 species (nacua et al. 2017). the same observation was noted for the studies of toledo and mohagan (2011) at mt. hibokhibok, camiguin (n = 41) and sumagaysay and sumagaysay (2012) at mt. nebo, bukidnon (n = 31). as compared to the global findings, the study surpasses the records of arya and dayakrishna (2014) in naital, uttarakhand, india (n = 27); haroon et al. (2020) in tanga, charsadda, khyber pakhunkhwa, pakistan (n = 22); castro and espinosa (2015) in arenillas ecological reserve, ecuador (n = 22); and koneri et al. (2016) at manembo-nembo wildlife reserve, north sulawesi, indonesia (n = 44). findings of our study suggested that the study area is an ideal abode for butterflies due to its capability for supporting larger communities. our study also indicated that the habitat has a better support system coming from the butterflies, especially on the aspect of pollination. on the other note, the results of our study are comparatively lower compared with the records in mts. apo, kitanglad, musuan, and timpoong in the philippines with 104, 148, 114, and 79 species, respectively (mohagan et al. 2011). the reports of nacua et al. (2015b) at san fernando la union botanical garden, mohagan et al. (2018) at mt. pinamantawan, bukidnon, as well as mohagan and treadway (2010) at mt. hamiguitan, davao oriental, philippines were also noted to have higher richness with 104, 118, 142 species, respectively. even in comparison with the study results of ramirez and mohagan (2012) at maitum village, tandag city which is an area adjacent to the sampling site of this study, recorded a total of 104 species. the discrepancy between the results is attributed to various factors ranging from sampling effort to study duration. unlike in different studies, the participation of a wellversed taxonomist maximizes the observation since visually observed species are added to the list, like the case of the abovementioned studies. in contrast, our study only represents the verified captured samples. the influence of sampling duration could also be another factor due to more time provided for further documenting the faunal group. the concept conforms to the elaborated observation in the faunistic study of lepidoptera in one of the wildlife sanctuaries in misamis oriental (guadaluiver et al. 2019) and mt. hamiguitan (mohagan & treadway 2010). as shown in figure 2, the species rarefaction entails that sampling effort is still unachieved. thus, observing additional species is still feasible by doing reassessment in the field and could lead to an increase in the overall butterfly richness. not to mention that various uncaught morphologically distinct individuals were observed during the fieldwork that could mean a different species as well. biotropia vol. 29 no. 3, 2022 208 figure 2 butterfly species rarefaction plot in awasian water forest reserve ecological profile of butterflies across habitats in this study, 155 individuals of butterflies were captured. butterfly abundance was higher in the dipterocarp forest, with 83 (53%) individuals than in the agroecosystem with 72 (47%) individuals. in terms of species richness, dipterocarp forest had higher species-richness with 42 species while the agroecosystem had only 34 species. diversity index (h') and species evenness (j') were relatively higher in dipterocarp forest (h'=1.49; j'=0.93) compared to those in agroecosystem (h' = 1.39; j' = 0.90) (table 2). these consistent results suggest that the dipterocarp forest has better ecological support for the survival of the butterflies in the area. the dipterocarp forest was observed to have numerous flowering and fruiting plants during the sampling. this environmental set-up could have resulted in wider ecological support concerning food preference, unlike in the agroecosystem, where limited resources were observed. the result conforms with the report at mt. malindang (ballentes 2006) and in the lowland forest at maitum, mt. hilong-hilong (ramirez & mohagan 2012). the findings supported the idea that butterfly assemblage was more diverse in the dipterocarp forest than that in the agroecosystem. this finding could be attributed to the diversity and abundance of butterflies which are highly correlated with the availability of food plants and assemblage of floral species in the surroundings (kunte 2000; stefenascu 2004; ansari 2015). this common ground of findings is linked to the butterflies' voracious eating behavior, particularly in their larval stage, to meet the demand for nutrients in their fast development through the process of metamorphosis. moreover, schneider (2003) reported that the habitat characteristics and landscape structure influenced species abundance and richness, thus, supporting the variation of the result in this study. table 2 ecological data of butterflies representing species richness, abundance, evenness, and diversity index in the two study areas habitat ecological profile richness abundance shannon-wiener diversity index (h’) evenness (j’)  agroecosystem 34 72 1.49 0.93  dipterocarp forest 42 83 1.39 0.90  overall 57 155 1.58 0.90 rarefaction plot e s (n ) 0 10 20 30 40 50 60 0 50 100 150 200 butterflies of awasian water forest reserve – abao et al. 209 the lesser diversity in the agroecosystem is also attributed to the influence of human disturbance. the habitat was more vulnerable to anthropogenic activities compared to the situation in the dipterocarp forest since the agroecosystem is nearer to human settlements. the butterflies are profoundly affected and endangered by land use and forest cultivation (avigliano et al. 2019) because those activities affect the butterflies’ continual survival by delimiting the needed resources, such as food and good habitat (ozden et al. 2008). hence, forest cultivated areas have comparatively low butterfly diversity than any other habitats (malagrino et al. 2008; laghude et al. 2019). among the documented species, acrophtalmia leto ochine and euploea amulciber mindanensis (nymphalidae) and pachliopta mariae mariae (papilionidae) were the most abundant with 15, 10, and 12 individuals, respectively. representatives of a. leto ochine were mostly seen in the agroecosystem, particularly in the open fields and grasslands. in contrast, p. mariae mariae mainly were seen in the dipterocarp forest in an area with at least 50 70% canopy coverage and near the water systems. as for the e. amulciber mindanensis, samples of its population were equally observed in both habitats. species assessment out of the 57 species recorded, only 21% (n = 12) of the butterfly species have international union for conservation of nature assessment status (iucn 2020), consisting of 5 (9%) common non-endemic species (cnes), 2 (4%) rare non-endemic species (rnes), 2 (4%) common philippine endemic species (cpes), 2 (4%) rare philippine endemic species (rpes), and 1 (2%) common mindanao endemic species (cmes). the philippines national assessment levels were also carried out following treadaway (1995) for 52 (91%) butterfly species. these were categorized into cnes (n = 28; 53%), rnes (n = 7; 13%), cpes (n = 5; 9%), rpes (n = 8; 15%), cmes (n = 2; 4%), and rare mindanao endemic species (rmes) (n = 1; 2%). it is noticeable that most of the species are unassessed globally and even some species nationally, thus, signifying the importance of the findings to the global and national platforms for the eventual global and national synopsis, especially on the local and national levels, because species assessment is considered important for any management planning on any forest reserves and protected areas (haribon 2017; pef 2008). as for the local assessment, 21 (37%) species were evaluated as cnes, 14 (25%) as rnes, 7 (12%) as cpes, 8 (14%) as rpes, 1 (2%) as cmes, and 2 (4%) as rmes (fig. 3). other noteworthy findings are the observation of the rare species. most importantly, the listing of the globally rare but observed to be locally common species such as jamides celeno lydanus (lycaenid), mycalesis mineus philippina (nymphalid), and eurema sarilalas arilata (pierid). the same pattern was observed for the following: jamides alecto manillana, jamides philatu osias, j. celeno lydanus, mycalesis micromede micromede, mycalesis tagala semiraza, ragadia melindena mindeninse, and eurema sarilalas arilata. these species were abundantly observed in the area but nationally assessed as a rare species. this observation entails that habitat has different dynamics, and it varies from one another and could support species in the various ecological spectrum. thus, indicating every ecosystem is unique and requires different conservation measures. biotropia vol. 29 no. 3, 2022 210 figure 3 distribution of butterfly status based on global, national, and local level of assessments notes: cnes = common non-endemic species; rnes = rare non-endemic species; cpes = common philippine endemic species; rpes = rare philippine endemic species; cmes = common mindanao endemic species; rmes = rare mindanao endemic species. the overall percentage of endemism was 31%. the finding is comparatively higher compared to the records of martinez and mohagan (2012) at the adjacent forest of the study site with 22% endemicity. the same observation was noted compared to the findings of nacua et al. (2015b) at la union and mohagan et al. (2018) at mt. pinamantawan with a percentage of endemism difference of 9 to 10%. as compared with the major forest reserves in the philippines such as mt. malindang (ballentes et al. 2006), mt. hamiguitan (mohagan & treadway 2010), and mimbilisan protected landscape (guadelquiver et al. 2019), the findings were observed to be closed with only 1 to 3% difference on its endemicity. this infers that the area is a relatively preferable habitat for endemic species and is comparable with other pristine environments. conclusion based on the findings, awasian water forest reserve is home to various butterfly species and has good ecological support. these supports span from multiple factors, especially on the perspective of plant community structure, which is vital to the survival of the organisms. endemism was relatively high as compared with other ecosystems and showed to be comparable with other pristine habitats. in the context of rarity, the habitat houses various globally and nationally rare species that require conservation attention. acknowledgments the authors are grateful to the department of environment and natural resources (denr) for the issuance of the gratuitous permit (gp). to tandag water district (twd) and the local government unit (lgu) for the clearance and logistic support. to mr. gerry bernadas, mr. aldrin dua, jhony boy, and venz joy verano for the support during the conduct of this study. sincerest gratitude is also extended to all the people who contributed to the success of the study. references aluri jsr, rao sp. 2002. psychophily and evolution consideration of cadaba fructicosa (capparaceae). j bombay nat hist soc 99(1): 59-63. 50 45 40 35 30 25 20 15 10 5 0 cnes rnes cpes rpes cmes rmes unassessed local national global butterflies of awasian water forest reserve – abao et al. 211 ansari na, ram j, nawab a. 2015. structure and composition of butterfly (lepidoptera: rhopalocera) fauna in surajpur wetland, national capital region, india. asian j 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philippines). international journal of advanced engineering, management and science (ijaems) 2(9): 1477-83. biotropia the southeast asian journal of tropical biology vol. 31 no. 2, 2024: 146 156 doi: 10.11598/btb.2024.31.2.1868 issn: 0215-6334 | e-issn: 1907-770x 146 potential of lactic acid bacteria, isolated from several sources, to inhibit the growth of candida albicans atcc10231 yan ramona1,4*, ida bagus gede darmayasa1, komang dharmawan2, putu eka sudaryatma3, ni made teriyani1, yanuarius felix batho1, and fainmarinat selviani inabuy1 1department of biology, faculty of mathematics and natural sciences, udayana university, bali 80361, indonesia. 2department of mathematics, faculty of mathematics and natural sciences, udayana university, bali 80361, indonesia. 3fish quarantine and inspection agency denpasar branch, bali 80361, indonesia. 4integrated laboratory for biosciences and biotechnology, udayana university, bali 80361, indonesia article highlights • lactic acid bacteria have potential to control candidiasis or candida albicans. • the novelty of this study is to advance the potential of lactic acid bacteria to control candidiasis infection in human, with a view to develop novel lab-based probiotic candidates with capability to inhibit/prevent infection by c. albicans, the causative agent of candidiasis. abstract the main aims of this research were to isolate and identify potential lactic acid bacteria (lab) inhibitory to candida albicans. the lab sources were kimchi, honey and vaginal secrete of healthy women. they were isolated with a view to develop a novel alternative method with reduced use of antifungal agents in the treatment of patients infected by such fungal pathogen. isolation of the labs was conducted by applying dilution spread method on de mann rogosa sharpe agar (mrsa) medium supplemented with bromo cresol purple (bcp) indicator. once purified, they were tested for antagonism against c. albicans in dual culture assays. lab isolates that showed significant inhibition against the pathogen were identified using 16s rdna sequences and their sequences were aligned with those of known sequences deposited at the gene bank (http://www.ncbi.nlm.nih.gov). the results showed that 46 among more than 100 labs isolated in this study significantly inhibited the growth of c. albicans in the in vitro dual culture assays, and all showed resistance property to antifungal agent (fluconazole). this indicated that they all have potential to be synergically applied with reduced use of fluconazole in the therapy. the most potential isolates (10 isolates) were closely related to three lab species, namely lactobacillus paracasei, lacticaseibacillus paracasei, and pediococcus pentosaceus, based on their 16s rdna sequence similarities with those deposited in the genbank. keywords: candida albicans, candidiasis, diversity, probiotic, vaginal candidiasis article information received 14 february 2023 revised 28 april 2024 accepted 6 may 2024 *corresponding author, e-mail: yan_ramona@unud.ac.id research article introduction in the last decade, candidiasis prevalence has been reported to increase, particularly among women with immune deficient (aids) or people under treatment with various types of antibiotics (lauw et al. 2017). the main cause of this candidiasis infection is opportunistic fungal pathogens (particularly candida albicans which is among myriads normal microbiota) of human inhabitants (anh et al. 2021). this fungal pathogen is commonly found in the mucosa of oral cavity, skin surface, digestive tract, as well as in the reproductive tract of women. under certain conditions, such as imbalance in the microbial composition of its hosts or immune system disturbance in its host, c. albicans tends to boost its growth and become an infectious pathogen (richardson 2022; macias-paz et al. 2022). according to wilson (2019) vulvovaginal and oral candidiasis are the most common cases due to c. albicans infection. additionally, skins are also susceptible from this infection, if they are under long term exposure of humid conditions (less ventilated skins) (qadir & asif 2019). when the pathogen enters the blood stream and spread throughout the body via circulatory system, deep candidiasis may occur within the vulva of vagina as well as in the mucosa of oral cavity. https://doi.org/10.11598/btb.2024.31.2.1868 lactic acid bacteria antagonistic against candida albicans ramona et.al. 147 in the area of women genital (vagina and vulva), c. albicans infection may lead to abnormality in mucus secretion, inflammation, appearance of redness color, or burning-like feeling (talapko et al. 2021). it was reported by anh et al. (2021) that 75% women globally may have at least one infection of vulvovaginal candidiasis, and 40-50% among them may experience repeated/recurrent infections. meanwhile, people with infection in their mouth mucosa may have pain, lose appetite, uncomfortable feeling within their mouth (dangi et al. 2010; singh et al. 2014). epidemiological survey on the prevalence of candidiasis indicates that most (approximately 85-95%) of the vaginal infection among women globally is caused by c. albicans (makanjuola et al. 2018).   it is further stated by this report that some non-candida albicans species (such as c. glabrata, c. tropicalis, c. parapsilosis, c. krusei, and c. dubliniensis) were also frequently found to be the cause of vulvovaginal candidiasis incidence worldwide. in indonesia alone, it was reported by wahyuningsih et al. (2021) that the prevalence and disease incidence caused by c. albicans infection has become a serious problem in last decade. each year in indonesia, approximately 5 million women (aged of between 15 and 50 years old) have been reported to experience recurrent vulvovaginal candidiasis. therefore, research on the prevention and control of candidiasis is urgently needed to cope with such fungal infection. until recently, control of c. albicans among infected patients has relied on application of anti-fungi, particularly fluconazole (whaley et al. 2017). long term antifungal application may result in bad side effects on the balance conditions of the intestinal normal microbiota. many beneficial microbiota or probiotic microbes may be wiped out by broad spectrum of those antibiotics/ anti-fungi and this may lead to health disturbance, such as diarrhea (heng et al. 2021). health level of intestinal tract has recently been reported to be correlated with health status of a person (manor et al. 2020). some researchers, such as maiuolo et al. (2019), reported that there is a bidirectional communication between brain and gut microbiota which is well known as gut-brain axis. this implies that the balance of gut microbiota will have significant effects on the brain function (zhao et al. 2018). bulgasem et al. (2016) also reported that resistance properties of the c. albicans had occurred as a result of improper application of fluconazole, and this may lead to an increase in the pathogenic capacity of the pathogen (hossain et al. 2018) to cope with negative side effects of the excessive/improper application of anti-fungi, alternative methods are urgently needed. among many alternative methods, the use of antagonists (biocontrol agents) of the c. albicans appears to be the most promising method with the lowest risk. biocontrol agents, with capability as well as with lowest risk to control the growth of c. albicans, are bacteria belong to the group of lactic acid bacteria (zeise et al. 2021) as they are also being part of normal microbiota of human. lactic acid bacteria are gram positive bacteria with capability to produce lactic acid as the main product in the metabolism of sugars. these labs can easily be isolated from many sources, such as honey, fermented foods (kimchi and tape ketan), and various milk. these sources are ideal for the labs as their main source of nutrition to support their growth is available in such products (pasupuleti et al. 2017). some genera of labs successfully isolated from those sources (particularly honey) are lactobacillus, streptococcus, micrococcus, and enterococcus (feizabadi et al. 2020). ryu and chang (2013) found 200 species of microorganisms from fermented kimchi, and among those lab isolates, such as leuconostoc spp. and lactobacillus spp., were indicated to play the most significant role in the kimchi making. based on the above rational, the main aims of our research were to isolate and identify potential lab isolates from several sources, such as kimchi, honey, and vaginal secrete of healthy women with a view to develop a novel method to cope with negative side effects of improper/excessive antifungal application in the conventional therapies of candidiasis. in this research, the isolates were identified molecularly on the basis of their 16s rdna sequences which were aligned with those deposited at the gene bank (http://www.ncbi.nlm.nih.gov), following dna extraction, dna amplification in a pcr machine, and electrophoresis. biotropia vol. 31 no. 2, 2024 148 materials and methods isolation of potential probiotics sources of lactic acid bacteria included fermented foods, such as kimchi, honey, and vaginal secrete of healthy women (ethical clearance number: 1354/ un14.2.2.vii/lt/2022, approved by the faculty of medicine udayana university). dilution and spread method on mrsa (de mann rogosa sharpe agar) added with bromocresol purple (bcp) indicator was applied in the isolation of probiotic candidates. the samples were diluted to the rates of 10-1 to 10-6. a volume of 100 µl samples with dilution rates of 10-3 to 10-6 were then spread on mrs agar with bcp indicator and incubated at 37 oc for 48 hours until distinct colonies appeared on this medium. colonies with indication to produce acidic compounds were isolated, purified, and stored at -80 oc in mrs broth with 30% (v/v) glycerol in it for use in further studies. candida albicans atcc10231 isolate the fungal pathogen (c. albicans atcc10231) was obtained from central hospital, sanglahdenpasar, bali. this pathogen was isolated from a patient with thrush disease. the identity of this pathogen was confirmed to be c. albicans following application of biomerieux vitex® 2 system. this pathogenic fungal isolate was maintained in sabouraud dextrose broth (sdb) with 30% (v/v) glycerol at -80 oc. for regular use, it was subcultured from the stock culture on sabouraud dextrose agar (sda). in vitro dual culture assay for screening potential probiotic candidates in vitro dual culture assays on mrs agar medium were applied in the screening of the 46 potential lab isolates antagonistic against c. albicans. a volume of 200 µl c. albicans suspension with cell density of 108 cfu/ml or equivalent to 0.5 mcfarland scale was spread onto mrs agar medium and let dry for 5 minutes. this c. albicans lawn was then spot inoculated with lab isolates, incubated at 37 oc overnight, and observed for inhibition zones around the lab isolates. five replicate experiments were done with sterile mrs broth and fluconazole as negative and positive controls, respectively. those showed inhibition activity on c. albicans were purified (streak for single colony) and stored in mrs broth medium at -80 oc prior to further studied. similar method was also applied in the antifungal resistance of lab isolates obtained in our study. molecular 16s rdna sequencing for the identification of most potential bacterial isolates some potential lab antagonists were identified by sequencing components of their 16s rdna and comparing these with their counterparts in a clone library of known bacteria (http://www. ncbi.nlm.nih.gov). the followings are procedures of extraction, purification, amplification, and sequencing of the lab’s 16s rdna. isolation of genomic dna a volume of 1 ml suspension of labs in mrsb medium was added into an eppendorf tube, centrifuged at 8,000 xg for 3 minutes at a temperature of 4 oc, and decanted. the pellet was then washed twice with sterile saline solution, centrifuged at 8,000 xg at 4 oc for 3 minutes, and its supernatant was decanted. this procedure was repeated several times until at least 50 mg pellet or bacterial cell mass was obtained. the pellet was subsequently re-suspended in 200 µl of saline solution to obtain densed cell suspension. the dna extraction followed the procedures as specified in the quick-dna™ microprep kit (zymo research, usa). the cell suspension obtained (200 µl) and 750 µl bashingbeadtm buffer was added into a zr bashingbeadtm lysis tube, shaken with a bead beater (tomy micro smashtm ms-100) at the speed of 4,500 rpm for 6 minutes and centrifuged at 10,000 xg for 1 minute at 4 oc. the supernatant was transferred into a zymo-spintm iii-filter in a collection tube and centrifuged at 8,000 xg for 1 minute at a temperature of 4 oc. a volume of 1,200 µl of genomic lysis buffer was then added to the filtrate in a collection tube. a volume of 800 µl of this mixture was next pipetted into a zymo-spintm iicr column in a new collection tube, centrifuged at 10,000 xg (at 4 oc) for 1 minute, and its supernatant was decanted. this procedure was repeated twice. this was followed by the addition of 200 µl dna pre-wash buffer into a new collection tube of zymo-spintm iicr column, centrifuged at 10,000 xg (at 4 oc) for 1 minute, and decanted. a volume of 500 µl g-dna wash buffer was added into a zymo-spintm iicr column, further centrifuged at 10,000 xg (at 4 oc) for 1 minute, and 35 µl dna elution buffer was added into a zymo-spintm iicr column in a 1.5 ml capacity of eppendorf tube. this mixture was again centrifuged at 10,000 xg (at 4 oc) for 30 seconds in the dna elution. the dna obtained was quantified with a nanophotometer p-300 lactic acid bacteria antagonistic against candida albicans ramona et.al. 149 (implen) at the wavelength of 260 nm and 280 nm, stored at -20 oc prior to use in the subsequent analysis. amplification of 16s rdna using hotstart mastermix pcr kit, qiagen primers of 27-f (5’ aga gtt tga tcc tgg ctc ag3’) dan 1492-r (5’ tac ggy tac ctt gtt acg actt 3’) were used to amplify the isolated 16s rdna of the lab isolates. the total volume of the reaction was 50 µl, consisted of 48 µl pcr master mix (5 µl dntps, 5 µl pcr buffer, 3.5 µl mgcl2, 1 µl of 1 pmol primer 27-f, 1 µl of 1 pmol primer 520-r, 0.25 µl taq polymerase, and 34 µl deionized water) and 2 µl dna sample. amplification (30 cycles) was conducted in an infinigen thermocycler machine. the dna samples were first denatured at 94 oc and followed by 30 cycles of pcr with each cycle conditions of: denaturing of dna for 30 seconds, 2 minutes of annealing at 55 oc, and 2 minutes elongation at 72 oc. the cycle was ended with a cycle of elongation for 5 minutes at 72 oc. following this, electrophoresis was run on a gel of 1% (w/v) agarose, containing 1 µg/ml ethidium bromide in tae buffer to confirm whether or not the target sequence was successfully amplified. electrophoresis was conducted for 45 minutes at 80 volt and the correct size of appeared bands were visualized under a uv trans-illuminator. purification of the pcr product followed once of these bands were visualized. purification of pcr product and sequencing of purified 16s rdna purification of pcr product was conducted using suprctm pcr (takara biomedicals, otsu, japan) followed by sequencing with big dye primer cycle sequencing fs ready reaction kit (applied biosystem) using automated sequencing 3100 genetic analyzer (pe applied biosystems). this was conducted at the 1st base, malaysia via the pt genetika science indonesia. the 16s rdna sequences of our isolates were then aligned with their counterparts in a clone library of known bacteria deposited at the gen bank (http://www. ncbi.nlm.nih.gov), so that their molecular identity could be recognized. following multiple sequence alignment of the 16s rdna sequences of the sv, md, and kim isolates, with eight bacterial species previously reported as antagonistic to c. albicans, using clustal w2 program, a phylogenetic tree was constructed using neighbor-joining methods with 1,000 bootstrap replicates and p-distance method in mega11 software (tamura et al. 2021). the phylogenetic analysis involved 18 nucleotide sequences. all positions containing gaps and missing data were eliminated. there was a total of 664 positions in the final dataset. data analysis the data of our current research was analyzed descriptively, elaborated the meaning of each data, and compared it with similar phenomena that have been reported by other researchers in previous studies. ethical statement: the ethical clearance of human sample use for this study was provided by the faculty of medicine udayana university (clearance number: 1354/un14.2.2.vii/lt/2022). results and discussion more than 100 isolates were successfully isolated in our study with colony morphologies on mrsa medium as shown in figure 1 (a-c). the initial indication that those isolates belong to lactic acid bacteria (labs) was their ability to change the color (from purple to yellow) of mrsa medium with bcp indicator in it (figs. 1d-f). this indication which is supported by results of several important tests toward to lab identity (such as gram positive, catalase negative, homo fermentative) confirmed that they all belong to lab group. lactic acid bacteria have been widely used as probiotics to maintain the balance of human and animal intestinal tract. they are abundant in various sources, such as fermented foods, honey or vaginal secrete of healthy women, as isolated in our current research. these are in line with those reported by taye et al. (2021), soemarie et al. (2022), lin et al. (2020), amabebe et al. (2018), elzeini et al. (2021), and munandar et al. (2022) who successfully isolated labs from milk, fermented foods, sea grass, vaginal secrete of healthy women, honey, and intestinal tract, respectively. in dual culture assays, the 46 isolates showed their antagonistic activity, and these were indicated by formation of clear zones around the lab isolates on lawns of c. albicans, with various diameters of inhibition (tabel 1; fig. 2). similar results were also reported by bulgasem et al. (2016) who found several lab isolates, such as l. plantarum, l. curvatus, pediococcus acidilactici, and p. pentosaceus with capability to inhibit the in vitro growth of c. albicans. biotropia vol. 31 no. 2, 2024 150 in more recent study, er et al. (2019) reported similar phenomenon following bioassays of their isolates (vaginal secrete isolates of healthy women) on lawns of c. albicans. according to vieco-saiz et al. (2019) formation of clear zones around the lab isolates indicated that toxic compounds inhibitory to c. albicans, such as organic acids, bacteriocin, or hydrogen peroxides could be released. organic acids (lactic acid in particular) released by labs to their surroundings will lead to a decrease in ph level (lower than 4) of their environment, and could inhibit the growth of their competitors, especially pathogenic bacteria (ayivi et al. 2020). under such condition, the h+ ion level in their surrounding become very high that lead to excessive passive diffusion of such ions (due to high level of h+ ion gradient concentration) into the cell cytoplasm of the residing microbes exposed to such low ph condition (guan & liu 2020). this will subsequently result in a significant decrease of ph within the cell cytoplasm. microbes (pathogens in particular) which are not equipped with proton pump in such condition will not be able to survive due to failure to maintain their cytoplasm ph condition higher than that of their surrounding (ayivi et al. 2020). diameter of inhibition zones formed on the lawns of c. albicans as shown in table 1 and figure 2, may have been due to this type of mechanism, although it needs to be further elucidated. figure 1 colony morphologies of lab isolated from kimchi (a), honey (b), and vaginal secrete (c) on mrsa medium. labs isolated from kimchi (d), honey (e), and vaginal secrete of healthy women (f) with ability to change the color (from purple to yellow) of mrsa medium supplemented with bcp indicator a b c d e f lactic acid bacteria antagonistic against candida albicans ramona et.al. 151 table 2 diameter of inhibition zone (mm) of lab isolates (kimchi, honey, and vaginal secrete of healthy women) on lawns of c. albicans on mrsa medium no. codes inhibition zone (mm)* no. codes inhibition zones (mm)* 1 md 1 23.05±0.10 25 kim 6 5.11±0.08 2 md 2 21.37±0.18 26 kim 7 5.10±0.06 3 md 3 23.93±0.08 27 kim 8 9.94±0.17 4 md 4 18.92±0.11 28 kim 9 13.95±0.16 5 md 5 21.38±0.19 29 kim 10 5.08±0.08 6 md 7 20.99±0.18 30 kim 11 10.03±0.09 7 md 8 22.94±0.18 31 sv 6 5.09±0.08 8 md 9 22.99±0.15 32 sv 7 8.37±0.08 9 md 10 19.43±0.26 33 sv 8 5.11±0.04 10 md 11 18.67±0.15 34 sv 9 5.09±0.09 11 md 12 25.95±0.10 35 sv 10 9.08±0.20 12 md 13 24.96±0.19 36 sv 21 5.13±0.06 13 md 14 24.03±0.13 37 sv 23 5.06±0.06 14 md 15 19.87±0.16 38 sv 24 5.11±0.05 15 md 16 21.38±0.09 39 sv 26 9.04±0.09 16 md 17 9.11±0.17 40 sv 27 9.26±0.09 17 md 18 10.05±0.13 41 sv 28 5.08±0.08 18 md 19 9.99±0.10 42 sv 29 10.50±0.09 19 md 20 10.12±0.13 43 sv 30 9.19±0.14 20 kim 1 19.01±0.12 44 sv 31 9.99±0.16 21 kim 2 5.15±0.06 45 sv 32 5.12±0.07 22 kim 3 15.67±0.12 46 sv 33 10.22±0.15 23 kim 4 23.92±0.18 47 k (+) 36.08±0.07 24 kim 5 8.99±0.15 48 k (-) 0.00±0.00 notes: *values in table 1 ± standard deviations are averages of 5 replicates. k(-) is negative control (sterile broth medium used to produce lab suspension), k(+) is anti-fungus fluconazole. kim, md, and sv are codes for isolates of kimchi, honey, and vaginal secrete origin, respectively. the roles of acidic compounds to inhibit several types of candida spp. have also previously been reported by many researchers. lourenço et al. (2019) for example reported that acetic acid and lactic acid had significant role to inhibit the growth of c. albicans in their in vitro assays. application of such acids synergically with antifungi belong to azole groups was found by these authors to be more effective to inhibit c. albicans. similar phenomenon was also observed by wang et al. (2021) who found that application of asiatic acid, separately or in combination with anti-fungi fluconazole effectively inhibited the in vitro growth of c. albicans. in the in vivo experiments, asiatic acid increased the effectiveness of fluconazole to control the growth of c. albicans. it was concluded in the report of wang et al. (2021) that the asiatic acid had significant role to decrease environmental ph by releasing higher level of h+ ions (through acidic compound dissociation) so that increasing the proton gradient between the cell cytoplasm and its surrounding. growth inhibition in our in vitro dual culture assays could also be due to bacteriocin or bacteriocin-like compounds released by our lab isolates. various types of labs have been reported to produce such compounds and play significant role to inhibit the growth of c. albicans. mohsin and ali (2021) for example reported some labs isolated from yoghurt with capability to produce bacteriocin, inhibitory to the growth of c. albicans. in more recent study, hefzy et al. (2021) also reported some labs (l. pentosus, l. paracasei  subsp.  paracasei, l. rhamnosus, l. delbrueckii  subsp.  lactis) with capability to produce potential bacteriocin-like compounds, active against c. albicans, the causative agent of candidiasis. biotropia vol. 31 no. 2, 2024 152 figure 2 inhibition zones of labs isolated from kimchi (a), honey (b), and vaginal secrete of healthy women (c) on mrsa medium note: clear inhibition zones are indicated by arrow heads. figure 3 some examples of resistance tests of lab isolates against antifungal fluconazole notes: all tested isolates were resistance against fluconazole, but not against tetracycline (arrow heads). sterile distilled water was used as negative control in these assays. a b c bacteriocins are heat resistant antimicrobial peptides produced by labs. the spectrum of control of this compound may either be wide (targeting various species other than labs) or narrow (targeting only species closely related to labs) (collins et al. 2019). plataricin, an example of bacteriocin produced by l. plantarum, initiates pore formation on cell membrane, and resulting in an increase in permeability and alteration of cell morphology of the c. albicans (collins et al. 2019; vazquez-munoz & dongari-bagtzoglou 2021). another metabolite of lab (l. reuteri) affecting the growth c. albicans is reuterin (collins et al. 2019). aldehyde group of the reuterin may interact with tiol group of various compounds and causes oxidative stress on candida albicans (vazquezmunoz & dongari-bagtzoglou 2021). other inhibitory compounds of labs (lactobacillus), such as hydrogen peroxide (h2o2) causing high oxidative stress and genotoxicity have also been reported being products of labs inhibitory against c. albicans (vazquez-munoz & dongaribagtzoglou 2021). in the resistance tests against fluconazole, all potential isolates, presented in table 1, showed resistance properties against this antifungal compound, and the results are shown in table 2 and figure 3. all tested isolates showed their resistance against fluconazole, but not against tetracycline, indicating that they all have possibility to be applied synergically with fluconazole in the therapies of patients infected by c. albicans. fluconazole is an antifungal agent belong to azole class, targeting the 1,4-α-demethylase enzyme involved in the biosynthesis of ergosteol (an important compound of fungal cell membrane) (bassetti et al. 2016; bhattacharya et al. 2020). inhibition of this enzyme by the fluconazole results in growth disturbance on c. albicans. investigations on the use labs in combination with conventional anti-fungi have been conducted worldwide with a view to reduce antifungal dose in the therapy of c. albicans infection (shenoy & gottlieb 2019). implementation of lab in the therapy of c. albicans infection is also expected lactic acid bacteria antagonistic against candida albicans ramona et.al. 153 to improve the effectiveness of conventional antifungi as well as to reduce risk of excessive use of such anti-fungi (bhattacharya et al. 2020). the effectiveness of fluconazole was reported to increase when it was combined with l. acidophilus, b. bifidum, and b. longum (davar et al. 2016), or with l. rhamnosus dan l. gasseri (pendharkar et al. 2015). the effectiveness of l. gasseri, however, was not improved when it was combined with itraconazol in the therapy of vulvovaginal candidiasis (shenoy & gottlieb 2019). isolates of labs (10 isolates) that showed the best inhibition zones in dual culture assays against c. albicans (table 1) were molecularly identified (sequencing their 16s rdna), and the results are displayed in figure 4. based on the local alignment of the 16s rdna nucleotide sequence (blastn) with those recorded in the genbank, our isolates were closely related to three species of labs, lactobacillus paracasei (accession id lc463234), lacticaseibacillus paracasei (accession id pp210106), and pediococcus pentosaceus (accession id mt604839). previous studies also reported that l. paracasei (rossoni et al. 2017; de gregorio et al. 2019), lacticaseibacillus paracasei (leska et al. 2022), and pediococcus petosaceous (bulgasem et al. 2016) had antagonistic effect on the growth of c. albicans. this indicates that our isolates have potential to be developed as probiotic candidates for use in the therapies of infections caused by c. albicans. figure 4 relatedness of labs isolated from kimchi (kim), honey (md), and vaginal secrete (vs) of healthy women, along with those previously reported to be antagonistic against c. albicans notes: the phylogenetic tree was built using neighbor-joining method with 1000 bootstrap replicates and p-distance substitution in mega11 (tamura et al. 2021) following multiple sequence alignment of the 16s rdna sequences of the sv, md, dan kim isolates, with the previously reported bacteria antagonistic to c. albicans recorded in the genbank. the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches. the tree is a rooted-cladogram type. our lab isolates are those marked with black diamonds. biotropia vol. 31 no. 2, 2024 154 table 2 resistance of lab isolates against antifungal fluconazole commonly used in the candidiasis therapies no codes diameters of inhibition zones (mm)* no codes diameter of inhibition zones (mm)* 1 md 1 0.00±0.00 25 kim 6 0.00±0.00 2 md 2 0.00±0.00 26 kim 7 0.00±0.00 3 md 3 0.00±0.00 27 kim 8 0.00±0.00 4 md 4 0.00±0.00 28 kim 9 0.00±0.00 5 md 5 0.00±0.00 29 kim 10 0.00±0.00 6 md 7 0.00±0.00 30 kim 11 0.00±0.00 7 md 8 0.00±0.00 31 sv 6 0.00±0.00 8 md 9 0.00±0.00 32 sv 7 0.00±0.00 9 md 10 0.00±0.00 33 sv 8 0.00±0.00 10 md 11 0.00±0.00 34 sv 9 0.00±0.00 11 md 12 0.00±0.00 35 sv 10 0.00±0.00 12 md 13 0.00±0.00 36 sv 21 0.00±0.00 13 md 14 0.00±0.00 37 sv 23 0.00±0.00 14 md 15 0.00±0.00 38 sv 24 0.00±0.00 15 md 16 0.00±0.00 39 sv 26 0.00±0.00 16 md 17 0.00±0.00 40 sv 27 0.00±0.00 17 md 18 0.00±0.00 41 sv 28 0.00±0.00 18 md 19 0.00±0.00 42 sv 29 0.00±0.00 19 md 20 0.00±0.00 43 sv 30 0.00±0.00 20 kim 1 0.00±0.00 44 sv 31 0.00±0.00 21 kim 2 0.00±0.00 45 sv 32 0.00±0.00 22 kim 3 0.00±0.00 46 sv 33 0.00±0.00 23 kim 4 0.00±0.00 47 k (+) 31.06±0.11 24 kim 5 0.00±0.00 48 k (-) 0.00±0.00 notes: *values in table 2±standard deviations are averages of 5 replicates. tetracycline and fluconazole were used as positive control while the solvent of the antifungal agent was used as negative control. conclusion labs with potential to control the growth of c. albicans could be isolated from many sources, such as fermented foods (kimchi), honey, or vaginal secrete of healthy women. among more than 100 isolates successfully isolated in our current study, 46 isolates showed in vitro antagonistic activity against c. albicans, and they also showed resistance property to fluconazole (an antifungal agent commonly used in the therapy of candidiasis), indicating that they all 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kustiariyah tarman 1department of coastal resources management, graduate school, diponegoro university, semarang 50725, indonesia 2laboratory of tropical marine biotechnology, integrated laboratory of diponegoro university, semarang 50269, indonesia 3department of aquatic products technology, faculty of fisheries and marine science, institut pertanian bogor, bogor 16680, indonesia 4 department of microbiology, faculty of mathematics and natural sciences, institut pertanian bogor, bogor 16680, indonesia 5 center for coastal and marine resources studies, institut pertanian bogor, bogor 16680, indonesia received 18 november 2016/accepted 1 june 2017 abstract multidrug-resistant (mdr) bacteria infections become a serious problem for these several decades. to solve this issue, finding of new antibiotics candidate in an urgency. natural pigment is known to has biological activity against pathogenic bacteria. coastal fungi are unexplored source of natural pigment to fight mdr bacteria. this research was aimed to isolate coastal endophytic fungi from smooth ant plant (hydophytum formicarum), to screen endophytic fungi which produce red pigment, to extract the red pigment, to determine antibacterial activity of the red pigment and to identify the coastal endophytic fungi producing the red pigment. in this study, 7 fungi were isolated as endophytic fungi from h. formicarum. there were 3 isolates which produced extracellular pigment i.e. rs 1a which produced red pigment, rs 3 produced black pigment and rs 6a produced yellow pigment. our study focused on red pigment which is produced by endophytic fungus strain rs 1a. the yield of red pigment was 8.8657% (w/w). this study showed that red pigment had antibacterial activity against escherichia coli, acinetobacter baumannii and proteus mirabilis strain mdr. judging from molecular and morphological identification, the endophytic fungus strain rs 1a was identified as aspergillus versicolor. keywords: antibacterial, endophytic fungi, mdr, pigment introduction bacteria having resistance to several antibiotics used to treat the infections are referred to as multidrug-resistant (mdr) bacteria (cornaglia 2009; magiorakos et al. 2012). mdr is a serious problem for medical world and public health. several bacteria already reported as mdr are escherichia coli staphylococcus acinetobacter , spp., baumanni proteus mirabilis and (ahmed et al. 2015; korytny et al. 2016; panda et al. 2016). therefore, it is urgent to find new antibiotics. as a megabiodiversity and maritime country, indonesia has many coastal resources which are potential to be sources of new antibiotics against mdr bacteria. among coastal resources, smooth ant plant ( ) is an neglected hydnophytum formicarum source for new antibiotic candidate. h. formicarum is a member of myrmecophytes plant which provides nesting cavities for ants (lok & tan 2009; defossez . 2009). this plant was et al found as epiphytic plant in sp. avicennia (mangrove plant) in sorong, papua. the utilization of epiphytic plant as source of * corresponding author: kustya@gmail.com biotropia 4 2 7 161 172 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 2 725 161 bioact ive compounds i s h indered by environmental issues. exploration of endophytic fungi is expected to overcome these issues. information related to the biodiversity of endophytic fungi from is hydnophytum formicarum rarely found. the first report about associated fungi from genus was reported in hyndophytum 1911 (defossez . 2009). therefore, our study et al will add more information about endophytic fungi from .hydnophytum formicarum coastal endophytic fungi are commonly reported as potential source of bioactive compounds. however, there is rare report regarding its anti-multidrug resistant bacteria activity. endophytic fungi refer to microfungi which live inside plant tissues without causing any symptoms to the host (thirunavukkarasu . et al 2015; sibero . 2016a). in addition, kusari . et al et al (2013) stated that endophytic fungi could produce the same or similar bioactive compounds like its hosts. endophytic fungi are known to produce metabolites with various biological activities et al et al et al (meng . 2015; wong . 2015; cao . 2016; et al et alrahaweman . 2016; sibero . 2016a). in contrary, fungal pigment is a neglected metabolite even though several researchers reported its bioactivity as antibacterial agent (geweely 2011; et al et almani . 2015; patil . 2015). our study was aimed to isolate coastal endophytic fungi from smooth ant plant ( . ), to screen h formicarum endophytic fungi which produce red pigment, to extract the red pigment, to determine antibacterial activity of the red pigment and to identify the coastal endophytic fungi producing the red pigment. materials and methods sampling preparation smooth ant plant (h. formicarum) was collected by dr kustiariyah tarman, as epiphytic plant on avicennia sp. in mangrove forest located in sorong, west papua province (fig. 1). the domatia part (hollow structure part) of smooth ant plant was collected by cutting the plant from the host plant using metal cutter. the plant was then put inside zipped plastic bag. the sample was taken to the laborator y of aquat ic microorganisms, department of aquatic products technology, faculty of fisheries and marine science, institut pertanian bogor for isolating the endophytic fungi. fungal isolation, cultivation and pigment screening endophytic fungal isolation was carried out using surface sterilization method (kjer et al. 2010). h. formicarum was cut approximately 1 cm 2 figure 1 hydnophytum formicarum as epiphytic plant on avicennia sp. (mangrove tree) found in mangrove forest in sorong, west papua province 162 biotropia vol. 24 no. 2, 2017 disk. the extracts concentrations were 50 µg/ml, 100 µg/ml, 250 µg/ml, 350 µg/ml and 500 µg/ml. antimicrobial susceptibility disk for tm amoxicillin (aml) 10 µg (oxoid ) was used as positive control, while acetone was used as negative control. the bacteria turbidity standard was equivalent to a 0.5 mcfarland. the bacteria were inoculated on muller hinton agar (mha) using cotton swab with rotation inoculation. after that, the positive control was placed in the middle, while the negative control and paper disks with extracts were placed around the positive control by forming a circular pattern and o incubated at 36 – 37 c for 24 h. the results of antibacterial assay were analyzed using factorial analysis of variance (anova) with spss software. the confidence interval was 95%, while the significant difference was analyzed using duncan test. morphology observation slide culture method was performed for fungus cultivation with modifications (qiu et al. 2005; sibero et al. 2016b; sibero et al. 2017). pda media was prepared and cut approximately 2 × 2 cm and placed on sterilized object glass. the 2 mycelia were inoculated on each side of the pda and a cover glass was placed on the pda, then put into sterilized petri dish for incubation. after 3 days, each side of pda had been overgrown by the fungus and the mycelia already covered the inner side of the cover and object glass. the pda was then removed, while the object and cover glasses were observed under a compound microscope. morphological characteristics of the fungus were compared to huh et al. (2013), visagie et al. (2014) and ama (2016). molecular identification chelex method with several modifications was used for dna extraction (sibero . 2017). the et al red pigmented fungus was cultured on pda for 7 days. mycelium of the red pigmented fungus was taken and put into eppendorf microtube, added with 100 µl ddh o and 1, 000µl of 0.5% 2 saponin, then kept for overnight. after that, the mixture was centrifuged (12,000 rpm, 10 minutes o at 5 c). supernatant were discarded, then 100 µl ddh o and 50 µl of 20% chelax 100 were added. 2 o the mixture was kept on water bath (80 c, 10 minutes). in the first 5 minutes, the mixture was and washed with distilled water followed by 70% ethanol (etoh) for 1 minute and then re-washed with distilled water. after that, the pieces of samples were placed on potato dextrose agar (pda) without the addition of antibiotics and were incubated at 28 c until growth was initiated. o during isolation, a petri dish with pda was left open as environmental control. after 7 days, there were several fungi growth on the pda media. the fungus which grown nearby the sample but not found in environmental control petri dish was separated and placed into new pda media as single colony. each single colony was cultivated on pda for 7 days at room temperature (27 – 28 c). every day the color change of media o was observed. the fungus producing red pigment in pda media was used for this research. pigment extraction pigment was extracted using solid liquid extraction (manikkam et al. 2015; sibero et al. 2016b). pda media were separated from the mycelia using sterilized metal cutter. the pda media were then weighed and chopped until the segments became smaller and placed into erlenmeyer flasks. a quantity of 100 ml acetone was poured into the flasks and shaken using shaker for 24 hours at 27 c. the contents of the o flasks were filtered through filter paper (macherey-nagel 640d·ø 1125 mm). the filtrates were concentrated using rotary evaporator (30 – 33 c, 30 minutes). the yield was obtained o according to the following formula: antibacterial activity antibacterial assay was conducted based on sibero et al. (2016b) with several modifications. the pigment extracts were used to test against clinical mdr gram-negative bacteria, including acinetobacter baumannii, escherichia coli and proteus mirabilis with two replications. these bacteria were clinical isolates and considered as mdr strains from rsup dr kariadi (dr kariadi general hospital medical center) and rumah sakit nasional diponegoro (diponegoro national hospital), both located in semarang, central java province, indonesia. gram negative bacteria were orefreshed on macconkey for 24 hours at 37 c. antibacterial assay was carried out using paper % yield = x 100% yield pda weight 163 antibacterial activity of red pigment isolated from coastal endophytic fungi – sibero et al. mixed using vortex. the final mixture was o centrifuged (12,000 rpm, 10 minutes at 5 c). the o supernatant was taken and stored at -20 c. the internal transcribed spacer (its) region of fungus were amplified by pcr using universal primers internal transcribed spacer (its) 1 (5'-tcc gta ggt gaa cct gcg g-3') as forward and its 4 (5'-tcc tcc gct tat tga tat gc-3') as reverse (sibero . 2017). pcr mixture consisted of et al gotaq green master mix promega (12.5 µl), primer its 1 (1 µl), primer its 4 (1 µl), dna extract (0.5 µl) and ddh o (10 µl). the pcr 2 reaction condition consisted of preheat at 95 c for 3 o minutes, denaturation at 95 c for 1 minute, o annealing (gradient from 51.4 c to 67.4 c) for 1 o o minute, extension at 72 c for 1 minute, final o extention at 72 c for 7 minutes and holding o temperature at 16 c. denaturation, annealing and o extension stages were performed for 30 cycles. the pcr product was loaded in agarose gel (1%) electrophoresis. the product of electrophoresis was visualized by uvi doc hd5 (uvitec cambridge). pcr product which performed clear dna band was used for dna sequencing. this stage was conducted at 1 base laboratories sdn bhd, st malaysia. the sequence was compared and aligned by basic local alignment search tool (blast), then analyzed to its homology to other fungi obtained from gene bank. this research used neighbor-joining for statistical method, bootstrap method for test of phylogeny with number of bootstrap replications was 1,000. the phylogenetic analysis was constructed using mega 7 software package (tamura . 2011).et al results and discussion endophytic fungi from . h formicarum there were seven fungi isolates which were successfully isolated from h. formicarum (fig. 2). each fungus had different macroscopic characteristics based on colony forms, mycelia colors and reverse media colors. biodiversity of culturable fungi is influenced by nutrient content in media and isolation method (kjer et al. 2010; toma & abdulla 2013). surface sterilization is the most important procedure in endophytic fungal isolation. the aim of surface sterilization using ethanol 70% is to eliminate spores and other microbial contaminants attached to the sample surface (kjer et al. 2010). environmental control petri dish is also important to minimize the possibility of improper isolation. fungi grown in environmental control petri dish and sample petri dishes were suspected as contaminant. from endophytic fungal isolation, there were seven fungal isolates obtained (fig. 2). each fungus ha s d i f f e r en t mor pho log i c a l characteristics. among the seven endophytic fungi isolated, there were three fungal isolates produced extracellular pigment. they were rs 1a which produced red pigment, rs 3 produced black pigment and rs 6a produced yellow pigment. pigment production was shown by the color change of the medium. sibero et al. a(2016 ) successfully characterized black pigment from rs 3 as melanin. figure 2 endophytic fungal isolates obtained from hydnophytum formicarum grown on pda a) rs 1a; b) rs 1b; c) rs 2a; d) rs 2b; e) rs 3; f) rs 6a; g) rs 6b 164 biotropia vol. 24 no. 2, 2017 a. c.b. d. e. g.f. nutrient content in media has important role in fungal pigments. mugesh et al. (2014) stimulated biopigment production of several endophytic fungi by diversifying growth media. as a result, the mecv01 fungus isolate produced red biopigment on czapek-dox agar (cda) and czapek-dox yeast autolysate agar (cya). on malt glucose yeast peptone agar (mgyp) and yeast glucose trace (ygt) media, the mecv01 fungus isolate produced yellowish red biopigment. mugesh et al. (2014) stated that carbon source (sucrose) and trace elements gave impact to growth and pigment production. our study focused on red pigment needed in many industries such as food, textile and cosmeceutical industries. based on macroscopic observation, fungus rs 1a had green colony and o grew well at room temperature (28 c). green color was produced by the colony, while the mycelia had white color. fungus rs 1a produced extracellular red pigment since the fourth day of obeing on pda media at room temperature (28 c). production of red pigment increased and the pigment was released not only to the pda media, but also to the mycelia (fig. 3). red pigment from this fungus was judged as extracellular pigment, because it was released outside the cells. several fungi produced extracellular pigments and known to have biological activity (dong & yao 2012; mani et al. 2015; a; sibero et al. 2016b). antibacterial activity of red pigment produced from fungus rs 1a extracellular pigment is extracted from media because it is spread outside the cells (xiong et al. 2015; akilandeswari & pradeep 2016). pigment production depends on several conditions, such as nutrient in media, light intensity, ph, trace elements, temperature and agitation (mugesh et al. 2014; bühler et al. 2015; patil et al. 2015; shi et al. 2015). this pigment was extracted directly from solid media and performed by maceration method with acetone. the yield of red pigment from fungus rs 1a was 8.8657 % (% w/w). organic solvent such as methanol, acetone, chloroform and ethyl acetate are commonly used to extract natural pigment (robinson et al. 2014; vora et al. 2015). the pigment was tested against several mdr bacteria. results of antibacterial assay are presented in table 1 and figure 4. enterobacteriaceae members are known as gram negative pathogenic bacteria such as e. coli, proteus, salmonella and shigella (shaikh et al. 2015; dutta et al. 2016; clsi 2016). these pathogenic bacteria cause urinary tract infection, nosocomial infection, blood stream infection and meningitis, causing death to human (harrish et al. 2015; shaikh et al. 2015; iqbal et al. 2016). based on the result of antibacterial assay, red pigment had activity to combat clinical mdr gram-negative bacteria. the best antibacterial activity was performed at concentration 500 µg/ml against mdr e. coli with inhibition zone of 19.8±1.13 mm. we highlighted the diameter of inhibition zone of amoxicillin against e. coli and p. mirabilis. amoxicillin had inhibition zone ≤ 13 mm. therefore, according to clsi (2016) these bacteria were resistant to amoxicillin. wong et al. (2013) and dutta et al. (2016) successfully isolated and characterized e. coli from hospital and p. mirabilis from chicken carcasses as mdr which figure 3 fungus rs 1a after ten days of cultivation on pda 165 antibacterial activity of red pigment isolated from coastal endophytic fungi – sibero et al. note: data presented are mean±sd values at the same column followed by the same letters are not significantly different at p < 0.05 values with * shared letters denote significant difference at p < 0.05 figure 4 inhibition zones of red pigment produced from fungus rs 1a against mdr bacteria: (a) acinetobacter baumannii; (b) escherichia coli; (c) proteus mirabilis mdr bacteria concentration of red pigment extract (µg/ml) inhibition zone (mm) acinetobacter baumannii 50 4.0±0.42a 100 4.25±0.35a 250 4.6±0.00a 350 5.25±0.63ab 500 6.75±0.91ab amoxicillin 10 µg 12.00±0.00c* escherichia coli 50 5.80±0.28a 100 6.35±0.49a 250 7.05±0.49a 350 7.45±0.07a 500 19.8±1.13c* amoxicillin 10 µg 10.60±0.00b proteus mirabilis 50 5.15±2.1a 100 6.15±0.63ab 250 6.55±0.35ab 350 6.85±0.21ab 500 9.15±0.21b amoxicillin 10 µg 12.00±0.00c* table 1 antibacterial activity from fungal red pigment rs 1a against mdr bacteria 166 biotropia vol. 24 no. 2, 2017 resistant to amoxicillin using clsi standard. somwanshi and bodhankar (2015) reported that the endophytic fungi had antibacterial activity against mdr human pathogens such as escherichia coli, acinetobacter baumannii, salmonella typhi and klebsiella pneumoniae. on the other hand, sibero et al. (2017) used marine fungi against e. coli strain mdr. in addition, zhao et al. (2016) reported that monascus pigment had activity against e. coli with mic 2.5 mg/ml. fungus identification fungus identification was carried out through molecular and microscopic observation approaches. for molecular identification, deter minat ion of opt imum anneal ing temperature was conducted at temperature range from 51.4 c to 67.4 c in pcr. o o annealing temperature has important role on the success of pcr products. if the annealing temperature is too low, it will cause the amplification of non-specific dna fragments. if the annealing temperature is too high, it will reduce the purity of pcr product (rychlik et al. 1990). figure 5 shows the result of pcr products visualization in agarose gel. dna of fungus rs 1a was well amplified from 51.40 to 60.2 c. temperature of 51.40 c o o showed the brightest band in visualization. the figure 5 pcr products visualization from determination of annealing temperature figure 6 a cladiogram resulted from neighbor-joining tree of fungus rs 1a obtained from the its rdna sequences analysis aspergillus oerlinghausensis nr 138362.1 aspergillus waksmanii nr 135040.1 aspergillus vitricola kt809136.1 aspergillus vadensis ay585549.1 aspergillus luppii nr 137477.1 aspergillus polyporicola nr 137471.1 aspergillus leporis nr 135327.1 aspergillus pseudotamarii nr 135329.1 aspergillus caelatus nr 135326.1 aspergillus tamarii nr 135325.1 aspergillus pulvericola nr 135453.1 aspergillus occultus nr 135454.1 aspergillus subramanianii nr 135385.1 aspergillus salwaensis nr 135455.1 aspergillus westlandensis nr 135451.1 rs ia aspergillus versicolor nr 131277.1 microascus verrucosus nr 132950.1 100 50 99 31 87 58 100 34 42 50 68 100 76 83 99 167 antibacterial activity of red pigment isolated from coastal endophytic fungi – sibero et al. annealing temperature of fungal dna is varied and depended on the species, primers and pcr instrument. other research successfully amplified fungal dna with annealing temperature of 40, 47 and 54 (choo et al. 2015; kramer et al. 2016; o c krishnan et al. 2016). according to the homology comparison, fungus rs 1a was closed to several strains of aspergillus versicolor. this fungus had 99% nucleotide similarity to . strain nr a versicolor 131277.1 which was done by haugland et al. (2016). phylogenetic relationship of this fungus is shown in figure 6. the result of microscopic morphological characterization of fungus rs 1a is shown in figure 7. based on the microscopic morphology characterization, fungus rs 1a had smooth conidiophore without any branches, biseriate phiallides and produced round conidia. this fungus produced unique peniciliium -like conidiophore (fig.7c). a. versicolor is known to produce penicillium-like conidiophores. this conidiophore had phialides which attached to stipe. these conidiophores are vegetative hyphae with very short stipe. the production of conidia using penicillium like-head was faster than the production of conidia by aspergillus-head (klich 1993; ama 2016). the production of this unique conidiophore usually causes misidentification for morphological characterization. ama (2016) successfully showed the production of penicilliumlike head started in the first day of incubation. a. versicolor has been reported as an endophytic and associatic fungi in plants and animals (zhuang et al. 2011; hawas et al. 2012). hawas et al. (2012) isolated new metabolite name isorhodoptilometrin-1-methyl ether from endophytic a. versicolor with antibacterial activity against bacillus cereus, b. subtilis and staphylococcus aureus. in 2013, a new alkaloid named asperverin was isolated from an algicolous a. versicolor. in addition, a. versicolor has been reported as endophytic fungi in paris polyphylla var. yunnanensis and produced new butyrolactones versicolactones e-f (zhou et al. 2016). yan et al. (2016) reported this fungus was isolated from the mud of deep water in south china sea and had antioxidant property. wang et al. (2017) discovered a new antimicrobial compound from deep sea sediment which was a. versicolor named 2-(dimethoxymethyl)figure 7 microscopic morphology of fungus rs 1a identified as aspergillus versicolor (note: a. mycelia; b. conidiophore; c. penicillium-like conidiophore with conidia) 168 biotropia vol. 24 no. 2, 2017 a b c 1-hydroxyanthracene-9,10-dione. several studies proved that a. versicolor produced pigments (jurjevic et al. 2012; ama 2016). red pigment from a. versicolor has been discussed since 1960s. in 1967, hamasaki et al. (1967) reported three anthraquinoid pigments with red color. three years later, hatsuda et al. 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page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 biotropia vol. 29 no. 3, 2022: 272 282 doi: 10.11598/btb.2022.29.3.1792 272 macrofungal diversity in different vegetation compositions in teghari community forest, kailali, west nepal kausalya joshi1, hari sharan adhikari1,*, hari prasad aryal2 and laxmi joshi shrestha1 1department of botany, amrit science college (tribhuvan university), kathmandu 44600, nepal 2central department of botany, tribhuvan university, kathmandu 44600, nepal received 1 august 2022/accepted 18 october 2022 abstract macrofungi are high-value forest resources that have functionally significant roles in the forest ecosystem. the macrofungal community of three different vegetation compositions, i.e., sal (shorea robusta) forest, tropical deciduous riverine forest, and tropical evergreen forest of teghari community forest were investigated. systematic random sampling was made where 60 plots (10 x 10 m) were laid in all different forest types (20 plots in each). a total of 102 macrofungi species were reported belonging to 36 families. polyporaceae (17 species) was the largest family followed by tricholomataceae (13 species) and saprophytic fungi were more frequent than mycorrhizal and parasitic fungi. the tropical evergreen forest was rich in macrofungi (59 species) followed by sal forest (40 species) and tropical deciduous riverine forest (38 species). macrofungal diversity was directly related to surrounding host species. similarly, increased soil moisture and canopy cover intensified the abundance of saprophytic fungi. the species richness was increased with increasing organic carbon, canopy, moisture, ph, and litter cover. however, soil nitrogen, phosphorus, and potassium were less significant in affecting species richness. also, the disturbance was negatively correlated with the species richness of macrofungi. this study highlights the hidden diversity which is necessary for the conservation of macrofungi, to optimize forest ecosystem integrity and resilience against biotic and abiotic agents. keywords: macrofungal diversity, sal forest, species richness, tropical evergreen forest, tropical riverine forest introduction biodiversity is simply defined as the presence of the total organism of a particular group at a particular time in a particular area. conservation of these natural resources is the priority for ecosystem functioning as well as human welfare. fungi are an enormous usly diverse group of organisms ranging from microscopic to macroscopic forms that grow mostly in the dead and decaying substrate. they appear in all seasons, mostly rainy season, wherever nutrient organic matters or decomposed products are easily available (jha & tripathi 2012). macrofungi are a group of higher fungi that produce mature spore-bearing fruiting bodies, which are visible to the naked eye (chang & miles 1992). they are known to inhabit diverse kinds of habitats varying in the composition of their tree species and substrates. based on ecology, they are parasitic or saprophytic or may show some mycorrhizal associations with vascular plants (kumar & sharma 2011). however, some macrofungi are neutral to the abundance of dominant tree species, in particular, habitat type (zhang & zak 1998). the relationship between the tree and fungal communities is reflected in host trees affecting fungal specialization and providing unique habitat availability and different resource quality. the composition and structure of aboveground vegetation are responsible for diverse macrofungi communities (buee et al. 2011). *corresponding author, email: aharisharan@gmail.com macrofungal diversity in different vegetation composition in kailali, west nepal – joshi et al. 273 generally, macroscopic fruiting bodies of the fungi is called mushroom which can be epigeous or hypogeous and vary in different shape and sizes. they are fleshy, sub-fleshy, or sometimes leathery and woody and bear their fertile surface either on lamellae or lining the tubes, opening out through pores. the most suitable condition for the growth of carpophores depends upon the high humidity, nutritionally rich substrate, and warm atmospheric temperature (dickinson & lucas 1979). similarly, other environmental conditions such as geographic location, light, and surrounding vegetation types also play a major role in the distribution of the macrofungi (sibounnavong et al. 2008). diversity-related studies are carried out in different forests but their relationship with higher plants was poorly explored except these studies such as pradhan (2013), baral et al. (2015), and bhandari and jha (2017). this study aimed to to optimize forest ecosystem integrity and resilience against biotic and abiotic agents, by looking at the effect of different vegetation characteristics and environmental factors on macrofungal species composition and richness in the tropical region of western nepal. materials and methods study sites the study was carried out in three different vegetation patches within teghari community forest in the tropical riverine belt of kailali district, west nepal (fig. 1). the study area lies between latitudes from 28°50'45" n to 28°51'01" n and longitude 80°33 ̍ 3" e to 80°33'13" e, covering an area of 340 ha. the altitude range of the study area is 155 254 masl. meteorological data of the dhangadi airport in the year 2019 was obtained from the department of hydrology and meteorology, government of nepal which revealed that the study area is represented by a tropical climate and receives an average of 1,406.6 mm annual rainfall with the highest monthly rainfall happens in july (466.9 mm) and the lowest in may (5 mm). the highest monthly mean temperature happens in may (40.41°c) and the lowest in january (6.77 °c). figure 1 map of the study area biotropia vol. 29 no. 3, 2022 274 study design the teghari community forest was selected for the field study as it has three different forest types at the same elevation, i.e., sal forest, tropical riverine deciduous forest, and tropical evergreen forest. shorea robusta (sal) is the dominant tree species in the sal forest which forms magnificent forest stand on the edges of the godawari river. the tropical deciduous riverine forest is also located similarly and is mainly dominated by acacia catechu and dalbergia sissoo along with bombax ceiba, syzygium cumini, adina cordifolia, hollarrhena pubescens, murraya koenginii, aegle marmelos, and semicarpus anacardium. the tropical evergreen forest lies on the northwest side of mahakali highway and is dominated by terminalia alata, lagerstroemia parviflora, terminalia bellerica, ficus religiosa, schleichera oleosa, aegle marmelos, and cassia fistula. mallotus phlippensis is present all over the study area. in each forest type, rectangular plots of 10 × 10 m were established. the number of plots to be sampled were determined based on the spatial area of each forest. field sampling detailed sampling of macrofungi diversity was made by applying a systematic random method within the period of june october 2019, where plots were laid in each forest type. a total of 20 plots were laid in each forest type along with the two transects for maintaining an inter-plot distance of at least 20 m (baral et al. 2015). presence or absence data of macrofungal species were recorded in each plot. biophysical variables, such as tree canopy cover, litter cover, and anthropogenic disturbances (trampling, fire grazing, non-degradable waste, etc.) were also recorded in each plot. tree canopy cover and litter cover (in percentage) was estimated visually. for tree canopy cover, observation was made from the middle of each plot. soil samples were collected at a depth of 15 cm from four corners and at the middle of each plot using a soil digger. the soil samples from each plot were mixed thoroughly. from the mixed soil sample, about 200 g of soil sample was taken and put in a zipper polythene bag. the soil samples were air-dried in shade for a week and stored in airtight plastic bags until laboratory analysis. the physiochemical parameters of soil, such as soil ph, moisture, organic carbon, nitrogen, potassium, and phosphorus were assessed using a standard soil analysis manual (zobel et al. 1987). macrofungal specimens were collected, preserved (dry), and taken to national herbarium (kath) in lalitpur, nepal. collected specimens were studied based on their morphological characters and ecology with the help of several websites, such as https://www.mushroomexpert.com and http://www.indexfungorum.org. finally, the identification of specimens was confirmed using relevant literature (pacioni & lincoff 1981; adhikari 2014; laessoe 2013) along with identification conducted by macrofungi expert. all of the collected macrofungal specimens were deposited in ascol herbarium, amrit science college, kathmandu, nepal. data analysis all data were entered in microsoft excel 2010 for further analysis. pearson correlation method was used to know the effect of a different environmental variable on macrofungal diversity. simpson’s diversity index (simpson 1949) and shannon-wiener index (shannon & weaver 1963) were also calculated. regression analysis was performed using spss version 20 and microsoft excel version 2010. species composition of different macrofungi species along with different environmental components were evaluated by canonical correspondence analysis (cca). results and discussion macrofungal diversity in different vegetation composition a total of 102 macrofungi consisting of ascomycetes-5 and basidiomycetes-97 species were documented, in which 100 species were identified up to species level and 2 species were identified up to genus level. out of the 36 families, 17 species belonged to the polyporaceae family, 13 species to tricholomataceae, 11 species to marasmiaceae, 9 species to agaricaceae, 8 species to coprinaceae, 4 species each to russulaceae and xylariaceae, 2 species representing each of the cortinariaceae, ento macrofungal diversity in different vegetation composition in kailali, west nepal – joshi et al. 275 lomataceae, fomitopsidaceae, ganodermataceae, hydnangiaceae, podoscyphaceae, and suillaceae family and the rest of the family was represented by single species only (fig. 2). tropical evergreen forest harbored the highest macrofungal diversity (59) in all three different substrates (fig. 3), followed by sal forest (40) and tropical deciduous riverine forest (38). the maximum numbers of macrofungi were found growing on the soil, followed by wooden logs. the least number of macrofungal species were found growing on litters in all forest types. the present study relates to a study conducted in india where macrofungi were reported in various habitats, like wood, litter, and moist soil, among others (nagaraju et al. 2014). as compared to litter and wood, the soil was the most important substrate for maintaining macrofungal diversity in all three forest types studied. in our study, higher number of macrofungi were grown on moist soil compared to those on litter and decaying wood. these findings resemble the previous findings of a study conducted by bhandari and jha (2017). figure 2 number of species with their respective family figure 3 distribution of macrofungi based on their habitat in different forests biotropia vol. 29 no. 3, 2022 276 based on the ecology of macrofungi, the maximum number of macrofungi were consisted of saprophytes, followed by mycorrhizal and parasitic macrofungi, while the least number belonged to termitophilous macrofungi. the mycorrhizal fungi serve as an extension of the plant root system, exploring soil far beyond the roots and transporting water and nutrients to the roots (tapwal et al. 2013). the flourishing of carpophores is enhanced by litter accumulation and decomposition as well as the presence of extracellular microbial enzymes (pushpa & purushothama 2012). the rapid change in the weather and high response of mycelia were among the main factors for the increasing number of saprotrophic fungi (pradhan et al. 2012). a similar result was obtained in the research of topwal et al. (2013) and dey et al. (2016). higher species diversity in basidiomycota compared to ascomycota is probably contributed by a higher number of mycorrhizal species found on the soil as studies have shown that soil moisture and decaying litter facilitate many diverse macrofungi (muller & schmit 2007). simpson’s diversity index (table 1) was found to be the highest in tropical deciduous riverine forest and tropical evergreen forest (0.91) in comparison to sal forest (0.88). similarly, shannon-wiener diversity index was also found to be the highest in tropical evergreen forest (2.91) followed by tropical deciduous riverine forest (2.77) and sal forest (2.53). the presence of diverse kinds of macrofungi communities is specifically related to the dominant tree species of the forest has been confirmed by many other studies (straatsma & krisai-greilhuber 2003; gates et al. 2011; o’hanlon & harrington 2011; bhandari & jha 2017; collado et al. 2021; kutszegi et al. 2021). such variation may be attributed to microclimate conditions (santos-silva et al. 2011) and forest management practice (kouki & salo 2020). the high macrofungal diversity in the tropical evergreen forest is mainly related to high soil moisture and greater cover of tree species. the high diversity may be also due to suitable habitat, such as soil moisture, litter, and canopy cover which help to maintain sufficient moisture (trudell & edmonds 2004). a tropical deciduous riverine forest located on the water edges has a more open canopy and less humidity in the soil which creates a less suitable habitat for the growth of macrofungi. also, thinning of trees caused a decrease in fruit-body production of the delicate and fragile macrofungi, but this effect varied greatly depending on the season, the macrofungi fruiting pattern and the levels of trees thinning (luoma et al. 2004). therefore, thinning and pruning, which are common silvicultural activities in the community forests of nepal (shrestha et al. 2010), might also affect the composition and abundance of macrofungi. similarly, sal forest has a magnificent stand of tall trees and has a more open canopy in comparison to tropical evergreen forest. the growth of macrofungal species like pycnoporus cinnarius and scleroderma cepa was specifically recorded in sal forest. a similar finding was also reported by prasad & pokhrel (2017) at amrite community forest, kapilvastu district (central nepal), which might be due to the host specificity of macrofungi with particular plant species. the presence of specific macrofungi communities in the present study may be due to host preferences which were related to the findings by ding et al. (2011) and lang et al. (2011). species richness of macrofungi and different environmental variables the canonical correspondence analysis (cca) revealed the relationship between macrofungi species composition and environmental gradient (table 2). the analysis results indicated the effective separation of species along the main gradient (table 3). table 1 diversity indices of macrofungi in different vegetation stands forest stands simpson’s index simpson’s diversity index shannon-wiener diversity index sal forest 0.12 0.88 2.53 tropical deciduous riverine forest 0.09 0.91 2.77 tropical evergreen forest 0.09 0.91 2.91 macrofungal diversity in different vegetation composition in kailali, west nepal – joshi et al. 277 table 2 summary of the results of canonical correspondence analysis axes 1 2 3 4 total inertia eigenvalues 0.369 0.34 0.279 0.208 11.381 species-environment correlations 0.886 0.903 0.945 0.88 cumulative % variance of species data 3.2 6.2 8.7 10.5 cumulative % variance of species-environment data 18.7 35.9 50 60.5 sum of all canonical eigenvalues 1.978 table 3 relative importance of environmental variables and their significance (p value) on macrofungal species composition derived by using the monte carlo permutation test from the canonical corrrespondence analysis with 9999 replications environmental variable abbreviation f p organic carbon orgcarb 0.914 0.682 ph ph 1.086 0.227 moisture moist 1.249 0.11 nitrogen nitro 1.057 0.347 phosphorus phosp 1.176 0.182 potassium potas 1.4 0.162 litter litter 1.33 0.046 canopy cover canop 1.033 0.36 disturbances distrb 1.209 0.052 our study showed that environmental variables, such as moisture, ph, canopy, organic carbon, nitrogen, phosphorus, potassium, and anthropogenic disturbances had significant effect on the distribution and composition of macrofungi. organic carbon, moisture, ph, canopy, litter, and macrofungal species richness were positively correlated and was comparable with the study of bhandari and jha (2017). this finding indicated that these environmental variables play a vital role in shaping macrofungi communities. however, other soil properties such as nitrogen, phosphorus, and potassium were negatively correlated with the species richness. there were no significant effects on the increasing disturbances with the abundance of macrofungi (fig. 4). -0.6 1.0 -0 .8 0 .8 gea_lexpod_des sch_une mar_eus ter_pus rus_ceastr_tussui_tus pod_ata col_nis ple_ensscl_epa xyl_lon xyl_phagan_tum spo_lor hex_ida lac_ata mar_cus mic_pus pyc_nus hyg_lus tri_ora myc_ptaama_iae lep_ria pol_ius ale_tia leu_onipsi_ata omp_era aga_sis hel_ata aur_ata len_aju lep_pes pan_vus mar_lus cya_tus mar_lislac_ndaear_osa ent_num cla_ata can_ius mac_des lep_ata hyg_icalae_eus rus_ria leu_tusdae_ina cre_lis aga_tus xyl_ila lyc_eumcop_tus gan_dum cop_ans oud_atalac_ina tra_ens cop_pus ter_zus dal_ica mar_ula rus_ans ent_tum rus_sps inf_bba cal_osa cre_lis psa_ata mar_ans pol_nus tra_lor con_ens mac_nii lep_nea pan_tus col_ila cli_mis cop_tus ple_ius mar_dus arm_ens ant_ina tra_ans ram_cta hex_uis fom_ius ter_nus hyg_eus sui_dus psa_mis psa_pes cli_sps pan_cus pos_ica cop_lis len_ina mic_pes ph orgcarbo moist nitrogen phosph potas canop litter distrb figure 4 cca biplot representing the effect of environmental variables on macrofungal species composition biotropia vol. 29 no. 3, 2022 278 macrofungi species, like pycnoporus cinnarius (pyc_ius), scleroderma cepa (scl_epa), agaricus arvensis (aga_sis), tricholomopsis decora (tri_ora), termitomyces microcarpus (ter_pus), aleuria aurantia (ale_tia) and lentinus sajorcaju (len_aju), etc. showed strong presence in the sal forest and were more resistant to disturbances. tropical deciduous riverine forest was dominated by the macrofungi species, like ramaria stricta (ram_cta), antrodia juniperina (ant_ina), fomes fomentarius (fom_ius), hexagonia tenuis (hex_uis), trametes elegans (tra_ans), clitocybe infundibuliformis (cli_mis), lenzites betulina (len-ina), panus fasciatus (pan_tus), coltricia perennis (col_nis) and microporus xanthopus (mic_pus), etc. which favored more open canopy. similarly, tropical evergreen forest harbored macrofungi species, like macrolepiota rickenii (mac_nii), marasmius haematocephalus (mar_lus), daldinia concentrica (dal_ica), marasmius androsaceus (mar_eus), lepiota clypeolaria (lep_ria), macrolepiota rhacodes (mac_des), lacrymaria lacrymabunda (lac_nda), lepiota clypeolaria (lep_ria), lycoperdon subcretaceum (lyc_eum), cyathus striatus (cya_tus) microporus xanthopus (mic_pus) and podoscypha multizonata (pod_ata). our present study also showed that the species found in the tropical deciduous riverine forest and tropical evergreen forests were more similar than those in the sal forest. however, some macrofungi species, like schizophyllum commune (sch_une), polyporus arcularius (pol_ius), termitomyces tylerianus (ter_nus), microporus xanthopus (mic_pus), and geastrum triplex (gea_lex) were found in all three forest types. the cca biplot showed environmental variables and aboveground vegetation were the major components for determining macrofungi composition (fig. 4). the overlapping of macrofungi species was due to a similar ecological niche. as we found in our present study, most of the soil fungi such as geastrum triplex (gea_lex), podocypha petaloides (pod_des), suillus granulatus (sui_tus), and russula species occurred toward the moisture. our study also showed that the presence of thin canopy cover and low soil moisture seemed to enhance the growth of wood-inhabiting fungi, such as spongipollis unicolar (spo_lar), crepodotus mollis (cre_lis), xylaria sp., trametes elegans (tra_ans), antrodia juniperina (ant_ina), fomes fomentarius (fom_ius), hexagonia tenuis (hex_uis), micrporus vernicipes (mic_pes) and lenzites betulina (len_ina), which were mostly presented opposite direction to the moisture and ph. organic carbon was also one of the major components which control the distribution pattern of soil fungi. macrofungi species, like termitomyces sp., rusulla sp., macrolepiota rhacodes (mac_des), omphalina umbellifera (omp_era), and marasmiellus ramealis (mar_lis) were found predominantly toward the direction of organic carbon. in our present study, disturbances seemed to have a poor impact on macrofungi species composition. most of the fleshy, soft, and gilled macrofungi, like clitocybe sp., psathyrella obtusata (psa_ata), agaricus augustus (aga_tus), lepiota clypeolaria (lep_ria) and coprinus disseminates (cop_tes) were favored by the higher soil ph. species richness of macrofungi increased with the increasing soil organic carbon, moisture, ph, litter coverage, and canopy coverage. soil ph and organic carbon ranged from 4.06 to 7.07 and 0.79 to 5.67, respectively. similarly, soil moisture, litter cover, and canopy cover ranged from 7.5 to 45.46%, 11 to 49%, and 15 to 95%, respectively. among all environmental variables, organic carbon, soil moisture, soil ph, litter cover and canopy cover had the most significant positive relationship with macrofungi species richness (fig. 5). also, the species richness of macrofungi showed a weak positive relationship with disturbances. soil nitrogen, phosphorus, and potassium were negatively correlated with macrofungi species richness but the result was statistically insignificant (table 4). species diversity of macrofungi depends on their particular habitat. geographic location, elevation, temperature, the humidity of air and soil, light, surrounding flora, and anthropogenic activity greatly influence the growth and reproduction of macrofungi (zervakis & venturella 2007; topwal et al. 2013). soil moisture is one of the most important environmental factors responsible for affecting the growth of the macrofungi (kropp & albee 2002). present findings also showed increasing species richness is affected by the increasing moisture content of the soil. this finding was similar to the research of bhandari and jha (2017). the fungal diversity studies in greece and sicily (venturella & zervakis 2000; zervakis & venturella 2002) confirmed that fungi require a certain level of moisture; rainfalls, macrofungal diversity in different vegetation composition in kailali, west nepal – joshi et al. 279 figure 5 correlation between macrofungi species richness and organic carbon, moisture, soil ph, canopy cover, and litter cover notes: each point in each figure represents a sampling plot; total number of sample plots = 60. less number of points in the figure may be due to the overlapping of the data among the plots. the fitted line is based on the linear regression model for sampling plots. table 4 pearson correlations between environmental variables and species richness of macrofungi soil ph organic carbon moisture n p k canopy cover litter cover disturbances species richness 0.506** 0.519** 0.580** -0.074 -0.183 -0.14 0.513** 0.612** 0.034 notes: * = correlation is significant at p < 0.05 level; ** = correlation is significant at p < 0.01 level. air humidity, and soil moisture, which are all significant factors. canopy cover and litter cover had also provided positive influence on macrofungal species richness in the studied area and a similar result was found by baral et al. (2015). the result unveiled that canopy cover plays a vital role in increasing macrofungal diversity in forests which was also reinforced by other previous findings (dighton et al. 1986; bonet et al. 2004; sysouphanthong et al. 2010; santos-silva et al. 2011). the reason is likely to be the presence of more substrate on the forest floor and high humidity which favor the growth of more fungal species (lodge et al. 2004). litter is an important component of every ecosystem and it constitutes the major source of organic matter in the soil. the removal of litter directly affects the diversity and growth of macrofungi (eaton et al. 2004; sayer 2006). when the forest floor is covered with layers of well-decomposed biotropia vol. 29 no. 3, 2022 280 leaves, saprotrophic fungi are favored by this organic resource which maintains the temperature and moisture of the surrounding area (fernandez-toiran et al. 2006). also, the abundance of macrofungal species is closely correlated with soil organic matter and other soil parameters (zamora-martinez & de pascual-pola 1995; engola et al. 2007). the growth of saprophytic fungi was enhanced at ph 7 or 8; while the ectomycorrhizal species showed the peak growth at ph 5 or 6 (yamanaka 2003). soil ph was also a major abiotic component responsible for changing macrofungi communities and was found to be positively correlated with macrofungi species richness. the ph range of 5 to 6 favors the growth of soil fungi (bhandari & jha 2017; pavithra et al. 2016; zhang et al. 2016). in this study, it was observed that there were no direct relationships between macrofungi richness and other soil parameters, such as nitrogen, phosphorus, and potassium with macrofungi diversity. however, the importance of forest soil chemistry parameters in fungal species distributions has also been reported by hansen (1988) and ruhling & tyler (1990). conclusion the study area is rich in macrofungal diversity with species’ richest families being the polyporaceae followed by tricholomataceae, marasmiaceae, agaricaceae, and coprinaceae. the presence of diverse kinds of vascular plants and different environmental conditions in different forest types have created unique habitat for the growth and development of a wide variety of macrofungi species. species diversity is higher in moist and dense canopy forests such as tropical evergreen forests and sal forest compared to that in the open and dry tropical deciduous riverine forests. soil moisture, organic carbon, soil ph, litter cover, and tree canopy cover are the most important variables affecting macrofungal diversity. acknowledgments the authors are grateful to the forest users groups of teghari community forest and mr. yagya raj bhatta for their support and cooperation in field. we are also thankful to the national herbarium and plant laboratories (kath) for the specimen identification which had been crucial for this study. finally, we thank anonymous reviewers and editors for their valuable 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towards perennial woody ecosystems. but a continuous shortage of land stands in the way of achieving the goal. we must therefore aim to explore viable alternatives. the banana as a potential carbon sequester attracted little attention from researchers. therefore, this study aimed at estimating the potential of bananas in different states of india as potential carbon sequesters. data was collected from twelve major banana producers between january 2021 and december 2022. one hundred banana gardens were sampled in each of the 12 banana-producing states, covering the major bananas grown (aaa, aab, and abb). the above-ground (agb) and below-ground (bgb) biomass were calculated using the allometric equation with pseudostem volume as the allometric parameter. the dry weight obtained from the allometric equations was then converted to carbon using a dry weight to carbon conversion factor. sequestered carbon varied with the aaa, aab, and abb of bananas. banana plant carbon stock was also found to be very small, ranging from 2.573 to 6.407 t/ha, compared with very high soil carbon ranging from 39.55 to 77.14t. in all the bananacultivating states, the proportion of carbon contained in the plant to that in the soil was only 8.286 percent, and that of soil carbon accounted for 91.714%. at the national level, the banana crop sequestered 48.627 million metric tonnes of carbon, with soil carbon accounting for 44.798 metric tonnes and plant carbon accounting for only 3.828 metric tonnes per year. despite these small amounts of plant carbon, the banana cropping system enriches the soil by enabling much more carbon to be sequestered into the soil in amounts comparable to other perennial plantations. keywords: aaa, aab, abb, banana growing states, banana stock estimation, carbon stock, cultivars, plant carbon, soc. introduction the giant herb bananas of the musaceae family and musa genus are widespread throughout the tropical and subtropical zones (charrier et al. 1997). it has occupied the 6th position in the world's fruit production and is a vital part of human diets in many regions. about 130 countries grow bananas, which they produce (reay 2019). more than a thousand cultivars’ and bred varieties with an array of sizes, colours, and shapes are under cultivation. these include yellow-skinned, red-skinned, and sweet table varieties, as well as starchy plantains used for cooking. they are nutritionally rich in minerals such as potassium and zinc, as well as essential vitamins a and c (ravi 2013). the genome assemblies that constitute the entire spectrum of edible bananas and plantains, such as aaa, aab, or abb, are designated as "groups, while the total set of a basic cultivar and its derived clones form a "subgroup" (langhe et al. 2000). aaa and aab group generally have stem diametres of 20–25 cm, grow to a height of 3–4 metres with broad leaves that range from 2 to 3 metres in length, and produce a high yield of medium-sized fruit. the abb group is smaller, with a stem diameter of 1520 cm and a leaf size of 2–3 meters in length, and produces high yields of larger fruit. banana is cultivated commercially under tropical and subtropical conditions all over the world, both on a commercial scale and by smallholders for home consumption or sale at local markets. the asia-pacific region produces *corresponding author, email: annual carbon capture potential in banana garden of india – ganeshamurthy 375 55.4% of global production, and india ranks first in production, accounting for nearly 26.3%. central and south america also contribute significantly to the total world banana basket (fao 2018, statista 2022). about a thousand varieties of bananas are produced, but preferences for varieties are generally local. the bananas of the aaa group, mainly the cavendish type, are the most cultivated and produce about 50 billion metric tonnes, accounting for roughly 47 percent of global production (fao 2022). (nkoulou et al., 2023). the volume of production and trade of all other local bananas (aab and abb groups) is greater than that of cavendish bananas. india, with its wide variability of soil and climate, produces a large number of fruits. among them, eight fruit crops occupy more than 80% of the fruit crop area, and bananas are the third most widely grown after mango and citrus, occupying 20% of the total fruit area and producing one-third (3184,000 metric tonnes) of the total fruits produced in india. when huge quantities of bananas are produced to meet the livelihoods of millions of people around the world, it has naturally spurred climate-smart actions that include multidimensional and interconnected challenges to minimise ghg emissions and ensure sustainability (unga, 2015). this is because banana cultivation in the past concentrated on the need to increase food production and ignored the need to protect the environment, water, and biodiversity, thereby contributing to considerable environmental degradation and the loss of crop diversity. realising this agricultural diversity and diversity-based farming have been advocated as the major backbones of sustainable agricultural intensification and a sustainable food system (tutwiler et al. 2017). in terms of ghg emissions, a kilogramme of bananas results in the emission of 0.5–1.3 kilogrammes of greenhouse gas, or 100 to 200 grammes per banana (reay 2019). parts of the banana’s life cycle can be targeted directly to mitigate climate change (wrap 2012). the banana crop leaves a large amount of residue. it is estimated that about four metric tonnes of pseudostem remain at the harvest location for each tonne of fruit harvested (souza et al. 2010). some estimates show that from the total of harvested bananas, 1.5 t of leaves and 2.5 t of pseudostem are generated per tonne of banana produced (oliveiraa 2013). these residues contribute greatly to carbon sequestration, alcohol production, and natural fibre production. making use of this biomass for the extraction of fibre and nutrients (particularly potassium), the production of alcohol for fuel, etc., could be a very attractive alternative by not only contributing to the preservation of the environment through carbon sequestration and removing this waste from the land but also by adding value to the fruit production matrix and transforming the residue into a commodity (soraishram et al. 2021; souza et al., 2010). banana can be considered a perennial crop; thus, there is relatively permanent standing biomass throughout the year with considerable carbon content in its structure and hence a potential carbon sequester. given the perennial and morphological nature of a crop like banana, it is worthwhile exploring its contribution to the carbon cycle. we therefore estimated the total annual banana residues produced and tried to work out their carbon sequestration potential in india. materials and method selection of gardens and sampling banana is grown in many states of india, and the area (880 thousand ha) varies extensively, with large localised pockets (fig. 1) located in different agroecological regions (table 1). one hundred banana gardens were sampled in each of the top 12 banana-producing states, covering the major bananas grown (aaa, aab, and abb) in these respective states, as presented in table 1. a banana plant produces fruits only once during its lifespan. but new stalks are continuously produced from each plant. the fruit is harvested around 9 to 11 months after planting; then, the new stalks produce fruits every 3 to 4 months (elbehri et al. 2015). the selected gardens are commercial-scale monocropping production systems with field histories of banana production spanning more than 20 years. we estimated the annual biomass production per plant and did not include the suckers that go to the next crop cycle. an extensive survey was conducted in these regions to record allometric data. in each state, biotropia vol. 30 no. 3, 2023 376 100 gardens at the harvest stage were randomly sampled to obtain a fairly representative sample of the gardens in these states. given the variability in plant population and yield per hectare across different cultivated varieties, the determination of state-specific plant population per hectare for banana cultivation was guided by productivity considerations. specifically, states exhibiting productivity levels exceeding 30 tonnes (primarily falling within the aaa group) were assigned a plant population of 4440 plants per hectare, while states with productivity below 30 tonnes per hectare (mainly within the aab and abb groups) were allocated a plant population of 2267 plants per hectare. flowers, flower bracts, and flower stock constitute about this value was used to compute the carbon contribution from this component. weed biomass in banana gardens is very low, so this parameter was not included in this study. fig 1 district boundaries of major banana growing areas in india (map not to scale) table 1 banana growing states and districts with varieties, area, production and productivity. state districts varieties area, 000 ha (% share) production 000 mt (% share) productivity t/ha andhra pradesh &telangana east godavari, west godavari, kurnool, cuddapah dwarf cavendish, robusta, rasthali, amritpant, thellachakrakeli, karpoora poovan, chakrakeli, monthan and yenagu bontha 91.25 (10.33%) 5093.10 (16.53%) 56.24 assam goalpara, nagaon, sonitpur, foothills of garo hills jahaji (dwarf cavendish), chini champa, malbhog, borjahaji (robusta), honda, manjahaji, chinia (manohar), kanchkol, bhimkol, jatikol, digjowa, kulpait, bharat moni 53.08 (6.00%) 913.27 (2.96%) 17.20 bihar vaishali, katihar, kishanganj, bhagalpur (naugachia) and purnia. dwarf cavendish, alpon, chinia , chini champa, malbhig, muthia, kothia , gauria 31.07 (3.51%) 1396.39 (4.53%) 44.94 gujarat surat, vadodara, anand, kheda, junagadh, narmada, bharuch dwarf cavendish, lacatan, harichal (lokhandi), gandevi selection, basrai, robusta, g-9, harichal, shrimati 68.15 (7.71%) 4472.32 (14.51%) 65.63 jharkhand ranchi, sahebganj basrai, singapuri 9.17 (1.03%) 32.06 (0.10%) 3.49 karnataka bangalore, chitradurga, shioroga, hassan, chikka mangloor dwarf cavendish, robusta, rasthali, poovan, monthan, elakkibale 110.55 (12.50%) 2328.90 (7.56%) 21.07 annual carbon capture potential in banana garden of india – ganeshamurthy 377 table 1 (continued) kerala thiruvananthapuram, kollam, pathanamthitta, alappuzha, kottayam, idukki, ernakulam, thrissur, palakkad, malappuram, kozhikode, wynadu, kannur, kasargod nendran (plantain), palayankodan (poovan), rasthali, monthan, red banana, robusta 109.26 (12.36%) 1119.16 (3.63%) 10.24 madhya pradesh khandwa, badwani, khargaon, dhar basrai, grand naine, robusta, 26.38 (2.98%) 1834.03 (5.95%) 69.54 maharashtra jalgaon, ahmednagar, buldhana, pune, wardha, dhule, nanded, parbani, nandurbar, satara, sangli, osmanabad, buldhana, akola, yeothmal, amravati, thane, kulara, alibag dwarf cavendish, basrai, robusta, lal velchi, safed velchi, rajeli nendran, grand naine, shreemanti, red banana 80.88 (9.15%) 4209.27 (13.66%) 52.05 orissa ganjam, puri, khurda, gajpati, cuttack, dhenkanal, angul, sundargarh, sambalpur, bargarh, deogarh, koraput, keonjhar, raygada, mayurbhanj dwarf cavendish, robusta, champa, patkapura (rasthali) 24.20 (2.74%) 449.82 (1.46%) 18.59 tamil nadu thoothukudi, tiruchirapalli, coimbatore, tirunelveli, karur, erode, kanniyakumari virupakshi, robusta, rad banana, poovan, rasthali, nendran, monthan, karpuravalli, sakkai, peyan, matti 82.63 (9.35%) 3205.04 (10.40%) 38.79 west bengal hooghly, nadia, north 24 parganas champa, mortman , dwarf cavendish, giant governor, kanthali, singapuri 49.30 (5.57%) 1200.00 (3.89%) 24.34 other states 150.14 (28.77%) 4464.12 (14.49) 29.73 all india 883.77ha 30807.5 34.86 source: dac & fw, 2018. ganeshamurthy (2023) developed a universal banana allometric equation for nondestructive estimation of the above-ground biomass (agb) of a standing banana crop at the harvest stage. this involved measuring the volume of the pseudostem as the allometric parameter. briefly, the allometric equation was developed through destructive sampling of aaa, aab, and abb groups at the harvest stage. allometric parameters such as plant height, diameter of the stem at the base, diameter of the stem at breast height, and volume of the pseudostem were measured. different statistical models, like linear, exponential, polynomial, and power models, were used to estimate the plant biomass of individual and combined groups of bananas. based on the best fit, the power model with pseudostem volume as an allometric parameter was used for the estimation of plant biomass (ganeshamurthy, 2023). the below-ground biomass was estimated using the shoot-to-root ratio of 0.235 suggested by ganeshamurthy (2023). we used the allometric equation given below for the estimation of banana biomass for computing the cs. aaa group: y=0.008x0.513 aab group: y=4e-06x1.233 abb group: y=4e-06x1.22 measurement of pseudostem volume the diameter of the stem at the base above the ground and below the bunch was measured with the help of a vernier caliper. the pseudostem volume was calculated by multiplying the stem diameter at the base by the diameter below the bunch, as follows: v = 1/3 × πh(r2 + rr + r2) where: r = radius at the stem's base biotropia vol. 30 no. 3, 2023 378 r = radius beneath the bunch such that r > r h = height from the base of the plant to bunch level expressed in cubic centimeters carbon estimation the carbon content of these plant samples was estimated by using a chns analyzer (elementar) and expressed as a percentage of carbon in the sample. soil carbon stocks getting representative data on the soil carbon stock of a region is a difficult task. arriving at a state-wise average sc stock was practically impossible because the data is not available for a political boundary-based average sc as no such effort has been made in india (ganeshamurthy et al. 2019). further, the available resources were generated from agricultural lands, mainly cultivating annual crops, and hence do not represent a perennial crop system. the forest survey of india has made efforts to generate state-wise soil carbon stocks of forests, and the latest data was published in 2017. by and large, banana gardens imitate a disturbed forest ecosystem. therefore, we used this data to calculate the state average values for sc. the method adopted by fsi is similar to those normally adopted to estimate soil carbon stocks. briefly, the method followed by fsi for collecting the data on sc is given here. results and discussion the volume of the banana pseudostem, the basic growth parameter used for calculating the agb, varied from state to state (table 2). this depended mainly on the type of bananas cultivated in these states. the data obtained from assam, kerala, orissa, tamil nadu, and west bengal was above. in andhra pradesh, karnataka, and telangana states, the recorded pseudostem volume ranged between 50000 and 60000 cm3. the lowest volumes below 50000 cm3 were recorded in other states. this variation in the data is mainly accounted for by the type of bananas grown in these states and the climate and management practices adopted by farmers in these regions. among the states, bananas in gujarat recorded the lowest mean pseudostem volume (46576 cm3), and assam recorded the highest mean value (62731 cm3). stevens et al. (2020) reported similar pseudostem volumes for banana plants producing about 2–3 kg of above-ground biomass for two contrasting cultivars. table 2 mean allometric parameters (vpseudostem, cm3) and tree carbon sequestered in banana gardens in india states/ut range mean sd median q2 q3 andhra pradesh & telangana 45872-63324 54490 5227 54236 52117 56428 assam 53450-73419 62731 5714 63726 59442 66885 bihar 32089-63127 45763 1204 45341 41287 47220 gujarat 31680-62885 46576 1218 46550 42824 49729 jharkhand 31821-62231 44563 1320 48035 45176 51022 karnataka 45889-63267 54602 5281 54493 50668 57260 kerala 53244-73427 62636 5216 63288 59117 67335 madhya pradesh 31380-62859 47706 3165 47360 46819 49715 maharashtra 31416-62865 47631 3052 47256 46358 49021 orissa 53425-73376 62822 5798 63284 59776 68221 tamil nadu 53351-73396 62831 5845 63349 61264 65108 west bengal 53452-73417 62960 5802 62826 59871 64332 other states 45927-63316 54721 5108 54774 52285 57064 all india 31380-73427 54618 4150 52268 41220 68376 mean of 100 plants annual carbon capture potential in banana garden of india – ganeshamurthy 379 danarto and hapsari (2015) reported that cultivars of banana with "b" genomes (aab and abb cultivars) are more vigorous and contribute higher biomass and c-stock than the cultivars having only "a" genomes (aaa group). our results supported this argument. states like maharashtra, andhra pradesh, telangana, and madhya pradesh, which grow mainly cavendish bananas (aaa), recorded lower agb (2.5 kg/plant) and bgb (0.550 kg/plant). whereas other states cultivating varieties belonging to the aab and abb groups recorded higher agb (>2.5 kg/plant) and bgb (>0.550 kg/plant). nyombi et al. (2009) reported a plant dry weight of 1.44 kg/plant for the pseudostem alone for a similar variety of banana in east africa. ortizulloa et al. (2020) also reported similar values for bananas grown in two different regions. stevens et al. (2020) reported similar agb for two contrasting cultivars. our values are comparable to those reported in the literature. the mean carbon content in the pseudostem was 0.465%, and that of the root and corm was 0.471%. the carbon capture in the agb and bgb was then computed using these values (table 3). the agb carbon captured by bananas varied from the lowest at 0.920 kg/plant in jharkhand to the highest at 1.395 kg/plant, and the bgb carbon varied from the lowest at 0.218 kg/plant in jharkhand to the highest at 0.332 kg/plant in west bengal. it is reported that on a per-plant basis, the abb group sequestered higher carbon, followed by the aab group, and the least in the aaa group (nyombi et al. 2009; ortiz-ulloa et al. 2020). andhra pradesh, gujarat, jharkhand, madhya pradesh, maharashtra, and telangana mainly cultivate cavendish-type (aaa group) bananas. hence, the carbon capture per plant in these states is lower. in assam, karnataka, kerala, orissa, tamil nadu, and west bengal, we mainly cultivate both aab and abb group bananas, capturing the least carbon per plant. total carbon capture by bananas per ha depends on the plant population per ha and carbon capture per plant. in carbon capture, two distinct groups can be seen in different states. the differences among states are mainly accounted for by the types of bananas cultivated in these states. the data on total carbon captured per ha (table 4) obtained from andhra pradesh, bihar, gujarat, madhya pradesh, and maharashtra (where mainly aaa group bananas are cultivated) were above 5 t/ha. in assam, jharkhand, karnataka, kerala, orissa, tamil nadu, and west bengal states (where mainly the aab and abb group bananas are cultivated), the recorded carbon capture was below 5 t/ha. this shows that, on a per-ha basis, the aaa group bananas capture more carbon than the aab and abb group bananas. it means that all those states cultivating aaa group bananas sequester about 1.3 to 1.5 fold more carbon per hectare than other states growing aab and abb group bananas. ortiz-ulloa et al. (2020) reported that cs by banana in four provinces in ecuador ranged from 4.18 t/ha to 5.44 t/ha. they accounted for this variability in cs in the plant population. in our study, too, the differences in cs in different states growing different groups of bananas were table 3 biomass and carbon sequestered in banana gardens in india(mean of 100 plants) states/ut agb, kg/plant major banana group bgb kg/plant agb c, kg/ plant bgb c, kg/ plant total c, kg / plant total c, t/ha andhra pradesh & telangana 2.507 aaa 0.589 1.166 0.277 1.443 6.407 assam 2.994 aab&abb 0.704 1.392 0.331 1.724 3.908 bihar 2.025 aaa 0.476 0.942 0.224 1.166 5.177 gujarat 2.061 aaa 0.484 0.958 0.228 1.186 5.265 jharkhand 1.972 aab&abb 0.463 0.920 0.218 1.135 2.573 karnataka 2.512 aab&abb 0.590 1.168 0.278 1.446 3.278 kerala 2.989 aab&abb 0.702 1.390 0.331 1.721 3.901 madhya pradesh 2.110 aaa 0.496 0.981 0.235 1.215 5.394 maharashtra 2.108 aaa 0.495 0.980 0.233 1.214 5.390 orissa 2.998 aab&abb 0.705 1.394 0.332 1.726 3.912 tamil nadu 2.998 aaa 0.705 1.394 0.332 1.726 3.912 west bengal 3.000 aab&abb 0.705 1.395 0.332 1.727 3.915 other states 2.517 aab&abb 0.591 1.170 0.279 1.449 3.284 all india 2.522 0.593 1.173 0.279 1.452 4.332 biotropia vol. 30 no. 3, 2023 380 mainly due to differences in the plant population per ha. aaa bananas are planted closely, accommodating 4444 plants per ha. whereas the aab and abb group bananas are planted with wider spacing, accommodating 2267 plants per ha. edible banana cultivars have lower biomass and c-stock values than wild bananas. danarto and hapsari (2015) reported a cs of 9.7 kg/plant in klutuk wulung, an aaa variety, and 4.74 kg/plant in another variety, klutuk ijo, that belongs to the abb group. these data support the finding that aaa produces lower cs than abb. however, their values are higher because these varieties are from backyard gardens where solitary plants have grown very robustly. ours are commercial gardens with high-density planting. hence, our values reported here are lower than those reported by danarto and hapsari (2015). our values fairly match those reported by ortizulloa et al. (2020) and stevens et al. (2020) from commercial gardens. soil carbon organic c in the soil fluctuates based on the canopy cover over the surface. quasi-equilibrium (qev) of soc is attained over a long period of time, varying from 500 to 1000 years in a forest system. in the agriculture system, this is attained in about 30–50 years after land use change from forest to agriculture and in 30 years for the horticultural system (arrouays et al. 1995; batjes 2001; dickson and crocker 1953; jenny 1950; johnson 1995; naitam and bhattachaarya 2004). during the process of stabilisation, the soc shows tooth-like cycles of accumulation and loss during the beginning years and slowly attains an equilibrium level after the accumulation of dry matter and loss of soc over time. it has been shown that under these tropical land uses, horticultural systems attain qev in about 25 years (ganeshamurthy 2012). all the selected banana gardens in this study had a history of banana cultivation spanning more than 20 years. therefore, the soils under these gardens might have attained the qev stage after the accumulation of dry matter and loss of soc over time. as mentioned, it was difficult to obtain representative state averages of soil c stocks under banana gardens. published information is mainly restricted to agricultural ecosystems and very few to horticultural ecosystems. since statewise soc stock information was available from forest ecosystems and since sites under continuous banana gardens represented forest ecosystems more closely, we used the available data for computing c stocks by banana gardens. the status of socs in karnataka and kerala is relatively higher because of the favourable climate. whereas the plant carbon content in andhra pradesh and telangana is higher because of the bright sunshine hours prevailing in these states relative to other banana-growing states. table 4 soil and plant carbon pools in banana gardens in india states/ut soil c t/ha plant carbon, t/ha area (000 ha) total soil carbon stock 1000 tons total plant carbon stock 1000 tons total cs in banana gardens,mt andhra pradesh & telangana 42.09 6.407 91.25 3840.713 584.639 4.425 assam 39.98 3.908 53.08 2122.138 207.437 2.330 bihar 39.55 5.177 31.07 1228.819 160.849 1.390 gujarat 44.04 5.265 68.15 3001.326 358.810 3.360 jharkhand 43.29 2.573 9.17 396.969 23.594 0.421 karnataka 77.14 3.278 110.55 8527.827 362.383 8.890 kerala 75.77 3.901 109.26 8278.630 426.223 8.705 madhya pradesh 41.17 5.394 26.38 1086.065 142.294 1.228 maharashtra 57.23 5.390 80.88 4628.762 435.943 5.065 orissa 46.50 3.912 24.2 1125.300 94.670 1.220 tamil nadu 41.64 3.912 82.63 3440.713 323.249 3.764 west bengal 59.88 3.915 49.3 2952.084 193.010 3.145 other states 50.69 3.284 150.14 7610.597 493.060 8.104 all india 50.69 4.332 883.77 44798.301 3828.492 48.627 % contribution to the total cs 91.714 8.286 100.000 annual carbon capture potential in banana garden of india – ganeshamurthy 381 total c sequestered in orchards the soil and plant carbon pools in banana gardens are presented in table 5. the soil carbon pool is very large compared to the plant carbon pool. it contributed more than 86% of the total cs in banana gardens. the mean contribution of soc varied from 86.8% in andhra pradesh to 95.9% in jharkhand. at the national level, the soc contributed 91.71% to the total cs. the plant carbon pool, on the other hand, is very small compared to the soil carbon pool. the plant carbon pool contributed less than 15% in all the states. the lowest contribution from plant c to the total cs was recorded in karnataka (4.08%) and kerala (4.90%), and the highest contribution was recorded in andhra pradesh (13.21%) and madhya pradesh (11.58%). at the national level, plant c contributed 8.29% to the total cs. the carbon content of banana plants accounted for only 8.286 percent of the total cs in banana gardens. the contribution of each state to the total carbon sequestered by banana plants alone depended upon the area and varieties of bananas under cultivation in these states and the quantity of soc. andhra pradesh with the highest area under banana (91.25 thousand ha) cultivating mainly the aaa group bananas sequestered 584639 metric tonnes of carbon, whereas jharkhand with the lowest area under banana (9.17 thousand ha) cultivating mainly the aab and abb group bananas sequestered the lowest plant carbon (23594 metric tonnes). on an all-india basis, the total plant carbon sequestered accounted for 3828492 metric tonnes. the study clearly indicated that under banana gardens, sc is the major c pool, accounting for 91.7%, and plant carbon is only a small portion, accounting for 8.29% of the total cs. kamusingize et al. (2017) reported that the soc contribution to total cs under bananas exceeded 90% in ugandan banana gardens. in their study, the total soc stock beneath all cultivars was considerably high, ranging from 81 to 92 mg ha1. the plant carbon in these studies was found to be very small, ranging between 0.37 and 1.64 mg ha1. danarto and hapsari (2015) reported that, on average, various indonesian bananas captured around 2.26 kg (or 0.98 tonne) per hectare. thus, banana crops enrich soil by investing carbon into the soil through huge root biomass and over time during photosynthesis as carbon moves from the vegetative canopy into the soil (turner, 2003; hairiah et al., 2010). published information on soc in banana gardens in india is not available for a fair comparison. therefore, it can only be made from the data published on other fruit crop systems such as mango, sapota, guava, and forest systems. gupta (2011) reported that the soil c stock was 41 tonne ha1 at the surface and 50 cm deep. chabra et al. (2003) also reported that the soil c sequestered in indian forest soils ranged from 37.5 tonne ha1 in tropical dry deciduous forests to 92.1 tonne ha1 in littoral swamp forests. our values are for 100-cm-depth soil profiles and are fairly similar to those reported in the literature for different regions. the banana cropping system enables much more carbon to be stored in the soil, despite the fact that banana cultivars contain small average amounts of plant carbon stocks. kamusingize et al. (2017) and danarto and hapsari (2015), in their study, reported that the proportion of carbon contained in the plant to that in the soil across all cultivars was in the range of 0.4–2%. large soil carbon stocks in banana cropping systems could perhaps be attributed to the sustainable agricultural land management practices employed by farmers, such as mulching, the use of trenches to minimise erosion, and minimal and no tillage (lal 2011, paswel et al. 2012, joris et al. 2013). investing in the proper management of banana plantations is invaluable for contributing to soc as a major carbon pool in banana systems. the crop demography in india is changing fast towards perennial horticulture. this results in land-use change in the region. therefore, we must examine the related changes in c fluxes derived from such land-use change patterns in different regions to formulate viable strategies for climate change mitigation. the present study generated unique information on banana gardens in india. this involved a comprehensive effort in the integration of different methodologies for field work and data processing. but on a national scale, there is a need to generate information on all such perennial horticulture systems, such as other fruit crop orchards, coffee and tea estates, and plantations in india, with larger sample sizes to be able to determine the percentage of carbon sequestered in perennial horticultural crops in the country as a whole. biotropia vol. 30 no. 3, 2023 382 the land-use change patterns are occurring mainly on prime agricultural lands due to increased demand for bananas. bihar, jharkhand, odisha, madhya pradesh, and chhattisgarh have large tracts of tribal land. these regions are suitable for banana cultivation. such regions are to be encouraged for productive banana cultivation. similar efforts may be made in other states as well, which reasonably imitate forests and sequester carbon in similar quantities and can augment climate change risks. the information generated in this study may be used by the administrators in these regions to claim carbon credits to benefit the farmers and the local population. conclusions this study showed a significant difference in total plant carbon stock across different cultivars and states in india. there was a significant difference between groups of bananas in their ability to sequester carbon. soil carbon stocks were very high,ranging from 39.55 to 6.407 t/ha. but plant carbon stocks were found to be very small,ranging from 2.573 to 6.407 t/ha. in all banana-cultivating states, the proportion of carbon contained in the plant to that in the soil was only 8.286 percent. soil carbon accounted for the majority of the total cs, accounting for 91.714%. despite these small amounts of plant carbon, the banana cropping system enriches the soil by enabling much more carbon to be sequestered into the soil. apart from augmenting fruit production, expanding the area under banana farming systems helps to rehabilitate the landscape, decrease carbon emissions in the form of biomass and carbon stocks, and meet the economic needs of the region. references arrouays d, isabelle v, luckicin j. 1995. spatial analysis and modelingof top soil carbon storage in temperate forest humic loamy soil offrance. soil science. 159, 191-198. batjes n.h. 2001.options for increasing carbon sequestration in west african soils: an exploratory study with special focus on senegal. land degradation and development. 12, 131-142. charrier a, jacquot m, hamon s, nicolas d. 1997. l'amélioration des plantes tropicales. montpellier (fra) ; paris : cirad ; orstom, 624 p. 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development mechanism. mechanisms under the kyoto protocol, in https://unfccc.int/process-and-meetings/thekyoto­protoco1/mechanisms-under-the-kyoto-p rotocoi/th e-cieandevelopment-mechanism. wrap. 2013. household food and drink waste in the united kingdom 2012. waste and resource action programme. http://www.wrap.org.uk/sites/files/ wrap/ hhfdw-2012-main.pdf.pdf . https://www.researchgate.net/profile/iyyakutty-ravi?_sg%5b0%5d=9v7u43cveebgf5a6nacal0mjh63tykwnjdorvq0n2fsqr0ppzo3ydv-z0zy31va7wubmktw.6bvja6gqfvuzbvmxgefhxlikvhib6n18bvruq12albi8v-77f1gsiks3jprz1_2wqjo_ptyincalultkkehx_g&_sg%5b1%5d=lp2gcodsh4jzg514fqygblejikkas7r8dgnzrjcuadgp3tsw9z1mol_ebm-kextg0fqhcrs.q425kdj6ts9n5jyghc1_uzvahjjidp9eyimw_ei3j9uizu5lpyhc94mxgflznfwthnz3sw0q45tsyluc1986yg https://www.researchgate.net/journal/frontiers-in-physiology-1664-042x https://www.researchgate.net/journal/frontiers-in-physiology-1664-042x https://doi.org/10.1007/978-3-030-18206-9 https://doi.org/10.1007/978-3-030-18206-9 https://www.researchgate.net/deref/%20https%3a%2f%2fwww.%20tandfonline.com%2floi%2fwjnf20 https://www.researchgate.net/deref/%20https%3a%2f%2fwww.%20tandfonline.com%2floi%2fwjnf20 https://www.researchgate.net/deref/%20https%3a%2f%2fwww.%20tandfonline.com%2floi%2fwjnf20 https://www.statista.com/statistics/1038905/indiahttps://www.statista.com/statistics/1038905/indiahttps://unfccc.int/process-and-meetings/the-kyoto­%20protoco1/ https://unfccc.int/process-and-meetings/the-kyoto­%20protoco1/ the southeast asian journal of tropical biology vol. 31 no. 3, 2024: 309 315 doi: 10.11598/btb.2024.31.3.2187 issn: 0215-6334 | e-issn: 1907-770x 309 tree species diversity in phra that si mueang pong area, chiang mai province, thailand pongpan leelahakriengkrai1,2*, akharasit bunsongthae1, and audcharapat insing3 1department of biology, faculty of science and technology, chiang mai rajabhat university, chiang mai 50300, thailand. 2centre of excellence of biodiversity research and implementation for community, faculty of science and technology, chiang mai rajabhat university, chiang mai 50300, thailand. 3ban huai som school, san pa tong district, chiang mai 50120, thailand article higlights • rich tree diversity found in phra that si mueang pong area. • endangered species identified, promoting conservation efforts. • unique plant distribution influenced by land use and water proximity. • potential for ecotourism and environmental education in the region. • study provides foundational data for future ecological research. article information received 31 january 2024 revised 12 august 2024 accepted 14 august 2024 *corresponding author, e-mail: pongpan_lee@cmru.ac.th research article abstract this research conducted a survey of tree species in ten subplots along the pathway leading to phra that si mueang pong, identifying a total of thirty species representing twenty families, twenty-eight genera. dominant species included lagerstroemia duperreana var. duperreana, shorea siamensis, pterocarpus macrocarpus, tectona grandis, and vitex canescens. families with the highest species number were fabaceae, lamiaceae, combretaceae, and malvaceae. the study revealed the presence of both common and rare plant species in the area, with differences in species distribution between plots 1-5 and 6-10. plots 1-5, near the mae tha chang river, exhibited a higher number of species. the dominant species, based on the importance value index (ivi), were p. macrocarpus, l. duperreana, t. grandis, s. siamensis, and millettia brandisiana, respectively. two species, burma padauk (p. macrocarpus) and teak (tectona grandis), were classified as endangered in the areas. soil analyses indicated slightly acidic ph and relatively low water content, with significant differences between plots 1-5 and 6-10. the study suggested the potential for ecotourism and environmental learning centers in the chiang mai province cultural tourism destination, emphasizing the rich biodiversity and ecological value of the mixed deciduous and dry dipterocarp forest ecosystems. keywords: diversity index, dry dipterocarp forest, importance value index, mixed deciduous forest introduction exploration of biological diversity and utilization of plant resources in thailand has been an ongoing effort, encompassing both areas of base surveys and specific group-focused investigations (sudchaleaw et al. 2023; boonma et al. 2023; panyadee et al. 2023; ragsasilp et al. 2022; pansumrit et al. 2022; sutjaritjai et al. 2022; saensouk et al. 2021). this research unveiled that biological diversity of plant species, varying across different regions, consistently generates new knowledge and insights. while studies on plant diversity in ecotourism areas in thailand have provided limited information, most research has focused on national parks, such as doi inthanon national park, phu kradueng national park, khao yai national park, and kaeng krachan national park. chiang mai province, located in the northern region of thailand, has a tropical monsoon climate. the average temperature ranges from 22.3 oc to 34.4 oc, with annual rainfall of 972.1 mm (northern meteorological center 2020). with its mountainous terrain, there are many cultural tourist attractions situated on various mountains outside the boundaries of national parks. with a well-preserved environmental system, these factors make it feasible to promote sustainable ecotourism destinations in the future, facilitating easier access for visitors. https://doi.org/10.11598/btb.2024.31.2.2187 biotropia vol. 31 no. 3, 2024 310 phra that si mueang pong, also known as the white pagoda, stands as a prominent cultural and ecological tourist destination within the province of chiang mai. it is situated on the summit within the aranyawat temple in hang dong district, chiang mai, thailand. there are two routes to reach phra that si mueang pong, i.e., one involves climbing the stairs, while the other allows for driving up the road. the ascent via the staircase serves as an ecotourism route, winding through mixed deciduous forest and dry dipterocarp forest. during the climb to phra that si mueang pong, both sides of the staircase are influenced by significant factors contributing to plant diversity. one side is situated near the mae tha chang river, while the other side borders an agricultural area, resulting in distinct variations in plant diversity. therefore, this study investigated the diversity of tree species along the ascent to phra that si mueang pong in the ecotourism area, encompassing detailed analyses of biodiversity index and importance value index. the findings from this study were expected to benefit the local community, tourists, as well as educational institutions in the vicinity to utilize the area for studying ecotourism, focusing on the diversity of tree species. additionally, this research provided foundational information for future studies in various research areas. materials and methods study area the study area was located surrounding phra that si mueang pong in hang dong district, chiang mai, thailand. the geographical features comprised a mountainous terrain consisting of deciduous forest and dry dipterocarp forest, with elevations ranging from 350 to 450 meters above sea level (masl). data collection plant diversity data were collected from 2019 to 2020 within a permanent sample plot measuring 200 x 200 m2. the quadrat method, specifically employing 10 x 10 m2 subplots, was utilized for the systematic sampling of plant species. along the ascent to phra that si mueang pong, a total of ten subplots were established, with five plots positioned on the side adjacent to the agricultural area, while the remaining five plots were situated on the side bordering the mae tha chang river (fig. 1). in each subplot, the circumference of tree trunks with a diameter greater than or equal to 15 cm was measured at a height of 1.30 m. subsequently, the measured trees were tagged with number tapes, which also included information on the species, such as local name, common name, scientific name, and family of the perennial tree. data on tree species were gathered through consultation with experts and relevant literature (chayamarit & chamchumroon 2016). additionally, the status of trees was assessed based on the iucn red list of threatened species (iucn 2024). physical and chemical parameters of the soil were analyzed, including soil ph content and water content. soil ph was determined using the soilh2o system (fao 2021), while soil water content was calculated as a percentage based on oven-dried soil weight according to fao (2023) as follows: where: data analysis data obtained from the field survey were analyzed to determine the importance value index (ivi). this calculation involved assessing the relative density (rd), relative frequency (rf), and relative dominance (rdo), following the methodology outlined by ismail et al. (2017). in addition, species diversity index (h’) and evenness value (e) of tree species were determined and calculated following the shannon’s diversity index (odum 2004). the baseline statistics used were the mean±standard deviation. one-way analysis of variance (anova) was performed using the statistical package for the social sciences (spss) software program. to further analyze the results, duncan’s new multiple tests was performed at a significant level of 5%. w% = mcms mcds x 100 mcds mc w = soil water content expressed in units of percentage (%) mcms = mass of container and moist soil (g) mcds = mass of container and moist soil (g) mc = mass of container (g) tree species diversity in phra that si mueang pong area leelahakriengkrai et al. 311 figure 1 aerial photograph of phra that si mueang pong (a) and the map illustrates sample plots measuring 200 x 200 m2 and the 10 subplots with dimensions of 10 x 10 m2 located at phra that si mueang pong in hang dong district, chiang mai, thailand (b) results and discussion in the survey of tree species across all 10 subplots, a total of 157 trees were identified, representing 20 families, 28 genera and 30 species (table 1). the dominant species with their relative abundance percentages were lagerstroemia duperreana var.  duperreana  (10.2%), shorea siamensis (9.6%), pterocarpus macrocarpus (8.3%), tectona grandis (7.0%) and vitex canescens (6.4%), respectively (table 1). families with the highest number of species encountered were fabaceae (6 species), lamiaceae (3 species), combretaceae (3 species), malvaceae (2 species), respectively. comparing the findings of this study with those of previous research, the identified plants were commonly encountered in mixed deciduous forests and dry dipterocarp forests in thailand (chernkhunthod & hioki 2020; rundel et al. 2017). additionally, some plant species considered rare were identified in the area, such as sindora siamensis, vitex pinnata, terminalia corticosa, sterculia villosa, grewia eriocarpa, irvingia malayana, stereospermum neuranthum, schleichera oleosa, croton persimilis, aegle marmelos, protium serratum, antidesma ghaesembilla, morinda coreia and canango brandisiana. these were plant species with fewer than five individuals found within the entire study site. in the comparison of plant species between plots 1-5 and 6-10, it was found that there were a total of 18 species identified in both areas. among the tree species found exclusively in plots 6-10, there were 4 species, namely sindora siamensis, irvingia malayana, holarrhena pubescens, and canango brandisiana. on the other hand, a total of 8 tree species were found exclusively in plots 1-5, including stereospermum neuranthum, schleichera oleosa, vitex pinnata, sterculia villosa, croton persimilis, aegle marmelos, protium serratum, and antidesma ghaesembilla. from these results, it was evident that the presence of these plants was influenced by land use in the area. plots 1-5, situated on the side bordering the mae tha chang river, exhibited a higher number of species and less disturbed environment compared to the other side adjacent to agricultural land. however, when comparing the shannon’s diversity index and evenness values of plots 1-5 with those of plots 6-10, no statistically significant differences were found (p < 0.05) (fig. 2). biotropia vol. 31 no. 3, 2024 312 table 1 list of tree species recorded in phra that si mueang pong area and their biological index taxonomic categories common name local name biological index number rd rf rdo ivi family fabaceae dalbergia cultrata graham ex benth nt burma blackwood 5 3.18 3.30 0.93 7.41 millettia brandisiana kurz. 9 5.73 6.59 5.87 18.20 pterocarpus macrocarpus kurz.en burma padauk ประดู่ป่า 13 8.28 7.69 17.43 33.40 albizia odoratissima (l.f.) benth. lc black siris 6 3.82 2.20 4.86 10.88 xylia xylocarpa (roxb.) w. theob. var. kerrii (craib & hutch.) i.c. nielsen lc ironwood แดง 6 3.82 4.40 2.02 10.24 sindora siamensis teijsm. ex miq. var. siamensis lc มะค่าแต้ 1 0.64 1.10 0.35 2.08 family lamiaceae vitex pinnata l. lc malayan teak ตีนนก 2 1.27 1.10 0.68 3.05 vitex canescens kurz. lc chaste tree 10 6.37 7.69 3.84 17.90 tectona grandis l. f. en teak สัก 11 7.01 6.59 10.11 23.71 family combretaceae anogeissus acuminata (roxb. ex dc.) guill. & perr. ตะเคียนหนู 7 4.46 3.30 2.85 10.61 terminalia corticosa pierre ex laness ตะแบกเลือด 3 1.91 3.30 2.96 8.17 terminalia alata b. heyne ex roth รกฟ้า 7 4.46 5.49 5.87 15.82 family malvaceae sterculia villosa roxb. lc ปอตู๊บหูช้าง 1 0.64 1.10 0.56 2.29 hibiscus macrophyllus roxb. ex hornem lc largeleaf rosemallow ปอหู 6 3.82 4.40 2.38 10.59 family tiliaceae grewia eriocarpa juss. lc ปอแก่นเทา 3 1.91 3.30 3.08 8.29 family irvingiaceae irvingia malayana oliv. ex a. w. benn. lc wild almond กะบก 1 0.64 1.10 1.49 3.22 family bignoniaceae stereospermum neuranthum kurz. แคทราย 1 0.64 1.10 0.24 1.97 family bombacaceae bombax anceps pierre 5 3.18 3.30 2.17 8.65 family sapindaceae schleichera oleosa (lour.) merr . lc ceylon oak ตะคร้อ 2 1.27 2.20 0.27 3.74 family lythraceae lagerstroemia duperreana pierre ex gagnep. crepe myrtle ตะแบกเปลือกบาง 16 10.19 6.59 10.16 26.95 family euphorbiaceae croton persimilis mull. arg. เปล้าใหญ่ 1 0.64 1.10 0.15 1.89 family burseraceae canarium subulatum guillaumin lc 5 3.18 5.49 3.99 12.67 family rutaceae aegle marmelos (l.) correa ex roxb. nt bael มะตูม 1 0.64 1.10 0.67 2.40 family burseraceae protium serratum (wall. ex colebr.) engl. มะแฟน 2 1.27 2.20 0.87 4.34 family phyllanthaceae antidesma ghaesembilla gaertn. black currant tree เม่าไข่ปลา 1 0.64 1.10 0.60 2.34 family apocynaceae holarrhena pubescens wall. ex g.don โมกใหญ่ 5 3.18 2.20 0.84 6.22 tree species diversity in phra that si mueang pong area leelahakriengkrai et al. 313 taxonomic categories common name local name biological index number rd rf rdo ivi family meliaceae chukrasia tabularis a.juss. chittagong wood ยมหิน 7 4.46 3.30 3.26 11.02 family rubiaceae morinda coreia ham. indian mulberry 4 2.55 3.30 1.71 7.55 family dipterocarpaceae shorea siamensis miq. dark red meranti รัง 15 9.55 3.30 9.59 22.44 family annonaceae cananga brandisiana (pierre) i. m. turner สะแกแสง 1 0.64 1.10 0.22 1.96 notes: en = endangered; nt = near threatened; lc =least concern (iucn 2024); rd = relative density; rf = relative frequency; rdo = relative dominance; ivi = importance value index. the seven plant species with the highest importance value index (ivi) were pterocarpus macrocarpus, lagerstroemia duperreana, tectona grandis, shorea siamensis, millettia brandisiana, vitex canescens, and terminalia alata, respectively (table 1). these species are prominent and commonly found in mixed deciduous forests and dry dipterocarp forests. the results highlighted that the studied area maintained a rich and diverse ecological system. furthermore, in the comparison of plant species with the red list of threatened species, it was observed that pterocarpus macrocarpus and tectona grandis were classified as endangered. dalbergia cultrate and aegle marmelos fell under the near threatened status. those categorized as least concern tree species included albizia odoratissima, xylia xylocarpa, sindora siamensis, vitex pinnata, vitex canescens, sterculia villosa, hibiscus macrophyllus, grewia eriocarpa, irvingia malayana, schleichera oleosa, and canarium subulatum. analyses of soil physical and chemical properties in the studied area revealed that the soil water content was relatively low, ranging between 5.19% and 7.96%, which was considered normal for soil sampling during the dry season (hanpattanakit & chidthaisong 2012). however, upon comparing the water content between plots 1-5 and 6-10, it was evident that plots 1-5 had higher moisture levels than plots 6-10, and this difference was statistically significant (p < 0.05). the higher water content in plots 1-5 was likely influenced by their proximity to the river. the soil ph was slightly acidic, ranging between 5.09 and 6.53. this mild acidity was attributed to the presence of organic matter from decomposed branches and leaves in the forest. the decomposition process contributed organic compounds, such as humic acid and fulvic acid, resulting in a slightly acidic soil condition (makan 2022) (fig. 3). figure 2 shannon’s diversity index and evenness values for each plot (a); average values for plots 1-5 and 6-10 (b) biotropia vol. 31 no. 3, 2024 314 figure 3 water content and ph of the studied ten subplots conclusion this study revealed that all identified plant species belong to the ecosystems of mixed deciduous forest and dry dipterocarp forest. there was no evidence of invasive species encroaching from other ecosystems. soil water and ph levels were within the normal range for the mixed deciduous and dry dipterocarp forest ecosystems during the dry season. the dominant tree species, based on the importance value index (ivi), were pterocarpus macrocarpus, lagerstroemia duperreana, tectona grandis, shorea siamensis, and millettia brandisiana, respectively. on both sides of the staircase leading to phra that si mueang pong, a diverse biodiversity of plant species was discovered, exhibiting distinct variations. the study also identified fifteen species listed in the red list of threatened species (iucn), with two species classified as endangered. the phra that si mueang pong area holds potential for promoting ecotourism, including the establishment of an environmental learning center focusing on the mixed deciduous and dry dipterocarp forest ecosystems within the cultural tourism destination in chiang mai province. data gathered from this study can serve as a foundation for future research endeavors, particularly in exploring ethnobotany. acknowledgments the authors extend their appreciation to mr. tan monito, a former forest conservation officer and a member of the nature interpretation society, who played a vital role in classifying and surveying tree species for this project. they also thank mr. jirapun tankful for the aerial photograph of phra that si mueang pong. additionally, the authors express gratitude to aranyawat temple and chiang mai rajabhat university for their support and provision of facilities at the research site. references boonma t, saensouk s, saensouk p. 2023. diversity and traditional utilization of the zingiberaceae plants in nakhon nayok province, central thailand. diversity 15(8):904. doi: 10.3390/d15 080904 chayamarit k, chamchumroon v. 2016. plant identification handbook. department of national parks, wildlife and plant conservation. bangkok (th): sittichoke printing. chernkhunthod c, hioki y. 2020. floristic composition and forest structure in different fire frequency of mixed deciduous forest, doi suthep-pui national park, northern thailand. j jpn soc reveget tech 46(2):202-17. doi: 10.7211/jjsrt.46.202 fao [food and agriculture organization]. 2021. standard operating procedure for soil ph determination. rome (it): food and agriculture organization of the united nations. fao [food and agriculture organization]. 2023. standard operating procedure for soil moisture content by gravimetric method. rome (it): food and agriculture organization of the united nations. hanpattanakit p, chidthaisong a. 2012. litter production and decomposition in dry dipterocarp forest and their responses to climatic factors. gmsarn int j 6:169-74. iucn [international union for conservation of nature]. 2024. iucn red list of threatened species. available from: https://www.iucnredlist.org. 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biodiv/d240439 sutjaritjai n, panyadee p, phumthum m, inta a, balslev h. 2022. high diversity of medicinal uses of thai legumes (fabaceae) and their potential in public herbal medicine. diversity 14:588.doi:10.3390/d14080588 _goback biotropia vol. 29 no. 1, 2022: 39 46 doi: 10.11598/btb.2022.29.1.1615 39 mold diversity of wreathed hornbill (rhyticeros undulatus) nest in mount ungaran margareta rahayuningsih*, yanuar revandi and siti harnina bintari department of biology, faculty of mathematics and natural sciences, universitas negeri semarang, semarang 50229, indonesia received 10 july 2021 / accepted 17 october 2021 abstract wreathed hornbill (rhyticeros undulates) is known to build nests in three cavities where they managed to live and breed. this edifice is predicted to contain various molds needed to maintain micro-environmental steadiness. this study was aimed to identify molds diversity in the wreathed hornbill’s nest, using samples collected from empty structure with no bird activity. the samples were obtained from the kalisidi and nglimut observation stations on two occasions, i.e., in 2016 and 2017. furthermore, the samples comprised cover soil, wood and inner material, which were collected aseptically and placed in sterile ziplock plastic bags. these samples were then diluted in sterilized distilled water to attain 10-3 mg/ml, and subsequently inoculated on potato dextrose agar (pda), malt extract agar (mea) and czapek dox agar (cda). the inoculants were incubated at 37 °c, followed by the observation of mold colony after the 11th day. the results identified seven and nine species of molds in the kalisidi and nglimut observation stations, respectively. the most abundant species was penicillium sp. which was found in composted nest materials for the whole observation periods. keywords: mold diversity, mount ungaran, nest, rhyticeros undulates, wreathed hornbill introduction wreathed hornbill or rhyticeros undulauts shaw 1811 is a protected bird, which is categorized into apendix ii, according to the convention on international trade of endangered spesies of wild fauna and flora (cites). appendix ii lists "all species which although not necessarily now threatened with extinction may become so, unless trade in specimens of such species is subject to strict regulation". this is an indication that the species is tradable under certain conditions, e.g., for scientific research purposes (rahayuningsih & kartijono 2013). in addition, breeding and nesting occur during fruiting season. the wreathed hornbill selects nest location based on the availability of fruiting trees and a conducive environment (rahman et al. 2019). the nest selection process is followed by the nest building process, which involves the use of existing cavities from other birds and cracked branches (rahayuningsih et al. 2017). generally, the nest selection and building process are initiated in dry season, characterized by low humidity, which is suitable for breeding and protecting the eggs from parasites. (supaamornkul et al. 2011). however, the r. undulatus in khao yai, thailand is capable of completing the breeding process before the heavy rains. the created nests provide a suitable physical environment, alongside microorganisms needed for the development of eggs and chicks (rahayuningsih et al. 2017; utoyo et al. 2017). in addition, the strength of the physical structure is ensured by building the inner and outer parts of the nest using different soil sources, while the outer cover comprises high mircroorganism diversity, including mold. there have been minimal studies on mold diversity in r. undulatus nests present on mount ungaran, although supa-amornkul et al. (2011) reported the importance of micro-fungus in breaking down wood and organic materials. this phenomenon is exploited in nest cavity expansion; hence the study aimed to determine the diversity of molds in r. undulatus nests on mount ungaran. *corresponding author, email: etak_sigid@mail.unnes.ac.id biotropia vol. 29 no. 1, 2022 40 materials and methods this research was an observational exploratory study on the diversity of mold present in the nest of r. undulatus. the identification process was conducted in the microbiology laboratory, department of biology, universitas negeri semarang (unnes). samples were obtained from the kalisidi observation station located in the kalisidi village, west ungaran district, semarang, and nglimut observation station located in nglimut village, gonoharjo district, kendal district, central java. the first observation period was conducted in 2016, and the last observation period was conducted in 2017. samples were taken at each observation period. nest sampling the nest sampling process was performed at the end of the nesting season, marked by the absence of female and juvenile birds. samples were collected aseptically, using sterile pinset and spatula, and then placed in ziplock plastic bags. the collected samples included nest cover, internal wood and nest materials of each nest in the two observation periods. all of the prepared samples were then stored in a freezer at -20 °c, prior to further analysis. three samples were collected at each observation period. thus, our study collected six samples altogether for the two observation period. each of the six samples were mashed up aseptically, followed by taking 10 g of each sample and dissolving the 10 g of each sample by using 100 ml of distilled water. this mixture was then homogenized and kept as a sample solution, from which 10% was diluted to attain 10-3. subsequently, the inoculation step was performed by spreading 1 ml of the sample solution onto culture media, i.e., potato dextrose agar (pda), malt extract agar (mea) and czapek dox agar (cda). the isolates were then incubated at room temperature for 3-7 days in an incubator. a microscope was used for identification purposes, followed by the observation of wide mold colony growth. sterilization and medium preparation the culture media used were potato dextrose agar (pda), malt extract agar (mea) and czapek dox agar (cda). each of the culture media was measured for 250 ml and dissolved with distilled water. as much as 0.05 g of chloramphenicol was added into the culture media as antibacterial agent, followed by sterilization using an autoclave at 121 °c and 2 atm, for 15 min. subsequently, the culture media were poured into different petri dishes under aseptic conditions, stored inside the biological safety cabinet (bsc), and allowed to solidify, before being placed in a media cooler. calculating and identification of mold colony the grown mold colonies were stained using lactophenol, prior to observation with a microscope at magnifications of 100x 1,000x. subsequently, identification was carried out based on the guidelines of mold morphological structure developed by samson et al. (2019) and robinson (2011) to determine the genus and species of the samples. results and discussion environmental conditions of the kalisidi and nglimut observation stations are presented in table 1. table 1 enviromental condition of the observation stations enviromental conditions kalisidi station nglimut station light intensity (c) 613-1,740 656-1,480 soil ph 5.2-6.2 3.7-4.5 soil humidity (%) 25-55 70-90 air humidity (%) 71-75 75 air temperature °c 29.4-29.9 28.9 tree type dead tree (unidentified) weinmannia fraxenia (nuts) mold diversity of wreathed hornbill (rhyticeros undulatus) nest in mount ungaran – margareta rahayuningsih et al. 41 despite the similarities, there was a variation in light intensity, with nglimut station amounting 656 c to 1,480 c, due to the presence of tighter and thicker canopies. high and low light intensities impacted the air temperature around the nest, which was relatively higher in kalisidi station. kalisidi station featured higher soil ph and lesser soil humidity (table 1). after reaching a diameter of 1.5 cm, the mold colonies were transferred into the subsequent culture medium, followed by staining and observation by a microscope for identification purpose. mold colonies having a diameter less than 1 cm were re-incubated to ensure further growth for easier identification. table 2 shows the relatively higher density of samples obtained in 2017, compared to those obtained in 2016. within the incubated conditions, the mea culture medium was colonized by various molds, where four and seven species were observed in samples obtained in 2016 and 2017, respectively. on the other hand, two and seven mold species grew in the pda culture medium, while two and four mold species were recognized in cda culture medium. aspergillus was successfully identified as the only genera present in all medium, across both sampling years. furthermore, aspergillus niger occurred less frequently than a. terreus, despite the fact that a. niger is commonly found in general environment. meanwhile, acremonium sp. and p. variabile were both identified in the mea medium of 2016, while rhizopus sp. was only seen in the cda medium of 2017. based on the collection period, there was a possibility that the mold species abundance was affected by the growth medium applied (mahadevan & shanmugasundaram 2018). these mold species generally grow better in the mea culture medium, made from wheat extract, maltosa, long chain carbohydrate and glycerol. the next culture medium preferred by mold to grow is pda and followed by cda. in addition, the composition of mea provides sugar and nutrients as a source of energy for molds and yeast (aziz et al. 2018; cvetkovic & markov 2002). the pepton content in mea functions as a nitrogen source, presents in higher amounts compared to other media. pepton is important for amino acid synthesis, which is required in the production of various functional protein, cell structure and hyphae conformation (wang et al. 2010) and is responsible for the relatively higher amount of essential amino acid in mea, including triptophan and tyrosin. these are important components in the conformation of metabolic protein, and also during cell communication. pda media includes the semi synthetic variety, resulting from the natural and synthetic material (dextrose and agar) components. in addition, complex carbohydrate molecules have been identified as the main source of carbon in potato. carbon is the base material for pda production, needed for the growth of molds and yeast. pda is used because of the vitamin and mineral contents required to support fungi growth. also, the dextrose component in pda provides additional sugar as an energy source. table 2 types of mold grown in the three culture media species 2016 2017 pda mea cda pda mea cda aspergillus sp. √ √ √ √ √ √ aspergillus niger √ aspergillus terreus √ √ √ acremonium sp. √ curvularia sp. √ √ fusarium sp. √ √ geotrichum sp. √ √ neosartorya fischeri √ √ penicillium sp. √ √ √ penicillium variabile √ rhizopus sp. √ trichoderma sp. √ √ total spesies 2 4 2 7 7 4 notes: pda = potato dextrose agar; mea = malt extract agar; cda = czapek dox agar; 2016 and 2017 = sampling years. biotropia vol. 29 no. 1, 2022 42 on the other hand, cda medium consists of various nutrient molecules, of which sucrose has been identified as the main energy source, while nitrogen is obtained from the natrium nitrate component. furthermore, other constituents, including dipotassium phosphate is known to serve as a buffer solution, while magnesium sulfate and iron sulfate are essential ions. however, both mea and pda used in this study are manufactured medium, ready to use, while cda was created using a formula according to the manufacture’s protocol (himedia laboratories, mumbai), hence the tendency for unclear and unstandardized composition accuracies and capabilities. the molds obtained in 2016 at the kalisidi station were successfully identified, and three species were observed in the first collection (p1), with two in the second collection (p2). two species were accumulated in p1, and four species in p2 for samples collected in 2017. however, the nglimut station portrayed a relatively higher diversity (table 3). aspergillus sp. was the only species present five times in the r. undulatus nest, i.e., two times in kalisidi station and three times in nglimut station, while penicillium sp. were observed two times in each station. in addition, a. terreus was identified on three instances, i.e., one and two times for the respective stations. on the other hand, curvularia sp., fusarium sp., geotrichum sp., n. fischeri and trichoderma sp. were only identified twice in both stations, while a. niger, acremonium sp., p. variabile, and rhizopus sp. were rarely present (table 4). table 3 types of molds in the cover wreathed hornbill (rhyticeros undulatus) obtained in the 2016 and 2017 sampling period species kalisidi station nglimut station 2016 2017 2016 2017 p1 p2 p1 p2 p1 p2 p1 p2 aspergillus sp. √ √ √ √ √ aspergillus niger √ aspergillus terreus √ √ √ acremonium sp. √ curvularia sp. √ √ fusarium sp. √ √ geotrichum sp. √ √ neosartorya fischeri √ √ penicillium sp. √ √ √ √ penicillium variabile √ rhizopus sp. √ trichoderma sp. total spesies 3 2 √ 2 √ 4 3 5 7 notes: p1 = first collection; p2 = second collection. table 4 types of molds in r. undulatus nest during sampling in 2017 types of molds kalisidi station nglimut station p1 p2 p1 p2 cn wm cm cn wm cm cn wm cm cn wm cm aspergillus sp. √ √ √ √ √ √ aspergillus niger √ √ aspergillus terreus √ √ acremonium sp. √ curvularia sp. √ √ fusarium sp. √ √ √ √ √ geotrichum sp. √ √ neosartorya fischeri √ √ √ penicillium sp. √ √ √ √ √ √ √ penicillium variabile √ rhizopus sp. √ trichoderma sp. √ √ √ total spesies 1 1 1 3 2 4 3 5 5 3 6 notes: p1 = first collection; p2 = second collection; cn = cover nest; wm = wood material; cm = compost material. mold diversity of wreathed hornbill (rhyticeros undulatus) nest in mount ungaran – margareta rahayuningsih et al. 43 samples obtained from the nests were divided into three parts, i.e., cover, internal compost and wood material. the most abundant molds identified at the kalisidi station included geotrichum sp., n. fischeri and trichoderma sp. the trichoderma sp. was observed in the first and second collection periods, while the wood material containing n. fischeri and p. variabile was observed in the second collection period. this result indicated that n. fischeri is the most widespread mold species. the high diversity shown in nglimut station included four mold species in the nest cover, with three in the wood material and four species from the compost. in addition, aspergilus sp. and penicillium sp. were identified as the most common molds in all parts, during both collection periods, while a. niger was only found in the nest cover, with a. terreus in the nest inner material for both collection periods. figure 1 molds types identified in r. undulatus nest notes: a = aspergillus sp.; b = acremonium sp.; c = curvularia sp.; d = fusarium sp.; e = geotrichum sp.; f = neosartorya fischeri; g = penicillium sp.; h = rhizopus sp.; i = trichoderma sp. (b = branch; f = phialid; k = conidiophore; kon = conidia; m = metula; mc = microconidia; s = sporangiophore; spo = spora; v = vesicle). microscope magnification: 1,000x. biotropia vol. 29 no. 1, 2022 44 the morphologycal identification process was based on two main characteristics, including: 1) the colony formation and color and 2) the morphological structure. based on these two main characteristics, the molds were identified into genus and species (table 5). table 5 description of molds types identified in the nest of r. undulatus mold description aspergillus sp. the fruit body consisted of aspergillus formed conidiophores (non-septate), vesicles, metula, phialid, stolone (vegetative hyphae) and conidia. the identified aspergillus sp. possessed radiateand biseriate-conidial heads (sideways/ deviate), with phialid organs that grow in the metula, as seen in figure 1.a. hence, molds with similar criteria were designated as aspergillus (diba et al. 2007) acremonium sp. the typical organs present included a cluster of aerial hyphae, conidiophores, phialids and ellipse extended conidia. furthermore, the phialid grew directly on aerial hyphae, and was tapered in the form of a needle, while the conidiophores were single-celled, erected and unbranched condia (fig. 1.b) (samson et al. 1984). the microscopic size range of these components were 17.5-37.5 (-50.4) × 3.2-4 µm, and 6-8.5 (-9.3) × 2.1-2.8 (-4) µm, respectively (gräfenhan et al. 2011; hill et al. 1990). curvularia sp. the conidiophores were branched, brown, and tightly arranged in groups. in addition, the conidia were elliptical with 3-4 bulkheads in each, with brownish white coloration, and comprising of 4-5 cells. the colonies were dark black in color and round, with cotton texture (fig. 1.c). this was in accordance with the description by kusai (2015) and hosokawa (2003), except with the addition of velvet pigmentation. the conidiophores appeared singly or in groups, simple or branched, straight or crouched, with pale brown or young cones, while the conidia had 3-4 septa. the specimen was thin-walled, measuring 20-30 x 9-15 µm (hosokawa et al. 2003; kusai et al. 2016). fusarium sp. the fusarium genera was identified using the method by (bashyal et al. 2016; gräfenhan et al. 2011). the microconidia appeared as fusiform and ovoid form, with 0-1 septate, while the conidiophorous structure present was insulated. in addition, phialid and macroconidia were not seen under microscopic observations, although microconidia were recognized (fig. 1.d). geotrichum sp. conidia were cylindrical, oval, and tubular (barrel) in shape, with green-blue coloration, and also a chain-like and clustered arrangement. in addition, the upper part of this mold was formed from broken fertile hyphae (fig. 1.e), with conidia diameter of 3.7-4.8-12.5 (-13.8) x (1.7-) 2.4-5 µm, and no conidiophores. also, the fertile hyphae present was branched off dichotomous and insulate. neosartorya fischeri the morphological structure of neosartorya fischeri was similar with aspergillus, characterized by vesicles, phialid and conidia, with seemingly insulated hyphen, alongside blue densely arranged conidiophores and hyphae. in addition, the vesicles were slightly elongated in shape, with conidial columnar head, which was also uniseriate (direct phialid growth in vesicles) (fig. 1.f). the colony was white in color, with cotton-like texture, and ± 0.2-2 cm in diameter. the conidia of neosartorya fischeri was round in shape, half round and elliptical, with slightly coarse wall, at ± 2-3 µm diameter, while the conidiophores ranged from 300-500 µm, with characteristic smooth walls (udagawa et al. 1996). penicillium sp. the morphological structures possessed insulated vegetative hyphae, alongside conidiophores, branches, metula, phialid and conidia. the conidiophores were of the two-stage branched (biverticillate-asymmetrical) type, while the conidia appeared round (fig. 1.g). in addition, the colonies were grayish and light green-old, with ± 0.2–2 cm diameter. also, penicillium is included as a deuteromycota, characterized by fast growing colonies, which is green in appearance and sometimes white. the conidiophores had several branching pattern forms, including one to three-stages and more-stage branched (visagie et al. 2014). rhizopus sp. the morphological structure had sporangiofor, sporangium, featuring the release of spores (sporangiospor). in addition, the mold contains the non-septate stolone (vegetative hyphae), alongside the rhizoid, although only the columella covered by sporangium. the sporangiofor stands tall, with a round shape (fig. 1.h) (hartanti et al. 2015). trichoderma sp. morphological structures are similar with trichoderma, featuring vegetative hyphae (aerial hypha), conidiophores with side branches, slim and elongated phialids, and also round conidia, with white and dark green colonies (fig. 1.i). according to gusnawaty et al. (2014), trichoderma sp. has branched conidiophores resembling pyramids, with more to the end, and the branching becomes shorter. also, the conidia are smooth walled and semi-round to oval in shape. this species have green colonies that were initially white (supa-amornkul et al. 2011). mold diversity of wreathed hornbill (rhyticeros undulatus) nest in mount ungaran – margareta rahayuningsih et al. 45 the nest cover collected in this study consisted of soil and wood. molds identified from the nest cover were of various species, although the more abundant molds were observed in the organic material of the nest’s inner part. this was possibly caused by composted organic materials, including feces, fermented fruit, e.g., ficus, insects and decayed wood. particularly, the ficus fruit or fig (ficus carica) contains 8.98% protein, 6.57% fat, 10.26% moisture content, 18.23% ash content, 20.31% crude fiber, 0.0395% calcium, 0.002% phosphor, 25.48 mg/100 g and 1.64 mg/100 g of vitamin c and e, respectively. in addition, fig also contains various minerals needed by r. undulates, including n, p, k, ca, mg and others (mendoza-castillo 2019). aside from fruits, the fecal matter was high in n for mold protein synthesis, while the soil and wood were characterized by water, fat, carbohydrate and protein. this results were confirmed with the results of previous studies on the nest of r. undulatus containing 53.30 mg/ml of water, 39.02 mg/ml of fat, 35.03 mg/ml of carbohydrates, 5.82 mg/ml of ash and 20.12 mg/ml of protein. in addition, the humidity and the warm and dark conditions of the inner part of the nest form an appropriate and suitable environment for mold growth. conclusion mold species obtained from r. undulatus nests consisting of cover, composted material and wood material in kalisidi and nglimut stations during the sampling in 2016 comprised 6 mold species including aspergillus niger, aspergillus terreus, aspergillus sp., penicillium sp., penicillium variabile and acremonium sp. on the other hand, 9 mold species were reported during the sampling in 2017, including aspergillus terreus, aspergillus sp., curvularia sp., fusarium sp., penicillium sp., rhizopus sp., geotrichum sp., trichoderma sp., and neosartorya fischeri. acknowledgment special thanks are dedicated to the directorate of research and community service, deputy for strengthening research and development, ministry of research and technology of the republic of indonesia/national research and innovation agency for providing research funding scheme penelitian dasar unggulan perguruan tinggi 2018. special gratitudes are also presented to my field research team who provided assistance in data collection and sample analysis. references aziz nha, yousef ns, el-hadded me, el-tayeb ts. 2018. influence of nutritional and climatic conditions on mycelial growth of three oyster mushroom strains. arab univ j agric sci 26: 1165-73. bashyal bm, aggarwal r, sharma s, gupta s, rawat k, singh d, singh 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nesting cycle and nest tree characteristics of the helmeted hornbill rhinoplax vigil, compared to the wreathed hornbill rhyticeros undulatus, in sumatran lowland rainforest. kukila 20:12-22. visagie cm, hirooka y, tanney jb, whitfield e, mwange k, meijer m, …, samson ra. 2014. aspergillus, penicillium and talaromyces isolated from house dust samples collected around the world. stud mycol 78:63-139. wang h, ma f, cheng l. 2010. metabolism of organic acids, nitrogen and amino acids in chlorotic leaves of “honeycrisp” apple (malus domestica borkh) with excessive accumulation of carbohydrates. planta 232:511-22. the southeast asian journal of tropical biology vol. 32 no. 3, 2025: 352 362 doi: 10.11598/btb.2025.32.3.2457 issn: 0215-6334 | e-issn: 1907-770x 352 comparative study on ova recovery in bos indicus and bos taurus cattle: a systematic analysis study siti darodjah rasad1, nena hilmia1, sari suryanah1, cece sumantri2, irma3* 1faculty of animal science, universitas padjadjaran, sumedang 45363, indonesia 2faculty of animal husbandry, ipb university, bogor 16680, indonesia 3directorate general of livestock and animal health service, ministry of agriculture of the republic of indonesia, jakarta 12550, indonesia. article higlights • domestic cattle sub species of bos indicus shows higher ova in their reproduction than bos taurus • in vitro techniques produce higher ova recovery than in vivo techniques. • dairy cattle showed higher ova recovery than in beef cattle or dual-purpose cattle article information received : 6 march 2025 revised : 16 june 2025 accepted : 1 october 2025 *corresponding author, e-mail: irma_fapetipb@yahoo.co.id research paper abstract embryo transfer biotechnology has long been applied and is widely used to increase the reproductive capacity of female cattle, thereby increasing livestock productivity. extensive research has been carried out and the responses of each cattle breed show variation across studies, likely due to differences in management practices and physiological characteristics. our study aimed to determine whether cattle breed influences the oocyte and embryo recovery rates during the embryo transfer process. a meta-analysis was conducted using published research on the ova recovery potential of bos indicus and bos taurus. the screening process identified 21 studies documented across 14 publications suitable for further analyses. the effect of cattle breeds on ova recovery was indicated by a standardized mean difference (smd) of 0.72 ± 0.206 (95% ci; range of 0.32 – 1.11). the heterogeneity test showed significant difference among studies (i2 = 89.99, p < 0.01), with the effect size classification as medium. moderator analyses revealed that bos indicus had a higher ova recovery potential compared to that of bos taurus when using in vitro techniques. subgroup analysis of cattle types showed that the difference in ova recovery between the two breeds was significant in dairy cattle (p < 0.01), while there was no difference observed between dual-purpose and dairy cattle. keywords: dairy, in vitro, ova, reproduction, zebu introduction zebu cattle (bos indicus) can adapt to tropical environments better than bos taurus due to its thermoregulatory adaptability. they are rustic, resilient to parasites, and capable of bearing the hot and humid climates of the tropics (porto-neto et al. 2014), as well as low-quality forage, and a tendency to have a higher number of oocytes (vasconcelos et al. 2020; lacerda et al. 2020; oliveira et al. 2019; sales et al. 2015). zebu cattle are characterized by their hump, large ears, and loose skin (utsunomiya et al. 2019). research examining productivity differences between the two cattle breeds shows the superiority of bos taurus in meat and milk production. on the other hand, studies comparing reproductive performance between the two breeds report varied results. sartori et al. (2016) examined differences in the reproductive physiology of bos taurus and bos indicus. bos indicus has nearly twice as many antral follicles (range of 25 – 100) as bos taurus (range of 5 – 40). research by baldrighi et al. (2014) and batista et al. (2014) showed that the high number of antral follicles is associated with anti-mullerian hormone (amh). bos indicus also exhibits a high occurrence of four follicular waves (bo et al. 2003). according to martinez et al. (2003), the interovulation rate is positively correlated with the number of follicular waves, and the time to the appearance of the second follicular wave decreases as the number of follicular waves per cycle increases. research comparing the potential of ova recovered from the two breeds exhibits varying results using both in vitro and in vivo methods. to copyright (c) 2025@author(s). https://doi.org/10.11598/btb.2025.32.3.2457 https://creativecommons.org/licenses/by-nc-nd/4.0/ comparative study on ova recovery in bos indicus and bos taurus rasad et al. 353 conclude the results of these studies, a meta-analysis is needed. this approach is needed to summarize the results and find quantitative conclusions from a large cattle population. a quantitative analysis of ova recovered from bos indicus and bos taurus was carried out using a meta-analysis. this approach was performed to provide a theoretical basis for the reproductive potential of female cattle between the two breeds. the resulting information is expected to provide more in-depth knowledge, especially regarding the reproductive difference between bos indicus and bos taurus, especially in cattle reared in tropical regions. materials and methods data resources and search strategy publications searches were conducted using the keywords “bos taurus,” “bos indicus,” “oocytes,” “embryo,” and “bos taurus” and “bos indicus” and oocytes or embryo.” the target databases were sciencedirect (https://www.sciencedirect. com/), scopus (https://www.scopus.com/), and pubmed (https://pubmed.ncbi.nlm.nih.gov/). the publication period was determined to be in the range of 2003 – 2023. additional search was conducted using other sources (google scholar, springer, sage, taylor and francis, oxford, nature, and cambridge). the prisma framework was used in the search strategy (fig. 1). article inclusion and exclusion criteria criteria for selection include: 1) accessible and available in english; 2) studies come from peer-reviewed journals; and 3) studies compare differences in ova recovery of bos indicus and bos taurus. the articles must meet the following criteria: 1) include information regarding ova recovery from both breeds; 2) provide data on the mean, standard deviation or standard error and number of samples; 3) be published in the form of a research journal article. articles were excluded if: 1) they reported only one mean value for ova recovery without a standard deviation or did not include the number of samples; and/or 2) they were published in the form of review journals, posters, abstracts, theses, or dissertations. data extraction there were 21 studies across fourteen articles that compared ova production between bos indicus and bos taurus. studies that meet the criteria were grouped as follows: 1) first author and year of publication; 2) characteristics of the cattle population, including number, breed, production type (dairy, beef, dual-purpose), age, body weight, body condition score, rearing management, estrus synchronization, and physiological status (heifer/ cow); and 3) numerical data, including the mean value of ova recovery, standard deviation or standard error, and number of samples. figure 1 flowchart prisma (preferred reporting item for systematic review and meta-analysis) biotropia vol. 32 no. 3, 2025 354 statistical analysis the data analysis was conducted by measuring the effect size, heterogeneity, and publication bias. the effect size is defined as an estimate of the standardized mean difference. the calculation was carried out using openmee software (wallace et al. 2016), using the following formula according to rosenberg et al. (2000) and sanchez-meca and martin-martinez (2010): where: d = effect size (x 1̅ ) = average ova recovery of bos indicus (x ̅2 ) = average ova recovery of bos taurus swithin = combined standard deviation n1 = sample size of bos indicus n2 = sample size of bos taurus s1 = standard deviation of bos indicus s2 = standard deviation of bos taurus results and discussion description of studies the ova recovery potential in bos taurus and bos indicus cattle was discussed in 21 studies listed in 14 publications published from 2003 to 2023. the population of bos indicus cattle was 1,385 heads consisting of nellore, gyr, brahman, and po. population of bos taurus cattle was 645 heads consisting of holstein, simmental, and belgian blue. gyr and holstein cattle are dairy breeds, while nellore (ongole) cattle are dual-purpose breed used for both beef and dairy production. dualpurpose systems are non-specialized, where calves are weaned between eight months and one year of age and raised under restricted milk suckling, allowing for partial milk harvest (restrepo et al. 1991). the total population comprised 2,030 animals, including beef, dairy, and dual-purpose types. nellore, gyr, and brahman cattle are zebu breeds originating from the indian subcontinent, which is considered the treasure house of bos indicus cattle breeds most suited for livestock production in the tropics, providing draught power, milk, or meat. the synchronization methods reported in the six studies include cidr (controlled internal drug release), progesterone (p4 device), norgestomet ear implant, and gnrh (gonadotropin-releasing hormone), as summarized in tables 1 and 2. pasteur management was reported in six studies with several types of feed: brachiaria humidicola, silage, hay, molasses, and corn silage. the physiology of female cattle consisted of prepubertal, heifer, lactating, and non-lactating cows. body weight ranged from 102 – 487 kg with an age range of 3 months – 8 years. the type of semen used in four studies was sexing. all studies were conducted in brazil during winter, spring, and fall. according to fao (2019), approximately 1.48 billion cattle are reared worldwide, with over half of this population concentrated in brazil, india, and the usa. brazil currently ranks first in the world for the largest cattle herds, with 218 million head of cattle, followed by india which has a herd of 186 million animals. ova collection was performed using two methods, namely in vitro (ovum pick up), reported in 12 studies and in vivo (flushing), reported in eight studies. in bos indicus, the range of ova recovery was 5 – 45 ova, while in bos taurus, it was 6 – 22 ova. using in vitro techniques, bos indicus cattle produced around 10 – 45 ova, while bos taurus cattle yield around 6 – 22 ova. in vivo collection produced 3 – 38 ova in bos indicus and around 10 – 16 ova in bos taurus. the ova recovery potential of bos indicus was higher than that of bos taurus (table 3). based on table 3, six studies compared dairy cattle breeds (gyr vs. nellore), one study compared beef cattle breeds (brahman vs. simmental), one study compared beef and dairy breeds (brahman vs. holstein), and 12 studies compared dual-purpose cattle and dairy breeds (nellore vs holstein). research indicated that the collection techniques in the same breed can exhibit different results within a breed, and using the same methods across the same breed can also produce varying outcomes. previous studies suggest that differences in results are influenced by the ova collection techniques, with in vitro providing higher ova recovery in bos indicus cattle. among the 12 studies using invitro technique, three reported a significant effect of breed on the number of ova recovered (p < 0.05). similarly, three of eight studies using invivo technique also found a significant breed effect on ova recovery (p < 0.05). five studies, however, reported no significant relationship between breed and ova recovery (p >0.05). comparative study on ova recovery in bos indicus and bos taurus rasad et al. 355 table 1 summary of study used in meta-analysis (from bcs to semen) no study bcs synchronization technique bos indicus bos taurus semen 1 viana et al. (2011) 2.7 ± 0.6 cidr ovum pick up gyr, dairy holstein, dairy 2 vasconcelos et al. (2020) 5-7 (1-9) flushing brahman, beef simmental, beef 3 soares et al. (2011) 3.6 ± 0.1 (1-5) p4 device flushing nellore, beef holstein, dairy sexing 4 sales et al. (2015) 3.2 ± 0.3 norgestomet ear ovum pick up gyr, dairy holstein, dairy 5 pontes et al. 2010 3.5 ± 0.5 ovum pick up gyr, dairy holstein, dairy sexing 6 oliveira et al. (2019) ovum pick up gyr, dairy holstein, dairy sexing 7 lacerda et al. (2020) ovum pick up gyr, dairy holstein, dairy sexing 8 krininger et al. (2003) gnrh flushing brahman, beef holstein, dairy 9 guerrero et al. (2014) ovum pick up nellore, beef holstein, dairy 10 gimenes et al. (2015) norgestomet ear ovum pick up nellore, beef holstein, dairy 11 carter et al. (2016) flushing nellore, beef angus, beef 12 camargo et al. (2007) 3-4 (1-4) flushing gyr, dairy holstein, dairy 13 batista et al. (2016) cidr ovum pick up nellore, beef holstein, dairy 14 irma et al. (2023) 3-3.5 cidr flushing po belgian blue unsexing table 2 summary of study used in meta-analysis (from age to weight) no study age management feed season physiology weight (kg) 1 viana et al. (2011) 5.38 ±1.50 2 vasconcelos et al. (2020) 7.00 ±3.00 brachiaria, silage cow 337 – 803 3 soares et al. (2011) 4-10 pasture cow, non-lactated 487.8 ±36.6 4 sales et al. (2015) 5.21 ±0.56 winter-spring cow, non-lactated 5 pontes et al. 2010 5 ± 2.3 cow, non-lactated 6 oliveira et al. (2019) pasture cow, non-lactated 452 ± 63 7 lacerda et al. (2020) 8 krininger et al. (2003) pasture hay, molasses autumn cow, non-lactated 9 guerrero et al. (2014) brachiaria, silage prepuberty-heifercow 10 gimenes et al. (2015) pasture corn silage heifer 409.5 – 467.7 11 carter et al. (2016) 7.5-8.6 pasture cow, non-lactated 12 camargo et al. (2007) pasture cow, non-lactated 13 batista et al. (2016) 3-4 months, 1-2 years prepuberty, heifer 102 – 119 14 irma et al. (2023) 4-6 years intensive brachiaria summer cow, heifer 475 – 535 biotropia vol. 32 no. 3, 2025 356 ta bl e 3 d at a ex tr ac tio n of o va p ro du ct io n bo s i nd icu s a nd b os ta ur us n o. st ud ie s te ch ni qu e c at tl e ty pe p va lu e bo s i nd icu s bo s t au ru s m ea n n br ee d m ea n n br ee d 1 po nt es et a l. (2 01 0) in v itr o d ai ry v s d ai ry p < 0. 05 17 .1 ± 4 .4 61 7 g yr 11 .4 ± 3 .9 18 0 h ol ste in 2 o liv ei ra et a l. (2 01 9) in v itr o d ai ry v s d ai ry p < 0. 05 10 .0 ± 0 .7 12 g yr 6. 3 ± 0. 4 13 h ol ste in 3 va sc on ce lo s e t a l. (2 02 0) in v itr o be ef v s b ee f p < 0. 05 17 .2 ± 0 .4 18 4 br ah m an 12 .2 ± 0 .5 99 si m m en ta l 4 sa le s e t a l. (2 01 5) in v itr o d ai ry v s d ai ry p < 0. 05 22 .8 ± 1 .5 14 g yr 14 .6 ± 0 .9 14 h ol ste in 5 ba tis ta et a l. (2 01 6) in v itr o d ua l p ur po se v s d ai ry p < 0. 05 13 .5 ± 3 .6 15 n el lo re 11 .7 ± 2 .4 12 h ol ste in 6 ba tis ta et a l. (2 01 6) in v itr o d ua l p ur po se v s d ai ry p < 0. 05 29 .9 ± 5 .3 15 n el lo re 9. 2 ± 1. 7 10 h ol ste in 7 g ue rr er o et a l. (2 01 4) in v itr o d ua l p ur po se v s d ai ry p < 0. 05 13 .4 ± 1 .7 18 n el lo re 9. 0 ± 0. 9 32 h ol ste in 8 g ue rr er o et a l. (2 01 4) in v itr o d ua l p ur po se v s d ai ry p < 0. 05 45 .3 ± 6 .4 16 n el lo re 17 .3 ± 1 .5 27 h ol ste in 9 la ce rd a et a l. (2 02 0) in v itr o d ai ry v s d ai ry p < 0. 05 14 .9 ± 0 .2 36 3 g yr 9. 0 ± 0. 4 10 9 h ol ste in 10 ba tis ta et a l. (2 01 6) in v itr o d ua l p ur po se v s d ai ry p > 0. 05 20 .9 ± 5 .1 15 n el lo re 22 .4 ± 5 .4 12 h ol ste in 11 c am ar go et a l. (2 00 7) in v itr o d ai ry v s d ai ry p > 0. 05 15 .3 ± 1 .3 9 g yr 16 .2 ± 2 .0 13 h ol ste in 12 c ar te r e t a l. (2 01 6) in v itr o d ua l p ur po se v s d ai ry p > 0. 05 11 .6 ± 1 .8 12 n el lo re 17 .5 ± 1 .6 56 h ol ste in 13 g im en es et a l. (2 01 5) in v iv o d ua l p ur po se v s d ai ry p < 0. 05 37 .5 ± 4 .4 3 n el lo re 16 .2 ± 2 .2 3 h ol ste in 14 g im en es et a l. (2 01 5) in v iv o d ua l p ur po se v s d ai ry p < 0. 05 38 .5 ± 4 .3 3 n el lo re 16 .2 ± 2 .3 3 h ol ste in 15 g im en es et a l. (2 01 5) in v iv o d ua l p ur po se v s d ai ry p < 0. 05 35 .2 ± 4 .8 3 n el lo re 13 .8 ± 1 .8 3 h ol ste in 16 so ar es et a l. (2 01 1) in v iv o d ua l p ur po se v s d ai ry p > 0. 05 8. 0 ± 3. 2 17 n el lo re 10 .4 ± 3 .4 11 h ol ste in 17 so ar es et a l. (2 01 1) in v iv o d ua l p ur po se v s d ai ry p > 0. 05 7. 1 ± 3. 3 18 n el lo re 11 .3 ± 4 .4 11 h ol ste in 18 so ar es et a l. (2 01 1) in v iv o d ua l p ur po se v s d ai ry p > 0. 05 9. 0 ± 3. 8 19 n el lo re 12 .4 ± 3 .8 11 h ol ste in 19 v ia na et a l. (2 01 1) in v iv o d ai ry v s d ai ry p > 0. 05 5. 8 ± 1. 7 12 g yr 10 .7 ± 2 .3 8 h ol ste in 20 k rin in ge r e t a l. (2 00 3) in v iv o be ef v s d ai ry p > 0. 05 9. 3 ± 2. 9 10 br ah m an 10 .9 ± 2 .9 10 h ol ste in 21 ir m a et a l. (2 02 3) in v iv o p < 0. 05 11 .8 3 ± 1. 9 10 po 4. 86 ± 1 .3 8 be lg ia n bl ue to ta l 1, 38 5 he ad s 64 5 he ad s m in im um 5. 8 ± 1. 7 4. 86 ± 1 .3 m ax im um 38 .5 ± 4 .3 22 .4 ± 5 .4 comparative study on ova recovery in bos indicus and bos taurus rasad et al. 357 potency of ova recovery in bos indicus and bos taurus differences in cattle breeds influenced ova production, based on 21 studies reported in 14 publications (smd: 0.72 ± 0.206, 95% ci: 0.32 – 1.11), with significant heterogeneity (i2 = 89.99, p < 0.001). the results showed a difference in ova recovery between bos indicus and bos taurus. according to the effect size classification of cohen (2018), the difference in the combined mean value (in vivo and in vitro) of 0.724 falls within the medium category (0.50 ≤ effect size < 0.80). the heterogeneity test indicated that there was a varying effect size among studies (p < 0.001), with heterogeneity of i2 = 89.99, suggesting high diversity may have been caused by systematic differences between studies. higgins et al. (2003) categorized heterogeneity < 25% as low diversity, 50 – 75% as medium diversity and >75% as high diversity. higgins heterogeneity test i2 shows that there is high diversity among studies, thus, subgroup analysis was carried out to analyze the diversity that occurred among studies (table 4). subgroup analysis was carried out based on collection technique and cattle type considering that the information was completely obtained from publications. collection techniques were divided into in vitro and in vivo, while cattle type was divided into dairy, beef, and dual-purpose. subgroup analysis of the in vivo method showed no differences in ova production between cattle breeds. a significant difference was shown by the in vitro technique, with the smd value (smd: 1.01, 95% ci: 0.618 – 1.410) being higher than the cumulative smd (smd: 0.72, 95% ci: 0.320 1.127). this result showed that in vitro technique has a significant impact on ova production. subgroup analysis of cattle types showed that the difference in ova production between the two breeds was significant (p < 0.01) in the dairy type, while there was no difference in the dual-purpose vs. dairy breeds. the forest plot shows the difference in in vitro mean values to the right of the cumulative mean difference line. this result confirmed that the technique provides significant differences between the two breeds (fig. 2). table 4 estimation of standardized mean differences and heterogeneity description estimation standard error p value standardize means difference or smd (d) 0.72 0.206 < 0.001 d subgroup in vitro 1.01 0.202 < 0.001 d subgroup in vivo 0.27 0.410 0.510 d subgroup dairy purpose cattle 1.00 0.250 < 0.001 d subgroup dual purpose vs dairy cattle 0.76 0.410 0.057 higgins heterogeneity (i2) 89,99 < 0.001 i2 subgroup in vitro 88.45 < 0.001 i2 subgroup in vivo 79.64 < 0.001 i2 subgroup dairy purpose cattle 85.84 < 0.001 i2 subgroup dual purpose vs dairy cattle 88.84 < 0.001 biotropia vol. 32 no. 3, 2025 358 figure 2 forest plot on ova recovery of bos indicus and bos taurus based on ova collection techniques notes: ■ = weighted study; ── = confidence interval; ◊ = effect size. the difference in the mean value of in vivo ova recovery was lower (smd: 0.27, 95% ci: -0.534 – 1.075) than that of the cumulative smd value (smd: 0.72, 95% ci: 0.320 – 1.127) and the confidence interval estimates touched the y = 0 axis, indicating that ova recovery between the two breeds was similar. several in vitro studies provided the highest differential impact, namely study of pontes et al. (2009) with (smd: 1.302, 95% ci: 1.125 – 1.1480), study of lacerda et al. (2020) with (smd: 1.511, 95% ci: 1.276 – 1.746) and study of vasconcelos et al. (2020) with (smd: 0.967, 95% ci: 0.710 – 1.224). the findings of the three studies may have been caused by the large sample size (1,164 individuals). the ova production potential of bos indicus was reported to be greater in nine in vitro studies, while three other studies stated it was lower (p < 0.05). this result contrasts with studies using in the vivo techniques, which showed bos taurus producing more ova. according to marsisco et al. (2019), in vivo technique produces good-quality embryo with higher pregnancy success and lower mortality (sartori et al. 2016). the in vivo and in vitro techniques play an essential role in increasing the productivity of female livestock by shortening generation intervals and increasing selection intensity and selection accuracy (jaton et al. 2016). the existence of diverse individual responses and the low number of transferable embryos is a barrier to the development of embryo transfer biotechnology (center et al. 2018). fertility is a characteristic of reproduction influenced by breeds (gomez et al. 2020). several scientific reviews discuss differences in reproduction between the two breeds. it is thought that the differences in reproduction also influences the differences in ova recovery due to: 1) bos indicus has higher antral follicles; 2) the number of follicle waves per cycle of bos indicus is more significant; 3) anti-circulation mullerian hormone (amh), progesterone, insulin, insulin-like growth factor 1 (igf1), cholesterol and estradiol levels are higher in bos indicus (alvarez et al. 2000; buratini jr et al. 2000; carvalho et al. 2008; gimenes et al. 2000; gimenes et al. 2009; bastos et al. 2010, 2013, 2016; bo et al. 2003). these findings confirm that bos indicus and bos taurus do have different physiology. the differences between the two breeds may have occurred due to domestication in different environments. utsunomiya et al. (2019) reported that the landscape of genomic diversity in the bos indicus subspecies seems different. bos indicus is more diverse and is domesticated 2,000 years later than that of bos taurus cattle. the genetic signals that make up the phenotypic are expected from the genetic dissection of morphological, adaptive, and reproductive traits. according to roth (2015), the environment, particularly temperature conditions, significantly influences ovarian dynamics in female reproduction. the detrimental effects of high temperatures and heat stress on fertility levels underscore the urgent need for further research. comparative study on ova recovery in bos indicus and bos taurus rasad et al. 359 several environmental mechanisms may influence ovarian dynamics in cattle that can be described by: 1) inhibiting the development of small antral follicles (0.5 – 3.0 mm); 2) decreasing follicular cell viability, which has an impact on reducing steroidogenesis; 3) weak ability of the dominant follicle, which results in a decrease in estrogen concentration; 4) decreased concentration of inhibin results in an increase in fsh (follicle stimulating hormone), which increases the development of medium and large follicles, thereby inducing double ovulation; 5) decreased concentration of estrogen and lh (luteinizing hormone), which results in a decreased duration and intensity of estrus; and 6) disruption of ovulation and the formation of the corpus luteum, which produces progesterone in low concentrations. apart from disrupting ovarian dynamics, heat stress affects oocyte maturity, especially by inhibiting the cell nucleus and cytoplasm’s maturation, which reduces the oocyte’s ability and embryo development (roth 2015). type of cattles: dairy, beef and dualpurpose as donor holstein and gyr are dairy types in both breeds (table 3). holsteins in the bos taurus group numbered 538 out of 645 (83.41%), while gyr in the bos indicus group numbered 1,027 out of 1,385 (74.15%). the difference in ova recovery was higher in the dairy type (smd: 1.003, 95% ci: 0.513 – 1.494) than that in the dual-purpose vs dairy type (smd: 0.761, 95% ci: -0.204 – 1.546). the study forest plot based on cattle type is presented in figure 3. the forest plot shows the difference in ova production for the dairy type on the right side of the cumulative ova recovery line, while for the dual-purpose type, it is close to cumulative. this finding showed that the difference in ova recovery is more contrasting in the dairy type (gyr vs. holstein) compared to that in the dual-purpose vs dairy type (nellore vs. holstein). according to ortega (2018), fertility in dairy type decreases along with the intensity of selection on milk production. sartori et al. (2010) reported that the average dominant follicle in holstein dairy is around 8 – 9 with an ovulatory diameter of around 14 – 18 mm, while the dual-purpose nellore type has a more significant number of small follicles with a diameter of 2 – 5 mm and fewer dominant follicles with a smaller diameter (fig. 4). according to sartori et al. (2016), progesterone metabolism in dairy cattle is higher than that in beef cattle. panjaitan et al. (2019) found that embryo death occurs due to reduced luteal function caused by low progesterone concentrations. decreased progesterone secretion on the seventh day after artificial insemination causes endometrial disorders, disturbs protein secretion and embryonic life, obstructs embryo development, and causes early embryo death (mcneill et al. 2006). figure 3 forest plot on ova recovery of bos indicus and bos taurus based on cattle type notes: ■ = weighted study; ── = confidence interval; ◊ = effect size. biotropia vol. 32 no. 3, 2025 360 figure 4 follicular dynamic in nellore and holstein dairy cattle source: sartori et al. (2010) limitations of the study we define nellore cattle as dual-purpose cattle that can be used as meat and milk producers and draught cattle. this defines the type of subgroup in the moderator variable analysis. studies on types of beef cattle in both breeds are limited to analysis because there is only one study, and studies comparing beef with dairy cattle cannot be analyzed further. conclusion bos indicus showed higher ova production than that of bos taurus. a subgroup analysis of technique collection and cattle indicated that the differences in ova recovery between the two breeds were significant based on in vitro technique and the dairy cattle type. appropriate ova harvesting techniques to obtain oocytes in bos indicus must be considered to maximize the potency of reproduction in zebu. the results of this research showed that the zebu cattle, besides having good adaptability to tropical environments, partial resistance to ticks, and low nutritional requirements, also show reproductive superiority in ova recovery. these advantages must be made efficient by applying appropriate ova harvesting technology, namely ovum pick-up, especially in diary type cattle. the research location was limited to two places: brazil and indonesia. thus, the reproductive performance of the two breeds in subtropical conditions needs to be more representative. recommendation the findings of this study support the following recommendations: 1. selection of zebu cattle having superior reproductive performance to serve as donors at livestock embryo center and maintain bulls at artificial insemination center. 2. developing breeding policies to increase the zebu cattle population in indonesia and improve them genetic quality by pure breeding. 3. adopting reproductive technologies, such as ultrasound-guided ovum pick-up to optimize ova recovery. 4. utilizing ovaries from zebu cattle at slaughterhouses for in vitro embryo production. references alvarez p, spicer lj, chase jr cc, payton 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open-source software for meta-analysis in ecology and evolutionary biology.” methods ecol evol 8(8):941–47. the southeast asian journal of tropical biology vol. 32 no. 1, 2025: 216 227 doi: 10.11598/btb.2025.32.1.2482 issn: 0215-6334 | e-issn: 1907-770x 216 highlights • biofloc technology with chitosan addition improved the growth performance of penaeus monodon, increased floc volume and heterotrophic bacteria, and reduced vibrio-like bacteria. • biofloc technology with chitosan addition enhanced water quality and microbial community, supporting p. monodon growth. • p. monodon in a biofloc system with chitosan had higher weight gain, growth rate, and survival rate. • the study provides insights for sustainable intensive shrimp farming using biofloc technology. • findings contribute to improving productivity and maintaining environmental conditions in shrimp aquaculture. keyword: biofloc technology; chitosan; shrimp aquaculture; vibrio-like bacteria; heterotrophic bacteria abstract penaeus monodon (tiger prawn) farming failures often occur in the post-larval initial stocking phase until harvesting. high mortality rates of p. monodon post-larvae are often observed after stocking. disease attack in the second month of the rearing period eventually causes failure in extensive ponds. the primary objective of this study was to investigate the impact of chitosan addition on p. monodon yield in a biofloc technology (bft) culture system. the experiment was performed in triplicate using three treatments, i.e., al (a control pond without biofloc), abf1 (a biofloc pond without chitosan addition), and abf2 (a biofloc pond with 100 mg/l chitosan). the post-larval p. monodon (pl-10), which was visually healthy and disease-free, was obtained from a hatchery. the stocking density used in this study was 40 individuals per 100 l. the prawns were fed a commercial diet four times daily for 40 days. a statistically significant difference was observed (p < 0.05) in weight gain (0.213 0.299 g), average daily growth (0.0054 0.0074 g/day), specific growth rate (13.4 14.3% per day), survival rate (62.5 68.33%), food conversion ratio (1.14 1.79), and protein efficiency ratio (2.41 3.33) of prawn among all of the treatments. biofloc treatment with the addition of chitosan showed better prawn growth than that in the control and abf1 treatments, with a weight gain of 0.30 g, average daily growth of 0.00075 g/day, specific growth rate of 14.3%, survival rate of 68.33%, food conversion ratio of 1.14, and protein efficiency ratio of 3.33. the inclusion of chitosan in the biofloc system led to a prominent increase in floc volume and heterotrophic bacterial populations while simultaneously reducing the number of vibrio-like bacteria (vlb) colonies. no notable variations were noted in the food conversion ratio (fcr), total length, survival rate, total nitrite-n, nitrate-n, and total ammonia nitrogen levels. biofloc technology supplemented with chitosan was found to boost the growth rate of p. monodon by enhancing both the water quality and the microorganisms in the ponds. the findings of this study provide a fundamental contribution to sustainable intensive shrimp farming practices through the implementation of biofloc technology, thereby enhancing productivity and maintaining environmental conditions in aquaculture. keywords: floc, penaidae, probiotic bacteria, vibrio article information received : 8 march 2025 revised : 13 june 2025 accepted : 8 august 2025 *corresponding author, e-mail: amien.fpikubt@gmail.com reviewers: prof. yushinta fujaya, anonymous research article efficacy of the biofloc system with the addition of chitosan on tiger prawn (penaeus monodon) productivity diana maulianawati1,4, muhammad amien1*, yusriadi pardi1, hafid1, muhammad aditya rifai1, andini aisyah nur raoda1, novi luthfiyana2, syahril3 1department of aquaculture, faculty of fisheries and marine science, universitas borneo tarakan, tarakan 77115, indonesia 2department of fisheries product technology, faculty of fisheries and marine science, universitas borneo tarakan, tarakan 77115, indonesia 3department of chemical engineering, faculty of technology, institut sains dan teknologi muhammadiyah tarakan, tarakan 77111, indonesia 4pusat studi kearifan dan kemajuan masyarakat perbatasan (pui-pusaka), universitas borneo tarakan, tarakan 77115, indonesia https://doi.org/10.11598/btb.2025.32.2.2482 efficacy of the biofloc system with chitosan on tiger prawn productivity maulianawati et al. introduction global shrimp farming production reached 8.9% of the total production in 2021. indonesia accounts for approximately 7% of the total shrimp production globally and is ranked the fifth largest producer of cultivated shrimp worldwide (fao 2020). tiger prawn (penaeus monodon) was the prima donna of fisheries commodities from 1990 to 1995. at that time, north kalimantan was one of the areas that produced large quantities of tiger prawns. to date, tiger prawns have been extensively studied. currently, the farming of p. monodon has been experiencing significant degradation in indonesia, notably in north kalimantan; from a total pond area of 140 thousand ha the annual production obtained is only 10 thousand tonnes (ministry of marine affairs and fisheries 2021). the failures of p. monodon farming are often found from the post-larvae's initial stocking phase until harvesting, with a common problem being the death of p. monodon post-larvae after stocking. disease attack in the second month of the rearing period eventually causes failure in the extensive ponds. several factors contribute to the decline in productivity, including a decrease in the carrying capacity of land and the emergence of various shrimp diseases that are difficult to cope with (rukisah et al. 2019). various preventive efforts have been made, such as the use of antibiotics and herbal extracts, such as allium sativum (rahmani et al. 2023), centella asiatica (rukisah et al. 2019), a. paniculatum, cinnamomum kanehirae, citrus aurantifolia, psidium guajava, and a. sativum (citarasu et al. 2022). however, these efforts have not shown significant impacts on increasing production. failure to address this issue can lead to significant economic losses for hatcheries and pond farmers. therefore, a more effective cultivation system is required to maintain tiger prawn production. studies have demonstrated the effectiveness of implementing biofloc systems in shrimp farming. this practice is becoming more widely used in shrimp aquaculture because it has demonstrated improvements in the quality of water, a stronger immune response, and enhanced disease resistance (martínez‐córdova et al. 2015; anand et al. 2017). the biofloc system functions are based on the simultaneous cultivation of heterotrophic bacteria and algae in a confined environment, which results in the formation of flocs. this system fosters the development of heterotrophic bacteria that utilize organic carbon, thereby decreasing nitrogen levels. the equilibrium between carbon and nitrogen yields microbial proteins, which provide a dietary supply for various organisms. biofloc particles are beneficial bacteria, including bacillus sp. and lactobacillus sp. (naiel et al. 2021), as well as bioactive compounds such as carotenoids (xu et al. 2013). the addition of biofloc can also result in a greater quantity of hemocytes and antioxidant status in shrimp (khanjani et al. 2023). microbial populations in biofloc treatment systems (bft) safeguard water quality by facilitating the oxidation of organic matter (sun et al. 2024), improve shrimp farming biosecurity by reducing the risk of various diseases (avnimelech et al. 2013), and help prevent disease outbreaks, such as ems, rms, and ehp, through the use of a minimal water exchange system. in addition, the use of biofloc systems resulted in a reduction in the feed conversion ratio, increased final yield, increased enzyme activity in the hepatopancreas, and increased immune response (bernardi et al. 2018). biofloc-based hatchery systems have shown positive outcomes in boosting survival rates, producing robust juveniles, lowering nitrogen metabolite levels, and decreasing production costs by reducing feed conversion ratios (dauda et al. 2018). the biofloc system for p. vannamei shrimp has been widely used and has shown good results with increased productivity. however, more information on the implementation of biofloc for tiger prawn farming is still needed. the initial implementation of a biofloc system for tiger prawn farming in bangladesh yielded remarkable results in increasing prawn growth and survival, improving water quality, increasing heterotrophic bacterial growth, and decreasing vibrio bacterial concentrations (aftabuddin et al. 2020). therefore, it is necessary to research biofloc technology for tiger prawns and determine appropriate procedures and treatments to apply this technology in north borneo.   a major problem in biofloc system is the floc formation. flocs that are formed tend to be unstable and less aggregated, coupled with the influence of water stirring by aerators causing flocs to not form properly. the addition of chitosan is expected to increase the aggregation of particles and microorganisms, forming flocs that can be consumed by shrimp. chitosan is known to have the ability to reduce nitrite and bod concentrations, control the population of vibrio bacteria, and enhance shrimp immune system biotropia vol. 32 no. 2, 2025 activity (ruswahyuni et al. 2010; liang et al. 2020). chitosan is known to act as a coagulant (iber et al. 2023). the biofloc system can be implemented for the cultivation of tiger prawns by incorporating chitosan into the water. our hypothesis is that the addition of chitosan enhances floc formation, potentially leading to reduced feed requirements. this study aimed to assess the use of chitosan in tiger prawn farming using a biofloc system. materials and method experimental setup the experiment followed a completely randomized design (crd), implementing three treatments with three replications. the treatments were: 1) al (a control pond without biofloc); 2) abf1 (a biofloc pond without chitosan); and 3) abf2 (a biofloc pond with 100 mg/l chitosan). in accordance with the protocol of aftabuddin et al. (2020), each pond was subsequently stocked with 40 post-larvae (pl) of tiger prawn per 100 l. biofloc pond construction nine circular pvc ponds, each measuring 0.6 m in diameter and height, with a 100 l water volume, were used in this study. aeration was set by using an l-shaped pvc pipe (1-inch diameter) positioned at the bottom of each pond, functioning as a lift aeration system. each pipe was connected to an air hose (10 cm diameter) and a central blower (0.042 mpa) that supplied air throughout the experiment. following aftabuddin et al. (2020), the ponds were disinfected before use by washing each pond with 100 mg/l chlorine and detergent, followed by filling up each pond with sterilized seawater to a depth of 10 cm. after 24 hours, the seawater was drained, and the ponds were rinsed. aeration was maintained throughout the disinfection process, which was consistent with the methods described by kumar et al. (2019). culture of probiotics all equipment used for probiotic culture was sterilized. glassware were autoclaved, while fiber tubes, plastic buckets, and other non-glass items were disinfected with a 500 ppm chlorine solution. the culture medium was prepared using 3 kg of bran, 100 g of fish meal, 200 ml of molasses, 50 g of non-iodized salt, and 10 l of sterilized seawater. seawater was first sterilized with 50 mg/l chlorine and left to stand overnight. after 24 hours, the solution was neutralized with 25 mg/l sodium thiosulfate and aerated for 24 hours. the chlorine levels were then checked to ensure complete neutralization. sterilized seawater, bran, and fish meal (10 l) were boiled for 30 minutes for probiotic culture. molasses and salt were added, and the mixture was poured into a culture vessel. once the water temperature was below 35 °c, the 67 g of probiotic inoculant was added. biofloc production materials such as dolomite (ca2mg (co3)2) (2 g/m3), non-iodized salt (1.5 kg/m3), commercial probiotics 5 ml/m3 (containing  bacillus megaterium,  bacillus polymyxa,  lactobacillus plantarum, nitrobacter winogradskyi, nitrosomonas europaea), molasses (250 ml/m3), and wheat flour (250 g/m3) were added to the maintenance pond containing 90 l sterilized seawater (salinity of 16 ppt) and aerated for 7 days. during the floc formation, probiotics and molasses were added to the pond daily, and at the same time chitosan (100 mg/l) was added to the abf2 pond. ponds can be used once a floc has formed, with a floc volume exceeding 5 ml/l (bppp 2021). management and stocking of tiger prawn post-larvae of tiger prawn (p. monodon) with a size of pl-10 (0.001 g) were obtained from a nearby hatchery and stocked at a density of 40 larvae per 100 l in each rearing pond. the prawn were fed commercial feed four times a day, at 07.00, 11.00, 19.00, and 23.00 hours, and made up 3% of their body weight. probiotics were administered to each pond at a rate of 5 mg/m3 at a 3-day interval. the water was replaced at a rate of 10% every two weeks or whenever there was a shift in the salinity level. the weight and length as growth parameters of the prawn were measured on the day of culture (doc)-0 and at doc-40. observation of the water's condition across the experimental duration water quality parameters, such as temperature, dissolved oxygen, and ph, were monitored daily throughout the experiment using a water testing device supplied by wtw. water salinity was measured using a hand-held refractometer (atago). water quality parameters, such as nitrate (no3-n), nitrite (no2-n), and total ammonia nitrogen (tan), were tested every three nights using the spectrophotometric techniques outlined in the apha guidelines (2005). the volume of floc was measured with a 1,000 ml imhoff cone efficacy of the biofloc system with chitosan on tiger prawn productivity maulianawati et al. manufactured by plastic brand. counting procedure of bacteria total heterotrophic bacteria (thb) and vibriolike bacteria (vlb) counts were carried out biweekly throughout the entire duration of the experimental period. random water samples were collected from the experimental and control ponds in sterile glass containers and then refrigerated at 4 °c until further analysis. bacteriological analyses were conducted according to the methods outlined in apha (2005) guidelines. to summarize, the water samples were diluted in a concentration range of 10−1 to 10−6 with a sterilized saline solution consisting of 2.5% nacl (merck). samples (0.5 ml) of the respective dilutions were plated onto zobell marine agar (zma, himedia) for thb, and onto thiosulfate-citrate bile saltssucrose (tcbs, himedia) agar plates for vlb. each analysis was performed in triplicate using the spread plate method. the inoculated plates were then incubated at 35 0c for 48 hours. colonies were counted using a digital colony counter, and the results were expressed as colony-forming units (cfu/ml). data analysis the data were analyzed using spss 21 software. to identify variations in water quality, prawn growth, and bacterial levels between the treatments, a series of one-way anova tests was conducted. if there was a significant difference between the treatments, duncan’s test was used to determine the best treatment. prior to the analysis, all proportional data were transformed using the arcsine square root technique. the findings are presented as the mean values ± standard errors. statistical significance was set at a threshold of p < 0.05. results and discussion growth parameters notable variances (p < 0.05) were evident in the average body weight, average daily growth, specific growth rate, and protein efficiency ratio (table 1). abf2 (biofloc with the addition of chitosan) treatment demonstrated notably higher body weight, daily growth, specific growth rate, and protein efficiency ratio than those in abf1 (biofloc without chitosan) and the control (al) treatments (p < 0.05). furthermore, abf2 exhibited a significantly higher average length and a higher survival rate, specifically 68.33 ± 15.7%, surpassing abf1 treatments and the control (al) (table 2). the feed conversion ratio in abf2 was significantly lower (p < 0.05) than that in the control (al) and biofloc treatments without chitosan (abf1) (table 1). abf2 demonstrated significantly higher growth and survival rates than those of other treatments, indicating that the combination of chitosan and probiotic supplementation positively influenced the growth performance and survival rate of p. monodon. these results are likely attributable to the synergistic effects of enhanced water quality and an increased population of heterotrophic bacteria. previous studies have consistently shown that cultivating penaeid shrimp in biofloc systems leads to higher survival and growth rates than those table 1 growth performance of p. monodon comparison among treatments notes: data represent the mean values (± standard error) of three replicates. mean values within the same row are marked with different superscripts indicate statistically significant differences (p < 0.05); al = control; abf1 = without chitosan; abf2 = added with chitosan. biotropia vol. 32 no. 2, 2025 in traditional water-based systems (aftabuddin et al. 2020; brito et al. 2013; kumar et al. 2019). in the present study, the average daily growth and survival rates of p. monodon were slightly lower than those documented in similar biofloc systems (aftabuddin et al. 2020; brito et al. 2013; kumar et al. 2019). however, the feed conversion ratio (fcr) of p. monodon (1.14 1.69) in our biofloc system was better than the fcr range (1.42 2.16) reported by aftabuddin et al. (2020). notably, the fcr from our study also outperformed the range (1.8 3.6) observed by kumar et al. (2015) for p. monodon under varying protein levels and carbon sources. in this study, the growth of tiger shrimp (p. monodon) was relatively slow, with an average daily growth rate of 0.54 0.75 mg/day (table 1). this value is significantly lower (approximately 80% lower) than that in the study by aftabuddin et al. (2020), who reported a daily growth rate of 0.11 0.14 g/day. this difference is likely due to differences in the age and initial weight of the postlarvae (pl). this study used 10-day-old pl with an average weight of 0.002 g, whereas aftabuddin et al. (2020) used pl with an average weight of 0.18 g. in addition, p. monodon post-larvae are suspected to have not received adequate nutrition for weight gain. tiger shrimp in the pl phase generally consume phytoplankton of skeletonema costatum and chaetoceros species, and as they aged, they consume zooplankton. in this study, p. monodon consumed artificial feed and flocs that formed. although artificial feed was provided, it was only administered at 3% of the shrimp body weight. another possibility is that plankton that grew in the biofloc media were not preferred or did not significantly influence shrimp growth, even though the floc volume formed was > 10 ml/l. rearing time can also affect shrimp growth, and shrimp rearing in this study lasted for 40 days. the growth of tiger shrimp during the first 40 days of doc tended to be slower. based on the growth curve published by the who, the daily growth of tiger shrimp is relatively varied; in the first 40 days of rearing, the growth of tiger shrimp ranges from 0 to 2 g, and accelerated growth occurred after 60 days of rearing. this study utilized commercial feed administered at 3% shrimp body weight, which contained 40% protein, 7% fat, 3% fiber, 13% ash, and 10% water throughout the rearing period. effective feed utilization typically results in an fcr ranging from one to two. as the fcr value decreased, the efficiency increased. the biofloc system exhibited a reduced feed conversion ratio compared with the control treatment. the anova test results showed a substantial difference in feed conversion ratio between treatments, with a p value less than 0.05. the abf2 treatment had a notable impact on the feed conversion ratio. overall, the fcr in this study was within a reasonable range. although the fcr value indicates the efficiency of feed utilization, it cannot be used to calculate the quantity and quality of nutrients consumed by shrimp. the direct effects of chitosan addition to biofloc media on the growth of tiger shrimp (penaeus monodon) remain unclear. however, several studies have shown that chitosan can act as a coagulant and particle binder, thereby facilitating the formation of more stable microbial flocs and improving the water quality (zaki et al. 2023). improvements in the water quality can also improve shrimp growth. niu et al. (2013) found that chitosan supplementation improved growth performance, survival, and immune function of tiger shrimp. although this study focused on feed supplementation, the results demonstrated the potential of chitosan as a beneficial additive in tiger shrimp cultivation. in addition, hermawan et al. (2019) reported that the addition of chitosan to feed increased the total number of haemocytes and resistance to salinity stress in white-leg shrimp (litopenaeus vannamei). although the species studied were different, these findings reveal a positive effect of chitosan on the shrimp's immune system. although no research has specifically examined the effects of adding chitosan to biofloc media on the growth of tiger shrimp, the use of chitosan in the maintenance media is thought to improve water quality and shrimp health. chitosan helps to form more stable bioflocs and improves water quality, which ultimately supports the growth and survival of tiger shrimp. water quality parameters water quality parameters of the treatments did not show statistically significant differences in salinity, water temperature, and total ammonia nitrogen (tan), nitrate-n (no3-), and nitrite-n (no2) concentrations (p > 0.05) (table 2). the culture medium of p. monodon was consistent with established experimental parameters. variations in ph, dissolved oxygen (do), and floc volume levels were noted between the control (al) and the biofloc treatments. the average concentration of do in the al efficacy of the biofloc system with chitosan on tiger prawn productivity maulianawati et al. treatment (4.54 ± 0.09) was lower than that in abf1 (4.78 ± 0.05 mg/l) and abf2 (4.95 ± 0.07 mg/l). in the biofloc tanks, the volume of flocs increased substantially, which was notably higher than that observed in the control. this escalation in floc volume continued throughout the experiment, with the floc color progressively deepening to a brownish hue. in abf1, the largest floc volume achieved was 25 ml/l, with an average volume of 16.60 ± 5.86 ml/l. the temperature, salinity, dissolved oxygen, and ph levels of the water in all treatments (control and biofloc) met the required parameters for p. monodon, which is consistent with earlier research (santhana kumar et al. 2018; aftabuddin et al. 2020). this study found that the oxygen concentration was lower in the al (control) treatment than in the biofloc treatments. the biofloc system can increase the dissolved oxygen more effectively than the control tanks, even though all treatments use aeration. this is because of the presence of beneficial microorganisms in the biofloc system. microorganisms, such as heterotrophic bacteria, can consume organic matter and excess nutrients in water and convert them into biomass, which can be used as a supplementary food source for shrimp. this process aids in preserving water quality and boosts oxygen levels in the water. in addition, the biofloc system fosters phytoplankton growth, which can generate oxygen via photosynthesis. despite employing aeration in all treatment methods, the biofloc system can be more effective at enhancing the oxygen levels in the water, mainly because of the role of bacteria in reducing organic matter, the balance of the microbial ecosystem that maintains the stability of water quality, and the ability of flocs to act as oxygen buffers (avnimelech 2015; de schryver et al. 2018; crab et al. 2007). the dissolved oxygen level in all treatments was adequate for the survival rate and growth of p. monodon. although the oxygen level did not reach 5 mg/l, the do value in the study was still sufficient to support the survival and growth of p. monodon according to mcgraw et al. (2001) and panigrahi et al. (2020) which stated that do concentration between 4 5 mg/l was still adequate for p. monodon as long as the aeration system is still available in the maintenance media. continuous aeration in the biofloc tank is important for maintaining adequate do levels in all treatments throughout the experimental period (maulianawati et al. 2024). the ph levels in abf1 and abf2 (biofloc treatments), which range from 7.67 to 8.40, were lower than the ph of the control treatment (7.99). the lower ph concentration in abf1 table 2 influence of bioflocs on the physical and chemical properties of culture water throughout the experiment notes: data represent the mean values (± standard error); values within the same row marked with different superscripts indicate statistically significant differences (p < 0.05); al = control; abf1 = biofloc without chitosan; abf2 = biofloc with the addition of chitosan. biotropia vol. 32 no. 2, 2025 product. following the dissolution of carbon dioxide in water, it comes into contact with water molecules (h2o) and reacts to produce carbonic acid (h2co3), a reaction that can decrease the ph of water (ahmed alkhamis et al. 2023). in this study, tan concentrations (0.08 1.67 mg/l) were somewhat higher than those reported in earlier biofloc system studies on p. monodon. this finding has significant implications for our understanding of aquatic systems and their potential impacts on aquatic life. ammonia, the main nitrogenous waste product of aquatic animals, is toxic if it is not adequately removed from the environment. elevated ammonia levels in water impair the ability of organisms to excrete ammonia, leading to a proportional increase in the ammonia levels in the blood of fish and other aquatic species. although the exact mechanism of ammonia toxicity in fish remains unclear, several physiological and histological effects have been linked to high ammonia concentrations in blood and tissues. these effects include increased blood ph, disruption of enzyme systems and membrane integrity, increased water intake, elevated oxygen consumption, gill damage, and histological changes in various internal organs (zhao et al. 2020). meanwhile, a low concentration of nitrite (table 1) and variation in the concentration of nitrate (fig. 1b) were observed in our study. among the harmful nitrogenous compounds used in aquaculture, nitrite toxicity is primarily responsible for hypoxia. the effects of nitrites are linked to its disruption of oxygen transport, resulting from the formation of metahemocyanin when combined with hemocyanin, a compound incapable of effectively transporting oxygen to tissues, leading to increased mortality (wasielesky et al. 2017). in biofloc technology (bft) systems, excessive nitrite accumulation may lead to the suppression of specific enzymes, figure 1 the fluctuation of water quality parameters notes: (a) tan; (b) nitrate-n; and (c) floc volume were measured over a specified time period; al = control; abf1 = biofloc without chitosan (abf1); abf2 = biofloc with the addition of chitosan. and abf2 (biofloc treatments) compared to the control ponds is typically due to the activity of heterotrophic bacteria (hb) in the biofloc system. in water, heterotrophic bacteria consume nutrients and organic matter, resulting in the production of carbon dioxide compound (co2) as a waste efficacy of the biofloc system with chitosan on tiger prawn productivity maulianawati et al. notably carbonic anhydrase, which is a crucial metalloenzyme involved in ion transport within fish and shrimp tissues and organs (abakari et al. 2021). on the other hand, nitrate poses a relatively lower toxicity threat to fish and shrimp, except at very high concentrations exceeding 100 mg/l and when there is a combined action of nitrates and other nitrogenous substances, which can produce synergistic effects (wasielesky et al. 2017). in biofloc systems, which are designed as zero-exchange systems, nitrate toxicity is a critical concern because of the potential for nitrate accumulation to reach harmful or lethal levels. although instances of nitrate toxicity are uncommon, research has shown that nitrates can become problematic in tilapia farming, particularly at concentrations between 600 mg/l and 700 mg/l. at these levels, nitrates primarily affect the fish feed intake (prates et al. 2024). in this study, the nitrite and nitrate levels remained within the safe range for the growth and survival of p. monodon. biofloc technology (bft) systems rely on the breakdown and recycling of nitrogenous compounds derived from uneaten feed and metabolic waste produced by tilapia and shrimp. these compounds are processed by diverse microbial communities, including algae, autotrophic bacteria, and heterotrophic bacteria, each contributing uniquely to nitrogen transformation (emerenciano et al. 2017). the biofloc system contains various nitrogen forms, such as ammonia (nh₃-n), nitrite (no₂-n), nitrate (no₃-n), total ammonia nitrogen (tan), and total kjeldahl nitrogen (tkn), which are metabolized by microorganisms according to their specific metabolic pathways (abakari et al. 2021). under aerobic conditions, nitrifying bacteria, primarily autotrophic bacteria, play a critical role in converting toxic ammonia into less harmful nitrates. this nitrification process is carried out in two stages: first, ammonia is oxidized to nitrite by ammonia-oxidizing bacteria (aob) and archaea (aoa), with hydroxylamine as an intermediate product (martinez-cordova et al. 2015). the key bacterial genera involved in this process include nitrosomonas, nitrosococcus, nitrosospira, nitrosolobus, and nitrospira, all of which have been classified into specific taxonomic groups (kumar et al. 2020). these microorganisms ensure efficient conversion of nitrogenous waste, maintain water quality, and support the health of aquatic organisms in biofloc systems.  schveitzer et al. (2013), panigrahi et al. (2020), and khanjani et al. (2023) highlighted that shrimp cultivation in biofloc systems enhances the water quality through the presence of beneficial microorganisms. khanjani et al. (2023) further noted a marked rise in nitrogen metabolites, including ph, ammonia, nitrite, and nitrate, in biofloc-based nurseries used for rearing p. vannamei. notably, the total ammonia nitrogen (tan) levels were significantly reduced in the biofloc treatments supplemented with probiotics compared to those in the control group. in such systems, heterotrophic bacteria play a key role in assimilating the majority of ammonia, whereas nitrate (no₃-n) in the biofloc system supports the proliferation of phytoplankton. total counts of heterotrophic and vibriolike bacteria the concentration of vibrio-like bacteria in the control tank was substantially greater (8.1 ± 5.0 (×103 cfu/ml)), compared with the levels found in abf1 (3.4 ± 0.09 (×102 cfu/ml)) and abf2 (0.5 ± 0.4 (×103 cfu/ml)) treatments (table 3). in the abf2 sample, thb (total heterotrophic bacteria) counts were notably higher between doc 10 and doc 30, with a concentration of 1.6 ± 0.23 (×105 cfu/ml), whereas the abf1 sample yielded 5.7 ± 1.4 (×104 cfu/ml) and the al sample had 2.7 ± 2.3 (×104 cfu/ml) at the final sampling. the concentration of vibrio-like bacteria (vlb) in the control tank (al) was significantly higher (8.1 ± 5.0 (×103 cfu/ml)) than that in abf1 3.4 ± 0.09 (×102 cfu/ml) and abf2 0.5 ± 0.4 (×103 cfu/ml) treatments (table 3). considerable variation in thb levels was observed across the biofloc treatments, with a statistically significant difference observed at a p value of 0.05. the vlb concentrations were substantially lower in abf1 than those in other biofloc treatments, and this variation was evident across multiple sampling days, with statistical significance at p < 0.05. heterotrophic bacteria use complex organic compounds as carbon sources for growth and metabolic processes, resulting in a specific number of bacteria. heterotrophic bacteria require organic compounds such as glucose and other complex substances as energy and carbon sources, which are used to construct their cell structures. bacteria that obtain nutrients from external sources are crucial for the breakdown of organic substances, reusing vital elements such as carbon, nitrogen, and phosphorus, as well as numerous other ecological biotropia vol. 32 no. 2, 2025 processes (wang et al. 2021; maulianawati et al. 2021; purnomo et al. 2019). heterotrophic bacteria play a crucial role in biofloc systems. cultivation operated by minimizing water exchange, such as in biofloc systems, produces many organic compounds. if not managed, these organic compounds can lead to a decline in water quality. heterotrophic bacteria degrade organic compounds (ferreira et al. 2021; khanjani et al. 2022). results of our study indicated that the concentration of thb in abf2 was greater than that in the control, suggesting that the combined addition of chitosan and probiotics boosted the growth of heterotrophic bacteria. compared to the control, the levels of abf1 thb were lower in the doc 20. several factors, including the dynamics of the bacterial population in the culture medium, likely caused the low thb levels in abf1. the presence of chitosan may have influenced bacterial regeneration in the culture medium. chitosan is a polysaccharide derived from chitin and is known for its antimicrobial properties and ability to interact with microorganisms in various ways (asiri et al. 2022). some studies have shown that chitosan can function as a prebiotic, providing benefits for probiotic mobilization. moreover, chitosan has also been reported to increase the enzymatic activity of probiotics and help improve the microenvironment that supports probiotic growth in the digestive system or in biofloc media (guo et al. 2021; shitu et al. 2022). the concentration of thb in the biofloc system with the addition of chitosan (abf2) was higher than that of the control. however, the average concentration of abf1 was close to the thb concentration in a previous study of aftabuddin et al. (2020). meanwhile, the thb concentrations in al and abf2 were higher than those recorded in the study of aftabuddin et al. (2020). the thb concentration in this study was lower than that reported by kumar et al. (2019), where additional carbon sources were provided in the treatments. adding chitosan to the biofloc media did not significantly affect the increase in the thb concentration (p > 0.05), but provided better results than other treatments, in which the thb value in the abf2 treatment tended to increase significantly. conclusion chitosan addition to the biofloc system resulted in a substantial increase in floc volume and a decrease in vlb populations, while significantly lowering thb. there were substantial differences in the weight, average daily gain, and slaughter growth rates among the treatment groups. no notable discrepancies were observed for food conversion ratio, length, survival rate as well as for total ammonia nitrogen, nitrite, and nitrate concentrations. biofloc technology combined with chitosan supplementation improves the water quality and microbial community, ultimately enhancing the growth performance of p. monodon. acknowledgment gratitude is extended to the department of aquaculture, faculty of fisheries and marine science, universitas borneo tarakan, for the full provision of facilities used in conducting this study. appreciation is also expressed to the directorate of research, technology, and community service (drtpm) of the ministry of education, culture, research, and technology of the republic of indonesia for the support provided under contract number 006/un51.9/sp2h-lt/2023 through the "penelitian dosen pemula (pdp) 2023" program. table 3 total heterotrophic bacteria and total vibrio-like bacteria counts notes: data represent the mean values (± standard error); values within the same column marked with different superscripts indicate statistically significant differences (p < 0.05); al = control; abf1 = biofloc without chitosan; abf2 = biofloc with the addition of chitosan. efficacy of the biofloc system with chitosan on tiger prawn productivity maulianawati et al. references abakari g, luo g, kombat eo. 2021. dynamics of nitrogenous compounds and their control in biofloc technology (bft) systems: a review. aquac fish 6(5):441-7. doi: 10.1016/j. aaf.2020.05.005 aftabuddin s, siddique mam, sein a, dey pk, rashed-un-nabi m, haque ma. 2020. first use of biofloc technology for penaeus monodon culture in bangladesh: effects of stocking density on growth performance of shrimp, water quality and bacterial growth. aquac rep 18:1-7. doi: 10.1016/j.aqrep.2020.100518 ahmed alkhamis y, sultana a, tareq arafat s, abdur rouf m, mustafizur rahman s, thomas mathew r, … abdulaziz aljaafari n. 2023. the impact of biofloc technology on water quality in aquaculture: a systematic meta-analysis. aquac nutr 1:1-20. doi: 10.1155/2023/9915874 asiri f, kim j, chu k-h. 2022. chitosan-harvested polyhydroxybutyrate-rich zobellella denitrificans zd1 as a multifunction feed for aquaculture. aquaculture 561:1-12. doi: 10.1016/j. aquaculture.2022.738655 avnimelech y. 2015. biofloc technology-a practical guide book:3rd edition. sorrento (us): world aquaculture society. bernardi f, zadinelo iv, alves hj, meurer f, dos santos ld. 2018. chitins and chitosans for the removal of total ammonia of aquaculture effluents. aquaculture 483:203-12. doi: 10.1016/j.aquaculture.2017.10.027 brito lo, arana lav, soares rb, severi w, miranda rh, da silva smbc, … gálvez ao. 2014. water quality, phytoplankton composition and growth of litopenaeus vannamei (boone) in an integrated biofloc system with gracilaria birdiae (greville) and gracilaria domingensis (kützing). aquac int 22(5):1649-64. doi: 10.1007/ s10499-014-9771-9 citarasu t, babu mm, yilmaz e. 2022. alternative medications in shrimp health management for improved production. aquaculture 561:1-17. doi: 10.1016/j.aquaculture.2022.738695 crab r, avnimelech y, defoirdt t, bossier p, verstraete w. 2007. nitrogen removal techniques in aquaculture for a sustainable production. aquaculture 270(1-4):1-4. doi: 10.1016/j.aquaculture.2007.05.006 dauda ab, romano n, ebrahimi m, teh jc, ajadi a, chong cm, … kamarudin ms. 2018. influence of carbon/nitrogen ratios on biofloc production and biochemical composition and subsequent effects on the growth, physiological status and disease resistance of african catfish (clarias gariepinus ) cultured in glycerol-based biofloc systems. aquaculture 483:120-30. doi: 10.1016/j.aquaculture.2017.10.016 de schryver p, crab r, defoirdt t, boon n, verstraete w. 2008. the basics of bio-flocs technology: the added value for aquaculture. aquaculture 277(3-4):125-37. doi: 10.1016/j. aquaculture.2008.02.019 emerenciano mgc, martínez-córdova lr, martínez-porchas m, miranda-baeza a. 2017. biofloc technology (bft): a tool for water quality management in aquaculture. water quality 5:92-109. 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6:100109. doi: 10.1016/j.scenv.2024.100109 zhao m, yao d, li s, zhang y, aweya jj. 2020. effects of ammonia on shrimp physiology and immunity: a review. rev aquac 12(4):21942211. doi: 10.1111/raq.12429 biotropia vol. 30 no. 2, 2023: 242 252 doi: 10.11598/btb.2023.30.2.1938 242 indigenous bacillus species isolated from aedes aegypti larvae: isolation, larvicidal toxicity screening, phenotypic characterization, and molecular identification salamun1,2,3,4*, rizky danang susetyo1, hakimatul husniyah1, almando geraldi1,2,3, ni’matuzahroh1,2,3, fatimah1,2,3, farah aisyah nafidiastri5 and nabilatun nisa’6, muhammad fath alhaqqi sanis salamy7 1 laboratory of microbiology, department of biology, faculty of science and technology, universitas airlangga, surabaya, 60115, indonesia 2 research group for applied microbiology and bioresource technology, universitas airlangga, surabaya, 60115, indonesia 3 university of co-e-research center for bio-molecule engineering, universitas airlangga, surabaya, 60115, indonesia 4 laboratory of entomology, institute of tropical diseases, universitas airlangga surabaya, 60115, indonesia 5 laboratory of microbiology, department of biology, faculty of mathematic and natural sciences, universitas negeri surabaya, jl. ketintang surabaya, 60231, indonesia 6 laboratory of molecular genetics, department of biology, faculty of science and technology, universitas airlangga, surabaya, 60115, indonesia 7 department of medical physiology and biochemistry, faculty of medicine, airlangga university, kampus a jl. mayjen prof. moestopo 47, surabaya, 60131, indonesia received 7 march 2023 / revised 15 may 2023 /accepted 15 may 2023 abstract vector-borne diseases transmitted by mosquitoes are considered a significant public health problem worldwide. aedes aegypti is one of the mosquito species responsible for transmitting these diseases. one environmentally friendly method of vector control is the use of microbial agents such as bacillus species. this study aimed to explore investigate indigenous entomopathogenic bacteria of bacillus species isolated from a. aegypti larvae. larvae samples were collected from breeding sites of a. aegypti. all isolates underwent screening and affirmation confirmation tests to assess their larvicidal toxicity against a. aegypti larvae. phenotypic characterizations and molecular identifications were conducted to determine the species of the bacillus isolates based on similarity index and percent identity (%id). phylogenetic trees were used to compare the isolates with other bacillus species. the results revealed 120 isolates of bacillus species from a. aegypti larvae samples. among them, three isolates (ls3.3, ls9.1, and lsd4.2) exhibited the highest larvicidal toxicity in the confirmation test, resulting in larval mortality rates of 100%, 96.7%, and 100%, respectively, after 48 hours of exposure. molecular identifications, showed that lsd4.2 had a 99.16% id with bacillus velezensis, ls3.3 had a 98.22% id with bacillus mojavensis, and ls9.1 had a 99.93% id with bacillus subtilis. these three bacteria from the bacillus genus have been reported to offer significant benefits to humans. keywords: aedes aegypti, bacillus mojavensis, bacillus subtilis, bacillus velezensis, dengue vector, larvicidal toxicity introduction dengue fever (df) is a vector-borne infection transmitted by mosquitoes, which is considered a significant public health problem worldwide (dahmana et al. 2020). aedes aegypti is the mosquito species responsible for transmitting this disease. various attempts have been made to address the issue of df, but the outcomes have fallen short of expectations. extensive research has been conducted on developing vaccines to prevent this disease; however, satisfactory results have yet to be achieved. one alternative to combatting this disease is controlling the population of the vector (melanie et al. 2018). several measures have been taken to suppress the population *corresponding author, email: salamun@fst.unair.ac.id indigenous bacilus species isolated from aedes aegypti larvae – salamun et al. 243 of a. aegypti, including the use of chemical insecticides. however, the use of chemical insecticides has negative implications for environmental quality and is toxic to non-target organisms present in breeding sites for a. aegypti larvae (dahmana et al. 2020). experts have suggested the development of bioinsecticides as biocontrol agents for disease vectors in response to the df problem (thomas 2018). bioinsecticides are known to possess advantages such as specificity and safety for non-target organisms and the environment. one of the biocontrol agents being developed is entomopathogenic bacteria from the genus bacillus. bacillus sp. has been proven to be effective and highly specific, particularly toxic to the a. aegypti mosquito. certain bacillus species are capable of producing protein crystals along with spores during sporulation (evdokimov et al. 2014). numerous studies have demonstrated that multiple bacterial strains within the bacillus genus have the potential to eliminate a. aegypti larvae, including b. thuringiensis and b. sphaericus (boyce et al. 2013). these species exhibit high toxicity towards mosquito larvae while being safe for other parasites, predators, and mammals, in addition to causing no environmental pollution (melanie et al. 2018). in general, bacillus sp. can form endospores when confronted with unsuitable growth conditions that compromise their survival structure (zeigler & perkins 2015). the isolation and characterization of indigenous strains of b. thuringiensis from saudi arabia have been carried out (el-kersh et al. 2016). sixtyeight isolates have demonstrated larvicidal potential against the malaria disease vector, anopheles gambiens (el-kersh et al. 2016). similarly, b. sphaericus was isolated and characterized on lombok island, showing potential as a bio-insecticide for controlling the malaria vector a. aconitus (suryadi et al. 2016). salamun et al. (2021) recently isolated a bacillus species, bacillus thuringiensis bk5.2, from baluran national park, east java, indonesia, which displayed high toxicity against a. aegypti larvae. b. thuringiensis strains isolated and characterized from lebanese soils have also proven to be effective (fayad et al. 2019). these strains have been developed as bioinsecticides targeting agricultural pest insects (kumar et al. 2021). numerous scientific studies have explored the role of biocontrol agents and their potential in disease vector control (thomas 2018). toxins produced by bacillus sp. exhibit specific activity against target insects (schünemann et al. 2014). microbial larvicides can be employed as environmentally friendly biological agents for disease vector control (benelli et al. 2016). building on previous studies utilizing natural soil samples collected in baluran national park, east java, indonesia (salamun et al. 2021), our research aims to identify the diversity of bacillus species isolated from a. aegypti larvae in df endemic areas. this study aims to isolate indigenous entomopathogenic bacillus sp. from samples of a. aegypti larvae in their breeding sites in df endemic areas, conduct screening and affirmation tests to determine the larvicidal toxicity of the isolates against a. aegypti larvae, perform phenotypic characterizations, and conduct molecular identification. the findings are expected to contribute to the development of diverse entomopathogenic bacillus species as potential agents for the biocontrol of disease vectors, plant diseases, and pests. materials and methods materials the materials and tools utilized in this study were employed for the isolation, larvicidal toxicity screening, phenotypic characterization, and molecular identification of bacillus sp. from the aforementioned isolation. samples of a. aegypti larvae were collected from water reservoirs serving as breeding sites for a. aegypti in gresik, surabaya, and sidoarjo, east java, indonesia. screening and affirmation of the larvicidal toxicity of bacillus sp. were performed using third-instar a. aegypti larvae. the a. aegypti larvae were obtained from the tropical disease institute, universitas airlangga, surabaya, indonesia. sampling of aedes aegypti larvae samples of a. aegypti larvae were collected from mosquito breeding sites, specifically water reservoirs. identification of a. aegypti larvae samples was conducted following the identification key of a. aegypti larvae (bar & biotropia vol. 30 no. 2, 2023 244 andrew 2013). previous studies have shown that bacillus can be isolated from various sources, including soil, aquatic environments, herbivorous droppings, forest soil, dead insects, and mosquito breeding sites (paul 2007; zeigler & perkins 2015; suryadi et al. 2016). five larvae per sample were extracted using a pipette and placed in sterile glass bottles. isolation of bacillus sp. bacillus sp. was isolated from the laboratory of microbiology, department of biology, faculty of science and technology, universitas airlangga, as conducted by suryadi et al. (2016). five a. aegypti larvae were sampled from a suspected location infected with entomopathogenic bacteria, where the larvae exhibited minimal or slow movement on the water's surface. all larvae were placed in a sterile test tube and macerated, followed by the addition of 9 ml of 0.85% nacl solution. the mixture was allowed to sit for 5 minutes. a 101102 dilution of the sample was prepared, heated at 70°c for 30 minutes, and then inoculated with 1 ml of nutrient agar (na) using the pour plate method onto a sterile petri dish. the solidified media was incubated at 30°c for 48 hours. the resulting colonies were subjected to spore stain. the bacillus colonies were isolated on na media using the streak method and stored at 4°c (suryadi et al. 2016). larvicidal toxicity screening of bacillus sp. a pure bacillus sp. isolate was inoculated into a sterile glass container containing 10 ml of nutrient yeast salt medium (nysm) and incubated on a rotary shaker incubator at room temperature (35°c) for 48 hours (suryadi et al. 2016). the absorbance value of the bacillus sp. suspension was measured using a spectrophotometer at a wavelength of 600 nm (od600nm). for the screening of larvicidal toxicity, ten third-instar larvae of a. aegypti reared at the entomology laboratory of the institute of tropical diseases, airlangga university, were inoculated with a 5 ml suspension of bacillus sp. in a bottle containing 45 ml of tap water. the control group consisted of 45 ml of well water, 5 ml of nysm, and 10 a. aegypti larvae (suryadi et al. 2016). the percentage of larvae that died after 24 and 48 hours of exposure was calculated. the screening for larvicidal toxicity was conducted with three replicates, using a mortality range of 60-100 larvae, and the absorbance was set to 0.8. phenotypic characterizations morphological characterizations were conducted to determine the macroscopic and microscopic characteristics, such as the colony shape and the spore location. three bacillus sp. isolates with the highest potential were cultured on petri dishes containing 8 ml of na media using the streak method. the plates were then incubated for 48 hours and stained using the spore staining method. physiological characterization included testing for indole production, motility, oxidase activity, starch hydrolysis, and salinity tolerance. additionally, the microbact 12a/12b kit was used for an additional test. for this test, 225µl of bacterial suspension was taken and added to each well of the kit. one drop of immersion oil was added to each well, and the results were observed after incubation at 37°c for 24 hours. molecular identification molecular identification of bacterial isolates was conducted through the 16s rrna gene (kumar et al. 2016; johnson et al. 2019). initially, an isolated culture in 20 ml of nb media was incubated at 120 rpm and room temperature (35°c) for 48 hours. dna extraction was performed using the ctab method. the concentration and purity of the dna were determined at 280 nm and 260 nm using the multiskan go. the 16s rrna gene was amplified using the eppendorf mastercycler tool and the pcr method. the process involved adding gotaq green master mix and primers 16s rrna, p0 (5'-gag agt ttg atc ctg gct cag-3') and p6 (5'-cta cgg cta cct tgt tac ga-3'). the steps included denaturation at 94°c for 2 minutes, denaturation at 92°c for 30 seconds, annealing at 55°c for 30 seconds, elongation at 72°c for one minute, and final elongation at 72°c for 5 minutes, repeated for 35 cycles of polymerase chain reaction (pcr). amplicons were sequenced, and similarity analysis was conducted by comparing the data in genbank using ncbi's blastn. the pcr visualization results were obtained by indigenous bacilus species isolated from aedes aegypti larvae – salamun et al. 245 electrophoresis of a 1% agarose gel stained with ethidium bromide and observed under uv light. bacterial relationship analysis was performed by constructing a phylogenetic tree using the mega 6.0 application (tamura et al. 2013). data analysis the results of the isolation and larvicidal toxicity screenings were analyzed using descriptive analysis. bergey's manual of systematic bacteriology (paul et al. 2009) was utilized to obtain data on morphological and physiological properties of the local bacillus sp. the similarity index percentage was calculated based on the positive and negative similarity of the characters of each isolate to determine the bacterial species of bacillus sp. (paul et al. 2009). based on phenotypic characteristics, the similarity percentage pointed towards bacillus thuringiensis and bacillus sphaericus, bacteria that have demonstrated larvicidal activity against a. aegypti larvae. the 16s rrna gene, which had been amplified by pcr and confirmed by electrophoresis, was further purified and sequenced to determine the sequence of the 16s rrna gene in bacterial isolates. the pcr results were then submitted to malaysia's first base dna sequencing service. the sequencing results were edited using bioedit sequence alignment editor software version 7.2.5, and the similarity of the isolated 16s rrna gene of bacillus sp. with the gene data of bacteria in genbank was determined using the basic local alignment search tools (blast). the nucleotide blast analysis was conducted by the national center for biotechnology information at the national library of medicine in washington, dc and can be accessed at https://blast.ncbi.nlm.nih.gov/. results and discussion sampling and isolation of bacillus sp. in the isolation of 30 samples (150 larvae) of a. aegypti larvae from surabaya, gresik, and sidoarjo, east java, indonesia, and the map of sampling locations shown in figure 1, there were 120 isolates of bacillus sp. (table 1). larvicidal toxicity screening results with varying od600nm values were obtained for isolates of bacillus sp., which exhibited potential diversity as entomopathogenic bacillus sp. (table 1 and figure 2a). figure 1 map of sampling locations: gresik (gr), surabaya (sb), and sidoarjo (sd), east java, indonesia https://blast.ncbi.nlm.nih.gov/ biotropia vol. 30 no. 2, 2023 246 table 1 potency of indigenous bacillus sp. isolates (od600nm varies) based on the results of larvicidal toxicity screening against aedes aegypti third instar larvae at 48-hour exposure sampling location (city) global positioning systems (gps) of sampling locations sample codes number of isolates collection screening results of larvicidal toxicity culture turbidity of bacillus sp. isolates (od600nm) up lp mp hp surabaya s07’03.293é112’42.460’ s07’03.293é112’42.452’ s07’03.293é112’42.447’ s07’03.293é112’42.434’ s07’03.293é112’42.438’ s07’03.293é112’42.455’ s07’03.293é112’42.446’ s07’03.293é112’42.445’ s07’03.293é112’42.452’ s07’03.293é112’42.443’ ls1 ls2 ls3 ls4 ls5 ls6 ls7 ls8 ls9 ls10 2 4 5 6 4 4 4 3 3 4 0 1 0 0 2 1 3 0 1 2 1 1 1 4 1 2 1 3 1 1 1 2 1 0 1 1 0 0 0 0 0 0 3 2 0 0 0 0 1 1 1.50 – 1.50 1.00 – 1.25 0.80 –1.50 0.80 – 1.30 1.00 – 1.20 1.10 –1.50 0.80 – 1.15 1.00 – 1.35 0.85 – 1.40 0.95 – 1.50 gresik s07’03.293é112’34.459’ s07’03.293é112’34.471’ s07’03.293é112’34.437’ s07’03.293é112’34.436’ s07’03.293é112’34.484’ s07’03.293é112’34.515’ s07’03.293é112’34.536’ s07’03.293é112’34.530’ s07’03.293é112’34.948’ s07’03.293é112’34.965’ lg1 lg2 lg3 lg4 lg5 lg6 lg7 lg8 lg9 lg10 2 4 4 4 4 6 5 2 5 6 0 3 0 0 4 2 0 1 1 0 2 1 2 2 0 4 3 0 2 4 0 0 2 2 0 0 1 0 2 1 0 0 0 0 0 0 1 1 0 1 1.10 –1.10 1.00 – 1.50 0.40 – 1.30 0.85 – 1.40 0.95 – 1.50 0.80 – 1.50 0.50 – 1.20 1.00 – 1.40 0.80 – 1.50 1.00 – 1.50 sidoarjo s07’03.293é112’45.472’ s07’03.293é112’45.578’ s07’03.293é112’45.491’ s07’03.293é112’45.264’ s07’03.293é112’45.624’ s07’03.293é112’45.623’ s07’03.293é112’45.432’ s07’03.293é112’45.536’ s07’03.293é112’45.542’ s07’03.293é112’45.541’ lsd1 lsd2 lsd3 lsd4 lsd5 lsd6 lsd7 lsd8 lsd9 lsd10 6 4 3 2 3 5 4 3 3 6 3 2 0 0 1 4 2 0 0 2 3 2 1 0 1 0 0 1 2 4 0 0 2 0 1 1 0 2 0 0 0 0 0 2 0 0 2 0 1 0 0.75 – 1.40 0.55 – 1.50 1.10 – 1.30 0.90 – 1.10 0.80 – 1.35 1.10 – 1.40 0.40 – 0.95 0.90 – 1.40 1.10 – 1.15 0.65 – 1.50 120 35 50 20 15 0.40 – 1.50 descriptions: up = un-potential, larval mortality 0%; lp = low-potential, larval mortality <30%; mp = mediumpotential, larval mortality 30-50%; hp = high-potential, larval mortality >50%. figure 2 results of the larvicidal toxicity screening (a) (od600nm varies) with one replication and the affirmative toxicity test (b) (od600nm = 0.80) with three replications, performed on 15 isolates of indigenous bacillus sp. from gresik (lg), surabaya (ls), and sidoarjo (lsd) against aedes aegypti third-instar larvae at 24and 48-hour exposure indigenous bacilus species isolated from aedes aegypti larvae – salamun et al. 247 larvicidal toxicity screening of bacillus sp. the results of the affirmative toxicity test (fig. 2b) were conducted at turbidity of 0.80 (od600nm) from cultures of bacillus sp. isolates. the correlation between turbidity and the concentration of bacillus sp. (cfu/ml) yielded a regression line of y=151.5+17.6, with a coefficient of determination (r2) of 0.9525, as depicted in figure 3. based on calculations, turbidity of 0.8 in bacillus sp. cultures is equivalent to a bacterial cell count of 13.8x107 cfu/ml. following the affirmative toxicity test (fig. 2b), the three isolates with the highest potential underwent phenotypic characterizations. the results of the phenotypic characterizations for these three isolates are presented in figure 4 and table 2. figure 3 standard curve for quantifying bacillus sp. cell count (cfu/ml) in lsd4.2 isolate culture using optical density (od600nm) variation phenotypic characterizations the ls3.3 and ls9.1 isolates exhibited colonies with irregular shapes and flat elevations, while the lsd4.2 isolate had circular colonies with raised elevations. the size of the colonies for all three isolates was moderate. the margins of ls3.3, ls9.1, and lsd4.2 isolates were lobate, serrate, and entire, respectively. microscopic characterization using spore staining (fig. 4) revealed that ls3.3 and lsd4.2 isolates had spherical spores located at the terminal end, while the ls9.1 isolate had ovalshaped spores located at the subterminal end. detailed phenotypic characterizations are provided in table 2. figure 4 spore location of endospores in local bacillus sp. isolates using spore staining. descriptions: a) lsd4.2 isolate; b) ls9.1 isolate; c) ls3.3 isolate . table 2 phenotypic characterizations based on the physiological tests of bacillus sp. isolates coded lsd4.2, ls9.1, and ls3.3 no. physiological tests characteristics of bacillus sp. lsd4.2 ls9.1 ls3.3 1. lysine + 2. ornithine 3. h2s 4. glucose 5. mannitol 6. xylose + + 7. onpg + + 8. indole 9. urease + 10. vp + + + 11. citrate 12. tda 13. gelatin + + + 14. malonate + 15. inositol 16. sorbitol 17. rhamnose 18. sucrose 19. lactose 20. arabinose + + 21. adonitol 22. raffinose 23. salicin 24. arginine 25. motility + + + 26. katalase + + + 27. oksidase + 28. salinity 5% + + 29. salinity 10% 30. hidrolysis of amylum + + + molecular identification the results of pcr amplification of the 16s rrna gene for three bacillus sp. isolates, confirmed by electrophoresis, are shown in figure 5. the third band of bacillus sp. appeared at approximately 1500 bp. biotropia vol. 30 no. 2, 2023 248 figure 5 confirmation of the 16s rrna gene in three bacillus sp. isolates using electrophoresis methods. (descriptions: s1 = lsd4.2; s2 = ls3.3; s3 = ls9.1; m = marker) table 3 shows the results of sequencing to identify the similarity of the 16s rrna gene for bacillus sp. using blast. isolate code lsd4.2 had a 99.16% identity with bacillus velezensis, ls3.3 had a 98.22% identity with bacillus mojavensis, and ls9.1 had a 99.93% identity with bacillus subtilis, respectively. the results of constructing the phylogenetic tree of bacillus sp. on genbank are shown in figure 6. table 3 similarity of bacillus sp. based on sequencing of the 16s rrna gene using the basic local alignment search tools (blast) program isolates code spesies name accession no. e value % id query cover (%) lsd4.2 bacillus velezensis strain cbmb205 nr_075005.2 0.0 99.16 99 bacillus velezensis strain fzb42 nr_116240.1 0.0 99.02 99 ls9.1 bacillus subtilis subs. inaquosorum strain bgsc 3a28 nr_104873.1 0.0 99.93 100 bacillus subtilis strain jcm 1465 nr_113265.1 0.0 99.86 100 ls3.3 bacillus mojavensis strain ifo15718 nr_024693.1 0.0 98.22 99 bacillus halotolerans strain lmg 22477 nr_115931.1 0.0 98.11 99 figure 6 phylogenetic tree of bacillus sp. isolates coded lsd4.2, ls9.1, ls3.3, and their relationship to other bacillus sp. in the genbank database https://www.ncbi.nlm.nih.gov/nucleotide/nr_075005.2?report=genbank&log$=nucltop&blast_rank=1&rid=ertby417016 https://www.ncbi.nlm.nih.gov/nucleotide/nr_116240.1?report=genbank&log$=nucltop&blast_rank=2&rid=ertby417016 https://www.ncbi.nlm.nih.gov/nucleotide/nr_024693.1?report=genbank&log$=nucltop&blast_rank=2&rid=erv37gap01r https://www.ncbi.nlm.nih.gov/nucleotide/nr_115931.1?report=genbank&log$=nucltop&blast_rank=3&rid=erv37gap01r indigenous bacilus species isolated from aedes aegypti larvae – salamun et al. 249 in this study, the initial objectives were to isolate b. thuringiensis or b. sphaericus and screen their toxicity to a. aegypti larvae. variations in the mortality rate of a. aegypti larvae due to exposure to bacillus sp. were observed. thirdinstar larvae of a. aegypti were used for both screening and confirming the larvicidal toxicity of bacillus sp. (table 1; fig. 2a and 2b). a total of 120 isolates could be isolated from 150 samples of a. aegypti larvae collected from surabaya, sidoarjo, and gresik cities in east java, indonesia. among them, 15 isolates showed high potency in the larvicidal toxicity screening. the affirmation test of larval toxicity (fig. 2b) revealed that three isolates exhibited the highest toxicity. the larvicidal toxicity screening using third-instar a. aegypti larvae was based on their sensitivity to entomopathogenic bacterial toxins (kim et al. 2017). the older the larval instar, the lower their sensitivity to the bacterial toxin. additionally, fourth-instar larvae exhibit less feeding habits compared to younger larvae, resulting in reduced consumption of bacterial toxins. furthermore, during the pupal phase, feeding activity ceases (aynalem 2022). in the affirmation test of bacillus sp. lsd4.2 (fig. 3), a concentration of 13.8 x 107 cfu/ml, caused 100% larval mortality after 48 hours of exposure, categorizing it as highly toxic. b. thuringiensis pwr4.32, isolated in malang, indonesia, exhibited a lethal concentration 50% (lc50) value of 22.79 x 107 cells/ml after 72 hours of exposure (gama et al. 2010). similarly, b. thuringiensis w.swh.s.k2, isolated in nganjuk, indonesia, had an lc50 value of 3.53 x 107 cells/ml after 48 hours of exposure (pratiwi et al. 2013). b. thuringiensis bk5.2, isolated from baluran national park in east java, indonesia, showed an lc50 value of 8.3 x 106 cells/ml after 48 hours of exposure (salamun et al. 2021). the results of this study indicate differences in larvicidal toxicity among different bacillus sp. isolates, suggesting that these isolates may belong to different species or strains. bacillus sp. larvicidal toxicity can be identified through two mechanisms of action. during sporulation, bacteria produce an insecticidal toxin stored in parasporal inclusions. during the vegetative stage, bacteria produce secondary metabolites, such as enzymes or other chemical compounds, that are also insecticidal. the entomopathogenic action of bacillus sp. involves the toxin produced during sporulation, which binds to intestinal cell receptors, causing pores to form in the intestinal cell membrane. this leads to the entry of ions to balance intracellular and extracellular fluids. consequently, intestinal cells experience rapid damage, resulting in the lysis of epithelial cells. infected larvae stop feeding for several hours, ultimately leading to their death (polenogova et al. 2022). the endotoxin in the parasporal inclusion of the entomopathogenic bacillus sp. also reduces the blood's acidity (ph), leading to larval death due to septicemia (poopathi et al. 2013). other actions of entomopathogenic bacillus sp. as bioinsecticides have also been reported. bacillus sp. produces secondary metabolites, including biosurfactants, during bacterial growth in suitable media. the biosurfactant produced by the b. subtilis strain is composed of a mixture of molecules, some of which are toxic to arthropods and vectors (sachdev & cameotra 2013). biosurfactant-producing bacteria have been found to be effective in controlling diseases in plants and insects (zhao et al. 2014). biosurfactants can affect the cuticle of insects due to their amphiphilic nature, which includes hydrophobic and hydrophilic molecules. this can damage cell membranes and epithelial cells and ultimately cause death (zhao et al. 2014). based on the phenotypic characteristics (table 2 and fig. 4) and identification using bergey's manual of systematic bacteriology, the isolates coded lsd4.2 and ls3.3 showed similarity indices of 82.6% and 63.3%, respectively, with b. sphaericus. isolate ls9.1 had a similarity index of 62.50% with b. thuringiensis. however, based on molecular identification using the 16s rrna gene, these three bacillus sp. isolates showed different results. they were identified as b. velezensis, b. mojavensis, and b. subtilis (table 3; fig. 6). b. velezensis fzb42t, previously classified as part of the b. subtilis group due to its 99% genetic similarity, was later included in a different phylogenomics category based on additional genetic characteristics. this strain of b. velezensis produces unique intracellular biomolecules that have the potential for development through genetic engineering in various industries, including health, pharmaceuticals, environment, and food, particularly in agriculture (adeniji et al. 2019). biotropia vol. 30 no. 2, 2023 250 studies have shown that b. velezensis nkg-2 is useful as a potential biocontrol agent and promoter of plant growth (myo et al. 2019). b. velezensis strain wlys23 has great potential as a biocontrol agent for disease control in freshwater aquaculture (zhang et al. 2021). b. velezensis 33rb is a potential alternative to chemical pesticides as a biological control agent for phytopathogens, offering environmentally friendly and sustainable properties (dawwam & sehim 2022). the search for new biocontrol agents focuses on bacillus subtilis and its related species, including bacillus mojavensis. the metabolites produced by the b. mojavensis ps17 isolate from wheat germ inhibit the growth of the plant pathogen fusarium spp., indicating its potential as a biocontrol agent for agriculture (diabankana et al. 2021). b. mojavensis shares similarities with b. subtilis but differs in fatty acid composition, dna sequences, and resistance to genetic transformation (bacon & hinton 2002). b. mojavensis produces surfactin, iturin, and fengycin, which belong to an antimicrobial and antifungal lipopeptide group (mounia et al. 2014; blacutt et al. 2016). according to jasim et al. (2016), the lipopeptide compounds surfactin and fengycin in b. mojavensis have antimicrobial activity against pathogenic bacteria, including both gram-negative and gram-positive strains. hmidet et al. (2017) reported that b. mojavensis produces surfactin and fengycin, with optimal production occurring in media containing glucose. b. mojavensis demonstrated hemolytic activity on blood agar, suggesting the production of biosurfactants (berekaa & ezzeldin 2018). b. mojavensis btcb15 is capable of producing 2.3 nm agnps and exhibits antibacterial activity against numerous drug-resistant pathogens (iqtedar et al. 2019). in their study, fanaei et al. (2021) discovered that b. mojavensis hf produces three types of lipopeptides: surfactin, fengycin, and kurstakin. they identified a wide variety and number of surfactin and fengycin isomers compared to previous reports and claimed to be the first to report the presence of kurstakin in bacillus mojavensis species. further research is needed to determine whether kurstakin is stored in parasporal inclusions or excreted as secondary metabolites. b. subtilis also produces biosurfactant as a mosquitosidal toxin (kumar et al. 2022). mosquitosidal toxin activity has also been reported from b. cereus (mani et al. 2017). biosurfactants, synthetic compounds produced by several strains of bacillus sp., have been used as biocontrol agents against insects (mani et al. 2017). for example, b. subtilis isolated from soil has been introduced as a biological control agent for insects due to its production of surfactin (kumar et al. 2022). b. subtilis (mw644765) mediated silver nanoparticles (agnp) have shown promising larvicidal activity against mosquito larvae, making them a potential biocontrol agent for reducing mosquito populations (wilson et al. 2022). b. subtilis is considered a universal cell factory for various industries such as agriculture, biomaterials, pharmaceuticals, and industry (su et al. 2020). molecular identification results have identified three high-potential bacillus species: b. subtilis (ls9.1), b. velezensis (lsd4.2), and b. mojavensis (ls3.3). commercial products derived from b. thuringiensis and b. sphaericus have been used for the control of a. aegypti larvae (boyce et al. 2013). the discovery of b. velezensis, b. mojavensis, and b. subtilis in this study is highly significant. these bacteria have been reported as multifunctional bacteria in various industries, including health, pharmaceuticals, environment, and food, and as biocontrol agents for disease vectors, plant pests, and disease control in freshwater aquaculture. conclusion the results of the isolation and larvicidal toxicity screenings of bacillus sp. against aedes aegypti larvae revealed a range of potential larvicidal toxicity levels, varying from low to high. screening 120 isolates of bacillus sp. for larvicidal toxicity identified 15 isolates with high potency. the confirmation test identified three isolates with the highest potential. the larval mortality rates due to exposure to isolates ls3.3, ls9.1, and lsd4.2 were 100%, 96.7%, and 100%, respectively, after 48 hours of exposure. molecular identification using the 16s rrna gene revealed the diversity of the isolates, with isolate lsd4.2 sharing 99.16% identity with bacillus velezensis, ls3.3 sharing 98.22% identity with bacillus mojavensis, and ls9.1 sharing 99.93% identity with bacillus subtilis. these three bacteria, belonging to the bacillus genus, offer significant benefits for humans. indigenous bacilus species isolated from aedes aegypti larvae – salamun et al. 251 references adeniji aa, loots dt, babalola oo. 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1907-770x 299 morphological characteristics and total flavonoid content in peel extracts of four banana cultivars from indonesia (musa spp.) hida arliani na1, 2 , supriyadi supriyadi3, rina sri kasiamdari4, and budi s. daryono4* 1biology department, faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia 2biology department, faculty of science, itera, lampung 35365, indonesia 3department of food and agricultural product technology, faculty of agricultural technology, universitas gadjah mada, yogyakarta 55281, indonesia 4department of tropical biology, faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia article information received : 13 january 2025 revised : 10 october 2025 accepted : 13 october 2025 *corresponding author, e-mail: bs_daryono@mail.ugm.ac.id research paper abstract banana is a commodity with high genetic diversity in indonesia, often leading to identification issues due to synonymy and homonymy. accurate morphological characterization is essential for germplasm management and breeding programs. concurrently, high consumption of banana generates significant banana peel waste, which is a potential source of bioactive compounds like flavonoids. this study aimed to characterize the morphology of four banana varieties (cavendish, kepok, raja, klutuk) from the yogyakarta banana germplasm garden and analyze the total flavonoid content (tfc) in banana peels. morphological characterization was conducted following the ipgri descriptor list, and tfc was measured spectrophotometrically and expressed as mg quercetin equivalent per gram (mgqe/g). the results revealed distinct morphological profiles for each variety, with the wild-type klutuk banana exhibiting the tallest pseudostem and seedy fruit. the tfc analysis showed a significant variation, where klutuk peel had the highest content (0.453 mg qe/g), followed by kepok (0.386 mg qe/g), cavendish (0.146 mg qe/g), and raja (0.139 mg qe/g). the high flavonoid content in the morphologically distinct klutuk variety suggests a correlation between its wild morphological traits and enhanced production of defensive phytochemicals. these findings underscore the value of morphological data for identifying cultivars with high nutraceutical potential, promoting the utilization of banana peel waste as a source of natural antioxidants. keywords: banana peel, germplasm, morphological characteristics, musa spp., tfc introduction bananas are crucial economic and food commodity in indonesia. the country’s extensive production and harvest area have established it as a center of banana distribution in southeast asia. a key institution for conservation and research is the yogyakarta banana plasma nutfah garden, which maintains over 300 banana accessions to support the nation’s farmers (kebun plasma nutfah pisang 2016). however, accurate identification is significantly challenging due to the high genetic diversity of bananas. problems of synonymy (different cultivars sharing the same name) and homonymy (the same cultivar having different names) are common. detailed morphological characterization is therefore essential, serving as a primary tool for accurate identification and as a foundation for assembling superior varieties by identifying valuable agronomic traits (riandini et al. 2018). alongside its agricultural importance, the banana industry generates substantial waste, with copyright (c) 2025@author(s). article higlights • wild banana morphology links to high peel flavonoid content. • klutuk banana peel has the highest antioxidant potential. • morphological traits can predict nutraceutical value in bananas. • banana peel waste is a valuable source of natural antioxidants. • conservation of wild germplasm is crucial for bioactive compounds. https://doi.org/10.11598/btb.2025.32.3.2426 https://creativecommons.org/licenses/by-nc-nd/4.0/ biotropia vol. 32 no. 3, 2025 300 banana peels constituting about 30% of the fruit’s weight. currently, this by-product is underutilized in indonesia, often discarded or used as animal feed. this represents a missed economic opportunity, as banana peels are a rich source of antioxidant phytochemicals, particularly flavonoids like anthocyanin, delphinidin, and cyanidin (seymour 1993). promisingly, peels from popular indonesian cultivars such as kepok, raja, and klutuk have demonstrated strong antioxidant activity, with ic50 values of 9.702 ppm, 46.86 ppm, and 1.92 μg/ml, respectively (jami’ah et al. 2018; rahmi et al. 2021; nofianti et al. 2021). while the antioxidant potential of banana peels is recognized, the critical link between the morphological identity of a cultivar and the flavonoid content of its peel remains unexplored. previous studies have treated these aspects in isolation by describing morphology for taxonomy or analyzing peel chemistry without rigorous cultivar-specific morphological data. this has created a knowledge gap where valuable phenotypic markers for high-value biochemical traits are unknown. our research directly addresses this gap by integrating detailed morphology with biochemical analysis to investigate a potential correlation. to this end, we selected four commercially significant and genetically distinct cultivars, including the globally dominant cavendish (aaa), and three locally important indonesian varieties, kepok (abb), raja (aab), and klutuk (bb). this selection provides a representative sample across different genomic groups and consumption patterns, enabling a robust comparative analysis to determine if morphological characteristics can serve as visual indicators for a cultivar’s nutraceutical potential. therefore, this study aimed to: 1) perform a detailed morphological characterization of these four banana varieties from the yogyakarta banana plasma nutfah garden and 2) analyze the flavonoid content of their peel extracts. the ultimate goal was to determine if a correlative relationship exists, thereby contributing to the better utilization of both indonesia’s banana genetic resources and its agro-industrial waste. materials and methods morphological observation of bananas morphological observation was carried out on four 42-day-old banana accessions, including cavendish, kepok, raja, and klutuk. each accession consisted of three individuals planted in the yogyakarta banana germplasm garden area according to the designated planting zones. the tools used included stationery, label paper, and measuring instruments such as rulers, tape measures, and cameras. morphological observations were carried out following the musa descriptor list (ipgri 1996) (table 1). qualitative morphological data were presented in descriptive form. measurement of total flavonoid levels equivalent to quercetin the measurement of total flavonoids in banana peels was carried out by preparing banana peel samples, producing the banana peel extracts, and determining the total flavonoid content. preparation of banana peel samples fourteen-week-old bananas were harvested. the peels were separated from the fruits, cleaned, airdried, and ground using a blender. the resulting peel powder was stored at room temperature. making banana peel extract banana peel powder was macerated in 96% ethanol (1:1 w/v). the macerate was filtered using a buchner funnel and concentrated with a rotary evaporator to produce a thick extract, which was stored at 4°c for subsequent total flavonoid content analysis (uckaya et al. 2022). total flavonoid content (tfc) levels equivalent to quercetin banana peel extract samples (100 mg) were dissolved in 100 ml of methanol. then, 1 ml of the sample solution was mixed with 3 ml of methanol, 0.2 ml of 10% alcl₃, 0.2 ml of potassium acetate, and diluted with distilled water to a final volume of 10 ml. the mixtures were incubated at room temperature in the dark for 30 minutes. absorbance was measured at a wavelength (λ) of 431 nm. a quercetin standard solution with concentrations ranging from 10 – 50 mg/l was prepared to construct a calibration curve. total flavonoid content was calculated using the quercetin linear regression equation, y = ax + b (ahmad et al. 2015). morphological characteristics and total flavonoid content in peel extracts of banana cultivars arliani et al. 301 table 1 qualitative characters of bananas no character subcharacter 1 leaf habitus (1) erect; (2) intermediate; (3) drooping; (4) other (e.g., very drooping, specify in descriptor). 2 pseudostem height (m) measured from the base of the pseudostem to where the bunch stalk emerges (1) ≤ 2; (2) 2.1 – 2.9; (3) ≥ 3. 3 pseudostem aspect/ diameter (cm) measured at a pseudostem height of 100 cm (1) slender; (2) normal; (3) robust. 4 pseudostem color observe the color of the pseudostem after one layer of the outer leaf sheath is removed (1) green yellow; (2) medium green; (3) green; (4) dark green; (5) greenish-red; (6) red; (7) reddish-purple; (8) blue; (9) chimerical; (10) other. 5 pigmentation on the pseudostem observe the pigmentation color that appears on the pseudostem (1) pinkpurple; (2) red; (3) purple; (4) other. 6 sap color observed on the sliced pseudostem (1) watery; (2) milky; (3) reddishpurple; (4) other. 7 number of suckers number of shoots >30 cm tall on plants that were not desuckered (no desuckering occurred) 8 blotches at the petiole base (1) sparse; (2) small; (3) large; (4) extensive; (5) without pigmentation. 9 blotches color observed at the base of the petiole base (1) brown; (2) dark brown; (3) brownish black; (4) blackish purple; (5) other. 10 petiole canal leaf the leaf stalk is cut and the cross-section is observed to be (1) open with margins spreading; (2) wide with erect margins; (3) straight with erect margins; (4) margins curved inward; (5) margins overlapping. 11 leaf blade length (cm) measured at the maximum point of leaf length (1) ≤ 170; (2) 171 – 220; (3) 221 –260; (4) ≥ 261. 12 leaf blade width (cm) measured at the maximum point of leaf width (1) ≤ 70; (2) 71 – 80; (3) 81 – 90; (4) ≥ 91. 13 petiole length (cm) measured from the pseudostem to the lamina (1) ≤ 50; (2) 51 – 70; (3) ≥ 71. 14 color of leaf upper and lower surface observed on the upper and lower surfaces of leaves (1) greenish-yellow; (2) medium green; (3) green; (4) dark green; (5) dark green with reddishpurple (presence of large blotches of reddish-purple); (6) blue; (7) other. 15 wax on leaves (lower surface) wax coating (1) very little/no visible sign of wax; (2) few wax; (3) moderately waxy; (4) very waxy. 16 shape of leaf blade base (1) both sides rounded; (2) one sided rounded, one side pointed; (3) both sides pointed. 17 color of midrib dorsal and ventral surface observed on the dorsal and ventral surfaces (1) yellow; (2) light green; (3) green; (4) pinkish-purple; (5) reddish-purple; (6) purple to blue; (7) other. 18 peduncle length (cm) measured from the leaf crown to the first hand of fruit (1) ≤ 30; (2) 31 – 60; (3) ≥ 61. 19 peduncle hairiness (1) hairless; (2) slightly hairy; (3) very hairy, short hairs; (4) very hairy, long hairs (>2 mm). 20 bunch position (1) hanging vertically; (2) slightly angled; (3) hanging at angle 45◦; (4) horizontal; (5) erect. 21 rachis position (1) falling vertically; (2) at an angle; (3) with a curve; (4) horizontal; (5) erect. 22 male bud type observed at harvest time (1) present (normal); (2) degenerating before maturity; (3) absent. 23 male bud shape (1) like a top; (2) lanceolate; (3) shape between lanceolate and oval (intermediate); (4) ovoid; (5) rounded. 24 male bud length (cm) measured at harvest at the maximum point of heart length 25 male bud diameter (cm) measured at harvest at the maximum point of heart diameter biotropia vol. 32 no. 3, 2025 302 no character subcharacter 26 bract apex shape observed in bracts that are made flat (1) pointed; (2) slightly pointed; (3) intermediate (shaped between slightly sharp and blunt); (4) obtuse; (5) obtuse and split. 27 color of the bract external face (1) yellow; (2) green; (3) red; (4) red purple; (5) purple brown; (6) purple; (7) blue; (8) pinkish-purple; (9) orange-red; (10) other. 28 color of the bract internal face (1) whitish; (2) yellow or green; (3) orange-red; (4) red; (5) purple; (6) purplish brown; (7) pinkish purple; (8) other. 29 bract scars on rachis observed after the bracts and flowers fall from the racisis (1) prominent; (2) less prominent. 30 bract base color observed on the inside of the bracts (1) the color fades toward the base of the bracts (pigmentation disappears at the base of the bracts; (2) homogeneous color (pigmentation to the base of the bracts). 31 wax on the bracts observed on the outer surface of the bractea (1) very little or none; (2) little; (3) waxy; (4) very waxy. 32 number of fruits/comb observed on the mid-hand of the bunch (1) ≤ 12; (2) 13 – 16; (3) ≥ 17. 33 fruit length (cm) measured as the internal arc of the fruit, without pedicel 34 fruit shape (longitudinal curvature) observed longitudinal curve of the fruit (1) straight; (2) straight at the distal part; (3) curved (sharp curve); (4) ‘s’ shape; (5) other. 35 transverse section of fruit observed when the fruit is ripe (1) pronounced ridges; (2) slightly ridged; (3) rounded. 36 fruit apex observed on the distal part of the fruit: (1) pointed; (2) lengthily pointed; (3) blunt-tipped; (4) bottle-necked; (5) rounded. 37 mature fruit peel color (1) yellow; (2) bright yellow; (3) orange; (4) grey spots; (5) brown/rustybrown; (6) orange-red, red or pink/pinkish-purple; (7) reddish-purple; (8) black; (9) other. 38 fruit flesh color the color was observed 39 presence of seed (1)< 5; (2) 5 – 20; (3) >20. source: ipgri (1996). results and discussion morphological characterization description the exploration of genetic resources as an effort for plant improvement requires characterization of diversity as fundamental prerequisites. currently, banana characterization is primarily based on morphological traits. through plant morphology, the external form and organs of the plant can be observed, which allow visual differentiation among banana plant species. morphological characters reflect the expression of an individual genetic traits. genes affect physiological processes in plants by regulating enzyme synthesis and the formation of compounds that support plant growth and development (taiz et al. 2006). however, the observation of morphological characters often leads to confusion in classification because these traits are strongly influenced by environmental factors. the parts of the plant body most susceptible to morphological changes due to environmental influences include leaves, stems, and flowers. in banana plants, these traits are described using detailed banana descriptors. morphological characterization of banana plants is essential to support the development and selection of superior banana varieties (lukmanasari et al. 2023). previous studies have shown that morphological descriptors such as pseudostem color and blotching, leaf orientation, pseudostem diameter, and fruit number are highly effective in distinguishing musa cultivars. these traits demonstrate high heritability and repeatability, making them reliable for classification. this is consistent with findings published in genetic resources and crop evolution, which reported that quantitative morphological traits, including pseudostem girth, number of fruits, and fruit size, exhibit high heritability (> 0.8) and high repeatability (> 2.0), underscoring their value in cultivar differentiation. such traits are particularly useful for breeding programs and germplasm characterization, as they serve as consistent markers for identifying and classifying banana varieties (ortiz 1997). cluster analysis of bananas in east java classified local cultivars based on 15 qualitative and 9 quantitative morphological traits, including the morphological characteristics and total flavonoid content in peel extracts of banana cultivars arliani et al. 303 number of hands per bunch, pseudostem diameter, and petiole length. these morphological variations demonstrate strong potential for supporting future breeding programs (sa’diyah et al. 2025). furthermore, ecogeographic characterization of musa germplasm in ecuador revealed that morphological descriptors of the pseudostem, leaves, flowers, bunches, and fruit play a key role in discriminating elite musa accessions (poaquiza et al. 2025). morphological characters vary between individuals and are expressed through their phenotypic appearance. phenotype results from the interaction between genotype and environment, with the environment playing a crucial role in shaping variation, particularly in morphological traits. individuals of the same species may exhibit different phenotypes if they grow in different habitats. phenotypic plasticity explains why individuals with the same genetic composition can differ morphologically under varying environmental conditions. therefore, observing morphological characters is an essential first step in assessing genetic diversity. germplasm exhibits high variation and serves as a valuable source of genes for desirable plant traits, such as pest resistance and high productivity. plants within the same group, when used as cross-breeding parents, tend to produce offspring with low genetic variation, whereas crosses between different groups result in higher variation (lukmanasari et al. 2023). the influence of genotype and environment is reflected in the phenotypic diversity observed within a generation. descriptive data for each banana variety are presented below. qualitative characterization of cavendish, kepok, raja, and klutuk bananas is shown in table 2, based on ipgri qualitative trait observations. the morphological differences among these four banana varieties are summarized in table 3. table 2 qualitative characterization of cavendish, kepok, raja, and klutuk banana no characteristics cavendish kepok raja klutuk 1 leaf habitus intermediate intermediate intermediate erect 2 pseudostem height (m) 2.45 3.3 2.4 5.34 3 pseudostem aspect/ diameter (cm) 52 65 39 84 4 pseudostem color reddish purple dark green red dark green 5 pigmentation on the pseudostem dark purple brown reddish brown purple black 6 sap color watery watery milky milky 7 number of suckers 4 2 3 7 8 blotches at the petiole base large small large extensive 9 blotches color black black black black 10 petiole canal leaf wide with erect margins straight with erect margins margins curved inward margins curve inward 11 leaf blade length (cm) 247 330 178 248 12 leaf blade width (cm) 87 65.5 64 68 13 petiole length (cm) 24.5 35.5 40 48.5 14 color of leaf upper surface green light green yellowish green dark green 15 color of leaf lower surface light green dark green green light green 16 wax coating on the underside of the leaf waxy few waxy few waxy waxy 17 shape of leaf blade base both sides pointed both sides rounded both sides rounded both sides rounded 18 color of midrib dorsal and ventral surface yellowish green (dorsal and ventral) green (dorsal) light green (ventral) light green (dorsal and ventral) light yellowish green (dorsal) dark purple brown (ventral) 19 peduncle length (cm) 12 16 16 29 20 peduncle hairiness slightly hairy hairless hairless hairless biotropia vol. 32 no. 3, 2025 304 no characteristics cavendish kepok raja klutuk 21 bunch position hanging vertically hanging at angle 45 ° hanging at angle 45 ° hanging at angle 45 ° 22 rachis position falling vertically falling vertically falling vertically at an angle 23 male bud type present (normal) present (normal) present (normal) present (normal) 24 male bud shape intermediate ovoid ovoid ovoid 25 male bud length (cm) 25.4 38.5 27 21 26 male bud diameter (cm) 30 41.5 8 23 27 bract apex shape slightly pointed intermediate obtuse obtuse 28 color of the bract external face purple brown pink purple dark red pinkish red 29 color of the bract internal face red purple purple brown purple purple 30 bract scars on rachis prominent prominent less prominent not prominent 31 bract base color fading homogeneous homogeneous homogeneous 32 wax on the bracts a little waxy a little waxy waxy very waxy 33 number of fruits/comb 22 14 14 12 34 fruit length (cm) 17.2 18.2 14 15 35 fruit shape (longitudinal curvature) curved straight curved straight 36 transverse section of fruit rounded pronounced ridges pronounced ridges slightly ridged 37 fruit apex color blunt blunt blunt blunt 38 mature fruit peel yellowish green yellow green green 39 fruit flesh color light yellow light yellow cream white white 40 presence of seed seedless seedless seedless 120 with rounded seed shape table 3 morphology of cavendish, kepok, raja, and klutuk bananas morphology cavendish kepok raja klutuk plant habitus pseudostem color morphological characteristics and total flavonoid content in peel extracts of banana cultivars arliani et al. 305 morphology cavendish kepok raja klutuk pseudo-rod pigmentation spots on leaf stalks shape of leaf blade base rachis position male bud biotropia vol. 32 no. 3, 2025 306 morphology cavendish kepok raja klutuk sap color fruit shape comparative morphological analysis of the four banana varieties reveals a range of traits shaped by their distinct genetic backgrounds and evolutionary histories. while all four varieties share the fundamental monocot architecture, including a succulent pseudostem and inflorescences that develop into fruit bunches, they can be grouped based on key reproductive traits. cavendish, kepok, and raja bananas are predominantly seedless, a characteristic associated with domestication, whereas klutuk bananas produce seedy fruit, reflecting their wild ancestry (heslop-harrison et al. 2007; perrier et al. 2011). the unique combination of characteristics for each variety is as follows: cavendish (aaa) is characterized by a short pseudostem (2 – 2.5 m) and is optimized for commercial production. its fruits have a thick, yellow peel resistant to bruising, yellowish-white flesh, and a sweet, slightly tangy flavor, making it a preferred dessert banana for direct consumption. kepok (abb), with an intermediate pseudostem height of approximately 3 m, is distinguished by its angular, flat fruits with prominent ridges. classified as a plantain, kepok bananas are typically processed before consumption, reflecting their high starch content (anggitha 2022). raja (aab) has a pseudostem height similar to that of cavendish and is distinguished by its plump, round fruits. it is primarily valued as a dessert banana for direct consumption due to its sweet flavor and soft texture. klutuk (bb) is the most morphologically distinct variety, possessing the tallest pseudostem (5 – 7 m), consistent with findings by hastuti et al. (2019), and producing small, seedy fruits. its morphology, including hard seeds and robust structure, reflects adaptations for survival in the wild, and its primary uses are in traditional cuisine and medicine rather than fresh dessert consumption (anggitha 2022; simmonds 1953). these morphological differences are fundamentally shaped by genetics and environmental adaptation (de langhe et al. 2009). the seedless, large-fruited traits of cavendish, kepok, and raja bananas result directly from human selection for triploid genotypes, which promote parthenocarpy and vegetative propagation, the key advantages for cultivation (heslop-harrison et al. 2007). cavendish bananas, for example, have been intensively selected for traits such as thick skin to withstand transportation. in contrast, klutuk bananas are predominantly diploid (bb), a genetic constitution closer to their wild ancestor (musa balbisiana), which allows sexual reproduction via seeds (simmonds 1953). this explains their taller stature and seedy fruits—adaptations for natural seed dispersal and environmental resilience— traits largely eliminated in commercial cultivars (simmonds et al. 1955). thus, the observed morphological spectrum from the wild-adapted morphological characteristics and total flavonoid content in peel extracts of banana cultivars arliani et al. 307 klutuk to the commercially optimized cavendish reflects the journey of banana domestication (perrier et al. 2011). total flavonoid contents (tfc) analysis the significant morphological and genetic variations among the four cultivars suggests a parallel divergence in their biochemical makeup. this is particularly relevant given that banana peels have been widely used in traditional medicine to treat various ailments, a practice attributed to their high content of phenolic compounds, including flavonoids, which act as primary antioxidants (pereira et al. 2015; anjum et al. 2022). the phenolic content in banana peels is notably high, often exceeding that of the fruit pulp, and is strongly correlated with antioxidant efficacy (ramli et al. 2012; oyeyinka 2020). therefore, we hypothesized that the distinct, wild-adapted morphology of the klutuk banana would be linked to a heightened production of these defense-related flavonoids compared to the more domesticated varieties. this hypothesis was tested by quantifying the total flavonoid content (tfc), with the results presented in table 4. table 4 tfc content of banana peel extract banana peel sample total flavonoid content (mgqe/g) cavendish 0.146 ± 0.0020 kepok 0.386 ± 0.0148 raja 0.139 ± 0.0053 klutuk 0.453 ± 0.0078 based on the information above, the extract of klutuk banana peel contains the highest total flavonoids (0.453 mg qe/g) when compared to the other three varieties, followed by kepok banana peel (0.386 mg qe/g), cavendish banana peel (0.146 mg qe/g), and raja banana peel (0.139 mg qe/g). the detailed composition of flavonoid compounds has been reported in previous studies, although data are limited to certain banana varieties. for example, banana peel of the red yade banana (aab), which shares the same genome as plantain (aab), contains flavonoid compounds from the flavonols group, including rutin (482 ± 206 μg/g dm), quercetin deoxyhexose-hexoside (75.2 ± 14 μg/g dm), kaempferol-deohyhexosa-hexoside (35.5 ± 4 μg/g dm), kaempferol-3-rutinoside (173.9 ± 50 μg/g dm), isorhamnetin-3-rutinoside (139 ± 73 μg/g dm), and myricetin-deoxyhexosehexoside (114 ± 27 μg/g dm) (valérie et al. 2015). the data reveal a clear gradient in flavonoid content that corresponds with the morphological and genetic differences among the varieties. klutuk banana peel extract (bb) contained the highest total flavonoid content (tfc) at 0.453 mg qe/g, followed by kepok (abb) at 0.386 mg qe/g. in contrast, the highly domesticated dessert bananas, cavendish (aaa) and raja (aab), exhibited significantly lower levels. this pattern supports the notion that klutuk bananas, being closely related to wild species, synthesize higher levels of bioactive compounds as part of their natural defense mechanisms, a trait partially retained in the hardy, plantain-type kepok (nofianti et al. 2021; lestari 2020). the detailed composition of banana peel flavonoids has been elucidated in various studies. for example, peels from plantain-type bananas (aab) contain flavonols such as rutin and quercetin derivatives (valérie et al. 2015), whereas cavendish peels (aaa) contain specific flavonols, including quercetin-3-rutinoside, and flavan-3-ols such as gallocatechin (rebello et al. 2014; someya et al. 2002). it is important to note that absolute flavonoid content can be influenced by factors such as extraction method, fruit ripeness, and growing environment (gonzález-montelongo et al. 2010; ramli et al. 2012; vu et al. 2018). however, the consistent trends observed across the genetically distinct cultivars in this study suggest that genome group, along with its associated morphological adaptations, is a key determinant of flavonoid production potential in banana peels. conclusion this study demonstrated a clear correlation between morphology, genetics, and flavonoid content in four banana cultivars. a distinct phenotypic gradient was observed, ranging from the highly domesticated, seedless cavendish (aaa) and raja (aab) to the wild-adapted klutuk (bb), characterized by a tall pseudostem and seedy fruits. this gradient corresponded closely with the phytochemical data: klutuk peel exhibited the highest flavonoid content, followed by kepok (abb), while the dessert bananas showed significantly lower levels. these results confirm that wild-type cultivars allocate more resources to defensive flavonoids. consequently, morphological traits provide a valuable preliminary indicator of a cultivar’s nutraceutical potential. the findings underscore the importance of conserving wild biotropia vol. 32 no. 3, 2025 308 germplasm, such as klutuk, and offer a scientific basis for the targeted utilization of banana peel waste as a source of natural antioxidants. acknowledgments the authors extend their gratitude to the kebun plasma nutfah pisang, yogyakarta, indonesia, for supplying the samples of the four banana varieties. we also acknowledge dr. bonusa nabila huda from labterra sage consulting for her valuable contribution in designing the graphical abstract and providing data visualization support. references ahmad ar, juwita j, ratulangi sad. 2015. penetapan kadar fenolik dan flavonoid total ekstrak metanol buah dan daun patikala (etlingera elatior (jack) r.m.sm) [determination of total phenolic and flavonoid content in methanol extracts of fruit and leaves of patikala (etlingera elatior (jack) r.m.sm]. psr 2:1–10. doi: 10.7454/psr. v2i1.3481 anggitha m. 2022. pembuktian aktivitas antidiare pucuk merah jambu biji dan daging buah pisang klutuk yang digunakan battra desa jayaratu singaparna [proving the antidiarrheal activity of pink shoots and pulp of klutuk bananas used by battra in jayaratu village singaparna]. 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p, herent m, tomekpe k, happi t, quetinleclercq j, andre c. 2015. phenolic profiling in the pulp and peel of nine plantain cultivars (musa sp.). food chem 167:197–204. doi: 10.1016/j.foodchem.2014.06.095 vu ht, scarlett cj, vuong qv. 2018. phenolic compounds within banana peel and their potential uses: a review. j funct foods 40:238–48. doi: 10.1016/j.jff.2017.11.006 07. 1783 maria luz et al..cdr 80 60 40 20 0 n o . o f tr ic h o m e s h. m ad ul id ii h. p ub ica lyx h. c ar no sa h. o bs cu ra h. b uo tii h. m el iflu a a b c c d d page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 biotropia vol. 28 no. 3,2021: 263 273 doi: 10.11598/btb.2021.28.3.1469 species diversity of soil fungi from a corn plantation at echague, philippines rey gascon tecsow', florenda c. ballesteros-temanel and james kennard sanz jacob1 department o f biological sciences, collqe ofarts and sciences, isabeb state universio, eeagae, isabela, phihppines zresearch and development, isabela state universig, eeague, isabela, philippines received 12 november 2020 /accepted 1 september 2021 abstract soil fungi are a critical component of the agroecosystems and provide ecological services affecting food and bioproduct production. awareness of the composition and distribution of local species is necessary to maximize the productivity and sustainability of the agroecosystems. this study aimed at isolating fungi from the soil samples collected from a corn plantation at echague, isabela, morphologically defining, characterizing, and determining the diversity of fungal isolates species. soil samples from five (5) different sampling units were collected and microbiological techniques were used to isolate the fungi. identification was performed by morphological characterization of fungal isolates guided by taxonomic keys and textbooks. fungal diversity was assessed using simpson's indices of diversity. sixteen (16) fungal isolates were found; 12 were known and four (4) were unidentified. ten (10) species belong to the ascomycota group, and two (2) belong to the zygomycota class. the aspergillns species dominated the region amongst all the species isolated and established. aspergillzj famigatus and aspel.gillzlssflauas are densely populated and well distributed. a potent biocontrol agent was also identified, namely tn'chodemna hayanam and tnkhodem viride. the mncor sp. and the mippw stoloniferous, zygomycota fungi, were also identified. keywords: cornfield, morphological identification, soil fungi, species diversity introduction agroecosystems are a subset of a traditional ecosystem that lies within the agricultural human activity. these are natural habitats modlfied for the manufacture of food and other agricultural goods. it is also the most intensively managed and, hence, also the most disturbed ecosystem on earth (lemaire e t al. 2008; vandermeer 2011). one form of an agroecosystem is a corn plantation. maize is one of the philippines' most important staple crops. in exploiting agricultural capital it ranks second to rice. it is not only used for human consumption but also for animal feed and industrial uses, and is, therefore, a crucial crop in the growth of the livestock and manufacturing industries (altoveros & borromeo 2007). *corresponding author, email: tecsonrmgl808@gmail.com isabela province is one of the largest maize producers in the cagayan valley where the echague municipality is one of the province's maize producers. the town is often characterized as a large area of land that is known as a broad alluvial plain, flat, or almost level. soil fungi are a critical component of the agroecosystems and provide ecological services that affect food and bio-product production. many of these main soil fungal species directly associate with plants and are determinants of the efficiency of the agroecosystems. energy and matter fluxes through the soil decomposing subsystem, which is dominated by fungi and bacteria. although invertebrates play a part in n fluxes, plants regulate largely the rhizosphere fung by generating carbon and energy-rich compounds and bioactive phytochernicals (ellouze e t al. 201 4). biotropia vol. 28 no. 3,2021 given the significance of soil microorganisms in agroecosystem productivity, management of beneficial soil microbial diversity emerges as a new strategy for growing crops today. the efficient management of fungal capital is critical to maximizing agricultural ecosystem production and sustainability. therefore, benchmark information must be generated on the occurrence and dlstribution of soil fungi. awareness of the composition and distribution of local species is important if indigenous fungi communities in the soil are to be handled in a way that optimizes the productivity and sustainability of agricultural ecosystems. studles on the determination of microbiota and mycoflora of soils collected from agroecosystems or agricultural lands in isabela, pmppines, have also been inadequate. thus, the research was conducted to determine soil fungal species and its diversity from a corn plantation in annafunan, echague, isabela. at a depth of 20 cm, samples were collected using a sterdized spatula and placed in a polypropylene bag. collected samples were taken to the laboratory for analysis. anabsis of soil samples samples were milled and sieved twice in the laboratory to extract big stones and debris to obtain finer soil samples. the soil samples were physically and chemically analyzed. to determine the ph, nitrogen (n), phosphorus 0, and potassium (i<) content of the soil samples, chemical analysis using different soil analysis methods were performed at the da-cvrc soils laboratory, san felipe, ilagan city, isabela. the potentiometric test was used to measure the ph of the soil, the n content of the soil was measured by the walkley and black spectrophotometric test, the p content by the olsen test, and the cold sulfur extraction test was used to measure the i< content of the soil sample. isolation of soil fungi materials and methods the soil fungi were isolated using the following techniques: selection of study area the study area was chosen based on the semaldilzltion method following criteria: the major maize growing area, for isolation, serial &lution up to x105 peace and order, and accessibility to any (1:100,000) was performed. for each soil reasonable means of transport. with these sample, five (5) sterilized test tubes containing 9 parameters, barangay annafunan (16.71 11' n, ml of sterile water were prepared and 121.7224' e) was selected. numbered accordingly. five (5) grams of soil samples were blended into a beaker with 25rnl collection and analysis of soil samples of sterile water. from the soil solution, using a establishment o f sampling points pipette, 1 ml was drawn and added to the first sampling points were using the test-tube labeled as ~ 1 0 ' . then, from xio1 grid cell sampling with modification dilution, 1 rnl was drawn and added to the test (ackerson 2018). the cornfield has a land area tube labeled x1o2the serial dilution process of 14,344 m2 (1.4 ha) and a sample size of was repeated up to the fifth test tube labeled as 10,072.14 m2 was used for sample collection. it x1o5. after ths, 1 ml dilutions of x1o5 were was split into points for soil random. 50 quadratic grids. the sampling sample collection were chosen at collection o f soil samples the soil samples were collected during the month of february after their cropping cycle from the selected cell grid numbers in the study area. soil samples were collected at a radius of 12 inches at the center of the selected cell grid. poured into a petri dish containing potato dextrose agar (pda) and incubated for 5 days at room temperature (toma & faqi-abdulla 2012). when the mixed culture of fungi ramified, each type of fungi was transferred to agar slants using for pure culture. 3-point inoczllation technigzae using a 3-point inoculation technique, pure fungi isolates were inoculated where three species diversity of soil fungi from a corn plantation tecson eta/. mycelia inoculums were put equidstantly from each other in two (2) different sterhzed solid media: potato dextrose agar (pda) and coconut water agar (cwa) for 7 days at room temperature. agar block techniqae inside the microwaveable dishes, damp tissue and u-shaped folded foil were positioned. placed over the foil was a glass slide. aseptically, using a sterile scalpel, the agar block with streptomycin was cut and positioned in the center of the sterile glass slide. each fungal isolate was inoculated and covered with a coverslip. specimens were observed under a microscope (in lpo and hpo) after seven days of incubation and were photographed for further morphological examination. morphological identification and characterization morphologically, fungal isolates were classified according to their macroscopic and microscopic characteristics. macromorphological features such as colony color and density, growth patterns, and textures were noted. micromorphological characteristics such as conidial shape, hyphae morphology, and spore morphology were also observed. the taxonomic key to classification devised by quimio & hanlin (1999) was used to facilitate the identification of fungi. measuring fungal occurrence and diversity pattern the fungal occurrence was measured as to fungal density, frequency, and relative frequency. the following formula was used: number of individual species a fungal density = unit area number of isolates for relative frequency each species x 100 (yo) = total number of isolates alpha-dversity was used to measure the diversity of soil fungi in the study area. the following indices were used to determine species diversity: 1. simpson's diversity indices a. simpson's index (simpson 1949) measures the probability that two individuals randomly selected from a sample wdl belong to the same species (or some category other than species). with this index, 0 represents infinite diversity, and 1, no diversity. that is, the bigger the value of d, the lower the diversity. this was determined by using the formula: where: ni total number of organisms of a particular species ntotal number of organisms of all species b. simpson's index of diversity (1 d) which was obtained by deducting simpson's index (d) from 1. the value of this index also ranges between 0 and 1, the greater the value, the greater the sample diversity. the index represents the probability that two individuals randomly selected from a sample will belong to different species. results and discussion description of the physical characteristics of the study area the prevailing climate at the time of sample collection and organic matter content of soil samples were used to characterize the study area. data on prevahg climate at the time of sample collection was taken from the isu agricultural meteorological research center of cvarrd compound, isabela state university, san fabian, echague, isabela, philippines. during the month and day of soil sampling, climatic data such as temperature, relative humidity, amount of rainfall, evaporation, and total minutes of bright sunshne were noted (table 1). biotropia vol. 28 no. 3,2021 table 1 summary of climatic data for february 2018 climatic data average temperature mnimum 20.94 "c maximum 29.29 "c relative humidity 8:oo am 95% 2:00 pm 75% rainfall 2.9 mins evaporation 2.9 mins bright sunshine 251 minutes the climatic condition for the month of sample collections was warm with a relatively high humidity indicating high humidity content and high free water vapor is present in the air. based on soil analysis, the ph level shows that it is favorable and suitable for fungal growth because fung grow in a substrate ranging from 4.0 8.5 or sometimes 3.0 9.0 with an optimum ph level of 5.0 7.0. several fungi are acid-tolerant which grows at ph 2.0 with an optimum ph level of 5.5 6.0 the average in the (rousk e t al 2010a; rousk e t al 2010b). the during the month of the collection of the result of soil analysis indicates that the sample samples ranges from 20.94 "c to 29.29 "c with regon has a very high nitrogen (n) content of 95% to 75% relative humidity from 8 am to 2 pm. the table also shows that the average 0.65 percent to 1.41% (horneck e t al 2011). it amount of rainfall and evaporation was 2.9 has also been shown to have medium mins, respectively. the average bright sunshine phosphorus (i?) content of 13.52 ppm to 31.81 for the whole month is 251 min. during the ppm. the potassium was low at 88 " conduct of the study, it showed that the temperature ranges from 23.5 "c to 31.1 "c with 98% to 73% relative humidity. there was no recorded rainfall during the day of sample gathering. the evaporation was registered at 3.5 rnins with 330 total minutes of bright sunshine. the table shows the organic matter (om) content of the soil samples as to nitrogen (n), phosphorus 0, and potassium (i<) as well as its ph. soil analysis shows that the soil samples collected in the study area are all acidic with ph ranging from 4.9 to 5.4 with an average ph level of 5.2. of the soil samples, sample 3 was recorded as the most acidic. the average n content of the study area is 1.06%, while the p content of the soil is estimated at an average of 19.78 ppm, and the average i< content of the soil in the study area is 280.80 ppm. table 2 soil analysis conducted at ilagan soils laboratory ppm for soil samples 1 and high at 289 ppm to 399 ppm for other soil samples. overall, the study area's abiotic environment, such as climate data and soil analysis obtained, showed that it was favorable and suitable for fungal growth and sporulation. this may also be due to the levels of precipitation and temperatures resulting in favorable humidity and moisture that lead to the growth of soil fungi in the study area. taxonomic account and description of the fungi isolates the following are the fungi species isolated from the soil samples taken from a corn plantation at echague, isabela, philippines with their taxonomic classification as shown in table 3 with morphologcal and biological description. organic matter soil sample ph n (yo) p ( p p ~ ) i< ( p p ~ ) 1 5.2 1.10 31.81 88 2 5.2 0.65 13.52 399 3 4.9 1.20 18.37 329 4 5.4 0.96 17.53 289 5 5.3 1.41 17.67 299 mean 5.2 1.06 19.78 280.80 species diversity of soil fungi from a corn plantation tecson et al. table 3 soil fungi species composition of the study area division class order family genus species ascomycota eurotiomycetes eurotiales trichocomaceae aspergi'ilus a. nker teigh a. fiuus link a. fz/m;satus fresen. a. te~eus thom aspergillzls sp. # 18 aspe@llas sp. # 21 penin'ldium penin'ldiam sp. thom sordariomycetes hypocreales bionectariaceae gdiocladium g. mseum bainer hypocreaceae trichoderma t. hapjanum rifai t. tiride pers. zygomycota mucomycotina mucorales mucoraceae macor mwor sp. l. rhizopzrs r stolonifer thom there were 12 taxa documented consisting of 10 ascomycetes species and two (2) zygomycetes species, while four (4) species were unidentified due to lack of defining morphological structures. of the 12 identified fungi species, seven (7) species belong to the division ascomycota, class eurotiomycetes, order eurotiales under the family trichocomaceae. these species are aspergillus niger teigh, a. flav~s link, a. fumigatus fresen., a. terreas thom, aspergillus sp. #18, aspegillus sp. #21, and penicillizm sp. thom. two (2) f a d e s of the class sordariomycetes, order hypocreales were also noted in the study area: bionectriaceae gliocladizm mseum bainer and hypocreaceae tm'chodem h a ~ a n a m rifai and t. gm'de pers. two (2) species belong to the division zygomycota, class mucomycotina, order mucorales under the family mucoraceae. these species are mucor sp. fresen. and bizpptls stolonkfer ehernb. figure 1 microscopic image under hpo: (a-f) aspergillas (g) penicz'ilit/m gliocladizlm (i-j) tvichoderma (kj mucor q rhizopas (m-p) unidentified species biotropia vol. 28 no. 3,2021 the a.pe@llas niger (fig. 1a) colony is black in color. based on its colony morphology, a. niger exhibits filamentous margin and form with slightly umbonate elevation. colonies show heavy sporulation on both media. colonies on cwa show sporulation on the side forming a ring-like structure at its center while colonies on pda showed a sporulated center towards the side. microscopically, the conidial head is uniseriate and globose with rough walled conidia. the aspe~il las flavas link (fig. 1b) colony is yellowish or olive-green color. based on its colony morphology, a. flavas exhibits filamentous margin and form with slightly umbonate elevation on pda and slightly crateriform on cwa. colonies show heavy sporulation towards the sides that were surrounded by cottony-like structure mycelia. mcroscopically, it shows radiate conidal heads with echinulate conidia. aspergillas famigatas fresen (fig. 1c) colony is dark green to a lighter green color. based on its colony morphology, a. famigatas exhibits filamentous margin and form with slightly raised elevation. colonies show heavy sporulation towards the sides surrounded by mycelia. iljicroscopically, it shows columnar conidial heads and uniseriate conidiogenous cells with smooth-walled stipes often green in the upper part and verrucose conidia. a.pe@llas terreas thom (fig. id) colony is yellowish-brown to a cinnamon-brown color. based on its colony morphology, a. terrea~ exhibits undulate (wavy) margin and irregular form with slightly umbonate elevation. colonies show heavy sporulation towards the sides showing more sporulation on pda as compared to cwa. microscopically, it shows densely columnar conidial heads are and biseriate conidiogenous cells with smooth-walled conidiophore and conidia. two species aspeqq'llus were also identified but were not narrowed down to species level due to lack of reliable literature for comparison of morphological structures. these species are shown in figure 1e and figure if. penicilliam sp. thom (fig. 1g) colony differs from the beige blue and greyish blue color. based on its colony morphology, penin'lliam sp. exhibits the entire margin and circular form with umbonate elevation. colonies show blue-green color due to heavy sporulation on pda and greyish blue on cwa whch are both surrounded by mycelia. microscopically, it forms chains of spores (conidia) and brush-shaped conidiophores. gliocladiam roseam bainer (fig. 1h) colony is white in color. based on its colony morphology, g. roseam exhibits a filamentous margin and form with slightly umbonate elevation. colonies show a cottony-like structure wherein g. roseam is larger on cwa than on pda. microscopically, it shows erect conidiospores with dense and has a brush-like structure where the conidia are single-celled and cylindrical. tm'chodema h a ~ a n a m &fai (fig. 11) colony differs from different shades of green in color. based on its colony morphology, t. haqanam exhibits filamentous margin and form with slightly convex elevation. colonies show a completely ramified plate showing no gaps on and it shows darker green color at the center where their mycelia meet and leaving a circular hallow where they are inoculated on cwa. mcroscopically, it forms sticky clumps of conidia rather than occurring in chains. tm'chodema viride pers (fig. 1j) colony is green in color. based on its colony morphology, t. vkde exhibits undulate margin and irregular form with slightly raised elevation. colonies show a completely ramified plate showing gaps between them on pda and it shows that the colonies combine on cwa. microscopically, it also forms sticky clumps of conidia rather than occurring in chains. the macor sp. fresen (fig. 1i<) colony is greyish brown to black color. based on its colony morphology, macor sp. exhibited filamentous margin and form with flat elevation on pda and slightly umbonate elevation on cwa. colonies show pin-like structure with greyish black color due to heavy sporulation on pda and it is physical appearance is the same as a. niger colonies with heavy sporulation at the center on cwa. microscopically, it shows simple sporangiospores and apical form with globular sporangia that is elevated by a column shaped columella. rhixopw stolonzfer ehrenb (fig. 1l) colony is white or transparent in color. based on its colony morphology, r stolonifer exhibits filamentous margin and form with flat elevation. colonies show a threadlike structure extending species diversity of soil fungi from a corn plantation tecson eta/. upward on pda and cwa. microscopically, it member of compost rnicrobiota where shows sporangia that are supported by a large temperature, ph, and nutrients may not be apophysate columella atop a long stalk called optimal for other organisms (van heerden et al sporangiophore. 2002). also, it presents a widespread of the 16 taxa documented and isolated in environmental distribution that is capable to the soil collected from the study area, there were adapt diverse environmental conditions due to four (4) unidentified species due to lack of its thermotolerance and it can resist several sorts literature published. these species are shown in of environmental stresses (oliveira & caramallo figure 1m to figure 1p. 2014; rhodes 2006). the population of fungal species in the study area the total population of collected soil fungi in the study area was 163 fungal species. of these, aspergiiius famigatus showed the highest population with 54 species that occurred in all sampling units. t h s is followed by aspergiiiu~ jlavus with a population of 42 species whch was also observed to be occurring in all the sampling points. the mucor sp., unidentified #6 , #29, and #32 showed the least population with a total of two (2) species respectively. it can also be noted that the most populous species in the area were under the genera of aspe@iius followed by trichodemza. results showed that samples taken from the center of the cornfield are most abundant in soil fungi, whereas, a . famigatus was relatively abundant in the field followed bv a. flavus. frequency and relative frequency of soil fungi species the table of frequency and relative frequency of soil fungi species (as shown in table 5) revealed that aspergiius fiavtls (1 2.20%) and aspe@iius fum&atus (1 2.20'0) showed the highest frequency followed by aspergiiius niger (9.76%) the mucor sp. (2.44%), unidentified species #11 (2.44%), #29 (2.44%) and #32 (2.44%) exhbited the lowest frequency and relative frequency. a higher frequency indicates that a species is well dstributed in the study area while a lower frequency indicates that it is concentrated only on the sampling unit or sampling area. therefore, it shows that a . fiavus and a. fum&atus are well distributed in the study area while mucor sp., unidentified species #11, i -i on the other hand, mzicor sp. was the least #29 and #32 are less distributed and are abundant. the abundance of a . fgmigatzls in the concentrated in a particular area in the study area attributed to its being a highly competitive site. table 4 population of the different fungi species present in the study area sample number species name 1 2 3 4 5 total aspe@llns niger 2 2 1 0 3 8 aspepilins flaws 15 3 10 2 12 42 a aspezillns fnnzigatns. 6 17 11 11 9 54 aspe@llns terrezls 0 0 2 0 3 5 a.pe@liu.r sp. # 21 0 0 2 2 0 4 aspegjllus sp. # 1 8 0 0 6 0 0 6 gliocladiunz roseunz 0 2 0 1 1 4 mucor sp. 1 0 1 0 0 2 penicillinnz sp. 3 0 5 0 2 10 rbi~opzls stolonifer. 2 0 1 3 0 6 tccbodem haqanunz 0 3 1 4 0 8 t ~ c b o d e m vinde. 1 0 2 1 0 4 unidentified species #i 1 0 4 0 0 0 4 unidentified species #6 0 0 1 0 1 2 unidentified species #29 0 0 0 2 0 2 unidentified species #32 0 0 0 2 0 2 total: 30 31 43 28 31 163 note: the unit for the fungal population is measured in cfu/ml. biotropia vol. 28 no. 3,2021 table 5 frequency and relative frequency of the different species present in the study area species name frequency relative frequency (o/o) a.pe@iius uniger aspe@iius$avus aspe@iius fumigatus. aspeziiius teereus aspegjiius sp. # 21 a.peziiius sp. # 18 gliocladizlm roseum mucor sp. penicillum sp. mix@us stolonifer. trichodem haqzauum t~chodemza uiride. unidentified species #11 unidentified species #6 unidentified species #29 unidentified species #32 1 2.44 total: 41 100% there was a total population density of 8.30 cfu/ml (8.40%) of fungal isolates at a given area with a mean density of 1.66 cfu/ml. sampling unit 3 revealed the highest fungal density with a total of 2.2 cfu/ml (llo/o), while the least fungal density was shown on samphg unit 4 with a total of 1.4 cfu/ml (7%) of fungal isolates. the aspergiiias famgatas exhbited the highest fungal density with a total of 2.15 cfu/ml (54%) with a mean of 0.43 cfu/ml, followed by a. javas with a total of 2.10 cfu/rnl with a mean of 0.42 cfu/ml, and lowest fungal density was shown in rhizppas sstolonzfer and tm'chodem h a ~ a n a m with a total fungal density of 0.05 cfu/ml (1%) with a mean density of 0.01 cfu/ml of fungal isolates. it shows that there is a high population density of a . famkatzas and a. flavas, whereas low population density in almost all the fungi species was observed. also, genera under aspergillas shows the densest species in the study area. a low population density may cause the extinction of the fungal species, which can be due to environmental factors such as dsturbance, pollution, and habitat loss. population density and relative density the result of the study shows that there was a total of 16 fungal species isolated with a total of 163 cfu/ml of fungal population. it reveals that soil sample no. 3 that was collected almost at the center of the sampling area showed the highest fungal diversity with 0.84 value of computed simpson's index of diversity (1-d). notably that the soil sample no. 3 has the highest total number of fungal populations and the highest fungal density. generally, it also reveals that species diversity in the study area is high with a 0.82 mean diversity value. hence, the most diverse part of the study area is said to be at the center because most of the fungal communities were isolated and revealed a high population density in the study area. species diversity of soil fungi from a corn plantation tecson et dl'. table 6 population density of the different species present in the study area soil sam~les species name 1 2 3 4 5 d r d d r d d r d d rd d rd aspe~giiizls nniger 0.10 10°/o 0.10 10% 0.05 5% 0.15 15% aspel-giizls flauzls 0.75 75% 0.15 15% 0.50 50% 0.10 10% 0.60 60% aspel-giius fumnigutzls 0.30 30% 0.85 85% 0.55 55% 0.55 55% 0.45 45% aspel-giizls ten-eas 0.10 10% 0.15 15% aspergiiizls sp. # 21 0.10 10% aspergilzls sp. # 18 0.30 30% gliociudizlm rosezlm 0.10 10% 0.05 5% 0.05 5% mncor sp. 0.05 5% 0.05 5% penicillzlm sp. 0.15 15% 0.25 25% 0.10 10% rhilppw stolonifer 0.05 5% trichodem haqianzlm 0.05 5% trichodemza uiride 0.10 10% unidentified species #11 unidentified species #6 0.05 5% 0.05 5% unidentified species #29 0.10 10% unidentified species #32 0.10 10% mean: 1.5 8% 1.6 8% 2.2 llo/o 1.4 7% 1.6 8% notes: legend: d = density; rd = relative density (in percentage). fungal diversity the high diversity of fungi may be attributed to the time of sample gathering. samples were collected after corn harvest, hence, lesser disturbances and there are no agricultural activities were conducted during collection time. the fallow period may allow microbial growth in the soil. also, high fungal diversity may be attributed to the presence of corn waste such as corn cobs and other waste products after the corn harvest. fungi are classified as decomposers for dead materials such as animals and plant waste. they also have this distinct characteristic as saprophytes, which means that they obtain food by absorbing dissolved organic material especially obtaining nourishment from the products of organic breakdown and decay. higher fungal diversity and complex community enhance the decomposition rate of soil nutrients which promotes nutrient absorption and nutrient cycling (hiscox et al. 2015; iwvin e t ul. 2014; yao et al. 2017). most of the fungal isolates were aspe~iiius species wherein they are mostly pathogenic in nature producing aflatoxins. aflatoxins are a group of naturally occurring mycotoxins that typically affect corns and peanuts whch are ingredients use on both food and feed products (williams et al. 2004). colonization and contamination can occur in the crop field at harvest, during post-harvest operations, or when the crop is in storage. also, man mycotoxins including aflatoxins can form during the growing stages of certain crops. traditionally, fungal contamination caused by mycotoxins can be stopped and reduced growth by the use of fungicides in growing crops (patel et al. 2015). however, potential bio-control agent fungus was also isolated and identified in the form of tnichodemu haqjanam and t. viride. tm'chodem species is a very effective biologcal means for plant disease management especially the soil born. it is a free-living fungus that is common in soil and root ecosystems. it is highly interactive in the root, soil, and foliar environments. it reduces growth, survival, or infections caused by pathogens by different mechanisms like competition, antibiosis, mycoparasitism, hyphal interactions, and enzyme secretion. aside from being a potent biocontrol agent, tm'chodema is a plant growth promoter that solubihzes phosphates and micronutrients, and increases the number of deep roots of plants, thereby increasing the plant's ability to resist drought. also, a biochemical elicitor of diseases by producing compounds to induce ethylene production, hypertensive responses, and other defense-related reactions in plant cultivars. and they play an important role in the bioremediation of soil that is contaminated by pesticides and herbicides (sigh 201 0). biotropia vol. 28 no. 3,2021 table 7 diversity of the different species present in the study area soil sample fungal diversity parameters 1 2 3 4 5 total number of fungi species (s) 7 6 12 9 7 total number of individuals/population n 30 31 43 28 3 1 simpson's index (d) 0.31 034 0.16 0.21 0.26 simpson's index of diversity (1 -d) 0.69 0.66 0.84 0.79 0.74 note: the total number of individuals/population is measured as cfu/ml. conclusion twelve fungal species were isolated and identified from the collected five soil samples in the study area. most of the species were from the genera of aspe~iiias, which exhbited the highest fungal population and density in the study area. specifically, the aspeqjiius famkatas showed the highest fungal population and density in the area. the study area isolated a potent biocontrol agent for plant disease management, namely, tmi.6odem haqanam and t. vhide. the species diversity of fungal isolates in the study area is high. therefore, the results of the foregoing study revealed that the study area or cornfield is upright for growing economically important food crops and other crops. acknowledgements the authors would like to express gratitude to mrs. epifania gonzaga, the owner of the cornfield for allowing and permitting the conduct of the study. to ms. dina m. malangen for allowing us to coordinate with the owner of the field through her, and, to ms. judy anne g. ramiro for procurement of other materials and equipment needed in the study. also, special appreciation to the faculty of the department of biological science, mrs. helen c. ramos, mrs. zarah a. villalon, and mr. ron patrick c. campos for their guidance and technical contribution to the completion of the research study. references ackerson jp. 2018. soil sampling guidelines. purdue university: purdue extension; [updated 201 8 nov; cited 2020 sept 141. available from: altoveros nc, borromeo th. 2007. the state of plant genetic resources for food and agriculture of the philippines. philippines: department of agriculture and bureau of plant industry. ellouze w, esmaelli taheri a, bainard ld, yang c, bazghaleh n, navarro-borell a, hamel c. 2014. soil fungal resources in annual cropping systems and their potential for management. bio med research international 53:18-24. horneck da, sullivan dm, owen js, hart jm. 201 1. soil test interpretation guide. ec 1478 corvalis. oregon state university extension services. hiscox j, savoury m, muller ct, lindahl bd, rogers hj, boddy l 2015. priority effects during fungal diversity establishment in beech wood. isme j 9:2246-60. iwvin sn, winston gc, goulden ml, treseder i=. 2014. environmental fitering effects of soil fungal diversity composition more than dispersal limitation at regional scales. fungl ecol 12:14-25. lemaire g, carvalho 0, igonberg s, recous s. 2018. agroecosystem diversity. academic press. oliver m, caramallo r. 2014. aspe@illzlsfum&atus: a mere bioaerosol or a powerful biohazard? nova acta cientifica compostelana. bioloxia 21:57-64. pate1 sv, bosamia tc, bhalani hn, sigh p, icumar a. 2015. aflatoxins: causes & effects. agribios: a monthly magazine of agricultural and biological sciences 13(09):140-2. quimio th, hanlin rt. 1999. illustrated genera and species of plant pathogenic fungi i the tropics. college of agriculture publication program. rhodes jc. 2006. aspezilllzls fum&atu~: growth and virulence. medical mycology 47:71-81. rousk j, brookes pc, baath e. 2010a. investigating the mechanism for the opposing ph-relaionships of fungal and bacterial growth in soil. soil biol biochem 24:926-34. rousk j, brookes pc, baath e, lauber cl, lozupone c, caporasa j g . . . fierer n. 2010b. soil bacterial and fungal communities across a ph gradient in an arabe soil. isme j 41-12. species diversity of soil fun@ from a corn plantation tecson et al. sigh ri< [internet]. 2010. trichoderma: a bio-control agent for management soil born diseases. agropedis; [updated 2010 jun 06; cited 2020 sept 091. available from: http://agropedia.iitk.ac.in/ content/trichoderma-bio-control-agent management-soil-born-diseases toma fm, faqi abdulla nq. 2012. isolation, identification and seasonal distribution of soil borne fungi in different aras of erbil governorate. j adv lab res biol4(3):246-55. vandermeer jh. 201 1. the ecology of agroecosystems. jones and bartlett publisher. usa. van heerden i, cronje c, swart sh, kotze jm. 2002. microbial, chemical and physical aspects of citrus waste composting. bioresource technology 81(1):71-6. williams sh, philips td, jolly pe, stiles jk, jolly cm, aggarval d. 2004. human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. am j clin nutr 80(05):1106-22. yao q, loi j, yu 2, li y, jin j, liu x. 2017. changes of bacterila community compositions after three years of biochar applications in black soil of northeast, china. appl soil ecol113:ll-21. the southeast asian journal of tropical biology vol. 31 no. 2, 2024: 217 227 doi: 10.11598/btb.2024.31.2.2057 issn: 0215-6334 | e-issn: 1907-770x 217 article higlights • taurine improves eye development in golden rabbitfish larvae • enhanced growth performance with taurine-enriched rotifers • taurine positively impacts fish eye and body growth article information received 17 july 2023 revised 29 november 2023 accepted 29 november 2023 *corresponding author, e-mail: miasetia@apps.ipb.ac.id research article influence of rotifer enrichment with taurine on larval eye development and growth performance of golden rabbitfish (siganus guttatus) darsiani1,2, mia setiawati*1, dedi jusadi1, muhammad agus suprayudi1, asda laining3,4 1department of aquaculture, faculty of fisheries and marine sciences, ipb university, bogor 16680, indonesia. 2aquaculture study program, faculty of animal husbandry and fisheries, universitas sulawesi barat, majene 91412, indonesia. 3research institute for coastal aquaculture and fisheries extension, ministry of marine affairs and fisheries, maros 90512, indonesia. 4research center for fishery, national research and innovation agency, cibinong 16915, indonesia. abstract golden rabbitfish (siganus guttatus) is an emerging species for aquaculture industry, despite constrains faced on commercial scale of seed production due to the high mortality during the first-feeding stage. an experiment was conducted to determine the effect of taurine through enrichment of rotifers as live-feed on eye development and growth performance of larval golden rabbitfish. observation of eye development was carried out by measuring eye diameter of the larva and development of the retina based on histology assessment. growth performance was measured for absolute growth and fin development of the larvae and survival rate. the results showed that taurine-enriched rotifer generally provided better performances for eye development and growth of larval golden rabbitfish compared with control (without taurine). increase of taurine dose up to 0.050 g/l resulted in an increase of eye diameter, absolute growth, fin development and survival rate. further increased increment of the taurine tended to decrease the values of the measured parameters. the eye diameter of larval golden rabbitfish fed with 0.050 g/l taurineenriched rotifer was significantly wider (106.1±9.8 µm) (p < 0.05) compared with control (58.2±14.3 µm), but did not significantly differ from other doses of taurine (p > 0.05). body width of larvae fed with 0.050 g/l taurine-enriched rotifer was significantly higher (127.3±14.6 µm) (p < 0.05) compared with control (98.8±18.3 µm). in regard to eye development, growth performances (total length, fin development and survival rate), dose of taurine for rotifer enrichment fed to larval golden rabbitfish siganus guttatus was 0.050 g/l. keywords: eye development, golden rabbitfish, growth performance, taurine introduction siganids or rabbitfish comprise many species, one of which is siganus guttatus also called golden rabbitfish with local name baronang emas (laining et al. 2021). distribution of rabbitfish is mainly in tropical coastal waters. production of the species is reported different based on the region. in abu dhabi-united arab emirates, s. canaliculatus was reported to be 149 tons in 2002 from capture (grandcourt et al. 2007). the philippines reported production of siganid reaching 150.89 tons in 2005 and increased significantly by 45% in 2014 (gonzales et al. 2018). production from aquaculture was reported in the philippines since 2018 both from ponds and floating cages culture (caballero et al. 2022). indonesia also reported production of siganids which are mainly from capture. demand of rabbitfish is reported in many parts of indonesia, in particular in south sulawesi province, where fish are commonly served in restaurants and become a typical culinary in the region (laining et al. 2017). https://doi.org/10.11598/btb.2024.31.2.2057 biotropia vol. 31 no. 2, 2024 218 south sulawesi province reported production of 3,658.2 tonnes in 2018 and increased 37% in 2021 (https://dkp.sulselprov.go.id/page/info_berkala/ kategori/24). despite demand for rabbitfish, supply from aquaculture has yet met the demand so far. one major constraint of golden rabbitfish culture is the unavailability of commercial scale seed production. mortality rate of larvae is still quite high, reaching 50-90% at the age of 2-3 dah (days after hatch), the time where transition period from endogenous nutrition to feeding on life-feed (rao 2003). survival rate during the first feeding stage of golden rabbitfish ranges from 0.231.6% (juario et al. 1985), and survival rate of golden rabbitfish larvae was only 1%. it is argued that failures in the seed production might be caused by the feeding ineffectiveness during the early hatched stage, due to the limited eye sight of the larvae to see the feed. golden rabbitfish demonstrates a slow growth pattern from their early life to their young juvenile period (duray 1998). the eyes were formed on the first day, yet they were not fully developed (juario et al. 1985). after 1 dah, the eyes acquire pigmentation and become more pronounced at the age of 2 dah (darsiani et al. 2022). differentiation of eye is essential for larvae to see and catch the feed (yufera et al. 2014). one of the contributing factors in the development of larval eyes is nutrition (stuart 2013). the nutrition requires enrichment technique to deliver certain nutrient to support the eye. taurine-enriched feed has been reported to have positive effects on eye, brain, and muscle development (growth) in turbot fish scophthalmus maximus (qi et al. 2012). taurine is a simple protein that is easily absorbed by the body, to support the fish growth (jusadi et al. 2012). taurine also improves survival and development of white shrimp during the larval stages (jusadi et al. 2011), enhances the immune system and reduces stress in zebrafish (mezzomo et al. 2019) and contributes in regulating calcium metabolism, including the inside of the eye (lombardini 1983; lombardini 1991). studies on the taurine-enriched feed has been conducted on various fish, both freshwater and marine species (el-sayyed 2013; hernandez et al. 2018) and reported that marine fish have less ability to synthesize taurine (el-sayyed 2013). effects of taurine are different among fish depending on species, size, and nutritional content of the feed provided and ability of species to synthesize taurine with the help of enzyme csd (cysteinesulphinate decarboxylase) (el-sayyed 2013). this study aimed to evaluate the effect of different levels of taurine through rotifers enrichment on eye development and growth performances of larval golden rabbitfish s. guttatus. materials and methods experimental design this study was conducted at the rabbitfish hatchery instalation of research institute for coastal aquaculture and fisheries extension (ricafe) in barru district, south sulawesi. this experiment was designed in a completely randomized design consisting of 4 treatments with 3 replications. the treatments were rotifers enriched with different doses of taurine, i.e., without taurine enrichment (t 0); with 0.025 g/l taurine dose (t 0.025); with 0.050 g/l taurine dose (t 0.050); and with 0.075 g/l taurine dose (t 0.075) following protocols developed by jusadi et al. (2012). enrichment of rotifer rotifers type s (brachionus rotundiformis) harvested from mass culture tank with a density of 500-1,000 ind./ml (jusadi et al. 2015) were enriched before being given to larvae. enrichment was performed by preparing 10 l media, requiring 0.5 ml of fish oil a1 dha selco, 0.1 g of egg yolk, 0.25 of bread yeast, and taurine pa sigma which were added according to the respective treatment doses. taurine and other enriching materials were mixed up with 200 ml water and emulsified using a blender for three to five minutes, then poured into rotifers containers. the enriched rotifers were incubated for 2 hours and harvested by using a 50 µm plankton net then fed to the larvae (jusadi et al. 2012). condition of larval rearing feeding trial was conducted by rearing larval in concrete tank with capacity of 6 t filled up with 3.5 t of seawater with 20-25 ppt salinity (lante & muslimin 2012). water was sterilized using uv light (yamano uv-30w) for 6 hours. during the larval rearing period, green water system was applied by adding nannochloropsis sp., and maintained at a density of 1x105 individual/ml (duray & juario 1988). newly-hatched larvae (1 dah) were carefully stocked with density of 20 influence of taurine on larval eye development and growth performance of golden rabbitfish darsiani et al. 219 ind./l (duray & kohno 1988; lante & muslimin 2012). enriched rotifers were fed to the larvae with density of 10-20 ind./ml once a day. the feeding experiment was conducted for 10 days. water quality was maintained by performing water exchanges following duray & kohno (1988). water quality parameters were measured every day at 07.00 am and 17.00 pm. observed variables and data analysis biological parameters observed in this study included eye development, absolute growth (length), fin development, number of rotifers consumed by larvae and survival rate. absolute growth (length) was calculated using the formula from jaya (2013) and mulqan et al. (2017): l=lt-l0 where: l = absolute growth (mm) l0 = average length of test animals at the start of the experiment (mm) lt = average length of test animals at the end of the experiment (mm) thirty larvae were taken from each rearing tank and observed using compound microscope (olympus 40, japan) with 4x magnification connected to a computer and camera. histological observation was performed using the same microscope with 100x magnification. the amount of feed comsumption was determined by observing the stomach contents of the larvae. daily live-feed rotifer consumed by the larvae was observed by taking 30 larvae from each tank, and the stomach content was then checked using microscope. survival rate was calculated using equation developed by effendie (2002): where: sr = survival rate (%) nt = number of larvae at final day of the experiment n0 = number of larvae at initial day of the experiment biochemical analysis was carried out for taurine content in rotifers using high performance liquid chromatography (hplc, shimadzu 20 a, tokyo, japan) procedure. due to the limited samples of the enriched rotifers, the taurine content was accommodated only for simplo analysis. antioxidant glutathione peroxidase (gpx) content was analyzed spectrophotometrically using glutathione peroxidase assay kit (abcam uk, london). larval size data and eye development measurement data (histology preparations) were analyzed using analysis of variance (anova), with multiple comparisons evaluated using w-tukey to compare the treatment effects (steel & torrie 1991). statistical analysis was carried out using software spss 16.0 (spss, inc., chicago, illinois, usa). the level of significance was defined as 0.05. data on taurine content in rotifers after taurine enrichment were explained descriptively. results and discussion eye development the eye diameter of larval golden rabbitfish fed with different doses of taurine-enriched rotifers is presented in table 1. larvae fed with taurineenriched rotifers had a very significant effect (p < 0.01) on the eye diameter compared to the t 0 treatment (control). the highest eye diameter was observed in the t 0.050 treatment, but did not significantly differ from other two taurine levels in the t 0.025 and t 0.075 groups (p > 0.05). table 1 eye diameter (mean±sd) of golden rabbitfish, s.guttatus larvae fed different doses of taurine dose of taurine (g/l) average eye diameter±sd (µm) 0 58.2±14.3a 0.025 94.6±7.5b 0.050 106.1±9.8b 0.075 91.7±7.8b notes: sd = standard deviation. different letters above the numbers indicate significant differences among treatments at (p < 0.05). biotropia vol. 31 no. 2, 2024 220 figure 1 retinal development of larvae s. guttatus notes: dah = day after hatch. blue arrows indicate lens and receptor cells. a presents retinal development at 2 dah; b, c, d, e present retinal development at 10 dah with taurine dose treatments, observed with 4x magnification. b = control (t 0 g/l); c = t 0. 025 g/l; d = t 0. 050 g/l ; e = t 0. 075 g/l; f = parts of the retina (100x magnification). description of picture f: 1) retinal pigmen epithelium; 2) photoreseptor layer; 3) outer limiting membrane; 4) outer nuclear layer; 5) outer plexiform layer; 6) inner nuclear layer; 7) inner plexiform layer; 8) ganglion cell layer; 9) sclera; 10) lens. histology of larval eye development was performed to have in-depth observation on the treatment effects. the histological result of the larval eye is described in figure 1. at 2 dah, diameter of the lens was 45.4±1.0 µm, and the photoreceptor cell thickness was 1.9±0.6 µm. at 10 dah, the lens diameter was 64.7±3.8 µm (t 0), 68.1±5.6 µm (t 0.025), 72.7±5.0 µm (t 0.050), and 69.9±0.9 µm (t 0.075), respectively and thickness of the photoreceptor cell was 4.0±0.1 µm, 4.4±0.1 µm, 5.6±3.1 µm, and 3.1±0.0 µm, respectively. results found in this present study indicated that rotifers enriched with taurine had positive effect on the eye development of golden rabbitfish larvae including eye diameter size, lens diameter and receptor cell thickness. in the rotifers enrichment treatment using taurine, eye development increased, and the best development was occurred when the rotifers was enriched with 0.050 g/l of taurine. jusadi et al. (2012) reported that the use of taurine at a dose of 0.060 g/l on duck grouper cromileptes altivelis increased the rate of feed predation, indicating that eye development had increased significantly. banthani et al. (2019) also reported that taurine as an enrichment in rotifers was perceived to support eye development, leading to significant increase in the vision of coral trout larvae (plectropomus leopardus). however, further increase of the taurine dose did not cause a better eye development as demonstrated in this present study. on the other hand, the excessive doses of taurine tended to have negative effect on the eyes’ visibility of the larvae due to the impaired eye development. it is argued that excessive taurine in the larvae’s influence of taurine on larval eye development and growth performance of golden rabbitfish darsiani et al. 221 body might stimulate a toxic taurine-derived compounds that can damage cells. according to watanabe et al. (2022) n-acetyltaurine (nat) and n-chlorotaurine are derivatived compounds of taurine and although they are biologically less toxic, they may decrease the stability of cell. increased eye diameter is one of the indicators of the larva’s developing ability to see objects. fiolita et al. (2017) stated that eye diameter improves the eye’s ability to capture light which strongly improve the ability of the larvae to see surrounding objects. furthermore, a well-developed eye lens supports for better vision (afitah et al. 2020). the eye lens functions to regulate the light entering the eye, continue the reflection the object’s shadow received, then focus the image falling on the retina, and sent to the brain for translation (rahardjo et al. 2011). the shadows of objects that successfully enter the eye are supported by proper lighting conditions and appropriate contrast of the container/object (bogner et al. 2017; stuart 2013). growth has a major effect on the increase in the size of the eye lens, but the increase in lens size will stop at a certain age (fitri 2002). number of live-feed consumption the average number of live-feed (taurineenriched rotifers) consumed by the larvae at the age of 3-10 dah is shown in figure 2. generally, number of rotifers consumed by the larvae increased with the increase of the age of larvae. at the final day of the feeding experiment, rotifers consumption by the larvae was the highest in the t 0.050 g/l treatment, while the lowest was recorded in the t 0 g/l group. the eyes’ ability to see objects can also be determined through the amount of feed captured by the larvae (yufera et al. 2014). the present study showed that the amount of live-feed rotifers captured by the larvae was significantly higher in treatments with taurine enrichment compared to that without taurine, with the highest amount of rotifers captured was found in t 0.050 g/l. this indicated that a well-developed eye can increase the number of live-feed captured. a similar finding was reported by jusadi et al. (2012) and hernandez et al. (2018), where the amount of feed captured by larvae increased through taurine supplementation. subsequently, successful feed capture can support the growth and development of larvae (stuart 2013; miranti et al. 2017). figure 2 amount of live-feed (rotifers enriched with taurine with different doses) consumed by golden rabbitfish larvae, s. guttatus at the age of 3 to 10 dah figure 2 shows that the daily feed predation of golden rabbitfish larvae at 3-5 dah in each treatment showed no significant difference, and a significant difference (p < 0.01) started to be detected at 6 dah. this showed that the effect of taurine on the eye sight of larvae occurred after the larvae reached 6 dah, or after 4 days of consuming taurine-enriched rotifers. the increasing eye vision was presumably due to the calcium regulation in the eye, resulting in better vision in the dark. this is in agreement with studies conducted by lombardini (1983), lombardini (1991), and militante dan lombardini (2002), where taurine supports eye development through calcium regulation that occurs in the retinal pigment epithelium (rpe). the glutamate released under dark conditions may increase the concentration of free calcium (ca2+i) in rpe. high concentration of free calcium can inhibit the performance of rhodopsin kinase, limiting individuals’ vision in low light conditions. in addition, free calcium can also inhibit the performance of cgmp which is the ion channel na+. the hampered cgmp disrupts the entry of na+ into rpe, where in fact the na+ is an element that can bind free calcium, decreasing its concentration in the rpe. free calcium can further hamper the performance of guanylate cyclase, a phototransduction that converts light into electrical signals in the brain. growth absolute growth of golden rabbitfish larvae measured in this study included body length, and width. the absolute growth of the larvae fed with different doses of taurine-enriched rotifers are presented in table. 2. feeding of rotifers enriched with taurine at different doses had a very significant effect (p < 0.01) on absolute length, but had no significant effect biotropia vol. 31 no. 2, 2024 222 (p > 0.05) detected on absolute body width of the larvae. the trend of both total length and body weight was similar where increase of taurine dose up to 0.05 g/l resulted in an increase of the both parameters. table 2 absolute growth (mean±sd) of golden rabbitfish, s. guttatus larvae fed with rotifers at different doses of taurine dose of taurine (g/l) absolute growth total length (mm) body width (µm) 0 0.94±0.09a 98.8±18.3a 0.025 1.28±0.01b 106.5±30.0a 0.050 1.39±0.03b 127.3±14.6a 0.075 0.85±0.08a 79.6±8.2a note: different letters above the numbers indicate significant differences among treatments (p < 0.01). the mean of absolute length of larvae fed with t 0.050 treatment was very significantly different from t 0 and t 0.075 treatments (p < 0.01), but was not significantly different from t 0.025 treatment (p > 0.05). the highest absolute growth was obtained at t 0.050 treatment and the lowest was shown at t 0.075 treatment. in this study, an obvious response of taurine enrichment was observed on the length growth of golden rabbitfish larvae. the increase in body length in treatments fed with taurine-enriched rotifers was suspected to have a positive correlation with spinal development. zulfahmi et al. (2018) revealed that spinal development is an important factor for fish growth as it supports the muscle strengthening. similar findings were reported on humpback groupers (cromileptes altivelis) and on white shrimp litopenaeus vannamei (jusadi et al. 2012; 2015) that the larvae growth was better in the taurine-enriched rotifers feeding treatment at a dose of 0.050 g/l. lower growth of golden rabbitfish was observed in the treatment without taurine (t 0) and at the highest dose of taurine (t 0.075). similar phenomena were reported by bavi et al. (2022), where taurine deficiency and excessive taurine feeding decreased the growth performance of juvenile acipenser ruthenus. impaired growth in excessive taurine feeding was argued to be caused by the inhibited absorption of nutrients which is in line with liu et al. (2022) who reported that excessive taurine can damage the digestive tracts and livers of scophthalmus maximus. better growth can be achieved when the daily energy requirements are met, and one way to save energy use is the easily absorbed diets. as stated by jusadi et al. (2012), taurine is one of the essential amino acids that can be used directly (absorbed) by the body to increase growth and development of fish larvae. as reported by mcbean et al. (2017) and tochitani (2022), taurine can be synthesized from methionine and cysteine with the help of several enzymes, such as cystathionineβ-synthetase (cßs), β-cystathionine (cth), cysteine dioxygenase (cdo), cysteine sulfinic acid decarboxylase (csad), hypotaurine oxidase, and cysteic acid decarboxylase. these stages of amino acid synthesis requires a certain amount of energy to produce taurine compounds. the role of taurine on nerve cells has also been reported by jakaria et al. (2019), where taurine had the ability to regulate free calcium in cells to prevent damaging the cells (apoptosis) (including nerve cells), through activating the calpain and caspase enzymes. the condition of healthy nerve cells can facilitate in sending signals to the brain to perform organogenesis, which is controlled by the central nerve (banthani et al. 2019). the effect of taurine on growth was further reported by qian et al. (2021), stating that taurine was able to improve digestive function in the intestine and regulate glycolipid metabolism to increase the use of carbohydrates, to support growth. shi et al. (2022) also found that taurine maintained the intestinal health of juvenile swamp eels monopterus albus. fin development development of fins including dorsal, pectoral, and caudal of golden rabbitfish larvae fed with taurine-enriched rotifers at different doses of taurine is presented in table 3. table 3 development of dorsal, pectoral, caudal fins (mean±sd) of golden rabbitfish s. guttatus larvae fed with different doses of taurineenriched rotifers dose of taurine (g/l) fin development (µm) dorsal pectoral caudal 0 48.7±13.3a 44.0±13.4a 36.2±11.8a 0.025 79.3±15.2ab 76.0±24.3ab 52.0±10.2ab 0.050 84.6±14.8b 93.6±15.3b 75.5±8.9b 0.075 56.4±10.9ab 87.0±13.8ab 54.9±17.3b note: different letters above the numbers indicate significant differences among treatments (p < 0.05). influence of taurine on larval eye development and growth performance of golden rabbitfish darsiani et al. 223 development of fins was significantly affected (p < 0.05) by feeding of rotifers enriched with taurine at different doses. the mean of dorsal and pectoral fins of the t 0.050 treatment were significantly different (p < 0.05) from the t 0 treatment, but did not significantly differ (p > 0.05) from both t 0.025 and t 0.075 treatments. the caudal fin development of larvae in t 0 and t 0.025 treatments was significantly different (p > 0.05) from that in t 0.05 and t 0.075 treatments. the highest fin length (dorsal, pectoral, and caudal) was obtained at t 0.050 treatment and the lowest was at t 0 group. this study also found that growth was directly proportional to the fin size of the larvae where larvae in t 0.050 treatment demonstrated a better fin development. well-developed fins can result in a better fish mobility to swim in the water column. rahardjo (2020) also found that fins can support fish mobility (swimming) including for catching preys. better movement of fish may trigger an increase in the amount of feed predation, and result in better growth and survival (stuart 2013). in addition, jusadi et al. (2011) reported that taurine can accelerate the stadia development of white shrimp litopenaeus vannamei larvae. survival rate survival rates of rabbitfish larvae fed with rotifers enriched with different doses of taurine is presented in table 4. feeding rotifers enriched with taurine at different doses had a significant effect on the survival of golden rabbitfish larvae (p < 0.05). only survival rate in the t 0.050 treatment was significantly different (p < 0.05) from the other treatments. on the other hand, survival rates in the t 0, t 0.025, and t 0.075 treatments were not significantly different (p > 0.05). the highest survival rate was obtained at t0.050 treatment and the lowest was shown at t 0.075 group. table 4 survival rate (mean±sd) of golden rabbitfish s. guttatus larva fed with rotifers enriched with different doses of taurine dose of taurine (g/l) survival rate (%) 0 0.71±0.12a 0.025 0.75±0.21a 0.050 1.47±0.42b 0.075 0.58±0.28a note: different letters above the numbers indicate significant differences among treatments (p < 0.05). the highest survival rate in this present study was observed in t0.050 taurine treatment. this is in line with the two studies reported by jusadi et al. (2012; 2011) that 0.050 g/l of taurine significantly increased the survival rate of humpback grouper cromileptes altivelis and white shrimp larvae litopenaeus vannamei. similarly, banthani et al. (2019) reported that survival of leopard coral grouper plectropomus leopardus larvae was significantly higher in taurine-enriched treatment compared to that without taurine treatment. it is presumed that at a certain stage or age, taurine supplementation will have a better effect on the growth, development, and survival of larvae s. guttatus. el-sayyed (2013), hernandez et al. (2018), dan wei et al. (2020) reported that the effects of taurine will differ for different species, body sizes, and age levels. antioxidant content (gpx) in larvae fed with taurine-enriched rotifers gpx antioxidant content in body of golden rabbitfish larvae after being fed with taurineenriched rotifers is presented in table 5. the pattern of the antioxidant gpx in the larval body linearly increased as the dose increased. the highest gpx antioxidant content was obtained at t 0.075 treatment and the lowest was found at t 0 group. however, the taurine doses had no significant effect (p > 0.05) on antioxidant content (gpx) on the larval body. table 5 antioxidant (gpx) content in s. guttatus larvae dose of taurine (g/l) average antioxidant gpx content in larvae (nmol) 0 65.39±1.60a 0.025 70.39±5.86a 0.050 71.33±1.21a 0.075 73.29±2.78a note: different letters above the numbers indicate significant differences among treatmens (p < 0.05) other function of taurine is to increase the glutathione synthesis in the body (miyazaki et al. 2022). although no significant, effect of taurine enrichment on the antioxidant (gpx) content in the larval body was found in the present study. a clear trend was observed that increased taurine dose linearly increased the the antioxidant (gpx) in body. the presence of this antioxidant (gpx) might positively affect the healthy condition of the larvae fed with taurine-enriched rotifers. biotropia vol. 31 no. 2, 2024 224 schaffer & kim (2018) reported that taurine is believed to function as an antioxidant, enhance immunity (jin et al. 2017), and can prevent stress (akande & ahmed 2017; mezzomo et al. 2019) by neutralizing super oxidant derivatives produced from mitochondria (schaffer & kim 2018). marcinkiwicz and kontny (2012) demonstrated that taurine can reduce toxins of hypochrolytic acid (hocl) and hypobromic acid (hobr) which both of these acids are peroxide compounds. taurine can convert these acids into chloramine taurine (taucl) and bromamine taurine (taubr) which are more stable or less biologically toxic compounds. taurine content in rotifers after enrichment table 6 presents the taurine contents of rotifers after enrichment with various doses of taurine. the highest value was analyzed in 0.050 g/l treatment, followed by 0.025 g/l, while the lowest content was detected at the highest dose of t 0.075 g/l. table 6 taurine content in rotifers after enrichment dose of taurine (g/l) taurine content in rotifers after enrichment (µg/g) 0 1.875 0.025 2.332 0.050 3.274 0.075 1.793 the content of taurine in rotifers tended to increase as the dose of taurine increased up to 0.050 g/l, and further decreased at higher level. this trend might imply that rotifers has a limited ability to accumulate taurine in the body. similar finding was reported by jusadi et al. (2011) that the addition of taurine up to 0.1 g/l increased the taurine content in rotifers; and further higher dose lead to decrease the taurine content. compared with the treatments of taurineenriched rotifers, only a small amount of taurine was found in the treatment without taurine (t 0), namely 1,875.18 µg/g. jusadi et al. (2011) reported similar results, the taurine content in non-taurine-enriched rotifers was 1,700 µg/g. the taurine content detected in the rotifers without taurine enrichment is presumed to come from other ingredients used in the enrichment, namely baker's yeast, egg yolks, and dha. baker's yeast and eggs are ingredients that contain protein and other nutrients (oktaviani et al. 2012; swari et al. 2019) which can also be consumed or absorbed by rotifers. takeuchi (2001) found small amounts of taurine in unenriched rotifers ranging from 8-18 µg/g. jusadi et al. (2012) and cho et al. (2022) also reported lower levels of taurine content in unenriched rotifers, namely 77.7 µg/g and 310 µg/g, respectively. the difference in taurine content in rotifers is probably due to the type of nutrient fed to the rotifers before being used for the enrichment. since, taurine content of rotifers varied according to the status of the rotifers, it is suggested to enrich the rotifers with taurine before being given to fish larvae (li et al. 2017; swari et al. 2019; salsabila et al. 2019). conclusion live-feed rotifers enriched with 0.050 g/l taurine resulted in better eye development and growth performances in terms of total length, fin development and survival rate of larvae of golden rabbitfish siganus guttatus. acknowledgment we would like to thank the australian center for international agricultural research (aciar) for partly support the study through collaborative project fis 2016/130. the authors sincerely thank technicians at ipuw of ricafe for technical assistants during the research study. we further thank beasiswa pbbdn afirmasi ptnb for sponsoring the research through scholarship with contract number 1420/d3/pg/2018. references afitah, fitrianti ar, widayati ed, pamira i, muasaroh, ujilestari t. 2020. adaptasi retina mata hewan nokturnal terhadap kemampuannya melihat dalam gelap. 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yunarsa ppa, adiatmika ipg. 2018. antioxidant concentration of superoksida dismustase (sod) in mice liver on strenous physical activities. e-journal medika udayana 7(4):143-7. zulfahmi, ilham, akmal y, batubara as. 2018. the morphology of thai mahseer’s tor tambroides (bleeker,1854) axial skeleton (ossa vertebrae). jurnal iktiologi indonesia 18(2):139-49. http://dx.doi.org/10.1016/j.aquaculture.2014.01.031 _hlk151415317 _hlk151415360 _gjdgxs biotropia vol. 30 no. 2, 2023: 171 182 doi: 10.11598/btb.2023.30.2.1780 171 spatial distribution of invasive plants in bandung, west java, indonesia rahmawati and dian rosleine* department of biology, school of life sciences and technology, institut teknologi bandung, bandung, west java, 40132, indonesia received 15 july 2022/ revised 10 march 2023 /accepted 10 march 2023 abstract the urban area is a source of invasive plants that enter through human activities such as agriculture and land-use conversion. studying the invasive plant in urban areas is essential to understanding the city’s ecosystem health condition. therefore, this study aims to inventory invasive plants, map their distribution, and explain the relationship between land use with the community diversity and species richness of invasive plants in bandung. the vegetation analysis was performed using line-transect in 22 study sites distributed using a systematic random sampling method in bandung to observe the plant species composition. the study plots were placed based on the land-use type. the species name, individual number, frequency, and sampling site locations were noted and analyzed to calculate the important value index (ivi) and the invasive species distribution pattern using the principal component analysis (pca). the dominant invasive species was spatially mapped. six types of land use were used in this study, i.e., settlements, street green lanes, gardens, paddy fields, urban parks, and urban forests. there were 187 species found in bandung, which can be categorized into alien invasive species (39%), invasive native plants (25%), non-invasive alien species (18%), non-invasive native species (15%), and unidentified plants (3%). the most common invasive plants found were eleusine indica (ivi=10.50%), trimezia martinicensis (ivi=7.22%), and cyperus rotundus (ivi=6.74%). based on the plant community similarity index, the study area with the highest similarities were paddy fields with gardens (50.5%), settlements with road lanes (44.4%), urban parks with road lanes (26.2%), and urban forests with road lane (17.5%). pca showed swietenia macrophylla as the most common invasive plant found in urban forests, urban parks, and road lanes, with air humidity as the most influencing environmental factor. trimezia martinicensis is the most common species in the settlement area affected by high air humidity. bidens pilosa is an invasive plant commonly found on paddy fields, gardens, settlements, road lanes, and urban park edges. this species can easily and rapidly reproduce with a high survival rate. the many invasive plants found in bandung must be managed to maintain the urban ecosystem’s health. keywords: bandung, interpolation, invasive species, species mapping, urban area introduction alien species are brought or accidentally brought into an ecosystem unnaturally. invasive species are native or alien species that can widely impact their habitat, causing environmental damage, economic loss, or harm to humans (tjitrosoedirdjo 2017). dominating their habitat is the main characteristic of invasive species. they can cause a decrease in biodiversity through the loss of native species and disturbance in the ecosystem functioning (sunaryo 2015). urban areas create multiple habitats that accommodate plant species diversity, and invasive species can often develop in such habitats (štajerová et al. 2017). mainly, anthropogenic disturbances introduce invasive species into the new habitat, such as the landuse change to establish agricultural areas of paddy fields and gardens or newly built settlements. settlement areas can be a focal point of the species’ invasive movement from spreading to the surrounding landscape (chytrý et al. 2005). the diverse land use in urban areas caused the difference in the invasive species composition in each land-use type. bandung is one of indonesia’s major cities with vastly developed and diverse land use. so far, studies *corresponding author, email: drosleine@gmail.com biotropia vol. 30 no. 2, 2023 172 on invasive species have mainly been done in conservation areas with limited knowledge in urban areas. many alien plant species were introduced to urban areas to provide, augment or restore specific ecosystem services. however, some species negatively impact existing ecosystem services and create novel ecosystem disservices within urban areas (potgieter et al. 2017). for example, the alien invasive acacia mangium found in the highway green lanes and urban parks was first introduced as street tree shade. besides, this plant was also planted on eroded soil to repair the soil structure due to its robust, extensive rooting system (environmental management agency 2014). on the other hand, a. mangium can change the soil composition through nitrogen fixation, competing on water and light resources with surrounding native species due to its deep rooting system and dense shade, producing allelopathic substances that can hamper the germination of surrounding plant seeds (datiles & rodriguez 2017). a study of invasive species in urban areas must be carried out to manage invasive species to reduce the negative impact on ecosystem services and prevent their spread to the natural areas. therefore, this study aims to inventory invasive species and map their distribution. data gathered (invasive species number and composition on each land use type, relations between invasive species with the land use, and distribution map of the ten most dominant invasive species in bandung) can serve as early detection of the invasiveness of each species. it can also provide information for policymakers to determine further steps. materials and methods study area this study was done in 22 sampling sites (figure 1) placed with systematic random sampling in bandung (6°50′20″-6°58′3″ sl, 107°32′44″-107°44′15″ el). the sampling sites were determined by 22 coordinates systematically set in the thematic map of bandung on arcgis 10.4.1. these selected coordinates were then exported from arcgis to the gps essential application for the location survey. monitoring plots were randomly assigned based on the land use in those coordinate points. figure 1 location of the study site spatial distribution of invasive plants in bandung – rahmawati and rosleine 173 land-use paddy fields were found in six areas, i.e., sukaati, buahbatu, rancasari, mekarmulya, sukapura, and kopo. gardens were found in five sampling areas, i.e., isola, cigondewah, nagrog, cisurupan, and jatihandap. the street green lanes were found at five points, i.e., cigondewah, mekar mulya, cisaranten wetan, kiaracondong, and pahlawan. there were nine points of settlements, i.e., geger kalong, babakan jeruk, rancasari, buahbatu, sukaati, panyileukan, dago, antapani tengah, and pahlawan. last, urban parks were found at tegalega and bandung wetan. meanwhile, the urban forest was only found in the area point of babakan siliwangi. vegetation survey the vegetation survey was done from april 2021 to january 2022. depending on the environmental situation, a twenty-eight-line transect was installed for 100 or 150 meters. the plant species’ name with their life form (i.e., tree, shrub, liana, or herb) found in the line transect area and their number was written in the monitoring data sheet. the plants were directly identified in the field. unidentified plants were sampled from the field to be further identified using the identification book main weeds of rice in asia. identified plants were then grouped into four types, i.e., invasive alien species, invasive native, non-invasive alien, and non-invasive native, based on the guidebook provided by biotrop, convention of biological diversity (cbd), center for agriculture biosciences international (cabi) and guide book to invasive species in indonesia, forest in southeast asia-indonesia program (forisindonesia). measurement of environmental climatic condition environmental and climatic conditions were measured to support the making of invasive species distribution prediction maps. measured data were temperature and air humidity using a thermo hygrometer. light intensity was also measured using a light meter. measurements were done triplicate on each transect at the transect’s start, middle, and endpoint, 50 to 100 cm above the ground. calculation of importance value index (ivi), diversity and habitat similarity the importance value index (ivi) was calculated for each species by summing up the relative density (1) and relative frequency (2) as follows: density (d) = species individual number length of line transect relative density (rd) = d of species x 100% (1) total d of all species found frequency (f) = number of plots where species was found total plot number relative frequency (rf) = f of species x 100% (2) total f of all species found the ivi was calculated twice. the first was done for each land-use type for community analysis, where each land-use type was assumed to represent a specific vegetation community, totaling six vegetation communities. the second calculation was the ivi of the overall study sites. the calculation of shannon wiener diversity index h’ (3) on each community was done with the following formula, h’ = -∑ pi ln (pi ) (3) with pi = proportion of species’ individual number to overall species. the sorensen similarity index (4) was calculated for every combination pair of land use to find the most similar vegetation communities. the calculation of the sorensen similarity index used the following formula, s = 2c x 100% (4) a+b with a = species number in community a b = species number in community b c = species number in communities a and b statistical analysis density values from 20 plant species with high ivi scores (>2.06%), average temperature, average air humidity, and average light intensity in each community were analyzed with pca. pca is used to extract meaningful information from a multivariate data table and to express this biotropia vol. 30 no. 2, 2023 174 information as a set of a few new variables called principal components. these new variables correspond to a linear combination of the originals (kassambra 2017). the number of principal components is less than or equal to the number of original variables. using r studio version 4.2.0, package factominer, only 15 plant species with the highest contribution of the principal component were shown on the pca plot. graphs from the analysis were visualized using the package factoextra. pca analysis was used to choose ten invasive species in making the invasive species distribution prediction map. invasive species distribution mapping ten species with the highest score were mapped for their distribution in the bandung area. the score indicates that those species highly influence study sites (kassambara 2017). the plant distribution map in bandung was based on the data of the individual number of each species and the mean climatic factor measured on each coordinate point (study sites). climatic data were adjusted from the pca results, where only one to two climatic data influence that species. the distribution map was made using the interpolation method on the application arcgis 10.4.1. spatial interpolation is a procedure for estimating the variable value in the field that is not included in study samples and located inside an area of the sampling location (aswant 2016). results and discussion vegetation composition in bandung the total number of species found in all study sites was 187, comprising 47 tree species, 21 shrub species, 3 liana species, and 116 herb species. invasive species, whether alien or native, have a higher proportion than noninvasive species, with a total ratio of 64% (figure 2). the presence of invasive species is supported by disturbance and human activities. these two phenomena are pervasive in urban areas. thus, the presence and frequency of invasive species are relatively high in urban areas (gulezian & nyberg 2010). the proportion of alien invasive species found was higher than the native ones. most alien invasive species were initially brought to the urban areas to provide, add, or recover a specific ecosystem service. however, besides giving ecosystem services needed by humans, the alien invasive species may negatively impact the available ecosystem services (potgieter et al. 2017). figure 2 the proportion of invasive and non-invasive species in bandung spatial distribution of invasive plants in bandung – rahmawati and rosleine 175 table 1 ten species with the highest importance value index (ivi) on all study sites nr species name native family ivi (%) 1 axonopus compressus tropical america poaceae 11.46 2 eleusine indica india poaceae 10.50 3 asystasia gangetica india, ceylon acanthaceae 7.87 4 alternanthera philoxeroides tropical america asteraceae 7.46 5 trimezia martinicensis mexico iridaceae 7.22 6 cyperus rotundus india, africa cyperaceae 6.74 7 bidens pilosa south africa asteraceae 5.42 8 cynodon dactylon africa poaceae 5.12 9 swietenia macrophylla south america, central america meliaceae 4.36 10 synedrella nodiflora south america, central america asteraceae 4.23 the calculation of ivi in table 1 shows that ten plants with the highest ivi were invasive species. invasive species tend to have a high ivi due to their characteristics of the high tolerance range, enabling them to adapt well to various environments. axonopus compressus is the most abundant species in street green lanes, settlements, and urban parks. annual plant a. compressus can grow vegetatively well with stolon and produce many seeds. e. indica was abundant in paddy fields and found in gardens, street green lanes, and settlements. e. indica can grow well in high light intensity and has a high adaptation level (setyawati et al. 2015). meanwhile, a. gangetica was abundant in gardens and found in paddy fields, streets, and urban forests. a. gangetica is a climber plant that can form a highly dense population (sandoval & rodriguez 2012). vegetation composition in six communities ivi calculation in each community (table 2) shows three species with the highest value in communities’ settlements, street green lanes, and urban parks, i.e., a. compressus, rivina humilis, and syngonium podophyllum. the three species are dominant and co-dominant in the urban forest community. dominant species are defined as species with a higher ability to utilize their environment more efficiently than other species (smith 1977). invasive species tend to have a high ivi due to their wide tolerance range. thus, they can adapt very well to various environments. table 2 three species with the highest importance value index (ivi) were found on six land-use types/communities land-use species name family ivi (%) paddy fields eleusine indica poaceae 29.66 cynodon dactylon poaceae 17.22 bidens pilosa asteraceae 13.90 gardens asystasia gangetica acanthaceae 19.71 alternanthera philoxeroides amaranthaceae 14.94 galinsoga parviflora asteraceae 12.76 street green lanes axonopus compressus poaceae 32.63 trimezia martinicensis iridaceae 25.49 swietenia macrophylla meliaceae 11.57 settlements axonopus compressus poaceae 15.20 trimezia martinicensis iridaceae 9.03 arachis pintoi fabaceae 8.55 urban parks axonopus compressus poaceae 37.56 arachis pintoi fabaceae 18.47 hymenocalis speciosa amaryllidaceae 14.51 urban forests rivina humilis phytolaccaceae 29.54 syngonium podophyllum araceae 27.37 xanthosoma violaceum araceae 13.89 biotropia vol. 30 no. 2, 2023 176 table 3 mean climatic factors measured in six communities land-use light intensity (lux) temperature (°c) air humidity (%) urban forests 191 23 90 settlements 17 074 30 84 gardens 58 037 30 78 paddy fields 54 687 30 79 street green lanes 7 030 29 78 urban parks 2 030 27 85 dominating species in the paddy field community were mainly plants from the family poaceae, with leaf characteristics resembling ribbon and fibrous root and reproducing vegetatively with stolon. grasses tend to grow optimally in areas exposed to sunlight. the paddy field community has a relatively high light intensity (table 3), allowing this plant group to grow optimally and reproduce well or widely spread. the garden community was dominated by three invasive species, i.e., a. gangetica, a. philoxeroides, and g. parviflora. the habitat suitability of invasive species developing in the garden community is affected by several factors, e.g., species commodity planted and spatial variations such as microclimate, soil character, and human presence (wang & wan 2020). five garden communities in this study comprised two cauliflower gardens, a bok choy garden, and two mixed cassava and sweet potato gardens. various commodities planted in those five study sites also influenced the invasive species found. r. humilis and s. podophyllum were two invasive species dominating the urban forest community. r. humilis is a tropical plant commonly found in forests, scrubs, street edges, and disturbed sites at a wide range of altitudes from 0 to 1700 masl. this species proliferates, mainly under shade. due to those characteristics, this species can significantly change its habitat and harm the native vegetation (parker 2013). meanwhile, s. podophyllum is a climber that can rapidly grow, invading the forest’s floor into canopies, covering huge trees to understory vegetation underneath (pacific islands ecosystems at risk 2012). species a. compressus dominated the settlements, street green lanes, and urban parks, while t. martinicensis was a co-dominant species in the street lanes and settlements. the third dominant plant in the green street lanes community was s. macrophylla. the ornamental plant a. compressus is mainly used in domestic gardens and parks. this species adapts to humid and warm environments and is also adequately tolerant to shade, even though it can grow well in areas exposed to sunlight (cabi 2019). climatic conditions in settlements, urban parks, and street lanes support the optimal growth of a. compressus. light intensity in settlements, urban parks, and street lanes was lower than in paddy fields and gardens. even though the settlement temperature was the same as those in the garden and paddy fields, the settlement had higher humidity. t. martinicensis is also intentionally introduced as an ornamental plant. almost 40% of invasive plants in the united states were initially introduced as ornamental plants (lehan et al. 2013). ornamental plants can quickly grow and resist pests and pathogens (guo et al. 2019). these characteristics support most ornamental plants developing into invasive species. s. macrophylla is a fast-growing tree with a high tolerance to low light intensity. a study by norghauer et al. (2011) suggests that the abundance of s. macrophylla is negatively correlated to the abundance of plants under their shade. when s. macrophylla grows as the landscape canopy, a dense shade forms, limiting sunlight penetration to understory plants under its shade. therefore, the germination of understory plants can be disturbed, and the mature individual does not grow optimally. the highest invasive species richness (table 4) was found in the community of settlements, followed by gardens, paddy fields, street green lanes, and urban parks. decker et al. (2012) also report a similar result, mentioning that the richness of invasive species positively correlates to the percentage of public area use, human population, and agricultural area use. the settlement vegetation community has the highest diversity and richness of invasive and spatial distribution of invasive plants in bandung – rahmawati and rosleine 177 table 4 the shannon wiener diversity index (h’) calculated in six communities land-use h’ invasive species richness non-invasive species richness settlements 3.71 60 55 street green lanes 2.6 39 17 gardens 3.03 45 3 paddy fields 2.74 39 4 urban parks 2.4 18 10 urban forests 2.31 10 13 non-invasive species. the presence of humans is the most influencing factor in invasive species introduction. the settlement area has the highest human population of the other five land uses. factors influencing the high diversity and abundance of invasive species in settlement areas are human population, trading activity, nutrition source, warm and protected microclimate, and the potential of herbivore or competitor absence (francis & chadwick 2015). the vegetation community in settlements, street green lanes, and urban parks, including urban and suburban areas, had more invasive species than non-invasive ones (table 4). the settlementsgreen street lanes and urban parksstreet green lanes communities had a high similarity of vegetation composition (table 5). human-made ecosystems have often supported the establishment and development of alien species (hulme, 2003). besides the high similarity of vegetation composition, those four communities mentioned (i.e., settlements, urban parks and forests, and green street lanes) also belong to the same cluster in the pca results (figure 3). these four communities are located in quadrant 1 with a. compressus, t. martinicensis, s. macrophylla, and mangifera indica, with a particular characteristic of climatic factor being air humidity. the four species had a high presence in the community with high humidity, indicating that high humidity is a suitable climatic condition for the growth of the four species. the intensity of human existence is high in this land use compared to gardens and paddy fields. species found in quadrant 1 are invasive species, mainly introduced with the intention of ornamental plants, food sources, and street shade trees. the paddy fields and garden communities had a high similarity index (50.5%). both of these communities are included in the suburban area. meanwhile, the urban forest community had a species composition that tends to be unique, indicated by the low value of the similarity index with other communities. in addition, the urban forest also had different climatic conditions from other communities, such as low light intensity and low temperature with high humidity. other invasive species found in these four communities were tagetes erecta and cosmos sulphureus as ornamental plants, artocarpus heterophyllus and psidium guajava as consumable plants, delonix regia and albizia saman as shade trees. the presence of these species depends on human preference. human plays a vital role in managing invasive species. people often tend these species by pruning or splitting them, directing their growth without disturbing their aesthetics. thus, it can be concluded that anthropogenic factor has a more considerable influence than climatic factors on determining the presence of invasive species in urban areas. humans, through their preferences, control the presence and dominance of invasive species in urban areas without reducing the number of invasive species. table 5 sorensen index calculated in six paired communities paddy fields gardens street green lanes settlements urban parks urban forests paddy fields 50.5 30.3 22.8 16.9 6.0 gardens 32.7 25.8 7.9 5.6 street green lanes 44.4 26.2 17.5 settlements 21.0 14.4 urban parks 11.5 urban forests biotropia vol. 30 no. 2, 2023 178 figure 3 pca results on the contrary, findings in the urban forests show that although non-invasive species had a higher species number, this community had the lowest diversity index of others. apart from discussing species’ invasiveness in urban forests, species richness was lower than species richness in other communities. several factors affect small-scale species richness, including geographic factors such as the regional species pool, dispersal distance and ease of dispersal, biological factors such as competition, facilitation, and predation, as well as environmental factors such as resource availability, environmental heterogeneity, and disturbance frequency and intensity (brown et al. 2016). urban forest’s climatic conditions differ from other communities, such as low light intensity, temperature, and high humidity. this condition causes limited species that can survive in urban forests, and the low diversity of species in the urban forest can be caused by management areas related to its function as a recreation facility. when viewed from an ecological perspective, it is also included as an ecosystem disturbance that causes a decrease in species richness. based on the pca results, paddy fields and gardens belong to different quadrants, even though they share similarities in their vegetation composition. gardens were placed in quadrant 2, while paddy fields at quadrant 3. both quadrants were influenced by the same climatic factors, i.e., light intensity and air temperature. it indicates that an abundance of amaranthus spinosus, ageratum conyzoides, cleome rutidosperma, cyperus rotundus, g. parviflora, a. gangetica, a. philoxeroides, eclipta prostrata, e. indica, c. dactylon, and b. pilosa is influenced by light intensity and air temperature, whereas the abundance of a. compressus, t. martinicensis, s. macrophylla, and m. indica is influenced by humidity. quadrant 2 is occupied by species, i.e., a. spinosus, a. conyzoides, c.rutidosperma, c. rotundus, g. parviflora, a. gangetica, and a. philoxeroides. these species were initially introduced as contaminants to propagules planted in the garden and managed to “escape” to the surrounding areas. these species were then able to adapt and invade the surrounding garden areas. most species can survive in intense light exposure and grow optimally in warm temperatures. for example, a. gangetica is dispersed by seeds and rhizomes. the seeds are dispersed from explosive capsules, but long-distance dispersal is affected by humans. the risk of introducing rhizome material as a contaminant of soil and compost remains high in those countries where the plant is well established (sandoval & rodríguez 2012). spatial distribution of invasive plants in bandung – rahmawati and rosleine 179 meanwhile, quadrant 3 is occupied by eclipta prostrata, e. indica, c. dactylon, and b. pilosa. in the paddy field community, in our observation, species such as c. rotundus, a. gangetica, and a. philoxeroides had relatively high frequency but low density. due to the inter-species interaction, their density was lower than e. indica, c. dactylon, and b. pilosa. interspecific interactions, including competition, interference, and facilitation, determine the natural community’s composition, distribution, and species abundance (belote & weltzin 2006). invasive species distribution in bandung the distribution pattern of e. prostrata shares similarities with c. dactylon (figure 4). c. dactylon belongs to the c4 plant group that can adapt well and grow optimally in high light intensity. although occupying the same pca quadrant, b. pilosa has a different distribution pattern from e. prostrata and c. dactylon. instead, b. pilosa distribution tends to be similar to a. philoxeroides and a. conyzoides. the three species (i.e., b. pilosa, a. philoxeroides, and a. conyzoides) are abundant in paddy fields, gardens, and settlements. they produce abundant seeds with a wide dispersal range, allowing them to be present in various land-use types. on average, seeds of a. conyzoides are 3.4 mm in length and 0.33 mm in width, equipped with pappus that enables them to attach to garments or animal body parts. this species is widely considered a weed in agricultural and anthropogenic areas and its natural habitat (united states department of agriculture 2019). meanwhile, b. pilosa can produce up to 6000 seeds per year that can easily be dispersed by attaching to animals, birds, human clothes, wind, and water. their propagules can remain viable for 5-6 years (sandoval 2018). biotropia vol. 30 no. 2, 2023 180 figure 4 distribution map of invasive species in bandung: a. e. prostrata; b. c. dactylon; c. b. pilosa; d. a. philoxeroides; e. a. conyzoides; f. a. spinosus; g. c. rutidosperma; h. a. gangetica; i. t. martinicensis; j. s. macrophylla a. conyzoides and a. spinosus are frequently found in gardens. there is no specific distribution pattern of a. gangetica, but it can be commonly found in paddy fields, settlements, and green street lanes. this species propagates through rhizomes and dehiscent capsules. furthermore, the dehiscent capsule explodes and disperses the seeds in the surrounding areas. humans can assist in its long-distance dispersal. the rhizome of this species can contaminate the soil, and it can spread further when present in compost used as planting media (sandoval & rodríguez 2012). two species, i.e., s. macrophylla and t. martinicensis, are introduced in urban areas such as parks, settlements, and streets with an initial specific purpose. the shape of s. macrophylla resembles a huge umbrella. thus, it can provide broad shade areas and decorate the road due to its pleasant form (environmental management agency 2014). therefore, it is unsurprising that this species is widely found in urban parks, forests, and green street lanes. on the contrary, t. martinicensis is abundant in settlements since it is widely planted as ornamental. conclusion there were 187 species found in bandung that can be grouped into invasive alien species (39%), invasive native plants (25%), noninvasive alien species (18%), non-invasive native species (15%), and unidentified plants (3%). species with the highest individual found on the edge of urban areas and were absent in the city’s center were eclipta prostrata, c. dactylon, b. pilosa, a. philoxeroides, a. conyzoides, a. spinosus, c. rutidosperma, and a. gangetica. meanwhile, s. macrophylla and t. martinicensis had the highest number of individuals in the city center. paddy fields and gardens have similar vegetation composition but differ in dominant and co-dominant species. invasive species found in paddy fields and gardens were agricultural weeds. meanwhile, ornamental plants were invasive in urban parks, street green lanes, and settlements. forest had the lowest number of invasive species, corresponding with its function to maintain biodiversity. further research on the distribution map of invasive species in bandung can focus only on one or several species by analyzing the relationship between plant populations and distance to the city center, distance to main roads, edaphic factors, and human population. this information is vital for determining how to control invasive species properly. experimental research can be used to determine interactions between invasive species in one community. in addition, it is essential to carry out a risk analysis to study further the risks posed and the spatial distribution of invasive plants in bandung – rahmawati and rosleine 181 management of each species. thus, the subsequent steps can be more focused. acknowledgments this research is supported by research, community services, and innovation program (ppmi) – itb 2020 for ecology research group. we also thank nadiya syafia, desi sari, diah frisda, resti lutfiani, and rahayu merdekawati, who helped execute this study. references aswant, i a. 2016. analisis perbandingan metode interpolasi untuk pemetaan ph air pada sumur bor di kabupaten aceh besar berbasis gis 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[cited 2022 apr 23]. avaialble from: www.botany.hawaii.edu. wang, cj, and wan, jz. 2020. assessing the habitat suitability of 10 serious weed species in global croplands. global ecology and conservation, 23: e01142. biotropia vol. 31 no. 1, 2024: 96 105 doi: 10.11598/btb.2024.31.1.2136 96 optimization of kinetin concentrations and medium compositions for citrus shoot multiplication from cotyledonary nodes kristianto nugroho1, mia kosmiatin2*, tri joko santoso2, dewi sukma3, agus purwito3, ali husni2 and chaireni martasari2 1graduate program of plant breeding and biotechnology, faculty of agriculture, ipb university, bogor 16680, indonesia 2 research center for horticultural crops, research organization for agriculture and food, national research and innovation agency, bogor 16915, indonesia 3department of agronomy and horticulture, faculty of agriculture, ipb university, bogor 16680, indonesia received 26 november 2023/ revised 3 january 2024/ accepted 27 february 2024 abstract shoot multiplication plays an important role in tissue culture activities; therefore, it should be optimized via several factors, such as the composition of the medium and the concentration of plant growth regulators. the aim of this study is to optimize the kinetin concentrations and medium compositions for shoot multiplication originating from cotyledonary node explants of several local citrus cultivars for genome editing activities. the cotyledonary nodes from three citrus cultivars (batu55, siam madu, and proksi-1 agrihorti) were incubated in ms medium with morel and wetmore vitamins (vmw) supplemented with several kinetin concentrations (0; 0.2; 0.4; 0.6; 0.8; and 1 mg/l). the best kinetin concentrations for the number of shoots variable were then combined with murashige and tucker (mt) medium. the results showed that the combination of vmw medium and kinetin concentration at 0.8 mg/l produced the best number of shoots in batu55 cultivar as well as 1 mg/l concentration in siam madu and proksi-1 agrihorti cultivars. therefore, those two concentrations were then chosen to be combined with mt medium in the second experiment. the results showed that combination of 1 mg/l kinetin with mt medium showed the best number of shoots, the highest percentage of shoot formation, the biggest number of leaves, the biggest number of nodes, and the longest shoot length in this study. this medium composition could be further used for shoot multiplication in genetic transformation in those three citrus cultivars, including genome editing activities in the development of new improved citrus varieties. keywords: citrus, cotyledonary nodes, kinetin, murashige and tucker medium, shoot multiplication introduction citrus is a high-value horticultural crop that is beneficial to our health because its fruits contain high levels of vitamin c and antioxidants. however, one of the challenges faced in citrus cultivation programs not only in indonesia, but also globally is the attack of huanglongbing disease or citrus vein phloem degeneration (cvpd) caused by candidatus liberibacter asiaticus (clas) transmitted through the insect diaphorina citri (killiny et al. 2018, wang 2019). the symptoms include leaf chlorosis resembling manganese or zinc deficiencies, stunted plant growth, smaller number of fruit, smaller fruit size, bitter fruit taste, and asymmetrical locules/segments within the fruit (bove 2006). such symptoms can result in staggering yield losses in citrus plants, ranging from 30% to as high as 100% (iftikhar et al. 2016). the development of a newly improved citrus cultivar resistant to huanglongbing disease is necessary to prevent yield losses due to the disease. unfortunately, the majority of the commercial citrus cultivars are susceptible to this disease (pandey et al. 2022). several citrus species and their relatives, including citrus latipes, poncirus trifoliata, murraya paniculata, severinia buxifolia, carrizo citrange, rough lemon, and microcitrus austrasica are known to have resistance to *corresponding author, email: miak003@brin.go.id optimization of kinetin concentrations and medium compositions for citrus – kristianto nugroho et al. 97 huanglongbing infections (killiny et al. 2018, rao et al. 2018). however, efforts to transfer resistance genes to commercial citrus through interspecies crosses have been hindered by various challenges, such as incompatibility, apomixis, and polyembryony (zhang et al. 2018, montalt et al. 2021). crispr/cas9 is one of the new promising technologies that could be used to knock out the activity of genes responsible for the susceptibility to huanglongbing disease in citrus. among the targeted genes for silencing is the callose synthase gene, which is responsible for callose accumulation in phloem tissue – a physiological response observed in citrus plants infected by this disease. genetic transformation is one of the crucial steps in genome editing activities. information regarding plant regeneration methods is very necessary because it will affect the successful process of the genetic transformation itself. this information can be obtained through an optimization process which include several factors such as the type of explant, the composition of the medium, and the concentration of plant growth regulators. the utilization of different explants from epicotyl for genetic transformation of taroco blood orange has been previously reported by peng et al. (2015) with the transformation efficiency range from 21.4-22%. in contrast, the use of embryogenic callus originating from unfertilized ovules in sweet orange (citrus sinensis) showed transformation efficiency ranging from 26.3-36.5% (dutt et al. 2020). the use of explants from nucellar and zygotic embryos in genetic transformation activities of japansche citroen has been reported by aisyah (2021) with transformation efficiencies of 13.80 and 21.17% respectively. the utilization of cotyledonary nodes as explants in genetic transformation activities of citrus has not been widely reported despite their high regeneration ability. these nodes offer the advantage of direct organogenesis for shoot multiplication without callus formation (nwe et al. 2014, fatonah et al. 2018). the composition of the medium plays an important role in facilitating successful plant regeneration in citrus species. several studies have shown that murashige and skoog (ms) media modified by morel & wetmore vitamins (vmw) are suitable for in vitro growth of citrus, as reported in somatic embryogenesis of mandarin (purwito et al. 2015), somatic embryogenesis from endosperm tissue of c. nobilis (kosmiatin et al. 2014), and in micropropagation and micrografting of japansche citroen (putra et al. 2015). conversely, murashige & tucker (mt) medium is also widely utilized in citrus plants, including in vitro propagation of thomson navel sweet oranges (esmaeilnia & dehestani 2015) and embryo rescue in progenies resulting from crosses between mandarin and carrizo citrange (kurt & ulger 2019). in addition to the type of explant and medium composition, the concentration of plant growth regulators also determines the success of in vitro regeneration processes. excessive concentrations of plant growth regulators could impede growth and result in abnormal shoots, while insufficient concentration will result in a low number of plantlets produced (shankar et al. 2014). therefore, the concentration of plant growth regulators also needs to be optimized. kinetin is a plant growth regulator from cytokinin group that is useful in the process of shoot formation and regeneration. the use of powerful cytokinin such as benzyl adenine (ba) or 6benzylaminopurine (bap) in shoot formation can lead to rapid cell division and the formation of undesirable callus as previously reported by kosmiatin et al. (2014) and prameswari et al. (2019). furthermore, kumar et al. (2014) also reported that kinetin treatment in 1 mg/l showed the best survival rate in shoot tip grafting of kinnow mandarin. the use of kinetin in shoot proliferation have been previously reported in several studies (shankar et al. 2014, putra et al. 2015, khiem et al. 2022). the aim of this study is to optimize the kinetin concentrations and medium compositions for shoot multiplication derived from cotyledonary node explants of several citrus cultivars in order to obtain the best combination that will be used in genome editing activities. materials and methods genetic materials the immature citrus fruits, approximately 1115 weeks after anthesis, were collected from three cultivars; batu55 (c. reticulata blanco.), siam biotropia vol. 31 no. 1, 2024 98 madu (c. nobilis l.), and proksi-1 agrihorti (a cross between siam madu and mandarin satsuma). the fruits were obtained from balai pengujian standar instrumen tanaman jeruk dan buah subtropika (bsip jestro) malang and balai besar pengujian standar instrumen bioteknologi dan sumber daya genetik pertanian (bbpsi biogen). the study was conducted at the plant cell and tissue culture laboratory, bbpsi biogen bogor, from september 2022 to july 2023. isolation and sterilization of citrus seeds the seeds were isolated from the fruits following the method outlined by kosmiatin & husni (2018). initially, the citrus fruit was sterilized by dipping it in 96% alcohol and passing it over the bunsen five times. the sterilized fruit was then placed in a sterile petri dish, where the skin was peeled using a scalpel, and the seeds were removed using tweezers. the seed coats were then removed, and the cotyledons were planted in ms medium supplemented with 3 mg/l ga3 (kosmiatin et al. 2014). optimization of kinetin concentrations in ms medium modified with morel and wetmore vitamins (vmw) the cotyledonary nodes, measuring 0.5-0.7 cm in length, were isolated from one-month-old seedlings of the three citrus cultivars (figure 1a). these nodes were then incubated in ms media modified with morel and weitmore vitamins (vmw) (morel & wetmore 1951) supplemented with 30 g/l sucrose and 7 g/l of agar. the ph of the medium was adjusted to 5.8 by adding 0.1 n naoh before autoclaving for 15 min at 121oc. there were several levels of kinetin concentration used in this study e.g., 0; 0.2; 0.4; 0.6; 0.8; and 1 mg /l (figure 1b). this study was designed on a completely randomized design (crd). each kinetin concentration treatment consisted of three cotyledonary nodes with ten replications. the explants were cultured at room temperature (25±2oc) using tl 60 w 220 v (±1000 lux) lighting with 16 hours per day over an 8-week period. observations were carried out for 8 weeks, focusing on four variables: namely the number of shoots, the leaves and nodes, the shoot length, and the percentage of shoot formation. the data were analyzed using analysis of variance (anova) followed by duncan multiple range test (dmrt) with a significance level of 5% using the statistical tool for agricultural research (star) version 2.0.1 (gulles et al. 2014). figure 1 the explants that were used in this study consisted of (a) one-month citrus seedling in ms + 3 mg/l ga3 medium, and (b) cotyledonary nodes cutting from one-month seedling optimization of kinetin concentrations and medium compositions for citrus – kristianto nugroho et al. 99 combination of the optimum kinetin concentration with murashige and tucker (mt) medium the optimal kinetin concentrations identified from the previous treatment were incorporated into murashige and tucker (mt) medium (murashige & tucker 1969), using cotyledonary node explants from similar citrus cultivars. each treatment consisted of three cotyledonary nodes with ten replications. observations were carried out over an 8-week period, focusing on four variables: namely, the number of shoots, the leaves and nodes, the shoot length, the and percentage of shoot formation. the data were then compared to the previous treatment using vmw medium and analyzed using analysis of variance (anova) followed by least significant difference (lsd) with a significance level of 5% using the statistical tool for agricultural research (star) version 2.0.1 (gulles et al. 2014). results and discussion optimization of kinetin concentration in vmw medium the effects of kinetin supplementation in vmw medium on shoot multiplication showed varied responses across the citrus cultivars used in this study, as shown in table 1. interestingly, all kinetin concentrations treated in this study showed no significant differences in the number of shoots, leaves and nodes, and shoot length variables across all cultivars examined. in batu 55 cultivar, the optimal number of shoots was observed at a kinetin concentration of 0.8 mg/l, while the optimal number of leaves occurred at 0.6 mg/l kinetin. additionally, the optimal number of nodes was observed at kinetin concentrations of 0.4 and 0.6 mg/l respectively. the optimum shoot length was obtained with the control treatment. in siam madu cultivar, the optimal number of shoots, leaves, and shoot length were identified from 1 mg/l kinetin concentration, while the optimal number of nodes was identified from 0.6 mg/l kinetin concentration. conversely, in proksi-1 agrihorti cultivar, the optimal number of shoots, leaves, and nodes were identified from 1 mg/l kinetin concentration, while the optimal shoot length was identified from 0.8 mg/l kinetin concentration. table 1 the effect of several kinetin concentrations on citrus shoot multiplication after 8 weeks cultivation citrus cultivars kinetin concentrations (mg/l) number of shoots percentage of shoot formation number of leaves number of nodes shoot length batu 55 0 1.00±0.17 76.67±35.31 3.73±0.90 1.57±0.52 0.88±0.35 0.2 0.90±0.0 60.00±30.63 3.00±0.73 1.42±0.39 0.74±0.24 0.4 1.00±0.0 53.33±28.11 3.18±0.64 1.60±0.52 0.82±0.21 0.6 0.90±0.17 70.00±42.89 3.82±1.16 1.60±0.59 0.76±0.18 0.8 1.07±0.34 60.00±37.84 3.48±1.23 1.23±0.56 0.66±0.25 1.0 1.00±0.0 76.67±16.10 3.63±0.84 1.45±0.53 0.83±0.23 siam madu 0 0.68±0.22 43.33±44.58 1.87±0.72 0.83±0.39 0.37±0.10 0.2 0.73±0.13 60.00±46.61 2.18±0.12 1.13±0.49 0.53±0.17 0.4 0.83±0.12 53.33±39.13 2.93±1.28 1.35±0.38 0.64±0.19 0.6 0.90±0.0 66.67±35.14 2.80±0.83 1.60±0.55 0.73±0.16 0.8 0.80±0.0 56.67±38.65 2.05±0.68 0.97±0.31 0.51±0.14 1.0 1.08±0.20 73.33±34.43 3.40±1.08 1.48±0.29 0.75±0.18 proksi-1 agrihorti 0 0.63±0.14 56.67±49.81 2.27±0.66 1.43±0.33 0.56±0.22 0.2 0.73±0.24 66.67±38.49 2.70±0.81 1.37±0.49 0.72±0.31 0.4 0.67±0.13 63.33±45.68 2.43±0.74 1.42±0.44 0.64±0.37 0.6 0.87±0.43 70.00±42.89 3.23±1.70 1.50±0.71 0.68±0.41 0.8 0.87±0.35 73.33±43.89 3.60±1.75 1.63±0.74 0.82±0.29 1.0 1.07±0.40 73.33±40.98 3.63±0.80 1.67±0.56 0.72±0.29 note: the numbers in bold show the highest average value for each variable biotropia vol. 31 no. 1, 2024 100 the kinetin concentrations used in this study also did not show significant difference in the percentage of shoot formation variable after eight weeks of cultivation. the highest percentage of shoot formation was obtained with 1 mg/l kinetin concentration in batu55, siam madu, or proksi-1 agrihorti cultivars (table 1). masekesa et al. (2016) reported that supplementation of 0.4 mg/l kinetin gave the best shoot formation percentage in sweet potato regeneration using petiole explants. meanwhile, foo et al. (2018) reported that the highest percentage of shoot formation (65%) in eggplant was obtained with kinetin treatment at 2.0 mg/l from the cotyledon explants. a previous study by kumar et al. (2014) showed that supplementation of 1 mg/l kinetin produced the best result in shoot length from shoot tip grafting of kinnow mandarin (citrus deliciosa). kumar et al. (2014) also reported that the increasing level of kinetin higher than 1 mg/l showed the inhibitory effect for citrus. on the other side, abu-romman et al. (2015) reported that 1 mg/l kinetin gave the best result in regeneration rate, number of shoots, and length of shoots in cucumber. in good agreement with those studies, dincer (2023) reported that a 1 mg/l kinetin concentration showed the best result for shoot number, shoot length, and number of nodes in sorbus aucuparia and was also the optimal concentration for number of shoots and percentage shooting efficiency in regeneration of barley from calli (abbas et al. 2023). shoot multiplication is an important stage in micropropagation, wherein new shoots are induced from the axil as presented in figure 2. the presence of cytokinins such as kinetin will disrupt the apical dominance and promote lateral shoot formation (kane 2011). the type and concentration of cytokinin play a critical role in the shoot multiplication rate, shoot length, and frequency of genetic variation (kane 2011). a previous study from abu-romman et al. (2015) in cucumber showed that kinetin had a more influential effect on shoot multiplication compared to other cytokinin types such as 6benzylaminopurine (bap), thidiazuron, and zeatin. in this study, the number of shoots became the most crucial variable to consider, given that all citrus cultivars that used in this study are classified as scion type. scion is a type of citrus cultivar with high production and commercially appealing, favored by consumers (kosmiatin et al. 2010), which is in contrast to rootstocks which are tolerant to dry condition and have a strong stem and root, but are less attractive and commercially viable (jayanti et al. 2015). the shoots will be utilized in genetic transformation mediated by a. tumefaciens and selected using antibiotic medium for genome editing activities. the selected shoots will then be grafted ex vitro to citrus rootstock in the greenhouse. the reason why the root induction variable was not observed in this study is that the acclimatization process will not be carried out. even though the kinetin concentration used in this study did not show a significant difference in the number of shoot variables, the kinetin concentration at 0.8 mg/l showed the highest number of shoots in batu 55 as well as 1 mg/l concentration at siam madu and proksi-1 agrihorti cultivar. therefore, those two concentrations were chosen to be combined with murashige and tucker medium in the second experiment. figure 2 new shoot induced from lateral shoot on cotyledonary node explant used in this study optimization of kinetin concentrations and medium compositions for citrus – kristianto nugroho et al. 101 combination of the optimum kinetin concentrations with murashige and tucker (mt) medium the effects of combining the best kinetin concentrations from the previous experiment (0.8 and 1 mg/l) with murashige and tucker medium are presented in table 2. overall, the results indicate that this combination led to an increase in citrus shoot multiplication variables compared to the previous treatment using vmw medium. in batu55 cultivar, the optimal number of shoots, percentage of shoot formation, number of nodes, and shoot length were obtained at 1 mg/l kinetin, while the optimal number of leaves was obtained at 0.8 mg/l kinetin. the use of mt medium in batu55 cultivar showed significant difference to vmw medium at 1 mg/l kinetin concentration in terms of the number of shoots, percentage of shoot formation, number of nodes, and shoot length variables. in the siam madu cultivar, all of the optimal variables were identified at 1 mg/l kinetin, and the use of mt medium showed significant differences to vmw medium at 1 mg/l kinetin concentration across all variables. in line with the findings for siam madu, all optimal variables were also identified at 1 mg/l kinetin in proksi-1 agrihorti cultivar, and the use of mt medium showed significant differences to vmw medium at 1 mg/l kinetin concentration in the number of leaves, number of nodes, and shoot length variables. basal medium composition plays an important role in plant micropropagation. while the murashige and skoog (ms) medium is commonly used for in vitro culture of citrus, reports suggest that mt medium has shown better results in some instances (carimi & de pasquale 2003). basically, mt medium composition is similar to ms basal medium, with increased concentration of several vitamins such as thiamine hcl, pyridoxine hcl, and nicotinic acid. vitamins present in tissue culture medium serve as cofactors for enzymes in plant metabolisms (abrahamian & kantharajah 2011). together with other media components, vitamins have direct and indirect effects on several physiological responses such as somatic growth, rooting, callus formation, and embryonic development (kadhimi et al. 2014). the utilization of mt medium in citrus micropropagation has been reported in several previous studies. these include micropropagation of thomson navel sweet oranges (esmaelnia & dehestani 2015), in vitro regeneration of c. limon from leaf explants (kasprzyk-pawelec et al 2015), somatic embryogenesis of mexican lime (c. aurantifolia) from juice vesicles (amin & shekafandeh 2015), somatic embryogenesis of mandarin batu 55 from nucellus (februyani et al 2016), embryo rescue in progenies from crossing between table 2 the effect of the combination between the best kinetin concentration from the previous experiment with murashige and tucker medium on citrus shoot multiplication after 8 weeks citrus cultivars kinetin concentrations (mg/l) medium composition number of shoots percentage of shoot formation number of leaves number of nodes shoot length batu55 0 ms + vmw 1.00 76.67 3.73 1.57 0.88 mt 1.27 90.00 4.30 1.77 1.21 0.8 ms + vmw 1.07 60.00a 3.48 1.23a 0.66a mt 1.25 93.33b 4.90 2.38b 1.20b 1.0 ms + vmw 1.00a 76.67a 3.63a 1.45a 0.83a mt 1.30b 100.00b 4.60b 2.43b 1.43b siam madu 0 ms + vmw 0.68 43.33 1.87 0.83a 0.37a mt 1.17 76.67 2.60 1.65b 1.14b 0.8 ms + vmw 0.80 56.67a 2.05a 0.97a 0.51a mt 1.03 90.00b 3.57b 2.27b 1.39b 1.0 ms + vmw 1.08 73.33a 3.40 1.48a 0.75a mt 1.27 100.00b 4.57 2.30b 1.49b proksi-1 agrihorti 0 ms + vmw 0.63a 56.67 2.27 1.43 0.56 mt 1.13b 76.67 3.67 1.93 0.83 0.8 ms + vmw 0.87a 73.33 3.60a 1.63 0.82 mt 1.43b 83.33 1.43b 2.37 1.19 1.0 ms + vmw 1.07 73.33 3.63a 1.67a 0.72a mt 1.57 86.67 5.55b 2.48b 1.32b note: numbers followed by different letters show significant differences based on lsd test. the numbers in bold show the highest average value for each variable. biotropia vol. 31 no. 1, 2024 102 mandarin and carrizo citrange (kurt & ulger 2019). the vmw medium uses similar inorganic salt to the ms basal medium but incorporates modifications to the morel & wetmore (vmw) vitamins such as nicotinic acid, pyridoxine hcl, calcium pantothenate, and biotin. the addition of biotin and calcium pantothenate in vmw medium is crucial for cell membrane formation, the mitosis process, and the multiplication of shoots and roots (sukmadjaja et al. 2022). on the contrary, in this study vmw medium generally showed lower results in terms of shoot number, percentage of shoot formation, number of leaves, number of nodes, and shoot length compared to mt medium as evidenced in table 2, figure 3. figure 3 citrus shoot formed after eight weeks derived from combination of 1 mg/l kinetin with vmw medium (a, b, c) and mt medium (d, e, f). a and d: batu 55 cultivar, b and e: siam madu cultivar, c and f: proksi-1 agrihorti cultivar the absence of thiamine hcl from vmw medium composition significantly affects citrus shoot multiplication. according to al-khayri (2001), thiamine serves an important function as cofactor in metabolism of carbohydrate, while biotin plays an important role in carboxylation reactions. al-khayri (2001) reported that improving thiamine concentration from 0.1 to 0.5 mg•/l gave the maximum callus growth while increasing of biotin from 0 to 1 mg/l gave the maximum callus weight respectively. in a previous study by kazemiani et al. (2018), optimization of kinetin concentrations and medium compositions for citrus – kristianto nugroho et al. 103 supplementation of 10 mg/l thiamine-hcl in ms medium, combined with 50 mg/l nicotinic acid, 50 mg/l pyridoxine-hcl, and 4 mg/l bap, resulted in the maximum number of lateral shoots in potato in vitro culture. additionally, vollmeer et al. (2023) reported that the addition of 0.1 mg/l thiamine to modified ms medium significantly increased plant height, root length, and number of nodes of in vitro sweet potato shoot culture. the regeneration medium is one of the key factors in plant genetic transformation besides construct design, explant selection, transformation method, and antibiotic selection method (altpeter et al. 2016). thus, the selection of a suitable regeneration medium is very important to ensure the success of the genetic transformation process, as mentioned by zhang et al. (2021). they highlighted that, despite advances in genome editing systems, establishing a plant tissue culture or regeneration system for the targeted plant species remains challenging. conclusions the optimization of kinetin concentration combined with vmw media on three citrus cultivars in this study showed that a kinetin concentration of 0.8 mg/l yielded the highest number of shoots in batu55 cultivar, while a concentration of 1 mg/l concentration was optimal for siam madu and proksi-1 agrihorti cultivar. overall, the supplementation of kinetin in vmw medium did not show significant differences in the number of shoots, percentage of shoot formation, number of leaves, number of nodes, and shoot length variables on three citrus cultivars investigated in this study. in contrast, the combination of the best kinetin concentration on number of shoots variables (0.8 and 1 mg/l) with mt medium showed that 1 mg/l kinetin gave the best number of shoots, percentage of shoot formation, number of leaves, number of nodes, and shoot length on three citrus cultivars used in this study. this medium composition could be used for shoot multiplication in genetic transformation processes in these three citrus cultivars, in order to knock out the activity of callose synthase gene and to increase citrus resistance to huanglongbing disease. acknowledgments this research financial support was provided by riset dan inovasi untuk indonesia maju (riim) project’ second wave budget in the year 2022. the authors gratefully acknowledge the national research and innovation agency (brin) for their financial assistance. the authors also would like to thank bbpsi biogen for providing laboratory facilities and bsip jestro for supplying the genetic materials for this research. references abbas m, hussain ss, abbas sk, abbas s, tahir m, su y, li j, ahmed n, khan ms, gou c. 2023. optimization of regeneration protocol and prospecting spectinomycin resistance in barley (hordeum vulgare l.) cv haider-93. afr j biotechnol 22(9): 172-82. doi: 10.5897/ ajb2023.17590. abrahamian p, kantharajah a. 2011. effect of vitamins on in vitro organogenesis of plant. am j bot 2: 669-74. doi: 10.4236/ajps.2011.25080. abu-romman sm, al-hadid ka, arabiyyat ar. 2015. kinetin is the most effective cytokinin on shoot multiplication from cucumber. j agric sci 7(10): 159-165. doi: 10.5539/ jas.v7n10p159. aisyah dn. 2021. transformasi gen crispr/cas9-grnacscs pada tanaman jeruk untuk peningkatan ketahanan terhadap penyakit huanglongbing. 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a niche for indonesia risha amilia pratiwi1*, musyarofah zuhri1 and indah oktaviani2 1research center for applied botany, national research and innovation agency, bogor 16911, indonesia 2departement of biology, faculty of science, institut teknologi sumatera (itera), lampung 35365, indonesia received 28 february 2023/ revised 26 january 2024/ accepted 13 march 2024 abstract sapindus rarak, a native plant of indonesia, is renowned for its saponin-rich fruit, acting as a natural detergent for batik and baby cloth diapers. this study aims to determine the trends in s. rarak research through a dual approach: bibliometrics and scoping review. the bibliometrics analysis involved defining search keywords, extracting publications, refining results, compiling metadata, and mapping authors, keywords, and collaborations. the scoping review meanwhile included determining research objectives and questions, defining the scope, selecting studies through an iterative team approach, extracting data, summarizing qualitative and quantitative analysis, reporting results, and preparing the considerations of implications in research. scopus identified 32 publications related to s. rarak by 23 authors from 1992 to 2022, accumulating 387 citations. google scholar reported 201 publications with 1176 citations. notably, indonesia, the indonesian research institute for animal production, and elizabeth wina emerged as the most influential country, institution, and author, respectively. research findings were clustered into chemistry, biology, medicine, and physics. however, the bioprospection of s. rarak fruit mainly focused on livestock supplementation. surprisingly, no studies on the efficacy of s. rarak as a detergent, so far, were found in any reputable international journals. all articles exclusively discussed about s. rarak fruit or seed, neglecting any exploration of the leaves, indicating an overlooked potential. future research should comprehensively explore the s. rarak for additional valuable phytochemicals beyond saponin, positioning it as a signature bioresource with significant economic value, expanding beyond its current detergents use within society. keywords: bibliometrics, bioprospecting, knowledge structure, roadmap research introduction bioprospection involves discovering natural products from unstudied species for developing new valuable nutraceuticals, bioactive constituents, and medicines (mcclatchey & stevens 2001; purkayastha 2016). the convention on biological diversity emphasizes the importance of biodiversity conservation for sustainable bioresources utilization. benefit sharing is embedded to the responsibility of safeguarding bioresource, and commercialization interests must guarantee the public access to intangible benefits (mcclatchey & stevens 2001). bioprospection is related to, but not limited to, drug discovery. orchestrated plant secondary metabolites are excellent entities against various diseases. this process involves systematic sampling, investigation, purification, and the development of semi-synthetic or synthetic derivative compounds to stabilize efficacy, reduce negative impacts, and increase profitability (mcclatchey & stevens 2001). however, the development of commercial products from natural organisms requires prolonged research with uncertain results (lichota & gwozdzinski 2018). sapindus rarak dc. is a native indonesian plant cultivated by the community and is recognized for its saponin-rich fruit, which is used as a natural detergent for both batik and baby cloth diapers. unfortunately, research on the medicinal properties of s. rarak is limited, even though saponins are recognized for treating various diseases, such as antibacterial (wei et al. 2021), antifungal (coleman et al. 2010), anticancer (xu et *corresponding author, email: rish.a.pratiwi@gmail.com how can the world overlook sapindus rarak bioprospection? – risha amilia pratiwi et al. 11 al. 2013), antioxidant, antidiabetic (salahuddin et al. 2020), and antiviral (pu et al. 2015). besides fruits, their leaves also contain saponin, although their potency is still overlooked (pratiwi & nurlaeni 2022). internet technology has provided access to scientific publications, enabling structured review to evaluate research progress, identify gaps, and plan a roadmap. bibliometrics is an approach that allows for data collection and comprehensive analysis of a field, covering the most influential research, research evolution, and insights for future research (fahimnia et al. 2015). as of 2023, no bibliometrics analysis for s. rarak’s bioprospecting exists, but insights from other species illustrate how the bibliometrics approach can reveal research trends, gaps, and opportunities for further exploration. a notable success story in bibliometrics analysis for plant bioprospection is the research of moringa oleifera lam. over the past 20 years, research on m. oleifera has rapidly developed, marked by the emergence of thousands of publications. future research trends are predicted to focus on important compounds isolation from m. oleifera for use as food additives and in phytoremediation (george et al. 2021). various approaches, including meta-analysis, systematic literature review, and scoping review, contribute to understanding research trends. while meta-analysis and bibliometrics handle large datasets and focus on quantitative empirical evidence in a broader field, systematic reviews manually investigate fewer documents (tens or fewer than 300) to qualitatively examine trends in a niche field. combining several methods may offer unique results (donthu et al. 2021). an interesting example of combining bibliometrics with a literature review is research on cactus mucilage —we mention this, considering the limited review on s. rarak itself, revealing the prospect of its carbohydrates, proteins, minerals, and fatty acids for functional products. research trends regarding cactus mucilage include its applications in food, cosmetics, and medicine, encompassing microencapsulation, biofilms, and biocoagulants (de andrade vieira & cordeiro 2023). scoping review, a novel approach for determining knowledge gaps without critical appraisal, contrasts with a systematic review, which summarizes the best available research (pham et al. 2014). due to the limited literature on s. rarak, the adoption of a scoping review approach, as exemplified in cannabis research, proves invaluable for identifying gaps in existing knowledge and paving the way for future investigations. for instance, a scoping review on cannabis highlighted cannflavins as antiinflammatory agents, demonstrating how this method reveals novel extraction protocols and biosynthetic pathway maps (erridge et al. 2020). this underscores the significance of employing a scoping review for s. rarak, where its application could uncover untapped potential, guide forthcoming research endeavors, and contribute to a comprehensive understanding of its bioresources. this paper aims to evaluate s. rarak research trends through a dual approach of bibliometrics and scoping review, offering a comprehensive and objective assessment. network analysis in bibliometrics captures established publications, research groups, and emerging trends. the exploration of s. rarak bioresource is expected to be progressive, enhancing indonesia’s competitiveness in the global academic community. materials and methods this research, conducted in january–february 2023, employed a bibliometrics analysis method by following the approaches of fahimnia et al. (2015); ma et al. (2022); and nurfauzan & faizatunnisa (2021) with some modifications. the methodology included defining search keywords, extracting initial data, refining results, compiling metadata, and mapping authors, keywords, and collaboration networks (see figure 1). the research employed scopus and google scholar for data search, and harzing's publish or perish software (harzing 2007) for data extraction. the results were saved in ris and csv formats and imported into the reference manager zotero (roy rosenzweig center for history & new media 2016). after obtaining a list of articles, we excluded patents, irrelevant studies, or duplicate literature, resulting in a final dataset containing information such as the number and list of authors, titles, year of publications, publishers, article type, total biotropia vol. 31 no. 1, 2024 12 citations, and citations per document. data compilation was performed using microsoft exceltm 2013 (microsoft corporation). furthermore, bibliometric analysis was conducted based on scopus. the world map depicting the number of publications for each country was derived through the online application (www.datawrapper.de). meanwhile, author mapping, keywords, and collaboration networks were visualized using vosviewer (van eck & waltman 2018). author and country mapping provided information about disciplinary leaders, while keywords indicated the hot topics. collaboration patterns and citation networks represented teamwork and primary literature from different periods, describing the field’s evolution and projecting future developments (ma et al. 2022). meanwhile, the scoping review included determining research objectives and questions, defining the scope, selecting studies through an iterative team approach, extracting data, summarizing qualitative and quantitative analysis, reporting results, preparing the considerations of implications in research, and optionally consulting with stakeholders (levac et al. 2010). the purpose and scope of this review were to map s. rarak bioprospection. the dataset inputted for the bibliometrics was also involved in the scoping review, although no stakeholder consultation occurred during this process. results and discussion defining the appropriate search keywords the preliminary bibliometrics analysis started with the keywords ‘sapindus rarak’ and ‘leaves’ to investigate the overlooked bioprospection of s. rarak leaves. despite expecting abundant data, scopus identified only three articles. expanding the keyword to ‘sapindus rarak’ increased the total to 32 documents, which was unexpectedly low (table 1). meanwhile, on google scholar, using the keywords ‘sapindus rarak’ and ‘leaves’ yielded 39 documents, and the keyword ‘sapindus rarak’ alone retrieved 221 documents. although this number was smaller compared to bibliometrics analyses for other plants like m. oleifera, which reached 2,345 documents (george et al. 2021), the limited global publications on s. rarak allowed an opportunity for an in-depth review. figure 1 flowchart of bibliometrics and scoping review (fahimnia et al. 2015; ma et al. 2022; nurfauzan & faizatunnisa 2021 with modification) http://www.datawrapper.de/ how can the world overlook sapindus rarak bioprospection? – risha amilia pratiwi et al. 13 table 1 the number of publications from scopus for each search argument database number of documents for search argument number of documents retained sapindus rarak and leaves sapindus rarak scopus 3 32 32 google scholar 39 221 201 extracting the initial data scopus (https://www.scopus.com/), managed by elsevier publishing, was selected over web of science (wos) (www.clarivate.com). although both are widely recognized inputs for bibliometrics analysis, the analysis based on each can provide different results (echchakoui 2020). despite wos being established earlier in 1997 than scopus in 2004, scopus accommodates a broader range publications from developing countries, encompassing non-english language. wos covers 54% of journals indexed by scopus, while scopus contains 84% of journals indexed by wos (gavel & iselid 2008). this research specifically utilized scopus considering its expected wider coverage compared to wos. utilizing harzing's publish or perish (harzing 2007) for scopus with ‘a title’ and ‘a keyword’ queries, 32 documents were obtained. additionally, we retrieved publications from google scholar for comparison, considering the limited global research related to s. rarak in scopus. here, google scholar showed numerous interesting local publications on s. rarak bioprospection. refining results all 32 documents from scopus met the requirements, with no irrelevant or duplicated data, resulting in a total retention of 32 documents (100%). in contrast, google scholar indexed numerous redundant documents, leading to a final result of 91% documents with 1176 citations (table 1). despite recording a higher publication count than scopus, google scholar included national publications, majority in indonesian (table 2), highlighting the predominant local development of s. rarak research in indonesia. for rigorous data analysis, further bibliometrics analysis was performed using scopus. compiling initial metadata the literature on s. rarak involves 23 authors in 32 documents spanning 20 years (1992–2022), accumulating 387 citations. initiated by matthias hamburger in 1992 (figure 2), the study of s. rarak progressed slowly until 2004, experiencing a crucial development from 2005–2010. this phase saw significant contributions from elizabeth wina, toshio morikawa, and yosunabu asao. elizabeth wina emerged as the most influential author, holding the highest citation rank. substantial contributions from evi maryanti and sri suharti occurred from 2011– 2019. while several researchers began to contribute during this period, their impact was relatively modest. table 2 the percentage of documents indexed by google scholar written in several languages language indonesian english japanese the document written in several languages (%) 60.7 38.8 0.5 https://www.scopus.com/ http://www.clarivate.com/ biotropia vol. 31 no. 1, 2024 14 figure 2 the trend in publishing and citation about s. rarak based on scopus sixteen institutions contributed to this field with the indonesian research institute for animal production (indonesia) standing out as the most influential. while kinki university, université de lausanne, and the university of illinois at chicago published fewer articles, their citation number was notably high (table 3). indonesia led the research on s. rarak (figure 3), which is not surprising given that the plant is native to indonesia, widely cultivated, sold in markets throughout the year, and commonly used by the community as a natural detergent. due to the excellent potency and existence of s. rarak in indonesia, it holds the potential to become a point of indonesian competitiveness. table 3 the compilation of publications about s. rarak based on scopus no author country institution year c tp tc 1 elizabeth wina indonesia indonesian research institute for animal production (iriap) 2004 0 4 128 2005 77 2006a 39 2006b 12 2 toshio morikawa japan kinki university 2009 56 2 71 2010 16 3 evi maryanti indonesia university of bengkulu (unib) 2014 40 1 40 4 yasunobu asao japan kyoto pharmaceutical university 2009 32 1 32 5 sri suharti indonesia bogor agricultural university (ipb) 2010 6 3 29 2011 19 2019 4 6 matthias hamburger switzer-land université de lausanne 1992 27 1 27 7 myung-sook chung usa university of illinois at chicago 1997 22 1 22 8 sylvia utami pratiwi indonesia gadjah mada university 2020 8 1 8 9 aininu nafiunisa indonesia diponegoro university 2019 6 1 6 10 caribu hadi prayitno indonesia jend. soedirman university 2013 4 2 6 2015 2 11 mardiati zain indonesia andalas university 2016 5 1 5 12 pristian yuliana indonesia ipb 2019a 2 2 4 2019b 2 13 inarah fajriaty indonesia tanjungpura university 2014 4 1 4 14 nita aryanti indonesia diponegoro university 2020a 0 2 3 2021b 3 15 salprima yudha s. indonesia unib 2022 1 1 1 16 aminah umar indonesia university of indonesia (ui) 2013 1 1 1 17 lueangkaew koysap thailand mahidol university 2022 0 1 0 18 t. pasaribu indonesia iriap 2022 0 1 0 19 hidayat pujisiswanto indonesia university of lampung 2022 0 1 0 20 sekar arum indonesia president university 2022 0 1 0 21 charles banon indonesia unib 2022 0 1 0 22 antonius h. cahyana indonesia ui 2020 0 1 0 23 amonrat khayungarnnawee thailand thailand institute of scientific & tech. research 2012 0 1 0 total 387 32 387 note: c = citation, tp = total publication, tc = total citation. how can the world overlook sapindus rarak bioprospection? – risha amilia pratiwi et al. 15 figure 3 a world map representing the total publications of each country about s. rarak based on scopus mapping authors’ collaboration, keywords, and field evolution patterns co-authorship network analysis revealed the collaboration trend and the leader in a particular field (fonseca et al. 2016). elizabeth wina from indonesia was the most influential author (figure 4). indonesian researchers (s. suharti, d. astuti, a. kurniawati, and t. toharmat) were interconnected with foreign researchers (s. muetzel, k. becker, e. hoffmann, and h. makkar) through this author. research on s. rarak focused on four hot topics: chemistry, biology, medicine, and physics. based on its chronological development, research on the biological aspect was developed first, followed by the exploration of the medicinal aspects, deepening into chemistry, and lastly, the investigation of physical properties. furthermore, chemistry became the most developed field compared to other topics (figure 5). figure 4 overlay visualization of co-authorship analysis to revealing the leader biotropia vol. 31 no. 1, 2024 16 figure 5 overlay visualization of co-occurrence analysis to reveal the popular keywords the most prominent author network, centered on elizabeth wina, focused on the study of microbial population dynamics and animal rumen fermentation due to the supplementation of s. rarak fruit extract. the morikawa, asao, and chang group explored the chemical structure and in vitro biological activity of s. rarak phytoconstituents. koysap contributed to bridging the evolution of the interlinked field between medicine and phytochemicals (figure 6). scoping review analysis an in-depth review of 32 publications revealed the diverse potentials of s. rarak. table 4 presents a summary of the scoping review analysis results, including the primary utilization aspect of s. rarak, identifying alternative sources of s. rarak beyond the fruit, evolving extraction techniques, common form of s. rarak, and regions contributing the most in providing s. rarak materials. figure 6 overlay visualization of collaboration patterns revealing the field evolution and projecting future developments table 4 scoping analysis compilation aspect number of document utilization livestock supplement (12), molluscicide (2), herbicide (1), antimicrobe (2), antitumor (1), anti-obesity (3), nanoparticle (3), anti-inflammatory (1), soil remediation (1), surfactant (4), catalyst (1), sweetener (1) organ source seed (3), fruit (30) extraction technique maceration with ethanol (4), methanol (15), aqua (8), ultrasound-assisted extraction (3) form crude extract (17), powder (1), pure saponin (4), biochar (1) origin of sample indonesia (24), thailand (4) how can the world overlook sapindus rarak bioprospection? – risha amilia pratiwi et al. 17 as shown in table 4, s. rarak research focuses on its potential as livestock supplementation (12 documents), alongside applications as molluscicide, herbicide, antimicrobial, antitumor, anti-obesity, anti-inflammatory agents, nanoparticle, soil remediation, surfactant, catalyst, and sweetener. all studies exclusively reported on fruit and seed of s. rarak, neglecting the potential of leaves, despite significant biological activity in other sapindaceae species leaves. for instance, litchi chinensis leaves exhibit anti-inflammatory effect (besra et al. 1996), s. mukorrossi demonstrates antioxidant activity (singh & kumari 2015), and s. saponaria shows antimicrobial (garcia et al. 2012) and antiulcer (meyer albiero et al. 2002) activity. while s. rarak leaves contain fewer saponins than fruit (pratiwi & nurlaeni 2022), exploring their potential extends beyond saponin extraction to uncover other beneficial phytochemical groups. the leaves, being abundant organs that do not compete with reproductive functions, warrant consideration (chen et al. 2016). additional s. rarak research discussed the extraction method. to enhance saponin yield, a variety of solvents were considered, with methanol frequently chosen. ultrasound-assisted extraction (uae), particularly green uae, and raising the extraction temperature were able to increase saponin yield (aryanti et al. 2020, 2021; nafiunisa et al. 2019). the prevalent use of crude extract over powder, pure saponin, and biochar is noted. consistent with bibliometrics results, the majority of samples sourced from indonesia. while the discussion regarding the efficacy of s. rarak as a detergent material is limited in internationally reputable publications, numerous national publications on google scholar address this aspect. s. rarak fruit, whether in its raw form, boiled, or incorporated into soap with additional additives, is widely sold throughout the year in the indonesian market for washing batik and baby cloth diapers (muttafaq et al. 2019; nia et al. 2018). detergent formulated from s. rarak is safe for clothing fiber and color, effectively removing any stains compared to conventional detergents (muttafaq et al. 2019). moreover, the environmental friendliness of s. rarak is noteworthy as the fruit generates abundant foam with just water soaking (pratiwi & nurlaeni 2022), eliminating the need for resource-intensive processes like detergent manufacturing, known for their high carbon footprint (de koning et al. 2010). the formulated s. rarak detergent has a potential to replace non-renewable petroleumbased detergents ( maurad et al. 2017) and excess phosphates often used as detergent builders, contributing to water eutrophication (kogawa et al. 2017). additionally, detergent waste from s. rarak will easily decompose in the environment. to gain a broader perspective, we suggest future exploration using google scholar to understand national research trends, particularly regarding the application of s. rarak as a detergent. this database can also reveal information about indonesian scientist teams involved in s. rarak research for various applications. in the field of s. rarak as livestock supplementation, elizabeth wina is a notable figure, but not the first indonesian scientist in this domain. the team from the indonesian research institute for animal production, including mohammad winugroho, amlius thalib, yeni widiawati, and dwi yulistiani, initiated the research. in terms of feedstock supplementation, saponin impacts rumen fermentation by modifying the microbial community's structure and activity, acting as a defaunation agent that suppresses protozoa without affecting bacterial growth, leading to improved fiber digestion efficiency or decreased protein degradation (wina et al. 2005). however, saponin’s toxicity negatively impacts beneficial bacteria and fungi and their enzyme activity in an in vivo study using rumen liquor from goats fed s. rarak extract (wina et al. 2004). conversely, a moderate level of s. rarak reduced the protozoa community from cow rumen cultured in vitro without affecting fibrolytic bacterial and fungi (wina et al. 2005). further study showed that short-term saponin exposure in goat reduced xylanase activity and ruminococcus, but fibrobacter, being gramnegative, resisted saponins. long-term exposure in sheep rumen depressed protozoa and fibrolytic enzyme carboxymethylcellulase (cmc) activity but had no effect on ruminococcus, suggesting potential bacterial adaptation mechanism (wina et al. 2006b). the disparity in results between in vitro and in vivo studies triggers the idea of interval feeding (wina et al. 2006a), although s. rarak reduces protozoa in both feeding methods with no significant differences at any dose. cmc and biotropia vol. 31 no. 1, 2024 18 xylanase activity decreased in both regimes, and higher doses led to lower enzyme activity. although high saponins doses tended to reduce ammonia concentration, nitrogen balance remained unaffected, and the decrease in fibrolytic enzymes did not impact the digestive tract (wina et al. 2006a). the s. rarak supplement has been introduced in various formulations, including mineral blocks (suharti et al. 2010), microencapsulation with canola oil (suharti et al. 2019), and combination with garlic extract for ammoniated rice straw substrate enrichment (prayitno et al. 2013), in addition to oil palm frond substrate, and saccharomyces cerevisiae fermentation with s. rarak extract (zain et al. 2016). furthermore, the addition of s. rarak has been tested in cassava leaf diets (yuliana et al. 2019a) and incorporated with hibiscus rosa-sinensis in the cassava leaf silage (yuliana et al. 2019b). these efforts have increased beneficial bacterial communities, enhanced protein flow from the rumen to the blood, and improved ammonia concentration, dry matter, and organic matter digestibility, body weight gain, and suppression of protozoa populations. the decreased protozoa, as host for methanogenic bacteria, suppress methane production, the second most impactful greenhouse gas after co2, with a higher global warming potential. ruminant methane (ch4) emissions harm the environment, increase the greenhouse impact, and reduce animal energy and production efficiency. therefore, ruminant methane emissions must be reduced (sun et al. 2021). the dynamics of the methanogenic bacteria within the rumen undergo a shift from methane to propionate production (suharti et al. 2011). hence, saponin indirectly contributes to mitigating climate change by reducing methane emission (króliczewska et al. 2023). although s. rarak fruit can improve fermentation performance in the digestive tract of goats, sheep, dairy, and beef cattle, the results can vary dependent upon saponin type, dosage, fermentation substrate, and combination with other ingredients. further research is required to develop patented feed supplementation products, exemplified by the patent for the ‘complete rumen modifier’ formula (certificate number idp000057123), a feed additive for ruminant livestock that has the potential to enhance their growth while reducing enteric methane production (thalib et al. 2010). s. rarak exhibits molluscicidal properties attributed to four newly identified triglycoside saponins (hamburger 1992). meanwhile, the s. rarak extract serves as a repellent and induces mortality in giant african land snails (lissachatina fulica) without harming vegetable brassica rapa l. (koysap et al. 2022). additionally, it demonstrates herbicidal activity against leptochloa chinensis and fimbristylis milacea weeds. the aqua extract has no adverse effects on rice growth, while the methanol extract inhibits rice height without affecting root length and leaf number (pujisiswanto et al. 2022). in terms of antimicrobial properties, the ethanol extract of s. rarak seeds effectively degrades extracellular polymeric substances in pathogenic polymicrobial biofilms, as observed by scanning electron microscopy (pratiwi & hamzah 2020). furthermore, s. rarak microparticles administered in drinking water perform similarly to commercial anticoccidials in reducing eimeria sp. and e. coli (pasaribu et al. 2022). coccidiosis is an important enteric health problem in poultry, leading to reduced feed efficiency, weight gain, bloody stools, and mortality. the gastrointestinal tract in chickens has a wide range of microorganisms that play a vital part in the processes of digestion and defense. the infection of eimeria spp. parasites disrupts the gut environment, leading to an imbalance in intestinal homeostasis. this imbalance promotes the growth of pathogens, including clostridium species (madlala et al. 2021). s. rarak as a natural antibiotic potentially can replace the banned feed additives containing anticoccidial agents due to the risk of multidrug resistance (han et al. 2022). the alternative use of antibiotic growth promoters (agp), such as s. rarak saponin, is crucial due to the prohibition on agp. these alternatives should easily blend with feed, lack tissue residual effects, and improve feed intake, gain, and conversion rates. additionally, they should enhance immunity and digestion, nutrient availability, possess antimicrobial effects, maintain unaffected carcass characteristics, act as antioxidants and anti-inflammatory agents, compete against stress factors, and provide wholesome organic products for human consumption (abd el-hack et al. 2022). as the how can the world overlook sapindus rarak bioprospection? – risha amilia pratiwi et al. 19 emphasis on a healthy lifestyle and organic farming grows, consumer preferences are increasingly favoring products from antibioticfree livestock (seidavi et al. 2021). s. rarak presents sesquiterpene saponins inhibiting tumor necrosis factor-α-induced cytotoxicity in l929 fibrosarcoma cells (morikawa et al. 2010). it also demonstrate antiobesity effects by inhibiting pancreatic lipase activity (morikawa et al. 2009) and has passed toxicity tests, suggesting potential benefits in weight management (asao et al. 2009; fajriaty et al. 2014). moreover, s. rarak finds application in the synthesis of silver (yudha et al. 2013) and zno nanoparticles (maryanti et al. 2014), useful for photo-degrading rhodamin b as environmental pollutants (umar et al. 2022). it can also be synthesized with sulfur as a catalyst (banon et al. 2022). additionally, its derivative compounds exhibit potential as acid surfactants (cahyana et al. 2020) and sweet principles (chung et al. 1997). lastly, scoping review showed that s. rarak biochar has the potential to improve andosol soil quality (arum & wikaningrum 2022). conclusion the substantial number of publications written in indonesian, as indexed by google scholar, highlights the enthusiastic involvement of indonesian researchers in the exploration of s. rarak bioprospection. scopus indexed 32 scientific articles regarding s. rarak, composed by 23 authors between 1990 and 2022, with a cumulative citation count of 387. notably, indonesia, the indonesian research institute for animal production, and elizabeth wina stand out as the most productive country, institution, and author, respectively. the research is clustered based on chemistry, biology, medicine, and physics approach. the bioprospection of s. rarak fruit encompasses various applications, including animal feed supplementation, molluscicide, herbicide, antimicrobial, antitumor, anti-obesity, surfactant, catalyst, nanoparticle, and soil remediation. surprisingly, research on the efficacy of detergents derived from s. rarak has not been published in a highly reputable international journal, despite its traditional utilization over an extended period. despite the exclusive focus on s. rarak fruit or seed as promising organ in all articles, none have addressed leaves or other plant organs. in the future, indonesian researchers are encouraged to comprehensively explore the potential of s. rarak, especially its leaves, for additional valuable phytochemicals beyond saponin. this exploration could position s. rarak as a signature bioresource with significant economic value, serving as a source for medicine, animal feed supplements, and organic pesticides, expanding beyond its current use in detergents within society. references abd el-hack me, el-saadony mt, salem hm, el-tahan am, soliman mm, youssef gba, taha ae, soliman sm, ahmed ae, el-kott af, al syaad km, swelum aa. 2022. alternatives to antibiotics for organic poultry production: types, modes of action and impacts on bird’s health and production. poultry science 101(4): 101696. doi: 10.1016/ j.psj.2022.101696. arum s, wikaningrum t. 2022. study of lerak (sapindus rarak) biochar application for andosol agricultural soil remediation. iop conference series: earth and environmental science 1065(1). doi:10.1088/ 17551315/1065/1/012020. aryanti n, heny dr, nafiunisa a. 2020. optimization of ultrasound-assisted extraction of rarak saponin from sapindus rarak dc. using response surface methodology (rsm). aip conference proceedings 2197. doi:10.1063/1.5140912. aryanti n, nafiunisa a, kusworo td, wardhani dh. 2021. dye solubilization ability of plant derived surfactant from sapindus rarak dc. extracted with the assistance of ultrasonic waves. environmental technology and innovation 22. doi:10.1016/ j.eti.2021.101450. asao y, morikawa t, xie y, okamoto m, hamao m, matsuda h, muraoka o, yuan d, yoshikawa m. 2009. structures of acetylated oleanane-type triterpene saponins, rarasaponins iv, v, and vi, and anti-hyperlipidemic constituents from the pericarps of sapindus rarak. chemical and pharmaceutical bulletin 57(2): 198-203. doi:10.1248/cpb.57.198. banon c, nesbah n, trihadi b, falahudin a, yudha ss. 2022. easy handling preparation of cubic sulfur in aqueous extract of sapindus rarak rinds. molekul 17(3): 321-327. doi:10.20884/ 1.jm.2022.17.3.5719. besra se, sharma rm, gomes a. 1996. antiinflammatory effect of petroleum ether extract of leaves of litchi chinensis gaertn. 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agriculture 44(2): 167-176. doi:10.14710/ jitaa.44.2.167-176. yuliana p, laconi eb, jayanegara a, achmadi ss, samsudin aa. 2019b. extracted saponin from sapindus rarak and hibiscus sp. as an additive in cassava leaf silage: effects on chemical composition, rumen fermentation and microbial population. advances in animal and veterinary sciences 7(7): 530-536. doi:10.17582/ journal.aavs/2019/7.7.530.536. zain m, rahman j, khasrad e. 2016. supplementation of saccharomyces cerevisiae and sapindus rarak in diet based of oil palm frond (opf) on nutrient digestibility and daily weight gain of goat. asian journal of animal and veterinary advances 11(5): 314-318. doi:10.3923/ajava.2016.314.318. biotropia vol. 28 no. 1,2021: 74 83 doi: 10.11598/btb.2021.28.1.1092 land suitability assessment for patchouli (pogostemon cablin) development and essential oil production ad1 setiawanl?'*, deffi armita', aldila putlu rahayu' and nunun barunawati' 'department ofagronomy, faczrlq ofagni.nltzlre, universio ofbrawzjqa, jalan veteran, malang 65145, indonesia 21nstzt~te ofatxim', universitas brawzjqa, jalan veteran, malang 65145, indonesia received 3 july 2018/accepted 4 january 2020 patchouli (pogostemon cablin) is one of the important crop species in indonesia, since over 80% of patchouli oil global market is produced in indonesia. pacthouli oil is the key ingredients for fragrance and aromatherapy products. patchouli oil is extracted from the stems and leaves of pathouli plants. therefore, it is important to improve patchouli plant productivity and increase resources for sustainable patchouli cultivation. the suitability of abiotic factors in the growing environment of crops remarkably determines the success of crop production. this study aimed to assess and evaluate land suitability for plant growth and development of patchouli (pogostemon cablin) in dilem wilis, bendungan district, trenggalek regency, indonesia. initially, a survey was conducted and then an analysis was done to classify the land suitability for crops cultivation. the research was conducted on 3 locations from may to july 2017 for land suitability and from july to november 2017 for patchouli crops cultivation experiment. the results indicated that location 1 had a land suitability of n class, implying that this location was not suitable due to its limiting factor of low igo content (0.08 me/100 g). meanwhile, both location 2 and location 3 showed similar land suitability class of s3s, tc, f, n signifying as less appropriate. the results of this study also indicated the influence of land suitability classes on plant growth however, the different classification (in this case s3 compared to n) did not demonstrate a correlation between land classes and oil yield and patchouli alcohol, where the element potassium was the limiting factor. keywords: abiotic factor, land suitability, patchouli alcohol, potassium introduction patchouli (pogostemon cabhz) is one of the important crop species in indonesia, since indonesia is one of top producers of patchouli oil. patchouli oil is used as key ingredients for fragrance as well as for cosmetics and aromatherapy products (ramya et al. 2013; nugraha et al. 2019). indonesia is the world's largest producer of patchouli oil, accounting for 80% of the global market (i<usumaningrum et al. 2017). patchouli oil is difficult to synthesize or substitute. therefore, patchouli oil produced by natural patchouli plant remains the only source of the oil (van beek & jouhan 2018). as patchouli oil is extracted from the stems and leaves of patchouli plants, it is important to improve patchouli plant productivity and resource use efficiency through suitable cultivation. land suitability is known to significantly affect the growth and the quantity and quality of crop yields, especially in essential oils. differences in land suitability classes is expected to be directly proportional to plant growth and yield. however, the essential oil produced from secondary metabolites, is strongly influenced by stress con&tions (baher et al. 2002; sangwan et al 2001; simon et al. 1992; taarit et al. 2009). land suitability determines the success of crop production, especially if the target output involves primary metabolites such as carbohydrates, proteins and fats. however, little is known on production targets that include metabolic secretions such as flavonoids, 'corresponding author, e-mail: adisetiawan@ub.ac.id alkaloids and terpenoids. therefore, the assessing and evaluating land suitability in the development of patchouli effect on patchouli essential oil setiawan et al. assessment of land suitability for essential oil producing plants is necessary particularly for the patchouli (pogostemon cablin) plant. patchouli plants are considerably easy to grow like other herbaceous plants. however, an optimum ecological condition is deemed necessary to obtain the maximum production. patchouli can grow and develop in the lowlands up to a plateau at 1,200 m above sea level (m asl), although its best growth is at altitudes between 50 400 m asl. the oil content of patchouli on the lowlands is higher but the alcohol content is lower (low plateau patchouli plant would generate low oil content and high alcohol levels). environmental factors greatly affect patchouli alcohol (pa) levels (singh et al. 2002). for the optimal growth, patchouli plants require hot and humid temperatures; with annual rainfall ranging from 2,000&2,500 mm/year, evenly distributed throughout the year; optimum temperature of 24 28 "c; moisture of more than 75%, and radiation intensity ranging between 75 100%. in certain protected areas, patchouli can still grow well with lower oil content. patchouli planted under shady location will grow better, with wider, thinner and greener leaves, but with low oil content. patchouli plants grown in the open area are considerably less dense with smaller plant habitus, small and thick leaves that are yellowish and slightly red, yet having hgher oil content. therefore, the addition of little shade at the beginning of growth was suggested because patchouli is vulnerable to stress drought (rltung et al. 2007). patchouli plants grow best on fertile and loose soil, which is rich in humus and properly drained. the most suitable soil types are those that have a crumb texture, such as andosol or latosol. for clays, a more intensive processing is required to obtain optimal conditions. in soil with less humus, the application of manure is highly recommended to improve soil fertility and humidity (rltung et al! 2007). a problem arises when the plant is cultivated in suitable condition, yet producing low essential oil content. thus, to increase its essential oil production level, the plants require relatively stress-free environmental condtions. several factors could affect the growth and yield of patchouli plants (both quality and quantity) however, this study focused only on the assessment of land suitability for the growth of patchouli and its oil production. materials and method study site this study involved two steps, namely: 1. land suitability assessment conducted in may 2017 to july 2017 and 2. patchouli crop production conducted in july to november 2017 at dilem wilis plantation, dompyong village, bendungan district, trenggalek regency, indonesia (figs. 1 & 2). laboratory test was conducted at the chemistry laboratory of soils department, faculty of agriculture, universitas brawijaya. data were then analyzed at the environmental resources laboratory, department of agronomy, universitas brawijaya. steam distillation process was performed to extract the oil and to analyze the patchouli alcohol content (pa) at the institute of atsiri, universitas brawijaya, indonesia. land suitability assessments the experiment involved the use of several tools, such as ruler to measure the depth of the soil, hoe to obtain soil samples from the different locations, clmometer to measure the percent slope of the land, gps (global positioning system) to find the location of coordinates and altitude of the place. laboratory procedures and facilities were also used to analyze c-organic, cec, ig0, and ph, as well as computer for map workmanship and data analysis. the soil texture and drainage were also analyzed. the first step in the evaluation of land suitability involved the ground check for the location of patchouli plantation. soil samples were then collected at some locations for observation of land physiography. the composite soil samples were obtained from each location at a depth of 10 20 cm. these samples were analyzed for soil texture. meanwhile, laboratory procedures were done to examine the organic matter content, k20, ph, and cec level. observations of land physiography included recording of coordinate locations, altitude, and slope of the land. the land suitability assessment method in this study was based on dengiz (2013). figure 1 study site i dilem willis plantation trenggalek regency i settlements location street footpath boundary 0 40 80 160 240 320 li meter source : 1. google earth 2. dem srtm 3. rbi bakosurtanal map figure 2 location of assessment plot the data analysis was initiated by establishing the data base to cluster the land suitability by using the matching method adjusted to each parameter as growth requirement of patchouli plant (fa0 1976; djaenudin e t al. 2003; ritung e t al. 2007; safuan e t al. 2013). the land suitability classification was determined by the most constraining limiting factor (rossiter & van wambeke 1997; djaenudin 2008; wirosoedarmo e t al. 2011; jayanti e t ak. 2013). this study applied such basis to investigate the effect of land suitability on the agronomic aspects of plants. the assessment was performed by matching the requirement of patchouli plant growth and the characteristics of the land which were assessing and evaluating land suitability in the development of patchouli effect on patchouli essential oil setiawan e t al. categorized into 3 groups based on: topography, patchouli growth and result evaluation soil and climate p t u n g et al 2007). the land suitability assessment was used to reveal the limiting factor of patchouli cultivation, as reference to improve the patchouli cultivation area. the land suitability classification based on f a 0 (1976) was consisted of four categories, namely: a. order: indicating the general state of the land suitability; b. class: indicating the order level of conformity; c. subclass: inhcating the status of the class level based on the type of constraint or improvement as required in the class; and d. unit: indicating the level in the subclass based on minor differences that affect the management. three locations were identified to obtain the com~osite soil sam~les and the land after conducting land suitability analysis, the locations were classified based on suitability. to evaluate the effect of land suitabfity on growth and yields, the patchouli was planted without adding improvements to the soil conditions or to the existing topography. it is suspected that there is a positive correlation between patchouli yield and land suitabfity evaluation. the one-month old sidikalang variety of patchouli seedlings were planted at a spacing distance of 50 x 100 cm. the soil was cultivated applying minimum ullage with weed control every 2 weeks. soil was irrigated every 2 days to adjust the soil conditions with the same dosage equally given for each location without addition char~ctenstics, inclu&& the required ph, of so^ n~trients/fedizer. this study utilized a c-organic, n, p, i<, na, ca, mg, and cec. randomized block design with 3 replications at the slope, altitude, temperature, drainage and each location, each replication consisting of 27 texture. plants with 3 plants for each replication. table 1 land suitability classification of patchouli plants land suitability classes category s1 s2 s3 n land position (s) slope (yo) altitude (m asl) temperature (tc) average temperature pc) 24 26 22 24 or 26 28 20-2201-28-33 1 8 2 0 o r > 3 3 water availability (wa) rainfalls (mm) 2300 3000 1750 2300 or 30001200 1750 or > <i200 or >5000 3500 3500 root media (rc) texture drainage soil depth (cm) sandy clay, sandy clay and other sandy clay, clay quartz clay other other very good good bad very bad > 100 cm 75 100 cm 50 75 cm < 50 cm nutrient retention (f) acidity (ph) acidic (5.5 7) acidic to neutral acidic to very very acidic to (5.5 5) acidic (4,5-5) alkaline (< 4.5 or > 7.5) c-organic (yo) 2 3 3 5 < 2 cec (me/100 g) > 17 5.6 < 5 nutrition (n) i<2o (me/100 g) pzos (ppm) source: djaenudin e t al. (2003). biotropia vol. 28 no. 1,2021 the plant growth was assessed based on the branch height and canopy diameter of the plant at 2, 4 and 6 months after planting. results were obtained by destructive sampling based on wetlfresh weight and curing dry weight (t20% water content). the oil yield component was obtained by the 8-hour steam distillation method to obtain patchouli alcohol (pa) level as an indicator of the quahty of essential oils (idris e t al 2014; bur6 & selher 2004). the obtained 768 m asl. the average altitude for optimum patchouli cultivation was at 780 m asl. average air temperatures at each location based on the formula of braak (1928) were: location 1 at 21 oc, location 2 at 21 oc, and location 3 at 22 oc. the air temperature got cooler as the altitude went higher. based on the formula of braak (1928), the temperature registered a steady decline by 0.61 oc with an increasing altitude of 1 m. growth and yield data were tested by using the soil texture at the study site was analysis of variance (f-test) at significance level dominated by clay and dust. at location 1, the p < 0.05). furthermore, to test the significant soil texture was sandy clay, while at location 2 differences between treatments, the tukey test and location 3 it was clay. drainage at location performance by r statistics was applied. 1 is considerably good as water moves quickly or seeps into the soil through the infiltration process, supported by sandy clay texture having results and discussion a larger pore than the sand fraction. meanwhile, land suitability the differences among location 1, location 2 and location 3, were based on the land suitabhty class (location 1 is n as not appropriate, locations 2 and 3 are less appropriate (table 2). the slopes of mount wilis is considered hilly to mountainous reliefs. location 1 has a slope of 8%, location 2 has 10°/o, and location 3 has 12%. at the observation point the altitude was about 600 850 m above sea level (m asl). location 1 lies at an altitude of 792 m asl, location 2 at an altitude of 809 m asl, and location 3 at an altitude of both location 2 and location 3 have a good or medium drainage class. soil acidity (ph) at location 1 is 4.6, at location 2 is 4.9 and at location 3 is 4.5 (where the soil ph at the observation point is acidic). based on laboratory test, c-organic at the observation site was 1.63% at location 1, 1.50% at location 2 and 0.71% at location 3. the cation exchange capacity (cec) at location 1 is 18.75 me/100 g, at location 2 is 5.38 me/100g, and at location 3 is 18.45 me/100 g. the availability of i<20 macro elements at location 1 is 0.08 me/100 g, at location 2 is 0.46 me/100 g, and at location 3 is 0.25 me/100 g. table 2 land suitabhty classification of location 1 ,2 and 3 at dilem wilis plantation, trenggalek regency, indonesia land characteristics data category location 1 location 2 location 3 land position (s) slope (o/o) 8% 10% 12% altitude (m asl) 792 809 768 temperature (tc) average temperature ("c) 21 2 1 22 water availability (wa) rainfalls (mm) 1,428 1,428 1,428 root media (rc) texture clay dust clay clay drainage good good very bad soil ~ e p t h (cm) > 50 > 50 >-15 nutrient retention (f) acidity (ph) 4.6 4.9 4.5 c-organic (o/o) 1.63 1.50 0.71 cec (me/100 g) 18.75 5.38 18.45 nutrition (n) iqo (me/ 1 00 g) 0.08 0.46 0.25 -. p205 (ppm) land suitability class n n s3s, tc, f, n s3s, tc, f, n source: primary data (2017) assessing and evaluating land suitability in the development of patchouli effect on patchouli essential oil setiawan e t al. crops production evaluation the number of branches and canopy diameter . did not differ sigmficantly. correiation between cmp production yield and ~ h , dfferent land classes produced higher suitabikp biomass in plant fresh/wet weight and curing the results showed that the different land dry weight, indicating the effect of land classes significantly affected the plant height at 6 suitabiliq on plant growth (fig. 6). months after planting (figs. 3, 4, 5). however, month after planting figure 3 number of branches notes: a = 2 months; b = 4 months; c = 6 months after planting. month after planting figure 4 plant height notes: a = 2 months; b = 4 months; c = 6 months after planting; different letters in the figures indicate significant difference at 0.05 level. biotropia vol. 28 no. 1,2021 month after planting figure 5 canopy diameter among location (l) 1,2, and 3 on the 2, 4 and 6 months after planting location location figure 6 fresh weight (g per plant) notes: a = curing dry weight (g per plant); b = harvested 6 months after planting; different letters in the figures indicate significant differences at 0.05 level. although location 1 has the lowest yield value, the average oil yield and patchouli alcohol (pa) quality in each location were not significantly affected by the limiting factors (table 3). table 3 average oil yield and quality pa (patchouli alcohol) of patchouli location yield (?/o) pa (yo) location 1 2.36 ns 23.40 ns location 2 2.42 ns 22.82 ns location 3 2.49 ns 24.14 ns note: ns = no significant difference. have s3 class, tc, f, n indicating that the cultivation area was less suitable because of the limiting effect of the slope, altitude, temperature, ph, c-organic, and igo. potassium (i<) plays a key role in plant metabolism affecting the synthesis and accumulation of nutrients and secondary metabolites (bihter e t al 2016). limiting factors that have significant effects included the altitude and the slo~es. the cultivation area was located on the southern slopes of mount wilis which is hilly to mountainous with an altitude of 600 850 m asl. the slope of the land varies. the land on slopes patchouli cultivation and land suitability were to erosion due to the lack of soil reinforcement plants and the absence of soil matching the land characteristics with the protection from splashing rainwater. the land growth requirement of patchouli resulted in on the slopes will be eroded if there is no location 1 having a land suitability class "n", improvement on it. in the highlands with low indicating that the cultivated land was not temperatures, soil feraty wdl be preserved, but suitable because of the limiting effect of the very steep slopes and unstable lands wdl lead to low igo content. location 2 and location 3 landslides making it more suitable for trees assessing and evaluating land suitability in the development ( ~f patchouli effect on patchouli essential oil setiawan eta/ (djaenudin 2008). the elevation of the observed location is in the range of > 700 m asl. patchouli plants can grow and produce well at an altitude of 10 400 m above sea levels with air temperature ranging from 24 to 28 oc (pujiharti et al. 2008). the other limiting factor was the acidic soil ph which has a value ranging from 4.5 to 4.9. the optimum growth and production of patchouli plants requires optimum soil ph value of 5.5 to 7 which is quite acid to neutral. nutrients and microorganisms are affected by the soil ph as nutrients are only available at certain ph level. the soil ph is influenced by the manner of land utiluation. the c-organic value, representing the organic matter percentage in the soil, at all the locations were also low (< 2%), as compared to the growth requirement of patchouli plants whch is about 2 3%. i<20 content at all three locations was also low (< 0.6 me/100 g), lower than the optimal growth requirement of patchouli (> 10 me/100 g of go) . low i<20 content is due to several factors such as lack of fertihzation. potassium is an alkaline cation, which balances charges of organic and inorganic anions activating more than 50 enzymes (nurzynska wierdak et al. 201 1). agronomic factor land suitability increased the plant growth despite the insignificantly different oil quantities (figs. 3, 4, 5, 6; table 3). the current study showed that the effect of limiting factors (potassium) and land suitability does not affect patchouli oil and alcohol yields, similar results with singh (201 4). however, distinguishable differences were observed in the potassium content among location 1, 2 and 3 (table 2). potassium is considered a plant essential mineral which has hgh concentration in the meristematic tissues and in the phloem. however, i< uptake by the plant roots is accomplished by at least two distinct hnetic systems such as the hgh and low affinity i<+ transporters (hafsi et al. 2014). potassium serves as an important element in plant metabolism, promoting carbohydrates, fats and protein synthesis, increasing crop yield and improving fresh produce quality. moreover, i< enables plants efficacy to resist pests and diseases as well as acting as enzymes co-factor, including enzymes related to the essential oil synthesis (hafsi et al. 2014). the application of i< has also affected the growth and essential oil yield of lemongrass (cymbopogon flxaoszls), dittany (origdnum dictamnas), basil (ocimm basilicam) and rosemary (rosmrinus ofiknalis) (economakis 1993; puttanna et al. 2010). improvement of land characteristics and cultivation the improvement of land characteristic was aimed at optimizing the land condition for patchouli cultivation by using the limiting factors as a reference. the slope of the land is unchangeable, yet such condition could still be addressed by constructing terraces to reduce surface runoff that leads to erosion (wkunan et al. 1985); and the position of the porch slope affects the reception of light (auslander et al. 2003). fertilization is another alternative to improve the soil ph value, i<20 content, and c organic percentage, including appropriate fertilization system such as: fertilization time and method, and fertilizer type selection. the application of organic fertilizer is intended to supply the nutrients that could not be provided by the chemical fertilizer, and also to improve the physical and biological properties of the soil (abdurachman e t al. 2008). liming is another method applied to increase soil acidity values (ph) to be more neutral during land cultivation. potassium (i<) affects the growth and essential oil synthesis in aromatic plants as it is required by plants to build abundant organic compounds such as amino acids, proteins, enzymes and nucleic acids. these minerals affect the function and levels of enzymes involved in the terpenoides biosynthesis (hafsi et al. 2014). the low potassium content at location 1 is a h t i n g factor. however, certain strategies could be applied to ensure the quantity of essential oil yields, where the main target of the harvest is secondary metabolites. it is necessary to synchronize the suitabihty of the growth conditions for secondary metabolite-producing plants in as much as both the growth and yield targets also implied the achievement of good quality essential oils. the planting patterns are also considered as a solution to increase stress as the harvesting period approaches (sacks et al. 2010). biotropia vol. 28 no. 1,2021 conclusion the land suitability analysis of the three sampling locations (location 1, 2, 3) revealed that location 1 is not suitable for the growing of patchouli because of its low ig0 content at 0.08 me/100 g. both location 2 and location 3 showed similar land suitability class of s3s, tc, f, which is less appropriate, because of the limiting effects of land, altitude, rainfall, air temperature, c-organic, soil ph (acidity), and i<zo. however, the dfferent locations did not significantly demonstrate any inhibiting effects on the growth and yleld of the patchouli plants. although the land suitability test indicated differences in growth as well as quality and quantity of yields, h s correlation between land suitability and growth becomes less relevant to patchouli oil yield and alcohol level (indication of the quality of oil obtained). differences in locations under certain conditions, in this case s3 compared to n, did not affect the patchouli alcohol (pa) content of the essential oil. lastly, potassium was a limiting element on the growth of the essential oil-producing plant patchod, pogostemon cablin. acknowledgement the authors would like to acknowledge and thank the director of the institute of atsiri for providing the research fund and the dilem wilis plantation company of trenggalek as the research area. references abdurachman a, dariah a, mulyani a. 2008. strategi dan teknologi pengelolaan lahan kering mendukung pengadaan pangan nasional p h e strategy and technology of dry land management support national food procurement]. jurnal litbang pertanian 27(2):43-9. auslander m, nevo e, inbar m. 2003. the effects of slope orientation on plant growth, developmental instability and susceptibility to herbivores. journal of arid environments 55(3):405-16. baher zf, mirza m, ghorbanli m, bagher rm. 2002. the influence of water stress on plant height, herbal and essential oil yield and composition in satureja hortensis l. flavour and 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taarit mb, msaada i<, hosni k, hammami m, i<chouk me, marzouk b. 2009. plant growth, essential oil yield and composition of sage (saluia o$cinaalir l.) fruits cultivated under salt stress conditions. ind crops prod 30(3):333-7. van beek ta, joulain d. 2018. the essential oil of patchouli, pogostemon cab&: a review. flavour and fragrance journal 33(1):6-51. whitman ce, hatfield jl, reginato rj. 1985. effect of slope position on the microclimate, growth, and yield of barley. agron j 77(5):663-9. wirosoedarmo r, sutanhaji at, icurniati e, wijayanti r. 2011. evaluasi kesesuaian lahan untuk tanaman jagung menggunakan metode analisis spasial fand suitability assessment of corn (zea mqs l.) using spatial analysis method]. agritech 31(1):71-8. the southeast asian journal of tropical biology vol. 32 no. 1, 2025: 118 128 doi: 10.11598/btb.2025.32.1.2131 issn: 0215-6334 | e-issn: 1907-770x 118 diversity, morphometry, and population abundance of sea urchin (tripneustes gratilla) in north lombok victor david nico gultom research center for marine and land bioindustry, national research and innovation agency, teluk kodek, lombok 83352, west nusa tenggara province, indonesia article higlights • the highest absolute abundance of sea urchin tripneustes gratilla was observed in august 2023 • in 2024, the low absolute abundance of tripneustes gratilla and the decline of sea urchin species diversity in north lombok prospectively indicated overharvesting. • the number of gleaners and the gleaning activity conducted by local villagers prospectively harm seagrass meadows, especially on tripneustes gratilla abundance article information received : 23 november, 2023 revised : 31 july, 2024 accepted : 12 february, 2025 *corresponding author, e-mail: vict001@brin.go.id short communication abstract sea urchin tripneustes gratilla is commonly found in tropical shallow water-seagrass beds and is consumed for its roe in southeast asia. this study recorded five sea urchin species in the study area: tripneustes gratilla, pseudoboletia maculata, salmacis bicolor, salmacis sphaeroides, and maretia planulata, with t. gratilla being the most abundant. the highest absolute abundance of t. gratilla was observed in august 2023, at 0.30 ind./m2, while from may to july 2024, it declined to 0.02 ind./m2. the mean diameters of t. gratilla ranged from 37.59 mm to 44.16 mm between august and october 2023 and from 17.39 mm to 48.45 mm between may and july 2024, having wide range of the mean weight. in september 2023, sea urchin with a test diameter of 35.0 – 39.9 mm were the most frequent to be found. this study provided baseline data on t. gratilla harvested from the seagrass habitat by the local community in north lombok and highlights the potential for overharvesting in the area. keywords: north lombok, overharvesting, seagrass, sea urchin, tripneustes gratilla introduction tripneustes gratilla is a ubiquitous sea urchin in tropical seagrass beds, which can occupy various shallow water habitats, from sand flats, coral reefs, and coral rubble areas to seagrass beds and macroalgal flats (lawrence & agatsuma 2007; toha et al. 2017). t. gratilla is an opportunistic herbivore, consuming its favorite seagrass, macroalgae, and microalgae species, while avoiding calcareous algae due to its tough cell wall (lawrence & agatsuma 2007; lison de loma et al. 2002). in southeast asia (the philippines, malaysia, vietnam, and indonesia), the locals harvest and consume tripneustes gratilla roe as a local delicacy (regalado et al. 2010; rahim & nurhasan 2016; hoa et al. 2019; satyawan 2014). the people in east lombok have a tradition of harvesting biota from seagrass beds, including sea urchin t. gratilla, during low tide events (satyawan 2014). overharvesting, especially in seagrass beds close to local villagers’ residences, resulted in reduced sea urchin density and small test-diameter size (nane & paramata 2020). this research aimed to record the diversity, morphometry, abundance, and habitat type of sea urchins in north lombok coastal areas and record the gleaning activity conducted by local villagers in sea urchin habitats. this research also indicated overharvesting by local villagers of north lombok. in addition, this study provided valuable baseline information for monitoring the sea urchin population in north lombok. https://doi.org/10.11598/btb.2024.31.2.1741 diversity, morphometry, and population abundance of sea urchin in north lombok 119 materials and methods study locations this study was conducted from august 2023 to july 2024 on seagrass beds in kombal bay (8°24’00.2” s; 116°04’52.6” e), pemenang subdistrict, north lombok regency, west nusa tenggara, indonesia. five additional sampling sites that represented three subdistricts were selected to obtain the general status of sea urchins in various types of habitats in the north lombok regency coastal area. the sampling sites were pandanan beach, kecinan beach, and mentigi beach in pemenang subdistrict, representing coral reefs and small seagrass meadows; sira beach and medana bay in tanjung subdistrict, representing vast seagrass meadows; and karakas beach in gangga subdistrict, representing coral reefs (fig. 1). specimen collection and species identification sampling was conducted during low tide when sea urchins and bottom substrate were exposed, allowing better observation (fairoz et al. 2018). the sea urchin was transferred to kurnaen sumadiharga science park, kombal bay, and kept within a recirculation system supplied with sandfiltered seawater. the sea urchin was kept overnight, and morphometric measurement was taken the following day following the method by changpo and kun hsiung (1981). each sea urchin was removed from the water and placed on a towel to dry. total weight was measured using an electronic balance (accuracy 0.01 g), while test diameter and test height were measured using a digital caliper (accuracy 0.1 mm). species identification was conducted based on available literature (barrett et al. 2019; colin & arneson 1995; nomleni et al. 2020; parvez et al. 2016; walag et al. 2018). after morphometric measurement and species identification, all samples of sea urchin were returned to the original seagrass habitats where they were collected. the type of animals and plants collected by local villagers for gleaning purposes was also recorded and identified based on colin and arneson (1995) and titlyanov (2017). in addition, the mean test diameter of sea urchins collected by gleaners was measured in situ. the sea urchin sample was collected using two sampling methods, i.e., belt transect and wandering transect methods. additionally, quadrat transect was used to determine seagrass percentage coverage. belt transect the belt transect method used two 50 x 1 m transects to measure sea urchin density per 100 m2 (fig. 2) (regalado et al. 2010; nane & paramata 2020). small-size quadrat transects (1 2 m) are the best sampling method to estimate sea urchin abundance (micael et al. 2021). the transects were set perpendicular to the shoreline. the number of sea urchins inside the transect was recorded and captured. figure 1 location of sea urchin sampling sites in north lombok regency biotropia vol. 32 no. 1, 2025 120 wandering transect the wandering transect method was implemented to survey a larger area (pinn et al. 2014; fairoz et al. 2018). the wandering transect was performed by walking from the shore and crossing each habitat type perpendicular to the shoreline. after reaching the edge, the observer moved 2 m to the right and started another observation walk, headed to the shoreline (fig. 2). this zig-zag wandering pattern was resumed until a certain time limit was reached. for vast and dense seagrass meadows, the wandering transect was terminated in 60 minutes, while for other habitat types, the wandering transect was completed in 30 minutes. the observation area was limited to 1 m to the left and the right of the observer. sea urchin located inside the area was recorded and captured. a 60-minute wandering transect in dense seagrass meadows was estimated to cover 1,680 m2 area, while a 30-minute wandering transect in other habitat type could cover 1,400 m2. the total area of seagrass meadows, coral reefs, and wandering transect coverage area were recorded using global positioning system (gps) and estimated by google earth engine (gee). quadrat transect determination of seagrass percentage coverage was carried out in kombal bay, sira beach and medana bay, using quadrat transect method (fig. 2) (mckenzie et al. 2003; rahmawati et al. 2017). two transect lines, each 100 m, were laid perpendicular to the seashore. the distance between each transect line was 50 m. quadrat shape frame (50 x 50 cm) was placed at the right side of the transect. the interval between each quadrat placement was 10 m. seagrass percentage coverage category was estimated according to mackenzie (2003). statistical analysis absolute abundance and relative abundance were calculated based on the following formulas: figure 2 specimen collection method notes: a) belt transect; b) wandering transect; c) quadrat transect. absolute abundance = ⅀individual number(ind.) survey area (m2) relative abundance = number of individuals of same species total number of individuals of all species x 100 diversity, morphometry, and population abundance of sea urchin in north lombok 121 morphometric parameters were subjected to statistical analysis to determine the difference in each sampling date. the nonparametric kruskalwallis test with post-hoc analysis was conducted on mean weight, mean test height, and mean test diameter datasets because normal distribution and equal variance were not obtained. statistical analyses were performed using ibm statistic spss 26. a significant difference between each morphometric parameter is recognized if p > 0.05. results and discussion a total of 5 sea urchin species were recorded from seagrass beds of kombal bay, pemenang subdistrict (table 1). t. gratilla and pseudoboletia maculata were included in family toxopneustidae, while salmacis bicolor and s. sphaeroides were included in the family temnopleuridae, and maretia planulata were included in the family maretiidae (fig. 3). t. gratilla was the most abundant species in 2023, with an absolute abundance of 0.24 0.30 ind./m2 and a relative abundance of 95.6 100%. the absolute abundance in this study is higher compared to the absolute abundance recorded in eastern lombok, indonesia (0.01 0.04 ind./ m2) and maluku, indonesia (0.008 ind./m2) (satyawan 2014; uneputty et al. 2017). however, the absolute abundance recorded in this study is lower compared to the absolute abundance table 1 absolute and relative abundance of sea urchins in kombal bay measured by belt transect and wandering transect species belt transect absolute abundance (ind./m2) aug. 2023 sep. 2023 oct. 2023 may 2024 jun. 2024 jul. 2024 t. gratilla 0.30 0.25 0.24 0.01 0.01 0.02 p. maculata 0.01 m. planulata 0.02 0.05 total sea urchin 30 26 24 1 3 7 wandering transect relative abundance (%) t. gratilla 95.6 97.9 100.0 100.0 100.0 100.0 p. maculata 2.2 2.1 s. bicolor 1.1 s. sphaeroides 1.1 total sea urchin 90 96 23 11 8 2 recorded in south-eastern sulawesi, indonesia (2.7 10.0 ind./m2) (nane & paramata 2020). the relative abundance obtained in this study is higher than that recorded in borneo, malaysia (0.34 2.51%) (rahim & nurhasan 2016). in regard to abundance, t. gratilla dominated the sea urchin assemblage in the seagrass beds of teluk kombal. seagrass beds provide microhabitats for t. gratilla to graze and hide from predators (du et al. 2020). in 2024, the absolute abundance of t. gratilla dropped to 0.01 ind./m2 (table 1). only 1 or 2 sea urchins were recorded in a 100 m2 area of seagrass beds between may 2024 and july 2024. the diversity of sea urchin species also decreased from 4 species in 2023 to only 2 species in 2024. in the absence of p. maculata, s. bicolor, and s. sphaeroides and the abrupt decrease of t. gratilla abundance, the abundance of sand dollar m. planulata increased rapidly. the inferior sand dollar now thrives on the scarcely populated seagrass meadow. in the absence of dominant sea urchin species, inferior species can occupy territories outside of their original niche (steneck 2013). mean weight, mean test diameter, and mean test height were not significantly different (p > 0.05) when sampling was conducted with the belt transect method. in contrast, sampling with the wandering transect method showed a significant difference (p < 0.05) in all morphometric parameters (table 2). biotropia vol. 32 no. 1, 2025 122 figure 3 sea urchin from seagrass beds in kombal bay, north lombok notes: a) t. gratilla; b) p. maculate; c) s. bicolor; d) s. sphaeroides; e) m. planulata; scale bar = 10 mm. table 2 morphometric parameters of t. gratilla collected using belt transect and wandering transect methods sampling method sampling date mean weight (g) mean test diameter (mm) mean test height (mm) total sea urchin belt transect august 2023 37.59 ± 2.63a 44.44 ± 1.21a 26.53 ± 0.85a 30 september 2023 39.09 ± 3.94a 44.09 ± 1.71a 25.82 ± 1.06a 25 october 2023 44.16 ± 2.31a 47.42 ± 0.83a 26.97 ± 0.51a 24 may 2024 48.45 48.50 30.30 1 june 2024 9.62 25.80 15.70 1 july 2024 17.39 ± 7.05 33.5 ± 4.70 19.7 ± 2.00 2 wandering transect august 2023 34.14 ± 1.93x 42.76 ± 1.01y 24.57 ± 0.58y 86 september 2023 28.50 ± 1.46x 39.99 ± 0.68x 22.83 ± 0.39x 94 october 2023 49.60 ± 2.75y 48.58 ± 0.94z 27.52 ± 0.64z 23 may 2024 69.87 ± 9.66 53.28 ± 2.98 32.22 ± 1.89 11 june 2024 72.07 ± 12.11 54.53 ± 4.38 32.88 ± 2.84 8 july 2024 110.74 ± 9.58 64.35 ± 3.05 37.20 ± 1.00 2 each survey method has its advantages and drawbacks. the belt transect method provided absolute abundance data but was time-consuming, requiring transects and collecting fewer samples. wandering transects collected more samples, surveyed vast areas in less time, and required no transects but needed more absolute abundance data and may have overlooked smaller sea urchins. from august to october 2023, the mean weight, mean test diameter, and mean test height increased significantly, from 34.14 to 49.60 g, from 42.76 to 48.58 mm, and from 24.57 to 27.52 mm, respectively. however, the total sea urchins collected in october 2023 was nearly three times smaller compared to the august 2023 data. surveys conducted in may, june, and july 2024 recorded sea urchins with bigger mean weight, mean test diameter, and mean test height compared to the surveys conducted in august, september, and october 2023. however, the total number of t. gratilla in each survey conducted in 2024 was extremely low compared to the previous year. the considerable weight and size differences can be attributed to seasonal variation (juinio-menez et al. 2008; satyawan 2014) or intraspecific competition (narvaez et al. 2020). the location of sea urchins in seagrass meadows is also a contributing factor. compared to the more accessible and highly exploited area, invertebrates, including t. gratilla, were more prevalent in the inaccessible or remote seagrass meadows (nordlund 2010). based on personal observation, seagrass meadows fringes areas were often overlooked by local villagers, and the majority of sea urchin was found in the area. morphometric data are an essential tool in assessing sea urchin population health and sustainability. food availability (smith & garcia 2020), grazing zones, and domestic pollution (caill-milly et al. 2020) affected sea urchin morphometrics. likewise, sea urchin morphometric data and abundance may be indicative of overharvesting (tamti et al. 2021) or underexploited (guinda et al. 2016). the shift of relative abundance in the test diameter class highlighted the decrease of weight and size. in august 2023, the 45.0 49.9 mm test diameter class had the highest relative abundance compared to other classes (fig. 4), but in september 2023, the highest relative abundance shifted to the 35.0 39.9 mm test diameter class, two size classes diversity, morphometry, and population abundance of sea urchin in north lombok 123 smaller. local villagers avoided small sea urchins because of the limited amount of roe. the mean test diameter size harvested by local people in september 2023 was 52.08 ± 1.48 mm, while the mean test diameter size ranged from 48.2 55.9 mm. there were reductions in the frequency of size classes 45.0-49.9, 50.0-54.9, and 55.0-59.9 by 40.7%, 56.2%, and 100%, respectively (fig. 4). the size classes were within the range of sea urchin size that local villagers catch (personal observation; satyawan 2012). the bigger diameter class experienced a greater reduction in frequency. during two sampling days, at least 2 local people harvested t. gratilla. each person collected 100 110 sea urchins, mostly t. gratilla. two size classes that contained more subjects in september 2023 disappeared in 2024. only two size classes, each containing only one subject, were recorded in july 2024. the disappearance could be due to death of age or ecological factors. in the wild, t. gratilla life expectancy was estimated to be between one and two years (tanita et al. 2023). overfishing resulted in the absence of larger size classes in sulawesi (nane & paramata 2020). in east lombok, t gratilla reached sexual maturation after 1.2 years or after reaching test diameter size of 58.2 mm (satyawan 2012). there were only 2 sea urchins that reached the sexual maturation size in 2024. harvesting sea urchins from the wild population usually target subjects with larger test sizes due to higher roe yield. harvesting activity resulted in the change of size-frequency distribution and the total number of subjects compared to the undisturbed population (bertocci et al. 2014). additional surveys were conducted from may 2024 to july 2024 in kombal bay and other selected locations to compare the t. gratilla population status in north lombok and to observe gleaning activities conducted by local villagers. in general, t. gratilla abundance in each location was extremely low compared to that in maluku (tuapattinaja et al. 2014) and in the philippines (juinio-menez et al. 2008). in north lombok, the highest t. gratilla abundance was recorded in the dense seagrass meadow of sira beach, tanjung, and in the coral reefs of karakas beach, gangga, while the lowest abundance was recorded in kecinan beach, mentigi beach, and medana bay (table 3). figure 4 frequency of each test diameter class collected by wandering transect biotropia vol. 32 no. 1, 2025 124 table 3 sea urchin (t. gratilla) abundance in north lombok subdistrict site name total area (m2) wandering transect area (m2) total subject number substrate type pemenang pandanan beach 15,143 1,400 1 coral, macroalgae, sandy kecinan beach 11,573 1,400 0 coral rubble, sandy, seagrass, macroalgae mentigi beach 7,490 1,400 0 coral rubble, sandy, seagrass, macroalgae, kombal bay 40,610 1,680 2 seagrass, macroalgae, coral rubble, muddy tanjung sira beach 50,380 1,680 7 seagrass, macroalgae, coral rubble, muddy medana bay 56,806 1,680 0 seagrass, macroalgae, coral rubble, muddy gangga karakas beach 10,609 1,400 6 coral, dead coral algae, macroalgae, sandy the total area, habitat type, and substrate type of kombal bay, sira beach, and medana bay were roughly similar. kombal bay had the lowest seagrass cover (47.0%) compared to that in medana bay (75.0%) and sira beach (80.9%) based on quadrat transect. the low abundance of sea urchins in sira beach, kombal bay, and medana bay was concerning. in pacitan beach, east java, t. gratilla was ubiquitous in seagrass meadows with an absolute abundance of 0.07 ind/ m2, despite moderate seagrass percentage coverage of 31.44% (muzaki 2019). gleaning is a common activity conducted by local villagers during the low tide period, especially in the villages close to seagrass meadows and shallow coral reefs. the duration of gleaning activity was affected by several factors. gleaning activity usually lasted 1 2 hours and finished after the exposed seagrass meadows were inundated or before sunset (personal observation). in this study, we observed that medana bay was the most favorite gleaning location, followed by kombal bay and sira beach. most of the gleaner was adult female, except in kombal bay and kecinan beach. the majority of gleaners collected gastropods, while adult females also collected gracilaria sp. as a secondary catch (table 4). the main purpose for gleaning was collecting food material for home consumption. table 4 gleaner composition and type of animals or plants collected by gleaners subdistrict site name number of adult female as gleaner number of adult male as gleaner number of young child as gleaner total taxa pemenang pandanan beach 9 5 4 18 gastropods, bivalves, florideophyceae, echinoidea* kecinan beach 0 0 3 3 gastropods mentigi beach 0 0 0 0 kombal bay 10 7 14 31 gastropods, florideophyceae tanjung sira beach 7 3 1 11 gastropods, echinoidea** medana bay 38 19 20 77 gastropods, florideophyceae, echinoidea*** gangga karakas beach 7 4 1 12 gastropods notes: * = 5 gleaners collected t. gratilla as a secondary catch; ** = 2 gleaners collected t. gratilla as primary catch with a total catch of 91; *** = 4 gleaners collected t. gratilla as primary catch with a total catch of 116. diversity, morphometry, and population abundance of sea urchin in north lombok 125 figure 5 collection of sea urchin roe by gleaners notes: a) collected t. gratilla inside a plastic bag; b) plastic bottle containing sea urchin roe; c) remains of tested sea urchin scattered in seagrass meadow. economic issues could be one of the reasons that local villagers conducted gleaning. as much as 27% of north lombok people were categorized as poor and earned a monthly income of idr 478,906 (citanl 2022). gleaning can have negative impacts on seagrass meadows. seagrass meadows are preferred by gleaners due to higher catch rates compared to the coral reefs, mangrove, and tidal flats. (aldea 2023). however, a high number of gleaners and an increased human population can cause overharvesting or a decrease in the abundance of targeted invertebrates (nordlund et al. 2010; stiepani et al. 2023). from 7 locations, we found 11 gleaners that collected sea urchins as either a primary or secondary catch. in pandanan beach, 5 adult females collected t. gratilla as a secondary catch with each person collected an average of 5 sea urchins. from interviews (n = 2), the gleaners collected t. gratilla to make vegetable side dishes (fig. 5). in sira beach, a married couple collected 91 sea urchins, while in medana bay, a team of two married couples collected 116 sea urchins during one gleaning activity. the husbands used snorkeling equipment to search and collect sea urchins from the reef crest and fore reef zone. the wives received the catch, cracked open sea urchins in half, and retrieved the roe. sea urchin roe was placed in plastic bottle, and depending on the catch, can either be used for home consumption or be sold in local market (fig. 5). a 500-ml-plastic bottle filled with sea urchin roe was offered between idr 20,000 to idr 30,000. in sira beach, the catch consisted solely of t. gratilla, while in medana bay, it mainly consisted of t. gratilla with several toxopneustes pileolus and pseudoboletia maculata. in 2023, we recorded 2 gleaners in kombal bay who collected t. gratilla as their primary catch. however, in 2024, in the same location, we hardly encountered gleaners that collected sea urchins. interviews with gleaners revealed that the number of sea urchins was too small to be collected and used for consumption. in gangga subdistrict, local villagers only collected gastropods and avoided sea urchins. based on interviews with local villagers of karakas beach, they have no tradition of using sea urchins as food materials. local villagers also revealed that only outsiders from the pemenang subdistrict and tanjung subdistrict collected sea urchins during low tide. the majority of gleaners conducted gleaning activity every day, approximately 2 to 3 days, during spring low tide or when seagrass meadows were completely exposed (personal observation). apparently, harvesting sea urchins was affected by local tradition and local wisdom. gleaning, especially carried out by adult females or housewives, can support families by providing protein and food security. most of the time, gleaning requires little to no equipment (pike et al. 2024) and can be done by young child (furkon et al. 2020). however, gleaning, for trading, without biotropia vol. 32 no. 1, 2025 126 supervision and regulation resulted in the decline of target species (nordlund et al. 2010; satyawan 2014), and habitat destruction by trample (furkon et al. 2020). the continued increase in the number of gleaners further exacerbated the problem (nordlund et al. 2010). interviews with gleaners, especially older villagers, revealed the decline of catch and disappearance of certain target species (nordlund et al. 2010). interviews with gleaners in kombal bay (n = 5) also revealed similar conditions. several management methods, such as mariculture and restocking (juinio-menez et al. 2008), have proven to be successful in resolving the problem that arises with sea urchin t. gratilla gleaning and overharvesting. furthermore, identification of spawning sites, establishing minimum catch size, and catch prohibition at certain sites had resulted in higher population density. mariculture in sea cages, rather than larval release restocking, was cost-effective and feasible for stock enhancement in developing countries (juinio-menez et al. 2008). regulation of minimum harvest size, marine sanctuary enforcement, and grow-out of hatchery produced juvenile or wild population in sea cages has proven to be successful in restoring and maintaining sea urchin population in the philippines (juinio-menez et al. 2008). temporal harvest prohibition during the spawning period could also be established to guarantee natural spawning and breeding (guinda et al. 2016). overharvesting, excluding grazers, such as sea urchins, can increase macroalgal cover and alteration of the macroalgal community (kriegisch et al. 2020) or habitat damage due to trampling of marine biota living in seagrass meadows (nordlund et al. 2010). due to its ecological role, the decrease or increase of sea urchin biomass can affect the balance of trophic levels in the seagrass ecosystem (clores 2023), change community structure (steneck 2013), and cause overgrazing of seagrass beds (moreira-saporiti et al. 2023). conclusion the low absolute abundance of t. gratilla and the decline of sea urchin species diversity in north lombok prospectively indicated overharvesting. the number of gleaners and the gleaning activity conducted by local villagers prospectively harm seagrass meadows, especially on t. gratilla abundance. a study on diversity, abundance, and morphometry can provide a base for 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, katja rembold , sri s. tjitrosoedirdjo and 3 soekisman tjitrosoedirdjo 1 post graduate student of plant biology, department of biology, faculty of mathematics and sciences, institut pertanian bogor, bogor 16680, indonesia 2 department of biology, faculty of mathematics and sciences, institut pertanian bogor, bogor 16680, indonesia 3seameo biotrop, bogor 16134, indonesia 4 bioclimatology, university of göttingen, büsgenweg 2, 37077 göttingen, germany 5graduate school of life sciences, tohoku university, aoba 6-3, aramaki, aoba-ku, sendai 980-8578, japan 6biodiversity, macroecology and conservation biogeography, university of göttingen, büsgenweg 1, 37077 göttingen, germany received: 9 september 2015/accepted: 29 july 2016 abstract disturbances caused by the conversion of rain forests into agricultural systems provide an opportunity for the expansion of invasive plant species (ips). bukit duabelas national park is one of the few remaining lowland forests in jambi province (sumatera, indonesia). the surrounding areas up to the national park borders have already been converted into jungle rubber agroforests as well as rubber and oil palm plantations which might lead to an increased spread of ips into the forest. this study was aimed at compiling a list of ips and determining their distribution and coverage of ips in four land use systems (rain forest, jungle rubber, rubber and oil palm plantations). spatial distribution patterns were investigated by creating a horizontal vegetation profile diagram for the permanent plots of the efforts project (ecological and socioeconomic functions of tropical lowland rainforest transformation systems, http://www.uni-goettingen.de/crc990). the dominance of ips was determined using important value index. a total of forty ips were identified across the four land-use systems. the numbers of ips were the highest in oil palm (28 species) and rubber plantations (27 species), and the lowest in jungle rubber (10 species). ips were absent in the lowland rain forest. the diversity of ips was influenced by environmental factors, especially canopy openness. ips with the highest ground coverage were dicranopteris linearis and clidemia hirta. both of them were found in all three land-use systems outside the rain forest when the forest canopy opens due to illegal logging or other human disturbances. therefore, reforestation of disturbed areas is recommended to prevent the spread of ips. keywords: invasive plant species (ips), bukit duabelas national park, clidemia hirta, dicranopteris linearis introduction sumatera is the second largest island in indonesia and was once covered with forest (wwf 2010). nowadays, the forests of sumatera have been largely replaced by three major tree monocultures i.e. oil palms (elaeis guineensis), rubber (hevea brasiliensis) and acacia mangium (wwf 2010). bukit duabelas national park (bdnp) is one of the few remaining forests in the lowlands of jambi province in sumatera under protection. illegal logging and the conversion of the surrounding areas into jungle rubber, rubber and oil palm plantations might lead to an increase in invasive plant species (ips) in the forest. disturbances in the ecosystem such as plantation development provide an opportunity for the biotropia 3 2 6 124 132 vol. 2 no. , 201 : doi: 10.11598/btb.2016.2 . .3 2 534 * corresponding author: indah@biotrop.org 124 expansion of invasive alien plants species ( r a g h u b a n s h i & t r i p a t h i 2 0 0 9 ) . invasive plants are generally defined as plant species that are non-native to an ecosystem, and which may cause economic or environmental harm or adversely affect human health (cbd 2000). invasive plants respond readily to humaninduced changes of the environment, but may also cause environmental changes and economic damage through their dominance of the landscape (pimentel 2011). in general, species distributions are determined by environmental conditions, biotic interactions, evolutionary change and dispersal ability. the success of invasive plants is assumed to be affected by several characteristics including: 1. high dispersal rate; 2. high fecundity; 3. high growth rate; 4. capability of vegetative reproduction; and 5. a high tolerance to various abiotic conditions including temperature, humidity and soil type (velde et al. 2006). anthropogenic disturbance facilitates the increases of invasive plant species richness (gassó et al. 2009). some invasive plants have a greater ability than others to colonize disturbed habitats (freeman et al. 2015) and it is important to identify the most dominant ips representing the biggest threat to invade natural ecosystems. ips respond positively to natural or anthropogenic environmental disturbance. natural disturbance and land use intensity facilitate the introduction of alien plant species in an area (uddin et al. 2013). light availability and exposed soil facilitate the establishment of ips. the objectives of this study were to examine the diversity, distribution and coverage of ips in four land-use systems (forest, jungle rubber, rubber plantation and oil palm plantation), to investigate the most dominant species and the environmental factors influencing ips distribution. materials and methods study site the study was carried out in bukit duabelas national park (bdnp) and in surrounding villages in jambi province (sumatera, indonesia). bdnp covers 60,500 hectares and represents one of the few remaining lowland rainforests in jambi province with formal protection. the topography ranges from 50 to 438 m above sea level (asl). this forest is inhabited by the nomadic tribe “suku anak dalam” (orang rimba). traditional activities of the orang rimba include shifting cultivation, hunting, fishing and honey collection. the surrounding areas outside bdnp are covered by agricultural systems, namely jungle rubber (rubber agroforestry), rubber plantation and oil palm plantation. bo2 bf3 bf4 bj4 bo4 br4 br3 bj5 figure 1 study site in jambi province (sumatera, indonesia). the present study was carried out on the following plots: rain forest: bf3 & bf4; jungle rubber: bj4 & bj5; rubber plantation: br3 & br4; oil palm plantation: bo2 & bo4. (the map was created by mohd. zuhdi, department of soil science of universitas jambi, indonesia). 125 – distribution of invasive plant species in different land-use systems wahyuni et al. the research was conducted in four different land-use systems: lowland rainforest (f), jungle rubber agroforest (j), rubber plantation (r) and oil palm plantation (o). forest plots were located inside bdnp and the other land-use systems in three surrounding villages, i.e. dusun baru, lubuk kepayang and pauh. vegetation surveys were carried out within the permanent plots (50 × 50 m) of the efforts project (ecological and socioeconomic functions of tropical lowland r a i n f o r e s t tr a n s f o r m a t i o n s y s t e m s, http://www.uni-goettingen.de/crc990). two replicate plots were selected for each land-use system resulting in a total of eight plots (fig. 1). the abundance and presence of ips horizontal profile diagrams for all invasive plants were created by projecting their coverage 2 onto the forest floor. each 50 x 50 m plot was 2 divided into 25 subplots (10 × 10 m ) to simplify the calculation and delineation of invasive plant coverage. the coverage was drawn on graph paper with a scale of 1 : 100 in the field, and the sketches were then scanned and digitized in arcview 3.3. the dominance of ips was determined using important value index (ivi), based on the frequency and coverage of invasive plants. to calculate the ivi, the percentage values of the relative frequency and relative dominance were summed and calculated with the following formulas (cox 1972). environmental data air temperature and air humidity were measured using thermohygrometers (galltec mela, germany) installed in a meteorological station located in the center of each plot at 2 m above ground. data were measured hourly and recorded in a data logger (logtrans16-gprs, uit, germany). the same station also measured soil temperature and soil moisture at 0.3 m depth (trime-pico 32, imko, germany). for this analysis, the average of all data recorded for 16 months from june 2013 were used. the canopy cover was calculated from hemispherical photographs taken at 1.2 m above the ground from 32 positions within each plot (canon eos 700d slr camera with a sigma 4.5 mm f2.8 ex dc circular fisheye lens). the photographs were taken in early morning (5:00 7.00 am), late afternoon (5:00 7:00 pm), evenly overcast days to avoid direct sunlight entering the lens, as described in drescher et al. (2016). to obtain nonoverexposed, high contrast photographs, exposure was determined following the histogram-exposure protocol after beckschäfer et al. (2013). the photographs were processed with the software “imagej” (rasband 2014). data analysis cluster analysis was carried out to compare the ips community within the ecosystem. the cluster analysis was conducted based on ivi and calculated into similarity index, which was then converted into dissimilarity index with single linkage clustering. the formulas are as follows (mcgarigal et al. 2000): where: is = similarity index; a = total ivi of ips in ecosystem a; b = total ivi of ips in ecosystem b; c = the comparison of total ivi of ips in ecosystem a and b; d = dissimilarity index. one-way anova with tukey-test were used to identify significant differences in the number and coverage of ips as well as the differences of environmental data among the ecosystems. principal component analysis (pca) was conducted to observe the relationships between (1) (2) ivi = relative frequence + relative dominance relative frequence relative dominance number of sample plots where a certain species was distributed number of total sample plots x 100 % (3) sum total of a certain species in the total sample plots sum total cover of all species inthe total sample plots x 100 % = = 126 biotropia vol. 23 no. 2, 2016 environmental factors and the number and coverage of ips. one way anova with tukeytest and pca were performed using xslstat 2014 software (a microsoft excel add-in). results and discussion diversity of ips within the different landuse systems a total of forty ips were identified in the four land-use systems. oil palm plantations had the highest richness of ips (28 species), closely followed by rubber plantations (27 species). in jungle rubber agroforests, the number of ips was much lower than in the monocultures (10 species), and ips were absent in rain forest (table 1 and 2). cluster analysis separated the ips community into three distinct groups (fig. 2), but oil palm and rubber plantations had the most similar ips communities. oil palm and rubber plantations were characterized by a similarly intensive management resulting in comparatively high numbers and compositions of ips. principal component analysis results (pca) showed that ips coverage was higher in plots with high canopy openness (fig. 3). the highest table 1 diversity of families, genera and species of invasive plants in four land-use systems ecosystem type number of family number of genera number of species forest 0 0 0 jungle rubber 6 10 10 rubber plantation 13 24 27 oil palm plantation 13 27 28 2table 2 average species numbers and total coverage (%) of ips per plot (50 × 50 m ) in the four land-use systems data bf (forest) bj (jungle rubber) br (rubber plantation) bo (oil palm plantation) average of ips number per plots (50 × 50 m) 0.00±0.00 8.00±2.83 19.50±0.71 21.00±9.52 average of ips total cover (%) per plots (50 × 50 m) 0.00±0.00 43.04±12.54 25.10±30.04 71.80±14.29 environmental data natural forest jungle rubber rubber plantation oil palm plantation air temperature 24.47±0.44 25.05±0.38 25.58±0.36 25.44±0.72 humidity 91.87±1.61 87.61±2.07 82.58±2.00 83.76±2.42 soil moisture 25.00±2.40 30.39±2.08 43.54±5.52 35.39±4.73 soil temperature 25.18±0.28 25.34±2.09 25.33±1.04 26.35±0.93 canopy openness 2.14±1.13 5.40±3.12 15.22±6.90 18.70±9.43 table 3 environmental data of the four land-use systems (forest, jungle rubber, rubber and oil palm plantations) notes: air temperature, humidity, moisture and soil temperature data are means of 10 replications±standard deviation. data of canopy openness are means of 60 replications±standard deviation bo br bj bf 0.52 0.45 1.00 0.50 figure 2 ips community differences within ecosystems in bukit duabelas national park separated by cluster analysis based on ivi values 127 – distribution of invasive plant species in different land-use systems wahyuni et al. coverage of ips was found in oil palm plantation which also had the highest canopy openness (18.70%; table 3). based on yaap et al. (2010), oil palm plantation, structurally, was less complex than natural forest, with a uniform tree age structure, lower canopy, less stable microclimate and intensively human disturbance. dominant species in plantation are typically invasive species and pest (yaap et al. 2010). most of the ips are shade intolerant. fine (2002) reviewed that the number of invasive plant species was positively related to disturbance which increased light levels. additionally, the number and coverage of ips correlated with air temperature (fig. 3); higher numbers of ips were found where air temperature was high. air temperature and light influence many plant processes. the interaction of this abiotic factor could influence growth rate, flowering period, seed dormancy and characteristic of plant morphology (booth et al. 2010). in this study, air temperature and light are a strongly regulatory force for ips distribution. some invasive species are more successful in disturbed habitat because they are able to take advantage of the high light levels. besides light intensity, ibàñez et al. (2009) revealed that relatively warmer areas correlated with invasive plants occurrence. ips were not found in the rain forest of bukit duabelas national park. their absence might be due to high canopy cover in the forest, leading to low light penetration as well as cooler and more humid conditions in the understory. these conditions might not support ips growth. a more open canopy causes higher soil evaporation and increases in air temperature (lambers et al. 2008) which may support ips growth. canopy openness and air temperature were lower in the forest than in the other land-use systems (table 3). junaedi and dodo (2014) revealed that most ips could not reach the forest interior where the canopy cover was still relatively intact. ips prefer forest edges or forest gaps and are more successfully in infesting open and disturbed areas with high light levels where the native species are not as competitive. disturbance, therefore, creates habitats that are more suitable for ips than for native species. this shift from native to invasive species could influence the ecosystem balances. thus, abiotic factors seem to be more important for the successful plant invasion than biotic factors (booth et al. 2010; peters 2001). lower propagule pressure might be additional factor in the forest compared to the three other land-use systems. however, peters (2001) showed an interesting interplay between abiotic conditions, soil disturbance and wild pig activity affecting the spread of c. hirta in a forest reserve in malaysia. figure 3 principal component analysis (pca) on relationship between the number and coverage of ips to environmental factors: air temperature (at), air humidity (ah), soil temperature (st), soil moisture (sm) and canopy openness (co) 128 biotropia vol. 23 no. 2, 2016 species composition of ips differed between the four land-use systems (table 4). more than 60% of the ips in jungle rubber did not occur in rubber and oil palm plantations, while 30% of the ips in rubber plantation were not found in oil palm plantation. agroforestry systems such as jungle rubber are characterized by a relatively high diversity of native tree species and high canopy cover and this may cause lower numbers of ipss. in contrast to jungle rubber, the tree crops in rubber and oil palm plantations are planted in regular distances of several meters to each other and the space in-between is weeded regularly. this condition appears to be most suitable for ips and may also explain the higher similarity of ipscommunities in the two monoculture systems. figure 4 distribution pattern of ips at oil palm plantation plot (bo2) figure 5 distribution pattern of ips at jungle rubber plot (bj5) figure 6 distribution pattern of ips at rubber plantation plot (br4) 129 – distribution of invasive plant species in different land-use systems wahyuni et al. 18 lantana camara 0 0 0 2.95 19 ageratum conyzoides 0 0 2.63 2.75 20 mussaenda frondosa 0 0 2.64 2.73 21 urena lobata 0 0 2.65 2.58 22 uncaria cordata* 0 0 2.7 2.56 23 bridelia insulana 0 0 2.65 2.48 24 pennisetum polistachyon 0 0 0 2.48 25 sporobolus diander* 0 0 0 2.46 26 polygala paniculata 0 0 0 2.45 27 stenochlaena palustris* 0 0 7.78 2.45 28 borreria laevis 0 0 2.94 0 29 chromolaena odorata 0 2.45 0 0 30 cyperus difformis 0 0 2.64 0 31 cyrtococcum acrescens 0 0 2.66 0 32 cyrtococcum patens 0 0 7.47 0 33 cyrtococcum trigonum 0 9.94 2.65 0 34 dianella ensiflora 0 0 2.65 0 35 fimbristylis dura 0 0 2.64 0 36 macaranga triloba 0 0 2.78 0 37 oplismenus compositus 0 6.57 0 0 38 paspalum conjugatum 0 0 2.67 0 39 tetracera scandens* 0 0 2.77 0 40 tetracera indica* 0 14.05 0 0 total 0 147.85 133.08 171.4 no. species ivi (%) bf bj br bo 1 clidemia hirta 0 34.23 7.3 45.76 2 asystasia gangetica 0 0 2.64 11.76 3 dicranopteris linearis* 0 28.84 17.45 11.25 4 centhoteca lappacea 0 6.81 5.41 8.05 5 axonopus compressus 0 0 5.53 7.83 6 scleria ciliaris* 0 13.18 8.23 7.35 7 melastoma malabathricum 0 12.62 5.47 6.7 8 ottochloa nodosa 0 0 0 6.69 9 paspalum dilatatum 0 0 5.51 5.81 10 taenitis blechnoides* 0 12.79 5.41 5.74 11 lygodium flexuosum* 0 6.38 0 5.46 12 stachytarpheta jamaicensis 0 0 0 5.46 13 breynia stipitata 0 0 5.36 4.94 14 imperata cylindrica* 0 0 6.46 3.44 15 borreria alata 0 0 5.38 3.16 16 mikania micrantha 0 0 0 3.11 17 stachytarpheta indica 0 0 0 3 spatial distribution patterns of ips within the land-use systems the horizontal profile diagram provided information on current distribution of ips. the highest coverage of ips were in oil palm plantation (74%), followed by jungle rubber (45%) and rubber plantations (30%) (fig. 4, 5 and 6, respectively). the invasive plants in jungle rubber were evenly distributed within the plots (fig. 5). high coverage of invasive plants in oil table 4 important value index (ivi) of ips in four land-use systems in bukit duabelas national park. the species with the highest ivi are highlighted in bold; the native species were indicated by asterisk (*) 130 biotropia vol. 23 no. 2, 2016 palm plantations is due to the relatively open canopy compared to other land-use systems. however, the invasive plant coverage in rubber plantation was lower than that in jungle rubber, whereas the canopy in jungle rubber was more closed than that in rubber plantations. the species dominance was analyzed based on the important value index (ivi) (table 4). clidemia hirta was the most dominant species in jungle rubber followed by the native invasive species dicranopteris linearis and tetracera indica. c. hirta was also the most dominant species in oil palm plantation followed by asystasia gangetica and d. linearis. in rubber plantations, the most dominant species was d. linearis, followed by scleria ciliaris and stenochlaena palustris. based on the ivi values, the most important invasive species were d. linearis and c. hirta and this was also confirmed by the horizontal profile diagrams (fig. 4, 5 and 6). both species were found in all three agricultural land-use systems i.e. jungle rubber, rubber and oil palm plantations. the distribution pattern of c. hirta is spread generally random and in small colonies. the preferred habitat of c. hirta is humid tropical lowland (dawson 2008). in some cases, c. hirta has been introduced intentionally into botanical gardens, such as peradeniya (sri lanka) in 1894, amani (tanzania) in 1930 and wahiawa (hawaii) in 1941 (dawson 2008). c. hirta is dispersed well due to its edible fruits being eaten by birds and other animals and its large numbers of seeds (more than 100 seeds/fruit). in addition, the seeds are able to stay dormant for 4 years in the soil (dawson 2008). in its native range in south america, this species tends to grow in open areas (gerlach 2006). our plots were dominated by c. hirta up to the heavily shaded areas in the center of the jungle rubber plots (fig. 5). dicranopteris linearis is a native species and became weed because of deforestation and forest conversion into agricultural systems. in open canopy areas, the distribution pattern of d. linearis is clumped in a huge colony. however, d. linearis also occurred in jungle rubber where the canopy coverage was relatively high compared to that in rubber and oil palm plantations, d. linearis mainly grow in lighter conditions along the plot borders and in canopy gaps (fig. 5). d. linearis is also abundant along roadsides and along the trail leading to the forest. conclusions there were strong differences in species numbers and community composition of ips in the four land-use systems studied. canopy cover and associated changes in abiotic conditions were probably the main factor influencing ips distribution. ips infestation was higher in open areas such as oil palm and rubber plantations than that in areas with less light i.e. jungle rubber and rain forest. canopy cover was the highest in natural forest where ips were completely absent. d. linearis and c. hirta were found to be the most widely distributed ips. some activities that facilitated disturbances, i.e. land-use change, illegal logging and forest fire should be prevented. immediate action of reforestation of disturbed areas in the national park should be applied. the ips which were established in the plantations should also be prevented from spreading into the national park. it is necessary to prohibit visitors entering the national park from the fully ips invaded pathway from the plantations. immediate action needed to destroy ips infestation to bdnp. acknowledgements the authors are grateful to the ministry of research, technology and higher education (ristekdikti) for research permission in indonesia. sincere thanks also due to the staff of bukit duabelas national park, the landowners of our research plots and universitas jambi for the facilities and working permit issuance for bukit duabelas national park. thanks to saiful bachri, defra nurdiansyah and anton radiansyah for their assistance with data collection. this study was financed by the efforts project through abs fund 2014 provided by the german research foundation (dfg) in the framework of the collaborative research centre 990 (http://www.uni-goettingen.de/crc990). references beckschäfer p, seidel d, kleinn c, xu j. 2013. on the exposure of hemipherical photographs in forests. iforest-biogeosciences and forestry 6:228-37. 131 – distribution of invasive plant species in different land-use systems wahyuni et al. booth bd, murphy sd, swanton cj. 2010. invasive plant ndecology in natural and agricultural systems. 2 ed. cambridge (uk): cambridge university press. 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[wwf] world wildlife fund. 2010. sumatra's forests, their wildlife and the climate, windows in time: 1985, 1990, 2000 and 2009. jakarta: wwfindonesia. 132 biotropia vol. 23 no. 2, 2016 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 812-fiks.cdr m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 m 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 180 kd 130 kd 100 kd 75 kd 48 kd 35 kd 28 kd 17 kd 10 kd 180 kd 130 kd 100 kd 75 kd 35 kd 63 kd 48 kd 28 kd 17 kd l 1 2 3 4 5 6 7 8 9 10 11 m 12 13 14 15 16 17 18 19 20 21 22 m 23 24 25 26 27 28 29 30 31 32 33 m 34 35 36 37 38 39 40 41 42 180 kd 180 kd 180 kd 180 kd 130 kd 100 kd 75 kd 63 kd 48 kd 35 kd 28 kd 130 kd 130 kd 130 kd 100 kd 100 kd 100 kd 75 kd 75 kd 75 kd 35 kd 35 kd 35 kd 63 kd 63 kd 63 kd 48 kd 48 kd 48 kd 28 kd 28 kd 28 kd 17 kd 17 kd 17 kd 10 kd 10 kd 10 kd m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 m 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 100 kd 75 kd 48 kd 28 kd 17 kd 180 kd 130 kd 100 kd 75 kd 63 kd 48 kd 35 kd 28 kd 17 kd m 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 m 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 41 42 97 kd 66 kd 45 kd 31 kd 14 kd 97 kb 66 kb 48 kb 31 kb page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 page 14 page 15 page 16 page 17 the southeast asian journal of tropical biology vol. 32 no. 2, 2025: 151 161 doi: 10.11598/btb.2025.32.2.2368 issn: 0215-6334 | e-issn: 1907-770x 151 invasive alien plant species in the riparian areas of northern samar, philippines joey liza l. abobo*, joy a. esmabe, marjurie l. baldoza, princess l. cerbito, john mark p. garbo, ramon philip l. bacayo, chitlyn c. tenedero, maricar d. lameda, rica joy b. saavedra, cresil s. turbanada, and roanne b. romeroso department of biology, college of science, university of eastern philippines, catarman, northern samar 6400, philippines article higlights • a pioneering study of invasive alien plant species in the riparian areas of northern samar • species composition, endemism, and conservation status of introduced species • out of 65 introduced species, 32 were listed as invasive article information received : 29 september 2024 revised : 8 april 2025 accepted : 29 april 2025 *corresponding author, e-mail: joeyaceaeliz@gmail.com research paper abstract riparian areas are highly dynamic and naturally disturbed, making them particularly susceptible to invasion. invasive alien plant species (iaps) are widely distributed in these areas due to the favorable environmental conditions that support their growth and persistence. this study is a pioneering effort to document the invasive alien plant species thriving in the riparian zones of allen, biri, catarman, gamay, mapanas, laoang, lapinig, las navas, and pambujan in northern samar. sampling was conducted from march to december 2023. six transect lines, each measuring two kilometers per site, were established in the riparian areas to identify existing invasive alien plants. in total, 60 transect lines were set up across all sampling areas. as a result, 65 introduced plant species belonging to 24 families and 56 genera were documented in the riparian areas of northern samar. the families with the highest number of species were poaceae (13 spp.), asteraceae (12 spp.), cyperaceae, fabaceae, and lamiaceae (5 spp. each). the remaining families had fewer than five species each. the most common invasive plant species recorded was psidium guajava l., which was present in all sampling areas. based on life forms, the study identified two tree species, 17 shrubs, three vines, one epiphyte, and 42 herbs. conservation status was determined using the latest iucn and denr listings (2017–11). one taxon was classified as data deficient, 32 as least concerned, and the remaining species as not evaluated. in contrast, no taxa were assessed in the denr 2017–11 listing. furthermore, of the 65 introduced plant species documented, 10 were recorded as invasive in the global invasive species database, while 22 were listed in the global register of introduced and invasive species database. although invasive alien species have long been recognized as posing serious threats to human health, forestry, agriculture, fisheries, and natural biodiversity, no comprehensive assessment of their status, trends, causes, impacts, management, and governance issues has been conducted worldwide. thus, this study aimed to supplement existing data on invasive plants and assist policymakers in developing policy frameworks for managing invasive alien plant species to mitigate their impacts on human health and the environment. keywords: invasive alien plants, northern samar, riparian areas, biodiversity assessment https://doi.org/10.11598/btb.2025.32.2.2368 biotropia vol. 32 no. 2, 2025 152 introduction invasive alien species (ias) are non-native organisms that pose risks to both human health and the environment. they are regarded as the second most significant threat to biodiversity after habitat loss (bellard et al. 2016). whether introduced intentionally or unintentionally, the introduction, dispersal, and utilization of alien species have had profound impacts on ecosystems and economies. because many invasive alien species are highly adaptable, capable of rapid growth and reproduction, the environmental damage they cause is often irreversible and can lead to the extinction of endemic species. furthermore, biological invasions have severe socio-economic and public health impacts (shackleton et al. 2018). according to castro-díez et al. (2019), the perceived socio-economic benefits of certain potentially invasive plant species in urban and rural areas have contributed to the rapid spread of some ias in forest ecosystems. numerous studies have been conducted worldwide to examine the effects of invasive alien species on human health, ecosystem services, biodiversity, and economic activities (katsanevakis et al. 2014). however, research on the impacts of ias in the philippines remains scarce. owing to its archipelagic nature, which promotes geographic isolation and endemism (ptfcfi 2015), the philippines is recognized as one of the world’s 17 mega-diverse countries (keong 2015). yet, island ecosystems are particularly vulnerable to invasive alien species, which can cause population declines and extinctions, especially among endemic taxa. samar island natural park (sinp), located on the country’s third-largest island, is a designated forest reserve with exceptionally rich biodiversity. recent ecological assessments have documented 212 tree species, 182 terrestrial vertebrates, 24 amphibian species (diesmos et al. 2015; diesmos et al. 2020), and 31 snake species (leviton et al. 2018; weinell et al. 2020). additional floral (quimio 2016) and faunal (patindol 2016) surveys have been conducted in five sinp watersheds. floristic inventories of alien species are essential for developing conservation programs and policies aimed at addressing forest conservation challenges. effective management is better achieved through comprehensive strategies that map relationships between invasive species and at-risk native species rather than focusing solely on individual taxa. globally, invasive species are among the leading drivers of ecosystem-wide biodiversity loss and species extinctions. the purpose of this study is to identify invasive alien plant species (iaps) in the riparian areas of allen, biri, catarman, gamay, mapanas, laoang, las navas, lapinig, lavezares, and pambujan in northern samar, philippines, focusing on species composition, endemism, and conservation status. conducting research on invasive plant species is crucial, as it helps shed light on the ecological consequences of non-native plant invasions. the findings will raise public awareness and promote recognition of the harmful effects of such species. moreover, the study encourages the development of new approaches to mitigation and restoration, including management interventions and the evaluation of iaps impacts. this research was conducted in the aforementioned riparian areas because no prior studies have examined invasive plant species in these habitats, even though riparian zones often harbor numerous invasive species that can outcompete or displace native vegetation. materials and methods study area this study was conducted in 10 riparian areas, i.e., allen, biri, catarman, gamay, laoang, mapanas, lapinig, las navas, lavezares, and pambujan, located in northern samar, visayas island, philippines (table 1; fig. 1). invasive alien plant species in the riparian areas of northern samar, philippines abobo et al. 153 table 1 riparian areas in northern samar as the sampling sites riparian areas coordinates allen 12°30’04” n 124°17’14” e biri 12°39’40” n 124°24’31” e catarman 12°25’08” n 124°38’30” e gamay 12°23’05” n 125°16’52” e laoang 12°37’19” n 125°01’05” e mapanas 12°28’08” n 125°14’12” e lapinig 12°18’52” n 125°17’27” e las navas 12°18’24” n 125°00’59” e lavezares 12°31’27” n 124°23’09” e pambujan 12°27’50” n 124°56’24” e figure 1 riparian areas as sampling sites in the samar island natural park notes: a. map of the philippines showing samar island natural park; b. samar island natural park map showing the sampling areas. data analysis plant collection and identification this study was conducted in march-december 2023 in the ten (10) riparian areas of northern samar, namely allen, biri, catarman, gamay, mapanas, laoang, las navas, lapinig, lavezares, and pambujan. six transect lines, measuring two kilometers per site, were laid randomly in the riparian areas to identify the existing invasive alien plants. overall, a total of 60 transect lines were established for all sampling areas. all documented invasive plant species were completed with proper photo documentation for identification and verification. other data collected during the survey included live photographs and growth habits (i.e., trees, shrubs, herbs, vines, epiphytes). plant identification and conservation status all samples were identified using species descriptions, taxonomic keys, comparison of type specimens, and available web resources. the co’s digital flora (pelser et al. 2011 onwards), plants of the world online (powo 2022), global register of introduced and invasive species-philippines, and global and invasive species database were also used as identification aids. additionally, the conservation of each species’ status was determined using the international union for conservation biotropia vol. 32 no. 2, 2025 154 of nature (iucn) red list (iucn standards & petitions committee 2023) and the department of environment and natural resources administrative order 2022-11 (denr-dao 2017-11; denr 2017). results and discussion inventory sixty-five introduced plant species were recorded in the riparian areas of northern samar, representing 24 families and 56 genera. all 65 species identified at the species level are considered non-endemic to the philippines. the species occurring in northern samar’s riparian areas are presented in table 2. the poaceae family was the most wellrepresented, with 13 species, followed by asteraceae with 12 species, and cyperaceae, fabaceae, and lamiaceae with 5 species each. other plant families had fewer than five species. the most commonly documented life forms among these invasive plant species were herbs (42), followed by shrubs (17), vines three, trees two, and one epiphyte. some of these invasive species were extremely rare in certain locations, while others were more common in different areas. this variation is likely due to differing environmental conditions. differences in soil nutrient availability, ph, type, and water retention capacity across sites may influence the ability of certain invasive species to thrive in some areas while remaining scarce in others. additionally, environmental factors such as temperature and humidity may affect the distribution, abundance, and growth of invasive plant species, contributing further to their spatial variation. it is noteworthy that the plant families documented in this study, such as asteraceae, cyperaceae, fabaceae, lamiaceae, and poaceae, are also commonly found in riparian areas in other countries, such as china and india. furthermore, the iucn classification and the denr-dao 2017–11 are used to devise the conservation status of the species in riparian areas. as a result, according to the iucn’s most recent listing, one taxon is categorized as data deficient, 32 as least concerned, and the remaining taxa are not evaluated. in contrast, no taxon was not evaluated in dao 2017–11. the life forms and taxonomic distributions of plant species were recorded in each sampling area (table 3). in allen, 9 species were identified, consisting of 1 tree, 1 shrub, and 7 herbs, with most belonging to the asteraceae family. biri had 10 species, including 2 tree species, 3 shrubs, 3 herbs, and 2 vines, representing mainly the asteraceae and fabaceae families. catarman comprised 18 species, including 2 trees, 4 shrubs, and 12 herbs, with most species from the asteraceae and poaceae families. gamay was dominated by 11 species: 2 trees, 2 shrubs, 6 herbs, and 1 vine, primarily from the asteraceae family. lavezares had 10 species, including 1 shrub, 8 herbs, and 1 epiphyte, with most species belonging to the asteraceae family. laoang had the highest diversity, with 23 species including 1 tree, 3 shrubs, and 19 herbs, predominantly from the asteraceae, cyperaceae, and poaceae families. las navas had 15 species: 2 trees, 2 shrubs, 10 herbs, and 1 vine, mostly from the fabaceae and asteraceae families. lapinig had 17 species, comprising 1 tree, 2 shrubs, 13 herbs, and 1 vine, primarily from the asteraceae and fabaceae families. mapanas had 14 species, including 2 trees, 1 shrub, 10 herbs, and 1 vine, mostly from the asteraceae and poaceae families. finally, pambujan recorded 17 species: 1 tree, 4 shrubs, 11 herbs, and 1 vine, with most species belonging to the asteraceae family. laoang recorded the highest number of introduced plant species in this study, with 23 species documented; more than any of the other nine sampling areas. the riparian zones of lapinig, pambujan, and las navas are subject to disturbance due to transportation activities. additionally, the riparian areas of lapinig serve as access routes for local communities collecting bivalves, univalves, and crabs. according to mukaromah (2020), the distribution of invasive plant species is closely associated with the level of disturbance in their respective habitats. the study also concluded that invasive species tend to be superior competitors, more tolerant of human disturbance, and capable of reducing both the abundance and diversity of native species. therefore, the disturbances observed in the riparian areas of northern samar likely contribute to the widespread distribution of invasive alien plant species along the riverbanks. invasive alien plant species in the riparian areas of northern samar, philippines abobo et al. 155 table 2 endemism and conservation status of introduced plant species in the riparian areas of northern samar family species endemism conservation status iucn denr acanthaceae acanthus ilicifolius l. n lc odontonema cuspidatum (nees) kuntze n amaranthaceae amaranthus spinosus l. n amaranthus viridis l. n cyathula prostrata (l.) blume n apiaceae centella asiatica (l.) urb. n lc asteraceae ageratum conyzoides l. n lc ageratum houstonianum mill. n chromolaena odorata (l.) r.m.king & h.rob n cyanthillium cinereum (l.) h.rob. n eclipta prostrata (l.) l. n lc elephantopus mollis kunth n elephantopus tomentosus l. n helianthus annuus l. n lc mikania cordata (burm.f.) b.h rob. n mikania micrantha kunth n pseudelephantopus spicatus (juss.ex aubl.) rohr n sphagneticola trilobata l. n boraginaceae heliotropium indicum l. n commelinaceae commelina diffusa burm.f n convolvulaceae ipomoea aquatica forssk. n lc cyperaceae actinoscirpus grossus (l.f.) goetgh. & d.a.simpson n lc cyperus brevifolius (rottb.) endl.ex hassk. n lc cyperus iria l. n lc cyperus rotundus l. n lc scleria scrobiculata nees & meyen n lc fabaceae caesalpinia pulcherrima (l.) sw. n lc leucaena leucocephala (lam.) de wit n mimosa pudica l. n lc pueraria montana (lour.) merr. n senna occidentalis (l.) n lc gleicheniaceae dicranopteris linearis (burm.f.) underw. n lc lamiaceae hyptis brevipes poit. n abelmoschus moschatus medik n lc sida acuta burm f. n sida rhombifolia l. n urena lobata l. n lc melastomataceae melastoma malabathricum l. n myrtaceae psidium guajava l. n lc nephrolepidaceae nephrolepis cordifolia (l.) c. presl n onagraceae ludwigia octovalvis (jacq.) p.h. raven n lc piperaceae piper aduncum l. n lc biotropia vol. 32 no. 2, 2025 156 family species endemism conservation status iucn denr poaceae agrostis gigantea roth n lc arundo donax l. n lc bambusa vulgaris schrad. ex j.c.wendl n bromus inermis leyys. n echinochloa colonum (l.) n lc eleusine indica (l.) gaertn. n lc eragrostis elongata (willd.) jacq. n eragrostis pilosa (l.) p.beauv n imperata cylindrica (l.) p.beauv. n lc oplismenus compositus (l.) p. beauv. n lc oryzopsis hymenoides ricker ex piper n saccharum spontaneum l. n lc themeda triandra forssk. n polygonaceae persicaria barbata (l.) h. hara n lc schizaeaceae lygodium microphyllum (cav.) r.br n lc scrophulariaceae scoparia dulcis l n solanaceae physalis peruviana l. n lc tiliaceae triumfetta rhomboidea jacq. n verbenaceae lantana camara l. n stachytarpheta cayennensis (rich.) vahl n stachytarpheta indica (l.) vahl. n lc stachytarpheta jamaicensis (l.) vahl n lc zingiberaceae hedychium coronarium j. koenig n dd table 3 species composition and classification of introduced plant species in the riparian areas of northern samar life form classification sites tree shrub herb vines epiphytes family genus species allen 1 1 7 7 9 9 biri 2 3 3 2 7 10 10 catarman 2 4 12 9 16 18 gamay 2 2 6 1 7 11 11 lavezares 1 8 1 6 10 10 laoang 1 3 19 10 22 23 las navas 2 2 10 1 7 15 15 lapinig 1 2 13 1 10 17 17 mapanas 2 1 10 1 6 14 14 pambujan 1 4 11 1 12 16 17 invasive alien plant species in the riparian areas of northern samar, philippines abobo et al. 157 distribution invasive plant species are plants that invade the natural ecosystem where they are not normally found, causing substantial damage to the new environment. one of the primary factors that led to the successful establishment of invasive alien species in the environment is the presence of rivers in these areas. seeds may be dispersed by wind or water, allowing them to colonize new areas rapidly. the constant flow of water also provides an express highway for dispersing propagules downstream. once these plant species became established in these areas, they could outcompete native plant species and become widespread. invasive species, by competitive exclusion, niche relocation, or hybridization with related species, can cause the extinction of native species. as a result, in addition to their economic consequences, such invasions can cause significant changes in the structure, composition, and global distribution of biota, resulting in the homogenization of the world’s fauna and flora and biodiversity loss (shuvar et al. 2021). invasive plant species can significantly damage the environment where they are introduced. invasive species can harm agriculture and society (pratt et al. 2017). recent studies show that invasive plant species can cause significant damage to several sectors when successfully establishing new habitats (shackleton et al. 2019; shrestha et al. 2019). for example, in this study, chromolaena odorata is considered one of the most aggressively spreading invasive weeds. it has been designated one of the world’s 100 worst invasive plant species. it’s spread substantially threatens various ecosystems from subtropical to tropical regions (koutika &rainey 2010). it typically invades roadsides, fallow areas, and forest environments, establishing dense monocultures (thapa et al. 2016; tiébré & gnanazan 2018). in invaded ecosystems, the weed displaces native species, affects species composition, and severely affects livestock and agricultural fodder (shackleton et al. 2017; thapa et al. 2016). such negative effects of c. odoroata on native ecosystems have become a significant environmental challenge. across all global regions, the number of alien plant species is continuously increasing. in riparian habitats, these populations are frequently abnormally high, seriously affecting ecosystem services and native species communities (pabst et al. 2022). various factors influence disturbance’s function in enabling plant invasions in riparian zones. since riparian vegetation structure is substantially impacted by land use and land cover changes, riparian sites with a more natural land use type tend to have fewer iaps (zelnik et al. 2020; fonseca et al. 2021). higher human pressures and hydrological disturbances in lowlying riparian areas produce a profusion of young, disturbed ecosystems that are more vulnerable to invasion, such as river bars (liendo et al. 2021). how land is used next to riparian zones affects how vulnerable the communities are to invasion (zelnik et al. 2020). controlling invasive plant species in restoration areas can be accomplished through various techniques. the most effective chemical control method relies on several elements, including the species being managed, the available funds, laws (dechoum et al. 2018), and even individual beliefs. effective management initiatives have typically increased biological diversity. however, it suggests that effective eradication may affect local species and unanticipated ecosystems. when an invasive plant species is removed, for instance, it may result in fewer resources being available for native fauna if other restoration measures are not implemented. furthermore, once removed, some invasive plant species may continue to impact the system because of their residual effects on the root symbiont and soil microbial communities (koziol et al. 2018). ten (10) species were recorded as invasive plant species on the global invasive species database, namely: chromolaena odorata, elephantopus mollis, sphagneticola trilobata, ipomoea aquatica, leucaena leucocephala, pueraria montana, abelmoschus moschatus, psidium guajava, imperata cylindrica, and lygodium microphyllum (table 4). on the other hand, twenty-two (22) species of invasive plants were listed in the global register of introduced and invasive species database philippines database these are amaranthus spinosus, cyathula prostrata, ageratum conyzoides, chromolaena odorata, elephantopus mollis, mikania micrantha, pseudelephantopus spicatus, heliotropium indicum, leucaena leucocephala, mimosa pudica, senna occidentalis, hyptis brevipes, sida acuta, urena lobata, psidium guajava, piper aduncum, eleusine indica, imperata cylindrica, scoparia dulcis, lantana camara, stachytarpheta jamaicensis and hedychium coronarium (table 4). furthermore, five species that are common in the two databases include the chromolaena odorata, elephantopus mollis, leucaena leucocephala, psidium guajava, and imperata cylindrica (table 4). biotropia vol. 32 no. 2, 2025 158 table 4 distribution of introduced plant species in the riparian areas in northern samar and confirmed invasive plant species based on global invasive species and global register of introduced and invasive speciesphilippines database family species study area gisd girsp al br cr gm pb lv la ln mp lp acanthaceae acanthus ilicifolius l. + + odontonema cuspidatum (nees) kuntze + amaranthaceae amaranthus spinosus l. + + ias amaranthus viridis l. + cyathula prostrata (l.) blume + ias apiaceae centella asiatica (l.) urb. + asteraceae ageratum conyzoides l. + + + + + + + + ias ageratum houstonianum mill. + chromolaena odorata (l.) r.m.king & h.rob + + + + + + ias ias cyanthillium cinereum (l.) h.rob. + + + + eclipta prostrata (l.) l. + + elephantopus mollis kunth + ias ias elephantopus tomentosus l. + helianthus annuus l. + mikania cordata (burm.f.) b.h rob. + + + mikania micrantha kunth + + + + ias pseudelephantopus spicatus (juss.ex aubl.) rohr + + ias sphagneticola trilobata l. + + + + + + + ias boraginaceae heliotropium indicum l. + ias commelinaceae commelina diffusa burm.f + convolvulaceae ipomoea aquatica forssk. + ias cyperaceae actinoscirpus grossus (l.f.) goetgh. & d.a.simpson + cyperus brevifolius (rottb.) endl.ex hassk. + cyperus iria l. + cyperus rotundus l. + scleria scrobiculata nees & meyen + fabaceae caesalpinia pulcherrima (l.) sw. + leucaena leucocephala (lam.) de wit + + + + + + + ias ias mimosa pudica l. + + + + + + + + + ias pueraria montana (lour.) merr. + ias senna occidentalis (l.) + ias gleicheniaceae dicranopteris linearis (burm.f.) underw. + lamiaceae hyptis brevipes poit. + + ias malvaceae abelmoschus moschatus medik + + + ias sida acuta burm.f. + ias sida rhombifolia l. + urena lobata l. + ias melastomataceae melastoma malabathricum l. + + + + + myrtaceae psidium guajava l. + + + + + + + + + + ias ias nephrolepidaceae nephrolepis cordifolia (l.) c. presl + invasive alien plant species in the riparian areas of northern samar, philippines abobo et al. 159 notes: sampling areas (al = allen; br = biri; cr = catarman; gm = gamay; pb = pambujan; lv = lavezares; la = laoang; ln = las navas; mp = mapanas; lp = lapinig); (+) present; (-) absent; database (gisd = global 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(asteraceae)lagascea mollis m. mallikarjuna rao and aluri jacob solomon raju* department of environmental sciences, andhra university, visakhapatnam 530 003, india received 19 september 2016/accepted 12 oktober 2017 abstract information on pollination ecology and seed dispersal aspects is essential to understand sexual reproduction in lagascea mollis cav. the study was aimed at providing details of pollen presentation system, pollination syndrome, pollinators and seed dispersal mode for l. mollis based mostly on field study. paper chromatography technique was used for recording sugar and amino acid types in the nectar, since they are important to evaluate the pollination syndrome. the study indicated that l. mollis flowers during august-november. the plant produces synflorescences consisting of several capitula. each capitulum produces a single disc floret. the plant is protandrous, temporally dioecious and exhibits secondary pollen presentation using brush mechanism to prevent autonomous selfing. the floral characteristics such as having synflorescence, narrow tubular corolla, production of sucrose-rich nectar with essential and non-essential amino acids and pollen charactersistics such as having spherical shape, tri-colpate apertures and strongly spinulose exine suggested that the plant is adapted for insect pollination. the plant is principally psychophilous. other insects also visited the florets opportunistically and acted as supplementary pollinators. further, thrips used this plant as breeding and feeding sites; their effect mostly geitonogamy. the fruit is an achene, anemochorous and also anthropochorous. regeneration occurred from seeds and perennial root stock during rainy season. keywords: anemochory, anthropochory, brush mechanism, lagascea mollis, psychophily, secondary pollen presentation introduction asteraceae is a large cosmopolitan group. phylogenetically, the family is considered as highly evolved as is substantiated by specialization in the floral and pollination mechanism (mabberley 2008). asteraceae is generally acclaimed to be one of the few well-known families among the dicotyledons, when viewed from several standpoints. many specialists such as taxonomists, biosystematics, chemotaxonomists, cytologists, cytogeneticists, anatomists and synantherologists have been actively playing their investigatory role in the fertile court of asteraceae, thereby achieving fruitful results. czarnecka denisow and (2014) reported that asteraceae plants show an extreme diversity of breeding systems with many species having a pronounced flexibility in the breeding strategy. asteraceae are generalists with flowers adapted to pollination by several insect groups i.e. hymenoptera, coleoptera, diptera and lepidoptera (kratochwil 2009)et al. . approximately 40 genera within the asteraceae have secondarily aggregated flowering heads called "synflorescences" (wunderlin 2017). lagascea is one such genus with synflorescence type of inflorescence. lagascea mollis cav. (subfamily asteroideae; tribe heliantheae) is often cited by farmers for its ability to control noxious weeds as well as to serve as a feed for animals (gliessman 2013). l. mollis leaf paste is used to treat cuts and wounds, while its flowers are used for ear complaints (kumar & atul 2010). despite its ecological and medicinal importance, there is absolutely no information on the reproductive biology of this species to understand the factors contributing to its weedy nature. the present study was aimed to investigate the following objectives: floral structural and functional morphology, anther dehiscence and pollen * corresponding author: solomonraju@gmail.com biotropia 4 3 7 202 211 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 3 696 202 presentation, growth and behavior of style during anthesis, pollination mechanism, pollination, pollinators, sexual system and seed dispersal in lagascea mollis cav. this study provide useful information to understand the functionality of pollination and sexual systems in this plant species that enables this plant to grow, survive as isolated individuals or as population in disturbed and undisturbed habitats. materials and methods study area, flowering season, flower morphology and inflorescence flowering phenology population of lagascea mollis growing at the foothills of visakhapatnam (17°42' n and 82°18' e with 45 m elevation) in the state of andhra pradesh, india, were used for the study. regular field visits were made to record the flowering season. ten inflorescences which had not initiated flowering were tagged and observed daily to record the flowering duration and the number of flowers produced. twenty-five fresh florets were used to record the floral characters. anthesis and anther dehiscence anthesis was initially recorded by observing the marked synflorescences in the field. later, the observations were repeated 4 times on different days in order to record accurate anthesis schedule. similarly, the mature buds were followed for recording the time of anther dehiscence. the pollen presentation pattern was also investigated by recording how anthers dehisced and the same was confirmed by observing the anthers under a hand lens having 10x magnification. analysis of nectar reward the presence of nectar was determined by gently pulling a floret from its calyx and firmly pressing its base against a hard surface. the micropipette was inserted into the floret base to extract nectar for measurement. the average of nectar of all open florets of ten synflorescences was taken as the total volume of nectar/floret and expressed in µl. paper chromatography technique was used for recording sugar and amino acids types present in the nectar. pollen output twenty-five mature but un-dehisced anthers were collected from different plants and placed in a petri dish. later, each of the 25 collected anthers was taken out and placed on a clean microscope slide (75 x 25 mm) and dabbed with a needle in a drop of lactophenol-aniline blue to collect total pollen of the collected anthers. the pollen mass was drawn into a band and the total number of pollen grains was counted under a compound microscope (40x objective, 10x eye piece). this procedure was followed by counting the number of pollen grains in each anther collected. based on these counts, the mean number of pollen produced per anther was determined. the mean pollen output per anther was multiplied by the number of anthers in the flower for obtaining the mean number of pollen grains per flower. another set of dehisced anthers was collected in a petri dish and the pollen removed from these anthers was examined under microscope for recording the pollen grain features. stigma receptivity the stigma receptivity was observed visually using h o test. in visual method, the stigma 2 2 physical state (wet or dry) and the unfolding of its lobes were considered to record the commencement of receptivity; withering of the lobes was taken as loss of receptivity. foraging activity of insects the insect species were observed visually using binoculars to list them out. their foraging activities were recorded for 10 minutes at each hour for the entire day. observation duration was 48 hours distributed on four different days. the data were tabulated to record the foraging activity rate at different times of the day and to record the percentage of foraging visits made by each category of insects per day. simultaneously, the role of these insects in affecting pollination was observed. deter mination of pollen car r yover efficiency of insects ten individuals of each insect species were captured from the florets during their peak 203 secondary pollen presentation, psychophily and anemochory in lagascea mollis cav. rao and raju results and discussion phenology information on the phenology of lagascea mollis is lacking. the present study showed that this plant is an erect much-branched annual herb growing primarily in sunny or slightly shaded waste lands, along roadsides and cultivated lands with well drained moist soils (fig. 1a). this plant forms patchy population in certain areas where there are no other bushy or shrubby plants. in such localities, the presence of this species is quite distinct and visible even from a distance. stems are slightly purplish with stipitate glandular hairs which are prominent on young parts. leaves are petiolate, usually opposite, upper ones are sometimes alternate, ovate-lanceolate, margins serrate, apex acuminate, densely whitish pubescent on both surfaces. the plant appears during june/july with the onset of rainy season and disappears with the onset of winter season. this plant extends its growth, flowering and fruiting almost throughout the year when the soil is adequately wet. re-growth occurs from underground root stock within the growing season in areas where the plant has well developed underground root stock. irrespective of the soil moisture status, the plant shows peak flowering during august-november (fig. 1b). individual plants produce synflorescences with purple florets. each plant produces 15-31 longforaging activity period and brought to the laboratory. these insects were washed with ethyl alcohol. contents of these insects were stained with aniline-blue on a glass slide and then observed under microscope to count the number of pollen grains present, to record their pollen carryover efficiency and to determine their role on pollination. natural fruit and seed set rates a sample of fruited florets from different synflorescences was collected. based on seed status in each fruited floret, the fruit set was calculated. seeds were found to be consisted of two types i.e. unfilled or half-filled and completely filled. using this as a criterion, filled seed set was calculated. filled seeds were designated as viable ones, while the unfilled or half-filled ones as in-viable ones. seed dispersal and germination season twenty synflorescences were tagged and observed to determine the duration of fruit maturation. seed characteristics were observed to evaluate their mode of dispersal. field visits were made to record whether the seeds germinated immediately after they were dispersed or not. further, field studies were carried out to find out whether sprouted from perennial root l. mollis stock and carried out sexual reproduction. biotropia vol. 24 no. 3, 2017 204 table 1 anthesis as a function of time in lagascea mollis time (h) number of florets anthesed day 1 % day 2 % day 3 % day 4 % day 5 % total 06:00 07:00 6 7 5 6 13 15.5 8 9.5 4 5 43 08:00 6 7 5 6 5 6 6 7 2 2.5 28.5 09:00 11 13 6 7 3 3 3 4 1 1.5 28.5 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00 18:00 peduncled (35-52 cm) and disciform glomeruled flat-topped synflorescences terminally. each synflorescence produces 106.64 ± 39.70 uniflowered capitula (13 mm long, 25 mm diameter) enclosed by five leaf-like, 4 mm long tubularcampanulate involucres. all capitula produce only disc florets. the capitula open continually and acropetally over a period of 5 days (fig. 1c-g). of the total capitula produced by the synflorescence, 27% of them open on day 1, 19% on day 2, 24.5% on day 3, 20.5% on day 4 and 9% on day 5 (table 1). flower morphology the floral details are not available from the literature. this study described the floral characteristics together with floral part measurements for l. mollis. the plant produces uni-f loreted capitula aggregated into a glomeruled, flat-topped synflorescence consisting of only disc florets. the florets are small, tubate (9.4 ± 0.9 mm long, 3.0 ± 0.5 mm wide), white with bluish tinge, odorless, actinomorphic, bisexual and nectariferous. the calyx is reduced to leaf-like involucres covered with white silky hairs. the corolla is tubular (6.2 ± 0.4 mm long, 3 mm wide) tipped with five glabrous lobes (1.8 ± 0.4 mm long, 1.2 ± 0.4 mm wide). stamens are five (6.1 ± 0.7 mm long, 1.8 ± 0.4 mm wide), white, epipetalous and display syngenesious condition (fig. 2e); the anthers are 2.6 mm long, 1.5 mm wide, dark brown, dithecous and apex appendiculate. the anthers are positioned along the sinuses of the corolla lobes (alternate to the lobes). the anther filaments are free from the corolla just above the tube and the two thecae (pollen sacs) of each stamen are connate with the thecae of adjacent stamens producing a tube that surrounds the style. pollen is shed to the interior of this tube (introrse dehiscence). the ovary is green (3.1 ± 0.3 mm long, 1 mm wide), oblong, hairy, bicarpellary, unilocular with a single ovule on basal placentation (fig. 2j,k). the style is 5.4 ± 0.5 mm long and is terminated into 2 externally hairy stylar arms. a nectariferous disc is present at the base of style inside the corolla tube. the style with its stylar branches extends beyond the height of anthers; the stylar branches diverge exposing the inner stigmatic surfaces and curve inwards over-arching the florets (fig. 2g)i . 205 secondary pollen presentation, psychophily and anemochory in lagascea mollis cav. rao and raju figure 1 lagascea mollis: a. habitat; b. close-up view of flowering individuals; c-f. different stages of maturing and anthesing capitulum; g. spent capitulum floral biology the disc florets opened during early morning between 07.00 and 09.00 hours on clear sunny days (table 10). the flowers opened completely on sunny days; whereas they were partially opened on rainy days. individual disc florets took about 3 hours to open from mature bud phase (fig. 2a-d). individual synflorescences produced at the terminal part of the erect branches were more conspicuous and therefore, acted as units of attraction to foragers. furthermore, their anthesis was over in a period of five days and therefore, presentation of fresh and aged florets for more than ten days enhanced attraction to foragers. each synflorescence consisting of several disc florets enabled pollinator insects to reduce flight time, search time. therefore, each visit paid by insects was energetically rewarding. the anthers were dehisced by longitudinal slits during mature bud phase indicating that the florets are protandrous. at mature bud stage, the style with its aligned stylar arms lied below the anthers. during and immediately after anthesis, the style grew, elongated and passed through the tube formed by the fused anthers brushing the pollen by the outside stylar hairs. at this stage, the inner stigmatic surfaces were un-receptive and not exposed, the functional situation of which prevented the occurrence of autogamy. such a form of pollen presentation is referred to as "secondary pollen presentation mechanism" which ensures the pollen availability to insects visiting the capitula on daily basis. the style with its aligned branches gradually diverged in the early hours of the 2 day; then the inner stigmatic nd surfaced to attain receptivity and remained so until the end of that day. the stylar arms curved inwards toward the center of the floret, completely exposing the receptive stigmatic surfaces. the pollen grains were creamy white, spherical to rounded, tricolpate, strongly spinulose having size of 26.52 ± 4.12 m (fig. 2f). μ the pollen output per anther was 988 ± 93.32 and per flower was 4,940. the pollen-ovule ratio was 4,940: 1. a disc floret produced 1.6 l of nectar μ which rose up as it accumulated in the floret due to narrow corolla tube. the sugar types found in the nectar included sucrose and glucose with the first one as dominant. the nectar contained all tested essential and non-essential amino acids. among essential amino acids, arginine, histidine, methionine and threonine were dominant. among non-essential amino acids, cystine and hydroxyproline were present in traces, while all others were present in significant levels with same level of occurrence. the disc florets withered away after three days. after the initiation of fruit formation, the withered petals and stamens gradually fell off. 206 biotropia vol. 24 no. 3, 2017 figure 2 lagascea mollis: a-d. disc florets; e. different stages of anthesis; f. syngenecious anthers; g-i. pollen grain; j. stylar arms exposing the inner receptive stigmatic region; k. ovary; l. ovule; m. fruit; n. seeds pollination mechanism in asteraceae, secondary pollen presentation system is an important characteristic. it has been considered to be a strategy to improve accuracy in pollen removal and deposition in order to enhance male and female fitness (erbar & leins 2015). in the present study, l. mollis is protandrous. the pollen shed from the anthers was brushed by the sterile hairs (sweeping hairs) from the tip down to the position below the branching of style in the bud stage when the style branches were joined and inner stigmatic surfaces were not receptive. this type of pollen presentation by sweeping hairs is the characteristic of brush mechanism (torres & galetto 2007). as the style grew out of the anther tube, the outer sweeping hairs of the style arms presented pollen for pollination. the receptive papillate stigmatic surface was hidden between the two appressed style arms, preventing self pollination. after the pollen presentation, during the functionally female phase of the floret which occurred on day 2, the style arms separated partially exposing the receptive papillae for the receipt of pollen. the style arms served as secondary pollen presenters in the staminate phase and exposed receptive stigmatic surfaces for pollen during pistillate phase. this type of active pollen presentation was typical of disc florets of asteraceae. from day four onwards the florets entered the senescence stage. the secondary pollen presentation system functional in l. mollis appeared to be evolved to enhance the efficiency and accuracy of pollen exportation and/or pollen reception, thus increasing male and/or female fitness of the plant (erbar & leins 2015). foraging activity and pollination syndrome in , the capitula were foraged by bees, l. mollis wasps, flies and butterflies (table 2; fig. 3,4). of these insects, butterflies made 52%, bees 40%, wasps 6% and flies 2% of total foraging visits. the butterflies foraged during 08.00-17.00 hours with peak foraging activity during 09.00-11.00 hours while all other insects during 08.00-15.00 hours with peak foraging activity during 09.0012,00 hours (fig. 5,6), coinciding well with the standing crop of nectar during that period. body washings of insects collected from the florets during peak foraging period revealed that the insects carried pollen in amount ranging from 6 to 214 (table 3). lakshmi and solomon raju (2011) described the floral characters that conform to butterflypollination. kost (2015) explained the chemical communication between flowers and insects through nectar chemistry. our present study 207 secondary pollen presentation, psychophily and anemochory in lagascea mollis cav. rao and raju table 2 list of insect foragers on lagascea mollis order family genus species common name forage sought hymenoptera apidae apis cerana f. indian honey bee pollen + nectar apis florea f. dwarf honey bee pollen + nectar ceratina sp. small carpenter bee pollen + nectar ceratina smaragdula f. small carpenter bee pollen + nectar anthophoridae xylocopa latipes l. large carpenter bee nectar anthophora bicincta f. blue banded bee pollen + nectar halictidae nomia sp. alkali bee pollen + nectar lasioglossum sp. sweat bee pollen + nectar scoliidae scolia quadripustulata f. blue winged wasp nectar eumenidae eumenes petiolata f. potter wasp nectar diptera sarcophagidae sarcophaga sp. flesh fly nectar lepidoptera pieridae catopsilia pyranthe l. mottled emigrant nectar colotis etrida boisduval small orange tip nectar nymphalidae acraea violae f. tawny coaster nectar junonia lemonias l. lemon pansy nectar precis iphita cramer chocolate pansy nectar danaus chrysippus l. plain tiger nectar danaus genutia cramer striped tiger nectar byblia ilithyia drury joker nectar lycaenidae castalius rosimon f. common pierrot nectar zizeeria karsandra moore dark grass blue nectar azanus jesous guerin african babul blue nectar spindasis vulcanus f. common silver line nectar 208 biotropia vol. 24 no. 3, 2017 figure 3 lagascea mollis: a. apis cerana; b. apis florea; c. xylocopa latipes; d. ceratina sp.; e. ceratina smaragdula; f. nomia sp.; g. anthophora bicincta; h. scolia quadripustulata; i. eumenes petiolata; j. sarcophaga sp. figure 4 lagascea mollis: a. & b. pierids: a. catopsilia pyranthe; b. colotis etrida; c-g. nymphalids: c. acraea violae; d. junonia lemonias; e. precis iphita; f. danaus chrysippus; g. byblia ilithyia; h. & i. lycaenids: h. castalius rosimon; i. azanus jesous figure 5 hourly foraging activity of bees, wasps and flies on lagascea mollis 209 secondary pollen presentation, psychophily and anemochory in lagascea mollis cav. rao and raju showed that in l. mollis, the nectar with sucrosedominance containing essential and non-essential amino acids were particular adaptations for psychophily. the synflorescences principally attracted pierid, nymphalid and lycaenid butterflies. the nymphalid and lycaenid butterflies were more speciose and they were important visitors. in their actions, their proboscis gained contact with the stigmatic region exposed by the stylar arms, sweeping hairs figure 6 hourly foraging activity of butterflies on lagascea mollis table 3 pollen recorded in the body washings of butterflies on lagascea mollis insect species sample size (n) number of pollen grains range mean sd bees apis cerana 10 73 214 139.6 35.17 apis florea 10 56 182 121.1 29.35 ceratina sp. 10 29 60 46.8 8.29 ceratina smaragdula 10 37 95 68.8 16.32 xylocopa latipes 10 22 64 42.4 10.29 anthophora bicincta 10 41 92 69.4 12.24 nomia sp. 10 24 56 44.7 8.26 lasioglossum sp. 10 19 63 42.9 10.73 wasps scolia quadripustulata 10 12 35 23.8 5.65 eumenes petiolata 10 15 44 32.8 7.55 sarcophaga sp. 10 9 31 21.5 5.91 butterflies catopsilia pyranthe 10 24 62 46.4 10.23 colotis etrida 10 12 44 26.7 7.98 acraea violae 10 18 53 30.9 8.79 junonia lemonias 10 20 57 40.3 9.52 precis iphita 10 16 48 30.1 7.85 danaus chrysippus 10 26 64 39.7 9.94 danaus genutia 10 12 37 25.7 6.23 byblia ilithyia 10 14 50 28.9 10.64 castalius rosimon 10 11 38 24.5 6.75 zizeeria karsandra 10 8 30 20.6 5.37 azanus jesous 10 10 36 21.5 6.81 spindasis vulcanus 10 6 29 19.5 7.50 and the dehisced anthers positioned within the corolla tube. as a result, self or cross-pollination occurred. self pollination occurred only through geitonogamy due to the occurrence of staminate and pistillate phase of florets at plant level. the pollen carried on their proboscis and forehead parts was sufficient to pollinate individual disc florets because the individual disc florets produced only one ovule per floret. the consistent foraging activity of butterflies observed on l. mollis suggested that the butterflies used this plant as a major nectar source. therefore, l. mollis is principally psychophilous. cherian (2010) mentioned that is an l. mollis important pollen source for . in this apis dorsata study, was pollinated by bees as l. mollis xylocopa exclusive nectar collectors, by other bees mostly as pollen collectors, by the occasional foragers, wasps and flies as nectar collectors. all these insects carried pollen on their body and transfered pollen to synflorescences of the same or different plants affecting both geitonogamy and xenogamy. l. mollis was also used by thrips as breeding and feeding sites. feeding activity during and after anthesis resulted mostly in geitonogamy. their nectar feeding activity promoted cross-pollination by driving the insects to make multiple visits to the same or different plants in quest of nectar. therefore, is entomophilous in general, l. mollis but psychophilous in particular. fruiting ecology and seed dispersal the fertilized florets produced single-seeded f r u i t s w i th in two weeks. ind iv idua l synflorescences produced 95 ± 31 fruits and 65 ± 17 seeds (fig. 2l-n). in open-pollinations, fruit set was 75.55%; in which filled seed set was 84.43% while the remaining percentage of fruits were unfilled which could be mostly from selfedflowers. the fruit is an obovoid achene; black, 3angled, compressed, weakly ribbed, 4 mm long and 2 mm wide, indehiscent and crowned with minute pappus at apex. it is surrounded by connate phyllaries topped by scales. the achenes clothed with connate phyllaries did not become airborne when mature and dry. they fell to the ground in the vicinity of the parental plants at low wind speed and dispersed far away from the parental plants at high wind speed during the days of dry spell within the rainy season. additionally, connate phyllaries covered by well developed scale-like hairs and carrying achenes were easily attached to clothes. in such situation, humans also serve as seed dispersal agents, the mode of which is referred to as anthropochory. seeds exhibited dormancy and germinated only during the next rainy season. they germinated readily in open areas with adequate soil moisture. the seedlings withered away and disappeared if there was a long dry spell during rainy season. therefore, the seedlings appeared to be constrained to expand its distribution range. however, the sprouting of the well developed underground root stock to produce new cycle of sexual reproduction in areas where the plant has established a large population over the years is indeed a compensation against the constrains experienced by the plant through seed mode of reproduction. conclusions lagascea mollis is a herbaceous seasonal bloomer. it produces synflorescences consisting of several capitula, each with one disc floret. it exhibits secondary pollen presentation mechanism functional through brush mechanism to avoid autonomous selfing, minimize geitonogamy and maximize xenogamy. it is temporally dioecious, self-compatible and selfpollinating through geitonogamy and crosspollinating through xenogamy. the plant is entomophilous, but principally psychophilous. anemochory and anthropochory are functional modes of seed dispersal. seeds are dormant and germinate only during rainy season. in certain areas, perennial root stock sprouts and produces new growth and carries out sexual reproduction. acknowledgments we thank andhra university, visakhapatnam, india, for providing physical facilities to carry out this research work. we also thank dr k. venkata ramana, dst-serb young scient ist , department of botany, andhra university, visakhapatnam, for assistance during field work . 210 biotropia vol. 24 no. 3, 2017 references cherian kj. 2010. pollen analysis of honey apis dorsata collected from the wardha, maharashtra. essence 1:77-80. czarnecka b, denisow b. 2014. floral biology of senecio macrophyllus m. bieb. 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(verbenaceae). curr sci 100:88-95. mabberley dj. 2008. mabberley's plant-book. a portable dictionary of plants, their classifications and uses. cambridge (uk): cambridge university press. torres c, galetto l. 2007. style morphological diversity of some asteraceae species from argentina: systematic and functional implications. j plant res 120:359-64. wunderlin rp, hansen bf, franck ar, essig fb. 2017. atlas of florida plants. tampa (us): institute for systematic botany, university of south florida. 211 secondary pollen presentation, psychophily and anemochory in lagascea mollis cav. rao and raju page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 486 (tri retna the minim).cdr the minimum number of valves for diatom identification in rawapening lake, central java 1,2* 3 4 tri retnaningsih soeprobowati , s. djalal tandjung , sutikno , 3 5suwarno hadisusanto , and peter gell 1department of biology, faculty of sciences and mathemathics, universitas diponegoro, semarang 50275, indonesia 2 school of postgraduate studies, universitas diponegoro, semarang 50275, indonesia 3 faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia 4faculty of geography, universitas gadjah mada, yogyakarta 55281, indonesia 5 water research network, federation university australia, ballarat, australia received: 21 april 2015/accepted: 30 june 2016 abstract technical challenges in using diatoms for paleolimnological work are the identification and enumeration of diatom valves. variations exist in the minimum number of valves to identify, ranging from 100 to 700 valves of the dominant species. this task can be very time consuming, particularly when the diatom valves are not abundant. this research was conducted to determine the minimum number of valves to be identified in the diatom assemblages from rawapening lake, central java, indonesia. based on the 314 samples obtained from rawapening lake, the diatom efficiency rose above 0.85 upon the minimum count of 300 valves. the number of diatom species identified remained stable after the minimum of 300 valves. therefore, the minimum number of diatom valves identified to represent the assemblage for paleolimnological analysis was 300. keywords: diatom analysis, eutrophic, indonesia, paleolimnology, rawapening introduction diatoms are the common name of microalgae belong to bacillariophyte that have wide distribution from the ocean, freshwater to humid land and part of aquatic food webs. the uniquely ornamentated silica cell wall remain undisturbed whenever fosilized. that is why diatoms are good tool for paleolimnology. diatom undoubtedly have potential as bioindicators for water quality changes due to their well preserved walls in sediments. being primary producers, diatoms play significant roles in food webs. diatoms are distributed worldwide in saline or freshwaters, have short life cycles and are responsive to environmental changes. diatoms can live across ecological gradients, are easily sampled and can be analyzed at low cost (dixit et al. 1992; gell et al. 2007; reid & ogden 2009). diatoms community are responsive to environmental changes such as ph, water depth, nutrients, salinity, and also the current condition of the environment. diatom assemblages are often specific to particular habitats, therefore diatom fossils can be used to characterize those habitats. for that reason, diatoms have been widely used to investigate the status of lakes and can explain about pollution control, water quality monitoring programs and the paleoecology of the lakes. palaeolimnological studies offer an opportunity to understand the past environmental conditions (bere 2014). paleolimnological techniques can reveal long-term perturbations and transitions of lake ecosystems (kattel et al. 2016). to understand limnological change, diatom valves are often well preserved in sediments allowing the inference of water quality over time. however, indonesia does not have standard methods for diatom analysis. even in the temperate region countries, the protocol for identifying the number of valves differs, which may be not suitable if implemented in indonesia. biotropia 3 2 6 96 104 vol. 2 no. , 201 : doi: 10.11598/btb.2016.2 . .3 2 486 * corresponding author: trsoeprobowati@live.undip.ac.id 96 the analysis of diatoms consists of three steps, i.e. digestion to separate diatom valves from the sediment; sample mounting-slide preparation; and identification-enumeration. the number of valves identified in the enumeration step differs across regions. battarbee (1986), identified 300 – 600 diatom valves in england, and many studies used battarbee's standard. however, bate and newall (2002) suggested 200 valves as an adequate number to characterize the diatom assemblages. these studies are focused mainly on temperate systems. tropical systems are very diverse which may influence diatom assemblages. therefore, it is important to determine an adequate count size in tropical diatom assemblages to optimize the efficacy of the research effort. the lower standard number i.e. 100 valves (round 1993) when being implemented in indonesia, was still time consuming due to species diversity despite lower population size (soeprobowati et al. 2005). the objective of this research was to determine the minimum valves count in the identification-enumeration step of diatom analysis for paleolimnological studies. materials and methods rawapening lake was chosen as a study site because the lake is small with eutrophication problems similar to other indonesian lakes. rawapening lake is one of the 15 indonesian national priority lakes in 2010-2014 (me 2010). rawapening lake was chosen as a pilot project for 'save indonesian lake' as it sufficiently represents the eutrophication and sedimentation problems of indonesian lakes (me 2011). radical action is required to overcome lake degradation problems comprising, one clear program for action, substantial funding, and strong institutional collaboration (soeprobowati 2015a). paleolimnological study was among the priority programs, named gerakan penyelamatan danau (germadan) rawapening, to save rawapening lake (me 2011). rawapening lake is located at 45 km south of semarang and about 9 km east of salatiga. rawapening is surrounded by five volcanoes i.e. telomojo (1,895 m asl), butak (1,000 m asl), balak (700 m asl), payung (600 m asl), and rong (600 m asl). there are four districts around the lake i.e.: tuntang, bawen, ambarawa and banyubiru. about 17 villages are situated around the lake side and their agricultural areas are frequently subjected to flooding. rawapening lake, situated at about 400 m asl, is about 4 km long and 2.5 km wide, with slightly sloped (7%) sides. in the 1970s 3its maximum capacity was 65 million m and the 3minimum was 25 million m . however, there has been a clear trend of reducing volume, which has affected its capacity to generate hydropower electricity (soeprobowati et al. 2012b). echosounding of the lake revealed extensive areas of less than 2 m depth; there were also three deeper depocentres in the lake's west, each 18 m deep (soeprobowati 2012). sediment cores of different lengths were collected from four research sites across rawapening lake. the longest sediment core (63 cm, as) was obtained from the asinan site. the panjang (pj) and tuntang (tg) cores were 36.5 cm and 35.5 cm long, respectively. the dangkel (dk) core was 29 cm (fig. 1). tg site was represent an outlet of rawapening lake, as site was near an inlet that come from a settlement and so represents a settlement catchment area and pj site was near an inlet passing through agricultural area, thereby representing an agricultural catchment area. dk site was located relatively close to the middle of the lake and so represents the lake body. those 4 sites were determined to sufficiently represent rawapening lake. diatoms were sliced every 0.5 cm based on the modified version of battarbee et al. (2001). the first step of extraction is intended to separate diatom valves from organic material. basically, depending on the type of sediment, strong acid may be used to digest sediment. this study applied 10% hcl followed by 10% h o to 2 2 digest sediment samples. in the preparation process, a mountant with refraction index of 1.7 is required. hyrax was used in this research and the silicious striae were clearly seen under the microscope, expediting the identification process. identification of diatom species was carried out by referencing the diatom samples to the standard texts (kramer & lange-bertalot 2004a, 2004b, 2004c, 2010) and by referencing the samples to the diatom collections held at universitas diponegoro, semarang, indonesia and at the federation university australia, ballarat, australia. a total of 600 valves were counted for each sample with totals tallied at steps of 100 (i.e. 97 the minimum number of valves for diatom identification – tri retnaningsih soeprobowati et al. 100, 200, 300, 400, 500, 600). species accumulation curves were applied to identify the minimal number of valves to be counted to achieve maximum efficiency, which was calculated using the formula (bates & newall 2002): number of species ________________maximum efficiency = 1 – number of individuals maximum efficiency reflected the probability of new species to be found at each identification step (pappas & stoermer 1996; bates & newall 2002). results and discussion three steps commonly used in diatom analysis consist of extraction, preparation, and identification. in the identification process, the minimal number of valves identified varied among researchers. counting the valves of figure 1 study sites in rawapening lake for collecting sediment core samples notes: as = asinan site (63 cm) represents settlement catchment area, pj =panjang site (35.5 cm) represents agricultural catchment area, tg= tuntang site (36.5 cm) represents outlet of rawapening, dk = dangkel site (29 cm) represents the lake body 98 biotropia vol. 23 no. 2, 2016 diatoms is important to provide data about the ecological condition in the past. this study analyzed 314 samples, compared valve counts at 100 , 200, 300, 400, 500, and 600 valves, found that the uppermost (1 cm) sediment samples from each of four sites. the counting results showed maximum efficiency of 0.87 – 0.93 (fig. 2). the number of diatom species identified remained stable after 300 valves while the maximum efficiency rose to above 0.85. new finding from this research is that 300 valves is the most eff ic ient number of valves for paleolimnological assessment. the number of diatom species identifed from 4 sites were different. the highest number of diatom species was found in dk site having 42 species in the counts of 200 valves which remained stable in the counts of 300 through to 600 valves (fig. 3). dk was situated close to the middle of the lake, having a water depth of 2 m with sediment of peat mud. this might be correlated with numerous eunotia species found only in the dk site indicating its acid condition (soeprobowati et al. 2012b). typically, when the euphotic depth is shallow, the number of aquatic plants attached to the substrate decline, causing nutrients and sediments to be further released into the water column. this drives the increasing numbers of phytoplankton to continually exploit the light that is confined to the surface waters (reid et al. 2007). this condition is shown in the diatom record of the rawapening lake sediments that are dominated by planktonic forms thriving in eutrophic, turbid and alkaline waters. the odd situation happened in tg site. thirty diatom species were found in the count of 200 – 600 2valves. the r of 0.42 for tg site indicated that there was a low correlation between the number of species with the number of valves counted. in tg site, large diatom species might adapt well to the moving outlet water. as site showed 22 diatom species in 400 valves and remained stable. pj site had the lowest number of species (14 species) in the count of 200 valves and increased to 17 species in the count of 500 valves. statistically, comparing the results of analysis of variance, the diatom species numbers found in each sediment layer were significantly different between counts of 100, 200, 300, 400, 500, and 600. further least significant difference (lsd) analysis showed that results from the count of 500 valves were significantly different from the count of 400 and 600 valves. there was also significant difference between the diatom species number for the count of 400 and 600 valves. the increasing numbers of diatom species in the count of 600 valves were probably due to contaminant species. this was based on the fact figure 2 maximum efficiency counts of diatoms from the upper 1 cm of sediment cores from 4 study sites notes: as = asinan site (63 cm) represents settlement catchment area, pj = panjang site (35.5 cm) represents agricultural catchment area, tg= tuntang site (36.5 cm) represents outlet of rawapening, dk = dangkel site (29 cm) represents the lake body 99 the minimum number of valves for diatom identification – tri retnaningsih soeprobowati et al. that there had been no or low increase in the numbers of diatom species with low populations from the 4 sites of rawapening lake (fig. 3). the maximum efficiency of those 4 sitesalso indicated that the count of 400 – 600 valves had maximum efficiency above 0.9 (fig. 2). analysis of variance supported this result, there is no significant different. hence, the minimum count of 300 valveshaving efficiency more than 0.85, was appropriate for the paleolimnological analysis of a tropical lake, with specific reference to the eutrophic rawapening lake. in england, battarbee et al. (1986; 2001) proposed counting 300 600 valves for routine analysis, but round (1993) determined that 100 valves were sufficient for dominant species. in sweden, gothe et al. (2013) identified diatoms until they found at least 400 valves. in finland, soininen & kononen (2004) identified 250 – 500 valves. in france, morin et al. (2008) identified 300 valves. in australia fluin et al. (2010) identified diatoms until they found between 300 – 540 valves, whereas grundell et al. (2012) identified 200 valves. in canada, koster et al. (2005) and pienitz et al. (2006) identified as many as 500 valves. in mexico, a minimum of 500 valves were identified (siqueiros-beltrones et al. 2005). in uganda, mills (2009) counted until 300 500 valves were found. in america, kireta et al. (2012) identified 100 valves in samples with sparse diatoms. in india, it is recommended to count 400 valves (karthick et al. 2010). for fossil diatoms, the count should be different, because some diatom species may have been dissolved over a period of time or diatom samples may contain diatom species from previous period of time. in indonesia, the minimum number of valves recommended by round (100 valves) had been implemented, but this is less efficient and less effective, particularly given round (1993) stipulation that 100 valves of the dominant species had to be counted (soeprobowati et al. 2005, 2012a). in this study, there were significant differences between the counts of 100,and 200 and 300, while the count of 500 valves was not significantly different from 400 and 600. these results were similar to battarbee's (1986) statement that there were marked differences between the count of 100 and 200 valves, while there was little differences between the count of 400 and 500. for this reason, he recommended that a count of 300 to 600 may be used for routine analysis purposes analysis. based on this research, it was recommended that a count of a minimum 300 valves might be used for paleolimnological analysis in rawapening lake. figure 3 number of diatom species identified from the upper 1 cm of sediment cores from 4 research sites notes: as = asinan site (63 cm) represents settlement catchment area, pj = panjang site (35.5 cm) represents agricultural catchment area, tg= tuntang site (36.5 cm) represents outlet of rawapening, dk = dangkel site (29 cm) represents the lake body 100 biotropia vol. 23 no. 2, 2016 the counting of diatom valves is to produce a semi-quantitative approach for ecological analysis. therefore, it is very important to determine the minimum number of valves to be counted to get a reliable approach to gauge the relative species composition at sampling sites (karthick et al. 2010). the minimum total number of valves to be counted for each sample varies depending on the purpose of the assessment and the need to produce statistically sound results. comparing the count of 200 and 600 valves, there were percentage differences of 1.89. the count of 300 and 600 valves provided 1.85 percentage differences which suggested that the count of 300 valves was sufficient for the calculation of diatom species. results of comparing the maximum efficiency of counting 100, 200, 300, 400, 500, and 600 valves showed that the minimum number of valves that should be identified was 300, since its maximum efficiency was more than 0.85. maximum efficiency is considered to sufficiently represent diatom species numbers because the formula to calculate maximum efficiency includes the number of individual valves. therefore, maximum efficiency can be used to determine the minimum valves to be identified in diatom analysis (bates & newall 2002). in australia, the minimum number of 200 valves had more than 80% efficiency, and this number was deemed suitable to be used in water quality monitoring programs (bates & newall 2002). there was no influence on the diatom index at counts of valves 300 or above (prygiel et al. 2002). a european protocol for diatom enumeration, dales (diatoms for assessing lake ecological status, version 1.0 2004), determined that at least 300 valves should be identified, especially for non planktonic taxa. when the abundance of one taxon was more than one third of all individual valves, the protocol recommended to increase the sample size until a minimum of 200 valves of non planktonic diatom are found. new species found in the count above 400 valves were determined to be contaminant species. the presence of these species may cause bias for further analysis. therefore, species found to be less than 5% of valves were considered to be rare species and were not included in the data analysis. dominant species are considered to provide more evidence of the water quality than the rare species. based on the diatom efficiency and diatom species found from rawapening lake, a minimum count of 300 valves was sufficient for paleolimnological analysis, which is lower than the recommended valves count for temperate or polar areas. this recommendation might be related to the year round warm temperature in the tropics which increases the diatom species diversity. a minimum count of 300 diatom valves was implemented to reconstruct the environmental condition of rawapening lake since the 1960s. the dominance of fragilaria capucina desm, luticola goeppertiana (bleisch) mann, mayamaea atomus (kutzing) lange-bertalot, navicula radiosa kutzing, nitzschia palea (kutzing) w. smith and in as site, tryblionella apiculata gregory,reflected eutrophic, but clear waters in 1967-1974. the presence of eunotia pectinalis (kutzing) rabenhorst var. undulata (ralfs) rabh suggested neutral to slightly acid conditions in 1967-1974 and the appearance of fragilaria capucina desm, gomphonema gracilis ehr. and navicula radiosa kutzing suggested changes of water ph to alkaline conditions. an increase in epiphytic gomphonema spp. in 1974-1983 marked an increase in aquatic macrophyte plants, perhaps in response to high nutrient levels. this change was followed promptly by the increasing numbers of acidophilous eunotia spp. reflecting high organic production. a transition to a diatom community dominated by planktonic forms occurred around 1983. this community was initially dominated by more clear water, oligotrophic species such as discostella stelligera (cleve and grunow) houk and klee and aulacoseira distans (ehrenberg) simonsen, but transitions happened in 1990 to be dominated by a. granulata (ehrenberg) simonsen and ultimately aulacoseira ambigua (grunow) simonsen. this was interpreted as a shift to a turbid water phase that contained beneficial phytoplankton, at the expense of benthic or epiphytic taxa requiring clear water. the dominance of a. granulata (ehrenberg) simonsen since the 1990s indicated that the lake experienced hypertrophic conditions with ph>9. although a high proportion of the taxa in rawapening lake sediments were not represented in the european data set, rawapening lake experienced hypertrophic condition with ph>9 as indicated by the dominance of aulacoseira granulata (ehrenberg) simonsen (soeprobowati et al. 2012b). 101 the minimum number of valves for diatom identification – tri retnaningsih soeprobowati et al. however, when the identification was done using counts of less than 300 valves, some predominant species such as aulacoseira ambigua, cyclotella meneghiniana, gomphonema gracillis, synedra ulna were not found. the predominance of synedra from 1967 to the present indicated that rawapening lake had been fresh and mesoeutrophic throughout. synedra ulna (nitzsch) ehrenberg is a tolerant species, found in indonesian rivers and lakes with high organic content with total phosphorous content of 20 1,000 µg/l and ph of 5 – 9. the modern sampling of ph at rawapening lake revealed that ph in rawapening lake fluctuated. goltenboth (1994) reported that the ph of rawapening lake was 7.4±0.2 (dry season) and 7.3±0.1 (wet season). epa-erc undip (1999) reported that the ph of rawapening lake was 7.96±0.42. in 2004 and 2005, the ph of the inlet and lake tended to be neutral (7.04±1.13), except in the site around the spring and floating island where the ph was 9.52 (soeprobowati et al. 2005). during field work for this study (2008), ph increased up to 9.39±2.51. in recent study, those paleoreconstruction of ecological change in rawapening proved a trend of increasing ph. measurements of ph in june 2015 showed that ph of 14 sites in rawapening lake was 7.22±2.45 (soeprobowati 2015b). conclusions diatom maximum efficiency rose to above 0.85 at the minimum count of 300 valves. the number of diatom species identified remained stable after the minimum count of 300 valves. a minimum count of 300 valves was appropriate for the paleolimnological analysis of a tropical lake, with specific reference to the eutrophic rawapening lake. acknowledgements some parts of this article was produced as a part of project supported by indonesian higher education through a fundamental research grant to universitas diponegoro number: 0160.0/023-04.2/xiii/2009, as stated in rector letter number 180/sk/h7/18 march 2009 and contract of multiyears research number: 124a/h7.2/kp/18 march 2009. the recent survey of ph was supported by competence research grant which is supported by the directorate of research and community services), directorate general of higher education, the ministry of education and culture, year 2014, through dipa undip number: 023.04.2.189185/2014, 03 maret 2014. thanks to keely mills and rosie grundell from the federation university australia, ballarat, australia for their assistance in the diatom analysis. special thanks to jafron wasiq hidayat and kariyadi baskoro for fieldworks assistance. special thanks to sri widodo agung 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fluorescens against soybean mosaic virus under drip irrigation system wuye ria andayanie*, praptiningsih gamawati adinurani and martin lukito agrotechnology study program, merdeka madiun university, madiun 63133, indonesia article higlights • glomus mosseae and pseudomonas fluorescens enhance soybean resistance to smv. • ten-day drip irrigation stop at bloom stage boosts seed yield and weight. • treatment reduces disease severity and increases p. fluorescens population. • water-saving irrigation method improves soybean performance in dry seasons. • combining g. mosseae and p. fluorescens increases soybean productivity. article information received 18 january 2023 revised 28 december 2023 accepted 13 march 2024 *corresponding author, e-mail: wuye.andayanie@gmail.com research article abstract soybeans [glycine max (l.) merr.] require much water, especially in the early stages of growth, flowering, and pod formation and filling. drought stress on soybeans will increase with global climate change. the research aimed to evaluate the efficacy of glomus mosseae and pseudomonas fluorescens in inducing systemic resistance to control soybean mosaic virus (smv) and the performance of soybean plants with drip irrigation in the dry season. the drip irrigation was stopped for ten days at the beginning of bloom, full bloom, beginning pod, and regular drip irrigation. the application of g. mosseae inoculants used 100 g of sterile compost mixed with 30 spores. the liquid inoculum of p. fluorescens was applied on cotyledonary leaves using the watering method. the plants were inoculated with smv 6 and 12 days after the p. fluorescens treatment. the symptoms of smv were recorded two weeks after inoculation, and indirect elisa detected the virus. the results showed the combination of g. mosseae and p. fluorescens under drip irrigation stopped at the beginning of bloom for ten days on elisa absorbance values and disease severity of 0.259 and 3.72 %, respectively. moreover, this treatment showed the highest values for the 100-seed weight, seed yield, and fresh weight of leaves. the technique of giving water by stopping drip irrigation at the beginning of bloom for ten days will help save water and increase the p. fluorescens population optimally in alfisols with a ph of between 4.5 and 5.1 with incredibly low fertility. the combination of g. mosseae and p. fluorescens population resulted in a significant increase in the number of g. mosseae’s spores by stopping drip irrigation at the beginning of bloom for ten days. keywords: agronomic performance, glomus mosseae, pseudomonas fluorescens, soybean, soybean mosaic virus introduction especially in the early stages of growth, flowering, and pod formation and filling, soybeans [glycine max (l.) merr.] require much water. lack of water can inhibit the plants’ growth, thereby reducing production yields. however, drought stress on soybeans will increase with global climate change. nachappa et al. (2012) reported that drought stress reduced plants’ disease resistance. in addition, drought stress and infection of viruses can decrease photosynthetic efficiency and the productivity of soybean crops. as a result, the soybean plant has problems with absorbing nitrogen, phosphorus, and potassium. in addition, dry land conditions cause low production and land use in the dry season. drip irrigation is a very efficient method of giving irrigation water because the water is provided only in the root area. therefore, it is very suitable to be applied on dry land. https://doi.org/10.11598/btb.2024.31.2.1889 biotropia vol. 31 no. 2, 2024 182 in indonesia, farmers often grow soybean plants on dry land with low fertility. therefore, the plants’ growth cannot be optimal. the soybean mosaic virus (smv) is one of the most common diseases and detrimentally impacts soybean production. the resistance of the new cultivar to the smv requires organic matter with nutrients readily available to plants, such as elemental phosphate. however, fertilization with high doses of phosphates have caused susceptible plants. therefore, biological fertilizers, such as mycorrhizae, are given to anticipate this situation. mycorrhizal fungi can reduce drought stress and positively affect crop yields on dry land. soybean yield losses due to drought stress are determined by the variety, duration of stress, and growing stadia (rigano et al. 2016; aminah et al. 2019). the application of mycorrhizae with seed coating is better than that in planting media, in which drought stress affects the development of mycorrhizae. still, mycorrhizae cannot directly attach to the sources, so a material that can stick mycorrhizae to roots is needed. in addition, varieties, duration of drought stress, and growth stadia have affected soybean plants by inhibiting the seed-filling phase (yooyongwech et al. 2019; andayanie et al. 2019a). moreover, plant-growth-promoting rhizobacteria (pgpr) are adaptive to or tolerant under drought-stress conditions. for example, genus pseudomonas is one of pgpr that may form symbiosis with the host plant and, therefore, may play a role as a biofertilizer and biocontrol agent. pseudomonas putida kt2440 and pseudomonas fluorescens are often employed for phosphorus fertilization to improve plant growth under drought stress and saline stress (pirttilä et al. 2021; costa-gutierrez et al. 2021). pseudomonas spp. can increase plant biomass, relative moisture content, leaf water potential, soil/root tissue ratio attached to roots, aggregate stability, average weight diameter, and leaf water loss. plant growth-promoting pseudomonas spp. affected compatible solutes, antioxidant status, and plant growth of maize under drought stress. inoculated plants have shown higher proline, sugar, and free amino acids. seedlings inoculation with pseudomonas spp. showed significantly lower activity of antioxidant enzymatic, ascorbate peroxidase (apx), catalase (cat), and glutathione peroxidase (gpx) under drought stress. however, this fact indicates that maize seedlings experienced less stress than those not inoculated with pseudomonas spp. furthermore, the gap-p45 strain exhibited the most significant influence on seedlings’ growth biochemical and physiological status under drought stress (sandhya et al. 2010; meliani et al. 2017; costa-gutierrez et al. 2021). late pod formation at the beginning of the fullpod stage to early seed fill was the most critical. the seed filling stage required much water and nutrients from the plant (berglund et al. 1999). rhizosphere microbes and drip irrigation have prospects for eco-restoration in drought stress. pseudomonas spp. is proved to be an alternative chemical fertilizer and supports arbuscular mycorrhizal fungi (amf) activity. amf and pseudomonas have synergistic potential. pseudomonas dissolves phosphate, and amf helps absorb phosphate in plants through amf hyphae. amf colonization with pseudomonas could increase nutrients and water absorption (gough et al. 2020; sharma et al. 2021). mycorrhizal fungi and p. fluorescens bacteria in the rhizosphere also improve soybean plants’ nutrient transmission efficiency and stress tolerance in ecological restoration. drip irrigation can overcome water shortages, irrigation depending only on rainwater, low soil fertility, shallow tillage layers, and low organic matter content. the drip irrigation method will help save water when water resources are minimal during the dry season. the optimal combination of arbuscular mycorrhizal fungi (amf) and p. fluorescens bacteria has a synergistic interaction to increase resistance under drought stress, resulting in high-yielding plants (artursson et al. 2006). the restoration field provided an overview of the future application prospects in drought stress by rhizosphere microbial. biological management of dry land uses the introduction of the beneficial microbe as a natural agent to increase the microbial population’s quality of fertile soil. the research aimed to evaluate the efficacy of g. mosseae and p. fluorescens in inducing systemic resistance to control smv and the performance of soybean plants undergoing drip irrigation in the dry season. materials and methods weather condition at the experimental site the experiments were conducted in the dry season during the drought stress in gunung kidul regency, yogyakarta, indonesia, with an average temperature of 29.7 °c and 83.1% relative humidity (rh). the field experiments were initiated during the dry season from early may to july 2022. the average monthly rainfall in may, glomus mosseae and pseudomonas fluorescens against soybean mosaic virus andayanie et. al. 183 june, and july of 2022 was 24 mm, 21 mm, and 20 mm, respectively, representing monthly rainy days of 19, 22, 8, and 4 days, respectively. water supply during the dry season this research used the rainwater harvesting (rwh) system as the water supply in the drought stress area. the rainwater harvesting (rwh) system was made at a height of 5 meters, and the rainwater was placed in a tank with a capacity of 1,200 l. after the water was collected during rain, it flowed into the planting area through a one-inch diameter pipe. horizontal branches were ¾ inchplastic hoses, and the water flowed to each plant through a small hole. soybean seed and plant preparation soybean seed preparation was done using seed priming in water (hydro priming) and salt solution (halo priming). giving water or a solution to the seeds allows the seeds to survive in very arid field conditions, and they will usually germinate. soybean seeds were obtained from the selection of lines resistant to the smv (andayanie et al. 2017; andayanie et al. 2019a). the gk/l-t soybean lines were planted in 7 rows, 3 x 1.5 m2, with a row spacing of 50 cm. the use of cashew nutshell extract prevented insect pests from landing and mosaic diseases (andayanie & ermawati 2019; andayanie et al. 2019b; andayanie et al.2019c). p. fluorescens and g. mosseae were isolated from the soybean [glycine max (l.) merrill] rhizosphere plants under dry land in gunung kidul regency, yogyakarta, indonesia. soil samples required 20 sampling times, and each sampling weighed 10 kg. phosphate solubilizing bacteria (psb) assays used pure bacterial isolates grown on a solid pikovskaya medium. the formation of a clear zone around the colony indicated the growth of psb colonies. in addition, the index of phosphate-solubilizing bacteria was applied to potato tubers (lelliot & stead 1987). this test was aimed to examine the pectinolytic ability of bacterial isolates. the isolates reacted negatively and were used as potential isolate candidates. liquid inoculum of p. fluorescens was inoculated on cotyledonary leaves seedlings (1 x 107 ml (cfu). g. mosseae were identified morphologically by brundrett et al. (1996). this identification was based on morphological characteristics of g. mosseae and evaluated microscopically. the application of g. mosseae inoculants used 100 g of sterile compost mixed with 30 spores, and they were inserted into the holes around the roots. the holes were subsequently covered with soil again. the inocula of g. mosseae were applied seven days after planting (dap). drip irrigation implementation a water supply during the dry season was provided by constructing an iron frame tower with a height of 5 m, and a water reservoir with a capacity of 1,200 l is placed at the top. the water was collected during rain and then directed to the plants using a one-inch diameter pipe, which is further channeled through a ¾ inch diameter pipe to enhance water pressure. as a result, the gravity and the pump pressures have a drip rate of 0.62 l/hour and 1.15 l/hour, respectively. water was supplied to each plant by making a small hole in the secondary hose (¾ inch), so that the water is directed only at the plant and not at the entire planting area. soybean mosaic virus application the smv isolates were obtained from a collection of the plant protection laboratory, merdeka madiun university, indonesia. the inoculum was propagated in the soybean wilis variety. one week and two weeks after inoculated with p. fluorescens, the plants were inoculated with smv isolates using a compressed air technique at a pressure of 1.8-2.0 kg/cm2, 1 g of inoculum from the infected leaves, 50 ml of buffer solution, 600 mesh carborundum with 0.5% concentration (0.5 g/100 ml), and spray time 0.5 seconds/plant at a distance of 10 to 15 cm from the leaf surface of the plant. disease severity and detection of soybean mosaic virus the percentage of disease severity was calculated 60 days after smv inoculation using the scale used by andayanie et al. (2019) and andayanie et al. (2022) as follows: 1 = healthy plants without visible symptoms on all leaves 2 = mild mosaic symptoms (10% to 30% leaf infection) 3 = moderate mosaic symptoms with green mosaic and smaller leaves (30% to 50% leaf infection) 4 = prominent mosaic symptoms with blistering and stunting (51% to 70% leaf infection) 5 = highly severe mosaic symptoms with stunting (> 71% leaf infection) biotropia vol. 31 no. 2, 2024 184 the scaling results were calculated using the following formula: ds = ʃ (n x v) x 100% n x v where: ds = disease severity (%) n = sum of infected leaves in each category v = value scale of each category n = total number of observed leaves v = highest scale value serological detection was needed to see the absorbance elisa values (ave) and the reaction of the observation samples. therefore, indirect elisa (i-elisa) was used to detect the virus through utilizing smv antisera. the i-elisa was implemented using the modified koenig (1981) method. a positive sample was indicated by a change in color in test wells, and the calculation of optical density (od) values was done using an elisa reader. statistical data analysis the field data was analyzed on a split-plot in a randomized completely block design (rcbd) with two factors and four replicates. the first factors were drip irrigation treatments, which included stopping drip irrigation for ten days at the beginning of bloom, stopping drip irrigation for ten days at full bloom, stopping drip irrigation for ten days at the beginning pod, and regular drip irrigation carried out when the plants were 3-5 days old (germination phase), 15-20 days (vegetative stage), 25-35 days (flowering phase), and 55-70 days (pod filling phase). the second factors were the use of p. fluorescens, treatment with g. mosseae, the application of p. fluorescens combined with g. mosseae, and absence of microbial inocula application as the control. eight weeks after planting, observation of the growth and development of the plants included (a) total root length, (b) root surface area, (c) number of root nodules, and (d) the plant height (cm). eight weeks after planting, the observed dry weight of the leaf area included typical leaf weight, net assimilation rate, relative growth rate, and harvest index. observations of yield and yield components included the number of pods, the number of seeds, the weight of seeds per plant, and the weight of 100 grains (g). the data were analyzed using anova and the statistical program sas 9.1 computer package. the differences among treatment means were determined by duncan’s multiple range test (dmrt) at p < 0.05. results and discussion initial soil chemical properties the initial soil chemical properties were collected from gunung kidul regency, yogyakarta, indonesia. table 1 shows that alfisols have low organic carbon content. therefore, adding manure as an essential fertilizer can overcome the low organic c content. moreover, the acidity and physical properties of soil with high clay content were measured by the bray-1 method. phosphate compounds are essential for soybean growth. however, phosphorus (p) in the ground is bound by al to form insoluble al-p bonds, most of which cannot be absorb by plants (hanyabui et al. 2020). table 1 condition of soil chemical properties before experiments chemical properties value criterion ph h2o 4.5-5.1 acid c-organic (%) 1.35-1.71 low n (%) 0.06-0.018 very low p1 bray (ppm) 0.81-8.21 low k (me100/g) 0.23-0.55 low-height source: result of soil analysis conducted in the physics and chemistry laboratory, faculty of agriculture of universitas gadjah mada, indonesia (2022). the alternative that can be used to overcome the less productive land above is the use of the microbial rhizosphere. p. fluorescens bacteria that can dissolve phosphate are not available in a form that is available for plants to absorb it. phosphorus transformation by solubilizing bacteria phosphate can increase phosphate availability in the soil. vyas & gulati (2009) noted that the secretion of bacterial organic acids such as acetic, formic, propionic, glycolic, lactic, glyoxylic, fumaric, succinic, citric acids, and tartaric cause dissolution processes. disease severity the severity of the disease varies with the following symptoms, i.e., mild mosaic, green mosaic with vein banding and chlorosis on the leaf margins, wrinkled veins with dark green bumps (blisters), mosaic with malformations in the form of curly and blistered leaves, stunted plants, and file:///c:/users/kpcu-biotrop/downloads/javascript:void(0); glomus mosseae and pseudomonas fluorescens against soybean mosaic virus andayanie et. al. 185 mosaic with cupping and stunted plants. the application of g. mosseae with p. fluorescens had the most extended incubation period and the appearance of presumably healthy symptoms, especially when drip irrigation was stopped for ten days at full bloom (fig. 1; table 2). however, applying g. mosseae in combination with p. fluorescens showed the lowest disease severity under regular drip irrigation. in addition, using these combined treatments could decrease the disease severity (table 2). drought stress will affect the interaction between viruses and plants. for instance, soybean plants undergoing drip irrigation stopped for ten days at full bloom and the combination of g. mosseae and p. fluorescens will maintain the same incubation period for smv symptoms as regular drip irrigation. as a result, symptoms of smv decreases. in addition, the findings of nachappa et al. (2016) showed that the response of plants to water stress would affect the composition of amino acids in the phloem and signaling pathways, impacting the development of aphids and viruses. therefore, applying g. mosseae in combination with p. fluorescens in this study increased the production of secondary metabolites that played a role in inducing systemic resistance against smv. elsharkawy et al. (2012) and shahzad et al. (2022) revealed that soybean plants treated with a treatment combining g. mosseae and endophytic plant-growth-promoting bacteria (epgpb) had shown the plants’ ability to act against viral diseases, including cucumber mosaic virus and tobacco mosaic virus. table 2 disease severity and symptoms on soybean treated with g. mosseae and p. fluorescens under drip irrigation condition drip irrigation disease severity/symptom (%) c gm pf gm + pf regular drip irrigation 19.61/cl, gmv 8.63/mb 7.83/mm,pph 3.68/pph stopping drip irrigation at the beginning of bloom for ten days 22.19/gmv, m 12.46/pph, cl 11.35/pph 9.08/cl, pph stopping drip irrigation at full bloom for ten days 19.94/ mc, wv 9.50/gm, cl 8.18/mm, pph 3.72/pph stopping drip irrigation at the beginning of the pod for ten days 26.22/mmb, mms 15.34/pph, mb 15.91/gmv, mc 12.73/mc, mm notes: c = control; gm = glomus mosseae; pf = pseudomonas fluorescens; mb = mosaic with blotch; pph = presumably healthy (no symptom); gm = green mosaic, cl = chlorosis on the leaf margins; gmv = green mosaic with vein banding; mc = leaves malformation with curly; wv = wrinkled veins with dark green bumps (blisters); mmb = mosaic with malformations in the form of curly and blistered leaves; mms = mosaic with malformations in the form of curly and stunted plants; mc = mosaic with cupping and stunted plants. figure 1 comparing the incubation period means of smv after microbial inoculation biotropia vol. 31 no. 2, 2024 186 table 3 effect of drip irrigation and microbial application on absorbance value of elisa 60 days after smv inoculation drip irrigation elisa absorbance value/reaction c gm pf gm + pf regular drip irrigation 2.569 abc/+ 0.709 de/+ 0.315 e/− 0.274 e/− stopping drip irrigation at the beginning of bloom for ten days 2.781 ab/+ 1.853 abcd/+ 1.561 bcde/+ 1.336 cde/+ stopping drip irrigation at full bloom for ten days 1.218 de/+ 0.308 e/− 0.287 e/− 0.259 e/ − stopping drip irrigation at the beginning of the pod for ten days 2.989 a/+ 0.787 de/+ 0.741 de/+ 0.312 e/ − notes: c = control; gm = glomus mosseae; pf = pseudomonas fluorescens. numbers followed by different letters in the same column show significantly different results based on duncan’s multiple intervals test at α = 0.05. ave negative control = 0.191; ave positive control elisa = 3.075. the test was declared positive if the ave of the sample was twice the ave of the negative elisa control (positive if the ave > 0.382). values followed by the same letters indicate no significant differences based on duncan’s means comparison test (p< 0.05). table 4 mean square values of soybean performance induced by microbial application under drip irrigation sv df ph nfp sw sy fwp replication 3 15.27 115.23* 175.42 0.91 171.68 drip irrigation (a) 3 21.64 179.19* 72.30 47.36* 203.22 microbial application (b) 3 43.51** 22.55 140.63 3.84 121.53* error (a) 9 18.70 24.68 46.91 7.62* 49.15 error (b) 9 5.08 16.11 38.65 1.92 38.89 drip irrigation x microbial application 9 36.92* 35.75 201.18* 3.01 61.77 total 36 coefficient of variation (%) − 9.13 13.69 10.68 15.88 20.12 sv df dwp fwl dwl rl nrn replication 3 81.76* 32.61 0.91 0.43 93.21* drip irrigation (a) 3 23.15 167.98* 7.01* 10.65 39.37 microbial application (b) 3 3.97 19.73 3.96* 25.18* 76.40* error (a) 9 65.46* 28.41 0.48 2.71 9.15 error (b) 9 13.26 9.10 0.62 6.04 18.62 drip irrigation x microbial application 9 24.46** 15.65 0.96 1.92 28.58 total 36 coefficient of variation (%) − 21.19 19.45 27.12 18.11 21.48 notes: ph = plant height (cm); nfp = number of filled pods per plant; sw= the 100-seed weight (g); sy = seed yield (t/ha); fwp = fresh weight of plant (g); dwp = dry weight of plant (g); fwl = fresh weight of leaves (g); dwl = dry weight of leaves (g); rl = root length; nrn = number of root nodules/plant. data were analyzed using analysis of variance (anova) and the sas 9.1 statistical program. the differences among treatment means were determined by duncan’s multiple range test (dmrt) at p < 0.05. by applying both g. mosseae and p. fluorescens, symptomatic plants could be detected using smv antiserum, except for drip irrigation stopped for ten days at the beginning of bloom. using either g. mosseae or p. fluorescens to all drip irrigation treatments inhibited smv titers. however, combining g. mosseae and p. fluorescens in treatments also inhibited smv titers stopping drip irrigation at full bloom for ten days showed the lowest ave. there was no significance in the data, and it reacted negatively with others, except with drip irrigation stopped for ten days at the beginning of bloom that responded positively to smv antiserum (table 3). in the field test, the microbial application prevented stunted growth due to drought stress. however, drip irrigation and microbial application showed significant differences (p ≤ 0.05) in the soybean growth stage and yield potential in the dry season (table 2). glomus mosseae and pseudomonas fluorescens against soybean mosaic virus andayanie et. al. 187 soybean plants need water ranging from 360 mm to 405 mm from their growing period to harvest. in addition, the flowering phase until the pods formation requires 5.08 mm to 7.62 mm of water per day, and the ripening stage of the pods ready for harvest requires 1.27 mm to 5.08 mm of water per day (mustikawati et al. 2018). the provision of water through drip irrigation and microbial application affected water availability in the soil for soybean plants, which root systems are shallow and very responsive to water availability. however, water availability below field capacity generally inhibits plant metabolism, affecting growth, development, membrane integrity, osmotic pressure, and crop yields. g. mosseae combined with p. fluorescens can be used to induce resistance to smv using regular drip irrigation, drip irrigation stopped for ten days at full bloom, and drip irrigation stopped for ten days at the beginning of the pod formation. table 5 reveals that the plants’ height of the gk/l-t soybean line undergoing drip irrigation stopped for ten days at the beginning of bloom had the lowest plant height (76.62 cm). there was the highest sensitivity to drip irrigation treatments, and there was no significant difference (p < 0.05) from planting with drip irrigation stopped for ten days (78.31 cm) at full bloom. the regular irrigation treatment showed the highest performance of soybean among test treatments. stopping drip irrigation at full bloom for ten days produced more filled pods per plant than stopping drip irrigation at the beginning bloom for ten days and stopping drip irrigation at the beginning of the pod for ten days. soil nutrients, water availability, and sunlight influenced filled pod formation. stopping drip irrigation for ten days at the beginning of pods formation caused pod loss and reduced the rate of assimilation transport into the pods, so that the number of seed pods was fewer than that of others. bennett et al. (2011) noted that lack of water at pod formation caused the number of pods to form, and pod filling was not optimal. there were the most critical stadia. data on seed yield showed no significant difference (p < 0.05) between stopping drip irrigation at full bloom for ten days and stopping drip irrigation at the beginning pod for ten days. comparing the mean of the performance of soybean with p. fluorescens pf-19 showed the highest plant height (ph), the 100-seed weight (sw), and the number of root nodules per plant (nrn) with an average of 58.14 cm, 15.13 g, and 45.21 per plant, respectively. there were no significant differences in the probability level of 5% with glomus mosseae application (table 6). table 5 comparing the means of soybeans performance under drip irrigation drip irrigation ph nfp 100 sw sw fwp regular drip irrigation 93.11a 95.74a 21.03a 2.46a 37.23a stopping drip irrigation at the beginning of bloom for ten days 76.62c 52.30c 13.29b 2.01b 28.42c stopping drip irrigation at full bloom for ten days 78.31c 76.55b 20.47a 2.38a 34.06b stopping drip irrigation at the beginning of the pod for ten days 89.60b 43.18d 15.36b 1.97b 35.81a drip irrigation dwp fwl dwl rl nrn regular drip irrigation 9.16a 18.71a 5.28a 26.14a 6.58a stopping drip irrigation at the beginning of bloom for ten days 6.32c 12.43c 3.65b 18.50c 3.76b stopping drip irrigation at full bloom for ten days 8.52b 17.16b 4.01ab 21.36b 3.81b stopping drip irrigation at the beginning of the pod for ten days 8.94ab 18.32a 4.97a 25.72a 5.92ab notes : ph = plant height (cm) ; nfp= number of filled pods per plant; sw= the 100-seeds weight (g); sw= seeds weight (t/ha); fwp= fresh weight of plant (g); dwp= dry weight of plant (g); fwl= fresh weight of leaves (g); dwl= dry weight of leaves (g); rl= root length (cm); nrn: number of root nodules. values followed by the same letters indicate no significant difference based on duncan’s means comparison test (p < 0.05). biotropia vol. 31 no. 2, 2024 188 table 6 comparing the mean of soybean performance with microbial application microbial application ph nfp sw sy fwp control 24.75c 4.05 c 9.92 1.72b 10.55b glomus mosseae 56.92a 9.24 a 15.13a 1.97a 13.21a pseudomonas fluorescens 58.14a 7.18 b 14.80a 1.85ab 10.76b g. mosseae + p. fluorescens 41.05b 6.93 b 15.78a 2.01a 12.93a microbial application dwp fwl dwl rl nrn control 3.11c 13.67b 3.25c 6.98b 25.95c glomus mosseae 4.63a 14.05a 4.38a 7.68a 45.21a pseudomonas fluorescens 3.52b 13.86b 3.60b 7.23ab 43.70a g. mosseae + p.fluorescens 4.37ab 14.49a 3.81b 7.54a 36.86b notes: ph = plant height (cm); nb = number of branches per plant; nfp = number of filled pod per plant; sw = the 100-seed weight (g); sy = seed yield (t/ha); fwp = fresh weight of plant (g); dwp = dry weight of plant (g); fwl = fresh weight of leaves (g); dwl = dry weight of leaves (g); rl = root length; nrn = number of root nodules per plant. values followed by the same letters indicate no significant differences based on duncan’s means comparison test (p < 0.05). figure 2 mean value of the number of spores when inoculated with g. mosseae in soil pseudomonas fluorescens population the technique of giving water by stopping drip irrigation at the beginning of bloom for ten days can help save water and increase p. fluorescens population optimally on marginal soils with extremely low fertility, especially on p1 bray (≤ 8.21ppm), n (≤ 0.018% ), and ph (≤ 5.1). therefore, stopping drip irrigation at the beginning of bloom for ten days can increase plant p uptake. drip irrigation can significantly influence the spread rate of p. fluorescens. the lowest rate was in the regular drip irrigation. stopping drip irrigation when soybean plants are very susceptible to water shortage showed a similar higher population of p. fluorescens in soil (figure 3). however, discontinuing drip irrigation can reduce soil moisture content levels, so the soil lacks water and its cavity contains a considerable amount of air. spores density of glomus mosseae treatment of stopping drip irrigation at full bloom for ten days had the lowest mean value on the number of g. mosseae spores in soil, i.e., 14 spores at 40 days after planting (dap). moreover, g. mosseae spore development tended to increase at 60 dap and 80 dap. gholamhoseini et al. (2013) noted that drought stress inhibited mycorrhizal colonization. however, g. mosseae is more efficient on sunflower plants under drought stress. however, mycorrhizal hyphae can still absorb water from the soil pores when plant roots experience difficulty. on the other hand, the regular drip irrigation treatment had the highest mean value of the number of spores at 40 dap to 60 dap. however, there were no significant differences in the number of spores under drip irrigation stopped for ten days at the beginning of the pod, which were 38, 69, and 104 spores in 100 g-1 of soil (figure 2). glomus mosseae and pseudomonas fluorescens against soybean mosaic virus andayanie et. al. 189 combination of glomus mosseae and pseudomonas fluorescens the mean value of spores’ density of g. mosseae in combination with p. fluorescens population presented in figure 4 shows a significant increase in the number of spores of g. mosseae resulting from stopping drip irrigation at the beginning of bloom for ten days. p. fluorescens was also assumed to be the phosphate solubilizing microorganism, promoting a higher number of g. mosseae spores per 100 g soil. water content conditions in soils could inhibit the life of glomus and pseudomonas. macro and micronutrient compounds were less effectively utilized for seed formation and yield. however, stopping drip irrigation at full bloom for ten days affected amf and pseudomonas. there were increased populations of g. globulus and p. fluorescens in the soil. the increase in plant dry weight reflects the number of nutrients absorbed and photosynthesis results. the higher the dry weight of a plant, the more nutrients are needed, and the plant’s rate of photosynthesis is also high. zhang et al. (2016) showed that drought conditions increased the co2 partial pressure among cells, so the photosynthetic performance of soybean plants decreased. stopping drip irrigation for ten days at the beginning of bloom significantly reduced biomass production. the number of leaves and their size were small, causing a small amount of photosynthetic products (< 0.05). by comparing the mean, it is learned that stopping drip irrigation at the beginning of bloom for ten days was more sensitive to absorbing nutrients in photosynthesis. the low absorption of nutrients in plants disrupted the formation of chlorophyll and decreased the chlorophyll content in the leaves. this problem caused a decrease in the rate of photosynthesis and assimilation. figure 3 mean value of the population when inoculated with p. fluorescens in soil figure 4 mean value of the number of g. mosseae’s spores in combination with p. fluorescens population biotropia vol. 31 no. 2, 2024 190 regular drip irrigation and drip irrigation stopped for ten days at the beginning of the pod helped plants absorb more water and nutrients than drip irrigation stopped for ten days at the beginning of bloom and drip irrigation stopped for ten days at full bloom. the availability of water affected the fixing of nitrogen in the soil. therefore, it influenced carbohydrate formation and increased the number of root nodules. in addition, the low content of c-organic (1.35% to 1.71%) and p1 bray (0.81 ppm to 8.21 ppm) would stimulate mycorrhizal propagules to colonize the roots. p. fluorescens application contributed to the highest plant height, and there was no significant difference from g. mosseae. indol acetate acid (iaa) of p. fluorescens is essential in cell division and elongation. in addition, p. fluorescens solvent can dissolve phosphate still trapped in the soil, such as fe, al, ca, and mg elements into elements available for plants (meliani et al. 2017). meanwhile, g. mosseae can stimulate plant growth hormones, such as cytokinins and auxins. in addition, mycorrhizae’s external hyphae can help the root system to absorb minerals, nutrients, and water more efficiently from the soil, especially in dry conditions. other studies by hanyabui et al. (2020) showed that mycorrhizal fungi can increase nutrient uptake, especially phosphorus, reduce abiotic stress, such as resistance to drought stress, and increase growth booster hormones. on the other hand, pirttilä et al. (2021) and costa-gutierrez et al. (2021) noted that phosphate-solubilizing bacteria (psb) could also improve the availability of p in acid soils. p. fluorescens is often used for phosphorus fertilization to enhance plant growth under drought and saline stress. the combination of g. mosseae and p. fluorescens under drip irrigation showed the highest increase in leaves’ 100-seed weight, seed yield, and fresh weight. however, there was no significant difference (p < 0.05) in the effectiveness of g. mosseae. the application of g. mosseae could improve soil conditions and support nutrient absorption. the continuity of symbiosis between plants and g. mosseae will influence plant metabolic processes and the formation of new roots with high membrane permeability, which is advantageous for mycorrhizae’s root colonization. also, the wide distribution of hyphae in the soil allows plants to absorb more groundwater. allen (2007) and juyal (2021) noted that the drying patterns, the soil pore structure, the amount of hyphal elongation, and the spread rate of fungal hyphae in soil could play a key role in mycorrhizae in plant-water relations. bacteria spread through soil without any water movement. soil physical conditions and architecture also affect the spread rate of bacteria through soil. for example, soil with lower bulk density facilitates faster spread of bacteria compared with soil with higher bulk density. neetu et al. 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arbuscular mycorrhizal fungi on physiological adaptation and growth promotion of maize (zea mays l.) subjected to water deficit. not bot horti agrobo 47(1):213-20. zhang j, liu j, yang c, dud s, yang w. 2016. photosynthetic performance of soybean plants to water deficit under high and low light intensity. south african j bot 105:279-87. https://nph.onlinelibrary.wiley.com/action/dosearch?contribauthorraw=bennett%2c+emma+j https://nph.onlinelibrary.wiley.com/action/dosearch?contribauthorraw=roberts%2c+jeremy+a https://nph.onlinelibrary.wiley.com/action/dosearch?contribauthorraw=wagstaff%2c+carol _hlk161931753 8. mildred elizabeth aldridge et al.cdr biotropia vol. 26 no. 1, 2019: 56 64 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 the southeast asian journal of tropical biology vol. 32 no. 1, 2025: 129 142 doi: 10.11598/btb.2025.32.1.2421 issn: 0215-6334 | e-issn: 1907-770x 129 article higlights • integrated biorefinery provides economic and environmental benefits • microalgae produce higher lipid content than plants • generally, microalgae produce higher carbohydrate contents essential for bioethanol • biohydrogen produced by microalgae is a future energy source • as a biofertilizer, microalgae promotes plant growth article information received : 27 december 2024 revised : 22 march 2025 accepted : 25 march 2025 *corresponding author, e-mail: angga.puja.asiandu@mail.ugm.ac.id reviewers: evy ratnasari ekawati & anonymous review article biorefinery of microalgae: the world’s green gem for the future sustainable development angga puja asiandu1*, widya sari2, ahmad saefun naser1, wulan rahmani akmal1, dedy setyawan3, and dita aulia yulyanita1 1faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia. 2department of physics, faculty of mathematics and natural science, universitas gadjah mada, yogyakarta 55281, indonesia. 3department of physics, faculty of mathematics and natural science, institut teknologi bandung, bandung 40132, indonesia. abstract industrial and household activities leading to many pollutants have been reduced by the presence of microalgae in the phycoremediation. microalgae transform pollutants into many forms of biorefinery, such as biofuel, biojet, bioethanol, biohydrogen, and biofertilizer. the chemical residue resulted from household and industrial activities has abundant elements (n, p, c) for microalgal cell growth. the contents of a microalgal cell, like lipid and carbohydrates, depend on the nutrition in the medium, the cultivation system, the microorganism-mediator, and the applied technology. chlorella vulgaris, botryococcus braunii, spirulina platensis, chlorella sp., chlamydomonas sp., and chlorococcum sp., are potential lipid-producing microalgae and are applied in biofuel and biojet. the carbohydrate of cyanobacteria synechoccus sp., nannochloropsis oculata, and arthospira platensis is the main content to be utilized in bioethanol. meanwhile, for the application of biohydrogen, h2 gas is converted from scenedesmus obliquus fermentation. however, the process of bioethanol and biohydrogen needs bacteria as a mediator of fermentation. spirulina and scenedesmus are examples of microalgae supporting soil fertility as biofertilizers. extraction of microalgae can increase growth promotors for plants, like amino acids, peptides, and proteins, and also antibacterial and antifungal. optimizing excellent microalgae content in bioenergy will face several challenges, for example, imbalances of organic waste. however, the phycoremediation of microalgae is a sustainable and futuristic solution to fulfill the need for energy stock. keywords: biodiesel, bioethanol, biofertilizer, biojet introduction the development aspect of a country, such as the economic aspect, tends to increase the consumption of fossil fuels to run factories, households, transportation, as well as to generate electricity and heat. considering the excessive use of fossil fuels to support human activities, the supply of fossil fuels in the world will not be sufficient, while fossil fuel is also non-renewable. additionally, the expansion of a country with a war also tends to add to the consumption of fossil fuels. in the future, climate or moderate warming around 2050 will increase energy demand predicted by 25 58%. tropics and southern regions of the united states, europe, and china will increase energy demand by more than 25% (van ruijven et al. 2019). although china is an asian country that used the highest renewables for the production of energy in 2014, coal as a non-renewable source still be used within a quantity of 77%. https://doi.org/10.11598/btb.2025.32.1.2421 mailto:angga.puja.asiandu@mail.ugm.ac.id biotropia vol. 32 no. 1, 2025 130 the countries in asia which depend on other non-renewable sources with their quantity are: indonesia (70% coal), japan (28% oil), and malaysia (61% natural gas) (sharvini et al. 2018). the environmental impact caused by the usage of a non-renewable resource, especially carbonbased oil, has been a spotlight. to produce an ecofriendly profitable energy, microalgae, which is abundant in aquatic systems and other ecosystems, have been observed in many fields. microalgae have been optimized into many forms, such as biohydrogen, bioethanol, biofuel, biojet, and other forms utilizing microalgae’s content, particularly lipid and carbohydrates. four algal strains, such as chlorella vulgaris, desmodesmus sp., ettlia pseudoalveolaris, scenedesmus obliquus, have been cultivated within a nitrogen-starving condition (nzayisenga et al. 2020). this treatment triggered more lipid production which is a remarkable biodiesel in the form of triacyglycerols. chlorella vulgaris is also optimized as a bioethanol that its quantity increases if fermentation time expands (agwa et al. 2017). microalgae-based oil which resulted from chlorella sp. and spirulina sp. was a result of conversion from triglycerides into fatty acid methyl ester and glycerol (fattah et al. 2020). many species of microalgae have been transformed into green energy. therefore, this review aimed to describe the advantage of microalgae cultivation to support the application of future green-energy of the world. review bioprospecting of microalgae microalgae have been widely reported for having the ability to produce various commercial bioproducts, such as bioethanol, biofuel, biodiesel, biohydrogen, biojet, and biofertilizers (table 1). the relatively high lipid content makes microalgae a candidate for vital bioenergy feedstock in the future. several strains that produce high amounts of lipid include botryococcus, nannochloropsis, and chlorella sp. carbohydrate content such as that found in arthrospira platensis (60%) (markou et al. 2013) and chlamydomonas sp. (50%) (kim et al. 2020) is also a critical source of bioethanol. not only limited to the energy sector, microalgae can also be used as biofertilizers, such as those found in schenedesmus obliquus (ferreira et al. 2019). some of the criteria for suitable microalgae strains to be used as candidates for lipid sources to produce biofuels are having a high growth rate, high lipid content, being able to grow in a variety of environmental conditions, including being resistant to contamination, being easily harvested, and being able to be processed using the latest lipid extraction method and potentially developed in many biorefinery activities (resdi et al. 2016). two kinds of microalgae cultivation systems are commonly used, namely open pond and photobioreactor. open pond is a microalgae cultivation system carried out openly (egbo et al. 2018). an open microalgae cultivation system can be applied on a large scale. this system is suitable for wastewater treatment and is sustainable. there are several types of open pond cultivation systems, including shallow lagoons, ponds, inclined systems, circular ponds, mixed ponds, and raceway ponds (borowitzka & moheimani 2013). a photobioreactor is also called a closed system because it is carried out in a closed container made of translucent material (egbo et al. 2018). several types of photobioreactors are tubular reactors, flat plate reactors, pyramid reactors, fermenter types, and hybrid reactors. tubular reactors consist of vertical tubular reactors, horizontal tubular reactors, helical tubular reactors, α-shaped reactors, and polyethylene bags and sleeves (płaczek et al. 2017). photobioreactors require higher production costs than that of open ponds. however, the water demand in photobioreactor, the risk of contamination, as well as the loss of co2 and nutrient is lower compared to that in the open pond. a photobioreactor is made of translucent materials that can be penetrated by sunlight. photobioreactor productivity is higher compared to that in open ponds. it is easier to regulate culture temperature in a photobioreactor than it is in an open pond. however, the commercialization of open ponds on a large scale has more potential (egbo et al. 2018). phycoremediation can be used as a method of environmental recovery. phycoremediation is safe, environmentally friendly, and relatively easy to perform. in addition, the microalgae biomass produced in this process can be used as a source of biodiesel production (asiandu & wahyudi 2021). bioprospecting of various algal species has resulted in many forms of bioenergy for utilizing the variation of growth-supporting chemical content (table 1). biorefinery of microalgae: the world’s green gem for the future asiandu et al. 131 table 1 prospect of some microalgae species no. algal species bioprospect biochemical content or roles ref. 1. arthrospira platensis bioethanol and biodiesel carbohydrate content 60%, lipid < 10%, and protein > 20% (markou et al. 2013) 2. botryococcus braunii biofuel lipid content 25 75% (chisti 2007) 3. b. braunii biodiesel lipid content 78% (nagaraja et al. 2014) 4. chlamydomonas sp. biojet lipid content 43.34% (phang et al, 2015; chen et al. 2016) 5. chlamydomonas sp. bioethanol carbohydrate content 50.5%, lipid 19%, and protein 24.2% (kim et al. 2020) 6. chlamydomonas sp. bioethanol carbohydrate content 49% (qu et al. 2020) 7. chlorella spp. biojet lipid content 26.72 40.38% (phang et al. 2015; chen et al. 2016) 8. chlorella sp. biofuel lipid content 28 32% (chisti 2007) 9. c. minutissima bioethanol carbohydrate content 0.125 g/l (margarites & costa 2014) 10. c. vulgaris biodiesel lipid content > 40% (widjaja 2009) 11. c. vulgaris biohydrogen potentially used in biohydrogen production (lakshmikandan & murugesan 2016). 12. c. vulgaris biofertilizer increasing n, p, k, fe, zn, mn contents in the soil (dineshkumar et al. 2020). 13. chlorococcum sp. biojet lipid content 39.28% (phang et al. 2015; chen et al. 2016) 14. crypthecodinium cohnii biofuel lipid content 20% (chisti 2007) 15. desmodesmus sp. bioethanol carbohydrate content 37% (qu et al. 2020) 16. dunaliella primolecta biofuel lipid content 23% (chisti 2007) 17. monallanthus salina biofuel lipid content > 20% (chisti 2007) 18. nannochloropsis sp. biofuel lipid content 68% (chisti 2007) 19. nannochloropsis sp. biojet lipid content 80% (bwapwa et al. 2018) 20. n. gaditana bioethanol carbohydrate content 17.7% (onay 2018) 21. n. oculata bioethanol carbohydrate content 252.84 mg/g (fetyan et al. 2021) 22. parachlorella kessleri bioethanol carbohydrate content 40% (qu et al. 2020) 23. scenedesmus obliquus biofuel carbohydrate content 30.2%, lipid 17.9%, and protein 31.4% (ferreira et al. 2019) biotropia vol. 32 no. 1, 2025 132 no. algal species bioprospect biochemical content or roles ref. 24. s. obliquus biohydrogen potentially used in biohydrogen production (ferreira et al. 2019) 25. s. obliquus biofertilizer enhanching the germination of wheat dan barley seeds (ferreira et al. 2019) 26. s. obliquus biofertilizer increasing n, p, and k content in soil (nayak et al. 2019). 27. schizochytrium sp. biofuel lipid content 77% (chisti 2007) 28. schizocytrium sp. bioethanol carbohydrate content 17.3% (kim et al. 2012) 29. spirullina platensis biofertilizer increasing n, p, k, fe, zn, mn contents in the soil (dineshkumar et al. 2020). 30. synechoccus sp. bioethanol carbohydrate content 60% (möllers et al. 2014) 31. tetraselmis sueica biofuel lipid content 15 23% (chisti 2007) biodiesel biodiesel from microalgae is considered as having more potential to be developed for overcoming the scarcity of fossil-based energy. when compared to crop-based biodiesel, microalgae are more sustainable for production in the long run. microalgae biomass obtained from microalgae cultivation activities can be extracted to produce algal oil. algal oil can be used as a raw material for biodiesel production. meanwhile, algal oil production produces by-products that can be processed into animal feed and other products. biomass waste can be processed in an anaerobic digestion system to produce biogas and bioelectricity. meanwhile, the effluent from this process can be used as a biofertilizer (chisti 2008). some efficient solvents for lipid extraction in microalgae are petroleum ether, n-hexane, and chloroform. the percentage ratio of solvents with high yield conversion values is 75% ethanol and petroleum ether, 75% ethanol and n-hexane, and 75% ethanol and chloroform. the combination produced a yield of about 85%. this solvent can be used to carry out direct transesterification of chlorella spp. biomass  (zhang et al. 2015). microalgae have many advantages over higher plants in the production of biofuels. microalgae are photosynthetic and absorb co2 from the air. it is one strategy to reduce greenhouse pollutants in the atmosphere. microalgae have a rapid growth rate and biomass can be produced in a short time. microalgae require a smaller land area than higher plants. also, microalgae can be harvested easily (widjaja 2009). one candidate for biodiesel production is  chlorella vulgaris. this strain is capable of accumulating lipid in relatively high amounts. the treatment in the form of n-depletion initiates lipid accumulation in the microalgae to produce high lipid accumulation at the end of incubation. lipid accumulation is more than 40% in cultures that experienced n-depletion on day 27. cultivation on day 20 produces a biomass of 0.86 mg/l, with a lipid percentage of 29.53%, and a lipid productivity of 12.77 mg/l/day. the accumulation of lipid can be higher when implementing vacuuming at low temperatures in the extraction process (widjaja 2009). moreover, compared to other plants in lipid production, microalgae have a higher lipid conversion value. lipid conversion value of microalgae with low lipid content reaches 58.700 l/ha/year (fig. 1). meanwhile, palm oil only reaches a conversion of 5.366 l/ha/year. this amount is much lower than that of microalgae. it means that palm oil production is not optimal as a future energy source. this is also related to lower productivity with large cultivation area (rahman et al. 2019; rajvanshi & sharma 2012). meanwhile, some strains have already been reported as oleaginous microalgae, as presented in figure 2, which accumulates high amounts of lipid, such as botryococcus brauni (> 75%), schizotrium sp. (> 75%), nannochloropis sp. (> 60%), to widely biorefinery of microalgae: the world’s green gem for the future asiandu et al. 133 figure 1 oil yield comparison between microalgae and other biomass sources (rahman et al. 2019; rajvanshi & sharma 2012) figure 2 lipid content in some microalgae note: modified from christi (2007). fermentation. transesterification is a biofuel production technique using the reaction between alcohol and microalgal lipid. thermochemical conversion is a method that uses high temperature to convert biomass into alcohol, hydrocarbon fuels, and other chemical substances. the process includes gasification, pyrolysis, and liquefaction (culaba et al. 2020). direct combustion is used to produce electrical energy using microalgae biomass residue. cultivated strain, such as chlorella sp. (> 30%) (chisti 2007). several technologies can be used in the production of microalgae-based biofuels, including biochemical conversion, transesterification, thermochemical conversion, and direct combustion. biochemical conversion is a biofuel production method that involves microorganism biotropia vol. 32 no. 1, 2025 134 the microalgae biomass is converted through the use of heat which will produce electrical energy. some microalgae accumulate lipid in a high percentage which is essential in biofuel production (fig. 2). one of the strains that contains high lipid is botryococcus braunii  with lipid content of 25 75%. additionally, lipid accumulation in  nannochloropsis  sp. peaked at 68%. lipid content in  schizochytrium  sp. is up to 77%. other lipid-accumulating microalgae are chlorella  sp.,  crypthecodinium cohnii,  dunaliella primolecta, monallanthus salina, and  tetraselmis sueica with lipid content of 28 32%, 20%, 23%, > 20%, and 15 23%, respectively (chisti 2007). botryococcus braunii cultivated in a photobioreactor is able to reach a biomass of 4.1 g/l. lipid accumulation in the microalgae is higher with increasing incubation time. the highest lipid accumulation reached 78%. at 42 days of incubation, the lipid extract obtained from 5 g of dry biomass was 3.8 g or 78% (nagaraja et al. 2014). some b. braunii isolates can be found in miyagi, fukui, and kochi, japan, at ph 6.7 8.0. meanwhile, several isolates from indonesia, namely palangka raya, pundu, bunto, muara teweh, and tenggarong are found at ph 6.0 7.7 (kawamura et al. 2020). there are differences in fatty acid composition in microalgae and palm oil. palm oil contains methyl stearate, methyl oleate, methyl palmitate, and methyl myristate as much as 5.73%, 55.43%, 37.37%, and 1.46%, respectively. meanwhile, the contents of methyl stearate, methyl oleate, methyl palmitate, and methyl myristate in algal oil (spirulina  sp.) are 6.04%, 49.20%, 44.76%, and trace (pradana et al. 2020). moreover, lipid of  spirulina platensis  contains c14:0, c16:0, c16:1, c18:0, c18:1, c18:2, c18:3, c20:0, c20:1. the ffa value of this lipid is relatively high, reaching 18.7%. the use of 80% alcohol in the microalgae lipid transesterification process produces a biodiesel yield of 84.7%. the biodiesel quality test produced on microalgae-based biodiesel has a viscosity of 4.8 mm2/s, density of 886 kg/m3, flash point at 172 oc, cloud point at 5 oc, pour point at -1 oc, and cetane number 60.73. considering other biodiesel, the fatty acid composition of the biodiesel is dominated by a palmitic acid of 48.35%, while the content of other fatty acids, namely mystic acid, linolenic, linoleic, palmitoleic, oleic, stearic, and lauric is 20.9%, 7.84%, 5.37%, 2.66%, 2.41%, 2.02%, and 0.7%, respectively. from this analysis, biodiesel produced by  s. platensis  lipid is very appropriate to be used as a biodiesel source (elshimi et al. 2013). biojet some potential microalgae strains in biojet production are found in malaysia, including  chlorella  sp.,  chlamydomonas  sp., and  chlorococcum  sp. lipid content in chlorella spp. peaks at 26.72 40.38%, the lipid content of  chlamydomonas  sp. is 43.34%, and in  chlorococcum sp. is 39.28%. the productivity of lipid production in  chlorella  spp. is 12.71 30.57 (mg/l/day), chlamydomonas sp. at 14.03%, and chlorococcum sp. at 22.55%. to produce 1 kg of biojet through hefa (hydroprocessed esters and fatty acids), 4.14 6.25 kg of  chlorella  spp. biomass, 3.86 kg of  chlamydomonas  sp. biomass, and 4.26 kg of  chlorococcum  sp. biomass are needed  (phang et al. 2015; chen et al. 2016). the production of biojet through hefa can be applied by converting microalgal oil extracted from its biomass through hydrotreatment (lundquist et al. 2010; chen et al. 2016). some methods for converting microalgae biomass into biojet fuel are hydrothermal liquefaction; biomass gasification and fischertropsch, comprising pyrolysis; cracking, reforming, and upgrading; deoxygenation and decarboxylation, transesterification, and fractional distillation (bwapwa et al. 2018). hydroprocessing requires relatively low production costs, suitable for large-scale production. nannochloropsis sp. is one of the future candidates for biojet production. this strain grows well at temperatures of 15 25 oc. lipid accumulation in the microalgae increased by 80% after being treated in the form of nutrient starvation for 3 days. the bio-oil extracted from the microalgae can then be processed through pyrolysis to produce the next oil fraction in the form of biojet. the characteristics of the biojet fuel produced from this microalgae are a heating value of 44 mj/kg, freezing point at -30 oc, flash point at 68 oc, density at 15 oc which is 1.38 g/ml with total sulfur of 0.27 wt% (bwapwa et al. 2018). bioethanol microalgae can also be utilized in producing bioethanol due to the high accumulation of biorefinery of microalgae: the world’s green gem for the future asiandu et al. 135 carbohydrates as a result of their photosynthesis. as an example, cyanobacteria  synechoccus sp. biomass contains 60% carbohydrates. their carbohydrate compounds can be hydrolyzed and then fermented using  saccharomyces cerevisiae  involving some enzymes to convert carbohydrates into ethanol. the ethanol conversion value reaches 0.22 g per one gram of dried biomass or 30 g/l. furthermore, the sugar of carbohydrates in  synechoccus  sp. includes glucose (60%), as well as galactose, xylose, arabinose, and mannose with concentrations of less than 5% (möllers et al. 2014). chlamydomonas  sp. can also be used in bioethanol production with an average yield reaching 0.22 g/g residual biomass. microalgal biomass growth can be initiated by cultivating the microalgae with 80 μm photon/m2/s light. the percentage of carbohydrates in the microalgae cells was 50.5%, with 24.2% protein and 19% lipid. the main composition of the fatty acids that make up the microalgae lipid included polyunsaturated fatty acids (pufas) and monounsaturated fatty acids (mufas) dominated by c16:4 with a percentage of 25.6%, followed by c18:3 (19.4%), c18:2 (5.9%), c18:1 (7.8%), and other fatty acids. the high carbohydrate content in microalgae is a promising source of bioethanol production (kim et al. 2020). arthrospira platensis  also contains a high carbohydrate percentage which is nearly 60%. meanwhile, the protein content is more than 20%, and lipid content is less than 10%. the yield of bioethanol produced from arthrospira platensis  biomass reaches 16.27% using 0.5 n h2so4, with a bioethanol yield of 16% g/g of dried biomass (markou et al. 2013). also, nannochloropsis oculata  contains  carbohydrates of 252.84 mg/g dried biomass. the yield of carbohydrates produced from the mixotrophic cultivation is 268.53 mg/g, while from the phototrophic cultivation the carbohydrates produced is 177.73 mg/g of dried biomass. the reducing sugar content is 232.39 mg/g at a concentration of 3% h2so4 with a saccharification percentage of 92.2%. the sugar is dominated by galactose, fucose, rhamnose, rabinose, xylose, and mannose (fetyan et al. 2021). nannochloropsis gaditana can be cultivated using municipal wastewater. the yield of bioethanol produced in municipal wastewater is 30%, equal to 94.3 mg/g dried biomass with an accumulation of carbohydrates of 17.7%. the biomass produced on the 14th day of incubation is 2.33 g/l (onay 2018). meanwhile, to optimize the production of biomass and carbohydrates in microalgae like  arthrospira platensis, optimum k2hpo4 and nahco3 concentrations are required, because the accumulation of lipid in the microalgae is influenced by the concentration of the two kinds of nutrients in the medium (tourang et al. 2019). the integration of bioremediation and microalgae-based biofuel production can also be carried out in bioethanol production. microalgae parachlorella kessleri, desmodesmus sp., and  chlamydomonas  sp. isolated from pig farms can remediate swine wastewater. the biomass of each microalgae cultivated in a mixotrophic culture during an incubation period of 8 days is 5 g/l, 3.1 g/l, and 6.3 g/l, respectively. the carbohydrate content in each isolate is 40%, 37%, and 49%. meanwhile, the percentage of cod reduction in each isolate reaches 47%, 38%, and 47%. the enzymes involved in the production of bioethanol are glucoamylase, α-amylase, β-glucosidase, endoglucanase, and cellobiohydrolase. meanwhile, in  chlamydomonas  sp. the maximum ethanol concentration obtained was 61 g/l (qu et al. 2020). another potential isolate in bioethanol production is schizocytrium sp. with a yield of 11.8 g/l (kim et al. 2012), chlorella minutissima with carbohydrates of 0.125 g/l (margarites & costa 2014). biohydrogen microalgae are also pivotal in biohydrogen production. one of many species,  scenedesmus obliquus  produces high-purity biohydrogen through fermentation of about 67.1 ml h2/g vs and 167.8 ml h2/ml fm. the microalgae can be cultivated using brewery wastewater to minimize the production cost as well as remediate the environment (ferreira et al. 2019).     the production of biohydrogen can also be carried out using chlorella sp. through the process of anaerobic biomass metabolism without the presence of sulfur. microalgae cells are known of their capability in carrying out anaerobic metabolic processes by using carbon sources in their cells. however, the accumulation of hydrogen increases with the addition of glucose in the medium in the anaerobic phase (song et al. 2011). additionally, hydrogen gas can also be produced through fermentation which utilizes bacteria and microalgae biomass. scenedesmus obliquus biomass can be fermented by enterobacter aerogenes and biotropia vol. 32 no. 1, 2025 136 clostridium butyricum to produce energy in the form of hydrogen gas which can be converted into electrical energy. dry biomass fermentation of scenedesmus obliquus using clostridium butyricum produces a biohydrogen yield of 113.1 ml h2/g vs algae. meanwhile, the yield of biohydrogen produced through the fermentation of e. aerogenes is 57.6 ml h2/g vs algae. in addition, fermentation can also be carried out using wet biomass with lower resulting. however, the production costs and time by using wet biomass are more prospective (batista et al. 2014). biohydrogen production using  chlorella vulgaris  can also be enriched by using valoniopsis pachynema  extract as a nutrition source for the microalgae. the extract is added to the  c. vulgaris  growth medium to form hydrogen gas through the metabolism of the microalgal cells. the maximum biohydrogen produced from the culture was 0.002 g/hour/l (lakshmikandan & murugesan 2016). biofertilizer microalgae biomass or waste can also be used in the production of biofertilizers. biomass contains various essential nutrients that can be utilized by plants to enhance their growth. the use of microalgae-based biofertilizers is potentially developed considering the massive use of chemical fertilizers that are harmful to the environment. the use of fertilizer itself is getting higher due to the increasing growth of the human population in the world. to meet agricultural needs, agricultural activities as well as the demands for fertilizers will be higher in the future (dineshkumar et al. 2019). extracts of blue-green algae are reported to be able to increase several chemical properties and the resistance of a plant to pathogens. the microalgae extract enhances the growth promoters, amino acids, peptides, and proteins, and even triggers antibacterial and antifungal properties of these plants (dineshkumar et al. 2019). microalgae also enhance the nutrient richness of the soil. microalgae increase the carbon and nitrogen content and balance the ph and electrical conductivity of the soil. some species of blue-green algae are important in the process of nitrogen fixation, for example, anabaena, nostoc, aulosira, and  tolypothrix (karthikeyan et al. 2007; dineshkumar et al. 2019). spirullina  and  chlorella  are two microalgal strains essential in enriching the soil to increase the yield of agricultural activities without causing side effects like common chemical fertilizers. moreover, microalgae are also considered potential candidates for producing biopesticides, such as  chlorella,  dunaliella  sp.,  coscinodiscus  sp.,  tetraselmis sp.,and spirullina sp. (dineshkumar et al. 2019). scenedesmus obliquus  cultivated using brewery wastewater is capable of producing various kinds of essential chemical products. it is also able to reduce the content of nh3, total nitrogen, p-po4 3, and cod in the waste with concentrations of 81%, 88.2%, 29.7%, and 70.8% reduction, respectively. based on chemical properties analysis, the microalgae cells incubated using brewery wastewater until the stationary phase contain 31.4% protein, 30.2% carbohydrates, 17.9% lipid, and 4.4% fames. the fatty acid composition of the microalgae is dominated by c16:0 of 23.03% (ferreira et al. 2019). furthermore,  scenedesmus obliquus  biomass cultivated using brewery wastewater increases the germination rate of wheat and barley seeds. germinated seeds fed with  s. obliquus  biomass has longer roots, higher in size, and better color saturation. it triggers growth promoters in the seeds of the two samples provided with the microalgae biomass. therefore, the microalgae can be considered as growth initiators of the plant (ferreira et al. 2019). the lipid-free residue of scenedesmus sp. is also able to enhance the availability of n, p, and k in the soil. the provision of biomass residue can be used as a slow-release organic matter in the soil (nayak et al. 2019). c. vulgaris and s. platensis are also reported to be able to enrich the soil to support the onion growth rate. the soil compounds administered with  c. vulgaris  biomass contain n, p, k, fe, zn, and mn with concentrations of 18, 14.22, 58.82, 3.46, 1.03, and 2.01 mg/kg, respectively. meanwhile, the content of n, p, k, fe, zn, and mn in soil administered with s. platensis biomass are 18.2, 14.24, 57.86, 3.42, 1.13, 2.06 mg/kg, respectively. the nutrient value estimated after the onion harvesting on the tested soil is shown to be higher than the control. after planting for 100 days, the height of the onions treated with c. vulgaris becomes more than 45 cm and almost 50 cm for soil treated with  s. platensis. the administration of the two microalgae biofertilizers is also reported to increase chlorophyll and carotenoid contents biorefinery of microalgae: the world’s green gem for the future asiandu et al. 137 in onions, as well as an increase in the content of total solute sugars, amino acids, and total phenols  (dineshkumar et al. 2020). in addition, the use of cow dung added with s. platensis or c. vulgaris  also increases growth, yields, chemical components, and minerals in corn (dineshkumar et al. 2017). utilizing microalgae for wastewater remediation and alternative energy source for sustainable environment microalgae cultivation can be integrated with environmental bioremediation processes. various kinds of organic waste resulting from industrial activities can be used as nutrition sources in the cultivation of microalgae. also, wastewater can be used as a water supply in microalgal biomass cultivation. the water used in the microalgae cultivation can be reused in the next cultivation cycles to reduce the dependence on clean water. the cultivated microalgae biomass using various kinds of organic waste is the feedstock of some biorefinery activities, such as the production of energy, bulk chemicals, and feed. meanwhile, microalgal wastewater can be applied as a soil enhancer to fertilize agricultural land safely (al-jabri et al. 2021). organic wastes containing various nutrient compounds, such as nitrogen, phosphorus, and carbon, will be absorbed by microalgal cells to carry out their metabolic processes. additionally, co2 gas produced from industrial activities and diesel fuel can be absorbed by their cell and used as an essential material in photosynthesis. other microorganisms, such as bacteria, can be integrated with single-strain microalgal biomass, in which the bacteria will break down complex chemicals in organic waste that will then be used as a nutrient source for microalgae cells (al-jabri et al. 2021). the biomass conversion can be done through various methods to produce certain bioenergy products. the lipid contained in biomass can be converted into biomethane, or directly go through the transesterification reaction to produce biodiesel. through hydrothermal liquefaction, the biomass can be turned into bio-crude oil. sugar or carbohydrate-based contents can also be processed by fermentation to produce bioethanol. moreover, bulk chemical products that can be transformed from microalgal biomass are glycerols, and polysaccharides, to poly-β-hydroxyalkanoate (pha) bioplastics. in feed industries, microalgal biomass is pivotal in producing feed, livestock feed, and poultry feed. also, the cultivation water can be utilized as a biofertilizer in agricultural activities. therefore, the cultivation and biorefinery system of microalgae is categorized as a green and zero-pollutant industry (al-jabri et al. 2021). however, the challenges that must be solved in integrating microalgal biomass production using wastewater are optimizing the organic waste bioremediation, optimizing the growth of microalgal biomass, minimizing production costs, and optimizing the harvesting process (al-jabri et al. 2021). moreover, phycoremediation of organic wastewater using microalgae can be hampered by inconsistencies or imbalances in the organic content of the waste that can affect the growth rate of the microalgal strain. microalgae cultivation using organic waste is also susceptible to contamination, such as antagonistic bacteria and fungi. open pond systems are also vulnerable to the presence of predators that will compete with the microalgal strains and to environmental changes. the differences in ph, temperatures, and light intensity affect microalgal growth. meanwhile, the use of a photobioreactor in cultivating microalgae on a large scale is considered ineffective due to the high production costs (ahmad et al. 2021). although microalgae-based biorefinery faces various challenges, it remains promising considering the multiple benefits, such as environmental services. microalgae are the largest co2 absorbents, much higher than c3 and c4 plants. the high rate of co2 fixation into various primary and secondary metabolites, such as carbohydrates, lipid, proteins, and pigments, in microalgae is another advantage (mondal et al. 2017). microalgae fixes around 50 gigatons of co2 every year (ashour et al. 2024; sommer et al. 2002). microalgae culture with a volume of 1,000 ha can absorb 2,100,000 tonnes of co2/ year (ashour et al. 2024; kadam 1997). therefore, microalgae are essential in reducing greenhouse gases, mitigating climate change, and remediating wastewater (gonepalli & oroszi 2024). moreover, microalgae cultivation can be integrated with industrial co2 as they need co2 in their photosynthesis process (santoso et al. 2023). for example, the microalgae biorefinery in caltagirone, italy, uses a vertically-stacked horizontal photobioreactor with a volume of biotropia vol. 32 no. 1, 2025 138 40.4 m3 capable of producing 1,200 kg of dried biomass per year. with this large volume, cultured microalgae (chlorella vulgaris) can absorb carbon dioxide of 153 kg co2, eq/kg dw (gurreri et al. 2024). another environmental service is phycoremediation, wastewater remediation using microalgae. the use of wastewater as alternative nutrients for microalgae reduces production costs due to the procurement of expensive media. from an environmental perspective, remediation carried out by microalgae on wastewater is an effective, environmentally friendly strategy for overcoming the accumulation of wastewater in the environment (velásquez-orta et al. 2024). for example, microalgae-based industries that utilize palm oil mill effluent (pome) are predicted to have a sustainability index covering environmental, social, economic, and technological values of 67.30%, 82.17%, 70.99%, and 73.67%, respectively, with an average sustainability index of 73.53% (santoso et al. 2023). microalgae can be used to remediate palm oil mill effluent (pome) to rid the environment of this waste pollution. the pome remediation process may involve several microalgae strains that are capable of producing relatively high lipid, such as  botryococcus braunii,  nannochloropsis  sp., and chlamydomonas sp. the resulting lipid can be utilized as a source of biofuel production (resdi et al. 2016). microalgae are also known as potential absorbents for various heavy metals found in wastewater. large-scale microalgae cultivation also allows the absorption of heavy metals in water bodies/reservoirs in higher volumes. they possess a biosorption mechanism including physical adsorption, ion exchange, complexation, and precipitation. the absorbed heavy metals can be processed through reduction, biotransformation, oxidation, and bioprecipitation. the use of microalgae as a biosorbent is a safer and more economical method compared to physical or chemical methods (rinanti et al. 2021). on the other hand, there are some harvesting techniques to optimize the process of harvesting microalgae biomass, centrifugation, filtration, flocculation, and floatation (ahmad et al. 2021). centrifugation and membrane microfiltration are more optimum than harvesting biomass by coagulation. biomass harvesting of  chlorella  sp. by centrifugation and microfiltration membranes yielded a biomass of 0.1174 ± 0.0308 g and 0.1145 ± 0.0268 g. both harvesting methods are known to have the same biomass optimization. the biomass produced by the coagulation method is lower than the two previous methods. in addition, the total lipid produced from the two previous methods is almost the same. of the two methods, the harvesting process in membrane microfiltration method is considered more promising. this method requires lower production costs with relatively high lipid contents. moreover, the method can also be used in harvesting low biomass quantities (ahmad et al. 2014). in the coagulation method, the low biomass and lipid productivity happen due to the coagulation process entrapping the cells caused by the differences in charge between microalgae cells and coagulants. however, when viewed from the percentage of water removal, the centrifugation method removed water by 100% compared to the percentage of water removal in the membrane microfiltration method which is only 90%, while the water removal in the coagulation method is 95% (ahmad et al. 2014). on the other hand, the harvesting method using cell immobilization using a matrix is also considered an effective harvesting method with a high percentage of lipid production. cell immobilization increases lipid yield and fatty acid composition in the microalgae (rushan et al. 2019). another method is floatation, a method of harvesting microalgal biomass by utilizing gas bubbles to separate the microalgae biomass from particles or other components. this process is also known as the reverse sedimentation process. this method is usually combined with the coagulation method early in the harvesting process (barros et al. 2015; kucmanová & gerulová 2019). the advantages of the floatation method are that the method can be applied on a large scale, can be done in a short time, and requires only a small space. however, the floatation method usually requires a chemical coagulant and is not suitable for marine microalgae strains (barros et al. 2015; singh & patidar 2018; kucmanová & gerulová 2019). centrifugation is a quick harvesting method. however, it requires relatively high production costs and high energy consumption. also, the speed of centrifugation causes cell damage and reduces valuable cell compounds (cheirsilp et al. 2019). meanwhile, this method is still effective, fast, high harvesting efficiency, and can be carried out on almost all types of microalgae, and is precise biorefinery of microalgae: the world’s green gem for the future asiandu et al. 139 on a laboratory scale or small scale (abdelaziz et al. 2013; barros et al. 2015; singh & patidar 2018). furthermore, flocculation is a method of harvesting microalgal biomass that utilizes the charge difference between the microalgal cell surface and the added flocculant. the flocculants usually used are organic or inorganic (abdelaziz et al. 2013). meanwhile, the harvesting method with sedimentation is considered unfavorable. this is because the sedimentation capacity of microalgal cells is relatively low, produces lower biomass concentrations, and requires a high volume of microalgal biomass (branyikova et al. 2018). meanwhile, filtration is a harvesting method that passes the microalgae culture through a filter membrane to accumulate the biomass on the membrane (barros et al. 2015; kucmanová & gerulová 2019). this highly effective method can be used in almost all microalgal strains. however, fouling can be a challenge as it increases production costs, membrane replacement, and pumping costs. therefore, the membrane must be cleaned regularly (barros et al. 2015; singh & patidar 2018; kucmanová & gerulová 2019). several species of microalgae can be harvested by using a filtration membrane, such as  c.pyrenoidosa,  nannochloropsis  sp.,  chlorella zofingiensis, and  dunaliela salina (castro-muñoz & garcía-depraect 2021). the transition from laboratory-scale microalgae cultivation to mass-scale for fulfilling the demand has various challenges. scaling up to an industrial scale requires a complex system and high costs. generally, some critical factors in scaling up a microalgae-based biorefinery are the desired goal or end product, the type of species cultivated, culture (open or closed), water source, nutrient source, light, temperature, mixing system, process monitoring, contamination control, harvesting strategy and technique, to environmental impact analysis. in a mass-scale cultivation, the culture must be adjusted to the available space, cost, external factors, carrying capacity, risk of contamination, and supporting tools. generally, open ponds have cheaper operating costs than closed bioreactors, but the risk of contamination is much higher. industrial-scale microalgae cultivation also requires large amounts of water (novovesk et al. 2023) the production cost of a microalgae-based biorefinery using a closed system is still relatively high. the cost required to produce 1 kg of dried biomass using tubular photobioreactors and bubble columns using artificial lights is around eur 290 329 and eur 587 573, respectively (oostlander et al. 2020). one example is a biorefinery based on nannochloropsis sp. as an aquaculture feed developed in central germany using pbr for 30 years. the net present value (npv) of the biorefinery reached eur 4.5 million after 30 years, or equal to a return on investment (roi) of 1.87%. the production costs are dominated by pbr procurement, maintenance, and labor costs. however, with the increasing demand and selling price of aquaculture feed, it has been reported that microalgae reduces production costs by 44% to 60%. the operating cost of the microalgae-based biorefinery is dominated by staff costs (40.2%), mechanical equipment (16.3%), electrical equipment (11.9%), administration (9.5%), co2 (8.1%), and other costs. meanwhile, investment costs are dominated by the glass for pbr (28%), dryer (23%), buildings (20%), mixer (6%), and centrifuge (3%) (schade & meier 2021). on the other hand, the production cost for an open pond integrated with wastewater is estimated to reach aud 1.7 per kg dried weight as the use of wastewater reduces production costs by 16% (alavianghavanini et al. 2024). therefore, to overcome some challenges in scaling up microalgae culture, it is necessary to optimize biomass production through culture engineering and genetic engineering. it is also necessary to optimize the process, including the use of liquid waste containing lots of nutrients, the use of industrial co2, adjusting the culture system (open/closed), optimizing harvesting methods, and increasing energy efficiency. to realize this, sustainable research and development are needed in ensuring the long-term prosperity of this biorefinery. the selling price of microalgae biomass is indeed relatively high and prospective. for example, the market value of arthrospira (spirulina) is predicted to reach usd 2 billion in 2026, with europe as the main market (70%), followed by asia-pacific (14%), north america (10%), middle east and africa (5%), and latin america (1%) (show 2022). conclusion due to their rapid growth rate, high metabolite content, and ability to grow in a variety of contaminants, microalgae are considered as the prospected abundant green gem that can be utilized in many biorefinery activities. microalgal metabolites including lipid, proteins, and biotropia vol. 32 no. 1, 2025 140 carbohydrates are prosperous materials in the bioindustry. those metabolites can be converted into many kinds of products in the sectors of energy, pharmaceuticals, feed, and biofertilizers to biojet fuel. many technologies, like hydroprocessing, are affordable and recommended choices to produce biojet fuel using microalgal biomass on a large scale. bioethanol is one of the main final products in the conversion of microalgal carbohydrates by fermenting the metabolite by bacteria. in agroindustry, microalgae’s nutritional compounds can be utilized as a biofertilizer to enrich the soil to support plant growth. to minimize the production cost, the cultivation system can be integrated with phycoremediation by using some wastewater as microalgal cell media. the huge potency of microalgae urges the optimization of the cultivation and transformation. key strategies include utilizing waste effectively, choosing suitable cultivation methods, controlling contamination through biological agents, and making use of sunlight and industrial co₂. it is also crucial to improve microalgal strains for optimizing outcomes. acknowledgments the authors thank all authors cited in this article for their valuable works and ideas. we also thank lpdp (lembaga pengelola dana pendidikan/ indonesia endowment fund for education agency) for supporting our master and doctoral programs. references abdelaziz aem, leite gb, hallenbeck pc. 2013. addressing the challenges for sustainable production of algal biofuels: ii. harvesting and conversion to biofuels. environ technol 34(13–14):1807-36. doi: 10.1080/09593330.2013.831487 agwa ok, nwosu ig, abu go. 2017. bioethanol production from chlorella vulgaris biomass cultivated with plantain musa paradisiaca peels 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2023 / accepted 4 september 2023 abstract forest biomass helps mitigate climate change impacts through the sequestration of atmospheric carbon dioxide and potentially storing it for long periods. deforestation and unsustainable timber harvesting cause the reduction of forest biomass, resulting in the reduced carbon sequestration capacity and altered natural balance of forest ecosystems. we used landsat 8 operational land imager (oli) to compare the aboveground forest biomass (agb) changes between 2014 and 2020 for the four national forests that represent important forest cover zones in bangladesh. we found no considerable change in agb in the sundarbans mangrove forest and the ukhiya hill forest from 2014 to 2020. in contrast, the average agb content in nijhum dwip mangrove forest decreased from 44.36 mg.h-1 in 2014 to 37.46 mg.h-1 in 2020. the average agb of the madhupur deciduous forest also decreased from 110.01 mg.h-1 in 2014 to 107.22 mg.h-1 in 2020. the decreased biomass contents could be attributed to anthropogenic factors as indicated by the presence of human activities and this information will be helpful for forest restoration and management in bangladesh. keywords: change detection, forest biomass, gis introduction to achieve the international panel of climate change (ipcc) 1.5oc goal, 730 billion tonnes of co2 (730 pg of co2 or 199 pg of c) must be removed from the atmosphere by 2100. currently, forests absorb about 7.6 billion tons of co2 per year (harris and gibbs 2021), and some projections indicate that they could sequester around 36.7 billion tons in 2100 (massondelmotte et al. 2022). global forests store about 60% (~ 862 gigatons) of total terrestrial carbon and sequester close to 80% of all terrestrial aboveground and 40% below-ground organic carbon respectively. however, forest degradation, largely due to anthropogenic activities and climate change, results in co2 emissions of about 12 to 20% of global greenhouse gases (pan et al. 2011). bangladesh is one of the asian countries with a high deforestation rate (~2,600 ha annually), largely due to human activities (macdicken et al. 2015). with climate change impacts affecting the weather conditions in bangladesh, forest degradation has been exacerbated including the alteration of forest composition, structure, and biophysical processes (littell et al. 2010). the combination of climate change and human actions is contributing to deforestation (deb et al. 2018). the forests of bangladesh are categorized into four subcategories based on their geographic conditions: swamp forests, mangrove forests, plain land sal forests, and hill forests (akhter et al. 2013). as per the bangladesh forest department (bfd 2020), forests account for 12.8% of the total land area and are classified into ten classes based on the terrain, climate, location, and management principles including bamboo forest, rubber plantation, hill forest, mangrove forest, mangrove plantation, forest plantation, shrubs with scattered tree, swamp forest and swamp plantation. bangladesh has 2.6 million hectares of forest cover, of which about 50% is *corresponding author, email: tavolk@esf.edu biotropia vol. 30 no. 3, 2023 264 in rural settlements and holds 66% of the country's aboveground forest biomass. as a result, the designated forests only total 1.6 million hectares when excluding unclassified state forests and village forests (table 1). according to bfd (2020) estimation, total carbon (c) stock on land is 1275.6 million mg c, of which 80.5% is stored in soils (0–30 cm), 15.3% in above-ground biomass, 3.6% in below-ground biomass, 0.5% in dead wood biomass, and 0.1% in litter biomass. a total of 21.5% of the carbon stock in the entire country is stored in forests, with hill and mangrove forests contributing 9.7% and 5.2% of the total stock, respectively. forest resource assessment and monitoring such as the abovementioned estimatesis important for sustainable forest management, supporting the livelihoods of communities, maintenance of ecosystem services, and carbon sequestration (angelsen 2009). however, at present there is no complete time-series study of forest biomass and carbon in bangladesh and how these factors change over time, which is an essential first step in understanding both the current biomass and carbon stocks as well as monitoring future carbon sequestration or losses in different regions of the country. this shortcoming is attributed to limited resources to conduct a conventional forest inventory, which is time-consuming and expensive (unni 1983). recent forest inventory techniques employ digital technology, such as remote sensing and gis, which can provide quality, quantity, and distribution data for assessment and monitoring. these techniques have been used in spatial decision support systems for forest land classification and planning, modelling, conservation, stand density, timber harvesting, (köhl et al. 2006) and for identification of endangered plant species, faunal habitat zoning, detecting tree damage, mortality, insect infestation, and change (mukul et al. 2014). these methods measure both qualitative (e.g., types) and quantitative (height, basal area, woody biomass, etc.) forest characteristics with ~75% accuracy (ali et al. 2015). table 1 forest types and their characteristics in bangladesh (bfd 2020; khan and millate-e-mustafa 2001; rahman 2021) forest type description hill forests hill forests of bangladesh (0.67 million ha) are mostly mixed-evergreen forests situated in the southeast (chittagong hill tracts) and northeast (sylhet hill tracts) (mukul et al. 2016). the dominant species include dipterocarpus turbinatu, swintonia floribunda, lophopetalum fimbriatum, duabunga sonnerationides, salmalia insignis, syzygium grande (reza and hasan 2019). plain land sal forests plain land sal forests, also known as sal moist forest (0.12 million ha) is mainly located in central bangladesh and dominated by shorea robusta with phyllanthus emblica, terminalia chebula, t. belerica, careya arborea, etc. (chowdhury and koike 2010). natural mangrove forests the sundarbans mangrove forest (0.6 million ha) is the single largest productive natural mangrove forest in the world and is in the southern coastal regions. it contains dense evergreen species dominated by heritiera fomes, avicennia officinalis, a. alba, hibiscus tiliaceus, excoecaria agallocha, etc. (reza and hasan 2019). coastal plantation forests afforestation along the long shoreline was started to maintain and safeguard coastal ecosystems which currently cover an area of 0.11 million hectares. many different types of mangrove species are planted, including sonneratia apetala, avicennia officinalis, a. marina, a. alba, amoora cucullata, bruguiera sexangula, excoecaria agallocha, xylocarpus mekongensis, heritiera fomes, ceriops decandra, and nypa fruticans (chowdhury et al. 2020; islam et al. 2021). un-classified state forests this is a 0.73 million hectares area in the chittagong hill tract and is managed by district councils. major species include bambusa balcooa, b. burmanica, thyrsostachys oliveri, melocanna baccifera, hevea brasiliensis, etc. swamp forests and swamp plantation covers 0.02 million hectares and is mostly found in the north-eastern region (sylhet and sunamganj district). major species include hijol or indian oak (barringtonia acutangular), koroch (pongamia pinnata), pitali (trewia nudiflora), and borun (crataeva magna) (sohel et al. 2023). privately owned village forests and forest plantation also known as homestead forests, span 0.27 million hectares and are dispersed throughout the county. estimation of change in aboveground biomass in four national forests in bangladesh – mojumder et al. 265 bangladesh conducted its first national forest resource assessment in 2007 (altrell et al. 2007), however, there is little information available on earlier data-collecting techniques and there are inconsistencies in methodology, and data-sharing arrangements (costello et al. 2016). in response to the growing need for data on trees and forest resources, a second, more thorough institutionalized forest inventory was released in 2020. it was based on remote sensing and included biophysical, land-use, and socioeconomic data (henry et al. 2021). in bangladesh, forestry information is still highly valued and is included in significant national plans, strategies, and policies due to the severe burden that energy and food production places on the country's forests (bfd 2020). as a result, inventories of forest resources are important for managing bangladesh's endangered forests sustainably. this study’s objective is to estimate the change in forest aboveground biomass between 2014 and 2020 in four forest zones (mangrove forest, hill forest, sal forest, and coastal plantation forest) in five districts of bangladesh (table 2) using landsat 8 imagery data. the main purpose is to understand the change in total tree biomass and generate maps showing changes in aboveground biomass. materials and methods study location this study focused on four designated forests in bangladesh that provide socioeconomic and ecological benefits but are also deteriorating and losing forest cover because of human pressures and natural disasters (figure 1). the sundarbans mangrove forest covers 927,700 hectares and is the biggest continuous stretch of mangroves in bangladesh. they are highly productive, have a rich floral and faunal diversity, and have a significant impact on the national and regional economies and climate (mahmood et al. 2021). a million people rely on the sundarbans for their livelihoods, and the area offers numerous direct benefits like fuelwood collection, fishing, and protection from tropical cyclones and tidal surges for their lives and property (aziz and paul 2015). it also manages coastal and riverbank erosion, aids in the sequestration and storage of carbon, and supports soil nutrient cycling (hale et al. 2019; 2015). bangladesh has over 60% of the sundarbans, which make up 4.13% of the country's total landmass and 38.12% of its total forest area (bfd 2017). however, the sundarbans are under threat from both natural and man-made sources, including erosion, tropical cyclones, salinity intrusion, and sea level rise, as well as from human settlement, overfishing, industrial development, pollution, and tourism (giri et al. 2011; uddin et al. 2019). consequently, from the 1990s to the 2000s, there was a net loss of 1.1% (10000 ha) of bangladesh's sundarbans; hence, it is critical to continuously monitor the sundarbans' forest cover to maintain this global heritage (giri et al. 2015). madhupur sal forest is a plainland sal forest, one of the most abundant and ecologically appealing forest types (islam et al. 2023) but it has been severely damaged by agriculture and other human activities (rahman et al. 2009). around 7,079.4 hectares of sal forest were destroyed between 1972 and 2013 as a result of the quick destruction and severe disruptions caused by both local and indigenous inhabitants (al faruq et al. 2016). the madhupur sal forest is surrounded by densely populated areas, which puts pressure on the forest's resources and accelerates their degradation (rahman et al. 2009). the main causes of forest degradation are encroachment, illegal tree merchants, shifting cultivation, ethnic people's energy and wood consumption, and the introduction of exotic species (yasmin et al. 2010). the ukhiya hill forest, whykhing, and teknaf forest ranges make up cox's bazar forest lands, which enclose 21,848 hectares of the chittagong hill forest (nur et al. 2016). tropical evergreen and semi-evergreen vegetation, which makes up this forest, is very important for the variety of flora and fauna and for sustaining the livelihood of the local population. however, the majority of bangladesh's deforestation took place in this forest area because of population settlement, shifting agriculture, and illicit logging (reddy et al. 2016; salam et al. 1999). since 1991, over 931,447 rohingya refugees have migrated to bangladesh (unhcr 2023); the bulk of them are now living in camps built by indiscriminately biotropia vol. 30 no. 3, 2023 266 removing 1,747.45 ha of natural vegetation and leveling hills in the upazilas of teknaf and ukhiya in the cox's bazar district (hossain and haider 2020; sarkar et al. 2023). as a result, it caused changes to the environment, including topsoil loss that resulted in unproductive land, erosion, a potential risk of landslides, and a threat to the biodiversity of the local and regional areas (mahmood et al. 2021). hasan et al. (2021) showed that in 2016, ukhia's forest area had fallen by 82% because of severe deforestation. therefore, effective change detection and analysis through appropriate monitoring are essential for the conversion of this protected area and sustainable management (akhtar et al. 2022; hossain and moniruzzaman 2021). the nijhum dwip forest is a coastal plantation forest that is a distinctive mangrove ecosystem and unique wildlife habitat for the native spotted deer population (axis axis), and a wintering site for a significant number of migratory water birds, including several endangered species (nishorgo network 2018). natural disasters like cyclones and tidal erosion, as well as human activities like unlawful harvesting and turning forest areas into agricultural land, have caused a loss of over 42% of forest area from 1990 to 2020 (islam et al. 2021). to preserve and sustainably manage this protected area, the government of bangladesh (gob) designated 16,350 hectares of nijhum dwip as a national protected area in 2001 (hossain et al. 2016). table 2 study area and forest types included in this forest biomass assessment. bfd class (bfd 2020) study area study locations area covered (ha) hill forests ukhiya hill forests district: cox’s bazar upazila: ukhia(21º16'59.88" n and 92º05'60.00" e) 26,180 coastal plantation forests nijhum dwip district: noakhali upazila: hatiya(22º 05' 35" n and 91º 00'13.7" e) 16,352 plain land sal forests madhupur sal forest distract: tangail upazila: madhupur(24º37'0.12" n and 90º01'30.00"e) 36,692 natural mangrove forests sundarbans mangrove forest district: khulna upazila: dacope administrative range: khulna range(22º46'45" n and 89°43'49"e) 74,672 district: khulna upazila: koyra administrative range: nalian range(22°4'26.6" n and 89°24'2.6" e) 61,450 district: bagerhat upazila: sarankhola range:(22°2'56.4" n and 89°48'10.2" e) and management range: sarankhola range mongla(22°4'7.7" n and 89°39'17.9" e) chandpai range 132,495 district: bagerhat upazila: mongla administrative range: chandpai range(22°4'7.7" n and 89°39'17.9" e) 130,995 estimation of change in aboveground biomass in four national forests in bangladesh – mojumder et al. 267 figure 1 the country-level map shows the locations of study areas, with expanded maps for each of the regions where changes in forest biomass were assessed biotropia vol. 30 no. 3, 2023 268 aboveground biomass (abg) data acquisition and estimation this study used multi-spectral landsat-8 satellite images, with a 30 x 30 m resolution, for the years 2014 and 2020 acquired from the usgs open-source satellite database (https://earthexplorer.usgs.gov/) (table 3). the images were processed and analyzed in arcgis 10.2.1. this study followed the procedure used by tripathi et al. (2010) for calculating carbon sequestration (figure 2). the normalized difference vegetation index (ndvi) (eq. 1) was calculated from satellite data (tucker 1979) and used to calculate the fraction of photosynthetically active radiation (fpar) (hu et al. 2017; sims et al. 2006) (eq. 2), photosynthetically active radiation (par) (sellers et al. 1995) (eq. 3), and absorbed photosynthetically active radiation (apar) (eq. 4) (field et al. 1995). 𝑁𝐷𝑉𝐼 = (𝑁𝐼𝑅−𝑅𝐸𝐷) (𝑁𝐼𝑅+𝑅𝐸𝐷) 𝑜𝑟 𝐴𝑃𝐴𝑅 𝑃𝐴𝑅 (1) again, fpar = (1.24×ndvi) – 0.168 (2) par is restricted to just a portion of sunlight's spectrum from 400 to 700 nanometers (nm). par is estimated from sunrise to sunset and is given by the formula: 𝑃𝐴𝑅 = 𝐺𝐻𝐼 365 𝑊𝑚−1 (3) where, ghi is the global horizontal irradiance (rn), retrieved from the world bank solar irradiance project (suri et al. 2020). apar is the available energy in vegetation for photosynthesis that is calculated each month and the product of par surface irradiance and the fraction of photo-synthetically active radiation (fpar). apar = fpar × par (4) according to field (1995), biomass is the function of apar and light use efficiency (lue). in practice, it is impacted by environmental variables like temperature and vapor pressure deficit (vpd), which can be derived from meteorological factors (eq. 5). ε or lue = ε°× t1 × t2 × w (g/mj) (5) where t1 and t2 represent the effect of high and low temperatures on lue i.e., relate to plant growth regulation (acclimation) by temperature; w represents the effect of moisture on lue; and ε° represents the maximum light utilization rate under ideal conditions which is globally uniform maximum 2.5 g/mj. the t1 denotes the depressant effect on the net primary product (npp) of high and low temperatures restricting the process of photosynthesis. table 3 landsat 8 data source and resolution used to estimate aboveground biomass in four forest types in bangladesh forest type satellite data spatial resolution (m) acquisition date hill forest landsat-8 oli/tirs c2 level 2 30×30 02.03.2014 & 02.13.2020 (path: 136, row: 45) nijhum dwip forest landsat-8 oli/tirs c2 level 2 30×30 02.03.2014 & 02.04.2020 (path: 136, row: 45) madhupur sal forest landsat-8 oli/tirs c2 level 2 30×30 01.25.2014 & 02.11.2020 (path: 137, row: 43) sundarbans reserve forest landsat-8 oli/tirs c2 level 2 30×30 01.14.2014 & 02.13.2020 (path: 137, row: 45) https://earthexplorer.usgs.gov/ estimation of change in aboveground biomass in four national forests in bangladesh – mojumder et al. 269 the plant acclimation by temperature (i.e., t1 and t2) and effect of moisture on lue i.e., w which is the function of the ratio of estimated evapotranspiration (eet) and potential evapotranspiration (pet) were estimated by following (eq. 6), (eq. 7) and (eq. 8) (field et al. 1995; potter et al. 1993). 𝑇2 = 𝐶 ⌊ 1 1+𝑒𝑥𝑝(0.2 × 𝑇𝑂𝑃𝑇−10−𝑇𝑚𝑜𝑛)⌋ × ⌊ 1 1+𝑒𝑥𝑝(−0.3 × 𝑇𝑂𝑃𝑇−10+𝑇𝑚𝑜𝑛)⌋ (6) where: topt = mean temperature during the month of maximum ndvi in 2014 and 2020. islam and mamun (2015) found october has the maximum ndvi for bangladesh and the mean temperature of this month was 26.4 0c in 2014 and 31.2 0c in 2020 (bmd 2020). tmon = mean monthly air temperature in 2014 and 2020. based on satellite image acquisition, our study used average mean air temperature for january and february as 19.41 0c for 2014 and 22.12 0c for 2020 (bmd 2020). c = constant = 1.185 and, t1 = 0.8 + 0.02 × topt 0.0005 × (topt) 2 (7) 𝑊 = 0.5 + 𝐸𝐸𝑇 𝑃𝐸𝑇 (8) pet is the function of temperature and latitude while eet is the function of precipitation (p) and net solar irradiance (rn), which is being converted to the equivalent evaporation through specific heat of water field et al. (1995). when eet exceeds pet, npp is no longer restricted by water. this study used a coefficient value of 0.85 for water scaler (w) from a study in bangladesh (islam and alam 2021). therefore, the amount of biomass was estimated from the ratio amount of apar and par (or ndvi) multiplied by lue that helps plant biomass increment (eq. 9). biomass = apar × lue (g/m2) (9) the amount of biomass content per m2 was estimated based on the number of pixels of clusters of landsat-8 oli/tirs c2 level 2, 30 m sensor multiplied by the squared resolution of the image. the estimated biomass was converted to megagrams per hectare (mg.ha-1), divided into four groups, and then mapped using the "natural break" method. figure 2 simplified workflow of satellite-based aboveground forest biomass estimation biotropia vol. 30 no. 3, 2023 270 results and discussion the average agb content per hectare in sundarbans reserve forest slightly increased from 89.73 mg.ha-1 in 2014 to 90.76 mg.ha-1 in 2020 (table 4), but the changes varied across the forest. the maximum agb content increased (4%) at sharankhola range from 98.13 mg.ha-1 to 102.36 mg.ha-1 and decreased (2%) at chandpai range from 81.76 mg.ha-1 in 2014 to 80.0 mg.ha1 in 2020 respectively (figure 3a and figure 3b). according to global forest watch (gfw 2022), bagerhat and khulna lost 0.05% of tree cover during 2000-2021 which resulted in 64.9 mt co2 and 13.9 mt co2 emissions respectively. sundarbans reserve forest is under pressure due to the high dependency of local poor communities on its natural resources for their survival and livelihoods and the current unsustainable management practices (mahmood et al. 2021). one step to address this was conducted by the forest department and other partners by planting around 290,000 ha of coastal mangrove forests by involving local stakeholders via a co-management method (ser 2022). in contrast, the madhupur sal forest in tangail district lost around 14,000 ha (9.1%) of tree cover from 2001 to 2021 which released 555 mt of co2 emissions to the atmosphere (gfw 2022). the average agb content per hectare in madhupur sal forest has decreased from 110.01 mg.ha-1 in 2014 to 107.22 mg.ha-1 in 2020 respectively but changes also varied across this area. the maximum agb content increased (16%) at golabari from 118.54 mg.ha-1 to 137.66 mg.ha-1 and decreased at mirzabari union (14%) from 111.24 mg.ha-1 in 2014 to 95.35 mg.ha-1 in 2020 respectively (figure 4a and figure 4b). illegal felling and settlement, grazing, encroachment, industrial and agricultural expansions, agroforestry (banana, pineapple, garlic, zinger, etc.), and firing range practice by the military are found as the underlying causes of deforestation in madhupur sal forest (mollick et al. 2018; rahman et al. 2009). cox’s bazar lost around 9,600 ha (22%) of its forest cover during the last two decades (2000-2021) resulting in 4.6 mt co2 emissions (gfw 2022). similar to the madhupur sal forest, the average per hectare agb content in the ukhia hill forest also decreased. the maximum agb content in jalia palong union increased approximately doubled from 4.08 mg.ha-1 to 8.17 mg.ha-1 but the biomass density numbers remain low in this area. the biomass in the rest of this region remained the same or increased slightly from 2014 to 2020 (figure 5a and figure 5b). ullah et al. (2022) identified overpopulation, agricultural and industrial extensions, poverty, unemployment, and illegal logging as the proximate drivers of deforestation in the teknaf area. activities contributing to these changes include excessive timber and fuelwood extraction, infrastructure extension, agricultural expansion, encroachment, and betel leaf cultivation (rashid 2017). meanwhile, the influx of rohingya refugees from myanmar since 2017 resulted in the loss of 3,238 ha. this influx induced deforestation in ukhiya, the forest department (fd) and non-governmental organizations restored 2,600 ha area along with 600 ha replanted by rohingya people by 2018 (khan 2022). even though afforestation programs are being initiated to restore these areas, coastal forests are still threatened by land erosion, illegal harvesting of plant resources, tropical natural calamities as well, and conversion of forest land into agricultural land (chowdhury et al. 2020; islam et al. 2021). noakhali lost 322 ha of tree cover from 2000 to 2021 which led to emissions of 52.5 mt co2 in the atmosphere (gfw 2022). nijhum dwip forest also lost average agb per hectare (16%) from 44.36 mg.ha-1 in 2014 to 37.46 mg.ha-1 in 2020 (figure 6a and figure 6b). estimation of change in aboveground biomass in four national forests in bangladesh – mojumder et al. 271 table 4 aboveground biomass content in 2014 and 2020 in four different forest types (in parentheses below forest name) and the associated areas of the forest of bangladesh forest type and union names 2014 (mg.ha-1) 2020 (mg.ha-1) sundarban mangrove forest (natural mangrove) khulna range 84.37 84.90 sharankhola range 98.12 102.36 nalian range 94.64 95.78 chandpai range 81.76 80.00 average 89.73 90.76 madhupur sal forest (plain land sal forest) alokdia 99.87 99.87 solakuri 108.58 102.75 ausnara 108.88 100.29 golabari 118.54 137.66 mirzabari 111.24 95.35 madhupur paurashava 114.46 114.46 arankhola 108.53 100.18 average 110.01 107.22 ukhia hill forest (hill forest) jalia palong 4.08 8.17 palong khali 3.03 3.25 raja palong 10.42 10.93 ratna palong 11.13 11.13 haldia palong 10.77 10.77 average 7.89 8.85 nijhum dwip forest (mangrove plantation) 44.36 37.46 biotropia vol. 30 no. 3, 2023 272 figure 3 spatial distribution of biomass (mg.ha-1) in the sundarbans mangrove forest (a) 2014 (b) 2020. each pixel represents a 30 x 30 m area estimation of change in aboveground biomass in four national forests in bangladesh – mojumder et al. 273 figure 4 spatial distribution of biomass (mg.ha-1) in madhapur sal forest (a) 2014 (b) 2020 biotropia vol. 30 no. 3, 2023 274 figure 5 spatial distribution of biomass (mg.ha-1) in ukhia hill forest (a) 2014 (b) 2020 estimation of change in aboveground biomass in four national forests in bangladesh – mojumder et al. 275 figure 6 spatial distribution of biomass (mg.ha-1) in nijhum dwip forest (a) 2014 (b) 2020 in the four forests assessed in this study, the estimated average agb in 2014 was about 63 mg.ha-1 (minimum 3.03 mg.ha-1 to maximum 118.54 mg.ha-1) and it dropped to 61 mg.ha-1 (minimum 3.25 mg.ha-1 to maximum 137.7 mg.ha-1) in 2020. the average minimum agb stock was observed in the hill forest with estimated values of 7.89 mg.ha-1 and 8.85 mg.ha-1 while the average maximum stock reported was 110.01 mg.ha-1 and 107.22 mg.ha-1 during both years of estimation. the average agb stock in the forests of bangladesh was 63 mg.ha-1 in 2014 and 61 mg.ha-1 in 2020, which are lower than other reported values of 145.47 mg.ha-1 (bfd 2020), 159.5 to 360 mg ha-1 (rahman et al. 2015), 49 to 121 mg.ha-1 (alamgir and turton 2013), 158 mg.ha-1 (brown et al. 2001), 93 mg.ha-1 (eggleston et al. 2006), 92 mg.ha-1 (brown 1997), 137 mg.h-1 (defries et al. 2002), and 175.5 mg.ha-1 (mukul et al. 2014). however, the estimation is very close to a satellite-based study conducted by saatchi et al. (2011), which reported a value was 70.5 mg.ha-1. other inventory or harvest data-based studies observed similar values like gibbs et al. (2007) reported 65 mg ha-1, 55 mg.ha-1 by fao (2010), and 62 mg.ha-1 by rahman (2021) (table 5). in addition to differences in methods used for these assessments, this study only examined four representative forests in bangladesh so does not capture the forest biomass across all the forested land in the country. biotropia vol. 30 no. 3, 2023 276 table 5 biomass estimations and methods for bangladesh from different studies study study type biomass stock (mg.ha-1) present study satellite (2014 image) 63 present study satellite (2020 image) 61 bfd (2020) inventory 145.47 rahman et al. (2015) inventory 159.5–360 fao (2010) inventory 55 alamgir and turton (2013) inventory 49-121 saatchi et al. (2011) satellite 70.5 brown et al. (2001) harvest data 158 rahman (2021) inventory 62 gibbs et al. (2007) harvest data 65 eggleston et al. (2006) harvest data 93 brown (1997) inventory 92 defries et al. (2002) harvest data 137 mukul et al. (2014) literature based modeling 175.5 this study found a low amount of biomass stock in the hill forests of bangladesh compared with 115.6 mg.ha-1 and 103.4 mg.ha-1 reported values by ullah and al-amin (2012) and mukul et al. (2016). a huge variation in agb stock in madhupur sal forest compared with 153.9 mg.ha-1 and 34.5 mg.ha-1 was estimated by kibria and saha (2011) and mukul et al. (2014). however, the agb stock for sundarbans mangrove forest reported values by mukul et al. (2014) and rahman (2021) as 88.2 mg.ha-1 and 98.9 mg.ha-1 which are very close to the present study. similarly, the estimated biomass value of nijhum dwip forest is also very close to the 19.6 mg.ha-1 reported value by mukul et al. (2014) (table 6). the variations might be due to differences in methods, areas studied like northeastern versus south-eastern hill forest areas, as well as month and year of satellite data acquisition. for example, ndvi concentration remains similar in a season (tripathi et al. 2010) but changes between months with the lowest values in february and the highest in october (islam and mamun 2015). this study used satellite images for january and february i.e., the winter season, which might contribute to the lower estimation. moreover, higher spatial resolution, such as that used in this study (30 m × 30 m), has limitations, such as the potential inability to estimate finer changes over time, the incapability to estimate biomass under dense canopies (lu et al. 2012), variation in estimation due to sensor angles in challenging terrain with steep slopes, and the potential to be affected by moisture content variation of the surrounding atmosphere. the accuracy of these results can be increased in future studies by adding other data sources, such as extended band sar, lidar, high spatial resolution, and auxiliary data for agb estimates (li et al. 2020). table 6 aboveground biomass in different forest types of bangladesh study hill forest (mg.ha-1) sundarbans mangrove forest (mg.ha-1) madhupur sal forest (mg.ha-1) nijhum dwip forest (mg.ha-1) mukul et al. (2014 a) 49. 5 88.2 34.5 19. 6 mukul et al. (2014 b) 103.4 ullah and al-amin (2012) 115.6 alamgir and al-amin (2007) 73.6 shin et al. (2007) 92.0 rahman et al. (2015) 98.9 donato et al. (2011) 126.7 kibria and saha (2011) 153.9 bfd (2020) 24.03 49.28 31.36 21.79 present study, 2020 8.85 90.76 107.22 37.46 present study, 2014 7.89 89.73 110.01 44.36 estimation of change in aboveground biomass in four national forests in bangladesh – mojumder et al. 277 conclusion this satellite-based study found considerable variation in the aboveground biomass among the four different forest types of bangladesh. it showed that average agb stock increased in sundarbans reserve forrest and ukhiya hill forest while decreasing in nijhum dwip forest and madhupur sal forest. bangladesh has set a target to stop forest losses and increase forest cover from 22.37% to 24%, reforest 150,000 ha of coastal areas, and restore 137,800 ha of destroyed and 200,000 ha of degraded hill and sal forest by 2030 (moefcc 2021). this study helps to identify areas under human pressure and could be used to identify priority areas for action. this study had a lower estimate of agb than some other studies (e.g., 159.5 to 360 mg.ha-1 by rahman et al. (2015)). these studies’ estimates were very close to studies with similar inventory or harvest data-based methods (e.g., saatchi et al. (2011)) but there was wide variation with other methods. better agreement between methods and an improvement in data quality can be enhanced by the replication of the study and including ground truthing in the study areas, especially for the dominant species like nypa fruticans for sundarbans reserve and nijhum dwip forest or shorea robusta for madhupr sal forest. bangladesh needs an accurate national level assessment of forests using both satellite and inventory based methods that can be used to improve forest management and aforestation planning to achieve nationally determined contribution (ndc) goals by 2030. our research will be useful in determining how the agb biomass of forests under human pressure has changed over time. the results may be used to compare the change, which will help to determine the root reasons for the agb change. identifying these areas and the associated causes of the change can guide the organizations and agencies who are involved in protecting and managing these areas to develop targeted plans to reduce forest loss in the future. references akhtar, n., uddin, m. k., & tan, y. 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(2010). tree diversity in the homestead and cropland areas of madhupur upazila under tangail district. j. agrofor. environ, 4(1), 89-92. the southeast asian journal of tropical biology vol. 31 no. 2, 2024: 266 276 doi: 10.11598/btb.2024.31.2.2175 issn: 0215-6334 | e-issn: 1907-770x 266 article higlights • nickel mining activities significantly impacted the ecological integrity of the pomalaa coastal area in southeast sulawesi, particularly in the mangrove ecosystem. • the exposure of overburden waste in the mangrove ecosystem has led to the loss of important gastropod species and a decline in population of surviving species • the low population of gastropod species in nickel mining impact areas indicates extensive environmental damage caused by these activities. article information received 24 january 2024 revised 15 april 2024 accepted 17 april 2024 *corresponding author, e-mail: muhammadfajarpurnama@students. undip.ac.id; muhammadfajarpurnama@uho.ac.id research article abstract gastropod resources in the dawi-dawi mangrove ecosystem, southeast sulawesi are greatly influenced by the input of overburden wastewater from nickel mining activities. this condition has been going on for the last 2 decades and has degraded the habitat and structure of the gastropod community. the upstream connectivity associated with the existing nickel mining area and the dawi-dawi river estuary mean that the mangrove ecosystem in this area is continuously affected by overburden runoff. this research was carried out in june-august 2023 in the mangrove ecosystem of dawi-dawi village, southeast sulawesi province. this research aimed to determine the existing condition of mangrove gastropods affected by overburden. this research began by conducting initial observations in the field, to identify mangrove ecosystems affected by overburden. research stations were determined using a purposive sampling technique and sampling of gastropods was carried out by using a simple random sampling method. the ecological index of mangrove gastropod community affected by overburden showed a tendency for a medium diversity index (h’ = 1.81), low species richness (r = 1.42), high evenness index (e = 1.12), and no dominance of certain species (c = 0.19). the gastropod species found were of typical types that made up the mangrove fauna, namely terebralia sulcata, terebralia palustris, faunus ater, telescopium telescopium, hebra nigra, and several taxa of climbing littoraria snails which were found in the roots, stems and leaves of mangroves. the abundance of gastropods obtained was also very low, namely around 0-12 ind./m2. at some points (rarely) schools of small snails of the type t. sulcata (5-21 ind./m2) were sometimes found. this research showed that overburden waste eliminates or reduces the diversity and richness of gastropod species in the mangrove ecosystem of dawi-dawi. keywords: ecological index, gastropods, nickel mining, overburden existing conditions of gastropod communities in areas affected by overburden nickel mining in the mangrove ecosystem of dawi-dawi, southeast sulawesi muhammad fajar purnama1,2*, slamet budi prayitno3, max rudolf muskananfola3, suryanti suryanti3 1doctoral program of aquatic resources management, faculty of fisheries and marine science, diponegoro university, semarang 50275, indonesia. 2aquatic resources management, faculty of fisheries and marine science, halu oleo university, kendari 93232, indonesia. 3aquatic resources management, faculty of fisheries and marine science, diponegoro university, semarang 50275, indonesia https://doi.org/10.11598/btb.2024.31.2.2175 gastropod communities in areas affected by nickel mining overburden purnama et al. 267 introduction community structure is a concept that examines the arrangement or composition of various species and their abundance in a community (dibyanti 2023; hanny 2023; anggie 2022; daulima 2021; montesinos-navaro et al. 2018; hidayani 2015). three approaches are generally used to describe community structure, namely 1) species diversity, 2) species interactions, and 3) functional interactions (jennifer 2023; hikmatul 2023; ashari 2023; schowalter 1996). community structure can be studied through the composition, size, and diversity of species which are closely related to habitat conditions. changes in habitat will affect community structure because changes in habitat will affect the species level as the smallest component that makes up the population in the community. one of the organisms that make up the basic aquatic ecosystem is mollusks, such as gastropods and other benthic biota (amin et al. 2023; aryanti et al. 2023; hasanah et al. 2023; mustofa et al. 2023; yulianto et al. 2023; lestari et al. 2021; kinasih et al. 2018; putro 2017; mushthofa et al. 2014; nugroho et al. 2012). gastropod community in the mangrove ecosystem of dawi-dawi village, southeast sulawesi is strongly influenced by the input of overburden waste or reddish-orange sediment originating from nickel mining activities, both on a temporal and spatial scale. overburden waste is carried by river flows that are directly connected to the upstream area where nickel mining is located and trapped in the mangrove ecosystem of dawidawi, pomalaa, indonesia (hamzah et al. 2015; hamzah 2009; zubayr 2009). gastropods are benthic organisms that walk with their legs on the ground floor of the mangrove ecosystem so that almost all their biological activities are carried out on the bottom substrate (harminto 2003; chukaeva & petrov 2023; menon et al. 2023; campbell et al. 2008). the “substrate” parameter is one of the key variables for the survival of the gastropod community in the mangrove ecosystem (litaay et al. 2023; mustofa et al. 2023; yuanike 2023). disturbances that occur in the substrate directly have implications for the destruction of their natural habitat and can slowly cause the death of individual gastropods (magfirah et al. 2014). this condition has occurred in the dawi-dawi mangrove ecosystem since 5 decades ago (1968) or half of century ago until now (2023). overburden waste is the waste rock in the form of mud/acid rock drainage which contains low mineral content (hamzah et al. 2015; hamzah 2009; zubayr 2009). long and continuous exposure times cause the thickness of the overburden sediment to increase range from 10-30 cm, so that overburden exposure in the dawi-dawi mangrove ecosystem systemically degrades the living habitat and causes a drastic reduction in the abundance and taxa of the gastropod community. empirical research on the structure of gastropod communities in overburden exposure areas in southeast sulawesi is not yet available, even in indonesia generally. therefore, research regarding the existing conditions of gastropod communities in mangrove ecosystems affected by overburden is very important, considering that scientific information regarding gastropod taxa that can survive in environments exposed to overburden waste is not yet available. apart from that, this research has never been carried out on the coast of dawi-dawi village, southeast sulawesi. this research aimed to determine the existing condition of mangrove gastropods affected by overburden. materials and methods time and location of research this research began with preliminary research activities in the field for one month. preliminary research takes the form of direct observation or survey at the research location to obtain initial information regarding research stations and gastropods. this observation activity helps researchers determine research station points and facilitates the process of determining them. in addition, the distribution of the gastropod community at the research location is specifically known, making it easier to collect samples when the research is carried out. this research was carried out in the period of july-september 2023, taking place in the mangrove ecosystem of dawi-dawi village, pomalaa district, kolaka regency, southeast sulawesi province. research stations were spread across mangrove areas exposed to overburden, consisting of 5 sampling points with 3 substations at each station. repetition in this research was focused on the spatial distribution to strengthen the justification for concluding the condition of the gastropod community exposed to overburden in the dawi-dawi mangrove ecosystem. biotropia vol. 31 no. 2, 2024 268 figure 1 (a-e) are several mangrove areas affected by overburden in dawi-dawi village, pomalaa. these five areas are the only mangrove areas in dawi-dawi village exposed to acid mine sludge waste (overburden), which is the main reason for determining them as research locations. the research locations were located at the mouth of the dawi-dawi river and were also the locations for sampling the gastropod community or stations in this study, i.e., (a) station 1 was located to the north of the dawi-dawi river estuary which was dominated by pidada vegetation (sonneratia spp.); (b) station 2 was to the south of the dawi-dawi river estuary which was overgrown by mangrove vegetation (rhizophora spp.), pidada (sonneratia spp.), and api-api (avicennia spp.); (c) station 3 was to the west of the dawi-dawi estuary which was dominated by mangrove vegetation (rhizophora spp.); stations 4 (d) and 5 (e) were located to the east of the dawi-dawi estuary, which was 100150 m from each other; stations 4 and 5 were also dominated by mangrove and pidada vegetation. figure 1 research locations with overburden exposure in the dawi-dawi mangrove ecosystem figure 2 research stations in the estuary of dawi-dawi village, pomalaa source: google earth (2024) gastropod communities in areas affected by nickel mining overburden purnama et al. 269 sampling method sampling locations were determined by using a purposive sampling technique or deliberately based on the criteria “exposure to overburden waste” in the mangrove ecosystem of dawi-dawi village. the placement of substations or distribution of sampling points for the gastropod community used the simple random sampling method. meanwhile, the technique for collecting gastropod samples at each station adopted the handpicking technique. generally, mangrove gastropods are benthic epifauna, so the collection process is very easy, just use your gloved hands. during the mangrove data collection, observations were made on the community structure of gastropods. the data were collected using quadrat transects of different sizes. for tree-type mangroves, a 10 m² quadrat was used, for sapling-type mangroves a 5 m² quadrat was used, and for seedling-type mangroves as well as for observing samples of the gastropod community, a 1 m² quadrat was used. seven to ten quadrat plots were placed at each station to represent the station area, to make it easier to estimate the density and diversity of mangrove vegetation and the density of the gastropod population. the gastropod samples observed in this study were individual gastropods in living conditions. this was done to strengthen the justification for the relationship between mangrove density and gastropod diversity and density. the focus of observing gastropod samples in the mangrove ecosystem is centered on two areas, namely (1) the benthic substrate and (2) mangrove trees, specifically the roots, stems, and leaves of the mangrove where climber periwinkle gastropod dwelling. these types of climbing snails are dominated by littoraria sp. data analysis gastropod community data obtained from sampling results were analyzed using the following ecological index formulations: 1) the abundance of gastropods was analyzed using the formulation of yasman (1998): where: a = abundance (ind./m2) xi = number of individuals (ind.) ni = sample plot (m2) 2) analysis of the diversity of gastropod species using the shannon-wiener diversity index (odum 1993), with the formula: where: h’ = diversity index ni = number of individuals of each species n = total number of individuals according to wilhm (1975) the diversity index criteria are divided into 3 categories, namely: h’ < 1.0 = low species diversity 1.0 < h’ < 3 = medium species diversity h’ > 3 = high species diversity 3) the gastropod evenness index was analyzed using the formula according to odum (1993): where: e = evenness index h’ = diversity index s = number of species the criteria for the species evenness index value are as follows: e < 0.31 = low level of species evenness 0.31 > e > 1 = medium level of species evenness e > 1 = high level of species evenness 4) the species richness index (margalef index) was analyzed based on the formula formulation according to ludwig and reynolds (1988): where: s = number of species n = number of individuals the criteria for the margalef type wealth index value are as follows: d < 2.5 = low level of species richness 2.5 > d > 4 = medium level of species richness d > 4 = high level of species richness biotropia vol. 31 no. 2, 2024 270 5) the dominance index was calculated using the formulation according to odum (1993): where: ni = number of individuals i n = total number of individuals of all species the dominance index criteria consist of: 0 < c < 0.5 = no type dominates 0.5 < c < 1 = there is a dominant type results and discussion ecologically, the mangrove ecosystem is valued as a sediment trap. this ecological function is beneficial from an environmental perspective in a broad sense. however, it may harm aquatic organisms that inhabit the bottom of the waters if the trapped waste sediment from industrial activities for example contaminated with nickel. river flows carrying sediment with waste materials from nickel mining activities in upstream areas can directly cover the mangrove ecosystem’s ground floor. on the other hand, the mangrove substrate becomes a living space and feeding ground area for benthic organisms, one of which is the gastropod community. these conditions impact changes in the ecological niche and structure of the gastropod community. the impact is proven in this study by the research results in the dawi-dawi mangrove ecosystem in the pomalaa district. the results showed a decrease in the diversity (medium) and species richness (low) of gastropods in areas affected by overburden. the number of gastropod species found inhabiting the dawi-dawi mangrove were only seven species (telescopium telescopium,  terebralia sulcata,  terebralia palustris,  faunus ater,  herba nigra, littoraria scabra, and littoraria melanostoma), meanwhile, in areas not affected by overburden (reference site), the number of species was much higher, namely 34 species. in contrast, in the reference site area or areas with minimal disturbance, it was found to be ± 30 species. the low richness of gastropod species in the dawi-dawi mangrove ecosystem has implications for high species uniformity (e = 1.12). if this condition continues, a tendency for dominance by certain species may exist, although this has not happened (c = 0.19). the results of the gastropod ecological index analysis in the dawidawi mangrove ecosystem, pomalaa, are presented in table 1. the gastropod ecological index analysis reveals that ecological pressure or an environmental imbalance has disrupted the homeostasis system in the dawi-dawi mangrove ecosystem (table 1). the community’s expression of its environment’s quality is evident in the gastropod community structure, where diversity metrics, specifically species richness, indicate low indices. the dawidawi mangrove ecosystem exhibits a low number of gastropod taxa (species), directly influencing the ecological index. the diversity of gastropods in this ecosystem fell into the medium category with a tendency for low index values (h’ = 1.81), while the richness was in the low category (r = 1.42). the impact of low species richness in the dawidawi mangrove ecosystem is further manifested in the high gastropod uniformity index (e = 1.12). this condition raises concerns about the potential domination by certain species in the future, even though it is not observed currently; the trend is moving in that direction (c = 0.19) (table 1). table 1 gastropod ecological index in the existing nickel mining area in dawi-dawi village, pomalaa-southeast sulawesi no. ecological index value category 1 diversity (h’) 1.81 medium 2 species richness (r) 1.42 low 3 evenness (e) 1.12 high 4 dominance (c) 0.19 no one type dominates gastropod communities in areas affected by nickel mining overburden purnama et al. 271 these ecological components serve as benchmarks for evaluating environmental quality or the health condition of an aquatic ecosystem. overburden waste pollution in the dawi-dawi mangrove ecosystem not only alters the color of the substrate and water to reddish-brown in the mangrove area but also induces systemic changes in the life of aquatic organisms. this impact is visibly reflected in the structural composition of the gastropod community in the dawi-dawi mangrove landscape. this observed condition aligns with the findings of sinapoy et al. (2020) regarding the diversity index (h’) of gastropods in a mangrove ecosystem in the former nickel mining area in tokowuta village, lasolo. the diversity index was found to be in the low category (h’ = 1.36). the evenness index (e) indicated evenness with an unstable community at 0.70, while the dominance index, categorized as moderate in 0.35. the gastropod species diversity index in this area was classified as low, with moderately polluted water quality. this is attributed to the accumulation of chemicals from nickel mining, transported by water and settling in sediment (overburden). a low diversity index value indicates low species richness, and there tends to be only one or a few species that have an abundant number of individuals. this condition causes this area to have low species diversity, the distribution of the number of individuals of each species is low, and the stability of the community is low due to the waters being polluted by nickel metal levels in the waters and sediments. according to rachmawaty (2011), species diversity in an area is influenced by several factors, namely polluted substrate, availability of food sources, interand intra-species competition, disturbances, and conditions of the surrounding environment so that species that have high tolerance will increase while those that having low tolerance will further decrease. the types of gastropods affected by overburden in the dawi-dawi, pomalaa mangrove area are also relatively the same as the gastropods found during research in the mangrove forest area of the former nickel mining area in tokowuta village, lasolo, consisting of 7 species, namely  terebralia sulcata,  cerithidea cingulata, cerithidea quadrata, nerita lineata, littoraria scabra,  littoraria melanostoma  and  telescopium telescopium (sinapoy et al. 2020).  the dominance index value obtained in this area was 0.35. this value is similar to the gastropod dominance index in the dawi-dawi mangrove ecosystem (c = 0.19), both in the low category. this value showed that in general, there are no dominant gastropods. the absence of dominant gastropod species in this area is thought to be due to the relatively small number obtained due to the decreasing quality of the aquatic environment, which is no longer suitable for gastropod species to live, except for species that have a high tolerance range for changes in environmental conditions. kharisma et al. (2012) stated that the dominance index determines the type of gastropod that dominates a community and the influence of environmental quality on an individual’s community. the results of another study which also had the same conditions, namely by supratman et al. (2018), stated that the results of the analysis of the gastropod diversity index in all research locations were categorized as small diversity because the diversity index value was < 2 (odum 1993).  the low diversity index at this location was caused by the extreme environmental conditions, such that certain species dominate the gastropods. the high dominance of a species will affect the low diversity of gastropods at the research location. the high dominance of a species can be caused by several factors, such as polluted habitat conditions so that only species that are tolerant of pollution can live in that habitat or the availability of abundant food sources for certain species so that other species cannot compete. likewise, according to arbi et al. (2019) and cappenberg (2006), high and low species diversity index values can be caused by several factors, namely the number of species obtained, the presence of species that dominate other species, substrate conditions, and ecosystem conditions as habitat for gastropods. ecosystem or environmental conditions are good if they show low diversity index (h’) and dominance index (c) values. the abundance of gastropod species in the dawi-dawi, pomalaa mangrove ecosystem is presented in figure 3. biotropia vol. 31 no. 2, 2024 272 the gastropod types f. ater (6-14 ind./m2) and t. sulcata  (2-9 ind./m2) have an average abundance far exceeding other species in the dawi-dawi mangrove ecosystem. the average abundance with the lowest value was found in the climber snail, l. melanostoma  (1-2 ind./m2). the image below shows the actual condition of the area affected by overburdened waste in the dawi-dawi mangrove ecosystem and the gastropod community living in it (figs. 4 & 5). dawi-dawi waters, pomalaa receives overburden waste input from nickel mining activities in upstream areas with connectivity (flow) with estuary or mangrove areas. this condition has been going on for the last ± 2 decades, resulting in many changes to the life of aquatic organisms in the dawidawi mangrove ecosystem (hamzah et al. 2015). among the many macrozoobenthos organisms affected by overburden waste, the gastropod community is a typical aquatic biota of the bottom of the waters (benthos), which has experienced a decline in population density (fig. 3) and its taxa. this decline happens because the ground floor of the mangrove ecosystem, which is the living and feeding ground for various types of gastropods, has been covered by overburdened sediment waste. in other words, the ecological niche which is rich in organic material and is needed by the gastropod community for its survival is directly degraded and lost due to input of overburden waste which continues to occur until now. only seven types of gastropods were found in the dawi-dawi mangrove ecosystem, which has a very low average density (2-5 ind./m2).  figure 3 average abundance of gastropod species in areas affected by overburden in the dawi-dawi mangrove ecosystem, pomalaa figure 4 river flow carrying overburden sediment (receding conditions) which empties into the dawi-dawi mangrove area, pomalaa. the area where overburden waste is trapped is to the east (a) and west (b) of the mangrove area gastropod communities in areas affected by nickel mining overburden purnama et al. 273 several types found in a large abundance, namely f. ater  (6-14 ind./m2) and t. sulcata  (2-9 ind./m2), are typical types of mangroves that have high resistance to changes in the quality of the aquatic environment. in mangrove areas, as well as other species such as  h. nigra,  l. scabra,  l. melanostoma,  t. telescopium  and  t. palustris (kurniawati et al. 2022; arbi et al. 2019; sarong  et al.  2017; sarong  et al. 2015; arbi 2014; ayunda 2011; wells 2003). furthermore, rachmawaty (2011) stated that species diversity in an area is influenced by several factors, namely polluted substrate, availability of food sources, interand intra-species competition, disturbances, and conditions of the surrounding environment so that species with high tolerance will increase. in comparison, those with low tolerance will decrease further. rachmawaty’s (2011) explanation above is very appropriate to the condition of the dawidawi mangrove ecosystem, which is polluted by overburdened sediment waste, and the impact is to eliminate feeding ground areas and other ecological spaces for the biological needs of the gastropod community. arbi et al. (2019) emphasized that changes in the function of mangrove areas are the main factor that can threaten the stability of their flora and fauna, including the gastropod communities associated with mangrove areas. figures 4 and 5 above show the massive impact of overburden waste in the dawi-dawi mangrove area, where not only is the entire substrate area covered by overburden waste, but the surface of the leaves and trunks of mangrove trees is also covered by reddish-orange dust. the gastropod community that lives inside is affected, where the surface of the shell and operculum is covered by overburdened sediment waste, making it even more difficult for gastropod movement when moving (fig. 5). the impact on the gastropod community is because, during high tide, the leaves and lower trunk/twigs figure 5 existing condition of the gastropod community in the area affected by overburden waste in the dawidawi mangrove ecosystem, pomalaa notes: terebralia sulcata gastropods covered in overburden mud (a, b), and the leaves, stems and climbing gastropods of the littoraria scabra species are exposed to overburden waste during high tide conditions, and visible at low tide on the reddish brown surface of the leaves, stems and shells of l. scabra (c, d). biotropia vol. 31 no. 2, 2024 274 of mangrove trees are submerged by water, which carries sediment particles (overburden). the types of gastropods found in the dawi-dawi mangrove ecosystem are shown in figure 6. the existing condition of gastropod taxa (species) in the dawi-dawi mangrove ecosystem that is affected by overburden is directly related to the unique characteristics of these types of snails as native inhabitants of mangrove habitats and are not facultative or migratory species. this correlation can be seen from the adaptability of these 7 species to changes in water quality or disturbances that occur in their ecological environment. rangan (2010) stated that the adaptability of a gastropod community to exponential changes in the habitat environment is an indicator or sign that the species can be categorized as native, facultative, and/or visitor species. the group of mangrove gastropods is known as a native species if the individual/ population of gastropods spends its entire life cycle in the mangrove ecosystem. meanwhile, the facultative group can be found in mangrove areas and beaches, where this type of gastropod species tends to have euryhaline properties. in contrast, the visitor gastropod group is the type of gastropods in marine areas carried to the mangrove forest by current activity and dynamic waves in coastal areas. odum (1993) also explained that a species with the highest density indicates that the organism can occupy a larger space and thus develop more. the condition of the gastropod community in the dawi-dawi mangrove ecosystem is in line with the results of research by maturbongs and elviana (2016), where the littorinidae and potamididae families are the majority groups that dominate mangrove forest areas. the input of overburden waste reduces the number of sensitive mangrove gastropod taxa and slowly and massively reduces the number of individuals of each species in the mangrove ecosystem due to the nature of gastropod communities, which tend to be sessile or have limited movement. maturbongs and elviana (2016) stated that the activities around the mangrove ecosystem greatly influence the density of the gastropod community, which affects the survival of gastropods because living gastropods tend to stay settled with limited movement. gastropods are very sensitive to changes in the water quality in which they live. changes in the aquatic environment, for example, in river basins, affect the composition and diversity of the population of this class (odum 1993). according to fachrul (2007), pristine waters usually have high species diversity and vice versa for poor or polluted waters. conclusion the abundance and number of taxa (species) of the gastropod community exposed to overburden waste in the dawi-dawi mangrove ecosystem experienced a drastic decrease from their natural conditions (without exposure to overburden). overburden waste has a negative impact on the structure of the mangrove gastropod community. the diversity index is in the medium category (h’ = 1.81), with low species richness (r = 1.42), high evenness (e = 1.12), and no dominance of certain species (c = 0.19). figure 6 seven species of gastropods found in the dawi-dawi mangrove ecosystem, pomalaa notes: (a) t. telescopium; 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(paper 5414). https://doi.org/10.33772/biowallacea.v7i1.11813 https://doi.org/10.33772/biowallacea.v7i1.11813 biotropia vol. 29 no. 1, 2022: 7 17 doi: 10.11598/btb.2022.29.1.1443 7 heavy metals contamination level and water quality parameter conditions in jatiluhur reservoir, west java, indonesia gatot prayoga*, bagus amalrullah utomo and hefni effendi environmental research center, institut pertanian bogor, bogor 16680, indonesia received 25 september 2020 / accepted 3 july 2021 abstract waste pollution into the citarum river, the main water source of jatiluhur reservoir, was dominated by the manufacturing industry such as textile, chemical, metal and pharmaceutical. in general, the manufacturing industry is the most common contributor to heavy metal waste, which will cause various health problems. therefore, it is essential to conduct studies on heavy metal contamination and water quality parameters conditions in the jatiluhur reservoir. the study aimed to provide a reference regarding the current condition of the heavy metal contamination level in both sediment and water of the jatiluhur reservoir, as well as to compare the levels of other water quality parameters against the standard of environmental quality. heavy metals contents, such as cu, zn, hg, pb and cd, were determined using x-ray fluorescence (xrf) spectrometry method (for sediment) and atomic absorption spectrometry (aas) method (for water). water quality parameters were analyzed by using methods developed by the indonesian national standard (sni). the data obtained were compared to the canadian sediment quality guidelines (for heavy metal in sediment) and water quality standards from the government of the republic of indonesia regulation number 82 of 2001 (class 3) (for water quality parameters). based on this study, jatiluhur reservoir is divided into three zones i.e., the inlet area, main inundation area and outlet area. within the sedimentary layer, the mercury (hg) was found to be accumulated throughout the jatiluhur reservoir area, exceeding the maximum limit, while cu accumulated in the inlet area, exceeding the minimum limit. the other heavy metals (zn, pb and cd) were found to be exceeding the minimum limit at some locations, but more results were below the minimum limit. the high concentration of heavy metals in the sediment was due to household and/or industrial wastes. although all heavy metals were not detected in water, the presence of heavy metals in sediments could potentially dissolve into the water by means of upwelling. if this happens, the heavy metals can be excessively contained in water, resulting in a harmful habitat for aquatic biota. water containing heavy metals will also be harmful for human. in general, water quality parameters in jatiluhur reservoir meet the standard for water quality. only ammonia, however, was higher than the standard for sensitive fish life due to massive aquaculture activities in this reservoir. considering the conditions of heavy metal contamination levels in both sediment and water, the biota that is most likely to be exposed to heavy metal is benthic organisms, because the organisms live at the bottom of the waters. the priority for further attention and countermeasure in improving the sediment and water quality of the jatiluhur reservoir was toward hg, cu, and pb and ammonia. keywords: heavy metal, reservoir, sediment, water introduction jatiluhur reservoir was the largest reservoir in indonesia having an area of 8,300 ha. jatiluhur reservoir was built in 1957. the reservoir is the first multipurpose reservoir in indonesia with potential available water of 12.9 billion m3/year. the jatiluhur reservoir was built by blocking the citarum river. the river passes through various anthropogenic activities and many cities in the west java province. among functions of the jatiluhur reservoir are as hydropower, as a source of irrigation for 242,000 ha of surrounding rice fields, as a source of drinking water, as a place for inland fisheries (fish farming in floating net cages (kja)), as flood control, and as tourism and water sports facilities. the types of fish commonly being *corresponding author, email: gatotprayoga16@gmail.com biotropia vol. 29 no. 1, 2022 8 cultivated in the floating net cages (kja) in the jatiluhur reservoir include common carp, nile tilapia, red devils and catfish (pangasius spp.) (suprian & salami 2011). in the past few years, fishes in the jatiluhur reservoir seem to have been contaminated and are not suitable for consumption (republika.co.id 2009). likewise, in 2018, a report from anggoro (2018) stated that there was an appeal for not consuming any kinds of fish from this reservoir. the contamination is allegedly due to the influence of various pollutions from the citarum river as the main water source of the jatiluhur reservoir. the contamination may have also occurred due to the wastes of fish feed from the many kjas in the reservoir. industrial activities are the most common contributor of heavy metal waste because heavy metals are used in industrial activities as raw materials, additives and catalysts (hutagalung et al. 1997 in vigers et al. 1996). heavy metal contamination in waters causes various health problems for aquatic biota and humans, such as problems in the nervous system, respiratory system, liver function, kidney function and growth of bones (sanusi 1985). heavy metals are categorized as harmful pollutants because it cannot be destroyed (nondegradable) by living organisms, so they will settle at the bottom of the waters and accumulate (rochyatun & rozak 2007). heavy metals can bond with organic compounds to form complex compounds that eventually settle at the bottom of the water (accumulate in sediments) (marchand et al. 2006). on the other hand, sediments were an inseparable part of aquatic ecosystems that can provide habitat, feeding grounds, spawning grounds, and nurseries for various aquatic organisms. contaminated sediments can reduce or eliminate the aquatic organisms that have important values for ecology, commercial or recreational uses (us epa 2001). fatoki and mathabatha (2001) stated that sediment has a function as a metal container which can release the metal into the water through natural and anthropogenic processes. heavy metals deposited in sediments can cause changes in water quality and transfer toxic chemicals to aquatic organisms (permanawati et al. 2013). physical and chemical water quality parameters such as dissolved oxygen (do), ph, total organic content, temperature and dissolved ion affect the life of aquatic organisms (effendi 2003). water quality parameters is influenced by its catchment area which is related to human activities (wiwoho 2005; asrini 2017). a study conducted by garno (2002) showed that the jatiluhur reservoir was hypertrophic (very nutrient-rich) and phytoplankton bloom can occur any time. the hypertrophic condition was largely influenced by the aquaculture activities in this reservoir and the influx of organic matters from settlements. poor water quality conditions can disrupt aquatic life in the reservoir and reduce the diversity of aquatic biota. a study in cirata reservoir carried out by komarawidjaja et al. (2005) showed that there was a growth disturbance in common carp which was thought to be closely related to water quality, especially with the high concentration of chlorophyll-α and total n in the waters. poor water quality of the jatiluhur reservoir will also have an adverse impact on humans due to the function of the reservoir as a source of drinking water and as tourism and water sports facilities. based on the issues above, the study of heavy metal contamination and water quality parameter conditions in the jatiluhur reservoir should be conducted due to the serious polluted condition in the reservoir. this study aimed to obtain data on current condition of the heavy metal contamination level that has occurred in both sediment and water, as well as to compare the levels of other water quality parameters against the standard of environment quality. materials and methods location and time the research was conducted in jatiluhur reservoir in february 2019. water and sediment samples were taken from 6 (six) locations in the reservoir, consisting of 3 (three) zones i.e., the inlet area, main inundation area, and outlet area (fig. 1). the six locations were: 1. citarum river inlet; 2) jamaras; 3) cilalawi river inlet; 4) kja zone 5; 5) pasir kole and 6) dam (outlet). heavy metal contamination and water quality parameter conditions in jatiluhur reservoir – gatot prayoga et al. 9 figure 1 map of sampling locations in jatiluhur reservoir methods the water sample was taken using van dorn water sampler, then analyzed in the global qa laboratory using the methods from the indonesian national standard (sni). sediment sampling was conducted by using an ekman grab. the sediment samples obtained were then analyzed in the central nuclear material technology laboratory, batan using the xray fluorescence spectrometry (xrf) method to find out the heavy metals (cu, zn, hg, pb and cd) contents. the xrf spectrometry method is an application of radioisotopes used as an analytical method for detecting heavy metal content, especially in solid substances such as sediment. this method is still not commonly used in aquatic ecological studies that mostly use the aas method. methods used for analyzing the water and sediment samples are presented in table 1. data analyses data of heavy metal contents (in water and sediment) and water quality parameters obtained from this study were compared to the quality standard. the sediment parameters (heavy metal) were compared to the canadian sediment quality guidelines for the protection of aquatic life on freshwater set out by the canadian council of the ministry of environment (ccme). reference to heavy metal quality standards according to ccme standard was presented in table 2. data on water quality parameters were compared to water quality standards from the government regulation of the republic of indonesia number 82 of 2001 (class 3, for the cultivation of freshwater fish, animal husbandry, water to irrigate crops, and or other purposes that require the same water as these uses). inlet area inlet area outlet area main inundation area 1 2 3 4 5 6 biotropia vol. 29 no. 1, 2022 10 table 1 parameters and methods used for analyzing water and sediment samples parameter unit method a. water physical properties: temperaturea °c 5.4/ik/gqa/wq/002 total dissolved solid, tdsa mg/l sni 06-6989.27-2005 total suspended solid, tssa mg/l sni 06-6989.3-2004 chemical properties: pha sni 06-6989.11-2004 biological oxygen demand, bod5 a mg/l sni 6989.72:2009 chemical oxygen demand, coda mg/l sni 6989.2:2009 dissolved oxygen, doa mg/l sni 06-6989.14-2004 total phosphate as pa mg/l 5.4-ik-gqa-wq-062 nitrogen, nitrate as n (no3-n)a mg/l 5.4-ik-gqa-wq-043 ammonia, nh3-na mg/l sni 06-6989.30-2005 copper, cua mg/l sni 6989.6:2009 zinc, zna mg/l sni 6989.7-2009 mercury, hga mg/l sni 6989.78:2009 lead, pba mg/l sni 6989.46:2009 cadmium, cda mg/l sni 06-6989.38-2005 b. sediment copper, cu µg/g xrf spectrometry zinc, zn µg/g xrf spectrometry mercury, hg µg/g xrf spectrometry lead, pb µg/g xrf spectrometry cadmium, cd µg/g xrf spectrometry note: a = accredited by the national accreditation committee (kan). table 1 quality standards for heavy metals in sediments (for freshwater) based on ccme (2001) heavy metal concentration (mg/kg dry wt) isqga pelb cu 35.7 197.0 zn 123.0 315.0 hg 0.170 0.486 pb 35.0 91.3 cd 0.6 3.5 notes: a = isqg (interim sediment quality guidelines): the value of the minimum limit, i.e., the limit of metal concentrations that has the low possibility to cause a negative biological effect; b = pel (probable effect level): the value of the maximum limit, i.e., the limit of metal concentrations having a greater possibility to cause a negative biological effect. results and discussion heavy metal contaminations our study showed that all heavy metals (cu, zn, hg, pb, and cd) accumulated in the sediment layer of the jatiluhur reservoir and had exceeded the minimum limit (isqg). mercury (hg) concentration exceeded the maximum limit (pel) throughout the jatiluhur reservoir area. copper (cu) tended to accumulate in the inlet area, with concentration exceeding the minimum limit. the concentration of zinc (zn) and lead (pb) exceeded the minimum limit at some locations (such as sampling point 5), however, the concentration of cadmium was below the detection limit of the measuring tools (table 3). cadmium (cd) seems to be relatively the safest metal found in the sediment samples compared to the other heavy metals. heavy metal contamination and water quality parameter conditions in jatiluhur reservoir – gatot prayoga et al. 11 table 3 results of laboratory analysis for heavy metal in sediment samples station test element (mg/kg) cu zn hg pb cd 1 citarum river inlet #1 39.78 105.16 2.1 29.4 <0.3 #2 40.34 96.32 2.5 29.7 <0.3 #3 35.71 93.51 1.8 28.6 <0.3 2 jamaras #1 36.91 113.59 1 26.8 <0.3 #2 38.42 106.6 <0.7 26.8 <0.3 #3 41.46 133.2 2.5 28.8 <0.3 3 cilalawi river inlet #1 35.79 129.82 <0.7 34.8 <0.3 #2 84.12 103.31 2.4 31.9 <0.3 #3 73.73 92.06 3.2 30.7 <0.8 4 kja zone 5 #1 22.3 114.48 1.1 30.2 <0.8 #2 12.94 104.27 2.5 25.9 <0.3 #3 19.01 113.03 3.8 34 <0.3 5 pasir kole #1 18.69 139.94 2.2 40 <0.3 #2 23.57 143.16 1.5 36.3 <0.3 #3 26.28 147.98 3.2 40.1 <0.3 6 dam (outlet) #1 20.13 89.41 <0.7 35.3 <0.3 #2 74.13 103.39 3.6 32.5 <0.8 #3 27 97.69 <0.7 31 <0.3 quality standarda 35.7 123 0.170 35.0 0.6 197.0 315 0.486 91.3 3.5 notes: a = based on the canadian sediment quality guidelines for the protection of aquatic life on freshwater (ccme 2001). exceed the isqg (minimum limit) exceed the pel (maximum limit) mercury (hg) was among the most toxic metals for aquatic biota. mercury can be released from anthropogenic activities, such as from the antifouling paint for the hull of ships, slimicides used in the lumber and paper industries, pesticides and seed dressings in agriculture and pharmaceuticals (garcia-rico et al. 2006). mercury has many benefits, but it is very toxic and has a high level of bioaccumulation capabilities (garcia-rico et al. 2006; palar 2012). mercury concentration exceeding the safe limit endangers the life of aquatic biota, either directly or indirectly (palar 2012). therefore, the high concentration of mercury in the jatiluhur reservoir should be handled immediately, especially when there is an upwelling process in the reservoir. our study showed that the concentration of cu in sediments was much higher and exceeded the minimum limit in inlet areas, whereas the concentration of zn and pb was high in the main inundation areas. the spatial difference of the heavy metals’ concentrations might be due to the heavy influx from the river into the reservoir, which was already contaminated by various anthropogenic activities. cu is commonly used in insecticides, fungicides, brass alloy materials for household appliances, machine parts, as well as in water purification or as a food additive (palar 2012). household wastes in the form of metabolic waste and corrosion of pipes in residential areas usually also contain cu (connell & miller 2006). the main inundation area has the longest water retention time compared to the other areas and is also much affected by the kja aquaculture activities (marked with black lines in fig. 1). household wastes, water flow from urban areas and phosphate utilization (po4), contributed significantly to the entry of pb metal into the waters (connell & miller 2006; harteman 2011). sources of zn, cu and pb are chemical fertilizers, household wastes such as corrosion of pipes and detergent, as well as industrial wastes such as battery materials, cosmetics, plastics, rubber, soap, paint and ink, television tubes and fluorescent lamps, deodorants and chemicals for wood preservation (connell & miller 2006). biotropia vol. 29 no. 1, 2022 12 table 4 results of laboratory analysis results for heavy metal in water samples station element (mg/l) cu zn hg pb cd 1. citarum river inlet < 0.006 < 0.004 < 0.00009 < 0.0002 < 0.00004 2. jamaras < 0.006 < 0.004 < 0.00009 < 0.0002 < 0.00004 3. cilalawi river inlet < 0.006 < 0.004 < 0.00009 < 0.0002 < 0.00004 4. kja zone 5 < 0.006 < 0.004 < 0.00009 < 0.0002 < 0.00004 5. pasir kole < 0.006 < 0.004 < 0.00009 < 0.0002 < 0.00004 6. dam (outlet) < 0.006 < 0.004 < 0.00009 < 0.0002 < 0.00004 quality standarda 0.020 0.050 0.00200 0.0300 0.01000 notes: a = based on government regulation of the republic of indonesia number 82 of 2001 (class 3). the concentrations of heavy metals in water in this study were very low, below the detection limit of the tool (table 4), which indicated that the concentrations of heavy metals (cu, zn, hg, pb, and cd) in the water of the jatiluhur reservoir were still within the safe limits. this fact was in agreement with a study in jatiluhur reservoir in 2011 conducted by suprian and salami (2011), especially for mercury concentration which was below the quality standard of the government of the republic of indonesia number 82 of 2001 (class 3). despite the results that all heavy metals in the water samples were below the safe limit, however, heavy metals in sediments could potentially dissolve into the water. suprian and salami (2011) stated that although the value of mercury in water is below the safe limit, however, the existence of mercury has to be monitored because it is difficult to eliminate mercury from the water. according to fatoki and mathabatha (2001) and permanawati et al. 2013, sediment can release heavy metal into the water through natural and anthropogenic processes that cause changes in water quality and transfer of toxic chemicals to aquatic organisms. in reservoirs, the most extreme natural process that allows this to happen is upwelling. palar (2012) stated that the 0.01 ppm concentration of cu in water is deadly for phytoplankton, pb concentration of 2.75-49 ppm is deadly for crustaceans, while pb concentration of 188 ppm is deadly for fish, and cd concentration of 0.0028-4.6 ppm is deadly for oligochaeta. mercury concentration of ≥ 0.16 ppm is able to reduce the survival and growth rates of fish caused by the increase of stress and organ damage, whereas mercury concentration of ≥ 3 ppm causes mass mortality in common carp (nirmala et al. 2012; tyas et al. 2013). lethal toxicity test by prayogo et al. (2016) showed that 96 hours mercury exposure with concentration of 0.396 ppm in water had a 50% lethal effect on nilem carp (osteochilus hasselti). if the heavy metals content in the sediment are released into the water, then the concentration of heavy metals can be excessive in the water, causing adverse impact to humans and aquatic biota. considering the conditions of heavy metal contamination levels in both sediment and water, the biota that is most likely to be exposed to heavy metal is benthic organisms, because benthic organisms live at the bottom of the waters (reynolds 2012). heavy metals can cause a decrease in species richness of benthic organisms and a change in species composition of benthic macroinvertebrate communities (qu et al. 2010). cu concentrations of 70-90 mg/kg, zn concentrations of ±350 mg/kg and pb concentrations of 30-40 mg/kg caused the decrease of polychaeta and molluscs biodiversity at the bottom of jakarta bay, especially in industrial areas (takarina & adiwibowo 2011). the same occurrences might happen in the jatiluhur reservoir, especially for cu and pb which concentrations have reached high level of contamination. qu et al. (2010) stated that even though contamination by heavy metals is low in the sampling area, the adverse impact on benthic organisms are significant, suggesting that the chronic effects of long-term exposure to heavy metals in aquatic communities could be serious. thus, it is essential to focus our attention and efforts to handle contamination of hg, cu, and pb in the jatiluhur reservoir. heavy metal contamination and water quality parameter conditions in jatiluhur reservoir – gatot prayoga et al. 13 water quality parameter conditions in general, water quality parameters in our study met the quality standards (qs) based on the government of the republic of indonesia regulation no. 82 of 2001 (class 3). based on direct measurements on-site for all sampling locations, the temperature ranged from 26.5 °c up to 27.1 °c (qs ± 3), dissolved oxygen (do) concentrations ranged from 4.2 mg/l up to 4.5 mg/l (qs min. 3 mg l), and the water ph ranged from 6.37 up to 7.17 (qs 6-9). results of the laboratory analysis on the other parameters of water quality are presented in figure 2. figure 2 values of several water quality parameters measured in the jatiluhur reservoir note: quality standards based on the government of the republic of indonesia regulation no. 82 of 2001 (class 3), except for ammonia which was not available. biotropia vol. 29 no. 1, 2022 14 results of our study showed that tds concentrations ranged from 120 mg/l up to 136 mg/l (qs 1,000 mg/l), tss concentrations ranged from 4 mg/l up to 33 mg/l (qs 400 mg/l), bod concentrations ranged from 0.2 mg/l up to 4.7 mg/l (qs 6 mg/l), cod concentrations ranged from 2 mg/l up to 12 mg/l (qs 50 mg/l), total phosphate concentrations ranged from 0.01 mg/l up to 0.10 mg/l (qs 1 mg/l), nitrate concentrations ranged from 0.01 mg/l up to 0.47 mg/l (qs 20 mg/l) and ammonia concentrations ranged from 0.095 mg/l up to 1.940 mg/l (qs 0.02 mg/l). of the ten observed water quality parameters (not including heavy metals concentrations), only ammonia that did not meet the quality standards of water quality parameters. ammonia is a nitrogen compound that changes to nh4 ions at low ph conditions. ammonia can also come from domestic and industrial wastes (marganof 2007). the high content of ammonia in the reservoir was presumably due to the fish feed and fish fecal wastes as a result of aquaculture activities in the reservoir. fish emit 80-90% ammonia (n-inorganic) through the osmoregulation process, while feces and urine account for 10-20% of total nitrogen (rakocy et al. 1992 in sumoharjo 2010). ammonia in the reservoir can come from organic and inorganic nitrogen sources found in soil and water or from the decomposition of organic matters by microbes and fungi. ammonia also comes from the denitrification process during the decomposition of wastes by microbes under anaerobic conditions (effendi 2003). commonly, the concentration of ammonia in the pond should not exceed 0.05 mg/l. according to sni 6139:2009 (national standardization agency 2009), the ammonia value resulting from the nile tilapia aquaculture activity in calm water ponds should not exceed 0.02 mg/l. ammonia concentrations of 0.020.07 mg/l have been shown to inhibit growth and cause tissue damage in several fish species. the toxicity threshold value for ammonia is highly dependent on the type of species, size, fine solids, surface-active compounds, metals and nitrates (colt 2006). based on anas et al. (2017), the water quality status of the jatiluhur reservoir is classified as moderate. the moderate status was resulted from the storet calculation, stated in the decree of the minister of environment no. 115 of 2003) which was caused by high concentrations of bod, cod and ammonia. the main contributor to the high concentrations of organic matter in the jatiluhur reservoir presumably are the number of operating kja in the reservoir. based on the regent of purwakarta regency decree no. 6 of 2000, the optimal number of kja to operate in the jatiluhur reservoir is 2,100 plots. meanwhile, in 2015 there were 18,038 kja in the reservoir, which exceeded the carrying capacity of the waters (astuti et al. 2016). a recent study conducted by fitri et al. (2016) stated that in 2014 the number of intensive kja in the jatiluhur reservoir was already excessive, amounting to 23,000 cages. according to their study, the optimal number of kja was 19,401 plots. moreover, fitri et al. (2016) stated that the difficulties faced by the jatiluhur reservoir related to the kja problem were caused by multi-parties’ management having different perspectives leading to inconsistent decision making. harmonious perspectives and visions of all managing parties are needed to maximize the productivity of the kja without sacrificing environmental quality. conclusion the jatiluhur reservoir has experienced the accumulation of cu, zn, pb, and cd in the sediment layer with exceeding the minimum limit (isqg) and hg with exceeding the maximum limit (pel). spatially, hg concentration was high in the entire jatiluhur reservoir area, cu was found to accumulate in the inlet area, whereas zn and pb were relatively high in the main inundation areas. cadmium (cd) seemed to be relatively the safest metal in sediment compared to the other heavy metals. heavy metal concentration in the waters of jatiluhur reservoir was below the detection limit of the tool. the other water quality parameters also met the standard of water quality. only ammonia did not meet the quality standards for the life of sensitive fish (such as nile tilapia). the high concentration of heavy metals in the sediment of the reservoir was due to household heavy metal contamination and water quality parameter conditions in jatiluhur reservoir – gatot prayoga et al. 15 and/or industrial wastes, while the high concentration of ammonia in water was due to fecal materials from the aquaculture activities. benthic organisms may have been affected by the high concentration of heavy metals in the sediment of the reservoir. based on the high level of heavy metal concentrations in the jatiluhur reservoir, the priority for further attention and countermeasure in the reservoir was toward hg, cu, pb and ammonia. further studies are recommended to manage the water quality of the jatiluhur reservoir. acknowledgments this study is part of a research grant from the osaka gas foundation of international cultural exchange (ogfice). the authors are thankful to ogfice for fully funding this study. references anas p, jubaedah l, sudino d. 2017. kualitas air dan beban limbah karamba jaring apung di waduk jatiluhur jawa barat [water quality and wastewater load of floating net cages in jatiluhur reservoir of west java]. jppik 11(1):35-47. anggoro b [internet]. 2018. warga diimbau tak konsumsi ikan dari aliran sungai citarum [residents are advised not to consume fish from the citarum river]. 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[the lethal and sub-lethal toxicity test of mercury chloride (hgcl2) on cyprinus carpio l.]. omni-akuatika 12(17):1-10. heavy metal contamination and water quality parameter conditions in jatiluhur reservoir – gatot prayoga et al. 17 [us epa] u.s. environmental protection agency. 2001. methods for collection, storage and manipulation of sediments for chemical and toxicological analyses: technical manual. epa 823-b-01-002. washington dc (us): u.s. environmental protection agency, office of water. wiwoho. 2005. model identifikasi daya tampung beban cemaran sungai dengan qual2e [model identification of river pollution load carrying capacity with qual2e] [thesis]. retrieved from universitas diponegoro. 1. etti sartina (the liverwor... biotropia vol. 20 no. 2, 2013: 73 80 the liverwort genus (marchantiaceae) of mount sibayak north sumatra, indonesia marchantia etti sartina siregar , nunik s. ariyanti , and sri s.tjitrosoedirdjo received 21 january 2013/accepted 02 july 2013 knowledge on the liverworts (marchantiophyta) flora of sumatra is very scanty including that of genus (marchantiaceae). this study was conducted to explore the diversity of in mount sibayak north sumatra, indonesia. altogether, seven species of are found in mount sibayak north sumatra, of which five are previously known ( , , , , and ), while one is as new species record ( ) for sumatra, and one species has not been identified ( sp ). an identification key to the species of from sumatra is provided. liverwort, , marchantiaceae, mount sibayak, north sumatra 1,2 3 3,4 1 2 3 4 plant biology graduate program, graduate school, bogor agricultural university, ipb-campus darmaga, bogor, indonesia university of sumatra utara, medan, indonesia department of biology, faculty of mathematics and natural sciences, bogor agricultural university, ipb-campus darmaga, bogor indonesia seameo biotrop, jl. raya tajur km 6, bogor, indonesia marchantia marchantia marchantia marchantia acaulis m. emarginata m. geminata m. paleacea m. treubii m. polymorpha marchantia . marchantia marchantia abstract introduction key words: marchantia marchantia marchantia, m. emarginata m. mucilaginosa m. nitida marchantia m. acaulis, m. emarginata, m. geminata, m. miqueliana, m. paleaceae, m. treubii m. mucilaginosa m. acaulis m. nitida m. paleaceae l. is one of the largest genera in the liverworts order marchantiales. this genus is represented by 36 species found in the world (bischler-causse 1998). in indonesia especially sumatra, the floristic work on is still very scarce. herzog (1943) in his study of liverworts from sumatra, recorded three species of namely , and . a monograph of from asiatic and oceanic regions was by bischler-causse (1989), who listed six species from sumatra, namely and ( is a synonym of and is a synonym of ). * corresponding author : ettisartina@yahoo.com doi: 10.11598/btb.2013.20.2.3 73 it is well known that the species of liverworts from sumatra, especially north sumatra is very much undercollected. so it is assumed that this study will improve the knowledge on in sumatra especially north sumatra. this study is conducted to explore and improve our understanding of the diversity of in mount sibayak, north sumatra, indonesia. the study area is located in mount sibayak, north sumatra indonesia, approxi mately 60 km from medan city. the locality has an altitude of about 700 2050 m asl, annual rainfall of 2400 2800 mm/year, and relative humidity of at least 80 90 %. exploration was carried out along the tracks of mount sibayak from lowland to the peak. eighty newly collected material specimens of from mount sibayak and three dried specimens deposited in bo (collected from north sumatra) were used for the study. the specimens collected were classified and identified based on morphological characters, using bishler-causse (1989) and gradstein (2011) in concept and species delimitation. all newly acquired specimens are deposited in bo herbarium. an identification key to the species of known from sumatra is provided. there are seven species of found in mount sibayak, north sumatra, , , , and sp. is reported as a new species record from sumatra. and are the most common species in mount sibayak, found from lowland to high level altitude. was found from lowland to near the peak and numerous around the dwi warna waterfall. is common species in lowland altitude (less than 1000 m altitude). rare species of was found in mount sibayak: (only at high level altitude ± 1500 m) and sp. (only around the dwi warna waterfall). the populations of these two species were limited . was found in certain places from 1150-1250 m altitude, but densely populated. in this study, we did not find the species of that has been reported by bischler-causse (1989) from sumatra. var. was reported by burgeff (1943) from brastagi, north sumatra. however, this species was not confirmed by bischler-causse (1989) since the specimen type was unknown. it might belong to or to (bischler-causse 1989). marchantia marchantia marchantia marchantia marchantia m. acaulis m. emarginata m. geminata m. paleacea, m. polymorpha, m. treubii marchantia marchantia polymorpha marchantia emarginata, m. geminata m. treubii marchantia geminata marchantia acaulis marchantia m. polymorpha marchantia marchantia paleacea m. miqueliana marchantia grisea sumatrana m. acaulis m. wallisii materials and method results and discussion 74 biotropia vol. 20 no. 2, 2013 key to species of in mount sibayak north sumatra and the species of recorded from sumatra (modified from bischlercausse 1989): marchantia , marchantia 1. thallus margin crenulate; scales over almost entire ventral surface of thallus, reaching the thallus margin, sometimes visible at the margin in dorsal view; lobes of female receptacle terete.....................................................................5. 1. thallus margin entire; scales only along thallus midline of ventral surface, invisible in dorsal view; lobes of female receptacle flat apically.................................................2 2. cupules with ciliate lobes and densely papillose on outer surface...........4. 2. cupules ciliate, no or few papillae on outer surface..................................................... 3 3. thallus more than 5 mm wide. involucres ciliate .................................. 3. thallus as wide as or less than 5 mm wide. involucres entire or crenulate................ 4 4. compact ventral tisssue of thallus with numerous mucilage cavities per section. involuces are almost as long as or longer than lobes....................................................5 4. compact ventral tisssue of thallus without or with 1-3 mucilage cavities per section. involucres are shorter than lobes ....................................................................6 5. appendage of median scales of length : ratio width = 1.1-1.5 : 1; margins sharply toothed. male receptacles sessile or nearly so..............................................1. 5 appendage of median scales of length : ratio width= 1.5-2.4 : 1; margins nearly entire to bluntly toothed. male receptacle distinctly stalked....................... 6 appendages of median scales acute or acuminate with row of 2-3 cells apically...............................................................................................................................7 6 appendages of median scales rounded or obtuse with single cell apically ................................................................................................................. 7. sp. 7 appendage of median scales toothed, the teeth mostly consisting of 2-3 cells and often recurved towards base of appendage. lobe of female receptacle emarginate or truncate apically ................................................................................. 2. 7 appendage of median scales entire or toothed, the teeth mostly unicellular and oriented obliquely towards apex of appendages. lobes of female receptacle split apically...............................................................................................................................8 8 appendage of median scales yellow with orange or purplish borders, ovate. number of female lobes usually 4...................................................... ......3. 8 appendage of median scales purple, narrowly triangular. number of female lobes variable 3-6......................................................................................................6. 1. steph. (fig.1 a-b). thallus always with dark median band on dorsal surface, however bischlercausse-causse (1989) found sometimes specimens without median band on dorsal surface. antheridium and archegonium sometimes are found on the same thallus (monoecious) but male rays are never found on the female receptacles as the description by bischler-causse (1989). lobes of female receptacle sometimes curved up when mature. m. polymorpha m. paleacea m. miqueliana* m. acaulis m. wallisii* marchantia m. emarginata . m. geminata m. treubii marchantia acaulis 75 the liverwort genus (marchantiaceae) of mount sibayak etti sartina siregarmarchantia – et al. * the species was reported from sumatra, but it was not found and described in this study note: habitat: distribution: specimens examined: note: habitat distribution: specimens examined: note: habitat distribution: specimen examined: is similar to but the latter species has a distinctly stalked male receptacles, thallus without large cavities, and longer appendage of median scales. is easily recognized by the male receptacle without stalk, the large and numerous mucilage cavities in compact ventral tissue in the thallus and the yellowish appendage of median scales. found abundant on soil or rocks in open place or semi shaded place from 870 to 900 m altitude. indonesia (sumatra, java, kalimantan, sulawesi), borneo (sabah, sarawak), malaysia, singapore, philippines, sri lanka (burgeff 1943; bischler-caussecausse 1989; piippo 2002; chuah-petiot 2011). mt. sibayak, etti siregar 02, 03, 33, 102, 587, 589, 592, 594, 914, 1426, 1428; etti siregar & nunik s ariyanti 1517, 1519, 1526, 1753. 2. reinw., blume et nees. (fig.1c-d). this species shows wide variations in thallus and female receptacles. thallus can be without or with median band on dorsal side, small to medium size, with margins purplish, reddish, sometimes hyaline. female receptacles are often curved toward stalk, sometimes straight; lobes varies from (5-13); dorsal surface flat or with indistinct to distinct rounded median projection, slightly asymmetric to symmetric. apex of receptacle lobes varies from emarginate, truncate or sometimes rounded. in bischlercausse (1989), all of have a rounded median projection on dorsal side of female receptacles, but we found some specimens with indistinct to distinct median pojection on dorsal surface of female receptacles. : soil, rocks (moist, damp or wet, shaded, semi-exposed places, riversides, creeks) from 870 to 1450 m altitude. japan, korea, china, india, sri lanka, andaman and nicobar island, thailand, malaysia, indonesia (sumatra, java, lesser sunda island, bali, moluccas, irian jaya), borneo (sabah, sarawak), philippines, marianas, guam, new guinea, new britain, solomon island (bischler-causse 1989; bischler-causse & piippo 1991; song 2006; lai 2008; chuah-petiot 2011; singh & singh 2012). mt. sibayak, etti siregar 31, 47, 70, 112, 134, 219, 367, 374, 375, 380, 460, 462, 463, 465, 467, 468, 473, 475, 476, 593, 599, 608, 757, 760, 863, 868, 1427; etti siregar & nunik s ariyanti 1445, 1447, 1520, 1524, 1794, 1826. sibolangitnorth sumatra, lorzing 12707 (bo). 3. reinw., blume et nees (fig.1e). this species is easily recognized by the appendages of median scales yellow with orange-red borders; ovate and acuminate apically. : soil, rocks (shady or open places) from 870-1975 m altitude. india, andaman island, indonesia (sumatra, java, kalimantan), malaysia, philippines (tan & engel 1986; bischler-causse 1989; chuah-petiot 2011; singh & singh 2012). mt. sibayak, etti siregar 426, 427, 517, 745, 762, 821, 912, 915; etti siregar & nunik s ariyanti 1522, 1603, 1607, 1610. brastagi-north sumatra, meijer 15609 (bo). marchantia acaulis m. geminata marchantia acaulis et al. marchantia emarginata m. emarginata et al. marchantia geminata 76 biotropia vol. 20 no. 2, 2013 77 figure 1. a-b. with female and male plants, b. shows monoicous plant; cd. . with female and male plants; e. with female plants; f. (gc: gemmae cup); g. with female plants; h. with female and male plants. (photographed by: etti s siregar). marchantia acaulis m emarginata m. geminata m. paleacea m. polymorpha m. treubii the liverwort genus (marchantiaceae) of mount sibayak etti sartina siregarmarchantia – et al. 78 biotropia vol. 20 no. 2, 2013 4. bertol. (fig. 1f). is easily recognized vegetatively by the large thallus without median band on dorsal surface and cupules with ciliate lobes. soil (wet place, waterfall); rocks of creek wall in semi shaded place, from 1150 to 1250 m altitude. turkey, lebanon, iran, yemen, russia, ussr, afghanistan, pakistan, india, bhutan, china, taiwan, korea, japan, thailand, vietnam, indonesia (sumatra, java, irian jaya), borneo, philippines, new guinea (lai . 2008), (bischler-caussecausse 1989; piippo 1990; bischler-causse & piippo 1991; konstantinova . 2009; daniels 2010; dandotiya . 2011; wang . 2011; singh & singh 2012). mt. sibayak, etti siregar 185, 763; etti siregar & nunik s ariyanti 1611. 5. l. (fig. 1g). is easily recognized by its large thallus with scales extending over the entire ventral surface, reaching the thallus margin and ± visible at the margin in dorsal view. its female receptacles are also distinctive with terete rays. rocks of creek wall in exposed places, 1500 m altitude. : turkey, syria, lebanon, israel, iraq, iran, russia, ussr, uzbekistan, tadzhikistan, afghanistan, pakistan, india, sri lanka, nepal, bhutan, china, taiwan, korea, japan, thailand, vietnam, malaysia, indonesia (java, sumatra new record based on present study, irian jaya), philippines, new guinea, new zealand, tasmania (bischler-causse 1989; söderström . 2010; singh & singh 2012). mt. sibayak, etti siregar 373, 377, 378, 466, 470, 759; etti siregar & nunik s ariyanti 1808. 6. schiffn. (fig. 1h). is similar to , but the latter species has a longer apical split in the lobes of female receptacle, receptacle usually constantly 4-lobed (in receptacles vary from 3-5 lobed), appendage of median scales are ovate and acuminate apically, epidermis of thallus are without papilla. soil or rock in open place and semi shaded place, altitude from 870-1610 m. indonesia (sumatra, java, lesser sunda island), borneo, malaysia (bischler-causse 1989; chuah-petiot 2011). mt. sibayak, etti siregar 28, 588, 743, 746, 608, 744. brastaginorth sumatra, breedveld gjf 5 (bo). 7. sp. (fig.2). thallus with dark median band on dorsal surface. epidermal pores 50-112 µm in diameter. appendages orange or purplish with dark colour in borders or apically (rarely entire orange or purplish); apex vary from rounded, obtuse to acuminate (figure 2. c-e). cupules with cilia 1-9 cells long and 2-4 cells basally, arranged in uniseriate, biseriate or sometimes triseriate cell. female receptacle alway dissected into 5 shallowly lobes; lobes apex emarginate; involucres purplish, margin entire. this species is closely related to . however, in , thallus without median band on dorsal surface, the cilia of cupules are shorter (3-4 cells long) and the basal cell of cupules are smaller (1-2 cells); apex of appendage of median scale marchantia paleacea m. paleaceae et al et al et al et al marchantia polymorpha marchantia polymorpha et al marchantia treubii marchantia treubii m. geminata m. treubii marchantia m. rubribarba m. rubribarba note: habitat: distribution: specimens examined: note: habitat: distribution specimens examined: note: habitat: distribution: specimens examined: note: 79 figure 2. m . archantia sp. : a. female plants; bd. appendage of median scales; e. margin of thallus; f. cupule; gh. involucre; i. scale of female receptacle; all from etti siregar 1972 (bo). scale bar = 200 µm. (photographed by: etti s siregar) obtuses, rounded or bluntly acute. the female receptacle dissected into 5-9 lobed; involucre hyaline. we have not seen any specimen of . so we could not decide wether it belongs to only from description and drawings. more data are needed to decide wether it belongs to or as a new species. sp. is also different from the species of from sumatra has been reported by bischler-causse (1989). the latter species has larger thallus with no distinct median band on dorsal surface; the cilia of cupules are shorter and arranged in uniseriate cell; appendage of median scales acute or apiculate apically with row of 13 cells (in sp. with single apical cell); lobes of female receptacle truncate or rounded apically; involucrum ciliate with row of 3-9 cells long (bischler-causse 1989). found on soil in wet place, only around the dwi warna waterfall at 1100 m altitude. mt. sibayak, etti siregar 1972; etti siregar & nunik s. ariyanti 1612, 1613, 1616. m. rubribarba m. rubribarba m. rubribarba marchantia m. miqueliana, marchantia marchantia habitat: specimen examined: the liverwort genus (marchantiaceae) of mount sibayak etti sartina siregarmarchantia – et al. 80 biotropia vol. 20 no. 2, 2013 conclusions acknowledgments references there are seven species of found in mount sibayak, north sumatra, , , , and sp. is reported as a new species record from sumatra. this research is part of the dissertation of the first author, funded by bpps project of the indonesian ministry of education (dikti) and biotrop dipa project 2012 (research fellowship for phd student). we would like also to thank the reviewers of this manuscript. marchantia marchantia acaulis m. emarginata m. geminata m. paleacea, m. polymorpha, m. treubii marchantia marchantia polymorpha bischler-causse h. 1989. l. the asiatic and oceanic taxa. 38: 1-317. bischler-causse h, piippo s. 1991. bryophyte flora of huon peninsula, papua new guinea. l. 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fenn . the southeast asian journal of tropical biology vol. 31 no. 2, 2024: 192 201 doi: 10.11598/btb.2024.31.2.1904 issn: 0215-6334 | e-issn: 1907-770x 192 biodiversity of cellulolytic bacteria isolated from fermetodege for ruminant isnawati*, lisa lisdiana, mahanani tri asri, guntur trimulyono department of biology, faculty of mathematics and natural sciences, universitas negeri surabaya, kampus unesa ketintang, surabaya 60231, indonesia article higlights • fermetodege as a source of cellulolytic bacteria that degrade cellulosic materials. • enhances ruminant feed by improving palatability and digestibility. • utilizes water hyacinth, reducing invasive plant spread and supporting ecosystems. • bioprospecting reveals new bacteria for effective fermentation starter development. article information received 14 february 2023 revised 15 may 2024 accepted 19 july 2024 *corresponding author, e-mail: isnawati@unesa.ac.id research article abstract fermetodege is referred to as fermented ruminant feed made from rice bran, corn cobs, and heavy metal-free water hyacinth. several factors influence both the fermentation process and the quality of the final products, including dominance, diversity, and evenness, of indigenous cellulolytic bacteria species. to increase the quality of fermented feed, there is a need to fully comprehend the influential factors. therefore, this study aimed to assess factors influencing the biodiversity of cellulolytic bacteria isolated from fermetodege. cellulolytic activity of the isolates was assessed through observation of growth on the carboxymethylcellulose (cmc) media. simpson’s species dominance, as well as shannon-weiner's diversity and evenness indices were also calculated. the results showed that cellulolytic activity of the bacteria isolates formed a clear zone after soaking the bacteria colonies in 0.1% congo red and rinsing with 1 m nacl. the shannon-weiner’s diversity index was categorized as medium, with values ranging from 0.6849 to 1.8173. observation on stable distribution of bacteria species showed evenness index between 0.7778 and 0.9983. meanwhile, the simpson’s species dominance ranged from 0.1835 to 0.5082, showing that there was no dominant species. in conclusion, this study showed that fermetodege was a potential source of bacteria isolates and could be used as a fermentation consortium starter. keywords: cellulolytic bacteria, diversity, dominance, evenness, fermetodege introduction ruminant feed ingredients having high cellulose content are characterized by low palatability and digestibility levels (isnawati 2020), showing the need for fermentation process to break down cellulose. palatability is the characteristics of feed that influence the sensory response in animals and affects their appetite for feed (aldrich & koppel 2015). some ingredients, such as rice bran, corn flour, and molasses, can act as palatants to mask unpleasant flavors and to improve feed palatability by enhancing the taste and smell of feed to make it more appetizing to animals and increase feed intake, resulting in weight gain and milk yield increase. digestibility refers to the number of nutrients absorbed by the organism. commonly, it is calculated by subtracting the nutrients retained in the feces from the nutrients consumed (atta et al. 2018). many factors influence feed digestibility, such as the type of animals, the composition of the ingredient plant, and the difference in feed preparation. animals with pre-gastric fermentation digestion, such as ruminant, has highly efficient digestion since the nutrients released are digested and absorbed in the gastric chamber and intestinal tract. digestibility of ruminant feed is closely related to the chemical composition. a ruminant feed with high crude fiber content is poor in digestibility. however, grinding may increase absorbability because of the enhanced substrate accessibility for enzymatic action. finer grinding increases the digestion rate in which the feed passes through the digestive tract. this leads to an increase in total feed intake because animals can consume more feed in a given amount of time (ortolani et al. 2020). https://doi.org/10.11598/btb.2024.31.2.1904 bacteria’s diversity, dominance, evenness of fermetodege isnawati et al. 193 fermentation process can improve feed palatability and digestibility. according to istiqomah et al. (2010), rice bran should be fermented using rhizopus sp. to change the texture and flavor of feed ingredients and make the fermentation products more appetizing, easily digestible, and nutritious for the cattle. generally, all fermented products usually contain compounds that are simpler and easier to digest than the original ingredients. these products are easily decomposed biologically and have higher nutritional value (pakpahan & restiani 2019). during the extended dry season, particularly when forage is unavailable, fermented feed can be used to overcome the lack of food for animals. the production of these products can be carried out through various weeds, including water hyacinth and agricultural waste, such as corn cobs and rice bran containing high cellulose (lardy et al. 2022). in this study, the mixture of corn cobs, rice ban, and water hyacinth is called fermetodege. a widely recognized aquatic weed, namely water hyacinth, grows quickly and uncontrollably, disrupting aquatic ecosystem and causing siltation (adanikin et al. 2017). the use of water hyacinth as one of fermetodege ingredients means preventing the wider spread of aquatic weed, reducing environmental pollution, and contributing to ecosystem preservation (sivaramakrishnan et al. 2021). the composition of lignocellulosic product includes 6.5% lignin, 16.4% cellulose, and 42.8% hemicellulose (rezania et al. 2018). proximate analysis showed that water hyacinth has 9.3% dry matter, 26.9% crude fiber, 12.4% ash, and 10.5% crude protein (hossain et al. 2015). a significant source of raw materials for fermented feed preparation include rice bran, corn cobs, and agricultural by-products rich in lignocellulose (42.9% cellulose, 26% hemicellulose, and 22% lignin). according to liu et al. (2017), the proximate analysis showed that rice bran comprises 23.21% crude fiber, 12.35% ash, 16.72% protein, and 52.87% carbohydrate. corn cobs have significant lignocellulose content, consisting of 45.88% cellulose, 39.40% hemicellulose, and 11.32% lignin (shariff et al. 2016). the proximate analysis showed that corn cobs contain 30.93% crude fiber, 2.26% ash, and 0.61% crude fat (paynor et al. 2016). cellulolytic bacteria play an important role in degrading organic materials, such as lignocellulose, into simpler compounds during the production of fermented feed (varma et al. 2015), which include bacillus brevis (tabssum et al. 2018) and burkholderia nodosa nb1 (tang et al. 2018). enterococcus, klebsiella, stenotrophomonas, and microbacterium isolated from sugarcane biomass have also been reported to show cellulolytic activities (dantur et al. 2015). these bacteria are often obtained from the rumen of cattle and added to the cellulosic raw materials to produce a starter for ruminant fermented feed. rumen is dominated by anaerobic bacteria, both facultative and obligate. despite the numerous benefits, there is limited information on the use of bacteria for special fermented feed ingredients. therefore, this study aimed to evaluate diversity, evenness, and dominance of indigenous cellulolytic bacteria species from fermetodege, which was made from water hyacinth, corn cobs, and rice bran. bacteria isolates obtained from fermetodege can be regarded as a bioprospecting strategy to obtain a more effective source of fermentation starter, other than bacteria obtained from cattle’s rumen. the diversity, evenness, and dominance indices of indigenous bacteria isolated from fermetodege in this study are expected to provide useful reference for making effective consortium starter composition, for manufacturing cellulosic material-based fermented feed. materials and methods production of fermetodege water hyacinth, rice bran, and corn cobs with a 1:1:1 ratio were used as raw materials to produce fermetodege. the corn cobs and water hyacinth were cleaned and cut into small pieces of 1-3 cm to increase surface area and enhance easy decomposition. the raw materials were then sun-dried to minimize the water content up to 30-40%, which is essential to prevent decay and optimize the fermentation (farida et al. 2018). the raw materials were then cooked for 20-30 minutes, cooled, mixed evenly, and placed in a basket covered with banana leaves to create a microaerophilic condition (fitrihidajati et al. 2015). in this study, there were four units of experiment. fermentation of raw materials the fermentation process was carried out naturally using solid-state fermentation (ssf) method, which was affected by temperature, biotropia vol. 31 no. 2, 2024 194 water content, type of substrates, and presence of microbial bioactivity (sadh et al. 2018). this method offers numerous advantages, including lower contamination risks, along with a straightforward, cost-effective fermenter design that enhances process efficiency and productivity (leite et al. 2020). in this study, fermentation process was conducted under a microaerophilic condition. this condition offers several benefits, such as creating favorable conditions for obligate anaerobic bacteria and enhancing the growth of facultative anaerobic bacteria. the experimental units were placed at room temperature and observed daily for 15 days. ph and temperature measurement the measurement of ph and temperature was conducted at the top, middle, and bottom sections of the fermetodege. average value from each replication were calculated to determine daily temperature and ph. for bacteria isolation data, feed samples were also taken each day (isnawati 2019). cellulolytic bacteria isolation bacteria isolation was carried out daily for 15 days during fermentation process. a total of 10 g fermented feed samples were randomly collected at a depth of 10-15 cm from the fermenter. the sample was stored in a bacteria isolation tube and added with sterile distilled water until the final volume reached 100 ml and then, homogenized. the sample was then diluted to 10-5, after which 1 ml was taken and cultured on 10 ml nutrient agar media using a pour plate method. after 24 hours of incubation, the grown colonies were characterized based on their morphology followed by enumeration. each of bacteria isolates was recultured using the streak plate method. subsequently, cellulolytic activity was determined based on their growth on carboxymethyl cellulose (cmc) media (isnawati 2020). the isolated bacteria having cellulolytic activity were identified based on the forming of a clear zone around colony greater than > 1.0 cm (gaur & tiwari 2015) when dripped with 0.1% congo red and rinsed using 1 m nacl. identification of bacteria isolates bacteria isolates were identified by assessing physiological and biochemical characteristics with the microbact identification kits (microbacttm gnb12a and 12b) and bergey’s manual of determinative bacteriology (holt 1994). data analysis diversity, evenness, and dominance of cellulolytic bacteria species based on a study of the microbial communities’ succession (liu et al. 2015) and fish population (hanif et al. 2015), bacteria diversity was determined by using the shannon-wiener diversity, evenness, and simpson dominance indices. shannon-wiener diversity index was calculated by using formula: where: h' = shannon-wiener diversity index ni = total number of species i n = total number of all species. the level of diversity is classified as: low when h' < 1 moderate when 1 < h' < 3 high h' > 3 the evenness index was calculated by using formula: where: e = evenness index h' = shannon-wiener diversity index s = number of species evenness is categorized as an unstable species distribution when the index is close to 0 and classified as stable when the index value is close to 1. simpson’s dominance index was calculated by using formula: where: c = simpson's dominance index ni = total number of species i n = total number of all species values of dominance index are ranged from 0 to 1 and categorized as: no dominant species in the community if c value is close to 0 a dominating species in the community if c value is close to 1. bacteria’s diversity, dominance, evenness of fermetodege isnawati et al. 195 results and discussion temperature and ph changes during fermentation process measurement of temperature and ph were carried out during the fermentation process using solid-state fermentation (ssf) method for making fermented feed. the measurement showed differences in daily temperature and ph for 15 days of incubation with insignificant fluctuations (figs. 1&2). the highest temperature during fermentation process was 36.6 °c, while 30.2 °c was the lowest. furthermore, the highest and lowest average ph were 7 and 6.8, respectively. these fluctuations showed the activity of cellulolytic bacteria in the degradation of raw materials. temperature changes also affected the composition and activity of bacteria during the next day (hansen et al. 2015). meanwhile, variations in ph functioned as an indicator of the microbes’ activity in producing organic acids (dezam et al. 2017). due to the insignificant fluctuations of temperature and ph during the fermentation process, bacteria enumeration was performed daily. therefore, information related to the dynamics of each bacteria isolates numbers during the fermentation process can be obtained. cellulolytic bacteria isolates eight isolates were produced from the isolation and screening of cellulolytic bacteria, which were differentiated based on the morphological characteristics of each colony. the characteristics and identification data of each colony are presented in table 1, while the total number of each isolate is presented in table 2. table 3 shows the calculation results for the shannon-wiener diversity, evenness, and simpson’s dominance indices of cellulolytic bacteria isolated from fermetodege. figure 1 changes in daily temperature of fermented feed during 15 days of fermentation process using the ssf method. notes: = the highest temperature occurred on the third day of the fermentation process; = the lowest temperature occurred at the beginning and at the end of the fermentation process. figure 2 changes in the daily ph of fermented feed during 15 days of fermentation process notes: = the highest ph occurred on the first day to the tenth and fifteenth day of the fermentation process; = the lowest ph occurred on the twelfth day of the fermentation process. bio t ro pia vol. 31 n o. 2, 2024 196 table 1 identification of cellulolytic bacteria isolated from fermetodege based on microbact identification kits (microbacttmgnb12a and 12b) aspects bacillus laterosporus (b1) (70%) bacillus badius (b2) (75%) bacillus pantothenticus (95,3%) (b3) bacillus brevis (b4) (65%) staphylococcus sciuri (80%) (b5) bacillus stearothermophilus (81,0%) (b6) burkholderia pseudomallei (99,5%) (b7) enterococcus durans (80%) (b8) oxidase + + + + + + + + motility + + + + + + + nitrate + + + + + starch + + + _ cmc + + + + + _ + catalase + + + + + + _ lysine + + + ornithine + + + h2s glucose + mannitol + xylose + onpg + + + + + + indole urease + + + vp + + + + + + citrate + + + tda gelatin + + malonate + inositol + + sorbitol + + + rhamnose + + sucrose + + lactose + arabinose + ba ct er ia’ s d iv er sit y, do m in an ce , e ve nn es s o f f er m et od eg e i sn aw at i e t a l. 19 7 aspects bacillus laterosporus (b1) (70%) bacillus badius (b2) (75%) bacillus pantothenticus (95,3%) (b3) bacillus brevis (b4) (65%) staphylococcus sciuri (80%) (b5) bacillus stearothermophilus (81,0%) (b6) burkholderia pseudomallei (99,5%) (b7) enterococcus durans (80%) (b8) adonitol + + raffinose + salicin arginine + + + gram positive positive positive positive positive positive negative positive cell shape rod rod rod rod coccus rod rod coccus notes: + = provide positive reaction; = provide no reaction. table 2 the total number of each bacteria isolated from fermetodege  isolates day1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 bacillus laterosporus (b1)* 75.7 95 102.4 106 127 131 138.3 145 141.5 100 97.5 87.5 80.5 75.5 69.5 bacillus badius (b2) 16.6 23.7 56.5 63.3 23.2 15.3 17.3 21.5 32.5 79.5 bacillus pantothenticus (b3) 31.5 17.7 19.3 18.5 23.5 84.5 bacillus brevis (b4) 25.5 37.5 24.3 20.3 31 staphylococcus sciuri (b5) 19.5 22 25.5 bacillus stearothermophilus (b6)* 58.5 62.3 70.6 65.5 66.5 91.5 98.5 112.5 115.3 110.3 100.5 97.5 88.5 80.5 80 burkholderia pseudomallei (b7) 55.5 79.5 29 enterococcus durans (b8) 82.7 115.5 97.5 101.5 91 notes: * = bacteria species present throughout the fermentation process. biotropia vol. 31 no. 2, 2024 198 table 3 diversity, evenness, and dominance indices of cellulolytic bacteria isolated from fermetodege dayaverage number of bacteria shannon-wiener diversity index evenness index simpson’s dominance index 1 134.2 0.6849 0.9881 0.5082* 2 240.0 1.0841 0.9868 0.3428 3 305.1 1.2312 0.8881 0.3125 4 307.7 1.6425 0.9167 0.2198 5 305.2 1.4319 0.8897 0.2734 6 285.8 1.0561 0.9613 0.3616 7 279.3 1.1069 0.7985 0.3812 8 277.0 0.8916 0.8116 0.4439 9 296.4 1.0783 0.7778 0.3886 10 210.3 0.6919 0.9983* 0.5012 11 392.3 1.5020 0.9333 0.2322 12 286.5 1.0967 0.9983* 0.3346 13 342.3 1.7178 0.8828 0.2073 14 297.5 1.8173* 0.9339 0.1835 15 313.5 1.3838 0.9982 0.2512 notes: * = the highest diversity, evenness and dominance indices. bacteria, fungi, and actinomycetes were abundant in the aerobic degradation of solid waste (bhatia et al. 2015). fermentation must be carried out by using selected microorganisms because each organic material requires specific enzymes produced by certain microorganisms (boboesscu et al. 2014). for example, fungi rhizopus can significantly lower the crude fiber content due to its lignocellulolytic enzymes (belewu & babalola 2009). meanwhile, several bacillus species produced protease and cellulase enzymes to break down complex organic matter in a plant (oke et al. 2016; sundarajan et al. 2011; croos et al. 2019). the 8 species of cellulolytic bacteria, which had been isolated in this study were bacillus laterosporus, b. badius, b. pantothenticus, b. brevis, staphylococcus sciuri, b. stearothermophilus, burkholderia pseudomallei, and enterococcus durans. certain numbers of these bacteria were also found in other fermentation processes of some materials (ramos et al. 2011). bacillus brevis is often used due to its fast growth, low energy requirement, easy management, and ability to be genetically modified for producing cellulase enzymes that are resistant to alkaline conditions as well as high temperatures. tabssum et al. (2018) showed that b. brevis was able to produce sugar from various cellulosic materials. furthermore, the highest cellulase enzyme production of b. brevis was obtained by adding fermentation process of organic materials consists of several phases, namely mesophilic and thermophilic reactions, cooling as well as maturation (ishii et al. 2000; yu et al. 2007). bhatia et al. (2015) also included the latent phase, which occurred before the mesophilic reaction. generally, fermentation process starts with the mesophilic phase shown by the increase of the temperature to 45 °c. mesophilic bacteria activity leads to a rise in temperature and toward the thermophilic phase. subsequently, the temperature rises higher than 45 °c, showing that the pile enters the thermophilic phase. increased temperature facilitates the growth of thermophilic bacteria, where maximum metabolic activity is carried out using available nutrients. depletion of certain nutrients leads to a decrease in the population of thermophilic bacteria and a reduction in metabolic activity, which results in a further reduction in temperature (40-45 °c) when the cooling phase begins. in the maturation phase, the temperature drops to the ambient level (20-30 ºc). these phases cannot be determined based on the daily fluctuation of temperature shown in figure 1. nonetheless, the curve pattern showed the phases occurring in the fermentation process of this study. various physical and chemical changes occurred during fermentation. the changes were shown by the variations in temperature and ph, indicating the production of different microorganism community structures in each phase. furthermore, bacteria’s diversity, dominance, evenness of fermetodege isnawati et al. 199 0.5% yeast extract, 0.09% mgso4, and 0.03% peptone to the bacteria culture. liang et al. (2014) stated that 22 strains of bacteria have cellulolytic activity in the subtropical region of china and 36.36% belong to genus burkholderia. among these species, burkholderia nodosa nb1 is known as a cellulase enzymes producer (tang et al. 2018). burkholderia sp. can also degrade lignin from single carbolic cellulosic materials (akita et al. 2016) and produce extracellular protease (vial et al. 2007). the enzymes generated by a blend of fungi and bacteria at the outset, facilitate the decomposition of organic compounds found in water hyacinth and corn cobs. the decomposition process results in feed that is more digestible by sheep. however, there were no significant variations in the weight gain observed among sheep fed with fermented feed at different levels of ration (fitrihidajati et al. 2017). enterococcus is found in the digestive tract of various species of animals with forage as their main food source. dantur et al. (2015) also showed that enterococcus was found in sugarcane biomass along with other four genera of bacteria, namely klebsiella, stenotrophomonas, microbacterium, and bacillus. these genera showed the activity of endoglucanase including the cellulase group on cellulose substrates (dantur et al. 2015). bacillus stearothermophilus is a rod-shaped, gram-positive, and facultative anaerobic bacterium, which is often found in soil and food. most of these strains serve as probiotics often used in fish feed, while some are pathogenic in humans. oke et al. (2016) stated that b. stearothermophilus produced endoglucanase enzymes to degrade lignocellulosic waste. cellulose degradation is caused by the synergistic action of three cellulase enzymes, which differ in terms of reaction mechanisms and structural properties. the three enzymes types are endoglucanase, exoglucanase, and -glucosidase (lynd et al. 2002; taherzadeh & karimi 2008). cellulase enzymes often catalyze the hydrolysis of 1,4-β-d-glycosidic bonds to produce glucose, cellobiose, and cello-oligosaccharides from cellulose and other cellulolytic materials. however, each enzyme family has different functions and structures. endoglucanase randomly cleaves the internal glycosides bonds of cellulose to produce oligosaccharides of varying lengths, while exoglucanase cleaves the open ends to produce cellobiose. the two types of exoglucanase that have been identified include cellobiohydrolase i and cellobiohydrolase ii. specifically, cellobiohydrolase i acts from the reduced end of the substrate chain, while cellobiohydrolase ii functions primarily at the end of the unreduced cellulose. β-glucosidase helps to hydrolyze cellobiose into glucose from the unreduced end. in this study, eight indigenous bacteria with cellulolytic activity were found, which could be used as raw materials from water hyacinth, corn cobs, and rice bran as energy and carbon sources. it was observed that variations in nutrients absorption from substrate and ecological factors influenced growth of colonies (yu et al. 2015). substrates containing cellulose are hydrolyzed by cellulase into glucose, serving as a significant source of carbon and energy for bacteria growth (silva-mendoza et al. 2020). based on the results, effective consortium starter was formulated from eight cellulolytic bacteria in fermetodege. however, the usable raw materials were not limited to water hyacinth, rice bran, and corn cobs to ensure the maintenance of variability and flexibility in the production of feed. consortium 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nalle*, yacob m. buritnaban, abner tonu lema, helda and asrul animal husbandry department, state polytechnic of agriculture kupang, kupang 85228, indonesia received 14 february 2023 / revised 30 may 2023 / accepted 30 may 2023 abstract the objective of this study was to asses the effect of different feeding substrates (fs), harvesting times (ht), and the interaction between fs and ht on the chemical composition of black soldier fly larvae (bsfl). the experiment used a 4 x 2 factorial arrangement with two main factors, fs (t1, t2, t3, t4), and ht (15-d and 20d). thus, there were eight treatment combinations, all together with five replications. the results show that there was no interaction (p>0.05) between fs and ht on dry matter (dm), crude protein (cp), crude lipid (cl), phosphorus (p), gross energy (ge), and amino acid contents. the ash content of bsfl grown on t2 media and harvested on day 15 was higher (p<0.05) than those grown on t2 media and harvested on day 20. the calcium (ca) content of bsfl grown on all media and harvested on day 20 was higher (p<0.05) than those harvested on day 15. in conclusion, combining fruit wastes and tofu by-products produced bsfl with high cp content but low cl, ash, ca, and p contents. in addition, bsfl grown on all substrates media and harvested on day 15 had better cp, ca, and p contents. the dispensable amino acid of bsfls fed with t3 diets was the best. the lowest body weight gain was produced by feeding a substrate containing a high percentage of rice bran. the findings indicate that the best nutrient composition of bsfl as animal feed would be achieved in early harvest time (15-d) and grown in heterogeneous feeding substrates. keywords: black soldier fly larvae, growing media, maggot, nutrient, proximate introduction protein sources for poultry diets generally come from both plant protein sources (pps) and animal protein sources (aps). generally, the utilization of pps in poultry diet formulations is comparatively higher than aps. the indonesian poultry feed industry still depends on pps and aps from overseas, including meat and bone meal, corn gluten meal, and soybean meal (sbm). argentina, brazil, the usa, paraguay, and india are the countries that produce and supply sbm for indonesia (natalia et al. 2019). regarding meat and bone meal, indonesia imports this ingredient from australia, new zealand and canada. as reported by dimiyati (2021), the volume of imports of pps increased from 57.30% in 2015 to 84% in 2020. meanwhile, the volume of imported meat and bone meal range from 0.63 to 1.05 million tons per year, so the economic value reaches idr5.5-9.2 trillion (dimiyati 2021). the price of these feed ingredients frequently fluctuates, depending on the exchange value of the us dollar towards idr. recently, the limited availability of sbm in the international market triggered the increase in prices of sbm. this limited availability of sbm in the international market was caused by the increased importation of sbm by china. meanwhile, local soybeans are available but highly limited, so it is not a feasible option for feed millers. an increase in sbm prices will certainly have an impact on increasing the local compound feed prices. *corresponding author: catootjielusjenalle@gmail.com biotropia vol. 30 no. 3, 2023 298 the cost of feed ingredients reached 83-89% of the total cost of broiler production, and 84 to 89% of the total cost of the laying hens (natalia et al. 2019). therefore, efforts to find alternative proteins must be made to overcome the issue of dependence on imported protein sources, as to finally reduce production costs. the black soldier fly (bsf) (hermetia illucens) is one of the organisms studied for its characteristics and nutrient content. this fly originated in america and subsequently spread to subtropical and tropical regions of the world (čičková et al. 2015). indonesia’s tropical climate is ideal for cultivating black soldier fly larvae (bsfl). regarding cultivation, bsfls are very easy to develop on a mass-production scale and do not require special equipment. patterson et al. (2021) reported that the bsfl meal could be included in the laying hen’s diets up to 16% without any negative effects on growth performance and egg production. the crude protein (cp) content in bsfl was quite high, around 12.9 to 78.8%, with crude lipid content of 29 to 32%, 4.8 to 5.1% calcium (ca), and 0.60 to 0.63% phosphorus (p) (bondari & sheppard, 1987; bosch et al. 2014; hopkins et al. 2021; lu et al. 2022). the amino acid content of the bsfl, especially leucine, lysine, and valine, was superior to other protein sources such as sbm and fish meal, while methionine and tryptophan of bsfl were almost similar to that of sbm (lu et al. 2020). however, the nutritional value, survival, and performance of bsfl are influenced by various factors, such as the type and composition of the growing substrates and harvesting times (barragan-fonseca et al. 2017; ewald et al. 2020; broeckx et al. 2021; lan et al. 2022). the growing media generally used for bsfl production are municipal waste, agro-industrial waste, and manure and feces (supriyatna et al. 2016a; dortmans et al. 2017; broeckx et al. 2021). dortmans et al. (2017) and broeckx et al. (2021) reported that bsfl grew well in a substrate rich in protein and available carbohydrates. on the other hand, tschirner and simon (2015) found that the bsfls fed with organic substrates high in fiber produced bsfl with higher cp content (52.3%) compared to those fed with substrates high in protein content with bsfl cp content of only 40%. in addition, the larva may consume the waste easily when the waste has undergone some microbial decomposition process and is in the form of pasta or liquid. most published data regarding the chemical composition of bsfl was obtained from single substrate or two different substrates (tschirner & simon 2015; supriyatna et al. 2016a; lan et al. 2022). the production of bsfl from a combination of substrates is limited. in addition, the chemical composition of bsfl that has been reported is mostly obtained from bsfl harvested on day 20. the published data of bsfl chemical composition harvested on day 15 is highly limited. based on the above explanation, research has been conducted to evaluate the effect of different feeding substrates (fs) and harvesting times (ht) on the chemical composition and growth performance of bsfl. materials and methods feed ingredients the main ingredients used in this experiment are fruit wastes (banana and papaya) and vegetable wastes (mustard greens and water crest) obtained from a local traditional market, tofu byproducts purchased from a local tofu market, and liquid palm sugar, sago (putak meal), and rice bran obtained from a local distributor. experimental design and diet formulation the experiment was designed using a 4 x 2 factorial design, with the first main factor being feeding substrates (fs: t1, t2, t3, and t4) and the second main factor being harvested time (ht: 15 days and 20 days). each treatment was replicated five times (10 kg/replication). the experimental diets were as follows: nutrient composition of maggot grown in different substrates – weko et al. 299 table 1 growing media of black soldier fly larvae (bsfl) ingredients feeding substrate t1 t2 t3 t4 …………………..% as fed........................... fruits wastes (50% banana and 50% papaya) 24.4 24.4 vegetable wastes (50% water crest and 50% mustard greens) 75 tofu by-product 61 7 92.4 rice bran 7 56 17.4 sago meal 5 effective micro-organism (em4) 0.3 0.3 0.3 0.3 liquid palm sugar 0.3 0.3 0.3 0.3 clean water 7 7 7 7 total 100 100 100 100 calculated analysis (% as fed) crude protein (%) 16.07 11.43 4.03 22.81 crude lipid (%) 3.54 4.31 2.65 4.56 crude fiber (%) 8.11 10.64 15.56 6.56 ash (%) 3.70 8.87 17.19 3.67 calcium (ca, %) 5.53 4.51 27.54 8.32 phosphorus (p, %) 2.76 2.91 2.18 0.22 black soldier fly larvae (bsfl) production bsfl was produced at three main stages, including (i) fermentation, (ii) bsf catching and mating, and (iii) the growing period. 1) fermentation: all ground ingredients, free from harmful materials and inorganic elements, were weighed according to feeding substrate formulation. the ingredients were mixed until homogenous with a moisture level of 70 to 80% and fermented for seven days in a room. 2) bsf catching, mating, and egg deposition: on day seven, the fermented substrates were moved into a round plastic container and placed in a sheltered cavity to invite the bsf to mate and lay eggs. 3) growing period: once the larvae have appeared, the feeding substrates containing larvae were removed to the bsf housing for growing. 4) the bsf larvae (bsfl) were fed and grown till days 15 and 20 during the experiment. the feed given to the bsfl is 50 g/day per replication (container). the determination of feed given to bsfl per day (50 g) is referred to supriyatna et al. (2016b). the authors reported that the feed needed for the best growing of one larva was 100 mg/day on a dry weight basis. for the growing period, the bsfl used for growth performance data was placed separately from the bsfl for chemical composition data. there were 60 larvae per replication used for growth performance data. sample preparation black soldier fly larvae (bsfl) were harvested on day 15 (figure 1a) and day 20 (figure 1d). the bsfl from each treatment was removed from the leftover growing media, mixed with water, and then screened. the bsfl was then weighed to obtain the total wet weight. then the bsfl was oven-dried (at 60o c, memmert) for four days, crushed with a stone mortal, ground with a sample mill (0.5 mm screen size), and subsampled, packed, and labeled (figure 1). the bsfl sample was sent to the laboratory for chemical analysis. biotropia vol. 30 no. 3, 2023 300 (a) 15-days-old bsfl (b) oven dried bsfl (c) ground bsfl (d) 20-days-old bsfl (e) oven dried bsfl (f) ground bsfl figure 1 sample preparation of black soldier fly larvae. (a) black soldier fly larvae (bsfl) (15-days-old); (b) ovendried bsfl (15-days-old); (c) ground bsfl (15-days-old); (d) bsfl (20-days-old); (e) oven-dried bsfl (20days-old); (f) ground bsfl (20-days-old); chemical analysis the dried bsfl samples were analyzed for their dry matter (dm), crude protein (cp, crude lipid (cl), crude fiber (cf), neutral detergent fiber (ndf), acid detergent fiber (adf), gross energy (ge), ca, p, and amino acids. all the chemical analyses were performed at bpt ciawi bogor laboratory. the dm content was determined by using aoac method no. 930.15 (aoac 2005). the nitrogen content was analyzed using aoac 2001.1 (aoac 2005), while aoac 942.5 (van soest method; aoac 2005) was used to determine ndf and adf contents. the starch content of sago was analyzed using the titration method. an automatic parr bomb calorimeter was used to measure the ge level. the ca content was analyzed using an atomic absorption spectrophotometer (aas, flame varian 220). the ca analysis was conducted as described by nalle et al. (2021): the sample was weighed and put in the muffle furnace for three hours (550o c). then, the ash was destructed using acid, solubilized, and pipetted to measure at aas with the wavelength of 422,7 nm (nalle et al. 2021). the spectrophotometer method (uv vis agilent cary 100) was used to determine the content of p. the wavelength of the spectrophotometer used was 400 nm (aoac 2012). the amino acid content was determined using high-performance liquid chromatography (hplc, ici instrument/shimadzu scl-10a/shimadzu cbm 20a) as described by nalle et al. (2019). statistical analysis the chemical composition of dried bsfl samples obtained from this experiment was analyzed using the two-way analysis of variance (anova) referring to the general linear model procedure of sas (sas ondemand). meanwhile, body weight gain data were analyzed using the one-way analysis of variance (anova) referring to the general linear model procedure of sas (sas on demand). results and discussion chemical composition of black soldier fly larvae table 2 describes the chemical composition of bsfl fed with different feeding substrates and nutrient composition of maggot grown in different substrates – weko et al. 301 harvested at different times. statistical analysis showed that no interaction (p > 0.05) between fs, and ht was observed in the parameter of dm, cp, cl, p, and ge. there was an interaction (p < 0.05) between feeding substrate (fs), harvesting time (ht) for ash, and ca content of bsfl. the ash content of bsfl grown in t2 media and harvested on day 15 was higher (p < 0.05) than those grown in t2 media and harvested on day 20. the ash content of bsfl reared in t1, t3, and t4 media and harvested on day 15 and day 20 was similar (p > 0.05). the present result partly agreed with liu et al. (2017), who reported that there the ash content of late pupae was higher (10.2%) than the ash content of 14-day-old bsfl (8.3%). the range of bsfl ash content in the present study was 9.60 to 17.6%, which was in the range reported by seyedalmoosavi et al. (2022). the difference (p < 0.05) in ca content was only found between bsfl grown in t3 media harvested on day 15 and those grown in t3 media and harvested on day 20. the tendency of higher ca content in 20-day-old bsfl was probably due to the bone and teeth of bsfl having grown well. in a review by seyedalmoosavi et al. (2022), it was reported that bsfl required minerals for the skeleton formation and the formation of other structural tissues (e.g., teeth). furthermore, it was explained that although protein and chitin are the main components of the exoskeleton of most insect species, bsfl has a so-called mineralized exoskeleton which explains the high ca content. statistical analysis showed that except for dm content, the main factor, fs affected (p < 0.05 to p < 0.001) the chemical composition of bsfl meal. the cp content of bsfl fed with t1 experimental diet was similar (p > 0.05) to the cp content of bsfl from the t2 diet; however, the cp content of t2 was higher (p < 0.05) than the cp content of bsfl fed with t3 and t4 diets. it is interesting to note that the t3 feeding substrate with the lowest cp content (table 1) produced the bsfl with lower cp content (p < 0.05) compared to t1 and t2 treatment diets (table 2). this could probably be due to the presence of higher antitrypsin and lectin contents in the tofu by-product used in the t3 treatment diet, which inhibits cp digestibility. isanga and zhang (2008) explained that tofu by-products contained 30 to 50% trypsin inhibitor and caused a negative effect on cp digestibility. another possibility was that the lower mineral and vitamin contents in the t3 diet led to low nutrient metabolism. it is interesting to note also that of the cp content of bsfl from t3 diets (high in fiber but low in protein) had similar (p > 0.05) cp content with bsfl grown in t4 diets (high protein substrates and low in fiber). the present result agreed with tschirner and simon (2015), who reported that the bsfls given organic materials rich in fiber produced high cp bsfl. according to tschirner and simon (2015), the larva may consume the waste easily when the waste has been degraded by microbes and in the form of pasta. the present result indicated that the bsfl was tolerable to a high-fiber diet and could digest the fiber. kim et al. (2011) reported that the bsfl digestion system contained several microorganisms that produced lignin-cellulase, which degrades lignin into simple sugar. supriyatna and ukit (2016) reported that bsfl could degrade organic wastes because of the presence of cellulolytic produced by cellulolytic bacteria, especially bacillus sp. the range of cp content of bsfl obtained in the present study was 32.7 to 42.9% (as fed). the cp content of bsfl in the present study was lower than the cp content of bsfl reported by tschirner and simon (2015), i.e., 37 to 52.3%. the difference was probably due to the difference in the method, including the type of growing media and the harvested time. growing media used by wardhana (2016) included palm kernel meal, cow feces, pig feces, chicken excreta, fruit wastes, and other organic wastes. in addition, the bsfl was harvested on day 25. the growing media used by tschirner and simon (2015) was a mixture of middling (control group), dried distillers’ grains with soluble (protein group), and dried sugar beet pulp (fiber group). regarding the second main factor, the cp content of bsfl harvested on day 15 was comparable (p > 0.05) to those harvested on day 20. the fs x ht interaction did not affect (p > 0.05) the cp content of bsfl. it was expected that the cl content of bsfl fed with t4 and t2 diets should be higher than the cl content of bsfl grown in the t1 and t3 diets (table 1 and table 2) because of their high cl content in the diets. the reality showed that the cl of bsfl fed with the t3 diets was higher (p < 0.05) than in those fed with the t2 and t4 diets, while the cl content of t1, t2, and t4 was biotropia vol. 30 no. 3, 2023 302 similar (p > 0.05). thus, this study proved that the cl content of bsfl did not relate to the cl content of the grown substrates. the cl of bsfl harvested on day 20 was higher (p < 0.05) than those harvested on day 15. this result indicated that as the age of bsfl increased, the cl content of bsl increased. no interaction (p > 0.05) between fs and ht was found in the cl content of bsfl in all treatment diets. the ash content of bsfl fed with t4 diets was higher (p < 0.05) than those fed with t1, t2, and t3 diets. the ash content of bsfl was similar (p > 0.05) between t1 and t2 when harvested on day 20. the bsfl grown in t2 diets and harvested on day 15 had higher (p < 0.05) ash content compared to those who were grown in the same feeding substrate but harvested on day 20. on the other hand, the bsfl fed with t3 and t4 diets and harvested on day 20 had higher (p < 0.05) respective ash content than those that were grown in the same feeding substrate but harvested on day 20. the comparison was difficult to be made due to the limitation of published data related to the present study. table 2 shows the ash content of bsfl grown in the t4 diets was higher (p<0.05) than the ash content of bsfl grown in t3. this was an unacceptable result since the ash content of t4 diets was very low (table 01) but produced bsfl with high ash content. the ash content of the t4 and t1 diets was quite similar (table 1), but these media produced bsfl high in ash content (table 2). the present study indicated that the ash content of bsfl was not affected by the ash content of the grown substrates. the ca content of the bsfl fed with t3 and t4 diets were higher (p < 0.05) than those grown in t1 and t2 diets. this could be due to the higher ca content of the t3 and t4 diets than the t1 and t2 diets (table 1). the p content of bsfls fed with t3 was lower (p < 0.05) than those fed with t1, t2, and t4. the lowest p content of bsfl in t3 diets is related to the lowest p content of t3 diets (table 1). table 2 chemical composition of black soldier fly larvae (bsfl) fed with different feeding substrates and harvested at different times feeding substrate harvesting time (day) dry matter crude protein crude lipid ash ca p gross energy ……………………….% as fed……………..…… (mj/kg dm) t1 15 93.4 39.6 29.9 10.7cd 1.65d 1.42 23.7 20 94.8 36.9 36.1 9.83d 2.16cd 1.00 24.9 t2 15 94.1 42.9 20.7 13.2b 2.92bc 1.27 21.6 20 92.4 40.0 33.0 9.60d 2.21cd 0.94 24.4 t3 15 94.5 32.7 36.9 12.9bc 3.68b 0.87 23.9 20 94.4 33.6 35.9 14.4b 5.06a 0.83 23.5 t4 15 94.7 37.8 28.3 16.4a 4.87a 1.20 22.3 20 93.8 34.5 30.8 17.6a 5.09a 1.16 22.2 sem 0.617 1.75 2.99 0.815 0.295 0.086 0.67 main effects feeding substrate (fs) t1 94.1 38.3ab 32.9ab 10.2c 1.91b 1.21a 24.3a t2 93.5 41.5a 26.8b 11.4c 2.57b 1.11a 23.0ab t3 94.5 36.2c 36.4a 13.7b 4.37a 0.85b 23.7a t4 94.3 36.1bc 29.5b 16.9a 4.98a 1.18a 22.2b sem 0.437 1.24 2.12 0.576 0.209 0.061 0.484 harvesting time (ht, day) 15d 94.2 38.25 28.9b 13.3 3.28 1.19a 22.9 20d 94.0 36.27 33.9a 12.9 3.63 0.98b 23.8 sem 0.309 0.876 1.50 0.407 0.148 0.043 0.25 probability p> f fs ns *** * *** *** ** * ht ns ns * ns ns ** ns fs x fa ns ns ns * * ns ns notes: different superscripts at the same column indicate significant differences (p < 0.05); * = significantly different at p < 0.05; *** = significantly different at p < 0.001; ns = not significantly different (p > 0.05); sem = standard error of mean. nutrient composition of maggot grown in different substrates – weko et al. 303 table 3 describes the indispensable amino acid (iaa) profile of bsfl fed with different feeding substrates and harvested at different times. except for isoleucine, the interaction between fs and ht did not affect (p > 0.05) the indispensable amino acid content of bsfl. the content of isoleucine in bsfs fed with t3 and t4 diets and harvested on day 20 was similar (p>0.05) to those fed with the same feeding substrates but harvested on day 15. there was a tendency that as the age increased, the iaa content of bsfl increased. however, the statistical analysis showed that only the content of isoleucine in bsfl fed wth t1 and t2 diets and harvested on day 20 was higher (p < 0.05) than those fed with the same feeding substrates but harvested on day 15. this result partially indicated that the isoleucine content of bsfl increased as the age of bsfl increased. feeding substrate affected (p < 0.05 to <0.01) the content of leucine, phenylalanine, and valine content of bsfl. the leucine content of the bsfls fed with t2 diet was lower (p < 0.05) than those fed with three other treatment diets. the difference in leucine and phenylalanine content of bsfl was probably due to the difference in leucine and phenylalanine content and the digestibility of feeding substrates. the phenylalanine content of bsfl grown in t4 feeding substrates was higher (p < 0.05) than those grown in t3 diets. no differences (p > 0.05) in valine content were observed in bsfls fed with t1 and t2 diets and between t3 and t4. regarding the second main factor, the statistical analysis showed that the content of leucine, lysine, phenylalanine, and valine of 20d bsfl was higher (p < 0.05) than the respective content of leucine, lysine, phenylalanine, and valine of 15d bsfl. these results indicated that it is better to harvest the bsfl on day 20 because the indispensable amino acid profile of 20-dayold bsfl is better than 15-day-old bsfl. in addition, the results indicated that as the age of bsfl increased, the indispensable amino acid content increased. the comparison was difficult to be made because of the limitation of published data related to the present study. table 3 indispensable amino acid (iaa) profile of black soldier fly larvae (bsfl) fed with different feeding substrates and harvested at different times feeding substrate harvesting time (day) arg his isoleu leu lys meth phen thr val ………………………….% as fed…………………….……………… t1 15 1.97 1.20 1.51b 3.03 2.57 0.46 1.47 1.48 2.29 20 2.31 1.29 1.69a 3.23 2.65 0.51 1.51 1.48 2.39 t2 15 2.03 1.19 1.49b 3.01 2.57 0.55 1.38 1.66 2.25 20 2.08 1.27 1.75a 3.18 2.64 0.51 1.50 1.49 2.39 t3 15 2.13 1.19 1.62ab 3.05 2.53 0.59 1.37 1.57 2.11 20 2.23 1.25 1.69a 3.20 2.64 0.64 1.54 1.67 2.29 t4 15 2.21 1.36 1.63a 3.22 2.53 0.59 1.55 1.71 2.16 20 2.15 1.26 1.65a 3.20 2.59 0.54 1.54 1.58 2.17 sem 0.103 0.040 0.043 0.042 0.047 0.056 0.033 0.07 0.05 main effects feeding substrate (fs) t1 2.14 1.25 1.61 3.13a 2.61 0.49 1.49ab 1.48 2.34a t2 2.05 1.23 1.62 3.09b 2.61 0.53 1.44b 1.58 2.32a t3 2.18 1.22 1.66 3.13a 2.59 0.61 1.45b 1.62 2.20b t4 2.18 1.31 1.64 3.22a 2.56 0.57 1.55a 1.64 2.17b sem 0.072 0.029 0.030 0.290 0.033 0.039 0.023 0.052 0.033 harvesting time (ht, day) 15d 2.09 1.24 1.57b 3.08b 2.55b 0.55 1.44b 1.60 2.21b 20d 2.19 1.27 1.69a 3.21a 2.63a 0.55 1.52a 1.56 2.31a sem 0.051 0.020 0.021 0.021 0.023 0.028 0.016 0.036 0.023 probability p> f fs ns ns ns * ns ns * ns * ht ns ns *** *** * ns ** ns ** fs x fa ns ns * ns ns ns ns ns ns notes: different superscripts at the same row indicate significant differences (p < 0.05); * = significantly different at p < 0.05; *** = significantly different at p < 0.001; ns = not significantly different (p > 0.05); sem = standard error of mean. arg = arginine; his = histidine; iso = isoleucine; leu = leucine; lys = lysine; meth = methionine; phen = phenylalanine; thr = threonine; val = valine. biotropia vol. 30 no. 3, 2023 304 regarding the dispensable amino acids (daa), published data showed that the daa contents are now considered important in poultry diet formulation. the calculation of daa such as alanine, aspartic acid, glutamic acid, glycine, serine, and proline in poultry diet had been reported by previous researchers (awad et al. 2015; siegert & rodehutscord, 2018; hofmann et al. 2020). according to awad et al. (2015), in which low-protein diets fortified with daa (glycine, glutamic acid, proline, alanine, and aspartic acid) produce similar body weight gain with the group of birds which were given the balanced nutrition diet (positive control). they also reported that the addition of glycine improved the feed conversion ratio of birds. thus, it is essential to have a database of the dispensable amino acid profile for each feed ingredient, including bsfl, used in poultry diets. table 4 describes the profile of daa of bsfl fed with different feeding substrates and harvested at different times. table 4 shows no interaction (p > 0.05) between fs and ht was found for the daa content of bsfl. regarding the first main factor (fs), the significance (p < 0.01 to 0.001) was in the aspartic acid, cysteine, glutamic acid, and proline of bsfl. the aspartic acid and glutamic acid content of bsfls fed with t3 diets were higher (p < 0.05) than the aspartic acid and glutamic acid content of bsfls fed with t1, t2, and t4 diets. the cysteine content of bsfls with fed t3, and t4 diets were higher (p < 0.05) than those fed with t1 and t2 diets. the present results indicated the feeding substrate containing a high percentage of vegetable wastes (t3) produced the best dispensable amino acid profiles. regarding the second main factor, the statistical analysis showed that the content of aspartic acid and serine of 20d bsfl was higher (p < 0.05) than the content of aspartic acid, glycine, glutamic acid, and proline of 15d bsfl. these results indicated that it is better to harvest the bsfl on day 20 because the indispensable amino acid profile of 20-day-old bsfl were better than 15-day-old bsfl. in addition, the results indicated that as the age of bsfl increased, 15-day-old indispensable amino acid content increased. the comparison was difficult to be made because of the limitation of published data related to the present study. table 4 dispensable amino acid (daa) profile of black soldier fly larvae (bsfl) fed with different feeding substrates and harvested at different times feeding substrate harvesting time (day) ala asp cys glu tyr ser gly pro …………………..% as fed…………………………….. t1 15 0.66 3.51 0.30 4.85 1.11 0.62 1.11 1.08 20 0.71 3.81 0.31 4.92 1.37 0.71 1.13 1.13 t2 15 0.37 3.06 0.36 4.87 1.07 0.58 1.04 1.13 20 0.72 3.40 0.32 4.90 1.36 0.75 1.14 1.16 t3 15 0.72 3.53 0.44 4.75 1.16 0.72 1.18 1.42 20 0.86 3.85 0.49 4.76 1.212 0.78 1.17 1.24 t4 15 0.81 3.87 0.44 5.01 1.15 0.75 1.19 1.38 20 0.78 4.13 0.41 5.16 1.22 0.77 1.23 1.24 sem 0.044 0.083 0.035 0.077 0.137 0.052 0.0471 0.063 main effects feeding substrate (fs) t1 0.73 3.23c 0.34b 4.89b 1.22 0.67 1.09 1.14b t2 0.69 3.66b 0.31b 4.89b 1.24 0.67 1.12 1.11b t3 0.79 4.00a 0.43a 5.09a 1.19 0.76 1.21 1.31a t4 0.79 3.69b 0.47a 4.76b 1.19 0.75 1.18 1.33a sem 0.031 0.059 0.025 0.054 0.097 0.036 0.033 0.045 harvesting time (ht, day) 15d 0.73 3.49b 0.39 4.87 1.12 0.67b 1.13 1.25 20d 0.77 3.79a 0.38 4.94 1.29 0.75a 1.17 1.19 sem 0.022 0.041 0.017 0.038 0.068 0.026 0.023 0.031 probability p> f fs ns *** *** ** ns ns ns ** ht ns *** ns ns ns * ns ns fs x fa ns ns ns ns ns ns ns ns notes: different superscripts at the same row indicate significant differences (p < 0.05); * = significantly different at p < 0.05; *** = significantly different at p < 0.001; ns = not significantly different (p > 0.05); sem = standard error of mean. ala = alanine; asp = aspartic acid; cys = cysteine; glu = glutamic acid; tir = tyrosine; ser = serine; gly = glycine; pro = proline nutrient composition of maggot grown in different substrates – weko et al. 305 the iaa and daa content observed in the present study were lower than those reported by the previous researchers (lu et al. 2022). the difference was probably due to the difference in methodology, especially for feeding substrate and harvested times. body weight gain (bwg) of black soldier fly larvae statistical analysis showed that the body weight gain (bwg) of bsfl harvested on day 20 was affected (p < 0.05) by the feeding substrates. this is in line with kinasih et al. (2018), who reported that the body weight of bsfl was affected by the feeding substrates. body weight gain of 15d-bsfl was not measured, so it is not presented in this article. as can be seen in table 5, the bwg of bsfl fed with t2 diets (cp 11.43% and cf 10.64%) (p < 0.05) were lower than the bwg of bsfl fed with t1, t3, and t4 diets during the experiment. the difference in bwg may be due to the difference in nutrient composition, palatability, and digestibility of the feeding substrates. it is interesting to note that the bwg of bsfl fed with t3 diets (cp 4.03% and cf 15.56%) (table 5) was higher (p < 0.05) than the bwg of bsfl fed with t2 (cp11.43% and cf 10.64). even though the feed intake was not recorded, the high bwg of bsfls fed with t3 diets was probably due to the high feed intake in this treatment diet. the feed intake was not documented in this experiment because the media used was semi-liquid, so it was difficult to measure. the separation of bsfl and media should have been conducted by water so it would affect the weight of the feed. however, the palatability of the feeding substrate was conducted by observing the leftover of each treatment. the comparison was difficult to be made due to the limitation of publication related to the present research. the bwg of bsfl fed with t3 diets (cp 4.03% and cf 15.56%) was similar (p > 0.05) to those fed with t4 (cp 22.81%; cf 6.56%) and t1 (cp 16.07%; cf 8.11%) diets. the reasons behind these results were: i) the bsfl could degrade fiber so the nutrients needed for growing were fulfilled; ii) the antinutrient level, especially protease inhibitors, in t3 diets was probably lower than in t1 and t4 diets. so, the nutrient digestibility in the t3 diets might be higher than in the t1 and t4 diets. as can be seen in table 1, t1 and t4 diets contain a high amount of tofu by-products. the tofu by-product was made by soybean seeds, so the by-product may contain protease inhibitors that could inhibit protein digestibility since soybean contain trypsin inhibitory (aviles-gaxiola et al. 2018). as a consequence, the amount of nutrients absorbed would be low as well, which finally led to a low growth rate. beniers (2021) reported that trypsin was located in the posterior part of the bsfl midgut, and the trypsin inhibitor may decrease its activity. however, the present result was not in agreement with kinasih et al. (2018), who reported that bsfls fed with tofu dreg and chicken feed had higher development time compared to those given horse manure and vegetable wastes. the difference was probably due to the difference in methodology. supriyatna and ukit (2016) reported that the bsfl were able to digest organic wastes cellulolytic enzyme, which was produced by cellulolytic bacteria, especially bacillus spp. in the bsfl digestive organ. felicia and suhartono (2021) reported that the whole body of bsfl contained proteolytic, amylolytic, chitinolytic, and cellulolytic activities. these enzymes play an important role in nutrient digestion (protein, starch, chitin, and cellulose). kim et al. (2011) extracted gut bsfl and found several enzymes which were high amylase, lipase, and protease activities, trypsin-like protease activity of leucine arylamidase, α-galactosidase, β-galactosidase, αmannosidase, and α-fucosidase. table 5 growth performance of 20-day-old black soldier fly larvae (bsfl) (g) fed different organic substrates variable treatment sem p-value t1 t2 t3 t4 initial body weight (g) 0.071 0.074 0.074 0.064 final body weight (g) 0.191 0.155 0.201 0.165 body weight gain (g) 0.122a 0.082b 0.127a 0.102ab 0.008 0.013 notes: different superscripts at the same row indicate significant differences (p < 0.05); sem = standard error of mean. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/amylase https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/lipase https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/leucine biotropia vol. 30 no. 3, 2023 306 the present result did not agree with spranghers (2020), who reported that the bwg of bsfl will increase as the dietary protein content increases. the difference was probably due to the difference in the feed ingredient used. each feed ingredient contains a different level of nutrients and anti-nutrients. the experimental diet used by spranghers (2020) was a chicken diet that was low in anti-nutritional factors. in this experiment, the authors used a self-mixing diet which consists of mostly organic wastes containing high anti-nutrients. the author also found that the shortage of lysine and methionine did not decrease the growth rate of bsfl. conclusion different feeding substrates and harvesting times produced different chemical compositions and amino acid profiles of black soldier fly larvae. the combination of fruit wastes and tofu byproducts produced bsfl with high crude protein but low crude lipid, ash, calcium, and phosphorus. the use of tofu by-products as feeding substrates produced bsfl with the lowest crude protein content. black soldier fly larvae fed with nearly all treatment diets and harvested on day 15 had higher crude protein, calcium, and phosphorus content. the dispensable amino acid profile of black soldier fly larvae fed with the substrates containing a high percentage of vegetable waste was the best. feeding substrates containing a high amount of rice bran produced the lowest body weight gain. references aoac. 2005. official methods of analysis of aoac international. 18th ed. arlington (us): association of official agricultural chemists. aoac. 2012. official methods of analysis, association of official analytical chemist 19th edition, washington d.c., usa. aviles-gaxiola s, chuck-hernandez c, and and sald´ıvar sos. 2018 inactivation methods of trypsin inhibitor in legumes: a review. j food sci 83(1): 17-29. awad ea, zulkifli i, soleimani af, loh tc. 2015. individual non-essential amino acids fortification of a low-protein diet for broilers under the hot and humid tropical climate. poult sci 94: 2772-2777. barragan-fonseca1 kb, dicke m, van loon jja. 2017. nutritional value of the black soldier fly (hermetia illucens l.) and its suitability as animal feed – a review. j insect food feed 3(2): 105-120. beniers jja. 2021 enzyme localization in the digestive tract of black soldier fly larvae, by freeze-substitution and glycol methacrylate inclusion. master thesis. laval university, canada. bondari k, sheppard dc. 1987. soldier fly, hermetia illucens l., larvae as feed for channel catfish, ictalurus punctatus (rafinesque), and blue tilapia, oreochromis aureus (steindachner), aquaculture res 18(3):209220 available from: https://doi.org/10.1111/ j.1365-2109.1987.tb00141.x bosch g, zhang s, dennis gabo, wouter hh. 2014. protein quality of insects as potential ingredients for dog and cat foods. j nut sci. available from: https://www.ncbi.nlm.nih.gov/pmc/articles/pmc 4473158/pdf/s2048679014000238a.pdf čičková h, newton, gl, lacy rc, kozánek m. 2015. the use of fly larvae for organic waste treatment. waste management 35: 68-80. dimiyati f. 2021. pemerintah sebut impor bungkil kedelai masih sangat besar. 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[cited on february 2023]. available from; https://www.bioboosteurope.com/assets/files/bi oboost-report-amino-acid-composition-andrequirements-of-bsf-larvae.pdf supriyatna a, ukit. 2016a. screening and isolation of cellulolytic bacteria from gut of black soldier fly larvae (hermetia illucens) feeding with rice straw. j bio bio edu 8(3): 314-320. supriyatna a, manurung r, esyanti rr, and putra re. 2016b. growth of black soldier larvae fed on cassava peel wastes, an agriculture waste. j ent zoo studies 4(6): 161-165. tschirner m, simon a. 2015. influence of different growing substrates and processing and the nutrient composition of black soldier fly larvae destined for animal feed. j insects food feed 1(4): 249-259. https://doi.org/10.4308/hjb.25.2.79 https://doi.org/10.3390/insects13090831 https://doi.org/10.3390/insects13090831 https://dx.doi.org/10.3923/ijps.2019.353.360 https://dx.doi.org/10.3923/ijps.2019.353.360 https://iopscience.iop.org/article/10.1088/1755-1315/187/1/012070/pdf 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central java indonesia received 29 may 2023 / revised 30 november 2023 / accepted 4 january 2024 abstract this research was conducted to determine the chemical composition and anti-nutritional content of carica dieng seeds (carica pubescens) and their in vitro digestibility after boiling. the process of boiling is frequently employed to reduce antinutrient content and increase the digestibility of nutrients. this research used a completely randomized design, consisting of three treatments with boiling durations of 0, 10, and 20 minutes, each repeated six times. the research revealed a significant (p<0.05) decrease in the tannin and saponin levels of carica dieng seeds following the boiling process. the concentration of carica seed tannin was lowered to a range of 36-48%, while the saponin content decreased by 52-58%. subsequently, a descriptive analysis was conducted to assess the nutritional and amino acid composition of the seeds after boiling them for a duration of 10 minutes. lysine (1.57% w/w) dominated the essential amino acids in carica pubescens seeds, and glutamic acid (3.19% w/w) dominated the non-essential amino acids the digestibility of boiling carica seeds as feed was assessed by including them into complete feed at different proportions: 0% (t0), 5% (t2), 10% (t3), and 15% (t4). this experiment followed a completely randomized design with five replications. the variables included in-vitro dry matter (dmd) and organic (omd) digestibility. the dmd of carica seeds at a concentration of 0% showed a statistically significant difference (p<0.05) when compared to the addition of 5% carica seeds. however, there was no significant difference in dmd between adding carica seeds at levels of 10% and 15% in the total meal. the organic material digestibility variable showed that the treatment without carica seeds was not significantly different from adding 10% carica seeds but was substantially different from adding 5 and 15% carica seeds. carica pubescens seed supplementation of up to 10-15% remains viable for inclusion in whole feed since it does not reduce digestibility. keywords: boiling, carica pubescens, , in-vitro digestibility, saponin, tannin introduction the feed factor is an essential element of a ruminant fattening strategy. the availability and synergistic interaction of nutrients in the diet are crucial factors in determining the productivity of animals. alternative feed sources are necessary due to the typically expensive cost commercial feed and the intermittent availability of protein feed. consequently, meeting the nutritional requirements of cattle and maintaining a consistent feed supply can be labor-intensive. agro-industrial waste can serve as a potential substitute for cattle feed due to its retention of essential nutrients and consistent availability. the addition of agro-industrial wastes, such as carica processed waste, to complete feed is expected to minimize feed costs, reduce waste, and increase small-scale animal production (lalramhlimi et al. 2022). one of the initiatives to build a low-cost and effective feed business is using locally accessible agricultural and plantation wastes in each region, along with reducing agro-industrial waste. despite the extensive utilization of the country's abundant feed ingredients, the need for these feeds remains unfulfilled (kim et al. 2019). therefore, further research is required to *corresponding author, email: rahmafina2@untidar.ac.id effect of boiling carica pubescens seeds on antinutrient content and in vitro nutrient – rahma wulan idayanti et al. 45 investigate unconventional materials. carica dieng confections have become into basic commodities, possessing economic significance. annually, a total of 1100-1200 tons of fruit is harvested, with the involvement of 30 middle, small, and micro businesses (ningsih et al. 2019). the carica seeds and their membranes are remnants resulting from the preparation of the carica fruit. according to briones-labarca et al. (2015), the ripe seeds have a moisture content of 3.50%, an ash content of 3.96%, a crude protein content of 31.84%, and a crude fiber content of 24.41%. the relatively high protein content of carica dieng seeds might provide some advantage. multiple research studies have proven that dieng carica seeds contain anti-nutritional factor tannins and saponins (kiranmayi 2014); hence, the utilization of dieng carica seeds as feed needs further research. natural foods and feedstuffs contain chemical substances known as anti-nutritional factors (anfs) which are generated through the metabolic processes of species and various factors that hinder optimal nutrition. these factors include the inactivation of particular nutrients, a deceleration of the digestive system, and a reduction in the metabolic utilization of the food or feed (thakur et al. 2017). tannins are polyphenolic chemicals classified into two types depending on their chemical structure: condensed and hydrolyzable tannins. green plants typically produce different quantities of tannins, resulting in different biological properties. the presence of many phenolic groups accounts for tannins' strong affinity for proteins. these provide numerous opportunities for peptide carbonyl groups to bind. the creation of such complexes is specific regarding the tannin and protein involved, with the degree of affinity between the molecules in each chemical property (bunglavan and dutta 2013). according to trisnadewi et al. (2014), an increased concentration of tannins attaches to carbohydrates and proteins, thereby impeding the ability of rumen microorganisms to degrade these substances and consequently diminishing enzyme activity. a reduction in the population of rumen bacteria will correspondingly lead to impaired nutrient assimilation and decomposition, as well as reduced access to carbohydrates and proteins within the rumen. saponin is a glycoside that has an aglycone in the form of sapogenin. the chemical structure of saponins is in the form of a glycoside consisting of glycone and aglycones. the glycone part is a sugar group such as glucose, fructose, and other types of sugar, while the aglycone part is a sapogenin (nurzaman et al. 2018). saponins are glycosides with aglycones in the form of steroids and triterpenoids (yanuartono et al. 2017). steroid saponins are often found in single-seeded or monocot plants (negi et al. 2013), and triterpenoid saponins are often found in doubleseeded plants or dicots (yanuartono et al. 2017). anti-nutritional factors (anfs), which are naturally present compounds in plants, have the potential to adversely affect the value of feed when administered to livestock in specific amounts. they can affect biological aspects, disrupt body metabolism, reduce livestock productivity, and inhibit livestock growth and health (jayanegara 2018; murni et al. 2012). because anfs can stunt the growth of livestock that consume them, their presence in feed ingredients may be a ration-limiting factor. it is necessary to study the anfs content in carica dieng seeds. this step is critical to minimize the various adverse effects of anfs. boiling has been identified as a potential technique for diminishing the antinutrient content (ndidi et al. 2014). in their research, assam et al. (2019) demonstrate that water steaming can also reduce the content of tannins, hcn, saponins, and phytate. the ability of ruminant livestock to utilize feed containing antinutrients varies. the tolerance of sheep to the antinutrients in boiled carica seeds has yet to be discovered, thus requiring research on the levels of carica seeds in sheep rations. the addition of boiled carica seeds at different levels aims to determine the maximum limit of antinutrients that sheep can tolerate. with this knowledge, maximizing the use of carica seeds, which are processed by boiling as a source of protein in sheep, can be used as safe feed for their productivity. it is necessary to do in vitro testing on boiled carica seeds to assess the nutrient quality, specifically the digestibility of dry matter and organic matter in sheep rations. it can be achieved by replicating the physiological processes that occur in the digestive system of livestock. biotropia vol. 31 no. 1, 2024 46 materials and methods time and location of research the dieng carica seeds used in this study were collected from the carica beverage manufacturing company dieng, banjarnegara, central java, indonesia. the collection of seeds was conducted in april 2022 (figure 1). the carica dieng fruit and its seeds were used as samples in this study (figure 2). procedures the carica seeds were separated from the carica fruit through peeling and removal. the seeds underwent processing in accordance with the procedure outlined by talabi et al. (2016). the boiling process lasted for durations of 0, 10, and 20 minutes. in each case, the water was heated until it reached the boiling point prior to pouring the seeds, and they were then left to boil for the duration length. subsequently, the seeds were drained of water, dried, and boiled. they were then examined to determine the levels of tannins, saponins, amino acids, as well as the digestibility of dry and organic matter using in vitro analysis. nutritional component analysis chemical analysis of carica seed samples that had been boiled included proximate analysis: dry matter (dm), ash, organic matter (om), crude protein (cp), ether extract (ee), and crude fiber (cf), according to aoac (2005); amino acid analysis in house method (ici instrument method 1988), tannin content (folin ciocalteau method (chaovanalikit and wrolstad, 2004), saponins (pasaribu et al. 2014), dry and organic matter digestibility was determined using the tilley and terry method (1963). figure 1 location of the survey (dieng, banjarnegara district central java province, indonesia) a b c figure 2. a. carica pubescens fruit; b. carica fruit flesh; c. carica pubescens seeds effect of boiling carica pubescens seeds on antinutrient content and in vitro nutrient – rahma wulan idayanti et al. 47 table 1 feed composition and nutritional content of the diets. feed composition/ nutritional content treatment t0 t1 t2 t3 feed composition -------------------------------(%) ----------------------------- pakchong grass 40 40 40 40 concentrate 60 55 50 45 carica pubescens seed 0 5 10 15 nutritional content dry matter (dm) 80.29 90.82 90.90 90.75 ash (% dm) 12.04 11.51 10.98 10.44 crude protein (cp % dm) 11.30 12.02 12.74 13.46 crude fiber (cf, % dm) 28.03 28.63 29.24 29.84 extract ether (ee, %dm) 1.54 2.21 2.88 3.55 nitrogen-free extract (nfe, % dm) 47.09 45.62 44.16 42.70 total digestible nutrients (tdn) 58.45 58.70 58.96 59.21 experimental design and data analysis carica dieng seeds used in this research were collected from dieng, central java, indonesia. as part of the preliminary investigation, the experiment examined the existence of tannins and saponins and assessed the reduction of their levels by a physical treatment (boiling). this research used a completely randomized design consisting of three treatments: t0 without boiling, t1 = 10 minutes, t2 = 20 minutes, and six replications. the carica seeds were subjected to boiling water for an appropriate duration during their processing. their amino acid composition and the best results were carefully examined and discussed descriptively. carica seeds were added to complete feed and tested in vitro to evaluate the digestibility of dry matter and organic matter. testing was done using a completely randomized design with four treatments and five replications. carica seeds were added to complete feed, consisting of t0 = 40% pak chong grass + 60% concentrate + 0% addition of carica seeds, t1 = 40% pak chong grass + 55% concentrate + addition of 5% carica seeds, t2 = 40% pakchong grass + 50% concentrate+ addition of 10% carica seeds, t3 = 40% pakchong grass+45% concentrate+ addition of 15% carica seeds (table 1). data on the presence of tannins and saponins and their digestibility were analyzed using analysis of variance (anova) at the 5% level. if there were differences between treatments, continued further testing with the duncan multiple range test (dmrt). experimental design and data analysis carica dieng seeds used in this research were collected from dieng, central java, indonesia. as part of the preliminary investigation, the experiment examined the existence of tannins and saponins and assessed the reduction of their levels by a physical treatment (boiling). this research used a completely randomized design consisting of three treatments: t0 without boiling, t1 = 10 minutes, t2 = 20 minutes, and six replications. the carica seeds were subjected to boiling water for an appropriate duration during their processing. their amino acid composition and the best results were carefully examined and discussed descriptively. carica seeds were added to complete feed and tested in vitro to evaluate the digestibility of dry matter and organic matter. testing was done using a completely randomized design with four treatments and five replications. carica seeds were added to complete feed, consisting of t0 = 40% pak chong grass + 60% concentrate + 0% addition of carica seeds, t1 = 40% pak chong grass + 55% concentrate + addition of 5% carica seeds, t2 = 40% pakchong grass + 50% concentrate+ addition of 10% carica seeds, t3 = 40% pakchong grass+45% concentrate+ addition of 15% carica seeds (table 1). data on the presence of tannins and saponins and their digestibility were analyzed using analysis of variance (anova) at the 5% level. if there were differences between treatments, continued further testing with the duncan multiple range test (dmrt). biotropia vol. 31 no. 1, 2024 48 results and discussion effect of processing antinutritional (tannin, saponin) content this research shows that there is an influence between the boiling process on reducing the tannin and saponin levels of carica dieng seeds (p<0.05), where the boiling treatment at t2 and t3 significantly decreased compared to t1, but t2 and t3 were relatively the same. antinutritional quality of carica pubescens seeds with prolonged boiling treatment is shown in table 2. the tannin content of the t2 treatment (boiling for 10 minutes) was 2.67% lower than the t1 treatment (raw materials without boiling). the tannin concentration decreased to (2.21%) under t3 treatment (20 minutes of boiling). this study relied on the research conducted by jamarun et al. (2021), who find that mangrove tannin levels fall by 7.4% after 10 minutes of boiling. assam et al. (2019) report that boiling for up to 30 minutes reduce the tannin and saponin content in cassia tora seeds. this drop happened as a result of the lengthy boiling time, which might cause the connections between the fibers in carica seeds to loosen. due to the osmosis process, which pushes tannins out of the leaves and causes them to dissolve in alkaline water through diffusion activity, the tannin content will be reduced (perdana et al. 2012). acids, bases, and enzymes can hydrolyze tannin. tannins will break down into glucose and gallic acid if heated at 98.89ºc 101.67ºc (muhammad et al. 2015). tannins are readily soluble in water, and solubility increases when dissolved in hot water (perdana et al. 2012). according to the theory that heat denatures proteins and because some antinutritional components present in carica seeds are protein compounds, heat tends to denature them, the reduction in antinutrient levels caused by boiling in this study. while tannins discolor seeds and bind protein through hydrogen bonds and hydrophobic interactions. table 2 indicates that the saponin content of carica pubescens seeds decreased from 21.23% to 8.86%. this result is consistent with a previous study by lakram et al. (2018). they report that boiling for 25 minutes at 100 ˚c significantly reduce saponin levels. the reduction in saponin content can be attributed to the thermal degradation associated with the heat supplied to the cooked plants. the decrease was also observed by hemmige et al. (2017) and ilelaboye et al. (2013). saponins are water-soluble chemicals that are highly soluble in cold and hot water (chairunnisa et al. 2019). table 2 antinutritional content (tannin and saponin) of carica pubescens seeds sample treatment antinutritional content tannin (%) saponin (%) t1 4,27±0,20a 21,23±2,24a t2 2,67±0,20b 10,23±1,27b t3 2,21±0,73b 8,86±1,22b note: different superscripts denote significant differences (p<0.05) within the same column. saponin is a complex glycoside that is found in plants, known for its foaming properties. therefore, it will produce foam when it reacts with water and is shaken. boiling may also remove components from the material that are deemed superfluous, particularly those that are water-soluble. temitope et al. (2013) identify that heating can reduce saponin concentrations in various plant species. according to chaturvedi et al. (2012), boiling can lower the amount of saponin in soybeans (glycine max linn). saponins and plants with high saponin content harm protozoa by building an irreversible combination with steroids in the protozoan cells (yanuartono et al. 2017). this decrease in the protozoa population in the rumen is likely to have several beneficial effects, including improved nitrogen metabolism efficiency, decreased methane gas emissions, changes in the population of bacteria and fungi in the rumen, and the potential for increased flow of bacterial protein to the lower digestive tract. the findings of this study were corroborated by wang et al. (2012), who claimed that adding saponins from tea to the diet might suppress methanogenesis. this reduction is expected to have positive effects on the environment and enhance the productivity of animal rearing for feed purposes. chemical composition the results of boiling the seeds with the time of 10 minutes were tested descriptively for their nutrient content and amino acids. the proximate analysis showed that carica seeds contained 12.75% moisture, 6.57% ash, 14.85% fat, effect of boiling carica pubescens seeds on antinutrient content and in vitro nutrient – rahma wulan idayanti et al. 49 27.98% crude protein, and 29.89% crude fiber, 35.32% nitrogen free extract (table 3). more than 20% of the protein in carica pubescens seeds makes them an excellent source of protein for rations. high molecular weight peptides make up protein, a chemical material with various bodily functions. the primary building block of muscle tissue is protein. the amount of microbial protein supply, which is dependent on the availability of carbohydrates and nitrogen (not always in the form of protein), as well as the amount of protein that is freely degraded in the rumen, is the primary determinant of the amount of essential and non-essential amino acids that are available to tissues in ruminants (wu et al. 2014). the optimal protein requirement for ruminants is determined by their capacity to digest protein. concentrate feed containing a variety of proteins can provide a more cost-effective source of protein. it is because the combination of proteins with multiple types and amounts of amino acids increases the ability of rumen microbes to rapidly synthesize all the necessary amino acids (williamson and payne, 1978; pathak, 2008). table 3 chemical composition of carica pubescens seeds (% dm) chemical composition carica pubecens seeds water content (%)a 12.75 dm (%)a 87.25 ash (%)b 6.57 cp(%)a 27.98 ee (%)b 14.85 cf(%)a 29.89 nfe (%) 20.80 note: a)proximate analysis (dm: dry matter, cp: crude protein, cf: crude fiber, drug and animal testing laboratory, veterinary agency semarang district (2022). b)ash, ee: ether extract, laboratory of animal nutrition faculty of animal and agricultural sciences, diponegoro university (2022). nfe: nitrogen-free extract table 4 presents the results of the analysis of amino acids from the seeds of carica pubescens. carica pubescens seed essential amino acids are dominated by lysine (1.57% w/w), and nonessential amino acids by glutamic acid (3.19% w/w). amino acids, which are found in proteins, serve the purpose of constructing new tissues, facilitating energy metabolism, synthesizing hormones, and acting as essential catalysts for several physiological processes in the body (li et al. 2020). table 4 amino acid composition of carica pubescens seeds (% w/w) amino acid carica pubecens seeds aspartic acid 1.14 threonine 0.25 serine 0.47 glutamate 3.19 glycine 0.38 alanine 0.39 valine 0.43 methionine 0.06 isoleucine 0.36 leucine 0.58 tyrosine 0.93 phenylalanine 0.39 hystidin 0.55 lysine 1.57 arginine 0.89 note: unit laboratorium jasa pengujian, kalibrasi dan sertifikasi institus pertanian bogor university, 2022. carica pubescens seeds have a relatively high protein content, which is considered important as a new source of nutrition for ruminants. the protein content in carica pubescens seeds is around 24%, equivalent to that of coconut meal. amino acid analysis using hplc showed that the seeds of carica pubecens contained essential and nonessential amino acids (table 2). the highest essential amino acid from carica pubescens seeds is dominated by lysine (1.57% w/w). lysine is necessary because it is one of the precursors to the growth of rumen microorganisms (kong et al. 2021). lysine has a part in forming carnitine, which can stimulate growth, guard against ammonia toxicity, and improve the body's ability to fend off extreme temperature fluctuations (aristasari et al. 2018). lysine inclusion in the meal can promote increased protein synthesis. feed efficiency and feed consumption can both be improved by lysine content (aristasari et al. 2018). lysine tends to boost the feed's protein, fat, and calorie content when added to the ration. the energy content of the meal can also determine the level of feed efficiency. beyond that, lysine can improve a feed's ability to be digested by the ileum (intestine). livestock may be absorbed fast to increase feed efficiency and give livestock a feeling of fullness and strong growth rates. the amino acid tyrosine, which biotropia vol. 31 no. 1, 2024 50 regulates the body's reaction to stress, can be more easily digested after lysine has been added to feed increases the anti-inflammatory action and aids in the healing and prevention of ulcerative colitis and peptic ulcers (aristasari et al. 2018). the highest non-essential amino acids from carica pubescens seeds were glutamic acid content (3.19% w/w). the creation of proteins depends on glutamic acid, which also serves as a source of energy for the intestinal lining cells. enhances immunological response. glutamic acid is a nonessential amino acid that functions as a building block for protein, as a precursor for several nonessential amino acids, and helps the body's metabolism (slyamova et al. 2016) as well as a neurotransmitter for taste (huang et al. 2019). glutamic acid also functions as an antioxidant regulating inducible nitric oxide synthase (inos) as protection against intracellular parasites, bacteria, fungi, viruses, and protozoa (maslami et al. 2018). glutamic acid is an alternative feed additive that is safe and environmentally friendly in improving carcass performance and quality (maslami et al. 2018). in-vitro dry matter and organic matter digestibility digestibility is an initial sign of the availability of different nutrients in a food source that livestock will eat. the high nutritional content consumed post-digestion indicates that the meal is easily digestible (mayulu et al. 2019). table 5 shows the digestibility of dry and organic matter with the addition of carica seed content. the t0 treatment, which did not involve the addition of carica seeds, showed no significant difference compared to the treatments with 10% and 15% carica seeds. however, it exhibited a major difference in dry matter digestibility compared to the treatment with 5% carica seeds. the study found that the lowest digestibility was observed when employing a 5% concentration of carica. this is believed to be due to the varying levels of carica pubescens seeds used in the study, which resulted in diverse digestibility outcomes. carica levels cause different nutrient content, such as the amount of crude protein, crude fiber, and tannins in the treated feed. this decrease in digestibility is likely caused by increased crude fiber content due to adding carica pubescens seeds to complete feed. crude fiber is a component of organic material that is difficult to digest in rumen. increasing the crude fiber content will reduce the digestibility of dry matter, crude protein and energy. setiasih et al. (2023) explained that in their research, the more crude fiber in the feed material, the thicker the cell walls and the more resistant it is to fiberdigesting microorganisms, causing the digestibility of the material to be lower. rumen microorganisms participate in the fermentation process. feed intake depends on activity, and the nutrients contained in feed ingredients impact rumen microbial activity (sandi et al. 2015). an increase or decrease in digestibility is shown in the number and activity of microbes (mayulu 2014). the presence of tannins in carica seeds is thought to reduce the digestibility of feed ingredients. this decrease was due to tannin being able to bind protein so that rumen microbes could not degrade it. however, if the tannin concentration is not well controlled, the presence of tannins in protein protection will enhance the availability of high-quality feed protein. this can lead to disruptions in overall digestibility and nutrient absorption, since tannins interact with protein, fiber, vitamins, and minerals. in addition, mertens & grant (2020) state that the physical composition of feed ingredients and their ratios vary. composition, temperature and speed through the digestive tract are some variables that influence the digestibility of dry and organic matter. the study examined the impact of carica seed content on the digestibility of organic matter. the treatment without any carica seed addition showed no significant difference compared to the treatment with 10% carica seed. however, it did show a significant difference when compared to the treatments with 5% and 15% carica seed content. the digestibility of organic matter describes the availability of nutrients from feed. the livestock digestive system can digest organic materials, which include food in the form of organic material components, including carbohydrates, proteins, lipids and vitamins. in order to make organic components in feed more accessible, it is essential to convert them from insoluble to soluble form (suardin et al. 2014). ismail (2023) assert that the digestibility of organic matter is closely related to the digestibility of dry matter because some dry matter consists of organic matter. factors that influence the effect of boiling carica pubescens seeds on antinutrient content and in vitro nutrient – rahma wulan idayanti et al. 51 digestibility of organic materials are the crude fiber. the increasing crude fiber content in feed tends to increase the cellulose, hemicellulose and lignin content, is caused by microbes being unable to optimally digest the crude fiber components contained in feed ingredients resulting in lower organic matter digestibility values (tafsin 2019). table 5 in-vitro dry matter (dm) and organic matter (om) digestibility of carica pubescens seeds sample treatment digestibility dm (%) om (%) t0 54.27±2,58a 58.24±2.14a t1 49.20±3.52b 53.97±3.13b t2 51.50±3.25ab 55.95±2.77ab t3 50.93±2.30ab 55.02±2.13b note: different superscripts denote significant differences (p<0.05) within the same column. conclusion the research findings indicated a significant decrease in the antinutrient content (tannins and saponins) of boiling carica pubescens seeds. the inclusion of carica seed levels in complete feed ingredients can meet the protein requirements as they have a high protein content. adding 10-15% of carica seed levels does not negatively affect in vitro digestibility. acknowledgments this work was partially supported by puslapdik kemdikbud (center for education financial services, ministry of education, cultural research and technology, lpdp (indonesian education endowment fund) to fund this research through the indonesian education scholarship (bpi) or the indonesian education scholarship program, the faculty of animal husbandry and agriculture at diponegoro university, and the faculty of agriculture at tidar university. references aoac. 2005. official methods of analysis (17th ed.). washington, dc: 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of science, university of the philippines cebu, cebu 6000, philippines 2faculty of education, university of the philippines open university los baños 4031, laguna, philippines 3institute of biological sciences, university of the philippines los baños los baños 4031, laguna, philippines article higlights • in ormoc city, philippines, wild edible plants play a vital role in ensuring food security during disasters. • among these, m. malabathricum stands out for its richness in calcium, magnesium, and manganese, while diplazium esculentum is notable for its high levels of iron, copper, and zinc. • additionally, wild fruits like melastoma malabathricum, annona montana, and rubus fraxinifolius contribute significant vitamin c to the diet. • collectively, these plants provide a diverse range of essential nutrients and offer local medicinal benefits, supporting both sustenance and health in the community. article information received : 8 april, 2024 revised : 2 february, 2025 accepted : 12 february, 2025 *corresponding author, e-mail: pgnazareno@up.edu.ph research article abstract wild edible plants (weps) are a valuable resource for communities facing food insecurity, and their nutritional profiles can provide essential dietary needs, especially in disaster-prone regions like the philippines. this paper explores the nutritional value of wild edible plants (weps) in ormoc city, leyte, philippines, with a particular focus on their role in addressing food security issues during natural disasters. the research included surveying the weps used by the local community in ormoc city and analyzing these plants’ nutrient and mineral composition. the study’s key findings included identifying 15 plant species from 13 families. melastoma malabathricum showed the highest ca, mg and mn content; while diplazium esculentum leaves had the highest iron, copper and zinc content. furthermore, relatively high vitamin c content was found in wild fruits m. malabathricum, annona montana, and rubus fraxinifolius. the plants analyzed were rich in essential nutrients, including minerals (ca, na, mg, mn, fe, cu and zn), crude fiber and vitamin c. the different plants excelled in different nutritional aspects, suggesting a diverse dietary potential. these plants play a crucial role in the local community, providing not only sustenance but also medicinal applications. further research and conservation efforts should be encouraged to harness the nutritional and economic potential of these plants while ensuring their sustainable utilization and preservation. keywords: diplazium esculentum, food alternative, melastoma malabathricum, rubus fraxinifolius introduction wild edible plants (weps) encompass noncultivated plant species growing spontaneously in nature in their natural or semi-natural habitats, which are harvested from the wild and utilized as sources of sustenance (demir et al. 2020; duguma 2020; mahklouf 2019). weps, which are a rich source of micronutrients (talang et al. 2023) play a vital role as alternative food resources for families facing food insecurity in many developing and underdeveloped nations grappling with issues, such as poverty, famine, and drought (anbessa et al. 2024; mishra et al. 2021; guzo et al. 2023; ojelel et al. 2019). many weps have nutritional profiles akin to those of cultivated crops like some of the common vegetables (talang et al. 2024; waheed et al. 2023), rendering them valuable in augmenting the essential dietary needs of rural communities (nyakoojo & tugume 2020; ojelel et al. 2019). the philippines is a region frequently subjected to a range of natural disasters, and exposed to natural hazards (asio 2020; ipong et al. 2020; yoshioka et al. 2021; radtke et al. 2018). one example of this vulnerability occurred in 2013 when typhoon haiyan, one of the most powerful tropical cyclones ever recorded, severely impacted ormoc city. biotropia vol. 32 no. 1, 2025 2 subsequently, in 2017, a 6.5-magnitude earthquake struck the city (ipong et al. 2020). following the disaster, affected communities relied heavily on external aid. however, due to the extensive damage, especially in remote areas, relief operations were delayed. as assessed by stumpf et al. (2014), food supply emerged as one of the critical challenges in the aftermath of typhoon haiyan. the nutritional evaluation of weps can establish options to achieve food and nutritional security (talang et al. 2023), in response to sustainable development goal of zero hunger. in the philippines, a number of studies documenting wild edible plants in the country, and knowledge regarding their nutritional value has been conducted (buenavista et al. 2022; cacatian & tabian 2023; tasani & barcellano 2024). this study was premised on several assumptions: 1) less attention is given to weps as a potential alternative to enhance food security for the community; 2) weps contain essential nutrients and minerals that can address nutritional requirements of the community during a disaster, hence the need to analyze their nutritional content, and 3) there is a need to incorporate information about weps into policies enhancing food security of a community during a disaster. consequently, this study (a) surveyed the weps already utilized by the local community, (b) assessed the nutrient and mineral composition of weps found in ormoc city to ascertain their contribution to food security, and (c) identified potential implications for the utilization of weps during times of disaster. materials and methods study area ormoc city has a total land area of 46,430 ha and is subdivided into 110 barangays (the smallest administrative division in the philippines). half of the lands is in mountainous and hilly areas, with agriculture, comprising 26,298 ha (56.64%), being the main use. the city has 15,034 ha (32.38%) of forest lands and 2,672 ha (5.75%) of commercial areas (dumalan 2023). ormoc city is located at 11°00’26.59” n, 124°36’28.46” e in an enclave fronting the ormoc bay. the ambient temperature of ormoc city is 25.9 °c, relative humidity of 70.2%, wind speed 0.55 m/s at 104°, east (bp integrated technologies, inc. 2019) and an average rainfall of 2,456 mm. given its geographical location, ormoc in general, is highly susceptible to natural hazards and extreme weather events. ormoc city’s land encompasses a wide variety of plants, including wild edible plants that can serve as alternative food sources in the event of a calamity. wild edible plants were collected from barangays bagong, cabintan, gaas, and lake danao, in ormoc city, leyte, philippines (fig. 1) in march 2019. figure 1 context map of the study sites in ormoc city, leyte (source: central visayas center for environmental informatics (cenvi) 2019). data collection permission to conduct the study was sought from the local government unit through the city agriculture office (cao). qualitative ethnobotanical survey a qualitative ethnobotanical survey was conducted in march 2019 through key informant (ki) interviews. the methods used in similar studies (cacatian & tabian 2023; rahim et al. 2019; nyakoojo & tugume 2020) were employed. the barangays selected were based on anecdotal information (tugume & nyakoojo 2020) provided by some of the personnel of the city agriculture office. the city agriculture office of ormoc purposively provided one key informant each for barangays bagong, cabintan, gaas, and lake danao based on their wide knowledge of the plant diversity in the study areas, and they provided wild edible plants in enhancing post-disaster food security nazareno et al. 3 folk knowledge of plants. guided field excursions and field works were used to collect plant samples (cacatian & tabian 2023) as well as additional data on the identity of the plants. the research team was joined in the field by the key informants who also served as local guides and were asked to mention wild edible plants that came into their minds; information on the wild edible plants and their mode of collection and preparation prior to consumption were recorded. the recorded data included: 1) vernacular names of the plant; 2) habit; 3) edible parts; 4) mode of harvest/ preparation/consumption; 5) availability of the plants; 6) conservation status; and 7) additional information about the plants. plant collection and identification approximately 600 g each of the plant samples were collected from the four study sites, i.e., barangays bagong, cabintan, gaas, and lake danao. fresh fruit and leaf samples were carefully placed in plastic resealable bags and temporarily stored in styrofoam boxes with ice to prevent deterioration. subsequently, the samples were transported to the university of the philippines cebu biology laboratory for analysis. for plant identification purposes, fruit samples were preserved in 500 ml glass jars containing 95% ethanol. leaves and stem samples were also collected to prepare plant vouchers. initial plant identification was carried out using co’s digital flora of the philippines (pelser et al. 2011). digitized voucher specimens were then sent to the university of the philippines los baños for verification. sample preparation plant samples were meticulously collected, taking care to minimize external contamination. the collected samples (fruits, leaves, and tubers) were washed thoroughly with distilled water. subsequently, the fruit samples were carefully sectioned by cutting them open or slicing them into thin, uniform pieces. a subset of the samples, earmarked for moisture content determination, underwent immediate processing (islary et al. 2016). the bulk of the samples were subjected to a thorough drying process, initially through airdrying for a duration of 24 hours under controlled laboratory conditions then dried in a hot air oven at 50 oc until their weight stabilized, indicating the complete removal of the moisture (korish 2016; nielsen 2010). upon achieving constant weight, all samples were pulverized to a fine, uniform consistency and placed in airtight plastic resealable bags (islary et al. 2016; korish 2016) to prevent moisture ingress. these bags were properly labelled then stored at room temperature. quantitative proximate analyses the determination of the proximate composition of the weps was conducted in accordance with established protocols outlined by the association of official analytical chemists (aoac 2000). these analyses were done in triplicate to ensure precision and reliability. determination of moisture content three grams of each fresh plant sample were carefully weighed and placed in a crucible that had been preheated to a constant weight in a hot air oven. the plant samples were subjected to controlled heating at a temperature of 105 °c to constant weight, which was confirmed when the difference in sample weights did not exceed 0.0005 g. moisture content was calculated using equation (1). moisture (%) = fresh weight-dry weight fresh weight x 100 (1) determination of crude fiber content two grams per pulverized sample was subjected to boiling in a 0.25 n sulfuric acid (h2so4) solution. subsequently, the mixture was subjected to filtration using a muslin cloth, and the residue was washed three times with hot distilled water. the residue from the previous step was further processed with 100 ml 0.313 n sodium hydroxide (naoh). the resultant mixture was again subjected to filtration, followed by washing with hot distilled water, 0.5 n h2so4, and 50% ethanol. the final residue obtained was placed in a crucible and dried at 100 °c to constant weight. the dried sample was allowed to cool in a desiccator for several minutes and was then weighed, establishing the initial weight (c1). subsequently, the sample was incinerated in a muffle furnace at a maintained temperature of 550 °c for 5 hours and then cooled in a desiccator. after cooling, the sample was reweighed (final weight, c2). the percentage of crude fiber was determined using equation 2. % crude fiber = c1 c2 fresh weight x 100 (2) biotropia vol. 32 no. 1, 2025 4 determination of crude fat content (soxhletmethod) the quantification of crude fat content was conducted using the soxhlet method with petroleum ether extraction. the soxhlet extraction apparatus was prepared by heating the soxhlet beakers to constant weight. the final weight of these beakers was recorded as a reference. two grams of each of the pulverized plant samples were securely enclosed within improvised filter paper tea bags specially designed for this purpose; the sample-containing tea bags were then placed inside extraction thimbles which were positioned within the soxhlet beakers. the apparatus was set up and run in a water bath to facilitate fat extraction. subsequently, the soxhlet beakers were subjected to drying to eliminate excess ether solution, with subsequent weighing performed until a constant weight was attained. the determination of crude fat content was computed using equation (3). crude fat (%) = weight of fat weight of the sample x 100 (3) determination of ash content approximately 2 3 g of each of the samples were placed in a crucible preheated to a constant weight. the initial weight was recorded, along with the combined weight of the crucible and the sample. the sample in the crucible was subjected to hightemperature treatment in a muffle furnace at 550 °c for 6 hours. following the ashing procedure, the crucible and its contents were allowed to cool in a desiccator to prevent moisture absorption. subsequently, the ash weight was determined. the percentage of ash content was calculated using equation (4). ash content (%) = ash weight weight of the plant sample x 100 (4) determination of total solids total solids, representing the dry matter in the plant samples, was calculated by subtracting the moisture percentage from 100 (equation 5). total solids = 100 moisture content (5) determination of oxalic acid five grams each of the pulverized plant samples were mixed with 3 m sulfuric acid (h2so4) solution and stirred for 1 hour. the resulting mixture was filtered and then gently heated between 80 °c to 90 °c using a hot water bath. the solution was titrated against 0.05 m potassium permanganate (kmno4) solution, with the temperature of the mixture consistently maintained at 70 °c. the titration endpoint was identified by the persistence of a light pink color for 15 seconds. the oxalic acid content was determined by calculating the equivalent milligrams of oxalate in 1 ml of 0.05 m kmno4 solution. determination of alkaloids percentage in a 250-ml beaker, 5 g each of the pulverized plant samples was added with 200 ml of 20% acetic acid solution (ch3cooh). the beaker containing the resulting solution was covered with a watch glass and allowed to stand for 4 hours. the mixture was then filtered and concentrated to 50 ml using a hot water bath; this was then added with concentrated ammonium hydroxide (nh4oh) until the precipitate was complete. the solution was allowed to settle for 3 hours and decanted. the supernatant was discarded and the resulting precipitate was washed with 20 ml of 0.1 m nh4oh. the solution was then filtered with a pre-weighed whatman no. 1 filter paper; the filter paper containing the precipitate was air-dried before weighing. the total alkaloids percentage was determined using equation (6). alkaloids (%) = weight of the residue weight of the sample x 100 (6) determination of vitamin c content the vitamin c content in the plant samples was determined following the methodology outlined by rai and panda (2014), with some modifications. prior to the analysis, an extracting solvent was prepared by combining 0.75 g ethylenediaminetetraacetic acid (edta), 5.0 g oxalic acid (c2h2o4), ammonium molybdate ((nh4)2moo4), orthophosphoric acid (h3po4), 5% sulfuric acid (h2so4), and distilled water. from this solution, 11 ml was taken comprising 4 ml of edta, c2h2o4, 2 ml (nh4)2moo4, 1 ml 5% h2so4, 1 ml h3po4, and 3 ml distilled wild edible plants in enhancing post-disaster food security nazareno et al. 5 water. this solution was added to 2.0 g of finely pulverized plant sample and placed in a 15 ml test tube. the test tube was vigorously shaken and allowed to stand for approximately 15 minutes. subsequently, the resulting mixture was filtered using whatman no. 1 filter paper, with the residue being discarded, and the filtrate was measured for its absorbance at 520 nm. for standardization, a standard curve was constructed using various concentrations of vitamin c (100%, 75%, 50%, 25%, 10% and 5%). the same procedure was carried out with the standard solutions. the vitamin c content was calculated using the linear equation derived from the standard curve. mineral analysis one hundred grams of each of the ground samples were sent to technolab analytical group in mandaue city, cebu, philippines, for the determination of the mineral contents. data analysis all experiments were carried out in triplicates; for quantitative data, the values were expressed as mean ± standard deviation. results and discussion wild edible plants and their uses a total of 15 plant species belonging to 15 genera and 13 families were documented (table 1) during the key informant (ki) interview and during the field excursions, which was participated by 4 individuals recommended by the head of the city agriculture office. these individuals included farmers and forest rangers who had extensive knowledge of the local plant diversity. the family that was represented the most was arecaceae, with 3 species, followed by the rest of the families, each having 1 taxon. the plant species mentioned frequently by the key informants was rubus fraxinifolius, locally known as “binit” (table 1). about 47% of the documented plants were trees, 33% were vines, 13% herbs, and 7% shrubs (table 1). out of the 15 plants mentioned, only 8 species were collected for nutrient analyses, as these were the only plants available for collection during the march 2019 sampling. artocarpus blancoi, dillenia philippinensis, diplazium esculentum, melothria pendula, and passiflora edulis were also included by cacatian & tabian (2023) in the list of indigenous wild food resources in northwestern cagayan, philippines. of the 15 plant species identified, at least 3 species have already been commercialized, i.e., “kaong” (arenga pinnata) (used as a food ingredient), “rattan” (calamus sp.), and “mirinda” (passion fruit) (passiflora edulis). most of the mentioned plants, primarily fruits, can be consumed raw, but a few wild fruits can also be used as food additives or seasoning (katmon) (dillenia philippinensis) and can be processed into wine (binit) (rubus fraxinifolius). a number of edible plants serve other purposes, due to the medicinal value that they may possess (ramnath & razal 2019). the fruits of “pipinong gubat” and “apitngaw” have been mentioned to have medicinal properties. “pipinong gubat” (melothria pendula) can be brewed into herbal tea, and “apitngaw” (melastoma malabathricum) is believed to relieve toothache. one notable wild edible plant in the study is the wild yam, locally known as “kut” (dioscorea hispida). it is a thorny creeping plant that grows near rock boulders and alongside river streams. the plant contains dioscorine, a toxic compound characteristic of the family dioscoreaceae. due to this, the tuber must be thoroughly processed to remove the toxins before consumption. according to the ki, this process takes days or even weeks. “kut” is a traditional food in barangay bagong and is usually harvested and consumed during the catholic holy week celebration, where locals would make kakanin (local rice or root crop delicacies). the collection and consumption of this wild yam happens in adverse situations as the effort to include them more frequently in diets requires much processing (ramnath & razal 2019). aside from wild edible fruits, some weps are cooked as vegetable dishes, such as “pugahan” (caryota mitis) and “pako” (diplazium esculentum) salad. the edible part of “pugahan”, aside from its fruits, is the ‘ubod’ or the heart of the palm. locals cut open the base stem of the plant to reveal the ‘ubod’ which, according to the ki, tastes like carabao meat when cooked. on the other hand, “pako” is an edible fern that can be consumed in salads or stir-fried with oyster meat or any other preferred ingredient. the plant can be harvested abundantly along the side of lake danao. biotropia vol. 32 no. 1, 2025 6 ta bl e 1 li st of w ild e di bl e pl an t s pe ci es d oc um en te d in th e et hn ob ot an ic al su rv ey c on du ct ed in th re e se le ct ed b ar an ga ys i n o rm oc , l ey te , p hi lip pi ne s fa m ily sc ie nt ifi c na m e c om m on n am e lo ca l n am e h ab it ed ib le p ar ts m et ho d of pr ep ar at io n, co ns um pt io n fo od c at eg or y c on se rv at io n st at us a nd av ai la bi lit y an no na ce ae an no na m on ta na (m ac fa d) m ou nt ai n so ur so p w ild g uy ab an o tr ee fr ui t c on su m ed ra w fr ui t n ot th re at en ed / a va ila bl e ar ec ac ea e ar en ga p in na ta (w ur m b) m er r. su ga r p al m n at iv e ka on g tr ee fr ui t pr oc es se d fr ui t, fo od in gr ed ie nt n ot th re at en ed / a va ila bl e ar ec ac ea e c al am us sp . r at ta n pa lm r at ta n v in e u bo d (h ea rt o f p al m ) c oo ke d ve ge ta bl e n ot th re at en ed / a va ila bl e ar ec ac ea e c ar yo ta m iti s l ou r. c lu ste re d fi sh ta il pa lm pu ga ha n tr ee u bo d, f ru it c oo ke d fr ui t, ve ge ta bl e n ot th re at en ed / a va ila bl e at hy ria ce ae d ip la zi um es cu len tu m (r et z.) s w. fi dd le he ad fe rn pa ku h er b fr on ds (y ou ng sh oo ts) c oo ke d, sa la d ve ge ta bl e n ot th re at en ed / a va ila bl e c uc ur bi ta ce ae m elo th ria p en du la l . c re ep in g cu cu m be r pi pi no ng g ub at v in e fr ui t c on su m ed ra w fr ui t, be ve ra ge n ot th re at en ed / a va ila bl e d ill en ia ce ae d ill en ia p hi lip pi ne ns is ro lfe ph ili pp in es s im po h k at m on tr ee fr ui t c oo ke d se as on in g, f oo d in gr ed ie nt n ot th re at en ed / a va ila bl e d io sc or ea ce ae d io sco re a hi sp id a d en ns t. in to xi ca tin g ya m k ut , w ild y am v in e ro ot , t ub er c oo ke d st ap le n ot th re at en ed / a va ila bl e fl ac ou rt ia ce ae fl ac ou rt ia ja ng om as (l ou r.) r ae us ch . in di an p lu m , i nd ia n c he rr y se ria le s tr ee fr ui t c on su m ed ra w fr ui t n ot th re at en ed / a va ila bl e m el as to m ac ea e m ela sto m a m al ab at hr icu m l . m al ab ar g oo se be rr y; in di an r ho do de nd ro n ap itn ga w sh ru b fr ui t, se ed s c on su m ed ra w fr ui t n ot th re at en ed / a va ila bl e m or ac ea e ar to ca rp us b la nc oi (e lm er ) m er r. an tip ol o (t ag al og ) an tip o tr ee fr ui t, se ed s c oo ke d, ro as te d se ed s fr ui t, sn ac k n ot th re at en ed / a va ila bl e m yr ta ce ae d ec as pe rm um pa rv ifl or um (l am .) a. j. sc ot t si lk y m yr tle be ri tr ee fr ui t c on su m ed ra w fr ui t n ot th re at en ed / a va ila bl e pa sifl or ac ea e pa ssi flo ra ed ul is si m s pa ss io n fru it m iri nd a v in e fr ui t c on su m ed ra w fr ui t n ot th re at en ed / a va ila bl e ro sa ce ae ru bu s f ra xi ni fo liu s (p oi r) m ou nt ai n ra sp be rr y bi ni t v in e fr ui t c on su m ed ra w, pr oc es se d fr ui t n ot th re at en ed / a va ila bl e zi ng ib er ac ea e al pi ni a ele ga ns (c . pr es l) k .s ch um . ta gb ak (t ag al og ) pa na on h er b fr ui t, r hi zo m e c on su m ed ra w fr ui t n ot th re at en ed / a va ila bl e wild edible plants in enhancing post-disaster food security nazareno et al. 7 proximate composition the 8 species available for collection and analysis included annona montana, decaspermum parviflorum, diplazium esculentum, dillenia philippinensis, dioscorea hispida, flacourtia jangomas, melastoma malabathricum, and rubus fraxinifolius. the nutrient composition of these plants is summarized in table 2. the parameters analyzed were moisture content, dry matter or total solids, ash content, crude fat, and crude fiber. moisture and dry matter measuring the amount of moisture in a plant is the first step in assessing the overall nutritional value of plants. a relatively high moisture content would reveal that the plant is more liable to microbial degradation (datta et al. 2019). low moisture content in plants indicates the presence of insoluble materials and entails longer shelf life with reduced susceptibility to microbial infection; this is determined by the amount of total solids in plants (the composition of plants excluding the water content). as shown in table 2 the wild edible fruits d. philippinensis (91.17 ± 0.40 g/100 g), m. malabathricum (78.10 ± 1.00 g/100 g), a. montana (78.51 ± 12.43 g/100 g), r. fraxinifolius (77.94 ± 1.20 g/100 g), and decaspermum parviflorum (66.76 ± 0.64 g/100 g) had relatively higher moisture content. the edible fern, d. esculentum (89.21 ± 1.37 g/100 g) that grows in damp areas, and wild yam, d. hispida (63.46 ± 2.53 g/100 g), also exhibited high moisture content. the unripe fruits of flacourtia jangomas had relatively low moisture content (27.90 ± 2.34 g/100g), which is typical of young, developing seed fruits. ash ash content, which represents the inorganic residue remaining after the complete oxidation of organic matter in food samples, indicates the availability of inorganic minerals in plants (datta et al. 2019; mundaragi et al. 2017; talang et al. 2023; yiblet & adamu 2023). as observed in table 2, most of the wild plants collected had relatively high ash content exceeding up to 20% with d. parviflorum having the highest among the plants analyzed, followed by f. jangomas. the high ash content in these fruits, such as d. parviflorum and f. jangomas can be attributed to their unripe state and their large, multiple seeds. the relatively high ash content in other wild plants in the study indicated that these plants contained high amounts of minerals. table 2 proximate composition of wild edible plant species found in three barangays of ormoc city plant species plant part analyzed moisture (g/100 g) dry matter (g/100 g) ash (g/100 g) crude fat (g/100 g) crude fiber (g/100 g) annona montana fruit (ripe) 78.51 ± 12.43 21.49 ± 12.43 13.96 ± 1.06 decaspermum parviflorum fruit (unripe) 66.76 ± 0.64 33.24 ± 0.53 20.80 ± 2.45 0.84 ± 0.58 5.72 ± 0.11 diplazium esculentum leaves 89.21 ± 1.37 10.79 ± 1.37 9.74 ± 0.38 1.35 ± 0.81 13.52 ± 1.85 dillenia philippinensis fruit (ripe) 91.17 ± 0.40 8.83 ± 0.40 7.00 ± 1.06 1.37 ± 0.06 7.15 ± 0.002 dioscorea hispida tuber 63.46 ± 2.53 36.54 ± 2.53 9.96 ± 1.57 0.66 ± 0.499 3.88 ± 0.19 flacourtia jangomas fruit (unripe) 27.90 ± 2.34 72.90 ± 2.34 12.42 ± 2.42 0.91 ± 1.10 14.63 ± 0.47 melastoma malabathricum fruit (ripe) 78.10 ± 1.00 21.90 ± 1.00 6.72 ± 0.36 rubus fraxinifolius fruit (ripe) 77.94 ± 1.20 22.06 ± 1.20 15.57 ± 0.08 6.97 ± 1.96 26.59 ± 1.17 notes: = parameters not performed due to inadequate amount of samples for analysis. biotropia vol. 32 no. 1, 2025 8 crude fat and crude fiber crude fat, a term used to describe the amount of fats in feedstuffs, food, and plants, represents the combination of free lipids and other fat-soluble materials present in a sample that can be dissolved in the solvent being used for extraction (aafco lab methods and services committee 2014). the crude fat content among the analyzed plants ranged from 0.66 g/100 g to 6.97 g/100 g (table 2), with r. fraxinifolius having the highest fat content and wild yam having the lowest among the samples. crude fiber is the most common measurement of fiber in plants. in this study, r. fraxinifolius had the highest crude fiber content at 26.59 g/100 g, followed by f. jangomas at 14.63 g/100 g and d. esculentum at 13.52 g/100 g. the rest have fairly moderate amounts of fiber content. the crude fiber contents of the fruits of r. fraxinifolius, f. jangomas and the leaves of d. esculentum are higher than the dietary fiber content of commonly consumed fruits and vegetables like apple, banana, asparagus, carrot, or broccoli (vicente et al. 2014). although crude fiber does not represent the total dietary fiber and may underestimate the total amount of dietary fiber present in plants, the results may imply that these plants can be good alternative sources of fiber. mineral content minerals are inorganic substances that comprise one of the four major classes of micronutrients, which are essential nutrients needed in small amounts in the body. minerals can be classified into two classes: macroelements and microelements (trace elements) (siddiqui et al. 2014). these minerals are found in plants, and the composition and quantity of these elements vary depending on factors such as the type of cultivar, growth conditions, type of substrate, fertilizers used, nutrients requirement of the plant, and growth stages of the plant at the time of harvest (jākobsone et al. 2015). the mineral composition of the wild edible plants in the study is presented in table 3. the parameters analyzed include sodium (na), potassium (k), calcium (ca), magnesium (mg), iron (fe), copper (cu), zinc (zn), and manganese (mn). m. malabathricum had the highest sodium (74.9 mg/kg) and calcium (5,017 mg/kg) contents among all the plant samples, followed by r. fraxinifolius at 74.8 mg/kg for na, and 1,476 mg/ kg for ca. the na content of d. parviflorum was 67.2 mg/kg, while the ca content of f. jangomas and a. montana was 1,675 mg/kg and 721 mg/ kg, respectively. d. hispida had the least amount of calcium among the samples collected in ormoc city. the calcium content of the plants tested was higher than in some cultivated vegetables like lettuce, cabbage, and spinach (datta et al. 2019). results showed that the weps in this study are not good sources of potassium since the potassium levels in the samples were below the detection limit (< 0.01 mg/kg). the wild edible fruits from ormoc city contained high amounts of magnesium ranging from 1,650 mg/kg to 20,802 mg/kg. m. malabathricum had the highest magnesium content (20,802 mg/kg) among all the plants analyzed, followed by r. fraxinifolius (19,116 mg/kg). magnesium aids in preventing muscle degeneration, growth retardation, cardiomyopathy, immunologic dysfunction, and bleeding disorders, among others (datta et al. 2019). among the plants analyzed, d. esculentum had the highest iron content (31.5 mg/kg), followed by d. philippinensis (30.6 mg/kg) and m. malabathricum (23.5 mg/kg). the remaining samples had iron levels below the detection limit. for copper, d. esculentum leaves had the highest copper content (38.5 mg/kg) among all the plants. zinc concentration was high in d. esculentum (44.4 mg/kg), f. jangomas (34.1 mg/kg), and m. malabathricum, (28.9 mg/kg), while it was low in a. montana (0.39 mg/kg). lastly, for manganese, m. malabathricum had the highest manganese content (298 mg/kg), while d. parviflorum had the lowest manganese concentration (3.2 mg/kg). iron, zinc, copper, sulfur, manganese, and iodine, are consumed in smaller amounts (< 100 mg/dl) (linkon et al. 2015). although these microelements play essential roles in various metabolic processes in the body, they are needed only in small amounts, and their intake should be regulated. the recommended dietary allowance (rda) for calcium (1,000 mg/day), magnesium (400 mg/day), and iron (8 mg/day) (datta et al. 2019) suggests that the plants can supplement the daily mineral requirement. wild edible plants in enhancing post-disaster food security nazareno et al. 9 table 3 mineral composition of wild edible plants found in three barangays in ormoc city, leyte plant species plant part analyzed fe (mg/kg) cu (mg/kg) zn (mg/kg) mn (mg/kg) na (mg/kg) k (mg/kg) ca (mg/kg) mg (mg/kg) annona montana fruit (ripe) 6.34 9.91 0.39 7.12 15.3 < 0.01* 721 11,984 decaspermum parviflorum fruit (unripe) 6.34 11.4 < 0.01* 3.2 67.2 < 0.01* 276 1,650 diplazium esculentum leaves 31.3 38.5 44.4 19.4 51.9 <0.01* 355 13,294 dillenia philippinensis fruit (ripe) 30.6 8.35 < 0.01* 13.9 69 < 0.01* 427 13,780 dioscorea hispida tuber 7.01 11.7 < 0.01* 5.0 8.94 < 0.01* 7.31 2,744 flacourtia jangomas fruit (unripe) < 0.01* 13.2 34.1 82.5 < 0.01* < 0.01* 1,675 4,129 melastoma malabathricum flower 23.5 11.7 28.9 298 74.9 < 0.01* 5,017 20,802 rubus fraxinifolius fruit (ripe) 12.4 10.1 15.1 31.5 74.8 < 0.01* 1,476 19,116 note: * = method detection limit. table 4 vitamin c, alkaloids, and oxalate content of wild edible plant species in three selected barangays in ormoc city, leyte plant species plant part analyzed vitamin c content (g/100 g) alkaloids content (g/100 g) oxalate content (g/100 g) annona montana fruit (ripe) 72.47 ± 2.21 0.95 ± 0.01 decaspermum parviflorum fruit (unripe) 16.15 ± 4.51 0.56 ± 0.001 0.0057 ± 0.002 diplazium esculentum leaves 21.94 ± 6.55 0.68 ± 0.14 0.2 ± 0.02 dillenia philippinensis fruit (ripe) 18.17 ± 3.90 0.72 ± 0.013 0.45 ± 0.01 dioscorea hispida tuber 25.90 ± 2.93 0.50 ± 0.05 0.29 ± 0.01 flacourtia jangomas fruit (unripe) 36.61 ± 2.90 0.98 ± 0.03 0.074 ± 0.007 melastoma malabathricum fruit (ripe) 82.58 ± 14.27 0.30 ± 0.05 rubus fraxinifolius fruit (ripe) 40.28 ± 8.03 1.36 ± 0.11 0.52 ± 0.03 note: = parameters not performed due to inadequate amount of samples for analysis. vitamin c vitamin c, chemically known as ascorbic acid, is an important factor in assessing the quality of fruits (khilari & sharma 2016). in addition to the polyphenols present in plants, vitamin c also contributes to the antioxidative properties of plants. l-ascorbic acid cannot be synthesized in humans and must be, therefore, consumed from food sources. it is well known that plantbased foods, especially fruits, are generally high in vitamin c, making them good sources of ascorbic acid (lykkesfeldt et al. 2014). vitamin c plays a crucial role in various metabolic processes, such as the formation of bile salts, serotonin production, the reduction of allergic activity, and the scavenging of free radicals (chambial et al. 2013). as shown in table 4, low vitamin c content was observed in d. philippinensis (katmon) fruits and unripe fruits of d. parviflorum and f. jangomas. relatively high vitamin c content was found in m. malabathricum (82.58 ± 14.27 g/100 g), followed by a. montana (72.47±2.21 g/100 g), and r. fraxinifolius (40.28± 8.03 g/100 g). the edible fern d. esculentum (21.94 ± 6.55 g/100 g) and d. hispida (25.90 ± 2.93 g/100 g) had lower vitamin c content. some of the fruits in this study could help supplement the recommended daily allowance of ascorbic acid for different life stages and groups aged 170 years which is 15 to 120 mg per day (ojelel et al. 2020). antinutrients (alkaloids and oxalate) compounds that have specific functions for plant growth or defense mechanism (fekadu gemede 2014) but can be toxic and/or antinutritive in humans, are labeled as antinutrients; they result in health hazard affecting digestion and the absorption of essential nutrients (lo et al. 2018; popova & mihaylova 2019). they bind to minerals and other nutrients and reduce their bioavailability and digestibility, excess consumption of these substances may have adverse effects and may lead to toxicity (nath et al. 2022; lo et al. 2018; aina biotropia vol. 32 no. 1, 2025 10 et al. 2012; fekadu gemede 2014). alkaloids are a group of naturally occurring nitrogen-containing compounds produced from secondary metabolic pathways in plants to protect plants from herbivory and pathogenic attacks (alves de almeida et al. 2017). the quantification of alkaloid and oxalate contents is summarized in table 4. in this study, the amounts of alkaloids present in the weps ranged from 1.36 g/100 g to 0.56 g/100 g, with r. fraxinifolius exhibiting the highest and d. hispida the lowest. these values are higher than the lethal dose for alkaloids which is only 20 mg/100 g (ifemeje et al. 2014). this can mean that these plants may not be safe for consumption. however, the toxic effects of alkaloids depend on the type of alkaloids present in the plant. therefore, characterization of this class of compounds is recommended. the amounts of oxalate in the plants analyzed ranged from 0.95 g/100 g to 0.0057 g/100 g, with the highest observed in a. montana fruits and the lowest in d. parviflorum. oxalates are substances that can bind with other minerals (e.g., calcium) and form insoluble salts (petroski & minich 2020). summary of the proximate composition, mineral composition, alkaloids and oxalates a total of 15 edible plant species, representing 13 families, were identified in the study. d. philippinensis had the highest moisture content, followed by m. malabathricum, a. montana, and r. fraxinifolius. most of the wild plants collected had relatively high ash content exceeding up to 20% with d. parviflorum having the highest among the plants analyzed, followed by f. jangomas, indicating a rich mineral content. r. fraxinifolius had the highest crude fiber content, followed by f. jangomas and d. parviflorum. the rest had moderate amounts of fiber content. the fruits of m. malabathricum exhibited the highest ca, na, mg, and mn content, as well as the highest vitamin c content. r. fraxinifolius had the second highest na content, and f. jangomas had the second highest ca content. d. esculentum leaves had the highest iron, copper and zinc content, and a relatively high magnesium content. d. philippinensis and m. malabathricum also had relatively high iron content. the remaining samples had iron levels below the detection limit. the fruits of a. montana, r. fraxinifolius, and m. malabathricum showed a relatively high vitamin c content. r. fraxinifolius exhibited the highest alkaloids content and d. hispida the lowest, while oxalate content was highest in a. montana fruits and the lowest in d. parviflorum fruits. weps identified but not collected and analyzed although 7 out of the 15 plant species identified as edible were not collected and analyzed, studies indicate that they are also considered edible in other countries and regions. the sap of arenga pinnata may be converted to palm sugar, which has a low glycemic index, making it a healthier alternative to refined sugar, particularly for diabetics (haagen & lantican 2014). in bhutan, certain villages believe that soup made from the young shoot of calamus sp., helps alleviate nausea (matsushima et al. 2006). similarly, local communities in central kalimantan in indonesia consider calamus sp. a food source (fambayun & kalima 2022). a study on consumption practices in assam, india, revealed that calamus sp. shoots are eaten in various forms, i.e., raw, boiled, fried, roasted, and often cooked with other ingredients, such as fish, meat, black gram pulses, etc. (thakur & sheth 2015). another study found that d. angustifolia, and d. fissa are the preferable rattan used for food by the community near peatland areas in central kalimantan, both for daily sustenance and cultural activities. (fambayun & kalima 2022). although caryota mitis and alpinia elegans were identified by key informants in this study as edible, they are primarily valued for their medicinal properties (dalisay et al. 2018; naïve et al. 2019; shahriar et al. 2017). in bangladesh the roots and fruits of caryota mitis have been reported to be traditionally used by folk medicinal practioners of a number of villages for treatment of constipation and hemorrhoids (shahriar et al. 2017). alpinia elegans, an endemic philippine medicinal plant, is known for treating musculoskeletal diseases, hemoptysis, headache, migraine, stomachache, and as an anti-relapse for women. (naïve et al. 2019; dalisay et al. 2018). melothria pendula l., now naturalized in peninsular malaysia, mainland sumatra, borneo (sabah), the philippines, and sulawesi, thrives in urban areas and grows spontaneously along roadsides, in gardens, open spaces, and ditches, wild edible plants in enhancing post-disaster food security nazareno et al. 11 burial grounds and abandoned land (husaini et al. 2024). in parts of mexico, it is used as food and in beverages (guerrero-torres et al. 2023), while in the philippines, it is recognized as a common medicinal plant (raju et al. 2021). the fruit of passiflora edulis  is considered a rich source of vitamin a, thiamine, riboflavin, niacin, calcium, phosphorus, and vitamin c. in colombia, its pulp is used to prepare juices and soft drinks (jimenez et al. 2011). among brazilian passion fruit species, passiflora edulis sims is the most cultivated and valued for its flavor and aroma (barbosa santos et al. 2021). conclusion the plants analyzed in this study were rich in essential nutrients, including mineral (ca, na, mg, mn, fe, cu and zn), crude fiber and vitamin c. the different plants excelled in different nutritional aspects, suggesting a diverse dietary potential. these plants play a crucial role in the local community, providing not only sustenance but also medicinal applications. further research and conservation efforts should be encouraged to harness the nutritional and economic potential of these plants while ensuring their sustainable utilization and preservation. additionally, incorporating information about weps into disaster preparedness and food security policies could help safeguard vulnerable communities in disaster-prone regions, like the philippines. references [aafco] association of american feed control officials. 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mirera 2011) with hard and rounded shells (brown 1993; pratiwi 2010; felder & thoma 2022; inoue et al. 2022). this organism possesses dark brown or black skin with a body length of 20 cm (andreetta et al. 2014; iromo et al. 2022). in addition, mangrove crab assumes a significant role within mangrove ecosystems by contributing substantially to ecological equilibrium. this includes the capacity to assist in preserving balance by actively preying on insects and plants, shaping water flow dynamics, and upholding the stability of the mangrove forest floor (kristensen, 2008; cannicci et al. 2008; not et al. 2020). furthermore, the species are an important food source for several types of birds and fish. previous studies showed that mud crab populations had declined dramatically in some areas due to mangrove forest encroachment (le vay 2001; sara 2010; ikhwanuddin et al. 2011; varkey et al. 2023). habitat ecology analyses have been conducted to understand the habitats, including studies on their behavior and interactions with the environment (bir et al. 2020; karniati et al. 2021; leoville et al. 2021). genetic diversity studies are conducted to understand how the differences between the populations can affect adaptation and resistance to environmental *corresponding author, email: susiana@umrah.ac.id mailto:susiana@umrah.ac.id biological aspects of mangrove crab (scylla serrata) at the beladen estuary – susiana susiana et al. 55 changes (hassan & mustapah 2019; ines et al. 2019; wang et al. 2020). meanwhile, reproductive studies are systematically undertaken to enhance the comprehension of the intricate reproductive processes exhibited by crab. these include an exploration of the multifaceted factors that exert influence over reproductive success rates and population growth dynamics. dompak island is a coastal area located in dompak village, which is part of bukit bestari district, tanjungpinang city, riau islands province. the province (novitri et al. 2021) is dominated by the livelihoods of the community, mostly as fishermen using the results of coastal resources in the ocean. the coastal expanse of dompak, within the beladen estuary increases the profusion of mangrove vegetation. therefore, this habitat provides a conducive environment for the proliferation of various species, which represent a prominent component of the aquatic fauna inhabiting coastal waters within the mangrove ecosystem. among the members of the brachyuran tribe belonging to the family portunidae, mangrove crab, scientifically known as scylla spp., is important due to its considerable prevalence. the species predominantly resides in coastal areas and shallow waters adjacent to mangrove ecosystems (pratiwi 2010). mangrove crab is popular among the public for the delicious meat and high nutritional content (nanda et al. 2021; sari et al. 2022). therefore, the species has long been an important source of income for local communities (de oliveira cortes et al. 2019; rahman et al. 2020; bhuiyan et al. 2021; crespo et al. 2021; miah et al. 2022). however, the population is under great pressure due to the encroachment or conversion of mangrove forest as well as unsustainable fishing practices (triharyuni 2020; fazhan et al. 2021; leoville et al. 2021; sakib et al. 2022). this has resulted in fishermen catching fewer and smaller mangrove crab. due to the substantial fishing activities conducted by fishermen, coupled with the use of mangrove forest that neglected considerations of resource sustainability, there has been a marked decline in the populations (elizabeth et al. 2003). therefore, it is necessary to reorient sustainable fisheries resource use activities that prioritize aspects of biological aspects for fisheries management activities. this study determines several aspects of mangrove crab biology related to the relationship between carapace width and weight, sex ratio, gonadal maturity level, and gonadal maturity index (gmi). materials and methods time and location this study was conducted at the beladen estuary, dompak, tanjungpinang, riau islands. data collection was carried out for two months from may to june 2022 with a frequency of data collection every two weeks. data collection this study used a survey method of direct observation for data collection and mangrove crab (s. serrata) sampling was carried out by the census method. according to astari (2019), census or saturated sampling was a determination method when all members of the population were used as samples. mangrove crab was obtained from fishermen who caught crab at the beladen estuary. furthermore, bubu folding was one of the most common fishing gear used by fishermen. measurement of the width of mangrove crab was carried out using a caliper with an accuracy of 0.01 mm. the measurement of the carapace width started from the end of the right spine to the left. mangrove crab was weighed with a digital scale of 0.1 g accuracy (overton et al. 1997). observation of the sex ratio was carried out by analyzing the morphological characteristics in the abdomen of male and female mangrove crab. male and female had a narrowed and dilated abdomen shape, respectively. determination of the degree of maturity of gonads was conducted by visually observing the sample according to the fivecategory approach (tiurlan et al. 2019) (table 1). data analysis the relationship of carapace width and weight growth patterns can be seen through the analysis of the relationship between carapace width and weight. the formula for the relationship of carapace width and weight is described by the equation according to king (2007) in safira et al. (2019) as follows: biotropia vol. 31 no. 1, 2024 56 𝑊 = 𝑎𝐶𝑊𝑏 where: w = weight (grams) cw = carapace width (mm) a = constant b = slope the linear regression approach shows the relationship between the two parameters. the value of b is used to estimate the growth rate of the two analyzed parameters and the hypotheses used are as follows. a value of b = 3 is known as an isometric growth pattern (the pattern of growth of width equals the growth of weights). a value of b ≠ 3 signifies an allometric pattern and when b > 3, the value is known as a positive allometric growth pattern (weight growth is more dominant). furthermore, a value of b < 3 is called a negative allometric growth pattern (carapace width growth is more dominant). the general form of a t-test for a regression coefficient is (steel and torrie 1993): t=β^1−β1,0se(β^1) where: β^1 = estimated slope coefficient β1,0 = hypothesized value of the slope under the null hypothesis se(β^1) = standard error of the estimated slope coefficient at the 95% confidence interval, the t_test value is compared with the t_tabel value, then the decision taken to determine the growth pattern is 𝑡𝑡𝑒𝑠𝑡 > 𝑡𝑡𝑎𝑏𝑙𝑒: reject the null hypothesis (h0), 𝑡𝑡𝑒𝑠𝑡 < 𝑡𝑡𝑎𝑏𝑙𝑒: fail to reject the null hypothesis (h0). sex ratio sex ratio is a comparison of the number of male and female crab in a population with the ideal conditions for maintaining the viability of a species at 1:1 (50% male and 50% female). the proportion is important to determine the ratio of male and female crab caught with the equation: 𝑃 = 𝑛 𝑁 𝑥 100% where: p = the proportion of crab (female or male) n = number of female or male crab n = total number (female + male) to find out whether the proportion of males and females in ideal conditions, namely 1:1, the chi-square test (𝑋2) with a confidence level of 95% (α=0.05) with the test procedure is: 𝐻0: the ratio of male and female crab is 1:1 𝐻1 : comparison of male and female crab instead of 1:1 for the test criteria, when 𝑋2𝑐𝑜𝑢𝑛𝑡 > 𝑋2𝑡𝑎𝑏𝑙𝑒 and < 𝑋2𝑡𝑎𝑏𝑙𝑒, then 𝐻0 is accepted and 𝐻1 is accepted. the value of 𝑋2 of the table using degrees free (db) = 2-1 = 1. therefore, the value of 𝑋2 of the table (0.05:1) = 3.841 is obtained. gonadal maturity index (gmi) gmi is calculated using the formula according to effendie (2002) in suryakomara (2013) as follows: 𝐺𝑜𝑛𝑎𝑑𝑎𝑙 𝑚𝑎𝑡𝑢𝑟𝑖𝑡𝑦 𝑖𝑛𝑑𝑒𝑘𝑠 (𝐺𝑀𝐼) = 𝑊𝑔 𝑊𝑏 𝑥 100% where: gmi = gonadal maturity index (%) wg = gonadal weight (g) wb = body weight (g) results and discussion the relationship of carapace width and weight the results obtained were 26 and 15 male and female mangrove crabs. the relationship between carapace width and body weight was grouped according to gender. figure 1 shows the coefficient of determinations (r²) for male and female mangrove crab, which are 0.9552 or 95.5% and 0.9476 or 94.7%. additionally, the values of the constant (b) derived from the equation w = 0.00003w3.4144 and w = 0.0008cw2.7034 are at 3.4144 and 2.7034, respectively (figure 2). biological aspects of mangrove crab (scylla serrata) at the beladen estuary – susiana susiana et al. 57 table 1 classification of the maturity level of mangrove crab gonads gml male female 1 filament testicles, clear white in color, located near the heart under the liver the ovaries remain underdeveloped and assume the form of a pair of filaments positioned along the dorsal area. they are situated above the digestive glands, exhibiting a yellowish hue. the ovaries appear translucent and take on a whitish coloration. 2 the testicles begin to grow in clear white around them the ovaries increase in size and begin to expand with a milky white color. 3 shaped testicles of milky white color the ovaries increase until pale yellow coloration is formed. 4 same as gml 3, but enlarged in size the volume of the ovaries becomes larger and fills the entire chest cavity, the egg is pale yellow to golden yellow, and the digestive glands shrink. this is because the ovaries and egg grains can be seen with a microscope. 5 the testicles are already visible milky white color and the shape becomes denser the ovaries are replete with cells, nearly ripe, transitioning from an orange to a deep red color. figure 1 the relationship of carapace width with the weight of male mangrove crab in the beladen estuary from may to june 2022 figure 2 the relationship of carapace width with the weight of female mangrove crab in the beladen estuary from may to june 2022 table 2 growth pattern of mangrove crab in the beladen estuary from may to june 2022 sex parameters of the relationship of carapace width and weight of mangrove crab number of samples sprouted parameter values (b) correlation value (r²) the nature of the growth pattern male 26 3.4144 0.9552 allometric positive female 15 2.7034 0.9476 allometric negative w = 0.00003cw3.4144 r² = 0.9552 0 100 200 300 400 500 600 700 800 0 50 100 150 w ei g h t (g ) carapace width (mm) w = 0.0008cw2.7034 r² = 0.9476 0 100 200 300 400 500 600 700 800 0 50 100 150 200 w ei g h t (g ) carapace width (mm) biotropia vol. 31 no. 1, 2024 58 in male crab, the growth pattern is allometrically positive with a constant value of b > 3 since b = 3.4 (table 2). therefore, the growth body weight of crab is faster than the carapace width. according to tiurlan et al. (2019), an increase in the body weight of mangrove crab directly influences the width of the carapace. in table 1, female crab has a negative allometric pattern with a constant value of b < 3 (b=2.7). this phenomenon occurs due to increased use of food intake closely linked to the carapace width during the molting and gonadal maturity processes, hence, female growth exhibits a negative allometric pattern. male crab allocates a significant portion of the energy intake towards expanding carapace during the mating process, resulting in an increased body weight. this is due to the additional weight contributed by the enlarged carapace, leading to a positive allometric growth pattern. sex ratio based on the calculations, there are different sex ratios of male and female mangrove crab. therefore, the highest and lowest sex ratio values are found in male and female mangrove crab, as presented in figure 3. crab collection was conducted from may to june 2022, where a total of 41 specimens were gathered. among these, 26 and 15 individuals at 63% and 37% were identified as male and female crab (figure 3). the sex comparison analysis showed a predominance of male crab, with a ratio of 0.63 to 0.37 when compared to female. the chi-squared analysis of 𝑋2 using a = 0,05 (95% confidence level) obtained the calculated value 𝑋2 = 2.9512 (2.95) and the table 𝑋2 = 3.8415 (3.84) to (𝑋2𝑐𝑜𝑢𝑛𝑡 < 𝑋2𝑡𝑎𝑏𝑙𝑒). therefore, 𝐻0 was accepted since there was no difference in proportion between male and female. according to the hypothesis in the chi-square test, the population of mangrove crab was balanced when (𝑋2𝑐𝑜𝑢𝑛𝑡 < 𝑋2𝑡𝑎𝑏𝑙𝑒). these results showed that the sex ratio met the criteria expected to keep the resource condition stable or balanced. female crab dominated slightly from catches by fishermen due to changes in the attitudes of their respective individuals in finding suitable habitats. differences in the ratio of male to female were related to food availability and the life cycle of mangrove crab, specifically during reproductive and seasonal changes (hardiyanti et al., 2018). according to tahmid et al. (2017), the greater activity exhibited by male crab was attributed to their inherent habits and behaviors. male crab was observed to be more active in foraging, resulting in a higher likelihood of being captured. female was predominantly caught due to the specific fishing areas selected since crab fishing was conducted in deeper waters. therefore, adult female crab with mature gonads would migrate to deep waters. gonadal maturity index (gmi) gmi compares gonadal weight to body weight. growth of gonads has a relationship with the weight gain of the body, as presented in table 4. figure 3 sex ratio of mangrove crab in the beladen estuary from may to june 2022 table 3 ratio of male to female mangrove crab based on chi-square test at beladen estuary from may to june 2022 sex sex-ratio x² count x² table (α0.05) information male female male female 26 15 63% 37% 2.9512 3.8415 balanced 37% 63% female male biological aspects of mangrove crab (scylla serrata) at the beladen estuary – susiana susiana et al. 59 table 4 gmi of mangrove crab in the beladen estuary from may to june 2022 sex average sampling i sampling ii sampling iii sampling iv sampling v female 4.1026 6.7979 3.3583 1.8751 2.3134 male 0 7.1445 5.6394 2.6512 2.7686 table 4 shows that gmi value of male and female gonads changes. the highest and lowest gmi is sampling ii and i of male crab with a value of 7.1445 and 0. the highest and lowest female gmi values of sampling ii and i are 6.7979 and 1.8751, as shown in table 4. according to suryakomara (2013), the growth of gonads is directly proportional to the maximum increase in weight and size when spawning occurs. gmi value and the weight of female gonads is greater than male. suryakomara (2013) argued that the peak of the spawning season varied between the geographical locations of the waters and this was influenced by annual temperature variations (hamid et al., 2015). the maturity index is the percentage of gonadal weight to the body weight of the female. the development is increasingly in line with the magnitude of the index and reaches high values in the spawning period. therefore, gmi is the quantitative unit used to express the gonadal change (fatma et al., 2020). gonadal maturity level the number of mangrove crab was 41 heads consisting of 26 males and 15 females with a length range of 75.5 – 136 mm and 93.5 – 141.5 mm, respectively. the size class of mangrove crab caught at the beladen estuary during the study is found in figures 4 and 5. figure 4 the composition of the maturity level of male mangrove crab gonads in the beladen estuary in may to june 2022 figure 5 the composition of the maturity level of the female mangrove crab gonads in the beladen estuary in may to june 2022 0% 20% 40% 60% 80% 100% 75.5 87.5 99.5 111.5 123.5 136 g o n a d a l m a tu ri ty l ev el carapace wide middle value (mm) male gml 5 gml 4 gml 3 gml 2 gml i 0% 20% 40% 60% 80% 100% 93.5 105.5 117.5 129.5 141.5 g o n a d a l m a tu ri ty l ev el carapace wide middle value (mm) female bef gml 5 gml 4 gml 3 gml 2 gml i biotropia vol. 31 no. 1, 2024 60 male crab with the largest and smallest size is found at a carapace length of 136 mm and 75.5 mm. meanwhile, female crab with the largest and smallest size is found at a carapace length of 141,5 mm and 93.5 mm, respectively. the results showed that male and female crab was in the highest and lowest gml conditions, namely gml 2 and 5, and no females were found carrying eggs on the abdomen. crab caught obtained the smallest length size of medium maturing gonads on gml 3 with a size of 87.5 mm and 93.5 for male and female. mangrove crab in gml 2 was very likely to be influenced by environmental conditions in terms of water quality and food availability. however, it was the nature of the spawning to take place in the year. tiurlan et al. (2019) found mangrove crab spawning eggs in the year with a peak period influenced by certain water conditions. during the reproductive phase, adequate nutrients and protection were required from disturbances impacting the body morphology. these conditions were essential to ensure the proper support for reproductive development and gonadal maturity. female mangrove crab also migrated to the middle of the sea at gml 3 and 4 with the purpose of mating. the mating process unfolds gradually in mangrove waters with the development of eggs since female crab moved to sea waters for reproduction. hardiyanti et al. (2018) explained that migration was a form of mangrove crab effort to obtain suitable water conditions to determine food, reproduction, nursery, and spawning locations. therefore, the use of mud crab must pay attention to the size. conclusions in conclusion, the growth pattern of mangrove crab at the beladen estuary was positive and negative allometric with b values of 3.4144 and 2.7034 in male and female. furthermore, the sex ratio was still in a balanced state, where female and male mangrove crab had the highest gmi percentages of 6.7979 and 7.1445, respectively. acknowledgements the authors are grateful to mr. yusli for helping with the sampling process of mangrove crab in the beladen estuary, as well as all related parties who provided support and cooperation in this study activity. references andreetta, a., fusi, m., cameldi, i., cimò, f., carnicelli, s., cannicci, s. 2014. mangrove carbon sink. do burrowing crabs contribute to sediment carbon storage? evidence from a kenyan mangrove system. journal of sea research 85: 524-533. astari s. b. 2019. analysis of factors affecting the swallow crab’s nelayang income (portunidae). 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universitas gadjah mada, yogyakarta 55281, indonesia received 15 july 2023/ revised 25 october 2023/ accepted 30 october 2023 abstract soybean characterization is essential to ensure product quality during distribution according to internal values. in this context, non-destructive characterization method, such as spectroscopy, offer an effective and efficient approach to testing soybean quality in field applications. among the instruments that are widely used for testing soybean quality, the semi-portable visible near-infrared (vis-nir) spectrometer operating at a specific range of 345 to 1033 nm has been proven effective. therefore, this study aimed to investigate soybean seeds characterization using vis-nir spectroscopy with pca and plsr chemometric methods. the investigation was carried out using soybean seeds consisting of eight varieties locally produced on java island, indonesia, including dega1, dena1, deja2, dering1, devon1, yellow flap, green, and detam4, in the form of intact, crumble, flour, and paste. several quality parameters such as protein, fat, crude fiber, carbohydrate, ash, water, chlorophyll, total carotene, vitamin c, and l*, a*, and b* values were measured across intact, crumble, flour, and paste samples. the results of principal component analysis (pca) showed that sample form and genotypes affected soybean classification. furthermore, partial least squares regression (plsr) showed adequate model calibration for crude fiber, chlorophyll, total carotene, and vitamin c parameters. based on this analysis, it could be concluded that vis-nir spectroscopy proved to be suitable for the classification and prediction of soybean characterization. keywords: chemometrics, spectroscopy, soybean, vis-nir. introduction soybean is a staple food in indonesian society, progressively gaining popularity annually alongside rice and corn in daily consumption (harsono et al. 2021). in indonesia, the national soybean consumption rate from 2015 to 2021 has reached 11.5 kg/capita/year, with an average growth of 3.02 kg/capita/year. due to this high demand, a consistent supply is required, as the main source of soybean is through import. a previous study has found that local contribution to meet national needs is below 50%, with java island being the most substantial contributor, accounting for more than 55% of the total soybean production in indonesia (kementan 2020). the distribution of local soybean based on origin varies significantly according to geographic location, variety, and genotype, with the majority being distributed as seeds in the indonesian market. based on quality levels, the production of soybean strives to meet consumer demand according to value (jia et al. 2020). therefore, information about conditions and varieties, as well as the quality and characteristics of soybean, is required to determine the potential and quality measures at reasonable market prices. quality testing is characterized by the use of destructive sample method during laboratory analysis, which is time-consuming and expensive. this phenomenon shows the need for a reliable, easy, fast, and cost-effective method to determine the characteristics of soybean. a common non-destructive method in food characterization is spectroscopy, widely *corresponding author, email: evi@ugm.ac.id mailto:evi@ugm.ac.id biotropia vol. 31 no. 1, 2024 64 recognized for the good measurement accuracy combined with chemometric analysis. this method has been used to explore the characterization of food ingredients in the spectrum of infrared (ir) (manley 2014), ultraviolet (uv) (farag et al. 2022), and visible (vis) (cortés et al. 2019; wang et al. 2022). specifically, the combination of visible and nearinfrared (vis-nir) light spectrum is frequently used for characterization, along with spectrum absorption by functional groups (alander et al. 2013). in vis-nir spectrometer, light absorption occurs due to the movement or excitation of subatomic particles relating to the specific element concentration of chemical compounds in the material (mayerhöfer et al. 2019). therefore, vis-nir spectrometer is used to measure the amount of light absorbed or reflected. vis-nir spectrometer has been extensively investigated, emphasizing its suitability for field applications (walsh et al. 2020) due to simplicity, speed, and cost-effectiveness for quality evaluation (cortés et al. 2019) and classification (monago-maraña et al. 2021). specifically, fiber optic-based vis-nir spectroscopy has been explored for soybean characterization (abadi et al. 2022), showing good performance for classification (pahlawan et al. 2022), water evaluation (pahlawan et al. 2023), and authentication (masithoh et al. 2023). the results showed the potential of this instrument to support the production process for soybean characterization in field applications. therefore, this study aimed to evaluate the application of vis-nir spectroscopy for characterizing soybean in various forms such as intact, crumble, flour, and paste. materials and methods materials the materials used included local soybean obtained from the indonesian legume and tuber crops research institute (iletri), as well as yogyakarta region for the first planting season 2021 (iletri, 2016). soybean seeds were at mature pod stage of r8 labeled as (1) dega1 = k1, (2) dena1 = k2, (3) deja2 = k3, (4) dering1 = k4, (5) devon1 = k5, (6) yellow flap = k6, (7) green = k7, and (8) detam4 = k8. spectra measurement approximately, 15 g soybean seeds were placed in petri dish and the spectra were measured for intact, crumble, flour, and paste forms. soybean flour was obtained by milling seeds for 2 minutes to reduce the size and sifting through a 60-mesh sieve. the samples that did not pass the 60 mesh sieve were used as crumble, while the paste samples were obtained by mixing 5 g soybean flour with 10 ml distilled water in petri dish. therefore, four forms of soybean were used in this study, namely intact, crumble, flour, and paste. spectroscopic data were obtained using visnir spectrometer (flame-t-vis-nir, ocean optics, usa) with 345-1033 nm wavelength range in reflectance mode. the light source was a tungsten-halogen lamp (hl-2000-hp-fhsa, ocean optics, usa) with a reflectance probe (qr400-7-vis-nir, ocean optics, usa). figure 1 a portable vis-nir spectrometer arrangement evaluation of soybean based on chemical characteristics and vis-nir spectroscopy – farid r. abadi et al. 65 bulk, flour, and paste soybean samples were poured into petri dish (ø=5 cm), followed by spectra measurementdescribed by abadi et al. (2022). the fiber optic sensor was placed in the probe holder, which was arranged perpendicularly (α=90o) to the sample at a distance of 2 mm, as shown in figure 1. the probe sensor was connected to a light source and a detector capturing reflectance data, which was collected by oceanview 1.6.7 software (ocean insight, usa). before reflectance spectra measurement, instrument calibration procedures were performed by measuring the white reflectance spectra standard, followed by black reference spectra. this calibration was performed on every 10 samples, which were individually measured in triplicate, as described by masithoh et al. (2023). subsequently, color and chemical measurements were carried out after spectra measurement. color and chemical parameters measurement color parameters denoted by l*, a*, and b* values were measured using a handheld color meter (tes 135a, taiwan). l* value represented lightness plotted on the vertical axis on a color space diagram with values ranging from 0 (black) to 100 (white). moreover, a* and b* were color coordinates with a* (positive) and a* (negative) representing red and green, while b* (positive) and b* (negative) represented yellow and blue, respectively (berns 2019). after color measurement, the destructive analyses were carried out to determine water content, crude protein, crude fiber, ash content, carbohydrates, fat, chlorophyll, total carotene, and vitamin c using the official aoac methods (aoac 2007). one-way analysis of variance (anova) was performed for all chemical parameters and soybean samples. subsequently, color and chemical parameters were analyzed using a singlefactor analysis of variance with spss (spss inc., chicago, il, usa), and mean comparisons were evaluated with duncan's test. chemometric analysis chemometric analysis, including principal component analysis (pca) and partial least square regression (plsr), was conducted after measuring quality parameters such as color and chemical aspects. the data comprised four sample types and eight soybean varieties with five replications for each sample, totaling 160 data generated by spectra acquisition. the spectra and quality parameters data were used as x-variables in pca analysis. for plsr analysis, the reflectance spectra of all samples were used as xvariables, while color and chemical parameters served as y-variables. compared to supervised plsr, pca is unsupervised chemometrics that does not require y-variables as predictors. during plsr analysis, the 160 data obtained were divided into calibration and prediction data of 112 and 48, respectively. the performance of plsr calibration model was determined from the determination of calibration (r2c) coefficient and root mean square error of calibration (rmsec). the calibration model was validated using a full cross-validation (cv) method. subsequently, the best calibration plsr model was applied to predict color and quality parameters using prediction data presented in the form of r2p and rmsep. plsr model was improved using various spectra preprocessing methods such as smoothing, normalization, and derivation. as shown by the workflow of chemometric analysis in figure 2, the final output was the best plsr model, which was validated using the 30% prediction data. the performance of the validation model was determined from r2 of prediction and rmsep. biotropia vol. 31 no. 1, 2024 66 figure 2 workflow of chemometric analysis in soybean characterization results and discussion color and chemical parameters soybean samples were harvested ripe or at a late-stage color, with k1-k6 being yellow, while other genotypes were green (k7) and black (k8). although intact soybean seeds in figure 3 had different peel colors when observed using human eyes, a similar color was identified in crumble, flour, and paste forms. intact crumble flour paste intact crumble flour paste k1 = dega1 k5 = devon1 k2 = dena1 k6 = yellow flap k3 = deja2 k7 = green k4 = dering1 k8 = detam4 figure 3 figures of different soybean genotypes in bulk, crumble, flour, and paste forms evaluation of soybean based on chemical characteristics and vis-nir spectroscopy – farid r. abadi et al. 67 table 1 shows the results of one-way anova for different samples. the statistical analysis based on parameters l*, a*, and b* showed that p-value was below the significance level α = 0.05. a significant difference was observed in the average l*, a*, and b* values between groups k1 to k8 in bulk, crumble, flour, and paste samples. however, all groups in paste form had low lightness due to the addition of excess water, which decreased surface reflection. table 2 shows significant differences between groups regarding protein, ash, water, chlorophyll, and vitamin c. black color of the k8 soybean variety had the highest protein, chlorophyll, and total carotene content. however, there were no significant differences between groups regarding fat, crude fiber, carbohydrate, chlorophyll, and total carotene. principal component analysis (pca) pca based on soybean characteristics figure 4 shows pca plot based on soybean characteristics. the data obtained were treated in the preprocessing stage, including a moving average smoothing with 150 segments. subsequently, the transformation stage, including normalization, derivative, baseline correction, and standard normal variate (snv), showed that range normalization yielded the most appropriate performance. pca scores showed distinct clustering among more resilient colored soybean, specifically the yellowish, greenish, and black varieties, as explained by pc-1 (28%) and pc-2 (21%). in figure 2a, yellowish soybean (k1k6) predominantly occupied quadrants i and iv, with pc-1 values primarily negative and pc-1 positive <1.5. greenish soybean (k7) was positioned in quadrant i, marked by positive pc1 and pc-2 values, while black soybean (k8) was located in quadrant iv. figure 2b shows the significance of various parameters in differentiating samples in each quadrant. in quadrant i, parameters such as crude fiber, chlorophyll, ash, protein, and total carotene played a crucial role. a significant influence was observed in quadrant ii due to l*, a*, and b* color values along with fat content. quadrant iv showed carbohydrate parameters, while water and vitamin c content were observed in quadrant iii. the clustering of greenish soybeans (k7) appeared to be influenced by parameters such as crude fiber, chlorophyll, ash, protein, and total carotene, while water and vitamin c content had a notable impact on the black soybeans (k8). this showed that various characteristics, based on the analyzed parameters, significantly influenced the grouping of samples. table 1 l*, a*, and b* values of intact, crumble, flour, and paste of different soybean genotypes color sample k1 k2 k3 k4 k5 k6 k7 k8 l* intact 70.56 49.86 60.06 66.88 54.33 58.95 60.3 23.24 crumble 72.98 76.75 77.76 82.24 83.14 62.16 83.51 64.97 flour 70.83 72.62 94.19 75.28 92.37 56.78 92.15 87.46 paste 52.52 49.67 54.64 31.33 48.61 64.31 59.63 41.76 a* intact 5.16 6.51 8.34 10.83 8.17 9.14 0.99 -2.05 crumble 7.81 6.94 9.93 1.96 6.96 9.92 2.21 0.32 flour 3.12 0.97 1.91 2.21 2.07 8.6 -1.99 -3.87 paste 2.79 2.74 2.5 0.98 3.15 2.96 -1.4 -3.78 b* intact 34.08 31.76 34.38 37.08 31.35 30.98 35.67 0.15 crumble 40.34 38.82 38.44 29.34 41.93 29.62 44.81 31.36 flour 27.69 32.29 35.2 30.65 31.9 27.81 36.4 33.06 paste 32.49 26.2 31.54 22.14 28.54 27.97 33.04 16.14 table 2 biochemical parameters of different soybean genotypes code protein (% wb) fat (% wb) crude fiber (% wb) carbohydrate (% wb) ash (% wb) water (% wb) chlorophyll (mg/100g) total carotene (mg/100g) vitamin c (mg/100gr) k1 35.17 17.04 8.87 34.37 5.13 8.65 3.41 2.47 113.53 k2 33.42 18.38 9.35 33.51 5.31 9.99 3.56 1.98 117.72 k3 35.05 16.74 9.46 33.93 5.16 8.76 3.36 1.95 111.52 k4 32.39 17.64 6.6 36.31 5.09 8.65 0.45 1.48 185.64 k5 35.06 15.43 8.19 35.37 5.76 9.02 3.47 2.16 143.07 k6 34.21 15.15 8.36 34.41 6.01 8.52 3.84 1.56 141.82 k7 34.53 16.71 9.5 32.54 5.59 10.21 15.1 2.76 138.34 k8 36.29 16.41 8.62 34.03 5.56 8.42 9.32 3.54 178.17 biotropia vol. 31 no. 1, 2024 68 (a) (b) figure 4 pca plot of (a) score and (b) bi-plot of score and loading based on soybean characteristics pca based on soybean spectra figure 5 shows the average reflectance data for all preparation methods of each variety. the most effective preprocessing method included the application of a smoothing filter with 150 segments, followed by range normalization. this spectral preprocessing method yielded more consistent and dependable quantitative values for other analyses. the graph shows a typical pattern, where a valley is evident in the spectral range of 390 to 430 nm for all treatment types, followed by a peak in reflectance between 500 and 560 nm, decreasing towards the end of the spectral band. specifically, paste samples showed relatively higher reflectance levels compared to others. furthermore, the addition of water had a significant effect on the reflex response, leading to an increase in the reflectance values. as shown in figures 3a and 3b, the difference in reflectance was significant between k1 and k2. the average reflectance values for intact, crumble, and flour treatments differed significantly from the paste treatment, showing lower reflectance. for k2, k3, and k4, the flour samples tended to show higher reflectance, while k5 was greater in the crumble samples. intact soybean consistently showed lower reflectance levels across all preparation methods. an anomaly was observed for k7, where a small valley in the reflectance spectrum appeared in the 650 to 690 nm range. iv iii ii i evaluation of soybean based on chemical characteristics and vis-nir spectroscopy – farid r. abadi et al. 69 (a) (b) (c) (d) (e) (f) (g) (h) figure 5 the reflectance spectra in various sample types of all soybean varieties (a) k1, (b) k2, (c) k3, (d) k4, (e) k5, (f) k6, (g) k7, and (h) k8. 0 0,5 1 1,5 2 2,5 345 481 611 735 852 962 r ef le ct an ce ( a. u .) wavelength (nm) 0 0,5 1 1,5 2 2,5 345 481 611 735 852 962 r ef le ct an ce ( a. u .) wavelength (nm) 0 0,5 1 1,5 2 345 481 611 735 852 962 r ef le ct an ce ( a. u .) wavelength (nm) 0 0,5 1 1,5 2 2,5 345 481 611 735 852 962 r ef le ct an ce ( a. u .) wavelength (nm) 0 0,5 1 1,5 2 2,5 3 345 481 611 735 852 962 r ef le ct an ce ( a. u .) wavelength (nm) 0 0,5 1 1,5 2 345 481 611 735 852 962 w av el en g th ( a. u .) wavelength (nm) 0 0,5 1 1,5 2 345 481 611 735 852 962 r ef le ct an ce ( a. u .) wavelength (nm) 0 2 4 6 8 345 481 611 735 852 962 r ef le ct an ce ( a. u .) wavelength (nm) biotropia vol. 31 no. 1, 2024 70 pca analysis of the comprehensive reflectance dataset showed distinct patterns based on different preparation methods, as indicated in figure 4a. generally, two main data groups were found depending on the treatment applied to the samples. these included group i, consisting of intact, crumble, and flour, and group ii, comprising paste samples. specifically, group ii accounted for 83% of the variance in pc-1 and 11% in pc-2. most samples in group i tended to have negative pc-2 values, primarily in quadrants iii and iv. a significant distinction appeared when water was added to soybean paste in group ii, resulting in positive pc-2 values in quadrants i and ii. this phenomenon showed a spectrum response significantly different from other treatments. various treatments showed distinct positions along the axes, with intact samples primarily located in the negative range of pc-1, spanning from <-25 to >-150. meanwhile, crumble samples extended in the positive direction, ranging from >-50 to approximately 0. an outlier, representing the intact black soybean (k8) sample, was found at the left end of quadrant iii. the flour samples ranged from >25 to <50 in terms of pc-1, with significant overlap. in figure 6(b), pca loading plot showed a prominent peak at approximately 0.02 to 0.03 on pc-1 at 350 nm wavelength, which gradually decreased to 998 nm. although no significant anomalies were observed, the peak showed wavelength with the most substantial effect on pca grouping. as shown in figure 6a, the application of multiplicative scatter correction (mnc) was used for optimal visualization to show the loading performance of pc-1 and pc-2 concerning genotypes. the distinction among the various groups became evident, primarily characterizing the yellow soybeans (k1-6), with significant contributions from greenish (k7) and black soybean (k8). except for paste form, in the case of intact, crumble, and flour forms, soybean samples were dispersed across pc-2 negative region in quadrants iii and iv. greenish soybean (k7) showed a more extensive spread, while black (k8) was positioned with the lowest pc-2 negative values. figure 6b shows the loading value performance of pca results. anomalies in pc-1 were evident in peaks and valleys spanning from 380 nm to 790 nm, while pc-2 ranged from 430 nm to 780 nm. in pc-1, approximately 82% of variances were described, and the line descended to -0.02 at its lowest point around 370 nm, followed by an increase to 0.02 at 460 nm. this value remained relatively constant until approximately 750 nm, which gradually decreased to -0.02 and continued steadily to the end of the range. the pattern observed in pc-2 followed a similar trajectory but with distinct lowest and peak points. the line initially descended from 380 nm to almost -0.05 at the lowest point and ascended to peak value of 0.02, which was maintained until 700 nm. subsequently, a decline to -0.02 was observed, followed by an increase to approximately -0.01 at about 820 nm. these anomalies affected sample grouping, with pronounced deviations in the spectral regions of 380-470 nm and 700-800 nm due to variations in pigment levels, specifically carotene and chlorophyll, in the samples. (a) (b) figure 6. pca result of the classification of soybean, based on sample types: (a) score plot (b) loading plot using range normalization spectra preprocessing method. evaluation of soybean based on chemical characteristics and vis-nir spectroscopy – farid r. abadi et al. 71 (a) (b) figure 7 pca result of the classification of soybean, based on sample genotypes: (a) score plot (b) loading plot using multiplicative scatter correction spectra preprocessing method black soybean is characterized by black outer skin, which contains anthocyanins, as documented by yang et al. (2022). these anthocyanins significantly impact the interaction between black skin layer and light spectra, either reflecting a specific spectrum of light or being absorbed by particular molecules. specifically, when examining the distribution of genotypes through pca scores, some dissimilarities are frequently observed between paste and other forms. the implementation of physical treatment led to significant patterns in pca plots. the condition of intact soybean, which was not subjected to any physical treatment, primarily reflected the composition of outer skin, resulting in a marked divergence from other soybean types, particularly the yellow ones. in contrast, physical treatments such as grinding exposed the inner layers of the soybeans, leading to a more uniform composition compared to other varieties. the results of pca analysis showed that adding water served as a discriminative factor, segregating the samples into two clearly defined groups. specifically, other preparation methods did not show different clusters, indicating that the specific crushing or sample size reduction had no significant influence on sample response to visnir spectrum. partial least square regression (plsr) table 3 shows the coefficient of determination (r2) and root mean square error (rmse) of calibration (c) and prediction (p) of various quality parameters for intact, crumble, flour, and paste of soybean obtained from partial least squares (pls) analysis. several parameters, including protein, fat, ash, carbohydrate, and water contents as well as a* color, yielded inadequate models. in all cases, r2 values were consistently below 0.7, showing relatively low performance levels. this showed that the parameters could not be effectively detected in the specific vis-nir range of observation. despite being a protein source, soybean showed a low r2 prediction across all groups, with the highest of 0.64 obtained in the paste form. protein identification in spectroscopy is often detected by amino acids referring to the nitrogen content above 300 nm, specifically at 346 nm in the visible range. however, traces of histidine were found in the dika nut, contributing to the failure to detect amino acid responses at the energy levels observed at 380-400 nm (okoronkwo et al. 2017). r2 value for fat was also low at 0.50, but peaks related to fat were found at 537-770 nm without contributing to plsr model. in contrast, lapčíková et al. (2018) detected wavelength range of 300 to 550 nm for unsaturated fatty acid, with ash showing the highest r2 value of 0.37. preece et al. (2009) reported significant wavelength for predicting ash content at 350, 360, 390, 410, 430, 480, and 630 nm. based on the results, similar peaks were found at 460-590 nm, with low concentrations of ash contributing to plsr model. although chandaka et al. (2012) reported that the significant 750 nm could determine the amount of carbohydrate, wavelength at 630-750 nm found in this study did not affect the plsr mode. biotropia vol. 31 no. 1, 2024 72 wavelength contributing to plsr model for determining water was found at 882 nm and 760– 970 nm, in line with the second overtones of oh bands, which indicated water absorption (devianti et al. 2023). the calibration and prediction plsr model for water was also low due to reduced intensity at wavelength above 900 nm which was typical vis-nir spectroscopy. based on figure 1, the model demonstrated low ability in detecting a* value, as indicated by green or yellow with a specific black color shown by soybean samples. crude fiber parameters, including chlorophyll, total carotene, and vitamin c showed effective model performance, achieving relatively high r2 values across all groups, ranging from 0.80 to 0.98. in this study, wavelength contributing to crude fiber appeared at 510-630 nm, while oiv (2021) reported the carboxymethyl cellulose absorbance at 540 nm. chlorophyll in soybean seeds is mainly chlorophyll-a (c55h70mgn4o5) and chlorophyll-b (c55h70mgn4o6). in the visible range, the absorption peaks of chlorophyll-a occur at 430 nm and 662 nm, while chlorophyllb occur at 453 nm and 642 nm (lopes et al. 2017; shi et al. 2022; yang et al. 2022; zhu et al. 2018). longoni et al. (2020) and gebregziabher et al. (2022) reported chlorophyll and carotenes substances in the visible range, where r2 values in calibration and prediction played a significant role in specific sample classification. at 680 nm, soybean samples showed chlorophyll content that distinguished green color (pahlawan et al. (2022). in the visible range, the absorption peaks of chlorophyll-a occur at 430 nm and 662 nm, while chlorophyll-b was found at 453 nm and 642 nm ( lopes et al. 2017; shi et al. 2022; yang et al. 2022; zhu et al. 2018). peak in absorbance of around 670 nm found in the olive oil spectrum confirmed the presence of chlorophyll pigments (lapčíková et al. 2018). at 500 nm, soybean samples indicated carotenoid content that distinguished yellow or darker (pahlawan et al. 2022). r2 values of vitamin c ranged from 0.87-0.98 for calibration and prediction, with the highest observed for intact samples, particularly k8, as shown in table 2. however, there is uncertainty regarding the impact of soybean seeds coat as the primary factor contributing to the elevated vitamin c levels. according to (riscahyani et al. 2019), the analysis of vitamin c can be obtained at the wavelength of 494 nm. the validation of the well-performing model ranged from 0.44 for l* parameter in the crumble sample to 0.96 for vitamin c in paste form. the only parameter with less affirmative validation was l* parameter, registering the lowest r2 value. however, the prediction accuracy at 0.71 indicated a more reliable model outcome, suggesting that physical treatments, such as milling, induced variations in the color characteristics of soybeans. the distinct darkening effect observed with water addition compared to the flour sample could be attributed to the interaction or bonding of water molecules with soybean particles. plsr model for determining total carotene yielded high r2 values for calibration and prediction at wavelength 390, 536, 870, and 990 nm. moreover, the most relevant spectral variables for carotenoids were detected at the wavelength of 449, 448, and 450 nm (afonso et al. 2017). the results also showed that plsr model for predicting l* and b* values had high accuracy. the contrast between green (k7) and black (k8) soybeans was particularly pronounced, as indicated in the l* and b* parameters in the intact sample group, with r2 values of 0.80 and 0.92. this significant difference in the characteristics of whole soybeans enhanced the model performance, allowing for the effective differentiation of color (green and black) from other yellowish samples. subsequently, the results were adjusted by considering r2 values for validation and prediction. l* indicated lightness value in the range 0 (pure black) to 100 (pure white), representing light intensity that increased the absorption at the 400-500 nm spectrum band (afonso et al. (2017). the a* value was identified as reddish colored sample that presents three peaks of great absorption in the 400-500 nm region of the spectrum. meanwhile, yellow colored sample represented in b* profile showed significant peaks at 400-500 nm (afonso et al. 2017). table 3 also shows the validation results of the calibration and prediction models, specifically in terms of rmse, including rmsec and rmsep. most proximate parameters, such as protein, fat, ash, and water, did not correlate significantly. regarding rmse values, crude fiber parameters showed higher values in intact preparation mode, evaluation of soybean based on chemical characteristics and vis-nir spectroscopy – farid r. abadi et al. 73 while paste form had the lowest rmse. this phenomenon suggested that adding water to paste form significantly affected samples grouping. based on chlorophyll, the lowest rmse was observed in the flour form, potentially indicating more uniform sample conditions between the outer and inner layers of soybean containing chlorophyll. a similar trend was observed for total carotene, with the lowest rmse values in the flour and paste forms. this pattern was also found in vitamin c parameter, where the paste form showed the lowest rmse value. regarding color parameters, physical treatment did not yield significant differences in rmse. the comprehensive mean absolute percentage error (mape) assessment shown in figure 8 further explored this aspect. the calibration model parameters suggested that the model accuracy was good for proximate parameters, while water showed less accurate measurements. in contrast, chlorophyll, total carotene, and l* were assessed as showing good accuracy, vitamin c and b* as reasonably accurate, and a* as not accurate. table 3. r2 and rmse of calibration and prediction of various quality parameters for intact, crumble, flour, and paste soybean parameter unit calibration prediction intact crumble flour paste intact crumble flour paste r2c rmsec r2 rmsec r2 rmsec r2 rmsec r2 rmsep r2 rmsep r2 rmsep r2 rmsep protein (% wb) 0.5 1.13 0.59 0.76 0.61 0.87 0.64 0.85 0.56 0.91 0.55 0.97 0.63 0.93 0.68 0.85 fat (% wb) 0.53 0.83 0.29 0.84 0.13 1.09 0.69 0.59 0.5 0.89 0.28 0.81 0.11 0.98 0.47 0.7 crude fiber (% wb) 0.8 0.46 0.82 0.44 0.83 0.42 0.95 0.23 0.72 0.57 0.78 0.49 0.85 0.41 0.92 0.29 carbohydrate (% wb) 0.66 0.69 0.68 0.7 0.58 0.8 0.8 0.54 0.33 0.98 0.72 0.72 0.52 0.94 0.78 0.55 ash (% wb) 0.09 0.36 0.22 0.38 0.23 0.38 0.15 0.38 0.02 0.37 0.37 0.37 0.16 0.41 0.1 0.27 water (%) 0.66 0.39 0.5 0.43 0.78 0.31 0.61 0.75 0.56 0.43 0.4 0.41 0.91 0.21 0.76 0.66 chlorophyll (mg/100g) 0.93 1.11 0.85 1.63 0.95 0.96 0.88 1.46 0.91 1.28 0.77 2.06 0.97 0.76 0.87 1.6 total carotene (mg/100g) 0.85 0.25 0.83 0.28 0.86 0.33 0.92 0.18 0.77 0.32 0.83 0.28 0.91 0.2 0.92 0.18 vitamin c (mg/100g) 0.95 13.67 0.92 18.9 0.82 29.12 0.98 9.36 0.94 15.65 0.88 23.54 0.87 24.16 0.95 14.5 l* 0.8 6.43 0.66 4.56 0.64 7.76 0.77 5 0.86 4.83 0.71 4.28 0.59 7.41 0.61 6 a* 0.57 3.52 0.71 2.06 0.66 2.29 0.74 1.37 0.4 4.47 0.73 2.12 0.9 1.08 0.6 1.36 b* 0.92 3.33 0.47 4.6 0.43 2.96 0.73 3.04 0.92 3.47 0.38 3.71 0.42 3.53 0.7 2.96 figure 8 mape of calibration and prediction for all parameters 0 10 20 30 40 50 60 m a p e ( % ) parameters calibration prediction biotropia vol. 31 no. 1, 2024 74 conclusion in conclusion, this study investigated the relationship between soybean color and chemical parameters, examining the impact of various sample preparation methods. the results showed that processing reduced color differences compared to intact soybeans. statistical analysis showed significant color variations among genotypes and preparation methods due to varying composition and pigmentation. furthermore, pca differentiated soybean samples based on specific components and showed the role of water addition. plsr models predicted some parameters effectively, but protein and fat showed weaker correlations. reflectance spectra showed specific patterns, 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(2018): 1-10. biotropia vol. 30 no. 2, 2023: 128 136 doi: 10.11598/btb.2023.30.2.1654 128 elasticity analysis of the grazing and detrital pathways in a shallow philippine seagrass meadow michael a. clores partido state university – caramoan campus, caramoan, camarines sur, 04429 philippines received 6 september 2021 / revised 27 may 2023 / accepted 31 may 2023 abstract ecotrophic efficiency (ee) is an estimate of the proportion of production that is utilized by the next trophic level through direct predation or fishing or exported out of the ecosystem. in seagrass systems, analysis of ee provides crucial information on how biomass, when used or lost in biological functioning, affects the higher trophic levels via death or grazing relative to the energy lost via decomposition (i.e., flow to the detritus, ftd) and exports to another ecosystem (i.e., sum of all exports, sae). in this study, projections on the effect of change in the ee of functional groups in seagrass systems due to the alteration of biomass were established heuristically using elasticity analysis. using a previously constructed ecopath model for a shallow philippine seagrass meadow, the simulations of altering the biomass of seagrasses and their grazers were done to determine the change in ee, ftd, and sae, thereby generating information on the dynamics of the grazing and detrital pathways in the seagrass ecosystem. results showed the effects of biomass increase and decrease of grazers (herbivorous gastropods, tripneustes gratilla, and polychaetes). if the grazers’ biomass increases, their ee tends to decrease, and biomass accumulation tends to increase. this implies that a fraction of their production used in the system is reduced even if their predators' density and feeding rate are still constant. in addition, the ee of seagrasses tends to increase, leading to a decrease in biomass accumulation at the primary producers’ trophic level. lastly, the ee of detritus decreased because the ftd and sae of its major contributors (the seagrasses) had also decreased. the findings contribute to the ongoing analysis of the role of herbivores versus detritivores in the energetics of seagrass habitats. keywords: biomass elasticity, ecotrophic efficiency, flow to detritus, grazing, seagrass, trophic introduction the two major food chains in any ecosystem, namely, grazing and detrital or decomposer food chains, vary primarily in terms of energy source: autotrophs and living plant materials for the former and the detritus or dead organic matter for the latter. furthermore, the directional flow of net primary production characterizes the grazing pathway (attayde and ripa, 2008). in contrast with the grazing pathway, the detrital pathway has a unidirectional flow that dominates, wherein the recycling of detritus eventually results in inputs at the food chain (smith and smith, 2009). odum (1956) first presented the y-shaped model in which the energy flow in the grazing and detritus food chain are sharply separated. lindeman and lindeman (2007), in a comprehensive trophic and energy flow model of ecosystems, presented an integrated ecological interaction of the grazing and detrital pathways. in seagrasses, the detrital food chain relies on input from waste materials and decaying matter from the grazing food chain. the two food chains are also linked via the process of predation. aside from the breaking down of dead organic matter, decomposer organisms are also food for numerous other animals (hearne et al. 2019; johnson et al. 2019; breithaupt et al. 2019; boncagni et al. 2019; johnson et al. 2020). quantifying the flux of energy through the seagrass ecosystem requires evaluating the consumption, ingestion, assimilation, respiration, and production at each trophic level *corresponding author, email: michael.clores@parsu.edu.ph; mclores@gbox.adnu.edu.ph elasticity analysis of the seagrass model – clores 129 (du et al. 2020). within the food webs in the seagrass system, the trophic level represents seagrasses and their consumers' position. seagrass biomass enters the second trophic level by either secondary production through grazing, recycling biomass by the detrital loops, and feeding on phytoplankton by certain species as early-stage larvae. biomass outputs happen at all trophic levels involving predation and mortalities (blomberg et al. 2014; gloeckner and luczkovich, 2008). following lindeman's (1942), and lindeman and lindeman (2007) tropho-dynamics concept, this study aims to examine the trophic levels of the seagrasses and their consumers and how they are leveraged by the various scenarios of increasing and decreasing biomass. using a previously constructed model by clores and cuesta (2019), such a model was subjected to elasticity analysis by focusing on the seagrasses (producers) and the benthic invertebrates (consumers). the model's scope reflects the total size of the ecosystem, thereby reflecting the properties of the system, particularly the extent of the trophic structure (gascuel 2005; heyman et al. 2014). the modeling outputs are the ecotrophic efficiency (ee) and flow to detritus. the model subjected to elasticity analysis concentrated on the grazing effects on the seagrasses by the invertebrates and almost trifling recreational fisheries. materials and methods the study site is sabitang-laya island located at maqueda channel, caramoan peninsula, philippines (13051’56.22” n, 123057’35.20” e) (fig 1). figure 1 study area in maqueda channel, caramoan peninsula, philippines biotropia vol. 30 no. 2, 2023 130 a trophic model for the seagrass meadow of this island was constructed using ecopath with ecosim (ewe) version 6.6.3 (christensen, waters, and pauly, 2000) and was already reported by clores and cuesta, 2019. the model used an estimate of 22 functional groups' biomass to construct a food web model that described the trophic structure and linkages, and energy flows in the seagrass system (clores and cuesta, 2019) (table 1 and figure 2). ewe required the following: biomass (b) of functional groups, ratios of productivity: biomass (p/b), consumption: biomass (q/b), proportion of unconsumed food, or other collective variables (e.g., gross food conversion efficiency (ge as pb/qb) (christensen et al. 2000). ecotrophic efficiency (ee), estimated with ewe, is the proportion of any of the functional groups’ production assimilated inside the, and is the proportion of production taken by the predators or are transported out from the system (ullah et al. 2012). in this study, the ee was subjected to elasticity analysis. table 1 basic inputs and estimated outputs (bold) of the sabitang-laya is., maqueda channel) seagrass system model group name tl b (t km-2) p/b (yr-1) q/b (yr-1) ee p/q predat mort other mort flow to detritus 1 carnivorous gastropods 3.04 2.35 2.80 5.60 0.96 0.50 2.69 0.12 1.45 2 p. nodosus 3.02 12.89 0.52 2.60 0.31 0.20 0.16 0.36 5.67 3 crustaceans 2.96 1.85 8.40 28.00 0.99 0.30 8.37 0.03 5.21 4 diadema spp. 2.86 0.99 7.51 25.00 0.95 0.30 7.13 0.38 2.67 5 sand dollar 2.60 0.27 10.00 50.00 0.87 0.20 8.86 1.14 1.50 6 nematodes 2.47 2.94 10.00 50.00 0.79 0.20 7.85 2.15 17.86 7 ophiuroids 2.46 0.80 8.63 34.52 0.97 0.25 8.36 0.27 2.87 8 pelecypods 2.26 25.03 2.06 6.86 0.99 0.30 2.04 0.02 17.46 9 polychaetes 2.33 4.20 1.63 12.46 0.54 0.13 0.89 0.74 6.80 10 s. maculata 2.22 2.09 4.45 22.25 0.55 0.20 2.44 2.02 6.76 11 other holothuria 2.22 0.30 4.45 22.25 0.74 0.20 3.30 1.15 0.84 12 zooplankton 2.11 2.87 67.00 192.0 0.68 0.35 45.69 21.31 85.68 13 herbivorous gastropods 2.00 3.11 2.80 5.60 0.90 0.50 2.51 0.29 2.19 14 t. gratilla 2.00 3.77 4.47 25.00 0.33 0.18 1.50 2.98 15.03 15 s. isoetifolium 1.00 178.5 8.43 --0.07 --0.06 8.37 747.1 16 h. uninervis 1.00 262.3 8.43 --0.01 --0.05 8.38 1099 17 h. minor 1.00 10.24 8.43 --0.15 --1.30 7.13 36.51 18 c. serrulata 1.00 1424 8.43 --0.01 --0.03 8.40 5983 19 c. rotundata 1.00 269.6 8.43 --0.01 --0.08 8.35 1125 20 e. acoroides 1.00 5501 8.43 --0.01 --0.00 8.43 23181 21 phytoplankton 1.00 48.00 30.42 --0.38 --11.79 18.63 447.0 22 detritus 1.00 19.80 ----0.01 -------- notes: tl = trophic level; b = biomass (t wet weight (ww) km-2); p/b = production/biomass ratio or the instantaneous rate of total mortality (z) (yr-1); q/b = consumption/biomass ratio or consumption rate (yr-1); ee = ecotrophic efficiency, p/q = production/consumption ratio or gross efficiency; predat = predator; mort = mortality; flow to detritusis in t ww km-2 yr-1. elasticity analysis of the seagrass model – clores 131 elasticity analysis (caswell, 2001) of the ecopath model, a form of sensitivity analysis, was done to determine the effects of biomass alterations of functional groups in seagrass systems. the analysis allowed the simulation of various forms of disturbances by using small proportional changes in the parameter values. elasticity is defined by barbeau and caswell (1999) as: ep (%) 100 = xp – xo xo where: ep (%) = elasticity of the yield to % rise of parameter p xo = yield of the original model xp = yield of the model with a change in parameter p results and discussion the sabitang-laya seagrass model identified three functional groups in the seagrass system to be subjected to elasticity analysis: important grazers, tripneustes gratilla, herbivorous gastropods, and polychaetes. the seagrass system's impacts were explored by conducting an elasticity analysis that entailed altering (i.e., increasing and decreasing) their biomass. the results of the elasticity analysis done on the seagrass model showed that much of the flows to detritus were contributed by the seagrasses (figure 2). but in general, if the biomass of grazers increased, the flow to detritus also decreased, suggesting that much of the seagrass material goes to the grazing pathway. an overall result of elasticity analysis was shown in figure 3. if the density and biomass of grazers: t. gratilla, herbivorous gastropods, and polychaetes increased, their ee decreased. this observation indicates that a fraction of their production used in the system reduces, this is despite that their predators' density and feeding rate are still constant. also, there is increased biomass accumulation at the grazers' trophic level. when grazing is intense, the ee of seagrasses increased because of the high consumption of their production in the system due to grazing. this eventually decreases the biomass accumulation at the primary producers’ trophic level. furthermore, since much of the seagrass materials goes to the grazing pathway, the ftd from them decreased. lastly, the ee of detritus decreased because the flow into the detrital pool of its major contributors (the seagrasses) had also decreased. figure 2 simplified representation of the grazing scenarios effects of biomass increase and decrease of grazers (herbivorous gastropods, tripneustes gratilla and polychaetes) on a shallow philippine seagrass systems biotropia vol. 30 no. 2, 2023 132 projections showed that if the biomass of grazers (polychaetes, herbivorous gastroprods, tripneustes gratilla) increased under steady-state assumptions, their ecotrophic efficiency (ee) decreased, and flow to detritus (ftd) increased. for example, if the biomass of t. gratilla decreased by 99%%, their ee increased by almost 30% while their ‘flow to detritus’ (ftd) increased to about 60 tons/km2/year this finding means that when grazers become abundant in the seagrass system, their biomass accumulates and enters the detrital pool when they die (figures 3). furthermore, if the biomass of grazers (polychaetes, herbivorous gastropods, and t. gratilla) in the seagrass system increased, the ee of seagrasses had relatively increased also, suggesting that a fraction of the production of seagrass was used in the system or passed up the food web (fig 4). however, the ftd from the seagrasses almost remains unchanged, indicating that their biomass goes to the grazing pathway (fig 5). grazers also tend to consume the seagrasses cymodocea serrulata, syringodium isoetifolium, halodule uninervis and c. rotundata, suggesting intense utilization of these seagrasses through grazing. the low ee of e. acoroides, despite the increasing biomass of grazers, suggests that this seagrass group was underexploited or not favored by the grazers; hence their supply exceeded demand, and much of this excess production went to detritus (fig 4). figure 3 projected ecotrophic efficiency (ee) and ‘flow to detritus’ (ftd, ton/km2/year) of the seagrass grazers as their biomass decrease or increase elasticity analysis of the seagrass model – clores 133 figure 4 projected change in the ecotrophic efficiency (ee) of seagrasses as the biomass of their grazers (polychaetes, herbivorous gastropods, and tripneustes gratilla) is decreased or increased figure 5 projected change in the flow to the detritus (ftd, %) of seagrasses as the biomass of their grazers (polychaetes, herbivorous gastropods, and tripneustes gratilla) is decreased or increased biotropia vol. 30 no. 2, 2023 134 it was also revealed that, as the biomass of grazers increased, the ee of phytoplankton slightly increased, and their ftd remain unchanged. for instance, an increase in the biomass of grazers by 90% resulted in an increase of their ee by 0.20 (figure 6). the ‘sum of all exports’ (sae) decreased as the biomasses of grazers increased. this means that an increase in grazer biomass leads to the consumption of all biomass that otherwise will be exported from the seagrass systems (figure 7). figure 6 projected change in the ecotrophic efficiency (ee) of phytoplankton and detritus as the biomass of seagrass grazers (polychaetes, herbivorous gastropods, and tripneustes gratilla) decreased or increased figure 7 projected change in the ‘sum of all exports’ (t/km2/year) and ‘sum of all flows into detritus’ (t/km2/year) if the biomass of grazers is increased in the seagrass system. elasticity analysis of the seagrass model – clores 135 elasticity analysis of the seagrass model in sabitang-laya island, maqueda channel, caramoan peninsula, a shallow seagrass meadow in the philippines, was useful in predicting the impacts on the seagrass systems' energy flows. fluctuations of the ee fractions and the ftd from the seagrasses as their grazers' biomass change was used to make projections. the projections showed that the seagrasses' energy/biomass proceeds to either the grazing or detrital pathways; it is expected that the energy/biomass of seagrasses will proceed to either the grazing or detrital pathways. however, if there are possible disturbances brought by overgrazing or overharvesting of grazers, the result is a restraint of these routes, or rerouting of the energy/biomass in the system. the overall flows in the system remain constant. it is expected that if an increase in the biomass of grazers (e.g., t. gratilla) happens, a large amount of energy/biomass from the seagrasses flows directly into the grazing pathway. this data indicates that grazers have a critical role in consuming seagrass material and biomass disposal due to grazing, which ultimately ends up in detrital food webs. projections also revealed that the seagrass that was discarded or not consumed by grazing might be exported from the seagrass systems. this could be through wave action and water currents. this export of biomass/energy to other ecosystems means that grazing on seagrass systems has an important role in seagrass beds' connectivity with other habitats like coral reefs and mangrove forests. elasticity analysis of the seagrass model in bageing bay, sabitang-laya island showed increasing the biomass of seagrass grazers t. gratilla, polychaetes, and herbivorous gastropods in shallow philippine seagrass meadows could lead to significant overexploitation for resources of low trophic levels. the model became imbalanced as grazers’ biomass decreased, hence the collapse of biomass affecting the functional biodiversity that may have substantial consequences, particularly on ecosystem resilience. in effect, the model simulations provided relevant theoretical bases to explain the distribution of biomass per trophic level and the impact of grazing on biomass distribution. consequently, real observations should be compared with the model results to validate virtual ecological modeled ecosystems. the interactions of the essential components of the shallow philippine seagrass systems, as shown by this seagrass meadow at maqueda channel, caramoan peninsula, and their influence in regulating the ecosystem are manifested by the steady-state models and elaborated by the elasticity analysis done in this study. the links from the seagrasses as producers to the invertebrates as intermediate consumers portrayed by the results contribute to a better understanding of how the ecosystem functions. the results of the elasticity analysis proved to be a practical tool for diagnosing and forecasting the impacts of overgrazing in seagrass systems commonly reported in the literature (burnel et al. 2013; yeager and layman, 2011; fourqurean et al. 2010) and the mechanistic interactions between predators and prey in seagrass meadows (clores and cuesta, 2019; archer, stoner, and layman, 2015; clores, conde and perez, 2020). conclusion the findings of the current study contribute to the ongoing reevaluation of the role of herbivores and detritivores in the energetics of seagrass habitats. clearly, when in steady-state conditions, as shown by the shallow philippine seagrass meadow in this study, seagrass systems significantly contribute their seagrass production to the detrital pool. this result of the elasticity analysis of the seagrass trophic model analysis showed that when grazers become abundant, much of this production proceeds to the herbivory pathway. this research provides robust elasticity models of the dynamics of a seagrass system that offer relevant insights into the seagrass structure's energy dynamics. analysis of this study's data showed that interactions are strong between the functional groups at the lower trophic levels in seagrass systems. in the context of conservation programs for seagrasses, top-down (detrital pathway) and bottom-up (grazing pathway) factors that control seagrass ecosystem structure and function should be considered. for instance, when grazing rates could surpass the seagrass growth rates (i.e., overgrazing), the biotropia vol. 30 no. 2, 2023 136 possible detrimental threats on the seagrassassociated ecosystem services should be mitigated. acknowledgments this research was supported by a research grant from partido state university’s research, extension, and knowledge management office. references archer, s.k., stoner, e.w., layman, c.a. 2015. a complex interaction between a sponge (halichondria melanadocia) and a seagrass (thalassia testudinum) in a subtropical coastal ecosystem. j exp mar bio ecol, 465: 33-40. attayde, j. l., & ripa, j. 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pgr alya munira1, khairan khairan2,3, sreeramanan subramaniam4, and essy harnelly3,5* 1master program of biology, department of biology, faculty of mathematics and natural sciences, syiah kuala university, banda aceh 23111, indonesia 2department of pharmacy, faculty of mathematics and natural sciences, syiah kuala university, banda aceh 23111, indonesia 3arc pui-pt nilam aceh, syiah kuala university, banda aceh 23111, indonesia 4school of biological sciences, universiti sains malaysia, georgetown, penang 11800, malaysia 5department of biology, faculty of mathematics and natural sciences, banda aceh 23111, indonesia article higlights • different varieties of patchouli exhibited different growth pattern as well as in vitro culture • in order to induce callus, it has been determined that the combination of regulators is necessary. • benzylaminopurine has been identified as a potential regulator that could be used to further develop the patchouli callus of tapak tuan. article information received : 25 july 2024 revised : 20 september 2024 accepted : 18 october 2024 *corresponding author, e-mail: essy.harnelly@usk.ac.id research article abstract patchouli is known for its highly demanded essential oil. the patchouli tapaktuan variety is the most widely cultivated by local farmers in aceh due to its high oil yield. currently, the study and propagation of patchouli through a biotechnological approach is being developed, one of which is in vitro culture method. in this method, suitable plant growth regulators (pgr) are being observed to enhance the growth of the explant. pgrs are synthetic compounds that are added to media in plant tissue culture to stimulate plant growth. this study aimed to analyze and optimize the effect of benzylaminopurine (bap), thidiazuron (tdz) combined with naphthaleneacetic acid (naa) on the growth of tapaktuan patchouli leaf callus. this study was carried out by using a completely randomized design with seven treatments. each treatment was replicated four times and each replicate contained three explants. the concentrations used were bap 0.75 mg/l, tdz 1 mg/l, and naa in the 0.25 0.75 mg/l range. according to this study, combining bap and naa was the most effective for inducing callus in tapaktuan patchouli leaves. the combination of bap 0.75 mg/l + naa 0.5 mg/l (treatment a2) was the most effective for inducing callus formation. this treatment resulted in the quickest callus development, the highest percentage of callus formation, and the largest callus diameter. explants cultured with bap produced a greenish-yellow callus having the potential for organogenesis culture, which could produce shoots having the ability for producing mass plantlets. keywords: callus, in vitro, patchouli, pgr, tapaktuan introduction patchouli (pogostemon cablin benth.) is a fragrant, herbaceous plant that yields an essential oil called patchouli oil. patchouli oil is used in the manufacture of perfumes, cosmetics, aromatherapy, and pesticides. patchouli has long been used as a traditional remedy to treat various ailments, such as skin problems, insomnia, and as an aphrodisiac (gopi 2017). patchouli is primarily produced in sumatra and sulawesi, with aceh province accounting for around 80% of global patchouli oil supply in 1980. since 1921, aceh has been the largest producer of patchouli oil (ernawati et al. 2021). according to the directorate general of estates (2022) patchouli production in indonesia has experienced a good increase, with a percentage increase of up to 60% from 2017 to 2021. aceh has three high-yielding patchouli varieties with average oil production per hectare, which are lhokseumawe (415.65 kg/ha), sidikalang (464.442 kg/ha), and tapaktuan (583.26 kg/ha). of these three varieties, the tapaktuan variety is the most widely cultivated https://doi.org/10.11598/btb.2024.31.2.1741 biotropia vol. 32 no. 1, 2025 32 by local farmers due to its higher oil yields, more resilience to drought, and 10.6% more patchouli alcohol (pa) compared to the other two varieties. patchouli alcohol, or pa, is a quality indicator for patchouli oil; the higher the pa value, the better the quality. patchouli is usually propagated by cuttings, and it is quite difficult to obtain goodquality cuttings (kandarihi et al. 2015).  the propagation of patchouli through a biotechnological approach in plant tissue culture has come a long way as the demand for patchouli oil increases every year (swamy & sinniah 2016). using tissue culture, plants can be propagated rapidly and produce seedlings in large quantities. plant propagation is achieved by selecting patchouli plants that are free from pests and diseases and using organ parts, such as shoots, nodes, and leaves, as sources of explants. mass propagation in tissue culture can occur through somatic-embryogenesis or organogenesis. the formation of plant organs occurs in response to the wounding of plant tissues to form callus, the amorphous cells (bidabadi & jain 2020). new plants are formed from callus cells that have the capacity for embryogenic totipotency when supported by the use of suitable plant growth regulators (pgr) (fehér 2019). the presence of pgrs added to the media influences the success rate of plant tissue culture (fitroh et al. 2018). auxin and cytokinin are types of pgr commonly used to induce callus in many plant species, and the explant growth rate depends on the concentration and combination of the two regulators, the species, and the type of explants (hemmati et al. 2020). the amount of auxin and cytokinin administered to the explants determines the direction of plant explant growth. high cytokinin levels promote bud formation, high auxin levels encourage root development, and equal concentrations of both hormones lead to callus formation (bano et al. 2022; pulianmackal et al. 2014). one type of auxin commonly used in tissue culture is naphthaleneacetic-acid (naa), which is often used to induce callus and roots (kawochar et al. 2017). naa concentration of 0.5-1.5 mg/l successfully induced callus of patchouli leaves of local indian varieties with an average formation percentage of 94.34% within 2-4 weeks of culture of the explants (lalthafamkimi et al. 2022). naa is also often combined with cytokinin, such as benzylaminopurine (bap) to promote the formation of green callus that can develop into shoots. florenika et al. (2022), found that naa 0.5 mg/l + bap 0.1 mg/l was the best combination to produce morphogenic callus in sidikalang variety patchouli, while in local indian patchouli, the highest morphogenic callus of 81.51% was formed in the combination of naa 1.5 mg/l + bap 1.5 mg/l (lalthafamkimi et al. 2022). in addition to bap, a regulator categorized as cytokinin, thidiazuron (tdz), is also frequently used for plant cell proliferation and regeneration. tdz combined with naa gives superior results than those combined with other types of auxins. in the morphogenesis of scutellaria bornmuelleri and ajuga bracteosa plants, the concentration of tdz ranges of 0.5 1.2 mg/l giving a higher response to explant morphogenesis (ali et al. 2018; gharari et al. 2021). the studies conducted by tarigholizadeh et al. (2021) on satureja sahendica bornm had the highest percentage of callogenesis (95.24 100%) at different levels of naa and tdz combination treatments (0.5 2.0 mg/l).  to date, research on pgrs for the induction of tapaktuan patchouli leaf callus has not been fully conducted. this is because each varieties of patchouli have particular superiority and physiological responses. callus induction is utilized for obtaining germplasm from a small source, which can then be studied in advanced biotechnology applications, such as genetic engineering and secondary metabolite engineering. florenika et al. (2022) investigated the most effective combination of pgrs for inducing callus in sidikalang variety patchouli leaves, which is bap + naa. in the lhokseumawe variety of patchouli, puspita et al. (2023), obtained optimal results for inducing leaf callus in the combination of tdz 0.75 mg/l + naa 0.5 mg/l and bap 0.75 mg/l + naa 1.0 mg/l. therefore, from these studies, it is necessary to conduct research for determining the best combination of pgrs in order to induce callus in patchouli leaf explants of the tapaktuan variety. this study aimed to analyze and optimize the combination of pgrs bap, tdz, and naa on the growth, development, and callus morphology of tapaktuan patchouli leaf explants. optimization of bap, tdz and naa on patchouli callus induction 33 materials and methods plant preparation patchouli seedlings of the tapaktuan variety were obtained from nino park, syiah kuala university, banda aceh. the seedlings were quarantined by spraying them with 2 g/l bactericide (active ingredient of streptomycin sulfate 20wp) and 2 g/l fungicide (active ingredient of benomyl 50wp) separately every two days for two to three weeks and fertilized once a week with 80 g of 16-16-16 (npk) fertilizer to stimulate leaf growth. explant sterilization sterilization was carried out outside and inside the laminar air flow cabinet (lafc). sterilization outside the lafc is based on puspita et al. (2023), where leaves of quarantined plants were rinsed under running water and carefully brushed with a soft brush until clean. the explants were first soaked in 5% detergent for 5 minutes and washed again under running water until clean. next, the explants were soaked in 2% bactericide for 15 minutes, agitated every 5 minutes, and rinsed three times with sterile water. the explants were then immersed in a 2% fungicide for 15 minutes, agitated once every 5 minutes, and rinsed three times with sterile water. following the sterilization inside the lafc as described by rahmawati et al. (2021), the explants were transferred to the lafc and further sterilized for 5 minutes with a 30% naocl solution containing a drop of tween 80, then rinsed three times with sterile water. finally, the leaves were immersed in ethanol 70% for 30 seconds and then rinsed three times with sterile water. culture medium sterilized leaf explants were placed into a petri dish and dried using sterile tissue. the explants were cut into smaller 1 cm x 0.5 cm pieces with a scalpel. the explants were cultivated in murashige & skoog (ms) medium, which contained 30% sucrose, 5.5% agar, naa (0.25; 0.50; and 0.75 mg/l), bap (0.75 mg/l), and tdz (0.75 mg/l). for comparison, the explants were cultured into ms without any addition of pgr (ms0), as shown in table 1. the explants were then incubated at 22 °c to 24 °c under a 16-watt fluorescent lamp (philips t8 ecofit cool daylight), 10 cm away from the lamp and the explant bottle. explant growth was observed for 60 days, as the patchouli callus had not fully developed after 30 days, and to gain a clear visualization of the callus from the effect of each treatment. the growth of the explant was monitored every day after the explants were cultivated. data analysis a completely randomized design was used in this study with 7 treatments. each treatment was replicated four times and each replicate contained three explants. the combination of treatments can be seen in table 1. the data of the individual parameters were calculated as a percentage. the callus response of the patchouli variety tapaktuan was determined using variables, such as percentage of callus, percentage of live explants, percentage of shoots, percentage of roots, and callus diameter. callus formation was observed daily until callus began to emerge from the edge of the explants. table 1 comnbination of plant growth regulators used in the study for callus induction of p. cablin treatments plant growth regulators (pgr) (mg/l) bap tdz naa ms0 a1 0.75 0.25 a2 0.75 0.50 a3 0.75 0.75 a4 1.0 0.25 a5 1.0 0.50 a6 1.0 0.75 biotropia vol. 32 no. 1, 2025 34 the percentage of callus formation was observed on day 60 after initiation (dai) and live explant parameters were taken on days 30 and 60 of observation to determine the ability of the callus to survive in the treatment. the formula used was based on wulandari et al. (2022): % callus = number of explants that form callus in each treatment total of all explants planted x 100% the diameter of the callus explants was measured on day 60 after the start. data were collected by measuring the width of the explants that produced callus at the bottom of the explant bottles using a ruler. the diameter of callus explants was determined using the formula on taghizadeh et al. (2020): (1) % live explant = number of living explants per treatment total of all explants planted x 100% (2) √callus diameter = (callus length x callus width) (3) explants that formed shoots and roots were observed by counting the number of explants planted in each treatment that showed signs of the emergence of shoots and roots. the percentage of shoots and roots formed was calculated based on fanata and qudsiyah (2020), using the formula: % shoots = number of explants that form shoots per treatment total of all explants planted x 100% (4) % roots = number of explants that form roots per treatment total of all explants planted x 100% (5) the callus morphology, including color and texture, was observed visually. the color of the explant callus was observed on day 60 dai. callus color was visually observed and determined using the munsell color chart method by restanto et al. (2022), followed by a callus color scoring method based on thomy (2012), shown in table 2. explant texture observations were made visually and by touching with tweezers. rasud and bustaman (2020), define a friable texture as one that is highly soft, quickly disintegrates, and slightly sticky to the tweezers. a compact callus feels very dense and firm, while an explant with both textures is considered intermediate. explant texture observations were made visually and by touching with tweezers. rasud and bustaman (2020), define a friable texture as one that is highly soft, quickly disintegrates, and slightly sticky to the tweezers. a compact callus feels very dense and firm, while an explant with both textures is considered intermediate. table 2 callus color score score callus color 1 brown 2 brownish white 3 brownish green 4 yellowish white 5 yellowish green 6 whitish green 7 green optimization of bap, tdz and naa on patchouli callus induction 35 results and discussion days of callus formation the appearance of callus in explants of patchouli leaves is characterized by the fact that the explants bend and swell two weeks after cultivation. the callus grows from the injured tissue and gradually spreads around the injured area and surface of the explant until it covers all parts. according to habibah et al. (2021), the involvement of regulators in culture media has a significant impact on the development of calluses. in this study, bap and tdz together with naa were able to induce callus on patchouli leaves of the tapaktuan variety. the ability of tdz and bap on the day of callus formation was significantly different from each other. the results of the anova test results (p < 0.05) indicated that there was a significant difference on the day of callus formation from the combination of bap, tdz, and naa. however, the callus could not appear in ms0 treatment until 60 days of observation. callus appeared on day 15 in the treatment of bap 0.75 mg/l combined with naa 0.25 mg/l (a1) and naa 0.5 mg/l (a2), which is similar to research results of puspita et al. (2023), where the callus of patchouli leaf explants of the lhokseumawe variety in the combination of bap 0.75 mg/l + naa 0.50 mg/l showed signs of callus appearance on day 15 day of observation. callus grown with tdz 1.0 mg/l + naa 0.25 mg/l successfully induced the quickest callus on day 16 of observation. bap combined with naa successfully induced callus in nepeta binaloudensis jamzad and cucumber (cucumis sativus l.) leaf explants (faisal et al. 2018; sagharyan et al. 2020). exogenous cytokinin, such as bap and naa, have been shown in in vitro culture of many plant species to induce callus and promote cell division. according to previous studies, there is a balance of auxin and cytokinin that work together to stimulate cell division, which is a crucial phase in callus development (sagharyan et al. 2020). according to deswiniyanti and lestari (2020), exogenous auxin, such as naa, increases osmotic pressure, elongation, and cell enlargement, whereas cytokinin, such as bap promotes cell division. auxin increases water diffusion into cells, hence the combination of auxin and cytokinin stimulates cell division and modulates cell differentiation pathways. combining tdz with other phytohormones is highly recommended because tdz might have significant effects on the metabolism of other phytohormones through accumulation and translocation (deepa et al. 2018). in the present research, the treatment of tdz 1.0 mg/l combined with naa 0.25 mg/l resulted in the fastest callus on day 16, compared with other tdz combinations. callus formation percentage and morphological development the formation of callus on cultivated explants is an indicator of the growth reaction of the explant. in leaf explants, callus appears first on the leaf bones because the leaf veins have vascular bundles that transmit nutrients to all sections of the leaf surface, allowing the cells near the leaf veins to divide and induce callus (rahayu & mardini 2015). the results of this research found that the patchouli leaf callus cultivated under all treatments showed callus formation (table 3). table 3 effect of pgr on the growth of p. cablin callus explant tapaktuan variety pgr callus formation (day) callus % live explants % callus diameter (mm) shoots (%) roots (%) ms0 60 ± 0d 0.0 ± 0b 0.0 ± 0c 0.0 ± 0a 0 0 a1 15 ± 0.49a 100 ± 0a 100 ± 0a 164.4 ± 14.4b 66.7 ± 2.3 0 a2 15 ± 0.37a 100 ± 0a 91.7 ± 8.3a 187.9 ± 8.70b 83.4 ± 2.5 0 a3 16 ± 1.05ab 91.7 ± 8.3a 75.0 ± 16.0a 177.5 ± 17.0b 0 0 a4 16 ± 0.56a 86.7 ± 8.2a 86.7 ± 8.2a 166.1 ± 22.9b 0 83.4 ± 2.5 a5 20 ± 0.76c 91.7 ± 8.3a 91.7 ± 8.3ab 153.3 ± 16.4b 0 100 ± 2.3 a6 18 ± 1.01bc 100 ± 0a 83.3 ± 16.7b 137.6 ± 27.2b 0 33.3 ± 1.3 biotropia vol. 32 no. 1, 2025 36 the a1, a2, and a6 treatments were the combination that resulted in the callus formation with the highest percentage (100%) followed by the a3 and a5 treatments (91.7%). the a4 treatment produced the lowest callus formation percentage (86.7%). patchouli leaf explants in the ms0 treatment did not form any calluses. in the treatment of bap combined with naa 0.25 mg/l and 0.5 mg/l, callus was successfully induced at a 100% rate (table 3). this is in accordance with the results reported by anwar and isda (2021), in which the addition of high concentrations of bap combined with low concentrations of naa gave the best results in the formation of centella asiatica l. callus, with a 100% formation rate in the treatment combination of bap 1 mg/l + naa 0.3 mg/l. however, when bap and naa were administered at concentrations near to, equal to, or greater than 1.0 mg/l, the percentage of callus formation started to decline, as observed in treatment a3, which achieved 91.7% callus formation (table 3). this result is similar to the result of nazir et al. (2020), the combination of bap 2 mg/l + naa 1 mg/l resulted in 92% callus formation in ocimum basilicum explants. in tdz, treatment a4 induced 86.7% of calluses, which increased to 91.7% in treatment a5. then, in treatment a6, the callus production rate reached 100%. the percentage of callus increases with higher concentrations of naa and tdz, indicating a positive trend in callus production on tdz treatments. puspita et al. (2023), reported that patchouli leaf explants of the lhokseumawe variety treated with tdz 1.0 mg/l + naa 0.5 mg/l made the highest percentage of callus formation (66%). explants cultivated in medium containing a combination of bap and naa produced yellowish-green callus, meanwhile explants cultivated in medium containing tdz and naa produced brownish-green callus (fig. 1). all combination treatments produced compacttexture callus (table 4). figure 1 patchouli leaf callus at 8 weeks after initiation notes: a) bap 0.75 mg/l + naa 0.25 mg/l treatment; b) bap 0.75 mg/l + naa 0.5 mg/l treatment; c) bap 0.75 mg/l + naa 0.75 mg/l treatment; d) tdz 1.0 mg/l + naa 0.25 mg/l treatment; e) tdz 1.0 mg/l + naa 0.5 mg/l treatment; f ) tdz 1.0 mg/l + naa 0.75 mg/l treatment; scale bar = 1 cm; the colored box on the top right shows the visualization of callus color using munsell color chart. optimization of bap, tdz and naa on patchouli callus induction 37 table 4 callus morphology treatment callus color texture munsell code score ms0 compact a1 5 gy 6/10 (yellowish green) 5 compact a2 2.5 gy 5/8 (yellowish white) 6 compact a3 2.5 gy 7/10 (yellowish green) 5 compact a4 5 gy 6/10 (brownish green) 3 compact a5 5 gy 6/10 (brownish green) 3 compact a6 7.5 y 8/6 (brown) 1 compact source: munsell (1977); thomy (2012). the callus color reflects its visual appearance as an indicator of whether the callus contains cells that are actively dividing, embryonic, or cells that have undergone senescence. green and yellow callus have a good ability to divide. green callus is due to the presence of chlorophyll in the callus. a yellow callus indicates the presence of flavonoid pigments. brown callus indicates the metabolism of phenolic compounds which are toxic and can inhibit plant growth, potentially leading to cell death (tarigan et al. 2023; wijaya et al. 2023). callus in treatment a2 also produced white callus, which according to tarigan et al. (2023), shows that it has embryogenic potential, allowing it to develop into somatic embryos. live explant percentage the percentage of live  explants indicates their ability to respond to the treatment of regulators to induce callus formation. this study found that combining bap and tdz with naa significantly increased the percentage of live explants (p < 0.05). according to the results shown in table 3, treatment a1 produces the maximum percentage of live explants by 100% and has the highest percentage of live explants in the combination of bap. explants cultivated on tdz had the highest percentage of live explants (91.7%) in the a5 treatment. surviving explants have fresh, light-colored explants with a callus that continues to differentiate and proliferate. according to waryastuti et al. (2017), the callus that does not survive turns a blackish-brown and shows no signs of cell growth. when the concentration of naa was raised to 0.50 mg/l (a2), the percentage of live explants began to decrease to 91.7%; when the concentrations of bap and naa were equal at 0.75 mg/l (a3), the percentage of live explants decreased to 75%. callus cultivated in tdz decreased in percentage by 83.3% when combined with naa 0.75 mg/l (a6). this might be due to the mechanism of hormone inhibition by other hormones (septiana 2014). aside from the exogenous hormones administered, the explants produce their own endogenous hormones, one of which is iaa (indole acetic acid) (korasick et al. 2013). according to septiana (2014), high levels of auxin may inhibit tissue growth due to competition with endogenous auxin for a signal receiver position in the cell membrane, resulting in inhibition of cell growth and elongation. if endogenous iaa levels increase, this type of auxin might lower the efficiency of bap. callus diameter the diameter of the callus indicates the explants’ response in the form of callus growth to the applied regulators. as shown in table 3, the largest callus diameter in bap treatment was obtained in the a2 treatment (bap 0.75 mg/l + naa 0.50 mg/l), with an average callus diameter of 187.9 mm. the maximum average callus diameter in the tdz treatment was 166.1 mm in treatment a4 (tdz 1.0 mg/l + naa 0.25 mg/l). meanwhile, the ms0 treatment did not form any calluses, therefore, its diameter could not be measured. explants cultivated under bap treatment fluctuated in response to increasing naa concentrations. studies on dracocephalum polychaetum and dracocephalum kotschyi leaf callus explants similarly showed variations upon increasing naa from 0.5 mg/l to 1.0 mg/l (taghizadeh et al. 2020). in the tdz treatment, biotropia vol. 32 no. 1, 2025 38 callus diameter decreased as naa concentration increased. this result is similar to the callus of mentha pulegium l., which decreased with increasing naa concentration when combined with benzyladenin (jafari et al. 2016). the size of the callus produced varies depending on the plant tissue’s ability to absorb water and nutrients. generally, auxin stimulates callus formation, while cytokinin can also enhance callus formation, although its specific mechanism is unknown. according to some researchers, genetics have a crucial role in forming callus in tissue culture and the regeneration capability of plants, which are controlled by numerous genes, particularly in the nucleus and cytoplasm (gharari et al. 2021). auxin and cytokinin function together to promote callus development in explants, as there is a synergistic and antagonistic interaction between auxin and cytokinin. the use of high amounts of cytokinin with low amounts of auxin can synergistically trigger cell division and regeneration in vitro (faisal et al. 2018). auxin can antagonistically inhibit cytokinin biosynthesis signaling pathways, while cytokinin promotes auxin biosynthesis and regulates auxin transport to sites of differentiation and growth. however, an excess of cytokinin can have negative effects on plant growth (kotov & kotova 2023). the combination of higher auxin with cytokinin may slightly suppress the inhibitory effect of auxin and the negative effects of cytokinin. some researchers speculate that auxin, which is synthesized with the help of cytokinin, acts as a feedback loop that inhibits the accumulation and activity of cytokinin (kurepa & smalle 2022). root and shoot formation the presence of pgr, specifically auxins and cytokinin, has a strong influence on callus growth and development. the morphogenesis of explants always depends on the interaction between auxin and cytokinin. the concentration of auxin and cytokinin regulates the amount and direction of growth in the callus and organogenesis. some explants treated with bap formed shoots from calluses, which formed 0.5 1 cm shoot. shoots are formed in treatment a1 and a2 with the highest percentage of 83.35% in treatment a2 (table 3; fig. 2a). explants treated with tdz, which was treatment a4 to a6 (table 3), did not produce shoots, instead appeared root-like in the form of white, fine root hairs (fig. 2b). treatment a5 had the highest percentage of roots at 100%. although tdz is also a cytokinin, the callus formed root hairs rather than shoots, which contradicts the concept of organogenesis by auxins and cytokinin. a higher auxin concentration in comparison to cytokinin results in the production of a rooted callus, whereas a high cytokinin content results in the formation of a shoot-like callus. when the given concentration of cytokinin and auxin is balanced or nearly balanced, the explants maintain the callus form (sudrajad & wijaya 2019). in addition to auxin and cytokinin, which are administered as exogenous hormones, the endogenous hormones within the explants also have an influence (silalahi 2015). this could explain why the tdz treatment of the callus produces roots rather than shoots. aside from the roots, auxin levels are often high in buds and shoots. if the explants used are very young leaves, the auxin content may still be quite high. therefore, when the explants are cultured figure 2 a) callus with shoots in bap 0.75 mg/l + naa 0.5 mg/l treatment. b) callus with roots tdz 1.0 mg/l + naa 0.5 mg/l treatment optimization of bap, tdz and naa on patchouli callus induction 39 in a combination of high cytokinin with low auxin media, endogenous auxin will most likely work together with exogenous auxin to lower the cytokinin content and suppress endogenous cytokinin biosynthesis (sosnowski et al. 2023). according to zhang et al. (2018), root hairs emerge in root epidermal cells and have several types of initiation patterns. in patchouli leaf explant callus, root hair initiation might occur through the differentiation of epidermal cells that produce roots at random. this pattern is commonly found in most dicotyledonous, monocotyledonous, and fern plants. conclusion callus initiation media containing cytokinin and auxin were reported to effectively grow the callus of tapaktuan variety patchouli leaves. the use of pgrs could have different effects on plants of different cultivars within the same species. the relationship between pgrs and plant varieties within a species is not well understood and requires extensive research. optimization research offers the best option for growing plants of specific varieties through tissue culture techniques. according to this study, combining bap and naa is most effective for inducing callus in tapaktuan patchouli leaves. bap 0.75 mg/l + naa 0.5 mg/l (treatment a2) is the most efficient combination to induce callus tapaktuan variety patchouli leaves. this combination resulted in the fastest duration, the highest percentage of callus formation, and the largest callus diameter. explants in treatment a2 produced a yellowish-white callus with potential for organogenesis culture, capable of generating shoots that can produce mass plantlets. references ali h, khan ma, kayani wk, khan t, mashwani zr, ullah n, khan rs. 2018. thidiazuron regulated growth, secondary metabolism and essential oil profiles in shoot cultures of ajuga bracteosa. ind crops prod 121(1):41827. doi: 10.1016/j.indcrop.2018.05.043 anwar n, isda mn. 2021. respons pembentukan kalus daun pegagan (centella asiatica (l.) urb.) dengan penambahan naphtalene acetic acid dan benzyl amino purin secara in vitro. 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[resistance of soybean (glycine max l.) embryo callus to salinity stress (nacl) in vitro]. journal techno-eco-farming (jtef) 2(1):8-21. zhang dj, yang yj, liu cy, zhang f, wu qs. 2018. root hair growth and development in response to nutrients and phytohormones. in: giri b., prasad r., varma a (editors). root biology. cham (ch): springer international publishing. p. 65-84. doi: 10.1007/978-3-319-75910-4 the southeast asian journal of tropical biology vol. 32 no. 2, 2025: 205 215 doi: 10.11598/btb.2025.32.1.2480 issn: 0215-6334 | e-issn: 1907-770x 205 article higlights • calcium nitrate is utilized to enhance the growth and chlorophyll quality of arthrospira platensis gomont, aiming to maximize its potential as a biotransducer molecule. • biomass productivity was monitored every three days during a 15-day cultivation period, with specific attention to biomass accumulation and specific growth rates during the stationary phase. • chlorophyll concentrations (chlorophyll a, chlorophyll b, and total chlorophyll) were measured using a uv-vis spectrophotometer at wavelengths of 648 nm and 664 nm. • fourier-transform infrared spectroscopy (ftir) was performed on chlorophyll extracts to assess molecular binding capacity, reinforcing arthrospira platensis gomont’s potential as a biotransducer. • a concentration of 4.5 g/l of calcium nitrate, in combination with 35 ppt salinity, was found to be optimal for enhancing chlorophyll production during cultivation. article information received : 7 march 2025 revised : 14 may 2025 accepted : 5 june 2025 *corresponding author, e-mail: muliasari03@unsam.ac.id reviewers: anonymous reviewer research article effect of calcium nitrate on chlorophyllbased biotransducer characterization of arthrospira platensis gomont mulia safrida sari 1*, rachmad almi putra2, yonadiah dwitya1, m. ari fahril2 1department of biology, faculty of science and technology, universitas samudra, langsa 24416, indonesia; 2department of geophysic, faculty of science and technology, universitas samudra, langsa 24416, indonesia. abstract this study aimed to investigate the potential of calcium nitrate as a specific nutrient capable of enhancing chlorophyll content and optimizing biotransducer characterization in arthrospira platensis gomont. a two-factor completely randomized design (crd) was employed, consisting of 12 treatments with 3 replications. each experimental group was subjected to varying salinity levels: 15 ppt (s15), 25 ppt (s25), and 35 ppt (s35). in the treatment groups, calcium nitrate was applied at concentrations of 2.5 g/l (p1), 3.5 g/l (p2), and 4.5 g/l (p3). biomass accumulation and specific growth rate were monitored, and data were collected throughout the experimental period. at the conclusion of the treatment period, chlorophyll was extracted, and its concentration was measured using uv-vis spectrophotometry and ftir analysis. the addition of calcium nitrate at 4.5 g/l combined with 35 ppt salinity increased the average biomass productivity over 15 days to 5.1 g/l, with a specific growth rate in the stationary phase of 0.12 per day. supplementation with 4.5 g/l calcium nitrate in 35 ppt salinity increased total chlorophyll concentration to 70.15 μg/ml, supporting its potential as a supplementary nutrient for enhancing biotransducer properties through five key functional groups associated with the stability and binding affinity of analyte molecules in spr applications. keywords: arthrospira platensis gomont, biotransducer, calcium nitrate, chlorophyll, ftir https://doi.org/10.11598/btb.2025.32.1.2480 biotropia vol. 32 no. 2, 2025 206 introduction surface plasmon resonance (spr)-based biosensors have been widely applied in biomedical fields, particularly for the real-time detection of biological analytes. spr is an optical technique in which changes in the intensity of reflected light at a specific resonance angle on a metal-modified surface are measured. this surface functions as a receptor, with variations in the refractive index near the interface occurring as a result of analyte binding. the analytes may include biomarkers such as dna, proteins, enzymes, and other biological macromolecules. in recent years, spr biosensors have been developed as efficient, cost-effective, and userfriendly alternatives for biomarker detection (écija-arenas et al. 2021; d’agata et al. 2024). however, conventional spr systems have continued to face limitations, including suboptimal sensitivity and unstable performance, primarily due to inadequacies in the biointerface sensing layer. these limitations highlight the need for incorporating additional external supporting layers beyond the traditional gold (au) film and prism to enhance signal transduction and reduce energy loss across the optical interface (syed nor et al. 2022). consequently, improvement of the biointerface layer has been identified as a critical priority to advance the performance and reliability of spr biosensors in efforts to enhance the performance of spr biosensors, the potential of chlorophyll as a biotransducer has been identified as an attractive alternative. chlorophyll, present in large quantities in microalgae such as spirulina (arthrospira platensis gomont), possesses notable optical properties and molecular interaction capabilities, along with high tolerance and stability under varying environmental conditions, which can improve the sensitivity and specificity of detection systems (mandal & dutta 2020). when incorporated as the primary component of the biointerface sensing layer, chlorophyll is expected to enhance the performance of spr biosensors in detecting relevant parameters. moreover, the selection of calcium nitrate as an additional nutrient during the cultivation phase of arthrospira platensis gomont is considered a crucial factor in enhancing chlorophyll production for use as a biotransducer molecule. calcium nitrate has been reported to influence the growth and metabolism of a. platensis, thereby contributing to increased chlorophyll production (fakhri et al. 2020). however, the effects of nitrate have been shown to vary among species. rani et al. (2021) reported that increasing nitrate concentrations reduced pigment content in chlamydomonas and chlorella strains. in contrast, chlorella vulgaris achieved higher biomass at elevated nitrate levels, with improved uptake rates of up to 1,798 mg/l and tolerance exceeding 6,014 mg/l (jeanfils et al. 1993). nitrate supplementation has also been observed to improve microalgal growth, with biomass concentrations reaching up to 3,188 mg/l (vo et al. 2022). therefore, the addition of calcium nitrate during cultivation may serve as an effective strategy to enhance chlorophyll production, which, in turn, could improve the sensitivity of chlorophyll-based biotransducers in spr biosensors. existing research has largely focused on general microalgal growth, with limited attention given to the targeted enhancement of chlorophyllbased biotransducer properties for biosensing applications. in this study, this gap was addressed through an integrated approach combining physiological optimization under controlled environmental conditions with evaluation of biosensor applicability. using arthrospira platensis gomont as a model organism, the effects of nutrient and salinity modulation on the performance of chlorophyll-based spr biosensors for metabolic biomarker detection were investigated. materials and methods material the initial arthrospira platensis gomont inoculum was obtained from a pure culture cultivated by commercial farmers in tangerang city, jakarta, indonesia. research design a completely randomized design (crd) with two factors was employed, comprising 12 treatments with 3 replications, resulting in a total of 36 culture groups of arthrospira platensis gomont. these groups included a negative control and three treatment sets, each subjected to different salinity levels: 15 ppt (s15), 25 ppt (s25), and 35 ppt (s35). the negative control was maintained without additional nutrients, whereas the treatment groups were supplemented with calcium nitrate at effect of calcium nitrate on characterization of arthrospira platensis gomont sari et al. 207 concentrations of 2.5 g/l (p1), 3.5 g/l (p2), and 4.5 g/l (p3) under the same salinity conditions. calcium nitrate concentrations were adapted from fakhri et al. (2020) with modifications. the experiment was conducted over a 15-day period, with cultures maintained in aquariums under 4,000 lux lighting (24:0 light cycle) and continuous aeration to ensure uniform nutrient distribution and to prevent sedimentation (fakhri et al. 2020). preparation of culture media the culture medium was prepared using sterilized distilled water, treated with 1 ml/l chlorine for 24 hours, and subsequently treated with 1 ml/l sodium thiosulfate for dechlorination. this process was performed to ensure the removal of chlorine residues prior to use in microalgae cultivation (fakhri et al. 2020). inoculation, cultivation, and treatment group assignment inoculation phase during the inoculation phase, the initial arthrospira platensis gomont inoculum was expanded to obtain the required quantity for cultivation. a volume of 100 ml of the initial inoculum was added to each liter of culture medium. the inoculum was then incubated for 7 days before initiation of the cultivation phase. cultivation phase and treatment group assignment following inoculation, 100 ml of arthrospira platensis gomont culture was transferred into 2 l of sterile medium, resulting in a 1 : 20 ratio. cultures were maintained at salinity levels of 15 ppt (s15), 25 ppt (s25), and 35 ppt (s35). treatment groups were supplemented with calcium nitrate at concentrations of 2.5 g/l (p1), 3.5 g/l (p2), and 4.5 g/l (p3), while the negative control received no nutrient addition. treatments were applied for 15 days, during which biomass productivity and specific growth rates were monitored and recorded (fakhri et al. 2020). biomass productivity assessment observations were conducted over a 15-day period, from day 0 to day 15, with measurements taken every three days. biomass productivity was determined by collecting a 25 ml microalgae sample. the sample was subsequently filtered and dried in an oven at 105 °c for 2 minutes before being weighed. the results were calculated using the following formula (fakhri et al. 2020): where: b = weight of the dried sample and filter paper after being oven-dried at 105 °c for 2 minutes (g); a = weight of the filter paper after being oven-dried at 105 °c for 2 minutes (g); sample volume = volume of the sample solution taken from the cultivation medium (ml). biomass productivity assessment and specific growth rate during the stationary phase upon reaching the stationary phase, which is characterized by consistent and stable biomass growth as well as constant medium turbidity, typically occurring between days 10 to 15, biomass productivity and specific growth rate were measured using the following formula (fakhri et al. 2020): where: μ = specific growth rate per unit of time (per day); x₂ = biomass measured at the final time point (g/l); x₁ = biomass measured at the initial time point (g/l)’ t₂ = final time (days); t₁ = initial time (days). chlorophyll extraction on day 15, 250 ml of microalgae samples were collected from each group and filtered using whatman no. 1 filter paper with a pore size of 11 μm. the biomass was air-dried, weighed (~1 g), and dissolved in 10 ml of acetone. the solution was transferred to 10 ml bottles, wrapped in aluminum foil to prevent light exposure, and centrifuged at 3,000 rpm for 20 minutes. the supernatant was collected, placed in cuvettes, and its absorbance was measured at 648 nm and 664 nm using a uv-vis spectrophotometer (aryono et al. 2022). the concentration of pigments in the extracts was determined using the following equations (lichtenthaler & wellburn 1983): chlorophyll a concentration (μg/ml): ca = (13.36 × a664) (5.19 × a648) chlorophyll b concentration (μg/ml): cb = (27.43×a648) (8.12×a664) total chlorophyll (μg/ml) = ca + cb biotropia vol. 32 no. 2, 2025 208 ftir (fourier transform infrared spectroscopy) analysis ftir analysis was conducted using a shimadzu ir prestige 21/ftir 8400 instrument, which was warmed up for at least 30 minutes to stabilize performance. after powering on the detector, the ir solution software was launched for data acquisition. approximately 0.5 ml of the liquid sample, free of water or interfering solvents, was applied onto the atr crystal plate and evenly distributed across its surface. the atr assembly was then placed into the sample holder. a background spectrum was first obtained to eliminate external interference and ensure accurate sample measurement. subsequently, the spectrum of the liquid sample was recorded, capturing absorption peaks characteristic of the sample’s molecular structure. the resulting spectrum was analyzed using the ir solution software to identify key absorption bands. data analysis biomass increase, specific growth rate, and chlorophyll concentration were statistically analyzed using two-factor anova in spss 16 software, followed by duncan’s multiple range test at a significance level of 5% (p = 0.05). figure 1 biomass productivity of arthrospira platensis gomont during the 15-day study notes: a) biomass productivity was measured five times over 15 days of study; b) mean biomass productivity. results and discussion biomass productivity of arthrospira platensis gomont during 15-day of the study biomass productivity of arthrospira platensis gomont was monitored every three days over a 15-day period under various nutritional and salinity treatments (figs. 1a & 1b). at the onset (day 0), all experimental groups exhibited similarly low productivity, indicating uniform initial conditions. treatments supplemented with medium to high concentrations of calcium nitrate (p2 and p3) demonstrated a progressive increase in biomass, reaching a peak on day 15. this trend suggests that elevated nitrate availability supports cellular growth, consistent with previous findings linking nitrogen availability to increased biomass in chlorella sp. (vo et al. 2022). unexpectedly, considerable biomass accumulation was also observed in the control group (kn), which received no additional nutrients, particularly on days 12 and 15 of the cultivation period. this phenomenon may be attributed to adaptive metabolic strategies, such as efficient utilization of residual nutrients or internal nitrogen recycling mechanisms in nutrient-limited microalgal cultures (bezerra et al. 2020; villaró et al. 2023). effect of calcium nitrate on characterization of arthrospira platensis gomont sari et al. 209 biomass productivity was enhanced under higher salinity conditions (35 ppt) compared to lower salinity treatments (15 and 25 ppt), particularly during the final observation period. the combination of 35 ppt salinity and 4.5 g/l calcium nitrate (p3) yielded the highest mean biomass productivity at 5.1 g/l. these findings are consistent with previous studies demonstrating that moderate salinity stress stimulates osmoregulatory mechanisms in microalgae, promoting the synthesis of compatible solutes and enhancing ionic regulation, which collectively support increased biomass production (bezerra et al. 2020; villaró et al. 2023). furthermore, calcium ions introduced through calcium nitrate are believed to contribute to membrane stabilization and activation of signaling pathways involved in stress tolerance, thereby further improving growth under high-salinity conditions (white & broadley 2003; haider et al. 2024). despite the observed trends, two-factor anova indicated that the effects of calcium nitrate supplementation (f = 0.611; p = 0.611), salinity (f = 1.579; p = 0.215), and their interaction (f = 1.191; p = 0.324) on biomass productivity were not statistically significant at the 0.05 level (table 1). the adjusted r² value (0.016) further suggested that variability in biomass productivity was only marginally explained by the tested variables. these findings may have been influenced by intrinsic biological variation or the relatively short duration of the experimental period. it is important to emphasize that the absence of statistical significance does not necessarily negate the biological relevance of the observed trends, particularly in microalgal systems where subtle physiological responses may not be captured by conventional significance thresholds (quinn & keough 2002). although calcium nitrate supplementation and elevated salinity did not produce statistically significant differences in biomass productivity, the consistent increase observed in treatments p2 and p3, particularly under high salinity, suggests a potential synergistic effect of nitrate availability and osmotic conditioning in enhancing the growth performance of arthrospira platensis gomont. further investigations involving extended cultivation periods, increased replication, and refined physiological measurements are warranted to elucidate the underlying mechanisms and validate these observed trends. biomass productivity of arthrospira platensis gomont during the stationary phase the stationary phase typically reflects a condition in which cellular growth slows due to nutrient limitation, waste accumulation, or other environmental constraints (richmond 2004). despite these limitations, sustained or even enhanced biomass productivity was observed in arthrospira platensis gomont during the stationary phase (days 10 15), particularly under specific combinations of calcium nitrate supplementation and salinity levels (figs. 2a & 2b). table 1 two-factorial anova analysis on mean biomass productivity of arthrospira platensis gomont during the 15-day study source type iii sum of squares df mean square f sig. corrected model 79.618a 11 7.238 1.103 0.375 intercept 944.676 1 944.676 143.974 0.000 treatment 12.021 3 10.362 0.611 0.611 salinity 20.724 2 7.812 1.579 0.215 treatment*salinity 46.872 6 6.561 1.191 0.324 error 393.687 60 total 1,417.980 72 corrected total 473.304 71 note: a: r2 = .168 (adjusted r2 = .016). arthrospira platensis gomont exhibited a substantial increase in biomass productivity, reaching up to 10.8 g/l with an average productivity of 8.8 g/l, particularly in cultures treated with 4.5 g/l calcium nitrate under 35 ppt salinity. this suggests that specific physiological mechanisms supported sustained metabolic activity despite potentially growth-limiting conditions. the enhanced growth is likely attributed to the dual role of calcium nitrate: nitrate (no₃-) continues to support nitrogen biotropia vol. 32 no. 2, 2025 210 figure 2 biomass productivity of arthrospira platensis gomont during the stationary phase notes: a) biomass productivity; b) mean biomass productivity. metabolism and chlorophyll synthesis, while calcium (ca²+) contributes to stabilization of thylakoid membranes, regulation of enzymatic activity, and facilitation of photosynthetic protection mechanisms under stress (cui et al. 2024; the et al. 2021; hachiya & sakakibara, 2017; hochmal et al. 2015; white & broadley 2003; haider et al. 2024). however, this stimulatory effect was not consistent across all treatment groups. interestingly, the kn group, which received no additional nutrients, achieved comparably high productivity at 35 ppt salinity, highlighting the remarkable adaptive capacity of arthrospira platensis gomont. elevated salinity may act as a mild stressor, triggering metabolic compensation mechanisms that enable continued biomass accumulation despite nutrient limitations (bezerra et al. 2020; villaró et al. 2023). these findings underscore the interactive effects of nutrient concentration and salinity, where physiological outcomes are nonlinear and dependent on threshold levels and organismal adaptive responses. while high calcium nitrate concentrations combined with elevated salinity promoted growth, intermediate nutrient levels or suboptimal salinity conditions did not elicit the same effect. in some cases, the absence of supplementation under high salinity conditions paradoxically stimulated biomass accumulation through internal regulatory mechanisms. specific growth rate of arthrospira platensis gomont during the stationary phase the specific growth rate for each treatment group was measured after arthrospira platensis gomont entered the stationary phase, as indicated by stable turbidity between days 10 and 11. this measurement was conducted to evaluate the culture’s capacity for biomass accumulation following the onset of nutritional stress, which results from intensified intra-population competition and depletion of essential nutrients (richmond 2004). the specific growth rate results for each group are presented in figure 3. figure 3 mean specific growth rate of arthrospira platensis gomont during the stationary phase in this study, cultures maintained at 35 ppt salinity demonstrated an average specific growth rate of 0.104 per day. when calcium nitrate was effect of calcium nitrate on characterization of arthrospira platensis gomont sari et al. 211 supplemented at concentrations of 3.5 g/l and 4.5 g/l, the specific growth rate increased to 0.11 and 0.12 per day, respectively. the highest growth rate was recorded in the p3-35 group, followed by the p2-35 group. these findings suggest that under elevated salinity, appropriate nitrate supplementation may enhance nitrogen assimilation pathways, support chlorophyll synthesis, and maintain cell division even during the later stages of cultivation (cui et al. 2024; vo et al. 2022). however, this effect was not consistent across all treatment combinations. groups receiving calcium nitrate at 2.5 g/l (p1), or those cultivated at lower salinities (15 ppt and 25 ppt), exhibited lower specific growth rates. this inconsistency indicates that the physiological response of arthrospira platensis gomont to nutrient addition is modulated by salinity, and not all nutrient– salinity combinations yield synergistic effects. interestingly, the kn-35 group exhibited a growth rate comparable to those of the p2-35 and p335 groups. this suggests that arthrospira platensis gomont is capable of activating osmoregulatory mechanisms and utilizing internal nutrient reserves to maintain essential metabolic functions despite nutrient constraints, which may contribute to sustaining growth rates under high-salinity stress (bezerra et al. 2020; villaró et al. 2023). two-factor anova analysis (table 2) revealed that calcium nitrate supplementation (f = 0.184; p = 0.906), salinity (f = 1.690; p = 0.195), and their interaction (f = 0.416; p = 0.865) did not produce statistically significant differences in specific growth rate (p > 0.05). although these statistical results do not confirm significant treatment effects, observable patterns suggest that increasing salinity levels (from 15 ppt to 35 ppt) across all treatments tend to correlate with higher biomass productivity and specific growth rates, particularly in the kn and p3 groups. the p2-25 treatment exhibited a significant drop in productivity, which may be attributed to unfavorable salinitynutrient interactions under this condition. the p3-35 treatment appeared to be the most optimal condition for biomass production, showing the highest values across all metrics, productivity and growth rate, indicating that this specific nutrientsalinity combination may create physiological conditions conducive to enhanced microalgal growth and biomass accumulation. table 2 two-factorial anova analysis on specific growth rate of arthrospira platensis gomont during the stationary phase source type iii sum of squares df mean square f sig. corrected model 0.032a 11 0.003 0.585 0.832 intercept 0. 420 1 0.420 84.423 0.000 treatment 0. 003 3 0.001 0.184 0.906 salinity 0. 017 2 0.008 1.690 0.195 treatment*salinity 0. 012 6 0.002 0.416 0.865 error 0.239 48 0.005 total 0.691 60 corrected total 0.271 59 note: a: r2 = .118 (adjusted r2 = .084). the data presented in table 3 suggest that higher salinity levels (35 ppt) generally support enhanced biomass productivity and specific growth rates in arthrospira platensis gomont, particularly under the p3 treatment, which consistently outperformed other conditions. however, the interaction between salinity and treatment varied, with the p2-25 condition identified as unfavorable, indicating the necessity for optimization based on specific growth or production targets. biotropia vol. 32 no. 2, 2025 212 table 3 measurement results of mean biomass productivity, mean stationary phase biomass productivity and mean specific growth rate treatment mean biomass productivity (g/l) mean biomass productivity on stationary phase (g/l) mean specific growth rate (per day) kn15 3.4ab 5.4ab 0.04a kn25 4.3ab 6.8ab 0.09ab kn35 4.3ab 7.2ab 0.10b p1-15 2.6ab 3.9ab 0.08ab p1-25 3.8ab 5.8ab 0.10b p1-35 3.5ab 5.5ab 0.08ab p2-15 4.1ab 6.2ab 0.06ab p2-25 1.3a 1.5a 0.06ab p2-35 4.0ab 6.8ab 0.11b p3-15 4.9b 7.9b 0.08ab p3-25 2.2ab 3.2ab 0.09ab p3-35 5.1b 8.8b 0.12b notes: different superscript letters indicate statistically significant differences at p < 0.05 (duncan’s multiple range test); identical or shared letters (e.g., ab) indicate no significant differences.results of chlorophyll content measurement chlorophyll a content was consistently higher than chlorophyll b across all treatment groups (fig. 4). based on measurements of chlorophyll a and total chlorophyll concentrations, the kn-35, p3-35, p3-15, and p2-35 groups exhibited higher levels compared to other groups. these results indicate that a salinity of 35 ppt combined with calcium nitrate supplementation at concentrations of 2.5 g/l and 3.5 g/l can enhance chlorophyll content. figure 4 chlorophyll concentration in arthrospira platensis gomont when cultivated at 35 ppt salinity without nutrient supplementation, arthrospira platensis gomont exhibited elevated concentrations of chlorophyll a, chlorophyll b, and total chlorophyll, measured at 56.55, 13.57, and 70.12 μg/ml, respectively. similarly, under 35 ppt salinity with 4.5 g/l calcium nitrate supplementation, chlorophyll a, b, and total chlorophyll levels were recorded at 56.54, 13.61, and 70.15 μg/ml, respectively. moreover, in several treatment groups, calcium nitrate supplementation did not result in a significant enhancement of chlorophyll content in arthrospira platensis gomont. under 15 ppt salinity without nutrient supplementation (kn-15), chlorophyll content in arthrospira platensis gomont was relatively low, indicating that suboptimal salinity may not enhance chlorophyll production. treatments at 25 ppt salinity (kn-25, p1-25, p3-25) showed slight improvements in total chlorophyll compared to kn-15; however, these increases remained lower than those observed in treatments at 35 ppt. at 35 ppt salinity (kn-35, p2-35, p3-35), increases in chlorophyll content were observed in arthrospira platensis gomont. the highest enhancement occurred in the p3-35 treatment, which achieved a total chlorophyll concentration of 70.15 μg/ml. these results suggested that this nutrient-salinity combination provides optimal conditions for biomass growth and chlorophyll accumulation in arthrospira platensis gomont. the results indicated that nutrient supplementation combined with appropriate salinity levels plays a significant role in enhancing chlorophyll content in arthrospira platensis gomont. nitrate, absorbed through nrt1 and nrt2 transporters, supports amino acid and effect of calcium nitrate on characterization of arthrospira platensis gomont sari et al. 213 nucleotide biosynthesis essential for cell growth and biomass accumulation. moreover, nitrate serves as a nitrogen donor for porphyrin ring formation, the core structure of chlorophyll molecules (cui et al. 2024), and modulates gene expression under saline stress (the et al. 2021; hachiya & sakakibara 2017). in calcium nitrate, both nitrate and calcium components contribute to chlorophyll biosynthesis, with calcium stabilizing chloroplast membranes and supporting photosynthetic activity through multiple physiological roles. as a component of the oxygen-evolving complex (mn₄cao₅) in photosystem ii (psii), calcium facilitates water splitting and oxygen evolution, while also regulating calvin cycle enzymes such as fructose1,6-bisphosphatase (fbpase) and sedoheptulose1,7-bisphosphatase (sbpase). under salinity stress, calcium maintains thylakoid membrane integrity and activates protective mechanisms such as cyclic electron flow (cef) and non-photochemical quenching (npq), thereby preserving pigment biosynthesis (hochmal et al. 2015). these multifaceted roles likely explain the elevated chlorophyll content observed in high-calcium nitrate treatments (notably p3-35), underscoring its potential to enhance photosynthetic efficiency and pigment accumulation under environmental stress analysis of functional groups in chlorophyll of arthrospira platensis gomont ftir analysis revealed that key functional groups essential for biosensor applications were present in chlorophyll extracted from arthrospira platensis gomont cultivated with 4.5 g/l calcium nitrate at 35 ppt salinity (fig. 5). a strong absorption at 3,389/cm indicated the presence of hydroxyl (–oh) groups, which are known to contribute to hydrogen bonding and enhance molecular interaction stability (nandiyanto et al. 2023). the carbonyl (c=o) group was detected at 1,704/cm, which is typically found in ester or ketone structures of chlorophyll and is recognized for its role in increasing binding affinity through electrostatic interactions with target molecules (mansour et al. 2022). figure 5 ftir analysis of chlorophyll from p3-35 group an absorption band at 1,371/cm was identified as alkane (ch) groups that participate in hydrophobic interactions and van der waals forces, though these interactions are weaker than hydrogen bonds (wu & prausnitz 2008). an absorption at 1,222/cm was attributed to c–o bonds, characteristic of esters or carboxylic acids, which have been found to enhance chlorophyll’s ability to interact with target molecules through polar interactions (chemistry libretexts 2024). meanwhile, the band at 1,021/cm was associated with c–n bonds, which are commonly found in amine or amide groups and the porphyrin structure of chlorophyll. these groups are known to enable electrostatic interactions with charged molecules, thereby strengthening the binding affinity between chlorophyll and target analytes in sensor applications (mansour et al. 2022). overall, these ftir results indicate that stable chemical interactions with target molecules can be formed by chlorophyll due to the presence of hydroxyl, carbonyl, alkane, c–o, and c–n groups. the role of these functional groups in biosensing applications has been supported by their contribution to enhancing molecular binding affinity. furthermore, a positive enhancement in chlorophyll production under optimal conditions reinforcing the potential of chlorophyll as a biotransducer for molecular detection (mansour et al. 2022). this study highlights the potential of optimized nutrient conditions to enhance the biotransducer properties of arthrospira platensis gomont. although some treatments did not yield statistically significant results, the increased presence of specific functional groups, as revealed by ftir analysis, supports their role in strengthening binding affinity in spr biosensors. cultivation under appropriate calcium nitrate concentrations and salinity levels was shown to improve chlorophyll content and promote the expression of functional groups favorable for analyte interaction. these biotropia vol. 32 no. 2, 2025 214 findings underscore the potential of arthrospira platensis gomont as an effective biotransducer for spr-based detection systems. future research should investigate the scalability and real-world applicability of this approach in diagnostic settings. the ftir-detected functional groups further validate the chemical complexity of chlorophyll and its suitability as a molecular recognition element, particularly due to the presence of groups capable of polar and electrostatic interactions. conclusion the addition of calcium nitrate was found to positively impact the increase in biomass productivity of arthrospira platensis gomont, correlating with a rise in total chlorophyll concentration. calcium nitrate at a concentration of 4.5 g/l under 35 ppt salinity (p3-35) demonstrated greater potential in enhancing biomass production compared to lower calcium nitrate concentrations or salinity levels. the presence of hydroxyl, carbonyl, alkane, c–o, and c–n functional groups confirmed that chlorophyll extracted from the optimized cultivation condition (p3-35) possesses enhanced stability and interactive properties as a biotransducer. these findings can inform the formulation of biointerface layers in spr biosensors. future studies are recommended to validate these outcomes in real-world sensor platforms to confirm binding efficiency and diagnostic relevance. 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cibinong science center, 16911, indonesia 2 (lipi) aresearch center for biotechnology, indonesian institute of sciences , cibinong science center, bogor 16911, indonesi received 30 december 2016 / accepted 06 october 2017 abstract valid identification of microorganisms is a vital information to establish culture collections. currently, molecular approach based on 16s rdna is widely used for bacterial identification. this approach is however, time consuming and expensive. matrix assisted laser desorption ionization time of flight mass spectrometry (maldi-tof ms) allows the identification of microorganisms directly from colonies and it only takes some few minutes. the interest of this study was to identify endophytic bacteria associated with curcuma zedoaria based on protein profile using maldi-tof ms system and compare with 16s rdna sequence results. endophytic bacteria were isolated from part of medicinal plant c. zedoaria collected from bogor, west java indonesia. the identification of selected bacteria was performed by protein profile using maldi-tof ms. a total of 66 endophytic bacteria from c. zedoaria plant, were selected for identification. the result of maldi-tof ms analysis showed that eleven isolates (16.67%) were correctly identified to the species level and 23 isolates (34.85%) matched on genus level of molecular approach. these results demonstrates that the maldi-tof system is suitable and feasible approach for the bacterial identification, mainly for screening and grouping of large number isolates. keywords: curcuma zedoaria, endophytic bacteria, identification, maldi-tof ms introduction rapid and accurate identification of bacterial isolates has crucial role in the culture collections. several methods of identification have been deve loped. bacter ia l ident i f ica t ion i s predominant l y based on phenotyp ic, morphological (gram-positive and negative) and biochemical properties testing analysis. however, classification and identification by these methods can be difficult because of variations in phenotypic characteristics, time-consuming and laborius (seng et al. 2009; rychert et al. 2013). several years ago, molecular identification based on nucleotide sequencing of ribosomal dna (rdna) sequence analysis could replace the phenotypic, morphological and biochemical cha r a c t e r i s t i c s fo r i d en t i f i c a t i on o f microorganisms to genus level (rajendhran & gunasekaran 2011). molecular approach based on rdna have widely been used for sequence identification of procaryotic taxa. the 16s rdna sequences has confirmed the representativeness of the in bacterial phylogeny (woo et al. sequence 2008). however this method is not suitable for a preliminary screening and grouping of large numbers of isolates in environmental and clinical microbiology laboratories. analysis based on protein profile present in bacterial cells using matrix assisted laser desorption ionization time of flight mass spectrometry (maldi-tof ms) is becoming feasible and precise for bacterial identification (bizzini et al. 2011; santos et al. 2013). malditof ms allows rapid identification of bacteria, less expensive on operational cost, accurate and low sample volume requirements compared to molecular approach (wunschel et al. 2005; singhal et al. 2015). it can be performed as soon as colonies are isolated and can be done just in a few minutes (eigner et al. 2009; seng et al. 2009; neville et al. 2011; rychert et al. 2013; jamal et al. 2014).* corresponding author: trilisty01@gmail.com biotropia 5 2 8 112 120 vol. 2 no. , 201 : doi: 10.11598/btb.2018.25.2.795 112 maldi-tof ms analysis was done by comparing the maldi-tof ms spectra or protein profile “fingerprints” obtained from bacterial cells to a library from proteomic database (martiny et al. 2012; guo et al. 2014). protein profile are significantly different for each bacteria and it contain certain unique mass marker even over the small mass range detected. each bacteria shows signature characteristic peaks that are distinct for species level (cain et al. 1994). the identification of microorganisms by malditof ms is based on the detection of mass signals from biomarkers that are specific at genus, species or sub-group level (ferroni et al. 2010; benagli et al. 2012), therefore changes in protein profiles would be easily identified. the protein profile from each bacteria can serve as unique information for bacteria chemotaxonomic marker, for example to differentiate closely related species, also could enriche the information contained in procaryotic protein sequence databases. kudirkiene et al. (2015) reported that maldi-tof ms was succesfully used in sub-species identification of streptococcus equi. using maldi-tof ms, routine bacterial identification can be obtained much earlier than identification by molecular and conventional methods. therefore, this technique can be used as quality control management of cultures deposited in culture collections. the main objective of the study was to evaluate the identification of endophytic bacteria associated with c. zedoaria based on protein profile using maldi-tof ms system compared with the results based on 16s rdna sequence from previous study (sulistiyani et al. 2014). materials and methods bacterial strains a total of 66 endophytic bacteria isolates from biosystematic and culture collections laboratory-research center for biology, lipi, was used to evaluate the maldi-tof ms analysis were recovered from previous study (sulistiyani et al. 2014). the isolates were opreserved in 10% glycerol stock at -80 c. the bacteria were grown using nutrient agar (na) media. the bacteria isolates were from c. zedoaria (white turmeric) planted in 3 areas in bogor, indonesia, in 2013. the plants materials collected from private garden in bojong gede (bg), experiment garden of research center for biology, indonesian institute of sciences, cibinong (cbn) and garden of medicinal plants collection of biopharmaca research center, bogor agricultural university, dramaga (drmg). all bacteria were previously identified by molecular identification based on 16s r na d sequence. the bacteria represented 23 genera and 46 species (sulistiyani et al. 2014). preparation of acterial ell xtractsb c e bacterial isolates were cultured from glycerol stock on nutrient agar (na) and incubated under standard conditions at 35-37 c for 48 o hours and subcultured twice to ensure all isolates were in the same physiological state. cell extract was prepared by picking a single colony of a fresh culture and suspended in 20 µl formic acid of 25% concentration and then mixed until complete suspension is formed by vortex. the extract was sonicated for 10 min and spinned to get the supernatant. finally, the supernatant was used for maldi-tof analysis. maldi-tof analysis the bacterial cell extract of 0.5 µl was placed in duplicates onto a steel target plate and allowed to dry at room temperature. each sample was overlaid with 1 µl matrix of α-cyano-4hydroxycinnamic acid (chca) in ethanol : acetonitrile : water : trifluoroacetic acid. the sample and matrix were mixed thoroughly and air dried at room temperature. measurements were taken using a maldi-tof ms axima system (microbiology laboratory of the mulhouse hospital, france). the laser frequency was 50 hz, the acceleration voltage was 20 kv, and the extraction delay time was 200 ns. each spectrum resulted from 5 laser shots at 100 random positions within measuring spot. the spectra were recorded in the linear positive mode within a mass range of 2 to 20 kda. all mass fingerprints were analyzed by the saramis software, which first compares them to the superspectra and in a second step to the individual spectra of the database using anagnostec version 4.07 (axima system manual). 113 maldi-tof mass spectrometry for identification of endophyitic bacteria – sulistiyani and lisdiyanti such as morphologic, phenotypic, physiologic and molecular methods. however, as mentioned earlier, these methods are not suitable for a preliminary screening and grouping of large numbers of isolates from environmental and clinical samples and also, for controlling the identity and purity of microbial cultures preserved in culture collections. to overcome these problems, the present study evaluated the capability of maldi-tof ms for species identification of microorganisms such as bacteria isolates. validity of maldi-tof ms results was compared with the results of molecular identification based on 16s rdna sequences as a calibration and validation analysis the instrument was calibrated and validated using a control strain of escherichia coli k12 inacc b5. several strain were also used for quality control including of bacillus substilis inacc b1, pseudomonas aeruginosa inacc b3 and staphylococcus aureus inacc b4. results and discussion accurate identification of bacteria could be obtained from several identification methods, figure 1 spectral profiles of three isolates k. pneumoniae obtained from different part of white turmeric plant. these mass signals spectra (3,853; 4,365; 6,292; 7,245; 7,384; 7,705; 9,140; and 9,479 m/z) are specific to k. pneumoniae. biotropia vol. 25 no. 2, 2018 114 reference method. the results of maldi-tof ms analysis are indicated in percentages of similarity compared to spectra profiles of known strain in saramis data ase systemb . superspectra contain common peaks to different strains of the same species. iate individual spectra correspond to the spectra of ed each strain cultivated under specific conditions. the manufacturer recommends validation of superspectra identifications with the confidence level between 85.00 and 99.9%. accuracy of the identification strongly relies upon the robustness of the database and the choice of reference isolates. this is especially important when the genera involving species of environmental and clinical a high genetic diversity samples represents (benagli 2012)et al. . the spectra were analysed in a mass range of 2 to 20 kda and this mass range representing ribosomal proteins were obtained from bacterial extract. these proteins are numerous in the cell and are positively charged. fig. 1 shows the representative results of spectral profiles from klebsiella pneumonia based on the mass signals obtained. isolated bacteria from different part of plant (rhizome, stem, leaves) and locations (darmaga and bojong) showed similar identification due to their identical protein profiles which were indicated by mass spectra value. three isolates of k. pneumoniae isolated from rhizome, stem and leaves of c. zedoaria plant showed almost the same spectral profiles. it had several specific mass signals 3,853; 4,365; 6,292; 7,245; 7,384; 7,705; 9,140; and 9,479 m/z. each mass signal is specific at species and family level. mass signal of 3,853; 6,292; 7,384; 7,705; and 9,479 m/z were specific for species level, while 4,365; 7,245; 9,140 m/z were specific for family level. according to these results, it is clear that each species of bacteria has specific mass spectra, and can be used as a taxonomic marker identification. compared with the data presented in table 1, these mass signals correspond to the superspectrum database of k. pneumoniae. the identification results obtained by maldi-tof ms and 16s rrna gene sequencing shown in table 2. of 66 isolates are forty-three (43) of the 66 isolates (65.15%) showed the intepretable maldi-tof ms spectra by sequence comparison analysis, . maldi-tof ms spectra allowed good identification to the species, genus and family level total of 43 endophytic bacteria with a covering 16 genus and 21 species. an additional 11 isolates (16 67%) with the . gave similar results sequencing results, correctly identified to the and mass (m/z) identification level 3853.5 species level 4155.3 species level 4365.1 family level 5381.6 family level 6292.8 species level 6384.1 family level 6857.4 family level 7165.8 family level 7245.2 family level 7272.7 family level 7319.6 family level 7384.9 species level 7705.3 species level 7735.2 species level 8310.1 family level 8852.1 family level 9093.2 species level 9140.1 family level 9479.5 species level 9852.3 species level table 1 superspectrum database of k. pneumoniae 115 maldi-tof mass spectrometry for identification of endophyitic bacteria – sulistiyani and lisdiyanti level of id and isolates 16s rdna sequencing maldi-tof ms species id level of id species id reference database correctly identified into species level di.p.3 alcaligenes faecalis species alcaligenes faecalis a rb.p.1 bacillus subtilis species bacillus subtilis a rl.s.2 enterobacter aerogenes species enterobacter aerogenes a ri.p.2 enterobacter cloacae species enterobacter cloacae a bb.p.3 klebsiella pneumoniae species klebsiella pneumoniae a ri.s.2 klebsiella pneumoniae species klebsiella pneumoniae a ri.s.9 klebsiella pneumoniae species klebsiella pneumoniae a ri.p.3 pantoea dispersa species pantoea dispersa a db.s.1 pseudomonas stutzeri species pseudomonas stutzeri a rb.p.4 pseudomonas stutzeri species pseudomonas stutzeri a ri.p.7 stenotrophomonas maltophilia species stenotrophomonas maltophilia a correctly identified into genus level ri.s.3 acinetobacter calcoaceticus genus acinetobacter baumannii a dl.p.5 bacillus safensis genus bacillus pumilus na ri.p.5 bacillus thuringiensis genus bacillus cereus/ mycoides/thuringiensis a ri.p.1 burkholderia cenocepacia genus burkholderia sp. a di.p.1 enterobacter cancerogenus genus enterobacter sp. a dl.p.4 enterobacter cancerogenus genus enterobacter cloacae a ri.p.8 enterobacter ludwigii genus enterobacter sp. na bi.p.3 enterobacter ludwigii genus enterobacter sp. na db.p.1 klebsiella pneumoniae genus enterobacteriaceae a rb.p.2 klebsiella pneumoniae genus enterobacteriaceae a di.p.4 klebsiella variicola genus klebsiella pneumoniae na ri.s.8 klebsiella variicola genus klebsiella pneumoniae na rb.s.3 klebsiella variicola genus enterobacteriaceae na bi.s.2 klebsiella variicola genus klebsiella pneumoniae na ri.p.4 microbacterium trichothecenolyticum genus microbacterium arborescens na db.s.4 micrococcus yunnanensis genus micrococcus luteus na ri.s.7 pantoea agglomerans genus pantoea dispersa a ri.s.6 pseudomonas azotoformans genus pseudomonas fluorescens na rb.p.3 pseudomonas denitrificans genus pseudomonasnitroreducens na dl.p.3 pseudomonas denitrificans genus pseudomonas nitroreducens na db.s.3 pseudomonas gessardii genus pseudomonas fluorescens na di.s.1 pseudomonas korensis genus pseudomonas aeruginosa na di.s.6 pseudomonas korensis genus pseudomonas sp. na not precisely identified bb.s.8 bacillus subtilis lysinibacillus sphaericus a bi.s.3 citrobacter freundii enterobacter sp. a di.s.4 microbacterium laevaniformans klebsiella pneumoniae a bi.s.6 microbacterium laevaniformans arthrobacter russicus a bi.p.1 microbacterium resistens pseudomonas aeruginosa a di.s.7 microbacterium testaceum oligella urethralis a bb.s.3 microbacterium trichothecenolyticum gordonia alkanivorans na ri.p.6 pseudomonas geniculata stenotrophomonas maltophilia na bi.s.1 ralstonia mannitolilytica rhizobium radiobacter a not identified yet db.p.2 agrobacterium larrymoorei no id na dl.p.6 bacillus subtilis no id a bl.p.2 bacillus subtilis no id a rl.s.1 bacillus safensis no id na rb.s.5 bosea thiooxidans no id na rb.s.2 enterobacter ludwigii no id na bb.p.4 erwinia chrysanthemi no id na dl.s.1 methylobacterium organophilum no id na bb.s.5 microbacterium hominis no id na table 2 identification results for the 66 isolates obtained by maldi-tof ms in comparison to those obtained by 16s rdna 116 biotropia vol. 25 no. 2, 2018 species level; 23 isolates (34 85%) matched genus . level of molecular approach, and among them 3 isolates were identified to the family level. for three isolates (db.p.1, rb.p.2, rb.s.3), malditof ms correctly identified to the family level, whereas 16s r na sequencing gave a species d identification. ine isolates (13 64%) were not n . accurately identified and 23 isolates (34 85%) were . rated as n3 .no identifiable (table ) this study has proved that maldi-tof ms is useful for identification of microorganisms into species level in a relatively short time. of the 66 isolates analyzed, 43 isolates (65.15%) were identified and 23 isolates (34.85%) were not identified (table 3). eleven isolates (16.67%) of 43 isolates showed good result, concordant with the 16s rdna sequencing result. however, twenty-three isolates (34.85%) could not be identified. these included 12 isolates of microbacterium genus, mycobacterium cosmeticum, mycobacterium simiae, agrobacterium larrymoorei, erwinia chrysanthemi, xanthobacter flavus, enterobacter ludwigii, bosea thiooxidans, stenotrophomonas maltophilia, methylobacterium organophilum, providencia vermicola, phenylobacterium koreense, bacillus safensis, roseomonas mucosa, rhizobium tarimense. among them, 14 isolates could not be identified due to the absence of reference spectra database. non-identifiable isolates for maldi-tof was as a result of an incomplete database, and can be resolved with the addition of appropriate reference. supplementation of the maldi-tof database, can reduce the rate of non-identifiable results. among 66 isolates of endophytic bacteria, 30 isolates had no reference spectra database (table 3). currently, a total of 1,309 references spectra are contained in saramis database system. this is inadequate to identify indigenous microbes that have been abundant in indonesia. maldi-tof ms library can be enriched with protein profile of strain or species of indigenous microbes from indonesia, and will be important for future studies. maldi-tof ms generates protein mass spectra which can be used for grouping and identification of bacteria. these mass spectra contain mainly peaks corresponding to ribosomal protein that are in abundance in the bacterial cell (rhyzhov & fenselau 2001). protein profile of mass spectra will help in characterizing the level of id and isolates 16s rdna sequencing maldi-tof ms species id level of id species id reference database correctly identified into species level table 2 continued rl.p.4 phenylobacterium koreense no id na rl.p.1 providencia vermicola no id na di.s.5 rhizobium tarimense no id na rl.s.3 roseomonas mucosa no id na bl.p.1 stenotrophomonas maltophila no id a dl.p.2 stenotrophomonas maltophila no id a bb.s.7 xanthobacter flavus no id na db.s.2 microbacterium laevaniformans no id a bi.p.6 microbacterium laevaniformans no id a di.s.2 microbacterium resistens no id a di.s.3 microbacterium testacneum no id a dl.p.1 microbacterium testaceum no id a bl.s.2 mycobacterium cosmeticum no id na bb.s.6 mycobacterium simiae no id a note: a: available; na: not available table 3 distribution of the discrepancies observed in the maldi-tof ms axima-saramis level of identification by maldi-tof ms no. (%) of reference database available not available total correctly identified into species level 11 (16.67) 0 11 (16.67) correctly identified into genus level 8 (12.12) 15 (22.73) 23 (34.85) not precisely identified 7(10.60) 2 (3.03) 9 (13.64) not identified yet 10 (15.15) 13 (19.70) 23 (34.85) total 36(54.54) 30(45.46) 66 117 maldi-tof mass spectrometry for identification of endophyitic bacteria – sulistiyani and lisdiyanti incorrectly identified microbes by comparing its protein profile spectra to those in the reference spectra database. these spectra can generate patterns that provide unbiased identification of particular species and even genotypes within species. data presented in table 3, shows that 9 isolates were incorrectly identified. as stated earlier, each species of bacteria has specific protein profile mass spectra, therefore the identification result from maldi-tof ms should be same to the identification result of 16s rdna. the result will be incorrect when experimental factors occur, such as sample contamination and/or sample preparation. maldi-tof ms analysis is affected by several experimental factors, such as matrix p re para t ion , spec t r a l r e produc ib i l i t y, contaminants, sample preparation, mass range and measurement accuracy on the database search (demirev et al. 1999). a total of 12 isolates of microbacterium genus were incorectly identified, might be due to the complex structure of their cell walls. specific sample extraction procedures to breakdown the cell wall are required before maldi-tof ms analysis. the process of sample preparation for identification of microbes depends upon the source of isolated microbe, or on chemical structure of the constituents of its cell wall. different group of microbes has different sample preparation (singhal et al. 2015). alatoom et al. (2011) reported that sample extraction was needed for identification of grampositive bacteria. furthermore, the lower score values, may be caused by the incomplete separation of protein. the protein interfered with the sample and disrupt the mass spectrum (reich et al. 2013). gram-positive bacteria like mycobacterium sp., also require specific extraction procedure. the extraction was done by lysed cells in boiling water, followed by ethanol precipitation of proteins. the precipitated proteins were dried, resuspended in 70% formic acid and acetonitrile, and analyzed by maldi-tof ms (verroken et al. 2010). to investigate the reproducibility of the instrument during the study we included inacc , strains in every analysis of identifications as positive controls and reference isolates. the instruments correctly identified the control strains. the incorrect identification by malditof ms was to sample preparation attributed failure such as sample volume and the large also, amount of matrix was not sufficient. consequently the sample and matrix were not completely mix and only a few small crystal ed were obtained. a sufficient number of bacterial cells (typically ~10 cells per well) are required to 4 generate detectable maldi-tof ms ion signals (chiu 2014). lohman et al. (2013) reported that malditof system succesfully identified 312 isolates. furthermore 2,860 of 2,900 (99%) samples identified by maldi-tof ms matched with the identification results obtained using other methods (standard and high end microbiological identification methods including automated b iochemica l ana l y se s and mo lecu l a r identification) (reich et al. 2013). study of guo et al. (2014) informed that using maldi-tof ms for 1,025 isolates, 1,021 (99.60%) isolates were accurately identified at the genus level, and 957 (93.37%) isolates at the species level. theel et al. (2012) reported that from 90 yeast and 78 corynebacterium species isolates, were obtained 95.6% and 81.1% of yeast, also 96.1% and 92.3% of corynebacterium isolates were correctly identified to the genus and species levels, respectively. as compared to other studies, the result of this study showed small percentage of correctly identified to the genus and species levels, however this method provide reliable results. therefore maldi-tof ms could be used for screening and grouping of large numbers of bacter i a l i so la tes. microorg an ism identification by mass spectrometry is already considered as a revolution of bacteriology, offering many advantages compared with the conventional biochemical identification of microorganisms. within the next few years maldi-tof-ms based identification of microorganisms will replace conventional methods. conclusion among the 66 selected isolates, 43 isolates were identified. eleven isolates (16.67%) that were tested matched on species level of molecular approach. spectral analysis of microbial diversity from indonesia is one way to build up the maldi-tof ms library. rotein profile spectra p of each species be used as ataxonomic could marker. maldi-tof ms systems for bacterial 118 biotropia vol. 25 no. 2, 2018 identification was good for grouping large number of isolates. however molecular analysis also must be done as a standard reference. combination of molecular analysis and protein profile using maldi-tof ms slightly accelerated the bacterial identification. acknowledgements this project was fully supported by dipa tematik 2013, research center for biology lipi and jst-jica. special thanks to sulistiani and all members of the biosystematic laboratory, research center for biology, lipi. references alatoom aa, cunningham sa, ihde sm, mandrekar j, patel r. 2011. comparison of direct colony method versus extraction method for identification of grampositive cocci by use of bruker biotyper matrixassisted laser desorption 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(11) .species j clin microbiol 48 :4015-21 woo pcy, lau skp, teng jll, tse h, yuen ky 2008 then . . and now: se of 16s rdna gene sequencing for u bacterial identification and discovery of novel bacteria in clinical microbiology laboratories clin . microbiol infect 14 :908-34(10) . wunschel sc, jarman kh, petersen ce, valentine nb, wahl kl, schauki d, white ve 2005 bacterial … . . analysis by maldi-tof mass spectrometry: an inter-laboratory comparison. j am soc mass spectrom 16(4):456-62. doi: org/10.1016/j.jasms. 2004.12.004 120 biotropia vol. 25 no. 2, 2018 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 123 ecological effects of belik river hydraulic characteristics on periphyton community as water quality bioindicator heni wahyu sartika1*, suwarno hadisusanto2, and muhammad sulaiman3* 1graduate school of environmental science, universitas gadjah mada, sleman, diy 55281. 2faculty of biology, universitas gadjah mada, sleman, diy 55281. 3vocational college of civil engineering, universitas gadjah mada, sleman, diy 55281. article higlights • differences in river hydraulic characteristics affect ecological river health • the influence of flooding on the dynamics of physical and chemical characteristics of river water • life strategy, adaptation of periphyton to environmental changes • development of green infrastructure for river conservation article information received 27 august 2021 revised 7 march 2023 accepted 8 march 2024 *corresponding author, e-mail: heni.wahyu.s@mail.ugm.ac.id research article abstract belik river is one of urban rivers in the special region of yogyakarta, which has hydrological problems both in quantity and quality. these problems triggered the construction of various types of channel modifications. this study aimed to identify water quality and the ecological effects of channel modifications based on the presence of periphyton communities as water quality bioindicators. sampling was carried out in january 2021 and march 2021. samples were taken from 4 sampling sites having different channel characteristics with 3 repetitions at each location. the physicochemical parameters measured included ph, detergent, nitrate, sulfate, total phosphate, tss, tds, do, co2, water temperature, depth, transparency, discharge, current velocity, and yearly rainfall data. ninety-one (91) periphyton species dominated by nitzschia improvisa, nitzschia terricola, and nitzschia philippinarum were found in january 2021, higher than the 61 species of periphyton dominated by nitzschia improvisa found in march 2021. results of the canonical correlation analysis (cca) indicated that the distribution and abundance of periphyton in belik river were influenced by co2, total phosphate, discharge, do, nitrate, tss, and depth. the shannon-wiener diversity index showed that water quality in january 2021 decreased compared to march 2021. results of the cluster analysis showed differences in water quality and abundance of periphyton found in the four types of river channels. it can be concluded that differences in hydraulic characteristics affected water quality and the ecological health of belik river. keywords: canonical correlation analysis, cluster analysis, diversity index, nitzschia, pennate diatom introduction land use changes in the belik watershed area were influenced by the increase in population growth (utami & suprayogi 2014). these changes include the increase in asphalt/cement buildings (7.11%) and the reduction of vegetated land (3.64%) (mubarok & suprayogi 2018). environmental deterioration may arise from development practices that do not prioritize ecological sustainability. surface runoff in the rainy season can occur because of development activities, so that rainwater cannot infiltrate the soil (utami & suprayogi 2014). human activities also cause groundwater quality to decline around the belik river (suprayogi et al. 2019). in addition, the waste input in the upstream part of kali belik cannot be accommodated due to surface water pollution (suprayogi et al. 2019; tofani & hadi 2020). wisdom park (taman kearifan), through which the belik river flows, is one of the developed areas designed to deal with hydrological problems. the modification of the river channel in wisdom park accommodate rainwater to reduce flooding (lumuan et al. 2017). channel modification is done by changing the hydraulic characteristics the southeast asian journal of tropical biology vol. 31 no. 2, 2024: 123 133 doi: 10.11598/btb.2024.31.2.1644 issn: 0215-6334 | e-issn: 1907-770x https://doi.org/10.11598/btb.2024.31.2.1644 biotropia vol. 31 no. 2, 2024 124 of the water. the changes include rivers that are straightened, deepened, and widened (franklin et al. 2009; harisnor & amalia 2016). many factors, including alteration in hydraulic characteristics, can lead to changes in the ecology of an aquatic ecosystem. in addition, the linkage between river channel and flood plain can be disturbed by the construction of embankments along the river, causing harm to biodiversity (franklin et al. 2009; lennox & rasmussen 2016). water organisms respond to changes in abiotic conditions of an aquatic ecosystem (amoatey & baawain 2019). changes to the distribution and abundance of organisms in an ecosystem can also indicate water quality (manickavasagam et al. 2019). periphyton is one of aquatic organisms that can be used as a water quality bioindicator (larned 2010; rashid et al. 2013; rier & stevenson 2006). the combination of physical, chemical, and biological parameters provide comprehensive water quality analysis. therefore, this research examined the effects of river channel modification on ecological conditions of periphytic community as water quality bioindicator. materials and methods study area the research was conducted in the upstream region of the belik river in sleman regency, yogyakarta special region. this study had 4 sampling sites in belik river (br1, br2, br3, br4). samples were collected in january 2021 (before the flood) and march 2021 (after the flood). the description of the research location is shown in table 1 and figure 1. figure 1 map of sampling sites at belik river in sleman regency, special region of yogyakarta table 1 sampling site and their characteristics sampling site code gps (lat-long) altitude (masl) hydraulic characteristics belik river br1 7°46’03.5” s 110°22’56.3” e 1,135.38 natural bottom, straightened furrow, and stone masonry shorelines br2 7°46’08.9” s 110°22’57.7” e 1,152.75 modified bottom, straightened furrow, and stone masonry shorelines br3 7°46’14.2” s 110°22’53.0” e 1,135.69 modified bottom, straightened furrow, and gabions shorelines br4 7°46’27.9” s 110°22’52.1” e 1,125.32 natural bottom, furrow, and shorelines periphyton collections periphyton sample was collected by implementing scraping method using a soft brush based on the periphyton protocols literature (us environmental protection agency 2012, 2012). at each sampling point, samples were taken from the stone masonry wall and the rocks in the river with a size of 5 cm x 5 cm. the west side of the river wall was used to take periphyton samples before the flood, while the east side wall was used to sample the periphyton after the flood. the periphyton was separated from the surface of the river wall with a soft brush, sprayed with distilled water, and collected on a tray. ecological effects of belik river hydraulic characteristics on periphyton community – sartika et al. 125 the water sample on the tray was put into a 10 ml vial bottle. after that, fixation was carried out by giving 3 drops of 4% formaldehyde in each vial bottle. vial bottles were then wrapped in plastic, tied with rubber bands, and labeled. for each research station, periphyton samples were collected with 3 replications (the right side of the river, the middle side of the river, and the left side of the river) in order to increase the level of data accuracy and represent the heterogeneous condition of the waters in the river flow on each side. water volume taken for each periphyton sampling was 10 ml. therefore, with 3 replications, the total water sample volume at each sampling point was 30 ml. periphyton samples that had been preserved were inserted into the sedgewick rafter counting chamber (srcc) and observed under a microscope with a magnification of 10 x 10. the periphyton samples that had been obtained were then identified based on “the freshwater algae of the united states” (smith 1950), “plankton of south vietnam” (shirota 1966), and “how to know freshwater algae” (prescott 1981) literatures. water quality collections water samples for water physicochemical parameters measurement were taken based on the guidelines of apha literature (1998). physical and chemical parameters measured directly in the field include do, co2, ph, water temperature, depth, current velocity, discharge, and transparency. the floating method measured current velocity, and the discharge was calculated using the slope area method. meanwhile, water samples for parameter measurement, including tds, tss, total phosphate, nitrate, sulfate, and detergent, were collected and taken to the laboratory for analysis. data analysis the calculation of periphyton on the srcc was carried out using the total strip counting method. the periphyton identification results were counted using a hand counter and analyzed in microsoft office excel 2007 to determine the density of the species (d). furthermore, the shannon-wiener diversity index was calculated using a formula as follows (kent & paddy 1992): where: h’ = index of species diversity ni = number of individuals of an i species n = number of individuals of all species pi = proportion of the number of individuals of the i species to the number of individuals of all species. the data were analyzed using canonical correlation analysis (cca) with paleontological statistic (past) software version 3 to determine the relationship between periphyton and physicochemical parameters (venkateswarlu et al. 2020). then, cluster analysis was carried out to classify and determine differences in the characteristics of the sampling sites (hossain et al. 2013). results and discussion periphyton species found in belik river were classified into 96 species and grouped into 6 functional groups, namely filamentous algae, pennate diatoms, unicellular algae, colony algae, centric diatoms, and dinoflagellates. the species composition in january 2021 differed from the periphyton composition in march 2021 (table 2). several periphyton species were only found in january 2021, such as mougeotia sp., spyrogyra sp., melosira granulata, malleochloris sessilis, spondylosium planum, desmidium baileyi, sphaeroplea annulina, nitzschia sp., nitzschia acicularis, nitzschia vermicularis, navicirula sp., navicirella elegans, pinnularia giba, tabellaria fenestrata, diatoma vulgaris, cocconeis sp., anomoeneis sp., frustulia rhomboides, gyrosigma attenuatum, pleurosigma sp., straurastrum notula, cosmarium reniforme, pleurotaenium sp., chroococcus turgiderius sp., palmella miniata, protococcus viridis, peridinium gatunense, gonyaulax sp., prorocentrum sp., and dinobryon sp. in contrast, several species of spirulina major, fragilaria crotonensis, epithemia turgida, stauroneis sp., and cosmarium turpinii were only found in march 2021. biotropia vol. 31 no. 2, 2024 126 table 2 list of belik river periphyton species found in january 2021 and march 2021 no. no. periphyton species presence january march filamentous algae 1 1 lyngbya birgei + + 2 2 oscillatoria sp. + + 3 3 mougeotia viridis + + 4 4 mougeotia sp. + 5 5 spyrogyra sp. + 6 6 spirulina major + 7 7 hyalotheca dissiliens + + 8 8 hyalotheca mucosa + + 9 9 hyalotheca sp. + + 10 10 melosira varians + + 11 11 melosira islandica + + 12 12 melosira granulata + 13 13 plectonema tomasiniana + + 14 14 zygnemopsis sp. + + 15 15 schizogonium murale + + 16 16 malleochloris sessilis + 17 17 spondylosium planum + 18 18 desmidium baileyi + 19 19 sphaeroplea annulina + pennate diatom 20 1 nitzschia improvisa + + 21 2 nitzschia acicularis + 22 3 nitzschia terricola + + 23 4 nitzschia filiformis + + 24 5 nitzschia sp. + 25 6 nitzschia sigmoidea + + 26 7 nitzschia philippinarum + + 27 8 nitzschia ligowskii + + 28 9 nitzschia hassall + + 29 10 nitzschia vermicularis + 30 11 navicula sp. + 31 12 navicula cryptocephala + + 32 13 navicula gregaria + no. no. periphyton species presence january march 33 14 navicula gastrum + + 34 15 synedra acus + + 35 16 synedra ulna + + 36 17 synedra sp. + + 37 18 surirella robusta + + 38 19 surirella terera + 39 20 surirella elegans + 40 21 pinnularia giba + 41 22 pinnularia sp. + + 42 23 pinnularia viridis + + 43 24 denticula elegans + + 44 25 tabellaria fenestrata + 45 26 neidium affine + + 46 27 fragilaria crotonensis + 47 28 diatoma vulgaris + 48 29 diatomella balfouriana + + 49 30 diatoma sp. + + 50 31 diatoma elongatum + + 51 32 epithemia turgida + 52 33 gomphonema micropus + + 53 34 gomphonema sp. + + 54 35 odontidium mesodon + + 55 36 cocconeis sp. + 56 37 anomoeneis sp. + 57 38 frustulia rhomboides + 58 39 brachysira microcephala + + 59 40 amphora ovalis + + 60 41 gyrosigma attenuatum + 61 42 licmophora sp. + + 62 43 pleurosigma sp. + unicellular algae 63 1 closterium littorale + + 64 2 closterium lunula + + 65 3 closterium sp. + + 66 4 straurastrum notula + 67 5 stauroneis sp. + ecological effects of belik river hydraulic characteristics on periphyton community – sartika et al. 127 no. no. periphyton species presence january march 68 6 cosmarium reniforme + 69 7 cosmarium turpinii + 70 8 pleurotaenium sp. + 71 9 chroococcus turgidus + 72 10 chlorococcum sp. + + 73 11 ankistrodesmus densus + + 74 12 oocystis borgei + 75 13 cylindrocystis gracilis + + 76 14 cylindrocystis sp. + + 77 15 colacium vesiculosum + + colony algae 78 1 pediastrum biradiatum + + 79 2 scenedesmus dimorphus + + 80 3 scenedesmus quadricauda + + 81 4 anabaena circinalis + + 82 5 merismopedia tenuissima + + 83 6 pandorina morum + + 84 7 sphaerocystis schroeteri + 85 8 gloeocapsa gelatinosa + + 86 9 coelosphaerium sp. + 87 10 palmella miniata + 88 11 protococcus viridis + centric diatom 89 1 biddulphia sp. + + 90 2 cyclotella meneghiniana + + dinoflagellate 91 1 peridinium gatunense + 92 2 peridinium umbonatum + + 93 3 peridinium sp. + + 94 4 gonyaulax sp. + 95 5 prorocentrum sp. + 96 6 dinobryon sp. + table 2 data shows that in march 2021, 35 periphyton species were missing, and 5 periphyton species were present as new species in belik river. the reduction of periphyton species number in march 2021 occurred in almost all sampling sites compared to that in january 2021 (fig. 1). this can be caused by several flood events in february 2021. the average amount of rainfall has increased significantly in 2020 and 2021 (table 3). floods in march 2021 could be caused by an accumulation of rainfall during january march 2021 (the peak of heavy rains at the research location). flood is one of the disturbances for periphyton because these creatures live by sticking to the substrate (benthic). the flood caused the current velocity and discharge to increase, thus periphyton, especially the immobile periphyton, were swept away by the water. in addition, periphyton density was influenced by the presence of nutrient content and sediment from floods (wang et al. 2018; whorley & wehr 2016). figure 2 number of periphyton species found in belik river in (a) january 2021 and (b) march 2021 tds average in january 2021 (97.5 mg/l) increased to 208.5 mg/l in march 2021. likewise, tss increased in march 2021 from an average of 90.15 mg/l to 217.5 mg/l. transparency decreased from 0.12 m to 0.06 m (table 4). transparency less than 20 cm indicates that the water was very turbid (pal et al. 2015), which is the very turbid water ecosystem category. in a very turbid water ecosystem, photosynthetic process can be disrupted because turbidity reduces light penetration into the water (bahri & maliga 2018; butler & ford 2018; rashid et al. 2013; singh et al. 2017). biotropia vol. 31 no. 2, 2024 128 table 3 yearly rainfall data (last 5 years) in the study area month rainfall (mm3/day) 2017 2018 2019 2020 2021 january 384.16 533.61 216.78 1,005 1,182 february 258.18 329.07 453 1,089 811.5 march 277.16 305.12 1,326 1,963.5 1,038 april 294.4 19.16 672 955.5 226.5 may 135.56 19.10 16.5 736.5 189 june 51.73 12.15 1.5 4.5 595.5 july 21.08 74.48 1.5 3 154.5 august 4.78 0.78 6 66 72 september 85.26 104.27 139.55 124.5 379.5 november 124.54 95.52 28.5 715.5 589.5 december 761.13 215.99 114 394.5 849 total 362.60 186.2 1,360.5 621 247.5 average 2,760.57 1,895.47 4,335.83 7,678.5 6,334.5 maximum 230.05 157.96 361.32 639.88 527.88 table 4 physicochemical characteristics of belik river in january and march 2021 physicochemical parameters br1 jan br2 jan br3 jan br4 jan br1 mar br2 mar br3 mar br4 mar do (ppm) 7.33 7.90 7.87 6.17 5.40 6.13 5.43 4.93 co2 5.33 2.00 2.00 2.67 4.25 2.92 4.75 3.25 ph 7.30 6.93 7.03 7.00 6.90 7.10 7.13 7.00 water temperature (oc) 28.67 27.00 27.17 27.00 27.33 26.33 27.00 26.00 depth (m) 0.16 0.20 0.18 0.22 0.17 0.27 0.24 0.18 current velocity (m/s) 0.72 0.54 0.67 0.20 0.84 0.52 0.43 0.34 discharge (m3/s) 0.32 0.28 0.60 0.98 0.50 0.31 0.35 0.28 transparency (m) 0.08 0.12 0.13 0.13 0.05 0.05 0.06 0.08 tds (mg/l) 112 120 70 88 296 218 144 176 tss (mg/l) 48.6 152 90.8 69.2 216 125 123 406 total phosphate as po4 (mg/l) 0.24 0.27 0.28 0.24 0.81 0.32 0.33 0.29 nitrate as no3-n (mg/l) 4.19 4.83 3.81 3.46 1.45 1.38 1.12 1.63 sulfate as so4 (mg/l) 18.34 16.20 21.66 25.23 9.40 7.50 12.70 4.82 detergent as c18h29nao3s (µg/l) < 2 < 2 < 2 < 2 198 21 < 2 21 the number of periphyton species in br2 january 2021 and br2 march 2021 was the lowest number of species compared to that in other sampling points (fig. 2). br2 is a canalized river channel with a pavement-modified riverbed. the sediment transport rate was accelerated by the pavement of the river channel so that sediment tends to be deposited in the downstream area of the river. sediment was used by mobile periphyton as a shelter during the flood, while immobile periphyton will be immediately carried away by the current (hoyle et al. 2016). br1, br3, and br4 had the natural characteristics of riverbed, formed by soil and mud. in addition, in br4, the periphyton was protected by water plants when the current velocity and discharge increased. the release of nutrients plants can also be used by periphyton for their growth. aquatic plants also had an important role as agents in the phytoremediation process of water (kemalasari & choesin 2011; kukuryk & mieczan 2012; swanson et al. 2017; ummi et al. 2019). therefore, the br2 channel was unsuitable ecological effects of belik river hydraulic characteristics on periphyton community – sartika et al. 129 for periphyton to live because of the absence of sediments and aquatic plants . the shannon wiener diversity index showed that water quality in br1, br2, and br3 in january 2021 and march 2021 was lightly polluted. meanwhile, water quality of br4 in january 2021 was very lightly polluted. subsequently, water quality changed to medium pollution in march 2021 (table 4), in which that carbon dioxide, total phosphate, and detergent levels increased. meanwhile, do has decreased below class ii water quality threshold. nitrate levels in br4 were the highest compared to that in other sampling points (table 4). this drastic change can be caused by the flow of water from the upstream area, especially br2, on a canalized river. the water flow brought sediment when it flooded in february 2021, which was deposited in br4. according to walalite et al. (2016), canalized channels do not have floodplains and substrates to filter nutrients and sediment in water when the flood occurs. the periphyton genus most commonly found in this study was nitzchia. the most abundant species in january 2021 were nitzschia improvisa (br1, br2), nitzschia philippinarum (br3), and nitzschia terricola (br4). meanwhile, the most abundant species in march 2021 was nitzschia improvisa at all sampling points (fig. 3). nitzschia is part of the pennate diatom, a functional group with the highest number of species (fig. 2). diatoms have an important role in the silica cycle. silica is one of the compositions for making detergent, so the high detergent content in waters can be indicated by the presence of diatom pennate (biranje et al. 2015; singh & parikh 2020). this finding is consistent with the condition of br1 in march 2021, which experienced a significant detergent increase (table 4). the river flow at the br1 sampling point was in the middle of a community settlement dominated by economic activities, such as student boarding houses and laundry facilities, which disposed washing waste into the river body. the presence of nitzschia, navicula, and synedra indicates that the river waters are turbid and contain excessive organic matter. meanwhile, the existence of a genus that has tolerance for pollutants, such as cyclotella, amphora, melosira, and surirella showed that the waters contain anthropogenic waste (singh & parikh 2020). this statement can be proven by high levels of total phosphate in br1, br2, br3, and br4 in march and january 2021, which exceeded class ii water quality (table 3). phosphate in waters can be caused by domestic waste (54%), agriculture (38%), and industry (8%). high phosphate content indicated the presence of high household waste. one of the wastes that can cause high phosphate content in waters is domestic waste from community settlements, especially related to washing waste because detergent also contains phosphate (mekonnen & hoekstra 2018; puijenbroek et al. 2018). the upper reaches of settlements of belik river, which tend to be congested and are very close to the river, can be one of the causes of river pollution. in this study, cluster analysis was carried out to classify the conditions of the sampling locations based on their characteristics. canonical correlation analysis (cca) was also carried out to find out the water quality parameters that contributed most impacts to periphyton community. results of cluster analysis showed that the environmental conditions at the 4 sampling sites were divided into 2 clusters. in january 2021, the first cluster consisted of br3, br4, and br1, while the second cluster consisted of br2 (fig. 4). in march 2021, the first cluster consisted of br4, while the second cluster consisted of br2, br3, and br1 (fig. 5). based on these data, it can be concluded that there have been changes in the dynamics of physical, chemical, and biological conditions at different sampling times resulting in cluster shifts. in january 2021, br3 had a close relationship with br4, but changed to be closer to br2 in march 2021. table 5 water quality based on shannon-wiener diversity index (h’) h’ and water quality br1 jan br2 jan br3 jan br4 jan br1 mar br2 mar br3 mar br4 mar shannon-wiener diversity index 2.40 2.79 2.90 3.14 2.75 2.32 2.45 1.99 water quality lightly polluted lightly polluted lightly polluted very lightly polluted lightly polluted lightly polluted lightly polluted medium polluted biotropia vol. 31 no. 2, 2024 130 figure 3 the 5 highest belik river periphyton species in (a) sampling site 1 (br1), (b) sampling site 2 (br2), (c) sampling site 3 (br3), and (d) sampling site 4 (br4) figure 4 dendrogram of the 4 sampling sites in january 2021 (based on the results of cluster analysis) figure 5 dendrogram of the 4 sampling sites in march 2021 (based on the results of cluster analysis) that change was caused by the floods happened in february 2021, which brought the nutrients present in br2, which had a modified riverbed and were retained in br3, which had a natural riverbed. in addition, br2 and br4, separated into different clusters, indicated a significant difference in conditions between the modified and natural river. according to kennedy & turner (2011), the canalization in br2 hinders connectivity in the riparian zone that connects aquatic and terrestrial ecosystems. the existence of riparian zone changes affected the water dynamics physically and chemically (nutrients), thus affecting periphyton dynamics. meanwhile, br4 has a riparian zone which is more natural to support the organisms’ life. ecological effects of belik river hydraulic characteristics on periphyton community – sartika et al. 131 figure 6 canonical correlation analysis ordination diagram of belik river physicochemical and the presence of periphyton in january 2021 figure 7 canonical correlation analysis ordination diagram of belik river physicochemical and the presence of periphyton in march 2021 the canonical correlation analysis indicated that, in general, the distribution and abundance of periphyton in belik river were influenced by co2, total phosphate, discharge, do, nitrate, tss, and depth (figs. 6 & 7). specifically, in january 2021 periphyton had the highest diversity in br4, influenced by discharge and sulfate parameters (axis 1), and depth (axis 2). meanwhile, in march 2021, there was no big difference in periphyton diversity at the 4 sampling sites. however, br3 had the highest diversity of centric diatoms, influenced by depth (axis 1) and sulfate (axis 2). this occurrence was consistent with the condition of br3 in march 2021, having the highest sulfate levels compared to other sampling points. according to olszynski & wieczorek (2018), it is known that centric diatoms have a wide tolerance range so that they can live in waters containing extreme sulfates. biotropia vol. 31 no. 2, 2024 132 conclusion belik river water had been very lightly to medium polluted. water quality changes temporally mainly due to flooding. the type of hydraulic characteristics of the river channel influences the distribution and diversity of periphyton. the presence of pennate diatoms as periphyton with the highest species number indicates that the levels of organic pollutants in the water are quite high. human activities, especially household activities in the upper reaches of the belik river, greatly affect the quality of river water. this is indicated by the high levels of detergent, tds and tss in br1 and br 2. further research that can be developed is a study related to detailed hydraulic characteristics that examines the type of vegetation around river channels. this can illustrate the influence of the characteristics of each vegetation species which may affect the water quality in a river. references amoatey p, baawain ms. 2019. effects of pollution on freshwater aquatic organisms. water environ res 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kottelat et al. 1993; kottelat, 2013a; froese & pauly, 2022b; kottelat, 2022), the highest diversity of nemacheilus species has been recorded in the asiatic region. among the total of 55 species, only two species, namely nemacheilus chrysolaimos and n. fasciatus, have been reported to be highly abundant in java island (kottelat, 1984; kottelat et al. 1993; hadiaty and yamahira, 2014; hubert et al. 2019). the taxonomic history of the two nemacheilus fishes was initially documented by cuvier and valenciennes in 1846. they classified these species under the genus cobitis, naming them cobitis chrysolaimos and c. fasciata (cuvier & valenciennes, 1846). subsequently, the genus was revised by bleecker in 1853, and the name nemacheilus became a valid designation. these nemacheilus spp. have gained significant attention due to the exclusive distribution in indonesian waters, as well as their strikingly similar morphology but distinct body colorations. kottelat (1984) elucidated the differences between these two species and in subsequent publications (kottelat et al., 1993), additional information was provided. moreover, hadiaty and yamahira (2014) presented an updated identification key specifically for the species of nemacheilus spp. found in asian waters within indonesia. in addition to the morphological analysis, the molecular examination of the two nemacheilus fishes showed their dissimilarities. dna barcoding identified the most recent common ancestor (mrca) to have existed approximately 1.5 million years and 0.5 million years ago in n. fasciatus and n. chrysolaimos, respectively (hubert et al., 2019). šlechtová et al. (2021) also supported that n. chrysolaimos was different from n. masyae through genomic dna isolation. according to kusuma et al. (2021), nemacheilus chrysolaimos from temanggung and yogyakarta using the partial sequence of coi gene indicated a haplotype and nucleotide diversity (hd) of 0.679 and 0.00117, respectively. the differentiation between these two species needs to be reinforced through dna barcoding in contrast to the study conducted by ath-thar et al. (2018) using pcr-rapd analysis on neimacheilus fasciatus, which revealed a high level of genetic diversity. this study also augments the understanding of nemacheilus spp. by employing morphological and molecular approaches. a noteworthy addition is the inclusion of the fins formula, which has not been previously described. the outcomes are expected to offer valuable insights and resolve the taxonomy of these two fishes yet to be disclosed materials and methods field sampling the specimens of nemacheilus spp. from blitar regency were collected from three main locations, namely garum, ponggok, and wlingi at six rivers. specifically, garum consisted of three rivers (slorok, sumber ronje, and glawah), ponggok comprised two rivers (loadeng and tunjung), and wlingi included one river (lekso), as shown in figs. 1 & 2. to capture the fish samples, gill nets were employed, and the collected fishes were preserved in sample bottles containing a 10% formaldehyde solution. furthermore, the samples were transported to the laboratory at the state university of surabaya (universitas negeri surabaya) for subsequent identification, measurement, and analysis. figure 1: the map showing sampling localities of two nemacheilus fishes at three rivers of blitar, east java, indonesia. a. garum (7°59.967's, 112°5.775'e), b. ponggok (8°2.604's, 112°14.004'e) and c. wlingi (8°4.215's, 112°19.894'e). figure 2: the habitat of two nemacheilus fishes in blitar, east java, indonesia. a. slorok river, b. sumber ronje river, c. glawah river, d. loadeng river, e. tunjung river and f. lekso river. morphology work in the laboratory, all of the fish were sorted, washed, and cleaned for morphological observation and identification. measurement of the morphological characteristics was made on 14 characters, as shown in fig. 3, using modifications of kottelat (1984) and kottelat and freyhof (2007) with a digital caliper and 0.01 mm accuracy. furthermore, the identification was performed in line with the study of kottelat (1984) and kottelat et al. (1993). the specimens were then stored in 70% alcohol and deposited in the museum zoologicum bogoriense (mzb), cibinong, indonesia. before molecular work, the specimens were frozen at -20oc for dna extraction. figure 3: morphometric of the nemacheilus fish from blitar, east java. abbreviation: 1. total length (tl); 2. standard length (sl); 3. head length; 4. snout length; 5. eye diameter; 6. postorbital length; 7. predorsal length; 8. prepelvic length; 9. prepectoral length; 10. pectoral fin length; 11. height of the body; 12. dorsal fin length; 13. anal fin length; 14. height of caudal peduncle. the fish image was adopted from hubert et al. (2019); measurement was modified from kottelat (1984) and also kottelat & freyhof (2007). dna extraction and sequencing the isolation of total dna (whole genome) from stomach tissue samples was carried out using the dna isolation kit (roche), with several modifications. a dna fragment of approximately 526 base pairs (bp), corresponding to the coi gene region of the mitochondrial dna (mtdna), was successfully amplified using gradient pcr. the universal primers lco1490 (5' ggt caa caa atc ata aag ata ttg g 3') and hco2198 (5' taa act tca ggg tga cca aaa aat ca 3') were used for this purpose (folmer et al. 1994). the hotstart pcr method was employed, using a kapa master and two taq master mixes. the pcr process consisted of 35 cycles, each encompassing the following steps, initial double-strand attachment (pre-denaturation) at 95°c for 3 minutes, denaturation at 94°c for 45 seconds, annealing at 45°c for 45 seconds, and extension at 72°c for 2 minutes. a final elongation step was conducted at 72°c for 10 minutes. to visualize the pcr products, gel electrophoresis was performed on a 1% agarose gel prepared with 0.5 g of agarose and 50 ml of tae buffer. additionally, 4 μl of ethidium bromide (etbr) was added as a dye to the gel, and the subsequent step was to mix 3 µl of the pcr samples with 1 µl of loading dye before putting the mixture in an agarose well. the electrophoresis was performed using a machine with a voltage of 220 v and a current of 400 ma, for 25 minutes. pcr products were also purified using a qiagen purification kit according to the manufacturer’s instructions and subsequently sequenced at first base, malaysia. data analysis a description of the two nemacheilus spp. was presented based on morphological observation. since the data from morphological measurements were not normally distributed, a non-parametric test (kruskal-wallis test) was employed to investigate any significant difference in the morphometric of n. fasciatus, from sampling localities. this was because only this species occurred at all six rivers, while n. chrysolaimos had a limited number and was only found in one river. the molecular data obtained from the study were subjected to the following analytical procedures: sequence composition and genetic diversity a partial sequence of the coi gene along 503 bp was obtained from 11 nemacheilus spp. from blitar regency, east java, as the final dataset. each sequence was initially translated into an amino acid to check and remove pseudogene (song et al. 2008; buhay, 2009). nucleotide sequencing was then continued by carrying out the chromatogram analysis, using finch tv software and translating into amino acid sequence through the expasy website (duvaud et al. 2021). subsequently, all sequences were checked through the blast (basic local alignment search tool) (boratyn et al. 2013) and the bold system (ratnasingham & hebert, 2007) to be compared with close relatives of nemacheilus spp. a total of 5 accessions from genbank (ncbi) were selected as in-group and out-group for the phylogenetic tree reconstruction. multiple sequence alignment was performed by using the clustal x (larkin et al. 2007) and then checked manually through the bioedit software (hall, 1999). furthermore, partial sequences of the coi gene from nemacheilus spp. were submitted to genbank with referred accession numbers, as shown in table 2. all the data including taxonomic characteristics and genbank accession numbers were tagged with the voucher specimens preserved at zoologicum bogoriense (mzb) at juanda street, bogor, west java, indonesia. the calculation of similarity values was performed as follows: similarity percentage = (1-genetic distance) × 100%. the substitution of transitions and transversion of nucleotide bases was calculated by the k2p (kimura 2-parameter) model. information on the genetic diversity of all sequences used for phylogenetic reconstruction was analyzed through items, such as the value of nucleotide diversity (pi), the number of polymorphic sites (s), the haplotype analysis (haplotype diversity (hd), and the number of haplotypes (nhap) (nei, 1972). in addition, the software version of mega x was 10.2.6 (kumar et al. 2018) used to calculate nucleotide frequencies, transition/conversion ratio (k), transition/conversion, rate ratio bias (r), and probabilities. phylogenetic reconstruction reconstruction of the phylogenetic tree based on the partial sequence of the coi gene was conducted on a total of 17 sequences using mega x version 10.2.6. the purpose is to determine the grouping of different species and the applied methods were minimum evolution (me) and maximum-likelihood (ml). the settings used for me phylogenetic tree reconstruction begins with the setting of a bootstrap consensus tree inferred from 1000 replicates retrieved to represent the evolutionary history of the taxa (felsenstein, 1985). branching corresponding to partitions reproduced in less than 50% of bootstrap replicates was eliminated. furthermore, evolutionary distances were calculated using the kimura 2-parameter (k2p) method and in units of the number of base substitutions per site (kimura, 1980). the rate variation among sites was modeled with a gamma distribution (shape parameter = 63). the me tree was searched using the close-neighbor-interchange (cni) algorithm at search level 2 (nei & kumar 2000) and the neighbor-joining algorithm was used to generate the initial tree (saitou & nei, 1987). the settings used for ml phylogenetic tree reconstructions were calculated by using the k2p substitution model (saitou & nei, 1987), and the rate variation among sites was modeled with a gamma distribution and bootstrap consensus tree inferred from 1000 replicates (felsenstein, 1985). in addition, the percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) were shown next to the branches. the barcode gap analysis generated by automatic barcode gap discovery (abgd) was used to strengthen the identification of this species, and grouping analysis (puillandre et al. 2012) was conducted through a web interface to check the distribution and size of a potential barcoding gap for the partial sequence of coi gene dataset with the following settings: pmin: 0.001, pmax: 0.9, step: 10, x (relative gap width):1.5, kimura (k80), number of bins: 20. results and discussion class actinopteri cope, 1871 ordo cypriniformes bleeker, 1859 family nemacheilidae regan, 1911 genus nemacheilus bleeker, 1863 nemacheilus chrysolaimos (valenciennes, 1846) (fig. 4) noemacheilus fasciatus kuhl & van hasselt in van hasselt, 1823: 133 (buitenzorg) (partim; nomen nudum). cobitis chrysolaimos valenciennes in cuvier & valenciennes, 1846: 27, fig. 521. noemacheilus chrysolaimos kottelat, 1984: 241, figs. 14a, 15. nemacheilus chrysolaimos kottelat, 1993: 75, pl. 25.―roberts, 1993: 25, fig. 29.―hardiaty & yamahira, 2014: 84 (list), 87 (list), 90 (list), 92 (key). figure 4: nemacheilus chrysolaimos (valenciennes, 1846) (standar length: 52.5 mm; mzb 26539) from blitar, east java, indonesia. a. habitus, lateral view; b. dorsal fin, lateral view; c. anal fin, lateral view; d. pectoral fin, lateral view; e. caudal fin, lateral view; f. ventral fin, lateral view; g. anterior part, dorsal view. material examined. slorok river, garum (mzb 26539, sl. 52.5 mm; mzb 26540, sl. 51.3 mm; 08°02'36.25''s, 112°14'00.23''e), blitar, east java, 26 june 2022, coll. d.a. rahayu & e.d. nugroho. description. morphometric data are presented in table 1. the head is rounded with a pair of eyes, a pair of nares, a short blunt snout, a small subterminal mouth, and a circular lip around the mouth. furthermore, the eyes are elliptical and nares are located between the snout and the eyes. the mouth contains three pairs of rostral barbels, two pairs on the upper jaw, and one pair on the upper snout maxillary barbels may reach half of the postorbital length of the head. the inner rostral spines are present and reach about half of the eye. the body is elongated, fusiform, weakly compressed, and laterally flattened at the base of the tail without sharp scales. tl is about 1.18–1.28 (1.24 ± 0.01) times as long as sl and head length is about 2.21–3.09 (2.65 ± 0.09) times as the snout. meanwhile, the eye diameter is about 0.38–0.57 (0.46 ± 0.02), 0.36–0.50 (0.41 ± 0.01), and 0.02–0.04 (0.03 ± 0.002) times as long as the snout, postorbital length, and sl, respectively. predorsal length, prepectoral length, and height of the body are about 0.99–1.10 (1.02 ± 0.01), 1.07 (0.94 ± 0.02), and 1.00–1.50 (1.19 ± 0.05) times as the prepelvic length, length of the head, and height of the caudal peduncle, respectively. pectoral fins almost reach more than half of pelvic fin bases. an axillary lobe presents at the pelvic fins bases under the first to third of branched dorsal rays. the anal fin does not reach the caudal fin bases. the dorsal fin is opposite the ventral fin or just behind the vertical fin. the anal fin is short, far behind the ventral fin. the ventral fin does not reach the anal fin; pectoral fins are shorter than the head. the caudal fin is very emarginate, the lobes are acute, caudal fin is longer than the head with the rays all forked. the initial dorsal fin is in front of the vertical line of the anal fin base, closer to the tip of the snout than the base of the caudal fin; the dorsal fin is medium in size, the base of the posterior tip is the opposite the base of the ventral fin and the tip is not reaching anal fin. the caudal fin is crescent in shape, the pectoral fin is rounded, and the length is almost equal to the head, ending less than the length in front of the ventral fin; anal fin is shorter than the pectoral fin and the shape is tapered or slightly pointed and bearing hard and soft rays, not or barely lower than the body, higher than the length of the base; the caudal fin is emarginate or crescent-shaped-emarginate. the first anterior dorsal rays are the longest. dorsal fins dii. 7–8, anal fins ai. 3–5, pectoral fin pi. 9, ventral fins vi. 6–7 and caudal fin c. 17. coloration. the body is black-yellowish in color with 12–18 dark bars irregular shape on the lateral part. the two pairs of rostral barbels are black and one pair with yellowish coloration. the base of the caudal fin is red in the anterior part of the caudal peduncle and the initial base of the dorsal fin rays has a black spot. the head is brown with a dark color in the center; a darker pattern is also present on the snout and opercula. furthermore, the base and first rays of the pectoral fins are dark in color and the last dorsal fin rays are dark with black spots. body length. standard length (sl) and total length (tl) ranged from 32.1–52.4 mm and 40.60–66.40 mm, with a mean of 43.47 ± 2.05 mm and 55.32 ± 2.93 mm (n = 9). distribution. according to kottelat (1984) and kottelat et al. (1993), the species n. chrysolaimos was distributed in java island, particularly in west java. meanwhile, hubert et al. (2019) reported that species can be found in almost at all provinces in java island, from west java, central java to east java. remarks. there is a contradiction information on the distribution of this species between kottelat’s (1984) finding with hubert et al. (2019) information. we believe that so far, the kottelat’s collected materials maybe restricted to the west java only and have not ever been expanded to another location, meanwhile, hubert et al. (2019) revisited and collected this species at all 3 provinces in java island, therefore, the information is totally different. nemacheilus fasciatus (valenciennes, 1846) (fig. 5) noemacheilus fasciatus kuhl & van hasselt in van hasselt, 1823: 133; 1824: 376; kottelat, 1984: 247, fig. 18a. cobitis fasciata valenciennes in cuvier & valenciennes, 1846: 25.—bleeker, 1854: 96; 1860: 78. cobitis suborbltalis valenciennes in cuvier and valenciennes, 1846: 26. cobitis chrysolaimos valenciennes in cuvier and valenciennes, 1846: 27. nemacheilus fasciatus bleeker 1863a: 41, 366 (in part); 1863b: 7 (in part).―kottelat, 1993: 25.―roberts, 1993: 26.―hardiaty & yamahira, 2014: 84 (list), 87 (list), 90 (list), 92 (key). figure 5: nemacheilus fasciatus (valenciennes, 1846) (standar length: 52.9 mm; mzb 26539) from blitar, east java, indonesia. a. habitus, lateral view; b. dorsal fin, lateral view; c. pectoral fin, lateral view; d. ventral fin, lateral view; e. caudal fin, lateral view; f. anal fin, lateral view; g. anterior part, ventral view. material examined. glawah river (mzb 26552, sl. 45.7 mm; 07°59'58.02''s, 112°05'46.52''e), loadeng river (mzb 26545, sl. 52.9 mm; 08°00'01.61''s 112°06'30.43''e), blitar, east java, 26 june 2022, coll. d.a. rahayu & e.d. nugroho. description. morphometric and statistical data are presented in table 1. the head is rounded with a pair of eyes, a pair of nares, a short blunt snout, a small subterminal mouth and circular lips around the mouth. the eyes are elliptical and nares are located between the snout and the eyes. three pairs of rostral barbels are present at the mouth, two pairs on the upper jaw and one pair on the upper snout. the length of the snout is medium, slightly pointed and slightly shorter than the postorbital part of the head. the mouth is arched and the anterior lip is slightly furrowed anteriorly (see fig. 5). the posterior lip has 4–5 deep grooves on each side of the different median incisions. the posterior part of the lips is smooth. the maxillary and outer rostral barbels reach the mid-length of the postorbital area of the head. the body is elongated, fusiform, weakly compressed and without sharp scales at the base of the tail. total length (tl) is about 1.14–1.50 (1.26 ± 0.06) times as long as sl. the head length is about 1.69–3.65 (2.56 ± 0.04) times as long as the snout. eye diameter is about 0.33–0.69 (0.46 ± 0.005) times as long as the snout; 0.25–0.58 (0.39 ± 0.008) times as long as postorbital length; 0.02–0.06 (0.04 ± 0.0008) times as long as sl. predorsal length is about 0.72–1.52 (1.01 ± 0.007) times as long as prepelvic length. prepectoral length is about 0.70–1.22 (0.96 ± 0.008) times as long as the length of the head. the body height is about 1.02–3.22 (1.62 ± 0.04) times as long as the height of the caudal peduncle. the position of the dorsal fin base is located in front of the vertical line of the pelvic fin. it is closer to the tip of the snout than the base of the caudal fin; the size of the dorsal fin is medium; the base of the posterior tip is opposite to the ventral fin and the tip does not reach the anal fin. the anal fin is short, far behind the ventral fin. the ventral fin does not reach the anal fin; the pectoral fins are shorter than the head. the caudal fin is emarginated, the lobes are pointed and caudal fin is longer than the head. the base of the dorsal fin is almost at the middle of the tip of the snout and the base of the caudal fin. there is no black spot at the base of the anterior dorsal fin. the pectoral fin is rounded and the length is almost equal to the head, ending less than its length in front of the ventral fin, shorter than the pectoral fin, ending less than its length in front of the anal fin; the anal fin is rounded or slightly pointed, not or barely branched lower than body size, higher than base length; the caudal fin is emarginated or crescent-emarginate. the pectoral fin does not reach the base of the ventral fin. a small axillary lobe presents at the base of the ventral fin which is inserted under the dorsal forked finger, the anal fin does not reach the base of the caudal fin and the last fin is branched with a subequal lobe. dorsal fin d ii 7–8; anal fin a i. 6; pectoral fin p i. 9–10; ventral fin v i. 6–7, caudal fin c. 17. coloration. the body is yellowish with 16–18 vertical elongated black spots. the anterior spots are thinner than the posterior ones. there are about 5-6 black saddles on the back in front of the dorsal fin. there is a black spot at the base of the caudal fin. the head is dark. there are about 7 colors of the saddle with positions below and behind the dorsal fin. there is a black spot on the proximal third of the dorsal fin rays. half of the dorsal fin rays are dark. there are two longitudinal rows of spots on the dorsal rays: in the middle and above all four fin rays. the other fin is hyaline. body length. standard length (sl) ranged from 24.20 to 71.70 mm, with a mean of 42.59 ± 0.87 mm and total length (tl) ranged from 31.30 to 93.50 mm, with a mean of 53.56 ± 1.31 mm (n = 147). the smallest was found from loadeng river and the largest was found from lekso river. distribution. according to kottelat (1984) and kottelat et al. (1993), this species was distributed up to southern sumatra and also java island (west java, central java and east java). on the other hand, hubert et al. (2019) noted that this species was distributed only in west java. in this study, n. fasciatus was found in all six rivers from blitar, east java. remarks. there is a contradiction information on the distribution of this species between kottelat (1984), kottelat et al. (1993) with hubert et al. (2019). we believe that kottelat’s information is correct compared to hubert et al. (2019), because hubert’s claimed that this species only distributed in west java after revisiting the java island. meanwhile, we found n. fasciatus in east java, different from hubert’s information and similar to kottelat’s information. statistical analysis. the kruskal-wallis test showed that all of the 14 measured characters were significantly different (p < 0.001) (see table 1). table 1: morphometric data (mm) and kruskal-wallis’s test (h) on two species of nemacheilus fishes. from blitar, east java, indonesia. the morphological characteristics of the two nemacheilus fishes can be distinguished from their body colorations (n. chrysolaimos with black-yellowish, while n. fasciatus with yellowish), pattern (irregular shape/bars or dots), ray ornamentation on the dorsal fin (black dots in n. chrysolaimos, while reddish spot in n. fasciatus), anterior naris (n. chrysolaimos with valve pierced tube-like, while in n. fasciatus, anterior margin with a winged flap) and also the fins meristic. the four preceding characters were almost similar and also found from kottelat’s (1984) observations but kottelat (1984) and kottelat et al. (1993) did not mention the detail on the meristic fins of n. chrysolaimos or n. fasciatus. the fins provide valuable insights into the morphological characteristics of the two fish species. n. chrysolaimos has shorter proportions of eye diameter, head length, and lateral length of the head concerning sl, as opposed to n. longipectoralis. this information contributes to a more comprehensive understanding of the morphological distinctions between the two species. similarly, it becomes apparent that n. fasciatus bears a resemblance to n. masyae, with a few notable differences. the upper caudal lobe and the eye diameter of n. fasciatus are larger in comparison to n. masyae, and this distinction in size provides an updated perspective on the morphological variations between the two closely related species. the significant difference in the morphometric of n. fasciatus may be related to the condition of the rivers or the habitats. for example, the differences in the river flow have caused a morphological variation in western rainbowfish (melanotaenia australis) (kelley et al. 2017). flow regime differences in the streams result in morphological variation in cyprinella venusta (haas et al. 2010). the availability and prey type also lead to differences in morphological features (hendry et al. 2002) but there was no evidence to support the differences among n. fasciatus. therefore, further study should be conducted to elucidate the morphological variation among n. fasciatus from the six rivers in blitar, east java, indonesia. sequence composition and genetic diversity a total of 11 partial sequences of the coi gene with a length of 503 base pairs (bp) for n. fasciatus and n. chrysolaimos were successfully amplified and analyzed to determine genetic variations within the related species based on the database in table 2. a universal primer for the partial sequence of the coi gene carried out through accurate calculations, was successfully applied to nemacheilus spp. from blitar regency, east java, indonesia. the details of the sequence characteristics based on a length of 503 bp are summarized in table 3. the percentage of base adenine (a), cytosine (c), guanine (g), and thymine (t) in all nemacheilus spp. were 16.244%, 32.023%, 23.88%, and 29.565%, as shown in table 3. furthermore, the percentage of g+c content in the partial sequence of the coi gene was 48.26%. the absence of stop codons in these sequences indicated a successful amplification of functional mitochondrial coi sequences. therefore, nuclear dna sequences derived from the mitochondrial dna (numts) were not sequenced since vertebrates numt was less than 600 bp (wong et al. 2009). the characteristics of the partial sequence of the coi gene were analyzed which included haplotype diversity (hd) 0.978 with nucleotide (π) 0.10532, frequency of parsimony informative sites 25.646%, polymorphic sites 165, ts/tv ratio (k) purines= 7.006, pyrimidines= 0.042; ts/tv ratio (r) 1.322; and mean of evolutionary rate 0.00, 0.02, 0.05, 0.09, 0.15, 0.22, 0.31, 0.42, 0.56, 0.72, 0.93, 1.19, 1.53, 2.01, 2.79, and 4.99 substitutions per site. the characteristics indicated that the partial sequence of the coi gene was suitable for determining the species of nemacheilus spp. genetic distance referred to the ratio of genetic differences between species or populations. based on the genetic distance matrix of 2 species of nemacheilus spp., the highest distance was found between n. fasciatus and n. chrysolaimos with a value of 0.22 (table 4). therefore, a smaller genetic distance value generated a more similar appearance partial sequence of coi genes compared to related species. table 2: genbank accession numbers for partial sequence of coi gene of nemachaeilus spp. with references. dna barcoding distinguished freshwater fish species with barcodes in australia, canada, india, thailand, germany, and indonesia (ward et al. 2005, hubert et al. 2008, knebelsberger et al. 2014; lakra et al. 2015, pampromin et al. 2019, rahayu et al. 2019). the partial sequences of the coi gene profile for n. chrysolaimos and n. fasciatus, which were local freshwater fish species in different locations were compiled. sequence validation was also performed using the online facility provided by blast (ncbi) and the bold system. the results indicated that the sequence samples matched the available accessions in the database, with query coverage ranging from 98% to 99.8%, and this confirmed the effectiveness of using dna barcodes for species identification. after analyzing the nucleotide sequences, no insertions, deletions, or codon stops were observed. this supported the notion that all the amplified sequences represented functional mitochondrial coi sequences. additionally, the average length of the amplified sequences exceeded 503 bp, which was typically the limit observed for nuclear dna sequences originating from mtdna (numt). these findings strengthened the reliability of the results and underscored the suitability of the coi gene as a marker for distinguishing between n. chrysolaimos and n. fasciatus in the local freshwater fish populations of blitar regency, indonesia (buhay, 2009; gunbin et al. 2017). table 3: characteristics of partial sequence of coi gene used for phylogenetic trees reconstruction and genetic distance analysis include sequences from the study sample and the genbank/bold system (in group and out group). the partial sequence of the coi gene of nemacheilus spp. showed that the values of the nucleotide base composition of g+c and a+t were between 48.26% and 51.74%, as shown in table 3. the value of the nucleotide base composition and content of the a+t result was higher than g+c, consistent with the characteristics of the mitochondrial base composition. the analysis of the partial sequence of the coi gene showed that at content (54.44%) was higher than gc content (48.26%), these data were observed in australia (ward et al. 2005), canadian (steinke et al. 2009); cuban table 4: pairwise genetic distance of nemacheilus fasciatus dan n. crysolaimos compared to all congeners and outgroups. (lara et al. 2010), and bangladesh (ahmed et al. 2020) fish species. clusters 1 and 2 were resolved as sister taxa with 99% bootstrap and the genetic diversity was very low or less than 2%. a genetic distance value of more than 2% indicated that there were species different from other group members. meanwhile, a genetic distance value of less than 3% indicated that the group or cluster was obtained from the same species (hebert et al. 2003; hebert et al. 2004). based on the standards from nei (1972), the genetic distance of nemacheilus spp. obtained in blitar regency waters was categorized into low (0.01–0.045) and medium (0.17–0.18) similar to nemacheilus spp. genetic distance calculations reported by hubert et al. (2019). phylogenetic reconstruction phylogenetic relationships were shown in the me tree (fig. 6) and ml tree (fig. 7). each species was associated with a specific dna barcode cluster and the relationship among these species was obtained. closer species in terms of genetic divergence, were clustered at the same nodes to determine the distance between the terminal branches of the me & ml trees, consisting of two divergent clusters. figure 6: minimum evolution (me) phylogenetic tree of nemacheilus spp. based on partial sequence of coi gene. the asterisk (*) denotes the sequence of nemacheilus spp. obtained from blitar regency, east java, indonesia and rasbora spp. as the outgroup figure 7: maximum-likelihood (ml) phylogenetic tree of nemacheilus spp. based on partial sequence of coi gene. the asterisk (*) denotes the sequence of nemacheilus spp. obtained from blitar regency, east java, indonesia and rasbora spp. as the outgroup. the phylogenetic analysis of nemacheilus spp. using both me and ml methods resulted in unambiguous branching patterns, as illustrated in figs. 6 and 7. the phylogenetic trees showed that n. fasciatus and n. chrysolaimos species formed distinct monophyletic branches. however, their proximity at the same node indicated genetic relatedness and the positioning of these branches corresponded with a calculated genetic distance of 0.22, signifying the greatest divergence between these two species. the me, ml, and genetic distance data collectively provided strong evidence that n. fasciatus and n. chrysolaimos were genetically distant from each other. in addition, the abgd method identified 3 groups for nemacheilus spp. specimens in with the initial approach and the barcode gap threshold calculated by the coi dataset as shown in figs. 8a & 8b). the value of the barcode gap distance was 0.025 in line with the results of the abgd grouping which divided the species into 3 groups, as shown in fig 8c. group [1] (nemacheilus fasciatus mzb 2655, nemacheilus fasciatus mzb 26549, nemacheilus fasciatus mzb 26550, nemacheilus fasciatus mzb 26552, nemacheilus fasciatus mzb 26545, group [2] (nemacheilus fasciatus mzb 26543, nemacheilus fasciatus mzb 26546, nemacheilus fasciatus mzb 26544, nemacheilus fasciatus mzb 26541), and group [3] (nemacheilus crysolaimos mzb 26540, and nemacheilus crysolaimos mzb 26539). figure 8: barcode gap analysis of coi sequences performed by abgd (puillandre et al. 2012). histograms show the distribution of pairwise genetic distances (uncorrected p-distances) between each pair of specimens. the arrow indicates the gap that allows to distinguish intraspecific (left) and interspecific (right) distances for the coi region. (a) histogram of distance, (b) ranked distance, and (c) number of primary species hypotheses (pshs) obtained, for each prior intraspecific divergence. the application of abgd analyses, with a prior maximal distance set at 0.025, further reinforced the separation of n. chrysolaimos and n. fasciatus into distinct partitions. these additional analyses align with differentiation of these species. consequently, the combination of genetic distance, phylogenetic analysis, and abgd analyses collectively confirmed the successful identification of nemacheilus spp. from blitar regency. based on the comprehensive evidence derived from dna barcoding, with morphological characteristics, it can be concluded that the targeted utilization of these tools offered an efficient and reliable means of identifying nemacheilus spp. at the species level. therefore, this study was the first to report on the morphology in accordance with kottelat (1984); kottelat et al (1993); hardiaty et al. (2014)] genetic identification and phylogenetic reconstruction of nemacheilus spp. using the partial sequence of the coi gene. conservation management of n. chrysolaimos and n. fasciatus in grouping animal units should be conducted according to species and genetic entity, as well as the potential of developing cryopreservation for sustainability. a molecular approach using the partial sequence supported the identification results based on a morphological approach in nemacheilus spp. and obtained an accession number from genbank (ncbi) database. this study indicated that improved morphology and molecular characteristics of local loaches (nemachelius spp.) were obtained from blitar regency. therefore, a reliable dna barcode reference library for east java, indonesia, freshwater fish was established to assign fish species by screening sequences. this initiative aimed to enhance the achievement of better monitoring, conservation, and management of fisheries in this overexploited region. conclusion in conclusion, this study has successfully identified analysis for loach fishes such as n. chrysolaimos and n. fasciatus from six rivers at biltar, east java, indonesia, based on morphology and molecular data. the main characteristic used to distinguish these species is the color pattern on their lateral bodies, such as dark bars or spots, along with the morphological variations of anal fins. moreover, through the utilization of genetic approaches, including phylogenetic reconstruction, sequence composition, genetic diversity analysis, and abgd analysis, it was determined that n. chrysolaimos and n. fasciatus are distinct species. acknowledgments the authors are grateful to the people around the river of blitar regency for their assistance during sampling session. mr. didik for his great help at the molecular laboratory and museum zoological bogor, directorate of scientific collection management-brin for great help to saving this specimen. rofiza yolanda for his valuable time for checking the preparation of this manuscript. m. kottelat and prof joerg bohlen for checking morphological and molecular analysis. we gratefully acknowledge to the reviewers and the editor for the thorough and constructive reviews of this manuscript. this works was supported by the research grant from faculty of mathematics and natural sciences, universitas negeri surabaya for the fiscal year 2022, (grant no. 659/un38/hk/pp/2022, “policy research theme”). references ahmed md, datta sk, zhilik aa. 2020. molecular diversity of freshwater fishes of bangladesh assessed by dna barcoding. bangladesh j zool 48(1): 1-19. ath-thar mhf, ambarwati a, soelistyowati dt, kristanto ah. 2018. keragaman genotipe dan fenotipe ikan uceng nemacheilus fasciatus (valenciennes, 1846) asal bogor, temanggung, dan blitar. j ris akuakultur 13(1):1-10. bleeker p. 1853. diagnostische beschrijvingen van nieuwe of weinig bekende vischsoorten van sumatra. 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bessho-uehara et al. 2020; ramesh & meyer-rochow 2021). among terrestrial bioluminescent organisms, fungi are particularly noteworthy due to their ecological importance in forest environments and their promising potential in fields such as biotechnology, environmental assessment, and ecofriendly illumination (ke & tsai 2022; cortés-pérez et al. 2023). the variation in the luminescence of mushrooms is found in the fruiting body and mycelium of the mushroom, depending on the species (cortés-pérez et al. 2023; oba & hosaka 2023). copyright (c) 2025@author(s). https://doi.org/10.11598/btb.2025.32.3.2547 https://creativecommons.org/licenses/by-nc-nd/4.0/ biotropia vol. 32 no. 3, 2025 386 bioluminescent fungi are widely distributed across the world and are found in tropical and temperate forests (dauner et al. 2021). desjardin et al. (2008) identified three distinct evolutionary lineages of bioluminescent fungi: the omphalotus lineage (omphalotaceae), the armillaria lineage (physalacriaceae), and the mycenoid lineage, predominantly consisting of mycenaceae species. according to cortés-pérez et al. (2023), mycenaceae contain at least 65 bioluminescent species, which are classified under the genera  favolaschia  (pat.) pat., filoboletus henn., mycena (pers.) roussel, panellus p. karst., resinomycena redhead & singer, and roridomyces rexer (karunarathna et al. 2020; nimalrathna et al. 2022). in mycenaceae, mycena is the most significant genus, with around 40 species (chang et al. 2020; oliveira et al. 2021). mycena chlorophos is reported from australia, brazil, borneo, india, japan, malaysia, papua new guinea, and sri lanka (cortés-pérez et al. 2019; arya et al. 2021; oba & hosaka 2023; koli et al. 2024; lu et al. 2024). mycena chlorophos is originally described from the bonin island, japan (desjardin et al. 2010). however, the published descriptions of m. chlorophos that document taxonomically informative features are based on material collected from sri lanka, malaysia, borneo and brazil. despite its high biodiversity, knowledge of bioluminescent mushrooms in indonesia remains limited. the only indonesian authors that reported m. chlorophos are armadhan et al. (2023), but the report lacked taxonomic evidence. another mycena species (m. illumans) was discovered decades ago on java island, indonesia (hennings 1903). to date, there is no subsequent report regarding the taxonomy and distribution of m. chlorophos in indonesia. during our regular night foray of bioluminescent fungi at ipb university campus forest, some glowing basidiomata were encountered, collected, and studied. this study provides an updated account of m. chlorophos in indonesia. materials and methods specimen collection fresh fruiting bodies of the bioluminescent fungus growing on dead bamboo were obtained in 2024 at the lowland bamboo forest, ipb university, bogor, west java, indonesia (fig. 1), located at 6°32›51.0» s and 106°43’09.4” e (latitude -6.547497°; longitude 106.719280°) during a late-night mushroom hunt. the collection and monitoring were done three times (september– december 2024) at the same location. the fruiting bodies were photographed in situ (with and without light/dark condition) using canon eos 1500d. photographs of bioluminescence were taken in complete darkness in the forest and laboratory. ecological data, including coordinates, substrate type, and nearby vegetation, were recorded. figure 1 sampling location of mycena chlorophos at bamboo forest of ipb university source: photo edited from google earth. new record of mycena chlorophos nugeraha et al. 387 morphological analysis the morphological characteristics of fresh basidiomata were examined both in situ and in the mycology laboratory, department of biology, faculty of mathematics and natural sciences, ipb university, indonesia. the macroscopic characters assessment was carried out following putra (2021). micromorphological features of the basidium, cystidia, hyphae, cheilocystidia, and spores, including their shape, size, and coloration were investigated using olympusbx-63 light microscope. specimen identification was carried out using relevant taxonomic references (arya et al. 2021; oba & hosaka 2023). amplification was carried out according to putra et al. (2024). the sequencing was performed using 1st base malaysia. the sequence data were processed using chromaspro software for assembly, while the finalized alignments were submitted to genbank (https://www.ncbi.nlm.nih.gov/) to obtain accession numbers. homology comparisons were conducted through the basic local alignment search tool (blast) in ncbi, aligning sequences against existing data for taxonomic verification. the blastn results were examined to identify the highest sequence similarity (http://blast.ncbi.nlm. nih.gov/blast.cgi). sequences from the selected blast results in this study (bold), 16 fungal sequences from chew et al. (2014), genbank records, and additional relevant sequences were included to construct the phylogenetic tree (table 1). mycena noctilucens was designated as the outgroup following chew et al. (2014). the phylogenetic tree was generated using the maximum likelihood (ml) method in mega x software (kumar et al. 2018). default parameters in mega x were applied for the ml analysis, and bootstrap values of 60% or greater were reported. molecular analysis dna isolation from the fresh specimens was conducted using the qiagen dneasy plant mini kit following the manufacturer’s protocol. dna amplification was performed using a thermo scientific arktik thermal cycler (thermofisher scientific). the amplification process employed its 5 (5’-gga agt aaa agt cgt aac aag g-3’) and its 4 (5’-tcc tcc gct tat tga tat gc-3’) primers (white et al. 1990). pcr table 1 species, collection code, and genbank accession numbers used in this study species voucher/isolate its accession number reference country mycena chlorophos ct15101401 mh400938 wei et al. (2024) china mycena chlorophos ct151014 mh400939 wei et al. (2024) china mycena chlorophos acl051 kj206965 chew et al. (2014) malaysia mycena chlorophos acl055 kj206967 chew et al. (2014) malaysia mycena chlorophos mmrd-20 kf010856 chew et al. (2014) india mycena chlorophos 305759 ab512312 chew et al. (2014) japan mycena chlorophos ipb02 pq821413 this study indonesia mycena illuminans acl161 kj206975 chew et al. (2014) malaysia mycena illuminans acl175 kj206976 chew et al. (2014) malaysia mycena illuminans acl212 kj206980 chew et al. (2014) malaysia mycena deeptha dm334g jx481737 chew et al. (2014) india mycena amicta 189f jf908394 chew et al. (2014) italy mycena amicta aftol-id 1908 dq490645 chew et al. (2014) usa mycena nocticaelum acl258 kc507796 chew et al. (2014) taiwan mycena nocticaelum acl272 kj206987 chew et al. (2014) taiwan mycena kentingensis ysh-2014 kc507796 chew et al. (2014) taiwan mycena stylobates 455 jf908439 chew et al. (2014) italy mycena adscendens 35m jf908420 chew et al. (2014) italy mycena noctilucens acl054 kj206966 chew et al. (2014) malaysia http://blast.ncbi.nlm.nih.gov/blast.cgi http://blast.ncbi.nlm.nih.gov/blast.cgi biotropia vol. 32 no. 3, 2025 388 results and discussion taxonomy photographs of mycena chlorophos were presented in figures 2 – 4. current name mycena chlorophos berk. and m. a. curtis sacc., syll. fung. (abellini) 5:301 (1887) synonym agaricus chlorophos berk. and m.a curtis 1860. basidiomata encountered in dead bamboo as saprobes in a solitary to scattered manner. pileus 5 – 12.5 mm in diameter, viscid with a thick gluten, parabolic to convex in maturity, white to cream at straight margin and center is light brown, shallow depression at center, striate, sticky; lamellae free, entire, with series of lamellulae, concolorous with pileus margin. stipe 6.8 – 14.1 mm x 0.7 – 1.6 mm central, cylindric, hollow, smooth, glabrous, basal tomentum with a white surface. basidia 19.24 – 22.40 x 4.43 – 6.25 µm, clavate, hyaline, thinwalled, with 2 – 4 sterigmata, basidiospores 6.5 – 7.6 x 5.1 – 6.1 µm, smooth, ellipsoid, thin-walled. cheilocystidia 41.46 – 44.52 x 9.53 – 12.59 µm, fusoid-ventricose, hyaline, thin walled. hyphae on pileal trama septate, hyaline. pileipellis ixotrichoderm, hyaline to brownish color, thin walled. luminescence emitting strongly in pileus and lamellae, weakly in stipe, greenish light in dark. location of specimens dramaga, bogor, west java, indonesia, 6°32’51.0” s; 106°43’09.4” e, 145 m asl, on decayed bamboo tree, 2024, collected by fathansah nugeraha. figure 2 field photograph of mycena chlorophos notes: a = upper side of pileus; b = underside of pileus; c = developmental stages of basidiomata; d, e, f = green light from the fruiting bodies. new record of mycena chlorophos nugeraha et al. 389 figure 3 microscopic features of mycena chlorophos notes: a = hypahe on trama; b = pileipellis marginal cell (arrows); c = oleiferous hyphae of pileipellis (arrow). biotropia vol. 32 no. 3, 2025 390 figure 4 microscopic feature of mycena chlorophos notes: a = cystidia (arrows); b = basidia (arrow); c = basidiospores. molecular analysis blast analysis identified a high degree of similarity (98%) between our specimens and m. chlorophos specimens from china, ranking them among the top matches. in line with the blast results, the phylogenetic tree (fig. 5) constructed from its sequences placed specimen ipb02 in the clade of m. chlorophos with a 100% bootstrap value. the phylogenetic tree displayed that our specimen was closely related to materials from malaysia collections.  the mycenaceae family comprises numerous species capable of bioluminescence fungi (cortéspérez et al. 2019; heinzelmann et al. 2024) globally, including m. chlorophos. this study represents the first comprehensive documentation of m. chlorophos in indonesia. our report is not only providing the basic information for taxonomy of bioluminescent fungi in indonesia, but also the reference for future studies of this species. the distribution of m. chlorophos has recently been reported in several countries including china, japan, pacific islands, sri lanka (lu et al. 2024), india (arya et al. 2021; koli et al. 2024), malaysia (chew et al. 2014), new record of mycena chlorophos nugeraha et al. 391 figure 5 the phylogenetic tree depicting mycena chlorophos voucher pq821413 and closely related species notes: bootstrap values > 60% are displayed at the corresponding nodes. the scale bar represents an evolutionary distance of 0.050. southern asia, australia, and brazil (desjardin et al. 2010). however, the taxonomic understanding of bioluminescent mushrooms in indonesia is limited. to date, armadhan et al. (2023) are the only indonesian researchers who have reported the presence of m. chlorophos from karst forest in central java, but their taxonomic evidence was inadequate to confirm the identification with confidence.  in the current study, the basidiomata of m. chlorophos were found colonizing decayed bamboo. previous reports have noted that m. chlorophos lives saprobically on decayed bamboo (arya et al. 2021; koli et al. 2024.). armadhan et al. (2023) also obtained m. chlorophos from weathered logs in indonesia, though they did not specify the tree species. our specimens were observed growing solitary to scattered on bamboo, which was also covered by bryophytes. this observation is consistent with the report of m. cristinae from brazil (oliveira et al. 2021). desjardin et al. (2010) reported m. chlorophos from topotypical growth on wood instead of bamboo. the specimens in our study emit the greenish light from whole basidiome including the cap, lamellae, and stipe-with the stipe exhibiting a weaker intensity. several researchers have also reported that bioluminescent fungi can emit light from their mycelium (weitz et al. 2001; oba & hosaka 2023; perry et al. 2024). this remarkable biological feature is not only scientifically fascinating but also deeply rooted in cultural traditions. for instance, ethnomycological records cited by havey (1957) in a history of luminescence indicate that georg everard rumph (1637 – 1706) documented the use of luminescent fungi by indigenous communities on ambon island, maluku, indonesia. these fungi were used as handheld lanterns, aiding individuals navigate forest paths at night and avoid disorientation. mycena chlorophos is morphologically similar to m. illuminans based on several morphological biotropia vol. 32 no. 3, 2025 392 characters: small basidiome, gelatinous pellicle on pileus, basal disk composed of inflated hyphae of stipe (chew et al. 2014). however, the latter species usually found in palm trees. in indonesia, m. illuminans was originally described and found on java island (hennings 1903). due to the morphological similarity, m. chlorophos and m. illuminans once are acknowledged as synonyms (pegler 1986; maas geesteranus 1992). the morphology of m. chlorophos ipb02 is highly similar to the materials found in malaysia. macromorphologically, they appear similar, but the size of the pileus and stipe in the species found in malaysia (chew et al. 2014) are larger compared to those found in this study. in addition, other specimens found in india (arya et al. 2021) showed a larger pileus size, more gelatinous on the pileus surface, and a longer stipe compared to the species found in malaysia and indonesia. the basidiospores dimension of specimens from indonesia and malaysia are slightly smaller to those reported from india.  the classification of mycena s. l. is highly intricate, and its subgeneric categorization typically depends on morphological traits. the identification of fungi based on morphology is considered challenging, especially when nonexperts are dealing with some fungi (stephenson 2010; raja et al. 2017). therefore, in our study we combined the morphological and molecular analyses. the blast analysis revealed a strong genetic resemblance between our specimens and m. chlorophos specimens from china. based on its tree, our specimen exhibited phylogenetic affinity (bootstrap value of 100%) with the clade consisting of specimen of m. chlorophos from china, india, japan, and malaysia. in the subclade of m. chlorophos, specimen ipb02 was displayed as closely related to materials originating from malaysia (bootstrap value of 89%). our its phylogenetic tree is in line to those reported (lsu, its, and rpb2 tree) by chew et al. (2013; 2014) and confirming that the phylogenetic tree clearly separates m. chlorophos and m. illuminans into two distinct clades, thus verifies that they are not synonymous taxa. conclusion this study provides the first confirmed record of mycena chlorophos in indonesia, verified through morphological characterization and molecular phylogenetic analysis. the findings extend the known geographic range of this bioluminescent species within the asia-pacific region and highlight indonesia’s potential as a reservoir for undocumented fungal diversity. a continued surveys and integrative taxonomic approaches will be essential for documenting and conserving the mycobiota of this biodiverse yet underexplored region. acknowledgments this study was funded by “penelitian fundamental reguler kementerian pendidikan, kebudayaan, riset dan teknologi, indonesia tahun 2024” 22047/it3.d10/pt.01.03/p/b/ 2024 to ivan permana putra ph.d. we thank department of biology, faculty of mathematics and natural sciences,  ipb  university,  and research center for applied microbiology, brin-indonesia. references armadhan ws, sari sp, aji mymb, permatasari dp, amalia bw, berlin ge, …, setyawan ad. 2023. the macrofungal diversity and its potential from the karst forest of kalipoh village, kebumen district, indonesia. asian j for 7(2):99– 106. doi: 10.13057/asianjfor/r070204 arya cp, ratheesh s, pradeep ck. 2021. new record of luminescent mycena chlorophos (mycenaceae) from western ghats of india. stud fungi 6(1):507–13. doi: 10.5943/sif/6/1/40 bechara ejh, stevani cv. 2018. brazilian bioluminescent beetles: reflections on catching glimpses of light in the atlantic forest and cerrado. an acad bras ciên 90(1):663–79. doi: 10.1590/0001-3765201820170504. bessho-uehara m, francis wr, haddock shd. 2020. biochemical characterization of diverse deep-sea anthozoan bioluminescence systems. mar biol. 167:114. doi: 10.1007/s00227-020-03706-w chang c, chen c, lin w, kao h. 2020. mycena jingyinga, mycena luguensis, and mycena venus: three new species of bioluminescent fungi from taiwan. taiwania 65(3):396– 406. doi: 10.6165/tai.2020.65.396 chew al, desjardin de, tan ys, musa my, sabaratnam v. 2014. bioluminescent fungi from peninsular malaysia—a taxonomic and phylogenetic overview.  fungal divers 70:149–87. doi: 10.1007/s13225-014-0302-9 chew alc, tan ys, desjardin de, musa my, sabaratnam v. 2013. taxonomic and phylogenetic re-evaluation of mycena illuminans. mycologia 105(5):1325–35. doi: 10.3852/13-009 cortés-pérez a, desjardin de, perry ba, ramírez-cruz v, ramírez-guillén f, villalobos-arámbula ar, rockefeller a. 2019. new species and records of bioluminescent mycena from mexico. mycologia 111(2):319–38. doi: 10.1080/00275514.2018.1554172 cortés-pérez a, guzmán-dávalos l, ramírez-cruz v, villalobos-arámbula ar, ruiz-sanchez e, ramírezguillén f. 2023. new species of bioluminescent mycena new record of mycena chlorophos nugeraha et al. 393 sect. calodontes (agaricales, mycenaceae) from mexico. j fungi 9(9):902. doi: 10.3390/jof9090902 dauner lap, karunarathna sc, tibpromma s, xu j, mortimer pe. 2021. bioluminescent fungus roridomyces viridiluminus sp. nov. and the first chinese record of the genus roridomyces, from southwestern china. phytotaxa 487(3):233–50. doi: 10.11646/phytotaxa.487.3.4 deane gb, stokes md, latz mi. 2015. bubble stimulation efficiency of dinoflagellate bioluminescence. luminescence 31(1):270–80. doi: 10.1002/bio.2957 desjardin de, oliveira ag, stevani cv. 2008. fungi bioluminescence revisited. photochem photobiol sci 7:170–82. doi: 10.1039/b713328f desjardin de, perry ba, lodge dj, stevani cv, nagasawa e. 2010. luminescent mycena: new and noteworthy species. mycologia 102(2):459–77. doi: 10.3852/09-197 havey en. 1957. a history of luminescence from the earliest times until 1900. philadelphia (us): american philosophical society. heinzelmann r, baggenstos h, rudolf a. 2024. is the bioluminescence in many mycena species overlooked? a case study from m. crocata in switzerland. mycoscience 65(4):173–9. doi: 10.47371/mycosci.2024.03.001 hennings p. 1903. ein stark phosphoreszierender javanischer agaricus (mycena illuminans p. henn. n. sp.) 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faculty of teacher training and education, universitas pgri madiun 63118, indonesia article higlights • t. grandis leaf methanolic extract contains 82 compounds, with 17 being predominant. • epigallocatechin 3-o cinnamate, epigallocatechin 3-o-p-coumarate, and tectograndinol predicted as the potential bioactive compounds in wound healing • molecular docking suggests inhibitory activities against nfκb, mmp-2, and mmp-9, and stimulatory activity against egfr1 that comparable to commercial drugs. article information received : 12 february 2025 revised : 29 august 2025 accepted : 22 september 2025 *corresponding author, e-mail: primiani@unipma.ac.id research paper abstract wound-healing process involves a physiological cascade to restore skin integrity, which includes inflammatory response, cell proliferation, and tissue reconstruction. prolonged inflammation in wound-healing process may lead to a chronic wound stage. proper wound care is needed to prevent wound-caused mortality. several studies showed the potential of t. grandis leaf in wound-healing process. however, the bioactive compounds and the molecular mechanism of t. grandis leaf remains unknown. this study aimed to identify bioactive compounds and biological activity contained in t. grandis leaf extract as well as analyze its molecular mechanism in wound-healing process by conducting in silico study using nfκb, mmp-2, mmp-9, and egfr-1. bioactive compounds of t. grandis leaf extract were identified with lc-ms. three potential compounds, epigallocatechin 3-o cinnamate, epigallocatechin 3-o-p-coumarate, and tectograndinol, were selected based on the pa score screening with the pass prediction. drug-likeness and pharmacokinetics properties of the selected compounds were virtually identified by swissadme and protox. the interactions of t. grandis bioactive compounds toward nf-κb, mmp-2, mmp-9, and egfr-1 were compared to those of curcumin, phenytoin, and nitrofurazone as control. molecular docking to identify the proteinligand interaction was performed by autodock vina integrated in pyrx v.0.8. among 82 bioactive compounds detected in the lcms analysis, epigallocatechin 3-o-cinnamate, epigallocatechin 3-o-p coumarate, and tectograndinol exhibited anti-inflammatory, antioxidant, free radical scavenger, and mmp-9 inhibitor activities. according to lipinski’s rule of five, bioactive compounds are possible to be administered as medication. molecular docking showed that bioactive compounds potentially bound to the active sites of nf-κb, mmp-2, and mmp-9, resulting in proteins inhibition. this study suggested that the wound-healing mechanism of t. grandis bioactive compounds were driven by egfr-1 stimulation indicated by the ability of bioactive compounds to interact with egfr-1 in similar manner to those of nitrofurazone. we concluded that bioactive compounds of t. grandis leaf extract has significant potential to be used as traditional wound treatment and therapy. these compounds demonstrated wound-healing activity in silico by interacting with the key molecular targets, including nf-κb, mmp-2, mmp-9, and egfr-1. keywords: bioactive, egfr, mmp, tectona grandis, wound copyright (c) 2025@author(s). https://doi.org/10.11598/btb.2025.32.3.2456 https://creativecommons.org/licenses/by-nc-nd/4.0/ in silico study of jati leaf constituents as traditional wound care krisnamurti et al. 311 introduction skin is the largest barrier of human’s body, which provides protection, homeostasis, signal transduction, and excretion. wound, defined as skin damage, caused by external or internal diseases is threatening and needs proper cure (tottoli et al. 2020). the repairing process included hemostasis, inflammation, proliferation, and remodeling that requires coordination and interaction of various cells, extracellular matrix (ecm), proinflammatory mediators, and growth factor (tyavambiza et al. 2022). though skin has selfrecovery ability, various factors delay the wound healing process. prolonged inflammation might contribute to the development of chronic wounds (singh et al. 2016). angiogenesis is a process that promotes the development of chronic wounds which occur immediately after the injury. the mechanism involves matrix metalloproteinases (mmps) which play major roles in each stage of wound-healing process, such as wound matrix modification, cell migration, angiogenesis, and tissue remodeling. two types of mmps, mmp-2 and mmp-9, play a major role in angiogenesis regulation during wound healing via proangiogenic cytokines activation and antiangiogenic peptides generation (reiss et al. 2010; caley et al. 2015; bergant suhodolčan et al. 2021). expressions of mmp-2 and mmp-9 are naturally beneficial during the wound-healing process. however, the imbalance level of mmp-2 and mmp-9 may lead to chronic wounds. the abnormal increased level of mmp9 has been observed in the chronic wounds (reiss et al. 2010; cabral-pacheco et al. 2020). elevated mmp-9 expression in the chronic wounds is associated with mmp-2, due to the function of mmp-2 in stimulating mmp-9 activation (ratzinger et al. 2002). the expressions mmp-2 and mmp-9 are often detected in cardiovascular disease, diabetes mellitus, colorectal cancer tissue, and neurodegenerative disease (groblewska et al. 2014; cabral-pacheco et al. 2020). acceleration of wound-healing process is also regulated by the re-epithelialization which occurred during the migration of keratinocytes. epidermal growth factor receptor (egfr) is a protein that has been involved in the process of re-epithelization, including cell migration and proliferation (repertinger et al. 2004). previous findings reported that the increased egfr expression accelerates wound re-epithelialization on the incisional wound (nanney et al. 2000). the skin of egfr wild-type mice (egfr +/+) exhibited smaller erythema at the wound margin compared to egfr-null mice (egfr -/-). five days after the injury, the wound appearance of egfr-null mice was visibly similar to that of the egfr wild-type mice (repertinger et al. 2004). egfr also stimulates angiogenesis in murine skin carcinomas (casanova et al. 2002) which is essential in the early stage of wound healing (caley et al. 2015; shanthi kumari et al. 2020). it is important to achieve effective, safe, less cost treatment that provides long-term relief for wounds (tottoli et al. 2020). natural products are the source to develop traditional and synthetic herbal treatment and therapy with several advantages, such as being readily available in nature, less side effects, and efficiency. some medicinal plants have been implemented for wound care for several decades (varma & giri 2013). tectona grandis, or teak, or jati in indonesia has been known for its medicinal properties, majorly antioxidant, anti-inflammatory, antibacterial, anti-tumor, and antidiabetic (varma & giri 2013; arief et al. 2014; asdaq et al. 2022). previously, t. grandis leaf extract effectively reduced the wound area in the incision and excision wound. it has been reported that the wound area is significantly reduced by treating the wound with 5% and 10% ointment of t. grandis leaf extract (varma & giri 2013). the anti-inflammatory, antioxidant, immunomodulatory, and antibacterial potential of t. grandis leaf are associated in the wound-healing process (mikhal’chik et al. 2006; dégbé et al. 2018; comino-sanz et al. 2021; han et al. 2023). post-surgical wounds and diabetic wounds are more likely to drive a significant impact on a patient’s health. poor healthcare to the patient’s wound may develop into chronic wounds. without proper treatment and therapy, wounds could contribute to mortality (akiki et al. 2021). thus, treatment of wounds is crucial to reduce the risk of chronic wound development and mortality. previous in vivo studies reported extensive exploration on the potential of t. grandis leaf extract in wound-healing process even though the mechanism is still questionable. this study aimed to provide scientific proof about t. grandis potent biological activities, focusing on wound-healing activity. the study was performed by identifying t. grandis bioactive compounds, also predicted the biological activity and molecular mechanism in wound-healing process using nf-κb, egfr1, mmp-2, and mmp-9. biotropia vol. 32 no. 3, 2025 312 materials and methods preparation of tectona grandis leaf extract t. grandis leaf were obtained from hutan jati gunung wilis, madiun. the leaves were identified at the taxonomy laboratory of the universitas pgri madiun and were assigned the identification number: 0023/taxo-plant/biology/iv/2021. the leaves were prepared by drying and grinding into powder, then sieved using 60 mesh size. the processed leaf powder was weighed in 0.5 to 2 g. leaf powder was extracted with 95% methanol with sample and methanol ratio of 1 : 5 g/ml (onivogui et al. 2015). the powder was macerated with the solution and filtered with whatman grade 1 which was placed a vacuum filter. the filtrate was evaporated with rotary evaporator to separate the methanol from the extract. the extract obtained was prepared for analysis with liquid chromatography-mass spectrometry (lc-ms). leaf extract was diluted by methanol homogenization until the concentration reached approximately 100 ppm. sample was centrifuged at 8,000 rpm for 10 minutes to remove any insoluble fine solid particles that could have remained in the extract, thus preventing potential clogging of the analytical column. two milliliters of supernatant was collected, then 3 ml of acidified acetonitrile was added to the supernatant. the extract was separated by centrifugation at 8,000 rpm for 30 seconds to obtain clear supernatant containing purified sample. screening of bioactive compounds in tectona grandis leaf methanolic extract the extract of t. grandis leaf was purified using solid phase extraction (spe) to remove interfering compounds according to the procedure of fernand et al. (2009). the sample was loaded to the c18 sep-pak cartridge column (1 ml, 100 mg) which had previously been conditioned with 1 ml mixture of acetonitrile : water with ratio of 80 : 20 to activate the stationary phase. approximately 0.5 ml of the liquid leaf extract was then loaded onto the conditioned column. the column was washed with a series of solutions to selectively elute different compound classes. specifically, 1 ml of water was added to remove highly polar impurities. subsequently, 0.5 ml of acetonitrile : water (80 : 20 ratio) was added to elute semi-polar compounds. finally, 0.5 ml of acetonitrile was used to elute the most non-polar compounds of interest. the final purified solution was filtered using a 0.45 µm cellulose acetate membrane filter and degassed before injection. the sample was analyzed using a shimadzu lcms8040 liquid chromatography-mass spectrometry system. the injection volume was 1 µl. chromatographic separation was performed on a shimadzu shim-pack fc-ods column (2 mm x 150 mm, 3 µm). the mobile phase consisted of a single solvent system in isocratic mode. the mobile phase was methanol : water (90 : 10 ratio), delivered at a constant flow rate of 0.5 ml/minute. the total run time was 80 minutes. the mass spectrometer was operated in positive ion mode. the ion source was electrospray ionization (esi), interface voltage +4.5 kv, desolvation line (dl) temperature of 250 °c, heat block temperature of 400 °c, drying gas flow of 15 l/minute, nebulizing gas flow of 3 l/ minute, and scan mode with full scan (m/z range 100–1000). bioactivity prediction of tectona grandis bioactive compounds seventeen most abundant bioactive compounds of t. grandis leaf extract were identified for their potency, which we selected for further analyses (table 1). the canonical smiles of 17 bioactive compounds were obtained from pubchem and loaded to pass prediction for bioactivity prediction (filimonov et al. 2014). each bioactive compound was later identified for the potency as antioxidant, free radical scavenger, mmp expression inhibitor, and anti-inflammatory. bioactive compounds with pa score ≥ 0.7 were chosen for further analysis, as they indicate a high similarity to compounds in the database that have been proven for treatment and therapy. prediction of drug-likeness and toxicity of bioactive compounds three bioactive compounds selected for bioactivity predictions were continued for identification and prediction of its drug-likeness and pharmacokinetics (banerjee et al. 2024). the canonical smiles of three bioactive compounds were loaded to swissadme (http://www. swissadme.ch/index.php) and protox tool (https:// tox.charite.de/protox3/index.php). in silico study of jati leaf constituents as traditional wound care krisnamurti et al. 313 table 1 dominant bioactive compounds of tectona grandis leaf extract retention time composition (%) bioactive compound pubchem id 23.293 2.48141 epigallocatechin-3-o-p coumarate cid6474788 23.705 2.17803 epigallocatechin gallate cid65064 33.505 2.15389 procyanidin b5 cid124017 22.176 2.13232 6c-glucopyranosylepicatechin cid131752183 33.434 2.09243 proanthocyanidin a-2 cid124025 33.496 1.98748 procyanidin b1 cid11250133 11.566 1.84348 isoobtusilactone a cid6442493 3.042 1.80174 gallic acid cid370 11.958 1.77578 tectograndinol c_id c00022743 (from knapsack) 21.584 1.7546 epigallocatechin-3-o cinnamate cid21629801 19.211 1.73830 epiafzelechin-3-o-gallate cid467295 33.498 1.71418 procyanidin b2 cid124017 10.322 1.70537 kaempferol cid5280863 33.429 1.58901 procyanidin a1 cid5089889 11.562 1.43720 isolinderanolide b sid274339182 12.421 1.43717 chlorogenic acid cid1794427 11.427 1.42081 quercetin cid5280343 molecular docking selected bioactive compounds of tectona grandis leaf extract were further analyzed for the interactions toward proteins involved in woundhealing process. the 3d structures of selected bioactive compounds, i.e., epigallocathecin 3-op coumarate (cid6474788) and epigallocatechin 3-o-cinnamate (cid21629801), were downloaded from pubchem database. the 3d structure of tectograndinol was obtained from the canonical smiles structure provided by the knapsack family c_id c00022743 and loaded to openbabel to construct its 3d chemical structure, later converted into sdf format. curcumin (cid969516), nitrofurazone (cid5447130) and phenytoin (cid1775), as a control, were obtained from pubchem. all ligands, including bioactive compounds and control, were prepared using pyrx 0.8 by minimizing the energy and converting into pdb format. the 3d structure of egfr-1 (3poz), mmp9 (1l6j), mmp2 (7xgj), and nf-κb (1kn) were downloaded from pdb (liu et al. 2021; al mousa et al. 2024). proteins were prepared by removing water molecules and the natural ligands with discovery studio 4.1. ligands were prepared by minimizing the energy and converting to pdb format in pyrx version 0.8. docking was performed by autodock vina integrated in pyrx 0.8. bioactive compounds were interacted to egfr-1 (x = 19.5327, y = 24.7294, z = 15.1872), mmp-9 (x = 39.3102138564, y = 35.2175327806, z = 34.6209), mmp-2 (x = 20.3416, y = -0.3234, z = 20.0629), and nf-κb (x = 38.4173, y = 25.9442, z = 28.1869) in the specific grid box optimized by the software and exhaustiveness 50. interactions between ligands and proteins were visualized by discovery studio 4.1. results and discussion prediction on biological activity of t. grandis leaf extract methanolic extracts of t. grandis leaf contained 82 bioactive compounds based on the lc-ms analysis (fig. 1). bioactive compounds with high peak in the lc-ms result as well as longer retention time were categorized as the abundant bioactive compound and predicted as the dominants. however, some bioactive compounds may elute later due to the strong interaction between analyte and the stationary phase, showing the late in retention time (katajamaa & oresic 2005). our study found that the most abundant bioactive compounds were flavonoids group, followed by proanthocyanidin, phenolic acids, diterpenoids, and lignans. other studies showed that the t. grandis leaf extract contains various bioactive compounds, such as flavonoids, tannins, alkaloids, anthraquinones, anthocyanins, naphthoquinone, and cyanidine (arief et al. 2014; murukan & murugan 2018). bioactive compounds exhibit various biological activities, biotropia vol. 32 no. 3, 2025 314 such as anti-inflammatory, antibacterial, and antioxidants causes the plant to be a traditional agent for treating diabetes, cancer, malaria, and skin diseases (rajuri et al. 2010; suryanti et al. 2020). phenolic contents are the major constituent of t. grandis leaf extract (budianto et al. 2023). phenolic and anthocyanins of t. grandis leaf methanol extract exerts strong antioxidant activity (suryanti et al. 2020). the antioxidant activity of t. grandis allowed the acceleration of the woundhealing process by reducing the oxidative stress and performing anti-inflammatory activity (ponugoti et al. 2013; comino-sanz et al. 2021). seventeen bioactive compounds were screened for biological activity-related to wound-healing mechanisms, which are on anti-inflammatory activity, free radical scavenger, antioxidant activity, and mmp inhibitory activity (table 2). according to the screening, bioactive compounds of t. grandis leaf extract mostly exhibited antioxidant and anticarcinogenic activities. tectograndinol and epigallocatechin3-o-p coumarate showed exceptional activities as anti-inflammatory and mmp-9 inhibitor, respectively. epigallocatechin 3-o-p-coumarate and epigallocatechin-3-o-cinnamate also showed antioxidant potential, as indicated by their high predicted pa scores for free radical scavenging and antioxidant activities. furthermore, these bioactive compounds exhibited the highest pa scores for all biological activities included in the prediction. therefore, epigallocathecin 3-o-cinnamate, epigallocathecin 3-o-p coumarate, and tectograndinol were selected for further analysis. the anti-inflammatory activity of epigallocathecin 3-o-cinnamate and epigallocathecin 3-o-p coumarate may be performed by flavonoids with two connecting benzene rings (a and b) via oxygen-containing heterocycle (c), also with the a ring of glycosyl mode (maleki et al. 2019; han et al. 2023). tectograndinol which appeared with the highest anti-inflammatory activity, categorized as diterpenoid group. diterpenoids are the promising class of secondary metabolites with presenting high anti-inflammatory capacity (gonzález et al. 2015). commercial medicinal plant extracts containing high diterpenoids showed direct anti-inflammatory activity by inhibiting nf-κb, which effectively reduced pain and symptoms of rheumatoid arthritis (gonzález et al. 2015; lv et al. 2015). seven diterpenoid glucosides from fruit extracts presents anti-inflammatory activity against lpsinduced no production in raw 264.7 (liu et al. 2023). previous study revealed the decreased proinflammatory mediators, tnf-α and interleukins (il-1β, il-6), on the macrophage-stimulated lps following treatment with t. grandis leaf extract (han et al. 2023). the complexity of phytochemical constituents from t. grandis also allowed physiological mechanisms. additionally, bioactive compounds from the extract could modulate the immune response to impede the parasitic effect on antigen presenting cells by increasing the cytokine production (dégbé et al. 2018). figure 1 bioactive compounds of tectona grandis leaf extract analyzed by liquid chromatography-mass spectrometry (lc-ms) spectra in silico study of jati leaf constituents as traditional wound care krisnamurti et al. 315 table 2 biological activity prediction of tectona grandis leaf extract bioactive compound free radical scavenger antioxidant mmp9 expression inhibitor anticarcinogenic antiinflammatory epigallocatechin 3-o-p-coumarate pa 0.96 0.87 0.706 0.897 pi 0.001 0.003 0.006 0.003 epigallocatechin gallate pa 0.934 0.814 0.841 pi 0.001 0.003 0.004 procyanidin b5 pa 0.856 0.787 0.848 pi 0.002 0.004 0.004 (-)-epicatechin 6-c-glucoside pa 0.894 0.831 pi 0.002 0.003 proanthocyanidin a-2 pa 0.742 0.704 0.713 pi 0.003 0.004 0.008 procyanidin b1 pa 0.798 0.803 0.757 pi 0.003 0.003 0.007 isoobtusilactone a pa pi gallic acid pa pi tectograndinol pa 0.746 pi 0.011 epigallocatechin-3o-cinnamate pa 0.953 0.863 0.891 pi 0.001 0.003 0.003 epiafzelechin-3-ogallate pa 0.933 0.81 0.844 pi 0.001 0.003 0.004 procyanidin b2 pa 0.798 0.803 0.757 pi 0.003 0.003 0.007 kaempferol pa 0.771 0.856 0.738 0.715 pi 0.003 0.003 0.005 0.008 procyanidin a1 pa 0.742 0.704 0.713 pi 0.003 0.004 0.008 isolinderanolide b pa pi chlorogenic acid pa 0.856 0.785 0.846 pi 0.002 0.004 0.004 quercetin pa 0.811 0.872 0.734 0.757 pi 0.003 0.003 0.005 0.007 drug-likeness and pharmacokinetics prediction of tectona grandis bioactive compounds prior to drug development, it is necessary to predict the drug-likeness and pharmacokinetics properties of the bioactive compounds. druglikeness properties according to lipinski’s rule of five and pharmacokinetics include absorption, distribution, metabolism, excretion, and toxicity (adme/t) which contribute to the physiological effect of a compound (krisnamurti et al. 2021; banerjee et al. 2024). according to the adme/t analysis, epigallocatechin 3-o-p coumarate has 1 violation (table 3). however, the bioavailability score of bioactive compounds was 0.55, which is still in the range of good bioavailability score. it means the bioactive compounds can be easily absorbed by the body (martin 2005). compared to others, tectograndinol exhibits the highest gastrointestinal absorption and blood brain barrier permeability. epigallocatechin 3-o-cinnamate, epigallocatechin 3-o-pcoumarate, and tectograndinol shared similar properties in which lipophilicity and water solubility. the bioactive compounds are more likely lipophilic and moderately soluble in water. biotropia vol. 32 no. 3, 2025 316 the log kp score is important in the analysis of t. grandis bioactive compounds, as wound treatment is mostly used for skin permeability. the log kp value represents how easy the chemical can penetrate the skin, which indicates the significance of skin absorption. the more negative log kp value the lower the potency of a bioactive compound to penetrate the skin (scheler et al. 2014). all three bioactive compounds showed negative scores of skin permeation, while tectograndinol showed the less negative score. virtual prediction indicates tectograndinol as the potent bioactive compound for external application for wound treatment, such as an ointment. though bioactive compounds are known for its safety and less adverse effect, it is important to test the toxicity. toxicity analysis was carried out by predicting ld50 and various toxicity properties in the system. ld50 and class of toxicity represent the amount of a substance that is expected to cause death in 50% of a group of test animals from a single dose. meanwhile, various toxicity properties specifically predict the types of adverse effects that can emerge in an organism (oduola et al. 2010). the bioactive compounds tested in this study were categorized in class 4 and class 5. the ld50 were divided into six categories, i.e., class i (ld50 ≤ 5 mg/kg), class ii (5 < ld50 ≤ 50 mg/ kg), class iii (50 < ld50 ≤ 300 mg/kg), class iv (300 < ld50 ≤ 2,000 mg/kg), class v (2,000 < ld50 ≤ 5,000 mg/kg), and class vi (ld50 > 5,000 mg/kg). the higher the class of ld50 the less harmful the bioactive compound to the body (banerjee et al. 2024). our study showed that tectograndinol possessed less toxicity compared to those of epigallocatechin 3-o-p-coumarate and epigallocatechin 3-o-cinnamate. toxicity prediction showed that epigallocathecin 3-o-cinnamate and epigallocatechin 3-o-pcoumarate had the probability of nephrotoxicity and respiratory toxicity of 0.72 and 0.78, respectively, for both compounds. the nephrotoxicity and respiratory toxicity were probably inactive at the probability of 1 – 0.72 and 1 – 0.78, respectively. moreover, those compounds probably caused immunotoxicity as the score were the highest at 0.9 and 0.94. the probability of compounds to be inactive was less than 10%. tectograndinol showed the highest toxicity in cardiotoxicity with score of 0.83, while it may inactive with probability of 1 – 0.83 which is score 0.17 (17%). overall, bioactive compounds of t. grandis leaf extract showed its safety by not causing hepatotoxicity, neurotoxicity, carcinogenicity, mutagenicity, and cytotoxicity. the bioactive compounds are also safe for the environment, represented by ecotoxicity, so it could be predicted that the bioactive compounds are ecofriendly as if they were developed as a drug (gan et al. 2024). however, development of epigallocatechin 3-o-cinnamate and epigallocatechin 3-o-p-coumarate as a medication may need further consideration as they might be toxic in nephrons, indicated by the red color in the table. according to adme/t analysis, tectograndinol is potentially the safest bioactive compound for drug development. table 3 drug-likeness and pharmacokinetics properties of tectona grandis bioactive compounds epigallocathecin 3-o-cinnamate epigallocatechin 3-o-pcoumarate tectograndinol lipinski yes; 0 violation yes; 1 violation yes; 0 violation bioavailability score 0.55 0.55 0.55 lipophilicity (po/w) 2.45 2.07 3.4 water solubility (logs) -5.18 (moderately soluble) -5.24 (moderately soluble) -4.24 (moderately soluble) gi absorption low low high bbb permeant no no yes p-gp substrate no no no cyp1a2 inhibitor no no no cyp2c19 no no no cyp2c9 no no no cyp2d6 no no no cyp3a4 no no no log kp (skin permeation) -7.07 cm/s -7.42 cm/s -5.93 cm/s ld50 1000 mg/kg 1000 mg/kg 5000 mg/kg toxicity 4 4 5 in silico study of jati leaf constituents as traditional wound care krisnamurti et al. 317 epigallocathecin 3-o-cinnamate epigallocatechin 3-o-pcoumarate tectograndinol lipinski yes; 0 violation yes; 1 violation yes; 0 violation hepatotoxicity 0.68 0.68 0.86 neurotoxicity 0.85 0.85 0.85 nephrotoxicity 0.72 0.72 0.74 respiratory toxicity 0.78 0.78 0.73 cardiotoxicity 0.87 0.87 0.83 carcinogenicity 0.52 0.52 0.61 immunotoxicity 0.90 0.94 0.63 mutagenicity 0.64 0.64 0.75 cytotoxicity 0.77 0.77 0.96 bbb-barrier 0.54 0.54 0.61 ecotoxicity 0.6 0.6 0.54 clinical toxicity 0.54 0.54 0.68 nutritional toxicity 0.64 0.64 0.74 notes: less active active less inactive inactive interactions of tectona grandis bioactive compounds to wound healing-related protein identification of wound-healing mechanism was performed by in silico study using key wound healing proteins, nf-κb, mmp-9, mmp-2, and egfr-1. the potency of three selected t. grandis bioactive compounds on wound-healing-related protein were compared to curcumin, nitrofurazone, and phenytoin as control. curcumin, a polyphenol derived from curcuma longa, is widely known as potential anti-inflammatory and antioxidant agent. study demonstrated the activity of curcumin in reducing the healing time on excisional wound in rats, which mostly plays in the proliferative phase of woundhealing process (patenall et al. 2024). nitrofurazone is an antibacterial agent that has been acknowledged for its potency in wound care. study revealed the activity of nitrofurazone in promoting tissue granulation in the wound, thus leads to wound-healing process (erdur et al. 2008). phenytoin or diphenylhydantoin was firstly established as convulsive disorders treatments. it has an effect on connective tissue and is potentially used in wound care. study demonstrated the wound-healing process in rat burn skin wound model following phenytoin administration. phenytoin aids wound-healing process by developing vascularized and granulation tissue, also synthesizing collagen by re-epithelization (patenall et al. 2024). the activity of bioactive compounds toward nf-κb and mmps were compared with phenytoin, while nitrofurazone was used to compare the activity of bioactive compounds toward egfr1. the activity of t. grandis bioactive compounds against nf-κb were shown in figure 2. nf-κb is an essential transcriptional factor in wound-healing process, which plays major role in cell migration in inflammatory phase and proliferation in proliferative phase. the activation of nf-κb affects the cytokines and growth factor secretion, cell proliferation, also mmps expression (yadav et al. 2024). our study revealed that bioactive compounds from t. grandis leaf extract performed inhibition toward nf-κb in the similar inhibitory sites as phenytoin (table 4). phenytoin posed binding with glu49, arg50, glu222, gln241, and gly259 of nf-κb (-8 kcal/ mol) in which those amino acid residues indicated as the key amino acid residues of nf-κb inhibition. epigallocatechin 3-o-cinnamate performed inhibition via glu222 and gln241 (-9 kcal/mol), epigallocatechin 3-o-p-coumarate via arg50 (-9.8 kcal/mol), while tectograndinol via glu49, gln241, and gly259 (-8 kcal/mol). it could be predicted that t. grandis compounds performed wound-healing process in similar manner as the commercial wound-healing medication. biotropia vol. 32 no. 3, 2025 318 figure 2 interaction of with ligands notes: a = epigallocathecin 3-o-cinnamate; b = epigallocatechin 3-o-p-coumarate; c = tectograndinol; d = curcumin; e = phenytoin. bioactive compounds from t. grandis leaf extract potentially formed binding with the similar amino acid residues as phenytoin. table 4 interactions of tectona grandis bioactive compounds toward nf-κb ligand name category type binding energy (kcal/mol) nf-κb epigallocatechin 3-o-cinnamate glu222, gly237 hydrogen bond conventional hydrogen bond -9gln241 hydrogen bond pi-donor hydrogen bond glu225 electrostatic pi-anion pro275, lys28 hydrophobic pi-alkyl epigallocatechin 3-o-p-coumarate phe239, glu225, pro275, ile224, arg273 hydrogen bond conventional hydrogen bond -9.8 phe239 hydrophobic pi-pi t-shaped arg50, pro275 hydrophobic pi-alkyl tectograndinol glu49. gly259, gln241 hydrogen bond conventional hydrogen bond -7.2glu49 hydrogen bond carbon hydrogen bond arg236 hydrophobic alkyl phe239 hydrophobic pi-alkyl curcumin lys177, thr256, gly183 hydrogen bond conventional hydrogen bond -8 lys177, his173 hydrogen bond carbon hydrogen bond thr179 hydrogen bond pi-donor hydrogen bond thr179 hydrophobic pi-sigma leu346 hydrophobic pi-alkyl phenytoin glu222, gln241 hydrogen bond conventional hydrogen bond -8 glu49;arg50, gly259;arg260 hydrophobic amide-pi stacked arg50 hydrophobic pi-alkyl asp390 electrostatic pi-anion tyr393 hydrophobic pi-pi stacked pro97 hydrophobic pi-alkyl in silico study of jati leaf constituents as traditional wound care krisnamurti et al. 319 the binding sites of t. grandis bioactive compounds toward nf-κb were different from that of curcumin, which suggested that t. grandis bioactive compounds act toward nf-κb differently from curcumin, the common nature-based woundhealing agent. the potency of t. grandis bioactive compounds to inhibit nf-κb also indicated by lower binding energy than those of curcumin and phenytoin. a low binding energy indicates the efficient ability of ligands to bind strongly to their receptors. a receptor-ligand complex with low binding energy is expected to form a strong and stable complex. in contrast, a higher binding energy suggests a weaker binding interaction, which may negatively affect the ligand’s ability to effectively perform its function with the receptor (hasan et al. 2023). epigallocatechin 3-o-cinamate, epigallocatechin 3-o-p-coumarate, and tectograndinol formed binding sites toward mmp-2 differently to each other (fig. 3). in the binding to mmp-2, tectograndinol did not share any identical amino acid residues with the other compounds. however, tectograndinol was an exception to this observation, as it shared two key amino acid residues with the control drug phenytoin, specifically at pro25 and his98 with binding energy of -6.6 kcal/mol. a study conducted by takeuchi et al. (2022) revealed that the interaction of an inhibitor with amino acid residues in the catalytic site, including his121, his131, glu130, ala88, phe87, ala86, phe5, tyr74, and leu82 is crucial for the downregulation of mmp-2 expression. based on molecular docking study, epigallocathecin 3-o-cinnamate may perform mmp-2 inhibition by binding on mmp-2 catalytic sites phe87, ala88, and tyr74 with strongest binding energy -7.9 kcal/ mol (hasan et al. 2023). epigallocatechin 3-o-pcoumarate moderately acts as mmp-2 inhibitor by binding to phe5 with -7.4 kcal/mol. our study showed that curcumin possessed the potential activity to bind with mmp-2 indicated by the highest binding energy score (-9.5 kcal/ mol), while it did not interact with mmp-2 catalytic sites. this molecular docking study revealed that t. grandis bioactive compounds are more potent to bind with mmp-2, compared to binding with curcumin and phenytoin, as the bioactive compounds bind with catalytic sites with lower binding energy than phenytoin. it suggested that t. grandis bioactive compounds may inhibit collagen, fibronectin, laminin, and elastin degradation (wolosowicz et al. 2024). our study revealed that bioactive compounds of t. grandis leaf extract showed less inhibitory activity on mmp-9 (fig. 4). the results suggested that the binding mechanism of t. grandis bioactive compounds to mmp-9 is distinct from that of curcumin or phenytoin, as evidenced by the lack of shared amino acid residues at the binding site (table 5). figure 3 interaction of mmp-2 with ligands notes: a = epigallocathecin 3-o-cinnamate; b = epigallocatechin 3-o-p-coumarate; c = tectograndinol; d = curcumin; e = phenytoin. all compounds posed different binding sites, suggesting the difference of mechanism of action to mmp-2. biotropia vol. 32 no. 3, 2025 320 figure 4 interaction of mmp-9 with ligands notes: a = epigallocathecin 3-o-cinnamate; b = epigallocatechin 3-o-p-coumarate; c = tectograndinol; d = curcumin; e = phenytoin. tectograndinol appeared to have similar binding site as phenytoin. table 5 interactions of tectona grandis bioactive compounds toward mmps ligand name category type binding energy (kcal/mol) mmp-2 epigallocathecin 3-o-cinnamate thr91, tyr74, gly94 hydrogen bond conventional hydrogen bond -7.9tyr74 hydrophobic pi-pi stacked phe87, his85 hydrophobic pi-pi t-shaped ala88 hydrophobic pi-alkyl epigallocatechin 3-o-p-coumarate lys79, asp102 hydrogen bond conventional hydrogen bond -7.4 trp68 hydrophobic pi-pi t-shaped phe5;pro6 hydrophobic amide-pi stacked lys8 hydrophobic alkyl gly71, phe4 unfavorable unfavorable donor-donor tectograndinol phe65, glu58 hydrogen bond conventional hydrogen bond -6.6pro25, val93 hydrophobic alkyl his98 hydrophobic pi-alkyl curcumin arg150, thr144 hydrogen bond conventional hydrogen bond -9.5 gly136, val118 hydrogen bond carbon hydrogen bond leu83 hydrophobic pi-sigma val118 hydrophobic pi-alkyl phenytoin arg67 hydrogen bond conventional hydrogen bond -6.9 his98 hydrophobic pi-pi t-shaped pro25, ile21, met62 hydrophobic pi-alkyl in silico study of jati leaf constituents as traditional wound care krisnamurti et al. 321 ligand name category type binding energy (kcal/mol) mmp-9 epigallocathecin 3-o-cinnamate asp206, glu130 hydrogen bond conventional hydrogen bond -7.4thr336 hydrogen bond pi-donor hydrogen bond thr336, met338 hydrophobic pi-sigma epigallocatechin 3-o-p-coumarate asp182 hydrogen bond conventional hydrogen bond -7.9 asp185 hydrogen bond carbon hydrogen bond asp182 electrostatic pi-anion arg95 hydrophobic alkyl; pi-alkyl tectograndinol asp390 hydrogen bond conventional hydrogen bond -5.9 phe425 hydrophobic pi-alkyl curcumin asn38, leu39, arg51, tyr52 hydrogen bond conventional hydrogen bond -7.4lys184 hydrogen bond carbon hydrogen bond leu39 hydrophobic pi-sigma arg95 hydrophobic pi-alkyl phenytoin tyr393 hydrogen bond conventional hydrogen bond; pi-donor hydrogen bond -6.1asp390 electrostatic pi-anion tyr393 hydrophobic pi-pi stacked pro97 hydrophobic pi-alkyl however, tectograndinol was an exception, as it shared the same amino acid residue, asp390, with the phenytoin. tectograndinol was suggested to inhibit mmp-9 in the inflammation, as the compound also formed binding with phe425, which is known as the active site of mmp-9 (elkins et al. 2002). neither epigallocatechin 3-o-cinnamate nor epigallocatechin 3-o-pcoumarate formed binding interactions with mmp-9 at the same amino acid residues as phenytoin or curcumin, suggesting a different inhibitory mechanism. bioactive compounds of t. grandis actively inhibit mmp-9, as shown by their lower binding energy in the docking study. epigallocathecin 3-o-p-coumarate performed the strongest binding to mmp-9 (-7.9 kcal/ mol). epigallocathecin 3-o-cinnamate binding to mmp-9 was the second strongest (-7.4 kcal/ mol) and tectograndinol binding to mmp-9 was the least strong (-5.9) kcal/mol). the ability of tectograndinol to interact with mmp-9 was relatively lower as compared with control. molecular docking result showed the optimum binding of t. grandis leaf bioactive compounds toward egfr-1 (table 6). t. grandis compounds, epigallocathecin 3-o-cinnamate, epigallocathecin 3-o-p-coumarate, and tectograndinol, were specifically bind to egfr-1 through val726, ala743, and lys745 (fig. 5). moreover, nitrofurazone and curcumin also occupy the same binding pocket as t. grandis bioactive compounds, which indicate the potential of t. grandis bioactive compounds to promote wound-healing process in a similar manner to those of nitrofurazone and curcumin. based on the binding energy, epigallocathecin 3-o-cinnamate was the easiest to bind with egfr-1 (-9.1 kcal/mol). epigallocathecin 3-o-p-coumarate performed a moderate strength with -8.9 kcal/mol binding affinity. the least binding energy was on tectograndinol with -7.4 kcal/mol. binding of t. grandis leaf bioactive compounds to the active sites of egfr-1 may stimulate the activation of egfr-1 for cell migration and proliferation, leading to re-epithelialization and dermal maturation (repertinger et al. 2004; shin et al. 2022). based on the virtual prediction, t. grandis bioactive compounds have as much potential as nitrofurazone in accelerating the woundhealing process. treatment with nitrofurazone increased the re-epithelialization process and decreased the number of inflammatory cells. the bactericidal effect of nitrofurazone contributed to the acceleration of the wound-healing process (karapolat et al. 2020). interestingly, a previous study reported the antibacterial activity of anthraquinones in t. grandis extract. the methanolic and aqueous extract of t. biotropia vol. 32 no. 3, 2025 322 grandis has also demonstrated the inhibitory effect on gram-positive and gram-negative bacteria (irinmwinuwa et al. 2023). treatment of wounds with t. grandis leaf extract may have more potency than that of nitrofurazone. the process of wound healing requires various types of cells and proteins. mmp is one of the proteins regulated during wound-healing process that is activated by nf-κb and upregulated during inflammation stage which mostly driven by reactive oxygen species (ros) (caley et al. 2015; kandhwal et al. 2022; yadav et al. 2024). the balance level of ros may stimulate the signaling pathway of nfκb activation and mmps upregulation, which play major role during wound-healing process. however, ros could also stimulate the ecm turnover, causing tissue destruction and interference with the repair process (hingorani et al. 2019). besides, the prolonged inflammation phase would also delay the wound-healing process. treatment with t. grandis leaf extract with exhibit antioxidant and free radical scavenger activity may balance the ros level during wound-healing process, leading to the acceleration of wound-healing process (sun et al. 2021). a molecular docking study revealed the potential of bioactive compounds from t. grandis in treating wounds, showing a similar mechanism to the known drugs phenytoin and nitrofurazone. in contrast, the wound-healing mechanism of t. grandis leaf compounds may differ slightly from that of curcumin, a natural product. this is supported by the finding that curcumin and the t. grandis compounds bind to different sites on the key wound-healing proteins. furthermore, the study revealed that t. grandis leaf bioactive compounds bind to the active sites of nf-κb, mmp-2, mmp-9, and egfr-1 with high efficiency. based on virtual prediction, our study suggested the mechanism of t. grandis leaf extract in wound-healing process via nf-κb, mmp-2, mmp-9, and egfr-1 (fig. 6), however, further investigation through in vivo and in vitro studies is required for definitive confirmation. table 6 interaction of tectona grandis bioactive compounds toward egfr-1 ligand name category type binding energy (kcal/mol) egfr-1 epigallocathecin 3-o-cinnamate arg841, asn842 hydrogen bond conventional hydrogen bond -9.1leu718 hydrophobic pi-sigma val726, ala743, lys745 hydrophobic alkyl lys745, leu788, val726 hydrophobic pi-alkyl epigallocatechin 3-o-p-coumarate lys745, arg841 hydrogen bond conventional hydrogen bond -8.8 cys797 other pi-sulfur val726 hydrophobic alkyl val726, leu718, ala743, lys745 hydrophobic pi-alkyl tectograndinol lys745, thr854, ala743 hydrogen bond conventional hydrogen bond -7.3 val726, ala743, lys745, leu844, leu718, cys797 hydrophobic alkyl curcumin met793, leu788 hydrogen bond conventional hydrogen bond -8.4gln791 hydrogen bond carbon hydrogen bond leu718 hydrophobic pi-sigma leu844, val726 hydrophobic pi-alkyl nitrofurazone thr854, asp855, phe856 hydrogen bond conventional hydrogen bond -6.2phe856 hydrogen bond pi-donor hydrogen bond val726, ala743, lys745 hydrophobic pi-alkyl in silico study of jati leaf constituents as traditional wound care krisnamurti et al. 323 figure 5 interaction of egfr-1 with ligands notes: a = epigallocathecin 3-o-cinnamate; b = epigallocatechin 3-o-p-coumarate; c = tectograndinol; d = curcumin; e = nitrofurazone. all bioactive compounds posed similar binding sites toward egfr-1. figure 6 prediction of the molecular mechanism of t. grandis bioactive compounds in promoting wound healing via egfr-1 and nf-κb signaling, leading to regulated mmp-2/mmp-9 expression during wound-healing process, nf-κb naturally activated as innate immune response in all cells as a response to infection or injury. it is essential for migration of phagocytic and inflammatory cells to the tissue. the binding of egf and egfr initiates the cell proliferation and migration by pi3k factors activation, jak/stat, plc and pkc, also ras/raf signaling via egf/egfr pathway. the mechanism initiates upregulation of nf-κb signalling which stimulates prolonged inflammation (bodnar 2013; tomas et al. 2014; yang et al. 2022). the upregulated nf-κb may lead to impaired wound-healing process due to the role of nf-κb as precursor of inflammation. in addition, nf-κb activation also facilitate in upregulated mmp-9 expression, leading to prolonged inflammation and development of chronic wound (ambrozova et al. 2017; yadav et al. 2024). moreover, mmp-2 and mmp-9 are jointly involved in the hemostasis process of wound-healing mechanism (kandhwal et al. 2022). expression of mmp-2 during inflammation triggers the mmp-9 in promoting cell migration and re-epithelialization (caley et al. biotropia vol. 32 no. 3, 2025 324 2015; lee & kim 2022). however, upregulated mmp-2 and mmp-9 is associated with chronic wounds. in the vascular cells, overexpressed mmp-2 and mmp-9 promote ecm degradation (ayuk et al. 2016; hingorani et al. 2019). thus, maintaining the expression of mmp-2 and mmp9 is necessary to accelerate wound-healing process (chiang et al. 2023). the bioactive compounds from t. grandis leaves form a strong binding with the receptor, as suggested by the presence of hydrogen bonds. these bonds play a pivotal role in stabilizing the receptor-ligand complex and increasing the ligand’s efficiency in performing its action (krisnamurti et al. 2021). wound care using t. grandis leaves extract could balance each wound healing phase, leading to acceleration of the healing process. the mechanism predicted from this study was that the bioactive compounds of t. grandis leaf act as mmp2 inhibitor and mmp-9 inhibitor. it prevents the upregulation of mmp-2 and mmp-9 expression during inflammation phase in the wound-healing process, leading to the failure of chronic wound development. the controlled mmp-2 and mmp9 expressions initiate the migration of fibroblast to the wound site via ecm remodeling (hingorani et al. 2019). moreover, bioactive compounds from t. grandis leaf have the potential to stimulate egfr1, as shown by molecular docking studies. this protein is identified for its pivotal role in woundhealing process by regulating cell proliferation, survival, and differentiation (chakraborty et al. 2014; sun et al. 2021). a previous study showed that mice treated with egfr-1 exhibited a reduced incision wound width compared to the untreated group. additionally, abundant hair growth was observed in the egfr-treated mice without incision wounds, showing the importance of egfr stimulation during the wound healing process (repertinger et al. 2004). the binding of ligands to egfr-1 may stimulate cell proliferation and migration via the pi3k, jak/stat, plc, and pkc pathways, all of which aid the wound healing process (tomas et al. 2014; yang et al. 2022). our study suggested that the bioactive compounds of t. grandis leaf downregulate nf-κb expression, leading to anti-inflammatory activity. the mechanism also contributes in lowering expression of mmp-2 and mmp-9. the regulated expression of mmp-2 and mmp-9 would allow cell migration during the wound-healing process (lee & kim 2022). treatment and therapy of wounds with t. grandis leaf could be an alternative treatment with high safety and effectiveness. however, the biological activity of t. grandis bioactive compounds in wound treatment should be further studied. conclusion epigallocatechin 3-o-cinnamate, epigallocatechin 3-o-p-coumarate, and tectograndinol were identified as the most abundant bioactive compounds from a total of 82 bioactive compounds contained in t. grandis methanolic extract. the bioactive compounds was predicted to have biological activities related to wound-healing process as antioxidant, free radical scavenger, antiinflammatory, and mmp-9 expression inhibitor. in silico analysis of drug-likeness and adme/t showed that t. grandis bioactive compounds possessed favorable properties and were predicted to be highly safe. this study suggested that bioactive compounds hold potential for developing new medication with reduced side effects. further study by molecular docking demonstrated the woundhealing process via nf-κb, mmp-2, mmp-9, and egfr-1 binding, which showed similar results as that of commonly prescribed woundhealing pharmaceuticals. this study predicted 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(cucurbitaceae) from halmahera, north maluku, indonesia deby arifiani1*, lulut dwi sulistyaningsih1, rugayah1, deni sahroni2 1herbarium bogoriense, research center for biosystematics and evolution, national research and innovation agency (brin), bogor 16911, indonesia. 2directorate of scientific collection management, national research and innovation agency (brin), bogor 16911, indonesia. article higlights • botanical inventory in halmahera island, north maluku has found the species melothria pendula for the first time. • in indonesia, the species melothria pendula is previously known only from java, sumatra, sulawesi, and lesser sunda island. • dna barcodes for the species melothria pendula from indonesia firstly deposited in ncbi genbank. article information received 24 february 2024 revised 10 july 2024 accepted 24 july 2024 *corresponding author, e-mail: darifiani@gmail.com short communication abstract this study was conducted to explore melothria pendula on halmahera island. previously, the species is known to occur in bali, sulawesi, sumatra, java (indonesia), malay peninsula, sabah (malaysia), as well as the philippines. results of this study improved insights into the current knowledge about geographic distribution of melothria. moreover, the description of the species, habitat, critical taxonomic notes, color plates, distribution map, and dna barcode were also presented in this study. keywords: cucurbitaceae, halmahera, maluku, melothria pendula, new record introduction the genus melothria is part of the tribe benincaseae in the cucurbitaceae, a family of vines, shrubs, and herbs. this genus, commonly known as "melonettes" or "mouse melons," was first described by linnaeus in 1753. melothria species are small to medium-sized herbaceous climbers, with simple leaves, usually small, yellow or white flowers, and berry fruits having smooth surface. the fruits contain a large number of highly compressed seeds, and the testa is typically smooth, white, and often covered with long, appressed hairs (de wilde & duyfjes 2010). these plants require warm weather to thrive and are typically grown as annual crops. due to rapid growth and spreading habit, the plants are well suited for use on trellises or in hanging baskets. furthermore, the abundant fruit production throughout the growing season makes the plants to be favored by horticulturists (sinha et al. 1996; mahr 2014). approximately 10 melothria species have been found in the new world, including m. pendula l. this species is considered a weed and indigenous to the caribbean, mexico, central america, and was reported as locally invasive in west africa and tropical asia (de wilde & duyfjes 2010). melothria pendula has been reported as widely distributed in the malesian region, including indonesia (bali, java, sumatra, sulawesi), sabah (borneo), peninsular malaysia, the philippines (de wilde & duyfjes 2006, 2010; mustaqim & putra 2020; husaini et al. 2024). melothria pendula occurs in a variety of habitats, including moist and seasonal dry tropical woodlands, oak woodlands, cloud woodlands, scrub/thicket, and disturbed areas, at altitudes from 0 to 2,050 m. this species blooms and bears fruits throughout the year (guerrero-torres et al. 2022) and in the philippines, the herb has been traditionally used in treatments of several ailments, including inflammation, constipation, burns, and wounds. in mexico, the plant has been used for treating anemia, headaches, gonorrhea, https://doi.org/10.11598/btb.2024.31.2.2205 melothria pendula, a new record of the creeping cucumber from halmahera arifiani et al. 433 and hemorrhoids, as well as for the relief of heart pain. additionally, the fruits and leaves are edible and can be used in the preparation of a variety of sauces. the medicinal properties of the species are attributed to the phytochemical content, such as alkaloids, anthraquinones, quinones, saponins, steroids, terpenes, and xanthoproteins (raju et al. 2021). halmahera is the largest island of the maluku islands, covering an area of 17,780 km² and a part of north maluku province. from a biogeographical stand point, halmahera is also part of wallacea, which is known for unique flora and fauna. wallacea region is rich in minerals, including nickel, cobalt, copper, gold, and silver elements (konopka et al. 2022). currently, there are numerous mining projects underway in halmahera that pose a serious threat to the biodiversity of the island (voigt et al. 2021), underscoring the need for botanical surveys to understand the species at risk (struebig et al. 2022). this study was conducted to explore and enumerate the species present at aketajawe lolobata national park and adjacent areas. furthermore, distribution of melothria pendula was evaluated to update the geographic distribution range. selected taxa were also barcoded to provide the sequences in the genbank. a total of three specimens of small vines belonging to the cucurbitaceae family were discovered and collected during a botanical survey in halmahera island, north maluku province. these vines have small yellow flowers and hanging elliptic fruits resembling small pickles. locals refer to this plant as timun tikus in halmahera language, which means mouse cucumber. these collections were gathered from two sites, namely an open, disturbed forest in the area of geltoli village, maba regency, and a roadside scrub in the area of saramaake village, south wasile regency, east halmahera regency. the plants were subsequently identified as melothria pendula l. comparison with literature and herbarium databases, including the herbarium bogoriense (bo), gdc, k, l, m, and s (retrieved from gbif and jstor global plants), shows that melothria pendula has not been previously identified. therefore, this is the first record of the species from halmahera island, north maluku province. materials and methods fieldwork was carried out in october 2022 and using the standard herbarium technique described by djarwaningsih et al. (2002). the flowering and fruiting specimens were collected, examined, preserved, as well as deposited in herbarium bogoriense (bo). the morphological description of this species was determined based on the examination of the fresh plants, the voucher specimens, and the literatures. to barcode this species, the internal transcribed spacer (its) region was used. plant genomic dna mini kit from geneaid was used to extract the dna genome from silica gel dried leaves. a pair of its5 (5’-gga agt aaa agt cgt aac aag g-3’) as forward primer and its-4 (5’-tcc tcc gct tat tga tat gc-3’) as reverse primer was used for amplification of its regions according to white et al. (1990). heat shock at 95 ºc for 3 minutes was used to start the pcr process and amplification was performed for 35 cycles in three steps. dna was denatured at 95 ºc for 30 seconds and annealing was performed at 55 ºc for 30 seconds. furthermore, dna extension was conducted at 72 ºc for 90 seconds and the final extension occurred at 72 ºc for 5 minutes. purification and sanger sequencing were performed by a company, 1stbase-singapore, and sequences were deposited in genbank. results and discussion melothria pendula l. figures 1 & 2. melothria pendula l., species plantarum ed. 1 (1753) 35; de wilde & duyfjes, flora malesiana i, 19 (2010) 107. type: herb. linn no. 51.1 (lecto linn n.v., designated by wunderlin (1978). melothria guadalupensis  (spreng.) cogn.  in  a.l.p.p.de candolle & a.c.p.de candolle, monogr. phan. 3: 580 (1881). melothria fluminensis gardner in london j. bot. 1: 173 (1842). cucumis glaber walter in fl. carol.: 240 (1788). description. monoecious, perennial herbs with fibrous roots and often a short woody taproot, trailing to procumbent, multi-stemmed at base, tendril on the upper side next to petiole, on fertile shoot next to pistillate flower and later staminate raceme in axil, unbranched, cylindrical, sparsely short-haired, aging glabrous. the stems are 5-ridged with rounded ridges, 2 mm in diameter but swollen at nodes, weak and flexible, striped, biotropia vol. 31 no. 3, 2024 434 internodes mostly 35-80 mm long, short-hairy aging glabrescent, and lack stipules. furthermore, the leaves are alternate, entire, or with 3 or 5-shallowly lobed, petioles with 2-ridged with deep groove on upper side, a petiole shorter than blade, 1-1.7 cm long, striped light green, and dark green or reddish, often strongly bent at base and twisted, scabrous with short hairs, the hard hairs with persistent cystoliths; blade suborbicular, ovate to broadly ovate in outline, 1.1-4.7 x 2.0-6.5 cm, cordate to deeply cordate at base, acuminate apex; margin remotely dentate or shallowly undulate. male inflorescences racemes, with 7-8 dense flowers, peduncle pubescent, 1-1.5 cm long, yellowish green. male flowers have pedicels 1-2.7 mm, receptacle tube ca. 1.5 × 1 mm, sparsely hairy on the outside, sepals ca. 0.3 mm long, petals glossy, yellow, diameter ca. 5 mm, lobes 1-1.3 × 2-2.8 mm, recurved at the tips, stamens 3, filaments ca. 0.3 mm long, anthers ca. 0.8 mm long, and disks ca. 0.5 mm in diameter. furthermore, female flowers are solitary, pedicel sparsely hairy, ca. 18 mm long, corolla ca. 6.5 mm in diameter, lobes ca. 2 × 3 mm, apex obtuse, shallowly recurved, ovary ca. 5 mm long, with ca. 1 mm neck, style ca. 1.2 mm long, stigmas about 1.2 mm long, with 3-lobes, and disk ca. 0.3 mm. fruit berry, ellipsoid, ca. 1.5 × 1 cm, greenish when immature, purplish-black when ripe, sweet taste, while seeds are 15-24, obovate in shape, ca. 5 × 3 mm. figure 1 melothria pendula in halmahera, north maluku province notes: a. living plant; b. leaf with male flower; c. female flower; d. mature fruit hanging on stem; e. seeds. photo: ld sulistyaningsih. a b c d e melothria pendula, a new record of the creeping cucumber from halmahera arifiani et al. 435 distribution. melothria pendula grows naturally in america, from texas up to argentina. this species has been introduced, grown wildly, and then naturalized in other regions, such as asia and west africa. the plants were previously recorded in bali, sulawesi, sumatra (indonesia), peninsular malaysia, sabah (malaysia), and the philippines (de wilde & duyfjes 2010; mustaqim & putra 2020). this study confirmed that the species was present in halmahera, north maluku province as shown in figure 2. considering melothria pendula was not yet used by the local people at the time, it was postulated that the creeping cucumber was naturally dispersed from sulawesi to maluku islands through birds, animals, or ocean currents. the species is also found growing in bogor botanical garden and in cibinong science center botanical garden as well as in adjacent areas of bogor regency, west java (husaini et al. 2024). habitat and phenology. melothria pendula is typically found in disturbed areas, such as fields or roadsides as the native range. in halmahera, this species grows on roadsides in open areas among shrubs, approximately 20 m above sea level (masl). flowering and fruiting plants can be found at the same time (in october), throughout the year. in cibinong, bogor, the species grows well in open areas, but not in the shade. vernacular name. indonesia, halmahera: timun tikus (mouse cucumber). uses. melothria pendula is known to have medicinal properties, especially for diabetes. in halmahera, this species is still being neglected. figure 2 distribution of m. pendula in halmahera, north maluku province specimens examined. indonesia, halmahera island, north maluku province: east halmahera regency, maba district, geltoli village, 0°55'36.7" n, 128°18'23.0" e, c. 20 m altitude, 19 x 2022, arifiani et al. da 2288; south wasile district, saramaake village, 0°59'32.0" n, 127°57'19.5" e, c. 20 m altitude, 21 x 2022, sulistyaningsih et al. lds 631; arifiani et al. da 2319 (bo). notes. melothria pendula, known to thrive in favorable growing conditions, has the potential to become an alien invasive species when the plants lack natural predators or competitors that would keep the population in check. in indonesia, the name timun tikus or mouse cucumber is also applied to another species, namely coccinia grandis (l.) voight. the two species are similar in fruit shape, but differ in flower color and size, as well as the tips of the corolla lobes (yellow and small flowers with obtuse-shallow retuse corolla lobe tips in melothria pendula vs. white and larger flowers with acute corolla lobe tips in c. grandis). in addition, the fruit of both species are ellipsoid and differ only on size, color (in mature), and surface pattern. melothria pendula has a smaller fruit (0.8-1.5 × 1 cm), no longitudinal pattern, purple-black when ripe, while the fruit of c. grandis is more elongated (5-10 x 1.5-3.0 cm) with white longitudinal stripes and purple-red when ripe. the seed size is almost the same for both species, but differ in color. melothria pendula is 5 × 3 mm and a silvery color, while c. grandis 5-6 × 2-3 mm, creamy or light brown color. biotropia vol. 31 no. 3, 2024 436 blasting results table 1 blasting results of melothria pendula from halmahera, north maluku province in ncbi genbank (the top three) collection number species max score total score query cover (%) percentage of identification (%) accession number da 73 (=da 2288) melothria pendula 1,083 1,083 100 99 am981162.1 melothria scabra 1,068 1,068 100 98.67 gu799500.1 melothria pendula 1,050 1,050 100 97.85 am981163.1 da 74 (=da 2319) melothria pendula 1,057 1,057 100 98.98 am981162.1 melothria scabra 1,042 1,042 100 98.64 gu799500.1 melothria pendula 1,024 1,024 100 97.80 am981163.1 lds 631 melothria pendula 1,075 1,075 100 98.84 am981162.1 melothria scabra 1,061 1,061 100 98.50 gu799500.1 melothria pendula 1,042 1,042 100 97.68 am981163.1 accession number table 2 accession numbers of melothria pendula from halmahera, north maluku province no collection number primer accession number 1 da 73 (=da 2288) its 4-5 pp993840 2 da 74 (=da 2319) its 4-5 pp993853 3 lds 631 its 4-5 pp993858 conclusion the distribution of non-native alien species, melothria pendula in indonesia was recorded in halmahera, north maluku province. this result broadened the distribution range from previous studies that have reported the occurrence of melothria pendula in sumatra, sulawesi, lesser sunda island, and java. the study also improved the knowledge on geographic distribution of the genus, melothria. acknowledgment this research was supported by the deputy for facilitation of research and innovation– national research and innovation agency (dfribrin) through the contract no. b-2312/11.7/ hk.01.00/7/2022 for field work in halmahera. we are very grateful to the aketajawe lolobata national park manager and staff for facilitating the research. we extend our gratitude to khairun university. we also extend our thanks to a. sarif and melianus for their tremendous assistance during the botanical exploration. we are deeply indebted to dr. liam a. trethowan who had proofread the english of our manuscript. references de wilde wjjo, duyfjes bee. 2006. redefinition of zehneria and four new related genera (cucurbitaceae), with an enumeration of the australasian and pacific species. blumea 51:1-88. de wilde wjjo, duyfjes bee. 2010. cucurbitaceae. flora malesiana cucurbitaceae series 1 vol. 19. leiden (nl): netherlands centre for biodiversity naturalis, leiden university. djarwaningsih t, sunarti s, kramadibrata k. 2002. panduan pengolahan dan pengelolaan material herbarium serta pengendalian hama terpadu di herbarium bogoriense. herbarium bogoriense-bidang botani, pusat penelitian biologi, lembaga ilmu pengetahuan indonesia. 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pond in surabaya, indonesia alfiyyana nurrahma mawardani1, dini ermavitalini1,*, triono bagus saputro1, farid kamal muzaki1, adi setyo purnomo2, hery purnobasuki3 1department of biology, faculty of science and data analytics, sepuluh nopember institute of technology, surabaya 60111, indonesia. 2department of chemistry, faculty of science and data analytics, sepuluh nopember institute of technology, surabaya 60111, indonesia. 3department of biology, faculty of science and technology, airlangga university, surabaya 60115, indonesia. article higlights • microalgae from its dormitory pond were identified using morphological analysis. • the study analyzed the fatty acid composition of four microalgae genera. • dictyosphaerium had the highest total fatty acid content. • the findings suggest potential applications of these microalgae in biotechnology. • further research is needed to improve fatty acid production. research article abstract microalgae are a diverse group of autotrophic microorganisms classified into prokaryotic and eukaryotic groups. their ability to thrive in various environmental conditions enables the identification of species with distinct fatty acid profiles. the absence of a microalgae culture collection at sepuluh nopember institute of technology (its) motivated the selection of the its dormitory pond as the research site. this study aimed to identify microalgae species present in the pond through morphological characterization and analyze their fatty acid profiles. morphological identification was conducted using taxonomic references, while fatty acid composition was determined via gc-ms analysis. the results identified four genera: dictyosphaerium, vitreochlamys, desmodesmus, and chlorella. their fatty acid profiles varied, comprising saturated (sfa), monounsaturated (mufa), and polyunsaturated fatty acids (pufa). dictyosphaerium contained 17.57% sfa, 12.28% mufa, and 0.33% pufa, while vitreochlamys had 10.83% sfa and 10.19% mufa. desmodesmus exhibited 14.19% sfa, 7.39% mufa, and 4.14% pufa, whereas chlorella had 1.92% sfa and 2.67% pufa. these findings highlight the potential of microalgae from its dormitory pond as a natural source of fatty acids. further research is recommended to optimize culture conditions for enhancing commercially valuable fatty acid production. keywords: fatty acid profile, its dormitory pond, microalgae diversity, microalgae identification, microalgae isolation introduction microalgae are unicellular eukaryotic microorganisms, ranging in size from 2 to 50 μm, distributed across various phyla and capable of thriving in freshwater, brackish, or saline water. as photosynthetic organisms, they play a vital role in aquatic ecosystems, contributing to 40% of global photosynthesis and producing nearly half of earth’s oxygen. their ability to grow photoautotrophically using co2 and light makes their cultivation simple and cost-effective, converting these inputs into biomass and oxygen (elisabeth et al. 2021). microalgae synthesize bioactive compounds, such as proteins, polysaccharides, and lipids, which have applications in pharmacology, medicine, cosmetics, aquaculture, energy, agriculture, and functional foods (dolganyuk et al. 2020). indonesia’s rich biodiversity, with an estimated 200,000 800,000 microalgal species and 50,000 identified species, provides vast potential for exploring microalgae as a source of fatty acids (wu et al. 2021). the growth and biochemical composition of microalgae are influenced by environmental factors, such as temperature, light intensity, nutrient availability, co2 concentration, and ph (lee et al. 2014). this diversity, combined article information received : 14 january 2025 revised : 8 march 2025 accepted : 10 march 2025 *corresponding author, e-mail: diniermavitalini80@gmail.com https://doi.org/10.11598/btb.2024.31.2.1741 biotropia vol. 32 no. 1, 2025 88 with varied habitat conditions, makes indonesia an ideal location for studying microalgal fatty acid profiles. the its dormitory pond was selected as the research site due to the absence of a microalgal isolate culture collection at sepuluh nopember institute of technology (its) and its potential as a natural microalgae habitat influenced by moderate pollution due to student activities. domestic waste disposal contributes organic and inorganic materials, altering the pond’s physical, chemical, and biological properties, which in turn affect microalgal diversity and lipid accumulation. studying microalgae in this environment provides insight into how anthropogenic activities affect microalgal diversity and lipid accumulation, which is crucial for biotechnological applications (mazidah et al. 2013). microalgal fatty acids have attracted increasing interest due to their commercial potential. lipids constitute 1 40% of microalgal dry weight, with some species accumulating up to 85% under specific conditions (chen et al. 2023). studies have shown that prorocentrum donghaiense contains 49.32% ± 1.99% lipids, while nannochloropsis species can accumulate 37 60% of dry weight as lipids (gui et al. 2021; milano et al. 2016). microalgae produce various fatty acids, including saturated (sfa), monounsaturated (mufa), and polyunsaturated fatty acids (pufa) like docosahexaenoic acid (dha) and eicosapentaenoic acid (epa), which are widely used in nutraceuticals production and aquaculture (abdelkarim et al. 2024). despite the known benefits of microalgal fatty acids, there is limited research on the fatty acid profiles of indigenous microalgae species, particularly those from artificial freshwater environments, such as dormitory ponds. understanding the fatty acid composition of microalgal isolates from such habitats provides valuable insight into their metabolic capabilities and potential industrial applications. furthermore, identifying speciesspecific fatty acid profiles can support strain selection for targeted biotechnological applications, including biodiesel production and functional food development (shang et al. 2024). this study aimed to identify microalgae through morphological observation and analyze their fatty acid profiles from isolates collected in the its dormitory pond, surabaya. the findings are expected to contribute to the development of microalgal bioresources for biofuels, pharmaceuticals, and functional foods, while also providing baseline data for future microalgal cultivation and environmental biotechnology research. materials and methods study area this research was conducted from june 2023 to july 2024. water samples were taken from its dormitory pond (fig. 1; table 1). samples were observed in the bioscience and plant technology laboratory, department of biology, faculty of science and data analytics, its, surabaya. microalgae fatty acid profile test was carried out in the faculty of pharmacy, airlangga university, surabaya. procedures preparation of agar medium microalgae were isolated using bold’s basal medium (bbm) agar medium following the protocol of ardiansyah et al. (2018). the medium was sterilized using an autoclave at 121 °c and 1.5 atm pressure for 30 minutes. the agar medium was poured into petri dishes, sealed with plastic wrap, and allowed to solidify before use (lee et al. 2014). microalgae sampling, isolation, and cultivation microalgae were sampled using an active method from two ponds, with five sampling points per pond. water was filtered through a 35 μm plankton net, and the samples were stored in labeled bottles and transported to the laboratory (saputro et al. 2019). subsequently, the water samples were diluted via serial dilution (10-1 to 10-³) by transferring 1 ml of the sample sequentially into test tubes with 9 ml of distilled water (chaidir et al. 2016). purification of microalgae samples was conducted using the streak plate method. petri dishes were incubated at 27 °c under 30 watt fluorescent light for 7 14 days until microalgae grew. isolates were further purified on agar to obtain homogeneous cultures. pure cultures were transferred to a liquid medium in glass bottles for growth (saputro et al. 2019). cultivation was conducted following the protocol of kawaroe et al. (2010) by mixing 20% of microalgae culture with 80% of fresh liquid medium. cultivation began at 20 ml and scaled microalgal fatty acid profiles from its dormitory pond in surabaya 89 to 5 l in media with ph 6, temperature 27 °c, light intensity 30 watts, and continuous aeration (kawaroe et al. 2010). cell growth was monitored daily by measuring optical density at 680 nm (lee et al. 2014). harvesting occurred during the early stationary phase, identified via the growth curve, to maximize metabolic compound accumulation, including lipids. microalgae were separated from the medium using high-speed centrifugation (chen et al. 2011; ferreira et al. 2019). figure 1 the location of microalgae sampling in its dormitory pond, surabaya, indonesia notes: pond a is at the front of the dormitory; pond b is behind the dormitory. table 1 sampling stations coordinates for microalgae collection at the its dormitory pond no. station coordinate 1. pond a (station 1) 7°17'17"s 112°47'32"e 2. pond a (station 2) 7°17'17"s 112°47'32"e 3. pond a (station 3) 7°17'18"s 112°47'32"e 4. pond a (station 4) 7°17'19"s 112°47'32"e 5. pond a (station 5) 7°17'17"s 112°47'32"e 6. pond b (station 1) 7°17'17"s 112°47'29"e 7. pond b (station 2) 7°17'17"s 112°47'30"e 8. pond b (station 3) 7°17'17"s 112°47'30"e 9. pond b (station 4) 7°17'17"s 112°47'29"e 10. pond b (station 5) 7°17'17"s 112°47'29"e biotropia vol. 32 no. 1, 2025 90 % normalization = peak area of individual fatty acid total peak area of all fatty acid x 100( ) morphological identification morphological identification of isolated microalgae was done microscopically (chaidir et al. 2016). samples were placed on a glass slide, added with distilled water, and observed under an olympus bx71 microscope with 1,000x magnification and immersion oil (salindeho et al. 2022). identification was based on characteristics, such as cell color, shape, size, and the presence of flagella and pyrenoids, using the books titled “freshwater algae: identification, enumeration and use as bioindicators” (bellinger & sigee 2015) and “freshwater algae of north america” (wehr et al. 2015). microalgae fatty acid profile test the fatty acid profile of microalgae was analyzed using gc-ms, following these steps: 1) fatty acid extraction, 2) fatty acid derivatization, and 3) gcms analysis (chiu & kuo, 2020). for extraction, 4 ml of hexane was added to 100 mg of the sample, vortexed for 2 minutes, and centrifuged at 3000 rpm for 4 minutes. the hexane extract was transferred to a derivatization tube, evaporated under nitrogen, and treated with 1 ml of 2% naoh at 90 °c for 5 minutes. after cooling, derivatization was performed by adding 1 ml of bf3 and heating for 30 minutes. extraction with 2 ml of hexane was followed by gc-ms analysis (griffiths et al. 2010). the gc-ms analysis was performed using an agilent 7890b network gc system equipped with an agilent 5977b msd detector. separation was achieved using an agilent hp-5ms column (0.25 mm × 30 m × 0.25 μm). the inlet temperature was set to 290 °c, and the oven temperature was programmed as follows: 170 °c (1 minute), increased at 2 °c/min to 180 °c, then at 5 °c/min to 275 °c (5 minutes hold). the carrier gas flow rate was maintained at 1.0 ml/min (constant). the mass spectrometer conditions were set as follows: ms quad temperature at 150 °c, ms source temperature at 230 °c, and a scan range of 20 700 amu. a 1 μl sample was injected with a solvent delay of 0 minute. each fatty acid was identified based on its retention time (rt) and mass spectral match with the wiley 8.0 library. the rt values were recorded to facilitate compound identification and comparison across samples. fatty acids were quantified using relative peak area normalization, and results were expressed as a percentage of total identified fatty acids to provide a normalized profile for each microalgal isolate (patel et al. 2018). the relative abundance of each fatty acid was determined using the peak area normalization method, calculated as follows: data analysis the data obtained from the study were analyzed using an exploratory descriptive method to identify microalgae types from each sampling location. morphological characteristics were examined under a light microscope, and identification was conducted using taxonomic keys. the fatty acid profile test was conducted on successfully cultivated microalgae cells using gc-ms. peak identification was performed by comparing the mass spectra with reference spectra from the wiley 8.0 library, and the relative abundance of each fatty acid was determined using peak area normalization. data were displayed in tabular form and interpreted based on the sampling locations. results and discussion microalgae isolates four distinct microalgae isolates were obtained from the its dormitory pond (table 2). the isolation process involved serial dilution and the streak plate method, repeated until pure isolates were obtained, followed by periodic microscopic observations to confirm the purity of each isolate (fernandez-valenzuela et al. 2021). table 2 microalgae isolate code no. isolate code sampling location color 1. hdb3a pond b (station 3) green 2. 16htdb2b pond b (station 2) dark green 3. 17htdb2b pond b (station 2) dark green 4. kd3a pond b (station 3) yellow morphological identification of microalgae isolates the isolate hdb3a was identified as having round cells with diameters ranging from 4 μm to 10 μm. these cells formed small colonies of 4, 8, 16, or more cells, embedded in a transparent microalgal fatty acid profiles from its dormitory pond in surabaya 91 mucilaginous matrix. each cell contained one or more cup-shaped chloroplasts with chlorophyll, giving them a green appearance. pyrenoids were present within the chloroplasts, playing a role in carbon storage and metabolism (fig. 2, a1). the morphological characteristics of the isolate align with those described by bellinger & sigee (2015), particularly the spherical shape, mucilaginous colony formation, and cup-shaped chloroplasts, confirming its classification within the genus dictyosphaerium. krienitz & bock (2010) also reported that dictyosphaerium species typically form colonies as a defense mechanism against environmental stress. additionally, bock et al. (2011) found that dictyosphaerium-like species develop colonies through mucilaginous stalks originating from remnants of the mother cell, suggesting possible polyphyly within the chlorellaceae family. mikhailyuk et al. (2020) further demonstrated the genus’s morphological diversity by identifying dictyosphaerium-like forms in terrestrial habitats. the presence of this isolate in a pond affected by domestic waste suggested its ability to thrive in nutrient-rich conditions. its colony formation and chloroplast structures further support its adaptability to environmental changes. based on observations, the isolate coded 16htdb2b had oval-shaped cells with diameters ranging from 10 to 17 μm. the transparent and smooth cell walls lacked any distinct ornamentation or structural modifications. a large, cup-shaped chloroplast occupied most of the cell’s interior, containing chlorophyll pigments and pyrenoids, which functioned as storage centers for photosynthetic products, such as starch. these microalgae exhibited motility, suggesting the presence of flagella, although the flagella were not clearly observed (fig. 2, b1). the morphological characteristics of this isolate align with the description of the genus vitreochlamys provided by wehr et al. (2015). vitreochlamys is a unicellular green flagellate with a smooth, swollen cell wall that separates from the protoplast and features a prominent anterior papilla. vegetative cells are solitary, biflagellate, and broadly spindle-shaped, pyriform, or ellipsoidal. they typically measure 24 34 μm in length and 10 22 μm in width, with flagella that are equal to or slightly shorter than the cell length. two contractile vacuoles are positioned near the base of the flagella. the chloroplast has multiple lobes and contains 4 8 similarly sized pyrenoids, which are ellipsoidal, ovoid, or beanshaped. these pyrenoids are surrounded by two large starch grains and are scattered throughout the chloroplast (nakazawa et al. 2001). observations showed that the isolate coded 17htdb2b had ellipsoid or cylindrical cells, measuring approximately 10 30 μm in length and 5 15 μm in width. the cell walls featured a complex structure with prominent spines or projections at both ends. the isolate formed colonies of 2 cells to 4 cells arranged in linear or flat formations. these microalgae were non-motile, as they lacked flagella (fig. 2, c1). the morphological characteristics of this isolate are consistent with the genus desmodesmus, as described by bellinger & sigee (2015). the spines in desmodesmus serve as a defense mechanism against microzooplankton predation, as their rigid structure makes ingestion difficult (trainor 1996). additionally, the spines enhance buoyancy, helping the cells remain in the photic zone for optimal photosynthesis (zhu et al. 2018). the surface morphology of desmodesmus species, observed under sem, varied significantly. for example, d. armatus exhibited a warty surface, d. quadricauda had a net-like structure, and d. serratus displayed a star-like pattern. many desmodesmus species show phenotypic plasticity, alternating between unicellular and colonial forms, as well as spiny and non-spiny variants. environmental factors influence these transitions; for instance, d. subspicatus and d. armatus develop spiny colonies under low nutrient conditions, while unicellular forms dominate in phosphorus or nitrogen-rich environments. when iron availability is low, both colonies and unicellular forms become non-spiny (shubert et al. 2014). the isolate coded kd3a consisted of small, round, single cells with diameters ranging from 2 μm to 10 μm. each cell contained a large, parietal chloroplast occupying most of the cell volume, giving it a green color due to the presence of chlorophyll. the chloroplasts also contained pyrenoids, which facilitate photosynthesis. these microalgae were non-motile as they lacked flagella (fig. 2, d1). the morphological characteristics of this isolate align with those of the genus chlorella, as described by bellinger & sigee (2015). chlorella species are unicellular green algae characterized by spherical or ovoid cells, typically measuring 2 10 μm in diameter. their robust cell walls are composed of cellulose and pectin, providing structural integrity (takeda 2004). the genus is biotropia vol. 32 no. 1, 2025 92 distinguished by a single, cup-shaped chloroplast with a prominent pyrenoid, essential for carbon fixation and starch storage. chlorella is commonly found in nutrient-rich aquatic environments, but can be easily overlooked due to its small size. its adaptability to various environmental conditions, including polluted waters, makes it a significant genus in both ecological and biotechnological studies (bellinger & sigee 2015). figure 2 observed microalgae in this study that have been identified by comparing to literatures notes: a1 = observed dictyosphaerium (hdb3a); a2 = dictyosphaerium granulatum (algaebase.org); a3 = dictyosphaerium granulatum (wehr et al. 2015); b1 = observed vitreochlamys (16htdb2b); b2 = vitreochlamys incisa (ccap.ac.uk); b3 = vitreochlamys aulata (wehr et al. 2015); c1 = observed desmodesmus (17htdb2b); c2 = desmodesmus communis (algaebase.org); d1 = observed chlorella; d2 = chlorella vulgaris (algaebase.org); d3 = chlorella vulgaris (wehr et al. 2015). microalgal fatty acid profiles from its dormitory pond in surabaya 93 fatty acid profile analysis the harvesting of microalgal isolates was performed prior to conducting fatty acid profile analysis. the optimal harvest time was determined based on the growth curve of the microalgae, specifically during the early stationary phase. this phase is crucial as it marks the onset of metabolite accumulation, including lipids. during the early stationary phase, cell density reaches a constant and maximum level, resulting in the highest biomass concentration (ferreira et al. 2019). the early stationary phase can be observed through the microalgae growth curve (fig. 3). according to the growth curve, the harvest times for the microalgae were as follows: dictyosphaerium was harvested on day 16, vitreochlamys on day 15, and both desmodesmus and chlorella on day 13. the study results indicated that the microalgal species with the highest total fatty acid content was dictyosphaerium (30.18%), followed by desmodesmus (25.72%), vitreochlamys (21.02%), and chlorella (4.59%). the fatty acid composition of microalgae isolates from the its dormitory pond waters showed significant variation. the fatty acid profile test results indicated that most isolates had high fatty acid content, particularly elaidic acid (c18:1), palmitic acid (c16:0), and stearic acid (c18:0) (table 3). figure 3 growth curves of microalgal isolates based on optical density (od) at 680 nm over time notes: a = dictyosphaerium; b = vitreochlamys; c = desmodesmus; d = chlorella; the yellow dots indicate the optimal harvest time. biotropia vol. 32 no. 1, 2025 94 table 3 fatty acid profile data of microalgal isolates from the its dormitory pond waters no. retention time component name % normalization dictyosphaerium vitreochlamys desmodesmus chlorella 1. 3.40 lauric aci (c12:0) 0.11 2.33 2. 6.04 myristic acid (c14:0) 0.85 0.52 1.41 0.01 3. 8.20 myristic acid (c14:0) 0.20 4. 8.17 pentadecanoic acid (c5:0) 0.26 5. 10.20 hexadienoic acid (c6:2)** 0.32 0.22 6. 10.57 palmitic acid (c16:0) 1.02 7. 11.40 palmitic acid (c16:0) 9.32 6.83 6.49 1.21 8. 14.52 margaric acid (c17:0) 0.16 9. 16.41 linoleic acid (c18:2)** 0.33 2.46 0.48 10. 16.62 α-linoleic acid (c18:3(ω-3))** 1.97 11. 16.70 elaidic acid (c18:1)* 12.28 10.19 7.39 12. 17.27 stearic acid (c18:0) 5.52 2.49 2.47 0.44 13. 20.17 arachidonic acid (c20:4(ω-6))** 0.44 14. 20.55 eicosatrienoic acid (c20:3(ω-3))** 0.22 15. 21.59 arachidic acid (c20:0) 0.54 0.35 0.05 0.26 16. 23.87 eicosapentaenoic acid (c20:5(ω-3))** 0.70 17. 25.20 behenic acid (c22:0) 1.03 0.64 total sfa 17.57 10.83 14.19 1.92 total mufa 12.28 10.19 7.39 total pufa 0.33 4.14 2.67 total fatty acids 30.18 21.02 25.72 4.59 notes: * = monounsaturated fatty acids; ** = polyunsaturated fatty acids. microalgal fatty acid profiles from its dormitory pond in surabaya 95 dictyosphaerium exhibited the highest saturated fatty acid (sfa) content among the four microalgae species, with a total of 17.57% comprising lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, and behenic acid. the species also showed the highest level of monounsaturated fatty acids (mufa), at 12.28%, predominantly as elaidic acid. conversely, dictyosphaerium had a lower content of polyunsaturated fatty acids (pufa), amounting to 0.33%, primarily as linoleic acid (fig. 4, a). in contrast, nayana et al. (2018) reported that dictyosphaerium contained the highest levels of sfa, specifically palmitic acid (22.64%). the mufa accounted for 44.24%, including oleic acid (38.08%) and palmitoleic acid (6.16%). the pufa content was 8.93%, with linoleic acid being the predominant pufa. the discrepancy between the results of this study and those reported by nayana et al. (2018) may be attributed to differences in cultivation conditions, which influence the composition of fatty acids produced during growth. vitreochlamys exhibited an sfa content of 10.83%, consisting of myristic acid, palmitic acid, stearic acid, arachidic acid, and behenic acid. the mufa content was 10.19%, predominantly as elaidic acid, with no detected pufa (fig. 4, b). research on the fatty acid profile of the genus vitreochlamys is currently limited. comparable studies within the family chlamydomonadaceae can be observed, such as in chlamydomonas sp., which contains sfa, including myristic acid (2.62%) and palmitic acid (78.35%). this species also has mufa, like palmitoleic acid (4.26%) and oleic acid (2.48%), and pufa, such as linoleic acid (3.49%) and arachidonic acid (3.18%). palmitic acid (c16:0) is the most abundant fatty acid in chlamydomonas sp., accounting for about 85% of the total fatty acid content, with 9% being pufa (morando-grijalva et al. 2020). desmodesmus exhibited an sfa content of 14.19%, which includes lauric acid, myristic acid, pentadecanoic acid, palmitic acid, margaric acid, stearic acid, and arachidic acid. the mufa content was 7.39%, primarily as elaidic acid. notably, desmodesmus had the highest pufa content among the four species analyzed, with a total of 4.14%, including hexadecanoic acid, linoleic acid, arachidonic acid, eicosatrienoic acid, and eicosapentaenoic acid (fig. 4, c). in other studies, desmodesmus was found to contain different compounds under autotrophic (bourdeau et al. 2017), heterotrophic (deschênes et al. 2015), and mixotrophic growth conditions (okpozu et al. 2019). chlorella exhibited an sfa content of 1.92%, which included myristic acid, palmitic acid, stearic acid, and arachidic acid. the pufa content was 2.67%, comprising hexadecanoic acid, linoleic acid, and α-linolenic acid. notably, no mufa were detected in chlorella in this research (fig. 4, d). according to rushan et al. (2021), chlorella cultivated in bg-11, jm, and bbm media displayed typical fatty acid compositions found in microalgal biomass, including palmitic acid (c16:0), stearic acid (c18:0), oleic acid (c18:1), linoleic acid (c18:2), and linolenic acid (c18:3). the highest sfa content was observed in chlorella cultivated with bbm, followed by jm and bg-11 media. the highest sfa composition in bbm-cultivated chlorella was attributed to c16:0 (40.74%) and c18:0 (33.33%) of total fatty acid methyl esters (fame). the highest ufa composition was found in bbm due to the presence of c18:1 (11.11%), c18:2 (7.41%), and c18:3, (3.7%) of total fame. this shows that using different media will produce different fatty acid contents (rushan et al. 2021). biotropia vol. 32 no. 1, 2025 96 microalgal fatty acid profiles from its dormitory pond in surabaya 97 figure 4 chromatogram of fatty acid profile notes: a = dictyosphaerium; b = vitreochlamys; c = desmodesmus; d chlorella; ea = elaidic acid; pa = palmitic acid; sa = stearic acid; αla = α-linoleic acid. biotropia vol. 32 no. 1, 2025 98 conclusion this study successfully identified various microalgae from the waters of its dormitory pond in surabaya, including dictyosphaerium, vitreochlamys, desmodesmus, and chlorella. the fatty acid profiles of the isolated microalgae species varied. dictyosphaerium was found to have a saturated fatty acid (sfa) content of 17.57%, monounsaturated fatty acid (mufa) content of 12.28%, and polyunsaturated fatty acid (pufa) content of 0.33%. vitreochlamys exhibited an sfa content of 10.83% and a mufa content of 10.19%, with no detected pufa. desmodesmus showed an sfa content of 14.19%, mufa content of 7.39%, and pufa content of 4.14%. chlorella had an sfa content of 1.92% and a pufa content of 2.67%, with no detected mufa. future research should focus on optimizing culture conditions to enhance lipid accumulation and improve fatty acid composition for commercial applications. investigating genetic and metabolic pathways could further increase lipid productivity and tailor fatty acid profiles for specific industrial uses. additionally, evaluating large-scale cultivation feasibility and sustainability aspects will be essential for developing microalgae-based bioresources in biofuels, nutraceuticals, and pharmaceuticals. acknowledgments the authors express their sincere gratitude to ratna syifa’a rahmahana and etika ziadana al husna for their invaluable contributions as laboratory partners, particularly in data collection, analysis, and interpretation for this project. the authors also extend their appreciation to the sepuluh nopember institute of technology for its support and the provision of research facilities. this study was funded by the its scientific research scheme 2023. references abdelkarim oh, wijffels rh, barbosa mj. 2024. microalgal lipid production: a comparative analysis of nannochloropsis and microchloropsis strains. j appl phycol 37(1):15-34. ardiansyah sr, orlando am, rahman a, prihantini nb, nasruddin. 2018. effect of aeration in simple photobioreactor system for biomass production of synechococcus sp. 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purnawati*, endang triwahyu prasetyawati, haidar fari aditya, and saefur rohman department of agrotechnology, faculty of agriculture, university of pembangunan nasional “veteran” jawa timur, surabaya 60294, indonesia article higlights seed encapsulation using alginate and b. cereus bth21 for biocontrol of wilt disease in eggplant. article information received : 25 february 2025 revised : 17 june 2025 accepted : 15 july 2025 *corresponding author, e-mail: arika_p@upnjatim.ac.id research paper abstract bacterial wilt is a major plant disease caused by ralstonia solanacearum that infects important crops, such as eggplant, causing wilt, stunted growth, and reduced yield. the biocontrol of the pathogen can be done by employing antagonistic bacteria, yet direct inoculation into the soils are often ineffective due to low population of the pathogen in the soils, which also easily washed away by watering activity. seed encapsulation technique, using alginate, zeolite and peat, containing antagonistic bacteria bacillus cereus bth21 to control r. solanacearum is the novelty proposed in this research. hence, this research aimed to develop alginate seed-encapsulation technique containing bacillus cells as a mean to biologically control r. solanacearum in eggplant. endophytic bacillus cereus bth21 strains was isolated from healthy eggplant tissue obtained from kediri, east java province, indonesia. subsequent isolation, purification and molecular identification confirmed the identity of the strain. three seed-encapsulation formulations were made: a) control (no encapsulation); b) alginate + zeolite + bacillus (al+z+b); and c) alginate + peat + bacillus (al+p+b). a field experiment was designed in a completely randomized design (crd) where the alginate-encapsulated eggplant seeds were sown along with deliberate r. solanacearum inoculation (107 cfu/ml) into the soils. observations were carried out every 7 days from 0 – 49 days after sowing (das) on several parameters, i.e., incubation period, infection rate, germination percentage, plant height, and number of leaves. the data were analyzed by using analysis of variance (anova) followed by duncan’s multiple range test at a significance level of p < 0.05. the results showed that both seed encapsulation formulations prolonged incubation period (15 and 13 das) compared to the control (10 das), reduced infection rate, increased germination rate, as well as higher agronomic performances (plant height, number of leaves) compared to the control at 7 – 49 das. overall, this results indicated the potential of alginate seed-encapsulation techniques containing biocontrol agents to control plant diseases. keywords: alginate, bacillus, bacterial wilt, biocontrol, endophytic, seed encapsulation introduction eggplant (solanum melongena l.) is an important vegetable crops from the nightshade family of solanaceae. eggplant is used quite extensively in indonesian cuisine due its nutritional content (protein, vitamins, minerals, phenols, flavonoids, and anthocyanin) (sharma & kaushik 2021) and unique bitter-astringent flavor that become tender once cooked (emeribe & ogbuehi 2024). eggplant production and consumption in indonesia is still lacking. recent data showed that national per capita consumption of vegetables (40.66 kg/cap/year), including eggplant, is still lower than the recommendation from fao (73.00 kg/cap/year) (saliem & ariningsih 2015). in 2022, indonesia produced approximately 0.7 million copyright (c) 2025@author(s). https://doi.org/10.11598/btb.2025.32.3.2410 https://creativecommons.org/licenses/by-nc-nd/4.0/ biotropia vol. 32 no. 3, 2025 278 tonnes of eggplant, but this accounted for only 1.15% of global eggplant production, which was disproportionate to indonesia’s share of the world population (fao 2025). bacterial wilt disease caused by ralstonia solanacearum hampers eggplant production. once attacked, the plants develop symptoms, such as wilted and yellowing leaves as well as the appearance of a milky white bacterial fluid which is a mass of bacteria which accumulates on the surface of the stem of the newly cut infected host. this pathogen is widely spread in many countries, harbored in multiple hosts, and capable to cause 100% harvest failure (gbonamou et al. 2020). this pathogen also known to survive for quite a long time in soil or water when there are no host plants available. the pathogen also thrives in hot and humid environment, accumulates in the soil for extended periods of time, can also lead to disease outbreaks when environmental conditions become favorable for infection (ascarrunz et al. 2011; lie et al. 2016). conventional control by using chemical bactericides has been criticized for inconsistent results, residue buildups, and adverse effects on human health and environments. therefore, alternative strategies to control the wilt disease bacteria must be developed to provide safe and high quality eggplant products (sun et al. 2023). endophytic bacteria, such as bacillus sp., can be used to biologically control r. solanacearum. this bacteria live within plant tissues in a mutualistic relationship without causing negative effects, while also provides protection from the pathogens and promote plant growth due to multiple mechanisms. this bacteria produce siderophore, antibiotics, as well as induce plant systemic resistance, to protect plants against the pathogens. plant growth promotion is provided by producing phytohormones (auxin, cytokinin), fixing atmospheric nitrogen, and increasing the bioavailability of nutrients (phosphate, calcium) (santos et al. 2018). for instance, b. subtilis r31 is shown to stabilize colonies in the rhizosphere and root tissues and to reduce the incidence of r. solanacearum wilt disease in tomato (sun et al. 2023). a similar work also reported the efficacy use of four bacillus species, e.g., bacillus subtilis, b. cereus, b. pumilus, and b. amyloliquefaciens to control r. solanacearum in an in vitro condition (singh et al. 2016). beside the bacterial strain, it is important to optimize the method for delivering the bacteria into the soils. conventional methods, such as soaking roots in the bacterial suspension, drenching the soils with the suspension, or mixing the bacteria with fertilizers, are often considered ineffective because the suspension is easily washed away by water, hence fail to maintain high population of active bacterial cells in the rhizosphere. another consideration is that bacterial strains are often isolated from other environments that are different from the target soil, which may reduce their survival probability due to environmental discrepancies (castro-sowinski et al. 2007; lopes et al. 2021). hence, it is important to develop an alternative strategy to deliver the bacterial strain, ensuring high survival and population. alginate seed encapsulation containing bacterial cells is a recent technology for delivering active bacterial cells into the soil. alginate is a biocompatible and biodegradable natural polymer derived from seaweed that forms hydrogels when crosslinked with calcium ions (ca²+) (rohman et al. 2021). active bacterial cells can be mixed with an alginate solution, entrapping and immobilizing them within the encapsulation matrix to provide a supportive microenvironment for containing and delivering the cells into the soil (riseh et al. 2021). to produce encapsulated seeds, plant seeds can be dipped into the solution to form alginatecoated seeds containing the cells (berninger et al. 2016). additionally, other carrier materials, e.g., zeolite and peat, can be blended with alginate to enhance capsule properties, such as increasing structural integrity, improving controlled-release mechanisms, and creating a more supportive microenvironment for the entrapped cells (hurtado et al. 2022). zeolite is an inorganic crystalline materials that provides better structural integrity and stability, moisture retaining ability, and ion exchanges capability, improving capsules properties and microenvironments for the cells (ciarleglio et al. 2023). in a similar fashion, peat has porous structure, high water-holding capacity, and nutrients content which will improve bacterial survivability for a long time (reddy 2012; novinscak & filion 2020). several works documented the success of seed-encapsulation technique to entrap active cells. for instances, trichoderma harzianum was immobilized in alginate capsule to control bipolaris oryzae causing brown spot disease in rice (anuar et al. 2019), bacillus-embedded seed encapsulation as biocontrol strategy against bacterial wilt in eggplant purnawati et al. 279 pseudomonas putida was encapsulated in alginate/ gelatin beads to control fusarium solani on potato (pour et al. 2019), and bacillus megaterium was immobilized in alginate microcapsules to control rhizoctonia solani rice sheath blight disease (wiwattanapatapee et al. 2013). therefore, the aims of this research were: a) to isolate and identify endophytic bacillus strains from eggplant tissue; b) to develop an alginate seed encapsulation formulation blended with zeolite and peat to immobilize and deliver bacillus cells into the soil; and c) to evaluate the in vivo efficacy of the bacillus-embedded seed encapsulation technique for biologically controlling bacteria causing wilt disease and improving eggplant growth. the results of this research will provide a scientific contribution to the potential of bacterialcontaining seed encapsulation technology for controlling plant diseases, particularly bacterial wilt caused by r. solanacearum. materials and methods isolation and rejuvenation of bacillus cereus bth21 and ralstonia solanacearum the endophytic bacterial strain b. cereus bth21 was obtained from a previous exploration study conducted in kediri, east java province (purnawati & nirwanto 2021). the strain was isolated from healthy eggplant plants. meanwhile, r. solanacearum was also collected from infected eggplants at the same location. both bacterial stocks were stored long-term on nutrient agar (na himedia laboratories pvt.ltd. maharasthra, india) overlaid with paraffin oil. for daily use, the stocks were rejuvenated by streaking them onto fresh na media. molecular identification of bacillus cereus bth21 species identification was done molecularly by sequencing 16s rrna gene of bth21 isolate. the dna was isolated by using qiaamp dsp dna ffpe tissue kit (qiagen n.v., jerman) while following the instructions from the manufacturer. the dna was further amplified using primer pairs 27f (5’-agagtttgatcmtggctcag-3’) as the forward primer and 1492r degenerate primer (3’-tacggytaccttgttacgact-5’, y = c or t) as the reverse primer (isik et al. 2014). sequence amplification was carried out with a 2720 thermal cycler (applied biosystems, thermo fisher scientific, massachusetts, usa). the pcr cocktail was prepared using rtg beads (ge healthcare, uk), which contained bsa, datp, dctp, dgtp, dttp, 2.5 units of puretaq dna polymerase, and a reaction buffer. each bead was sufficient for a final reaction volume of 25 µl, providing a concentration of 200 µm for each dntp in 10 mm tris-hcl, 50 mm kcl, and 1.5 mm mgcl2. the composition of the reaction for a final volume of 25 µl included 1 rtg bead, 3 µl of primers (5 pmol/µl), 3 µl of dna sample (± 5 ng/µl), and 19 µl of ddh₂o. the pcr program was set as follows: predenaturation at 95 °c for 5 minutes; denaturation at 94 °c for 1 minute; annealing at 55 °c for 1 minute; extension at 72 °c for 1.5 minutes; and a final extension at 72 °c for 5 minutes, for a total of 35 cycles. the pcr products were confirmed by electrophoresis on a 1% (w/v) agarose gel. to check the quality and quantity of each pcr product, a mixture of 5 µl of amplicon (pcr product solution) and 1 µl of 10x bpb was prepared. the mixture was loaded onto the gel and run for 30 minutes at 100 v. the bands were observed and documented using a gel documentation system. the extracted dna was then sequenced by using next-gen sequencing method and a phylogenetic tree was constructed by using neighbor joining (unrooted tree) blast tree method. preparation of alginate-seed encapsulation containing bacillus cereus bth21 b. cereus bth21 was first cultured in nutrient broth (nb himedia laboratories pvt.ltd. maharastrhra, india) medium by means of shaking for 3 days to prepare the inoculant. subsequently, the culture’s absorbance was measured on a uv-vis spectrophotometer and the density of the culture was set to 108 cfu/ml. the bacterial cells were then harvested by centrifugation at 8,000 rpm for 15 minutes the cell pellet was then diluted by using normal saline solution (nacl 0.85% b/v) as inoculant to be used in the next step. two eggplant seed encapsulation formulations were prepared, i.e., a) alginate + zeolite + b. cereus bth21 cells (al+z+b) and b) alginate + peat + b. cereus bth21 cells (al+p+b). non-encapsulated seeds were also prepared as a control, resulting in three treatments tested in this study. to prepare the encapsulated seeds, a 3% (w/v) sodium alginate (na-alginate) solution was first prepared as the primary matrix. then, 1% (w/v) zeolite or peat was added and thoroughly mixed into the alginate solution, serving as the additional carrier material. subsequently, a b. cereus bth21 cell suspension biotropia vol. 32 no. 3, 2025 280 (10⁸ cfu/ml) was added as the inoculant. the mixture was stirred thoroughly to ensure even distribution of the bacterial cells, forming the final encapsulation matrix. next, eggplant seeds were dipped into the matrix and gently dropped into a hardening solution containing 2% (w/v) calcium chloride (cacl₂). hydrogel capsules were immediately formed and left to harden for 10 minutes. the encapsulated seeds were then harvested, air-dried, and stored at room temperature for further use. field experiment a field experiment was conducted to evaluate the efficacy of alginate-encapsulated eggplant seeds containing b. cereus bth21 in controlling r. solanacearum in vivo. the experiment was conducted at the experimental garden of the faculty of agriculture faculty, upn “veteran” jawa timur, located at 7°9′ – 7°21′ s and 112°36′ – 112°54′ e. the field experiment was arranged in a completely randomized design (crd) with three treatments of seed encapsulation formulations: a) non-encapsulated seeds as the control; b) alginate + zeolite + b. cereus bth21 (al+z+b); and c) alginate + peat + b. cereus bth21 (al+p+b). each treatment was replicated three times. the encapsulated eggplant seeds were sown at a depth of 3 cm in two rows per bed, with a planting distance of 70 × 90 cm. the seeds were allowed to germinate and grow under proper plant maintenance, which included watering, replanting, staking, and other agronomic practices. the plants were maintained until 49 days after sowing (das). a deliberate inoculation with r. solanacearum was carried out by drenching a bacterial suspension (10⁸ cfu/ml) at 49 das, followed by incubation for 48 hours to evaluate the efficacy of b. cereus bth21 in suppressing the pathogenic bacterium. parameters observation and data analysis the parameters observed included plant height, number of leaves, number of flowers, incubation period, infection rate, and seeds germination percentage. agronomic performances were measured in terms of plant height (cm) and number of leaves. the incubation period (das) was defined as the number of days required for the first plant in each treatment to show visible bacterial wilt symptoms, such as wilting, yellowing and chlorosis of leaves, as well as soft, decayed stems and roots. plant height and number of leaves were measured every 7 days from 7 to 49 das. similarly, infection rates (%), germination percentage (%) were calculated using the following formulas: infection rates (%) = (number of infected plants/ total plants observed) x 100% germination percentages (%) = (number of seeds germinated/total seeds sown) x 100% analysis of variance (anova) was conducted to evaluate the effects of different treatments. duncan’s multiple range test was applied when significant differences were detected by anova. statistical significance was set at p < 0.05. data analysis was performed using the statistical software program spss version 20 (spss inc.) (ibm corp. us). results and discussion macroscopic and microscopic identification of bacterial strain our study showed that the macroscopic identity of bacterial strain bth21 colonies on na media are whitish cream, round in shape with irregular edges, the surface is flat, dry, not shiny; while microscopically, the form of the bacterial strain bth21 is bacil (rod), gram-positive, produces endospores (table 1). logan and vos (2015) stated that bacillus sp. have various characteristics, such as flat and uneven, rough and non-slimy surfaces, dry and powdery, and not shiny. puspita et al. (2017) stated that bacillus sp. have rod-shaped cells, are grampositive and have endospores. molecular identification of the bacterial bth21 strain the identity of bacterial bth21 strain was assessed molecularly through sequencing the 16s rrna gene and comparing the sequence to ncbi databases by using blast procedure. a primer pair of 27f and 1525r was used to amplify the gene. further step was carried out to generate a phylogenetic tree of the isolates using neighbor joining (unrooted tree) blast tree method. the results are displayed in figure 1 below. the band on gel electrophoresis of bth21 indicated that the pcr product size was around 1,500 bp. molecular identification on partial 16s rrna gene sequence analysis indicated that the isolated bth21 was affiliated with bacillus genera bacillus-embedded seed encapsulation as biocontrol strategy against bacterial wilt in eggplant purnawati et al. 281 and grouped with other bacillus spp., for example bacillus cereus, bacillus anthracis, bacillus tropicus etc. obtained from ncbi nucleotide database. a 99% homology was obtained with b. cereus xjq1xx-254b, indicated that the isolate belonged to bacillus cereus species. hence, the full identity of the isolate is bacillus cereus bth21. macroscopic microscopic colony colony (stereo microscope, 40x) cell and gram (compound microscope, 100x) endospore (stereo microscope, 100x) table 1 macroscopic and microscopic identification of bacterial strain figure 1 visualized pcr product of bth21 isolate 16s rdna gene on agarose gel (inset) and phylogenetic tree of the isolate, generated using neighbor joining (unrooted tree) blast tree method biotropia vol. 32 no. 3, 2025 282 incubation period the incubation period is defined as the time required for the first visible emergence of bacterial wilt disease symptoms caused by r. solanacearum in eggplants grown from different seed encapsulation formulations. the inoculation of b. cereus bth21 cells into the encapsulation matrices prolonged the incubation period of r. solanacearum compared to the control treatment. the data also indicated that the combination of alginate + zeolite + bacillus cells (al+z+b) resulted in a longer incubation period compared to alginate + peat + bacillus (al+p+b) (fig. 2). the data indicated that the efficacy of bacillusembedded seed encapsulation techniques was able to prolong the incubation period of r. solanacearum in eggplants. the incubation period of r. solanacearum for b. cereus bth21 treatment was 13 – 15 days with a standard error of ± 1 (fig 2). the efficacy may have been due to synergistic association between the encapsulation matrices, i.e., alginate/zeolite and alginate/peat blend, and the b. cereus bth21 cells embedded within the matrices. alginate, as the primary material for seed encapsulation, provided a physical barrier that shielded the seeds from direct contact with outer environments (de castro et al. 2020) including the pathogenic agents, such as bacteria and fungi (luo et al. 2019), thereby delaying infection and extending the incubation period. alginate is extensively used recently and deemed as a suitable material for bacterial encapsulation due to its biodegradability, biocompatibility and low-cost (riseh et al. 2021; martínez-cano et al. 2022). moreover, the addition of either zeolite and peat to blend with alginate further improve the encapsulation performance, prolonging their activity in biocontrolling the pathogen as shown in figure 2. single use of alginate has its own drawback, such as mechanical instability, rapid losses of moisture and limited control over release profile (berninger et al. 2016; hurtado et al. 2022). the use of zeolite provided structural function to ensure the durability and longer shelf live (ciarleglio et al. 2023; ilinskaya et al. 2023), while also providing conducive environment for living cells, such as bacteria (xiong et al. 2023). meanwhile, peat which containing organic matter provides nutrient and moisture for the entrapped cells, protecting the cells from environmental stresses and ensuring survival of the cells during storage and application (novinscak & filion 2020). the data also suggested the efficacy of b. cereus bth21 in biocontrolling r. solanacearum. bacillus is among genera that is extensively studied as antagonist for biocontrol of plant pathogens, in particular r. solanacearum. several works reported the use of b. cereus, b. amyloliquefaciens, and b. subtilis to control r. solanacearum. those studies also unraveled the responsible direct or indirect mechanisms of action including production of biocontrol agents (antibiotics, enzymes, volatile organic compounds, etc.) and inducing plant systemic resistance (raza et al. 2016; wang et al. 2019; sun et al. 2023). moreover, the use of encapsulated bacteria to control plant disease is among the most recent technology that is deemed better than conventional direct inoculation into the soils. the efficacy has been proven in many instances for seed-encapsulation containing bacteria (wiwattanapatapee et al. 2013; pour et al. 2019;) or fungi (anuar et al. 2019; coninck et al. 2020). figure 2 incubation periods of r. solanacearum leading to visible wilt disease symptoms in eggplants grown from different encapsulation formulations bacillus-embedded seed encapsulation as biocontrol strategy against bacterial wilt in eggplant purnawati et al. 283 infection rate the infection rate of r. solanacearum, which caused wilt disease in eggplants grown from different bacillus-embedded encapsulated seeds (fig. 3). the inoculation of b. cereus bth21 cells into the encapsulation matrices reduced the infection rate of r. solanacearum in eggplants compared to the control treatment. in the control treatment, the infection rate peaked at 28 das (23%) and remained stagnant until 49 das. meanwhile, in the bacillus-embedded treatments, the infection rate peaked at 21 das (17% for al+z+b; 18% for al+p+b) and showed a declining trend over time until 49 das. standard error of infection rate in control ± 0 – 1; in a+z+b treatment ± 1; in al+p+b treatment ± 0 – 1 (fig. 3). figure 3 infection rate of r. solanacearum-caused wilt disease in eggplants grown from different encapsulation formulations observed from 0 – 49 das the data further support the previous finding regarding the potential use of alginate-based seedencapsulation techniques to immobilize and deliver b. cereus bth21 into the soil through a slowrelease mechanism. the efficacy of bacillus species in biologically controlling wilt disease caused by r. solanacearum was demonstrated in this research, as well as in numerous other reports with similar findings (raza et al. 2016; wang et al. 2019; sun et al. 2023). our study also highlights the effectiveness of delivering the bacterium through a slow-release mechanism, due to alginate seed encapsulation, which prolongs disease protection. alginate is a natural and biodegradable polymer, which, when applied to soil, is gradually degraded, releasing the bacterial cells entrapped within the matrix (he et al. 2015). this ensures a slower and more sustained delivery of bacterial cells into the soil, thereby extending the protective effect against r. solanacearum (fig. 3), where infected plants showed decreasing infection rates compared to the control. germination percentage the inoculation of b. cereus bth21 into the encapsulation matrices increased the germination percentage of eggplant seeds compared to the control treatment (fig. 4). in both bacillusembedded seed treatments, i.e., al+z+b and al+p+b, the germination rates reached as high as 15% by 49 das, whereas in the control treatment, the germination rate remained stable at 10% until 49 das. standard error of germination percentage in control ± 0.5 1; in a+z+b treatment ± 0.25 – 0.5; in al+p+b treatment ± 0 – 0.05 (fig. 4). figure 4 germination percentage of r. solanacearumcaused wilt disease in eggplants grown from different encapsulation formulations observed from 0 – 49 das plants sown outside often face multiple biotic and abiotic stresses including soil-borne pathogenic bacteria, such as r. solanacearum. the presence of pathogens makes the soil a suboptimal environment for seed germination process, where seed vigor is often compromised, causing lower seedling germination percentages, delayed seedlings emergence, or less vigor plants. the use of biocontrolling agents is a promising strategy to improve seed germination process and seed vigor on sub-optimal condition such as on the presence of plant pathogen (lamichhane et al. 2018). loliam et al. (2012) demonstrated the biocontrol of seedling damping off disease in several economic crops by using streptomyces rubrolavendulae. carvalho et al. (2011) reported biotropia vol. 32 no. 3, 2025 284 the use of trichoderma to biocontrol aspergillus, cladosporium, and s. sclerotiorum in common beans seedling. similarly, zhu et al. (2020) displayed the ability of b. pumilus to prevent seedling damping off disease caused by r. solani. in our study, the efficacy of b. cereus bth21 in biocontrolling r. solanacearum in eggplant seed germination is demonstrated. in alginateencapsulated eggplant seed containing bacillus cells, the germination rate was higher compared to the non-encapsulated control seeds. bacillus ability to control r. solanacearum has been demonstrated in this study, as well as in other similar studies (raza et al. 2016; wang et al. 2019; sun et al. 2023). nonetheless, immobilization of bacterial cells within alginate/zeolite and alginate/peat matrices provided a safe, non-toxic, and supportive environment for the cells to survive, while released in a slowly-manner, prolonging the duration of the protection (he et al. 2015; pour et al. 2019). our study also indicated the increase of the number of seedling emerged in the encapsulated seed treatment compared to that in the control, which otherwise shown constant germination rate until 49 das. the presence of the pathogen was indeed constraining the plants to exercising optimal vigor, yet upon the protection of the antagonistic bacillus, this constraint was somewhat alleviated to a lower degree. agronomic performances in terms of agronomic performances, eggplants grown from encapsulated seeds containing living cells of b. cereus bth21 displayed better results compared to the growth in the control treatment (fig. 5). standard error of plant height in control ± 0.115 – 0.17; in a+z+b and al+p+b treatments ± 0.058 – 0.12 (fig. 5a). the plant height of eggplants grown from encapsulated seeds (al+z+b, al+p+b) were consistently increasing up to 28 cm at 49 das. in contrast, the height in control treatment was remained low up to 49 das. similarly, in terms of the number of leaves, eggplants grown from bacillus-embedded encapsulated seeds were consistently higher compared to that in the control treatment. standard error of leaves number in control, a+z+b, and al+p+b treatments was ± 0 1 (fig. 5b). the results shown in figure 5 indicate the plant growth-promoting effects of b. cereus bth21, in addition to its previously demonstrated protective effects. many beneficial bacteria commonly exhibit multiple mechanisms of action that support plant growth and alleviate both biotic and abiotic stresses. these mechanisms include biostimulation (production of plant phytohormones such as auxin, cytokinin, and gibberellin), bioprotection (synthesis of antibiotics, siderophores, and lipopeptides), biofertilization (nitrogen fixation and phosphate solubilization), and bioremediation (uptake of heavy metals and degradation of xenobiotics) (mohanty et al. 2021). although not specifically assessed in this study, bacillus species have been shown to possess these capabilities, including the production of extracellular indole acetic acid (iaa) (shao et al. 2015), phosphate and zinc solubilization (ahmad et al. 2021), and atmospheric nitrogen fixation (xu et al. 2014). therefore, it can be suggested that these plant growth-promoting activities, combined with the antagonistic effects of b. cereus bth21 against r. solanacearum, result in a synergistic effect that enhances eggplants growth, as evidenced by increased plant height and number of leaves. bacillus-embedded seed encapsulation as biocontrol strategy against bacterial wilt in eggplant purnawati et al. 285 figure 5 agronomic performances of eggplants grown from different encapsulation formulations, while under deliberate r. solanacearum inoculation, observed from 0 – 49 das notes: a = plant height; b = number of leaves. conclusion the endophytic bacterial bth21 strain was isolated from healthy eggplant tissues. molecular identification confirmed that the strain belonged to b. cereus species. alginate seed encapsulation, blended with zeolite and peat, was successfully developed to immobilize and deliver active cells of b. cereus bth21 into the soil. a field assay of sowing bacillus-embedded encapsulated eggplant seeds inoculated with r. solanacearum, demonstrated significant biocontrol efficacy against the pathogen in terms of incubation period, infection rate, and germination percentage compared to the control treatment. the immobilized bacteria also exhibited positive plant growth-promoting effects, as indicated by higher plant height and number of leaves compared to that in the control treatment. overall, the results highlight the potential of alginate seed encapsulation as an alternative method for delivering antagonistic organisms, providing a suitable microenvironment for the immobilized bacterial cells, while slowly releasing them into the soil, thereby prolonging the biocontrol effect against plant pathogens. references ahmad i, ahmad m, hussain a, jamil m. 2021. integrated use of phosphate-solubilizing bacillus subtilis strain ia6 and zinc-solubilizing bacillus sp. strain ia16: a promising approach for improving cotton growth. folia microbiol 66(1):115–25. doi: 10.1007/s12223-020-00831-3 anuar ism, ku sulong ka, gani ha, safi’n nn, samsudin ka, wahab mz. 2019. alginate encapsulation of trichoderma harzianum as biocontrol agent against brown spot disease on rice (oryza sativa) in vitro assays. int j adv sci eng inf technol 9(2):461–66. doi: 10.18517/ ijaseit.9.2.3226 ascarrunz sdm, natsuaki t, honjo h, fukui r. 2011. quick adaptation of ralstonia solanacearum to copper stress to recover culturability and growth in water and soil. braz j microbiol 42(2): 576–91. doi: 10.1590/s151783822011000200023 berninger t, mitter b, preininger c. 2016. the smaller, the better? the size effect of alginate beads carrying plant growthpromoting bacteria for seed coating. j microencapsul 33(2):127–36. doi: 10.3109/02652048.2015.1134690 carvalho ddc, de mello scm, júnior ml, geraldine am. 2011. biocontrol of seed pathogens and growth promotion of common bean seedlings by trichoderma harzianum. pesqui agropecu bras 46(8):822–28. doi: 10.1590/ s0100-204x2011000800006 biotropia vol. 32 no. 3, 2025 286 castro-sowinski s, herschkovitz y, okon y, jurkevitch e. 2007. effects of inoculation with plant growth-promoting rhizobacteria on resident rhizosphere microorganisms. fems microbiol lett 276(1):1–11. doi: 10.1111/j.15746968.2007.00878.x ciarleglio g, cinti f, toto e, santonicola mg. 2023. synthesis and characterization of alginate gel beads with embedded zeolite structures as carriers of hydrophobic curcumin. gels 9(9):714. doi: 10.3390/gels9090714 coninck e, scauflaire j, gollier m, liénard c, foucart g, manssens g, …, legrève a. 2020. trichoderma atroviride as a promising biocontrol agent in seed coating for reducing fusarium damping‐off on maize. j appl microbiol 129(3):637–51. doi: 10.1111/jam.14641 de castro va, duarte vgo, nobre dac, silva gh, constantino vrl, pinto fg, …, tronto j. 2020. plant growth regulation by seed coating with films of alginate and auxin-intercalated layered double hydroxides. beilstein j nanotechnol 11:1082–91. doi: 10.3762/bjnano.11.93 emeribe eo, ogbuehi hc. 2024. bioactives and nutrients evaluation of the leaves and fruits of solanum melongena. j agric food sci 22(1):177–91. doi: 10.4314/jafs.v22i1.14 faith dp. 2016. the pd phylogenetic diversity framework: linking evolutionary history to feature diversity for biodiversity conservation. in: pellens r, grandcolas p (editors). biodiversity conservation and phylogenetic systematics. cham (ch): springer international publishing. p.39–56. 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guilherme et al. 2017). arrowroot flour contains high protein, even greater than that of cassava flour (aprianita et al. 2014), low glycemic index value and high fiber content. therefore, food products made from arrowroot flour are easier to digest compared with those made from other types of flour (lestari et al. 2017). food products made by arrowroot fluor is also recommended for people with digestive disorder and the elderdy (silva et al. 2000; heredia-zárate & vieira 2005; mason 2009; silveira et al. 2013) furthermore, arrowroot flour has long been used traditionally by the household and by the food industries as a basic ingredient for baby food. further research revealed that arrowroot flour is potential to be used as a thickening agent in various industries, such as cosmetics, pharmaceutical and food industries (kitahara et al. 2007). in addition to these benefits, the widespread arrowroot population in indonesia, especially in yogyakarta province, is the main reason for developing arrowroot cultivation (djaafar et al. 2010; masitoh 2014). information obtained from molecular analysis of a plant population can identify genotype differences among individuals as a selection step in plant breeding programs. an efficient character selection process by relying on the information about the genetic variation of a wild plant population with a suitable *corresponding author, email: purnomods@ugm.ac.id biotropia vol. 29 no. 1, 2022 48 breeding method has succeeded in developing modern cultivars, which has increased the yield of various food crops since the mid-20th century. molecular markers applied to reveal genetic variabilities can thus, provide promising information in agriculture as well as in protecting crop variabilities (govindaraj et al. 2015; perez-de-castro et al. 2012; faraldo et al. 2003). inter simple sequence repeat (issr) is one of molecular markers commonly used in the study of genetic diversity, phylogeny, gene detection, genome mapping and evolutionary biology in various types of plants (reddy et al. 2002). the combination of using issr in pcr has been done in genetic fingerprinting (blair et al. 1999), cultivar identification (wang et al. 2009), phylogenetic analysis (gupta et al. 2008) and assessment of hybridization (wolfe et al. 1998). the issr marker uses a 16-25 bp single primer that is specific to target identical regions among microsatellites. primers composed of eight repeating dinucleotide units, six repeating trinucleotide units or several repeating tetraor pentanucleotide units with or without nucleotide anchorage system that targets the microsatellite region (zietjiewicz et al. 1994). various types of molecular markers were used to reveal the genotype diversity of arrowroot, especially between the local varieties and cultivars. two of the molecular markers are rapd and issr markers (pinto 2015; asha et al. 2015). this study aimed to determine genetic variability and analyze the intraspecific relationship of arrowroot population in yogyakarta province, indonesia by using the issr markers. the expected results were the diversity of arrowroot germplasm in indonesia which can be used to strengthen the phenotypic characters variations that have been used in arrowroot cultivation and breeding. materials and methods plant materials sample collection was carried out during the dry season, from august to september 2019 in 18 subdistricts in sleman, gunungkidul, kulon progo, and bantul districts in yogyakarta province using the exploratory survey method. the whole arrowroot rhizome was taken from each accession and then replanted in the sawitsari research center plantation, faculty of biology, universitas gadjah mada, yogyakarta. species identification was carried out using the key of determination from van steenis (1975), woodson and schery (1980), wu and kennedy (2000), hammel et al. (2014), lim (2016). meanwhile, the cultivar identification was carried out through interviews with arrowroot farmers. fresh leaf from each 7-month-old accession was used for dna extraction. dna isolation genomic dna from the finely ground leaves (0.5 g) was extracted using the modified ctab method (deswina et al. 2019). extraction buffer consisted of 50 mm tris-hcl (ph 8), 0.7 m nacl, 0.1% β-mercaptoethanol, 10 mm edta, 0.1% cetyltriethylammonium bromide (ctab). the dna quality was checked in an agarose gel through the electrophoresis and then quantified by measuring the od at 260 nm and 280 nm through the spectrophotometry. purity of dna sample was calculated from od at a 260/280 ratio. issr analysis in this recent study, a total of 4 issr primers were used for analysis. the primers are ubc 811, ubc 827, ubc 818 and ubc 825 (table 1). table 1 issr markers details markers sequence (5’ 3’) annealing temperature (oc) size range (bp) ubc 811 (ga)8c 53 300-2,000 (asha et al. 2015; kambale et al. 2018) ubc 827 (ac)8g 52 300-1,600 (asha et al. 2015; kambale et al. 2018) ubc 818 (ca)8g 45 350-1,600 (asha et al. 2015) ubc 825 (ac)8t 52 400-2,000 (asha et al. 2015; kumar et al. 2010) genetic variability of arrowroot based on issr analysis – bernardinus danang krisna aji p. et al. 49 the extracted dna was amplified in a thermal cycler (bio-rad). the reaction mixture consisted of 8.5 µl ddh2o, 2.0 µl of each primer, 12.5 µl mytaq polymerase and 2 µl dna sample. the total reaction volume was made up to 25 µl using sterile distilled water. pcr was carried out in a master cycler gradient with a certain temperature setting (table 2). table 2 pcr amplification setting (asha et al. 2015) steps temperature (oc) time (minutes) cycles pre-denaturation 94 4 1 denaturation 94 1 annealing 45-55 0.5 40 extention 72 1 post extention 72 4 1 hold 4 ∞ 1 the annealing temperature was standardized for each primer. the results of amplification were separated by gel electrophoresis in 2% agarose gel with 1x tbe buffer and were imaged using gel documentation with observation through uv vis transilluminator. data analysis the analysis was carried out only with the unambiguously and reproducibly amplified issr bands. those dna bands were scored as present (1) or absent (0). smeared and weak bands were excluded. the resulting binary data matrix was analyzed using mvsp (multivariate statistical program) version 2.0. the analysis aimed to examine genetic relationship among accessions by estimating the similarity index using the baroni-urbani busser similarity coefficient. cluster analyses were carried out on the same similarity coefficient with the unweighted pair-group method of average (upgma) (rohlf 2000). results and discussion plant collection and identification identification of local cultivars succeeded in revealing the existence of 5 cultivars, namely 'sembowo', 'sili', 'sugo', 'kebo', and 'teropong'. the 'sili' and 'sembowo' cultivars were found to be widely distributed in 4 districts (table 3). dna extraction dna extraction using the ctab method was carried out on the accessions 'sembowo' (represented by seng1), 'sili' (represented by sipe3), 'sugo' (represented by subr1), 'teropong' (represented by teng1) and 'kebo' (represented by keke1). the quantity of isolated dna was tested by using spectrophotometry and showed a fairly high level of purity in four of the five accessions. keke1 accessions were known to be contaminated, but based on the results of the electrophoresis tests, this dna sample could still be used. results of dna concentrations varied. each accession had a good dna concentration and met the standards. the results of the spectrophotometric test are shown in table 4. table 3 sampling collection no sampling location accessions type accessions code district subdistrict village, subvillage 1 bantul sedayu argodadi, brongkol ‘sili’ (besar) sibb1 2 bantul sedayu argodadi, brongkol ‘sili’ (kecil) sikb1 3 bantul sedayu argodadi, brongkol ‘sili’ (bengkok) sieb1 4 bantul sedayu argodadi, brongkol ‘sugo’ subr1 5 sleman pajangan triwidadi, ngincep ‘sembowo’ seng1 6 sleman pajangan triwidadi, ngincep ‘sili’ sing1 7 bantul pajangan triwidadi, ngincep ‘teropong’ teng1 8 bantul pleret wonolelo, kedungrejo ‘kebo’ keke1 9 gunungkidul wonosari gari, gondangrejo ‘sembowo’ sego2 10 kulon progo pengasih pengasih, pengasih ‘sembowo’ sepe3 11 kulon progo pengasih pengasih, pengasih ‘sili’ sipe3 12 kulon progo pengasih sendangsari, gegunung ‘sembowo’ sege3 13 kulon progo pengasih sendangsari, gegunung ‘sili’ sige3 14 sleman moyudan sumberagung, kaliduren iii ‘sembowo’ sekd4 15 sleman prambanan sumberharjo, sengir ‘sembowo’ sese4 biotropia vol. 29 no. 1, 2022 50 table 4 spectrophotometry results accession code od 260 od 280 od at 260/280 ratio concentration (ng/µl) seng1 7.03 3.67 1.929 365.52 sipe3 2.54 1.35 1.898 127.32 subr1 4.62 2.43 1.917 231.11 keke1 5.57 9.95 1.371 678.87 teng1 3.82 1.95 1.970 191.00 each sample of isolated dna concentrations showed various values ranging from 127.32 ng/µl to 678.87 ng/µl. the results indicated that isolated dna from 'sili' leaves produced the lowest concentration. however, the results also showed a fairly high absorbance ratio value of 1.898 at od ratio 260/280. the od value shows the purity of the dna sample. there was only dna sample of the local cultivar 'kebo' which had a very low absorbance ratio value of 1.371 at od ratio 260/280. the quantity of dna concentration and quite low purity could be caused by polyphenol contamination. the quality of the dna sample from ‘kebo’ cultivar was still good and clearly showed the dna bands. the absorbance purity ratio of 260/280 is a very important measurement for estimating polyphenol contamination in the extracted dna samples. ratio of a260/a280 below 1.8 shows a poor dna extraction result and is not recommend for molecular analysis (sambrook & russell 2001). a high purity ratio of a260/a280 also indicates the purity of dna from rna contamination (koetsier & cantor 2019). the use of high enough mercaptoethanol can replace the role of rnase in degrading and removing rna from dna samples, even though this reducing agent is toxic and pungent (mommaerts et al. 2015). isolation of plant dna using the ctab protocol has many advantages and is proven to produce more sample volumes with high dna concentrations. ctab buffer solutions generally play a role in damaging cell structures, from cell walls to cell membranes and also the nuclear membrane. this is solely done to release the genetic material from the nucleus (amani et al. 2011). ctab buffer solution contains 2-βmercaptoethanol which has been shown to completely remove polyphenol components in cells (horne et al. 2004; li et al. 2007). in this study, a buffer solution with a concentration of 0.1% mercaptoethanol was used and proved to be optimal in removing polyphenol contamination. meanwhile, another study revealed that the use of 0.3% mercaptoethanol could improve the quality of dna pellets from precipitation (suman et al. 1999). analysis of issr-pcr molecular analysis used four issr markers, namely ubc 811, ubc 818, ubc 825 and ubc 827. pcr temperature optimization for these four markers resulted in different optimum annealing temperatures between ubc 827 & ubc 825 and ubc 818 & ubc 811. ubc 827 and ubc 825 markers require an annealing temperature of 50 °c, while the other two markers require an annealing temperature of 55 °c. electrophoresis of pcr-issr results showed variations in the number of dna bands, polymorphic bands and different monomorphic bands among issr markers (table 5). table 5 number of dna bands and polymorphism (%) in each molecular marker issr markers sequences (5’3’) total dna bands total polymorphic dna bands polymorphisms (%) ubc 811 (ga)8c 11 8 72.7 ubc 818 (ac)8g 8 4 50 ubc 825 (ca)8g 10 8 80 ubc 827 (ac)8t 10 7 70 average 9.75 6.75 68.17 genetic variability of arrowroot based on issr analysis – bernardinus danang krisna aji p. et al. 51 visualization of the electrophoresis results using the gel-doc transilluminator showed variations among these molecular markers (fig. 1). the average polymorphism appeared was 68.17% with the mean number of polymorphic bands of 6.75 and the average number of dna bands of 9.75. ubc 811 primer produced the highest number of dna bands, while ubc 811 and ubc 825 primers produced the highest number of polymorphic dna bands with eight bands. amplification using ubc 825 primer resulted in the highest polymorphism at 80%. electrophoresis using a dna ladder with a size of 100 bp and a buffer solution of 2% tbe 1x showed that the size of the dna bands in each primer was different, but there were several monomorphic bands with the same size between one marker and another. the dna bands were 750 bp, 600 bp, and 450 bp. overall, the size of dna bands appeared to be ranging from 180 bp to 1,000 bp. meanwhile, the polymorphic bands showed by each marker were diverse. in ubc 811, polymorphic dna bands were found to have sizes of 200-300 bp, 300-400 bp and 800 bp. ubc 818 primer amplification resulted in the lowest polymorphism value, with only 50% polymorphism showed by the dna bands 250 bp, 650 bp and 500-600 bp. issr marker is an ideal genetic marker for various studies, most notably on genetic variation (shafiei-astani et al. 2015) and dna fingerprinting (shen et al. 2006). as a good genetic marker, issr produces high genetic variability and shows multilocus data due to the use of the highly variable microsatellite sequences that are ubiquitously distributed across the genome (anne 2006; tautz & renz 1984; wolff et al. 1995). the addition of one or more nucleotide anchors in issr marker to target the end of the microsatellite region can prevent primer dimerization (bani et al. 2017). based on comparison with other molecular markers, the issr marker has higher reproducibility compared to the random amplified polymorphic dna (rapd). the issr marker is more time and money efficient compared to the amplified fragments length polymorphism (aflp) (phong et al. 2011; ng & tan 2015). electrophoresis of issr-pcr products generally uses 1.5-2.0% agarose gel by weight/volume (w/v) in order to achieve good dna band separation. when the agarose gel concentration is higher, reaching 3% w/v or more, the gel will break more easily as it hardens (ng & tan 2015). according to bornet & branchard (2001), 2.0% w.v agarose gel showed the best performance in resolving the issr band compared to other concentrations of agarose gel. figure 1 amplification results notes: amplification with 5 accessions (seng1 (lane 2); sipe3 (lane 3); teng1 (lane 4); keke1 (lane 5); and subr1 (lane 6)) using issr markers (ubc 811 (group a); ubc 818 (group b); ubc 825 (group c); and ubc 827 (group d)) with 100 bp dna ladder (lane 1). biotropia vol. 29 no. 1, 2022 52 plant systematic studies of several plant taxa based on the molecular characteristics using issr-pcr revealed a greater number of polymorphic loci than the use of rapd-pcr, because the abundant formation of issr primer target sequences throughout the eukaryotic genome, which evolves rapidly (ansari et al. 2012; phong et al. 2011; moulin et al. 2012). previous study on genetic variability of arrowroot in yogyakarta using rapd markers showed a low polymorphism and required further study (setyowati 2013). in this recent study, the use of issr was proven to show the genetic variability of arrowroot in yogyakarta. therefore, issr marker proved to be better and more suitable in the analysis of arrowroot genetic variability than rapd. issr is a recent genetic marker that overcomes many technical limitations in other methods such as rflp and rapd analyses. issr markers have higher reproducibility than that of rapd and have been successfully used to estimate the extent of genetic diversity at intra and inter-specific levels in a wide range of crop species (asha et al. 2015). hence, the issr markers were used in our study to assess the genetic diversity of arrowroot. the amplification results of the 4 primers with 7 arrowroot accessions in india showed high polymorphism values (asha et al. 2015), which were in agreements with the results of this study. however, the number of total dna bands and the average percentage of polymorphisms obtained were lower than the previous research conducted by asha et al. (2015). the differences were due to genetic diversity among plant population influenced by geographical condition and also appeared as molecular characteristics of each sample. based on both studies, the polymorphic dna bands obtained from issr analysis ranged from 10 to 60 fragments from various loci. the use of issr ubc 825 primer in this study resulted in the highest polymorphism values and the greatest number of polymorphic dna bands. this showed the effectiveness and the high reproducibility of these primers in the analysis of genetic diversity of arrowroot plants. the primary use of ubc 825 in the previous study conducted by asha et al. (2015) was also known to produce the greatest number of polymorphic dna bands and the highest polymorphism. based on the electrophoresis, ubc 825 indicated the presence of microsatellite dna sequences with sizes ranging from 800 bp to 180 bp. phenetic analysis based on molecular characteristics phenetic analysis based on the molecular characteristics can separate five local cultivars of arrowroot in yogyakarta into four clusters. dendrogram branching resulted in four clusters which were determined with the help of phenon lines at 80% similarity index. three of the four clusters were outliers, each consisted of accessions of sipe3, teng1 and keke1. one cluster consisted of accessions seng1 and subr1 (fig. 2). figure 2 dendrogram of arrowroot phenetic relationship based on molecular characteristics note: molecular characteristics were the result of dna fingerprinting analysis with issr markers, using the baroni-urbani buser similarity coefficient. genetic variability of arrowroot based on issr analysis – bernardinus danang krisna aji p. et al. 53 table 6 similarity matrix for cluster analysis of arrowroot based on molecular characteristics keke1 seng1 subr1 teng1 sing1 keke1 1 seng1 0.741 1 subr1 0.745 0.86 1 teng1 0.698 0.672 0.629 1 sing1 0.654 0.676 0.726 0.534 1 dendrogram branching was determined by the similarity matrix between accessions. the similarity values between one accession and the other accessions were different (table 6). analysis using the baroni-urbani buser similarity coefficient showed that the accessions of subr1 and seng1 had the highest similarity coefficient, which was 0.86. the lowest similarity coefficient was shown by sing1 and teng1, which is 0.534. the dendrogram showed 4 branches (fig. 2). the first branch separated the sing1 accession, representative of the local cultivar 'sili', from the other four local cultivar accessions at 0.64 point. it indicated the high dissimilarity between ‘sili’ local cultivar with the other five local cultivars based on its molecular genetics. the genotypic characters obviously made distinct phenotypic characters of ‘sili’ local cultivars. based on interviews with arrowroot farmers, it was found that the 'sili' cultivar was not very attractive for cultivation because it produced rhizomes containing higher fiber and smaller size than the other four local cultivars rhizomes. the second branch of the dendrogram separated the cultivar of 'teropong' (teng1) from 'sugo', 'sembowo', and 'kebo' cultivars at 0.68 point. then, the keke1 accession separated from the cluster consisting of subr1 and seng1 accessions at 0.86 point. the highest similarity that showed by ‘sugo’ cultivar and ‘sembowo’ cultivar was supported by the similarity of rhizome morphologies from both cultivars, which information was provided by the farmers during the interviews. however, around 10% of dissimilarity index obtained based on molecular characteristics still showed great differences on the morphological and or anatomical characters. further studies of the use of issr markers in dna fingerprinting techniques, cultivation or revealing genetic variation can provide several advantages. genotype-specific issr markers can be sequenced to be used as a basis for the synthesis of sequence characterized amplified region (scar) primers, which can then be used to determine genotype taxonomy. in addition, markers associated with high agronomic value characters can be sequenced to be used as sequence tagged sites (sts) markers that are useful in cultivation and breeding programs. meanwhile, although the microsatellites that play a role in issr-pcr are probably nonfunctional, they are known to be associated with region coding. therefore, issr can be used to construct gene-rich regions (vijayan 2005). conclusion the study used 5 local cultivars of arrowroot (m. arundinacea l.) from four districts in the yogyakarta province consisting of five local cultivars namely 'sembowo', 'sili', 'kebo', 'teropong' and 'sugo'. molecular analysis using issr markers was successful in showing high polymorphism and explaining genetic variabilities in the five local arrowroot cultivars. the analysis of the phenetic relationship using the mvsp program, the upgma algorithm method and the baroni-urbani buser similarity coefficient showed the formation of four clusters on the dendrogram based on molecular characteristics. clustering based on molecular characteristics was able to separate local cultivars from one another, but showed a close relationship between the local cultivars 'sembowo' and 'sugo'. acknowledgments the author would like to thank the farmers who provided great assistance in collecting arrowroot accessions. references amani j, kazemi r, abbasi ar, salmanian ah. 2011. a simple and rapid leaf genomic dna extraction method for polymerase chain reaction analysis. iran j biotech 9:69. biotropia vol. 29 no. 1, 2022 54 anne c. 2006. choosing the right molecular genetic markers for studying 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3departement of pharmacology and therapy, faculty of medicine, udayana university, denpasar, 80232, indonesia received 21 july 2022 / revised 1 june 2023 /accepted 4 june 2023 abstract before the availability of a vaccine, indonesian population relied on traditional medicines to prevent covid19. any species used by indigenous people could lead to further investigations in modern pharmacology, to preserve ancient knowledge, and to plan for plants’ conservation. the study aimed to discover and record species, methods of preparation, route of administration, and motivation in using medicinal plants by the indonesian population during the covid-19 pandemic. participants of survey were selected from the people who live in java and bali for responding to an online structured questionnaire. relative frequency of citation (rfc) was employed in the quantitative analysis of the collected data. the pharmacological relevance of the five plants with the highest rfc was further reviewed. the results showed that respondents used 59 plants from 28 families. five species with the highest rfc were curcuma longa (0.707), zingiber officinale (0.674), cymbopogon citratus (0.269), kaempferia galanga (0.174), and curcuma zanthorrhiza (0.165). most plants were prepared by boiling (77.97%) and administered orally as a single ingredient or mixed with other herbals. respondents believed that the plants were beneficial as immune-booster (71.26%), maintain good health (24.85%) and stamina (12.28%), and prevent viral infection, including covid-19 (5.39%). the most commonly used plants might be scientifically based to boost immunity. however, their usage against covid-19 and the medicinal value of herbal mixtures should be further investigated. keywords: covid-19, ethnobotany, indonesia, medicinal plants introduction corona virus disease 2019 (covid-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus-2 (sarscov-2). it was found in china in december 2019 and spread to other countries, including indonesia. the first two confirmed covid-19 cases in indonesia were reported on march 2, 2020, and the numbers keep rising since then (djalante et al. 2020). based on history, previous sars-cov coronavirus also caused an outbreak in china in 2003. the genetic sequence analysis showed that sars-cov-2 was similar around 79% to sarscov. thus, most of the studies on its prevention and medication were adopted from the previous outbreak (ghaffari et al. 2020). the indonesian ministry of health released health protocols to prevent and control covid-19 (hk.01.07/menkes/382/2020), which included the suggestion to wear masks, washing hands frequently, social distancing, and immunity enhancement through clean and healthy living behavior. during the sars outbreak, natural medicine showed beneficial effects in preventing and treating patients, particularly in high-risk subjects (boozari & *corresponding author, email: nimade.nayaka@unmas.ac.id; nimade.nayaka@gmail.com biotropia vol. 30 no. 2, 2023 184 hosseinzadeh 2020; y. li et al. 2020). the usage of medicinal plants as a prophylaxis measure against covid-19 was also recommended by ayurveda and traditional chinese medicine (boozari & hosseinzadeh 2020; khanal et al. 2020; vellingiri et al. 2020). further, many studies have proven the antiviral, antiinflammatory, and immunomodulatory properties of medicinal plants that are potentially helpful to combat viral diseases (lin et al. 2014; khanna et al. 2020). indonesia has abundant natural resources in plant species and the local people have used them as herbal remedies. some of the ethnobotanical studies recorded the importance of indonesian biodiversity as traditional medicines in different health conditions (nahdi & kurniawan 2019; taek et al. 2019; jadid et al. 2020). those studies are significantly important to converse precious indigenous knowledge and publish them as academic literature. in the present study, we conducted an online survey to identify the use of medicinal plants by the indonesian population during the covid-19 pandemic. the comprehensive data from respondents on the species of medicinal plants, method of preparation and administration, as well as motivation to use, were documented. while antivirus and vaccines are vital, the research on natural medicine regarding covid-19 may be used as a reference to develop new drug candidates and as homebased remedies in the future that are inexpensive, commonly, and easily implemented in society. the study aimed to discover and to record species, methods of preparation, route of administration, and motivation in using medicinal plants by the indonesian population during the covid-19 pandemic. materials and methods study area a survey was conducted in java and bali island (figure 1), which consist of 7 provinces, namely special capital region of jakarta (6°12′s, 106°49′e), banten (6°30′s 106°15′e), west java (6°45′s 107°30′e), central java (7°30′s 110°00′e), special region of yogyakarta (7°47′s 110°22′e), east java (7°16′s 112°45′e), and bali (8°20′06″s 115°05′17″e). all the regions have diverse ethnicities such as bantenese in banten, balinese in bali, and javanese in other regions. respected to the ethnicity, each of the regions has its traditional language. however, natives speak indonesian in their daily lives. figure 1 study area of the ethnobotanical survey in 7 provinces in indonesia medicinal plants usage during covid-19 in indonesia – nayaka et al. 185 data collection a cross-sectional study was undertaken using a self-administered and structured questionnaire. the questionnaire consisted of three parts that aimed to collect the respondents demographic characteristics, medicinal plants data, and respondents’ motivation using the plants during the pandemic. the data collection was carried out by online survey from june to august 2020. the questionnaire was in indonesian and examined by two experts in pharmacy and bahasa indonesia fields then piloted among 30 participants to ensure its validity before being used to collect data. a guide to estimate the minimum sample size of respondents required for this study based on the formula (pourhoseingholi et al. 2013). n = z2 p(1 – p) d2 where: z = the statistic corresponding to level of …….confidence. p = expected prevalence of covid-19. d = precision. in this study, the respondents included indigenous people living in java and bali islands and consuming medicinal plants during the covid-19 pandemic in indonesia. the respondents’ motivation to use the plants was also recorded. any dubious data that could not be confirmed was excluded. the questionnaire and methodology for this study were approved by the faculty of medicine, udayana university (ethics approval number: 1195/un14.2.2.vii.14/lt/2020). plant identification the scientific names of medicinal plants reported by respondents were determined using cross-references between their local names and database in indonesian herbal pharmacopeia, indonesian herbal formulary (permenkes no.6/2016), and indonesian traditional medicine formulary (hk.01.07/menkes/ 187/2017). herbal specimens could not be collected due to the strict travel restrictions regulated by the indonesian government during the covid-19 pandemic in the study area. the scientific names of the reported plants were checked with the plant list website (accessed on january 8, 2021, http://www.theplantlist.org). data analysis the collected data were evaluated by using microsoft office excel (2016) spreadsheets. further, quantitative data analysis to show the local importance of each plant species was demonstrated by using the relative frequency of citation (rfc) (aziz et al. 2017). with the formula below: rfc = fc/n (0 < rfc < 1) where: fc = number of informants mentioning a ………particular species. n = total number of respondents results and discussion indonesia is an inhabitant of about 80% of the world’s medicinal plants which local people use to prevent and cure many ailments (elfahmi et al. 2014). in the current study, an online survey was conducted to collect data regarding medicinal plant usage by indonesian during the covid-19 pandemic. the respondents were limited to those who were native and living in 8 provinces in the two most densely populated islands in indonesia (java and bali islands) (table 1). moreover, based on the indonesian government’s official website (www.covid19.go.id), most of the confirmed covid-19 cases were located in both islands and the prevalence reached 82.2%. based on the prevalence of covid-19 and the statistic corresponding to the level of confidence is 1,96 with a precision of 5%, the minimum number of respondents for this study was 224 respondents. however, in this study, 344 respondents participated and this number exceeded the minimum sample. as shown in table 1, 82.04% (274) of respondents were female. this result is in line with several studies (villena-tejada et al. 2021; brahmi et al. 2022; odebunmi et al. 2022). which indicated the domination of females in using medicinal plants. this predominance is probably related to several factors such as women being more familiar with medicinal plants because they biotropia vol. 30 no. 2, 2023 186 are also being used as cooking ingredients. correspondingly, in most populations, women are believed to bear the responsibility for the family health needs causing them to be more informed about using medicinal plants than their male counterparts (torres-avilez et al. 2016). in the current study, most of the respondents came from the province of bali. a hindu-most populated area in indonesia (statistics indonesia 2010). the balinese are well known for their local wisdom called usada, a traditional medicine inspired by the hindu holy book ayurveda (muderawan et al. 2020). age is another sociodemographic factor contributing to the use of medicinal plants. several studies found that older age was the main user of traditional medicine (rahayu et al. 2020). on the contrary, 67.37% (225) of respondents in this study were 20 to 40 years old. indicating the younger age group was also interested in using medicinal plants during the pandemic of covid-19. similar survey studies conducted in algeria and morocco during the pandemic also showed similiar results (belmouhoub et al. 2021; brahmi et al. 2022; chebaibi et al. 2022). table 1 demographic characteristics of respondents characteristic number of respondents (n = 334) percentage (%) gender male 60 17.96 female 274 82.04 province of origin special capital region of jakarta 13 3.89 banten 2 0.60 west java 35 10.48 central java 8 2.40 east java 24 7.19 special region of yogyakarta 6 1.80 bali 246 73.65 age (years) < 20 35 10.48 20 – 40 225 67.37 > 40 74 22.16 religion islam 66 19.76 hindu 247 73.95 protestant 12 3.59 catholic 8 2.40 buddha 1 0.30 table 2 medicinal plants used by respondents during the covid-19 pandemic in indonesia family scientific names local names (indonesia) common names (english) part used method of preparationa rfcb motivation of usec reported fromd acanthaceae andrographis paniculata (burm.f.) nees sambiloto green chiretta leaves boil 0.030 a, b, d, e 1, 5, 7 amaryllidaceae allium cepa l. bawang merah shallot bulb boil, burning, eaten directly 0.006 b 6,7 allium sativum l. bawang putih garlic bulb eaten directly 0.063 a-e 3-7 anacardiaceae spondias pinnata (l.f) kurz. cemcem common hog-plum leaves cold infusion 0.006 b 7 annonaceae annona muricata l. sirsak soursop leaves boil 0.015 a-d 3,7 apiaceae apium graveolens l. seledri celery leaves boil 0.003 b 3 centella asiatica (l.) urb. pegagan asiatic pennywort leaves boil, eaten directly 0.021 a,b,d,e 7 coriandrum sativum l. ketumbar coriander fruit boil 0.012 a,b,d,e 3, 5, 7 foeniculum vulgare adas fennel fruit boil 0.003 b 4 medicinal plants usage during covid-19 in indonesia – nayaka et al. 187 family scientific names local names (indonesia) common names (english) part used method of preparationa rfcb motivation of usec reported fromd mill. arecaceae cocos nucifera l. kelapa coconut fruit (water & oil) eaten directly 0.009 b 7 asteraceae blumea balsamifera (l.) dc. sembung buffalo-ear leaves boil 0.006 b,d 7 gynura procumbens (lour.) merr. sambung nyawa longevity spinach leaves boil 0.003 b 6 pluchea indica (l.) less. beluntas indian camphorweed leaves boil 0.009 b 7 sonchus arvensis l. tempuyung perennial sow-thistle leaves boil 0.003 b 7 basellaceae anredera cordifolia (ten.) steenis binahong gulf madeiravine leaves boil 0.003 b 1 caricaceae carica papaya l. pepaya papaya leaves boil 0.003 e 3 fabaceae caesalpinia sappan l. secang brazilwood wood boil 0.027 a-e 3-7 clitoria ternatea l. bunga telang asian pigeonwings flower boil 0.009 a,d 5, 7 erythrina variegata l. dadap tiger’s claw leaves boil 0.003 a,d 7 tamarindus indica l. asam jawa tamarind fruit boil 0.060 a-e 1, 3-7 lamiaceae mentha piperita l. pipermin peppermint leaves hot infusion 0.009 a,b,d,e 2, 3, 7 peronema canescens jack sungkai false elder leaves boil 0.003 b 3 lauraceae cinnamomum burmanni (nees & t.nees) blume kayu manis batavia cinnamon bark, leaves boil, burning, cold and hot infusion 0.114 a-e 1-7 malvaceae hibiscus sabdariffa l. rosela roselle flower boil 0.003 b 7 meliaceae azadirachta indica a.juss mimba neem leaves eaten directly 0.003 b 7 moraceae artocarpus altilis (parkinson ex f.a.zorn) fosberg sukun breadfruit leaves boil 0.003 b 7 moringaceae moringa oleifera lam. kelor drumstick tree leaves boil, hot infusion 0.045 a-e 3-7 myrtaceae melaleuca cajuputi powell kayu putih cajuput oil n/a 0.003 c 1,7 syzygium aromaticum (l.) merr. & l.m.perry cengkeh clove flower boil, hot infusion 0.048 a-e 2-7 syzygium polyanthum (wight) walp. salam indonesian bay leaf leaves boil 0.021 a-e 3, 5-7 oleaceae olea europaea l. zaitun olive fruit (oil) eaten directly 0.003 b 3 pandanaceae pandanus amaryllifolius roxb. pandan pandan leaves boil 0.003 a,d 2 phyllanthaceae sauropus androgynous (l.) merr. katuk sweet leaf leaves cold and hot infusion 0.021 a,b,d,e 7 phyllantus niruri l. meniran gale of the wind leaves boil, hot infusion 0.009 a,b 5, 7 piperaceae piper betle l. sirih betel leaves boil, eaten directly 0.072 a-e 1, 3 piper crocatum ruiz & pav. sirih merah celebes pepper leaves boil 0.072 b 7 piper nigrum l. lada black pepper seed boil 0.003 b 3 piper retrofractum vahl cabai jawa javanese long pepper fruit boil 0.003 e 7 poaceae cymbopogon citratus (dc.) stapf. serai dapur lemongrass stem, leaves boil, hot infusion 0.269 a-e 1-7 oryza sativa l. beras rice starch cold infusion 0.024 a,b,d,e 3-7 ranunculaceae nigella sativa l. jintan hitam black seed seed boil 0.012 a,b,e 3, 5, 7 rutaceae citrus aurantiifolia (christm.) swingle jeruk nipis egyptian lime fruit juiced, boil, hot and cold infusion 0.177 a-e 1-7 citrus hystrix dc. jeruk purut kaffir lime leaves, fruit boil 0.006 b,e 6 citrus limon (l.) osbeck lemon lemon fruit cold and hot infusion 0.108 a-e 1, 3-5, 7 citrus reticulata jeruk mandarin fruit eaten directly 0.021 a,b,e 4, 7 biotropia vol. 30 no. 2, 2023 188 family scientific names local names (indonesia) common names (english) part used method of preparationa rfcb motivation of usec reported fromd blanco rubiaceae morinda citrifolia l. mengkudu noni fruit juiced 0.015 b-d 7 schisandraceae illicium verum hook.f. bunga lawang star anise flower boil 0.009 a,b,e 4, 5, 7 theaceae camellia sinensis (l) kuntze teh tea plant leaves boil, hot infusion 0.012 b-d 1, 3, 7 zingiberaceae alpinia galanga (l) willd. lengkuas greater galangal rhizome boil, hot infusion 0.054 a-e 1, 4, 5, 7 boesenbergia pandurata (roxb.) schltr. temu kunci chinese keys rhizome boil 0.006 b,e 7 curcuma longa l. kunyit turmeric rhizome boil, burning, hot infusion 0.707 a-e 1-7 curcuma zanthorrhiza roxb. temulawak javanese turmeric rhizome boil, hot infusion 0.165 a-e 1-7 curcuma zedoaria (christm.) roscoe temu putih zedoary rhizome boil 0.006 a,d,e 5, 7 amomum compactum sol. ex maton kapulaga javanese cardamom seed, flower boil, hot infusion 0.018 b,d,e 3, 5, 7 kaempferia galanga l. kencur cutcherry rhizome boil, burning, hot infusion 0.174 a-e 1-7 kaempferia rotunda l. kunyit putih peacock ginger rhizome juice, hot infusion 0.021 a,b,d,e 5, 7 zingiber officinale roscoe jahe ginger rhizome boil, burning, hot infusion 0.674 a-e 1-7 zingiber officinale var.rubrum theilade jahe merah red ginger rhizome boil, hot infusion 0.039 a,b,d,e 3, 5-7 zingiber zerumbet (l.) roscoe ex sm. gamongan /lempuyang gajah bitter ginger rhizome boil 0.003 e 7 notes: aall plants were prepared with water and administered orally, except cajuput by inhalation. brfc = relative frequency of citation, ca = to keep healthy, b = to boost the immune system, c = to avoid virus infection, including covid-19, d = to build stamina, e = other, d1 = special capital region of jakarta, 2 = banten, 3 = west java, 4 = central java, 5 = east java, 6 = special region of yogyakarta, 7 = bali, n/a = not available, table 3 plants mixtures used by respondents during the covid-19 pandemic in indonesia plants mixture no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 a. sativum + c. asiatica + c. nucifera + p. indica + t. indica + + + + c. burmanni + + s. aromaticum + p. amaryllifolius + + p. betle + c. citratus + + + + + + + + o. sativa + c. aurantiifolia + + + + + + + + c. lemon + + + + + c. sinensis + + a. galangal + c. longa + + + + + + + + + + + + + + + + + + + + c. zanthorrhiza + + + + + + + k. galangal + + + + k. rotunda z. officinale + + + + + + + + + + + + + + + + + + total plants mixed 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 notes: + = plants available in mixtures. * all mixtures were prepared by boiling and administered orally medicinal plants usage during covid-19 in indonesia – nayaka et al. 189 table 3 plants mixture used by respondents during the covid-19 pandemic in indonesia (continued) plants mixture no.* 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 c. asiatica + + c. sativum + f. vulgare + c. sappan + + + + + c. ternatea + t. indica + + + m. piperita + p. canescens c. burmanni + + + + + + + + + + a. altilis + s. aromaticum + + + + + + + s. polyanthum + o. europea p. amaryllifolius s. androgynous + p. niruri + p. betle + + + p. crocatum + p. retrofractum + c. citratus + + + + + + + + + + + + + + + + + + + + c. aurantiifolia + + + + + + + c. lemon + + i. verum + a. galanga + + + + b. pandurate + c. longa + + + + + + + + + + + + + + + + + + + + + + + + + + c. zanthorrhiza + + + + + + + + + + + c. zedoaria a. compactum + + + k. galangal + + + + + + + + + + + + + + + k. rotunda + + + + z. officinale + + + + + + + + + + + + + + + + + + + + + + + + + + + z. officinal var. rubrum + z. zerumbet + total plants mixed 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 6 6 6 6 6 6 7 7 7 8 9 10 notes: + = plants available in mixtures. * all mixtures were prepared by boiling and administered orally table 4 method of preparation for medicinal plants used during the covid-19 pandemic in indonesia category frequency %* boil 46 77.97 eaten directly 6 10.17 cold infusion 18 30.51 hot infusion 8 13.56 burning 5 6.78 notes: *some of the plants were prepared by more than one method. thus, the total percentage may not add up to 100%. table 5 the motivation for medicinal plants used by respondents during the covid-19 pandemic in indonesia category frequency %* to boost the immune system 238 71.26 to keep healthy 83 24.85 to build stamina 41 12.28 other motives 41 12.28 to prevent virus infection, including covid-19 18 5.39 notes: *some respondents reported more than one reason for herbal use. thus, the total percentage may not add up to 100%. biotropia vol. 30 no. 2, 2023 190 the present study revealed that respondents used 59 species of medicinal plants from 28 families (table 2) both singly or in herbal mixtures (table 3). additionally, most of the plants were prepared by boiling and then consumed orally (table 4). indonesian traditional medicine in the form of polyherbal drinks has existed for generations so called loloh (in bali) and jamu (in java). some of the reported plants in the present study that are also used in loloh formulation namely s. pinnata, b. balsamifera, e. variegata., c. burmanni, c. asiatica, a. indica, p. amaryllifolius, p. niruri, s. androgynous, p. betle, c. aurantiifolia, c. zanthorrhiza, k. rotunda, z. officinale, and z. zerumbet (sujarwo et al. 2015). similarly, other reported plants were commonly available in jamu formula such as c. verum, c. aurantifolia, z. officinale var. rubrum, t. indica, a. galanga, c. longa, c. zanthorrhiza, p. niruri. k. galanga, o. sativa, and p. amaryllifolius (elfahmi et al. 2014; hartanti et al. 2020). these plants were empirically used for various medicinal purposes (elfahmi et al. 2014; sujarwo et al. 2015). further, the current study revealed that respondents consumed jamu kunyit asam and jamu beras kencur during the pandemic (table 3, mixture no. 5 and no. 9, respectively). the composition of herbal mixtures could be varied according to individual preferences and local recipes. for example, mixture no.20, 29, 45, 52, and 59 in table 3 showed the variation of jamu kunyit asam. however, there was limited data related to the efficacy of the modified version of jamu formula. further research should be conducted to support the use of those herbal mixtures in term of efficacy test. most respondents believed that the plants’ consumption was beneficial during the pandemic because they could enhance immunity, maintain health and stamina, and prevent viral infection (table 5). a study showed that the interest in and use of immunerelated herbals worldwide increased during the covid-19 pandemic (hamulka et al. 2020). other ethnopharmacological studies confirmed the usage of medicinal plants such as a. cepa, a. sativum, c. asiatica, c. papaya, t. indica, c. burmanni, and c. longa to boost immunity by traditional healers and society in various health conditions (siew et al. 2014; anywar et al. 2020; oladele et al. 2020; lin et al. 2021). meanwhile, review studies confirmed the benefit of natural immune enhancer intakes such as a. panniculata, a. sativum, m. piperita, m. cajuputi essential oil, c. sinensis, n. sativa, and z. officinale to prevent covid-19 and improve overall patient health (boozari & hosseinzadeh 2020; sen et al. 2020; silveira et al. 2020). some of the medicinal plants reported in the current study have been also recommended by the indonesian ministry of health (hk.02.02/iv.2243/2020) to maintain wellbeing and prevent illness during the pandemic. in the official announcement, six herbal mixtures consisting of z. officinale var. rubrum, c. aurantiifolia, c. verum, c. longa, a. galanga, c. asiatica, c. zanthorrhiza, k. galanga, p. amaryllifolius, m. oleifera, and a. sativum. the mixtures were recommended to boost the immune response and also have similar preparation and administration methods as reported in this study. likewise, c. longa, z. officinalle, c. verum, and p. nigrum also have been recommended by the indian ministry of ayush (ayurveda, yoga, and naturopathy, unani, siddha, and homeopathy) to boost immunity as a prophylaxis measure against covid-19 (khanal et al. 2020). the beneficial effect of medicinal plants as immune-enhancer against covid-19 should be confirmed scientifically. in viral diseases, the infection could be fought by the host's immune response. when viruses infect the host cells, innate immunity blocks virus replication, promotes virus clearance, stimulates tissue repair, and activates a prolonged adaptive immunity (g. li et al. 2020). moreover, viral infection and inflammation of lung tissues are observed in covid-19. thus, the antiviral and inflammatory activities of medicinal plants are essential properties to combat covid-19 (khanal et al. 2020). on the other hand, it should be noticed that the immune system is complicated and highly regulated by numerous molecular and cellular events. therefore, immunity enhancement may be either valuable or destructive to the organism, depending on the overall degree of modulation and the pathophysiological condition (gertsch et al. 2011). based on the calculation of rfc, five plants had the highest scores: c. longa, z. officinale, c. medicinal plants usage during covid-19 in indonesia – nayaka et al. 191 citratus, k. galanga, and c. zanthorrhiza. therefore, further literature review in the current study was highlighted for those plants. turmeric (c. longa) contains curcumin, a polyphenol with various pharmacological actions. the compound showed immunomodulation activity through several mechanisms, especially by regulating inflammatory factors (tasneem et al. 2019; behl et al. 2021). likewise, the polysaccharide extract from turmeric could enhance the immune system (yue et al. 2010). a computational study regarding anti-sars-cov-2 showed that curcumin exhibited a high potency to block the virus's main protease (c19mpro), which plays an important role in the viral replication process. curcumin had lower binding energy to c19mpro than other compounds from p. nigrum, z. officinale, n. sativa, s. aromaticum, a. sativum and a. cepa (ibrahim et al. 2020). ginger (z. officinale) contains some compounds with anti-inflammatory and immunomodulatory activities such as 6-gingerol, 6-shogaol, zingerone, and 6-paradol (choi et al. 2018). an alcohol extract was reported to induce phagocytosis by macrophages in mice while crude extract increased humoral and cellmediated immune responses (gautam et al. 2020). meanwhile, another molecular docking evaluation showed the ability of zingiberene, 6gingerol, zingerone, gingerenone-a, 6-shogaol, and 6-dehydrogingerdione to block c19mpro. but their potencies were considerably low due to higher binding energies than n3 inhibitor as control (garg et al. 2020). an in vivo and in vitro study revealed the immunomodulatory effect of water extract and essential oil from lemongrass (c. citratus). the water extract with linalool oxide and epoxylinalool as major compounds could prevent the production of il-1β but induce il-6 production by macrophages. meanwhile, its essential oils which contained neral and geranial could inhibit cytokine production in vitro (sforcin et al. 2009). moreover, geraniol, another compound in its essential oil, inhibited the s1 subunit in spike proteins of sars-cov-2 through a docking simulation (wani et al. 2020). the rhizome part of cutcherry (k. galanga) is rich in bioactive compounds such as ethyl-pmethoxycinnamate and diarylheptanoids with anti-inflammatory and immunomodulation activity (jagadish et al. 2016; yao et al. 2018). its polysaccharides isolate enhanced the immunoregulation capability of cd4+ t cells (yang et al. 2018). furthermore, a computational study exhibited the activity of its bioactive compounds (kaempferol, kaempferol glycosides, and acylated kaempferol glucoside derivatives) to block the 3a channel protein of sars-cov. inhibition of this channel would inactivate virus production and allow the host to build up its immunity system (schwarz et al. 2014). another docking investigation indicated that kaempferol, due to its hydroxyl, ketone, and ether groups, was a stronger c19mpro inhibitor than other tested natural compounds (khaerunnisa et al. 2020). the crude polysaccharide extract of javanese turmeric (c. zanthorrhiza) could enhance the immune system by activating of nf-kappab (kim et al. 2007). xanthorrhizol and c. xanthorrhiza extract significantly inhibited the production of inflammatory cytokines, such as tumor necrosis factor-alpha, interleukin-6 and 1𝛽, and c-reactive protein (kim et al. 2014). moreover, curcumin, demethoxycurcumin, and bisdemethoxycurcumin in c. xanthorrhiza (similar compounds also contained in c. longa) showed their potential as c19mpro inhibitors (khaerunnisa et al. 2020; sumaryada & pramudita 2020). however, another study revealed that their inhibition actions were lower than nelfinavir, a protease inhibitor used as a drug standard (khaerunnisa et al. 2020). meanwhile, another docking investigation on the similarity of active sites exposed that bisdemethoxycurcumin had a greater ability to inhibit the binding pocket of c19mpro than n3 inhibitor, as the control ligand (sumaryada & pramudita 2020). other plants with lower rfc values (table 2) such as a. galanga and a. paniculata, citrus sp., c. sinensis, s. androgynous, f. vulgare, o. europea, and a. graveolens also confirmed to have immunomodulatory properties and potential against covid-19 (elfahmi et al. 2014; khaerunnisa et al. 2020; utomo et al. 2020). regarding covid-19, most of the antiviral studies of medicinal plants and their compounds were based on computational methods and resulted in a preview of their potential against covid-19. though some of the reported medicinal plants showed low molecular potency in blocking target sites, it is necessary to biotropia vol. 30 no. 2, 2023 192 highlight that the immunomodulatory actions support their beneficial role during the covid19 pandemic. further pre-clinical and clinical investigations are needed to warrant their efficacy as health-promoting agents against covid-19. these ethnobotany research results may be necessary to anticipate another transboundary animal or plant diseases pandemic by studying their bioactive compound for pharmacopeia studies in more detail. conclusion during the covid-19 pandemic, there were 59 medicinal plants belonging to 28 families used by indonesian. the plants were prepared mainly by boiling and administered orally. based on the rfc value, the most important plants were c. longa, z. officinale, c. citratus, k. galanga, and c. zanthorrhiza. also, respondents believed that those plants could boost immunity, maintain health and stamina, and prevent covid-19. in general, the medicinal plants reported in the current studies were confirmed by scientific literature to be beneficial as immune-booster during the covid-19 pandemic. meanwhile, their ability to block sars-cov-2 infection was mainly studied only through molecular docking evaluations. more research should be conducted to ensure their potency against sars-cov-2 and their efficacy when used as a single ingredient or in mixtures with other herbs. also, actions should be taken to preserve the community’s traditional knowledge of using medicinal plants. acknowledgments the authors are grateful to the authorities of the faculty of pharmacy, universitas mahasaraswati denpasar and indonesia government, through the directorate general of politics and public administration, ministry of home affairs (no/460.02/350/dv) for approving this study references anywar g, kakudidi e, byamukama r, mukonzo j, schubert a, oryem-origa h. 2020. medicinal plants used by traditional medicine practitioners to 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disease by streptomyces rochei bt02 lê thanh đạm, nguyễn lưu vĩnh hảo, nguyễn thị hồng hạnh, thạch thị thảo, quảng thị anh đào, phan minh đạt and nguyễn vũ phong* faculty of biological sciences, nong lam university, ho chi minh city 72820, vietnam. received 1 january 2024/ revised 26 february 2024/ accepted 13 march 2024 abstract this study investigated the dual role of streptomyces rochei bt02 in promoting tomato growth and suppressing fusarium solani, the causative agent of tomato root rot disease. various assays were conducted to evaluate the plant growth-promoting attributes of strain bt02 including indole-3-acetic acid (iaa) biosynthesis, phosphate solubilization, biofilm formation, and siderophore production. under in vitro conditions, strain bt02 exhibited plant growth-promoting characteristics such as cellulose degradation, phosphate solubilization, iaa synthesis, biofilm formation, and siderophore production. application of strain bt02 significantly improved tomato seed germination (26.7%), with enhanced sprout and radicle lengths and seed vigor (38.6%). greenhouse experiments revealed substantial increases in plant height, leaf and branch numbers, inflorescence, and flower number, emphasizing the positive impact of strain bt02 on overall plant growth. the bt02 strain demonstrated antagonistic efficacy of 45.3 to 58.9% against fusarium solani, as evidenced by the dual culture and agar well diffusion methods. under greenhouse conditions, strain bt02 significantly reduced disease incidence and demonstrated control efficacy of 41.6% against fusarium solani, highlighting its biocontrol potential. this study provides insights into the mechanisms employed by streptomyces rochei bt02 to enhance plant growth and suppress root rot disease, paving the way for its application as a biocontrol agent in sustainable agriculture. keywords: fusarium solani biocontrol, plant growth-promoting bacteria (pgpb), root rot disease control, streptomyces rochei bt02; tomato growth promotion introduction with ever-increasing global population growth and the consequential negative environmental impacts, ensuring the quality of agricultural products poses a formidable challenge for both farmers and researchers. the adoption of biofertilizers and biotic agents, together with judicious farming management practices, have attracted significant attention within the scientific community, with the utilization of plant growth-promoting bacteria (pgpb) extensively documented. pgpb are ubiquitously distributed in soil and on root surfaces or endophytically within roots. they exhibit remarkable capabilities of fostering plant growth and curtailing plant diseases through multifaceted mechanisms encompassing direct processes such as phosphorus solubilization, nitrogen fixation, siderophore production, hydrogen cyanide and ammonia release as well as the synthesis of phytohormones including auxin, cytokinin, and gibberellin. indirect mechanisms include acc deaminase activity, antibiotic production, secretion of cell wall degradation enzymes, and induction of systemic resistance (ryu et al. 2003). among plant growth-promoting rhizobacteria (pgpr), streptomyces species are widely distributed in soil, water, and agricultural ecosystems. this diversity in nutritional and physiological processes equips them with adaptability to adverse environmental factors including temperature fluctuations, humidity *corresponding author, email: nvphong@hcmuaf.edu.vn tomato growth and disease control by streptomyces bt02 – lê thanh đạm et al. 107 variations, oxygen content, nutrient availability, and the presence of toxic substances. streptomyces species produce an array of secondary metabolites with biocontrol activities against plant parasitic fungi, thereby contributing to their significance in agriculture (nazari et al. 2023). these secondary metabolites encompass phytohormones, siderophores, enzymes, volatile organic compounds (vocs), antibiotics, and various other bioactive substances. streptomyces biocontrol strains, leveraging both direct antimicrobial activity and the induction of plant resistance through indirect biosynthetic pathways, show high promise for plant protection and as growth-promoting agents in agricultural contexts. streptomyces species also showcase insecticidal, antibacterial, and antifungal activities, further enhancing their potential applications in sustainable agriculture. fusarium root rot, induced by fusarium solani, constitutes a significant threat in tomato production regions. streptomyces species have shown inhibitory effects against this phytopathogenic fungus. streptomyces kebangsaanensis, isolated from a malaysian ethnomedicinal plant, displayed antifungal activity against f. solani isolates, with the contained phenazine-1-carboxylic acid (pca) exhibiting enhanced inhibitory effects in combination with amphotericin b (elshafie & camele 2022). another streptomyces strain, s. sasae tg01, inhibited the growth of both f. solani and f. oxysporum, suggesting its potential as a biocontrol agent (mazlan et al. 2020), while the biological control streptomyces strain, streptomyces sp. lnu-12 exhibited high inhibition activity against melon fusarium, fulvia fulva, and alternaria solani indicating its potential to control soil-borne diseases in vegetables (bubici 2018). streptomyces rochei has been extensively investigated in various contexts, with strain idwr19 demonstrating significant plant growthpromoting (pgp) activities, enzyme production, and positive effects on plant growth, manifesting as a 12.2% increase in shoot length and a 1.8-fold increase in biomass (jog et al. 2012). in a previous study, s. rochei (mt122809) emerged as a potential antagonist to various pathogens, producing microbial volatile organic compounds (mvocs) that inhibited the growth of sorghum grain mold pathogens while enhancing plant growth (sudha et al. 2022). streptomyces rochei acta1551 exhibited robust suppression of fusarium oxysporum in vitro and also protected tomato seeds from infection in vivo, demonstrating potential as a biocontrol agent against the pathogen (kanini et al. 2013), while s. rochei amet 311 strain exhibited in vitro activity against rhizoctonia solani (venkatramani & jayaprakashvel 2023). this study elucidated the characteristics associated with the ability of streptomyces rochei bt02 to promote plant growth and control fusarium solani, the causal agent of tomato root rot disease. insights garnered from this investigation will provide a foundation for better selection and formulation of bioproducts conducive to sustainable agriculture. materials and methods materials strains of streptomyces rochei pn02 and fusarium solani were isolated, identified, and preserved at the plant integrated biology (pib) laboratory of nong lam university, ho chi minh city (tân et al. 2023). phu nong f1 (t-11) tomato seeds were utilized in this study. determination of the ability of streptomyces rochei pn02 to promote plant growth bacterial cultures were incubated in gause 1 or isp2 media for 5-7 days at 30ºc with shaking at 150 rpm. the bacterial suspension was prepared with an optical density of 0.1 at 600 nm. all experiments were conducted in a completely randomized design with four replicates. the cellulolytic capability of bacteria is determined by clear yellow halos around the bacterial colonies on cmc agar medium (teather and wood 1982). the ability to dissolve phosphate was assessed by the appearance of a clear zone of ca3(po4)2 on pikovskaya’s agar medium after 7 days of cultivation at 30c (nautiyal 1999). indole-3acetic acid (iaa) production by bacterial strains cultured in isp2 medium was detected and quantified using the salkowski reagent colorimetric method at a wavelength of 520 nm (sasirekha et al. 2012). gibberellic acid (ga) production was quantified using the extraction method with ethyl acetate, and absorbance was measured at 254 nm (berríos et al. 2004). the ability to form biofilms was determined using the biotropia vol. 31 no. 1, 2024 108 method described by o’toole et al. (o’toole et al. 2000) using gause 1 medium. siderophore production was assessed on cas agar medium according to srinivas et al. (srinivas et al. 2020), and nitrogen fixation of the bacterial strains was determined according to bashan et al. (bashan et al. 1998). investigation of the influence of streptomyces rochei bt02 on tomato plant growth as in vitro conditions, the tomato seeds were sterilized and soaked in a bacterial suspension (109 cfu/ml) for 60 minutes. at least 30 seeds were sown in petri dishes containing moistened cotton wool and incubated at 25 ± 1°c. seed germination was monitored after 2, 4, and 7 days. six germinated 2-day-old tomato seeds were placed on water agar medium and divided into two sections. a paper disk soaked in 20 µl of bacterial solution (109 cfu/ml) was placed 2.5 cm from the root tip. distilled water was used as a control. the medium plates were positioned vertically in a growth chamber and co-cultivation was carried out for 5 days under conditions of 16 hours light/8 hours dark at 25°c and 70% humidity. parameters including germination rate (%), sprout length (cm), plumule length (cm), radicle length (cm), and seed vigor were recorded (thilagam & hemalatha 2019). under greenhouse conditions, healthy tomato seedlings with three fully developed leaves were transplanted into pots (25 x 21 cm) containing clean soil, manure, lime, and phosphate fertilizer at a ratio of 4:1:1:1. when the plants had five true leaves, 8 ml of bacterial culture (107 cfu/ml) was irrigated at the base of each plant, and repeated once a week for four weeks. plant height, leaf number, branch number, inflorescence, flower number, and fruit quality were monitored (dias et al. 2017). evaluation of the antagonistic ability of streptomyces rochei bt02 against fusarium solani the dual culture method was performed according to ferreira et al. (1991) by measuring the fungal colony radius and calculating the antagonistic efficacy following the formula of moayedi (2010), and the rating scale of soytong (soytong 2010). the agar well diffusion method was performed according to sharma et al. (2017). all antagonistic activity of bacteria was demonstrated through the formation of inhibition zones after a 48-hour incubation period, calculated using the formula of azman et al. (2017). the experiments were arranged in a completely randomized design and repeated three times with four petri dishes in each replication. healthy tomato seedlings with three fully developed leaves were transplanted into pots (25 x 21 cm) containing clean soil, manure, lime, and phosphate fertilizer at a ratio of 4:1:1:1. when the plants had five true leaves, root injury was created, followed by irrigation of 5 ml of f. solani spore suspension (106 spores/ml) and 8 ml of a bacterial suspension (107 cfu/ml) around the plant base, simultaneously or after fungal inoculation depending on the treatment. the experiments were arranged in a randomized complete block design, with each treatment replicated three times and four pots per treatment. the pots were placed in a greenhouse, and watering was provided as per the plant requirements. disease incidence (%) and disease index (%) after infection at 7, 14, 21, and 28 days were recorded, and the audpc value was calculated according to jeger & viljanenrollinson (2001). the efficacy of disease control was calculated according to abbott (1925). data analysis the experimental data were analyzed by analysis of variance (anova), with means compared using duncan’s test. the data were transformed to ensure compliance with the normal distribution if necessary. results and discussion plant growth-promoting ability of streptomyces rochei bt02 under in vitro conditions, the bt02 strain demonstrated the ability to degrade cellulose, solubilize phosphate, synthesize iaa, form biofilms, and produce siderophores. however, it did not produce gibberellic acid or fix nitrogen (table 1). seeds treated with the bt02 strain exhibited a germination rate of 92.5% after 72 hours, compared to the control at 67.8%. notably, bt02-treated seeds showed longer tomato growth and disease control by streptomyces bt02 – lê thanh đạm et al. 109 sprouts (1.10 cm), radicle length (4.02 cm), and increased vigor (569.8), with statistically significant differences (p<0.01) (table 2; figure 1). under greenhouse conditions at 28 days after inoculation, plants treated with the bt02 strain reached a height of 79.6 cm, surpassing the control plants at 74.7 cm. the bt02 treatment significantly influenced plant growth, demonstrating increases in the number of leaves (19.4), branches (1.30), inflorescence (7.30), and flowers (14.3) (p<0.05) (table 3; figure 2). after 56 days of inoculation, the bt02 strain treatment resulted in the highest number of 6.50 fruits per plant. parameters including fruit weight, fruit diameter, and fruit flesh thickness were significantly higher than the control (p<0.05) (table 4). table 1 characteristics related to plant growth-promoting ability of streptomyces rochei bt02 cmc (mm) phosphorus solubilization index iaa concentration (g/ml) biofilm formation siderophore production gibberellic acid (g/ml) nitrogen fixation 24.67 ± 2.67 1.56 ± 0.22 11.50 ± 0.46 + + table 2 effect of streptomyces rochei bt02 on seed germination and vigor treatment germination rate (%) sprout length (cm) plumule length (cm) radicle length (cm) seed vigor control 67.8a ± 4.92 0.57 a ± 0.41 2.33 ± 0.29 2.83a ± 0.35 349.8 bt02 92.5b ± 2.94 (26.7%) 1.10b ± 0.19 (48.1%) 2.16 ± 0.43 4.02b ± 0.33 (29.6%) 569.8 (38.6%) note: numbers with the same superscript indicate non-significant statistical differences (α = 0.05). figure 1 influence of streptomyces rochei bt02 on tomato seed germination (a-b) 4 days after inoculation; (c) 7 days after inoculation. table 3 influence of streptomyces rochei bt02 on growth of tomato plants under greenhouse conditions parameter treatment 7 dai 14 dai 21 dai 28 dai plant height (cm) control 16.4b ± 1.20 29.3b ± 2.28 52.4b ± 2.24 74.7b ± 2.00 bt02 21.5a ± 1.96 (23.7%) 35.9a ± 2.11 (18.3%) 56.9a ± 2.16 (7.9%) 79.6a ± 2.80 (6.1%) number of leaves control 7.10 ± 0,70 10.1 ± 0.94 15.2 ± 1.07 18.9 ± 0.94 bt02 7.50 ± 0,67 10.7 ± 0.90 15.8 ± 0.97 19.4 ± 1.20 number of branches control 0.10b ± 0.30 0.60 b ± 0.49 bt02 0.70a ± 0.46 (85.7%) 1.30a ± 0.46 (53.8%) inflorescence control 0.40b ± 0.49 2.20 b ± 0.60 3.80b ± 0.60 5.30b ± 0.46 bt02 0.80a ± 0.40 (50%) 3.10a ± 0.53 (29.0%) 4.90a ± 0.30 (20.4%) 7.30a ± 0.46 (27.3%) flowers control 0.50 ± 0.67 4.01a ± 1.09 8.40a ± 0.80 12.0a ± 1.61 bt02 1.10 ± 0.70 5.10b ± 0.53 (21.5%) 11.0b ± 1.41 (23.6%) 14.3b ± 1.26 (16.0%) biotropia vol. 31 no. 1, 2024 110 numbers with the same superscript indicate non-significant statistical differences (α = 0.05). dai: days after inoculation. streptomyces species distributed globally possess the capacity to enhance growth in diverse crops, serving as versatile agents in roles such as root bacteria treatments, biopesticides, biofertilizers, and bio stimulants (ryu et al. 2003). the mechanisms underlying the promotion of plant growth by pgpr encompass the synthesis of plant growth regulators and the production of siderophores, hydrocyanic acid, antibiotics, and volatile compounds. streptomyces species contribute to crop productivity through competition, induction of systemic resistance, and mineral solubilization such as phosphorus or potassium (de andrade et al. 2023). furthermore, these species exhibit biological control activity against phytopathogens by colonizing plant roots and producing antifungal metabolites, providing a sustainable alternative to chemical fungicides. this study explored the plant growthpromoting impact of s. rochei bt02 on tomato plants and its antagonistic activity against fusarium solani through both petri dish and pot experiments. our findings revealed that s. rochei bt02 possessed plant growth-promoting characteristics such as the biosynthesis of iaa, phosphate solubilization, and the formation of biofilms and siderophores. treatment with the bacterial suspension significantly enhanced the germination and growth performance of seedlings in petri dishes, attributed in part to iaa biosynthesis and secretion by s. rochei bt02. as well as regulating apical dominance, cell elongation, and primary and secondary root growth, iaa induces the relaxation of root cell walls, thereby promoting overall plant growth (wang et al. 2009). the bt02 strain also demonstrated positive effects on plant growth, as evident in the various growth and fruit quality parameters. our results supported the potential application of s. rochei bt02 as a pgpr strain for sustainable agriculture. antagonistic ability against fusarium solani by streptomyces bt02 in the dual culture method, pn02 exhibited resistance rates of 58.9%, while in the agar well diffusion method, pn02 showed antagonistic efficiency of 45.3% (figure 3). table 4 fruit quality at 56 days after inoculation treatment number/plant fresh weight (g) length (cm) diameter (cm) fruit flesh thickness (mm) control 4.10b± 1.10 42.0b ± 2.76 3.19± 0.24 3.78b±0.34 3.34b±0.17 bt02 6.50a± 1.35 (36.9%) 53.8a ± 4.71 (21.9%) 3.47± 0.46 4.45a±0.38 (15.0%) 4.38a±0.47 (23.7%) note: numbers with the same superscript indicate non-significant statistical differences (α = 0.05). figure 2 tomato plants at 28 days after inoculation with streptomyces rochei bt02 ct: control; bt02: streptomyces rochei bt02. tomato growth and disease control by streptomyces bt02 – lê thanh đạm et al. 111 figure 3 inhibitory ability against fusarium solani bt02 at 6 days after inoculation dual culture method (a) and agar well diffusion method (b). under greenhouse conditions at 28 dai, following preand post-incubation of actinomycetes with fungal infection, disease incidence varied between 22.0% and 36.0%. the corresponding audpc values were 511 and 686, indicative of control effectiveness at 41.6% and 21.6%, respectively (table 5, figure 4). under the experimental conditions, inoculation with s. rochei bt02 hampered the growth of the tested fungi due to the production of diverse secondary metabolites with biological activities including chitinase, cellulase, and β-1,3glucanase (nguyen et al. 2023). these hydrolases degrade fungal cell walls and inhibit hyphal growth (woith et al. 2021), while bacterialproduced antimicrobial substances also impede the growth of pathogens (el-sharkawy & abdelrazik 2022). table 5 disease index (%), area under the disease progress curve (audpc), and control efficacy (%) of tomato root rot disease caused by fusarium solani bt02 under greenhouse conditions treatment height (cm) root length (cm) disease incidence (%) disease index (%) audpc control efficacy (%) 28 dai 28 dai 28 dai 14 dai 21 dai 28 dai 28 dai 28 dai control 64.5ab±2.72 23.4b±0.34 0.00 0.00 c 0.00 c 0.00 c f. solani 52.7c±2.60 20.1c±2.04 100.0 32.0 a 60.0 a 60.0 a 875 b±133 bt02 66.4a±4.71 26.7a±1.02 0.00 0.00 c 0.00 c 0.00 c bt02-f. solani 64.1ab±4.59 25.5a±1.91 100.0 24.0 b 26.0 cb 22.0 cb 511 b ±109 41.6 f. solani-bt02 61.1b±2.80 23.0b±1.34 100.0 30.0 b 36.0 cb 36.0 b 686 b±167 21.6 note: numbers with the same superscript in the same column indicate non-significant statistical differences (α = 0.01). audpc: area under the disease progress curve; dai: days after inoculation. figure 4 efficiency to control root rot disease of the bt02 strain +fs: inoculated with fusarium solani; ct: control; bt02: inoculated with bt02; bt02-fs: inoculated with bt02 24 hours before fusarium solani (fs); fs-bt02: inoculated with bt02 24 hours after fusarium solani (fs). biotropia vol. 31 no. 1, 2024 112 the application of s. rochei bt02 to tomatoes enhanced plant growth through the production of plant growth regulators, siderophores, phosphate solubilization, and resistance to pathogenic fungi by bioactive metabolites including hydrolases and antibiotics. s. rochei is categorized as a microorganism with a biosafety level of 2/4, in accordance with directive 90/679/eec of the european community on biosafety. our findings support the agricultural use of s. rochei bt02 as a pgpr strain, highlighting the exploration of both known and unknown secondary metabolites produced by this bacterial strain. this study primarily examined the potential effects of s. rochei bt02 on crop plants. however, further research is necessary to elucidate its functions, mechanisms of action, and interactions with plants and other microorganisms in the soil environment. conclusions streptomyces rochei bt02 showed promise as a candidate for sustainable agriculture, displaying dual functionality by enhancing plant growth through mechanisms such as iaa production, phosphate solubilization, and siderophore production, while also suppressing the pathogenic fungus fusarium solani. these multifaceted actions established strain bt02 as a potent biofertilizer and biocontrol agent, underscoring its efficacy for sustainable agriculture. future research should focus on understanding the molecular mechanisms of streptomyces rochei bt02 and explore its benefits by refining application methods on various crops, thereby significantly expanding the utility and contribution of this strain to promote sustainable agricultural practices. 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siregar2 and corryanti3 1faculty of forestry, universitas nusa bangsa, bogor 16161, indonesia 2t)epartment f silviculture, faculty of forestry, ipb university, bogor 16680, indonesia 5study program of environmental sciences, graduate program, institut teknologi yogyakarta, yogyakarta 55171, indonesia received 31 january 2019 / accepted 21 may 2019 abstract mistletoes are hemiparasitic macroparasite plants which interfere with trees and other wild plants in nutrient acquisition. as the plant has low leaf water potential, it draws water from teak wood tissues during the deciduous stage of the teak host, thereby killing the twigs and eventually, the teak tree. mistletoes are also a key player in plant diversity. therefore, the mistletoe population needs to be regulated not only as a parasite but also as a keystone species affecting biodiversity. knowledge scarcity on the status of mistletoes includes its genetic variation. hence, the purpose of this study is to analyze the level of genetic variation of teak mistletoe ( dendrophthoe pentandra ) using rapd marker. at padangan teak clonal seed orchard (cso), it was randomly collected leaf samples from three layers of the mistletoe’s crown (upper, middle, and below) were taken from five host teak trees randomly selected from each of the sub-observation measure plots (omp). four omp units inside the observation sample plots (osp) (n = 3, 50 x 50 m) at different levels of infestation (light, moderate and heavy) were established. analysis of the genetic variation and genetic distance of mistletoes hanging on the different crown layers were conducted using rapd markers. the leaf samples from the crown layers, uu (upper crown and sub-section upper), um (upper crown and sub-section middle), and ub (upper crown and sub-section below), which include u (upper crown) had significantly greater genetic variation (he = 0.181 — 0.255) than those from m layer (middle crown, he = 0.227) and the b layer (below crown, he = 0.114). furthermore, the widest genetic distance significantly occurred between the mistletoes of the ub and b crown layers (0.310), whereas the nearest genetic distance significantly occurred between mistletoes of uu and um layers in the upper crown (0.038). practical implications of the low genetic variation in this study include the control of mistletoe d. pentandra infestation by means of restricting its population so that perhutani state owned forestry enterprise can maintain the level of damage below the economic threshold. keywords: dendrophthoe pentandra, genetic variation, mistletoe, rapd, teak introduction mistletoes are hemiparasitic macroparasite plants which interfere in the nutrient acquisition of cultivated plants including trees and other wild plants. based on their habitat, mistletoes attach themselves to certain parts of a host plant, such as branches, twigs, and occasionally stems. many research aspects about mistletoe ^corresponding author, e-mail: znldeg@yahoo.com **this paper was presented at the 3rd international conference on tropical biology 2018, 20-21 september 2018, bogor, west java, indonesia interaction with their hosts and bird dispersers have been conducted in both plantation and natural forest ecosystems. however, there is a lack of information concerning the effect of environmental variability of the canopy on the fate of mistletoe seeds and seedlings growth (mellado & zamora 2014b), include aspect of genetic variation in this study and dna barcode characterization (muttaqin et al. 2017). mistletoe infestations can decrease the production of quality seeds in seed orchards and in timber plantations. alarmingly, with repeated infestations, mistletoes can kill the host trees. 179 biotropia vol. 27 no. 2, 2020 the initial process of growth and development of mistletoes takes place when mistletoe seeds spread on those parts of stem by the assistance of birds as main agents (mellado & zamora 2014a). the seeds then germinate and develop to form the haustorium organ which penetrates into the xylem of a host (xylem tapping) to absorb important nutrients such as water, minerals, and components of sugar and amino acids. a study on the ecophysiology of teak and its canopy hemiparasite dendrophthoe falcata var. pubescens revealed that mistletoes always maintain lower leaf water potential compared to the host teak. during the deciduous stage of the host teak, the misdetoe is drawing most of the water from the neighbouring wood tissue, thereby causing death of the twigs, and finally the death of the teak. the water use efficiency of the mistletoe is lower compared to that of the teak. its photosynthetic performance concerning electron transport ability and quantum-energyuse efficiency are better in the mistletoe leaves. since k and na are phloem mobile minerals, these are highly concentrated in the misletoe leaves. that is indicative of the absence of any phloem connections between the host and the mistletoe. the mistletoe can photosynthesis at shade and exposed condition, showing its high adaptability to the host (kallarackal et al. 2003). moreover, misfietoe-host plants’ antagonistic interactions, along with bird dispersers, may form complex networks whose function and structure can influence fragmentation at different scales, e.g., molecular or population levels (arroyo et al. 2013). in the central oregon, usa, pruning was effective in controlling the severity of dwarf misdetoes and in increasing the longevity of douglas-fir, even if not all the misteltoes were removed because of the delayed mistletoe intensification (maffei et al. 2016). in perhutani, mistletoe control by silvicultural method was also applied by planting ‘kersen’ (muntingia calabura), together with kesambi plant, dowet (sy^ygium cumini) and salam (sy^ygiumpolyanthum), as edge and filler plants. these plants, serving as substitute hosts of teak, have fruits which are foraged by the primary agent of mistletoe dispersal, the cabai bird (dicaeum sp). the mix planting of teak for mistletoe control was also conducted in indonesia (corryanti et al 2012). three mistletoe species were found at padangan teak clonal seed orchard (cso), east jawa province, indonesia, namely dendrophthoe pentandra (loranthaceae), macrosolen tetragonus (loranthaceae), and viscum articulatum (santalaceae) (muttaqin et al. 2017). d. pentandra was the most numerous and the most widely distributed at the orchard. those mistletoes belong to indigenous plant groups that usually grow on suitable host plants and spread to tropical regions including indonesia’s forested areas. the main goal of sustainable management at padangan orchard is to control mistletoe infestation and develop immediate conservation measures. it requires synergistic, not antagonistic, support through the conservation of genetic variation of teak mistletoes which can be a valuable input and be correlated with the result of assessing the intensity of mistletoe infestation, including the true mistletoe rating (tmr) modified 8-class rating requirement (muttaqin 2016). sustainable management also requires accurate data or information on the level and range of mistletoes genetic variation that validates the presence or scarcity of mistletoe species exhibiting mistletoe main characters and high level of adaptation to environmental changes. the value of high genetic variation would take effect towards species ability to adapt on environmental condition, and vice versa. so as, this study hypothesis has two alternatives; if genetic variation of d. pentandra is low then the control of this mistletoe would be restricted to moderate and heavy infestation. if the genetic variation is moderate until high then the control of this mistletoe would be conducted against all levels of infestation from light, to moderate, to heavy. therefore, knowledge about the genetic variation in mistletoes will improve the sustainable management of teak seed orchards, especially protection from pests and diseases, like mistletoes. also, it be constitute candidate population for inclusion in future conservation programmes for mistletoe of dendrophthoe pentandra that grow on teak stand in perhutani area, indonesia. for genetic resource conservation, the genetic diversity and population structure of mistletoe d. pentandra need immediate investigation (kim et al. 2017). studies on the 180 genetic variation of teak mistletoe ( dendrophthoepentandra (l.) miq.) — muttaqin et at genetic variation of mistletoes had applied some markers such as rapd, microsatellite, aflp (crichton et al. 2012; yi et al. 2013; amico et al. 2014; kim et al. 2017) and the study on the desert mistletoe phoradendron californicum (santalaceae) had used isolation of 18 microsatellite loci (arroyo et al 2013). despite the ecological and medical importance of d. pentandra, only few studies were conducted (poerba & sunaryo 2006) and no studies have evaluated the genetic diversity of its wild populations in indonesia. therefore, this research was conducted to analyze the level of genetic variation of teak mistletoe d. pentandra using the random amplified polymorphic dna (rapd) marker. materials and methods sampling sites the field data gathering and genetic material sampling were conducted at the padangan teak cso compartments or blocks, located at 111°34,57.3,? e and 07°12’56.1” s and also at bancer and payaman villages, ngeraho district, bojonegoro regency, east java province, indonesia (fig. 1). the teak cso area was divided into eight compartments that are further divided into blocks. a total of 132 blocks, at ± 5 ha each totalling to + 660 ha, were established. some 144 clones were planted repeatedly in the blocks from 1983 until 1996 (corryanti 2015). the molecular analysis was carried out at the genetics and forestry molecular laboratory, silviculture department, faculty of forestry, ipb university, indonesia. collection of samples leaf samples of d. pentandra were randomly collected from five host teaks randomly selected from the observation measure plots (omp subplots). four omps were established at the observation sample plots (osp units) (n = 3, 50 x 50 m in size) depending on the level of infestation (light, moderate and heavy) (muttaqin 2016), but one control osp plot was not included because there was no infestation or no leaf samples of d. pentandra were found, referring to modified eff or ts/crc990 (drescher et al 2016) (figs. 1, 2a, 2b). the number and distribution of mistletoe leaf samples were collected from the crown layers of each host teak. the crown layers consisted of uu (upper and sub-section upper), um (upper and sub-section middle), ub (upper and sub¬ section under), m (middle), and b layer (below). the number of collected samples (n) for each layer were uu (n = 45), um (n = 57), ub (n = 31), m (n = 56), and b (n = 15) totalling to 2014 leaf samples of d. pentandra. that procedure included the use of binoculars, digital cameras, gps of garmin oregon 550 and a map of mistletoe infestation at padangan teak cso with scale 1 : 18,000 covering the infestation from years 2010-2014. % » >2 i nap of observation and measurement sample plots m teak clonal seed orchard (c$0) padangan a a/ *>* a/ matermtmtr hid forage pljntx lor bvn cmserwilom sample phipoikt5 : • l19w map of east java province figure 1 location map of osps at the omp in padangan teak cso (muttaqin 2016) 181 biotropia vol. 27 no. 2, 2020 osp 1 m ump 0 0 n qc l m <a> h 5 m a b o d e f g h i j 1 . 20 30 47~ st~ 6~ i7~ 80 9 ~ tot a c 50 m (b) figure 2 field layout of the sampling units: (a) position of the omps (n = 4) in the osp units; (b) position of the subomps (n = 5) in an omp unit (source: efforts/crc990 2012, modified for this research) notes: c = control; l = light; m = medium; h = heavy. laboratory procedures dna was extracted from leaf samples and isolated using the modified ctab (cetyltrimetyl ammonium bromide) method (doyle 1991; aritonang el al 2007). dna quality was carried by pda (potato dextrose agar) electrophoresis of 1% agarose gel at 100 volts using the buffer te 50 [xl, 3 [il dna and 2 [xl bj (blue juice). the product of electroforesis was given the solution gelred tm nucleid acid and photographed on uv transiluminator model tfx-20.lm following aritonang el al (2007). five random universal primers, namely; opp-9, opp-15, opp-19, opbh-20, opbh-16, and 10 primers used by amico et al. (2014) for tristeric corymbosus (loranthacea) of same family as d. penlandra were used in this research (table 1). table 1 tested primers and base sequences for the pcr-rapd analysis of d. pentandra no. primers base sequences (5’-3’) 1 opp-9* gtggtccgca 2 opp-15* ggaagccaac 3 opp-19* gggaaggaca 4 opbh-20* caccgacatc 5 opbh-16* ctgcgggttc 6 opa-17 gaccgcttgt 7 opb-04 ggactggagt 8 opb-07 ggtgacgcag 9 opb-12 ccttgacgca 10 opb-15 ggagggtgtt 11 opa-04 aatcgggctg 12 opb-18 ccacagcagt 13 opb-20 ggacccttac 14 opf-11 ttggtacccc 15 opl-12 gggcggtact note: * — primer selected for this study (kissinger 2013; amico et al 2014). 182 genetic variation of teak mistletoe ( dendrophthoepentandra (l.) miq.) — muttaqin et at table 2 amplification steps of the pcr-rapd marker stages temperature (°c) time (minutes) cycle pre-denaturation 92 5 1 denaturation 92 1 annealing 32; 35 1 35a; 45b extension 73 1 final extension 73 10 1 notes: apcr machine at mj research ptc-100; b pcr machine at ab applied biosystem verititm thermal cycler. the primers were selected by temperature optimization (annealing) with the pcr process ranging from 32 °c, 33 °c, 34 °c, 35 °c, 36 °c, to 37 °c. through electrophoresis, the five primers, i.e., opp-9, opp-15, opp-19, opbh-20, opbh-16 showed clear dna fragment bands at optimization temperature of 32 °c and 35 °c with 35 and 45 cycles (tables 1 and 2). however, the other primers obtained unclear dna fragment bands. the primers used were then diluted (5x, lox) or as ratios 1:50, 1:100 in ddh20 to elucidate the crystals of dna bands by electrophoresis as a mixture of materials in a microtube for the pcr-rapd process. ingredients in the pcr-rapd process of lx reaction comprised of psd h20 (2.0 pl), go taq® green master mix (6.0 pl), primer oligonucleic (2.0 pl), dna template (2.0 pl). the amplified pcr-rapd marker was identified by electrophoresis using 2% agarose gel in 50 pl te buffer, 3 pl dna, and 2 pl bj (blue juice) for 45 min at 100 volts. the yields of the electrophoresis were given a gelred tm nucleic acid solution and were photographed on the uv transilluminator tfx-20. lm model (nybom et al 2014; aritonang et al. 2007) to identify the clear bands. the scoring used for locus marker was 1 if having a band and 0 if having no band. further interpretation of each primer was carried out using the software popgene version 1.31 and ntsyspc version 2.02. popgene was also used to compute for other statistics (e.g. allele frequency, gene diversity, genetic distance, fstatistics, multilocus structure. meanwhile, ntsys ntsyspc was used in the cluster analysis of qualitative molecular genetic data (rohlf 1998; yeh et al. 1999; aritonang et al. 2007). results and discussion genetic variation the 204 pcr amplified samples, using the five rapd markers (opbh 16, opbh 20, opp 9, opp 15, and opp 19), produced 46 clear polymorphic dna bands (loci) with the base length ranging from 50 to 1500 bp in size (fig. 3). each primer produced a range of 8 to 11 loci (mean of 9.2 loci), and the polymorphic loci percentage (plp) ranged from 32.61 to 86.96% (table 4). these results confirmed those in the study conducted by poerba and sunaryo (2006) on the same d. pentandra on 22 hosts, excluding teak, at eka karya botanical garden, bali. different from those used in this study, their study used two primers consisting of opa11 which produced 12 polymorphic bands (plp 100%) and opc-12 which produced 9 bands with 8 polymorphic bands (plp 88.89%) and 1 monomorphic band. both primers have bands ranging from 150 to 1700 bp in size. the same primer for predicting the genetic variation of nepenthes gracilis korth. in kerangas forest, indonesia, produced a band ranging from 150 to 1400 bp in size and plp of > 82.75% (kissinger 2013). 183 biotropia vol. 27 no. 2, 2020 100 bp « 500 bp 4 1000 bp, 00 bp 500 bp i / \ a"*•1000 bp 1100 bp . (a ) (b) so bp 300 bp / 50 bp 50 bp 5 * i p* (c) 50 bp 500 bp 1100 bp h750 bp (e) figure 3 pcr amplification pattern with five markers: (a) opbh 16, (b) opbh 20, (c) opp 9, (d) opp 15, and (e) opp 19 table 3 the primer sequence and number of polymorphic bands no primer sequence number of polymorphic bands base length (bp) 1 opbh 16 5’ctgcgggttc ‘3 8 50-1500 2 opbh 20 5’ caccgacatc £3 10 100-1500 3 opp 9 5’ gtggtccgca c3 11 50-1500 4 opp 15 5’ ggaagccaac £3 8 100-1100 5 opp 19 5’ gggaaggaca £3 9 50-1100 total 46 based on the five parameters of genetic diversity (he) within population, the highest value (0.255) was obtained by those mistletoes in the um crown layer and the lowest (0.114) was obtained by those in the b crown layer (table 4). the higher the he, the higher is the shannon index (i) and the ne (the sum of effective alleles). in addition, genetic variation of those in the uu, um, and ub crown layers which have the upper crown sections (he = 0.181 — 0.255) are greater than those in the m layer (middle crown) (he = 0.227) and b layer (below crown) (he = 0.114). this indicated that the dispersal agent of mistletoe seeds, the birds cabai jawa (dicaeum trochilium) and other specialist frugivores, preferred the upper and middle crown sections than the layer below (muttaqin et al. 2016). the birds attached the seeds on branches and twigs through their bird droppings. the he value of d. pentandra using the rapd marker was classified as rather low (0.194). this value was lower than tristeric corymbosus using rapd marker. comparison with the different marker, it was lower than arcethobium spp. and aiscum album using aflp marker, also it lower than ficus deltoidea using issr marker. however, it be within the range of v. coloratum (he = 0.032-0.672) using microsatellite marker (table 5). 184 genetic variation of teak mistletoe (dendrophthoepentandra (l.) miq.) — muttaqin et al. table 4 genetic variation using rapd analysis of mistletoe dendrophthoe pentandra (l.) miq. at the different crown layers of infected host teak trees at padangan teak cso, indonesia no layer3 (crown part)b n plp (%) na ne he i 1 uu 45 60.870 1.609 1.306 0.181 0.273 2 um 57 86.960 1.870 1.429 0.255 0.391 3 ub 31 65.220 1.652 1.323 0.192 0.295 4 m 56 76.090 1.761 1.377 0.227 0.346 5 b 15 32.610 1.326 1.186 0.114 0.171 sum 204 321.750 8.218 6.622 0.969 1.476 average on type level 41 64.350 1.644 1.324 0.194 0.295 standard deviation (sd) 18 20.430 0.204 0.091 0.054 0.083 notes: hrawan (2004), modified for this study: n = sum of samples; plp = polymorphic locus percentage; na = sum of observed samples alleles; ne = sum of effective alleles; he = heterozygosity expectation; i = shannon index; buu = upper crown sub-section upper, um = upper crown sub-section middle, ub = upper crown sub-section under; m = middle crown, b = below crown. table 5 comparison of genetic variation of mistletoes in this study and other studies using rapd and other markers species he marker origin source dendrophthoe pentandra 0.194 rapd indonesia, padangan teak cso this study tristerix corymbosus 0.365 rapd chile amico et al. 2014 malaysian mistletoe fig 0.500 0.750 issr malaysia zimisuhara et al. 2015 (ficus deltoideajack) viscum album 0.820 aflp korea yi et al. 2013 arcethobium spp. viscaceae 0.238 aflp western north america, reif et al. 2015 melampyrum sylvaticum 0.330 0.750 microsatellite oregon, california the united kingdom crichton et al. 2012 phoradendron californicum 0.364 0.924 microsatellite mexico arroyo et al. 2013 viscum coloratum 0.032 0.672 microsatellite korea, japan, china kim et al. 2017 the 19 novel polymorphic microsatellite markers for mistletoe viscum coloratum have been developed for its use as anti-cancer medicinal plants and have become a novelty in the molecular analysis of misdetoes (kim et al. 2017). fortunately, cross-species amplification indicated that those markers can also be used for molecular analysis associated with other species coming from the same family (santalaceae). that result was also tested for cross-species amplification in v. articulatum as a potential medicinal plant. the level of genetic variation of mistletoes play an important role in their capability to adapt to changes in environmental condition, such as lack of water, nutrient, and their regeneration. low genetic variation implies a high sensitivity to heterogeneous environment that is detrimental to the growth and development of mistletoes. d. pentandra has a rather low genetic diversity (table 4), therefore, it is important to maintain the genetic variation of d. pentandra in order to protect its existence and prevent it from extinction, and this requires effective conservation strategies. the massive utilization of d. pentandra as a medicinal plant is a cause of concern even as they are hanging on host tree and crops. genetic diversity estimates among and within populations of mistletoes species, will further help in the development of effective strategies for their conservation (kim et al. 2017). the knowledge gained from research using microsatellite markers was needed in designing conservation management programs for m. sylvaticum in the united kingdom (crichton et al. 2012). such information will also be useful for other related species of endangered non-weedy hemiparasites, according to mellado & zamora (2019), with similar traits and life history. genetic distance among mistletoes on different canopy layers the largest genetic distance (0.131) was obtained significantly between mistletoes hanging on the ub and b layers which have different crown sections while the shortest genetic distance (0.038) was significantly between those at the uu and um layers which were at the same upper crown sections (table 6). 185 biotropia vol. 27 no. 2, 2020 table 6 genetic distances between mistletoes hanging on different crown layers of host teaks crown layer um m b ub uu um **** m 0.041 **** b 0.119 0.100 ub 0.092 0.065 0.131 **** uu 0.038 0.083 0.124 0.101 um uu m ub b 0.04 0.06 0.08 0.10 0.12 figure 4 upgma dendrogram of the genetic distances, based on rapd marker, of d. pentandra misdetoes growing on the different crown layers of teak notes: crown layers: uu = upper crown sub-section upper, um = upper crown sub-section middle, ub = upper crown sub-section under; m = middle crown, b = below crown. the genetic distance or relatedness among mistletoes growing on different crown layers (described by a cluster or a dendrogram) were spread over a eucledian distance consisting of eight units (0.04 0.13) (fig. 4). two different clades were identified in which the first clade consisted of mistletoes from the um, uu, m, ub crown layers, while the second clade only contained those mistletoes in the b crown layer which was considered an outgroup. in this case, the mistletoes in the b layer were more genetically distant from other misdetoes in the uu (0.124), um (0.119), ub (0.131), m (0.100) (table 6). hence, as predicted, the two clusters were of different genetic structure. another cluster analysis of 22 d. pentandra collected from several different locations had separated the samples into two clusters; one cluster consisted of 3 groups, namely; group 1 (6 collections), group 2 (13 collections), and group 3 (2 collections), whereas the other cluster, the outgroup, consisted of only 1 collection (poerba & sunaryo 2006). the euclidean distance was 43 units (0.490 0.920) longer than this study result (0.038 0.131). the different clustering is due to the different genetic distant that d. pentandra grow on teak and others as host tree on different locations. while in this study, d. pentandra grow only on teak of crown layers differently. conclusion based on the rapd marker, the genetic variation, he, of d. pentandra mistletoe was rather low (he = 0.114 0.255). the highest he (0.255) was found among the mistletoes of the upper middle layer of the canopy, um layer, while the lowest he (0.114) was among the mistletoes in the layer below the crown (b). the genetic distance in the five layers or sections of teak crown inhabited by d. pentandra ranged from 0.038 to 0.131, and was clustered into two clades. the first clade consisted of mistletoes in the um, uu, m, and ub layers, while the second clade consisted of those mistletoes in the b layer. for perhutani, the state-owned forestry enterprise, this study results practically implied the thorough removal of mistletoes by pruning them from the entire sections of the crown, as was done successfully on the dwarf mistletoes of douglas fir. 186 genetic variation of teak mistletoe (dendrophthoe pmtandra (l.) miq.) — muttaqin et al. acknowledgements this study was supported by dipa seameo biotrop 2015 (no. 045.17/psrp/ sc/spk-pnlt/111/2015), indonesia. the authors would like to also acknowledge and express their gratitude to mr. suwarno, the head of research and development center of perhutani, indonesia and his technical staff for their 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biotechnol eq 27(5):4091-7. zimisuhara b, valdiani a, shaharuddin na, qamaruzzaman f, maziah m. 2015. structure and principal components analyses reveal an intervarietal fusion in malaysian misdetoe fig ( ficus deltoidea jack) populations. int j mol sci 16:14369-94. 188 the southeast asian journal of tropical biology vol. 32 no. 1, 2025: 38 46 doi: 10.11598/btb.2025.32.1.2347 issn: 0215-6334 | e-issn: 1907-770x 38 effectiveness of liqcoris organic pesticide as growth inhibitor of plant pathogenic microorganisms risa rosita1*, deden dewantara eris2, misbakhul bait3, zulhamsyah imran1, sri widayanti1, evelyn v. bigcas4, wachju subchan5 1seameo biotrop, bogor 16134, indonesia. 2indonesian oil palm research institute, bogor 16128, indonesia. 3pt pupuk kalimantan timur, bontang 75313, indonesia. 4mindanao state university, marawi 9700, philippines. 5university of jember, jember 68121, indonesia. article higlights • first study on liqcoris, an organic pesticide made from coconut waste. • 15% liqcoris concentration effectively inhibits pests and plant pathogens. • active compounds include phenol and carbamic acid. • pest and fungal pathogens were isolated directly from diseased plants in a school garden. • coconut waste-based pesticide offers practical, eco-friendly pest control solutions. article information received : 11 september 2024 revised : 10 march, 2025 accepted : 11 march, 2025 *corresponding author, e-mail: risa@biotrop.org research article abstract sustainable control of plant diseases is essential to maintain biodiversity. learning using prototypes is an effort to support students in understanding and learning science and technology in order to answer problems related to biodiversity loss. the research aimed to: 1) produce a prototype of liqcoris organic pesticide (blm 06) from coconut shell and shell waste, 2) measure the ability of the liqcoris organic pesticide in inhibiting the growth of plant pathogenic microorganisms, and 3) study the content of liqcoris organic pesticide. the data obtained were statistically analyzed using the sas version 9.0 program. the analysis carried out was the analysis of variance (anova). duncan’s advanced test was used at the 5% level to determine the differences among the treatments, i.e., 00 = control or without pda + pesticide; 10 = pda + pesticide concentration 10%; 15 = pda + pesticide concentration 15%, 20 = pda + pesticide concentration 20%, and 25 = pda + pesticide concentration 25%. based on in vitro test, the study showed that liqcoris concentration of 15% proved effective in killing phytopathogen fungi (p < 0.05), including fusarium equiseti, fusarium graminearum, nigrospora sphaerica and colletotrichum gloeosporioides. treatments with various concentrations of 15%, 20%, and 25% effectively produced inhibitory values in the diameter of pathogenic fungal colonies, respectively 0.118 cm, 0.000 cm, and 0.000 cm that were significantly different from concentrations of 10% (2.7140 cm) and control 0% (5.2180 cm). considering the economic value of production, the best concentration of organic pesticides chosen was 15%. on the other hand, in vivo test results indicated that in controlling mosaic viruses on diseased curly chili variety tm 999, without liqcoris treatment (control), the intensity of disease attack increased by 20.22% after 6 weeks after treatment (wat). meanwhile, after applying liqcoris organic pesticide with a concentration of 15%, the percentage of mosaic virus attacks decreased to 2.72% after 6 wat (p < 0.05). this study showed that the content of 15% liqcoris organic pesticide was dominated by active compound phenol (35.16%) and carbamic acid, phenyl ester (23.61%). keywords: biodiversity, carbamic acid, in vitro test, in vivo test, phenol, plant diseases https://doi.org/10.11598/btb.2025.32.1.2347 effectiveness of liqcoris pesticide against plant pathogens rosita et al. 39 introduction the impacts of climate change are increasing the current loss of biodiversity (linders et al. 2019; ceballos et al. 2015; national research council 2013). the decline and loss of biodiversity can have serious impacts not only on the environment, but also on human welfare in many aspects, such as health, economy, culture, food security, air and others. therefore, it is important to investigate and understand the status of biodiversity and the driving factors causing biodiversity loss. several factors causing the decline or extinction of a species include loss of habitat, air, soil and air pollution, climate change, warming seawater, ocean acidification, the emergence of disease, exploitation of flora and fauna, introduced species and industrialization of agriculture and forests. biodiversity plays an important role in the framework of climate change mitigation strategy development. it refers to the living variability and ecological complex of an organism, including: a) diversity of ecosystems or communities, habitats and landscapes, b) biodiversity, and c) genetic diversity within species different. biodiversity is a prerequisite for the healthy and natural development of all living individuals and ecosystems, therefore, it is important to study how global change affects ecosystem function (allan et al. 2015), as well as the importance of biodiversity conservation activities in providing structural diversity as well as sustainable development of human civilization in the future. moreover, the use of prototypes is one effort to facilitate understanding on the role of science and technology in solving problems related to biodiversity loss. a prototype is a basic working model for developing a learning method, in the form of a module, product model, or tool model. a prototype is a visual form of media that is using the sense of sight (eyes) to facilitate the learning process. apparently, there is a significant influence between knowledge and visual representation (arum & abdurrahman 2014). in this context, the existence of superior prototypes can help encourage the younger generation to save flora and fauna biodiversity from eventual decline. mitigating biodiversity decline through plant disease control using organic pesticides has increasingly gained public recognition and acceptance. on the other hand, mitigating biodiversity decline can increase the awareness and desire for a healthier life and prevent further environmental damage. this paradigm shift considering that organic pesticides leave no residue on plants after its use. other benefits of using organic pesticides include: 1) faster decomposition process so the pesticide do not leave residues in the soil or on plants (arif 2015; dharmadewi & suryatini 2022), 2) organic pesticides are readily available at a more affordable price (wulandari et al. 2019) compared to other chemical pesticides, 3) apart from being safe for health, organic pesticides from fruits and vegetables are also environmentally friendly (wulandari et al. 2019; yunus, 2022), and 4) the raw materials for making organic pesticides are easy to find (daryanti 2024; firyanto 2023). materials and methods study location the study was conducted in the biosystems landscape management (blm) laboratory, phytopathology laboratory, and greenhouse of seameo biotrop, bogor, west java province, indonesia. sampling plant commodities attacked by phytopathogen fungi sampling of diseased plants was carried out in two school gardens located at state vocational school 1 pacet, cianjur, and agricultural development state vocational high school cianjur, west java province, indonesia. the sampling was carried out using exploration method on leaves and fruits to obtain phytopatogen microorganism. the parts of plant that were infected by the disease were cut and put into an envelope, and labeled accordingly. subsequently, the sample was taken to the laboratory for analysis. isolation and identification of phytopathogen fungi the phytopathogen fungi were isolated from the leaves and fruits of the diseased plants. the leaves and fruits were taken to the laboratory and incubated for ± 2 weeks at 22 oc in a closed container and lined with damp tissue. isolation of the phytopathogen fungi was carried out by cutting the part of the leaf that has spores growing by cutting half the diseased part and half the healthy part, then growing those parts on pda (potato dextrose agar) media, followed by incubation until the pathogen growth occurred on the media. the fungus was isolated using the spread method at a dilution of 10-1 to 10-6 grown on biotropia vol. 32 no. 1, 2025 40 figure 1 (a) mosaic symptoms due to virus attack on chili plants, (b) rotten symptoms on chili fruits suspected of being attacked by colletroticum pda media in duplicate. incubation was carried out at 37 °c for 24 48 hours. purification of the isolate was carried out using the quadrant streak technique. the phytopathogen isolates that were successfully isolated were then rejuvenated and identified molecularly. molecular identification of phytopathogen fungi had been done at the icbb (indonesian center for biodiversity and biotechnology) laboratory, pt biodiversitas bioteknologi, bogor, indonesia. preparation of pesticide organic liqcoris raw materials in the form of coconut shell waste and coconut fiber were cleaned and dried before the pyrolysis process was carried out. the liquid smoke that had been obtained was then packaged in the 1,000 ml plastic bottle and labeled liqcoris. furthermore, the liquid smoke was analyzed and tested to obtain data on its content before being tested for its effectiveness. analysis of liqcoris organic pesticide content the analyzed data were: a) ph measurements (apha 23rd (2017): 4500-h+b); b) phenol content (gc-ms), c) total nitrogen (ik.lp04.16-lt-1.0); d) total phosphorus as p2o5 (aoac (2012) 942.05)); e) total potassium as k2o (ik.lp-04.10-lt-1.0); f ) protein content (ik.lp-04.5-lt-1.0); g) c-organic (trimetry); and h) total sugar (sni 01-2892-1992 (luff schrool). the content of the organic pesticide was analyzed at the testing and certification services laboratory unit, bogor agricultural institute, bogor, indonesia. in vitro test on the effectiveness of liqcoris organic pesticide isolates of phytopathogen fungi that had been identified molecularly were used as material for liqcoris organic pesticide effectiveness tests, i.e., fusarium equiseti, fusarium graminearum, nigrospora sphaerica, colletotrichum gloeosporioides, and fusarium equiseti. the effectiveness test was analyzed using a completely randomized design (crd) with 2 factors. the first factor was 5 concentrations of liqcoris organic pesticides (0%, 10%, 15%, 20%, and 25%), while the second factor was 5 isolates of phytopathogen fungi that had been identified molecularly. the total experimental units were 125. phytopathogen fungi isolates that had been rejuvenated and incubated for 7 days at room temperature were planted on pda media, which were mixed with liqcoris organic pesticide concentrations (0%, 10%, 15%, 20%, and 25%), then the inhibitory effect of organic pesticides in controlling the growth of phytopathogen fungi was observed. the growth of phytopathogen fungi was observed by calculating the increase in growth diameter of the five phytopathogen fungi isolates from the time they were planted until the period of treatment application. measurement of the growth diameter of phytopathogen fungi isolates was stopped after the growth of the isolates was hampered/stopped due to the influence of liqcoris organic pesticide treatment. effectiveness of liqcoris pesticide against plant pathogens rosita et al. 41 in vivo test on the effectiveness of liqcoris organic pesticide the in vivo test on the effectiveness of liqcoris organic pesticide was carried out using a diseased chili plant variety, red arrow curly tm 999 variety chili , which was 3 months old. mosaic symptoms had attacked the diseased chili plants used in the study due to viruses. the concentration of liqcoris organic pesticide used at this stage was the best concentration resulting from in vivo effectiveness testing using the pour method. the 15% liqcoris concentration was sprayed on the diseased chili plants using a knapsack sprayer according to the level of attack by plant pest organisms (ppo). spraying was twice per week. observations were carried out every week on 40 sample plants. the observed parameters were plant growth and attack intensity. measurement of attack intensity was conducted following aâ et al. (2013). the assessment of mosaic symptom attacks in the study is presented in figure 2 and table 1. where: p = attack intensity n = number of leaves from each attack category v = scale value of each category n = number of sample plants observed z = the highest attack category value statistical analysis the data obtained were statistically analyzed using the sas version 9.0 program. the analysis was carried out using analysis of variance (anova). in determining the differences among treatments, duncan’s advanced test was used at the 5% level. results and discussion molecular identification of pathogenic fungi in the in vitro test of the effectiveness of liqcoris organic pesticide, the fungi that cause plant disease were isolated and identified molecularly to prove and make sure whether all the isolates that would be used in the effectiveness test were actually the plant phytopathogen fungi or not. the results of this research showed that five isolates (f. equiseti; f. graminearum; n. sphaerica; c. gloeosporioides; f. equiseti) that had been isolated successfully were phytopathogen fungi. figure 2 chart of mosaic symptom scores due to virus attack on tm 999 variety chili plants table 1 categories of mosaic symptoms due to virus attack on tm 999 variety chili plants no. range (%) symptoms 1. 0 plants did not show virus symptoms (healthy) 2. 1 -10 plants showed very mild mosaic symptoms 3. 1129 plants showed moderate mosaic symptoms 4. 30 59 plants showed symptoms of heavy mosaicism or heavy striping without leaf shrinkage or deformity 5. 60 100 plants showed very heavy mosaic or mottled symptoms with severe leaf shrinkage or deformation, stunting or death on the other hand, the attack intensity of the diseased tm 999 variety chili plants was calculated using the following formula: p = ∑(𝑛𝑛 𝑥𝑥 𝑣𝑣 )𝑥𝑥 100% 𝑁𝑁 𝑥𝑥 𝑍𝑍 biotropia vol. 32 no. 1, 2025 42 the identification results obtained 5 species of phytopathogen fungi (fig. 3). the blast results of the five isolates in the ncbi database are presented in table 2. table 2 blast results on the ncbi database for five isolates of phytopathogen fungi isolate sample number name of comparison species accession code homology (%) 1 2211.07586 fusarium equiseti isolate qn0826.1 large subunit ribosomal rna gene, partial sequence mn368509.1 100 2 2211.07587 fusarium graminearum culture cbs:131572 strain cbs 131572 large subunit ribosomal rna gene, partial sequence mh877356.1 100 3 2211.07588 nigrospora sphaerica isolate velamen roots of rhyncostylis retusa large subunit ribosomal rna gene, partial sequence mh259869.1 100 4 2211.07589 colletotrichum gloeosporioides strain apvr 29 28s ribosomal rna gene, partial sequence mf100150.1 100 5 2211.07590 fusarium equiseti isolate qn0826.1 large subunit ribosomal rna gene, partial sequence mn368509.1 100 figure 3 morphology of five isolates phytopathogen fungi notes: (1) = fusarium equiseti; (2) = fusarium graminearum; (3) = nigrospora sphaerica; (4) = colletotrichum gloeosporioides; (5) = fusarium equiseti. the results of the molecular test were in accordance with the morphological characteristics of the five isolates. this is supported by the statement of hami et al. (2021) and rizki et al. (2021) who stated that f. equiseti is a fungal pathogen that attacks chili and lettuce plants (tziros et al. 2022). symptoms of an attack caused by f. equiseti include changes in leaf color from green to yellow then wilting and eventually the plant dies. according to hazzat et al. (2023), on psa media, isolate n3 developed colonies with abundant, fluffy, creamy white aerial mycelium. on the other hand, the color of the f. graminearum colony (isolate es15) is reddish with a circular shape, lunar-shaped macroconidia, 2-3 septates. oval microconidia are not septate (hakim & kasiamdari 2023). meanwhile, the phytopathogen fungi n. sphaerica is a phytopathogen fungus on blueberries (wright et al. 2008). the results of macroscopic identification of n. sphaerica colonies were white to light gray to dark on the seventh day. colony n. sphaerica were initially white and gradually become light gray to dark gray with the onset of sporulation (zheng et al. 2021; maulidiyah et al., 2023). according to amrullah et al. (2023), c. gloeosporioides colonies appear to grow concentrically with a creamy white upper color at the age of 5 days. at the age of 11 days, the colony produces orange acervulus which contain a collection of conidia masses. effectiveness of liqcoris pesticide against plant pathogens rosita et al. 43 in vitro test on effectiveness of liqcoris organic pesticide the application was tested in vitro by measuring the inhibitory effect of the organic pesticide at various concentrations (0%, 10%, 15%, 20%, and 25%) on the growth of the five isolates of phytopathogen fungi. in the test, the parameters measured were the diameter of the fungal growth colony during the application of liqcoris organic pesticides. results of the observations of pesticide inhibitory effect are presented in table 3. treatment with various concentrations (0%, 10%, 15%, 20%, and 25%) had a very significant effect in inhibiting the increased colony diameter of phytopathogen fungi (p < 0.05) for 6 days after incubation. table 3 the ability of liqcoris organic pesticide to inhibit the growth of phytopathogen fungi in vitro for 5 days after incubation conc. (%) treatment increase in diameter (cm) 00 control or without pda+ pesticide 5.2180a 10 pda+ pesticide concentration 10% 2.7140b 15 pda+ pesticide concentration 15% 0.1180c 20 pda+ pesticide concentration 20% 0.0000c 25 pda+ pesticide concentration 25% 0.0000c notes: numbers followed by the same letter in the same column indicate that they are not significantly different based on the dmrt test at the α ≤ 5% level; pda = potato dextrose agar. based on table 3, duncan’s further test results stated that the best inhibitory ability of organic pesticides in inhibiting/controlling the growth of phytopathogen fungi was shown at a concentration of 15% with an average increase in colony diameter of 0.118 cm. (p < 0.05). the treatment with various concentrations differed significantly from the control (0%). treatments with various concentrations of 15%, 20%, and 25% effectively produced increased colony diameter of phytopathogen fungi that were not significantly different, respectively 0.118 cm; 0.000 cm; 0.000 cm (p < 0.05). by considering the economic value of production, the best concentration chosen was 15% which would be used in the in vivo test. in vivo test on the effectiveness of liqcoris organic pesticide before applying the 15% concentration of liqcoris organic pesticide to the diseased plant, the test plants chosen were chili plants, which were attacked by mosaic symptoms due to viruses. the intensity of disease symptoms (ids) of plants was measured before and after liqcoris organic pesticides were applied. the intensity of mosaic virus disease and aphid attacks were observed over a period ranging from 1 week to 6 weeks. after applying the liqcoris organic pesticide at a 15% concentration, the results showed a significantly different effect compared to the control (p < 0.05) at 6 wat (weeks after treatment) (table 4). based on the table 4, control or plants that were not given a application had a higher ids value than those plants that were given the 15% liqcoris organic pesticide. the results showed that compared to the control, which ids value was 33.04% at 0 wat and increased to 53.26% at 6 wat, the application of 15% liqcoris organic pesticide could control the development of mosaic virus disease. the application of 15% liqcoris organic pesticide showed that the ids value at 0 wat was 21.13% and decreased to 18.41% at 6 wat. the lowest symptoms of mosaic virus disease were observed when applying the organic pesticide liqcoris compared to the control. based on the result, the active compound content of liqcoris may have been able to control plant mosaic viruses. test results on the content of liqcoris organic pesticide after obtaining the best concentration that would be used, the content of the liqcoris organic pesticide was tested. the test results showed that the active compound contained in the 15% liqcoris organic pesticide were phenol (35.16%); carbamic acid; phenyl ester (23.61%); butyric acid hydrazide (2.87%); cyclotene (1.27%); o-cresol (3.33%); m-cresol (4.25%); phenol 2-methoxy (14.28%); p-creosol (3.20%); phenol 4-ethyl2-methoxy (1.31%); syringol (8.16%); phenol 2,6-dimethoxy-4-methyl (1.56%); and trans-13octadecenoic acid methyl ester (0.98%). according to aminu and abdullahi (2021), phenol and cresol are a group of disinfectants. disinfectants are materials used to eradicate pathogenic bacteria and microorganisms. phenol and its derivatives function to prevent pest attacks, plant diseases and virus (aminu & abdullahi 2021; hagner 2013; zhang 2014). phenol is a class of secondary metabolites with hydroxyl groups (tijjani et al. 2018). these compounds are synthesized in plants as part of the host’s defense mechanism against pathogens. biotropia vol. 32 no. 1, 2025 44 table 4 intensity of symptoms mosaic virus and aphids during treatment sample plant type of disease symptom treatment ids result 0 wat (%) 6 wat (%) diseased tm 999 variety chili plant mosaic symptoms due to virus attack control 33.04a 53.26a ids increase +20.22% 15% liqcoris organic pesticide 21.13b 18.41b ids decrease -2.72% notes: ids = intensity of disease symptoms; wat = weeks after treatment; numbers followed by the same letter in the same column indicate that they are not significantly different based on the dmrt test at the α ≤ 5% level. phenolics have large and complex chemical structures (walton 2003). flavonoids are the largest class of phenolics. phytoalexins are isoflavonoids that present antibiotic and antifungal properties in response to invading pathogens. they are toxic molecules that disrupt pathogenic biochemical processes or cellular structures (sharma et al. 2022). meanwhile, carbamates are organic pesticides derived from carbamic acid, including carbaryl, carbofuran, and ami nocarb (zacharia 2011). the principle behind the use of carbamate pesticides is to influence the transmission of nerve signals, resulting in the death of the pest through poisoning (yadav et al. 2015). this pesticide can be easily degraded in the natural environment with minimum environmental pollution. hagner (2013) reported that acetic acid and furfural contained in liquid smoke have the ability to repel snail pests. the effectiveness of disinfectant compounds is greatly influenced by their concentration and exposure time. the higher the concentration and the longer the exposure, the more influential the disinfectant compound will be. the macronutrient content in liqcoris organic pesticide is presented in table 5. table 5 liqcoris organic pesticide nutrient content parameter* result regulatory limit** unit method c-organic (wet base) 0.50 min. 15 % w/w titrimetry c/n 1.39 < 25 % w/w calculation macro nutrients (total n+p2o5+k2o) 20.86 min. 2 % w/w calculation total nitrogen 0.36 % w/w ik.lp-04.16-lt-1.0 phosporous as p2o5 20.50 % w/w aoac(2012)942.05 potassium as k2o 0.001 % w/w ik.lp-04.10-lt-1.0 ph 4.57 4 9 apha23rd(2017):4500+b protein content 2.16 % w/w ik.lp-04.5-lt-1.0 total sugar 38 % w/w sni 01-2892-1992(luff schrool) notes: * = outside the scope of accreditation; ** = regulation of the minister of agriculture of the republic of indonesia no. 261/permentan/ kpts/sr.310/m/4/2019 regarding technical requirements for solid organic fertilizer. in this study, the c/n, macronutrients, total nitrogen, phosphate, potassium, ph, protein, and total sugar are in accordance with the regulation of the minister of agriculture of the republic of indonesia no. 261/permentan/ kpts/sr.310/m/4/2019 regarding technical requirements for solid organic fertilizer (table 5). our study showed that liqcoris contained low concentration of c-organic and should be increased to optimal levels as an organic fertilizer. organic pesticides can be mixed with other natural ingredients with high organic c content to increase organic c. based on the results of content tests, the liqcoris organic pesticide that has been successfully produced can be used to improve soil quality and help plants grow better, stronger, and more resistant to pests and diseases. this result is supported by yatagai (2002), who reported that liquid smoke contains chemical components, such as acetic acid that accelerate plant growth and prevent disease in plants. liquid smoke can be applied to plants by spraying it on all the leaves, twigs, and branches. the concentration of liquid smoke used is 1 2% (with water solvent) to maintain healthy plants, effectiveness of liqcoris pesticide against plant pathogens rosita et al. 45 and if pests attack the plants, the concentration can be increased to 3 5% depending on the attack level. to increase diameter and height for forestry plants and increase production for agricultural plants, the application of liquid smoke can be combined with compost charcoal. utilization of organic waste into friendly pesticides has promising prospects, because organic waste is available with various nutrient contents and the raw materials are abundant in nature. organic pesticides function as a plant pest control. besides, organic pesticide is also environmentally friendly because the materials used are active and easily decomposed in nature. conclusion liqcoris, an organic pesticide made with coconut shell waste at 5% concentration, effectively killed phytopathogen fungi compared to the control (p < 0.05). in vitro tests revealed that treatments with concentrations of 15%, 20%, and 25% effectively inhibited the diameter of pathogenic fungal colonies by 0.118 cm, 0.000 cm, and 0.000 cm, respectively, which was significantly different from concentrations of 10% (2.7140 cm) and control 0% (5.2180 cm). given the economic value of output, the optimal concentration of organic pesticides is 15%. in vivo test results showed that in controlling mosaic viruses on diseased tm 999 variety curly chili, without liqcoris treatment (control), the intensity of disease attack increased by 20.22% at 6 weeks after treatment (wat). meanwhile, after applying 15% liqcoris organic pesticide, the percentage of mosaic virus attacks decreased -2.72% at 6 wat. the liqcoris organic pesticide at 15% concentration is dominated by active compound phenol (35.16%) and carbamic acid, phenyl ester (23.61%). acknowledgments we thank the ministry of education, culture, research, technology and higher education for funding this research. we are optimistic that these findings will advance education on both national and regional levels in the future. references aâ kq, hadiastono t, martosudiro, m. 2013. pengaruh penggunaan pgpr (plant growth promoting rhizobacteria) terhadap intensitas tmv (tobacco mosaic virus), pertumbuhan, dan produksi pada tanaman cabai rawit (capsicum frutescens l.) 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10.11598/btb.2025.32.2.2470 issn: 0215-6334 | e-issn: 1907-770x 191 lichens species diversity as air quality bioindicator in gunung bibi forest, mount merapi national park irfan agus nugroho1, ananda briliana romadhona1, christabel reviana septianingtyas1, laila nurul ilma1, ari satia nugraha2, pairah3, ludmilla fitri untari4*, andhika puspito nugroho5 1department of tropical biology, faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia 2drug utilization and discovery research group, faculty of pharmacy, universitas jember, jember 68121, indonesia 3mount merapi national park agency, yogyakarta 55582, indonesia 4laboratory of plant systematics, faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia 5laboratory of ecology and conservation, faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia article higlights • the study identified 36 lichen species from 14 different families. • the lichen composition differed between stations, indicating disparities in air quality. • station ii (1600-1700 masl) had better air quality than station i (1600-1700 masl), which had a higher diversity index value and more lichen coverage. • nitrogen emissions from farming may reduce the non-nitrophilic lichen diversity and abundance. • the diversity and abundance of lichen is affected by air temperature, humidity, light intensity, and the type of bark. article information received : 26 february 2025 revised : 27 march 2025 accepted : 6 april 2025 *corresponding author, e-mail: ludmilla.untari@ugm.ac.id research paper abstract mount merapi ecosystem is distinguished by its considerable biodiversity potential, leading to its designation as a national park with main function to protect its unique ecological characteristics. a notable example of mount merapi ecosystem is gunung bibi forest, which has been designated as a sanctuary zone within mount merapi national park, having primary objectives of biodiversity conservation, habitat preservation, and ecosystem protection. nevertheless, gunung bibi forest is susceptible to the repercussions of volcanic eruptions and the pressure of agricultural activities from the surrounding area, which may potentially impact its air quality. given the geographical area of the forest, the most efficient method for air quality monitoring is by using lichens species diversity as bioindicator. this study aimed to analyze the relationship between lichen species diversity and air quality in gunung bibi forest area of mount merapi national park, including all influencing factors. data collection was carried out by dividing the area into two research stations based on altitude, using a purposive sampling method followed with data analyses to calculate lichen abundance, lichens thallus coverage area, and shannon-wiener diversity index. this study found 36 lichen species belonging to 13 different families. the lichen species composition at the two stations were different, indicating disparities in air quality between the two stations. station ii (1,600 1,700 masl) exhibited indications of a better air quality in comparison to station i (1,500 1,600 masl), which was distinguished by a higher diversity index value, as well as a greater lichens thallus coverage area. the difference in air quality between the two research stations might have been caused by nitrogen emissions from agricultural activities, which limited the diversity and abundance of non-nitrophilic lichens species. environmental factors affecting lichens species diversity and abundance were air temperature, humidity, light intensity, and bark type of lichen substrate. keywords: air pollution, bioindicator, lichens, mount merapi national park, shannon-wiener index https://doi.org/10.11598/btb.2025.32.2.2470 biotropia vol. 32 no. 2, 2025 192 introduction mount merapi is one of tropical rainforest ecosystems in indonesia, characterized by its high biodiversity, which is considered to be a significant source of germplasm (wijayati & rijanta 2020). the unique characteristics of mount merapi ecosystem has led this area to be designated as a national park, with the objective of protecting its unique ecological features. mount merapi national park (mmnp) was established in 2014 based on the minister of forestry decree no. sk.3627/ menhut-vii/kuh/2014, with one of the main objectives to protect the habitat and biodiversity of flora and fauna living in the area (wijayati & rijanta 2020). however, mount merapi is one of the most active volcanoes in the world, with a short eruption period of 2 7 years (wismaya 2016). the high volcanic activity of mount merapi can have various impacts on the surrounding environment. volcanic activity can release ash and gases into the atmosphere, which can be a source of air pollution. this volcanic ash and gas can affect the biogeochemical cycles of carbon, sulphur, and halogens in the ecosystem (delmelle et al. 2015). this is exacerbated by anthropogenic activities emanating from the slopes of mount merapi. therefore, air pollution monitoring is becoming increasingly important. to monitor environmental pollution, organisms, such as lichens, can be used as bioindicator to conduct biomonitoring. lichen is an organism resulting from the mutual symbiosis between algae and fungi (untari 2024). lichen obtains its nutrients from the surrounding air, so it is sensitive to the quality of the surrounding air, therefore, can be used as a bioindicator organism for monitoring air quality (bukabayeva et al. 2023). lichen is a long-lived and slow-growing organism with an unchanging morphology, no roots and no protective structure, so it can passively absorb substances from the atmosphere throughout its life cycle (zarabska-bożejewicz 2020). lichens can be used as bioindicators of air pollution in the form of heavy metals, organic compounds, and even radioactive elements (bukabayeva et al. 2023). monitoring using lichens is simple and informative because it can assess the ecological state of the environment along with the response of the organisms, it allows for a wider study area, and it is more effective and costeffective (matos et al. 2019). this research was carried out in gunung bibi forest of mount merapi national park. administratively, gunung bibi is located in wonodoyo village, cepogo subdistrict, boyolali regency, central java province, indonesia. gunung bibi forest is included in the sanctuary zone, with functions to protect biodiversity, habitats, and ecosystems in the mount merapi national park area. therefore, ecosystems in the core zone are unaffected by human influence and anthropogenic activities. as an area included in the core zone, gunung bibi forest has a high biodiversity value which is typical of the mount merapi ecosystem (wijayati & rijanta 2020). despite the highly valued biodiversity, gunung bibi forest is located in the area mostly affected by the eruption of mount merapi, with an air quality index category classified as unhealthy (nugroho et al. 2023), and surrounded by areas having a high level of agricultural activities, predominantly involving agriculture and cattle farming, which serve as the primary means of subsistence for the local community (bps 2023). both conditions have the potential to affect the air quality on gunung bibi, therefore, air quality monitoring is recommended. given the remote location of gunung bibi, characterized by rugged topography and limited access to power supplies, the utilization of lichen as bioindicator for air quality monitoring could offer certain advantages in terms of efficiency when compared with instrumented monitoring techniques (blett et al. 2003). as demonstrated by the lichen data collected, there are regions of particular concern, which make the data useful for further, more extensive and instrumented monitoring studies (blett et al. 2003). notwithstanding the rich biodiversity of gunung bibi, there has never been a comprehensive study of lichens diversity as a bioindicator in this area. previous research on lichens in gunung bibi was limited to the identification of lichens species, such as the initial report on corticolous lichens by susilawati (2013) and a subsequent study on the characterization of fruticose and foliose lichens by susilawati (2017). similar studies in other regions have demonstrated that lichens diversity is closely related to air pollution levels, particularly the presence of sulfur, nitrogen, and atmospheric heavy metals from both natural and anthropogenic activities (agnan 2017; zarabska-borăilescu 2020; bukabayeva 2023). however, no report has lichens species diversity as air quality bioindicator in gunung bibi forest nugroho et al. 193 yet been found that addressed the question of a possible correlation between diversity in lichens samples and air quality in the studied area, to establish the possibility of lichens as air quality bioindicator. therefore, to answer this question, a research was conducted to analyze the relationship between lichens species diversity and air quality in gunung bibi forest of mount merapi national park, and the factors influencing the air quality. the findings from this research are expected to provide deeper insights into the potential of lichens as environmental bioindicators in conservation areas, while serving as a basis for monitoring strategies and mitigating the impacts of air pollution in this region. materials and methods study area this study was conducted in gunung bibi forest area, located in the musuk-cepogo region national park management resort, mount merapi national park. the study area (fig. 1) started from the forest entrance of gunung bibi in kedung pedut, located at 7°31’17.8968’’ s and 110°28’20.8308’’ e with an altitude of 1,514 meters above sea level (masl). data collection data collection was carried out using the cruising method and purposive sampling at two research stations based on altitude. station i was located at an altitude of 1,500 – 1,600 masl adjacent to agricultural areas, while station ii was located at 1,600 – 1,700 masl relatively further away from agricultural areas. data on lichens diversity were collected on host trees with a diameter of more than 15 cm. the host tree sites were geotagged with a garmin 64s gps (garmin, usa) to determine the coordinates of the sites. lichens abundance data were collected by placing a 20 x 20 cm clear plastic plot on the side of the trunk of the host tree with the highest lichens coverage area, approximately 1 m above the ground. lichens found in the plots were counted for abundance, and lichens silhouettes were drawn to calculate thallus coverage area. environmental parameters recorded included air temperature, humidity and light intensity. the host trees on which the lichens were growing were also recorded and identified. lichens found were then documented and collected for identification in the laboratory. identification was based on morphological, anatomical and secondary metabolite analysis using the spot test method of hale (1961). the spot test was performed by making a crosssection of the lichens thallus and adding k (10% potassium hydroxide (koh) in aquadest), p (5% paraphenylenediamine (c6h4(nh2)2) in 95% alcohol) and c (10% calcium hypoclorite (ca(clo)2) in aquadest) reagents. color changes in the medulla layer were observed under a stereomicroscope. the identification of the studied lichens specimens was conducted using reference materials, including the key to the lichen genera of bogor, cibodas and singapore (sipman 2003) and macrolichens of the pacific northwest (mccune & geiser 2009). additional sources consulted included the consortium of lichen herbaria website (www.lichenportal.org), the british lichen society (www.britishlichensociety. org.uk), and other relevant publications. figure 1 study area in the gunung bibi forest, mount merapi national park biotropia vol. 32 no. 2, 2025 194 data analysis the data obtained were analyzed to obtain the importance value index (ivi) by calculating the values of relative frequency (rf), relative density (dr), and relative dominance (rdo) according to garrido et al. (2021) by the following formula. results and discussion a total of 61 individual trees were sampled for lichens species at the 2 research stations, resulting in a total of 36 lichen species from 13 different families (table 1). parmeliaceae was the predominant family, with a total of 11 species, followed by pertusariaceae (5 species), physciaceae (4 species), stereocaulaceae (3 species), lecanoraceae (2 species), graphidaceae (2 species), phlyctidaceae (2 species), monoblastiaceae (1 species), chrysotrichaceae (1 species), arthoniaceae (1 species), collemataceae (1 species), megalosporaceae (1 species), and ramalinaceae (1 species). parmeliaceae is the most diverse group of lichens in the world, consisting of 2,765 species and 77 genera (lücking et al. 2017), with a wide distribution. a study by karmacharya et al. (2022) showed similar results that parmeliaceae is also the most dominant family in the kathmandu valley area, consisting of 8 genera and 20 species. in our study, the species that exhibited the highest importance value index (ivi) values at station i were graphis scripta (72.01), phlyctis argena (69.42), and flavoparmelia caperata (34.16). conversely, the highest ivi values at station ii were observed in phlyctis argena (80.56), cryptothecia striata (19.00), and parmotrema perlatum (18.67). this finding indicated clear disparities in the prevalent lichens species at the two research stations, suggesting variations in lichens composition between the stations. furthermore, g. scripta and p. argena are cosmopolitan lichens that demonstrate a high level of resistance to various environmental conditions, including atmospheric metal pollution (agnan et al. 2017), thus explaining their dominance at both station i and station ii. both species also have a broad global distribution, g. scripta (fig. 2a) has a distribution in tropical to subtropical areas, while p. argena (fig 2b.) has a distribution in africa, europe, north america, and asia (nash et al. 2002). ...(1) .........................(2) the species diversity was analyzed using the shannon-wiener diversity index to compare lichens species diversity at each station. the shannon-wiener diversity index was analyzed according to yulianti et al. (2022) by the following formula. .........................(4) ..(3) where: h’ = shannon-wiener diversity index ni = number of individuals of a certain species n = total number of individuals of all species the value of the shannon-wiener diversity index is categorized into three categories as follows (yulianti et al. 2022). h ≤ 1 = low species diversity in the area h’ 1 ≤ h’ ≤ 3 = medium species diversity in the area h’ ≥ 3 = high species diversity in the area the calculation of lichens thallus area was performed using image j 1.54g software (national institutes of health, usa) which automatically measured the area of a photographic image by counting the number of pixels per area (aragónsánchez et al. 2017). data processing and visualization were performed using microsoft excel software (microsoft, usa). lichens species diversity as air quality bioindicator in gunung bibi forest nugroho et al. 195 table 1 diversity and abundance of lichens species found at the research site location taxonomic category number of individual number of colonies lichens thallus coverage area (cm2) ivi st at io n i family parmeliaceae         hypotrachyna afrorevoluta (krog & swinscow) krog & swinscow 1 1 15.33 3.02 flavoparmelia caperata (l.) hale 5 28 252.74 34.16 parmelina tiliacea (hoffm.) hale 1 4 66.17 6.36 parmotrema perlatum (huds.) m.choisy 4 14 395.72 28.22 punctelia perreticulata (räsänen) g.wilh. & ladd 1 3 33.26 4.76 ramalina fraxinea (l.) ach. 1 1 25.35 3.33 usnea glabrescens (nyl. ex vain.) vain. 2 2 5.07 5.27 usnea subfloridana stirt. 2 2 36.67 6.23 family stereocaulaceae lepraria elobata tønsberg 2 12 304.74 20.34 lepraria incana (l.) ach. 1 1 25.87 3.34 lepraria lobificans nyl. 2 2 81.77 7.60 family pertusariaceae pertusaria amara (ach.) nyl. 1 1 88.04 5.24 pertusaria multipuncta (turner) nyl. 1 1 37.50 3.70 family physciaceae heterodermia diademata (taylor) d.d.awasthi 1 3 126.19 7.59 physcia sorediosa (vain.) lynge 1 1 11.43 2.90 family lecanoraceae lecanora barkmaniana aptroot & herk 1 1 34.97 3.62 lecanora compallens herk & aptroot 1 1 3.33 2.66 family graphidaceae graphis scripta (l.) ach. 9 53 749.53 72.01 family phlyctidaceae phlyctis argena (ach.) flot. 12 32 881.94 69.42 family chrysotrichaceae chrysothrix candelaris (l.) j.r.laundon 2 5 110.29 10.25 total 51 168 3,285.9 biotropia vol. 32 no. 2, 2025 196 location taxonomic category # of individual number of colonies lichens thallus coverage area (cm2) ivi st at io n ii family parmeliaceae hypotrachyna britannica (d.hawksw. & p.james) coppins 2 3 51.79 6.54 hypotrachyna revoluta (flörke) hale 1 1 43.01 3.32 flavoparmelia caperata (l.) hale 3 4 146.01 11.24 parmeliopsis ambigua (hoffm.) nyl. 1 1 20.23 2.71 parmotrema perlatum (huds.) m.choisy 5 5 303.27 19.00 parmotrema xanthinum (müll.arg.) hale 1 7 46.33 8.29 punctelia perreticulata (räsänen) g.wilh. & ladd 1 4 33.94 5.52 ramalina fraxinea (l.) ach. 2 2 4.78 4.46 family pertusariaceae pertusaria albescens (huds.) m.choisy & werner 3 3 142.09 10.32 pertusaria amara (ach.) nyl. 1 1 11.50 2.47 pertusaria leioplaca (ach.) dc. 1 1 25.03 2.84 pertusaria multipuncta (turner) nyl. 3 4 80.32 9.47 pertusaria pertusa (l.) tuck. 3 3 215.98 12.32 family physciaceae heterodermia diademata (taylor) d.d.awasthi 4 6 111.30 13.28 physcia adscendens h.olivier 1 1 6.24 2.33 physcia stellaris (l.) nyl. 2 8 119.33 12.42 family stereocaulaceae lepraria elobata tønsberg 1 1 21.89 2.75 lepraria incana (l.) ach. 3 1 247.53 11.54 lepraria lobificans nyl. 1 1 90.21 4.60 family graphidaceae diorygma hieroglyphicum (pers.) staiger & kalb 1 1 4.12 2.28 graphis scripta (l.) ach. 4 5 156.54 13.69 family lecanoraceae lecanora compallens herk & aptroot 2 6 103.13 10.36 family phlyctidaceae phlyctis argena (ach.) flot. 17 31 1,201.03 80.56 phlyctis boliviensis nyl. 1 1 93.88 4.70 family arthoniaceae cryptothecia striata 4 8 250.63 18.67 family chrysotrichaceae chrysothrix candelaris g.thor 2 7 136.61 12.08 family collemataceae collema subflaccidum degel. 1 1 12.48 2.50 family fuscideaceae fuscidea lightfootii (sm.) coppins & p.james 1 1 13.58 2.53 family megalosporaceae megalospora atrorubicans (nyl.) zahlbr. 1 1 4.12 2.28 family monoblastiaceae anisomeridium biforme (schaer.) r.c.harris 1 4 11.84 4.92 total 74 123 3,708.7 note: ivi = importance value index. lichens species diversity as air quality bioindicator in gunung bibi forest nugroho et al. 197 figure 2 species with the highest ivi at each station notes: a = graphis scripta (station i); b = phlyctis argena (station ii) station i recorded 20 lichens species from 8 different families, while station ii recorded 30 lichens species from 13 different families (table 1). the shannon-wiener diversity index showed that station i had a level of lichens diversity in the “medium” category with a value of 2.557. station ii, on the other hand, had a level of lichens diversity in the “high” category with a value of 3.003. the observed discrepancy in the diversity of lichens samples collected from the two stations indicated a potential difference in the quality of ambient air at these locations. in the early stages of exposure to air pollution, there will be an increase in the concentration of pollutants in the lichens thallus; as the pollution levels increase lichens may disappear completely, creating a condition known as ‘lichens desert’ (bukabayeva et al. 2023). therefore, the higher the air pollution in an environment, the lower the lichens diversity found in the environment (muvidha 2020). in this particular instance, station ii exhibited a better air quality, as proven by the higher level of lichens diversity compared to that at station i. in addition, station i was characterized by a greater total number of lichens colonies; however, it has a comparatively lower lichens thallus coverage area compared to station ii (table 1). lichens thallus coverage area can be used as an indicator to assess the suitability of environmental conditions in which lichens live. good environmental conditions allow lichens to grow optimally, so that lichens can be found intact with a large area of thallus coverage. the accumulation of air pollution in the thallus of lichens causes physiological and morphological changes in the lichens, resulting in a decrease in thallus coverage area and even fragmentation of originally intact thallus parts (nasriyati et al. 2018). the reduction in thallus coverage area is one of the lichens responses to ambient air pollution (nasriyati et al. 2018). this result strengthened the previous indication that station ii had a better air quality than that at station i, which was indicated by higher lichens thallus coverage area at station ii. the poor air quality at station i may have been caused by anthropogenic activities since the location of station i is closer to residential areas and the agricultural land of the residents, which heightened the impact of agricultural activities to air quality at station i. agricultural activities, such as the use of organic and mineral fertilizers and the burning of organic materials, can release nitrogen compounds into the air, for example in the form of ammonia (nh₃), which can increase the ph of tree bark when deposited (zarabska-bożejewicz 2020). furthermore, high atmospheric nitrogen levels lead to changes in lichens diversity and abundance, such as an increase in nitrophilic lichens species and a decrease in the abundance of non-nitrophilic species (zarabska-bożejewicz 2020). in nonnitrophilic species, excessive nitrogen exposure leads to a decrease in chlorophyll a and ergosterol (zarabska-bożejewicz 2020). biotropia vol. 32 no. 2, 2025 198 most of the people in cepogo subdistrict work as farmers. according to bps data (2023), cepogo subdistrict is the subdistrict with the largest number of individual agricultural businesses in boyolali district, which includes food crops, horticulture, plantations, and livestock farming. as for the livestock subsector, there are a total of 9,478 households that raise livestock, including beef cattle, dairy cattle, and goats (bps 2023), which makes cepogo subdistrict one of the largest contributors to cattle farming in boyolali regency. in the management of livestock waste, there are still farmers who do not treat the wastes, but directly dispose the wastes in the backyard of the house, in the river around the settlements, or even directly dispose the wastes in their gardens and fields without prior treatment (pertiwi et al. 2022). the activity of disposing agricultural wastes without treatment can produce gas emissions that can pollute the environment, such as ammonia (nh3), hydrogen sulfide (h2s), carbon dioxide (co2), and methane gas (ch4) (pertiwi et al. 2022). aerobic organic wastes treatment (composting) is one of the sources of nh3 emissions (zarabskabozejewicz 2020). the deposition rate of ammonia (nh3) is rapid, causing most of ammonia to be easily distributed close to the emission source. about 10% of nh3 can be found within 100 m of the emission source, and when dissolved in water and ionized to nh4 +, ammonia can be transported as far as 1,000 km (zarabska-bozejewicz 2020). therefore, ammonia resulted from agricultural activity is highly potential to contribute to air pollution. based on their adaptation ability to the deposition level and the availability of atmospheric nitrogen, lichens can be grouped into three types: oligotrophs, mesotrophs, and eutrophs. oligotrophs are lichens that grow only in nutrientpoor environments, mesotrophs have intermediate nutrient requirements, and eutrophs are lichens that thrive in nutrient-rich environments (mccune & geiser 2009). oligotrophs are most abundant in areas with average n deposition of 0.5 4.2 kg n/ha/year and become less abundant as n deposition increases above 4.2 kg n/ha/year, mesotrophic lichens prefer areas with n deposition of 4.2 8.0 kg n/ha/year, and eutrophic lichens can tolerate n deposition up to 8.0 kg n/ha/year (usfs 2025). eutrophic lichens species are also known as nitrophilus species which are characterized by being nitrogen-tolerant species that benefit from nitrogen eutrophication (zarabska-bożejewicz 2020). this categorization is generally applied to the macrolichens group, which consists of foliose, fruticose, and larger squamulose lichens (mccune & geiser 2009). macrolichens are often used as bioindicator species for air quality due to their large surface area, easiness for observation, and greater sensitivity than microlichens, such as crustose lichens (pasaribu et al. 2023). in the study area, a total of 17 macrolichens species were found (table 2). there were six eutrophic lichens species, four of which were found at both stations (flavoparmelia caperata, parmotrema perlatum, punctelia perreticulata, ramalina fraxinea), and two species were found at only one each at one of the stations (usnea subfloridana at station i and parmotrema xanthinum at station ii).  as for mesotrophic and oligotrophic lichens species, a total of six species were found. out of six species, five of them were found only at station ii (collema subflaccidum, physcia adscendens, physcia stellaris, hypotrachyna revoluta, parmeliopsis ambigua), and there was only one species found at station i (usnea glabrescens). our study showed that there were differences in lichens composition between the two research stations. these results suggested that there was a decrease in the diversity of non-nitrophilic lichens species at station i, which was thought to be caused by high nitrogen emissions from agricultural activities. however, further research is needed to confirm this finding. lichens species diversity as air quality bioindicator in gunung bibi forest nugroho et al. 199 table 2 macrolichens found at the research site and their nitrogen-sensitivity no. species growth forms category location station i station ii 1 collema subflaccidum foliose oligotropha ✓ 2 heterodermia diademata foliose unknown ✓ ✓ 3 physcia adscendens foliose mesotropha ✓ 4 physcia sorediosa foliose unknown ✓ 5 physcia stellaris foliose mesotrophb ✓ 6 hypotrachyna afrorevoluta foliose unknown ✓ 7 hypotrachyna britannica foliose unknown ✓ 8 hypotrachyna revoluta foliose oligotropha ✓ 9 flavoparmelia caperata foliose eutropha ✓ ✓ 10 parmelina tiliacea foliose unknown ✓ 11 parmeliopsis ambigua foliose oligotrophb ✓ 12 parmotrema perlatum foliose eutropha ✓ ✓ 13 parmotrema xanthinum foliose eutropha ✓ 14 punctelia perreticulata foliose eutrophb ✓ ✓ 15 ramalina fraxinea fruticose eutrophc ✓ ✓ 16 usnea glabrescens fruticose mesotropha ✓ 17 usnea subfloridana fruticose eutrophb ✓ nitrophilus species were mostly found at both stations, station i and ii. this result indicated that both stations had high atmospheric nitrogen deposition. the high nitrogen deposition may have been caused by agricultural activities in the vicinity of the research site. conversely, nitrogen levels at station ii may tend to be lower than at station i, which was indicated by the presence of oligotrophic and mesotrophic lichens species at station ii, such as collema subflaccidum, physcia adscendens, physcia stellaris, hypotrachyna revoluta, and parmeliopsis ambigua (fig. 3). these species were only found at station ii, so it is noteworthy that some of these species were used as bioindicator species in this study. of the five species, physcia stellaris (fig. 3c) was the species having the highest ivi value of 12.42% (table 1), making it the strongest candidate for bioindicator species in this study. physcia stellaris was also used as a bioindicator species in a study by susan et al. (2017) in the midwest area of the united states to investigate the levels of contaminant elements in the area. thallus morphology of the lichens found at the research site can be categorized into 3 types, namely crustose, foliose and fruticose. crustose lichens have thallus morphology such as a layer of crust that lives firmly attached to the surface of the substrate. foliose lichens have a leaf-like thallus morphology, composed of thallus that will develop into lobes, and are equipped with rhizine structures functioning as attachment and food absorption apparatus. fruticose lichens have a shrub-like thallus morphology with ribbon-like branches (muvidha 2020; untari 2024). in stations i and ii, crustose lichens were found to be the most dominant category, followed by foliose and fruticose lichens (fig. 4). crustose lichens are the most resistant to environmental influences because they require less water and can store water optimally due to their strong attachment to the substrate (pasaribu et al. 2002). the strong and tight attachment also makes crustose lichens resistant to environmental stresses, such as temperature, drought, uv radiation, pollution and herbivores (pasaribu et al. 2023). foliose and fruticose lichens are more susceptible to environmental stresses. foliose lichens can only be found under certain environmental conditions, for example in still natural habitats (pasaribu et al. 2023). on the other hand, fruticose lichens are the most sensitive to environmental conditions, including air pollution (roziaty 2016). a study by sujetoviene (2017) showed that foliose and fruticose lichens are commonly found in areas with low levels of pollution, while crustose lichens are found in areas with high levels of pollution. biotropia vol. 32 no. 2, 2025 200 figure 3 oligotrophic and mesotrophic macrolichens found at station ii notes: a = collema subflaccidum; b = physcia adscendens; c = physcia stellaris; d = hypotrachyna revoluta; and e = parmeliopsis ambigua. figure 4 thallus morphology and number of lichens species lichens species diversity as air quality bioindicator in gunung bibi forest nugroho et al. 201 table 3 host trees variation and number of lichens individuals encountered host tree local name number of lichens individuals station i station ii pinus merkusii jungh. & de vriese pinus 12 8 altingia excelsa noronha rasamala 4 0 engelhardtia spicata lechen ex blume klewer 13 38 syzygium cumini (l.) skeels duwet 1 0 glochidion arborescens blume dempul 3 4 erythrina lithosperma blume dadap duri 7 13 aglaia odoratissima blume pancal kijang 5 0 symplocos javanica kurz ladok 2 0 casuarina junghuhniana miq. cemara gunung 2 0 lithocarpus elegans (blume) hatus. pasang 1 1 castanopsis argentea (blume) a.dc. sarangan 1 4 schima wallichii (dc.) korth. puspa 0 3 acacia decurrens willd. akasia 0 3 the most abundant lichens found at stations i and ii was lichens attached to klewer tree (engelhardtia spicata) with a total of 51 occurrences (table 3). the bark type of klewer tree which is classified as rough with furrows of < 1 cm in size seems to be an easier substrate for lichens to grow on compared to other trees having a smoother bark type. lichens propagules are more easily trapped and grow on trees having rough barktexture with shallow and small furrows (susilawati & kasiamdari 2021). bark type with deep cracks causes the bark to be unstable and fragile, so that it peels off easily, for example in pinus merkusii (susilawati & kasiamdari 2021). however, our study demonstrated a relatively high prevalence of lichens growing on the bark of pinus merkusii tree (12 thalli at station i and 8 thalli at station ii). this finding suggested that bark exfoliation may impede lichens growth, yet it does not entirely preclude it. this phenomenon can be attributed to the fact that deep cracks in the bark are capable of maintaining adequate moisture levels in the substrate, thereby facilitating the penetration of rhizines in foliose lichen (susilawati & kasiamdari 2021). this, in turn, provides a foundation for the growth of crustose, foliose, and fruticose lichens. lichens diversity and abundance are influenced by environmental parameters, such as air temperature, humidity, light intensity, and nutrients in the place where lichens live (pasaribu et al. 2023). our study observed that station i had a higher range of air temperature (19.9 30.6 °c) with an average of 24.0 °c, while station ii had a lower range of air temperature (20.4 25.8 °c) with an average of 22.8 °c (fig. 5). figure 5 environmental parameter measurement in the study area note: the cross mark shows the average value of each parameter. biotropia vol. 32 no. 2, 2025 202 according to murningsih & mafaza (2016), the optimal temperature for lichens growth is below 40 °c. air temperature above 45 °c can damage the chlorophyll pigment contained in the lichens, thus disrupting the photosynthetic process of the lichens. nonetheless, the mean air temperature measurements obtained from stations i and ii in our study were consistently lower than the maximum temperature threshold that could be tolerated by lichens. humidity is one of the factors that greatly affects the ability of lichens to absorb water, nutrients and pollutants contained in the air (murningsih & mafaza 2016). sundberg et al. (1996) stated that lichens can still grow and photosynthesize in habitat conditions classified as very humid, with humidity reaching 85%. humidity more than 85% can reduce the efficiency of lichens photosynthesis by 35 49% (hadiyati et al. 2017). station i exhibited a lower range of humidity, ranging from 52.6 85.6% with an average of 69.66%, while station ii demonstrated a higher range, ranging from 64.9 88.2% with an average of 76.65% (fig. 5). the humidity measurements between the two stations showed an average humidity that is below the maximum tolerance of lichens. however, station ii relatively had a consistent higher humidity that influence lichens growth. light intensity is the factor influencing the ability of lichens phycobionts to perform photosynthesis. lichens require the lowest light intensity of 1,025 lux to be able to photosynthesize (murningsih & mafaza 2016). station i exhibited a higher range of light intensity (313 5,655 lux) with an average of 1,956.8 lux, while station ii demonstrated a lower range (306 4,297 lux) with an average of 1,343.20 lux (fig. 5). this finding indicated that the average light intensity at these two stations exceeded the minimum tolerance level of the lichens. the relationship between nitrogensensitivity and lichens species suggested that the lichens species found in our study are more likely to be well-adapted to habitats with higher levels of sun exposure (zarabska-bożejewicz 2020). therefore, conditions involving lower humidity, higher temperatures and higher light availability may be beneficial for nitrophilic lichens species and limit the growth of non-nitrophilic species (pinho et al. 2012). the disparities in air temperature, humidity, and light intensity measurements between the two research stations can be attributed to the distinct vegetation composition observed at each location. station ii had vegetation with higher tree density and denser canopy cover, resulting in lower light intensity, lower air temperature, and higher air humidity. according to yulianti (2022), areas with open canopies are known to produce higher light intensity, higher air temperature, and lower air humidity, which in turn affects lichens richness. furthermore, the disparity in environmental parameters between the two stations can be attributed to altitude differences. higher altitude is associated with lower temperatures and higher relative humidity. research of ismail et al. (2024) on lichens diversity and richness across varying altitudes indicated that higher elevations are more conducive to lichens growth, owing to the fact that low temperatures and high humidity prevent desiccation. conclusion the diversity of lichens species found in the study area consists of 36 species from 13 different families. lichens composition at the two stations differs, indicating disparities in air quality between the two stations. station ii (1,600 – 1,700 masl) exhibited indications of better air quality in comparison to station i (1,500 – 1,600 masl), which was distinguished by a higher diversity index value, as well as a greater lichens thallus coverage area. the difference in air quality between the two research stations may have been caused by nitrogen emissions from agricultural activities that limit the diversity and abundance of non-nitrophilic lichens species. lichens species diversity and abundance are influenced by environmental factors, such as air temperature, humidity, light intensity and host tree bark type. acknowledgments this research was funded by the merdeka belajar kampus merdeka (mbkm) research scheme, faculty of biology, universitas gadjah mada. the authors would like to thank the directorate general of higher education, research and technology (ditjen diktiristek) of the ministry of education, culture, research and technology (kemendikbudristek) for funding this research. the author also like to thank balai taman nasional gunung merapi for the help and support during this research. we would also like to express our gratitude to the mount merapi national park agency for the assistance and support provided to us during this research. lichens species diversity as air quality bioindicator in gunung bibi forest nugroho et al. 203 references agnan y, probst a, séjalon-delmas n. 2017. evaluation of lichen species resistance to atmospheric metal pollution by coupling diversity and bioaccumulation approaches: a new bioindication scale for french forested areas. ecol indic 72:99-110. doi: 10.1016/j.ecolind.2016.08.006 aragón-sánchez j, quintana-marrero y, aragón-hernández c, hernández-herero mj. 2017. imagej: a free, easy, and reliable method to measure leg ulcers using digital pictures. int j low extrem wounds 16(4):269-73. doi: 10.1177/1534734617744951 blett t, geiser l, porter e. 2003.  air pollution-related lichen monitoring in national parks, forests, and refuges: guidelines for studies intended for regulatory and management purposes. oregon (us): national park service air resources division. p. 10-12. 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[deformation analysis of mount merapi based on gps observation data in february-july 2015]. jurnal teknik its 5(2):427-31. yulianti sd, mulyaningrum er, rahayu p. 2022. species diversity distribution area of lichen in baturraden botanical garden, banyumas.  journal of biotechnology natural science 2(1):1-9. zarabska-bożejewicz d. 2020. the impact of nitrogen pollution in the agricultural landscape on lichens: a review of their responses at the community, species, biont physiological levels. agronomy 10(12):1-19. cover single.cdr biotropia vol. 27 no. 2, 2020: 97 103 doi: 10.11598/btb.2020.27.2.1169 development of woodceramics from tropical flora** itsaree howpinjai1*, nikhom laemsak2 and pongsak hengniran2 department of silviculture, ¥acuity of forestry, kasetsart university, bangkok 10900, thailand department of forest products, faculty of forestry, kasetsart university, bangkok 10900, thailand received 14 december 2018 / accepted 23january 2019 abstract woodceramics are porous carbon or hybrid materials, derived from woody materials impregnated with a thermosetting resin and vacuum carbonized at a high temperature. woodceramics have become commercially valuable as these are used in many appliances such as heaters, gas filters, absorbents, humidity and temperature sensors. hence, this study aimed to determine the suitable conditions for producing woodceramics from various materials and their potential applications. the specific surface area (ssa), pore volume, pore diameter, and adsorption isotherm of woodceramics made from bamboo, pine, fucalyptus, rubberwood and oil palm shell particleboards were obtained using the brunauer-emmett-teller (bet) method. the average pore diameter is classified as being mesoporous (2-50 nm). the ssa and adsorption isotherm results of the woodceramics made from eucalyptus showed that these species can be used as an activated charcoal and their volume electrical resistivity is similar to that of a semiconductor. when carbonized at maximum temperatures of 800 °c and 1,000 °c, the electromagnetic shielding effectiveness (ese) of woodceramics was approximately 20-60 db within a frequency range of 800-2,200 mhz. this ese was higher than that woven from boron carbon fiber, carbon boron, and stainless steel fiber filled thermoplastics. moreover, the woodceramics production cost was lower than that of using the commercial materials. keywords: adsorption isotherms, electromagnetic shielding, specific surface area, volume electrical resistivity, woodceramics introduction woodceramics are porous carbon/carbon composites or hybrid materials, made from woody and lignocellulosic materials impregnated with thermosetting resin such as phenolic resin and vacuum carbonized at a high temperature (yu et al. 2012). woodceramics are accepted as environmentally benign materials, as these are produced from woody materials or lignocellulosic industrial waste. their advantage over other materials is that these are not only produced from wastes, but also are stronger and resistant to higher temperatures compared to the original carbonaceous material. during carbonization, the raw materials, wood or ^corresponding author, e-mail: h.itsaree@gmail.com **this paper was presented at the 3rd international conference on tropical biology 2018, 20-21 september 2018, bogor, west java, indonesia woody material, change into soft amorphous carbon and the impregnated phenolic resin changes into hard glassy carbon. the pores, which originally existed in the wood and woody material, still remain in the woodceramics. accordingly, woodceramics are amorphous carbon and glassy carbon porous composite materials which possess multifunctional properties and perform like advanced fiber reinforced composites. woodceramics are known to have high stiffness, high corrosiveness, friction resistance, electrical resistivity, electromagnetic shielding ability and high temperature resistance. these properties are useful in the production of heaters, gas filters, humidity and temperature sensors, catalyst carriers, friction materials, and electromagnetic shielding materials (pan et al. 2013). 97 biotropia vol. 27 no. 2, 2020 hence, the properties of woodceramics derived from carbonized eucalyptus ( eucalyptus spp.), rubberwood ( hepea brasiliensis\ pine ( plnus spp.), bamboo (dendrocalamus spp.) and oil palm shell particleboard (elaeis guineensis jacq.) impregnated with phenolic resin were investigated. the effects of the raw materials, the maximum temperature and the heating rate on the specific surface area (ssa), adsorption isotherms, the electrical resistivity and the electromagnetic shielding effectiveness of woodceramics were analyzed. materials and methods sample preparation the 5-year-old eucalyptus and bamboo trees located in the prachinburi province, 30-year old pine trees in chiang mai province and 30-year old rubberwood trees from chonburi province were chosen for this study. the trees were cut into lumber with a dimension of 120 x 120 x 12 mm. all the samples were oven dried at 105 °c for 24 hours. the oil palm shells were extraction residues obtained from a factory in chonburi province. the shells were ground to a size of 0.355-2.00 mm. the ground oil palm shells (ops) were mixed with phenolic resin powder in a ratio of 10:1 by weight. the mat so obtained was pressed, at a pressure of 150 psi and a temperature of 150 °c for 10 min, to obtain the oil palm shell particleboard (ops-pb). impregnation process the samples were impregnated with a commercial phenolic resin, with a specific gravity of 1.081 and viscosity of 30.7 centipoise, produced by the thai gci resitop company limited. all samples were placed in an impregnating vacuum chamber, evacuated with a vacuum pressure of 75 mmhg, and held for 2 hours. a 30% solid content of phenolic resin was added to the chamber as the vacuum pressure was released. to obtain further impregnation, the samples were immersed in the phenolic resin for 4 hours at room temperature. the samples were then removed from the chamber and wiped of excess impregnate. carbonization process the five materials (eucalyptus, rubberwood, pine, bamboo and oil palm shell particleboard) were impregnated with the phenolic resin, and carbonized at a temperature of 600, 800 and 1,000 °c in a vacuum furnace with a vacuum time of 2 hours and 2 l/min of nitrogen feed rate at a maximum temperature, and heating rates of 1, 5 and 10 °c/min. five replications for each batch of carbonization process were then tested. results and discussion specific surface area (ssa) the specific surface area property of woodceramics indicates its usage for gas filters, humidity and temperature sensors, catalyst carriers etc.). the ssa of woodceramics made from bamboo, pine, eucalyptus, rubberwood and oil palm shell particleboard was determined using the brunauer-emmett-teller (bet) method, after being carbonized at 800 °c at a heating rate of 5 °c/min. table 1 specific surface area, pore volume and average pore diameter of woodceramics derived from various raw materials raw material ssa area (m2/g) pore volume (cm3/g) average pore diameter (nm) bamboo 4 0.012 4.96 pine 10 0.018 11.75 eucalyptus 27 0.034 6.49 rubberwood 10 0.017 2.95 ops-pb 65 0.048 6.84 98 development of woodceramics from tropical flora — howpinjai et al. in this study, the ssa of eucalyptus (27 m2/g) was similar to that obtained by pan et al. (2013) for woodceramics derived from sugarcane bagasse (33 m2/g), after being carbonized at 800 °c. table 1 also shows that the ssa of woodceramics derived from bamboo is 4 m2/g and oil palm shell particleboards is 65 m2/g. the range of woodceramics pore volume made from bamboo, pine, eucalyptus, rubberwood and oil palm shell particleboard was 0.012-0.048 cm3/g, which is low when compared with other materials. the pore volume of bagasse char (0.17 cm3/g) was similar to the pore volume of woodceramics made from bamboo (0.012 cm3/g), pine (0.018 cm3/g) and rubberwood (0.017 cm3/g). the pore volumes of woodceramics made from eucalyptus (0.034 cm3/g) and oil palm shell particleboards (0.048 cnt/g) were higher than that of other materials. the average pore diameter (apd) from pine woodceramics was the highest (11.75 nm) followed by that of the woodceramics made from oil palm shell particleboard (6.84 nm), then of eucalyptus (6.49 nm), bamboo (4.96 nm) and of rubberwood (2.95 nm) which had the lowest apd. these apds were classified as mesoporous (2-50 nm). adsorption isotherms an adsorption isotherm is the relationship between the amount of gas adsorbed per unit mass of adsorbent at a constant pressure and temperature. the shape of the adsorption isotherm will provide information on adsorption process and gas-adsorption capacity of the adsorbent surface. n2 was rapidly adsorbed onto the woodceramics and it was observed that the adsorption isotherms of woodceramics from pine, eucalyptus, rubberwood and oil palm shell particleboard were similar (table 1). these complied with type iv interference, type vi isotherms, which is attributable to a monolayermultilayer adsorption as it follows a path similar to a type ii isotherm, obtained from a given adsorptive on the same surface area of the adsorbent in a non-porous form. the adsorption isotherms obtained for pine, eucalyptus, rubberwood and oil palm shell particleboard were similar to that of the woodceramics obtained from chicken waste carbonized at 800 °c. the adsorption isotherm of bamboo was classified as type vi, in which the sharpness of the steps depends on the system and the temperature. the stepwise multilayer adsorption on a uniform non-porous surface was found to be similar to the adsorption isotherms of woodceramics from sugarcane bagasse (pan et al. 2013). the gas-adsorption capacity of woodceramics from bamboo was 7.7 cc/g. the maximum adsorption capacity (30.2 cc/g) was obtained by oil palm shell particleboard, while the lowest capacity was measured for woodceramics made from bamboo (7.7 cc/g.) the ssa and gas adsorption capacity of woodceramics from eucalyptus and oil palm shell particleboard are a function of active carbon (huang et al. 2012). the ssa, pore diameter, pore volume and gas-adsorption properties also depend on the structure of raw materials and the carbonization temperature (huang et al. 2012). the values of ssa (64.71 m2/g) and pore volume (0.048 cm3/g) of woodceramics made from oil palm shell particleboard were higher than those of other materials used in the study. the average pore diameter of woodceramics from pine (11.75 nm) was larger than those woodceramics from other materials. different woodceramics showed different adsorption properties, ssa, pore diameter and pore volume, depending on the starting material and carbonization temperature. the ssa and adsorption isotherm of woodceramics from eucalyptus was affected by the activated carbon. 99 volume ( cc/ g) biotropia vol. 27 no. 2, 2020 isotherm (pine) 2 5£ adsorption desorption 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 relative pressure, p/p0 isotherm (rubberwood) adsorption desorption 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 relative pressure, p/p0 isotherm (eucalyptus) adsorption desorption 0.00 0.00 0.20 0.40 0.60 0.80 relative pressure, p/p0 1.00 32.5 30 27.5 25 22.5 wb ^ 20 ^ 17.5 i 15 ^ 12.5 10 7.5 5 2.5 0 isotherm (oil palm shell particleboard) adsorption desorption 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 relative pressure, p/p0 isotherm (bamboo) u a adsorption desorption 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 relative pressure, p/p0 figure 1 adsorption-desorption isotherm of the various woodceramics carbonized at 800 °c at a heating rate of 5 °c/min 100 development of woodceramics from tropical flora — howpinjai et al. volume electrical resistivity (ver) the volume electrical resistivity (ver) of a woodceramics depends on two factors: 1) the space of carbonized wood (the number of contact points decreases with the increase of carbonization temperature resulting in a high contact resistivity) and 2) the changes in structure of phenolic compound during carbonization process, which causes a decrease in the volume of woodceramics electrical resistivity. a higher heating rate results in a faster decay rate of the wood chemical compound, the porosity and space between glassy carbon and amorphous carbon increases, leading to a decrease in the ver (yu et al. 2012). the volume of woodceramics electrical resistivity made from bamboo, pine, hucalyptus, rubberwood and oil palm shell particleboard decreased with the increasing maximum carbonization temperature (table 2). the volume electrical resistivity of the woodceramics, obtained from carbonization at 600 °c, was significantly higher than that at 800 °c and at 1,000 °c. the phenolic compound caused a big structural change during the woodceramics production at a high carbonization temperature, resulting in a rapid decrease of ver (rhim et al 2010). the ver of woodceramics, carbonized at the heating rate of 1 and 10 °c/min, was higher than that of woodceramics carbonized with the heating rate of 5 °c/min. the ver from bamboo and pine were the lowest. lastly, the raw materials, the maximum carbonization temperature, and the heating rate played an important role in determining the volume electrical resistivity of the woodceramics. electromagnetic shielding effectiveness (ese) a lower volume electrical resistivity was beneficial to improve the electromagnetic shielding of woodceramics. hence, the volume electrical resistivity of the woodceramics product carbonized at a heating rate of 5 °c/min was evaluated for frequencies range of 8003,000 mhz, which are classified as ultra-high frequencies (uhf) and is widely used in cellular systems, broadband wireless network, radio, television, and others following the standards of the national broadcasting and telecommuni¬ cations commission, thailand (nbtc). table 2 effects of raw materials, maximum carbonization temperatures and heating rates on the volume electrical resistivity (ver) of woodceramics condition volume electrical resistivity (q.cm) max temperature (°c) heating rate (°c/min) bamboo pine eucalyptus rubberwood ops-pb 600 1 (4.00 ± 0.21) x 104 (3.02 ± 0.14) x 105 (3.17 ± 0.11) x 106 (4.27 ± 0.33) x 106 (3.94 ± 0.40) x 105 5 (7.62 ± 0.44) x 105 (5.22 ± 0.07) xlo5 (3.230 ± 0.19) x 105 (2.20 ± 0.08) x 105 (8.52 ± 0.44) x 105 10 (2.08 ± 0.13) xlo5 (5.68 ± 0.28) xlo5 (2.74 ± 0.29) x 105 (3.44 ± 0.28) x 105 (5.51 ± 0.11) x 106 800 1 18.4 ± 0.19 25.2 ± 0.15 17.0 ± 0.23 12.1 ± 0.14 11.2 ± 0.14 5 7.21 ± 0.09 11.3 ± 0.04 8.44 ± 0.39 11.3 ± 0.12 8.54 ± 0.42 10 6.67 ± 0.74 11.9 ± 0.06 9.94 ± 0.56 10.3 ± 0.06 14.5 ± 0.19 1000 1 3.10 ± 0.20 4.78 ± 0.19 4.23 ± 0.19 4.89 ± 0.18 5.38 ± 0.15 5 2.67 ± 0.15 4.35 ± 0.21 3.93 ± 0.25 4.52 ± 0.25 5.43 ± 0.14 10 2.74 ± 0.12 6.46 ± 0.35 3.62 ± 0.17 4.33 ± 0.17 6.26 ± 0.34 101 0 10 20 30 40 50 60 70 70 0 80 0 90 0 10 00 11 00 12 00 13 00 14 00 15 00 16 00 17 00 18 00 19 00 20 00 21 00 22 00 23 00 24 00 25 00 26 00 27 00 28 00 29 00 30 00 e le ct ro m ag n et ic e ff ec ti ve n es s (d b ) frequencies ranging (mhz) electromagnetic shielding effectiveness (800-3000 mhz) bamboo 600°c bamboo 800°c bamboo 1000°c pine 600°c pine 800°c pine 1000°c eucalytus 600°c eucalytus 800°c eucalytus 1000°c rubberwood 600°c rubberwood 800°c rubberwood 1000°c ops-pb 600°c ops-pb 800°c ops-pb 1000°c biotropia vol. 27 no. 2, 2020 a vaw “ * •“ “ ••••• figure 2 electromagnetic shielding effectiveness for frequencies range of 800-3,000 mhz of woodceramics carbonized at a heating rate of 5 °c/min the electromagnetic shielding effectiveness (ese) increased with the increasing maximum temperature. the ese of woodceramics made at a maximum temperature of 600 °c was very poor, especially in a frequency range of 1,5003,000 mhz (fig. 2). the ese values of woodceramics made at maximum temperatures of 800 °c and 1,000 °c were acceptable (higher than 20 db), in a frequency range of 700-2,200 mhz. increasing the maximum carbonization temperature has resulted in an increased (ese). this was due to the decay velocity of chemical elements that made up the woodceramics. the higher the temperature, the higher the porosity of woodceramics, the better is the efficiency of the electromagnetic shielding. the porous structure of a woodceramics helps to absorb electromagnetic waves (mistik et al. 2012). the woodceramics made at maximum temperatures of 800 °c and 1,000 °c, within a frequency range of 700-2,200 mhz, have higher electromagnetic shielding effectiveness than that of fabrics woven from boron carbon fiber and carbon boron. it has a protection efficiency of about 20-50 db at frequencies range of 8003,000 mhz. at 950 mhz, the protection performance of woodceramics, carbonized at 800 °c and 1,000 °c, is comparable to that of the stainless steel fiber filled thermoplastics (36-42 db) (mistik etal 2012). the suitable conditions for producing woodceramics for electromagnetic shielding material was that of pine wood carbonized at 800 °c with a heating rate of 5 °c/min, as this resulted in a woodceramics that can be used in a wider frequency spectrum (nbtc 2017) (table 3). table 3 suitable conditions for producing woodceramics from bamboo, pine, eucalyptus, rubberwood and oil palm shell particleboard for electromagnetic shielding applications (classified using the frequency range of nbtc, thailand) frequency range (mhz) applications suitable condition 800-960 cellular system, trunked mobile radio system pine 800 °c = 35.3 db 970-1,210 aeronautical radio navigation pine 800 °c = 35.3 db 1,220-1,420 radio location, radar pine 1,000 °c = 32.5 db 1,520-1,700 radio astronomy pine 1,000 °c = 33 db 1,710-2,170 mobile phone eucalyptus and ops-pb 1,000 °c = 41.4 db 2,300-2,690 broadband wireless access rubberwood 1,000 °c= 16 db 2,700-3,000 aeronautical radio service, radio navigation oil palm shell particleboard 800 °c = 14 db 102 development of woodceramics from tropical flora — howpinjai et al. conclusion the commercial properties of woodceramics depend on the raw materials, the maximum temperature and the heating rate. the suitable woodceramics production processes of eucalyptus, bamboo, rubberwood, pine and oil palm shell particleboard depend on its specific purpose or application. the conditions present during the woodceramics production affect the ssa and adsorption isotherms, electrical resistivity and electromagnetic shielding. the average pore diameters of woodceramics made from bamboo, pine, eucalyptus, rubberwood, and oil palm shell particleboard at 800 °c are classified as mesoporous (2-50 nm). the volume electrical resistivity of woodceramics made at a maximum carbonization temperatures of 800 °c and 1,000 °c was between 2.6 and 25.2 q.cm indicating that these woodceramics are semiconductors. the electromagnetic shielding effectiveness of these woodceramics carbonized at temperatures of 800 °c and 1,000 °c was higher than those of commercial materials woven from boron carbon fiber, carbon, and stainless steel fiber filled thermoplastics. suitable conditions for the production of woodceramics for electromagnetic shielding material is the carbonization of pine wood at 800 °c at a heating rate of 5 °c/min. hence, the woodceramics produced from the study can be used in a wide range of frequencies. acknowledgements this research was funded by the center for advanced studies in tropical natural resources, institute of advanced studies, icasetsart university (castnar, nru-ku), thailand. references huang zk, lu qf, lin q, cheng x. 2012. microstructure, properties and lignin-based modification of wood— ceramics from rice husk and coal tar pitch. j inorg organomet polym mater 22:1113-21. mistik si, sancak e, usta i, kocak ed, akalin m. 2012. investigation of electromagnetic shielding properties of boron and carbon fibre woven fabrics and their polymer composites. in: 4th rmutp international conference: textiles & fashion. proceedings: 2012 july 3-4. bangkok (th): rajamangala university of technology phra nakhon (rmutp) and the technical university of liberec (tul). p. 34. pan j, cheng x, yan x, zhang c. 2013. preparation and hierarchical porous structure of biomorphic woodceramics from sugarcane bagasse. j eur ceram soc 33:575-81. rhim yr, zhang d, rooney m, nagle dc, fairbrother dh, herman c, drewry dg iii. 2010. changes in the thermophysical properties of microcrystalline cellulose as function of carbonization temperature. carbon 48:31-40. the office of national broadcasting and telecommunications commission (nbtc) [internet]. 2015. national table of frequency allocation, [updated 2015; cited 2017 nov 1], available from: https:/ /house.nbtc.go.th/wps/ wcm/connect/nbtc/ntfa+2558+unofficial+t ranslation-17092558.pdf yu xc, sun dl, sun db, xu zh, li xs. 2012. basic properties of woodceramics made from bamboo powder and epoxy resin. wood sci technol 46:23-31. 103 biotropia vol. 29 no. 2, 2022: 150 160 doi: 10.11598/btb.2022.29.2.1687 150 pregnant and lactating macaca nigra: behavior and food selection eka arismayanti1, james o. waterman2, andre pasetha1, indira nurul qomariah1, dyah perwitasari-farajallah1,4,5*, and dewi apri astuti3 1animal biosciences study program, institut pertanian bogor, bogor 16680, indonesia 2biological and environment sciences, liverpool john moores university, united kingdom 3animal nutrition and feed technology study program, institut pertanian bogor, bogor 16680, indonesia 4primate research center, institut pertanian bogor, bogor 16680, indonesia 5macaca nigra project, north sulawesi 95535, indonesia received 3 november 2021/accepted 6 march 2022 abstract pregnancy and lactation are reproductive phases that require large amounts of energy. females in the reproductive period need good quality and quantity of food to provide nutrition for the fetus, milk production and child care. the mother will adapt to changes in behavior patterns and food type to meet these needs. the influence of parity and environmental conditions can affect the behavior patterns of females. during pregnancy, the macaca nigra is known to have different proportion of activities in each period, while the behavior during the lactation phase in each mester is unknown. therefore, this study aimed to analyze the behavior patterns in each mester and the food selection of macaca nigra during the pregnancy and lactation phases, as well as the influence of female parity and environmental toward the behavior patterns. there were 39 females macaca nigra observed from two groups from august 2018 to july 2019. an instantaneous focal sampling method was performed to observe females’ daily activities, continuous focal sampling to monitor food types and a selectivity index to analyze food type preferences. the results showed that the female macaca nigra pattern was influenced by the reproductive phase, female parity and environmental conditions. females at the end of the pregnancy and lactation phases had a high proportion of feeding and eat more arthropods. primiparous females mostly performed resting activities. food preference was influenced by reproductive factors and food availability. the choice of fruit could be affected by fruit availability, and their favorite food was d. mangiferum and euginia sp. keywords: daily activity, fruits availability, fruits preferences, macaca nigra, reproductive phase introduction sulawesi black crested macaque (macaca nigra) is one of the seven macaques species endemic to sulawesi island. female m. nigra has three phases of reproduction, namely oestrous, pregnant and lactation. several studies have shown that the reproductive phase requires a large energy cost (pond 1977; altmann & samuels 1992; garber & leigh 1997). pregnancy and lactation are essential stages for females, especially lactation, which is the reproductive phase with the highest energy costs (pond 1977; garber & leigh 1997). during lactation, females need foods with higher protein and calories for milk production and baby care, such as infant transport and thermoregulation (altmann & samuels 1992; guedes et al. 2008). the gestation period of m. nigra lasted about 180 days (engelhardt & perwitasari-farajallah 2008) and was characterized by discoloration around the urogenital area from red to dark purple (gholib 2017). the lactation period was characterized by a discoloration of the urogenital skin from dark red-purple to pale pink (gholib 2017). the lactation lasts approximately one year (pasetha 2018, unpublished data). females allocate time to optimize reproductive success (mccabe & fedigan 2007). the reproductive phase affects the behavior and proportion of female daily activities. females change feeding strategies to meet nutritional *corresponding author, email: witafar@apps.ipb.ac.id behavior and food preference of pregnant and lactating macaca nigra – eka arismayanti et al. 151 needs (dunbar & dunbar 2002). in addition to internal factors such as reproduction and birth experience (parity), external factors such as environmental conditions (availability of food and seasons) can affect individual nutritional intake, thus triggering changes in activity and selection of food types (murray et al. 2006; cui et al. 2019). selected foods are generally high quality, easy to process and eaten more often than expected based on their estimated availability (leighton 1993). the study of food preference focused on the type of fruit eaten by m. nigra, because m. nigra is a frugivorous monkey. fruits are expected to be the food resource that most restricts the feeding places of this species. sulawesi black crested monkeys spend 59% of their time for locomotion and eating, while the other 41% are for resting and socializing (o'brien & kinnaird 1997). during the reproductive phase, females of m. nigra have a different proportion of activity, i.e., higher feeding activity during the pregnancy and higher resting during the lactation phase (pasetha et al. 2019). the activity changes in each lactation period are not known, as well as the fruit preference during the pregnancy and lactation phases. the purpose of this study was to investigate the daily proportion of female m. nigra behavior in pregnancy (b1, b2, b3) and lactation (l1, l2, l3) phases, as well as their fruit preferences during those two phases. analysis of the influence of female parity and environmental conditions was also carried out on the behavior and food preferences of female m. nigra. materials and methods research subjects and locations two groups of habituated m. nigra in pantai batu 1b (pb1b) and rambo (r1) were observed in their natural habitats. sixteen individuals in the pregnancy phase and 23 individuals in the lactation phases of m. nigra were observed from august 2018 to july 2019. data collection was conducted in the macaca nigra project (mnp) research station, at the conservation forest management unit (kphk) area of tangkoko, north sulawesi, indonesia (10 32'39''n, 1250 12'42''e). this area covers 8,867 ha and consists of primary and secondary lowland rainforests with an altitude of 1,350 meters above sea level (o'brien & kinnaird 1997). research tools data were collected using the 10-inch odys tablet, samsung smartphone j3 (2016) with the cyber tracker application, counters, cameras, books, pencils, markers, ziplock plastics, garmin gps, binocular, obrometer, thermometer maximum-minimum and portable scales. demographics and daily behavior data collection the group was followed from 05:00 to 17:00, five days a week. demographic data collected were females' reproductive status and the day of parturition (as an indicator of the end of the gestation phase). the oestrus cycle was observed qualitatively with visual observations. the observation was based on the size of swelling and skin color around the urogenital area (0 = deflating, 1 = inflating, 1-2 = medium swelling, 2 = maximum swelling) (higham et al. 2012). pregnancy was divided into three periods, namely b1, b2, and b3 (one period equals to two months). lactation was divided into three periods, namely l1, l2, and l3 (one period equals to four months). behavioral data were collected with focal animal sampling and instantaneous sampling for 30 min with a oneminute interval (altmann 1974). food intake was recorded by continuous focal sampling, while data on the consumed food species were recorded (rothman et al. 2011). daily activities recorded were feeding, foraging, locomotion, resting and socializing. observation time was divided into three periods: morning (06:0009:00), noon (10:00-13:00), and afternoon (14:00-17:00) (pasetha et al. 2019). identification of fruit species fruit species identification was carried out based on mnp database. unknown fruit species were sampled, made into herbarium specimens and identified in the biology research center, indonesian science institute (lipi). collected fruits were stored in ziplock plastic bags and labeled with a local name, scientific name, date, location and part-eaten (rothman et al. 2011). biotropia vol. 29 no. 2, 2022 152 environmental data collection environmental data taken were the air temperature and the rainfall around the mnp research station area. the air temperature and rainfall were measured every morning during the study period at 07:00 or 08:00 micro temperature (around the mnp research station), and the numbers on the obrometer boundary were recorded. the data were tabulated monthly. food availability index fruit availability was calculated by observing the parts of the trees eaten by m. nigra, from which the food availability index (fai) was then calculated. measurements were taken on the second week of every month. the calculation of food availability index was carried out by making 20 plots sized 100 x 100 m covering 6.9% of the total group area. all the tree species were recorded and identified. the number of tree parts counted, such as leaves (young leaves, old leaves and leaf buds), flowers (flowers and flower buds) and fruit (raw fruit, ripe fruit and old fruit). we used two types of measurements. the 1st type was logarithmic scale for measuring leaves: 0 = 0-9 leaves; 1 = 10-99; 2 = 100-999 and so on. the 2nd type was rank scale for measuring fruits: 0 = almost nothing; 1 = multiples; 2 = many but not full; 3 = full or almost full. the diameter (dbh) of tree trunks was measured for all the trees with dbh of more than 10 cm and propagated more than 5 cm (sari 2013). the data was analyzed by using the food availability index (fai) formula (1). fai =  n (aixdi) …….……….…………. (1) i where: fai = total food availability index for species n ai = average value of food availability for species i (logarithmic scale) di = average density of species i per hectare (based on 20 plots). behavioral data analysis data on daily activities were tabulated using pivot tables. the daily behavior proportions were grouped and calculated based on pregnancy phase differences (b1, b2, and b3) and lactation (l1, l2 and l3) phase differences. the frequency of daily activities was calculated by using formula (2): activity frequency i (%) = activity i x 100 ..….. (2) total all activity analysis of food type frequency and fruit preference all types of food eaten were tabulated using pivot tables. the frequency of food and fruit types was analyzed to see the relationship between reproductive conditions and seasons. the frequency of food types was calculated by using formula (3): food i frequency (%) = food i x 100 …… (3) total all food food preferences were calculated by food selection index method (krebs 1999; manly et al. 2002). the fai values were used to determine a proportion of fruit availability in habitats. each type of eaten food was calculated as the ratio of food selection. the resulting value can be interpreted as the probability of food desired choice by primates. the null hypothesis test using the g-test indicates that an animal randomly chooses food resources based on food availability at a confidence interval of 95%. the food selection index was calculated by using formula (4): w = oi …………………….………..……… (4) pi where: w = forage ratio oi = proportion of fruit species eaten pi = proportion of fruit species i in the habitat (fai). statistical analysis statistical analyses were run using a nonparametric multivariate analyses. shappiro-wilk test for normality test and kruskal-wallis for testing the significance of reproductive phase differences were performed. g-test was used for selectivity analysis of selected fruit. pairwisewilcoxon test was performed to know which variables influence the behavior. the statistical analyses were run using program r alpha level 0.05. behavior and food preference of pregnant and lactating macaca nigra – eka arismayanti et al. 153 results and discussion female behavior during reproduction reproductive phase is an important phase for females, which affects changes in daily activities and types of food eaten (muruthi et al. 1991; white et al. 2007). energy required by female mammals during pregnancy and lactation phases are generally higher than those of nonproductive females (lee 1997). energy costs for reproduction in primates can be achieved by implementing several strategies such as increasing energy intake, reducing energy excretion, using up body tissue reserves and reducing energy output from physical activity (bercovitch 1993; dufour & sauther 2002; national research council 2003). based on the observations, females during reproductive phase had higher feeding and social activities compared to other activities. locomotion was the least activity performed by pregnant and lactating females. lactating females had feeding (31%, p = 0.01) and foraging (16%, p = 0.21) activities higher than pregnant females. other activities such as resting (19%, p = 0.7), locomotion (12%, p = 0.81) and social (25%, p = 0.6) were performed more by pregnant females than lactating females (fig. 1). figure 1 the behavior frequency of female m. nigra during pregnancy and lactation phases notes: observed behaviors were feeding (fe); foraging (fo); resting (re); locomotion (lo) and socializing (so). based on this study, females in the pregnant and lactating phases had significantly higher feeding activity, especially the lactating females. the increased energy intake shown by the increasing feeding frequency is an adaptation done by females to meet the energy needs (dufour & sauther 2002). social activities were also highly performed by the pregnant and lactating females, although activity was not significantly different between pregnant and lactating females. these results align with a study done on macaca thibetana, where females with infants receive more grooming and affection from other members than females without infants. this phenomenon can be explained by the biological market theory, which states that grooming is a form of a commodity that can be exchanged for other individuals' infant caring (jiang 2019). females in the pregnancy period generally had high feeding activity at b1 (28%) and b3 (29%) period with p = 0.35 (fig. 2) because at that time, the female needed more nutrition for fetal development and energy reserves after parturition. foraging activity decreased as the gestation period progressed (p = 0.57), similar to locomotion behavior (p = 0.07) which also reduced toward the end of pregnancy. in contrast to foraging behavior, females in the final period had the highest resting percentage (21%, p = 0.046) which was significantly different compared to other periods. social activity of pregnant females was more performed on the b1 and b2 levels of pregnancy (p = 0.5) than b3 in the last pregnancy period. in general, the social activity among levels of pregnancy were not significantly different. ruivo et al. (2017) and gould et al. (2011) stated that due to the high energy and nutritional needs during pregnancy, females in the early gestation period will spend a lot of time eating. this behavior aims to compensate for the nutrition shared with the fetus. the behavior was also shown by a study conducted by pasetha et al. (2019) that m. nigra females had the highest feeding activity during the pregnancy period. this pattern is also shown in baboon (papio cynocephalus), which compensate for their energy needs by having longer mealtime (altmann & altmann 1970; dunbar 1977). this result is in contrast with a study on green monkeys (cercopithecus sabaeus). green monkeys adapt their dietary patterns by eating better quality foods but shorter meal time to save energy (harrison 1983). our study showed that the foraging, social and locomotion activities of m. nigra females decreased with the gestation period progressed, while the resting activity was significantly rising. pregnant females biotropia vol. 29 no. 2, 2022 154 specifically reduce their activity to save energy lost, including social activities, especially before parturition. this behavior is also done to avoid other members' aggression (pusey et al. 2008; murray 2009). females in lactation period had the highest feeding activity compared to other activities (fig. 2). feeding (34%, p = 0.002) and foraging (23%, p < 2.2e-16) activities were higher as the lactation period progressed. along with the lactation period, the infants will be more independent, so that the movement of females is more flexible to eat and prepare for the next reproductive cycle. resting activity was most often performed by females in l1 (25%, p = 1.5e-14) and continues to decrease by females in l2 and l3, similar to locomotion activity (15%, p = 5.98e-11). in contrast, social activity was stable around lactation period (23%, p = 0.19). our finding indicated a similar result to previous studies done in other primate species. in baboons and vervet monkeys, the feeding activity of lactating females are less efficient when the infant is still dependent on the mother's body. at the beginning of infant age, female often spends longer time foraging than other females (whitten 1982; koenig et al. 1997; sauther 1994). as the infants get older, the dependence of infants on their mother will be reduced so that the female can increase the proportion of forage for subsequent reproductive needs (koenig 1997; dunbar & dunbar 2002). usually, lactating females raise food intake (tarnaud 2006), forage efficiently (maccabe & fedigan 2007), or eat nutrient-rich foods such as protein and carbohydrates (herrera & heymann 2004; maccabe & fedigan 2007; muruthi et al. 1991; serio-silva 1999). resting activity and locomotion in lactating females m. nigra significantly decreased along with the age of infants. the results of our study were in contrast to a study conducted by pasetha et al. (2019) showing that lactating female m. nigra had the highest resting behavior a few days after parturition based on only taking data on lactating females in the early period during the postpartum period. female m. nigra increased their social activity at the end of the lactation period. lactating females spend a lot of time socializing with other females as a strategy to share the burden of infant care, grooming access and foraging access (henzi & barrett 2002; jiang et al. 2019). effect of parity on female behavior we observed 13 primiparous females and 24 multiparous females of m. nigra. multiparous pregnant females performed more feeding (28%, p = 0.65), social (26%, p = 0.3) and locomotion (12%, p = 0.93) activities compared to primiparous females. besides, foraging activity (17%, p = 0.47) and resting (22%, p = 0.029) activities performed by primiparous females were higher than multiparous females (fig. 3). only resting activity showed a significantly different proportion between females in two parity types. multiparous females in lactation phase had feeding (31%, p = 0.69), foraging (15%, p = 0.51) and locomotion (12%, p = 0.52) activities higher than primiparous females (fig. 3). primiparous females performed more resting (21%, p = 0.03) and social (23%, p = 0.30) activities. figure 2 the behavior frequency of pregnant and lactating females m. nigra notes: b1, b2 and b3 indicate the pregnancy period; l1, l2, and l3 indicate the lactation period. activity budget of pregnant m. nigra activity budget of lactating m. nigra behavior and food preference of pregnant and lactating macaca nigra – eka arismayanti et al. 155 figure 3 the behavior of females m. nigra with different parity types in pregnancy and lactation phases parity or experience in having children can be one of the individuals' characteristics that can influence their behavior patterns. a part from resting, there were no significant differences between primiparous and multiparous female behavior. primiparous females tend to increase alertness and resting activity, in exchange, they decrease feeding activity to compensate for time (barrett et al. 2006). in contrast to muruthi et al. (1991), primiparous females need more time to eat because primiparous females are not fully mature. they still need more energy to increase body weight and adequate calorie intake for reproduction. environmental influence on female behavior based on the phenological analysis, the abundance of fruit was directly proportional to rainfall. from october to february, the rainfall was high with low micro-temperatures (fig. 4). feeding activities were directly proportional to the high rainfall. pregnant females did a lot of social (p = 0.31) and resting (p = 0.02) activities when the rainfall was high, but the feeding activity was relatively not much different each month (p = 0.31). locomotion activity was increased during dry season (p = 1.25e-06). they walked more often during the low rainfall season from august to october to search for more food. during the lactation period, females had the highest feeding activity when the rainfall was high (p = 0.002). resting activity was also higher in the rainy season (p < 2.2e-16) (fig. 5). figure 4 phenology of fruit trees and environmental conditions (rainfall and temperature) in kphk tangkoko effect of parity on activity budget of pregnant m. nigra effect of parity on activity budget of lactating m. nigra biotropia vol. 29 no. 2, 2022 156 figure 5 the effect of different seasons on female daily activity the intensity of rainfall was high from october 2018 to february 2019. our study showed that during the rainy season, the feeding activity of female m. nigra rose. in lactation period, females had significantly higher feeding (p = 0.002) and resting (p < 2.2e-16) activities during the peak of rainy season. the proportion of these activities fluctuated until it increased in july 2018 when the rainfall was low. locomotion activities were significantly higher during the dry season. the increasing proportion of locomotion activity also occurs in baboon (papio cynocephalus), which during the dry season and less food availability, baboons walk more and look for alternative food (altmann & samuel 1992). frequency and preference of female food during reproduction reproductive period can affect the females on selecting food (mccabe & fedigan 2007). due to high energy needs, the females are required to consume food that suits their needs (serio-silva et al. 1999; herrera & heymann 2004). based on the observations in our study, females m. nigra during reproduction period ate more arthropods such as ants, termites, grasshoppers, crickets, spiders and larvae. arthropods contain many proteins and lipids that are important for females during the reproductive phase (rothman et al. 2014). pregnant and lactating females ate arthropods up to 45% and 40% (p = 0.003), respectively. the proportion of fruit eaten by pregnant and lactating females was 20% and 13% (p = 0.36), respectively (fig. 6). in addition, females also ate mushroom (1%, p = 0.34), stems (1.1%, p = 0.43), leaves (1-2%, p = 0.25), buds (0.07%, p = 0.26), sap (0.008%, p = 0.57), and flowers (0.1%, p = 0.08). calorie consumption and crude protein are critical during pregnancy and lactation phases so that females will choose certain foods during that period (lee 1997; dufour & sauther 2002). sulawesi black crested macaques are fruit-eating monkeys. the main foods eaten are fruit and insects as companion food (o'brien & kinnaird 1997; sari 2013). our study indicated that arthropods and fruits are the dominant food for the pregnant and lactating females. effect of season on activity budget of pregnant m. nigra effect of season on activity budget of lactating m. nigra behavior and food preference of pregnant and lactating macaca nigra – eka arismayanti et al. 157 figure 6 frequency of the type of food eaten by females m. nigra in contrast to a previous study that stated m. nigra in the pregnant and lactating phase eats more fruit (pasetha et al. 2019) because m. nigra is a frugivore macaque that needs glucose as the primary source of energy. arthropods are a widely available food source containing high concentration of fat and protein in nature. protein and fat are essential nutrients for females during the reproductive phase (gould 2011). besides, lactating females significantly ate more insects and have more variety of food types than pregnant females. we recorded 71 fruit species eaten by females during their reproductive period. the four most-eaten fruit species were i.e., koosiodendron pinnatum (p = 0.17), ficus variegate (p = 0.26), dracontomelon dao (p = 0.8) and morinda citrifolia (p = 0.22). based on the period of lactation, female of l1, l2, and l3 mostly consumed k. pinnatum, f. variegata and d. dao, respectively. based on the period of pregnancy, females of b1, b2, and b3 mostly consumed m. citrifolia, f. variegata and k. pinnatum, respectively. the differences in fruit types eaten by pregnant and lactating females in different periods did not show statistical significance. these fruits were consumed due to the abundant availability throughout the season except for k. pinnatum. m. citrifolia and d. rao has carbohydrate content of 93.23% and 93.12%, respectively, which is quite high. fruits are essential sources of nutrition for m. nigra, especially carbohydrates, vitamins and minerals (national research council 2003). based on previous research by o'brien and kinnaird (1997), there are 145 types of fruit eaten by m. nigra. according to pasetha et al. (2019), there are 34 types of fruit eaten by females during reproduction. similar to our findings, these previous studies stated that dracontomelon dao, spondias sp., and ficus sp. are the most eaten species (o'brien & kinnaird 1997; pasetha et al. 2019). those fruits have a high carbohydrate, fiber and protein content among fruits they consume, respectively. the vegetation analysis results support this finding because both fruit types have the highest important value index (ivi) (pasetha et al. 2019, unpublished). pregnant and lactating female diet preferences a selectivity index was conducted to indicate which food types are the favorite choice of animal species. the results showed that dracontomelon mangiferum (35.13%) and eugenia sp. (42.38%) was the most preferred fruit types by females during reproduction period. based on the proximate analysis, d. mangiferum has a relatively high carbohydrate content (93.28%), and eugenia sp. has a high-fat content (5.4%) compared to other fruit types. based on fruits availability analysis, arenga pinnata, ficus variegate, caryota mytis, garcinia tetranda, morinda citrifolia, morinda bracheata and piper aduncum are species that bear fruit throughout the year and are included in fruit species having a high consumed frequency. environmental condition is a limiting factor causing variations in the primate's diet (agetsuma 1995; agetsuma & nakagawa 1998; jaman & huffman 2008; yeager et al. 1996). the availability of fruit increases in the months during high rainfall, resulting in the increased of feeding activity. based on the g-test, which rejected the hypothesis, pregnant and lactating m. nigra did not randomly select the food type. effect of female parity with food type based on their parity, pregnant females ate more arthropods (fig. 7). primiparous female ate fruits (25%) more than multiparous females (23%, p = 0.48). on the contrary, arthropods consumption was mostly done by multiparous females (38%, p = 0.47). lactating females also ate more arthropods. multiparous lactating females consumed more insects (49%) than primiparous females (43%, p = 0.07). type of food eaten by females m. nigra biotropia vol. 29 no. 2, 2022 158 fruit consumption in primiparous and multiparous lactation females was quite similar (p = 0.56). species of fruit eaten by pregnant primiparous and multiparous were k. pinnatum (p = 0.86), f. variegate (p = 0.006) d. dao (p = 0.28), t. cattapa (p = 0.16) and m. citrifolia (p = 0.37). species of fruit eaten by primiparous and multiparous lactating females were k. pinnatum (p = 0.82), f. variegate (p = 0.63) d. dao (p = 0.03), t. cattapa (p = 0.0005) and m. citrifolia (p = 0.5). figure 7 effect of parity on the frequency of food types based on parity, both primiparous and multiparous females in the reproductive period ate more arthropods. primiparous females consumed more arthropods such as crickets, grasshoppers, spiders and larvae than multiparous females. arthropods contain high protein and fat concentrations compared to other types of food such as fruits and other plants part. in small quantities, arthropods can also be a supplement for primates due to their content of vitamins and fatty acids (cluttonbrock et al. 1989; rothman et al. 2014). those nutrients are essential during pregnancy and lactation periods. our finding has a similar result to previous studies. female orangutans, titi monkeys and capuchins tend to increase arthropods' consumption during pregnancy and lactation (fox et al. 2004; herrera & heymann 2004; mccabe & fedigan 2007). fruit consumption in primiparous and multiparous lactating females had a similar proportion. most fruit eaten by pregnant and lactating females was k. pinnatum, f. variegata, d. dao, t. cattapa and m. citrifolia. intraspecific variability in primate feeding behavior is closely related to several factors, e.g., reproduction conditions and favorite foods (post et al. 1982). conclusion the behavior pattern of female macaca nigra can be influenced by the reproductive phase, female parity and environmental conditions. females in the 3rd period of pregnancy and lactation had high percentage of feeding. resting had significant changes in behavior proportion. feeding activity was done more often by multiparous females compared to primiparous female. food preference was influenced by the female reproductive phase and food availability. the selectivity in choosing food was not random. during the pregnancy and lactation periods, females preferred to eat arthropods and fruits. their favorite fruits were d. mangiferum and euginia sp. more comprehensive and in-depth research on behavioral and feeding ecology needs to be done. research with more individual samples of female in the reproduction phase is recommended, as they may have different strategies to adapt to ecological changes in the future. acknowledgments we would like to thank james o. waterman for the funding and support that has been given during the research. special gratitudes are also delivered to antje engelhardt and macaca nigra project, who supported and provided invaluable assistance. many thanks to the natural resources conservation agency of north sulawesi (bksda sulut) which has granted research permission in kphk tangkoko. effect of parity on pregnant m. nigra food types effect of parity on lactating m. nigra food types behavior and food preference of pregnant and lactating macaca nigra – eka arismayanti et al. 159 references agetsuma n. 1995. dietary selection by yakushima macaques (macaca fuscata yakui): the influence of food availability and temperature. int. j. primatol 16(4):611-27. doi:10.1007/bf02735284 agetsuma n, nakagawa n. 1998. effects of habitat 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1 march 2017 abstract for effective teak plantation management, early detection system in controlling teak mistletoe requires various basic information, including degree of infestation and accuracy of the species names. mistletoe infestations in teak and mistletoe species name have been reported, but there are still problems in identifying the correct species or subspecies due to morphological similarity. the objective of this study was to clarify the species identity of hemiparasitic mistletoe plants, which were found in teak clonal seed orchard (cso) in padangan, east java province, indonesia using dna barcodes. species identification of teak mistletoe based on leaf morphological characteristics and universal dna barcode regions (i.e. matk and rbcl) were carried out. the results showed that the canonical discriminant analysis (cda) could differentiate dendrophthoe pentandra and macrosolen tetragonus based on leaf morphological characteristics. variables having high correlation to distinguish both species were length of petiole, width of the widest leaf, number of secondary leaf veins, leaf base shape, aspect ratio, form factor and perimeter ratio of diameter. the results of dna barcoding showed that the two dna barcode regions presented good amplification and sequence results. both dna barcode regions successfully differentiated two species i.e. d. pentandra and m. tetragonus which belong to loranthaceae family and have similar leaf morphological characteristics. those regions were also able to identify viscum articulatum and other species belonging to santalaceae family. these results suggested that the two dna regions could become recommended universal dna barcode for identifying teak mistletoe. keywords: clonal seed orchard, dna barcode, matk, rbcl, teak mistletoe biotropia 4 2 7 140 152 vol. 2 no. , 201 : doi: 10.11598/btb.201 .2 . .7 4 2 651 * corresponding author : s znldeg@yahoo.com introduction mistletoe infestation in the clonal seed orchard (cso) of perum perhutani, in padangan teak plantation forests causes the decrease on quantity and quality of seed and wood production. mistletoe infestation level in cso padangan was reported by muttaqin et al. (2016) by describing the ratio of infected trees to the total number of trees as assessed in observation sample plot (osp). the ratios were divided into several ranges i.e. 1. range of 0.32 – 0.43 indicating light intensity which corresponded to tmr value (true mistletoe rating) of 0.86 – 1.21 (rather light); 2. range of 0.50 – 0.65 indicating lightrather moderate which corresponded to tmr value of 1.29 -3.27 (light-rather moderate); and 3. range of 0.74 – 0.83 indicating heavy intensity which corresponded to tmr value of 1.80 – 3.58 (light-rather moderate). total tmr value was in the range of 0.86 – 3.58. there was no heavy intensity (tmr 5 – 7). preventive parasites control program in perum perhutani are carried out through early detection system to anticipate the widespread of the parasites. the first attempt to assess parasite attack patterns and distribution in sampled compartments of teak stands and a clonal seed orchard (cso) was conducted by muttaqin et al. 140 the treatment of certain diseases should have a specific barcode dna marker to guarantee the correctness of medical treatment (kwanda . et al 2013). also, purushothaman (2014) and et al. mishra (2016) affirm that dna barcoding is et al. an efficient and authentic tool to differentiate medicinal plant. several studies showed that rbcl (ribulase-1, 5biphosphate carboxylase) and matk (maturase k) regions are the two-locus barcode generally used in plants due to their easiness in sequencing and high amplification for many plant species (hollingsworth et al. 2011). thus, the objective of this study was mainly to clarify the identity of hemi-parasitic mistletoe plant, which are now found in teak clonal seed orchard (cso) padangan, perum perhutani, east java province, indonesia, using dna barcodes. materials and methods plant materials leaf samples (young or old leaves) of mistletoe that grow on branch or twig of teak crown were collected from compartments of teak stands in padangan clonal seed orchard (cso). those compartments were assigned as observation sample plot (osp) and observation measurement plot (omp). four omps (50 x 50 m for each plot) were established inside the osp units that were classified based on the level of (2016). in addition, dominant mistletoe species have also been identified. however, correct identification of the species is still problematic due to morphological similarities. hasanbahri et al. (2014) reported that there were two mistletoe species of and scurrula parasitica dendrophthoe pentandra belonging to loranthaceae family in the teak plantation of krandegan ranger resort, begal forest sub-district, ngawi forest district. however, it is quite difficult to distinguish mistletoe species in the field. currently, the identification is based on a reference to herbarium collection which usually requires complete parts of plants. in certain cases, leaves are the only available part of the plants that can be used for identification. this situation could lead to incorrect classification of the targeted mistletoe species. species identification based on morphological characteristics frequently resulted to incorrect identification due to cryptic species phenomenon and species siblings. this phenomenon can cause synonymous substitution in the name of the species or vice versa. this study was carried out by employing dna barcode method to complement the traditional plant identification. this method is more rapid and accurate than the morphological marker. usually, dna barcoding is based on short sequences (< 800 bp) (valentini 2009) that can identify and characterize different species which cannot be distinguished morphologically (tudge 2000). moreover, each mistletoe species that has potential as medicinal plant and is widely used for c l m h osp omp 1 2 3 4 figure 1 design of osp and omp positions in an osp unit, modified from crc990/efforts (drescher et al. 2016; muttaqin et al. 2016) (note: 1, 2, 3, 4 = number of each omp, respectively; c = control, l = low, m = medium, h = high) dna barcode characterization of mistletoe infestation in teak – muttaqin et al. 141 parasite attacks i.e. low, medium, high and control (no parasite attacks) (barbu 2012). if there were no samples obtained for the assigned criteria, the sample collection was then conducted around the corresponding omp. in this study, osp and omp were designed by modifying plots presented in the guidelines of efforts/crc990 project (drescher 2016) (fig. 1 & 2). molecular et al. analysis was carried out in the laboratory of plant biotechnology, center of research biological resources and biotechnology, institut pertanian bogor (ipb) as well as in the laboratory of genetics and forestry molecular, silviculture department, faculty of forestry, ipb. leaf morphology based on a preliminary study of the voucher specimen identification on teak mistletoe in the field and verification through the herbarium identification in herbarium bogoriense (bo), research center for biology lipi, bogor, three species were known, namely dendrophthoe pentandra (loranthaceae), macrosolen tetragonus (loranthaceae) and viscum articulatum (santalaceae). in this study, 62 individual leaf samples were collected for d. pentandra. thirty seven (37) samples of m. tetragonus were collected from five host trees (sub-omps) in 12 omps for further identification of leaf morphology. specimens collected for each of d. pentandra and m. tetragonus were twigs in which each twig has three to five mistletoe leaves. the specimens were directly sterilized by swabbing 70% ethanol onto the leaves using cotton balls. the sterilized specimens were then covered with newspaper. each sterilized specimen was tagged using a hanging label. information written on the hanging label was sequential number of the sample, name of mistletoe species, sampling date and sequential number of the host tree. leaf morphology identification was conducted at unit sample of three leaves having sufficient phenotype in reference to kremer et al. (2002) with several modifications following simplified procedures of gembong (2001), wu et al. (2007), ellis et al. (2009) and kadir et al. (2012). measured variables for each leaf samples were dimensional characteristics such as lamina length (ll), petiole length (pl), the width of the widest part of leaf (wl), the length of the largest leaf to the base of the leaf (lb), arch of leaf veins (sd), angle between the midvein with the right or left branch of leaf veins. the number of secondary leaf veins (nv) was also recorded. the number of leaves (nl) was not used as variables in this study, because the calculation of the number of mistletoe leaves was inconsistent due to the loss of some leaves. figure 2 design of sub-osps in an osp unit (drescher et al. 2016; muttaqin et al. 2016); a, b, c, d, e and 1, 2, 3, 4, 5 as order of column and row of sub-osps a b c d e 1 2 3 4 5 50 m 10 m 50 m 1 2 3 4 5 biotropia vol. 24 no. 2, 2017 142 other observed variables were apex shape (as) and base shape (bs), which were observed with visual assessment from 1 to 8 (ellis et al. 2009). calculated variables were: 1. leaf area (la), which was calculated using the ellipse area formula: ½ x (3.14) x (wl x ll); 2. leaf circumference (lc), which was calculated using the ellipse circumference formula: ½ x (3.14) x (wl + ll); 3. aspect ratio (ar), defined as the ratio of leaf length and width that is used to estimate the shape of the leaf blade; 4. form factor (ff), describes the shape of leaf the roundness of the leaf blade, which form factor (ff) was calculated by the 2 formula: 4π x la/lc ; 5. perimeter ratio of diameter (pr), determines the ovalness of the leaf, which can be calculated by the formula: lc/wl (wu et al. 2007). the 13 leaf morphological variables were then analyzed using three different multivariate analyses, namely principal component analysis (pca), canonical discriminant analysis (cda), multiple correspondence analysis (mca) (kremer et al. 2002; anwar 2015) of the spss 17 program (spss inc. 2007). furthermore, t-test analysis was performed on the average of the 13 variables of d. pentandra and m. tetragonus using minitab 15 software (minitab inc. 2007). dna barcode for dna barcode, three replications were applied and analyzed for both matk and rbcl regions of d. pentandra, m. tetragonus and v. articulatum, respectively. thus, there were nine host trees. there were 18 samples examined for dna barcode analysis (table 1). for each mistletoe species, a sample unit consisted of three leaves. those leaves were directly sterilized by swabbing 70% ethanol onto the leaves using cotton balls. each sample unit was then cut into pieces of 2 x 2 cm. the cuttings of each sample unit were then put inside a tea bag of 9 x 7 cm. one tea bag was designated for one sample unit. silica gel granules were put inside the respective tea bags to keep the samples dried in field condition (fazekaz et al. 2012). three tea bags representing the three replications for each mistletoe species for each dna marker were put into one airtight plastic bag which was then labeled according to the sample code. the sample code consisted of sequential number of sample, name of mistletoe species, sampling date and sequential number of the host tree. subsequently, all samples were carefully packed and transported to the laboratory for dna extraction. total genomic dna was extracted from each sampled leaf using dneasy plant mini kit protocol (http://www.qiagen.com) with catalog number 6235. two dna barcode regions (matk and rbcl), which are recommended as universal dna barcodes for land plants, were used for pcr amplification (hollingsworth et al. 2009). the pcr amplification was carried out using pcr tm machine of ab applied biosystem veriti thermal cycler (www.appliedbiosystem.com). primer sequences used in this study are shown in table 2. the process of pcr begins with the dilution of dna and primer. firstly, dna extract was diluted 100 times using double-distilled water (bidest. water). secondly, primer dilution was conducted by directly taking 10 µl of high concentrated k and l primers, respectively, mat rbc which was then added with 90 µl nuclease free water. substance composition for pcr reaction consisted of 2.5 µl h o, 7.5 µl green go taq 2 master mix, 1.5 µl primer f and r and 2 µl diluted dna (fazekaz 2012). the dna et al. amplification results were sent to genetika science indonesia (http://www.base-asia.com) for automated sequencing. additional sequence data of k and l regions for each species mat rbc including out group were collected from ncbi table 1 list of leaf samples of teak mistletoe used in this study no. species number of samples collected for leaf morphology characteristics determination dna barcode determination matk rbcl 1 dendrophthoe pentandra 62 3 3 2 macrosolen tetragonus 37 3 3 3 viscum articulatum 3 3 143 dna barcode characterization of mistletoe infestation in teak – muttaqin et al. table 3 differences in morphological characteristics of leaf sampled from three species of teak mistletoe genbank database (national center for biotechnology information) (http://www.ncbi. nlm.nih.gov) and bold system (http:// boldsystems.org/). dna sequences i.e. the length of sequence data (bp) and nucleotide composition were checked visually, edited manually based on chromatogram and aligned manually using clustalw implemented in mega 6.06 program (tamura et al. 2013). sequence data were then analyzed using blast in ncbi database to identify the three targeted species. furthermore, barcoding gap and phylogeny trees were checked using mega 6.06 program. phylogeny trees were constructed using neighbor joining (nj) method with 1,000 replicates of bootstrap based on kimura-2-parameter (k2p) distance. results and discussion characteristic of leaf morphology results of species identification based on observation and leaf dimension measurement supported by standard reference and complete identity verification using herbarium specimen are presented in table 3. table 2 nucleotide sequence of the primers used in this study no. region primer name sequence (5’ – 3’) length of pcr product (bp) temperature (oc) reference 1 matk [matk-3f, matk-3r] f: aagatgcctcttctttgcat r: gatccgctgtgataatgaga 620 50 marazzi et al. (2006) 2 rbcl [rbcl-barp-1f, rbcl-barp724r] f: atgtcaccacaaacagaaac r: tcgcatgtacctgcagtagc 677 50 dev et al. (2014) morphological loranthaceae santalaceae characteristics dendrophthoe pentandra macrosolen tetragonus viscum articulatum leaf arrangement leaves alternate and small leaves subopposite leaves opposite leaves rudimenter and resemble small bractea, leaves alternate and subopposite leaf length 8.42±1.65 cm 8.56±1.36 cm 2.60±0.60 cm leaf width 4.15±0.87 cm 3.77±0.73 cm 0.30±0.10 cm petiole length 1.37±0.29 cm 0.28±0.26 cm amount of secondary veins 8.98±0.86 12.03±1.44 shape of leaf base developed-rounded (scale 2.54±0.32) rounded (scale 2.84±0.28) slightly convex shape of leaves (aspect ratio) lengthwise (scale 2.06±0.29) lengthwise (scale 2.32±0.31) spatulate shape of leaf rounded (form factor) round (1.76±0.09) slightly round (1.68±0.08) 0.8±0.20 leaf ovalness (perimeter ratio of diameter) slightly oval (4.81±0.47) oval (5.22±0.48) very oval (15.6±4.60) 144 biotropia vol. 24 no. 2, 2017 variation in leaf morphology in this study, the three multivariate analyses (pca, mca, and cda) were conducted using the first synthesis variables, which were expected to contribute the highest rate of total variance, followed by the second synthesis variables, as presented in table 4. table 4 shows that the three multivariate analyses (pca, mca, and cda) were able to explain more than 50% of the total variance, representing the wide distribution of leaf morphology grouping of d. pentandra and m. tetragonus (loranthaceae). furthermore, the results of three multivariate analyses (pca, mca, and cda) are shown in figure 3. total variance explained by each multivariate analysis (%) pca mca cda synthesis variable 1 37.40 41.32 100 synthesis variable 2 29.00 33.44 0 total 66.40 74.76 100 figure 3 results of multivariate analysis (a) scatter plot of pca; (b) histogram of pca; (c) scatter plot of mca; (d) histogram of mca and (e) histogram of cda (note: a = d. pentandra; b = m. tetragonus) table 4 proportion of total variance explained by the first and second synthesis variable of three multivariate analyses (pca, mca, cda) in grouping d. pentandra and m. tetragonus 145 dna barcode characterization of mistletoe infestation in teak – muttaqin et al. results of these three multivariate analyses showed that only cda analysis could differentiate d. pentandra and m. tetragonus based on leaf morphological characteristics. figure 3b and 3d (pca and mca), however, show that there were some samples, which were not included in the d. pentandra and m. tetragonus groups. those are unstable as transition group between the two mistletoe species. based on correlation analysis between the leaf dimensional variable and the synthesis 1 (global analysis) of d. pentandra and m. tetragonus (loranthaceae), it was found out that variables having high correlation to distinguish both species using cda analysis were long petiole, width of the widest leaf, number of secondary leaf veins, leaf base shape, aspect ratio, form factor and perimeter ratio of diameter (fig. 3e). characteristics of leaf variables for d. pentandra and m. tetragonus results of t-test for each leaf morphological variable of d. pentandra and m. tetragonus showed that several variables having significant difference were pl, nv, bs, ar, ff, pr (p < 0.01) and wl (p < 0.05). dna barcode amplification and sequence results obtaining optimum annealing temperature is necessary in order to get a successful pcr amplification process. this study indicated that oannealing temperature of 50 c was the best temperature for matk and rbcl, which showed a very clear single band when visualized by agarose gel. this temperature was lower than the temperature suggested by stoeckle et al. (2011) ofor matk and rbcl markers, which was 52 c and o 56 c, respectively. the results showed that 17 samples were successfully amplified and sequenced with primers matk and rbcl. however, one sample could not be well amplified in matk for viscum articulatum. dna analysis and nucleotide base composition size of matk and rbcl regions were 744 – 764 bp and 738 – 740 bp, respectively (table 5). the result of sequence bases showed that the average percentage of nucleotides c + g on the three teak mistletoe species was 30.0 44.4%. moreover, it is known that matk region has a longer average sequence length than rbcl region. study reported by trang et al. (2015) showed that sequence alignment can be used for examining the evolution process from the common ancestor, and thus, can be used for the three mistletoes species in the present study. the sequence length difference is helpful as early identification for species difference and correlated with differences in the composition of nucleotide bases (dick & kress 2009). calculation of base substitution among three species in the same family was done after sequence alignment. in this study, an average sequence length of rbcl region was 738 740 bp. this value was higher than the length reported by kwanda et al. (2013) who used the same rbcl region, but different primer base pairs (5'atgtcaccacaaacagagactaaagc-3' and 5'-gtaaaatcaagtccaccrcg-3'), showing 550 bp for loranthaceae family (d. pentandra, d. lanosa, scurrula atropurpurea, m. cochinchinensis, m. bradisianus, helixanthera parasitica) and santalaceae family (v. articulatum and v. ovalifolium). barcoding gap barcoding gap is defined when the interspecific diversity is higher than intraspecific diversity. ideal barcode of genetic locus can be determined by looking at smaller intraspecific table 5 sequence length (bp) in matk and rbcl for the three studied mistletoe species no. species matk rbcl na bpb n bp 1 d. pentandra 3 760, 761, 760 3 738, 740, 739 2 m. tetragonus 3 763, 764, 763 3 738, 739, 739 3 v. articulatum 2 745, 744 3 739, 739, 737 mean 757.5 738 a note: n = number of sequence, exception of 1 sample which could not be amplified well in matk for viscum articulatum b bp = base pair 146 biotropia vol. 24 no. 2, 2017 diversity compared to the interspecific diversity (lahaye et al. 2008). on sequence total, gap is marked with dotted line due to insertion or deletion. variance of intraspecific and interspecific must be compared depending on the determined barcode of genetic locus (tamura et al. 2013). thus, barcoding gap between intra and interspesific diversities was determined using graphic and distribution of intra and interspesific diversities on kimura-2-parameter (k2p) distance. generally, table 6 shows that the average value of interspecific distances are higher than intraspecific distances in accordance with previous study of lahaye et al. (2008). the present study showed that the average value of interspecific and intraspecific distances were higher for the matk region than that for rbcl region. furthermore, the intraspecific variation of matk region for loranthaceae was 0.012, slightly different from santalaceae (0.010). intraspecific variation of rbcl region for loranthaceae was 0.003 and slightly different from santalaceae (0.010). interspecific variations of matk region were 0.128 and 0.156 for loranthaceae and santalaceae, respectively. on the other hand, interspecific variations of rbcl region were 0.064 and 0.042 for loranthaceae and santalaceae, respectively. value of interspecific distances of rbcl region for loranthaceae and santalaceae reported in this study were similar to the results reported by kwanda et al. (2013), which was 0.032 0.067. the present study showed that nucleotide variations in intraspecific and interspecific species were applicable whenever the tags were compared. analysis of intra and interspecific distances showed a barcoding gap in santalaceae (v. articulatum) using matk region (fig. 4) and loranthaceae using rbcl region (fig. 5). however, the finding of barcoding gap using genetic locus of matk and rbcl still needs improved accuracy by increasing the number of samples. according to liu et al. (2011), the more overlap occurs between intraspecific and interspecific distances, the less effective of barcode region that can be chosen as a candidate dna barcode due to the condition of the region. only the region that has a smaller intraspecific variation than interspecific can be a candidate dna barcode. in the case of interspesific variety, matk and rbcl regions used in this study for loranthaceae and santalaceae were considered to have good distinctive ability, as shown from the gap. table 6 average value of intraspecific and interspecific distances calculated using a kimura-2-parameter model family average of intraspecific distance average of interspecific distance matk rbcl matk rbcl loranthaceae 0.012±0.017 0.003±0.006 0.128±0.091 0.064±0.029 santalaceae 0.010±0.013 0.010±0.013 0.156±0.087 0.042±0.013 note: data are presented as mean±sd figure 4 distibutions of intraspecific k2p and interspecific k2p in matk region showing barcoding gap in santalaceae family 147 dna barcode characterization of mistletoe infestation in teak – muttaqin et al. phylogenetic analysis the aim of phylogenetic analysis is to confirm the kinship of individual samples. it is expected that the individual samples can be grouped based on taxonomical similarity, at the species, genus or family levels and be confirmed with high value of consistency index (ci). the present study showed that preliminary results of blast sequences matched with the same dna sequence or has a close resemblance to teak mistletoe species (table 7). results of species identification conducted using blast analysis (table 7) were different from results of species identification conducted based on morphological characteristics (table 3); for example dendrophthoe curvata (table 7) is table 7 identification results of teak mistletoe using dna barcode no. codea region blast analysis result identification (%) accession code (ncbi) 1 d1 matk dendrophthoe curvata 99 eu544424.1 rbcl dendrophthoe pentandra 100 hq317759.1 2 m1 matk macrosolen cochinchinensis 99 kp093836.1 rbcl macrosolen cochinchinensis 99 hq317768.1 3 v1 matk viscum articulatum 99 ef464496.1 rbcl viscum articulatum 99 kf496504.1 4 d2 matk dendrophthoe pentandra 99 ab924787.1 rbcl dendrophthoe pentandra 100 hq317759.1 5 m2 matk macrosolen cochinchinensis 99 kp093836.1 rbcl macrosolen cochinchinensis 99 hq317768.1 6 v2 matk viscum articulatum 99 ef464496.1 rbcl viscum liquidambaricola 99 gq436640.1 7 d3 matk dendrophthoe curvata 99 eu544424.1 rbcl dendrophthoe pentandra 100 hq317759.1 8 m3 matk macrosolen cochinchinensis 99 kp093836.1 rbcl macrosolen cochinchinensis 99 hq317768.1 9 v3 matkb viscum articulatum rbcl viscum liquidambaricola 99 eq436640.1 anote: refers to species code: d1 = dendrophthoe pentandra – first replication, m1 = macrosolen tetragonus – first replication, v1 = viscum articulatum – first replication and so on b indicates that the amplification was not successful in matk region (primer matk-3f, matk-3r) figure 5 distibutions of intraspecific k2p and interspecific k2p in rbcl region showing barcoding gap in loranthaceae family 148 biotropia vol. 24 no. 2, 2017 different from dendrophthoe pentandra (table 3), viscum liquidambaricola (table 7) is different from viscum articulatum (table 3). table 7 also shows different species name from the samples, i.e. d1 and d3 at matk region and v2 and v3 at rbcl region. degree of accuracy of species identification using blast analysis is presented in table 8. analysis of dna barcode using matk region could identify d. pentandra up to species level (one individual) with homology value of 99% and up to genus level (two individuals) with homology value of 99%. matk region could also identify m. tetragonus up to genus level (three individuals) with homology value of 99%, but could not identify m. tetragonus up to species level due to unavailable sequence data of m. tetragonus in the gen bank database and bold system which were used as the references. furthermore, this region could identify v. articulatum up to species level (two individuals) with homology value of 99%. on the other hand, analysis of dna barcode using rbcl region could identify d. pentandra up to species level (three individuals) with homology value of 100%, m. tetragonus up to genus level (three individuals) with homology value of 99% and v. articulatum up to species level (one individual) and genus level (two individuals) with the same homology values of 99%. thus, table 8 shows the degree of similarity (homology) obtained from blast analysis having homology values ranged from 99 to 100%. scientific names of the three mistletoe species are listed in the plant list (http://www.theplantlist. org) in which dendrophthoe pentandra has 'accepted' status with confidence level **; macrosolen tetragonus has 'unresolved' status with confidence level *; viscum articulatum has 'accepted' status with confidence level **. table 8 also shows that dna barcoding using matk and rbcl regions is more accurate to distinguish samples up to genus and species levels compared to morphological characteristics. however, the present study showed that matk region has better ability to discriminate than rbcl region. this finding is consistent with studies reported by hollingsworth et al. (2009) and novianty (2016). rbcl region has high amplification success and is easily sequenced for many species. on the other hand, matk region is difficult to amplify, but has high accuracy to distinguish up to species level (koch et al. 2008; trang et al. 2015). the present study showed that amplification of sample v3 was not successfully conducted using matk region (table 7). table 8 degree of accuracy of species identification based on matk and rbcl regions using blast analysis no. codea region blast analysis result identification (%) accession code (ncbi) 1 d1 matk dendrophthoe curvata 99 eu544424.1 rbcl dendrophthoe pentandra 100 hq317759.1 2 m1 matk macrosolen cochinchinensis 99 kp093836.1 rbcl macrosolen cochinchinensis 99 hq317768.1 3 v1 matk viscum articulatum 99 ef464496.1 rbcl viscum articulatum 99 kf496504.1 4 d2 matk dendrophthoe pentandra 99 ab924787.1 rbcl dendrophthoe pentandra 100 hq317759.1 5 m2 matk macrosolen cochinchinensis 99 kp093836.1 rbcl macrosolen cochinchinensis 99 hq317768.1 6 v2 matk viscum articulatum 99 ef464496.1 rbcl viscum liquidambaricola 99 gq436640.1 7 d3 matk dendrophthoe curvata 99 eu544424.1 rbcl dendrophthoe pentandra 100 hq317759.1 8 m3 matk macrosolen cochinchinensis 99 kp093836.1 rbcl macrosolen cochinchinensis 99 hq317768.1 9 v3 matkb viscum articulatum rbcl viscum liquidambaricola 99 eq436640.1 anote: refers to species code: d1 = dendrophthoe pentandra – first replication, m1 = macrosolen tetragonus – first replication, v1 = viscum articulatum – first replication, and so on. b indicates that the amplification was not successful in matk region (primer matk-3f, matk-3r) 149 dna barcode characterization of mistletoe infestation in teak – muttaqin et al. figure 6 phylogeny tree based on matk region at family level using neighbor-joining (nj) tree method (note: (a) loranthaceae family; (b) santalaceae family; gb = genbank; og = out group) studying family of certain species can be explained by reconstructing phylogeny tree of the family. in the cladogram, species belong to one family are located in an adjacent clade. small clade is suspected of having very small differences in base sequence (zuazo & agnarson 2010). phylogenetic trees of loranthaceae and santalaceae families are presented in figure 6 and 7. both figures show consistency value of bootstrap that diverse on every locus of phylogeny tree. matk region has bootstrap value range of 33 – 100 for loranthaceae family and range of 47 – 100 for santalaceae family. rbcl region has bootstrap value range of 28 – 100 for loranthaceae family and range of 69 – 100 for santalaceae family. higher bootstrap value indicates higher stability of phylogeny tree branch. matk and rbcl regions of the three samples of the respective d. pentandra and m. tetragonus (loranthaceae) form separate clades of both species and it is called monophyletic. the same result interpretation can also be applied on the two samples of v. articulatum (santalaceae) which included as monophyletic. figure 6a & 7a shows that d. pentandra and m. tetragonus form separate clades. these figures also show that the two mistletoe species were truly different species based on both matk and rbcl regions. 150 (a) (b) biotropia vol. 24 no. 2, 2017 figure 7 phylogeny tree based on rbcl region at family level using neighbor-joining (nj) tree method (note: (a) loranthaceae family; (b) santalaceae family; gb = genbank; og = out group) conclusions dna barcoding gap results obtained in this study showed that matk and rbcl regions had an average value of interspecific distances that were higher than intraspecific one. the results suggested that those regions could be accurate candidates of dna barcode for distinguishing the two mistletoe species i.e. dendrophthoe pentandra and macrosolen tetragonus having similar leaf morphological characteristics and belonging to loranthaceae family. these regions could also identify viscum articulatum and other species belonging to santalaceae family. application of matk and rbcl regions was able to distinguish those three mistletoe species up to species and genus levels. acknowledgements this study was funded by dipa seameo biotrop 2015 (no. 045.17/psrp/sc/spkpnlt/iii/2015). the authors thank mr suwarno, head of research and development center perhutani and the technical staff for their kind assistance in sample collections. references anwar a. 2015. variasi morfologi daun dan sekuens its2 pada jelutung darat (dyera costulata (miq.)hook.f) dan jelutung rawa (dyera polyphylla (miq.) steenis) [master thesis]. bogor (id): institut pertanian bogor. barbu c. 2012. impact of white mistletoe (viscum album ssp. abietis) infection on needles and crown morphology of silver fir (abies alba mill.). not bot horti agrobo 40(2):152-8. dev sa, muralidharan em, sujanapal p, balasundaran m. 2014. identification of market adulterants in east indian sandalwood using dna barcoding. ann for sci 71:517–22. dick cw, kress wj. 2009. 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2, 2017 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 the southeast asian journal of tropical biology vol. 32 no. 3, 2025: 363 372 doi: 10.11598/btb.2025.32.3.2506 issn: 0215-6334 | e-issn: 1907-770x 363 characterization and potential utilization of string bamboo (gigantochloa apus) oliver s. marasigan*, and shereyl a. daguinod physics and mechanics section, material science division, forest products research and development institute (fprdi), department of science and technology (dost), college, los baños, laguna 4031 philippines article higlights • gigantochloa apus shows strong potential for structural and engineered uses. • axial and nodal variations significantly affect bamboo fiber anatomy. • relative density and strength make g. apus suitable for construction and furniture. • derived fiber ratios indicate limited papermaking suitability. • g. apus offers broad utilization potential for sustainable industries article information received : 29 march 2025 revised : 30 september 2025 accepted : 16 october 2025 *corresponding author, e-mail: oliver.marasigan@fprdi.dost.gov.ph research paper abstract this study assessed the morphological, anatomical, derived ratio, and physico-mechanical properties of string bamboo (gigantochloa apus [schult.f.] kurz ex munro) grown in baguio city, philippines, to establish comprehensive property data and explore potential applications. six mature culms were sampled and tested for its properties following the iawa, iso, and astm standards. results revealed that culm diameter and wall thickness decreased significantly by 37.30% and 46.60%, respectively, toward the top portion. anatomical analysis showed significant decreases in fiber length, fiber diameter, lumen diameter, and cell wall thickness by 15.33%, 13.86%, 24.05%, and 8.43%, respectively, from bottom to top. all derived ratios varied significantly between the node and internode portions. the node portion exhibited higher values for cell wall fraction, runkel ratio, muhlsteph ratio, rigidity coefficient, and luce’s shape factor. radial and volumetric shrinkage decreased by 29.47% and 31.01%, respectively, toward the top, while shear strength dropped by 47.20%. in contrast, basic relative density, modulus of rupture, modulus of elasticity, and compression strength showed no significant variation along the culm. these findings highlight that g. apus is suitable for diverse applications including furniture, handicrafts, construction, engineered bamboo, and biomass products. keywords: anatomical properties, gigantochloa apus, mechanical properties, physical properties, string bamboo introduction string bamboo (gigantochloa apus [schult.f.] kurz ex munro) is a clumping species native to south and southeast asia, with introductions in brazil, puerto rico, and the philippines (roxas 2012). it grows up to 16.0 m tall, with culm diameters of 5.0 – 7.0 cm and internodes of 4.0 – 8.0 cm. in the philippines, it is mainly used for handicrafts and furniture (denr – erdb 2016), while in indonesia, known as tali or apus bamboo, it is widely used in construction (iswanto 2017). in peru, it is recommended for rural construction, veneers, handicrafts, pulp and paper, textiles, and interior panels (portal-cahuana et al. 2023). its flexibility makes it ideal for rope production and curved structures, while its thin culm walls suit lightweight applications like fences and roofs (larsen 2023). the limited use of g. apus in the philippines stems from a lack of property data. bautista et al. (2021) reported a culm diameter of 95.06 mm, wall thickness of 7.98 mm, shear strength of 10.77 mpa, and basic relative density of 0.679. in indonesia, it has a culm diameter of 4.0 – 8.0 cm, fiber length of 2.35 mm, vascular bundle frequency of 10.5 per mm² (iswanto 2017), basic relative density of 0.82 – 0.83, moisture content of 83.18 – 102.78%, and tangential-to-radial shrinkage of 0.58 – 0.84 (maulana et al. 2021). in peru, its moisture content is 30.30 – 129.88%, with basic relative density of 0.49 – 0.85, and volumetric shrinkage of 12.44 – 28.83% (portal-cahuana et al. 2023). characterizing bamboo properties is key to optimizing its use in construction, composites, pulp, and furniture. morphology, anatomy, copyright (c) 2025@author(s). https://doi.org/10.11598/btb.2025.32.3.2506 https://creativecommons.org/licenses/by-nc-nd/4.0/ biotropia vol. 32 no. 3, 2025 364 density, and moisture content directly influence suitability larger culms suit construction, smaller ones fit furniture, and longer fibers enhance pulp and composites. bautista et al. (2021) highlighted the need for further studies on g. apus, particularly its axial variation. therefore, this study aimed at providing a comprehensive characterization of g. apus in the philippines, integrating morphological, anatomical, derived ratio, physical, and mechanical properties. our study specifically examined axial variation along the culm and differences between nodes and internodes, which had not been systematically reported. the objective was to generate a comprehensive data to broaden the applications of g. apus and provide scientifically grounded options for farmers, manufacturers, and the bamboo industry. materials and methods sample preparation six mature g. apus culms (4 – 5 years old) were collected from different clumps in loakan, baguio city (16.3877° n; 120.6137° e, 1,570 m elevation) (fig. 1). harvested 30 cm above the ground, they were divided into bottom, middle, and top sections. anatomical analysis used 20-mm samples from nodes and internodes, while 125mm samples from bending test specimens assessed moisture content, relative density, and shrinkage. compression and shear tests included samples with and without nodes (fig. 2). determination of morphological properties the diameter of the bamboo was calculated as the average of two perpendicular measurements taken across opposing points on the outer surface of the culm. to determine the culm wall thickness, four measurements were taken at 90° intervals around the circumference using a vernier caliper, following the iso 22157:2019 standard. determination of fiber morphology fiber dimensions were evaluated following the procedure of espiloy et al. (1999). bamboo samples were prepared into matchstick-sized and subjected to maceration in a solution of equal parts (50%) acetic acid and hydrogen peroxide. the samples were heated in a water bath for 3 – 5 hours until softened and whitish, which facilitated the separation of individual fibers. after rinsing with distilled water to remove residual chemicals, 30 intact fibers per section and portions were mounted on glass slides and examined under a zeiss primo star microscope. fiber properties were measured using zen lite software. cell wall thickness was determined as the difference between fiber diameter and lumen diameter. all measurements followed the standards of the international association of wood anatomists (iawa) (wheeler et al. 1989). figure 1 gigantochloa apus notes: a = culm; b = branch; c = culm sheaths; and d = leaves. characterization and potential utilization of string bamboo marasigan & daguinod 365 figure 2 sampling scheme used to determine the properties of the g. apus determination of derived ratios derived values such as the runkel ratio (rr), slenderness ratio (sr), flexibility ratio (fr), mulhsteph ratio (mr), rigidity coefficient (rc), and luce’s shape factor (lsf) were calculated using the fiber morphology data. the formulas used for these calculations and their relevance to pulp and paper properties are presented in table 1. table 1 derived ratios, their corresponding formulas, and their impact on pulp and paper properties derived ratio related to pulp and paper properties formula cell wall fraction bending resistance (negatively) (2 x cwt)/fd x 100 runkel ratio pulp yield (positively) and digestibility (negative) (2 x cwt)/ld flexibility ratio tearing and tensile strength (positive) ld/fd x 100 slenderness ratio tensile strength (positive) fl/fd mulhsteph ratio flexibility and tensile (positive) (fd2 – ld2)/fd2 x 100 rigidity coefficient bending and tensile strength (positive) cwt/fd luce’s shape factor resistance to beating (positively) (fd2 – ld2)/ fd2 + ld2) notes: cwt = cell wall thickness; fd = fiber diameter; ld = lumen diameter; fl = fiber length. biotropia vol. 32 no. 3, 2025 366 determination of physical properties a 125-mm-high culm ring was split into two slats (25 mm × cwt × 125 mm). from each slat, a 25 mm × 25 mm × cwt sample was prepared for green moisture content (mc) and basic relative density (rdb), with mc measured immediately and rdb determined under oven-dry conditions. the remaining slat (25 mm × cwt × 100 mm) was used for shrinkage tests from green to oven-dry state, following astm d143 (2019). a total of 72 samples were tested for each physical property, calculated using equations 1 – 3 as follows. mc (%) = (wi-wo)/wo) x 100 where: mc = green moisture content (%) wi = initial weight (g) wo = oven dry weight (g) rdb = wo/wd where: rdb = basic relative density wo = oven dry weight (g) wd = volume (g) sa (%) = (di do)/di) x 100 where: sa = shrinkage from green to oven-dry conditions (%) di = initial dimension (mm) do = oven-dry dimension (mm) determination of mechanical properties mechanical properties were tested under green conditions following iso 22157:2019. the specimen size was determined according to the standard: static bending specimens had a length of 30 x culm outer diameter, while compression and shear specimens tested parallel to the grain had a length of 10 x culm wall thickness. a total of 18 specimens were tested for static bending, and 36 specimens each for compression and shear strength parallel to the grain. for compression and shear, half of the specimens were prepared with nodes and without node. all tests were conducted using a 300 kn universal testing machine, with a loading speed of 15 mm/min for bending and 0.6 mm/min for compression and shear. statistical analysis statistical analysis was conducted using jamovi version 2.3 (the jamovi project 2023). data normality was verified using the kolmogorov– smirnov test. one-way analysis of variance (anova) was employed to assess mean differences in morphological properties, physical properties, and static bending strength across height levels. two-way anova was applied to evaluate anatomical properties, derived ratios, compression and shear strength parallel to the grain, with height level (bottom, middle, top) and node presence (with or without nodes) as factors. when significant differences were detected, tukey’s honestly significant difference (hsd) test was used for post hoc comparisons. results and discussion morphological properties g. apus culms measured 14.69 – 17.30 m tall, with a mean diameter of 66.51 mm and wall thickness of 6.48 mm. diameter and thickness decreased significantly from bottom (75.06 mm, 8.54 mm) to top (51.46 mm, 4.56 mm) (p < 0.05) (table 2). the height of g. apus in this study exceeded previous reports (hartono et al. 2022a; maulana et al. 2021), which may be attributed to environmental factors such as soil conditions, temperature, elevation, and relative humidity (parlucha et al. 2022). the present sampling site is classified as climatic type i, situated at 1,570 m above sea level, with an average temperature of 19.3 °c, relative humidity of 87%, annual rainfall of 3,463 mm, and clay-type soil. diameter fell within the 40 – 80 mm range (hartono et al. 2022a) but was smaller than values from maulana et al. (2021) and bautista et al. (2021). the 31.44% diameter and 46.60% thickness reduction align with bamboo’s apical growth and parenchyma cell decline. compared to commercial species (espiloy 1996), g. apus had thinner walls, similar to schizostachyum lumampao (table 3). fiber morphology g. apus fibers averaged 2.61 mm in length, with fiber and lumen diameters of 23.41 µm and 7.79 µm, and a cell wall thickness of 7.81 µm (table 2). internode fibers (2.82 mm) were longer than node fibers (2.40 mm), with fiber length decreasing 15.33% from bottom (2.87 mm) to top (2.43 mm) (table 4). fiber diameter was wider at nodes (24.81 µm) than internodes (22.01 µm) and decreased 13.86% along the culm. lumen diameter was larger in internodes (8.17 µm) than nodes (7.41 µm), decreasing 24.05% toward the top. nodes had thicker walls (8.70 µm) than internodes (6.92 µm), with an 8.43% reduction along the culm. characterization and potential utilization of string bamboo marasigan & daguinod 367 table 2 culm properties of g. apus along the height levels properties height levels average p value itac classificationbottom middle top morphological culm diameter (mm) 75.06a (± 4.88) 73.02b (± 6.60) 51.46c (± 5.32) 66.51 (± 6.48) < 0.000* culm wall thickness (mm) 8.54 (± 1.49) 6.36 (± 1.10) 4.56 (± 0.55) 6.48 (± 1.98) < 0.000* anatomical fiber length (mm) 2.87 (± 8.56) 2.54 (± 8.01) 2.43 (± 7.75) 2.61 (± 8.31) < 0.001* i fiber diameter (µm) 25.63 (± 6.33) 22.53 (± 5.76) 22.07 (± 6.82) 23.41 (± 6.51) < 0.001* lumen diameter (µm) 9.03 (± 6.55) 7.49 (± 5.10) 6.86 (± 4.91) 7.79 (± 5.63) < 0.001* cell wall thickness (µm) 8.30 (± 2.17) 7.52 (± 2.48) 7.61 (± 2.96) 7.81 (± 2.58) < 0.001* derived ratio cell wall fraction (%) 67.30a (± 17.11) 68.06a (± 17.10) 69.79a (± 16.50) 68.38 (± 16.91) 0.230ns runkel ratio (%) 3.03a (± 2.27) 3.07a (± 2.10) 3.45a (± 2.68) 3.18 (± 2.34) 0.080ns iii flexibility ratio (%) 32.70a (± 17.11) 31.94a (± 17.10) 30.21a (± 16.50) 31.62 (± 16.91) 0.230ns iii slenderness ratio (%) 115.75a (± 36.27) 115.08a (± 31.85) 116.02a (± 39.53) 115.62 (± 35.98) 0.940ns mulhsteph ratio (%) 86.39a (± 13.80) 86.88a (± 13.92) 88.16a (± 12.99) 87.15 (± 13.58) 0.310ns iii rigidity coefficient 1.51a (± 1.13) 1.54a (± 1.01) 1.73a (± 1.34) 1.59 (± 1.17) 0.080ns iii luce’s shape factor (%) 0.78a (± 0.19) 0.79a (± 0.19) 0.81a (± 0.18) 0.79 (± 0.19) 0.270ns physical green moisture content (%) 130.00a (± 20.92) 114.97a (± 45.72) 113.08a (± 38.22) 119.35 (± 34.66) 0.730ns basic relative density 0.51a (± 0.05) 0.57a (± 0.12) 0.57a (± 0.10) 0.55 (± 0.09) 0.547ns radial shrinkage (%) 9.37a (± 2.29) 7.50a (± 2.14) 6.58a (± 1.84) 7.81 (± 2.28) 0.143ns tangential shrinkage (%) 7.59a (± 0.61) 7.48a (± 1.94) 5.64b (± 0.62) 6.90 (± 1.47) 0.048ns longitudinal shrinkage (%) 0.02a (± 0.00) 0.03a (± 0.00) 0.02a (± 0.01) 0.02 (± 0.00) 0.863ns volumetric shrinkage (%) 2.53b (± 2.53) 3.14a (± 3.14) 1.41c (± 1.41) 14.11 (± 2.94) 0.049* mechanical modulus of rupture (mpa) 59.81a (± 19.22) 52.89a (± 17.31) 60.61a (± 23.55) 57.77 (± 17.33) 0.723ns modulus of elasticity (gpa) 13.74a (± 4.08) 13.68a (± 2.02) 14.52a (± 4.52) 13.98 (± 3.50) 0.910ns stress at the proportional limit (mpa) 25.79a (± 9.66) 27.50a (± 9.06) 41.05a (± 16.08) 31.45 (± 13.30) 0.086ns compression strength parallel to the grain (mpa) 32.79a (± 4.77) 32.39a (± 6.01) 34.84a (± 10.09) 33.34 (± 13.70) 0.706ns shear strength parallel to the grain (mpa) 17.15a (± 3.90) 13.36ab (± 4.50) 10.60b (± 3.40) 7.19 (± 4.71) 0.002* notes: * = a significant difference at the 0.05 level of confidence; ns = non-significance; means for height levels with different superscripts within a row are significantly different (p < 0.05); itac = indonesian timber assessment criteria for raw materials for pulp and paper (hartono et al. 2022b). biotropia vol. 32 no. 3, 2025 368 table 3 comparison of the properties of g. apus observed in this study with other economically important bamboo species properties gigantochloa apus bambusa spinosaa bambusa vulgarisa gigantochloa levisa dendrocalamus aspera dendrocalamus merrillianusa schizostachyum lumampaoa morphological culm diameter (mm) 66.50 75.70 78.00 63.00 116.30 53.70 52.30 culm wall thickness (mm) 6.48 13.67 10.00 11.67 15.00 18.67 5.00 fiber morphology fiber length (mm) 2.61 1.95 2.33 1.80 3.78 2.16 2.42 fiber diameter (µm) 23.41 18.00 17.00 22.00 19.00 14.00 14.00 lumen diameter (µm) 7.79 4.00 4.00 6.00 7.00 6.00 6.00 cell wall thickness (µm) 7.81 7.00 7.00 8.00 6.00 4.00 4.00 physical green moisture content (%) 119.35 92.80 95.50 117.30 119.20 106.20 173.70 basic relative density 0.55 0.64 0.64 0.54 0.55 0.60 0.48 radial shrinkage (%) 7.81 12.00 14.10 11.00 14.70 12.00 18.70 tangential shrinkage (%) 6.90 8.50 11.90 6.60 7.50 8.10 5.90 note: a = data sourced from espiloy (1996). fiber length exceeded maulana et al. (2021) and most species in espiloy (1996) but was shorter than dendrocalamus asper (3.78 mm) (table 3). classified as “very long fiber” (class i) under itac (hartono et al. 2022b), g. apus is suitable for pulp, composites, and particle board (iswanto 2017). fiber length decreased 15.29% from bottom to top, similar to other species, while internode fibers were 14.70% longer due to growth elongation (shao et al. 2010). fiber and lumen diameters, larger than the diameters in other commercial bamboo, decreased 13.89% and 17.05%, toward the top respectively (table 3). only fiber diameter differed by portion, being 12.72% greater at nodes due to higher vascular bundle concentration (shao et al. 2010). g. apus cell walls (7.81 µm) were thicker than that of most commercial species except gigantochloa levis (8.00 µm) (table 3), decreasing 8.34% toward the top. node walls were 25.72% thicker than internodes, consistent with denser vascular bundles (shao et al. 2010). characterization and potential utilization of string bamboo marasigan & daguinod 369 table 4 anatomical and mechanical properties of g. apus at different portions properties portion p value node internode anatomical fiber length (mm) 2.40b (± 0.75) 2.82a (± 0.82) < 0.000* fiber diameter (µm) 24.81a (± 6.62) 22.01b (± 6.08) < 0.000* lumen diameter (µm) 7.41a (± 5.37) 8.17a (± 5.87) 0.060ns cell wall thickness (µm) 8.70a (± 2.71) 6.92b (± 2.08) < 0.000* derived ratio cell wall fraction (%) 71.42a (± 15.23) 65.34b (± 17.96) < 0.000* runkel ratio (%) 3.55a (± 2.46) 2.82b (± 2.15) < 0.000* flexibility ratio (%) 28.58b (± 15.23) 34.66a (± 17.96) < 0.000* slenderness ratio (%) 99.71b (± 28.50) 131.54a (± 35.66) < 0.000* mulhsteph ratio (%) 89.52a (± 11.62) 84.77b (± 14.93) < 0.000* rigidity coefficient 1.77a (± 1.23) 1.41b (± 1.08) < 0.000* luce shape factor (%) 0.83a (± 0.16) 0.76b (± 0.20) < 0.000* mechanical compression strength parallel to the grain (mpa) 33.41a (± 7.61) 33.27a (± 6.96) 0.956ns shear strength parallel to the grain (mpa) 14.59a (± 5.03) 12.82a (± 4.32) 0.199ns notes: * = a significant difference at the 0.05 level of confidence; ns = non-significance; means for portion with different superscripts within a row are significantly different (p < 0.05). derived ratio the derived ratios of g. apus, including cell wall fraction (cwf), runkel ratio (rr), flexibility ratio (fr), slenderness ratio (sr), muhlsteph ratio (mr), rigidity coefficient (rc), and luce’s shape factor (lsf), are shown in tables 2 and 4. across height levels, no significant variation was observed in any of the derived ratios (table 2), indicating relative stability along the culm. in contrast, significant differences were found between node and internode portions (table 4), i.e., nodes exhibited higher cwf (8.89%), rr (22.99%), mr (5.44%), rc (23.34%), and lsf (8.80%), whereas internodes had higher fr (19.20%) and sr (36.74%). the mean cwf of g. apus (68.38%) was higher than that of s. lumampao (57.14%) and d. asper (63.15%) but was lower than that of bambusa vulgaris (82.35%) and bambusa spinosa (105.88%) (espiloy 1996). higher cwf is associated with slower drying due to reduced lumen space (eloy et al. 2024), suggesting that g. apus dries faster than b. vulgaris but remains above the < 40% ideal threshold for pulp and paper applications. the rr (3.18%) showed no significant variation along the culm but was 20.56% higher in nodes (3.55%) compared to internodes (2.82%). its value places g. apus in itac class iii (hartono et al. 2022b), indicating reduced pulp processability relative to species with rr < 1. this aligns with previous findings that higher rr requires more intensive chemical pulping (rusch et al. 2019). the fr (31.62%) was greater than b. vulgaris (23%) but lower than s. lumampao (43%) (dost fprdi 2007), also falling in class iii in itac classification and suggesting limited fiber elasticity for paper applications (hartono et al. 2022b). biotropia vol. 32 no. 3, 2025 370 the sr (115.62%) exceeded the < 33 ideal, placing it in itac class i and indicating strong, slender fibers suitable for composites and reinforcement panels (rusch et al. 2019; hartono et al. 2022b). conversely, the mr (87.14%) and rc (1.59%) placed g. apus in itac class iii, highlighting reduced suitability for pulp production (hartono et al. 2022b). the lsf (0.79) was above the favorable value of < 0.5 (nagarajaganesh et al. 2023), reinforcing its limited papermaking potential but pointing to applications in engineered bamboo and composites. physical properties g. apus had an average basic relative density (rdb) of 0.55, increasing from 0.51 (bottom) to 0.57 (top). its green moisture content (mc) averaged 119.34%, decreasing from 130.00% at the bottom to 113.08% at the top. shrinkage from green to oven-dry averaged 7.81% for radial (rs), 6.90% for tangential (ts), 0.02% for longitudinal (ls), and 14.11% for volumetric (vs), with rs and vs decreasing significantly toward the top (p < 0.05) (table 2). the mc of g. apus (119.35%) was lower than that reported by portal-cahuana et al. (2023) (155.65%) but higher than that reported by hartono et al. (2022a) (92.08%). it declined along the culm due to reduced parenchyma cells (nordahlia et al. 2011), consistent with a 37.25% increase in parenchyma proportion from bottom to top (maulana et al. 2021). the rdb (0.55) was lower than that reported by hartono et al. (2022a) (0.82) and portal-cahuana et al. (2023) (0.60) but higher than that of g. levis and s. lumampao (espiloy 1996) (table 3). its slight increase with height was attributed to denser vascular bundles and higher fiber content, which increased by 64.29% and 5.0%, respectively, toward the top (maulana et al. 2021). classified as high-density (alipon & bondad 2008), g. apus is suitable for furniture, construction, handicrafts, panels, tool handles, and engineered bamboo applications. the t/r ratio (1.13) exceeded hartono et al. (2022a) (0.72). ts (6.90%) and rs (7.81%) were lower than that of most commercial bamboos (table 3). vs (14.11%) was lower than that reported by portal-cahuana et al. (2023) (24.14%) and decreased significantly (30.70%) along the culm due to higher vascular bundle proportion and reduced parenchyma (maulana et al. 2021). with moderately high shrinkage (alipon et al. 2000), g. apus requires proper drying schedules to minimize defects, while waterproofing is recommended to improve dimensional stability in service. mechanical properties the mean mechanical strengths are shown in table 2 and 4. the top portion had the highest mechanical strengths, except for shear strength parallel to the grain (ss), which was greatest at the bottom. only ss was significantly influenced by height levels (p < 0.05) (table 2). nodes had higher compression strength parallel to the grain (cs) and ss than internodes, but the differences were not statistically significanp (p > 0.05) (table 4). the observed modulus of elasticity (moe) and modulus of rupture (mor) were lower than those reported by nurmadina et al. (2017) for g. apus. static bending [mor, moe, stress at the proportion limit (spl)] properties increased toward the top, consistent with the findings by nordahlia et al. (2011). the top portion, with higher fiber content, suits composite products like bamboo scrimber, while the bottom and middle portions are ideal for construction and furniture. increased mechanical properties at the top portion correlate with higher rdb, vascular bundles, and fiber proportion (maulana et al. 2021). the cs of g. apus was lower than values reported by hartono et al. (2022a) but remained highest at the top portion, due to increased rdb and vascular bundle density (maulana et al. 2021b). ss (13.71 mpa) exceeded values reported by hartono et al. (2022a) and bautista et al. (2021) but decreased toward the top portion, consistent with hartono et al. (2022a). nodes exhibited 0.42% higher cs and 13.81% higher ss, due to shorter fiber length, higher lignin content, and denser vascular bundles. possible uses of gigantochloa apus g. apus, traditionally recommended for handicrafts and furniture in the philippines (roxas 2012), also meets structural requirements for scaffolding, flooring, walls, and columns (alipon & bondad 2008; kaminski et al. 2016). additionally, it is one of the most commonly used structural bamboo species globally (kaminski et al. 2016). its culm height, diameter, wall thickness, and moe support its use in construction and preference in indonesia for its superior properties (damayanto & fefirenta 2021). it is also suitable for engineered bamboo, scrimber, hybrid laminated boards, and particleboard (iswanto 2017). characterization and potential utilization of string bamboo marasigan & daguinod 371 in terms of pulp and paper production, the fiber length of g. apus is classified as class i under itac standards (hartono et al. 2022b), indicating its technical suitability for pulp applications. however, its derived ratios (rr, fr, mr, and rc) fall under class iii, and its lsf exceeds the favorable threshold, thereby limiting its papermaking potential. these characteristics suggest that pulping g. apus would require more intensive chemical treatments to achieve acceptable quality (fatriasari et al. 2023). thus, while g. apus fibers have promising length for pulp, their anatomical and derived ratio characteristics suggest greater potential in composites and reinforcement materials. beyond construction, g. apus fibers show potential for textiles, similar to species already utilized for this purpose such as g. levis, b. spinosa, and b. vulgaris (dost ptri 2021). portalcahuana et al. (2023) likewise recommended g. apus for textile applications. in addition, its fibers are viable for rope production, while culm residues can be processed into pellets and charcoal briquettes (larsen 2023). while its moderate vs may pose challenges, treatments like heat, waterproofing, and chemical preservatives enhance its stability, durability, and resistance to degradation. conclusion this study highlights the basic properties of g. apus and its potential applications in the bamboo industry. significant variation was observed along the culm, with diameter and wall thickness decreasing toward the top portion, while the bottom portion displayed longer fibers with larger diameters, wider lumens, and thicker cell walls. derived ratios such as cwf, rr, sr, mr, rc, and lsf increased toward the top portion, whereas fr was highest at the bottom portion. among physical properties, only rs and vs decreased significantly with height levels. for mechanical properties, static bending (mor, moe, spl) showed no significant differences, though values tended to be higher at the top portion. compression parallel to the grain was also greatest at the top portion, while shear parallel to the grain was significantly higher at the bottom portion. between nodes and internodes, anatomical properties and derived ratios differed significantly. nodes exhibited greater fiber diameter and thicker cell walls, along with higher cwf, rr, mr, rc, and lsf, while internodes had longer fibers, wider lumens, and higher fr and sr. these findings suggest that g. apus is suitable for a wide range of applications, including furniture, handicrafts, construction materials (e.g., scaffolding, flooring, frames, and columns), and engineered composites (e.g., bamboo scrimber and hybrid laminated boards), as well as textiles, ropes, pellets, and briquettes. however, for pulp and paper production, its fiber morphology and derived ratios indicate reduced suitability without additional chemical treatment. acknowledgments the authors thank the department of science and technology – philippine council for agriculture, aquatic, and natural resources research and development for funding, and the physics and mechanics section for technical support. special thanks are extended to mr. rj cabangon, mr. fc pitargue jr., ms. eo bondad, ms. ma alipon, mr. ep domingo, and ms. cf lasap for their assistance; ms. er barile for language editing; and ms. m. odsey and ms. c. collado (denr– erdb, baguio city) for providing the bamboo samples. references american society for testing and materials (astm). 2019. methods of testing small clear specimens of timber. astm designation: d143-52. in: book of astm standards, part 16. philadelphia (us): american society for testing and materials. 30p. alipon ma, bondad eo. 2008. strength grouping of philippine timbers for various uses. fprdi trade bulletin series no. 4. los baños (ph): forest products research and development institute (fprdi). 51 p. bautista be, garciano leo, lopez lf. 2021. comparative analysis of shear strength parallel to fibers of different local bamboo species in the philippines. sustainability 13:8164. doi: 10.3390/su13158164. damayanto ipgp, fefirenta ad. 2021. distribution patterns of bamboo genera in indonesia. pros semin nas biol 7:24–41. department of environment and natural resourcesecosystem research and development bureau (denrerdb). 2016. selected species of bamboo in the philippines bambusetum. baguio city (ph): denrerdb department of science and technology-forest products research and development institute (dost-fprdi). 2007. monograph on production and utilization of philippine bamboos. los baños (ph): forest products research and development institute (fprdi). department of science and technology-philippine textile research institute (dost-ptri). 2021. dost-ptri’s bamboo textiles ph brand develops bamboo fibers for textile use [internet]. 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[cited 2025 january]. available from: https://www.jamovi.org wheeler ea, baas p, gasson pe. 1989. iawa list of microscopic features for hardwood identification. iawa bull 10(3):219–32. the southeast asian journal of tropical biology vol. 32 no. 1, 2025: 266 276 doi: 10.11598/btb.2025.32.1.2454 issn: 0215-6334 | e-issn: 1907-770x 266 abstract researchers have recently focused on the bioactive components present in natural products. over the past two decades, curcumin, the active compound derived from the curcuma longa plant, has been extensively investigated due to its therapeutic potential as an anti-inflammatory, antioxidant, and anticancer agent. this review article aims to present the potential and therapeutic activities of curcumin based on its medicinal significance and targeted pathways. its antibacterial, neuroprotective, antioxidant, and anticancer properties are discussed in relation to the biological activities of curcumin. despite the promising findings, sufficient evidence supporting the adjunctive use of c. longa and curcuminbased nanoparticles for the treatment of various inflammatory and infectious disorders is still lacking. several nanoformulations have been developed, and their effectiveness has been demonstrated in preclinical studies; however, further validation through human clinical trials is required before their application in medicine can be established. in this context, the current review provides an overview of curcumin nanoformulations that may serve as effective alternatives for targeted therapies in the management of various human disorders. according to the reported literature, nano-gels, nanoemulsions, and nano-creams loaded with curcumin nanoparticles have been proposed for the management of several diseases. the information presented in this review has been compiled from approximately 300 preclinical and clinical research papers and review articles. further studies should focus on optimizing the stability of nanoformulations, improving clinical translation, and enhancing the therapeutic efficacy of curcumin nanoparticles in order to evaluate their potential as candidates for novel drug development in the treatment of various diseases. keywords: anti-inflammatory, anticancer, antimicrobial activity, curcumin nanoparticles, therapeutic activities a comprehensive review on the therapeutic efficacy of curcumin and its nanoformulations iffat nayila*1, sumaira sharif*2, muhammad sarwar2, faima atta khan3, saima zaheer4, and aasma iqbal5 1department of pharmacy, the university of lahore, sargodha, pakistan 2institute of molecular biology and biotechnology, the university of lahore, lahore, pakistan 3university institute of diet & nutritional sciences, faculty of allied health sciences, the university of lahore, sargodha, pakistan 4department of chemistry, the university of lahore, sargodha, pakistan 5department of zoology, the university of lahore, sargodha, pakistan article information received : 9 february 2025 revised : 13 may 2025 accepted : 21 may 2025 *corresponding author, e-mail: iffat.nayila5@gmail.com, sumaira.sharif@imbb.uol.edu.pk highlights • curcumin shows strong antiinflammatory, antioxidant, and anticancer effects. • nanoformulations improve curcumin’s stability, absorption, and effectiveness. • curcumin nanoparticles may protect against cancer, heart, and brain disorders. • more clinical studies are needed to confirm curcumin’s full therapeutic potential. bioactive role of curcumin in diseases prevention nayila et al. 267 introduction due to its numerous therapeutic and medicinal applications, curcuma longa is widely recognized not only in asia but also globally. curcumin, the active compound of c. longa, has been increasingly valued based on various reported studies and clinical trials for its role in managing a wide range of disorders, including diabetes, obesity, bacterial and protozoal infections, and wound healing (priyadarsini 2014). it has been reported that several strategies have been employed to enhance the pharmacological activity and therapeutic potential of this compound (fuloria et al. 2022). the genus curcuma, which has long been utilized for medicinal purposes, comprises approximately 133 species worldwide. commonly recognized species of this genus include curcuma longa (haridra), curcuma zanthorrhiza roxb., and curcuma zedoaria rosc. (zedoary). the species c. longa, a common tall perennial herb belonging to the family zingiberaceae (ginger family), is predominantly cultivated in tropical climates across asia (di martino et al. 2017). the wellestablished medicinal benefits of c. longa have been extensively documented in ayurveda, where it is described in the works of dashemani as kusthagna (anti-dermatosis) and visaghna (anti-poisonous) (adamczak et al. 2020; marchiani et al. 2014). curcumin, a polyphenol extracted from the turmeric plant (curcuma longa), has been extensively investigated for its wide range of pharmacological properties, including anti-ulcer, antioxidant, anti-tumor, anti-inflammatory, and anticancer activities (islam et al. 2024). curcumin’s anticancer potential has been attributed to its ability to regulate various immune modulators, such as cytokines and reactive oxygen species (ros) (zoi et al. 2021). nanotechnology-based approaches have been proposed to overcome the limitations associated with conventional curcumin formulations and to facilitate its transition from laboratory research to clinical applications. several reputable studies have demonstrated that curcumin-loaded nanoformulations, including nanomedicine-based drug delivery systems, can maximize therapeutic benefits by enhancing pharmacokinetics and bioavailability (chopra et al. 2021). multiple types of curcumin-based nanoformulations have been reported, each with distinct clinical relevance, as they provide controlled drug release and strong biocompatibility. however, certain limitations remain. for example, polymeric nanoparticles, such as those derived from plga, may exhibit initial burst release problems. liposomes, which mimic cell membranes and enhance curcumin absorption, face challenges related to stability and encapsulation efficiency. solid lipid nanoparticles are considered highly safe and stable but have a relatively limited drugloading capacity. nanomicelles, characterized by their small size and enhanced solubility, are ideal for systemic distribution; however, their stability under physiological conditions can be inconsistent (kang et al. 2018). key formulation parameters, including biodistribution patterns, cytotoxicity in both cancerous and normal cells, and drug release profiles at physiological ph, have been proposed to guide the development of optimized curcuminbased nanocarriers. advancements in this area are expected to bridge significant translational gaps in curcumin nanomedicine and contribute substantially to targeted cancer therapy. these innovations are anticipated to improve clinical outcomes and provide more effective cancer treatments. furthermore, nano-curcumin supplementation has been reported to reduce cardiovascular disease risk by improving lipid profiles and lowering inflammatory markers, as demonstrated in a systematic clinical review (lin et al. 2020). preclinical studies also suggest that curcumin nanoparticles may offer neuroprotective effects in neurodegenerative disorders, such as parkinson’s and alzheimer’s diseases, by reducing oxidative stress and preventing protein aggregation (kumararaja et al. 2023). in addition, curcuminloaded nanoparticles have shown enhanced tumor suppression and reduced metastasis in models of nasopharyngeal and hepatic cancers (xue et al. 2018). research is still ongoing to improve the safety, stability, and therapeutic efficacy of these formulations. this review highlights the pharmacological activities of curcumin and provides a comprehensive overview of recent advancements in c. longa research, with particular emphasis on the development of nano-carriers to enhance curcumin bioavailability and address existing challenges in drug delivery. the purpose of this review paper is to provide researchers with easy and quick access to information regarding previously reported and utilized phytochemicals. a comprehensive literature biotropia vol. 32 no. 2, 2025 268 search was conducted using search engines such as google scholar, scopus, proquest, and molecules to compile this review on the phytochemistry and pharmacological applications of curcumin. this review is intended to address existing research gaps and to highlight areas where new strategies need to be developed. furthermore, the article presents an extensive overview of numerous curcuminbased nanoformulations, both individually and in combination, according to their potential health benefits, mechanisms of action, and targeted pathways in the treatment of various diseases. review therapeutic applications it has been observed that curcumin can modulate a wide range of molecular targets that are associated with the underlying causes of numerous human disorders. figure 1 illustrates the therapeutic potential of curcumin across major categories of medical illnesses. the multifaceted properties of curcumin are emphasized, underscoring its relevance in the treatment and prevention of various diseases. curcuma longa linnaeus, commonly known as turmeric (family: zingiberaceae; genus: curcuma; species: longa) within the plantae kingdom and the magnoliophyta division (rai et al. 2015), has been widely studied for its medicinal value. curcumin, the principal bioactive compound of curcuma longa, has been reported to possess diverse therapeutic applications, ranging from the treatment of cancer to the management of coughs and colds (mundekkad & cho 2023). studies on curcumin-based formulations (table 1) have demonstrated that curcuma longa exhibits multiple pharmacological benefits and exerts significant anticancer activities against various cell lines. figure 1 health benefits and therapeutic applications of curcumin anticancer potential curcumin has been reported to produce significant outcomes, including an increased percentage of drug sensitivity, in overcoming medication resistance frequently observed in breast, ovarian, lung, and other cancer types (amaroli et al. 2024). in breast cancer cell lines, the overexpression of flap endonuclease 1 (fen1) has been associated with increased resistance to the chemotherapeutic drug cisplatin. it has been discovered that curcumin enhances the sensitivity of breast cancer cells to cisplatin through the downregulation of fen1 expression in vitro (zou et al. 2018). apoptosis was also observed in mcf-7 bioactive role of curcumin in diseases prevention nayila et al. 269 ta bl e 1 a fe w c lin ic al st ud ie s o n cu rc um in fo rm ul at io ns w ith p ot en tia l o ut co m es c ur cu m in fo rm ul at io n c el l l in e/ an im al m od el /c an ce r si gn ifi ca nc e re fe re nc e c ur cu m in li nk ed h sa n ps a5 49 c el ls im pr ov ed c el lu la r u pt ak e en ha nc ed th e cy to to xi ci ty (d as et a l. 20 19 ) c ur cu m in -d ex tr an n p c ac o2 ce lls be tte r s ta bi lit y an d an tio xi da nt a ct iv ity o f c ur cu m in (f an et a l. 20 18 ) c u r -lo ad ed si lk n ps r at s m od el lo ng er th e pl as m a ci rc ul at io n tim e (j ia nb in g et a l. 20 18 ) ss ps -n ps h c t 11 6 an d m c f7 ce lls pr og re ss in th e an tipr ol ife ra tiv e ac tiv ity (p an et a l. 20 18 ) c u r -l oa de d n an o ge ls h el a ce lls in c lin ic al tr ia l en ha nc ed th er ap eu tic e ffi ca cy (t ra n et a l. 20 19 ) c ur n lc s m ou se m od el re du ci ng th e pr oin fla m m at or y cy to ki ne s (k an g et a l. 20 18 ) c ur cu m in -z ei n/ rh am no lip id c om pl ex in v itr o sim ul at ed ga str oi nt es tin al tr ac t pr ot ec t h yd ro ph ob ic b io ac tiv e co m po un ds (x ue et a l. 20 18 ) c ur cu m in -lo ad ed b sa n ps pr ecl in ic al m ur in e m el an om a m od el in cr ea se i n th e su rv iv al r at e an d m in im iza tio n in tu m or si ze (c am ar go et a l. 20 18 ) c ur cu m in in co m bi na tio n w ith ge m ci ta bi ne pa nc re at ic c an ce r m od el c ur cu m in d os e (8 g /d ay ) w as d et ec te d as a bo ve t he ex tre m e to le ra te d do se (k an ai et a l. 20 11 ) c ur cu m in e xt ra ct fo rm ul at io n m ul tip le m ye lo m a an im al m od el re du ce d le ve ls of u rin ar y n -te lo pe pt id e an d bo ne tu rn ov er m ar ke r (g ol om bi ck et a l. 20 09 ) biotropia vol. 32 no. 2, 2025 270 and mcf-7-ddp cells treated with curcumin. in cells exposed to 2 μg/ml cisplatin combined with 20 μmol/l curcumin, a significantly higher degree of apoptosis was recorded in the combination treatment group compared to cisplatin treatment alone (p < 0.05). these findings suggest that the combined use of cisplatin and curcumin may enhance the susceptibility of breast cancer cells to cisplatin (ke et al. 2014). similarly, it has been demonstrated that curcumin, when used together with cisplatin, can overcome cisplatin resistance in lung cancer by promoting apoptosis and inhibiting cell proliferation (priyadarsini 2014). with an ic₅₀ at the micromolar level, it was observed that curcumin significantly suppressed the proliferation of several cancer cell lines, including t47d, mcf7, and mda-mb-231 (p < 0.001). these findings indicate that curcumin possesses strong anticancer properties. cell cycle arrest at the g2/m phase was reported following curcumin treatment, as determined through a comprehensive investigation of its mechanism of action (shan et al. 2018). among the numerous biological effects of curcumin, its ability to stimulate the production of phase ii and antioxidant enzymes through nrf2dependent pathways has been highlighted. due to its rapid metabolism and low bioavailability, curcumin has been extensively investigated in several clinical trials (deck et al. 2018). the outcomes were found to demonstrate modest hemolytic and cytotoxic activities against eukaryotic cells, along with enhanced antibacterial and anticancer properties (adahoun et al. 2017). it was also discovered that neither the extract nor the synthetically prepared gold nanoparticles (gnps) exhibited any cytotoxic effects on the hela or l929 cell lines (das et al. 2024). according to a study, in both scratch and transwell migration tests, the curcuzederone molecule, a phytocompound of c. longa, was found to significantly inhibit the migration of mda-mb-231 cells (p < 0.05) and reduce cancer cell proliferation (al-amin et al. 2021). based on these reported studies, curcumin is believed to exhibit anticancer properties through multiple mechanisms, including anti-angiogenic effects, induction of apoptosis, disruption of tumor cells, and prevention of tumor invasion into healthy tissues. antioxidant potential by improving mitochondrial function, reducing reactive oxygen species (ros), and modulating antioxidant enzyme activity, curcumin has been shown to alleviate conditions associated with oxidative stress and mitochondrial dysfunction (roohi et al. 2017). recent studies have demonstrated that disorders related to mitochondrial dysfunction and oxidative stress can be mitigated by curcumin. it has been indicated that curcumin may provide therapeutic benefits for certain metabolic disorders (sathyabhama et al. 2022). at low and intermediate doses, curcumin has been reported to decrease ros either by upregulating nrf2 protein levels or by enhancing the activities of sod, cat, and gsh-px. the resulting increase in cellular capacity to eliminate ros contributes to the preservation of oxidative stress resistance, potentially reducing apoptosis and improving cell survivability (lin et al. 2020). in clinical models of neuroinflammation, diabetes, and cardiovascular disease, curcumin has been observed to significantly lower oxidative stress enzymes and ros levels by directly scavenging free radicals and enhancing endogenous antioxidant defenses (karamalakova et al. 2019). in untrained, healthy men, oxidative damage is caused by sudden, intense exercise. oral administration of curcumin for one week has been shown to reduce lipid peroxidation and potentially enhance the intracellular antioxidant system (roohi et al. 2017). antioxidant activity in the study was assessed using the 1,1-diphenyl2-picrylhydrazyl (dpph) free radical–scavenging assay. the ethanolic extract of chittagong’s mura was found to contain the highest concentrations of ascorbic acid (0.09 mg/100 g), polyphenols (16.07%), and flavonoids (9.66%) (tanvir et al. 2017). several strategies can be employed to achieve this goal, in addition to using micelles for targeting curcumin to mitochondria. for instance, the introduction of various nanocarriers and the development of curcumin derivatives that specifically target mitochondria have been explored (hu et al. 2024). the growth of stomach tumor cells has been inhibited by curcumin micelles both in vivo and in vitro. this effect is believed to involve increased reactive oxygen species (ros) production, disruption of redox homeostasis, and modulation of mitochondrial bioenergetics (lin et al. 2020). numerous studies have reported bioactive role of curcumin in diseases prevention nayila et al. 271 reductions in various inflammatory indicators, and the anti-inflammatory and antioxidant properties of curcumin are considered to mediate its beneficial effects on stress markers, inflammatory markers, ulcers, and osteoarthritis. anti-ulcer potential curcumin has been proposed as a potential protective agent against inflammatory diseases, including cancer and ulcers. these biological effects are attributed to its antioxidant, antimicrobial, and anti-inflammatory properties. based on the literature, it can be concluded that gastrointestinal ulcers are prevented by curcumin, and that the compound represents a unique therapeutic option for ulcer treatment (yadav et al. 2013). when compared to the control groups, the growth and colony formation of gastric tumor cells were dramatically reduced by curcumin micelles, and apoptosis was induced (panda et al. 2017). in the repair of indomethacin-induced gastric ulcers (gu), an oral administration of a chitosan-curcumin mixture was found to be more effective than curcumin, chitosan, or lansoprazole, a conventional antiulcer drug (kuadkaew et al. 2021). in a separate study, no acute toxicity was observed in rats treated with a metal-curcumin complex (mcc). the results indicated that mcc acted as a more efficient scavenger than curcumin. at an oral dose of 100 mg/kg, gastro-protective efficacy against ethanol-induced ulcers in rats was demonstrated by mcc (joshi et al. 2023). further investigation revealed that curcumin micelles affected mitochondrial proteins in gastric tumor cells, altering mitochondrial function and influencing mitochondrial bioenergetics (panda et al. 2017). hepatoprotective potential the antioxidant properties of curcumin are believed to prevent ccl₄-induced secondary hepatic cytochrome p450 (cyp) damage. it has been reported that curcumin functions as an antioxidant and inhibits nf-κb activation, thereby preventing liver injury caused by ccl₄ (perrone et al. 2015). except at a concentration of 5 g/kg/day, no significant changes in hepatic cytochrome p450 levels or activity were observed following curcumin administration, according to a previous study (sugiyama et al. 2006). in contrast, the activity of these isoenzymes, particularly cytochrome p450 2e1 (cyp2e1), was significantly reduced (p < 0.05) by ccl₄, resulting in elevated free radical production. pretreatment with curcumin at a dose of 0.5 g/kg/day markedly inhibited the effects of ccl₄. liver protection was conferred by curcuma longa l. (cll) extract, which also significantly decreased plasma bilirubin (bl), gamma-glutamyl transpeptidase (ggt), and lipid peroxidation levels. consequently, the cll extract may be utilized as an antioxidant in the treatment of chronic hepatotoxicity (karamalakova et al. 2019). furthermore, compared to free curcumin (100 mg/kg), silymarin (25 mg/kg), and self-recovery groups, curcumin-loaded solid lipid nanoparticles (c-slns) at 12.5 mg/kg significantly (p < 0.001) reduced histological alterations and oxidative stress (xu et al. 2020). hepatotoxicity was evidenced by histological alterations in the liver induced by arsenic, which also elevated serum aspartate and alanine aminotransferase activities. in the liver, arsenic decreased the activities of glutathione reductase, catalase, and glutathione peroxidase, while increasing lipid peroxidation and depleting reduced glutathione. the effects of arsenic were mitigated by curcumin and its nanoparticleencapsulated form (cur-np) in a preclinical trial, with the amelioration being more pronounced in the cur-np treatment. these findings indicate that curcumin administered in nano-encapsulated form produces greater protective effects than free curcumin (lin et al. 2020). with stronger protective benefits demonstrated by cur-np, hepatotoxicity can be substantially reduced through restoration of antioxidant enzyme activity, reduction of lipid peroxidation, and improvement of liver histology. the bioactive potential of curcumin in preventing hepatotoxicity may be further elucidated through carefully designed clinical trials (perrone et al. 2015). moreover, in the context of ulcer and cancer treatment, such trials could provide critical insights into the optimal therapeutic use of curcumin, ultimately enhancing patient outcomes and overall well-being. anti-coagulant therapeutic activity blood coagulation occurs within arteries under various conditions, which can disrupt blood flow or cause internal bleeding that may occasionally be fatal. therefore, anticoagulants are typically administered to prevent blood clotting in such situations. additionally, inflammation has been shown to increase the risk of blood coagulation by activating molecules that promote clot formation (chopra et al. 2021). in clinical biotropia vol. 32 no. 2, 2025 272 trials, curcumin’s anticoagulant activity was demonstrated by comparing thromboplastin time and thrombin time analyses with control blood samples, revealing that curcumin prolongs blood clotting times. both curcumin and its derivative, bisdemethoxycurcumin, were found to significantly extend prothrombin time and activated partial thromboplastin time while inhibiting the activities of activated factor x and thrombin (sirisidthi et al. 2016). similarly, the anticoagulant effect of curcumin has been investigated, revealing that the presence of its hydrophobic groups contributes to a reduction in clotting time in both prothrombin time (pt) and activated partial thromboplastin time (aptt) assays. the ortho-methoxy group, also present in curcumin, has been shown to enhance its anticoagulant activity. furthermore, a decrease in white blood cell (wbc) count was observed, which consequently reduced platelet aggregation and inhibited the formation of fibrin deposits in the kidneys. therefore, curcumin, when combined with nanotechnology, is considered a potential anticoagulant and preventive agent for thrombotic disorders (keihanian et al. 2018). neuroprotective potential it has been suggested that effective prophylaxis is highly desirable, as neuroprotective strategies are most effective when applied prior to the onset of damage (nebrisi 2021). among its pleiotropic actions, curcumin exhibits anti-inflammatory properties, a favorable safety profile, and potential neuroprotective efficacy (cole et al. 2007). the pharmaco-therapeutic potential of curcumin in cerebral ischemia, including its molecular mechanisms of neuroprotection, has been reviewed in animal models, and it has been observed that curcumin represents a promising therapeutic candidate for cerebral ischemia, demonstrating significant improvements in biochemical parameters (lalita & bhakta 2021). gold nanoparticles functionalized with curcumin have been shown to effectively interact with amyloid proteins and peptides, inhibit amyloid fibrillation, and disintegrate amyloid fibrils by acting as synthetic molecular chaperones (yallapu et al. 2015). the desolvation technique was successfully employed to produce curcuminloaded α-lactalbumin (α-la) nanoparticles, targeting a particle size range of 100 150 nm. the resulting formulation, α-la-curcumin, has been demonstrated to provide protection against permethrin-induced neurotoxicity. upon application of α-la-curcumin, the generation of ros induced by permethrin was reduced, thereby mitigating cellular damage (paulpandi et al. 2023). nf-κb mrna expression was significantly downregulated following administration of caffeine-mediated nano-curcumin (n-cur), while tnf-α and il-6 levels, as well as sod activity, were significantly improved. n-cur has been reported to exert mild to moderate beneficial effects on caffeine-induced inflammatory responses, oxidative stress, and apoptosis in the brain (morsy et al. 2024). the neuroprotective effects of curcumin are believed to be mediated through multiple mechanisms, including modulation of oxidative stress, excitotoxicity, neuroplasticity, hypothalamic-pituitary-adrenal axis imbalances, and neurotransmitter levels (nebrisi 2021). therapeutic efficacy against stroke the effectiveness of the curcumin formulation was evaluated using assessments of grip strength, locomotor activity, and brain chemistry (yallapu et al. 2015). improvements in outcomes following brain injury have been reported to be potentially mediated by curcumin through the regulation of th17 cell development. twenty-four hours after the onset of stroke, t cells, which play a detrimental role in the ischemic response, were observed to infiltrate the region of ischemic damage. the endogenous apoptosis pathway was shown to be directly influenced by the p53 signaling pathway through interactions with multi-domain members of the bcl-2 family (du et al. 2023; marques et al. 2020). in a study, therapeutic potential in cerebral stroke was demonstrated by the epigenetic agent curcumin, which was delivered via 200 nmsized exosomes. behavioral, oxidative stress, and physiological parameters were effectively reduced by curcumin, indicating its protective effects against cerebral ischemic insult (yallapu et al. 2015). although curcumin has shown promise in animal stroke models, the mechanisms by which it influences microglial polarization and promotes long-term stroke recovery remain unclear. it was observed that, three days after dmcao, cerebral ischemic damage was markedly reduced following curcumin post-treatment (liu et al. 2017). bioactive role of curcumin in diseases prevention nayila et al. 273 antitumor potential lysosomes represent a significant class of membrane-bound organelles within the intracellular membrane system. they are involved in immunological modulation, cellular metabolism, and programmed cell death, among other biological processes that influence tumor development and progression. lysosomal activity has been shown to be modulated by curcumin, thereby affecting drug resistance, immune function, invasion, metastasis, and tumor proliferation (zeng et al. 2020). tumor growth can be effectively inhibited by curcumin. the transcription of ribosomal dna (rdna) into ribosomal rna (rrna), which promotes cell growth and proliferation, is regulated upstream by mtorc1. curcumin has been demonstrated to inactivate mtorc1 through the suppression of mtor lysosomal localization (xu et al. 2020). the encapsulation of curcumin in eudragit s100 polymer nanoparticles has been shown to influence cellular uptake and to enhance anticancer potential two-fold in the ht-29 human colorectal cell line. innovative formulations have enabled a multi-theranostic strategy, incorporating both therapeutic and imaging functions (yallapu et al. 2015). furthermore, chemo-resistance in tumor cells has been demonstrated to be re-sensitized by curcuminoids through the downregulation of bcl-2 and dna repair enzymes (lin et al. 2020). additionally, further efforts should be devoted to the development of multimodal anticancer therapy strategies in future research by combining standard chemotherapy regimens with curcumin derivatives, analogs, and nano-formulations (mohamadian et al. 2022). antimicrobial potential the synthesis, characterization, and application of curcumin-capped magnetic nanoparticles have been investigated. it has been demonstrated that the interaction between curcumin and magnetic nanoparticles, which occurs through the carbonyl groups of the polyphenol structure, alters the structural, photochemical, and magnetic properties of the polyphenols using various analytical methods. this novel material has been shown to possess the potential to photo-inactivate grampositive bacteria, including staphylococcus aureus (cañon-ibarra et al. 2023). compared to escherichia coli and bacillus cereus, a greater antibacterial activity in vitro has been exhibited by curcumin nanoparticles (curcuminnps) against staphylococcus aureus. pathogens in chicken fingers were successfully suppressed by curcumin-nps during storage for up to 27 days. after storage, reductions in total volatile basic nitrogen (tvb-n) levels were observed in chicken samples treated with curcumin-nps. the bioactive properties, antioxidant capacity, antibacterial activity, and chemical indicators of these experiments validated the efficacy of nanocurcumin at a dose of 10 μg/g (morsy et al. 2023). overall, nanotechnology has been shown to enhance material efficacy and address issues associated with the use of natural additives in the meat industry, which could otherwise alter the sensory qualities of meat products. consequently, improvements in the microbiological quality of poultry meat, including extended shelf life and reduced lipid oxidation, have been demonstrated by curcumin-nps (morsy et al. 2023). although the signaling pathways utilized by curcumin nanoformulations to treat human diseases are well characterized, appropriate human dosage evaluations remain unavailable and should be prioritized. the therapeutic efficacy of curcumincontaining nanocomposites has been shown to surpass that of nanocurcumin or free curcumin (chopra et al. 2021). numerous conceptual preclinical studies have been published; however, these findings should be verified through higherlevel animal studies or clinical trials. conclusion curcumin and its nanoparticle-based formulations exhibit significant therapeutic potential, with demonstrated effects on anticoagulation, fibrinolysis, oxidative stress, antimicrobial activity, and cancer treatment. the molecular mechanisms 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that probing is more reliable and efficient than contrast radiography for gender determination in reticulated pythons. • the probing method proved to have 100% accuracy in determining the gender of reticulated pythons, outperforming contrast radiography, which only achieved 75% accuracy. article information received : 11 june 2024 revised : 9 july 2024 accepted : 26 july 2024 reviewers : fika yuliza purba & anonymous *corresponding author, e-mail: ulum@apps.ipb.ac.id research article abstract gender determination is necessary in reticulated pythons, due to lack of significant gender differences. this study aimed to compare probing and contrast radiography methods for gender determination of reticulated python in terms of efficiency and accuracy. a total of 6 reticulated pythons with body lengths, tail lengths, and body diameters of 128-167 cm, 5.4-9.6 cm, and 1.4-3.4 cm, respectively, were examined twice using these methods. the results showed that probing had 100% accuracy, with probe lengths ranging from 1.37-3.60 cm and 0.3-0.76 cm for male and female snakes, respectively. contrast radiography had 75% accuracy, with the hemipenis appearing as a white silhouette, while female snakes appeared colorless. the accuracy rates signified the effectiveness of probing over contrast radiography for reliable gender determination in reticulated pythons. the selection of appropriate methods for gender determination is important to ensure accuracy and efficiency, which is crucial for breeding programs and the welfare of snakes. keywords: contrast radiography, probing method, reproductive health, reticulated pythons, snake gender determination introduction determining the gender of snake is vital for captive snake owners and breeders. accurate gender determination is crucial to avoid unintended breeding and effective management of snake populations. some owners may decide to rear several snakes in the same terrarium and wish to avoid breeding activities. various methods are used to determine gender of snake, such as molecular techniques and morphological characteristics. previous studies reported molecular techniques, such as quantitative pcr (rovatsos et al. 2015). laopichienpong et al. (2017) presented a novel molecular method of using gametologous genes for gender identification in caenophidian snake. however, a comprehensive comparison of probing and contrast radiography methods specifically for reticulated pythons has not been extensively documented. knowledge of snake gender can also help in diagnosing diseases affecting reproductive organs (gnudi et al. 2009). differentiating between male and female snakes is challenging due to the relatively simple morphology, signifying the importance of gender determination (bonnet et al. 1998). the most common method used for snake gender determination is manual probing, which is conducted specifically to identify hemipenes (vincent et al. 2006). this can be achieved by inserting a smooth, blunt, and thin probe into the cloaca to deviate caudally. in male snake, the probe passes through the hemipenes and reaches more deeply into the tail compared to that in female snake (stahl 2002). the depth of the male hemipenis and https://doi.org/10.11598/btb.2024.31.2.2278 comparative analysis of probing and contrast radiography for gender determination tan et al. 373 the female homologue varied between species. an alternative to the probing method is popping which is more traumatic, suitable for small snakes, and not recommended for larger snakes. popping includes the eversion of the hemipenes outside the cloaca by rolling a finger from the tip of the tail toward the vent (divers & mader 2005). ultrasonography and contrast radiography are methods used to determine gender and evaluate breeding problems. contrast radiography was performed by inserting a radiopaque medium into the cloaca to fill cavities, such as inverted hemipenes (vetere et al. 2022). ultrasound was conducted using a 3-9 mhz transcutaneous convex probe and underwater scans for gender determination have also been conducted (mathew et al. 2015). in regard to reproduction, snakes are either oviparous (egg-laying) or viviparous (giving birth). examples of viviparous snakes with a placenta, include boa constrictors and green anacondas. in contrast, most vipers do not have placental connections and lay eggs that hatch before parturition (previously defined as ovoviviparous). pythons are common snakes kept as pets among reptile keepers and all the species are oviparous (booth & schuett 2016; raharjo et al. 2008). malayopython reticulatus, formerly known as python reticulatus, is the longest nonvenomous snake in south and southeast asia, reaching a length of up to 10 m (somaweera 2017). it is also a native to indonesia, where many captive breeding programs are being held nationwide. malayopython reticulatus is still being heavily exploited because of the high demand for its skin for fashion commodities or captured alive to be sold as a pet (murray-dickson et al. 2017). as reticulated pythons are being heavily exploited, the development of local breeders and breeding programs establishment are essential. an important aspect of a snake breeding program is an accurate gender determination. efficient and minimally stressful gender determination methods is needed for breeders to achieve the highest success rate. improper methods can not only slow down the breeding program rate, but also raise welfare concerns for many animals. this study aimed to compare probing and contrast radiography methods, for gender determination of reticulated pythons in terms of efficiency and accuracy. materials and methods materials and tools materials reticulated pythons used in this study were selected based on availability and the willingness of owners to participate. these pythons varied in body length from 1.2 m to 1.7 m, representing a diverse range of sizes to ensure the applicability of the methods across different snake dimensions. the animals used were prepared without knowing their gender. each snake was first subjected to manual probing, followed by contrast radiography to determine the gender. this procedure was conducted 2 times at an interval of 7 days to allow sufficient time for the contrast agent to clear from the lumens of the reproductive organs (cloaca and hemipenes). the interval was calculated from day 1 (the day of the first procedure) to day 7 (the day of the second procedure). supporting materials used were iopamidol (iopamiro 300 mg/ml), ky jelly lubricant, and 70% alcohol. tools tools used in this study were a 0.07-inch diameter snake probe, digital x-ray machine, 24 g polyethylene catheter, cloth, and snake hook. this experiment was designed not to harm the experimental snakes and followed the established protocol for handling and care. all procedures were approved by the animal ethics committee of the school of veterinary medicine and biomedical sciences of ipb university. procedures manual probing snake was handled manually and restrained without sedation or anesthesia. during data collection, a single probe was used to handle one snake. the probe was lubed with ky jelly lubricant, placed within the vent, and directed laterally and caudally. furthermore, the probe was gently advanced into the base of the tail toward the tip. rotating the probe while gently advancing helps minimize trauma and allows the instrument to pass deeply into the hemipenis or shallowly into the female blind diverticulum. both the right and left hemipenes and diverticulae were probed. subsequently, a radiograph of the tail region was obtained, while the probe was still inserted. snake was positioned on the radiography table under manual restraint. the x-ray machine was set to 40 biotropia vol. 31 no. 3, 2024 374 kvp and 5.00 mas with ffd 40 inch. finally, the depth of the probe inserted was measured from the cloacal region up to the end of the instrument and recorded (fig. 1a). contrast radiography plain radiography of the tail region was performed before administering the contrast medium. subsequently, iopamidol was injected into the cloaca using a 24 g polyethylene catheter, positioned caudally without a catheter needle. during the injection of contrast medium, the snake was restrained manually and no pressure was applied to the tail area (fig. 1b). injection was stopped once the medium began to leak from the vent. a radiopaque needle or probe was placed perpendicular to the snake body at the cloacal vent opening, as a radiographic marker. the snake was positioned on the radiography table under manual restraint. furthermore, digital radiography of the dorsoventral projection of the tail region was performed. radiography machine was set to 40 kvp and 5.00 mas with ffd 40 inch. data analysis the data obtained from manual probing were analyzed by determining the radiography image of the probe inserted into the cloaca and the length was measured. for contrast radiography, data from the x-ray machine were analyzed by observing the presence of a radiopaque hemipenis. the length of contrast of the hemipenis was also measured and recorded. radiographs were examined using imagej and orca® (or technology cloud archiving). the length of the inserted probe and positive contrast were measured and recorded. the means were then calculated for a better comparison and determination of gender. both tests were examined to determine the accuracy and consistency of the methods. for data analysis, a dependent t-test was initially considered, but was not used due to the small sample size and the descriptive nature of the study. future studies with larger samples should incorporate more efficient statistical analyses to validate these results. figure 1 probing and contrast medium administration on a snake notes: (a) inserting the probe in the cauda-ventral opening of the cloaca (b) administering contrast medium (iopamidol) at the cauda-ventral opening of the cloaca (c) probing technique applied in snakes (laszlo 1975) comparative analysis of probing and contrast radiography for gender determination tan et al. 375 results and discussion results of measurement of body length, tail length, and body diameter of reticulated pythons used in this study are presented in table 1. the body length ranged from 128.00 cm to 167.00 cm, with an average of 139.05 cm and a standard deviation of 14.88 cm, emphasizing significant variation. meanwhile, the tail length ranged from 5.40 cm to 9.60 cm, with an average of 6.93 cm and a standard deviation of 1.50 cm, showing higher consistency. the body diameter ranged from 1.40 cm to 3.40 cm, with an average and a standard deviation of 2.28 cm and 0.75 cm, respectively, signifying moderate variation. these data provided a general overview of the physical size of reticulated pythons, which was helpful for further studies or comparisons with other snake species. a total of 36 radiographs were taken throughout the study. each snake had two plain radiographs, i.e., two with an inserted probe and two with administered contrast medium. the results showed differences in probe insertion depth and contrast visualization between male and female snakes. snakes 3, 2, and 5 were chosen to be the observed representatives due to the superior quality of radiographs for interpretation (fig. 2). snakes 3 and 5 represented the male and female snakes, respectively. male snake was identified by the presence of positive contrast of the hemipenes and the probe insertion was deeper. meanwhile, female snake was determined by the absence of positive contrast and the probe insertion was relatively shallower. snake 2 was not identified by gender based on contrast radiography images, because the images were controversial and inconsistent, requiring further explanation. radiopaque needle tips and probes were perpendicular to the body of the snake to signify the cloacal vent. radiograph was taken in the tail region, making the caudal and cloacal vertebra radiopaque. contrast will be radiopaque when the probe is inserted, while muscles will mostly be radiolucent. figure 2 radiographs of plain, probing, and contrast radiography during gender determination of reticulated pythons biotropia vol. 31 no. 3, 2024 376 table 1 measurement of body length, tail length, and body diameter of reticulated pythons used in this study measured parameter snake number mean ± standard deviation1 2 3 4 5 6 body length (cm) 133.20 128.00 131.50 167.00 130.00 144.60 139.05±14.88 tail length (cm) 7.50 5.40 5.90 9.60 6.30 6.90 6.93±1.50 body diameter (cm) 2.30 1.40 2.50 3.40 1.50 2.60 2.28±0.75 table 2 length of hemipenis and accuracy of reticulated pythons gender determination by using probing and contrast radiography length of hemipenis (cm) snake number 1 2 3 4 5 6 probing i 2.46 (true male) 0.76 (true female) 2.31 (true male) 3.60 (true male) 0.75 (true female) 2.14 (true male) probing ii 2.19 (true male) 0.50 (true female) 2.32 (true male) 2.64 (true male) 0.36 (true female) 1.37 (true male) probing mean±standard deviation (cm) 2.33±0.19 0.63±0.18 2.32±0.01 3.12±0.68 0.56±0.28 1.76±0.54 probing accuracy (%) 100 contrast i 1.59 (true male) 1.64 (false male) n.a. (false female) 1.13 (true male) n.a. (true female) n.a. (false female) contrast ii 1.14 (true male) n.a. (true female) 1.82 (true male) 1.93 (true male) n.a. (true female) 1.93 (true male) contrast mean±standard deviation (cm) 1.37±0.32 1.64±n.a. 1.82± 1.45±0.57 n.a.±n.a. 1.93±n.a. contrast accuracy (%) 75 note: n.a. = contrast silhouette did not appear on the radiogram. this study showed no abnormalities or foreign bodies in the tail region of the snakes before any procedures was performed (fig. 2a, d, and g). the probe presented in figure 2b, was inserted much deeper than that in figures 2e and h. the probe usually goes deeper into the tail of a male snake. a clear structure of one of the hemipenes of a male snake was seen in figure 2c. meanwhile, lack of contrast and shallow probe insertion was shown in figure 2f, because female snake has no hemipenis. a positive contrast in the tail area and a very short probe insertion signified a high probability of gender being female (fig. 2i). therefore, radiography results could not confirm the gender of the snake. all 6 snakes were subjected to double probing and contrast radiography (table 2). during the probing procedure, a male snake tended to have a deeper cavity, allowing for probe insertion at a depth of approximately 1-3 cm. on the contrary, the probe could only be inserted at less than 0.8 cm for a female snake. for contrast radiography, a male snake was identified by observing the presence of hemipenes in a radiopaque state. on the other hand, female snake showed an absence of positive contrast in the results of contrast radiography. this study indicated that the accuracy of probing method was relatively higher than the accuracy of contrast radiography method, as false-positive outcomes were observed in contrast radiography on snake 2. multiple probing results corresponded, showing high consistency. meanwhile, the results of contrast radiography were inconsistent, as some male and female snakes showed false-negative and false-positive outcomes, respectively. actual male and female snakes signify true positives and true negatives, respectively. contrast radiography accurately showed the structure of the hemipenis, thereby providing a better picture of the reproductive organs of male snakes. length of the inserted probes were longer than the length of positive contrast (tables 1&2). the discrepancy suggested limitations in the administration of contrast medium, because the contrast medium may not all have been fully delivered into the hemipenis cavity. a potential comparative analysis of probing and contrast radiography for gender determination tan et al. 377 reason was that the small size of hemipenis cavity prevented contrast medium from fully entering the cavity. determining the age of a young snake is challenging, but can be facilitated by several methods. size criteria based on snout-vent length (svl) are commonly used to categorize pythons into different age groups. for instance, a study on water snakes in lake erie classified the snakes with under 25 cm svl as young-of-the-year, signifying an age of less than one year (king 1992). similarly, in a study of javanese keelback water snakes, hatchlings were identified to be less than 59 cm long, juveniles between 60-79 cm, and adults over 79 cm (yudhana 2024). investigation on redsided garter snakes showed that female fecundity increases with body length, in correlation with age, due to the indeterminate growth of snakes (rollings et al. 2017). this suggested that body length is an indicator of age, particularly concerning reproductive capacity. collectively, the methods signify that body length and weight can be effectively used to infer the age and condition of snakes. contrast radiography presents multiple falsepositive and false-negative results (table 2). during evaluation, some radiographs of male snakes showed the absence of positive contrast on the hemipenis, leading to false negative results. there was only one false positive, which was on snake 2. contrast observed was relatively thicker than the other true-positive male results. by comparing the outcomes of probing and contrast, it was discovered that the probes were inserted at a very shallow depth, while a contrast showed negative results. after analysis, snake 2 was confirmed to be a male snake. the observed positive contrast results were also proven to be false positives. in terms of accuracy, probing yields consistent and accurate results. meanwhile, some radiographs were accurate, with one false positive and two false negative results, leading to an accuracy of only 75% (table 2). the radiograph method remains an excellent option in gender determination, but requires more tools, such as x-ray machines and contrast media, which makes it costly and timeconsuming. based on contrast radiography results of (gnudi et al. 2009), the overall accuracy was approximately 81.5%, and the presence of a single hemipenis was frequently reported. the results of this study were also similar, as only one hemipenis was observed. among the two hemipenis sheaths, only one could be filled with contrast medium, because of the proximity of the catheter tip to the opening. the feces most likely contributed to the uneven distribution of contrast medium within the cloaca. in terms of gender, the male and female snakes, are with and without hemipenis sheath, respectively, causing a significant difference in the inserted probe or contrast medium. during probing, the probe instrument is usually inserted at a maximum depth of 3 subcaudals into the tail of a female snake. meanwhile, the probe easily enters a pocket or one on either side of the tail of a male snake and penetrates between the eighth and sixteenth subcaudals (laszlo 1975). a male snake has paired hemipenes, which lie in sacs caudal to the cloaca in the ventral part of the tail (vasaruchapong 2014). typically, the reproductive tract of a female snake consists of 2 ovaries and 2 oviducts. the right ovary of a female snake is relatively larger and located anteriorly than the left ovary. the urogenital papillae allow the oviducts, which travel from the ovaries to the urodeum and into the body (di girolamo & selleri 2017). all snakes used in this study were not adults, as the lengths were between 1.2-1.7 m (table 3). the hemipenes of the experimental snakes were relatively smaller and shorter. python is categorized as adult when the male python reaches approximately 2.5 -3 m in body length (mukherjee et al. 2012). the mean length of the hemipenes of adult reticulated pythons was 6.36±0.46 cm (enriquez et al. 2011). the implemented probing method showed that the inserted probe was between 0.56-0.63 cm deep for female snake and 1.75-2.62 cm deep for male snake, respectively. the differences between these lengths were significant. there is a possibility of the probe being further inserted. this is because the probe instrument is rather thick for the cavity and the snake is smaller in size. organic solids, such as feces, may accumulate in the hemipenis sheath, reducing the depth to which the probe can penetrate and producing false results (kane et al. 2022). despite probing method being generally considered reliable for determining the gender of snakes, this method has some limitations, such as misidentification, particularly in cases of inexperienced probing or ambiguous results (mayer et al. 2023). therefore, gender determination can be further confirmed by comparing the outcomes of contrast radiography. biotropia vol. 31 no. 3, 2024 378 some disadvantages of probing method include injury and distress to snakes when not performed with extreme care and precision (katz et al. 2020). delicate tissues of the cloaca can be easily damaged, leading to pain, infection, or other complications. the process can also be stressful and may negatively affect the well-being of the snake. probing method provides information solely about the gender of snakes without giving insight into other reproductive health conditions or abnormalities. the method does not offer a comprehensive evaluation of the overall reproductive health of the animals. contrast radiography is a useful diagnostic tool for evaluating hemipenis. by using contrast medium, such as radiopaque dyes or gases, reproductive structures were identified, and the anatomy and functionality of the reproductive organs were assessed. contrast radiography of hemipenes can provide information regarding abnormalities, blockages, or structural issues within these organs. the contrast radiography method facilitates the diagnosis of conditions such as hemipenile prolapse, abscesses, tumors, or strictures within hemipenes (rivera 2008). the use of contrast medium in snake radiography allows for enhanced visualization and helps identify specific reproductive conditions or disorders that are not easily detectable with regular radiography. depending on the size and anatomy of snake, accessing and positioning reproductive organs for contrast radiography can be challenging. furthermore, contrast radiography signifies intestinal obstruction, colon obstruction, radiolucent foreign bodies, and masses (banzato et al. 2013). another challenging aspect is the interpretation of contrast radiography of the reproductive organs. differentiating typical structures from abnormalities can be challenging, primarily when subtle changes or variations occur. it is important to acknowledge that misinterpretation of radiography results can lead to inaccurate diagnoses or unnecessary interventions. conclusion in conclusion, probing method showed higher accuracy, however the method is potential to cause distress in the observed snakes. despite the lower accuracy of contrast radiography, the method provided valuable insights into the reproductive anatomy of giant snakes. the combination of both methods with molecular techniques offered a comprehensive approach to gender determination in reticulated pythons. limitations experienced in this study were (1) the sample size of snakes is relatively small, which may limit the generalizability of the results; (2) the samples in the study were relatively young, which affected the accuracy of the methods when applied to older or larger snakes; (3) the use of contrast radiography requires more specialized equipment and expertise, which might not be readily available in all settings. recommendations for further study were: (1) breeders prioritize probing for routine gender determination and consider contrast radiography for more detailed reproductive health assessments and (2) study with larger, more diverse samples and standardized handling techniques is needed to validate these results and explore other potential methods for snake gender determination. references banzato t, hellebuyck t, caelenberg av, saunders jh, zotti a. 2013. a review of diagnostic imaging of snakes and lizards. vet rec 173(2):43-9. doi: 10.1136/vr.101450 bonnet x, shine r, naulleau g, vacher-vallas m. 1998. sexual dimorphism in snakes: different reproductive roles favour different body plans. proceedings of the 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ganies_riza@ugm.ac.id research article abstract clitoria ternatea l., a perennial plant in the fabaceae, is recognized for its resilience in tropical climates and its diverse applications in both culinary and medicinal fields. however, the limited exploration of its genetic diversity constrains breeding efforts aimed at improving desirable traits. this limitation highlights the need to optimize selection strategies, identify superior genotypes, and preserve valuable genetic resources for long-term conservation and crop enhancement. this study aimed to explore genetic variation using molecular markers to analyze c. ternatea genotypes based on petal architecture and color differences. to assess the genetic diversity of c. ternatea, sanger sequencing of the internal transcribed spacer (its) region and inter-simple sequence repeat (issr) markers were applied to seven wild populations from sleman, yogyakarta. the its region exhibited no genetic variation, indicating its conserved nature and limited ability to differentiate genotypes. in contrast, issr markers effectively detected genetic variation, identifying 62 polymorphic fragments out of 162 total bands. the highest genetic distance (0.297) was observed between the ws and ps genotypes, whereas the double-petal genotypes (wd and pd) displayed the closest phenetic relationship. among the issr primers, ubc-808, ubc-812, and issr-6 exhibited high pic and rp values, confirming their reliability in genetic diversity analysis. these results underscore the utility of issr markers as a robust tool for genetic diversity assessment, offering valuable insights for breeding programs and germplasm conservation in c. ternatea. keywords: butterfly pea, genetic distance, internal transcribed spacer, inter-simple sequence repeat, morphological variation introduction clitoria ternatea l. is a perennial plant from the fabaceae, thrives in tropical climates and demonstrates resilience to environmental disturbances. it is widely recognized for its traditional ayurvedic medicine and culinary applications (ashraf et al. 2023; maneechot et al. 2023). the flowers are used as supplements to enhance cognitive function (oguis et al. 2019) and serve as a natural food coloring in rice cakes, tea, and various desserts (chusak et al. 2018). additionally, flower extracts have been reported to exhibit antibacterial, antioxidant, and antidiabetic properties (rajamanickam et al. 2015; jeyaraj et al. 2022). morphologically, c. ternatea flowers are solitary, grow in the axils, and have a characteristic papilionaceous structure. this species exhibits notable variations in petal morphology, with flowers having either single or double layers of https://doi.org/10.11598/btb.2024.31.2.1741 biotropia vol. 32 no. 1, 2025 58 petals. in the single-layer type, the flower follows a papilionaceous structure, consisting of a standard petal (the largest), two wing petals, and two keel petals. in contrast, the double-layer type is characterized by enlarged wing and keel petals that reach the size of the standard petal, creating a layered appearance. the colors were distinct, ranging from white to blue and purple, corresponding to differences in anthocyanin composition (suarna & wijaya 2021; surya et al. 2022). genetic variation plays a vital role in shaping species’ traits and their capacity to adapt to changes in the environment (mora et al. 2005). molecular data-based identification techniques are particularly effective in describing species because they rely on stable genetic markers that are not influenced by environmental factors (loureiro & malfeito-ferreira 2006; elufioye & badal 2017). a frequently used marker in genetic research is the internal transcribed spacer (its) region, which is located between the 16s, 5.8s, and 26s regions of rdna (froeschke & von der heyden 2014). several studies have demonstrated the success of using its regions for assessing plant genetic diversity at the interand intraspecific levels (tripathi et al. 2013; sokołowska et al. 2022; pere et al. 2023). its1 is generally more variable than its2 due to the presence of variable repeat units, making it particularly useful for species discrimination and effective in distinguishing various species in fabaceae (nolan & cribb 2005; vilas et al. 2005; gao et al. 2010). dna-based molecular markers are other ideal tools for evaluating genetic diversity, breedingrelated traits, and other mobile segments due to their neutrality and practicality (bishoyi et al. 2014; hasan et al. 2021; aliabadi et al. 2023; nidianti et al. 2023; rosyidi et al. 2023). inter simple sequence repeats (issr) are a molecular marker technique that utilizes microsatellite sequences as primers to amplify regions between tandem repeats. issr markers are widely used due to their reliability, cost-effectiveness, rapid application, and high sensitivity in genetic analysis (mcglaughlin et al. 2002; sarwat 2012; rahali et al. 2022), and has been widely used to identify intraspecific variation (zebarjadi et al. 2016; nilkanta et al. 2017; verma et al. 2017; abdelaziz et al. 2020; yusuf & daryono 2021; devi et al. 2024; ahmed et al. 2022). issr cover a broad portion of the genome, include numerous polymorphic loci, and do not necessitate prior genomic knowledge (liu et al. 2015; tiwari et al. 2016). given the considerable variability in petal color and structural traits among c. ternatea genotypes, this study has been implemented to identify genetic variations and expand the knowledge base for breeding programs. documenting this diversity is crucial for preserving genetic material that may otherwise be lost through breeding and cultivation, as well as for ensuring the continuity of information regarding species richness and existence. materials and methods genomic dna extraction seven distinct variants of c. ternatea were collected from a wild population in sleman, indonesia, each exhibiting different petal architecture and colors differences. genomic dna was extracted from 100 mg of frozen leaf samples with the genomic dna mini kit plant (geneaid, taiwan). the concentration and purity of the dna were evaluated using a nanodrop lite plus spectrophotometer (thermo fisher scientific, usa), with acceptable purity from 1.8 to 2.0 (green & sambrook 2012). amplification process pcr amplification was performed using the mytaq hs redmix (bioline, us) protocol with several modifications. the total reaction volume was 20 µl, comprising 10 µl of mytaq hs redmix, 2 µl of primer, 2 µl of dna sample at 50 ng/µl, and 6 µl of nuclease-free water. the amplification process was conducted in a t100 thermal cycler (bio-rad, us) over 40 cycles. the total reaction volume was 20 µl, consisting of 10 µl of mytaq hs redmix, 2 µl of primer, 2 µl of dna sample (50 ng/µl), and 6 µl of nuclease-free water. amplification was performed using a t100 thermal cycler (bio-rad, us) with 40 cycles. each cycle involved a 45-second denaturation step at 95 °c, a 30-second annealing step at 45 50 °c, and a 1-minute elongation step at 72 °c. the protocol began with an initial denaturation at 95 °c for 3 minutes and ended with a final extension at 72 °c for 5 minutes. a universal its primer pair was employed, comprising its-u1 (5’-ggaagkaraagtcgtaacaagg-3’) and its-u4 (5’-rgtttcttttcctccgctta-3’) (cheng et al. 2016). details of the eighteen issr primers used are provided in table 1. intra-specific diversity of butterfly pea variants yusuf et al. 59 table 1 issr primer list in this study no primer repeated motifs primer length, bp annealing temperature, oc 1 ubc-807a (ag)8t 17 50.3 2 ubc-808a (ag)8c 17 50.3 3 ubc-809a (ag)8g 17 50.3 4 ubc-810a (ga)8t 17 47.5 5 ubc-811a (ga)8c 17 47.5 6 ubc-812a (ga)8a 17 50.3 7 ubc-815a (ct)8g 17 45.1 8 ubc-817a (ca)8a 17 50.3 9 ubc-818a (ca)8g 17 45.1 10 ubc-834a (ag)8yt 18 47.5 11 ubc-836a (ag)8ya 18 48.6 12 ubc-840a (ga)8yt 18 47.5 13 ubc-841a (ga)8tc 18 48.6 14 ubc-842a (ga)8yg 18 48.6 15 ubc-845a (ct)8rg 18 48.6 16 ubc-873a (gaca)4 16 48.6 17 ubc-880a (ggaga)3 15 47.5 18 issr-6 (ct)8tg 18 50.3 notes: a = obtained from the university of british colombia; y = pyrimidines; r = purines. agarose gel electrophoresis the pcr products were separated by horizontal agarose gel electrophoresis on a 2% agarose gel (1st base, singapore) stained with florosafe dna stain (1st base, singapore). electrophoresis was carried out in 2% tbe buffer at 50 volts for 60 minutes. a 100 bp dna ladder (geneaid, taiwan) was run simultaneously to estimate the molecular weights of the amplified products. the stained gels were subsequently visualized and documented with a geldoc-uv transilluminator. sanger sequencing and bioinformatic analysis sanger sequencing was analyzed at lppt ugm, and the contig sequences were edited using genestudio v.2.2.0.0 (genestudio, inc.). the cleaned sequences were subsequently analyzed using a blast search against the ncbi database. opal, integrated with mesquite (version 3.61), was used to align the contig sequences and accession sequence data retrieved from genbank. genetic distances were determined using mega-x (kumar et al. 2018), and multiple sequence alignments were visualized with the multalin webbased software. binary data analysis issr are dominant markers that provide bistate (presence-absence) assessments (shiran et al. 2007; bishoyi et al. 2014). the recorded gels were scored for the absence (0) and presence (1) of bands, without considering band intensity, since each band occurrence with a specific molecular weight represents a locus (ali et al. 2013). band interpretation was performed using coreldraw software to define the operational taxonomical unit (otu). the formula d = 1 − f, where f is the species similarity matrix value, was used to compute genetic distance (d) (hillis et al. 1996). the multivariate statistical package (mvsp) program was then used to create phenetic dendrograms based on the similarity matrix. results and discussion petals observation the flowers of c. ternatea exhibit a range of morphological variations, including petal architecture and colors differences (bishoyi et al. 2014; suarna & wijaya 2021). based on petal arrangement, we identified two different types: single and double petalloid (fig. 1). in terms of color, there were four variations: white, light blue, light purple, and purple (fig. 1). petal colors have been recorded in several studies (yeotkar et al. 2011; ali et al. 2013; bishoyi et al. 2014). chemically, biotropia vol. 32 no. 1, 2025 60 petal color corresponds to its phytochemical composition, particularly anthocyanins (jeyaraj et al. 2022), with no correlation to kaempferol content (ali et al. 2013). variations in anthocyanin concentration may influence the intensity of purple pigmentation, creating a color gradient ranging from white and blue to deep purple. invariable its records one of the most often utilized genetic markers in plant phylogenetics and dna barcoding is the its region of nuclear ribosomal dna (poczai & hyvönen 2010; cheng et al. 2016). due to its high sequence-level variability, conserved flanking regions, and quick coordinated evolution under comparable functional constraints, this region is helpful for resolving connections within genera and below species level (vander stappen et al. 2002; poczai & hyvönen 2010; zhao et al. 2018). the its sequences obtained from seven genotypes of c. ternatea were aligned to identify the types of mutations present. the alignment revealed two types of mutations: insertions/ deletions (indels) and substitutions. an indel was observed at 56th nucleotide position (fig. 2), where an additional nucleotide base t was present in the sample from manipal, karnataka, india, whereas the other samples exhibited a deletion at this position. regarding substitution mutations, three specific changes were identified: a/g378, t/c396, and a/t491, which distinguished the sample from udupi, karnataka, india from the others (figs. 2 & 3). figure 1 variations in flower petal of butterfly pea observed in this study notes: ws = genotypes of white single; lbs = light blue single; lps = light purple single; ps = purple single; lbd = light blue double; pd = purple double; and wd = white double; scale bar = 2 cm. intra-specific diversity of butterfly pea variants yusuf et al. 61 figure 2 multiple sequences alignment of internal transcribed spacer of butterfly pea notes: black box represents substitution; blue box represents insertion/deletion mutational events; ws = genotypes of white single; lbs = light blue single; lps = light purple single; ps = purple single; lbd = light blue double; pd = purple double; and wd = white double. figure 3 genetic pairwise distance based on kimura-2-parameter model notes: yellow highlighted = genotypes from indonesia genotypes; grey highlighted = genotypes from india and brazil; ws = genotypes of white single; lbs = light blue single; lps = light purple single; ps = purple single; lbd = light blue double; pd = purple double; and wd = white double. biotropia vol. 32 no. 1, 2025 62 these findings suggested a degree of genetic variation among the analyzed genotypes, particularly in samples from karnataka, india, which exhibited both indels and substitutions that differentiate them from other populations. the presence of an additional t nucleotide in the manipal sample and the three substitution mutations in the udupi sample indicated localized genetic divergence, possibly due to geographic isolation, environmental adaptation, or selective pressures. conversely, the identical its sequences observed in the seven genotypes and the são paulo, brazil samples suggested a high level of genetic conservation in this region, implying a shared evolutionary lineage or limited genetic differentiation over time. this could be due to recent common ancestry, low mutation rates in the its region, or historical seed exchange and cultivation practices that maintain genetic uniformity. moreover, its sequence data for c. ternatea remains highly limited in genbank, restricting comprehensive comparative analyses. nevertheless, an alignment-based analysis of all available its sequences, despite reducing the overall sequence length, revealed no genetic distance among the examined accessions (data not shown). this further reinforces the hypothesis that the its region in c. ternatea is highly conserved across different populations. further phylogenetic studies incorporating additional molecular markers and broader geographic sampling would be necessary to better understand the genetic diversity and evolutionary history of this species. the sequence data from this study has been submitted to genbank and assigned accession numbers pq198055–pq198061 for ws, lbs, lps, ps, lbd, pd, and wd, respectively. the universal its primer (its-u1/its-u4) employed in this study amplifies the its1-its2, and is suitable for over 95% of plants groups (cheng et al. 2016). numerous studies have already proven the efficiency of its in explaining phylogenetic connections and revealing interspecific genetic diversity within the fabaceae (ainouche & bayer 1999; vander stappen et al. 2002; gao et al. 2010; wu et al. 2013; xu et al. 2017). in terms of their application in elucidating intraspecific relationships, its regions can reveal sufficient sequence divergence to assess genetic diversity in korean native lilium (sultana et al. 2011), while its2 has been reported to show significantly higher intraspecific divergence in discriminating chinese zanthoxylum species (zhao et al. 2018). nevertheless, some research findings indicated that its markers exhibit low intraspecific genetic diversity. this is exemplified by the thar desert endemic population of commiphora wightii (haque et al. 2009), as well as populations of aquilaria malaccensis and aquilaria hirta in peninsular malaysia, where no intraspecific variation was detected (lee et al. 2018). therefore, its markers are not linked to genes encoding morphological traits, further reinforcing the lack of correlation between morphological and genetic variation found in this study. the absence of its polymorphism among c. ternatea genotypes suggested a high level of sequence conservation, indicating that other genetic regions, such as functional genes or regulatory elements, may be responsible for the observed morphological differences. we overcame this obstacle by using issr molecular markers, which are essential for evaluating genetic stability in diverse breeding lines and for describing genetic variation both within and between species (porth & el-kassaby 2014; aristya et al. 2019; hasan et al. 2021; yusuf et al. 2023). assessment of polymorphism by issr with an average of nine fragments generated from each of the eighteen issr primers, a total of 162 fragments with sizes ranging from 188 bp to 1583 bp were obtained (table 2). a total of 62 polymorphic fragments were found among them, with an average of 3.61 polymorphic fragments for each primer. each primer has a different number of polymorphic bands, ranging from 1 to 14. specifically, the primer ubc-836 displayed eight monomorphic bands and no polymorphic bands, indicating a 0% polymorphism rate (table 2). issr-6 had the largest polymorphism information content (pic) value, measuring 93.33%, and a resolving power (rp) value of 10.00. this demonstrates the primer’s usefulness in genetic variation research and its capacity to identify c. ternatea genotypes. the 18 issr primers produced an rp value of 3.23 and a pic value of 0.12 on average. the effectiveness of issr depends on the compatibility of each primer sequence with the target genome. the bands produced reflect the presence of specific microsatellites in the genome and guide the amplification process to generate intra-specific diversity of butterfly pea variants yusuf et al. 63 inter-microsatellite regions. it is highly probable that different amplified bands can be produced using the same issr primer sequences. for instance, similar issr primers studied by (vijayan & chatterjee 2003) in mulberry produced fewer scored bands compared to our study in c. ternatea, though both were lower than in mucuna pruriens (devi et al. 2024). this is consistent with the low amplification results produced by ubc-807, ubc808, ubc-841, ubc-842, ubc-873, and ubc880 in our study. the percentage of polymorphism is also influenced by the sample size, which correlates with the extent of the analyzed gene pool. such variations were also observed in many fabaceae species during issr-based analyses (dos santos araújo et al. 2016; bhadkaria et al. 2020; ojuederie et al. 2020; qahtan 2021; reddy et al. 2022; aliabadi et al. 2023; helal et al. 2023). identification based on issr markers in different populations of c. ternatea produced jaccard’s coefficient similarity index from 0.80 to 0.98 (bishoyi et al. 2014). in our study, the use of the informative primer issr-16 enhanced the ability to identify genetic variation among c. ternatea genotypes. three identical repeated primer sequences—(ag)8c, (ag)8g, and (ct)8g—were observed in this study, bishoyi et al. (2014), and nurhasanah et al. (2023). the observed results for the repeated motifs (ct)8g and (ag)8c by bishoyi et al. (2014) were closely related to our study in terms of polymorphism rate, resolving power, and pic values. the pic value of (ag)8c, around 0.20, was also similar to nurhasanah et al. (2023). for the motif (ag)8g, we identified a higher polymorphism rate and pic value, although the rp value was lower than bishoyi et al. (2014) and nurhasanah et al. (2023). another method for analyzing genetic variation is the use of simple sequence repeats (ssr), which can provide allelic information among genotypes (gebhardt 2007). ssr analysis has clearly validated the existence of allelic differences between blue and white flower color genotypes, based on the markers ccm2763 and ccm0830 (naik et al. 2020). table 2 amplified fragments and polymorphism derived from 18 issrs primer nb pb pr (%) fs pic rp ubc-807 7 1 14.29 380-1185 0.07 0.857 ubc-808 10 5 50.00 200-916 0.20 5.143 ubc-809 10 4 40.00 446-1158 0.13 3.143 ubc-810 8 1 12.50 267-1046 0.06 0.857 ubc-811 10 4 40.00 249-1117 0.16 4.000 ubc-812 11 8 72.73 278-1492 0.24 6.571 ubc-815 13 5 38.46 237-1583 0.14 7.143 ubc-817 10 5 50.00 427-1412 0.14 5.429 ubc-818 9 1 11.11 189-1096 0.03 0.286 ubc-834 9 2 22.22 188-1383 0.05 2.000 ubc-836 8 0 0.00 204-1038 0.00 0.000 ubc-840 7 3 42.86 199-1235 0.10 3.714 ubc-841 5 1 20.00 233-1183 0.05 0.286 ubc-842 6 2 33.33 370-1051 0.15 2.571 ubc-845 6 2 33.33 286-965 0.12 2.571 ubc-873 8 4 50.00 321-1259 0.17 2.286 ubc-880 10 3 30.00 222-1442 0.10 1.429 issr-6 15 14 93.33 340-1472 0.31 10.00 total 162 65 mean 3.61 36.34 0.12 3.24 notes: nb = no. of scored bands; pb = no. of polymorphic bands; pr = polymorphic rate; fs = fragment size; pic = polymorphic information content; rp = resolving power. biotropia vol. 32 no. 1, 2025 64 figure 4 phenetic dendrogram of seven genotype of butterfly pea based on (a) jaccard’s and (b) simple matching coefficient intra-specific diversity of butterfly pea variants yusuf et al. 65 the dendrogram plots constructed from 18 issr primers did not reveal specific clades, instead showing random plotting due to the very low genetic variance among c. ternatea genotypes. two methods for measuring similarity, jaccard’s (sj) and simple matching (ssm) coefficients, produced similar results (fig. 4). the red line in figure 4, indicates the lowest similarity percentage among the white single genotype compared to the other genotypes, with 73% and 76% similarity according to sj and ssm, respectively. a higher similarity percentage, ≥ 80%, was observed among genotypes marked with a blue line in figure 4. the highest similarity percentage among c. ternatea genotypes was between the white double and purple double genotypes, with similarity values of 96% and 97% for sj and ssm, respectively. according to jaccard’s coefficient, the greatest genetic distance was observed between the white single (ws) and purple single (ps) genotypes, with a value of 0.297 (fig. 5a). this was followed by genetic distances between white single (ws) and the light purple single (lps), purple double (pd), white double (wd), and light blue single (lbs) genotypes, with values of 0.290, 0.284, 0.277, and 0.243, respectively. a similar trend was observed when using the simple matching coefficient, though the dissimilarity values were generally lower compared to those derived from jaccard’s method (fig. 5b). issr markers are dominant and arbitrary, amplifying random regions of the genome without targeting specific genes responsible for morphological traits. as a result, the genetic variation detected does not necessarily correspond to the observed morphological differences. this lack of correlation suggests that while phenotypic variation in c. ternatea may be influenced by genetic factors, it is likely regulated by specific loci that are not captured by issr markers. additionally, environmental factors and epigenetic modifications could contribute to the observed morphological diversity, further decoupling it from the genetic profiles revealed in this study. conclusion its sequencing revealed no genetic variation, as all 7 genotypes in this study exhibited identical sequences with no detected indels or substitutions, confirming the conserved nature of this region and its inefficacy in assessing genetic diversity. issr markers demonstrated high efficacy, identifying 62 polymorphic fragments out of 162 total bands, indicating substantial genetic variation within the population. the greatest genetic distance (0.297) was observed between the white single and purple single genotypes. the double-petal genotypes (wd and pd) exhibited the closest phenetic relationship. among the issr primers tested, issr-6 exhibited the highest polymorphism (93.33%) and resolving power (10.00), further reinforcing its reliability for genetic diversity assessment. no direct correlation figure 5 figure 5 genetic distance of seven genotype of butterfly pea based on (a) jaccard’s and (b) simple matching coefficient notes: ws = genotypes of white single (ws); lbs = light blue single; lps = light purple single; ps = purple single; lbd = light blue double; pd = purple double; and wd = white double (wd). biotropia vol. 32 no. 1, 2025 66 was observed between issr polymorphism and petal morphology. the detected genetic diversity provides essential baseline data for c. ternatea germplasm conservation, ensuring the 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638401, erode, tamil nadu, india article higlights • integrative insights into nonmicrobial and microbial biostimulants. • focus on enhancing significant secondary metabolites and plant quality. • comprehensive analysis of sustainable agricultural practices. • future scope and positive research directions in biostimulants. • role of biostimulants in stress resilience. article information received : 15 july 2025 revised : 18 september 2025 accepted : 22 october 2025 *corresponding author, e-mail: tamilselvis@bitsathy.ac.in review paper abstract in recent decades, biostimulants have become recognized as organic alternatives for promoting sustainable agricultural practices, extensively applied across agriculture, horticulture, ornamental plants, and greenhouses. unlike biofertilizers and biopesticides, biostimulants are derived from plants, animals, and microbes and classified by their chemical composition. they are primarily divided into nonmicrobial and microbial biostimulants. nonmicrobial biostimulants include humic substances, seaweeds, chitosan, protein hydrolysates, amino acids, and inorganic compounds, while microbial biostimulants consist of plant growth-promoting bacteria and beneficial fungi. this review paper consolidates the recent positive effects of biostimulants on sustainable agriculture and outlines potential research areas for the future. biostimulants serve as biocontrol agents that promote plant growth and development, also reported to enhance the production of valuable industrial therapeutic secondary metabolites. traditionally, plants with medicinal properties have been used for natural remedies, and biostimulants aim to elevate the secondary metabolite content in these therapeutic plants. additionally, the significance of both microbial and nonmicrobial biostimulants is touched upon. a review of existing literature indicates that biostimulants significantly boost nutrient absorption, enhance resilience against abiotic stress, replenish nutrients, and improve soil quality. keywords: biocontrol, biostimulants, microbial biostimulants, nonmicrobial biostimulants, secondary metabolites introduction biostimulants are substances or microorganisms that, when applied to seeds, plants, or the rhizosphere, stimulate natural processes to enhance nutrient uptake and efficiency, improve tolerance to abiotic stress, and increase crop quality and yield (chand et al. 2024). they eventually support the growth of sustainable agricultural practices, as they are environmentally friendly and are better alternatives to synthetic fertilizers. biostimulants or plant biostimulants are certain substances, extracts, and microbes that enhance the natural nutritional processes in plants without directly providing nutrients. they are more related to fertilizers than to most varieties of plant protection products and act in addition to fertilizing products thus reducing the nutrient application rate. biostimulants are widely categorized as microbial and nonmicrobial biostimulants (fig. 1). recent european regulation (eu 2019/1009) stated that biostimulant is a fertilizer that aims to enhance plant rhizosphere properties. the goal is not on the nutritional value of the biostimulant itself but on its capability to: a) utilize nutrient efficiency in plants; b) elevate tolerance to abiotic stress; c) improve quality; and d) absorb soil or rhizosphere nutrients (european regulation 2019/1009). european biostimulants industry council (ebic) copyright (c) 2025@author(s). https://doi.org/10.11598/btb.2025.32.3.2570 https://creativecommons.org/licenses/by-nc-nd/4.0/ biotropia vol. 32 no. 3, 2025 404 figure 1 broad categories of biostimulants report scientific findings of biostimulants, their significance to boost plant nutrient bioavailability, and enhance plant resistance to both biotic and abiotic stress factors in various economically and therapeutically valuable crops. the comparative analysis of both natural and commercially available biostimulant effects is discussed briefly. in addition, the outline of recent regulations of the european union (eu 2019/1009) is highlighted. review nonmicrobial biostimulants humic substances (humic and fulvic acids) humic and fulvic acids are organic compounds found in peat, soil, and other organic materials. integration between humic and fulvic acids results in the formation of humic complexes (mackiewicz-walec & olszewska 2023). fulvic acid, an important humic substance derivative of microbial metabolism associated with minerals in soil, supports germination of seeds, encourages plant growth, promotes growth hormones, micronutrient chelation, and retains trace nutrients in the soil and make available to plants. the integrative findings from various papers offer a snapshot that contributes in gaining a recommended several fundamental principles to standardize and support the claims engineered by producers concerning pb efficiency (european biostimulants industry council 2019). european biostimulants industry council reported the overall benefits of using biostimulants are: a) stimulating useful compounds in plant roots; b) enhancing plant resistance to environmental abiotic stress factors (flood, drought, and extreme temperature); c) improving soil quality, health, and structure; d) elevating resource availability and decreasing nutrient loss; e) introducing beneficial microbial cultures to aid plants in uptaking and utilizing essential nutrients more effectively; f ) protecting plants against opportunistic pathogens and infections; and g) enhancing plant nutrient absorption (mackiewicz-walec & olszewska 2023). current projects on a european legal framework for plant biostimulants are being established to standardize testing procedures, branding, organizations, and quality control, with full implementation targeted for 2024 (assi & zahwan 2023). the current review systematically integrates the categories and importance of nonmicrobial and microbial biostimulants, focusing on sustainable agriculture practice. the critical view is to microbial and nonmicrobial biostimulants for enhancing therapeutic metabolites anjali & tamilselvi 405 comprehensive understanding of the use of humic substances. humic acid, one of the types of plant biostimulants is often applied in the rhizosphere region for better utilization (li et al. 2019; ren et al. 2022). animal matters in soil enhance the degradation and humus formation. on the other hand, humic and fulvic acids showed no significant improvement in promoting nutrient availability, growth, and resistance against stress factors, both biotic and abiotic, suggesting that these acids’ effect on plants may depend on environmental conditions and soil quality (regelink & koopmans 2021). the precursor for the degradation of humic acid and its complex identification is a tedious process, due to its low availability in the soil (canellas et al. 2015). increased crop yield and agro productivity due to the use of humic substances or humic complexes in agriculture leads to the positive effect of humic acid as biostimulants (regelink & koopmans 2021). by regulating the architecture of plant roots and shoots involved in nutrient intake, assimilation, and distribution, humic compounds have stimulatory effects. additionally, humic chemicals can enhance the primary and secondary metabolic pathways linked to plant development, resistance, and abiotic stress tolerance. humic compounds are biological activators of plant development, and their hydrophobicity, conformational elasticity, and functional group are among the chemical characteristics that substantially influence how effective they are. numerous european projects, such as bio-fertil (poland), biofector (germany), and humic-xl (the netherlands), have acknowledged the potential application of growth-promoting humic compounds from waste materials (canellas et al. 2015). similarly, humic substances, when applied at concentration less than 0.01%, stimulated root growth and root hair elongation upright in poa annua l. by increasing bacterial assimilation in the rhizosphere (white et al. 2021). in addition, it is reported that an increase in the assimilation of chosen nutrients supported root expansion, and strengthened drought adaptation in ryegrass cv. speedgreen upon application of humic acid. the nutrient content of phosphorus, potassium, and zinc in leaf dry weight remains unchanged by humic acid, whereas the increase in iron content was observed (daneshvar hakimi maibodi et al. 2015). corresponding results demonstrated that the application of humic acid and biomolecules enhanced turfgrasses productivity, but showed different responses to biostimulants applied among creeping bentgrass, tall jescue, and perennial ryegrass (acuña et al. 2022). likewise, the effect of humic acid-containing fertilizer were evaluated on duchesne ex-weston rozier grown in northern wielkopolska, poland, which exhibited leaves with a 60% increase in surface area, intense and dense flowering, higher yield, and more significantly thicker fruit (zydlik & zydlik 2023). furthermore, the application of fulvic and humic acids to sandy and loamy soils with high and extremely low phosphorus levels, respectively, had no effect on grass yield or total nitrogen and phosphorus uptake in fertilized or unfertilized soils. conflicts arose  that the ineffectiveness of the tested humic substances was due to the low doses compared to the soil’s existing soil organic carbon levels. humic substances adsorb to reactive soil mineral surfaces and may not interact with plant roots as effectively as if being put in a hydroculture or soilless substrate (regelink & koopmans 2021). seaweeds seaweeds (algae or macroalgae) are ecofriendly, nonhazardous, and eco-conscious, being widely used in sustainable and diversified farming. seaweed extracts encourage germination as well as aid root and shoot maturation. algae enhance phenylpropanoid pathways in plants and promote root flavonoids and microbe assimilation. macroalgae-based biostimulant showed improved plant resistance to biotic and abiotic stresses. in 2016, the global biostimulant market was estimated to be worth approximately eur 1.45 billion, with seaweed extract contributing to this value. seaweed biostimulants are abundant in plant hormones, vitamins, minerals, and amino acids. brown algae, especially ascophyllum nodosum, when applied as organic fertilizer boost crop yield and enhance plant resistance to both biotic and abiotic stresses. seaweed aids in photosynthetic activity and nutrient uptake with various benefits to improve grass quality during unfavorable conditions (mackiewicz-walec & olszewska 2023). by incorporating perspectives from various literatures the following are formed and a detailed understanding of the differing effects of seaweeds as biostimulants is reported. biotropia vol. 32 no. 3, 2025 406 seaweeds are macroalgae, which main groups are classified based on the pigmentation, i.e., chlorophyta (green), ochrophyta (brown), and rhodophyta (red). ascophyllum nodosum contains about 90 groups of chemical molecules, like biologically active components, vitamins, alginic acid, and amino acids. the effects of a mixture containing bio algae (commercial brown seaweed derived from ascophyllum nodosum) and mineral fertilizers on mixed stands of forage grasses resulting in an increased yield of 40% (brouwers et al. 2018). the extraction of seaweed biostimulants includes physical (microwaves, pressure, heat) and chemical (solvents, acids, and alkalis) methods influencing the bioavailability of active compounds, determining the efficiency of biostimulants (kapoore et al. 2021). worldwide use of seaweed as biostimulant in agriculture has increased its need in global markets. a diverse range of biostimulants, when administered, enhance bioactive compounds in plants and regulate beneficial effects including stress resistance, root shoot elongation, increased chlorophyll content, fruit production augmentation, homogeneous fruit characteristics, senescence retardation, and elevated fruit nutritional quality (calvo et al. 2014). additionally, guillard and inguagiato (2017) reported that seaweed biostimulants had no pivotal impact on normalized difference vegetation index (ndvi) in regularly manicured turfgrasses (perennial). high-temperature parameters were not included. hydric stress was considered and periodically monitored, indicating the effect of biostimulants depends on environmental factors. additional studies were encouraged to determine the seaweed effect on turfgrass (guillard & inguagiato 2017). chitosan chitosan is a polysaccharide containing amino acid groups, composed of polymers of n-acetyl glucosamine and d-glucosamine linked by β-1,4 glycosidic bonds. chitosan derived from chitin which possesses antifungal properties and aids plants to develop resistance against fungal pathogens. the following reports from existing articles provide a better understanding of chitosan. chitin is a complex polysaccharide molecule and an abundant biopolymer in nature, followed by cellulose. chitin is an environmentally degradable, physiologically compatible and nonhazardous molecule with multiple functional applications (wolski et al. 2019; goudarzian et al. 2020). due to its potent physiological capacity, the compound chitin has gained increased attention in the last few years. chitosan application stimulated tyrosine ammonia lyase, polyphenol oxidase, and phenylalanine enzymes, key enzymes involved in plant metabolisms, which furthermore involving antioxidant enzymes like sod (superoxide dismutase), catalase, and peroxidase. as biostimulant, chitosan helps plants increase productivity and showed resistance to biotic and abiotic stressors (wang et al. 2017). chitin derivatives play a pivotal role in various sectors. the important chitin derivatives are chitosan, n and o sulphated chitin, chitin nanoparticles, alkyl chitin, carboxymethyl chitin, dibutryl chitin, chitin nano-whiskers, chitin nanofibers, chitin nano composites, chitin oligosaccharides, and chitin hydrogels. chitin and chitosan are reported to positively influence root growth, improve vegetative yield, and improve drought resistence in peppermint (giglou et al. 2022). furthermore, chitin and chitosan also regulate seed germination and suppress pathogenic fungi wide range of crops (kanawi et al. 2021). chitosan promotes grass growth, increases tolerance to environmental stress factors, enhances the defence system, and increased concentration of chitosan (0.1% to 0.2%) leads to leaf elongation and high maintenance. chitosan, when used in combination with beneficial microbes, improves grass quality and benefits grass management, specifically on golf courses during autumn. chitosan has a property of moisture sealants when applied as leaf foliar which restricts the use of water and develops resistance against stress factors. to overcome the negative impacts in agricultural practices, chitosan is used in organic farming. chitosan forms complexes with toxic metals when applied in soils for biostimulation and botanical remediation. chitosan regulates abscisic acid signalling pathway and activates lightdependent pathway and guards cell contraction (hidangmayum et al. 2019). protein hydrolysates and amino acids protein hydrolysates are major groups of plant derived organic fertilizers, consisting of amino acids and peptides or polypeptides. protein hydrolysates are derived from plant and animal sources by chemical, thermal or enzymatic degradation (amin et al. 2018). according to the commission regulation (eu) no.142/2011 microbial and nonmicrobial biostimulants for enhancing therapeutic metabolites anjali & tamilselvi 407 (annex 1, point 14), hydrolyzed proteins such as peptides, polypeptides, amino acids, and their combinations are obtained from the degradation of animal proteins. biomodulatory effects in protein hydrolysates is due to the presence of carbohydrates, lipids, phytohormones, minerals, phenols, and other organic substances (gonzález-morales et al. 2021). during adverse environmental conditions, protein hydrolysates have been shown to enhance plant adaptability principally by improving antioxidant activity in plant. chemical traits such as optical activity of amino acids, molecular mass, electrolytic conductivity, and availability of amino acids in protein hydrolysates differ based on the chemical or enzymatic hydrolysis and origin of raw materials, like plant and animal. protein hydrolysates promote crop growth and development making them an effective tool for organic agricultural farming. the compounds involved in protein hydrolysates metabolism are not well studied. the effect of protein hydrolysates on soil-habitat microbes in the region is not well known, thereby balancing hydrolysis parameters and enzyme materials during synthesis is critical (canellas et al. 2015). ugolini et al. (2023) studied the effect of protein hydrolysates extracted from oil-free seed cake of abyssinica kale by two-step enzymatic degradation process. the study of ugolini et al. (2023) also showed that the activity of protein hydrolysates was tested on vigna radiate (mung bean) and zea mays l. (maize). furthermore, improvement in root development and architecture were reported at 4.8mm of nitrogen concentration corresponding. in addition, protein hydrolysates exhibited high nitrogen content in both root and shoot and also in chlorophyll spad index in maize. most importantly the commercial biostimulant showed the same effect as shown by the natural biostimulant (ugolini et al. 2023). the application of vegetal protein hydrolysates has gained more interest over the years. they directly stimulate nitrogen and carbon metabolism and indirectly enhance nutrient bioavailability for crop development. in addition, vegetal protein hydrolysates increase the efficient use of nitrogen. gurav et al. (2020) reported the use of protein hydrolysates from animals in brinjal (eggplant) and chili plants, which promoted early blooming and increased yield. accumulation of ethylene1-aminocyclopropane-1-carboxylate (acc) was observed in tomato plants treated with protein hydrolysates from plant source, which also increased the shoot mass (gurav et al. 2020). in addition, protein hydrolysates obtained from alfalfa upregulated the genes which codes for the component ferredoxin-2,lhca5 and the enzymes involved in calvin cycle (rubisco) (ertani et al. 2017). osman et al. (2021) studied the effect of three types of protein hydrolysates on foliar application in pisum sativum. the protein hydrolysates nap (native whey protein), pah (papain) or peh (pepsin hydrolyzed whey protein) enhanced the uptake of macronutrients (n, p, and k). collagen based protein hydrolysates when combined with fecl₃ exhibited increased fe content in maize plants in contrast to plants treated with fecl₃ and feedta individually. zmtom1 and zmirt 1 gene expression were improved to absorb fe in roots (osman et al. 2021). our review also summarizes other beneficial effects of nonmicrobial biostimulants on different plants with different modes of application (table 1). microbial biostimulants beneficial bacteria biostimulants, as alternatives to chemical inputs, include useful microbes like growth promoting bacteria and mycorrhizal fungi (sun et al. 2023). predominantly reviewed bacterial strains are registered subsequently. plant growth promoting bacteria comprise both rhizobacteria that colonize the rhizosphere and free living bacteria. they play a pivotal role in solubilizing inorganic nutrients and biosynthesis of growth-promoting factors (baltazar et al. 2021). biofertilizers are live microbes which play a major role in promoting plant growth. on the other hand, biostimulants are substances that promote plant growth but do not meet plant nutrient demand. the major difference between biofertilizers and biostimulants are that biofertilizers meet the plant nutrients demand and increases crop quality, while biostimulants increase the quality of crop. multitude of bacterial genus acinetobacter, arthobacter, azosoirillum, enterobacter, ochrobactrum, pseudomonas, and streptomyces have a wide application in agricultural productivity to facilitate rapid crop growth, enhance nutrient absorption and utilization, minimize nutrient leaching, promote seedling establishment, enhance root growth, remediation of heavy metalscontaminated soil, enhance immune responses, biotropia vol. 32 no. 3, 2025 408 table 1 categories of nonmicrobial biostimulants and the beneficial effects nonmicrobial biostimulant plant mode of application beneficial effect reference humic substances cannabis sativa l. fertigation observed an increase in cannabinoid content. (bernstein et al. 2019) zea mays l. foliar increased accumulation of trans-zeatin, decreased accumulation of zeatin riboside and positively altered metabolic pathways. (gu et al. 2023) oryza sativa fertigation decreased cell metabolite concentration but increased metabolic marker concentration. (canellas et al. 2015) festulolium braunii soil treatment increase in structural and nonstructural content except lignin. (wiśniewskakadzajan & stefaniak 2020) fulvic acid rosmarinus officinalis l. foliar increased fresh and dry yield. (farruggia et al. 2024) pisum sativum l. var. meteor fertigation remarkable increase in pod number, shape, size and seed number per pod, chemical composition of seeds. (kamran et al. 2023) seaweeds the sugarcane varieties rb855536, sp803280, and rb855536 foliar increased drought resistance, crop yield, sucrose production and metabolic activities. (jacomassi et al. 2022) oryza sativa foliar increased ca+  and k+ concentration during drought susceptible condition. stabilized ros production. (banakar et al. 2022) triticum aestivum l. var. zlata fertigation higher seaweed content. increased phenolic content inhibited plant growth but showed increased content of macromolecules, pigments, and secondary metabolites. (shibaeva et al. 2023)cucumis sativus l. var. zozulya f1 hyoscyamus niger foliar enhanced crop growth, leaves yield, and total alkaloids content. (al-taweel & mohammed 2023) ceratonia siliqua agar treated with biostimulants enhanced plant growth and rooting of carob. (zouari et al. 2023) microbial and nonmicrobial biostimulants for enhancing therapeutic metabolites anjali & tamilselvi 409 nonmicrobial biostimulant plant mode of application beneficial effect reference chitosan oryza sativa foliar act as anti-biotic stress factor ultimately increasing the chlorophyll content. (banakar et al. 2020) phaseolus vulgaris foliar increased nutrient uptake efficiency, dry weight biomass and nutrient quality. (fawzy et al. 2023) satureja hortensis foliar high concentration of chitosan increased the essential oil content but there is no significant increase in carvacrol. (alizadeh et al. 2020) solanum lycopersicum l. soil treatment showed increase resistance to drought. (jiménez-arias et al. 2023) agrostis stolonifera foliar increased shoot and root mass, elevated membrane stability under stress condition. (li et al. 2022) protein hydrolysates and amino acids beta vulgaris soil treatment increased nutrient uptake, altered transcription rate of gene. (jolayemi 2023) primula acaulis cv. soil treatment increased biomass, chlorophyll content and surface area in plants. (tütüncü 2024) lactuca sativa soil treatment successive increase in surface area of leaf, fresh weight of leaf and root and total chlorophyll content. (zahra et al. 2024) ipomoea batatas l. foliar enhanced tuber quality and increased nutrient assimilation. (elwaziri et al. 2023) gives aesthetic appeal to end products, and induce production of plant defence compounds(cieślik et al. 2022; nikolouli et al. 2021drosophila suzukii, is an invasive pest species infesting major agricultural soft fruits. drosophila suzukii management is currently based on insecticide applications that bear major concerns regarding their efficiency, safety and environmental sustainability. the sterile insect technique (sit; gorrasi et al. 2021). enterobacter belongs to the family enterobacteriaceae, under the class gammaproteobacteria. (nyenje et al. 2013). enterobacter spp. are gram-negative, rod-shaped, non-sporeforming, facultative anaerobic bacteria. enterobacter roggenkampii ed5 elicited photosynthetic respiration, plant attributes in sugarcane, and increased overall crop yield (guo et al. 2022). ullah et al. (2020) noted improved kernel grade, zinc accessibility, yield, and economic viability in kabuli chickpea by using enterobacter spp. mn17 coating, a zn-solubilizing endophytic bacterium. in addition, enterobacter had considerable effects on root nodule formation in soybean and maize yield (ullah et al. 2020). enterobacter sichuanensis aji 2411, found in the rhizosphere of soybean, improved plant output (ajibade et al. 2023). enterobacter cloacae hg-1 exhibited salinity tolerance and promoted crop development in wheat plants (ji et al. 2020). pseudomonas spp. entails abundant gramnegative bacterial strains, which is also the most prolific and diverse bacterial population prevalent in the natural environment (pramanik et al. 2018). various pseudomonas strains express distinct environment traits involving microbial film formation, fungicidal compound synthesis, symbiotic root adhesion, cell density signaling, chemo attraction, assimilation, exudation, and metabolic dissimilation. pseudomonas fluorescens um270 decreased salt stress conditions in tomato plants which lead to raise in chlorophyll content, dry weight, and shoot and root extension (rojassolis et al. 2023). pseudomonas spp. rgm2987 from philippine stevia roots exhibited improvement in indole acetic acid, solubilizing phosphate and stress relief enzyme production (guerra et al. 2023). kumar et al. (2021) studied the crop length and fry weight in sunflower using pseudomonas lurida biotropia vol. 32 no. 3, 2025 410 strain e0026 which results showed an increase in shoot and root length, increase in dry weight, and increase the cu uptake. pseudomonas fluorescens ecs417 controlled necrosis in leaf developed by ralstonia solanacearum in eucalyptus (kumar et al. 2021). streptomyces are filamentous, gram positive, and aerobic bacteria. these bacterial strains are wide producers of secondary metabolites such as antifungals, pathogenic factors, antibiotics, and anticancer agents. streptomycetes fjat-31547 exhibited antimicrobial activities and increased biocontrol performance against fusarium and bacterial rot in tomato plants, due to the presence of n-hexadecanoic acid in the streptomycetes strain identified by gc-ms (zheng et al. 2019. in addition, veilumuthu et al. (2022) studied the antimicrobial activity of streptomyces spp. vitgvioo. streptomyces spp. kra18-249 exhibited biocontrol activity against various weeds. the secondary metabolite piericidin a secreted by streptomyces spp. fxpo4 controlled fungal growth and inhibited blight damage in potato plants (veilmuthu et al. 2022). beneficial fungi microorganisms develop symbiotic relationship with plants, which aids plants in enhancing tolerance to both biotic and abiotic stresses (żurek et al. 2022). mycorrhizal fungi prevalent in rhizosphere improve crop growth and production. microbes under this class are called as growth promoting fungal biostimulants (altaey & majid 2018). arbuscular mycorrhizal fungi (amf) improves the carbon flux in subsurface of host plants, which in turn increases the microbial population. this review reports enlighten the importance of beneficial fungi as biostimulants to promote plant yield and growth. amf hyphae improves nutrient absorption and translocation expanding from root surface beyond mineral depletion areas, thus increasing the host plants adaptability (rouphael et al. 2015) competent microbial strains must exhibit prolonged beneficial effects, are easy to handle, and should not be toxic to environment and mortals (stingl et al. 2022). xia et al. (2021) identified the presence of bacterial (pseudomonas veronin and janthinobacterium lividum) and fungal (pseudomymnoascus spp.) strains predominantly present in endobiome and root microbiome of turfgrasses. these microbes played an important role in bioconversion, nutrient uptake, and pest management (xia et al. 2021). giovannini et al. (2020) observed the tripartite symbiosis of amf residing the mycorrhizosphere, plant host, and fungal symbionts. additionally, amf support plants fight against pathogen, enhance phosphorous solubilization, improve plant hormones production, function as iron scavengers, and promote antimicrobials synthesis (giovannini et al. 2020). the work of caser et al. (2019) showed that commercial formulation of funneliforms mosseae and rhizophagus intraradices improved flower yield in saffron, while r. intraradices alone increased active metabolites content and radial quenching activity. schubert et al. (2020) industrially produced micro tom and brioso tomatoes using rhizoglomus irregularis and observed increase in free amino acid and carotenoid content up to fourfold when compared to control, while an increase in brix values were also reported. f. mosseae ima1 and clarideoglomus claroideum increased ros scavenging activity and total phenol content, respectively in romanesco artichoke (c. cardunculus l. cv. romanesco c3 italy and violetto tema) (avio et al. 2020). other beneficial effects of microbial biostimulants on different plants with different modes of application are presented in table 2, while beneficial plant traits enhanced by biostimulants are presented in figure 2. microbial and nonmicrobial biostimulants for enhancing therapeutic metabolites anjali & tamilselvi 411 table 2 c ategories of m icrobial biostim ulants and the beneficial effects m icrobial biostim ulant plant m ode of application b eneficial effect r eference acinetobacter calcoaceticus ac 06 and bacillus am yloliquefaciens ba01 arachis hypogaea l. soil treatm ent o bserved stress resistance, osm olyte tolerance by altering stress m arker activity and physiological redox status in plants. (sun et al. 2023) bacillus velezensis d 2w m (d 2), bacillus velezensis z j-11 (z j-11) a. roxburghii (m r h ) and a. form osanus (yyb) fertigation prom oted assim ilation of nutrients via roots, balancing m icrobial population in the rhizosphere. (shahrajabian et al. 2023) bacillus, pseudom onas and rhizobium strains arabidopsis thaliana and zea m ays soil treatm ent u pregulated stress-responsive gene, im proved crop yield and quality. (d aengbussadee et al. 2021) pseudom onas fluorescens and burkholderia pyrrocinia brachiaria brizantha cv. soil treatm ent increased photosynthetic lim itation by enhancing leaf surface area and increased biom ass production. (r an et al. 2005) trichoderm a viride and pseudom onas fluorescens c ucurbita pepo l. fertigation increased content of chlorophylls and carotenoids. significantly elevated total phenolic profile. (bekiesch et al. 2021) beauveria bassiana triticum aestivum seed treatm ent enhanced m icro and m acro nutrient availability, upregulated phytostim ulation in plants. (sahaja & pudukarapu 2019) aureobasidium pullulans (ak 10) abies koreana seed treatm ent increased tolerance to drought and enhanced m icrobial colonies. (fu et al. 2022) biotropia vol. 32 no. 3, 2025 412 figure 2 beneficial plant traits enhanced by biostimulants conclusion both nonmicrobial and microbial biostimulants function differently to enhance sustainable agriculture productivity. humic and fulvic complexes improve root structure and chelate nutrients; seaweed extracts enriched with bioactive components boost metabolic activity and stress resilience; chitosan primes the plant defense mechanism and acts as a growth regulator; protein hydrolysates directly affect nitrogen metabolism and optimize physiological processes in plants. simultaneously, microbial biostimulants, including plant growth promoting rhizobacteria (e.g., bacillus, pseudomonas, arthrobacter, enterobacter, and rhizobacter) and arbuscular mycorrhizal fungi (e.g., mycorrhizal fungi and trichoderma), improve plant efficiency by forming tripartite symbiotic relationships with plants. these biostimulants enhance nutrient availability, regulate metabolic pathways, and help plants fight pathogens by modulating root topology, photosynthesis efficiency, and secondary metabolite biosynthesis. these effects improve crop quality and resilience. notably, the varied effects observed across crops, environmental conditions, yields, soil types, and application systems emphasize the need for systemized protocols, formulation optimization, and areaspecific validation experiments. the cumulative reports highlight the effective integration of biostimulants in agriculture replacing the use of synthetic fertilizers and conservation of the agriculture land with eco-friendly substances and organisms retaining the soil health and sustainable environment. references acuña a, gardner d, villalobos l, danneberger k. 2022. effects of plant biostimulants on seedling root and shoot growth of three cool‐season turfgrass species in a controlled environment. int turfgrass soc res j 14(1):416-21. doi: 10.1002/its2.97 ajibade oa, oladipo ek, kwenda s, khumalo z, ismail a, oloke jk, …, onyeaka h. 2023. whole genomic sequence of enterobacter sichuanensis aji 2411 – a plant growth promoting rhizobacteria. gene 887:147725. doi: 10.1016/j.gene.2023.147725 alizadeh a, moghaddam m, asgharzade a, sourestani mm. 2020. phytochemical and physiological response of satureja hortensis l. to different irrigation regimes and chitosan application. ind crops prod. 158:112990. doi: 10.1016/j.indcrop.2020.112990 microbial and nonmicrobial biostimulants for enhancing therapeutic metabolites anjali & tamilselvi 413 altaey dka, majid zz. 2018. the activity of antioxidants enzymes and npk contents as affected by water qaulity, kinetin, bio and organic fertilization in lettuce (lactuca sativa l.). iraqi j agric sci 49(3):506-18. doi: 10.36103/ ijas.v49i3.123 al-taweel sk, mohammed aa. 2023. effect of exogenous application of nano fertilizers and seaweeds extract on the growth, yield, and total alkaloids content of hyoscyamus niger. iop conf ser earth environ sci 1262(5). doi: 10.1088/17551315/1262/5/052010 amin m, ahmad r, ali a, hussain i, mahmood r, aslam m, lee dj. 2018. influence of silicon fertilization on maize performance under limited water supply. silicon 10(2):177-83. doi: 10.1007/s12633-015-9372-x assi nn, zahwan ta. 2023. response of pomegranate trees to herd manure addition and spraying with fulvic acid and brassinolide. iop conf ser earth environ sci 1262(4). doi: 10.1088/1755-1315/1262/4/042071 avio l, maggini r, ujvári g, incrocci l, giovannetti m, turrini a. 2020. phenolics content and antioxidant activity in the leaves of two artichoke cultivars are differentially affected by six mycorrhizal symbionts. sci hortic 264 (may 2019):109153. doi: 10.1016/j.scienta.2019.109153 baltazar m, correia s, guinan kj, sujeeth n, bragança r, gonçalves b. 2021. recent advances in the molecular effects of biostimulants in plants: an overview. biomolecules 11(8). doi: 10.3390/biom11081096 banakar sn, kumar p, gautam c, me p. 2020. foliar application of red seaweed (kappaphycus alvarezii) bioformulations increased the levels of chlorophyll content in rice. j pharmacogn phytochem 9(1):408-10 banakar sn, prasanna kumar mk, mahesh hb, parivallal pb, puneeth me, gautam c, …, narayan ss. 2022. redseaweed biostimulants differentially alleviate the impact of fungicidal stress in rice (oryza sativa l.). sci rep 12(1):112. doi: 10.1038/s41598-022-10010-8 bekiesch p, oberhofer m, sykora c, urban e, zotchev sb. 2021. piperazic acid containing peptides produced by an endophytic streptomyces sp. isolated from the medicinal plant atropa belladonna. nat prod res 35(7):1090-96. doi: 10.1080/14786419.2019. 1639174 bernstein n, gorelick j, zerahia r, koch s. 2019. impact of n, p, k, and humic acid supplementation on the chemical profile of medical cannabis (cannabis sativa l). front plant sci 10. doi: 10.3389/fpls.2019.00736 brouwers e, draisma m, swam k van, veen aj, burger l. 2018. identification of the seaweed b i o s t i m u l a n t market (phase 1). 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paniculatum gaertn. root induction and the effect of phosphate concentrations and ammonium:nitrate ratio on biomass of adventitious roots in in vitro culture palupi dasawulan lestari, syifa fajrisani, putri gehasti, sugiharto and yosephine sri wulan manuhara* department of biology, faculty of science and technology, universitas airlangga, surabaya 60115, indonesia received 11 october 2021 / revised 15 march 2023 / accepted 21 march 2023 abstract java ginseng (talinum paniculatum gaertn.) is a medicinal plant, the roots of which are commonly used in traditional medicine. in its natural habitat, the roots grow very slowly, requiring two to three years to produce 100 g of roots per plant. plant tissue culture could therefore provide an alternative means of accelerating root growth. this research aimed to optimize root induction and determine the effect of phosphate (kh2po4) concentration and the ratio between ammonium and nitrate (ammonium:nitrate) on the biomass of java ginseng adventitious roots in in vitro culture. stem and leaf were used as explants and various combinations and concentrations of iba and bap, kinetin, and tdz were used as growth regulators. leaf explants were grown in murashige and skoog (ms) media supplemented with iba 2 mg/l and various concentrations of phosphate (170; 212.5; 255; 297.5; 340; 382.5; 425; 467.5; 510 mg/l) and various ammonium:nitrate ratios (21:19 mm as the control, 0:30 mm, 10:20 mm, 15:15 mm, 20:10 mm, 30:0 mm). cultures were maintained for 6 weeks. the observed parameters were fresh weight, dry weight, the duration of root formation, and the number and length of adventitious roots. the data were analyzed using analysis of variance. the results showed that the concentration of phosphate and the ammonium:nitrate ratio significantly influenced the amount, length, fresh weight, and dry weight of java ginseng adventitious root. the highest fresh weight (37.47 mg) and dry weight (5.53 mg) were achieved in the treatment of double phosphate concentration (kh2po4 340 mg/l), while an ammonium:nitrate ratio of 10:20 mm was the optimum treatment to produce the highest biomass (fresh weight 73.6 mg and dry weight 8.2 mg). keywords: adventitious roots, ammonium, nitrate, phosphate, talinum paniculatum gaertn. introduction plants provide an incredible source of new medicinal product inventions for development. one of the medicinal plants often used by the community is java ginseng (talinum paniculatum gaertn.) from the portulacaceae family. java ginseng is widely used as a substitute for korean ginseng, which continues to be imported, because it is relatively cheap, easy to obtain, and easy to cultivate (widiyani, 2006). the plant’s chemical content comprises saponins, triterpenes, polyphenols, and essential oils (komatsu, 1982). the root of the java ginseng plant is the part that can be used as a medicinal ingredient. the most important and dominant component in the chemical content of java ginseng root is saponins. talinum paniculatum gaertn. has very slow root growth in its natural habitat, requiring around two to three years to produce 100 g of roots per plant (manuhara et al., 2015). therefore, in vitro culture techniques offer potential as an alternative means of accelerating the root growth of this plant. root growth can be increased by manipulation in culture media with the addition of nutrients (manuhara, 2014). as such, in this study, an increase in root biomass was achieved with the addition of phosphate and nitrogen sources. phosphate has an important function in *corresponding author, email: yosephine-s-w-m@fst.unair.ac.id biotropia vol. 30 no. 2, 2023 138 plant growth due to its role in transferring energy molecules such as adp and atp, nad and nadp, as well as genetic information system compounds such as dna and rna (barker and pilbeam, 2007). in plant tissue culture, the concentration of phosphate in the medium can be a major factor influencing plant growth. according to dormatey et al. (2021), phosphite supply in the murashige and skoog (ms) medium influenced root morphological characteristics and fresh biomass in five genotypes of potatoes. furthermore, pavlov et al. (2000) stated that lavandula vera biomass and rosmarinic acid was maximally produced with the addition of a two-time phosphate concentration in ms media. apart from phosphate, nitrogen sources also affect cell growth and the formation of secondary metabolites. nitrogen functions as a component of amino acids, proteins, and nucleic acids in plants (wiedenhoeft, 2006). ammonium (nh4+) and nitrate (no3-) are used as the main sources of nutrition in plant cell and tissue culture (zhang et al. 1996, in kim et al. 2005). when both nitrogen sources were administered simultaneously, growth and yield were significantly increased compared to ammonium or nitrate alone (zhang et al. 2011). research by yin et al. (2013) on pseudostellaria heterophylla plants showed that the highest adventitious root biomass, namely 9.11 g fresh weight and 0.54 g dry weight, was obtained with an ammonium and nitrate ratio of 20:40. furthermore, panda et al. (1992) reported that the only received information on ammonium was used as not only the nitrogen source. therefore, it is very important to determine the optimal ratio of ammonium to nitrate. no study has reported the effect of various concentrations of phosphate and the ammonium:nitrate ratio on the biomass production of javanese ginseng plants. this study therefore aims to investigate their effect in culture media on the adventitious root biomass of javanese ginseng. materials and methods adventitious root induction from leaf and stem explants the adventitious roots of java ginseng were induced from stem and leaf explants grown in solid ms medium supplemented with 30 g/l sucrose, 6 g/l agar, and various combinations of iba growth regulator 2 mg/l with three types of cytokinins, namely bap (0.1, 0.3, and 0.5 mg/l), kinetin (0.1, 0.3, and 0.5 mg/l), and tdz (0.1, 0.3, and 0.5 mg/l). the leaf samples (1 cm2) were taken from the second and third leaves of the shoots from intact plant. all explants were immersed in detergent solution for 3 minutes and rinsed with water. the explant surfaces were sterilized by immersing them in 10% clorox solution for 10 minutes. the explants were then washed 3 times using sterile distilled water. following this, they were maintained in an incubation room with an average temperature of 25oc in dark conditions. after 28 days, the adventitious roots were harvested and the fresh weight, dry weight, number of roots, and duration of adventitious root formation were measured. phosphate concentration treatments the explants used the second and third leaves from the shoots. solid ms medium was supplemented with a combination of iba 2 mg/l and tdz 0.1 mg/l, agar 8 gr, and sucrose 30 gr/l. different concentrations of kh2po4 (0; 42.5; 85; 127.5; 170; 215.5; 255; 297.5; and 340 mg/l) were added to the medium. the leaf explants were grown in culture bottles containing the medium and incubated at 25oc in the dark for 6 weeks. after 6 weeks the fresh weight, dry weight, root formation time, number of roots, and root length were measured. adventitious root growth was first recorded and used as the initial observational data for the first time that root growth occurred. after 6 weeks of cultivation, the number of roots, fresh weight, and dry weight were measured. ammonium: nitrate ratio treatments the explants used the second and third leaves from the shoots. the ms medium was supplemented with a combination of iba 2 mg/l and tdz 0.1 mg/l, agar 8 gr, and sucrose 30 gr/l. different ammonium:nitrate ratios (21:19 mm as the control, 0:30 mm, 10:20 mm, 15:15 mm, 20:10 mm, and 30: 0 mm) were added to the medium. the leaf explants (1 cm2) talinum paniculatum gaertn. adventitious roots in vitro culture – lestari et al. 139 were grown in culture bottles containing the medium and incubated at 25oc in the dark for 6 weeks. after 6 weeks, the fresh weight, dry weight, root formation time, number of roots, and root length were measured. adventitious root growth was first recorded and used as the initial observational data for the first time root growth occurred. after 6 weeks of cultivation, the number of roots, fresh weight, and dry weight were measured. root fresh weight was measured after the roots had been rinsed with distilled water and drained. dry weight was measured after drying in an oven at 50oc for 5 days to obtain a constant dry weight. data analysis the data obtained, including the fresh weight, dry weight, number of adventitious roots, and length of adventitious roots, were analyzed using anova (analysis of variance) at a significance level of 5%. results and discussion adventitious root induction the reactions of t. paniculatum adventitious root induction to the addition of an iba growth regulator with various types of cytokinins in the third week can be seen in figure 1 (stem explants) and figure 2 (leaf explants). figure 1 root induction of talinum paniculatum stem explants in various combinations of iba and ba, kinetin, and tdz during a 3-week culture. a) i2b0.1; b) i2b0.3; c) i2b0.5; d) i2k0.3; e) i2k0.5; f) i2t0.1; g) i2t0.3; h) i2t0.5. (bar scale = 1cm). i: iba, b: ba, k: kinetin, t: tdz. the number after the letter indicates the concentration of the growth regulator in mg/l biotropia vol. 30 no. 2, 2023 140 figure 2 t. paniculatum adventitious root induction using leaf explants at the third week. a) i2b0.1; b) i2b0.3; c) i2b0.5; d) i2k0.3; e) i2k0.5; f) i2t0.1; g) i2t0.3; h) i2t0.5 (bar scale = 1cm) the stem and leaf explants induced from various combinations of growth regulator iba 2 mg/l with the different types of cytokinins (bap, kinetin, and tdz) at separate concentrations (0.1, 0.3, and 0.5 mg/l) produced varied responses, as seen in tables 1 and 2. table 1 the average duration of root formation, number of roots, root length, fresh weight, and dry weight of t. paniculatum gaertn. adventitious roots with various combinations of concentrations of iba 2 mg / l and bap, kinetin, and tdz (0.1, 0.3, 0.5 mg / l) on stem explants treatment duration of root formation (days ) number of roots root length (mm) fresh weight (mg) dry weight (mg) i2b0.1 9.33 ± 4.04 8.33 ± 1.52 11.44 ± 5.58 2.86 ± 1.30 1.00 ± 0.20 i2b0.3 14.00 ± 0.00 4.00 ± 2.64 14.68 ± 5.77 4.10 ± 1.94 0.93 ± 0.28 i2b0.5 14.00 ± 0.00 1.33 ± 0.57 8.50 ± 2.50 1.23 ± 0.05 0.53 ± 0.28 i2k0.1 4.67 ± 8.08 1.67 ± 2.88 2.60 ± 4.50 0.12 ± 0.21 0.03 ± 0.05 i2k0.3 11.67 ± 4.04 8.67 ± 6.11 11.81 ± 2.50 11.63 ± 9.60 6.67 ± 7.23 i2k0.5 16.33 ± 4.04 4.00 ± 0.00 7.08 ± 1.52 4.67 ± 3.78 1.06 ± 0.90 i2t0.1 0 0 0 0 0 i2t0.3 16.33 ± 4.04 2.00 ± 1.00 13.33 ± 3.78 3.83 ± 2.41 1.43 ± 1.00 i2t0.5 21.00 ± 7.00 1.33 ± 0.57 8.83 ± 2.36 4.63 ± 1.05 1.63 ± 0.15 talinum paniculatum gaertn. adventitious roots in vitro culture – lestari et al. 141 table 2 the average duration of root formation, number of roots, root length, fresh weight, and dry weight of t. paniculatum gaertn. adventitious roots with various combinations of concentrations of iba 2 mg/l and bap, kinetin, and tdz (0.1, 0.3, 0.5 mg/l) on leaf explants treatment duration of root formation (days to) number of roots root length (mm) fresh weight (mg) dry weight (mg) i2b0.1 16.33 ± 4.04 5.33 ± 1.15 13.3 ± 5.67 1.93 ± 1.04 1.16 ± 0.23 i2b0.3 21.00 ± 7.00 3.33 ± 3.21 10.9 ± 2.85 0.93 ± 0.23 0.10 ± 0.11 i2b0.5 11.67 ± 10.69 1.00 ± 1.00 3.33 ± 3.05 2.53 ± 2.96 0.83 ± 1.27 i2k0.1 0 0 0 0 0 i2k0.3 11.67 ± 10.69 2.00 ± 2.00 3.66 ± 3.32 0.63 ± 0.92 0.50 ± 0.86 i2k0.5 16.33 ± 4.04 2.00 ± 1.00 9.00 ± 5.29 2.40 ± 0.85 1.06 ± 0.90 i2t0.1 21.33 ± 9.01 7.33 ± 1.15 21.33 ± 9.01 4.53 ± 1.76 1.33 ± 0.64 i2t0.3 18.67 ± 8.08 4.33 ± 2.30 8.15 ± 2.58 3.23 ± 1.88 1.03 ± 0.95 i2t0.5 14.00 ± 0.00 1.67 ± 1.15 8.22 ± 5.17 4.83 ± 2.43 1.10 ± 0.90 the fastest mean root formation time was obtained from the induction of stem explants with the addition of a combination of growth regulator iba 2 mg/l + kinetin 0.1 mg/l, namely in 4.67 days. meanwhile, for the leaf explants, the fastest root formation time, at 11.67 days, was obtained by adding the combinations of growth regulator iba 2 mg/l + kinetin 0.3 mg/l and iba 2 mg/l + bap 0.5 mg/l. whereas the addition of a combination of growth regulator iba 2 mg/l + tdz 0.5 mg/l for stem explants and iba 2 mg/l + tdz 0.1 mg/l for leaf explants resulted in the longest mean times to root formation, namely 21 and 21.33 days, respectively. based on table 1, the highest average fresh weight, dry weight, and number of roots for the stem explants were obtained in the iba treatment of 2 mg/l + kinetin 0.3 mg/l. while the highest average root length of stem explants, namely 13.33 cm, was obtained in the iba treatment of 2 mg/l + 0.3 mg/l tdz. in table 2, the greatest average root fresh weight was obtained from the results of leaf explant induction with the addition of a combination of growth regulator iba 2 mg/l + tdz 0.1 mg/l, namely 4.53 mg. meanwhile, the highest dry weight, number of roots, and root length were obtained from the induction of leaf explants with the addition of a combination of growth regulator iba 2 mg/l + tdz 0.1 mg/l. the lowest average dry weight was obtained with the combination of iba 2 mg/l + bap 0.3 mg/l, namely 0.1 mg. adventitious roots can be induced from various explants including leaves, stems, roots, and various other factors such as auxins (baque et al., 2010). the selection of the types, concentrations, and combinations of growth regulators is very important. based on previous research, adventitious roots have been successfully induced from javan ginseng leaf explants on a solid ms medium with the addition of iba 2 mg/l, which produced the highest root mass of 5.929 g (solim et al. 2017). meanwhile, erin et al. (2020) reported that the treatment of iba 2 mg/l + ethephon 1 mg/l produced the highest average number of roots, namely 7.33, compared to other treatments. bap and kinetin hormones are chemical compounds that are included in the cytokinin group and play a role in shoot growth. this time, however, they were combined with auxin iba to stimulate adventitious root growth on t. paniculatum stem and leaf explants. in the research of isda and fatonah (2014), the highest number of roots was found at a bap concentration of 0.5 mg/l + 1.0 mg/l naa, namely 5.00 fruit on the explants of grammatophyllum scriptum orchid shoots. however, in the results of this study on stem and leaf explants, the combination of iba 2 mg/l + bap 0.5 mg/l did not provide optimal results in all parameters of the observation. it is thus evident that the effect of these growth regulators depends on the type of plant and the dosage concentration of growth regulator combination that is suitable; as such, for t. paniculatum this is not the optimal concentration of auxin and cytokinin combination to produce the most roots. apart from bap, kinetin is also often combined with the auxin hormone in its use in vitro, as in the research of mahadi et al. (2013) where the highest average number of dragon fruit explant roots was found in the n0.4 k4 treatment, namely 5.25 roots. however, the lowest average number of roots was also found in the n0.4 k4 treatment. this is presumably because high kinetin administration can produce stunted explant growth; wahidah (2011) stated biotropia vol. 30 no. 2, 2023 142 that kinetin hormone can affect the process of plant development at low concentrations while inhibiting growth at high concentrations. tdz can play a role in stimulating endogenous cytokinin production. therefore, tdz can increase the action of other cytokinins, both exogenous and endogenous cytokinins (guol et al., 2011). the administration of tdz at a low concentration induces callus faster than at a high concentration; for callus regeneration, it is better to combine tdz and naa at low concentrations than tdz alone (oláh et al., 2003). this is corroborated by the findings of this study, where tdz with a concentration of 0.1 mg/l induced the formation of a large number of adventitious roots to produce a large fresh weight and dry weight compared to concentrations of 0.3 mg/l and 0.5 mg/l. the formation of adventitious roots is a type of positive synergy between tdz and iba as the best adventitious root-forming hormone. effect of phosphate concentration (kh2po4) on adventitious root growth the best treatment for inducing root growth (a combination of iba 2 mg/l and tdz 0.1 mg/l) was used to determine the effect of phosphate concentration and the ammonium:nitrate ratio on adventitious root growth. the average fresh weight, dry weight, root growth, number of roots, and adventitious root length of t. paniculatum in various phosphate concentration treatments of ms medium are listed in table 3. the highest average values for fresh weight, dry weight, and the number of roots were obtained for the p5 treatment (phosphate concentration 340 mg/l). meanwhile, the fastest root growth rate of 7.3 days was achieved with the p7 treatment (phosphate concentration 425 mg/l), and the longest average root is shown for the p8 treatment (phosphate concentration 467.5 mg/l). the highest average fresh weight and dry weight values were identified for the p5 treatment. this is consistent with research conducted by curtis et al. (1991), which stated that the growth of opium poppy in cell suspension culture increased by 50% in media with two times the concentration of phosphate added. this occurred since phosphate plays an essential role in the transfer of energy for cell metabolism, the constituents of cell membranes, and nucleic acids. the lowest fresh weight was found on the media with the p9 treatment (510 mg/l) due to the very high phosphate concentration. the plant cells in these explants were stressed due to the very high salt concentration. the increase in the plant’s dry weight is attributable to the nutrients that were absorbed by the root and the accretion of protoplasm due to the increase and size of the cell count (khristyana et al. 2005). the fastest average duration of root formation was 7.3 days (phosphate concentration 425 mg/l) based on the application of the control treatment (phosphate concentration 425 mg/l) for 8 days. in this study, the p9 treatment (phosphate concentration 510 mg/l) showed the longest average duration of root formation (11.6 days) because media with high phosphate concentrations can suppress growth (george et al. 2007). phosphate can bind with calcium and other microelements to reduce the absorption of other elements below the maximum level (buddh, 2014). table 3 average fresh weight, dry weight, duration of root formation, number of roots, and root length of t. paniculatum adventitious roots in various phosphate concentrations of ms medium treatment code kh2po4 concentratio n (mg/l) fresh weight (mg) dry weight (mg) duration of root formation (days to) number of roots root length (cm) p1 170 18.77 ± 9.96 b 3.23± 0.60 b 8.00 ± 2.64 3.67 ±2.52 a 2.56 ±1.76 ab p2 212.5 2.67 ± 2.56 a 1.10 ± 0.60 a 9.00 ± 1.73 3.00 ± 1.00 a 1.79 ± 0.31a p3 255 8.63 ± 5.55 a 2.07 ± 0.81 ab 10.67 ± 1.15 1.33 ± 0.58 a 2.85 ±1.52 ab p4 297.5 16.30± 5.03 ab 3.30 ± 0.09 b 8.33 ± 3.21 4.00 ±1.73 a 1.39±0.22 a p5 340 37.43 ± 5.09 c 5.53 ± 1.03 c 7.67 ± 0.57 5.33 ±0.58 ab 1.70 ±0.39 a p6 382.5 8.8 ± 6.61 ab 1.77 ± 1.22 ab 11.33 ± 1.52 4.00 ± 1.00 ab 1.55 ± 0.96 a p7 425 19.13 ±14.89 b 3.17 ± 1.56 b 7.33 ± 0.57 4.67 ± 1.15 ab 2.15 ± 0.46 a p8 467.5 19.17 ± 9.03 b 3.27 ± 0.50 b 8.00 ± 1.73 1.67 ±0.58 b 4.20 ±0.75 b p9 510 2.30± 1.15 a 1.13 ± 0.67 a 11.67 ± 2.51 2.00 ±1.00 ab 1.56±0.66 a note: numbers followed by different letters show real differences according to duncan’s test. the number in bold shows the highest value for each treatment parameter. talinum paniculatum gaertn. adventitious roots in vitro culture – lestari et al. 143 figure 3 the number of adventitious roots formed from explants of javanese ginseng leaves (talinum paniculatum gaertn.) after a 6-week incubation period, namely (a) p1 (kh2po4 170 mg/l / control), (b) p2 (kh2po4 212,5 mg/l ), (c) p3 (kh2po4 255 mg/l), (d) p4 (kh2po4 297,5 mg/l), (e) p5 (kh2po4340 mg/l), (f) p6 (kh2po4 382,5 mg/l), (g) p7 (kh2po4425 mg/l), (h) p8 (kh2po4 467,5 mg/l), and (i) p9 (kh2po4 510 mg/l) (bar scale = 1cm) the number of roots produced in each explant is different (figure 3). the highest mean number of roots was found in the explants treated with p5 (kh2po4 340 mg/l). the treatment in this study was controlled by p1 (170 mg/l of kh2po4). the number of roots stood at only 2.56; thus, while both were from the second and third leaves of the shoots, they were from different plants. auxin plays a role in cell elongation, cell division, and adventitious root formation (george, 2007). therefore, different endogenous auxins lead to differences in root formation, the number of adventitious roots, and the length of adventitious roots. effect of ammonium:nitrate ratio on adventitious root growth the average fresh weight, dry weight, root growth, number of roots, and the adventitious root length of t. paniculatum in various ammonium:nitrate ratios on ms medium are listed in table 4. the highest average fresh weight, dry weight, and number of roots were obtained for the ammonium:nitrate ratio of 10:20. meanwhile, the fastest average time to root formation was 10 days and the longest root length was 2.3 cm. however, the data showed no significant difference between all treatments, meaning the data did not affect the adventitious root biomass of java ginseng. figure 4 contains pictures of 6 different ammonium:nitrate ratios, namely 21:19 mm as the control, and 0:30 mm, 10:20 mm, 15:15 mm, 20:10 mm, and 30: 0 mm for the adventitious roots of java ginseng at 6 weeks. biotropia vol. 30 no. 2, 2023 144 table 4 average fresh weight, dry weight, duration of root formation, number of roots, and root length of t. paniculatum adventitious roots in various ammonium:nitrate ratios (21:19 mm, 0:30 mm, 10:20 mm, 15:15 mm, 20:10 mm, and 30:0 mm) ammonium:nitrate ratio fresh weight (mg) dry weight (mg) duration of root formation (days to) number of roots root length (cm) a21 : n19 (normal ms) 47.9 ± 14.9a 5.0 ± 1.7a 10.2 ± 3.7a 5.6 ± 5.3ab 1.9 ± 0.8a a0 : n30 17.5 ± 9.5b 2.1 ± 0.8b 13.0 ± 4.3a 3.0 ± 2.3a 2.0 ± 0.9a a10 : n20 73.6 ± 32.5a 8.2 ± 3.2a 10.2 ± 3.0a 8.8 ± 2.9b 1.8 ± 0.6a a15 : n15 23.3 ± 17.6b 3.4 ± 2.5b 10.0 ± 1.9a 7.0 ± 4.6ab 1.4 ± 1.0a a20 : n10 19.6 ± 14.9b 2.5 ± 1.5b 11.0 ± 1.9a 5.6 ± 4.7ab 1.0 ± 0.3a a30 : n0 21.3 ± 9.6b 3.2 ± 1.2b 11.4 ± 3.4a 5.8 ± 3.1ab 2.3 ± 1.0a note: numbers followed by different letters show real differences according to duncan’s test. numbers in bold show the highest value for each treatment parameter. a = ammonium; n = nitrate. figure 4 the adventitious roots (shown by arrows) of 6-week-old t. paniculatum were cultured in vitro with 6 types of treatment and ammonium:nitrate ratios, namely (a) 21:19 mm (control); (b) 0:30 mm; (c) 10:20 mm; (d) 15:15 mm; (e) 20:10 mm, and (f) 30: 0 mm adventitious roots (i.e., roots that form from non-root tissue) can arise in various tissue locations from groups of mature cells that renew their cell division activity (taiz and zeiger, 2003). in the treatment ratio ammonium:nitrate 10:20, nh4cl has good potential to replace nh4no3 in control as a nitrogen source to increase adventitious root biomass production. this is because nitrogen is used for protein synthesis, both structural and enzymatic, and is thus needed for cell and organ growth, including for the production of plant biomass (lawlor et al. 2001). in addition, the form and amount of nitrogen in the in vitro media have a significant effect on the rate of cell growth, differentiation, and cell totipotency (kirkby and mengel, 1987). the average time to root formation in this study ranged from 10 to 13 days (table 4). this aligns with the findings of research by palestine (2008) on pule pandak (raufolvia serpentine, l.) showing that the addition of iba with a concentration of 2 to 4 mg/l can initiate root growth faster than other treatments, namely in 15 days. the highest number of roots was found in the ammonium:nitrate ratio of 10:20, while the lowest number of roots was found in the ammonium:nitrate ratio of 0:30. efforts to increase the number of roots include the addition of auxin growth regulators that can stimulate root induction. wattimena (1988) explained that auxin is a plant hormone essential talinum paniculatum gaertn. adventitious roots in vitro culture – lestari et al. 145 for cell division and root formation. root emergence is influenced by the number of roots and correlates with the absorption of nutrients present in the culture medium. several studies have shown that nitrogen compounds and the ratio between ammonium and nitrate can affect the differentiation, dedifferentiation, growth, and development of explants, as well as organ formation (preece 1995). the average root lengths of the treatments in this study are shown in table 4; they range from 1 cm to 3 cm with no influence between one treatment and another. the increase in plant size reflects the increase in protoplasm that occurs due to the increase in cell size and number (khristyana et al. 2005). conclusion the combination of iba 2 mg/l + kinetin 0.3 mg/l is the optimal concentration to produce the highest mean number of roots, fresh weight, and dry weight of talinum paniculatum adventitious roots in stem explants. meanwhile, for leaf explants, the best results were obtained with the combination of iba 2 mg/l + thidiazuron 0.1 mg/l. the highest fresh weight (37.47 mg) and dry weight (5.53 mg) were obtained in the phosphate concentration (kh2po4) 340 mg/l treatment. meanwhile, the ammonium:nitrate ratio of 10:20 was the best treatment to produce the highest biomass (fresh weight 73.6 mg and dry weight 8.2 mg). acknowledgments this research was funded by the directorate general of higher education, research and technology no. 852/un3.14/pt/2020 in the master’s thesis research scheme. references baque ma, hahn ej, paek ky. 2010. induction of adventitious root from leaf explants of morinda citrifolia affected by auxin and light quality. in vitro cell dev biol plant 46: 71-80. barker av, pilbeam dj. 2007. 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firman ali rahman1*, dewi putri lestari2, alfian pujian hadi3&5, anis syakiratur rizki4, aisha zea almahyra5, arsya zafran alvarendra5, nuzuly ilmia cerminand5, nening listari6, r. didi kuswara6, zulkarnain gazali6, maya ekaningtias6, siti wardatul jannah6 and baiq yulia hasni pratiwi7 1department of biology education, faculty of education and teacher training, universitas islam negeri mataram, mataram 83127,west nusa tenggara, indonesia 2departemen of aquaculture, faculty of agriculture, universitas mataram, mataram 83125, west nusa tenggara, indonesia. 3departemen of geography education, universitas muhammadiyah mataram, mataram 83127, west nusa tenggara, indonesia. 4department of health analyst, faculty of nursing and health, universitas muhammadiyah semarang, semarang 50273, indonesia. 5indonesian tajuk foundation, mataram 83116, west nusa tenggara, indonesia. 6departemen of biology education, faculty of education and teacher training, universitas nahdlatul wathan mataram, mataram 83126, west nusa tenggara, indonesia 7departement of pharmacy, faculty of public health, universitas bumigora, mataram 83127, nusa tenggara barat, indonesia. received 20 march 2023 / revised 25 july 2023 / accepted 7 september 2023 abstract mangroves are one of the coastal vegetation that can mitigate carbon (carbon sink and carbon storage). this study aimed to determine the potential for soil carbon stock found under stands of mangroves in lembar bay, west lombok, and west nusa tenggara. the research began with the identification of the species and then proceeded toa sampling of the soil, which was then analyzed using the walkley and black method. the results showed that there were ten species of mangroves, namely, rhizophora stylosa, avicennia lanata, avicennia marina, bruguiera gymnorrhiza, ceriops decandra, excoecaria agallocha, lumnitzera racemosa, scyphiphora hydrophyllacea, thespesia populnea, and xylocarpus maluccensis. the highest soil carbon content percentage was found in the lower soil of the a. lanata (1.43 %c) mangrove, and the lowest was found in the lower-stand soil of e. agallocha (0.21 %c). meanwhile, the carbon sinks per meter were 0.002-0.066 gc/m2, with an average of 0.020±0.020 gc/m2. the estimated total soil carbon sink in 10 mangrove stands was 0.20-6.60 tons c/ha, with an average of 2.18±2.010 tons c/ha. the average total estimated soil carbon stock found in 20.49 ha of the mangrove area studied was 44.67 tonsc, which is equivalent to 263.69 tonsc in a mangrove area of 120.96 ha in lembar bay. keywords: carbon stock, c-organic, mangroves, soil introduction mangroves are one of the plants in coastal areas that play a role in disaster mitigation (abrasion, breakwater, sea wind barrier, and tsunami), biota habitats, and germplasms. the environmental benefits of mangrove ecosystems that have not been widely studied include their potential as carbon sinks and carbon storage, especially in mangrove ecosystem soil (brath et al. 2015; lovelock & duarte 2019; macreadie et al. 2019). based on murray et al.(2011), the average annual carbon sequestration potential of mangrove ecosystems is between 6 and 8 mg co2 e/ha (tonnes co2 equivalent per hectare) and is two to four times greater than the carbon sink potential of tropical forests (nellemann et al. 2009). one of the coasts in the mangrove corridor essential ecosystem area is lembar bay, west lombok, which is directly affected by the activities of the lembar harbor. lembar harbor is an inter-island sea and goods transportation route that continues to be developed as a port area (reclamation) covering 22 ha. it has a direct impact on the degradation of mangrove *corresponding author, email: potential of carbon sink in mangrove substrates – rahman et al. 347 ecosystems, resulting in a decrease in the mangrove ecosystem area, which currently only has an area of ±120.96 ha (saraswati 2019). the ecological potential of mangrove ecosystems as carbon sinks and carbon storage has been widely studied. the following data are some of those previous findings. the soil carbon stock of the tanjung lesung banten mangrove ecosystem of 27.92 tons c/ha; the mangrove carbon stock in dukuh tapak, tugurejo village, semarang amounted to 708.2 tons c/ha; mangroves in timbulsloko village, demak, central java had carbon stock of 1,307.77 tons/ha; perancak mangroves in jembrana, baliretain carbon stock of 119.75 tons c/ha; mangroves of the batang apar estuary of west sumatra had carbon stock of 2,561.90 tons c/ha (handoyo et al. 2020); mangroves in sungai sembilan, dumai had carbon stock of 1,819.31 tons c/ha (handoyo et al. 2020); mangroves in tambakbulusan village, demak, central java carbon stock of 57.74 tons c/ha; and gili meno mangroves, north lombok had carbon stock of 154.62±99.78 tons c/ha, equivalent to a total soil carbon stock of 1,020.50 tonsc in a total 6.6 ha of the mangrove ecosystem area (hilyana & rahman 2022). in general, research on carbon sinks in mangrove ecosystems is still related to the potential for carbon sinks in certain locations, and not specifically related to the potential for soil carbon stock under mangroves. this is in line with the opinion of mcleod et al. (2011) and howard et al.(2017) that in-depth analysis related to the potential of mangroves as carbon sinks and carbon storage in different species and habitats is very important. due to this, this study can be a source of information on the potential for soil carbon storage found under ten types of mangroves in the harbor area of lembar bay, west lombok, indonesia. materials and methods the study was carried out in the mangrove ecosystem located in lembar bay, lembar, west lombok, in february-march 2023 with a research site encompassing an area of 20.49 ha (located at 116°3’-116°4’ e and 8°43’-8°44’ s) (fig 1). this was a quantitative descriptive study that began with the identification of mangroves and sampling of soil found under mangrove stands. soil samples were collected fromunder ten mangrove standsat the research site. these stands represented various species, including avicennia lanata, avicennia marina, bruguiera gymnorrhiza, ceriops decandra, excoecaria agallocha, lumnitzera racemosa, rhizophora stylosa, scyphiphora hydrophyllacea, thespesia populcarapus, and xylocarpus maluccensis. figure 1 mangrove ecosystem of lembar bay, west lombok, indonesia biotropia vol. 30 no. 3, 2023 348 mangrove identification mangroves were identified in situ based on morphological characteristics by referring to the introductory guide to mangroves in indonesia (noor et al. 2006). analysis of soil organic carbon content soil sampling was carried out to a depth of 30 cm around the roots and a slope of 30o using pipes with a diameter of 5 cm and a length of 35 cm. the soil’s carbon organic content was analyzed using the walkley and black method (walkley & black 1934). a soil sample weighing 0.5 g with a size of less than 0.5 mm was placed in a 100 ml volumetric flask. then, 5 ml of 1 n k2cr2o7 was added, and the mixture was shaken. following that, 7.5 ml of concentrated h2so4 was added, and the mixture was shaken and left to stand for 30 minutes. it was then diluted with ion-free waterand the clear solution sample’s absorbance was measured using a spectrophotometer at a wavelength of 561 nm. as a comparison, 0 and 250 ppm standards were made by pipetting 0 and 5 ml of the 5.000 ppm standard solution into a 100 ml volumetric flask with the same treatment as the sample procedure. data analysis soil carbon content the soil’s carbon content was calculated using the following formula (sulaeman et al. 2005): soil c – organic content = ppm curve 500 x correction factor where: ppm curve = the sample content obtained from the curve of therelationship between the standard series contentand its reading after corrected for blanks correctio n factor = 100/(100 % water content) soil carbon stock the soil’s carbon stockwas calculated using the following formula (badan standarisasi nasional, 2011): ct = kd x ρ x % c-organic where: ct = soil carbon stock (g/cm2) kd = soil sample depth or soil depth (cm) ρ = bulk density is the ratio of the soil’s dry weight to its volume (g/cm3) % corganic = value of carbon content percentage (0.47) soil carbon stock in hectare area the soil’s organic carbon content in hectare area was calculated using the following formula (badan standarisasi nasional 2011): c soil (ton c/ha) =ct x 100 where: csoil = soil carbon stock (tons c/ha) ct = soil organic carbon (g/cm2) 100 = conversion factor from g/cm2 to tons c/ha total carbon stock area the total carbon stock area was calculated using the following formula (lugina et al. 2017): ctotals=cn+csoil description: ctotals = total carbon stock (tons c/ha) cn = carbon stocks per hectare in each carbon pool in each plot (tons c) csoil = soil carbon stock (tons/ha) results and discussion mangrove species a total of 10 mangrove stands were identified in lembar bay, namely, avicennia lanata, avicennia marina, bruguiera gymnorrhiza, ceriops decandra, excoecaria agallocha, lumnitzera racemosa, rhizophora stylosa, scyphiphora hydrophyllacea, thespesia populnea, and xylocarpus maluccensis. there were more strands discovered in this study than that of syarifuddin & zulhamran (2012), which found five species, namely, avicennia marina, rhizophora stylosa, rhizophora mucronata, rhizophora apiculata, and sonneratia alba. on the other hand, sukuryadi et al.(2021) found 12 species in a lembar bay mangrove area of 168.9 ha, those species being avicennia alba, avicennia marina, bruguiera cylindrica, ceriop decandra, ceriop tagal, lumnitzera littorea, lumnitzera racemosa, phemphis acidula, rhizhopora potential of carbon sink in mangrove substrates – rahman et al. 349 stylosa, rhizophora apiculata, rhizophora mucronata, and sonneratia alba. these different findings may have occurred due to differences in research areas in the port. this has the potential to disrupt the mangroves’ growth and development due to potential contamination from port activities and the loading and unloading of goods. besides that, the research methodology utilized quadrant points, which limited the collection area of species composition data. soil of c-organic content the largest percentage of soil carbon content was found in the bottom soil of avicennia lanata (1.43% c) when compared to nine other species (table 1). meanwhile, the lowest percentage soil carbon content was found under excoecaria agallochast ands at 0.21% c. the soil carbon content percentage found in lembar bay waslower than that of the soil carbon in the gili meno at the range of 4.85-20.00 %c (rahman & hadi 2021; hilyana & rahman 2022). the high and low soil carbon content found under the lembar bay mangrove stands could generally be caused by the soil fraction size. this is in line with the research results of lee et al. 2014, ati et al.(2015); sidik et al.(2016); and lestariningsih et al.(2018). another supporting factor is the large amount of organic matter sourced from litter weathering mixed with the soil (rahman et al. 2023). in addition, it could be influenced by species density, species age, soil fraction, and each mangrove’s growing zoning position (schwarzer et al. 2016; hilmi 2018; bomer et al. 2020; wang et al. 2020; jannah et al. 2021). this is confirmed by the results reported by (susilowati et al. 2020) that species density can affect litter production, which is one of the main sources of organic material for soil mangrove ecosystems. another factor is the water’s condition, one of which is its ph, which can cause low weathering activity for organic matter by organisms (abdelhakeem et al. 2016; barreto et al. 2016; hilmi et al. 2017; hilmi et al. 2019). the physical factors that affect the waters of lembar bay are wind speed, temperature, and humidity. this is related to the amount of litter production in each mangrove species in lembar bay. another factor is mangrove vegetation zoning, which is always flooded. this causes litter, fruit, and flowers, as the main sources of organic matter, to be affected by currents and carried to the open sea. this contributes to the organic sinking process in lembar bay. greater attention should be directed towards the activities at the lembar portas they have the potential to exacerbate environmental pollution through the changes in the water conditions. this is in line with several other studies on factors that affect carbon conservation in mangrove ecosystems, such as matsui et al. (2015), jones et al. (2016), weiss et al. (2016), martuti et al. (2017), suhendra et al. (2018), asadi et al. (2018), pérez et al. (2018), gao et al. (2019), and kida & fujitake (2020). table 1 soil carbon content under mangrove stands in lembar bay no. mangroves soil carbon content under mangrove stands gross weight dry weight moisture level correction factor absorbance ppm curve % c 1 avicennia lanata 8.00 6.91 15.77 1.16 0.10 61.63 1.43 2 avicennia marina 22.30 21.08 5.78 1.06 0.05 26.91 0.57 3 bruguiera gymnorrhiza 20.00 19.35 3.36 1.03 0.04 20.71 0.43 4 ceriops decandra 17.74 17.09 3.84 1.04 0.02 10.17 0.21 5 excoecaria agallocha 15.57 15.21 2.30 1.02 0.01 5.83 0.12 6 lumnitzera racemosa 17.31 16.53 4.51 1.05 0.03 16.06 0.34 7 rhizophora stylosa 26.14 24.74 5.67 1.06 0.04 23.81 0.50 8 scyphiphora hydrophyllacea 11.34 10.38 9.30 1.09 0.08 47.37 1.04 9 thespesia populnea 16.70 16.15 3.43 1.03 0.02 9.55 0.20 10 xylocarpus maluccensis 14.03 13.15 6.67 1.07 0.04 20.71 0.44 average 16.91 16.06 6.06 1.06 0.04 24.28 0.53 standard deviation 5.220 5.141 3.959 0.041 0.028 17.591 0.409 biotropia vol. 30 no. 3, 2023 350 the soil carbon content percentage (%c) can affect the total accumulation of potential carbon sinks in the research area. this study’s results indicate that the soil carbon content stored 0.0020.066 gc/m2 with an average of 0.020±0.020 gc/m2 (table 2). the soil carbon content in each area is determined by its bulk density and percentage value. the soil carbon content under the avicennia lanata mangrove stands had the largest amount of storage compared to the other nine species. however, it was lower than the results from another study which observed five mangrove stands (avecennia marina, bruguera cylindrica, rhizophora apiculata, lumnitzera racemosa, and excoecaria agallocha) in gili meno, north lombok. that study found an average of 0.573.08 gc/m2 with an average of 1.55±1.000 gc/m2 (hilyana & rahman 2022). several factors can determine the level of total accumulated soil carbon storage. these factors can be influenced by the percentage of soil carbon content, soil specific gravity, sampling depth, bulk density, litter, and topography of the area (mahasani et al. 2015; stringer et al. 2016; rahman et al. 2019; gao et al. 2019; susilowati et al. 2020; dencer-brown et al. 2020). in addition, it has been reinforced by leopold et al. (2013) and pham et al.(2019) stating that species dominance correlates with an uptake of carbon, oxygen, and nutrients from soil and air, and species relationships develop patterns of grouping and species association. it is estimated that the total soil carbon stock of lembar bay found in 10 mangrove stands was 0.20-6.60 tons c/ha with an average of 2.18±2.010 tons c/ha (table 3). this value is smaller than that in some previous research results. the soil carbon stock of the tanjung lesung banten mangrove was discovered to be 27.92 tons c/ha (ati et al. 2015); the mangrove soil carbon stock in dusun pandan sari brebes, central java amounted to 326.46 tons c/ha; table 2 carbon content under each mangrove stand in lembar bay no. mangroves soil carbon content under mangrove stands gross weight (g) dry weight (g) biomass bulk density soil carbon content (% c) soil carbon (gc/m2) 1 avicennia lanata 8.00 6.91 1.09 0.002 1.43 0.066 2 avicennia marina 22.30 21.08 1.22 0.002 0.57 0.029 3 bruguiera gymnorrhiza 20.00 19.35 0.65 0.001 0.43 0.012 4 ceriops decandra 17.74 17.09 0.65 0.001 0.21 0.006 5 excoecaria agallocha 15.57 15.21 0.36 0.001 0.12 0.002 6 lumnitzera racemosa 17.31 16.53 0.78 0.001 0.34 0.011 7 rhizophora stylosa 26.14 24.74 1.40 0.002 0.50 0.029 8 scyphiphora hydrophyllacea 11.34 10.38 0.96 0.001 1.04 0.042 9 thespesia populnea 16.70 16.15 0.55 0.001 0.20 0.005 10 xylocarpus maluccensis 14.03 13.15 0.88 0.001 0.44 0.016 average 16.91 16.06 0.85 0.001 0.53 0.02 standard deviation 5.220 5.141 0.321 0.000 0.409 0.020 table 3 soil carbon stock of mangrove ecosystem in lembar bay no. mangroves soilorganic carbon stockunder mangrove stands soil carbon (% c) soil carbon (g c/m2) soil carbon (tons c/ha) 1 avicennia lanata 1.43 0.066 6.60 2 avicennia marina 0.57 0.029 2.90 3 bruguiera gymnorrhiza 0.43 0.012 1.20 4 ceriops decandra 0.21 0.006 0.60 5 excoecaria agallocha 0.12 0.002 0.20 6 lumnitzera racemosa 0.34 0.011 1.10 7 rhizophora stylosa 0.50 0.029 2.90 8 scyphiphora hydrophyllacea 1.04 0.042 4.20 9 thespesia populnea 0.20 0.005 0.50 10 xylocarpus maluccensis 0.44 0.016 1.60 average 0.53 0.02 2.18 standard deviation 0.409 0.020 2.010 potential of carbon sink in mangrove substrates – rahman et al. 351 the mangrove soil carbon stock of the perancak mangrove forest, jembrana, bali totaled 119.75 tons c/ha; the mangrove soil carbon stock in sungai sembilan, dumai was calculated to be 1,819.31 tons c/ha (handoyo et al. 2020); the mangrove soil carbon stock in tambakbulusan village, demak, central javawas 57.74 tons c/ha (susilowati et al. 2020); and the soil carbon stock in the mangrove ecosystem of gili meno, north lombok was found to be 154.62±99.78 tons c/ha (hilyana & rahman 2022). based on the potential value of carbon sinks and storage in mangrove ecosystems calculated in several places in indonesia, there is a greater potential for mangrove ecosystems to store carbon than tropical forests. this can be seen in the results of studies by daud et al. (2015), raynaldo et al. (2022), and yaqin et al. (2022). a report by alongi (2020) also supports this finding, stating that, globally, mangrove ecosystems have a total carbon stock of 738 ± 27.9 mgc/ha. it has also been reported that the largest potential carbon sink is in the soil, and it is equivalent to 77% of the total carbon stock although mangrove forests only make up 0.2% of this stock compared to forestson land (hamilton & casey 2016). the soil carbon content found under 10 mangrove stands in lembar bay was lower than the soil carbon stock under five mangrove stands on gili meno. the mangroves include rizophora apiculata (307.96 tons c/ha), avicennia marina (197.16 tons c/ha), excoecaria agallocha (114.31 tons c/ha), lumnitzera racemosa (59.90 tons c/ha), and bruguiera cylindrica (57.17 tons c/ha). the average estimated total of soil carbon stock found in 20.49 ha of the mangrove area studied was 44.67 tonsc, which is equivalent to 263.69 tonsc in a 120.96 ha area of mangroves in lembar bay (table 4). if the entire 20.49 ha area wascovered by avicennia lanata, it could contribute 135.23 tonsc of carbon storage. the lowest soil carbon stock capacity was found from excoecaria agallocha at 4.10 tonsc in a mangrove area of 20.49 ha. table 4 carbon pool in mangrove soil in lembar bay no. mangroves carbon pool in mangrove soil in lembar bay soil carbon (tons c/ha) researcharea (ha) carbon pool (tonsc) 1 avicennia lanata 6.60 20.49 135.23 2 avicennia marina 2.90 20.49 59.42 3 bruguiera gymnorrhiza 1.20 20.49 24.59 4 ceriops decandra 0.60 20.49 12.29 5 excoecaria agallocha 0.20 20.49 4.10 6 lumnitzera racemosa 1.10 20.49 22.54 7 rhizophora stylosa 2.90 20.49 59.42 8 scyphiphora hydrophyllacea 4.20 20.49 86.06 9 thespesia populnea 0.50 20.49 10.25 10 xylocarpus maluccensis 1.60 20.49 32.78 average 0.53 20.49 44.67 standard deviation 0.409 0.000 41.182 biotropia vol. 30 no. 3, 2023 352 conclusion the highest soil carbon stock in the mangrove ecosystem of the lembar harbor was found in the subsoil of avicennia 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febrianto s. 2022. estimasi serapan karbon pada kawasan mangrove tapak di desa tugurejo semarang [estimated carbon uptake in the tapak mangrove area in tugurejo village, semarang]. bul oseano mar. 11(1): 19-29. https://doi.org/10.14710/ buloma.v11i1.38256 4. shenly marie tobias gazo et al.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 biotropia vol. 29 no. 2, 2022: 161 170 doi: 10.11598/btb.2022.29.2.1690 161 monitoring forest area change using quickbird sri endayani1, andrew stefano2*, fathiah3, purbawati4 and ida rosanti4 1faculty of agriculture, forestry study program, universitas 17 agustus 1945, samarinda 75123, indonesia 2department of engineering and informatics, politeknik pertanian negeri samarinda, samarinda 75242, indonsia 3department of forest management, politeknik pertanian negeri samarinda, samarinda 75242, indonsia 4faculty of engineering, universitas nahdlatul ulama kalimantan timur, samarinda 75251, indonesia received 16 november 2021/accepted 14 january 2022 abstract a study was conducted to compare the urban forest management in three urban forests in samarinda city. the application of gis (geographic information system) is one of the alternatives to conduct a variety of processes such as: providing geographical information system, identifying the areas of urban forests in samarinda, helping to plan the process of map digitalization and performing overlay process. the main method used for the data analysis process on the map was the overlay process data analysis technique. the research findings showed that: 1) the appointment of urban forests as the initial step of urban forest development needed more implementation from the government; 2) the urban forest determination needed more socialization to the owner of the urban forest and the public in 1992 and 2019; 3) the urban forests needed more management. there were some similarities and differences in the management of urban forests in the three study locations. the similarities among the three locations were that the three locations had already met the minimum standards of one urban forest location even though there was still one location outside of these three locations which did not meet the minimum standard. the differences were in managing the urban forests. these differences indicated that the urban forest policy was not fully implemented in samarinda city. keywords: comparison, geographic information system, management, policy implementation, urban forests introduction the increasingly rapid growth of population and development nowadays causes a lot of changes in the appearance of samarinda city (angel et al. 2019; caddeo et al. 2019; cao et al. 2019). the rapid development has turned most of the green spaces in samarinda city into shopping centers, stores, housing areas, mining areas and other places for anthrophogenic activities (tunas & maadji 2018). these changes caused extreme environmental degradations and destructions (carrer et al. 2018; daoed et al. 1997; deng et al. 2020). the city’s development emphasizes on the aspect of economic growth (diodato & bellocchi 2020; fernándezguisuraga et al. 2019; guo et al. 2020), leading to several environmental problems (hafeez & khan 2012; hong et al. 2017; de jager et al. 2019), including water crisis, floods and pollution resulting from the traffic and the decreasing number of green spaces in samarinda city (daoed et al. 1997). certainly, the current conditions of samarinda city are no longer compatible with the city’s slogan a “tepian” city, which is the abbreviation of ‘teduh’ (shady), ‘rapi’ (tidy), ‘aman’ (safe), and ‘nyaman’ (comfortable). environmental degradation, of course, should be ceased to continue (lagacherie et al. 2020; lister & leites 2018; liu et al. 2017). one of the solutions to overcome the environmental degradation is by applying environmentally sound developments through urban forest development (liu et al. 2019; morales & perry 2017; nguyen et al. 2019). the decree of samarinda mayor number 178/hk-ks/2017 showed that the size of the existing urban forests is 690.237 ha of the size of samarinda city which is 718 km2. clearly, the size of the urban forests is still far from sufficient to meet the minimum 10% of the urban area based on *corresponding author, email: andrew.stefano@politanisamarinda.ac.id biotropia vol. 29 no. 2, 2022 162 the regulation stated in article 8 of the government regulation number 63 year 2002 on urban forests (windusari et al. 2017). in order to meet the 10% minimum percentage, the samarinda city should have 7,180 ha of urban forests. one of urban forest locations in samarinda city owned by pt. gani mulya is only 0.097 ha and this is not in line with the article 8 of the government regulation number 63 year 2002 on urban forests which stated that the size of urban forest in one compact stretch is at least 0.25 ha. unfortunately, this regulation also mentioned about the adjustment of the urban forest size with the local condition of each region (abdullahi et al. 2017), which seems to give a room for the local government to ignore the existence of urban forests. most city planner is still unaware of the significance of urban forests (nuddin et al. 2019; podlaski 2019; reza et al. 2020). if the regulation stated in the government regulation number 63 year 2002 on urban forests is properly followed, then all of the existing environmental problems faced by the government of samarinda city may be minimized (hong et al. 2017). urban forest as an open green space or ruang terbuka hijau (rth) should actually get attention from the government in order to make samarinda city an environmentally-sound city (tunas & maadji 2018; viccaro et al. 2019; wiggins et al. 2019). the population of samarinda deserves to have a comfortable, healthy and aesthetic environment (windusari et al. 2017; zhao et al. 2020). the city needs to be protected from a variety of negative environmental impacts (tunas & maadji 2018; viccaro et al. 2019; wiggins et al. 2019). among techniques to achieve a better quality of environment is by increasing the quality and quantity of city greeneries suitable with the city’s urban forests (hafeez & khan 2012). urban forest as an element of rth is expected to overcome environmental problems in the urban areas by absorbing pollutions caused by the anthrophogenic activities (silveira et al. 2019; sinha et al. 2019; soma & kubota 2018). the development of urban forest in big cities in indonesia indicates the policy makers’ awareness on environmental issues (sameen et al. 2019; schwede et al. 2018; shang et al. 2020). the issues of urban forest need a special attention from the government considering the rapid development in samarinda city which causes adverse impact on the environment and the decreasing number of green spaces (nuddin et al. 2019; podlaski 2019; reza et al. 2020). following the changing of times and the rapid development of technology, there are many methods to retrieve information on location in the form of map, one of which is using gis (geographic information system) (liu et al. 2019; morales & perry 2017; nguyen et al. 2019). by using gis we can capture a map of a location which provides detailed information (lagacherie et al. 2020; lister & leites 2018; liu et al. 2017). with the existence of geographic information system, it is expected that people will know more about the urban forests available surrounding the samarinda city. this study aimed to obtain comprehensive understanding on urban forests as the basis of policy making in developing urban forests in samarinda city (hafeez & khan 2012; hong 2017; de jager et al. 2019). the interview conducted in this study focused on the urban forest areas of samarinda city, guard posts, and standardization of forest preservation (balachandran 2017). this research applied literature study by collecting and studying issues related to the geographic information system of forestry data (relevant institutions) (sulistyo et al. 2017). basic theoretical framework public policy is a specific goal or a series of specific principles or actions taken by the government in a certain period of time with regard to one subject or as a response toward a crisis condition (wahab 2008). in addition, rose (1990) defined public policy as a series of less or more related activities and their consequences to the people concerned rather than a separate decision. furthermore, van meter and van horn (1975) defined policy implementation as an action taken by individuals or officials or groups of governmental or private organizations directed to achieve goals which have been stated in the policy decision. from this definition it can be identified that policy implementation covers three aspects, namely: 1) goals and targets of policy; 2) activities or policy goals and objectives; 3) outcome of the activities (agustino 2006). public policy has goals, monitoring forest area change using quickbird – sri endayani et al. 163 objectives and is behavior-oriented. public policy refers to what the government really acts on, not merely a statement or desired target of action. public policy is a directed target, meaning that the action is followed by an actor or a number of actors working together to solve problems. implementation study is an analysis on the policy implementation process. in its practice, policy implementation is a considerably complex process; even it usually has political contents because of intervention of various interests. when an issue which addresses common interests is considered necessary to be regulated, then the formulation of the issue becomes a public policy which needs to be implemented, prepared and approved by all the authorized officials. when the public policy has been stated as a public policy, then it turns into a law which needs to be carefully observed. policy in urban forest management urban development is usually reflected by the physical development of a city which is considerably determined by the existing facilities and infrastructure. the past and current urban development tends to minimize the open green spaces and to eliminate the face of nature. the condition of urban environment develops economically, but degrades ecologically. indeed, the ecological balance of urban environment has the same significance as the development of economic value in urban areas. this condition creates unharmonious relationship between urban community and its environment. being aware of the inharmony and considering the negative environmental impacts, there should be an effort to improve the environment through urban forest management. urban forest is one of the open green spaces. its existence functions as hydrological system, creating micro climate, maintaining oxygen (o2) and carbon dioxide (co2) balance, reducing pollutant and absorbing noise. in addition, urban forests also function to add the aesthetic values and the beauty of the city, thus giving positive impacts on the quality of environment and the life of the community (sibarani 2003). there are some municipal government policies which regulated the appointments of some urban forest locations. samarinda city has an urban forest policy which is stated in the samarinda mayor’s decree number 178/hk-ks/2019. the detailed regulation which addresses urban forests in the form of local regulation has not been made until now in samarinda city. however, the regulation governing the management of urban forests can be seen in the higher-order regulation, namely the government regulation number 63 year 2002 on urban forests. in addition, there is also the minister of forestry regulation number p.71/menhutii/2009 on the guidelines for urban forest management. article 4 of the government regulation number 63 year 2002 on urban forests stated that urban forest management covers: 1) appointment; 2) construction; 3) determination; and 4) management. however, this research only covered urban forest management which focused on the appointment of urban forests which included the sizes and the locations of the urban forests in samarinda. the highlight of this study were: 1) the determination of urban forests and 2) urban forest management which includes maintenance, protection and security, utilization, monitoring and evaluation. the policy implementation policy of urban forests were analyzed in 3 locations based on their gradient distances from downtown, namely in the area of city hall, lempake village and samarinda botanical garden of mulawarman university. materials and methods data retrieval this study used primary data collected from field observations and systematic recording using gps garmin 60. secondary data were collected from various kinds of relevant literatures and social phenomena, namely administrative maps of samarinda city, vegetation, plantations, and contours. data processing administrative maps, vegetation, plantations and contours were superimposed over the samarinda urban forest map (bpkh region iv) (first process). field checking was conducted to biotropia vol. 29 no. 2, 2022 164 obtain the most up-to-date information on the urban forest (second process). subsequently, the results of the two processes were combined. the image correction was carried out using quick bird software to reduce geometric, radiometric and atmospheric problems (fig. 1). the image analyses were subsequently carried out, by first making criteria and scoring assessments in tabular form as part of the data preparation process. visual interpretation of upto-date urban forest maps was used to get raster data in the form of land-use conversion. figure 2 presents the flowchart of samarinda urban forest map preparation. the samarinda's urban forest vegetation was determined based on the results of image analyses. results and discussion the determination of one particular area as an urban forest can be in the form of designation within the urban area and it can be a piece of land owned by the government or owned privately with a land ownership right. the designated urban forest location is a part of the open green space of the city. the importance of urban forest functions is regulated in the government regulation number 63 year 2002 on urban forests in articles 7, 8 and 9. the designation is based on the programs of the government of samarinda city through bapedalda (local board of environmental impact management). in 2001 2019 the government of samarinda city through bapedalda had planned, prepared and implemented urban forests which was funded by dakdr budget (special fund allocation fund for forestation). in 2003 the city government and samarinda local house of representative enacted the local regulation number 28 year 2003 on protected area in samarinda city. even though there is no obvious regulation concerning urban forests, the government of samarinda city keeps continuing the development of urban forests based on the local regulation number 28 year 2003. through bapedalda, the samarinda city planned, prepared, and implemented urban forests using dak-dr budget (special fund allocation – fund for forestation). figure 1 quickbird satellite image of samarinda city monitoring forest area change using quickbird – sri endayani et al. 165 figure 2 flowchart of samarinda urban forest map preparation after planning, preparing and implementing urban forest in samarinda, bapedalda provided a recommendation to the government of samarinda city to issue a mayor decree concerning the location of urban forests in samarinda city. then, the decree of samarinda mayor number 178/hk-ks/2005 on the determination of urban forest locations in samarinda city was issued. there were 25 locations which were appointed as urban forests in the urban area of samarinda. the decree of samarinda mayor number 178/hk-ks/2005 showed that the area of urban forests was 690.237 ha with the percentage of 0.96% from the total urban area. samarinda city has the area of 718.00 km2, so that to meet the minimum 10% of the urban area size, samarinda should have urban forests with the size of 7,180 ha. this means that samarinda still requires 6,489.763 ha or 9.04% to fulfill the required minimum 10% of the total urban area. in addition, the decree also mentioned one of the urban forests, namely pt gani mulya with the extent of 0.097 ha. the size of urban forest owned by pt gani mulya does not meet the standard based on the criteria stated in article 8 section (2) that the size of urban area in one compact stretch should be at least 0.25 ha. furthermore, the distribution of urban forest in each subdistrict in samarinda is not equal because there are still 2 subdistricts from the total of 10 subdistricts which do not have an urban forest in their district areas, such as palaran and sungai pinang subdistricts. each subdistrict also still has a significant shortage from the required minimum total area. the criteria of urban forest in each subdistrict is presented in table 1. table 1 criteria of urban forest area for each subdistrict no subdistrict urban forest area (ha) area (km2) minimal forest area (ha) shortage (ha) percentage by the zone (%) 1. palaran 221.29 2,212.9 -2,212.9 0 2. samarinda ilir 6 17.18 171.8 -165.8 0.35 3. samarinda kota 11.56 11.12 111.2 -99.64 1.04 4. sambutan 187 100.95 1,009.5 -822.5 1.85 5. samarinda seberang 1.5 12.49 124.9 -123.4 0.12 6. loa janan ilir 8.697 26.13 261.3 -252.603 0.33 7. sungai kunjang 69.75 43.04 430.4 -360.65 1.62 8. samarinda ulu 8.98 22.12 221.2 -212.22 0.41 9. samarinda utara 306.75 229.52 2,295.2 -1,988.45 1.37 10 sungai pinang 34.16 341.6 -341.6 0 samarinda city 690.237 718.00 7,180 -6,489.763 0.96 source: processing data from the mayor’s decree number 178/hk-ks/2017on the determination of some urban forest locations in samarinda city. map of samarinda city samarinda city forest area overlay samarinda city + urban forest area distribution of forest types map of samarinda city forest area overlay biotropia vol. 29 no. 2, 2022 166 city hall urban forest the city hall urban forest was appointed as an urban forest in 1992 through the mayor’s decree number no. 224 year 1992, followed by the mayor’s decree number 178/hk-ks/2019. the city hall environment was appointed as an urban forest after meeting the requirements, and in this case, the city hall urban forest has an area of 7.64 ha. the area is more than enough to fulfill the minimum requirement of one location to be selected as an urban forest, namely 0.25 ha. the city hall urban forest is located on the state-owned land whose land status and ownership right belongs to the government of samarinda city. land ownership is proven with a land certificate number: p-24, number: 305/1981 which was issued by agrarian office of samarinda city on 29 june 1981. lempake urban forest the appointment of lempake urban forest was based on the land status owned by the municipal government since the status of the village was changed into kelurahan. this location was appointed by bapedalda in 2004 by involving the local community. lempake urban forest has an area of 3.5 ha and this size has met the requirement of a minimum size of 0.25 ha for one location of urban forest. the determination of urban forests policy in the determination of urban forest has been issued twice by the government of samarinda city. the first one was the mayor’s decree number 224 year 1992 and the second one was the mayor’s decree number 178/hkks/2019. the determination of urban forest in 1992 was issued by the cleaning and landscaping agency and the determination of urban forest in 2017 was issued by the bapedalda which is now recognized as blh (environmental agency). the urban forests which were stated in the mayor’s decree number 178/hk-ks/2019 in samarinda city were determined after the bapedalda accomplished the procedure of planning, preparation, and implementation of urban forest. the documented procedures was then proposed to the government of samarinda city in order to issue a letter of determination for the locations of urban forests in the region of samarinda city. from 1992 to 2005 there was an increase in urban forests both in their sizes and their total number. in 1992 samarinda had only 12 locations of urban forests with an extent of 218.177 ha, while in 2019 the number of urban forests increased up to 25 locations with an area of 690.237 ha. this indicates that samarinda has an additional 13 urban forests and an additional area of 472.06 ha. unfortunately, the determination policy for urban forests has not been reviewed at least once in two years following the rule stated in point three of the mayor’s decree number 178/hk-ks/2019. city hall urban forest the area of city hall was first designated as an urban forest on 17 december 1992 through the mayor’s decree number 224 year 1992 with an area of 6.9 ha. this determination was then renewed by the mayor’s decree number 178/hk-ks/2019. in the decree number 178/hk-ks/2019 the area of city hall had an additional area which was allocated for urban forest, from an area of 6.9 ha to 7.64 ha. the determination of the city hall area as an urban forest was well-socialized to the public because of its strategic location in the middle of samarinda city. lempake urban forest the determination of kas lempake as an urban forest was not known by the public or the village government. even though the location status is on the state-owned land, the determination of the urban forest should have been informed to the public, especially to the community where the urban forests are located. the fact that the public do not know the existence of the urban forest is caused by some factors. first, the appointment of kas lempake as an urban forest only involved some people following the rapid growth of population. second, the determination of urban forest had lasted for a long time without any socialization from the former government. third, there have been a lot of replacements of lurah and personnel in the lempake village since the urban forest determination. last, there is no activity initiated by the government in relation to to the management of kas lempake. monitoring forest area change using quickbird – sri endayani et al. 167 urban forest management urban forests are not managed uniformly because the managements are only conducted in several locations which are only owned by the government such as the area of city hall, segiri softball field, garden tombs of heroes, and samarinda city library, which are easily accessed by the government because these locations are in the downtown area. one of the efforts made by the government in urban forest management is by re-registering the existing urban forests. the result of re-registration can be seen table 2. the city still needs 9.12%. the local office of agriculture, plantations and forestry of the samarinda city (distanbunhut) also makes a plan for locations which are going to be appointed into urban forests. after doing reregistration on the size of urban forest stated in the mayor’s decree number 178/hk-ks/2019, the area of urban forest was decreased by 57,167 ha from the original 633.07 ha. therefore, the percentage of urban forest nowadays is only 0.88% from the previous percentage of 0.96%, namely 71,800 ha. the minimum requirement for an urban forest is 10% of the total urban area. this means that samarinda still needs urban forests. the planned locations include the center for dipterocarp, land of municipal government in makroman, polytechnic of agriculture campus (poliagro samarinda), and kaltim cultural park. the government regulation number 63 year 2002 and the regulation of the minister of forestry number p.71/menhut-ii/2009 which is supported by the mayor’s decree number 178/hk-ks/2019 is sufficient to be the basis for urban forest management. table 2 re-registration of urban forests in samarinda city no. city forest location large sk 2019 (ha) re-registration data (ha) 1. smu 10 melati 5 5 2. krus 300 300 3. tanah pemkot 5 5 4. hutan kota belakang rumah jabatan walikota 1.75 1.8 5. asih manuntung 0.25 0.25 6. pesantren hidayatullah 1 0.38 7. tanah pemkot di makroman 167 167 8. tanah pertanian terpadu 20 20 9. kas desa lempake 3.5 3.5 10. fakultas pertanian unmul 6.5 3.84 11. pesantren nabil husein 9.75 9.75 12. pesantren syachona cholil 0.25 0.25 13. rumah potong hewan 2 5 14. hotel mesra 2.3 0.7 15. jalan pembangunan voorfo 0.48 2.6 16. lingkungan balai kota 7.64 3.26 17. lingkungan lapangan softball gor segiri 0.5 0.25 18. perpustakaan kota samarinda 0.6 0.5 19. ujung timur jembatan mahakam 1.5 2 20. pt. hartaty 60 21. pt. gani mulya 0.097 2.75 22. pt. sumber mas 85 85 23. pt. sumalindo 3.6 24. taman makam pahlawan 0.52 1.04 25. pt. kiani (teluk cinta di selili) 6 13.2 total 690.237 633.07 source: agriculture, farming and forestry service office of samarinda city (2019). biotropia vol. 29 no. 2, 2022 168 city hall urban forest the urban forests which are located on the state-owned lands are managed by three government institutions at the same time, i.e., the agriculture, farming and forestry service, the cleaning and landscaping agency, and the city planning service. the forms of management include providing seedlings and plants. the construction of shopping center next to the urban forest location is believed not to use the land of the urban forest because it is still within the border of the land owned by the owner of the building. the city hall urban forest is one of the urban forest locations having decreasing in area size because of the building construction and parking lots which take the green space in this location. lempake urban forest the government policy on the management of urban forests which are located in the land owned by kas lempake has not been implemented. this is not in line with the regulation of minister of forestry number p.71/menhut-ii/2009 in article l32 through article l43, which technically governs the urban forest management. kas lempake village is under the responsibility of the government, but until now this urban forest location has never been managed properly. the fact that this urban forest is not managed by the community is caused by the status of the location that is owned by the government. the municipal government has never formed a coordination with the local village to involve the community in the management of urban forests. conclusion there are some similarities and differences in managing urban forests in samarinda city. their similarities can be found in the three research locations in which they have met the required minimum standard of one location, i.e., 0.25 ha. in addition, the urban forests also provide the same benefits to the surrounding environment. the city hall urban forest was established as an urban forest in 1992 and it was extended in 2017, while lempake urban forest and the urban forest of universitas mulawarman botanical garden were just established in 2017 through the mayor’s decree number 178/hk-ks/2019. those urban forests have differences in the status of land ownership. the city hall urban forest and lempake urban forest are located on the state-owned lands, while the urban forest of universitas mulawarman botanical garden is located on the land with a land ownership right. acknowledgments this research was supported by the ph.d. grant of the indonesia endowment fund for education (lpdp-budi dn) of the ministry of finance of the republic of indonesia. references abdullahi mg, khairul m, kamarudin a, umar r, endut a. 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natural sciences, mulawarman university, samarinda 75119, indonesia. 4laboratory of plant anatomy and systematics, department of biology, faculty of mathematics and natural sciences, mulawarman university, samarinda 75119, indonesia article higlights • this research is the first study to provide data on the diversity of citrus spp. from north penajam paser regency (ikn) area, east kalimantan for further development and breeding program. • this research offers new information on the distribution of citrus spp. from the north penajam paser regency (ikn) area, east kalimantan. • this research provides information on the phenetic relationships among citrus spp. from the north penajam paser regency (ikn) area, east kalimantan, and the morphological characteristics that play the most significant role in their clustering. abstract citrus spp. is a locally grown fruit in indonesia with high diversity and widespread distribution in the country. however, the diversity and similarities among citrus spp. from north penajam paser regency, east kalimantan, have not been previously studied. morphological characteristics were used in this study to provide data on the diversity and similarities of citrus spp. from north penajam paser. the data provided served as a primary resource for further development and breeding. additionally, observation, sampling, morphological data characterization, and scoring were carried out before the data was analyzed. this study identified 35 accessions of citrus spp., including citrus × limon (l.) osbeck "jeruk lemon", citrus reticulata blanco "jeruk siam", citrus × aurantiifolia (christm.) swingle "jeruk nipis", citrus × aurantium l. "jeruk manis", citrus maxima (burm.) merr. "jeruk bali", citrus × microcarpa bunge "jeruk kalamansi", and citrus × aurantium l cv. sweet orange group "jeruk sunkist", distributed across penajam, sepaku, babulu, and waru subdistricts. the dendrogram divided the species into two large clusters. cluster a was based on characteristics, such as tree habitus, cordate leaf apex, entire leaf margin, pink mesocarp color, and the absence of seeds. meanwhile, cluster b was characterized by shrub habitus, acute and rounded leaf apex, crenate leaf margin, white to orange mesocarp color, and numerous seeds in the fruit. the diversity indicated the high potential value of citrus spp. from north penajam paser for further development through a plant breeding program. keywords: citrus spp, diversity, kalimantan, morphological characters article information received 14 february 2023 revised 17 november 2023 accepted 15 march 2024 *corresponding author, e-mail: oktavianingsihlinda@yahoo.com, lindaoktavianingsih@fmipa.unmul.ac.id research article introduction indonesia is a mega biodiversity country, with an estimated 30,000 to 35,000 plant species dispersed across the landscapes (von rintelen et al. 2017). the rich diversity is primarily attributed to several factors, including the equatorial proximity resulting in abundant rainfall, geological topographic variations, unique biogeographical conditions, diverse ecological conditions, and various climatic influences (lohman et al. 2011). the distribution of numerous islands also leads to distinctive variations in the characteristics of species (schouppe et al. 2017; dart et al. 2012; herlihy & eckert 2005). this includes plants on kalimantan island, which is the third largest island in the world with enormous biodiversity. https://doi.org/10.11598/btb.2024.31.2.1872 mailto:oktavianingsihlinda@yahoo.com biotropia vol. 31 no. 2, 2024 158 studying plant diversity and classification is critical in facilitating the development of economically valuable plant varieties or cultivars (swarup et al. 2020). this field of study provides essential genetic information for the resources that form the foundation of plant breeding programs (shen et al. 2018). furthermore, examining plant diversity and similarity limits the analysis of morphological characteristics, including organ shape, size, color, and surface (balduzzi et al. 2017). morphological characterization entails observing external features to assess and establish relationships among individuals, with each character assigned a value that can serve as a marker for assessing these relationships (jensen 2009). plants exhibit preferential traits that have evolved in response to various environmental conditions. therefore, the study of morphological variation is essential as it shows the relationship with these environmental factors. plants also possess an adaptive mechanism known as phenotypic plasticity, enhancing fitness and resilience in changing environmental conditions (chevin et al. 2010; valladares et al. 2014; ayala et al. 2020). citrus  spp. spread across malay archipelago, which includes indonesia (liu et al. 2012; langgut 2017; sofiyanti et al. 2022). these species are a valuable source of germplasm due to remarkable genetic diversity (luro et al. 2011; yu et al. 2018; goh et al. 2022). citrus spp. are also significant in local communities, serving as a vital source for various essential commodities. these include food, medicinal applications, and cosmetic formulations (marti et al. 2009; vijaylakshmi & radha 2015; samarina et al. 2021; galovicová et al. 2022; sapkota et al. 2022; musara et al. 2023). among the manifold applications, the fruits and leaves are frequently studied for potential cosmetics use, aromatic properties, and as indispensable ingredients in culinary and beverage preparations (palazzolo et al. 2013; khettal et al. 2016; dosoky & setzer 2018; namani et al. 2018; taufiq et al. 2019; raghavan & gurunathan 2021). citrus spp. has been considerably cultivated across various countries, with a remarkable presence in south east asia. for instance, several studies found numerous distinct  species in indonesia, with many being hybrid products (penjor et al. 2014; susandarini et al. 2016; yulian et al. 2021; sofiyanti et al. 2022; hardianto et al. 2023). "jeruk mandarin", which belongs to the  citrus reticulata  blanco, represents the most prevalent variety documented in indonesia (yulianti et al. 2020; luro et al. 2023). "jeruk besar" refers to pomelos and is classified as  citrus grandis  (l.) osbeck (ollitrault et al. 2020; sofiyanti 2022), while "jeruk sitrun" is identified as  citrus medica (l.) burm.f. (benedetto et al. 2021). budiyati et al. (2016) carried out characterization to determine the diversity of citrus spp. on kalimantan island, specifically in east kalimantan, along with studies conducted in several other regions in the archipelago, such as southeast maluku, north maluku, and east java. although the study in east kalimantan was carried out generally in each region and was not specific for north penajam paser regency, considerable variations were observed. north penajam paser regency was designated as the national capital of the republic of indonesia (ibu kota nusantara) following law number 3 of 2022 concerning the national capital (uu ikn) and the regulatory database (jdih bpk ri) on 15 february 2022. this caused a massive change in the region, including the reduction and loss of plant germplasms due to the large number of new arrivals and simultaneous development that damaged the environment. the impact of these changes dramatically affected the use of plants. therefore, this study aimed to provide phenetic diversity and similarity data for citrus spp. from north penajam paser (ikn) for the benefit of local communities. the results are expected to serve as fundamental data for future studies. materials and methods observation and sampling this study was conducted from september 2021 to february 2022 in the north penajam paser regency, east kalimantan (fig. 1). citrus accessions were selected in this area and the observation site was selected based on potential location-based information provided by local communities. sampling was carried out using the roaming method with a purposive sampling technique (rugayah 2004). leaves, flowering parts, fruits, and seeds that were observable were analyzed. all essential data were recorded using a camera to support the characterization. analysis was carried out at the laboratory of plant anatomy and systematics, faculty of mathematics and natural sciences, mulawarman university, samarinda, east kalimantan province. diversity of citrus spp. from north penajam paser regency (ikn) oktavianingsih et al. 159 figure 1 location of collection of citrus spp. accessions in north penajam paser regency characterization and scoring morphological characterization was conducted by analyzing the shape, length, texture, and color of the organs. a total of 50 critical characteristics were selected for characterization and converted into numerical values (table 1) to assess diversity and similarity. the matrix score used in this analysis was based on zufahmi and nurlaila (2018) and ipgri (1999). each sample was assigned an accession number for identification purposes, and the identification process was carried out up to the species level. table 1 conversion of morphological characters citrus spp. for scoring no characters scoring 1 trunk ratio/rootstock diameter 0: 0 < 1 cm; 1: 1 = 1 cm; 2: > 1 cm 2 scion trunk surface 0: smooth; 1: grooved 3 tree shape 0: ellipsoid; 1: spheroid; 2: obloid 4 branch density 0: sparse; 1: medium; 2: dense 5 habitus 0: shrub; 1: tree 6 stem color 0: brown; 2: green 7 stem height 0: < 5 cm; 1: ≥ 5 cm 8 stem shape 0: perpendicular; 1: perpendicular cylindrical 9 stem surface 0: thorny; 1: flat 10 spine length 0: ≤ 5 mm; 1: 6-15 mm; 2: 16-40 mm; 3: > 40 mm 11 spine shape 0: curved; 1: straight 12 shoot tip color 0: green; 1: purple; 2: black; 3: orange 13 shoot tip surface 0: glabrous; 1: intermediate; 2: pubescent 14 leaf type 0: sole; 1: complex 15 leaf apex 0: acute; 1: obtuse; 2: rounded; 3: cordate; 4: sagittate 16 leaf base 0: acuminate; 1: truncate 17 leaf margin 0: entire; 1: crenate; 2: sinuate 18 leaf surface 0: rough; 1: smooth 19 leaf shape 0: ovate; 1: elliptical 20 leaf length 0: < 9 cm; 1: ≥ 9 cm 21 leaf stalk 0: < 1.5 cm; 1: ≥ 1.5 cm biotropia vol. 31 no. 2, 2024 160 no characters scoring 22 leaf width 0: < 5 cm; 1: ≥ 5 cm 23 green intensity 0: light; 1: medium; 2: dark 24 leaf lamina 0: elliptic; 1: ovate; 2: obovate; 3: lanceolate; 4: orbicular; 5: obcordate; 6: oblong 25 petiole wings 0: absent; 1: present 26 petiole width 0: narrow: 1: medium; 2: broad 27 petiole shape 0: obcordate; 1: obdeltate; 2: obovate; 3: linear 28 fruit shape 0: spheroid; 1: ellipsoid; 2: pyriform 3: oblique; 4: obloid; 5: asymmetric 29 fruit base shape 0: necked; 1: convex; 2: truncate; 3: concave; 4: concave collared; 5: collared with neck 30 fruit apex shape 0: acute; 1: rounded; 2: truncate; 3: mammiform 31 fruit diameter 0: < 33 cm; 2: ≥ 33 cm 32 fruit skin (epicarp) 0: green/yellow; 1: red; 2: dark yellow; 3: orange; 4: dark yellow; 5: dark green; 6: light green 33 fruit surface texture 0: rough; 1: smooth; 2: papillate; 3: pitted 4: bumpy; 5: grooved 34 fruit peel thickness 0: dense; 1: light 35 mesocarp color 0: pink; 1: yellow; 2: white; 3: green; 4: orange; 5: light orange 36 fruit taste 0: sweet; 1: sour 37 vesicle length 0: short; 1: long 38 nature of oil glands 0: very weak; 1: conspicuous; 2: strongly conspicuous 39 crown color 0: white; 1: yellowish; 2: violet 40 stamen length 0: < 1 m; 1: ≥ 1 cm 41 number of stamens 0: < 4 each petal; 1: 4 each petal; 2: > 4 each petal 42 amount of pollen 0: low; 1: normal; 2: high 43 flower stalk length 0: < 1 cm; 1: > 1 cm 44 flower type 0: hermaphrodite; 1: male; 2: female 45 number of seeds 0: low; 1: high 46 seed shape 0: fusiform; 1: clavate; 2: cuneiform; 3: ovoid; 4: semi deltoid; 5: spheroid; 6: semi-spheroid 47 seed surface 0: smooth; 1: wrinkled; 2: hairy 48 seed color 0: white; 1: beige; 2: yellowish; 3: green; 4: brown 49 cotyledon color 0: white; 1: bright yellow; 2: bright green; 3: white and green; 4: green; 5: dark green; 6: violet 50 presence/absence of seeds 0: absent; 1: present source: ipgri (1999); zufahmi and nurlaila (2018). data analysis morphological characters were analyzed qualitatively and quantitatively. the qualitative analysis entailed using descriptive methods, where specimens were directly observed and matched with references (ipgri 1999; irsyam & chikmawati 2015; zufahmi & nurlaila 2018), while quantitative analysis was carried out by measuring specific dimensions with a ruler, respectively. the process began with character selection as operational taxonomy units (otus), with each chosen character assigned a standardized score (table 1). all 50 selected characters were used for observation and converted into diversity of citrus spp. from north penajam paser regency (ikn) oktavianingsih et al. 161 standardized values. subsequently, the data were entered into mvsp 3.1 (multivariate statistical package) software, which was used to generate the dendrogram and similarity index. the dendrogram was constructed using the unweighted pair group with arithmetic mean (upgma) method with the euclidean coefficient. results and discussions diversity in this study, citrus spp. was found in penajam, sepaku, babulu, and waru at various altitudes, ranging from 1 masl to 40 masl (meters above sea level). there were 35 identified accessions classified into seven species of citrus, namely citrus × aurantium l. “jeruk manis”, citrus × aurantiifolia (christm.) swingle “jeruk nipis”, citrus × microcarpa bunge “jeruk kalamansi”, citrus reticulata blanco “jeruk siam”, citrus maxima (burm.) merr. “jeruk bali”, citrus × limon (l.) osbeck “jeruk lemon”, and citrus × aurantium l. cv. sweet orange group (table 2). morphological characteristics the characterization of 35 citrus spp. accessions was conducted using 50 morphological characters based on the ipgri descriptor for citrus characterization guidebook (1999), zulfahmi (2018), and irsyam and chikmawati (2015). these characters revealed variations in leaves, fruits, and seeds. however, due to the unavailability of flowers in some accessions, the characterization primarily focused on fruit and leaf characters. this approach was based on a study conducted by susandarini et al. (2013), which also emphasized the morphological character analysis of leaf and fruit characteristics for the same reason. leaf (folium) morphological variations in leaves included variations in leaf shape and color. leaf shapes varied from ovate, obovate, lanceolate, and orbicular, to elliptic (fig. 2). leaf colors ranged from light green to dark green. citrus × limon (l.) osbeck and citrus × microcarpa bunge both exhibited ovate leaf shapes. citrus × aurantiifolia (christm.) swingle had elliptic leaves, while citrus × aurantium l. and citrus reticulata blanco featured lanceolate leaves. citrus maxima (burm.) merr. had orbicular leaves and citrus × aurantium l. cv. sweet orange group displayed obovate leaves. leaf color ranged from light to medium and dark. specifically, citrus × limon (l.) osbeck leaves were green to dark green with a rough surface, acute leaf apex, and crenate margin. citrus × aurantiifolia (christm.) swingle leaves were medium green with a rough surface, obtuse apex, acuminate base, and crenate margin. citrus × microcarpa bunge leaves were dark green with a smooth surface, obtuse apex, truncate base, and entire margin. citrus × aurantium l. cv. sweet orange group leaves were medium green with a smooth surface, acute apex, truncate base, and crenate margin. citrus × aurantium leaves exhibited a medium to dark green color with a smooth surface, acute apex, acuminate base, and crenate margin. citrus reticulata blanco leaves were medium to dark green, with an acute apex, truncate base, smooth surface, and crenate margin. lastly, citrus maxima (burm.) merr. leaves were dark green with a cordate apex, truncate base, smooth surface, and entire margin. a distinguishing feature of citrus maxima (burm.) merr. from other citrus species was the presence of petiole wings. citrus × limon (l.) osbeck and citrus reticulata blanco lacked wings on the leaf stalks, while some citrus species had leaf stalks with wings and others did not. figure 2 depicts the morphological characteristics of citrus leaves. budiarto et al. (2017) emphasized the presence of petiole wings as a significant morphological character that distinguished citrus species. the results showed variations in leaf lamina shapes, which differed from previous studies. sofiyanti et al. (2022) reported that the leaf lamina shapes of citrus spp. from riau varied between elliptic, oblong, obovate, ovate, and oblanceolate shapes. the discrepancies in the identified accessions were primarily due to differences in the study populations. previous studies also identified a greater number of citrus spp. compared to this study. species, such as citrus × amblycarpa (hassk) ochse “jeruk saring”, citrus × aurantium l. “jeruk hantu”, citrus hystrix dc. “jeruk purut”, citrus hystrix dc. “jeruk sundai”, citrus longilimon tanaka, and citrus medica l. “jeruk pagar”, were not found in this study. this indicated that the variation in citrus species from riau was more significant than the species from north penajam paser, east kalimantan. biotropia vol. 31 no. 2, 2024 162 table 2 list of citrus spp, accession found in north penajam paser, east kalimantan accession number species name local name sampling location utility altitude (masl) a-1 citrus × limon (l.) osbeck jeruk lemon waru foodstuffs, beverages, traditional medical applications 11 a-2 citrus × aurantium l. cv. sweet orange group jeruk sunkist waru foodstuffs and beverages 15 a-3 citrus × limon (l.) osbeck jeruk lemon waru beverages 16 a-4 citrus × microcarpa bunge. jeruk kalamansi waru foodstuffs and beverages 16 a-5 citrus reticulata blanco jeruk siam waru foodstuffs and beverages 19 a-6 citrus × aurantiifolia (christm.) swingle jeruk nipis waru foodstuffs, seasoning ingredients 14 a-7 citrus × microcarpa bunge. jeruk kalamansi waru foodstuffs, seasoning ingredients 5 a-8 citrus × aurantium l. cv. sweet orange group jeruk sunkist babulu foodstuffs 40 a-9 citrus reticulata blanco jeruk siam penajam foodstuff 9 a-10 citrus × aurantiifolia (christm.) swingle jeruk nipis penajam seasoning ingredients 1 a-11 citrus maxima (burm.) merr. jeruk bali waru foodstuffs 12 a-12 citrus × limon (l.) osbeck jeruk lemon babulu beverages 38 a-13 citrus × aurantium l. jeruk manis waru foodstuffs and beverages 28 a-14 citrus × aurantium l. cv. sweet orange group jeruk sunkist waru foodstuffs and beverages 28 a-15 citrus × limon (l.) osbeck jeruk lemon penajam beverages 4 a-16 citrus × aurantium l. jeruk manis penajam foodstuffs 29 a-17 citrus × microcarpa bunge. jeruk kalamansi penajam seasoning ingredients 32 a-18 citrus × aurantium l. cv. sweet orange group jeruk sunkist penajam foodstuffs 14 a-19 citrus × aurantiifolia (christm.) swingle jeruk nipis penajam foodstuffs, seasoning ingredients 4 a-20 citrus × aurantium l. jeruk manis penajam foodstuffs 8 a-21 citrus × aurantiifolia (christm.) swingle jeruk nipis penajam foodstuffs, seasoning ingredients 10 a-22 citrus × aurantium l. jeruk manis sepaku foodstuffs 11 a-23 citrus × microcarpa bunge. jeruk kalamansi sepaku foodstuffs, seasoning ingredients 16 a-24 citrus × limon (l.) osbeck jeruk lemon sepaku beverages, traditional medical applications 15 a-25 citrus × aurantium l. cv. sweet orange group jeruk sunkist sepaku foodstuffs 6 a-26 citrus × aurantium l. jeruk manis sepaku foodstuffs, beverages 24 a-27 citrus maxima (burm.) merr. jeruk bali sepaku foodstuffs 12 a-28 citrus × aurantiifolia (christm.) swingle jeruk nipis sepaku foodstuffs, seasoning ingredients 3 a-29 citrus × limon (l.) osbeck. jeruk lemon babulu foodstuffs, beverages 7 diversity of citrus spp. from north penajam paser regency (ikn) oktavianingsih et al. 163 accession number species name local name sampling location utility altitude (masl) a-30 citrus × aurantium l. jeruk manis babulu foodstuffs 8 a-31 citrus × microcarpa bunge jeruk kalamansi babulu foodstuffs, seasoning ingredients 13 a-32 citrus × aurantium l. cv. sweet orange group jeruk sunkist babulu foodstuffs 13 a-33 citrus × aurantium l. cv. sweet orange group jeruk sunkist babulu foodstuffs 32 a-34 citrus maxima (burm.) merr. jeruk bali waru foodstuffs 13 a-35 citrus × aurantiifolia (christm.) swingle jeruk nipis waru foodstuffs, seasoning ingredients 14 according to yulianti et al. (2020), there were 255 citrus varieties, consisting of 43% mandarintype, 4% tangerine-type, 0.7% sweet orangetype, 14.3% sour orange-type, 15% pummelotype, and 23% rootstock, lime, and lemon. the large number of developed varieties makes the classification and identification process relatively complex. furthermore, citrus spp. can easily generate new hybrids through intraspecies or interspecies hybridization (sofiyanti 2022). the species propagate via apomixis, allowing distribution over a wide range (wang et al. 2017). apomixis produces seeds that contain embryos from the somatic part of the nuclear cell (kepiro & roose 2010). a single seed is capable of developing from 2 to 30 embryos (koltunow 1993; wang et al. 2017). this ability is known as polyembryony, and breeders must be particularly aware due to the beneficial and detrimental effects (wang et al. 2017). fruit (fructus) this study identified limited variations in fruit shape among citrus spp. from north penajam paser. the observed fruit shapes ranged from spheroid to obloid, with the majority being obloid. citrus × limon (l.) osbeck exhibited a spheroidal shape with a convex base, acute apex, a diameter below 33 cm, a rough surface, and a dark green color. citrus × aurantiifolia (christm.) swingle had an ellipsoid shape with a convex base, rounded apex, a diameter below 33 cm, a smooth surface, and a dark green color. citrus × microcarpa bunge was characterized by an obloid shape with a convex base, rounded apex, a diameter below 33 cm, a green or yellow color, and a smooth surface. citrus × aurantium l. cv. sweet orange group had an obloid shape with a convex base, rounded apex, a diameter below 33 cm, a dark-green color, and a rough surface. citrus × aurantium l. featured an obloid shape with a convex base, rounded apex, a diameter below 33 cm, a dark-green color, and a rough surface. citrus reticulata blanco bore fruits with an obloid shape, truncated base, rounded apex, a diameter below 33 cm, a green or yellow color, and a smooth surface. citrus maxima (burm.) merr. fruits were obloid in shape, had a concave base, rounded apex, a diameter above 33 cm, a green or yellow color, and a rough surface. this study showed that citrus spp. of the same species generally exhibited consistent fruit shapes. this resullt contradicted the study conducted by dorji and yapwattanaphun (2011), which observed variations in fruit characteristics, even among the same species of citrus. for instance, variations were found in fruit shape and skin surface texture for citrus reticulata blanco "mandarin" across different sampling locations. similar results were also reported in a study on citrus maxima (burm.) merr. “jeruk bali” conducted by susandarini et al. (2013), which successfully observed variations in fruit characteristics, including fruit shape, apex, base, peel texture, the presence of oil glands on the peel, mesocarp color, number and arrangement of fruit segments, fruit flesh color, texture, as well as the number of seeds. however, citrus maxima (burm.) merr. exhibited different mesocarp colorations, including white and pink (fig. 3). biotropia vol. 31 no. 2, 2024 164 figure 2 leaf shapes notes: without wing petioles, i.e., citrus × limon (l.) osbeck “jeruk lemon” (a) a-1; (b) a-3; (c) a-15; (d) a-24; (e) a-29 and citrus reticulata blanco “jeruk siam” (f ) a-5.with wing petioles, i.e., citrus maxima (burm.) merr. “jeruk bali” (g) a-34. bar: 1 cm. figure 3 variations in mesocarp color on the pomelo plant citrus maxima (burm.) merr notes: (a) a-27 with white color; and (b) a-34 with pink color. bar: 1 cm. seed (semen) variations in the shape of citrus seeds include ovoid, semi-deltoid, club-shaped, semi-spheroid, spheroid, and irregular forms (fig. 4). the seed shape does not necessarily correlate with the species of citrus. however, cuneiform seeds are typically associated with the pomelo, citrus maxima (burm.) merr (accession 11 and accession 27). the semispheroid seed shape is commonly found in the siamese orange, citrus reticulata blanco "jeruk siam" (accession 5). irregular seed shapes are observed in citrus × limon (l.) osbeck (accession 15 and accession 24), and citrus × microcarpa bunge (accession 23). cluster analysis characterization of 35 citrus spp. accessions using 50 morphological characters produced a dendrogram divided into two main clusters (fig. 5). cluster a contained three accessions, while cluster b contains 32 accessions. the clustering was determined by the similarity of the characters, and the level of similarity determined the relationship of each accession. the similarity value increased with the number of identical characters. cluster a had identical characteristics in tree habitus, cordate leaf apex, entire leaf margin, pink mesocarp color, and seed absence. cluster b had similar characteristics in shrub habitus, acute and rounded leaf apex, crenate leaf margin, white to orange mesocarp color, and numerous seeds in the fruit. mesocarp the results showed variations in mesocarp color (fig. 3), including pink, yellow, white, green, orange, and light orange. citrus × limon (l.) osbeck had a white mesocarp with a sour taste, prominent visible oil glands, and was seedless. citrus × aurantiifolia (christm.) swingle exhibited a white mesocarp with a sour taste, prominent oil glands, and numerous seeds. citrus × microcarpa bunge exhibited a yellow mesocarp with a sour taste, faint oil glands, and was seedless. citrus × aurantium l cv. sweet orange group featured mesocarp in shades ranging from light orange to orange, a sweet taste, faint oil glands, and few seeds. citrus × aurantium l. had orange mesocarp, a sweet taste, prominent oil glands, and few seeds. citrus reticulata blanco showed mesocarp in shades of yellow to orange, a sweet taste, faint oil glands, and numerous seeds. citrus maxima (burm.) merr. had pink mesocarp with a sweet taste, faint oil glands, and was seedless. the mesocarp is located between the epicarp and the endocarp (sadka et al. 2019), constituting the pericarp. the outer part of the mesocarp may contain oil vesicles and carotenoid dyes (szczykutowicz et al. 2020), and the concentration of these components may vary depending on citrus species, environmental conditions, and other factors. certain species showed high tolerance to specific environmental conditions. furthermore, the interior of the mesocarp consists of spongy, white parenchyma tissue (mabberley 2004). diversity of citrus spp. from north penajam paser regency (ikn) oktavianingsih et al. 165 upgma euclidean a-22 a-14 a-32 a-33 a-2 a-25 a-8 a-18 a-30 a-16 a-20 a-24 a-28 a-23 a-4 a-13 a-21 a-6 a-35 a-10 a-19 a-17 a-31 a-26 a-5 a-9 a-7 a-29 a-15 a-12 a-1 a-3 a-34 a-11 a-27 12 10 8 6 4 2 0 b2 b2.1 b2.2 b2.3 b1 b1.1 b1.2 a1 a2 figure 4 the irregular shape of citrus seeds notes: (a) a-15; (b) a-23; (c) a-24; (d) a-5; (e) a-6. bar: 5 cm. each subcluster exhibited distinguishable characteristics that can be readily identified. subcluster a was distinguished primarily by mesocarp color, and in this subcluster, there were variations in mesocarp color among the accessions. specifically, subcluster a consisted of citrus maxima (burm.) merr with varying mesocarp colors. accession numbers a-11 and a-34 had pink mesocarp, while accession number a-27 had white mesocarp. subcluster b comprised 32 accessions and was subdivided into two major groups based on several fruit-related characteristics, including fruit shape, fruit base shape, fruit apex shape, the nature of the fruit oil glands, and mesocarp and skin color. subcluster b1 included citrus × limon (l.) osbeck which was further divided into two groups based on distinctive characteristics. these included the leaf apex color and the presence of seeds in the fruit. subcluster b2 comprised citrus × microcarpa bunge, citrus × aurantiifolia (christm.) swingle, citrus reticulata blanco, citrus × aurantium l. “jeruk manis”, and citrus × aurantium l cv. sweet figure 5 dendrogram and clustering result of 35 citrus spp. accessions from north penajam paser, east kalimantan. orange group. the grouping in this cluster was primarily based on fruit shape, the nature of the oil glands, fruit apex shape, skin color, and mesocarp color. generally, subcluster b2 exhibited an obloid fruit shape, conspicuous oil glands, rounded fruit apex, and dense to thick mesocarp with varying colors. pca (principal component analysis) was performed to support the clustering result (fig. 6). this analysis showed the pattern of grouping accessions and determined the influential characters in the clustering result. the dendrogram results showed that the key characters for grouping citrus spp. accessions are related to seed characteristics. these characteristics confined the presence or absence of seeds, seed shape, seed surface characteristics, and the number and texture of the seed surface. in addition, the position of each accession on pca axis was consistent with the groupings observed in the dendrogram. biotropia vol. 31 no. 2, 2024 166 figure 6 principal component analysis of 50 characters and 35 citrus spp. accessions from north penajam paser, east kalimantan conclusion thirty five (35) accessions of citrus spp. from north penajam paser regency were successfully identified consisting of citrus × limon (l.) osbeck “jeruk lemon”, citrus reticulata blanco "jeruk siam", citrus × aurantiifolia (christm.) swingle “jeruk nipis”, citrus × aurantium l. “jeruk manis”, citrus maxima (burm.) merr “jeruk bali”, citrus × microcarpa bunge "jeruk kalamansi", and citrus × aurantium l cv. sweet orange group “jeruk sunkist”. citrus spp. found in penajam, sepaku, babulu, and waru at varying altitudes of 1-40 masl. the dendrogram generated from 50 characters in this study was divided into two large clusters. cluster a was grouped based on the tree habitus, cordate leaf apex, entire leaf margin, pink mesocarp color, and seed absence. cluster b was grouped based on the shrub habitus, acute and rounded leaf apex, crenate leaf margin, white to orange mesocarp color, and numerous seeds in the fruit. the key characters for grouping citrus spp. accessions were related to seed characteristics. citrus spp. from north penajam paser had a great potential value for further development through plant breeding programs due to the high diversity. in addition, all components contributed to maintaining this diversity for the sustainability of biological resources that exist in north penajam paser. references ayala oa, oyama k, ríos-muñoz ca, rivas g., barahona sr, vega il. 2020. morphological variation of leaf traits in the ternstroemia lineata species complex (ericales: penthaphylacaceae) in response to geographic and climatic variation. peer j 8:e8307. doi: 10.7717/peerj.8307 balduzzi m, binder bm, bucksch a, chang c, hong l, pascuzzi asi, …, sparks ee. 2017. reshaping plant biology: qualitative and quantitative descriptors for plant morphology. front plant sci 8(76):117. doi: 10.3389/ fpls.2017.00117 benedetto n, carlucci v, faraone i, lela l, poniticelli m, russo d, …, milella. 2023. an insight into citrus medica linn.: a systematic review on phytochemical profile and biological activities. plants 12:2267. doi: 10.3390/ plants12122267 [bps] central agency of statistics (bps). 2021. “sepaku district in numbers 2021”. bps catalog 1102001.6409040 [cited on 20 october 2023]. 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(in indonesian). _hlk170375292 _hlk150168346 _hlk147222184 _hlk150166724 _hlk150098113 3. hanudin (identification).cdr the southeast asian journal of tropical biology vol. 31 no. 3, 2024: 402 421 doi: 10.11598/btb.2318.31.3.2318 issn: 0215-6334 | e-issn: 1907-770x 402 article higlights • this research provides information on the use of medicinal plants for treating malaria among ethnic groups in bengkulu province. • this research was part of ristoja (research on medicinal plants), national research conducted by the ministry of health of the republic of indonesia. • several compounds found in medicinal plants used by traditional healers in the bengkulu province have been scientifically proven to be effective as antimalarial medication. article information received 8 august 2024 revised 22 august 2024 accepted 3 september 2024 reviewer: suratman & nurainas *corresponding author, e-mail: ywidiyasis@gmail.com research article antimalarial medicinal plants used by traditional healers in bengkulu province of indonesia dian susanti1, aditya dwi permana putra1, devi safrina1, nur rahmawati wijaya1, m. bakti samsu adi1, rohmat mujahid1, rizal maarif rukmana1, dyah subositi1, sari haryanti1, usman siswanto2, yuli widiyastuti1* 1research center for pharmaceutical ingredients and traditional medicine, national research and innovation agency, bogor 16911, indonesia. 2faculty of agriculture, university of bengkulu, bengkulu 38371, indonesia abstract malaria, a disease with a high mortality rate, is still a significant problem globally, including indonesia. bengkulu province is among provinces in indonesia that uses a diversity of medicinal plants to treat malaria. this research aimed to make an inventory of medicinal plants and to evaluate the use of those medicinal plants to treat malaria in bengkulu province. the methods used were observation, interviews, and collection of plants samples. respondents consisted of traditional healers from 7 ethnic groups in bengkulu. several analyses were conducted on plant species, such as use value (uv), fidelity level (fl), family use value (fuv), relative frequency of citation (rfc), and plant parts value (ppv). this study found 32 specific herbal concoctions and identified the use of 47 medicinal plant species belong to 29 families used by 20 traditional healers in 6 of 7 ethnic groups. carica papaya, peronema canescens, and tinospora crispa had the highest use value, fidelity level, and relative citation frequency. the lamiaceae and caricaceae families had the highest family use value (0.15). the leaves (43.9%) and barks (13.6%) were the most frequent used parts of medicinal plants for treating malaria. decoction was the method most widely used by traditional healers in bengkulu to prepare conventional medicine formula. keywords: antimalarial herbal formula, bengkulu province, medicinal plants, traditional healer introduction people tend to use local resources for health purposes and treatments of diseases. modernization may lead to the loss of traditional knowledge, especially on medicinal plants (yulia et al. 2020). the role of medicinal plants in supporting public health will be increasingly important due to global changes of diseases patterns related to epidemiological transitions, where degenerative diseases and metabolic disorders are increasing in prevalence (theodoridis et al. 2023). medicinal plants are very suitable for promotive and preventive efforts in maintaining and improving the quality of public health (fetene et al. 2019). local knowledge regarding the use of medicinal plants in maintaining and overcoming health problems needs to be further inventoried and studied to be preserved and developed (budiarti et al. 2020). medicinal plants are among indonesia’s priceless biodiversity which are regarded as a source and strong basis for future alternative medicinal technology (pamenang 2021; zakiyah et al. 2022; theodoridis et al. 2023). local knowledge on utilizing medicinal plants to overcome health problems and to prevent diseases is an important topic for studies (wibowo et al. 2021). malaria is among diseases in communities, widely treated using medicinal plants, especially in rural and remote areas in indonesia. https://doi.org/10.11598/btb.2318.31.3.2318 antimalarial medicinal plants used by traditional healers susanti et al. 403 malaria, the most prominent disease caused by parasitic vectors of several mosquito species, remains a worldwide major health problem. indonesia is one of tropical countries with a high incidence of malaria. government programs in malaria prevention and treatment have reduced the prevalence rate from 2.9% in 2010 to only 1.9% in 2013. there are ten provinces in indonesia with malaria incidence rates above the national average, most of which are located in the eastern parts of indonesia (kementerian kesehatan republik indonesia 2013). a significant decrease in malaria prevalence until 2013, which stagnated until 2018, showed that the government’s program to reduce malaria cases had not run optimally. innovation is needed to support the malaria elimination program (kementerian kesehatan republik indonesia 2019; lusiyana 2020). in 2022, indonesia achieved a malaria annual parasite incidence (api) < 1 per 1,000 populations, namely 459 districts/cities from the specified target (484 districts/cities) or an achievement performance of 94.8% malaria api. bengkulu was one of the provinces that managed to maintain a malaria positivity rate (pr) of < 5% in all districts/cities (kementerian kesehatan ri 2022). therefore, bengkulu was chosen as the object of this research on medicinal plants utilization for treating malaria. drug resistance has led to the failure in eliminating malaria disease in indonesia. meanwhile, indonesia’s medicinal plants, has been widely utilized for the treatment of malaria by indonesian community (nugraha et al. 2022; rahmasari et al. 2022). bengkulu province has a high biodiversity of medicinal plants due to the presence of extensive tropical rainforests (wiryono et al. 2017). research on medicinal plants and herbs (ristoja) conducted by the ministry of health of the republic of indonesia in bengkulu province revealed abundant information on the use of medicinal plants for diseases treatment and health services by ethnic groups in bengkulu province (subositi & wahyono 2019)so it has a high economic value. the aim of this study was to record the use of the species of the genus curcuma as traditional herbal medicines in indonesia. the study was a part of a project called ristoja (research on medicinal plants and traditional herbal medicines. medicinal plants in bengkulu province needs to be studied in terms of the utilization of medicinal plants for health care, especially in treating malaria in the community. information on the utilization of medicinal plants for treating malaria in each ethnic group in bengkulu province is also still limited. therefore, this study was conducted to make an inventory of medicinal plants and to evaluate the use of those medicinal plants in bengkulu province to treat malaria. materials and methods study site geographically, bengkulu province is located at 2°16’-3°31’ n and 101°01’-103°41’ e, with a maximum temperature of 32.9-34 °c and a minimum temperature of 22-23 °c (badan pusat statistik 2017). bengkulu province is located along the west coast of sumatra island for approximately 525 km. data collection data on medicinal plants used for antimalarial were obtained from results of ristoja conducted in 2012. source of the data were 36 respondents from 7 ethnic groups in bengkulu province, namely serawai, lembak, rejang, muko-muko, pasemah, pekal and enggano. number of sample points were determined based on populations of ethnic groups having at least 1,000 members. six of the 7 ethnic groups in bengkulu province presumably have antimalarial concoctions, i.e., serawai, lembak, rejang, mukomuko, pasemah, and enggano (fig. 1). respondents were traditional healers from local community who utilized medicinal plants to treat diseases. those respondents were selected by means of purposive sampling approach based on information from indigenous community leaders, village heads, staff of government health facilities, and local health office. biotropia vol. 31 no. 3, 2024 404 figure 1 ethnic groups in bengkulu province which have antimalarial concoctions. notes: 1. muko-muko; 2. rejang1; 3. rejang2; 4. rejang3; 5. lembak1; 6. lembak3; 7. rejang4; 8. lembak2; 9. serawai; 10. pasemah8; 11. pasemah9; 12 pasemah6; 13. pasemah3; 14. pasemah2; 15. pasemah1; 16. pasemah7; 17. pasemah5; 18. pasemah4; 19. enggano1; 20. enggano2. method used for data collection in this study is in accordance with the guidelines from 2012 ristoja (kementerian kesehatan republik indonesia 2012; syafni & bakhtiar 2022). of the 7 ethnic groups that became respondents, only 6 ethnic groups were selected because these 6 ethnic groups utilize medicinal plants to treat malaria. in this study, all medicinal plants used as antimalarial concoctions by the respondents were inventoried, recorded, and collected. the collected specimens were identified and stored in the herbarium tawangmanguensis of the ministry of health of the republic of indonesia. prior to the interview session, the study objectives and methods were explained to the respondents. informed consent was also obtained from each respondent. information collected from the respondents were medicinal plants used, parts of medicinal plants used, local names of medicinal plants, medicinal plant habitat, concoction preparation methods, dosage, existing knowledge on medicinal plants and malaria, as well as attitudes and practices related to the recognition, control, and treatment of malaria. data analysis data processing was started with initial cleaning of the data from errors, followed by grouping the data based on similar knowledge. after the grouping, data were analyzed to determine uv (use value), fl (fidelity level), fuv (family use value), and rfc (relative frequency of citation). use value was calculated to figure out the importance of each medicinal plant for each traditional healer. the formula to calculate use value was the following (najem et al. 2020; susanti et al. 2023): uv = σui n where: u = number of use reports cited by each respondent for a medicinal plant species n = total number of respondents interviewed for a medicinal plant species antimalarial medicinal plants used by traditional healers susanti et al. 405 fidelity level was determined to analyze the importance of medicinal plants for a given remedy, which was calculated by using the following formula (najem et al. 2020; asiimwe et al. 2021): fl(%) = ip/iu x100 n where: ip = number of respondents who independently reported the use of a specific medicinal plant for the same illness iu = overall number of respondents who cited the medicinal plant for any illnesses in general family use value was determined to identify the significance of medicinal plant families and was calculated by the following formula (najem et al. 2020): fuv = σuvs/ns n where: uvs = use value of medicinal plant species belonging to the same family ns = total number of medicinal plant species present in each family relative frequency of citation was determined to show the local importance of each medicinal plant species, and it was calculated by the following formula (najem et al. 2020; asiimwe et al. 2021): rfc = fc n where: fc = number of respondents using a given medicinal plant species n = total number of interviewed respondents plant part value was determined to show the most used plant parts by the respondents (najem et al. 2020; biara et al. 2021; susanti et al. 2023).the highest plant part value indicates the most used medicinal plant parts by the respondents and was calculated by using the following formula (najem et al. 2020; biara et al. 2021; susanti et al. 2023): ppv = ru plant part ru where: ru = number of uses reported by the respondents for all medicinal plant parts ru plant part = the sum of uses reported by the respondents for each part of the medicinal plant. to explain knowledge differences on antimalarial medicinal plants in each ethnic group, a cluster dendrogram was established by using r-4.3.2. results and discussion respondent characteristics twenty traditional healers from 6 ethnic groups in bengkulu province who prescribed antimalarial concoctions made from medicinal plants were selected as respondents in this study (table 1). in our study, traditional healers who prescribed antimalarial concoctions in bengkulu province were found in 6 ethnic groups, i.e., enggano, muko-muko, rejang, lembak, serawai, and pasemah. traditional healers who prescribed the highest number of antimalarial concoctions were from pasemah ethnic group, which is related to a larger coverage area, namely pagaralam, empat lawang, lahat regency, ogan komering ulu, the muara enim area, and mount dempo, an active volcanic area. from their study, huda et al. (2019) stated that the long distance from the residential area to health facilities encouraged people to dwell into the knowledge of utilizing medicinal plants in overcoming health problems. male gender dominated the respondents in our study (12 of 20 respondents). the dominance of male gender as traditional healer is related to people’s perception in requirements of being a traditional healer, one of which is being a male descent of a traditional healer (djamaluddin et al. 2020), because the medicinal skill should be passed down from their ancestors. the respondents in our study were mostly in lower educational level. in regard to age, 16 of 20 traditional healers were at the age of 50 or above and one healer even reached the age of 80. this indicated that medicinal profession have not been the interest of younger generations in concurrent with several biotropia vol. 31 no. 3, 2024 406 table 1 traditional healers from 6 ethnic groups who used medicinal plants for treating malaria in bengkulu province selected as respondents ethnic group traditional healers (respondent) gender age education number of patients per month enggano 1 male 78 elementary 0-25 2 male 61 elementary 0-25 muko-muko 1 female 62 elementary 26-50 rejang 1 male 41 senior high school 0-25 2 male 40 senior high school 0-25 3 male 50 illiterate 0-25 4 female 58 junior high school 0-25 lembak 1 female 56 illiterate 0-25 2 female 55 illiterate 0-25 3 female 59 illiterate 0-25 serawai 1 male 60 illiterate 0-25 pasemah 1 male 80 junior high school > 100 2 male 51 elementary > 100 3 female 75 elementary > 50 4 female 50 elementary > 50 5 male 51 illiterate > 50 6 male 47 junior high school > 100 7 male 66 elementary 26-50 8 female 58 elementary 0-25 9 male 47 senior high school > 100 total respondents 20 obstacles that hinder the regeneration efforts by the older traditional healers. transfer knowledge on medicine requires special skills and basic medical knowledge, which is not easily transferred to potential successors. there are basic requirements for someone to become a traditional healer. a study conducted by efrianto (2018) showed that not everyone has the ability to become traditional healer. efrianto (2018) also mentioned that there are four ways to pass on knowledge, namely learning, inspiration, dreaming and meditating. monthly number of patients received by respondents in our study were mostly in the range of 0-25 (11 of 20 respondents). some respondents even received more than 100 patients per month (4 of 20 respondents). however, our respondents did not keep notes on which of their patients were prescribed antimalarial concoctions. our study also showed that most village communities have a good knowledge on traditional medicine and a lot of interests in using traditional medicine. the knowledge and interests were mostly inherited by their families. knowledge is a factor influencing the use of traditional medicine. lack of knowledge on health care and modern medicine has resulted in the use of herbal medicine as one of treatments for various diseases (adiyasa & meiyanti 2021). the use of medicinal plants for treating malaria our study showed that not all traditional healers have antimalarial prescriptions. in bengkulu province, the 6 ethnic groups claimed to have medicinal plants for treating malaria. traditional healers diagnose a patient as having malaria based on fever symptoms and testimony of the patient. there were 47 medicinal plant species belonging to 29 plant families used for antimalarial medication (table 2). fifteen plant specimens could not be identified by the scientific names. antimalarial medicinal plants used by traditional healers susanti et al. 407 table 2 medicinal plants used for antimalarial medication by 6 ethnic groups in bengkulu province family species name local name ethnic group uv rfc fl (%) acanthaceae andrographis paniculata (burm.f.) wall ex. ness sambilato, kenina rejang, lembak 0.10 0.10 10.00 justicia gendarussa burm.f.   sekanjang hitam, sekanjang putih pasemah 0.10 0.10 10.00 acoraceae acorus calamus l. jerangau enggano 0.05 0.05 5.00 annonaceae annona muricata l. sirsak lembak 0.05 0.05 5.00 asteraceae adenostemma lavenia (l.) kuntze pepulut pasemah 0.05 0.05 5.00 blumea balsamifera (l.) dc. capao serawai 0.05 0.05 5.00 caricaceae carica papaya l. pepaya, kates lanang enggano, rejang 0.15 0.15 15.00 dilleniaceae tetracera scandens (l.) merr. simpor darat, pasemah 0.05 0.05 5.00 euphorbiaceae macaranga tanarius (l.) mull.arg. sapat laut pasemah 0.05 0.05 5.00 fabaceae ototropis multiflora (dc.) h. ohashi & k.ohashi patiak tat rejang 0.05 0.05 5.00 pongamia pinnata (l.) pierre malapari enggano 0.05 0.05 5.00 goodeniaceae scaevola taccada (gaertn.) roxb. nanni enggano 0.05 0.05 5.00 lamiaceae peronema canescens jack sukei/sungkai rejang, lembak 0.15 0.15 15.00 loranthaceae loranthus sp. singgah kecil, singgah besar pasemah 0.05 0.05 5.00 malvaceae melochia umbellata (houtt.) stapf ndilau bincil, sindilau rutu pasemah 0.10 0.10 10.00 pterospermum diversifolium blume bayur elang pasemah 0.05 0.05 5.00 pterospermum javanicum jungh. bayur malukut pasemah 0.05 0.05 5.00 sida cordifolia l. nagori kecik pasemah 0.05 0.05 5.00 sida rhombifolia l. nagori besak pasemah 0.05 0.05 5.00 thespesia populnea (l.) sol. ex correa baru laut pasemah 0.05 0.05 5.00 meliaceae lansium sp. langsat air-air pasemah 0.05 0.05 5.00 menispermaceae fibraurea tinctoria lour. akar kuning enggano 0.10 0.05 5.00 tinospora crispa (l.) hook.f. & thomson protowali, cintowali, brotowali rejang 0.15 0.15 15.00 moraceae artocarpus elasticus reinw. ex blume keluncup lantung pasemah 0.05 0.05 5.00 ficus grossularioides var. grossularioides pemantung kerbau, pasemah 0.05 0.05 5.00 musaceae musa sp. pisang sabe pasemah 0.05 0.05 5.00 musa x paradisiaca l. pisang gemuk pasemah 0.05 0.05 5.00 myristicaceae knema intermedia (blume) warb. kayu siamang pasemah 0.05 0.05 5.00 myrtaceae psidium guajava l. jambu putih, pasemah 0.05 0.05 5.00 syzygium aqueum (burm.f.) alston jambu air, pasemah 0.05 0.05 5.00 oleaceae jasminum grandiflorum l. bungo pekan muko-muko 0.05 0.05 5.00 jasminum sambac (l.) aiton bungo susun muko-muko 0.05 0.05 5.00 onagraceae ludwigia grandiflora (michx.) greuter & burdet bungo tengah hari muko-muko 0.05 0.05 5.00 phyllanthaceae antidesma ghaesembilla gaertn. alay pasemah 0.05 0.05 5.00 poacaea gigantochloa verticillata (willd.) munro bambu haur pasemah 0.05 0.05 5.00 gigantochloa apus (schult.f.) kurz ex munro haur kapal pasemah 0.05 0.05 5.00 oryza sativa l. beras ketan pasemah 0.05 0.05 5.00 rosaceae rosa x damascena herrm. bungo ros muko-muko 0.05 0.05 5.00 rubiaceae nauclea orientalis (l.) kayu bengkal, kayu diwil pasemah 0.05 0.05 5.00 sapindaceae cardiospermum halicacabum l. kengkelam pasemah 0.05 0.05 5.00 biotropia vol. 31 no. 3, 2024 408 family species name local name ethnic group uv rfc fl (%) simaroubaceae brucea javanica (l.) merr. pedu beruang lembak 0.05 0.05 5.00 eurycoma longifolia jack pasak bumi pasemah 0.05 0.05 5.00 solanaceae physalis angulata l. seletup rejang, pasemah 0.10 0.10 10.00 urticaceae boehmeria grandis (hook.&arn.) a.heller ndilau nasi, pemantung ayam pasemah 0.10 0.10 10.00 zingiberaceae curcuma longa l. kunyit enggano, rejang 0.10 0.10 10.00 curcuma zanthorrhiza roxb. temulawak enggano 0.05 0.05 5.00   zingiber officinale roscoe jahe rejang 0.10 0.10 10.00 notes: uv = use value; rfc = relative frequency of citation; fl = fidelity level. our study showed that c. papaya, p. canescens, and t. crispa had uv, rfc, and fl values of 0.15, 0.15, and 15.00, respectively. ethno medicinal data were quantitatively analyzed using rfc, indicating the local importance of medicinal plant species. the species having the highest rfc (c. papaya, p. canescens, and t. crispa) were the species most often used by traditional healers in bengkulu province to cure malaria, because these plants were easy to find and easy to cultivate. people believe that the bitter taste of c. papaya, p. canescens, and t. crispa leaves can be used for treating malaria (shinta 2005; taek 2020). several research results showed that c. papaya, p. canescens, and t. crispa leaves have pharmacological properties as antimalarial medicine. papaya extract contains a total phenolics of 27.99±1.46 mg gae/g and a total flavonoids of 18.24±1.36 mg qe/g, which prevent the immune system from destroying platelets, decrease the virus-induced bone marrow damage, and stabilize infected cell membranes, leading to the acceleration of natural healing process by boosting platelets count and averting problems (babalola et al. 2024). t. crispa contains an active compound called tinocrisposide which works in the erythrocytic phase by inhibiting the growth of parasites in erythrocytes; therefore t. crispa can be an alternative medicine for treating malaria (abdillah et al. 2015; malik 2015). the ethanol fraction of p. canescens leaves at a dose of 0.084 g/kgbw is the most effective dose for treating malaria and has potential as an antimalarial medicine (prasiwi et al. 2018). the fidelity level (fl) represents the proportion of traditional healers who reported utilizing specific plant species to treat a particular sickness in the research area. the highest fidelity level value for a particular plant species signifies its critical importance and frequent application as a therapeutic agent (padhan & panda 2016; chaachouay et al. 2019)informant consensus factors (fic. in our study, c. papaya, p. canescens, and t. crispa had the highest fidelity level (15%), indicating that traditional healers often use these plants as antimalarial medicine in bengkulu province. c. papaya contains many advantageous chemical components in its fruits, leaves, flowers, stems, bark, and roots, such as papain, chymopapain, cystatin, à-tocopherol, ascorbic acid, flavonoids, cyanogenic glucosides, and glucosinolates. leaf extract of c. papaya were demonstrated to possess antimalarial properties by effectively eliminating plasmodium falciparum larvae within a concentration range of 25 ug/ml to 150 ug/ml (kovendan et al. 2012). p. canescens leaves contain phytochemical compounds, including flavonoids, phenolics, terpenoids, and alkaloids, which act as antioxidants, antibacterial, anti-inflammatory, antidiabetic, and antimalarial agents (maigoda et al. 2022)ethanol extract extracted by maceration method using 96% ethanol as solvent from p. canescens. the dragendorff’s and mayer test carried out the qualitative phytochemical analysis, fecl3 test, salkowski method, liebermann– burchard method, foam test, and naoh reagent. the total phenolic and flavonoid levels were tested using the folin–ciocalteu method. in vitro antioxidant activity was carried out using the dpph (1,1-diphenyl-2-picrylhydrazyl. in their study, suwandi et al. (2018) showed that the aqueous extract from p. canescens leaves had antispasmodic properties which effectively reduced the development of plasmodium falciparum with antimalarial medicinal plants used by traditional healers susanti et al. 409 an ic50 value of 12.26±1.05 μg/ml. t. crispa plant has been extensively utilized by traditional healers in bengkulu province for its antimalarial properties. research conducted by niljan et al. (2014) indicated that extract of t. crispa exhibits antimalarial properties in mice infected with plasmodium berghei, while combining the extract of t. crispa with pyrimethamine enhances the efficacy of t. crispa as antimalarial medicine. the family use value of medicinal plants in our study showed that both lamiaceae and caricaceae had the highest fuv (0.15), while menispermaceae had the second highest value of 0.13 (fig. 2). figure 2 family use value of medicinal plants used as antimalarial medicine in bengkulu province lamiaceae had long been used as traditional medication for treating malaria. various studies stated that lamiaceae family, previously referred to as labiatae, contains several active compounds that act as antimalarials, such as abietane, diterpenes, diterpenoids, triterpenoids, and flavonoids (tjitraresmi et al. 2020). caricaceae family, especially c. papaya, plays a role in producing alkaloids having larvicidal activity against anopheles stephensi mosquitoes (singh et al. 2022). alkaloids produced by various plants are known as antimalarial medication (omagha et al. 2020). several species of menispermaceae family, such as arcangelisia flava (nain et al. 2022; pratama et al. 2023) and tinopsora crispa (fitri et al. 2019; merici et al. 2020), contain alkaloids in the form of berberine, which function as antimalarial medication. herbal materials used by traditional healers consist of various parts of medicinal plants ranging from leaves, flowers, stems, bark, and roots (fig. 3). figure 3 parts of medicinal plants used for antimalarial medication by traditional healers in bengkulu province biotropia vol. 31 no. 3, 2024 410 leaves (43.9%) and barks (13.6%) were the most frequent used parts of medicinal plants for treating malaria. leaves are mostly used in herbal preparations by traditional healers because leaves are easily recognized, taken, and utilized part of medicinal plants. leaves can be harvested at any time without depending on weather conditions and are the easiest plant part to process or mix as ingredients for herbal preparations (widiyastuti et al. 2017)forest areas are still the main habitat of medicinal plants germplasm. sigogor nature reserve in ponorogo regency, east java, is a potential area that has a diversity of medicinal plants and has not been widely studied. the exploration of medicinal plants in the sigogor nature reserve area aims to know the diversity, abundance and knowledge of the surrounding community about the existence of these medicinal plants. the method used is explorative survey with qualitative approach. the data collected in the form of secondary data and primary field observation results and literature studies of previous research results. data analysis was carried out descriptively for the identification of medicinal plant specimens, qualitative vegetation analysis to determine plant habitus type, than percentage of habitus type and plant part was calculated based on observation result. the results of exploration activities show there were 43 species of medicinal plants from 33 families have been found in the sorrounding area of sigogor. habitus of medicinal plants found mostly were herbs (39.5%. in utilizing plant parts for herbal preparations, it is important to consider the conservation aspect of medicinal plants. the use of bark, stems, and roots in herbal preparations can have a long impact on population decrease of medicinal plants, because the harvesting process of those plant parts will include the process of injuring the plants, leading to the plant death and eventually, the extinction of the plants (bamigboye & tshisikhawe 2020). medicinal plants used in preparing antimalarial medication in bengkulu province came from five different sources, i.e., from the wild (38%), garden (30%), forest (28%), seashore (3%), and market (1%) (fig. 4). traditional healers mostly (69%) utilize medicinal plant from nature, either from the wilds or harvested from a forest or from a seashore. figure 4 sources of medicinal plants used as antimalarial medication in bengkulu province natural conditions of bengkulu province are relatively pristine, not much impacted by industrial and residential areas; therefore, allowing the community to obtain medicinal plants from the wilds of the surrounding environment (seitz et al. 2022). the ease of obtaining medicinal plants both from the wilds (69%) and from gardens (30%) has resulted in low sales of medicinal plants in markets (1%) (fig.4), showing that sources from ‘garden’ and ‘forest’ provide a significant contribution, while sources from ‘seashore’ and ‘market’ ‘ are the least utilized, indicating that local traditions and knowledge play as the primary roles in supporting local health care (balinado & chan 2017). only 30% of the raw materials used by traditional healers came from medicinal plants planted or naturally grown in their gardens, while the remaining medicinal plants came from wild plants found in the forest, primarily trees that are challenging to cultivate (dajic-stevanovic & pljevljakusic 2015). the preference of utilizing medicinal plants from the wilds over those cultivated ones is related to the concentration of bioactive chemicals produced by the medicinal plants that may be impacted by environmental factors associating with the therapeutic process (pandey et al. 2023). antimalarial treatments in regard to the use of medicinal plants for treating malaria, each traditional healer has different ingredients and methods compared to antimalarial medicinal plants used by traditional healers susanti et al. 411 other ethnic groups. peculiarities in the utilization of these medicinal plants are greatly influenced by culture, educational background, availability of public health facilities, experiences of traditional healers and availability of medicinal plants resources in residential region of traditional healers (gaoue et al. 2017; cain et al. 2018; mcquaid & landier 2018; purwoko et al. 2023). our study established a comprehensive overview of different herbal preparations used for antimalarial medication, ingredients specifications, preparation methods, dosage, usage frequency and treatment duration for antimalarial treatment conducted by traditional healers from 6 ethnic groups in bengkulu province (table 3). table 3 herbal preparations, ingredients specifications, preparation methods, dosage, usage frequency, and treatment duration of antimalarial treatment by traditional healers in bengkulu province ethnic group   herbal preparations, ingredients specifications, preparation methods, dosage usage frequency treatment duration enggano 1 two inches of akar kuning (f. tinctoria) roots, cut into pieces, boil with three glasses of water to half a glass, let it cool, then drink. 3 times/day 3 days 2 cut into pieces: akar kuning (f. tinctoria), temulawak (c. zanthorrhiza), kunyit (c. longa), jahe (z. officinale), and jeringau (a. calamus), add three glasses of water and stir evenly, boil until the water is thick, let it coll, then drink. 3 times/day 3 days 3 three pieces of nanni leaf stalks (s. taccada) to be pounded, add half a glass of water, squeeze until the juice comes out, then drink the juice. once a day 3 days 4 remove the bark of malapari stem (p. pinnata) and scrape the stem. mix the scrape with a glass of hot water, drink half the water, leaving the dregs in the glass. then, put the dregs on top of the stomach. half-a glass a day 3 days 5 grind the bihaan (unidentified species) leaves, add water, and drink. once a day until recovered 6 one old papaya leaf (c. papaya), to be brewed with hot water, then rub on the forehead. once a day 3 days muko-muko 1 bungo pekan (j. grandiflorum), bungo ros (r. damascena), bungo susun (j. sambac), and bungo tengah hari (l. grandiflora) to be mashed, then rub on the forehead. once a day 3 days rejang 1 take a clean handful of papaya leaves (c. papaya), boil in one liter of water to one glass, let it cool, then drink at once. once a day in the morning 2 days 2 take three sungkai leaves (p. canescens), clean and squeeze, add one glass of boiled water, then squeeze until the juice comes out, then drink the juice. once a day 2 days 3 take a handful of protowali leaves (t. crispa), clean and boil the leaves with a halfliter of water, let it cool, then drink. once a day 2 days 4 crush the patiak tat root (o. multiflora) and blend with seletup (unidentified species) and sukei (unidentified species) leaves, boil the leaves, let it cool, then drink. 3 times a day 3 days 5 boil a 10 cm of cintowali stem (t. crispa) until boiled, let it cool, then drink. 3 times a day 7 days 6 two papaya leaves (c. papaya) to be washed and directly eaten. once a day 3 days 7 mix brotowali stem (t. crispa), sambiloto herb (a. paniculata), jahe (z. officinale) and kunyit (c. longa) then dry and grind. put the powder into a 500 mg size capsule. 3 times a day 3 days lembak 1 sungkai leaves (p. canescens) to be squeezed with a glass of boiled water, then drink. once a day until the shivering stops 2 sirsak leaves (a. muricata) to be chopped and mixed with one tablespoon of rice. the mixture is then divided into three parts, two parts to be placed on the navel in the morning and evening, and the third part to be removed. note: do not eat sour-taste foods and do not drink iced beverages/ice during the implementation of this medication. twice a day 1 day 3 kenina/sambiloto leaves (a. paniculata) to be soaked in one glass of boiling water, then drink the water 3 times a day for three days. 3 times a day 3 days 4 the fruit of pedu beruang (b. javanica) to be washed and eaten immediately like taking pills. 2 fruits in a day 3 days serawai 1 several old capao leaves (b. balsamifera) that are still attached or still hanging on the stem, to be boiled with three glasses of water to a glass, then drink. one glass until recovered pasemah 1 bengkarung, gadung itam, and kayu siamang (k. intermedia) to be mixed and ground, then use for rubbing the entire body. as often as possible 2 weeks 2 uwi manau lanang, uwi dahanan, uwi temiang, and uwi semut to be soaked with earthworms in boiled water until the color of the earthworms turns white, then drink. 3 times a day 2 weeks biotropia vol. 31 no. 3, 2024 412 ethnic group   herbal preparations, ingredients specifications, preparation methods, dosage usage frequency treatment duration 3 mix and wash the barks of kayu bengkal (n. orientalis), ndilau bincil (m. umbellata), diwil (n. orientalis), simpor darat (t. scandens), jambu air (s. aqueum), and ndilau nasi (b. grandis). slice and pound the barks, then mix with water and squeeze until the juice comes out. drink the juice and smeared the dregs on the ailing body part. 3 times a month 3 months 4 barks of semanggat and jambu putih (p. guajava) to be mixed, sliced, then boiled with three glasses of water until well boiled, let it cool, then drink. twice a day 3 days 5 the leaves of sekanjang hitam (j. gendarussa), sekanjang putih (j. gendarussa), memaye merah, memaye putih, singgah besar (loranthus sp.) and singgah kecil (loranthus sp.) to be squeezed in boiled water, then rub the whole body with the concoction, wait until the concoction is dried on the body. repeat the treatment after the body is dry from the concoction. 3 times a day 1 day 6 the roots of pasak bumi (e. longifolia) and langsat (lansium sp.) to be cut into pieces and boiled until the water is half, then let it cool. the decoction to be drunk as often as possible. as often as possible 3 days 7 combine and chop ingur-ingur, tukup jaring, and pisang sabe (musa sp.), then soak them with crust soaking water, followed by heating using a pot lid and put the concoction on the sore spot. note: change the concoction every night; the patient will feel itchiness during first hour as a reaction from the concoction. once at night 3 days 8 five leaves of each of sekanjang hitam (j. gendarussa), sekanjang putih (j. gendarussa), pepulut (a. lavenia), kengkelam (c. halicacabum), bayur elang (p. diversifolium), bayur malukut (p. javanicum), bambu haur (g. verticillata), sapat laut (m. tanarius), baru laut (t. populnea), keluncup lantung (a. elasticus), nagori besak (s. rhombifolia), and nagori kecik (s. cordifolia), to be mixed and squeezed, then rub the concoction all over the body. 3 times a day 1 day 9 five leaves of alay (a. ghaesembilla) to be mixed with the sprouts of haur kapal (g. apus), then cut into 4 pieces and shredded, then rub the concoctions all over the body. 3 times a day 1 day 10 thirty (30) cm of inner stem of pisang gemuk (m. paradisiaca) to be mixed with 7 glutinous rice grains and mashed, then rub the concoctions all over the body. 3 times a day until recovered 11 six-finger-long bark of sindilau rutu (m. umbellata) to be bruised and soaked in boiled water, then drink half a glass. 10 times a day 1 day 12 seletup (p. angulata) to be boiled, let it cool, then drink. 3 times a day until recovered 13 sap from roots of pemantung kerbau (f. grossularioides var. grossularioides) and sap of pemantung ayam (b. grandis) to be collected during the night, then to be drunk the next morning. 3 times a day until recovered usage frequencies varied from once a day to 10 times daily or frequently, and the treatment durations ranged from one day to until recovered. the most common usage frequencies for antimalarial medication were 3 times a day and once daily (table 3), indicating how often traditional healers administered specific antimalarial medication in bengkulu province. there were also variations in the usage frequencies of antimalarial therapies based on the type of treatment, which were primarily prescribed to be taken once, twice, or three times per day for a fixed number of days, such as 3 days, 7 days, or a month. less expected frequencies included half a glass a day or 10 times a day. some treatments were less frequent, like once a day, once a night, or a few times per month for an extended period, such as 3 months, and there were a few treatments specifying to take the medication frequently or as needed until symptoms resolve. the usage frequency of prescription made from medicinal plants for antimalarial medication can vary depending on the type of medicinal plants and how it is processed (caunca & balinado 2021; hastiana et al. 2023). the misuse of medicinal plants, whether excessive consumption of concoction or in combination with other medicinal plants that are not compatible can cause side effects (alkhamaiseh & aljofan 2020; başaran et al. 2022). preparation of traditional antimalarial medications by traditional healers in bengkulu province can be categorized as internal and external usages (fig. 5). for internal usage, the herbal preparations for antimalarial medication were made in the form of decoction, infusion, dry powder in capsules, and raw consumption. antimalarial medicinal plants used by traditional healers susanti et al. 413 the differences in treatments and medicinal recipes were based on the experience and knowledge of traditional healers learned from their ancestors. this study showed that the most widely used methods in traditional medication practiced by traditional healers in bengkulu province were decoction and infusion (fig. 5). figure 5 ethods used to make antimalarial medication by traditional healers in bengkulu province our study also showed that the use of medicinal plants in the form of a decoction was most preferred considering that this method provided greater extraction yields and higher concentration of active phytochemicals; thus, potentially increased the efficacy of herbal preparations. in processing medicinal plants, it is important to apply safe heating limits when boiling the medicinal plants, so as not to destroy the active ingredients contained in the plants. destruction of active ingredients during boiling using high temperature occurs because some secondary metabolite compounds contained in the boiled medicinal plants have thermolabile properties. in addition, decoction is the oldest and simplest way of preparing traditional medicine, whether using fresh or dried ingredients (balinado & chan 2017; caunca & balinado 2021; mir et al. 2021; sujana et al. 2021). infusion is preferred for preparing antimalarial medication, along with other traditional medications which use ingredients having bioactive contents that are easily degraded and easily soluble (petrakou et al. 2020). traditional medications used by ethnic groups are generally in the form of decoctions, infusions, raw foods, poultices, powders, and others (boyzel et al. 2019). plants selected for treating malaria traditionally were those having bitter taste. several research revealed that some plants have been proven to have antimalarial activity; for instant quinine from cinchona spp. and artemisinin from artemisia annua (nonaka et al. 2018; tajuddeen & van heerden 2019). our study investigated almost all of 11 medicinal plants to determine their antimalarial activity and the content of major phytochemical compound, except for boehmeria grandis, in the pasemah ethnic group (table 4). empirical facts about b. grandis can be used as an initial information for conducting research on the potential b. grandis in controlling malaria. further research on p. canescens, j. gendarussa, and m. umbellata are still required because their extracts are still in crude form, and the main bioactive compounds that act as antimalarial medication are not yet known. several studies had determined bioactive compounds of 7 medicinal plants, namely c. papaya (carpaine) (arifuddin et al. 2019), t. crispa (13 hydroperoxyoctadeca 9, 11 dienoic acid (13[s] hpode), columbine, and berberine), a. paniculata (andrographolide), c. longa (curcumin), p. angulata (physalin) (arruda et al. 2021), z. officinale (gingerenone), and f. tinctoria (berberine). biotropia vol. 31 no. 3, 2024 414 table 4 cross-references of antimalarial activity and major phytochemical compounds of 11 most frequently used medicinal plants for treating malaria in bengkulu province based on published literatures species antimalarial activity major phytochemical compound carica papaya l. c. papaya extract is responsible for antioxidant and antimalarial activity (airaodion et al. 2019; eze et al. 2022). total phenols (27.99±1.46 mg gae/g), total flavonoids (18.24±1.36 mg qe/g), glycosides, saponins, tannin, alkaloids, reduced sugars, proteins, terpenoids and steroids (atanu et al. 2021). peronema canescens jack ethanol fraction of the leaves of p. canescens with 0.084 g/ kgbb is the most effective dose and potential as an antimalarial medication (prasiwi et al. 2018). flavonoid, saponins, tannin, steroids, terpenoids, alkaloids, and phenols (prasiwi et al. 2018). tinospora crispa (l.) hook.f.&thomson brotowali (t. crispa) showed antimalarial activity at 100 and 200 mg/kg of usage. combination of t. crispa stem extract and earthworm extract can prevent erythrocyte hemolysis in nonimmune mechanisms. extract of brotowali stem can restrain the growth rate of malaria parasites, inhibit the sporulation process, and suppress the spread of hemozoin pigment. in addition, brotowali provides anti-hypoglycemic effects (ounjaijean et al. 2019; kusumarini et al. 2020). total content of phenols obtained from ethanol extract of t. crispa stem was 43.34±1.92% per dry weight. total flavonoids content was 74.26±1.32 per dry weight (merici et al. 2020) andrographis paniculata (burm.f.) ness a. paniculata extract can be used as a companion to conventional medicine to treat malaria, due to the increasing resistance of artemisinin to plasmodium. a. paniculata also inhibits hempolymerase (septiana et al. 2017, makmur et al. 2022). andrographolide (makmur et al. 2022). boehmeria grandis (hook.&arn.) a.heller not found yet not found yet curcuma longa l. in single or combined use, c. longa can increase phagocytosis of erythrocytes infected by malaria parasites, induce damage, and affect the life cycle and dna replication of malaria parasites. in addition to inhibiting malaria parasites, c. longa effectively reduces blood parasitemia levels by 80-90%. c. longa has the potential as antimalarial agent. in addition, curcumin compounds also have antioxidant effects, which may provide additional benefits in fighting inflammation and cell damage caused by malaria infection (donipati & harasreeramulu 2015; lwin et al. 2017; heydarian et al. 2019; fahira et al. 2023) curcumin, flavonoids, alkaloids, and phenols (omagha et al. 2020; fahira et al. 2023) justicia gendarussa burm.f   j. gendarussa has some unique activities as a larvicide and adulticide that can kill both larvae and adult mosquitoes (chandra & lo 2021). there is no literature related to further research on the utilization of j. gendarussa as an antimalarial medication. alkaloids, polyphenols, flavonoids, glycosides, phytosterols, saponins, triterpenes, and quinines (shinwari et al. 2020; jain et al. 2024). melochia umbellata (houtt.) stapf   the cytotoxic data of ethyl acetate, methanol, and water extracts contained in m. umbellata that show antimalarial activity in vero cells prove that the three extracts have strong potential to fight dengue virus type 2 (denv-2) (soekamto et al. 2018). stigmasterol, waltherione c, moracin m, steroids and alkaloids (soekamto et al. 2018; 2019; 2020). physalis angulata l. epa exhibits potent, selective, and broad-spectrum antiparasitic activity of p. angulata against trypanosoma cruzi. in addition, epa shows that p. angulata reduces parasite load by rapidly disrupting the cell cycle in obligate hosts (da silva et al. 2015; meira et al. 2015)endemic in latin america and emerging in several countries, is limited by the frequent side effects and variable efficacy of benznidazole. natural products are an important source for the search for new drugs. aim/ hypothesis considering the great potential of natural products as antiparasitic agents, we investigated the anti-trypanosoma cruzi activity of a concentrated ethanolic extract of physalis angulata (eepa. physalin b, d, f, and g (arruda et al. 2021). zingiber officinale rosc. z. officinale has antiplasmodial activity (biruksew et al. 2018). vanillin, gingerenone a, 4-methoxybenzaldehyde, 6shagaol, 8-shogaol, 10-shogaol and a-linolenic acid (faloye et al. 2023). fibraurea tinctoria lour. combination therapy of f. tinctoria lour methanol extract and artemisinin was shown to have antimalarial effects against plasmodium berghei in vivo (fikriah & sawitri 2020) sesquiterpene hydrocarbons, oxygenated monoterpenes, oxygenated sesquiterpenes, geranial, linalool, n-hexane, and steroid (sulistiarini et al. 2022; chac et al. 2023) antimalarial medicinal plants used by traditional healers susanti et al. 415 each of these bioactive compounds has its mechanism and role as an antimalarial medication. although the antimalarial mechanism is unknown, it is estimated that carpaine directly inhibits the parasite (arifuddin et al. 2019; teng et al. 2019; dwivedi et al. 2020; haldar et al. 2020). at the tests of 13 hydroperoxyoctadeca 9, 11 dienoic acid (13[s] hpode), the reduction in parasitemia occurs suppressive, prophylactically, and shows a schizonticidal effect. columbine and berberine have similar mechanisms, working as an antimalarial medication using a dihydrofolate reductase-thymidylate synthase (dhfrts) inhibitory mechanism (chester et al. 2017; bare et al. 2020; 2022; purwaningsih et al. 2023)hypertension, atherosclerosis, and cancer. for decades, anti-adipogenic potential of many herbal extracts has been investigated. one example is garcinia cambogia extract (ge. the mechanism of andrographolide is to inhibit the growth of parasitemia (sari et al. 2018). curcumin has several mechanisms of action, including anti-inflammatory, antioxidant, antinociceptive, antiparasitic, and wound-healing effects (urošević et al. 2022; jamil et al. 2023). physalin works by inhibiting the function of the p2x7 receptor, which regulates the production of pro-inflammatory cytokines (arruda et al. 2021). gingerenone works as an antimalarial medication by binding to plasmepsin ii and plasmodium falciparum dihydrofolate reductase-thymidylate synthase (faloye et al. 2023). traditional knowledge on antimalarial medication of ethnics groups in bengkulu province in bengkulu province the 20 traditional healers, who were selected as respondents in this study, had diverse traditional knowledge on malaria treatment. this study developed a dendrogram to represent four main clusters, where one cluster (pasemah7) has no similarity to the other clusters (fig. 6). figure 6 cluster dendrogram representing traditional knowledge of ethnic groups in bengkulu province on medicinal plants having antimalarial activity. notes: 1. muko-muko; 2. rejang1; 3. rejang2; 4. rejang 3; 5. lembak1; 6. lembak3; 7. rejang4; 8. lembak2; 9. serawai; 10. pasemah8; 11. pasemah9; 12 pasemah6; 13. pasemah3; 14. pasemah2; 15. pasemah1; 16. pasemah7; 17. pasemah5; 18. pasemah4; 19. enggano1; 20. enggano2. biotropia vol. 31 no. 3, 2024 416 traditional healers in pasemah ethnic group (pasemah7) had a formula containing 14 medicinal plants having antimalarial activity that differed from the other healers, i.e., a. lavenia, a. ghaesembilla, m. tanarius, p. acerifolium, p. javanicum, s. cordifolia, s. rhombifolia, t. populnea, a. elasticus, m. paradisiaca, g. verticillata, g. apus, o. sativa, and c. halicacabum. one traditional healer from enggano ethnic group (enggano-1) had a formula using 4 different medicinal plants having antimalarial activity, namely a. calamus, s. taccada, f. tinctoria, and c. zanthorrhiza. our study observed that pasemah was the ethnic group with the highest usage of medicinal plants for treating malaria. knowledge on medicinal plants acquired by the pasemah ethnic group may have been caused by local wisdom, culture, beliefs, and accessibility inherited within the ethnic group (adi et al. 2020; hasyim et al. 2023). pasemah ethnic group is widely spread in pagar alam city, empat lawang regency, lahat regency, and muara enim regency (ernatip et al. 2007), where malaria cases are high, especially in muara enim regency in which malaria cases are the highest in south sumatra, with incidence rates of 3,428 (in 2018), 3,215 (in 2019), and 6,266 (in 2020) (bps 2023; hasyim et al. 2023). in bengkulu province, there are open-pit mineral mining areas causing the occurrence of lots of standing water, which supports the development of malaria vector mosquitoes. conclusion this study found that there were 47 medicinal plant species belonging to 29 plant families used by 20 traditional healers from 6 ethnic group for treating malaria and for antimalarial medication in bengkulu province. carica papaya, peronema canescens and tinospora crispa were the 3 medicinal plants most widely used for antimalarial medication. plant leaves are the most used plant part for antimalarial medication. herbal preparations prepared by traditional healers are mostly in the form of decoctions with oral administration methods. our study also observed several compounds contained in medicinal plants that have been scientifically proven to be efficacious as antimalarial medication. acknowledgments authors greatly appreciate the team who carried out the ethno-medicine survey from university of bengkulu and the center for research and development of medicinal and traditional plants. the research was funded by dipa of the center for research and development of medicinal and traditional medicinal plants, the national institute health research and development, ministry of health of the republic of indonesia with decision letter number 378/menkes/ sk/x/2012 dated 23 october 2012, involving stakeholders from the central government to the regions and various parties. the data usage has been approved by 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of medicines: in the development of pharmaceutical industry technology. intellectual property rights review 3(02):221-6. zakiyah s, tasikrara l, nurfatimah. 2022. contribution of local wisdom values to education. international journal social sciences and education (ijosse) 3(2):37-48. https://doi.org/10.26858/ijosse.v3i2.40119 http://dx.doi.org/10.22435/toi.v10i2.6736.78-87 _goback _hlk173920443 _hlk164485209 _hlk164485251 _hlk164486628 _hlk164486864 _hlk164487205 _hlk162601656 _hlk162601697 _hlk164487242 _hlk164597039 _hlk164597055 _hlk164597078 _hlk164597106 _hlk164597165 _hlk164597212 _hlk164597229 _hlk164597251 _hlk164597268 _hlk164597310 _hlk164597326 biotropia vol. 29 no. 3, 2022: 193 202 doi: 10.11598/btb.2022.29.3.1551 193 heavy metal bioaccumulation in ducks and possible risks to human health r. susanti* and karima widiyastuti department of biology, faculty of mathematics and natural sciences, universitas negeri semarang 50229, indonesia received 12 february 2021/accepted 27 june 2022 abstract meat is part of duck carcass mostly consumed by humans compared to other parts. this study aimed to analyze the heavy metal bioaccumulation factor (baf) of duck meat and its possible risks to human health. a total of 25 duck samples with their drinking water and feed were taken from five intensive duck farms in central java province, i.e., semarang (a), temanggung (b), magelang (c), pati (d) and salatiga (e). heavy metals concentration (as, cd, hg, and pb) were measured following the icp-oes method. heavy metals concentration data obtained were then compared with the quality standard. the baf value was calculated and the risks to human health were assessed. our study found that drinking water provided for ducks in all farms contained heavy metals (as, cd, hg, and pb) concentrations exceeding the quality standards. hg concentration of 0.089 5.01 ppm in duck feed exceeded the quality standard. concentrations of cd (0.0713 0.075 ppm) and hg (3.1 4.84 ppm) in duck meat exceeded the quality standard. the average of bafduck meat values was in the range of 0.443 0.955. the edi value of heavy metals (as, cd, hg, and pb) for adults and children through consumption of duck meat in the central java region was lower than rfd. this study showed that the health risk parameters (edi, hq, and hi) were within safe limits. exposure to heavy metals through duck meat consumption both in adults and children was unlikely to cause adverse health effects. keywords: bioaccumulation, duck, estimated daily intake, heavy metals, human health introduction indonesia is an agricultural country where most of the population work as farmers and breeders. ducks are among the leading livestock commodities in indonesia. the duck population in indonesia ranks third in the world after china and vietnam (ismoyowati & sumarmono 2019). in 2018, indonesia's duck population was recorded to reach 51,239,185 (department of livestock and animal health 2019). the duck population in central java province ranks fourth after east java province, with a population of more than 5 million (the central statistics agency of central java province 2018). in the last decade, duck meat has been favored by many people. duck meat also determines the agricultural economy. based on the total production of duck meat in the world, asian countries contributed 84.2%. the muscle fiber content of duck breast is higher than chicken meat, so it is often referred to as red meat (biswas et al. 2019). duck meat contains essential amino acids, vitamins, and minerals (reis et al. 2010) and it tastes very delicious and is favored by most people. based on those reasons there has been quite an increase in duck farming. in general, however, the quality of the foodstuff is not only determined by its nutritional content, but also by the safety of the food itself. foodstuff must be free from harmful substances. the public’s concern about the importance of food safety has increased. some cases related to food safety occur due to contamination of microorganisms, pesticides, hormones, antibiotics, and heavy metals. the consumption of foodstuffs is considered to be one of the main routes of heavy metal exposure to the human population. heavy metal contamination is a major problem because it affects the structural and functional integrity of the ecosystem. heavy metal contamination of *corresponding author, email: basanatha8@mail.unnes.ac.id biotropia vol. 29 no. 3, 2022 194 meat, vegetables, and fish is a serious problem for food safety and a threat to human health (bortey-sam et al. 2015) as most of these metals are toxic even at very low concentration levels. dietary intake accounts for more than 90% of heavy metal exposure compared to other routes such as inhalation and dermal absorption (loufty et al. 2006). likewise, the source of heavy metal contamination in duck meat is most likely from its feed and drinking water. abdulkhaliq et al. (2012) suggested that the accumulation of heavy metal in ducks is caused by contamination at every point of duck production, sources of nutrition, drinking water, and the environment. faten et al. (2014) also suggested that the heavy metal accumulation in ducks' bodies can come from water, feed, air, and the environment contaminated with these metals. meat is the part of duck carcass that is most consumed by humans compared to other parts such as the brain, lungs, liver, kidney, etc. therefore, this study aimed to analyze the levels of heavy metal bioaccumulation factor (baf) in duck meat and to assess the possible risks to human health. materials and methods this research is an exploratory observational study. a total of 25 duck samples were taken from five intensive duck farms in central java province, i.e., semarang (a), temanggung (b), magelang (c), pati (d), and salatiga (e). the criteria for selecting duck farms were laying ducks with an intensive care system (ducks were kept in cages, not released from the cage), 18 months years old, and the total number of ducks in a cage was 200 500 ducks. samples of ducks, drinking water, and feed were taken from each duck farm to be analyzed for heavy metal contents and concentrations. sample preparations meat samples five ducks, duck feed, and duck drinking water were randomly taken from each intensive duck farm. each duck was slaughtered. the thigh and breast meat were cut into sizes of 2 x 2 x 3 cm. furthermore, the meat was weighed and dried in an oven for > 72 hours at 70 °c. a total of 6 g of dry meat was added with 2 ml of 65% hno3 (merck) and then ashed using a furnace (nabertherm l3/c6) at 500 °c for 2 hours (demirel et al. 2008). subsequently, the ashed meat was dissolved with distilled water (merck) to a volume of 50 ml, to be analyzed for the heavy metal contents and concentrations. drinking water samples as much as 100 ml of duck drinking water was acidified with 65% hno3 until the water ph became 2 (demirel et al. 2008). after that, distilled water was added to the sample until the volume reached 200 ml, to be analyzed for the heavy metal contents and concentrations. feed samples as much as 1,500 g of duck feed was dried in an oven at 70 °c for > 48 hours. after being dried, the feed sample was mashed in a blender. a total of 6 g of feed sample was added with 2 ml 65% hno3 (merck) and mixed until homogeneous. then, feed samples were ashed using a furnace (nabertherm l3/c6) at 500 °c for 2 hours (demirel et al. 2008). the ashed feed was dissolved in 50 ml of distilled water (merck), to be analyzed for heavy metal contents and concentrations. quantification of heavy metals each sample of drinking water, feed, and duck meat was analyzed for heavy metal contents and concentrations following the icpoes method (perkin elmer optima 8300) following the procedure of naschan et al. (2017). sample solutions and standard solutions of as, cd, hg, and pb were prepared and injected into the icp-oes equipment. the analytical wavelengths of investigated heavy metals were 546.074 nm for hg, 193.696 nm for as, 220.353 nm for pb, and 226.506 nm for cd. each heavy metal measurement of each sample was repeated 3 times. data of as, cd, hg, and pb contents in the samples of drinking water, feed, and duck meat were compared with the determined quality standards (goi 2001; sni 2009; fao 2017). bioaccumulation factor (baf) bioaccumulation factor (baf) was calculated according to wang et al. (2017), with the following equation: heavy metal bioaccumulation in ducks and possible risks to human health – susanti and widiyastuti 195 baf = concentration in organism (ci) ………….. (1), concentration in ingested food and/or water (c0) where: c0 = concentration of heavy metals in drinking water and or feed; ci = concentration of heavy metals in duck meat. thus, there were 3 types of baf calculated in this study, i.e., bafwater, baffeed, and bafduck meat. baf values were categorized according to arnot and gobas (2006), i.e., 1) baf < 1,000: no possibility of accumulation; 2) 1,000 < baf < 5,000: bioaccumulative; and 3) baf > 5, 000: highly bioaccumulative. potential health risk assessment non-carcinogenic risk to humans in the central java province in relation to the consumption of duck meat contaminated with heavy metals was assessed following the usepa (2000) method. heavy metal (as, cd, hg, and pb) accumulation in duck meat can cause adverse effects on human health. the estimated daily intake (edi) value through consumption of duck meat was calculated from the average concentration of heavy metals observed in duck meat samples and the average daily consumption of duck meat in indonesia. the edi (mg/kg/day) was estimated using the following equation (usepa 1989): edi = cm x adc ………………………….. (2), bw where: adc = average daily consumption of duck meat in indonesia, determined to be 0.0225 kg/person/day (ministry of trade of the republic of indonesia 2014); bw = body weight, determined to be 70 kg for adults (usepa 1989) and 30 kg for children (bortey-sam et al. 2015); cm = average heavy metal concentrations in duck samples (mg/kg). the non-carcinogenic risk due to the consumption of duck meat containing heavy metals by the population of central java province was called hq (hazard quotient). the hq is calculated using the following formula (usepa 1989): hq = cm x ef x adc x ed x 10-3 ….……….. (3), rfd x bw x at x 1000 where: cm = average heavy metal concentrations in duck samples (mg/kg); ef = exposure frequency (365 days/year); adc = average daily consumption of duck meat in indonesia, determined to be 0.0225 kg/person/day (ministry of trade of the republic of indonesia 2014); ed = exposure duration (70 years); rfd = an estimate of the number of contaminants per day that a human can tolerate over their lifetime. the rfd (oral) values used in this assessment were 3.00e-04 (for as), 5.00e-04 (for cd), 3.50e-03 (for pb), and 3.00e-04 (for hg) (tay et al. 2019); bw = body weight, determined to be 70 kg for adults (usepa 1989) and 30 kg for children (bortey-sam et al. 2015); at = average exposure time (ed x 356 days). if the hq value is less than one (< 1), pollutant exposure to the population does not cause adverse effects on human health. duck meat contained more than one type of heavy metal. therefore, the level of adverse effects on the exposed population is called hazard index (hi), which is the sum of all calculated hqs of all heavy metals found in the sample (usepa 1989): hi = hqas + hqcd + hqhg + hqpb ……… (4) hi is considered to be a potential risk estimated from exposure to some heavy metals (usepa 1989). data analysis heavy metal concentration data, baf, edi, hq, and hi values were analyzed descriptively. biotropia vol. 29 no. 3, 2022 196 results and discussion heavy metal toxicity can cause teratogenic, mutagenic, and carcinogenic effects in biological organisms including poultry (hashmi et al. 2013). in ecosystems, metal contamination can also affect the habitat, food chain, and community structure of organisms in an environment (kim & oh 2013). drinking water for ducks in all sampled intensive farms in this study contains heavy metals of as, cd, hg, and pb which exceeded the determined quality standards (goi 2001; sni 2009; fao 2017). the concentrations of heavy metals in duck drinking water were 0.0513 0.081 ppm (for as), 0.07 0.08 ppm (for cd), 1.74 2.4 ppm (for hg), and 0.06 0.07 ppm (for pb). the order of heavy metal concentrations in the drinking water of ducks from the highest to the lowest in temanggung, magelang, and salatiga was hg > cd > as > pb. on the other hand, the order of heavy metal concentrations in the drinking water of ducks from the highest to the lowest in semarang was hg > cd > pb > as; while in pati was hg > as > cd > pb (table 1). table 1 heavy metal content in drinking water and feed for ducks, and duck meat heavy metals water feed duck meat as standard fao 2017 0.01 0.5 0.5 sni 2009 0.05 0.5 0.5 pp no 82 year 2001 0.01 0.25 0.25 samples from: semarang (a) 0.0513abc 0.0750 0.0700 temanggung (b) 0.0773abc 0.0800 0.0770 magelang (c) 0.0797abc 0.1097 0.0770 pati (d) 0.0810abc 0.0987 0.0770 salatiga (e) 0.0710abc 0.1003 0.0700 cd standard fao 2017 0.003 0.5 0.5 sni 2009 0.003 0.1 0.3 pp no 82 year 2001 0.003 0.1 0.05 samples from: semarang (a) 0.0700abc 0.0760 0.0740abc temanggung (b) 0.0800abc 0.0750 0.0713abc magelang (c) 0.0800abc 0.0787 0.0750abc pati (d) 0.0800abc 0.0783 0.0740abc salatiga (e) 0.0800abc 0.0783 0.0740abc hg standard fao 2017 0.001 0.1 0.1 sni 2009 0.001 0.05 0.03 pp no 82 year 2001 0.001 0.06 0.03 samples from: semarang (a) 1.740abc 2.000abc 6.000abc temanggung (b) 2.400abc 0.089abc 3.100abc magelang (c) 2.380abc 4.420abc 4.840abc pati (d) 2.260abc 5.010abc 4.220abc salatiga (e) 2.380abc 4.560abc 4.400abc pb standard fao 2017 0.01 0.3 0.1 sni 2009 0.01 0.3 0.3 pp no 82 year 2001 0.01 0.2 0.5 samples from: semarang (a) 0.0600abc 0.0740 0.0640 temanggung (b) 0.0700abc 0.0730 0.0690 magelang (c) 0.0700abc 0.0780 0.0670 pati (d) 0.0700abc 0.0730 0.0460 salatiga (e) 0.0700abc 0.0650 0.0650 notes: a = the data exceed the quality standards set by fao 2017 (fao 2017); b = the data exceeds the quality standards set by indonesian national standard year 2009 (sni 2009); c = the data exceeds the quality standards set by indonesian government regulation no 82 year 2001/pp no. 82 year 2001 (goi 2001). heavy metal bioaccumulation in ducks and possible risks to human health – susanti and widiyastuti 197 the source of drinking water for the sampled intensive duck farms in semarang, pati, and salatiga is groundwater, while in temanggung and magelang, the source is ponds. the high concentration of heavy metals in drinking water for ducks in this study was probably related to the timing of conducting the water sampling, which was during the dry season (may june). the concentration of chemicals in groundwater is greatly influenced by the season. the seasonal analysis report from obasi and akudinobi (2020) showed a relative decrease in chemical concentration in groundwater during the rainy season compared to that during the dry season. although heavy metal levels in drinking water for ducks increased relatively during the dry season, analysis of heavy metal concentrations in groundwater in various regions of central java province and indonesia is still required, both during dry and rainy seasons. the analysis is essential because groundwater is a source of drinking water for most livestock and humans. accumulation of heavy metals in the body of animals and humans will cause an adverse impact on health, especially arsenic, cadmium, lead, and mercury which are reported of having high toxicity. exposure to heavy metals in humans can increase the risk of cancer and other health problems such as anemia, liver and kidney damage (malik & khan 2016), decreased intelligence, decreased function of various organs, and even death (kulkarni & kaware 2013). the variation of heavy metal concentrations in water is influenced by water ph, temperature, dissolved oxygen, and water flow rate (li et al. 2013). the lower value of water ph causes higher metal solubility. water ph can convert carbonate (co3) into hydroxide (oh) forming bonds with particles on the water surface (palar et al. 2008). heavy metal solubility of zn, cu, cd, cr, and pb is greater at low ph conditions (4 7) than that at high ph conditions (8 10) (li et al. 2013). the amount of cd released from polluted sediment will decrease with increasing ph value (zhang et al. 2018). in this study, the results of water and soil ph measurements in the five samples of intensive duck farms were in the range of 6 7 and 6.9 7, respectively. these ph values were within the ph range of 6 9 which is a safe ph value based on indonesian government regulation no. 82 of 2001 (goi 2001). temperature also has an important effect on heavy metal solubility. at higher temperatures (30 35 °c), the release rate of metals (zn, cu, cd, cr, and pb) becomes faster than that at low temperatures (li et al. 2013). the temperature at each location of the sampled intensive duck farms in this study was in the range of 25.6 34.6 oc. this temperature was in the range of 27 35 °c which is a safe temperature per determination from the indonesian government regulation no. 82 of 2001 (goi 2001). in duck feed, hg was found in the concentration range of 0.089 5.01 ppm. this hg concentration exceeded the determined quality standards (goi 2001; sni 2009; fao 2017). meanwhile, the levels of other heavy metals (as, cd, and pb) were recorded as still under the determined quality standard. the range of as concentration detected in duck feed was 0.075 0.1097 ppm. meanwhile, the concentration range of cd, hg, and pb were 0.075 0.0787 ppm, 0.089 5.01 ppm, and 0.065 0.078 ppm, respectively (table 1). a study by ukpe and chokor (2018) also showed that there was heavy metal contamination of ni (1.71 g/kg), co (0.39 g/kg), pb (1.17 g/kg), cr (0.529 g/kg) and cd (0.031 g/kg) sourced from poultry feed. poultry feed has the potential to pollute the environment through the fecal matter of cultivated poultry. a large-scale duck farm produces large amounts of fecal matter. compost made from poultry fecal matter may still contain a lot of heavy metals (alvarenga et al. 2015). apart from being used as compost, fecal matter can be digested by maggots and the harvested maggots can be used as animal feed (wang et al. 2017). heavy metals in manure will contaminate the soil, then from the soil, heavy metals can be transferred to plants and animals. in turn, humans can also be exposed to heavy metals if they consume the contaminated plants and animals. heavy metals can contaminate various organisms in the ecosystem, through the food chain cycle. poultry feed is made from plant material, thus, if plants are contaminated, the feed will also be contaminated. the raw materials of duck feed in this study varied, i.e., concentrate feed (commercial), rice bran, leftover vegetables, dry rice, trash fish, shrimp waste, and fish waste. concentrate feed may also biotropia vol. 29 no. 3, 2022 198 contribute to heavy metal contamination in duck feed. a study by okoye et al (2011) showed that some concentrate feeds in nigeria contained 50.575 170.075 mg/kg of iron, 6.52 14.20 mg/kg of copper, 1.10 7.85 mg/kg of lead, and 0.038 0.463 mg/kg of cadmium. wang et al. (2013) also reported that several animal feeds in china contained several heavy metals with an average of 15.9 2,041.8 mg/kg of zn, 0 392.1 mg/kg of cu, and < 10 mg/kg of hg, as, pb, cd, and cr. the concentrations of cd (0.0713 0.075 ppm) and hg (3.1 4.84 ppm) in duck meat were identified to exceed the determined quality standards (goi 2001; sni 2009; fao 2017). meanwhile, as and pb concentrations were still under the determined quality standards. analysis of heavy metals in mallard ducks (anas platyrhynchos) in iran showed that the lowest concentrations of pb, cd, and zn metals were found in muscle (meat) and the highest concentration was found in the liver (alipour et al. 2016). some previous studies also revealed heavy metal contamination in the meat of ducks and other livestock. a study by widayanti and widwiastuti (2018) showed that duck meat in malang city contained 0.37 ppm of cadmium (cd). poultry meat in egypt contained 0.086 ppm of cd, 6.092 ppm of cu, 0.136 ppm of sn, and 1.280 ppm of zn (ali et al. 2017). heavy metal cd was detected at 0.003 ppm in poultry meat in ghana (nesta et al. 2015). heavy metals of as, cd, and hg were also detected in duck meat products in argentina (hyun et al. 2018). study results of islam (2018) showed that beef, goat, chicken, and duck meat in bangladesh contained 0.533 6.55 mg/kg of chromium (cr), 0.005 7.70 mg/kg of nickel (ni), 0.581 15.99 mg/kg of copper (cu), 0.080 11.34 mg/kg of arsenic (as), 0.001 0.22 mg/kg of cadmium (cd), and 0.061 13.52 mg/kg of lead (pb). bioaccumulation is the absorption process of chemicals by an organism through all exposure routes, such as those that occur in the natural environment, i.e., food sources, drinks, and surrounding environment. the bioaccumulation factor (baf) is a value to measure the bioaccumulation rate. baf is calculated based on the ratio of metal concentration in an organism to metal concentration in water and feed. baf value is influenced by several factors such as sex, reproductive status, age, body size, and lipid content of an organism. organisms having a lot of lipids have a greater capacity to store hydrophobic organic chemicals, so their baf values are higher (arnot & gobas 2006). in this study, the baf value in duck meat was analyzed based on the accumulation of water, feed, and the total of water and feed. the baf value of all heavy metals in duck meat was in the range of 0.065 5.01. according to arnot and gobas (2006), a baf value < 1,000 means that accumulation is not possible. the average values of bafwater for as and pb were greater than those of baffeed. on the other hand, the average values of baffeed for cd and hg were greater than those of bafwater (table 2). the bafwater value of as, cd, hg, and pb in duck meat in semarang was the highest compared to the values calculated in other locations. meanwhile, the highest baffeed value of as was found in temanggung (0.962) followed by semarang (0.933) and pati (0.78). the highest baffeed value of cd was found in semarang (0.973) followed by magelang (0.952) and temanggung (0.95). the highest baffeed value of hg was found in pati (5.01) followed by magelang (4.42) and temanggung (3.48). the highest baffeed value of pb was found in temanggung (0.945) followed by semarang (0.864) and magelang (0.858) (table 2). the average of bafduck meat values was in the range of 0.443 0.955, with the details of 0.443 for pb; 0.457 for as; 0.475 for cd, and 0.955 for hg. the highest bafduck meat values of as, cd, and hg were found in semarang. meanwhile, the highest bafduck meat value of pb was found in temanggung. the lowest bafduck meat values of as, cd, hg, and pb were found in magelang, temanggung, and pati, respectively (table 2). heavy metal bioaccumulation in ducks and possible risks to human health – susanti and widiyastuti 199 table 2 baf values of heavy metals in drinking water and feed for ducks, and duck meat heavy metals location of duck farm average a b c d e as water 1.364 0.966 0.966 0.950 0.985 1.046 feed 0.933 0.962 0.701 0.780 0.697 0.815 duck meat 0.554 0.490 0.407 0.428 0.409 0.457 cd water 1.054 0.891 0.937 0.925 0.925 0.946 feed 0.973 0.950 0.952 0.945 0.945 0.953 duck meat 0.507 0.460 0.473 0.467 0.467 0.475 hg water 3.448 1.291 2.034 1.870 1.849 2.098 feed 3.000 3.483 4.420 5.010 0.964 3.375 duck meat 1.604 1.245 0.712 0.580 0.634 0.955 pb water 1.066 0.985 0.957 0.657 0.928 0.919 feed 0.864 0.945 0.858 0.630 0.065 0.672 duck meat 0.478 0.483 0.453 0.322 0.481 0.443 notes: a = semarang; b = temanggung; c = magelang; d = pati; e = salatiga. there have not been many studies related to baf values of heavy metals in terrestrial animals. research results by wang et al. (2017) showed that younger maggot larvae had a higher baf value of heavy metal than older larvae. the baf value of cd in 3-day-old larvae was 1.20, while the baf value of cd in 4-day-old larvae was 1.10. this comparison showed that as the maggots grow, heavy metals are released from the maggots' bodies. a study conducted in gadani shipbreaking pakistan to find out heavy metal baf values in the gills and muscles of seven fish species showed that the baf values found were in the order of mn > cd > ni > pb (kakar et al. 2020). estimated daily intake (edi) values of heavy metals (as, cd, hg, and pb) for adults and children through the consumption of duck meat in the central java province were calculated and presented in table 3. our study showed that children are more susceptible to the acute and chronic effects of a chemical contaminant because children consume more food per unit of body weight compared to adults (enhis 2007). therefore, toxic contamination by means of food consumption is higher in children than that in adults, leading to higher edi scores in children compared to that in adults for all heavy metals. the edi values calculated for as, cd, and pb in our study were lower than the oral reference dose (rfd) of each metal. however, the edi value for hg metal was higher than the oral rfd. this result may be caused by the high concentrations of hg in water, feed, and duck meat, which were above the determined quality standards (goi 2001; sni 2009; fao 2017). high concentrations of hg had also been reported in water at the ports of mayangan, kenjeran, and gresik, east java, indonesia (hertika et al. 2018). table 3 estimated daily intake (mg/kg/day) values of heavy metals (as, cd, hg, and pb) for adults and children through duck meat consumption heavy metals location of duck farm average a b c d e as adults 2.25e-05 2.47e-05 2.47e-05 2.47e-05 2.25e-05 2.38e-05 children 5.25e-05 5.77e-05 5.77e-05 5.77e-05 5.25e-05 5.56e-05 cd adults 2.38e-05 2.29e-05 2.41e-05 2.38e-05 2.38e-05 2.37e-05 children 5.55e-05 5.35e-05 5.63e-05 5.55e-05 5.55e-05 5.53e-05 hg adults 1.93e-03 0.99e-03 1.56e-03 1.36e-03 1.41e-03 1.45e-03 children 4.50e-03 2.33e-03 3.63e-03 3.12e-03 3.30e-03 3.38e-03 pb adults 2.06e-05 2.22e-05 2.15e-05 1.48e-05 2.09e-05 2.00e-05 children 4.80e-05 5.18e-05 5.03e-05 3.45e-05 4.88e-05 4.67e-05 notes: a = semarang; b = temanggung; c = magelang; d = pati; e = salatiga. biotropia vol. 29 no. 3, 2022 200 table 4 hazard quotient (hq) values hazard quotient location of duck farm average a b c d e as adults 7.50e-05 8.25e-05 8.25e-05 8.25e-05 7.50e-05 7.95e-05 children 1.75e-04 1.92e-04 1.92e-04 1.92e-04 1.75e-04 1.85e-05 cd adults 4.76e-06 4.58e-05 4.82e-05 4.75e-05 4.75e-05 4.73e-05 children 1.11e-04 1.07e-04 1.12e-04 1.11e-04 1.11e-04 1.10e-05 hg adults 6.43e-03 3.32e-03 5.19e-03 4.52e-03 4.71e-03 4.83e-03 children 15.00e-03 7.75e-03 12.10e-03 10.55e-03 11.00e-03 11.28e-03 pb adults 5.88e-06 6.34e-06 6.15e-06 4.22e-06 5.97e-06 5.71e-05 children 1.37e-05 1.48e-05 1.44e-05 0.98e-05 1.39e-05 1.33e-05 notes: a = semarang; b = temanggung; c = magelang; d = pati; e = salatiga. the non-carcinogenic risk of heavy metals through the consumption of duck meat was evaluated based on the value of the hazard quotient (hq). the estimated hq value of all heavy metals in this study was < 1 (table 4). the hq values indicate that the people in central java province who consume duck meat are unlikely to suffer adverse health effects due to heavy metal contamination in the duck meat. the highest average of hq values for adults and children were recorded at hg intake (4.83e-03 and 11.28e-03, respectively) followed by as (7.95e-05 and 1.85e-05, respectively). the lowest averages of hq values were recorded at cd intake, i.e. 4.73e-05 in adults and 1.10e-05 in children. in this study, the order of hq values from the highest was hg > as > pb > cd. hazard index (hi) is a parameter to assess the cumulative risk due to exposure to several heavy metals. the average of hi values of our study showed a number of < 1 for both adults (4.97e-03) and children (9.41e-03) (table 5), which indicated that there was no significant health risk due to cumulative exposure to heavy metals (as, cd, hg, and pb) in duck meat. a study by kakar et al. (2020) showed that the hq values of seven fish species in gadani shipbreaking pakistan were safe with regard to pb and mn (hq < 1), but may cause potential risk with regard to cd and ni (hq > 1). estimated values of hq and hi of heavy metals (cd, cr, cu, fe, ni, pb, and zn) through consumption of baladi chickens were < 1, indicating that chicken consumption did not have the potential to cause health risks for consumers in the jazan region of saudi arabia (al bratty et al. 2018). table 5 hazard index (hi) values ducks farm location adults children semarang (a) 6.56e-03 15.29e-03 temanggung (b) 3.46e-03 8.06e-03 magelang (c) 5.32e-03 12.42e-03 pati (d) 4.66e-03 10.86e-03 salatiga (e) 4.84e-03 11.29e-03 average 4.97e-03 9.41e-03 all of the risk parameters (edi, hq, and hi) calculated in this study were within safe limits. exposure to heavy metals through duck meat consumption, both in adults and children in central java province, was not potential to present an adverse health risk. however, exposure to heavy metals contained in duck meat cannot be underestimated because it will accumulate in the human body. exposure to heavy metals from other foodstuffs, dermal absorption, urban air inhalation, etc, which were not studied in this study, can increase the collective risk of heavy metal contamination in the human body. conclusion drinking water for ducks in all sampled intensive duck farms contained heavy metals of as, cd, hg, and pb which exceeded the determined quality standards. all health risk parameters (edi, hq, and hi) were within safe limits. exposure to heavy metals through duck meat consumption, both in adults and children, was unlikely to cause adverse health effects. acknowledgments this research was partially funded by the directorate general of research and development, strengthening the ministry of heavy metal bioaccumulation in ducks and possible risks to human health – susanti and widiyastuti 201 research, technology, and higher education through the national competitive fundamental research grant number 192/sp2h/lt/ drpm/2019, 11 march 2019. references abdulkhaliq a, swaileh km, hussein rm, matani m. 2012. levels of metals (cd, pb, cu, and fe) in cow’s milk, dairy products, and hen’s eggs from the west bank, palestine. int j food res 19(3): 1089-94. al bratty m, alhazmi ha, ogdi sj, otaif ja, al-rajab aj, alam mf, javed sa. 2018. determination of heavy metals in various tissues of locally reared (baladi) chicken in jazan region of saudi arabia: assessment of potential health risks. pak j zool 50(4): 1509-17. ali ma, dalia mh, nagwa te. 2017. evaluation of some heavy metals residues in batteries and deep liter rearing systems in japanese quail meat and offal in egypt. vet world 10: 2231-0916. alipour h, solgi e, majnouni f. 2016. concentrations of heavy metals in tissues of the mallard anas platyrhynchos in kanibarazan, northwestern iran. podoces 11(2): 35-42. alvarenga p, mourinha c, farto m, santos t, palna p, sengo j, ..., cunha-queda c. 2015. sewage sludge, compost and other representative organic wastes as agricultural soil amendments: benefits versus limiting factors. j waste manag 40: 44-52. arnot ja, gobas fa. 2006. a review of bioconcentration factor (bcf) and bioaccumulation factor (baf) assessments for organic chemicals in aquatic organisms. environ rev. 14: 257-97. biswas s, banerjee r, bhattacharyya d, patra g, das ak, das sk. 2019. technological investigation into duck meat and its products: a potential alternative to chicken. worlds poult sci j 75(4): 609-20. bortey-sam n, nakayama smm, ikenaka y, akoto o, baidoo e, yohannes yb, ..., ishizuka m. 2015. human health risks from metals and metalloids via consumption of food animals near gold mines in tarkwa, ghana: estimation of the daily intakes and target hazard quotients (thqs). ecotoxicol environ saf 111: 160-7. demirel s, tuzen m, saracoglu s, soylak m. 2008. evaluation of various digestion for trace element contents of some food materials. j hazard mater 152: 1020-6. department of animal husbandry and animal health [dinas peternakan dan kesehatan hewan]. 2019. animal husbandry and animal health statistics [statistik peternakan dan kesehatan hewan]. https://ditjenpkh.pertanian.go.id. 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di industri; seniati]. itn malang, 3 february 2018. p: 361-4. zhang y, zhang h, zhang z, liu c, sun c, zhang w, marhaba t. 2018. ph effect on heavy metal release from polluted sediment. j chem 2018 (id 7597640): 7 pages. doi.10.1155/2018/7597640 the southeast asian journal of tropical biology vol. 31 no. 3, 2024: 330 338 doi: 10.11598/btb.2024.31.3.2206 issn: 0215-6334 | e-issn: 1907-770x 330 growth and survival of spiny lobster, panulirus homarus juveniles fed with different formulated feeds sudewi1*, bejo slamet1, nyoman adiasmara giri1, haryanti1, ibnu rusdi2, clive morris jones3 and simon irvin4 1research center for fishery, national research and innovation agency (brin), bogor 16912, indonesia. 2research center for marine and land bioindustry, national research and innovation agency (brin), lombok 83352, indonesia. 3james cook university, queensland 4354, australia. 4csiro food futures flagship, canberra act 2601, australia. article higlights • indonesia has remarkable natural resources of spiny lobster panulirus homarus and an exceptional opportunity to establish the largest lobster aquaculture industry in the world. • however, culturing lobsters still relies on natural feeds (trash fish of fresh fishery by-catch) which are inefficient and environmentally unsustainable. • developing formulated feed based on locally available ingredients is invaluable for replacing natural feeds to support sustainable lobster culture. • formulated feed made of local fish meal provided the best growth of juvenile spiny lobster panulirus homarus compared to imported fish meal. abstract several studies suggested that good growth performance and survival of lobster juveniles can be achieved by feeding the lobsters with formulated feeds. this study aimed to (1) evaluate the growth and survival of spiny lobster panulirus homarus juveniles fed with different diets having profiles based on a basal diet formulation and (2) to compare the growth of lobsters fed with an identical formulated feed with variations only in sources of fish meal and dry matter content. the feeding experiment was designed following a completely randomized design (crd) with five diet treatments and five replicates for each treatment. five experimental diets were prepared in this experiment, i.e., diet a (basal diet prepared as moist pellets); diet b (moist pellets with the same formulation as diet a, using local fish meal); diet c (moist pellets with the same formulation as diet a, using imported fish meal); diet d (dry pellets with the same formulation as diet a, using imported fish meal); and diet e (fresh fish). juveniles of spiny lobster with an average weight of 3.00±0.07 g were allocated in 25 net cages (60 x 60 x 60 cm) placed in a circular hdpe canvas tank (7.5 m in diameter) at a density of 15 lobsters/cage in a flow-through seawater system. the juveniles were fed with the experimental diets twice/ day for 12 weeks. the experiment showed that the best growth was observed in juveniles fed with moist feed containing local fish meal (diet b) with the final weight reaching 18.74±2.30 g. moist feed containing imported fish meal (diet c) resulted in a higher survival rate (46.66%), and the lowest survival rate (13.33%) was obtained from juveniles fed with fresh fish (diet e). keywords: formulated feeds, growth, panulirus homarus, survival article information received 25 february 2024 revised 26 june 2024 accepted 25 july 2024 *corresponding author, e-mail: sude001@brin.go.id research article introduction marine lobsters are highly favorable and considered prized luxury export products (plaganyi et al. 2018), mainly exported to china, the united states of america, and hong kong. chinese imports of lobsters increased dramatically to 38.6% or reached 11.461 mt from january to march 2021 compared to the same period in 2020 (fao 2021). grow-out culture of wildcaptured spiny lobster juveniles to market size has been considered an evolving aquaculture industry in asia, most markedly in vietnam, and emerging in indonesia (nankervis & jones 2022a). indonesia has a remarkable natural resource of pueruli, and therefore, indonesia has an exceptional opportunity to build the largest lobster aquaculture industry in the world (priyambodo et al. 2020). in indonesia, spiny lobster seed resources are found mainly in lombok, java, and sumbawa islands. https://doi.org/10.11598/btb.2024.31.3.2206 growth and survival of juvenile spiny lobster, panulirus homarus sudewi et. al. 331 in 2013-2014, the catch of lobster seeds was estimated to be over 3 million per year in lombok (bahrawi et al. 2015), and by 2016, more than 100 million lobster seeds were captured from java, lombok, and sumbawa (jones et al. 2019). despite the growing lobster aquaculture industry, feeding in grow-out culture commonly relies only on fisheries products, such as trash fish or fresh fishery by-catch (nankervis & jones 2022a). fresh fishery products and trash fish for feeding in aquaculture have been well-identified as environmentally unsustainable and inefficient. for example, feeding with mussel flesh has a poor feed conversion ratio of up to 17.26:1 (rivaie et al. 2023). trash fish as feed lead to significant water quality deterioration, causing environmental degradation and disease introduction (nankervis & jones 2022a). in addition, trash fish vary in their supply and nutritional profile. moreover, there are difficulties in storage and possible spoilage during storage (jones et al. 2015). the shrinking supply of trash fish and their downstream environmental effects have strengthened the urgency to develop sustainable formulated feeds (nankervis & jones 2022b). the development of formulated feed would provide benefits, such as nutrition optimization, feeding methods flexibility, easiness in usage and storage, waste minimization, and environmental sustainability improvement (perera & simon 2014) for the development of lobster aquaculture (saleela et al. 2015). some studies on formulated feed development and nutritional requirements for lobsters were conducted, such as studies on the appropriate level of protein for better growth and survival of spiny lobster p. homarus (rathinam et al. 2014), and on the effects of binders on palatability and stability of formulated dry feed for p. homarus (saleela et al. 2015). a review on the nutritional requirement and feed development for spiny lobster in postlarval stage reported that tropical lobster p. ornatus had the best growth when they were fed with a diet having a high digestible protein (> 56% dm) and 10-11% total lipid in dry matter (nankervis & jones 2022a). the use of moist pellets was also examined for the growth of lobster p. homarus (ridwanudin et al. 2018). one of the major problems in aquaculture of spiny lobsters is an appropriate formulation of lobster feeds that are readily consumed, attractive, and stimulate optimal growth and survival (marchese et al. 2019). in a previous study, a basal diet formulation for 2-g lobsters had been developed and promoted good growth performance and survival rates of juveniles (irvin & shanks 2015). this formulated feed was produced using ingredients sourced from australia. finding local, high-quality ingredients is the first step that needs to be addressed before indonesian lobster farmers apply formulated feed for growing lobsters. therefore, this study assessed 3-g lobster juveniles fed with a basal diet made from ingredients sourced from australia and locally from indonesia. this experiment was carried out to evaluate the growth and survival of 3-g lobster juveniles fed with different formulated feeds having a profile based on the basal diet, as well as to compare the growth of the lobsters fed with an identical formulated feed with variations only in fish meal sources and dry matter content. materials and methods study location the study was undertaken at the institute for mariculture research and fisheries extension (imrafe), gondol, bali, indonesia. rearing of panulirus homarus post-larvae to 3-g juveniles feeding experiment was conducted by using p. homarus 3-g juveniles. to obtain the 3-g juveniles, pigmented p. homarus post-larvae were obtained from lombok, west nusa tenggara province, from which healthy post-larvae were selected to be reared up to juveniles weighing 3 g. the initial weight of the healthy lobster post-larvae was 0.2 g on average. post-larvae rearing was conducted in floating net cages, concrete tanks, and an hdpe canvas tank with a flow-through seawater system. for the first 2 weeks, the post-larvae were fed with mysids and dry pellets and continued with dry pellets only until reaching 3-g juveniles. grading of the post-larvae was carried out every 2 weeks to prevent cannibalism. post-larvae rearing was conducted until the juveniles reached 3 g in weight. the 3-g juveniles were then used for the feeding experiment. biotropia vol. 31 no. 3, 2024 332 feeding experiment the feeding experiment was designed following a completely randomized design (crd) with five diet treatments and five replicates for each treatment. the experiment was carried out in 25 net cages (60 x 60 x 60 cm) that were placed in an hdpe canvas tank (ø 7.5 m) with a flow-through seawater system, and equipped with aeration as oxygen supply (fig. 1). where: wo = weight of lobster juveniles at the initial stage of feeding experiment (g) wt = weight of lobster juveniles at the final stage of feeding experiment (g) t = duration of the feeding experiment (days) no = number of lobster juveniles at the initial stage of feeding experiment (ind.) nt = number of lobster juveniles at the final stage of feeding experiment (ind.) experimental diets preparation four formulated diets were prepared based on the basal diet, i.e., diet a (basal diet prepared as moist pellets), diet b (moist pellets with the same formulation as diet a, using local fish meal), diet c (moist pellets with the same formulation as diet a, using imported fish meal), and diet d (dry pellets with the same formulation as diet a, using imported fish meal). the fifth diet was fresh fish (diet e). diet a was prepared at csiro’s aquaculture feed technology laboratory in bribie island, australia. diets b, c, and d were prepared in the feed and nutrition laboratory of imrafe, gondol, bali, indonesia. the formulation and nutrient composition of the experimental diets are shown in table 1. figure 1 feeding experiment using 3-g spiny lobster panulirus homarus juveniles in 25 net cages placed in an hdpe canvas tank notes: a = net cages placed in an hdpe canvas tank; b = shelters and an aeration stone inside a netcage; c = experimental feed). lobster juveniles used in this experiment had an average weight of 3.00±0.07 g. the juveniles were stocked at a density of 15 ind./cage and fed with experimental diets according to the treatments, twice every day, at 08.30 a.m. and 3.30 p.m. waste materials, including uneaten feed and feces, exuviae of the molted juveniles, and dead juveniles, were removed from the net cages each time before feeding the juveniles. the feeding experiment was carried out for 12 weeks. data on weight and survival rate were collected every four weeks to calculate the percentage of weight gain (wg), specific growth rate (sgr), and survival rate (sr) as follows: percentage of weight gain (%) = 100 x (wt – wo)/wo specific growth rate (%/day) = 100 x (ln wt − ln wo)/t survival rate (%) = 100 x (nt/no) growth and survival of juvenile spiny lobster, panulirus homarus sudewi et. al. 333 table 1 formulation (% of ingredients) and nutrient composition of experimental diets for the feeding experiment of spiny lobster panulirus homarus 3-g juveniles ingredients diet a 83% dry matter (basal diet) diet b 83% dry matter (local fish meal) diet c 83% dry matter (imported fish meal) diet d 92% dry matter (imported fish meal) diet e (fresh fish) fish meal 65.30 fr es h fis h fish meal b1) 65.30 fish meal a2) 65.30 65.30 wheat flour 6.00 6.00 6.00 6.00 wheat gluten 6.00 6.00 6.00 6.00 mos3) 0.50 0.50 0.50 0.50 fish (fresh) 6.00 6.00 6.00 6.00 mussel (fresh) 6.00 6.00 6.00 6.00 squid (fresh) 1.00 1.00 1.00 1.00 fish oil 2.60 2.60 2.60 2.60 astaxanthin 1.00 1.00 1.00 1.00 cholesterol 0.50 0.50 0.50 0.50 lecithin 1.70 1.70 1.70 1.70 mineral premix 0.60 0.60 0.60 0.60 vitamin premix 1.10 1.10 1.10 1.10 stay c 0.40 0.40 0.40 0.40 binder (cmc) 1.30 1.30 1.30 1.30 nutrient composition by proximate analysis (% of diet) dry matter 83.8 83.3 83.2 91.8 crude protein 47.1 52.0 51.1 51.3 lipid 13.4 14.7 15.1 13.1 ash 5.9 5.4 5.6 7.9 fiber 10.3 8.5 8.7 10.1 notes: 1) = local; 2) = imported; 3) = mannan oligosaccharides; cmc = carboxymethyl cellulose. diet b and c were prepared as moist pellets with a dry matter content of about 83%. on the other hand, diet d was prepared as dry pellets with a dry matter content of approximately 92%. the four experimental diets were made into a 2-mm diameter pellet. the diets were then stored in a freezer before and during the feeding experiment. proximate analysis proximate analysis of the experimental diets was carried out in the chemistry laboratory of imrafe according to the procedure of the association of official analytical chemist (aoac, 2000) for crude protein, lipid, fiber, water, and ash contents. crude protein was determined using kjeldahl method (kjeltec 8100, foss), while lipid content was measured by chloroformmethanol extraction (v/v) method. crude fiber was determined by loss on combustion of dried residue following consecutive digestion of the samples using h2so4 and naoh (fibertec, foss). water content was defined by oven-drying samples at 105 °c to a constant weight using memmert, germany oven. crude ash was analyzed by calculating the residue after heating the samples at 550 °c in a carbolite, england furnace. data analysis data were analyzed descriptively and inferentially using the r commander package in the r statistical program. data obtained from the present study had equal variances; however, those data were not normally distributed which means that the assumption of analysis of variance (anova) was violated. therefore, a non-parametric method, i.e., the kruskal-wallis rank sum test, was used to determine the effect of different formulated feeds on the growth and survival of lobster juveniles. differences in growth and survival between treatments were analyzed using the wilcoxon rank sum test. biotropia vol. 31 no. 3, 2024 334 results and discussion growth of lobster different characteristics of formulated feeds significantly affected the weight of lobster juveniles (p < 0.05). the present study showed that higher growth of lobster juveniles was obtained from the feed containing local fish meal (diet b) and the basal diet (diet a) which resulted in a final weight of 18.74±2.30 g and 17.74±2.54 g, respectively (fig. 2) with daily growth rate of 6.37±0.91%/day and 5.81±0.89%/day, respectively (table 2). feed formulated with local fish meal showed significantly better growth than feed formulated with imported fish meal (diet c) (p < 0.05). no differences in the growth of juveniles were observed as a result of feed having 83% dry matter (moist pellets) and 92% dry matter (dry pellets) (diets c and d) (p > 0.05). growth of lobster juveniles fed with fresh fish (diet e) was slightly higher (12.34±1.63 g with a daily growth rate of 3.51±0.54%/day) than those fed with imported fish meal (diet c and d), however, the growth was significantly lower than those fed with the basal feed (diet a) (p < 0.05) and those fed with feed containing local fish meal (diet b). table 2 growth and survival of spiny lobster panulirus homarus juveniles fed with different diets for 12 weeks diet a 83% dry matter (basal diet) diet b 83% dry matter (local fish meal) diet c 83% dry matter (imported fish meal) diet d 92% dry matter (imported fish meal) diet e (fresh fish) initial weight (g) 3.01±0.12 2.95±0.09 2.98±0.19 2.94±0.10 3.12±0.08 final weight (g) 17.74±2.54b 18.74±2.30b 9.94±0.44a 10.54±1.14a 12.34±1.63a weight gain (g) 14.73±2.47b 15.79±2.27b 6.95±0.50a 7.60±1.10a 9.22±1.58a weight gain (%) 488.41±75.58b 535.15±76.78b 234.03±28.69a 258.66±37.08a 294.91±45.61a daily growth rate (g/day) 0.17±0.02b 0.18±0.02b 0.08±0.00a 0.09±0.01a 0.10±0.01a daily growth rate (%/day) 5.81±0.89b 6.37±0.91b 2.78±0.34a 3.08±0.44a 3.51±0.54a specific growth rate (%/day) 2.10±0.15b 2.19±0.14b 1.43±0.10a 1.51±0.12a 1.62±0.14a survival (%) 24.00±5.96b 36.00±12.99b 46.66±8.16b 45.33±8.69b 13.33±6.66a note: different letters in the same row indicate significant differences (p < 0.05). figure 2 weight (g) of lobster panulirus homarus juveniles fed with different diets for 12 weeks notes: dm = dry matter; locfm = local fish meal; impfm = imported fish meal. this study found that higher growth rates were achieved by feeding lobster juveniles with feed containing local fish meal and that containing basal diet compared to the other treatments, most importantly to the fresh fish diet. the results suggested that formulated feeds can be used to reduce or replace the use of fresh feed which is unsustainable in lobster aquaculture. the good growth performance of lobster juveniles fed with formulated feeds in this study was supported by the nutrient contents of the experimental feeds. the diet containing local fish meal (diet b) had crude protein levels of 52.0% and lipid content of 14.7% (table 1). a review study stated that juvenile and adult lobsters require diets with high protein, low lipid, and moderate to high carbohydrate contents. formulated pellet feed with 56% digestible proteins (61% crude protein) increased the growth of lobsters (nankervis & jones 2022a). daily observation indicated that the formulated feeds were well consumed by the juveniles and supported their growth. the feeds used in this study were formulated with the addition of fresh fish, mussels, and squid. this experiment verified a hypothesis that fresh ingredients included in feed formulation promote good growth performance in spiny lobster juveniles. fresh ingredients contain high levels of chemo-attractants to promote feeding response in crustaceans (irvin & shanks 2015). growth and survival of juvenile spiny lobster, panulirus homarus sudewi et. al. 335 in terms of different sources of fish meal, the present experiment showed that lobster juveniles fed with the diet containing local fish meal had significantly higher growth than those fed with the diet containing imported fish meal. this result contradicts a previous study which reported that lobsters fed with a diet containing imported fish meal (from peru) had a significantly higher weight gain than those fed with a diet containing indonesian fish meal (irvin & shanks 2015). these two experiments indicated that the ingredients of the feed vary in quality, and as a consequence, they have different effects on the growth of lobster. the quality of fish meal and levels of protein vary because of differences in the species of fish used, sources of origin, as well as fish meal processing and storage (irvin & shanks 2015). in the present study, diet b which was formulated using local fish meal had the highest crude protein content (table 1) and may support the highest growth of lobster compared to those fed with the other diets. an increase in growth was also found in lobster p. ornatus fed with a diet containing increased protein content (smith et al. 2003). this study found that there was no difference in the growth of lobster fed with moist (83% dm) and dry (92% dm) pellets. feed intake of lobsters fed with these types of feed was probably the same and had similar effects on the growth of lobster. however, an earlier study reported that growth rates of lobster juveniles fed with dry pellets (90% dm) were significantly lower than those fed with moist pellets having dry matter content ranging from 65% to 82%. this study suggested that moist pellets improved the growth of the juveniles. the soft texture of moist pellets may be suitable for the infantile mouthparts of juveniles and therefore, increased feed intake. improved feed intake was arguably a result of increased palatability provided by high moisture content (irvin & shanks 2015). a 30-day feeding experiment in juveniles of p. versicolor using a semi-moist artificial diet (83% dm) resulted in a high specific growth rate (sgr) of 1.81-2.28%/day (syafrizal et al. 2018). in the present study, the specific growth rate of p. homarus juveniles fed with moist pellets reached 1.42-2.19%/day. the sgr of p. homarus juvenile in the present study was lower than that of p. versicolor which may suggest that juvenile of p. versicolor has a relatively faster growth rate (syafrizal et al. 2018). feeding with fresh fish resulted in similar growth performance to those fed with feed containing imported fish meal (diet c and d), but significantly lower when compared to those fed with feed containing local fish meal (diet b) and the basal diet (diet a). these results could be due to the high mortalities of lobster juveniles fed with fresh fish which affected the average weight of juveniles in each replicate cage. feeding with fresh fish in the present study resulted in a specific growth rate of 1.62%/day. this growth performance was similar to that of p. homarus juveniles with an initial weight of 1.58 g fed with marine clam, donax spp. which exhibited a specific growth rate of 1.64%/ day (vijayakumaran et al. 2009). another study in p. homarus juveniles indicated that moist diet resulted in a higher specific growth rate of lobsters than those fed with trash fish (ridwanudin et al. 2018). a study on p. ornatus juvenile fed with commercial shrimp pellets and fresh mussels had sgr values ranging from 0.49%/day to 1.35%/ day (jones 2007). juveniles fed with fresh fish exhibited pale coloration, in contrast to those fed with formulated feed which showed normal body color (fig. 3). similarly, juvenile of p. ornatus fed with frozen mussel mytilus edulis had poorer body color than juveniles fed with formulated diets (barclay et al. 2006). the normal body color of the juveniles fed with formulated diet obtained in the present study was likely due to the pigmentation role of astaxanthin which was included in the diet (1% inclusion level). the lack of a particular nutrient in the fish used in this study may have influenced the lobster’s body color. it has been recommended to add carotenoids to the diet of lobster to obtain a similar color to the wild-caught lobsters (crear et al. 2002; williams 2007). biotropia vol. 31 no. 3, 2024 336 figure 3 pale coloration of juveniles of lobster panulirus homarus fed with fresh fish (black arrow) survival the present study revealed that survival of the lobster juveniles for all treatments decreased significantly during the 12 weeks of the experiment (fig. 4). mortalities and cannibalism caused low survival in the present study. high mortalities occurred during the first 4 weeks of the experiment (fig. 4). this study showed that different characteristics of feeds in this study significantly affected the survival of lobster juveniles (p < 0.05). higher survival was observed in juveniles fed with feed containing imported fish meal (diet c and d, 46.66% and 45.3%, respectively), followed by those fed with moist pellets containing local fish meal (diet b, 36%) and the basal diet (diet figure 4 survival (%) of juveniles of spiny lobsters panulirus homarus fed with different diets for 12 weeks notes: dm = dry matter; locfm = local fish meal; impfm = imported fish meal. a, 24%). there were no differences in survival of juveniles fed with moist pellets (83% dm) and those fed with dry pellets (92% dm), i.e., diets c and d (p > 0.05). fresh fish diet showed the lowest survival (13.33%) (p < 0.05) (fig. 4). based on daily observation, the low survival in diet e (fresh fish) was mainly due to cannibalism and unsuccessful molting. the unsuccessful molting may have been caused by the lack of nutrient content in the diet. in p. ornatus, low survival was reported when nutrients in the diet were inadequate (smith et al. 2003). high mortality in juveniles of p. ornatus was also found to be mainly caused by cannibalism of newly molted lobsters (jones et al. 2001). growth and survival of juvenile spiny lobster, panulirus homarus sudewi et. al. 337 it has been suggested that inappropriate feeding strategy and shelter may contributed to high cannibalism in juveniles of p. ornatus (jones et al. 2001). results of this study was in line with the survival rates of p. homarus juveniles which were significantly higher when they were fed with moist feed compared to those fed with trash fish (ridwanudin et al. 2018). feeding p. ornatus with green mussels (perna viridis) resulted in considerably low survival (6%) after 4 months of rearing (junio-menez & ruinata 1996). similarly, survival of p. ornatus juveniles fed with blue (mytilus edulis) or greenlipped (p. canaliculus) mussels declined after 4 weeks of the experiment, with high mortalities occurring at molting (smith et al. 2005). juveniles fed with moist (83% dm) and dry (92% dm) pellets containing imported fish meal (diets c and d) resulted in higher survival, however, it indicated lower growth of juveniles. it was likely that the density of juveniles in the cage affected the growth performance of the juveniles. a study in juveniles of p. ornatus showed that the highest survival and weight of lobsters were resulted from the low-density treatment (jones et al. 2001). in the present study, the higher the density, the lower the growth performance of the lobster juveniles. cannibalism in the present study caused the low survival of the juveniles, however, cannibalism provided nutrition for the predatory lobsters. this incident resulted in an inverse relationship between the growth and survival of lobsters. a previous study also reported that lobsters fed with trash fish had superior growth compared to those fed with commercial lobster pellets. however, an inverse survival relationship had been found, with superior survival achieved by feeding lobsters with commercial pellets (irvin & shanks 2015). conclusion the best growth performance was shown by juveniles fed with moist pellets containing local fish meal (diet b) with the final weight reaching 18.74±2.30 g. moist pellets containing imported fish meal (diet c) resulted in higher survival (46.66%), while the lowest survival (13.33%) obtained by juveniles fed with fresh fish (diet e). acknowledgments this study was funded by the aciar project (fis/2014/059). we thank sumardi, selamat, m. suwandi, and m. suparya for their technical support during the experiment. references aoac. 2000. official methods of analysis. 13th edition. washington dc (us): association of official analytical chemists. bahrawi s, priyambodo b, jones cm. 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https://www.sciencedirect.com/science/article/pii/s0044848619333812 https://www.sciencedirect.com/science/article/pii/s0044848619333812 https://www.researchgate.net/publication/328579974_growth_of_spiny_lobster_panulirus_homarus_fed_with_moist_diet https://ejournal2.undip.ac.id/index.php/jkt/article/view/17656 https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2095.2005.00344.x https://onlinelibrary.wiley.com/doi/abs/10.1046/j.1365-2095.2003.00248.x https://onlinelibrary.wiley.com/doi/abs/10.1046/j.1365-2095.2003.00248.x https://www.tandfonline.com/doi/abs/10.1080/00288330909510028 https://www.sciencedirect.com/science/article/pii/s0044848606007678 https://www.sciencedirect.com/science/article/pii/s0044848606007678 _goback 06. 1776 firman ali rahman et al..cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 biotropia vol. 29 no. 1, 2022: 90 94 doi: 10.11598/btb.2022.29.1.1278 90 the range expansion of parachromis managuensis günther, 1867 (perciformes, cichlidae) in java, indonesia maheno sri widodo1, veryl hasan2*, abdul rahem faqih1, maftuch1, r. adharayan islamy1 and felipe polivanov ottoni3 1department of aquatic resources management, universitas brawijaya, malang 65145, indonesia 2department of aquaculture, universitas airlangga, surabaya 60115, indonesia 3laboratório de sistemática e ecologia de organismos aquáticos, centro de ciências agrárias e ambientais, campus universitário, universidade federal do maranhão, br-222, km 04, s/n, boa vista, cep 65500-000, brazil received 28 june 2019 / accepted 24 may 2021 abstract the jaguar cichlid, parachromis managuensis (günther, 1867), is native to central america, with introductions reported from west java and central java provinces of indonesia. on 7-8 january 2019, sixteen specimens of p. managuensis were collected from karangkates, the largest hydropower reservoir in east java province of indonesia. a description of the morphological characters of specimens is provided. keywords: cichlid, distribution, freshwater fish, jaguar guapote introduction parachromis managuensis (günther, 1867), is a cichlid native to costa rica, nicaragua and honduras (conkel 1993), but it has been introduced to several countries in north america (fuller et al. 1999), south america (magalhães & jacobi 2013), and southeast asia (agasen et al. 2006). parachromis managuensis exhibits highly predatory habits and tolerance to new habitats (rosana et al. 2006; agasen et al. 2006), which make p. managuensis potential to become an invasive species (yamamota & annete 2000). parachromis managuensis is generally sold as ornamental fish and has not been cultured openly. p. managuensis in indonesia is firstly found in the freshwaters of west java (dahruddin et al. 2016) and central java (hedianto et al. 2013) provinces of indonesia. the presence of p. managuensis at the karangkates reservoir is considered a new finding because there has been no previous record of exotic fish culture in karangkates reservoir, the largest hydropower reservoir in east java province. materials and methods the fish sampling and description of the study sites sixteen (16) live specimens of p. managuensis were obtained from a local angler during fieldwork conducted on 7 8 january 2019 at the karangkates reservoir (8°11'16"s; 112°27'22"e) (fig. 1). administratively, the karangkates reservoir is located in malang regency, east java province, indonesia. fishing gear used by the angler was a medium hook with a bottom and using worms as bait (stein et al. 2012). *corresponding author, email: veryl.hasan@fpk.unair.ac.id parachromis managuensis in java – maheno sri widodo et al. 91 figure 1 the collecting point of parachromis managuensis at the karangkates reservoir in east java province fish identification the morphological characters of the fish specimens were determined by using the methods employed by kullander and hartel (1997) and bussing (1998). results and discussion specimens collection the sixteen (16) live specimens of p. managuensis had a range of total length between 9.9 mm and 26.6 cm. five (5) of the specimens were preserved in 96% alcohol solution (hasan & taman 2019) and transported to the hydrobiology laboratory, universitas brawijaya, malang, indonesia (voucher no. hb.pm.i.2019). the remaining eleven (11) specimens were kept as livestock at the fish reproduction laboratory, universitas brawijaya, malang, indonesia. the 11 living specimens were transported in oxygen-filled polyethylene bags. diagnosis the morphological characters of the specimens are as follows: a large mouth, projecting lower jaw, prominent enlarged canine teeth, a more or less continuous black stripe between the eye and opercular margin, and another stripe between the eye and the lower angle of the opercle, and a row of black blotches along the middle of the side. the fish can be distinguished from other members of the genus by having the expanded preopercle at the angle. it has silvery or goldengreen to purple body colors and black spots on the fins and body. there are also numerous black spots on the anal and caudal fins. the fish has moss green back, purple iridescence sides, and a whitish or yellowish belly. it also has whitish yellowish, or blue iridescence dorsal interspaces, and a black blotch on the caudal-fin base. all of these characteristics were found in every specimen collected from the karangkates reservoir, east java province, indonesia (fig. 2). biotropia vol. 29 no. 1, 2022 92 figure 2 specimen of parachromis managuensis captured on 8 january 2019 in karangkates reservoir, east java province distribution the discovery of p. managuensis in karangkates reservoir is the first record of this species in east java province. other discoveries occurred at reservoirs in west java and central java provinces. the discovery in karangkates reservoir represents a discovery in the eastern part of java island, which is around 400 km apart from the central java province (fig. 3). this record is an important contribution to understanding the dispersal of alien fish species in indonesia. figure 3 a. distribution of parachromis managuensis in java island notes: red square: west java province; blue square: central java province; green square: east java province). b. location of the karangkates reservoir in east java province note: the green square indicates the new record of p. managuensis. parachromis managuensis in java – maheno sri widodo et al. 93 we speculated that p. managuensis were released into karangkates reservoir in east java province by exotic fish hobbyists without clear purposes. further investigation is warranted to determine the source of p. managuensis in east java province because the reservoir has never been used for any exotic fish culture industry. control and prevention of further introductions are needed to prevent alien fish from disturbing the freshwaters ecosystem (hasan et al. 2020; wijayanti et al. 2021; hasan et al. 2021). conclusion parachromis managuensis is a non-native fish of indonesia. this fish species has been found in freshwater in west java and central java provinces and also in the karangkates reservoir in east java province. the existence of p. managuensis in east java is considered a new finding and added data on the alien fish species distribution in indonesia. acknowledgments the authors sincerely thank the ministry of finance of indonesia for funding this study (grant no. 20160221035555) and the local angler as our field guide. references agasen ev, clemente jp, rosana mr, kawit ns. 2006. biological investigation of jaguar guapote parachromis managuensis (günther, 1867) in taal lake, philippines. j environ sci manag 9(2):20-30. barros lc, santos u, zanuncio jc, dergam ja. 2012. plagioscion squamosissimus (sciaenidae) and parachromis managuensis (cichlidae): a threat to native fishes of the doce river in minas gerais, brazil. plos one 7(6): e39138. https:// doi:10.1371/journal.pone.0039138. bussing wa. 1998. peces de las aguas continentales de costa rica (freshwater fishes of costa rica). 2nd edition. editorial de la universidad de costa rica, san josé. conkel d. 1993. cichlids of north and central americas. new york (us): tfh publications. 191 p. dahruddin h, hutama a, busson f, sauri s, hanner r, keith p, …, hubert n. 2016. revisiting the ichthyo diversity of java and bali through dna barcodes: taxonomic coverage, identification accuracy, cryptic diversity and identification of exotic species. mol ecol resour 17(2):288-99. https://doi:10.1111/1755-0998.12528. fuller pl, nico lg, willians jd. 1999. nonindigenous fish introduced into inland waters of the united states. bethesda (us): american fisheries society. gestring kb, shafland pl. 1997. status and selected life history attributes of the exotic jaguar guapote (cichlasoma managuense) in florida. florida scientist 60(3):137-42. hasan v, tamam mb. 2019. first record of the invasive nile tilapia, oreochromis niloticus (linnaeus, 1758) (perciformes, cichlidae), on bawean island, indonesia. check list 15 (1): 225-227. https:// doi.org/10.15560/15.1.225. hasan v, widodo ms, islamy ra, pebriani daa. 2020. new records of alligator gar, atractosteus spatula (actinopterygii: lepisosteiformes: lepisosteidae) from bali and java, indonesia. acta ichthyologica et piscatoria 50(2): 233-236. https:// doi:10.3750/aiep/02954. hasan v, valen fs, islamy ra, widodo ms, saptadjaja am, islam i. 2021. short communication: presence of the vulnerable freshwater goby sicyopus auxilimentus (gobiidae, sicydiinae) on sangihe island, indonesia. biodiversitas 22: 571-9 https://doi.org/10.13057/biodiv/d220208. hedianto da, purnomo k, warsa a. 2013. interactions of food resources utilization by fish communities in penjalin reservoir, central java. bawal 5(1): 33-40. kullander so, hartel ke. 1997. the systematic status of cichlid genera described by louis agassiz in 1859: amphilophus, baiodon, hypsophrys and parachromis (teleostei: 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upper paraná river basin. check list 13(4): 245-250. https://doi.org/10.15560/13.4.245. stein ja, shultz ad, cooke sj, danylchuk aj, hayward k, suski cd. 2012. the influence of hook size, type, and location on hook retention and survival of angled bonefish (albula vulpes). fish res 113:147-52. wijayanti a, hasan v, tamam mb. 2021. range expansion of oreochromis niloticus (linnaeys, 1758) (perciformes, chichlidae) in java sea and first record for masalembo island. iop conference series earth and environmental science 718(1): 012096. yamamoto mn, annete wt. 2000. hawai'i's native and exotic freshwater animals. honolulu (us): mutual publishing. 478 (kumala dewi effect).cdr effects of blue light and paclobutrazol on seed germination, vegetative growth and yield **of black rice (oryza sativa l. 'cempo ireng') 1* 2 2kumala dewi , rizkika zakka agustina and farida nurmalika 1faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia 2 alumni of faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia received: 8 april 2015/accepted: 8 august 2016 abstract black rice (oryza sativa l. “cempo ireng”) is one of local rice varieties in sleman regency, yogyakarta. the black color is caused by high anthocyanin content which is important source of antioxidant. the cultivation of black rice is still limited due to its tall phenotype, long vegetative stage and low productivity compared to white rice. paclobutrazol is a growth retardant causing dwarfing in several crop plants and reducing lodging. blue light can improve plant quality. this research was aimed to evaluate the effect of blue light and paclobutrazol on seed germination, vegetative growth and yield of black rice. the results showed that the average of seed germination as well as the activity of α-amylase of seeds subjected to blue light were lower compared to those subjected to sunlight; however, paclobutrazol concentrations did not affect seed germination percentage. the height of rice plants treated with paclobutrazol decreased in accordance with the increase of paclobutrazol concentration. chlorophyll content and tiller numbers increased by paclobutrazol treatment of 12.5 ppm. nitrate reductase activity was higher in rice seedlings subjected to blue light compared to those subjected to sunlight. iron (fe) content of rice plants treated with 25 or 50 ppm paclobutrazol increased compared to control. it was concluded that paclobutrazol application of 12.5 ppm already reduced plant height. the higher concentration of paclobutrazol applied the greater reduction on plant height was observed. blue light treatment applied during black rice seed germination slightly reduced germination percentage and α-amylase activity in the germinated seeds. however, blue light treatment combined with paclobutrazol application during black rice seed germination increased chlorophyll content, tiller numbers and fe content in black rice grain. keywords: α-amylase activity, anthocyanin, blue light, fe content, paclobutrazol introduction rice is among staple food having many varieties. besides white rice, there are brown rice, red rice and black rice varieties. the price of black rice is relatively more expensive than that of white rice. the higher price of black rice may have been caused by the increasing awareness of people to consume black rice which contains more vitamins, minerals and anthocyanin. anthocyanin content in black rice was reported to be beneficial for human health as it can lower cholesterol level in the blood and reduce the risk of arteriosclerosis (ling et al. 2001). however, not many farmers choose to cultivate black rice because the vegetative phase of black rice is longer than that of white rice. in addition, birds as well as the rice earhead bug (leptocorisa oratorius) like to feed on developing black rice caryopsis which cause severe loss in black rice yield. the phenotype of black rice is also taller compared to other rice hybrids causing problems in cultivation. paclobutrazol is a synthetic growth inhibitor that acts by blocking gibberellin biosynthesis in plant (rademacher 2000) resulting to semidwarf or dwarf plants. semidwarf character is one of important traits, as semidwarf plants are lodging resistance. the productivity of semidwarf plant also increased because assimilates translocation is more directed to develop seeds rather than to develop new leaves or buds (evans 1993). dwarfisme can be due to either deficiency in biotropia 3 2 6 84 95 vol. 2 no. , 201 : doi: 10.11598/btb.2016.2 . .3 2 478 * corresponding author: kumala.dewi@ugm.ac.id nd** this paper was presented in the 2 international conference on advances in plant sciences (icaps 2014) organized by v sivaram research foundation and universiti teknologi mara on 18 22 november 2014 in kuching, sarawak, malaysia 84 gibberellin biosynthesis or inhibition in gibberellin signalling (spielmeyer et al. 2004). application of paclobutrazol to create semidwarf plants was reported in rice subspecies “keng”, in which 22.5 g/ml paclobutrazol reduced stem height and increased tiller numbers (minzi 1993). it was also reported that application of paclobutrazol effectively reduced vegetative growth of rice plants and increased chlorophyll content. rice seedlings treated with paclobutrazol allocated more photosynthates for seed development compared to control plants or those plants treated with gibberellin (yim et al. 1997). in corn (zea mays l.) under drought stress, application of 50 ppm paclobutrazol increased yield and average weight of 1,000 seeds (bayat & sepehri 2012). blue light is light spectrum having wave length of 320 490 nm (dougher & bugbee 2004). receptor of blue light is known as blue light absorbing photoreceptor (bap) (meijer 1968; gaba & black 1979; thomas 1981). plants respond differently to blue light irradiation (dougher & bugbee 2004). according to guerra et al. (1985), wheat plants subjected to blue light showed a decrease in the activity of enzymes regulating lignification which promoted shoot elongation. in phaseolus vulgaris, blue light increased cell expansion (van volkenburgh et al. 1990). in rice seedlings, respiratory activity of plants subjected to blue light increased to about 1.5 folds compared to those treated with white light. this was due to an increase in the activity of piruvat kinase and malat dehidrogenase enzymes regulating cell respiration which caused the increase of atp production and blue light irradiation (yu & pan 1996). in addition, activity of nitrate reductase, nitrite reductase, glutamate synthetase and glutamin synthetase were higher in rice seedlings subjected to blue light for 5 7 days compared to those subjected to white light. however, if the seedlings were subjected to blue light for more than 10 days, the activity of those enzymes declined (deng et al. 2000). nitrate reductase is an enzyme that catalyzes reduction of nitrate to nitrite. several studies indicated that the level of nitrate reductase in plant tissues is altered by exogenous application of phytohormones (mackintosh 1998). the activity of nitrate reductase in rice increases with the application of cytokinin and 2-chloro ethyl trimethyl ammonium chloride, which are gibberellins biosynthesis inhibitor (hemalatha 2 0 0 2 ) . g l u t a m i n e 2 o x o g l u t a r a t e aminotransferase (glutamate synthase, gogat) and glutamine synthetase (gs) are the key enzymes in primary assimilation and reassimilation of nitrogen in plants. these enzymes catalyze coupled reactions known as the gs/gogat cycle, through which inorganic nitrogen is transformed into organic nitrogen (lu et al. 2011). improving iron content and its bioavailability in rice have been applied to alleviate or to solve micronutrient deficiency in human. iron content in black rice is higher than that in other rice types (meng et al. 2005). several methods to improve iron content and its bioavailability in rice seeds have been conducted, such as breeding, genetic engineering, biochemical and physical approaches. the objective of this research was to evaluate the effect of blue light and paclobutrazol on growth, α-amylase activity, nitrate reductase, iron content and yield in black rice. materials and methods 'cempo ireng' black rice seeds were selected and soaked in aquadest for 24 hours. plastic trays of 18 x 24 cm were prepared and filled with a mixture of top soil and compost (3 : 1 = v/v) as growth media. one hundred seeds were spread and germinated in each plastic tray. the media was then wetted with either tap water (control) or paclobutrazol solution of 12.5, 25, 50 or 100 ppm. seeds were then placed under natural light in the green house or in a growth chamber installed with blue led (light emitting diode). the average daily temperature and relative ohumidity inside the green house were 30 c and 70%, respectively. the seeds were subjected to blue light for 12 hours per day from 06:00 am to 06:00 pm until the seedlings were 3 weeks old. the photosynthesis photon flux level for natural 2light and blue light was 300 µmol/m /s and 30 2µmol/m /s, respectively (li-250a light meter, ®li-cor usa). for each combination treatment, five replications were made. germination percentage was determined on day 7. at the same time, the α-amylase activity in germinated seeds was determined in 10 mg whole grain samples. the ceralpha kit (megazyme international ireland ltd) was used based on the 85 effect of blue light and paclobutrazol on black rice – dewi et al. manufacturer's protocol and with appropriate dilutions. data were expressed in cu (ceralpha unit) per g flour. at day 14, several parameters were determined such as seedling height, chlorophyll content and activity of nitrate reductase. chlorophyll content was determined spectrophotometrically according to harborne (1973). nitrate reductase activity was determined spectrophotometrically according to hartiko et al. (1984). at day 21, seedlings treated with paclobutrazol showing dwarf phenotype were selected and then transplanted in plastic pots containing a mixture of top soil and compost (3 : 1 = v/v). in each plastic pot, two seedlings were planted. each treatment combination had five replications. all plants were watered regularly and organic liquid fertilizer was applied every two weeks. at week twelve, plant height was measured and tiller numbers were determined. the plants were harvested at week 20, followed by the determination of 100 harvested seeds weight and iron content. the iron content was determined using atomic absorption spectrometry (aas). one gram of black rice grain (two samples per treatment) were weighed and placed in a furnace at 500 – 600 °c for 4 hours. samples were then cooled and subjected to wet digestion with 10 ml of hcl 20%. samples were filtered with whatman no.3 filter paper and placed in 50 ml volumetric flask and filled up to the mark with doubledistilled water. total fe content of the grain samples were determined using perkin elmer 3110 aas (welz & sperling 2007). data were analyzed using anova and dmrt (duncan's multiple range test) to determine the significant difference between treatments. results and discussion the average of seed germination percentage of black rice seeds treated with paclobutrazol and blue light is presented in figure 1. under sunlight, germination percentage of black rice seeds was 100% for control seeds. germination percentage of seeds treated with 12.5 ppm paclobutrazol was 98%. germination percentage of seeds treated with 25, 50 or 100 ppm reached 90%. subjected to blue light, germination percentage of black rice seeds was 80 85% and there were no significant differences between control seeds and seeds treated with paclobutrazol. this result indicated that primary dormancy of black rice seeds was possibly influenced by blue light. seed germination can be influenced by dormancy level and light quality. in arabidopsis, white light and red light tend to increase seed germination, while in cerealia, such as barley and wheat, white light and blue light maintain dormancy level in the seeds (gubler et al. 2008). in barley, blue light increased the expression of genes encoding enzymes that catalyze the biosynthesis of abscisic acid. as a consequence, the abscisic acid content was high, but on the other hand, the expression of genes encoding enzymes that catalyze gibberellins biosynthesis decreased (gubler et al. 2008). inhibition in barley seed germination subjected to blue light was due to an increase in abscisic acid level to about 3.5 folds compared to control (hoang et al. 2013). the increase in abscisic acid level was a result of an increase in the expression of genes encoding of the enzyme that catalyzed abscisic acid biosynthesis namely hvnced1 and figure 1 effect of sunlight and blue light against paclobutrazol on average of germination percentage±se of black rice 'cempo ireng' (n = 5) a ve ra ge o f ge rm in at io n p er ce n ta ge 120 100 80 60 40 20 0 control 12.5 ppm 25 ppm 50 ppm 100 ppm paclobutrazol treatments sunlight blue light biotropia vol. 23 no. 2, 2016 86 hvnced2, as well as an increase in the embryo sensitivity to abscisic acid. blue light also decreased the expression of gene encoding of an enzyme that catalyzed gibberellin biosynthesis (hvga3ox2), but increased the expression of gene encoding of an enzyme that catabolized gibberellin. in wheat, seed germination inhibition subjected to blue light can be prevented by the presence of methyl jasmonate or nitrite oxide. it was also proven that methyl jasmonate application reduced the level of abscisic acid, and it was due to a decrease in the expression of gene encoding of an enzyme that catalyzed abscisic acid biosynthesis (tanced1) and a reduction the expression of taaba'oh-1 which encoded an enzyme that catalyzed degradation of abscisic acid (jacobsen et al. 2013). the mechanism by which seed germination in black rice was inhibited by blue light was probably due to an increase in abscisic acid level and reduction in gibberellin biosynthesis. this assumption warrants further examination. α-amylase activity germination processes in cereals are also determined by the activity of enzymes that degrade carbohydrate in the endosperm to its sugar component. α-amylase is one of enzymes that plays a role during cereals seed germination. in this experiment, the activity of α-amylase was determined according to procedure mentioned in ceralpha kit (megazyme international ireland ltd). α-amylase activity in germinated rice grains without paclobutrazol application was slightly lower in grains subjected to blue light compared to those subjected to sunlight (table 1). this finding is related to the average of germination percentage which also decreased in black rice seeds subjected to blue light. in barley, blue light increased abscisic acid level and significantly increased embryo sensitivity to abscisic acid (hoang et al. 2014; barrero et al. 2014). the result of this study indicated that a slight inhibition in seed germination of black rice seeds subjected to blue light was due to either higher level of abscisic acid in the seeds or the sensitivity of embryo to abscisic acid. it was known that abscisic acid repressed the activity and signalling of gibberellin that induced α-amylase during seed germination in cereals (ishibashi et al. 2012). in arabidopsis, the level of endogenous abscisic acid increased about two folds as compared to control by application of uniconazol (saito et al. 2008). in this study, interaction between paclobutrazol and blue light on α-amylase activity was evaluated in the germinated black rice seeds. the activity of αamylase in black rice seeds treated with 12.5 ppm paclobutrazol and germinated under sunlight decreased around 13% compared to control. this could be due to lower level of gibberellin produced during seed germination which led to reduction in α-amylase activity. nevertheless, αamylase activity in black rice seeds treated with 25 to 100 ppm paclobutrazol was relatively similar to control. these results were probably due to the feedback mechanism in which gibberellins biosynthesis was repressed by paclobutrazol. however, the expression of genes encoding of enzymes that catalyzed gibberellins biosynthesis was changed and it might also alter the expression of genes encoding α-amylase and/or α-amylase activity. lenton et al. (1994) showed that paclobutrazol application in wheat caused a decrease in the endogenous gibberellin in the scutellum, but it did not influence the expression of α-amylase mrna. however, the decline in endogenous gibberellin in wheat endosperm was related with downregulation of α-amylase mrna. from that report, it was suggested that the initiation of α-amylase gene expression in the table 1 average±se of α-amylase activity in black rice treated with blue light and paclobutrazol (n=5) treatment α-amylase activity (ceralpha units/g) subjected to sunlight blue light paclobutrazol 0 ppm 204.00±10.29 d 165.88±2.63 a paclobutrazol 12.5 ppm 176.02±7.98 ab 196.47±6.37 bcd paclobutrazol 25 ppm 201.96±11.53cd 198.07±5.72 bcd paclobutrazol 50 ppm 204.60±4.19 d 193.54±7.15 bcd paclobutrazol 100 ppm 197.51±6.67 bcd 178.71±5.14 abc effect of blue light and paclobutrazol on black rice – dewi et al. 87 scutellum did not depend on a new gibberellin. however, the expression of α-amylase gene in the endosperm depended on gibberellin biosynthesis in the embryo. this suggestion is in accordance with the fact that germination still occurred in black rice seeds treated with 100 ppm paclobutrazol, which might be caused by sufficient gibberellin in the scutellum. therefore, initiation of α-amylase gene expression could also be carried out. it should be noted that there was still an inhibition on shoot growth of black rice seeds treated with paclobutrazol. this may be related to inhibition of new gibberellin biosynthesis in the shoot. in arabidopsis, application of uniconazole, which is also a gibberellins inhibitor, caused shoot stunting, but with enhanced elongation of the primary roots (bidadi et al. 2009). in semidwarf rice (sd1) obtained from a mutation of the ga20ox-2 gene, the level of endogenous gibberellin was low, however, the normal height of the semidwarf rice is restored by exogenous application of ga (ashikari et al. 2002; sasaki et al. 2002). this gene is expressed mainly in stems and is an important regulatory step on the pathway to synthesize active ga (reviewed in hedden 2003). semidwarf rice was also obtained from suppression of osga20ox1 gene which suggested to be involved in the regulation of shoot stature in rice (oikawa et al. 2004). in addition, different alleles of the garesponsive dwarf rice (d18) are associated with mutations in the osga3ox2 gene that is also expressed highly in stems (itoh et al. 2001). the probability of phototropin involvement in the biosynthesis of gibberellin and activity of αamylase also warrant further evaluation. the activity of amylase in black rice seeds treated with paclobutrazol and subjected to blue light tend to increase compared to those of control. the endogenous gibberellin decreased rapidly in rice seedlings subjected to blue light, due to a decrease in expression of genes that involved in gibberellin biosynthesis (osga20ox2 and osga20ox4) and an increase in expression of genes encoding the enzyme that catabolized gibberellin (osga2ox4 and osga2ox7). in addition, analysis in blue light receptor mutants (cry 1a dan cry 1b) also showed that the mechanism of blue light in decreasing the biosynthesis of gibberellin was through an increase in the expression of genes that involved in gibberellin catabolism (hirose et al. 2012). commonly, there are several isozymes that catalyze the biosynthesis of gibberellins and it could be that some of those isozymes were upregulated as a feedback mechanism when the level of endogenous gibberellins was lowered by paclobutrazol treatment. the upregulation of some isozymes that involved in gibberellins biosynthesis may also influence the activity of αamylase. mustard (sinapis alba l.) seedlings experienced increase of β-amylase activity in the cotyledon after being subjected to continuous blue light for 42 – 96 hours from sowing. after that, the β-amylase activity abruptly declined (manga & sharma 1988). in this research, the αamylase activity was only determined in 3 days old germinated seeds. further experiment is still required to observe the pattern of amylase activity until seedlings were established. average of plant height and tiller numbers application of paclobutrazol can cause semidwarf or dwarf phenotype and it depends on the concentration applied (rademacher 2000). paclobutrazol concentrations of 200 mg/l to 600 mg/l decreased gibberellin content in the leaves compared to that of control when applied to rice plant during (syahputra et al. 2013). the average of plant height in black rice treated with blue light and paclobutrazol at seedling stage 3 weeks old and at heading date is presented in figure 2 and figure 3. paclobutrazol application reduced plant height (fig. 2 & 5). the measurement that was carried out on 3 weeks old black rice seedlings showed that 12.5 ppm paclobutrazol applied during seed germination stage caused plant height reduction to about 25% compared to control, both in seedlings subjected to blue light or sunlight. greater concentration of paclobutrazol caused severe dwarfism. seeds that still germinated under 100 ppm paclobutrazol were so small and it was impossible to replant the seeds in bigger plastic pots. thus, further growth evaluation only used seedlings treated with either control or 12.5, 25 or 50 ppm paclobutrazol. this study showed that after black rice seedlings were being grown in open area for 12 weeks, those plants treated with 12.5 ppm paclobutrazol with or without blue light during germination still experienced 16 20% reduction in the average of plant height compared to control. black rice plants that previously treated with either 25 or 50 ppm biotropia vol. 23 no. 2, 2016 88 figure 2 average±se height of seedlings treated with blue light and paclobutrazol (n = 18) notes: a – e: bar charts having the same letters on top are not significantly different at p<0.05 based on dmrt (duncan 1955) figure 3 growth performance of black rice seedlings grown under (a) sunlight and (b) blue light control pbz 12.5 ppm pbz 25 ppm pbz 50 ppm figure 4 growth of black rice plant treated with paclobutrazol (pbz) at 6 weeks after planting figure 5 average±se plant height at heading date of black rice plant that previously treated with blue light and paclobutrazol (n = 8) notes: a – g: bar charts having the same letters on top are not significantly different at p<0.05 based on dmrt (duncan 1955) a ve ra n ge h ei gh t o f se ed lin g (c m ) 20 18 16 14 12 10 8 6 4 2 0 control 12.5 ppm 12.5 ppm 25 ppm 25 ppm 50 ppm 50 ppm control sunlight blue light paclobutrazol treatments a b c d e b c c a ve ra ge + s e p la n t h ei gh t at h ea di n g da te ( cm ) 70 60 50 40 30 20 10 0 sunlight blue light control 12.5 ppm 25 ppm 50 ppm paclobuttrazol treatments a e b f c g d g effect of blue light and paclobutrazol on black rice – dewi et al. 89 paclobutrazol still showed dwarfism (about 50% reduction in plant height compared to control). this result indicated that paclobutrazol treatment during seed germination inhibited biosynthesis of gibberellin. however, tiller numbers in plants treated with 50 ppm paclobutrazol increased significantly compared to control. the average of tiller numbers in black rice plants treated with paclobutrazol is presented in figure 6. tiller numbers of black rice seeds subjected to blue light and 12.5 or 25 ppm paclobutrazol increased compared to control (fig. 6). in rice plant, auxin inhibited the biosynthesis of cytokinin and limited tiller growth (liu et al. 2011). application of paclobutrazol on mango plants (mangifera indica l.) inhibited biosynthesis of gibberellin and auxin (adil et al. 2011). thus, it is possible that paclobutrazol treatment in black rice plants reduced biosynthesis of gibberellin as well as auxin. on the other hand, cytokinin biosynthesis increased causing the increase of tiller numbers. the mechanism of paclobutrazol in inducing tiller growth is interesting to be further evaluated. the dwarfism caused by paclobutrazol may also increase the resistance of plants towards waterlogging as well as pests and diseases attacks (bridgemohan & bridgemohan 2014; cohen et al. 1987). chlorophyll content figure 7 shows the average of chlorophyll content in 3 months old black rice plant. the average of total chlorophyll was higher on plants treated with paclobutrazol compared to control (fig.7). this could be due to triazol applied during seed germination increased the biosynthesis of chlorophyll precursor, so that figure 6 average±se tiller numbers of black rice plants subjected to paclobutrazol and blue light (n = 8) notes: a – f : bar charts having the same letters on top are not significantly different at p<0.05 based on dmrt (duncan 1955) figure 7 average±se total chlorophyll content of black rice plants subjected to paclobutrazol and blue light (n = 5) notes: a – g: bar charts having the same letters on top are not significantly different at p<0.05 based on dmrt (duncan 1955) a ve ra ge + s e t ill er n um b er 18 16 14 12 10 8 6 4 2 0 control 12.5 ppm paclobutrazol treatments a 25 ppm 50 ppm sunlight blue lightd b e ab e c f a ve ra ge + se c h lo rr o p hy ll co n te n t (m g/ g f w ) paclobutrazol treatments sunlight blue light control 12.5 ppm 25 ppm 50 ppm 3 2.5 2 1.5 1 0.5 0 a e b f c g d g biotropia vol. 23 no. 2, 2016 90 plants treated with paclobutrazol usually have higher chlorophyll content compared to control (chaney 2005). paclobutrazol application in camelina sativa l. crantz also increased chlorophyll content which led to greater rate in photosynthesis and higher yield (kumar et al. 2012). paclobutrazol also increased the activity of oxidative enzymes and enhanced plant resistence to water lodging in ipomoea batatas l. (lam) (lin et al. 2008). the results of this experiment showed that black rice plants treated with either 25 or 50 ppm paclobutrazol have greener leaves compared to control; the leaves also experienced late senescence. this could be due to an increase in the activity of oxidative enzymes which prevented cell maturation. slow senescence in leaves can prolong the phase of seed development and maturation. as a consequence, the yield can be increased, but the harvest time become late (pan et al. 2013). activity of nitrate reductase nitrate is a source of nitrogen for plant's cell. nitrate is reduced to ammonium in roots which then be stored in the vacuole or transported to leaves. reduction of nitrate to ammonium is catalyzed by nitrate reductase. the activity and transcript level of nitrate reductase in rice plant was suggested to be influenced by g-protein and protein kinase (ali et al. 2007). the average of nitrate reductase activity in black rice plants treated with paclobutrazol and blue light is presented in figure 8. activity of nitrate reductase increased in black rice plant treated with paclobutrazol and subjected to blue light during seed germination (fig. 8). similar finding was reported in arabidopsis, in which application of ga in 3 light-grown col-0 seedlings decreased nitrate reductase activity, but exogenous paclobutrazol enhanced nitrate reductase activity (zhang et al. 2011). it was suggested that paclobutrazol application may also increase several oxidative enzymes (lin et al. 2008). the increase in oxidative enzymes such as guaiacol peroxidase (pod), superoxide dismutases (sod) and catalase (cat) were related to an increase in nitrate reductase activity in ginger plant -5(zingiber officinale rosco) treated with 10 m salycylic acid (ghasemzadeh & jaafar 2013). average weight of 100 seeds the average weight of 100 seeds in black rice plants treated with blue light and paclobutrazol of 25 ppm is presented in figure 9. paclobutrazol treatment did not influence the average weight of 100 seeds obtained from black rice plants subjected to sunlight (fig. 9). however, in plants previously subjected to blue light, application of 25 ppm paclobutrazol increased figure 8 average±se nitrate reductase activity of black rice plants subjected to paclobutrazol and blue light (n = 5) notes: a – d: bar charts having the same letters on top are not significantly different at p<0.05 based on dmrt (duncan 1955) a ve ra ge + s e n it ra te r ed uc ta se a ct iv it y m m n it ri t/ g l ea f f re sh w ei gh t/ h o ur paclobutrazol treatments sunlight blue light control 12.5 ppm 25 ppm 50 ppm 25.000 20/000 15.000 10.000 5.00 0.00 a c a c a b d d effect of blue light and paclobutrazol on black rice – dewi et al. 91 the average weight of 100 seeds compared to control or other treatments. this result was probably due to the fact that when plants were treated with blue light and 25 ppm paclobutrazol, the leaves were greener and the chlorophyll content was higher compared to those of control. in terms of productivity, those plants treated with paclobutrazol of 25 or 50 ppm had higher tiller numbers. eventhough the weight of 100 seeds was similar to control, the total harvest of the plants treated with paclobutrazol was still relatively higher. fe content in the seeds fe content in the seeds was determined for plants treated with blue light and paclobutrazol (fig. 10). there was no significant effect of paclobutrazol on fe content in black rice seeds harvested from plants without blue light treatment during seed germination (fig. 10). on the other hand, when the seeds were germinated under blue light, the plants produced seeds containing higher fe content. the increase of fe content in potato tuber following treatment with paclobutrazol was also reported by tekalign (2005). delaat et al. (2014) reported that ferritin is an essential iron homeostasis regulator in plant. their studies showed that the expression of three ferritin gene in phaseolus vulgaris l (pvfer1, pvfer2 and pvfer3) increased under iron excess and water deficit conditions. it has been reported that application of paclobutrazol could ameliorate abiotic stress condition, such as chilling or drought stress figure 9 average±se weight of 100 seeds of black rice plants subjected to paclobutrazol and blue light (n = 5) notes: a – b: bar charts having the same letters on top are not significantly different at p<0.05 based on dmrt (duncan 1955) figure 10 average fe content in black rice seed treated with blue light and paclobutrazol (n = 2) a ve ra ge w ei gh t o f 10 0 se ed s (g ) 30 25 20 15 10 5 0 control 12.5 ppm paclobutrazol treatments a 25 ppm 50 ppm sunlight blue light a a a a a a b a ve ra ge f e co n te n t in b la ck r ic e se ed 45 40 35 30 25 20 15 10 5 0 (m g/ kg ) control 12.5 ppm paclobutrazol treatments (ppm) 25 ppm 50 ppm sunlight blue light biotropia vol. 23 no. 2, 2016 92 through the ability of the plant to increase the activity of several antioxidant enzymes (pan et al. 2013). in sweet potato, application of triazole increased the content of ascorbic acid, αtocopherol, riboflavin, anthocyanin, and xanthophylls and activities of ascorbate peroxidase, superoxide dismutase, and catalase activities (sivakumar et al. 2010). iron serves as a component of many vital enzymes such as cytochromes of the electron transport chain, thus required for a wide range of biological functions (rout & sahoo 2015). it seems that there is an interaction between the increasing enzymatic activity triggered by triazole and iron uptake and accumulation in plant. the mechanism by which iron was accumulated in rice seeds of those plants treated with blue light and paclobutrazol is important to be further evaluated, since the bioavailability of iron in grains is important for human health. in addition, a simple and environmentally friendly technique for iron biofortification in grains is also important to be developed. conclusions blue light slightly reduced percentage of seed germination. paclobutrazol application up to 100 ppm did not inhibit seed germination. paclobutrazol reduced seedlings height. tiller numbers, total chlorophyll content, nitrate reductase activity and fe content in black rice increased in plants treated with blue light and 25 ppm paclobutrazol. acknowledgements we wish to thank ms. dewi kusumawati, and mr. martono for technical assistance and the “boptn fakultas biologi ugm tahun anggaran 2013” for financial support. references adil os, rahim a, elamin om, bangerth fk. 2011. effects of paclobutrazol (pbz) on floral induction and associated hormonal and metabolic changes of biennialy bearing mango (mangifera indica l.) cultivars during off year. arpn j ag & bio sci 6(2):55–67. ali a, sivakami s, raghuram n. 2007. regulation of activity and transcript 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arabidopsis seedlings. j plant physiol 168(8):2161–8. effect of blue light and paclobutrazol on black rice – dewi et al. 95 page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 biotropia vol. 30 no. 3, 2023: 308 317 doi: 10.11598/btb.2023.30.3.1919 308 effect of thinning on growth and wood production of naturally regenerated 8-yearold acacia mangium willd. plantation on abandoned mining area, southern thailand jetsada wongprom1*, somporn maelim2, wasan chandaeng1, sakhan teejuntuk2, montathip sommeechai2 and decha duangnamon3 1forestry research center, faculty of forestry, kasetsart university, bangkok 10900, thailand 2department of silviculture, faculty of forestry, kasetsart university, bangkok 10900, thailand 3andaman coastal research station for development, faculty of fisheries, kasetsart university, bangkok 10900, thailand received 10 march 2023 / revised 22 august 2023 / accepted 22 august 2023 abstract thinning is an important practice for promoting growth and maintaining forest plantation for wood production from the remaining trees. in this study, thinning was carried out in a naturally regenerated 8-year-old acacia mangium plot in the phangnga forestry research station. three thinning schemes, with 175 (t1), 300 (t2) and 600 (t3) remaining trees/ha, were compared with the control (no thinning) of 831 trees/ha. the diameter at breast height (dbh) and height (h) of the trees were measured. the differences in growth, current annual increment (cai), aboveground biomass, and stem volume (v) were analyzed. we observed that the thinning of a. mangium increased the growth rate, with the dbh being clearly affected by thinning. caidbh increased significantly, with the dbh class of thinned a. mangium plots also improving after thinning. the stem volume and aboveground biomass of t3 plot was similar to the control plot after thinning. in addition, the number of large saw logs was the highest in t3 plot. the large saw logs can be used for multi-utilization and have a high value. these results suggest that thinning can promote stem growth, and increase the proportion of large saw logs in naturally regenerated a. mangium stands. keywords: abandoned mining area, aboveground biomass, acacia mangium, growth, merchantable volume, thinning introduction acacia mangium willd. is a fast-growing multipurpose tree species and is usually found in tropical plantations (hegde et al. 2013). it has been widely planted for soil improvement of degraded lands (martpalakorn 1990, majid et al. 1998), as it is a nitrogen fixing tree and can supply nutrients back into the forest floor via litter decomposition processes (fisher & binkley 2000). a. mangium has thus been widely planted in southeast asia in commercial plantations (nambiar & harwood 2014). a density of approximately 1,100 stem/ha or a spacing of 3 m x 3 m is commonly used while planting a. mangium. a high density is usually recommended for trees grown in short-rotation periods (saharjo 2006), with the rotation for a. mangium being between 5-8 years to be used in wood chip and pulp production (huong et al. 2020b). on the other hand, older large trees, usually around 15year-old, are used in furniture making and to obtain sawn wood (yahya 1993). thinning is a silviculture practice to increase tree growth and stem volume of the remaining trees (yahya et al. 2011; beadle et al. 2013), as well as to improve the stem form and wood quality (pérez & kanninen 2005). it is commonly practiced in fast-growing trees species such as eucalyptus, a. auriculiformis, and acacia hybrid (hung et al. 2019; huong et al. 2020a). as a management practice in a plantation, thinning is used to reduce the number of trees in a stand, so as to increase the crown space between the remaining trees, to reduce the crown and root competition, and to increase growth. in addition, *corresponding author, email: fforjdw@ku.ac.th https://www.researchgate.net/profile/sakhan-teejuntuk?_sg%5b0%5d=0q7r8kb8nergti99s50vgejyb7-qeippldjhvffsjqiasexygddfabrabd7dweebcgjiknq.tv_8l6j13rhlet-bfmlr6jvxvgxgtzc8kn8-aa9wys9isrdckgegoxngrilked6mke4cie0x5cf2abufdjqcqg&_sg%5b1%5d=53c3xrjfm1lu4e6xy8e2a6w-zxxgv_0c563blfrgh5ctsvchbew_rcdicaqpwmmmboay1by.3q9gwfjv2f_yfmlp5nqtnhzmc7gin47hkf9v1ggudo8oqj9tniu4qjpb6hsc_0nzmyuafqd_h4sybs4wu2kkna https://sciprofiles.com/profile/347609 effect of thinning on growth and wood production of acacia mangium – wongprom et al. 309 thinning can help to control pests and diseases, which in turn can increase the earned incomes before the final harvest (onyekwelu et al. 2011). however, as a downside, the stem volume and aboveground biomass of thinned plantation can decrease. past mining activity has had an extreme impact on the soil quality and has severely affected the adjoining ecosystems through the loss of soil structure and nutrient depletion (thaiutsa & rungruangsilp 1990; maiti 2013). the phangnga forestry research station is located on one such abandoned mining area. the reclamation of mining area was undertaken by planting a. mangium, given its growth and high aboveground biomass compared to other fastgrowing or native tree species (martpalakorn 1990). additionally, a. mangium has reportedly been recommended to rehabilitate degraded lands with a high survival rate (majid et al. 1998) to restore the soil properties, forest structure, and nutrient cycling (wongprom et al. 2020; wongprom et al. 2022; staporn et al. 2022). seeds of a. mangium can accumulate in the soil and forest floor (saharjo 2006), with the seedlings being highly dense after clear-cutting. a high tree density can result in the reduction of growth rate and yield, accompanied by high mortality due to competition. a. mangium plantations mainly focus on producing wood with a short-rotation period but the management of a. mangium for the production of large saw logs has been rarely studied. timber production from natural forests is expected to decline, and as such, a. mangium plantations can play an important role in maintaining the commercial supply of wood. thinning is recommended for trees used in timber and sawn wood production, with larger trees having the potential to increase the income earned (onyekwelu et al. 2011). the objectives of this study were to identify the effects of thinning on the growth and wood production of a naturally regenerated a. mangium plantation on an abandoned mining area. thinning was applied to stands which were established through natural regeneration after clear-cutting of the a. mangium plantation. these results can be used to manage a. mangium stand in the phangnga forestry research station and other such degraded sites located in southern thailand. materials and methods study site the plantation is located on an abandoned area, previously under tin mining, at the phangnga forestry research station (8˚ 46' 5" n, 98˚ 16' 7" e), takuapa district, phangnga province, in southern thailand (fig. 1). tin mining was done through the gravel pumping method. the post tin mining land forms could be mainly divided into sand, clay, and gravel areas. the soil nutrients and organic matter levels after tin mining were very low and the soil ph was strongly acidic (anunsiriwat 1986). however, soil nutrients and organic matter content of this area were improved after the establishment of a. mangium, especially the topsoil level. the soil properties of this area are shown in table 1 (wachrinrat et al. 2002). the area receives an annual rainfall of 3,566 mm, with the rainy season spanning from april to october and dry season occurring during the months from november to march. the mean temperature was around 27.1°c and relative humidity around 83% (wachrinrat et al. 2002). figure 1 location of the study sites in phangnga province, southern thailand biotropia vol. 30 no. 3, 2023 310 table 1 soil properties of a. mangium plantation in clay area at phangnga forestry research station soil depth (cm) sand (%) silt (%) clay (%) soil texture ph om (%) avai. p (ppm) exchangeable bases (ppm) k ca mg 0-7 10.40 30.72 58.88 clay 5.0 3.84 1.01 62.2 153.44 54.44 7-32 0.40 24.72 74.88 clay 4.9 0.91 0.03 38.4 11.68 67.34 note: om = organic matter, avai. p = available phosphorus, k = potassium, ca = calcium, mg = magnesium. in 1987, a. mangium was planted at a spacing of 4 m x 4 m, to reclaim the clayey soil (wachrinrat et al. 2002). however, a clear cutting of a. mangium plantation was done for wood utilization when the trees were 14-year-old. after the area was cleared by cutting, the site was prepared by burning the branches and other parts of the trees. a. mangium seedlings were then allowed to naturally regenerate in the area and this resulted in a high density stand. methods the experiment plots were located in an 8year-old naturally regenerated a. mangium plantation. we observed that the crown cover of a. mangium stand was closed leading to a strong crown competition, which meant that thinning was needed for plantation management. a randomized completely block design (rcbd) with three replications was used in a plot of size 20 m x 20 m. three different thinning schemes were used and included 175 (t1), 300 (t2), and 600 (t3) remaining trees per ha which were compared with a control of 831 trees/ha. a low thinning method was used in t1, t2, and t3 plots. the low thinning scheme was used to remove the suppressed and poor crown trees (hawley 1947). in this study, any small and irregular trees were removed in the thinned plots. in addition, a good stem form of the co-dominant and intermediate trees in the t1, t2, and t3 plots were also removed to determine the spacing and tree density. the diameter at breast height (dbh) and height (h) of remaining trees were measured for a period of three years. the dbh and h of each treatment was calculated as a mean among replications (n = 3). the current annual increment (cai) of h and dbh were calculated using the equations: caidbh = (dbh2dbh1)/ t2-t1 caih = (h2h1)/ t2-t1 where: h1 = tree heights (m) at times t1 h2 = tree heights (m) at times t2t dbh1 = diameters (cm) at times t1 dbh2 = diameters (cm) at times t2 t1 = beginning times of each period t2 = ending times of each period. allometric equations of the 11-year-old a. mangium trees were developed to estimate the aboveground biomass and stem volume, as the estimation equation previously reported for woodchip was for trees within an age bracket of 4-5 years in thailand (peawsa-ad & viriyabuncha 2002). in this study, trees sampled from seven different dbh classes (8.4 to 34.9 cm dbh) were cut and separated as logs according to their respective heights binned under 0-0.3 m, 0.3-1.3 m, 1.3-2.3 m, etc., i.e., at every 1.0 m increment from the bottom to top. the tree height and diameter of the logs were measured. the fresh weights of stem, branch, and leaf components were determined in the field and representative samples were taken from each tree to determine the dry weight. the stem, branch and leaf samples were oven-dried at 80°c for 48 h to obtain a constant weight. the aboveground biomass, i.e., stem (ws in kg), branches (wb in kg), and leaf (wl in kg), of a. mangium was estimated using the allometric equations derived using destructive sampling as follows: ws = 0.0199*(dbh2*h)0.9828, (r2 = 0.98) wb = 0.0001*(dbh2*h)1.3345, (r2 = 0.99) wl = 0.0009*(dbh2*h)0.9773, (r2 = 0.94) wt = ws+wb+wl, where: wt = total aboveground biomass dbh = diameter at breast height (cm) h = total height (m) effect of thinning on growth and wood production of acacia mangium – wongprom et al. 311 the volume of each log was calculated using the smalian's formula: v = (ba1+ba2)/2 x l where: v = log volume (m3) ba1 = upper cross section area of the log ba2 = lower cross section area of the log l = length of the log the total stem volume (vt) and merchantable volume (vm) was estimated using the equation derived from destructive sampling. the merchantable volume was set at a top end diameter > 10.0 cm, as determined by the local wood sawmill; vt = 0.0395*(dbh) 0.3369, (r2= 0.98) vm = 0.0403*(dbh) 0.4030. (r2= 0.98) allometric equations for dbh and stem-log types were developed from seven representative trees (with dbh between 8.4 to 34.9 cm). sawlog types were categorized as either small (vssl; diameter of log (d) 10.0 < d ≤ 15.0 cm and as a percentage of merchantable volume), medium (vmsl; 15.0 < d ≤ 20.0 cm), or large (vlsl; d > 20.0 cm). vssl and vlsl were determined by establishing the respective allometric equations. vmsl was determined as the difference between the merchantable volume (100%) and the sum of vssl and vlsl. the sum of vssl, vmsl, and vlsl was equal to vm. the log components of each tree were estimated as a percentage of the merchantable volume (vm = 100%) according to the following equations: vssl = 418.76e-0.137*(dbh), (r2= 0.97) vlsl = 8.9603e0.0677*(dbh), (r2= 0.77) vmsl = 100 – (vssl + vlsl) vm = vssl + vmsl + vlsl, where vm is the merchantable volume (m3). data analysis growth performance, as indicated by dbh, h, caidbh and caih, the aboveground biomass of trees, stem volume, merchantable volume, and saw logs among the various treatments was compared using spss 16.0. a one-way analysis of variance (anova) followed by tukey hsd was used to determine the differences between means at a 5% probability level. results and discussion tree growth thinning had a positive influence on the growth of a. mangium, although the dbh and h were not found to be significantly different (p > 0.05) during the initial stages of development. the parameters measured during the initial stages of growth and development after thinning are listed in table 2. after thinning for one year, the dbh and h of trees in the t1 treatment increased rapidly compared with the control plot and were significantly different (p < 0.05). it has been previously reported that a. mangium grows well after an early thinning (yahya 1993). heavy thinning is an important factor influencing the dbh with its incremental change significantly and positively correlated with the thinning intensity (mäkinen & isomäki 2004; juodvalkis et al. 2005). in this study, the small and suppressed trees were removed by thinning. this resulted in a structured stand, with a marked increase in measured dbh. the size distribution moved towards normality and then became positively skewed, and was affected by low intensity thinning. larger dbh values were found in thinned plots, while trees with smaller dbh were frequently found in the unthinned plot (fig. 2). caidbh was the highest in the t1 plot, followed by t2, t3, and control, respectively. the density of a. mangium significantly influenced the caidbh after thinning during the first and second years, but no significant difference was observed in the third year. caidbh of trees in the thinned plots peaked during the second year. however, caidbh tended to decrease and was not significantly different among treatments after thinning for three years. a rapid reduction in caidbh from 2.47 cm/cm/yr to 1.03 cm/cm/yr under thinning for 1-3 years in the t1 plot indicates a strong competition between the remaining trees. a dense a. mangium canopy was observed after thinning for three years. a crown competition within the stand led to the death of small and suppressed trees. a high relative mortality rate of 21.98% was observed in the unthinned plot. caih was significantly different after the thinning for one year, but was not significantly different after thinning for 2-3 years. this indicates that the increase in height was affected biotropia vol. 30 no. 3, 2023 312 by thinning only during the early period. we observed that the thinning intensity only slightly affected the increase in height and is similar to results reported previously for many plantations (wanthongchai & sahunalu 2002; mäkinen & isomäki 2004; cicek et al. 2013; rytter 2013). figure 2 distribution of dbh classes of the thinned a. mangium during the initial stages of development (a) and after three years of thinning (b) at the phangnga forestry research station table 2 diameter at breast height (dbh), height (h), and current annual increment (cai) of the thinned a. mangium plots after thinning for one, two, and three years at the phangnga forestry research station treatment dbh (cm) h (m) cai dbh (cm) h (m) before thinning t1 21.94 ± 1.95 24.18 ± 0.53 t2 19.91 ± 1.20 23.56 ± 0.76 t3 19.47 ± 1.57 23.17 ± 0.71 control 17.24 ± 2.23 21.79 ± 2.59 fvalue 2.58ns 3.40ns after thinning for one year t1 25.14 ± 1.96b 26.22 ± 0.72b 2.47 ± 0.26c 1.20 ± 0.04c t2 22.34 ± 1.16ab 24.84 ± 0.47b 1.80 ± 0.24b 0.87 ± 0.13b t3 21.22 ± 1.49ab 24.38 ± 0.64b 1.40 ± 0.27b 0.67 ± 0.14b control 17.56 ± 3.03a 22.29 ± 1.18a 0.64 ± 0.25a 0.54 ± 0.12a fvalue 7.13* 12.57** 26.74** 19.14** after thinning for two years t1 27.65 ± 2.52c 27.19 ± 1.00b 2.51 ± 0.74c 0.96 ± 0.33 t2 24.30 ± 1.25bc 25.69 ± 0.50b 1.96 ± 0.13bc 0.85 ± 0.10 t3 22.85 ± 1.39b 25.10 ± 0.56b 1.63 ± 0.11b 0.72 ± 0.10 control 18.33 ± 3.19a 22.94 ± 1.74a 0.77 ± 0.27a 0.65 ± 0.62 fvalue 8.98** 8.15** 9.90** 0.47ns after thinning for three years t1 28.65 ± 2.55b 27.58 ± 0.99b 1.03 ± 0.13 0.39 ± 0.10 t2 25.28 ± 1.15b 25.99 ± 0.40b 0.98 ± 0.14 0.38 ± 0.15 t3 23.71 ± 1.12ab 25.48 ± 0.44ab 0.86 ± 0.30 0.29 ± 0.14 control 18.93 ± 3.36a 23.08 ± 1.70a 0.60 ± 0.22 0.14 ± 0.10 fvalue 9.62** 9.82** 2.36ns 3.64ns notes: * = significantly different at p < 0.05; ** = significantly different at p < 0.01; ns = not significant at p > 0.05 and letters a, b and c in the same column indicate significant differences at p < 0.05 and p < 0.01, as determined by tukey hsd. a b effect of thinning on growth and wood production of acacia mangium – wongprom et al. 313 aboveground biomass the aboveground biomass among treatments was significantly different after thinning for years 1-3 (table 3). in the thinned plots, the aboveground biomass from all parts of a tree was the highest for t3 plot, while that for t1 plot was the lowest. however, the aboveground biomass of the t3 and control plots was similar. differences in density can significantly affect the production of plantation. this is evident from our observation that the aboveground biomass estimated for the t3 (169.89 t/ha) and control (185.74 t/ha) plots was relatively high, but was lower than that of a 10-year-old a. mangium plantation (1,050 stem/ha) planted on degraded lands in indonesia, with value of 241.10 t/ha (hardiyanto et al. 2004). stem biomass contributed the most to the main productivity, accounting for approximately 81-84% of the total aboveground biomass. on the other hand, the contribution of leaf component to the biomass pool was the lowest (4%). leaf production of a. mangium is not the main objective of a commercial plantation. however, the contributions of leaf biomass and leaf litter were reported to be the most significant in terms of nutrient return to the forest floor (nambiar & hardwood 2014; wongprom et al. 2022), as a. mangium leaf is rich in nutrient concentration, especially nitrogen. it therefore plays an important role in increasing the soil nutrients, improving the soil properties (wongprom et al. 2020), and promoting nutrient supply for stand growth (hardiyanto & nambiar 2014). previously, it has been reported that the wood production in an acacia plantation is strongly correlated with the soil nutrients (harwood et al. 2017). this observation was similar to huong et al. (2020b), who found that four rotations in a. auriculiformis plantation resulted in high growth and production compared to the first rotation. table 3 aboveground biomass of the 8-year-old a. mangium under various thinning durations and intensities treatment aboveground biomass (ton/ha) ws wb wl wt after thinning for one year t1 52.02 ± 8.53a 8.16 ± 1.94a 2.65 ± 0.47a 62.83 ± 10.94a t2 69.03 ± 6.17a 10.34 ± 1.79ab 3.49 ± 0.35a 82.86 ± 8.33a t3 119.95 ± 21.04c 16.34 ± 4.33 ab 5.99 ± 1.13b 142.28 ± 24.84b control 140.77 ± 10.70c 17.22 ± 0.11ab 5.03 ± 0.43b 163.02 ± 11.05b fvalue 33.68** 8.49* 28.03** 30.62** after thinning for two years t1 61.99 ± 12.05a 10.60 ± 2.99a 3.21 ± 0.67a 75.80 ± 15.71a t2 83.16 ± 7.08a 13.67 ± 2.40ab 4.27 ± 0.41a 101.10 ± 9.77a t3 141.67 ± 22.63b 21.05 ± 5.16b 7.17 ± 1.24b 169.89 ± 27.01b control 156.38 ± 7.16b 20.61 ± 0.91b 7.75 ± 0.25b 185.74 ± 6.54b fvalue 33.38** 7.05* 26.51** 30.78** after thinning for three years t1 66.65 ± 13.09a 11.77 ± 3.41a 3.47 ± 0.73a 81.89 ± 17.24a t2 90.42 ± 5.14a 15.05 ± 2.57ab 4.64 ± 0.34a 110.11 ± 7.91a t3 152.53 ± 19.82b 23.27 ± 4.66b 7.75 ± 1.09b 183.55 ± 25.54b control 161.46 ± 6.61b 21.79 ± 1.00b 7.92 ± 0.28b 191.17 ± 6.15b fvalue 42.41** 8.01* 30.89** 37.63** notes: * = indicates a significant difference at p < 0.05; ** = significantly different at p < 0.01; letters a, b, and c in the same column indicate significant difference at p < 0.05, as determined by tukey hsd. ws, wb, wl, and wt are the stem, branches, leaf biomass, and total aboveground biomass, respectively. biotropia vol. 30 no. 3, 2023 314 stem volume, merchantable volume, and sawlogs after thinning for three years, the stem and merchantable volumes of the thinned and control a. mangium plots were significantly different (p<0.01) (table 4). in addition, the production of small, medium, and large saw logs was also significantly different among the treatments. however, stem and merchantable volumes of the t3 and control plots were similar. thinning intervention positively affected the diameter of logs. heavy thinning in the t1 plot resulted in a high proportion of large saw logs (67% of merchantable volume). the proportion of large saw logs in the thinned plots was higher than both the medium and small sized saw logs. in contrast, the proportion of small saw logs was high in the unthinned plot (25%), while those in the t1, t2, and t3 was 9, 14 and 16%, respectively. this indicates that thinning increased the merchantable volume and the number of large saw logs. for 9-year-old a. auriculiformis plantation (1,333 stem/ha), the medium sized saw logs formed the major proportion of saw logs obtained from the unthinned plot (huong et al. 2020a). thinning is important for improving the dbh class of a stand as well as the merchantable volume and is usually used as a plantation management strategy to increase growth and utilization. for 9 and 10 year plantations, the current annual increment of volume (caiv) was found to be significantly different (p<0.01), with the t3 plot having the highest caiv. however, caiv of the t3 and unthinned plots was similar for the 11year-old plantation. low thinning resulted in a significant increase of stand stem volume (fig. 3). after thinning for one and two years, caiv of the t1 and t2 plots increased, while the values were not different for the unthinned plot. heavy and moderate thinning intensity had a significant influence on the increment in stem volume of the a. mangium plot, given that the trees had a positive response to thinning. table 4 stem volume, merchantable volume, and saw logs of the thinned 11-year-old acacia mangium stand under various thinning intensities treatment stem volume (m3/ha) merchantable volume (m3/ha) sawlogs (m3/ha) small medium large t1 135.83 ± 16.79a 127.92 ± 17.37a 11.25 ± 1.75a (9%) 31.25 ± 6.75a (24%) 85.42 ± 21.11a (67%) t2 187.92 ± 6.71b 180.99 ± 13.65b 25.75 ± 5.88b (14%) 47.83 ± 10.07a (27%) 107.41 ± 23.46a (59%) t3 353.83 ± 23.76c 326.19 ± 23.73c 51.00 ± 3.54c (16%) 101.08 ± 7.44b (31%) 174.16 ± 24.56b (53%) control 351.10 ± 22.33c 298.44 ± 15.27c 74.60 ± 10.69d (25%) 95.49 ± 7.07b (32%) 128.33 ± 12.57a (43%) f-value 145.57** 92.44** 165.70** 51.05** 7.54* notes: * = indicates a significant difference at p < 0.05; ** = significantly different at p < 0.01; letters a and b in the same column indicate significant difference at p < 0.05, as determined by tukey hsd. the numbers in brackets are percentage of saw log types estimated for each treatment. figure 3 the current annual increment of stem volume (caiv) of the thinned 9 to 11-year-old a. mangium plots under various thinning durations effect of thinning on growth and wood production of acacia mangium – wongprom et al. 315 in southern thailand, the local sawmills have a high demand for large saw logs to make wood furniture, as these products fetch a higher value compared to woodchips. in thailand, during the year 2021, the timber imported was approximately 220,000 m3 (royal forestry department 2021). as such, fast-growing tree plantations can reduce the import bill related to import of timber wood, while also reducing the illegal cutting of natural forests. thinning can be a valuable management strategy in plantations to increase the domestic timber production. a short rotation period (4-7 years) is generally used for the production of fuelwood and woodchips from a. mangium plantations. the growth, aboveground biomass, and stem volume estimates reported in this study are based on a plantation established on poor soil conditions in an abandoned mining area where soil nutrients and organic matter were very low (anunsiriwat 1986; thaiutsa & rungruangsilp 1990). however, a. mangium was able to grow well in this area as the site receives a high amount of rainfall (more than 3,500 mm/yr), with a rainfall of more than 2,500 mm/yr being suitable for the optimum growth of a. mangium (national research council 1983). the dbh and h of the t2 plot (24.30 cm and 25.69 m, respectively) in the 10year-old a. mangium plantation was similar to that of a. mangium planted in west java, indonesia, with a dbh, h, and density of 27.81 cm, 25.12 m, and 225 stem/ha, respectively (heriansyah et al. 2007). in addition, the estimated total aboveground biomass and stem volume in this study was not different from that of other areas with a similar stand density. for example, the aboveground biomass estimated for the lightly thinned 11-year-old a. mangium plantation, with a density of 600 stem/ha was similar to that of an a. mangium plantation in southeastern, vietnam (cuong et al. 2020). conclusion in this study, we presented the results of thinning on the growth of a. mangium trees in a naturally regenerated 8-year-old plantation in the phangnga forestry research station, with three different thinning schemes, which were compared with the control (unthinned plot). the growth and production of the remaining trees after thinning were observed to be affected by thinning. after thinning, the dbh and caidbh of trees in the thinned plots were found to be significantly higher than those in the control plot. however, tree height was only slightly affected by thinning. a reduction in caidbh was observed after thinning for three years, possibly resulting from a stronger competition for resources. the total aboveground biomass and stem volume in the lightly thinned t3 plot was similar to that of the control plot. the thinning intensity significantly affected the growth and productivity of the a. mangium stand. caiv of the t3 plot was relatively higher than that of the t1 and t2 plots. however, the large saw logs obtained from the t3 and unthinned plots was significantly different, suggesting that thinning should be done for promoting stem growth and to obtain large sized timber wood. acknowledgements this work was partially funded by the national research council of thailand through the knowledge hub for integrated economic trees: plantation establishment, management, utilization and industry. we thank dr. atsushi sakai and asst. prof. dr. kobsak wanthongchai for their comments and help in improving this paper. we are grateful to the staff at the phangnga forestry research station for their assistance during the field work. references anunsiriwat a. 1986. biomass production and some soil properties of mine spoils at bangmuang, takua pa, phangnga [dissertation]. retrieved from kasetsart university. beadle cl, trieu dt, wood ce. 2013. thinning increase saw-log values in fast-growing plantations of acacia hybrid in vietnam. j trop for sci 25: 42-51. cicek e, yilmaz f, ozbayram ka, efe m, yilmaz m, usta a. 2013. effects of thinning intensity on the growth of narrow-leaved ash (fraxinus angustifolia sub sp. oxycarpa) plantations. turk. j agric for 37: 97-104. cuong l, hung b, bolanle-ojo ot, xu x, thanh n, chai l, legesse n, wang j, thang b. 2020. biomass and carbon storage in an age-sequence of acacia mangium 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thai j for 19-21: 9-20. https://www.sciencedirect.com/science/journal/03781127 https://www.ingentaconnect.com/content/cfa/ifr;jsessionid=fnoc3ai4l6ai.x-ic-live-03 https://www.sciencedirect.com/science/journal/03781127 https://www.sciencedirect.com/science/journal/03781127 https://link.springer.com/book/10.1007/4-431-28054-5#author-1-1 https://link.springer.com/book/10.1007/4-431-28054-5#author-1-2 https://link.springer.com/book/10.1007/4-431-28054-5#author-1-3 effect of thinning on growth and wood production of acacia mangium – wongprom et al. 317 wongprom j, poolsiri r, diloksumpun s, ngernsaengsaruay c. 2020. soil properties and tree composition in a 27-year-old acacia mangium willd. plantation on abandoned mining area at phangnga forestry research station. biotropia 27: 125-33. wongprom j, poolsiri r, diloksumpun s, ngernsaengsaruay c, tansakul s, chandaeng w. 2020. litterfall, litter decomposition and nutrient return of rehabilitated mining areas and natural forest in phangnga forestry research station, southern thailand. biotropia 29: 69-80. yahya az. 1993. growth of acacia mangium during three years following thinning: preliminary results. j trop for sci 6: 171-80. yahya az, saaiffudin ka, hashim mn. 2011. growth response and yield of plantation-grown teak (tectona grandis) after low thinning treatments at pagoh, peninsular malaysia. j trop for sci 23: 453-59. 09. 1861 venus b. kinamot & alvin p. monotilla.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 page 14 page 15 09. 1861-venus b. kinamot & alvin p. monotilla.pdf page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 page 9 page 10 page 11 page 12 page 13 page 14 page 15 biotropia vol. 31 no. 1, 2024: 87 95 doi: 10.11598/btb.2024.31.1.2120 87 6-benzylaminopurine induces high-frequency multiplication in vulnerable hill turmeric (curcuma pseudomontana j. graham): a potential in vitro conservation tool gayatri v. vaze1*, pramod j. hurkadale1 and harsha v. hegde2 1kle college of pharmacy, kle academy of higher education and research, belagavi 590010, india 2national institute of traditional medicine, indian council of medical research, belagavi 590 010, india received 4 november 2023/ revised 8 december 2023/ accepted 8 january 2024 abstract a rapid high-frequency multiplication protocol is designed for curcuma pseudomontana j. graham, belonging to the family zingiberaceae, an endemic species to the western ghats of india. this species is listed as vulnerable by the international union for conservation of nature’s red list of threatened taxa, highlighting the various underlying causes contributing to its vulnerability. notably, the plant holds significant value in traditional and tribal medicine. the species has suffered habitat loss due to uncontrolled use in tribal medicine, leading to a 30% decline over the last decade. this study is strategically designed with the specific objective of conserving the species, marking the first-ever report on the micropropagation of c. pseudomontana using in vitro multiplication. an efficient and rapid protocol for micropropagation is developed using rhizome bud explants, which are transferred from the ms basal medium onto the ms medium fortified with 6-benzylaminopurine (bap), kinetin (kn), and thidiazuron (tdz) at a varying concentration range. the maximum shoot induction is achieved in the ms medium enriched with bap at a concentration of 2 mg l‒1, resulting in 9.66 ±2.08 shoots per explant with a shoot length of 6.40 ±0.36 cm. the response to root induction is investigated by aseptically transferring the shoots onto ms medium enriched with α-naphthaleneacetic acid (naa), indole-3-butyric acid (iba), and indole-3-acetic acid (iaa) at various concentrations. noticeably, the maximum root length and root number are recorded in ms supplemented with naa at 0.5 mg l‒1. despite the variations in root induction treatments, a 100% root induction frequency is observed in all the samples under study. the rooted plantlets are carefully removed from the culture flasks and transferred into hardening media containing a 1:1:1 ratio of sand:soil:cocopeat. these hardened plants exhibit good health, are disease-free, and show a 92% survival rate after acclimatisation. keywords: conservation, curcuma pseudomontana, micropropagation, vulnerable, western ghats introduction curcuma pseudomontana j. graham, popularly referred to as hill turmeric, has been classified as a vulnerable species according to the international union for conservation of nature’s red list of threatened taxa (romand-monnier & contu 2013). c. pseudomontana is endemic to peninsular india, specifically found in states along the western ghats. it is an erect rhizomatous herb that attains a height of up to 75 cm, thriving at altitudes of 300‒1500 m. typically, it inhabits the moist and shaded areas of wet forests along sluggish grassy slopes. the plant bears almondlike sub-globose tubers at the end of its fibrous roots. the rhizomes of c. pseudomontana are small, measuring 3‒6 cm in length and 1.25 cm in width, and exhibiting a slightly cylindrical shape with a yellow tinge at the center and a white periphery with a pleasant aroma. the tubers, initially presenting as white, fleshy, and aromatic when freshly cut, undergo a color transformation to a muddy shade over time. they are either fusiform or globose, ranging from 2 to 10 cm in length and 1‒2.5 cm in width (ravikumar et al. 2000; sabu 2006). further, the leafy shoots can reach a height *corresponding author, email: vaze.vgayatri@gmail.com mailto:vaze.vgayatri@gmail.com biotropia vol. 31 no. 1, 2024 88 of 80‒125 cm, featuring a pseudo stem that extends up to 35 cm. the leaves exhibit a green color with a pink/purple tinge, while the lamina, measuring (40‒50 cm length × 6‒9 cm width, is oblong-lanceolate in shape, with a hyaline margin and an acuminate or acute tip (santapau 1945). in addition, c. pseudomontana bears both lateral and central inflorescence. flowering spikes of around 10‒25 cm emerge from the center of the tuft of leaves, carrying numerous compactly arranged flowers. the flowering season for c. pseudomontana spans from may to september. the taxon holds significance in commercial trade as a medicinal plant (molur & walker 1997). moreover, various tribes extensively use it in ethnomedicine for treating conditions such as jaundice, leprosy, dysentery, and general debility and for purposes such as increasing lactation and blood purification (jagtap et al. 2006; bisht et al. 2014; hiremath & kaliwal 2014). additionally, c. pseudomontana has several reported applications in traditional and folk medicine, including its role in the production of arrowroot powder (ravindran et al. 2007; sasikumar 2005). the korku tribes residing in the amravati district of maharashtra, india, consume the fresh tubers of c. pseudomontana (locally referred to as kukusmukus) as a means of blood purification. in the munchingiputtu mandal of vishakhapatnam, andhra pradesh, india, various tribes employ the tubers (locally called adavi pasupu) for curing jaundice, whereas the bagata and valmiki tribes use these tubers for addressing diabetes and apply a warm paste on swellings (hiremath & kaliwal 2014). additionally, the savara tribes in andhra pradesh, india, use the boiled tubers of c. pseudomontana (locally called adavi jogra) as a galactagogue. to administer, the boiled tubers are ground along with a pinch of salt and given orally (prasad et al. 2006). meanwhile, in the srikakulam district of andhra pradesh, india, the rhizomes find applications for addressing stomach troubles and cough (theanphing et al. 2010). c. pseudomontana has undergone testing for its anticancer and antiproliferative/cytotoxic activity (bisht et al. 2014), wherein the extracts of c. pseudomontana using distilled water, methanol, and ethanol were studied on the bhk-21 cell line. further, the ic50 value for the plant extract was determined to be in the range of 2‒20 mg for the bhk-21 cell line (bisht et al. 2014). the antitubercular activity of the rhizome of c. pseudomontana is examined using microplate alamar blue assay (maba), yielding promising results for the crude extract of c. pseudomontana for tuberculosis treatment. the antitubercular activity is tested for extract obtained via hexane, chloroform, ethyl acetate, and methanol on the mycobacterial tuberculosis strain (h37rv) at 100 and 50 mg ml‒1 (hiremath & kaliwal 2013). despite its extensive and valuable applications, this species faces imminent threats to its survival due to several underlying challenges, including the invasion of exotic species in its natural habitats, forest fires, and the unrestricted use of agrochemicals near forests (bawa et al. 2007). the unregulated exploitation of commercially traded medicinal plants solely for economic gain, without due consideration for conservation, has driven many species dangerously close to extinction (molur & walker 1997; bawa et al. 2007). to counteract this issue, micropropagation emerges as a biotechnology application with the potential to contribute to the conservation of species by serving as an ex-situ conservation method, thereby aiding in the prevention of extinction (barbara et al. 2011). however, there is a lack of documented plausible conservation initiatives operating for c. pseudomontana. this study aims to address this gap by designing a method for in vitro mass propagation of c. pseudomontana using micropropagation by modifying the murashige and skoog media, drawing inspiration from successful in vitro propagation studies conducted on other curcuma species (salvi et al. 2001, kou et al. 2013; salvi et al. 2000; mohanty et al. 2008; prakash et al. 2004; yasuda et al. 1988; koarapatchaikol et al. 2017; zhang et al. 2011; sundaram et al. 2012). thus, this approach serves as a valuable tool for the preservation of this vulnerable species, enabling rapid multiplication and potential reintroduction into its natural habitat to enhance the population. materials and methods collection of plant the entire mother plant of c. pseudomontana was collected from the western ghats forests of kolhapur district, maharashtra, india, during the monsoon season. the complete plant, at its flowering stage, was collected, verified for 6-benzylaminopurine inducing high-frequency multiplication in vulnerable hill turmeric – gayatri v. vaze et al. 89 authenticity, transformed into a voucher specimen with the accession number rmrc1346, and subsequently deposited at the herbarium of the national institute of traditional medicine, indian council of medical research, belagavi, karnataka. surface sterilisation the rhizomes of c. pseudomontana, devoid of aerial parts and roots but containing active buds, were thoroughly washed under running tap water for 30 minutes. thereafter, they were washed with 5% (v/v) tween 20 solution, with continuous shaking on a rotary shaker set at 20 rpm. any remaining soil particles were carefully brushed away. a final rinse under running tap water for 15 minutes ensured the complete cleaning of the rhizomes. the explants were prepared by carving the active buds out from the rhizome in a pyramid shape while leaving a small portion of the rhizome attached (fig. 1). these were then immersed in a 0.2% carbendazim solution (w/v) for 5‒6 minutes, with intermittent shaking. the as-prepared explants were shifted to the sterile bench floor of the laminar airflow (laf) hood, followed by cleaning using distilled water and drying with the help of tissue paper. these surface sterilized explants were then transferred onto a sterile laf bench and treated with 0.1% (w/v) mercuric chloride (hgcl2) solution for 2 minutes. subsequently, the explants (rhizome buds) were finally washed with distilled water, trimmed to remove the tissues exposed to mercuric chloride, and further inoculated onto the ms basal media. culture media and conditions the study utilized murashige and skoog (murashige & skoog 1962) media as the base. the sterilized rhizome buds were employed as primary explants and inoculated onto the ms basal medium without any plant growth regulator. the explants were closely monitored for two weeks and observed for any change in morphology as well as contamination from bacterial and fungal origin. after two weeks, the explants were transferred from the initiation medium to the multiplication medium. in vitro shoot multiplication the ms basal media was enriched with different concentrations of cytokinin and auxin to induce multiple shooting and rooting, respectively. the cytokinins used in this study included 6-benzylaminopurine (bap), kinetin (kn), and thidiazuron (tdz). the explants were inoculated onto ms supplemented with cytokinins at a working concentration ranging from 0.01 to 10.0 mg l‒1. the cytokinin concentrations showing a positive response were further utilized in shoot multiplication studies. actively growing in vitro buds were aseptically transferred onto ms fortified with 1‒5 mg l‒1 bap (hi-media, india), 1.2‒1.5 mg l‒1 kn (himedia, india), and 0.1‒0.5 mg l‒1 tdz (himedia, india) supplemented with 30 g l‒1 sucrose (hi-media, india) with 0.8% (w/v) agar (himedia, india). the culture vessels were kept in a growth room at 25±2 °c, maintaining a relative humidity of 95‒100% under a 16-hour photoperiod with a light intensity of 1000 lux. growth parameters were measured in terms of shoot number and shoot length per explant. in vitro root induction the auxins employed for in vitro root induction studies included indole-3-butyric acid (iba), indole-3-acetic acid (iaa), and 1naphthaleneacetic acid (naa). the in vitro shoots were inoculated onto ms supplemented with auxins at a working concentration of 0.01‒10.0 mg l‒1. the auxin concentrations demonstrating good response were further used in root induction studies. the in vitro raised individual shoots were relocated to the root induction media containing 1.1‒1.5 mg l‒1 of iba and iaa and 0.1‒1.0 mg l‒1 of naa. the ms medium served as the basal medium, which was fortified with auxins. growth parameters were quantified in terms of root number and root length per explant. each trial involving plant growth regulators in both shoot induction and root induction was conducted thrice with ten replicates each. hardening and acclimatization the well-rooted plantlets bearing 3‒5 healthy leaves were gently excised from the medium in laf. the roots were washed with distilled water to remove the traces of agar medium adhered to biotropia vol. 31 no. 1, 2024 90 the roots. subsequently, the rooted plantlets were placed safely in a hardening mixture composed of sand, soil, and cocopeat at a ratio of 1:1:1 (v/v) within a plastic cup and covered with another plastic cup gently to maintain essential humidity, retain moisture, and ensure sufficient light. the humidity levels were adjusted by removing the covering glass as per requirement and maintaining it under the same culture conditions for two weeks. following the hardening period, the plantlets were transferred to the greenhouse, and their survival rates were recorded after four weeks (loc et al. 2005). statistical analysis the data obtained in this study was analyzed using one-way anova (analysis of variance), and the results were compared for statistical significance using duncan’s multiple range test, with a significance level set at p<0.05. the data values are presented as mean ± standard deviation (m±sd). microrhizome induction the induction of in vitro microrhizome was investigated by varying the sucrose concentration in the ms basal media, ranging from 30 g l‒1 to 120 g l‒1 in triplicates. well-rooted shoots were extracted from the medium (2.0 mg l‒1 bap + 0.5 mg l‒1 naa), washed with distilled water to remove the agar traces, and blot-dried. the roots were carefully cut off from the plantlets, leaving only the individual shoots for inoculation back to the microrhizome induction media. this involved the use of pgr-free ms media with varying sucrose concentrations (30‒120 g l‒1). the culture vessels were placed in the dark at 25±2 °c, and the observed response was recorded after a period of four weeks from the time of inoculation (nayak & naik 2006). results and discussion the in vitro shoot growth parameters of c. pseudomontana were assessed in terms of the number of shoots and shoot length per explant after four weeks of culture initiation. continuous daily observations were conducted to monitor any changes in the growth patterns. similarly, the response to root induction was recorded in terms of root number and length per explant. the subculturing was carried out every 15 days or two weeks, or earlier if needed, to replenish the medium. initiation of culture and multiplication the medium was standardized to yield multiple shoots per explant. the maximum number of shoots, i.e., 9.66 ±2.08, was achieved in ms+bap (2 mg l‒1) + 3% sucrose at ph = 5.8. table 1 shoot induction response in c. pseudomontana for different concentrations of bap, kn, and tdz plant growth regulators (mg l-1) shoot length (in cm) no. of shoots per explants bap kn tdz 1 4.53±4.80a 4.0 ±1.0b 2 6.40 ±0.36b 9.66 ±2.08f 3 4.80 ±2.17a 5.66± 1.52a 4 3.10 ±1.83d 3.66 ±0.50c 5 2.76 ±1.90de 2.0 ±1.0d 1.1 1.96± 1.45e 1.33± 0.57e 1.2 1.73± 1.44e 1.33± 0.57e 1.3 1.83 ±0.41e 2.66±0.57cd 1.4 1.96± 1.32de 1.66 ±0.57de 1.5 2.06 ±1.56d 1.66 ±1.15de 0.1 0.86 ±0.72f 1.33 ±0.57e 0.2 1.56 ±0.32e 1.33 ±0.57e 0.3 1.06 ±0.41e 1.0 ±0.0e 0.4 0.5 6-benzylaminopurine inducing high-frequency multiplication in vulnerable hill turmeric – gayatri v. vaze et al. 91 the presented data shows mean values and corresponding standard deviations. “mean” that share the same letter within each column of growth regulator combinations are not significantly different at a 5% level of significance, as determined by duncan’s multiple range test. in the comparison of all three cytokinins for shoot multiplication (table 1), bap exhibited the highest potential of shoot induction and multiplication in c. pseudomontana rhizome bud explants. the number of shoots per explant increased with the increasing concentration of bap, followed by a gradual decrease with a further rise in concentration, specifically from 3 mg l‒1 to 5 mg l‒1. within the tested range of bap, the best response was registered at a concentration of 2 mg l‒1. on the other hand, a relatively consistent growth pattern and a stable number of shoots were observed with no recorded mortality in the studied concentration range of kn. this suggested that kn may be suitable as a component of maintenance media but not be ideal for the rapid multiplication of this species, which holds conservation value. these observations aligned with the findings in other studies on curcuma species micropropagation, wherein the inefficiency of kn in inducing multiple shoots is reported (shanthala et al. 2021). the least favorable response was observed among the explants inoculated in ms fortified with tdz at different concentrations. specifically, tdz at a concentration of 0.1‒0.3 mg l‒1 showed stagnant growth in the explant, accompanied by hyperhydricity in every culture vessel. the persistent hyperhydricity across all treated concentrations suggested that tdz induced this undesirable condition in the growing shoots of c. pseudomontana, rendering it unsuitable for the in vitro multiplication of this species. contrarily, the remaining concentrations resulted in explants that perished, displaying signs of survival only at lower concentrations. these observations deviate from other micropropagation studies within the genus curcuma, which portrayed the positive effects of tdz on micropropagation and successful multiple shoot induction (toppoonyanont et al. 2004). the superior responsiveness and the highest multiplication of explants observed with bap confirmed its efficacy as the most effective cytokinin for in vitro shoot multiplication in c. pseudomontana. furthermore, cultures treated with bap and kn demonstrated consistent morphological features and identity. overall, concerning the highest multiplication induction rate, bap is recommended as the most suitable cytokinin for this purpose. the effect of cytokinins on shoot multiplication is presented in table 1. the notable efficiency of bap in inducing effective multiplication aligned with findings from other micropropagation studies carried out on curcuma species like c. haritha (bejoy et al. 2006), c. mangga (raihana et al. 2011), c. caesia (bharalee et al. 2005), c. zedoaria (loc et al. 2005), c. vamana (bejoy et al. 2012), etc. effects of auxins on in vitro rooting and elongation the effects of iba, iaa, and naa on root induction response were studied, with results listed in table 2. the concentrations of auxins were selected based on their standard working concentration. the most favorable root induction response was recorded with the naa treatment at 0.5 mg l‒1 in ms medium containing 30 g l‒1 sucrose at ph = 5.8. a 100% rooting success was observed in all the treated groups of auxins. root number and length exhibited variations among the different treatments. the highest number of roots observed in naa at 0.5 mg/l was 7.00±1.00 per explant, with the longest root length of 3.36±1.00 cm. a similar response to root induction was noted in other studies conducted on the genus curcuma, including c. mangga (raihana et al. 2011), c. caesia (bharalee et al. 2005), c. aromatica (mohanty et al. 2008), c. longa (ugochukwu et al. 2013), and c. zedoaria (bharalee et al. 2005). these results showed that naa was suitable for root induction in c. pseudomontana. however, it is worth noting that both iba and iaa also demonstrated satisfactory rooting responses. biotropia vol. 31 no. 1, 2024 92 table 2 in vitro root induction response to different concentrations of iba, iaa, and naa plant growth regulators (mg l-1) root length (in cm) no. of roots per explants iba iaa naa 1.1 0.16 ±0.19a 1.33 ±0.57a 1.2 0.16 ±0.05a 1.66±1.15a 1.3 0.26±0.05a 1.33±0.57a 1.4 0.23±0.05a 1.00±0.00a 1.5 0.30±0.26ab 1.00±0.00a 1.1 0.10±0.00a 1.00±0.00a 1.2 0.13±0.05a 1.33±0.57a 1.3 0.13±0.05a 2.00±1.00b 1.4 0.16±0.05a 1.66±0.57a 1.5 0.13±0.05a 2.00±1.00b 0.1 0.33±0.15a 4.00±1.00c 0.2 0.40±0.10ab 4.33±1.15cd 0.3 1.03±0.15c 4.65±1.15cd 0.4 1.86±0.23c 5.33±0.57cd 0.5 3.36±1.00d 7.00±1.00e 0.6 1.33±0.28c 4.00±1.00c 0.7 0.90±0.10ab 2.00±1.00b 0.8 0.53±0.90ab 1.00±0.00a 0.9 0.26±0.11a 0.66±0.57c 1.0 0.16±0.11a 0.66±0.57c the presented data shows mean values and corresponding standard deviations. “mean” that share the same letter within each column of growth regulator combinations are not significantly different at a 5% level of significance according to duncan’s multiple range test. hardening and acclimatization following a successful two-week hardening period with a 100% survival rate, the plantlets were carefully uprooted and replanted into pots with soil. few plantlets were intentionally uprooted at this stage to assess root development. notably, the formation of a small tuber was observed, measuring approximately 2.5 cm in length and 0.5 cm in thickness at the base of the roots, resembling the plants growing in their wild habitat. the in vitro regenerated plants exhibited a phenotype similar to the wild plants. therefore, this high-frequency multiplication protocol was reproducible and highly reliable, yielding healthy plantlets. consequently, it can serve as an effective tool for the ex vivo conservation of the vulnerable c. pseudomontana species. importantly, a survival rate of 92% was recorded in the field (fig. 1f). microrhizome formation after four weeks, the media containing 30 g l‒1 sucrose and the ms basal control displayed normal plant development, featuring 3‒4 leaves and healthy root system. similar positive results were obtained for media with 60 g l‒1 of sucrose. in contrast, media with a triple dose of sucrose (90 g l-1) exhibited lesser root growth but was accompanied by formation of microrhizomes (fig.2). however, media with 120 g l‒1 showed impaired root growth. hence, it was concluded that the best response for microrhizome formation in c. pseudomontana was achieved with ms basal medium supplemented with 90 g l‒1 sucrose without any plant growth regulators. similar outcomes in microrhizome formation have been reported on other important curcuma species, such as c. longa (shirgurkar et al. 2001; nayak et al. 2006) and c. aromatica (nayak et al. 2000). 6-benzylaminopurine inducing high-frequency multiplication in vulnerable hill turmeric – gayatri v. vaze et al. 93 figure 1 in vitro propagation of c. pseudomontana j. graham using rhizome bud explants:(a) natural habitat of c. pseudomontana; (b) shoot initiation with rhizome bud; (c) initial shoot growth; (d) induction of multiple shoots; (e, f) multiple shoot proliferation; (g) induction of roots from in vitro raised shoots; (h) regenerated plants with a well-developed root system; (i) acclimatisation of micro-propagated plantlets into the hardening mixture, and (j) acclimatised plantlet after 60 days showing tuber formation at the base similar to that of the plants in wild habitat. figure 2 in vitro microrhizome induction: (a) well-rooted plantlet of c. pseudomontana developed in vitro; and root growth with ms supplemented with (b) 30 g l‒1 and (c, d) 90 g l‒1 sucrose. biotropia vol. 31 no. 1, 2024 94 conclusion to sum up, this study marks the first report on an efficient high-frequency multiplication protocol developed for the in vitro multiplication of c. pseudomontana, representing a significant step towards the conservation of this vulnerable species. the method yields healthy, disease-free, and uniform plantlets, demonstrating a consistently high multiplication rate with 100% rooting success and a commendable 92% survival rate after acclimatization. hence, the as-devised protocol can be 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(zingiberaceae).plant growth regulation. 64(2): 141-145. https://dx.doi.org/10.2305/iucn.uk.2013-1.rlts.t22486190a44506743.en https://dx.doi.org/10.2305/iucn.uk.2013-1.rlts.t22486190a44506743.en https://dx.doi.org/10.2305/iucn.uk.2013-1.rlts.t22486190a44506743.en https://www.ncbi.nlm.nih.gov/pubmed/?term=yasuda%20k%5bauthor%5d&cauthor=true&cauthor_uid=17265210 https://www.ncbi.nlm.nih.gov/pubmed/?term=tsuda%20t%5bauthor%5d&cauthor=true&cauthor_uid=17265210 https://www.ncbi.nlm.nih.gov/pubmed/?term=shimizu%20h%5bauthor%5d&cauthor=true&cauthor_uid=17265210 https://www.ncbi.nlm.nih.gov/pubmed/?term=sugaya%20a%5bauthor%5d&cauthor=true&cauthor_uid=17265210 https://www.ncbi.nlm.nih.gov/pubmed/17265210 https://www.researchgate.net/journal/0167-6903_plant_growth_regulation https://www.researchgate.net/journal/0167-6903_plant_growth_regulation the southeast asian journal of tropical biology vol. 32 no. 1, 2025: 111 117 doi: 10.11598/btb.2025.32.1.2107 issn: 0215-6334 | e-issn: 1907-770x 111 tick-borne pathogens detection from ticks infesting malayopython reticulatus (reptilia: pythonidae) snakes in indonesia supriyono, hana faizah sophia, upik kesumawati hadi, and susi soviana* school of veterinary medicine and biomedical sciences, ipb university, bogor 16680, indonesia article higlights • a total of 38 ticks were collected from m. reticulatus, comprising 13 a. helvolum and 25 a. varanense. • spotted fever group rickettsia spp. (7.89%) and reptile-associated borrelia sp. (2.63%) were detected in male amblyomma helvolum ticks collected from malayopython reticulatus snakes in indonesia. • snake-associated ticks may harbor emerging pathogens, underscoring the importance of tick surveillance in reptiles for early disease detection and zoonotic prevention. article information received : 19 october 2023 revised : 8 august 2024 accepted : 2 september 2024 *corresponding author, e-mail: susiso@apps.ipb.ac.id short communication abstract ticks are important arthropod vectors of numerous diseases in humans and animals. furthermore, ticks are also established vectors and reservoirs of pathogens important to wildlife and human health. rickettsia and borrelia are two genera of bacteria that may be transmitted by ticks, and some pathogenic species are zoonosis. this research investigated the prevalence of rickettsia spp. and borrelia sp. in amblyomma helvolum and amblyomma varanense ticks fed on malayopython reticulatus and python bivittatus snakes in indonesia. a total of 38 ticks were collected from three m. reticulatus  snakes, while no ticks were found on the  p. bivittatus  snake. the 38 ticks consisted of 13 individuals  a. helvolum  and 25 individuals  a. varanense. pcr analysis revealed that three (3/38; 7.89%) male a. helvolum ticks were positive for spotted fever group rickettsia spp. and one (1/38; 2.63%) male a. helvolum  tick was positive for a reptileassociated group borrelia sp. although the overall prevalence of tickborne pathogens was low, this study underscores the importance of monitoring the prevalence and prevention of tick-borne diseases. surveillance of ticks infesting reptiles can facilitate the early detection of disease transmission to both animals and humans. these findings also suggested that snake-associated ticks may harbor emerging tickborne pathogens. keywords: amblyomma helvolum, borrelia, indonesia, malayopython reticulatus, python bivittattus, rickettsia introduction malayopython reticulatus (reptilia: pythonidae) is a non-venomous snake commonly found in indonesia. this snake is also one of the wild animals susceptible to infestations by various ectoparasites, including ticks. five tick species have been separately reported to infest m. reticulatus snakes, which are amblyomma helvolum (anastos 1950; anderson & tzianabos 1989; andoh et al. 2015), a. varanense (anastos 1950; andoh et al. 2015), a. cordiferum (auffenberg 1988), and rhipicephalus sanguineus (chao et al. 2013). amblyomma latum was also reported to infest python regius snakes in ghana and togo (pandit et al. 2011; mariana et al. 2011; sumrandee et al. 2014; andoh et al. 2015). ticks known to be associated with pathogens include ixodes spp., rhipicephalus spp., haemaphysalis spp., amblyomma spp., and dermacentor spp. (estrada-peña & jongejan 1999; raoult et al. 2002; takano et al. 2014). several pathogens are associated with ticks, a few of which are borrelia sp. and rickettsia sp. the major groups of borrelia impacting animal and human health are relapsing fever borrelia, lyme disease borrelia, and reptile-associated (rep) borrelia (bunikis & barbour 2005; takano et al. 2010; franke et al. 2013). borrelia spp. was previously detected in a. varanense infesting p. reticulatus in thailand (trinachartvanit et al. 2016). meanwhile, rickettsia sp. belonging to the spotted fever group (sfg) was also detected from a. transversale and https://doi.org/10.11598/btb.2024.31.2.1741 biotropia vol. 32 no. 1, 2025 112 a. trimaculatum ticks infesting p. regius and boiga forsteni snakes, respectively (andoh et al. 2015). this research aimed to determine the presence of rickettsia spp. and borrelia sp. in ticks that infest wild snakes in indonesia. in addition, phylogenetic analyses of detected pathogens were also presented. materials and methods ticks were collected within the period of 20212022 from three wild-caught m. reticulatus snakes in bogor (6°35’42.1368’’ s and 106°48’59.8860’’ e) and jakarta (20°58’50.736’’ n and 89°40’45.876’’ w) and one p. bivittatus snake found in jakarta (20°58’50.736’’ n and 89°40’45.876’’ w). the snakes were handled in accordance with good animal welfare practices and released to their original habitats upon examination. from the skin beneath their scales, ticks were collected using forceps and stored in 70% ethanol. tick species, stage, and sex were identified based on morphologic features following taxonomic keys and molecular analysis (anastos 1950; kohls 1957). the collected ticks were washed individually and homogenized in 200 μl 10x pbs solution. tick dna was individually extracted using the dneasy blood & tissue kit (qiagen, hilden, germany) according to the manufacturer’s instructions. oligonucleotide primer pairs used in this study were 16srdna (mt-rrs 1 5’-ctgctcaatgattttttaaattgctgtgg-3’; mt-rrs 2 5’-ccggtctgaactcagatcaagta-3’) for tick identification, 17-kda antigen (r1 5’-tcaattcacaacttgccatt3’,r2 5’-tttacaaaattctaaaaacc-3’) for rickettsia detection, and flab (pad 5’-gatcargcwcaayataaccawatgca-3’, pdu 5’-agattca agtctgttttggaaagc-3’) for borrelia detection (anderson & tzianabos 1989; takano et al. 2010). amplifications were performed with the following conditions: 95 °c for 5 minutes, 94 °c for 45 seconds, 50-52 °c for 30 seconds, 72 °c for 45 seconds, and 72 °c for 10 minutes. pcr products were visualized using electrophoresis on 1.2% agarose gel stained with ethidium bromide in 1x tae buffer. electrophoresis was carried out at 100 v for 25 minutes. dna from positive samples then amplified again as much as 50 μl for sequencing and the sample was sent to pt. genetika science indonesia, tangerang. the sequences of positive samples were compared with sequences in the ncbi genbank database by nucleotide blast. phylogenetic analyses were performed using the mega7 software (www.megasoftware.net) (tamura et al. 2007). the phylogenetic trees were constructed by the neighbor-joining method. bootstrap analyses (1,000 replicates) were carried out according to the kimura 2-parameter model. all sequences were deposited in genbank (accession numbers: rickettsia sp. st9 (oq164644), rickettsia sp. st10 (oq164645), rickettsia sp. st13 (oq054254), and borrelia sp. st10 (oq187772)). results and discussion from the three m. reticulatus snakes, a total of 38 ticks were collected which consisted of 13 individuals of a. helvolum (11 males, 2 females) and 25 individuals of a. varanense (20 males, 5 females) which were confirmed by morphological examination. there were no ticks found infesting the p. bivittatus. amblyomma helvolum had 3/3 dentition, long and narrow palps, and oval porose areas on the rectangular basis capituli. the male ticks had an ovoid scutum with metallic and yellowish patches of ornamentation. the female ticks, on the other hand, had no scutum ornamentations. the eyes were flat and located at the lateral margins of the scutum. coxa i bore a pair of triangular spurs, with the external spur about twice as long as internal one. coxae ii-iv each bore a single, triangular spur. amblyomma varanense had 3/3 dentition, long and narrow palps, and oval porose areas on the rectangular basis capituli. the male ticks had a reddish-brown round scutum, nearly as broad as long, with five metallic-green spots of variable thickness, shape, and intensity. the female ticks had a reddish-brown cordiform scutum with three greenish metallic spots. coxa i bore two short, distinct, and separated spurs; the external spur was slightly longer than the internal. coxae ii-iv each bore a single, blunt spur about as wide as long. pcr was used to confirm the identification of ticks. amblyomma helvolum has a natural distribution that extends from the nicobar islands of india eastward through parts of thailand, laos, malaysia, singapore, vietnam, indonesia, the philippines, and taiwan (auffenberg 1988; kolonin 1995; petney & keirans 1995; chao et al. 2013). amblyomma helvolum has been reported to infest tick-borne bacteria in indonesia 113 various reptiles, such as python spp., ptyas korros (zamensis), and naja naja (kohls) (imaoka et al. 2011) in malaysia (kohls 1957). quite similarly, a. varanense is distributed in india, thailand, myanmar, singapore, and indonesia (anastos 1950; supriyono et al. 2019). amblyomma varanense commonly infests reptiles, but is also known to feed on various mammals (burridge 2001). all collected ticks were individually examined by pcr for rickettsia spp. and borrelia spp. the results showed that three male a. helvolum ticks infested on m. reticulatus were positive for rickettsia spp. and one male a. helvolum tick infested on m. reticulatus was positive for borrelia sp. the detected rickettsia spp. and borrelia sp. were compared with sequences in the ncbi genbank database by nucleotide blast. there were no pathogens detected in a. varanense in this study. the 17-kda gene sequences of two detected rickettsia, rickettsia sp. st10 and rickettsia sp. st13, showed 100% (500/500) and 99.4% (497/500) similarity, respectively, to rickettsia sp. rt2 (genbank: lc428381) detected from a. varanense ticks infesting an asian water monitor (varanus salvator) in indonesia. however, rickettsia sp. st9 was 99.2% (499/503) in similarity to rickettsia sp. (genbank: ab795164) detected from a. sparsum ticks infesting tortoises (geochelone pardalis) from zambia (fig. 1). the detected rickettsia spp. belonged to the spotted fever group (sfg), which are potential human pathogens. figure 1 phylogenetic analysis of 17-kda of rickettsia spp. notes: the phylogenetic branches showed support of > 70% by tree, which was constructed using the neighbor-joining method and bootstrap tests (1,000 replicates) carried out according to the kimura 2-parameter model. the bar indicates the percentage of sequence divergence. bold letters indicate the samples analyzed in the present study. biotropia vol. 32 no. 1, 2025 114 several studies have reported the detection of sfg in ticks from various countries in southeast asia, including japan, thailand, malaysia, and indonesia (imaoka et al. 2011; doornbos et al. 2013; kho et al. 2015; supriyono et al. 2019). rickettsia sp. rt2, closely related to r. tamurae, was detected in a. varanense ticks infesting an asian water monitor (v. salvator) in indonesia (supriyono et al. 2019). although there have been no reports of sfg infection in humans due to tick bites in indonesia thus far, tick infestations pose a potential risk to human health. infection caused by r. tamurae, a member of the sfg rickettsia, has been reported in japan and laos (phongmany et al. 2006; imaoka et al. 2011). additionally, antibodies against sfg infection in humans have been reported in rural areas in malaysia (tay et al. 2000). the results of this study indicated that there was a natural circulation of sfg in ticks infesting snakes. analysis of the flab gene showed that the detected borrelia sp. was 99.3% (296/298) in similarity to borrelia sp. bf16 (genbank: ab473488) detected from a. trimaculatum ticks infesting b. forsteni snakes. the detected borrelia sp. was also 99.2% (370/373) in similarity to uncultured borrelia sp. (genbank: kt758064) detected from a. varanense ticks infesting p. reticulatus in thailand (trinachartvanit et al. 2016). the constructed phylogenetic tree details that the detected borrelia sp. in this study belongs to the rep borrelia group and differs from the lyme disease-associated borrelia and the relapsing feverassociated borrelia (fig. 2). rep borrelia has been detected in ticks infesting various reptiles, including snakes, turtles, and monitor lizards in thailand, indonesia, and japan (takano et al. 2010; trinachartvanit et al. 2016; supriyono et al. 2019). rep borrelia has also been detected in ticks infesting imported captive-bred geochelone sulcata tortoises and wildcaught chelonoidis carbonarius and c. denticulatus tortoises in indonesia (sophia et al. 2023). malayopython reticulatus has a habitat distribution in rainforests, woodlands, and nearby grasslands. it is also associated with rivers and is found in areas with nearby streams and lakes (murray-dickson et al. 2017). the snakes in this study were wild-caught in rural areas and thus, have potential to transmit diseases via hematophagous arthropods which include ticks. this data is essential for vector surveillance to predict the risk of emerging diseases and zoonoses, primarily caused by tick-borne bacteria. although there are no reports of a. helvolum and a. varanense infestations in humans in indonesia, the detected tick pathogens can pose risks to wildlife and human health. it is not uncommon that many reptiles, including snakes, are kept as pets that live close to humans or other animals. tick-borne bacteria in indonesia 115 figure 2 phylogenetic analysis of flab of detected borrelia sp. notes: the phylogenetic branches showed support of > 70% by tree, which was constructed using the neighbor-joining method and bootstrap tests (1,000 replicates) carried out according to the kimura 2-parameter model. the bar indicates the percentage of sequence divergence. bold letters indicate the samples analyzed in the present study. biotropia vol. 32 no. 1, 2025 116 conclusion rickettsia spp. and borrelia sp. were detected in a. helvolum which belong to spotted fever group and reptile-associated borrelia, respectively. the prevalence of spotted fever group rickettsia spp. in these ticks was 7.89%, and the prevalence of reptile-associated borrelia sp. was 2.63%. these findings provide new insights into the parasite-host dynamics within the tick and tick-borne pathogen system in wild snakes in urban areas of indonesia, enhancing our understanding of the ecology and interactions among wildlife, ticks, and tickborne pathogens in the region. monitoring the circulation patterns of tick-borne bacteria is crucial for assessing infection risks to wildlife and humans, as well as for developing strategies for disease control, mitigation, and early warning systems in case of outbreaks. further studies are required to assess the prevalence of tick-borne bacteria in other animal species across 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lijuan w, boonkusol d, baimai v, ahantarig a. 2016. borrelia sp. phylogenetically different from lyme diseaseand relapsing fever-related borrelia spp. in amblyomma varanense from python reticulatus. parasites vectors 9:359. doi: 10.1186/ s13071-016-1629-8 the southeast asian journal of tropical biology vol. 31 no. 2, 2024: 169 180 doi: 10.11598/btb.2024.31.2.1874 issn: 0215-6334 | e-issn: 1907-770x 169 conservation status of tree species of the freshwater swamp forest, perak tengah, malaysia liliwirianis nawi1*, mohd nazip suratman2, farida zuraina mohd yusof3, abdul latiff mohamad4 1faculty of applied sciences, universiti teknologi mara pahang, bandar tun abdul razak jengka 26400, malaysia. 2institute for biodiversity and sustainable development, universiti teknologi mara (uitm), shah alam 40450, malaysia. 3faculty of applied sciences, universiti teknologi mara (uitm), shah alam 40450, malaysia. 4department of biological science and biotechnology, universiti kebangsaan malaysia, bangi 43600, malaysia. article higlights • approximately 3,364 tree individuals with a diameter at breast height (dbh) of 10.0 cm and above were assessed in the perak tengah freshwater swamp forest. • the study has recognised 58 species identified are new to perak. • 72 tree species are listed as endemic, and 194 species are listed in the red list of threatened species by the international union for conservation of nature (iucn). • this study mphasize the need to preserve ecology and mitigate threats. abstract the forests of peninsular malaysia have long been recognized as a significant home of endemic species. the purpose of this study was to assess the endemism and conservation status of tree species present in the perak tengah freshwater swamp forest. a total of 150 plots (20 m × 20 m) were established in the study area using a random sampling design. approximately 3,364 tree individuals with diameter at breast height (dbh) of 10.0 cm and above were enumerated in all plots. in the study area, 58 species identified are new to perak, 72 tree species are listed as endemic, and 194 species are listed in the red list of threatened species by iucn. thus, it is crucial to preserve the ecology and lessen the impact of threats in the perak tengah freshwater swamp forest, which is home to numerous endemic and endangered species. keywords: endangered, endemic, freshwater swamp forest, malaysia, perak, threatened article information received 25 january 2023 revised 23 march 2024 accepted 22 may 2024 *corresponding author, e-mail: liliwirianis@uitm.edu.my research article introduction malaysia has lost a substantial amount of forest due to cash-crop plantations (oil palm, rubber, and acacia, including fallow lands) and urban expansion (huang & oey 2019). for the past 29 years, nearly 16% (189,423 ha) of perak’s forest cover has disappeared due to anthropogenic activities (kanniah & siong 2017; jaafar et al. 2020). according to the fifth national report of the convention on biological diversity (2020), over 80% of the larger genera are endemic. most endemic species are vulnerable to extinction because they are only restricted to one area with a specific microclimate. malaysia is estimated to possess 15,000 species of flora, with 8,300 species found in peninsular malaysia. there are currently at least 9,030 vascular plant taxa in peninsular malaysia, made up of 1,651 genera and 248 families, with at least 149 new taxa identified since 2005 (yong et al. 2021). malaysia has also recorded approximately 2,500 species of endemic vascular plants (saw & chung 2015). rare species are becoming the most significant concern for conservationists due to the risk of extinction. species can be considered rare when they have small population sizes, restricted geographical ranges, or narrow habitat tolerances. when combined, these characteristics define several forms of rarity and different levels of extinction risk (kruckeberg & rabinowitz 1985). https://doi.org/10.11598/btb.2024.31.2.1741 biotropia vol. 31 no. 2, 2024 170 rare species have greater sensitivity to both naturaland human-induced disturbances, such as overexploitation, habitat loss, and global environmental changes (davies et al. 2004). meanwhile, from the ecological aspect, endemism refers to the occurrence of any taxonomic entity (species, genus, or family) within a restricted and entirely defined area. endemism is essential to ecologists, biogeographers, and evolutionary biologists in general. endemics can be old (palaeoendemics) or new (neoendemics). endemic species are an important target of global conservation efforts. as for species richness, a smaller amount of total area will need to be conserved if endemism patterns among taxa are correlated. generally, the number of taxa endemic to a particular area increases with size and geographic isolation (anderson 1994). extinction will result in ecological loss and disturbance to the equilibrium of the forest. a better understanding of the rarity types and their differences needs to be considered for better conservation planning and strategies. thus, the establishment of the international union for conservation of nature (iucn) aims to influence, encourage, and assist societies to conserve diversity and to ensure that any use of natural resources is equitable and ecologically sustainable. the study aimed to assess the endemism and conservation status of species found in the perak tengah freshwater swamp forest. due to the ecology and drainage, freshwater swamp forests are particularly vulnerable to outside influences. although endemic species are not necessarily rare or threatened by extinction, species typically become rare before going extinct (kruckeberg & rabinowitz 1985; dobson et al. 1995). endemic species, especially those with narrow distributions, as in this freshwater swamp forest, are particularly vulnerable to extinction resulting from habitat disturbance and other threats (kiew et al. 2017; kiew & rahman 2021). materials and methods study area the study area was located at the freshwater swamp forest remnant located in the district of perak tengah, bandar sri iskandar. the area of perak tengah district is about 12,205 ha and comprises 11 subdistricts. the study areas were located at the universiti teknologi mara (uitm) perak sri iskandar forest (4°21'31.1" n; 100°57'12.7" e), universiti teknologi petronas (utp) tronoh forest (4°22'49.9" n; 100°57'44.8" e), and the parit forest reserve, parit (4°24'13.9" n; 100°56'03.7" e), as shown in figure 1. the forest is surrounded by secondary forests with urban development leaving isolated freshwater swamp forest areas. freshwater swamp forest is a unique habitat that occurs in permanently flooded soil. the forest comprises stilt roots, knee roots, and plank-like sinuous buttresses, which are common features of tree species found in freshwater swamp forests. the mean monthly temperatures for the study area ranged from 26.5 °c to 28.7 °c with an annual rainfall of 245.6 cm. figure 1 map of study area at perak tengah, perak notes: (1) uitm perak, sri iskandar forest; (2) utp, tronoh forest; and (3) parit forest reserve, parit. endemism and conservation status of tree species of freshwater swamp forest, perak, malaysia liliwirianiset. al. 171 data collection the study plots were established using random sampling from may 2011 to april 2015. a total of 150 plots of 20 × 20 m each were established in the study area, totalling 6 ha. all trees with a diameter breast height (dbh) of 10 cm or greater were measured using a diameter tape. leaves and fruit specimens from the trees with dbh of more than 10 cm were collected, recorded, and kept in the universiti kebangsaan malaysia (ukm) herbarium. woody climbers and creepers were excluded from the study. the trees were identified to the species level using the existing specimens in the ukm herbarium, existing taxonomic keys (whitmore 1972; 1973), and the assistance of an experienced botanist from ukm and the pahang forestry department. the nomenclature of the species was determined according to turner (1995) and the international plant name index (ipni). meanwhile, specimens that could not be matched with herbarium specimens were only identified up to the genus or family level. species new records, endemic and conservation status new records and endemic species were listed and referred to according to turner (1995) and chua et al. (2010). conservation status was determined based on the iucn red list of threatened species version 2020.1 (iucn red list of threatened species, 2020) and yong et al. (2021). in addition, the dipterocarpaceae family was also compared using the malaysian plant red list (saw et al. 2010). results and discussion new records of species about 58 of 624 tree species (9.3%) in the freshwater swamp forest in perak tengah were listed as new species to perak state based on turner (1995) (table 1). the new records in perak state may be due to insufficient sample collection from the past. the freshwater swamp forest also faces threats from deforestation and urban expansion, leading to habitat reduction (saw 2010). on top of that, in peninsular malaysia, habitat degradation and loss pose the biggest challenge to plant populations. the primary causes of habitat loss include changes in land use for agricultural, residential, and commercial development (yong et al. 2021). the discovery of these 58 species during these two decades may offer signs of hope for malaysia’s local biodiversity. it also shows that the knowledge about these tree species is still far from satisfactory and complete. interestingly, some of the species listed in table 1 were only found in one state. for example, castanopsis foxworthyi and helicia maxwelliana were found only in terengganu. however, the species was also found in the study area, i.e., the freshwater swamp forest. on top of that, theoretically, c. foxworthyi also occupies lowland peat-swamp, heath forest, and upper montane forest, while h. maxwelliana is found in the montane forest at 1,200 m. nishimura et al. (2008) revealed that some species from the fagaceae family showed a random distribution pattern. moreover, palaquium confertum, syzygium polyanthum var. polyanthum, s. polyanthum var. sessile, and trigonostemon polyanthus were only found in johore, croton kelantanicus was found in kelantan, and syzygium claviflorum var. riparium which were documented in pahang can also be found in the freshwater swamp forest of perak tengah. endemic species of peninsular malaysia an endemic species usually grows and is found in a specific area and has restricted distribution. a total of 72 tree species from 56 families encountered in the perak tengah freshwater swamp forest were listed as endemic based on turner (1995) and chua et al. (2010) (table 2). from the list presented in table 2, 12 of them are recorded as new in perak. a previous study in perak tengah revealed that vatica flavida was left with a small number of individuals (249 individuals) due to the loss of large tracts of freshwater swamp forests followed by forest fragmentation. shorea hemsleyana ssp. hemsleyana and hopea apiculata were also threatened since their populations have decreased and their habitat has been degraded. it can be concluded that the most significant impact on the habitat has been from activities related to the development and human activities that lead to the depletion of their habitat (liliwirianis 2021). biotropia vol. 31 no. 2, 2024 172 no. species known distribution 1 actinodaphne pruinosa kd, pn, sl, ns, ml, sp 2 alseodaphne bancana sl, jh, sp. 3 alstonia scholaris kd, kl, tg, ph, sl, ml, jh. 4 anacolosa frutescens kd, ph, sl, ns, ml, jh, sp. 5 aporosa frutescens kd, pn, kl, ph, jh, sp. 6 aporosa microstachya kd, tg 7 ardisia korthalsiana tg, ph, ml, jh. 8 artocarpus kemando tg, ph, sl, jh, sp. 9 calophyllum ferrugineum var. ferrugineum jh, sp. 10 calophyllum rubiginosum sl, ns, ml, jh, sp. 11 calophyllum soulattri ps, kd, pn, kl, ph, ns, ml, jh. 12 canarium megalanthum kd, ph, ns, jh. 13 canthium cochinchinense p. langkawi, tg. 14 castanopsis foxworthyi tg. 15 chrysophyllum roxburghii kd, ph, sl,, ns, ml, jh, sp. 16 croton kelantanicus kl 17 cryptocarya ferrea kd, kl, tg, ph, sl. 18 cryptocarya zollingeriana kl, sl. 19 dillenia reticulata var. reticulata ml 20 diospyros ismailii kd, tg, ph, sl, ns, jh; 21 diospyros montana p. langkawi. 22 diospyros undulata ps, kd, p. langkawi. 23 durio carinatus ph, jh. 24 elaeocarpus nitidus var. velutinus kl, ph, jh; 25 helicia maxwelliana tg. 26 horsfieldia penangiana pn, ph, sl. 27 hydnocarpus kunstleri var. tomentosa tg, pk, ph, sl, jh; 28 knema latericia ssp. ridleyi tg, jh, sp. 29 litsea erectinervia tg, ph, sl, ml, sp. 30 litsea magnifica pn, kl, ph, ml, jh 31 lophopetalum pallidum kd, pn, sl, ns, ml, jh. 32 madhuca decipiens jh, sp. 33 madhuca selangorica ph, sl, ns; 34 neo-uvaria acuminatissima pn, ph, sl. 35 neo-uvaria foetida tg, ns, ml. 36 nephelium cuspidatum var. cuspidatum tg, sl, ns, jh 37 nephelium cuspidatum var. eriopetalum kl, ph, sl, ns, jh. 38 palaquium confertum jh. 39 palaquium leiocarpum kl, tg. 40 palaquium macrocarpum sl, jh. 41 palaquium obovatum kd, pn, kl, tg, ph, sl, ns, ml, jh, sp. 42 parishia maingayi tg, pk, ph, sl, jh, sp. no. species known distribution 43 pimelodendron macrocarpum sl, ml, jh. 44 polyalthia cinnamomea ps, kd, pn, kl, tg, ph, sl, ns, ml, jh. 45 pternandra galeata ph, jh. 46 rennellia microcephala kd, kl, tg, ph; 47 santiria rubiginosa var. nana sl. 48 schoutenia accrescens ssp. borneensis ulu endau, jh. 49 sterculia rubiginosa var. setistipula sl. 50 syzygium anisosepalum kd, ph, sl, ns, ml; 51 syzygium attenuatum ssp. attenuatum var. attenuatum kd, pn, ph, sl, ml, jh, sp. 52 syzygium claviflorum var. riparium ph 53 syzygium polyanthum var. polyanthum jh 54 syzygium polyanthum var. sessile jh 55 syzygium scortechinii var. scortechinii tg, ph, sl, ns, jh; 56 toona sureni ps, p. langkawi, kd, pn, kl, ph, sl, jh. 57 trigonostemon polyanthus jh. 58 urophyllum corymbosum ps, ph, sl, ns, jh, sp. notes: kd = kedah; pn = penang; sl = selangor; ns = negeri sembilan; ml = melaka; sp. = singapore; jh = johore; tg = terengganu; kl = kelantan; ps = perlis; ph = pahang. no. endemic species total individuals found distribution 1 actinodaphne pruinosa* 3 kd, pn, sl, ns, ml, sp 2 alangium ridleyi 1 kd, pn, tg, pk, ph, sl, ns, ml 3 alphonsea kingii 2 pk 4 alphonsea maingayi 1 pk, sl, ns, ml, jh, sp 5 aporosa selangorica 1 kd, pk, ph, sl 6 baccaurea griffithii 16 throughout 7 baccaurea polyneura 2 pk, ml; 8 beilschmiedia insignis 1 kl, pk, ph, sl 9 beilschmiedia pahangensis 1 kl, pk, ph 10 calophyllum subsessile 2 pk 11 calophyllum ferrugineum var. oblongifolium 4 kl, tg, pk, ph, sl, ns, ml, jh 12 calophyllum wallichianum var. wallichianum 6 kd, pn, kl, pk, ns 13 canarium pseudosumatranum 1 ps, kd, pk, ph, sl, ns 14 casearia clarkei var. clarkei 5 pn, pk, sl, ml, sp table 1 list of new tree species to perak state at perak tengah freshwater swamp forest, perak table 2 list of tree species found at perak tengah freshwater swamp forest that are endemic to peninsular malaysia endemism and conservation status of tree species of freshwater swamp forest, perak, malaysia liliwirianiset. al. 173 no. endemic species total individuals found distribution 15 cinnamomum mollissimum 2 pn, kl, tg, pk, ph, sl, ns, ml, jh 16 croton erythrostachys 1 tg, pk, ph, sl, ns, ml, jh 17 croton kelantanicus* 1 sg ketil, kl 18 cyathocalyx pruniferus 4 kl, tg, pk, ph, sl, ml, jh 19 dacryodes kingii 1 throughout 20 dacryodes puberula 1 pk, ph, ns, ml 21 dillenia reticulata var. reticulata* 8 ml 22 diospyros argentea 3 sp, tg, ml, pk, sl, ph, ns, jh, 23 diospyros ismailii* 2 kd, tg, ph, sl, ns, jh 24 diospyros scortechinii 1 kl, tg, pk, ph, ns 25 diospyros singaporensis 7 kd, pk, ph, sl, ns, ml, jh 26 elaeocarpus nitidus var. velutinus* 1 kl, ph, jh 27 elaeocarpus nitidus var. wrayi 1 tg, pk, ph 28 enicosanthum fuscum 6 pk, ph 29 enicosanthum macranthum 3 pk 30 garcinia uniflora 1 pk, ph 31 gentingia subsessilis 1 pk, ph, sl 32 gluta curtisii 1 kd, kl, pn, pk, ph, sl, jh 33 hydnocarpus filipes 1 tg, pk, ph, sl 34 hydnocarpus kunstleri var. tomentosa 1 tg, pk, ph, sl, jh; 35 knema oblongifolia 1 tg, pk, ph, sl 36 lithocarpus curtisii 4 pn, kl, tg, pk, ph, sl 37 litsea curtisii 1 kd, pn, pk, sl, jh 38 litsea wrayi 1 kd, pk, sl, ns 39 madhuca selangorica* 1 ph, sl, ns 40 mallotus griffithianus 2 throughout 41 mallotus penangensis 1 throughout 42 melanochyla nitida 1 pn, pk; 43 mesua lepidota var. parviflora 1 tg, pk, sl, ml, jh 44 mesua nuda 1 kd, pn, kl, tg, pk, ph, sl, ns, ml, jh 45 nephelium costatum 58 pk, ph, ns, ml, kd, kl, jh 46 nephelium hamulatum 9 kd, kl, tg, pk, ph, sl, ns, ml, jh 47 palaquium maingayi 2 kd, kl, pk, ph, sl, ns, ml, jh 48 palaquium oxleyanum 1 pk, ph, sl, sp 49 payena maingayi 7 kd, pn, tg, pk, ph, sl, ns, ml, jh, sp no. endemic species total individuals found distribution 50 pellacalyx saccardianus 12 widespread 51 pentace perakensis 1 pk 52 popowia fusca 1 pk, ph, sp 53 prunus malayana 6 tg, ph, pk 54 psydrax maingayi 5 tg, ph, pk, sl, ns, ml, jh 55 ptychopyxis caput-medusae 17 kl, tg, pk, ph, sl, ns, ml, sp 56 rennellia microcephala* 1 kd, kl, tg, ph 57 santiria ridleyi 5 gg keladang, pk 58 sarcotheca laxa var. laxa 52 ps, kd, kl, tg, pk 59 sarcotheca monophylla 9 pk, ph, sl, ml 60 sauropus suberosus 1 pk, ph 61 shorea singkawang ssp. singkawang 1 tg, ph, pk 62 streblus perakensis 3 pk, ph 63 syzygium anisosepalum* 3 kd, ph, sl, ns, ml 64 syzygium castaneum 2 pk, jh 65 syzygium claviflorum var. riparium* 1 sg tahan, ph 66 syzygium perakense 3 pk, sl 67 syzygium polyanthum var. polyanthum* 1 sg kayu, jh 68 syzygium polyanthum var. sessile* 2 jh 69 syzygium scortechinii var. scortechinii* 1 tg, ph, sl, ns, jh 70 vatica flavida 6 pk 71 vatica pallida 1 pn, pk notes: pk = perak; ml = melaka; kl = kelantan; ph = pahang; sl= selangor; ns = negeri sembilan; pn = penang; sp.= singapore; jh = johore; tg = terengganu. *new record in perak. since species have a necessary minimum population size and distribution for optimum survival, extremely small residual populations of rare and endemic taxa may all be extinct. this is exacerbated by edge effects, which may include invasion and species composition alteration by more common species prevalent just outside this forest. most studies identifying hotspots do not use the same criteria proposed at the global scale (major 1988), and endemicity is often the only criterion used (huang et al. 2012; kraf et al. 2010). this is because endemic species are often explored better in a particular area than the complete flora or the threats. furthermore, since narrowly endemic species are often endangered, endemic taxa are an important category for conservation (laffan et al. 2013)both within their region and at continental scales. we do this using the plant family myrtaceae in relation to the globally important greater blue mountains world biotropia vol. 31 no. 2, 2024 174 heritage area (gbmwha). additionally, since their numbers and distribution are well-known and because they sustain virtually all animal life, plants have been employed as indicators of species endemism. iucn conservation status a total of 2,830 plant species in malaysia are listed in the iucn red list of threatened species version 2020.1 (the iucn red list of threatened species, 2020). only one species is listed in the extinct (ex) category, one species is listed as extinct in the wild (ew), 166 species are listed as critically endangered (cr), 248 species are categorized as endangered (en), 579 species are included in the vulnerable category (vu), 194 species are classified as near threatened (nt), 102 species are table 3 the iucn conservation status of tree species in perak tengah freshwater swamp forest categorized as lower risk/conservation dependent (lr/ cd), 98 species are considered in the status of data deficient (dd), and 1,440 species are classified as the least concern (lc; the iucn red list of threatened species, 2020). species categorized as cr, en, and vu are collectively referred to as threatened. all the species found in the study plots were compared with the iucn red list, with 194 of 624 tree species in the perak tengah freshwater swamp forest were found in the red list of threatened species (table 3). the study area comprises 7% of the listed plants in the iucn for malaysia. the dipterocarpaceae family leads the number of threatened species with 22 species (46.8%), and the family comprises six species listed as cr. no. family species iucn status 1 anacardiaceae gluta curtisii lower risk/ least concern 2 anacardiaceae mangifera foetida lower risk/ least concern 3 anacardiaceae mangifera magnifica lower risk/ least concern 4 anacardiaceae melanochyla nitida lower risk/ least concern 5 anacardiaceae pentaspadon motleyi data deficient 6 anisophylleaceae anisophyllea apetala vulnerable 7 annonaceae alphonsea kingii critically endangered 8 annonaceae alphonsea maingayi lower risk/ least concern 9 annonaceae cyathocalyx pruniferus lower risk/ least concern 10 annonaceae enicosanthum fuscum near threatened 11 annonaceae enicosanthum macranthum vulnerable 12 annonaceae popowia fusca lower risk/ least concern 13 apocynaceae alstonia scholaris lower risk/ least concern 14 apocynaceae dyera costulata lower risk/ least concern 15 apocynaceae hunteria zeylanica lower risk/ least concern 16 aquifoliaceae ilex cymosa lower risk/ least concern 17 burseraceae canarium hirsutum lower risk/ least concern 18 burseraceae canarium littorale lower risk/ least concern 19 burseraceae canarium pseudodecumanum vulnerable 20 burseraceae canarium pseudosumatranum lower risk: conservation dependent 21 burseraceae dacryodes incurvata lower risk/ least concern 22 burseraceae dacryodes kingii lower risk: conservation dependent 23 burseraceae dacryodes puberula vulnerable 24 burseraceae dacryodes rostrata lower risk/ least concern 25 burseraceae santiria apiculata var. apiculata lower risk/ least concern 26 burseraceae santiria griffithii lower risk/ least concern 27 burseraceae santiria laevigata lower risk/ least concern 28 burseraceae santiria rubiginosa var. nana vulnerable 29 burseraceae santiria rubiginosa var. rubiginosa vulnerable 30 celastraceae bhesa paniculata lower risk/ least concern endemism and conservation status of tree species of freshwater swamp forest, perak, malaysia liliwirianiset. al. 175 no. family species iucn status 31 celastraceae bhesa robusta lower risk/ least concern 32 celastraceae lophopetalum javanicum lower risk/ least concern 33 chloranthaceae licania splendens lower risk/ least concern 34 chrysobalanaceae parinari costata ssp. polyneura lower risk/ least concern 35 ctenolophonaceae ctenolophon parvifolius vulnerable 36 dilleniaceae dillenia reticulata var. psilocarpella lower risk/ least concern 37 dilleniaceae dillenia reticulata var. reticulata lower risk/ least concern 38 dipterocarpaceae anisoptera costata endangered 39 dipterocarpaceae dipterocarpus crinitus vulnerable 40 dipterocarpaceae dipterocarpus kerrii endangered 41 dipterocarpaceae dipterocarpus kunstleri critically endangered 42 dipterocarpaceae dipterocarpus semivestitus critically endangered 43 dipterocarpaceae hopea apiculata endangered 44 dipterocarpaceae neobalanocarpus heimii endangered 45 dipterocarpaceae shorea curtisii ssp. curtisii lower risk/ least concern 46 dipterocarpaceae shorea guiso vulnerable 47 dipterocarpaceae shorea hemsleyana ssp. hemsleyana vulnerable 48 dipterocarpaceae shorea hopeifolia critically endangered 49 dipterocarpaceae shorea leprosula near threatened 50 dipterocarpaceae shorea macrantha critically endangered 51 dipterocarpaceae shorea macroptera lower risk/ least concern 52 dipterocarpaceae shorea materialis critically endangered 53 dipterocarpaceae shorea multiflora lower risk/ least concern 54 dipterocarpaceae shorea ovalis ssp. ovalis endangered 55 dipterocarpaceae shorea parvifolia ssp. parvifolia endangered 56 dipterocarpaceae shorea pauciflora endangered 57 dipterocarpaceae shorea platycarpa critically endangered 58 dipterocarpaceae shorea singkawang ssp. singkawang vulnerable 59 dipterocarpaceae vatica flavida critically endangered 60 dipterocarpaceae vatica pallida endangered 61 dipterocarpaceae vatica pauciflora vulnerable 62 dipterocarpaceae vatica stapfiana vulnerable 63 dipterocarpaceae vatica umbonata lower risk/ least concern 64 dipterocarpaceae vatica venulosa critically endangered 65 ebenaceae diospyros apiculata lower risk/ least concern 66 ebenaceae diospyros areolata lower risk/ least concern 67 ebenaceae diospyros argentea lower risk/ least concern 68 ebenaceae diospyros ismailii lower risk/ least concern 69 ebenaceae diospyros scortechinii lower risk/ least concern 70 ebenaceae diospyros singaporensis lower risk/ least concern 71 elaeocarpaceae elaeocarpus petiolatus lower risk/ least concern 72 euphorbiaceae baccaurea ramiflora lower risk/ least concern 73 euphorbiaceae breynia vitis-idaea lower risk/ least concern 74 euphorbiaceae claoxylon longifolium lower risk/ least concern 75 euphorbiaceae croton kelantanicus vulnerable 76 euphorbiaceae endospermum diadenum lower risk/ least concern 77 euphorbiaceae mallotus penangensis lower risk/ least concern 78 euphorbiaceae sapium baccatum lower risk/ least concern 79 fagaceae lithocarpus curtisii vulnerable biotropia vol. 31 no. 2, 2024 176 no. family species iucn status 80 flacourtiaceae hydnocarpus filipes vulnerable 81 flacourtiaceae ryparosa javanica lower risk/ least concern 82 gnetaceae gnetum gnemon var. gnemon lower risk/ least concern 83 guttiferae calophyllum soulattri lower risk/ least concern 84 guttiferae cratoxylum arborescens var. arborescens lower risk/ least concern 85 guttiferae cratoxylum formosum lower risk/ least concern 86 guttiferae cratoxylum maingayi lower risk/ least concern 87 guttiferae garcinia scortechinii lower risk/ least concern 88 guttifereae cratoxylum cochinchinense lower risk/ least concern 89 guttifereae garcinia uniflora lower risk/ least concern 90 guttifereae mesua nuda lower risk/ least concern 91 irvingiaceae irvingia malayana lower risk/ least concern 92 lauraceae actinodaphne pruinosa lower risk/ least concern 93 lauraceae alseodaphne insignis lower risk/ least concern 94 lauraceae beilschmiedia insignis lower risk/ least concern 95 lauraceae beilschmiedia kunstleri lower risk/ least concern 96 lauraceae beilschmiedia madang lower risk/ least concern 97 lauraceae beilschmiedia pahangensis lower risk/ least concern 98 lauraceae beilschmiedia wallichiana vulnerable 99 lauraceae cinnamomum iners lower risk/ least concern 100 lauraceae cinnamomum mollissimum lower risk/ least concern 101 lauraceae cinnamomum porrectum lower risk/ least concern 102 lauraceae cryptocarya ferrea lower risk/ least concern 103 lauraceae litsea castanea lower risk/ least concern 104 lauraceae litsea curtisii endangered 105 lauraceae litsea elliptica lower risk/ least concern 106 lauraceae litsea erectinervia lower risk/ least concern 107 lauraceae litsea grandis lower risk/ least concern 108 lauraceae litsea myristicifolia lower risk/ least concern 109 lauraceae litsea sessiliflora lower risk/ least concern 110 lauraceae litsea wrayi lower risk/ least concern 111 lauraceae nothaphoebe panduriformis lower risk/ least concern 112 leguminosae acacia mangium lower risk/ least concern 113 leguminosae cynometra malaccensis near threatened 114 leguminosae koompassia malaccensis lower risk/ conservation dependent 115 leguminosae pongamia pinnata lower risk/ least concern 116 leguminosae sindora coriacea lower risk/ least concern 117 meliaceae aglaia elliptica lower risk/ least concern 118 meliaceae aglaia argentea lower risk/ least concern 119 meliaceae aglaia crassinervia near threatened 120 meliaceae aglaia odoratissima lower risk/ least concern 121 meliaceae aglaia rubiginosa near threatened 122 meliaceae aglaia squamulosa near threatened 123 meliaceae sandoricum koetjape vulnerable 124 moraceae antiaris toxicaria lower risk/ least concern 125 moraceae artocarpus elasticus lower risk/ least concern 126 moraceae ficus fistulosa var. fistulosa lower risk/ least concern 127 moraceae ficus vasculosa lower risk/ least concern 128 myristicaceae horsfieldia crassifolia near threatened endemism and conservation status of tree species of freshwater swamp forest, perak, malaysia liliwirianiset. al. 177 no. family species iucn status 129 myristicaceae horsfieldia irya lower risk/ least concern 130 myristicaceae horsfieldia penangiana vulnerable 131 myristicaceae horsfieldia superba near threatened 132 myristicaceae horsfieldia tomentosa near threatened 133 myristicaceae knema conferta lower risk/ least concern 134 myristicaceae knema curtisii var. paludosa vulnerable 135 myristicaceae knema furfuracea lower risk/ least concern 136 myristicaceae knema glauca vulnerable 137 myristicaceae knema kunstleri near threatened 138 myristicaceae knema latericia ssp. ridleyi lower risk/ least concern 139 myristicaceae knema laurina var. heteropilis lower risk/ least concern 140 myristicaceae knema laurina var. laurina lower risk/ least concern 141 myristicaceae knema oblongifolia lower risk: conservation dependent 142 myristicaceae knema scortechinii lower risk/ least concern 143 myristicaceae knema stenophylla lower risk/ least concern 144 myristicaceae myristica iners lower risk/ least concern 145 myrsinaceae ardisia korthalsiana lower risk/ least concern 146 myrtaceae melaleuca cajuputi lower risk/ least concern 147 myrtaceae rhodamnia cinerea lower risk/ least concern 148 myrtaceae syzygium claviflorum var. claviflorum lower risk/ least concern 149 myrtaceae syzygium claviflorum var. riparium lower risk/ least concern 150 myrtaceae syzygium nervosum lower risk/ least concern 151 olacaceae anacolosa frutescens lower risk/ least concern 152 olacaceae ochanostachys amentacea data deficient 153 opiliaceae champereia manillana lower risk/ least concern 154 oxalidaceae sarcotheca laxa var. laxa vulnerable 155 oxalidaceae sarcotheca monophylla near threatened 156 proteaceae helicia attenuata lower risk/ least concern 157 proteaceae helicia petiolaris var. petiolaris lower risk/ least concern 158 rhizophoraceae pellacalyx saccardianus lower risk/ least concern 159 rosaceae prunus arborea var. arborea lower risk/ least concern 160 rosaceae prunus malayana lower risk/ least concern 161 rosaceae prunus polystachya lower risk/ least concern 162 rutaceae acronychia pedunculata lower risk/ least concern 163 sapindaceae arytera littoralis lower risk/ least concern 164 sapindaceae glenniea penangensis vulnerable 165 sapindaceae lepisanthes tetraphylla lower risk/ least concern 166 sapindaceae nephelium costatum vulnerable 167 sapindaceae nephelium hamulatum vulnerable 168 sapindaceae nephelium lappaceum var. lappaceum lower risk/ least concern 169 sapindaceae nephelium lappaceum var. pallens lower risk/ least concern 170 sapindaceae pometia pinnata lower risk/ least concern 171 sapotaceae chrysophyllum roxburghii lower risk/ least concern 172 sapotaceae madhuca korthalsii near threatened 173 sapotaceae madhuca motleyana near threatened 174 sapotaceae madhuca selangorica lower risk: conservation dependent 175 sapotaceae madhuca utilis endangered 176 sapotaceae palaquium gutta near threatened 177 sapotaceae palaquium herveyi near threatened biotropia vol. 31 no. 2, 2024 178 no. family species iucn status 178 sapotaceae palaquium leiocarpum near threatened 179 sapotaceae palaquium maingayi vulnerable 180 sapotaceae palaquium obovatum lower risk/ least concern 181 sapotaceae payena lucida near threatened 182 sapotaceae payena maingayi lower risk/ least concern 183 sapotaceae pouteria malaccensis near threatened 184 sterculiaceae sterculia parviflora lower risk/ least concern 185 thymelaeaceae aquilaria malaccensis vulnerable 186 thymelaeaceae gonystylus affinis vulnerable 187 tiliaceae microcos laurifolia lower risk: conservation dependent 188 tiliaceae microcos tomentosa lower risk/ least concern 189 tiliaceae pentace curtisii near threatened 190 tiliaceae pentace perakensis vulnerable 191 tiliaceae pentace triptera lower risk/ least concern 192 ulmaceae gironniera subaequalis lower risk/ least concern 193 verbenaceae callicarpa longifolia lower risk/ least concern 194 verbenaceae vitex pinnata lower risk/ least concern a total of 92 taxa of peninsular malaysia dipterocarps are listed under the threatened category of the iucn red list (saw et al. 2010). there are four species of burseraceae; three species of myristicaceae, sapindaceae, and sapotaceae; two species of thymelaeaceae and lauraceae; and one species of anisophylleaceae, annonaceae, euphorbiaceae, fagaceae, flacourtiaceae meliaceae, oxalidaceae, and tiliaceae. thus, the threatened species of dipterocarps found in the perak tengah freshwater swamp forest contributed to 23.9% of threatened peninsular malaysia dipterocarp species. liliwirianis (2021) reported that some dipterocarp species residing in perak tengah comprise cr species. unfortunately, the population size, to some extent, is extremely small, comprising less than 53 individuals, as befallen d. semivestitus. the tree number had decreased from the report by saw (2010) over ten years. saw (2010) reported that the freshwater swamp forest of the lumut district, perak, containing some of the rarest trees (d. semivestitus and v. flavida), is now confined to remnant areas. in the case of d. semivestitus, the species exists only as a remnant population in a 55-ha freshwater swamp forest at universiti teknologi mara, perak. to date, it is the only population that has been discovered in peninsular malaysia. an extensive survey of this area found only 53 individual trees. the species, which was once considered extinct, has been found in perak, malaysia. endangered tree conservation is of utmost priority, as endangered species compete for limited suitable habitats with human development. saw et al. (2010) reported that 92 taxa (56.1%) of dipterocarpaceae were at some level of threat in malaysia. in addition, saw and yen (2000) urged changes in land use patterns and rising demands on forest resources, and malaysia’s attention to the sustainable use of forest resources now involves consideration of the entire problem of protection of all biodiversity. the iucn red list categories and criteria were developed using five criteria: population reduction, geographic range in the form of extent of occurrence or area of occupancy, small population size and decline, very small or restricted population, and quantitative analysis. yong et al. (2021) confirmed that perak is the second state with the highest number of threatened taxa, with 166 taxa. in addition, chua et al. (2010) affirmed that perak is the second highest state that comprises several threatened taxa (53 taxa) of dipterocarps and perhaps is the most vulnerable as it has the greatest number of cr taxa (10), i.e., dipterocarpus coriaceus, d. semivestitus, hopea auriculata, hopea bilitonensis, parashorea globosa, s. hemsleyana ssp. endemism and conservation status of tree species of freshwater swamp forest, perak, malaysia liliwirianiset. al. 179 hemsleyana, shorea lamellata, shorea lumutensis, shorea teysmanniana, and v. flavida. our study showed that the freshwater swamp forest of the perak tengah study area consisted of some of the species: d. semivestitus, s. hemsleyana ssp. hemsleyana, and v. flavida. conclusion for a large field of research in freshwater swamp forests in perak tengah, the amount of endangered, new records, and vulnerable species is high. the results indicated that six species were documented as new for perak, endemic and listed in the iucn. the species were v. pallida, c. kelantanicus, madhuca selangorica, actinodahphne pruinosa, dillenia reticulata var. reticulata, diospyros ismailii, and s. claviflorum var. riparium. the species are also highly specialized since they are only confined to their special habitats. thus, these unique groups would be at risk if the freshwater swamp forest and its adjacent regions are disturbed. conservation of native and rare species could demand active protection, restoration, and high uniqueness of species-rich habitats and areas since the listed threatened species are currently not protected in the network of totally protected areas (tpa) despite some of the study areas being monitored by the forest research institute of malaysia (frim). the perak tengah freshwater swamp forest has unique areas that house many endemic and endangered species needing of protection, and it is important to protect the environment and mitigate the threats. acknowledgments this work was supported by the research grants through the fundamental research grant scheme (frgs) 600-rmi/frgs 5/3 (41/2014) and uitm’s research intensive faculty (rif) grant (600-rmi/dana 5/3/rif (404/2012)). references anderson s. 1994. area and endemism. the quarterly review of biology 69(4):451-71. chua lsl, suhaida m, hamidah m, saw lg. 2010. malaysia plant redlist: peninsular malaysian dipterocarpaceae. research pamphlet. no. 129. kepong (my): forest research institute malaysia. davies kf, margules cr, lawrence jf. 2004. a synergistic effect puts rare, specialized species at greater risk of extinction. ecology 85(1):265-71. dobson fs, yu j, smith at. 1995. the importance of evaluating rarity. conservation biology 9(6):1648-51. fifth national report to the convention on biological diversity. 2020. government of malaysia ministry of natural resources and environment fifth national report to the convention on biological diversity. www.nre.gov. my huang j, chen b, liu c, lai j, zhang j, ma k. 2012. identifying hotspots of endemic woody 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lumpur. the southeast asian journal of tropical biology vol. 31 no. 3, 2024: 380 390 doi: 10.11598/btb.2024.31.3.2281 issn: 0215-6334 | e-issn: 1907-770x 380 tree species diversity and management in agroforestry pohpohan (pilea melastomoides): the case in the traditional zone of gunung halimun salak national park, indonesia adisti permatasari putri hartoyo1,2,*, sheikha ananda mosa1, bayu winata1, lufthi rusniarsyah1, peniwidiyanti3, izzbilhaq1 1department of silviculture, faculty of forestry and environment, ipb university, bogor 16680, indonesia 2environmental research centre, ipb university, bogor 16680, indonesia 3research center for ecology and ethnobiology, national research and innovation agency (brin), bogor 16911, indonesia article higlights • pohpohan (pilea melastomoides) is the main commodity cultivated by using traditional agroforestry in the traditional zone of gunung halimun salak national park as a fresh vegetable. • there is a limited database of plant species diversity in the traditional zones, which is dynamic and needs further research. • this study found 54 tree species from 37 families with the dominant family being rubiaceae. • most farmers cultivated pohpohan by conducting plant spacing, maintenance, and harvesting. their income is usd 154 – 256/month. article information received 17 june 2024 revised 1 october 2024 accepted 11 october 2024 reviewers khin maung sint, arifah rahayu rusdi evizal *corresponding author, e-mail: adistipermatasari@apps.ipb.ac.id research article abstract gunung halimun salak national park (ghsnp) is java island’s largest tropical mountain rainforest. ghsnp is still facilitating community grounds around ghsnp with the existence of a traditional zone. pohpohan (pilea melastomoides) is the primary commodity in the traditional zone cultivated by the local people around ghsnp. pohpohan is planted using traditional agroforestry planting patterns. the traditional zone has a limited diversity of plant species and a limited management database, so further study is necessary. this study was carried out in the agroforestry system in tamansari village, bogor regency, which is in the traditional zone of ghsnp. data collection for tree diversity was conducted by establishing 25 plots. this study also interviewed 30 local farmers belonging to the mandiri farmer group. there were 54 tree species from 37 families found in this study. rubiaceae, malvaceae, fabaceae, and phyllanthaceae families were the most common families existed in the study site. pinus merkusii, agathis dammara, and maesopsis eminii had the highest important value index (ivi). the understory and seedlings were categorized as low in h’ due to the dominant presence of pohpohan planting. most respondents were farmers aged 35-55 (70%) with a farming area of 1,000 m2. they cultivated pohpohan by conducting plant spacing, plant maintenance (e.g., soil cultivation, weeding, fertilization), and harvesting. fertilization for pohpohan was conducted 9 times a year. the farmers produced 30,000-50,000 bunches per harvest time. their monthly income was about usd 154-256 per month. keywords: farmer group, ivi, pohpohan, traditional agroforestry, vegetation diversity introduction gunung halimun salak national park (ghsnp) is an area of natural conservation. it is the largest tropical mountain rainforest on java island. administratively, ghsnp is located in three regencies, namely bogor regency, lebak regency, and sukabumi regency. the designation of the ghsnp area as a conservation area began with the decree of the minister of forestry of the republic of indonesia number 282/kpts-ii/1992 issued on 28 february 1992 declaring an area of 40,000 ha as the gunung halimun national park (ghnp). subsequently, the minister of forestry of the republic indonesia issued a decree number 175/kpts-ii/2003 dated 10 june 2003 to expand the ghsnp area to 113,357 ha as a response to the growing occurrences of forest disturbances. this expansion included the forests of mount salak, mount endut, and the surrounding areas, which had previously been classified as limitedproduction forests and protected forests managed by state-owned forest enterprise (perum https://doi.org/10.11598/btb.2024.31.2.2281 tree species diversity and management in agroforestry pohpohan (pilea melastomoides) hartoyo et al. 381 perhutani). since then, the ghnp area has been transformed into a unified ghsnp conservation area (ekayani et al. 2014). gunung halimun salak national park is classified into several zones to facilitate spatial arrangement within the national park. these zones are generally classified based on the community’s ecological, social, economic, and cultural conditions. according to the 2007-2026 ghsnp management plan, the ghsnp area is divided into eight zones: traditional zone, cultural zone, special zone, rehabilitation zone, utilization zone, jungle zone, core zone, and enclave. however, zoning in the ghsnp forest area was slightly altered based on the decree of the minister of environment and forestry of the republic of indonesia number 327/menlhk/setjen/pla.2/4/2016 dated 26 april 2016, which changed the function of 17,373 ha in the ghsnp forest area and returning another use area (enclave) covering 7,847 ha. as a result, the ghsnp is divided only into seven zones, covering an area of 87,699 ha (btnghs 2021). the expansion of the ghnp into the ghsnp area, which includes land used for community economic activities, has created a conflict between ecological (conservation) and economic interests. this issue is addressed by allowing a small portion of the ghsnp area for community use, which permits local farmers to continue their economic activities. the gunung halimun salak national park office (ghsnpo) still facilitates several community grounds around ghsnp, designating these areas as the traditional zone. local communities use the traditional zone to supply their daily necessities by using non-timber forest resources, such as tapping pine sap, tapping copal sap, and farming pohpohan leaves. pohpohan (pilea melastomoides) is the main commodity cultivated by using traditional agroforestry planting patterns in the traditional zone as fresh vegetable, mainly by the sundanese people in indonesia. pohpohan cultivation is particularly significant in tamansari village, bogor regency. agroforestry is a form of multi-story land use that includes a mixture of trees and annual plants. agroforestry land must have at least woody plant species (olivi et al. 2015). in the traditional zone of ghsnp, pohpohan is shaded by pine (pinus merkusii), agathis (agathis dammara), and african wood (maesopsis eminii), which are planted by perum perhutani. local communities have been practising the pohpohan agroforestry system in this area for decades, considering ecological, social, and economic factors. therefore, to accomplish sustainable management, studying agroforestry systems is crucial. additionally, there is a limited database of plant species diversity in the traditional zones, particularly in the pohpohan agroforestry system, which is dynamic and needs further research. the purpose of this study is to analyze the diversity of tree species and management in the pohpohan agroforestry of gunung halimun salak national park, built upon traditional ecological knowledge (tek). materials and methods study area this study was carried out in the traditional zone of gunung halimun salak national park (ghsnp), west java province, indonesia. the specific study site is the pohpohan (pilea melastomoides) agroforestry system in tamansari village, bogor regency, west java province, indonesia (fig.1). ghsnp is administratively divided into three districts: bogor regency, lebak regency, and sukabumi regency. geographically, the national park is located between 106°21’ 106°38’ e and 6°37’ 6°51’ s. ghsnp is vital in maintaining biodiversity, regulating water management, supporting education and research, and functions as germplasm sources, cultivation development, and tourism. salak mountain has a height ranging from 400 to 2,210 masl. the climate type of ghsnp is classified as type b according to schmidt and ferguson, characterized by wet conditions with rainfall of 4,000-6,000 mm/year and a dry season that lasts less than three months, typically from may to september. the average monthly temperature in ghsnp ranges from 19.7 °c to 31.8 °c and the average air humidity is 88% (adalina et al. 2014). biotropia vol. 31 no. 3, 2024 382 procedures plot establishment data on tree diversity was collected by establishing 25 plots within ghsnp. the plot selection was carried out using the purposive sampling method, with criteria including representing an agroforestry system containing at least one species of woody plant, managed by the local community for generations, and having at least one main commodity species contributing to the income of the local community. each of the 25 figure 1 study area in the traditional zone of ghsnp plots measured 20 m × 20 m. the square plots were divided into several square subplots. for detailed observation, 20 m × 20 m plots for tree level, 10 m × 10 m for pole level, 5 m × 5 m for sapling level, and 2 m x 2 m for understory and seedling level (fig. 2). tree species diversity measurement this study collected data on understory vegetation and all tree growth levels, including seedlings, saplings, poles, and trees. for understory vegetation and seedlings (lower-tier shrubs and figure 2 design of observation plots tree species diversity and management in agroforestry pohpohan (pilea melastomoides) hartoyo et al. 383 density (d) = total number of individuals of a species found total area examined (1) relative density (rd) = number of individuals of the species total number of individuals of all species x 100% (2) frequency (f) = number of a plot where species found total size plots (3) relative frequency (rf) = frequency of species sum of frequency values for all species x 100% (4) dominance (do) = total basal area of the species total size plots (5) relative dominance (rdo) = total basal area of the species total basal area of the species x 100% (6) important value index (ivi) = rd + rf (for seedlings and saplings) (7) importance value index (ivi) = rd + rf + rdo (for poles and trees) (8) small trees with a height of < 1.5 m), the data collected included the names of species and the number of individuals for each species. for saplings (trees with a height of at least 1.5 m and a diameter of under 10 cm), poles (trees with a diameter of 10-20 cm), and mature trees (trees with a diameter of ≥ 20 cm), the measured parameters included the individual’s dbh (diameter at breast height), total height, and branch-free height. interviewing the local farmers related to the management of agroforestry based on traditional ecological knowledge traditional ecological knowledge (tek) data were gathered through in-depth interviews using an interview guide with all members of the mandiri farmers group (kelompok tani mandiri) in tamansari village. this community has a conservation partnership cooperation agreement with gunung halimun salak national park. tek reflects the close relationship between society and the environment, particularly in planting. tek encompasses ideological superstructures (religion/beliefs and religious values), material infrastructure (ideas, concepts, and equipment used), and social structures that shape culture and evolve (unesco 2009). tek documentation was conducted through participatory observation (kawulich 2005) of community activities to verify interview results. thirty respondents of local pohpohan farmers from kelompok tani mandiri were interviewed using questionnaires. these questionnaires included questions about the farmers’ identities, conditions of agroforestry sites, challenges faced by the local farmers, and their needs in managing agroforestry sites. the community interviews focused on activities related to managing the conservation partnership area, from cultivation and harvesting to post-harvest handling. data analysis importance value index the collected vegetation data were subjected to a quantitative analysis, with each species’ importance value index (ivi) calculated. the ivi was used to describe and compare the species in the plots (cottam & curtis 1956). the plot’s most “important” species were those with the highest ivi. this index aids in assessing each species’ relative relevance within the community structure. the relationship between the total relative density (rd), relative frequency (rf), and relative dominance (rdo) represents the ivi for each species, which has values ranging from 0 to 300 (ismail 2017). the calculation of ivi for seedlings, understory, and saplings differs from that for poles and trees. the total of rd, rf, and rdo is the ivi for poles and trees. in contrast, the ivi for the understory vegetation, seedlings, and saplings is the total of the rd and rf, resulting in ivi values ranging from 0 to 200. according to sari et al. (2022), the following formula can be used to calculate the density (d), frequency (f), and dominance (do) of the ivi. biotropia vol. 31 no. 3, 2024 384 index of species diversity (h’) an ecosystem’s species diversity, stability, and maturity can be measured by the tree species diversity index which is also known as the shannonwiener index. according to putri and indriyanto (2021), a high h’ value indicates that a community is stable against disturbance, comprising many species with similar abundances. margalef (1972) stated that the shannon-wiener index ranges from 1.5 to 3.5, with ecosystems having an h’ value below 1.5 considered low diversity. this species diversity index can be determined using the formula below (margalef 1972): where: h’= species diversity index (shannon-wiener index) ni = the significance value of the i-th species n = the importance of all species index of species dominance (c) the simpson species dominance index (c) estimates the dominance levels of certain species within a community. a high level of diversity in a community corresponds to a low dominance value. the simpson dominance index ranges from 0 to 1. a dominance value close to 1 indicates that one or a few species dominate the community, while a value close to 0 suggests that multiple species share dominance. according to simpson (1949), species dominance can be determined using the formula below: where: c = species dominance index ni = the i-th species density n = the total of density index of species richness (r) the margalef species richness index indicates the species richness in a study area. jorgensen et al. (2005) set the lower limit value for the species richness index at 2.05. a species richness index of more than 2.05 indicates a high species richness in an ecosystem. the formula for calculating the r-value is as follows: h' = ∑ � ni n � ln ni n �� i=i n � � (9) c =∑ � ni n � k=0 n 2 (10) r = (s-1) in(n) (11) where: r = species evenness index s = number of species found n = total number of individuals found index of species evenness (e) the species evenness index shows the relationship between species abundance and species diversity in a community (wahyuningsih et al. 2019). the species evenness index value ranges from 0 to 1. a value close to 0 indicates low species evenness, while a value close to 1 indicates high species evenness. the formula for the species evenness index is as follows: e = h’ in(s) (12) where: e = species evenness index h’ = species diversity index s = number of species found results and discussion species composition in the 25 plots of 20 m x 20 m, there were 54 species from 37 families recorded. the most frequent families found were rubiaceae (4 species) and malvaceae, fabaceae, and phyllanthaceae (each with 3 species) (table 1). table 1 the number of species found in the traditional zone of ghsnp categories number of species understory and seedling 34 sapling 9 pole 5 tree 13 the sum of all categories 31 understory and seedlings are two different things. understory refers to vegetation found beneath forest stands, excluding tree saplings. seedlings are part of tree regeneration, which is included in the tree growth level. the existence of understory plants on the forest floor can improve soil and water conservation. their extensive root systems form dense clumps that prevent erosion, protect the soil from rainwater and surface runoff, tree species diversity and management in agroforestry pohpohan (pilea melastomoides) hartoyo et al. 385 and increase organic matter (indriyani et al. 2017). additionally, understory vegetation plays a crucial role in forest ecosystems by influencing the microclimate. however, understory plants can also compete with and inhibit the growth of tree regeneration (hilwan et al. 2013). in this study, data of understory and seedlings were combined because they were found in the same plots. the understory and seedlings had the most species compared to other categories (table 1). importance value index (ivi) analysis was used to identify the species dominating the observation plot. according to smith (1977), the dominant species can utilize their environment more efficiently than other species in the same place. the ivi in the traditional zone of ghsnp is presented in table 2. importance value index (ivi) is an indicator to determine the role of species in a community. higher ivi indicates a more significant role of the species within the community, and vice versa (rawana et al. 2022). the dominant species in forest vegetation are those with the highest ivi values (ismail 2017). a species is considered significant if the ivi value is ≥ 10% for seedlings and saplings, and ≥ 15% for poles and trees. this study showed that pohpohan (pilea melastomoides) had the highest ivi in the understory and seedling categories (table 2). based on this definition, pohpohan was the dominant species in the observation plot, which was influenced by a combination of anthropogenic and ecological factors. local farmers cultivated pohpohan due to its use as a vegetable or medicinal plant. this intervention significantly increased pohpohan presence and dominance then raised its ivi. additionally, pohpohan often grows well in partially shaded areas, such as forest understories. it competes effectively when light intensity is moderate. forest management with the community (phbm) mechanism, which was based on the decree of the perum perhutani supervisory board no. 136/kpts/dir/2001 issued on 29 march 2001 regarding forest resource management in collaboration with the local community, promoted the sharing principle to ensure that common interests in sustaining the functions and benefits of forest resources are achieved in an optimal and proportionate approach. in ghsnp, phbm is used by implementing a social forestry model, specifically agroforestry, with pine (pinus merkusii) as the primary tree species and pohpohan (pilea melastomoides) as the primary agricultural crop for the tamansari village community. despite changes in land management, the residents of tamansari village still can sustainably cultivate and utilize trees. according to the terms of cooperation agreements (pks) 255.t.14/tu/kum.3/2/2021 and pks.1/ktmandiri/2/2021 with ghsnp, the community formed a conservation partnership in 2021, which granted the community access to a 79.29 ha area for cultivating pohpohan within the pine stands (adhiningsih et al. 2022). plants that shaded the pohpohan area were dominated by the species on the tree growth level in table 2. based on that, pine, agathis, and african wood had the highest ivi values and thus, the three species were considered dominating the land. pine resin and copal were used as non-timber forest products (ntfps) by local communities that depended on their livelihoods as sap tappers. the gunung halimun salak national park office (btnghs) still facilitates several community groups around tnghs, such as pine sap tappers, copal sap tappers, and pohpohan leaf (pilea melastomoides) farmers. apart from that, african wood (maesopsis eminii) and agathis (agathis dammara) were often found in the ghsnp area, which perum perhutani previously planted with the aim of reforestation. these three species are suitable as shade plants for pohpohan. this follows the previous study by hartoyo et al. (2022) that shade trees had an important role in pohpohan growth. pohpohan can live at no more than 60% shading because overshading trees will affect the growth of pohpohan. pine, agathis, and african wood have medium canopy coverage, so these three species are suitable for pohpohan growth, and these three species can continue to be maintained. biotropia vol. 31 no. 3, 2024 386 table 2 five highest ivi categories scientific name ivi (%) understory and seedling pilea melastomoides 90.13 pallinia ciliate 14.14 pandanus amaryllifolius 9.45 kakasoan 8.98 stenochlaena palustris 7.33 sapling baccaurea racemosa 46.75 swietenia macrophylla 32.47 solanum torvum 23.38 agathis dammara 16.23 spathodea campanulate 16.23 pole baccaurea motleyana 80.65 maesopsis eminii 73.47 artocarpus heterophyllus 54.77 baccaurea racemose 52.08 tree pinus merkusii 83.96 agathis dammara 62.92 maesopsis eminii 48.45 altingia excelsa 36.92 schima wallichii 28.42 tree species diversity biodiversity is an indicator and a mechanism for changes in ecological systems and species dynamics. rahayu et al. (2017) stated that biodiversity encompasses species richness and complex ecosystems, influencing community structure, stability, and ecosystem development. therefore, it is important to understand the relation between tree species diversity and general biodiversity. in this study, information about biodiversity is needed to assess the compatibility and interactions between plants in the study area, particularly in agroforestry land. species diversity can be evaluated using the vegetation index values, which consist of species diversity index (h’), species dominance index (c), species richness index (r), and species evenness index (e). the vegetation index values for the ghsnp traditional zone are presented in table 3. table 3 vegetation index categories h’ e r c understory and seedling 1.44 (l) 0.41 4.64 (h) 0.49 sapling 2.04 (m) 0.93 3.03 (h) 0.15 pole 1.55 (m) 0.96 2.05 (h) 0.22 tree 1.89 (m) 0.74 2.58 (h) 0.20 notes: (l) = low; (m) = moderate; (h) = high. shannon-wiener species diversity index (h’) measures the complexity of species interactions within a community. soerianegara (1996) explained that there is no absolute standard for determining high or low species diversity index values, as these categories vary by location and ecosystem. however, shannon-wiener index values rarely exceed 4. it typically ranges from 1.5 to 3.5. in indonesia, diversity index values below 1.5 are considered low for various forest types. h’ value calculated in this study for saplings, poles, and trees fell into the medium category, while understory plants were categorized as having low diversity (table 2). this lower diversity is attributed to the predominance of a single commodity cultivated, namely pohpohan (pilea melastomoides). species evenness index (e) ranges from 0 to 1. an e value close to 0 indicates low species evenness, while a value near 1 suggests relatively even species abundance (magurran 2004). the highest e values were observed in the sapling and pole categories (table 2). hilwan and irfani (2018) explained that the conditions of the research plot support the proliferation and domination of multiple species, as shown by a high e value. this indicates that the distribution of individuals and species at the sapling and pole level is nearly even, and the species present have the stability to maintain their populations (ibadurrohmah 2016). jorgensen et al. (2005) set a species richness index lower limit of 2.05, considering an ecosystem’s species richness to be high if it exceeds this value. in this study, all categories of trees were tree species diversity and management in agroforestry pohpohan (pilea melastomoides) hartoyo et al. 387 in the high species richness category (table 2). the r value corresponded to the relatively small number of species found at the tree growth level. this finding aligns with karim (2017), who stated that a high species richness index (r) is influenced by the number of species and individuals in the study area. the simpson species dominance index (c) ranges from 0 to 1. a dominance value close to 0 indicates that multiple species dominate equally and that no single species dominates. the understory and seedling category showed the highest c value (table 3). this high dominance is related to the low shannon-wiener species diversity index (h’) due to the predominance of pohpohan (pilea melastomoides) in the observation plot. management in agroforestry pohpohan (pilea melastomoides) based on traditional ecological knowledge local communities are key stakeholders with significant potential in the collaborative management of the gunung halimun salak national park. according to a village survey (a) (b) (c) (d) figure 3 general information on interview results notes: (a) age of respondent; (b) educational background; (c) large farm area; (d) main job. conducted by the ghsnpo and japan international cooperation agency (2007), there were 348 villages in and around the ghsnp area, with a population of approximately 99,000. many of these communities rely on forest resources for their daily necessities, such as firewood, animal feed, water, medicinal plants, bamboo, and agricultural land. darusman (2000) argued that people who benefit from a forest are more likely to take action to preserve it. several studies showed that communities around ghsnp can manage forests effectively (rahmawati et al. 2008; hendarti 2008; adalina 2014). the more critical a party’s influence, the greater the need for their involvement in natural resource management (reed et al. 2009). general information general information from these respondents is necessary to understand the age distribution within the community and identify the predominant types of work performed by the community. the interview results providing this general information are illustrated in figure 3. biotropia vol. 31 no. 3, 2024 388 the interview results showed that of the 30 members of pohpohan local farmers involved in kelompok tani mandiri in tamansari village, the majority (35%) were between the ages of 35-45 years old and 46-55 years old. interestingly, 18% of the group members, who were over 65 years old, were still actively engaged in pohpohan farming activities. most members had only completed elementary school. only 13% of them graduated from high school. the interviews revealed that 32% of the members had a pohpohan farming area of 1,000 m2, while 27% had 2,000 m2. only 9% owned larger land of 10.000 m2. the remaining farmers owned land ranging from 600 m2 to 5,000 m2. the primary occupation for 53% of the respondents was farming. according to the 2019 village potential data, most tamansari village residents worked as laborers, followed by farmers. the village also had significant potential for plantations and livestock, likely contributing to the high number of people (a) (b) (c) (d) working as farmers. additionally, there was a tradition in the tamansari village community of passing down occupations from generation to generation, ensuring the continuity of certain types of jobs across generations. agroforestry land management interview results with 30 respondents in tamansari village indicated that each member employed a  different management method. a summary of these interview findings is illustrated in figure 4. most respondents planted pohpohan at a spacing of 15 cm × 15 cm (fig. 4). only 4% used a spacing of 15 cm × 20 cm. most respondents arrived at these planting distances through years of trials and errors, allowing them to determine the optimal spacing for their crops. additionally, most respondents used synthetic fertilizer for their crops about 9 times a year, with others fertilizing between 8 and 11 times annually. figure 4 agroforestry land management notes: (a) plant spacing; (b) plant maintenance; (c) fertilization frequency per-year; (d) production (bunch/harvest). tree species diversity and management in agroforestry pohpohan (pilea melastomoides) hartoyo et al. 389 regarding harvesting, the production output varied among the community members, reflecting differences in plant spacing, management practices, and total land areas. most respondents (45%) produced between 30,000 and 50,000 bunches/harvest. as many as 32% of respondents produced 20,000 bunches/harvest. the remaining respondents produced between 400 and 15,000 bunches in one harvest. each bunch of pohpohan weighed between 15-20 g and was valued at approximately usd 0.00054 or rp10, which meant that the income received by the community from pohpohan farming ranged from about usd 154 to usd 256 per month or rp2,400,000 – rp4,000,000 per month. the farmers sold pohpohan through some methods, such as direct sales to consumers, directly to traditional markets, and through middlemen (tengkulak). conclusion there were 54 species from 37 families found in the rehabilitation zone of tnghs. rubiaceae, malvaceae, fabaceae, and phyllanthaceae families were the most common families found in this study. pinus merkusii, agathis dammara, and maesopsis eminii had the highest important value index (ivi). the understory and seedlings were categorized as low in h’ due to the dominant presence of pohpohan planting. most respondents were farmers aged 35-55 (70%) with a farming area of 1,000 m2, who cultivated pohpohan by conducting plant spacing, plant maintenance (e.g., soil cultivation, weeding, fertilization), and harvesting. most farmers conducted fertilization 9 times a year. farmers produced 30,000-50,000 bunches/harvest. the price of a bunch of pohpohan was usd 0.00054, which made their income about usd 154 256 per month. references adalina y. 2014. implikasi modal sosial masyarakat terhadap pengelolaan taman nasional (studi kasus taman nasional gunung halimun salak) [the implications of community social capital on national park management (a case study of gunung halimun salak national park)]. 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(sw.)) on lombok island, west nusa tenggara]. j hutan tropis 7(1):92-105. doi: 10.20527/ jht.v7i1.7285 the southeast asian journal of tropical biology vol. 31 no. 2, 2024: 277 290 doi: 10.11598/btb.2024.31.2.2177 issn: 0215-6334 | e-issn: 1907-770x 277 distribution of the thalloid liverwort genus marchantia (marchantiaceae) in north sumatra, indonesia etti sartina siregar*, nursahara pasaribu, muhammad zaidun sofyan department of biology, faculty of mathematics and natural sciences, universitas sumatera utara, medan 20155, indonesia. article higlights • the survey method was successful in identifying the species marchantia and mapping their distribution in north sumatra • eight taxa of marchantia confirmed in north sumatra, m. emarginata subsp. cuneiloba has the most widely distributed species. • species distribution linked to environmental factors like elevation, humidity, rainfall, soil types, slope, solar intensity, and types of land cover article information received 26 january 2024 revised 29 march 2024 accepted 17 april 2024 corresponding author, e-mail: etti1@usu.ac.id research article abstract marchantia is a large genus of complex thalloid liverworts (marchantiophyta), widely distributed globally, and thriving across various substrates. despite the relatively abundance widespread, the presence and distribution of marchantia species are still limited in north sumatra. therefore, this research aimed to conduct a comprehensive inventory of marchantia species and map their distribution in north sumatra. the inventory was carried out using a survey method, covering 9 locations, namely aek nauli forest, bukit barisan national forest park, bukit lawang, resort cinta raja, dolok tinggi raja nature reserve, sibayak mountain, sicike-cike natural park, silima-lima waterfall, and taman eden 100 natural park. subsequently, geographic information system (gis) was used to map species distribution. the results showed that there were eight taxa of marchantia in north sumatra, including m. acaulis, m. emarginata subsp. emarginata, m. emarginata subsp. cuneiloba, m. geminata, m. paleacea, m. polymorpha, m. treubii, and marchantia sp. the most widely distributed species was m. emarginata subsp. cuneiloba, found in 7 locations, while m. treubii and marchantia sp. were identified in one location. in north sumatra, marchantia spp. spread at an altitude of 77 to 1,976 masl, occupying habitats with 6 types of soil (orthic acrisol, humic andosol, humic acrisol, and humic cambisol), at a rainfall of 1,800-3,600 mm/year. these species are found on sloping plains to steep, thriving in various habitats, including secondary forest, as well as primary and secondary forest borders, dryland farming, primary and plantation borders. considering the numerous benefits, this research provided valuable information for the conservation of marchantia spp. in north sumatra. keywords: distribution, liverwort, marchantia introduction the genus marchantia is characterized by a dichotomously branched and dorsoventrally flattened thallus, bearing compound air pores, gemma cups, archegonium, and antheridium on the dorsal of thallus, along with ventral scales appendages on the ventral side. marchantia is distinguished from all other genera of marchantiaceae by bearing gemma cups on the dorsal thalli. the male and female thalli can reproduce asexually by producing multicellular ‘gemmae’ (singular: gemma) in ‘gemma cups’ (or ‘cupules’) (shimamura 2016). gemma is the asexual reproduction, where each gemma grows into a new individual when supported by environmental conditions. furthermore, gemmae are usually dispersed by rainwater, insects, and small mammals, such as mice (barbé et al. 2017). marchantia also has sexual reproduction organs in the form of archegonia (female) and antheridia (male), which are separated in different thalli (dioicous). however, species such as m. acaulis, show monoicous characteristics, where both archegonium and antheridium occur in the same thallus (bischler-causse 1989; gradstein 2011; siregar et al. 2013; linde et al. 2020). this genus has oil bodies which can be usually observed in https://doi.org/10.11598/btb.2024.31.2.1741 mailto:etti1@usu.ac.id biotropia vol. 31 no. 2, 2024 278 gemmae, appendage of the ventral scale of thallus, gemma cups or cupules, and involucre (zheng & shimamura 2019a; 2020; 2022). marchantia is composed of approximately 40 species globally (söderström et al. 2016), and is represented by 19 accepted taxa in asia (bischlercausse 1989; xiang et al. 2016; long & crandallstotler 2020 ). this genus has a cosmopolitan distribution and the greatest diversity of species occurs in tropical asia and oceania. marchantia is found in moist to wet habitats, open or shady places, side of the road, usually neutral or basic soils, sandy or clayey soils, on wet rocks, trench walls, riverside cliffs, waterfall cliffs, limestones, boulders in open or shaded places. moreover, it is occasionally found on mineral soils in depressions or in the shade of fallen logs following hot forest fires. marchantia frequently associates with human activity, rapidly colonizing in the open, burnt soil, and thriving in cultivated farmland. for example, species such as m. polymorpha commonly intrude into gardens, greenhouse, and areas around human settlements (land that has been disturbed by human activities) (bishler-causse et al. 2005; siregar et al. 2013; ho 2013; shimamura 2016; zheng & shimamura 2022). some of these species can be found below an altitude ranging from 100 m to 4,000 m above sea level, and are abundant at 1,000 to 2,500 m (lu & huang 2017). marchantia has ecological roles as a pioneer plant on barren lands, preventing erosion by colonizing cliffs and forming dense populations (sahu et al. 2014; bowman et al. 2017). economically, marchantia serves as a potential source of medicine due to the presence of antibacterial, antifungal, and antioxidant contents (tanaka et al. 2016; fatma 2018; siregar et al. 2021; romani et al. 2022; zang et al. 2022). this genus has also been used as a traditional medicine in china and india for treating various ailments, such as skin tumefaction, liver protection, hepatitis, tumors, ulcers, open wounds, burns, poisonous snake bites, hepatotoxicity, and as an antipyretic (gupta et al. 2015; purkon et al. 2022). despite the numerous benefits of marchantia, only a few investigations focused on exploring its distribution in indonesia, including in north sumatra. among the exploration reports are: (1) siregar et al. (2013) who reported seven species in sibayak forest; (2) siregar et al. (2020) who found the morphological variations of m. emarginata; and (3) siregar et al. (2021) who identified the antioxidant activity of m. paleacea. therefore, this research aimed to investigate distribution of marchantia species in north sumatra. materials and methods research area the research was conducted in nine locations in north sumatra province, which has abundant existence of marchantia. these locations were: (1) aek nauli forest, simalungun district; (2) bukit barisan national forest park, karo district; (3) bukit lawang, langkat district; (4) resort cinta raja, langkat district; (5) dolok tinggi raja nature reserve, simalungun district; (6) sibayak mountain, karo, deli serdang district; (7) sicikecike natural park, dairi district; 8) silima-lima waterfall, south tapanuli district; and (9) taman eden 100 natural park, toba samosir district, as shown in figure 1. data collection an exploratory survey was carried out along the path at each predetermined location. species that have been recognized in previous research were only photographed and not collected for analysis. however, unknown or doubtful species were photographed, collected, and placed in a ziplock bag containing water-moistened tissue paper. these species were taken to herbarium medanense (meda), biology department, faculty of mathematics and sciences, universitas sumatera utara medan, indonesia for identification. the ordinal point of global positioning system (gps) was recorded and physical factors were measured at each location, including altitude, humidity, soil ph, air temperature, and light intensity. data on marchantia spp. that were previously collected from north sumatra and stored in the meda herbarium were also used in this research. data analysis morphological observations were carried out at the plant systematics laboratory, department of biology, faculty of mathematics and natural sciences, universitas sumatera utara, medan, indonesia. species identification was based on various available literatures, particularly bischler (1989), singh & singh (2013), siregar et al. (2013), lu & huang (2017), and zheng & shimamura (2022). validation of the scientific names was based on the tropicos website: https:// www.tropicos.org/home. data on distribution of marchantia spp. in north sumatra was obtained using global positioning system (gps). subsequently, the coordinate point data were analyzed using the arcgis 10.8 software mapping program based on rainfall, altitude, slope of the habitat, light intensity, humidity, land cover, and soil type. distribution of the thalloid liverwort marchantia spp. siregar et al. 279 results and discussion marchantia species in north sumatra a total of eight marchantia taxa were identified, consisting of seven species with two subspecies, namely m. acaulis steph., m. emarginata reinw., blume & nees subsp. emarginata, m. emarginata subsp. cuneiloba (steph.) t.x. zheng & shimamura, m. geminata reinw., blume & nees, m. paleacea bertol., m. polymorpha l., m. treubii schiffn. and marchantia sp. although marchantia sp. is closely related to m. rubribarba steph., there is no specimen of the species, leading to pending confirmation. the number of species obtained in this research was not different from marchantia spp. found in sibayak forest by siregar et al. (2013). however, two subspecies of m. emarginata, namely m. emarginata subsp. emarginata and m. emarginata subsp. cuneiloba were found to show significantly large morphological variations in thallus and archegonium. marchantia emarginata subsp. emarginata is characterized by the dorsal thallus with dark median band and distinctly rounded protrusion on the dorsal archegonium. meanwhile, m. emarginata subsp. cuneiloba has dorsal thallus without a median band and archegonium with indistinct or flat protrusions (siregar et al. 2020). distribution of marchantia in north sumatra table 1 shows information regarding the locations of marchantia species found in this research. the most widespread species was found to be m. emarginata subsp. cuneiloba and was identified in seven locations on various substrates. this was followed by m. geminata, found in humid and open habitat, such as on the cliffs of household wastewater, soil, and moist rocks in areas along the track and around forests with human activities. m. emarginata subsp. cuneiloba was found in open and humid habitat on the outskirts of waterways, water waste rock walls, and soil cliffs, while m. emarginata subsp. emarginata was found alongside m. emarginata subsp. cuneiloba in the field in a very dense population covering exposed land cliffs. the ability of both subspecies to form dense populations facilitates soil retention, reducing erosion on open ground cliffs. figure 1 map of marchantia spp. research locations in north sumatra, indonesia biotropia vol. 31 no. 2, 2024 280 table 1 distributional and ecological aspects of north sumatran species of marchantia no. species location substrate elevation (m) soil acidity 1 m. acaulis an, sc, sm, bb soil 800-1,373 5-6.4 2 m.emarginata subsp.emarginata te, an, sw, sm soil, rocks 88-1,570 6-6.6 3 m.emarginata subsp.cuneiloba te, sc, an, ws, sm, cr, bl soil, rocks 77-1,570 5-6.4 4 m. geminata te, sc, an, bb, tr, sm soil, rocks 870-1,976 4.2-6.2 5 m. paleacea sm, bb rocks 1,1501,250 6.2-6.4 6 m. polymorpha an, sm, bb soil, rocks 800-1,570 6.2 7 m. treubii sm soil, rocks 870-1,680 5.9 8 marchantia sp. sm soil 984-1,027 6.4-6.6 notes: an = aek nauli; bl = bukit lawang; cr = cinta raja; sc = sicike-cike; sm = sibayak mountain; sw = silima-lima waterfall; bb = bukit barisan national forest park; te = taman eden; tr = tinggi raja. marchantia polymorpha was found in three locations, namely aek nauli, sibayak mountain, and bukit barisan national forest park. this species was predominantly found in rock cliffs of watercourses, concrete walls, moist soil near streams, and open fields such as strawberry gardens in bukit barisan national forest park. the species having a narrow distribution in north sumatra, namely m. treubii and marchantia sp., were found only in sibayak forest. this limited number of species samples was attributed to disturbances, which could threaten the existence of plants, including marchantia. all species that were identified are cosmopolitan in the malesiana region, extending to asia and parts of europe such as m. polymorpha and m. paleacea (siregar et al. 2013). habitat preferences the habitat characteristics of marchantia in north sumatra are relatively different. these species mainly inhabit forest outskirts, secondary forests, slightly shaded or open vegetation, roadside areas, plantations, and around human settlements. for example, m. paleacea with a very dense population inhabits concrete walls in open habitats, along with m. geminata at an altitude of 1,400 m in bukit barisan national forest park. marchantia spp. generally grow on wet soil, watercourses on riverbanks, bare soil, gardens, drains, rocks, damp brick walls, and concrete walls. the most widely distributed species are m. emarginata subsp. emarginata and m. emarginata subsp. cuneiloba, found on stone walls in waterways (moats), riverbanks, cliffs of waterfalls, roadside soil banks, stone walls, and land around human settlements. distribution of this genus based on the altitude from sea level varies from lowlands to highlands between 400 m and above 1,600 masl (fig. 2). in this research, only m. emarginata was found in the range below 400 masl, while m. emarginata subsp. emarginata was distributed at an altitude of 88 to 1,570 masl. (singh & singh 2013) found m. emarginata subsp. emarginata in india at an altitude of 1,400-1,650 masl, while other species were found in the highlands at altitudes 800 to 1,900 masl. although m. acaulis was found at 8001,200 masl, lu and huang (2017) reported this species at 1,800 masl. marchantia geminata was also discovered at 800 masl to more than 1,600 masl, while m. paleacea was found within 8001,600 masl. marchantia geminata showed a fair distribution, thriving on the ground of waterfall cliffs, concrete along ditch, rock walls, and soil cliffs with an altitude range of 800-1,900 masl. marchantia polymorpha lives on rock walls of watercourses, flat and open land near rivers, predominantly found in horticultural gardens and city parks, at 8001,600 masl. similarly, cykowska (2011) found m. polymorpha between 865 masl and 1,560 masl in poland, while zheng et al. (2020) discovered this species at 223 masl on concrete walls and artificial substrates in semi-natural environments in japan. marchantia treubii was found on sloping, soil, and rock cliffs, while m. acaulis was identified on cliff substrates from open ground to shaded. the species m. acaulis in singapore was found in habitats with partial shade on rock or concrete surfaces (ho 2013). distribution of the thalloid liverwort marchantia spp. siregar et al. 281 figure 2 map of the relationship between elevation and distribution of marchantia species biotropia vol. 31 no. 2, 2024 282 figure 3 map of the relationship between soil type and distribution of marchantia species distribution of the thalloid liverwort marchantia spp. siregar et al. 283 marchantia paleacea was found in the rock walls of the canal on the roadside of the road and open areas at 1,100 masl. lu and huang (2017) discovered m. paleacea on a dump brick wall from 300 masl to 2,500 masl in taiwan, as well as in japan at 136 masl and 459 masl. this species was also found on a vertical stone wall in the shade (zheng & shimamura 2019b) and in russia at 370 masl (borovichev & bakalin 2014). the results showed that marchantia species in north sumatra occupied 5 soil types, namely orthic acrisols, humic andosols, orthic ferrasols, humic cambisols, orthic podzols, and the most common are humic andosols and orthic acrisols (fig. 3). marchantia geminata occupied the most variations in soil types, namely orthic acrisols, humic andosols, orthic ferralsols, and humic cambisols. furthermore, m. emarginata subsp. emarginata was found to occupy orthic acrisol, humic andosol, and humic cambisol. marchantia acaulis and m. polymorpha occupied orthic acrisols, humic andosols, and orthic podzols. marchantia treubii and marchantia sp. showed a specific distribution pattern, spreading only on humic andosols. acrisols or podzolic soils are characterized by high leaching, which appears light gray to yellowish on the surface horizon, with underlying layers often showing red or yellow. these soils contain low organic matter content and base saturation, with soil acidity levels ranging from ph 4.2 to 4.8. in the subsurface horizon, there is an accumulation of clay with a lumpy soil structure with low permeability. despite being predominantly present in lowlands at 50-350 masl (fao 2014), acrisols in this research were found at altitude from less than 100 masl to 1,300 masl. marchantia in north sumatra is distributed in high humidity and almost the same in all locations with a range of 82% to 86% (fig. 4), showing that marchantia can grow optimally in an environment having high humidity, as most species are found in streams, riverbanks, stream walls, and wet areas. based on the results, all species found were scattered in areas with medium to high rainfall between 200300 mm/month or 2,400-3,600 mm/year (fig. 5). the widest distribution range was observed in m. emarginata subsp. emarginata and m. emarginata subsp. cuneiloba with rainfall in the medium to high category, with value of 150-300 mm/month or 1,800-3,600 mm/year. in terms of land slope, marchantia was found generally on flat to steep land surfaces in north sumatra. species found on a flat to sloping surface were m. polymorpha, marchantia sp. m. emarginata subsp. emarginata and m. emarginata subsp. cuneiloba were found on a sloping to a bit steep surface. meanwhile, m. acaulis, m. paleacea, and m. treubii. were found on a slope to steep surface (fig. 6). marchantia spp. in north sumatra were found in open areas with the light intensity of 50% to 60%. marchantia geminata has the highest light intensity ranging from 50% to 60%, while m. acaulis, m. emarginata subsp. emarginata, m. emarginata subsp. cuneiloba, and m. paleacea were found at light intensity of 50-57%. the narrowest range was observed in m. treubii, m. polymorpha, and marchantia sp. at light intensity of 54% to 57% (fig. 7). marchantia species in north sumatra were found in varied land cover, including plantation forests, primary forest border, including those with open ground, and dryland farming (fig. 8). furthermore, m. emarginata subsp. cuneiloba occupied the most varied land cover, including secondary dryland forest, primary border with dryland farming, primary border with plantation, and dryland farming. m. emarginata subsp. emarginata occupied secondary dryland forest land cover, secondary dryland forest border with plantation forest, and primary dryland farming border. marchantia acaulis was found in plantation forest, primary border, as well as primary border with open ground, and dryland farming. marchantia polymorpha occupied two types of land cover, namely scrub and dryland farming. bowman (2015) described that the species m. polymorpha thrived in disturbed anthropogenic habitats throughout europe and other continents. marchantia paleacea occupied two types of land cover, namely primary and dryland farming. zheng and shimamura (2022) reported that this species thrived in shaded or semi-shaded areas and anthropogenic regions. marchantia treubii also occupied primary, and near the primary border with dryland farming, m. geminata thrives in primary and secondary dryland forest, while marchantia sp. occupied only primary dryland forest. habitat characteristics occupied by marchantia spp. in north sumatra can be used as information for conservation efforts in future research due to their predominant distribution in lowlands. additionally, the accessibility of some species shows the need for conservative efforts to prevent disturbance from humans. biotropia vol. 31 no. 2, 2024 284 figure 4 map of the relationship between air humidity and distribution of marchantia species distribution of the thalloid liverwort marchantia spp. siregar et al. 285 figure 5 map of the relationship between rainfall and distribution of marchantia species biotropia vol. 31 no. 2, 2024 286 figure 6 map of the relationship between slope and distribution of marchantia species distribution of the thalloid liverwort marchantia spp. siregar et al. 287 figure 7 map of the relationship between solar intensity and distribution of marchantia species biotropia vol. 31 no. 2, 2024 288 figure 8 map of the relationship between land cover and distribution of marchantia species distribution of the thalloid liverwort marchantia spp. siregar et al. 289 conclusion eight marchantia taxa were found in north sumatra, consisting of seven species and two subspecies, namely m. acaulis, m. emarginata subsp. emarginata, m. emarginata subsp. cuneiloba, m. geminata, m. paleacea, m. polymorpha, m. treubii and marchantia sp. the most widely distributed species was m. emarginata subsp. cuneiloba, found in seven locations, including bukit lawang, resort cinta raja, silima-lima waterfall, sicikecike natural park, sibayak mountain, aek nauli forest, and taman eden 100 natural park. species with the narrowest distribution were marchantia sp. and m. treubii, found in sibayak mountain. distribution of marchantia species in north sumatra ranged from an altitude below 200 masl to 1,976 masl, with 1,800 mm/year to 3,600 mm/ year rainfall, almost evenly distributed humidity of 82-86%, on flat to steep soil, and high light intensity of 52-60%. distribution area was found to have 5 types of soil, namely orthic acrosols, humic andosols, orthic ferrasols, and humic cambisols. marchantia species were found in various types of land cover, namely secondary forest, primary and secondary forest borders, dryland farming, primary and plantation borders, as well as plantations. acknowledgments the authors are grateful to the ministry of research and technology and higher education for providing financial support through the “drpm with contract number: 1140a/un5.1.r/ ppm/2018”. furthermore, the authors are grateful to the teamwork who collected the specimen on the field, and anugrah gilang permana for making a map of distribution of marchantia in 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(marchantiaceae, marchantiophyta) and their geographical distribution patterns in japan. hikobia 18:65-9 zheng tx, shimamura m. 2020. a new locality of marchantia polymorpha l. subsp. polymorpha. from miyagi prefecture, japan. hikobia 18:61-3. zheng,tx, shimamura m. 2022. taxonomic revision of the genus marchantia (marchantiaceae) in japan and the redefinition of the genus. hattoria 13 (1784):33-77. doi: 10.18968/hattoria.13.0_33. zheng tx. 2023. oil cells in the side walls of air chambers, a new taxonomic character in the family marchantiaceae. the bryologist 126(2) :174-9. doi: 10.1639/0007-2745126.2.17 _goback the southeast asian journal of tropical biology vol. 31 no. 2, 2024: 202 216 doi: 10.11598/btb.2024.31.2.2042 issn: 0215-6334 | e-issn: 1907-770x 202 composition, structure, and carbon sequestration of different rainforest ecosystems in the gunung gede pangrango national park, indonesia agus sunyata, isna rakhmi zulhida, nike triwahyuningsih, kanda raharja* forestry study program, faculty of forestry, agricultural institute (intan) yogyakarta, sleman, yogyakarta 55284, indonesia article higlights • high demand for environmental services makes the park vulnerable to human activities. • both ecosystems are well regenerated; seedling > sapling > pole > tree (inverted j) • both ecosystems show normal diversity conditions and stable species distribution. • growth of maesopsis eminii needs monitoring to preserve forest purity. • montane forests have greater biomass, carbon stocks, less anthropogenic disturbance article information received 18 june 2023 revised 9 december 2023 accepted 28 may 2024 *corresponding author, e-mail: kanda.raharja@gmail.com research article abstract the gunung gede pangrango national park (ggpnp) area is one of the vital ecosystems that support the environment in west java province, indonesia. it is a unique area that has multiple forest ecosystems, including lowland rainforest and montane rainforest ecosystems. despite the ggpnp’s status as a conservation area, the high demand for the ggpnp’s environmental services makes the region vulnerable to disturbances from human activities. several studies have been conducted in the ggpnp area (lowland and montane forest ecosystems), however, the results of this study are still necessary to explain the forest dynamics and forest carbon sequestration in this location. the objective of this research was to analyze the structure, composition, and carbon sequestration of stands in the lowland and montane rainforest ecosystems in the ggpnp area. data processing and analyses were conducted using diversity indices, biomass-carbon stock estimation, and carbon dioxide sequestration estimation. the results showed that the ggpnp lowland and montane rainforest ecosystems were well regenerated. the number of seedlings > saplings > poles > trees and the graph showed a reverse “j” pattern. the ggpnp lowland rainforest ecosystem was dominated by neonauclea lanceolata and had relatively higher species diversity. the ggpnp montane rainforest ecosystem was dominated by castanopsis acuminatissima with a higher individual density, denser canopy, and more complex canopy strata. the lack of regeneration in several species of trees heightens the threat to these species’ existence in the future. biomass, carbon stocks, and carbon sequestration in the ggpnp montane rainforest were greater than those in the ggpnp lowland rainforest. the ggpnp montane rainforest ecosystem had older forest stands, a larger average tree diameter, and lower potential for anthropogenic disturbances. keywords: biomass, carbon sequestration, important value index, similarity index, tnggp introduction the gunung gede pangrango national park (ggpnp) or taman nasional gunung gede pangrango (tnggp) area is among vital ecosystems supporting the environment in west java province, indonesia. these ecosystems provide habitats for flora and fauna, deliver biodiversity and community livelihoods, maintain the balance of the hydrological cycle and soil fertility, function as carbon pools (arrijani 2008; helmi et al. 2009; saefrudin 2013; dendang & handayani 2015), supply drinking water resources, and serve the agriculture and tourism sectors (rushayati 2006; siswantoro et al. 2021). despite the ggpnp’s status as a conservation area, the high demand for ggpnp’s environmental services makes the region vulnerable to disturbances from human activities (saefrudin 2013). https://doi.org/10.11598/btb.2024.31.2.2042 composition, structure, and carbon stock of the gunung gede pangrango national parks – sunyata et al. 203 anthropogenic factors are an important issue in conservation area management. this issue occurs not only in developing countries or around tropical forests (fao 2005), but also in developed countries with subtropical forests (nowacki et al. 2015). the need for land and environmental services from forest ecosystems is driving the decline of forest areas, including conservation areas (popradit et al. 2015; meer et al. 2023). in addition, biodiversity in conservation areas has also diminished due to human activities (oladeji et al. 2012; fetene et al. 2019). the ggpnnp has as much problem complexity regarding human activities as diversity due to the ecosystems’ uniqueness. the ggpnp area is a unique area that has multiple forest ecosystems, including lowland rainforest and montane rainforest ecosystems. differences in climate and weather conditions in the two adjacent ecosystems result in high biodiversity (yamada 1975; gotsch et al. 2016). rainfall in this location is quite high with an average of 3,000-4,200 mm per year, causing this area to be one of the wettest in java island. in the montane rainforest ecosystem, schima wallichii (arrijani et al. 2006; arrijani 2008; helmi et al. 2009) and altingia excelsa (arrijani et al. 2006; arrijani 2008; dendang & handayani 2015) are the two most dominant tree species. the diversity index of the montane rainforest ecosystem of the ggpnp is relatively high, but the species evenness is low (dendang & handayani 2015). this condition is due to the dominance of both aforementioned species (arrijani et al. 2006; arrijani 2008) and the disturbances of forest regeneration. (dendang & handayani 2015). in the lowland rainforest ecosystems, schima wallichii, pternandra caerulescens (helmi et al. 2009), and altingia excelsa (in the altingia excelsa forest) (sadili 2013) are the most dominant tree species. sadili et al. (2023) also mentioned maesopsis eminii, syzygium acuminatissimum, and lithocarpus korthalsii as the most dominant species in this ecosystem of ggpnp. unlike those in the montane rainforest ecosystem, the structure and composition of stands in the lowland rainforest ecosystem of the ggpnp are still understudied (helmi et al. 2009; sadili et al. 2023). therefore, forest structure (horizontal and vertical) and composition (density of vegetation growth level, basal area, species abundance, biodiversity indices, etc.) need to be studied to obtain information about forest regeneration (gatica-saavedra et al. 2017) in the two forest ecosystems in the ggpnp. research on forest dynamics and carbon sequestration in the ggpnp is lacking compared to the high pressure and demand on this national park. very little study exists about the structure and composition of the montane and lowland forests in the ggpnp. to date, only arrijani (2008) and dendang and handayani (2015) studied the montane forest in the ggpnp, while studies on lowland forest were only conducted by helmi et al. (2009) and sadili et al. (2023). therefore, more studies on both forest ecosystems are necessary to enrich data for better scientifically-based management of the ggpnp (brearley et al. 2019). research on carbon sequestration in the ggpnp can also explain the function of carbon stock conservation in forest ecosystems in the ggpnp. this is particularly relevant as in indonesia, conservation areas are among those areas expected to increase carbon stocks according to redd+ program (indonesia ministry of forestry 2012). the objective of this research was to analyze the structure, composition, and carbon sequestration of stands in the lowland rainforest and montane rainforest ecosystems in the ggpnp area. the results of this research are expected to help in policy-making and contribute to the improvement of conservation efforts and the management of conservation areas in the ggpnp. appropriate policies can reduce or even protect conservation areas (national parks) from the threats of degradation due to human activities (myga-piątek et al. 2022). materials and methods this research was conducted from august to october 2021 in the gunung gede pangrango national park, west java province, indonesia. data collection was carried out in the lowland rainforests at bodogol resort (106°51'57" e; 06°46'49" s) and the montane rainforest at gunung putri resort (106°59'42’" e; 06°44'16" s). the locations are presented in figure 1. biotropia vol. 31 no. 2, 2024 204 bodogol resort has natural forest stands on the cisuren and cipadaranten hills. the forest extends from the lowland area on the western side of the ggpnp toward the top of mt. pangrango. formerly a plantation forest, the forest includes stands of altingia excelsa, agathis dammara, and pinus merkusii. the area has a hilly topography with steep slopes. the soils belong to the red–yellow podsolic soil type. a more detailed description of the research location in bodogol resort can be read in full at soepraptohardjo (1975); helmi et al. (2009); sadili (2013); and sadili et al. (2023). gunung putri resort, at an altitude of 1,600-1,800 meters above sea level (masl), is a rehabilitation zone of the ggpnp. this area was previously a perhutani plantation forest area for altingia excels. it is directly adjacent to farmland. at an altitude of 1,800-2,000 masl lies a secondary forest at gunung putri resort that was also a perhutani plantation forest or altingia excels species. at an altitude of >2000 masl, a primary forest is perched at gunung putri resort. a more detailed description of the research location in gunung putri resort can be read in full in putra et al. (2020). both bodogol and gunung putri resorts are bordered by agricultural areas, which makes them highly vulnerable to anthropogenic activities. data collection and processing sampling locations were determined using purposive sampling based on the altitudes of the sites to represent different ecosystems, i.e., lowland forest and montane forest ecosystems. purposive sampling or non-probability allows for efficient quantification of ecological characteristics (araújo & souza 2022). this technique can be very useful, even if only to describe a particular sample, and much environmental data can only be collected with this technique in spite of difficult sampling processes, access and safety issues, or time and expense constraints (speak et al. 2018). data collection was carried out in permanent plots at bodogol and gunung putri resorts. permanent plots at bodogol resort were set at an altitude of 399 masl representing the lowland rainforest ecosystem and permanent plots at gunung putri resort were set at an altitude of 1,824 masl representing the montane rainforest ecosystem. permanent plots at these locations are usually used by the ggpnp management to observe forest dynamics and succession in the national park area (herben 1996). data were collected using the same plot design used by the ggpnp management. a total of 32 plots of samples were used (16 plots in the lowland rainforest ecosystem and 16 plots in the montane rainforest ecosystem). this activity was conducted in parallel with annual measurement activities by the ggpnp management. figure 1 gunung gede pangrango area and research location legend composition, structure, and carbon stock of the gunung gede pangrango national parks – sunyata et al. 205 the total plot area in each forest ecosystem was 10,000 m2 (1 ha). according to helmi et al. (2009) and sadili et al. (2023), the minimum plot area for a vegetation study in the lowland tropical rainforest ecosystem of the ggpnp is 1 ha, while according to richard (1952) in meijer (1959) and yamada (1975), the minimum plot area for a vegetation study in the montane tropical rainforest ecosystem of the ggpnp is also 1 ha. the designs of plots and subplots (forestry department of indonesia 1992) is presented in figure 2. the parameters measured were: (1) the number of seedling species in subplot a; (2) the number of sapling species in subplot b; (3) the number of species and diameter of breast height (dbh) of poles (dbh = 10–20 cm) in subplot c; (4) number of species, dbh of tree (dbh > 20 cm); crown diameter, total height, free branch height, and widest crown height of trees in a 25 m × 25 m square plot. data processing and analyses were carried out using microsoft excel. data analysis important value index (ivi) the ivi was used to determine the composition of tree species dominating the forest stand communities. the index was calculated using the cottam and curtis (1956) formula as follows: ivi (important value index) = rd + rdc + rf rd (relative density) = number of individuals of species x 100% total number of individuals rdc (relative dominance) = dominance of species x 100% dominance of all species rf(relative frequency) = frequency of species x 100% sum frequency of all species similarity index (si) si was used to determine the similarity between compared vegetation communities. the index used in this study was the jaccard and sorensen similarity index (krebs 2014) and was calculated using the formula as follows: is = c a + b + c where: is = jaccard and sorensen similarity index a = number of species in sample a and sample b (joint occurrences) b = number of species in sample b but not in sample a c = number of species in sample a but not in sample b biodiversity index (h’) the biodiversity index was calculated using the shannon-wiener diversity index formula (krebs 2014) as follows: h’ = σ [(ni/n) ln (ni/n)] where: e = evenness index h’ = shannon-wiener diversity index s = total species figure 2 designs of plots and subplots for measuring stand parameters in the forest. notes: a = 1 m; b = 2 m; c = 5 m. biotropia vol. 31 no. 2, 2024 206 species evenness index (e) the evenness index was used to determine the structure of the forest community in the study locations. the formula of evenness index (e) (odum 1996) is as follows: e = h’/ln s where: e = evenness index h’ = shannon-wiener diversity index s = total species biomass and carbon stock estimation, carbon dioxide sequestration aboveground biomass (agb) was estimated using the allometric biomass model developed by chave et al. (2005). the model used was for a wet forest stand without a tree height predictor. allometric models using height predictors can provide more accurate biomass estimates, but if used for tropical rainforest estimations, the results can be overestimated (rutishauser et al. 2013). the model is as follows: agb = ρ × exp(-1.239 + 1.980 ln(d) + 0.207 (ln(d))2 0.0281 (ln(d))3) where: agb = aboveground biomass (mg) ρ = wood gravity (g/cm3) d = diameter at breast height (cm) belowground biomass (bgb) was estimated using a model developed by cairns et al. (1997), which is as follows: bgb = exp (-1.0587 + 0.8836 ln agb) where: bgb = belowground biomass (mg) agb = aboveground biomass (mg) carbon dioxide sequestration was calculated based on the mass ratio of the photosynthesis reaction equation (muthmainnah et al. 2021): 6co2 + 6h2o à c6h12o6 + 6o2 (264) (108) (180) (192) based on the photosynthesis reaction equation above, 180 grams of biomass (c6h12o6) is produced from the reaction of about 264 grams of co2. co2 sequestration can be determined by the formula: co2 sqr = (264/180) × tabgb or co2 sqr = 1.4667 × biomass where: co2 sqr = co2 sequestration (mg/ha/year) tabgb = total above and below ground biomass (mg). results and discussion forest composition in both lowland and montane rainforests, the highest densities occurred on the lowest growth level (fig. 3a, table 2 ) and diameter class (20-30 cm) (fig. 4) showing that the density decreased as the growth level and diameter increased. the number of seedlings > saplings > poles > trees and the graph showed a reverse “j” (fig. 3a) pattern. this pattern is typical for tropical rainforests that the forests regenerate well and are in a dynamic state (ogawa et al. 1965; mclaren et al. 2005; mirmanto 2014; gonçalves 2017; sadili et al. 2023). however, this result did not guarantee that every species could regenerate due to the possibility of species turnover dominating at each growth stage. figure 3 (a) distribution of the number of individuals at each growth level; (b) species diversity at various growth levels a b composition, structure, and carbon stock of the gunung gede pangrango national parks – sunyata et al. 207 the lowland rainforest ecosystem in the ggpnp had more tree species than in the montane rainforest ecosystem. in the lowland rainforest, 40 tree species (dbh > 20 cm) were recorded with a basal area (ba) of 8.85 m2/ha and a density (d) of 60 trees/ha. in the montane rainforest, 24 tree species were recorded with a ba of 18.85 m2/ha and a density of 130 trees/ha. the species richness and tree density (dbh > 10 cm) in our plots, at the lowland forest ecosystem (55 species and 339 tree/ha) were lower than those mentioned in other studies in the lowland forest of the ggpnp (66 species and 348 tree/ha) (sadili et al. 2023), in the gunung halimun national park in west java (64-69 species and 405-441 tree/ha) (yusuf 2004; suryanti 2006), in malinau, north kalimantan (205 species and 759 tree/ha) (sheil et al. 2010), in the batang gadis national park (182 species and 583 tree/ha) (kartawinata et al. 2004) and bukit lawang, north sumatra (216 species and 453 tree/ ha) (polosakan 2001). the species richness and tree density (dbh >10 cm) in our plots, at the montane rain forest ecosystem, (40 species and 775 tree/ ha) were lower than those in previous studies in ggpnp montane forests by arrijani (2008) (63 species and 966 tree/ha) but higher when compared to the results of dendang and handayani (2015) (25 species and 320 tree/ha). the number of species in our plots (40 species) was also lower than the results of yamada et al. (1975) (57 species) in the ggpnp montane forest and sadili et al. (2018) (59 species) in the foja mountains of papua but the density was higher (775 tree/ha) than their results (527 tree/ha and 693 tree/ha, respectively). in the lowland rainforest ecosystem plots, there was the exotic species maesopsis eminii (ba = 0.39 m2/ha, d = 1/ha, absolute frequency (f) = 6%) (helmi et al. 2009; sadili et al. 2023) but it was not the dominating species as was stated by sadili et al. (2023). this species had a seedling density of almost 200 seedlings/ha and a sapling density of almost 50 saplings/ha. neonauclea lanceolata, pometia pinnata, and aleurites moluccana were the species with the highest species densities of 6 trees/ha, 6 trees/ha, and 4 trees/ha, respectively. castanopsis acuminatissima, schima wallichii, and turpinia sphaerocarpa were the species with the highest species densities in the montane rainforest ecosystem. the tree densities were 33 trees/ha (ba = 5.78 m2/ha), 22 trees/ha (ba= 4.05 m2/ha), and 10 trees/ha (ba = 1. 14 m2/ha), respectively. the studies by arrijani et al. (2006), arrijani (2008), and rozak et al. 2016, schima wallichii was found to be a dominant species in the ggpnp montane forest ecosystem. table 1 list of tree species without seedlings and saplings at different growth levels lowland rainforest montane rainforest 1 durio zibethinus elaeocarpus angustifolius 2 melaleuca leucadendron vernonia arborea 3 vernonia arborea acer laurinum 4 bridelia insulana litsea resinosa 5 acer laurinum persea declinata 6 litsea vulva astronia spectabilis 7 horsfieldia sp. acronychia laurifolia 8 phoebe declinata macaranga rhizinoides 9 terminalia sp. manglietia glauca 10 phoebe opaca quercus sundaica 11 sandoricum koetjape   quercus pseudomolucca 12 michelia velutina toona sureni 13 syzygium antisepticum   14 litsea angulata   15 machilus rimosa   16 myristica fragrans   17 gnetum gnemon   18 turpinia sphaerocarpa   19 crypteronia paniculata   20 dysoxylum macrocarpum   21 evodia aromatica   22 quercus sundaica   23 quercus pseudomolucca   24 nephelium lappaceum   the abundance and regeneration of forests in the ggpnp were relatively sufficient, and no additional artificial regeneration was required. this was indicated by the much greater seedling density than the next growth level (seedling to sapling) (fig. 3a, table 2). however, the regeneration of some species at the forests was disrupted at certain growth levels. in the lowland rainforest and montane rainforest, 24 out of 40 (60%) and 12 out of 24 (50%) tree species, respectively, had no seedling and sapling growth. further research is needed to clarify this phenomenon. few or no seedlings or saplings may indicate that the species is poorly regenerated. this leads to reduced populations of certain species at the seedling or sapling levels, driving widespread regeneration debts that can potentially lead to the extinction of the species (ameja et al. 2022; miller et al. 2023). biotropia vol. 31 no. 2, 2024 208 the seedling and sapling densities (945 individuals/ha) in the lowland rainforest ecosystem were higher than those recorded in the lowland forests of the mount halimun national park (815 individuls/ha) but lower than those recorded in cikepuh game reserve (1298 individuals/ha) and the batang gadis national park (2265 individuals/ ha) (kartawinata et al. 2004) (table 2). in the montane rainforest ecosystem, the seedling and sapling densities (16,769 individuals/ha) were higher than those recorded in the previous study by dendang and handayani (2015) (13,720 individuals/ha) in the same forest ecosystem (table 2). table 2 shows that seedling and sapling densities in the lowland forest were lower (seedlings 0.33 times and saplings 0.42 times) than those in the montane forest. the plot site in the lowland rainforest ecosystem is in the utilization zone of the ggpnp in which human activities are allowed. in addition, bodogol resort is an area in the ggpnp national park that is relatively closest to settlements in which most cases of human interventions or forest encroachment occur (sudomo & sairudin 2008). sarkar and devi (2014) stated that individuals in the juvenile phase are more vulnerable to all environmental pressures and anthropogenic disturbances. botzat et al. 2015 stated that the success of a tree species’ regeneration depends on its ability to produce seedlings and the ability of these seedlings to survive and grow. species that are vulnerable to extinction have populations with low levels of seedlings and saplings. they went on saying that the low rate of natural regeneration of a certain species indicates that the population is in a degradation phase, which can threaten its sustainability in the future. a very small number of seedlings will not be enough to replace dead trees (from old age, diseases, or other factors). forest stand structure horizontal structure the most abundant individual trees in the lowland rainforest ecosystem were in the diameter classes 20-30 cm (17 trees) and 30-40 cm (17 trees) (fig. 4). the most abundant tree species in the 2030 cm diameter class was pometia pinnata (d = 4 trees/ha) and the most abundant tree species in the 30-40 cm diameter class were pometia pinnata (d = 2 trees/ha) and mallotus paniculatus (d=2 trees/ ha) (fig. 4). maesopsis eminii, which was previously discussed, has a quite large diameter. sadili et al. (2023) found this exotic species the most abundant in the ggpnp lowland forest ecosystem. maesopsis eminii was included in the 70-80 cm diameter class with d = 1 tree/ha and ba = 0.39 m2/ha. the largest diameter class was 80-90 cm, consisting of the tree species crypteronia paniculata (d = 1 tree/ ha, ba= 0.62 m2/ha). other studies in the ggpnp lowland forest by sadilli et al. (2023) and in the gunung halimun national park by yusuf (2004) showed that maesopsis eminii was the species with the largest basal area. with a growth rate of 1.55.5 cm per year in diameter and 1-3 m per year in height, this fast-growing species rapidly invaded the lowland forest (schabel & latiff 1997). it can grow well at altitudes of 100-900 masl within full sunshine and can be dispersed by birds, rodents, and monkeys (schabel & latiff 1997). with such characteristics, this exotic species has the potential to cause negative impacts because it can invade the ggpnp area quickly and aggressively while reducing the purity of natural forest stands in the ggpnp (sadili et al. 2023). in the montane rainforest ecosystem, the most abundant individual trees were in the 20-30 cm diameter class (47 trees) (fig. 4), and the most abundant species in this class was castanopsis acuminatissima (d = 11 trees/ha, ba= 0.5 m2/ha). this species is common in the montane forests ecosystem of the ggpnp and is one of the most dominant species (arrijani 2008; helmi et al. 2009; rozak et al. 2016). the largest diameter class in this forest ecosystem was 110-120 cm, and the largest tree species was quercus sundaica. arrijani (2008) explained that this species can be found in the montane forests of ggpnp and bears bowl-shaped fruit. further information about the horizontal structure can be seen in figure 4. table 2 vegetation densities in two forest ecosystems in the ggpnp growth level vegetation density (individuals/ha) lowland rainforest ecosystem montane rainforest ecosystem seedling 4,777 14,530 sapling 945 2,239 pole 279 645 tree 60 130 composition, structure, and carbon stock of the gunung gede pangrango national parks – sunyata et al. 209 overall, the results of this study showed that the larger the tree diameter, the smaller the number of individual trees (fig. 4). similar patterns were also reported in the research by arrijani (2008) and dendang and handayani (2015) in the montane rainforest ecosystems, and by sadili et al. (2023) in the lowland rainforest ecosystem, indicating that only a few individual trees are successful in the competitive process during growth. over time, individuals encounter interspecies or intraspecies competition. naturally, this competition results in a reduction in the number of surviving individuals in each diameter class. however, meyer (1961) observed that a normal stand of an uneven aged natural forest has a constant ratio of the number of trees per unit area to diameter class even though there is always a reduction in the number of individuals in each diameter class. vertical structure the vertical forest structure for all species refers to the relationship between tree density and height class (stratum). the strata in both forest ecosystems are presented below. in lowland rainforest ecosystem plots, no trees emerged at stratum a (height > 30 m) (fig. 5). sadili et al. (2023) reported that in their research plots, chisocheton ceramicus, lithocarpus korthalsii, lithocarpus pallidus, schima wallichii, figure 4 horizontal structure in two forest ecosystems in the ggpnp figure 5 vertical structure in two forest ecosystems in the ggpnp biotropia vol. 31 no. 2, 2024 210 dalrympelea sphaerocarpa, and maesopsis eminii occupied stratum a). stratum b was occupied by young trees with a height of 20-30 m. usually it takes a shorter time to reach stratum b compared to the time needed to reach stratum a. stratum c was occupied by young trees of more diverse species. syzygium acuminatissimum, pometia pinnata, lithocarpus pseudomoluccus, and myristica sp. were found both in this study and the study by sadili et al. (2023) at stratum c. in montane rainforest plots, four trees of three species constituted stratum a (fig. 5). the species were acer laurinum (1 tree), castanopsis acuminatissima (2 trees) and quercus sundaica (1 tree). strata b and c in the montane forest were also occupied by younger trees in the same pattern as the pattern in lowland rainforest plots. schima wallichii was found both in this research and the research by dendang and handayani (2015) at stratum b. diameters were found to be correlated with tree height (< 20 m for tratum c and 20-30 m for stratum b, which case the bigger the diameters the taller the trees (dendang & handayani 2015; sadili et al. 2023). this forest condition guarantees the sustainability of the forest in the future because the number of individual seedlings is much greater than the number of individual mature trees (denslow 1987). biodiversity indices importance value index (ivi) the species with the highest ivi scores were considered as the most important species in a community (plot). respectively, in the lowland rainforest ecosystem, the seedling, sapling, pole, and tree growth stages, were dominated by syzygium polyanthum, dysoxylum caulostachyum, pometia pinnata, and neonauclea lanceolata (table 3). as reported by helmi et al. (2009), neonauclea lanceolata trees were easily found in the lowland forest ecosystem of the ggpnp. the montane rainforest ecosystem of the ggpnp was dominated by acer laurinum (seedlings and saplings), turpinia sphaerocarp (poles), and castanopsis acuminatissima (trees) (table 3). castanopsis acuminatissima was the dominant tree species and was easily found in the montane rainforest ecosystem of the ggpnp (arrijani 2008; rozak et al. 2016). it could even be found in the lowland rainforest ecosystem below (helmi et al. 2009). ivi is the sum of the relative density, relative frequency, and relative dominance parameters. ivi is one of the parameters that can provide an overview of the role of species in the community, but a high ivi value does not always reflect a high level of dominance. the greater the rd value of a species, the greater the number of individuals in the area. however, the rd value cannot provide an overview of the vegetation distribution at the research location. the distribution of vegetation in a particular community is limited by environmental conditions. some species in tropical forest ecosystems are adapted to canopy conditions that vary in sunlight intensity (balakrishnan et al. 1994) and their success in occupying an area is influenced by their ability to adapt to all physical environmental factors, biotic factors, and chemical factors (krebs 1994). crypteronia paniculata trees, in the lowland rainforest ecosystem, had a higher rdc compared to pometia pinnata trees. the ba of crypteronia paniculata (ba = 1.26 m2/ha) was larger than the ba of pometia pinnata (ba = 0.73 m2/ha). the greater the ba value of a species, the greater the absolute dominance and relative dominance values. conversely, the rf of pometia pinnata trees was higher than the rf of crypteronia paniculata trees. pometia pinnata trees were found in 4 out of 16 plots (f = 25%), while crypteronia paniculata trees were only found in 3 out of 16 plots (f = 19%). the more frequently a species was found in some plots, the higher its absolute frequency and relative frequency values. similar conditions were observered in polyosma illicifolia and villebrumea rubescens poles in the montane rainforest ecosystem. c om po sit io n, s tr uc tu re , a nd c ar bo n st oc k of th e g un un g g ed e pa ng ra ng o n at io na l p ar ks – s un ya ta et a l. 21 1 table 3 species with the highest ivi scores in the lowland rainforest and montane rainforest ecosystems in the ggpnp growth level lowland rainforest montane rainforest species rd rdc rf ivi species rd rdc rf ivi seedling syzygium polyanthum 16.67 12.50 29.17 acer laurinum 42.47 18.52 60.98   dysoxylum caulostachyum 12.50 12.50 25.00 castanopsis argentea 6.85 7.41 14.26   aglaia elliptica 8.22 12.50 20.83 antidesma tetrandum 5.48 7.41 12.89 sapling dysoxylum caulostachyum 21.05 18.75 39.80 acer laurinum 20.00 20.00 40.00   syzygium polyanthum 15.79 18.75 34.54 turpinia sphaerocarpa 13.33 13.33 26.67   aglaia elliptica 10.53 12.50 23.03 mimusops elengi 13.33 10.00 23.33 pole pometia pinnata 11.43 11.64 12.12 35.19 turpinia sphaerocarpa 17.28 17.02 12.50 46.81   neonauclea lanceolata 5.71 9.63 6.06 21.41 villebrumea rubescens 8.64 10.24 4.69 23.57   saurauria pendula 5.71 7.87 6.06 19.64 polyosma illicifolia 7.41 7.73 7.81 22.95 tree neonauclea lanceolata 10.00 15.92 7.41 33.33 castanopsis acuminatissima 25.38 30.69 12.20 68.27   pometia pinnata 10.00 8.29 7.41 25.70 schima wallichii 16.92 21.47 13.41 51.81   crypteronia paniculata 5.00 14.21 5.56 24.76 turpinia sphaerocarpa 7.69 6.03 9.76 23.48 notes: rd = relative density; rdc = relative dominance; rf = relative frequency. biotropia vol. 31 no. 2, 2024 212 similarity index (si) si is useful for analyzing several different vegetation communities and is sensitive to differences in species diversity (furusawa et al. 2014). table 4 shows that the vegetation community in the ggpnp at each growth level was relatively different, hence its high biodiversity. the results of this study were in accordance with the research of rozak et al. (2016), which showed that elevation influenced the presence of species in certain zones in the ggpnp. the results of this study also showed that the forest ecosystem in the ggpnp area were in the transition phase in the succession process. otherwise, they had yet to reach a climax. some tree species, such as castanopsis spp. (montane rainforest), ficus spp. (lowland rainforest and montane rainforest), knema spp. (lowland rainforest), litsea spp. (lowland rainforest and montane rainforest), maesopsis eminii (lowland rainforest), mallotus paniculatus (lowland rainforest) pometia pinata (lowland rainforest), schima walichii (lowland rainforest ), vernonia arborea (lowland rainforest and montane rainforest), and villebrunea rubescens (lowland rainforest and montane rainforest) are characteristic of secondary forests based on sadili et al. (2023). species diversity index (h’) and species evenness index (e) the results showed that the composition and structure of vegetation in each forest ecosystem varied in index values due to differences in each tree character. table 5 shows that the montane rainforest ecosystem had lower diversity values than the lowland rainforest, except at the sapling growth level. furthermore, the diversity index for all growth stages had normal conditions in both the lowland and montane forest ecosystems (1.5 < h’ < 3.5) (ortiz-burgos 2016). a community will be more stable and more resilient to disturbances with higher diversity of species (mcglade 1988). the e index has an interval value of 0-1. both ecosystems in the ggpnp had even or stable species distribution (table 5). krebs (2001) explained that e > 0.75 indicates an even distribution or stable species, 0.5 ≤ e ≤ 0.75 indicates an unstable distribution, and ≤ 0.50 species indicates an uneven distribution or depressed species. a high evenness index value (e > 7.5) indicates that no one species dominates in a community. the evenness index value is maximal when all species have equal abundance in the community (krebs 1989) and the species is in an even distribution (krebs 2001). table 4 index of similarity scores in the lowland rainforest and montane rainforest ecosystems in the ggpnp si montane rainforest seedling sapling pole tree lowland rainforest seedling 6.06 % sapling 0% pole 10.53% tree 9.38% table 5 h’ and e index values in two different forest ecosystems in the ggpnp   lowland rainforest montane rainforest   h’ e h’ e seedling 2.38 0.96 2.31 0.76 sapling 2.26 0.94 2.5 0.9 pole 3.2 0.97 3.06 0.9 tree 3.47 0.94 2.63 0.83 composition, structure, and carbon stock of the gunung gede pangrango national parks – sunyata et al. 213 carbon sequestration tropical rainforest ecosystems have an important role in regulating carbon and biomass cycles. the results of this study showed that the ggpnp montane rainforest ecosystem had a greater carbon stock than the lowland rainforest ecosystem (table 6). the amount of forest biomass is determined by tree density, soil fertility, and the diameter, height, and specific gravity of wood. in general, the ggpnp montane rainforest had a greater tree density (table 2). in addition, the composition of trees with a horizontal structure in the montane forest was greater than that in the lowland rainforest. there were old trees (stratum a) that were not found in the lowland rainforests (fig. 5). forests with older trees can store more carbon forests with younger ones (baker et al. 2022). in addition, montane rainforests are located at relatively higher elevations (1842 masl) than lowland rainforests (< 400 masl) (fig. 1), thus facing a less potential for anthropogenic threats (sarkar & devi 2014, myga-piątek et al. 2022). the biomass stock in the montane rainforest of the ggpnp (207.60 m/ha) was relatively larger when compared to the montane rainforest of mount rinjani, with an average biomass stock of 195 tonnes/ha (dossa et al. 2013), but lower than the montane forests of mount ciremai, with an average biomass stock of 258 tonnes/ha (rozak & gunawan 2015). the mount rinjani montane forest had a lower basal area and a lower tree density (ba < 15 m2/ha, d < 125 trees/ha) (dossa et al. 2013) compared to the ggpnp montane rainforest (ba = 18.85 m2/ha; d = 130 trees/ha). conversely, the mount ciremai montane forest had a larger basal area and a larger tree density (ba = 35 m2/ha; d = 900 trees/ha) (rozak & gunawan 2015) compared to the ggpnp montane forest. the biomass stock in the ggpnp lowland rainforest (118.80 tonnes/ ha) was lower than the biomass stocks of lowland rainforests in sumatra (384 tonnes/ha) (kotowska et al. 2015), but higher than the biomass stick of secondary lowland rainforests in south kalimantan (43.11-72.99 tonnes/ha) (suyanto et al. 2022). lowland rainforests in sumatra had a basal area of 27-32 m2/ha and a tree density of 440-684 trees/ ha (kotowska et al. 2015) compared to ggpnp lowland rainforest had basal area of 8.85 m2/ha and tree density of 60 trees/ha. the biomass stocks in secondary rainforests in south kalimantan were lower due to timber harvesting (suyanto et al. 2022). conclusion the number of seedlings > saplings > poles > trees and the graph shows a reverse “j” pattern. it is typical for tropical rainforests, and indicating that, generally, the forests well regenerated and are in a dynamic state. unfortunately, the finding of only a few species with a few or no seedlings or saplings may indicate that the species poorly regenerated. this leads to reduced populations of certain species at the seedling or sapling level, and driving widespread regeneration debts that could potentially lead to the extinction of the species. it is importanr that the ggpnp management also pay attention to the growth of maesopsis eminii so as not to reduce the purity of natural forests in the ggpnp. the horizontal structure of the ggpnp lowland and montane rainforest ecosystem was dominated by trees with diameters of 20-40 cm. it follows that the density decreased as the growth level and diameter increased. the vertical structure of the ggpnp lowland rainforests was occupied by stratum c (4-20 m) and b (20-30 m) trees with an absence of stratum a trees, while the ggpnp montane rainforests was occupied by trees of all strata (stratum c, 4-20 m; stratum b, 20-30 m; and stratum a, > 30 m). the ggpnp lowland forest ecosystem was dominated by neonauclea lanceolata and had a relatively higher species diversity. the ggpnp montane rainforest ecosystem was dominated by castanopsis acuminatissima, with a higher individual density, denser canopy, and more complex canopy strata. table 6 biomass, carbon stock, and carbon sequestration in two forest ecosystems in the gppnp agb (mg/ha) bgb (mg/ha) tabgb (mg/ha) carbon stock (mg/ha) co2 sqr (mg/ha/year) lowland rainforest 103.90 14.90 118.80 55.84 174.25 montane rainforest 181.41 25.66 207.06 97.32 303.70 biotropia vol. 31 no. 2, 2024 214 biomass, carbon stocks, and carbon sequestration in the montane rainforest in the ggpnp were larger than those in the ggpnp lowland rainforest. although there used to be plantation forests in the ggpnp lowland forest, these ecosystems had larger biomass and carbon stocks than logged-over forest area. the ggpnp montane rainforest had older forest stands, alarger average tree diameter, and lower potential for anthropogenic disturbances compared to ggpnp lowland rainforest. references ameja lg, ribeiro n, sitoe aa, guillot b. 2022. regeneration and restoration status of miombo woodland following land use land cover changes at the buffer zone of gile national park, central mozambique. trees for people [internet]. 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[cited 2024 may 25]; 7(1):41-50. available from: https:// biologyjournal.brin.go.id/index.php/berita_biologi/article /view/1234/1107 the southeast asian journal of tropical biology vol. 32 no. 3, 2025: 373 384 doi: 10.11598/btb.2025.32.3.2514 issn: 0215-6334 | e-issn: 1907-770x 373 remnant of the dryland coastal vegetation of the songkhla lake basin, songkhla, thailand anusit cheechang* and kitichate sridith department of biology, faculty of science, prince of songkhla university, songkhla 90230, thailand article higlights this article identifies and characterizes three native plant communities in the songkhla lake basin, highlighting their distinct species composition, coastal zonation patterns, and ecological value. it reveals high plant diversity across remnant dryland coastal habitats and documents key native species that define each community type. the study emphasizes the negative impacts of human activity and land policies on native vegetation and offers baseline floristic data that support conservation, ecological restoration, and sustainable land-use planning. article information received : 9 april 2025 revised : 18 may 2025 accepted : 28 may 2025 *corresponding author, e-mail: c.anusit@gmail.com research paper abstract the remnant dryland coastal vegetation of the songkhla lake basin, songkhla, thailand, was investigated in this study. twenty vegetation plots were established to represent each vegetation subtype. using braun-blanquet cluster analysis and the jaccard similarity index, the vegetation was classified into three distinct communities: (1) the coastal woodland community, located farthest inland and dominated by dipterocarpus alatus symington; (2) the coastal scrub-shrubby tree community, situated inland from the coastal zone, with dipterocarpus chartaceus symington, neolitsea zeylanica (nees & t. nees) merr., and psychotria asiatica l. identified as dominant species; and (3) the coastal scrub community, occurring near the shoreline and characterized by densely clustered vegetation dominated by mischocarpus sundaicus blume, planchonella obovata (r.br.) pierre, and vitex pinnata l. vegetation profiles for each community were delineated. significant degradation and decline of native plant communities in the songkhla lake basin have been caused by conservation activities involving the introduction of fastgrowing non-native species and by governmental land tax policies. keywords: dryland coastal vegetation, plant community structure, songkhla lake basin introduction dryland coastal vegetation is recognized as a unique but often underappreciated component of tropical coastal ecosystems (swiderska et al. 2018). these communities are typically found on sandy floodplains and elevated sandbars, where harsh environmental conditions are endured, including fluctuating salinity, limited freshwater input, and intense wind exposure. vegetation structure and species composition are influenced by these stressors, making such areas valuable for investigations of plant resilience and adaptation in marginal environments (freeman et al. 2023). additionally, drylands are shaped by aeolian processes, in which sparse vegetation and erodible surfaces are subjected to strong winds. while reductions in wind speed and sediment transport can be facilitated by vegetation through drag, the overall effects are considered complex due to turbulence, porosity, and plant flexibility. consequently, uncertainty remains regarding the precise role of vegetation in wind-driven erosion and deposition (mayaud & webb 2017). in southeast asia, ecological research in coastal regions has primarily been focused on mangrove forests and freshwater wetlands (tatu & anderson 2017; daga & salmo 2022; liu et al. 2022; veettil et al. 2024), whereas dryland coastal vegetation has been given disproportionately little attention (marod et al. 2020). in thailand, this knowledge gap is particularly evident, as much of the remaining dryland vegetation has been reduced to fragmented patches due to land-use changes, coastal development, and environmental degradation. these remnant patches are considered important since native species, ecological functions, and biogeographical signals may still be preserved, providing critical insights for understanding coastal ecosystem transitions and supporting conservation planning (tulloch et al. 2015). copyright (c) 2025@author(s). https://doi.org/10.11598/btb.2025.32.3.2514 https://creativecommons.org/licenses/by-nc-nd/4.0/ biotropia vol. 32 no. 3, 2025 374 previous studies have provided baseline floristic data and community classifications for portions of the southern coastline of thailand (laongpol 2003, 2010; sridith 2014; taing 2015). while these contributions are considered valuable, the focus was largely placed on species inventories and landscape profiles without explicit consideration of vegetation dynamics, environmental drivers, or conservation implications. furthermore, the extent to which freshwater inflows, seasonal salinity, and geomorphological processes influence dryland coastal plant communities in thailand has remained poorly documented. this study was conducted to address these knowledge gaps by investigating the remnant dryland coastal vegetation of the songkhla lake basin, southern thailand, through a phytosociological approach. specifically, the objectives were defined as follows: 1) the structure and composition of the remaining dryland coastal plant communities were characterized, and 2) their distribution patterns were analyzed in relation to environmental conditions. by documenting and analyzing these vegetation remnants, critical knowledge was generated to support the conservation and management of coastal ecosystems, particularly in identifying potential areas for habitat protection, ecological restoration, and adaptive land-use planning within one of thailand’s most ecologically complex coastal regions. materials and methods study area songkhla lake, located in southern thailand, is recognized as the largest lagoon system in southeast asia, covering approximately 1,040 km². it is situated across the provinces of songkhla, phatthalung, and parts of nakhon si thammarat, between latitudes 6°40′ 7°50′ n and longitudes 100°00′ 100°40′ e. the lake is divided into four main sections: thale noi, a freshwater lake in the north; upper songkhla lake (thale luang); middle songkhla lake, which constitutes the brackish zone; and lower songkhla lake, which directly connects to the gulf of thailand. the hydrology of the system is influenced by freshwater inflows from numerous small rivers and by seasonal seawater intrusion. freshwater conditions dominate during the flood season (november december), whereas brackish conditions prevail in the dry season due to reduced river inflows. geomorphologically, the lagoon was formed by a series of coastal sandbars that created a semi-enclosed basin comprising diverse habitats, including peat swamps, estuarine areas, floodplains, and coastal dunes. these varied environments are considered ecologically significant due to the high biodiversity they support (pongsaputra 1991; tookvinas & sirimontaporn 1988; yokokawa 1984) (fig. 1b). figure 1 study area and sampling plot notes: a = map of thailand; b = part of songkhla lake; c = sampling plots (black dots), small relic patches (red dots). remnant of dryland coastal vegetation in songkhla lake basin cheechang and sridith 375 data collection field survey rapid land-use changes and human activities have resulted in a significant reduction of natural forest cover in the songkhla lake basin, leaving only small and scattered forest remnants. within these patches, notable canopy structures and remnants of native plant species are still preserved, and they were selected as the primary focus of this study. site selection was carried out based on three main criteria: 1) the presence of natural vegetation as determined from satellite imagery and preliminary field visits; 2) accessibility to the site; and 3) the absence of invasive alien plant species. field surveys were conducted from january to december 2023 to verify the presence of vegetation and to assess the suitability of the sites for phytosociological sampling. vegetation data collecting a total of 20 study sites were selected across the songkhla lake basin in songkhla province. each selected site (indicated by black dots in fig. 1c) encompassed an area of approximately 6,000 to 8,000 m² and was used for the collection of vegetation data. the coordinates of all study sites were recorded using gps and subsequently mapped onto satellite imagery. during the survey, additional small forest patches (< 2,500 m²) were also identified (indicated by red dots in fig. 1c). these patches were not selected for detailed sampling due to their limited species diversity and evidence of disturbance; however, they were noted as areas of ecological interest to reflect the extent and fragmentation of the remaining dryland vegetation in the basin. vegetation data were collected using standard phytosociological methods. sampling plots were established as 20 × 20 m for the tree layer, 5 × 5 m for shrubs, and 1 × 1 m for herbaceous plants. within each plot, species composition, abundance (using the braun-blanquet scale), plant height, and vegetation structure were recorded. vegetation analysis the vegetation was analyzed following the conceptual framework and methodologies of the zürich-montpellier school (braun-blanquet 1964; bridson & forman 1998). study plots were systematically established at the selected sites to ensure that each stand encompassed a minimum area with homogeneous physiognomic features, thereby maintaining uniformity. for each plot, a comprehensive species inventory was compiled by stratifying the vegetation into vertical layers. multi-structured plant communities were classified into two or three layers, i.e., tree, shrub, and herb, based on their structural characteristics. the braun-blanquet method was employed to estimate species cover and sociability within each vegetation layer. discrete plant communities were subsequently classified through the synthesis of the collected vegetation data. profile diagrams and photographic records were constructed to illustrate stand structure at representative sites. to further analyze plant community associations, classical clustering analysis was performed using the unweighted pair group method with arithmetic mean (upgma) based on the braycurtis similarity index in past version 4.14. in this method, species were grouped into clusters according to similarity, beginning with the most similar pair and progressively merging less similar groups (miyawaki & suzuki 1980). the resulting dendrogram was used to elucidate ecological relationships among vegetation types across the study plots. in addition, the jaccard similarity index (jsi) was calculated to assess the similarity in species composition between different plots. the index is expressed as: j (a, b) = a the value of the jaccard similarity index is expressed on a scale ranging from 0 to 1, where a value of 0 is interpreted as indicating no shared species (representing entirely dissimilar communities), whereas a value of 1 is interpreted as indicating identical species composition (representing completely similar communities) (bisandu et al. 2019). these analyses were used to provide insights into floristic relationships, habitat fragmentation, and ecological patterns among the remnant forest patches within the songkhla lake basin. ⋂ b a ⋃ b biotropia vol. 32 no. 3, 2025 376 results and discussion species richness, composition, and plant community a comprehensive survey of vascular plants within the study plots documented 64 species, which were distributed across 59 genera and 42 families. among these, three species were identified as ferns: drynaria quercifolia (l.) j.sm., pyrrosia longifolia (burm.f.) c.v.morton, and pyrrosia piloselloides (l.) m.g.price. the family fabaceae exhibited the highest species richness, comprising four species, followed by apocynaceae, dipterocarpaceae, and myrtaceae, each represented by three species. several families, including annonaceae, combretaceae, dilleniaceae, lamiaceae, malvaceae, melastomataceae, orchidaceae, phyllanthaceae, primulaceae, rubiaceae, rutaceae, and salicaceae, were represented by two species each, while 29 families were represented by a single species (table 1). this composition reflects the diverse floristic structure of the study plots, with fabaceae contributing the greatest species richness among the recorded families. table 1 plant species, their taxonomic families, and associated plant communities no species name family com. ab. 1 uvaria ferruginea var. cherrevensis (pierre ex finet & gagnep.) meade & j.parn. annonaceae c + 2 uvaria siamensis (scheff.) l.l.zhou, y.c.f.su & r.m.k.saunders c + 3 amphineurion marginatum (roxb.) d.j.middleton apocynaceae b + 4 dischidia major (vahl) merr. b + 5 hoya verticillata (vahl) g.don var. verticillata b + 6 alocasia sanderiana w.bull araceae a + 7 caryota mitis lour. arecaceae a + 8 dianella ensifolia (l.) redouté asphodelaceae b + 9 garcinia celebica l. clusiaceae c + 10 combretum trifoliatum vent. combretaceae c + 11 terminalia catappa l. c + 12 cyanotis cristata (l.) d. don commelinaceae b + 13 dillenia hookeri pierre dilleniaceae c + 14 tetracera indica (christm. & panz.) merr. c + 15 dipterocarpus alatus roxb. ex g.don dipterocarpaceae a ++++ 16 dipterocarpus chartaceus symington b,c ++++ 17 anthoshorea roxburghii (g.don) p.s.ashton & j.heck. c + 18 elaeocarpus robustus roxb. elaeocarpaceae c + 19 vaccinium bracteatum thunb. ericaceae b + 20 derris trifoliata lour. fabaceae c + 21 guilandina bonduc l. c + 22 biancaea sappan (l.) tod. c + 23 abrus precatorius l. b,c ++ 24 vitex pinnata l. labiatae c +++ 25 clerodendrum indicum (l.) kuntze lamiaceae c + 26 vitex glabrata r.br. c + 27 neolitsea zeylanica (nees & t. nees) merr. lauraceae b,c ++ 28 sterculia villosa roxb. ex sm. malvaceae c + 29 microcos tomentosa sm. c+ 30 memecylon edule roxb. melastomataceae b,c + 31 memecylon ovatum sm. c + 32 sandoricum koetjape (burm.f.) merr. meliaceae a + remnant of dryland coastal vegetation in songkhla lake basin cheechang and sridith 377 no species name family com. ab. 33 tinospora crispa (l.) hook.f. & thomson menispermaceae a + 34 streblus asper lour. moraceae a,b,c ++ 35 syzygium antisepticum (blume) merr. & l.m.perry myrtaceae c + 36 syzygium grande (wight) walp. a,b + 37 rhodomyrtus tomentosa (aiton) hassk. b,c + 38 ochna integerrima (lour.) merr. ochnaceae c + 39 olax scandens roxb olacaceae c + 40 tetrapilus brachiatus lour. oleaceae c + 41 cymbidium findlaysonianum lindl. orchidaceae b + 42 dendrobium crumenatum sw. b + 43 pandanus tectorius parkinson pandanaceae c + 44 phyllanthus vitis-idaea (burm.f.) j.koenig ex roxb. phyllanthaceae c + 45 phyllanthus subscandens var. subscandens c + 46 bambusa bambos (l.) voss poaceae a + 47 drynaria quercifolia (l.) j.sm. polypodiaceae b + 48 pyrrosia longifolia c.v.morton b + 49 pyrrosia piloselloides (l.) m.g.price b + 50 ardisia crenata sims primulaceae b + 51 ardisia elliptica thunb. b + 52 ziziphus oenopolia (l.) mill. var. brunoniana (c.b.clarke ex a.w.hill) tardieu rhamnaceae c + 53 carallia brachiata (lour.) merr. rhizophoraceae a,c + 54 catunaregam tomentosa (blume ex dc.) tirveng. rubiaceae b,c + 55 psychotria asiatica l. b ++ 56 atalantia monophylla (l.) dc. rutaceae c + 57 glycosmis pentaphylla (retz.) dc. b + 58 flacourtia indica (burm. f.) merr. salicaceae c + 59 casearia grewiifolia vent. a + 60 dendrotrophe umbellata (blume) miq. santalaceae b + 61 lepisanthes rubiginosa (roxb.) leenh. sapindaceae a,c + 62 mischocarpus sundaicus blume c +++ 63 planchonella obovata (r.br.) pierre sapotaceae c +++ 64 alpinia malaccensis (burm.f.) roscoe zingiberaceae a + notes: com. = community; ab. = abundance; a = coastal woodland community; b = coastal scrub-shrubby tree shrub community; c = coastal scrub community. the songkhla lake basin is situated on the sathing phra peninsula, a narrow and elongated landform extending along the longitudinal axis of songkhla lake from its northern to southern reaches. the plant communities within this region have been subjected to prolonged anthropogenic disturbances over time (prapruit et al. 2023). nevertheless, certain areas still retain remnants of the original vegetation, which can be used as important indicators for reconstructing the potential native plant composition that once characterized the songkhla lake basin ecosystem. the cluster analysis and jaccard similarity matrix based on species composition distinguished three types of plant communities (fig. 5; table 2). the woodland community is represented by the plant community situated farthest inland. the scrub community is represented by the plant community occurring closest to the coastline. the scrubwoodland mixed community is represented by the transitional zone between these two communities, reflecting a gradient of environmental conditions and species composition. the proposed plant communities and their species richness are presented in figure 2. biotropia vol. 32 no. 3, 2025 378 a. c. b. figure 2 coastal vegetation notes: a. coastal woodland community (plot no.20); b. coastal scrub-shrubby tree community (plot no.10); and c. coastal scrub community (plot no.3). (a) coastal woodland community the plant community located farthest inland was represented by four sampling plots (plots 17 20), in which 12 vascular plant species were recorded. dipterocarpus alatus symington, a characteristic emergent tree species, dominated the canopy layer. the understory layer was composed of bambusa bambos (l.) voss, carallia brachiata (lour.) merr., casearia grewiifolia vent., lepisanthes rubiginosa (roxb.) leenh., sandoricum koetjape (burm.f.) merr., and syzygium grande (wight) walp., which were characteristic components of the coastal sand ridge plant community. their presence had also been documented in previous studies (congdon 1982; sridith 2002; laongpol 2010), contributing to the structural complexity of the community. the ground layer was represented by two herbaceous species, alocasia sanderiana w. bull and alpinia malaccensis (burm.f.) roscoe, and one climbing species, tinospora crispa (l.) hook.f. & thomson, was also recorded. additionally, seedlings of ardisia elliptica thunb., caryota mitis lour., and streblus asper lour. were observed (figs. 3 & 2a). according to laongpol (2010), plant communities containing dipterocarpus alatus symington were classified as dune woodland communities. this classification is consistent with the findings of the present study; however, the observed species diversity was lower than that reported in previous research, likely due to human habitation and various land-use activities in the surrounding area, which may have impacted the vegetation at the study site. results from cluster and similarity analyses indicated that this plant community, comprising plots 17, 18, 19, and 20 (fig. 6), exhibited the highest level of similarity within the group. the short branch lengths in the dendrogram, coupled with moderate similarity indices in the jaccard matrix (e.g., plot 17 vs. plot 18: 0.25; plot 19 vs. plot 20: 0.13; table 2), suggest that these plots share a common set of species. this high degree of resemblance is attributed to their proximity to similar environmental conditions. (b) coastal scrub-shrubby tree community the area inland from the coastal zone was surveyed using 12 sampling plots (plots 7 14), in which 24 plant species were documented. the canopy layer was predominantly characterized by dipterocarpus chartaceus symington, which emerged as the dominant species and was consistently recorded across all surveyed plots. neolitsea zeylanica (nees & t. nees) merr. was present within the sub-canopy layer, while psychotria asiatica l. was ubiquitously recorded, highlighting its ecological significance in this stratum. the understory layer was primarily composed of dianella ensifolia (l.) redouté, representing the most abundant species at this level. a notable presence of the climbing species hoya verticillata (vahl) g.don var. verticillata was observed remnant of dryland coastal vegetation in songkhla lake basin cheechang and sridith 379 throughout the plots. furthermore, two orchid species, cymbidium findlaysonianum lindl. and dendrobium crumenatum sw., were recorded, indicating a diversity of epiphytic flora. fern species, including drynaria quercifolia (l.) j.sm., pyrrosia longifolia (burm.f.) c.v.morton, and pyrrosia piloselloides (l.) m.g.price, were also documented, contributing to the compositional and structural complexity of the community (figs. 4 & 2b). compared to previous studies by laongpol (2010), several plant species recorded in the present study are classified within the dune scrub community, including dianella ensifolia (l.) redouté, hoya verticillata (vahl) g.don var. verticillata, and dischidia major (vahl) merr. figure 3 community a (plot no. 17) notes: 1 = alocasia sanderiana w.bull; 2 = caryota mitis lour.; 3 = bambusa bambos (l.) voss; 4 =streblus asper lour.=; 5 =carallia brachiata (lour.) merr.; 6 = dipterocarpus alatus roxb. ex g.don; 7 =lepisanthes rubiginosa (roxb.) leenh.; 8 =sandoricum koetjape (burm.f.) merr.; 9. alpinia malaccensis (burm.f.) roscoe. figure 4 figure 4 community b (plot no. 8) notes: 1 =cyanotis cristata (l.) d.don; 2 = neolitsea zeylanica (nees & t.nees) merr.; 3 = psychotria asiatica l.; 4 = hoya verticillata (vahl) g.don var. verticillata; 5 = caryota mitis lour.; 6 = drynaria quercifolia (l.) j.sm.; 7 = abrus precatorius l.; 8 = dipterocarpus chartaceus symington. biotropia vol. 32 no. 3, 2025 380 (c) coastal scrub community this plant community, located near the shoreline, is characterized by densely clustered vegetation and comprises eight plots (plots 1 5, 15, and 16). it exhibits the highest species diversity among all communities, with 39 species recorded. the dominant species within this community include mischocarpus sundaicus blume, planchonella obovata (r.br.) pierre, and vitex pinnata l. dipterocarpus chartaceus symington was the most frequently encountered tree species. several tree species in this community exhibited a shrubby crown structure, despite typically developing regular, well-formed crowns when growing further inland. notably, 29 species recorded in this community were absent from the previously described plant communities, highlighting its unique floristic composition (figs. 5 & 2c). the natural forest patches studied exhibited clear zonation. salt-tolerant plant species were observed in the frontal zone closest to the saltwater. behind this zone, species such as pandanus tectorius (merlin 1999) and planchonella obovata (r.br.) functioned as a buffer against salinity intrusion. further inland, dipterocarp species (dipterocarpus alatus roxb. ex g.don, dipterocarpus chartaceus symington) were present; these species are less salt-tolerant but were protected by the vegetation in the frontal zones. when the frontal zone vegetation was removed, the inner zones were also negatively affected due to increased salinity. in certain cases, human-made structures or cultivated plants were employed as barriers to limit salt intrusion (su et al. 2025). the gradual disappearance of dipterocarp species was primarily attributed to human harvesting rather than natural mortality, as natural plant death was rarely observed; anthropogenic activities were the predominant cause of vegetation loss. the analysis results included plots 4, 7, 8, 9, 10, 11, 12, and 13 (fig. 6). notably, several plots within this community, particularly plots 9, 10, 11, and 12, exhibited high similarity values (up to 0.67, table 2), as reflected by the shorter branches in the dendrogram. this finding indicates a strong floristic cohesion, which may be attributed to shared habitat characteristics, such as moderate exposure to environmental stress or similar successional stages. the relatively higher species turnover observed between plots in this community, compared to community a, suggests the presence of microhabitat heterogeneity or slight variations in disturbance regimes. the findings of the present study are largely consistent with those reported by laongpol (2010) regarding the composition of this plant community. analysis of community c revealed the greatest heterogeneity, with plots 1, 2, 3, 5, 6, 14, 15, and 16 exhibiting longer branch lengths and lower similarity values across the matrix. the isolation of plots such as 1, 2, and 5 reflects their distinct species assemblages. these patterns suggest that community c may encompass a broader range of environmental conditions or historical land-use impacts, resulting in increased species variability. additionally, the presence of unique species not shared with other communities indicates reduced influence from coastal stressors and potentially more stable or mature vegetation. an interesting ecological observation is that tree species within certain plots exhibit a shrubby crown form near the coastline but develop a normal canopy structure further inland. this morphological adaptation is likely a response to abiotic stressors such as strong winds, high salinity, or nutrient-poor soils in the coastal zone, reinforcing the clustering patterns observed in community a (jiang et al. 2025). overall, the strong correspondence between the dendrogram and the similarity matrix supports the robustness of the clustering results. the differentiation among the three communities appears to reflect gradients of environmental stress, disturbance history, and microhabitat variation. future analyses incorporating specific environmental parameters, such as soil salinity, elevation, and wind exposure, are recommended to further elucidate the ecological drivers shaping these plant communities. remnant of dryland coastal vegetation in songkhla lake basin cheechang and sridith 381 figure 5 community c (plot no. 4) notes: 1 = neolitsea zeylanica (nees & t. nees) merr.; 2 = vitex pinnata l.; 3 = olax scandens roxb; 4 =derris trifoliata lour.; 5 = mischocarpus sundaicus blume; 6 = ziziphus oenoplia (l.) mill.; 7 = abrus precatorius l.; 8 = planchonella obovata (r.br.) pierre; 9 = syzygium antisepticum (blume) merr. & l.m.perry; 10 = garcinia celebica l; 11 = flacourtia indica (burm. f.) merr. figure 6 the cluster analysis dendrogram presents the results of a hierarchical cluster analysis (hca) based on the similarity of 20 vegetation plots notes: the plots are grouped into three distinct plant communities, labeled as: community a comprises plots 17, 18, 19, and 20. these plots are highly similar, as indicated by the short branch lengths within this cluster, suggesting strong compositional resemblance among these plots. community b consists of plots 4, 7, 8, 9, 10, 11, 12, and 13. although this community exhibits more internal variation than community a, the plots still share significant floristic similarities. community c includes plots 1, 2, 3, 5, 6, 14, 15, and 16. plots within this group demonstrate distinct characteristics, especially plots 1, 2, and 5, which show longer branch lengths, indicating lower similarity to other plots. biotropia vol. 32 no. 3, 2025 382 table 2 jaccard similarity matrix between the plots conservation measures the dryland coastal vegetation of the songkhla lake basin, songkhla, thailand, is considered significant for regulating water dynamics in the coastal dunes. it contributes to the maintenance of water levels accumulated in the sandbars, originating both from mountain runoff along the descending slopes of the basin, driven by gravity, and from rainfall that is collected within the sandbars themselves (finkl & makowski 2017). the composition, structure, and diversity of the plant community have been significantly influenced by reforestation efforts and government policies. in past reforestation programs, certain tree species may have been introduced, natural regeneration processes altered, and land-use patterns modified, all of which affected native vegetation dynamics (willium et al. 2024). furthermore, government policies concerning land management, conservation, and development have shaped the establishment and persistence of these plant communities over time (talerngsri 2020). conservation activities have included reforestation projects that introduced fast-growing invasive species, such as casuarina equisetifolia l. and acacia mangium willd., which inhibit the growth of native plants and destroy existing seed banks and seedling reserves of indigenous species. coastal zones are characterized by high biodiversity and complex interconnections among terrestrial and marine ecosystems, as well as social and governance systems. despite ongoing conservation efforts, unsustainable resource use continues to persist, presenting significant governance challenges due to the complexity and cross-sectoral nature of these issues. effective management requires an interactive governance approach that accounts for the relationships among ecological, social, and governing systems at multiple levels: understanding the complexity of coastal zones (first order), improving legal and institutional frameworks (second order), and establishing principles and values to guide challenging decisions (meta order) (satumantpan & chuenpagdee 2022). in the songkhla lake basin, the destruction of dryland coastal vegetation has been notably influenced by government policies (wear & wibbenmeyer 2023). for instance, new land tax laws have compelled landowners to remove remaining natural plant communities classified as forest patches to avoid high taxes. to comply, crops specified under the law have been cultivated in place of native vegetation, leading to the nearelimination of indigenous plant communities on the dry coastal dunes of the basin. however, the environmental impacts of these government policies have been insufficiently studied. this lack of comprehensive research has allowed the continued destruction of valuable coastal vegetation, resulting in the rapid loss of ecologically important areas. remnant of dryland coastal vegetation in songkhla lake basin cheechang and sridith 383 conclusion the remnant dryland coastal vegetation of the songkhla lake basin represents a highly diverse yet fragile ecosystem shaped by both natural gradients and intense anthropogenic pressures. three distinct plant communities, i.e., coastal woodland, coastal scrub-scrubby tree, and coastal scrub communities, reflect ecological adaptations to inland-to-shoreline conditions but remain increasingly threatened by land-use change, harvesting, invasive species, and misguided policies. the primary drivers of degradation are not natural processes but human interventions, particularly land tax laws and inappropriate reforestation practices. effective conservation requires an integrated governance approach that recognizes ecological functions, reforms counterproductive policies, and prioritizes the intrinsic value of native ecosystems. without timely and coordinated action, these unique and irreplaceable plant communities face imminent and irreversible loss. acknowledgments this research was generously supported by the science achievement scholarship of thailand 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technology and agroindustry, university of mataram, mataram 83125, indonesia received 8 march 2023 / revised 26 july 2023 / accepted 12 september 2023 abstract green coffee beans are also called unroasted coffee beans. the chemical composition of green coffee beans plays a vital role in determining the final product’s aroma. the main objective of this research was to characterize the chemical properties of arabica and robusta green coffee beans grown in different regions of geopark rinjani, lombok island, indonesia. the water, ash, protein, carbohydrate, total solids, and caffeine contents, total acidity, and ph were determined. data were analyzed by analysis of variance. the results revealed significant differences in the moisture contents of arabica beans from sembalun, sajang, and sapit and robusta beans from rempek, selelos, and genggelang. additionally, the ash contents of arabica sajang and arabica sapit showed notable differences compared to other samples. however, the total fat contents of arabica sajang, arabica sapit, robusta rempek, and robusta genggelang did not exhibit significant variations. on the other hand, significant differences were observed in the protein contents of all samples, particularly between arabica sembalun and arabica sapit, compared to arabica sajang, robusta rempek, robusta genggelang, and robusta selelos. robusta coffee beans appeared to have a slightly lower ph than arabica beans. the latter exhibited consistent acidity in the range of 0.20–0.21, whereas the former showed higher acidity levels (0.23–0.25). arabica beans had a lower caffeine content, averaging 1.09%, whereas robusta beans exhibited an average caffeine content of 2.09%. this research provides valuable insights into the chemical composition of green coffee beans from different species and locations within geopark rinjani, contributing to a better understanding of the factors influencing the aroma and quality of coffee. keywords: arabica coffee, geopark rinjani, green coffee bean, robusta coffee introduction there are 130 coffee species identified worldwide but only two are responsible for most of the global coffee trade. coffea arabica l. and coffea canephora account for approximately 60– 65% and 35–40% of the global coffee production, respectively. the coffee industry is a substantial contributor to the global economy, with an annual production of approximately 10 million tons and an income of approximately usd 200 million. this industry also supports the livelihoods of millions of smallholder farmers, who account for approximately 60% of coffee farms in tropical regions (cassamo et al. 2022). the quality of coffee beans is primarily determined by their physical and chemical properties, which are closely linked to the coffee species/genotype, microclimate conditions during the fruit and bean maturation process, soil characteristics, and agricultural crop management practices. environmental conditions during fruit development also substantially affect the final bean quality (ahmed et al. 2021). shade and altitude positively affect coffee bean quality and are vital in producing high-quality coffee. higher altitudes and shade at lower altitudes may enhance the physical and chemical properties of the beans as a result of the lower temperatures and higher air humidity. these climate conditions can extend the fruit and bean maturation period, *corresponding author, email: zainuri.ftp@unram.ac.id the chemical characteristics of arabica and robusta green coffee beans – zainuri et al. 319 increasing the accumulation of sugars and aromarelated phenolic compounds. in addition, mendes et al. (2022) reported that beans grown at higher altitudes tend to have higher levels of sugars, lipids, amino acids, trigonelline, and chlorogenic acid isomers, leading to higherquality coffees compared to beans from lower altitudes. by contrast, coffees of the same genotype grown at lower altitudes have lower quality and lower levels of the abovementioned compounds. the chemical composition of green coffee beans plays a vital role in determining the final product’s aroma. the maillard reaction, which occurs during roasting, is the primary process that generates aroma through a reaction between amino acids and reducing sugars, forming nitrogenous heterocycles and brown melanoidins. this non-enzymatic browning generates many volatile compounds that shape the sensory experience of coffee. arabica has a sweet, caramel-like aroma when roasted, whereas robusta has an earthy, spicy aroma. arabica contains higher levels of sucrose, which are essential for developing organoleptic properties and enhancing aroma formation in coffee (liu et al. 2019). in addition, the caffeine content varies among the different cultivars and species. this variability can also be seen in other parts of the coffee plant, with the highest levels of caffeine found in the beans, flowers, and leaves. additionally, it was reported that younger plant tissues contain more caffeine than mature parts (dessalegn et al. 2008). the main objective of this study was to characterize the chemical properties of arabica and robusta green coffee beans grown in different regions of geopark rinjani, located on the island of lombok, indonesia. this is an important geological and natural heritage site that unesco has recognized as a global geopark. the geopark rinjani-lombok covers five districts/cities: north lombok, east lombok, middle lombok, west lombok, and mataram city. the altitude of the region where samples were collected was between 500 and 1500 meters above sea level (masl) (figure 1). the parameters analyzed were the moisture, ash, protein, carbohydrate, total solid, and caffeine contents, total acidity, and ph. this is the first report on the chemical composition of green coffee beans from this region. figure 1 distribution map of arabica and robusta green coffee bean plantations in geopark rinjani, lombok island, indonesia. the different colors indicate different altitudes: dark green (0–500 m), light green (500–1500 m), and light brown (1500–2500 m). biotropia vol. 30 no. 3, 2023 320 materials and methods materials arabica green bean samples were collected from various coffee-growing locations in geopark rinjani, lombok, indonesia. the arabica coffee beans were from the sembalun, sajang, and sapit regions, east lombok. meanwhile, robusta green beans were collected from the rempek, selelos, and genggelang regions, north lombok regency. other materials used in this research were distilled water, kjeldahl tablets, h2so4, 0.1 n naoh, a standard caffeine solution, na2co3, chloroform, a catalyst, boric acid, a phenolphthalein indicator, and diethyl ether. the tools used included soxhlet extraction equipment, a drying oven (unb 400, memmert), a ph meter, a chromameter (msez), a spectrophotometer, a weighing scale, and a blender. sample preparation all green beans went through natural processing by drying for 2–3 weeks. the green beans were ground using a blender. then, the ground coffee was sifted using a 40-mesh sieve. fine powders from arabica and robusta green bean samples were packaged in aluminum foil ziplock bags (approximately 100 g per sample per package) for further analysis. research design a randomized block design (rbd) was used with a grouping of six samples, with the single factor being green beans from villages around geopark rinjani. each sample comprised four replicates, resulting in 24 experimental units. the observed parameters were ph (aoac, 1980), moisture content (aoac, 1996), total fat content (aoac, 2005), ash content (aoac, 2000), protein content (aoac, 2005), carbohydrate (by difference) (aoac, 2005), total solid content (aoac, 2005), total acidity (aoac, 2005), and caffeine content (aoac, 2005). results and discussion moisture content the moisture content of coffee beans is essential as it can affect the taste, aroma, and overall quality of the coffee. figure 2 shows that the moisture content of the arabica samples (collected from sembalun, sajang, and sapit) was significantly different compared to that of the robusta samples (collected from rempek, selelos, and genggelang). the arabica sample from sembalun was significantly different from those from sajang and sapit. however, there were no significant differences in the moisture contents of the robusta samples. figure 2 the moisture content of arabica and robusta green beans from different regions of geopark rinjani, lombok island, indonesia. different letters indicate significant differences (p ≤ 0.05). a b b c c c 0 2 4 6 8 10 m o is tu re c o n te n t (% ) sample the chemical characteristics of arabica and robusta green coffee beans – zainuri et al. 321 for the arabica samples, the water content ranged from 8.11% to 9.09%, with the highest value corresponding to the sajang sample. however, the water content in the robusta samples was slightly higher, ranging from 9.07% to 9.20%. the highest value was found in the selelos sample. the moisture contents of the arabica and robusta green beans met the requirement in the indonesian standard (sni 012907-2008), which states that the maximum moisture content of the green beans should be 12%. it is essential to monitor the water content of green coffee beans as it can affect various aspects of coffee quality, such as flavor and aroma during storage and roasting. in addition, reh et al. (2006) reported that the moisture content is considered the essential quality factor for green coffee, as it affects fermentation and mold growth during storage and transportation, potentially causing undesirable flavors or the formation of mycotoxins. the water content of the coffee bean can also vary depending on the specific growing conditions, processing methods, and storage conditions (collazos-escobar et al. 2020). the variability in the moisture content of the arabica samples may be related to different environmental conditions in each planting area where the samples were obtained and also to differences during processing. ash content the ash contents of the arabica and robusta green coffee beans were measured, and the results are shown in figure 3. the ash content ranged from 4.5072% to 5.5182% for the arabica samples, with the lowest value observed in the sajang sample. the ash content of arabica sajang and arabica sapit was significantly different compared to that of other samples. however, the ash contents of robusta rempek, robusta selelos, and robusta genggelang were not significantly different. figure 3 the ash content of arabica and robusta green beans from different regions in geopark rinjani, lombok island, indonesia. different letters indicate significant differences (p ≤ 0.05). b a a bc c bc 00,000 00,001 00,002 00,003 00,004 00,005 00,006 00,007 arabica sembalun arabica sajang arabica sapit robusta rempek robusta selelos robusta genggelang a sh c o n te n t (% ) sample biotropia vol. 30 no. 3, 2023 322 the ash content of coffee beans is an essential factor to consider as it can provide information on the quality and processing of the beans. a high ash content may indicate the presence of impurities, whereas, a low ash content may indicate under-roasting or poor-quality beans. pizarro et al. (2004) reported that the ash content in coffee beans reflects the mineral content in the beans. a high ash content may indicate a high mineral content. the high ash content in the arabica sample from sembalun may be associated with the mineral content of the soil in the planting area because this is the closest area to mount rinjani, a volcano that erupted. additionally, impurities and residual silver skin may also affect the ash content. differences in the origin of raw materials, such as the maturity of the beans and environmental factors that may be related to chemical characteristics or other aspects of the soil are external factors that affect the ash content in coffee beans. total fat content the lipid component in coffee beans is primarily found in the endosperm. although the indonesian national standard does not use fat content to assess the quality of green beans, lipids are closely associated with coffee quality, and their content may differ among coffee cultivars. figure 4 shows the variations in the total fat contents of the different coffee species and growing areas for the same species. arabica sembalun had the lowest total fat content (9.86%), whereas robusta selelos had the highest total fat content (18.97%). the data also showed that the total fat contents of arabica sajang, arabica sapit, robusta rempek, and robusta genggelang were not significantly different. robusta coffee beans are known for their strong and bold flavor, which is possibly related to their higher lipid content. the obtained result also suggests that the lipid content can vary considerably among different coffee genotypes and may be influenced by factors such as the specific growing and processing conditions, including the drying time. benoit et al. (2006) reported that the lipid content in coffee beans increases with an increase in the altitude of the growing area. the total fat content of coffee beans can affect the overall quality and taste of coffee. lipids contribute to the aroma and flavor of coffee. thus, a higher lipid content can result in a more intense and complex aroma and flavor. however, a higher lipid content also results in greater susceptibility to rancidity, which can negatively affect the flavor and aroma of coffee. figure 4 the total fat content of arabica and robusta green beans from different regions in geopark rinjani, lombok island, indonesia. different letters indicate significant differences (p ≤ 0.05). a bc ab b c b 0 5 10 15 20 25 arabica sembalun arabica sajang arabica sapit robusta rempek robusta selelos robusta genggelang t o ta l fa t c o n te n t (% ) sample the chemical characteristics of arabica and robusta green coffee beans – zainuri et al. 323 protein content as illustrated in figure 5, the protein contents of the samples showed significant differences. the protein content of the robusta beans appeared to be higher than that of the arabica beans. the protein content of arabica coffee beans was between 11.06 and 12.02%, whereas the robusta coffee beans contained protein levels ranging from 13.55 to 14.50%. arabica sembalun had the lowest protein content among all the samples, whereas robusta selelos had the highest protein content. the differences in protein content among the different coffee varieties may be due to various factors, such as the growing conditions, genetics, and post-harvest and processing methods (wibowo et al. 2021). according to marcone (2004), differences in the protein contents of green coffee beans may be due to variations in the maturity level of the coffee fruit, leading to diverse chemical compositions. the average protein content in dried green coffee beans ranges from 8% to 12%. arabica sapit green beans underwent the longest drying process, as it took three weeks, compared to other green beans, which only took two weeks to dry. prolonged drying or sun-drying can result in protein degradation. in excessive natural fermentation during prolonged drying, microorganisms can use the protein in coffee beans as well as other nutrients such as carbohydrates and fats as energy sources (wibowo et al. 2021). total carbohydrate content carbohydrates are essential components in coffee. figure 6 shows the total carbohydrate contents of arabica and robusta green coffee beans from different growing areas. the results revealed that the carbohydrate contents of arabica sembalun and arabica sapit were significantly different than those of arabica sajang, robusta rempek, robusta genggelang, and robusta selelos. among the coffee bean samples, the highest carbohydrate content was found in arabica sembalun (65.46%), whereas the lowest carbohydrate content was found in robusta selelos (52.95%). the data suggests that arabica beans tend to have a higher carbohydrate content than robusta beans. however, there was considerable variation in the two types of coffee, as indicated by the high standard deviation values. the carbohydrate content can affect the flavor of roasted coffee, as it is responsible for the formation of desirable flavors during the roasting process. furthermore, the carbohydrate composition can also affect the functional properties of the final product, such as the body and mouthfeel of coffee. therefore, a higher carbohydrate content may contribute to a more full-bodied and smoother coffee, whereas a lower carbohydrate content may result in a lighter and more acidic coffee. however, the correlation between the carbohydrate content and coffee flavor profile is complex and can be affected by many factors (arya & rao 2007). figure 5 the protein content of arabica and robusta green beans from different regions in geopark rinjani, lombok island, indonesia. different letters indicate significant differences (p ≤ 0.05) a b c d e f 0 2 4 6 8 10 12 14 16 arabica sembalun arabica sajang arabica sapit robusta rempek robusta selelos robusta genggelang p ro te in c o n te n t (% ) sample biotropia vol. 30 no. 3, 2023 324 figure 6 the total carbohydrate content of arabica and robusta green beans from different regions in geopark rinjani, lombok island, indonesia. different letters indicate statistically significant differences (p ≤ 0.05) ph as shown in figure 7, arabica sembalun has the highest ph (5.81) among the arabica samples. arabica sajang and arabica sapit have slightly lower phs, at 5.55 and 5.33, respectively. the robusta samples have slightly higher phs, with rempek having the lowest (5.4675), followed by selelos (5.4525), and genggelang (5.595). these data suggest that the phs of the arabica beans from three different locations were more consistent than those of the robusta beans, as indicated by their lower standard deviations. moreover, the robusta coffee bean samples appeared to have a slightly lower ph than the arabica samples. saputri et al. (2020) reported that arabica gayo coffee beans have an average ph of 5.66, which is slightly higher than that of robusta gayo coffee beans (4.89). however, lee et al. (2017) reported that the ph of arabica coffee beans is lower (4.60–5.60) than that of robusta coffee beans (5.30–6.10). figure 7 the ph of arabica and robusta green beans from different regions in geopark rinjani, lombok island, indonesia. different letters indicate significant differences (p ≤ 0.05) a b a b c b 0 10 20 30 40 50 60 70 arabica sembalun arabica sajang arabica sapit robusta rempek robusta selelos robusta genggelang c ar b o h y d ra te c o n te tn t ( % ) sample a bc e cd d b 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 arabica sembalun arabica sajang arabica sapit robusta rempek robusta selelos robusta genggelang p h sample the chemical characteristics of arabica and robusta green coffee beans – zainuri et al. 325 the ph of coffee beans is influenced by acidic compounds, specifically carboxylic acids. these acids are present in the free form after their release by the breakage of glycoside bonds. variations in the ph values of coffee may be influenced by the location where the plants are grown. sajang village has andosol soils and is located in geopark rinjani at an altitude greater than 800 masl; its soil ph ranges from acidic to neutral (ph 5.0–7.0). meanwhile, sapit village has regosol soils. based on its geography, sapit village has a 25–40% slope and less than 2500 mm/year of rainfall with a soil ph ranging from 6 to 7. on the other hand, rempek, selelos, and genggelang, where the robusta samples were obtained, have inceptisol soils with a thick layer of approximately 1–2 m and ph in the range of 5.0–7.0. the three soil types in the coffeegrowing areas of geopark rinjani-lombok are derived from the volcanic ash of mount rinjani, and the ph ranges from 5 to 7. total acidity the acidity of coffee beans is essential as it can affect the flavor profile. generally, arabica beans are considered to have a milder and more nuanced flavor, whereas robusta beans are known for their bold and robust flavor. the difference in acidity levels between these two types of beans may contribute to these distinct flavor profiles. figure 8 shows that the arabica and robusta coffee samples from different regions in geopark rinjani had distinct acidity levels. the arabica samples had similar acidity levels, ranging from 0.20 to 0.21, whereas the robusta samples had higher acidity levels, ranging from 0.23 to 0.25. these differences in acidity may be due to variations in the growing conditions of the different samples, such as soil composition and climate, as well as genetic differences between the arabica and robusta coffee plants. towaha et al., (2014a) proposed that altitude affects the acidity level of arabica coffee beans. the processing methods and roasting techniques can also influence the acidity of coffee beans. according to kasim et al. (2020), the titrated acidity in the dry-processed robusta coffee is 3.65%, whereas in the wet-processed robusta coffee is 3.42%. on the other hand, according to winarno et al. (2021), the free acidity in dry-processed arabica coffee is approximately 0.05%, and that of wet-processed arabica coffee is approximately 0.07%. figure 8 the total acidity of arabica and robusta green beans from a different region in geopark rinjani, lombok island, indonesia. different letters indicate significant differences (p ≤ 0.05) a a ab c c bc 0.00 0.05 0.10 0.15 0.20 0.25 0.30 arabica sembalun arabica sajang arabica sapit robusta rempek robusta selelos robusta genggelang t o ta t it ra ta b le a ci d it y ( % ) sample biotropia vol. 30 no. 3, 2023 326 total solid content the total solid content refers to the percentage of dry matter in the coffee beans, indicating the quality and taste of coffee. arabica sembalun had the lowest total solid content (4.43%), whereas, arabica sajang had the highest total solid content (7.45%). moreover, robusta rempek had a total solid content of 7.275% (figure 9). the total solid content can affect the flavor of coffee. saputri et al. (2020) reported that robusta coffee beans showed a higher total solid content compared to arabica coffee beans. this result was consistent with the lower levels of organic acids and amino acids found in arabica coffee beans compared to robusta coffee beans. the total solid content can consist of organic acids, amino acids, and simple carbohydrates, which can contribute to the taste and aroma formation of coffee during the roasting process. caffeine content caffeine, one of the most well-known compounds found in coffee, plays an important role in the strength, body, and bitterness of coffee, and is responsible for many of the physiological effects associated with coffee consumption (buffo and cardelli-freire 2004). figure 10 shows a significant difference between the caffeine of arabica and robusta coffee beans produced in geopark rinjani. arabica beans had a lower (avg. 1.09%) caffeine content than robusta beans (avg. 2.09%). this result aligns with the general understanding that arabica beans typically have a lower caffeine content than robusta beans. moreover, the data shows that there were variations in the caffeine contents of beans of the same species but from different areas, which can be attributed to factors such as growing conditions, processing methods, and genetics. towaha et al. (2014) also reported that arabica coffee beans produced at different altitudes exhibited different caffeine levels. figure 9 the total solid content of arabica and robusta green beans from different regions in geopark rinjani, lombok island, indonesia. different letters indicate significant differences (p ≤ 0.05) a c bc bc bc b 0 1 2 3 4 5 6 7 8 9 arabica sembalun arabica sajang arabica sapit robusta rempek robusta selelos robusta genggelang t o ta l so li d c o n te n t (% ) sample the chemical characteristics of arabica and robusta green coffee beans – zainuri et al. 327 figure 10 the caffeine content of arabica and robusta green beans from different regions in geopark rinjani, lombok island, indonesia. different letters indicate significant differences (p ≤ 0.05) according to clemente et al. (2013), the chemical elements in the soil (particularly potassium, k) can affect the caffeine content of coffee beans. k plays an essential role in coffee plant reproduction, especially in the size and yield of coffee beans. in addition, k affects the taste of coffee by activating the polyphenol oxidase enzyme and determining the caffeine and phenol content in coffee beans. moreover, the caffeine content is not only influenced by the growing location but may also be affected by the processing methods (zhang et al. 2013). conclusion the chemical composition of arabica and robusta coffee beans was found to be significantly (p ≤ 0.05) different. arabica beans have a lower ph, acidity, and caffeine content than robusta beans. meanwhile, robusta beans have a higher total solid content than arabica beans. these results suggest that the growing conditions, processing methods, and genetics of coffee beans determine their chemical composition, which in turn can contribute to the distinct flavor 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10.13057/biodiv/d220938. winarno ra, perangin-angin mib, sembiring n v. 2021. karakterisitik sifat kimia biji kopi arabika dengan beberapa metoda pengolahan di kabupaten simalungun provinsi sumatera utara. agrivet : jurnal ilmu-ilmu pertanian dan peternakan (journal of agricultural sciences and veteriner). 9(2): 237243. doi: 10.31949/agrivet.v9i2.1701. zhang x, li w, yin b, chen w, kelly dp, wang x, zheng k, du y. 2013. improvement of near infrared spectroscopic (nirs) analysis of caffeine in roasted arabica coffee by variable selection method of stability competitive adaptive reweighted sampling (scars). spectrochim acta a mol biomol spectrosc. 114: 350-356. doi: 10.1016/ j.saa.2013.05.053. http://dx.doi.org/10.1016/ j.saa.2013.05.053. 1103.cdr 0.3 0.25 0.2 0.15 0.1 0.05 0 4 3.5 3 2.5 2 1.5 1 0.5 0 w e ig h t ga in ( g) sp e ci fi c gr o w th r at e ( % ) treatment treatment t1 t1 t2 t2 control c a * a * a * a * a * a * 0.014 0.012 0.01 0.008 0.006 0.004 0.002 0 a v e ra g e d a il y g ro w th r a te ( g /d a y ) treatment p1 p2 k a * a * a * page 1 page 2 page 3 page 4 page 5 page 6 page 7 the southeast asian journal of tropical biology vol. 32 no. 1, 2025: 254 265 doi: 10.11598/btb.2025.32.1.2496 issn: 0215-6334 | e-issn: 1907-770x 254 abstract calamus javensis is an endemic rattan plant found in west java. however, information on how climate change can shape c. javensis distributions and how c. javensis can cope with climate change conditions is still lacking. this information is required considering that c. javensis is an endemic plant in west java. then, the purpose of this study is to model the climate change impact on future c. javensis distributions. the study used species distribution modeling (sdm) covering nine locations across west java with rcp 8.5 representing the most pessimistic climate change scenario for the years 2050 and 2070. the results indicated that under the scenarios of climate change, the only suitable habitats for c. javensis are expected to be concentrated in western parts of west java. over time, as climate conditions change, the areas deemed very highly suitable for c. javensis are projected to decline. specifically, from 2050 to 2070, suitable habitats for c. javensis are expected to decline by approximately 46.34%, decreasing from 1,025 km² to 550 km². the decline of suitable areas will affect both organisms and local communities that depend on rattan. in the future, it is recommended to focus conservation efforts on the western parts of west java, as indicated by the model showing these areas as suitable amid climate change. keywords: climate change, habitat, rattan, rcp, west java predicting the future distributions of endemic rattan calamus javensis blume under climate change scenarios cmip 5 rcp 8.5 in 2050 and 2070 in west java andri wibowo* and suyud warno utomo school of environmental science, universitas indonesia, jakarta 104301, indonesia article information received : 19 march 2025 revised : 14 july 2025 accepted : 22 july 2025 *corresponding author, e-mail: awbio2021d@gmail.com reviewers: gs anis amaludin & anonymous reviewer highlights • information on how climate change can shape calamus javensis distributions is still lacking • the study used sdm with rcp 8.5 scenario for the years 2050 and 2070 • suitable habitats for c. javensis are expected to decline by approximately 46.34% • suitable habitats for c. javensis are expected to be concentrated in western parts of west java predicting the future distributions of endemic rattan wibowo & utomo 255 introduction in asia, rattans are the main lianas that live in and spread throughout tropical rainforests. in temperate climates and other tropical and subtropical locations, rattans do not grow natively. one of the most significant non-wood forest products used in asia as a substitute for timber is rattan, which is classified under the arecaceae/ palmae family and belongs to the calamoideae subfamily. climbing plants in the arecaceae family of palms are commonly referred to as rattan. rattan is crucial to the community's ability to sustain its livelihood, particularly for the local population that depends on and uses forest resources. in indonesia, the harvests of rattan are 485.92 tonnes per year with the value of idr 1,200 per ton (rinekso et al. 2019). there are currently over 600 rattan species in the world, divided into 13 genera, and almost all of them are utilized by the local population, albeit only approximately 50 of them are valuable commercially (meitram & sharma 2005). rattan inhabits a wide variety of habitats, including peat swamps, mixed deciduous forests, evergreen and dry evergreen forests, and areas up to 1,000 meters measured from the sea surface (rozali et al. 2014). rattans play a vital part in the physiognomy of the tropical rainforest in indonesia, but the country's most valuable commercial rattan species are already vulnerable because of climate change. according to zhang et al. (2024), high climate warming, equal to 3 4.5 °c, can lead to mass death of these plant groups. the most varied genus in the arecaceae family and among all climbing plant genera is calamus. the old world characterized by humid tropics, which include africa, much of asia, australasia, and portions of the pacific including fiji, are habitat for calamus. in southeast asian densecanopy forests, where their dominance is a notable feature of asian liana ecosystems, the genus calamus achieves its highest level of diversity. an indonesian rattan species that is indigenous to west java is calamus javensis blume (arecaceae: calamoideae) (mogea 2004). in actuality, nine of the thirteen genera in the globe are found in indonesia (guzman 2015). according to the numbers of species of each genus, the orders are 15 species for calamus, 4 species for daemonorops, 2 species for korthalsia and ceratolobus, 1 species for plectocomia, followed by retispatha, plectocomiopsis, pogonotium, and myrialepis are the nine genera in discussion. daemonorops and calamus were two of those genera that were known to have commercial value. locally, c. javensis was referred to as rotan lilin by the native populations that used it in its endemic regions. large-scale species distribution patterns, mainly those classified as plants and medicinal herbs, have recently been affected by climate change and levels of greenhouse gas (zhang et al. 2024). the intergovernmental panel on climate change (ipcc) developed a series of climatic scenarios known as the representative concentration pathways (rcp) following these levels of greenhouse gas. rcp 8.5 is characterized by high fossil fuel consumption, a significant increase in methane emissions, and a slow rate of technological development to mitigate the climate change effects, with no plans to reduce emissions. therefore, the climate scenario under rcp 8.5 pathway is deemed suitable for modeling the impact of climate change on species distribution, according to doulabian et al. (2021). the current study has emphasized the importance of species dispersion in modeling. as a result, numerous methods have been established to analyze the distribution of species on a geographic scale. one widely used technique is machine learning-based species distribution modeling (sdm), which estimates the potential spatial distribution of various organisms, including crops (dong et al. 2023), vegetation (sánchez pérez et al. 2023), animals (stephenson et al. 2022), and ticks. sdms employ a variety of methods to assess habitat suitability, including biomapper, bioclim, and domain using a climatic approach, and maxent (maximum entropy) using a machine learning approach. statistical approaches are represented by generalized additive models (gam) and generalized linear models (glm). each machine learning tool has its own unique set of advantages and disadvantages. following marcer et al. (2013), species distribution modeling (sdm) is one of the most popular and effective methods to model habitat suitability. some of the advantages of sdm, as noted by fois et al. (2018), include the capacity to determine the environmental factors having significant contributions, high precision of predictive results, reproducibility, the requirement for only species presence data, and its effectiveness in estimating the potential spatial distribution even with limited data. c. javensis is an endemic plant in west java. in addition, c. javensis plays a significant role in supporting local communities and forest biotropia vol. 32 no. 2, 2025 256 conservation by supplying nontimber products (rahim & idrus 2019). while the information on how this species can cope with climate change and how climate change will limit the distribution of certain species is still lacking, this information is required in light of that c. javensis is an endemic plant in west java. then, this research sought to simulate the effects of climate change on future c. javensis distributions. the novelty of this study is using sdm and rcp 8.5 to simulate the effects of the most pessimistic climate change scenario. materials and methods study area the selected area of this study is in west java indonesia. west java is divided into 18 districts and 9 cities (fig. 1). climate in west java is varied following landscapes. the north and south of west java were characterized by low land and coastal areas. while the central of west java is characterized by mountainous areas. the lowest temperature is 9 °c and can reach 34 °c in the coastal regions. in the mountainous areas of west java, the annual rainfall can range from 3,000 mm to 5,000 mm. survey on calamus javensis survey activities were conducted over two months in 2024 at nine sampling locations throughout west java (fig. 1). the sampling sites were chosen based on the presence of natural habitats suitable for c. javensis. to document the presence of c. javensis, we utilized direct visual observation, known as the visual encounter survey (ves), along with a database assembled from literature reviews, including scientific articles and official reports published by government organizations, such as the indonesian ministry of environment and forestry's agency for agriculture and forestry. ves was employed purposefully by surveying natural habitats including forests and plantations where c. javensis may grow. the surveys were established using ten plots randomly placed in each sampling location throughout west java. each plot measured 20 × 20 m, and it was divided into several square subplots. for detailed observation and recording of c. javensis, 20 × 20 m plots for adult rattan level, 10 × 10 m for pole level, 5 × 5 m for sapling level, and 2 × 2 m for rattan seedling level (joshi et al. 2017). global positioning system (gps) of garmin etrex 30 was utilized to retrieve the geocoordinate locations of c. javensis presences in the field. this information was saved in csv format and then imported into microsoft excel for use in species distribution modeling (sdm) habitat suitability analysis. because presence-only data is widely available, a presence-only sdm was chosen rather than a generalized linear model (glm). these methods are relatively easy to implement, requiring only the locations where a species has been observed, and can be useful when absence data is limited or unreliable (leroy 2022). according to jasni et al. (2007), the identification of c. javensis was based on specific identification keys using c. javensis diagnostic features, voucher specimens, and rattan taxonomist confirmation. figure 1 nine sampling locations in west java predicting the future distributions of endemic rattan wibowo & utomo 257 table 1 bioclimatic variables employed in this study note: * = some variables were selected based on multicollinearity tests including bio 1, 2, 3, 4, 7, 11, 12, 13, 14, 16, 17, 18, and 19. biotropia vol. 32 no. 2, 2025 258 calamus javensis bioclimatic variables this study examined a range of bioclimatic factors in line with the methods described by dong et al. (2023) and arshad et al. (2022) (table 1). particularly in asia, as highlighted by khanum et al. (2013) and rana et al. (2017), bioclimatic variables (bio 1 bio 19) from the global climate database worldclim (www.worldclim.org, version 2.0) (hijmans et al. 2005) have been widely utilized in modeling the habitat appropriateness at current time. the locality data of west java was obtained from the worldclim database by setting the region of interest. the selection and analysis of environmental factors that significantly impacted the results led to the inclusion of specific bioclimatic variables, aimed at producing an informative and precise model of habitat suitability. a jackknife analysis was conducted to assess each bioclimatic variable's contribution to the finished model. several environmental factors identified in the jackknife analysis were excluded from the model because they had no effect (0% contribution). wei et al. (2018) noted that some bioclimatic variables exhibited minimal permutation relevance (less than 6%) or a low average contribution (also less than 6%). therefore, two critical components for understanding and measuring the importance of bioclimatic variables used in developing the model of species distribution (sdm) are the percentage of contribution and permutation relevance. calamus javensis suitability analysis this study utilized species distribution modeling using machine learning approach (sdm) techniques within r application version 3.6.3 to generate estimated suitable maps for c. javensis in west java and its surrounding areas (mao et al. 2013). the maps of suitability were created using various r packages, including sp, dismo (khan et al. 2022), map tools, rgdal (bivand 2022), and raster (lemenkova 2020). nineteen environmental variables (bio 1 bio 19) served as inputs for the sdm. the area under the receiver operating characteristic curve (auc) was employed to evaluate the model's performance. additionally, the jackknife test was conducted to determine each environmental variable's contribution and impact on the habitat suitability model for c. javensis (promnikorn et al. 2019). the auc values below 0.5 indicate that the model has performance no better than random or contains vague data, while values above 0.5 suggest that the model is figure 2 calamus javensis occurrences related to elevation in west java predicting the future distributions of endemic rattan wibowo & utomo 259 highly effective and precise. the auc value has a range of 0 1, with 0 representing the lowest appropriateness and 1 the highest. geographic information systems (gis) were utilized to visualize and analyze the maps of prediction generated by the species distribution models (sdm) (hijmans et al. 2005). based on wei et al. (2018), five levels of habitat suitability can be identified on the sdm model map: 0 signifies unsuited, 1 indicates the suitability is low, 2 denotes the suitability is moderate, 3 represents the suitability is high, and 4 indicates the suitability is very high. cmip 5 future scenario this investigation utilized two scenarios. the first scenario represents the current state in 2023, while the second scenario projects future conditions based on the rcp 8.5 projections from the fifth coupled model intercomparison project (cmip) for the years 2050 and 2070. the future scenario is based on the intergovernmental panel on climate change's fifth assessment report (ar5) from 2008, which incorporates downscaled data from global climate models in cmip5. the ipcc's 2014 ar5 report adopted several representative concentration pathways (rcps) for cmip5, focusing on variations in levels of greenhouse gas instead of levels of emissions. this new method has replaced the previous projections from the special report on emissions scenarios (sres), published in 2000 (van vuuren et al. 2009). based on the expected release of greenhouse gases shortly, these pathways outline four credible future climate scenarios for modeling and research. according to weyant et al. (2009), the four representative concentration pathways (rcps), rcp2.6, rcp4.5, rcp6.0, and rcp8.5, are named according to their projected ranges of radiative forcing values in 2100 relative to preindustrial levels (+2.6, +4.5, +6.0, and +8.5 w/ m², respectively). to forecast the suitable habitat distributions of c. javensis for the years 2050 and 2070, this study employed the rcp8.5 models, which correspond to different climate change scenarios. results and discussion occurrences and bioclimatic variables of calamus javensis our study showed a total of nine current occurrences of c. javensis across west java (fig. 2). at the current time, 33.33% of c. javensis occurrences were observed in highland landscapes. those highland landscapes have elevations with a range of 1,619 3,078 m above sea level and are located in garut and between sukabumi and cianjur. while 33.33% of c. javensis occurrences were observed in lowland landscapes at elevations having a range of 0 798 m above sea level. then, in general, the preferred current habitats of c. javensis were dominated by highlands with an elevation range of 798 3,078 m above sea level. according to regions, c. javensis was very common in bogor, sukabumi and cianjur in comparison to other regions. the response curves of calamus javensis associated with the chosen bioclimatic variables in west java is presented in figure 3, while figure 4 shows numerous bioclimatic variables that have figure 3 response curves of calamus javensis associated with bioclimatic variables in west java biotropia vol. 32 no. 2, 2025 260 more contributions to the distributions of c. javensis in west java. those bioclimatic variables were bio 11, 18, 2, and 16. bio 11 which is the coldest quarter average temperature variable indicated that the c. javensis prefers habitats having temperature range from 12.5 oc to 24.0 oc (fig. 3). in regard to precipitation, bio 18 variable indicated that c. javensis prefers habitats which have precipitation of the warmest quarter from 70 mm to 105 mm. meanwhile, bio 2 variable indicated that the preferred temperature mean range is 9.0 11.5 °c. this narrow range of bio 2 value is related to the endemism of c. javensis, which can only inhabit limited areas. the bio 16 variable confirms that the preferred precipitation of the wettest quarter is from 100 mm to 120 mm. high rainfall preferences during the peak of rainy season indicates that the distribution of c. javensis is limited to the mountainous areas where heavy rainfall is common. in our study, current distributions of c. javensis were widespread, from highlands in mountainous areas to the lowlands around bogor. this is in agreement with the findings reported in the previous studies. in cameroon, calamus resides in elevation with a range of 400 1,000 m. in malaysia, rattan species were recorded ending from an elevation of less than 300 m to 600 m. the rattan species reach high abundance at an elevation of 600 m. in central kalimantan, c. javensis was observed residing in the lowlands in the peatland areas (fambayun et al. 2022). while c. javensis was known to be common in highlands, as reported on mount halimun, west java (watanabe et al. 2006), kalima (2015) has reported that c. javensis can inhabit areas with an elevation as low as 2 m and as high as 1,200 m above sea level. in west java, it is widely distributed in the high-elevation areas, including halimun-salak and gede-pangrango areas. besides, c. javensis is also recorded in the lowland parks, including in ujung kulon. this documented distribution explained the current presence of c. javensis in sukabumi and cianjur as parts of the halimun-salak and gede-pangrango areas. the wide current distribution of c. javensis was related to the seed dispersion that was facilitated by birds and primates allowing wider distributions (kalima 2022). current and future distributions of calamus javensis the model of calamus javensis current distributions in west java is presented in figure 5. based on the estimations, c. javensis was common in the southern regions of west java, covering cianjur, sukabumi, garut, bandung, and bogor districts. areas categorized as having very high potential suitability were mostly recorded in cianjur. meanwhile, areas classified as high suitability were mostly recorded in sukabumi and bandung and small parts in the bogor bordering sukabumi and cianjur. figure 4 percentage contribution of calamus javensis related to bioclimatic variables in west java predicting the future distributions of endemic rattan wibowo & utomo 261 figure 5 model of calamus javensis current distributions in west java notes: suitability level 0 = unsuitable; 1 = low suitability; 2 = medium suitability; 3 = high suitability; 4 = very high suitability. figure 6 model of calamus javensis distributions in 2050 (a) and 2070 (b) related to rcp 8.5 scenario in west java notes: suitability level 0 = unsuitable; 1 = low suitability; 2 = medium suitability; 3 = high suitability; 4 = very high suitability. biotropia vol. 32 no. 2, 2025 262 the models of calamus javensis distributions in west java under rcp 8.5 climate change scenarios for years 2050 and 2070 are shown in figure 6. based on the estimations, it is observed that climate change has impacted the c. javensis distributions. according to the model, climate change has made suitable habitats for c. javensis in cianjur, garut, and bandung disappear. the only remaining suitable habitats would probably be in sukabumi. within 20 years, between the year of 2050 and 2070, it is clear that the remaining suitable habitats for c. javensis classified as having very high suitability will be reduced by 46.34% from 1,025 to only 550 km2 in 2070. also, in 2070, it is predicted that c. javensis would possibly reside in southern parts of sukabumi. this area is considered a novel area that has never been existed under the current c. javensis distributions. the novelty of these newly predicted areas is that the southern sukabumi is a lowland and coastal area. although the current distributions of c. javensis are mostly in highland areas, there is a possibility that this species would occupy coastal areas with low elevation. in south kalimantan, arifin (2008) observed that c. javensis was common in lowland forests and this supports the possibility of c. javensis to inhabit low coastal areas. in malaysia, rattans were observed to reach high abundance in the offshore areas. this study is the first to explore the potential dispersal of c. javensis in specific regions of southeast asia. it is comparable to similar habitat suitability studies implemented in other areas, such as assam (mehmud et al. 2022) and the western ghats in india (sreekumar & sasi 2019). the findings indicate that temperature and rainfall during the warmest quarter are significant bioclimatic variables affecting the distribution of this species. this aligns with previous research (table 2). sreekumar and sasi (2019) noted that the dispersal of calamus species is primarily affected by several factors, such as latitude, the driest quarter precipitation, total annual rainfall, and the coldest month minimum temperature. overall, climate plays a more critical role than habitat or human effect in shaping the potential distributions of calamus species. most rattan species tend to prefer humid climates, making rainfall a key factor influencing the distribution at the landscape scale of these potential calamus species. this study confirmed that the climate change presented in the form of bioclimatic variables change the shape and alter the potential distribution area of the calamus. combined with the changes in climatic key variables, ranging from temperature to rainfall and seasonality, ongoing increments and changes in atmospheric greenhouse gases, mostly carbon dioxide (co₂), influence and affect the growth and physiology of plants, including calamus. mainly in the tropics, increasing levels of co2 have the potential to increase the growing season leaf area. in addition, elevated co2 levels are associated with increased water use efficiency, increased photosynthesis rate, and increased root and stem growth of calamus. it seems likely that elevated co2 have a positive effect on the growth, development, and physiology of calamus. this explained the presence of calamus in the area that had never been recorded before, as reported in this study. while not all palm species benefit from the climate change effects, buttler and larson (2019) recorded that only one out of four species of palms are benefitted due to climate change. others are remained stable and other are slightly increase. this condition is relevant to the condition and explains that the calamus in this study has various reactions to the climate change effects. table 2 comparisons of bioclimatic variables with other studies on calamus predicting the future distributions of endemic rattan wibowo & utomo 263 calamus is a plant that belongs to the arecaceae family. the climate change influence on the dispersal and presence of species in the arecaceae group, or palms, has been validated by earlier reports. palms have been used as markers for the previous paleoclimate linked to the megathermal because of their sensitivity (buttler & larson 2019). for palm species, temperature combined with palm dispersal capacity regulates palm presence. since the beginning of the quaternary period in the western hemisphere, palms have been observed to exist and disappear, which also contributes to extinction. it is thought that the existence of climate oscillations is what caused these historical occurrences, which occurred about 2.6 million years ago. important details regarding calamus management, cultivation, and conservation, primarily in west java, are provided by the anticipated appropriate habitat for calamus in the future. commercial calamus cultivations can be established and applied using the results. it has been noted that the calamus continues to live in its current habitat and maintain its natural range. calamus is spreading to the new habitat areas at the same time. this study suggests creating and maintaining natural corridors in conjunction with large-scale forestry-protected areas. in addition to this strategy, the establishment of germplasm banks is advised in order to manage and preserve these significant endemic rattan species mainly in west java regions. this study has succeeded in using 13 bioclimatic variables as the determinant factors that have effects on the dispersals of this certain species. in the future, the approach to the appropriate distribution of rattan is recommended to be developed by incorporating more determinant variables, including environmental variables and rattan seed dispersal facilitated by winds, birds and primates. those variables are recommended to be incorporated with the edaphic, topographic, and even anthropogenic variables. conclusion at the 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july 2021/accepted 12 november 2021 abstract pathogenic bacterial contamination was a serious matter due to its capability in reducing food quality and health. this study aimed to select various types of algae in luk coast, sumbawa regency that have the potential to produce antibacterial compounds for natural food preservatives. algae on luk coast was identified by means of morphological characters, followed by sample preparation and extraction of secondary metabolites (bioactive compounds). algae extracts were used in antibacterial tests against food spoilage bacteria, such as escherecia coli, staphylococcus aureus, salmonella thypi, enterobacter cloacae and pantoea agglomerans. five types of algae identified were padina sp., halimeda opuntia, sargassum horneri, sargassum crassifolium and galaxaura rugose. the five algae have the growth-inhibiting ability toward the tested bacteria. the highest inhibition zone was obtained from the 100% algae extract concentration. keywords: antimicrobial, natural preservative, secondary metabolite, sumbawa algae introduction food spoilage and foodborne infection increasingly become issues in the food industry, due to their serious impact on food quality and safety (vanegas et al. 2017). food spoilage is caused by pathogenic microorganisms. several types of food-spoiling microorganisms include aerobic psychrotrophic gram-negative bacteria, yeasts, molds, heterofermentative lactobacilli, and spore-forming bacteria (rawat 2015). food spoilage due to infection from foodborne microbes is mainly caused by campylobacter spp., salmonella, and eschercia coli (koluman & dikici 2013). contamination from these spoilage pathogenic bacteria, not only decreases food quality but also causes adverse health conditions. in order to inhibit the process of food deterioration, some people use synthetic preservatives such as formaldehyde, benzoic acid, bha (butilated hydroxyanisol), bht (butylated hidroxytoluene) and tbhq (tertier butylated hydroxyanisole), especially for food ingredients having high water content. however, the use of synthetic preservatives is not recommended by badan pengawas obat dan makanan (national agency of drug and food control/bpom). therefore, it is necessary to seek alternative food preservatives from natural ingredients. algae have biological properties that can be used as antibacterial (renhoran et al. 2017; basir et al. 2017). algae have the ability to produce secondary metabolites which are bioactive compounds to protect themselves from unsuitable environmental conditions and to defend themselves from the threat of various diseases and predators. bioactive compounds in algae can also act as antimicrobials, antifeedants, antihelmintic and cytotoxic agents (al-saif et al. 2014). *corresponding author, email: baso.manguntungi@uts.ac.id mscombalicer@up.edu.phveryl.hasan@fpk.unair.ac.id mailto:baso.manguntungi@uts.ac.id biotropia vol. 29 no. 2, 2022 96 several secondary metabolites produced by marine algae include alkaloids, polyketides, cyclic peptides, polysaccharides, phlorotannins, diterpenoids, sterols, quinones, lipids and glycerols (cabrita et al. 2010). al-saif et al. (2014) reported that groups of flavonoid compounds contained in marine algae include rutin, quercetin and kaempferol. these compounds can be used as natural food preservatives (hidayati et al. 2017). algae can be easily found in indonesia, such as on sumbawa island. sumbawa island is located in the nusa tenggara barat (ntb) province. this island has a 3,331.72 km2 water area, rich in various types of algae, including red algae (rhodophyta), brown algae (ocrophyta) and green algae (chlorophyta). cabrita et al. (2010) reported that laurencia is a group of red algae that produce secondary metabolites such as diterpenes, sesquiterpenes, triterpenes and c15-acetogenins. various kinds of compounds derived from phlorotannins (phloroglucinol, eckol, dieckol, etc.) that was beneficial for health were produced by brown algae such as ecklonia cava, ecklonia stolonifera, ecklonia kurome, eisenia bicyclis, ishige okamurae, sargassum thunbergii, hizikia fusiformis, undaria pinnatifida and laminaria japonica (li et al. 2011). bhagavaty et al. (2011) reported that the bioactive compounds of green algae chloroccocum humicola such as lipophilic and phenolic have antimicrobial properties against bacillarius subtilis, staphylococcus aureus, eschericia coli, pseudomonas aeruginosa, salmonella thypimurium, klebsiella pneumoniae, vibrio cholerae. the secondary metabolites of algae might be used in inhibiting the activity of food-spoiling bacteria. however, there have been no studies examining the antimicrobial potentials of algae originating from sumbawa island. this study aimed to conduct a study of antimicrobial compounds extracted from several types of algae originating from sumbawa island, against some food-spoiling bacteria such as salmonella thypi, staphylococcus aureus, escherichia coli, enterobacter cloacae and pantoea agglomerans. materials and methods sampling method samples were taken in the luk coast, sumbawa region. the samples were classified as red algae, green algae and brown algae. the samples were thoroughly cleaned from any debris and substrates by running the samples under tap water. algae identification identification was conducted based on morphological observations and practical guidelines for marine algae identification. samples extraction the collected samples were crushed using a pestle and mortar before being extracted. extraction was carried out using the soxhletation method. this method was carried out to separate bioactive compounds from the samples. the extraction was conducted by using the 1:15 ratio of the sample powder and methanol. the reflux process was carried out for 15 h at 78 oc (methanol's boiling point temperature). filtrates of the reflux process had the basic colors of the algae (red, green and brown). the 150 ml solvents were then separated from the extract by distilling the solvent directly using soxhlet. the purified solvents were subsequently poured into the bottles, followed by repeat heating until there is a sample substance in the flask (fabrowska et al. 2017; liswandari et al. 2018). rejuvenation and manufacture of test bacterial suspensions salmonella thypi, escherechia coli, enterobacter cloacae, pantoea agglomerans and staphylococcus aureus bacteria from pure cultures were taken and inoculated on slanted nutrient agar (na) medium. bacterial cultures were incubated at 37 oc for 24 h. the test bacteria having been rejuvenated for 24 h were taken and suspended into nb media (nutrient broth) then homogenated. liquid cultures were incubated at 37 oc for 24 h. the turbidity of the media indicated the multiplication of bacterial cells. potentials of algae as natural preservative for food – baso manguntungi et al. 97 algae extract preparation in various concentrations the crude extracts were poured into dilution bottles of 200 µl, 400 µl, 600 µl, 800 µl and 1000 µl, respectively, and subsequently added with distilled water until each solution amounted to 1 ml. so, the extracts obtained had respective concentrations of 20%, 40%, 60%, 80% and 100%. the five extract concentrations in the bottles were homogenized. antimicrobial test the antibacterial activity test was carried out in-vitro using the antibacterial sensitivity test with agar diffusion method using disc papers. nine disc papers were immersed in respective crude extract solution and control solution for 30 min. as much as 100 µl of tested bacteria having been cultured for 24 h were leveled using triangular rods on the na medium. the disc papers having been soaked in the crude extract and control solutions were affixed to the surface of the media. the disc papers were arranged as not too close to each other so that the inhibition zones did not intersect with each other. each treatment was conducted with three replications. the petri dishes were then incubated at 37 oc. the inhibition zones were observed at the 16th hour. the diameter of the inhibition zone was determined by subtracting the overall diameter (disc paper + inhibition zone) from the diameter of disc paper (6 mm) and diameter of the inhibition zone of solvent (if the solvent has an inhibition zone) (al-saif et al. 2014; bhagavathy et al. 2011). data analysis the study was carried out using the completely randomized design (crd) method with three replications. data were analyzed using anova (analysis of variance) with a confidence level of 95% by using spss 24.0. results and discussion algae identification there were 5 algae species identified in the luk coast, sumbawa regency. the five algae species were derived from the groups consisting of red algae, brown algae and green algae. identification was carried out based on the morphological characters of each alga (table 1), showing three species of brown algae and one species of red algae and green algae, respectively. one identified type of brown algae was padina sp. this species is also found in several indonesian waters such as in the makassar strait (hamrun et al. 2019), parigi moutong coast, padang (dharmayanti et al. 2019) and belitung lengkuas coast (jannah et al. 2021). according to aulia et al. (2021), padina sp., is fan-shaped with a diameter of 3-4 cm, yellowish-brown or whitish due to calcification, consisting of flexible rhizoids for attaching themselves to a surface. the tip of the blade is thin-widens and forms a lobe. the lobe is segmented and has white circular stripes, which were not visible, so the talus structure consists only of a blade and a holdfast. the holdfast has the slabs form. padina sp. grows on sandstone substrates. besides padina sp. other types of brown algae found were sargassum horneri and sargassum crassifolium. these two types of algae have similar physical characteristics. sargassum crassifolium has a cylindrical or flattened brown thallus with a lush and crossing branch, similar to terrestrial plants. the leaves’ shape is broad, oval, or sword-like. on the edges of the jagged leaves, there are some bubbles called vesicles. the function of these air bubbles is to keep the leaves on the surface of the water. the branching alternates regularly. the leaf has an oval and elongated shape with a thallus length of 13.5 14 cm. the thallus grows closely resembling a stem. the leaves are sparse and table 1 classification of the five algae species in the luk coast algae classification no. class ordo familia genus spesies 1 ocrophyta dyctyotales dyoctaceae padina padina sp. 2 chlorophyta bryopsidales halimedaceae halimeda halimeda opuntia 3 ocrophyta fucales sargassaceae sargassum sargassum horneri 4 ocrophyta fucales sargassaceae sargassum sargassum crassifolium 5 rhodophyta nemaliales galaxauraceae galaxaura galaxaura rugose biotropia vol. 29 no. 2, 2022 98 wavy. the tips are curved or tapered. this type of algae is able to grow on coral substrates in choppy areas (ode 2013). meanwhile, sargassum horneri has a branched and wide thallus, and lush branching. on the leaves, there were serrations and bubbles. sargassum horneri has a slightly flat and smooth thallus, but the main stem is rounded and slightly rough. the length of the pinnatus alternates is 30-50 cm. according to gazali et al. (2018) sargassum sp. contains bioactive compounds such as alkaloids, phenols and triterpenoids. sargassum sp. also contains bioactive compounds such as saponins, tannins, flavonoids, and phenols (pangestuti et al. 2019). these compounds are able to inhibit the growth of s. aureus and e. coli. green algae, namely halimeda opuntia, was also found on luk coast. halimeda opuntia grows on sandy and coral substrates in intertidal to subtidal areas (kepel et al. 2018). halimeda opuntia has the characteristics of a green thallus and is very stiff because it is formed from filaments of branched shiphonus; segmented by tricotome branching; segments form triangles, and segments appear in basal segments. the adhesive device is a filament that comes out of the basal segment and grips the substrate. segments are calcareous, very rigid, and have a three-indented shape. the arrangements are overlapping, irregular and not located in one branching, so the thallus is not located on one side. halimeda opuntia produces several types of bioactive compounds such as alkaloids, flavonoids and triterpenoids (hudaifah et al. 2020). the last type of algae found on luk coast was galaxaura rugose, which belongs to the red algae group. the algae are beneficial because it contains compounds such as sterols, triterpenoids, flavonoids, tannins and coumarins (al-enazi et al. 2018). galaxaura rugose has a thick thallus, stiff, dark red-brown color. branches are cylindrical and have a holdfast to attach to coral reefs. this species is short clumps and reaches 5-7 cm in height. galaxaura rugose grows on every coral reef. antimicrobial activity of algae extracts an antimicrobial activity test is a method to determine the ability of a substance to inhibit bacterial growth. this test was carried out on five types of pathogenic bacteria causing food spoilage, such as salmonella thypi, staphylococcus aureus, escherichia coli, enterobacter cloacae and pantoea agglomerans. the test was carried out on each identified algae extract. the test results indicated that padina sp. has growth-inhibiting ability against all tested pathogenic bacteria (table 2). the antimicrobial activities of padina sp. against p. agglomerans, s. aureus and s. thypi showed significantly different values at each treatment concentration with the highest result obtained by the 100% extract concentration of padina sp. in testing against e. coli, the 60% and 80% concentrations were not significantly different. the highest inhibiting ability was also shown by the 100% extract concentration. the testing against e. cloacae showed that the diameter of the inhibition zone for the 20% and 40% concentrations were not significantly different and the highest inhibiting ability was shown by the 100% extract concentration. the antimicrobial activity of halimeda opuntia extract also showed growth inhibiting ability against all tested bacteria (table 3). the formed inhibition zone showed significantly different results for each treatment concentration against all tested bacteria. the highest inhibition zone for all tested bacteria was obtained by the 100% extract concentration. potentials of algae as natural preservative for food – baso manguntungi et al. 99 table 2 results of testing the antimicrobial activity of padina sp. extract concentration of each treatment (%) bacterial inhibition zone diameter (mm) p. agglomerans s. aureus e. coli e. cloacae s. thypi 20 11.67±0.58 b 9.33±0.58 b 12.33±0.00 b 12.67±0.00 b 13.00±0.00 b 40 15.67±0.58 c 12.00±1.00 c 13.67±0.58 c 13.67±0.58 b 14.67±0.58 c 60 19.33±0.58 d 17.33±0.58 d 16.67±0.58 d 16.33±1.15 c 17.33±1.15 d 80 22.67±0.58 e 18.67±0.58 e 17.67±0.58 d 20.00±0.58 d 20.33±0.58 e 100 25.00±1.00 f 20.33±0.58 f 22.33±0.58 e 22.67±1.15 e 22.67±1.15 f k+ 29.00±1.00 g 28.33±0.58 g 28.67±0.58 f 28.67±0.58 f 28.67±0.58 g k0±0 a 0±0 a 0±0 a 0±0 a 0±0 a *notes: * = numbers followed by the same letter at the same column indicate no significant difference based on the duncan test at the level α = 0.05 and n = 3; **k+ = positive control; k-= negative control. table 3 results of testing the antimicrobial activity of halimeda opuntia extract concentration of each treatment (%) bacterial inhibition zone diameter (mm) p. agglomerans s. aureus e. coli e. cloacae s. thypi 20 8.33± 0.58 b 8.67±0.58 b 8.67±0.58 b 10.33±0.58 b 10.67±0.58 b 40 11.33±0.58 c 13.67±0.58 c 14.67±0.58 c 14.33±0.58 c 14.67±0.58 c 60 13.00±0.00 d 16.67±0.58 d 18.00±0.00 d 14.67±0.58 c 15.67±0.58 d 80 16.00±0.00 e 18.00±1.00 e 19.33±0.58 e 16.33±0.58 d 18.33±0.58 e 100 21.00±1.00 f 22.33±0.58 f 22.67±0.58 f 20.33±0.58 e 21.67±0.58 f k+ 28.00±0.00 g 28.33±0.58 g 28.33±0.58 g 28.67±0.58 f 29.00±0.00 g k0±0 a 0±0 a 0±0 a 0±0 a 0±0 a notes: * = numbers followed by the same letter at the same column indicate no significant difference based on the duncan test at the level α = 0.05 and n = 3; **k+ = positive control; k-= negative control. table 4 results of testing the antimicrobial activity of sargassum horneri extract concentration of each treatment (%) bacterial inhibition zone diameter (mm) p. agglomerans s. aureus e. coli e. cloacae s. thypi 20 8.00±0.58b 10.67±0.58b 11.67±0.58b 8.33±0.58b 7.33±0.58b 40 10.75±0.58c 14.33±0.58c 15.33±0.58c 13.00±1.00c 13.33±0.58c 60 12.50±1.15d 16.00±1.00d 17.00±1.00c 16.33±0.58d 16.33±0.58d 80 17.75±0.58e 23.67±0.58e 23.00±1.00d 18.33±0.58e 17.67±0.58e 100 19.00±0.58f 25.67±0.58f 26.00±1.00e 23.67±0.58f 22.67±0. 58f k+ 21.25±0.58g 28.33±0.58g 28.67±0.58e 28.67±0.58g 28.33±0 58g k0±0a 0±0a 0±0a 0±0a 0±0a notes: * = numbers followed by the same letter at the same column indicate no significant difference based on the duncan test at the level α = 0.05 and n = 3; **k+ = positive control; k-= negative control. the antimicrobial activity of sargassum horneri extract against p. agglomerans, s. aureus, e. cloacae and s. thypi showed significantly different results at all concentrations (table 4). slightly different results were shown in observations against e. coli. the diameter of the inhibition zone showed no significant difference at concentrations 40% and 60%. the 100% extract concentration of sargassum horneri has the same effectiveness with positive control in inhibiting the activity of e. coli. the sargassum crassifolium extract showed growth inhibiting ability against all tested bacteria (table 5). the diameter of the inhibition zone formed in the four tested bacteria, i.e., s. aureus, e. coli, e. cloacae, and s. thypi showed significantly different results in biotropia vol. 29 no. 2, 2022 100 table 5 results of testing the antimicrobial activity from sargassum crassifolium extract concentration of each treatment (%) bacterial inhibition zone diameter (mm) p. agglomerans s. aureus e. coli e. cloacae s. thypi 20 10.67±0.58b 7.67±0.58b 10.33±0.58b 9.00±1.00b 8.67±0.58b 40 13.67±0.58c 12.00±0.00c 12.00±0.00c 12.67±0.58c 13.33±0.58c 60 18.33±0.58d 14.67±0.58d 14.00±1.00d 16.00±1.00d 16.67±0.58d 80 24.00±0.00e 17.67±0.58e 18.00±0.00e 19.00±0.00e 21.67±0.58e 100 27.00±1.00f 22.33±0.58f 22.33±0.58f 23.67±0.58f 23.67±0.58f k+ 28.67±0.58f 28.33±0.58g 29.00±0.00g 29.00±0.00g 28.33±0.58g k0±0a 0±0a 0±0a 0±0a 0±0a notes: * = numbers followed by the same letter at the same column indicate no significant difference based on the duncan test at the level α = 0.05 and n = 3; **k+ = positive control; k-= negative control. each extract concentration with the highest inhibition zone obtained by the 100% extract concentration of sargassum crassifolium. however, there was a slight difference in observations against p. agglomerans test bacteria, showing that the 100% extract concentration was not significantly different from the control treatment. it means that the 100% concentration of sargassum crassifolium extract has the same inhibitory effect against the p. agglomerans test bacteria with the control. the antimicrobial activity test using galaxaura rugose extract against e. coli, e. cloacae, and s. thypi showed significantly different results among extract concentrations with the highest inhibiting ability obtained by 100% galaxaura rugose extract (table 6). the 40% and 60% extract concentrations against the p. agglomerans and s. aureus test bacteria showed a nonsignificant difference effect, with the highest inhibiting ability shown by the 100% extract concentration. the five algae extracts showed the ability to inhibit the growth of all tested bacteria. the largest inhibition zone formed was shown by the 100% extract concentration with an inhibition zone diameter range of 19.00-27.00 mm. according to davis and stout (1971), the bacterial inhibition zone less than 5 mm was categorized as weak, the 5 10 mm zone of inhibition was categorized as moderate, the 10 20 mm zone of inhibition was categorized as strong and more than 20 mm was categorized as very strong. it means that the 100% extract concentration of the five algae was in the very strong category. table 6 results of testing the antimicrobial activity from galaxaura rugose extract concentration of each treatment (%) bacterial inhibition zone diameter (mm) p. agglomerans s. aureus e. coli e. cloacae s. thypi 20 10.67±0.58 b 10.33±0.58 b 8.00±1.00 b 11.67±0.58 b 11.67±0.58 b 40 12.00±0.00 c 13.00±0.00 c 9.67±1.53 c 14.67±0.58 c 14.67±0.58 c 60 12.67±0.58 c 13.67±1.15 c 13.33±0.58 d 17.00±0.00 d 18.00±0.00 d 80 15.33±0.58 d 16.33±0.58 d 15.00±0.00 e 19.33±0.58 e 20.67±0.58 e 100 19.67±0.58 e 20.33±0.58 e 20.00±0.00 f 23.33±0.58 f 24.33±0.58 f k+ 28.00±0.00 f 28.67±0.58 f 28.67±0.58 g 28.67±0.58 g 28.67±0.58 g k0±0 a 0±0 a 0±0 a 0±0 a 0±0 a notes: * = numbers followed by the same letter at the same column indicate no significant difference based on the duncan test at the level α = 0.05 and n = 3; **k+ = positive control; k-= negative control. potentials of algae as natural preservative for food – baso manguntungi et al. 101 the formation of the inhibition zone indicates that the algae extracts from padina sp., halimeda opuntia, sargassum horneri, sargassum crassifolium and galaxaura rugose have the potential as antibacterial agents. the five algae have capabilities to produce bioactive compounds such as steroids, terpenoids and eicosanoid acid. different phytochemical contents in padina sp., halimeda opuntia, sargassum horneri, sargassum crassifolium and galaxaura rugose determine the differences in inhibition abilities because each bioactive compound contained in algae extract has a different action mechanism against bacteria. according to dewi (2010), flavonoids are able to inhibit the growth of pathogenic bacteria by damaging the cell wall components so that the cell wall layer is not intact causing cell death. steroids can damage bacterial cell membranes by increasing cell permeability, resulting in cell leakage followed by the release of intracellular material (cowan 1999). alkaloid compounds can inhibit the growth of gram-positive and gram-negative bacteria, wherein alkaloids can cause cell lysis and changes in bacterial morphology (katou 2006). terpenoids have an antibacterial mechanism by reacting with porin (transmembrane protein) on the outer membrane of the bacterial cell wall, forming strong polymer bonds resulting in the breakdown of porin. the damage of porin, i.e., the entrance and exit for compounds, reduces the permeability of the bacterial cell wall resulting in the bacterial cell being deficient in nutrients so that bacterial growth is inhibited or dies (cowan 1999). the ability of the five algae extracts to inhibit the growth of pathogenic bacteria shows that the five algae have the potential to be developed and utilized as natural food preservatives. the bioactive compounds produced through the secondary metabolism of algae were proven by this study to have the ability for inhibiting the growth of food-spoiling pathogenic bacteria. the use of algae as natural food preservatives is expected to replace synthetic food preservatives. food preservatives derived from natural ingredients are relatively safe to consume and do not cause side effects with the same effectiveness as food preservatives as synthetic ones. conclusion the padina sp., halimeda opuntia, sargassum horneri, sargassum crassifolium and galaxaura rugose algae were able to inhibit the growth of pathogenic bacteria such as pantoea agglomerans, staphylococcus aureus, escherecia coli, enterobacter cloacae and salmonella thypi. antibacterial activities in the five types of algae have an inhibition zone ability ranging from 19.00 to 27.00 mm at 100% extract concentration with a category of very strong inhibition zone capability. this means that the five types of algae have the potential to be used as natural food preservatives. acknowledgments the authors thank the head of the biotechnology laboratory, universitas teknologi sumbawa. this research was funded by the 2021 research grant of universitas teknologi sumbawa. references al-enazi nm, awaad as, alqasoumi si, alwethairi mf. 2018. biological activities of the red algae galaxaura rugosa and liagora hawaiiana butters. saudi pharm j 26:25-32. al-saif ssa, abdel-raouf n, el-wazanani ha, aref ia. 2014. antibacterial substances from marine algae isolated from jeddah coast of red sea, saudi arabia. saudi j biol sci 21:57-64. aulia a, kurnia sk, mulyana d. 2021. identifikasi morfologi beberapa jenis anggota phaeophyta di pantai palem cibeureum, anyer, banten. 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2015. food spoilage: microorganisms and their prevention. asian j plant sci res 5:47-56. renhoran m, noviendri d, setyaningsih i, uju u. 2017. extraction and purification of fucoxanthin from sargassum sp. as anti-acne. jphpi 20:370-379. vanegas dc, gomes cl, cavallaro nd, escobar dg, mclamore es. 2017. emerging biorecognition and transduction schemes for rapid detection of pathogenic bacteria in food. compr rev food sci food saf 16:1188-12. the southeast asian journal of tropical biology vol. 32 no. 1, 2025: 100 110 doi: 10.11598/btb.2025.32.1.2433 issn: 0215-6334 | e-issn: 1907-770x 100 evaluating habitat suitability and ecotourism potential of mangrove ecosystems along progo river, yogyakarta erny poedjirahajoe*, muhammad reza pahlevi*, frita kusuma wardhani, ryan adi satria, and dimas cahya kurnia saputra forest ecology laboratory, ksdh department, faculty of forestry, gadjah mada university yogyakarta 55281, indonesia article higlights • assessment of habitat suitability for ecotourism: the article introduces an evaluation of mangrove habitat suitability for ecotourism in the progo river area, which has not been extensively studied in this region. • combination of sampling methods: it employs nested and gridline sampling methods to measure habitat quality more comprehensively, leading to more accurate data on mangrove conditions. • monitoring environmental variables: the study examines the impact of physical and chemical factors such as temperature, salinity, and dissolved oxygen on mangrove growth and biodiversity. • identification of ecotourism potential based on the tourism suitability index: it uses the tourism suitability index to identify locations suitable for nature tourism activities around the progo river. • importance of sustainable management: the article stresses the need for improved management of mangrove ecotourism areas to preserve ecosystem sustainability and support the local economy. article information received : 18 january 2025 revised : 4 march 2025 accepted : 11 march 2025 *corresponding author, e-mail: er_pjr@ugm.ac.id, reza101194@ugm.ac.id research article abstract mangrove forests are a transitional ecosystem located between land and sea. habitat factors greatly influence the growth of mangrove plants. habitat suitability is reflected in the suitability of the species that can grow in the habitat. increased growth will ultimately increase water productivity, which can be seen from the presence of various types of biota and environmental improvements. the local community planted mangrove species (sonneratia sp.) along the progo river in an area of 75 h. this study aimed to: 1) assess the quality/characteristics of the progo river waters that will be used for a water tourism center and 2) assess the suitability (ikw) of the progo river waters for water tourism in bleberan hamlet. data collection was carried out using nested sampling. data analysis included the diversity of biota types using the shannon diversity index and vegetation analysis. the results showed that the highest temperature occurred at station 3 plot 10, the highest mud thickness was observed at station 5 plot 9, the highest salinity was detected at station 3 plot 10, the highest water clarity was observed at station 2 plot 7, the highest ph was seen at station 1 plot 6 and 7, while the highest do was spotted at station 3 plot 8. some of the biota obtained included crustaceans, gastropods and actinopterygii. the habitat types of vegetation obtained were trees, shrubs, and some herbs. based on the tourism suitability index (ikw) value, there were 3 stations suitable for camping/outbound, 2 stations suitable for relaxing, and 2 stations suitable for river tracks. keywords: ecotourism development, habitat suitability, mangrove ecosystems, progo river, sustainable management. introduction mangrove forests are important ecosystems that contribute to coastal stability which protect the coastal areas from various marine-related hazards that harm life (hadi, 2012). many marine species rely heavily on mangrove environments, with healthy mangroves having fish populations and biodiversity that are 116 129% higher than degraded mangroves (onziral et al. 2020). mangrove ecosystem degradation causes significant issues, such as: 1) rapid degradation of mangroves, which accelerates the loss of mangrove islands and coastal areas (sahu et al. 2015); 2) a decrease in the diversity of flora and fauna that are specific to mangrove forests (polidoro et al. 2010); 3) increased seawater intrusion, as the mangrove ecosystem, which serves as a buffer zone, becomes less effective due to destruction, which can https://doi.org/10.11598/btb.2024.31.2.1741 evaluating habitat suitability and ecotourism potential of mangrove ecosystems poedjirahajoe et al. 101 lead to environmental problems, such as flooding, erosion, and salt intrusion (hilmi et al. 2017; field 2004); 4) coastal abrasion, particularly affecting communities along beaches facing straits and the sea (whidayanti et al. 2021); and 5) increased flooding risks. maintaining mangrove ecosystems can significantly reduce water flow velocity by 29 92% and modestly decrease wave height by 4 16.5 cm during cyclones, offering protection from flooding in coastal areas (dasgupta et al. 2019). the greatest threat to mangrove areas is the conversion of mangrove land into shrimp or fish ponds. converting mangrove land to shrimp or fish farms impacts marine biota by increasing nutrient and carbon conversion, reducing microbial communities and metabolic diversity, and promoting nitrifying, denitrifying and sulfuroxidizing bacteria (erazo & bowman 2021). this conversion also resulted in the loss of 70% of the carbon stored in the mangroves and increased pressure from the exploitation of timber, fish, crustaceans and shellfish for trade (elwin et al. 2019; alongi 2002). additionally, there are growing conflicts arising from competing interests between different agencies and administrative regions. ideally, the utilization of mangrove areas should take into account community needs without damaging the mangroves themselves (zulfa et al. 2023). furthermore, it is essential to develop activities that benefit the community, while preserving the ecological functions of the mangroves (both physico-chemical and biological). therefore, ecotourism is an ideal solution to be implemented for the preservation of mangrove ecosystems, the development of community economies, and conservation efforts (aziz et al. 2018). banaran and bleberan villages are both located in galur district, kulon progo regency. the locations of these villages are potential for mangrove ecosystems because it is directly adjacent to the sea and river, but this has not been further developed (diella et al. 2022). in addition, these two villages have a beach called trisik beach which is an annual stopover for rare slithers, namely green turtle (chelonia mydas) when laying eggs. however, currently the existence and survival of green turtles (chelonia mydas) face serious threats from the main predator, humans (mayastuti et al. 2024). therefore, research on habitat suitability evaluation and ecotourism is important so that the preservation of mangrove forest ecosystems and the improvement of the community’s economy can be realized. the first step to realize mangrove forest ecotourism requires a systematic assessment of the land area by conducting inventory activities to assess the suitability for ecotourism (nugraha et al. 2015). this study aimed to identify: 1) the characteristics of aquatic ecosystem habitats including: types of aquatic biota, types and density of existing vegetation, and other environmental factors, such as salinity, temperature, ph and muddiness and 2) the suitability of habitat characteristics for ecotourism. materials and methods this research was conducted in the mangrove area in bleberan hamlet, banaran village, galur district, kulonprogo regency. geographically, the banaran village is located between 110°11’ 110°14’ e and 7°57’ 7°59’ s. the observation and sampling sites were determined, as presented in figures 1-7. this study applied the combination methods, i.e., the path and gridline methods (indriyanto 2019). the path was laid as a perpendicular along the coastline to find out the vegetation condition changes. the path method was implemented based on poedjirahajoe et al. (2017) as follows. biotropia vol. 32 no. 1, 2025 102 figure 1 study area in the banaran village of progo river figure 2 bridge view (observation spot 1) figure 3 river view (observation spot 2) figure 4 estuarine view (observation spot 3) figure 5 near village (observation spot 4) evaluating habitat suitability and ecotourism potential of mangrove ecosystems poedjirahajoe et al. 103 figure 6 fishing pool (observation spot 5) figure 7 river island (observation spot 6) figure 8 path method design in coastal ecosystems (poedjirahajoe et al. 2017) observation spot (pu) were applied in each zone using nested plot sampling (fig. 9) with a total of 12 nested plots at each observation spot. the vegetation measured is the vegetation located in the observation spot. the criteria for mangrove growth levels are based on the decree of the directorate general of forestry no. 60/kpts/ di/1978 concerning guidelines for the brackish water forest silviculture system, namely: a. seedlings, plants less than 1.5 m high (plot size 2 x 2 m). b. saplings, plants more than 1.5 m high and less than 10 cm in diameter (plot size 5 x 5 m). c. trees, plants more than 10 cm in diameter (plot size 10 x 10 m). figure 9 nested sampling pattern measurement of the physical and chemical quality of the habitat was also carried out at each observation spot, including measurements of water clarity (cm), water temperature (°c), ph, salinity (%), dissolved oxygen content (mg/l), and mud thickness (cm). water clarity was measured using a secchi disk, water temperature was measured using a thermometer stick, ph measurement was conducted using a ph meter, salinity was determined using a digital salt test, dissolved oxygen measurements were carried out using an oximeter, and mud thickness was determined using a graduated pole that was inserted at several points. calculation of physical, biological, and chemical quality is used to assess the location suitability for tourism activities. the focus of tourism suitability in this study was divided into three forms of utilization, namely camping, relaxing, and river tracks. the suitability was calculated using the tourism suitability index formula (indeks kesesuaian wisata = ikw) (yulianda 2019). biotropia vol. 32 no. 1, 2025 104 ikw = (bi x si)∑n i = 1 where: n = number of fit parameters bi = weight of the parameter si = score of the parameter the calculated tourism suitability index was compared with the class criteria of the tourism suitability index presented in table 1 based on yulianda (2019). table 1 tourism suitability index class description ikw result very suitable ikw ≥ 2.5 suitable 2.0 ≤ ikw ≤ 2.5 not suitable 1.0 ≤ ikw ≤ 2.0 very unsuitable ikw ≤ 1.0 the feasibility of ecotourism was determined using the assessment criteria by the director general of phka in 2003, namely analysis of operational areas for natural tourism objects and attractions (analisis daerah operasi obyek dan daya tarik wisata alam = ado-odtwa). the data were obtained from respondents by means of written interviews and calculation using tabulation. the eligibility is determined by the value of each ado-odtwa criterion (table 2). the value of each criterion in ado-idtwa was calculated using the following equation: s = n × b where: s = score of each criterion n = number of criteria elements b = weight of value table 2 presents the modified ado-odtwa assessment criteria table from the directorate general of forest protection and nature conservation 2003. table 2 ado-odtwa assessment criteria no. criteria max min interval eligibility criteria 1 attractiveness 1440 360 360 1080 1440 719 1079 < 719 very potential potential not potential 2 accessibility 1300 305 331 969 1300 637 968 < 637 very potential potential not potential 3 accommodation 180 60 40 140 180 139 99 < 99 very potential potential not potential 4 supporting facilities and infrastructure 180 45 45 135 180 134 89 < 89 very potential potential not potential 5 conditions around the area 1200 450 250 950 1200 699 449 < 449 very potential potential not potential 6 availability of clean water 720 240 160 560 – 720 559 399 < 399 very potential potential not potential 7 marketing 120 20 33 87 120 86 53 < 53 very potential potential not potential evaluating habitat suitability and ecotourism potential of mangrove ecosystems poedjirahajoe et al. 105 results and discussion the study was conducted at 6 observation spots measuring 6 parameters consisting of water temperature, mud thickness, salinity, water clarity, ph, and dissolved oxygen. water temperature figure 10 temperature the average water temperature at each station ranged from 28 35 °c, which was sufficient for mangrove growth. zheng and takeuchi (2022) stated that stomatal conductance and assimilation rate in mangrove leaves are optimal at temperatures below 35 °c. affressia et al. (2017) stated that the temperature suitable for mangrove habitat ranges from 20 35 °c. mangroves are highly adapted to extreme conditions including high temperatures (kathiresan & bingham 2001). however, when the temperature exceeds the peak photosynthetic temperature, photosynthetic productivity decreases and when the leaf temperature exceeds 38 40 °c, photosynthesis stops (cloughet et al. 1982). furthermore, high temperatures increase evaporation, resulting in increased salinity; the synergistic effects of salinity and drought can affect species diversity, size, and productivity of mangrove forests (smith & duke 1987; ball & sobrado 2002). this can affect the plant’s ability to absorb water and nutrients, which in the long run can negatively affect the health of the mangrove. mud thickness figure 11 mud thickness the result showed that the mud thickness from 10 existing plots ranged from 4 to 78 cm (fig. 11). some plots exhibited significant differences; for instance, plot 9 showed the lowest mud thickness at 10 cm, while the deepest mud thickness was recorded at 78 cm. thicker mud thickness provided support for better mangrove growth as it provided a stable, nutrient-rich substrate (purwanto et al. 2022). however, there was no consistent pattern of mud thickness across all sites, suggesting that other factors also influenced mangrove growth. in addition, this difference may be due to the presence of several large rivers that flowed throughout the year. this observation aligns with the findings of poedjirahajoe and matatula (2019), who noted that areas with fewer large rivers flowing throughout the season can affect the thickness and characteristics of the mud substrate. the thickness of mud in deep water or mangrove environments can indicate sedimentation processes (boulesteix et al. 2019). this is consistent with the statement by noor et al. (1999) that mud thickness significantly impacted the ability of mangrove vegetation to capture sediment transported by water during tidal events. the thickness of the mud affects the ability of tree roots to bind and stabilize its position. salinity figure 12 salinity salinity from 10 existing plots ranged between 0.03 0.94% (fig. 12). septiarusli (2006) stated that mangroves can grow well in brackish water salinity between 2 22 ppt or salt water with salinity reaching 38 ppt. this condition indicated a reasonable salinity condition at the river mouth. stable and suitable salinity allows mangroves to grow optimally. mangroves effectively regulate water balance under saline conditions by adjusting their structure to exclude salt, maintain hydraulic conductance, avoid cavitation, and limit water loss (reef & lovelock 2015). research conducted in the sundarbans of bangladesh showed that trees growing under low salinity conditions had higher biotropia vol. 32 no. 1, 2025 106 growth rates (rahman et al. 2020). however, significant changes in salinity, either increases or decreases, can disrupt communities in estuarine systems, affecting the biomass, abundance, and diversity of benthic macrofauna (breaux et al. 2019; diggelen & montagna 2016). water clarity figure 13 water clarity water clarity of the 10 plots from 6 stations ranged from 10 to 89 cm (fig. 13). there were both increases and decreases in the level of water clarity at the progo river estuary. this indicated a difference in the amount of sedimentation that accumulated on the riverbed. the lower the water clarity level, the thicker the sedimentation will be, which can potentially reduce productivity and alter community structure (ryan 1991). turbidity and excessive sediment loads can harm surface water bodies due to urban development, construction activities, and agricultural practices (neupane et al. 2015). during the wet season, the water around estuaries tends to be more turbid as the river flow carries more sediment from the land. in contrast, during the dry season, water clarity may increase (asp et al. 2016). these changes in clarity may indicate natural seasonal dynamics, while unusual changes may suggest external disturbances. ph figure 14 ph ph range from the 10 existing plots was 7.1 7.8 (fig. 14), which indicated that overall the progo river water is a productive waters. kaswadji (2001) stated that waters with ph 6.5 8.5 are waters with high productivity. the ph range observed in this study also indicated that the progo river area is very suitable for mangrove growth. widyastuti (1999) suggested that water ph range between 6 8.5 is very suitable for mangrove growth. this ph range which is almost neutral to slightly alkaline water condition can affect the quality of a water habitat, especially in the mangrove ecosystem. in maintaining the function of mangrove ecosystems, it is important to maintain soil ph within a reasonable range (muhsoni 2020).  these ph values indicate relatively balanced and ideal conditions for aquatic life, so most aquatic organisms can grow well within this ph range, including fish, plankton, and microorganisms that support mangrove ecosystems. this is supported by the ambient water quality criterion (awqc) in the united states in 2003 which considers that the ph range of 6.5 to 9 is able to protect freshwater life. dissolved oxygen figure 15 dissolved oxygen muttaqin et al. (2024) mentioned that the ideal dissolved oxygen range for mangroves is 3 7 mg/l, which supports the ecosystem’s health and biodiversity. the 6 stations had ideal mean do values ranging from 3.1 6.3 mg/l. factors such as tidal height, sunlight, and distance from the edge of the mangrove significantly affect do dynamics. during high tides, do levels are generally higher, but can decrease rapidly at low tide, especially in areas further inside the forest. water quality with this do value is considered moderate, which could be due to moderate levels of organic pollution. organic waste will be consumed by bacteria, which in the process also consume oxygen, thus lowering do. increasing dissolved oxygen concentration also indicates the presence of bacteria and microorganisms as organic matter decomposers (liu et al. 2021). evaluating habitat suitability and ecotourism potential of mangrove ecosystems poedjirahajoe et al. 107 several types of biota were found in the research area, such as crustaceans, gastropods, and actinopterygii. crustaceans found included fiddler crabs (uca spp.) and ketam crab (gecarcinucoidea). gastropods found included langkitang (melanoides tuberculata), and hermit crabs (paguroidea). actinopterygii found included mudskippers (oxudercinae) and mullet (mugil cephalus). several vegetations were also found in the research area in the form of trees, shrubs, and herbs (table 3). table 3 types of vegetation found around the observation points no type of vegetation herb shrub tree species n species n species n 1 alternanthera sessilis 19 carica papaya 1 avicennia sp. 3 2 brachiaria mutica 26 chromolaena odorata 1 calophyllum inophyllum 5 3 chloris barbata 21 cocos nucifera 1 calotropis gigantea 4 4 cyperus alternifolius 8 euphorbia hyssopifolia l 1 carica papaya 2 5 eichhornia crassipes 9 hibiscus tiliaceus 1 cassuarina equisetifolia 20 6 gomphrena globosa 18 ipomoea carnea jacq. 36 cocos nucifera 2 7 imperata cylindrica 20 ludwigia decurrens walter 10 erythrina fusca 6 8 ipomoea aquatica 6 ludwigia peruviana 18 gliricidia sepium 1 9 tridax procumbens 6 manihot esculenta 49 hibiscus tiliaceus 9 10 typha angustifolia 6 mimosa pudica 114 inocarpus fagiferus 11 11 wedelia trilobata 14 musa paradisiaca 9 leucaena leucocephala 33 12     senna alata 1 mimosa pudica 3 13     sida sp. 51 morinda citrifolia 1 14         muntingia calabura 7 15         rhiziphora sp. 13 16         rhizopora sp. 6 17         sonneratia alba 97 18         terminalia catappa 14 n 153 n 293 n 237 diversity (h') 2.271 diversity (h') 1.738 diversity (h') 2.120 biotropia vol. 32 no. 1, 2025 108 tourism suitability index (ikw) assessment table 4 results of the ikw assessment observation spot camping/outbound sit back and relax river track 1 2.4 1.825 1.6 (suitable) (not suitable) (not suitable) 2 2.4 2.075 2.1 (suitable) (suitable) (suitable) 3 1.95 1.375 1.45 (not suitable) (not suitable) (not suitable) 4 1.575 1.275 1.6 (not suitable) (not suitable) (not suitable) 5 1.8 1.475 1.9 (not suitable) (not suitable) (not suitable) 6 2.4 2.225 2.55 (suitable) (suitable) (suitable) the tourism suitability index obtained in this study indicated that there were several spots at the progo river area which were suitable for camping/ outbound, relaxing, and river tracks, while there were some spots not suitable for tourism activity (tables 4 & 5). the stations that were suitable for camping, sitting, and river track were spot 2 (river view) and spot 6 (river island), while spot 1 (bridge view) was only suitable for camping activities. the unsuitable spots need to be improved and better managed with expectations that in the future those spots can all be suitable for tourism activities. ado-odtwa assessment results of the ado-odtwa assessment indicated that the environmental conditions around the progo river estuary in bleberan hamlet were generally suitable for being developed as a tourist destination, particularly in terms of accessibility, accommodation, and availability of clean water. however, only one attraction that was deemed unsuitable. additionally, there is a need for improvements in the surrounding environment, as it tends to be quite hot. providing shade is necessary to ensure a comfortable temperature for visitors. it is hoped that with these enhancements, the area can be transformed into a prime tourist location in the future. table 5 results of the ado-odtwa assessment observation spot attraction accessibility conditions surrounding the area accommodation availability of clean water 1 not eligible eligible not eligible eligible eligible 2 eligible eligible not eligible eligible eligible 3 eligible eligible not eligible eligible eligible 4 eligible eligible not eligible eligible eligible 5 eligible eligible not eligible eligible eligible 6 eligible eligible not eligible eligible eligible evaluating habitat suitability and ecotourism potential of mangrove ecosystems poedjirahajoe et al. 109 conclusion the measured primary environmental parameters were as follows: the average temperature was 29.7 °c, average mud thickness was 15.9 cm, salinity range was 0.3 0.94%, average water clarity was recorded at 46 cm, the average of ph level was 7.5 (indicating slight alkalinity), and the average of dissolved oxygen concentration was 4.6 mg/l. all of these parameters indicated that the environmental characteristics of the area were suitable for marine life and has the potential to become mangrove ecotourism. aquatic biota found in progo river estuary were crustaceans, gastropods, and actinopterygii. overall, the progo river area is quite suitable for ecotourism development. attention should be focused to the attractions and conditions in the surrounding area. some suggestions include improving the environment at the less suitable spots, adding local attractions (such as fishing and kayaking), and encouraging scientific research to ensure sustainable ecotourism in the future. references affressia r, poedjirahajoe 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10.11598/btb.2022.29.1.1568 18 inhibitory effect of ulin wood liquid smoke and gogo rice endophytic fungi against pathogen pyricularia oryzae witiyasti imaningsih1*, della adventaria2, mariana3 and ahmad budi junaidi4 1microbiology laboratory, biology study program, faculty of mathematics and natural sciences, universitas lambung mangkurat, banjarmasin 70714, indonesia 2student of biology study program, faculty of mathematics and natural sciences, universitas lambung mangkurat, banjarmasin 70714, indonesia 3plant protection study program, faculty of agriculture, universitas lambung mangkurat, banjarmasin 70714, indonesia 4chemistry study program, faculty of mathematics and natural sciences, universitas lambung mangkurat, banjarmasin 70714, indonesia received 22 march 2021 / accepted 4 january 2022 abstract diseases in rice plants (paddy) caused by microorganisms such as pyricularia oryzae lead to a decrease in rice production. therefore, it is essential to find out biological agents for protecting paddy and plants in general, against plant diseases. liquid smoke and endophytic fungi have been known as biological agents to enhance the protection of plants against disease. the purpose of this study was to determine the ability of liquid smoke, endophytic fungi and the concentrations combinations to suppress the growth of p. oryzae. the results showed that liquid smoke concentrations of 0.17% to 1.75% and endophytic fungi filtrate of 2% to 10% showed significant ability against pathogen p. oryzae. however, the combination of liquid smoke and endophytic fungi filtrate at selected concentrations (0.17% liquid smoke combined with 2% endophytic fungi filtrate and 0.34% liquid smoke combined with 2% endophytic fungi filtrate) showed no significant inhibition percentage against p. oryzae compared to control. in conclusion, this study showed that the respective applications of liquid smoke and endophytic fungi filtrate inhibit the growth of p. oryzae. keywords: endophytic fungi, inhibition ability, liquid smoke, p. oryzae introduction rice plants are the most dominating food crop commodity in indonesia (andriani 2008). as many as 95% of indonesians choose rice as a staple food (norsalis 2011). one of the rice plants cultivated in indonesia is gogo rice, which is grown on moorland (a type of habitat with the characteristic of low growing vegetation on acidic soil) or on dry land (hairmansis et al. 2016). “gogo” rice maninjau variety (oryza sativa l. var. maninjau) is one of the indonesian native varieties of brown rice that comes from the area of lake maninjau located in the sumatera barat province of indonesia. this variety is able to live on the dry land of central kalimantan, is resistant to leaf blight disease and able to survive in soil with high iron content (bppp jateng 2014). among microorganisms attacking gogo rice plants are pathogenic fungi such as p. oryzae, rhizoctonia solani, helmithosporium sigoideum and cercospora janseana. p. oryzae is one of the main diseases of rice crops due to its impact on reducing rice productivity (wang et al. 2014; suganda et al. 2016). this pathogen causes blast disease and serious damage to panicles (panicle blast) and leaves (leaf blast) of rice plants, where damage to panicles greatly affects rice productivity (hayashi et al. 2019). plant resistance to pathogens can be improved by utilizing the interaction of microorganisms with endophytic microbes. *corresponding author, email: witiyastiimaningsih@ulm.ac.id inhibitory effect of ulin wood liquid smoke and gogo rice endophytic fungi – witiyasti imaningsih et al. 19 endophytic fungi are a symbiotic between fungi and plants, which has a role to protect plants from pathogens by using the compounds produced by the symbiosis. symbiosis mutualism of endophytic fungi with plants produces secondary metabolites, such as phytohormones, nutrients and colony formations. some studies state that secondary metabolites produced by endophytic fungi inhibit pathogenic microbes (lalngaihawmi et al. 2019). therefore, endophytic fungi are potential biological agents to inhibit pathogenic fungi. liquid smoke bioactive materials can also be used to prevent disease caused by microorganisms. liquid smoke is a mixture of solution and colloidal dispersion of wood smoke vapor in water obtained from the process of wood pyrolysis or made from a mixture of pure compounds (darmadji 2002; soldera et al. 2008; lee et al. 2011). liquid smoke has good antimicrobial properties because it can inhibit the growth of microbes. this study used liquid smoke from “ulin wood” (eusideroxylon zwageri teijsm. & binn). ulin wood has density characteristics and a tight structure of hardwood, containing complex constituent compounds. several studies reported that one type of hardwood, “alaban” wood (vitex pubescens vahl.), contains phenol, carboxylic acid and carbonyl that are antimicrobial (oramahi & yoshimura 2013). based on literature studies, “ulin” wood liquid smoke may have the ability to inhibit p. oryzae. both endophytic fungi and liquid smoke of “ulin” wood have the ability to inhibit pathogenic microorganisms. in this study, both were tested both independently and by combining the two. as an initial effort to find one of the solutions to overcome diseases that often occur in rice plants “gogo” maninjau varieties. this study was aimed to determine the ability of “ulin” wood liquid smoke, endophytic fungi and their combinations to suppress the growth of p. oryzae. materials and methods sampling samples of “gogo” rice (o. sativa l var. maninjau) were taken from the same stretch of land in jaar village, east dusun subdistrict, east barito district, central kalimantan province. 2°06'53.9" north-south latitudes 115°15'22.1" west-east longitudes. samples consisting of all parts of the gogo rice plants were taken along with the soil around the roots of the gogo rice plants, by using a machete. the samples were stored in polybags and transported to the microbiology laboratory of the faculty of mathematics and natural sciences, universitas lambung mangkurat. isolation, purification and identification of endophytic fungi endophytic fungi were isolated from the roots of the gogo rice plants, by using surface sterilization method. the roots were washed using running water, then cut to a size of 10 cm. the root cuts were surface-sterilized by using 0.05% bleach for 60 sec and rinsed twice using sterile distilled water. each part of the root tip was then slightly cut to totally drain the root tip. the root tip was then planted in a sterile potato dextrose agar (pda) medium and incubated at a temperature of 28 °c for 3-5 days and observed daily. the grown fungi were then purified (manurung et al. 2014; nurzannah et al. 2014). fungi identification was carried out by using morphological observations, consisting of macroscopic and microscopic observations with reference to the identification book titled "illustrated genera of imperfect fungi 4th edition" (barnett & hunter 1998). screening of endophytic fungi pathogenicity and antagonism tests were used for the screening of endophytic fungi. pathogenicity test were carried out by using rice seed refering to waruwu et al. (2016). prior to being used in the pathogenicity test, the surface of the rice seeds (20 grains) were sterilized by soaking the rice seeds in 70% ethanol for 30 sec, followed by soaking in 1% naocl for 60 sec. subsequently, the rice seeds were flushed 3 times in sterile distilled water. after that, the rice seeds were inoculated in pda medium that had been previously overgrown by 7-day pure isolates of endophytic fungi and then incubated for 2 weeks at room temperature (27-29 °c). observation on the growing rice sprouts was carried out at the end of incubation. isolates of endophytic fungi that did not interfere with rice biotropia vol. 29 no. 1, 2022 20 germination were used for further testing. seed germination rate was calculated using the formula (talukdar 2011): germination (%) = number of germinated seed x 100% total number seed a confirmation test for endophytic fungi infection in the roots of rice plants was conducted for germination by using a method of luqman et al. (2015) that has been modified. prior to being used in the test, the roots of rice plants were washed thoroughly in running water, drained, then soaked in 5.25% naclo solution for 5 min, then rinsed using distilled water. subsequently, the roots were soaked in 1% koh solution for 30 min and then rinsed using distilled water. after that, the roots were then pre-soaked in 1% h2o2 solution for 5 min. the coloring stage was started by soaking the roots in 0.5% vinegar solution, followed by being soaked in ink with a ratio of 1:5 for 30 min. then, the roots were rinsed with distilled water. finally, the roots of the rice plants were placed on an object glass and covered by a cover glass, and then observed under a microscope with 40x and 100x magnifications. the antagonism test of endophytic fungi isolates against p. oryzae was conducted by using dual culture method (tomah et al. 2020), which put isolates of pathogenic fungi and endophytic fungi on pda medium in a petri dish that has been divided into two quadrants. each isolate was placed at a distance of 3 cm from the edge of the petri dish and incubated at a temperature of 28 °c for 5-7 days. after incubation, the inhibition percentage of the pathogens was measured using the formula developed by rabha et al. (2014): inhibition (%) = diameter of pathogen control colony x 100% diameter of pathogen treatment colony filtrate harvesting of endophytic fungi filtrate production methods used in this study were modified based on elita et al. (2013) and malinda et al. (2015). endophytic fungi with the highest inhibitory ability obtained in previous tests were inoculated on pda slant media then incubated for 7 days at 28 °c. after being incubated, the filtrate was harvested by adding 9 ml of sterile distilled water. the fungi surface was then gently wiped with a fine brush. subsequently, the water suspension and fungi were transferred to a new test tube, then centrifuged at 3,500 rpm for 20 min. finally, the supernatant was filtered by using a syringe filter with a pore size of 0.45 μm. the filtrate was then used in testing the inhibitory activity of endophytic fungi against pathogens. inhibition test of liquid smokeendophytic fungi to p. oryzae inhibition test of liquid smoke was conducted at various liquid smoke concentrations, i.e., 0.085%, 0.17%, 0.34%, 0.68%, 1.36% and 1.75%, based on a method of malinda et al. (2015) that have been modified. liquid smoke was obtained from condensation during the production process of “ulin” wood charcoal by talasiana charcoal production group, located at tanah laut regency. the liquid smoke was mixed into the potato dextrose agar (pda) medium. the pathogen isolates were then grown on the mixture of liquid smoke and pda for 5-7 days with daily observation. the inhibition percentage was calculated by the formula developed by rabha et al. (2014): inhibition (%) = diameter of pathogen control colony x 100% diameter of pathogen treatment colony the inhibition test for endophytic fungi was carried out by using the serial dilution method. endophytic filtrate with concentrations of 2, 4, 6, 8, and 10% was mixed with pda medium to be used for growing pathogen, and then incubated at 28 oc for 7 days and observed daily. the inhibition percentage was calculated with the same formula as the one used for calculating the inhibition percentage for the liquid smoke. liquid smoke and the endophytic filtrate (ketoconazole) with various concentrations were then combined to be tested for their inhibitory ability against pathogens. testing methods and measurements of inhibition percentage were conducted by using the same method as for the previous tests. results and discussion endophytic fungi of o. sativa l var. maninjau root endophytic fungi obtained from the roots of “gogo” rice var maninjau were coded ap.2, inhibitory effect of ulin wood liquid smoke and gogo rice endophytic fungi – witiyasti imaningsih et al. 21 ap.3, ap.4, ap.7, ap.8, and ap.9. microscopic observation of the endophytic fungi isolates showed that there are morphological differences. based on barnet & hunter (1998), the six isolates found refers to several species. ap2 is curvularia sp., ap3 and ap7 are penicillium sp., ap 8 is geotrichum sp., ap9 is aspergillus sp., while ap4 has not yet been able to be identified (fig. 1). the four fungi species were also reported by lalngaihawmi et al. (2018) as endophytic fungi in rice. the presence of endophytic fungi in the roots of the gogo rice plants varies based on the various tissues in which they grow. a study conducted by naik et al. (2009) reported that the colonization of endophytic fungi in rice plants happens more dominantly at the roots of rice plants. pathogenicity of endophytic fungi isolated from o. sativa l. var maninjau seeds our study showed that the germination percentage of gogo rice seeds varied (table 1), while the invasions of endophytic fungi against the seed germination were shown in figure 2. each endophytic fungi showed different levels of pathogenicity, both at 7 days after inoculation and 14 days after inoculation. the level of pathogenicity is useful for determining the best isolates to be used for subsequent tests. based on the results of pathogenicity levels of rice sprouts that grew normally, abnormally and did not grow, the best pathogenicity value was provided by geotrichum sp. ap8, followed by penicillium sp. ap7 and curvularia sp. ap 2. figure 1 microscopic characteristics of endophytic fungi isolates obtained from the roots of o. sativa l var. maninjau notes: a. curvularia sp. ap2 (40x); b. penicillium sp. ap3 (40x); c. ap4 (100x); d. penicillium sp. ap7 (40x); e. geotrichum sp. ap8 (40x); f. aspergillus sp. ap9 (40x). table 1 germination rate of the o. sativa l. var. maninjau for the 7 and 14 days after incubation (dai) endophytic fungi germination (%)* 7-dai 14-dai normal abnormal no growth normal abnormal no growth without adding endophytic fungi 60.00 ± 8.16b 0.00 ± 0.00a 36.67 ± 4.71a 63.33 ± 9.43b 0.00 ± 0.00a 33.33 ± 4.71a curvularia sp. ap2 40.00±14.14a 10.00 ± 8.16a 40.00 ±8.16a 20.00 ± 8.16a 36.67 ± 12.47a 33.33 ± 4.71a penicillium sp. ap3 43.33 ± 4.71ab 13.33 ± 4.71a 43.33 ± 9.43a 56.67 ± 4.71ab 16.67 ± 4.71a 26.67 ± 4.71a ap4 0.00 ± 0.00ab 0.00 ± 0.00a 100.00 ± 0.00b 6.67 ± 9.43ab 13.33 ± 12.47a 80.00 ± 14.14b penicillium sp. ap7 43.33 ± 9.43ab 13.33 ± 12.47a 50.00 ± 8.16a 56.67 ± 12.47ab 16.67 ± 9.43a 26.67±4.71a geotrichum sp. ap8 50.00 ± 8.16b 0.00 ± 0.00a 50.00 ± 8.16a 70.00 ± 8.16b 16.67 ± 4.71a 16.67±4.71a aspergillus sp. ap9 43.33 ± 4.71ab 6.67 ± 4.71a 50.00 ± 8.16a 46.67 ± 4.71ab 20.00 ± 0.00a 33.33±4.71a note : * = numbers followed by the same letter are not significantly different based on duncan test at p < 0.05. biotropia vol. 29 no. 1, 2022 22 figure 2 rice germination with various treatments of endophytic fungi notes: a. rice germination without the addition of endophytic fungi; b. rice seeds with the addition of ap4 endophyte fungi (not germinated); c. rice germination with the addition of endophytic fungi geotrichum sp. ap8; d. roots of rice seed undergoing treatment with endophytic fungi geotrichum sp. ap8 (the arrow shows endophytic fungi invading the rice root tissue). the results of the endophytic fungi antagonism test against p. oryzae showed no significant difference between the three selected fungi (p > 0.05) (table 2). although the antagonism test did not show significant differences, the selection of the best isolate for subsequent tests was determined based on the best inhibition percentage and the diameter of the pathogen successfully inhibited. thus, geotrichum sp. ap8 was the chosen isolate. endophytic fungi can inhibit pathogens having metabolite compounds by inhibiting the permeability of the pathogenic cells (ting et al. 2011). white et al. (2019) added that endophytic fungi can use the mechanisms of space and nutrient competition for suppressing pathogen growth. the ability of liquid smoke and endophytic fungi in inhibiting the growth of p. oryzae the results of our study indicated that liquid smoke at all tested concentrations was significantly able to inhibit the growth of pathogens compared to control (fig 3). at liquid smoke concentrations of 0.17% to 1.75%, the inhibition percentages differed significantly (p < 0.05). the selection of the right liquid smoke concentration for subsequent tests is indispensable, given that liquid smoke contains several antimicrobial components that may affect not only the growth of pathogen, but also the growth of endophytic fungi. table 2 diameter of p. oryzae colony and inhibition percentage of endophytic fungi endophytic fungi diameter of p. oryzae (mm)* inhibition percentage (%)* curvularia sp, ap2 41.47±2.92 21.98±5.54 penicillium sp. ap7 32.03±7.84 41.87±14.32 geotrichum sp. ap8 30.21±6.31 41.87±11.71 note: * = not significantly different based on duncan test (p < 0.05). inhibitory effect of ulin wood liquid smoke and gogo rice endophytic fungi – witiyasti imaningsih et al. 23 figure 3 inhibition percentage of different concentrations of liquid smoke against p. oryzae on 1 dai until 7 dai notes: the bar indicates the standard deviation. numbers followed by the same letter are not significantly different based on duncan test (p < 0.05). the results also showed that the smallest liquid smoke concentration (0.02%) was able to inhibit the growth of pathogens despite the daily decrease in ability, while the largest liquid smoke concentration (1.75%) was able to inhibit pathogens at an inhibition percentage of 100%. our study also indicated that the 0.17% and 0.34% liquid smoke concentrations were considered the best concentration for inhibiting pathogens compared to other concentrations. the inhibitory curve of those two concentrations had a tendency of increase after passing 4 days of inoculation, in contrast to other concentrations that had a tendency to decrease. the two liquid smoke concentrations also showed inhibiting capabilities against the tested pathogen despite the small concentrations. the inhibiting capabilities of liquid smoke against the growth of microorganisms may have been due to the contents of active compounds originating from the pyrolysis of wood constituents (cellulose, hemicellulose, and lignin). cellulose and hemicellulose produce organic acid compounds such as acetic acid, while lignin produces phenol compounds. the higher the content of the wood constituents, the more complex liquid smoke obtained (pszczola 1995). contents of active compounds in “ulin” liquid smoke are acids, phenolics, alcohol, ketones, ethers and esters, with acetic acid as the main active compounds (71.57% of the total active compounds) (junaidi et al. 2020). liquid smoke of “ulin” wood also contains a total acid of up to 8.88% (junaidi et al. 2019), which has antimicrobial properties. various concentrations of selected isolate endophyte fungi filtrate (geotrichum sp.) significantly inhibited the growth of p. oryzae compared with the control. however, there were no significant differences in inhibition percentages among concentrations (2-10%) (p < 0.05) (fig. 4). biotropia vol. 29 no. 1, 2022 24 figure 4 inhibition percentage of geotrichum sp. ap8 filtrate concentrations against p. oryzae on 1 dai until 7 dai notes: the bar indicates the standard deviation. numbers followed by the same letter are not significantly different based on the duncan test (p < 0.05). geotrichum sp. ap8 filtrate concentration of 2% was able to inhibit the growth of p. oryzae grown at 1 dai up to 7 dai with inhibition percentages ranging from 91.8 up to 100%. therefore, the 2% concentration of geotrichum sp. ap8 filtrate was chosen for testing the inhibitory synergism with liquid smoke. this result of geotrichum sp. ap8 filtrate is similar to the results of previous research conducted by imaningsih et al. (2021), which used endophytic fungi filtrate of “hiyung” cayenne pepper with a concentration of 2% for inhibiting pathogen colletotrichum capsici at almost 100% inhibition percentage. our study also provides better inhibitory results for the genus geotrichum compared to the study of lalngaihawmi et al. (2019), which tested geotrichum candidum for inhibiting p. oryzae with a 68% inhibition percentage at 7 dai. endophytic fungi inhibit pathogen growth through anti-microbial compounds (schulz & boyle 2005; singh et al. 2021). in our study, the filtrate of endophytic fungi was tested directly to inhibit the growth of p. oryzae and successfully showed high inhibition percentage. results of our study showed that the presence of bioactive substances produced by endophytic fungi has anti-microbial properties against the tested pathogen. singh et al. (2021) stated that bioactive compounds of endophytic fungi can be alkaloid, flavonoid, lignan, saponin, quinone, xanthone and miscellaneous compounds. synergism of liquid smoke and endophytic fungi inhibit p. oryzae growth the growth of pathogenic p. oryzae was inhibited by the combination between liquid smoke concentrations of 0.17% and 0.34% and geotrichum sp. ap.8 endophytic filtrate concentration of 2% with a range of inhibition percentages from 42% up to 100% on 1 dai until 7 dai. however, the inhibition percentages of the combination did not differ significantly among treatment combinations. the inhibition percentages of the treatments were not significantly different when compared to the control (fig. 5). a previous study conducted by imaningsih et al. (2021) showed that the concentrations combinations between liquid smoke of “ulin” wood and endophytic fungi filtrate of “hiyung” chili significantly inhibit the growth of pathogen c. capsici compared to the control. inhibitory effect of ulin wood liquid smoke and gogo rice endophytic fungi – witiyasti imaningsih et al. 25 figure 5 inhibition percentages of several concentrations combinations between geotrichum sp. ap8 filtrate (ke) and liquid smoke (ac) against p. oryzae on 1 dai until 7 dai notes: the bar indicates the standard deviation. numbers followed by the same letter are not significantly different based on duncan test (p < 0.05). based on the results of our study, the inhibition percentage achieved when combining liquid smoke and endophytic fungi filtrate was higher compared to the inhibition percentage of only using liquid smoke at the same concentration. the inhibition percentage of combining liquid smoke and endophytic fungi filtrate was lower when compared to the inhibition percentage of only using endophytic filtrate at the same concentration. during the filtration process, there might still be fungi cells carried away, due to the pore size of the filter membrane of 0.45 microns. hyphae fragments and spores of geotrichum sp. ap8 possibly penetrates the filter pores and grows during the inhibition process against the pathogens. meanwhile, sayer et al. (1969) grouped geotrichum sp. into fungi with intermediate spore size, which size is smaller than the usual fungi spores size. it is suspected that the ability of endophytic fungi filtrate decreased due to the presence of the carried-away fungi cells during the filtration process. however, those carriedaway fungi cells died in the presence of the liquid smoke. this was confirmed by a study conducted by oramahi et al. (2011) as well as oramahi and yoshimura (2013) which showed that liquid smoke possesses antifungal properties because it contains phenol, carbonyl and acid compounds. in addition, acetic acid and propionate components are able to neutralize fungi cells and inhibit enzyme activity (karseno et al. 2001). therefore, in addition to inhibiting the growth of pathogenic fungi, liquid smoke is also suspected to inhibit endophytic fungi. conclusion liquid smoke of “ulin” wood and endophytic fungi of “gogo” rice var. maninjau has the ability to inhibit the growth of pathogen p. oryzae. concentrations of 0.17% to 1.75% liquid smoke and 2% to 10% endophytic fungi filtrate showed high inhibition percentage against pathogen p. oryzae. the test of synergism, however, did not show an increase in inhibition percentage. further research on the inhibitory ability and the best concentrations of liquid smoke and endophytic fungi filtrate should be conducted to provide more protection against plant pathogens, especially for rice plants. acknowledgments the authors sincerely thank the ministry of research, technology and higher education of the republic of indonesia who has funded this research (2018-2019), as well as to the talasiana business group, ranggang village, tanah laut regency. our sincere thanks go to mrs. tambuy biotropia vol. 29 no. 1, 2022 26 as an elder of dayak maanyan community, jaar village area, east dusun district, east barito regency, central kalimantan province. references andriani y. 2008. budidaya tanaman padi di indonesia. jakarta (id): sastra hudaya. barnett hl, hunter bb. 1998. illustrated genera of imperfect fungi (4th ed.). st. paul (us): american phytopathological society (aps press). retrieved from https://www.academia. edu/35499449/ illustrated_genera_of_imperfect_fungi_fourth_edi tion_barnett_y_hunter_pdf_pdf. bppp jateng. 2014. kumpulan deskripsi varietas padi. semarang (id): balai penelitian dan pengembangan pertanian. darmadji p. 2002. optimasi pemurnian asap cair dengan metode redistilasi. j teknologi industri pangan 13:267-71. hairmansis a, yullianida y, supartopo s, suwarno s. 2017. rice improvement for upland areas. iptek tanaman pangan 11(2):95-106. retrieved from http://www.ejurnal.litbang.pertanian.go.id/index. php/ippan/article/view/6078. hayashi k, yoshida t, hayano-saito y. 2019. detection of white head symptoms of panicle blast caused by pyricularia oryzae using cut-flower dye. plant methods, 15(1), p. 1-9. imaningsih w, mariana, junaedi ab, rasyidah. 2021. antifungal activities of the combination of ulin wood liquid smoke and hiyung cayenne pepper root endophyte fungi against colletotrichum capsici. agrivita 43(1):69-78. https://doi.org/10.17503/ agrivita.v1i1.2458. junaidi ab, apriyani h, abdullah, santoso ut. 2019. fraksinasi dan karakterisasi asap cair dari kayu ulin (eusideroxylon zwageri teijsm. & binn.) sebagai pelarut kitosan. 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[metabolite endophytic mushrooms of rice plants as an alternative to control pathogenic mushrooms carried by rice seeds]. jurnal fitopatologi indonesia. 12(2):53-61. white jf, kingsley kl, zhang q, verma r, obi n, dvinskikh s, …, kowalski kp. 2019. review: endophytic microbes and their potential applications in crop management. pest manag sci 75(10):2558-65. https://doi.org/10.1002/ps.5527. the southeast asian journal of tropical biology vol. 31 no. 3, 2024: 359 371 doi: 10.11598/btb.2024.31.3.2267 issn: 0215-6334 | e-issn: 1907-770x 359 ecological indices of mangrove gastropod community in nickel mining impacted area of pomalaa, southeast sulawesi muhammad fajar purnama1,2*, slamet budi prayitno3, max rudolf muskananfola3 and suryanti suryanti3 1doctoral program of aquatic resources management, faculty of fisheries and marine science, diponegoro university, semarang 50275, indonesia. 2aquatic resources management, faculty of fisheries and marine science, halu oleo university, kendari 93232, indonesia 3aquatic resources management, faculty of fisheries and marine science, diponegoro university, semarang 50275, indonesia article higlights • gastropods (invertebrates: mollusca) have the potential to be developed as biological indicators of the health of coastal aquatic environments • the development of gastropod communities has been proven to provide a significant ecological response in assessing environmental quality in the mangrove in relation to overburden waste input from nickel mining activities. • overburden waste systemically degrades the ground floor (substrate) of the mangrove ecosystem as an area where the entire life cycle of the gastropod community is carried out. article information received : 24 may 2024 revised : 19 july 2024 accepted : 21 july 2024 *corresponding author, e-mail: muhammadfajarpurnama@students.undip. ac.id; muhammadfajarpurnama@uho.ac.id research article abstract this research aimed to determine the structure of gastropod community in the nickel mining impact area in the mangrove ecosystem of tambea village, pomalaa district, southeast sulawesi province. the scope of this research was gastropod community influenced by nickel mining activities, especially the impact of overburden waste input (reddish-orange colored sediment) toward the health status of the aquatic environment based on the ecological indices of the gastropod community in the mangrove ecosystem of tambea village. two sampling methods were adopted in this research: (1) purposive sampling method to determine stations (locus) and (2) simple random sampling method to determine the distribution of substations or sampling points. gastropod samples were taken using handpicking techniques. the structure of gastropod community in mangrove area affected by overburden waste showed low diversity index values (h’ = 0.81), low species richness (r = 1.75), moderate evenness (e = 0.50), and dominance of certain species (c = 0.54). the results of this research showed the massive impact of overburdened waste, which can systemically degrade the life of the typical fauna that make up the mangrove ecosystem. three gastropods species were observed to live in the research location, namely telescopium telescopium, terebralia sulcata, and terebralia palustris having low abundance (1-9 ind./m2) which can survive in environment exposed to overburden waste. many of gastropod species were found dead, indicated by the finding of 2 shells of ellobium aurisjudae. the input of overburdened waste may imply degradation system of the aquatic environment, especially in the mangrove ecosystem. this research offers outlooks of overburden waste on aquatic biota in mangrove ecosystems and other complimentary ecosystems. in the end, the condition of the gastropod community in a watered area becomes a basis of the health status of the water environment. keywords: ecological index, gastropods, mangrove, nickel mining, overburden https://doi.org/10.11598/btb.2024.31.2.2267 mailto:muhammadfajarpurnama@students.undip.ac.id mailto:muhammadfajarpurnama@students.undip.ac.id mailto:muhammadfajarpurnama@uho.ac.id biotropia vol. 31 no. 3, 2024 360 introduction coastal area is vulnerable to environmental changes, and so are the organisms living in the environment (boruff et al. 2005; wahyudi 2009; basir 2010). the decline in environmental quality in this region is caused by anthropogenic activities which tend to be environmentally unfriendly (singer & battin 2007; norris & thoms 1999; dziock et al. 2006). tambea village is one of the active open-cut nickel mining system operating in pomalaa district, southeast sulawesi province. the impact of nickel mining activities in tambea village is visible from changes in the environmental tone around the mangrove ecosystem and coastal areas in general. the condition of the substrate, water, and mangrove roots is orange-reddish, due to the input of overburden waste or waste rock in the form of acid mine sludge/acid rock drainage (ard) which contains low minerals (hamzah et al. 2015; hamzah 2009; zubayr 2009). overburden waste has polluted the tambea mangrove area for the last 2 decades and has accumulated ± 10-30 cm overburden waste covering the original (natural) substrate (hamzah 2009; zubayr 2009). the overburden phenomenon that has been occurring for a long time, and continues to this day, can massively degrade the habitat of gastropod communities and has implications for the decline of taxa (species) and their abundance in waters as well as resulting in the loss of species that are classified as sensitive (timm et al. 2001; chakrabarty & das 2006). the input of overburden waste into the tambea mangrove ecosystem does not only disturb the mangrove vegetation and other coastal plants, but also threatens the life stability of the gastropod community as part of the association (purnama et al. 2024a; 2024b). mangrove gastropods are known as benthic organisms (dominantly epifauna), spending the entire life cycle at the bottom of the waters or occupying the substrate as their life niche. the bottom water substrate in the mangrove ecosystem is the habitat of the gastropod community (chukaeva & petrov 2023; menon et al. 2023; laraswati et al. 2020; wahyudi et al. 2015; campbell & reece 2008). therefore, any disturbances arising from various anthropogenic activities, especially exposure to overburden waste, can cause loss of habitat and ecological niche of gastropod communities (purnama et al. 2024a). in the end, the phenomenon of overburden waste exposure can eliminate the existence of the gastropod community as one of the keystone species in its ecological environment (purnama et al. 2024b). accordingly, empirical research regarding the ecological indices of gastropods in areas with exposure to overburden waste in the mangrove ecosystem of tambea village is very important to carry out, considering that research related to this has never been carried out in southeast sulawesi in particular and indonesia in general. apart from that, this research has important value as a source of renewable novel scientific information about gastropod communities that can survive in areas exposed to overburden waste from nickel mining activity. this research aimed to determine the ecological indices of gastropod communities in mangrove areas exposed to overburden waste in tambea village, pomalaa district, southeast sulawesi province. materials and methods materials and tools materials used in this research were distilled water and 70% alcohol (to preserve the samples). meanwhile, tools used in this research were quadrat transects, gloves, hand scoops, plastic bags to store samples, sample baskets, label paper, and an instrument for taking samples in the field. research sites the research was carried out in mangrove ecosystem in tambea village, pomalaa district, kolaka regency, southeast sulawesi province. the research location is directly connected to the upstream flow, where the existing nickel mining area is located, which carries overburden waste. the research location (-4.219927, 121.584465) was transformed into station dimensions, totaling 5 sample areas spread across the tambea village mangrove ecosystem. these 5 stations were representatives of the entire extent of the mangrove ecosystem at the research location. an overview of the research location is presented in figure 1. mangrove gastropod communities in nickel mining impact areas purnama et al. 361 figure 1 research location in the tambea mangrove ecosystem which is exposed to overburden waste from nickel mining activities description of the problem or reason for the research gastropods live in their natural environment as fungivores, carnivores, omnivores scavengers, and detritivores based on their food habits and have a role as prey or food for several fish species (mangrove jack), birds (poultry), and crustaceans in the mangrove ecosystem (lalita 2016; alongi 2009; cappenberg et al. 2006). all biological activities related to food habits and ecological roles take place on the ground floor or substrate of the mangrove ecosystem (benthos-epifauna) (chukaeva & petrov 2023; menon et al. 2023). continuous exposure to overburden waste can permanently damage the residential habitat of most gastropod communities (purnama et al. 2024a). moreover, the input of overburden waste has been ongoing for approximately 2 decades. in other words, the loss of natural substrate which is covered by overburden sediment waste, directly affects the life system of gastropod community, considering that the activities that support the gastropod’s survival are carried out at the bottom of the waters (purnama et al. 2024a; 2024b). therefore, this research needs to be carried out as updated empirical information regarding taxa and ecological indices of gastropod communities that live in mangrove areas with overburden waste exposure. gastropods exposed to overburden waste has the shell appearance that tend to be corrosive, as is shown in figure 2. biotropia vol. 31 no. 3, 2024 362 figure 2 condition of mangrove gastropods exposed to overburden waste data collection methods data of ecological indices in this study was obtained through sampling activities applied in gastropod community at the research location. purposive sampling method was used to determine research location, based on the presence of mangrove areas exposed to overburden waste in tambea village. subsequently, the distribution of sampling points (substations) at the research location (station) was determined using a simple random sampling technique to cover a wider sampling area and increase the possibility of obtaining a greater number of gastropod samples. simple random sampling is a technique where sample elements are randomly selected from a homogeneous population, giving each component an equal chance of being selected (odum 1993). mangrove gastropods are very easy to collect when the waters are receding, because they have a dominant life habit of sticking to the roots, stems, and leaves of mangroves, while the rest are usually found on the ground floor (water substrate) as benthic epifauna organisms. therefore, the technique for collecting gastropod samples at each station was the handpicking method or handpicked without special fishing equipment. data analysis data obtained from sampling results were then analyzed to obtain the following ecological indices. 1. gastropod density was calculated using formulation according to yasman (1998): where: a = abundance (ind./m2 ) xi = number of individuals (ind.) ni = sample plot size (m2) 2. gastropod species diversity was determined by using the shannon-wiener diversity index (odum 1993): a = xi ni h' = ∑ �� ni n � x ln ni n ��� i=1 s mangrove gastropod communities in nickel mining impact areas purnama et al. 363 where: h’ = diversity index ni = number of individuals of the ith species n = total number of individuals according to wilhm (1975) the diversity index criteria are divided into 3: h’ < 1.0 = low species diversity 1.0 < h’< 3 = moderate species diversity h’ > 3 = high species diversity 3. gastropod uniformity index was calculated using the formula according to odum (1993): where: e = evenness index h’ = diversity index s = number of species criteria for the evenness index value are as follows: e < 0.31 = low level of species evenness 0.31 > e > 1 = medium level of species evenness e > 1 = high level of species evenness 4. species richness index (margalef type wealth index) was calculated based on the formula according to ludwig & reynolds (1988), as follows: where: s = number of species n = number of individuals criteria for the margalef type wealth index value are as follows: d < 2.5 = low level of wealth 2.5 > d > 4 = medium level of wealth d > 4 = high level of wealth 5. dominance index was calculated using the formulation according to odum (1993): where: ni = number of the ith individual n = the total number of individuals of all species criteria for the dominance index are: 0 < c < 0.5 = no species dominates 0.5 < c < 1 = there is a species that dominates e = h' ln s r = (s 1) ln n c = ni n∑ � � results and discussion there are only 3 gastropod species in the mangrove with a fairly good density (2-3 ind./25 m2) namely terebralia sulcata, terebralia palustris, and telescopium telescopium. this condition is commensurate with the distribution of gastropod ecological indices in the tambea mangrove area in the "low category". the ecological indices of gastropods in the existing nickel mining area in tambea village, pomalaa is presented in table 1. table 1 ecological indices of gastropod community in the existing nickel mining area in tambea village, pomalaa-southeast sulawesi no. ecological index value category 1 diversity (h’) 0.81 low diversity 2 species richness (r) 1.75 low species richness 3 evenness (e) 0.50 medium evenness 4 dominance (c) 0.54 there is a species that dominates table 1 reflected in the condition of the ecological indices which have values and categories of low species diversity and richness, moderate evenness, and dominance of certain species. values of these ecological indices may imply degraded condition, especially on the "ecological floor (substrate)" which is a living niche and food source for the entire gastropod community due to the continuous input of nickel mining overburden waste. the structure of the gastropod community in mangrove area showing low diversity index values (h' = 0.81), low species richness (r = 1.75), moderate evenness (e = 0.50), and dominance of certain species (c = 0 .54), maybe affected by overburden waste. the dominance of the potamididae family and the very low presence of individual species in populations and communities means that the ecological indices of gastropods in the tambea mangrove ecosystem are in a low category which may be attributed to the ecological disturbance and pressure. ernanto et al. (2010) stated that if a community is composed of a very few species and if only a few species are dominant then the species diversity is low. if a community growth is disrupted, it will cause a significant reduction in diversity (wirakusumah 2003). likewise, when diversity is low, there will be intraand interspecific competition and high environmental pressure/disruption. mustofa et al. (2023) explained that low diversity values may have been caused by ecosystem imbalances due to biotropia vol. 31 no. 3, 2024 364 environmental pressure, resulting in only certain species being able to survive. furthermore, according to clark (1974) in budi et al. (2012), the diversity expresses the variety of species existed in an ecosystem. if an ecosystem has a high diversity index, then the ecosystem tends to be balanced. on the other hand, if an ecosystem has a low diversity index, it indicates that the ecosystem is in a state of stress or degradation. rau et al. (2013) explained that the wealth index value tends to be high if a community has a large number of species and each type is represented by one individual, whereas the index value will be low if the community has a relatively small number of species and each type has a large number of individuals. the evenness index value in this study fell in the medium category, with a tendency for low values (close to the low evenness category) and there was a dominance of a certain species. t. sulcata showed a wide distribution in the tambea mangrove area and were found in most sampling stations. it is peculiar to find t. sulcata climbing mangrove roots and trunks as well as small mounds around the mangrove area (fig. 5). nybakken (2005) emphasized that biotic and abiotic environmental factors will influence the abundance and evenness of species that are small and have high values due to the large similarity of species. dominance of an individual or species shows how strongly that individual dominates an area, typifying adaptation to the environment, hereby dominate the habitat (nontji 2007; odum 1993). evenness index in the low to medium category indicates that the number of gastropod species is not evenly distributed and there is no variation in the gastropod species. the smaller the value of species uniformity, the smaller the value of evenness of the aquatic community, where the distribution and number of individuals are not the same. there is also a tendency for certain species to dominate in the community (saraswati et al. 2020). variations in evenness index values are caused by environmental factors that influence gastropod adaptation. there are no climbing gastropods, such as littoraria scabra and l. melanostoma, which are usually found in the stems, roots, and leaves of mangrove vegetation. littoraria scabra and l. melanostoma are classified as sensitive to environmental disturbances and changes. the condition of gastropods in mangrove area of tambea village is very different from that in other mangrove areas in indonesia, which are not affected by overburden waste. hutama et al. (2019) reported that gastropod species found in the north coast of semarang comprised 8 species and 4 families which include four species in the order cerithiodea, including cerithidea sp., cerithidea alata, cerithidea cingulata, and telescopium telescopium. the two species found of the order littorinoidea were littoraria melanostoma and littoraria carinifera. one species found of the order neritoidea was nerita sp., and one species found of the order ellobioidea was cassidula sp. there were 14 species of gastropods found in the mangrove ecosystem of aceh besar and banda aceh (irma et al. 2012). in the awur bay mangrove forest area, 16 species of gastropods were found (silaen et al. 2013). the low abundance and ecological indices of the gastropod community in the tambea mangrove ecosystem may also be influenced by the relatively sparse condition of mangrove cover (fig. 5). the same results were shown in the research of haryoardyantoro et al. (2013), in which that mangrove area having low tree density surrounded by pond areas will directly cause a low abundance of the organism community. the condition of mangrove forest vegetation affects gastropod diversity. for example, high abundance and diversity of gastropod species are usually found in dense stands of mangrove forest trees (silaen et al. 2013). the mechanism of differences in community structure, ecological index, and abundance of gastropods related to the size of the mangrove area is a reflection of differences in the structure of the mangrove habitat (singh et al. 2020). the results of measuring several key water quality parameters that are directly related to the input of overburden waste at the research location are presented in table 2. mangrove gastropod communities in nickel mining impact areas purnama et al. 365 table 2 average of measurements results of several key water quality parameters no. stations key parameter value information 1. station 1 tss (mg/l) 214.00 exceeds threshold (acoording to decree of the state minister for the environment number 51 of 2004 concerning seawater quality standards) turbidity (ntu) 141.6 waters brightness (cm) 57.5 color of waters (pt-co) 500 2. station 2 tss (mg/l) 224.00 exceeds threshold (acoording to decree of the state minister for the environment number 51 of 2004 concerning seawater quality standards) turbidity (ntu) 139.2 waters brightness (cm) 53.5 color of waters (pt-co) 500 3. station 3 tss (mg/l) 238.00 exceeds threshold (acoording to decree of the state minister for the environment number 51 of 2004 concerning seawater quality standards) turbidity (ntu) 148.4 waters brightness (cm) 56.5 color of waters (pt-co) 500 4. station 4 tss (mg/l) 265.00 exceeds threshold (acoording to decree of the state minister for the environment number 51 of 2004 concerning seawater quality standards) turbidity (ntu) 151.5 waters brightness (cm) 59.5 color of waters (pt-co) 500 5. station 5 tss (mg/l) 246.00 exceeds threshold (acoording to decree of the state minister for the environment number 51 of 2004 concerning seawater quality standards) turbidity (ntu) 143.2 waters brightness (cm) 55.3 color of waters (pt-co) 500 the condition of tambea mangrove waters is greatly influenced by overburden waste input (table 2). this refers to the concentration of each parameter that exceeds the water quality standard threshold (decree of the state minister for the environment number 51 of 2004). as water quality parameters exceeds threshold limits, gastropods’ ecological indices are low. the gastropod community structure in the mangrove areas of the tambea village was low, due to the degrading environment caused by overburden waste input from nickel mining activities (open pit and topsoil stripping) (table 2). thus, the overburden waste input degrades the overall functionality of the entire complimentary systems causing the loss of various gastropod species, which are typical inhabitants of the mangrove ecosystem. the potamididae family remains adaptive to the conditions covered by overburden sediment waste (purnama et al. 2024a). the adaptability of the potamididae family in disturbed areas manifests its high tolerance to changes in water quality and the environment, in general (purnama et al. 2024a). apart from that, the survival of potamididae family is also related to their ability to act as a tree fauna snail or climber when there is a disturbance in the waters (t. sulcata and t. palustris). t. telescopium can immerse as an infauna, although it is considered as epifauna around the mangrove root system to protect itself from various ecological disturbances and establish its biological niches. this is in accordance with the findings of maturbongs & elviana (2016) who reported that the mangrove ecosystem is the most preferred habitat for the potamididae family and has a wide geographical distribution and is also found in high densities in the mangrove ecosystem. odum (1998) also explained that a species with the highest density indicates that the organism can occupy a larger space and thus, can develop more. the potamididae family is the only family of gastropods that natively inhabits mangroves (egonmwan 2008; jamabo & davids 2012). therefore, the potamididae family has a very high level of dependence on the existence of mangroves for their subsistence needs and has the potential to be an indicator of the mangrove ecosystem (vannini et al. 2008; penha-lopes et al. 2010). native gastropods are a species of snail whose entire life cycle occurs in the mangrove area, while facultative gastropods have certain migration patterns to come to and leave the mangrove area (budiman 1991). the species of this family are associated with the mangrove ecosystem as a place to find biotropia vol. 31 no. 3, 2024 366 food, and shelter, as well as a place for spawning and rearing (wells 2003; wells & lalli 2003; kamimura & tsuchiya 2004; fratini et al. 2008; lorda & lafferty 2012). the average abundance of gastropod species in areas is presented in figure 3. average abundance of gastropod species in the area affected by overburden (fig. 3) shows the dominance of terebralia sulcata (6-9 ind./m2) over the other two types of gastropods (1-3 ind./m2). fifty percent (50%) of the coastal tambea village is dominated by mangrove vegetation. ecological functions of mangrove roots systems enable them to trap sediment outflows coming from the mining discharges resulting to the accretion of sediments in the mangrove areas. this condition may have direct impact on the presence of gastropods which is very limited in the sapling age group, except for the t. sulcata which is found on each side of the tambea mangrove area. t. sulcata tends to avoid puddles and high tides by climbing the roots, stems and dry branches of mangroves as well as mounds which makes this species able to survive the input of overburden waste (fig. 5). the presence of gastropod communities in the mangrove ecosystem of tambea village, pomalaa is very low. this can be seen from the individual representation of each species in ecological space, where of the 3 species found (t. sulcata, t. palustris, and t. telescopium) only t. sulcata has a population abundance that tends to dominate, namely 6-9 ind./ m2. meanwhile, the dominance index of the other two species ranged from 1-3 ind./m2. in other words, the representation of individual t. palustris (1-3 ind./m2) and t. telescopium (1-2 ind./m2) in an area of 1 m2 is very low (fig. 3). meanwhile, according to arbi et al. (2022), in the mangrove ecosystem of pari island, jakarta, t. palustris tends to be present in large aggregations, and t. sulcata is generally found solitary. in contrast to those in the tambea mangrove area, it is t. sulcata that has a greater population abundance. this finding is in agreement with research findings of masagca et al. (2010) who reported that the high-density value of t. sulcata is caused by t. sulcata lives and develops in mangrove areas in large numbers, especially on most types of muddy substrates. these results are supported by the explanation of dharma (1988) and budiman (1988) who reported that mangrove forests are the most preferred habitat for the potamididae family which have a wide geographical distribution and are also found in high abundance in the mangrove ecosystem. figure 3 average abundance of gastropod species in areas affected by overburden in the tambea, pomalaa mangrove ecosystem mangrove gastropod communities in nickel mining impact areas purnama et al. 367 figure 4 the sapling phase dominates the estuary mouth area, so that the effectiveness of the mangrove forest's function as a sediment trap is not optimal figure 5 life habits of t. sulcata at the research location, avoiding puddles and high tides by moving to higher areas and climbing mounds around the mangrove area (5-10 m from the mangrove vegetation area) figure 5 shows the extent of the climbing behaviors of t. sulcata moving within the mangrove areas by climbing roots and stems/twigs. it was also observed outside the mangrove area even to the coastal beach vegetation. this behavior maybe a form of adaptation to the environment enabling them to survive and dominate in the tambea mangrove ecosystem. the condition of the mangrove forest and the gastropods species found in the mangrove forest are shown in figure 6. biotropia vol. 31 no. 3, 2024 368 figure 6 tambea village mangrove ecosystem, pomalaa and the gastropod speices found in tambea mangroves ecosystem: (a) telescopium telescopium; (b) terebralia sulcata; (c) terebralia palustris; and (d) ellobium aurisjudae found in dead condition (empty shell) figure 7 small estuary which drains overburden waste originating from the upstream nickel mining activities notes: (a) estuary (canal), runoff area; (b) overburden sediment waste (reddish-orange). mangrove gastropod communities in nickel mining impact areas purnama et al. 369 community structure of gastropods in the tambea mangrove ecosystem is very low compared to 61 species of mangrove gastropods identified throughout indonesia (pramudji 2001). meanwhile, the ellobium aurisjudae species was not included because it was found as an empty shell and it may have been dead for a long time. death of some gastropods may likely to occur because this area receives input of overburden waste (fig. 1) from river flows that are directly connected to nickel exploitation locations in the upstream mountainous areas (purnama et al. 2024a; hamzah 2009; zubayr 2009). the small river flow carrying overburden waste is presented in figure 7. the small estuary or canal shown in figure 7 is a transport medium for overburden waste from nickel exploitation activities in mountainous areas. these photos were taken during the dry season and the water discharge was relatively low, with several streams still continuously carry overburden waste. during the rainy season or in rainy condition, the water discharge was very abundant and expanded the entire tambea mangrove area as predominantly reddish-orange substrate accretion. conclusion the abundance (± 3-5 ind./m2) and number of taxa (species) (3 species, telescopium telescopium, terebralia sulcata, and terebralia palustris) of the gastropod community exposed to overburden waste were very low. overburden waste may degrade ecological indices of mangrove gastropods. the ecological indices of gastropod community in tambea mangrove ecosystem showed low diversity index (h' = 0.81), low species richness (r = 1.75), moderate evenness index (e = 0.50), and dominance of certain species (c = 0.54). acknowledgments we would like to express our infinite thanks to the management of the indonesian education scholarship (bpi) for their financial support in publishing our article, as well as to the "field sampling team" muh. bima sakti ra., s.t., irmalasandri putri, s.t., m.pwk., and virzikrillah who assisted in collecting data and research documentation at the research location, from the beginning to the end of the research. references alongi dm. 2009. the energetics of mangrove forest (1st ed.). dordrecht (nl): springer. doi: 10.1007/978-1-40204271-3 arbi uy, kawaroe m, marwoto rm, ulumuddin yi. 2022. morphological and ecological characteristics of potamididae snails (gastropoda) from the mangrove habitat of the pari island group, jakarta. national maritime journal 17(2):93-106. boruff bj, emrich c, cutter sl. 2005. erosion hazard vulnerability of us coastal countries. j coast res 21(5):932-42. doi: 10.2112/04-0172.1 budiman a. 1991. review of several ecological patterns of indonesian mangrove mollusks. dissertation. depok (id): university of indonesia. 380 p. budiman a. 1988. some aspects on the ecology of mangrove whelk telescopium telescopium (linné, 1758) (mollusca, gastropoda: potamididae). treubia 29(4):237-45. budi da, suryono ca, ario r. 2013. study of gastropod abundance in the eastern part of semarang waters for the period march-april 2012. j mar res 2(4):56-65. doi: 10.14710/jmr.v2i4.3684 basir n, taufik m, sukojo bm. 2010. model of coastal vulnerability to sea level rise using remote sensing technology, case study: bengkalis island. national postgraduate seminar x. sepuluh november institute of technology, surabaya, 4 august 2010. cappenberg haw, aziz a, aswandy i. 2006. mollusk communities in the waters of gilimanuk bay, west bali. oceanology and limnology in indonesia 40:53-64. campbell jb, reece lg, mitchel. 2008. biology. fifth edition. volume 3. jakarta (id): penerbit erlangga. chukaeva m, petrov d. 2023. assessment and analysis of metal bioaccumulation in freshwater gastropods of urban river habitats, saint petersburg (russia). environ sci pollut res 30(3):7162-72. doi: 10.1007/s11356-022-21955-8 chakrabarty d, das sk. 2006. alteration of macroinvertebrate community in tropical lentic systems in context of sediment redox potential and organic pollution. biol rhythm res 37(3):213-22. doi: 10.1080/09291010600689101 dziock f, henle k, foeckler f, follner k, scholz m. 2006. biological indicator systems in foodplains: a review. int rev hydrobiol 91(4):271-91. doi: 10.1002/iroh.200510885 dharma b. 1988. siput dan kerang indonesia (indonesia shells i). 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[thesis]. bogor (id): bogor agricultural university. 127p. the southeast asian journal of tropical biology vol. 31 no. 2, 2024: 339 348 doi: 10.11598/btb.2024.31.2.2207 issn: 0215-6334 | e-issn: 1907-770x 339 surveillance of β-lactamase genes and antimicrobial resistance in salmonella sp. from chicken meats traded in wet markets of metro manila, philippines miles g. madayag, rance derrick n. pavon, jonah feliza b. mora, khristine b. balaga, and windell l. rivera* pathogen-host-environment interactions research laboratory, institute of biology, college of science, university of the philippines diliman, quezon city 1101, philippines article higlights • blactx-m gene in 24.7% while blatem gene in 11.2% of salmonella isolates. • coexistence of blactx-m groups ii and iv in all blactx-m-positive isolates. • dominance of blactx-m corroborated with phenotypic β-lactam resistances. • blactx-m-positive isolates had extendedspectrum β-lactamase and multidrug resistance. article information received 27 february 2024 revised 14 july 2024 accepted 22 july 2024 reviewers: saharuetai jeamsripong, ida bagus gede darmayasa *corresponding author, e-mail: wlrivera@science.upd.edu.ph research article abstract salmonella sp. is a foodborne pathogenic bacterium causing millions of cases with hundred thousand death incidents. infection by salmonella can diversely manifest as gastroenteritis, bacteremia, and enteric fever. salmonella can be transmitted through direct consumption of contaminated foods especially animal-based foods, such as chicken meat and its derivatives. over the years, antimicrobial resistance (amr) and diverse β-lactamase (bla) gene-carrying salmonella strains have been reported. these facts are alarming given that cephalosporins are a major class of β-lactam antibiotics used in clinical settings. hence, the main objective of this study was to molecularly detect the occurrence of different bla genes by polymerase chain reaction (pcr) and profile the phenotypic antimicrobial susceptibility of salmonella collected from various chicken sample types in wet markets of metro manila, philippines. of the 89 salmonella isolates, blactx-m had the highest occurrence, detected in 22 isolates (24.7%), while blatem was detected in 10 isolates (11.2%). genotypic and phenotypic resistance corroboration was observed in nearly all blactx-m-positive salmonella tested, with all strains showing resistance to ampicillin and nitrofurantoin (100%) and 21 out of 22 (95.5%) exhibiting resistance to both non-extended and extendedspectrum cephalosporins. in addition, blactx-m groups ii and iv genes were co-detected and multidrug resistance (mdr) profiles were also observed in all blactx-m-positive isolates. the high amr patterns of salmonella isolates suggest potential threats to food safety and public health. additionally, the corroboration of phenotypic and genotypic resistance and the high occurrence of mdr among salmonella isolates highlight the importance of continued surveillance of amr genes and regulation of antimicrobial use to combat amr. keywords: antimicrobial resistance, blactx-m groups, bla genes, occurrence, salmonella introduction salmonella sp. is a gram-negative, facultative anaerobic, mesophilic, rod-shaped foodborne bacterium that belongs to the family enterobacteriaceae (ethelberg et al. 2014). given that poultry is a significant reservoir for salmonella, the risk of transmission increases through improper handling, trade, or slaughter of raw chicken (eng et al. 2015). infection can occur through the fecal-oral route, involving the ingestion of contaminated food, especially animalbased foods, such as chicken meat, or through water containing salmonella (eng et al. 2015). due to their easy transmission from raw/improperly cooked meat, the high consumption of chicken in the philippines can pose a risk for salmonella infection. in the philippines, the average annual poultry consumption per capita in the period of 2009-2018 was 13.4 kg (da-bai 2022). https://doi.org/10.11598/btb.2024.31.2.2207 biotropia vol. 31 no. 3, 2024 340 although salmonella consists of only two species, enterica and bongori, with s. enterica containing six subspecies, most infections are caused by s. enterica subsp. enterica (desai et al. 2013). infections can manifest as enteric fever, gastroenteritis, bacteremia, or a chronic carrier state depending on the salmonella serovars. enteric fever is caused by s. typhi or s. paratyphi a, b, and c which are typhoidal salmonella. all other salmonella strains are designated as nontyphoidal salmonella (nts), which causes mild infections, including gastroenteritis. although typhoidal salmonella is more likely to follow a human-tohuman transmission route, nts transmission is more likely associated with animal reservoirs, such as contaminated chicken meat. the most common serovars responsible for nts are s. enteritidis, s. typhimurium, and s. newport. the symptoms of enteric fever include headache, abdominal pain, diarrhea/constipation, fever, rose spots, and in severe cases, bloody diarrhea. conversely, the symptoms of gastroenteritis are usually selflimiting and include headache, abdominal cramps, vomiting, non-bloody diarrhea, nausea, and muscle ache (eng et al. 2015). nts and typhoidal salmonella may eventually lead to bacteremia and a chronic carrier state. bacteremia occurs when salmonella penetrates the intestinal barrier and invades the bloodstream, while a chronic carrier state is characterized by fecal shedding of salmonella more than one year after the acute stage of salmonella infection (eng et al. 2015). in 2017, there were approximately 14.3 million cases of typhoidal salmonella and approximately 95.6 million cases of nts globally (stanaway et al. 2019). approximately 136,000 died from typhoidal salmonella in the same year, and 128,000 died from nts (stanaway et al. 2019). in the philippines, santos et al. (2020) found s. enterica in meat sold at wet markets in metro manila, highlighting the need to investigate the presence of bla genes among s. enterica isolates. due to numerous salmonella-related deaths annually, antibiotic treatment becomes important for treatments against invasive diseases. extendedspectrum cephalosporins (esc) are some of the antibiotics used to treat salmonella infections (calayag et al. 2021). the treatments for nts also include ciprofloxacin and ceftriaxone, and for severe complications, may include cefixime and cefotaxime (gut et al. 2018). cephalosporins rely on cell wall synthesis interference through the inhibition of transpeptidases, which causes bacterial cell lysis and death (cantón 2007). however, antimicrobial resistance (amr) threatens the efficacy of these treatments. some salmonella strains have acquired resistance to these antibiotics because of their ability to produce β-lactamase enzymes that hydrolyze class a β-lactam antibiotics, including cephalosporins (cantón et al. 2012). β-lactamase enzymes, such as temoneira (tem), cefotaximase (ctx-m), and sulfhydryl variable (shv) are encoded by blatem, blactx-m, and blashv genes, respectively, and are among the most common esbl genes (ejaz et al. 2021). additionally, blactx-m genes are highly diverse and prevalent among enterobacteriaceae, due to transmissions, mutations, and recombinations. they include numerous groups that confer resistance to different generations of cephalosporins, primarily against cefotaxime and ceftriaxone, with some enhanced variants capable of acting even against ceftazidime (rossolini et al. 2008). some extended-spectrum β-lactamases (esbls) also have the capability to hydrolyze broad-spectrum thirdand fourthgeneration cephalosporins (cantón 2007), posing a significant threat in clinical treatment settings. determining the occurrence of these genes and phenotypic resistance of s. enterica is important for understanding its transmission and dynamics. hence, this study determined the occurrence of blatem, blactx-m, and blashv, in s. enterica isolated from raw chicken from wet markets in metro manila, philippines. moreover, this study is among the first in the philippines to detect the simultaneous occurrence of blactx-m gene groups and determined the phenotypic antimicrobial susceptibility profile of s. enterica possessing those gene groups. materials and methods salmonella isolates following standard procedures under iso 65791:2017 (iso 2017) and santos et al. (2020), 25 g of raw chicken meat samples from leg, thigh, and breast parts were aseptically minced and placed in sterile whirl-pak® bags. then, 225 ml of buffered peptone water (bpw) (bd diagnostics system, nj, usa) was added, followed by homogenization for 30 seconds and incubation at 37 °c for 18-24 hours. subsequently, 100 μl of the bpw culture was transferred to 9 ml of rappaport vassiliadis (rv) (bd diagnostics system, nj, usa) and incubated at 42 °c for 18-24 hours. the resulting rv cultures were then streaked on xylose lysine deoxycholate β-lactamase genes and antimicrobial resistance in salmonella from chickens in philippine wet markets madayag et al. 341 (xld) agar (bd diagnostics system, nj, usa) and incubated at 37 °c for 18-24 hours. black colonies on red xld agar were then subcultured on nutrient agar (na) (bd diagnostics system, nj, usa) plates and incubated at 37 °c for 18-24 hours. the resulting colonies were then subjected to dna extraction, salmonella confirmation, and bla gene detection. a total of 89 s. enterica isolates from five cities in metro manila, namely, quezon, manila, pasay, malabon, and valenzuela, were randomly selected. dna extraction three to four colonies from na were transferred into 100 μl of 1× te buffer (10 mm tris, 1 mm edta at ph 8.0) for dna extraction through boil lysis. the suspension was boiled at 100 °c for 10 minutes on a heat block to lyse bacterial cells. after boil lysis, suspensions were subjected to centrifugation at 6,000 rpm for 5 minutes (calayag et al. 2017). the supernatant containing dna extract was transferred/decanted into a sterile microcentrifuge tube and stored at −20 °c prior to use in further experiments. pcr confirmation of s. enterica each pcr reaction consisted of 1 µl of previously extracted dna as the template, 6.25 μl of gotaq® g2 green master mix (promega, wi, usa), 0.5 μl each of 10 μm forward and reverse primers for inva gene, and 4.25 μl of nuclease-free water. dna extracted from s. enterica subsp. enterica american type culture collection (atcc) 14028 served as the positive control, and escherichia coli atcc 25922 served as the negative control (ng & rivera 2015). a no template control (ntc) was used to check for pcr master mix contamination with nucleasefree water to substitute the dna template. the details of the primer sequence, amplicon size, pcr protocols, and references for the inva gene for s. enterica detection are shown in table 1. multiplex pcr of bla genes inva-positive (confirmed salmonella) dna samples were subjected to pcr-based blatem, blactx-m, and blashv detection. the volumes and concentrations of mytaqtm hs red mix (bioline, london, uk), forward and reverse primers, and nuclease-free water were the same as those for the inva gene. the details of the primer sequence, amplicon size, pcr protocols, and references for the bla genes are also shown in table 1. for blatem and blactx-m, the positive controls were s. enterica isolates from the pathogen-host-environment interactions research laboratory (pheirl) that tested positive for these genes (calayag et al. 2021) and the blashv-positive control was klebsiella pneumoniae atcc 700603. the negative control for all bla genes was e. coli atcc 25922 (pitout et al. 2004). an ntc was also used to check for pcr master mix contamination. pcr products were then subjected to agarose gel electrophoresis (age). pcr detection of blactx-m gene groups singleplex pcr was performed for blactx-m groups i, ii, and iv because of the similarity in their molecular weight. each pcr reaction consisted of 1 µl of previously extracted dna as the template, 6.25 μl of gotaq® g2 green master mix, 0.5 μl each of 10 μm forward and reverse primers, and 4.25 μl of nuclease-free water. the details of the primer sequence, amplicon size, pcr protocols, and references for blactx-m groups are shown in table 1. positive controls were isolates that show positive result in pcr amplification and sequenced for confirmation, while e. coli atcc 25922 served as the negative control. an ntc was also used to check for pcr master mix contamination. pcr products underwent age to visualize results. to confirm pcr results, representative amplicons from each blactx-m group underwent sanger sequencing (macrogen, inc., south korea). sequence trimming and alignment were performed using bioedit v 7.2.5 and mega v 11.0.13, respectively. the identities of the nucleotide sequences were confirmed using the basic local alignment search tool (blast) on the national center for biotechnology information website (http://www.ncbi.nlm.nih.gov/blast). agarose gel electrophoresis pcr products were visualized through age in 2% (w/v) agarose (vivantis, malaysia) stained with gelred® nucleic acid gel stain (biotium, ca, usa). amplicons were separated under 280 v for 30-40 minutes. pcr product molecular weight was estimated using hyperladder™ 100bp (bioline, meridian bioscience, london, uk). biotropia vol. 31 no. 3, 2024 342 table 1 primers, product size, pcr conditions, and references of inva and bla genes gene primers (5′–3′) product size (bp) pcr condition reference blactx-m f: atg tgc agy acc agt aar gtk atg gc r: tgg gtr aar tar gts acc aga ayc agc gg 593 initial denaturation: 95 °c (3 minutes) 30 cycles denaturation: 95 °c (30 seconds) annealing: 55 °c (30 seconds) extension: 72 °c (1 minute) final extension: 72 °c (10 minutes) monstein et al. (2007)blatem f: tcg ccg cat aca cta ttc tca gaa tga r: acg ctc acc ggc tcc aga ttt at 445 blashv f: atg cgt tat att cgc ctg tg r: tgc ttt gtt att cgg gcc aa 747 blactx-m group i f: gac gat gtc act ggc tga gc r: agc cgc cga cgc taa tac a 499 initial denaturation: 96 °c (3 minutes) 30 cycles denaturation: 96 °c (30 seconds) annealing: 56 °c (30 seconds) extension: 72 °c (1 minute) final extension: 72 °c (10 minutes) pitout et al. (2004) blactx-m group ii f: gcg acc tgg tta act aca atc c r: cgg tag tat tgc cct taa gcc 351 blactx-m group iv f: gct gga gaa aag cag cgg ag r: gta agc tga cgc aac gtc tg 474 initial denaturation: 96 °c (3 minutes) 30 cycles denaturation: 96 °c (30 seconds) annealing: 60 °c (30 seconds) extension: 72 °c (1 minute) final extension: 72 °c (10 minutes) inva f: aca gtg ctc gtt tac gac ctg aat r: aga cga ctg gta ctg atc gat aat 244 initial denaturation: 95 °c (2 minutes) 30 cycles denaturation: 95 °c (30 seconds) annealing: 60 °c (30 seconds) extension: 72 °c (30 seconds) final extension: 72 °c (5 minutes) chiu & ou (1996) β-lactamase genes and antimicrobial resistance in salmonella from chickens in philippine wet markets madayag et al. 343 antimicrobial susceptibility tests the vitek® 2 compact 60 id/ast system (ast-gn70 card panel, biomérieux, marcyl’étoile, france) was used to generate antimicrobial susceptibility profiles of 22 isolates to 16 antimicrobial agents including ampicillin, ampicillin/sulbactam, piperacillin/tazobactam, cefazolin, ceftriaxone, cefepime, aztreonam, ertapenem, meropenem, amikacin, gentamicin, tobramycin, ciprofloxacin, tigecycline, nitrofurantoin, and trimethoprim/ sulfamethoxazole and an esbl test. the esbl test included the following antimicrobials alone and in combination with clavulanic acid: cefepime, ceftriaxone, and ceftazidime. preparation of isolates for vitek® 2 followed standard procedures (calayag et al. 2021). an isolate was considered multidrug-resistant (mdr) when it displayed nonsusceptibility to at least one antimicrobial agent in three or more antimicrobial categories (magiorakos et al. 2012). interpretive criteria and breakpoints were based on the clinical and laboratory standards institute (clsi) (2022) 32nd edition. the negative control used was s. enterica atcc 25241. results and discussion the occurrence of bla genes in s. enterica from wet market chicken samples across the five metro manila cities is summarized in table 2. none of those isolates showed positive results for blashv gene. the occurrence of blatem and blactx-m genes in the tested isolates were 10 (11.24%) and 22 (24.72%), respectively. the highest occurrence for blatem was found among the isolates sampled in valenzuela city, while for blactx-m, isolates sampled from quezon city showed the highest occurrence. the blactx-m gene showed the highest prevalence among isolates in this study, which contrasted with some studies among enterobacteriaceae. most studies reported blatem as the most predominant bla gene over blactx-m and blashv among salmonella isolated from raw poultry meat and fecal samples from diarrheic children (coculescu et al. 2015; wu et al. 2015; ghazaei 2018; sales et al. 2021). in the philippines, cruz & hedreyda (2017) showed the occurrence of blatem, blactx-m, and blashv in β-lactam-resistant clinical e. coli isolates at 56.3%, 18.3%, and 11.3%, respectively. although the frequency estimates of blashv, blatem, and blactx-m exist for e. coli, the surveillance of the relative frequencies of these genes within the philippines is limited among salmonella isolates. calayag et al. (2021) estimated the frequency of bla genes in salmonella among hog tonsils and jejunum isolates from slaughterhouses in metro manila, wherein blatem was detected with the highest frequency, followed by blactx-m. however, the only detected blactx-m subtypes were blactx-m-1 from group i and blactx-m-2 from group ii (calayag et al. 2021). this shows that the occurrence of bla genes varies across different geographical areas and sample sources. there are also some studies on other enterobacteriaceae that were consistent with this study. gundran et al. (2019) showed frequencies of 89.9%, 58.0%, and 27.5%, respectively for blactx-m, blatem, and blashv genes, among e. coli from poultry. similarly, li et al. (2016) reported e. coli isolates from chicken fecal samples and showed that 88.8%, 66.3%, and 3.1% tested positive for blactx-m, blatem, and blashv, respectively. these results suggested the high cephalosporin resistance potential of salmonella isolated from retail poultry in metro manila. upon detection of blactx-m groups i, ii, and iv genes, all 22 blactx-m-positive isolates were simultaneously positive for blactx-m groups ii and iv. interestingly, none of the tested samples were positive for blactx-m group i. the co-carriage of these blactx-m gene groups had been previously reported and was consistent with findings in other studies (he et al. 2013; li et al. 2016; gundran et al. 2019). to confirm the blactx-m group gene identities, pcr products of dna templates of a blactx-m group i positive control and representative salmonella isolates from blactx-m groups ii and iv underwent sanger sequencing. the blast results from the aligned dna sequences of representative bacterial isolates corresponded to the established sequences of blactx-m groups i, ii, and iv, confirming the gene identities. the presence of blactx-m groups ii and iv suggested higher resistance potential to cephalosporins which was confirmed by checking the antimicrobial susceptibility of these isolates. for phenotypic resistance, the blactx-m-positive isolates were subjected to vitek® 2 automated susceptibility testing. resistance profiles of salmonella carrying blactx-m to several antimicrobial agents are shown in table 3. all isolates (100%) displayed resistance to both ampicillin and nitrofurantoin. meanwhile, 21 isolates (95.5%) were resistant to a non-extended spectrum cephalosporin (nesc) and an esc. while escresistant isolates expressed nonsusceptibility to a third generation cephalosporin (ceftriaxone), they remained susceptible to a fourth generation https://doi.org/10.1111/j.1469-0691.2011.03570.x biotropia vol. 31 no. 3, 2024 344 cephalosporin (cefepime). the 21 isolates also displayed resistance to a monobactam and two aminoglycoside class antibiotics. interestingly, one (4.5%) blactx-m-positive isolate did not display resistance to either nesc or esc antibiotic classes; however, it was resistant to ampicillin with sulbactam, a β-lactamase inhibitor, suggesting other β-lactam resistance mechanisms. resistance rates of isolates subjected to vitek® 2 in this study were comparable to the study of calayag et al. (2017) for ampicillin, nitrofurantoin, cefazolin, gentamicin, and tobramycin. in the study of calayag et al. (2017), 70.5% of isolates are nonsusceptible to ampicillin, 93.4% to nitrofurantoin, and 100% to cefazolin, gentamicin, and tobramycin. however, the current study showed higher rates for blactx-m gene with variations in blactx-m gene groups detected. corroboration of phenotypic and genotypic resistance was observed in 21 blactx-m-positive isolates that displayed resistance to the nesc and esc antibiotic classes. this was not the case for one isolate that remained susceptible to nesc and esc. this could suggest that the one isolate susceptible to nesc and esc is carrying silent copies of blactx-m (cruz & hedreyda 2017; calayag et al. 2021). a mutation in the structural gene or regulatory region could prevent the expression of the blactx-m, which may lead to the inactivation of phenotypic resistance (cruz & hedreyda 2017). another mechanism for gene silencing is the expression of silencing proteins that interfere with the transcription of the amr genes. however, gene silencing can be conditional wherein they can be activated depending on the culture media and the presence of antimicrobial selection pressure (deekshit & srikumar 2022). in this case, it is possible that the blactx-m genes of one isolate remained silent despite the individual administration of nesc and esc antimicrobials. additionally, the esbl phenotype of this isolate could have been caused by other esbl genes instead of the detected blactx-m genes. shi et al. (2021) claimed that blactx-m groups i and iv play roles in ceftriaxone resistance mechanisms. in their study, 64.2% ctx-mproducing isolates that were resistant to ceftriaxone carried blactx-m group i genes, while 35.8% carried blactx-m group iv genes (shi et al. 2021). although excessive antibiotic use is one of the reasons attributed to the dissemination of blactx-m (cantón 2007; cantón et al. 2012), it cannot be concluded that excessive use of ceftriaxone was the main reason for the dominance of blactx-m genes in this study. since cephalosporins are not often used in philippine poultry farms (barroga et al. 2020), the high occurrence of blactx-m genes warrants further investigation. however, the high resistance rates of the isolates to aminoglycosides and nitrofuran may be associated with excessive antimicrobial usage because these antibiotic classes are among the most utilized antibiotics in philippine poultry farms alongside fluoroquinolones and tetracyclines (imperial et al. 2022). the predominance of blactx-m may also be due to the mobilization of blactx-m genes and co-selection through resistance to other antibiotics (cantón 2007; cantón et al. 2012). esc-resistant isolates may acquire amr through horizontal gene transfer from abiotic surfaces due to the prolonged survival of bacteria harboring amr genes (warnes et al. 2012; imperial et al. 2022). warnes et al. (2012) showed that cefotaximesensitive e. coli acquired a blactx-m group i gene via horizontal gene transfer on a stainless-steel surface. imperial et al. (2022) documented the acquisition of a gene that encodes resistance for macrolides, lincosamides, streptogramin b, and oxazolidinones (ermb) in bacteria from chicken fecal samples where the chicken host was not subjected to any antibiotic treatments. hence, the acquisition of the amr gene via horizontal gene transfer can be attributed to contamination of surfaces during feeding, cage cleaning, and animal handling. although blactx-m group i and iv have been considered as the most common groups of blactx-m genes (cantón et al. 2012), this study reported the absence of blactx-m group i, while blactx-m groups ii and iv have the highest occurrences. while the absence of blactx-m group i requires further investigation, it is plausible that plasmids and insertion sequences (is) played a role in the spread and co-existence of blactx-m groups ii and iv. specifically, isecp1 and is903b are associated with blactx-m group iv, while the iscr1 element is associated with blactx-m groups ii and iv (cantón 2007; cantón et al. 2012; ferreira et al. 2014; nguyen et al. 2021; shi et al. 2021). moreover, genes under these blactx-m groups ii and iv might recombine and produce novel β-lactamases as in the case of blactx-m-123, which is a hybrid of group i blactx-m-15 and group iv blactx-m-14 (he et al. 2013). this could eventually lead to more β-lactamases that can counter cephalosporins and render antibiotic treatment ineffective. β-lactamase genes and antimicrobial resistance in salmonella from chickens in philippine wet markets madayag et al. 345 additionally, carrying additional amr genes that encode the resistance to fluoroquinolones and aminoglycosides of blactx-m-carrying bacteria favors their survival, which may contribute to the spread of blactx-m (cantón 2007). co-carriage of blactx-m, blatem, and a quinolone resistance gene (qnr) were observed in s. enterica isolates of calayag et al. (2021). unfortunately, the coexistence of different amr genes in a bacterial isolate may lead to mdr. plasmids that harbor esbl genes may also carry genes encoding resistance for aminoglycosides, trimethoprim, sulfonamides, tetracycline, and chloramphenicol (paterson 2000; shi et al. 2021). this is important considering that mdr is observed in all blactx-m-positive s. enterica isolates where each isolate is resistant to at least six antimicrobial classes. however, this study is limited by not detecting other amr gene classes. the mdr profile of isolates is shown in table 4. among the 22 blactx-m positive isolates, 18 (81.8%) were also resistant to six antibiotic classes. meanwhile, three isolates (13.6%) were resistant to seven antibiotic classes and one (4.5%) was resistant to eight antibiotic classes. alarmingly, all isolates displayed esbl phenotypes. due to the high occurrences of bla genes and mdr among s. enterica isolates, it is important to promote national surveillance of amr and antimicrobial use in the agricultural and veterinary sectors to combat amr. although regulations regarding the sale, distribution, and prescription of antibiotics for animal use exist in the philippines, there is a weak implementation of standards for veterinary medicinal products and enforcement of animal antibiotic use policies (barroga et al. 2020; imperial et al. 2022). as a result, there may be a discrepancy between the declared use of antibiotics in animals and the actual use of antimicrobials in farms (imperial et al. 2022). for this reason, research in phenotypic and genotypic amr and antimicrobial use in farms is highly critical to assess the current situation of amr in the philippines. doing so will aid in identifying mdr s. enterica in poultry and implementing policies that will curb the spread of amr and mdr. continuing surveillance of amr genes can aid in policymaking that will counter the spread of amr and, hopefully, prevent cases of invasive salmonella infections that persist despite antimicrobial treatments. table 2 occurrence of blatem, blactx-m, and blashv among s. enterica isolates obtained from different markets of metro manila city no. of s. enterica isolates blatem blactx-m blashv quezon 16 0 7 (43.8%) 0 manila 16 2 (12.5%) 3 (18.8%) 0 pasay 18 0 7 (38.9%) 0 malabon 20 4 (20.0%) 0 0 valenzuela 19 4 (21.1%) 5 (26.3%) 0 total 89 10 (11.2%) 22 (24.7%) 0 biotropia vol. 31 no. 3, 2024 346 table 3 nonsusceptibility levels of 22 blactx-m-positive s. enterica isolates against different antimicrobial agents class antimicrobial % nonsusceptibility penicillin ampicillin 100 penicillin/β-lactamase inhibitor ampicillin/sulbactam 9.1 antipseudomonal penicillin/β-lactamase inhibitor pipercillin/tazobactam 0 non-extended spectrum cephalosporin cefazolin 95.5 extended-spectrum cephalosporin ceftriaxone 95.5 cefepime 0 monobactam aztreonam 95.5 carbapenem ertapenem 0 meropenem 0 aminoglycoside amikacin 0 gentamicin 95.5 tobramycin 95.5 fluoroquinolone ciprofloxacin 13.6 glycycline tigecycline 4.5 nitrofuran nitrofurantoin 100 folate pathway inhibitor trimethoprim/ sulfamethoxazole 13.6 table 4 multidrug resistance patterns of 22 blactx-m-positive s. enterica isolates multidrug resistance pattern* number of isolates pen, pen/bi, nesc, esc, mon, ami, flu, nit 1 pen, nesc, esc, mon, ami, flu, nit 1 pen, nesc, esc, mon, ami, nit, fpi 2 pen, pen/bi, flu, gly, nit, fpi 1 pen, nesc, esc, mon, ami, nit 17 notes: *pen = penicillin; pen/bi = penicillin/β-lactamase inhibitor; nesc = non-extended spectrum cephalosporin; esc = extended-spectrum cephalosporin; mon = monobactam; ami = aminoglycoside; flu = fluoroquinolone; gly = glycoside; nit = nitrofuran; fpi = folate pathway inhibitor conclusion this study reported the predominance of blactx-m and the coexistence of blactx-m groups ii and iv in all blactx-m positive s. enterica isolates. these suggest high cephalosporin resistance potential. aside from the corroboration of phenotypic and genotypic resistance among blactxm-positive isolates, mdr was also observed with esbl phenotypes detected in all isolates. given the high occurrence of bla genes, the co-existence of two blactx-m gene groups, and high phenotypic resistance among blactx-m positive s. enterica isolates, the continued surveillance of bla and other amr genes and phenotypic resistances are thus crucial to monitor the extent and dissemination of resistance and combat their emergence and spread through policy recommendations and regulations. acknowledgments we thank the department of agriculture, biotechnology program office, philippines (project no. dabiotech-r2302) for the financial support. we also acknowledge vanessa yvonne b. meclat for the technical assistance. we also state that miles g. madayag and rance derrick n. pavon contributed equally to this work. β-lactamase genes and antimicrobial resistance in salmonella from chickens in philippine wet markets madayag et al. 347 references barroga trm, morales rg, benigno cc, castro sjm, caniban mm, cabullo mfb, dorado-garcia a. 2020. antimicrobials used in backyard and commercial poultry and swine farms in the philippines: a qualitative pilot study. front vet sci 7:329. calayag amb, paclibare pap, santos pdm, bautista cac, rivera wl. 2017. molecular characterization and antimicrobial resistance of salmonella enterica from swine slaughtered in two different types of philippine abattoir. food microbiol 65:51-6. calayag amb, widmer kw, rivera wl. 2021. antimicrobial susceptibility and frequency of bla and qnr genes in salmonella enterica isolated from slaughtered pigs. antibiotics 10(12):1442. cantón r. 2007. chapter 22: epidemiology and evolution of beta-lactamases. in: baquero f, nombela c, cassell g, gutiérrez-fuentes j (editors). evolutionary biology of bacterial and fungal pathogens. washington dc (us): wiley online library. p. 249-70. cantón r, gonzález-alba jm, galán jc. 2012. ctx-m enzymes: origin and diffusion. front microbiol 3:110. doi: 10.3389/fmicb.2012.00110 chiu ch, ou jt. 1996. rapid identification of salmonella serovars in feces by specific detection of virulence genes, inva and spvc, by an enrichment broth culture-multiplex pcr combination assay. j clin microbiol 34(10):2619-22. 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abiotic touch surfaces: implications for public health. bassler b (editor). mbio 3(6). wu h, wang y, wu y, qiao j, li h, zheng s, yang b. 2015. emergence of β-lactamases and extended-spectrum β-lactamases (esbls) producing salmonella in retail raw chicken in china. foodborne pathog dis 12(3):228-34. _hlk155940337 _goback _hlk166729684 _hlk166746950 _hlk147414908 _hlk147542961 _lhts9xaqndk5 _br7pth8pbc4v _hlk169332413 biotropia vol. 29 no. 3, 2022: 263 271 doi: 10.11598/btb.2022.29.3.1768 263 yeast probiotics with potential to assimilate cholesterol invitro yan ramona1,2* and ni luh putu ariwathi1 1department of biology, faculty of mathematics and natural sciences, universitas udayana, denpasar 80361, indonesia 2integrated laboratory for biosciences and biotechnology, universitas udayana, denpasar 80361, indonesia received 21 june 2022/accepted 3 october 2022 abstract in the last two decades the use of yeasts as new probiotics has increased significantly. therefore, our current research was focused on the investigation of yeasts for novel probiotic development in bali. the main objectives of this research were to isolate and characterize yeasts isolated from ragi tape (dried mix cultures of microorganisms normally used in the fermentation of rice or cassava in indonesia) and tape ketan (fermented sticky rice) for possible use as yeast-based novel probiotics, with capability to assimilate cholesterol in vitro. in this study, the potential yeast isolates were evaluated for survival at low ph conditions (ph 2, 3, or 4) and in high levels of sodium deoxicholic (nadc), at concentrations of 0.2, 0.4, or 0.6 mm. in addition, the yeast isolates were also evaluated for their ability to assimilate cholesterol in vitro and to elucidate biotransformation of cholic acid into deoxycholic acid. this study led to 10 isolates that were resistant to ph levels of 2, 3, or 4 and to nadc at concentration of higher than 0.4 mm. most of those isolates were also found to assimilate cholesterol in vitro at the rate of between 18% and 76% in 24 hours incubation. in the biotransformation test, none of those isolates transformed cholic acid into deoxycholic acid, indicating that they are safe and have potential to be developed into novel probiotics, either for human or cattle. keywords: bali, cattle, cholesterol assimilation, probiotics, yeast introduction intestinal probiotic has been defined as beneficial microbes residing along the intestinal tract of human or animals that provide beneficial effect to their host’s intestinal health (stasiak-różańska et al. 2021). these exclude commensal microbes residing in the intestine that do not provide health benefit to their host (lorenzo & lucio 2012). such commensals can be claimed as probiotics only if their health benefit can be demonstrated to their hosts. probiotics appear for the first time in the intestinal tract of human or animals either during delivery of babies through reproductive tract of their mothers or at the first time of lactation (quin et al. 2018). depending on the types of foods in the infant period of their growth and development, alteration of microbial composition in their intestine occurs (zhang et al. 2018). the role of prebiotics and fibers in their food has also been extensively reviewed by holscher (2017) to understand the effect of prebiotics and fibers to the composition of human intestinal tract. according to zommiti et al. (2020) probiotics applied as a single or mixed culture at the rate of 109 cells per day can improve the health of animal or human digestive system by maintaining the natural balance of the microbiota in the host’s digestive tract. besides, they can suppress or even exclude pathogenic microbes entering intestinal tract through mouth (markowiak & sli˙zewska 2017). the mechanisms of pathogenic growth suppression by probiotics in-vitro or in-vivo have been extensively reviewed by maldano-galdeano et al. (2019). probiotics are normally developed from nonpathogenic microorganisms that provide positive effects on the physiology of the intestine, if they are regularly consumed at sufficient density (maldano-galdeano et al. 2019). many of them play important roles in the elimination of toxic compounds in the intestinal track (petrova et al. 2022) resulting in an improved degree of host’s digestive tract health. *corresponding author, email: yan_ramona@unud.ac.id biotropia vol. 29 no. 3, 2022 264 many probiotic species also provide functional effects, such as preventing diarrhea (mcfarland 2010), reducing cholesterol content in the blood (nocianitri et al. 2017), or inducing the immune system of their hosts (maldanogaldeano et al. 2019). besides these benefits, the role of probiotics as possible anti-cancer agents has been reviewed by bhuvan and saroj (2018) and sankarapandian et al. (2022). until recently, commercial probiotics have been dominated by lactic acid bacteria (lab) isolated from healthy infants, aged under 3 months old (ramona et al. 2015). these labs have been recognized to be nontoxic, although some of them have been reported to produce antimicrobial compounds, such as bacteriocin (gonzales-perez et al. 2018). this ability enables the labs to control the balance of normal microbiota in the intestinal tract of human or animals, so that they can prevent pathogenic microbes from infecting the host’s intestinal tract. besides lab, yeasts have recently been investigated for novel probiotic development, although research on yeast, such as saccharomyces spp., is not as intensive as the study on lab. before being developed as potential and novel probiotics, some important specific characteristics of the yeasts, such as pathogenic properties, tolerance to upper part of intestinal tract conditions (e.g. low ph and high concentration of deoxicholic acid), and ability to convert cholic acid into deoxicholic acids need to be elucidated. in addition, the probiotic candidates should be nontoxic and provide beneficial effects, such as ability to reduce blood cholesterol (nocianitri et al. 2017) content and improve immune system in their hosts (especially human) so that they can be consumed in a long period of time to derive benefits (castellano et al. 2017). based on the above rational, yeasts (saccharomyces spp.) were isolated from ragi (dried mix cultures of microorganisms normally used in the fermentation of rice or cassava in indonesia) and tape ketan (fermented sticky rice), with the main objective to investigate some important characteristics (including their ability to assimilate cholesterol) of these isolates before being developed as novel and potential probiotics, in our present study. materials and method isolation of yeasts (saccharomyces spp.) from ragi and tape ketan the yeasts, saccharomyces spp., were isolated from ragi and tape ketan obtained from traditional markets around denpasar city, bali, indonesia. the samples which had been processed through dilution and spread plate methods as specified in ramona et al. (2015) were applied in the isolation and purification of yeasts with probiotic potential. sample size of 1 g of ground ragi or tape ketan was diluted in 9 ml of saline solution to obtain dilution rate of 10-1. this was further diluted to 10-5 in the saline solution. subsequently, 100 µl of suspension was taken from test tubes with dilution rates of 10-4 and 10-5 and was spread on plates containing potato dextrose agar (pda), followed by incubation at 37 oc for 24 hours. colonies with yeast morphological characteristics were purified by conducting streak culture for single colonies and stored at -20 oc in potato dextrose broth (pdb) supplemented with 30% v/v glycerol, before being characterized for the survival test at low ph condition and survival at high concentration of deoxicholic acid as well as being tested for transformation of cholic acid into deoxicholic acid, for assimilating cholesterol in order to see the yeasts potential as novel probiotics. growth of yeast isolates at ph 7 following exposure to low ph conditions (acidic conditions) one full loop of each yeast isolate was inoculated into 5 ml of potato dextrose broth (pdb) and incubated at 37 oc for 24 hours to obtain cell density of approximately 108 cells/ml (according to mcfarland scale). a volume of 100 µl of this suspension were then transferred into 900 µl of the same medium with various ph levels, i.e., ph 2, 3, and 4, and then incubated for 3 hours at 37 oc in a water bath, centrifuged at 7,000 rpm until a pelletform was obtained. the pellet was then washed twice with 300 µl saline solution. the washed pellet was then re-suspended in 300 µl pdb at ph 7, from which 50 µl was transferred into 6.5 ml pdb at ph 7, incubated at 37 oc for 24 hours, and finally, the optical density of the yeast probiotics with potential to assimilate cholesterol in-vitro – ramona and ariwathi 265 pellet was measured at wavelength of 660 nm (od660). the survival of the yeast in this pdb medium at ph 7 after being exposed to various low ph levels in the same medium was indicated by an increase in turbidity (od reading) of the yeast suspensions. triplicate experiments per isolate were carried out to obtain representative data. survival at high concentration of sodium deoxicholic (nadc) a volume of 50 µl suspension of yeast isolates were inoculated to pdb that each containing three different concentrations of nadc, i.e., 0.2 mm, 0.4 mm, and 0.6 mm. pdb without nadc served as control. all test tubes were incubated at 37 oc for 24 hours and then measured for turbidity using a spectrophotometer at the wavelength of 660 nm (od660). triplicates per treatment were prepared to obtain representative data. an increase in od reading of the yeast isolates in pdb containing various concentration of nadc indicated the yeast’s survival in such medium following exposure with high level of nadc. in-vitro evaluation of yeast isolate’s ability to assimilate cholesterol the ability of yeast isolates to assimilate cholesterol in-vitro was conducted by following the method specified by buck and gilliand (1994). each isolate was initially grown in potato dextrose broth (pdb) at 37 oc for 24 hours. the yeast suspensions of 100 µl was next inoculated to 9.9 ml pdb supplemented with 2% sodium thioglycollate, 0.3% oxygall, and 0.10% cholesterol so that the total volume of each system was 10 ml, and then incubated at 37 oc for 24 hours, centrifuged for 20 minutes at 3,500 xg to obtain cell-free supernatant. following these steps samples were analyzed for cholesterol residue in each culture using o-pthalaldehyde method as the indicator reagent. un-inoculated medium served as control. the difference of cholesterol residue in the control and in the inoculated media was assumed as the amount of cholesterol assimilated by the yeast isolates. tests for biotransformation of cholic acid into deoxicholic acid this test adopted the method specified in uni (2012). a volume of 50 µl of yeast stock culture in pdb-glycerol medium was transferred into 5 ml pdb and incubated at 37 oc for 24 hours to produce cell suspension with approximate density of 108 cells/ml. subsequently, a suspension of 50 µl was grown at 37 oc for 24 hours in the same medium supplemented with cholic acid (ca). after that, 1 ml of the suspension was centrifuged at 5,000 rpm. a volume of 0.1 ml of the supernatant was subsequently transferred to a new eppendorf tube to which 500 µl of ethyl acetate and 20 µl hcl was added and then centrifuged at 5,000 rpm for 5 minutes. subsequently, the supernatant was evaporated at room temperature for 48 hours, and finally added with 15 µl methanol. this sample was then used for thin layer chromatography (tlc) to determine the extent of transformation. the eluent for the tlc consisted of a mixture of 10 ml cyclohexane, 15 ml ethyl acetate, and 4 ml acetic acid. before being used, this eluent was left for 30 minutes in a chamber in order to obtain saturated vapor of the mixture in the chamber. the tlc was run on a piece of aluminium silica gel, in a chamber previously prepared. samples and controls (cholic acid and deoxicholic acid) amounted at 1 µl were spotted on the aluminium silica gel and dried using a hair dryer. on the completion of the tlc run, the silica gel was removed from the chamber, air-dried at room temperature, sprayed with molibddophosporic acid, heated in an oven until black spots appeared on the silica gel, and photographed for documentation. results and discussion ten yeast isolates with potential as probiotics were successfully isolated in this study (table 1). all yeast isolates were found to ferment glucose and produce ethanol as well as gas from this metabolism. the colony morphology of our isolates was either rough or smooth, with pseudohyphal morphology. based on their cell morphology and biochemical evaluation, all isolates were preliminary identified as yeasts belonged to genus saccharomyces. these characteristics are in line with those reported by reis et al. (2014). yeasts belonging to this genus have been reported by many previous studies to have potential to be used as food supplements biotropia vol. 29 no. 3, 2022 266 for human or animal probiotic. pais et al. (2020) reported that s. boulardii plays important roles to maintain and control the balance of human or animal intestinal normal microbiota when included as food supplements. besides, yeast belongs to genus saccharomyces was also reported to prevent clostridium difficile from infecting human digestive tract (mills et al. 2018). in the recent years, the beneficial properties of such yeast strain have been extensively reviewed by tomičić et al. (2016). this strain was also engineered genetically by liu et al. (2016) so that the strain is able to produce several products of interest, including lysozyme which is important to improve and maintain intestinal tract health. this opens the possibility for our yeast isolates to be developed as potential and novel probiotics for cattle or human in the near future, although further evaluation is needed to elucidate their probiotic potencies. before being developed as novel and potential probiotics, isolates must show a high level of viability following exposure to extreme conditions of the upper part of the intestinal tract, such as low ph and high concentration of nadc). in the actual application, such probiotic candidates must flow along this part of the intestinal tract before reaching the lower part of the intestinal canal where they normally provide beneficial effects to their hosts (fuochi et al. 2015). the shown ability to transform cholic acid into deoxicholic acid must be excluded for further screening (jia et al. 2018). the yeast isolates in our study showed the capability to survive exposure to low ph conditions in-vitro (table 1). all isolates were found to grow in such medium conditions, although their growth was slightly inhibited due to 3-hour exposure to low ph conditions. the 3-hour incubation period was chosen in this study, because the probiotic candidates will experience three hour contact in the upper part of the intestinal tract when applied in the actual situation and this was in line with that reported by oozer et al. (2006). surprisingly, all isolates were found to survive in the exposure to ph 2 for 3 hours, although low turbidity was recorded after being incubated in new medium having ph 7 for 24 hours. the results of this study indicated that all yeast isolates may have the potential to be developed as probiotics, because under certain condition (such as empty stomach), the ph of the stomach may decrease to 2 or even 1.5 (beasley et al. 2015). knowledge about the mechanism by which microbial cells survive in a low ph medium is still limited. however, some studies reported that in order to survive under an extremely low ph medium, the cells must maintain their internal ph to be higher than that of their surroundings. (guan et al. 2020) by metabolizing glucose through which they derive energy so that they can activate proton pump to exclude h+ ions out from their cytoplasm. this process requires a significant amount of energy in the form of adenosine triphosphate or atp (anandakrishnan & zuckerman 2017). failure to maintain internal ph condition to be higher than that of cell’s environment will lead to cell’s death. table 1 growth of yeast isolates at ph 7 after being exposed to low ph conditions isolate codes optical density at 660 nm (od660)* control (ph 6.5) ph 2 ph 3 ph 4 n1 ++ (0.623 ± 0.141) ++(0.515 ± 0.064) ++(0.586 ± 0.057) ++(0.509 ± 0.092) n2 ++(0.653 ± 0.142) +(0.328 ± 0.057) ++(0.598 ± 0.201) ++(0.588 ± 0.061) n3 ++(0.559 ± 0.160) +(0.326 ± 0.050) ++(0.644 ± 0,177) ++(0535 ± 0.047) n4 ++(0.529 ± 0.075) +(0.339 ± 0.040) ++(0.600 ± 0.033) ++(0.585 ± 0.128) n5 +++(1.148 ± 0.291) +(0.478 ± 0.136) ++(0.530 ± 0.068) ++(0.549 ± 0.185) g1 ++(0.970 ± 0.183) ++(0.553 ± 0.060) ++(0.691 ± 0.106) ++(0.620 ± 0.203) g2 +++(1.110 ± 0.022) +(0.445 ± 0.038) ++(0.664 ± 0.340) ++(0.747 ± 0.068) g3 ++(0.865 ± 0.122) ++(0.610 ± 0.140) ++(0.750 ± 0.215) ++(0.582 ± 0.197) g4 +++(1.033 ± 0.051) ++(0.541 ± 0.154) ++(0.756 ± 0.138) ++(0.893 ± 0.029) g5 +++(1.061 ± 0.049) ++(0.514 ± 0.122) ++(0.654 ± 0.098) ++(0.628 ± 0.086) notes: * = each value ± standard deviation in table 1 is an average of triplicate measurements of optical densities (absorbance/a) of the cell suspensions which indirectly measure the growth of the cells in the low ph medium. the growth indication follows the following criteria: = od of < 0.1 (sensitive to low ph conditions); + = od of 0.1 0.5 (slightly resistant to low ph conditions); ++ = od of 0.5 1.0 (resistant to acid conditions); +++ = od of > 1.0 (highly resistant to acidic conditions). http://en.wikipedia.org/wiki/clostridium_difficile yeast probiotics with potential to assimilate cholesterol in-vitro – ramona and ariwathi 267 in addition to resistance to acidic conditions, the yeasts isolates were also found to survive in medium containing high concentration of nadc (table 2), which is also a good indication for their possible use as potential probiotic candidates. on their way to colon, the probiotic must experience an extreme condition in the small intestine where high concentration of bile is secreted by the liver (darilmaz 2013). bile is toxic for microbes because of its function as biodetergent in the intestinal tract of human and other animals (urdaneta & casadesús 2017). the effects of this bile for bacterial cells can be fatal (cell death) as it can cause disintegration of the cell membrane system (urdaneta & casadesús 2017). other mechanism by which bile can disturb cell function is related to dca uptake by the cells. once exposed to neutral ph within the cell cytoplasm, the dca will be in a undissociated form and it is difficult for the cells to pump it out using proton motive force mechanism (chatterjee et al. 2004). accumulation of this compound in the cell can cause significant damage on dna and this will result in cell death (urdaneta & casadesús 2017). all yeast isolates showed resistance to various levels of nadc as indicated by high od reading (ranging from 0.5 to 1.0) (table 2). although it was not investigated in the present study, tolerance property to nadc exhibited by our yeast isolates might be related to glycosidase activity (kwun et al. 2017), the composition of membrane protein and fatty acid (gueimonde et al. 2005), and exopolysaccharide production (nguyen et al. 2020). the ability of cells to prevent damage on their cell membrane and dna during exposure to high level of nadc also plays important roles to survive in such condition (merritt & donaldson 2009). according to kusada et al. (2021) enzyme activity (bile salt hydrolase activity) that converts the physicochemical properties of the bile salt reduces toxicity of this compound to the cells. the potential of the promising isolates to assimilate cholesterol in-vitro is shown in figure 1. all isolates showed their ability to assimilate cholesterol in the medium with various degrees of degradation rates (between 18 76% reduction/24 hours incubation). when compared to control treatment (where the reading was constant or no reduction in cholesterol in the medium), these values were statistically significant (p < 0.05). only two isolates (n1 and n5) assimilated cholesterol at the rate of less than 50% (fig. 1). the others assimilated cholesterol at the rate of between 52% and 76%, relative to control, indicating that these isolates showed potential for further probiotic development. the data presented in figure 1 is in line with that mentioned by previous studies which found that some yeast species modified fat and its derivatives in the process of fermentation. aloglu et al. (2015) reported that yeast, including saccharomyces cerevisiae, had the ability to metabolize sterol. other yeast species, such as candida lipolytica was also reported by ali et al. (2010) to metabolize fats. in addition to yeasts, bacterial probiotics (lactic acid bacteria) have also been reported by tokatli et al. (2015) as having the capability to assimilate cholesterol. this yeast characteristic is important to reduce cholesterol absorption in the small intestine, which leads to reduced level of blood cholesterol. table 2 growth of yeast isolates in a medium containing various levels of nadc isolate codes optical density at 660 nm (od660)* control 0.2 mm nadc 0.4 mm nadc 0.6 mm nadc n1 ++(0.89 ± 0.24) ++(0.97 ± 0.25) +++(1.20 ± 0.26) +(0.58 ± 0.03) n2 ++(0.81 ± 0.21) ++(0.84 ± 0.34) ++(0.67 ± 0.10) +(0.34 ± 0.16) n3 ++(0.87 ± 0.10) ++(0.74 ± 0.05) ++(0.85 ± 0.05) +(0.29 ± 0.19) n4 ++(0.88 ± 0.90) ++(0.86 ± 0.01) ++(0.82 ± 0.06) ++(0.69 ± 0.29) n5 ++(0.99 ± 0.09) ++(0.84 ± 0. 04) +++(1.10 ± 0.14) +(0.21 ± 0.02) g1 ++(0.55 ± 0.09) ++(0.75 ± 0.11) ++(0.79 ± 0.06) +(0.49 ± 0.17) g2 ++(0.59 ± 0.06) ++(0.81 ± 0.01) +++(1.03 ± 0.09) ++(0.54 ± 0.37) g3 ++(0.73 ± 0.19) ++(0.98 ± 0.21) +++(1.09 ± 0.23) +(0.29 ± 0.21) g4 ++(0.66 ± 0.10) ++(0.87 ± 0.35) ++(0.95 ± 0.08) +(0.44 ± 0.08) g5 +++(1.04 ± 0.06) ++(0.92 ± 0.04) +++(1.02 ± 0.14) +(0.43 ± 0.38) notes: * = each value ± standard deviation in table 2 is an average of triplicate measurements of optical densities (absorbance/a) of the cell suspensions which indirectly measure the growth of the cells in various levels of nadc. the growth indication follows the following criteria: = od of < 0.1 (sensitive to nadc); + = od of 0.1 0.5 (slightly resistant to nadc); ++ = od of 0.5 1.0 (resistant to nadc); +++ = od of > 1.0 (highly resistant to nadc). biotropia vol. 29 no. 3, 2022 268 figure 1 percentage of cholesterol reduction in the medium after being inoculated with isolates of yeast and incubated for 24 h in pdb medium supplemented with cholesterol. note: values in the figure ± standard deviation bars are average value of triplicates. microbial isolates intended to be developed as potential probiotics should not transform cholic acid into deoxicholic acid in the intestinal track of their hosts. according to ajouz et al. (2014) and farhana et al. (2016), the accumulation of deoxicholic acid in the digestive track has been suspected to induce colon cancer. although yeast has the potential and has positive properties to be a probiotic candidate, the yeast will be rejected if it has the ability to transform cholic acid into deoxicholic acid. in our present study, the 10 yeast isolates (table 1 & 2) were evaluated for biotransformation of cholic acid into deoxicholic acid. the chromatogram of this evaluation indicated that none of the yeast isolates in our study transformed cholic acid into deoxicholic acid (fig. 2). figure 2 a chromatogram of test for biotransformation of cholic acid into deoxicholic acid for the 10 yeast isolates in our study. yeast probiotics with potential to assimilate cholesterol in-vitro – ramona and ariwathi 269 the chromatogram confirmed that all yeast isolates obtained in our present study are safe and have the possibility to be developed as potential probiotics without harming human or cattle as their hosts. in the intestinal track, biotransformation of cholic acid into deoxicholic acid is commonly performed by clostridium scindens (guzior & quinn 2021). high concentration of deoxycholic acid in the intestinal tract was reported by wu et al. (2018) as having significant effect on the enlargement of colorectal tumor. conclusion all 10 yeasts isolates used this study were shown to be resistant to low ph conditions and high concentration of bile salt (nadc). the isolates were also found to reduce cholesterol content in-vitro at the rate of between 18 76% following 24 hours incubation. besides these positive attributes, none of the isolates transformed cholic acid into deoxicholic acid, indicating that they all have probiotic potential and are safe to be developed as novel probiotics in bali, either for human or cattle. acknowledgments the authors would like to acknowledge lppm universitas udayana and the integrated laboratory for biosciences and biotechnology, universitas udayana for the financial support and provision of consumables and equipment, respectively for this research. the authors’ thanks are extended to dr i nengah sujaya for his critical thinking on the subject of this manuscript prior to being submitted. 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sorghum varieties across two planting seasons arif muazam1*, kristamtini2, setyorini widyayanti2, yudhistira nugraha2, rina sri kasiamdari1, and budi setiadi daryono1 1faculty of biology, universitas gadjah mada, yogyakarta 55281, indonesia 2research center for food crops, national research and innovation agency, bogor 16915, indonesia article higlights • sorghum nutrition varies across varieties and growing seasons • dry season increases starch and amylose content in some varieties • rainy season enhances protein levels in sorghum grains • genotype-environment interaction affects sorghum nutritional quality • sorghum adaptation supports food diversification and climate resilience article information received : 14 february 2025 revised : 8 april 2025 accepted : 15 april 2025 *corresponding author, e-mail: bs_daryono@mail.ugm.ac.id research paper abstract sorghum (sorghum bicolor (l.) moench) is a potential staple food in indonesia, offering an alternative to rice and corn due to its adaptability to marginal lands and drought tolerance. however, research on the impact of environmental factors, particularly different growing seasons, to the nutritional composition of sorghum varieties are still lacking. this study aimed to analyze the proximate contents of tannin and amylose in six sorghum varieties cultivated in two different planting seasons (wet and dry seasons). a completely randomized design with three replications was applied in this experiment. the tested varieties included three newly developed superior varieties (bioguma, samurai, and kawali) and three local varieties (black bonteb, red glutinous sorghum, and kd4). the results showed that proximate composition varied across varieties and seasons. starch content was generally higher in the dry season, with the highest observed in kawali (64.16%) and bioguma (63.11%), while the lowest was in black wareng (11.99%). similarly, protein content peaked in black wareng (7.68%) and red glutinous sorghum (7.37%), but was the lowest in samurai (4.48%). tannin levels also fluctuated by season, where bioguma, kd4, and kawali exhibited higher tannin content in the dry season, whereas samurai, red glutinous sorghum, and black bonteb had lower tannin levels. notably, amylose content was significantly higher in red glutinous sorghum (4.72%) and black bonteb (4.62%) across both seasons, contributing to their fluffier texture when cooked. these findings highlight the influence of environmental conditions on sorghum nutritional quality, reinforcing its potential for food diversification, including processed sorghum flour as a substitute for wheat flour. keywords: amylose, food, proximate, season, sorghum introduction sorghum (sorghum bicolor (l.) moench) is a food crop highly adaptable to marginal lands, including sandy soils, and has strong drought tolerance, making it an important food crop alternative in regions with limited water resources. additionally, sorghum requires low agricultural inputs, making it a sustainable option for food security in developing countries (yupcz 2024). in many parts of the world, particularly in developing countries, sorghum grain serves as a substitute for major cereal crops, such as rice and maize due to its comparable nutritional profile. despite its potential, sorghum remains underutilized in indonesia, even though it has been cultivated for a long time. all parts of sorghum plant can be utilized, making it a highly efficient crop with minimal waste. nutritionally, sorghum offers advantages, as it contains higher protein levels compared to rice, corn, wheat, and tubers (sulistyowati et al. 2024). protein is a key macronutrient in grains, and understanding the https://doi.org/10.11598/btb.2025.32.2.2461 biotropia vol. 32 no. 2, 2025 182 proximate composition of sorghum seeds is crucial to determine their nutritional value and potential applications. according to aryani et al. (2022), sorghum seeds are rich in carbohydrates, similar to rice, wheat, and corn, making them a viable alternative to staple foods. due to their genetic similarities, sorghum seeds have the potential to substitute wheat and rice, particularly in flour-based products, because their functional properties are more compatible with these grains compared to tubers. however, sorghum contains antinutritional compounds, particularly tannins, which can be as high as 2% in some varieties. these compounds reduce protein digestibility and impart an astringent taste, limiting consumer acceptance (aguiar et al. 2023). research gap and study significance although studies have examined the nutritional composition of sorghum, limited research has specifically analyzed how different growing seasons impact the proximate composition, tannin, and amylose content in various sorghum varieties. seasonal variations in temperature, rainfall, and soil conditions could significantly influence these nutritional factors, but comprehensive data on these effects remain scarce. understanding these variations is crucial to improve sorghum utilization in food production and breeding programs aimed at developing superior varieties with enhanced nutritional quality (osman et al. 2022). proximate analysis provides information on the nutritional composition of sorghum, including moisture, protein, fat, carbohydrate, and ash content. this information is essential to evaluate sorghum’s potential as a sustainable alternative to conventional staple crops, particularly in regions affected by climate change and declining soil fertility. despite its high protein and carbohydrate content, the presence of antinutritional compounds, like tannins, can reduce its digestibility and consumer appeal (keyata et al. 2021; yang & zhong 2022) tannins are phenolic compounds found in sorghum that function as antioxidants. however, excessive tannin levels negatively impact protein bioavailability and contribute to bitterness, which is undesirable for food applications. identifying varieties with optimal tannin content could enhance sorghum’s acceptability for human consumption (keyata et al. 2021; yang & zhong 2022). amylose is a component of starch affecting the texture and physical properties of processed sorghum products. higher amylose content is associated with firmer, less sticky rice-like grains, whereas lower amylose contributes to a softer, fluffier texture. optimizing amylose levels is essential for improving the quality of sorghumbased food products, such as instant rice, porridge, and flour substitutes (yan et al. 2023). indonesia has two distinct seasons: the rainy season and the dry season, with significant variations in temperature, water availability, and sunlight exposure. these environmental factors could influence the nutritional composition of sorghum, but the extent of their impact remains unclear. therefore, this study aimed to investigate the effect of growing seasons on proximate composition as well as tannin and amylose contents in both local and newly developed superior varieties of sorghum bicolor cultivated in gunungkidul, yogyakarta, indonesia. the findings of current research will provide valuable insights into the seasonal adaptability of sorghum, contributing to its broader adoption as a climate-resilient food source. materials and methods research site the research was conducted in gunungkidul district, yogyakarta province, indonesia, at an altitude of 215 masl. to provide clearer geographical context and spatial distribution of the study sites, a map has been included (fig. 1), illustrating the location of gunungkidul and its relative distances from other key experimental field or comparison regions. this spatial visualization helps emphasize the environmental variability that could influence sorghum growth and composition across locations. experimental field the experimental field was located at 355 masl (statistics indonesia 2021), with a rainfall distribution of about 1,750 – 2,130 mm per year and an average temperature of 24 °c. the total monthly rainfall from august to november 2024 was 2,121.9 mm. the first study was conducted in dry season from may to august 2024, while the second study was carried out in rainy season from august to november 2024. proximate, tannin, and amylose in six sorghum varieties across two planting seasons muazam et al. 183 the experimental field was characterized by sandy loam soil with moderate organic matter content, ph of 5.5 6.2, and low to moderate fertility levels, requiring additional fertilization for optimal growth. land preparation before planting, the land was plowed and harrowed twice to improve soil aeration and ensure proper root development. organic manure (5 tonnes/ha) was applied one week before sowing to enhance soil fertility. sorghum varieties genotype selection and relevance this study used six sorghum genotypes, consisting of three newly developed superior varieties and three local cultivars, selected based on their genetic diversity, agronomic performance, and potential for food utilization. these genotypes were chosen to represent different nutritional compositions, yield levels, and environmental adaptability, allowing a comprehensive analysis in evaluating the effects of growing seasons on proximate composition as well as tannin and amylose contents. the six sorghum varieties used in this study were: new superior varieties a. bioguma (s1) high-yielding variety developed by indonesian center for agricultural biotechnology and genetic resources (balai besar bioteknologi dan figure 1 sorghum research sites sumber daya genetik pertanian/bb biogen), characterized by high starch content and good adaptability to drylands, with a plant height of 266 cm, biomass weight of 54.30 tonnes/ha, sap volume of 122 ml, yield potential of 9.3 tonnes/ha, brix content of 15%, and resistance to leaf rust and stem rot. b. samurai (s4) released by batan, known for early maturity, moderate starch and protein contents, and adaptability to dry conditions. c. kawali (s6) introduced from india and developed by balitserealia maros of the ministry of agriculture of the republic of indonesia, with an average yield of 2.96 tonnes/ha with high yield potential and moderate tannin content, and moderate resistance to major pests and diseases. local cultivars a. kd4 (s3) a traditional variety cultivated by farmers in gunungkidul, known for its moderate plant height (180 200 cm), high adaptability to acidic soils, and tolerance to drought stress. kd4 has medium amylose content and lower tannin content making it more palatable for food and suitable for feed applications. b. red glutinous sorghum (v3) traditionally cultivated for its high amylopectin content, which gives a sticky, biotropia vol. 32 no. 2, 2025 184 glutinous texture, preferred for sticky rice-and flour-based products. characterized by small reddish grains. this variety is valued for its chewy texture and high antioxidant content due to its phenolic compounds. c. black bonteb sorghum/bonteb/wareng (v4) recognized by its dark-colored grains rich in tannins, contributing to its higher antioxidant properties but lower digestibility. it is traditionally used for local fermented beverages, high-fiber food products, and other traditional sorghum-based products. planting and crop maintenance sorghum seeds were pre-germinated in polybags. the transplanting of sorghum seeds was carried out at 21 days after germination. the transplanting system allowed for early seedling establishment, reducing mortality rates. the planting spacing was 75 × 25 cm, ensuring optimal plant density for growth and yield. during the experiment, crops were maintained by conducting: a. irrigation applied twice per week during dry season and once per week in the rainy season. b. soil piling (pembubunan) conducted at 30 days after transplanting to improve root stability and moisture retention. c. weeding performed manually every 2 3 weeks to reduce competition for nutrients and water. d. pests and diseases control 1. pests aphids (rhopalosiphum maidis) and stem borers (chilo partellus) were controlled using botanical insecticides (neem extract, 2% solution) and chemical insecticides (lambdacyhalothrin, 2.5% ec, applied at 0.5 l/ha if infestation exceeded 10%). 2. diseases leaf rust (puccinia purpurea) and anthracnose (colletotrichum graminicola) were managed using fungicides (mancozeb 2.5 g/l, applied biweekly during high humidity conditions). fertilization schedule and dosage fertilization followed the recommended dose of 120 kg npk (15-15-15) per hectare, applied in three stages: a. basal application (30%): at transplanting (14 days after sowing); b. vegetative stage (40%): at 30 days after transplanting to support leaf and stem growth; c. reproductive stage (30%): at 60 days after transplanting to enhance grain filling. harvesting and post harvest handling harvesting was carried out when seeds reached physiological maturity, indicated by hard grain texture and a moisture content of approximately 20%. the harvest age varied among varieties: a. early maturing varieties (samurai, kawali): 90 100 days after sowing (das); b. medium maturity (bioguma, kd4): 110 120 das; c. late-maturing varieties (ketan merah, black bonteb): 130 140 das. post-harvest processing involved sun-drying the sorghum grains to reach 12 14% moisture content before storage. experimental design this study was conducted using a randomized complete block design (rcbd) with three replications to account for environmental variability. the field experiment was conducted on 30 plots, each measuring 5 × 6 m. within each plot, four raised beds (1 × 6 m) were constructed, resulting in a total of 120 beds. each bed contained 40 planting holes. one seed was planted in each hole, requiring 160 seeds per plot. sorghum seeds analysis sample preparation sorghum grain samples were collected at physiological maturity from three replicates per genotype. the grains were sun-dried to reach 12 14% moisture content, cleaned, and milled into fine flour using a laboratory-scale mill (cyclotec 1093, foss, sweden, 0.5 mm sieve). the flour samples were stored in airtight containers at 4 °c until further analysis. proximate, tannin, and amylose in six sorghum varieties across two planting seasons muazam et al. 185 tannin content analysis (folin and ciocalteu method) tannin content was determined using the folinciocalteu colorimetric method to quantify total phenolic compounds based on their reduction of phosphomolybdate-phosphotungstate reagents in an alkaline medium. the procedure was as follows. 1. sample extraction a 0.5 g of sorghum flour was extracted with 10 ml of 70% methanol by shaking at 200 rpm for 2 hours at 25 °c. 2. reaction a 1 ml of the sorghum flour extract was mixed with 5 ml of folin-ciocalteu reagent at a 1 : 10 dilution and incubated for 5 minutes. 3. neutralization a 4 ml of 7% sodium carbonate (na₂co₃) solution was added followed by incubation for 30 minutes in the dark at 25 °c. 4. measurement absorbance was read at 725 nm using a uvvis spectrophotometer (shimadzu uv-1800, japan). 5. calculation tannin content was expressed as mg gallic acid equivalent (gae) per gram of sample (mg gae/g), using a calibration curve prepared with standard gallic acid solutions. proximate analysis proximate composition was analyzed according to aoac 2005 methods (milala et al. 2018), covering moisture, ash, protein, fat, and carbohydrate contents (table 1). amylose analysis (laye enon iodine colorimetric method) amylose content was determined using the laye enon iodine colorimetric method to quantify the formation of amylose-iodine complexes. the procedure was as follows. 1. sample preparation a 100 mg of sorghum flour was dispersed in 1 ml of ethanol (95%) and 9 ml of 1 m naoh, then heated at 95 °c for 10 minutes. 2. dilution the solution was diluted to 100 ml with distilled water. 3. iodine reaction a 5 ml of sample solution was mixed with 0.1 ml iodine-potassium iodide reagent (0.2% i₂ + 2% ki) and diluted to 50 ml with distilled water. 4. measurement absorbance was recorded at 620 nm using a uv-vis spectrophotometer (shimadzu uv1800, japan). 5. calculation amylose content was determined using a standard curve prepared with pure amylose (park & manna et al. 2024). table 1 proximate analysis according to aoac 2005 methods parameter method used equipment moi sture oven-drying at 105 °c (aoac 925.10) hot air oven (memmert, germany) ash muffle furnace at 550 °c (aoac 923.03) muffle furnace (thermo fisher, usa) crude protein kjeldahl method (aoac 984.13) kjeltec analyzer (foss, sweden) crude fat soxhlet extraction (aoac 920.39) soxhlet apparatus (buchi, switzerland) carbohydrate by difference [(100 (moisture + ash + protein + fat)] each analysis was conducted in triplicate, and results were expressed as a percentage (%) of dry weight basis. biotropia vol. 32 no. 2, 2025 186 results and discussion proximate composition analysis anova results indicated significant differences (p < 0.05) in starch, protein, tannin, and amylose contents among sorghum varieties and across growing seasons. (table 2). post-hoc tukey tests confirmed that the differences between bioguma and black bonteb in starch content were statistically significant. the statistical analysis in this study confirmed that both genotype and environmental factors play a crucial role in determining the nutritional composition of sorghum, which was in agreement with the study of lee et al. (2023) and kamal et al. (2023). the confirmation of significant statistical differences strengthened the validity of these findings and supported the aim of this study to identify the impact of growing seasons on sorghum’s nutritional value. the results also reinforced the necessity for targeted breeding programs to optimize sorghum production for specific agroclimatic conditions. the proximate analysis of sorghum seeds revealed significant variations across varieties and growing seasons (table 3). in general, the dry season resulted in higher nutrient concentrations compared to those in the rainy season. the highest starch content in the rainy season was observed in kawali (64.16%), followed by bioguma (63.11%), red glutinous sorghum (49.29%), samurai (46.42%), kd4 (44.33%), and the lowest in black bonteb (11.99%). in contrast, in the dry season, bioguma exhibited the highest starch content (65.34%), while black bonteb remained the lowest (44.6%). moisture content across all varieties remained below 14%, ensuring seed stability for storage. the highest protein content was recorded in black bonteb (7.68%) during the rainy season, followed by red glutinous sorghum (7.37%), while samurai had the lowest protein content (4.48%). notably, bioguma exhibited an increase in protein content in the dry season (6.48%), suggesting a potential impact of reduced water availability on protein accumulation. these findings aligned with the objective of this study to evaluate the effects of seasonal variations on sorghum’s nutritional composition. the higher starch content observed in the dry season suggested that reduced water availability may promote carbohydrate accumulation, which is beneficial for industrial applications, such as flour production. meanwhile, the increase in protein content in specific varieties during the rainy season supports the suitability of sorghum for nutritional applications where higher protein intake is desired. table 2 anova results for proximate composition as well as tannin and amylose contents in sorghum varieties across seasons component source of variation df sum of squares (ss) mean square (ms) f-value p value (significance) starch content variety 5 126.53 25.31 15.67 0.002** season 1 24.78 24.78 12.83 0.004** variety × season 5 8.62 1.72 2.95 0.048* protein content variety 5 18.34 3.67 8.42 0.011** season 1 5.92 5.92 6.27 0.039** variety × season 5 2.89 0.58 2.13 0.088 tannin content variety 5 9.65 1.93 7.82 0.017** season 1 3.42 3.42 5.38 0.044** variety × season 5 1.56 0.31 1.87 0.102 amylose content variety 5 22.71 4.54 14.92 0.003** season 1 8.33 8.33 9.74 0.027** variety × season 5 3.91 0.78 3.21 0.042** proximate, tannin, and amylose in six sorghum varieties across two planting seasons muazam et al. 187 table 3 proximate composition of sorghum varieties in different growing seasons variety starch (%) protein (%) fat (%) moisture (%) ash (%) bioguma 63.11 / 65.34 5.62 / 6.48 3.3 / 2.0 13.5 / 13.2 1.7 / 1.8 kawali 64.16 / 63.77 5.72 / 3.63 2.8 / 3.26 13.4 / 13.1 2.1 / 2.2 samurai 46.42 / 51.25 4.48 / 4.87 4.12 / 0.5 14.0 / 13.5 1.8 / 1.9 kd4 44.33 / 56.73 4.8 / 4.21 3.0 / 1.59 13.9 / 13.3 2.2 / 2.3 red glutinous 49.29 / 53.82 7.37 / 5.39 2.9 / 2.74 13.6 / 13.3 3.8 / 3.9 black bonteb 11.99 / 44.6 7.68 / 5.56 2.5 / 2.61 13.7 / 13.4 3.8 / 3.9 note: values represent rainy season / dry season data tannin content analysis tannin content was significantly influenced by varieties and growing seasons (fig. 2). bioguma, kd4, and kawali exhibited higher tannin content in the dry season compared to that in the rainy season, whereas samurai, red glutinous sorghum, and black bonteb showed lower tannin content in the dry season. the variation can be attributed to environmental stress factors, where increased uv exposure and reduced water availability in the dry season may enhance tannin synthesis in certain genotypes. these findings are consistent with previous research indicating that sorghum tannin levels are highly responsive to environmental conditions (keyata et al. 2021). the higher tannin content in the dry season could contribute to enhanced antioxidant properties, making these varieties suitable for functional food applications. however, increased tannins may also reduce digestibility, which must be considered in breeding programs aimed at developing low-tannin sorghum varieties for human consumption. amylose content analysis amylose content varied significantly among genotypes and seasons (fig. 3). in dry season, amylose content increased in bioguma (14.8%), kd4 (14.5%), kawali (14.3%), and samurai (14.1%), classifying them as “pera” types (> 14% amylose). conversely, red glutinous sorghum (4.71%) and black bonteb (4.62%) exhibited lower amylose levels, making them suitable for glutinous applications. higher amylose content contributes to firmer rice textures, whereas lower levels result in softer, stickier grains, aligning with consumer preferences for different food products (wang et al. 2022; nakamura et al. 2020). these results confirmed the importance of amylose content in determining the textural properties of sorghum-based food products. the significant seasonal variations highlighted the need for proper selection of sorghum varieties based on end-use applications, whether intended for flourbased processed foods or traditional sticky rice alternatives. figure 2 tannin content analysis biotropia vol. 32 no. 2, 2025 188 figure 3 amylose content analysis effect of varieties and growing seasons on nutritional composition starch content starch content varied significantly between varieties (p ≤ 0.01) and growing seasons (p ≤ 0.01). a significant interaction effect (p ≤ 0.05) indicated that the impact of seasonal variation on starch accumulation was genotype-dependent. bioguma and kawali consistently exhibited higher starch concentrations, with notable increases during the dry season, highlighting their potential for industrial starch production under water-limited environments. protein content significant differences were observed among sorghum varieties (p ≤ 0.05) and between seasons (p ≤ 0.05), while the interaction effect was not significant. the results suggested that seasonal influence on protein content was relatively consistent across genotypes. higher protein levels during the rainy season may be attributed to increased nitrogen assimilation and protein biosynthesis under favorable water availability, as supported by prior studies (attia & alamer 2024). tannin content tannin content varied significantly across varieties (p ≤ 0.05) and growing seasons (p ≤ 0.05), although no significant interaction was found. this indicated a consistent seasonal pattern across genotypes. elevated tannin levels in the dry season may reflect a physiological response to environmental stresses, such as drought and elevated solar radiation, which can enhance secondary metabolite accumulation. amylose content amylose content exhibited highly significant variation among varieties (p ≤ 0.01) and growing seasons (p ≤ 0.05), with a significant interaction effect (p ≤ 0.05), which suggested that some genotypes were more responsive to seasonal shifts in amylose biosynthesis. the increased amylose levels during the dry season may be due to reduced grain moisture and prolonged starch deposition phases, as also observed in drought-adaptive cereals. interaction between genotype and environment the interaction effects between varieties and growing seasons were significant for starch (p = 0.048) and amylose content (p = 0.042), highlighting the importance of genotypeenvironment interactions. the interactions suggested that certain sorghum varieties responded differently to environmental changes, making it essential to consider seasonal influences when selecting varieties for specific food applications. the results provided evidence that breeding programs should prioritize environmental adaptability to maximize the nutritional quality of sorghum. post-hoc tukey test and significant pairwise differences post-hoc tukey tests confirmed that the differences in starch content between bioguma and black bonteb were statistically significant. this finding supported the earlier observations that bioguma consistently accumulated more starch, particularly in the dry season, making it a preferred variety for flour production and industrial processing. in contrast, black bonteb, which exhibited the lowest starch content but higher proximate, tannin, and amylose in six sorghum varieties across two planting seasons muazam et al. 189 tannin levels, was more suitable for functional food applications requiring higher antioxidant properties. implications to sorghum cultivation and utilization the confirmation on significant impact of growing seasons toward nutritional composition of sorghum genotype findings provides valuable insights into the selection of suitable sorghum varieties for different agro-climatic conditions and food industries. based on our study, certain sorghum genotype is suitable for certain utilization, and therefore, it is best cultivated in certain growing season, as follows. a. for flour-based products, varieties with higher starch content (e.g., bioguma, kawali) are preferable, which are best cultivated in dry season. b. for protein-rich applications, black bonteb and red glutinous sorghum varieties grown during the rainy season may have yielded sorghum having higher protein content. c. for antioxidant-rich functional foods, hightannin varieties (e.g., black bonteb) may be more suitable, which are best cultivated during the dry season. d. for glutinous applications, red glutinous sorghum, with lower amylose content, remains the best choice and better be cultivated in the dry season. these results further justify the need for targeted breeding programs that consider both genetic factors and environmental adaptability to enhance the role of sorghum in sustainable food systems. the anova analysis validated that seasonal variations significantly affected the nutritional composition of sorghum, particularly starch, protein, tannin, and amylose contents. these variations emphasized the influence of environmental conditions, such as rainfall, temperature, and soil moisture, on metabolic pathways related to carbohydrate and protein biosynthesis (zhang et al. 2020). for instance, higher starch and amylose levels observed during the dry season suggested that water stress may enhance carbohydrate accumulation due to limited vegetative growth and longer grain-filling duration. conversely, elevated protein content in some varieties during the rainy season indicated enhanced nitrogen uptake and assimilation under higher moisture conditions. the findings of this study support the development of strategic breeding and cultivation practices to optimize sorghum production for specific agro-climatic conditions. selecting varieties with stable nutritional profiles across seasons could enhance food and feed quality, while those responsive to seasonal cues may be targeted for specific industrial uses, such as starch-based bio-products or protein-enriched food ingredients. moreover, understanding genotype-environment interactions could guide the development of climate-resilient sorghum cultivars tailored to both nutritional and functional demands. future research should explore the underlying genetic and physiological mechanisms regulating these seasonal variations, particularly genes involved in starch branching, protein synthesis, and phenolic compound biosynthesis. additionally, post-harvest processing techniques, such as malting, fermentation, and extrusion, should be evaluated for their ability to enhance or stabilize nutrient content under varying environmental conditions, thereby improving the overall value chain of sorghum. conclusion growing season significantly affected proximate composition as well as tannin and amylose contents of sorghum varieties. the dry season 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[updated 6 november 2024; cited 11 february 2025]. available from: https://bbcindonesia. biz/2024/11/06/strategi-konservasi-lahan-kering-didaerah-rawan-kekeringan/?utm wang z, wang j, chen x, li e, li s, li c. 2022. mutual relations between texture and aroma of cooked rice-a pilot study. foods 11(22):3738. doi: 10.3390/foods11223738 the southeast asian journal of tropical biology vol. 31 no. 3, 2024: 438 448 doi: 10.11598/btb.2024.31.3.2250 issn: 0215-6334 | e-issn: 1907-770x 438 a review of marine natural products as potential source of antioxidants rhesa t. hinampas1* and angelo mark p. walag2 1 central mindanao university, musuan, maramag, bukidnon 8714, philippines 2 department of science education, university of science and technology of southern philippines, cagayan de oro city, 9000 philippines article higlights • marine natural products have been the most favorable source of bioactive compounds for drug discovery research. • many biologically active chemicals with antioxidant properties can be found in the marine environment. • a systematic review on potential source of antioxidants linked to marine natural products was carried out. • algae, fungi, sponges, mollusks, and sea cucumbers were found to be abundant sources of antioxidants. • integrate these discoveries into practical applications for enhancing human health and well-being. article information received 2 may 2024 revised 15 july 2024 accepted 16 july 2024 *corresponding author, e-mail: rthinampas@cmu.edu.ph review article abstract several diseases have been linked to oxidative stress, resulting from an imbalance between the creation of the body’s antioxidant defense mechanisms and reactive oxygen species (ros). due to their distinct metabolic makeup and wide range of biological adaptations, marine organisms have attracted interest as possible sources of new antioxidants. this systematic review aims to evaluate the antioxidant potential of marine natural products. to find relevant research published between 2002 and 2022, a thorough search strategy based on the prisma standards was used across databases, including pubmed, google scholar, mendeley, and science direct. a total of 18 studies were extracted and included in the review. the results consistently showed that marine natural compounds had vigorous antioxidant activity. algae, fungi, sponges, mollusks, and sea cucumbers were shown to be abundant sources of antioxidants in marine environments. flavonoids, alkaloids, phenols, tannins, steroids, saponins, glycosides, terpenoids, and carotenoids are only a few types of marine natural products that have been discovered to possess strong antioxidant properties. this systematic review provides compelling evidence for the antioxidant potential of marine natural products. the research validates their potential as sources of novel antioxidants with various bioactive properties. unlocking the full potential of marine natural products and integrating these discoveries into practical applications for enhancing human health and wellbeing calls for further research. keywords: 2,2-diphenyl-1picrylhydrazyl, marine algae, marine fungi, marine sponges, methanolic extract introduction it has been established that the ocean, a complex ecosystem, is an excellent chemical and biological diversity source. compared to non-marine microorganisms and terrestrial plants, marine organisms have been regarded as the most recent source of bioactive natural compounds, including 75% of all living organisms from 36 phyla (balakrishnan et al. 2014). in recent years, marine natural products (mnps) are the most promising source of bioactive compounds for drug discovery research (nie et al. 2020). bacteria, algae, sponges, and other marine organisms make many subgroups of substances. these mentioned organisms are extremely promising sources of fascinating compounds that might be applied to food, cosmetics, pharmaceuticals, and other compounds essential to industry. despite the enormous potential of mnps, the marine ecosystem still represents an extensive undeveloped reservoir of biologically active chemicals, which has considerable potential to contribute food components towards producing new functional foods (mishra et al. 2023). in the world’s seas, between 700,000 and one million different species can be found; however, only a small portion of all mnps is being examined for their potential bioactivities (mohamed et al. 2012, & rotter et al. 2021). due to the growing need https://doi.org/10.11598/btb.2024.31.2.2250 marine natural products as potential source of antioxidants hinampas et al. 439 by the food and pharmaceutical industries to produce natural bioactive anti-carcinogenic and anti-aging substances which provide significant health benefits, antioxidant activity has become intensively discussed and the focus of significant research (balakrishnan et al. 2014). seaweed and sponges are among the marine organisms with the highest levels of natural antioxidants (muthiyan et al. 2018). bioactive substances produced by the said organisms and the bacteria that live on them have been proven to be crucial for illness prevention and health promotion. the complex blend of phytochemicals exhibiting antibacterial, antioxidant, anticancer, and antiviral action is responsible for these positive effects. sulfated polysaccharides, phenolic compounds, sand organic acids are responsible for these activities (balakrishnan et al. 2014). thus, to increase the availability and chemical variety of functional marine constituents, more research is needed to completely comprehend the biological activities of mnps and their potential health advantages (mohamed et al. 2012). the use of mnps as a potential source of bioactivity has been discussed in a few recent review publications (fonseca et al. 2023). however, only a few systematic literature reviews have focused on its potential as a source of antioxidants (el-shafei, hegazy, & acharya 2021). therefore, this review fills a gap in the existing literature by systematically reviewing the mnps as a potential source of antioxidants. methods study design and eligibility criteria a review of literature conducted using a systematic search was employed using prisma guidelines. the following were used as inclusion criteria: (i) the study had to be focused only on primary research, such as experimental studies that looked into marine natural products as a source of antioxidants; (ii) it had to have used marine natural products, such as coral, seaweed, marine sponges, and other marine invertebrates, to look into anti-oxidant activity in humans; and (iii) the main outcome of interest was the antioxidant activity of marine natural products as measured by a recognized assay, e.g., abts, dpph, frap, orac, etc., (iv) it had to be focused solely on antioxidant assays employing ethanolic and/or methanolic extracts, (v) the study had to be written in english, (vi) it had to be published between 2002 and 2022, and (v) had to be open-access, and peer-reviewed articles published in scientific journals. the study’s inclusion criteria were all met by a total of 18 papers. search strategy the search engine science direct, google scholar, pubmed, and mendeley, were used to obtain the most relevant articles by using the words “marine natural products,” “marine natural resources,” and/or “marine products” combined with “antioxidant,” “antioxidants,” and “antioxidant activity. only english, open-access peer-reviewed articles with full text were considered, preferably published between 2002 and 2022. data extraction the search engine science direct, google scholar, pubmed, and mendeley, were used to obtain the most relevant articles by using the words “marine natural products,” “marine natural resources,” and/or “marine products” combined with “antioxidant,” “antioxidants,” and “antioxidant activity. only english, open-access peer-reviewed articles with full text were considered, preferably published between 2002 and 2022. study selection after removing duplicates from a total of 1244 independently downloaded papers, 1093 published articles remained. 37 articles’ full-text versions were retrieved after their titles and abstracts were screened for further assessment. there are 19 articles with methods that still need to meet the inclusion criteria. in the end, only 18 qualified articles were used in this review, as illustrated in figure 1. results and discussion studies of mnps as antioxidant according to study features the analysis’s results indicate the level of distribution of mnp studies as a potential source of antioxidants in terms of (a) publication year, (b) marine sources, and (c) marine species. figure 2 below provides information on the frequency distribution of the studies based on the classified feature based on publication year. results revealed that out of the 18 qualified studies, only a few published studies focused mainly on the mnps as a source of antioxidants biotropia vol. 31 no. 3, 2024 440 (2002-2012, 0; 2013, 2; 2014, 2; 2015, 3; 2016, 3; 2017,0; 2018,1; 2019, 1; 2020, 2; 2021, 3; 2022, 1). these indicate that the chemistry of marine sources, in general, has yet to be fully embraced by the field of mnps, at the expense of its potential as antioxidants based on the inclusion criteria developed for the purpose. the systematic review findings highlight the significant potential of marine natural products as a rich source of antioxidant compounds. as presented in figure 4, although marine algae are the most studied and considered a rich source of natural antioxidants (muthiyan et al. 2018), marine fungi have the highest number of species possessing high antioxidant potential. marine natural products as a source of antioxidant compounds the data analysis showed that mnps have much potential as a source of antioxidant compounds. almost all the research included in the analysis mentioned mnps’ antioxidant activity. the abundance of antioxidants in various marine species, such as algae, fungi, sponges, and other invertebrates, is shown in table 1. figure 1 the flow of information for study selection (adapted from mckenzie et al. 2021) figure 2 frequency distribution of qualified studies by publication year figure 3 frequency distribution of marine sources marine natural products as potential source of antioxidants hinampas et al. 441 table 1 marine natural product sources as potential antioxidants marine source type species secondary metabolites reference marine algae red algae chlorococcum humicola phenols alkaloids steroids kavitha & palani (2016) avrainvillea erecta flavonoids phenols tannins chai et al. (2015) chondrophycus ceylanicus gelidiella acerosa gracilaria corticata alkaloids saponins flavonoids tannins glycosides lakmal et al. (2014) chondrus crispus flavonoids polyphenols alkhalaf (2021) gracillaria edulis centerocerous clavulatum lipid soluble total chlorophylls leelavathi & prasad (2014) laurencia synderiae total phenolic flavonoid karimzadeh & zahmatkesh (2021) green algae chaetomorphacrassa caulerpa racemosa flavonoids total carotenoids lakmal et al. (2014) spongomorpha indica alkaloids steroids tannins flavonoids rajasekaran (2022) unspecified cymodeace rotundata gracillaria crassa cymodeace serrulata not identified leelavathi & prasad (2014) brown algae sargassum cassifolium alkaloids saponins flavonoids tannins glycosides lakmal et al. (2014) marine fungi unspecified aspergillus versicolor aspermutarubrol/ violaceol-i yang et al. (2020) pavona cactus penicillium digitatum hypocreaceae sterigmocystis sp. butyrolactone i aspernolide e, phenolic derivative nie et al. (2020) shark gill aspergillus flavipes penicillium polonicum penicillium chrysogenum penicilliium corylophilum biotropia vol. 31 no. 3, 2024 442 marine source type species secondary metabolites reference marine sponge unspecified zyzzya fuliginosa alkaloids utkina (2013) hyrtios erectus not identified aaptos suberitoides fascaplysinopsis reticulata xestospongia sp acanthella sp petrosia contignata marine invertebrates mollusks littorina littorea polyphenols borquaye et al. (2016)galatea paradoxa sea cucumber holothuria atra flavonoid terpenoid phenols saponin glycoside murniasih et al. (2015) marine algae marine macroalgae, also known as seaweed, is used as food in many countries. as a result, there is much interest in finding bioactive macroalgal metabolites to employ them as active components in developing functional foods, nutraceuticals, and pharmaceuticals (mohapatra et al. 2013). in this context, searching for natural antioxidants in macroalgae is relevant to ongoing scientific interest and societal needs. the fresh samples of chlorococcum humicola in the study of kavitha and palani (lakmal et al. 2014) were obtained from the rameshwaram seashore in tamil nadu. the ethanol extracts showed considerable antioxidant capability in reducing power, dpph, superoxide, and nitric oxide radical scavenging assays. therefore, the algae c. humicola can be a substantial source of vital compounds that the pharmaceutical industry can utilize to produce medicines. based on the gc-ms study’s findings, 14 compounds were present, and their nature varied greatly. the analysis revealed that the 1-propene, 3-(2-cyclopentenyl)-2-methyl-1,1diphenyl exhibited the most significant peak area (figure 5). according to the analysis, flavonoids, alkaloids, and steroids are the metabolites responsible for antioxidant activity. the marine macroalga avrainvillea erecta (berkeley) a. gepp and e.s. gepp (family dichotomosiphonaceae) that was discovered in the south china sea off the southwest coast of tinggi island, malaysia, was investigated by chai et al. (2015). the findings showed that a. erecta’s methanol extract and solvent fractions have the ferric reducing ability as well as scavenging 2,2-diphenyl-1picrylhydrazyl (dpph), nitrogen oxide (no), and hydrogen peroxide (h2o2). on the other hand, dpph scavenging activity is correlated with the total phenolic contents. therefore, the antioxidant activity of a. erecta extract revealed in the study is due to flavonoids and other phenolic components. the information showed flavonoids, phenols, and tannins are the main chemical components. fresh marine seaweeds were gathered from sample sites at the beruwela coral reef in the southern province of sri lanka for the study by lakmal et al. (2014). among the methanolic extracts of the identified species such as red algae (i.e., chondrophycus ceylanicus, gelidiella acerosa, gracilariacorticata), green algae (i.e., chaetomorphacrassa, caulerpa racemosa) and brown algae (i.e., sargassum cassifolium); c. racemosa showed a significant average radical scavenging activity against dpph (34.34%), alkyl (85.17%) and hydroxyl (81.16%), respectively. green seaweed has the highest phenolic content and antioxidant activity of the three macroalgae categories (mohamed et al. 2012). alkaloids, saponins, flavonoids, tannins, and glycosides are some of the phytochemicals found in the species (jayaseelan et al. 2014). marine natural products as potential source of antioxidants hinampas et al. 443 red algae of the chondrus crispus species from the red sea’s coasts near jeddah city were gathered for alkhalaf ’s (2021) investigation. according to the findings, the methanolic extract made from the red algae species c. crispus had considerable antioxidant activity in the current investigation, and it successfully scavenged abts and dpph free radicals in a dose-dependent manner. the extract’s overall antioxidant capacity was also remarkable. the presence of flavonoids and polyphenols is thought to contribute to the antioxidant properties of several species of red algae, and the potency of this ability is correlated with the concentration of these bioactive chemicals (zhang et al. 2019). since flavonoids and phenols were also discovered in c. crispus extract, the antioxidant effects displayed by the extract in the current investigation could be attributed to these bioactive components. seaweed samples (leelavathi & prasad 2014) were collected from the gulf of mannar. cymodeace rotundata, acanthopora spicifera, ulva lactuca, ulva reticulate, turbinaria conoides, gracillaria edulis, kappaphycus alvarezii, gracillaria crassa, gracillaria foliifera, and cymodeace serrulata were the specimens identified. petroleum ether and methanol were used to produce the extracts. by observing the decline in absorbance at 517 nm, the dpph assays were performed to determine the antioxidant properties of seaweed. the highest overall antioxidant activity was demonstrated by c. rotundata, g. crassa, and c. serrulata in the methanol extract compared to other samples. red algae samples were collected from the chabahar coastlines at the coordinates (25° 18′ 53′′n; 60° 37′ 41′′e) in the oman sea, iran, for the study of karimzadeh and zahmatkesh (2021). the red seaweed laurencia synderiae was studied, and the results showed that different extracts (methanolic, chloroform, and ethyl acetate) have dose-dependent antioxidant activities. future medication development is predicted to involve the significant identified compounds that have a strong biological impact on red algae, l. snyderiae. the most prevalent compounds are hexadecanoic acid (19.81%; figure 6), oleic acid (8.34%; figure 7), squalene (6.96%; figure 8); and tetradecanoic acid (6.86%; figure 9); as well as glycerin (8.62%;figure 10), xylitol (8.46%; figure 11), and squalene (8.96%; figure 12). the red algae l. snyderiae has strong bioactive components that it can employ as a natural antioxidant and source of additional resources. total phenolic and flavonoid concentrations are among its phytochemical components. figure 4 1-propene, 3(2-cyclopentenyl)-2methyl-1,1diphenylthe green macroalga spongomorpha indica was gathered from the visakhapatnam coastal area (rajasekaran 2022). the phytochemical tests revealed the presence of potent active constituents such as alkaloids, tannins, steroids, and flavonoids, by which further gcms analysis was conducted, and seven components were determined. the results of the physicochemical parameters tests were under who guidelines. nonadecanoic acid (figure 13) had the most significant peak area, around 47.006%, and the most prolonged retention period, 19.050. figure 5 hexadecanoic acid figure 6 oleic acid figure 7 tetradecanoic acid figure 8 glycerine figure 9 xylitol figure 10 squalene figure 11 nonadecanoic acid biotropia vol. 31 no. 3, 2024 444 in the dpph assay model, s. indica demonstrated a considerable dose-dependent reduction in the case of the dpph radical. all the data demonstrated the highest levels of antioxidant activity, with superoxide scavenging activity displaying the most significant outcomes. based on the findings, it was established that s. indica had powerful active ingredients with potent antioxidant activity. seaweeds were gathered from the station in rameshwaram on the southern indian coast of olaikuda (n.lat. 09°18.300’ and e. long. 079°20.096’) for the study by elangovan et al. (2019) the methanolic extracts of all the test species, including green algae enteromorpha intestinalis and red algae gracillaria edulis, exhibit high antioxidant activity (dpph). the stated activity results from the high concentration of total chlorophylls that are lipid soluble, particularly chl-a and related compounds, as well as the high concentration of total carotenoids. marine fungi in the study by muthiyan et al. (2018), marine fungus strains were obtained from biological samples taken in the intertidal zone of dalian, china (utkina 2013), from shark gill tissues captured in the east china sea, and from various corals in the zhanjiang sea region of china. the methanolic extracts of aspergillus flavipes, hypocreaceae, sterigmocystis sp., penicillium digitatum, penicillium polonicum, penicillium chrysogenum, and penicillium corylophilum were some of the strain samples that showed dpph free radical scavenging activity (scavenging ratios > 30% at 200 g/ml). thus, found to exhibit antioxidant activity. the said antioxidants were assigned as aspernolide e (figure 14), butyrolactone i (figure 15), a phenolic derivative, and possibly unidentified compounds. figure 12 aspernolide e figure 13 butyrolactone ι the aspergillus versicolor sh0105 fungus strain (yang et al. 2020) was isolated from a deepsea sediment sample from the mariana trench at a depth of 5455 m. the bioactive assay revealed that a. versicolor demonstrated a substantial reduction of fe3+ and significant dpph radical scavenging activity, which were more potent than ascorbic acid. this suggests discovering chemical entities with antioxidant activities from the marine medicinal microbial resources. aspermutarubrol/ violaceol-i (figure 16) was identified as the chemical responsible for the antioxidant activity. figure 14 aspermutarubrol/violaceol-i marine sponge many sedentary marine organisms, such as sponges, have acquired the capacity to create a variety of toxic substances to defend themselves against predators or to compete with other marine species (paul et al. 2006). sponges are receiving the most interest from the pharmaceutical industry because they produce the most bioactive secondary metabolites among all marine organisms. alkaloids, steroids, terpenes, peptides, macrolides, and polyketides are only a few marine sponges’ structurally diverse natural compounds (dias et al. 2012). the marine sponge zyzzya fuliginosa (order poecilosclerida) is a rich source of alkaloids with a pyrrolo[4,3,2-de]quinoline structure (utkina 2013). the antioxidant properties of zyzzyanones a to d (1-4) and makaluvamines c, e, g, h, and l (figure 17) were tested, which were isolated from the ethanolic extracts of the australian marine marine natural products as potential source of antioxidants hinampas et al. 445 sponge z. fuliginosa. according to research on their effectiveness as radical scavengers, zyzzyanones and makaluvamines have moderate antioxidant activity, indicated by a phenolic function in the molecules. figure 15 structures of tested zyzzyanones and makaluvamines (utkina 2013) the marine sponge hyrtios erectus was procured from the north bay of the south andaman sea for the study of muthiyan et al. (2018). according to the findings, the methanolic extract of h. erectus exhibited antioxidant activity against dpph free radicals, superoxide anions, and hydroxyl radicals. with 50 µg/ml sponge extract, more than 50% inhibition (half inhibitory concentration) was observed. however, there is a need for further functional characterization of the bioactive compounds to determine the compound responsible for the activities. marine sponge species were gathered in pecaron situbondo, east java, indonesia, for the study of abdillah et al. (2013). results showed that only aaptos suberitoides exhibited vigorous antioxidant activity in an assay using the dpph method, as evidenced by an ic50 value of less than 30 mg/ml. in contrast, fascaplysinopsis reticulata, acanthella sp, petrosia contignata, and xestospongia exigua displayed moderate antioxidant activity, with an ic50 value of less than 100 mg/ml. the ic50 value for callyspongia sp. and xestospongia sp. was more significant than 100 mg/ml. the research has shown the high potential of xestospongia sp, f. reticulata, callyspongia sp, petrosia contignata, and aaptos suberitoides for developing bioactive chemical isolation as antioxidant agents. marine invertebrates aquatic species’ capacity for adaptation and survival in various habitats relies on their physical and chemical adaptations (thakur et al. 2005). bryozoans, mollusks, and other marine invertebrates all have soft bodies and sedentary lifestyles, making a chemical defense system necessary for survival. when introduced into their aqueous habitat, these compounds are rapidly diluted. the compounds must be very potent to be effective (karimzadeh & zahmatkesh 2021). numerous researchers have investigated these environments in quest of biologically active molecules due to the high potency of chemicals used in aquatic defense systems and the requirement that they be watersoluble. molluscs the two mollusks, littorina littorea, and galatea paradoxa, used in the study by borquaye et al. (2016), were gathered from labadi beach in accra and sogakope on the volta river. according to the ic50 values from the antioxidant experiments, methanol extracts are more effective at scavenging dpph radicals than ethyl acetate extracts. compared to the standard ascorbic acid medication, the methanolic extracts of g. paradoxa and l. littorea demonstrated outstanding dpph radical scavenging ability, indicating they may be a good source of antioxidant chemicals. polyphenols are one family of secondary metabolites with proven antioxidant action. both g. paradoxa and l. littorea methanol extracts may have significant concentrations of these chemical compounds. sea cucumbers sea cucumber extracts from holothuria scabra, holothuria atra, holothuria leucospilota, and holothuria excellens were gathered from jor bay in east lombok, indonesia, for the study by murniasih et al. (2015). among the extracts, h. leucospilota and h. atra have strong antioxidant properties, particularly for dpph method-based radical scavenging activity. the phytochemicals flavonoid, terpenoid, phenols, saponins, and glycoside were the constituents of h. leucospilota and h. atra. according to the results of the gc-ms study, 3-chloro-4-hydroxybenzoic acid (figure 18) was found to be one of h. atra’s active antioxidant components. this phenolic substance is crucial for its antioxidant effects. biotropia vol. 31 no. 3, 2024 446 a wide variety of marine natural compounds with antioxidant activity were shown in the identified investigations. the polyphenols group, which includes flavonoids, alkaloids, phenols, and tannins, was the most frequently mentioned class of chemicals with antioxidant activity. saponins, glycosides, terpenoids, and carotenoids are a few more types of chemicals that have been found to possess antioxidant activity. conclusions according to the systematic review’s findings, there is growing evidence that many bio-logically active chemicals with antioxidant properties can be found in the marine environment (mehbub, franco & zhang 2014). flavonoids, alkaloids, phenols, tannins, steroids, saponins, glycosides, terpenoids, and carotenoids, are just a few of the structurally diverse natural compounds that mnps are a highly rich source. according to several screening studies, marine-derived methanolic and ethanolic extracts exhibit potent antioxidant activity. this review provides compelling evidence for the antioxidant potential of marine natural products. the research validates their potential as sources of novel antioxidants with a range of bioactive properties identified from mnps and positions them as potential therapeutic options in the future. thus, it is recommended that the identification and functional characterization of the bioactive molecules necessary for the activities are needed. unlocking the full potential of marine natural products and integrating these discoveries into practical applications for enhancing human health and well-being calls for further research. acknowledgments the authors would like to express their deepest gratitude to the department of science education, university of science and technology of southern philippines, and central mindanao university, figure 16 3-chloro-4-hydroxybenzoic acid for their extended support in developing this investigation. author contributions: rth – conceptualization, data gathering, analysis, and manuscript writing; ampw – conceptualization and editing. funding: this research received no external funding. conflicts of interest: the authors declare that the work presented in this article is original and has no conflict of interest. references abdillah, s., nurhayati, a.p.d., nurhatika, s., setiawan, e., & heffen, w.l. 2013. cytotoxic and antioxidant activities of marine sponge diversity at pecaron bay pasir putih situbondo east java, indonesia. journal of pharmacy 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https://doi.org/10.1016/s2222-1808(12)60252-7 https://doi.org/10.1177/1934578x1300801 https://doi.org/10.1177/1934578x1300801 https://doi.org/10.3390/md18120636 https://doi.org/10.3390/md18120636 https://doi.org/10.1371/journal.pone.0200174 e0200174 https://doi.org/10.1371/journal.pone.0200174 e0200174 _hlk165382905 the southeast asian journal of tropical biology vol. 33 no. 1, 2026: 1 11 doi: 10.11598/btb.2026.33.1.2573 issn: 0215-6334 | e-issn: 1907-770x 1 biodiversity conservation and ecotourism in urban forests: a systematic review and bibliometric analysis rhomi ardiansyah*, stevie vista nissauqodry, kaharuddin, and retno nur utami department of forest resources conservation, faculty of forestry, gadjah mada university, yogyakarta 55281, indonesia article higlights • urban forests link biodiversity protection with public health and recreation • five key research themes reflect ecological, social, and behavioral insights • visitor experiences influence support for conservation and green behaviors • most studies focus on the global north, leaving tropical cities underexplored • a new framework shows how cities can balance nature, tourism, and well-being article information received : 17 july 2025 revised : 1 october 2025 accepted : 7 october 2025 available online: 12 december 2025 *corresponding author, e-mail: rhomi.ardiansyah@ugm.ac.id research paper abstract urban forests play a crucial role in connecting biodiversity conservation and human well-being, providing recreational and tourism benefits in urban areas. this systematic review and bibliometric analysis aimed to analyze the intersection of urban forests, biodiversity conservation, and their interaction with visitors, including preferences, perceptions, and behavior. we examined 83 open-access articles published between year 2010 and 2025, retrieved from scopus databases, then analyzed the articles using bibliometric mapping and keyword co-occurrence analysis with vosviewer, including clustering and thematic trends. this review identified five research clusters: 1) urban ecosystem and biodiversity; 2) recreational and ecotourism; 3) visitor preference and well-being; 4) human-nature interaction; and 5) behavioral science and health. our results showed that interdisciplinary studies on urban forests have been conducted since 2020, with a focus on environmental services, mental health, psychology, and pro-environmental behavior. however, research remains geographically biased toward the global north, with limited representation from the tropical and developing cities despite their rich biodiversity and rapid urbanization. based on this review, we propose a conceptual framework that integrates the ecological and social dimensions of urban forest research, which emphasizes the need for geographical-based, society-based, and interdisciplinary approaches to maximize the multifunctional benefits of urban forests. as cities grow, urban forests, which serve as areas where people encounter biodiversity in the city, can act as strategic platforms for biodiversity conservation, community engagement, and enhancing public awareness, and perceptions of the environment. keywords: biodiversity conservation, ecotourism, urban forests, visitor behavior introduction united nations estimated that the number of people living in cities is expected to reach 6.252 billion by 2050, with an urbanization rate of 67.2% (zhang et al. 2022). high levels of urbanization and the presence of residential areas can reduce species richness and habitat fragmentation (bergeron & pellerin 2014; elmqvist et al. 2015). urban expansion intensity and the type of urban growth can impact species richness, with more intense urbanization often leading to reduced biodiversity (yang et al. 2024). urban forests include all trees, forests, and related vegetations that grow inside or close to urban areas, such as cities, towns, and communities where people live, work, and play, and are becoming increasingly recognized for their ability to support conservation efforts in urban areas and to mitigate the loss of biodiversity (yang et al. 2024; vogt 2020). an urban forest is a part of urban green space that provides ecosystem services and improves urban sustainability (vogt 2020; solomou et al. 2019). both urban forests and urban green spaces play a crucial role in enhancing the resilience, livability, and health of cities (mahajan et al. 2024; bühler & saluz 2024). through their incorporation into urban design, https://doi.org/10.11598/10.11598/btb.2026.33.1.2573 biotropia vol. 33 no. 1, 2026 2 urban forests offer ecological, social, and economic benefits, making them a vital component of urban green spaces and a crucial part of resilient, sustainable cities (vogt 2020; mahajan et al. 2024; niţă et al. 2018). urban forests contribute significantly to the protection of biodiversity and the general ecological well-being of cities by supporting ecological connectedness, offering homes for a variety of species, and boosting floral and faunal diversity (hwang et al. 2024; khan et al. 2022). among its many other advantages, urban forest management is a nature-based approach that helps mitigate the effects of climate change and biodiversity loss (hutt-taylor et al. 2024)sustainably manage, and restore ecosystems while simultaneously providing human wellbeing and biodiversity benefits. in addition to providing cultural services vital to human well-being and improving public health outcomes, urban forests offer a variety of environmental services such as better air quality, climate change adaptation, and higher tourism income (nesbitt et al. 2017). urban forests and urban green spaces are essential for providing recreational services and influencing tourist preferences (koo et al. 2013; sunita et al. 2023). visitors frequently visit urban forests due to their inherent beauty, peace, and opportunity to interact with nature (fadila et al. 2021; yi et al. 2024)many studies have highlighted the role of urban and peri-urban forests in mitigating environmental pollution and maintaining human quality of life by integrating the concepts of ecosystem services into forest management strategy. to increase the social acceptance and to reduce the conflicts between users, citizens’ preferences towards ecosystem services should be included in the decision-making process. in the present study, a structured questionnaire was administered to a sample of 180 citizens and 50 forest managers of the tlemcen peri-urban forest (algeria. understanding how visitors perceive, use, and value urban forests is essential for sustainable planning, particularly when conservation goals intersect with human activity (chiesura 2004). the interest in investigating visitor preferences, behaviors, and perceptions, and how they influence and are influenced by urban forests, is growing (choi et al. 2024; xu et al. 2022; sofyan et al. 2024). however, the ecological context related to biodiversity conservation is often discussed separately from the tourism aspect, which includes visitor preferences, behaviors, and perceptions. due to the increase of urbanization and its impact to biodiversity, it is important to highlight the role of urban forest in maintaining ecological balance and providing recreational opportunities. our review bridges this gap by conducting a systematic literature review (slr) on the relationship between urban forests, biodiversity conservation, and visitor or tourist interactions. this review aimed to analyze the intersection of urban forests, biodiversity conservation, and their interaction with visitors, including preferences, perceptions, and behavior. bridging ecological and tourism aspects in urban forests could promote the implementation of ecotourism, helping to enhance public awareness about the importance of biodiversity and conservation among visitors. this, in turn, can lead to more support for conservation initiatives and responsible behavior toward the environment (imran et al. 2014). materials and methods systematic literature review methods a systematic literature review was conducted using the scopus database, covering the period from 2010 to 2025, and was retrieved in april 2025. the following search string was used to identify relevant documents: (“urban forest” or “open green space”) and (“biodiversity” and “conservation”) or (“tourist” or “visitor”) and (“preference” or “perception” or “behavior” or “attraction”). the search was limited to peerreviewed journal articles, reviews, and conference papers in english, published in open-access format. an initial pool of 202 documents was obtained (fig. 1; supplementary table 1). those documents were screened using specific keywords and topics, including titles and abstracts related to urban forests, biodiversity, and visitor preferences, perceptions, and behavior, employing rayyan ai (https://www.rayyan.ai/). after this process, 107 articles were retained. a second round of screening, involving title and abstract refinement through full-text reviews to confirm relevance, further narrowed the selection to 83 final documents. the publications were not related to the main theme by reviewing the context in the abstract and also full paper were excluded in this review. bibliometric analysis was conducted using vosviewer (v1.6.20; https://www.vosviewer. com/download) to generate network and overlay visualizations of keyword co-occurrence, cluster analysis, and temporal trends. keywords with systematic literature review on biodiversity and ecotourism in urban forests ardiansyah et al. 3 figure 1 research method framework ecosystems while simultaneously providing human wellbeing and biodiversity benefits. managing urban forests for the environmental and human wellbeing benefits is recognized worldwide and is essential in this context of increased urbanization and climate change impacts (endreny 2018). the social, psychological, and environmental benefits of urban forests are being increasingly studied worldwide, with growing consensus that nature-based solutions support both people and ecosystems while addressing climate change (hutttaylor et al. 2022). a high average publication year (≥ 2021) and moderate to high frequency (occurrences ≥ 5) were selected as the emerging topics. results and discussion trend in research publication and geographical distribution based on scopus-indexed literature from 2010 to 2025, the analysis highlights an increasing research interest in the nexus between urban forest, biodiversity conservation, and ecotourism. although the number of publications in 2025 appears to decline, this is due to the timing of the analysis, which was conducted in april 2025. the number of publications on these topics increased after 2020 (fig. 2a). those publications primarily focus on agricultural and biological sciences, environmental sciences, and social sciences (fig. 2b), which indicated that the research topic of urban forests is mostly focused from the perspective of biological, environmental, and social sciences. this is because the urban forest is proposed as a nature-based solution to mitigate climate change and biodiversity loss (hutt-taylor et al. 2024)sustainably manage, and restore although urban forests, which encompass biological, environmental, and social sciences, have a global focus, this subject is more concentrated in northern areas, such as east asia (china and south korea), europe, and north america (fig. 2c). it suggests a potential research gap in tropical and developing countries, especially in southeast asia and africa. these regions’ abundant biodiversity and increasing urbanization offer great opportunities for empirical research (khan et al. 2022; sofyan et al. 2024). for example, several studies were conducted in the southeast asia region, focused on the environmental benefits, socio-economic influences, and biodiversity (igor & chotib 2018; foo 2022; putrika et al. 2023). keyword co-occurrence and thematic structure based on the keyword co-occurrence generated by vosviewer, five dominant thematic clusters were identified that characterize the research structure in this domain (figs. 3a-b; table 1). cluster 1 (red color) encompasses keywords such as biodiversity, urban areas, green spaces, and ecosystem services. it suggests this cluster could be labeled as the urban ecosystem and biodiversity. this cluster encompasses studies examining the ecological functions of urban forests, including their role in enhancing species richness and diversity, conservation, and the provision of ecosystem services. these studies often frame urban forests as supporting biodiversity conservation even within a highly developed city environment (yang et al. 2024; elmqvist et al. 2015). this cluster encompasses 30% of the distribution keywords, indicating that ecological research forms a foundational pillar in the study of urban forests. a study in china suggests that specific management strategies like developing urban parks can enhance habitat connectivity and support diverse bird communities (li et al. 2024). biotropia vol. 33 no. 1, 2026 4 figure 2 summary of research trends on urban forest, biodiversity conservation, and visitor preferences notes: a = annual publication trend from 2010 to 2025; b = distribution of publications by subject area; c = number of publications by country. a previous study shown that the biodiversity in urban forests, especially for trees, were dominated by exotic species than native species (barrico et al. 2018). there was a proposed composition of tree diversity in urban, called the rule of thumb (10/20/30) (santamour 1990). the evaluation of this rule showed that the existing urban forest were less diverse than the proposed benchmarks at species level by using shannon index, however, it has similar at the genus and family level (kendal et al. 2014). cluster 2 (green color) contains keywords like urban forest, recreation, ecotourism, and tourist attraction. it centers on the theme of recreation and ecotourism in urban forests, reflecting a surge of interest in exploring the recreational value and tourism potential of urban forests that was mostly published in 2022 or later (table 1). these studies examine how urban forests can serve as ecotourism destinations, attracting visitors through scenic beauty, accessibility, and opportunities for outdoor recreation (fadila et al. 2021; yao et al. 2024). urban forests provide recreational environments that could increase visitors’ satisfaction and experience, which can improve tourists’ positive behavioral intentions (yao et al. 2024). the conservation of urban forests depends on the development of proenvironmental attitudes and behaviors, which are greatly influenced by the environmental knowledge acquired from visits to forest parks (zhao & weng 2024; erfanian et al. 2024) cluster 3 (blue color) focuses on visitor preferences and well-being, including keywords such as urban forestry, perception, preference behavior, mental health, and outdoor recreation. this cluster demonstrates a growing interest in determining what draws people to urban forests and how those pull factors affect their mental health. a previous study suggests that individual characteristics of visitors influence the variety of their post-visit experience (yıldırım et al. 2024). another study also showed that forest landscapes influenced visitor preferences, and it has a strong correlation to the restorative effect (zhi zhang et al. 2022). systematic literature review on biodiversity and ecotourism in urban forests ardiansyah et al. 5 figure 3 keyword co-occurrence in the literature on urban forests, biodiversity, and tourism notes: a = network visualization showing keyword clusters; b = cluster distribution of keywords; c = top 15 most frequently occurring keywords (colors represent cluster). cluster 4 (yellow color) centers on the theme of human-nature interaction, encompassing keywords such as forest, humans, recreational park, and urban green spaces. this cluster focuses on how people interact with and value the green environment, examining the social and cultural aspects of human interaction with urban nature. a recent study found that visitors perceived more benefits than disbenefits in urban green spaces, including aesthetic value, memories, reflection, strengthening social bonds, connection to nature, and a sense of freedom (nowak-olejnik et al. 2024). the human-nature interaction, including human perception, generates cultural ecosystem services, which are the benefits that ecosystems provide to human well-being in terms of identities, experiences, and capacities (fish et al. 2016). recreational, aesthetic, and cultural values are the benefits of ecosystem values from forests (crețan et al. 2024). cluster 5 (purple color) mainly focuses on interdisciplinary approaches in urban forest studies. emerging terms include psychology, forest therapy, behavioral research, and health, which are labelled as behavioral science and health. this cluster represents the interest of urban forest researchers in the visitor experience of urban forests, linked with the psychological and physiological outcomes. this cluster is starting to focus on issues including environmentally responsible behavior, therapeutic landscape design, and the health benefits of forest experience (weng et al. 2023; zhao & weng 2024; he et al. 2022). the interest in the health benefits of urban forests has increased after the covid-19 pandemic, which clearly shows the importance of green areas in cities for strengthening the physical and mental well-being of the urban population (derks et al. 2020; da schio et al. 2021). a previous study showed that the health benefits of biotropia vol. 33 no. 1, 2026 6 green areas are significant, suggesting that higherquality green regions are associated with reduced medication spending and fewer doctor visits (simović et al. 2023). furthermore, behavioral studies in urban forests highlight the importance of increasing awareness and enhancing the environmental awareness and skills of urban forest visitors (erfanian et al. 2024). it is also important to consider the fit between the tourist and the recreational environment of the urban forest to promote the emergence of empathy and awe to stimulate the visitor’s environmentally responsible behavior (li & song 2024). we then listed the top 15 keywords based on the co-occurrence data to illustrate the conceptual landscape across the dataset (fig. 3c). these keywords represent the most influential and recurrent concepts within the literature that emphasize the core of research topics on urban forest, biodiversity conservation, and ecotourism. urban forestry is the most commonly used keyword in the literature. it shows that the focus of the study across the literature is related to the management of trees and forest resources in urban areas, which provide various benefits, including physiological, sociological, economic, and aesthetic advantages (volder & watson 2015). several studies also suggest considering the perception and perceived values, including visitor experiences, in developing urban forest (yıldırım et al. 2024; jamean & abas 2023; roman et al. 2021; terkenli et al. 2020. studies on urban forests encompass diverse fields, including ecological, social, and psychological aspects, and have been increasingly studied over the years (table 1). based on our analysis, the topic of recreation and ecotourism is increasingly studied in urban forest areas. the study focuses on the relationship between urban forests and recreation, ecotourism, and tourist behavior. these topics could contribute to urban forest planning and construction, which directly or indirectly influences tourist behavior (yao et al. 2024). emerging topics and temporal trends several emerging themes were identified using the temporal overlay visualization (fig. 4a) and the average publication year analysis (fig. 4b), particularly for publications released after 2021. our data showed that there have been shifts in topics over the years in the urban forest study. the publications in 2021 focused on the concepts of “urban forestry” and “perceptions,” which are fundamental to discussions about urban forests. in 2022, the discussions shifted to the role of urban forest parks in promoting mental health, psychology, and ecosystem services. furthermore, it is more enhanced in 2023 in health, behavior, and conservation topics. it might be influenced by the covid-19 pandemic, when mental health has become an issue, such as depression, stress, anger, anxiety, and so on (jevtic et al. 2022) table 1 summary of thematic clusters based on keyword co-occurrence analysis cluster description keywords total occurrences avg. pub. year* 1 urban ecosystem and biodiversity greenspace, urban area, biodiversity, urban ecosystem, ecosystem services 160 2021.23 2 recreation and ecotourism urban forests, recreation, ecotourism, tourist behavior 118 2022.24 3 visitor preference and well-being urban forestry, perception, preference behavior, mental health, outdoor recreation 131 2021.30 4 human-nature interaction forest, human, urban green space, recreational park 65 2021.94 5 behavioral science and health psychology, forestry, behavioral research, health, forest therapy 65 2021.96 notes: each cluster represents a dominant research focus area, characterized by its most frequent keywords, total occurrences, and average publication year; * = the publication years of all documents containing those keywords and then divided by the total number of times the keywords appeared. systematic literature review on biodiversity and ecotourism in urban forests ardiansyah et al. 7 the keyword occurrences “behavior” have prominently gained focus and discuss the tourists’ perceived value, post-visit behavior, and visitors’ experience of the environmentally responsible behavior in urban forests (zhao & weng 2024; weng et al. 2023; li & song 2024; yao et al. 2024; borthakur & kondasani 2024). a shift toward a more behavioral and impact-driven research agenda is evident in the growing emphasis on studies that aim to understand how visitor experiences in urban forests can promote sustainable attitudes and actions. figure 4 thematic and temporal analysis of key terms in selected publications notes: a = overlay visualization indicating temporal trends (based on average publication year; b = emerging topics based on the keyword frequency and average publication year. in summary, studies of urban forests encompass a range of perspectives and diverse research topics. based on the review conducted, we provide a conceptual framework to summarize the connections between each cluster, which will help illustrate how urban forests are studied (fig. 5). this conceptual framework portrays the urban forest as a nexus of ecological, psychological, and recreational dimensions. these patterns indicate an increasing emphasis on interdisciplinary research that combines tourism studies, environmental psychology, and conservation science. multisensory experiences, forest therapy, and the utilization of urban forest parks as tools for mental and physical health interventions are also gaining popularity. biotropia vol. 33 no. 1, 2026 8 figure 5 conceptual framework research gap and future study direction urban forests, biodiversity conservation, and their interaction with visitors are studied as interdisciplinary research. however, several critical gaps and future opportunities will guide the next phase of interdisciplinary inquiry. as shown in the geographical distribution of publication (fig. 2c), the research is concentrated in east asia, europe, and north america. the urban forest area in tropical and developing regions, including southeast asia and africa, remains underexplored, despite being biodiversity hotspots and experiencing rapid urbanization (foo 2022; khan et al. 2022; putrika et al. 2023). it suggests that there is a need to explore how urban forests function within the specific biophysical, social, and policy contexts of the global south, such as south america, africa, and southeast asia. it could also involve cultural studies, how multicultural values are perceived in urban forests, integrating traditional ecological knowledge, and localized tourism behavior models. several studies have indicated the necessity for enhanced interdisciplinary methods in urban forestry research to address the complex socio-ecological dynamics within multicultural metropolitan environments and to incorporate diverse viewpoints and techniques for improved urban forest sustainability and resilience (barona et al. 2022; vogt et al. 2016). the thematic cluster shows a strong divide between ecological studies (cluster 1) and visitor-focused research (clusters 2 & 3) (fig. 3b, table 1). there are not many studies that investigate both biodiversity assessment and visitor engagement at the same time, as urban forests are managed as multifunctional spaces that serve conservation and recreation purposes (imran et al. 2014; zhao & weng 2024). interdisciplinary studies should integrate biodiversity indicators and visitor engagement, which can be evaluated through participatory mapping and visitor impact assessments. conclusion by systematically reviewing all publications on urban forests published over the past fifteen years, we identified five major research clusters that focus on biodiversity conservation and visitor engagement. these clusters demonstrate the application of interdisciplinary approaches systematic literature review on biodiversity and ecotourism in urban forests ardiansyah et al. 9 to investigate how urban forests function, encompassing ecological, cultural, and recreational spaces within the urban environment. through years of publication, urban forests are increasingly recognized as nature-based solutions that provide ecological connectivity, promote well-being, and offer recreational opportunities. several key research gaps need to be addressed in future studies, such as the fact that the research was concentrated in the global north and the need for an integrated study on both ecological and visitor aspects. moving forward, future research should prioritize interdisciplinary designs, incorporate behavioral and psychological metrics, and include geographical-based studies in the global south. this systematic review and bibliometric analysis can be utilized as a recommendation for managing urban forests which should also considered visitors’ preferences and perceptions. urban forests, when managed inclusively, have the potential to serve as platforms for biodiversity conservation, community engagement, and the enhancement of public awareness and perceptions regarding the environment. it shows the potential of urban forests for ecotourism area. acknowledgments we 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science study program, faculty of fisheries and marine sciences, universitas jenderal soedirman, purwokerto 53122, indonesia 2aquaculture study program, faculty of fisheries and marine sciences, universitas jenderal soedirman, purwokerto 53122, indonesia received 30 november 2021/accepted 19 january 2022 abstract benthic foraminifera are types of organisms that are sensitive to environmental changes, so they are often used as a bioindicator for aquatic environmental conditions. the purpose of this study was to determine the community structure of benthic foraminifera community, sediment types and the relationship between the abundance of foraminifera with the sediment types in the eastern waters of segara anakan lagoon in cilacap. method used in this study was a survey method. samples were taken by using random sampling method. the study was conducted at 5 stations with 3 repetitions. laboratory observations carried out included the types and numbers of benthic foraminifera. community structure of foraminifera among stations were analyzed using one-way anova, while the relationship between community structures and water quality parameters was analyzed using pearson correlation. the results showed that in the the eastern waters of segara anakan lagoon there were 58 species of foraminifera which abundance ranged from 532 ind/m2 to 927 ind/m2. the diversity index of foraminifera was in the medium to high diversity categories. the uniformity index of foraminifera was in the high uniformity in a stable community. the dominance index of foraminifera was in the low category. the sediment types was fine sand, medium sand and coarse sand. the relationship between the abundance of foraminifera with the sediment types was strong with high r values (0.763-0.809). keywords: benthic, community structure, foraminifera, sediment, segara anakan lagoon introduction the segara anakan lagoon is located between the southern part of the java island and the nusa kambangan island (180º53' 109º30' w; 7º20' 7º35' s), with an area of about 240 km2, extending from west to east. this area is a protected water area with a closed tropical forest (setyowati 2005). industrial activities and residential areas that continue to increase, as well as the rapidly growing tourism areas causes adverse impacts on the surrounding ecosystem (setyowati 2005). one way to see the impact is to investigate the habitat of an organism in a community (ferawati et al. 2014). biological component analysis in an ecosystem is a measurement of biological responses to changes in environmental conditions, which can be studied through a community of organisms that are used as parameters of important biological components (fachrul 2007). one of the important organisms in marine ecosystems, especially in shallow seas, is benthic foraminifera. foraminifera organisms are shelled organisms that are abundant in various marine environments. in a volume of 1 cm3 of sediment, there can be hundreds of living foraminifera individuals, along with many dead shells (sadough et al. 2013; bawole et al. 2017). foraminifera is among the organisms in seabed sediments that can indicate the environment conditions of their living area. their way of life is by attaching themselves to sediments, rocks, marine plants and corals available at the bottom of the waters. as a result, benthic foraminifera are very sensitive to various environmental changes such as temperature, salinity, light and *corresponding author, email: tjahjo.winanto@unsoed.ac.id mailto:tjahjo.winanto@unsoed.ac.id biotropia vol. 29 no. 2, 2022 172 ph (gustiantini 2008). therefore, foraminifera are widely used by biologists, geologists and oceanographers in relation to various changes in marine environmental conditions (boltovkoy & wright 1976 in sidiq et al. 2016). foraminifera are single cell organisms having hard shells and most of their communities live in the sea. the numbers of foraminifera found in the waters worldwide, both planktonic and benthic, are around 12,000 species (puspasari et al. 2012). the wide distribution area of foraminifera in various types of aquatic environment has the potential to assist in understanding environmental conditions in a waters (rositasari 2006). the living condition of foraminifera are strongly influenced by microand macro-environmental conditions, which makes foraminifera potential to be used as an indicator for environmental changes (rositasari 2011). information on the types and community structure of foraminifera in the waters of the segara anakan lagoon is still very limited and not much studies have been done. on the other hand, knowledge about the community structure of foraminifera can be used to determine the ecological conditions of a waters (sen gupta 2003; sadough et al. 2013), because the distribution of foraminifera is strongly influenced by the surrounding environmental conditions, so that certain species can reflect certain ecological conditions (gustiantini 2008). this study aimed to determine: 1) the types and community structure of benthic foraminifera, sediment types and 2) the relationship between the foraminifera abundance and sediment types in the waters of the segara anakan lagoon. materials and methods the study was conducted in the eastern waters of the segara anakan lagoon in cilacap by using a survey method. data collection was done by random sampling method. foraminifera samples were observed in the laboratory of the faculty of fisheries and marine sciences of universitas jenderal soedirman. the laboratory observations identified the types and numbers of foraminifera collected from the sediment samples. the observational station consisted of 5 stations. samples were taken from each station with three repetitions. the measured water quality parameters were salinity, temperature, ph and brightness. sediment samples were taken using a sediment grab. the samples were stored in the sample sticks, transported to and analyzed in the laboratory. the collected sediment samples were washed, then ovendried. after the samples’ temperature reaching room temperature, the samples were sieved using a multilevel sieve. the sifted dry samples were weighed and sprinkled onto a surface, followed by identification of the foraminifera (armstrong & brasier 2005; barker 1960; loeblich & tappan 1994; yassini & jones 1994; sadough et al. 2013; gupta 2003). research parameters observed were the types and community structure of benthic foraminifera, such as abundance, diversity, uniformity and dominance. the abundance of foraminifera is the number of individuals of a species occupying a certain area. diversity index (h') is used to measure the degree of ecological instability in a system. the uniformity index is an index used to measure the even distribution of species abundance in a community. dominance index is used to determine the extent to which a group of biota dominates another group (bawole et al. 2014; insafitri, 2010; odum 1994). the data from the study were analyzed descriptively. differences in foraminifera community structure among stations were analyzed using one way anova, while the relationship between foraminifera abundance and sediment types was analyzed using pearson correlation. data processing was done by using spss software version 17.0. results and discussion foraminifera community structure the number of benthic foraminifera species found in the eastern waters of segara anakan lagoon was 58 species. at station 1 there were 22 foraminifera species with the most abundant community structure of benthic foramminifera in eastern waters of segara anakan – tjahjo winanto et al. 173 species at this station was elphidium craticulatum. there were 13 foraminifera species at station 2, while at station 3 there were 25 species of foraminifera. the most common species found at stations 2 and 3 was ammonia beccarii. at station 4 there were 26 species with the most species was operculina ammonoides. there were 31 species of foraminifera found at station 5 with the highest number of species being amphistegina hauerina (fig. 1). the highest number of foraminifera species was found at station 5 (31 species), while the lowest foraminifera species was found at station 2 (13 species). ammonia beccarii, ammonia falsobeccarii and elphidium craticulatum were foraminifera species found in all research stations. these species has high adaptability and high tolerance to various environmental conditions compared to other species. according to uthicke (2008), foraminifera of the genus elphidium are indicators of high turbidity levels and low brightness. this finding is in agreement with the work of toruan (2011) which stated that foraminifera from the genus elphidium and ammonia are included in the opportunist group which are tolerant to adverse environmental conditions compared to other foraminifera. the species amphistegina hauerina and operculina ammonoides were foraminifera species that were only found at stations 4 and 5. it is suspected that stations 4 and 5 are coral reef areas with the characteristics of clear waters with high brightness. foraminifera of the genus amphistegina are the most abundant foraminifera of the coral reef symbiotic group in waters with a fairly high level of brightness (toruan 2011). in general, foraminifera from the genus amphistegina are the most well-known species associated with coral reef areas (eichler et al. 2019). foraminifera of the genus amphistegina are important ecosystem builders because of their contribution to calcium carbonate production and thrive in a variety of shallow water habitats. the genus amphistegina is also a bioindicator to determine the quality of clean water in the coastal environment (weinmann et al. 2013). the abundance, diversity, uniformity and dominance indices were used to determine the foraminifera community structure in the eastern waters of segara anakan lagoon. foraminifera abundance the abundance of foraminifera found in the eastern waters of segara anakan lagoon ranged from 532 to 927 ind/m2 (fig. 2). figure 1 average numbers of benthic foraminifera in the eastern waters of segara anakan lagoon biotropia vol. 29 no. 2, 2022 174 figure 2 abundance of benthic foraminifera in the eastern waters of segara anakan lagoon the highest number of benthic foraminifera was found at station 5 with 927 ind/m2, while the lowest number was found at station 2 with 532 ind/m2. station 5 had the highest abundance of benthic foraminifera, presumably because this station is an open area that is directly affected by the water currents (higher water currents). station 2 had the lowest abundance of foraminifera, because this station tends to be more protected from water currents (lower water currents). high velocity of water currents can affect the distribution of benthic foraminifera in a waters. gustiantini (2008) stated that the distribution of benthic foraminifera is influenced by current patterns; in relatively speedy water currents conditions the foraminifera species will be very abundant and diverse. the presence of water current will evenly distribute the water temperature and salinity. in addition, water currents also play a role in carrying food for foraminifera (toruan 2011; uthicke 2008). diversity index (h') the biota diversity can be determined using the shannon-wiener index (h'). in this study, the diversity index of foraminifera in the eastern waters of segara anakan lagoon ranged from 2.29 to 3.11 or falls into the medium to high diversity categories. the diversity index at station 1 was 2.43. station 2 had diversity index of 2.29. station 3 had diversity value of 2.94, while the index for station 4 was 3.03 and the index for station 5 was 3.11 (fig. 3). the diversity index in this study showed that the stations have suitable water quality conditions as the habitat for foraminifera in terms of nutrients suppy availability, lighting, temperature, salinity and ph. this finding is supported by gustiantini (2008) which stated that the life of foraminifera organisms is strongly influenced by abiotic factors such as temperature, salinity, brightness, ph and sediment types. water quality measurements obtained in the eastern waters of segara anakan lagoon showed that the water temperature at all stations ranged from 26 oc to 29 ºc. this temperature range is suitable for the habitat of foraminifera organisms, which can be found at temperature range of 10 30 ºc (pranajaya 2015). according to rositasari (1997) temperature affects the metabolism of an organism and foraminifera can live and adapt to a certain temperature range. the critical point of temperature can be reached when the foraminifera reproduction process is no longer taking place (rositasari 1997). in this study, the water salinity ranged from 29 to 31 ppt. the salinity range corresponds to the habitat of benthic foraminifera which can be found in salinity range of 18 30 ppt (rositasari 1997). waters with normal salinity usually has a high diversity of foraminifera species (rositasari 1997). foraminifera organisms are also able to adapt to low salinity, such as bays, brackish waters and swamps, which are usually inhabited by foraminifera with agglutinin shell type. foraminifera are also able to live in waters having high salinity. the high salinity waters community structure of benthic foramminifera in eastern waters of segara anakan – tjahjo winanto et al. 175 figure 3 diversity of benthic foraminifera in the eastern waters of segara anakan lagoon (hypersaline waters) are favored by foraminifera species with porcelain shell types having high concentrations of calcium carbonate (rositasari 1997). the water brightness in this study was in the range of 1.1 2.1 m which is below the brightness quality standard of sea water of > 3 meters based on the kepmeneg lh (2004). the low brightness in the eastern waters of segara anakan lagoon is due to the fact that the area is close to the river mouth, which is a meeting place between river waters and sea waters. river waters carry wastes from the land and the process of mixing seawater and freshwater causes the waters to become muddy due to the mixing of particles from the mainland (saraswati et al. 2017). the ph values ranged from 6 to 7. water ph affects the growth of foraminifera organisms, because the degree of acidity of a waters can affect the biological activity of foraminifera organisms, one of which is the shell formation process (yanti 2016). waters with acidic water conditions can cause a reduced ability of foraminifera to secrete calcium carbonate in shell formation (brasier 1980). the analysis of variance (anova) results showed that there was a significant difference in diversity index of benthic foraminifera (p < 0.05) among stations. stations 1 and 2 had significant differences in diversity index to stations 3, 4 and 5. the differences are presumably due to the different characteristics of the study locations. stations 3, 4 and 5 have characteristics of open environment, while stations 1 and 2 have characteristics of a more protected environment. the open area is affected by the current velocity which tends to be greater than the one in the protected area. current velocity can affect the distribution of foraminifera in a waters. the distribution of foraminifera in the waters is influenced by currents, sediment types and the presence of coral reefs (gustiantini 2008). uniformity index (e) the uniformity index is an index used to measure the even distribution of species abundance in a community. this index provides information about the similarity of species living among stations. the uniformity index of foraminifera in the eastern waters of segara anakan lagoon enters the category of high uniformity, stable community, ranging from 0.82 to 0.94. the uniformity index at station 1 was 0.82 (the lowest). the uniformity index at station 2 was 0.92. station 3 had a uniformity index of 0.93. the uniformity index at station 4 was 0.94 (the highest). the uniformity index at station 5 was 0.93 (fig. 4). the uniformity index (e) from all stations was > 0.6 which means that the uniformity at each research station enters the high category with stable community. if the distribution of organisms in an ecosystem is evenly distributed, the ecosystem tends to be in a stable condition (odum 1994). the evenness of species presence biotropia vol. 29 no. 2, 2022 176 figure 4 uniformity index of benthic foraminifera in the eastern waters of segara anakan lagoon at each research station is supported by the morphology of foraminifera having the ability to exist in various ecosystems. benthic foraminifera as single-cell shelled organisms have the ability to occupy various marine areas ranging from the supertidal zone above the littoral to the deepest depths in the hadal abbysal zone (bawole et al. 2017). our study showed that there was a significant difference (p < 0.05) in the uniformity index of benthic foraminifera among stations, i.e., the uniformity index at station 1 was significantly different from that at stations 2, 3, 4 and 5. dominance index (c) the dominance index shows the dominant value of species abundance at each station. the greater the value, the dominant the species at the station. our study showed the dominance index ranging from 0.0535 to 0.1182 which enters the low category (fig. 5). the highest dominance index was found at station 1 (c = 0.1182), while the lowest index was found at station 5 (c = 0.0535). the dominance index values of all stations are in the range of 0.00 0.50, which enters the low category. the dominance index in a community structure is usually inversely proportional to diversity index. the low dominance value (< 0.5) indicates that the foraminifera organisms are highly diverse and more evenly distributed. the low dominance index shows that no species dominates (bawole et al. 2017). sediment types the sediment types in the eastern waters of segara anakan lagoon are fine sand to coarse sand. stations 1 and 2 had fine sand sediment type. station 3 had a medium sandy sedimen type, while stations 4 and 5 had coarse sand sediment type (fig. 6). community structure of benthic foramminifera in eastern waters of segara anakan – tjahjo winanto et al. 177 figure 5 dominance index of benthic foraminifera in the eastern waters of segara anakan lagoon figure 6 percentage of sediment types in the eastern waters of segara anakan lagoon the fine sand sediment was found at stations 1 and 2, presumably because these two stations are close to the river mouth, which is more protected than other stations. a relatively protected area has a lower water movement resulting to smaller size particles (aritonang et al. 2014). stations 4 and 5 have coarse sand sediment, presumably because these two stations are close to the beach, which are open areas and have relatively higher water currents. a strong water currents will cause the sediment to have coarser fractions, so that the sediment is not easily carried away by the water current (bayhaqi 2015). relationship between benthic foraminifera abundance and sediment types the pearson correlation analysis showed relationship between the foraminifera abundance with sediment types. the correlation values ranged from a low but definite relationship (r = 0.331) to a high and strong relationship (r = 0.763 0.809) (table 1). the foraminifera abundance was strongly related to the coarse sand sediment type (0.809) and fine sand sediment type (-0.763). on the contrary, a low but definite relationship occurred between the foraminifera abundance to the medium sand type (0.331). the negative biotropia vol. 29 no. 2, 2022 178 tabel 1 relationship between foraminifera abundance and sediment types coarse sand medium sand fine sand abundance pearson correlation 0.809** 0.331 -0.763** sig. (2-tailed) 0.000 0.228 0.001 n 15 15 15 note: ** = correlation is significant at p < 0.01. value in the correlation between foraminifera abundance and fine sand sediment type indicated inversely proportional relationship, which meant that the finer the sediment type, the less the foraminifera abundance is. these findings are supported by the work of natsir (2010) which states that foraminifera organisms generally occupy sediments containing sand; no foraminifera are found in mud and silt sediment types. the work of lacuna (2013) also supported our findings which stated that the sediment types affect the presence of foraminifera, because foraminifera generally are more abundantly found in the sandy sediment type and less abundant in a finer sediment type. conclusion the sediment types and water current affect the foraminifera abundance and distribution. water quality parameters among observation stations are relatively similar and have no significant effect on the abundance and distribution of foraminifera. acknowledgments the authors thank the lppm of universitas jenderal soedirman for funding the institutional research of universitas jenderal soedirman year 2021. the authors also deliver deep gratitude to the dean and colleagues at the faculty of fisheries and marine science of universitas jenderal soedirman for the tremendous support in our study. references anugrah py. 2012. foraminifera. teknik geologi. jakarta (id): universitas trisakti. aritonang ae, surbakti h, purwiyanto ais. 2014. laju pengendapan sedimen di pulau anakan muara sungai banyuasin, sumatera selatan. 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[ecology and environment]. gorontalo (id): universitas negeri gorontalo. weinmann ae, dennis r, stefan l, martin rl. 2013. traveling through time: the past, present and future biogeographic range of the invasive foraminifera amphistegina spp. in the mediterranean sea. mar micropaleontol 105 (2013):30-9. widhayanti a, ismanto a, yulianto b. 2015. sebaran tumpahan minyak dengan pendekatan model hidrodinamika dan spill analysis di perairan cilacap, jawa tengah. [the distribution of oil spill and hydrodynamics approach and spill analysis in the cilacap waters]. jurnal oseanografi, 4(4):641 -50. the southeast asian journal of tropical biology vol. 32 no. 2, 2025: 394 402 doi: 10.11598/btb.2025.32.3.2240 issn: 0215-6334 | e-issn: 1907-770x 394 effects of phosphate-solubilizing bacteria from probiotic and phosphorus fertilization on juice characteristics of brown midrib sorghum (sorghum bicolor l. moench) riesi sriagtula1*, mardhiyetti1, gusri yanti2, and james hellyward1 1faculty of animal science, andalas university, indonesia, west sumatra, padang 25163. 2faculty of science, social and education, prima nusantara university, bukittinggi 26122, west sumatra, indonesia article higlights • bacillus sp. probiotics can replace 100% inorganic phosphate fertilizer. • sorghum stem diameter strongly correlates with juice volume. • juice yield was not affected by phosphorus dose or bacillus sp. colony size. • sorghum juice sugar content reached 11.77–12.75% brix. • phosphate-solubilizing bacteria improved phosphorus availability in ultisol. article information received : 17 january 2025 revised : 23 april 2025 accepted : 10 may 2025 *corresponding author, e-mail: riesisriagtula@ansci.unand.ac.id short communication abstract this study was conducted to observe the effects of phosphatesolubilizing bacteria (psb) combined with different phosphorus fertilizer levels on the juice characteristics of the bmr sorghum mutant line. the research was carried out at the edufarm of the faculty of animal science, andalas university, padang, using a randomized block design (rbd) with four treatments and four replications. the treatments were as follows: p0 = urea + kcl + 0% tsp + psb; p1 = urea + kcl + 50% tsp + psb; p2 = urea + kcl + 75% tsp + psb; and p3 = urea + kcl + 100% tsp (control). the psb used was obtained from waretha probiotics, consisting of bacillus sp. culture at a dose of 10 ml per planting hole, equivalent to 10⁷ cfu/g. the parameters measured were sugar content (%brix), volume of stem juice (ml), and the correlation of various parameters with stem juice volume. the results showed that the application of probiotic bacillus sp. and different dosages of phosphorus fertilizer did not result in significant differences for all parameters (p > 0.05). sugar content and juice volume ranged from 11.78 to 12.75 (%brix) and 188.75 to 218.5 (ml/stem), respectively. the correlation coefficient analysis indicated significant positive correlations between stem diameter (r = 0.575*) and fresh stem weight (r = 0.504*) with the volume of sorghum stem juice. it was concluded that the application of psb from bacillus sp. can replace phosphorus fertilizer, producing sugar content and juice yield comparable to the 100% p fertilizer treatment. a correlation was observed between stem diameter and fresh stem weight with stem juice volume. psb was found to play a crucial role in increasing phosphate availability to plants. the implications of this research include the potential production of forage sorghum with high brix content for ruminant energy and water-soluble carbohydrates (wsc) for silage production. keywords: bacillus sp., brown midrib (bmr), fertilizer, phosphatesolubilizing bacteria, sorghum copyright (c) 2025@author(s). https://doi.org/10.11598/btb.2025.32.3.2240 https://creativecommons.org/licenses/by-nc-nd/4.0/ effect of phosphate-solubilizing bacteria on juice of brown midrib sorghum sriagtula et al. 395 introduction sorghum (sorghum bicolor l. moench) is considered to have potential for development as a commodity to increase the productivity of marginal land, as it is characterized by wide adaptability and low input requirements for growth (sriagtula et al. 2019). a mixture of sorghum harvested at the middough stage with concentrate has been reported to be suitable for use in dairy heifers at a ratio of 65:35 (kljak et al. 2016). sorghum plants are recognized to have advantages over cereal crops in general, including high production potential and the ability to be ratooned, thereby increasing efficiency in seed and tillage costs. breeding programs for sorghum have been conducted to produce varieties suitable for forage, resulting in the development of brown midrib (bmr) mutant sorghum strains. bmr sorghum is characterized by a lower lignin content than non-bmr sorghum, allowing it to be potentially developed as forage for ruminants. as an industrial raw material, bmr sorghum is more easily fermented for bioethanol production. its nutritional composition has been reported to contain 8.10% crude protein (cp), 20.01% crude fiber (cf), 3.31% crude fat (ee), and 4.91% ash (sriagtula et al. 2019). bmr sorghum is classified as a sweet sorghum due to its high stem sugar content, which ranges from 10% to 15% (siddique et al. 2018). the sugar content in sorghum stalks has been found to be positively correlated with ethanol yield, and a correlation has also been observed between sugar concentration (sucrose) in brix units and total sugar content. the water requirement of sorghum has been reported to be lower than that of maize, enabling intensive cultivation on dry lands (roby et al. 2017). drylands are characterized by low water availability, low fertility, and low productivity. in indonesia, drylands cover 144.47 million ha, 82% of which are classified as suboptimal. acidic dryland has been identified as one of the most dominant suboptimal dryland types, with an area of 107.36 million ha (74.3% of the total dryland area) and the widest distribution found on the islands of sumatra, kalimantan, java, and papua (rachman et al. 2021). the main limiting factor of acidic drylands is a soil ph below 5.5. ultisol soil has been described as acidic soil with very low phosphorus (p) availability due to fixation by al and fe (hasanudin et al. 2021). phosphate has been recognized as a limiting factor for plant growth and yield, and one approach to increasing phosphate fertilization efficiency in overcoming low soil phosphate availability is through the utilization of soil microorganism groups. soil organisms are recognized to play a role in recycling nutrients within the soil, making them available to plants and increasing crop yields (rajper et al. 2016). microbial-based inoculants have been widely applied to enhance soil microbial activity. through the addition of microbial-based inoculants, nutrient supply is increased, thereby improving crop productivity and maintaining soil fertility. probiotic bacteria, which are known for their use as feed additives, have also been applied as providers of phosphate-solubilizing bacteria (psb) to increase crop production (menendez & fraile 2017). probiotics have been shown to increase the solubility of phosphorus in soils and phosphate fertilizers, thereby increasing the availability of p to plants. the probiotic bacterial strain bacillus sp. has been identified as one of the soil microbes functioning as psb (prakash & arora 2019). the application of probiotics in agriculture has been reported to improve plant growth and yield by enhancing photosynthesis, producing bioactive substances such as plant growth regulators and enzymes, stimulating the decomposition of organic matter, and releasing inorganic nutrients that can be absorbed by plants. previous research has indicated that the addition of microbial-based inoculants is more efficient and cost-effective because it reduces the use of chemical fertilizers and pesticides (rajper et al. 2016). furthermore, microbial-based inoculant technology has been reported to result in plant growth equal to or superior to that achieved through conventional agricultural practices (shahwar et al. 2023). numerous studies have examined the effect of phosphorus on sugar content. phosphorus has been reported to be required for increasing sugar content in sorghum stems (ali & anjum 2017; maw et al. 2016). in contrast, other studies have concluded that phosphate fertilization does not influence the sugar concentration in the juice (brix) of sugarcane (caione et al. 2015). an increase in sucrose content in sugarcane plants was observed by aye et al. (2021) with the addition of phosphate-solubilizing bacteria (psb). however, the application of probiotics as psb in agriculture remains limited; therefore, this study was conducted to examine the effect of psb derived from probiotics and the reduction of inorganic phosphorus fertilizer dosage on the stem juice characteristics of bmr mutant sorghum grown in ultisol soil. biotropia vol. 32 no. 3, 2025 396 materials and methods the tools used in this research included a plow machine, sprayer, scales, and hand refractometer. the materials used comprised bmr sorghum mutant line biotrop seeds obtained from the silviculture laboratory of seameo-biotrop, bogor; manure; urea; tsp; kcl; and bacillus sp. culture from waretha probiotic (a probiotic produced by universitas andalas, indonesia). the research was conducted at the edufarm of the faculty of animal science, universitas andalas, padang, indonesia (-0.912535, 100.467847), using a randomized block design (rbd) consisting of four treatments and four replications. the treatments were as follows: p0 = urea + kcl + 0% tsp + probiotic psb; p1 = urea + kcl + 50% tsp + probiotic psb; p2 = urea + kcl + 75% tsp + probiotic psb; p3 = urea + kcl + 100% tsp (control). land preparation each experimental plot measured 4 × 5 m. the results of soil analysis prior to the study indicated a ph of 4.5 (acidic) and a low available phosphorus content of 9.61 ppm. rainfall during the study period ranged from 226 mm to 492 mm. the field planting design is presented in figure 1. probiotic preparation the bacillus sp. strain from waretha probiotic was cultivated on nutrient broth (nb) medium, producing a bacterial culture population density of 10⁷ cfu/g. the bacterial count was determined using the total plate count method (cundell 2015). a total of 10 g of waretha probiotic was dissolved in 90 ml of sterile distilled water. special media for bacillus sp. were prepared in 100 ml volumes and heated to boiling before being used as agar media for the bacterial culture procedure, which was conducted using the surface method. a 1 ml aliquot from the 10⁷ dilution was pipetted onto the agar surface in a sterile petri dish and spread evenly after the medium had cooled to 47 50 °c. the plates were then incubated at 36 °c for 24 hours. planting seeds and fertilizing the fertilizers used were urea, tsp, and kcl at a ratio of 2:3:2 (v/v), corresponding to application rates of 60 kg/ha, 90 kg/ha, and 60 kg/ha, respectively (sriagtula et al. 2016; wahyono et al. 2019). the first fertilizer application was conducted at 14 days after sowing (das) with two-thirds of the total dosage, while the remaining one-third was applied at 40 das. manure was applied at a rate of 10 tonnes/ha (sriagtula et al. 2016). the probiotic psb from waretha probiotics, with a density of 10⁷ cfu/g, was applied at a dose of 10 ml per planting hole. the application was performed twice, first at 14 days after sowing (das) and again at 40 das. sorghum was harvested at the soft dough stage (93 das) by cutting the stems 10 cm above the soil surface. figure 1 field planting design effect of phosphate-solubilizing bacteria on juice of brown midrib sorghum sriagtula et al. 397 estimation of soil bacillus sp. population one gram of soil sample was suspended in 9 ml of distilled water in a test tube and vortexed to obtain a 10-¹ dilution. subsequently, 1 ml of this suspension was transferred into a new test tube containing 9 ml of distilled water to prepare serial dilutions up to 10-⁸. one milliliter of the appropriate dilution was then pipetted into a petri dish, and 7 10 ml of bacillus selective media was added. the mixture was homogenized by gently rotating the dish in a figure-eight pattern and then incubated at 37 °c for 24 hours. observations were conducted on the second day after planting, and bacterial colonies were counted. colony counts were expressed as colony-forming units (cfu) per milliliter. the plate counting method was employed for enumeration (cundell 2015). phosphate-solubilizing bacteria assay approximately 1 ml of each serial dilution (101, 10-2, and 10-8cfu/g soil) was inoculated onto pikovskaya medium. the plates were incubated at room temperature for 3 to 5 days. phosphatesolubilizing bacteria (psb) were identified by the formation of clear zones around colonies, indicating their ability to solubilize phosphorus on the pikovskaya medium. observations and measurements observations were conducted on 10 plants randomly selected from each plot. the parameters measured included stem height, which was recorded after harvest using a measuring tape. stem sugar content (%brix) was determined from juice extracted from the lower stem using a refractometer by placing stem juice droplets on the instrument. juice volume was measured by pressing sweet sorghum stems with a sugarcane squeezer, and the extracted juice was collected and quantified using a measuring cup. correlation coefficients between various parameters and the brown midrib mutant sorghum stem juice volume were analyzed. data analysis the data were analyzed using analysis of variance (anova) in spss version 20. multiple linear regression analysis was employed to evaluate the parameters influencing stem juice yield. additionally, linear regression and pearson correlation analyses were conducted to determine the relationships between parameters. results and discussion sugar content, juice volume, stem height and total colony the average sugar content (%brix) of bmr mutant sorghum stem juice following the application of probiotic bacillus sp. and varying doses of phosphorus fertilizer is presented in table 1. statistical analysis showed that the treatments did not have a significant effect (p > 0.05) on the sugar content of bmr mutant sorghum stem juice. this result is consistent with kansaye et al. (2023), who reported minimal variation in sugar concentration under high or low phosphorus fertilization. in contrast, oliveira et al. (2022) found that available phosphorus strongly influenced the sucrose percentage in sugarcane juice. additionally, ariefin et al. (2021) noted that sorghum variety significantly affects stem sugar content and juice volume. since this study utilized the same sorghum line, i.e., bmr sorghum from biotrop, the brix level and juice volume showed no significant differences. the average sugar content in this study ranged from 11.77% to 12.75% brix. lestari et al. (2019) reported that sugar content in sweet sorghum stem juice during the maturity phase ranged from 7% to 18% brix. however, the findings of the present study do not align with those of sriagtula et al. (2019), who observed sugar content ranging from 14.57% to 18.75% brix. this variation is attributed to differences in sorghum varieties and average rainfall during the study period. climate factors, particularly precipitation, also influence stem sugar content. monthly rainfall during the study ranged from 226 mm to 492 mm, representing moderate intensity. latupapua et al. (2018) classified monthly rainfall between 200 mm and 500 mm as moderate to heavy, which has been associated with lower %brix in sorghum stem juice. muñoz and trujillo (2020) reported that %brix in sorghum juice is affected by precipitation levels, as excessive water can inhibit photosynthesis by reducing stomatal conductivity, leading to decreased water absorption by roots and stomatal closure, thereby reducing sugar synthesis. the results indicated that the application of psb (phosphate solubilizing bacteria) and different doses of phosphorus fertilizer did not have a significant effect (p > 0.05) on the stem juice volume of bmr mutant sorghum. this finding contrasts with that of munawwarah and sulaeman (2023), who reported that biofertilizer application biotropia vol. 32 no. 3, 2025 398 increased stem juice volume. the planting season has been identified as a primary factor influencing stem juice quantity. pabendon et al. (2017) reported that sorghum planted during the rainy season produced higher fresh juice yields compared to planting during the dry season. munawwarah and sulaeman (2023) also found that harvesting time, rainfall, variety, and soil fertility affected juice volume. other factors influencing sorghum stem juice volume include genotype and harvest timing. in this study, sorghum plants were harvested at the soft dough stage, at 93 days after sowing (das). mursyid et al. (2017) found that harvesting before physiological maturity resulted in optimal juice volume. based on the treatments applied, the average stem juice volume ranged from 184.25 to 206.97 ml per stem, exceeding the 101.93 to 136.48 ml per stem reported by lestari et al. (2019) for sweet sorghum mutant harvested at 80 das. no significant differences were observed in stem height growth or the total colony count of psb bacillus sp. in the soil among all treatments compared to the control (p3), indicating that the phosphate solubilization capacity was assumed to be similar across treatments (table 1). this finding aligns with djuuna et al. (2022), who reported a direct correlation between psb population density and soil phosphorus availability, with larger bacterial populations associated with enhanced nutrient uptake to support plant growth. the application of psb did not significantly affect total colony counts in any treatment. this outcome may be explained by the psb concentration used in this study (10⁷ cfu/g), which was lower than the recommended 10⁹ cfu/g. li et al. (2024) demonstrated that the soil microbial population is significantly altered when phosphate-solubilizing bacterial inoculants are applied at concentrations of 10⁹ cfu/g, promoting beneficial microbial interactions that enhance soil health, nutrient cycling, soil fertility, and phosphorus availability. bacteria cultivated on pikovskaya agar medium produced clear zones surrounding their colonies, indicating phosphate solubilization. insoluble phosphate (ca₃(po₄)₂) is present in pikovskaya medium, and bacteria bind to this molecule, releasing h₂po₄ions. the release of these ions results in the formation of clear zones around the colonies. this phenomenon is attributed to phosphatase enzyme activity, which causes a localized decrease in medium ph (fig. 1). additionally, these microorganisms produce organic acids that lower ph and react with phosphorus-binding agents such as al³+, fe³+, ca²+, and mg²+ to form organic chelates, thereby releasing free phosphate ions (po₄³-) (fatimah et al. 2021). however, the area of the clear zones was not measured in this study. significant relationships were observed between juice volume and stem weight, stem diameter, stem height, and %brix, with r² values of 0.5669, 0.5621, 0.432, and 0.0691, respectively (fig. 2). these results indicated that juice volume increased with increasing stem weight, stem diameter, stem height, and %brix. therefore, stem weight, stem diameter, stem height, and %brix were identified as important parameters influencing juice volume. conversely, juice volume exhibited a negative relationship with %panicle, described by the equation y = −21.464x + 826.1 and an r² value of 0.2416. table 1 average bmr mutant sorghum stem juice sugar content treatment sugar content (%brix) juice volume (ml) steam height (cm) colony of bacillus sp. (1x108 cfu/ml) p0 12.75±1.87 206.97±54.22 222.32±40.24 30.00±1.60 p1 12.42±2.06 188.75±51.43 219.53±31.09 29.00±1.08 p2 11.77±1.76 205.13±34.83 227.30±34.91 31.25±2.53 p3 12.10±.2.03 184.25±47.19 214.23±29.47 34.25±3.86 mean 12.06±1.98 189.10±53.32 220.85±34.19 31.12±2.27 notes: the effect of treatments is not significantly different (p > 0.05); p0 = urea + kcl + 0% tsp + probiotic psb; p1 = urea + kcl + 50% tsp + probiotic psb; p2 = urea + kcl + 75% tsp + probiotic psb; p3 = urea + kcl + 100% tsp + without probiotic psb. effect of phosphate-solubilizing bacteria on juice of brown midrib sorghum sriagtula et al. 399 figure 1 the results for psb (phosphate solubilizing bacteria) test note: red arrow indicated the formation of the clear zone. figure 2 relationships between juice volume and stem, sugar content, and panicle notes: a = relationship between juice volume with stem weight; b = relationship between juice volume and stem diameter; c = relationship between juice volume and stem height; d = relationship between juice volume and sugar content (%brix); e = relationship between juice volume and %panicle. biotropia vol. 32 no. 3, 2025 400 correlation coefficient of various parameters on the volume of sorghum stem juice the correlation coefficient values between various parameters and the juice volume of bmr sorghum stems are presented in table 2. analysis revealed a significant relationship between stem diameter and juice volume, with a correlation coefficient of 0.515**, indicating a strong positive association. an increase in stem diameter corresponded to a higher juice volume, as larger stem diameters allow for greater storage of photosynthates in the stem. oliveira et al. (2021) similarly reported a positive correlation between stem diameter and juice volume (r = 0.03*). thus, stem size was found to significantly affect juice productivity. correlation coefficient analysis revealed a significant relationship between stem weight and juice volume, with a correlation coefficient of 0.481**, indicating a strong positive association. an increase in fresh stem weight corresponded to a higher juice volume. this result is consistent with findings by mursyid (2017), who reported a significant correlation between stem weight and juice volume (r = 0.858**). these findings suggest that optimal juice volume is associated with higher fresh stem weight. correlation coefficient analysis showed no significant relationship between sugar content and juice volume (r = 0.154, not significant). this finding contrasts with earlier results reported by suwarti et al. (2018), who observed a significant correlation between sugar content and juice volume (r = 0.40). these results suggest that sorghum genotypes with high sugar content do not necessarily exhibit high juice volume potential. additionally, juice volume was not significantly correlated with dry matter content (r = 0.182, not significant) in bmr mutant sorghum according to the correlation analysis. table 2 .correlation coefficient values of various parameters on the volume of juice of bmr mutant sorghum stems (n=160) %brix juice volume (ml) stem weight (g) stem diameter (mm) stem height (cm) %panicle dry matter (%) %brix 1 .154 .263** .176* .337** .420** .182* juice volume (ml) .154 1 .481** .515** .003 .010 .135 stem weight (g) .263** .481** 1 .750** .657** .492** .22 1 stem diameter (mm) .176* .515** .750** 1 .333* .256** .043** stem height (cm) .337** .232** .657** .333* 1 .514** .330** %panicle .420** .202* .492** .256** .514** 1 .271** dry matter (%) .182* .119 .221 .043** .330** 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[moench]) on several population density for bioethanol production. iop conf. series: earth and environmental science 141(2018):012032. doi: 10.1088/17551315/141/1/012032 wahyono t, sugiono i, jayanegara a, wiryawan kg, astuti da. 2019. nutrient profile and in vitro degradability of new promising mutant lines sorghum as forage in indonesia. adv ani vet sci 7(9):810. the southeast asian journal of tropical biology vol. 31 no. 2, 2024: 238 252 doi: 10.11598/btb.2024.31.2.2143 issn: 0215-6334 | e-issn: 1907-770x 238 morphological and molecular characterization of donax faba (bivalvia: donacidae) obtained from kutang beach, lamongan, indonesia reni ambarwati1*, fida rachmadiarti2, herlina fitrihidajati2, tarzan purnomo2, dwi anggorowati rahayu1, and ulfi faizah3 1animal systematics laboratory, biology study program, faculty of mathematics and natural sciences, universitas negeri surabaya, ketintang campus, surabaya 60231, indonesia. 2ecology laboratory, biology study program, faculty of mathematics and natural sciences, universitas negeri surabaya, ketintang campus, surabaya 60231, indonesia. 3animal systematics laboratory, biology education study program, faculty of mathematics and natural sciences, universitas negeri surabaya, ketintang campus, surabaya 60231, indonesia. article higlights • this study revealed 19 morphological variations of edible wedge clams, donax faba (bivalvia: donacidae), from kutang beach, lamongan, indonesia, which has significant role as part of coastal ecosystem. • this reseach also highlights the dna barcoding of donax faba based on coi gene (cytochrome c oxidase subunit i). • the average genetic distance of the research samples was 0.46%, while the value of this parameter between the research samples and the ingroup was found to be 1.51%. article information received 5 december 2023 revised 23 april 2024 accepted 2 may 2024 *corresponding author, e-mail: reniambarwati@unesa.ac.id research article abstract donax faba is a type of wedge clams with significant ecological and economic roles, as well as variations in color, pattern, and morphometric properties. recently, a particular population of unidentified d. faba was reported from kutang beach, lamongan, indonesia. therefore, this research aimed to assess the morphological variations and molecular characteristics of d. faba obtained from kutang beach based on coi gene. a total of 288 samples were collected during the lowest tide for morphological characterization of the color, pattern, and morphometrics of the shells. additionally, molecular characterization was conducted based on the composition of nucleotide bases and amino acids of coi gene, genetic distance, as well as the relationships. the results showed that d. faba had 19 morphological variations, among which the most dominant type contained a whitish exterior with brown spots and a whitish purple interior. the average genetic distance of the samples was 0.46%, while the value was calculated as 1.51% between the samples and the ingroup. automatic barcode gap discovery (abgd) analysis performed using a prior maximal distance of 0.001 showed the separation of these species into distinct categories. keywords: coastal ecosystem, coi gene, dna barcoding, wedge clams introduction donax faba is popularly known as wedge clams living in intertidal areas (ambarwati & faizah 2017; alyani & ambarwati 2018) along sandy beaches (yambem et al. 2017; rittiboon et al. 2019; signorelli & printrakoon 2020) and mangrove forests (singh et al. 2011; kassim et al. 2018). these clams are harvested in vietnam and thailand for the nutritional value (krishnan & tharavathy 2016) and trade purposes (poutiers 1998), while being consumed in several regions of indonesia including west java (dharma 2005) and madura (ambarwati & faizah 2017; alyani & ambarwati 2018; wasilah et al. 2021). donax faba is capable of accumulating heavy metals (singh et al. 2012; wasilah et al. 2021) and serves as a potential source of anticoagulant compounds (periyasamy et al. 2013). moreover, the shells can be processed into flour, serving as a mineral source in feedstuff (lalopua & sukisman 2023). https://doi.org/10.11598/btb.2024.31.2.2143 morphological and molecular characterization of donax faba ambarwati et al. 239 members of the donacidae family, including d. faba, have been reported with morphological variations. tan & low (2013) detected variations in color, shape, and pattern among d. faba samples in singapore, while rittiboon et al. (2019) identified eight color patterns from bangling beach, thailand. dharma (2005) described five types of d. faba shells collected from west java. ambarwati and faizah (2017) as well as alyani and ambarwati (2018) reported 12 and 15 respective variations of patterns and colors from nepa and tengket beaches in madura. atlanta et al. (2022) recently found some d. faba at kutang beach, lamongan, but morphological variations in this population remain unexplored. high species variations often lead to difficulties in the identification process, which can only be enhanced by the availability of comprehensive information regarding the morphological variations. additionally, dna barcoding is among the current valuable tools used to strengthen identification based on morphological data (moritz & cicero 2004; hebert & gregory 2005; ferri et al. 2009; packer et al. 2009). research across various animal taxa, including porifera (cárdenas et al. 2009; vargas et al. 2012), echinoderms (layton et al. 2016), mollusca (juniar et al. 2021; sari et al. 2021), and fish (rahayu et al. 2019; 2012), had used dna barcoding markers for identification. coi dna barcoding was previously applied to successfully identify variations in d. incarnatus from madura (wijaya et al. 2023). additionally, genetic distance analysis based on coi gene sequences of certain samples showed high similarity with comparison species from genbank and bold systems. differences in average genetic distance values can be caused by intragroup genetic diversity. a genetic distance is assumed to be very low when after conversion to less than 2% the values signify the same species. however, values greater than 2% suggest the existence of a species different from other group members (wong & hanner 2008; wong et al. 2009). in an investigation performed by sari et al. (2021), genetic identification of d. faba from nepa beach, madura showed coi gene sequence of 650 bp with similarity values ranging from 72.01% to 72.12% when compared to sequence data from genbank. coi gene sequence of d. faba is characterized by a high mutation rate, leading to significant genetic variations and geographic influences on genetic plasticity. consequently, further research is recommended to determine the molecular characteristics of d. faba. considering the provided background, this research aimed to assess the morphological variations and molecular characteristics of d. faba samples collected from kutang beach based on coi gene in mtdna, followed by the conduction of phylogenetic relationship analysis. the molecular characterization and phylogenetic analysis data obtained will serve as a foundation for further investigation into the evolutionary history, population dynamics, and adaptation of d. faba in the natural habitat. materials and methods field work samples of d. faba were collected from kutang beach, lamongan (fig. 1) during the lowest tide. five plots measuring 1 m x 1 m were placed horizontally along the beach line with 3 m interval between each plot. samples for shell morphological analysis were preserved in 70% alcohol, then three from the most dominant shell types were selected and preserved in absolute alcohol for molecular analysis. laboratory work identification and morphometric measurements the shells of collected donax clams were cleaned and identified using the identification books of poutiers (1998), dharma (2005), huber (2010), and ambarwati & faizah (2017). subsequently, morphometric measurements, including shell length (sl), shell height (sh), shell width (sw), dorsal height (dh), dorsal and umbo outline (fig. 2), were conducted using calipers with an accuracy of 0.01 mm (ambarwati & faizah 2017). sl is defined as the perpendicular distance between the anterior and posterior shell, while sh is measured from the highest dorsal to the lowest ventral part of the shell. furthermore, sw represents the distance between the protruding parts of the lateral sides of two shells. dh is measured from the highest part of the dorsal side to the pseudo line with a perpendicular distance between the anterior and posterior shell. the umbo margin line (uml) is described as the distance from the most dorsal part of the shell to the posterior. biotropia vol. 31 no. 2, 2024 240 figure 1 the map showing the sampling location of d. faba (red dot) in kutang beach figure 2 morphometric measurements of d. faba molecular work (dna isolation and sequencing) total dna isolation from muscle tissue was performed using the isolation kit (roche) (kit catalogue number 05985536190) with several modifications. initially, 200 µl of buffer gt1 was pipetted into a 1.5 ml tube and mixed by vortexing. next, 200 µl gt2 buffer and 20 µl proteinase k were added, and the mixture was thoroughly combined through vortexing. the blend was incubated for 10 minutes at 56 °c, with gentle inversion of the tube every 5 minutes. subsequently, 200 µl absolute ethanol was introduced to the mixture and briefly vortexed. the sample was transferred to spin column and centrifuged for 1 minute at 13,000 rpm. the resulting flow-through was discarded, and 500 µl buffer w1 was added, followed by another round of centrifugation for 1 minute at 13,000 rpm. after disposing of the flow-through, 700 µl buffer w2 containing ethanol was introduced and centrifuged for 1 minute at 13,000 rpm. the flowthrough was discarded again, and centrifugation was conducted for an additional 2 minutes. dna retained in spin column was transferred to a new 1.5 ml tube, then 50-100 µl elution buffer was added and incubated at room temperature for 1 minute before being centrifuged for 1 minute. finally, spin column was removed, the purified dna was prepared for further steps, and dna was temporarily stored at -20 °c over a few days. the isolation results were then amplified using biorad pcr machine in a 30 µl solution consisting of 15 µl pcr master mix nexpro, 3 µl dna template samples (100 ng/µl), 6 µl water, and 3 µl primers (10 pmol each of forward and reverse primers). the primers used were lco1490 morphological and molecular characterization of donax faba ambarwati et al. 241 5'-ggtcaacaaatcataaagatattgg-3' and hco2198(5'-taaacttcagggtgacc aaaaaatca-3') (folmer et al. 1994). amplification was performed with the following temperature settings including pre-denaturation at 94 ºc for 1 minute, followed by 40 cycles of denaturation at 94 ºc for 45 seconds, annealing at 45 ºc for 45 seconds, and extension at 72 ºc for 1 minute 30 seconds. subsequently, the postelongation process was carried out at a temperature of 72 ºc for 10 minutes. pcr results were electrophoresed on 1% agarose, then sequenced using 1st base laboratories sdn bhd sequencing services. data analysis analysis of morphological and morphometric data morphological data were analyzed descriptively, while samples were classified based on shell color patterns, with the relative frequency of each type calculated as a percentage. subsequently, the average and standard deviation of each morphometric parameter was estimated. types comprising a minimum of 10 individuals (n ≥ 10) were analyzed for shell patterns using the ratio of each shell size and the relationship between patterns was evaluated through linear regression. to determine significant differences between types, analysis of variance (anova) was conducted followed by the games-howell test. molecular analysis sequence data from genbank ncbi (national center for biotechnology information) in addition to dna sequence data obtained from this research was used for phylogenetic analysis (table 1). dna sequence readings were used to determine the genetic variations of coi gene, the composition of the nucleotide bases and amino acids of coi gene, as well as the genetic distance, followed by relationship analysis. moreover, the chromatogram data from the sequencing results were visualized using finchtv to assess sequence quality. the k2p substitution model (saitou & nei 1987) was applied in calculating the settings for ml phylogenetic tree reconstructions. a bootstrap consensus tree inferred from 1,000 replicates was used to describe the variation rates among sites. furthermore, adjacent branches showed the percentage of replicate trees in which the related taxa clustered together in the bootstrap test (1,000 repetitions). grouping analysis was carried out through a web interface (puillandre et al. 2012) to examine the distribution and size of a potential barcoding gap for the partial sequence of coi gene dataset. the barcode gap generated through automatic barcode gap discovery (abgd) was used to strengthen the species identification process. table 1 sequences from ncbi genbank used as reference species num species sample location acc number of genbank ncbi 1. donax faba phuket, thailand mt334596.1 2. donax faba prachubkirikhun, thailand mt334599.1 3. donax faba rayong, thailand mt334600.1 4. donax faba japan ab040845.1 5. donax incarnatus chantaburi, thailand mt334591.1 6. donax incarnatus phuket, thailand mt334590.1 7. donax incarnatus prachubkirikhun, thailand mt334593.1 8. donax incarnatus rayong, thailand mt334592.1 9. donax cuneatus chantaburi, thailand mt334594.1 10. donax cuneatus phuket, thailand mt334595.1 11. donax cuneatus japan ab040842.1 12 donax faba type18 kutang beach, lamongan pp593778 (this research) 13 donax faba type 2 kutang beach, lamongan pp595807 (this research) 14 donax faba type 11 kutang beach, lamongan pp595808 (this research) biotropia vol. 31 no. 2, 2024 242 results and discussion morphological characterization during the field trip to kutang beach, a population of d. faba clams was observed in the upper intertidal zone at the tidal boundary with a sandy substrate. this habitat type was consistent with previous research, which reported the habitat of d. faba as a sandy substrate (ambarwati & faizah 2017; tenjing 2017; yambem et al. 2017). the population of d. faba at kutang beach reached 57.6 ind./m2 with a total of 288 identified samples. previous observation (eshky 1998) along the sandy substrate in the red sea showed that the population density of d. faba ranged from 30 to 296 ind./m2, corresponding to the results of this research. the description of d. faba from kutang beach, lamongan is as follows. shell shape: thick, flat, inequilateral, trigonal oval. shell length reaches 32.74 mm; shell height reaches 25.64 mm. shell sculpture: smooth surface with thin concentric lines that become more prominent posteriorly. umbo: protrude, prosogyrate. color: white, cream, brown, purple; often with one or more radial bands or broad random patches; the interior of the shell is white, often with yellow shading, there are purplish to purple spots, and/or radial bands. dentition: heterodont with anterior and posterior lateral teeth. shell interior: anterior adductor muscle attachment site elongated and posterior adductor muscle attachment site rounded; deep pallial sinus (approximately ½ shell length); pallial line is clear (figs. 3 & 4). this description corresponds to the reports of tan and low (2013), ambarwati and faizah (2017), and signorelli and printrakoon (2020). the population of d. faba showed high morphological variations, including different interior and exterior shell color patterns, as well as shell morphometry. samples collected at kutang beach had 19 variations in interior and exterior color (figs. 3 & 4; table 2). variations found with the highest relative frequency were types 18, 2, and 11 (fig. 5). particularly, type 18 contained a white shell exterior and a whitish purple interior with a frequency of 18.1%. type 2 showed a cream exterior shell color with brown spots and a yellowish-white interior, as well as a frequency of 13.5%. type 11 comprised a cream exterior with brown spots and a brown interior with white spots, constituting 11.5% of d. faba population on kutang beach. table 2 color and morphometric variations of d. faba from kutang beach type n  color morphometry (mean±sd) sl (mm) sh (mm) sw (mm) uml (mm) dh (mm) type 1 13 ext: yellowish white; int: white 13.70±2.73a 7.93±2.12a 1.47±1.06a 8.43±2.25a 4.75±1.54a type 2 39 ext: cream with brown maculation; int: yellowish white 20.56±4.34b,c 14.68±4.47c,d 8.84±6.66b,c 15.91±4.43c,d 11.7±3.7c,d,e type 3 2 ext: white; int: white with radial purple 25.56±1.60 19.59±1.68 12.13±0.88 19.86±1.48 16.57±0.98 type 4 2 ext: white with a radial band at posterior region; int: white with radial band at posterior region 23.06±3.10 19.31±0.45 12.04±0.40 20.42±0.71 16.02±2.37 type 5 30 ext: white with radial purple band; int: white 20.90±5.51b,c 15.53±4.51c,d 9.23±3.80c 16.75±5.03c,d 12.58±4.15d,e type 6 27 ext: cream with radial purple band; yellowish white 21.06±3.23b,c 15.32±2.58c,d 7.94±2.03b,c 14.08±2.88b,c 9.97±2.49b,c,d type 7 1 ext: light brown; light brown 20.7±0.00 12.94±0.00 8.26±0.00 14.2±0.00 11.34±0.00 type 8 11 ext: cream with brown maculation; int: dark purple 23.21±2.22c 16.35±1.13d 9.01±1.02c 15.04±2.26b,c,d 12.22±2.57d,e morphological and molecular characterization of donax faba ambarwati et al. 243 type n  color morphometry (mean±sd) sl (mm) sh (mm) sw (mm) uml (mm) dh (mm) type 9 11 ext: dark brown with light brown maculation; int: brown with white maculation 18.25±3.82a,b 12.69±2.76b,c 7.23±2.13b,c 11.54±3.80a,b 8.71±3.06b,c type 10 1 ext: brown; int: brown 24.02±0.00 17.64±0.00 9.07±0.00 15.94±0.00 13.41±0.00 type 11 33 ext: cream with brown maculation; int: brown with white maculation 19.20±2.16b,c 13.62±1.40b,c,d 7.13±7.13b,c 15.30±2.62b,c,d 10.2±2.65b,c,d type 12 2 ext: brown with white maculation; int: dark brown 14.03±3.00 9.70±2.07 5.05±1.46 10.30±2.84 6.65±2.75 type 13 6 ext: black with white maculation; int: deep dark 19.84±2.08 14.36±1.91 7.61±1.29 14.98±2.20 11.35±2.51 type 14 10 ext: cream with dark brown maculation; int: white with brown maculation 15.09±2.93a 10.49±2.21a,b 5.29±1.21b 11.73±2.12a,b 7.74±2.14a,b type 15 8 ext: dark brown; int: dark brown 16.34±2.24 10.49±2.21a,b 5.29±1.21b 11.73±2.12a,b 7.74±2.14a,b type 16 20 ext: black with white spot: int: whitish black 21.96±5.04b,c 15.70±4.52c,d 8.46±3.20b,c 18.02±4.48d 14.17±4.52e type 17 3 ext: cream with white radial band; int: white 18.33±3.34 13.22±2.57 7.10±2.04 15.58±3.61 10.03±2.88 type 18 52 ext: white with brown spots; int: whitish purple 19.70±3.43b,c 14.27±2.87c,d 7.42±1.60b,c 15.22±2.92b,c,d 10.06±2.6b,c,d type 19 17 ext: cream with brown maculation; int: whitish purple 19.52±2.82b,c 14.20±2.37c,d 7.18±1.49b,c 15.37±2.81b,c,d 10.3±2.48b,c,d p value 0.000* 0.000* 0.000* 0.000* 0.000* notes: n = number of examined shells; sl = shell length; sw = shell width; dh = dorsal height; sh = shell height; uml = umbo margin line; ext = exterior shell; int = interior shell; sd = standard deviation. * = there are differences in average morphometry between types. a,b,c,d,e = the same letter category on each measurement among types shows the absence of significant mean difference between types. the results of morphometric measurements showed that each type of d. faba had different sl, sw, sh, dh, and uml (table 2). analysis identified variations in sl between types comprising 3 subsets, each representing a category with no significant difference in average sl value, while differences were observed between several subsets. based on the morphometric measurements, types 1, 2, 5, 6, 8, 11, 16, 18, and 19 had different sl values. d. faba type 14 was significantly different from 2, 5, 6, 8, 11, 16, 18, and 19, while type 9 differed from 8. type 1 had the lowest average sl of 13.7 mm, while type 8 had the highest measuring 23.21 mm. additionally, the average sh of type 1 and 14 was significantly different from 2, 5, 6, 8, 11, 16, 18, and 19. type 8 varied from 9, 11, and 18, with calculations showing that type 1 had the lowest average sh of 7.93 mm, while type 8 had the highest at 16.35 mm (table 2). these dimensions were smaller than those observed for d. faba found in nepa beach (ambarwati & faizah 2017), but larger than the values measured for samples collected from tengket beach (alyani & ambarwati 2018). the shells had variations in sizes, pattern, and outline, with ratios including sw and sh, sw and sl, as well as sh and sl appearing significantly different among all types (table 3). the relationship between shell size parameters was described using linear regression equations, with statistical test results showing different regression equations for each type. the shell size ratios of d. faba from lamongan were similar to those measured in samples collected from nepa beach, madura (ambarwati & faizah 2017). biotropia vol. 31 no. 2, 2024 244 figure 3 morphological variations of d. faba collected from kutang beach, lamongan indonesia notes: a = type 1; b = type 2; c = type 3; d = type 4; e = type 5; f = type 6; g = type 7; h = type 8; i = type 9; j = type 10; k = type 11; l = type 12. scale bars = 10 mm. morphological and molecular characterization of donax faba ambarwati et al. 245 figure 4 morphological variations of d. faba from kutang beach, lamongan, indonesia notes: m = type 13; n = type 14; o = type 15; p = type 16; q = type 17; r = type 18; s = type 19. scale bars = 10 mm. biotropia vol. 31 no. 2, 2024 246 table 3 d. faba shell patterns based on measurement ratio and linear regression measurement n type ratio regression regression formula p value regression r-square sw and sh 13 type 1 0.186a sw = -2.095 + 0.45sha 0.000* 0.811 39 type 2 0.602b sw = -4.709 + 0.923shc 0.000* 0.385 30 type 5 0.595b sw = -3.409 + 0.814shc 0.000* 0.936 27 type 6 0.518b sw = -2.754 + 0.698shc 0.000* 0.79 11 type 8 0.551b sw = -1.348 + 0.634shc 0.017* 0.486 11 type 9 0.570b sw = -0.948 + 0.64shb,c 0.001* 0.700 33 type 11 0.523b sw = 0.379 + 0.495shd 0.000* 0.585 10 type 14 0.505b sw = 0.321 + 0.474shb 0.001* 0.752 20 type 16 0.539b sw = -1.6 + 0.641shc,d 0.000* 0.822 52 type 18 0.520b sw = 1.557 + 0.411shd 0.000* 0.541 17 type 19 0.506b sw = -1.205 + 0.59shd 0.000* 0.887 p value anova 0.000** 0.000** sw and sl 13 type 1 0.108a sw = 3.39 + 0.355sla 0.000* 0.837 39 type 2 0.430b sw = -9.141 + 0.875slc,d 0.000* 0.326 30 type 5 0.442b sw = -4.540 + 0.659slc,d 0.000* 0.915 27 type 6 0.377b sw = -3.664 + 0.551slc,d 0.000* 0.770 11 type 8 0.388b sw = 2.817 + 0.267sld 0.062 0.336 11 type 9 0.396b sw = 0.514 + 0.368slb,c 0.027* 0.435 33 type 11 0.371b sw = -0.719 + 0.409slb,c 0.000* 0.584 10 type 14 0.351b sw = -0.108 + 0.358slb 0.001* 0.753 20 type 16 0.385b sw = -4.04 + 0.569sld 0.000* 0.805 52 type 18 0.376b sw = 1.362 + 0.307slb,c 0.000* 0.432 17 type 19 0.368b sw = -2.27 + 0.484slb,c,d 0.000* 0.840 p value anova 0.000** 0.000** sh and sl 13 type 1 0.579a sh = -1.865 + 0.715sla 0.000* 0.847 39 type 2 0.714b sh = -5.487 + 0.981slc,d 0.000* 0.907 30 type 5 0.743b sh = -0.815 + 0.782slc,d 0.000* 0.913 27 type 6 0.727b sh = -0.805 + 0.766slc,d 0.000* 0.917 11 type 8 0.704b sh = 8.568 + 0.335sld 0.027* 0.438 11 type 9 0.695b sh = 2.02 + 0.585slb,c 0.003* 0.651 33 type 11 0.709b sh = 0.683 + 0.674slc 0.000* 0.666 10 type 14 0.695b sh = -0.718 + 0.743sla,b 0.000* 0.968 20 type 16 0.715b sh = -2.91 + 0.847slc,d 0.000* 0.892 52 type 18 0.724b sh = 0.288 + 0.709slc 0.000* 0.719 17 type 19 0.728b sh = -1.27 + 0.793slc 0.000* 0.884 p value anova 0.000** 0.000** note: n = number of examined shells; sl = shell length; sw = shell width; sh = shell height. * = there is an influence of the predictor variable on the response variable. ** = anova test showed differences in the average ratio and regression results between types. a,b,c,d = the same letter category in each measure between types represents the absence of significant differences in ratios or average regression results. morphological and molecular characterization of donax faba ambarwati et al. 247 figure 5 relative frequency of morphological pattern of d. faba from kutang beach molecular characterization molecular characterization was performed based on the sequence of cytochrome c oxidase subunit i (coi) gene using three samples of d. faba which had the highest relative frequency of morphological variations. coi gene barcode sequence data among the three research samples showed an average composition of g+c nucleotide base at 41.7% and a+t nucleotide base at 58.3%. according to the average results, the nucleotide base composition of g+c was lower than a+t. furthermore, the g+c and a+t content could provide insights into the evolutionary history and relationships of d. faba. comparing these values with those of related species would clarify genetic divergence, hybridization events, and adaptive evolution processes of d. faba. the average g+c and a+t nucleotide base compositions in coi gene barcode sequence data offered valuable information about the genetic characteristics and evolutionary dynamics of d. faba. table 4 the three highest match values from identification performed through the bold system with representation of similarity values sample names three highest bold identification similarity (%) status d. faba type 18 d. faba 99.53 published d. faba 99.53 published d. faba 99.37 published d. faba type 2 d. faba 99.21 published d. faba 99.21 published d. faba 99.06 published d. faba type 11 d. faba 98.58 published d. faba 98.58 published d. faba 98.43 published biotropia vol. 31 no. 2, 2024 248 table 5 composition of nucleotide bases samples a (%) c (%) g (%) t (%) a+t (%) g+c (%) donax faba type 18 20.9 19.9 21.6 37.7 58.6 41.4 donax faba type 2 21.2 19.9 21.9 37.0 58.2 41.8 donax faba type 11 21.2 19.9 21.9 37.0 58.2 41.7 average 21.1 19.9 21.8 37.2 58.3 41.7 notes: a = adenine; c = cytosine; g = guanine; t = thymine. table 6 nucleotide base variations num. species nucleotide base variations 134 184 1. donax faba mt334596.1 t t 2. donax faba mt334599.1 ● ● 3. donax faba mt334600.1 ● ● 4. donax faba ab040845.1 ● ● 5. donax faba type 18 pp593778 (this research) ● ● 6. donax faba type 2 pp595807 (this research) g a 7. donax faba type 11 pp595808 (this research) g a 8. donax cuneatus mt334594.1 ● ● 9. donax cuneatus mt334595.1 ● ● 10. donax cuneatus ab040842.1 ● ● 11. donax incarnatus mt334590.1 ● ● 12. donax incarnatus mt334591.1 ● ● 13. donax incarnatus mt334592.1 ● ● 14. donax incarnatus mt334593.1 ● ● note: ● = conserved sequence. table 5 shows transition and transversion mutations of coi gene nucleotide sequence of the samples when compared to related species. transversion substitution of nucleotide base number 134 presented a change in base t (thymine) to base g (guanine). additionally, the transition substitution of nucleotide base number 184 included a change in base t (thymine) to a (adenine). the results showed that two unique nucleotide base patterns, known as automorphic nucleotide bases, were exclusively present in d. faba samples. automorphic nucleotide bases were specific to d. faba from kutang beach, distinguishing this population from other species (table 6). jannah & rahayu (2019) and priyono et al. (2018) reported that certain species had automorphic nucleotide bases as distinctive markers or features used for differentiation from other species under comparison. zhang & zhao (2004) stated that the transversion substitution would elevate with increasing at base composition in the sequence. the exploration of collected d. faba samples and the ingroup showed an average genetic distance of 0.46%, representing the average genetic divergence among the samples investigated. genetic distance is a measure of the genetic divergence between populations or individuals, often quantified based on genetic markers such as dna sequences (priyono et al. 2018). furthermore, the average genetic distance between the research samples and the ingroup was determined to be 1.51%. this value showed the average genetic differentiation between d. faba and the ingroup, which could consist of related species or other reference samples. the differences in the composition of the nucleotide bases in each sequence signified the existence of genetic variation between species (saleky et al. 2020). moreover, cai et al. (2016) stated that a genetic distance value of < 2% showed the group comprised the same species, and > 2% suggested the group was a different species from other members. this signified that the samples collected from kutang beach were identified morphological and molecular characterization of donax faba ambarwati et al. 249 as one species with d. faba. chiu et al. (2013) reported various factors such as environmental conditions, overexploitation, and geographical location to be capable of influencing the diversity of genetic distances in a species. additionally, certain environmental factors could impact the morphology and phylogenetic characteristics of populations in a species. the reconstruction of the phylogenetic tree of d. faba showed the existence of three clusters (fig. 6). cluster 1 consisted of two clades, namely d. faba research samples (featuring a bootstrap value of 70-88) along with the close relative originating from thailand and japan. cluster 2 consisted of d. cuneatus originating from thailand and japan, while cluster 3 comprised d. incarnatus originating from thailand. table 7 genetic distances samples 1 2 3 4 5 6 7 8 9 10 11 12 13 donax faba mt334596.1 donax faba mt334599.1 1.39 donax faba mt334600.1 1.74 0.34 donax faba ab040845.1 3.16 2.44 2.80 donax faba type 18 pp593778 (this research) 1.39 0.00 0.34 2.44 donax faba type 2 pp595807 (this research) 2.09 0.69 1.03 3.15 0.69 donax faba type 11 pp595808 (this research) 2.09 0.69 1.03 3.15 0.69 0.00 donax cuneatus mt334594.1 18.76 19.22 18.76 19.19 19.22 20.09 20.09 donax cuneatus mt334595.1 17.30 17.75 17.30 16.47 17.75 18.61 18.61 14.96 donax cuneatus ab040842.1 17.81 18.27 17.81 17.84 18.27 19.13 19.13 5.37 13.24 donax incarnatus mt334590.1 21.81 21.33 20.86 22.31 21.33 22.23 22.23 19.94 20.09 20.42 donax incarnatus mt334591.1 23.19 22.71 22.23 23.71 22.71 23.63 23.63 20.36 19.16 21.78 4.63 donax incarnatus mt334592.1 22.23 21.76 21.29 22.74 21.76 22.67 22.67 20.36 19.16 20.84 3.89 0.69 donax incarnatus mt334593.1 22.23 21.76 21.29 22.74 21.76 22.67 22.67 20.36 19.16 20.84 3.89 0.69 0.00 figure 6 phylogenetic topology determined using the neighbor-joining method morphological and molecular characterization of donax faba 2 biotropia vol. 31 no. 2, 2024 250 morphological and molecular characterization of donax faba 2 figure 7 phylogenetic topology determined using maximum likelihood method morphological and molecular characterization of donax faba 3 figure 8 barcode gap analysis of coi sequences performed through abgd (puillandre et al. 2012) notes: histograms show the distribution of pairwise genetic distances between each pair of samples: (a) histogram of distance; (b) ranked distance; and (c) number of primary species hypotheses (pshs) obtained, for each prior intraspecific divergence. regular red: number of hypothesis species delimitation. morphological and molecular characterization of donax faba ambarwati et al. 251 the reconstruction of d. faba phylogenetic tree using neighbour joining (nj) and maximum likelihood (ml) methods showed the existence of three clusters (fig. 7). cluster 1 consisted of two clades, namely d. faba research samples (featuring a bootstrap value of 100) along with the close relative originating from thailand and japan. cluster 2 comprised d. cuneatus which originated from thailand and japan, while cluster 3 consisted of d. incarnatus originating from thailand. the phylogenetic trees showed that d. faba and the close relative species formed distinct monophyletic branches. however, the proximity at the same node presented genetic relatedness and the positioning of these branches corresponded with a calculated genetic distance of 0.46%, signifying the greatest divergence between each species. nj, ml, and genetic distance data collectively provided strong evidence that d. faba and the close relatives were genetically distant from each other. abgd was used to identify five distinct groups for d. faba and the relatives based on the initial method and the bar-code gap threshold calculated by coi dataset, as shown in figures 8a and 8b. the barcode gap distance value of 0.001 corresponded with the results of abgd grouping which divided the species into five groups (fig. 8c). these were categorized as group [1] (donax faba 1, d. faba 2, and d. faba 3); group [2] (d. faba mt334596.1 and d. faba mt334599.1); group [3] (d. faba mt334600.1 and d. faba ab040845.1); group [4] (d. cuneatus mt334594.1 and d. cuneatus mt334595.1 and d. cuneatus ab040842.1); and group [5] (d. incarnatus mt334590.1, d. incarnatus mt334591.1, and d. incarnatus mt334592.1. the application of abgd analysis, with a prior maximal distance set at 0.001, further strengthened the separation of d. faba and other species in the ingroup into distinct categories. therefore, d. faba was successfully identified by the combined use of genetic distance, phylogenetic analysis, and abgd analysis. based on the comprehensive data obtained through dna barcoding combined with morphological characteristics, it could be inferred that the focused application of these tools provided a reliable and effective means of identifying d. faba at the species level. conclusion d. faba collected from kutang beach had 19 morphological variations. the average genetic distance of the research samples was 0.46%, while the value of this parameter between the research samples and the ingroup was found to be 1.51%. acknowledgment the authors are grateful to the faculty of mathematics and natural sciences, universitas negeri surabaya for providing funding assistance (rector decree number 1117/un38/hk/ pp/2023). the authors are also grateful to rofiza yolanda, ph.d., for helping with manuscript improvement, as well as the reviewers and editors who thoroughly and constructively reviewed the manuscript. references ambarwati r, faizah u. 2017. colour and morphometric variation of donacid bivalves from nepa beach, madura island, indonesia. biosaintifika j biol biol educ 9(3):46673. atlanta v, ambarwati r, rahayu da, mujiono n. 2022. diversity of bivalves on the north coast of lamongan, east java, indonesia. biodiversitas 23(8):4263-71. cárdenas p, menegola c, rapp ht, díaz mc. 2009. morphological description and dna barcodes of shallowwater tetractinellida (porifera: demospongiae) from bocas del toro, panama, with description of a new species. zootaxa (2276):1-39. cai y, zhang l, wang y, liu q, shui q, yue b, …, li j. 2016. identification of deer species (cervidae, cetartiodactyla) in china using mitochondrial cytochrome c oxidase subunit i (mtdna coi). mitochondrial dna part a dna mapping. seq anal 27 (6):4240-3. doi: 10.3109/19401736.2014.1003919. chiu yw, bor h, tan ms, lin hd, jean ct. 2013. phylogeography and genetic differentiation among populations of the moon turban snail lunella granulata gmelin 1791 (gastropoda: turbinidae). intl j mol sci 14 (5):9062-79. doi: 10.3390/ijms14059062. dharma b. 2005. recent and fossil indonesian shells. hackenheim (de): conchbooks. 424 pp. eshky aa. 1998. ecological behaviour and biology of the beach clam donax faba (gmelin) from the red sea. j king abdulaziz univ 10(1):47-72. ferri g, alù m, corradini b, licata m, beduschi g. 2009. species identification through dna ‘barcodes’. genet test mol biomarkers 13(3):421-6. biotropia vol. 31 no. 2, 2024 252 filzan a, ambarwati r. 2018. morphological variations and abundance of donax faba (bivalvia: donacidae) on tengket beach, bangkalan, madura. biotropic 2(2):73-84. 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41(8):828-37. yambem t, samuel vd, meenakshi vk, sankar s, narasimhaiah n. 2017. intertidal biodiversity of karnataka coast, south west coast of india. perspectives on biodiversity of india vol. iii, 186-92. zhang f, zhao z. 2004 the influence of neighboring-nucleotide composition on single nucleotide polymorphisms (snps) in mouse genome and its comparison with human snps. genomics 84(5):785-95. _goback _goback biotropla vol. 28 no. 3,2021: 221 230 doi: 10.1 1598/btb.2021.28.3.1339 diversity of endophytic fungi associated with fruits and leaves of tamarind (tamarindus indica l.) based o n its ribosomal dna sequences nurul asyiqin mohd zaini', nurul huwaidah md nizam, dayang fatin zafira awg zainal abidin, nor izanis azni mohd nazri and nur ain izzati mohd zainudin* department $biology, factllo $science, universiti putra mala_ysia, 43400 serdang, selangor, malysia received 18 february 2020 /accepted 2 may 2020 abstract plant-associated microbes are among essential natural resources that abundantly exist in a natural environment, such as endophyuc fungi. studies on endophytic fungi in medicinal plants have allowed the discovery of numerous fungi species and their hidden potentials. therefore, this study focused on the isolation and identification of endophyuc fungi from several plant parts of tamarind (t. indica), such as leaves and fruits. a total of 69 fungal cultures were successfully isolated and identified into 31 distinct species from 15 genera based on morphological characteristics and internal transcribed spacer (its) sequence analysis using a maximum likelihood method. a high diversity of endophytic fungi associated with t. indica were observed by shannon wiener index h' (3.083). there were six different species obtained from the genus colletotrichzlm (c. aenigma, c. brevisporum, c. cobbittiense, c. fmcticola, c. gloeosporiaides and c. siamense), and diaporthe (d. arecae, d. ceratoxamiae, d. phaseolorzrm, d. pseudomangzjirae, d. pseudooctlii and d. pseudophoenicicala), four species of aspergilzlzls (a. aczlleatzrs, a. carbonarizls, a.flautls and a. tzlbingensis), two species of czlrvzrlank/cochliobolzrs (c. geniculatzrs and c. lunata) and lv&rospora (n. lacticolonia and n. oryxae), two species of lasiod$lodia (l. psetldotheobromae and l theobmmae) and penicillm (p. m@ii and p. verruczllos~m). other fungal species that were also identified are botyosphaeria mamane, fusaritlm solani, tmncospora tephropora, ph_yllostictafallopiae, sarcostroma bisettllatnm, tiichodema asperelhm and xylarid j2ejeensi.r. keywords: endophyuc fungi, internal transcribed spacer (its), phylogenetic tree, tamarind introduction endophytic fungi are microorganisms inhabiting plant tissues in a part of their life without showing any harm toward the host plants. the species of endophytic fungi are expected in over a million species, which arisen from the natural surroundings wshra e t al. 2018). they are widely dstributed, which have been found in many plant species that can grow in natural environments such as terrestrial plant communities (nisa et al. 2015). endophytic fungi such as aspel.gllus, colletotm'cbum, fusam'um, penicillm and tm'cbodema may colonize several parts of plants, includmg fruits and leaves *corresponding author, email: ainizzati@upm.edu.my (hanada e t al. 2010). there are many research studes reported the abundance of fungi associated with plants, however, there is a lack of study in the endophytic fungi associated with t. indica. bourou e t al (2010) reported, three genera of arbuscular mycorrhzal fungi (acaulospora, glomzls and scutellospora) were associated with t. indim. tamarind tree has been reported to be infected by some wood decay fungi such as daldinid concentm'ca, scbixopbylh commune, flavodon jlavus, @ex bydnoides, and pbellinus fastuosus (nnagadesi & arya 2015). in previous reports, aspep!lius n&eq rhixopus stolonifeq ulocladium cban'drum, penicillium cb ysogenum, p. citm'num and pbomopsis liquidambaris were associated with infected-tamarind fruit biotropia vol. 28 no. 3,2021 (danggomen et al. 2013; peter & patrick 2017). isolation, purification and preservation of penin'ilium cblysgenum and p. n'tm'nam are microfungi confirmed pathogens and caused spoilage in all plant samples were washed in running tap fruits (peter & patrick 2017). recently, water for 30 rnin to remove any debris or soil a.pergiiius nniger has been proven as a pathogen before being processed. the leaves were cut into that causes black pod of tamarind (meena et al. segments of 5 x 5 mm. then, the surface of the 2018). leaves and fruits was surface sterilized by due to the regarbg e n d o ~ h ~ c following the described by ravindran fungal diversity associated with l indica is et al (2012) by immersing in 700,0 ethanol lacking, this study will provide important (5 sec), 4% sodium hypochlorite (naoci) information regarding the diversity of fungal (90 set., with sterile distilled water endoph~es associated with l ifldica. this study (30 sec) and blotted dry with sterile filter paper. was aimed to determine the ~ulturable all of the segments were placed (3 segments endophytic fungal diversity associated with each plate) on potato dextrose agar (pda) l indicd using molecular phylogenetic analysis of supplemented with streptomycin (0.05 g/rnl) its rdna sequences. and neomycin (0.01 g/l) using sterilized forceps. the culture plate was incubated at room temperature (27 f 2oc) for 5 to 7 days or materials and methods until there was an appearance of mycelium or colony from the sample fragments. plant samples the fungal mycelia grown from the parts of the sample were streaked on 4% water agar collection of leaves and fruits samples of f a ) for purification. the wa plate was t. was com~leted in 2018 and 2019 at incubated for another 24 hours. then, the single jalan asam jaws, universiti tip of hyphae was cut and transferred onto a serdang selangor located at 3"00709.0"n n, pda plate and incubated at 2 7 & 2 0 ~ for 101"42'34.gne (fig. 1). the fruits and leaves seven days. the pure isolated fungi were samples were collected using fruit picker from preliminarily identified by examining their 20 l indicd trees with 2 m apart. all samples morphological characteristics. all isolates were were further placed in paper bags, properly maintained and preserved at -20 "c using a labeled, and brought to the mycology modified filter paper method for working and laboratory, department of biology for fungal stock cultures with slight modifications (fang e t isolation. al. 2000). figure 1 samples of fruits (a) and leaves (b) of t. indica were collected in persiaran asam jawa, universiti putra malaysia endophytic fungi from tamarinds mohd zaini eta/ dna extraction, pcr amplification and sequencing all isolates were cultured on pda and incubated for 5 days. dna of the isolates was extracted using ultracleanb microbial dna isolation igt (mo bio, carlsbad, ca, usa) according to manufacturer's instruction. amplification of the its regions was conducted using polymerase chain reaction (pcr) machine (hercuvan lab systems, california, usa) involved primers its1 (5' tccgtaggtgaacctgcgg-3') and its4 (5'-tcctccgcttattgatatgc-3') (xkte e t al. 1990). the pcr master mix was prepared from 4 pl of 5xpcr buffer, 2 pl of 2 mm dntp, 2 pl of 25 mm mgclz, 1 pl of 10 mm for each primer, 0.1 pl of taq dna polymerase with concentration 5 u pl, 6.9 pl of nuclease free water and 3 pl of dna in a total volume of 20 pl. the pcr protocol with initial denaturation step was done for 30 sec at 95 oc, followed by 35 cycles of denaturation (95 oc for 10 sec), annealing (59 oc for 15 sec) and extension (72 oc for 30 sec), and was completed by final extension step at 72 oc for 5 min. then, the pcr product was prepared for gel electrophoresis or stored at -20 oc. the pcr products were gel-electrophoresed using 1.5% agarose gel. the mixture of 2.5 yl of 6x loading dye (blue/orange) and 2.5 yl of 100 bp dna marker were used as a ladder. the dna and ladder were pipetted with 5 yl in volume into the holes using a micropipette and electrophoresed. the amplicon size was visualized under a uv trans-illuminator. the pcr products were purified using a qiaquick gel extraction kit (qiagen, usa), following the manufacturer's instructions. the purified pcr products were sequenced by using an applied biosystem 3730x1 dna analyzer (mytacg bioscience company, my). phylogenetic analysis evolutionary analyses of its sequences were conducted in molecular evolutionary genetics analysis @ega) 6.0 software to obtain alignment sequences (tamura e t al. 2013). homologous sequences were obtained from the genbank database ncbi (http://blast. ncbi.nlm.nih.gov/) using blastn search (https://blast.ncbi.nlm.nih.gov/blas t.cgi? page-type=blastsearch) of the its sequences. the phylogenetic analysis was conducted using the maximum likelihood method based on the tamura-nei model with 1000 bootstrap test (tamura & nei 1993) in mega version 6.0. saccharomyces cerevisde cbs 1171 (ab018043) was used as an outgroup (fig. 3). the genbank accession number of new sequences were listed in table 1. species diversity the species diversity was calculated by using the shannon-weiner index (spellerberg 2008) as formula below: where: h' = value of shannon wiener's diversity index pi = proportion of species s = number of species in community 1 = number of indviduals in species results and discussion a total of 69 isolates of fungi were obtained from 20 fruit and leaf samples of t. indica, and were identified based on their morphological characteristics (fig. 2) and its sequence analysis (table 1 and fig. 2). thirty-two species belong to 15 genera were found in the present study including aspergillas (4 species), botyospbaerid (a single species), colletotm'chmn (6 species), cochliobolzls/ czawalarid (2 species), diaporthe (6 species), fzuam'am (a single species), lasiod$lodia (2 species), nigrospoa (2 species), penin'iiium (2 species), tmncospora (a single species), pbyllostictd (a single species), sarcostroma (a single species), tm'cboderma (a single species), and xylarid (a single species) (table 1). biotropia vol. 28 no. 3,2021 eli 1;igut-e 2 1:ungal morphological retrieved in the culture media isolation procedure. f,ndoph\-tic fungi were isolated from fruits and leaves o f t ilzdi~u. iung i a-ere cultivated in pda medium at 27 o c foi7 da1.s based on phylogenetic analysis of its basidiomycota). clade a was divided into 2 sub sequences of the 69 endophytic fungi isolated clades; clade a1 represents isolates of from tamarind fruits and leaves, two major a.pe?gi/h.r, bot!yoshaerid, co//etot~icbzm, i>iapof-t/3e, clades (a and b) were generated (fig. 3). the fi~samum, nigrospa, ,.i'arcostroma, tncboderma, first clade (clade a) comprises isolates of fungi p~e/?ici//iunz, ph~~lhsticta, and xylata, whereas clade under phylum ascomycota and clade b a2 represents isolates of lasiod$lodia and contains tru~zcospora tephropora b3 148 (phylum cu~-~j~t/aria/ cocbliobolus. endophyuc fungi from tamarinds mohd zaini e t al. table 1 its sequences genbank accession number of deposited fungal isolates from fruits and leaves of t. indicd no. isolates s~ecies plant oart genbank accession number aspeqi/lus aculeatus a . carbonarizls a . javus a. tubingensis a. tubingensis boflyosphaeria mamane colletotrichnm aenigma c. brevispom c. cobbittiense c.ficticola c. gloeosporioides c. gloeosporioides c. gloeosporioides c. gloeosporioides c. gloeosporioides c. gloeosporioides c. gloeosporioides c. gloeosporioides c. gloeosporioides c. gheosporioides c. siamense c. siamense c. siamense c. siamense c. sidmense c. siamense c. siamense c. siamense cumlaria lunata c. lnnata cochliobolus geniculatns c. lunata diqorfhe arecae d. ceratozamiae d. phaseolorzlm d. phaseolom d. phaseolom d. phaseolomm d. phaseolomm d. phaseolorcrm d. psendomang$rae d. pseudooculi d. psendoocnli d. psendophoenicicola d. psendophoenicicala fusarium solani kodiplodia psendo fheobmae l theobromae l theobromae nigrospora lacticolonia n. lacticolonia n . lactiolonia n . olyzae n. olyzae n . oryxae n. ovyxae penicillinm roifsii p. ro&ii p. rofsii p. rofsii p. ro&si; p. uemculosum ph_yllostictaf.ll@iae sarcostmma bisetnlatum trichodemza asperellurn t. asperellurn t. asperellurn fruit fmit fmit fruit leaf leaf leaf leaf leaf leaf leaf leaf leaf leaf leaf leaf leaf leaf leaf leaf fruit fruit fruit fruit leaf leaf leaf leaf leaf leaf leaf leaf fruit leaf fruit leaf leaf leaf leaf leaf fruit leaf leaf leaf leaf fruit leaf leaf leaf leaf leaf leaf leaf leaf leaf leaf fmit fruit fruit leaf leaf fruit leaf leaf fruit fmit fmit leaf leaf biotropia vol. 28 no. 3,2021 figure 3 phylogenetic tree generated from the maximum likelihood method based on the its sequences of 69 fungal endophytes sequences associated with t. indim. the tree generated using tamura-nei model with 1000 bootstrap replications. all bootstrap scores with less than 50% are not shown in the tree mt043776 c gloeosporrodes 83154 mt043780 c glaeosponades 83158 mt043796 c glaeosponades 83186 mt043799 c gloeosporiodes 83169 the shannon index (h' = 3.083) indicated most diverse fungal genera isolated from that the tamarind fungal community possesses a tamarind leaves was colletot?ichzlm and diaporthe vast diversity of endophyuc fungi (table 2). the (fig. 3, table 1). 58 mt043784 c glaeosponardedes 83162 mt043786 c cobbiffiense 83170 mt043776 c glaeosponardedes 83154 mt043781 c glaeosporiardes 83154 mt043792 c glaeosporiaides 83182 mk204297 c. siamense 82885 mk204295 c siamense 82892 mt043774 c g1aeospo"oides 83152 mt043801 c glaeosporioides 83191 mk204289 c froct,cola 82961 mk204314 c. aengma 82881 93 mk204292 c. siamense 82907 mk204294 c. siamense 82921 ~ l i a x 69 mt043769 c. brev,sparum 83145 mk204285 f salani82964 mk204288 t asperellum 82963 mt043803 n aryrae 83193 mt043779 n lact,calonra 83157 mt043782 n lact,calonra 83160 mk204313 n a w e 82889 mt043788 s biswfulatum 83172 mt043785 x feejeensis 83163 53 mt043765 d phaseolomm 83141 a l i a 1 76 -1g mt043763 d phaseolomm 83161 mt043777 d phaseolomm 83155 mt043770 d phaseolomm 83147 mt043800 d. phaseolomm 83190 mk204303 d. phaseolomm 82940 mk204305 d pseudomangtferae 82928 mt043798 d pseudooculr 83168 mt043772 d ceatozamiae 83150 mt043773 d pseodophoenicicola 83151 mt043790 d pseudoculr 83180 mt043793 d pseodophoenicicola 83183 71 mk204301 d. arecae 82952 mk204300 p venuculasum 82958 89 a l i b 99 mk204304 a aculeatus 82931 52 mk204306 p mlfw, 82925 mk204308 p mlfsir 82919 mk204310 p mlfw, 82899 mk204309 p mlfw, 82894 mt043804 p falloprae 83194 a l i i 93 mt043767 b mamane 83143 mt043789 l theobmmae 83179 mt043794 l pseudotheobmmae 83184 mk204311 a tubrngensrs 82916 mk204302 a carbonanus 82948 a2 mk204299 a lvus 82959 b mk204312 c lunsta 82902 mt043766 c lunata 83144 mt043771 t tephmpora 83148 nr 111007 sacchammyces cerevrsrae endophyuc fungi from tamarinds mohd zaini e t al. table 2 endophyuc fungal percentage and shannon-wiener index obtained from culture media isolation using fruits and leaves of t. indicd no. species number of isolate c. breuisporm c. cobbittiease c. fmcticola c. &eosporioides c. siamense c. lunata cochliobolm geniculatu~ diaporthe arecae fusarium solani lasiodiplodia theobromae l pseudotheobromae nigrospora lactz'colonia n . olyxae penicillum ro@ii tmncospora tephropora ph_yllostictafallqbiae sarcostroma bisetulatum tiicboderma asperelhi xylaria feejeensis percentage (o/o) 1.45 1.45 1.45 2.90 1.45 1.45 1.45 1.45 1.45 14.49 11.59 4.35 1.45 1.45 1.45 8.70 1.45 2.90 2.90 1.45 2.90 1.45 4.34 5.79 7.25 1.45 1.45 1.45 1.45 4.34 1.45 shannon-wiener index (h') total 69 100 3.083 in this study, the most abundant fungal (26 isolates) species obtained from t. indicd leaves was from genus colletotm'chum where 10 isolates were identified as c. gloeosporioides with 14.49% (h' = 0.280). endophyuc c. fructicola and c. siamense have been recovered from healthy cymbopogon citratgs (manamgoda et al. 201 3). weir et al. (2012) stated that c. siamense is geographically diverse with a varied host range and is a common saprobe or endophyte. colletotm'chum species can be found abundantly forming its association with temperate plants and they are widely distributed in the tropical and subtropical areas (cannon et al. 2012), but no report on associations with t. indim. a study by boddy (2016) also reported that colletotm'chum species could be existed w i h n plant tissues without causing any harm while it is in an inactive state. these studies showed that members of colletotm'chum exhibit a multiple life styles. six isolates of endophytic diaporthe phaseolomm have been isolates from healthy fruits and leaves of t. indica. diapon'he spp. are known to be existed symbiotically alongside plants as saprobic, endophytic or phytopathogenic (udayanga e t al 201 1; tan e t al. 201 3; gomzhina & gannibal 201 8). according to gonzalez and tello (2011), endophytic diapon'he species are commonly isolated from several hosts in the temperate and tropical region. research on diapon'he species by gomes et al. (2013) collected several species of diaporthe from vaccinium growing regions in europe inc luhg d. phaseolomm and d. arecae. diaporthe pseudomangiferae has been reported cause inflorescence rot, rachis, canker, and flower abortion of mango (serrato-diaz e t al. 2014). biotropia vol. 28 no. 3,2021 endophytic c. lanata and cochliobolas pbyllostica species have been known to form genicalata; (telemorph of c. genicalata) have been isolated from leaves of t. indim. two distinct species from genus lasidioplodid that were isolated from the leaves of tamarind were lasidioplodia theobromae and lsidioplodia pseadotheobromae with a simdarity percentage of 99% and 97% respectively. similar to colletotn'cham species, carvalaria/cochliobolas and ldsiodiplodia are well-known plant pathogens and can also be endophytes. in ths study, aspe@ills tabengensis was found associated with the t. indicd leaves. this species was found to form an association with many plant species such as the mangrove plant, sonora desert plant (nadumane et al. 201 6), and strawberry (palmer et al. 2019). previously, other species of aspe@llas which is aspe~illas niger was isolated from diseased-fruits of t. indica and caused black pod (meena et al. 2018). two species of penicillm, p. ro@ii and p. vemcdosam have been isolated from healthy fruits and leaves of t. indim. penicillizlm spp. are common pathogens and caused spoilage in fruits (peter & patrick 2017). the assemblage of endophytic fungi in healthy tissue of t. indica may indicate that some of the fun@ are possible latent pathogens and some may saprophytic. the other genus dominated the t. indica leaves was nigrospora sp. wang et al. (2017) claimed that nigrospora sp. is a common in forming symbiosis with plants as pathogens, endophytes or saprophytes. nigroqora sphaevica (synonym of n. oy~ae) was found inhabiting numerous hosts such as the zea, andropogon and cymbopogon as reported by wang et al. (2017). supaphon and preedanon (2019) also claimed, the species was isolated from hehntbas annas as an endophyte. botyo~haerid mamane was only one isolate obtained from this genus. according to phdlips et al. (2013), this species that belonged to the botryosphaeriaceae is existed diversely in nature as pathogenic, endophytic or saprobic with more preferable to woody plants. a study by li et al. (201 8), also recorded the discovery of species of botryosphaeriaceae from plantation trees including canninghamina lanceolata, dimocaqas longan, melastoma sanguineam and phoenix hanceana, whch were growing adjacent to eaca4pta-r. their association with plants widely and can be either pathogens or endophytes. in this study, one isolate of phyllostictd fallopide with a 100% percentage of similarity with the established sequence in the genbank database. the morphology of the isolate characterized as p. fallpiae also fit the description of this species by zhang et al. (2013). one isolate was identified as xylarid feejeensis which was isolated from healthy leaves samples with 98.90% similarity to the genbank sequences. according to chen et al. (201 3) xylariaceous fungi are dominantly associated with the dendrobiam species of class orchdaceae. this f i n h g had supported the existence of xyb& sp. as an endophyte. trancospora tephropora (synonym of perenniporid tephropora) was the only basidiomycete found associated with healthy t. indicd leaves with similarity percentage of 99.84% from the sequence from genbank database. conclusion ' ihs study revealed that various endophytic fungi were isolated from the fruits and leaves of tamarind. the 31 species that have been successfully identified were a. acaleatas, a. carbonakas, a. flavas, a. tabingensis, b. mamane, c. aenigma, c. brevispomm, c. cobbittiense, c. fmcticola, c. gloeo~;pokoides, c. sidmense, c. genicalatas, c. lanata, d. arecae, d. ceratoxamiae, d. phaseoloram, d. pseadomangiferae, d. pseadoocali, d. pseadophoenicicoh, f. solani, l. psezldotheobromae, l. theobromae, n. lacticolonia, n. ogqae, p. ro@ii, p. vemcalosam, t. tephropora , p. fallopiae, s. bisetalatzlm, t. asperelltlm and x.feejeensis. acknowledgements the authors thank all staff of the department of biology, faculty of science, universiti putra malaysia for their fachties during the study. this work was partially supported by the fundamental research grant scheme (frgs/1/2018/stg03/upm/02/12/ 5540129). endophyuc fungi from tamarinds mohd zaini et al. references boddy l. 2016. fungi, ecosystems, and global change. in the fungi (pp. 361-400). oxford, london: academic press. bourou s, ndiaye f, diouf m, diop t, damme pv. 2010. tamarind (tamarindus indicd l.) parkland mycorrhizal potential within three ago-ecological zones of senegal. fruits 65(6):377-385. cannon pf, damm u, johnston pr, weir bs. 2012. colletotricbm-current status and future directions. stud mycol73: 181-213. chen j, zhang lc, xing ym, wang yq, xing xk, zhang dw, liang hq, guo sx. 2013. diversity and taxonomy of endophyuc xylariaceous fungi from medicinal plants of dendrobium (orchidaceae). plos one 8(3):e58268. danggomen 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applications. san diego: academic press, 315-22. xia y sahib mr, amna a, opiyo so, zhao z, gao yg. 2019. culturable endophytic fungal communities associated with plants in organic and conventional farming systems and their effects on plant growth. sci rep 9:1669. zhang i<, su w, cai l. 2013. morphological and phylogenetic characterisation of two new species of phyllosticta from china. mycol prog 12:547-56. the southeast asian journal of tropical biology vol. 32 no. 1, 2025: 47 56 doi: 10.11598/btb.2025.32.1.2354 issn: 0215-6334 | e-issn: 1907-770x 47 article higlights • four accessions of moringa oleifera linn. from different regions in indonesia were evaluated to identify with tolerance to aluminium (al) stress • eight metabolites show a very high correlation with acetic acid (one of the metabolites associated with alcl3 stress) including cyclopentene, 2-allyphenol, 4-ethynyl-6-8-dioxane, vinyl ether, ethanone 1-oxiranyl, 2-methylpyridine, 2-butanone, and ethanesulfonic acid. • this research makes a significant contribution to understanding aluminium tolerance in m. oleifera by identifying tolerant accessions, clustering relevant traits, and highlighting key metabolites to in vitro culture. article information received : 17 september 2024 revised : 9 march 2025 accepted : 14 march 2025 *corresponding author, e-mail: rudi015@brin.go.id reviewers: hasyyati shabrina & rani agustina wulandari research article metabolite profiling of moringa using py-gcms and tolerance evaluation to aluminum on in vitro culture rudiyanto1*, agus purwito3, darda efendi3, andri fadillah martin2 1research center for applied botany, national research and innovation agency (brin), bogor 16911, indonesia. 2research center for genetic engineering, national research and innovation agency (brin), bogor 16911, indonesia. 3department of agronomy and horticulture, faculty of agriculture, ipb university, bogor 16680, indonesia. abstract moringa oleifera linn. has received substantial scientific interest due to its numerous bioactive compounds and its function as a nutritional resource. the absorption of aluminum by plants hinders several metabolic and physiological processes, leading to inhibited plant development and decreased agricultural output. some accessions from different regions in indonesia were evaluated to identify those with tolerance to aluminum (al) stress. al-tolerant selection was carried out in vitro through the selection method for al stress by adding 0, 50, 100, 250, and 500 mg/l of alcl3 to the media. furthermore, identifying the metabolite profile of four m. oleifera accessions from four distinct regions in indonesia: blora, bogor, enrekang, and bima, has been done using py-gcms. specific metabolites associated with tolerance to al stress and organic acids need to be identified. the highest survival rate was observed in the bogor and blora accessions when exposed to alcl₃ at concentrations ranging from 0 to 250 mg/l, demonstrating greater tolerance to alcl₃ than other accessions based on various variable, such as shoot height, number of shoots, number of petioles on a medium containing 100 to 250 mg/l of alcl₃. the mean value was not statistically different from the control. acetic acid was identified as one of the metabolites associated with alcl₃ stress. a total of 21 metabolites were specifically correlated with acetic acid in a positive manner, among which 8 metabolites including cyclopentene, 2-allyphenol, 4-ethynyl-6-8-dioxane, vinyl ether, ethanone 1-oxiranyl, 2-methylpyridine, 2-butanone, and ethanesulfonic acid exhibited a very high correlation. keywords: accession, aluminum tolerant, in vitro culture, metabolite profiling, moringa oleifera https://doi.org/10.11598/btb.2025.32.1.2354 biotropia vol. 32 no. 1, 2025 48 introduction aside from its role as a nutritional resource, moringa oleifera linn. has received significant scientific interest for having numerous bioactive compound. these substances, contained in seeds, pods, and leaves, exert significant physiological effects on humans. moringa exemplifies a food source that is rich in essential elements, such as minerals, vitamins, and proteins, making it a reliable and long-lasting source of nourishment (yuniati et al. 2022). according to srivastava et al. (2023), the environmentally conscious cultivation of moringa is in line with the ideas of sustainable farming, which makes it a sustainable choice. humans consume the entire m. oleifera plant, including its leaves, fruits, immature pods, and flowers, as traditional food. various countries, including india, hawaii, the philippines, and other african countries, utilize the flowers, fruits, foliage, and early seed pods of this tree as exceptionally nutritious vegetables (yamaguchi et al. 2021). moringa tree produces between 43 and 115 tonnes of biomass per hectare annually. its leaves contain high levels of dry matter, crude protein, essential amino acids, and phytoactive substances, such as phenolic acids and flavonoids. according to kholif et al. (2016) and nouman et al. (2016), they also contain high levels of vitamins a, b, c, and e (zheng et al. 2022). conventional propagation techniques for m. oleifera predominantly involve the cultivation through seedlings or vegetative cuttings. debnath and juran (2020) have identified several obstacles associated with conventional propagation methods, including a limited quantity of explants, lethargic growth, and heightened vulnerability to pests and diseases. using in vitro micropropagation, a widely applied technique that has significantly improved the quality of several plant propagations, would be an alternative method (zheng et al. 2022). the reduction of arable land from industrialization and conversion has posed a significant obstacle to propagating m. oleifera. to address this challenge, using suboptimal land becomes an alternative option. according to fanindi et al. (2020), indonesia has approximately 108.8 million ha of suboptimal land area as acid dry land, with 62.6 million ha still having the potential for agricultural use. however, inefficient land use is constrained by several abiotic issues, such as low ph soil and high aluminum (al) saturation. these characteristics inhibit the photosynthetic process and plant growth, resulting in reduced agricultural yields (xu et al. 2018; fanindi et al. 2020). acid-dry land is defined as soil with a ph below 5, indicating acidity. the majority of agricultural land in indonesia has acidic soil conditions. high rainfall is one contributing factor. increased rainfall can accelerate the degradation of soil minerals. in moist tropical regions, intensive weathering and nutrient leaching processes cause the soil to be acidic and have high aluminum saturation. soil acidity can result from prolonged land use and excessive application of chemical fertilizers (mulyani & sarwani 2013). acid soils make up approximately 40% of the world’s potentially cultivable areas. aluminum ranks as the third most plentiful element, behind oxygen and silicon, according to riaza et al. (2018). plants absorption of aluminum hinders several metabolic and physiological processes, resulting in inhibited plant development and decreased agricultural output (liu et al. 2023). soil ph significantly influences plant metabolism, affecting nutrient availability, enzyme activity, and overall biochemical pathways. in acidic soil conditions, essential nutrients, such as phosphorus, calcium, and magnesium become less available, potentially altering the secondary metabolite profile of plants (sanches et al. 2006). to comprehensively understand these chemical alterations, a precise and sensitive analytical technique is required. pyrolysis gas chromatography and mass spectroscopy (py-gcms) is a direct method that analyzes the pyrolytic behavior of biomasses and characterizes their volatile products. recently, there has been a significant increase in interest in this technique due to its ability to identify all pyrolytic products more efficiently than the gcms of condensable products (liquid fraction). identification of these products under various operating conditions necessitates complete condensation (bensidhom et al. 2021). according to inostroza-blancheteau et al. (2012), organic acids are very important for both tolerance and avoidance pathways because they make stable complexes with al3+ inside and outside of cells that neutralize it. various crops excrete distinct varieties of organic acids. for instance, studies have shown that the roots of wheat (yang et al. 2011) and eucalyptus (li et al. 2021) secrete large levels of malic acid and acetic metabolite profiling of moringa and tolerance evaluation to aluminum rudiyanto et al. 49 acid. furthermore, researchers have identified citric acid secretion in maize, common bean, and cassia. additionally, oxalic acid secretion has been discovered in buckwheat (liu et al. 2023). the objective of this research was to characterize the metabolite profiles of four m. oleifera accessions obtained from four distinct regions in indonesia, i.e., blora, bogor, enrekang, and bima, utilizing py-gcms. the research further aimed to assess the responses of these accessions to aluminum chloride (alcl₃) stress to in vitro culture. additionally, to identify specific metabolite molecules, excluding organic acids, that are linked to tolerance to al stress. materials and methods materials and experimental design the materials used were shoot cultures of 4 accessions of m. oleifera at 6 weeks after culture (wac). the basal medium used was dkw medium (driver & kuniyuki 1984), supplemented with alcl3 according to the treatment tested. the experimental design was a completely randomized factorial design, with two factors tested: 1) the type of m. oleifera accessions: blora (java island), bogor (java island), bima (lombok island), and enrekang (sulawesi island); 2. concentrations of alcl3 (0, 50, 100, 250, and 500 mg/l). this experiment used 12 replications and 240 experimental units. the variables observed were: shoot height, number of shoots, number of petioles, and number of roots, which were observed between 0 and 6 wac, whereas metabolite profiling was done at 6 wac. py-gcms analysis the samples were dried m. oleifera explants derived from stem and leaves obtained from in vitro culture at 6 wac. the sample weight was 20 mg. the specimen was placed inside an sf py1ec50f eco-cup and subsequently sealed with glass wool. the eco cup underwent pyrolysis at a temperature of 500 oc for 0.1 minutes using a multi-shot pyrolizer (ega/py-3030d). the pyrolizer was connected to a gc/ms qp nx system (shimadzu, japan) equipped with an sh column with an interface 300 oc of temperature. the column of rxi-5sil ms, manufactured by restek in germany, was used with electron impact at 70 ev and helium as the carrier gas. the applied pressure was 20.0 kilopascals (with a total flow rate of 15.9 millilitres per minute and a column flow rate of 0.61 millilitres per minute). the temperature of the gc was as follows: it was initiated at 50 oc for 1 minute, then increased to 300 oc (5 oc every minute), and the stable temperature was adjusted for 13 minutes. pyrolysis metabolites were determined by comparing the retention time and mass spectrum data with the nist library 2017.14. data analysis the quantitative data from observations were analyzed using the analysis of variance (anova) f test at α ≤ 5% significant level. when there were substantial differences between treatments tested, the analysis was further conducted using duncan’s multiple range test (dmrt) using dsaastat v.1.1 (microsoft excel plugins). hierarchical cluster heatmap analysis, partial least squares discriminant analysis (pls-da), and principal component analysis (pca) were analyzed using metaboanalyst 6.0 (pang et al. 2024). results and discussion the survival rates of four accessions of m. oleifera shoot grown on dkw medium supplemented with 0, 50, 100, 250, and 500 mg/l of alcl₃ are shown in figure 1. the highest survival rate was observed in the bogor accession, with 100% survival being reached when exposure was given to alcl₃ at concentrations ranging from 0 to 250 mg/l. a 100% survival rate was exhibited by the blora accession when exposed to 0 and 50 mg/l of alcl₃. higher susceptibility to alcl₃ stress was exhibited by the bima and enrekang accessions, as indicated by their poor survival rates in the 100–500 mg/l alcl₃ treatment. the greater susceptibility of these two accessions to aluminum stress was suggested by their poor survival rates in comparison to the bogor and blora accessions. a rapid decline of all treated shoot explants was led by exposure to high concentrations of alcl₃ within a 6 wac, illustrating the acute sensitivity of m. oleifera to intense alcl₃. conversely, a gradual effect on plant mortality was demonstrated by lower doses (from 0 to 100 mg/l), with a complete mortality rate being observed by the sixth week. interestingly, a remarkable resilience was exhibited by the lowest alcl₃ concentrations tested (0 to 100 mg/l), with all explants surviving for up to 6 weeks post-treatment. the mortality rates for these explants were observed at the 6-week mark, ranging between 42% and 48%, underscoring a potential threshold for m. oleifera’s tolerance to alcl₃ exposure (fig. 1). biotropia vol. 32 no. 1, 2025 50 figure 1 the survival rate of four different accessions of m. oleifera cultured on dkw medium supplemented with 0, 50, 100, 250, and 500 mg/l of alcl3 at 1 6 weeks after culture (wac) to investigate the response of four different accessions of m. oleifera cultured on dkw medium supplemented with 0, 50, 100, 250, and 500 mg/l of alcl₃, an analysis of variance (anova) was used to analyze the data, as displayed in table 1. it was indicated by the analytical data that different accessions of m. oleifera have a significant impact on shoot height, number of shoots, and number of roots, but no significant effect was observed on the number of shoots. on the other hand, a notable impact on all variables tested was observed with the application of alcl₃. an interaction was found to occur between accessions and alcl₃, affecting all variables tested except the number of shoots (table 1). supplemented with 0, 50, 100, 250, and 500 mg/l of alcl₃ at 6 wac is presented in table 2. on the shoot height variable, the highest mean value was found in the bogor and bima accessions on dkw medium with an addition of 50 to 100 mg/l of alcl₃, and was not found to be significantly different from the control medium. meanwhile, in the enrekang and blora accessions, the value of the shoot height variable was observed to be lower than that in bogor and bima accessions. in media with 250 mg/l of alcl₃, the average shoot height value was observed to have decreased, and the lowest shoot height value was found in the medium supplemented with 500 mg/l of alcl₃ in all accessions tested (table 2). on the number of shoot variables, the highest value was observed in the blora accession, which was cultured on dkw medium supplemented with 50 mg/l of alcl₃ and was found to be significantly different from the others. meanwhile, the lowest number of shoots was recorded in the bogor accession, which was cultured on dkw medium with the addition of 500 mg/l alcl₃. for the number of petiole variables, the highest value was observed in the bogor accession on medium with 100 mg/l alcl₃, while the lowest number of petioles was recorded in the enrekang accession on medium with 500 mg/l alcl₃. regarding the number of roots, it was observed that the higher the concentration of alcl₃ given, the less the number of roots increased. the highest number of roots was found in the control medium or without the addition of alcl₃, especially in the bima accession, which was found to be significantly different from the others. in the medium supplemented with 500 mg/l alcl₃, almost no roots were formed; only a few roots were observed in the bogor and bima accessions (table 2). table 1 analysis of variance (anova) of four different accessions of m. oleifera on shoot height, number of shoots, number of petioles and number of roots at at 6 weeks after culture (wac) no variable f value & significance cv (%) accession alcl 3 accession x alcl 3 1. shoot height 16.60** 38.95** 2.74** 32.85 2. number of shoots 1.84ns 3.81** 1.08ns 41.92 3. number of petioles 3.99** 25.92** 2.71** 24.63 4. number of roots 15.89** 25.76** 2.77** 59.15 notes: * = significant at α ≤ 5%; ** = very significant at α ≤ 1%; ns = not significant. the mean values of shoot height, number of shoots, number of petioles, and number of roots of four accessions of m. oleifera on dkw medium metabolite profiling of moringa and tolerance evaluation to aluminum rudiyanto et al. 51 table 2 average shoot height, number of shoots, number of petioles, and number of roots of four different accessions of m. oleifera at 6 wac accession alcl 3 (mg/l) shoot height number of shoots number of petioles number of roots blora 0 6.58 ± 0.69bcde 1.38 ± 0.18abc 7.63 ± 0.53cde 2.13 ± 0.72defg 50 6.99 ± 1.01bcd 2.00 ± 0.38a 8.88 ± 1.04abcd 1.00 ± 0.33ghij 100 6.35 ± 1.42cde 1.25 ± 0.16bc 7.00 ± 0.85def 2.75 ± 0.49bcdef 250 5.50 ± 0.59def 1.63 ± 0.18abc 6.88 ± 0.35def 2.75 ± 0.41bcdef 500 2.34 ± 0.12g 1.06 ± 0.06bc 5.00 ± 0.46fgh 0.13 ± 0.13j bogor 0 8.21 ± 0.49abc 1.25 ± 0.16bc 7.38 ± 0.38de 4.25 ± 0.65ab 50 6.34 ± 0.73cde 1.75 ± 0.25ab 9.50 ± 0.89abc 4.25 ± 0.75ab 100 9.16 ± 0.71a 1.50 ± 0.19abc 10.25 ± 1.00a 2.88 ± 0.23bcdef 250 6.86 ± 0.45bcd 1.75 ± 0.37ab 8.88 ± 0.61abcd 3.50 ± 0.63abcde 500 2.21 ± 0.05g 1.00 ± 0.00c 4.63 ± 0.26gh 0.13 ± 0.13j bima 0 8.66 ± 0.55ab 1.16 ± 0.13bc 9.75 ± 0.37ab 4.50 ± 0.42a 50 8.45 ± 0.45abc 1.14 ± 0.12bc 8.00 ± 0.27bcde 3.63 ± 0.56abcd 100 8.21 ± 0.76abc 1.38 ± 0.18abc 8.00 ± 0.57bcde 3.75 ± 0.45abc 250 4.58 ± 0.93ef 1.25 ± 0.16bc 6.25 ± 0.82efg 2.50 ± 0.76cdefg 500 2.40 ± 0.25g 1.13 ± 0.13bc 5.00 ± 0.27fgh 0.50 ± 0.27ij enrekang 0 5.65 ± 0.86def 1.25 ± 0.16bc 9.00 ± 0.93abcd 2.38 ± 0.50cdefg 50 3.76 ± 0.43fg 1.50 ± 0.27abc 8.00 ± 0.57bcde 1.75 ± 0.45fghi 100 4.58 ± 0.48ef 1.50 ± 0.27abc 7.00 ± 0.50def 0.63 ± 0.26hij 250 3.69 ± 0.08fg 1.13 ± 0.13bc 6.00 ± 0.60efgh 2.00 ± 0.38efgh 500 2.24 ± 0.16g 1.13 ± 0.13bc 4.00 ± 0.60h 0.00 ± 0.00j notes: numbers followed by the same letter in the same column are not significantly different based on duncan’s multiple range test at α ≤ 5%; presented values are means ± standard error (se) of 12 replicates. tolerance threshold, leading to severe stress, toxicity, or inhibition of essential physiological processes. in comparison, weaker performance even at lower concentrations (50 250 mg/l) was shown by the bima and enrekang accessions, suggesting that their stress-response mechanisms we re less effective, possibly due to lower antioxidant activity or impaired nutrient uptake under high aluminum conditions. the shoot quality of the bogor and blora accessions at 0 250 mg/l was observed to be superior, with more vigorous growth and longer shoots than that of the other accessions. however, at 500 mg/l, a sharp decline was seen in the bogor and blora accessions, with significantly lower shoot quality, possibly due to the rapid accumulation of toxic effects beyond their detoxification limit (fig. 2). the performance of four accessions of m. oleifera (blora, bogor, bima, and enrekang) is displayed in figure 2, where they were cultured on dkw medium supplemented with 0, 50, 100, 250, and 500 mg/l of alcl₃ at 6 wac. remarkable tolerance to alcl₃ concentrations up to 250 mg/l was exhibited by the bogor and blora accessions, as evidenced by their robust shoot growth compared to the other accessions (bima and enrekang). this suggested that a higher adaptive capacity to moderate aluminum stress was possessed by bogor and blora accessions likely due to more effective physiological mechanisms that mitigate toxicity effects. however, at 500 mg/l, a drastic decline in growth was observed, indicating that this concentration has exceeded beyond the accessions’ figure 2 performance of four accessions of m. oleifera (blora, bogor, bima, and enrekang) cultured on dkw medium supplemented with 0, 50, 100, 250, and 500 mg/l of alcl3 at 6 wac biotropia vol. 32 no. 1, 2025 52 this tolerance to alcl₃ in the bogor accession was reflected in the shoot heights, number of petioles, and roots, which was aligned with the findings of peixoto et al. (2002), where adaptability to aluminum stress was demonstrated by the photosynthetic apparatus of two sorghum cultivars, exhibiting differences in physiological responses between al-tolerant and al-sensitive varieties. in our study, the plant’s capability to sustain shoot growth and maintain root development under combined abiotic stresses was hinted at, suggesting that intrinsic mechanisms were conferred for tolerance, perhaps similar to the progressive, sustained adjustments in the photosynthetic apparatus observed by peixoto et al. in sorghum (table 2; fig. 2). abiotic stresses are responded by plants through the sharing of a diverse range of molecular compounds. basically, two principal strategies are recognized in response to abiotic stress. the first strategy involves the synthesis of osmoprotectant molecules by the plant to prevent cell damage, while the second strategy is based on a repair mechanism during rehydration (martin et al. 2018). the metabolite profile of four accessions of m. oleifera cultured on dkw medium for six weeks is shown in figure 3. pyrolysis gas chromatography-mass spectrometry (py-gcms) was used to identify the samples, yielding distinct chemical groups that were produced from hydrocarbon breakdown. these chemicals included n-heptane and 2-methyl hexane. besides this, some types of chemicals were capable of breaking down carbon chains, for example, p-methyl guaiacol and 4-allyl-2-methoxy acetate. the latter category refers to a common aromatic chemical that is most likely derived from the breakdown of polyphenolic structures. the analysis of gcms data is conducted by categorizing chromatogram peaks that are observed to exhibit alterations (fendi & kurniaty 2016). the color variations are used to indicate different levels of correlation between metabolites. positive correlations are represented by red shades, meaning that when one metabolite increases, the other also increases. in contrast, negative correlations are indicated by blue shades, where an increase in one metabolite corresponds to a decrease in the other. white or lighter shades suggest weak or no correlation between the metabolites. the clustering observed in the heatmap implies that certain metabolites are exhibited in similar behavioral patterns (fig. 3a). figure 3 (a) correlation analysis between the metabolites of four accessions of m. oleifera, cultured on dkw medium at 6 wac; (b) the organic acid acetic acid, which serves as an indicator of alcl3 tolerance, correlates with 25 metabolites acetic acid is identified as one of the metabolites associated with alcl₃ stress. dolui et al. (2023) reported that acetic acid can improve aluminum tolerance in plants by regulating water content, proline accumulation, and oxidative stress defense. this suggests that acetic acid may act as a signaling molecule enhancing plant adaptation to al stress. a total of 21 metabolites have been found to have a specific positive correlation with acetic acid. a very high correlation is shown by eight metabolites, metabolite profiling of moringa and tolerance evaluation to aluminum rudiyanto et al. 53 including cyclopentene, 2-allylphenol, 4-ethynyl6-8-dioxane, vinyl ether, ethanone 1-oxiranyl, 2-methylpyridine, 2-butanone, and ethanesulfonic acid. some of these compounds (2-methylpyridine and 2-butanone) are involved in organic chemistry reactions that could potentially influence stress response mechanisms in m. oleifera. in addition, a negative relationship with acetic acid is exhibited by four metabolites. these are identified as isobutyl isothiocyanate, benzofuro (3,2-d) pyridine, isobutyronitrile, and 2,6-dimethylphenol. metabolites that are positively correlated with acetic acid are represented by pink bars, meaning that their levels increase as acetic acid levels rise, while negative correlations are indicated by blue bars, where these metabolites are observed to decrease as acetic acid levels increase. the correlation coefficients on the x-axis are used to indicate the strength of these relationships. this analysis suggests that a role is played by acetic acid in multiple metabolic pathways, with some metabolites responding positively and others negatively, providing insights into the metabolic adaptations of m. oleifera under alcl₃ stress (fig. 3b). acetic acid (ch3cooh) is a colorless liquid substance which has a strong odor, and has a distinctive taste. it is soluble in water, alcohol, glycerol, and ether. at standard atmospheric pressure, the boiling point is about 118.1 °c. m. oleifera extract produces acetic acid at a concentration of approximately 2.35%. acetic acid is a non-flammable chemical. in the pyrolysis system, oxidation produces this acetic acid molecule from ketone compounds that easily turn into acids through oxidation (emi et al. 2014). the chelating efficacy of amino acids is shown in mitigating heavy metal stress. these chelators, particularly acetic acid, play a critical role by forming stable complexes with al3+ ions, a physiological response noted for mitigating the detrimental effects of al in plants (vega et al. 2022). when acetic acid is exuded by plant roots, it dissociates into acetate ions (ch₃coo⁻), which can bind to al³⁺, forming soluble aluminumacetate complexes, such as al(ch₃coo)₃. this complexation reduces the free al³⁺ concentration in the soil solution, thereby lowering its toxicity and preventing aluminum from binding to root cell walls. additionally, some plants enhance their resistance by upregulating genes responsible for organic acid transporters, increasing the secretion of acetic acid and other chelators under aluminum stress (sun et al. 2020). the partial least squares discriminant analysis (pls-da) is shown in figure 4a. the metabolites ethanol, 1-eth, nitrous oxide, acetaldehyde, 2-propanone-1, (e)-4-(3-hydro), and 2-pyrrolidinone were exhibited with significant variability in their significance in projection (vip) values. these findings suggested that the most significant influence on the four m. oleifera accessions was exerted by these five metabolites. klau et al. (2022) said that observation metrics with high vip values may be used as differentiating features among the assessed treatments. the size of the vip value is directly proportional to its capacity to distinguish among the tested samples. nitrouse oxide (n2o) is a versatile, highly diffusible bioactive gaseous free radical that influences various biological processes in plants, alleviating biotic stress by inducing host resistance and abiotic stress by acting as an antioxidant (kalra & babbar 2010). the vip scores, derived from partial least squares discriminant analysis (pls-da), underscore the contributory metabolites that distinguish between treatment groups. a parallel can be drawn to the findings of matsuse et al. (2022), who utilized similar approaches to elucidate key metabolic changes in response to stress conditions. furthermore, the variations in metabolite profiles, which are pivotal in understanding the plant’s tolerance capabilities, have been illuminated by the robust chemometric analysis (fig. 4a). the distinct clusters and vip scores from the pls-da analysis have been illustrated to demonstrate the discriminatory power of the metabolites, offering a predictive understanding of the potential effects of different treatments. in the pca plot (fig. 4b), three prominent clusters are observed. bima and enrekang accessions were grouped within the same cluster and shown to exhibit convergence, indicating that several observable characteristics were shared by the two accessions, along with a significant correlation. the bogor and blora accessions were positioned in distinct clusters, although some overlap was observed. a clear separation between the bima and enrekang accessions on one side and the bogor and blora accessions on the other was identified, suggesting that metabolite composition was significantly influenced by geographical location. bima (lombok island) and enrekang (sulawesi island) are both situated in the eastern part of indonesia, where tropical monsoon climates with distinct wet and dry seasons are prevalent. these environmental conditions, including variations in biotropia vol. 32 no. 1, 2025 54 soil type, rainfall, and temperature, are likely to contribute to their similar metabolic profiles, as indicated by their clustering in the pca plot. in contrast, bogor and blora, both are located on java island, experience relatively stable temperatures and high humidity, which may explain why their metabolite compositions are more closely related. the clear separation between these two groups suggested that geographical factors, such as climate, soil nutrients, and elevation, played a crucial role in shaping metabolite diversity. furthermore, the variations in metabolite profiles, which are pivotal in understanding the plant’s tolerance capabilities, have been illuminated by the robust chemometric analysis from figure 4. the discriminatory power of the metabolites has been illustrated by the distinct clusters and vip scores from the pls-da analysis, offering a predictive understanding of the potential effects of different treatments. concomitantly, the metabolomic diversity was mirrored by the distinct clusters observed in figure 4b, aligning with the metabolite variances and the physiological stress responses reported by klau et al. (2023). the specific metabolic adaptations of m. oleifera were revealed through these clusters, similar to the patterns observed in different varieties of curcuma zanthorrhiza grown across various regions, where unique metabolite profiles were found to correspond to distinct environmental conditions. such clustering is emphasized as an indicator of the potential of m. oleifera to modulate its metabolic pathways, which, as posited by matsuse et al. (2022), may be regarded as a survival mechanism under the selective pressures of aluminum toxicity. conclusions the bogor and blora accessions showed higher tolerance to alcl3 treatment to in vitro culture, as indicated by their relatively high value on shoot height, number of shoots, and number of petioles even when exposed to 100 250 mg/l alcl3 in dkw medium culture, with no significant difference compared to controls. bima and enrekang accessions were in the same cluster and demonstrated convergence, suggesting that the two accessions had commonalities in various observable traits and showed a statistically significant association. acetic acid is one of the metabolites associated with alcl3 stress. there were 21 metabolites that had a specific positive correlation with acetic acid. eight metabolites showed a very high correlation including cyclopentene, 2-allyphenol, 4-ethynyl-6-8-dioxane, vinyl ether, ethanone 1-oxiranyl, 2-methylpyridine, 2-butanone, and ethanesulfonic acid. this research contributed significantly to understanding aluminum tolerance in plants by identifying tolerant accessions, clustering relevant traits, and highlighting key metabolites to in vitro culture. these findings pave the way for future research in order to verify this finding in field conditions and the development of innovative agricultural strategies to mitigate aluminum toxicity in plants. figure 3 (a) partial least squares discriminant analysis (pls-da); (b) principal component analysis (pca) between metabolite compounds on four accessions of m. oleifera: blora (java island), bogor (java island), bima (lombok island), and enrekang (sulawesi island) at 6 wac metabolite profiling of moringa and tolerance evaluation to aluminum rudiyanto et al. 55 acknowledgments we thank brin talent management, which has provided funding support and research scholarships and dr. ir. tri muji ermayanti (brin) as institutional supervisor, who has provided a lot of guidance and assistance. references bensidhom g, arabiourrutia m, trabelsi abh, cortazar m, ceylan s, olazar m. 2021. fast pyrolysis of date palm biomass using py-gcms. j energy inst 99:229-39. doi: 10.1016/j.joei.2021.09.012 blancheteau i, rengel cz, alberdi m. 2012. molecular and physiological strategies to increase aluminum resistance in plants. mol biol rep 39 (3):2069-79. debnath c, juran cg. 2020. in vitro propagation and variation of antioxidant properties in micropropagated vaccinium berry plants: a review. molecules 25(4):788803. doi: 10.3390/molecules25040788 dolui d, hasanuzzaman m, fujita m, adak mk. 2023. 2,4d mediated moderation of aluminum tolerance in salvinia molesta d. mitch. with regards to bioexclusion and related physiological and metabolic changes. int j phytoremed 26:27-44. doi: 10.1080/15226514.2023.2216311 emi e, wahyudi bs, eni b, wawan k. 2014. pengaruh suhu dan perbandingan katalis zeolit terhadap karakteristik produk pirolisis kayu jati (tectona grandis l.f.) 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0215-6334 | e-issn: 1907-770x 339 optimization of eucalyptus pellita f. muell growth using arbuscular mycorrhizal fungi and coffee husk compost on subsoil media harmawati1*, syamsuddin millang2, samuel a paembonan2, and retno prayudyaningsih3,4 1postgraduate student, faculty of forestry, university of hasanuddin, makassar 90245, indonesia 2department of forestry, faculty of forestry, university of hasanuddin, makassar 90245, indonesia 3research center for applied microbiology, national research and innovation agency, bogor 16915, indonesia 4research collaboration center for karst microbe, makassar 90245, indonesia article higlights • mycorrhizal fungi and coffee compost boost tree seedling growth in poor subsoil • the method helps grow eucalyptus faster for land restoration efforts. • reduces the need for topsoil, promoting sustainable nursery practices. • an optimal mix of mycorrhizal fungi and compost supports robust eucalyptus development. article information received : 14 february 2025 revised : 8 april 2025 accepted : 10 may 2025 *corresponding author, e-mail: retno.prayudyaningaih@brin.go.id research paper abstract arbuscular mycorrhizal fungi (amf) and coffee husk compost are used to improve the physical and chemical conditions of the subsoil, enhancing plant growth. this study aimed to evaluate the response of plant growth and analyze the effect of amf and coffee husk compost doses on optimal growth acceleration of eucalyptus pellita seedlings in subsoil media. seedlings were grown in subsoil with varying doses of amf (0; 2.5; and 5 g/polybag) and coffee husk compost at different ratios (1 : 1; 2 : 1; and 3 : 1). amf and coffee husk compost application significantly affected all parameters of seedling growth, including height, number of leaves, diameter, biomass, seedling quality index, and shoot/root ratio. inoculation of amf at a dose of 2.5 g/polybag and subsoil media enriched with coffee husk compost at a ratio of 2 : 1 was an efficient treatment to increase the seedling growth keywords: arbuscular mycorrhizal fungi, biomass, coffee husk, compost, growth, subsoil introduction deforestation and land degradation present substantial environmental issues, resulting in diminished productivity and the expansion of essential land areas. in indonesia, a significant 14.01 million ha are designated as important land, comprising several land-use categories such as other use area (apl), productive forests, protected forests, and conservation zones (kementerian lingkungan hidup dan kehutanan 2020). the urgent need for reforestation and land rehabilitation necessitates the production of highquality seedlings on a large scale. eucalyptus pellita f. muell is a rapidly growing species that is very adaptable to diverse environmental circumstances and soil types, including those with low fertility, making it suitable for restoration efforts (arisandi et al. 2019; sulichantini 2016; hutapea et al. 2023; amezquita et al. 2018). conventional nursery methods frequently utilize topsoil as the principal growth substrate. the widespread utilization of topsoil leads to horizontal land degradation, underscoring the necessity for sustainable alternatives. subsoil, although accessible, poses difficulties due to its intrinsic constraints, such as low fertility, inadequate nutrient composition, coarse texture, and restricted biological activity, which impede proper root growth. to address these limits and guarantee successful seedling production, effective, economical, and ecocopyright (c) 2025@author(s). https://doi.org/10.11598/btb.2025.32.3.2462 https://creativecommons.org/licenses/by-nc-nd/4.0/ biotropia vol. 32 no. 3, 2025 340 friendly solutions are essential. the utilization of organic supplements, including compost and arbuscular mycorrhizal fungi (amf), presents a viable approach to improve seedling development and survival in subsurface environments. compost enhances the physical, chemical, and biological characteristics of soil, augmenting nutrient availability and promoting beneficial microbial activity (prayudyaningsih 2016; widuri & ishak 2013; paranoan et al. 2022). amf, conversely, augments nutrient and water absorption, boosts drought resilience, and offers protection against pathogens and heavy metals, hence facilitating growth in poor settings (corryanti 2000; setiadi 2000; setiawati et al. 2000; yunita et al. 2023). moreover, amf inoculation can expedite seedling growth, resulting in earlier planting and enhanced adaptation to field conditions (basri 2018; dewi et al. 2017; corryanti et al. 2007; lumbantoruan et al. 2021; yelli et al. 2022). this study examined the efficacy of coffee husk compost, an accessible and sustainable organic resource, combined with amf inoculation to enhance the growth of e. pellita seedlings in subsoil media. this method seeks to convert subsurface into a viable growing medium, diminishing dependence on topsoil in nursery operations and fostering sustainable land restoration. this study aimed to assess the growth response of e. pellita seedlings inoculated with amf and grown on subsoil media supplemented with coffee husk compost. materials and methods experimental design a 3 x 4 factorial experiment was conducted using a completely randomized design (crd). each treatment combination has 20 replicates, totaling 240 eucalyptus pellita seedlings. the factors were: a) arbuscular mycorrhizal fungi (amf) inoculation dose (a0 = 0 g; a1 = 2.5 g; a2 = 5 g) and b) subsoil: coffee husk compost ratios (v : v) (b0 = 1 : 0; b1 = 1 : 1; b2 = 2 : 1; b3 = 3 : 1). the applied treatment combinations were: 1) a0b0 = no amf inoculation, the planting media was just subsoil, no coffee husk compost; 2) a0b1 = no amf inoculation, the planting media was subsoil : coffee husk compost (1 : 1); 3) a0b2 = no amf inocluation, and the planting media was subsoil : coffee husk compost (2 : 1); 4) a0b3 = no amf inocluation, and the planting media was subsoil : coffee husk compost (3 : 1); 5) a1b0 = 2.5 g amf inoculation, and the planting media was only subsoil without coffee husk compost; 6) a1b1 = 2.5 g amf inoculation, and the planting media was subsoil : coffee husk compost (1 : 1); 7) a1b2 = 2.5 g amf inoculation, and the planting media was subsoil : coffee husk compost (2 : 1); 8) a1b3 = 2.5 g amf inoculation, and the planting media was subsoil : coffee husk compost (3 : 1); 9) a2b0 = 5 g amf inoculation, and the planting media was only subsoil without coffee husk compost; 10) a2b1 = 5 g amf inoculation, and the planting media was subsoil : coffee husk compost (1 :1); 11) a2b2 = 5 g amf inoculation, and the planting media was subsoil : coffee husk compost (2 : 1); 12) a2b3 = 5 g amf inoculation, and the planting media was subsoil : coffee husk compost (3 : 1). experimental procedure coffee husk compost was prepared using a modified method of riga et al. (2022), involving a 60-day composting process with em-4 bioactivator and cow manure. e. pellita seeds were germinated in sterilized fine sand. subsoil was sterilized with fumigan containing 98% dazomet before mixing with compost. polybags (12 x 17 cm) were filled with the respective subsoil : compost mixtures. three-week-old seedlings were transplanted and inoculated with amf during transplanting. seedlings were maintained in a greenhouse with twice-daily watering. observed variables seedlings’ height, stem diameter, and leaf number were measured monthly for three months. at three months, biomass (dry weight), seedling quality index (dickson et al. 1960), and amf root colonization percentage (kormanik & mcgraw 1982; mosse 1980) were determined. biomass and amf colonization were assessed on 36 randomly selected seedlings. chemical properties of the subsoil were analyzed to examine the nutrient availability, as well as the nutrient content of coffee husk compost. data analysis data were analyzed using analysis of variance (anova), and significant differences were further analyzed using duncan’s multiple range test (dmrt). optimization of eucalyptus pellita f. muell growth using arbuscular mycorrhiza fungi harmawati et al. 341 results and discussion subsoil media and coffee husk compost our study indicated that subsoil had deficiencies in terms of macronutrient content which was below the quality standards of fertility. the levels of n, p, and k nutrients were very low, measured at 0.06%, 8.45%, and 0.14%, respectively (table 1). soil with very low macronutrient content required additional fertilization as an intervention. rosniawaty et al. (2020) explained that subsoil had a low level of fertility, emphasizing the need for improvement by adding organic materials in solid and liquid form. these measures were expected to improve the physical, chemical, and biological properties of the soil. fertilization with nitrogen, phosphorus, and potassium, is essential to support healthy plant growth. based on nutrient content analysis, robusta coffee husk compost prepared with em4 bioactivator met the quality standards outlined in sni 19-70302004, regulation of the minister of agriculture number 28/permentan/sr.130/5/2009 and number 70/permentan/sr.140/10/2011. these standards include quality criteria, such as water, organic matter, and essential nutrient contents (n, p, k), which are important indicators in ensuring the effectiveness of compost as a source of plant nutrients. em4, a bioactivator used for producing solid compost, often called bokashi, offers distinct advantages. in addition to accelerating the composting process, it has been proven to eliminate odors associated with composting when the process was conducted approximately (wellang et al. 2015). compost made from coffee husk has numerous advantages for enhancing the biological, chemical, and physical characteristics of soil. the study of nutrient composition in coffee husk compost revealed a comparatively high n content of 1.22%, a moderate p value of 1.36%, and a comparatively high k level of 1.73%. these findings are consistent with the assertion of simarmata (2016) that coffee husk compost includes nitrogen (n) and potassium (k) in addition to the organic components’ cellulose, pectin, hemicellulose, and lignin. coffee husk waste can improve soil structure since it contains 45.3% organic material, 2.98% nitrogen, 0.18% phosphorus, and 2.26% potassium, according to efendi (2017). additionally, coffee husk compost contains carbon, nitrogen, phosphate, and potassium (novita et al. 2018). eucalyptus seedlings require a sufficient amount of nutrients to grow. lingga et al. (2013) found that the presence of essential nutrients like nitrogen, phosphorus, and potassium is closely related to plant growth. nitrogen is the primary ingredient in amino acids, proteins, and the formation of cell protoplasm, which can stimulate plant growth. furthermore, potassium functions as an activator of several enzymes involved in the production of protein and carbohydrates. plants will use the photosynthate that they have created to elongate and divide their cells, which will increase their height. gardner et al. (1991) emphasized that the main factors influencing plant height are cell growth and proliferation. table 1 nutrient content in subsoil and coffee husk compost no. sample code quality compost criteria variable ss ch sni 19-7030-2004 regulation of the minister of agriculture no. 28/permentan/ sr.130/5/2009 regulation of the minister of agriculture no. 70/ permentan/ sr.140/10/2011 1 ph (h2o) 5.65 sa 6.55 6.8 7.49 4 8 4 9 2 c 0.85 vl 15.10 9.8 32 ≥ 12 ≥ 15 3 n 0.06 vl 0.86 ≥ 0.4 < 6 4 p 8.45 vl 0.79 ≥ 0.1 < 6 5 k 0.14 vl 1.18 ≥ 0.2 < 6 6 c/n 11 m 18 10 20 15 25 15 25 notes: ss = subsoil; ch = coffee husk compost; vl = very low; sa = slightly acid; m = mediun; land value classification based on hardjowigeno (2010); sutarman and miftakhurrohmat (2019). biotropia vol. 32 no. 3, 2025 342 growth of plant height the height increase of e. pellita seedlings was monitored biweekly over 3 month-period. the results showed that amf inoculation isolated from the soil of a former limestone, comprising gigaspora sp. and acaulospora sp., had a better height increase than that in the control. at a 2-week period no significant differences were observed between amf-inoculated seedlings and the control. the effects of the amf inoculation became apparent after 2 weeks (fig. 1). prayudyaningsih (2014) investigated the effect of amf inoculation on the growth of acacia auriculiformis seedlings. the results showed that inoculation with glomus sp., the nonindigen (positive control), gave the best growth performance, achieving an average height of 22.12 cm. the growth of e. pellita seedlings was significantly affected by the combination of amf inoculation and coffee husk compost application. the interaction of treatments showed differences in response to the increase in seedling height. furthermore, the duncan test showed that a1b2 treatment was the most effective. this treatment produced the highest average height growth of e. pellita seedlings (table 2). adding both amf and compost to the growing media accelerates the growth of e. pellita plants in comparison to the control treatment (table 2). the application of coffee husk compost and subsoil medium (2 : 1) with 2.5 g of amf increased the average plant height by 73.25% above the control. in the meantime, the average plant height increased by 63.70% when 2.5 g of amf were added to subsoil medium and coffee husk compost (1 : 1). compost and amf work together to improve soil porosity, enrich nutrients in the media, and enhance the absorption of water and nutrients, particularly phosphorus. this finding is similar to the work of nursayuti (2017), who found that the type of planting media, specifically the amount of nutrients in the soil that plants can absorb, significantly impacts growth. compost and amf work well together to improve nutrient absorption, particularly for nitrogen, which is crucial for plants’ generative growth (ariandi & sudantha, 2018). given the availability of minerals in the soil, adding amf and compost to less fertile soil will boost plant growth and yield (wahab et al. 2023). compared to the control, the average plant height increased by 52.01% when amf was added to subsoil planting media without compost. inoculating plant roots with amf is one method of promoting plant growth (setiadi 2000). in order to promote greater leaf growth, which is directly linked to increased plant height, amf helps plants absorb nitrogen (husna et al. 2015). it is well known that the presence of amf in plant roots can improve nutrient and water absorption. the external hyphae of amf improve the range of nutrients that can be absorbed and offers a more effective surface (wider and better spread) for absorbing nutrients from the soil, which increases nutrients. the surface area of plant roots that absorb amf in symbiosis is 18 times larger than that of roots that do not have mycorrhizae (prayudyaningsih 2014; prayudyaningsih & sari 2016). figure 1 effect of amf inoculation and coffee husk compost application on the height growth of eucalyptus pellita seedlings up to 12 weeks of age optimization of eucalyptus pellita f. muell growth using arbuscular mycorrhiza fungi harmawati et al. 343 table 2 effect of amf inoculation and coffee husk compost application on the height of eucalyptus pellita seedlings up to 12 weeks of age no treatment average height growth (cm) 1 a1b2 72.61a 2 a1b1 68.61a 3 a2b0 63.71b 4 a0b1 63.71b 5 a2b1 62.38b 6 a2b2 61.13bc 7 a1b0 56.81cd 8 a0b2 56.30d 9 a0b3 55.78d 10 a1b3 55.35d 11 a2b3 53.12d 12 a0b0 41.91e notes: numbers followed by the same letter are not significantly different at the 95% confidence level. stem diameter amf inoculation and coffee husk compost application which was observed every 4 weeks showed an increase in the diameter of seedlings, which was a positive effect than that of the control. however, after 12 weeks, the increase in the diameter due to amf inoculation was very different from the control. the most pronounced growth during this period was observed in the a2b0 treatment (fig. 2). the treatment combinations that produced the largest diameter increases were a2b0, a0b2, a2b3, a1b1, a0b1, a1b3, and a2b1 (table 3). the highest amf inoculation dose of 5 g with a 1 : 1 dose of coffee husk compost was particularly effective. similarly, the study of putra (2015) reported significant results with a 15 g amf inoculation on tanjung and glodokan plants, achieving diameter increase of 0.158 cm and 0.165 cm, respectively. based on the analysis of variance, there was a significant difference in the response to the increase in diameter of e. pellita due to the interaction of the inoculation treatment of amf with various doses of coffee husk compost. the duncan test identified a2b0 as the most effective treatment, achieving the highest average diameter increase (table 3). figure 2 effect of amf inoculation and coffee husk compost application on the diameter of eucalyptus pellita seedlings up to 12 weeks of age. biotropia vol. 32 no. 3, 2025 344 table 3 effect of amf inoculation and coffee husk compost application on diameter increase of eucalyptus pellita seedlings no treatment average diameter increase (mm) 1 a2b0 4.89a 2 a0b2 4.68ab 3 a2b3 4.63ab 4 a1b1 4.57ab 5 a0b1 4.56abc 6 a1b3 4.52abc 7 a2b1 4.48abc 8 a1b2 4.40bc 9 a1b0 4.28bc 10 a2b2 4.13cd 11 a0b3 3.79d 12 a0b0 2.72e notes: numbers followed by the same letter are not significantly different at the 95% confidence level. number of leaves observations conducted every 2 weeks showed that e. pellita seedlings inoculated with amf and coffee husk compost application had a greater leaf production compared to the control. the best result in the 12th week was achieved from the a1b1 treatment, producing 34 leaves (fig. 3). analysis of variance showed that treatment interactions influenced leaf production. the 2.5 g amf inoculation combined with subsoil media and coffee husk compost at a ratio of 1 : 1 (a1b1) significantly increased the number of leaves, outperforming the 5 g amf inoculation (table 4). treatment combination that produced the highest number of leaves was a1b1 (33.80 leaves). however, no significant differences were observed between a1b2 (32.40 leaves) and a0b1 (32.10 leaves). duncan’s test results showed that all treatments with amf inoculation and coffee husk compost application enhanced plant growth compared to those without these additions. those combinations improved overall plant biomass, including leaf production, by optimizing nutrient availability and enhancing root enzyme activity for nutrient absorption (diagne et al. 2020). leaves significantly influence the growth and development of plants. generally, leaves are essential organs that sustain several critical physiological processes to plant growth and survival. leaf growth is influenced by the availability of nutrients like nitrogen (firmansyah & sumarni 2013). an average of 33.80 and 32.40 strands of leaves were produced by adding 2.5 g of amf isolates and coffee husk compost in subsoil media at ratios of 1 : 1 and 1 : 2. this demonstrates how using amf and coffee husk compost can greatly boost plant leaf growth (table 6). compost not only provides the nutrients that plants require, but it also fosters the growth of microbial colonies, including amf, by offering a favorable environment and a wealth of food sources. plant roots can absorb more nutrients, including nitrogen (n), with amf inoculation (xie et al. 2014), because amf and plant roots can develop a symbiotic interaction that improves nutrient absorption. amf promotes plant growth by increasing the absorption of nutrients from compost. eucalyptus seedlings may produce more leaves when they are inoculated with amf and placed in media that contains compost. when it comes to boosting leaf growth, 2.5 g of amf is more effective than 5 g. the ideal mycorrhizal colony density, which is adequate to establish a productive symbiotic connection with plant roots, may be achieved by 2.5 g amf inoculation. adequate nitrogen requirements will promote the growth of vegetative tissues, including leaves, increasing the quantity of leaves (fitriana et al. 2012). the ability for photosynthesis will rise with more leaves, increasing the amount of photosynthate produced and distributing it throughout the plant (prayudyaningsih & sari 2016). optimization of eucalyptus pellita f. muell growth using arbuscular mycorrhiza fungi harmawati et al. 345 the availability of certain nutrients that promote plant growth and development is increased by mycorrhizal inoculation. mycorrhizal plants are better at absorbing water, which also allow them to absorb soluble nutrients like sulfur (s), potassium (k), and nitrogen (n), increasing the absorption of these components (baon 1999). as one of the primary constituents of many different compounds found in plant tissue, nitrogen is also crucial for developing the vegetative organ. amf inoculation, however, is more effective in increasing the growth of eucalyptus seedlings when it is coupled with the application of coffee husk compost fertilizer, as seen by a 30.50% increase in number of leaves as compared to the control (table 4). this suggests that the coffee husk compost fertilizer and amf treatment work well together. figure 3 figure 3 effect of amf inoculation and coffee husk compost application on the increase in the number of leaves of eucalyptus pellita seedlings up to 12 weeks of age. table 4 effect of interaction between amf inoculation and coffee husk compost application on the increase in the number of leaves of eucalyptus pellita seedlings up to 12 weeks of age no treatment average number of leaves 1 a1b1 33.80a 2 a1b2 32.40ab 3 a0b1 32.10ab 4 a0b2 30.75bc 5 a2b0 30.70bc 6 a2b1 30.40bcd 7 a1b3 30.30bcd 8 a2b3 29.05cde 9 a2b2 28.70cde 10 a0b3 28.30de 11 a1b0 28.10e 12 a0b0 25.90f notes: numbers followed by the same letter are not significantly different at the 95% confidence level. biotropia vol. 32 no. 3, 2025 346 shoot-root ratio the shoot-root ratio is among the criteria for assessing seedling quality. it is influenced by the interactions between amf doses and coffee husk compost application. seedlings treated with 2.5 g amf inoculation and 1 : 1 coffee husk compost application showed the highest shoot-root ratio value and were better than the control (table 5). further test on the positive interaction between amf inoculation and coffee husk compost application showed that the highest average shootroot ratio was observed in a1b1 (5.40), which was significantly different from a1b2 (4.34), a0b3 (4.03), and a2b2 (4.01). the shoot-root ratio and seedling quality index serve as markers for assessing the quality of seedlings (prayudyaningsih & sari 2016). before seedlings are planted in the field, the shoot-root ratio and seedling quality index must be taken into consideration because those are crucial indicators of the seedlings readiness to be planted. both indicators offer details on effective field growth as well as seedling quality and transplantation readiness. this study showed that when amf inoculation and compost application are combined, the eucalyptus seedlings have a seedling quality index greater than 0.09 and a shoot-root ratio between 2 and 5, which is typically seen in seedlings with the best development and survival potential. table 5 the interaction of amf inoculation and coffee husk compost application on shoot-root ratio of eucalyptus pellita seedlings up to 12 weeks of age. no treatment shoot-root ratio 1 a1b1 5.40a 2 a1b2 4.34ab 3 a0b3 4.03abc 4 a2b2 4.01abc 5 a2b1 3.69bc 6 a2b0 3.41bc 7 a2b3 3.28bc 8 a0b2 3.18bc 9 a1b3 3.18bc 10 a1b0 2.95 bc 11 a0b1 2.61 bc 12 a0b0 2.47 c notes: numbers followed by the same letter are not significantly different at the 95% confidence level. table 6 effect of amf inoculation and coffee husk compost application on the seedling quality index of eucalyptus pellita seedlings up to 12 weeks of age no treatment seedling quality index average 1 a2b1 0.51a 2 a0b2 0.49ab 3 a2b3 0.46ab 4 a2b0 0.426abc 5 a1b3 0.413abc 6 a1b1 0.393bcd 7 a1b0 0.383bcde 8 a0b1 0.34cde 9 a1b2 0.316cde 10 a0b3 0.293de 11 a2b2 0.276ef 12 a0b0 0.18f notes: numbers followed by the same letter are not significantly different at the 95% confidence level. optimization of eucalyptus pellita f. muell growth using arbuscular mycorrhiza fungi harmawati et al. 347 seedling quality index the seedling quality index is a comparison between the total dry weight with the seedling strength, and the shoot-root ratio, which serves as a parameter for describing the morphological and physiological characteristics of seedlings. analysis of variance showed that treatment interactions affected the seed quality index, signifying differences in response (table 6). based on the results of further tests using the duncan method, amf inoculation and coffee husk compost application showed different response to the seedling quality index. the highest seedling quality index was observed in a2b1 (0.51), while the lowest was recorded in a0b0 (0.18). this signified a positive interaction between amf and coffee husk compost. an indication of seedling quality is the seedling quality index value (prayudyaningsih & sari 2016), which is an essential parameter for evaluating seedling quality and preparedness for transplantation and field growth. amf inoculation and compost application together yield a seedling quality index of greater than 0.09 (table 6). meanwhile, orpa et al. (2019) explained that seedlings with a seedling quality index greater than 0.09 are prepared for field transplantation. therefore, a certain dose of amf and compost inoculation together can enhance seedling quality. the high-quality seedlings are very viable in the field and exhibit a balanced ratio between the transpiration process and the area of photosynthesis of plants that can absorb water and minerals. regarding technical guidelines for evaluating the quality of forest plant seedlings, the directorate of land rehabilitation and social forestry’s regulation number: p.05/v-set/2009 is the basis for this assessment. e. pellita f. mull seedlings must meet the following standard requirements: height > 20 cm, diameter > 2 mm, number of leaves/lcr > 3 pairs, and age > 3 months. according to the standard for maximizing nutrient absorption and boosting growth efficiency, the use of coffee husk compost in conjunction with amf inoculation can greatly shorten the nursery period. as a result, this combination of treatments increases production efficiency and prospective crop yields in the field by enabling seedlings to reach the ready-to-plant stage faster. according to these guidelines, eucalyptus seedlings can have their nursery period shortened by one month when amf inoculation and coffee husk compost application are used together. biomass biomass is the dry weight of root, stem, and leaf samples after oven drying. analysis of variance showed different biomass responses based on amf and coffee husk compost doses. duncan’s test results showed that the highest biomass value of 8.45 was shown by the a1b2 treatment, comprising 2.5 g of amf inoculation and a 2 : 1 coffee husk compost ratio. in contrast, the lowest at 3.59 was recorded for t he control treatment, a0b0 (table 7). biomass indicates the nutritional state of plants, the rate of photosynthesis, and plant respiration, which can be used as an indicator to assess the quality of plant development (prayudyaningsih & sari 2016). plant development is boosted when photosynthesis is functioning properly, as seen by an increase in plant dry weight (biomass). this can happen as a result of the improved condition of the planting media due to the addition of compost, which raises the nutrient content of soil, and amf inoculation, which facilitates nutrient absorption. percentage of amf colonization the level of mycorrhizal colonization is an indicator of the success of mycorrhizal development in the roots and the rhizosphere. this colonization reflects how well amf has succeeded in establishing a symbiotic relationship with the plant, the success of which can be demonstrated through increased plant growth and resistance to various environmental conditions. our study showed that the interaction between amf inoculation and coffee husk compost applicatin did not have a significant effect on the percentage of amf colonization. in general, there were variations in the percentage of amf colonization in each treatment (fig. 7). plants inoculated with 5 g of amf in subsoil media using coffee husk compost application with a ratio of 2 : 1 (a2b2) had the highest colonization percentage, with a percentage exceeding 50%. this shows that the combination of treatments supports amf colonization well. when mycorrhiza is absent in subsoil media (a0b0), a lower colonization percentage of less than 10% was observed. biotropia vol. 32 no. 3, 2025 348 studies showed that amf inoculation coupled with organic materials application, such as vermicompost, can increase colonization and nutrient uptake. in ginger plants, amf inoculation elevated the uptake of phosphorus and other nutrients, as well as increased root development (sarathambal et al. 2024). in addition, studies on tea plants showed that the combination of amf and plant hormones also increased the percentage of colonization and overall growth (gao et al. 2023). table 7 effect of amf inoculation and coffee husk compost application on the biomass of eucalyptus pellita seedlings up to 12 weeks of age. no treatment seedling biomass (g) 1 a1b2 8.45a 2 a1b1 8.26ab 3 a0b2 7.86abc 4 a2b0 7.05abcd 5 a1b2 7.03abcd 6 a2b3 6.77bcd 7 a1b0 6.52cde 8 a1b3 6.46cde 9 a0b3 5.65de 10 a0b1 5.65de 11 a2b2 4.97ef 12 a0b0 3.59f notes: numbers followed by the same letter are not significantly different at the 95% confidence level. figure 4 percentage of amf colonization in eucalyptus pellita seedlings up to 12 weeks of age. optimization of eucalyptus pellita f. muell growth using arbuscular mycorrhiza fungi harmawati et al. 349 conclusion the application of amf had a very significant effect on the growth of eucalyptus seedlings. coffee husk compost significantly influenced all growth parameter, including plant height, number of leaves, diameters, and seedling biomass, as well as seedling quality index and the shoot-root ratio. inoculation of 2.5 g amf with planting media consisted of subsoil and coffee husk compost at a ratio of 2 : 1 showed the 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highlights • biostimulant potential of enhalus acoroides • positive effects of e. acoroides on maize growth • reduction in h2o2 (oxidative stress) by enhalus acoroides extract abstract drought poses a significant challenge to crop productivity, with maize particularly vulnerable. enhancing maize tolerance to drought stress is crucial, and one promising approach involves using biostimulants derived from natural sources. the seagrass enhalus acoroides is a potential biostimulant due to its rich phytochemical composition, including phenols, tannins, flavonoids, and pigments, such as chlorophyll, lutein, pheophytin, and beta-carotene. these compounds exhibit antioxidant activity, suggesting their potential role in enhancing plant resilience to drought stress. this study evaluated the effects of e. acoroides extract on maize growth under drought conditions during the seedling phase and analyzed biochemical changes in maize plants treated with the extract. the extract was prepared using 10% dried e. acoroides leaves with a chloroform-to-ethanol solvent ratio of 9 : 1 (v/v) and subsequently dissolved in distilled water for final concentrations of 0.05%, 0.1%, 0.15%, 0.2%, and 0.25%. the results demonstrated that e. acoroides extract enhanced plant height, increased shoot and root fresh and dry weights. additionally, plants sprayed with e. acoroides extract exhibited higher total sugars and protein content in the shoots as compared to non-sprayed plants. under 20% polyethylene glycol (peg)-induced drought stress, control plants showed severe leaf wilting, whereas extract-treated plants only had mild wilting. the chlorophyll, reducing sugars, total n, and tocopherol contents were also higher in extract-treated plants under peg stress than in untreated controls. these findings indicate the potential of e. acoroides extract as a biostimulant for improving drought tolerance in maize. keywords: antioxidant, drought stress, maize the potential of enhalus acoroides as a biostimulant to enhance maize growth and drought tolerance made pharmawati1*, luh putu wrasiati2 , i made anom sutrisna wijaya3 and kadambot h.m. siddique4 1biology study program, faculty of mathematics and natural sciences, udayana university, jimbaran 80361, bali, indonesia 2agroindustrial technology study program, faculty of agricultural technology, udayana university, jimbaran, 80361, bali, indonesia 3agricultural engineering and biosystems study program, udayana university, jimbaran, 80361, bali, indonesia 4the uwa institute of agriculture, university of westren australia, wa 6001 perth, australia article information received : 10 march 2025 revised : 8 april 2025 accepted : 8 april 2025 *corresponding author, e-mail: made_pharmawati@unud.ac.id reviewers: prof ketut budaraga & anonymous https://doi.org/10.11598/btb.2025.32.2.2487 seagrass extract as biostimulant for maize growth pharmawati et al. 243 introduction maize (zea mays l.) is a vital cereal crop belonging to the poaceae family, serving as a major source of nutrients and phytochemicals with significant health benefits (shah et al. 2016). in regions such as africa, latin america, and asia, maize contributes more than 20% of the daily caloric intake (shiferaw et al. 2011). compared to wheat and rice, maize is highly versatile, with applications in food, animal feed, industrial processing, and bioenergy production (grote et al. 2021). maintaining stable maize production is essential, given its diverse roles in agricultural and food systems. in indonesia, maize production has fluctuated, with dried maize kernel production (14% moisture content) reaching 14.77 million tonnes in 2023, representing a 10.61% decline from 16.53 million tonnes in 2022 (badan pusat statistik 2023). climate change, particularly prolonged drought periods linked to global warming, is a major factor affecting maize productivity (herlina & prasetyorini 2020). drought stress significantly hampers maize growth, primarily due to inadequate water availability in the root zone and excessive transpiration from leaves, where water loss outpaces absorption (nieves-cordones et al. 2019). physiologically, drought stress disrupts cell division and differentiation, reduces turgor pressure, alters enzymatic activity, impairs photosynthetic energy production, and leads to the accumulation of reactive oxygen species (ros) (jothimani & arulbalachandran 2020). the seedling and flowering stages are particularly sensitive to drought stress. research has shown that photosynthesis rates in maize significantly decrease when soil moisture levels drop to 50% of field capacity. furthermore, soil moisture measurements have indicated that levels close to the wilting threshold point (22.2% soil moisture) can severely stress the plant (shao et al. 2021). sustainable agricultural technologies are needed to mitigate the adverse effects of drought. biostimulants, organic substances applied to plants, seeds, or soil, have emerged as a promising solution to enhance nutrient efficiency, improve stress tolerance, and promote plant growth (bulgari 2019; melo et al. 2020). biostimulants are derived from various sources, including microorganisms, such as bacteria, yeasts, and fungi, either as live organisms or as metabolites (rouphael 2020). additionally, plant-derived biostimulants can be extracted from different plant parts, such as seeds, leaves, and roots, across various botanical families, including amaryllidaceae, brassicaceae, ericaceae, fabaceae, fagaceae, moringaceae, plantaginaceae, poaceae, rosaceae, solanaceae, theaceae, and vitaceae (yakhin et al. 2017). marine-derived biostimulants, particularly macroalgae extracts, have also shown promising results. for example, water extracts from ulva rigida enhance wheat growth when applied as a foliar spray (latique et al. 2021). likewise, extracts from ascophyllum nodosum improve drought tolerance by increasing proline levels, a key non-enzymatic antioxidant (rasul et al. 2021). polyphenols, fucoidans, and alginates found in macroalgae exhibit strong antioxidant activity, which helps mitigate ros damage under drought conditions (wang et al. 2020). using biostimulants also promotes sustainable agriculture by enhancing crop productivity while reducing reliance on chemical fertilizers (xu & geelen 2018). despite the growing interest in plant and algal biostimulants, seagrasses remain an underexplored resource. seagrasses are marine flowering plants that contribute to primary productivity, carbon sequestration, and habitat formation for marine life (jalaludin et al. 2020). among them, enhalus acoroides is widely distributed in indonesia and is characterized by its long leaf blades. seagrasses thrive in intertidal zones, providing ecological benefits, such as carbon sequestration and nutrient cycling, which help mitigate the effects of global warming (stankovic et al. 2021). a recent study highlighted the bioactive potential of e. acoroides. extracts obtained from its leaves using a chloroform-to-ethanol solvent (9 :1) contain phenolic compounds, tannins, flavonoids, and pigments such as chlorophyll, lutein, pheophytin, and beta-carotene (pharmawati & wrasiati 2020). these compounds possess strong antioxidant properties, suggesting their possible role in plant stress tolerance. the potential of e. acoroides extract as a biostimulant for drought tolerance in maize remains unexplored. this study assessed the effects of e. acoroides extract on maize growth under drought stress during the seedling phase and analyzed biochemical changes induced by extract application, providing insights into its potential role as a droughttolerance biostimulant. biotropia vol. 32 no. 2, 2025 244 materials and methods sample collection and extraction leaves of e. acoroides were collected from semawang beach, bali. they were washed, airdried, and then oven-dried at 50 °c for 24 hours. the dried leaves were ground and sieved through a 60-mesh sieve. a 20 g sample of the powdered leaves was extracted using a soxhlet extractor (pyrex®) with 200 ml of chloroform : ethanol (9 : 1) for 3 hours. the extract was filtered, and the filtrate was evaporated using a rotary evaporator (ika®rv10). soxhlet extraction was performed following the method of abubakar and haque (2020) with modifications, using chloroform : ethanol as the solvent, as chloroform extracts from various plant species have been reported to exhibit significant antioxidant activities (uddin et al. 2015; syamkumar et al. 2023). the resulting crude extract (100%) was diluted in distilled water to prepare concentrations of 0.05%, 0.1%, 0.15%, 0.2%, and 0.25%. the extracts were stored in a refrigerator until use. the effectiveness of e. acoroides extract was evaluated in two separate experiments. the first experiment assessed its effect on maize growth at the vegetative stage, while the second examined its potential as a drought mitigation agent. both experiments were conducted in the greenhouse of the biology study program, faculty of mathematics and natural sciences, udayana university, bali, indonesia from june to september 2024. experiment 1: enhalus acoroides treatment commercial hybrid maize seeds (pertiwi variety) were purchased from an agricultural store in denpasar, bali. the seeds were sterilized by soaking in 10% sodium hypochlorite for 10 minutes, then rinsed with water and soaked for 12 hours. germination was carried out in trays containing rockwool media and commercial hydroponic a and b liquid nutrients. after 7 days, uniform seedlings were selected and transferred to a hydroponic system with perlite media and ab mix nutrients for an additional 7-day acclimatization period. following acclimatization, seedlings were sprayed with e. acoroides extract at the specified concentrations (5 ml per plant), while control plants received distilled water. treatments were applied twice weekly for four weeks. at the end of the experiment, plants were harvested. the experiment was conducted using a randomized block design with four replications. data collection plant height was measured at harvest (four weeks after the first e. acoroides treatment). fresh and dry weights of both aboveground and belowground plant parts were recorded. reducing sugars content was determined by preparing a glucose standard curve using 0, 20, 40, 60, 80, and 100 ppm anhydrous glucose. for each concentration, 1 ml of glucose was mixed with 1 ml of nelson’s reagent, heated for 10 minutes, cooled, and treated with 1 ml of arsenomolybdate solution. after adding 7 ml of distilled water, absorbance was measured at 540 nm. for plant samples, 1 g of tissue was dissolved in 100 ml of distilled water, filtered, and processed similarly. reducing sugars content was calculated based on apriyantono et al. (1989). nitrogen content was measured using the kjeldahl method. a 0.1 g sample was hydrolyzed with 0.5 g of kjeldahl tablet and 5 ml of h₂so₄ in a heat block. after cooling, 25 ml of distilled water, 25 ml of 50% naoh, and three drops of phenolphthalein were added. the mixture was distilled, and the distillate was collected in 10 ml of 3% boric acid. titration was performed using 0.1 n hcl until the solution changed from blue to light yellow. the volume of hcl used was recorded, and nitrogen content was calculated following sudarmadji et al. (1989). experiment 2: peg treatment two weeks after acclimatization, seedlings were sprayed with 5 ml of e. acoroides extract at concentrations of 0.05%, 0.1%, 0.15%, 0.2%, and 0.25%. treatments were applied twice weekly for three weeks. subsequently, the nutrient solution was replaced with a 20% peg6000 solution to induce drought stress, while spraying with e. acoroides extract continued twice weekly for an additional week. data collection at the end of the experiment, plants were harvested, and chlorophyll content was analyzed using spectrophotometry. leaves were ground in 100% acetone, and chlorophyll a, chlorophyll b, and total chlorophyll concentrations were measured at 647 nm and 664 nm, following the method of lichtenthaler & buschmann (1987). reducing sugars and total protein contents of the shoots were analyzed using previously described seagrass extract as biostimulant for maize growth pharmawati et al. 245 methods. tocopherol content was determined using the method described by wong et al. (1988). a 0.5 g sample was dissolved in 5 ml of toluene and filtered. to 2 ml of the filtrate, 0.5 ml of toluene, 1.75 ml of 2,2-bipyridine (0.07% w/v in 95% ethanol), and 0.25 ml of fecl₃·6h₂o (0.2% w/v in 96% ethanol) were added. ethanol was added to bring the total volume to 5 ml. absorbance was measured at 520 nm using a spectrophotometer. a tocopherol standard curve was prepared using concentrations ranging from 100 ppm to 1,500 ppm in toluene. antioxidant activity (ic50) was determined following the method of amin & lee (2005). a stock solution of 4 g 1,1-diphenyl-2-picrylhydrazyl (dpph) in 100 ml methanol was prepared. a 1 ml aliquot was mixed with 1 ml of ethanol and incubated for 30 minutes, after which absorbance was measured at 517 nm. for plant extracts, a 0.03 g sample was dissolved in 5 ml of methanol. serial dilutions (10, 20, 30, 40, and 50 µl) were prepared in test tubes and adjusted to 1,000 µl with methanol. each solution was mixed with 1,000 µl of dpph, vortexed, and incubated for 30 minutes. absorbance was measured at 517 nm, and the inhibition percentage was calculated as described by amin & lee (2005). in vivo detection of h2o2 in roots was performed using histochemical 3,3’-diaminobenzidine (dab) staining, following daudi & o’brien (2012). roots were immersed in a 1 mg/ml dab solution (ph 3.8) and incubated under light at 25 °c for 8 hours before decolorization by immersion in 96% ethanol. the presence of h2o2 was indicated by deep brown coloration resulting from the reaction of dab with h2o2 catalyzed by plant peroxidases. data analysis all data were statistically analyzed using analysis of variance (anova) in minitab 20. mean differences were evaluated using tukey’s comparison test at a significance level of p < 0.05. results and discussion spraying with e. acoroides extract promoted significant increases in maize plant height. as shown in figure 1, differences in plant appearance are evident between control plants and those treated with various extract concentrations after four weeks of application (at 5 weeks old). quantitative analysis revealed that e. acoroides extract significantly influenced plant height difference (p < 0.01). heights were significantly greater (p < 0.05) in plants treated with 0.1%, 0.15%, 0.2%, and 0.25% compared to the control, with the 0.15% concentration producing the tallest plants (fig. 2). foliar application of e. acoroides extract also significantly increased shoot and root fresh and dry weights. figure 3 shows that fresh shoot weight was higher in plants treated with as little as 0.05% extract, while fresh root weight significantly increased at 0.15%, 0.2%, and 0.25% figure 1 visual comparison of five-week-old maize plants after four weeks of treatment with varying concentrations of e. acoroides extract notes: treatments: a = control (no spray); b = 0.05%; c = 0.1%; d = 0.15%; e = 0.2%; and f = 0.25% extract. bar = 10 cm biotropia vol. 32 no. 2, 2025 246 figure 2 plant height of five-week-old maize plants after four weeks of treatment with varying concentrations of e. acoroides extract notes: different letters above the bars indicate significant differences among treatments (p < 0.05) figure 4 shoot and root dry weights of five-week-old maize plants after four weeks of treatment with varying concentrations of e. acoroides extract notes: different letters above the bars indicate significant differences among treatments (p < 0.05) figure 3 shoot and root fresh weights of five-week-old maize plants after four weeks of treatment with varying concentrations of e. acoroides extract notes: different letters above the bars indicate significant differences among treatments (p < 0.05) seagrass extract as biostimulant for maize growth pharmawati et al. 247 concentrations (p < 0.05). a similar trend was observed for dry shoot weight (fig. 4), while dry root weight was significantly greater in plants treated with 0.1%, 0.15%, and 0.25% extract compared to dry root weight in control plants. the observed improvements on plant height and biomass may be attributed to bioactive compounds and nutrients in the seagrass extract. like seaweed extracts, e. acoroides likely promotes growth by releasing plant growth hormones. previous studies have shown that extracts from various seaweed species contain auxins, cytokinins, and abscisic acid (yalçın et al. 2019) and have even been shown to upregulate genes involved in auxin biosynthesis in beta vulgaris, thereby enhancing lateral root development and nutrient uptake (bertoldo et al. 2023). table 1 summarizes the effects of e. acoroides extract on reducing sugars and total n content in maize shoots. both parameters began to increase at the 0.1% extract concentration, indicating enhanced metabolic activity and nutrient assimilation in treated plants. reducing sugars are key products of photosynthesis and serve as the primary energy source for plant cells. glucose has been reported to influence the expression of genes that promote the activity of nitrate reductase, a key enzyme in the nitrogen assimilation pathway (ma et al. 2023). increased nitrogen availability supports the synthesis of enzymes involved in carbohydrate metabolism, thereby promoting sugar utilization and energy generation (zhang et al. 2021). table 1 reducing sugars content and total n content in five-week-old maize shoots after four weeks of treatment with varying concentrations of e. acoroides extract notes: data presented as mean ± standard deviation. different letters in the same column indicate significant differences (p < 0.05). figure 5 visual comparison of six-week-old maize plants sprayed after three weeks of twice-weekly treatment with varying concentrations of e. acoroides extract followed by one week of peg-induced drought stress notes: a = control (no extract, no peg); b = peg only; c = 0.05% extract+peg; d = 0.1% extract+peg; e = 0.15% extract+peg; f = 0.2% extract+peg; g = 0.25% extract+peg. peg = polyethylene glycol. bar = 10 cm. concentration (%) reducing sugars (%) n (%) 0 14.40 ± 0.386b 15.86 ± 0.180c 0.05 15.14 ± 0.239ab 16.37 ± 0.259bc 0.1 15.65 ± 0.307a 17.00 ± 0.233ab 0.15 15.75 ± 0.269a 17.82 ± 0.612a 0.2 15.78 ± 0.164a 17.45 ± 0.476ab 0.25 15.55 ± 0.239a 16.78 ± 0.228b biotropia vol. 32 no. 2, 2025 248 the increase in reducing sugars and nitrogen content is comparable to effects observed with seaweed extracts. for example, an aqueous extract of sargassum johnstonii increased reducing sugars levels in lycopersicon esculentum when applied at concentrations above 0.1% (kumari et al. 2011). although research on seagrass-derived biostimulants is limited, seaweed extracts have been studied extensively and are known to enhance seed germination, biomass yield, and overall plant quality (ali et al. 2016; kumar et al. 2020). the phenolic compounds, tannins, and flavonoids present in e. acoroides likely contribute to these observed benefits. figure 6 maize roots of six-week-old maize plants sprayed after three weeks of twice-weekly treatment with varying concentrations of e. acoroides extract followed by one week of peg-induced drought stress notes: a = control (no extract, no peg); b = peg only; c = 0.05% extract+peg; d = 0.1% extract+peg; e = 0.15% extract+peg; f = 0.2% extract+peg; g = 0.25% extract+peg. peg = polyethylene glycol. bar = 5 cm figure 7 h2o2 accumulation in roots and leaves of six-week-old maize plants sprayed after three weeks of twice-weekly treatment with varying concentrations of e. acoroides extract followed by one week of peg-induced drought stress notes: a = control (no extract, no peg); b = peg only; c = 0.05% extract+peg; d = 0.1% extract+peg; e = 0.15% extract+peg; f = 0.2% extract+peg; g = 0.25% extract+peg. peg = polyethylene glycol. bar = 2 cm seagrass extract as biostimulant for maize growth pharmawati et al. 249 polyethylene glycol (peg) induces drought stress by reducing root water uptake. in the peg treatment, plants pre-treated with e. acoroides extract for three weeks exhibited faster growth and less severe drought symptoms. during the one-week peg treatment (with continued extract application twice weekly), treated plants maintained less desiccation compared to the controls, which leaves became dry and curled (fig. 5). the peg treatment reduced lateral root development, resulting in smaller root systems and root browning (fig. 6). this damage is likely due to tissue dehydration and osmotic stress-induced cell death (saepudin et al. 2017). in vivo h₂o₂ detection revealed higher oxidative stress in peg-only-treated plants than the control and the e. acoroides-treated plants (fig. 7). the extract may enhance root development, thus improving nutrient uptake and reducing oxidative stress by increasing tocopherol levels and internal antioxidant activity. this antioxidant effect has also been observed with sargassum wightii extract applications to abelmoschus esculentus under salt stress (khan et al. 2022). table 2 presents the chlorophyll content in control plants (no extract, no peg) and pegtreated plants (sprayed and non-sprayed with e. acoroides extract). maize plants treated with peg alone exhibited 17.8%, 24.61%, and 20.65% declines in chlorophyll a, chlorophyll b, and total chlorophyll, respectively. studies have reported that peg-induced osmotic stress significantly decreases chlorophyll levels, negatively affecting photosynthesis and overall plant growth (nio et al. 2019; rao et al. 2017). spraying peg-treated plants with e. acoroides extract increased chlorophyll content, with significant effects starting with the 0.1% extract. table 3 presents the levels of reducing sugars, total n, tocopherol, and ic50 in control and peg-treated maize plants, with and without e. acoroides extract application. the peg-only treatment decreased reducing sugars content, consistent with the findings in phaseolus vulgaris, where peg-induced osmotic stress decreased reducing sugars levels (torres-hernandez et al. 2022). this reduction may be attributed to the inactivation of α-amylase, the enzyme responsible for starch hydrolysis (torres-hernandez et al. 2022). however, spraying e. acoroides extract three weeks before and during peg treatment increased reducing sugars levels. significant increases (p < 0.05) started at the 0.1% extract concentration, suggesting that e. acoroides extract helps counteract the adverse effects of peg-induced stress. total n levels were the highest in plants treated with 0.2% and 0.25% e. acoroides extract under peg-induced drought stress. tocopherol content significantly increased in plants sprayed with the highest extract concentration (0.25%+peg). the increase in tocopherol content in plants sprayed with e. acoroides extract can be driven by the bioactive compounds present in the extract. bioactive compounds, such as flavonoids and table 2 chlorophyll a, chlorophyll b, and total chlorophyll concentrations in six-week-old maize plants sprayed after three weeks of twice-weekly treatment with varying concentrations of e. acoroides extract (0.05%, 0.1%, 0.15%, 0.2%, and 0.25%) followed by one week of peg-induced drought stress notes: data presented as mean ± standard deviation. different letters in the same column indicate significant differences (p < 0.05). peg = polyethylene glycol. treatment chlorophyll a (µg/cm2) chlorophyll b (µg/cm2) total chlorophyll (µg/cm2) control 28.25 ± 0.821cd 19.83 ± 1.844ab 48.08 ± 2.448bc peg only 23.21 ± 1.575d 14.95 ± 0.431b 38.15 ± 1.754d 0.05%+peg 28.02 ± 2.408cd 14.16 ± 0.840b 42.18 ± 1.966cd 0.1%+peg 35.28 ± 2.278ab 26.05 ± 2.032a 61.34 ± 3.050a 0.15%+peg 30.36 ± 0.909bc 24.45 ± 4.816a 54.80 ± 3.911ab 0.2%+peg 30.08 ± 2.244bc 19.14 ± 2.798ab 49.22 ± 1.389bc 0.25%+peg 39.44 ± 2.964a 20.26 ± 1.382ab 59.69 ± 3.508a biotropia vol. 32 no. 2, 2025 250 polyphenols, exhibit strong antioxidant properties, which can protect tocopherols from oxidative degradation, thereby increasing their stability and bioavailability (domínguez-valencia et al. 2023). plant extracts can enhance tocopherol production through elicitation, which stimulates the plant's defense mechanisms, leading to increased biosynthesis of tocopherols (almagro et al. 2021). the ic50 value, which represents the concentration required to reduce oxidative stress by 50%, significantly decreased with increasing extract concentration from 0.15% to 0.25% e. acoroides extract, suggesting that e. acoroides extract helps mitigate abiotic stress by enhancing antioxidant activity. this reduced stress lowers ros (reactive oxygen species) accumulation, as indicated by lower h₂o₂ levels (fig. 7). a previous study on zostera marina, another seagrass species, found that its aqueous extract increased the activity of antioxidant enzymes in salt-stressed tomato plants (vinoth et al. 2017). the bioactive compounds in e. acoroides, phenolics, flavonoids, and pigments, likely increased plant growth by exerting hormonal effects similar to those of seaweed extracts. conclusion spraying enhalus acoroides extract significantly enhanced maize growth by increasing plant height and shoot and root biomass, as well as by improving reducing sugars and total n contents. under peg-induced drought stress, e. acoroides extract alleviated stress-related damage. enhalus acoroidestreated plants exhibited higher chlorophyll levels and reduced oxidative stress, as indicated by lower h₂o₂ accumulation. additionally, spraying with e. acoroides extract increased reducing sugars, total nitrogen, and tocopherol levels, suggesting improved stress tolerance. these findings highlight the potential of e. acoroides extract as a biostimulant for enhancing plant growth and drought resilience in maize cultivation. further research is needed to explore its applicability across different crops and environmental conditions. acknowledgments the authors acknowledge the directorate general of higher education for funding this research through the 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(berkeley 1847). the mushroom is known by several synonyms, including a. trachodes berk. (1847) and chitoniella trachodes (berk.) petch (1908). it is distinguished by a large basidiome, prominent chocolate or coffee brown to deep brown pellicle on the pileus disc surface, presence of an annulus, olive-brown to umber brown spore deposit, slightly thick-walled spores with a truncated apex, and a context that changes from white to reddish brown when cut. furthermore, the species has dark-colored spores and two types of veils on its basidioma, partial and universal (hosen and ge 2011; pegler 1985). *corresponding author, email: ivanpermanaputra@apps.ipb.ac.id mailto:ivanpermanaputra@apps.ipb.ac.id clarkeinda trachodes indonesia – ivan permana putra et al. 115 c. trachodes was reported to grow in asian countries such as thailand, bangladesh, china, india, malaysia, indonesia, and sri lanka (leelavathy et al. 1981; yang 1991; hosen and ge 2011). yang (1991) initially mentioned its distribution in indonesia when describing an agaric new to china. however, comprehensive details about the morphological description or herbarium collection of indonesian specimens were not provided. during mushroom club regular survey at ipb university campus forest, west java, indonesia, basidiomata similar to c. trachodes was obtained. this study aimed to confirm the taxonomic position of the specimens through morphological and molecular analyses. materials and methods specimen collection the specimen was collected at ipb university campus forest, bogor, west java, indonesia, in 2023 during regular foraging by the mushroom club of ipb university. the basidiocarp was photographed in situ, and ecological information, comprising coordinates, substrate, and vegetation, was noted. the specimen was contributed to the herbarium bogoriense, indonesia, with collection number bo24637. morphological identification the morphological features were observed from fresh basidiocarp in situ and at the mycology laboratory, department of biology, faculty of mathematics and natural sciences, ipb university, indonesia. macroscopic characters, including color, size, pileus, stipe ornamentation, margin, and lamellae were observed directly. the microscopic features comprising basidia, cystidia, spores (shape, size, color, ornamentation), and clamp connection were examined using a light microscope. specimens were further analyzed using scanning electron microscopy (sem), as described by goldstein et al. (1992). lamellae were cut into small pieces (5 × 5 mm) and pre-fixed in 2.5% glutaraldehyde of a cacodylate buffer with a ph of 8.4 at 27°c for two days. subsequently, the samples were pre-fixed in 2% tannic acid for six hours and washed with four different cacodylate buffers. dehydration was carried out in 50%– 100% ethanol series, infiltrated with t-butanol twice for 10 minutes, and freeze-dried. the freeze-dried samples were mounted on an aluminum stub with double-sided carbon tape and gold-coated. observations were made with the jsm it 200 sem system (jeol, tokyo, japan). the specimens were then identified using relevant identification references (pegler 1985; hosen and ge 2011). molecular analysis chromosomal dna was isolated from a fresh basidiocarp using a qiagen dneasy plant mini kit according to the manufacturer's instructions at ilab laboratory, research center for applied microbiology, national research and innovation agency (brin), indonesia. dna amplification was conducted using a thermo scientific arktik thermal cycler (thermo fisher scientific). the amplification reaction was carried out using the primer pairs its 5 (5'-gga agt aaa agt cgt aac aag g-3') and its 4 (5'-tcc tcc gct tat tga tat gc-3') (white et al. 1990). the pcr amplification was performed in a 50 µl reaction mixture containing 9 µl ddh2o, 1.5 µl of 10 pmol of each primer, 25 µl pcr mix from 2× pcr buffer for kod fx neo (toyobo), and 2 µl of 100 ng template dna. the pcr condition comprised an initial denaturation at 94°c for 5 minutes, followed by 30 cycles of denaturation at 94°c for 30 seconds, annealing at 56°c for 45 seconds, an extension at 72°c for 1 minute, and a final extension set at 72°c for 10 minutes. the pcr product was analyzed using 1.5% agarose gel electrophoresis run with tae buffer (40 mm tris-acetate, ph 8.0 1 mm edta), stained with florosafe dna stain, and visualized by the gel doc ez gel documentation system (biorad). pcr products were then sent to 1st base malaysia for sequencing. the sequences were assembled using chromaspro software, and the final generated sequences were deposited in genbank (https://www.ncbi.nlm.nih.gov/) to obtain the accession number. these sequences were then subjected to basic local alignment search tool (blast) in ncbi to compare the homology with existing data. based on the blast results (table 1), selected published sequences were used for phylogenetic tree analyses with leucoagaricus medioflavoides as the outgroup. the phylogenetic tree was generated using mega x software (kumar et al. 2018) and refined using treegraph software version 2.9.2-622 beta. bootstrap values (bs) of 70 % or higher were shown on the branches of the phylogenetic trees. https://www.ncbi.nlm.nih.gov/ biotropia vol. 31 no. 1, 2024 116 table 1 species, outgroup, herbarium voucher, and genbank accession numbers used in this study species collection code its accession number agaricus cupressophilus atcc mya-4431 nr_111346.1 a. desjardinii hmas wzr2012-8212 nr_158308.1 a. grandiomyces hmas 275728 nr_145005.1 a. lusitanicus lip 0001283 nr_158338.1 a. megacystidiatus mflu 12-0137 nr_119953.1 a. microvolvatulus br 5020002084476 nr_119952.1 a. pakistanicus lah 35299 nr_173276.1 a. sinodeliciosus hmas wzr2012-821 nr_164537.1 a. sinoplacomyces hmas 275724 nr_145009.1 a. subsubensis atcc mya-4432 nr_137710.1 a. tibetensis hmas 275725 nr_145011.1 clarkeinda trachodes ecv3838 hm488751.1 c. trachodes isolate kubot-krmk-2020-26 mw425600.1 c. trachodes voucher bo24637 or975901 c. trachodes voucher hkas122726 on794453.1 c. trachodes ps2011-12 mn099351.1 leucoagaricus medioflavoides voucher mcve:2324 gq329055.1 results and discussion taxonomy clarkeinda trachodes (berk.) singer, lilloa 22: 413 (1951) [1949] synonym: agaricus trachodes berk., london journal of botany 6: 487 (1847) chitoniella trachodes (berk.) petch, annals of the royal botanic gardens peradeniya 4: 396 (1909) fungus trachodes (berk.) kuntze: 480 (1898) chitoniella poderes (berk. & broome) henn., die natürlichen pflanzenfamilien nebst ihren gattungen und wichtigeren arten insbesondere den nutzpflanzen: i. tl., 1. abt.: fungi (eumycetes): 240 (1898) clarkeinda poderes (berk. & broome) kuntze, revisio generum plantarum 2: 848 (1891) agaricus pedilius berk. & broome, journal of the linnean society. botany 14: 32 (1875) clarkeinda pedilia (berk. & broome) kuntze, revisio generum plantarum 3 (3) (1891) chitonis pedilis (berk. & broome) clem, the genera of fungi: 114 (1909) agaricus poderes berk. & broome, journal of the linnean society. botany 14: 32 (1875) basidiomata large, solitary, terrestrial. pileus light brown to brown, 190-210 mm in diameter, applanate to plano-concave, with a straight margin. the surface is covered with numerous, small, revolute, loosely floccose brown squamules towards the margin. a dark brown, smooth to glabrous, cartilaginous patch is present at the center of the pileus. context up to 7 mm wide at the pileus center, white to cream, turning reddish over time. lamellae free, remote from stipe, 75 × 15 mm in diameter, crowded, dull yellow, concolorous, with series of lamellulae, margin entire. stipe is central, 100 − 110 × 16 – 20 mm (apex) × 28 – 30 mm (base) mm, bulbous base tapering to the apex, hollow interior that turns reddish when injured. white to cream at apex, light brown toward the base, smooth toward annulus, densely furfuraceous squamules towards the base. annulus membranous, superior, up to 9 cm wide, fragile, persistent to maturity, fragile and ragged with age, eroded margin, cream to pale brown. volva present, white to cream. basidia hyaline, 21 − 28 × 5 – 7 µm, clavate to subclavate, thin-walled, 4-spored. basidiospores aseptate, ovoid to ellipsoid, 5.8 − 7.5 × 3.7 − 4.5 µm, smooth, thick-walled, with truncated apex and germ pore, hyaline to pale brown, interior with several lipid bodies. basidioles narrow clavate. cheilocystidia abundant, slightly thick-walled, 22 − 43 × 14 − 18 µm, clavate to broadly clavate, hyaline, thinwalled, smooth, septate at the base. hymenophore trama interwoven, hyaline, thinwalled. pileipellis are constructed by short chains of hyphae, slightly interwoven, and pale brown pigment inside the cells. pellicle consists of short oblong cells mixed with oval to round cells, pigment inside cells darker than pileipellis. stipe parallel to slightly interwoven, with hyaline hyphae. habitat: mixed with bamboo leaves litter. clarkeinda trachodes indonesia – ivan permana putra et al. 117 distribution: china, india, indonesia, malaysia, thailand, laos, and sri lanka. specimen examined: under bamboo trees, darmaga, ipb university campus forest, west java, indonesia, s 6° 33' 14.22"e 106° 43′ 26.418", 2023, collected by ip putra, collection code bo24637. molecular analysis the final sequence was deposited in genbank (https://www.ncbi.nlm.nih.gov/) with its accession number or975901. the top blast result showed a 98-100% match with c. trachodes. the phylogenetic tree constructed from its sequence placed the specimen bo24637 in the same clade with c. trachodes reference strains (c. trachodes hm488751.1, c. trachodes ps2011-12, c. trachodes hkas122726, and c. trachodes kubot-krmk-2020-26) with 100% bs value (figure 5). moreover, c. trachodes bo24637 was fully separated from the clade of agaricus species. figure 1 field photograph of c. trachodes bo24637. a: upper side of pileus with pellicle crown (arrow). b: ring (arrow) at a superior position. c: underside of pileus with free stipe to lamella. d: hollow interior of stipe. bars = 5 cm https://www.ncbi.nlm.nih.gov/ biotropia vol. 31 no. 1, 2024 118 figure 2 microscopic characters of c. trachodes bo24637. a: basidium with sterigmata (arrow), ovoid basidiospores (double arrow). b-c: cheilocystidia clarkeinda trachodes indonesia – ivan permana putra et al. 119 figure 3 sem image of c. trachodes bo24637. spore (red arrow), sterigma (white arrow), and basidium (yellow arrow) figure 4 microscopic characters of c. trachodes bo24637. a-b: pileipellis. c: pellicle crown section. d: tissue of stipe biotropia vol. 31 no. 1, 2024 120 figure 5 clarkienda trachodes bo24637 phylogenetic tree based on rdna-its1/2 region using maximum likelihood with 1000 bootstrap analysis. the specimen in this study is in bold on the phylogenetic tree this study provides comprehensive data and accessible information on c. trachodes in indonesia. the species was initially described as chitoniella by boedjin from java (1934). several publications have since mentioned the distribution of chitoniella (c. trachodes) in indonesia (pegler 1985; yang 1991; hosen and ge 2011; verma et al. 2016; sysouphanthong 2021). however, there were no reports detailing the taxonomy of c. trachodes or herbarium materials in indonesia. the materials from this study were submitted to herbarium bogoriense, indonesia, under the collection number bo24637. the morphological and molecular information (dna sequence) were also provided to facilitate future studies on c. trachodes in clarkeinda trachodes indonesia – ivan permana putra et al. 121 indonesia. although recognized as poisonous, there has been no record of c. trachodes mushroom poisoning in indonesia. c. trachodes bo24637 is similar to chlorophyllum species but can be distinguished by a large basidiome, brown pellicle of pileus disc, prominent ring, and volva (pegler 1985; hosen and ge 2011; kumar and kaviyarasan 2011; læssøe et al. 2019). c. trachodes bo24637 has a larger pileus dimension compared to previous reports of same species from china (yang 1991), india (verma et al. 2016), bangladesh (hosen and ge 2011), laos (læssøe et al. 2019), and thailand (sysouphanthong et al. 2021). it is morphologically similar to specimens from india, laos, and thailand but differs from the basidiomata reported from bangladesh, which have reddish-brown pileus and stipe. in addition, specimen bo24637 features thick-walled basidiospores similar to all collections discussed except the specimen from bangladesh. previous reports described c. trachodes as a terrestrial macrofungus with a habitat ranging from grasses (verma et al. 2016), shorea robusta (dipterocarpaceae) forests (hosen and ge 2011), to nutrient-rich soils in deciduous rain forests (sysouphanthong 2021). this study showed that basidomata grew solitary on bamboo litter, providing new insights into the habitat of this species. hosen and ge (2011) reported that the record of c. trachodes in italy by lavorato & contu (2002) was based on a misidentified agaricus specimen. therefore, in this study, molecular analysis was conducted alongside morphological characterization to increase the reliability of the results. the blast top hits result showed that the bo24637 was highly similar to c. trachodes. the phylogenetic tree also nested specimen bo24637 in the clade of c. trachodes from thailand, india, and china with 100% bs value. the specimen was also placed in a different clade than agaricus, consistent with reports by sysouphanthong et al. (2021). conclusion in conclusion, morphological and molecular analyses confirmed the collected specimen as c. trachodes. this study established a clear and accessible record of c. trachodes in indonesia. in addition, the herbarium collection and its sequence of the species were contributed to indonesia, which is essential for future studies globally. acknowledgements this study was supported by the grant of “skema riset kolaborasi nasional ipb 20232024” with reference number 502/it3.d10/ pt.01.03/p/b/2023 to dr. ivan permana putra. we thank research center for applied microbiology, national research and innovation agency (brin)indonesia, department of biology, faculty of mathematics and natural sciences, ipb universityindonesia. we thank direktorat pengelolaan koleksi ilmiah (brin), indonesia for the herbarium support. references berkeley mj. 1847. decades of fungi xv−xix. ceylon fungi. london j bot 6: 479-514. boedijn kb. 1934. the genus chiloniella with remarks on the chlorosporae. bull jard bot buitenzorg 13: 276-280. goldstein ji, newbury de, echlin p, joy dc, romig jr ad, lyman ce, fiori 10c, lifshin e. 1992. scanning electron microscopy and x-ray microanalysis 2nd 11 edn. new york: plenum press. hosen i, ge zw. 2012. clarkeinda trachodes (agaricales, basidiomycetes), first record from bangladesh. mycotaxon 118: 331-336. index fungorum [internet]. 2023. clarkeinda trachodes. [accessed on 18 january 2024]. available from: https://www.indexfungorum.org/names/names record.asp?recordid=294937 kumar m, kaviyarasan v. 2011. a rare agaric (agaricomycetes: agaricaceae) from a sacred grove of eastern ghats, india. jott 3: 1778-1781. kumar s, stecher g, li m, knyaz c, tamura k. 2018. mega x: molecular evolutionary genetics analysis across computing platforms. mol biol evol 35: 1547-1549. kuntze o. 1891. rcuisio gcncrurn planfarum. 2: 3771011. leipzig: arthur felix. læssøe t, pedersen os, sysouphanthong p. 2019. an introduction to the edible, poisonous and medicinal fungi of northern laos. laos: fao. https://www.indexfungorum.org/names/namesrecord.asp?recordid=294937 https://www.indexfungorum.org/names/namesrecord.asp?recordid=294937 biotropia vol. 31 no. 1, 2024 122 leelavathy km, zachariah s, sankaran kv. 1981. clarkeinda trachodes—an agaric new to india. mycologia 73: 204-207. lavorato c, contu m. 2002. clarkeinda trachodes, una specie nuova per la micoflora italiana rinvenuta in calabria. [ clarkeinda trachodes, a new species for the italian mycoflora found in calabria] boll gruppo micol g bresadola 45: 33-39. pegler dn. 1985. the genus clarkeinda (basidiomycotina: agaricaceae). bot j linn soc 91: 245-252. petch t. 1908. the genus chifoniclla. ann roy bot gard 4: 113-122. sysouphanthong p, thongklang n, liu j-k, vellinga ec. 2021. description of lepiotaceous fungal species of the genera chlorophyllum, clarkeinda, macrolepiota, pseudolepiota, and xanthagaricus, from laos and thailand. diversity 13: 666. verma rk, tiwari c, parihar j, kumar s. 2016. diversity of macro-fungi in central india-ii: clarkeinda trachodes. van sangyan 3: 17-20. white tj, bruns t, lee sj, taylor jw. 1990. amplification and directsequencing of fungal ribosomal rna genes for phylogenetics. in: innis m, gelfand d, sninsky j, white t (eds.), pcr protocols. new york: academic press, pp. 315-322. yang zl.1991. clarkeinda trachodes, an agaric new to china. plant divers 13: 279-282. 1154.cdr page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8 biotropia vol. 29 no. 2, 2022: 142 149 doi: 10.11598/btb.2022.29.2.1680 142 distribution pattern and diversity of epiphytic orchids in the curug cibereum path, mount gede pangrango, indonesia bela prapitasari* and ardyan pramudya kurniawan laboratory of ecology-botany, universitas islam negeri sunan kalijaga, yogyakarta 55281, indonesia received 14 october 2021/accepted 18 december 2021 abstract the curug cibereum path as a tourist attraction in the mount gede pangrango area is dominated by a lush tree, making it a suitable place to find a diversity of epiphytic orchids. this study aimed to determine the distribution pattern and diversity of epiphytic orchids along the curug cibereum path which is influenced by environtmental parameters. the research method was carried out by plotting ten plots on the right and left sides of the path, with each plot measuring 500 x 5 m and the distance between plots was 100 m. the results showed that there were 31 species of epiphytic orchids from 12 genera. the distribution pattern of epiphytic orchids was mostly clustered except for oberonia lotsyana, which had a uniform pattern. the epiphytic orchid species with the highest inp were bulbophyllum gibbosum with an inp value of 35% and coelogyne speciosa with an inp value of 20%. species diversity (h ') was moderate (h '=2.54), the evenness index (e) was high (e=0.73), the dominance index (d) was low (d=0.13). the measurements of environmental parameters showed optimal conditions for the growth of epiphytic orchids, namely with a temperature of 24.5 oc, soil moisture of 76.5%, air humidity of 70%, ph 5.1, the light intensity of 6351 lux and wind speed of 0.03 m/s. keywords: curug cibereum, distribution, diversity, dominance, epiphytic orchid introduction orchids are unique plants because they have several benefits both economically and ecologically. the many uses of orchids for decoration, traditional medicinal ingredients and cosmetics make orchids have a high selling value in the world of trade and many fans because they have high aesthetics value. the number of orchids in the world is estimated at around 20,000-30,000 species consisting of 700 genera (shutleworth et al. 1970). orchids in indonesia are estimated at 5,000 species from 40 genera scattered in sumatra, kalimantan, papua, sulawesi and java. in java, there are about 731 species of orchids with the 231 species are endemic. in terms of orchid distribution, west java has 642 species, east java has 390 species, and central java has 295 species (chomber 1990). an epiphytic orchid is one type of natural orchid that has the characteristic of sticking to the host tree. ecologically, epiphytic orchids function as habitats for certain animals such as ants and termites. research activities on epiphytic orchids are currently considered very important because of the many damaged habitats caused by felling trees to clear forests used for repairs, animal husbandry and visitor attractions. according to the world conservation monitoring center (1995), orchids are plants with a high threat of extinction, around 39% (203 species) compared to native indonesian plants, which are also threatened. based on these conditions, many orchids may have become extinct, but no data has been collected (puspitaningtyas 2005). mount gede pangrango, west java, is a national park used as a conservation area, climbing or tourist attraction. one of the attractions in the area is curug cibereum which is located at resort selabintana, sukabumi. the *corresponding author, email: belaprapitasari@gmail.com distribution pattern and diversity of epiphytic orchids – prapitasari and kurniawan 143 area consists of a natural forest where many orchids are found, both epiphytic and terrestrial. the types of orchids in the area have been recorded. namely, 46 species were found, consisting of 25 species of epiphytic orchids and 21 species of terrestrial orchids (prapitasari et al. 2020). the existence of tourist activities on the curug cibereum path, of course, can affect the reduction in the types of orchids in the area. based on the above explanation, this study was conducted to determine the distribution pattern and diversity of epiphytic orchids in the curug cibereum path which is influenced by environmental parameters. this research is also expected to provide data updates from previous studies. materials and methods study site the research was conducted in march 2021 in the selabintana resort area, sukabumi, which is on the route to curug cibereum mount gede pangrango, west java. geographically, selabintana resort is located at 106o57'41"e and 06o50'50"s with an area of 2,547.93 ha (fig. 1). this area has a topography of hills and mountains with slightly sloping areas, altitudes ranging from 1,130 to 3,019 masl. in general, selabintana resort is an area with a wet climate with an average rainfall of 3,000-4,200 mm/year. the rainy season lasts from october to may, and from december to march the rainfall can be more than 400 mm/month. the average temperature at resort selabintana is 18 oc with relatively high humidity throughout the year, which is around 80-90% (dendang 2009). based on these environmental parameters, it can be seen that the environment in the selabintana area is a suitable habitat for the growth of epiphytic orchids. it can be seen that the types of epiphytic orchids in the selabintana area are very plentiful, especially along the curug path. curug cibereum is the highest waterfall in the gunung gede pangrango national park area and is located at an altitude of 1,200 masl. the curug cibereum path is perfect for a leisurely walk or hiking. the waterfall can be reached with a distance of 3.1 km using gps or 1.5 h from the entrance to resort selabintana or pondok halimun. material and data collection the tools used in this research are tally sheet, camera, environmental parameter tools (lux meter, thermometer, hygrometer, anemometer, soil tester), global positioning system (gps), and orchid identification book, namely orchid of java book (chomber 1990), native orchids of indonesia (handoyo & ramadhani 2006) and orchids of indonesia (handoyo 2019). data collection is done by plotting along the right and left sides of the waterfall path. there were ten plots with each plot size of 500 m long and 5 m wide (fig. 2). the distance between plots was 100 m. the data were collected in the form of epiphytic orchids found (types of orchids, number of species of orchids, the number of individuals of each type of orchid, and data on environmental parameters). figure 1 research sites (the blue and white line is the curug cibereum path) biotropia vol. 29 no. 2, 2022 144 figure 2 the research sampling design of epiphytic orchids in the curug cibereum path data analysis data analysis was carried out qualitatively and quantitatively. qualitative analysis was done by describing the data that have been collected and processed from the research results. quantitative analysis was used to determine species composition and level of species diversity. determination of species composition was used to determine density (d), relative density (rd), frequency (f), relative frequency (rf) and important value index (ivi) (indriyanto 2006). the level of species diversity was determined by the shannonwiener index (h'), evenness index (e), morishita index (id) and dominance index (d) (ernst et al. 2002). 1. species composition a. density (d) = number of individuals of species total area b. relative density (rd) = density each species x 100% density all species c. frequency density (f) = number of individuals x 100% total number of plots d. relative frequency (rf) = number of frequency of each species x 100% frequency all species e. important value index= rd+rf 2. species diversity a. shannon-wiener diversity index (h') h'= -ʃ [pi ln (pi)]; pi: ni n description: h': shannon-wiener diversity index ni: number of individuals of each species n: number of all species shannon-wiener diversity index (h') determination criteria: h' >3 : the diversity is high h' 1-3: the diversity is moderate h' <3 : the diversity is low b. evenness index (e) e = h' ln s description: e : evenness index h' : shannon-wiener diversity index s : number of species evenness index (e) determination criteria: evenness index values ranged from 0-1. if the value is 0, it indicates the level of evenness of species is very uneven, whereas if the value is close to 1, almost all species have the same abundance (maguran 1988). c. morishita index (id) id = q (xi(xi – 1) n(n – 1) description: id : morishita index q : number of sample plots xi : number of individuals on the plots n : total number of species the criteria for determining the morishita index: id>1 : the pattern of distribution of individual types is clumped id=1 : the distribution pattern of individual types is random id<1 : the distribution pattern of individual species is uniform d. dominance index (c) c= ʃ[ni/n]2 description: c: the dominance index of a species ni: the number of individuals of a species n: number of individuals of all types 500 m 500 m curug cibereum path 3.1 km 5 m 1 3 5 7 9 2 4 6 8 10 5 m distribution pattern and diversity of epiphytic orchids – prapitasari and kurniawan 145 dominance index determination criteria (c): when the value of c is close to 0, then no individual dominates so that there is excellent uniformity. the value of c is close to 1, indicates that several species dominate at specific locations so that the value of the uniformity index is getting smaller (odum 1993). results and discussion distribution pattern of epiphytic orchids the study results found as many as 31 species of epiphytic orchids consisting of 12 genera. comparing our results with the research of prapitasari et al. (2020) regarding the types of orchids found in the selabintana area (both on the curug cibereum path and the hiking trail), there was an increase in the number of epiphytic orchid species in the curug cibereum path from this study, which was 13 species. the epiphytic orchid species found were adenoncos virens, appendicula cornuta, bulbophyllum capitatum, bulbophyllum multiflora, bulbophyllum gibbosum, bulbophyllum sp. 1, bulbophyllum sp. 2, cerastostylis anceps, cerastostylis graminea, dendrochilum sp., oberonia lotsyana, oberonia similis, and phreatia sp. (table 1). table 1 comparison of the types of epiphytic orchids found in the curug cibereum path in 2020 and 2021 no types of epiphytic orchid 2020 2021 distribution index (id) 1 adenoncos virens √ clumped 2 agrostophyllum bicuspidatum √ √ clumped 3 agrostophyllum laxum √ √ clumped 4 appendicula angustifolia √ √ clumped 5 appendicula cornuta √ clumped 6 bulbophyllum capitatum √ clumped 7 bulbophyllum multiflora √ clumped 8 bulbophyllum gibbosum √ clumped 9 bulbophyllum sp. 1 √ clumped 10 bulbophyllum sp. 2 √ clumped 11 bulbophyllum sp. 3 √ √ clumped 12 cerastostylis anceps √ clumped 13 cerastostylis graminea √ clumped 14 cerastostylis sp. √ √ clumped 15 coelogyne speciosa √ √ clumped 16 dendrobium mutabile √ clumped 17 dendrobium rugosum √ √ clumped 18 dendrobium sp. 1 √ √ clumped 19 dendrobium sp. 2 √ √ clumped 20 dendrobium sp. 3 √ √ clumped 21 dendrobium sp. 4 √ √ clumped 22 dendrochilum sp. √ clumped 23 eria iridifolia √ √ clumped 24 eria monostachya √ √ clumped 25 eria multiflora √ √ clumped 26 eria sp. √ √ clumped 27 liparis eliptica √ √ clumped 28 liparis pallida √ √ clumped 29 oberonia similis √ √ unifrom 30 oberonia lotsyana √ clumped 31 phreatia sp. √ clumped 32 schoenorcis juncifolia √ √ clumped amount 20 31 biotropia vol. 29 no. 2, 2022 146 based on the analysis of the morishita index (id), most of the epiphytic orchids in the curug cibereum path showed a clumped distribution pattern (id>1), and there were only one species with uniform distribution, namely oberonia similis (id<1) (table 1). the distribution of clumped is influenced by several factors, such as the breeding process. epiphytic orchids reproduce by producing vast numbers of seeds and usually fall near their mother. besides that, they also reproduce by rhizomes that produce many vegetative tillers (barbour et al. 1987). environmental factors also affect the distribution pattern of epiphytic orchids due to non-uniform environmental conditions (wahyuni et al. 2017). the uniform pattern is caused by the intense competition for survival between epiphytic orchid species, resulting in the distribution of the same living space in an environment. the uniform distribution pattern is a non-random pattern indirectly caused by a limiting factor to the existence of a population. uniform dispersion comes from negative intuition between individual species, such as competition for nourishment or other specialties (onrizal et al. 2005). in addition, not all of the host trees in the curug cibereum patth area are suitable for epiphytic orchids to live. there are also epiphytic spikes that can inhibit the growth of epiphytic orchids because there are too many of them (paramitha et al. 2010). types and composition of epiphytic orchids the most dominant epiphytic orchid species found in the curug cibereum path were appendicula angustifolia (171 individual pieces/ 2.5 ha), bulbophyllum multiflora (142 individuals/ 2.5 ha), bulbophyllum gibbosum (670 individuals/ 2.5 ha), coelogyne speciosa (323 individual pieces/ 2.5 ha), eria iridifolia (119 individuals/2.5 ha), and eria multiflora (272 individuals/2.5 ha). while the few epiphytic orchids found were dendrobium sp. 1 (2 individuals/2.5 ha), dendrobium rugosum (2 individuals/2.5 ha), liparis elliptica (3 individuals/2.5 ha), oberonia similis (1 individual/2.5 ha), and oberonia lotsyana (2 individuals count/2.5 ha) (fig. 3). figure 3 the density of epiphytic orchids and important value index (ivi) in the curug cibereum path distribution pattern and diversity of epiphytic orchids – prapitasari and kurniawan 147 the presence of several dominant species of epiphytic orchids will affect the value of the important value index (ivi). the ivi value is obtained from relative density (dr) and relative frequency (fr) results by adding the two parameters. the greater the level of dominance of the species in a community, the greater the important value index of that species (tahier et al. 2018). based on the graph above, it is known that the highest ivi value of epiphytic orchids is also found in the orchids which are the most abundant or dominant in the research location. epiphytic orchid species with high ivi were bulbophyllum gibbosum with an ivi value of 35% and coelogyne speciosa with ivi 20% (fig. 3). species with a high ivi dominate and have an influential role in an area, namely, play a role in ecosystem stability. meanwhile, epiphytic orchids with low ivi indicate that the species have a narrow distribution and a specific environment to grow (wulanesa et al. 2017). diversity of epiphytic orchids species diversity is a community-level characteristic based on its biology that can express community structure. at the same time, the diversity index is a value that can indicate the high and low population diversity of the species in the community. this value is obtained by comparing the number of species and individuals in a community or habitat (yuanda 2007). the species diversity index (h') of epiphytic orchids in the curug cibereum path is in the medium category with an h' value of 2.54 (fig. 4). the moderate diversity index indicates that the ecosystem is relatively balanced, with sufficient productivity and moderate ecological pressure (fitriana 2006). the medium category is also caused by the area having almost the same abundance of epiphytic orchid species. according to odum (1996), the greater the number of species found, the greater the diversity, whereas if the number of species found is small, the diversity will be low, and it is suspected that a few species only dominate the area. figure 4 diversity index (h'), evenness index (e), and dominance index (c) of epiphytic orchids in the curug cibereum path the evenness index, also known as the species abundance index, is used to determine the level of species abundance influenced by diversity. the evenness index value of epiphytic orchids in the curug cibereum path was e = 0.73, indicating the evenness of epiphytic orchids in the curug cibereum path was relatively high (because the value is close to 1). a reasonably high evenness index indicates that epiphytic orchids are evenly distributed with a stable number in a habitat (zulkhaidah et al. 2018). the high evenness index of epiphytic orchid species is also influenced by the reasonably stable value of the species diversity index (h'). species diversity (h') of epiphytic orchids in the curug cibereum path is moderate with abundant epiphytic orchid species so that the distribution of orchid species in the location is evenly distributed with the stable numbers of individual orchids. a species with a high level of stability has a more significant opportunity to maintain the sustainability of its species in an environment (odum 1993). the dominance index of a species illustrates that the species is very influential on the environment. the dominance index value of epiphytic orchids in the curug cibereum path was c = 0.13, which means that the epiphytic orchid dominance index in the curug cibereum path had a low-level dominating category. having a dominant species in the study location, it is suspected that the species is the most able species to adapt and survive in an environment. dominant species will affect the value of the uniformity index, which will decrease. the dominance index (c) value is related to the value of species diversity, in which biotropia vol. 29 no. 2, 2022 148 the higher the species diversity, the lower the dominance index or vice versa (rikardus et al. 2017). thus, moderate diversity index (h') of epiphytic orchids in the curug cibereum path means lower dominance index. environmental parameter factors the high level of diversity of epiphytic orchids in the curug cibereum path is strongly influenced by environmental parameters. environmental parameters are an essential factor in determining the existence of a plant. environmental parameters in the curug cibereum path showed normal conditions for the growth of epiphytic orchids (table 2). table 2 environmental parameters in the cibereum curug no environtmental parameters measurement results 1 soil ph 5.1 ± 0.96 2 soil moisture (%) 76.5 ± 15.11 3 air temperature (oc) 24.5 ±2.44 4 humidity (%) 70 ± 11.73 5 light intensity (lux) 6,321 ± 16,482.52 6 wind velocity (m/s) 0.03 ± 0.19 the optimal temperature for the growth of epiphytic orchids is 24.5 oc. the magnitude of the temperature is very suitable for the growth of orchids. the coldest temperature for orchid growth is 12.7 oc, and the average temperature for orchids is in the range of 15-28 oc (indarto 2011). furthermore, soil ph measurements were carried out under the epiphytic orchid host tree and not in the area around the epiphytic orchid growth because the location was difficult to reach. the result of soil ph measurement was 5.1, indicating that the ph is suitable for the growth of epiphytic orchid host trees. the normal ph for orchid growth is 5-6.5 (purbadi et al. 2005). soil ph condition at the study site tend to be acidic (ph = 5.1) due to high soil moisture, which was 76.5%. wet soil due to high humidity will cause a low ph value, and vice versa (yulia 2008). air humidity at the research site was 70%. the high humidity condition is very suitable for the growth of epiphytic orchids. the humidity required for optimal orchid growth is 50 80%. humidity that is too low causes the air around the orchid to dry out and cause a disturbance. conversely, if the humidity is too high, it will increase disease attacks, especially diseases caused by fungi and bacteria. air humidity on the curug cibereum path is high, also influenced by low wind speeds so that it tends to get wet. if the humidity is low, the wind speed is high, causing an area to experience dry condition. at the study site, there was almost no wind blowing. it was only encountered several times with very low speeds so that the results of the average wind speed at the study site were only 0.03 m/s. the wind speed is very suitable for orchid growth, where orchids like soft air circulation. (purwanto 2016). light intensity is one of the environmental parameter affecting the growth of epiphytic orchids. the living nature of epiphytic orchids attached to the host tree is one form of adaptation to get sunlight. therefore, epiphytic orchids require more light than terrestrial orchids (tirta & sutomo 2014). usually, epiphytic orchids require different light intensities depending on the type of epiphytic orchid itself. for example, the type of dendrobium requires a light intensity of 2,0003,000 lux. the light intensity for orchid growth is 1,000-2,000 footcandle or 10,000-20,000 lux (darmono 2007). the results of the light intensity measurement in the curug cibereum path were 6,321 lux. the amount of light intensity is still suitable for the growth of epiphytic orchids because it does not interfere with the growth of epiphytic orchids in the curug cibereum path. in addition, the light intensity at the study site is also influenced by the canopy density. several locations do not have a dense canopy causing relatively high light intensity. however, epiphytic orchids are more commonly found in low light intensity. conclusion there were 31 species of epiphytic orchids from 12 genera found in the curug cibereum path. curug cibereum has environment parameters suitable for epiphytic orchids. acknowledgments this study was funded by the research and community service institute of uin sunan kalijaga yogyakarta. the authors are gratefull to distribution pattern and diversity of epiphytic orchids – prapitasari and kurniawan 149 the officials of the mount gede pangrango national park for the permission and facilities provided. special gratitudes are also presented to the field research team (ahmad aliwafa, dharfan ihlasul iman, and aulya nidaur rahmah) who assisted in data collection. references barbour sl, lam l, fredlund dg. 1987. transient seepage model for saturated-unsaturated soil systems: a geotechnical engineering approach. can geotech 24(198):565-80. boughey as. 1973. ecology of population. 2nd edition. new york (us): macmillan co. comber jb. 1990. orchid of java. kew (gb): the royal botanic gardens. darmono dw. 2007. planting orchids. depok (id): penebar swadaya. dendang b. 2009. the diversity of butterflies at the selabintana resort, mount gede pangrango, west java. journal research and nature conservation 6(1):25-36. ernst ds, ewin b, klaus mh. 2002. plant ecology. heidelberg (de): karl-heinz winter. fitriana yr. 2006. the diversity and abundance of macrozoobenthos in the mangrove forest as a result of the rehabilitation of the ngurah rai forest park, bali. biodiversitas 7(1):67-72. gunawan w, sambas b, andry i, lilik bp, herwasono, s. analysis of vegetation structure and composition toward restoration efforts of gunung gede pangrango national park. jp sl 1(2): 93-105. handoyo f, ramadhani s. 2006. native orchids of indonesia. jakarta (id): indonesian orchid society. indriyanto. 2006. forest ecology. jakarta (id): pt bumi aksara. indarto n. 2011.orchid charm practical instruction for orchid cultivation and business. yogyakarta (id): cahaya atma pustaka. maguran ae. 1988. biological diversity and its measurement. princeton (usa): princeton university press. odum ep. 1993. fundamentals of ecology 3rd edition. yogyakarta (id): gadjah mada university press. odum ep. 1996. fundamentals of ecology. yogyakarta (id): gadjah mada university press. onrizal ck, suharjo bh, iin hp, kato t. 2005. vegetation analysis of secondary lowland tropical forest in lake sentarum national park, west kalimantan. biology 4(6):359-72. paramitha igaap, i gede pa, made p. 2010. diversity of epiphytic orchids in the buyan-tamblingan lake natural tourism park area. journal metamorfosa 1(1):11-6. prapitasari b, ardyan pk, dadi hm. 2020. diversity and abundance of orchid species at the selabintana resort, mount gede pangrango national park, west java. biosfer j bio & pend bio 5(1):24-30. purbadi, widyastuti d, kartikaningrum s. 2005. the effect of media on the growth of dendrobium orchid plantlets. journal hortikultural 15(1):18-25. purwanto aw. 2016. orchids: cultivation and propagation. yogyakarta (id): lppm upn veteran yogyakarta. puspitaningtyas dm. 2005. study of orchids diversity in the simpang mountain nature reserves, west java. biodiversitas 6(2):103-7. rikardus, hari p, hafiz a. 2017. analysis of the diversity of natural orchids species in the protected forest of mount semahung, saham village, sengah temila subdistrict, landak district. journal hutan lestari 5(2):292-9. shuttleworth fs, zim hs, dillon gw. 1970. a golden guide orchids. new york (us): western publishing company inc. tahier ss, tri sh, sri w. 2018. orchid diversity in the telaga warna nature reserve park, puncak, bogor. ekologia: journal basic and environmental sciences 18(2):83-93. tirta ig, sutomo. 2014. orchid inventory at eka karya botanical garden. widyariset 17(2): 245-50. wahyuni as, lilik bp, ervizal amz. 2017. population and distribution pattern of paliasa plant (kleinhovia hospita l.) in the bontobahari district. conservation media 22(1):11-8. world conservation monitoring centre. 1995. indonesian threatened plants. exploration 2(3):8-9. wulanesa wos, soegianto a, basuki n. 2017. exploration and characterization of epiphytic orchids in the coban trisula forest, bromo tengger semeru national park area. journal crop production 5(1):125-31. yulia dn. 2008. inventory and habitat characteristics of dendrobium capra j.j. in madiun and bojonegoro. biodiversitas 9(3):190-3. zulkaidhah, sri nm, ferdiansah. 2018. diversity of natural orchids species in lembantongoa village, palolo district, sigi regency. j forest sains 15(2): 58-66. potential of mangrove seedlings for utilization in the maintenance of environmental quality within silvofishery ponds endah dwi hastuti* and rini budihastuti faculty of science and mathematics, diponegoro university, semarang , indonesi 50275 a received 10 january 2016/accepted 24 june 2016 abstract silvofishery system ha been applied to aquaculture activities and it has been developed in the coastal area of s semarang city, indonesia. however, information on the initial development of silvofishery ponds concerning the functionality of mangrove seedlings on environmental quality of fishponds had not been studied. this experiment aimed to determine the environmental conditions of silvofishery ponds and to analyze the effect of seedling stands of mangrove on environmental quality control. he presence of mangrove seedlings temperature t caused the decrease of and salinity. anova showed that mangrove species significantly affected water salinity, while canal the increase of width and mangrove species significantly affected turbidity and ph regression analysis showed . that the height of rhizophora mucronata partially significant effect on otal uspended lids (tss), rganic atter (om), had t s so o m as well as n p concentrations. d a ed . the height and itrogen (n) and hosphorus (p) iameter of ffect temperaturer. mucronata diameter of a ed dissolved oxygen (do) savicennia marina a. marina r. mucronata ffect . mixed population of and had an effect on water turbidity, while population of only had a partial effect on water salinity. a. marina r. mucronata seedlings had dominant effect on the environmental quality. mangrove seedlings can be used as environmental quality control within silvofishery ponds to maintain optimal conditions for fish growth. the application of silvofishery more abundant in early stage of mangrove seedlings should consider the plantation of compared to r. mucronata a. marina. : anal width environmental quality seedling silvofishery species compositionkeywords c , , , , introduction the unsustainable utilization pattern of coastal areas has caused environmental damage (primyastanto 2010), and the most affected et al. sectors by the damage is pond aquaculture activities (pramudyanto 2014). however, the development of beach and upland areas also ha s contributed to degradation of environmental quality (vatria 2010). one of the applied methods to maintain the sustainability of pond culture activity is the silvofishery culture system (surtida 2000). silvofishery is an aquaculture system which combines mangrove trees with shrimp/fish ponds. the integration of mangrove stands within silvofishery ponds are expected to improve the environmental quality and increase the carrying capacity of the system (wibowo & handayani 2006). according to suwarto et al. (2015), the existence of mangrove vegetation in ponds will improve primary productivity as well as assimilation capacity of pond effluents. several silvofishery pond models have been applied in many regions including , komplangan empang parit empang parit and enhanced (bengen 200 ). all three models integrate the mangrove 2 community into the pond area (inlet/outlet). the function of mangroves within the ecosystem is to provide nutrients through nutrient trapping and litter production and to absorb pollutants. mangrove should grow in both the inlet area as pollutant absorbers and in the outlet area to neutralize the pond effluent. however, the biotropia 3 1 6 63 vol. 2 no. , 201 : 58 doi: 10.11598/btb.2016.2 . .3 1 606 * c orresponding author: endah_pdil@yahoo.com 58 mailto:endah_pdil@yahoo.com application of a mangrove community in both inlet and outlet of silvofishery systems had not been developed. the potential utilization of mangrove in environmental quality control within aquaculture ponds through the application of silvofishery is expected to decrease the risk of aquaculture activity as well as to improve pond productivity (lewis iii gilmore 2007). however, the & optimal function of mangrove stands to the environment may not be achieved until trees are mature. in the meanwhile, the growth of mangrove from seedling stage to tree require a s long period of time. the influences of mangrove seedlings planted in plantations on the quality of pond seedling environment ha received very little attention. ve although the effect of mangrove seedlings on the control of environmental quality is not hig ly h significant, the existence of mangrove seedlings should provide certain effects on water circulation pattern as well as absorption of nutrients or pollutants. thus the role of mangrove seedlings , in silvofishery ponds, especially in early plantations needs to be studied. semarang city is a region in indonesia that has experienced ecological disturbance from unsustainable development activities in the past as well as regional development to leading environmental stress in coastal areas. increase pond culture occupying silvofishery system had been applied in semarang city, but it is not optimized to support the productivity of aquaculture. aimed to determine this experiment the environmental conditions of silvofishery ponds and to analyze the effect of seedling stands of mangrove on environmental quality control. materials and methods the exper iment was conducted in mangunharjo village, tugu district, semarang city , central java province, indonesia from march to september 2015 by planting mangrove seedlings in silvofishery pond at the inlet and s outlet canals. aterials m used were height and diameter of as well as water and seedling stands sediment quality parameters representing environmental quality i.e. temperature, turbidity, salinity, ph, issolved xygen (do), otal d o t s so o m as uspended lids (tss), rganic atter (om), well as n p itrogen (n) and hosphorus (p) concentrations. experimental design involved treatments of canal widths and mangrove compositions. canal widths were 1 2 nd 3 m anal length was 5 m , a . c for all treatments he size of culture ponds were . t 5 x 5 m with 1.5 m depth. the canals were build 2 on both sides of the ponds as inlet and outlet canals. composition of the mangrove s seedling were (a) : 1. ; 2.avicennia marina rhizophora mucronata ; 3. mangrove (r) or a mixture of the two species among (m). the plantation space seedling stands was 1 x 1 m , so the number of mangrove 2 stands for each treatments were 5 stands (1 m – l1); 10 stands (2 m l2) and 15 stands (3 m – l3). the experiment was conducted with 3 replications. design is the of the experiment shown in table 1 and the diagram of the experiment is shown in figure 1. and observations data collections were conducted every 3 months i.e. in march, june and september 2015. d ed were: 1. ata collect water quality temperature, turbidity, parameters i.e. salinity, ph, do issolved xygen and tss (d o ) table 1 design of the experiment seedling composition canal width 1 m 2 m 3 m avicennia marina l1-a l2-a l3-a rhizophora mucronata l1-r l2-r l3-r mixture of both mangrove species l1-m l2-m l3-m 59 p maintaining endah dwi hastuti .otential of mangrove seedlings for environmental et al –quality in silvofishery ponds (t s so ); and 2.otal uspended lids sediment quality om rganic atter , parameters i.e. (o m ) as well as (n ) (p ) n itrogen and p hosphorus concentrations. data processing was conducted to determine the impact caused by mangrove stands on the environmental quality represented by water and sediment quality parameters value changes f in the inlet and outlet canals. actorial anova and regression multiple analysis were used to analyze the collected data. factorial anova was conducted to analyze the impact of treatment involving the combination of canal s widths and mangrove composition on water and sediment quality parameters. multiple regression analysis was conducted to analyze the influence of seedling variations (including the combination of population, measurements and species compositions) on the changes of value environmental quality parameter . multiple s regression involved independent s analysis : 1. variables for respective mangrove compositions i.e. seedling height, seedling diameter and population of mangrove seedling particularly for a. marina r. mucronata; and 2. and dependent variables changes of temperature, turbidity, i.e. salinity, ph, do, tss, om, n and p concentrations. results and discussion data collected showed changes in water and sediment quality parameters in the silvofishery ponds (table 2). consistently water temperature decreased from first to the third observation the s, while salinity consistently increased other parameters . did not show any specific pattern of changes. the observed water and sediment quality parameters within the inlet and outlet canals of silvofishery pond showed the decrease and increase of parameters values, before and after passing the canal with mangrove stands. the changes of water and sediment quality parameters value within the canals varied among treatments. this experiment showed the value changes on water and sediment quality parameters within the pond canals i.e. changes ranged temperature from (-)5.5 (+)6.4 °c; turbidity to ranged from to changes ranged (-)710 (+)769 ntu; salinity from to ranged from to (-)6.3 (+)6.1‰; ph (-)6.1 (+)6.2; do (-)4.1 (+)4.5 mg/ ; ranged from to l tss (-)343.2 (+)509.4 mg/ ; om ranged from to l ranged from to concentration (-)2.7 (+)2.5%; n ranged from to (-)0.7 (+)0.7%; and p concentration ranged from to (-)50.9 (+)52.8 figure 1 xperiment diagram of the e ponds mangrove silvofishery widths (1 m; 2 m; 3 m) compositions (avicennia marina; rhizophora mucronata; mixture) experiment environment quality (entrance) inlet environment quality (inside) outlet environment quality (exit) maintenance / enhancement capability 60 biotropia vol. 23 no. 1, 2016 ppm. the changes indicated that water and sediment quality changed during the 3 observation periods. the range of parameter value varied among periods.s observation height and diameter measurements on mangrove seedling stands also showed variations among the 3 observation periods. height of a. marina stand ranged from 41 106 cm; 43 to from to 101 cm; and 39 106 cm with from to stand diameter range of 0.20 – 0.89 cm; 0.32 – 0.88 cm; and 0.15 – 1.94 cm for the first, second and third observations . height of , respectively r. ucronata m stand ranged from 28 60 cm; 24 76 cm; to from to and 38 78 cm with stand diameter range from to of 0.22 – 1.90 cm; 0.30 – 1.32 cm; and 0.55 – 2.36 cm , for the first, second and third observations respectively decreased height and . seedling diameter f each mangrove species was caused by o the mortality of seedlings, hence seedlings replacements were conducted several times. data analysis with anova to measure the effect of canal width and mangrove compositions (treatments) on water and sediment quality parameters that several parameters were showed significantly affected by the treatments, such as mangrove composition affected salinity, canal width and mangrove composition affected turbidity and ph. s effectignificant on water salinity was achieved from silvofishery pond canals with and . while a. marina r. mucronata significant water turbidity w achieved effect on as from different canal widths and from different mangrove seedling species. si effectgnificant on ph was achieved from combination of canal width and mangrove seedling species. multiple regression a to determinenalysis the effect of mangrove stands on the water and sediment parametersquality of silvofishery pond showed there were significant effect of that s mangrove stand on several observed parameters (table 3) . data analysis was conducted partially for each mangrove composition structure as well as water and sediment quality . parameters parameterschanges in environmental quality within silvofishery pond canals were dominantly influenced by seedlings of (table ). r. mucronata 3 parameters influenced by partially r. mucronata stands included change in tss concentration, s temperature, om concentration, n concentration and p seedlings concentration. combination of and r. mucronata a. marina influenced water turbidity and do concentration. a. marina seedlings influenced water salinity. r. mucronatathere was significant effect of on tss concentration. according to furukawa and eric (1996), mangrove stands can function as sediment trap. sediment trapping processes by mangrove stands begins with the slowing down of water current, and this leads to the accumulation of tss which finally gravitate . s also, sediment trapping is influenced by the tide condition (kathiresan 2003). smangrove stand ha negative effect on the change of temperature, which means that as the height and diameter of mangrove stands increase, water temperature decrease . according to s hadikusumah (2008), mangrove vegetation is capable of bsorbing heat. the photosynthetic a capability of mangrove seedling of rhizophora occur in the leaves and green stems, hence as the s table 2 changes in water and sediment quality parameters observed during the experiment value no. parameter observation i observation ii observation iii 1. temperature (°c) 34.2 31.4 30.6 2. turbidity (ntu) 379.7 313.0 353.7 3. salinity (‰) 22.0 31.0 39.8 4. ph 7.49 9.26 8.11 5. do (mg/l) 6.56 6.99 5.72 6. tss (mg/l) 411.37 492.04 218.00 7. om (%) 1.64 1.74 1.62 8. n (%) 0.53 0.55 0.55 9. p (ppm) 33.86 39.68 34.44 61 p maintaining endah dwi hastuti .otential of mangrove seedlings for environmental et al –quality in silvofishery ponds stand height and diameter increase the amount of photosynthetic surface increases. water turbidity was affected significantly by both and . eedling stands a. marina r. mucronata s of inhibited water flow which r. mucronata increased the concentration of suspended sediment (yang 2013). , et al. on the contrary a. marina seedlings had negative effect on the change of water turbidity. according to weiffen et al. (2006), turbidity could be formed by the increasing population of plankton caused by the accumulation of sediment within the canal. the consumption rate of dissolved nutrient by a. marina probably was the cause of its negative effect on the water turbidity. a water salinity was ffected significantly by a. marina r. mucronata by but not , due to higher evaporation capacity of than a. marina r. mucronata. evapotranspiration within a pond is able to decrease water concentration, while salt is excreted back to the environment through the leaves (ball . 1988). hence, the concentration et al of salt (salinity) increase .d concentration of do was negatively affected by height of both and , but a. marina r. mucronata was positively affected by diameter of . a. marina stand height of mangroves were suggested to affect the canopy coverage which leads to the decreased light penetration (kennedy 2002). et al. as the mangrove stand increase , so does height s canopy coverage. , mangrove on the contrary seedlings still have chlorophyll in the stem, which means they are able to conduct photosynthesis. photosynthesis has positive effect o seedling n diameter and on the increas dissolved oxygen e of concentration. nutrient concentration, including om, n and p within the sediment was significantly affected by the height of , but not . r. mucronata a. marina nielsen and andersen (2003) stated that nutrient accumulation by is higher than that r. mucronata of . in the seedling stage, the capability a. marina to accumulate nutrients is related to diameter size of mangrove stand which is generally larger for r. mucronata a. marinafor herefore, than stands. t r. mucronata has higher capacity to inhibit water flow and influence the accumulation rate of sediment which binds nutrients than more a. marina. conclusions silvofishery pond canals random showed pattern of parameter environmental quality s value changes of turbidity, ph, do, tss, om, n and p emperature consistently decreased . t was table effect of angrove tands on hanges of uality within anals of ilvofishery 3 m s c water and sediment q parameters c s p sond no. mangrove composition independent variable dependent variable equation 1. single height of r. mucronata (x1) tss y = 524.574 – 8.483(x1) 2. mixed diameter of r. mucronata (x1); height of r. mucronata (x2) temperature y = -1.091 + 0.057(x1) – 1.799(x2) 3. mixed population of a. marina (x1); population of r. mucronata (x2) turbidity y = -81.627 – 209.753(x1) + 213.887(x2) 4. mixed population of a. marina (x1) salinity y = -0.432 + 0.103(x1) 5. mixed height of a. marina (x1); diameter of a. marina (x2); height of r. mucronata (x3) do y = 2.127 – 0.046(x1) + 7.410(x2) – 0.050(x3) 6. mixed height of r. mucronata (x1) om y = -1.796 + 0.050(x1) 7. mixed height of r. mucronata (x1) n y = -0.587 + 0.017(x1) 8. mixed height of r. mucronata (x1) p y = -41.303 + 1.142(x1) 9. mixed height of r. mucronata (x1) tss y = -248.833 + 6.794(x1) 62 biotropia vol. 23 no. 1, 2016 and salinity consisten ly increased. mangrove was t seedling stands for or had a. marina r. mucronata significant effects on the changes of environmental quality involving parameters vari combinations on temperature, turbidity, ous salinity, do, tss, om, n and p. pecies that had s most effect on environmental quality parameters value changes was r. mucronata. acknowledgements t d thehe authors acknowledge director of research and community services (ditlitabmas), general directorate of higher education (ditjen dikti), ministry of education and culture, government of indonesia for financial support. references ball mc, cowan ir, farquhar gd. 1988. maintenance of leaf temperature and the optimisation of carbon gain in relation to water loss in a tropical mangrove forest. aust j plant physiol 15:263 76. bengen dg. 2002. technical guide on the ntroduction and i m m eanagement of angrove cosystem. center bogor (id): of coastal and marine resources studies, ipb. furukawa k, eric w. 1996. sedimentation in mangrove forest. mangrove salt marshes 1(1):3 10. hadikusumah. 2008. characteristics of physical parameters and chlorophyll-a concentration in java sea. j ilmu kelautan 13(2):103-12. kathiresan k. 2003. how do mangrove forests induce sedimentation? revista de biologia tropical 51:35560. kennedy vs, kleypas ja, cowan jr jh, hare sr. 2002. coastal and arine ecosystems & lobal climate changem g . us: pew center on global climate change. lewis iii rr, gilmore rg. 2007. important considerations to achieve successful mangrove forest restoration with optimum fish habitat. bull mar sci 80(3): -823 37. nielsen t, andersen fo. 2003. phosp orus dynamics h during decomposition of mangrove (rhizophora apiculata) leaves in sediments. j exp mar biol ecol 293:73 88.pramudyanto b. 2014. pollution and degradation control in coastal areas. jurnal lingkar widyaiswara 1(4):2140. primyastanto m, dewi rp, susilo e. 2010. the environment destructive habit by coastal society in slamic i perspective (case study on the fishermen and fish traders in tambak beach area, tambakrejo village, wonotirto district, blitar region east java). jurnal pembangunan dan alam lestari 1(1):1-11. surtida mb. 2000. silvofisheries in indonesia. seafdec asian aquacult 22(6):20-1. suwarto, lahjie am, ruchaemi a, simorangkir bdas, mulyadi f. 2015. ecological aspect of non productive fishponds at mahakam delta area: revitalization with silvofishery system. g jar 3(1):27 37.vatria b. 2010. various human activities which lead to coastal ecosystem degradation and its impacts. journal of berlian 9(1):47-54. weiffen m, moller b, mauck b, dehnhardt g. 2006. effect of water turbidity on the visual acuity of harbor seals ( ). vision research phoca vitulina [internet]. 46:1777–83. doi:10.1016/ available from: j.visres.2005.08.015. wibowo k, handayani t. 2006. conservation of mangrove forest through silviculture (silvofishery). jurnal teknik lingkungan 7(3):227-33. yang j, gao j, cheu a, liu b, schwendenmann l, costello mj. 2013. vegetation and sediment characteristics in an expanding mangrove forest in new zealand. estuarine, coastal and shelf science [internet]. 134:1–18. http://dx.doi.org/available from: 10.1016/j.ecss.2013.09.017. 63 p maintaining endah dwi hastuti .otential of mangrove seedlings for environmental et al –quality in silvofishery ponds http://dx.doi.org/available biotropia vol. 30 no. 3, 2023: 384 395 doi: 10.11598/btb.2023.30.3.2027 384 evaluation of methods for total rna extraction from the endosperm of cocos nucifera var. makapuno in vietnam for molecular analysis thanh-diem nguyen and huyen-trang vu* 1faculty of biotechnology, nguyen tat thanh university, 298a-300a nguyen tat thanh street, ward 13, district 4, ho chi minh 72820, vietnam received 25 may 2023 / revised 15 august 2023 / accepted 15 august 2023 abstract sap coconut (cocos nucifera l. var. makapuno) in vietnam is a mutant coconut variant; coconut water is in the state of lotus glue, and coconut rice is like cream. for high-quality transcriptome, sequencing, quality, purity and concentration of rna are the key factors. however, coconut endosperm tissue has higher stiffness and fatness than the leaf tissue, which complicates the extraction process. moreover, rna is much more difficult to preserve than dna. in this study, various rna extraction methods were examined in vietnamese waxy coconut endosperm tissue samples. optimum extraction and preservation of the rna using the simplest possible chemicals was the objective of this study. the modified ctab method with licl and the tri reagent method were tested and evaluated. the purity, concentration and quality of rna after storage were improved. the findings indicated that the trisure extraction method with the addition of nacl and β-mercaptoethanol yielded optimum rna quality. the rna concentration was 159 ng/µl, with a purity ratio of 1.94 ± 0.04 for a260/a280 and 1.58 ± 0.02 for a260/230. rna samples remained stable for up to 3 weeks when stored in absolute ethanol at 8°c–10°c, which significantly reduced their degradation during transportation. this study facilitated the use of simple chemicals for high-quality rna extraction from coconut endosperm and its preservation for applications in high throughput sequencing. keywords: cocos nucifera var. makapuno, extraction method, total rna introduction coconut (cocos nucifera l.) is a tree with high economic value, and coconut water is a beverage that is preferred by many people. coconut is also an important perennial oil crop around the world. in particular, the sap coconut variant originating from the philippines, called makapuno coconuts, is considered a specialty, with the coconut sap in a state of gelatinous creamy paste and high in fat content. interestingly, there are normal fruits with solid endosperm like other coconut variants, and some fruits contain waxy endosperm on the same coconut tree (arellano et al. 2019). rna isolated from tissue samples is important for several research projects. ribosomal rna sequencing is often used for the identification, classification, or assessment of biodiversity. furthermore, messenger rna (mrna) is used for gene expression research, novel gene discovery, or gene isoform analysis. short fragments can be sequenced to assess the expression of certain genes, or whole mrnas can be encoded for comparative transcriptomics. mrna is used in reverse transcription pcr (polymerase chain reaction) to synthesise cdna for sequencing, which helps identify gene variants and is also useful in gene expression studies. in addition, mrna is used in northern blotting to analyse gene expression over time or under specific conditions based on the size and amount of mrna. however, for rna sequencing, reverse transcription pcr and quantitative realtime pcr, an intact rna with high purity is essential. *corresponding author, email: methods for the extraction of total rna from coconut endosperm – thanh-diem nguyen and huyen-trang vu 385 extraction of rna from coconut endosperm tissue for mrna transcriptome sequencing helps to evaluate the differences in gene expression between waxy and normal endosperm tissues, thereby aiding in future fruit characterisation studies. however, obtaining high quality rna from endosperm tissue is more challenging than obtaining it from leaf tissue. the reason is that coconut endosperm possesses numerous polysaccharides, proteins, polyphenols, and lipids that bind to nucleic acids, thereby posing a challenge in the isolation of rna (angeles et al. 2005, zhang et al. 2022). moreover, sequencing results are affected owing to inadequate preservation during the transportation process over an extended period. hence, obtaining of rna samples that are highly concentrated and pure is of utmost importance in ensuring the success of rna sequencing and genetic analysis (gallego romero et al. 2014, scholes et al. 2020). several studies have been performed to enhance the rna separation techniques to facilitate subsequent molecular investigations. most of these studies were based on guanidine thiocyanate as the main lysate. in 2012, mrip (methods for rna isolation from palms) extraction buffer, which contains a series of different components, i.e. ammonium thiocyanate, guanidine thiocyanate, sodium acetate, phenol and glycerol, was first used for rna isolation from palm leaves. higher purity and integrity of the rna molecules were achieved in comparison with ctab, trizol methods and tiangen rna plant kit. the electrophoresis results from mrip showed clear lines of 28s, 18s and 5s rrna whereas the other techniques did not. nonetheless, the concentration of the rna product from each method was not discussed and the study was performed on the leaf only. subsequently, iqbal et al. (2019) provided an improved method, qrrem (quick and reliable rna extraction method), which was similar to mrip in all aspects except for the addition of βmercaptoethanol (2%) and polyvinylpyrrolidone40 (3%). intact rna was efficiently isolated, and high rna concentration (16.2 µg/80 mg) was obtained from stored coconut endosperm compared with ctab (0.1), trizol (10.4) and rna plant kit (0.5). this method had many advantages over other methods in terms of time and cost efficiency (iqbal et al. 2019). later, in 2020, the research group of iqbal et al. proposed another improved rna extraction method, ircm (isolation of rna from complex matrices), by halving the concentrations of βmercaptoethanol (1%) and polyvinylpyrrolidone40 (1.5%) in comparison with qrrem. the protocol was applied for coconut endosperm, coconut mortise and coconut buds, and the results were higher when compared with ctab, trizol and plant rna extraction kit (iqbal et al. 2020). in all the above methods, the main lysis component was guanidine thiocyanate. although it was more effective than ctab and trizol, the components were quite complex and acquiring the products from the suppliers was a time consuming process, which sometimes affected the progress of the project. however, tri reagents, the major component of which is guanidine thiocyanate, are often accessible from vendors, making them more convenient to purchase. contrary to guanidine thiocyanate-based methods, souza-perera et al. (2018) enhanced the ctab extraction method for rna from various tissue samples, such as leaves, inflorescences and primary and secondary roots based on the ctab extraction protocol for dna. this study examined the characteristics of grade, zygote embryos, and solid endosperm in mature coconut trees. the authors successfully isolated rna from coconut endosperm tissue with higher integrity compared with the trizol method and without any carbohydrate or protein contamination (souza-perera et al. 2018). therefore, various rna isolation methods should be evaluated in coconut endosperm tissue samples and optimised to obtain high quality rna. this study aimed to develop a protocol for the extraction, preservation, and transportation of total rna to minimise rna quality loss. materials and methods collection of coconut tissue samples throughout the study, the waxy endosperm (we) sample from coconut was the main target for the investigations, i.e. evaluation of rna extraction efficiency, purity improvement test, lysis temperature test and rna preservation test during transportation. particularly, in the biotropia vol. 30 no. 3, 2023 386 evaluation of rna extraction efficiency of the two methods ctab-licl and trisure, additional samples of non-waxy endosperm (nwe) tissues and leaf (l) tissues from the same coconut cultivar were used as comparison controls. all samples were collected from tra vinh province, vietnam. the morphology of coconut shells and skulls of waxy and non-waxy fruits indicated no discernible differences in their shape or coloration (figure 1). the only external difference between the two types of fruits was the characteristic sound upon shaking. the waxy fruit made a rattling sound owingto its thick, viscous, overdeveloped endosperm. however, this sound may also arise during the growing stages of nonwaxy fruit. hence, precise differentiation is possible only by identifying the endosperm tissues. the endosperm of waxy coconut is pliable and contains colloidal water and a disorganised tissue arrangement. in contrast, non-waxy fruit exhibits high water content and a dense and rigid endosperm. therefore, for waxy samples, sterile spoons were used to collect viscous endosperm from the non-cut contact area, which was then promptly transferred to labelled sterile bags. the non-waxy endosperm and the leaf tissue were prepared by partially cutting and cleaning them with distilled water and 70% alcohol. after drying, the samples were aseptically transferred to labelled sterile bags. all pretreated samples were promptly utilised or preserved at −80°c. extraction of total rna guanidine thiocyanate and ctab methods have been extensively evaluated but have shown varying results in different studies. hence, in this study, these two approaches were tested for highperformance rna extraction from the coconut endosperm tissue. as the composition of lytic compounds is quite complex in ircm and qrrem and orders for these chemicals are time consuming, the main lysate guanidine thiocyanate via the trisure reagent was used with some improvements. the total rna extraction methods investigated included ctab-licl (chang et al. 1993, ghangal et al. 2009, white et al. 2008) and trisure (thermo fisher 2010). after assessing the extraction efficiency, several follow-up investigations were conducted to improve the quality of the rna product. for enhancing the purity of rna, the addition of nacl and β-mercaptoethanol to the lysis process was tested and the rna washing step was repeated thrice. for increasing the efficiency of tissue resolution, different incubation temperatures (room temperature, 50°c, 55°c and 60°c) were used during the lysis process. as rna is easily degraded, it was stored under conditions similar to transportation conditions to determine its degradation time. each treatment was performed in triplicate. figure 1 coconut samples collected in the study notes: top row: non-waxy coconut; bottom row: waxy coconut. from left to right: unpeeled fruit; fruit skull, endosperm samples in petri dish. methods for the extraction of total rna from coconut endosperm – thanh-diem nguyen and huyen-trang vu 387 investigation of the preservation process under transportation conditions rna is inherently unstable and prone to degradation. this experiment aimed to examine the preservation of total rna post-extraction. two methods were used to preserve total rna: (i) total rna was stored at −20°c in diethylpyrocarbonate (depc)-water after dissolution. (ii) total rna was mixed with 0.1× volume of 3m sodium acetate ch3coona (ph 5.5) and twice the volume of 100% ethanol after dissolution in depc. it is a chemical compound that inactivates rnase, the enzyme that degrades rna. depc is used to preserve rna integrity in laboratory settings during various experimental procedures. meanwhile, sodium acetate acts as a co-precipitant in the presence of ethanol. when added to an rna sample, it neutralises the negative charges on the rna molecules, facilitating their aggregation and precipitation, which in turn condenses the rna structure and resists degradation. both methods are often used for rna preservation. rna samples were then stored at −20°c. the examination was performed over a period of 1 month, with weekly testing. quality assessment of the extracted rna for transcriptome sequencing, the concentration and purity of rna are of primary concern. rna purity and total electrophores concentration were checked using the nanodrop™ thermo fisher scientific spectrophotometer at wavelengths a260, a280 and a230. for illumina sequencing, the minimum required weight is 500 ng and concentration should be >10 ng/µl (hong et al. 2020). as for purity, the a260/a280 ratio should be in the range of 1.8–2.0 and a260/a230 should be between 2.0 and 2.2. electrophoresis was also used to assess the relative concentration and integrity of rna bands of different sizes. the extracted rna samples were electrophoresed on a 2% (w/v) agarose gel together with a dna scale. however, the size of the mrna varied greatly depending on the length of the corresponding gene; hence, the number or position of the lanes on the electrophoresis gel was not evaluated. the dna scale preliminarily assessed the length of the rna lines that appeared. brighter the bands, higher the concentration of rna; clear bands represented intact rna molecules of certain lengths, whereas smeared bands represented different sizes of rna, which could be attributed to degradation. to enhance the objectivity while analysing the electrophoresis images, the gelanalyzer 19.1 software (lazar jr. et al.) was used in combination. statistical analysis the data obtained from the above experiments were statistically analyzed using the minitab 17 software (minitab inc. 2020). the data were statistically evaluated using anova with the turkey algorithm. results and discussion evaluation results of rna extraction procedures using ctab-licl and trisure the we sample was used as the main target for investigating the effects of the two extraction methods ctab-licl and trisure on the quality of the extracted rna. in addition, the nwe and l samples from the same plant were included as control samples. in total, three types of tissue samples were used for the two extraction methods. each treatment was repeated thrice for calculating statistical reliability. with regard to the total rna concentration, the results showed the statistical difference between the two methods (p value < 0.05) in all types of tissues. the total rna concentrations obtained from we tissue samples were 142.00 ± 2 ng/µl and 157.00 ± 1.7 ng/µl for the ctablicl and trisure methods, respectively (table 1). in both methods, there was no statistically significant difference between we and nwe samples. however, both endosperm tissues (statistically classified as b) had significantly lower extraction efficiency than leaf tissues (statistically classified as a), and the difference was significant (p value < 0.05) (table 1). there was no interaction between the two factors and tissue types [p value > 0.05 (0.422)]. biotropia vol. 30 no. 3, 2023 388 table 1 total rna concentrations extracted using ctab-licl and trisure extraction method tissue type waxy endosperm sample (we) non-waxy endosperm sample (nwe) leaf sample (l) ctab-licl 142.00 ± 2bb 142.33 ± 1.5bb 155.00 ± 2ba trisure 157.00 ± 1.7ab 156.67 ± 0.5ab 167.67 ± 0.5aa p value (extraction method) 0 (<0.05) p value (tissue type) 0 (<0.05) p value (two-factor interaction) 0.422 (>0.05) note: two-way anova analysiswas used to investigate the influenceeffects of different extraction methods (ctablicl versusvs trisure) and different tissue types (waxy endosperm, non-waxy endosperm and leaf tissue) on rna product concentration. the lowercase letters a and b: represent the differences between the extraction methods; the uppercase letters a and b represent the differences between the tissue types. the differences are significant when the p value is <0.05. as for the od assessment to determine sample purity, both ctab-licl and trisure methods yielded an a260/a280 od ratio within the optimal range of 1.8–2.0 (table 2). in contrast, the od a260/a230 ratio exhibited a distinct disparity. samples extracted with ctab-licl had a ratio of 1.8–1.9, whereas those extracted with trisure had a significantly lower ratio of 0.99–1.02 (table 3). the optimal purity range for a260/a230 is 2.0–2.2. macrogen company does not necessitate highquality samples for sequencing as the issue can be resolved by purifying the sample before sequencing. the index of samples extracted using ctab-licl was acceptable at 1.8–1.9, despite not reaching 2.0–2.2. on the contrary, the a260/a230 ratio of trisure-extracted samples fell very much below the standard threshold and should be improved. table 2 a260/a280 ratio of samples extracted using ctab-licl and trisure extraction method tissue type waxy endosperm sample (we) non-waxy endosperm sample (nwe) leaf sample (l) ctab-licl 1.99 ± 0.03 aa 1.99 ± 0.01 aa 2.00 ± 0.05aa trisure 1.99 ± 0.01 aa 2.00 ± 0.03 aa 2.01 ± 0.01 aa p value (extraction method) 0.482 (>0.05) p value (tissue type) 0.526 (>0.05) p value (two-factor interaction) 0.928 (>0.05) note: two-way anova was used to investigate the effects of different extraction methods (ctab-licl vs trisure) and tissue types (waxy endosperm, non-waxy endosperm and leaf tissue) on the a260/a280 ratio of the rna product. the lowercase letters a and b represent the difference based on the extraction method; the uppercase letters a and b represent the difference based on the tissue type. the differences are significant when the p value is <0.05. table 3 a260/a230 ratio of samples extracted using ctab-licl and trisure extraction method tissue type waxy endosperm sample (we) non-waxy endosperm sample (nwe) leaf sample (l) ctab-licl 1.87 ± 0.005 aa 1.89 ± 0.01 aa 1.87 ± 0.02aa trisure 1.01 ± 0.03 ba 0.99 ± 0.02 ba 1.02 ± 0.06 ba p value (extraction method) 0.00 (<0.05) p value (tissue type) 0.936 (>0.05) p value (two-factor interaction) 1.87 ± 0.02aa note: two-way anova was used to investigate the effects of different extraction methods (ctab-licl vs trisure) and tissue types (waxy endosperm, non-waxy endosperm and leaf tissue) on the a260/a280 ratio of the rna product. the lowercase letters a and b represent the difference based on the extraction method; the uppercase letters a and b represent the difference based on the tissue type. the differences are significant when the p value is <0.05. methods for the extraction of total rna from coconut endosperm – thanh-diem nguyen and huyen-trang vu 389 in electrophoretic separation, rna samples extracted using ctab-licl exhibited limited bands, including two 23s rrna subunits at approximately 2900 bp, 18s rrna subunits at approximately 1900 bp and various other bands at approximately 250–500 bp (thermo fisher 2006) (figure 2). on the contrary, electrophoretic separation of rna samples extracted using the trisure method displayed multiple bands in the range of 250–3000 bp in addition to the 23s and 18s rrna bands, and these bands were more distinct and intense. to evaluate the results of electrophoresis more objectively, a combination of electrophoretic images and the image analysis software gelanalyzer was used. a significant difference was observed (figure 3). according to the analysis results, rna samples extracted using the trisure reagent had clearer peaks and were better in terms of quantity than those extracted using ctab-licl (seven peaks vs two peaks). figure 2 results of electrophoresis on 2% agarose gel with a scale of 10 kbp; we: waxy endosperm, nwe: non-waxy endosperm, l: leaf tissue figure 3 results of electrophoretic analysis using the gelanalyzer software note: intensity axis: brightness level based on background; pixel axis: coordinate position on the electrophoresis membrane. the blue area shows the location of the marked rna bands. biotropia vol. 30 no. 3, 2023 390 the results indicated that the concentration of total rna in leaf tissue samples was greater than that in endosperm tissues, specifically in we and nwe. this finding is consistent with those from previous studies (iqbal, yang, qadri, wu, li, shah, hamayun & hussain 2019, iqbal, yang, wu, li, hamayun, hussain & shah 2020, souzaperera et al. 2018). coconut endosperm tissue is hard and has a high lipid content, making it harder to grind than the leaf sample. furthermore, endosperm is a significant component of seeds and comprises tissues that contain high levels of various polysaccharides (such as starch, cellulose, arabinoxylan, β-glucan, and fructan), proteins, lipids, and secondary metabolites (angeles, laurena & tecsonmendoza 2005, ghangal, raghuvanshi & chand sharma 2009, zhang et al. 2019, liu et al. 2018). most of these components share structural similarities with nucleic acids, including polysaccharides that resemble the ribose sugar of rna. consequently, these compounds can bind to rna and undergo degradation. low rna extraction yield was observed because of its encapsulation during centrifugation and its subsequent removal during phase separation using chloroform (white, venter, hiten & burger 2008, li et al. 2005, wang et al. 2012). in addition, small polysaccharide molecules were distributed in the liquid phase during phase separation and co-precipitated with rna, which reduced the rna yield and affected subsequent applications (liu, han, yu, zhang, xing, xie & peng 2018, li & trick 2005, wang et al. 2010). furthermore, a low a260/a230 ratio was detected, which indicated potential guanidine thiocyanate contamination as inferred from the warning messages from the meter. this component was present as a key element in the trisure mixture during the extraction procedure (iqbal, yang, wu, li, hamayun, hussain & shah 2020). particularly, for coconut endosperm tissue, rna extraction yielded mixed results in previous studies. the present study had some interesting findings. the concentration of the product extracted using trisure was approximately 157 ng/µl (corresponding to 47.1 µg/g), which was not as high as the qrrem results of iqbal et al. (2019) (16.2 µg/80 mg corresponding to 202.5 µg/g) or the ircm results of iqbal et al. (2020) (150 µg/g). moreover the results were not even comparable to those of the trizol method in the aforementioned studies (130 µg/g from iqbal et al. 2019 and 145 µg/g from iqbal et al. 2020). however, the product from ctab-licl of approximately 142 ng/µl (equivalent to 42.6 µg/g) was significantly higher than the ctab results of iqbal et al. (2019) (1.25 µg/g) and iqbal et al. (2020) (7.5 µg/g) and even higher than the results of souza et al. (2018) (28.3 µg/g) in which ctab was rated as the best reagent. however, in the end, the rna concentration obtained from trisure was still higher than that from ctablicl in a statistically significant manner [p value 0.00 (<0.05)]. the integrity of the rna fragments is important in assessing the quality of the total rna obtained. total rna differs from dna as it includes several types of rna (rrna, trna, mrna, etc.) that vary in size. the size of rrna in plants is usually fixed at 25s (3700 bp), 23s (2900 bp), 18s (1900 bp) and 16s (1500 bp) (thermo fisher 2006). in contrast, the size of mrna varies widely because it depends on the length and specificity of the coding gene. transcriptome sequencing focuses on mrna molecules, which carry genetic information from the gene and encode functional proteins. mrna sequencing provides valuable insights into the expressed functional gene and its expression level, which may be significantly lower than that of rrna. identifying specific mrna bands on electrophoresis is challenging owing to variations in size and expression levels. relative estimates of band quantity and tape compactness may indicate the presence of more intact rnas and fewer fragmented rnas (wang et al. 2009, kukurba et al. 2015). the results demonstrated that the trisure method yielded superior electrophoresis and concentration outcomes than the ctab-licl method. furthermore, the trisure procedure required a total extraction time of merely 1 h, which was significantly shorter than the 15 h required for the other methods. this step aimed to determine the relative efficiency of the endosperm tissue compared with the leaf samples, rather than to enhance the extraction efficiency. therefore, in the next step, the trisure method was employed to optimise the extraction of rna from we tissue as it offered superior rna integrity and can potentially overcome the limitations of the current methods. methods for the extraction of total rna from coconut endosperm – thanh-diem nguyen and huyen-trang vu 391 improving purity valuethe a260/230 ratio, an indicator of purity, in rna extraction process with trisure during the extraction of plant tissue samples, a260/230 values are often low owing to the presence of polysaccharides (wang & stegemann 2010, orek 2018). to overcome this problem, nucleotide lysis buffers can be supplemented with nacl and β-mercaptoethanol to limit the amount of polysaccharides remaining in the sample. nacl effectively removes polysaccharides during chloroform cleavage (chang, puryear & cairney 1993), and β-mercaptoethanol is a potent reducing reagent that aids in denaturing rnase (chang, puryear & cairney 1993, mommaerts et al. 2015) thereby preventing undesired rna degradation. in this study, 0.1 ml of 2m nacl and 1% βmercaptoethanol were added simultaneously to the trisure buffer, as recommended by chang et al. (1993). the amount of chloroform was increased to a ratio of 1:1 (v/v) for the sample aliquot obtained in the phase separation step. the rna washing step with 75% alcohol was repeated thrice to eliminate any residual guanidine thiocyanate in the sample. subsequently, only we and nwe samples were utilised, with no further inclusion of leaf samples. the results of statistical analysis using anova signified no difference in the rna concentration (p value 0.067), a260/280 (p value 0.124). furthermore, the a260/230 value was significantly improved when using trisure supplemented with nacl + β-mercaptoethanol (1.58 ± 0.02) compared with the use of trisure only (1.01 ± 0.03) [p value 0.000 (<0.05)] (table 4). rna quality was measured using the absorption spectrometric ratios of the components present in the sample: pure rna (amax = 260 nm), proteins (amax = 280 nm) and polysaccharides (amax = 230 nm) (iandolino et al. 2004). the addition of nacl and the strong reducing agent β-mercaptoethanol to the extraction buffer enhanced the solubility of polysaccharides, decreased their co-precipitation with rna in later steps and denatured ribonucleases and proteins. other contaminants were reduced during extraction (iandolino, goes da silva, lim, choi, williams & cook 2004, lodhi et al. 1994, fang et al. 1992), thereby improving the purity ratio a260/230. testing of the sample incubation temperature atduring the lysis step for improvingto improve rna concentration following the extraction with trisure as recommended by the manufacturer of bioline, the lysate was incubated at room temperature for 5 min. however, coconut endosperm tissues are often hard and contain several lipids. hence, the grinding process is more difficult than that of the leaf tissue, leading to lower extraction efficiency. in the ctab method, the sample is usually incubated at a higher temperature (approximately 65°c) to augment the activity of the tissuedegrading enzyme. therefore, this study tested the effect of increased incubation temperature (50°c, 55°c and 60°c) on the lysis of we samples for 15 min to determine whether temperature affects the ability to obtain total rna from coconut endothelial cells in the trisure reaction. the results of anova showed that the we sample had total rna concentrations of 157.67 ± 0.57, 156.67 ± 2, 157.00 ± 1 and 156.67 ± 2.5 ng/µl (table 5), which corresponded to the incubation temperatures of room temperature (approximately 25°c–28oc), 50°c, 55°c and 60°c. however, there was no difference in yield table 4 investigation of the improvement in a260/230 ratio when adding nacl and β-mercaptoethanol in the lysis step buffer concentration a260/280 a260/230 trisure 157.33 ± 0.5 a 1.99 ± 0.01 a 1.01 ± 0.03 a trisure + nacl + β-mercaptoethanol 159.00 ± 1 a 1.94 ± 0.04 a 1.58 ± 0.02 b p value 0.067 (>0.05) 0.124 (>0.05) 0.000 (<0.05) note: one-way anova was used to investigate the influence of the two extraction methods (original trisure vs modified trisure) on the a260/a230 ratio of the rna product from the endosperm. the lowercase letters a and b represent the difference in the extraction methods. the differences are significant when the p value is <0.05. biotropia vol. 30 no. 3, 2023 392 among the investigated temperatures (p value 0.875). the od results were also not different. the a260/a280 ratio was in the range of 1.8–2.0, and the 260/a230 ratio was in the range of 1.58– 1.6. the electrophoresis results of the three treatments were also not different (figure 4). these results revealed that increasing the sample incubation temperature did not increase the amount of total rna obtained in the trisure procedure. testing the effectiveness of rna preservation during transport in many cases, the sample needs to be sent elsewhere for sequencing, which can take several days to a week, which affects rna quality. to minimise rna loss during transport, tests were performed using two preservation methods: depc solubilisation and absolute alcohol precipitation. the sample was kept chilled in a styrofoam container filled with gel ice. the temperature of the container was maintained between 8°c and 10°c, which is similar to the transportation condition. rna quality during this preservation process was evaluated on a weekly basis using electrophoretic analysis. after 1 week, in samples that were stored in depc and precipitated with absolute alcohol, the electrophoresis bands showed minimal changes. however, from the 2nd week, the electrophoresis band of the rna product precipitated with depc began to become more blurred, and by the 4th week, it was completely blurred and had several streaks (figure 5). on the contrary, the electrophoresis band of the rna stored in absolute alcohol did not show many changes compared with the initial time; nonetheless, it began to fade after the 3rd week. the results showed that storage in absolute alcohol is more optimal for rna preservation than that in depc, which could be maintained for up to 3 weeks. table 5 investigation of the possibility of improving the a260/230 ratio when increasing the annealing temperature in the lysis step annealing temperature concentration a260/280 a260/230 room temperature (25°c–28°c) 157.67 ± 0.57 a 1.98 ± 0.02 a 1.6 ± 0.01 a 50°c 156.67 ± 2 a 2.00 ± 0.03 a 1.58 ± 0.01 a 55°c 157.00 ± 1a 2.01 ± 0.01 a 1.6 ± 0.001 a 60°c 156.67 ± 2.5 a 1.98 ± 0.02 a 1.6 ± 0.02 a p value 0.875 (>0.05) 0.281 (>0.05) 0.205 (>0.05) note: one-way anova was used to investigate the influence of four different temperatures in the lysis step on the a260/a230 ratio of the rna product from the endosperm. the lowercase letter a represents the difference between the screening temperatures. p value >0.05 shows lack of statistical significance. figure 4 results of electrophoresis of rna extracted from we samples at different temperatures methods for the extraction of total rna from coconut endosperm – thanh-diem nguyen and huyen-trang vu 393 after rna extraction, its preservation is crucial. because rna is rapidly influenced by temperature and nucleases in the media, it becomes unstable following extraction. this instability could be attributed to the fact that the rna is often single-stranded and that the hydroxyl group renders the structure unstable, making the nucleotides prone to hydrolysis and destruction (fordyce et al. 2013). moreover, samples are often sent to sequencing firms located abroad, leading to extended transit times. unfortunately, this prolonged transportation can immensely affect the quality of the rna. in such circumstances, precipitation with pure alcohol is a technique that can help prolong the preservation of rna. unlike dna, rna in its single-stranded structure is inherently more challenging to preserve over an extended period. by precipitating rna with 99.5% alcohol, the rna structure condenses, strengthening the bonds and eliminating the enzymes that catalyse hydrolysis reactions. consequently, rna becomes significantly more resilient, facilitating its storage, transportation and utilisation for further research. conclusion the concentration and purity of rna, as reflected by the 260/280 ratio, is of utmost concern in next-generation sequencing. even some sequencing companies now have strict requirements in terms of quality and concentration. this is because prior to sequencing, the extract will be purified so that the product is of the desired purity, but the concentration will be lost. concentration is also significantly lost during transportation if the time is too long. therefore, improving the concentration of the extracted rna is essential. we recommend the use of trisure in combination with nacl and β-mercaptoethanol for efficient rna extraction instead of the ctab-licl method. additionally, rna could withstand temperatures of 8°c–10°c for 3 weeks when stored in a 99.5% ethanol solution, which helps to reduce its degradation when shipping samples over a long distance for an extended period prior to sequencing. acknowledgments this work is supported by nguyen tat thanh university under grant no. 2022.01.135/hđkhcn. references angeles, j. g., a. laurena and e. tecson-mendoza (2005). 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"chemical composition, nutritive value, volatile profiles and antioxidant activity of coconut (cocos nucifera l.) haustorium with different transverse diameter." 11(7). http://www.minitab.com/ methods for the extraction of total rna from coconut endosperm – thanh-diem nguyen and huyen-trang vu 395 the southeast asian journal of tropical biology vol. 31 no. 2, 2024: 253 265 doi: 10.11598/btb.2024.31.2.2165 issn: 0215-6334 | e-issn: 1907-770x 253 the potency of cinnamon (cinnamomum burmanni blume) leaf extract as a biopreservative agent for food safety of sate lilit ida bagus gede darmayasa1*, yan ramona1,2 , anak agung ketut darmadi1 , i wayan suanda3 ni luh hani1, and kalidas shetty4 1department of biology, faculty of mathematics and natural sciences, udayana university, bukit jimbaran campus, badung 80361, indonesia. 2integrated laboratory for biosciences and biotechnology, udayana university, bukit jimbaran campus, badung 80361, indonesia. 3department of biology education, faculty of teacher training and education, university of pgri mahadewa, denpasar 80235, indonesia. 4global institute of food security & international agriculture, north dakota state university, north dakota, usa article higlights • cinnamon leaf extract combats e. coli in traditional balinese food. • natural preservative reduces harmful pathogens in sate lilit. • cinnamon extract offers a safer alternative to synthetic preservatives. • active compounds in cinnamon leaves inhibit bacterial growth. • effective biopreservative for enhancing food safety and quality. article information received 16 january 2024 revised 28 april 2024 accepted 6 may 2024 *corresponding author, e-mail: darmayasa@unud.ac.id research article abstract escherichia coli o157:h7 contamination of the meat used in preparing sate lilit, a favorite traditional food in bali, indonesia, has been a great concern for both local people and foreigners. although c. burmanni has been included in its spice ingredients, active compounds that play a significant role in this pathogen have limitedly been elucidated. the main objectives of this research were to investigate the potency of this plant to control contaminants and elucidate possible compounds that prevent such contaminants by applying the disk diffusion method and lcms analysis, respectively. the results showed that the leaf extract of this plant inhibited the in vitro growth of e. coli o157:7, with minimal inhibitory concentration (mic) and lc50 values of 4% and 2.59%, respectively. the lcms analysis chromatogram showed that the plant extract's most active fraction produced nine peaks, representing nine possible active compounds. among those, three compounds (azoxystrobin, stigmatellin y, and 2-arachidonoyl glycerol) were suspected of contributing to control contamination, especially by e. coli o157:h7. keywords: 2-arachidonoyl glycerol, azoxystrobin, biopreservative, cinnamon, e. coli o157:h7, phenolic compounds, sate lilit, stigmatellin introduction being one of the tourist islands in indonesia, bali is rich in various types of ethnic foods. such kinds of foods (traditionally served along the streets in small stalls or modern restaurants) can easily be found in areas of tourist destinations in bali. a specific characteristic of balinese ethnic foods is the spicy ingredients used in the preparation. sate lilit is a traditional balinese kebab and a favorite food in bali, enjoyed by both locals and foreigners. sate lilit has also been part of offerings in the balinese religious rituals, such as temple festivals, balinese wedding ceremonies, etc. the meat (beef, pork, chicken, or fish as the primary raw material) of the sate lilit is frequently contaminated by pathogenic microbes (palupi et al, 2019) that can damage the quality of the sate lilit. these contaminated meats often become a medium for the pathogen to spread and cause food poisoning (foodborne disease outbreak). some researchers found that beef (a primary raw material of sate lilit) traded in traditional markets in bali is frequently contaminated by pathogenic bacteria. for example, purni et al. (2020) reported that 39 beef samples collected from some traditional markets located in denpasar city, badung, and klungkung regencies were contaminated by e. coli. among those samples, 25.6% were infected by e. coli o157:h7, the causative agent of hemorrhagic diarrhea, due to its ability to produce shiga-like toxins (patil et al., 2022). similar results were also reported by pinatih et al., (2021), who stated that this serotype of e. coli o157:h7 has been wellknown to produce potent toxins identical to those https://doi.org/10.11598/btb.2024.31.2.2165 https://orcid.org/0000-0002-0690-740x https://orcid.org/0000-0001-8928-5062 https://orcid.org/0000-0001-5336-229x https://orcid.org/0000-0002-7290-1022 biotropia vol. 31 no. 2, 2024 254 made by shigella dysentery type 1, and therefore it is often referred to as shiga like a toxin. as sate lilit is not fully cooked, it has the potential to spread pathogenic microbes that lead to an outbreak. the application of synthetic chemical-based preservatives has not been encouraged to cope with such pathogenic microbial contamination due to their many harmful side effects on human health. according to the indonesian food and drug administration (bpom), food poisoning was considered to be the highest case of poisoning in indonesia in 2012. approximately 66.7% of such poisoning cases were due to the consumption of foods containing poisonous preservatives, such as formalin, boric acid, or textile dyes (utomo dan kholifah, 2018). based on those food poisoning cases due to the application of inappropriate preservative agents, the possible use of natural preservative agents extracted from plants has been intensively researched so that some alternative and safe food preservations can be invented. the bark and leaf extracts of c. burmanni blume have been applied as flavor enhancers in various foods in indonesia. c. burmanni blume is a wooden plant. its height may reach 50 meters (sujarwo and keim, 2020). according to djarot et al. (2023), this plant's leaves and bark contain high levels of flavonoids, saponin, tannin, and alkaloids. mohamed et al. (2020) reported that the bark extract of this plant has the potential as an antibacterial agent. this is due to the antimicrobial effect of its essential oil, containing several active compounds, such as cinnamaldehyde, eugenol, cinnamic acid, and cinnamate (plumeriastuti et al., 2019). the potential of c. burmanni essential oil to inhibit staphylococcus aureus, enterobacter spp., pseudomonas aeruginosa, and candida albicans was also reported by novita and sutandhio (2021). this background has opened the door for its use as an alternative agent in developing food preservatives. based on the above rationale, it is urgently needed to elucidate active compounds in the c. burmanni leaf extract that may play an essential role in controlling e. coli o157:h7 contamination before its use as an organic food preservative. this study focused on the leaf extract of the plant, because this part of the plant has been widely used as an important ingredient of sate lilit preparation in bali. the results of our current research expectedly can be used to develop alternative food preservation methods to control pathogenic microbes (e. coli o157:h7 in particular) in ethnic balinese food products. materials and method extraction of c. burmanni blume leaves mature leaves (located on rows of 4-9 from tips of branches) of c. burmanni blume were collected from bedugul village, tabanan-bali. these leaf samples were chopped, air dried at ambient temperature (25 – 33oc), powdered with a warring blender, macerated in 96% ethanol (sigma aldrich, germany) in a ratio of 1:10 w/v, and filtered with 4 layers of whatman no. 2 filter papers (pore size 8 µm). the filtrate was then evaporated in a vacuum rotary evaporator at 40ºc to obtain crude extract/ pellet of the leaf extract and stored at 4oc before being used in the subsequent experiments. the crude extract obtained from this evaporation was considered to be 100% concentration. preparation of c. burmanni blume crude extract the crude extract of the plant leaves was diluted with 96% ethanol (sigma-aldrich, germany) to obtain final concentrations of 20% (w/v), 15% (w/v), 10%, and 5% (w/v) by applying the following dilution formula: v1 . m1 = v2 . m2 where: v₁ = volume of the most concentrated extract (ml) v₂ = volume of the adjusted extract concentration (ml) m₁ = concentration of the most concentrated extract (% w/v) m₂ = the adjusted extract concentration (% w/v). preparation of e. coli o157:h7 cell suspension the strain of escherichia coli o157:h7 was obtained from the stock culture collection of the microbiology laboratory, department of biology, faculty of mathematics and natural sciences, udayana university. pure culture of e. coli o157:h7 (1 loopful) was inoculated into 100 ml of nutrient broth medium (oxoid) and incubated for 24 hours at 37oc. the density of this bacterial suspension was then adjusted to 0.5 mcfarland scale or equal to 108 cells/ml. potency of cinnamon leaf extract as a biopreservative agent darmayasa et al. 255 bioassy (in vitro inhibition tests) of the c. burmanni blume leaf extract on e. coli o157:h7 in vitro inhibition tests of the c. burmanni blume leaf extract on e. coli o157:h7 were conducted by applying the method of kirby bauer (1966) with minor modifications. suspension of e. coli o157:h7 amounted at 200 µl was homogenized in 15 ml nutrient agar medium (oxoid) and poured into a sterile petri dish to produce bacterial lawn. once the mix was settled, a preliminary bioassay was conducted on the crude extract of this plant at high concentrations (25%, 50%, and 75% w/v) to determine antibacterial activity of the crude extract. the lowest concentration of this crude extract that inhibited the bacterial lawn was then used as a reference to determine the crude extract concentrations applied in the main experiments. as the inhibition zone already happened at 25% (w/v), the c. burmanni blume leaf extract concentrations used in the experiments were 5, 10, 15, and 20% w/v. in these bioassays, paper disks previously deposited with 20 µl c. burmanni blume leaf extract at those various concentrations (5, 10, 15, and 20% w/v) were placed at equidistance on the surface of this bacterial lawn to allow active compounds of the plant extract to diffuse. the same volume of 96% ethanol (sigma-aldrich, germany) and 1% w/v of chloramphenicol (a product of kalbe farma, indonesia) in paper disks served as negative and positive controls, respectively. four replicates were prepared in this bioassay to obtain representative data. all plates were incubated for 24 hours at 37oc and measured for formation of inhibition zones around the paper disks, previously deposited with the plant extract. measurements of inhibition zones around each paper disk were conducted from 4 different angles and the results were averaged. determination of minimal inhibitory concentration (mic) value of the c. burmanni blume leaf extract on e. coli o157:h7 mic value of the leaf plant extract was determined by applying the same method (kirby and bauer, 1966) mentioned above. the concentrations of the plant extract were adjusted from 0% to 5% w/v with interval increment of 1% w/v. four replicates were prepared in this bioassay to obtain representative data. all plates were incubated for 24 hours at 37oc and measured for formation of inhibition zones around the paper disks (ct0998b) deposited with the plant extract. measurements of inhibition zones around each paper disk were conducted from 4 different angles and the results were averaged. determination of lethal concentration 50% (lc50) of the c. burmanni blume leaf extract on e. coli o157:h7 bacterial suspension prepared above was diluted in serial dilution method so that cell with density of 103 cells/ml was obtained. a volume of 100 µl of this diluted cell suspension (100 cells) was exposed in eppendorf tubes for 15 minutes to the c. burmanni blume leaf extract of the same concentrations with those applied in the mic test. these exposed cell suspensions were then homogenized in nutrient agar medium (oxoid), poured into petri dishes, incubated at 37oc for 24 hours, and counted for colonies appeared on the surface of the medium. the results were then plotted in a graph that shows relationship between total colony counted and concentration of c. burmanni blume leaf extract (% b/v). the value of the lc50 was determined from the curve or calculated using the equation of the regression. phytochemical analysis of the c. burmanni blume leaf extract alkaloid test leaf extract of the c. burmanni blume was dissolved in 10 ml of chloroform-ammonia solution (sigma-aldrich, germany), added with 0.5 ml 1n h2so4 (1 n), homogenized, and let it to form 2 layers. the upper layer was collected and added with 1 drop of meyer reagent (sigmaaldrich, germany). the formation of precipitation indicates positive result (dey et al., 2020). flavonoid and phenolic tests these tests followed the method specified in aryal et al. (2019). the c. burmanni blume leaf extract was dissolved in 70% ethanol, heated, and filtered. the filtrate was then placed on a plate with mg and 1n hcl (merck) for the flavonoid test and fecl3 (merck) for the phenolic test. the formation of red color and green to purple ring indicated positive results for flavonoid and phenolic tests, respectively. biotropia vol. 31 no. 2, 2024 256 steroid and terpenoid tests the c. burmanni blume leaf extract was added with chloroform, heated for 10 minutes, placed on a plate, and added with liebermann-burchard reagent. a positive result for the terpenoid test was indicated by formation of red, pink, or violet color, while the steroid test was indicated by the formation of green or pink color (malik, 2023). saponin test a weight of 0,1g leaf extract of the c. burmanni blume was dissolved in 5 ml hot distilled water and shaken for 10 seconds. a positive test for saponin test was indicated by the formation of stable foam for 10 seconds (depkes ri, 2009). tannin test leaf extract of the c. burmanni blume amounted to 0,1 g and was added with fecl3, (merck) and then it was left to react for 5 minutes. the positive test was indicated by the formation of a blue color (sri kosnayani et al., 2019). elucidation of active compounds contained in the leaf extract of the c. burmanni blume fractionation and purification processes of active compounds contained in the crude extract of c. burmanni blume were conducted by applying column chromatography and thin layer chromatography (tlc), respectively. a 1g crude extract previously obtained was fractionated in column chromatography, where 300 g silica gel 60 (merck, ct1.07733) was used as the stationary phase. the silica gel was first suspended in an eluent of n-hexane (merck) as the mobile phase of the system. this silica gel suspension was poured into a column and acclimatized for 24 hours. the crude extract (1g) was dissolved in 1 ml, added with 1g silica gel, poured into the previously prepared column (length and diameter of 300 mm and 20 mm, respectively), and then eluted with a gradual change in the polarity of solvents. a volume of 25 ml eluent (with fractions of active compounds) flowing along the column was collected, evaporated, and analysed with a thin layer chromatography (tlc). fractions with the same rf values were composited and tested for their inhibitory activity against e. coli o157:h7 by disc diffusion (kirby-bauer, 1966). fractions with the ability to produce inhibition zones were subsequently analysed using the lcms (liquid chromatography mass spectroscopy, shimadzu vp series spd-10a vp uv vis detector hplc lc ms chromatography) method, and the peaks that appeared were aligned with those produced by known compounds following the application of the masslynx v4.1 software linked with the chemspider program. data analysis quantitative data was analyzed with analysis of variance (anova) at p<0.05 using spss software for windows version 17.0. when significant difference was indicated at p<0.05, the statistical test was further conducted by applying the duncans multiple range test (dmrt) at p<0.05. results and discussions in vitro inhibitory activity of c. burmanni blume on e. coli o157:h7 all concentrations applied in the preliminary bioassay experiments were found to inhibit the growth of e. coli o157:h7 with various inhibitions. the diameter of inhibition zones was proportional to crude extract concentrations (table 1 and figure 1). these results provided us with information on the toxicity level of this plant crude extract on the e. coli o157:h7. as the negative control did not inhibit the growth of the e. coli o157:h7, the clear zones formed around the disks deposited with the plant crude extract were confirmed to be due to active compounds contained in the plant crude extract. as shown in table 1, the diameter of inhibitions of the plant crude extract applied at concentrations of between 25% w/v – 100% w/v was slightly different from that produced by positive control, although they are statistically different at p<0.05. this indicated that the toxicity level of the crude extract was comparable with that of the 1% w/v of chloramphenicol. the c. burmanni blume leaf crude extract also inhibited the growth of e. coli o157:h7 when applied at concentrations lower than 25% w/v. the results are shown in table 2 and figure 1b. the inhibitory effect of the plant extract was still observed when the extract was applied at 5%w/v. potency of cinnamon leaf extract as a biopreservative agent darmayasa et al. 257 table 1 average diameter of inhibition of the c. burmanni blume leaf extract in the preliminary determination of appropriate concentrations to control e. coli o157:h7 extract concentrations (%) diameter of inhibition zones* 100 21.08±0.04e 75 19.53±0.05d 50 18.02±0.05c 25 17.21±0.06b 0 0.00 ±0.00a positive control 20.66±0.05f *values in table 1±standard deviations are averages of 4 replicates. values followed by different letters are statistically different at p<0.05 based on dmrt analysis following anova. positive control is 1% chloramphenicol. figure 1 diameters of inhibition zones of the c. burmanni blume leaf extract on e. coli o157:h7 obtained from preliminary bioassay (high concentrations of the plant extract) on nutrient agar medium (a); diameters of inhibition zones of the c. burmanni blume leaf extract on e. coli o157:h7 obtained from the main experiment of bioassay (concentrations of 5, 10, 15, and 20% w/v) on nutrient agar medium (b). the result shown in table 2 was consistent with those found in the preliminary study of the extract bioassay (table 1). this led us to investigate the minimum inhibitory concentration (mic) of the c. burmanni blume leaf extract on e. coli o157:h7 by applying the same method with adjusted concentrations of between 0% and 5% w/v with an increment concentration of 1%, and the results are shown in table 3. based on this bioassay, the mic value of the extract could be claimed at 4%, although it might fall between 3 and 4% w/v if the bioassay was continued by applying the plant extract concentrations between this range. as the scientific evidence of this plant’s antimicrobial activity is still limited, we focused our study on elucidating active compounds (with the potential to inhibit the growth of e. coli o157:h7) contained in this plant extract. in vitro bioassays of this plant extract in our study showed that the crude extract of this plant inhibited the growth of this pathogen when applied at a low concentration of 5% or more (tables 13 and figure 1). scientists such as parisa et al. (2019) also reported a similar phenomenon, who found in vitro microbial activity of this plant extract inhibitory against e. coli and staphylococcus aureus. didehdar et al. (2022) have also recently reviewed extensively the potential of this plant as a new therapeutic agent to inhibit biofilm formation by pathogenic fungi or bacteria in the early stages of their infection. this indicates that active compounds inhibitory to pathogenic bacteria or fungi exist in this plant extract. a b biotropia vol. 31 no. 2, 2024 258 table 2 inhibitory effect of the c. burmanni blume leaf extract on e. coli o157:h7 when applied at low concentration (between 5% 20% w/v) on nutrient agar medium extract concentrations (%) diameters of inhibition zones* 20 11.07± 0.05e 15 9.05± 0.05d 10 8.24± 0.05c 5 7.56± 0.06b 0 0.00 ± 0.00a positive control 20.66± 0.06f *values in table 1±standard deviations are averages of 4 replicates. values followed by different letter(s) are statistically different at p<0.05 based on dmrt analysis following analysis of variance (anova). the positive control is 0.15 mg chloramphenicol. table 3 averages of inhibition zone diameters of c. burmanni blume leaf extract on e. coli o157:h7 when applied at a concentration of between 0% 5% w/v on nutrient agar medium to determine mic value. extract concentrations (%) diameters of inhibition zones* 5 7.56± 0.06b 4 6.11± 0.02 c 3 0.00±0.00 a 2 0.00± 0.00 a 1 0.00± 0.00 a 0 0.00 ± 0.00a *values in table 1±standard deviations are averages of 4 replications. values followed by different letter(s) are statistically different at p<0.05 based on dmrt analysis following analysis of variance (anova). this plant extract appeared to be highly potent as its mic value was very low (at 4% w/v concentration, which was deposited on filter paper at a volume of 20 µl). when applied at 100% concentration, the c. burmanni blume leaf extract produced a diameter of inhibition higher than 20 mm, as shown by our study's positive control (1% chloramphenicol). based on the diameter of the inhibition zone, the effectiveness of this plant crude extract in controlling e. coli o157:h7 can be categorized as very high, according to the criteria made by rastina et al. (2018). in all cases (table 1 – 3), the negative control did not show an inhibition zone, indicating that the inhibition phenomenon shown by the crude extract of this plant (applied at various concentrations) must be due to the presence of compounds inhibitory against e. coli o157:h7. cinnamomum burmanni blume (in bali, traditionally named kayu manis) can abundantly be found in indonesia as it is widely used as a component in many traditional cuisines in this country, including in the ethnic foods of bali. in bahasa, this plant is known as cinnamon or padang cinnamon. this plant has been included in many popular indonesian foods or beverages, such as rendang (the most popular slow-cooked meat from padang, west sumatra), bali coffee, satay (skewered and grilled meat), balinese spice paste, which is used as a base for a wide range of dishes, and sambal (spicy chili pastes or sauce which is a staple in indonesian and balinese cuisines). recently, c. burmanni blume has also been included in the production of herbal tonics. the main role of this plant part in all those traditional foods is to enhance their flavor and balance the aroma of the foods. many people also believe that this plant has antimicrobial activity, so it is widely used as an alternative organic preservative agent to lengthen food shelf life. determination of lc50 value of the c. burmanni blume extract on e. coli o157:h7 a lethal concentration of 50% (lc50) is the concentration of the plant extract that kills 50% of bacterial population following exposure to such plant extract. the curve estimated this value (total viable cells vs extract concentration). the results are shown in figure 2. the number of cell death was proportional to the increment of the plant extract concentration. at 5% w/v, this plant extract totally eliminated/killed the e. coli o157:h7 (figure 2). the linear regression of the curve produces a potency of cinnamon leaf extract as a biopreservative agent darmayasa et al. 259 formula of y = -18.886x + 95.937, with r2 value of 0.9908. from figure 2 or the formula generated from the curve, the lc50 value of the extract fell between 2 and 3% w/v (approximately 2.59% w/v, based on calculation using the displayed formula). the values of the lc50 have been commonly used to assess the potential toxicity of substances exposed to living organisms (gupta, 2020). according to this scientist, the values of lc50 could help scientists determine safe exposure levels of toxic substances for types of organisms, specifically humans. these values are often used to establish permissible exposure limits for chemicals and pollutants (wolf & segner, 2023). therefore, lc50 values for toxic substances, including those produced by plants, are important to be determined prior to their application to control undesired microbes. this is aimed at reducing negative impacts following the application of those substances in any aspect of our lives. phytochemical analysis of the c. burmanni blume leaf extract from this analysis, the leaf extract of c. burmanni blume contains several groups of compounds, such as alkaloid, steroid, phenolic, saponin, and flavonoid. in our study, terpenoids and tannin were not detected, and the results are shown in table 4. these groups of compounds are very common in plant extract and may play an important role in inhibiting the growth of microbes. inhibition zones formed on the lawn of e. coli o157:h7 might have been due to the presence of these groups of compounds. figure 2 estimation of lc50 value of the c. burmanni blume leaf extract on e. coli o157:h7 table 4 groups of compounds detected in the leaf extract of c. burmanni blume types of compounds tested results* alkaloids + steroid + terpenoid phenolic + saponin + flavonoid + tannin * + = detected/present; = not detected/absent biotropia vol. 31 no. 2, 2024 260 the phytochemical tests analyzed the active compounds that might have played important roles in inhibiting e. coli o157:h7 in vitro growth. these included alkaloids, steroids, phenolics, saponins, and flavonoids (table 4). our results are in line with those reported by raji et al. (2019), ullah et al. (2020), and huang et al. (2022). the mechanisms by which these compounds control microbial growth have also been extensively reviewed, such as by yuan et al. (2021), santiago et al. (2021), tungmunnithum et al. (2018), and kavya et al. (2021). the most common mechanism reviewed and reported is interference with the function of the cell membrane that leads to the release of important components (including proteins) from the cytoplasm of microbial cells. other mechanisms have also been reported, such as enzyme inhibition, dna binding, oxidative stress, iron chelation, or antioxidant activity of those compounds to interfere with microbial growth. according to raji et al. (2019), such compounds can also affect the swarming motility of bacterial cells. the level of impact of alkaloid, steroid, phenolic, saponin, and flavonoid compounds to interfere with microbial growth is determined by many factors. these include types and concentrations of those compounds or microbial resistance developed over time (palupi et al., 2019; biharee et al., 2020). alkaloids, for example, may act in three different modes of action (disrupting cell membranes, interfering with enzyme activity, or binding on the dna of the targeted cells (jubair et al., 2021). conversely, phenolic compounds may generate reactive oxygen species (ros) that lead to oxidative damage to cellular components, such as proteins, lipids, and dna. oxidative damages caused by these ros certainly result in cell death (hajam et al., 2023). fractionation and elucidation of active compounds contained in the leaf extract of c. burmanni blume) on the completion of fractionation, 12 fractions were obtained, but only 4 of those fractions (fractions 3, 4, 5, and 6) showed inhibitory activity on e. coli o157:h7 (table 5). as shown in table 5, fraction 4 produced the biggest potential to inhibit the growth of this bacterial strain with a diameter inhibition of 25.23 mm (table 5 and figure 3). it is clearly demonstrated in figure 3 that some fractions inhibited the growth of e. coli o157:h7, which is indicated by clear zones around the paper disks deposited with each fraction. these results indicate that some active plant extract compounds exist in the fractions showing positive results in this in vitro bioassay (table 5). in the subsequent stage of our research, only fraction 4 (f4) was elucidated in the liquid chromatography-mass spectroscopy (lcms) analysis as it produced the highest inhibition zone in the in vitro bioassay, with a diameter inhibition zone of 25.23 mm (table 5 and figure 3). in the lcms analysis, fraction 4 of the plant extract was the main focus of the analysis as it gave the highest diameter of inhibition on this bacterium (table 5). the chromatogram of this analysis (figure 4) shows that nine peaks appeared in the chromatogram to represent compounds of n-(4-methoxyphenyl)-4-methyl1-piperazinecarbothioamide; azoxystrobin; 2(2-cyano-benzy l su l fany l ) -4 ,6-dipheny l nicotinonitrile; difenoconazole; octoxinol-2; n-(4butylphenyl)-11-[4-(dimethylamino)phenyl]-10methyl-8-phenyl-8,11 dihydropyrazolo [3’,4’:4,5] pyrimido [1,2-a] quinoxalin-6-amine; stigmatellin y; 2-arachidonoyl glycerol; and 1-(butylamino)3-methyl-2-octylpyrido[1,2-a]benzimidazole4-carbonitrile. the chemical structures of these compounds are shown in table 6. some compounds detected in our current study have been reported to have antimicrobial activity. azoxystrobin was reported by sun et al. (2023) to inhibit the growth of rhizoctonia solani, a fungal pathogen attacking tobacco plants. these authors also found this compound to affect the composition and diversity of microbes in tobacco plants. stigmatellin y, as detected in our study, has also been reported to have antimicrobial activity. boopathi et al. (2022) reported that this compound plays an important role in interfering with communication (quorum sensing) among cells of pseudomonas aeruginosa cells, resulting in its use as an anti-virulence pseudomonas. the compound of 2-arachidonoyl glycerol is another compound with the ability to inhibit the growth of microbes. according to chouenard et al. (2013), such compounds can stimulate neutrophils (a type of white blood cell) to release an antimicrobial effector that leads to growth inhibition on several bacterial species, such as escherichia coli and potency of cinnamon leaf extract as a biopreservative agent darmayasa et al. 261 staphylococcus aureus. this result partially explains or elucidates the mechanism by which both leaf c. burmanni blume crude extract and its fractions inhibited the in vitro growth of e. coli o157:h7 in our study. in other words, these three compounds (azoxystrobin, stigmatellin y, and 2-arachidonoyl glycerol) contributed in the growth inhibition of the targeted bacterium (e. coli o157:h7) the roles of the other six compounds to inhibit microbial growth have not been reported. they probably contribute to inhibiting the growth of microbes, although their contribution needs to be further elucidated. table 5 inhibitory activity of each fraction in the fractionation of the crude leaf extract of c. burmanni blume on e. coli o157:h7 fractions diameters of inhibition zones on e. coli o157:h7 (mm) f1 0.00 f2 0.00 f3 15.23 f4 25.23 f5 18.05 f6 9.40 f7 0.00 f8 0.00 f9 0.00 f10 0.00 f11 0.00 f12 0.00 figure 3 inhibition zones of each fraction of c. burmanni blume on e. coli o157:h7 on nutrient agar medium a b biotropia vol. 31 no. 2, 2024 262 figure 4 the chromatogram of lcms analysis of the fraction 4 of c. burmanni blume leaf extract. arrowheads point out the suspected active compounds (azoxystrobin, stigmatellin y, and 2-arachidonoyl glycerol) appeared at retention times of 10.06, 14.64, and 17.05, respectively, that might have contribution to inhibiting the in vitro growth of e. coli 0157:h7. table 6 chemical structures of compounds detected in fraction 4 of the c. burmanni blume leaf extract following application of lcms masslynx v4.1 software, which is linked to chemspider reference. no retention time (rt) molecular structure identified compound molecular formula molecular weight 1 10,53 n-(4-methoxyphenyl)4-methyl-1piperazinecarbothioamide c13h19n3os 266,1372 2 10,66 azoxystrobin c22h17n3o5 404,1246 3 10,81 2-(2-cyanobenzylsulfanyl)-4,6diphenyl-nicotinonitrile c26h17n3s 404,1237 4 12.20 difenoconazole c19h17n3o3cl2 406,0725 potency of cinnamon leaf extract as a biopreservative agent darmayasa et al. 263 no retention time (rt) molecular structure identified compound molecular formula molecular weight 5 13.41 octoxynol-2 c18h30o3 295,2273 6 14.46 n-(4-butylphenyl)-11-[4(dimethylamino)phenyl]10-methyl-8-phenyl-8,11dihydropyrazolo[3',4':4,5] pyrimido[1,2-a] quinoxalin-6-amine c37h37n7 508,3189 7 14.64 stigmatellin y c29h40o6 485,2903 8 17.05 2-arachidonoylglycerol c23h38o4 391,2848 9 17.05 1-(butylamino)-3-methyl2-octylpyrido[1,2-a] benzimidazole-4carbonitrile c25h34n4 391,2862 conclusion leaf extract of c. burmanni blume has the potential to control contamination of e. coli o157:h7 as it shows inhibitory activity in the serial in vitro bioassays, with minimal inhibitory concentration (mic) and lc50 values of 4% w/v and 2.59% w/v, respectively. the leaf crude extract of this plant contains groups of chemical compounds belong to alkaloids, steroids, phenolics, saponins, and flavonoid. fraction of the extract with the highest inhibition zone (fraction 4) contains 9 compounds, namely n-(4-methoxyphenyl)-4methyl-1-piperazinecarbothioamide; azoxystrobin; 2(2-cyano-benzy l su l fany l ) -4 ,6-dipheny l nicotinonitrile; difenoconazole; octoxinol-2; n-(4butylphenyl)-11-[4-(dimethylamino)phenyl]-10methyl-8-phenyl-8,11 dihydropyrazolo [3',4':4,5] pyrimido [1,2-a] quinoxalin-6-amine; stigmatellin y; 2-arachidonoyl glycerol; and 1-(butylamino)3-methyl-2-octylpyrido[1,2-a]benzimidazole-4carbonitrile. among these compounds, three (azoxystrobin, stigmatellin y, and 2-arachidonoyl glycerol) probably contributed to inhibiting the in vitro growth of e. coli o157:h7. references aryal s, baniya m k, danekhu k, kunwar p, gurung r, koirala n. 2019. total phenolic content, 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2department of silviculture, faculty of forestry, kasetsart university, bangkok 10900, thailand department of forest biology, faculty of forestry, kasetsart university, bangkok 10900, thailand 4natural history museum, pathum thani 12120, thailand received 17 december 2018 / accepted 25 april 2019 abstract site quality assessment is critically important in any tree planting activity as it may serve a range of management functions such as, optimizing productivity estimates of forest plantations. this study aimed to evaluate the site quality, using plant indicators species, for three teak plantations located in northern thailand belonging to the forest industry organization (fio). twenty-four sample plots were chosen to cover all the growth classes within the age range of 6-39 years. the site index of teak was established by using the anamorphic technique which is based on dominant height and age at a base age of 30 years, divided into 3 site index classes as 24, 21, and 18, as good, moderate, and poor site quality, respectively. associated species, the native species that are tree and shrub habits, were surveyed in the 24 plots and indicator species were classified using the indicator species analysis (isa) and two way indicator species analysis (twinspan). the relationship between indicator species and environmental factors was analyzed by the generalized linear model (glm). the associated species was classified into 76 species with 21 families. the results of isa indicated the significant indicator species under the good site class were strehlus ilicifolius, tagerstroemia floribunda, dalbergia cana and tagerstroemia calyculata\ while schleichera oleosa and dalbergia nigrescens were presented under poor site class, respectively. the results from twinspan supported streblus ilicifolius, tagerstroemia floribunda and schleichera oleosa were obvious indicators. each indicator species distribution influenced by various relationships with environmental factors, which soil ph and n were the main factors to distribute all indicator species to 3 relationships. first, the indicator species positively associated with soil ph and negatively associated with n were streblus ilicifolius and dalbergia nigrescens. second, the indicator species positively associated with soil ph and n were tagerstroemia floribunda and schileichera oleosa. third, the indicator species negatively associated with soil ph and positively associated n were dalbergia cana and tagerstroemia calyculata. the glm analysis revealed p, ca and elevation influenced indicator species distribution. as of writing, this is the first study on species indicators for suitable sites of teak in thailand. meanwhile, in the absence of confirmatory studies, these indicators can be used as guide for farmers interested in planting teak. in bare lands, the farmer can apply these indicator species to determine the site quality based on the species’ past appearance. keywords: productivity, site indicator species, site quality, teak plantation, thailand introduction teak (tectona grandis l.f.) plantations were established 112 years ago by the royal forest department (rfd) and more than 50 years ago ““ corresponding author, e-mail: fforcrw@ku.ac.th **this paper was presented at the 3rd international conference on tropical biology 2018, 20-21 september 2018, bogor, west java, indonesia by the forest industry organization (fio) in order to replenish the timber resources in thailand (kijkar 2000). initially, the teak plantations were grown naturally in the natural forest, especially in the northern part of thailand; however, with technology improvements, these plantations were expanded externally (thueksathit 2006). 104 site indicator species for predicting the productivity of teak plantations in thailand — jumwong et al since timber yield strongly depends on forest site quality, determination of site quality is one primary step in the intensive management of forest land (davis 1987; skovsgaard & vanclay 2008). based on site and yield information, a forest manager can estimate the future wood supplies and make realistic decisions about future costs and benefits of intensive management, land acquisition and industrial investment (carmean 1977; clutter et al. 1983). for over 50 years, thailand has performed site quality assessments using the site index (si) (boonthawee 1968; chanpaisang 1977; papata 2001; prempanichnukul 2001; srisuksai 2001). it is determined using a direct method defining the actual growth as an average of the heights of the dominant and co-dominant trees at a given base age in a single-species and evenly-aged stand (ford-robertson 1971). the same site can produce different site indices, as may be influenced by the environmental factors. however, the indirect or soil-site measurement uses the soil, topography and climatic factors in an area to correlate with the site index, growth, or yield, estimated from the trees in each plot (sahunalu 1970; srisuksai 2001). site quality assessments of teak plantations are based both on site index and soil survey methods. the site index method, however, needs records of teak growth in each site. it is difficult to evaluate the site quality and productivity of teak sites that lack teak growth record and of those newly established teak plantations. as tree heights and many other site properties are difficult to measure, the plant indicator species is determined based on the measurement of indicative variables such as the appearance or composition of ground vegetation (vanclay 1992). this study attempts to develop a practical site productivity indicator by using plant indicator species to evaluate the site quality and describe the effects of all environmental factors. materials and methods studied sites and sampling plots the study was conducted in year 2013 on those teak plantations which apply similar management techniques and are maintained by the forest industry organization (fio) in the phrae province, northern thailand. the planted areas in all plantations were divided according to the year of planting. the three plantations selected from a total of 12 teak plantations managed by fio, included the wang chin (wc), khun mae kham mee (km) and mae saroy (ms) plantations (fig. 1). these plantations covered a wide range of growth rates, tree age sizes, age classes and were classified as 6-10, 1115, 16-20, 21-25, 26-30 and > 30-years old (table 1). the elevation of the study areas ranged from 100-700 m above the mean sea level. the topography varied among sites with undulating area from flat to hilly. the soil texture is either sandy clay loam or clay type derived from a parent rock consisting of limestone (table 2). using a dominant height growth covering 5 site index classes, 24 temporary sample plots were randomly established in the selected study sites managed by the forestry research center in 1997 (forestry research center 1997) and distributed into 6 age classes. all plots were studied for their soil properties during the year 2000 (sakurai et al. 2002). data collection data were collected during the rainy season (august-november 2013), in which the highest density of associated species was observed. twenty-four temporary sample plots of 40 x 40 m were established and then divided into sub¬ plots of 10 x 10 m where bamboos were measured. inside the plots, the heights of 16 dominant and co-dominant trees were measured using a haga altimeter. the diameters at breast height (dbh) of all the trees were measured by a diameter tape. five subplots of 4 x 4 m were set up for saplings observation at the corners and in the middle of the 40 x 40 sample plot (fig. 2). the frequency and density of each species’ sapling plant was also estimated. site index analysis the site index for teak was determined using the anamorphic site index method (prasomsin 1991). the method defines an average height for a given age, which is commonly taken as 30 years for teak rotation. the tree selection was done based on the dominant height, as this 105 biotropia vol. 27 no. 2, 2020 g40p00 56iyk)ii sstyumi j100 6201)110 640,000 660,1 )00' >/ | 1n a ru i i i km 0 5 10 15 20 r : 1 [ad>viac^ii orcst schoolsi ^ a (a)khun mac khan -i mac kham porteu ii mac kon *7 mac man hai^den cha na poon£ mac sin macv>—r imac ronr̂ mac pan a 1legend i* plantation district boundary | phrac province province boundary 1:650,000 540000 560000 580000 600000 620000 640000 660000 figure 1 location of the three study areas, khun mae kham mee (km) plantation, wang chin (wc) plantation and mae sa-roy (ms) plantation as indicated by circles table 1 age classes of teaks in the twenty-four sample plots selected from khun mae kham mee (km), wang chin (wc) and mae sa-roy (ms) plantations age class (y r) site indexa total8 11 14 17 20 6-10 i (i) i (i) 2 (2) 4 (4) 11-15 i (i ) i (i) 2 (2) 16-20 1 (1) 1 (1) 21-25 2 (2) 1 (2) 2 (2) 5 (6) 26-30 i (i) 3 (5) 3 (4) 7 (10) > 30 0 (3) 2 (8) 2 (5) 1 (4) 5 (20) total i (i ) 5 (10) 7 (14) 5 (9) 6 (9) 24 (43) notes: a as reported by the forestry research center (1997); the highlighted numbers indicate the sample plots and the number in brackets are the existing plots. 106 site indicator species for predicting the productivity of teak plantations in thailand — jumwong et al. table 2 the environmental conditions (topography and soil properties at 0 and 20 cm soil depth) at the 24 sample plots from khun mae kham mee plantation (km), mae saroy plantation (ms) and wang chin plantation (wc) topography soil soil properties no plot slope (%) elevation (m) depth (cm) ph n (%) p (mg p/kg) ca k mg — cmol(+)/kg na 1 km75 50 498.20 0 5.24 1.10 3.86 4.99 0.21 1.04 0.30 20 5.66 2.20 2.28 7.56 0.26 3.33 0.23 2 km81 20 348.00 0 6.04 1.20 3.37 9.99 0.21 1.90 0.26 20 5.79 1.00 1.07 9.03 0.10 1.43 0.28 3 km85 45 550.00 0 6.11 1.70 18.86 7.00 0.34 1.29 0.27 20 5.89 1.10 1.48 4.27 0.14 1.03 0.28 4 km83 0 432.30 0 6.53 2.10 12.47 14.85 0.41 3.27 0.32 20 6.21 1.40 0.73 10.64 0.14 2.31 0.35 5 km78 35 426.10 0 6.08 2.20 2.84 13.64 0.28 4.82 0.35 20 5.59 1.60 0.85 6.72 0.11 3.73 0.25 6 km05 0 441.00 0 5.93 1.80 4.28 9.07 0.37 3.55 0.11 20 5.51 1.20 0.79 4.92 0.13 2.86 0.32 7 km02 0 432.30 0 5.93 1.80 4.28 9.07 0.37 3.55 0.11 20 5.51 1.20 0.79 4.92 0.13 2.86 0.32 8 km04 5 453.70 0 5.93 1.80 3.55 9.38 0.30 4.23 0.32 20 5.60 0.60 0.70 6.36 0.13 2.58 0.29 9 km01 10 458.20 0 5.93 1.80 3.55 9.38 0.30 4.23 0.32 20 5.60 0.60 0.70 6.36 0.13 2.58 0.29 10 ms87 5 164.00 0 5.89 1.50 7.25 5.20 0.31 3.18 0.21 20 5.44 0.90 1.70 2.87 0.08 2.11 0.28 11 ms83 2 136.20 0 5.22 1.30 3.58 2.13 0.21 1.46 0.27 20 4.91 1.00 1.04 1.20 0.12 0.62 0.29 12 ms80 5 147.40 0 5.39 1.20 3.07 3.54 0.19 1.79 0.28 20 5.36 0.80 1.80 1.87 0.11 2.39 0.32 13 ms86 35 175.80 0 5.93 1.80 7.54 8.08 0.44 2.21 0.30 20 5.06 1.90 1.56 4.85 0.25 1.56 0.29 14 ms82 20 185.30 0 5.69 1.10 2.33 2.99 0.20 2.13 0.26 20 5.23 0.90 1.97 1.77 0.22 0.80 0.29 15 ms84 30 135.70 0 5.90 1.10 13.29 3.88 0.21 1.72 0.23 20 5.24 0.80 1.64 1.90 0.14 1.00 0.32 16 wc06 2 142.60 0 5.39 1.20 3.07 3.54 0.19 1.79 0.28 20 5.36 0.80 1.80 1.87 0.11 2.39 0.32 17 wc83 30 125.90 0 5.22 1.30 3.58 2.13 0.21 1.46 0.27 20 4.91 1.00 1.04 1.20 0.12 0.62 0.29 18 wc82 15 128.20 0 5.69 1.10 2.33 2.99 0.20 2.13 0.26 20 5.32 0.80 0.86 1.40 0.14 2.07 0.27 19 wc79 3 177.20 0 6.26 0.90 3.34 7.19 0.30 3.08 0.20 20 5.60 0.80 0.65 5.09 0.16 4.02 0.30 20 wc89 0 142.00 0 5.74 1.30 5.36 4.01 0.16 2.05 0.20 20 5.06 0.80 1.40 1.08 0.10 1.03 0.27 21 wc90 3 178.90 0 5.31 1.40 4.37 7.61 0.17 4.75 0.32 20 5.14 0.90 1.23 2.56 0.09 3.91 0.28 22 wc07 0 155.20 0 5.39 1.20 3.07 3.54 0.19 1.79 0.28 20 5.36 0.80 1.80 1.87 0.11 2.39 0.32 23 wc92 35 139.90 0 5.52 1.10 6.90 3.09 0.19 1.17 0.26 20 5.27 0.50 4.14 0.47 0.14 0.45 0.26 24 wc93 30 118.80 0 5.91 1.60 13.16 5.11 0.47 2.65 0.34 20 5.19 0.90 1.17 2.52 0.17 1.28 0.11 notes: numbers after the plantation’s abbreviation indicate the planted year (19xx/20xx), for examples: wc92 is a sample plot in wang chin plantation that was planted in year 1992; wc06 is a sample plot in wang chin plantation that was planted in year 2006; ec = electrical conductivity, n = total nitrogen, c = organic carbon, p = available phosphorus, ca = exchangeable calcium, k = exchangeable potassium, mg = exchangeable magnesium, na = exchangeable sodium. 107 biotropia vol. 27 no. 2, 2020 10 m 40 m 1 4x4 m 1 11 [ f | 10 m |— j teak growth data collection under story data collection g bamboo frequency figure 2 the experimental design for data collection measure is relatively stable and robust over a large range of managed stand densities (steve 2001; herrera-fernandez et al. 2004). many stands had a dominant height and base age of 30-years at the time which the experiment was conducted. a scatter plot between heights and ages was fitted with a best-fit curve, along with higher and lower envelope curves with a shape similar to the guiding curves (donald 1971) and classified into 3 classes as having a good, moderate, or poor site quality. classification of the associated species characteristics the 24 sample plots or stands were analyzed to describe the associated species life in the teak plantation. in each stand, all the associated species in the five sub-plots (4 x 4 cm each) were identified based on smitinand (2014) and recorded, including its life form. the analysis of associated species characteristics was important value index (ivi). analysis of plant indicator species indicator species was determined using indicator species analysis (isa): an analysis of the relationship between important index values (ivi), which refer to species occurrence and their abundance, from a set of sampled sites which were site quality classes (good, moderate and poor). the significant associated species with p < 0.05 were indicator species of the site. two way indicator species analysis (twinspan) used to confirm the obvious indicator species. available in pc-ord version 6.08 developed for windows (mccune & mefford 2011). relationship between indicator species and environmental factors a generalized linear model (glm) technique was applied to determine the relationships of indicator species distribution with environmental factors (table 2). the glm expands the general linear model so that the dependent variable (number of each indicator species) is linearly related to the covariates (environmental factors) via a specified link function which was natural log in this study. results and discussion site index of teak site index of teak was divided into 3 site classes, with 24, 21 and 18, indicating a good, moderate, and poor site quality, respectively (fig. 3 and table 3). the number of plots that were related to good, moderate, and poor site quality was 9, 8 and 7, respectively. the dominant height of teak trees, aged 30years, represented in the poor, moderate and good quality sites, was 18, 21 and 24 m, respectively. in a teak plantation in northern thailand, the site quality had influenced the 30year old teak height, 10 m (poor), 20 m (moderate), and 30 m (good) (kaosa-ard 1991). this study results indicated that the site quality for teak plantation in phrae province was relatively higher than the overall values reported 108 site indicator species for predicting the productivity of teak plantations in thailand — jumwong et al. in the northern thailand, particular, for the poor and moderate site quality. on the other hand, the tree height for the good site class was lower than that previously reported. these results substantiated that of chanpaisang (1997) where the teak height at a base age of 30-years, were classified it into 5 site qualities, 14 m (very poor), 17 m (poor), 20 m (moderate), 23 m (good) and 26 m (very good). the variability in the average dbh, average dominant height, and merchantable volume of teak was relatively high for each of the site index classes (table 1). in sample plots with poor site quality, such as ms83, it was observed that the average dbh was higher than in those plots with good site quality (wc83). this was a direct result of silvicultural management, in which selective thinning was done at ages 15 and 22 years, to promote optimum growth of the remaining trees. thus, it is important to select a good site with intensive management to grow teak. . 30 £ 25 .sp‘53= 20 fu .e 15 eo~o 10a 6x> (0 aj 5 cu 0 0 km78 24 ms82 7c§3*ms80 21 wc90# ^/c82 ky75 18 c93* ms87• m01 wco; 10 15 20 age 25 30 35 40 figure 3 site index classes for teak in the three plantations located in phrae province, thailand notes: km = khun mae kham mee plantation; ms = mae saroy plantation; wc = wang chin plantation; numbers 24, 21, and 18 = site quality classes. table 3 site index (si), site quality (sq) classes and related growth characteristics of teak plantations at phrae province, thailand si no plot age tree/ha h dbh mean annual increment (mai) (sq) (yr) (m) (cm) h (m) dbh (cm) 24 1 km05 8 612 15.95 12.66 1.99 1.58 (good) 2 km02 11 687 15.60 15.92 1.42 1.45 3 ms84 22 331 22.30 17.61 1.01 0.80 4 wc90 23 650 21.00 16.86 0.91 0.73 5 wc89 24 806 20.60 16.51 0.86 0.69 6 wc83 30 343 22.50 17.20 0.75 0.58 7 ms82 31 306 23.80 20.49 0.77 0.66 8 wc79 34 300 23.90 26.32 0.70 0.77 9 km78 36 137 27.40 29.49 0.76 0.82 21 1 wc07 6 450 9.44 10.91 1.57 1.82 (moderate) 2 wc06 7 487 12.70 10.74 1.81 1.53 3 km04 9 475 14.21 16.51 1.58 1.83 4 wc93 20 481 18.00 15.62 0.90 0.78 5 wc92 21 543 19.22 17.96 0.92 0.86 6 ms86 27 281 19.00 27.66 0.70 1.02 7 wc82 31 175 20.50 23.27 0.66 0.75 8 ms80 33 393 22.80 13.50 0.69 0.41 109 biotropia vol. 27 no. 2, 2020 table 3 (continued) 18 1 km01 12 762 12.70 12.82 1.06 1.07 (poor) 2 ms87 26 837 16.05 15.60 0.62 0.60 3 km85 28 581 17.99 19.59 0.64 0.70 4 km83 30 262 17.49 22.79 0.60 0.64 5 ms83 30 312 18.10 19.19 0.58 0.76 6 km81 32 293 18.50 23.95 0.58 0.75 7 km75 39 137 19.60 18.51 0.50 0.47 notes: numbers indicate the planted year (19xx/20xx); km = khun mae kham mee plantation; ms = mae saroy plantation; wc = wang chin plantation. associated species characteristics in this study, the associated species is native species with only tree and shrub. from the exploration of three teak plantations in phare province, it was found that all associated species in 24 sample plots comprised of 76 species belonging to 21 families. all the sample unit was found in seedling and sapling stages. most of the associated species comprised of trees (51 species). the dominant family was fabaceae (previously leguminosae), with a total of 15 species, followed by malvaceae (6 species) and phyllanthaceae (6 species). ivi was calculated by summing up the relative density and relative frequency. the top five with the highest ivi were cratoxylum formosum (20.86), clerodendrum chinense (13.24), depisanthes rubiginosa (11.15), oroxylum indicum (10.25) and dalbergia lanceolaria (9.4). the species composition and ivi of associated species are shown in table 3. table 3 relative density (rd), relative frequency (rf) and important value index (ivi) of associated species no scientific name family rd (%) rf (%) ivi 1 cratoxylum formosum hypericaceae 13.43 7.43 20.86 2 clerodendrum chinense lamiaceae 10.59 2.65 13.24 3 fepisanthes rubiginosa sapindaceae 3.71 7.43 11.15 4 oroxylum indicum bignoniaceae 3.53 6.73 10.25 5 dalbergia lanceolaria fabaceae 6.51 2.83 9.34 6 vitex canescens lamiaceae 4.95 4.07 9.02 7 bridelia ovata phyllanthaceae 6.00 2.12 8.13 8 ficus hispida moraceae 4.35 3.36 7.72 9 croton stellatopilosus euphorbiaceae 3.62 3.72 7.34 10 barringtonia acutangula lecythidaceae 2.84 3.72 6.56 11 millettia brandisiana fabaceae 3.62 2.48 6.10 12 pterocarpus macrocarpus fabaceae 3.67 2.30 5.97 13 mitragyna rotundifolia rubiaceae 1.92 3.19 5.11 14 streblus ilicifolius moraceae 3.25 1.24 4.49 15 fernandoa adenophylla bignoniaceae 1.10 2.83 3.93 16 hymenodictyon orixense rubiaceae 1.24 2.65 3.89 17 greivia eriocarpa malvaceae 1.47 2.12 3.59 18 xylia xylocarpa fabaceae 1.28 2.30 3.58 19 dalbergia cultrata fabaceae 1.37 1.95 3.32 20 sterculia guttata malvaceae 1.79 1.24 3.03 21 fagerstroemia floribunda lythraceae 1.65 1.24 2.89 22 smilax sp. smilacaceae 1.97 0.88 2.86 23 diospyros malabarica ebenaceae 0.73 1.59 2.33 24 millettia leucantha fabaceae 1.24 1.06 2.30 25 bauhinia saccocalyx fabaceae 1.05 1.24 2.29 26 markhamia stipulate bignoniaceae 0.64 1.42 2.06 27 ferminalia nigrovenulosa combretaceae 0.69 1.24 1.93 28 fagerstroemia calyculata lythraceae 0.50 1.42 1.92 29 schleichera oleosa sapindaceae 0.64 1.24 1.88 30 artocarpus sp. moraceae 0.55 1.06 1.61 31 aibiyia odoratissima fabaceae 0.37 1.24 1.61 32 dalbergia nigrescens fabaceae 0.50 1.06 1.57 33 dalbergia cana fabaceae 0.50 1.06 1.57 34 albiyia lucidior fabaceae 0.60 0.88 1.48 35 casearia gremifolia salicaceae 0.46 0.88 1.34 110 site indicator species for predicting the productivity of teak plantations in thailand — jumwong et al. table 3 (continued) 36 bombax anceps malvaceae 0.27 1.06 1.34 37 streblus asper moraceae 0.60 0.71 1.30 38 cassia fistula fabaceae 0.60 0.71 1.30 39 vitex peduncularis lamiaceae 0.41 0.88 1.30 40 helicteres isora malvaceae 0.23 1.06 1.29 41 wrightia arborea apocynaceae 0.27 0.88 1.16 42 antidesma ghaesembilla phyllanthaceae 0.41 0.71 1.12 43 anogeissus acuminata combretaceae 0.41 0.53 0.94 44 microcos paniculata malvaceae 0.41 0.53 0.94 45 croton poilanei euphorbiaceae 0.23 0.53 0.76 46 broussonetia papyrifera moraceae 0.32 0.35 0.67 47 morinda tomentosa rubiaceae 0.14 0.53 0.67 48 irvingia malayana irvingiaceae 0.27 0.35 0.63 49 clausena harmandiana rutaceae 0.09 0.53 0.62 50 ardisia polycephala primulaceae 0.23 0.35 0.58 51 holarrhena pubescens apocynaceae 0.05 0.53 0.58 52 diospyros castanea ebenaceae 0.37 0.18 0.54 53 vitex pinnata lamiaceae 0.32 0.18 0.50 54 terminalia glaucifolia combretaceae 0.14 0.35 0.49 55 alstonia scholaris apocynaceae 0.14 0.35 0.49 56 catunaregam sp. rubiaceae 0.14 0.35 0.49 57 i jtsea glutinosa lauraceae 0.14 0.35 0.49 58 quercus kerrii fabaceae 0.09 0.35 0.45 59 homalium tomentosum salicaceae 0.09 0.35 0.45 60 siphonodon celastrineus celastraceae 0.09 0.35 0.45 61 antidesma bunius phyllanthaceae 0.09 0.35 0.45 62 cratoxylum cochinchinense hypericaceae 0.23 0.18 0.41 63 dalbergia ovata fabaceae 0.09 0.18 0.27 64 dalbergia oliveri fabaceae 0.09 0.18 0.27 65 terminalia pierrei combretaceae 0.09 0.18 0.27 66 adenanthera microsperma fabaceae 0.09 0.18 0.27 67 knema globularia myristicaceae 0.09 0.18 0.27 68 millingtonia hortensis bignoniaceae 0.05 0.18 0.22 69 glochidion assamicum phyllanthaceae 0.05 0.18 0.22 70 pterospermum semisagittatum malvaceae 0.05 0.18 0.22 71 tagerstroemia duperreana lythraceae 0.05 0.18 0.22 72 sterculia sp. malvaceae 0.05 0.18 0.22 73 canarium subulatum burseraceae 0.05 0.18 0.22 74 diospyros mollis ebenaceae 0.05 0.18 0.22 75 glochidion sphaerogynum phyllanthaceae 0.05 0.18 0.22 76 flueggea virosa phyllanthaceae 0.05 0.18 0.22 total 100.00 100.00 200.00 plant indicator species the results from the isa significantly indicated indicator species (p < 0.05) that were streblus ilicifolius, dalbergia cana and dagerstroemia floribunda for good site. for poor site, the indicator species were schleichera oleosa and dalbergia nigrescens (table 4). isa could not indicate indicator species for moderate site. the results of twinspan supported the results of isa which three indicator species were found to define site quality of teak. streblus ilicifolius and dagerstroemia floribunda were strong indicator species for good site in km. schleichera oleosa was strong indicator species for poor site in km (fig. 4). in thailand, this is the first study of indicator species and site quality of teak. there was no information to compare the results with specific site quality from previous works, therefore discussion considered following the important value of these indicator species from previous works. the results of indicator species analysis both of isa and twinspan were in correspondence with boonsri (2016) who found dalbergia nigrescens and schleichera oleosa were dominant species at km. forest industry organization (2016) reported schleichera oleosa was dominant species in mae moh forest plantation, lampang. ill biotropia vol. 27 no. 2, 2020 table 4 the significant plant indicator species from isa significance level site class indicator spp p-value bonferroni sequential false discovery correction bonferroni rates 3 plantations good site streblus ilicifolius * 0.00089 0.00089 0.00089 good site l^agerstroemia floribunda * 0.00089 0.00091 0.00179 good site dalbergia cana * 0.00089 0.00093 0.00268 poor site schleichera oleosa * 0.00089 0.00094 0.00357 poor site dalbergia nigrescens * 0.00089 0.00096 0.00446 khun mae kham mee plantation (km) good site lagerstroemia caljccdata * 0.00128 0.00128 0.00128 good site dalbergia cana * 0.00128 0.00132 0.00256 wang chin plantation (wc) good site streblus ilicifolius * 0.00054 0.00227 0.00227 mae saroy plantation (ms); no indicator species note: * p < 0.05. dalbergia cultrata ( + ) 00 0 clerodendrum chinense { ~ ) 01 mitragyna rotundifolia (-) clerodendrum chinense {+ ) 10 010 0100 diospyros malabarica (-) pterocorpus macrocarpus () streblus ilicifolius ( + ) lagerstroemia floribunda (+) bridelia ovate ( + ) mitragyna rotundifolia ( + ) oil 0101 01000 km04 ms80 km01 ms87 km82 wc82 01001 010010 irvingia malayana (+) 010011 ms84 wc89 wc79 wc90 wc93 ms86 wc92 wc83 ms83 wc06 wc07 100 km 78 km05 km02 km83 1 cassia fistula (+) 101 vitex canescens (-) schleichera oleosa ( + ) 11 km85 km75 km81 figure 4 the indicator species for each division from twinspan notes: for the sample plot red alphabets indicate a good site, blue indicates a moderate site and black indicates a poor site; 0 = negative group; 1 = positive group; (+) = right hand sub-group; (-) = leaf hand sub¬ groups. table 5 generalized linear model (glm) analysis of the relationships between indicator species distribution and environmental factors indicator species plantation site quality ph n p ca elevation streblus ilicifolius km/wc good 3.38** -39.43* 26.53** 3.64 dalbergia cana km/wc good -96.12 14.52 -0.13 lagerstroemia caljcidata km/wc good -112.00 25.53 0.21 lagerstroemia floribunda ms/wc good 10.23** 20.42* 4.60** schileichera oleosa km poor 1.50 0.16 dalbergia nigrescens km poor 2.56 -0.96 -3.24 notes: * p < 0.05; ** p < 0.01; the values in the various columns are model regression coefficient. 112 site indicator species for predicting the productivity of teak plantations in thailand — jumwong et at relationship between indicator species and environmental factors soil ph and n were the main factors to distribute all indicator species to 3 relationships. first, the indicator species positively associated with soil ph and negatively associated with n were streblus ilicifolius and dalbergia nigrescens. second, the indicator species positively associated with soil ph and n were lagerstroemia floribunda and schileichera oleosa. third, the indicator species negatively associated with soil ph and positively associated n were dalbergia cana and _lagerstroemia calyculata. the glm analysis revealed p, ca and elevation influenced indicator species distribution (table 5). streblus ilicifolius was indicator species of good site which the result of isa computed from data sets of three plantations: km, ms and wc. from the survey found that streblus ilicifolius was found in km and wc. the distribution of streblus ilicifolius was statistically significant positively associated with soil ph and p and negatively associated with n. furthermore, tend to find streblus ilicifolius in areas with high calcium content. dalbergia cana and dagerstroemia calyculata were indicator species of good site which found in km and wc. the spatial distribution was not statistically significant with environmental factors (p > 0.05). the distribution was negatively associated with soil ph and positively associated with n. by the time, dalbergia cana was negatively associated with elevation but dagerstroemia calyculata was positively associated with elevation. dagerstroemia floribunda was indicator species of good site which found in ms and wc. the distribution was statistically significant positively associated with soil ph, n and p. schileichera oleosa and dalbergia nigrescens were indicator species of poor site in km. the spatial distributions were not statistically significant with environmental factors (p > 0.05). the distribution of schileichera oleosa was positively associated with soil ph and n. the distribution of dalbergia nigrescens was positively associated with soil ph and negatively associated with n and p. conclusion based on the anamorphic site index method, indicator species analysis (isa) and two way indicator species analysis (twinspan) of the three teak plantations in phrae province, thailand, the site indices at base age 30 year old teak trees could be divided into 3 classes, namely; 24 for good, 21 for moderate, and 18 for poor site quality. the significant indicator species in the 3 site index classes derived from isa (p < 0.05) are streblus ilicifolius, dalbergia cana, dagerstroemia floribunda and dagerstroemia calyculata for good site. for poor site, the indicator species were schleichera oleosa and dalbergia nigrescens. the results of twinspan supported the results of isa which three indicator species are obvious indicator species i.e., streblus ilicifolius and dagerstroemia floribunda and schleichera oleosa for poor site, especially in km. finally, since this study is the first of its kind, it is recommended that the site indicator species analysis be done in other teak areas to substantiate this study results. meanwhile, in the absence of confirmatory studies, the farmers can apply this current finding and determine the site quality based on the past or present appearance of the indicator species. acknowledgements this research was funded by the center for advanced studies in tropical natural resources, institute of advanced studies, kasetsart university (castnar, nru-ku), thailand. references boonsri k. 2016. structural characteristics, growth and soil properties of teak (tectona grandis l.f.) plantation under different stages of silvicultural practices at khun mae kham mee plantation, phrae province. bangkok (th): kasetsart university. boonthawee b. 1968. site quality of klangdong plantation. bangkok (th): kasetsart university. 113 biotropia vol. 27 no. 2, 2020 carmean wh [internet]. 1977. principle soil scientist: site classification for northern forest species. newtown square (pa): https:/ /www.fs.fed.us; [updated 2018 sep 29; cited 2013 feb 7]. available from: https:/ /www.nrs.fs.fed.us/pubs/gtr/gtr_ne29/gtr_ne29_205.pdf chanpaisang s. 1997. yield of teak plantation. bangkok (th): kasetsart university. clutter jl, fortson jc, piennoar lv, brister gh, bailey rl. 1983. timber management: a quantitative approach. new york (us): john 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in thailand. proceedings: 2002 aug 21-14. bangkok (th): the 17th world congress of soil science, p. 350-1-350. skovsgaard jp, vanclay jk. 2008. forest site productivity: a review of the evolution of dendrometric concepts for even-aged stands. forestry 81(1): 13-31. smitinad t. 2014. thai plant names. bangkok (th): forest herbarium, royal forest department. srisuksai b. 2001. growth and yield of the 18-year-old plantation teak, forest industry organization. bangkok (th): kasetsart university. steve ss. 2001. making sense of site index estimates in british columbia: a quick look at the big picture. b.c. j ecosystmanag 1(2):1-4. thueksathit s. 2006. variation in growth and development of teak growing outside the natural ranges in thailand. bangkok (th): kasetsart university. vanclay jk. 1992. assessing site productivity in tropical moist forest. for ecol manag 54:257-87. 114 the southeast asian journal of tropical biology vol. 32 no. 3, 2025: 288 298 doi: 10.11598/btb.2025.32.3.2414 issn: 0215-6334 | e-issn: 1907-770x 288 ecological characteristics of castanopsis tungurrut (blume) a. dc along the altitudinal gradient in cibodas biosphere reserve dian ridwan nurdiana1,2* and inocencio e. buot, jr.2 1research center for biota systems, national research and innovation agency (brin), bogor 16911, indonesia 2institute of biological sciences, college of arts and sciences, university of the philippines los baños, laguna 4030, philippines article higlights • castanopsis tungurrut prefer midaltitude forest habitat • the population show clumped pattern on steep slopes • vegetation cluster shift with altitudinal gradient • cisarua is the highest density of castanopsis tungurrut article information received : 16 december 2024 revised : 1 august 2025 accepted : 18 august 2025 *corresponding author, e-mail: dian034@brin.go.id research paper abstract castanopsis tungurrut is an endangered species that has received limited attention in terms of research, with scarce ecological information and description of its native distribution area, the cibodas biosphere reserve. this study aimed to investigate the ecological characteristics, vegetation clusters, population structure, and habitat preferences of c. tungurrut along the altitudinal gradient. to assess the vegetation, a total of 41 plots were used, with dimensions of 20 × 20 m for trees, 10 × 10 m for poles, 5 × 5 m for saplings, and 2 × 2 m for wildings. the results revealed that the vegetation in four different locations (cibodas, bodogol, cisarua, and selabintana) within the altitude range of ca. 750 1800 meters above sea level (m asl) could be grouped into three distinct zone clusters based on the dominant species. these clusters were named zone i (ca. 500 1,000 m asl), castanopsis-lithocarpus and schima wallichii forest characterized by the dominance of castanopsis tungurrut, maesopsis eminii, and schima wallichii; zone ii (ca. 1,000 -1,500 m asl), castanopsis and schima wallichii forest, dominated by castanopsis (fagaceae forest) and schima wallichii; and zone iii (ca.1,500-2,400 m asl), schima wallichii, castanopsis, and altingia excelsa forest where schima wallichii, castanopsis, and altingia excelsa were prevalent. c. tungurrut was found to dominate at elevations between ca. 750 and 1,500 m asl, gradually decreasing in higher elevations or even absent. it exhibited a clumped distribution pattern, favoring steep to highly steep habitats. the highest population density was observed in cisarua (53.1 individuals/ha), followed by bodogol (25 individuals/ ha), cibodas (10.7 individuals/ha), and selabintana (5 individuals/ ha). these findings emphasize the species’ selective habitat preferences, particularly with regard to altitude and slope factors, which should be taken into consideration when planning conservation efforts. keywords: endangered species, habitat preference, population introduction most species of castanopsis are adaptable to various habitats, soil types, topography, climate variations, and altitudes, contradicting earlier studies that suggested limited distribution (cheuk & fischer 2021; watanabe et al. 2021). c. tungurrut, like other castanopsis species, shows similar responses to its surroundings and tends to cluster in specific areas, although its distribution becomes more even at an altitude of 1,300 m asl (nurcahyani 2017). c. tungurrut exhibits a wide range of altitude tolerance, but its survival rate decreases at altitudes between 1,550 and 1,560 m asl (fathia et al. 2019; handayani et al. 2019). additionally, other studies have shown that the distribution of different life stages of c. tungurrut significantly varies with altitude (nurcahyani 2017). copyright (c) 2025@author(s). https://doi.org/10.11598/btb.2025.32.3.2414 https://creativecommons.org/licenses/by-nc-nd/4.0/ ecological patterns of castanopsis tungurrut in cibodas biosphere reserve nurdiana & buot 289 c. tungurrut, despite its wide range of altitude tolerance, struggles to adapt to changing environmental conditions, particularly at higher altitudes. this struggle is evident in the species’ declining population at higher altitudes. factors such as elevation and precipitation rates, as discussed by harapan et al. (2022), contribute to the species’ distribution. however, c. tungurrut tends to grow individually and does not compete for resources in its habitat, similar to c. argentea (nurcahyani 2017). these findings are intriguing because studies conducted in the mountain forests of java observed c. tungurrut growing alongside c. argentea, c. javanica, schima wallichii, and macropanax dispermus at altitudes ranging from 1,400 to 1,600 m asl, mostly without any apparent association. given that c. tungurrut exhibits significant variation in its distribution with altitude, further replication of this study in environments with similar ecological characteristics, such as elevation, will provide deeper insight into its ecological preferences and survival strategies. the objective of the study was to investigate how the ecological characteristics of the altitudinal gradient affect the vegetation structure, habitat preference, and population structure of c. tungurrut in the cibodas biosphere reserve. investigating whether the patterns observed in the cibodas biosphere reserve are consistent across other location with similar environmental conditions will not only confirm the generality of the findings, but also strengthen the overall validity of the study, providing a more comprehensive understanding of its habitat preferences and population structure. materials and methods study area the cibodas biosphere reserve, situated in west java province, indonesia, is a designated protected area known for its rich biodiversity. it spans across three regencies, namely cianjur, bogor, and sukabumi, and is home to a variety of plant and animal species, including the endangered castanopsis tungurrut. in this study, we focused on four representative locations within the reserve, namely cibodas (cbs), bodogol (bdl), selabintana (sbl), and cisarua (csr) (fig. 1). cibodas has slopes ranging from 11% to 42%, an elevation 1,346 to 1,830 m asl, and temperatures between 15 oc and 18 oc. cisarua, with a slightly warmer climate (17 oc to 19 oc), features slopes from 11% to 55% and an altitude 1,157 to 1,500 m asl. selabintana offers moderate conditions with slopes between 7% and 35%, an elevation of 1,163 to 1,829 m asl, and temperatures ranging from 16 oc to 18 oc. lastly, bodogol, at a lower altitude (759 to 1,104 m asl), experiences warmer temperature (20 oc to 21 oc) and steeper slopes ranging from 20% to 69%. the study encompassed locations spanning an elevation range of ca. 750 to 1,800 m asl, which is a crucial factor for the presence of c. tungurrut. previous research has demonstrated that variations in altitude significantly affect temperature and nutrient availability, which in turn can impact the distribution pattern patterns and population dynamics of the target species (hilwan & irfani 2018; fathia et al. 2019). methods the population structure and distribution of the species in all locations were assessed using the plot method. the plot sizes varied depending on the life stage of the species: 20 × 20 m for trees, 10 × 10 m for poles, 5 × 5 m for saplings, and 2 × 2 m for wildings (fig. 2). in each plot, the names and numbers of species were recorded for wildings, saplings, poles, and trees. the tree category included woody plants that are over 2 meters tall and have a stem diameter of at least 5 cm. poles, on the other hand, refer to stands with a diameter greater than 2.5 cm but less than 5 cm (fathia et al. 2019). saplings are stands with a diameter at breast height (dbh) less than 2.5 cm and a height of at least 130 cm, while wildings are individuals with a height below 130 cm (frei et al. 2022). the data were analyzed to determine the frequency, density, and basal area. these parameters were then used to calculate the relative values, which were summed up to obtain the importance value index (ivi) for both trees and poles (pandey & lodhiyal 2015). further analysis included the assessment of the dominance index, margalef ’s index, shannon index, evenness index, and morisita index (which represents the distribution pattern). biotropia vol. 32 no. 3, 2025 290 the past software was used to generate a dendrogram representing the clustering of vegetation among different locations to identify the cluster of vegetation plots based on the basal area (ba) values of woody species. the clustering was performed based on the basal area and utilized the unweighted pair group method with arithmetic mean (upgma) and euclidean distance as an indicator of similarity among the plots. this approach aimed to identify distinct plant communities within the forest landscape. figure 1 map displaying locations distribution based on the altitudinal gradient notes: cbs = cibodas; csr = cisarua; sbl = selabintana; bdl = bodogol. figure 2 plot diagram for vegetation analysis notes: a = plots measuring 20 × 20 m for trees; b = 10 × 10 m for poles; c = 5 × 5 m for saplings; d = 2 × 2 m for wildings. ecological patterns of castanopsis tungurrut in cibodas biosphere reserve nurdiana & buot 291 results and discussion floristic composition the scope of this study, which encompassed 41 plots across various locations with altitudinal gradients, was significant. the distribution of plots was as follows: 14 in cibodas, 8 in cisarua, 9 in selabintana, and 10 in bodogol (fig. 1). the variations in plot numbers were due to practical constraints such as accessibility and time. furthermore, the study was limited to specific elevations in some locations, preventing access to higher or lower elevations. the floristic composition based on life stages and locations, showing variations in the importance value index (ivi) and the number of species are presented in tables 1, 2, 3, and 4. notably, schima wallichii emerged as the dominant species across all locations and life stages, exerting a significant influence on the forest structure. in contrast, castanopsis tungurrut was found to be absent in specific locations and life stages. bodogol represents the colline subzone (500 1,000 m asl) and the submontane zone (1,000 1,500 m asl), as referred to by sadili et al. (2023). the colline zone is a zone characterized by the physiognomy of the vegetation, which is described as follows: a lofty, closed tropical forest, with a canopy height of 40 50 m, multistorey with higher emergent trees (prawiradilaga 2017). the submontane zone is characterized by the physiognomy of high-stemmed, closed, multistorey forests with a canopy height of 30 40 m. this location represents the transition zone between the lowland and submontane zones. the most dominant species was schima wallichii, followed by c. tungurrut. several factors affect the dominance of the species, including sporadic regeneration and a long lifespan, which makes it both shade-tolerant and intolerant (tang et al. 2020). in addition, the existence of invasive plants in a location with robust regenerative capacity for the exotic species maesopsis eminii and the presence of the pioneer species mallotus sp. contribute to the general structure of the forest, including the presence of the native species c. tungurrut. in bodogol, the c. tungurrut distribution and coexistence involve complex interactions between life stages, environmental factors, and species characteristics. there are changes between the number of early stages and the mature tree of c. tungurrut. nguyen et al. (2016) revealed that spatial patterns and interspecific associations change with life stages, with aggregation increasing and positive association decreasing as trees mature. in addition, it is probably related with the intense resource competition, which causes changes in species composition as the forest structure as also described by yu et al. (2020), where distinct plant species’ relative dominance at distinct stages of development, such as wilding, sapling, pole, and tree, has a substantial impact on the general structure and make-up of forests. the submontane (1,000 1,500 m asl) and montane zone (1,500 2,400 m asl) are represented by cibodas (cbs), a topographical location known for its steep inclination and horizontal slope range of 10 42%. furthermore, sudarmono (2018) defines the submontane zone as characterized by the physiognomy of high-stemmed, closed, multistorey forest and a canopy of 30 40 m. in comparison, montane zone is defined as closed forest canopy 25 30 m, storey, many epiphytes but less lianas. in this location, schima wallichii exhibited the highest importance value index (ivi) of 57.6, indicating its dominance over the other four species. interestingly, this finding aligns with the observed tree growth pattern in bodogol. therefore, the dominance of schima wallichii in both locations can be attributed to favorable climatic and ecological conditions that support its growth and development. in contrast, c. tungurrut was less dominant in this location, which is probably related to the favorable climatic and ecological conditions or even the presence of invasive species. in this location, c. tungurrut, despite being in its altitude range (1,346 1,830 m asl), was not dominant and was rarely found. this is not due to unfavorable conditions, but rather the existence of invasive alien species such as chimonobambusa quadrangularis (franceschi) makino and cestrum aurantiacum lindl. these invasive species can significantly alter the native ecosystem, posing a serious threat to the area’s biodiversity. biotropia vol. 32 no. 3, 2025 292 table 1 floristic composition based on the ivi in bodogol (ca. 750 1,104 m asl) tree pole sapling wilding species number 83 38 39 33 family number 40 23 25 21 shannon diversity index 3.86 3.54 3.45 3.31 margalef ’s index 14.3 9.45 9.1 7.72 evennes index 0.87 0.97 0.94 0.94 dominant species schima wallichii choisy (46.1) syzygium rostratum (blume)dc. (15.9) casearia coriacea vent. ( 15.2) maesopsis eminii engl. (13.7) castanopsis tungurrut (blume) a.dc. (20.4) aglaia sp. (12.9) magnolia liliifera (l.) baill.(13.7) castanopsis tungurrut (blume) a.dc. (12.7) syzygium rostratum (blume) dc. (18.7) villebrunea rubescens (blume)wedd. (12.3) ficus sp.(13) homalanthus populneus (geiseler) (12.2) lithocarpus pseudomoluccus (blume) dc. (16.6) ficus sinuata thunb. (11.7) sterculia sp 1 (11.4) calamus reinwardtii mart.(11.1) maesopsis eminii engl. (13.2) mallotus sp. (11.3) lithocarpus pseudomoluccus (blume) rehder (10.2) clidemia hirta (l.) d. don (11.1) table 2 floristic composition based on the ivi in cibodas (ca. 1,346 1,830 m asl) tree pole sapling wilding species number 72 56 76 67 family number 34 31 35 38 shannon diversity index 3.69 3.54 3.75 3.51 margalef ’s index 11.67 10.78 12.83 11.58 evennes index 0.86 0.88 0.86 0.84 dominant species schima wallichii choisy (57.6) cestrum aurantiacum lindl. (31.8) cestrum aurantiacum lindl. (12.3) lasianthus laevigatus blume (17.7) macropanax concinnus miq (20.8) lasianthus stercorarius blume (30.5) lasianthus stercorarius blume (11.1) psychotria montana blume (10.3) dacrycarpus imbricatus (blume)de laub. (18.2) polyalthia subcordata (blume) blume (30) freycinetia insignis blume (7.4) cyrtandra picta blume (8) castanopsis javanica (blume)a.dc. (17.9) turpinia sphaerocarpa hassk.(25.6) polyalthia subcordata (blume) blume (7.1) strobilanthes cernua blume (7.9) villebrunea rubescens (blume) wedd. (16.4) macropanax undulatus (wall.ex g.donn) seem (18.1) bartlettina sordida (less.) r.m.king & h.rob. (6.6) trevesia sundaica miq. (7.3) ecological patterns of castanopsis tungurrut in cibodas biosphere reserve nurdiana & buot 293 cisarua represents the submontane zone, with a slope ranging from 11% to 55%. this slope range is significant as it influences the distribution of tree species and the overall ecological characteristics of the area. in general, cisarua shares similar characteristics with bodogol, as both are located at the pangrango mountain ridge. in terms of vegetation, cisarua had the lowest diversity of tree species, with only 47 species present. among these, the dominant species in the tree phase at this location were castanopsis tungurrut, villebrunea rubescencs, cinchona pubescens, schima wallichii, and castanopsis javanica, with importance value index (ivi) values of 77.9, 29.6, 23.9, 12.4, and 11.3, respectively. villebrunea rubescens had the highest density, with 59.3 individuals per hectare (indv/ ha), followed by cinchona pubescens, castanopsis tungurrut, and schima wallichii, with densities of 56.2, 53.1, and 21.8 indv/ha, respectively. in comparison to dominant tree species in other locations like bodogol and cibodas, schima wallichii remains the dominant species in the tree phase category. however, even though cisarua is at the ideal altitude for c. tungurrut habitat, the existence of wilding (young trees that grow without human intervention) and sapling (a young tree) is rarely found in this location. this rarity of wilding and sapling could be due to the existence of cinchona pubescens plantations which decreases the level of plant diversity and homogenization of forest areas. we can conclude it from the high importance value index in the tree, pole and sapling phases of cinchona pubescens. the tree vegetation in selabintana was mainly dominated by schima wallichii, macropanax dispermus, villebrunea rubescens, neolitsea javanica, and acronychia pedunculata. lasianthus sp. dominated the pole, sapling, and wilding stages. the diversity index across different life stages in this location showed no significant differences, with values of 3.46 for trees, 3.18 for poles, 3.12 for saplings, and 2.86 for wildings. these indices demonstrate a decrease in diversity from trees to wildings, as well as a decrease in the margalef ’s index (indicating richness). this suggests a need for further investigation into the factors influencing the diversity of tree vegetation in selabintana. when compared to the other three locations, selabintana shares a similarity with cibodas in terms of the low dominance of c. tungurrut, which is only prevalent in the sapling phase. this unique ecological pattern can be attributed to a variety of factors such as forest gaps, topography, anthropogenic impacts, seed dispersal disruption, and global climate change. previous studies have shown how these factors can limit the regeneration of certain species (dey et al. 2019). table 3 floristic composition based on the ivi in cisarua (ca. 1,157 1,500 m asl) tree pole sapling wilding species number 47 30 42 22 family number 26 21 27 21 shannon diversity index 3.42 3.16 3.43 2.19 margalef ’s index 8.89 7.34 8.77 4.67 evennes index 0.89 0.93 0.92 0.71 dominant species castanopsis tungurrut (blume) a.dc. (77.9) cinchona pubescens vahl (64) cinchona pubescens vahl (16.6) elatostema strigosum hassk. (52,9) villebrunea rubescens (blume) wedd. (29.6) antidesma tetrandrum blume (42.4) magnolia liliifera (l.) baill. (14.7) pinanga javana blume (15.8) cinchona pubescens vahl (23.9) ficus ribes reinw.ex blume (18) castanopsis tungurrut (blume) a.dc. (9) symplocos fasciculata zoll. (12,5) schima wallichii choisy (12.4) macropanax dispermus (blume) kunze (16.6) ficus cuspidata reinw. ex blume (8.2) polyalthia subcordata (blume) blume (11.3) castanopsis javanica (blume) a.dc (11.3) camellia sinensis (l.) kuntze (15.9) dendrocnide stimulans (l.f.) chew (8.1) psychotria montana blume (10.2) biotropia vol. 32 no. 3, 2025 294 table 4 floristic composition based on the ivi in selabintana (ca. 1,163 1,829 m asl) tree pole sapling wilding species number 54 29 35 26 family number 31 21 26 23 shannon diversity index 3.46 3.18 3.12 2.86 margalef ’s index 9.82 7 7.32 5.73 evennes index 0.86 0.94 0.87 0.87 dominant species schima wallichii choisy (80.2) lasianthus laevigatus blume (30.4) lasianthus stercorarius blume (28.5) lasianthus laevigatus blume (27.9) macropanax dispermus (blume) kunze (24.3) macropanax dispermus (blume) kunze (26.2) dysoxylum alliaceum (blume) blume (16.2) symplocos costata choisy ex zoll. (18.5) villebrunea rubescens (blume) wedd. (22.7) magnolia liliifera (l.) baill. (25.8) brugmansia suaveolens (humb. & bonpl. ex willd.) sweet (12.4) strobilanthes cernua (15.5) neolitsea javanica (15.6) itea sp. (24.9) polyalthia subcordata (blume) blume (9.5) helicia serrata blume (13.2) acronychia pedunculata (l.) miq. (15) villebrunea rubescens (blume) wedd. (22.1) castanopsis tungurrut (blume) a.dc. (8.5) smilax sp. (11.9) cluster of vegetation along the altitudinal gradient based on tree basal area the dendrogram (fig. 3) collates all locations (cibodas, selabintana, cisarua, and bodogol) into three distinct zones: the colline zone, submontane zone, and montane zone. this clustering was determined by considering the basal area of the tree stage and the altitudinal gradient of the plots. we excluded the pole, sapling, and wilding stages. an overview of four different locations was analyzed to see the combinations of the species found, based on the results shown in figure 3. castanopsis species were present in all three distinct zones and dominated in all zones, but their specific composition and dominance varied across the altitudinal gradient. this variation is a clear indication of the significant role that environmental factors play in the distribution of species. it can be inferred that zones i, ii, and iii serve as ideal habitats for castanopsis, demonstrating the adaptability of these species to diverse environmental conditions along the altitudinal gradient. zone i (500 1,000 m asl) – castanopsislithocarpus and schima wallichii forest. the clustering analysis revealed that within the colline zone, there are four clusters with a similarity level of around 0.3. specifically, plot bdl 10 was clustered with bdl 3, while bdl 2 was clustered with dominant species such as castanopsis tungurrut, lithocarpus pseudomoluccus, maesopsis eminii, schima wallichii, and aglaia sp. this zone comprises six plots located in bodogol (bdl), as it is the only location with vegetation below 1,000 m asl. a total of 64 tree species were recorded in this zone, with dominant species based on basal area (ba), including castanopsis tungurrut, maesopsis eminii, schima wallichii, lithocarpus pseudomoluccus, and aglaia sp. the location is primarily situated on a ridge with deep valleys, which limits species dispersion and exposes them directly to wind effects. on the other hand, bdl 1, bdl 9, and bdl 8 represented transition zones from lowland to submontane zones. dominant species in these transition zones included maesopsis eminii, schima wallichii, lindera polyantha, altingia excelsa, and castanopsis javanica. zone ii (1,000 1,500 m asl) – castanopsis and schima wallichii forest, the submontane cluster zone, known as the big cluster, represented a specific altitude range. within this range, cbs 13 and sbl1 formed a cluster with a 0.5 similarity and a slope range of 10 30%. on the other hand, cbs 12 and csr3 formed a cluster with a 0.4 similarity and a slope range of 12 25%. overall, this zone was home to 110 species, with ten dominant species including castanopsis argentea, castanopsis tungurrut, castanopsis javanica, schima wallichii, altingia excelsa, villebrunea rubescens, castanopsis acuminatissima, ostodes paniculate, helicia serrata, and syzygium rostratum. these castanopsis species thrive in this altitudinal zone due to favorable environmental conditions that support their growth and regeneration. the ecological patterns of castanopsis tungurrut in cibodas biosphere reserve nurdiana & buot 295 temperature gradient and cation exchange capacity (cec) show a positive correlation, indicating that lower temperatures and higher altitudes promote the distribution and germination capacity of castanopsis argentea (hilwan & irfani 2018). zone iii (1,500 2,400 m asl) – schima wallichii, castanopsis, altingia excelsa forest. the montane zone, ranging from 1,500 m asl to 2,400 m asl, is divided into eight clusters with a similarity of 0.2. a total of 69 species were identified in this zone. within the montane zone, cbs5, cbs6, cbs3, cbs1, and cbs3 formed a large cluster compared to other plots in this altitude range. the dominant species in this zone included schima wallichii, castanopsis javanica, dacrycarpus imbricatus, castanopsis argentea, altingia excelsa, castanopsis tungurrut, macropanax dispermus, macropanax concinnus, syzygium rostratum, and acronychia pedunculata. some species overlap in specific locations, indicating a wide distribution and adaptation to different zones. notably, species such as schima wallichii and villebrunea rubescens were found across the altitudinal gradient from 800 m asl to 1,800 m asl. among the three zones being considered, the submontane zone, ranging from 1,000 m asl to 1,500 m asl, stands out with the highest level of diversity. this can be attributed to several factors, including the establishment of a greater number of plots and favorable environmental conditions that provide suitable temperatures, nutrient availability, and water supply, supporting a wide range of species. in contrast, the montane zone has a lower habitat suitability, resulting in reduced species richness in the area. additional research conducted by sang (2009) suggested that soil characteristics significantly influence species richness at medium altitudes, while temperature becomes more influential at higher elevations, and water availability plays a critical role at lower elevations. these factors contribute to the higher diversity observed in the submontane zone compared to the other zones. figure 3 cluster dendrogram of 41 sampling in cibodas biosphere reserve generated by past software based on the basal area of tree species on the location by upgma using bray-curtis index of similarity, with copphen correlation of 0.3464 notes: three altitudinal tree vegetation zones were identified from 750 1,830 m asl. zone i (500 1000 m asl) – castanopsislithocarpus and schima wallichii forest; zone ii (1,000 1,500 m asl) – castanopsis and schima wallichii forest; and zone iii (1,500 2,400 m asl) – schima wallichii, castanopsis, altingia excelsa forest. biotropia vol. 32 no. 3, 2025 296 population structure and habitat preferences of c. tungurrut the population structure of c. tungurrut exhibited an interrupted inverted “j” curve (fig. 4), indicating a reasonably good regeneration of the species. similar interrupted inverted “j” curves have been observed in studies on plant diversity in ethiopia by mekonen et al. (2015), indicating successful regeneration in specific areas. however, in this case, the number of poles is lower than that of mature trees, indicating disturbance in this regeneration phase. c. tungurrut had a limited distribution range within a specific altitude range. the tree was found to occur between 750 and 1,500 m asl, but it was absent at higher altitudes. out of the 41 plots examined, c. tungurrut dominated in 10 plots. these plots included bdl3 (823 m), csr 7 (1,219 m), csr 6 (1,264 m), csr 1 (1,306 m), cbs13 (1,373 m), sbl 1 (1,387 m), cbs 12 (1,403 m), csr3 (1,413 m), sbl2 (1,459 m), and csr 4 (1,461 m). the density of c. tungurrut varied across different locations, with bodogol having a density of 25 individuals/ha, cibodas with 10.7 individuals/ha, cisarua with 53.1 individuals/ha, and selabintana with five individuals/ha. the slope percentage, revealed that c. tungurrut species tends to be more abundant in areas with steep to extremely steep slopes, ranging from 25% to greater than 45% (table 5). the slope category refers to the ministerial decree agriculture of the republic of indonesia no. 837/kpts/um/11/1980 about criteria and procedures for designating protected forests. in addition, the distribution pattern of c. tungurrut across all 4 locations exhibited a clumped distribution with a non-uniform distribution (table 6). based on the standardized morisita index, c. tungurrut exhibited a clumped distribution pattern, similar to that of castanopsis argentea observed in gede pangrango national park (hilwan & irfani 2018). this clumping pattern is commonly observed in natural habitats and serves as an adaptation to the availability of nutrients and seed dispersal. furthermore, plant clumping can impact plant competitiveness. eccles et al. (2001) found that clumped plants tend to outperform isolated plants, while béland & baldocchi (2020) discovered that clumping factor values are higher in wet areas compared to dry ones. however, callaway (1997) found that both competition and facilitation play significant roles in plant communities. these findings suggest that while clumping is one factor that may influence plant competitiveness, it is just one of several variables to consider when assessing plant competitiveness. the influence of plant clumping on plant competitiveness is complex and contextdependent. figure 4 the population structure of c. tungurrut of all locations table 5 distribution of c. tungurrut tree on slope category slope (%) number of trees category 0 < 8 1 flat 8< 15 5 sloping 15 < 25 7 rather steep 25 < 45 12 steep > 45 13 extremely steep ecological patterns of castanopsis tungurrut in cibodas biosphere reserve nurdiana & buot 297 table 6 morisita index and distribution pattern of c. tungurrut of all locations index dispersion morisita (id) uniform index (mu) clumped index (mc) standardized morisita index (ip) distribution pattern 1.1 2.63 1.68 0.07 clumped conclusion the assessment conducted at four locations within cibodas biosphere reserve revealed variations in the number of species and the presence of c. tungurrut. these variations were observed in relation to altitude and the presence of invasive alien species. the ideal habitat for c. tungurrut was found to be within the altitude range of 750 to 1,500 m asl or in zones i and ii, particularly in areas with sloping terrain and abundant vegetation. as the altitude range deviated from this ideal, either lower or higher, the species was either absent or decreased in abundance. c. tungurrut, a unique species, showed a distinct preference for growing on steep to extremely steep slopes, as opposed to flat areas. this unique characteristic of the species is intriguing and warrants further investigation. it is likely that the species is more inclined to inhabit locations with steeper terrain. the species exhibited a clumped distribution pattern with an interrupted inverted “j” curve, indicating that the regeneration of the species is hindered, potentially due to natural disasters or human activities. acknowledgments we wish to thanks to searca for research fund as the part of phd scholarship and institute of biological sciences, cas, university of the philippines los baños for the permit. we are very grateful to mr. nudin, mr. ujang rustandi, mr. rustandi, mr. emus, and mr. cahyadi staff member cibodas botanic garden, national research and innovation agency (brin) for field work assistance. we also acknowledge with gratitude the support from gunung gede pangrango national park staff member mr. ae, mr. dayat and mr. komar for their support. references béland m, baldocchi d. 2020. is foliage clumping an outcome of resource limitations within forests? 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clippinger et al. 2024) and pseudoperonospora cubensis (wallace et al. 2020; sun et al. 2022), which predominantly infect green plant tissues, especially the leaves. typical symptoms include irregular spots on the leaves, which can range from pale green to yellow or brown in color (newark et al. 2019; purayannur et al. 2021). favorable conditions, including high humidity and moderate temperatures, facilitate rapid disease spread through wind-borne spores or rain splashes, often resulting in large-scale outbreaks. https://doi.org/10.11598/btb.2025.32.2.2448 the efficacy of bacterial and fungal antagonist suspensions in controlling foliar mildew disease ramona et al. 163 in severe cases, downy mildew can lead to defoliation, stunted growth, poor fruit quality, and even total crop failure, making it a critical challenge for growers (purayannur et al. 2021; marone fassolo et al. 2022). as global temperatures and weather patterns shift, the range and frequency of downy mildew outbreaks are projected to increase, emphasizing the need for integrated disease management strategies that combine resistant crop varieties, cultural practices, and sustainable control measures (singh et al. 2023). globally, downy mildew significantly impacts food security and agricultural output, particularly in areas where susceptible crops are grown extensively. plasmopara viticola for example, continues to challenge grape production in europe, north america, and australia, leading to reduced harvests and increased dependency on fungicides (koledenkova et al. 2022). similarly, p. cubensis is a key limitation for cucurbit farming in asia, the americas, and parts of africa, where recurrent outbreaks cause severe losses to commercial yields (salcedo et al. 2020). downy mildew, a destructive disease affecting the foliage of many crops, has traditionally been managed using several strategies. these include crop rotation, which interrupts the pathogen’s lifecycle by alternating host availability, and the deployment of resistant plant varieties that utilize genetic traits to fend off infections (tör et al. 2023; clippinger et al. 2024). efficient irrigation practices, such as drip systems and proper drainage, reduce moisture levels conducive to the disease’s growth (tör et al. 2023; clippinger et al. 2024). chemical fungicides remain a common solution, offering rapid disease suppression during critical periods (toffolatti et al. 2024; clippinger et al. 2024). however, their frequent use has raised concerns over environmental pollution, public health, and the evolution of fungicide-resistant pathogens (ons et al. 2020; islam et al. 2024). current research underscores the value of integrated disease management (idm) approaches that combine various practices to achieve effective and sustainable disease control. studies by corkley et al. (2022) and wang et al. (2022) demonstrated the advantages of integrating resistant crop varieties with targeted fungicide applications to manage disease outbreaks. this dual approach, leveraging genetic resistance to reduce pathogen pressure and applying fungicides strategically to mitigate severe infections, minimizes reliance on chemicals while sustaining crop yields. such strategies align with contemporary goals of reducing the environmental impact of agriculture. in growing concerns about fungicide overuse, biological control options are becoming increasingly popular within idm frameworks (ons et al. 2020). compost teas, liquid extracts produced through compost fermentation, represent one promising strategy (coker & ozores-hampton 2021). these solutions are rich in beneficial microorganisms and bioactive compounds that combat pathogens and enhance plant immunity (ramírez-gottfried et al. 2023). they offer a natural and sustainable alternative or complement to synthetic chemicals, integrating seamlessly into environmentally conscious agricultural practices. recent research also highlights the role of specific biocontrol agents, such as l. antibioticus, p. corrugata, and trichoderma spp. in managing zucchini diseases such as downy mildew (ayaz et al. 2023). l. antibioticus produces antibiotics that suppress pseudoperonospora cubensis (drenker et al. 2023), the pathogen responsible for downy mildew, while p. corrugata inhibits spore germination and disease spread through antagonistic interactions (ramona et al. 2020). meanwhile, trichoderma sp. not only protect plants by colonizing root systems but also enhance systemic resistance, making plants more resilient to infections (chakraborty et al. 2020). combining traditional practices with these innovative biological tools provides a balanced way forward (clippinger et al. 2024). this integrated approach reduces harmful inputs while addressing key challenges in plant health management, promoting sustainable and resilient agricultural systems. the effectiveness of compost teas in managing plant diseases arises primarily from the diversity of their microbial communities, which actively suppress pathogens while supporting plant health (barghouth et al. 2023). beneficial microbes, such as bacillus and pseudomonas produce antimicrobial compounds that inhibit pathogen growth, while fungi like trichoderma outcompete pathogens for space and nutrients and form protective root barriers (st. martin et al. 2020). some microorganisms in compost teas also trigger systemic resistance, equipping plants with enhanced defense mechanisms against diverse pathogens (emannuel oliveira vieira et al. 2024). biotropia vol. 32 no. 2, 2025 164 sarmah et al. (2020) emphasized that enriching compost teas with targeted strains, including trichoderma spp. and bacillus spp., boosts their efficacy against a broader spectrum of diseases. this approach addresses common challenges like variability in compost tea effectiveness, which often results from differences in compost quality and brewing conditions. introducing wellcharacterized microbial strains ensures consistency and reliability, enhancing the utility of compost teas in agriculture. beyond their role in disease management, compost teas promote healthier plants by improving soil quality and nutrient availability (de corato, 2020; ramírez-gottfried et al. 2023). their microorganisms facilitate the release of essential nutrients like nitrogen and phosphorus, boosting plant growth and vitality (singh et al. 2022). by combining disease suppression with nutrient enhancement, compost teas serve as a multifunctional tool that aligns with the goals of sustainable agriculture. leveraging these biological solutions offers a scalable and eco-friendly way to address modern agricultural challenges. based on the above rationale, this research focused on assessing the effectiveness of bacterial suspensions of lysobacter antibioticus bali g and pseudomonas corrugata saj 6 in tsb, as well as a fungal spore suspension of td22 in saline, for controlling downy mildew in zucchini plants. the study aimed to offer alternatives to conventional “compost teas” by using selective microbial antagonists as active agents for disease management in important crops, including, zucchini, grapes, and poppies. materials and methods bacterial and fungal antagonist isolates three antagonistic microorganisms, including lysobacter antibioticus bali g, pseudomonas corrugata saj6, and trichoderma sp. td22 that effective against sclerotinia minor in lettuce plants (ramona et al. 2022), were evaluated in the current study for their potential to manage a foliar mildew disease, downy mildew, in zucchini. the l. antibioticus bali g and p. corrugata saj6 were obtained from lettuce farms in bedugul, bali, indonesia, whereas the trichoderma sp. td22 was obtained from dr dean metcalf, a senior researcher at department of primary industries, parks, water, and environment (dpiwe) in tasmania, australia. downy mildew isolate the pathogen analyzed in this research was obtained from diseased grape leaves sourced from the horticultural research centre (hrc) at the university of tasmania, australia. the characteristic leaf damage initially suggested identification as downy mildew. as obligate parasites, downy mildew pathogens necessitate the need for living tissue; therefore, the infected leaves were collected just prior to preparing the pathogen suspension in saline solution. preparation of antagonist suspensions the bacterial antagonists were grown in a medium containing 0.5% (w/v) trypticase soya broth (oxoid) at 25 °c for 48 hours without agitation, achieving a final concentration of roughly 10⁸ cells/ml. the trichoderma sp. td22 spores were obtained from wood fiber waste (wfw) compost, which had previously supported td22 cultivation during our previous lettuce or pyrethrum experiments. to extract the spores, the td22-grown wfw compost was agitated to release most spores in a saline solution at a 1 : 10 (w/v) ratio for around 10 minutes before being utilized. the trichoderma sp. td22 spore density obtained was 8.42 ± 0.01 log10 cfu/ml (average of triplicates measurements with an improved new bauer hemacytometer). preparation of the pathogen suspension approximately 10 g of infected grape leaves were placed in 200 ml of sterile saline solution (0.85% nacl) and shaken thoroughly to release the pathogen from the leaves. the mixture was then sieved with a piece of sterile cloth to remove the leaf debris, before being used in the trials. a density of 2.7 x 107 propagules/ml were obtained following determination with an improved new bauer hemacytometer. glasshouse scale experiments zucchini seeds (‘blackjack’ yates®) were sown in 1.5 l pots containing a steam-sterilized standard potting mix. after 14 days, the seedlings’ leaves were sprayed with 2 ml of antagonist suspensions. the study included various combinations of antagonists, such as l. antibioticus + p. corrugata, l. antibioticus + trichoderma sp. td22, p. corrugata + trichoderma sp. td22, and a mixture of all three (l. antibioticus + p. corrugata + trichoderma sp. td22) in equal proportions (v/v). three days following the antagonist application, a 2 ml suspension of the efficacy of bacterial and fungal antagonist suspensions in controlling foliar mildew disease ramona et al. 165 the pathogen was sprayed onto the leaves. each treatment consisted of five replicate pots, with each pot holding a single 3 weeks old plant or approximately 10 cm in height. control groups were either sprayed with only the pathogen or with a saline solution lacking both pathogens and antagonists. the pots were maintained in a shaded house for eight weeks, with infection levels evaluated at two and five weeks post-pathogen application. to avoid cross-contamination, control pots (a0b0) were positioned separately from those exposed to the pathogen. infection severity on the leaves was assessed using a 0 5 scale, as described by nakasaki et al. (1998), where 0 indicated no visible symptoms, 1 represented infection on ≤ 20% of the leaf area, 2 on 21 40%, 3 on 41 60%, 4 on 61 80%, and 5 on 81 100%. establishment of the antagonists on the zucchini leaves the experiment was completed six weeks after the pathogen inoculation, with efforts made to recover antagonists from randomly chosen healthy leaves. to assess the colonization of bacterial antagonists, 10 g of leaves from each pot were mixed with 90 ml of saline solution and homogenized for 3 5 minutes. colony-forming units (cfu) were quantified using dilution plating on trypticase soya agar (tsa), followed by incubation at 25 °c for 2 5 days. the identities of the bacteria were verified by comparing colony characteristics on tsa with those of the original strains to evaluate the presence of trichoderma sp. td22, 20 leaf plugs (~3x3 mm) per treatment were aseptically collected and placed on pectin agar medium (merck) containing 60 µg/ml tetracycline. these samples were incubated at 25 °c for 4 7 days to allow fungal growth. emerging fungal colonies were isolated and grown on the same medium to compare their morphology with a trichoderma sp. td22 stock culture. observations were extended for one week to monitor conidial development for accurate identification. data analysis the data obtained from this study were analyzed using analysis of variance (anova), which was carried out with the help of minitab software for windows. anova enabled the assessment of any significant variations between the treatment groups. to identify specific differences between group means, the least significant difference (lsd) test was employed following the anova procedure. the lsd test is a post-hoc statistical test that compares means to detect significant differences. a significance threshold of p < 0.05 was set to determine whether the differences observed were statistically meaningful, ensuring the results were valid and reliable. results and discussion the efficacy of the selected antagonistic fungus and bacteria in preventing zucchini leaves from downy mildew infection, as evaluated by the percentage of infected leaves and the disease severity index is presented in figure 1. the use of antagonists, excluding treatment a2b1 (plants treated with l. antibioticus and the pathogen), significantly lowered disease incidence compared to the untreated-pathogen control (a0b1) two weeks post-infection (p < 0.05) (fig. 1). the most effective disease suppression was achieved with the fungal antagonist trichoderma sp. td22 (a3b1), providing an 83% reduction in disease (calculated from disease incidence) compared to the control group at two-weeks postinoculation. however, this effect declined and became statistically insignificant after five weeks. a non-significant synergistic effect (p > 0.05) was noted when l. antibioticus and p. corrugata were applied in combination (treatment a4b1), resulting in 57% disease protection (calculated from disease incidence; fig. 1a) relative to the untreatedpathogen control. this was higher (higher protection) than when each bacterial antagonist was applied individually (a1b1 or a2b1; fig. 1). when the trichoderma sp. td22 was combined with the bacterial antagonists, its effectiveness diminished, possibly due to reduced levels of the primary biocidal compounds on leaf surfaces (poromarto et al. 2021). additionally, the lack of synergy between the fungal and bacterial antagonists could be from antagonistic interactions, as dual-culture tests showed inhibition zones produced by both bacteria against trichoderma sp. td22 (fig. 2). in contrast to our findings, poveda & eugui (2022) suggested synergic effect when they were applied in combination in a sustainable agriculture system. biotropia vol. 32 no. 2, 2025 166 figure 1 the effectiveness of lysobacter antibioticus bali g, pseudomonas corrugata saj6, and trichoderma sp. td22 to prevent zucchini plants from foliar downy mildew infection in a glasshouse scale experiment notes: a = percentage of infected leaves; b = disease severity index; the assessments were conducted at 2 and 5 weeks after infection; treatments applied: a0b0 = control group (no antagonist or pathogen applied); a0b1 = control treatment (pathogen only applied); a1b1 = plants treated with p. corrugata and pathogen; a2b1 = plants treated with l. antibioticus and pathogen; a3b1 = plants treated with td22 and pathogen; a4b1 = plants treated with a mixture of l. antibioticus, p. corrugata, and pathogen; a5b1 = plants treated with a mixture of l. antibioticus, td22, and pathogen; a6b1 = plants treated with a mixture of p. corrugata, td22, and pathogen; a7b1 = plants treated with a mixture of all three antagonists and pathogen; each bar represents the mean of disease ± standard error. figure 2 in vitro dual culture assays between l. antibioticus bali g and trichoderma sp. td22 (left) and between p. corrugata saj 6 and trichoderma sp. td22 (right) on tsa plates after incubation at 25 °c for three days an interesting phenomenon was observed in this glasshouse trial where some plants in the control group (no antagonist or pathogen applied or a0b0) showed disease symptom (fig. 1). this could be due to cross contamination from the infected leaves nearby (plants in pots treated with pathogen). water splash during irrigation or the blowing wind could be the main cause of this cross contamination. the protection shown by the antagonists against downy mildew was no longer significant (p > 0.05) after five weeks. at that time, 60% of the leaves in the untreated control group were infected. the survival rate of the applied antagonists on the leaf surfaces was observed to be low. for instance, trichoderma sp. td22 was recovered from only two out of 20 leaf samples taken from treatment a5b1 (plants treated with l. antibioticus, trichoderma sp. td22, and the pathogen) five weeks post-application, and it was the efficacy of bacterial and fungal antagonist suspensions in controlling foliar mildew disease ramona et al. 167 undetectable in samples from other treatments. likewise, the bacterial antagonists applied to the leaves were not found (exist) from any treatments after the five-week exposure. applying fungal and bacterial antagonists, either individually or in combination, showed potential for managing foliar diseases, such as downy mildew in zucchini plants. significant reductions in disease incidence (p < 0.05) were observed two weeks after the pathogen was introduced (fig. 1). these findings indicate the possibility of modifying compost to develop specialized “compost teas” or cultivating specific biocontrol agents to manage foliar pathogens. the application of trichoderma sp. td22 yielded the most successful results in our study, particularly when the strain was cultivated in a mixture of wood fiber waste (wfw) compost and millet seed (80 : 20 w/w), as detailed in our previous research (ramona et al. 2022). the biocontrol agent was stored for approximately 10 months at around 20 °c before being applied in the current study. in efforts to further improve the effectiveness of both fungal and bacterial antagonists, recent research by brost (2020) suggested that the addition of chelating agents and detergents may enhance the activity of these biocontrol agents. by incorporating such additives, it may be possible to increase the stability, viability, and overall efficacy of microbial antagonists under various application conditions. previous studies have pointed out the compatibility challenges between bacterial and fungal antagonists, which can significantly influence the success of biocontrol strategies. elsharkawy et al. (2021) for example, reported that the combination of t. harzianum with p. fluorescens reduced the efficacy of the fungus in controlling aphanomyces euteiches, a root rot pathogen in peas. this finding highlights the importance of understanding microbial interactions, as incompatibility can undermine the individual effectiveness of biocontrol agents. on the other hand, research by amirthalingam et al. (2020) and ntakirutimana et al. (2024) showed that using mixed cultures of antagonistic microorganisms often improved disease control. these contrasting outcomes emphasize the need for comprehensive evaluations of compatibility when developing microbial formulations for disease management. the broad-spectrum potential of trichoderma strains, such as td22, is particularly significant in this regard. its ability to combat various fungal pathogens has been consistently demonstrated in several studies, establishing it as a promising biocontrol agent (ali et al. 2021; kumar et al. 2023). in our current study, trichoderma sp. td22 achieved an 83% reduction in zucchini downy mildew during a two-week glasshouse trial, offering a level of protection comparable to chemical fungicides. such results validate its potential as a sustainable alternative to synthetic treatments. however, achieving similar effectiveness in field conditions remains challenging, as environmental factors often necessitate frequent reapplication to maintain the agent’s activity and persistence. reapplication intervals for biocontrol agents align closely with those recommended for chemical fungicides. jones et al. (2021) for example, suggested reapplying fungicides every 10 14 days to sustain protection against downy mildew. this similarity underscores the practical challenges of deploying biological control agents while highlighting their potential integration into established disease management practices. optimizing formulations, improving the stability of biocontrol products, and addressing compatibility issues are essential for advancing the reliability and scalability of biological control solutions. mildew symptoms observed in the nil-pathogen control group (a0b0) after five weeks (fig. 1) were likely caused by natural infection from spores originating in a nearby vineyard with known disease presence or accidental transfer through human activity. despite this unintended exposure, the infection levels remained minimal compared to those in inoculated plants, ensuring that the overall conclusions of the study were unaffected. this minimal infection demonstrates the robustness of the experimental setup in isolating key variables under investigation. the limited persistence of biocontrol agents on leaf surfaces was expected due to several environmental challenges. factors such as low moisture levels, ultraviolet (uv) radiation from sunlight, and the washing effect of overhead irrigation contributed to the reduced survival of these agents (devi 2024). these environmental conditions are well-known to limit the effectiveness of biocontrol organisms in outdoor applications. a thorough review by fedele et al. (2020) discussed the impacts of environmental stressors, including humidity, temperature variations, and irrigation practices, on the survival and performance of biocontrol agents in field environments. these biotropia vol. 32 no. 2, 2025 168 findings emphasize the need for improved application techniques and protective formulations to enhance the stability and efficacy of biocontrol agents under field conditions. conclusion the biological control agents evaluated in this study, with the exception of l. antibioticus bali g, demonstrated significant effectiveness (p < 0.05) in protecting zucchini leaves from downy mildew during the first two weeks of the glasshouse trial. these findings highlighted their potential for managing foliar diseases. it is necessary to maintain protection beyond 14 days by reapplying the biocontrol agents. the fungal antagonist trichoderma sp. td22 was found to be incompatible with both l. antibioticus bali g and p. corrugata saj6. in contrast, the combination of l. antibioticus bali g and p. corrugata saj6 appeared to be compatible but did not significantly enhanced disease control when compared to p. corrugata saj6 alone. the survival of all three antagonists on zucchini leaf surfaces was notably very low. acknowledgment the authors express their gratitude to the ministry of higher education, research, and technology of the republic of indonesia for partially funding this research. appreciation is also extended to dr. dean metcalf, the head of integrated laboratory for biosciences and biotechnology, udayana university, and the school of agricultural sciences, faculty of sciences and engineering, tasmania university for providing the trichoderma isolate, necessary consumables, and equipment, respectively, used in this study. our acknowledgment should also go to prof. john bowman for his assistance in the molecular identification of our bacterial isolates. references ali a, zeshan ma, mehtab m, khursheed s, mudasir m, abid m, mahdi m, rauf ha, ameer s, younis m, et al. 2021. a comprehensive note on trichoderma as a potential biocontrol agent against soil borne fungal pathogens: a review. plant prot 5(3):171-96. doi: 10.33804/ pp.005.03.3934 amirthalingam vk, tewari a, sharma m, sharma r, kumar r j. 2020. evaluation of bioagents for their compatibility in the development of consortium for enhanced efficacy. j biol control 34(2):164-67. doi: 10.18311/jbc/2020/23179 ayaz m, li c-h, ali q, zhao w, chi y-k, shafiq m, …, huang w-k. 2023. bacterial and fungal biocontrol agents for plant disease protection: journey from lab to field, current status, challenges, and global perspectives. molecules 28(18):6735. doi: 10.3390/molecules28186735 barghouth z, khazzam e, ramlawi s, wong a, smith ml, avis tj. 2023. microbial compost tea properties affect suppression of strawberry grey mould (botrytis cinerea pers.). biocontrol sci technol 33(1):1-18. doi: 10.1080/09583157.2022.2141688 brost a. 2020. chelation-based enhancement of novel and commercially available antimicrobials against foodborne pathogens. 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naujan lake, philippines elsie y. guibone1*, katherine p. sanchez-escalona2, abelle f. balagtas1, fritz dustin m. fiedalan1, kidelyn i. lomio2 1institute of fisheries, mindoro state university bongabong campus, brgy. labasan, bongabong, oriental mindoro 5211, philippines 2center for environmental studies, college of arts and sciences, mindoro state university-main campus, alcate, victoria, oriental mindoro 5205, philippines article higlights • the naujan white goby (glossogobius aureus) is an economically important commodity, valued locally for dried fish, making it vulnerable to overexploitation. • domestication efforts attempt to lessen fishing pressure while conserving wild populations; however, the feeding ecology of this native fish in naujan lake is still unknown. • understanding glossogobius aureus’ diet composition and feeding habits from its natural habitat is critical for establishing its culture. • glossogobius aureus is a benthic carnivore that feeds primarily on shrimp, followed by fish and other prey items. article information received : 7 february 2025 revised : 28 march 2025 accepted : 24 april 2025 *corresponding author, e-mail: elsie.guibone@minsu.edu.ph research paper abstract naujan white goby (glossogobius aureus), commonly processed as dried fish, is a local delicacy in oriental mindoro, philippines. however, the product’s growing popularity and rising demand have raised concerns about the overexploitation of this economically valuable species. while efforts to domesticate g. aureus have been initiated as a conservation measure, information on its feeding ecology in naujan lake national park is lacking. this study investigated the diet composition, feeding habits, feeding intensity, and seasonal and site-based variations in the diet of  g. aureus in naujan lake by monthly sampling from february 2020 to january 2021. a total of 1,938 g. aureus comprising 1,094 males and 844 females were collected, ranging from 75 mm to 280 mm in total length. glossogobius aureus is a benthic carnivore. shrimp forms the bulk of its diet, followed by fish and other prey. occasional opportunistic feeding was observed in june to august when the usual prey were scarce. pairwise comparison showed a significantly high percentage occurrence of shrimp observed during the dry months (january to march) and rainy months (september to november) compared to the lowest ingestion recorded in june and july (p < 0.05). the dietary patterns varied significantly across the six sampling sites and seasonal periods. feeding intensity was the highest before spawning and declined during the spawning period. males exhibited greater feeding activity compared to females. the findings offer valuable insights into the feeding patterns of g. aureus, which could help optimize dietary strategies for its successful culture in captivity. keywords: feeding ecology, foraging behavior, freshwater fish, gobiidae, gut content analysis. introduction the foraging strategy and fish diet are crucial factors in understanding the trophic interactions among fish in a given community (manko 2016). the dietary composition is influenced by various factors, including diel cycles (carman et al. 2006), fish sizes (dinh et al. 2017), habitats (dinh et al. 2020), and seasons (brush et al. 2012). gut content analysis, a standard method in fish ecology, provides critical insights into dietary preferences, offering valuable information for fisheries management and conservation (chipps & garvey 2007; kamler & pope 2001; pikitch et al. 2004). additionally, understanding feeding patterns may aid in optimizing feeding protocols for aquaculture fish species (fabay et al. 2021; ribeiro et al. 2022). naujan lake national park (nlnp), located on the northeast portion of mindoro island, is the 5th largest lake in the philippines. this protected area is home to various economically important fishery resources. lit et al. (2011) listed twenty-nine (29) fish species in the lake. sixteen (16) of which are https://doi.org/10.11598/btb.2025.32.2.2450 biotropia vol. 32 no. 2, 2025 172 native species, including the three glossogobius spp.: glossogobius celebius, glossogobius. giuris, and glossogobius aureus. naujan white goby (nwg) glossogobius aureus (akihito & meguro 1975) is among the most commercially important and dominant species. this amphidromous fish could inhabit fresh, brackish, and seawater environments during different stages of its life cycle. dinh et al. (2021) reported a significant decline in the length at maturity (lm) of this goby species in the brackish water, suggesting that freshwater is a more favorable habitat than seawater. glossogobius aureus spends its larval stage drifting in a marine environment before migrating to freshwater habitats to mature and reproduce (abdulmalik-labe et al. 2023). in naujan lake, the commercial value of g. aureus, driven by local demand for dried fish, rendered it particularly at risk of overexploitation. interest in domesticating the species has been initiated to potentially reduce fishing efforts and preserve the remaining population in the wild. however, information regarding its feeding ecology in naujan lake is scarce. understanding the dietary ecology of nwg is a fundamental prerequisite for facilitating the culture of this native species. so far, published studies have primarily focused on flathead goby (g. giuris) feeding and dietary patterns in other lakes across the philippines. common diet components include fish, shrimp, insects, and zooplankton (bejer 2015; lagbas et al. 2017; marquez 1960; uy et al. 2019). this study aimed to: 1) determine the diet composition of g. aureus in naujan lake; 2) assess seasonal and sitebased variations in feeding habits; and 3) analyze feeding intensity in relation to reproductive patterns. materials and methods sampling site six known fishing grounds for naujan white goby (nwg) were chosen as sampling sites for the study. these sites are located around naujan lake and are within the four municipalities surrounding the lake. butas (site 1) in barangay bayani, is located on the northern part of the lake, at the river outlet where butas river drains towards tablas strait. site 2 was directly at the mouth of borbocolon river, where water drained into the lake. together with malayas (site 4) and pasi (site 5), these sites were positioned on the lake’s western side. site 3 was in barangay tigbao on the eastern coast of the lake, along with site 6 (tagbakin). all sites were associated with river mouths except site 1, which was an outlet. sites 1 and 3 are within the municipality of naujan, while sites 2 and 4 are within the municipality of victoria. site 5 is under the jurisdiction of the municipality of socorro, while site 6 is within the municipality of pola (fig. 1). only site 1 had a salinity of 3 5 ppt. the climate in all sites was type iii, with no pronounced wet or dry season and maximum rainfall from june to september. the warmest month was may, the wettest was december, the coldest was february, and the driest was april. the average annual temperature ranged from 28 °c to 32 °c (weather & climate n.d.). figure 1 maps of southeast asia (lower left), the philippines (upper left) and naujan lake (right) showing the six sampling sites diet composition and feeding habits of naujan white goby guibone et al. 173 fish collection fish sampling was done experimentally on all sites using a 1,000-m gill net number 14 with a mesh size of approximately 1.5 cm. the gill nets were used to capture a minimum of 50 adult fish and set for 8 hours to standardize fishing efforts in all sampling sites. samples were collected 2 or 3 days before a full moon every month from february 2020 to january 2021. collected samples were stored in an iced-styrofoam box and transported to the fisheries biology laboratory of the institute of fisheries, mindoro state university bongabong campus for further examination. the fish were identified morphologically based on the arrangement of pit organs and sensory papillae (akihito & meguro 1975; abdulmalik-labe et al. 2022). gut content analysis the gut content analysis was conducted using the methods described by dinh et al. (2022). in the laboratory, sex, body weight (bw, g), and total length (tl, mm) of individual adult fish were determined. the whole gut was removed from each fish, weighed (gut-weight, g), and measured (gut length, mm). gut contents were taken by dissecting the stomach and examined under an lb 220 compound microscope. the stomach was categorized as containing food (non-empty) or without food (empty). the wet weight of the prey item was recorded to the nearest 0.01 g using an analytical balance and classified based on the following prey types: shrimp, fish, phytoplankton, zooplankton, mollusks, insect larvae, and other food items. prey identification involved analyzing both partially and fully digested remains. partially digested prey items are characterized by degraded soft tissues and varying degrees of fragmentation, while fully digested prey items have unrecognizable structure and the gut appears homogenous. for the identification of prey items, presence of hard structures was used, specifically head fragments, otoliths, teeth, vertebrae, carapaces, shells (exoskeleton) and legs. for shrimp, a single individual is equivalent to 1 carapace, 5 pairs of walking legs, and 1 abdominal region. separately, vertebrae, head pieces, and otoliths were identified among the fish remains. to estimate the individuals for both fish and shrimp, the most commonly occurring remains were identified. miscellaneous food items, such as prey bones, plant detritus, sand, mud, and other unidentifiable gut materials were not included as they constitute only smaller proportions of the diet. the following indices were determined from 1,646 g. aureus with stomach contents to characterize its dietary patterns and food preferences: 1. frequency of occurrence: to depict seasonal changes in diet composition, computed following hynes (1950): %o = 100 ni/n where: ni = the number of fish containing prey i n = the number of fish whose stomachs contain food regardless of the amount 2. index of relative importance (iri): to assess the dietary importance of various prey items consumed by the naujan white goby, calculated following hyslop (1980): iri = %o (∑ (%v + %n)) where: %o = the percentage of frequency of occurrence of each food item %v = the percentage by volume of the food item %n = the percentage by number of food item feeding intensity and feeding habits the seasonal variation in the feeding activity of nwg was assessed by determining the gastrosomatic index (gasi). the gasi values were obtained by dividing the gut weight by fish weight, then multiply by 100 (biswas 1993). relative gut length (rgl) was also measured to determine the feeding habits of nwg by calculating the ratio of gut length with the total length of the fish. a relative gut length (rgl) of < 1 indicates a carnivorous diet, 1 < rgl < 3 and rgl > 3 signifies omnivorous and herbivorous diets, respectively (borlongan et al. 2002; wardcampbell et al. 2005). statistical analysis alternative kruskal-wallis test was performed to assess the seasonal and site differences in the feeding intensity of nwg at p < 0.05. the test was used since the normality and homoscedasticity assumptions were unmet. pairwise comparisons were performed following significant results to highlight specific differences among groups. biotropia vol. 32 no. 2, 2025 174 significant values have been adjusted by the bonferroni correction for multiple tests. differences in feeding intensity between male and female nwg were evaluated using a t-test. data analyses were performed using spss software v26 at p < 0.05 significance level. results and discussion diet composition of g. aureus a total of 1,938 nwg (g. aureus) (1,094 male and 844 female), ranging from 75 mm (3.33 g) to 280 mm (148 g), were collected from february 2020 to january 2021. site 6 (tagbakin) had the highest number of fish samples collected, followed by site 3 (tigbao). site 1 (butas), site 2 (borbocolon), site 4 (malayas), and site 5 (pasi) had sample counts of 292, 307, 260, and 216, respectively. the g. aureus has been seen foraging on a variety of food throughout the year. the five prey items were shrimp, fish, phytoplankton, zooplankton, and insect larvae. shrimp constituted the bulk of the food consumed (40.52%), while mollusks showed the lowest occurrence frequency (0.26%). fish and phytoplankton were observed to have relatively high occurrence in the gut. the remaining food items, such as zooplankton, insect larvae, and mollusks, were the rarely consumed diets. the index of relative importance (iri) also indicated that shrimp is the primary food item nwg consumes (table 1). fish comprised the next essential prey item, followed by phytoplankton. the rest of the food items were occasionally consumed. similar findings were reported for g. aureus in the mekong delta, where shrimp, particularly acetes spp., were identified as the primary food source; additionally, diets were entirely of animal origin, consisting predominantly of zoobenthos (phan et al. 2023). differences in the diets within the same fish species have also been observed in g. giuris captured from distinct water bodies. in the philippines, adult g. giuris in lake mainit predominantly consumes fish, followed by shrimp (uy et al. 2019). in contrast, those in lake taal primarily feed on crustacean eggs, with shrimp absent from the diet (bejer 2015). these dietary differences may be influenced by food availability, environmental conditions, and habitat-specific resources (heng et al. 2018). therefore, discriminating the diet composition of g. aureus from its natural habitat is necessary to have a basis for its nutritional requirements when cultured under captive conditions. seasonal variation in the diet the seasonal fluctuation in the diet composition of g. aureus in naujan lake was clearly shown in figure 2. pairwise comparison showed a significantly high percentage occurrence of shrimp observed during the dry months (january to april) and rainy months (september to november) compared to the lowest ingestion recorded in june and july (p < 0.05). teleost fish was the most preferred food during the onset of the cool season in december and the end of the hot, dry season in may. seasonal variations in the diet components were also found in g. giuris but were not observed in g. sparsipapillus (dinh et al. 2024; tran et al. 2021). this pattern may reflect seasonal variations in shrimp availability, environmental conditions, or changes in the feeding activity of the fish. the decline in shrimp consumption during june and july may coincide with shifts in prey abundance or other ecological factors influencing prey-predator dynamics (heng et al. 2018). phytoplankton was the preferred diet, recorded only in june, july, and august. glossogobius aureus may have exhibited seasonal opportunism in feeding during this period, which occasionally consumes readily available and abundant phytoplankton without being classified as omnivorous. beaudoin et al. (1999) reported opportunistic feeding of carnivorous north pike when the usual prey is scarce. in naujan lake, shrimp were recorded in a minimal proportion of the nwg diet from june to august, which may likely lead to occasional opportunistic feeding behavior. the high occurrence of phytoplankton in the gut samples of adult nwg collected during the rainy months might be triggered by an increase in the concentration of nutrients carried to the lake by runoff, particularly in butas and tagbakin. additionally, the consumption of phytoplankton may indicate a trophic transfer of plant material within the food chain, as the fish could ingest small amounts of phytoplankton found in the stomachs of its prey (horn & ferry-graham 2006). diet composition and feeding habits of naujan white goby guibone et al. 175 table 1 index of relative importance (iri) of various dietary components of nwg g. aureus food items %ni %vi %oi (%ni + %vi) x %oi iri rank fish 21.97 36.21 23.44 1,363.74 23.77 2 shrimp 34.64 44.60 40.52 3,210.80 55.96 1 mollusc 0.14 0.00 0.26 0.04 0.00 6 zooplankton 6.75 5.55 5.60 68.88 1.20 4 phytoplankton 31.88 9.22 25.61 1,052.57 18.35 3 insect larvae 4.61 4.42 4.58 41.36 0.72 5 notes: %ni = percentage by number of food item; %vi = percentage by volume of the food item; %oi = percentage of frequency of occurrence of each food item; (%ni + %vi) x %oi = sum of the percentage by number and volume, multiplied by the percentage of frequency of occurrence of each food item); iri = index of relative importance. figure 2 frequency of occurrence (%o) of food items consumed by glossogobius aureus (n = 1,646) in naujan lake from february 2020 to january 2021 biotropia vol. 32 no. 2, 2025 176 (shukla 2009). the burrowing goby parapocryptes serperaster also showed a similar trophic strategy (dinh et al. 2017), differing from the specialist feeding behavior of goby  economidichthys pygmaeus (gkenas et al. 2012). moreover, the presence of sand, mud, and detritus in the diet of g. aureus collectively indicates benthic feeding, a characteristic commonly observed in the gobiidae family (macinnis & corkum 2000; parkinson & booth 2011). from 1,938 gut samples, 1,646 (85%) contained food and 292 (15%) were empty. this percentage of empty stomachs is lower relative to the 65.36% reported for g. sparsipapillus (tran et al. 2021a). variations in the rate of empty stomachs may be linked to differences in spawning events or reproductive cycles. the food intensity of g. aureus was observed based on the gastrosomatic index (gasi) and gut food volume taken monthly. sanchez-escalona et al. (in press) identified march, june, and december as the peak spawning months for male g. aureus and june and november for females. the highest percentage of empty stomach of nwg was recorded in march in males and june in females, coinciding with the spawning season of g. aureus in naujan lake. the lowest percentage of empty stomachs of nwg was observed from november to january for both sexes. there was no significant variation (t-test, p = 0.60) in the mean gasi values in both sexes; however, males generally feed more actively than females. the mean gasi estimates showed that females forage intensively in march, april, and october, while in males, a high feeding activity was observed in april, september, and november (fig. 4). the gasi values were observed to be high before spawning months and decreased during spawning season in both sexes. a similar result was observed in the feeding intensity of g. guiris in bangladesh, which showed maximum feeding during the off-breeding season while minimum during the spawning period (hossain et al. 2016). the findings may suggest possible biological or behavioral differences that are not strongly linked to the gasi values. halting food intake during most of the spawning period is likely associated with previous build-up and stored energy reserves from high feeding intensity during pre-spawning periods. the suppression of feeding activity may also have been due to less space in the body cavity for food (hoar et al. 1983). however, as males do not bulge as much as females, the male’s body cavity was likely able to provide more space for food than the female’s site-based variation in diet composition shrimp dominated the diet composition of nwg across the six sampling sites; however, a significant variation in the proportion of other dietary items was recorded (fig. 3). dietary components of samples from site 6 (tagbakin) significantly varied with that of sites 3, 4, and 5, respectively (p < 0.05). site 1 (butas) had significantly lower proportions of fish in the diet than site 3 (tigbao) and site 4 (malayas). however, site 4 (malayas) samples exhibited a more diverse diet with the occurrence of all the six food items identified in the gut. the findings may be attributed to the diversity and availability of food sources across the sampling sites. additionally, competition for food, manifested in diet overlaps, was observed among fish species (das et al. 2018; matern et al. 2021). insect larvae were absent from the diets of fish sampled at site 1 (butas) and site 2 (borbocolon) but were present in varying proportions at the other sampling sites. the limited occurrence of insect larvae in deep waters may explain their absence in the stomachs of the samples from sites 1 and 2, which are both deep sites. meanwhile, mollusks were observed only in site 4 (malayas). their distribution may be influenced by substrate type, water depth, and hydrochemical parameters (bespalaya et al. 2021). phytoplankton abundance in the stomach of g. aureus was relatively high in site 1 (butas), site 2 (borbocolon), and site 6 (tagbakin), but relatively lower occurrence in site 4 (malayas). all sites are associated with rivers with varying flows. among the rivers, malayas (site 2) has the biggest flow. phytoplankton growth is always associated with nutrient inputs from runoff but also results from complex dynamics of water flow, light penetration, and temperature (lu et al. 2023). the presence of phytoplankton in the stomach of all samples, irrespective of sites, indicates they are part of the regular diet of the species. specifically, the consumption of benthic forms, as gobies are benthic feeders. feeding intensity and food habits with a preference for shrimp and fish and the ability to exploit various prey types, nwg is potentially classified as a carnivorous generalist feeder. it showed a highly carnivorous diet correlated with the computed relative gut length of 0.631. because meat is easier to digest, carnivorous fishes often have short, more or less straight intestines diet composition and feeding habits of naujan white goby guibone et al. 177 figure 3 frequency of occurrence (%o) of food items consumed by glossogobius aureus across the six sampling sites in naujan lake figure 4 gastrosomatic index (gasi) of male and female glossogobius aureus from naujan lake (maklad et al. 2016). male nwg grazed more actively than females during the breeding season. in naked goby (gobiosoma bosc), males forage less intensively during the breeding season (d’aguillo et al. 2014). males of the species are in charge of egg guarding and territory defense, reducing their opportunity for foraging activity and lowering their feeding intensity. differences in feeding intensity between sexes of a species, thus, may be the reproductive status (rideout 1999) and the member’s investment in parental care (d’anguillo et al. 2012). biotropia vol. 32 no. 2, 2025 178 conclusion the gut content analysis revealed that naujan white goby (glossogobius aureus) is a carnivorous fish, with shrimp and fish being the most important prey items in the diet. due to its ability to utilize a variety of prey types, g. aureus is considered a generalist feeder, with occasional opportunistic feeding behavior on benthic organisms like insect larvae and plankton. the mean gasi estimates showed that males and females had low feeding activity during peak spawning months of nwg in naujan lake. this study provides baseline information for future fish nutrition research for nwg, which could optimize dietary strategies for the aquaculture production of this native species. further research is needed to examine gut contents of other life stages, such as larvae and juveniles, to determine dietary ontogeny. the use of fatty acid analysis, molecular approaches, and isotope analysis may also be explored to have a deeper understanding of the trophic relationships and interactions within food webs, including the identification of dietary components. this study is the first to assess the food and feeding habits of g. aureus in naujan lake. the findings will contribute to a deeper understanding of the species’ ecological role and provide a foundation for sustainable aquaculture and conservation initiatives. acknowledgments the authors wish to extend their gratitude to the department of science and technology mimaropa (dost-mimaropa) and the department of science and technology philippine council for agriculture, aquatic and natural resources research and development (dostpccaard) for funding the research. special thanks are also extended to the fishermen for their assistance during sample collection and vincent jay h. gado for creating the map of the study sites. references abdulmalik-labe op, eduardo ajl, quilang jp. 2023. genetic diversity and divergence among native and translocated populations of the golden flathead goby glossogobius aureus (gobiiformes: gobiidae) in philippine lakes.  plos 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(n.d.). naujan, oriental mindoro, philippines climate. available from: https:// weatherandclimate.com/philippines/oriental-mindoro/ naujan the southeast asian journal of tropical biology vol. 32 no. 3, 2025: 328 338 doi: 10.11598/btb.2025.32.3.2457 issn: 0215-6334 | e-issn: 1907-770x 328 molecular analysis of waxy gene markers in sorghum crosses kd4 and bonteb gunungkidul arif muazam1, kristamtini2, setyorini widyayanti2, yudhistira nugraha2, rina sri kasiamdari1, and budi setiadi daryono1* 1postgraduate program, faculty of biology, gadjah mada university, yogyakarta 55281, indonesia 2research center for food crops, national research and innovation agency, bogor 16915, indonesia article higlights local sorghum crosses show unique fixation of the waxy starch gene allele only wxc allele is expressed, while other waxy alleles are not detected waxy allele expression strongly relates to low amylose grain quality marker-based selection supports breeding of soft-textured sorghum findings enhance sorghum use for food, feed, and industrial applications article information received : 12 february 2025 revised : 20 august 2025 accepted : 4 september 2025 *corresponding author, e-mail: bs_daryono@mail.ugm.ac.id research paper abstract sorghum (sorghum bicolor (l.) moench) is a food crop that exhibits resilience to extreme environmental conditions and has the potential to be developed as an alternative food source. the quality of sorghum seeds is significantly influenced by the starch composition in the endosperm, which is regulated by the waxy (wx) gene. this gene has several major alleles, namely wxa, wxb, and wxc, which play roles in the synthesis of amylopectin and amylose. this study aimed to analyze the expression of wx alleles in the crosses of sorghum cultivars kd4 and bonteb gunungkidul. the main method used was the molecular marker-based pcr method. dna was extracted from the leaves of 30 individual f2 sorghum progeny samples using a slightly modified ctab method. pcr reactions were performed with specific primers for each allele, and the amplification results were analyzed using 1.5% agarose gel electrophoresis. several statistical analyses were performed to ensure results significance, i.e., a) chi-square test: to determine relationships between waxy allele expression with genetic segregation within cross populations; b) allele frequency analysis: to determine distribution of waxy genotypes within populations by comparing counts showing expressions of wxa, wxb, and wxc; and c) pearson correlation test: to evaluate relationships between waxy gene expression with specific agronomic traits (e.g., amylose content). the main findings of our study showed that only the wxc allele exhibited a clear amplification band, while wxa and wxb did not show any significant expression. this indicates that the wxc allele plays a dominant role in starch synthesis in this cross, while wxa and wxb are likely not expressed due to genetic or epigenetic regulatory mechanisms. these findings provide a brief summary of sorghum breeding efforts aimed at producing varieties with superior waxy starch characteristics. further studies are needed to understand the regulation of wx gene expression and its potential implications for molecular selection, ultimately enhancing sorghum quality for both food and industrial applications. keywords: genetics, local, plant breeding, sorghum, waxy genecopyright (c) 2025@author(s). https://doi.org/10.11598/btb.2025.32.3.2457 https://creativecommons.org/licenses/by-nc-nd/4.0/ molecular genetics of sorghum crosses kd4 and bonteb gunungkidul muazam et al. 329 introduction sorghum (sorghum bicolor (l.) moench) is a strategic food crop that contributes significantly to global food security, particularly in tropical and subtropical regions. its advantages include drought tolerance, high water-use efficiency, and the ability to thrive on marginal soils compared with other cereals such as rice and wheat (xie et al. 2022). sorghum also provides high nutritional value, being rich in carbohydrates, proteins, dietary fiber, and bioactive compounds such as polyphenols and antioxidants (tian et al. 2023). these attributes make sorghum a promising candidate for food diversification, especially under increasingly variable climate conditions (mutisya et al. 2023). however, one of the main limitations to its wider adoption as a staple food is that sorghum has relatively dry and firm grain texture compared with those of rice (lu et al. 2022). the eating quality of sorghum is largely determined by the ratio of amylose to amylopectin in the grain. amylose, a linear glucose polymer, contributes to hardness and dryness, while amylopectin, a branched polymer, produces a softer and stickier texture (yano et al. 2020). therefore, manipulating starch composition has become a key objective in sorghum breeding programs aimed at improving palatability. the waxy gene (wx) is the primary genetic factor controlling amylose synthesis. it encodes granulebound starch synthase (gbss), and mutations at this locus result in reduced or absent amylose content. such variants, termed waxy sorghums, are more acceptable to consumers in many asian countries due to their softer texture (boyles 2017; zhou et al. 2021; tian et al. 2023). several waxy alleles, including wxa, wxb, and wxc, have been reported to influence amylose levels, although their expression varies depending on genotype and environment (wang et al. 2023). wardhani and wirnas (2024) documented considerable genetic diversity for amylose content in sorghum crosses (pulut 3 × soraya 3), demonstrating the potential of allele-based selection to generate waxy lines. hence, molecular mapping and characterization of wx alleles are essential steps in sorghum improvement. molecular markers, particularly pcr-based assays, provide accurate identification of waxy alleles compared with conventional phenotypic screening and can accelerate breeding progress (lu et al. 2022). this approach allows breeders to detect waxy genotypes early in plant development, increasing selection efficiency (wang et al. 2023). beyond breeding, waxy sorghum has broad potential in the food industry. waxy starch improves product quality in applications such as noodles, bread, and other processed foods requiring superior gelatinization and textural properties (zhou et al. 2021; tian et al. 2023). thus, the development of waxy sorghum varieties could enhance both consumer acceptance and industrial utilization. in this study, we analyzed the expression of wx alleles (wxa, wxb, and wxc) in kd4, bonteb gunungkidul, and their progenies. in addition, we examined the relationship between allele expression and amylose content to provide a comprehensive understanding of how wx alleles contribute to starch quality. these findings are expected to support sorghum breeding programs in developing varieties with improved eating quality and industrial potential. materials and methods materials and equipment this study was conducted at the laboratory of genetics and plant breeding, faculty of agriculture, gadjah mada university. leaf and seed samples from crosses between kd4 and bonteb gunungkidul were used as dna sources. dna was extracted using a modified ctab (cetyl trimethyl ammonium bromide) protocol. specific primers targeting wxa, wxb, and wxc were used for allele-specific amplification. standard pcr reagents (buffer, dntps, taq dna polymerase, and mgcl₂) were employed, and pcr (polymerase chain reaction) products were separated by 1.5% agarose gel electrophoresis. a nanodrop spectrophotometer was used to measure dna quality and concentration prior to amplification. sample collection fresh leaf tissues were collected from 30 individual f2 sorghum progeny derived from the cross between kd4 and bonteb gunungkidul for molecular analysis. genomic dna was extracted using a slightly modified ctab method (doyle & doyle 1990) to improve purity and yield, while seeds were stored for further evaluation. biotropia vol. 32 no. 3, 2025 330 dna extraction genomic dna was isolated using a slightly modified ctab method (doyle & doyle 1990) to improve purity. the procedure involved grinding leaf tissue in liquid nitrogen, incubating in ctab extraction buffer at 65 °c, separating phases with chloroform–isoamyl alcohol, precipitating dna with isopropanol, and washing with 70% ethanol. the resulting dna was dissolved in te buffer and stored at -20 °c. dna quality and concentration were assessed using a nanodrop spectrophotometer at 260/280 nm. pcr amplification pcr was conducted to amplify waxy genes (wxa, wxb, wxc) using specific primers (table 1). the reaction mixture (25 µl) consisted of pcr master mix, dna template (50 ng/µl), forward and reverse primers (10 µm each), and ddh₂o. amplification was performed in a thermal cycler with an initial denaturation at 95 °c for 5 minutes, followed with 35 cycles of denaturation (95 °c, 30 seconds), annealing (50 – 60 °c, 30 seconds), and extension (72 °c, 1 minute). a final extension at 72 °c for 10 minutes was applied before the samples were stored at 4 °c. gel electrophoresis and visualization pcr products were resolved by 1.5% agarose gel electrophoresis in tbe buffer at 100 v for 45 minutes. gels were stained with ethidium bromide or sybr safe and visualized under uv light. banding patterns were compared with positive and negative controls to confirm allele-specific amplification. data analysis electrophoresis results were analyzed using imagej or gelanalyzer software to quantify band intensity. data were summarized as gel images, tables, and allele distribution charts. statistical analyses included: (a) chi-square analysis to assess segregation patterns; (b) allele frequency analysis to estimate genotype distribution; and (c) pearson correlation to examine relationships between waxy allele expression and amylose content. these analyses provided insights into the effectiveness of molecular marker-based selection for waxy sorghum improvement. results and discussion pcr analysis results electrophoresis results indicated that only the wxc allele exhibited clear amplification bands, while both wxa and wxb showed no significant amplification (fig. 1). this pattern was consistent across all 30 f2 samples analyzed, confirming that wxc was the only allele expressed in the studied populations. the results suggest several underlying factors that could contribute to this observation, which is often associated with primer specificity, genetic mutations, and pcr optimization issues (yang et al. 2013). one primary consideration is the specificity and binding efficiency of the primers used during the polymerase chain reaction (pcr). primers are short sequences of nucleotides that anneal to specific regions of the dna template to initiate amplification. if the primers are designed based on sequences unique to the wxc allele, they may not effectively bind to the wxa and wxb alleles due to sequence variations, leading to preferential amplification of the wxc allele. a study by teng et al. (2012) emphasized the importance of meticulous primer design to ensure that all target alleles are equally recognized and amplified during pcr. table 1 specific primers for identification of waxy genes in sorghum allele type primer (5’-3’) temperature/ annealing (°c) band size (pb) reference wxa f1:cgtggcgagatcaaactcta 60.0 non waxy: 523 wang et al. (2023)f2:ggcctggattcaatgttctt waxy: 615 r:gcagctggttgtccttgtag wxb f:cgaccgtgtgttcattgaccac 61.0 non waxy: 1,281 wang et al. (2023) r:ttgttcagtgccttgcctcg waxy: 745+537 wxc f:gctggttctgagtgcaaca 58.5 non waxy: 523 wang et al. (2023)r1:acttcttcttgccagtgacc waxy: 615 r2:acttcttcttgccagtgacg molecular genetics of sorghum crosses kd4 and bonteb gunungkidul muazam et al. 331 genetic variations or mutations within the wxa and wxb alleles could also impede primer binding or the amplification process. for instance, single nucleotide polymorphisms (snps) or insertions/ deletions (indels) in the primer binding sites can reduce the efficiency of primer annealing, resulting in weak or absent amplification signals for these alleles. zhang et al. (2019) reported that specific snps in waxy genes significantly affected the amplification efficiency of different alleles in rice and sorghum, underscoring the need to account for such variations in experimental design. the quality and quantity of the dna template used in the pcr can significantly influence amplification outcomes. degraded dna or insufficient template amounts can lead to suboptimal amplification, particularly for certain alleles. ensuring high-quality dna extraction and quantification is crucial for obtaining reliable and reproducible results across all target alleles (shin et al. 2015). pcr conditions, including annealing temperature, magnesium ion concentration, and cycle number, play pivotal roles in amplification efficiency. suboptimal conditions may favor the amplification of one allele over others. therefore, optimizing these parameters is essential to achieve balanced amplification of multiple alleles. wang et al. (1995) suggested that techniques such as gradient pcr can help determine the optimal annealing temperatures for primers, thereby enhancing the amplification of all target alleles. in our study, the exclusive amplification of the wxc allele observed in the electrophoresis results is likely due to a combination of factors, including primer specificity, allelic variations, dna template quality, and pcr conditions. addressing these aspects through careful experimental design and optimization can lead to more balanced and accurate amplification of the wxa, wxb, and wxc alleles (pedersen et al. 2007). statistical analysis of sorghum molecular and agronomic data 1. chi-square test (χ²) the chi-square (χ²) test was employed to determine whether genetic segregation in the f1a and f1b cross populations adhered to the expected mendelian inheritance ratios. this statistical test is widely used in genetic studies to assess the goodness-of-fit between observed and expected distributions, thereby evaluating deviations that may indicate underlying genetic factors such as dominance effects, epistasis, or selection biases (mcdonald 2014). hypotheses: • h0: genetic segregation follows expected ratios (e.g., 1 : 2 : 1 for heterozygotes). • h1: genetic segregation does not follow expected ratios. observed data: • wxa: 0 (not expressed) • wxb: 0 (not expressed) • wxc: 4 (kd4, bg, f1a, f1b) figure 1 pcr analysis results for the examined sorghum leaf samples biotropia vol. 32 no. 3, 2025 332 expected ratio: if adhering to ratio of 1 : 1 : 2: • wxa: 1 • wxb: 1 • wxc: 2 formula used for calculating chi-square χ² = ∑(o-e)2/eχ² = ∑e(o-e)2 where: o = observed data e = expected ratio the results obtained were: • for wxa: (0-1)2/1 = 1(0-1)2/1 = 1 • for wxb: (0-1)2/1 = 1(0-1)2/1 = 1 • for wxc: (4-2)2/2 = 2(4-2)2/2 = 2 total χ² = 4 since χ2 = 4 > χ table 2 (5.991 for df 2, α = 0.05), this result suggests that the observed genetic segregation does not deviate significantly from the expected mendelian ratio at the 5% significance level. however, despite failing to reject the null hypothesis, the observed data indicate an apparent absence of wxa and wxb, with exclusive amplification of wxc. this deviation could be attributed to: a. dominance effects of the wxc allele if wxc exhibits a dominant expression pattern, it may suppress or mask the amplification of wxa and wxb, leading to a skewed distribution of phenotypic traits (zhang et al. 2020). b. technical or biological constraints issues such as primer specificity, dna degradation, or selective expression due to environmental factors may contribute to the lack of amplification in wxa and wxb (shin et al. 2015). c. epistatic interactions potential genetic interactions between waxy alleles might influence expression levels, causing suppression of wxa and wxb in favor of wxc (wang et al. 2022). the chi-square test confirmed that genetic segregation in the studied sorghum samples does not significantly deviate from mendelian expectations at the α = 0.05 level. however, the exclusive amplification of wxc suggests that additional genetic or molecular factors may be influencing allele expression. further studies incorporating molecular markers, gene expression analysis, and controlled breeding experiments are necessary to elucidate the exact mechanisms governing wxc dominance and the suppression of wxa and wxb. 4. allele frequency analysis allele frequency analysis is a fundamental approach in population genetics to determine the distribution of genetic variants within a given population. it provides insight into the inheritance patterns of specific genes and helps identify selective advantages or genetic bottlenecks affecting allele prevalence (hedrick 2019). in this study, the allele frequencies of the waxy (wx) gene variants (wxa, wxb, wxc) were analyzed to assess their distribution in the cross populations (kd4, bg, f1a, and f1b). the results obtained were: a) wxa: 0/4=00/4=0 (0%); b) wxb: 0/4=00/4=0 (0%); and c) wxc: 4/4=14/4=1 (100%). the exclusive presence of the wxc allele in the analyzed sorghum samples suggests several possible genetic and evolutionary factors influencing allele distribution: a. selection pressure favoring wxc the fixation of wxc at 100% frequency suggest a selective advantage in the studied sorghum lines. the wxc allele might be associated with beneficial agronomic traits, such as improved starch composition or higher yield, leading to positive selection over other alleles (tian et al. 2009). given that waxy starch is often preferred in food and industrial applications, it is possible that breeding programs have indirectly selected for this allele, resulting in its predominance (wang et al. 2020; maung et al. 2021). b. genetic drift and founder effects the absence of wxa and wxb could also be attributed to genetic drift, especially if the population underwent a bottleneck effect or molecular genetics of sorghum crosses kd4 and bonteb gunungkidul muazam et al. 333 was derived from a limited number of parental genotypes. in small breeding populations, certain alleles may be lost due to random genetic drift, leading to fixation of a single allele over multiple generations (falconer & mackay 1996). c. dominance and epistatic interactions if wxc exhibits strong dominance over wxa and wxb, it may mask the expression of these alleles, preventing their detection in the analyzed samples. additionally, epistatic interactions between genes regulating starch biosynthesis may play a role in the observed allele distribution (zhang et al. 2021). further investigation into gene expression patterns and regulatory mechanisms is necessary to confirm whether such interactions influence wxc dominance. d. pcr and electrophoresis detection limitations it is also important to consider technical limitations in detecting wxa and wxb. the lack of amplification for these alleles could be due to inefficient primer binding, sequence variations at primer sites, or low template dna concentrations (shin et al. 2015). repeating the analysis with alternative molecular markers or sequencing approaches could validate these findings and rule out technical biases. the observed fixation of the wxc allele has significant implications for sorghum breeding and starch quality improvement. since wxc confers a waxy starch phenotype, its exclusive presence may indicate a targeted selection for this trait in breeding programs (paterson et al. 2009). the complete absence of wxa and wxb suggests that traditional non-waxy alleles have been eliminated in these specific sorghum lines, possibly due to human-driven selection for improved processing and culinary properties (tian et al. 2011). future research should focus on expanding the genetic pool to determine whether wxa and wxb alleles exist at low frequencies in related sorghum populations. additionally, transcriptomic and proteomic analyses could provide insights into how gene expression differences contribute to the predominance of wxc at the phenotypic level. allele frequency analysis of the waxy gene in the studied sorghum populations revealed a complete fixation of the wxc allele (100%) and the absence of wxa and wxb. this phenomenon suggests that wxc may confer a selective advantage or has been subject to a strong genetic drift or breeding selection. while these findings provide valuable insights into the genetic architecture of starch biosynthesis in sorghum, further molecular investigations are needed to confirm the underlying mechanisms driving wxc fixation. 3. pearson correlation test the pearson correlation test was employed to assess the relationship between waxy (wx) gene expression and amylose content in the studied sorghum samples (table 2). pearson’s correlation coefficient (r) measures the strength and direction of a linear relationship between two continuous variables, providing insights into genetic interactions influencing starch biosynthesis (rodgers & nicewander 1988). this test is commonly used in plant genetics to evaluate the impact of specific gene expressions on biochemical traits such as starch composition (huang et al. 2020). table 2 evaluated relationships between expressions of waxy genes and amylose content sample allele expression amylose content (%) kd4 wxc 19 bg wxc 2 f1a wxc 10 f1b wxc 12 biotropia vol. 32 no. 3, 2025 334 hypotheses: • h0: no relationship exists between expressions of waxy genes and amylose content. • h1: a relationship exists between expressions of waxy genes and amylose content. the pearson correlation coefficient was calculated using formula: where: x = wxc expression (constant value of 1 across all samples). y = percentage of amylose content given that wxc expression is invariant (always 1), a direct pearson correlation calculation would yield an undefined result, as standard deviation in the independent variable is zero. this result suggests that while the presence of wxc is necessary for waxy starch production, it alone does not determine the variation in amylose content. instead, post-transcriptional regulation, environmental influences, or additional genetic factors may be involved (tian et al. 2009). results of pearson correlation test suggest: a. lack of correlation due to uniform wxc expression since all samples expressed wxc, a direct statistical correlation could not be established between the presence of wxc and amylose levels. this indicates that the presence of wxc alone does not dictate amylose content but rather interacts with other regulatory elements affecting starch biosynthesis (zhang et al. 2021). b. post-transcriptional and environmental effects studies have shown that the influence of waxy gene on amylose synthesis is regulated at multiple levels, including transcriptional control, post-translational modifications, and enzymatic activity modulation (hirano et al. 2018). variations in amylose content across samples could result from environmental factors such as temperature, soil conditions, or water availability, which influence starch biosynthesis pathways (asante et al. 2019). c. potential influence of other genetic loci the observed variation in amylose content suggests the involvement of modifier genes or allelic interactions that regulate the degree of wxc expression or its enzymatic activity. previous research in cereal crops has identified secondary genes affecting starch biosynthesis, such as ssiia and gbssi, which contribute to differences in amylose levels even when wx alleles are expressed (wang et al. 2020). the findings underscore the importance of considering additional genetic markers and environmental conditions when breeding for starch composition traits. while wxc expression is essential for waxy starch production, achieving desired amylose levels requires a broader selection strategy incorporating regulatory genes and agronomic practices (shin et al. 2015). the pearson correlation test could not establish a direct statistical relationship between wxc expression and amylose content due to the invariant nature of wxc expression across samples. however, the variation in amylose content suggests that there may be several factors beyond wxc presence, such as genetic modifiers, post-translational regulation, and environmental influences, which play significant roles in starch biosynthesis. future studies using genome-wide association studies (gwas) or transcriptomic analysis could provide deeper insights into the regulatory networks governing amylose synthesis in sorghum. visualization of waxy allele expression the uniform expression of wxc across all samples further supports its essential role in waxy starch production, whereas the non-expression of wxa and wxb suggests that these alleles may not be actively contributing to starch biosynthesis in this genetic background (table 3). this finding aligns with previous studies that have established wxc as a key determinant in controlling amylose biosynthesis in cereals (tian et al. 2009; hirano et al. 2018). the absence of wxa and wxb expressions suggests that these alleles may not be actively involved in starch biosynthesis within this specific genetic background, possibly due to genetic regulation, allele-specific expression patterns, or epigenetic modifications. molecular genetics of sorghum crosses kd4 and bonteb gunungkidul muazam et al. 335 functional role of wxc in starch biosynthesis research in cereals such as rice, maize, and sorghum has shown that the waxy gene encodes granule-bound starch synthase i (gbssi), the enzyme responsible for amylose synthesis in endosperm cells (tian et al. 2009). variations in the wx locus, including allelic differences in wxa, wxb, and wxc, lead to differences in enzyme activity and starch composition (zhang et al. 2021). the predominant expression of wxc in this study supports the hypothesis that it is the main contributor to waxy starch synthesis in the analyzed sorghum genotypes. potential explanations for the nonexpression of wxa and wxb 1. genetic silencing the non-expression of wxa and wxb may be attributed to regulatory elements that suppress transcription under specific genetic backgrounds (wang et al. 2020). previous study has shown that gene expression in the waxy locus can be influenced by upstream regulatory sequences or trans-acting factors that preferentially activate wxc over other alleles (asante et al. 2019). 2. epigenetic modifications dna methylation and histone modifications are known to regulate gene expression in cereals (zhang et al. 2018). it is possible that wxa and wxb undergo methylation or chromatin remodeling, preventing their transcriptional activation while allowing wxc to be expressed. 3. allelic expression preference some plants exhibit allele-specific expression due to dominance interactions or alternative splicing mechanisms. this could explain why wxc is consistently expressed while wxa and wxb remain inactive (hirano et al. 2018). 4. gene structural variations studies in rice and maize have reported that mutations, insertions, or deletions in the promoter or coding regions of wx alleles can disrupt their expression (zhang et al. 2021). it is plausible that wxa and wxb contain structural differences that render them non-functional in this sorghum cross. implications for waxy sorghum development understanding the differential expression of wx alleles is essential for breeding waxy sorghum varieties with desired starch properties. since wxc is consistently expressed, it serves as a reliable genetic marker for selecting waxy sorghum lines, which are valuable for food and industrial applications. however, further investigation is needed to determine whether wxa and wxb are truly nonfunctional or if their expression could be induced under different conditions (wang et al. 2020). future studies should integrate transcriptomic, epigenetic, and genome-editing approaches to elucidate the regulatory mechanisms governing wx allele expression in sorghum. implications for sorghum breeding these results hold significant implications for sorghum breeding programs aiming to develop varieties with specific starch properties: 1. selection of waxy sorghum varieties given that wxc is the only expressed allele, breeders can use this marker for selecting waxy sorghum varieties with reduced amylose content, which is desirable for food and industrial applications (wang et al. 2020). 2. regulation of starch biosynthesis the lack of wxa and wxb expression suggests potential gene silencing mechanisms or allelic interactions that warrant further investigation through transcriptomic and epigenetic studies (hirano et al. 2018). 3. genetic improvement strategies understanding the molecular regulation of wxc could facilitate targeted genetic modifications, table 3 waxy allele expression in the sorghum sample sample wxa wxb wxc kd4 + bg + f1a + f1b + notes: + = gene expression; = no expression. biotropia vol. 32 no. 3, 2025 336 such as crispr-based gene editing, to optimize starch composition in sorghum (zhang et al. 2021). the exclusive amplification of the wxc allele observed in this study provides important implications for both genetic understanding and breeding strategies of sorghum bicolor (l.) moench. segregating populations of sorghum often show significant genetic variability in amylose and yield-related traits, as documented by trikoesoemaningtyas et al. (2024), indicating that waxy gene alleles can segregate differentially and determine starch quality. in our study, the fixation of wxc is consistent with these findings, suggesting strong selection or genetic drift leading to allele predominance. furthermore, the absence of wxa and wxb may be the result of allelic silencing, epistatic interaction, or breeding history. similar patterns of allele loss and fixation have been reported in segregating populations in indonesia, particularly when breeding pressure favored specific quality traits (lestari et al. 2024; munarti et al. 2022). these results highlight the potential of wxc as a reliable marker for the development of waxy sorghum. from an agronomic perspective, waxy allele fixation should be considered alongside variability in other traits such as lignin content, stay-green genes, and biomass quality. astuti et al. (2024) demonstrated that agronomic variability among sorghum genotypes with different lignin levels could be exploited for both food and non-food purposes, while munarti et al. (2022) emphasized the role of stay-green genes in crop resilience. this integration shows that waxy allele expression, combined with other genetic factors, can strengthen both yield stability and quality improvement. at a broader level, studies in kazakhstan by bogapov et al. (2024) have shown that sweet sorghum genotypes can simultaneously provide high value for food, feed, and energy, stressing the importance of breeding materials with multiple functional traits. thus, the fixation of wxc in our population not only benefits starch quality but also aligns with global breeding trends aiming at multifunctional sorghum cultivars. overall, this study supports the hypothesis that wxc allele expression is a critical determinant of waxy starch properties in sorghum, and its consistent detection in kd4, bonteb, and their f1 populations strengthens its role as a target in molecular breeding programs. our findings further indicate that the consistent expression of wxc is correlated with relatively low amylose content in kd4, bonteb gunungkidul, and their progenies. this supports the role of wxc as a determinant of starch quality, particularly in producing waxy sorghum types with desirable grain texture. the absence of wxa and wxb expression suggests possible gene silencing or allelic regulation, which warrants further investigation. future studies should focus on quantitative expression analysis (qpcr or rna-seq) to explore the regulatory mechanisms of wx alleles, as well as environmental influences such as temperature and water availability that may affect starch biosynthesis. additionally, phenotypic evaluations of starch properties should be integrated with molecular findings to strengthen marker-assisted selection strategies in sorghum breeding. conclusion this study provides a comprehensive understanding of the role of the waxy gene in sorghum and its relationship with amylose content. the dominant expression of wxc suggests its crucial involvement in starch biosynthesis, while the absence of wxa and wxb expression raises questions regarding their regulation. future research should explore the genetic and environmental factors influencing waxy gene expression to enhance sorghum breeding strategies for improved starch characteristics. acknowledgments the authors gratefully acknowledge the financial research support from the research center for food crops of the national innovation research agency (brin) and the faculty of biology, gadjah mada university (ugm) which facilitated the design, data acquisition, and analysis of this study, also for their invaluable technical assistance in sample collection and fieldwork. their contributions were essential in ensuring the success of this research. additionally, we acknowledge all individuals and institutions that provided materials and resources necessary for the completion of this study. the funding body had no role in the study design, data analysis, interpretation, manuscript preparation, or the decision to submit this paper for publication. molecular genetics of sorghum crosses kd4 and bonteb gunungkidul muazam et al. 337 references asante md, offei sk, hall aj, gracen ve. 2019. influence of environmental factors on waxy and non-waxy sorghum starch quality. food sci 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abandoned mining area in southern thailand** jetsada wongprom* vacuity of forestry, kasetsart university, bangkok 10900, thailand received 17 december 2018 / accepted 16 september 2019 abstract dominant dipterocarp trees hold a significant importance in the ecology and economics of the declining tropical forests of asia. of equal importance is the restoration of these dipterocarp forest using the technique of matching species with silvicultural practices. the study aimed to investigate the effect of the acacia mangium nurse trees on the survival and growth of six dipterocarp species planted on abandoned mining areas in the phangnga forestry research station, thailand, namely; dipterocarpus alatus roxb., dipterocarpus gracilis blume, hopea odorata roxb., shorea gratissima (wall, ex kurz) dyer, shorea roxburghii g. don, and parashorea stellata kurz. the approximately 1.5-year-old dipterocarp seedlings were planted at a 6-year-old a. mangium plantation cover and at open plots. the survival rate, diameter at root collar (do), total height (h) and the relative growth rate (rgr) of do and fi of the seedlings were compared. soil samples at the open and the a. mangium plots were collected and its physical and chemical properties were analyzed. the results indicated that the survival rates of the dipterocarps planted at the a. mangium plot were higher than those at the open plots. the survival rates (75100%) of the 1 to 3-year-old d. alatus, h. odorata, s. gratissima, s. roxburghii, and p. stellata were relatively high at the a. mangium plot, but not for d. gracilis. the do of s. roxburghii, d. alatus, h. odorata, and p. stellata were higher at the a. mangium plot. however, the do and h of s. roxburghii, d. alatus, and h. odorata at the open plot were high and similar to that of the a. mangium plot. rgrdo and rgrh of s. roxburghii, d. alatus, and h. odorata were high both at the a. mangium and the open plots. generally, the dipterocarp seedlings were growing better under the a. mangium nurse trees, thus suggesting the possibility of their improved survival and growth in mined out areas. moreover, the findings also suggest that s. roxburghii, d. alatus, and h. odorata can be planted at open areas with poor soil conditions and extreme environments. lastly, thinning interventions are also needed to improve the growth of dipterocarp trees. keywords: dipterocarp species, growth performance, mining area, phangnga forestry research station introduction dipterocarp trees mostly dominate the asian tropical rain forests (ghazoul 2016). these species are widely planted for forest ecosystem maintenance and for the restoration of degraded lands (kettle 2010; yeong et al. 2016; budiharta et al. 2018). with the diminishing non-timber forest products, these dipterocarp trees have become an important component of the rural livelihoods. ^corresponding author, e-mail: fforcrw@ku.ac.th **this paper was presented at the 3rd international conference on tropical biology 2018, 20-21 september 2018, bogor, west java, indonesia illegal logging and conversion of forests to agricultural lands, mainly for oil palm and rubber, plantations has dramatically resulted in deforestation and forest loss (appanah et al. 2016; ghazoul 2016). hence, forest restoration has become one the options left throughout the region of southeast asia (appanah et al. 2016). dipterocarp trees have been widely planted for commercial, conservation, and agroforestry purposes as well as forest restoration (sakai et al. 2014; appanah et al. 2016; ghazoul 2016). however, those trees planted on open and degraded lands has low survival and growth rates (hattori et al. 2013). forest rehabilitation with dipterocarp trees generally plant nurse 115 biotropia vol. 27 no. 2, 2020 trees using the line, strip, and gap methods (norisada et al 2005; hattori et al 2013; sakai et al 2014; budiharta et al. 2014). providing nurse trees for dipterocarp trees significantly influenced the growth and survival of dipterocarp seedlings (norisada et al. 2005; sakai et al. 2014). moreover, environmental factors such as light, air temperature and soil properties also improved and thereby, enhanced the growth performance of dipterocarp trees (norisada et al. 2005; sakai et al. 2014). forest restoration using native tree species with appropriate silvicultural practices significantly contribute in the recovery of forest structure, diversity, and ecological services by improving tree growths and enhancing natural regeneration. a. mangium is a fast-growing, nitrogen fixing tree commonly planted in commercial plantations and used for rehabilitation of degraded lands in southeast asia, including thailand (norisada et al. 2005; sakai et al. 2011). compared with other fast-growing and native tree species, its survival and growth rates on abandoned mining area are relatively higher (martpalakorn 1990). forest plantations do not only provide wood, but also simultaneously improve the ecosystems. soil properties, nutrient cycling and biodiversity have been reportedly improved and restored after plantation establishment (parrotta 1999; lanuza et al. 2018), particularly under a. mangium plantation. soil nutrients and organic matter contents were remarkably increased (kongchum et al. 2016). globally, mining operations have severely impacted the environment, particularly, the soil properties, resulting to very low soil nutrient levels with strong acidic soil ph. the soil structure becomes unsuitable for planting tree species (oktavia et al. 2015). in addition, mining also destroys the existing vegetation. hence, the restoration of previously mined areas has been undertaken on many sites using the plantation approach (bohre & chaubey 2014; kongchum et al. 2016). however, the forest structure and diversity levels are usually low. as such, plantation using mixed species are then recommended for restoration of degraded lands, including at post-mining sites (lamb 2014), but the survival and growth rate of seedlings depend much on the tree species, environmental factors (norisada et al. 2005; sakai et al. 2014; lestari et al. 2019), and silvicultural practices (sakai et al. 2011; sakai et al. 2014). to recover the forest structure, diversity and functions, the restoration of mined out areas using indigenous tree species has been going on for a long time. moreover, tree selection for such degraded lands and appropriate management intervention for nurse trees, such as thinning, are such important steps needed to promote the growth of native trees (sakai et al. 2014). understanding the influence of silvicultural practices on the growth performance of dipterocarp trees is very critical in any restoration program. thus, the objectives of this study were to (1) investigate and compare the survival and growth performance of dipterocarp seedlings planted at a 6-year-old a. mangium plantation and at open plots in a mined out area and, (2) to investigate the influence of thinning the a. mangium stand on the growth of dipterocarp seedlings growing on mined out areas. materials and methods study site and experimental plot the experimental plot was located in an abandoned tin mine at the phangnga forestry research station, thakuapa district, phangnga province, thailand. the province experiences the rainy season during april-november and the dry season during december-march. its mean annual rainfall is 3,649.4 mm per year and the relative humidity is around 83.7% (wongprom et al. 2013). the post mined area has a high bulk density and very low soil nutrient levels (kongchum et al. 2016). species using completely randomized design with three replications, six species of approximately 1.5-year-old nursery-raised dipterocarp seedlings were planted both at the open and a. mangium plots (fig. 1). the species chosen were dipterocarpus alatus roxb. ex. g. don, d. gracilis blume, hopea odorata roxb., shorea gratissima (wall, ex kurz) dyer, s.roxburghii g. don, and parasborea stellata kurz. thirty dipterocarp seedlings were planted in a 15 x 15 m plot with a spacing of 3 x 3 m at the open 116 growth performance of dipterocarp species — wongprom plot, and thirty dipterocarp seedlings at the a. mangium plot were planted with a spacing of 3 x 3 m between the rows of 6-year-old a. mangium. the original spacing of the a. mangium plot was 3 x 3 m. weeding and liana cutting were practiced two times a year. growth performances of 1 to 3-year-old dipterocarp seedlings after planting, including the survival rate, diameter at root collar (do), and total height (h), were measured. at the a. mangium plot, the number of 7year-old a. mangium trees was reduced to 50% using the row thinning method. the crown cover at the a. mangium plot was evaluated before and after thinning by using hemispherical photographs taken at 5 points by a digital camera (nikon coolpix 4500 with a fisheye lens) set on a tripod at 1.2 m above the ground. the cover was analyzed using hemiview 2.1 (delta-t device ltd.). moreover, diameter at breast height (dbh), height, density and crown cover of the 6 to 8-year-old a. mangium trees were also measured (table 1). dg+am ps+am dg+am sg+am ps+am da+am ho+am sr+am sr+am da+am ho+am sg+am dg+am ho+am ps+am sr+am da+am sg+am . o -o-o-o-oo-a-o-a-o-a— o-a-o-a-o |* o a 0 * o * 0 * i 0 * 0 * 0 * o a 0 * 0 i i o a o i o a o l i a a o a o a o a o a 6 t 3 m q a q a q a q a o a q l 6 _ _ 1-3 nh -o— o o o o— o— o 15 m 1 5 m h ps dg da sg ps ho da ho dg dg sr sg ho sg sr sr da ps a a a a 3 r a a-l _la 1 5 m h 1 5 m figure 1 layout of dipterocarp seedling planted at the open and at the a. mangium plots notes: am = acacia mangium, dg = diptertocarpusgracilis, da = dipterocarpus alatus, sr = shorea roxburgii, sg = .shoreagratissima, ho = hopea odorata, ps = parashorea stellata)\ filled triangle (jk) = dipterocarp seedlings and the open circle (#) = a. mangium. table 1 density, diameter at breast height (dbh), height (h), and crown cover of the 6 to 8-year-old a. mangium plot growing on the mined out area in phangnga forestry research station age (year) density (tree/ha) mean dbh (cm) mean h (m) crown cover (%) remark 6 943.75 14.14 13.89 71.16 7 471.87 15.60 14.46 23.92 thinned a. mangium 8 425.00 16.86 15.76 117 biotropia vol. 27 no. 2, 2020 soil properties soil samples were taken from two soil depths, i.e., topsoil (0-10 cm) and subsoil (20-30 cm), using a split tube sampler at three random locations from both the open and a. mangium plots. soil physical properties including soil particle and soil texture, as well as soil chemical properties were measured. organic matter content (om) was analyzed using the walkley and black's rapid titration method. the available phosphorus (p) was extracted using the bray ii method (bray & kurtz 1945) and was analyzed using a spectrometer. exchangeable potassium (k), calcium (ca) and magnesium (mg) were extracted using 1 n ph 7.0 ammonium acetate (nh4oac) and analyzed using an atomic absorption spectrometer (estefan et al 2013). soil moisture of the topsoil and subsoil, both at the open and a. mangium plots, were measured monthly for 1 year after planting of the dipterocarp trees. the collected soils were oven-dried at 80 °c for 48 hours to constant weight. data analyses relative growth rate (rgr) of do (rgr do) and h (rgrh) was calculated using the formula: rgrdo = (in d02 in d01) / (t2 tl) rgrh = (in h2 in hi) / (t2 tl) where dol and do2 are diameter at root collar and hi and h2 are height at times tl and t2. the survival rate, do, h, rgrdo, and rgrh of the six dipterocarp seedlings, both at the open and a. mangium plots, were compared and tested using the one way analysis of variance (anova) with the duncan's multiple range test (dmrt). results and discussion soil properties most of the soil particles at the experimental plots were more than 80% sand particles. soil texture at both the open and a. mangium plots were of a loamy and sandy types for the topsoil and subsoil, respectively, while the soil ph was strongly acidic. the soil nutrient levels in the topsoil layer, including the exchangeable k (21.92 ppm), ca (109.52 ppm), mg (18.38 ppm), and available p (2.33 ppm), were very low (table 2), except om content (2.50%), which was at a moderate level (usda 1967). these low levels of soil nutrients in such areas are a result of intense mining. however, the levels of exchangeable k, ca, mg, p and om content at the a. mangium plot were higher than those in a abandoned mining area (kongchum et al. 2016). nitrogen fixing trees, like a. mangium, help improved the soil properties of degraded lands, particularly in increasing the soil organic matter and nutrient content (kongchum et al. 2016). survival rate survival rates of the dipterocarp seedlings significantly differed among the species, both at the open and a. mangium plots. one year after planting, most of the dipterocarp seedlings at the open and a. mangium plots survived, ranging from 75 to 100% indicating a high survival rate. however, survival rate of the 2 and 3-year-old dipterocarp seedlings at the open plot decreased (fig. 2). three year old s. roxburghii had the highest survival rate (62.22%) followed by h. odorata (44.44%) and d. alatus (33.33%), while p. stellata, s. gratissima and d. gracilis did not survive at all (100% mortality) table 2 physical and chemical properties of soil at the open and a. mangium plots at phangnga forestry research station, southern thailand plot soil depth soil particle (%) soil ph exchangeable (ppm) available p om (cm) sand silt clay texture k ca mg (ppm) (%) a. mangium 0-10 86 7 7 ls 4.4 21.92 109.52 18.38 2.33 2.50 20-30 91 2 7 s 5.5 6.34 43.58 7.36 1.73 0.57 open 0-10 82 10 8 ls 5.1 37.74 339.40 18.94 2.04 2.41 20-30 90 3 7 s 5.2 11.48 94.46 14.76 0.87 0.16 notes: ls — loamy sand, s — sandy. 118 growth performance of dipterocarp species — wongprom 100 90 80 ? 70 2 60 et50 et £ 40 s 30xn 20 10 0 ab b b^b b b b b b a survival b b b » b ab a a b t\ b ab a c c c a b open plot | am plot 1year-old open plot | am plot 2year-old open plot | am plot 3year-old dg eda eho qsg sr eps figure 2 survival rates of dipterocarp trees planted at the open and at m. mangium (am) plots notes: dg = diptertocarpus gracilis, da = dipterocarpus alatus, sr = shorea roxburgii, sg = shorea gratissima, ho = hopea odorata, ps = parasborea stellate^different letters above bars indicate significant differences at p < 0.05 among species by dmrt. however, the dipterocarp seedlings in the a. mangium plot had higher survival rates, between 75% and 100%, except for d. gracilis (fig. 2). thinning of the a. mangium stand resulted in the mean crown cover being reduced from 71.16 to 23.92%. the thinning may have negatively influenced the survival rate of p. stellata and d. gracilis seedlings. stronger light intensity in the open areas negatively affected the survival as well as the growth of many dipterocarp seedlings (hattori et al. 2009). the subsequent reduction in the sunlight intensity due to the canopy of the a. mangium resulted in the high survival rate of the dipterocarp seedlings (kenzo et al. 2011). although s. roxburghii, h. odorata and d. alatus had moderate survival rates in the open plot, these can still be planted in open areas with poor soil conditions. a study in southern thailand, showed that these dipterocarp species are drought and stress tolerant and can be planted on degraded sandy soil in open areas moreover, their high survival rates, especially s. roxburgii, ranged between 78% and 97%, when planted in an open area in a disturbed dry evergreen forest in northeastern thailand (niamrat & marod 2005). when a. roxburgii was planted in a grassland interspersed with bushes, its survival rate was also reportedly high (93%) (ang et al. 2003). hence, rehabilitation of degraded lands using dipterocarp seedlings is better done alongside nurse trees as these can create a microclimate suitable for the survival and growth of the seedlings. after a. mangium was thinned by 50%, the survival rate of d. gracilis and p. stellata decreased gradually from 75 to 36.67% and 98.33 to 75%, respectively, after 3 years. the higher mortality of d. gracilis and p. stellata may have been caused by stronger light intensity. light intensity distinctively affected the survival rates of dipterocarp seedlings planted on degraded lands (norisada et al. 2005; sakai et al. 2014). high soil temperature and water stress, result in higher mortality, especially in drought conditions (norisada et al. 2005). the sandy soil type in the study area expectedly held a very low water storage in dry season (fig. 3), despite the fact that soil moisture significantly influences the establishment of a seedling (hattori et al. 2013). in this study, soil moisture at a. mangium plot was higher than that in the open plot, indicating that a. mangium can reduce water transpiration from the soil. the crown cover and high amounts of litterfall from a. mangium create a microclimate preferable for the establishment of dipterocarp seedlings under a mixed plantation. as the light requirements of dipterocarps depend on the species (sakai et al. 2014; hattori et al 2009), d. gracilis and p. stellata behavior showed that these may thrive well in darker conditions. thus, to enhance the dipterocarp seedlings survival, thinning of a. mangium may be delayed for mixed plot of d. gracilis and p. stellata. lastly, thinning intensity is equally important in the enrichment planting of dipterocarp seedling (sakai et al. 2014). 119 s o il m o is tu re ( % ) biotropia vol. 27 no. 2, 2020 topsoil subsoil 24.00 20.00 16.00 12.00 8.00 4.00 ~ <• > }•*•••* •> jfan. mar. may jul. sep. nov. ian. mar. may jul. sep. nov. month month open plot— am plot figure 3 soil moisture in the topsoil and subsoil levels at the open and a. mangium (am) plots during a one-year period after planting of the dipterocarp seedlings diameter at root collar (d0) and height (h) of the dipterocarp seedlings diameter at root collar (do) and height (h) of the six dipterocarp seedlings, for the years 1 to 3, significantly differed among the species in the open plot. d0 and h of d. alatus were the highest in the open plot after the first year of planting. moreover, do of d. alatus (6.04 cm) was significantly higher than that of a. roxburghii (4.07 cm) and h. odorata (2.95 cm), but their heights (h) did not differ at ages 2 and 3 years. the d. alatus seedling experienced a good growth in the open area and its stem diameter (7.20 cm), at 30 cm above the ground, was larger than those of the dipterocarp seedlings reported by sakai et al. (2014). however, do and h of the 1 to 3-year-old a. roxburghii and h. odorata seedlings experienced a relatively higher growth at the open plot indicating their preference for brighter light conditions. h. odorata and a. roxburghii seedlings had good growths and were both tolerant to drought, suggesting that these can be planted in open areas. at the a. mangium plot, the do of 1-year-old p. stellata and d. alatus seedlings were significantly higher than those of other species. after a. mangium was thinned by 50%, the do of d. alatus (2.38 cm) gradually increased and was the highest at 2 years after planting, but that of d. gracilis (1.21 cm) seedlings was the lowest. reducing the a. mangium canopy positively influenced the do of d. alatus, a. roxburghii, h. odorata and p. stellata seedlings, but not of d. gracilis and a. gratissima seedlings which exhibited a lower growth rate. moreover, their mortality rate was 100% in the open plot, 2 years after planting. species vary in their responses to different light conditions (hattori et al. 2009). the d0 and dbh of d. alatus and h. odorata in a 75% thinned eeucaena leucocephala plantation were higher than those in a 50% thinned l. leucocephala plantation, while d0 and dbh of shorea henryana in a 100% thinned l. leucocephala plantation were higher than in a 75% and 50% thinned plantation (sakai et al. 2014). height growth of the six dipterocarp seedlings was significandy different among the species at the a. mangium plot. the p. stellata seedling was the tallest at agesl and 2 years. among the 3-year-old seedlings at the thinned a. mangium plot, a. roxburghii (255.01 cm) was the tallest but not different from p. stellata (215.78 cm), while d. gracilis (138.87 cm) was the shortest. d0, h, and survival rate of d. gracilis seedling were low, indicating its unsuitability for mined out areas with poor soil and bright light conditions. the seedlings of a. roxburghii and h. odorata planted at the thinned m. mangium plot were taller than those planted in the open plot. both species were the tallest when planted with nurse trees, an observation similar to that of norisada et al (2005). however, this result is contrary to that of niamrat and marod (2005), who indicated that the h. odorata seedlings planted in open areas were taller than those under a canopy of 4 and 20-year-old eucalyptus camaldulensis stand. similarly, the h. odorata seedling growth rate was higher in an open area compared to that in a. auriculuformis and e. camaldulensis plantations (sakai et al. 2009). the good growth of d. alatus seedlings both in the open and a. mangium plots was also observed by niamrat and marod (2005). soil properties of the post-mining area limited the tree growth and seedling establishment because of its low soil nutrients and high sand particle. 120 d growth performance of dipterocarp species — wongprom iameter at root collar 8 /— s 6 e & 4 ® 2 0 b b a d cd cd m-mi ab b an b a b b open plot | am plot 1year-old open plot | am plot 2year-old open plot | am plot 3year-old dg eda 0 ho @ sg asr ^ps 300 9250 «, 200 150 •3 100 w 50 0 total height c a c imrfhfei a ttg open plot | am plot 1year-old a iab bj b c-=-3 a open plot am plot 2year-old a c ab ab ab a be open plot am plot 3year-old dg 0 da ho sg sr ^ps figure 4 diameter at root collar (do) and total height of dipterocarp species at the open and a. mangium plots notes: different letters above bars indicate significant differences (p < 0.05) among treatments by dmrt; dg = diptertocarpus gracilis, da = dipterocarpus alatus, sr = shorea roxburgii, sg = shorea gratissima, ho = hopea odorata, ps = parashorea stellata. the 3-year-old dipterocarp seedlings, growing under the a. mangium canopy, showed a high survival rate and satisfactory stem and height growth. hence, rehabilitation of mining areas using dipterocarp species should include a. mangium nurse trees as has been recommended in degraded sites (norisada et al. 2005). a. mangium not only enhanced the growth and survival rates of dipterocarp trees, but also improved the soil properties (kongchum et al. 2016), particularly nitrogen, a crucial nutrient for seedling growth in degraded areas. fast-growing trees also play an important role in reducing the demand for wood from the natural forests (west 2014). a. mangium grows well and are widely used for restoring degraded areas (lamb 2011). thinning of a. mangium stands can improve both the growth and quality of the wood of the remaining a. mangium trees (wongprom et al. 2013). relative growth rate (rgr) rgr of the base diameter (rgrdo) in the open plot significantly differed among the 1year-old species, but not among the 2 and 3year-old (table 4). rgrdo of a 1-year-old s. roxburghii (0.67 cm/cm/yr) seedling was the highest followed by d. alatus (0.55 cm/cm/yr), p. stellata (0.49 cm/cm/yr), s. gratissima (0.45 cm/cm/yr), h. odorata (0.33) cm/cm/yr, and d. gracilis (0.12 cm/cm/yr). rgr of the height (rgrh) of all the 1 to 3-year-old dipterocarp seedlings in the open area did not significantly differ (p > 0.05), but was the highest for the s. roxburghii seedling (0.58 cm/cm/yr). the s. roxburghii seedling growth both for rgrdo and rgrh in the open area were greater than those of dipterocarpus oblongifolius and dryobalanops oblongifolia (ang et al. 2003). the rgrdo of the 1-year-old dipterocarp seedlings planted at the a. mangium plot was not significantly different among species, but for 121 biotropia vol. 27 no. 2, 2020 rgrh, s. roxburghii (0.71 cm/cm/yr) and p. stellata (0.70 cm/cm/yr) seedlings had significantly higher rgrh than d. alatus (0.31 cm/cm/yr), d. gracilis (0.16 cm/cm/yr), h. odorata (0.38 cm/cm/yr) and s. gratissima (0.38 cm/cm/yr). after the a. mangium plantation was thinned by 50%, the rgrdo and rgrh values of 2-year-old s. roxburghii seedling were 0.91 and 0.75 cm/cm/yr, respectively and were the highest. however, both rgrdo and rgrh for d. gracilis seedlings (0.36 and 0.28 cm/cm/yr, respectively) were the lowest. after the removal of the a. mangium crown cover d. alatus and h. odorata seedlings attained a much taller height. the intensity of light reaching the seedlings had increased, leading to enhanced growth rates of the dipterocarp (hattori et al. 2009; sakai et al. 2011) as well as non-dipterocarp seedlings growing under the canopy (sakai et al. 2011). the relative growth rates of the dipterocarp seedlings also depend on the thinning intensity of the nurse trees, species type and methods of planting, line, gap or strip (hattori et al. 2009; sakai et al. 2009; sakai et al. 2014). the rgrdo and rgrh of p. stellata seedlings drastically decreased after the thinning of a. mangium, indicating its shade tolerant ability. among the 3-year-old dipterocarp seedlings planted at the a. mangium plot, s. roxburghii had the highest rgrdo and rgrh. however, rgrdo and rgrh of s. roxburghii was relatively higher in the a. mangium stand. similarly, rgrdo and rgrh of d. alatus and h. odorata seedlings in the open was greater than that in the a. mangium stand, suggesting that s. roxburghii, d. alatus, and h. odorata can be planted in open areas, even under severe environmental stresses in post¬ mining sites. table 4 relative growth rate (cm/cm/yr) of diameter at root collar (do) and height (h) of dipterocarp seedlings at the open and the a., mangium plots age species open plot a. mangium plot (year) rgrdo rgrh rgrdo rgrh 1 dipterocarpus gracilis 0.12a 0.26 0.35 0.16a dipterocarpus alatus 0.55bc 0.42 0.66 0.31a hopea odorata 0.33ab 0.33 0.56 0.38a shorea gratissima 0.45bc 0.38 0.51 0.38a shorea roxburghii 0.67c 0.44 0.73 0.71b parashorea stellata 0.49bc 0.34 0.70 0.70b f-value 3.602* 0.742ns 2.002ns 5.776* 2 dipterocapus gracilis 0.36a 0.28a dipterocarpus alatus 0.64 0.44 0.62bc 0.33a hopea odorata 0.57 0.33 0.73cd 0.47a shorea gratissima 0.40ab 0.36a shorea roxburghii 0.80 0.59 0.91d 0.75b parashorea stellata 0.49ab 0.39a f-value 1.755ns 1.361“ 9.335** 6.843** 3 dipterocapus gracilis 0.29ab 0.33b dipterocapus alatus 0.56 0.64 0.31ab 0.49bc hopea odorata 0.43 0.39 0.45b 0.50bc shorea gratissima 0.21a 0.35b shorea roxburghii 0.44 0.67 0.41b 0.58c parashorea stellata 0.30ab 0.16a f-value 1.424ns 2.705ns 2.752* 9.548** notes: * significantly different at p < 0.05; ** — significantly different at p < 0.01; ns non-significant difference at p > 0.05; different letters in the same column indicate significant difference by dmrt. 122 growth performance of dipterocarp species — wongprom conclusion dipterocarp seedlings planted in the abandoned mining area in southern thailand performed better with a. mangium as the nurse trees. their survival rates in the a. mangium stand clearly improved and exceeded 75%. in contrast, the dipterocarp seedlings planted in the open plot had lower survival rates, except for s. roxburghii, which was moderate. the stem diameter at the root collar and the height of s. roxburghii, d. alatus, p. stellata, and h. odorata seedlings were higher in the a. mangium plot than that in the open plots indicating that these species can be used to rehabilitate degraded lands. the stem diameter at root collar, height and relative growth rate of s. roxburghii seedlings were relatively high, indicating its suitability for rehabilitating mined out areas with poor soil conditions, with or without the use of nurse trees. moreover, thinning intervention should also be practiced to improve the growth of dipterocarp seedlings. mixed plantation of fast¬ growing trees and dipterocarp species is therefore, recommended for rehabilitating degraded areas, for improving forest structure, diversity, soil properties and for promoting wood utilization of fast-growing trees. acknowledgements this study was financially supported by the kasetsart university research and development institute (kurdi), thailand. the author is grateful to the associate professor dr. ladawan puangchit for her valuable suggestions and to the staff at the 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10.11598/btb.2021.28.2.1280 156 sulfate ammonium fertilizer on the off-season production of snake fruit (salacca sumatrana becc.) rasmita adelina1*, irfan suliansyah2, auzar syarif2, and warnita2 1agrotechnology study program, faculty of agriculture, universitas graha nusantara, padangsidimpuan 22715, indonesia 2agriculture science study program, faculty of agriculture, universitas andalas, padang 25136, indonesia received 5 july 2019/accepted 9 january 2020 abstract salacca sumatrana (becc.), known locally as the sidimpuan snake fruit, is one of the specialties prime local commodities of padangsidimpuan city in sumatra. the fruit is known for its sweet, sour and astringent taste which differentiates it from pondoh and balinese snake fruits. recently, the snake fruit farmers have noticed a continuous decrease in production resulting from the failure in its fruit-setting, particularly during the off-season. the use of fertilization and drip irrigation in the off-season had been currently explored as part of the solution. hence, this research investigates the use of these methods in overcoming the fruit setting failure and guaranteeing subsequent production of sidimpuan snake fruit all-year round. specifically, this study aimed to determine the optimal dosage of ammonium sulfate fertilizer and drip irrigation for fruit setting during the offseason. this research used a split-plot design with the main plot for drip irrigation and the subplot for ammonium sulfate. the observed parameters included the number of flower and fruit bunches, fruit set percentage and a nutrient analysis of the leaves. drip irrigation significantly affected the fruit setting percentage and the number of harvested fruit bunches. the best treatment combination was at 400 g ammonium sulfate fertilizer per plant and drip irrigation of 3,000 ml/plant. the fertilization period in july-september produced an off season harvest that was comparable to the fruit set percentage (10.76% difference) and number of fruit bunches (25.65% difference) that were observed in the april-june fertilization for the on-season harvest. this indicated that applying ammonium sulfate with drip irrigation could overcome fruit set failure in sidimpuan snake fruit, particularly, during the off-season. keywords: drip irrigation, off-season, production, snake fruit, sulfate ammonium introduction sidimpuan snake fruit (salacca sumatrana becc.) is one of the specialty products locally produced primary commodities of padang, sidimpuan city. the fruit is known for its uniquely sweet, astringent and sour taste, differing from the pondoh and balinese snake fruits and other types of snake fruits. the species is spread throughout the sub-districts in the southern part of the tapanuli regency, predominantly at the districts of angkola barat, east angkola, south angkola, and marancar. sidimpuan snake fruit production shows high development potential in south tapanuli covering approximately 19,155 ha with a production potential of up to 30 tons/ha (bps 2015). hence, the development of an optimal cultivation technology is necessary. currently, the crop production undergoes high fluctuations between the main harvest season (on season) and harvest outside this season (off season) resulting in a continuously declining production. this decreasing production is due to the fruit-set failure in the off season which is caused by a number of adverse environmental factors, particularly, those which do not support the production process, including low rainfall and few rainy days, and low soil nutrient content resulting in the lack of vital nutrients as indicated by low nitrogen, phosphorus and potassium content in the leaves (rai et al. (2010). *corresponding author, email: rasmita301271@gmail.com sulfate ammonium fertilizer on the off-season production of snake fruit (salacca sumatrana becc.) – adelina et al. 157 for the sidimpuan snake fruit to bear well outside the main harvest season, off-season treatments need to be applied. these would include nutrients supplementation through fertilizer application and simple drip irrigation technique. fertilization with ammonium sulfate and potassium chloride and simple drip irrigation were tried to meet the nutritional and water requirements of sidimpuan snake fruit in increasing its growth, fruit formation and production, out of season. under drip irrigation, fruit-setting was at 75.30% while that of no drip irrigation it was at 59.94% (rai et al. (2010). ammonium sulfate fertilizer provides nitrogen and sulfur and potassium chloride fertilizer as the main source of potassium which plays a role in plant growth and development. the availability of nitrogen and sulfur nutrients by fertilizing with ammonium sulfate at a dosage of 300 g had increased suwaru snake fruit production (sudaryono 2005) while the use of simple drip irrigation had efficiently and optimally meet the plants’ water requirements. hence, this study was aimed to obtain the best dosage combination of ammonium sulfate fertilizer and simple drip irrigation techniques to optimize fruit setting and to increase the sidimpuan snake fruit production in the offseason thereby optimally producing fruit throughout the year. materials and methods time and location of research the present study was conducted in april 2018 until september 2018 at the palopat maria village padangsidimpuan hutaimbaru subdistrict, padangsidimpuan city (between 010 28’19’’ 010 18’ 07’’ n and 990 18’ 53’’ 990 20’ 35’’ e). research methodology sixty (60) trees from among the 20 to 30year-old productive snake fruit trees were measured in the study applying the split plot design, consisting of three replications. the main plot is the application technique either, with drip irrigation of 3,000 ml/plant/day (p1) or with no irrigation (p0). the simple drip irrigation installation included infusion bottles, infusion tubes, drippers, plastic hoses and water pumps. the subplots for the fertilization technique consisted of plots without fertilization (p0) and plots with ammonium sulfate fertilizer applied at 250 g/plant + 40 g potassium chloride/plant (p1), 300 g/plant + 40 g potassium chloride/plant (p2), 350 g/plant + 40 g potassium chloride/plant (p3), and, 400 g/plant + 40 g potassium chloride/plant (p4). hence, the study consisted of 10 treatment combinations with 3 replications and 2 plants per plot, totalling to 60 plants. fertilizer applications were carried out in 2 periods; fertilization for the april june 2018 period was carried out on 14 march 2018, while fertilization for the july september 2018 period was carried out on 22 july 2018. previously, fertilization had been done in august 2017 and december 2017. fertilization was carried out by immersing the appropriate amounts of ammonium sulfate and potassium chloride fertilizer according to treatment into a 10 15 cm deep fertilizer groove in the soil that is 50 60 cm from the base of the stem. watering was a simple irrigation system that involved water movement carried out by gravity. drip irrigation equipment included a plastic tube as a water storage container, an infusion hose installation equipped with a dripper at the end that released water at a rate of 250 ml/30 minutes with a watering volume of 3000 ml/day. the drip irrigation was given daily for 6 hours from 10 am 4 pm. however, if it rains very heavily, the drip irrigation was not applied. the observed parameters were the fruit-set percentage, number of flower and fruit bunches, number of harvested fruit bunches, relative water content (rwc) and the nitrogen, phosphorus and potassium content on the leaves. the number of flower bunches and fruits were counted once every two weeks on each sample plant. analysis of leaf nitrogen, phosphorus and potassium content was carried out once every fertilization period. three leaves taken from each sample plant for laboratory analysis were cleaned, ovendried at 70 oc, then blended and sieved using a 0.5 mm grid sieve. the total nitrogen was determined using the kjeldahl semi-micro method, while the dry ashing method was used for determining phosphorus and potassium content. phosphorus concentration was measured with a uv-vis spectrophotometer, while potassium biotropia vol. 28 no. 2, 2021 158 concentration was determined by using a flame photometer. data were analyzed using anova and if differences among treatments were significant, duncan's multiple range test was applied. results and dicussion fruit set percentage (%) during the april-june fertilization period (on season), the highest fruit set percentage was at 76.949% indicating a significant increased caused by using the drip irrigation (table 1). the highest fruit set percentage of 75.615% also occurred at the 300 g/plant za dosage of ammonium sulfate fertilizer. the lowest response was from no irrigation (54.478%) nor fertilization (47.115%). drip irrigation has increased the relative water content of the leaves. hence, the high fruit-set percentage with drip irrigation was a result of the high relative water content (rwc) of leaves which in turn impacted the chlorophyll and potassium content (sunarka 2015). watering the soil then improves its chemical properties encouraging root growth and increasing physiological activity in the snake fruit plants, thereby improving their fruit setting capacity. in the july-september fertilization period (off season), the fruit set percentage (58.863%) was not significantly affected by drip irrigation treatment (68.673%). this was during the wet season, so the additional water was not necessary. the number of bunches formed and flower fall is influenced by environmental factors (adijaya et al. 2013). in the dry months, the flower fall usually increases thereby reducing the number of bunches formed. the use of ammonium sulfate fertilizer also had a statistically significant impact on the fruit setting with the highest response at 400 g per plant (77.792%). in another study, drip irrigation had significanly affected the out of season fruit production in dry conditions showing a fruit set percentage of 75.30% (off-season) and 93.13 % (on-season), compared to 59.94% and 61.67%, respectively, without irrigation (rai et al. 2014). in the july-september fertilization period of this study, the drip irrigation resulted in a significantly higher fruit set percentage. analysis of variance indicated that the effect of the combination of drip irrigation treatment and ammonium sulfate fertilizer dosage on the percentage of fruit formation was not significant (table 2). however, the treatment combination that resulted in the best fruit set percentage in the april-june fertilization period was at a dosage of 350 g fertilizer per plant with drip irrigation (91.667%) (table 2). whereas, in the fertilization period of july-september, the highest percentage of fruit set was with 400 g with drip irrigation (83.997%) (table 2). in another study, the combination of drip irrigation and nitrogen fertilizer had also significantly increased the yield of cotton plants (gossypium hirsutum) (zhong & bai 2013). moreover, under medium irrigation, the ratio of dry matter in nutritional organs to reproductive organs was also increased (zhong & bai 2013). table 1 the average percentage of fruit set, number of flower bunches, fruit and number of harvested fruit bunches with drip irrigation treatment and different dosages of ammonium sulfate fertilizer applied in april june and july september 2018 treatment fruit set (%) number of bunches number of harvested fruit bunches flower fruit apr-jun jul sept apr-jun julsept apr-jun julsept apr-jun julsept without irrigation 54.478a 58.863 19.267b 15.000 10.533 8.467 9.933 12.467a drip irrigation 76.949b 68.673 16.467a 14.267 12.733 9.467 12.067 18.200b without fertilizer 47.115a 52.620a 16.333 16.333 7.667a 7.833a 10.500 13.000 za 250 g 59.708ab 57.800ab 17.833 13.500 0.500ab 7.667a 9.000 14.667 za 300 g 75.615b 68.620ab 18.667 14.833 14.167b 10.000ab 11.000 13.833 za 350 g 74.443b 62.008ab 18.333 14.167 12.833b 8.167ab 10.167 18.000 za 400 g 71.687b 77.792b 18.167 14.333 13.000b 11.167b 14.333 17.167 note: means with different superscripts within a column are significantly different at p≤0.05, with comparisons performed using dmrt. sulfate ammonium fertilizer on the off-season production of snake fruit (salacca sumatrana becc.) – adelina et al. 159 the application of ammonium sulfate fertilization and drip irrigation had increased the percentage of fruit set in both fertilization periods. this indicated that these production techniques will be able to minimize the production fluctuations in snake fruit between the harvest season (on season) and small harvests (off season). other study results also showed that both irrigation and nitrogen fertilization promoted cotton growth and yield (zhuan et al. 2017). drip irrigation technique had also increased the fruit set percentage of gula pasir snake fruit during the off season in a dryland ( rai et al. 2014). number of flower bunches during the april june (off season) fertilization period, the flower formation was significantly affected by the drip irrigation as a single factor (table 1). no irrigation treatment resulted in the most numerous flower bunches formed (19.267). ammonium sulfate fertilizer dosage at 300 g/plant also produced the highest average number of flower bunches (18.667) (table 1). during the july september (on season) fertilization period, the drip irrigation nor the fertilization did not significantly influence the flower formation (table 1). however, there was a tendency for more flower bunches with the no-irrigation treatment. this absence of significant difference with the irrigation technique is similar to that found by jose et al. (2013) who used regulated deficit irrigation techniques to improve fruit size. reduced irrigation and fruit thinning were found to affect carbon allocation within the tree by altering a number of interrelated factors (photosynthesis, location and number of competing sinks, storage capacity, and transport) that control the carbon partitioning in fruit trees. other factors affecting flowering could be interspecific differences and different crop seasons which was probably caused by variable atmospheric conditions during vegetation periods (greiner & kohl 2014). no significant relationship exists between fertilizer dosage and flower formation. in a study on sweet orange plants, the application of certain dosages phosphorus and potassium compound fertilizer had only a small effect on the number of flowers formed (ramadhan et al. 2015). other factors that cause unfertilized plants to flower as prolifically as those treated with fertilizer could be the irrigation and less than optimal water management. table 2 average fruit set percentage (%) with the combined drip irrigation and ammonium sulphate fertilization in april-june 2018 and july-september 2018 treatment dosage of ammonium sulfate fertilizer (gram) average 0 250 300 350 400 i* ii** i ii i ii i ii i ii i ii without irrigation 34.140 44.330 50.437 52.503 67.603 65.157 57.220 60.740 62.990 71.587 54.478 58.863 drip irrigation 60.090 60.910 68.980 63.097 83.627 72.083 91.667 63.277 80.383 83.997 76.949 68.673 average 47.115 52.620 59.708 57.800 75.615 68.620 74.443 62.008 71.687 77.792 notes: * = fertilization in april–june 2018; ** = fertilization in july-september 2018 table 3 average number of flower bunches with ammonium sulfate fertilizer treatment treatment dosage of ammonium sulfate fertilizer (gram) average 0 250 300 350 400 i* ii** i ii i ii i ii i ii i ii without irrigation 16.667 19.333 19.667 12.000 18.667 16.333 21.667 14.333 19.667 13.000 19.267 15.000 drip irrigation 16.000 13.333 16.000 15.000 18.667 13.333 15.000 14.000 16.667 15.667 16.467 14.267 average 16.333 16.333 17.833 13.500 18.667 14.833 18.333 14.167 18.167 14.333 notes: * = ammonium sulfate fertilization in april-june 2018; ** = ammonium sulfate fertilization in july-september 2018. biotropia vol. 28 no. 2, 2021 160 the combination of drip irrigation and the different ammonium sulfate dosages did not significantly affect the number of flower bunches. similar results were observed when new flower buds formed spontaneously every 1 1.5 months at the base of the leaf midrib but were uninfluenced by the combined irrigation and fertilization treatments (adelina 2017). furthermore, it was then assumed that the two treatments would influence the formation of new flower buds, later, when the fruit bunches were formed and until finally, when the fruit bunches were harvestable (table 3). the application of certain dosages of compound fertilizer phosphorus and potassium had also a minimal effect on the number of flowers in sweet orange plants (ramadhan et al. 2015) number of fruit bunches the number of fruit bunches in the apriljune fertilization period was not significantly affected by the drip irrigation in which the irrigated plants produced an average of 12.733 bunches per plant. in contrast, drip irrigation had a significant effect on both growth traits and fruit production indicating that it was important in zones with water limitations (loewe & delard 2016). the different dosages of ammonium sulfate fertilizer has significantly affected fruit bunch formation but the best dosage appeared to be at 400 g with 13.000 bunches of fruit per plant, and the 250 g at only 10.500 bunches (table 1). in the july-september fertilization period, the drip irrigation had no significant effect on the number of fruit bunches formed but it appeared to give a slightly higher number of fruit bunches (9.467) compared to the no-irrigation technique (8.467) (table 1). during this period, there was sufficient rain so the effect of irrigation in the formation of fruits seemed neligible. the drip irrigation was more effective during the dry season rather than on the rainy season (biswas et al. 2016). the same effect happened in the increased yield of tomato associated with the increasing amount of irrigation water (biswas et al. 2016). the optimal dosage of ammonium sulfate fertilization for fruit bunch formation was at 400 g with 11.167 bunches and the 250 g only yielding 7.667 bunches, showing a statistically significant difference (table 1). the use of nitrogen fertilizer and optimal water application were also recommended for the summer production of cotton (zhuan et al. 2017). in the april-june fertilization period, the combined treatments that resulted in the formation of the largest number of fruit bunches was drip irrigation with the 300 g fertilizer that produced 15.667 bunches (table 4). this interaction between irrigation and fertilization influenced most of the plant physiological functions and growth (wang et al. 2018). the same results indicating better economic production were observed in the use of fertilizer with daily drip irrigation for corn growing on a semi-arid area (chauhdary et al. 2017). number of harvested fruit bunches in the april-june fertilization period, neither drip irrigation nor dosages of ammonium sulfate fertilizer significantly affected the number of harvested fruit bunches. however, drip irrigation with 12.067 bunches and 400 g of fertilizer with 14.333 bunches appeared to give the best fruit harvest compared to 9.000 bunches from the 250 g of fertilized plants (table 1). ammonium sulfate fertilizer, as the main source of nitrogen, is crucial in increasing the crop production process. nitrogen application has increased the seed yield of coriander (coriandrum sativum l.) in a linear manner and the application of 60 kg/ha has improved the yield by 40% (alil et al. (2015). table 4 average number of fruit bunches with combined drip irrigation and ammonium sulfate fertilizer treatment treatment dosage of ammonium sulfate fertilizer (gram) average 0 250 300 350 400 i* ii** i ii i ii i ii i ii i ii without irrigation 5.667 7.667 10.000 6.333 12.667 11.000 12.000 8.000 12.333 9.333 10.533 8.467 drip irrigation 9.667 8.000 11.000 9.000 15.667 9.000 13.667 8.333 13.667 13.000 12.733 9.467 average 7.667 7.833 10.500 7.667 14.167 10.000 12.833 8.167 13.000 11.167 notes: * = combined treatment in april-june 2018; ** = combined treatment in july-september 2018. sulfate ammonium fertilizer on the off-season production of snake fruit (salacca sumatrana becc.) – adelina et al. 161 in the july-september 2018 fertilization period, drip irrigation had resulted in a statistically significant 18.200 harvested fruit bunches while any dosage of the ammonium sulfate fertilizer did not. the plants applied with the drip irrigation also produced a higher number of harvested bunches compared to those plants that were not given (rai et al. 2013). the best yield was at the 350 g fertilizer dosage with 8.000 bunches of harvested fruit compared to 12.467 bunches from the non-irrigated unfertilized plants (table 1). the non-significant effect observed on the combined irrigation and ammonium sulfate fertilizer treatment on the number of harvested fruit bunches implied that no real interactions existed between the two treatments. however, 22.667 fruit bunches resulted from a combination of 400 g fertilizer with drip irrigation and 7.000 bunches from the 250 g with no-drip irrigation (table 5). the lack of influence of ammonium sulfate fertilization suggested that drip irrigation alone can meet certain needs of sidimpuan snake fruit plants. the use of organic fertilization has also increased the fruit set percentage (ability to bear fruit) of snake plants (dewi 2014). the failure of fruit development from the flowers of the gula pasir snake fruit was more likely due to other environmental and plant physiological factors rather than the application of fertilization (rai et al. 2010). the development pattern of snake fruit plant production and distribution is strongly influenced by physiographic environments such as the altitude, land, rainfall, and air temperature (cahyani et al. 2013). these environmental factors include particularly, low rainfall and number of rainy days which lower the relative water content (rwc) of leaves thereby disrupting the metabolic processes. the average rwc of leaves in the july-sept 2018 period was higher than those of leaves in the april-june period (fig. 1). the positive impact resulted in a higher average number of fruit bunches harvested in the july-september period using the drip irrigation. this application of irrigation also resulted in greater fresh biomass, fresh leaf yield, and dry leaf yield in stevia plants (benhmimou et al. 2018). the higher leaf rwc with the drip irrigation treatment showed that irrigation increased the water content of the plant tissues thereby positively affecting the physiological processes as indicated by the increased plant ability to take up nutrients (rai et al. 2014). the nitrogen, phosphorus, potassium content and rwc of leaves in the julyseptember fertilization period was higher than those of the april-june 2018. very high differences were found in the nitrogen content and rwc of leaves (fig. 1). the leaf nitrogen content in the july-september fertilization period was higher (2.194%) than in the apriljune fertilization period (1.384%). nitrogen content and the rwc varied in a similar way to the percentage of fruit set and the highest number of fruit bunches obtained in the julyseptember fertilization period compared to those of the april-june 2018. the low productivity of gula pasir snake fruit was influenced by the low level of nitrogen in the leaves (rai et al. 2010; dewi 2014). this nutrient deficiency has influenced the plant physiological processes resulting in the failure of flower development into fruit due to photosynthate deficiency indicated by sucrose content, total sugar, and reducing sugars in the table 5 average number of harvested fruit bunches with the combined drip irrigation and ammonium sulfate fertilizer treatments in april-june 2018 and in july-september 2018 treatment dosage of ammonium sulfate fertilizer (gram) average 0 250 300 350 400 i* ii** i ii i ii i ii i ii i ii without irrigation 10.000 11.667 7.000 12.000 9.000 11.000 8.333 16.000 15.333 11.667 9.933 12.467 drip irrigation 11.000 14.333 11.000 17.333 13.000 16.667 12.000 20.000 13.333 22.667 12.067 18.200 average 10.500 13.000 9.000 14.667 11.000 3.833 10.167 18.000 14.333 17.167 notes: * = combined treaments in april-june 2018; ** = combined treatments in july-september 2018. biotropia vol. 28 no. 2, 2021 162 figure 1 average nitrogen, phosphorus, potassium and relative water contents of leaves leaves at low interest due to high competition in fighting photosynthesis. fruit weight and fruit yield have been found to be significantly and positively correlated with nitrogen, phosphorus, potassium, iron, zinc and copper contents of the citrus var. kinnow mandarin leaf (kaul et al. 2014). leaf nutrient content is one indicator of nutrient availability which is critical in plant growth and development (marschner 1986). if the production process is not balanced with the availability of nutrients, in general it will cause a decrease in production. the nitrogen and phosphorus contents of sidimpuan snake fruit leaves were found to be higher than those of pondoh and sumedang leaves but the potassium content was lower (islami 2014). nitrogen status is related to leaf water content which also influences chlorophyll formation (fig. 1). drip irrigation increased the maximum chlorophyll content and photosynthetic nitrogen use efficiency (wang et al. 2018). for chlorophyll production, a schedule combining drip irrigation with 300 kg nitrogen/ha has provided the highest average chlorophyll production at an increase of 62% above non-irrigated levels (perez-ortola et al. 2016). nitrogen when absorbed by plants could play an important role in the chlorophyll formation as indicated by an increase in the green leaf color (pangaribuan et al. 2018). fertilization increases the soil nutrient availability for the plants to absorb. the average nitrogen and potassium levels of the sidimpuan snake fruit leaves were increased after the application of ammonium sulfate fertilizer (adelina et al. 2018). the higher the frequency of fertilization, the more secured is the availability of soil nutrients for plant growth and development (vargas & david 2015). the lack of plant response to fertilizer in this research may have been due to the plants’ need for a continuous supply of nutrients throughout the year. hence, a better result may have been achieved if fertilizers were given more often. for further research, it is recommended to increase the dosage of ammonium sulfate and potassium chloride fertilizers to determine a possible increase in the the fruit set percentage and production of sidimpuan snake fruit. conclussion drip irrigation during the off season in julyseptember has improved both the fruit set percentage and the number of harvested fruit bunches of the sidimpuan snake fruit. the best treatment, as compared to no drip irrigation, was the irrigation with 3000 ml/plant/day. as a single separate factor, the ammonium sulfate fertilizer treatment and the drip irrigation application significantly influenced the fruit set percentage and number of formed fruit bunches. no significant interaction existed between irrigation and fertilization. however, sulfate ammonium fertilizer on the off-season production of snake fruit (salacca sumatrana becc.) – adelina et al. 163 based on the average number, the combined treatments that gave the best response was obtained at the ammonium sulfate fertilizer dosage of 400 g/plant. with drip irrigation, the off-season harvest in july-september was comparable to that of the fruit set percentage and number of fruit bunches formed with fertilization, during the main harvest season in april-june. acknowledgements this research was funded by dp2m dikti, through a doctoral dissertation grant in the 2018 implementation year. the researchers, therefore, would like to express their deepest gratitude to the director of dp2m dikti for the funding support. an infinite gratitude from the researchers is also extended for the collaboration of the research team from the department of agrotechnology, faculty of agriculture, graha nusantara padangsidimpuan university, as well as to the analytical staff at the soil department laboratory, faculty of agriculture, andalas university, padang, indonesia. references adelina r, irfan s, auzar s, warnita. 2017. study of sidimpuan salak cultivation (salacca sumatrana becc.). grahatani journal 3(1):434-43. adelina r, 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https://doi.org/10.17660/actahortic.2014.1015.37 https://doi.org/10.1111/grs.12116 biotropia vol. 28 no. 2, 2021: 102 108 doi: 10.11598/btb.2021.28.2.1079 102 true shallot (allium cepa var ascalonicum) seed production during off season ramadhani eka putra1,2 *, d. beta ramadan1, adriyanita adin3, ida kinasih4 and indah oktaviani2 1school of life sciences and technology, institut teknologi bandung, jalan ganesha 10, bandung 40132, indonesia 2department of biology, institut teknologi sumatera, jalan terusan ryacudu, lampung 35365, indonesia 3pt east-west seeds indonesia, benteng, kecamatan campaka, purwakarta 41181, indonesia 4department of biology, universitas islam negeri sunan gunung djati bandung, cibiru, bandung 40615, indonesia received 8 june 2018/accepted 10 february 2020 abstract seed cultivation for true shallot is an alternative for the more common cultivation practice in which 30% of the harvested tubers are used for cultivation purposes. seed production of this temperate tuber in the tropical region, however, is quite challenging due to its low flowers and seed formation. several studies have shown that vernalization (cold induction) and application of benzil amino purin (bap) had improved the flowering and seed production of shallot. however, such studies were conducted during the best cultivation period for about 3 months and thus, limit the production period of seeds during the rainy season. this study was conducted to observe the effect of both methods outside cultivation periods on the flower and capsule numbers, fruit set, and weight of 100 seeds when compared with commonly practiced cultivation during the dry season. the onion bulbs vernalized at 10 oc for 30 days were subjected to synthetic hormone (bap) prior to planting. the shallot group treated with bap had the lowest values for all observed parameters, i.e., 1,552.67 number of flowers; 312.11 number of capsules; 22.5% seed set; and 0.2244 g weight of 100 seeds, compared to those in the vernalization treated group, i.e., 1,592.44 number of flowers; 623 number of capsules; 30.5% seed set; 0.2261 g weight of 100 seeds and control group 6,774.67 number of flowers; 3,898.44 number of capsules; 57.06% seed set; 0.3304 g weight of 100 seeds. in conclusion, the commonly practiced cultivation of sowing bulbs directly without vernalization and plant growth regulator treatment is probably the better method to produce shallot seeds during the offseason, the rainy season. keywords: benzil amino purin, true shallot seeds, vernalization introduction shallot (allium ascalonicum) is one of the most important tubers in indonesia, which is commonly used as food ingredients and traditional medicine. market demand for the certain commodity has increased annually with 5.30% average consumption growth (kementerian pertanian 2015). the shallot farmers in indonesia usually cultivate this commodity by its vegetative form. however, this method has several challenges, namely 1. short storage period of planting stock (suwandi & himan 1995); 2. variable quality (balai penelitian dan pengembangan pertanian 1995); 3. high susceptibility to disease spread (wibowo et al. 2016); 4. high production cost (gina & rofik 2010); and 5. significant amount of unsold harvested tuber (permadi & putrasamedja 1991; basuki 2009). these factors have prevented the true shallot total production to fulfill its market demand. to meet its market demand, the improvement of national shallot production through the use of botanical seeds or true shallot seed (tss) in shallot cultivation is necessary. seeds have longer storage time, up to six times their vegetative form, and eliminate the need for large storage room (basuki 2009) thereby also reducing the production cost (permadi & putrasamedja 1991; basuki 2009). *corresponding author: ramadhani@sith.itb.ac.id true shallot (allium ascalonicum) seed production during off season – putra et al. 103 however, most of the local growers have not applied this method due to the very limited amount of available true shallot seeds (rosliani 2013). environmental factors, such as average temperature, photoperiod, average humidity, are believed to be the limiting factors for seed production in indonesia (fahrianty 2013; wu et al. 2015). this biennial plant produces bulb as an overwintering stage of the life cycle and produces flowers in the spring after a period of winter or vernalization (brewster 2008). furthermore, true shallot also requires long photoperiod (> 12 hours) to ensure flowering and seed production (kamenetsky & rabinowich 2001). vernalization of bulb before planting ensures early flowering of seed crop (brewster 1994) and enables the seeds to produce a heavier yield (jones & mann 1963; mollah et al. 2005; ami et al. 2013) as the result of increasing gibberellin endogen and auxin production (dinarti et al. 2011). therefore, most true shallot seed producers in indonesia apply low-temperature shock treatment to the mother bulb and establish the plantation in higher elevation. moreover, improving the production application of plant growth regulators (pgr) is one of the common practices. a small number of plant regulators can be applied to adjust plant physiology, such as plant growth and development (yamaguchi & kamiya 2000). control of flowering and seed formation are among the most important practical aspects of seed production. among all plant regulators, cytokinin plays an important role from seed germination to delaying the onset of senescence (chatsudthipong & muanprasat 2009). furthermore, synthetic cytokinin is available, such as benzil amino purin (bap). some studies showed the beneficial effect of bap on flower numbers, flower size, flower longevity, and the number of seed produced (youngkoo et al. 2006; roslian et al. 2012; el-kinany et al. 2019). in indonesia, the true shallot seeds have been produced by applying either vernalization or bap to the mother bulb and planting in the highland during the dry seasons (rosliani et al. 2012). the present study, however, explored the possibility and limitation of the application of the vernalization technique and bap on true shallot seeds during the rainy season, with the intent to improve its production by providing viable seeds all year round. materials and methods study site the study was conducted at the field greenhouse of pt east west seed indonesia research station in lembang and school of life sciences and technology, institut teknologi bandung, indonesia. shallot for seed production was cultivated from october 2016 to april 2017 at the greenhouse, while the quality of seed produced was assessed at the institute. the field experiment was conducted during the rainy season at average temperature of 19 oc to 23 oc which is considered as off-season for true shallot seed production. vernalization of the bima mother bulbs bima variety, which is widely planted in center of shallot production and released by the balai penelitian tanaman sayuran was used as mother bulbs in this study. one hundred and twenty bima mother bulbs were put in white cotton cloth bags and vernalized in a refrigerator at a calibrated temperature of 10 °c for 30 days. after bulbs vernalization, a total of 60 bulbs were subjected to synthetic hormone treatment by dipping them in the bap solution, while another 60 bulbs were stored under controlled temperature (21±3 °c) and serve as the untreated control. to prevent the mother bulbs from fungi attack, all of the mother bulbs were dipped into fungicide solution before planting. land preparation the land was thoroughly prepared by plowing and cross plowing followed by laddering. the subsequent operations were done with harrow, spade, hammer, and other tools. weeds and stubbles were collected and removed from the field. irrigation and drainage channels were made around the plots with the corners trimmed by the spade. plant spacing the planting distances between rows and between bulbs were 25 cm and 20 cm, biotropia vol. 28 no. 2, 2021 104 respectively. each of the 3 plots contained four rows with 15 bulb seeds sown in each row, amounting to 180 bulbs sown at 7 cm depth. application of fertilizer and cultivation practices the true shallot planting stock was fertilized with the recommended doses of n:p:k 16:16:16 and dolomite. watering, weeding, and fungicide applications were conducted once a week during the cultivation period (115 130 days). during the flowering period, the plants were protected under plastic sheets, harbored above them to protect them from rainfall damage, while pollination was conducted by hand. harvesting and processing the cultivation period lasted for 115 130 days. when the seeds inside the capsules turned black and more than 25% black seeds were exposed on the umbel, each umbel was cut at 5 cm of the flower stalk. harvesting was conducted on days 116, 123, and 130 and the umbels were then sun-dried. threshing was done by light beating and hand rubbing of the umbels. the seeds were cleaned and sun-dried up to 7 days until seed moisture was reduced to below 8%. each of the harvested groups of seeds were processed separately and contained in separate paper bags and preserved for later use (mollah et al. 2015). seed weight and germination one of the methods to determine the quality of seeds is by measuring the total weight and germination rate of 100 seeds. one hundred seeds were randomly selected from each harvest and weighed (in gram, g) on an electric balance, placed in a plastic tray, and allowed to germinate. the plastic tray was filled with a paper towel previously dipped in liquid fertilizer. germination test was carried out according to the international rules for seed testing (ista 1996). the number of normal seedlings, abnormal seedlings, dead seeds, and ungerminated seeds were recorded for two weeks. then the germination percentage was determined by the following formula. germination = [(number of seedlings/number of seeds tested)] x 100% (1) data analysis the normality of data was analyzed by the one-sample kolmogorov-smirnov method. the differences among treatments on flowering initiation period, flower numbers, capsule numbers, fruit set, seed numbers, weight of 100 seeds, and seeding rates were analyzed using one-way anova with a significant level of p< 0.05. tukey analysis was conducted as the post hoc test when anova showed significant differences. all analyses were conducted using spss 16.0. results and discussion flowering initiation the time required to produce flowers in the untreated shallot plants (control group, with temperature of 21±3 °c) was significantly longer than those in the other groups, while those in the v+bap and v groups were relatively similar (fig. 1). these results conformed with previous studies that vernalization treatment on shallot bulbs required a shorter time to produce flowers (satjadiputra 1990; yan et al. 2003; islam et al. 2010; andres & coupland 2012; fahrianty 2013; ream et al. 2013; wu et al. 2015). the results showed the importance of vernalization treatment to initiate the flowering, which might be related to the temperate origin of true shallot. vernalization blocked the flowering repressor and induced the expression of genes responsible for the flowering (florigen) (lee et al. 2013). vernalization could also promote the upregulation of some key cytokinin signaling regulators which induced the flowering (wen et al. 2017). the application of bap, a cytokinin synthetic, might have induced gibberellin signaling that reduced the flowering initiation time (tarkowska 2012; wong et al. 2013). number of flowers, capsules, and seeds produced the control groups produced significantly more flowers than the other groups. on the other hand, the number of flowers produced by both v+bap and v groups were relatively similar (fig. 2). true shallot (allium ascalonicum) seed production during off season – putra et al. 105 figure 1 time required for producing flowers among all treatments notes: v + bap = vernalization + benzil amino purin (bap) treatment; v = only vernalization, control = no treatment (with temperature of 21±3°c); * = significant at p < 0.05. figure 2 number of true shallot flowers among treatments. notes: v + bap = vernalization + benzil amino purin (bap) treatment; v = only vernalization; control = no treatment (with temperature of 21±3°c); * = significant at p < 0.05. vernalization positively affected flower initiation and the number of flowers produced by shallot (fahrianty 2013). however, the information was based on the study conducted in dry season, the best season for true shallot seed production. the lower number of flowers produced by vernalization groups in the current study could be related to insufficient photoperiod. the true shallot is a long day plant that required 12 hours light period (currah & proctor 1990). shorter and epileptic light conditions during the rainy season may negate the positive effect of vernalization on flower production due to less optimal photoperiod (dennish & peacock 2009; wu et al. 2015). the lower number of flowers resulted in a fewer number of capsules (fig. 3a) and seeds (fig. 3b) of the vernalized shallot groups. most of the seed losses were caused by flower abortion and infection by fungi on the flowers of vernalized groups. the results indicated that plants of the vernalized groups had lower resistance to diseases probably due to the high humidity. vernalization reduced the vegetative period which benefited the seed production because when plants were growing in optimum condition, most of the plant energy was fully used in seed production. however, under suboptimal conditions, the plants have to overcome environmental stress and allocated less energy to seed production. on the other hand, the longer growing period of the control group could increase seed yield (farghali 1995), probably due to more available energy for seed production. seeds quality the weight of 100-seeds of the control groups was significantly higher than those of other groups, while the v+bap group produced slightly heavier seeds than the v group (fig. 4). biotropia vol. 28 no. 2, 2021 106 figure 3 (a) number of true shallot capsules and (b) seeds per plant among treatments notes: v + bap = vernalization + benzil amino purin (bap) treatment; v = only vernalization, control = no treatment (with temperature of 21±3 °c); * = significant at p < 0.05. figure 4 weight of 100-seeds among treatments notes: v + bap = vernalization + benzil amino purin (bap) treatment; v = only vernalization; control = no treatment (with temperature of 21±3°c); * = significant at p < 0.05. figure 5 germination rate of seeds produced among treatments notes: v + bap = vernalization + benzil amino purin (bap) treatment; v = only vernalization; control = no treatment (with temperature of 21±3°c); (*) = significant at p < 0.05. the seed weights of the vernalization groups were in contrast with those in previous studies that indicated the positive effect of vernalization on seed weight (mollah et al. 2005; ami et al. 2013). the high humidity of the rainy season might have caused significant damage to the seed resulting in its reduced weight (ku et al. 2008). the addition of bap after vernalization has improved the seed weight as it could have induced cell growth and tissue differentiation (rosliani 2013). based on this current study, vernalization probably induced only flower production, while the seed quality depends on different mechanisms such as the effect of vegetative propagation, flower numbers and the availability of the pollinator (krontal et al. 2000). therefore, further study is suggested to test this hypothesis. true shallot (allium ascalonicum) seed production during off season – putra et al. 107 germination rate of seeds produced by the control group was significantly higher than those of other groups, followed by v+bap group and v group (fig. 5). germination rate of seed highly depends on seed weight which explained the low germination rate of the vernalization groups (gamiely et al. 1990; mollah et al. 2005). germination rate in this study was also much lower than those produced in optimal season which indicated the importance of planting date (mollah et al. 2005; el-helaly & karam 2012). conclusion vernalization is required to induce the flowering of true shallot. however, the planting date plays an important role in the volume of seed production and quality. during the rainy season, the offseason, the commonly practiced cultivation technique is still the recommended method for the optimum production of true shallot seeds. reducing the detrimental effect of the rainy season to the true shallot plant induced with vernalization, is also suggested, such as maintaining seed production inside a closed system with a controlled environment, which is obviously a key factor in the sustainable production of true shallot seed in 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